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Microbiological transformation of conessine. II
421
IdICROBIOLOGICALTEAWSPORMATIOW OF COWESSIWE. II. A.F. Urx, H.C. Beck, W.F. van der Waard and J. de Flines
Research Laboratories of the Royal Netherlands Fermentation Industries Ltd., Delft, Holland Received April 4, 1966 (Note: "MicrobiologicalTransformationof Conessine. III" appeared in the September 1966 issueof Steroids.)
ABSTRACT Incubation of4conessine with Stach botr s arvi ora Hughes yielded A -conenin-J-one ZiiZ+&%o$&+cononin-J-one. Fermentation with Gloeoa orium fructi num '*he yielded a mix oonessine and lia-hydroxyconessine.
In our first publication on the microbiological transformation of conessine (1) we reported the conversion of conessine (I) into
A 4-conenin-3-one by
Gloeosporium cyclaminis and Hypomyces haegtatococcus. Since then the microbiological hydroxylations of conessine by Aspergillus ochraceus (2) and Cunninghamella echinulata (3) have been reported. We now wish to describe the transformation of conessine with Stachybotrys narvispora Hughes and Gloeosporium fructigenum f. americana KrQer. Stachybotrys parvispora has not yet been reported as a microorganism capable of hydroxylating ster
belongs to the family of Dematiaceae, which includes
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several genera that are able to hydroxylats many steroids1 compounds at the eleven carbon atom, e.g. Curv-ularia(4), Helminthosporium (5),
Stamphylium (6) and
Stachylidium (7). Incubation of conessine with Staohybotrys parvispora Hughes yielded a mixture of II and III (scheme I). Thin layer and paper ohromatograms of ssmples taken during the incubation showed the conversion of I into II and thence of II into III. Fermentation of II with Stachybotrys narvispora Hughes or Asnergillus ochraceus also yielded III. Compound II was identical with A4 -conenin-j-one and III proved to be the Ala-hydroxy derivative of II. The infrared and ultraviolet absorption spectra of III showed the presence of a hydroxyl group and a
A4-j-one
system. Chromium
trioxide oxidation of III in acetic acid yielded IV identical with A4-conenine-3, ll-dione (8). Since III was easily acetylated with pyridine-acetic anhydride at room temperature the hydroxyl group must possess the a-orientation. This was also deduced from the nuclear magnetic resonance spectra and the molecular rotations, In the NMR spectra (Jin p.p.m,) the C,,-proton signal was found at 3.66. After deuterium exchange this signal consisted of a triplet of doublets (Jax,ax E 10 cps; Jax,ae = 4 cps) which demands an axial (p) orientation of the C,,-proton (two axial and one
Oct.
1966
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Schetne I
423
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equatorial protons in a-positions), The same conclusion was reached on the basis of molecular rotatory contributions, Among the A4-3-ketosteroids (9,lO) the lla-hydroxylated derivatives have lower and the lip-hydroxy and 11-0x0 derivatives have higher 5
values than the parent compounds. Therefore,
by this analogy the hydroxyl group in III was also assigned the a-configuration,
Several Gloeosporium species have already been described for the microbiological conversion of steroids, e.g. 2. foliicolum
(11)
and g. kaki (12) convert several
steroids into their lla-hydroxy derivatives, while g. olivarum (13) on incubation with progesterone or Reichstein's compound S yields the A’
derivatives.
We have already described the conversion of conessine into A4 -conenin-3-one with s0 cgclaminis (1>0 Incubation of conessine with Gloeosporium fructigenum go americana KrEger yielded a mixture of three monohydroxylated conessines V, VI and VII which were separratedon an alumina column, The melting points,, specific rotations and WMR data for compounds V and VI were in good agreement with those reported for 7a- and 7p-hydroxyconessineby Kupchan et al, (2) who obtained these substances by incubation of conessine with A. ochraceus, Oxidation of V and VI with chromium trioxide yielded the 7-ketone (X) also obtained by Kupchan -et -0 al
Oct. 1966
STEROIDS
(2) and Patterson artal e (3).
425
This ketone was likewise
obtained by allylio oxidation of conessine (14). Chemical shift data for the C-19 methyl group and the coupling constants for the C6-H signals in the KKK spectra for V and VI (15*16) support the assignments of orientation mado by the earlier investigators (2,3).
Kupchan et al. (2) reported a third product, the structure of which was not elucidated. We expected this product to represent lla-hydroxyconeasinebecause of the reported ability of 9. ochraceus (17) to convert steroids into their lla-hydroxy derivatives. However, the physical constants of this product differed markedly from those of the third metabolits (VII) reported in this paper, which proved to be lla-hydroxyconessinc.Observations leading to the suggestion that VII might represent lla-hydroxyconessinewere as follows: oxidation of VII furnished a saturated 6-membered ring ketone (3 max CHCL3 1695 ~r”)~ which on reduction with lithium aluminum hydride yielded IX as the major product as well as a small amount of VII. The 16~1 ratio (estimated by paper chromatography) of the two reduotion products repreeents exactly what would have been expected from such reduction of an 11-ketone. The KKE speotzum of VII (at 100 MC after deuterium exchange) showed a triplet of doublets (Jax ae= 4 cps) similar to the Cll-H signal 9 in
III. The chemical shift for the C,g-methyl group was
426
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also in good agreement with structure VII. In view of the fact, however, that the molecular rotation contributions for VII, IX and X did not lend support to the above structural assignment we have synthesized lla-hydroxyconessine.According to the method of Johnson et al. (19,20), lla-hydroxy-A4-conenin-j-one was converted into 3-dimethyl-amino-11a-hydroxy-A395conadienine (XI), Reduction of the enamine with sodium borohydride yielded a mixture of 3a- end 3fldimethylamino-lla-hydroxy-A5-conenine. The more polar 3@epimer was found to be identical with VII.
EXPERIMENTAL Melting points were determined on a "Mehi" melting point apparatus according to Tottoli, Nuclear magnetic resonance spectra were taken in deuterated chloroform at 60 (Varian A60) and 100 Mc (Varian HR 100) with tetramethylsilane as an internal standard. Infrared absorption spectra were recorded on a PerkinElmer 221 spectrophotometer with NaCl prism, Ultraviolet spectra were determined on a Zeiss PMQII spectrophotometer. Paper chromatograms were run on Whatman no. 1 using the system butanol-acetic acid-water (4~1~5 by volume, upper phase). Thinlayer chromatograms were run on alumina coated glass plates (alumina G; MERCK A,G.), using the systems benzene-acetone-25s ammonia solution (19rl:l by volume; upper phase) and chloroform-methanol (99tl by volume), The chromatograms were developed with iodine vapour or Mayer's reagent (21). loFermentation of conessine with Stachybotrys parvispora Hughes (22), A strain of EStachyblOt~~e~u~d~~o~~h~~leB obtained from the N entraa cultures" (Baarn, Holland) was inoculated from an oatmeal agar slant into Erlenmeyer flasks containing a 2s cornsteep solids-2% glucose nutrient medium
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427
(20-20 medium). After three days shaking at 26O on a rotary shakia machine (250 ropomoO stroke 2.5 cm) this culture f 1.5 1) was used to inoculate 70 1 of a 0,5$ cornsteep solids-0,5k glucose (5-5) medium in a 100 1 stainless steel fermentor, equipped with stirrer and air inlet, Incubation took place at 260 with stirring (140 r.p,m,) and aeration (80 1 of air/min,) for 16 hrs. after which time this culture was used to inoculate 2000 1 of a 5-5 medium in a 4000 1 stainless steel fermentor. After 24 hrs, of incubation at 260 under conditions of agitation and aeration, 300 g of conessine (1) were added as a solution in dilute sulfusic acid adjusted to pH 3.5-4. Thin layer chromatography of a sample 70 hrs. after the addition of the substrate indicated a complete conversion of I into II and III. The culture broth was filtered after acidification to pH 3, the filtrate was adjusted to pH 10 and extracted three times with 1/3 volume of methyl isobutyl ketone, The combined organic extracts were concentrated in vaouo to 20 1 whereupon the solution was extracted with dilute sulfuric acid. The combined aoidio extracts were adjusted to pH 10 and again extracted with methyl isobutyl ketone. The final extract was evaporated to d ness under reduced pressure. The residue (230 Q r was dissolved in 200 ml of acetone whereupon II crystallized, The product was filtered, washed with cold acetone and dried. The yield was '54.5Q of a product with m.p, 107-1080, After recrystallizationfrom acetone there were obtained 42,3 g with m.p. 108.5-109,5*, A mixed melting point with an authentic sample of A4-oonenin-3-one showed no depression. The infrared spectra (KBr and CHC13) were identical to those of A4-conenin-3-one. The mother liquors of the first and second crystallization were combined and evaporated to dryness. The residue was chromatographed on an alumina column. After combining the matching fractions and evaporation of the solvent there were obtained another 94 g of II and 53 g of III, III could not be obtained crystalline and was converted into the hydriodide, After several crystallization8from methanol-acetone there were obtained 39.5 g with m,p. 231-232*. The salicylate could also be obtained crystalline. Anal. calcd. for ~22H34N02J: C, 56,05; H, 7.22~ N, 2.97; J, 26.96. Found: C, 55,75; H, 7.36; N, 2,89; J, 26.79. Salicylate m.p, 246-247** Calctd,for C29H3gN05: C, 72.35; H, 8.11; N 2,91. Found: C, 72.30; H, &28; I?,2,91. The free base (a light yellow glass) has czIl, + 1380 (C = 1.0, (XX,); I&,+ 473°;xCH30H 241 rnli J-
(c
= 14,600)
“‘I3 ;v max
3600,
=
2785,
1665:?610 and 1033
OIU’‘;
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p.p.m, , after deuterium exchange) 1.28 (C,9-H3), 1.03 (doublet, C21-H3)g 1,97 (doublet, C18-H), 2.32 (N-CH3), 3.00 (doublet, C18-H), 3.66 (multiplet C,,-H), 5.75 (C4-H).
NMR (6 in
2.Fermentation of n4-conenin-3-one with Stachybotrys parvispora Hughes A strain of Stach hot parvispora Hughes was inoculated from an oatmea agar slant into an + Erlenmeyer flask containing 700 ml of a 20-20 medium. After three days shaking at 260 on a rotary1shaking machine (250 r.p.m., stroke 2.5 cm) this culture was used to inoculate (5%) 100 ml of a 5-5 medium in a 500 ml Erlenmeyer flask. After 24 hrs, shaking at 260 15 mg of n4-conenin-3-one (II) were added as a solution in dilute sulfuric acid adjusted to pH 3.5-4. 24 Hrs. after the addition of the substrate the culture broth was adjusted to pH 10 and extracted once with 100 ml of methyl isobutyl ketone. Thin layer and paper chromatography of the extract showed a complete conversion of II into III, 3,Fermentation of n4-conenin-3-one with Aspergillus oohraceus An inoculum was prepared by inoculation of a strain of 4s er llus ochraceus from an oatmeal agar slant intoayer an flask containing 100 ml of a 20-20 medium. After three days shaking at 260 on a rotary shaking machine (250 r.p.me, stroke 2.5 cm) the resulting culture was used to inoculate (5s) 100 ml of a 5-5 medium in a 500 ml Erlenme er flask. After 24 hrs shaking at 26 0 15 mg of /\%-conenin-3-one were added as a solution in dilute sulfuric acid adjusted to pH 3.5-4. After 24 hrs shaking at 260 the culture broth was adjusted to pH 10 and extracted with methyl isobutyl ketone. Thin layer and paper chromatography of the organic extract showed a complete conversion of II into III. 4.~4-coneninB-3.11-dione (IV) 343 mg of III were dissolved in 10 ml of acetic acid. To this solution was added a solution of 200 mg of CrO in 5 ml of acetic acid. The mixture was kept 3 overnight at room temperature, after which excess CrO 3 was destroyed with methanol. The reaction mixture was evaporated to dryness under reduced pressure, The residue was suspended in 1B I?aOH and the resulting suspension extracted with methylene dichloride. The organic extract was washed with water,
Oct. 1966
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429
evaporated to dryness and the residue was chromatographsd on an alumina column, The fractions containing IV were combined and the solvent Wats evaporated. Crystallization of the residue from heptane yielded 205 m of &Iconenine-3,13-dione (IV); m,p, 161-7620; 77E B + 2700 (c = 1.0, CHC13); MB+ 920°;hC2H50H 239 rniTt = 15,700); max viE'3 2788, 1665 and 7620 cm-l. Anal. calcd. for C22H3,R02: C, 77042; H, 9.09; N, 4.70, Found: C, 77.32; H, 9,04; N, 4.17. An authentic sample of a4-conenine-3,1l=dione (obtained from Dr. J.F. Kerwin of Smith Kline and French Lab. U,S.A,) has mop. 757-1610; fz __ 7, + 2680 (CHC13);~c2H50H 239 my (t = 16,000); mixed melting max point with IV 15%7610; XR spectra (KBr and CHC13) were identical with those of IV.
1 g of III was dissolved in 4 ml of pyridine and 2 ml of acetic anhydride and left for 2 hrs at room temperature. The reaction mixture was then evaporated to dryness under reduoed pressure, the residue dissolved in 50 ml of methyl isobutyl ketone and the solution washed three times with 2 ml of a 0.1s ammonia solution and once with 4 ml of water, The organic layer was evaporated to dryness in vacua and the residue chromatographed on an alumina column. The fractions containing IIIa were combined and the solvent was evaporated. The residue, a colourless glass, could not be obtained crystalline; the yield was 860 mg, V CHc13: 1739, 1665 and 1610 cm-'. max 6, Fe~entation of conessine with Gloeosporium frtactigenum f, americana mr A strain of Gloeos orium fructi enum f. americana bbozr SchimmelKriigerobtained from the culturesf*(Baarn, Holland) was inoculated from an oatmeal agar slant into Erlenmeyer flasks containing a 20-20 nutrient medium. After three days shaking at 26* on a rotary shaking machine (250 r.p.m., stroke 2,5 cm) this culture (6 1) was used to inoculate a stainless steel fermentor containing 280 1 of a 5-5 medium. After 26 hrs of incubation at 260 under conditions of agitation and aeration 56 g of conessine (I) were added as a solution in dilute sulfuric acid adjusted to pH 3.5-4. Thin layer ahromatograpby of a sample 24 hrs after the addition of
S:4
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430
the substrate indicated a complete conversion of I into V, VI and VII. The culture broth was worked up in the same way as described before. The crude product (33 g) was chromatographed on an alumina column. The matching fractions were combined, the solvent evaporated and the residues crystallized several times from benzene. The following pure products were obtained: 7a-hydroxyconessine (V) 4.6 g; m.p. 176,5-178O; &7,, -59.1° (c = 0.77, CdC13);V;f;13 3605, 2785, 1660 and 1035 cm"; NMR (6 in epom., after deuterium exchange) 0.91 (C19-H3)P 1.04 Pdoublet, C2,-H3), 1.88 (doublet, C18-H), 2.20 (N_CH3), 2.28 (N (CH3)2), 2.98 (doublet, C18-H), 3@95 (multiplet, C7-H); 5.61 (doublet, Cg-H). Anal. calcd. for C24H40N20r C, 77.42; H, 10.75; N, 7.52. Found: C, 77.62; H, 10.82; N, 7.22. 7p-hydroxyconessine (VI) 8.5 g; m.p. 210-213O;
&ID
+
34.7O
(c = 0.94,
CHC13);V;;z13,
3600,
2785,
1675 and 1038 cm"; NMR (6 in after deuterium exchange) 0.98 (C1g-H3), 1.05 PiE;mbiet,C21-H3), 1.88 (doublet, C18-H), 2019 (N-CH3), 2.32 (N(CH3)2), 3.00 (doublet, C18-H), 3.87 (multiplet, C7-H), 5.33 (doublet, C6-H). Anal, found: C, 77.60; H, 10693; N 7.38. lla-hydroxyconessine(VIIj 4,3g; m.p. 171.5-172.5 O; cE&, + lo (c = 1.0, CHC13); MBD+ 3,7O; yCHc1 max 3 3600, 2785, 1655 and 1033 cm"; NMR (6 in p.p.m,, after deuterium exchange) 1.03 (doublet, C21-H3), 1.10 $9 -H3 >, 1.94 (doublet, C,8-H), 2.19 (N-CH3), 2.28 (N(CH3)2), 2.98 (doublet, C,8-H), 3.71 (multiplet, C -H), 5.43 (doublet, 0%H). Anal. found: &I 77.62; H, 10.82; N, 7,40.
7.7-oxoconessine (X) a0 Oxidation of V, To a suspension of lo8 g of V in 100 ml of acetone was added a solution of 500 mg of Cr03 in 7 ml of 3N H SO at such a rate that the addition was 2 4 complete within 30 minutes. After the addition excess reagent was destroyed with methanol, water was added
Oct. 1966
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and the solution adjusted to pH 9.5. The mixture was extracted with methyl isobutyl ketone and the organic extract evaporated to dryness. The residue was crystallized several times from benzenq; ield 1.08 g of 7-oxoconessine (X); m.p. 160-162; /p_7y ,,-59.50 (c = LO, CHc13);XCH3OH 237 rnq (t= 12,000);J7cHc13, max max 2785, 1665 and 1630 cm-lo Anal, calcd. for C24H38N20: C, 77.84; H, 10.27; N, 7.57. Found: C, 77,92; H, 10.28; N, 7.53. b. Oxidation of VI In the same way as described above 1.8 g of VI was oxidized to yield 0.97 g of 7-oxoconessine;m.p. 158-1600; @_TD-58.30 (c = 1.0, CHC13)ACH30H 237 rnp (&= 11,500). The infrared spectra (KBr % CHCl,) were identical with those of the oxidation prod;ct of V. co Oxidation of conessine. 1 g of conessine was dissolved in a mixture of 70 ml of acetic acid and 35 ml of acetic anhydride. To this solution were added 970 mg of anhydrous Na2Cr04 in small portions. During the addition the temperature was kept below 3T00 After the addition of the chromate the mixture was kept at a temperature between 300 and 400, The reaction was followed by thin layer chromatography,, After 109 hrs the reaction mixture was evaporated to dryness in vacua,,The residue was dissolved in water and extracted with methyl isobutyl ketone after pH adjustment to 10, The organic extract was evaporated to dryness under reduced pressure and the residue (0.9 g) chromatographed on an alumina column, The fractions containing X were combined and evaporated to dryness. The residue was crystallized from methanol-water yielding 100 mg of 7-cxoccnessine;m.p, 156-1580, Crystallization from acetone yielded 80 mg of product; m,p. 157-1590; &_&
-57.6O (c = 1.0, CHC13);XCHJoH3 237 rnp max (f= ll,OOO), The infrared spectra (KBr and CHC13) were identical with those of the oxidation products of V and VI.
8.11-oxoconessine (VIII) To a solution of 500 mg of VII in 50 ml of acetic acid were added 1.69 g of Na2Cr207 and the
432
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mixture was kept 2 hrs at 400. The reaction mixture was then evaporated to dryness in vacua and the residue worked up in the same way as described above. The yield was 340 mg of ll-oxoconessine (VIII); m,p. 129-130°; &?_l, + 56O (c = 100, CHCl$g
MB i- 207O;$i!jl3
1695 cm", Anal. found: C, 77.91; H, 10,29; N, 7,50, 9.118-hydroxyconessine
(IX)
100 mg of ll-oxoconessine (VIII) dissolved in 5ml of anhydrous ether were added to a solution of 200 mg of LiAlH4 in 50 ml of anhydrous ether, The mixture was refluxed for 1 hr after which time excess reagent was destroyed with ethyl acetate. Water was added to the reaction mixture, the ether layer separated, washed with water and dried. After evaporation of the solvent the residue was crystallized from eth 1 acetate yielding 50 mg of ll@-hydroxyconessine (IXT ; m,p, 18O-1820; + 6.4O (c = 0.94p CHC13); MB i- 24Oo &q-J 10.~-dimethylamino-lla-hydroxy-~395-conadienine (XI) --730 mg of lla-hydroxy-A4-conenin-3-one, 2 g 5 mg of p-toluene sulfonic acid, of anhydrous MgSO nd 2 ml of dimethylamine were mixed 10 ml of toluene 4' in a cooled glass tube,, After replacing the air by nitrogen the tube was sealed and heated for 18 hrs at 80". The contents of the tube were then evaporated to dryness in vacua and the residue was crystallized from acetone. The yield was 477 mg of 3-dimethylamino11a-hydroxy-A3s5-conadienine (XI); m,p. 264-2670; x CH30H 271.5 rnp (6s 18,0OO)~~"~?3 max max 1612 and 1043 cm-lo
3605, 2792, 1642,
ll.l!a-hydroxyconessine -I_ 460 mg of XI were dissolved in 9,2 ml of dioxane, To this solution were added under nitrogen 690 mg of NaBH and thereafter 9.2 ml of acetic acid, The mixture was $ efluxed for 1 hr under nitrogen after which dilute alkali was added and the mixture extracted with methyl isobutyl ketone. The organic extract was evaporated to dryness under reduced pressure and the residue chromatographed on an alumina column, After combining the matching fractions and evaporation of the solvent there were obtained 134 mg of 3a-dimethylaminolla-hydroxy-45-conenine as a yellow oil and 285 mg of lla-hydroxy-conessine; m.p, 169-170.50; /z __ 7B + 2O (c = 1,04, CHC13).
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Anal. foundt C, 77.33; H, 10.76; N, 7.47. A mixed melting with VIII showed no depression. An infrared spectrum in CHC13 was identical with that of VII. ACKNOWLEDGEMENT The autors wish to thank Dr. A. Melera of VARIAN A.G. Switzerland, for the recording and interpretation of the NMR spectra, Dr. J.F. Kerwin of Smith Kline and French Laboratories, U.S.A, for a sample of A4-conenine3,11-dione, Miss V. Roest for the measurements and interpretation of the infrared spectra, Miss II.van Waateringe and Mr. H. van Welzen for their skilful assistance. REFERENCES 1. Part I: Flines, J. de, Marx, A.F., Waard W.F. van der and Sijde, D. van der, TETRAHEDRON LETTEHS 1257 (1962). 2. Kupchan, S.M., Sih, C.J., Kubota, S., and Bahim, A.M., TETRAREDRON LETTERS 1767 (1963). 3. Patterson, E.L., Andres, W,W,, and Hartman, R.E.,
EXPERIENTIA 20, 256 (1964)o 4. Shull G,M., and Kita, D.A., J. AM.CHl%SOC. 77,763 (1953j;
Djerassi, C., and H.J., Werder, F. v,, Bork, H,H., Metz 6 Btickner, K., TETRAHEDRON LETTERS 21 [19;b), Schneider, W.P,, and Murray, H.C., CHEM.AND IND. 1163 (1960); Zetsche, K., NATURWISS. 8, 407 (1961); Werder, F. v., Briickner,Kop Bork, K.ik ., Mete, H., 2110 (1962). Hampel, B., and Mannhardt, H.J. BER. & 762 (1960); S.Kondo, E., NIPPON NOGEIKAGAKU KAISHI & Tsuda, K., Asai, T., Iizuka, H., Tanaka, T.,Nakamura, M Shirasaka M and Naito, A., U:&.':::%'2;&, 839 <19&l,:' 6.British Patent 767,361 (1957). 7.Davisson, J.W., U.S. Patent 2, 830, 937 (1956); Shirasaka, M., CHEM.PHARM,BULL.2, 203 (1961). 8.Kerwin, J.F., Wolff, M.E,, Owings, F.F., Lewis, B.B,, Magnani, A. Karash, C,, and Georgian, V., ?a%(&M. 3, 3628 (1962).
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g.Kline, W., DETERMINATION OF ORGANIC STRUCTURE BY PHYSICAL METHODS,,Editors E,A, Braude and F,C, Nachod, Acad. Press. Inc., New York, 1959, vol. I, p, 73. lO.Mathieu, J.P., and Petit, A,, CONSTANTES SELECTIONNEES, POUVOIR ROTATOIRE NATUREL I, SteroTdes, Masson and Co., Paris 1956. E o ,, NIPPON NOGEIKAGAKU KAISHI a, ll,Kondo, E. 759 (1960f.and MaSUo9 12,Shirasaka M., and TsuArutasM,, CHEMO PHARM. BULL. 2, 159 (19613. 13.Kondo E. 847
and Masuo, E,, NIPPON NOGEIKAGAKU KAISHI BP
d96d.
140Tschesche, R,, and Ockenfels, H., BER, a9
2316
(1964>.
15,Jackman, L,M.,,NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY, Pergamon Press, New York, 195g9 p0 87. 16.%ircher, R., HELV,CHIM. ACTA 469 2055 (19631, 17,Dulaney, E.L., Sta ley, E,C,, and Lavac, C,H,, MYCOLOGIA a9 464 i) 1955); Karow E,A., and Petsiavas, D,N,, IND,ENG.CHEM, & and Wall M.E., 2213 f1956)* Kenney H,E, J.AM.CHEM, $OC. 80,95568 $%":;dE~;;689 (196Oj; Weaver, E.A., Kenney, H,E,, and Wall, MOE., APPL. MICROBIOL. & 345 (1960);; Wehrli, H,, Cereghetti, M,,Schaffner, K,, Urech Jo9 and Vischer, E,, HELV,CHIM. ACTA 44, 1927 (1961j. 18,Fieser, L,F,, and Fieser, M,, STEROIDS, Reinhold Publishing Corp., New York, 1959, p0 269. 19,Johnson, WoS., Bauer, V,J., and Franck, R.W,, TETRAHEDRON LETTERS 72 (196% 20.Marshal1, J,A. and Johnson, W,S., J, ORG. CHEM,28, 421 (1963). 21.Tschesche, R., and Petersen, R,, BER. $I9 1719 Sl~~~~lc~fa~~r;iio~.1,~U4~ton,R,B., and Keutman,EeHo, 0
0
0
--9
/e
22.We also found that Stachybotrys arvis ora could %+--9 progesterone hydroxylate Reichstein's compound and 17a-hydroxyprogesteroneat the Ila-position, 0 Present address R.V,O,-T.N.O., Rijswijk,Holland (Netherlands),
IdICROBIOLOGICALTEAWSPORMATIOW OF COWESSIWE. II. A.F. Urx, H.C. Beck, W.F. van der Waard and J. de Flines
Research Laboratories of the Royal Netherlands Fermentation Industries Ltd., Delft, Holland Received April 4, 1966 (Note: "MicrobiologicalTransformationof Conessine. III" appeared in the September 1966 issueof Steroids.)
ABSTRACT Incubation of4conessine with Stach botr s arvi ora Hughes yielded A -conenin-J-one ZiiZ+&%o$&+cononin-J-one. Fermentation with Gloeoa orium fructi num '*he yielded a mix oonessine and lia-hydroxyconessine.
In our first publication on the microbiological transformation of conessine (1) we reported the conversion of conessine (I) into
A 4-conenin-3-one by
Gloeosporium cyclaminis and Hypomyces haegtatococcus. Since then the microbiological hydroxylations of conessine by Aspergillus ochraceus (2) and Cunninghamella echinulata (3) have been reported. We now wish to describe the transformation of conessine with Stachybotrys narvispora Hughes and Gloeosporium fructigenum f. americana KrQer. Stachybotrys parvispora has not yet been reported as a microorganism capable of hydroxylating ster
belongs to the family of Dematiaceae, which includes
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several genera that are able to hydroxylats many steroids1 compounds at the eleven carbon atom, e.g. Curv-ularia(4), Helminthosporium (5),
Stamphylium (6) and
Stachylidium (7). Incubation of conessine with Staohybotrys parvispora Hughes yielded a mixture of II and III (scheme I). Thin layer and paper ohromatograms of ssmples taken during the incubation showed the conversion of I into II and thence of II into III. Fermentation of II with Stachybotrys narvispora Hughes or Asnergillus ochraceus also yielded III. Compound II was identical with A4 -conenin-j-one and III proved to be the Ala-hydroxy derivative of II. The infrared and ultraviolet absorption spectra of III showed the presence of a hydroxyl group and a
A4-j-one
system. Chromium
trioxide oxidation of III in acetic acid yielded IV identical with A4-conenine-3, ll-dione (8). Since III was easily acetylated with pyridine-acetic anhydride at room temperature the hydroxyl group must possess the a-orientation. This was also deduced from the nuclear magnetic resonance spectra and the molecular rotations, In the NMR spectra (Jin p.p.m,) the C,,-proton signal was found at 3.66. After deuterium exchange this signal consisted of a triplet of doublets (Jax,ax E 10 cps; Jax,ae = 4 cps) which demands an axial (p) orientation of the C,,-proton (two axial and one
Oct.
1966
STEROIDS
Schetne I
423
8:4
STEROIDS
424
equatorial protons in a-positions), The same conclusion was reached on the basis of molecular rotatory contributions, Among the A4-3-ketosteroids (9,lO) the lla-hydroxylated derivatives have lower and the lip-hydroxy and 11-0x0 derivatives have higher 5
values than the parent compounds. Therefore,
by this analogy the hydroxyl group in III was also assigned the a-configuration,
Several Gloeosporium species have already been described for the microbiological conversion of steroids, e.g. 2. foliicolum
(11)
and g. kaki (12) convert several
steroids into their lla-hydroxy derivatives, while g. olivarum (13) on incubation with progesterone or Reichstein's compound S yields the A’
derivatives.
We have already described the conversion of conessine into A4 -conenin-3-one with s0 cgclaminis (1>0 Incubation of conessine with Gloeosporium fructigenum go americana KrEger yielded a mixture of three monohydroxylated conessines V, VI and VII which were separratedon an alumina column, The melting points,, specific rotations and WMR data for compounds V and VI were in good agreement with those reported for 7a- and 7p-hydroxyconessineby Kupchan et al, (2) who obtained these substances by incubation of conessine with A. ochraceus, Oxidation of V and VI with chromium trioxide yielded the 7-ketone (X) also obtained by Kupchan -et -0 al
Oct. 1966
STEROIDS
(2) and Patterson artal e (3).
425
This ketone was likewise
obtained by allylio oxidation of conessine (14). Chemical shift data for the C-19 methyl group and the coupling constants for the C6-H signals in the KKK spectra for V and VI (15*16) support the assignments of orientation mado by the earlier investigators (2,3).
Kupchan et al. (2) reported a third product, the structure of which was not elucidated. We expected this product to represent lla-hydroxyconeasinebecause of the reported ability of 9. ochraceus (17) to convert steroids into their lla-hydroxy derivatives. However, the physical constants of this product differed markedly from those of the third metabolits (VII) reported in this paper, which proved to be lla-hydroxyconessinc.Observations leading to the suggestion that VII might represent lla-hydroxyconessinewere as follows: oxidation of VII furnished a saturated 6-membered ring ketone (3 max CHCL3 1695 ~r”)~ which on reduction with lithium aluminum hydride yielded IX as the major product as well as a small amount of VII. The 16~1 ratio (estimated by paper chromatography) of the two reduotion products repreeents exactly what would have been expected from such reduction of an 11-ketone. The KKE speotzum of VII (at 100 MC after deuterium exchange) showed a triplet of doublets (Jax ae= 4 cps) similar to the Cll-H signal 9 in
III. The chemical shift for the C,g-methyl group was
426
STEROIDS
8:4
also in good agreement with structure VII. In view of the fact, however, that the molecular rotation contributions for VII, IX and X did not lend support to the above structural assignment we have synthesized lla-hydroxyconessine.According to the method of Johnson et al. (19,20), lla-hydroxy-A4-conenin-j-one was converted into 3-dimethyl-amino-11a-hydroxy-A395conadienine (XI), Reduction of the enamine with sodium borohydride yielded a mixture of 3a- end 3fldimethylamino-lla-hydroxy-A5-conenine. The more polar 3@epimer was found to be identical with VII.
EXPERIMENTAL Melting points were determined on a "Mehi" melting point apparatus according to Tottoli, Nuclear magnetic resonance spectra were taken in deuterated chloroform at 60 (Varian A60) and 100 Mc (Varian HR 100) with tetramethylsilane as an internal standard. Infrared absorption spectra were recorded on a PerkinElmer 221 spectrophotometer with NaCl prism, Ultraviolet spectra were determined on a Zeiss PMQII spectrophotometer. Paper chromatograms were run on Whatman no. 1 using the system butanol-acetic acid-water (4~1~5 by volume, upper phase). Thinlayer chromatograms were run on alumina coated glass plates (alumina G; MERCK A,G.), using the systems benzene-acetone-25s ammonia solution (19rl:l by volume; upper phase) and chloroform-methanol (99tl by volume), The chromatograms were developed with iodine vapour or Mayer's reagent (21). loFermentation of conessine with Stachybotrys parvispora Hughes (22), A strain of EStachyblOt~~e~u~d~~o~~h~~leB obtained from the N entraa cultures" (Baarn, Holland) was inoculated from an oatmeal agar slant into Erlenmeyer flasks containing a 2s cornsteep solids-2% glucose nutrient medium
STEROIDS
Oct. 1966
427
(20-20 medium). After three days shaking at 26O on a rotary shakia machine (250 ropomoO stroke 2.5 cm) this culture f 1.5 1) was used to inoculate 70 1 of a 0,5$ cornsteep solids-0,5k glucose (5-5) medium in a 100 1 stainless steel fermentor, equipped with stirrer and air inlet, Incubation took place at 260 with stirring (140 r.p,m,) and aeration (80 1 of air/min,) for 16 hrs. after which time this culture was used to inoculate 2000 1 of a 5-5 medium in a 4000 1 stainless steel fermentor. After 24 hrs, of incubation at 260 under conditions of agitation and aeration, 300 g of conessine (1) were added as a solution in dilute sulfusic acid adjusted to pH 3.5-4. Thin layer chromatography of a sample 70 hrs. after the addition of the substrate indicated a complete conversion of I into II and III. The culture broth was filtered after acidification to pH 3, the filtrate was adjusted to pH 10 and extracted three times with 1/3 volume of methyl isobutyl ketone, The combined organic extracts were concentrated in vaouo to 20 1 whereupon the solution was extracted with dilute sulfuric acid. The combined aoidio extracts were adjusted to pH 10 and again extracted with methyl isobutyl ketone. The final extract was evaporated to d ness under reduced pressure. The residue (230 Q r was dissolved in 200 ml of acetone whereupon II crystallized, The product was filtered, washed with cold acetone and dried. The yield was '54.5Q of a product with m.p, 107-1080, After recrystallizationfrom acetone there were obtained 42,3 g with m.p. 108.5-109,5*, A mixed melting point with an authentic sample of A4-oonenin-3-one showed no depression. The infrared spectra (KBr and CHC13) were identical to those of A4-conenin-3-one. The mother liquors of the first and second crystallization were combined and evaporated to dryness. The residue was chromatographed on an alumina column. After combining the matching fractions and evaporation of the solvent there were obtained another 94 g of II and 53 g of III, III could not be obtained crystalline and was converted into the hydriodide, After several crystallization8from methanol-acetone there were obtained 39.5 g with m,p. 231-232*. The salicylate could also be obtained crystalline. Anal. calcd. for ~22H34N02J: C, 56,05; H, 7.22~ N, 2.97; J, 26.96. Found: C, 55,75; H, 7.36; N, 2,89; J, 26.79. Salicylate m.p, 246-247** Calctd,for C29H3gN05: C, 72.35; H, 8.11; N 2,91. Found: C, 72.30; H, &28; I?,2,91. The free base (a light yellow glass) has czIl, + 1380 (C = 1.0, (XX,); I&,+ 473°;xCH30H 241 rnli J-
(c
= 14,600)
“‘I3 ;v max
3600,
=
2785,
1665:?610 and 1033
OIU’‘;
STEROIDS
428
8:4
p.p.m, , after deuterium exchange) 1.28 (C,9-H3), 1.03 (doublet, C21-H3)g 1,97 (doublet, C18-H), 2.32 (N-CH3), 3.00 (doublet, C18-H), 3.66 (multiplet C,,-H), 5.75 (C4-H).
NMR (6 in
2.Fermentation of n4-conenin-3-one with Stachybotrys parvispora Hughes A strain of Stach hot parvispora Hughes was inoculated from an oatmea agar slant into an + Erlenmeyer flask containing 700 ml of a 20-20 medium. After three days shaking at 260 on a rotary1shaking machine (250 r.p.m., stroke 2.5 cm) this culture was used to inoculate (5%) 100 ml of a 5-5 medium in a 500 ml Erlenmeyer flask. After 24 hrs, shaking at 260 15 mg of n4-conenin-3-one (II) were added as a solution in dilute sulfuric acid adjusted to pH 3.5-4. 24 Hrs. after the addition of the substrate the culture broth was adjusted to pH 10 and extracted once with 100 ml of methyl isobutyl ketone. Thin layer and paper chromatography of the extract showed a complete conversion of II into III, 3,Fermentation of n4-conenin-3-one with Aspergillus oohraceus An inoculum was prepared by inoculation of a strain of 4s er llus ochraceus from an oatmeal agar slant intoayer an flask containing 100 ml of a 20-20 medium. After three days shaking at 260 on a rotary shaking machine (250 r.p.me, stroke 2.5 cm) the resulting culture was used to inoculate (5s) 100 ml of a 5-5 medium in a 500 ml Erlenme er flask. After 24 hrs shaking at 26 0 15 mg of /\%-conenin-3-one were added as a solution in dilute sulfuric acid adjusted to pH 3.5-4. After 24 hrs shaking at 260 the culture broth was adjusted to pH 10 and extracted with methyl isobutyl ketone. Thin layer and paper chromatography of the organic extract showed a complete conversion of II into III. 4.~4-coneninB-3.11-dione (IV) 343 mg of III were dissolved in 10 ml of acetic acid. To this solution was added a solution of 200 mg of CrO in 5 ml of acetic acid. The mixture was kept 3 overnight at room temperature, after which excess CrO 3 was destroyed with methanol. The reaction mixture was evaporated to dryness under reduced pressure, The residue was suspended in 1B I?aOH and the resulting suspension extracted with methylene dichloride. The organic extract was washed with water,
Oct. 1966
STEROIDS
429
evaporated to dryness and the residue was chromatographsd on an alumina column, The fractions containing IV were combined and the solvent Wats evaporated. Crystallization of the residue from heptane yielded 205 m of &Iconenine-3,13-dione (IV); m,p, 161-7620; 77E B + 2700 (c = 1.0, CHC13); MB+ 920°;hC2H50H 239 rniTt = 15,700); max viE'3 2788, 1665 and 7620 cm-l. Anal. calcd. for C22H3,R02: C, 77042; H, 9.09; N, 4.70, Found: C, 77.32; H, 9,04; N, 4.17. An authentic sample of a4-conenine-3,1l=dione (obtained from Dr. J.F. Kerwin of Smith Kline and French Lab. U,S.A,) has mop. 757-1610; fz __ 7, + 2680 (CHC13);~c2H50H 239 my (t = 16,000); mixed melting max point with IV 15%7610; XR spectra (KBr and CHC13) were identical with those of IV.
1 g of III was dissolved in 4 ml of pyridine and 2 ml of acetic anhydride and left for 2 hrs at room temperature. The reaction mixture was then evaporated to dryness under reduoed pressure, the residue dissolved in 50 ml of methyl isobutyl ketone and the solution washed three times with 2 ml of a 0.1s ammonia solution and once with 4 ml of water, The organic layer was evaporated to dryness in vacua and the residue chromatographed on an alumina column. The fractions containing IIIa were combined and the solvent was evaporated. The residue, a colourless glass, could not be obtained crystalline; the yield was 860 mg, V CHc13: 1739, 1665 and 1610 cm-'. max 6, Fe~entation of conessine with Gloeosporium frtactigenum f, americana mr A strain of Gloeos orium fructi enum f. americana bbozr SchimmelKriigerobtained from the culturesf*(Baarn, Holland) was inoculated from an oatmeal agar slant into Erlenmeyer flasks containing a 20-20 nutrient medium. After three days shaking at 26* on a rotary shaking machine (250 r.p.m., stroke 2,5 cm) this culture (6 1) was used to inoculate a stainless steel fermentor containing 280 1 of a 5-5 medium. After 26 hrs of incubation at 260 under conditions of agitation and aeration 56 g of conessine (I) were added as a solution in dilute sulfuric acid adjusted to pH 3.5-4. Thin layer ahromatograpby of a sample 24 hrs after the addition of
S:4
STEROIDS
430
the substrate indicated a complete conversion of I into V, VI and VII. The culture broth was worked up in the same way as described before. The crude product (33 g) was chromatographed on an alumina column. The matching fractions were combined, the solvent evaporated and the residues crystallized several times from benzene. The following pure products were obtained: 7a-hydroxyconessine (V) 4.6 g; m.p. 176,5-178O; &7,, -59.1° (c = 0.77, CdC13);V;f;13 3605, 2785, 1660 and 1035 cm"; NMR (6 in epom., after deuterium exchange) 0.91 (C19-H3)P 1.04 Pdoublet, C2,-H3), 1.88 (doublet, C18-H), 2.20 (N_CH3), 2.28 (N (CH3)2), 2.98 (doublet, C18-H), 3@95 (multiplet, C7-H); 5.61 (doublet, Cg-H). Anal. calcd. for C24H40N20r C, 77.42; H, 10.75; N, 7.52. Found: C, 77.62; H, 10.82; N, 7.22. 7p-hydroxyconessine (VI) 8.5 g; m.p. 210-213O;
&ID
+
34.7O
(c = 0.94,
CHC13);V;;z13,
3600,
2785,
1675 and 1038 cm"; NMR (6 in after deuterium exchange) 0.98 (C1g-H3), 1.05 PiE;mbiet,C21-H3), 1.88 (doublet, C18-H), 2019 (N-CH3), 2.32 (N(CH3)2), 3.00 (doublet, C18-H), 3.87 (multiplet, C7-H), 5.33 (doublet, C6-H). Anal, found: C, 77.60; H, 10693; N 7.38. lla-hydroxyconessine(VIIj 4,3g; m.p. 171.5-172.5 O; cE&, + lo (c = 1.0, CHC13); MBD+ 3,7O; yCHc1 max 3 3600, 2785, 1655 and 1033 cm"; NMR (6 in p.p.m,, after deuterium exchange) 1.03 (doublet, C21-H3), 1.10 $9 -H3 >, 1.94 (doublet, C,8-H), 2.19 (N-CH3), 2.28 (N(CH3)2), 2.98 (doublet, C,8-H), 3.71 (multiplet, C -H), 5.43 (doublet, 0%H). Anal. found: &I 77.62; H, 10.82; N, 7,40.
7.7-oxoconessine (X) a0 Oxidation of V, To a suspension of lo8 g of V in 100 ml of acetone was added a solution of 500 mg of Cr03 in 7 ml of 3N H SO at such a rate that the addition was 2 4 complete within 30 minutes. After the addition excess reagent was destroyed with methanol, water was added
Oct. 1966
STEROIDS
431
and the solution adjusted to pH 9.5. The mixture was extracted with methyl isobutyl ketone and the organic extract evaporated to dryness. The residue was crystallized several times from benzenq; ield 1.08 g of 7-oxoconessine (X); m.p. 160-162; /p_7y ,,-59.50 (c = LO, CHc13);XCH3OH 237 rnq (t= 12,000);J7cHc13, max max 2785, 1665 and 1630 cm-lo Anal, calcd. for C24H38N20: C, 77.84; H, 10.27; N, 7.57. Found: C, 77,92; H, 10.28; N, 7.53. b. Oxidation of VI In the same way as described above 1.8 g of VI was oxidized to yield 0.97 g of 7-oxoconessine;m.p. 158-1600; @_TD-58.30 (c = 1.0, CHC13)ACH30H 237 rnp (&= 11,500). The infrared spectra (KBr % CHCl,) were identical with those of the oxidation prod;ct of V. co Oxidation of conessine. 1 g of conessine was dissolved in a mixture of 70 ml of acetic acid and 35 ml of acetic anhydride. To this solution were added 970 mg of anhydrous Na2Cr04 in small portions. During the addition the temperature was kept below 3T00 After the addition of the chromate the mixture was kept at a temperature between 300 and 400, The reaction was followed by thin layer chromatography,, After 109 hrs the reaction mixture was evaporated to dryness in vacua,,The residue was dissolved in water and extracted with methyl isobutyl ketone after pH adjustment to 10, The organic extract was evaporated to dryness under reduced pressure and the residue (0.9 g) chromatographed on an alumina column, The fractions containing X were combined and evaporated to dryness. The residue was crystallized from methanol-water yielding 100 mg of 7-cxoccnessine;m.p, 156-1580, Crystallization from acetone yielded 80 mg of product; m,p. 157-1590; &_&
-57.6O (c = 1.0, CHC13);XCHJoH3 237 rnp max (f= ll,OOO), The infrared spectra (KBr and CHC13) were identical with those of the oxidation products of V and VI.
8.11-oxoconessine (VIII) To a solution of 500 mg of VII in 50 ml of acetic acid were added 1.69 g of Na2Cr207 and the
432
8:4
STEROIDS
mixture was kept 2 hrs at 400. The reaction mixture was then evaporated to dryness in vacua and the residue worked up in the same way as described above. The yield was 340 mg of ll-oxoconessine (VIII); m,p. 129-130°; &?_l, + 56O (c = 100, CHCl$g
MB i- 207O;$i!jl3
1695 cm", Anal. found: C, 77.91; H, 10,29; N, 7,50, 9.118-hydroxyconessine
(IX)
100 mg of ll-oxoconessine (VIII) dissolved in 5ml of anhydrous ether were added to a solution of 200 mg of LiAlH4 in 50 ml of anhydrous ether, The mixture was refluxed for 1 hr after which time excess reagent was destroyed with ethyl acetate. Water was added to the reaction mixture, the ether layer separated, washed with water and dried. After evaporation of the solvent the residue was crystallized from eth 1 acetate yielding 50 mg of ll@-hydroxyconessine (IXT ; m,p, 18O-1820; + 6.4O (c = 0.94p CHC13); MB i- 24Oo &q-J 10.~-dimethylamino-lla-hydroxy-~395-conadienine (XI) --730 mg of lla-hydroxy-A4-conenin-3-one, 2 g 5 mg of p-toluene sulfonic acid, of anhydrous MgSO nd 2 ml of dimethylamine were mixed 10 ml of toluene 4' in a cooled glass tube,, After replacing the air by nitrogen the tube was sealed and heated for 18 hrs at 80". The contents of the tube were then evaporated to dryness in vacua and the residue was crystallized from acetone. The yield was 477 mg of 3-dimethylamino11a-hydroxy-A3s5-conadienine (XI); m,p. 264-2670; x CH30H 271.5 rnp (6s 18,0OO)~~"~?3 max max 1612 and 1043 cm-lo
3605, 2792, 1642,
ll.l!a-hydroxyconessine -I_ 460 mg of XI were dissolved in 9,2 ml of dioxane, To this solution were added under nitrogen 690 mg of NaBH and thereafter 9.2 ml of acetic acid, The mixture was $ efluxed for 1 hr under nitrogen after which dilute alkali was added and the mixture extracted with methyl isobutyl ketone. The organic extract was evaporated to dryness under reduced pressure and the residue chromatographed on an alumina column, After combining the matching fractions and evaporation of the solvent there were obtained 134 mg of 3a-dimethylaminolla-hydroxy-45-conenine as a yellow oil and 285 mg of lla-hydroxy-conessine; m.p, 169-170.50; /z __ 7B + 2O (c = 1,04, CHC13).
Oct.
1966
STEROIDS
433
Anal. foundt C, 77.33; H, 10.76; N, 7.47. A mixed melting with VIII showed no depression. An infrared spectrum in CHC13 was identical with that of VII. ACKNOWLEDGEMENT The autors wish to thank Dr. A. Melera of VARIAN A.G. Switzerland, for the recording and interpretation of the NMR spectra, Dr. J.F. Kerwin of Smith Kline and French Laboratories, U.S.A, for a sample of A4-conenine3,11-dione, Miss V. Roest for the measurements and interpretation of the infrared spectra, Miss II.van Waateringe and Mr. H. van Welzen for their skilful assistance. REFERENCES 1. Part I: Flines, J. de, Marx, A.F., Waard W.F. van der and Sijde, D. van der, TETRAHEDRON LETTEHS 1257 (1962). 2. Kupchan, S.M., Sih, C.J., Kubota, S., and Bahim, A.M., TETRAREDRON LETTERS 1767 (1963). 3. Patterson, E.L., Andres, W,W,, and Hartman, R.E.,
EXPERIENTIA 20, 256 (1964)o 4. Shull G,M., and Kita, D.A., J. AM.CHl%SOC. 77,763 (1953j;
Djerassi, C., and H.J., Werder, F. v,, Bork, H,H., Metz 6 Btickner, K., TETRAHEDRON LETTERS 21 [19;b), Schneider, W.P,, and Murray, H.C., CHEM.AND IND. 1163 (1960); Zetsche, K., NATURWISS. 8, 407 (1961); Werder, F. v., Briickner,Kop Bork, K.ik ., Mete, H., 2110 (1962). Hampel, B., and Mannhardt, H.J. BER. & 762 (1960); S.Kondo, E., NIPPON NOGEIKAGAKU KAISHI & Tsuda, K., Asai, T., Iizuka, H., Tanaka, T.,Nakamura, M Shirasaka M and Naito, A., U:&.':::%'2;&, 839 <19&l,:' 6.British Patent 767,361 (1957). 7.Davisson, J.W., U.S. Patent 2, 830, 937 (1956); Shirasaka, M., CHEM.PHARM,BULL.2, 203 (1961). 8.Kerwin, J.F., Wolff, M.E,, Owings, F.F., Lewis, B.B,, Magnani, A. Karash, C,, and Georgian, V., ?a%(&M. 3, 3628 (1962).
8:4
STEROIDS
434
g.Kline, W., DETERMINATION OF ORGANIC STRUCTURE BY PHYSICAL METHODS,,Editors E,A, Braude and F,C, Nachod, Acad. Press. Inc., New York, 1959, vol. I, p, 73. lO.Mathieu, J.P., and Petit, A,, CONSTANTES SELECTIONNEES, POUVOIR ROTATOIRE NATUREL I, SteroTdes, Masson and Co., Paris 1956. E o ,, NIPPON NOGEIKAGAKU KAISHI a, ll,Kondo, E. 759 (1960f.and MaSUo9 12,Shirasaka M., and TsuArutasM,, CHEMO PHARM. BULL. 2, 159 (19613. 13.Kondo E. 847
and Masuo, E,, NIPPON NOGEIKAGAKU KAISHI BP
d96d.
140Tschesche, R,, and Ockenfels, H., BER, a9
2316
(1964>.
15,Jackman, L,M.,,NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY, Pergamon Press, New York, 195g9 p0 87. 16.%ircher, R., HELV,CHIM. ACTA 469 2055 (19631, 17,Dulaney, E.L., Sta ley, E,C,, and Lavac, C,H,, MYCOLOGIA a9 464 i) 1955); Karow E,A., and Petsiavas, D,N,, IND,ENG.CHEM, & and Wall M.E., 2213 f1956)* Kenney H,E, J.AM.CHEM, $OC. 80,95568 $%":;dE~;;689 (196Oj; Weaver, E.A., Kenney, H,E,, and Wall, MOE., APPL. MICROBIOL. & 345 (1960);; Wehrli, H,, Cereghetti, M,,Schaffner, K,, Urech Jo9 and Vischer, E,, HELV,CHIM. ACTA 44, 1927 (1961j. 18,Fieser, L,F,, and Fieser, M,, STEROIDS, Reinhold Publishing Corp., New York, 1959, p0 269. 19,Johnson, WoS., Bauer, V,J., and Franck, R.W,, TETRAHEDRON LETTERS 72 (196% 20.Marshal1, J,A. and Johnson, W,S., J, ORG. CHEM,28, 421 (1963). 21.Tschesche, R., and Petersen, R,, BER. $I9 1719 Sl~~~~lc~fa~~r;iio~.1,~U4~ton,R,B., and Keutman,EeHo, 0
0
0
--9
/e
22.We also found that Stachybotrys arvis ora could %+--9 progesterone hydroxylate Reichstein's compound and 17a-hydroxyprogesteroneat the Ila-position, 0 Present address R.V,O,-T.N.O., Rijswijk,Holland (Netherlands),