411 CHARTI CH,R
CH3
I
COCH2R,
I
H-C-OAc
H-C-OH
- dPH1 HO
I
2a, R = H
R, la, R,=R,
4
b , R - OAc
H b , R,=OAc; R ? = H c , R,=H; R2=CHl =
\
CH
CHJ
I
HO-C-H
HO-&H
- &
NHCH,]
HO 6a, R=NH.
HO
////
CH,OH
HO--C-H
I
HO-C-H
b, R = NHCH, c, R = NHCIH, d, R=NH(n-CJH-) e, R =NH(n-C,H,,) f , R = NH(CH.),OH g, R =KH(CH,),NHL h, R=N(CH,). i , R,h'Zoore, Tetrahedron, 16, 187 (1961).
l l a , L'Oa b. 200
12a. L'Oa b 20p
I
.-\cO- C-H
I H I
CH I HO-C-H
pyyw 14
CH I
{yyxm
13
H0-C-K I
_(HI
).
6h
-4cetylation of 6b gave :L diacetate amide 13 in which o n e hydroxy group did not renct, an observation w i i sistent with thc presence of the hindered, tertiary 17hydroxy group. Stepwise methylation of 6b with methyl iodide gave a diniethylamirio compound 6h slid a quaternary ammoriiuni iodide 14. The quantitative conversion of 14 t o the starting epoxide 3a by sodium
methoxide in methanol followed by acetylation establishes the structural integrity of the pregnane system. It also eliminateb the possibility of epoxide migr:itioii.2b Such a niigratioii would result in the conversion of the 16,17a-epoxy-20P-hydroxy system into a 1Ga-hydroxy-17P,20P-epoxy-li-isopregtiane.Amine addition to the rearranged epoxide would be expected t o give a lBa,20~-dihydroxy-l7~-aminopregnane, TI hich x\-ould not be converted to the 16,17a-epoxy-2O@-hydroxy system on methylation and deamination as desrrihed above nor would it have a hindered hydroxy group. Biological Evaluation-The amines were broadly screened but only those categories showing the most interesting activities will be considered here. Xntibacterial activity against Diplococcus pneumoniaeZi was observed with the ethylamino compound 6c, the S-niethylpiperazino compound 61, the "-hydroxymethylamino compound 7, and the 20a-hydroxymethylamino compound 4. The ethylamino compound 6c and the S-methylpiperazino compound 61 also showed complete immobilization (death) of the protozoan Tetrahymena geleii,28 while the dimethylamino compound 6h, the 21-hydroxy-methylamino compound 7, and the 20a-hydroxy-methylamino compound 4 killed about half of the organisms. Hypotensive RCtivity, in pentobarbital-anesthetized dogs with cannulated femoral arteries,29 was shown by the ethylamino compound 6c, the n-propylamino compound 6d, and the methylethylamino compound 6i. =Inalgesic activity, as measured by the writhing mouse assayJ3Owas noted with the following compounds: ethylamino 6c, n-propylamino 6d, ethanolamino 6f, P-aminoethylamino 6g, diethylamino 6h, niethylethylamino 64 piperidino 6j, X-methylpiperazino 61, 21hydroxymethylamino 7,4,5-dihydromorpholino 8b. Experimental Section3I 16,17a-Epoxy-5-pregnene-3p,2O-diol3,20-Diacetate (2a and 3a).-To a stirred suspension of 200 g of 16,17a-epoxy-3p-hydroxy-5-pregnen-20-one ( l a ) in 2 1. of methanol containing 20 ml of 10% aqueous NaOH was carefully added I 1 g of NaBH4. After 1 hr the suspension was cooled and 20 ml of acetic acid was added. Precipitation with water gave 184 g of a mixtiire of 20a- and 206-diols and this was stirred with 450 ml of cold pyridine. Filtration gave 45 g of crude 20a-diol which was acetylated n i t h acetic anhydride and pyridine giving 54 g of diacetate. One crystallization from acetone gave 27 g (llyc)of pure 2a: mp 166-167.5" (lit.'8 mp 168"); [ c ~ ] D-17.5' ~ ~ (e 1, E t O H ) : principle nmr peaks a t 58 (18-H), 63 (19-H), 76, 83 (21-H), 122 (26) G . B. Payne, J . Org. Chem., 27, 3819 (1962). (ai) T h e compounds n e r e placed directly on t h e surface of blood agar plates which had been inoculated with the test organism. .\fter a n incuhation period of 24 h r a t 36O active compounds shoaed a clear zone, free of hacterial growth, around the compound. (28) Approximately 5 mg of compound was added t o 1.0 mi of a 24-hr ciilture and t h e effect was measured after 24 lir a t room temperature. (29) T h e amines were administered intravenously a t 5 mgjkg. .\ctive compounds showed a t least a 207, decrease in blood pressure lasting 5 min or longer in 50% or more of the dogs. T h e method is described by A. L. A. I3oura and -1.F. Green in "Evaluation of Drug hctivities: Pharmacometrics," D. R. Laurence and A. L. Bacharach, Ed.. Academic Press Inc., New York, N. T., 1964, Chapter 19. (30) This test is a modification of t h e procedure of E . T. Eckhardt, F. Cheplovita. 31. Lipo, and W.11.Govier, Proc. SOC.Ezptl. B i d . M e d . , 98, 186 (1958). One hour following the oral administration of 60 mg or less of compound each mouse was challenged with t h e intraperitoneal administration of 0.2 ml of 0.5% aqueous HCI. T h e compound is rated active if a t least 20% of t h e animals d o not show the writhing response. (31) Melting points were taken on a Thomas-Hoover melting point apparatus and are corrected. N m r spectra, were taken with a Varian A-60 instrument using CDCla a s solvent unless otherwise indicated. T h e data are given in c y c l ~ sper second downfield from internal LIeaSi.
(acetate CH3's), 200 (16-H), 260-290 (3-H), 318, 325, 329, 336 (20-H), 315-330 (6-H). Anal. Calcd for C2:,TT,,0:,: C, 72.08; IT, S.71. Foiiiid: C, i 2 . 0 8 ; JI, 8.44. The pyridine filtrate was treated with acetic anhydride and, after standing overnight, at, room temperature, was added to wat,er, precipitating 165 g of crude 206-acetate. Crystallization from ether gave 88 g (35%) of piire 3a. The analyzed sample was crystallized from acetone-hexane and had the following properties: mp 136-137.5", partial recrystallization arid complete D (e 1, EtOH): nnir fiision 147-148" (lit.18mp 150'): [ c Y ] ~ ~0.00 peaks at 33 (18-H), 62 (19-H), 62, 69 (21-H), 121 (%acetate), 124 (%&acetate), 104 (16-H), 260-200 (3-H), 315-330 (6-H), 328, 335, 342, 348 (20-H). Bnal. Calcd for C25H3605:C, 72.08; H, 8.71. Found: C, 72.07; H, 8.82. 16,17~~-Epoxy-5-pregnene-3p,20,21 -triol 3,20,21-Triacetate (2b and 3b).-The reduction of 30 g of 16,17a-epox~-3p,21-dihydrox~--5-pregnen-20-one21-acetate ( l b ) with 6 g of NaT3Ill was carried out as described above. The product, being i i o i i crystalline, was extracted into ethyl acetate, isolated, and aretylated with acetic anhydride i l l pyridine to give 36 g of ci,iide triacetate. Foiir crystallizations from acetone-hexane gave 1 3 ~ (c 1, ICtOII): g ( 3 . 5 $ ) of piire 3b: mp 179-181.5°: [ a I z 6$25.5" nmr peaks at .54 (IS-H), 62 (l9-H), 122, 127 (acetate methyls), 202 (16-11), 225, 228, 237, 243, 245, 247, 253, 258 (21-H), 260-290 (3-H), 31t5-327 (6-H), 341, 346, 348, 3-53 (2P-H). Anal. Calcd for C2,H,,0j: C, 68.33; IT, S.Oi. Foiind: C, 68.61; 13, 8.14. The mother liqiior was allowed to slowly evaporate a t room temperatiire and from the residual crystalline mass nodiiles of prisms were removed by hand. Foiir crystallization^ from acetone-hexane gave 1.1 g ( 3 % ) of piire 2b: mp 12R.5-13I0; [ C Y ] ~ ~-61" D ( e 1, Et,OH); nmr peaks a t 5S (18-H), 62 (lg-TT), 121, 123, 124 (acetate methyls), 197 (16-H), 230, 243, 252, 263, 266, 276, 278 (21-H), 2.55-290 (3-H), 315-327 (6-H), 332, 336, 341, 343 (20-H). Anal. Calcd for C9;HqR07: _. "" . C., 68.33: H. 8.07. Foiind: C. -, 68.31; H, 7.84. 16,17~~-Epoxy-6-methyl-5-pregnene-3/3,2Op-diol (3c).-The reduction of 20 g of 3p-hvdroxy-l6,17a-epoxy-6-methyl-5-pregnen20-one with 1.1 g of NaRH4 in 0.20 1. of methanol containing 2 ml of 10% aqiieous NaOH was carried oiit, as described above. The crude product was crystallized five times from acetonehexane, giving 8.0 g (40Yc) of pure 312: mp 183.5-190.5'; [aIz7n -52' (e 1, EtOH); nnir peaks a t 50 (l%H), 57 (19-H), 60, 67 (21-H), 93 (6-CH3), 197 (16-H), 190-220 (3-H), 258, 264 (20-H). Anal. Calcd for CgzH3403: C, 76.26; H, 9.89. Foiind: C, 76.15; H, 9.70. The Addition of Amines to 16.17-Eooxv-20-hvdroxv-5-oree. - nenes.-A 10-g quantity of the appropriate epoxide and 100 ml of amine with 1 g of p-toluenesulfonic acid monohydrate were heated eit>hera t reflux or in a bomb. The excess solvent was largely removed by evaporation and the re*idue was washed with water. The crude amine was either crystallized directly (isolation D ) or was converted to its hydrochloride (isolation H ) . The hydrochlorides were prepared by adding HC1 in 2-propanol or in water to an ether solution of the amine. The amine hydrochlorides were separated as a solid or an aqiieoiis solution or a.: both and the free amines were regenerated by adding XaOH to a solution in water or methanol. No attempt was made to find optimum conditions for the amine preparations. T h e yields are calciilated from the weight of the analyzed sample. The pertinent data are summarized in Table I. 16p-Dimethylamino-5-pregnene-3~,17a,20p-triol (6h).-A solution of 5.00 g of 16p-methylamirio-~-pregnene-3~,lia,20ptriol (6b)in 0.50 1. of methanol containing 0.10 1. of CHJ and 10 g of ?;aHC03 was refluxed for 6 hr. The solvents were largely distilled under vacuum and the product was precipitated with watei.. Four crystallizations from aqueoiis methanol gave 2.85 g (5*5TC) of 6h, mp 161.5-176.5'. Anal. Calcd for C?3H39N03:C, 73.16; H, 10.41; N, 3.71. Found: C, 72.88; H, 10.35; ?T, 3.64. 16p-MethylethyIamin0-5-pregnene-3~,17a,2O~-trioI (6i).-To a stirred suspension of 1.50 g of LiAlH4 in 0.20 1. of T H F was added 5.00 g of N-acetyl-l66-methylamiiio-5-pregnene-36,17ol,20~-triol 3,20-diacetate (13) and t,he mixtiire was refluxed for 7 2 hr. The cooled reaction mixtiire B-as treated with 0.30 1. of ethyl acetate and 0.10 1. of saturated aqueoiis Rochelle salt solution. The layers were separated and the aqueous phase was washed with ,
I
~
- -
445 TABLE I1 XMR
Compd
160-Substit
9 6a 6b 6c Gd Ge Of
PE %KShO F
1 6 8 - A M I h - O - 1 7 c u , 2 0 - D I H ~ ~ ~ O ~ Y - 5 - P K E G 2 r EIENS PERDEUTEKIO.~CETIC-4CID
C-18
Shifta
C-19
Oh Gi
52 63 64 64 64 64 65 67 67 68
11 12 12 12 12 13 1.i 13 16
63 63 64 64 64 64 64 64 63 64
6j
67
15
64
6k
67
13
61
63
11
OR
3
8a 8b
iXHCH3' NHCHZd n N
Od
U
64 63 64
Shift
-C-21-7
c-20-
68 79 80 78 81 80 82 83 84 86
74 86 87 87 87 87 88 88 92 92
1
8tj
92
63
0
85
92
63
0
83
NCHi
255
248 267 267 268 270 260 269 267 27 1 272
268
27-3
269
276
89
2.57
264
174
RIiscellaneoiis Compounds 64 80 86 52 79 86
258 2,i9
264 266
166 166
26.5
272
31
84
91
XHCHIe 63 63 33 63 i.) 81 4 NHCH,' A pohitive valiie denoteb a downfield shift from the corresponding 16-H compowid 9. ard. e 6-CH3. 5,B-Dihydro. e 21-Hydroxy. / 20a-Hydroxy. 3-
Anal. Calcd for C24H3603: C, 77.37; H, 9.74. Found: C, 77.43; H, 9.85. 17~~,20p-Dihydroxy-4-pregnen-3-one Acetonide (12b).-A 500mg sample of 17a,20p-dihydroxy-4-pregnen-3-one*~ was converted to its acetonide as described for the preparation of the 2001-acetonide 12a. T h e yield of pure acetonide 12b wa3 343 mg (61%,),mp 193-194",33 [ c Y ] * ~ D+54O (c 1, CHCL). L4nul. Calcd for C24H3603: C, 77.37; H, 9.74. Found: C, 77.32: H. 9.70. N-Acetyl-16p-methylamino-5-pregnene-3p,l7~,2Op-trio~ 3,20Diacetate (13).-Treatment of 1.00 g of 16B-methylamino-5pregiieue-3~,17~,2Op-triol(6b) with a mixture of 10 ml of pyridine and 5 ml of acetic anhydride for 18 h r a t room temperature gave, after precipitation with water, 1.32 g, mp 205-213', of crude product. Two crystallizations from acetone-hexane gave 1.01 g (75%)ofpure 13: mp 214-217'; kz:'32.76p (17-OH); [ a ]-55' ~ (c 1, CHC13); nmr peaks a t 52, 54 (18-H), 63 (19-H), 69, 75 (21-H), 123 (acetate methyl..), 127, 134 (S-acetyl), 174, 180
(NCHI). Snal. Calcd for C28H43N06: C, 68.68: H, 8.85; X, 2.86. Found: C, 68.84; H, 9.07; N, 3.05. 16p-Dimethylamino-5-pregnene-3p,17~,2Op-triolMethiodide solution of 1.04 g of 16P-dimethylamino-5-pregnene3p,17a,20p-triol (6h) in 20 ml of nitrobenzene and 5 ml of CH3I was refluxed for 3 hi. The product precipitated and was collected on a filter after cooling in ice and was recrystallized from methanol-ethyl acetate giving 0.54 g (38yc)of 14: mp 229-234' dec; nmr peaks (in pyridine) a t 64 (19-H), 74 ( l & H ) , 103, 109 (31-H), 215 (16-H), 225 (XCH,). Anal. Calcd for C2,H4?ISO3: C, 55.48; H, 8.15; I, 24.43; S, 2.70. Found: C, .55.34; If, 8.00; I, 24.62; S, 2.62. ( 14).-A
-7
242 260 261 263 263 262 263 26 1 263 263
r
236 256 237
239
26 1
273 278
278
166
184 170
282
164 168 Cps at 60 hlc iisiiig hIeeSi as internal stand-
The Conversion of 16p-Dimethylamino-5-pregnene-3p,l7a,2Op-triol Methiodide (14) to 16,17a-Epoxy-5-pregnene-3p,20& diol 3,ZO-Diacetate (3a).-A solution of 100 mg of methiodide 14 and 100 mg of sodium methoxide in 10 ml of methanol was kept a t room temperature under Ns for 4 hr. The reaction mixture was partitioned between ethyl acetate and water, the ethyl acetate layer was washed with water and after drying (XazS04) was concentrated under vacuum. The crystalline residue (62 mg) was acetylated with acetic anhydride in pyridine. The yield of acetate was 68 mg, mp 132-134', and thin layer chromatography 5howed a single spot migrating a t the same rate as 3a. Recrystallization from acetone-hexane gave 52 mg (6SCc from 14) of epoxide 3a, mp 135-136', identical with authentic 3a by mixture melting point and a comparison of infrared spectra.
Acknowledgment.-The author is grateful to A h . 11. G. Scaros for carrying out the pressure reactions, to Mr. J. D. Choi for the hydrogenations and to l l r . C. Pitzer for preparing some of the reference compounds. The microanalyses mere performed by Nr. E. Zelinski and eo-workers and A h . A. J . Damascus and his coworkers provided the spectral data. The antibacterial and antiprotozoal data were provided by Dr. R. Nuir, the hypotensive data by l I r . R. Jacobs, and the analgesic data by Mr. D. Knapp and Air. J . Westney. Our thanks are also extended to Dr. R. H. Bible, Jr., for many helpful discussions of the nmr data and to Dr. L. J . Chitin for his counsel on qtereocheniiral problems.
