BIJIJ-HOI,et al.
1048 Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 11
6-Amino Derivatives of Stigmastanol and Cholestanol H. PIXHAS, 9.LOISEAC,A. KRIXORIAX-MANOUKIAN, D. COURMARCEL, Laboratoires Laroche Navarron, Levalloia, 9.9, France
K.P. Buu-HoI,* AND P. JACQUIGNON Institut de Chimie des Substances Naturelles du C.N.R.S., Gif-sur-Yvette, 91, France
Received February $2, 1971 A number of new 6-@-aminoand 6-p-(B,iV-dialkylaminoalkyl)amino derivatives of stigmastanol and cholestanol have been prepared by reductive amination of the corresponding 6-keto sterols or reduction of Schiff bases from the same ketones. Several of these amines produce a decrease in the blood cholesterol level in normal rats.
p-Sitosterol (stigmast-5-en-3/3-01) is known to possess some degree of hypocholesterolemia-promoting activity on oral administration and this effect has been ascribed to a partial inhibition of intestinal resorption of dietary cholesterol.’ Recently, other structural analogs of cholesterol such as trihydroxy derivatives of both stigmastane2 and cholestane3 have been reported to show the same type of pharmacologic activity. But with all these compounds very high dose levels are required; and 17-amino derivat’ives of androstane, some of which are very a ~ t i v eshow , ~ side effects due to their estrogenicity. It was therefore of interest to investigate other types of amino steroids, and for this purpose we synt,hesized a number of 6-amino derivatives of stigmastane and cholestane (I1 and IV), which are readily accessible from /3-sitosterol and from cholesterol v i a Sp-hydroxy6-oxo-5a-stigmastane (I) and 3p-hydroxy-6-oxo-5acholestane (111). These two last ketones were either converted with primary amines into Schiff bases which werc then catalytically hydrogenated, or submitted directly to catalytic reductive a m i n a t i ~ n . ~ The primary amines used were either monoamines (n-PrNHz, phenethylamine, and glycine) or diamines (p-dimethylaminoet hylamine, 0-diet hylaminoet hylamine, and 7-dimethylaminopropylamine) . Ketone I11 was obtained by treatment of 3p-acetoxy-6-nitro-A5-cholestene (prepared by Dodson and Riegel’s method6) with Zn powder in AcOH;’ a similar reaction sequence was used for the preparation of ketone I from 3p-acetoxy-6-nitroA5-stigmastene. The axial configuration of the amino group assumed for the various amines thus prepared is derived from an extension, to reduct’ive amination of ketones I and 111, of the rule of “rear attack” as applied to the catalytic hydrogenat,ion of sterically hindered ketones.* This assumption was confirmed by the synthesis of 3Phydroxy-6/3-(2-diethylaminoethyl) amino - 5 a - cholestane (IV, R = CH2CH2NEt2)and its 6a epimer, by condens(1) See ref in: T. A I . Lin and K. K . Chen, Fortschr. Araneimittelforsch., 1, 125 (1959). (2) 11. Pinhas, French Patent 7466 M (June 28, 1968). ( 3 ) Y. Aramiki, T. Kobayashi, Y. Imai, S.Kikuchi, T. Maksukana, and K. Kamazawa, J . Atheroscler. Res., 7 , 653 (1967). (4) R . E. Counsell, P . D. Klimstra, R . E. Ranney, and D . L. Cook, J . .\fed. I’harm. C h e m . , 6 , 720 (1962); R. E. Counsell, P. D. Klimstra, and K . E. Ranney, i b i d . , 5, 1224 (1962); P. I). Iilimstra, R. E. Ranney, and R. E. Counsel], ibid., 9 , 323 (1966). ( 5 ) G. Maume and C. Baron, Bull. SOC.C h i m . Fr., 4508 (1967). (6) R . M. Dodson and B. Riegel, J. Ore. C h e m . , 13, 424 (1948). (7) J. Mauthner and W. Suida, M o n n t s h . Chem., 24, 648 (1903). (8) L. F. Fieser and 31. Fieser, “Natural Products Related to Phenanthrene,” 3rd ed, Reinhold, 1949; L. F . Fieser, Ezperientia, 6, 312 (1950); T. F. Gallagher and T. H. Kritcliersky, J . A m e r . C h e m . Soc., 78, 882 (1950).
0 I
I1
I11
IV
ing P-diethylaminochloroethanewith, resp, 3P-hydroxy6p-anlino-5a-cholestane (IV, R = H) and its 6 a epimer, both of which are known.Q A similar structure determination was made for 3p-hydroxy-6j3-(2-diethylaminoethyl)amino-5 a-stigmastane (11, R = (CH&NEtz), which proved different from its 6s epimer. The chemical data concerning the monoamines (free bases and monohydrochlorides) and diamines (isolated as dihydrochlorides) obtained are given in Table I. Pharmacological screening for hypocholesterolemiainducing activity consisted of an evaluation of the decrease in serum cholesterol content of rats treated orally (9) C. W. Shoppee, D. E. Evans, and G. H. R. Summers, J . Chem. Soc., 690 (1955); B. G. Ketcheaon and A. Taurine., Can. J . Chem., 88, 981 (1960).
Joicrnal of Medicinal Chemistry, 1971, Vol. 14, h'o. 11 1049
HYPOCHOLESTEROLEMIC AMlNOSTEROlDS
NEW AMINES
TABLE I1
TABLE I DIAMINES 11 A N D I v
EFFECTSO N SEHUM CHOLESTEROL
AND
\Veight
MP,
R
Compd
0Co.b
of liver.
Formulac,d Dose. mg/k8
1
Compdo
per day
2*50
2 3 4 5 6
2 Control 3 Control 4 Control
7
1
8 9 10 11 12 Bases were 0 Mps were taken with a Keichert microscope. recrystd from aq EtOH, hydrochlorides and dihydrochlorides from EtOH-Eta0 (1:3). Purity of all samples was confirmed by tlc (silica gel, spot detection with either Draggeiidorf's All compds anal. satisfactorily reagent or 1: 1 Acto-KSO,. Free base, mp for C, H, CI. * Free base, mp 202-205". 168'.
N,
for 6 days with the substance under test, in comparison with the untreated controls, and taking clofibrate (2-pchlorophenoxy-2-methylpropionic acid Et ester) and 3,5,&trihydroxystigmastane as reference substances. Results, recorded in Table 11, showed that several of the amines and diamines produced a significant decrease in serum cholesterol levels; however, this effect was accompanied by significant reduction of the weight of the liver and increase in the weight of the adrenals. This suggests that the steroid amines and diamines investigated hefe induce hypocholestorolemia in normal rats via some interference in cholesterol and lipid metabolism rather than by preventing intestinal absorption of dietary cholesterol.
Experimental Section
Control 6 Control 7 Control S Control 10 Control 11 Control 12
230
Serum cholesterol g/l.
0 . .io 0.70 0.63 0.XG
2.iO 125 2.50 12.5 2.N 12.5 2.50 2.50
12.i 2.iO 125 2.i0 12.i 2.50
Control
1.07 0.99 0.73 0.70 0.7s 1.06 0.93 0.99 0.x7 0.68 0.7s 0.77 1.04 0.90
0.74 0.99 0.81 0 .i3 0.74 0 . .i9 0..52
g/100 g
of I J U ~ V j v t rat
3.33 3.93 2.42 3.70 3.92 3.54 3.27 3 , 0.i 23s 3.62 3 . .1i :I. 79 3.40 3.17 2.ss 3.2.i 3.62 3,:Is 2.97 3.79 2.76 231 3.17 3.06 2.6s 3 . O!) 3.84 3.70 3.44 :{. 93
Weight of adrenals. mg/100 g of body w t rat
23 16 15 14 1s 16 16 21 1G 1s 23 l!) 16 1.i
16 19 14 19 32 19 24 31 20 24 33 23
0.66 0.s7 14 0.86 14 Control 2.iO 0.72 14d 16 0.70 16 Control 12 .i n , 7.5 15c 4.1x 16 17 0.72 2.iO 4.90 3.64 Coli t rol 0.w 14 For identificatiyn of compds, see Table I. b All conipds i n the form of their hydrochloride or dihydrochloride except for 4, 13, 14. 3B-Ilydroxy-6-oximi1~~3a-st igmastane. 3B-Acetoxy-6fl-amino-5a-stigmastane. Clofibrate. 13'
2.50
crystd from EtOH-Et20 as colorless prisms; mp 21G-21So, yield, 20c?,. Anal. ( C ~ ~ H & I Z N ~ O . ~C, I I 11, ~ OC1. ) A. Chemistry. 3p-Hydroxy-64-arllino-5a-stigmastane (11, ~P-Hydroxy-Ga-(2-diethylaminoethyl)amino-5a-cholestane R = H).-A soln of 2 g of the oxime of ketone I in 90 ml of glacial was prepd i n the same way; its dihydrochloride crystd from AcOH was reduced catalytically ig the prescnce of 0.2 g of Ptbz EtOIl-EtzO as colorless prisms, mp 2O0-21Oo. A n d . (c;JIisla t FjO" and a t normal pressure. 'After 'the reduction was comCI2NZO.O.5H&) C, 11, CI. A similar method was applied for the pleted (48 hr) the s o h was filtered, the solvent was vacuiim distd, prepn of the 2 epimeric diamines (6 and 11 i i t Tables I aiid 11). the product was dissolved i n HzO, and the amiiie was pptd with 2 Reductive Amination of Ketones I and 111.--A soli1 of ketone N aq NaOH and taken up in PhlI. The corresponding hydroI or 111 (0.1 mole) and a large excess of the :ippropriate amine chloride was prepared by treatment with IlCl gas in Et&; over(0.23 mole) i i i 400 ml of abs Et011 coiitg 1 g of loc; Pd/C was all yield, 70%. hydrogenated a t 60-90" under pressure (100 kg/cmz), with vigor38-Hyd~oxy-6~-amino-5a-stigmastane.-A s o h of 2 g of the ous stirring. The reduction lasted ca. 24 hr; the sol11ohtd was oxime of ketone I in 250 ml of n-PrOH was quickly treated with filtered hot, the EtOH distd off, and the residue recrystd o r coil2 g of Na, and the mixt was maintained a t boiling poiiit u n t i l total verted into the mono- or dihydrochloride; yield, 65-7W ;. disappearance of the Na. After cooling, Et20 (250 ml) was The reaction could be performed i i i 2 steps: prepn of the added, and the product was shaken with 3000 ml of If,O. Schiff base by refluxirig R CsHs solit of ketone I or 111 (0.1 The org layer was dried (Na2S04)and HCI gas i i i E t & was added mole), the primary amine (0.25 mole), aiid one drop of AcOH to form the hydrochloride; the n-PrOIi was vacuum distd, aiid S hr, under azeotropic eliminatioii of H20 i n a l)eail-St ark for aq the cryst residue was washed with Et,O and recrystd from apparatus; the solvent was distd off, aiid the crude, visroris EtOH to give the stigmastane hydrochloride i n 63';; yield, as Schiff base was takeii up i n Et011 ntld c;it:ilytic.ally hydrogencolorless prisms, mp 239-261'. Anal. (C*~13j4CIN0.H~O)H, ated as above; yield, 60-70c/b. N. The free base crystd from aq EtOH as colorless prisms, B. Pharmacology.-The animals used were male \Vist ar rats mp 175-176'. Anal. ( C ~ ~ H u N 0 ~ 0 . ' 5 H C,~ H, 0 ) N. weighing ca. 200 g atid fed a ilornial diet; the substniices tested 38-Hydroxy-6a- (Z-diet~ylaminoethyl)amino-5a-stigmaswere administered orally (gavage) for a period of 6 days. The tane.-A s o h of 1 g of 38-hydroxy-6a-amino-5a-stigmastane rriid serum cholesterol was measured by means of the Zlatkis, el al., 1 g of freshly distd 24iethylaminochloroetharie in 50 ml of anhyd colorimetric methodlo (CIJFc-IIzSO,; absorption hnnd :it >6O COHO was refluxed for 24 hr with a few drops of Et3N, the CsHs mfi after 30 min). The pharmacohigical assays were pei.fornied was then distd off, the residue was takeii up i n 200 ml of Et,O, and on batches ( i f 20 rats per dose atid per suhstni1c.e. the Et20 s o h was extd with dil HCI. The aq layer was basified and the diamine was taken up in EtlO, dried (NazS04),and coii(10) .\. Zlatkis. B. Zak, and .\. J. Boyle, J . Lob. Clin. .lied., 41, 486 verted into its dihydrochloride by means of HCI gas. This salt (1953).
c,
