HYPOSTEROLEMIC (TRIFLUOROMETHYL)BIPHENYLYLOXYETHYLPYRROLIDINE 993
September 1968
pure sample of 4-acetamido-4'-methylsulfonylbiphenyl, recrystallized from glacial AcOH, melted a t 267-268'. Anal. (Ci6HijN03S) C, 13, N, S. The 4-acetamido group of the biphenyl derivative described above was hydrolyzed by refluxing 1.89 g (0.01 mole) of 4-acetamido-4'-methylsulfonylbiphenyl in 20% HC1 (100 ml) for ca. 20 hr. The reaction mixture was then filtered hot and the clear Y NaOH solution. The filtrate m-as made strongly basic with 10 i crude, hydrolysis product was collected by filtration and recrystallized (AleOH); 1.4 g (82%), mp 203-205". Anal. (C13H13NO,S) C, H, N, S. Diazotization of the hydrolysis product was accomplished by adding an aqueous solution of XaNOg (1.6 g in 4 ml of HgO) dropwise (20 min) to a vigorously stirred, cooled (0-5") suspension of 4-amino-4'-met~hylstdfonylbiphenyl (2.3 g, 0.01 mole) in glacial AcOII (15 ml) and concentrated H,SOa (15 ml). When diazotization was complete, the excess HSO, was decomposed by the cautious addition (30 min) of urea a t 0-j". The cold solution of the diazonium sulfate was then added slowly (20 min) to a refluxing H,SOI solution (40 wt 5;) and, after addition was complete, the reation mixture was allowed to reflux an additional 15 min. Dilution of the acidic solution with HZO afforded a crude solid which was collected by filtration, dissolved in 1 S NaOH (20 ml) and refiltered. Acidification of the clear filtrate using a n excess of 1 AYHzSOI furnished a precipitate which was washed with two 75-ml portions of HgO and then air dried. The yield of crude 4-hydroxy-4'-methylsulfonylbiphenyl obtained following the procedures outlined above melted at 1S9190"; 1.7 g (75%). The ir absorption bands were as expected and the intermediate was used in the final step without further purification. Following method A, 1.9 g (0.01 mole) of the sodio derivative of 4-hydroxy-4'-methylsulfonylbiphenyl a a s allowed to react with 1.3 g (0.01 mole) of 2-pyrrolidinylethyl chloride in refluxing DJIF. .4fter a 60-hr reflux period, the reaction mixture was
cooled, filtered, and concentrated to a semisolid residue. Trituration of the crude yield with two 50-ml portions of HzO yielded an insoluble product which was taken up in C& (75 ml). The C6H6 solution was decolorized (charcoal), dried (NazS04), and then treated with excess HCI gas. The HC1 salt which precipitated was dissolved in HzO (100 ml), decolorized (charcoal), and filtered, and the clear, acidic filtrate was made basic (excess 1.0 AYNaOH). The desired product (32)which separated from the basic solution was collected and recrystallized (C&), 1.0 R(41%), mp 153-154'. 1-{2- [p-(p-Bromophenyl)anilino]ethyllpyrrolidine (15).-To a sus~ensionconsisting of 6.2 g (0.025 mole) of the lithio derivative of 4-amin0-4'-bromobip~enyl in dry toliiene (100 ml) was added 3.4 g (0.025 mole) of 2-pyrrolidinylethyl chloride and the reaction mixture was refluxed 22 hr. The LiCl was removed by filtration and the clear filtrate was concentrated to a semisolid residue which was dissolved in an Et20 (50 ml) and C ~ H (30 A ml) solution. The solution was then treated with an excess of dry HCl gas and the crude precipitated HCl salt was collected and dissolved in a minimum amount of HnO. A gray-white solid separated from the acid solution after addition of an excess of aqueous KOH solution (10 'V). The solid isolated in this manner was recrystallized once (heptane); 3.8 g (447,), mp 141-142'.
Acknowledgment.-We wish to thank Dr. S. Gordon and his group for the evaluation of these drugs. We are also indebted to l l r . L. Brancone and associates for the microanalytical results, Sir. W. Fulmor and associates for nmr spectral analyses, and A h . C. Pidacks and his group for their invaluable assistance in resolving the isomeric mixtures described herein using liquidliquid partition chromatographic techniques.
-
1 { 2- [ 4'- (Trifluoromethyl) -4- biphenylyloxy ] e thy1 } pyrrolidine.
A Potent Hyposterolemic Agent SANUEL GORDON A N D WALTER P.CEKLEI~IAK Toxicology and Experimental Therapeutics Research Sections, Lederle Laboratories, Ameracan Cyanamid Company, Pearl River, AVew York Received X a r c h 14, 1968 The oral hypocholesteremic compound, 1-( 2-[4'-(trifluoromethyl)-4-biphenylyloxy]ethyl)pyrrolidine (boxidine),' was studied in rats, mice, monkeys, and dogs and found to be active in all species. I n the rat, the species studied most intensively, i t was active a t a dose of 0.000370 in the diet, equivalent to the ingestion of approximately 0.3 mg, kg of body weight. Triglycerides and phospholipids were reduced as well. Boxidine was ten times as active as trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexanedihydrochloride,* 1000 times as active as clofibrate,3 and 50 times as active as t r i p a r a n ~ l . ~It inhibited the biosynthesis of cholesterol a t the 7-dehydrocholesterol stage but its primary mechanism of action may be the inhibition of sterol absorption.
Sustained interest in hypocholesteremic agent's has resulted in a plethora of reports on compounds which lower blood cholesterol level by inhibiting its synthesis at various stages in the biosynthetic pathway. Most of these compounds exert their inhibitory action a t the desmosterol stage,4-6 whereas t~~ans-l,4-bis(2-chlorobenzylaminomet'hy1)cyclohexane dihydrochloride inhibits a t 7-dehydro~holesterol.~In the course of our (1) T h e name of this compound was approved by the U. S. Adopted Names Council; J . Am. M e d . Assoc., 203, 143 (1968). (2) AY9944, Ayerst Research Laboratories, Montreal, Canada. (3) Atromid-S@. ethyl a-(p-chlorophenoxy)isobutyrate,Ayerst Research Laboratories, Montreal, Canada. (4) T . R. Blohm and R. D. MacKenaie, A r c h . Biochem. Biophys.. 85, 245 (1959). ( 5 ) E. W. Cantrail, R . Littel. S. M .Stolar, W. P. Cekleniak. H. J. Albers, S. Gordon, and S. Bernstein. Steroids, 1, 173 (1963). (6) R. E. Cousell, P. D. Klimstra, and R. E. Ranney. J . M e d . P h a r m . Chem., 5, 720 (1962).
search for means of lowering serum and tissue cholesterol, a class of compounds was discovered whose hyposterolemic activity may be due indirectly to the formation of T-dehydrocholesterol and directly to the inhibition of sterol absorption. A preliminary study of these compounds has been reported8 and the details of synthesis have been described. We wish to report on some biological studies done on a representative member of this class, 1-( 2 - [4'-(trifluoromethyl)-4-biphenylyloxy]ethyl}pyrrolidine (boxidine, 35) in the series described by Bach, et aL9 (7) D. Dvornik, M. Kraml, J. Dubuc. M.Givner, a n d R. Gaudry, J . Am. C h e m . Soc., 85, 3309 (1963). (8) F. L . Bach, J. C. Barclay, F. Kende, a n d E. Cohen. l5lst National JIeeting of American Chemical Society, Pittsburgh, P a , .\larch 1966, Abstracts, p 18P. C h z m . Therap., 2 , 178 (1967). (9) F. L. Bach, J. C. Barclay, F. Kende, and E. Cohen, J . J f e d . Chem.. 11, 987 (1968).
995 HYPOSTEROLEMIC (TRIFLUOROMETHYL)BIPHENYLYLOXYETHYLPYRROLIDINE
September 1968
TABLE IV EFFECT O F BOXIDINE O N SERUM LIPIDSO F RATS5 BT VARIOUS TIMEINTERVALS ,--l
Dose, 5% of diet
week-----. Cholesterol
---2
% of control
mg 5%'
0 89 i 12c 8.5 f 3 . 4 0.0001 60i8.2 0.0003 25 1 2 . 8 0.001 12 i 1 . 0 0.01 1 8 f 1.9 0.03 a Male, CFE strain, 125
89 =k 3 . 8 72zt3.3 44~t6.7 21 f 1 . 9 1 2 & 1.9 7 f 1.4 5 g initial weight.
+
Final body nt, g 285c 17 . 2 283 5.3 264 6.4 270 & 4 . 8 156 k 4 . 6 176 * 8 . 0
* *
v.4RIOUS
Adrenal w t , mg 43.3 3 Z 2 . 4 4 6 . 6 zt 1 . 9 55.8 i2.1 7 4 . 1 13 . 0 88.0 f 5 . 3 9 4 . 1 1 4.2
--
7 -
5% of control
mg %'
95 67 28 13 20
EFFECTSO F Food intake, Dose, 70 gjrat,' of diet day 0 18.2 0,0001 18 2 0.0003 16.2 0,001 16.8 0.01 9.1 0.03 11.0
weeks--Cholesterol
Cholesterol, mg %
7-Dehydrocholesterol, mg %
-4
Total % of control
83 i 7 . 8 0 96 78 f 2 . 5 2 f0.2 81 50 4 . 3 73 11 f 1 . 2 49 49 18 f 1 . 0 24 23 f 1 . 9 18 3 f 1.5 12 =I= 1 . 0 13 5 f1.4 23 8 17 f 0 . 9 Uncorrected for 7-dehydrocholesterol.
TABLE V LEVELSO F BOXIDINE I N RATP
Adrenal wt/ body wt, mg/100g 15.13Z0.64 1 6 . 5 10 . 6 1 2 1 . 1 10 . 8 2 2 7 . 3 3Z 0 . 9 8 5 6 . 1 1 2.6 53.5 f 1.0
Adrenal stero1.b mg/g 2 8 . 5 11 . 8 2 2 . 9 11 . 5 2 1 . 0 11 . 7 1 0 . 1 1 1.2 4.4 + 1.0 3.2 1 0 . 4 3
Liver wt, g 1 2 . 8 10 . 6 4 1 2 . 8 10 . 3 3 1 2 . 4 10 . 3 6 13.7F0.44 8 . 2 i.0.37 9 . 0 f 0.43
AFTER
weeks --Phospholipids--
4 FVEEKS
Liver wt/ body wt, g/lOOg 4 . 4 10 . 1 1 4 . 8 zk0.04 4.7 f 0 . 0 6 5.0 1 0 . 0 8 5.2 1 0 . 1 6 5.1F0.08
+
mg 7%
% of control
169 f 13.4 95 f 2 . 7 91 1 5 . 3 96 160f 15.1 95 79f5.0 83 109i: 12.2 65 57f6.4 60 113 f 6 . 4 67 26f3.1 27 61 f 8 . 2 36 27f4.4 28 86f8.7 51 Mean f standard error, six per group.
OF
ADMINISTRATION
Liver sterol,b mg/g 2 . 4 10 . 1 1 2 . 3 3ZO.08 2 , l i 0.08 1 . 8 i. 0 . 0 3 1.4f0.08 1 . 4 3Z0.03
a Male CFE strain, 123 f .i g initial weight. These are the same animals used in Table IV. terol. c Mean + standard error, six per group.
7-dehydrocholesterol could not be detected. S o effect o n body weight was observed. Time and Dose Response in Rats.-The hyposterolemic dose response in the rat at intervals of 1, 2, and 4 weeks and the effects of serum phospholipids and triglycerides are shown in Table IV. As can be seen from the data, the compound a t 0.0003% in the diet was active in reducing lipid levels. The actual quantities of 7-dehydrocholesterol and cholesterol were determined in the serum samples obtained at 4 weeks (Table IV). The results indicate that relatively small amounts of the former appear, whereas relatively large amounts of cholesterol disappear so that one cannot sap that the 7-dehydrocholesterol replaces cholesterol. There is, in fact, an absolute reduction of total sterol with compound administration. The effects of different levels of boxidine on various organs and their lipid content are shown in Table V. Adrenal sterol was markedly reduced and adrenal hypertrophy was noted. Liver lipids and perhaps sterols were unchanged. Examination of liver sterols by glpc revealed increasing quantities of 7-dehydrocholesterol with increasing dose levels. Hence, if the sterol analyses had been corrected upwards, they would have been approximately the same as controls. Food intake was reduced at the high dose levels which mas subsequently reflected in reduced body, liver, and testes weights as compared with controls. It is of interest, however, that the ratio of testes or liver to body weight was increased, probably due to the marked decrease in body weight. Three Months Feeding Study in Rats.-To protect the animals from the pharmacologic effects of boxidine resulting in marked sterol depletion, they were fed ground Purina Laboratory Chow supplemented with 1% cholesterol o.5Y0cholic acid. This group served as the control. A corresponding group of animals was fed the same supplemented diet with the addition of boxidine a t a level of 0.0370. After 3 months, tail blood samples for hematological examination were
% of control
mg %
c
7
--Triglycerides--
Liver lipids, mg/g 80.0 i 0 . 1 1 81.4F 1 . 6 7 3 . 4 i. 1 . 8 7 9 . 3 12 . 0 76.3 F 2.9 74.0 1 3 . 0 b
Testes wt, g
3.1i.0.06 3.1=!=0.09 3.0 f 0.04 3.110.03 2 . 6 ztO.08 2.7 ztO.06
Testes w t / body w t , g/100 g 1 . 0 3Z 0 . 0 3 1.0 F0.02 l . l i 0.04 l . l i 0.04 1 . 6 10.03 1.5 f0.04
Uncorrected for 7dehydrocholes-
obtained under light ether anesthesia and the animals were sacrificed by decapitation. Various organs were removed, weighed, and examined grossly. Organ weights are shown in Table VI and the hematology study in Table VII. There was no significant difference between treated and control animals. The large livers in both treated and control animals were TABLE VI EFFECTSO F BOXIDINE O N BODYA N D ORGAX WEIGHTS" OF RATS AFTER 3 h I O N T H S O F ADMINISTRATION
Controls
0.03% boxidine'
Final body weight, gc 365.0 + 5 . 8 d 343.0 f 6 . 5 Liver, g 22.5 f 1 . 0 21.5 f 1 . 2 Adrenal glands, mg 59.2 f 3.2 57.2 f 2 . 5 2.96 f 0.10 2.78 =t0.13 Kidneys, g Heart, g 1.13 f 0.07 1.09 f 0.05 Testes, g 2.70 i- 0.05 2.84 i 0.08 Seminal vesicles, g l.lOf0.05 1.00 f 0.07 a All organs were wet weight. b One death in this group. Male CFE rats, 50 f 5 g initial weight, six per group. All ani0.570 cholic acid supplemented mals were o n a 1 % cholesterol diet for 13 weeks. Mean f standard error.
+
TABLE TI1 EFFECTOF BOXIDINE O N THE HEMITOLOGY O F THE R ~ T ~ AFTER 3 ~ I O ~ XOF TH ADMIXISTRATIOX S Control
0.03% boxidine
Serum sterol, mg yo 219 f 3Sb 211 f 16 Hemoglobin, g/100 ml 12.4 i 0.29 12.3 f 0.50 Hematocrit, yo 34.3 f 1 . 3 33.2 i 1 . 2 White cells, lO3/mm3 7 . 8 i 0.4 8.8 f 1.1 Red cells, l06/mm3 6.06 f 0.20 5 . 7 5 f 0.21 Differential count, % 11.8 f 2 . 1 11.6 f 1 . 3 Seg neutrophil 81.5 f 2 . 4 84.0 f 1 . 4 Lymphocytes Band cells 0 0 Eosinophils 0.2 f .2 1 f0 . 4 6 . 3 f 1.5 3 . 4 =I= 0.8 Monocytes hl yelocytes 0 0 0 Same animals and conditions as in Table VI. b Mean f standard error.
prol):it)l\ tluc t o the m:irhecl iiiereuw iri liver yterol : u i d lipidh resultiiig fro111 the cIiolesterol-suppleiiietite(1 diet Thi- t j p c of diet is iiecchharj to prevent lethal t cholesterol depletion observcvl with long-term high regimen Of particular ititere-t ib the observation that : d r c w d g h d erilargement w:ih not found in thiy iriht:tiicc although thew aninid, received verj high dohe\ ( i f cornpourid. Thib ~ v o u l dsuggest that adrenal glalicl 1ij perp1:isi:i observecl with boxidinc (Table V) waociutcd with imii (1 :tdren:il sterol depletion. 1 (Tablc VI) :ldren:il size na. normal. Similar findings h a w heen reported with t ripar:mol in which rnarlied adrenal hypofunction x i * csorrcctecl by the addition of cholebterol t o the diet. Ih
Relative Potency as Compared to Known Hypocholesteremic Agents.- .4 c'omparisori of hypocholctc1reniic potency in rats \\a- made nith f i aus-l,4-bis(Zchloroberiz! laniiriornet hj1)cyclohex:uic dih? drochloritlr ( 1 ) . trip:ir:inol (2), arid clohbrwte (3) i L t vnrious do 111 t l l v r:tt -1s slio\iti i t i I.'igurc 2. boxidi proxiiii:ttelj t r i i tinirs :is :rtivc, : i > 1. 50 tin1 'L- 2, a i d 1000 tiineh a* active :i\ 3. Mechanism of Action. 111 *onic preliiniiiar\ experii i i o i i t h o i l thc abwrptioii of stcrols in the rat, it vaq cihscrved that 11 heri hoxiditie u:i- added t o \terolwpplcnicntcd dieti tlicre ercb 1ritlic:itioris of iiihibitioii oI :ibwirpticiti. I t h:i. beril o u r c~\perienre that the> et I t mcayurcmeti t itidicxt ivc (if' xhsorptior I (11' diet:rrj .trrol \\:I\ elrvutcd l i i w stciol. I ,i- our rriterioit, o u r iiiitial -iridic- i1idic:it c d e [ i o diffcw~ice bet\\ ccii thc :iborptioii of 7 c~liole-tci~ol rid cliolcztcrol i r i the rat (T:tblc VILI) vas ill,)
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