The Effect of Substituted Carboxylic Acids on Hepatic Cholesterogenesis'
cfyc-liceyteni ranging froni cyclopropane to cyclohesane were synthesized. The synthetic methods employed were ( a ) rondensation of the rorresponding aryl acetonitrile with the appropriate a,w-dibromide of the hydrocarbon and ( b ) yuasi-Favorski1 rearrangement of the corresponding aralkyl bromo ketone under strongly basic conditious. \\-hen ~-biphenylyl-l-brt,mocyrlopentvl ketone (1TIa) \vas treated under quasi-Favorskii conditions, an '.XI)riomal" produrt was ohtained. The structure of this compound was assigned based on its spectral properties xiid confirmed by unequivocal synthesis. \Vhile some of the substituted c.arbosylic mids inhibited caholesterogenesis from niev:iloniite i n nit liver honiogenntes, none eshibitecl :my :~ntihprrc:holesterr,lelnic.effect i n nephr~it i c s
rats.
rl'h(>first observation that pheiiylacetic acid derivativcs iiiight bc useful as cholesterol lowering agents was itiadc by C'ottet, et al.? l h e y claimed that a-pheiiylbutyric acid (Ia) was cliiiically cffectivc in the treatiiiciit of I.iypercholcstci.ole~iiia. Thcse results were not coiifiriiicd by other ~ o r k c r s . ~Stchhcrg arid his collaborator9 and Garattini, ef u / . , ~havc1 show1 that the conipouiid exerts its inhibitory effect at the acetate activatioii stage. Possible iiiterfereiicc with fatty acid iiietabolisrii coupled with the weak aiitihypercholesteroleiiiic activity render this agent of doubtful therapeutic valuc. A dcfiiiite advance in the direction of producing potriit and more selective hypocholesteroleiiiic agents was rcvealed by the following findings. When th(. 0-phcny1 group was replaced hy a 4-hiphenylyl nioicty, the coiiipouiids produced were iiiore active and caused appiwiublc inhibition in the cholesterol biosynthetic pathway at some post-mevalonate step.g 2-(4-Biphciiylyl)-l-liexeiioic acid"(1b) is a highly active nieriiher of this class aiid has been shown to be active in vivo as an aiitagoiiist to the first phase of Triton-induced hppcrcholcsteroleniia in the ratL2as well as an antagonist
I.
to dietary liypercholesteroleiiiia in dogs. l 1 Iitmlts iii the clinic with 2-(4-bipheriylvl)-4-hexe1ioic acid (It)) have bceii variable. In an at tempt to obtain coiiipounds having spccific liypocholel-~tcroleiliicactivity inhibiting the biosynthesis a t a post-iwvalonate stage, suhstitutcd carboxylic. acids aiid cwboxylic acid derivatives of the type' I1 (It = 4-biphenylyl or 2-fluorenyl) n-ert' synthesized. 'l'hrsc. coiiipounds were prepared by the two general methods showi ill Chart I. J
c, I 1 = 4-biphenylyl; n = 4 I\->I, It = 4-biphenylyl: 11' = OH; 72 h , R = 4-biphenyI\I; 11' = OH; n c. R = 4-hiphenylyl; 11' = OH; n t i , I{ = 4-hiphenvlvl; 13' = YH?; n
= 2 = :3 = 4 = 4
'OH 11
\
Ta, It = phenyl; It' = ethyl I), It = 4-biphenylyl; l e ' = CH,CH=CHCHj
1
('HZRP \ r E TIIOT)
TI, I t
4-t)iphenyl~-l01 '2-fluoren yl =
.I,
11,
151.5i).
iiii 1). SteinberL: and I ) . S. i'redricksirn, 1 ' i . o ~ .Sac. E x p t l . B i d . .\fed.. 90, 2 3 2 (19.54). im. B ~ o p h y s .Acta, 17, 592 (1938). ( 7 ) It. Masters a n d I). Steinberg. B ( 8 ) H. Garattini, C . blorpurgo. B. 3ltirelli, K. Paolett,i, a n d N. Passerini, A r c h . I n t e r n . Pharmacodyn., 109, 400 (1957). avormina arid SI. Gibhs, J . .lm. Chem. Soc., 79, 788 (1957), \$'ridit, I'roc. S o c . E x p t l . B i d . .Ifmi., 96, 364 (1957). v a l i i q i , I C . llassarani. I).Nardi. I,. hIauri, F. Barzaghi. :LII I 1'. .\luriteeazza. J . .lm.Chem Soc ,81. 2.ilj.1 (19.59).
=
4
=
5
IBr
(1) P a r t V of a series entitled ".iyents Affecting Lipid hletabolisiii." I'or p a r t I Y ( f . I ) . I ) a o r n i k , 31. lirariil. .I. Iltibiir., 11. Givner, a n d R. Gaudry, .I. .tm. C ' / ~ ~ t nS .o c . , 8 6 , 3309 i1963). ( 2 1 . A i i t l i i x to w l ~ o n i iniiuiries sliorilii h e addressed; I l e g a r t n i e n t (if < ' ! i v ~ t ~ i s t r1y- r,i i i c,rsity of Ottniua. OttawL, Panada. f:i!J . C o t t e t . .A. l I a t I i i v a t , ani1 .I. Redel. I'ressr .%fed.. 62, 930 ( 1 0 R S J . ( 4 1 I ) . S. Fredric,ksoii i l t i r l 1). SteinhPrs, Circulation, 15, 391 (1H57). (.i] 1'. (;ran,le, ,J. T, .Anderson, ;tiid A . Keys, .l.letah. Clin. >;xpll., 6, 1.54
n n
"
(CHZ)~
qC)'
OH __t
c ~ ,
(8,
a,
(['Hj),,
'(.02R'
IS
\'I11
R
:
R
= =
11, li P,
-
(CH,) R
'COR YII
as in V I b, as in 1'1 as in T'I
-
a, 11 = 4-b1phenjl\l; t1 = 1 b, It = 4-biphenylvl; n = ,i c, It = Zfluorenyl; n -- .i
4-biphenyl) I, 11' 2-fluorenyl; R' 4-hiphenylyl; R'
= = =
,OH
C
'"
H, n = 5 H; n = 3 CH2CH2?;(C2H n =
.)
(12) S. G a r a t t i n i . It. I'aoletti. L. 13izzi, E. Grossi, and It. \-ertim ill ".%gents Affec tine Lipid lletabolisni." S. Garattini a n d R. I'aoletti, l : < l , , Elsevier Piiblisiiinp Co., .Amst~rdarii,1961, p. 14-1. (13) 11. Xlassaioli, 0 . ('ennaino, G . Ijalbo, a n d N. Masera, R o l l . S o i . Piemontese C h ~ r .30, , :3 (1960). 114) I;. Tescori, G m z . I r i t ~ m ..ifed. C'hir., 63, 2447 f19ZJ). (15) E. Sahbadiiii s n i i 11. Gazzaniga. dfinerua .Wed., 40, 154:7 fI!l:!):.
505 1- (4-Biphenylyl)- 1-cyanocyclopropane (IIIa) , -cyclobutane (IIIb), and -cyclopentane (IIIc) were prepared16 by the treatment of ethylene dibroniide, 1,3dibroniopropane, and 1,4-dibromobutane, respectively, with the disodium salt of p-phenylbenzyl cyanide anion.17 Hydrolysis of the resulting nitriles gave the corresponding carboxylic acids (IVa-e). The hydrolysis of IIIc also gave amide ITTdin addition to the acid IT'c. An alternative route to the desired carboxylic acids involved the corresponding aralkyl ketones (VIa-c). These ketones were obtained by Friedel-Crafts reaction of the acid chlorides of the appropriate alicyclic carboxylic acids with the aromatic hydrocarbons. The bromo derivatives of these ketones underwent quasiFavorskii18 rearrangement on treatment with powdered sodium hydroxide in boiling xylene. I n the case of 4-biphenylyl-1-bromocyclohexyl ketone (VIIb) the desired l-(4-biphenylyl)cyclohexanecarboxylic acid'g (VIIIa, 34y6)was obtained with the conconiitant formation of a large amount (43y0) of 4-biphenylyl-l-hydroxycyclohexyl ketone'bb (IXa). The bromo kctone (VIIc) having a 2-fluorenyl substituent gave a poorer yield of the corresponding carboxylic acid (VIIIb) in the quasi-Favorskii reaction. Here again a substantial amount of hydroxy ketone (IXb) was isolated. When 4-biphenylyl-1-broinocyclopentyl ketone (VIIa) was treated under quasi-I;avorskiI conditions18 none of the expected 1-(4-bipheiiylyl)cyclopentanecarboxylic acid could be isolated. Instead, a compound was obtained in 54% yield which absorbed a t 286 mp ( E 20,600) in ultraviolet light (expected absorption would be a t 256 nip) and exhibited bands a t 1680, l i O O (C=O absorption) and in the region 3500-2500 (COOH) ciii.-l in its infrared spectrum. Based on the spectral and analytical data this product was identified as 6oxo-6-(4-biphenylyl) hexanoic acid (Xb) , This structural assignnient was confirmed by a n unequivocal synthesis of Xb. When Z-carbornethoxyvaleryl chloride mas treated with biphenyl in the presence of aluminum chloride, the keto ester Xa was isolated. Hydrolysis of this substance gave an acid which was identical in all respects with the keto acid Xb obtained froin the reaction of 4-biphenylyl-1-bromocyclopentyl ketone (VIIa) under conditions18 for quasiFavorskii rearrangement. CsH,C&L
CICO(CH2)&OOCH3 AICI:,
0
0
p-C6HSCGH4CO(CH2)4COOR /I
Xa,R=CH3 b,R=H
-+ = c;= (-J VIIa
The mode of formation of acid X b is worthy of comment. One possible pathway to this product is depicted below. It appears that the rate of quasiEavorskiI rearrangement in the cyclopentyl series must be niuch slower relative to that in the case of the cyclo(16) F. H. Case, J. A m . Chem. Soc., 66, 715 (1934). (17) (a) R. Lesser, Chem. Abstr.. 82, 4798 (1938); German Patent 658,114 (1938); (b) G. Cavallini and E. Massarani, Italian Patent 600,214; Chem. Abslr.. 55, 16,486 (1961). (18) (a) Cf. C. L. Stevens and E. Farkas, J. Am. Chem. Soc., 74, 5352 (1952); (b) E. E. Smissman and G. Hite, ibid., 81, 1201 (1959). (19) N. H. Croniwell and P. H. Hess, ibid.. 82, 136 (1960).
/
hexyl compound, to allow the forniation of coinpound X b in over 50% yield. It is probable that these rate differences result from the inherent stereochenlical differences between the five- and six-nieinbered ring systems. Biological Methods. A. Cholesterol Biosynthesis Inhibition. -The ability of the test compounds to inhibit the incorporation of mevalonate-2-CI4 into cholesterol by rat liver homogenate5 was estimated as previously described.*O B. Antihypercholesterolemic Effect.-This effect was measured in rats that had been made hypercholesterolemic by inducing nephrosis o m daily administration of the aminonucleoside of puromycinz1 ( l i . 5 mg./kg. s.c.) for 8 days. Test compounds were administered daily (s.c.) from day 3 to 12 and their ability t o prevent the rise in serum cholesterol n-as taken as a measure of their antihypercholesterolemic effect. Serum cholesterol levels were measured using the method of Pearson, et aL2*
Results and Discussion The results of in vitro inhibition are presented in Table I. 2-(4-Biphenylyl)-4-hexenoicacid (Ib), our reference compound, inhibited cholesterol biosynthesis 91% at 1 X 10-3X final but was inactive a t 1 X AI. In our series of compounds only three (Xb, YIIIa, and VIIIc) were as potent as Ib, inhibiting 86, 94, and 98%, respectively, a t 1 X 10-3JI final. At 0.3 X 10-4M final, VIIIc was devoid of activity. TABLEI INCOKPORATIoN O F >~EV.4LON.4TE-2-C14I K T O CHOLESTEROL BY RATLIVERHOMOGEXATES
EFFECT OK
n CRCOR' u
(CH,),
% inhibition No.
R'
R
n 2 3 4 4 5 5 5
at 1 X 10-3 .If, final 21 7 0 0
1Vb IVc IVd VIIIa VIIIc YIIIb
4-Biphenylyl OH 4-Biphenylyl OH 4-Biphenylyl OH 4-Biphenylyl NHz 4-Biphenylyl OH 4-Biphenylyl OCHzCHzN(CzHs)z 2-Fluorenyl OH
Xb Xa Ib
p-CeHaCsH4CO(CHz)aCOOH 86 ~ - C ~ H ~ C ~ H I C O ( C H ~ ) ~ C O O C H ~32 p-CsHsCaHaCHCH2CH=CHCHa 91 (0 at 1.0 X 10-4 M )
IVa *
PO
94 98 (0 at 0.5 X 10-4 M ) 0
Miscellaneous Compounds
I
COOH
Compound VIIIa, being among the most active in the series in vitro and being available in sufficiently large amounts, was tested as an antihypercholesterolemic agent in rats rendered nephrotic and hyper(20) L. G. Humber, SI. Kraml, and J. Dubuc, Bioch,em. Pharmacal., 11, 755 (1962). (21) C. Chappel and K. Voith, private communication. (22) S. Pearson, S. Stern, and T. H. McGavack, Anal. Chem.. 26, 813 (1953).
rtiolcstc~roleiiii~~ i i i the i i m i i i ~ i *diwribed above.. 2(l-13iphciiyIyl)-l-hesc.lioic acid was thc refcreiicc coni~ O U a Ji d~ the ivsiilts are prcseiited i l l Table 11. .As caii tw heen, the roiitrol aiiiiiials are iiiarkedly hypercholesteroleniic, the levels having risen to the 600700 ing.,'lOO nil. range froiii a normal of approximately 100 11ig. 100 nil. 2-(l-Biphe1iyly1)-4-hexenoic acid was activc at both thc 40 and 20 iiig. kg. dosage schedules. droppiiig cholesterol lcvcl:, 3i and 1gG;, respcctiwly Elowcvcr J'IITa wab iiiactivc i n this in i wo tebt \Thili> soiiie wnipouiidb in this series arc active ? t i c i t r o as cholesterol biosyithr. irihibitors, this activity is not reflected zn 1 i i o as cvidenced by the failure of I'ITIa to dcpresa cilevatcd rholesterol Icv~ls. Coni-
Experimentalzri 4-Biphenylyl Cyclopentyl Ketone (VIa). --A solution of cyi,liiI)c.iitariec.:trbciiiyl chloride (Va, 24.9 g.) arid biphenyl (26.5 g.) i n dry carbon disulfide (80 nil.) ddetl over :t period of 3 0 r u i n . to a stirred suspensioii of srihytlrous aluminum chloride (26.5 g.) in wrboii disulfide (SO nil.) a t 0". The reaction was quenched by riririg t,he mixture into wat8erarid ice containing concentrated drorhlorir acid. This was extracted with et,her and the excats were washed with dilute sodiuni hicarbonate solution :ti111 x n w t t e r . The ether solutioii \vas dried arid evaporated t o dryness leaving :in oily solid (41.2 g . ) whicsh, after crystallieatitrri froni ethaiiol, had i1i.p. 36-78', A,,,,,, 2% ins ( E 2.?>000). The crudc producat c-oiild he used without, further pnrificaation f'cir the nest sttq). An aiislytic:ii s:iiiiple preps rc,:igeii t ' tiad 111.11. iiI --ii;3' : A,,,., f ~ l l l . 1.
.It~tfi.
M.44;
(:al~tl. IO^ C'isHisO:
(',
H,7 . 2 5 .
1-Bromocyclopentyl Ketone (VIIa).--Crude +hientyl ketone (VIa, 10.0 g . ) was dissolved in acaetic :ic*itl(01 nil.) and heated to 85'. Hromine (6.4 g.) &-asadded and i l i p solutioii decolorized iinmediatelg. 11ore bromine was then added until a faint yellow cdor persisted and the solution was k q ) t at 85" for 30 iriin. The cooled solution was poured into cold xvater :inti tlie guriirriy iii:tteri:il which precipit.ated was extract,ed wit ii ether. After xvashing the ertracts with sodium hicarbonate : i n ( I water, the ether solution was dried and the solvent removed. 'I'ht: krruwn sirup obtained was chronlatographed on (300 K . 1. Elution with petroleuni ether gave biphenyl (2.01 g.) and I I I ~cahnnging to petrolriirii et,her--lwnzene ( 4 : l ) the bromo ketone (23) l l e l t i n g points were determined o n a Tliiele-Dennis a p p n r a t u s and a w vurret.tei1. Infrared spectra were recorded on a Perkin-Elmer (.\lode1 21) s1,ertrophotonirter equipped \rith sodium chloride optics. Ultraviolet qgrctra were t a k e n in ethanol w i t h a Beckinan fMoi1el Dk-1 recordinp in~ t r u r n e n t . I'lorisil (60-100 rnesli, I'loridin (10.)a n d silica gel (Ilavison. gradt. !42:3, 100-200 meshj were used for c1iromatoprar)hy Petroieiiin etiier refers t,ii t h e fraction with h.p. :10-60". Organic extracts were d r i d o v e r :in113 ( I r i i i i p inagnesiiiin w i f a t e ami T I I P solx-rnra were r e ~ n o r e d iindrr vi,l',l,ln,.
( 2 3 ) I.. F'. Fiewr i n "Experiments i n Organic ('lirriiistrx," I ) . c'. Heat11 ( ' , > r n ~ m n vI>r,ston, , hIass., 1857, p. 88.
ritiii
July, 19G-l
C.4RBOXYLIC
ACIDSI N
carbon disulfide (60 ml.). The r e a d o n mixture was cooled in an ice-water bath during the addition, and stirring was continued for 15 min. a t room temperature. After pouring the reaction mixture into cold 1:3 hydrochloric acid-water, the layers were separated and the aqueous phase was extracted with ether. The carbon disulfide solution and ether extracts were combined and washed with 5clc aqueous sodium hydroxide solution and water. .4 yellow solid (18.7 9.) was isolated after the organic solution had been dried and subsequent,ly evaporated to dryness. An analyt'ical sample of methyl 6-0~0-6-(4-biphenylyl)hexanoate(Xa) 285 nip ( e 23,900); u::: 1737 (ester melted a t 108-109"; A,, carbonyl), 1685 (carbonyl adjacent to aromatic ring) cm.-', and was obtained after three crystallizations froni acetone-hexane. Anal. Calcd. for C19H&: C, 77.00; H, 6.80. Found: C, 77.06; H, 6.86. Methyl 6-oxo-6-(4-biphenylyl)-hexanoate(Xa, 1.0 g.) prepared above was dissolved in methanol (25 ml.) and 5% aqueous sodium hydroxide solution (5 ml.) was added. This solution was refluxed for 1 hr., cooled, diluted with water, and extracted with ether. The aqueous extract was acidified (hydrochloric acid) and then extracted with ether. Working up in the usual way gave a white solid (0.87 g.) which after one crystallization from 287 m p ( e 26,150); 1700 benzene melted at, 162-163'; A,, and 1680 em.-'. Comparison of infrared spectra and a mixture melting point showed that this compound ( X b ) was identical with the acid obtained in the quasi-Favorskii rearrangement of 4-biphenylyl 1-bromocyclopentyl ketone (VIIa). 2-Fluorenyl Cyclohexyl Ketone (VIc).-A solution of fluorene (25.0 g.) and i.yclohexanecarbony1 chloride (23.0 g , ) in anhydrous carbon disulfide (80 nil.) was added over a period of 40 min. to a stirred mixture of anhydrous aluminum chloride (30.0 g.) and carbon disulfide (100 ml.) cooled in an ice-water bath. After the addition was completed, the cooling bath was removed and stirring was continued a t room temperature for 30 min. Working up in the usual way gave a crude product which after one crystallization from methanol afforded needles (23.5 g.), n1.p. 150-152". A second crop (8.2 g.), n1.p. 147-150°, was also obtained. An analytical sample melted at 15-152'; A,, 297 (E 23,900), 317 nip ( e 26,350); 1676 cm.-l. Anal. Calcd. for CYOHZOO: C, 86.92; H, 7.29. Found: C, 86.80; H, 7.15. 2-Fluorenyl 1-Bromocyclohexyl Ketone (VIIc).-2-Fluoreny1cyclohexyl ketone (IIc, 10.0 g.) was dissolved in chloroform (100 ml.) and warmed gently while a solution of bromine (5.8 g.) in chloroform (40 nil.) was added over a period of 45 min. with stirring. The volume of the solution was concentrated, methanol was added, and the product crystallized as plates (11.0 g.), m.p. 118-124'. Two more crystallizations from chloroform-methanol icalsample, m.p. 121-122; A,, 242 ( e 8570), 318 mp 1675 (carbonyl adjacent to aromatic ring) cm. -1. Anal. Calcd. for CnoHlsBrO:C, 67.60: H, 5.38; Br, 22.50. Found: C, 67.57: H, 5.61; Br, 22.75, 22.72. Quasi-Favorskii Rearrangement of 2-Fluorenyl 1-Bromocyclohexyl Ketone (VIIc).-A solution of 2-fluorenyl l-bromocyclohexyl ketone (VIIc., 9.5 g.) in dry xylene (45 ml.) was added to a suspension of powdered sodium hydroxidelab (34 9.) in refluxing xylene (400 ml.) over 20 min. with vigorous stirring. The reaction mixture was stirred and refluxed for 2 hr., cooled to room temperature, and washed with water until washings were neutral. A gummy material precipitated a t the interface and was collected by filtration. This could now be dissolved in water and after washing the aqueous solution with ether it was acidified with hydrochloric acid. A red solid (2.2 g.) precipitated which was collected hy filtration. The basic aqueous extracts above were found to contain only t,races of acid. Chromatography of the red solid over silica gelz3and elution with benzene-chloroform (24:l) gave a white solid (0.52 g.) which after 4 crystallizations from aqueous methanol gave analytically pure 1-(2-fluoreny1)cyclohexanecarboxylic acid (VIIIb), m.p. 207-209'; A,, 270 ( e 29,200), 295 (9380), 307 niH (13,100); v:: 1692 (carboxylic acid) cm.-'. Anal. Calcd. for C?OH?,O~: C, 82.15; H, 6.89. Found: C; 81.96; H, 6.92. The neutral xylene solution above was dried and evaporated to dryness. One crystallization of the residue from benzene-hexane gave a substance (2.0 g.) identified as 2-fluorenyl l-hydroxycyclohexyl ketone (IXb), m.p. 131-135'. An analytical sample had m.p. 140-141"; A,,,314nir (E 25,800); u:: 3610 (bonded 0-H), 3470 (nonbonded 0-H), 1671, 1657 (carbonyl adjacent t o aromatic ring) cm. -1.
:u
YE:
507
HEPATIC CHOLESTEROGESESIS
Anal. Calcd. for C20H2002: C, 82.15; H, 6.89. Found: C, 82.43; H, 6.76. 1 -( 4-Biphenylyl)-l-cyanocyclopropane(IIIa).-p-Phenylbenzyl cyanide (15.0 9.) was dissolved in anhydrous ether (250 ml.) and sodamide (6.5 g.) was added to the solution. This was refluxed with stirring and after a short time a red color appeared which became very intense after 0.5 hr. At this point ethylene dibromide'e (14.65 g.) dissolved in dry ether (20 ml.) was slowly added over a period of 15 Inin. Refluxing with stirring was cont,inued for 9 hr. and then the reaction mixture was poured over crushed ice and extracted with ether. The combined extracts were washed with water, dried, and evaporated to dryness. A red solid (14.9 g.), m.p. 80-88", was obtained which after several crystallizations from acetone-hexane afforded an analytical 256 nip ( e 25,800); Y;>Z 2240 (nitrile sample, m.p. 108": A,, group) cm.-'. Anal. Calcd. for CI6Hl3N: C, 87.64; H, 5.98; N, 6.39. Found: C, 87.65, H, 6.09; N, 6.62. 1-(4-Biphenylyl)cyclopropanecarboxylic Acid (IVa).-1-(4Biphenyly1)-1-cyanocyclopropane(IIIa, 5.0 9.) was dissolved in ethanol (70 ml.) and to this was added a solution of potassium hydroxide (20 g.) in water (20 ml.). The reaction mixture was refluxed for 20 hr., cooled, and diluted with water. After extraction with ether, the ether solution was in turn extracted with 5% aqueous sodium hydroxide solution. The first basic extract was a clear red solution but after a few sec. a white solid precipitated and was collected by filtration. This solid was dissolved in water and on acidification with dilute hydrochloric acid, 1-(4biphenylyl)cyclopropanecarboxylic acid (IVa, 1.97 g.), n1.p. 215219O, precipitated as a white solid. An analytical sample prepared by t'wo crystallizations from aqueous ethanol was obtained 255 nip ( e 24,300); uZx1690 as fine needles, m.p. 218-221"; A,, (carboxylic acid ) cni.-'. Anal. Calcd. for C16H1402: C, 80.64; H, 5.92. Found: C, 80.79; H, 6.10. 1-(4-Biphenylyl)-l-cyanocyclobutane(IIIb).-p-Phenylbenzyl cyanide (15.0 g.), dissolved in dry ether (250 nil.), was condensed with 1,3-dibromopropane (15.8 g.) in the presence of sodamide (6.0 g.) under the same conditions16as those used in the preparation of IIIa. The reaction was quenched by pouring the mixture over crushed ice. An emulsion formed which was broken by filtration and the solid (5.5 g.) which was collected was found to be crude I I I b (nitrile band a t 2240 em.-' compared to 2265 cm.-l in p-phenylbenzyl cyanide). Separation of the layers in the filtrate and extraction of the aqueous layer with ether resulted in the isolation of a reddish brown oil (11.7 g.) which was also identified as crude IIIb. An analytical sample was obtained by chromatography on Florisil*3 and crystallizat,ion from acetone254 m p ( e 22,400); P:::'~ 2240 hexane, m.p. 115-116'; A, (nitrile group) em.-'. Anal. Calcd. for CliHlaN: C, 87.51; H, 6.48. Found: C, 87.64; H, 6.52. l-(4-Biphenylyl)cyclobutanecarboxylic Acid (IVb).-Crude 1-(4-biphenylyl)-l-cyanocyclobutane(IIIb, i.1 g.) was dissolved in ethanol (100 ml.) and this solution was added to a solution of potassium hydroxide (28.3 g.) in water (28 nil.). The reaction solution was refluxed for 20 hr., cooled, diluted with water and extracted with ether. Acidification of the aqueous solution with dilute hydrochloric acid precipitated a white solid (1.73 g.), m.p. 121-136'. One crystallization froni aqueous methanol raised the melting point to 146-150". An analytical sample melted a t 246 m p ( e 21,700); 150-154'; A, 1695 (carboxylic acid) em.-'. Anal. Calcd. for C17H1602: C, 80.92: H, 6.39. Found: C, 80.65; H, 6.32. 1-(4-Biphenylyl)-l-cyanocyclopentane (IIIc).-p-Phenylbenzyl cyanide (15.0 g.), dissolved in anhydrous ether (250 ml.), was condensed with 1,4-dibromobutane (16.85 g.) in the presence of sodamide (6.0 g.) under the same conditions16as those used in the preparation of IIIa. The crude product (18.9 9.) on crystallization from hexane gave a first crop (8.50 g.), m.p. 75-81', and a second crop (3.47 g.). An analytical sample melted a t 91-93'; A,, 252 ( e 28,800), 258 mp (29,200); u:!::"'~ 2238 (nitrile group) cm. -l. Anal. Calcd. for ClsH1,!V: C, 8i.41; H, 6.93: K, 5.66. Found: C, 87.70; H, 7.04; N, 5.83, 5.77. Hydrolysis of I-(4-Biphenylyl)-l-cyanocyclopentane (IIIc).Crude 1-(4-biphenylyI)-l-cyanocyclopentane(IIIc, 8.5 g.) was dissolved in ethanol (120 ml.) and added to a solution of potassium hydroxide (33 9.) in water (33 ml.). The reaction solution
uzx
was refluxed for 16 lir. aiid t,tien cxroled. ljilutioii w i t h water precipitated needles (5.19 g.) w1iic.h were identified its 1-(4-biphenyly1)cyclopentanecarboxamide(IVd), n ~ p14'3-1 . 5 I O after onv allization from acetone. An analytical sample melted R I 1510; x,,; 257 nip ( E 23,400); v , 1692 (amide) mi:-'. i l n n l . Calcd. for CISHIQNO: ( 2 , 81.4i; IT, i.22: S , 5.2,k. F o ~ n d :C, 81.80; 13, 7.20; X, 5.28, 5 . 2 i . The above filtrate was extrarted with ether and acidification of the nyucous solution gave only trams of :tc:idic material. The combined et,lier extracts were washed \vir11 water, dried, an(l rvnporateti to dryness leaving a yellow arriorphous solid (2.62 g. ). 'This solid was suspended in it 5f!; aqiieous solution (50 nil.) of sodium hydroxide and reflused for 1 hr. After cooling, tlie undipsolved solid (crude amide) was separated by filtration and ttir filt,rat,ewas acidified with dilute Iiydrocliloric acid. Fktractioii with ether and working u p in the usir:il way gave 1-(4-biphenylyl)-
cyclopentanecarboxylic acid [IVc) as a Xvliite solid (0.21 g.) \vhic.li riielted a t 187-189" after one crystallization from aqueous nieth:triol. -411aiialyticd sample melted a t 200--201': A,,,,,, 256 nip ! e 24,200); ugt Iti% icsrboxylic acid) cni. - 1 .+trtal. C:tlctl. fiir CIYHL702: C, S l . 1 5 ; l i , ti.;'. ~ ~ ' ~ I ~ (I ' I: I SI).%; €1, 6.9s.
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Acknowledgment. --The authors gratefully adman ledge the techniral awistance of l r r . H. Luz and 111.. 1: (:uthrie aiid thank Dr. G I'apineau-Couture and h i aw)c*iatesfor aiialyt iral and spect 1 a1 data. They also their appreciatioii to 1)r. I). I)\ 01 iiih illl(i t o nr. r, G ~ I for ilc.ipfui ~ suggestlolls ~ ~ tillti d i s ( ~ u s i ~ oduriiig i i ~ t lie (*ourieof this n ork.
A Correlation of Drug Concentration with Sterol Biosynthesis Inhibition in the Liver1 P. I\. FIGDOR, 15. ( ' , SCHRETBER. 11. B. STERBIUS, P. P. MOORE,A Y D REX PIXSOY, JR.
The time course of sterol biosynthesis inhibition of t v o Z,:~-diplieriylac.ryloni~riles which block tlie conversion The data thus obtained are cmrrelated with drug concentration in the liver and with the effects of these cornpounds on sterol biosynthesis of desrnosterol to cliolesterol is cornpared with triparanol in orally treated rats. 111
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the relative amounts of labeled desmosterol arid choThe liver is regarded as a primary site for the synlesterol in the liver and to measure a t the same time the thesis as well as the metabolism of cholesterol.? That drug content of the liver, This report describes ttii a feed-hack mechanism is operatiw i i i the liwr has experiment in which the drug content of the liver wa+ 1)eeii shown by cholesterol-feeding experiments which correlated with its effect as indicated by the comparisorr produce higher than normal concentratioiis of cholesof radioactive liver sterols following mevaloiiate-2-("J terol in the liver. These livers have a reduced capacity injection. For this purpose two compounds, 2 , : ; t,o synthesize new ~ h o l e s t ' e r o l . ~Chemical ,~ agents his [p-(2-diethylaminoethoxy)phenyl]acrylonitrile ( I ) also affect' liver cholesterol. Estrogens Trhich effectively and trans-3-[p-(2-diethylaminoethoxy)pherigl]-~-pheiilower serum cholesterol have been shown to alter yl-%pentenenitrile ( T I ) , 9 were c o m p a i ~ ~n-it11 l ti+ both liver sterol content and the ability of these livers paranol (Chart I ) . to synthesize p hole sterol."^ Triparanol, a sterol biosynt'hesis inhibitor, does not materially change liver CHART 1 sterol concentration, but does alter liver sterol comI. posit'ion. Desmosterol, which is present, in minute amounts iii normal rat liver, replaces much of the cholesterol in the livers of triparanol-treated a ~ i i m a l s . ~ , ~ 111 the course of studies of sterol biosynthesis irihihit,orsYthat block the conversioii of desmosterol to cholesterol, we became iiiterested ill correlating drug concentratioii at the site of action with degree of inhibition. By admiuisteiiig a labeled precursor to animals pretreated u-ith the drug we hoped to deteriiiiiie the extent, of biosyiithesis inhibitioii by measuring ( 1 1 Based o n a preaentrttion a t the l 4 l n d S a t i o n a l M e e t i n s of t h e Anierir a n Chemical Society, Atlantic C i t y , S . J., Seyjt. 10. I9fi2. 1 2 ) n. K r i t r h e v s k v , "Cholesterol." .Jolin \T-iley a n d Sons, I n c . , S e a Turk. X. T..1958, pi>. 7 5 , 115. (31 1. I). I'rantz, ,Ir., El. >. W i n e i d e r , : i n d 1%. T. Hinkle:nan, J . B i d . C ' h ~ m ,2, 0 6 , 46,; (1954). 141 l1orvevt.r. i t hiis also been shorvn i n inore recent experiments that in,i n i r e d liver chdesteropenesis does not always correlate with liver sterol cont e n t : A I . 1 ) . .';perstein a n d 31. .J. Gut.at, .I. Clin. I n i e r f . . 39, 642 (ISGO). . I3oyd a n d LT. 13. 3 f r ( i i i i r e , Biochem. J . , 62, 19p (1956).
,711 (1964).
TRIPARANOL Et Et:NCH,CH, Et Et
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