13. 12. DEWALD A N D I
~ ~ 1 ~ 1 l A ~ ~ .\c"l'I\.I'l'Y ~ 1 C ~ 4 1 , ()I*' 13R.\lIYKISIS
.\S.AI.(l(;>
I:I.oIIcIIv-
Peptide
constrictor :irtivity," mima pic
Ilypotensivi, activity," giiinen ~ i i x
9-llesarginine bradykiiiin 9-Citrulline bradykinin 9-Histidine bradykinin ' /?O Bradykinin 1 1 'I H. 0 . J. Collier, J. Iiolgate, 11.Scbactcr, nnd 1'. (;. Sliorh brief lowering of ley, Brit. J . f'hartnucoi., 15, 290 (1060). blood pressure ttiroiigli vasodilation and increase of perme:ibility of skin capilla,ries. with 13.6 g. 10.03 mole) of curhobenzoxy-L-phenyl:~l~Iiinep nitrophenyl ester. The mixture was stirred Overnight :it 35" to yield 16.3 g. (S7C;,) of colorless needles, m.p. 156-1.5!3", [ ( Y ] % -40.5" (c 1, dimethylformamide). A n d . Calcd. for C3SH40N408:C, 65.18; H, 6 . 2 5 ; N, S.fi$). Found: C, 65.12; HI 6.03; X, 8.87. Carbobenzoxyglycyl-L-phenylalanyl-L-seryl-L-prolyl-~~-phenylalanine Methyl Ester (III).-Carbobenzoxy-L-phenylalanyl-1,seryl-L-prolyl-L-phenylalaninemet'hyl ester ( 15.5 g., 0.024 molri was hydrogenated in niethanol in the presence of 20',i Pd-G i n the usual manner. The reaction mistlire wits filtered and ev:~porated in vacuo. The oil was dissolved in i 5 1111. of warm ctliyl acetate and S.0 g. (0.034 mole) of carbobenzoxyglgcine p - n i h phenyl ester was added. The mixture was stirred a t 35' for 2 days; it was washed Xyith sodium carbonate, dilute hydrc~chloric acid, and saturated sodiurii chloride. The solution XV:LS uoncentr:tted to ca. GO Inl. and ether ~ : t added s to yield 13 g. (777,) of colorless solid, m.p. 105-10So. It was recrystallized from methanol-water, 111.p. 112-114°, [ e ] ? a ~-32.4" ) (c 1, tiirn~?thylforinnmide). h a l . Calcd. for C3:H43Nj09: C, ij3.31; H, ii.17; N, !J.W. Found: C, 63.19; H, 6.08; S , 10.13. Carbobenzoxy-L-prolyIglycyl-L-phenylalanyl-~-seryl-~-prolyl1.-phenylalanine Methyl Ester (IV).-Car~)obenzosyglyc?.l-r.phenylalanyl-r~seryl-r~-prolyl-~-plienyl:~lsnirie methyl ester ( I 2 g., 0.017 niole) was 1iydrogen:ited in nieth:inol in the preacnc-c oi io(,*;P&C. 'rhc solid, w1iic.h W:IS obtained 1)y filtering t l i e aitalyst, : r i d evaporating in L'UCUO, weighed 9.3 g. It WLS dissolved in GO I n l . of rliinet~hylforrnairiide:tnd 6.5 g. (0.0175 iliole) C I ~ c,nrbobenzosy-L-proline p-nitrophenyl ester wis :iddcd. 'rlw inixture was wirrtied :it ii.5' for 3 d:iya. It n-:ts diluted wit,ll ethyl acetate, and xished successivt:ly with :iclueous sodiuiii cmhonate, dilute hydrochloric. wid, :tnd satur:itetl sodium (rli11)ride solutions. The solut,ion was coricent,r:ited to ? ( I . 60 nil. :inti diluted with ether to yield 10.3 g. (79'2,) of :I gr:inul:ir s,)licl, ri1.p. 147--lr5O0. An :inalytical snrriplc \vas recryst,:illizcd frclli, :I cei.onitrile-ether, 1n.p. 153-1;5". A n d . Calrd. for C.&,,N,O,n: ii3.1-1; 11. 0.:31 ; S : 10.:3?. Found: C, (i3.02; H, 6.54; N, 10.39. Carbobenzoxynitro-~-arginyl-r,-prolyl-~-prolylglycy~-~-phen~lalanyl-0-acetyl-L-seryl-L-prolyl-L-phenplalanineMethyl Ester (V).--Carbol~enzosp-~-prolylglycyl-~-phennyl-~-seryl-~~prcilyl-i,-phenyl:tlanine methyl rst,cr ( 3 g., 0.00373 niole) WRS dissolved in 50 rnl. of g1ac:i:tl acetic acid rontaining 10 g. of :mhytlro\is hytlrogen bromide. The n-iixture 'ims kept at room temperaturc 1.5 hr. and poured into 600 ml. of dry ether. The hygroscopic: solid was collected, washed with ether, :inti dried in v n v i ( o . 'Ftw hydrohrornitlc (3.Ci g.) wis diswlvetl in 15 1111. of diixi(:tliylforiiiaIiiide. l ' h e solution was c~iciledto 5" : I n d 1.4 nil. of tri':is added. The niistiire was filtered :Ind t o thc tldod 1 4 g. (0.003; rnolc) c>f (~:irl)~~Iw~iz~ixy nitro-i,(I,
51
BRADYKININ ANALOGS
January, 1964
CHARTI Cbz = carbobenzoxy; DCCI = Dicyclohexylcarbodiimide Pro-Phe-OMe
Cbz-Ser-Ns
I
I
C bz- Gly-0 + n
OMe (11)
Cbz-Phe -&r-Pro-PheHaPd
0 0 N 0 2 Cbz-Gly-Phe-Ser
Cbz -Pro-
OMe (111)
-Pro-PheHdPd
NOz Cbz-A?g -Pro-OH
C bz- Pro- Gly -Phe -Ser
H
O
-Pro -Phe -OMe
(IV)
HBr/AcOH
DCCI
NOz
-~
N
O
Z
I
J
Ac
I - Ser-Pro-Phe
Cbz-Arg-Pro-Pro-Gly-Phe
-0Me
(V)
1. NaOH 2. H d P d
Arg-Pro-Pro-Gly-Phe-S@r-Pro-Phe
(VI)
CHARTI1 Cbz -Phe -
0
I
- a NO2
X-OMe I
J Cbz -Pro
-
Cbz -Phe-X-OMe
(VII)
HBr/AcQH
A, X = His B, X = Cit
I
4 Cbz -Phe-Ser-N3
Cbz-Pro-Phe-X-
OMe
(VIIIA, B)
i HBr/AcOH
-0 U
Cbz-Pro-Gly
N
O
'
Cbz -Phe-Ser
-Pro-Phe-X-
OMe
(IXA, B)
I HBr'AcoH
i.
Ac
N0z
I
I
Cbz-Arg-Pro DCCI
Cbz-Pro-Gly-Phe-&r-Pro-Phe-X-OMe
I
NOz
I
C bz-Arg-Pro-Pro
NOz
Ac
I
-Gly -Phe-Ser-Pro-Phe
-X-OMe
(XIA, B)
lNaoH
I Cbz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-X
(XIIA, B)
IH'/Pd Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-X
(XIIIA, B)
(XA, B)
52 Carbobenzoxy-L-phenylalanyl-L-seryl-L-prolyl-~-phenylalanyl~iryinyl-i.-proline,~ 0.75 g. of diryclohexylcnrbodiimide, and 0.5 g. L-histidine Methyl Ester (IXA~.-Carl~ohenzosy-r.-l,rtri?.l-i.p-nitrophenol. The solution was kept a t 5" overnight and t,hen ~~tienylalanyl-~.-histidine niethyl estrr j 7.Ii g,, 0.014 i t i o l c , ) \\;IS warmed at 45" for 10 hr. The rencation iiiixture was filtered, ifissolved in 100 i i i l . of g1:tci:il acetic :ic.itl ciint:iining 1.5 g. tlr) diluted wit,ti ethyl acetate, and washed SII hydrogen l)r,l acetate siiluticin was e v a p prot1uc.i 1v:is tlissolvcd in 30 nil. of diiiirttiylforiii~iiiiido,I ~ I ) I ) ~1I1~1 or:tted in V ~ C I I Oto yield 3.5 g. of :iniorphous solid, ni.p. S3-!)Oo. 4', ; t i i d trexted witti 7 nil. of triethy1:iniine. .1Ie:inwhile wrhii. I d . Calcd. for C:,jH;IS1201a:C, 57.83; H, 6.27. Fiiiind: lierizoxy-i,-plienylal:~nyl-L-serineazide \vas prrp:iredK froni 1; g. C, %.OS; H, 7.20. Carbobenzoxynitro-L-arginyl-L-prolyl-L-prolylglycyl-~-phenyl- of the hytirazidc in 1.3) inl. of (*oldethyl :ivet:ite. 'This dil~eptitli, azide ivas :ictdeti t o tlie filtered soliitiiin of i ~ - ~ ~ r ~ ~ l y l - ~ . - ~ ~ l ~ e n y l alanyl-L-seryl-L-prolyl-L-phenylalanine(IV).-The protec+xl ~ ~ l a n ~ l - i , - l ~ i s it ii~i~1iy1 ~ i ~ iextcr e :in8 1tr:pt overnight, :it, -7". 'I'liiL cic*t:ipeptide above (2.7 g. 0.025 mole) was dissolved in I5 nil. i i f riiisturc wiis wushcvl witti water, nqueous sodiurir bic.;iri)on:itc~. illethanol and treated with 5 ml. of sodiuni hydroside f u r L' ; m i dilutc, Iiydrciohloric* acid; thcl ~ t l i y lawtate solrition \Y:IR tir. at room t,eniperature. The solution n-as evaporated in oacito, tlrird iivt'r iriagnesiuni sulfate anti ewporated in e'(icl/o t o yithld the residue was triturated with 70 Inl. of water and filtered froni (j.0 g. of :I glnss. The pentapeptide was not crystallized. 1.7 g. of insoluble material. The filtrate vas acidified to precipCarbobenzoxy-L-phenylalanyl-L-seryl-L-prolyI-~,-phenylalanylitate :t gum which solidified on standing to yield 0.63 g.. 1n.p. L-citrulline methyl ester (IXB) was prepared in the. S:III~P n1:tn75-78", [ a1 2 3 -46.7" ~ (c 0.52, diniethylfiirrii:iriiide). ner frorn fi g. IJ f c~:ir~)obenzosy-r,-~)~ieiiy~:iianyl-~,-serine hydrazide . I nu/. Calcd. for CjlHGi-U1201d.H?O: C, 36.70: H, 6 . 2 3 ; mil 7.3 g.. (if 1~:irl~ohenzosyl-i~-~~rolyl-~-p~ieriy1ul~inyl-i.-1~itrulliric~ S , 1,;.26. Found: C, 56.26: H, 6.91; S , 15.2ti. ), I I I . ~11!)-152° . frcini rnt:thnniil, r~-iirginyl-L-prolyl-L-prolylglycyl-L-phenylalanyl-~-seryl-i~-nietliyl ester t r i yieltl 4.6g. [a]".'ii - 4 l . S ' I '' I , diriiethy1foriii:iiiiidej. prolyl-L-phenylalanine (VI).-The aforesaid octapeptide (450 rrig.) was hydrogenated in acetic acid-niethanol over platinurn .Inti/. ('nlctl. f ~ C,~lH5~XiOl,~: ~ r ( I , Ol.40: 11, 6.41; N. 12.24. Found: (', (jl.22: H, 6.50: N,12.0.7. black in the usual manner t.o yield 350 nig. of water-soluble solid [a]?'1) -80.5" (c 1, N acetic acid). It w a s shown to be honln('arbobenzoxy-L-prolylglycyl-I.-phenylalanyl-O-acetyl-L-seryIL-prolyl-L-phenylalanyl-L-histidine Methyl Ester (XA).-C:irhigeneous by paper electrophoresis. t~enzosy-~~-ptieny~~ilanyl-~.-seryl-~~-prolyl-~,-pheny~alnn~l-r~-his ti.lno/. Calrd. for C41HslNl101n. CH3C00H 2H,O: (1, H, 6.95; 3 , 75.41. Found: C, 55.04: H,6.67; S , 15.7 diiie methyl ester (5.4g., 0.0066 mole) w a s dissolved in 7 5 nil. oi glaviul :icetic* :wid containing 13 g. of dry hydrogen hroniide. Carbobenzoxy-L-phenylalanyl-ph histidine Methyl Ester' (VIIA).-To a solution of 25 g. (0.06 mole) of cwlmbenzosy-LThe solution W;IY kept at, 2.5' for 1.5 hr. mid then poured into 500 ~ of dry (Jtlicr. T h t ! scdid \vas idlrc~ted,washed with dry phenylalanine p-nitrophenyl ester in T O nil. of d i n ~ e t t i y l f r ~ r n i a n i i ~ l ~nil. ether, an(! dried I'n riii:/w. The priidui.t (5.0 g.) \vas dissolved in 25 was added 14.5 g. (0.06 mole) of 1.-histidine niethyl ester dihq-drorid. (if diiiiethylforrii:tiiiide, i w i l e t l to A', and treated \vit,li 2.8 i d . chloride and 17 nil. of triethj.l:iriiine. 'The iiiisture was stirred :it room temperature for 20 hr. and filtered. The fi1tr:ite was diluted of triethyluniinct. T h c l rnisture was filtered and to the filtrate wit,h 2,50 nil. of et,hyl acetate and washed with water, aqueous \\-;is added L'.!) p. (0.0068 mole) of iwbohen p-nitrciphenyl ester. 'Thc1 inistiire ~v:is siirrc sotiiuni carbonatr, and aqueous saturated sodiuni rtiliiridv. days :in11 then diluted with ethj.1 uc.ct:ite. The solutiiin \ The ethyl itcetate solution was dried over anhydrous niagnesiuni sulfate and wncentrated to CQ.75 nil. 1)ilution with petroleum washed wit,h :icluciius pnt:issiuni carbonate and saturated sodi ~ t h r rprecipitated the (,rude dipeptide. It wis recrystallized chloride solutions, dried over rnagnesiriin sulfate, and evapor:ited From i,tilorr)forni-petroleum ether, 24.8 g. (I)?,; )>ni.1). 1 21-124', in m c i / o . siilitl TTW crystallized froin methanol-\v:iter t o yield 4.:3 p. ( 6 2 ' ; ) of wlorless sdid, 1ii.11. 1X2--IXI0, [ r ~ ] ' ~ ~ i ) [ a l 2 3 i i - 10.4° (c 2, diniethylformaniide) -.Incii. Calcd. for C?1H.'6S40:,:C!) 6 3 . H: 3.K2: S , 12.44. - . I O . a~ ( c I , tliriirtliylfc~rminiid~~~. I'ountl: C, 64.03; H, 5 . T O : ?;, 12.58. .Im/. ('u11,cl. for Cj~Hj!,S.,OI,.HJ): C, 60.30; €I> (j.17; . tainetl 26 g. ( S O r ; ) , ni.1). lS7--190a. i m ) ? " i i - 4 3 " ( I . 1 , dinirthyl.IRQ/. c'dcd. fur C ~ l l - l ~ ; X 1 6 0C:,, ~ 57.40; : I[, ti.10: S , If\.4(5. f i irnianiide). E'ouiid: c', 37.64; €1, 6 . U ; S , 15.97. .lnol. Calcd. for C29H37XjOi: C!, 61.35; H, (i.57: N, 12.31. Carbobenzoxynitro-L-arginyl-r,-prolyl-L-prolylglycyl-~~-phenylb'cxinrl: C, 61.50; H, 6.56: S , 12.19. alanyl-0-acetyl-L-seryl-L-prolyl-L-phenylalanylI,-ci trulline methyl ester (XIB 1 \vas prepared in the foregoing nimnor frcirn 1 .It? g. (0.0025 mole\ o f cnrl~obcnzos~iiiirci-i.-:irg.in~l-i.-~~riilin~~ I I ~ ) It, P. Roissonnaa. S t . ( ; i i t t i i i i i n , a n d l'-.\. ,Ia,iiiennii(l, flrli. A\7
'I'ticb
.-P.
; X I I:. 1 1 . X i w 1 : i i d w : r r i ~ l11. . \ , l l v \ f ~ ; i I < iihid., , 26
January, 1964
THYROMIMETICS. 111
53
and ~-prolylglycyl-~phenylalanyl-0-acetyl-~-seryl-~-prolyl-~ ~-Arginyl-~-prolyl-~-prolylglycyl-~-phenyla~any~-~-sery~-~phenylalanyl-L-citrulline methyl ester hydrobromide (2.2 g.). prolyl-L-phenylalanyl-L-histidine (9-Histidine Bradykinin) The crude solid solidified from acetonitrile to yield 1 4 g., m.p. (XIIIA) .-Carbobenzoxynitro-L-arginyl-L-prolyl-L-pro1 y 1g 1y c y 1~ (c 0.5, dimethylformamide); lit.2 m.p. 118-120", [ a I z 3-54.4" ~phenylalanyl-~-seryl-~-prolyl-L-phenylalanyl-~-histidine (250 ~ (c 0.99, dimethyl155-160' (sintering 115'), [ a ] 2 0-61.3" mg.) was hydrogenated in glacial acetic acid-methanol over formamide). palladium black in the usual manner. The mixture was filtered 4 n a l . Calcd. for CslH&15017.3HiO: C, 54.25; H, 6.49; and evaporated in ~ a c u o . The residue was dissolved in water, N, 15.56. Found: C, 54.32; H, 6.35; X, 15.02. filtered, shell frozen, and lyophilized to give 220 mg. of a solid Carbobenzoxynitro-~-arginyl-~-prolyl-~-prolylglycyl-~-phenylwhich gave a single spot on electrophoresis in acetate buffer, pH alanyl-L-seryl-L-prolyl-L-phenylalanyl-L-histidine (XIIA).5.6, 3 hr. a t 30 ma. The protected nonapeptide methyl ester (2.4 g., 0.002 mole) was Anal. Calcd. for C ~ O H ~ ~ NCHlCOOH.4HzO: I~O~I. C, 52.20: dissolved in 20 ml. of methanol and 2.5 ml. of N sodium hyH, 6.46; N, 17.75. Found: C, 52.59; H, 6.76; N, 15.77. droxide was added. The mixture was allowed to stand 2.5 hr. L-Arginyl-L-prolyl-L-prolylglycyl-L-phenylalany1-L-seryl-Land was evaporated in vacuo. The residue was partitioned prolyl-L-phenylalanyl-L-citrulline (9-Citrulline Bradykinin) between 75 ml. of water and 75 ml. of ethyl acetate. The aqueous (XIIIB).-Hydrogenation of 500 mg. of the protected citrulline layer was separated and acidified with 117 hydrochloric acid to nonapeptide in the same manner yielded 275 mg. of water-soluble precipitate a small amount of oil which solidified, 0.3 g., m.p. ~ (c 0.85, i v acetic acid), lit.* [ c u I z z ~ -88.7' solid, [ a I z 2-79" 140" with preliminary softening. (c 1,-\I acetic acid). Anal. Calcd. for C5~H7&150~6.HzO:C, 56.27; H, 6.11; N, 16.97. Found: C, 56.02; H, 6.28; h', 16.94. Anal. Calcd. for C ~ O H ~ ~ N I ~ O I ~ . ~ C HC,~ 51.70; COOH.~H~~: Carbobenzoxynitro-~-arginyl-~-prolyl-~-pro~ylg~ycyl-~-phenylH, 7.08; N, 15.65. Found: C,51.00; H, 6.8; N, 15.40. alanyl-L-seryl-L-prolyl-L-phenylalanyl-L-citrulline (XIIB) was prepared by hydrolysis of 1 g. of the protected methyl ester in Acknowledgment.-We are indebted to Mr. C. E. methanolic sodium hydroxide to yield 0.65 g. of amorphous solid, Childs and his staff for the microanalyses and to Mrs. m.p. 125-130°. Carola Spurlock and Mrs. Vivien Lee for the optical Anal. Calcd. for C ~ S H ~ ~ N ~ ~ O I ~C,. H55.38; Z O : H, 6.31; rotations. N,16.70. Found: C,55.46; H, 6.62; N, 15.74.
Thyromimetics. 111. The Synthesis and Relative Thyromimetic Activities of Some 4'-Ethers of Iodinated Thyronines and Thyroalkanoic Acids BENJAMIN BLANK,CYRUS31. GREESBERG, AND JAMES F. KERWIS Research and Development Division, Smith Kline and French Laboratories, Philadelphia, Pennsylvania Receiued August $1, 1963 4'-Methyl, ethyl, and p-diethylaminoethyl ethers of several thyroxine-like compounds were prepared by treating suitable intermediates with base and dialkyl sulfates or p-diethylaminoethyl chloride. Protecting groups were hydrolyzed to yield the desired ethers (IIa-f, 1-a-d, and VIa and b) which were screened for their ability to lower plasma cholesterol levels in rats fed a cholesterol-cholic acid diet. Interesting compounds were studied further for their ability to stimulate oxygen consumption and/or to increase heart weight. A comparison of these results with the values from the cholesterol-lowering - screen gave some indication of whether the compounds had a desired separation of activities.
During the study in our Laboratories of various classes of t hyroxine-like compounds for their thyromimetic activities, it was noted that by controlling the administered dose of the methyl ether of 3,3',5-triiodothyropropionic acid a good separation betmeen calorigenic and antigoitrogenic responses could be obtained in the rat.' This initial observation coupled with the report by Herman, Lee, and Parker2 that 4'-methyl ethers of thyromimetic substances often possess a large separation between the minimum effective hypocholesteremic dose and the dose which causes weight loss in animals prompted us to prepare a few examples of methyl ethers in an earlier study.3 These results were encouraging enough that several additional 4'-methosy compounds have now been screened for their cholesterol-lowering activity. In addition, two 4'-ethyl ethers were prepared to determine what effects a larger alkyl group in the 4'-position would have on biological activity. Moreover, since the preparation of P-diethylaminoethyl esters of several iodinated thyroalkanoic acids resulted in compounds with good hypo(1) C . AI. Greenberg, L. F. Mansor, C. A. Bocher, H. L. Saunders, a n d J. F. K e r a i n , Endocrinology, 70, 365 (1962). (2) R. G . Herman, C. C. Lee, and R. Parker, A r c h . Intern. Pharmacodyn., 199, 284 (1961). ( 3 ) B. Blank, F. R. Pfeiffer, C. M.Greenberg, and J . F. Kerwin, J. M e d . Chem., 6 , 560 (1963).
cholesteremic activity, the cholesterol-lowering activity of some represent at ive 4'-p-diet hylaminoe t hyl et hers was determined. It was hoped that a study of these types of structures would reveal compounds that would prove to be potent hypocholesteremic agents in man at doses which would cause no calorigenic or cardiac distress. The methyl and ethyl ethers of thyroalkanoic acids (IIa-f) used in this study were prepared by treating the requisite acids (I) with either dimethyl or diethyl sulfate in the presence of aqueous base.3 Diethylaminoethyl ethers (IV) were prepared from the requisite thyroalkanoic acid ethyl esters (111) on treatment with sodium met hoxide and p-diethylaminoet hyl chloride. These intermediates (IV) were then converted to the desired ethers (V) upon hydrolysis with hydrochloric acid. In some instances the intermediate esters (IV) were isolated, purified, and characterized as their hydrochlorides. In other instances they were hydrolyzed directly to V. The methyl ethers VIa and b of 3,3',5-triiodo-n and L-thyronine were prepared via their S-benzoyl methyl esters as indicated by Tomita, et aL4 (4) K. Tomita, H. A. Lardy, D. .Johnson, a n d 4. Kent, J. E d . Clem.. 296, 2981 (1961).
