Journal of Medicinal Chemistry, 1970, Vol. I S , ‘Vo./t
XOTES and acylated as previously described3 with 260 mg (0.90 mmol) of Boc-Gly-Gly-Gly (I) and 186 mg (0.90 mmol) of DCCI in 20 ml of purified D1\IFIO since Boc-Gly-Gly-Gly was not soluble in CHzC12. Complete acylat’ion was achieved using a single 12-hr reaction. The peptide was cleaved from the polymer and hydrogenated as described3 giving 125 mg. The crude peptide was dissolved in .5 ml of 0.05 ill NH40Ac-l A l AcOH and applied to a 2.5 x 100 cm column of Sephadex-C25-SE (NH4+) packed in the same buffer. The column was eluted a t 34 ml/hr with a gradient of NHdOAc in 1 Ai‘ AcOH using a concentration change of 3.3 X 10-4 M/ml starting from 0.05 ?if. The effluent was monitored at 280 mp and the fractions from the center of the main peak were pooled and lyophilized giving 40 mg. After furt,her purification via the picrate salt2s11there was 30 mg of white powder, homogeneous 011 tlc, RfV: 0.57, RfVII: 0.33 (detected with Pauly reagent). A472-hr acid hydrolysate had the following amino acids: Gly, 2.99; Tyr, 0.96; Ile, 1.00; His, 1.00; Pro, 1.00; Phe, 0.99; peptide content, 81yc. A 48-hr acid hydrolysate incubated with L-amino acid oxidase (Worthington)3 had the following amino acids: Gly 2.23, Tyr 0.00, Ile 0.00, His 0.06, Pro 1.00, Phe 0.00.
VAoc-Tyr-Ile-His-Pro-Phe (IV).-Boc-Tyr(Bz1)-Ile-His(Bz1)Pro-Phe-polymer3 (0.9 g, 0.15 mmol), was deprotected and acylated as before with 155 mg (0.60 mmol) of Boc-~Aoc(11)and 123 mg (0.60 mmol) of DCCI in 20 ml of CHZCL. The peptide was cleaved from the polymer and hydrogenat’ed as previously described3 giving 140 mg. This crude product was disolved in 5 ml of 0.1 ;M XHdOAc-1 M AcOH and applied to a, 2.5 X 100 cm column of Sephadex-C25-SE (“4’) packed in the same buffer. The column v a s eluted at, 34 ml/hr with a gradient of NHdOAc in 1 M A .4cOH using a concentration change of 2.0 X M/ml starting from 0.1 N. The effluent’was monitored a t 280 mp and the fractions from the main peak which were homogeneous on tlc in solvents VI and VII were pooled and lyophilized to give 60 mg. ilfter further purification via the picrate salt,2a11there was 47 mg of chromatographically homogeneous material, RrVI: 0.70, RfVII: 0.32 (detected with Pauly reagent). A 72-hr acid hydrolysate had the following amino acids; V ~ O C 1.00; , Tyr, 0.97; Ile, 1.02; His, 1.04; Pro, 1.00; Phe, 0.99; peptide content, 96%. p4oc emerged from the short (5.3 cm) column of the analyzer 20 ml after arginine and had a color value which was 23y0 that of leucine. A 48-hr acid hydrolysate incubated with L-amino acid oxidase3 had the following amino acids: ~ A o c ,0.80; Tyr, 0.00; Ile, 0.00; His, 0.09; Pro, 1.00; Phe, 0.00.
Acknowledgment.--We are grateful to Dr. D. Nitecki for her helpful discussions during the course of this work. (10) A. B. Thomas and E. G . Rochow, J. Amera Chem. Soc., 1 9 , 1843 (1957). (11) Both peptides described in this paper gave gummy picrate salts n.hich were decomposed very slowly by the ion exchange resin AG 1 X 2 (acetate) in 1 M AcOH. I n this case, the procedure described in ref 2 was modified so t h a t the precipitated salt was dissolved in 1 ml of AcOH, HzO was added until slightly turbid then t h e AG 1 X 2 (acetate) was added. (12) These values were corrected for t h e amount of racemization occurring when a mixture of these amino acids was subjected t o the same hydrolytic conditions.
Thyroxine Analogs.
XIX.
3,5-Dimethyl-3’-isopropyl-DL-thyronine EUGENE C. JORGCNSEX A N D JEREYY WRIGHT Department of Pharmaceutical Chemistry, School of Pharmacy, University of Californza, S u n Francisco Medical Center, S u n Francisco, California 94122. R e c t i d Decenabcr SO, 1969
The iodine atoms of the thyroid hormones, L-thyroxine (L-T~, 1) and 3,5,3’-triiodo-~-thryonine (L-T~,2), (1) Paper X V I I I : E. C. Jorgensen and J. Wright, J. Med. Chem., 18, 367 (1970). This research was supported by Research Grant A M 4 4 2 2 3 from the National Institute of Arthritis and Metabolic Diseases, E. S. Public Health Service.
R
/
745
COOH
1. R = R’=R” = 1 2,R = R’ = I ; R ” = H 3, R = Me; R’ = I ; R” = H 4, R = Me; R’ = i-Pr; R” = H 5. R R‘ = R ’ = Me
have been replaced, in part, by alkyl or aryl groups, with ret’entioriof hormonal activity.2 Hormonal activity is also retained by a partial or complete replacement of I by other halogen atoms, particularly Br13or by a combination of Br and alkyl groups, e .g. , 3,5-dibromo-3’-isopropyl-~-thyronine. The i-Pr group substitution produces an analog more potent than t’he parent compound when it replaces t,he 3’-I atom of L-TB(~).’~,~ i-Pr group replacement of the 3,5-I atoms of L-T~, however, results in loss of hormonal activity.l*j The Me group is the only nonhalogen substituent yet reported to be capable of replacing the 3,?5-iodines of L-T3 with retention of thyroxinelike properties. 3,5-Dimethyl-3’-iodo-~~-thyronine (3) has been reported to show about 3% the activity of L-T1 in the rat antigoiter assay.6 Because of the uniqueness of this finding, it was felt desirable to carry out an independent resynthesis and biological evaluation of 3. The establishment or rejection of a n essential role for halogen in the hormonal actions of L-T~ and L - T ~ could aid in a better understanding of the events which initiate the hormonal response. Thus, the heavy atom perturbation theory, whereby I participates in energy transfer mediated by its excitability to the long-lived highly reactive triplet state,’ could not be valid if a completely alkyl-substituted thyronine proved to be hormonally active. Because of the activities of 3,5-diiodo-3’-isopropyl-~-thyronine (5001200% L-T~) and of 3,5-dirnethyl-3‘-iodo-~~-thyronine (3, 3% L-T~),3,5-dimethyl-3’-isopropyl-~~-thyronine (4) was selected for synthesis as a potentially active halogen-free thyronine derivative. Previous studies indicated that 3,5,3’5’-tetrarnethyl-~~-thyronine (5) was inactive,* or possessed questionable weak activity.6 A reevaluation of 5 a t high dose levels in the antigoiter assay was therefore included in the present study. (2) (a) E. C. Jorgensen, N. Zenker. and C. Greenberg, J. Biol. Chem., 386, 1732 (1960); (b) E. C. Jorgensen, P. A. Lehman, C. Greenberg, a n d N. Zenker, ibid., 337, 3832 (1962); (c) E. C. Jorgensen and J. A. W. Reid, J . M e d . Chem., 8, 533 (1965); (d) B. Blank, F. R. Pfeiffer, C. M . Greenberg, and J. F. Kerwin. i b i d . , 6, 554 (1963); (e) C. M. Greenberg. B. Blank, F. R. Pfeiffer, and J. F. Pauls, Amer. J . Physiol., 306, 821 (1963); (f) C. S. Pittman, H. Shida, a n d S. Barker, Endocrinology, 68, 248 (1961); S. B. Barker, M. Shimada, and M. hlakiuchi, ibid., 76, 115 (1965); (9) M. Wool, V. S. Fang, and H. A. Selenkom, ibid.. 78, 29 (1966): (h) C. M. Buess, T. Giudici, N. Kharasoh, W. King, D. D . Lawson, and N. iK.Saha, J. Med. Chem., 8, 469 (19651. (3) M. V. Ivlussett and R. Pitt-Rivers, Metab. Clin. Ezp., 6, 18 (1957). (4) (a) E. C. Jorgensen a n d J . R . Nulu, J . Pharm. Sci., 68, 1139 (1969): (b) R. E. Taylor, Jr., T. T u , S. B. Barker, and E. C. Jorgensen, Endocrinology, 80, 1143 (1967). (5) T. Matsuura, T. Nagamichi, K. Matsuo, a n d 8. Nishinaga, J . Med. Chem., 11, 899 (1968). (6) E. C. Jorgensen a n d R . A. Wiley, ibzd., 6, 1307 (1962). (7) (a) G. Cilento a n d M . Rerenholc. Biochim. Biophys. Acta, 94, 271 (1965) ; (b) A. Szent-Gyorg>i, “Bioenergetics,” Academic, New York, N . Y.,1957, pp 24, 27. (8) (a) C. 8.P i t t m a n , H. Shida, and 8. B. Barker, Endocn’nology, 88, 248 (1961); (b) H. J. Bielig and G. LWael, Justus Liebigs Ann. Chem.. 608, 140 (1957).
Tlormoiinl blocking action i 4 :I p o 4 h l c propert> of ci~lalog-, p:irticulurl> in those I\ i t l i .tertwclieniic.al similarity t o the natural hormone., combined \\ itli the potential for htrong receptor ai.ociation., and lo\\ tiiological activitv. The alkyl thyronine. 4. TI ah therctor(. tested a i :in antagonist to L - T ~in the antigoiter :t-.a\ . Butyl :~,3-diiodo-~-h?.dro~ybeIi~oat~~ (6) liaz hrru \\ell characterized for i t \ mtagoni-tic effects u t i L-T,in rodent 0, consumption Since no reports cxiyt on the :intagonistic propertie- of 6 i n the nntigoitcr test, thi. compound n a- included in the present h,ological stud! . ?'he 3yritlietic route used i- ,slion II 111 Scheme I . Attempt< to condense the di:ir> liodoniuni halidc 8 \\ ith S-wet! 1-:3,5-dimethyl-m-tyrosine etli>.l +ter (9) i t 1 the presence of Cu powder and Et3S,"' or u-ith I\O-t-Bu,fi yielded only 2-isopropy1-4-iodoanisole, u ~ i d no diaryl ether. Therefore, N-acet,vl-3,3-dimethyltJrusine (10) \\a\ converted into its S a halt nith SaO l l e . .\leOH \\ as removed, and the condensation nith tlie diaryliodonium halide 8 was carried out i n DMSO. The crude N-acetyl diaryl ether v a s c.iterified ~ i t hEtOH to facilitate purification as the AT;tc.et\l eth\l ester (12). Hydroljsii n-ith HT in AcOH J irlded S,T,-dimeth>l - ~ ' - i ~ o p r o ~ ~ ~ - D L - t l i ~ , r(4). ( ~ n i r-4i e similar beries of reactions afforded 3,5-dimethyl-l)i,tliyronine (13) nhich wah iodinated in aq I < t S H 3t i ) virld 3,5-dimethyl-3'-iodo-~~-thyronine (3). Biological Results (Table I) and Discussion. . \ t t hrer different dose levelq arid i n tu, o independeiit 3 ,?j-dimethy1-3 '-iodo-r)~-thryc mine (3) wa5 co 11i r i its thyroxine-like activity in the rat antigoiter tent level of 4-57; the activity of i,-TAI\ a4 found, in good agreement \\ith the previou. 1 ("porth of about 3Cr,. :3,5-Dimethyl-3'-isopropyl-1)1~thyronine (4), however. was completely inactive, a:3qpj33'.5'-t etramethyl-r)~-th! roiiine (5) \\ hen t est ed
H:S = B Y 8.R = r-Pr: S = I 7. R =
/
('OOR'
kl P
9. R'=Et IO. K' = H XILA
\
/
/
R
Me
C'OOEt
3
at dose level\ Iiiglier than those previously used. Tliu*:. t u o compound^ containing qtructural element- hicli permitted T,-like activit? , a\ long as halogen \\a- preqerit in the molecule. \yere completely devoid of TJilie effects \\hen halogen waz absent. This indicates that lialogen, or at least i~ structural element more halogenlike than the alkyl group, is an essential compoiient for tli! roid hormonal activit! . Further, it appears that the location of the halogen, 011 either the phetiolic or tlie alanine-bearing ring, is iiot critical. An alter-
.loui.nul of Metlicinnl Chamislr!~,1970, 1'01. 13, .Yo. $
N O T ICs
747
3,5-Dimethyl-4-( 3-iodo-4-hydroxyphenoxy)phenyl-~~-alanine nat,e possibility is that the halogens on t'he alanine(3).-3,S-Dimethyl-i,r,thyroiiiiie6 l 5 (13) wa\ ohtaiiied in thr hearing ring arc uniquely essential for the direct horbame manner a> 4, by HI-AcOH hydrolysis of 11. Iodination of monal effect, since 3,5-dimet~hyl-3'-iodo-~~-thyronine120 mg (0.4 mmol) of 13 in 10 ml of 337, EtSH2 was carried out6 (3) might be producing its T4-like actions by a n indirect with 101 mg of ISin 0.2 g of aq KI to yield 125 mg (707,) of 3, mp 213' dec (Lit.6 212-214" dec). dnal. (C,iH1JS04.H,0) C, mechanism. Possible indirect actions in the intact H, I. tem used, include inhibition of enzyme systems inactivating tlic thyroid hormones, such as the Acknowledgments.-We thank Mr. Ahmad Parvez deiodinase3, or the displacement of hormone from t;issue for the synthesiz of butyl 3,3-diiodo-4-hydroxybenzoate, binding sites. The possibility of an indirect action of and for a'sistaiire in the roiitliict of riit :intigoiter 3 is currently under study. nssa>.s. :3.;i-Dimethyl-3'-isopropyl-DL-thyronine(4) was inactive ns an antagonist to L-T, in the antigoiter assay. (1.5) .I. Dihljo, L. Stephenson, 1'. \Valkpr, and \\'. E;. \Yarhiirton, .I. Butyl 3,5-diiodo-4-hydroxybenzoate(6) was also inC h m . S o r . , 28-15 I l H A I ) . :xtive :LS a n antagonist to L - T in ~ the antigoiter assay, ( ~ v ( ~:it i i the liigli molar dose ratio of :io0 t o 1. Experimental Sectionlz
Central Nervous System Activity of
S-Acetyl-3,5-dimethy1-4-( 3-isopropyl-4-methoxyphenoxy )Ethyl 1-Naphthylalkylcarbamates phenyl-DL-alanine Ethyl Ester (12).-iV-Acetyl-3,S-dimethyltyrosine'^ (6.8 g, 0.027 mol) was dissolved in 33 ml of 1.17 S SaObIe in AIeOH. The MeOH was removed under reduced pressure and the residual K a salt dried in vacuo at 27". Anhydrous DMSO (30 ml) was added, folloved by 16.5 g (0.03 mol) iodide. Zd After stirof di- (3-i-propy1-4-met~hoxypheilyl)iodonium ring overnight at l l O o , the solvent, was removed by distillat,ionand the residue dissolved in BcOEt and extract,ed with dilute NaOH. The aq ext,ract was acidified, and extracted with AcOEt. Bfter Our interest in the field of naphthalene chemistry, drying (JlgSOa) the AcOEt was removed ririder reduced prersure together Ivith the recent availability of primary l-naphm d the residue disolved in 200 ml of dry CHC1, cont~ainirig0.9 thylalkylamirie~,ihas led us to synthesize an extenhive g of p-tolueriesulfonic acid and 8 ml of abs EtOH. The solution was heated under reflux for 24 hr, HgO produced being removed series of ethyl 1-naphthylalkylcarbamates of structure by passage through a molecular sieve. The solvent was removed I, in which R was H , or alkyl, or aminoalkyl; R1 u a s under reduced pressure, the residue was dissolved in EhO, then T\"C02Et or CH,SHC02Et; KAA n-as a tertiary amino washed with 1 0 5 Na2COj,10% NaOH, H:O, and dried (AlgSO,). group; n = 2-4. The nen substances (Table I) were Refrigeration of the filtered solution yielded 0.8 g (77,) of small crystals, which were recrystallized from AIeOH-EteO: m p 180obtained from the corresponding amines by reaction 181'; t,lc (BuOH: 3IeOH: AcOH; HuO, 9 : l : l : l ) one spot, Ri 0.88; rimr spectrum was as expected.14 Anal. ( C Z ~ H ~ ~ NC, O;) H, K. S-Acetyl-3,5-dimethy1-4-( 4-methoxyphenoxy)phenyl-~~alanine Ethyl Ester (ll).-This compound was prepared in the same manner as 12 from 6 g (0.02,5 mol) of S-acety1-3,5-dimethylRCR,(CHL),N I tyrosine (10) dissolved in 30 ml of 1.17 S NaOlIe in MeOH. After removal of llIeOH and dissolution of the residue in 50 ml of DMSO, a total of 31.3 g (0.075 mal) of di-(4-methoxypheny1)iodonium bromideI5 (7) was added in portions over 48 hr to t'he stirred solutioii maintained at 90". After filtration and evaporaI tion of solvent, the rebidue was dissolved in AcOEt and extract,ed with 10% KaOH. The aq extract was acidified, ext,racted with AcOEt and the AcOEt extract dried (lIgSo4) and evaporated uiider reduced pressure. The residual N-acetyl with excess C1C02Et, the reaction time depending on acid was esterified as described for 12, to yield 860 mg (97G)of a the steric hindrance of the amines. colorless oil which was homogeneous by tlc and showed the exAll of the new compounds were submitted to an inpected spectral characteristics (nmr, ir, uv). The oil was hydrovestigation of their CNS activity which included studies lyzed to yield t,he amino acid 13 without further purification. 3,5-Dimethyl-4-( 3-isopropyl-4-hydroxyphenoxy)phenyl-~~-a~a- of behavioral eff ects,2 action on pentobarbital sleeping nine Hydriodide (I).-The S-acetyl ethyl ester (12, 0.427 g, time,3 and a n a l g e t i ~antic~nvulsant,~ ,~~~ antidepressant 0.001 mol) was heated under reflux (N2atmosphere) for 4 hr with (reserpine antagonism) ,fi and antitremor (oxotremorine 10 ml AcOH and 1.5 ml 4 7 5 HI. The free amino acid mras too antago~iism)~ actions. I n all the experiments, the soluble in HSOfor isolation by isoelectric precipitation at p H 5.5. drugs were tested in mice at the oral dose of 100 mglkg. T h e solution was evaporated imder reduced pressure t o yield t'he HI salt, which crystallized as needle? from H2O (0.40 g, SOCr,): Of all the substances tested 8, 12, 17, 20,21,26, and mp 2,X-260" der: t l c (BuOH: IIeOH: AcOH: HzO, 9:l:l:l) one spot. d n d . ( ( & f f S 6 1 ~ 0 4C ) : calcd, 50.97; found, 50.44; I-T? (1) G . Pala, A. Donetti. C . Turba. and S.Casadio. J . M e d . Chem., 13, N.
/*-, \*/'
(12) Melting points (corrected) were determined with a Thomas-Hoover capillary melting point apparatus. Microanalyses were performed by t h e Microanalytical Laboratory, Dept. of Chemistry, University of California, Berkeley, Calif. Nmr spectra were obtained in CDCla on a Varian A-60.4 (MetSi). Where analyses are indicated only by symbols of t h e elements, analytical results obtained for those elements were vithin 0.4% of the theoretical values. (13) E. C. Jorgensen and R . .i,FViley, J . I'hnrm. Sci., 62, 122 (1963). (14) P. A. Leliman and E. C . Jorgensen, Tetrahedron, 21, 363 (1965).
6 6 8 (1970). (2) 5. Irwin, Communication a i t h e Gordon Researoh Conference on Medicinal Chemistry, New London, N. H., b u g 3-7, 1959, (3) E.Marazzi-Uberti, C. Turba, and C . Bianchi. J . Pharm. Sei.. 66,1105 (1966). (4) C. Bianchi and J. Franceschini, Brit. J . Pharmacol. Chemother., 9, 280 (1954). (5) J. Y.P. Chen and H. Beckman, Science. lis, 631 (1951). (6) S.Garattini, -4. Giachetti. A. Jori, L. Pieri, and L. Valzelli, J . Pharm. Phurmacol., 14, 509 (1962). (7) A . Burger, "Selected Pharmacological Testing Methods," Vol. 3, Marcel Ilekker, Inc., Kew York, N. Y., 1968, p 337.
