May, 1963
DERIVATIVES OF ( -)-trans-2,3-E~oxrarrcc1~1~ ACID
a-Chloroacid Chlorides of Substituted Benzilic Acids.-3hIethylpheny1 phenyl-, 3,4-dimethylphenyl phenyl- and 3,4methylenedioxyphenyl phenylchloroacetyl chlorides3: I n these cases, distillation under reduced pressure led t o decomposition of the final products. They were therefore freed from ether, subjected t o high vacuum for 15 min., and employed xvithout further purification. Phenylcyclopentylchloroacetyl Chl~ride.~-Phenylcyclopentyl:lcaetir acid (10 g.) gave 9.8 g. (787,) of chloroacid chloride, b.p. 124" (0.5 mm.) f n a l . Calcd. for C13H14C120:CI, 27.68. Found: C1, 27.80. Ester Hydrochlorides of Substituted Hydroxyacids in Table 11. 3-N,N-Dimethylaminopropylisothiouroniunichloride hydrochloride s a s obtained in 83' yield from 3-chloro-5,X-dimethylpropylamine hydrochloride by the method of Albertson and Clinton,*1m.p. 159-161'. dnaI. Calcd. for CcHliCl?N3S: C, 30.77; H, 7.32. Found: C, 30.94, H, 7.16. 2-N,N-Dimethylaminopropanethio121 was used in the ethereal solution obtained on extraction. Compounds 110 through 118 in Table I1 were prepared by identical procedures viliich may be described by the synthesis of (21) N. F. Albertson and R . 0 . Clinton, J . A m . Chem. S o e . , 6 7 , 1222 (194.5).
233
3-N,N-dimethylaminopropylthiolbenzilatehydrochloride according to the method of Kolloff, et al.8 T o a solution of 8 g of diphenylchloroacetyl chlorided in 50 ml. of anhydrous ether, \vas added the ethereal extract from the alkaline hydrolysis of 7 g. of 3-S,Sdimethylaminopropyl isothiouronium chloride hydrochloride; an oil precipitated immediately. After refluxing for 1 lir , the reaction mixture was cooled and the ether was decanted The residual oil was heated on the steam bath for 15 min. with 100 nil. of n ater and one drop of concd. hydrochloric acid. t-pon being cooled and made basic with sodium carbonate, a white solid separated. Several crystallizations from ethanol led to m.p 86-87". When hydrogen chloride was passed into A solution of the solid in anhydrous ether, 1.8 g. ( 1BCb)of a white solid formed, 11hich melted at 180-182" after several crystnllization from ethanol-ether . Compounds 119, 120 and 121 were prepared from the corresponding a-cliloro derivatives by a procedure \\ liicli may be illustrated for 2-N,N-diethylaminoethyl phenylcyclohexylthiolglycolate hydrochloride. The a-chlorester hydrochloride ( 2 3 g.), dissolved in a minimum amount of nater, was refluxed for 2 Iir , made dlstinctly alkaline M ith sodium carbonate solution, itnd extracted with ether. The dried ethereal solution was acidified with ethereal hydrogen chloride to give a solid uhicli, \\hen crystallized from a mixture of acetone, methanol, and ether, weighed 2.0.5 g. ( 8 6 5 ) m p. 174-11.5 5".
Derivatives of ( -)-trans-2,3-Epoxysuccinic -4cid and Some of their Biological Effects l l a x W. MILLER .l/erlical Research Labomtories, Charles Pfiterand Po., I n c . , Groton, C'onn.
Received October 29, 1962 Syninietrical esters and amides as well as the nitrile have been prepared from the mold metabolite, ( - ) - t r a m ?,&epoxysuccinic acid. Opening of the oxirane ring in epoxj-succinic acid and its methyl ester with aninionia and amines t o form e,.ythro-p-hydroxy-L-aspartic acid and S-substituted analogs is discussed and some products are described.
The three isomers of epoxysuccinic acid (transracemate and cis-meso) have been related to the corresponding tartaric and chloronialic acids. The cis-meso form has been prepared by hydrogen peroxide oxidation of benzoquinone4 and by nitric acid oxidation of the macrolide antibiotic, carbomycin,j and both the cis isomer and the trans racemate can be prepared by tungstate-catalyzed hydrogen peroxide oxidation of, respect,ively, maleic and fumaric acids.6 To obtain the pure (-)-trans isomer, however, a fermentat'ive preparation was used since fairly high yields had been reported from t k 2 fermentation of glucose with d s p e r yillus jumigat~s.~-'O We observed yields from this mold of over 20 g. '1. of fermentation broth or a 40% molar conversion calculated from the glucose moiety of' ( 1 ) R. Kulin and F. Ehel, Ber., 68, 926 (1925). (2) R . Kuhn and R . Zell, ibid., 59, 2514 (1926). ( 3 ) R . Iiuhn and T. Wagner-Jauregg, i b i d . , 61, 513 (192b). (1) E. Weitz, H.Schohbert, and H. Seibert, ibid., 68, 1166 (1935). ( 5 ) R. B. Woodward, Angezo. Chem., 69, 50 (1957). (6) G.B. Payne and P. H. Williams, J . 0 ~ g C. h e m . , 24, 54 (1959). ( 7 ) J. H. Birkinshaw, -4.Bracken, and H. Raistrick, Biochem. J . , 39,7 0 (1945). (8) J. hloyer, U. S. Patent 2,674.561, Sept. 8, 1950 (to Secretary of Department of Agriculture). (9) IT. Martin and J. Foster, J . Bacteriol., 70, 405 (1955). (10) It is interesting t h a t fumagillin another Aspergillus fumigatus metabolite, contains two epoxide groups: [J. Landquist, J . Chem. Soc., 4237 (19561, and D. 9. Tarbell, R . 11. Carman, D. D. Chapman, K. R. Huffman, and N. .J. l\lcCorkindale, J . Am. Chem. Soc., 8 2 , 1005 (1960)l.
the sugar in the crude molasses used. Other inolds, particularly Penicillium itiniferum and Jlorzilia jmmosa also produce this acid."-'j The oxiraiie ring as substituted in epoxysuccinic acid is less reactke to acidic reagents than many epoxides, l6 permitting selective reactioiis a t the carboxyl groups. Thus, either free epoxysucciiiic acid or its slightly soluble bariuni salt, the form in vhich the acid was isolated from fermentation broths, could he esterified in alcohols with sulfuric acid catalyst. .I number of the esters so prepared are listed in Table I. K h e n (-)-trans-epoxysuccinic acid \vas heated \\-ith hydrochloric acid in methanol, dimethyl er.~jtlz~-o-chloromalate was formed. Treatment of either this ester or dimethyl epoxysuccinate with cold aqueous ammonia led to high yields of the slightly soluble (-)-transepoxysuccinainide. Similarly other amides were pre(11) K. Sakaguohi, T. Inoue, and Y.Tada, J . A g r . Chem. S o c . J a p a n , 13, 241 (1937); Proc. I m p . Acad. (Tokyo), 13, 9 (1947). (12) Ii. Sakaguchi, T. Inoue, and Y.Tada, J . .4gr. Chem. SOC.J a p a n , 14, 362 (1938). (13) K.Sakaguchi and T. Inoue, i b i d . , 14, 1517 (1938). (14) K. Sakaguchi, T. Inoue, and S. Tada, Zentr. Bakf. Parasiferik., I1 .Ibt., 100, 302 (1939). (15) I(. Sakaguchi and T. Inoue, J . 4 g i . Chem. SOC. J a p a n 16, 1013 (1940). (16) For quantitatire determinations of the rates of cleavage of a number
of epoxysuccinic acid derivatives in pyridinium hydrobromide -acetic acid a t 30° see hl. W.Miller, J . O r g . Chem., 2 8 , 1148 (1963).
12 l!I
pawd by iiitcractioii of dimethyl cyoxybuccinate i~ ith t n o iiiolc, of a primary amine in suitable solvent3 (l'ahlv IT). 'l'hc. aromatic esters aiid amide:: dion-11 i i i the table? \\tire prepared b y TI ay of (-)-trnns-epoxysucciiiyl cxhloiidc 11hich was ot)taiiied hy treatment of ( - ) (r.trns-cposysucciiiic acid with phosphorus peiitachloride and rapid distillatioil in a tn in-hulh flask. -1lthough inert in niaiiy acidic circunistaiices, the oxiraiie ring of epoxysuccinic acid was opened rapidly i i i a(liicoiis alkali. When (-)-tmts-epoxysucciiiic acid wah armed ~5 ith aqueous aniinoiiia, for example, 3-hydroxyaspartic acid formed. Of the four possible ihonicrq of this amino acid (racemates of the erythro aiid ot the threo forms) an erythro isomer was considered prot)ahlc since amines are kiion i i generally to opeii uiiconiplicated epoxide rings b y a siiiiple S s 2 mechanism i 5 ith iiiverhion of configuration. Our isomer has esseiitially the same specific rotation ( [ L Y ] ? ~ D+49", c 0.143 S i i i hydrochloric acid) as the eryfhro-@-hydroxy-L-
I" 1 1
I itly hy traiiaatiiiiiatioii t x t ~ w e i i glutamate and ( oglycolate ( [ a ] % +.',lo f 2'. c 1.59 111 A\7hydrochloric acid.)" It alm seein* to c~)rrc>~poiid to the "r~-clnti-hydrosyaspartLC acid" prepared long ago'&by hcatiiig chloronialic acid n-ith amnioiiia, a I I od iiot mtirely stereospecific. ' \T'hile other 5yiithe h a w becii reported lately,?" these also have lieen liiocheniical 111 iiatui~'or i t i r - o l \ ~ isomer separatioii. a i d t.he stereospecific route troin ( - )-frans-eposSsucc.iiiic acid i5 the method of choiw for prepariiig pure eruthrc,-P-hpdroxy-L-aspart ic acid Pancreatic digest.; of casein contain erythro-d-hy-
a-partir acid prepai(d
(17) IT. J . Ballarli, J . Bioi. C'hem., 229, -4H7 l l i 1 5 i ) . (18) H. Jl. Dakin, ihid., 48, 273 (1921). (19) 11. L. K o r n g u t h and l f . .I. Sallncli, .4wh. H i o c h e m . Biupltjis.. 91, 3 ! l
(1960). ( 2 0 ) 51. (::Ll.cia-Hernnnil~,z a n d 1,:. I i u n . Biu~,Ihim.Biophys. . l c t o . 24, 7 8 (1937). f ? l ) C . S. Franklin. .I. C h e f n . S u r . . , ,4709 (1960). (22) D. E:. RIetzler, J. B. Longenecker, and E. E. dnell. .J. .lm. Cheni. Yo?.. 76, 639 (10.54).
DERIVATIVES OF (-)-trans-2,3-E~ox~svcc1s1c ACID
May, 1963
233
TABLE I11 S,X',?~"'-TRI.~~.K~L-~-HYDROXYASP~ R?;HCOCHOHCH(SHR)CoSHR RT~~~II~E~, Carbon, % Calcd. Found
Yield,
K
31.p.. O C
70
Met hyla Allyl 0 t )-I Decyl Octndecyl Phenyl
125-126 136-137 120-122 119.5-120.5 111-112 234-237.5 (s-230)
9s 80 60 56 97 s3
(4
[ C Y ] ~ ~+60" D
Formula
CiHijN303 C13H?1X308 C28HgN30J C31H69?;30a CdiiiN303 Cz?H?1XaOd
44.43 58.41 69,5l 71.90
44.10 58.37 69.50 71.52
...
...
70.38
70.64
Hydrogen, 5% Calcd. Found
Nitrogen, 70 Calcd. Found
7.99 7.92 11,SS 12.25 ... 5.64
7.75 8.17 12.03 11.81
22.21 15.72 8.69 ...
...
...
1.73 11.19
4.75 11.x1
5.53
21.67 16.07 8.69
(c 1.0 in ethanol).
droxy-L-aspartic acid,?3aiid in several biological systems studied it is the active diastereoi~oiner.'~I n studies on nitrogen fixation a hydroxyaspartic acid, isomer unspecified, was isolated from cultures of azotobacter cells gronii with elemental iiitrogeii as the sole nitrogen s o ~ i r c e . ~The ~ iZmaniia phalloides toxin, phallacidin, is distinguished from the related phalloidin by the presence of erythro-P-hydroxy-D-aspartic acid as a coinpoiient of the cyclic polypeptide structure instead of r,-threonine. ?5 26 Hydroxyaspartic acid has been shown to cause bacteria to mutate,*' and it inhibits competitively the enzyme aspartate aminopherase.20 Although good yields of diamides were obtained by coiltrolled addition of two moles of amine to dimethyl eposysuccinate, three or more moles of a primary amiiie reacted with epoxysuccinic esters to form 1substituted P-hydroxyaspartic amides (Table 111). The triphenyl analog was prepared by heating a solution of epoxysucciiiic acid in aniline t o reflux for a short period. Treatment of S,S',S"-triphenyl-P-hydroxyaspartamide with methanolic hydrogen chloride yielded a half methyl ester, C,H5SHCOCH(SHC6Hb)CHOHCOOCH3, the amine salt inhibiting esterification at the proximate amide group. ,Uthough epoxysuccinic acid is a much stronger acid than succinic acid (epoxysuccinic acid Kig = 1.2 X lo-?. succinic acid K:5 = 6.4 X lop5)it is not very toxic. Selected toxicity data are shown in Table IT.'.
epoxysucciiiamides also showed hypnotic effects, the H \'
R
/
c---C
\o/
/ \
R'
COSH?
octyl derivative, for example, causing a mild decrease in motor activity with a duration of 90 minutes in mice. The novel structure (- )-trans-epoxysuccinoiiitrile was prepared in about 50Toyield by carefully controlled heating of (- )-trans-epoxysuccinaniide with phosphorus oxychloride. I t was an effective miticide, 0.35YGemulsions giving effective coiitrol of Tetranychzis atlanticus. S,?;'-Bis-72-decylepoxysuccinamide was a specific insecticide for the pea aphid, an economic pest on several legume crops. Propargyl (- )-trans-epoxysuccinate showed notable antifungal activity n-ith niininium inhibitory coiiceiitratioiis at 100 pg. ml. against Trichophyton sulfuricum, Phiolophora t'errucosa, the Aspergillus jlarus-oryzae group, Pzillularia pulliilans and Cladosporium herbarium. erythro-P-Hydroxy-Laspartic acid was active against Gram-negative bacteria on Witkin synthetic medium, some examples of niinimum inhibitory concentrations ( p g . nil.) being : Escherichia coli 6.25, Aerobacter aerogenes 6.25, Proteus m l g a r i s , 1.56, Pseudomonas aeriiginosa 6.25.
Experimental29
(24) A . I. Virtanen and N. Saris, Suomen Kem., 30B, 100 (1957). ( 2 5 ) T. Wieland a n d H. W. Schnabel, Ann., 657, 218 (1962). (26) The antibiotic telomycin has been shown to contain t h e amino acid erythro-P-hydroxy-L-leucine [J. C. Sheehan, K. Xlaeda, A. K. Sen, and J. A. Stock, J . A m . Chem. S o c . . 84. 1303 11962)l. (27) J. B. Clark, Pror. Okla. Acad. Srz., 34, 114 (1953); C h e m . A h t i . , 49,
Only a few typical or key experiments :ire reported, most of the esters and amides having been prepared by conventional terhniques. All preparations involving amines were carried out in a C02-free nitrogen atmosphere. The production of ( - )-trans-epox?succinic acid by an improved strain of Aspergillus jumigatus Fres. was accomplished using media based on those described in earlier publications (e.g.,*)16). Isolation of Barium Epoxysuccinate.--A 130 gal. batch of fermentation whole broth was adjusted to a pH of 1.5 with conrd. hydrochloric acid, 4.56 kg. of cellulose filter aid (Super-Cel) added, and the mixture filtered through a small ]"plate filterpress. T o the filtrate, with stirring, was added 10.94 kg. of barium carbonate. The pH was adjust'ed to i with sodium hydroxide pellets, and stirring continued for 10 hr. In this way 13.82 kg. (dry wt.) of barium epoxysuccinate was obtained. Bariumanalysis (as BaSOe): valcd., 51.4; found, 51.2%. Conversion of Barium (- )-trans-Epoxysuccinate to (-)-transEpoxysuccinic Acid.-A 100 g. sample of barium eposysuccinate, assaying 85% purity, was suspended in 100 nil. of dry tetrahydrofuran and the temperature maintained a t 5-10" while 1S.5 ml. of concd. sulfuric acid was added dropwise with stirring during 2 . 5 hr. The barium sulfate was removed, xi-ashed with fresh cold solvent, and the filtrate evaporated to give 40 g. of dry, tan crystalline epoxysuccinic acid, assaying 955i purity (95% yield), as well as 5.25 g. of a gummy residue. One recrystallization from dry dioxane gave nearly pure white crystals with little loss, m.p. 182-184". When acetone was used as the acidification solvent,
83731 (1955). ( 2 8 ) R. S. Shelton and K. W. Wheeler, U. S. Patent 2,493,090 (19:O); Chem. Abstr.. 44, 2552 (1950).
(29) hfelting points were determined on a calibrated Kofler micro hot stage.
TABLE IV SELECTED TOXICITIES Compound
( - )-t,ans-Epoxysuccinic acid Dimethyl ( - )-trans-epoxysuccinate
X,S '-Bis-n-decylepoxysuccinamide Y,K ' , S"-Tridecyl-8-hydroxyaspartamide ( - )-t/ ans-Epoxysuccinonitrile
Toxicity (acute oral LDjo)
> 3 . 0 g./kg. (mouse) > 2 . 5 g./kg. (mouse) >3 0 g./kg. (mouse)
>3.0 g./kg. (mouse) 0 025 g./kg. (rat)
Mild hypnotic activity has been reported for dialkylglycidamides of the type shown below. 28 Several (23) H. J. Sallach and 31.L. Kornguth, Biochim. B i o p h v s . A c t a , 34, 582
(1959).
May, 1963
FATEA X D RELATIOXSHIP OF S-METHYL TO ACTIVITY OF ~ I O R P H I S E
This niaterial gelled in most organic solvents, but n a s recrystallized from about 5 1. of dioxane 3 times and once from 3.5 1. of chloroform to obtain white waxy crystals, m.p. 120-122". Other (*loseanalogs shoiFed the same tendency to gel in most organic solvents a t conventional concentrations.
Acknowledgments.-~~-T;e are grateful to Dr. D. B. Seeley for the fermentatiye preparation of the epoxy-
23i
succinic acid, to Dr. S. Y. P'aii for measuring hypnotic activities, to joseph liaiser for toxicit!, determinations, to Dr. Arthur English for the antifungal tests, and t o Charles Scott for technical assistance. Insecticide tests were done at the Wisconsin Alumni Research Foundation.
Some Aspects of the Fate and Relationship of the N-Methyl Group of Morphine to its Pharmacological Activity'" CHRISTIAS EL IS OX,'^ HEXRY W. ELLIOTT, MELVISLOOK,A X D HEYRYRAPOPOKT Ilcpartinent of Phaiinacoloyy a n d Expei~nientalTherapeutzcs, I'nzcerszty of ('alifornzu Merlzcal Centei. San Ficinciaco, ('ulifoinia und Department of C'hemistry, Cnzterszty of Californza, Re?keley, ('alzfoinia Receizied J u l y 28, 1968 The chemical synthesis of S-trideuteriomethylnormorphine is described. Pharmacologically it is less potent than morphine although its duration of action is unaffected. Compared with morphine its X-demethylation bv rat liver microsomal enzymes occurs less readily and requires a larger energy of activation. A larger K , indicates t h a t it is less strongly bound to the enzyme. The V,'s of rat liver X-demethylating enzymes were found to be inversely proportional to the biological potencies of the drugs, whereas the K , increases as the potency increases. Diminished capacity of the enzymes of tolerant rats t o S-demethylate morphine was shown to be due to reduced availability of enzymes rather than alteration in enzyme structure. Both the 1- and d-isomers of analgesics apparently are demethylated by the same microsomal enzymes. The enzymes from male and female rats are probably identical. By two separate methods i t was shown t h a t nalorphine inhibited the Sdemethylation of morphine noncompetitively. The results obtained with rat liver microsomal enzymes fail to support either Beckett's theory on the mechanism of analgesia or Axelrod's theory on the development of tolerance to some of the actions of these drugs. It is concluded that these enzymes are not suitable as models by which these theories may be judged.
The mechanism by n hich narcotic agents produce analgesia is not known nor has any satisfactory mechanistic explanation been presented for the development of tolerance to some of their actions. Recently, t n o theories have been advanced which have implied that K-dealkylation of these agents is essential to these phenomena. Beckett, ct ul.,? believe that not the mere presence of these drugs a t the receptors in the brain, but the subsequent Ii-demethylation which occurs there constitutes the first step in the reaction sequence that leads to analgesia. Kalorphiiie is thought to antagonize the actions of narcotic analgesics by virtue of a greater affinity for the receptor sites plus a much slower Kdealkylatioii therein. Axelrod was prompted to study the effects of tolerance to narcotic analgesics on the capacities of liver microsomal enzymes from rats to demethylate these drugs by the finding that the enzymes are capable of S-dealkylating various drug^.^ He found considerable reduction in the capacities of the livers from tolerant rats to S-demethylate morphiiie and other analgesics which exhibit cross tolerance to morphine (1) (a) This iniestigation >\as supported i n part b> research g r a n t B-570 from the Kational Institutes of Health a n d in p a r t bs the United States Atomic Energ) Commission (b) Portions of the d a t a included in this report n e r e taken from a thesis submitted b y C Elison t o the Graduate School of the Uniiersity of California in partial fulfillment of the requirements for t h e Ph D degree in comparatiie pharmacology a n d toxicoloxs F Casy and N J Harper J Pharm Phajmacol 8 , 874 (1956) ( 3 ) S C Mueller and G A Miller, J Bid Chem 202, 579 (19%) (4) B i X LaDu L G. Gaudett iX Trousoff, and B B Brodie 1b7d 214, 741 (1955). ( 5 ) J kxelrod, Sczence 124, 263 (1956)
When nalorphine was administered x i t h morphine. the diminution in enzymic activity of the liver was significantly less than when the tolerant rats received morphine alone. hxelrod suggested that the continual interaction of these agents with the enzymes that Sdealkylate them inactivates the enzymes. SimilarIy he inferred that the continual interaction of these drug5 with their receptors in the central nervous system may inactivate the receptors. In other words, if the liver microsomal enzymes are used as models for the receptors in the brain it follows that tolerance may occur as a result of unavailability of receptor sites. Substitution of X-trideuteriomethylmorphine for morphine in in vitro and in iitro studies should proride a direct test of the theory of Beckett, et czl.,'j on the nature of binding to the central nervous system receptors as mell as on the niechanism of narcotic analgesia. The two conipounds differ u-ith respect to the zero-point energies of the C-H and C-D bonds as well as the masses of the tTvo methyl groups, but should combine with identical receptors both in the central nervous system and the S-demethylating enzymes. Since the theory is based on an inter-related consideration of stereochemical configuration, physicochemical properties, and the enzymatic ?r'-dealkylation, coniparisoiis of the i n vico potencies, the rates of enzymatic Ii-demethylation, the energies of activation for ?;-demethylation, and the Michaelis constants of the Xdemethylating enzymes for these substrates, have been made to obtain inforniatioii for judging the theory. Since the degree of ionization of the basic groups is (6) A. H. Beckett and A. F. Casy, J l'haim
Phormacol
6, 986 (1954)
