July 196s
(305
?;OTES
action.6 We had previously reported that ethyl ohydroxyphenoxyacetate (la) undergoes a facilitation of hydrolysis through intramolecular catalysis, the lactone 2 being a probable intermediate in the process.7 It was presumed that esterification of a drug with o-hydroxyphenoxyacetic acid (lb) would give a labile derivative, whose rapid breakdown in vivo might reason-
SCIIEJIE
OCH2CH-CH2
+
1 RCOOH
\
H
la, R = CLHS b,R=H
@:ri9
1b
2
ably be expected. One of the hydrolysis products, lb, is relatively nontoxic and had been used in the past, as the calcium or sodium salt, as an antipyretic.* We felt it mould be of interest to compare the intensity and duration of action of a drug derivative of l b with respect to other aryloxyacetates of the same drug, in order to gain some basic knowledge on the “latentiating” capacity and mechanism of these ester derivatives. A comparison between aryloxyacetates and other esters might also be useful. The muscle relaxant 3-o-toloxy-l,2-propanediol (me~ ~ c H ; C H O H C H , O H
3
phenesin, 3) was chosen as the model drug for this study. The compound is characterized by an extremely short duration of action, due to its rapid in vivo oxidation to P-(o-to1oxy)lactic acid.g Conversion of the drug into the 1-acid succinate,10 1-carbamate,” or 1-nicotinate12 has been effected in an attempt to prolong its action by protecting the labile 1-hydroxy group, but these derivatives are not entirely satisfactory, so that a new long-acting ester might be of interest. Chemistry.-The esters (listed in Table I) \\ere prepared (Scheme I) by reaction of 1,2-epoxy-3-(0to1oxy)propane (4) with the appropriate acid (procedure A), or by reaction of mephenesin (3) with the appropriate acid chloride in pyridine solution (procedure B), or, in the case of the o-hydroxyphenoxyacetate (7), by reaction of the lactone 2 nith 3 (procedure C). The p-amino esters were obtained from the corresponding nitro derivatives by catalytic hydrogenation. Several attempts to prepare mephenesin mono-onitrobenzoate by procedure A were unsuccessful. Treatment of 3 with o-nitrobenzoyl chloride (procedure B) gave only the bisester 15, even when 3 was used in excess. Treatment of 4 with p-nitrobenzoic acid gave (6) D. Gould, L. Finckenor, E. B Hershberg, J. Cassidy, a n d P.L. Perlman, J . A m . Chem. Soc., 79, 4472 (1957). ( 7 ) S. R I Kupchan a n d hl. F. Saettone, Tetrahedron, 18, 1403 (1962). ( 8 ) P. Leheau a n d G . Courtois, “ T r a i t e de Pharmacie Ctiimique,” Vol 2, Masson & Cie., Paris, France, 1946, p 1390. (9) R. F. Riley and F. M.Berger, Arch. Bzochem. 20, 1959 (1949). (10) F. M. Berger a n d R. F. Riley, J . Pharmacol. Erptl. Therap., 96, 269 (1949). Lott a n d E. Prihyl, U. S. P a t e n t 2,609,386 (1952); (b) F. 11. Berger. J . Pharmacol. Erptl. Therap , 104, 468 (1952) (12) T. I. F e n d a n d R. H. Broti-Kahn, U. 5. Patent 2,750,391 (1956).
5
the monoester 11, while attempts at l)rel)aring the inme ester by procedure B gave, in analogy with the 1)revious case, only the bisester 13. Partial acid hydrolybis of 13 gave 11, whereas 15 resisted attempts at hydrolysis. It mas assumed that the primary ester \vas formed in all procedures. As a matter of fact, (a) ring opening of propylene oxide derivatives by acids occurs to give preferentially the primary ester, which may be formed directly, or through acyl m i g r a t i ~ n ; ’ (b) ~ the primary rather than the less reactive secondary alcohol function of 3 should react with the acid chloride or with 2. The proposed structure was confirmed through oxidation of the esters 6 and 11, selected as representatives of the series : both compounds were readily transformed, under mild conditions, into the corres1)onding esters of l-hydroxy-3-(o-toloxy)~1ropan-2-oiie( 5 ) . A study of the noncatalysed hydrolysis of all esters was carried out in aqueous acetone at looo, following the method described in ref 7. The half-life for the hydrolysis of 7 was ca. 5.5 hr (us. ea. G hr for la), while all other esters were practically unchanged after 24 hr under the same conditions. The facile hydrolysis of esters of l b was thus confirmed. Pharmacology.-All compounds listed i n Table I were screened for paralyzing activity in mice, at four dose levels, using groups of three male albino mice (Swiss SAT) for each dose level. The animals vere injected intraperitoneally at dose levels of 100, 200, 400, and 800 mg kg, and observed for 2 hr. Only the phenoxyacetates 6-9 were found to possess mephenesinlike activity and nere selected for further study. Mephenesin salicylate (10). was devoid of muscle relaxant activity at the 300-500-mg/kg levels, but exhibited a weak sedative action. The pharmacology of this compound is being investigated further in this laboratory. The esters 11-16 were practically devoid of niephenesinlike activity except at toxic doses (1000-1.500 mg/kg). I t is interesting to observe that oxidation of the active ester 6 to the corresponding ester of l-hydroxy-3-(0toloxy)prol)an-2-orie (17) results in total loss of pharmacological activity. Compound 18 was also inactive. To our knowledge, %oxo derivatives of mephenesin had never been prepared and tested before. In Table I1 are recorded the methods and results of a preliminary biological investigation on the four active compounds. Our intent was to provide comparative (13) A. Weissherger, “ T h e Chemistry of Heterocyclic Compounds; Heterocyclic Compounds nitti Three and Four-Membered Rines,” P a r t 1. Interscience Publisliers, Inc., Sex\ 1-ork, N. T.,1964, pp 366-382.
It,
[I 11
II I1 I1 I1 11
I: I: 1: I1
d:tt:t, under cquivnleiit condition:,, o t i the intctisitp iuitl cluratioii of activity- (rotating rod test ; bee footnote h, Tahle 11) :~ndon the toxicity of our compounds nitli rvhl)ect to mephenesin and mc1)heiiesin carbamate.
Discussion 'Hie rc5ults olitaiiied n i t h tlie ester:, 6-9, :tiid the rc,lntivii ~)hariiiacological iiiertiiem of tlie other Iilicncbiii esters prepared iii this ztudy, confirm thc I)rcviously published observatioiit on aryloxyacetic ;tcidz and their inherent abilit) to impart a longer duration of pharmacologic activity to the respective drug ( > * t u \ . Iiideed, the duratioii of action of mephenesin I)hcnox>acetate:, iucre~iseifrom 1.5 to 3 time. over that of iiiepheriesin carbnrixiti~. The intensity of actioii of the ~~heiioxyacetate cite lightly decreases as coni1):tr(d \vith the cnrbnnintc ester, however. Interc y t itigl\ , the testohtcroiie ar~1osy:tlkurioater have l~eeii t,el)ortcd6to have given maxiina as high as three time:, th:it of the pro1)ioiinte iuid tuicc that of other c o ~ i v ~ ~ i i m
i
-
SOTES
July 19(% Experimental Section
Chemicals.-Phenoxyacetic acid and the corresponding acid rhloride were prepared as described by AIameli, et 0-Hydroxyphenoxyacetic acid lactone (2) was prepared from the corresponding acid as described by Liidewig.15 p-Chiorophenoxyacetic acid and the corresponding acid chloride were prepared according to RIintori and Stephen.I6 l,%-Epoxy-3-(o-toloxy)propane (4) was prepared as iiidicatfd by Chizhevskaya, et a[.," and was purified by distillation under reduced pressure, bp 132-135" (10 mm). All other starting materials were commercially available products, purified by crystallization to constant melting point. Procedures for the Preparation ot'the Esters. A.-Compounds 6, 8-11, and 16 m r e prepared from 4 and the appropriate acid, following the procedure given by Petrow, ei a1.,'8 for the preparation of mephenesin benzoate. B.-Compounds 6, 8, 13, 15, and 16 were prepared by treatment of mcphencsin (3) with the appropriate acid chloride. Treatment of 3 hvith o- or p-nitrobenzoyl chloridc gave only the hisester 15 and 13, respectively, even n-hen the reactants were used in equimolar amounts. As an example, the preparation of 8 is reported. A mixture of 3 (5.0 g, 2 i mmoles), p-chlorophenoxyacetyl chloride (5.6 g, 27 mmoles), and anhydrous pyridine (10 ml) was heated 1 hr at loo", then was poured into cold water. The mixture was extracted with ether, the ethereal extract was washed with water, 105; ?Ja&O3, and water, dried (lIgSOI), and evaporated t,o give an oil which crystallized from benzeiiepetroleum ether to afford pure 8 in 40% yield. C.-The ester 7 x a s prepared by treatment of 3 with the Iactoue 2 as follows. A mixture of 3 (9.1 g, 0.05 mole) and 2 ( i . 5 g, 0.05 mole) was heated a t 130' for 2 1 hr. The resulting syrupy material afforded, on crystallization from benzene-petroleum ether (hp 60-80"), 5.9 g ( 2 8 % ) of ester, which was purified by further crystallization from the same solvent mixture. D.-Compounds 12 and 14 were obtained from 11 and 13, respcctively, by catalytic reduction over PtOl in dioxane, as follo~m. A solution of the compound (5.0 g) in anhydrous dioxane (80 ml) was hydrogenated a t normal pressure until the theoretical amount of H1 had been adsorbed. The catalyst \\-as filtered off and the solvent was evaporated a t reduced pressure; the oily Ilized from anhydrous ether. Oxidation of 6 and 11.-The oxidation of 6 and 11 to the corresponding l-hydroxy-3-(o-tolosy)propan-Z-one derivatives 17 and 18 was carried out as follows. To a solution of the compound (3.0 mmoles) in acetone (10 ml, previously distilled over ICvInOr) was added dropwise an 8 -Ysolution of CrOa in H,S04 (1.3 ml),l9 while stirring and cooling a t 5'. The mixture was theti dilrited with H%O,and the solid which separated was collected, washed with 10%; Sa.$203and H,O, dried, and cq-stallized from EtOH. Acid-Catalyzed Hydrolysis of 13 to 11.-A solution of 13 (1.0 g) in 95% EtOH (10 ml) was treated with several drops of concentrated HCI, then was heated 1 hr under reflux and poured into H20. The solid which separated gave on crystallization from CsH6 0.41 g (60Yc)of pure 1 1 , * O mp 98-99'. Kinetic Experiments. Hydrolysis of 6-11 in Aqueous Acetone.-The kinetic experiments were performed with an electrically controlled oil bath (100 i 0.01"), using analytical grade acetone, purified by reflux over KUnOl, desiccation over K2C03, and fractionation. Solutions (0.1 J I ) of the compoinids in aretone containirig 405;, 1120 by voltime were heated at 100" iiiaealed 10-nil ampoules. The rates of reaction were measured by titration of successive ampoules, removed after appropriate intervals. with standard alkali (phenol red indicat,or). The hydrolysis of 7 was found to be first order in ester up to '30C,, completion: K = 3.5 X 10-6 (14) (a) E. Mameli, E. Gambetta, a n d G. Rimini. Gaiz. Chim.I t a ( , , SO, 170 (1920); (13) E. Mameli, ibid., S6, i 6 3 (1926). (15) H. Ludewig, J . P r d t . Chem., 6 1 , 345 f1900). (16) T. H. Minton and H. Stephen, J . Chem. Soc., 1 2 1 , 1600 (1922). (17) I. I. Chizhevskaya, Z. B. Idel'chik, L. A . Yakimovich, and I(. S. Shadurski, Vestsi Akad. S u a u k Belaururk. S S R , Ser. F i z . - T e k h . A ' u ~ u k ,115 (1957); Chem. Abstr., 59, 9009 (1958). (18) V. Petrow, 0. Stephenson, a n d A . &I.Wild, J . Pharm. Phurmacol., 12, 37 (1960). (19) Prepared according t o C. Djerassi, R . R . Engle, and .i. Bowers. J . Ore. Chem. 21, 1547 (1956). (20) The reaction very prol)ahly involves hydrolysis of t h e primary ester function, followed b y acyl miaration t o give the a-monoester 11. For similar cases of acyl mieration in diol monoesters, see, e . g . , G. Berti, F, Bottari, and B. lIacchia, A n n . Chim. ( R o m e ) , 52, 1101 (lrJ62).
sec-l, half-life ca. 5.5 hr. 811 other esters were not appreciably hydrolyzed after 24 hr.
Acknowledgment.-The authors wish to express their gratitude to Professor L. Beani, Institute of Pharmacology, Cniversity of Pisa, and to Dr. S. Tellini, Guidotti Pharmaceutical Laboratories, Pisa, for stimulating discussions.
Reduced Derivatives of Methotrexate' 8.B. HUR\\ITZ~ AND R.L. KISLIUK~ Department of Pharmacology, R i f t s rniuersity School of Medicine, Boston, Xassachusetts 02111 ReceiGed March 25, 1968
It has been reported that 5,6,7,8-tetrahydromethotrexate (111) is a more potent folic acid antagonist than methotrexate (I) for Streptococcus faecalisj4Pediococcus ~erevisiae,~ niice,j chicks,6 and dogs.' When a niet hod developed for separating dihydrofolate and tetrahydrofolates was applied to I11 it mas observed that the material was actually a mixture of dihydromethotrexate (11) and 111. Some properties of the purified derivatives are reported here. The reduced material showed two major peaks on diethylatrinoethylcellulose chromatography. It was shown spectrally that the peak eluted first was 111 and the second 11. They accounted for 39 and 52% of the total absorbing material, respectively. The absorption maxima are shifted 10 mp toward longer wavelengths as compared with the corresponding aminopterin der i v a t i v e ~ . ~The extinction coefficients at maximum absorption were assumed to be the same as for aminopterin derivatives.g 111 and I1 are less potent than I as inhibitors of dihydrofolate reductase but more potent as inhibitors of thymidylate synthetase (Table I). In every system tested I1 was more inhibitory than 111. I11 is most likely a mixture of diastereoisomers resulting from the addition of hydrogen to carbon G. The contributiorl of each diastereoisomer t o the inhibition is not known. Experimental Section Compound I, provided by Lederle Laboratories, Pearl Iliver, N. Y., was purified by diethylaminoethylcelliilose chromatography as described for aminopterin.9 Hydrogenation was carried out in AcOH using PtOl catalyst.1° The reduced material was filtered wider H, and washed with ether." (1) This work \vas supported I)?. the National Science Foundation (Grant N o . GB-1890) and the U. 9. Public Health Service (Grant N o . 6 R 0 1 G X
118il). (2) U. S.PulAic Health Trainee in Biochemical Pliarmacology (Graduate Training Grant No. 5 T l G > l - i 6 5 ) . (3) Leukemia Society Scholar. (4) R . L . Kisliuk. Sature, 188, 584 (1960). (5) J. A . R. Mead, J. h1. Venditti, A. W. Rchrecker, h. Goldin, and R. L Kisliuk, ibid., 189, 937 (1961). (6) R . L. Kisliuk and 11. R. S. Fox. Arch. Biochem. B i o p h y s . . 99, 534 (1961). (7) R . L. Iiisliuk, J. Jankowski, and H. Kahaner, Tufts Folia M e d . , 8, 117 (1962). (8) C. K . Matheirs and F. M. Iluennekens, J . B i d . Chem., 235, 3304 (19fiO). (9) R.L. Kisliuk and 31. D. Levine, ibid.. 239, 1900 (1964). (10) B. L. O'Dell, J. 31. Vandenhelt, E. 6 . Bloom, and J. J. Pfiffner. J . A m . Chem. S o e . , 69, 250 (1947). (11) R . L. Iiisliuk, J . B i d . Chem., 227, 805 ( 1 9 5 2 .
