738
Some Nuclear-Substituted Derivatives of w3lethylclopa'
AND
DOROTHEA ilUlXFP
The -vnthe+ of 2- and 6-melhyl, 2-rhloro, and 2-fluoro derivaiivc, (Jf a-meihyldopa ha. bem perfornictl. T h e i r conipoiiiidq have been tested a i inhibitorq of dopa decarbouylase, tyrosine hydrouylaw. : i i i ( I of varioui virii+ zn c~ztro. The 2-chloro nnalog is n better hog kidney dopa decarboxylase inhibitor than a-methyldopa.
Derivntivc' of dopa have been tested for various i)iologic:-tl activitie5. notably 3s inhibitors of dopa dcr8arboxyl:ises with a sequel in the control of elevated hlood as virustatic a g e r ~ t s ,etc. ~ , ~ TT'c were interested in exploring the composite effect of steric hindrance,both i n the benzene nucleus and in the side rhnin, on the prolongation or intensification of some ot t hesc activities which may result from the protectioii of side-cham degradation. Therefore, 2- arid 6-methyl-, ?-chloro-, and 2-fluoro-substituted derivatives of ainethyldopa were synthesized, essentially by the inei hod used by Stein, et d.,; in the preparation of a-niethyldopa, starting from the respective chloromethyliwxtroles. These step', along with various niodificaf ions, are outlined in the Experiniental Section. Biological and Biochemical Tests.--2-Aniino-2-nietl~yl-3-(3,3-dihydroxy-2-tolyl)l,rol,ionic acid (28), its 3,4tlinicthyl ether (22), 2-aniino-2-niethyl-3-(4,5-dihydroxy-2-toly1)propionic acid (33), its 4.5-dimethyl ether (26), 2- amino-%methyl- 3 - ( 2 - chloro- 3,4- dihydroxyI)heriyl)propionic acid (30), and 2-aniino-2-rnethyl-3~2-fluor0-3,4-dihydroxy~~heriyl)prol)ionic acid (32) were tc.zted in several in citro systeiiis. Dopa decarboxylase inhibition te were carried out \\ ith hog kidney dry powder under conditions described 111 the &;xperiniental Section. l'hc r e d t s , coinpiled iir Table I. 5how that 30 is ii hettcr inhibitoi*than CY1llethyltlop:l.
scribed i i i the Experimental Section. Only U L - ~ fluoi,o-a-methyldopa (32) caused an iriliibition of 24Yc. Antiviral tests, carried out by Dr. E. Furasawn, qhowed that :it the niaxiniuni nontoxic doses 28 (23 ,ug ' nil), 33 (12 pg,~nilj.and 30 (25 pg/inl) failed to show inhibitory activity against Columbia SI< virus (ItS.1 typc) in Ehrlich aacites tunior cells,6 and at 5 ,ug/nil against LClL virus (RS-2 type), T'accina (IHD, neurotropic strain) (IIXA type), and A4denovim> typr 12 (DSA typc) in KB cell tissue culture. Experimental Section' Substituted ChloromethyIveratroles.-Following a study by T'avon, et uL.)$cwiicerning reaction rates of the chloromethylntion with chloromethyl ether, the chloromethylation of 3-me1,hylveratrole was performed a t 34-35" for 7 hr, yielding 64Tc of 2metliyl-3,4-dimethoxybenzyl chloride as compared with 42% found previously. I n a similar fashion, 3-chloro-9 and 3-fluoro4-chloromet,h~-lveratrole3 were prepared. 4-Chloromethyl-5-methylveratrole.--A solution of 16.0 g (0.105 mole) of 4-niethylveratrole and 16.8 g (0.21 mole) of chloromethyl ether in 16.0 g of glacial acetic acid was warmed at 34-36' with stirring for 6 hr. T h e mixture was quenched with 200 ml of ice-water, the separated oil was extracted three times with ether, arid the ether solution was washed (10% NaHCOt, FI,O), dried (SanS04), and evaporated. A fraction of the residual oil boiling a t 93" (0.1 mm) (8.0 g, 40%) showed a correct aiialysis (Table 11) for the desired chloromethyl compourid :tiid exhibited the expected infrared spectrum. T h e position of tlie c:hloroniethyl groiip was established by oxidation of the c~~mpoiind with aqneous IiJlnOc to 6-methylveratric acid, mp 145-116°,10n s well a i by converting i t to 6-methylveratraldehyde, nip 70-51","~'~ h p $-OS" (0.05 mm),l2 by heating Jvith dinietliyl sulfoxide i n t h r presence of NaHC03 as :in acid :icceptor.13
:iftcr hhloruniet ti~l-.i-ineth~-lvcrntlole had distilled, a higlirr h i l i n g fraction which solidified i n the receiver was collected. illized from et h:inol with the :iid of charcoal, 111p l"l.5--122". (fi) I:. riirasa~va.\\'. Ci;tting, and A . Furst, C h e m o t h e r a p t n , 8, 91 (1964).
Tyrosine hydroxylase inhibition tests were carried out by Dr. Edith G. NcGeer; the methodology is de(1) Supported b ~ Grants . SU-1445, XlH-03663, and GXI-001882 from tlie Xational Institutes of Health, IT. S. Public Health Service. (21 For a comprehensive review, see. .4. R . Patel and A . Burger, l'ropr. / ) r u g Ires., 9 , 223 (1966). 13urger, J . < l m ,L'lirm. SOC., 79, 4365 (1.957J. Coyne, anrl G. Janssen, .I. M e d . Chum., 6, 014 ( I W ) . . 13ronn~r.ani1 Iistctlof 2.0 1:irIy (see A ) , >-ielded nzyl vthyl ether ns a colorlttss miitiilc licluid, 1111 135-140' (i ). .1raa/. c:ll(d for IO:: 57.26; 11, 6.53. Fo1intl: (I,
appe:ired :is 9.0 g of aii oil, hp 144-15:3' )lidified i i i the receiver, and was itleiiticnl w i t h 2 - 1 ~ h l o r 1 i - 3 , 4 - t ~ ~ m e t ~ ~ 1 i s y p ~ i e i i y ~as~ iobtaiiied ~ ~ e t 1 1 iin i ~ dimethJ.1 lr~~c siilfiixide mccliiim. Iii diomne, wit11 or withorit c,ntaIyt,ic mioiltits of NnI, i i n i~Ii:tiiged st:irtiiig materid \ w s recovered. ~~-(3,4-Dimethoxyphenyl)acetylacetonitriles.--Ti~ :I hot soht i o i i of sodiuni ethoside prepared from 0.12 y-:itom of K n in 40 rill of :rholute ethaiid w i s added a solution I J f 0.04 mole iif tlita iiit,rilr in 14.2 g (0.16 mole) of niihydrous ethyl acetate. h f t c r reflusiitg for 4 hr the mixture, which cwiitniiied :L heavy prwipitute of the sotliiini rall, m i 5 allowed to s t a i d a t 25" f i r 14 hr, anti the precipi1,:ite W:LS filtered off, wished with arihydroiis ethyl aiaetni,e and ether, :inti dried. A solution of the sodiuni s:dt i n ti0 nil r ~ f1%-ater\V:LS cooled nntl acidified with glnrinl :iceti(* :icaid. .liter stirring for nhoiit 15 m i l l the oil which had separated o i i t was eslrncted with ether, and the ether extract was w : i s h e ~ I ( I T 2 0 ) atid dried (Na2SOh). Ilemoval of the ether followed Iiy tlistil1:iiioii of tlie resitliial oil give the ncetS.l:ic'eloiiitriles 2 4 c-olorle.;~viscoiis Iiquidb which i tallizcd ?lowly o t i shtidiiig. 1iifr:irecl ipectr:i of tlirse conipoiirid? :ire li.;ted in Talilr 111. icrti
%-ClIa 2x1 2-F 6-CII,
3100-3650 3050-3'700 3050-3700 3100-3700
2225 2220 2220 2210
1760 1758 1760 173Cl
Substituted 3,4-Dimethoxyphenylacetones.--'l'he viscou.i 0ketoiiitriles xere warmed slightly to make them rniibile before mixing them with IIzSO4. To a cold ,solution of 39 ml of I12POa :tiid 10 ml of water was added wih stirring 0.075 mole of the ketonitrile. The reaction mixture was warmed with stirring over a 10-min period to the temperature indicated below for each case, and maintained at that temperature as directed:'* for the 2-methyl derivative, OO", 10 min; 2-chlor0, looo, 10-15 r i i i r i ; &methyl, looo, 20 min; 2-fliior0, go", 20 min. The reaction mixtitre was cooled and diluted with 120 ml of water and w:irnietl at 95-100" wit,h d r r i i t g for about 3 hr. It was then c~ooleti, diluted with water, and extracted with ether. After w:ishiitg (diliite KaIICOa, T1,O) and drying (NanS04),the et'her
, l(i)
L. Friedman and 11. Shechtpr, .I. Oru. Chem., 26, 8 i i (1060). ( 1 7 ) For experiments conducted i n methanol, e / . R. 11. F. l l a n s k e an11 1. 1,:. 1,rdingtiam. Can. .I. l i e s . . 17B,14 (1039). ( 1 8 ) TTydrolq-sis a t 80° did n u t rprnove t i l e cyano yroiii, c o m ~ , l e t e l ~ ,
September 1966
CEPHALOSPORIK ASTIBIOTICS. V
solution. A mixture of 0.1 ml of this homogenate, 0.1 ml of 0.2 M phosphate buffer, p H 6.4 (containing the test compound where indicated), and 0.1 ml of a mixture of 1.5 ml of labeled tyrosine, 0.6 ml of water, and 0.9 ml of tyrosine (26.6 pglml) was incubated. Final compound concentrations were M a t which concentration DL-phenylalanine, DL-p-fluorophenylalanine, and D L ~ methyl-rn-tyrosine decreased the conversion to about 25% of the control. Only 32 showed some activity (247, decrease of conversion) but less than a-methyldopa.
74 1
Acknowledgments.--We are grateful to Dean Windsor Cutting and Dr. E. Furusawa of the Department of Pharmacology, I-niversity of Hawaii, for the antiviral tests, and to Dr. Edith G. AIcGeer, Kinsmen Laboratory of Neurological Research, The University of British Columbia, who carried out the determinations with tyrosine hydroxylase.
Chemistry of Cephalosporin Antibiotics. V.' Amides and Esters of Cephalothin2 ROBERT R. CHAUVETTE AND EDWIN H. FLYNN T h e Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, I n d i a n a Received March 21, 1966 Chemical alteration of cephalothin ( I ) which might lead to orally active derivatives v,-as investigated by preparing a number of C-4 carboxyl modification^.^ Iwmerization of the double bond in the thiazine ring was encountered under many conditions of amidation and esterification, giving riie to A*-cephalosporins which were completely devoid of antimicrobial activity.
7-(Thiophene-2-acetamido)cephalosporanic acid (I) is an outstanding member of a series of cephalosporins prepared some time ago in these laboratorie~.~There are many reports of its desirable antibacterial act'ivities against gram-posit'ive and gram-negative organisms and penicillin-resistant staphylococci in the laboratory and in clinical p r a ~ t i c e . ~As cephalothin and many of it's analogs lack oral efficacy, an investigation of the effect of modification a t the C-4 carboxyl group on oral absorption and biological activity was undertaken. Both amides and esters were considered. An interesting speculation was the possibility bhat amides derived from a cephalosporanic acid and an amino acid might' cross the intestinal wall and be cleaved in the body. Although simple esters, like the methyl ester, are known to possess diminished antibiotic activity compared to the free acids,4 the possibility exist's that more easily hydrolyzable esters (by enzymatic or chemical means) might exhibit significant in vivo activit'y. A therapeutic advantage might be anticipated from derived compounds if the structural environment of the carboxyl group is a bar to absorption through the gast'ric or intestinal walls. Activity could be inherent in the derivative or be produced as a result of enzymatic cleavage t'o the parent compound after absorption has occurred. Gasbric acidity, often a negative influence in oral absorbability of penicillins, would seem to be an unlikely fador in cephalosporin absorption because of the relatively good acid st,ability of this class of antibiotics. Objectives similar to these are not (1) Paper I V : E. Van Heyningen and C. K. Brown, J . ?fed. Chem., 8 , 174 (1965). (2) Cephalothin is the generic name for 7-(thiophene-2-acetamid0)cephalo-
sporanic acid; cephalothin sodium salt, KeflinB. (3) For naming and numbering of the cephalosporins, see R. l3. Morin, B. G. Jackson, E. H. Flynn, and R . IV. Roeske, J . A m . Chem. Soc., 84, 3400 (1962). (4) R. R. Chauvette, E. H. Flynn, B. G. Jackson, E. R. Lavagnino, R. R. Morin, R. A. Mueller, R. P. Pioch, R. W. Roeske, C. I$-.Ryan, J. L. Spencer, and E . Van Heyningen, "Antimicrobial Agents and Chemotherapy," American Society for Microbiology, Detroit, Mich., 1962, p 687. ( 5 ) W. S. Boniece, W. E. Wick, D. H. Holmes, and C. E. Redman, J . Bacterid., 84, 1292 (1962): T. Chang and L. \Veinstein, ibid., 8S, 1022 (1963); R . S.Griffith and H. R. Black, J . A m . M e d . Assoc., 189, 823 (1964).
uncommon in the literature of penicillin chemistry.6 A second motivation for this work was provided by a recurring need for an easily cleaved blocking group for the carboxylic acid in cephalosporin synthetic chemistry. This paper reports the chemistry involved in amidations and esterifications of 7-(thiophene-2-acetamido)cephalosporanic acid (I). To form peptides from a cephalosporin required that the carboxyl at C-4 be appropriately activated for acylation of a protected amino acid. Kefkens, et UZ.,' have demonstrated that S-hydroxyphthalimide condenses with carboxylic acids, in the presence of a carbodiimide, to give oxyphthalimide esters that are suitable intermediates in peptide synthesis. Using their conditions, I was treated with K-hydroxyphthalimide t o yield the expected cephalosporanoyloxyphthaliniide (111) in respectable yield. The isolable products from reaction of I11 with a number of amines, however, were nct the anticipated A3-cephalosporinamides. With ethyl glycinate, for example, 111gave a good yield of IV in which the thiazine ring double bond had completely isomerized to the A 2 position, Ready isomerization to A2-cephalosporins acconipanied many of the reactions included in this study. Identification of these A 2 isomers was possible from the characteristics which follow. (1) The ultraviolet absorption near 260 m l , which is correlated with p-lactam double bond conjugation in the normal A3-cephalosporin ring system, is lacking. (2) I n the nmr spectra, lone protons at C-2 and C-4, visible as single peaks near r 3.6 and 5.0, respectively, replace the methylene protons adjacent to the sulfur, evident as doublets centered near r 6.4 and 6.7 in the normal A3cephalosporin series. Further, the centers of the single-proton quartet and the single-proton doublet (6) A . l3. A . Jansen and T. J. Russell, J . Chem. Soc., 2 1 2 i (1965); D. A. Johnson, J . A m . Chem. Soc., '76, 3636 (1953); H. F. J. 1IcDuffie and D. E. Cooper, U. S. P a t e n t 2,650,218 (1959). (7) G. H. L. Nefkens, G. I. Tesser, and R. J. F. N l \ a r d , Rec. Trnu:. Chzm., 81, 683 (1062).
