NOTES
September 1969 tagonizes strychnine I n the present series myorelaxant activity was present in those compounds with depressant activity. At 100 mg/kg (oral and ip) 1 and 2 completely protected'" against st,rychnine-induced convulsions while 3, 4, arid 5 protected only one of four experimental animals. At 500 mg/kg (ip) 1 and 2 partially protected all and completely protect'ed three of four test animals. Although 1 and 2 were shown by comparison experiments to have the same order of potency as meprobamate, the sedative properties of the former compounds preclude any pointed consideration of their muscle relaxant properties. Sone of the compounds in this series antagonized the parkinson-like tremors induced by tremorine-">'* or pentylerietet mzole-induced convulsions. Acknowledgments.-This work was supported in part by t'he U. S. Public Health Service Research Grant KB 7548. (7) F. 11.Rerger and W. Bradley. Brit. J . PharmacoZ., 1, 265 (1946). (8) T. L. Kerley, A. B. Richards. R. W. Beglep, B. E. Abreu, and L. C. Weaver, J . Pharmacol. Exp. Ther., 132, 360 (1961). (9) D. L. Trepanier, V. Sprancmanis, and J. E.E;ii!e, J . .Wed. Chem., 9, 753 (1966). (10) G. L. Hassert. Jr.. J. W.Pontsiaks, D. Papondrianos. V. C. Burke, and H. X. C r a r e r , Tozicol. A p p l . Phnrmacol.. 3, 726 (1961). (11) G. 31. Everett, L. E . Blockus. and I. M, Shepperd, Science, 124, 79 (1956). (12) D. L. Trepanier, P. E. Krieger, and J. N. Eble, J . M e d . Chem., 8 , 802 (1965).
Isolation and Identification of Cephalexin from Human Urine T.
s. CHOU
925
criteria of the chromatography-bioautography are not necessarily specific, it was desirable to secure definitive evidence that the antibiotic was excreted unchanged. This paper reports the direct isolation arid complete identification of cephalexin from urine after single cephalexin doses. The urine samples were obtained from a group of ten normal volunteers, who after fasting overnight were given an oral dose of 500 mg of cephalexin. Urine specimens were then collected during 0-6 hr after drug administ,ration, the total volumes were measured, and all aliquots mere frozen for microbiological assay via the cup-plate method using Sarcirta lutea to establish the cephalexin concentration and the total cephalexin contained in urine as shown in Table I. TABLE Ia Volunteer no.
Volume, ml
Cephalexin, pg/ml
Total Cephalexin, mgb
2700 333 598 1620 551 1225 1260 209 563 1340 44 150 550 917 8 578 1995 9 539 1890 10 1480 533 14,i8 470 Average Many groups of normal volunteers' urine samples had been tested; this table was used as a typical example. T h e oral dose was 500 mg. * Actual dose of cephalexin in the 250-mg capsule was 265 i.11 mg. 1 2 3 4 5 6 7
190 370 450 19.5 420 295 600 290 285 360
To isolate the cephalexin excreted in the urine and establish its identity, ion-exchange chromatography was employed. The cephalexin in urine can be isolated via Received February 17, 1969 its anion using AG2-X8 resin in acetate form or via its cation using Dowex 50 W-X2 resin in sodium form. Cephalexin, 7-(~-c~-amino-~~-phenylacetamido)-3-However, the anionic resin was better than the cationic methyl-3-cephem-4-carboxylic acid is a broad-spectrum resin since no complication by salt formation was encephalosporin antibiotic. The oral administration of countered. The eluted fractions from the column were followed by means of the ninhydrin test, uv absorption maximum a t 262 m ~ paper , ~ chromatography-bioaue c ; N H p & H l tography, and microbiological assays. Only those fractions with a positive ninhydrin test and highest absorbCOOH ance ( A ) were combined and used for crystallization. Two crystalline forms of cephalexin, namely, cephalexin doseq of cephalexin in mice' as well as in human volunhydrochloride and cephalexin anhydrate (acetonitrile teers* demonstrated nearly complete absorption, high solvated) were obtained, and their identity mas estabserum and urine concentration, and antibacterial lished by ir, uv, and nmr spectra and by direct comactivity. Experiments conducted on cephalexin-14C in parison of X-ray diffraction patterns with respective rats and mice3 indicated that unchanged cephalexin-14C authentic samples. In addition, tlc also revealed that it was the only radioactive substance appearing in urine had the same R f value as cephalexin, and bioautography or bile. Absorption and excretion at the administered also indicated that their activities against Sarcina dose level was also essentially quantitative. Human lutea were comparable.6 The fractions with lower urine after single cephalexin doses was shown via paper absorbance a t 262 mp were not used for crystallization chromatography-bioautography techniques4 to contain of cephalexin. Instead they were assayed to establish only one substance active against Sarcina lutea with an the total content of cephalexin in all the fractions from Rf value identical with that of cephalexin. Since the the column as shown in Table 11. The total content of The Lillr/ Research Laboratories, Eli Lilly and Conzpanp, Indianapolis, Indiana 46206
(1) X' E Wick, A p p l Mlrcrobzol , 15, 765 (1967) (2) R. L Perkins, H. N Carlisle. a n d S Saslan, A m e r . J . W e d . Scz., 256, 122 (1968) ( 3 ) Personal communication from H R. Sullivan, Lilly Research Laboratories. (4) Personal communication from \I7 E Wick, Lilly Research Laboratories
( 5 ) R. R. Chauvette, et al.. J . Am. Chem. Soc., 84, 3401 (1962), showed t h a t t h e ultraviolet maximum near 260 mp was attributed t o the C=CNC=C chromophore of t h e ring. ( 6 ) T h e microbiological assay of t h e isolated cephalexin anhydrate using the cup-plate method had a n average value of 1043 pg/mg, compared with standard sample of cephalexin a t 1000 pg/mg.
reph:tlexin in these fractiorih ~v:i\ al-o calcu1:ited from the slope' of a standard curve of conce~itrationof rephalexin t s . absorbance :it the 262-mp peak. From columns 8 :ind 10 in Table 11, it is apparent that the :way valur. aiid the calculated value do adequate1.irepreserit the level of cephalexin in uririe and cephalmiri w i s truly excreted unch:iiigcd. t tu)
Experimental Section Preparation of the Ion-Exchange Column.---The anionic re-it), Hio-liad AG2-XX (100-200 mesh, 10 g 1 wii; traii>ferred froin the Cl- form to the -OH f o r m h y treating with 200 ml of 1 .\SaOII. It was theri washed frer ( i f alkali with deionized H 2 0 t o ahoiit pII 6.7 arid poiired i i i t o :I 1.0 X :ZO ciii c.oliimii. The resiii was hack waahed to auaiige the particles acmrding t o sizes arid the flow rate waq adjiisted t o 2 ml,'niiit (0.14 ml in1 of w r t resin per miti). h c O H i l *T) w:is then rill1 throiigh the coliim~i iiritil the etfliienl wits p H 2.,i or Ir-s. It was theri wa$hed free of AcOlI with H?O and 1hen wit 11 0.1 S S a O X c iuitil the effliieiit was €)€I8.2. The cwliimti wt* i w d y for the experiment. drscribed below.
Separation and Identification of Cephalexin Excreted from Vrine.---.i hiimati voliinteer's itriiie sample (4.50 ml, microay' 1140 pg 1111, totti1 c~otiietit.?l:Z ing) collected in after orti1 adminiitration of t w o 2Xi-iiig capGii1e.: of c*ephalexiri n*ah chilled aiid ceiitrifriged. To an aliyiiot of nil of the siipernatalit was :idded 20 rtig ( i f ri~'e:i.;eaud the solrition K ~ S stirred vigoroii,-ly. The 1111 o E t hr sc~lritioiiwas raised aftel, >tirririg for B few riiitiiiies xiid l)roilgIit t1:rc.k ~ ~ o i i t i n i i ~ ~to i i ~t hl ry isoelecatric point of ceph:tlexitt :rt pI1 4.5 with t h e dropwise itdtiition of (viiicentrated ITCI i i t i t i l 1 tic? 1111 t l o loiiger as alterecl. The solrit i o i i was r t ? t t i rifiiged :irid the siipernatiint was poiireti i i i i i i the ion-exchaiige cc~lruiin pwparcd :rl)ove. It, w a ~ :theti elrited x i t h 1 ATAc011 :iiid the v:irioii,* fv:irtioii* were c~ollwtetl i i i showti in Talile 11. The fractions wcre ev:il\i:ited b y the followiiig testa: I I V maxirii;a :it 262 in@, pnper cahroniatography -bioaritography!h : ~ n dl l ~ . . The ~ rebiilti of iiv at)sorptioris anti mici~~biological :tssays are shown in Table 11. The tlc (Ri0.67) a n d hioaiitography tHf 0.60) o f fr:ic~tioii.: I1 \'I showed oiily o w active q i o t tigainst S . intea. Fracttioris 111a n d I\. were ci)inbiiied arid evaporaied t o tiryiie.-iltider variuim at room temperatiire. The solid residiie wa? ,tallired i n dilrite HCl-lIeCPi t,o give 1X rlig of cephalexin anhydrate (hIeCN solvated). In a separate experiment, the S. /utea active ehieiit-: from thc coliimii were evaporated to dryne.qs and crystallized in n-PrO€IG (,oilcentrated HC1 to give cephalexin hydrochloride in crystalline t'cirm. Both the cephalexin anhydrate aiid cephalexin hydrovhloride were identified by ir, iiv, n n d ntnr spertra arid by direct ~
~
~
~
~
.
.
( 7 ) Tile slope o i t h e standard r ' i i r r e \vas 1 7 . 1 I.1 ( m l ) ~ n a l . (8) T h e sample (2--5 mg) v a s sl)ot,ted on a strip of V'hatman So. 1 papei (4ti.4 x 19 cm) and was elnted by a descending solvent system composed of 13uOH-AcOI1-HzO (3: 1 : 1 ) . .Iiter the solvent front 1iad reached about 2.64 cnl irom the other end of the paper it was air dried and laid on top of a n agar plate seeded with suitable microorcanism for 10 min Ilefore t h e plate was incubated ol-ernight. T h e Rr values of the antibiotics were calculated irom the clear zone shown on the plate. Roth cephalexin standard and the nrine sample before a n d after t h e column chromatography shoxed an identical mpot v l t h Rr 0.60. (9) T h e tic utilized t h e Urinkmann precoateil silica gel plates iplaslic sheet? mere used when a bioautograph was needed) wit,h fluorescent indicator and the solvent system \vas MeCK-HzO (3: I ) . T h e cephalexin stanrlarrl :itit1 1lIc, nrine fractions showed one spot with F:r 0.67 in tlc.
Iliamides of Cvclohutane-l.l-dicarbo\? l i v A c - i t 1
