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 ~ ~toi i t~hlry 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
NOTES
September 1969
927
T.\RLEI : DIAMIDES O F CYCLOBCT.IXE-~,~-DIC \RROXTLIC ACID CONHR
Gross effect'
s s
s ?;
1 2
1;
8
8
-@I
R'd
I 8 1 2 2
1 8 9 3 :3
63
232
A
C~OH~~N~OL
N
N
0 9
75
258
A
ClsHleC12Xz02
N
N
1 2
Drug dose was 1000 mg/kg orally. N = no effect, S = slight reduction in spontaneous a Corrected. b A, acetone; P, pentane. motor activity, subjectively graded and compared with control animals receiving medium only. Drrig dose was .500 mg/kg orally. R' = (dirig pentobarbital sleep time)/(pentobarbital sleep time). Pentobarbital dose, 50 mg'kg ip administered 30 min after test drug.
+
true potentiation and becomes significant when greater than 1.5. The fact that 1 is act,ive as a depressant but inactive as a barbiturate potentiator lends some support to the suggestion that there is functional independence of sites for the two types of a ~ t i v i t y . ~ Two of the compounds, 1 and 2, were also tested for anticonvulsant6 (pentylenetet'razole antagonism), antist,rychnine lethality,' and antitremorhe* e f f e ~ t s . ~ Seither showed antagonism to pent'ylenetetrazole- or strychnine-induced convulsions. However, they completely protected 40-607, of the test animals from tremorine-induced tremors at an oral dose of 1000 mg/k Experimental Section Microanalyses were performed by Midwest, Microlab Inc., Indianapolis, Ind. Where analyses are indicated only by elemental symbols, analytical results for those elements were within &0.4% of theoret,ical values. Preparation of Diamides.-The methods used in synthesizing diamides of cyclobiitaiie-1,l-dicarboxylic acid were essentially t,he same as those reported for producing diimides of that acid.' stallizat,ion solvents and yields are reported in Table 1.
Acknowledgments.-This work was made possible by research Gra,nt SB-7548 of the National Institutes of Health, U. S. Public Health Service.
OCH, I
This paper describes the synthesis and vasodilators. pharmacological properties of a number of new subkituted chromonbs (111-XII) which were prepared as potential coronary vasodilators (Table I). I n addition to the fact that 111-XI1 share the characteristic features of active c h r o m ~ n e s ,these ~ ? ~ compounds could also be considered khellin analogs in which the furane ring of the khellin molecule (I) is not a part of the rigid furanochromone structure, but instead is attached to the chromone molecule through an ether linkage as in VI, VII, and XI. Chemistry.-2-Furyl-7-hydroxychromone (11, R = 2-furyl) a as synthesized from 2,4-dihydroxyacetophenone and 2-furoyl chloride by a standard three-step p r ~ c e d u r e . ~The synthesis of 2-aryl-7-hydroxychromones (11, R = C6Hj and C6H40CH3-4) is already described in the literature.5
(6) E. Soaje-Echayiie and R . K. S. Lim, J . Pharmacol. Ezptl. Therap.. 138, 224 (1962). (7) G. L.Hassert, Jr., J. 15.. Pontsiaka, D. Papandrianos, J. C. Burke, and 13. S . Craver. Tozicol. A p p l . P ~ L w ~ R c3,o 726 ~ . , (1961). (8) T. L. Kerley. A . B. Richards, R. W. Begley, B. E. Abreu, and L. C. \Veaver, J . Pharmacol. Ezptl. Therap., 132, 360 (1961).
I1
Substituted Chromones as Coronary Vasodilators D. S . BARIANA Research Department, Abbott Laboratories Ltd., Montreal, Canada Received Januaru 13, 1969
Khellin or 2-methyl-5,8-dimethoxyfuranochromone (I) and some of its analogs are known to be coronary
The chromones I1 (R = C&, C6H40CH3-4,and 2-furyl) were treated with various chloromethyl intermediates and acid chlorides to give the desired compounds 111-XII. (1) R. Charlier, "Coronary Vasodilators," Pergamon Press Inc., New York, N. Y., 1961, P 84. (2) G . Jongehreur, Areh. I n t . Pharmacodyn. The?., 90, 384 (1952). (3) Koninklijke Industrieele Maatschappij voorheen Noury a n d van der Lande N. V., Dutch Patents 70,267 and 70,268 (June 16, 1952); Chem. Abstr., 47, 64458,h (1953). (4) D . Donnelly, R. Geoghegan, C. O'Brien, E. Phillin, and T. 9. Wheeler, J. ,Wed. Chem., 8, 872 (1965). ( 5 ) W.Baker, J. Chem. S o c . , 1381 (1933).
