SOTES
September 1968
1067
Notes ucts can be prepared simultaneously or separately. Thus the mild acidic hydrolysis a t 30" for 96 hr (pH 4.4-3.5) gave 11. If the hydrolysis was performed a t a higher temperature (50") and/or with a longer reaction time, lactone I11 was also produced. Desacetylcepha8.K L K O L J ~ loglycin was separated from unreacted 1 and formed lactorie I11 by chromatography on a cellulose column. The Lilly Reseatch Laboratories, Elz Lilly and Companu, Indianapoh, Indzana 46206 Hydrolysis of I with HCl for 24 hr a t 30" (pH 1.4) gave only I11 isolated as the hydrochloride. I11 can be made Received Match 26, 1868 by lactoriization of I1 with HCl. Desacetylcephaloglycin also could be prepared by enzymatic deacetylaPrevious publications' from these laboratories detion of I according to the procedure described by Janscribed the synthesis and the antibiotic activity of sen, et aL6 cephaloglycin (I) . 2 I n continuing investigations the The course of reaction can be followed by tlc and stability of this antibiotic has been studied. I n parnmr spectroscopy. The acetyl group in I showed a ticular, attention has been directed toward the selective singlet a t T 7.9 which gradually disappeared as hydrolhydrolysis of cephaloglycin, since it has been shown that ysis proceeded, and a new signal for the acetate anion the desacetyl derivative of cephaloglycin is a metabolic was observed at 7 Additional information product present in the blood and urir1e.j I t was also which was useful in the identification of products was shown to have antibacterial activity. For this reason obtained from the signals due to the exocyclic CH2 it was necessary to establish the degree of therapeutic (see Table I). A tlc method was developed primarily effectiveness of desacetylcephaloglycin (11) arid to to aid in identification arid separation of miutures. determine it5 role in the treatment of infections. ThereColumn chromatography fractions were analyzed by tlc fore, our attention and efforts were directed to the using cellulose plastic sheets. The leading spot havirg preparation and isolation of I1 in pure form. an Rr of 0.8 was identical with lactone 111. The middle Cleavage of the acetate group in cephalosporins has spot (Rf 0.7) was starting material I. The slow moving been successfully carried out by enzymatic h y d r ~ l y s i s . ~ spot, desacetylcephaloglycin, had an R, of 0.5. The The same group in A2-cephalosporins mas hydrolyzed separation on tlc cellulose plastic sheets was used as with dilute alkali, since it has been reported that A 2 the basis for development of a method of isolation of the compounds are relatively more stable under alkaline components from the reaction mixture by column conditions. chromatography. The crude mixture was chromatoI t has been now found that, in an acidic medium, graphed over cellulose. The column was developed cephaloglycin is converted to desacetylcephaloglyciIi with aqueous acetonitrile a t 3". The appropriate (11) and desacetylcephaloglycin lactone (111) (Scheme fractions were collected and, after removing the solvent, (I). By controlling the reaction conditions both proddesacetylcephaloglycin was isolated as an amorphous powder and crystallized from aqueous acetonitrile. SCHEME I Isolation of I1 presented considerable difficulty C , > H , C H C O NNH ~/ %> because of its chemical instability and physical properINH, 0 ties. I n a crystalline form desacetylcephaloglycin CH,OCOCH, displayed good stability, but in solution stability mas COOH very dependent on p H and temperature. The lactone I was formed easily with traces of acid or a t higher temperatures. The free amino group in the lactone, because of its basic properties, is another source of instability. When the lactone was isolated as a free base, it polymerized rapidly. l l o s t probably the lactone and 1 or lactam functions were attacked by the amino group forming a polymcr. The lactone I11 was isolated in the form of a hydrochloride salt. Desacetylcephaloglycin (11)and the corresponding lactone (111)were characterI1 I11 ized by ir, uv, and nmr spectra, elemental analyses, arid optical rotations. Bacillus subtzlis and Sarcina lutea bioautographs for (1) (a) Paper S : J. W.Chamberlin and J. B. Campbell, J . M e d . Chem., 10, 966 (1967); (b) J. L. Spencer, E . 11. Flynn, R. W.Roeske, F. Y . Siu, and I1 and I11 displayed single, biologically active comR . B. Chauvette, ibid., 9, 746 (1966); W.E. Wick a n d W.9. Boniece, A p p l . ponents. I n the course of in vitro studies3 desacetylMicrobial., 18, 248 (1965). cephaloglycin mas found to have essentially the same ( 2 ) Cephaloglycin is t h e generic name given t o 7- [(D-l-amino-Z-phenyl)acetamido 1-3-acetoxymethyl-3-cephem-4-carboxylicacid. broad-spectrum antibiotic activity as cephaloglycin" Chemistry of Cephalosporin Antibiotics. S I . Preparation and Properties of Desacetplcephaloglycin and Its Lactone
(3) Results will be published b y W.E. Wick. (4) (a) J. D ' h . Jeffery. E . P. h h r a h a m , and G. G. F. Newton, Biochem. J . , 81, 591 (1961); ( h ) E. Van Heyningen, J . .Wed. Chem., 8 , 22 (1965). ( 5 ) J. D. Cocker, B. R . Cowley, J. S . G . Cox, S. Eardley, G. I. Gregory, .J. Ii. Lazenh\-, .IG .. Long, J . C. P. Sly, and G. A Somerfield, J . Chem. Soc., 5015 (IY65).
( 6 ) E. F . Jansen. R. J a w , and L. R . MacDonneIl. Arch. Biochem., 16, 415 (1947). (7) (a) .I.B. Taylor, J . Chem. Sac., 7020 (1965): (b) J. D. Cocker, S. Eardley, G. I. Gregory, % I . E. Hall, and A . G . Long, ibid., 1142 (3966).
SOTES
September 19G8 Acknowledgment.-The author is grateful to W. E. Wright for valuable discussions; to G. Jlaciak and his staff for the microanalyses; to H. Boaz, D. 0. Woolf, L. A. Spangle, and their associates for spectral data and titrations; to W. E. Wick and J. L. Ott for biological data; to c. T. Pugh for bioautography; and to A. I. Ellis for technical assistance.
1069
MR’ 3, R = H ; R’ = 0 4, IZ = t-CaHg; R’ 5, R = tC411g; R’
= =
8, R
=
f-CJ~9; R’
= ’’
R
=
f-C4119;
0 KOH
Cassiaine Analogs. V.’ A Distant Analog of Cassaine SOLJ. Dauv
AXD
ROBERTL. C L ~ R K E
Sterling-ll’inthrop Research Institzite, Rensselaei‘, .Yew I’ork 12144 Receiz’ed .lIarch 18, 1968
Extending our efforts in the search for a cardiac stimulant, we have prepared another analog related to the Erythmphleu?u alkaloid cassaine (1). 2 We felt ,COOCH2CH2N(CH3),
“CH,
1
HO
H 2
that if the group =CHCOOCH,CHzn’(CH& of 1 were replaced by the equatorially oriented -NHCOOCH,CH,N(CH,),, perhaps we could obtain a cardiac stimulant having a much longer duration of activity. The ease of i n vivo hydrolysis of the ester group might be related to duration of activity. It was hoped that the equatorial Configuration of the side chain would approximate the configuration demanded by the double bond of the natural product and that the unshared electron pair on the nitrogen might substitute for the electron character of the double bond. I n order to test this hypothesis, we have prepared the carbamate
2. The hydroxyl group of the starting material 3 was protected as its t-butyl ether derivative 43 while the necessary transformations were made at C-13. The oxime 5 of 4 was reduced chemically ( S a , EtOH), a procedure which allows assignment of the major product as the equatorial ( a ) amine 6 and the minor product as the axial (p) amine 7. S o t being crystalline, these amines mere characterized by their conversion in the next step of the sequence t o crystalline 2(1) For paper IV sre R . L. Clarke, 9. J. Daum, P. E. Shaw, T. G. Brown, Jr.. G . E. Grohle\vski, and W.V. O’Connor, J . M e d . Chem.. 10, 593 (1967). ( 2 ) See F. Erjavec a n d 3. Adamic, Arch. Intern. Pharmncodyn., 155, 251 (1Y65): E. L.lIcCawle?, A ~ k d o i f k5. , 101 (19.551, and references therein. (3) H. C. Bayerman a n d G. J. Heisznolf, Kec. Trau. Chim.,84, 203 (1965).
9,
=
\rr
R’ =
10, R = t-C4Hs; R’
11, R
XHCOOCH,CH~C~
SIICOOCH~CH~C~ (( ‘H
,NHCOOCH,CH~K(CH,), =
FICCO; R’ =
I’
\U
,NHCOOC€I,C€I~N(CH~)Z ’
\H
chloroethyl carbamates 8 and 9. Reaction of 8 with dimethylamine afforded the dimethylaminoethyl carbamate 10 which was characterized as its hydrochloride salt. The t-butyl ether group in 10 was cleaved by treatment with trifluoroacetic acid; the compound isolated was the trifluoroacetate 11. This ester was hydrolyzed with methanolic S H 4 0 H at room temperature to give 2-dimethylaminoethyl dl-S-(1,2,3,4,4aa,4b,5,6,7,SJSaa,9,lO,lOnp-tetradecahydro-7p-hydroxy4bfi-methylphenanthr-2a-yl)carbamate (2) in good yield. Biological Te~ting.~-Compound 2, upon intravenous administration in the dog a t a dose level of 4 mg ’kg, produced an increase of 307, in ventricular contractile force, accompanied by a blood pressure drop of Ouabain at a dose level of 0.03 mg/kg or cassaine (1) at a dose level of 0.04 mg/kg produces an increase in ventricular contractile force of 20YGwithout concomitant lowering of blood p r e ~ s u r e . ~ Experimental Section6
dI-7p-1-Butoxy-3,4,4aa,4b,5,6,7,8,8aa,9,10,1 Oag-dodecahydro-4bb-methyl-2( la)-phenanthrone 2-Oxime (5).-A solution of 6 g ( 2 5 mmoles) of hydroxyphenanthrone 37 in 150 ml of CH,C12 was treated with 1 ml of BF3 etherate and 1 ml of anhydrous H1P04. The latter was prepared by the addition of a calculated amount of P?Oj to 85yGH3PO4. Isobutene (150 ml) was added t o t,he solution at 10’. The solution was shaken in the Parr shaker for 5 hr. The reaction mixture was poured into 200 ml of 2 S NHIOH, the layers were separated, and the aquqppus layer was Fashed with 200 ml of CHZC1,. The organic lagers were combined and dried (Xa2SO4). The solvent was removed under reduced pressure t o yield an oil that was chromatographed on 300 g of silica gel. Ether eluted 4.i g of dl-70-t-butoxy-3,. 4,4aa,4b,5,6,7,8,8aa,9,lO,lOa~-doderahydro-4b0 -methyl 2( 1H)phenanthrone ( 4 ) as an oil that would not crystallize. The oil,
-
(4) For details of t h e experimental methods of evaluation see R . L. Clarke, IT. V. O’Connor, J . .lied. Chem., 10, 582 (196i). ( 5 ) We wish to thank Mr. William V. O’Connor f o r t h e biological testing.
S. J. Daum, P. E. Shaw, T. G . Brown, Jr., G . E. Groljlewski, and
(6) All melting points a r e corrected. I r spectra were recorded on a PerkinElmer Infrared spectrophotometer, Model 21. T h e silica gel used for column chromatography (100-200 mesh) was obtained from t h e Davison Co.. Baltimore, bld. Silira gel G, purchased from Brinkmann Instruments, Inc., was used for thin layer chromatography. JI-here analyses are indicated only by symbols of t h e elements analytical results obtained for those element? were within ;tO.4% o f ttir t!reoretical aaliies. (7) A. J. Daum, I’. E. %inn.. and R . I,. Clarke, J . 078. ChPm., 32, 1427 (1967).
