E v Iter icii iri SO.
coli
I !I
1"; 25
2
s
li
?;
I
x a
1'1 =
s 100 partial inhibition, S
= iio
artivity a t 500 pg 'nil.
filtered :ifid worked iip iii the iisiial niaiiiier. The resilltirig coiiip ~ ~ i i t i(free ~ l s base or 2HCl salt) were purified by crystalhatioil.
Acknowledgment.--The authors \\-ish t o th:mI; 1 h . Robert' Strauss, Miss Ariiie JIcBurney, and A h . Beverly Slowicki for the microbial assays.
3 l - Sulfato-N2-isonicotinylhydrazine. A Potential Metabolite of Isoniazid'
During studies on the metabolism of the antituberculous isoniazid (INH), in animals3 and we considered the possibility that, one of the group of acidlabile urinary metabolites was an ^\'-sulfate conjugate of IKH. This was suggested by earlier in v i u 0 ~ 7and ~ i.n vitro7 studies, using various laboratory animals, in which arylamines such as aniline and 1- and 2-naphthylamine were conjugated with sulfate to form highly acidlabile sulfamates. Since 1K.H is acetylated by thc same pigeon liver enzyme that acetylates arylamines,b :L similar parallel activity seemed possible for sulfate conjugation. If highly acid labile, an INH-sulfate (1) Supported in part b y U. S. I'ulilic
H e a l t h Service Grant .21-03682. ( 2 ) Komedical Research, Life Sciences Division, Btanford Researall Institiire, XIenlo P a r k , Calif. 94026. (:O , I . I I . Peters and V. E. lIayes, A r , , l t , l ! t l e r , i . l ' t i i i r m o c o d y n , , 169, 340 lIU6li). (1) (a) .I. H. l'eters, li. Y. Xiller, anLl 1'. 1 3 r o \ ~ nA , i d . B i o c h e n . , 12, 37il (1$165): ( b ) .I. Phurmarol. L'zptl. T h e r a p . , 160, 298 (1965). (5) 1'. 13o,vland, r). hlanson, and S. F. I). Orr, Bioriiem. J . , 66, 117 ) I). \'. I'arke. i 6 i d . , 7 7 , 4!i3 l I 9 6 0 ) . ) ( a ) .\. 13. Roy. Biorhim. ~ ' 3 i o p h y . q ..Ictii, 30, 1!13 (1'358): i l l ) B ./., 74, 49 (1960). ( 8 ) .J. \ \ . .lenne ani1 1'. 1 ) . 13oycr, Wiochiin. B?opl,U,>..
coiljugate would contribute t o the incompletrly itlciit Ified acid-labile fraction of urinary I S H nietabolitr.;. Therefore, \\e synthesized the I S H analog of :iryl sulfamates, Si-sulfato-S2-i~o~ii~otiriylhydr:tzir~e, l)y procedures employed previou4y.i It was found to t)e atable to the mild acidic conditiolis known to hydrolyzr the pyruvic and a-lietoglut :uic acid hydrazone.: of I S H 3 and the aryl sulfaniatehj (pH 1.0, room Icmper:iture). However, it could be bplit quantitatively t o I N H by heating at 43" in 1 N HCl for 24 hr, conditioiiy developed for the hydrolysih of :icetylisoniazid t o ISH \pithout the decomposition of ISH.4a When thii liytlrolysis procedure wa5 applicd to urine collectrd froni dogs receiving I S H in itudies reported previously, no increase of I N H content could be detected. Tliew results indicated that the sulfate conjugate n-:is not :I metabolite of I X H ill the (log. Thereforr W P tc>stctl the vi~oit:ihility of t h c iulia1 e compound iii h u h r quent experiment-. For preliminary te on tho toxicity of thii c.oi11single doses of 43.1 m g 1.g pound, we ndmini.t iv (equivaleiit to 25 mg of ISH'lig) to six 26-day-old Ho1tzm:tn female r:& Similar groups of untrc:ittd id n i t * receiving 2%5nip of I S H 'kg served :I- COIISo iinto\wrd rcxction- were noted in t lie rai iiig either I S H O r thc ISH-sulfate con,iug:itc. The ruts J v c w weighed inimdintely before irijrctioii :tiid daily for t h e next 9 days. Necropsiea \ w r r p ~ r formed aftcr killing the aiiimuls with CHCld, \v1th bpecial atteiition being given t o the liver, kidney,, :mI \pleen, which were' cxamirid internally a i w ~ l :is l Yternally. So gros;.; chaiigc~from the untreutrd r:itw r e iiotcd ill tlw t rcJiitctl groups. In utlditioii, t i o toxic effects of the conipouiitl> \yere noted in the growth uf the animal-, :t.z cvideiicecl by nearly identical weight gains in the trc:itr(l :itid i u i t reated groups cluriiig lh(. btudy period T o determine t h e 211 i > i wyt:ibility of the IXH--uhat(~ cwijugate, iiijcci et1 four yourig adult female b(a:Igltu clogs (weight riiiige. 5 . 2 cJ.7 kg) with doseh of 9 . ( j
-
(1
mg/kg iv (equivalent to 5 mg of INH/kg), successively for 3 days and collected urine during each 24 hr following the protocol used earlier.3 Aliquots of the urine collections were analyzed for I?rTHl9for total hydrolyzable INH, and for total hydrolyzable isonicotinic acid derivatives. lo Table I
Experimental Section13
N1-Sulfato-NZ-isonicotinylhydrazinePotassium Salt.-Redis-
tilled chlorosulfonic acid (5.0 g, 43 mmoles) was added dropwise to dry pyridine (50 ml) maintained a t 15' in a stoppered flask. Solid I N H (5.9 g, 43 mmoles) was added in small portions with gentle agitation. After 1 hr the mixture was allowed to warm to room temperature. After 23 hr the orange-red solution was poured into 100 In1 of 1 N KOH. Following extraction with three 100-ml portions of EtZO, the aqueous phase was evaporated to one-half volume on a steam bath, without the product appearTABLE I ing after cooling. -4pH check showed the solution to be weakly URINARY EXCRETIOX OF RELATED COMPOUNDS BY DOGS acid. Therefore it was alkalinized with additional KOH, yielding R IWCEIVINGN~-SULFATO-N~-ISONIC~TIKYLHYDR.IZINE a copious precipitate. The mixture was reheated to dissolve POT.\SSIUM SALT(9.6 mg/kg) INTRAVENOUSLY the precipitate and allowed to stand overnight a t 5'. The crude -Mean %" of the admind dose in 24-hr urinesproduct (9.0 g, 86%) was crystallized from 8070 MeOH. The Total yield was 4.6 g (417"). A small sample was dried to constant No. of Total hydrolyzable weight a t 65' over PnOs; mp 170-180". Anal. (CaHaKKaOS. treathydrolyzable isonicotinic 0.5H20) C, H, S; N: calcd, 15.9; found, 16.4. Dog ments INHb INHC acidd The compound was easily separated from I N H by descending A 3 0.0 (0.0-0.2) 97.1 (89.5-102) 97.3 (91.0-103) paper chromatography3 (Ri of INH, 0.51; of product, 0.25). B 3 0.0 (0.0-0.0) 94.3 (88.4-103) 96.0 (84.6-105) On paper chromatograms, the product could be detected by uv C 2 0.0 (0.0-0.0) 104 (97.0-110) 100 (93.5-107) absorption or with color reagents specific for unsubstituted pyriD 3 0 . 0 (0.0-0.0) 93.2 (80.8-104) 95.6 (78.6-110) dine N.3 Tests with reagents for unsubstituted hydrazine N were negative. Hydrazine content was found to be 104% of theory a Mean of the indicated niimber of successive daily treatments. by a procedure for total hydrazine.14 Total hydrolyzable isol'alues in parenthesis are the ranges observed. b lteasured by nicotinic acid contentlo was 10lyo of theory. Tests of the revanillin procedure9 before hydrolysis. Measured by vanillin covery of the INH-sulfate conjugate from urine by the procedure procednreg after hydrolysis in 1 S HC1, 24 hr at 45'. d Measured for I S H 8 yielded less than 0.5% of the theoretical I N H content. by isonicotinic acid procedure'O after sealed-tube hydrolysis ( 5 A' HCI, 3 hr, 120'). (13) Melting points were taken on a Fisher-Johns apparatus and are un-
presents the results obtained. It is apparent that the INH-sulfate conjugate was not split in the body since no I N H could be detected in the urine. Qualitative paper-chromatographic examination of aliquots of urine3 yielded only one I N H derivative at an Rf (0.25) identical with that of the injected compound. Furthermore, the nearly quantitative recovery of the dose administered as either total hydrolyzable I N H or total hydrolyzable isonicotinic acid derivatives indicates that the INH-sulfate conjugate was excreted unchanged in the urine. These results indicate that the INHsulfate conjugate is chemically and metabolically stable like the noncaloric sweetening agent, sodium I\'-cyclohexylsulfamate. l 1 Table I1 compares the anti-
corrected. XIicroanalyses were performed by Clark Microanalytic Laboratory, Urbana. Ill. Where anslyses are indicated by the symbols of the elements, analytical results obtained for those elements were within *0.4% of the theoretical values. (14) J. H. Peters, Am. Reu, Respirat. Diseases, 81, 485 (1960).
Preparation and Antibacterial Activity of a-(5-Tetrazolyl)benzylpenicillin J. RI. ESSERY Research Division, Bristol Laboratories, Division of Bristol-Myers Company, Syracuse, N e w York 13901 Received March 17, 1969
A recent publication from these laboratories2 discussed some examples of the replacement of the carboxyl group by the comparably acidic 5-tetrazolyl TABLE I1 groupSv4 in biologically active compounds. Such a ANTIMYCOBACTERIAL ACTIVITY OF substitution has now been made in an antibiotic withX~-SULFAT~-N~-I~ONICOTINYLHYDRAZINE AGAINST out loss of antibacterial potency. The Beecham M . tuberculosis VAR.hominis 5159 ~ N HI?Rva D group has disclosed5 that the semisynthetic penicillin, -Min inhib concn, pg/mldisodium a-carboxybenzylpenicillin (carbenicillin), exDrug 5159 HsiRv hibited activity against certain Pseudomonas and N1-Sulfato-N 2-isonicotinylProteus species high enough t o warrant clinical inhydrazine potassium salt 12.8b 12.8b vestigation.'j A similar order of in vitro activity was INH 0.05 0.05 shown by the analogous compound dipotassium a-(5a Tests were carried out in Dubos liquid medium, incubation tetrazoly1)benzylpenicillin (IV) which was prepared a t 37' for 9 days. Equivalent to 6.6 pg of IKH/ml. according to the reaction sequence depicted in Scheme I. Chemistry.-Treatment of 5-benzyltetrazole (I) with mycobacterial activity of the INH-sulfate conjugate 2 equiv of n-butyllithium followed by carbonation with that of INH.12 It is clear that this I K H derivaafforded a-(5-tetrazoly1)phenylacetic acid (11). This tive possesses only a small fraction of the activity of acid, an analog of phenylmalonic acid, was readily IX'H. (9) J. R. Maher, J. If.Whitney, J. S. Chambers. and D. J. Stanonis, A m . Rev. Tuberc. Pulmonary Diseases, 76, 852 (1957). (10) J. H. Peters. A m . Rev. Respirat. Dzseases. 83, 153 (1960). (11) (a) J. P. Miller, L. E. M. Crawford, R. C. Sonders, and E. V. Cardinal, Biochen. Biophys. Res. Commun., 36, 153 (1966); (b) J. D. Taylor, R. K. Richards, and J. C. Davin. Proc. Soc. Ezptl. Bid. &fed.. 78,530 (1951). (12) We are indebted t o Dr. R. C. Good for these determinations.
(1) This compound. and related ones, have been described by J. XI. Ess e w , U. S. Patent 3,427,302(1968). (2) P. F. Juby. T. 1%'. Hudyma, and XI. Brown, J . M e d . Chem., 11, 111 (1968). (3) F. R. Benson, Chem. Rev.. 41, 1 (1947). (4) R. M. Herbst, "Essays in Biochemistry," John Wiley and Sons. Inc., New York, N. T.,1956,p 141. (5) P. Acred, D. M. Brown, E. T. Knudsen, G. N. Rolinson, and R. Sutherland, Nature, 216, 25 (1967). (6) W. Brumfitt, A. Percival, and D. A. Leigh, Lancet, 1, 1289 (1967).
