Antitumor, Cytotoxic, and Antibacterial Activities of Tenuazonic Acid

Determination of Exposure to the Alternaria Mycotoxin Tenuazonic Acid and Its Isomer allo-Tenuazonic Acid in a German Population by Stable Isotope Dil...
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TENUAZOXIC AND TETRAMIC ACIDS

July 1965

483

Antitumor, Cytotoxic, and Antibacterial Activities of Tenuazonic Acid and Congeneric Tetraniic Acids'

Merclc Sharp & Dohme Research Laboratories, Rahway, .Yeus Jprsey

Receibed Sovember 23, 1964 Sodium L-tenuazonate and several congeneric tetramates were tested for antitumor activity against the human adenocarcinoma (HAdl ) growing in the embryonated egg. The D - d l 0 isomer of potassium tenuazonate was no more than one-fourth as active as sodium Ltenuazonate, and because the optical purity of the n-allo isomer was unknown its relative activity could not be further defined. The D-isomer was inactive. Congeners substituted at the C-5 position had less than 5% of the antitumor activity of sodium L-tenuazonat'e. The N-methyl substituted 3-acetyl-5-sec-butyltetramate was 15yc as active as sodiiim Lteniiazonate. Other siibstitutions a t S-1 and substitutions at C-3 resulted in tetramates which were not active against HAdl. Cytotoxic activities of the C-5 substituted tetramates against K B cells correlated with antitrimor activity in the egg. Siibstitutiori at the X-1 positiou in some instances resulted in enhanced cytotoxic activity. Antibacterial activity against Bacillus 7negatari7rm did not correlate with antitumor activity. The antibacterial activity of tenuazonate salts R-as not stereospecific. The sribstitiition of a benzyl grorip for t,he N-hydrogen greatly enhanced antibacterial activity.

Utilization of the huiiian tumor-egg host system for testing potential aritituiiior agents led to the discovery of the antituiiior properties of tenuazonic acid (I).2 Compounds structurally related to tetiuazoiiic acid mere synthesized in the -\Ierrl; Sharp cC- Dohiiie Research Laboratories3 arid were tested for aritituiiior activity in the huiiiari tumor-egg host systeiii. The coinHg=+om CZHSCH

I

CH3

1

H I

pounds were tested also for cytotoxic activity against KB cells, and for activity against Bacillus megafekm. The results of studies comparing the activities of tenuazoriic acid and the related compounds in the three different test systems are reported here.

Materials and Methods

several levels. A volume of 0.06 nil. of test solution was placed on a filter paper disk (9.5 mm.) which was applied to the surface of a synthetic, glucose-citrate-salts-agar medium (part A, KzHPOI 7.0 g., K H 2 P 0 43.0 g., sodium citrate 0.5 g., Mgso4. 7 H ~ o0.1 g., (INH,)?SOd 1.0 g., Bacto agar (Difco Laboratories) 20.0 g., deionized water 800 ml., adjust to p H 7.0; part B, glucose 2.0 g., deionized water 200 ml. Parts A and B were autoclaved separately a t 120" for 15 min. and combined prior t o use.) The synthetic glucose-citrate-salts-agar rnediurn (10 ml.) was seeded with about 3 x 106washed spores of R. megaterium and poured into 100-mni. assay plates. Plates were incubated for 24 hr. a t 37" and then zones of inhibition were measured. The level of each compound producing a zone of given diameter was compared with t'he level of sodium L-tenuazonate found on the same test day t o produce the same size zone.

Results Comparison of the L-, D-, and D-allo Isomers of Tenuazonate Salts.-Table I compares the activities of salts of the L-, D-, and n-allo (isotenuazoiiic+) isomers of TABLE I L4CTIVITIES O F SALTS

T E N C . 4 Z O i Y I C .4CID .IG.IINST

Human Tumor-Egg Host System.-The human adenocarcinoma ( H A d l ) 4was used in these studies. The procedures for testing compounds in the human tumor-egg host system have been described p r e v i ~ u s l y . ~ Cell Culture System.-Cell cytotoxicity measurements were made as previously described2 using Eagle's K B cell carcinoma.6 Cytotoxic activities were expressed by numbers ranging from 4 (complete cell dest,ruction) to 0 (no cell damage). Cytotoxic readings L'S. concent,r:ttion were plotted on semilogarithmic paper for estimation of the cytotoxic end point ( C E ) (corresponding to a Cytotoxicity reading of 2). The relat'ive cytotoxic activity of each compound was based on a comparison of its CE value with that of sodium L-tenuaxonate determined on the same day. Microbial System.-Compounds were tested against B. megaterium by the agar plate diffusion method. Test compounds were dissolved in 0.05 Jf phosphate buffer a t pH 6.0 and when necessary, the solutions were adjusted to pH 6.0 by the addition of 0.1 N HCl or 0.1 S NaOH. Each compound was tested a t (1) This investigation was supported, in p a r t , under National Institutes of Health Contract N o . SA-43-ph-3057, by t h e Cancer Chemotherapy S a t i o n a l Service Center, National Cancer Institute, Department of Health, Education and Welfare. U. S. Public Health Service, Bethesda. Md. (2) C. 0. Gitterman, E. L. Dulaney, E. A. Kaczka, G. W. Campbell, D. Hendlin, and H. B. Woodruff, Cancer Res., 24, 440 (1964). (3) S. A. Harris, L. V. Fisher, and K. Folkers, J . M e d . Chem., 8 , 478 (1965). (4) H. W. Toolan, Cancer Res., 17, 418 (1957). ( 5 ) C . 0. Gitterman. E. L. Dulaney, E. A. Kaczka. D. Hendlin. and H. B. Woodruff, Proc. S O C Ezpll. . B i d . ?Wed.. 109, 852 (1962). (6) H. Eagle, ibid., 89, 362 (1955).

O F THE L-,

Isomer

Dose, mg./egg

D-,

AIiD D - U l b

--7c Deaths

grow-th inhibitionrumor Embryo

9/12 31 0.8 0.48 7/12 13 0.29 1/12 12 0.17 1/12 3 - 11 0.10 2/12 0.06 0/12 -1 0 4/20 ... 8/12 27 L (Xa) 0.7 (Twt 2) 0.42 3/12 9 0.25 2/12 1 0 . 15 3/12 1 0.09 n/i? 2 0 4/20 ... c 4.8 1/6 i 1.2 0/6 -18 -1 0.3 2/6 0 3/20 ... D - d o (K) 2.5 7/8 WI" i (Test 1) 0.6 1/ 8 0 2/20 ... D-allO ( K ) 2.0 2 /a 25 (Test 2) 1.0 0/8 31 0.5 1 /S -4 ... 0 1/20 Inhibitory value derived from only one survivor. L

(Ka) (Test 1)

-

a

ISOMERS O F

HAdl IiY THE EMBRYONATED EGG

98 9i 79 62 72 22 ...

98 86 81 62 47 ... 9 21 - 29 ...

[sola 68

.. 93 83 61

...

I

n IO 20 30

I 2 3 4 C Y T O T O X I C I T Y DEGREE

40

ZONE SIZE, mm.

Figure l.--C'ytotosic activities o f I,-, I)-, and u-do-tenuazonic acid salts against KB cellh: espt. 1, A, 1)-tenuazonic acid ( K j : H, ~ - enuazonic 1 acid ( S a )j expt. 2,O. D - d o - t eniiazoriic acid ( K ): II~,1,-teniiazonic arid ( Y d .

acid. The r,-isoiiier was highly inhibitory lo the tunior at a dose of 0.1 mg. /egg and was toxic. to tlic eriibryo at 0.48 tiig. egg (7 12 deaths). The I)isotii(1i did not i i i h i b i t tuiiior gron th iior wai i t toxic2 to the e i i i h i y o at tlic liighc~stlevel testcxd, 4.8 iiig. 'egg. The u-a//o isoiiiw at 2 L iiig. 'cgg nab toxic. (718 deaths) atid at 0.6 iiig. egg inhibited tiuiior growth 68% Iir a waoiid t cst, thc r)-a/lo isoilier at 0 5 ing. egg i r i liibited tunior growl h 60 It \vas toxic at 1.0 lllg egg, a lei el whic*li iiihib c~lllblyogron t l l 3 1 C ; % although tic) d e a t h O N ~ I I I ' Ic d . E ' r o t i i thv data I I I Table I EDEovaluc for hodiuiir r,-teiiuazoiiate n a y oytiiiiatrd l o be 126 y 'egg. Based o i i the approxiriiate dohe whic*h inhibited H.ldl t iiiiioi gron t h (XFL. t l i v i)-dlo i h o i i i t ' r was 2.jCc a. artiw a i sodiutii L-t(>riuazotiat(' and 1h(1 i)-isoiii(-'r \vas lesq thatr t muazotiic

t h c h

ithesis of teiiuazoiiic. acid raremizaiioii (mati o(*vur w i l h t h c forination of thr roircspoiiditig diast ~~rc~oisoiiier. Ai caoiiipai isoii of diflei eiit saiiipleb oi tciinazoriatcs qhowed that high aritituiiioi activity tias a-+o(~iatctl~ v i t l iliigh wgat ix.11 rotatiotr cTal)l(~11) T.\HI.E

IT

E i h -//egg

9 - o',

- 16.5 -- 110

126 (104-153)'' il 9-h'J 215 (18G-249)" 1, [Ykl) \I-(; -8 10 1000 Activity relative t o that

Ri,h

%

10 100 150 40 60 ... 350 10 10 5 5