January 1907
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After thawing, the volume was measured, a i d theii the uriiie was filtered through a Seitz filter immediately before determinatioii. This was effected with the twofold dilution method, i i i broth, using a strain of Bacillus lateosporus particularly seii3itive to nitrofuran drugs. Excretion was recorded as cumulative per w i t of administered substance.
SCHEME I
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1.” i l l iiitio antibacterial activity, I I I C data are reported in Table I. By comparison of the various AIIC of I and 11, it appears that the activity of the tmo materials is of the same order of magnitude, although for some bacterial strains I1 iz superior to the polymer. EDjovalues for in vico antibacterial activity of I and I11 are 840 and 345 mg kg. respectively (confidence limits for 96y0 probability are 316-1369 nig/kg for I and 272-438 mg’kg for 111); this clearly shows a systemic action of the polymer much lower than that of the reference compound. Urinary excretion of molecular species endowed with antibacterial activity after injections of I and I1 is shonri in Table 11. An obvious delay of elimination i+ ob-erved in the case of the polymer: antibacterial power is observed in urine also at 72 hr, nhereas for I1 it stops a t 24 hr.23 When a similar experiment was tried by oral administration, it showed that I was completely unabsorbed and n o antibacterial activity 11 ab found in urine. Acknowledgments.-We thank Professor G. Giaconiello for suggestions and help during the execution of this work. ITe also thank A h . D. Tricoli for technical aid in the experimental work. (23) €1. K Reckendorf, R G Castringius, and H K bpingler, dnttmzc~obzul Agents Chemotherapy, 1962, 531 (1963).
Fluorene Derivatives for Antitumor Activity’” KRISHNA C . AGRAWAL’~ Phortrwceulical Chemistry Research Luboralory, Uniuersity of Florzda, Gainesville, Florida
Receiced J u l y 5 , 1966
Tn-o compounds 2,7-di(niethanesulfonyloxy)fluorenc (V) and dimethyl 2,7-fluorenedisulfonate (TII), were prepared for arititunior screening. I1ethanesulforiyl esters have been reported2to be active cancer chemotherap u t i c agents. Busulfan, 1,4-di(methanesulfonyloxy) butane, is a well-known representative of this type of alkylating agent. I n order to determine if replacement of the alkane chain of four carbon atoms by a ring system such as fluorene would produce compounds with antineoplastic activity, compound T’ was made. Compound VI1 was made t o determine the effect of reversing the ester group (S linked to the ring instead of to 0). Compound V was synthesized by the action of methanesulfonyl chloride on 2,i-dihydroxyfluorene (IT’) in pyridine solution (Scheme I). The synthesis of IT7 was attempted by diazotization of 2,7-diammofluor(1) (a) This investigation n a a supported Public Health PerIice Kesearch Grant C 1-08186 from the National Cancer Institute (b) Department of Pharmacologq , I ale Unil ersitq School of Medicine, h e n Hal en, Connecticut ( 2 ) T H Goodridge 11 ‘r l l a t l i e r 11 h Harmon, and R 1’ I3ratLe1, ( ‘ u u e r Cimrrmtherupy l l ~ p l, 9, 78 (lQb0)
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ene followed by hydrolysis; colored condensation products resulted. The synthesis, therefore, was carried out from dipotassium 2,i-fluorenedisulfonate (I), of proved structure.3 During alkali fusion of I, the ring was broken a t C-9 t o give 4,4’-dihydroxybiphenyl-2carboxylic acid (11) which on dehydration with zinc chloride gave 2,i-dihydroxy-9-fluorerione (III). Proof of structure of I1 was obtained by decarboxylation (heating with lime) to the known 4,4’-dihydroxybiphenyL4 Compound 111, on reduction by hydrazine hydrate and potassium hydroxide, gave IT. Compound VI1 was prepared from I which was first converted to its dichloride (VI) using phosphorus pentachloride. By the action of sodium and methanol, \’I gave the corresponding dimethyl ester (YII). Biological Results.-Compound VI1 was administered a t a dosage of 50 m g / k g each alternate day to three tumor-bearing, ChF1/Jax mice. The tumorcurves for these mice are shown in Figure 1A. In the case of two niice there was quite rapid regression of the tumor. The tumor areas of 106 and 128 mm2 were reduced to 37 and 72 mm2 on the 9th and 11th day of the administration, respectively, when both the mice died. In the third, the mouse-tumor area of 137 was reduced to 90 nim2 on the 2lst day which was followed by death. The death of all three mice indicated that the toxicity of the compound was high; the dose was reduced to 25 n i g k g . Figure 1B shows the tumorgrowth curves on administration of 23 mg/kg of VII. During the time of drug administration the tunior growth seemed to be inhibited. When the drug was stopped, the tumor size increased rapidly, comparable to the controls (Figure 2 ) . After a period of 2 weeks, the tumors of treated mice tended to ulcerate arid to expel a (*oreof necrotic tissue, after which, regression of the tumors occurred. Compound IT,a t a dosage of 300 mg kg, also showed inhibited growth patterns (see E’igure 3) when compared with controls. These mice also survived with ulceration and expulsion of necrotic tissue. In contrast to these results, two of the control mice died within 21 days due to excessive tumor growth. The third control mouse, however, survived by expelling the core of necrotic tissue. The number of animal- in these experiments is too sniall for conclusive results, but they do iii( 3 ) C Courtot, A n n Ctiim., 1 4 , i(1930) (4) \ I. b e i a s t q a n o i , Zh I ’ r i i l K h i m
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Experimental Section5
cussions and to the Slom-Ketteriiig Institute for Cancer Research, S e w York, S . Y., for screening results. Thanks are due the American Cancer Society for an allotment from the University of E'lorida Institutional Grant IS-G2E arid 66-7.
2,7-Dihydroxy-9-fluorenone(III).-4,4'-I)ihydroxybipheiiyl-2carboxylic acid3 ( 5 g ) was piilverized with 10 g of anhydrous ZiiClp. The mixtiire wah heated to 200" for 10 min while stirring conataiitly, cooled, and deconiposed i n water. The precipitate was filtered aiid dried t o yield 4.0 g (87Ci), mp 325-328". Recrystallization from aqiieoii- alcohol gave dark red crystah, mp :3:8" (lit,.3nip 338'). 2,7-Dihydroxyfluorene (IV).-Compoiind I11 ( 2 g ) was dissolved i n 40 nil of hot diethJ-lene glycol. Hydrazine hydrate (8.57, 10 ml) wits added, and the mixture vias refllixed at 120' for 1 hr. KOH (4g ) was then added, and t,he temperature was raised to 200-20,5°, Heating was continued withoiit a condenser for 2 hr. The reactiou mixtiire way then cooled, treated wit,h 30 ml of cold wnter, aiid acidified with 1%ml of concentrated HC1 to precipitate I\-: yield 1.7 g (00.9:;), mp 262-263'. Three recrystallization> from aqueoiis alcohol gave white flakes, mp 269270'6 (lit. mp 283"' aiid 249-250"*). .Inu/. Calcd for CI3H1,,0p: C, 78.7s; H, 5.05. Fouiid: C, 78.67; 11, 5.12. 2,7-Di(methanesulfonyloxy)fluorene (V).-To compound I T ( I g ) in 15 ml of pyridiiie, 2 ml of methanesiilfonyl chloride was added slowly while s t iyriiig a t 0 ' . hfter refrigeration overnight. the coiitent,s wei'e poured into 100 ml of cold water. The resulting precipitate >~-a.filtered, wa-hed with cold water, aiid dried. ltecrystallizatioii from a mixtiire of CHCI, and alcohol gave long, colorless needle., m p l(i6-1GT0, yield 1.5 g ( 8 4 5 ) . Anal. Calcd for C,jHl~O&: C, 50.85: H, 3.98; S, 16.08. Found: C, 5 0 . 8 8 : H, 2,7-Fluorenedisulfo I) w a d made by mixing I (20 g) with I'Cli (31 g ) and heating over a steam bath for 2 hr. The mixtiire was decomposed in ice, filtered, washed with cold water, mid dried iinder vacuiim. It was recrystallized from ethylene dichloride, mp 2%;" (lit.9 mp 22.5'). Dimethyl 2,7-Fluorenedisulfonate (VII).-To sodium (0.9 g, PO mmoles) i n 101) ml of aiihydrolis methanol, 1-1 (7.3 g, 10 mmoles) was added in small portioiis at, room temperature, T h e precipitate wa. collected, and the filtrate was evaporated rinder vaciiiim. The residiie was combined with the original precipitate, and the whole waa .tirred with 25 ml of cold water and filtered. ltecrystallizatioii from methaiiol gave white microneedles, mp 1!)3-194", yield 4.2 g (GO";). d n a / . Calcd for CljH1&6&: C, 50.85; H, 3.98; S, 18.08. Foiind: C, 50.88: 11, 4.12: S,18.29. Animal Experiments.-Male, CAFl/Jax mice were employed in this study. Three weeks prior to administration of the compoiind-, the mice received a siibaxially transplant of a keratinizing sqiiamoiis cell The mice were divided into groiiph of three. Oiie group consiited of control animals which received 0.2 ml ip of a 0.5";solution of methylcellulose, the vehicle for iiijectoii, each alternate day. Compound \-, at dobe> of 25 and 30 mg/kg aiid compound \TI, at a dose of 500 mg/kg, were admiiiihtered t o three groiiph of animals. The treatment schediile was coiitiiiiied for 21 days by injecting the compounds on alteriiate days. Oil the day of drug administratioii, estimates of the size of the t,umors were made. Tumor areas ("2) were plotted against the number of dags. .Inimal weights were also recorded on the same days and plotted. After termiiiatioii of the treatment, tiinior sizes were estimated for The animals were further observed for a period of 5 weeks to determine the iirimber of survivals among t,reated l and c o i i ~ r oaiiimalr.
Acknowledgment.-The author h h e . to expre>- hi. 41ic*ere:~pl)reriatioiito Dr. 1'. E.Ray for helpful d i ( 5 ) 11eltinp points \yere determined on a Thomas-Hoover capillary apparatus and are recorded as obtained. Analyses were performed by Ycliwarzkopf 3Iicroanalytical Laboratory, \\-oodside 7 7 , N. Y. (6) Discrepancies in the melting point of compound I V seems to be due t o differences in purity. Tlir pnrity of compound I11 is also a n important factor in \ t i p final 1,iiritv o f I \ : I o w p r nielting points resulted from the use of crude 111. ( i )C". I). Kenitdewi and 11. .\warn, A c n i i . K e p . P o p u l u r p Kornine, S f i s i l i i Cerrrfriri Chim., 4 , 5i flY.56j. (8) .\. Barnes and R. \V.Faessinger, .I. O r g . C h m . , 26, 4544 (lY61). (9) C. Courtot and R . Geoffroy, C o m p t . Rend., 178,2259 (lY24). (10) Line a , stomach carcinoma originally obtained from the animal a u p i ~ l yand researcli units of t h e British Empire Cancer Campaign.
New Alkylating Agents D e r i v e d from Uiaziridine' Cs I B I Sz L X T L I , Z. F. CHMIELEWICZ, IUD T. J. B L R D O S ~
Depm tmenl of J f e d m n a l Chemzstry, School of Phnrmacy, State CnTLeiszty of A\7ew170ik at H,cffalo, Buffalo, ,Yew Y o l k 14214 RecezLed July 11, 1966
Several of the best known biological ;ill;ylating ageiith that were found effective as antineoplastic drugs3 and/or insect chei~iosterilants~ contain aziridine (ethylenimine) rings as their reactive functional groups. I n this laboratory there has been particular interest in the syntheRis and study of a series of ring-substituted bis(1-aziridiny1)phosphinylurethaiiq ("dual antagonists"); as well as such other ring-substituted aziridine derivatives6 that might be capable of undergoing Sx1type reactions under biological conditions.7 Diaziridiries (I) were shown recently to undergo acid hydrolysis by an Sx1 mechanisms which must involve ring opening with the formation of a carboriiuin ion (11). It wah thought that diaziridiiics might act as
biological alkylating agents if their ring-opening reaction is sufficiently 'iactivatedfJby electroIi-attracting Ry substituents similar to those that were proved effective in the case of the aziridiries (C=O, P+O, P-4, etc.). It seemed, therefore, of interest to prepare some diaziridinyl analogs of the ring-substituted bis(1-aziridinyl)phosphinylurethanj and tri-(1-aziridi1iyl)phosphine oxide3 series of known chemotherapeutic agents. While on the basis of analogy6 arid rationale,' the corresponding derivatives of 3,3-dialkyldiaziridines would seem to be the most desirable ones; unfortunately, such diaziridine rings (derived from ketones) are known to be unstable toward acylating reagent~.g>~O
~
(1) This investigation was supported by Grant C.3-06695 from t h e National Cancer Institute, U. S.Public Health Service, Hethesda, Sld. (2) T o whom inquiries should be directed. (3) See, e . g . , D. A . Karnofsky and F. Bergel in "Chemotherapy of Can1964, p p cer," P. L. Plattner, Ed., Elsevier Publishing Co., Xew l-ork, N. I-., 3-18 and 21-31. (4) A . B. Rorkorec, Science, 137, 1034 (1962). K. Barua, Z . F. Chmielewicz, G . E . Crevar, J. P . Dailey. S.Divald, and Z . 13. Papanastassiou, J . I'hurm. Sci.. 64, 187 (1965); (b) T. J. Bardos, 2. I?. Chmielewicz, and C. K . h-avada, thad., 64, 399 (1965). (6) 2. F. Chmielexicz, T. J . Bardos, h. Sepaloff, h. SIunson, and J. L. Ambrus, Pror. Am. Assoc. Cuncer Res., Denver, Colo., M a y , 1966, 7, 14 (1966). (7) (a) T. J. Bardos, Biorliern. P h w i n , i m / . , 11, 256 (1962); (hl T . .I. Hartlos, N. Ilatta-Guptn, 1'. Ild>lmrn, and 1). .I. Trixrle, .I. .Ilvd. C t i r ~ n ~8.,. 167 (1965). ( 8 ) C. Szantay and E . dcliinitz, Chem. Ber., 96, l i s 9 (1462). (9) E. Schmitz. in A d l a n . Heterocyclic Clirm., 2 , 83 (1963). (10) H. J. Abendroth, A n g e u ~ .Chern., 73, Si (lYS1).
