September 1966
TUMOR-LOCALIZING AGENTS
pressure of 2.8 Irg/cm2 (40 psi). The pressure drop after 90 min indicated that the reduction was 92% complete. Fresh catalyst (0.25 g ) was added and shaking under hydrogen was continued for 15 min with no additional hydrogen absorption. The mixture was filtered by suction under nitrogen and the pale yellow filtrate, which started to turn dark immediately, was acidified with dry HC1. The precipitated amine hydrochloride was filtered by suction under nitrogen and immediately dried under vacuum. The green-gray powdery product (9.4 g, 81%) was very hygroscopic and on exposure to air turned instantly to a black t a r ; it was used in the subsequent preparation without further purification. Sodium nitrite (1.4 g, 0.02 mole) in 3 ml of water was added dropwise during 10 min to a stirred and cooled (0-5") solution of 7.8 g (0.02 mole) of crude X,Ku'-bis(2-methanesulfonoxyethyl)p-phenylenediamine hydrochloride in 11 ml of fluoroboric acid (48-50Yc). The black tarry mixture was stirred an additional 10 min a t 5" and the water layer was decanted from the sticky
Tumor Localizing Agents.
729
tar. The tar was dissolved in 60 ml of acetonitrile, stirred with decolorizing charcoal, and filtered, and ether was added to the filtrate (20 ml) until crystallization srarted. The mixture was cooled a t -15" for 70 hr and then filtered by buction to give 3.5 g of brownish green crystals, mp 135' dec. The product was recrystallized twice from the Fame solvent mixture (with charcoal treatment) to give 1.7 g (20%) of dull yellow crystals: mp 135' dec; pE$eurban 6ud) 3030-2950, 2230, 2180, 1590, 1520, 1350, 1175 cm-'. Anal. Calcd for C12H~YBF~?J~O&: C, 31.94; H, 4.02; N, 0.31. Found: C, 31.43; H, 4.08; N, 9.56. 07
Acknowledgment.-We t'hank Dr. Wilson 31. Whaley for his contributions and encouragement in the initial phases of our program, Mrs. Frances Potts Fernandes for t'echnical assistance, and Dr. G. Richard Handrick for helpful discussions.
Radioactive Iodofluorenaminesulfonic Acids'
KRISHNAC. AGRAWAL AND FRANCIS E. RAY Pharmaceutical Chemistry Research Laboratory, University of Florida, Gainesuille, Florida Received April I S , 1986 T n o compounds, sodium ru'-2-(3-iodofluoreriyl)acetamido-7-sulfonate-l~11 (TI) and N,N'-2,7-(3-iodofluorenylene)bisacetamido-6-s~lfonic-~~~I acid (XI), were studied for distribution of radioactivity in tumor-bearing mice. The results clearly indicate that both compounds tend to localize in tumor tissue to a greater extent than in many organs or muscle. Compound VI gave the best ratios (concentration in tumor/concentration in tissue) a t the end of 8 hr after injection. I t showed a statistically significant difference a t 0.01 level of probability with liver, kidney, stomach, muscle, and blood, and with spleen a t 0.05 level of probability.
For some years, work in these laboratories has been aimed a t finding a compound that would display preferential affinity for tumor tissue; if this compound were made radioactive, it could be used in the diagnosis I and therapy of internal cancer. Our previous studies have shown that certain 35S-labeled derivatives of fluorenesulfonic acids have an affinity for the tumor tissues2 But these compounds did not always give a favorable ratio of uptake of 35S-labeledcompound by tumor to other organs, especially liver and kidney. concentration to be most suitable for further investigaRecently we have elaborated3 the fluorenesulfonic acid tion. molecule by introducing an additional moiety, the basic 3jS-labeled compounds, while useful for animal examino group, to facilitate the protein binding of the perimentation, have the disadvantages associated with compound. These new flu~renaminesulfonic-~~S acids low-energy 6 emission (0.168 mev) for clinical use. localized in tumor to a greater extent than in vital It was, therefore, felt that the labeling of these potenorgans such as kidney, liver, and spleen. One of these tially interesting compounds with I3lI (a y and p compounds, sodium N-2-fluorenylacetamido-7-sulfonate emitter) might combine ease of detection and estimation (I), gave a favorable ratio (concentration in tumor/ with even a possible therapeutic dose of radiation concentration in the tissue) with liver after 16 hr. derived from the compound itself since 1311-iodide is The favorable ratios with kidney, spleen, and blood used in the therapy of thyroid carcinoma. The use of increased with increased time. This indicated that the a y-emitting isotope could make possible the visualizaconipound is eliminated less readily from the tumor tion of tumor tissue by photoscanning. Our attention tissue than from the vital organs: it shows the affinity was focused on the iodination of fluorenaminesulfonic of the substance for the tumor tissue. The second coniacids because the fluorenaniine molecule has been found pound, r\',S'-2,7-fluorenylenebisacetaniido-3-sulfonateto retain iodine despite metabolic processes.s This 35S(11),gave better ratios of about 4.0 or more at the should be a great advantage over such compounds as end of 8 hr with the vital organs. These compounds tetrasodium 2-methyl-3-halo-1 :4-naphthohydroquinone have shown sufficient relative and absolute tumor tissue diphosphate in which the substituted halogen atom at the 3 position was quickly removed from the hydro(1) Thls investigation was supported b y U. S . Public Health Service G r a n t C.4 08186 from t h e National Cancer Institute. quinone ring.6 In the present work, therefore, we ( 2 ) (a) M. F. Argus, Brzt. J . Cancer, 7 , 273 (1953); (b) &I. F. Argus a n d K. Hewson, %bad., 8 , 698 (1964). (3) F. E. R a y a n d K. C. Agrawal, Cancer Res., i n press. (4) H. hI. Dyer a n d H. P. hIorris, J . S a t l . CancerInsl., 17, 6 7 7 (1956).
( 5 ) H. hI. Dyer, ihzd.. 16, 11 (195s). (6) D. H. hIarrian a n d D. R. AIaxwell, Brit. J . Cancer, 10, 739 (1956).
2
\
2. Ac,O
Fuming
-
-
CH CONH w 2 H C O C H 3 C H 3 C O N H w N H C O C H 3 H03S
A I
liavct synthesized the iodinated fluoreriairiiriesulfoiiic acids using 1311. It could be that the addition of iodine atom (at. wt 131) to t'he molecule might, alter the propcrt'ies of the parent compound. Therefore, biological st,udies of t,hese compounds were undertaken. Chemistry.--Sodium S-2- (3-iodofluoreny1)acetamido-7-sulfonate (VI) was prepared by sulfonation of S2-(3-iodofluorenyl)acetamide (V,see Scheme I). Direct iodination of S-2-fluorenylacet~amido-7-sulfoni~:acid (I) was unsuccessful. The synthesis of the intermediate V was achieved by t'he mercuration of 2-fluorenamine (111) to give a 2-amino-3-acetoxymercurifluorenc complex. ' &etylation of this product with acet.ic mhydride a t room temperaturc yielded 2-acetyl:~niirio-3-acetoxyrnercurifluorene (IV). Replacement of the acetoxymercuri group by iodine (I3lI) gave the intermediate V. Direct iodination of 2-fluorenamine produces a 7-iodo derivative. but mercuration has been shown to occur a t the 3 positioii.' S,X'-2,7-(3-Iodofluorenylene) bisacetai~iido-(i-sulforiic acid (XI) was obtained by sulfonation of S,S'-2,7-(3iodofluoreiiy1ene)bisacetamide (X). ,Ittempts to obtain compound XI by iodination of I1 were unsuccessful. ('ompound X also could not be made by the iodinatiou of VIII. Compound V was, therefore, riitrat'ed with fuming nitric acid to give iX-2-(:3-iodo-7-nitrofluorenyl):icetamide (VII). Hydrogenation in the presence of platinum arid by hydrazine hydra,t,e in presence of cxtalytic Itaney nickel or palladized charcoal dehalogenated the molecule to give S-2-(7-aminofluorenyl):wtaniide. The latter compound on acetylation gave S,IZ"-2,7-fluorenylenebisacetamide (VIII). This reaction gave proof that nitratioii in IV t'ook place a t the i position. Reduction with s t m n o u s chloride arid ,
t i l ) E. 11. Hiintress. I different tinic iiitervalb following intraprritoncal injection of 17 arid XI are givc~ni n Tables I aiid TI, respectively. Thc ratioc of cotweritration of the conipound in tunior t o the c*oncentr:itioii in tissue arc also given. Timi intervals b e t w e n the injection of Ilabeled conip0u1idi :ind Idling of the :miinals w w rhoscn to hr the i:me as in the case of noniodinatc~cl ronipouncls so that iiiaxiiiial comparison could be niadc* to determine the optimum molecular size for t uiiioi localization. C"onnpound VI localized in tunior ltIiiio~1 four times as iiiuc1.i :it 8 hr as after 6 hr, while the revc !vas true for liver, liver having four times ti5 iiiuc~li(*on(witration at 6 hr as at 8 hr. The ratio of the coric*ciitration of radioactivity bet 1 1 tumor and liver thc.ti rcmained relatively constant up to 16 hr. Thr ( ~ ~ i c * e i i tratiori of radioactivity decreased i n all the tissues exrcpt the tumor while going from (i to 8 hr. Thus, thr. ratio> which were not favorable at 6 hr improved to ;t great extent at 8 hr. Thiu inchrease i n ratios indicates thr) selec4ve localization of VI i i i the neoplastic tissw. These observationi \%ereoiiiiilar lo those i i i the cabc ol tioriio~linatetlc ~ o ~ ~ i p o u hut r i t l ~the ratios of the iodiriat~l c~onipouritl IWIP hitter. I3y 16 lir, eliniination of t h v i-;ttlio:wtivity a p p c ~ ; r r c ~10l hc iii(~regeneral than d e r t i ~ t ' . T h c h ratio> \ v i t li I\idnry x i t l spleen \\ c~rci i i ( w : L w l :it
TL~.WH-LOCALIZIXG AGENTS
September l O U U
TABLE I TUMOR TISS
C O M P I K I S 0 N O F ‘THE CONCEXTRATION O F R A D I O A c m m Y I N
FOLLOWING ISTRAPERITONE.4L 6 hr
7 -
‘rissue
THE
CONCENTR.ITION IN O T H E ITISSUES ~
I N J E C T 1 0 3 O F S O D I C M ~ - 2 - ( 3 - I O D O F L U O R E ~ Y L ) . ~ C E T A M I D O - 7 - S L . L ~ E - 1 3 1 1( V I ) 8 hr 16 )lr
-
Rb
Ca
o
731
-----
___-C‘L
LC
€25
---
--___
tC
C“
Rb
tC
‘r!unor 25.9 04.7 27.1 0.40
