NEWJ COMPOULN]DS FOR CANCER. RESEARCH.
THE SYNTHESIS AND> METAIBOLIIC STUIDYI
OF ISOTOPIC DERIVATIVES OF FiLUORENE
MARY FRANCES ARGUS
A DISSERTATION PR~sENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
lA Ir his1 kltYisAsndiaBar t adams $be~O I
eahrtb stehes t eques her armaLttede to ar. Psast
TABLEg OF CONTENTS
ACK(NE0KLEDENT.~ ......................~r..************* 1 1
LIS OF TABLES.......................r +rrrs. *************
I. INTRODUICTION. .. r... ..... .. .. r *** ******************* 1
A1. Chemie41 Carrcinogenesia......l........*. ......... 1
B. Chemical Detection and Therapy of Cancr..,....... 10
II. CARCINlOGENIC DERIVATIVES OF 2P-AKIN0FLU0RZEE
A. S~tatenat of the Prl~oblem....... r................ 20
B. Procedure................................... S
Q. ap)8perimenal Detals ................... ... r.... 22
1. 2-Witr'n~rotherene-N .......................... 22
b. 2-Acetyllaminetheren dirchty from
P I8bl8t81C8.******** ***********************
III. TIDEMTAOISME DISTRIBUTION01 MSEXCHETIONW
GP 2p- OLU9~ENESoU~w~LFOMAIDFI0RRS4 N
RAT....*rri.. ,.....~...r+...... .................. 26
A. Stabamen1t of the~ Pro~blem.... ...r ~ c ..*.*........ 26
B.1 Baperimental Detaila....*....................... 27
C. Res~ults........................................ - 33
ET. THE~ DErErIRIOar OF E8Da uM FLucrBEMB kpa
2,7-DIBULFCHATB8 II MICg anranr A
TfRAM8S;PIARTAELBJ sTCOMAC CARIROBlAW............. 1b
A. 84temt et the Frdblem....................... 41
1. Btperimeasa~t~l Dettts................. 45;
1. I~diadum .u~w1aeea-87isalthate-8 ...... L5
2. AaImal ~studie........................ 46
BDR~A~LPHIICAL SEARCH................... ..................6
IJS OF TABLES
I. Metrkibu~on of Radi~oactivityl ta the
aart Polreda 0mit AdmnrE agoiono
a ~~rolUHArArMT EdtWZ~idnianvensm e.e s*** *******e**
II. MartriboUton of Radionstivity in
Tmr~or-bearn MIos Followingl nt~ra
venous Injetion' of Disedium Theuoren-
III. Ilation of ther Concentration of
Badioactivityr in Tmor Tissue to
the Consentration to Obber Tirssues
Pollowin a Sinrle InJection of Labeed
Ooop~md o r~or-br~ci0 1Y ***************,., .* ***
Th atoddent of aancer is intserested in tbs origin, the charao-
teristics, the deveoprmms ad the control et neOPZ1latia gC~row. If
srtudy la liited to ~those ~sars which~ arise rpenaneously, the oppoic
turnity for std is lieewise limited, It io greplat vatlue therefore,
that mthoe~ of itadtiag Samore experimentall in annsmi. hav been
devised. This not only pemib ts a abdyo the pawaeasrr e~t Szefnefm-
tion tem the nomral to the oancerurea tsaae, butb eappies rrcar m ianfr
the~ investigation of di~agnotie aad thelrapeatic agents
A. Chatea Ciaroingensis
It is knLOm that a number of physical, obesical ad biolo~ica~l
agents a capable of intaoing~ cuanrags growth in vAaariou fissues of
rantmal ad plants. These ge~nts are of twe genelral bypes those
wh~ich ~wecnsider foreign to the body~ and eall extarinsa P~agens ad
those predaeed by the o~drgns itelf, which we clss a intfirinet
agents. Crtata~ rspattle eleas of ehemicrlaerl mpunds hav been show
to be estataste ageae eapable of earaer praUotetin
S~evrl earetoaegenia ageabal wer kneanI tem allateal observe-
tion long befor the extension of iavestigatisas be the laborratoy
Perhas th first ws ther demr~iption by Pebt (63)1 in ansland, et scro
$a care~ams in man expsed to coasant~r gooabse with soot. WCith the
development of th eal~ tar industry duriLng t aineteenth century an
iaorease in the nber of skin ~Canoer was noted in worker in this
indusrtry. Thae earninrogsaen ic eot of coa tar was establaised whetn
tamgirwa sa Icohikamn (90) reported, An 1918~, that contin~uous ~paid-
ing of rabbita ears with tar led to the appearance of earninama.,
The anaoeeasta search for the pecifie constituentt~~~~~ttttt~ active in
ana tar nas achieved b a gronp of Lon onsientists under te lead~e -
abi'p oIReaaf Kenwy(36) Pad Cook (17). The active inredient as found
tor be 3,t-~b~anpyram. As a mtter of fact, th first earciangenie
polyoyaelie rrdrocarbon to1 be described ras 1,2,5;,hribeasanthracene
reported byr Clar (10~). Further modifications of the boeaanthracoen
anoleas led to8 the synthsis Pand biological testing of mnmronoe re-
lated compounds. Particular interest was areased when one of the uane
actn ive a the caroaiogenic trgdrocarbons, 20aethy~laholatf~nthrnewas
prepared trer bile saida (16).;
It~ has been sha that these compounds are detaditied and
made noncaroinogenio~ daring mtabolianr byr~ t a~ddM in ofhdroxy1
groapa to specific positions to th moleade3 (12139).
The frequent occrarene etL cancer of th hinaa= A n orkers
to the aniline dp industryT let to the stuidy of various chemiaOls An
this group. Toshida (91-) ta Jarpan foud that both ingestion Pad sub-
cataneous tarrjection of e-eminoasro~talaen resulted to the devlopmnt
of hepatmasa in rate rad mise. IPn 1937 ~inesita (37) reported that
p-dimethplaninoanaba~nsen wh included in the diet of rate prodnest
liver cancer to these anmals.r Since that tiae mayrp compounds of
this cjlJas have been found to be earninogenie and a studly atl~ th
rrals$1m o fw 4bre u Ishe.
sn satuesa harpseams~ to setsas been# eastung savemented b7
..s..n.-eas, I, wnan, r4e me Qam ()19,8,88) observes no sis)
emrpenab, kwh~n Saepor~ ate in the die at m, predagd aliUple
dmesr ebbt Smars, elo~n mthes saggesta that emis Anget im
a&~s 1t Melshob ($)~ rdtst iasyrbr==e atr~ ar~ sata
at 4 ag. pe~~ day~ t a aegettebsrl tsat se oat tat,
alasrPrdea atn easn headset at fea mi teta develop bothII~d
eaiwurreaas mee5 twu askqo teums liver earetaems,
Bielsehowaky reporbed that 2-eaety~la~miothoeene wa inreffoo-
thes when injeted suboutansoaljy. He believed that such tiess ews
not; capable atI bgdrolising off the acetyll gra to form 2 sinfinorrne
which he considered to be the active comppounrd. Cortirnedl painrting et
epithelial tisame with a-em~sinetheren resulted Sa liver Samore to all
et five rats tested. These won experiments cannot be compared di~dretly
however, sines the mode at application, nUamlyr sudbontaneous and oufan-
coas,1 dif~fered fr each composed.
In a recent study~L Marris, DhniL cand johnson (53) have shownr
that both Osiainoflorren sa 2-acetylamina~o1soree are capable of
producing tumore in a varietyr of sites after ingestion sa after paint-
ing. Sevn eat of eleven Ipts devloped Sumor followlagl paintings
with 2-emaionthrne, while fiive ent et elen developed tames follow
ta fusding of~ the san ae opound, Ininting with 2.eaetylamianfluaren
resulted in Smoora in seven out of nine rate, and ora ingestion of
this aompound by tenity reta resulted in Swave developing bumore.
Armstrong and Bonser (3) abudied the effect of feeding 8-acety-
aminthra~+tarne b five strains of atnee nd tenard that this created to
th production of Sumers As the Mnadam, liver, breast, forestansch,
thyrid, rnaol pelvis ea teal~e genital trad.,
Xlopes (b1), a ~angland, has reporbed the induction t ar brain
Samor in a rat by including 2reatylaminethereas~ in the diet. This
is ulln queai nee milnan t Samrs ofnre tissu are11 usull U prO*
duced in aies and rets by direct taplanrtation of a oerninogenic hydro-
asbee kar dr tr ala sat assel (ap). bd hiur relts agn~a show
wdesrpreat teetdme et Smr0 g~rowh to varis a~rgans wlashal th
somewatalry, mmars paraagge sa yd tLLgan~Lrlds mAth liver.
Thesel sampesu evidmas a the Mgh earetagemt solivity
emnubctln by 8.eestysremnseme Aswe le t extnsi~Ive~ stuyo this
sampena to detam as ast steaes a ~ steae a the 1 mIsels
reponsthl dr itra sette
Ir12ar, al~ls, acadlase wombr (la) asve mad seeLes to
detamta it the -We b brb g to M*** =<=Hn= AsL essemb~la
to~ ~ ~ ~ ~~~~~~b te eiese sivt aasws r b-ata r~u Ieemsest s
seplata the -O~g by 4-B es Sa, e....p..e.nes...nqu.. 484~ ae
asrlttttttttttttttttttt th urdse....use o e.w ..1...1 ibr .sa., .....r sae
ear dash 4tsame. Bababian of .g. ier the *Oh- a in 3-se~tat-
..e..lma...shrg..r- & ageaL Essag asQ ~ acivt teamsts)~1
thes twe Stasae, wdhryle~q Aseri a saI -0- brdg as ).ae~Stat
ImammaJhammanwa u g1 paiUrrtl lray r_ asasue the asiitt the male-l
agrs to these awrpetr. Salth the eunua empeant $=uetlaime
1semapn, horrwner pna ait H U@se seapenda had aar eredmgual
sativiy temrd the live. The smrs pmaQn.4 by al thes empetlads
appeJ asetfor to elabb maths aitte Meb balealagt at as, emeImab.
LIll- ea Ipimiabclphei a deskative~ Sa wh tb S -Qge
Isese to~r+ aheems, preeent amr sa~ as merC rads, earf east
lirwr me vrsese sm ab orr man r me. W them~ttttt~~~~ttta rrsers aperse w
Sttttttttttttttttto see b mones te a bestmsa or tie empalrtm
I[n 19148 Pinek (61) postalated that 2,2 -dirantylm~ino-9,9**r
bif~~loorludne II, is the essential intemeR1diate in earningemaIs
A~lthr considerable dificuoltr ~this empoung an~d i-ts rednobion product,
2,28-rldercCaisootylina.,9bitnuorgy1, IIII were pr~epred (83). Morris"
and Dubnik (51) tested thses derivativesr on rauts in f eedingq experiments
exc~eedng periods of 30 weks. They found 2,2 **dicetyliamin-9, '-
bithuory1 to be ineffotie while 2,2 *.diaoebylamino=9,9**biflooryrlie
done had very litt~a sBtivity. Thes facts east doubt on th posa1-
bility at the conmpunds betag intermediate.
HCoCHN NHCOCM, *CO CHN? N~HCOCH,
The ony metabolit~ of 2..acetylamiunothre that be been re-
ported is 7-M-esp~aeylamianeo~naen (6). This oaspound is only
weakly carehno(Cento. Th qrustbian ariser wheter~ the ?**positon anat
'be avaiable for hydrampat in order folr the compound to clerb
powerrful. crarinogento sactionr I an attemp8 to answr~ this question
46* Torposition of 2.a~Ceflraintantherene has been blooked witfh chlorine
and todine (67, 82), pareimia~ry report by Marcri (So) iadisases
that these eaapounds are not oarcinogento, althouh rserarl brain ab-
seesses were found when 7iode.2-acetylLmneflooone wasu fed. In
light et Blsabougbyssl ask thi Wald Sa~teat that as sett
apaqran Is agemseasy ta Metlp~astaes Who laterpenitim at agg
berame a agdream at the mateam, a Sa T..twa ~3re-mn mine
1raemem, sadness beh doe not dsrtroy asw e farelassnttt as em-
penaAd (3). In eres todtwamA a ttrages1 ~i aoolld kbe laepse
wash sustar iresu~s t,7-d~aolpamman-sea ha be prepared (YI)
sat Saital tests SaMen ts 15 A aOL meeaciv easetang tha
As assr peas 14 sheel bea m tes~ SMS lasts saad sentake
(71,79) hav reporkbted ts$ashishloretlraer, sad 2,74ip~ri~r
eas embibt a greter reseithit a the ast lea greap the 1se9ase
itself Famr abia RS~ walA s probablytas the Satandaebtea a a
halse late the Tystrta at 3.ee~j-~agmnrfluerns 3d rlals sea
nv te as~r at LraneSp aitbb M~ epseat mat i asaaaaaaaaaaaaaaaaaaa sativit of Me
amaplans gorep is Me al tapenat tser to He earr~reaegnttt a
sta appenseless, )hn T-shole- ad Tteae-aw~lreanunsr---
shea~ld sho greater nl~b or eaeteenssai than~ r 8.nesi nu.4m mma.
Stam th ts to ae the eas iS As rrr~ tabaet t Mask has eystakdr
an importaa 1pela As anginttag th evidentU rly ..aa so et h
In e id**Aag the~~ ~ pea hla tesimr ef th seeby greap la
a-lecs ra-a-a-wea., wel qurfiam airse same sennver bem to
tb ree mtet a assessary st Sa 69mor
m"an th the p~iaren aperweawrb, flueome, sat Ma1 m~st pbredst
lnramoremns, as samense (ap)I wast a-alssensasome i suete~ b be
eakly~J careinogesnto wh fed (53). Hetre ages is to eidens tas the
madem of application is not unimlporitat ainee six ea of te rata
developed cancr from painting with 2-nitzwfloosene Mile t1~wout of
nine sets developed tumorei after ingestian of the compond. 2-kLnino-
floorane o h ighl activel although maybe asomewha less than 2-ace~ty-
wamfine orane (528,53,89), bu this may be dnu to He different solu-
gMrs (31) has prerand N0-(2-f~ol~O)J~rerl),n rand >e(2-thr-
enygl)-heminrisuveamed in hope that their greater slubility to wa~tertttttttt~~~~~~~~
over 2-mianthebor~renewold prodceaw mre16 rapid caroinogento response.
Tests of these compoundr Ireported ~byJ huabl (80) abow the compoundab
to be eareinogening) but the relativelyr large raounts adinistered
i~e. dr ag. per da fo 288 daysr, do not lend themselves to any direct
comparison witbh stallar experiments involvng 24minetherene
Btelehowaky (fs) has reporbed that teethyleminnethosn rand
2-dimethyl~aminefloozwne are less earnnogeniea $bOan2minetheorene.
A photometrio mesthod et estimation of 2-~eaetJyaminetherene
in the $1sames sad xasrets of anmals~~~~~~~~~~~~~~~~ adenh~Ctered this compound has
been used byr wett~ll and Morrs (8Q6). Their procedure tavelesr
hydrolyisis o thef treamine, dissotisation, sa coupliag with B alt
to give a colored compound. a this methBod, however, onrly~ 32 of the
ingested dose of 2-eatylriamnuofluren is aoeounted for (55), snartes-
ing tha in the anias1l lebodymtablie are formed which involve re-
moval of themne grM oup or which tie upp the nitrogen in awah a man-
aer that it cannot be hydrolisd to) a primary aine.u Deacetyliatio
waMl be a p~rrese Ath cenMi be assmet to paesdsr the iautogmr a
cmaneru~rr stuaeshS hlav bam man onl tee udiee~
sad 4eayraema-mes1e (Q), abs Ma$l Meykb~ a
P*J-baIMr-sy-rrr doe Sa plae As the mS (%,4). IAs per ea
resplatry 001 ntWba 6 hows, whil ea 40 perII me la esend Sa
A-~~hennempeda namma gag alrrumnandfamaidaftwawmm- g, Egp
seeme **) ****r are to r..asy rmuse iram awmuls ansmes
gI gg, whll Obr haw51 Crp As rrmewe w$M dttlema5r. go em
same ss amu to smove asr sea (ascapapon) g~asp. It wa p~e-
ateam as i envemrri or as ra ... is aI .. ***=@7 su onP
eartagetef t of thes fsse derivaLves at Er i-@==aP waM
very dist~ly with the eas at amueal at theseCCC~~~~CCCC~~~~CCC vasees geps.
rr~ so CH
in haranny with these predictions Mobrria (50) reports that
prelimnnary experlasnts indicate that the 2**benso#l ompound to con-
siderarbly less sobive than 2-eaetylaminoflonaera whle the tosyl
derivative la noncrccrinogenia.
Th pictures la still ineomplete and furrther abd of the
carcinogenio sechanisms of theorene derivatives to necessary At th
important mole of these ompounda la chemical earninogenesis is to
Ib. Chem~oalP Detection sa thcrapy of Canoer
The need for ar method of early diagnosis and treatment of
internal cancer is aes urgent today to the case of gastric earlninama
Cancer of the stacach, a disease predominating in the majle in a ratio
of about 2 to4 ~to the most eama of all cUarinamas in the h1uman,
amusling almoat lC0I000 deaths every yea in the United States and
mirrm u3p appr~oxmatelyT one-tounrth of all the death due to caneer (1).
Two msthods that have been used meat Wnocesatull to the
trea~tme~8nt of malignant neoplasms are surgery and radjliation in gastrie
earciness, however, these methods find only limited efsectiveness since
their anecess depends primerl~y on diagnares of the disease in th
early stages prior to the developmsfent ofetastases. Cancer of the
stPoch is ar ** ilent ** disease which usually does ae display alram*
Aug apptoms mat well ardvanced. ICn fact, as symptoms rse worded
to 25-30 per cent at the antopeled persrons dying of this disease (36),
a ofac whih make earl diagosi almost taposible. On the other
home, asrsrlle eareamm lne ~a nsem to assetastes easy saals
rital oalrrgn at Las be< asb as Ip modes, Iliww, leag, she ES As
alddesh tha r alple mthed te~ r tedagemel~s et easy gast~ris eman
asutwesea saw rass ar b as mus** r onessel assoder sa semesal
seeb rare nsnwoesses, Uraim r eemnta ass n rea'twly expnsie Thi
ansate ageles ther earl b see. ~L T~II~
ras~ a=eeps o~k r ses to b sreathe asta k mns or
weassu or eal~rneem sat b Cer o g deea as war seem kSary
Z~It rsa~le seald be heads et m ada~alat~ b Sma
beets, weld 1sel~r s with a certata dereer at eaesit Sa as
waitpen agepleau, sat it sedissetiv at availtbr kA be Sasseeatal
Aate eagh meerias, the aidsh we be useta An~ a despeakr saA
eat~tea avanale~ to th~ mak~ a presea whicha wedd adapt ~thmealv
well to as) pbnan o we sasses s a sesse seesW or assame. alesh
and av (8) pens eek, beenew, as tbss em sher~raag Peepat a
bbet miters wrl4 probatty measesitate theCrJl~ deig at a atd toP
tha this seald b so....ps and by tastatea a pless at pheteggphle
12a or ai sm1 gag~gnl set As plc Iriml2 Pat rb bashe etU a
be introdneed into the stomach and the radioactivity mesasured. This
incolrnvenienc cold be r im~nated if the shor6-ang, bet a emibbrs
wer reosltritd to therapeutic ueseand the long-range, gammaP-zay
emit~ters employed for diagnostics work.
TPhere a~re a onsiderable nulmber of know instancses here
elements localise in oarbtain organsr and tissues writh a high degree
of selectivity. Ou~ntaaing examples ane the deposition of calaim
and phosphorus in bone aA She uptakce of iodine by the thyroid.
Studies on the use~ of rdiophosphorrs in tebr reatment of
leueakia and lymsphosarooma have been carried out. Unfortunately~ bone
takes up larger auronts of th eleament than do the malignant cells
(36). selverstone eg rX (70) recently rwporbedi on the deposition of
P in brain tlmora, It wa necessary in this csee to devise a
minialdre (kiger counter that oould be inserted dizechlyr into brailn
tirasm, because the attactiuive sam of this baet emitter ia only a
fe sentimbers. Good differentials between normal and pathological
areas were obtrained ~Locairsation of P in samaryv and test~ieular
tuaprs ha rrals beern ported (69).
adiLolodine has been used extensively to the investigation
of the thyroid phfreiolog~. HAmiltonb and Soley (27) have assaured
the nptake of radiootdine by the tttttttttttttttttttthoi A patients by placing a
Geiger-Maeller counting tube against the neak~, directly over the faCtth
mas of the gland. Steven st et al. 7) hav~estudied the distribution of
i in nioe with and without mamarynP enswincam, 150ple, after Angeo-
tion of radioactiveo sodium iodide. DLiferenees t the distribution of
taJ pwraredm eprtm~l ass sta we (Tt7), 45temases Sa @alw em
ta~est at am Ppseses wer rs brlomer~ ratate to ea~r strterease t
nalism a bomnrea(ka elmt a bemk rlted~e4 antnsru
by a~d~sy gb g. (t0,t1,82). IS As~ measrsiteml tWake rp by beas
amese, ha Cbe esrasseem an enmeu passasituesu ms t be
in amous as erasate& emsue r iUr kno w to eeitm Use a asshb
ar tases, seek as sediar assr;ume~aphmmipharee sett 3-tate
LlrnaLbrl merposess ee grate (fepam), cad setir wham..ahan1u~-
sasat (rahattl 8kLadm).
Inr attemptingf~f t develop a tes tCO to~ eagg Hdosymn.4 a
gasri emeruo gRee sat asselate () a~aleJtttttttttttttttttttt~ a ree .1@...*4
(sa~bulan smiosdr satcsaat sal+ltappld). Wslel aeMY a these
wastond appliarbe to Me em~ aty deestaa gastr~ ==14====****
test et pairmbs ada aegl asrtsl se~ldity
Althoughb as lsealwsatt a the 4.minalf stenal In mitp
masL Masam As hoem to be lawalved, basp (10) reportd the
sensiass oikmr men to~ irsemsnewsa as sensasseeau o sense
sat speate dose a senat saate aae maatgsatr
such as t4 '-di~amindipnhagylaulfoni~t d and ltangy aultailic acid.
Wilth furrther investilgatiton, complete inhibition of growth of spon-~
taneusJ tumre la four of four mice was obtained by Yethylene Eles,
6r,6 5-d~gia~~b~midphaltaided cad tWhe~u~aadiphmaphaw ether (11).
Stevens et 1. (76) have rported a hish local conoontratlon
in caoers tiseme of atiappraisin amnrkistered at al aitt distantl
te the t~oer and tolowerd by reeated soose~ in5ections. 01soos
increase the laotio sold femation byr the cancer Stasse and the
su~ltarpy~rasin w preeipitated pisreamaly because it is less solable
at sold pI Shan at pa 7.L.
Severl charaoterbistic of the sulonamide llnkae~ p~rompted
Epay and Softe (66) t, co~ntimetttt~~~~~~tttttt inve~istigato of these compounds for
their di~ama~t~ie and therrapeati potentttt~~~~~ttttttialt ies. Appuently the
saltonramide linkage A not a~ttaked ly enayme. In daddtion to l~oa-
ising in Oertain tissues srolanamide hv been abomn t redese th
efotive vitamin intaker etr anima by supmressin the intesrtinal
fle, and this rednetion1 of essenP~tial vtamIn in thetttttttt~~~~~~~~~ 45stb of tumor-
bearing atoe is kmwn to ihibit the gnrrowth aiofumo (10), Maad
Soffer proposed the ~ino~rporatio of Sa hto thee lreadyL promisi
comlponda. TIrhis aborbrrange bet a hitter wou;ld no~t e ayexrt porssble
additionalP Sharapentio effoots, bart weald also offer a ~mea of denteo-
Stan Procedures for the preparartion of mnumrona compounds of this
type ar ie, bu as yt no anim1 eperimn tsm a ha been~ earieid eat
Ex~Ltensiv inv~etstigat~io hav been asd conceringE th possibe
seletiv loclistion of certain dtpes in Saro tissue. Reesea (60e)
and latrfC Bblater (7) rreprted a bePAstita attSt at 79Isara EXL\n
on eafrtia ta$ore. Ther dea a dje reQuired howeware prWetd to be
rather teme cand the treatment aewerely stained He~ skn et~ a
As ***str a 1peI, nau....a (as) meese au eares wasr
see toe k Pwannocess by swers. Theser risassag hav bee eob
times ~ somro ownra saaastqtee (sO~a,1ss).
Zha 1939, Dpumlta-Splds (t)) reportd estC T=.18th (base
Pur) and sabrr peedy dittashle d(pass iagete intufnessly
lesalrr Iked~ aikw r Sa peatnseras eat 45maptabnt o tess gr
sas inr ma, treans ad ensues. arousbase scans ass1 aLa
aSewarte no nassag or Passmapen sa rrwa sa wn ns ~b earrsa
bersIprem tasseed seaeem sat earr eme cand furher~ anteded a
eteam t essapslagr thatn l P4U sailOeth 1sealIsto at
as a~ re emem sa nhrot as a or arr sale o huma sweets pe.
senes resr o ~r -14-* asspism (16).
meeman g gI, sa 1911, ~~pasupe to see seen ask nse
IseUleasrul sh aOP tmOr a a veh l forT ambatease that sa
peassue assapeans agems tr the entr me sma (at). The pe**
paret getsaiwPasst empeads what wrer mistM- t b h14ahly eme
seestl As the estemsw or assal emers. assaunately a
methdsr at propeaatte sat emesd te~amla at the empa- were as
separte ud, ea the l work assa been saRbetmatUtd,
ashl et gOeper (8,93) Iangete fliateu sale at washes
4ee seena s aP~pen a, treasss av 1ee 6a sat easmsate son
into tumor-bearing aise. These compoudnd we to be need in the
therapy of cancer in arnnjuntion width alow neutrons, since th ire-
pacb of such a neut;ron on a Ilithirm rtoam leads to a nuclear reactionr
with a high energy release. The lithiun salts of the (yes wenre sup-r
posed to localise selectively to the tumrore, thereby prodnetaig a
high conoontration of lithima in the region wlhaze a madinam erdiation
effect wa desired. As might have been expected, however, the lithe*
to salts of these dyes dissociated frapidly~ i ~the body and thae
litGhiurm ions did not remain with the dye moiety.
The first synthesis of a radioactive dye was reported by
Tobin and mbor (79). They prepared zedioactive ditbramo-Trytpan B15*
and dibrame-Eva na Sh braelasting o~tolidine with Br~U and cou
ling the resulting comapound wilth H acid or Chicage acid. An inveosti-
gation of the distribntian of these dyee in tmo~Pr atoe, however,
showed that th liver took up ab twices as much radioactivity as
thstue mr. WAine these findings east same doubt on the selectivity
of dye uptake by SamoreLI more and hi assooiates point outs that
* th significant fact raeminst that $be collaid permeaees Into and
therefore radiates turmor tissue wherever th tissue may Vbe, and no
matter how widesprsed the metastases are n (47).
The work of Wbor eand Tobin showed clearly tha determination
of radioactdite compounda la tissues with Geiger counters is both
siapler and more emobt than the chemical or colorimetrie analyrrsi
which had previously been empsloyed in de~teminingy the localisation
of dyes. Eleah and Ray (8) point out that most obsserers newly
estimated the dita bLaten e the j~ Ope tor asespLe sat Meoreseep
~laspeea~i r@iel rweltal in may Isers~peagles Greety as pree-
ease at de cran be sa mee seatt4 Sa lig~r~h r ea~ret Stses
ergus rrsee a ksasy eates amsd emer Stases than A dask argan
e8a@ a livr o r p3sea. Lthedr the stagesee)Ps haunwar, (pe as
stage Sa th settee'a-eadethe1Aa spate but assel i the Mdmar.
The we at a treese shame rl t****Se tase dii~lthealta sat Pard~s
~as measotnw eame (as ) was seems as sons~ ue s
astslasse, rwe ass neessass rrdsses~ ter M~assentrr wrki ~a
ar@ em be s~aeastead Sak te~ eag ate L~ copeds at a imr 1Seg
Sb eek d Bar (8) n~bresgts ta r Mth a L510-1$2 at 8 depa as
tame spgreattees~ sal propere a aga at sealtdeb derivtie
Thisa Ida me $shmrp ~a tre r Saestgatere As as) t~e
Esrl~lws and e-wbse (bD)> ***$Lag the attest at 1est (pae ,
eatP not seem srdustglrlyq ~attlrm$ empea atI this seri
Sumor-bearing mine receiving thi compound, he was abl to~s determine
the distribution in the animal body. WOhile significant concentrra-
tion of th dye in the tumor was noted, the liver, kidney and spleen
also showed a comparable affinity for th dye.
Yoore (t8) studied the localisa~i~on ofEL fluozesrein in a wide
variety of carcinomas sad earcomas, neing as a criterion of uptake
luorescence under ultraviolet light. Lesions of the central nor
ous system took up the compound with an accuracy ~ofi t ozzler of 90
per cent. The clinical application of this fact wars greatly increased
by incorooration of I'at into the fluorescein molecule thu permitting
measurements to be made outside the skull, quite independent of opera-
tion procedures (L9,*78).
An extensive series of acidic and basic dyee tagged with
radioactive II~ or 8I have been prepared by Wyersrr and are being
investigated for their selective, affinity for various types of trans-
planted and spontaneous mouse tumors (57L58).
Stevens si l C"4. ) hav reently studied the distribution of
radiactive iodinated Tryrpan ElseYI one of th compounds first prepared
by Bloch and pay (8)), This compound ~ws admninis~t~ere intravenously to
mine bearing suboutaneous implants of tumor 15opia. The concentration
of radioactivity in tuo tissue asu several times greater than that
in skeletal muscle or skin but considerably less than in the liver,
spleen or kidneys.
If a compound could be found which would display a greater
attin itor the, tumor than for such vital organs as the liver and
kwdmag abile at the emew Mae monashing the thesble satie at
eenesateattest tar ad su~1 rrseen@Sa matel dIsplapd Tappran
anrs, such~ ra sme nrp weald swaysannss th schemes o seemess
disguest sat tha~gag at gastate se thrreg ~the met f he
ACINgiO~gEIC D]ERIVATIVES OF 2-AMINQFLUagREH
A. Statement et tbe Prohlm
As sa An Part U1s 2-so*SylP~amifloo~rens (AAF) hsbeen
definitely established as an active carcinogen, prodnoing a varietyl
at tumorsa t the anias1bodyI a numerous sitee often tar removed
r the point et ap~ss (5,87,88). Th1es cabracteristics or
the compound ask it tuselh for studying somes of~ the fndsametal
factors rat carcinogenesis.
A1 satisfactory meothod for ite quantitative eatimation bas
th antaajl body to necessary foar eneh a study. A photoamtrio metshod
of estimation has bee previotusy used, whic involves brirolysis of
ALAF to 2 therenuo a s (AF), dissotisation, and coupling with ar kmnon
naphrthol derivative to gv ar colored compound (86). Mr this method,
huowevr, only a 32 per cent recovery of AAF to obtained, indicating
that in a ve abort ae twro*hizzla of the materil no longer has a
dissotisable po~rmay ~anim group (55).
BRecetl, prepartion and investigation of th radioactive
carmingens, IE-acetylaminoothrne-94I aA 2-soetylminefloorene-,
e-C0 I hae greatlyr facilitated and metabolio abadies of AAF, Shaoe
wel over 90 per cen of th rad~driont t may be s~oaounted~~~~0000~~~~00 for As
the animal boy thse compoun~ds brae permitted a study at th pat
way taknby the l~ororane radical and the asety1 grasp of the
nob se t asr miness as.. bror, ss yes s as es
ialad. As, ha bem iradiate bteam a disasw mm ada greep to
prrrrrrs age~ swever~U by Bla mahe astestee theso, either
tha th attegm stem Se a 1s~pagb 858, BAS kAM nweav fam
the Senarrm minetsrl a tha th pirima ~sala attgm has bee
3awdled .....a...e..n. psI~dfy briy betag oebd be a seaseda
a UITarss amta gea. Tlbs seegat peasiblit mY ~ILawlw the
by~P, the sometime a AF~ PPith ppswte sed($)
The Supe~rtmag at ~ Hallag a at d whi welt eass a
decaim to be~ awa et beaus rbthe tse i~ altmtie is evrm
brtel at AAF-r wie~ a 2abala atteg stem Ale emiA etn
Sa he alm %*7 m thresmemodrtae. las asseta I
rrl~~i~ hP~ d itP~I L ~f~~) bl )11~~ i~t~j~ S
audisseth Lam at ateg As amsst* the heav Aaees ur k
anr oisr t ~~~a~eerr-=-a as i n tol posstat to este-
1aerms audle eir whs thetttttttt~~~~~~~~~ arttae ge sapgage the meae,
cheag whteh oedre ASr 4...7 ** e disset*****
The usual asthod of preparation of AAF As the nitration of
therene to 2-nrit3nronnore (NF), redutobon with So ea 78 perr cent
othanol to A (39), rad setyILatim w iatha settandride. Senrage
of the high cost of )8 eampounds, th ordinary methods at nitra-
tion which use 20011300 per cent exsose caneantrated nitric acid
could ae be employed. in ~addiion, the N1 nitrio acid As a dilate
(appraoxiaste2y iM) solabimo, so it wass necessaryT t workP out a satis-
factory method of eliminating mtrer rad of astag equarl molar quanti-
ties of therenel and HI Oa* This wras ooomrplishd by employing
seestic anhydride as a solvent and dehjdrating~ an rand concentra~~tttt edtttt~~~~~
8480 as a dahydrating aget rand catalyst. Th"e felZd based on nitric
raid wlas 78.9 per eens1 ras compard to a alculated 21.6 per centa by
pevourrcrs methods (39).
Since the he intemediates, eF and AF,1 have also been shomn
to be careoinoento (5,53), sap~les oft these eresr isolated 6lr s~tudy.
A pr~oacs wase also woRked ea for a one-step reduction and
sootylartion at N to AAF. TPhis Ipurocedure conserves both ti es sa
rregen~ts, although it does not substantllyr improve the Jrield as
C. Expeurintal Detaill
1.2" irfloan* Flueorene, 1I0.4 sp. (0.063 moles),
we dissolved to acetic anbcr~drid, 131.t al. (1.39 moles), which wa
ass. no saleson as Lrsses lo SSana 86. m (o.efsp sle)
evr~ a period at 1 hour dar~a At@ a t-aperslwo me kept be*
emea 50.40-. fnon, ---*-see skaters adSa 5 ax. (0.*8 anxe),
3ws adde dropeaeat $$to pdbrese wi@ Me star toat~- sa t at
dpae ashed by stirring wina merk eat 1 per em$ aedimp seeat
mail awapwlg me mrlb pota was 15k a asha (3) tones 155-156 I
so yiel as 78.9 per cen
3.3 P. (0.055 males), sad TS Per em4~ ethamel 1101 at. to ste
au ladded a dast, )) p.l GaS%1, 1.1 p. (As 1.5 al. et wate)l
eat habreel, 1 p., me haste to nark sub a sterberaor. gtIl
as stature assan t ranw~P, tbn 2sk asu r>hrb= wn a retrym
Stian t keep S orkl embat waH, Mast Hestw as bened ~
easy ~As a hears, so assto p., as assa aI~ddae ad serimsasi~
seatt~agd fror an additional t hears. H he$ tlrater, Ate~b me re-
mewed by muana a a filter salt cans 111muSant ga pareause to
avElld seatset~ Ith air, wa ~adat to 600 al. et st to paeeipitat
the rtte pee~ms4 Uo alsta posab m 186I Ia Ena 3) Som
157.5r al as gaz as 78. per s.
3. 2AoetyluadnuotherewNl t*Amin~onsol~re- B i g.
(0.027 moles), was dissolved to 8 al. barnseas, and beraed to reths.
Acetie sale~dride, "1 al. (0.04b moles)) was radde dropwise rad the
mixture reflued ge0 ain~tea. Th prodneat prec~ipitar~~~~~~~ttttttte ead oaoling
rad asc reesrpetalised dissolving~ A hot 95E per cent etheanl,
adding arter uatil Surbid, til2triag hob, sad allowingr th prodnot
to orgetallisea the melting point was 191 g1 Parrai-Eoshite ea
19iktitorova (62) reported 186 ) Morris and westall~ (55) save 196( I
the yrield ~wa 90 per cent. As an exrbrnanus nitric sold wass used,
the compound abould contain 62 ato per cent at 8 .l
ii. 8-Acoelrcaolamioflure directly farm 2*nit~rothenrea.
01setal seeble sold, 250 al. (0.03 maoles), 2-frnitrothrene, $ p.
(0.02)I moles), ~En dust, 20 p.o cand alaorool, fE OI* were heated
meaas of a ai~oraur to rethz with vigorrou shaking After hard
ref~lumng te 8 hourea more Sn dust, 10 p,l w added and th alim
turei refluxed an additioaal 2 hou~rs. Actio salkgaride, 10 al.
(0.017 molea), was ardded dropout rand the ratninga continued 1 more
hour. The sa ur as tiltered hot rad thle product precipitatd b
addition at9 the fitrate to 700 al. et arter sad rearystllnIsedd as
described aboves a.p. 190.501 Jied, 66 per cent.
Ai ranaceaul sylnthesis t the carelnogena, 2-nritrofluoremes
2 theonurene, and 2-a~e47flaminoofluoren lrabled with isotople
been reported Qs$ Iarg perQteam at Hew eaireapg, meaused both
as a asd a dsamoCusan assueson pass fbreagh Me i, U1r e
resal~o ala sappeda the theor that a 2sag peaktrom etw the llatte
at the anniange As preams inr the~~~ffffff~~~~~fffff M~sas at te at toa r$mm
that is lae datestshts ty r badissetest~a metho
THE METABOLISI, DISTRIBUTION AN EXCRETION
CF 2-p-CGIDENESULFORMIDOFLU0RENE-6~n~ I THIE RAT
A.+ Sta~ttttttttttttttemet~tt of th Problem
Recently the preparation (6) and investigations ($6,86) of
the radioactive forms, 2-acetylamilnofluorone**9-C ~and 2-acetyl-
aminmofluaoredC have greatly rexteded our kn~owledges of th
metabolism of 2**ac~reby~lamisnetor (AAF) Among the coDBC1Hri0BS
estaiblished is that 6 per cent of the 8 ingested in thse cetyl*
labeled derivative is expelled as respirartory COI within 6 hoars,
while more than Ir0 per cen was found in the breath afe 88 hers
This shows that descectylation of IAAF in the animal bodyr ca take
The obeseration by Morris e6 al., ($2) ta $L-aMino-
fluorane (AF) is also caroinogenie raised the question whether con-
rersion to the tre amine is a necessaryr prelUde to tumor incidence.
in general, the acetyrl group As readily removed from arnastio
ramines. If th acetyl group of ~AAF could be replaced with groulps
hich ar lsess readily hydrolised or not nremovd at all, th study of
such derivatives would throwo lih on the subject. It wa predicted
thatl it conversion to thes free uaata is a necessary~ step, the onl
oinogenicity of such derivativeB roald vary directlyJ with the eas
of remaoal of these group. The bebnsoy1 gru la4 hyddrlised wth
astateelay rne an empes io kano to remove on fiees areap
(ccanace,so). seoomsasty, swen~l~r~n~aso~r- -*2n ene on ampeea n.
..se...sanet--ab wre~ p~rparea
IZ unsJ hada ah we p~lreeesen, arrse (iO) superts was
th 3Eknsofl empom8u ~I enesidesehl les settw than
AilI r, 451. ~on tes derivet is m~oneseansense.~
ane tme~~~~~~~~tttttttttt att~I~ric~atr of aparalsis o as tF Seuap
start te arbsorlpian and meaboia m at this~~ffffff~~~~~fffff derroivh to each an
extent that it is rlrar~u ts~amad ===mar team Me-r mibdy ? To
endvrw to answ this question Sep-tokenesu-urk es ......
(Ta A) as ~admnses b mas sea U eepn knes aI t **r
ardiesetivityj t ear ma sat mrets The wime sat fee wre~ aJ
-=4***1 t debemise it on empounta at waters mtablts absaga
a. supersmata assans1
re~Fassema h~ldaplegas~ad (Hal~mea) stst ass wit
** ****s bear mateb or >So On. are emptlpre. ases (mkp.
195.15p for th Mrst st seems ~rems ap. I16161 Ser the Mi
expe~rmac) rrme a* A ther Ass a a w~ eaem el salat ~ten 0 ag.
SEb A pse al. *(1. Malatstmaste was by stomeh tabe.~~ aseat
was tastatt is ouse prie t sesiig Ral. de at the TS AP.b 8
oeasant oil sol~hiabe satl the penettest mahatte tes (Perasw Ba
bOwn) through the w...4.ne.* e tbs emper~mm4 Th analsr we
almlet meer at all SUme.
Following tnretmalt, each rat was~ placed in an espeelalp
construeted eage which faslitated thwe separaion lad colletiono of
urine rad feees~ sramples. The floor of the ager mes hartre~ cloth
(4m, assh) 'belwwhc wdas ar fine-meh sreen tray( for the collection
of teces. Under this wass ar nnel through whe the artae wass col-
leoted into a graduated containr. The the--eakh tbray was replaced
rther each defrootion to prevet the wrine tzam coming tnocontact
with the fsooes.
The anal~ were ke~pt in an air-conditioned room at go P
oept toa the onee of the third run where utnavoidbPPle soaditions
necess~ita~~~~~tttttt edt~t keeping th animal at a roca ~temperature ofC rabout 27 .
Feaea samplesr we weighed imedia~tey ~following *011***
Sta sa than air dried.
Inl th first run arine at feees saples ea~llectedffff~~~~ffff~~~ fo r
~51 hour period wresr stadio. The antal was dep~ived a t food at
8) raa and the TS AP solution adinisteard at 8 aa the following8
day., The animal wa uner~ observat~Ion betweenl the firPt rad twert~h
hours, tbe Swnmtytearth sad thirty-sixth hours, ta the fot
eighth rad fiittetourth heaurs, dartrag whih intenrals ine rand
fooes wren meaurredl AmeIatelyt on emtar1an. Collective samples
for the 12 erw periods be~ee the twlltsh an twedefear~ h houmrs
rad the thi8pr~lty-x rand forby**ighth hour were made.
The socnd ran lad1 was tnreted at 8 pg. rand adaen sa
tooe sraples wrer collected as described above, but for th lit
howr periods alternate to those of -the fis run. At the s~izty
evi~ty~ epeast.I The~ enr as exose, the Latt rrentrtP me Mit
wthb a sebsseway satSh hen bw eC allowed t pmp So hi setlt
a assessd snattagei embe semsssa 1 mi e 1.15 sessm emlate.
The 5.7 ma. or mosta ~se~nses me contateas t seaunts reme
almamb tear plams. The sotssatH tm~asnSatla
eat larg lateera sma~l~ad the seem were ebtalad Qumaitatrey.
Inigatura were plead as fallegel 8 age #Wspartr be thetttttt~~~~~~~tttttt espha-
seal alve, to (5 .. apara) ,ie atre to14 as Iblet sO1P Passeter
Sw at th jamettom et th 11sm sat the ur~aseatn sel, sad eas ea
the rree~m as eeg to tbs ame as peaathle The temple gastee
Astes~tas1 tre ms, amovat hopag l Ulgturre Satat. RF embs
Stag ~ behe be ligatuire a the pdPled aphias~ter at tlrr **~
eseal. gamest the Qbree neglas were sepaaste4. I~ ame ws gretalk
allbad sa u n eL pened sad seape tree of eabeat, these se~atents
emple we~re m~ah weigh d set 21ud to dry fa ai.
~Dartag the thatd sea Satividul Wrae sad fees samples was
ea31sete for a ~ap or parte4. A tas tweaspeert~h hea tCe bas
adl **=a==* etL an ueme, mal~l sateass me bree1 itestsa we
remove as baers. te livw sat kldee a t~ this sa l wag Ma
remove w1as weignet
The var~ea sample eat orgee wea Se pOPeredI fo audie
satUivit detbrdlerstse to th telled ag asr. Ate drgage t
teem, sat abaseh sat1 Saesta eekaasra mopinr wre smed to a
iyampene Sa aL mrtar, A susrpenerm at meh to 15 sessm agenesses
(containin 5 drops of the wetting agent, Tergitol, to each 25 alt.
of solution) was then prepared by aizing in a aring blendor far
thirty minutes. Fifty al. of sedu hydroxide solution wa used
for each 10 g. of sample., A few drop of capry1 alcohol was added
toE prevent foaming. Th suspension wa allowed tor stand twenty-four
hours at go.1 The mixture wras then brought to roam~ tempseatuma and
again agitated in a Waring blendorr A 1 al. portion was placed on
a shallow alum~inum planches and allowed toJ dry at roanr temperabare.
The liver and kidneys wer prepared one-half as can~centrasttttttttedt~~~t by
macerating the tissue in a Waring blendor in the presence of 19Z
sodhall hydroxide solution (25 al. or solution to to g. or tionue).
Planchets were prepared from this suspension as described above.
One m1. portions of the urine, blood plasman and blood sel samples
were plated dinlctlyr on planchets.
Radioactivity measurements wrere made in an internal-type
counter (Q-gas chamber and Nuclear Inhstrurmnt and Chemical Corpora-
tio Soaler, Unit Model 162) with an efficiency of tS pe cent
Th 8A hard an activity of L9,670 counts per min. per agr Bach
sampsle was counted fo three te minute intervals and $b not counts
per minute above background recorded for each sarmple,
the concentration of TS AF in these samples was determined
by direct comparison with abandard planchets prepared in the same
panner and can~taning known concentrations of TS AFI Peoes stand-
ards were used for dertermining the faces, cand stomach and intes-
thel contenba; liver standards for the liver, kidney and blood
ealla ad wtarcs ~ rabadad $e $ art m at beet plasm assples.
]> eade to allatast earnestic the the6~ dee i a 8 aS t 4~he taa
were cented eas th sm daqr a the suples. The Ytel amans ot
W A pwream in the IhaeP~ sample me saleltd bp altipying~ the
assentatiesa by th teta w~tab at the sample The tetl hies va-
as as slaulatd a She basis at 6.71 at per 100 gm. bod weihtl (al).
The preeadare used nar the es~tiatia at AF ia $be wrle a
the phetameri asthe et We~tl saat arts (6). Ti authed
empep exreatiesi~~~~iiii~~~~iii byr rageen bg aprass with Wasreeblerie asl@
Alasettasties and reap~iag with I salt. Passwr I.4m**y xpadama
earned ea ea TSAP sala~tims As e~La abowed th the apdae~pa~
p~~lrecdre sd to set espsh~le a breaklag St s~rCalt**** 1Sakage.
Sarbsequent phetamet~~riealysts at the wIPae~ ...PPIn us rr sat
eat allm~anting hperelO sts sat $bas pamtLttag es~ate a free AF.
Utrine, obtatant ao ran salml a the sam stata Pal ae tod whic
t al. at es~asem oi hd beenlr- n anmeS ret teested to the sam
m~anaar as he~ expe.rmatl emle sat agd as a htmakll Asl the sa~lr
made aPnalysts. ~adards seatstalag varylag kness asesetmat
et trehl prearet AF (m~p. 187) per al. at wAB WWae ade ap.
These manr sarbjesta t the same procedure a the epramatal
samp~le sad a +rstenan earv pleSSlag ag. AF peor l. wrrae agtast
optgical d~ensity m gprepred Fa this Ibart th eeaegetaa$5m
et Ue experiment imple as obtasnd ather theirr eptic desrity
as det~amind. grim tr Cl the ul asaae as L~ ebblam m~r be sa
eeriadite fam Isabreated salmls.~b The~ I ~ intr amb mpd Apr the
photometrio anal sis me Bookmaan Quarts Specatrophotomater, Model
SI~terial for the isotope dilation experiments mes obtained
ia the folliming mnner.r~ The suspension of third run fooes in 1%
sodium hydroXide was evaporated in air to dryess A s~amle of TBAF
whrich had previoal~y been allowed to stand a caomprable period at
time in ip sodium hydramide ~was found to to unatffeted. The dried
feceers s then extacte~~td with 10 al. Portions of acetfoans until no6
activity wasr detectable in the reside. Ten ag., of the acrtive resir
due obtained by evaporation et the acetone filtrartes wa successive-
ly rarys~t~alisd with 110 ag. of base TSALP tre 70P ethenal. iTh
rearyrstallisations were carried eat, I a t~areds if al.,, gpraduted
centrifuge tube by solution in a minians a boilagw 70) etbhaol;i
filtering hotly sa then allowing be cool alowly to room temperatures
for ) hours. After 8 hours at $ tbe material wa centritagd and
the expenatant defeated. The plreciptate w ~ase dried at 1009
and weaighed. One al. of acetone mes added for each 10 ag. of psl
sipitate se oneforurth a*gl. et tis solution plated on a planehet..
In1 this asoner i2.5 ma. of te maternal as~ cgunrtttttttttttttttttm each stirr Pight
realrstallisations were carried eat. The active aoterial from the
uParin was obtained by evaporating th pooled es samples to dry-
asess in air and extractttt~~~~~~ttttttt~ ng wh tbhree 10 al. portions of seetona and
two 10 al, porbians of 70% ethanal. This wa rearyrstoallised with
base TSALP as describd for the aost~rial obtained from the fooes.,
Itsotope dilation ex~perimenPts of the active material from the arine
Mef base =*rlv***T-*le=2**=** wre also ar~nst ea$ Th ba~se m
penga me aeclye arber5 msr iamb tratie together te Boo hease
hrang abomb Amr wehmse.+ Th serystallisatim proedur me as,
ram u ~as esrid beters excpt that 950 ebenal me ased as the us.
aspatantata a di and s~ta a ther solven for the ~preptarao et
We test for sadleservtrr Sa th taergents saltate ~16 a
tsmaf~ poletY saml ofDI artm am the fies and seeent Frm amr-s
srainta J~ J, ros. sense Sala per al. annous te adatason of
t al. Y sedia seetate ~, hepg me adgagbe to A dth I IOL epr 11 Mao
The 1Pales was them bewah to 80O al. wth water. Ftitees al. at
h...ldus. weaga (0.068C r knr-4@= d~adwebler~de l 0.16 1 ML)
me adde d stw the pg 8RP whsremeabliebst st@h M aeate aesLtate
Ather 10 atastes, O 10 mi a Itw seam med~ded After steadin swr~
alght, He blL==<@=O salta me selleted, ai d~rls sat cobDIP
of rr atstloutag ader~atabas im at S AF As gCive Sn rabe I.
firresstm asr r b em rwea SI) hoser sd It my b emes tha
abrile 67 per em$ et the stra~~~~~~~~~~aaaaaaaaaal a eagetd t the fees, sely 0.
Per cet appeared ia the radar.
Th $5m for Heseme ~a end ws extended t 66 hees. At
the set e his 4tas asrL radU~tentit a datesta~ble to the best.l
o c C we w oooo
$8 :L8 8 & 8
. . .
O e O O
S Et 8 S ;8 IIi
e e e a e o
~a, cn d till
new mea sun amemsr Orra aO s meesa sa as a satdeae hS1 m
rwealad asL the steseh Ovw 90 pehr ehdbe laattS
1~~aemas r bme uselrsalLIQ b bee eempletelyumsse ct m onIP
blee atr 66 hoLuse th dttrbuto etk d AF, me sumtan as an Ast e-
Two p3e r ea feiae~d is th seemeh the ma kl~l 3L Iassa~ae emaa
pe r~ ses, d the large lateta 30 per ems, The lver had abou
1 pef emb me ther kidasyles tasa 0~ .10 peret. The bleet pJlama
aew showe deflatt evidmres at law eunemligata at the em~pant
1Bth anae Sa~ Uod htest ee1 This agresMt theI iattage at Iards
eat Westirall ($5), atmas all th AAF dateted Sa th beet bp disms
tob Sa the hb4r~ emartsm as 10$. per emb.IIC This Irror on $b
peatftwo aide is jraSt abeat $bs eme a Sha at Hew prcein ampl
ea the neganwr sid
The peak to CMe~~~~~~~~CCCCCCCCCC riede..~ar Sa Cbe fees gameI a 16 hears
as aheat 6 bearrs ama t agedually damp ath, arrris ad liwesta
($6) ~test~ Ibt as) peak As H semagawsa at dieSuabl AAF
Sa tLe as~ wrrme em as as 4.6 hear perslet
in Frigrue t a camparison of the distribution of TS APl and
AA in the rat is made. Th data ter AA mas determined on the
basis of Masotisable attrogen (55,56), on the souirityr or the 0 .
labeled compound (6), rand on pase spectorographie ana~ysis of
After 16 hours aYrris and Westral (f;S) found 745~ per eons
at the Angested dosle of AAF abill rsmening la tsh stomach. Af 2
hours, abant 6: per nt TBAF wa found in the stomach, but noea n..,
gained in this organ at 66 hours.
Using a dose of 16 ag. AAF per 100 ga. of rat, which wa
comarat~ble to the dose of TSAF, arria and We~talll (56) found that
28-65 per sons nas .iseamted to the w$.ne, as compared with 0.3: per
cent of our materials atD 24 haours. Using the radioaotive AAFI 6-7
per cent of the nsteria was recovered in the wilne after a 6 hour
period. The greatest park of this cmpound, 75 per owns, wase aall
to the stomeoh, Over 1 pe c ent of the AAF wa found to the kidneyr,
w~hseure less than 0.1 per cent of the to aa1copound was t this
orglan. This is to e exlpected, An view at the anceh higher proportion
of AA eliminated in th wrine.
Th carrier experiments carried eat on the saberial aliminated
by the intestine sh~owed it to be anchanged TSAF, aaisnrtion of the
materal exarated in the urinea, whoever, showed that it had been
metbolised, the original cwormond not being present.
Wlhen h wrine wsas subjected to the naditted~ diasotisatoln
anld coupling procedureIII~~II~~IP (86) it was found that f~re AF wse present as
r 1 ___ __ __
1 I 1 I
0 5 10 15 20 25 3060
65 70 75
W TSAF BY Sm"; 24 HOURS
El AAF BY C'*; 6 HOURS
IIII AAF BY DIAZOTTIZATO)N; 16 HOURS
Imi AAF BY DIAZOTTIZATION; 24HOURS
M AAF BY N'ss 18 HOURS
0.3 OS 0.9
Ingas 1. A egprtaea~ at the dIsttate at 'PWAF sat AA
to the eagne adr emeet at $ba saS. Th seals ~bf r th le
pleam, Stra rJ and~ seesear hLasee pepaguall
0.148 per cent of th ingested TSAF, IS As thus evident that al the
compound seooonted for to th urine mne la the emrpletely hyldrolised
In an attempt to determine whart other metabolite at TSAF mse
present in the urrine, th aruarynr inorrgnie sultate aes precipitated
as besidine sulfarte, No radioactive inor~ganio saltaek wa ~Prese~t.
The hgdrolysis of TSAF would be expected to Jield, An addk.
Stion to AF, ptokensstu~ulonae. Crrier exrperiments serrned ea with
thi aeamound failed to establish its presence. Slither befor e o
after hydrolgsis, the ptolrueeulto~nio said moaisty woas mobabolised.
Oyridtion of n areawtsti methyl groulp to the oujabz racid to a known
assabolio procees. IS As poessthl that the other metabolite of~ TSAP
As p-rloulobenoe acd.
Sincer these studioe s ow that over 90 per cenrt of th ingested
T8AF i excreted unehanged in the teees, Atl~ ma bs suggested that th
compeand s naoneareogad~e beeuse it; is not absorObed to entriotent~n
conenotrations by organs sarat likely to be attahesd. The following
facts, howevrl contrardict this idea. Th amount of radioactive LAAF
in the livrer at 6 hears asr Q.7 per cent, which to oamprable with
the TSAP val~u (1.0 per cent) at the end~ of 21shous. Afterii~~~iii~~~ g hours,
howver, harris eat Westrall (55) found only 0.2 per ount of the AAF
in the liver by dissotisation, This different to the two values for
AAF is signitioanat. T the first case, the AF wase determined in teras
of th C The second value la based on dissotisable AiF aviarble by
extractian and hy~drolyst. The~ ract that less is racotuned for by
et AAF to $be Hear~ L met th resals ati~ alherUl embtIBa Wga
ba$ As a --y*- eat the stah e th empema la Sh livr.
5.4 as emr te dest~rsre is presse s me, therP to hasrrp
somessasses m fr aa ipl eager parted a am )ban AF. I~ eaSU
were espsLe et eaaar~ preashina, A ha rrisettete embae6 rL
Sht argan t dslaF Y bShl sativity Ai probab asp~sas te r
assL lack as onesagemensp~rI asr~ an u as1U tar, lshr asU a m
state egmpea sa is ee$ meral t**~r Sa the lir.pr
lAwtherP aeenrth asslt is ShsS Sh samenteetteesY at both
TBAF eat AA in H e ble p~ams are equ~rl, Neis sd WeeSUe2 (5%)
repr 100 pg. AAF pe 100 at. Heat pI ms, 16 hows afterP Sagetism
We fema this sum level (100 ps. TSAF per 100 al. beet l m) even
after als hows.
The ques~ata stallttt~~~~~ttttt~~~~~ wosa seen r to meU pitry erarnesa
gea, eat whether enverels a salbsta~a momta a a derrivativ to
th te adae lsr aL measesary peatde to wi5eawretegn s An obj***
Smto r b s theory is She evidea tha AF Is the purimr eretae=
gas. n is knew ea a vrsea or seemste mas re aestatates
aE gg as addistem, ustso, 0eat and am (ap) weprt Mat La Mer
as r~ to a sishtl a 3rlrower-ess onsagr 41an ~Ur1 a tees whic
wealdl aeat ct ha be respect if AAF As assive#3j sa~ly Manghse
stea to AF Uve tteetss te1AFeaAt miea
the ~pr mr~Jy l easaee as ar Sa abab t the AF me supllis,
provided that A~ could be formd in who from the other derivativ~es
Yl~et eve i this were the ease, the necessityJ exists for the hydroly
sis of a fl6uenwn derivative ~to Af prior to meetyla~tion.
Go the other hand, Morriaet al~. (51r,86) have shown that AAF
urndergoes deacetylation~i tot animal body The lower activity of
AF~ compared to AAF in the sat is not too indicative of primrpairyA
activity.e Animal s ingesting $.dissetyt2landnotheren (di-AAF) develop
tumore in a shorter time than is neceessary for either AF or AAF (53).
It CanotID be infer~l~rednevrtheless, that dI-AAF isl the primaryr car-
oinogent The ltower activityr of AF in the 55 maJT b due o soluE-
bil~ity differences. Sine A1F is conseiderbly more soluble than
AAF (85), it mlar be exrated m~or quick~lyl hence, th tassues are
not exposed as long as with AAF, which is exoreted at a rate depend-
ing onl its hydrolysia to AF. FRthemore,~ Wilson, gk al. (89~) point
eat th in the anose AF is possible ~mos active than AAPI since it
shows ar alightly shorter period of inenbation.
Fromr the eddenae# available at present, it does not asee
possible to reach a final conclusion An this matter however, the
results of these expeorimnts wth T8 fAF support the theory that unless
a floorene derivatieir ca be converted in anlbetanrtial uaorunts to the
o~rganis to the free ashesl th substance will not be oaronnogenio (615).
Zhk 2 pat& Rp ReT metf au~d stalel agat emely deteake
sad Mesapy et gastate same has be petated est. Mate~rsals ti
ear uLeemen~tast to aor bests, wa 1sealta ndaU a hearts de
ma years. Th Saeaspeatibaa on at diame rs at*==* lite such em.
pounse uses wel be wanhrl sa to seag==** ane tretma or amsta
81,98.93). Tobsa rat merre (P) wer e a~rS 4* synaesh a
turpa Rea. An AavesI ged a et bb aser dpe As tamsr a Lrm, h
shouet Ma$ He lve tek ap aheat Stee u asb me the rad~risagTl
as as Smar (Ir7). These week ar~~wares, (astesses e~auty as
litssemianattt~~~tt~~~ttt omo aseov empeandsU I ~Y~to ~ Masses sdteL este moder
to both M~apl sad mnage ~ emt thetttttt~~~~~~~tttttt shia ar salar ImetrI ~
analyds Aleh but Presteady kbee east S dessmissawY the 1s~Yeat
Stan o at res
Bloah sa Ba (8) selected I to labl a nmber of anno-
and bis-ao dyeer. This elemen writh a half-life of 8 days~ has a
rradiion suitable t biologilcal wrik and en be synthesised into
organio eapounds with a ther linkarge. Recetly~i~ Stevns gt al
(Th) studied th distribabienr of radiactive todia~ted Trpan Bae,
one et te compoun~ds first PrePared by Roch and Ray. Thirs pen
pound was a~dmnistered in~t~reravenus to mioe being suboataneus
imrplant ei~t $mor 150pia. Thwe oncentraion et radio~ativity to
Sranor tissue wa several timesr g~rea~te thoa that in ake~letal musol
or skin, 'bab coniderably less than to the liver, spleen or kild~ney
It a compound could ~be found which would displayr a greatr
afiat te the Samr than for suh vital organs as the liver and
kidnes~, while at ther same Ma maisntainia favo~rable rbie of
~ooeantrations in Sumor and surrounding tsissu, it weald g~retly
Inozease the chanoes of soounzte diagosis and thera~py of gasitrie
cancer. Since radionatiiviy offerrs a ready mran of detction, the~
need for a colord der~ivativ no longer exists sa other Sylpes of
Xlabed coaompsnade a be studied.
Several characterriaties of the sultonramde linage~ endow
these substnance writh diagnoshio and therarpetio potentialities.,
So far as cold be found to the literature the wrk reported in
Part III (66) dear~ibes the only ass of in who hydro~ysis of a
sulto~namde ilinkag and this hgdroljsis occurred to the extent of
on], 0.5 per cent. The ea ninkage wh~aich oeanrred in the colmpouds
of Tobin ea Mboor, and RLoch and Ry is quite susephible to easy-
m~ la Hste. ZEm sediat to ~ Hleamiss in eartat Manan (9176)
sa~ltaamdae av been sham toa seme the atteo~n tive tba intaba
o r ansmrsr sapiuppeea th satemato nonr ass tas seemtouse
tabbase on saewh or tumer (te). asy an sorte (66) popoe~d the
taSrrpape~n atC 8 tabe Uee alrrea pmisla8 empead This
abs~ebleeage bet mitr wal as4 ea~y er4 posethi adAS4Seas1
theapeatte Jattet, but wald ae after -au son atdeecim
StageY eartta JP~ atteegm ~ deba ies thereas barknew t
pea mes~L Sm b As IO rare siae to $baasl- body, is is puaims
alrea bbpeeem~al. Itsekd Mawe drivtie emetma ea or~~
t Fob~~~r enpe byt ... ..4* as seS able~p~rid at cmga
goes to Trypran Ew or Igea Rae b sd ~be etaas. Is these em
pheapleamathea (Augema) gepbl ~ sl She seed~Blp)gehe a 1Aahage~
No HqsSQNH O)H
the~ possibe cro~mpuds which could be successthil~ly cn-
densed writh florone-2,7-disulfony1 chloride are by as mesans lImited
to the naphthylaminre sultonio asida, ad disaltonamide derivatives at
1sorene with a; ride rnge an varietyl etd physical properties an be
The selection e a derivative with te best possible physical
qualities~ for specific afflinity to canner C6trasa pn~resnt a pzablem.
Sucah a large~ moecu~le with numerous functionail group makebsI it difi-i
ealt be debemrine what speoitic alteration in structlu rehould be
made to obtain th de~ired tumror localiatiton. ~It the maoleule to
built up ste br s~tep it shouldl be possible be detemine wrich stnrao-
tSure contribute to the desired properbies and whichae responsibe
for the objectional quatities.
The tendemnafl sreture gspa whieb a melesal of Sht typ
costsl he brn ap is Raerwreme7~r~nt,.isal ate sett Werar ea di-
seatm theresl-(,7teesonatet~ (a,7-sta ) has bee pr~epar
ant its 4tatrib~zUtion ~ain kl rmelerta mise ivtiga~~~~~ttttttted.~t
1. piness.~ 1Pa.....q7~a~lsy-ases..s- n -,PI so en.
(0.05 mles), toete with rannamewate 5,8, dB al. (1.80 mise)l
sentainta Sas Sa wak I01, 0.3 al. (8 m.)r wa wamed ea ast
kse. Asser one--lair heAr me nameOneDP essmerea. The selatte
Mhie precpi~tat foameA ~anmlalat- 1* was ad to disslv MeI
panredst sa H salatten1Lm was tseda ay andlesolvd seeidae by
iitui~telasr Seag an ttalr tc. He predst as preelpitated
on alew eassim o r sel se~ + ang s. nsersata~bntsnt wre~
emris was Lq assment a prodnes sa a massu at benta uses
Asse paQreds t arratllise me stead wa 9.ht pe rw seat baed
theren sea l7.71 per cent base a BaB Qgg salter analyster ga
17.53 per sons as anu eneouses raise ~to) 15 pr eaO S. Th
pealaidina drivtiv salU a at 386 U th rlag peta e the al-
strong ablesseeu meslZat6L a gerte an mehe (tS) teams
a~ssas iaro Onrssem Pa,7.isexr ur enesse.~
2. nimal studies. Fiver to six week~ old 85main A (Bars
Barbor) nise were employed as trwen third generation hosts fo thetttttt~~~~~~~tttttt
sbaxiarn inv transplantation ofJ a leratinising equtamous cell caroi-
nom (Line A, stamsob carrcinosts originally obtained tfre The ~Aim1p
Supply and Research Units of the British Empire Cancer Campaign) .
Whe th tumore wonr 10 day old, each mouse m adm iniste ed, ~by
tail vein injection, 0.25 al. of saline solution aonnetining 4.0 ag.
2,7-FDBas having a specific activity of 39,660 onsts per ala per
ag. Base experimanta As which the ansmi.l received twice this con-
centration of th compound ~failed tao reveal anyq ~taricit y sympataas.,
The annrmls we sacrificed at 2-, 8-, and 32-hour interval
followring trea~tmentt~~~~ttttt and pooled samples ;he a anim'al wer used for
eacrh determination. T2he stee were ansthetised wIth Ne~bual 10
minutes beforeU~ the Slae death and 'blood removed by~ hertr punctuer
prior to death. A1 per cen art solatian of sodium amlat (0.$ al.
to 1 al. blood) wa used as an antioagulaa$.
Th tumor, lIvers spleen, 'kidaeys, stomach with coabants and
leg anacoles were rmnoved, Amediately woeihed and suspended in a 1 penr
cent sodiumP bydraziide solution cent~fnine 1I al. of Tarital per
250 al. one a of this solution w as sd for each too as. sissue.r
The snapension wa called 0ost~and b8 hour at E. The mistanr m
then brought to race tmperature and Shorough~Ly haaegenised for 80
minutes. A t al. porbilon mes pl2aced on a shallowr aluminum planchet
and allowed to dlry at r~oao temperature. One al. samples of blood
wrer plated dizectl on plralunehta
anne~Searte mammes wrre mee s so sautern.
center (Qlgas eBbmber ~at Mas~Te Eastrmen eat Chemen~ COrposett
seatr, a tt god Y ide1) dte sa attlesarr at Iwg pr em$ yas
per rmammes aban bekgreatseedd rrar eac wh Uaml,
The ememb~att at ,7456~ A thes samples a debap
aule b7 dimet~ eempaste dtM ~srtandlaspleaebst pnrropne to a
empma.d Liver sesatard nd usewed Mr the lirwa remr sple seth
nrJ50 ments atsa&ad tery ~the meals aat rs sat ab~bt blee
asadsj fa the blet IWnl en r to e2SLa eaesu ter the
deear at Sa*, ~ th ata a ts w~as sente emteam day asl ther
at 0.2 al. p~ e~ kag. bod wlt (40).
The a4irtribution at selittenet it the #args rad Sbssma
Table1 zz. I Inw~ reas aftel~tr adeass estsmas emmasm o
41EsT4 ia the tar (0.ift ag. pe so. Steam)u wasMar tha
g~ras a that ienat in Ierabl usele sat mee ~the Olim~ tha A
tlw ln~r~Bin ~wrrrr~r ~ LUL~ YI\I ~ d as
Cone. in Mgrs. oa
Per am. Tissue Yge. 1I
am 10. Emood Recoveredr Recovreld
TwoI hours s
Blood 0.16, .0.331 3.531
Liver 0.930 0.295 2.95b
fi~dney 0.136 0.066 0466
spleen o0.06 0.2 0.26r ~z
Stamach + er 0.16) 0.067 0.67
Tumor 0.152 0.058 0.58
Leg muscless 0.035 -
Blood 0.079 0.17r 1 .7L
Livrn o0.0So odoe8 "1 oe
Miasysa 0.062 0.035 0.35
spleen 0.030 0.011 0.11
stomach + or 0.077 0.088 0.28
Tumor 0.058 0.016 0.16
I~g masoles 0.052 ~- --
Thirtp-two houra a
Blood 0.00Q 0.006r 0.06
Liver 0.007 0.011 0.11
EldAsys 0.026 0.011 01
Spleen 0.005: 0,col oot
Stamach er 0.028 0.006 0.06
Ttamar 0.0186 0.006 0.06
Iss meelesn 0.000 -- -
DISTRI13TION1 OF ADICACTIVITY QIN
TUMBOR-BBARINGQ MICB F~UQIJWN INTRATEN~OUS ,,
INJECTION OF D]BODIUM~ FIDnORENE*2,7-IBZUI.POATE..
(5.0 as. 4,7-rua*~ sn 0.25 al. saLsn)
anm ror ea Ir amammL~rrz o
M~DfICI~RWIVWI W80 TUArm TWU O THB
OMWRMATIWJ W WHRl TWWWSE FWT~AWZD~ A
88W.15 TIDJBRIW Q Itrllrm OgADWAD TO TPIICImnarm gasC
as r I *
t,l48 TappmI Rae5 Sal Rbws2*
t has. EA bra. Gkt has. 86 amr,
* ow+mtvt se iss sa~ wei asts name
++ Ather Ll heeseas sa nesettUr ar temr atle assi t1 m
detstbl Sa the mr~us1 be@
soaosantd for in the liver and kid~neye these raines wer less than
fo the tmomr tissue. The blood showed a slightly higher concentrar-
tim (0.1616 ag. per a.) tha did the tumor.
At~he 8 honrse th locallnisat of 2,71-FD8 a As the Smar~ w
abill greaterf~~~ffff~~~fff~~~ than to the liver, spleen or m~oole ba slightly ]less
thea n to th kidneye and blood.
The ditstibution abardies after 32 ho~ur rrevel that none at
this radioactive ccanpound rem~faind Sa the mascoe tismsue The com-
enrtration et 2,7-IE6ae in te fpamr wa over three and one-half
blaea that in thse pleen antd two and ohabslt Stae that in th liver.
The idnes~ still abowed a higher localisation than the tumor, bu
the concetration in th bood was ne onrly one-ninth that to the
tumor Thes ratios ~re given ta Table III. Comprarrble rtios ager
for radioactive todinarted Trpan Rue ws obtained fkrm th work et
Steven a al. (T)) the d~istriburtionl studies for i -Wie due 2
Con~tretng thew distribution at 2,7-lDB wih that of I *1
labeled Trypan hue and Nie PRae 28, it is evidenrt that the th~oesen
deriative has a only maintacind but has bettered the Irtio of con-t
centrartions in Samor and msenle Birssue This ratilo to 4.Asti tr the
2,7-FDB tr be hers athe injection. The ratio Sor Nile R~ue 2B is
3.61 51 afte 2.5; hOare~ while Stevens and associates report a 3.33 51
ratio tepr Trpan~ 81n at 24 hears. Tlhir;ptwtto hours afe in~jection
of 2,7-DBIl consaidzrshle radioactivity rud d in the tomer whie
As meu massetame sa she rsess Balisr tera ms noseseearty
awon at as hte ass~aeasses in nob use at as dgas ttd
to~ tea lealianita studies As asr fee Stat eaeb vial rga a
as ther, kndness sat Isple .takettt~~~~~ttttt~~~~ up leage Quatt~ee at toL dp
seomsat i ser to Ie nvr as sa as smer ~4 hows ane~ sagusa.
Thisr sam~ still peressess an~ 5 dears a tas tsms eflbrStannu
study me arde. With thisrr sam empem (be kiamp sat splea m s
ema~trate eww three~ t$$ms~tttttt~~~~~ttttt as meh at te (p a did the tear.
at ad-Fas. At 8 a StU hars er~ as admps 2.4 a ass r
$5m me the lesalwtias~ atf tbsu Reum dervaiv grete~ l the
liver $ban Sa th osmr, sad af~ te LrSr hea 1Ye I.mn..mat me
2abeka empent Oan did He Sameb ab all at to~ $$m Saberva
th~ias dataLP ter Ub7*as ats emare thr lity wbAM 81 tr '
Opttttttttttttttttttthae for WYUe Ra RS.~ Atrte 2.5 heseth kiny showe ab
2 hears the ~ emnaenso of the 2aseem detrati me ).98 Mass
geater to the DSmo r ta Sal th lkidse.Te teeteema
of radiooacbivity in tumpor tissue over the concentration in~ he pleenr
at 2.r houra s "I.46 for Nile Blue 28, while this same ratio was 2.2t
and 3.60 at 2 and 32 hours for 0,7-FD1S The proportion of labeled
com~poulnd in the tumsor as campared to the liver wa about the eas for
the two ompounds atr 2 and 2.5 hourre (1117 for 2,7-FDSas and 1.37 for
NVile Blue 2B)I hoevr this ratio mPore than doubled for the fluorsea
derivative atk 32 hours.
The anall percentages of radioactivity accounted for in the
animal body indicate that 2,7l-FDs1 t readily uecreted. This would
be expected sines the disulonic acid groups impart a hig degree of
solubility to the fluorsen molseule. Such aP fairly rapid excretion
is a definite advantage over the high molecular weight. Trypan Blue
which remains in the animal body after $ days in concentrations only
alight.1y less than at 2L hours. On th other hand Nile BLue 2B to
exoreted sto rapidly that non remains in th an~imal body after 24
hours while th fluorene compound is still detectable la the tumor
at 32 hours,
lV coamparison wit th dyes studied, 2,7-FDS1) has great~ly ine
areased the ratio of locarlisation in tumor tissue compared to lier
kidneys, spleen aal blood while. at the ease time increasing the ~faree-~
able balance between tumaor and musrcle tissue. it, is retained by the
animal body for a length of tim suffricint too enable detection and
trea~tmert; of malignant tisse neithout persisting to high canoontrations
for a prolonged period. The ratio between tumpor and kidney theave of
1.12 a t 2srr hers fals 0.69 at 32 hows, In Part ZI (46) is ras
tomad that a sr~baliamt deirivat et ao~rea asr a~lt~ate b
$be kidasy to mall aments. It is possbe hs the bL rrainrable
satbie om4 wth the 8,7-lrD emuld be grreal Sptrevd by eaa=
veirbigS Itake~ a IJault-th.
This flur~arr nsmpeaaA, themateae, ofers a panreisag basis
rapan whih saltaamide delroivate my be ba~llsst tasterd in Smors
at L2aealisatie a 2,7-@# As th stamah ad itsemes.I
evry ease the emeeratrasti of audiesetivi~ ity the s~mseh Ias e
pamble to or highr akan Cbs$ A the Smar Staea the 1sbatd em-
penab me abbdmistted bq Satnwaveou Asatten~, iS As aseer y that At
be aweatd by M e stomh well be rwesA I gastr~s seatet.
sreawtim et a higbZ+ seSA empeata is not is agramm$~ with~ $be re
eats obtalanrd to stense OnL thetttttt~~~~~~~tttttt allataStem at dJe starts byf $be
gast~~rie and. fingaban sat TisseherI (3) Anwastgate a larg ag
bar et deS~ ad tomatfb tha ba only emsu seeretd by the s~ae wre
ths empable et settag as baste~r (pes. iart s ld or amhetge dyesr
st~dudie wo as$ aliasasta by the getrie Stane
St~wma sad earkerrl~rs (Th) teent that audirsethe iettane
Trpsa Rae al* leeaised to th stareh to emerantanstemsl emperaht
be that to Sager Stases but anslh less tuan S tbs Ule kidsy sa
splem WIkle Trypn Emb is sa sed (p bp vtamw at A5 salteate
aid gamp, it also oeantrat bute Amb~marl gepe, Mrmwenr,
splitting of the aso linkage with the formation of amaino groups is
know to take place in the animal body The radioactive iodine could
hav been directed two the stomach by a fragment of the dye made more
basic byr this cleavage of the aso linkage. However, in view of the
results reported above for 2,7-FDPS it may be necessary to revise
present ideas of the secretoryr mechanism of the stomach.
,1. A pathesi is desclribet wateh tserpenate isetepte
at3weges (18) tate the malsele et Ye eartaeees kitratheea
sastrats the htherte rwaknewl ta a the, ati~wege darrag assal u
3. The ditsribabin et radianativiy me rstate Sa the
orprgans sat emrets e the ses as a-, Sir, spad 6bew tatera
fallerrag exal a~attatten6i~ ea stage1 ~ des ~at memureangat
the *...4 t ome i to as usr (1.* pero one) ass to r baasms
(100 pg. per 100 al, p1'am) me empeLbl to Me ammSllk at 8.seetph
admthereasp tomt S allmilar expr~umats. Th anneareSsagentatty
at -~ 8-g.4o sesut===<*krr s, thebrfrs, is ae$ oased by~ Sa
eatti~iate enaembruratta et the empenaA~ As the live (a p~rj^*=<=*=
atte te1Pr MeequmairnftwasStas tamers) or As the hised.
Ovr 90 per ee6 at the Angested des as Sead to be askin*See le**
emage swashD asr eastLrratbassal rswee on7 0.95 per eat a
ter Sa the arias me la th temr at m..amdammeas.u thds s~tLaat
wembrse win bbe relaruly lars* amentp, a~ppenalmbel )0 per seat
or as earnineass r -seests.4-frlasum appealrta As H adae.
3. A, synthesis isl described which incorporates radioactive
sulfar (Sas) into the molecules of disedina fluorene-2,7-dsuioaulont
The distribution of radionativity in the tissues of turmo~tearing
mice ftollowing a single injection of this compound ~ws studied at
2=, k and 32-hour intervals. The ratios of concentration~ et5 radio-
activity in trrmor tissue tor the concentration in other tissues were
detewrmied. My comparisoon wibth similar ratios for I -labeled
Tryrpan Bluae and Mil Blue SS, the thorone compound has greatly in-
oreased the ratio of localisation in tumor tissue comapared to livler,
kidney, spleen, blood and rsscole. It offes~ a promising basis upo
which aulfonamido derivatives may be buillt and tested in Sumor
diagnoals an therapy.
1. L.Y.* Aebamar satl 4. del, angrate, I ar Mag ~uuests Tawaissn,
sal Pangwrents l Th .9. amlrbg gapear fat.Lats, Spis, p. $15.,
1. LJ. Argesr rat F.E. Wa, onr ~ appeared. 1a~~r ELR (1951).
5* Ir, dluses.s..*F Brk.~l 1P. Iso~ar. pt. gs 1 (lphis).
6. F. Waleshowatre Meebm. J.. gs SW (1911).
t. H. Watae, kpub met Wee~rbeaeh Sga 81015 (18).r
8. 8.8. Meek an F.B. an, r. gas, amear End.. ys 61 (19316).
P9. Q.. eh, LI Ibehttt 8J. FlamCin W. Shapta sat I.lf. Stat
asy, answeenesrale r Is ba (1945).
10. 0. asplrant, Iebem.~* J.. go 1 (1938).
13. B. baepleat AA. OLrdute r.5. loween ad B. age ebm. In go
15. J. Mr, gesg., u s Pc 5*,sd 137 (1939)*H~f f
I6. IAW ~1, goek seab. aw getmd, I. She.~a Sun a RIO. 1U 3
18. C. Courtos rad R. Geotthro, Qumpt, awad., 1 8 2250 (19261).
19r A.J. CaOr, lelt rlroa rad Ie DT$$85 CARDSP Brrrgealth %S 6L
20, Hl.C. Duldley, J. Am. QChm Soo., g 3822 (1950),
21. B.C. DudSLey O a;lddox satd H.C. LaBue, Jr Phazaaeol. mptl.1
T.,]mass Ms 135 (19b9).
22, H.C. Dudley, Ot%. Mn rand I.E. Henry, j. PharanOel. Emgt,1.
TMrp 98s 105 (1905),
23. F. DuPran-sReolds, iAm i. Canoes~ gs 98 (1939),
261. H.M. Dyer, H.E. Bros rad H.P. Morris, Canoer Research 11 a
2$. S.J. Farris and J.Q. Griffit~h (eds.)L r* Bh at ha Laboratbory
Investigation, J.B. ILppineott Comppy Pblidmiphia,
1919, p. 41).
86.I. E.E. Goldman, Beikr. kslla. Chir. 88s 109 (1909).
27. J.O. Hamiltan rad Y.P. Soley, Am. J. Phpio1., 1; 135 (who0).
28. P.W. Harries Cancer Researsh, Xls 88 (196r7).
9p. i. ~Iania sat D. Marisl, Canoer asseazrah, Sgo 617 (1986).
30. M. Bes, J. ath. glact... 51 309 (1920).
31. C.H.W. Hirs, J. ~A. Che. Soc., lgol 189) (1949).
32. o. H~oebt~tset, arit, J. Caneer~, 15 391 (196r7).
33. R.C. fhgsebs rand M.B. Vissober, J. Gen. Payatal., 18s 695 (1935).
3k. A.C. Try a Biology ofCanoen Science, 1061 No. 275
(N9ovember It, 1967).
35. ae.L. IreMay, i. Iath, ames., got 233 (1926).
36. Jplx, EseARyICL meaah As130 C1 Ma).
37. x, Ismeans, peak Rs th.. Ma~na. 11o 455 (193).
1p, as, an, ** Orloramat* aramma,* ax, rz, John any ..4L amasl
as,, anr mRE, 19PIe, p. bL7.
40. M.B. Letsdr PPI 0al~ ab sat, LA.reiser games agaeargh. t
Irl. a~v. seen, aggIV as~ ap* (1965).
lit. LI. kLeates, lre.bn Mkr. See. 8. A hp) (1989).
I3. LO.~ YI11st, A.J. Mllarr- R.B. Sandle sat 1C.E. agese
genesr J ae eek go S (149).
Irk. a.. anwr ass e.. Ir-aman gensaBwarea ZI say (14).U
Us. J.A. anar~ ass e.A amemem, anasr amourrh. Is ~asp 096).
ig$ J.. all, R.W. Bapp sad LO. WLlekr, Ogeagel~rr J aere).
Ag~r 7.8. Moose L.H.I Webts eab 8.. mb Rher J Iac.Driattange
lb s 181 (1943). rtl~ C~I)
Ble8. a w. e, gg1.apg gort 130~1 047). 1 0
sat ed.k Butth, uJ O PlbSpenage fs. IIAMWIB 1ARMF go 1(1991).
Se. a.P. ~ mewis (astwe -** **-). i ~r
St .P ands E~csat 0. Wth, gaea aese. s S (95)
los 1801 (1950).
Sb. ar.P. Morris, awa, waisharLe ad Sj.w WeBiaburger, Canour Researah,~
rot 620 (1990) .
SS. n1.P. Yorri rand a~2a. wetran~ J. was, once Inst., es 129 (1968).
56. Bsa, unrrir rad a.a, westtall, aOer Rsarch k~, 10 506 (1950),
$7, .0,Myer Cner~ eearch,~ to 612 (1969).
Sa. wlrO. usersl, caOIr f~Seaah, los ask3 (1950).
$9. W.J.P. etksh, aree trar. rhia.l 67r 329 (1948).
60. C.L. O1ake rand G.H. arnrack, J. Path. Bact., gs 372 (1960).
61. L.A. Pinek, IAnn. NJr'. Acad. Soi., a ) (19k8).
62. B.A. Persi-eshite rad 0.1(. rikiforova, J. Apple Chem.l 1
63. P. Post, The chirnrgcl wlorks, Zs 255 (1783).
62. F.a. Bay rad M.P. Argru, Creer Beaeareh, g 718 (1951).
65. 9,9, s rand C.B. Geiser, Cancer Rese~arch g 616 (1950).
66. P.E. May rad L. Beffer, J. _Op. Chem.. 11s 1037 (1950) .
67. Pr.D~ Ra ad E. We~iaborrger (Privsae consendeation).
68. R. aoosen, peassehe me, Woohanehr., goe $38 (1923).
69. Ros wt, J. Somnrtino rad R. Talows J. Urol.1 go 792 (1950).
70. 8, 0elversfton, A.E. Solomona ad W.H, Sweet8, J. Am. xesd. Assoo,,
1,,g 277 (1929).
TS. A. Sie~glits, '1M Zt 1232 (1920).
72. A.* Siegits rand J. SohWtake, blr, Ast 2072 (1921).
73. BA.I 81oviter, Canoer REseareh, g677 (1949),
74., C.D. Stevens,~~tttt~~~tttt~~~ A. In*,r P.H. 84ewrptl P.11. Qurkiai an P.R. Gilsonr,
Cancer Researeh, IgO 139 (91969).
tS, rD SC~a aIm, s.A. m1~a. P~f. qIPPws rd ses 9. asrt,
sieaserp assrearl. 10 155 (195e).
76. Qa mU, aseans h Qulatia, W.A Idakea t ~J AM.br askk
TS DL. Talkeen, J.D. TayLsr ass g.I. Balesse, Westmentee. La
79. L.'hL eaJ ana IJ. as, 3. GIbta. Ine~aa en, go (194).
81., A.T. rassm F. Keyse sat Y, asem Deuseb me.,
-***,,aw~ gro 830 (1919).
St. x.wisus, 5,brp Am, (prJ see, g. U 1758 (1950).
815. r~a. waishmageor, l.lI. wekerger ad a.P. Iperswas I._ It.. ganger
Pa, 11 tST (1951).
SS. r.r. Wsraull J. ast. ganour DDass., io 8 (1965).
86. aIg wetri l sat LP. ar~s JJ.t. gas eaer sees.. go 1 (1967).
87. atg. wslaso, F. Dea rds se.J. can, planerb yeaea is95
88. r~ll. ansea P. Begas aas A.J glas, g~renueer Aseaea WLe
ap r. aswise, F. pem and A.J. gas, gameII I+Carek. Lo )L5
90. IgPsangin ad g. IchmJano, J. Cancer peeazwh,, rs t (1918).
91. P, Ioshida, Omn gs~ 495 (1935~).
92. P.Ar. Sah1 and F~.. Cooper, godialgy, goP 67) (1961).
93. P.Ai. Zrlh1 ad Fr.S. Coope~r, agLc~ c ga 6 (1961r),
nIo autho~ r mep ko sa rr team e, on asty 16)
1926, she as raleatersea at me idEar smanasta eases a ass
deseph 1Eg Sebal Iate, askn B.SR. degree wit a lrr a shm
tstay we awasd~es ages em ass, Prm me onless at seas
salt Jsep~ewhemble M--Ma@kt, A 195. r th aes
tue yearrs abe~n meenagt gr adnt see~uk rdy at the~~~ffffff~~~~~fffff galversitj a
rme-M, me sweeived $ba M.. degase~ Qlargeate C~d amis ry
tbs$ iasttattian. Mear Ar es me eaInstreate at Cmsry~ a
aga~n, cae13ge, astweity Magangu, ier the sardmi year,
spier-spisp Es Baptabwe 191ip, she enteret as Gadat Sabsel
oi r noastrrdate or Ruses whe re shea hrls a asses asse
wants analt h rnwashp Si aa~ Imer aweshi She is a esadldt
f~ar thedegrer lat 1eater et Phileepay sa ~re masr* 98
This disserbatio n was prepared rader t direction of
thel Chiairma of the *Mdelapdnkl Supeurviso Committee and has
been approved by al mebers of th CommIt~ tee t wa smibbed
be( the Owedrrate Counc~il and rws apptrove asr partial tu~lfilmen
et the requirements forP the dege of Dbotor of Philosophy,
]pebruary It 1952
,I, J ,.'/ChS