Studies on Fluorinated PyrimidinesII. Effects on Transplanted Tumors*

CHARLESHEIDELBERGER,Lois GRIESBACH,BETTYJo MONTAG,DOROTHYMOOREN,OLIVIACRUZ

(MeArile Memorial Laboratory, The Medical School, University of Wisconsin, Madison 6, Wis.)

R. J. SCHNITZERANDE. GRUNBERG

(Hoffmann-LaRoche, Inc., Nvtley, N.J.)

In 1954, Rutman, Cantarow, and Paschkis reported an increased utilization of labeled uracil inprimary induced rat hepatoma as compared withnormal liver (22); similar observations with thetransplanted Flexner-Jobling carcinoma, togetherwith some studies on the mechanism of this effect,were reported from this laboratory (15). This increased utilization of uracil for nucleic acid biosynthesis in tumors may be a general phenomenon,but it is not specific for tumors, since intestinalmucosa showed a high uptake of this compound(15). Nevertheless, in view of the meager knowledge that we now possess about the characteristicbiochemical lesions of cancer, it appeared worthwhile to devise and synthesize a potential blockingagent very closely related to uracil, even thoughliterally dozens of pyrimidine analogs had beenfound to be inactive. During the early stages ofthis work we were encouraged by the report ofWelch and his colleagues ot the tumor-inhibitoryactivity of 6-azauracil and uracil-6-niethylsul-fone (12).

It appeared plausible that replacement of ahydrogen atom by a fluorine atom in the pyrimidine ring should lead to an analog of considerablepotency. This belief was based upon the profoundalterations in biological effects brought about byfluorine substitution in various unrelated series ofcompounds: the high toxicity of fluoroacetate (4),the amino acid-inhibitory properties of p-fluoro-

* This work was supported in part by a grant-in-aid of theWisconsin Division of the American Cancer Society; a grant,C-2832, from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service; and by a grant-in-aid from Hoffmann-LaRoche, Inc. A preliminary accountof this work appeared in Proc. Am. Assoc. Cancer Research, 2(3) :212, 1957. Paper No. I in the series is (14).

Received for publication October 25, 1957.

phenylalanine (1), the increased biological potencyof fluorinated steroids (11) and fluorinated azodye hepatocarcinogens (19), and the nerve toxicityof diisopropylfluorophosphate and related compounds (17) serve as illustrations of this point. Itseems likely that the biological activity of a number of these compounds stems from the great stability of the carbon-fluorine bond, and the verysmall 'Van der Waal's radius of the fluorine atom

(1.35 A), which is very close to that of hydrogen(1.2 A) and considerably less than those ofchlorine and bromine (1.80 and 1.95 A) (20). Thesmall size of the fluorine atom apparently permitsthe antimetabolite to occupy active sites on enzymes, often in preference to normal substrates,and the stability of the carbon-fluorine bond apparently prevents the attachment of groups to thesubstituted carbon atom of the metabolite.

Of the three possible positions for the substitution of the fluorine, carbons 2, 6, and 5, the latterwas chosen, because the fluorine is on the samecarbon as the methyl group of thymine, and it wasanticipated that this analog might interfere withthe formation or utilization of thymine. Accordingly, a collaboration was started with Dr. RobertDuschinsky and Mr. E. Pleven of Hoffmann-LaRoche, Inc., the result of which was the successful synthesis of 5-fluorouracil and of related5-fluoropyrimidines (10). 5-Fluorouracil turnedout to have considerable biological activity, andan introductory paper, published jointly withscientists of Hoffmann-LaRoche, described itseffects on several transplanted tumors, a numberof bacteria, and some biochemical and metabolicfindings (14). The effects of the fluorinated pyrim-idines shown in Chart 1 against several transplanted mouse and rat tumors are described inthis paper, and some metabolic and biochemical

305

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306 Cancer Research Voi. 18, April, 1958

studies are reported in the accompanying reports(3,4,8).

MATERIALS AND METHODSSource of compounds.—Allfluorinated pyrimidines and their

derivatives were obtained from Hoffmann-LaRoche. OPSPA(N-Oxapentamethylene-N', N"-diethylenethiophosphoramide)

was prepared in this laboratory, and the other compounds wereobtained from commercial sources.

Sarcoma 180.—FemaleSwiss mice, weighing approximately25 gm., obtained from Taconic Farms, Germantown, N.Y.,were used to maintain this tumor, originally obtained fromDr. Ii. 3. Teply and Mr. B. E. Kline of the Wisconsin AlumniResearch Foundation. We also acknowledge the courtesy ofDr. Donald A. Clarke of the Sloan-Kettering Institute forsending us his line of Sarcoma 180. Tumor fragments from8-day-old tumors, about 1 mm. in diameter, were transplantedunder aseptic conditions, with trocars, bilaterally into theaxillary region of groups of five to ten mice. The drugs were

OH

HO

NH,

OOH HO

5-FLUOROURACIL 5'FLUOROOROTIC 5-FLUOROCYTOSINE

ACID

FU FO FC

RO 2-9757 R02-9945 RO 2-9915

CHART 1.—Structures and abbreviations of fluorinatedpyrimidines.

usually administered by intraperitoneal injection for 7 successive days, starting 1 day after transplantation. The micewere weighed, and the spherical tumors were measured withcalipers in three diameters on the 7th, llth, and 14th days.The volumes were calculated according to the formula N =4/Sirr3 and corrected by a factor of 2 obtained empiricallyfrom the weight-volume comparison given in Chart 2.

Flexner-Jobling carcinoma.—Thistumor was obtained fromDr. G. A. LePage and was maintained in 150-gm. female albinorats, purchased from the HolUmann Rat Co. of Madison,Wis. The 10-14-day-old tumor was minced, two volumes ofsterile saline were added, and 0.25 ml. of the suspension wasinjected subcutaneously into the right and left axillary regions.The rats were weighed, and the cylindrically-shaped tumorswere measured in three diameters with calipers on the 9th,16th, and 22d days. The volumes were calculated according tothe formula V = ir/l(di)(dì)(d^),with an empirical correctionof 1.4 as obtained from the experiment given in Chart 2.

755 Mammary adenocarcinoma.—This tumor was obtainedfrom Dr. L. J. Teply and Mr. B. E. Kline of the WisconsinAlumni Research Foundation. It was maintained in 18-gm.female BDFi mice, obtained from the R. B. Jackson MemorialLaboratory, Bar Harbor, Maine. Twelve-day-old tumors wereused as the donor material, which was transplanted withtrocars as described for Sarcoma 180. The mice were weighed,the tumors were measured on the 8th, llth, 15th, and 18thdays, and the volumes were calculated as described for Sarcoma 180. The tumor remained well differentiated with a glandular histological structure.1

Ehrlich osciles carcinoma.—Thistumor was obtained fromDr. G. A. LePage and was maintained in the same mice used

1 Private communication from Dr. S. S. Sternberg.

for Sarcoma 180. Mice bearing 8-day-old transplants weresacrificed, and 1 ml. of sterile saline was injected into the peritoneal cavity. The suspension of cells was withdrawn with thesame syringe, placed in a sterile centrifuge tube, and centri-fuged for 2 minutes in a clinical centrifuge. The packed cellswere suspended evenly in 20 times their volume of sterilesaline, and 0.15 ml. of this suspension was injected intraperi-toneally into recipient mice. This inoculum is equivalent toabout 5,000,000 cells. The mice were weighed 3 times weeklyand their survival times recorded.

A 5-fluorouracil-resistant line of this tumor was developedby successive transplantation of cells from mice treated withfluorouracil daily for 7 days, starting 1 day after transplantation. The transplantation to recipients was done 1 day aftercessation of treatment, according to the following schedule:80 mg/kg/day for four generations, no treatment for two generations, 40 mg/kg/day for one generation, no treatment fortwo generations, 80 mg/kg/day treatment for seven generations.At this point resistance was obtained, which was maintainedto the 40th generation without further drug treatment. Theresistance was lost between the 40th and 45th generations.

Resistance to 5-fluoroorotic acid was not successfullyachieved as a result of the following treatment: 105 mg/kg/1*day after transplantation for eight generations, 10 mg/kg/7days2 for four generations.

LISIO leukemia.—Theparent line and a 6-mercaptopurine-resistant line of this tumor were kindly supplied by Dr. L. W.Law of the National Cancer Institute. The tumor was maintained in ascites form in female BDFi mice and transplanted

1,000VOLUME F-J

5,000 10,000 15,000

abo sbo7boVOLUME S-180

900

CHART2.—Correlation of tumor weights and volumes inSarcoma 180 and Flexner-Jobling Carcinoma. X = S-180, O =Flexner-Jobling carcinoma.

every 6 days, with the same procedure for both lines. Thedonor mice were sacrificed, and the skin was peeled back fromthe abdominal wall. A suspension of 0.1 ml. of cells was removed with a sterile syringe from the upper quadrant anddiluted with 9 parts of sterile saline. Of this suspension, 0.1ml. was injected into recipient mice, which were weighedthrice weekly and their survival times recorded.

Novikqff hepatoma.—Thistumor, obtained from Dr. V. R.Potter, was maintained in the female albino rats described forthe Flexner-Jobling carcinoma. The tumor was transplantedintraperitoneally every 6 days and was dissected from the

2Throughout this and succeeding papers, the notationx mg/kg/y days means x mg/kg/day for y days.

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HEIDELBERGERet al.—Studies on Fluorinated Pyrimidines. II 307

intestinal tract of donor rats, minced, and diluted with 4 partsof sterile saline; and 0.2 ml. was injected intraperitoneally intorecipient rats. These were weighed thrice weekly and theirsurvival times recorded.

Yoshida sarcoma.—Thisascites tumor was obtained fromDr. G. C. Mueller and maintained in female rats. Transplantation was carried out on 7-day-old tumors, by the injection of2—4ml. of sterile saline into the peritoneal cavity and withdrawal of the cells. Following centrifugation, the packed cellswere suspended in 9 parts of sterile saline, and 0.25 ml. wasinjected into rats, which were weighed, and their survival timesrecorded.

Whenever possible, animals dead of ascites tumors wereautopsied for confirmation.

Sarcoma A-l.—This tumor was originally induced by thesubcutaneous implantation of 1,2,5,6-dibenEanthracene (MissM. Buck) and has been maintained in serial passage in Swissalbino mice at Hoffmann-LaRoche since December, 1954. Thetumor was transplanted by trocar, was measured 11 and 18days afterwards, and the volumes were computed.

E 0771 mammary adenocarcinoma.—Thistumor was maintained by trocar transplantation in BAF/1 mice; measurements were made on the 15th day.

Walker 256 carcinosarcoma.—Thistumor was implantedinto the right groin by trocar in Wistar rats. The animals weresacrificed on the 15th day, and the tumors were excised andweighed.

X-radiation experiments.—In these experiments the micewere quieted with 15 mg/kg of chlorpromazine (which wasshown not to affect the tumor growth in the doses employed),and placed in cylindrical shields made of J-inch lead. A slotin the cylinder permitted the tumor and surrounding skin tobe irradiated, while secured with small clamps, with shieldingof the rest of the body. The radiation was delivered in a singledose, calibrated with an ionization chamber, of 115 r/min.The conditions were 175 kv with a 0.5-mm. Cu and 0.1 mm.Al filter at 10 ma. and 31.7 cm. focal distance.

RESULTSToxicity of 5-fiuorouracil and 5-fluoroorotic acid

for albino mice.—Toxicity studies were carried out

in albino mice from a single colony. Groups of sixto ten animals of 18—22-gm.body weight were usedfor every dose. They were kept in suspended wire-bottomed cages at a constant temperature of21°-23°C.

The compounds were dissolved in sterile saline,in the case of 5-fluoroorotic acid after the additionof 1-2 drops of concentrated (8 per cent) solution

of Na2CO3/ml, and were administered by theintravenous, intraabdominal, subcutaneous, andoral route. The amount of drug calculated for thebody weight of the individual mice was containedin 1.0 ml/20 gm, except that, for the intravenousinjection, a volume of 0.5 ml. was given.

Toxicity of single doses.—The toxicity of boththe 5-fluorouracil and the 5-fluoroorotic acid ischaracterized by delayed death, such as has beenobserved with other carcinostatic compounds. Forthis reason, a comparatively long observation timeof 10—20days was necessary, during which periodthe mice were weighed every 2d-3d day.

Table 1 shows the results of injection of single

doses of the two fluoro derivatives expressed asthe LD60.

5-Fluorouracil was, as a rule, better toleratedthan 5-fluoroorotic acid, particularly with theintravenous and, to a certain degree also, with thesubcutaneous injection. Single oral administrationof 5-fluoroorotic acid was, however, less toxic than5-fluorouracil.

The outstanding toxic symptoms produced bythe compounds were diarrhea and a progressiveloss of weight, generally between 15 and 40 percent of the initial weight. The animals died, following toxic doses, after average periods of between 6 and 12 days, in a state of complete emaciation, although their stomachs in all instances contained food. After tolerated doses of the compounds, either the weight remained constant orgains up to 24 per cent and occasionally more werenoted at the end of the observation time.

TABLE 1

TOXICITYOFSINGLEADMINISTRATIONOF5-FLUOROURACILAND5-FLUOROOROTIC

ACIDINALBINOMICELDw (MO/KO)*

Intra- Intraperì- Subcu- PerCOMPOUND venous toneal taneous os

5-Fluorouracil >2SOf 188 233 1895-Fluorooroticacid 94 188 190 532

* Calculated according to Reed and Muench (21).

t Temporary loss of weight of 17 per cent.

Toxicity of repeated doses.—The long duration

of the toxic effects makes it understandable thatrepeated administration required a substantial reduction of the doses. The maximal dose of 5-fluoro-uracil which could safely be given once daily for10 successive days was 25 mg/kg by the subcutaneous, intra-abdominal, or oral route. The corresponding dose of 5-fluoroorotic acid was 10-12.5

mg/kg for the same routes of administration.Toxic symptoms or loss of weight were rarely observed after these doses. The figures are in goodagreement with those given by Wong and Benson(25).

There was also no essential difference in theamount of drug tolerated when the compoundswere given continuously to mice by diet. The diet,which consisted of finely ground GLF Big Red dogpellets containing 0.02 per cent 5-fluorouracil or0.005 per cent 5-fluoroorotic acid, produced no, oronly insignificant, loss of weight if consumed overa 16-20-day period. These doses correspond rough

ly to 50 mg/kg/day and 12.5 mg/kg/day.Albino rats tolerated daily subcutaneous injec

tions of 20-25 mg/kg of either 5-fluorouracil or5-fluoroorotic acid given on 14 consecutive days.

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308 Cancer Research Vol. 18, April, 1958

The histopathology observed after toxic dosesof 5-fluorouracil has been briefly outlined by Wongand Benson (25). The pathological changes consisted mainly of hemorrhages in lungs and intestines and a marked depression of the hematopoi-etic tissue of the bone marrow. Bollag (2) also observed extensive damage of the myelo-, lympho-,and thrombopoietic tissues of mice and rats afterrepeated injections of 30-40 mg/kg, whereas eryth-ropoietic cells were involved to a lesser degree.

Our studies, which were limited to tissues ofmice, confirmed these observations. 5-Fluorouracilas well as 5-fluoroorotic acid produced extensivehemorrhages of the lungs after lethal doses of these

S-180

FU 25 MG/KG/7 FO 12 MG/KG/7

(105)

7 II 14DAYS

(50)

14

VOL.

1000

750-

500-

250-

0

+ 2

AWT. 0

-2-

-5-

CHART3.—Effects of 5-fluorouracil and 5-fluoroorotic acidon Sarcoma 180. X = controls; O = treated. Volume (cu.mm.), and body weight change (gm.) vs. days after transplantation. The numbers on the graph (cf. .28) are the ratiosof the treated to control tumor volumes.

compounds. A further characteristic finding wasthe cessation of mitotic activity in the mucosa oíjejunum and ileum which was accompanied by regressive changes in the epithelium of the villi.These changes were observed after parenteral andoral administration, but were absent if small, welltolerated doses of the compounds were given.

TESTS WITHTRANSPLANTEDTUMORSAll animals, unless otherwise stated, were

treated with the drug by intraperitoneal injectionsonce daily for 7 days, starting 1 day after transplantation of the tumor. The justification for theuse of tumor volumes for the estimation of thesize of S-180 and the Flexner-Jobling carcinoma,together with the empirical correction factorsneeded to convert the calculated volumes intoweight equivalents, is shown in Chart 2. The use

of volumes instead of the weights of dissectedtumors has the advantage of enabling a growthcurve to be constructed, which often provides considerably more information than estimation of thetumor size at a single time interval after treatment.

Sarcoma 180.—Theeffects of 5-fluorouracil and5-fluoroorotic acid on the growth rate of Sarcoma180 are shown in Chart 3, which represents a composite of seventeen experiments and 105 mice for5-fluorouracil and eight experiments and 50 micefor 5-fluoroorotic acid. Although the usual considerable variation in tumor sizes among experiments was encountered, the results were usuallyquite consistent among individual experiments.Thus, no attempt has been made at a statisticalanalysis of the variation. It will be seen that bothcompounds significantly retarded the growth ofthis tumor, 5-fluorouracil being more effective andless toxic then 5-fluoroorotic acid, as indicated bythe tumor volumes and weight changes. That thetumor inhibition was not primarily due to weight

VOL.

1000-

750-

500-

250-

0

0AWT.

-5-

-8-

STARVATION EXPERIMENTS-180 (IO) F-J (5)

16,000-

8,000-

4,000-

0-50'

7 II 14 DAYS

O-20

.71

15 21

CHART4.—Effects of starvation and drugs on Sarcoma180 and Flexner-Jobling carcinoma growth. Tumor vols,(cu. mm.) and body weight change (gm.) vs. days after transplantation. X = controls; O = FU at 25 mg/kg/7 days; A =starvation.

loss or caloric restriction is clearly demonstratedin Chart 4, in which the effect on the tumor of5-fluorouracil, which caused a body weight loss of2 gm. at 7 days, is compared with that in a groupof mice starved to a weight loss of 8.4 gm. Theeffect of 5-fluorouracil treatment for 7 days, starting with established 9-day-old tumors, is shownin Chart 5. It will be noted that from 9 until 30days the volumes of the treated tumors remainedessentially constant, while the controls increased.Beyond 30 days, the tumors ulcerated badly, werenibbled on, and regressed in both groups. The

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HEIDELBERGER et al.—Studies on Fluorinated Pyrimidines. II 309

number of mice that died in the treated group wassignificantly less than that in the controls. It isevident that in this tumor 5-fluorouracil was acarcinostatic rather than a carcinolytic substance.5-Fluoroorotic acid had less effect on establishedtumors.

A comparison of the effects of 5-fluorouracil and5-fluoroorotic acid on S-180 under various conditions of dosage and modes of administration isgiven in Table 2. It is evident that there was nosignificant difference in the effect of 5-fluorouracilagainst this tumor whethei' it was injected intra-peritoneally, subcutaneously, or intramuscularly.On the other hand, administration by stomachtube was considerably less effective. It also appears that a single daily dose of 25 mg. was moreeffective than two daily injections of 12.5 mg/kg.With 5-fluorouracil, repeated doses had a greatereffect than a large, single dose, whereas with5-fluoroorotic acid these two dose schedules appeared to be approximately equivalent.

The combination of 5-fluorouracil with severalother drugs has been investigated. As will be seenfrom Table 3, effects that appear to be additiveonly were obtained from the combination of5-fluorouracil with 5-fluoroorotic acid, azaserine,A-methopterin, OPSPA, and urethan. However,effects that appeared greater than additive wereobserved when 6-mercaptopurine and 5-bromoura-ul were combined with 5-fluorouracil.

When a course of treatment with 5-fluorouracilwas combined with a single local x-radiation ofestablished S-180, complete disappearance of alltumors was observed, as shown in Chart 6. Thetumors in the group treated with a relatively lowdose of 5-fluorouracil continued to grow, and inthe group that received only the x-ray the tumormaintained its original size. The effectiveness ofthis combination suggests the desirability of clinicaltests of chemotherapy with 5-fluorouracil, together with local roentgen radiation. Comparableresults were obtained with the 755 mammaryadenocarcinoma.

755 Mammary adenocarcinoma.—A summary ofour experience with this tumor is given in Chart 7,in which it is seen that 5-fluorouracil and 5-fluoroorotic acid were about equally effective. However,this inhibition was considerably less than that produced by 6-mercaptopurine. Treatment of theestablished tumor with 5-fluorouracil produced anarrest of growth but no regression, an effect comparable to that observed in S-180.

Sarcoma A-l.—As indicated in Table 4, thistumor was inhibited to a marked extent by both5-fluorouracil and 5-fluoroorotic acid, the formerproducing a significant number of mice in which

no tumor had appeared at the time of death or the18th day after transplantation. However, bothcompounds were inferior to 6-mercaptopurine forthis tumor. In a comparison of the two fluorinatedpyrimidines, it is evident that, when the drugswere given thrice weekly for 2 weeks, 5-fluoroorotic acid was somewhat more effective than5-fluorouracil, although somewhat more toxic, asindicated by the weight losses. When treatmentwas started 3 days after transplantation, 5-fluorouracil stopped the growth of the tumor, but noregressions were produced.

E 0771 mammary adenocarcinoma.—The datapresented in Table 5 show that 5-fluorouracil pro-

20-

o _1500'

1000 -

500-

ESTABLISHED S-180

FU 25 MG/KG/7

NO. ALIVE

*****»<-**„,

VOLUME

,07

9 II 14•FU-}

18 21 30 DAYS

AWT,

CHART5.—Effectsof S-fluorouracil on the established Sarcoma 180. Volume in cu. mm.; weight change in gm.

duced an inhibition of the growth of this tumor,but to a lesser extent than did 6-mercaptopurine.

Flexner-Jobling carcinoma.—This rat tumor, inwhich OPSPA produces complete regressions ofestablished tumors with a single injection (13),was affected only slightly by 5-fluorouracil and notat all by 5-fluoroorotic acid, as shown in Chart 8.With 5-fluorouracil there was a pronounced effectat 9 and 15 days after transplantation; but withthe usual 7-day treatment the effect wore off, andbetween the 15tb and 22d days the treated tumorsgrew as fast as the controls. That the initial effectobserved was not due to caloric restriction isshown by the experiment in Chart 4, in whichstarvation to a weight loss of 20 gm. did not affectthe growth of the tumor. Treatment of rats withestablished tumors produced a slow-down, but notarrest, in the growth rate of the tumor.

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TABLE 2

COMPARISONOFMODESOFADMINISTRATIONOFDRUGSONS-180Tumors measured on llth day

GroupControls (10)5-Fluorouracil5-Fluorouracil5-Fluorouracil

Controls (5)S-Fluorouracil

Controls (5)5-Fluorouracil

K

S-Tluoroorotic

' Ratio T/C volumes

Dose(mg/kg/day)

15, 1 time daily, i.p.25 (given in 2 doses/day) i.p.25, by stomach tube

25, intraperitoneal25, subcutaneous25, intramuscular25, by stomach tube

150 for 1 day25 for 7 days

105 for 1 day15 for 7 days

volumes of treated tumorsvolumes of control tumors '

Bodywt.change(gm.)-2.8-3.5-3.5-4.7

+2.0-1.1-1.0+0.2-0.9

-1.5-0.3-3.8-1.6-5.4

Volume(cu. mm.)

102492

1735782862296

118

436113

•-in248204

RatioT/C

volumes*

.09

.17

.5«

.08

.03

.02

.41

.26

.04

.57

.47

TABLE3COMBINATIONEXPERIMENTS,SARCOMAiso, MEASUREDONHTHDAY

GroupControls5-Fluorouracil

a

6-Mercaptopurinea

Fluorouracil+6-Mercaptopurine

Fluorouracil+6-Mercaptopurine

Controls5-Fluorouracil5-Fluoroorotic5-Fluorouracil

+5-Fluoroorotic

Controls5-FluorouracilAzaserine5-Fluorouracil

+Azaserine

Controls5-FluorouracilOPSPA5-Fluorouracil

+OPSPA

Controls5-FluorouracilUrethan5-Fluorouracil

+ Urethan

Controls5-Fluorouracil5-Bromouracil5-Fluorouracil

+5-Bromouracil

Controls5-FluorouracilA-methopterin5-Fluorouracil

+A-methopterin

Dose(mg/kg/day)

201015

7.5201510

7.5

15101510

155

155

104

104

12300

12800

15501550

251.5

251.5

Body wt. change(gin-)

+ 1.1-1.8+ 1.6+1.4+2.9-0.1

+2.0

+2.4-0.4-2.0-4.6

+1.2-0.3-0.1+0.4

+ 1.0-0.2+0.6-1.3

-0.6+0.4-0.8-1.4

+1.0+0.4-0.3-1.7

+0.1-2.3-0.5+1.3

Volume(cu. mm.)

454176254

62270

16

106

488442508196

412220160266

407337332244

1000214586332

56222452443

545108355284

RatioT/C volumes

.39

.56

.14

.59

.04

.22

.911.04

.40

.53

.39

.65

.83

.82

.60

.21

.53

.33

.41

.93

.08

.20

.65

.52

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HEIDELBERGER et al.—Studies on Fluorinated Pyrimidines. II 311

When 5-fluorouracil was used in combinationwith other compounds, the results shown in Table6 were obtained. The 5-fluorouracil combinationswith azaserine and A-methopterin were less effective against this tumor than the individual drugs;the combination with urethan appeared to giveadditive results. The combination of 5-fluorouracilwith 6-mercaptopurine, 5-bromouracil, andOPSPA affected the tumor in more than an additive fashion, under these conditions.

Walker 256 carcinosarcoma.—When 5-fluorouracil and 5-fluoroorotic acid were compared withtriethylene melamine (TEM), they were found to

be much inferior to the alkylating agent in inhibiting the growth of this tumor (Table 7). Whengiven at relatively high doses, 5-fluorouracil wassomewhat more effective than 5-fluoroorotic acid.There was little effect upon established tumors.

755 TUMOR (21)

FU 20MG/KG/7 FO IOMG/KG/7

CHART 6.—Effects of drugs and roentgen radiation onSarcoma 180 and 755 mammary adenocarcinoma. Volume (cu.mm.) and body weight change (gm.) vs. days after transplantation. X = controls; O = 2000 p(S-180), 1500 r(755); A =5-fluorouraeil, 20 mg/kg/7 days; n = combination of samedoses of x-ray and FU.

CHART7.—Effectsof 5-fluorouracil and 5-fluoroorotic acidon 755 Mammary adenocarcinoma. Tumor vol. (cu. mm.) andbody weight change (gm.) vs. days after transplantation;X = controls; O = treated.

Ehrlich ascites carcinoma.—The effects of 5-fluorouracil and 5-fluoroorotic acid on the survival ofmice given inoculations of the Ehrlich ascites carcinoma are shown in Chart 9. In this tumor the5-fluorouracil was more potent than 5-fluorooroticacid, and 18 per cent of the mice survived tumor-free, as determined grossly and by transplantationattempts. Saline was injected 'intraperitoneally

Dos«(mg/kg/day)

TABLE4EFFECTS

OFDRUGSONSARCOMAA-iFREQUENCY2X5ÃŽ

2X52X52X52X3#

2X32X32X3SubcutaneousBODY

WT.CHANGE,llth

day(Km.)+4.5

-2.7+1.1+2.6+2.4+1.6

-1.6-3.3-1.5+3.6injectionsTillth

day(<637

20(.03)§162 (.25)272 (.43)42(.07)900260

( .29)270 (.30)198 (.22)65 (.07)

TUMOH VOL.

18th day(cu. mm.)

TCMOR-GROUP*

Controls +4.5 637 1660FU 50 2X5Î -2.7 20(.03)§ dead 5/10FU 25 2X5 +1.1 162 (.25) 659(.40)§ 5/23FU 12.5 2X5 +2.6 272 (.43) 1220 (.74) 3/206-MP 50 2X5 +2.4 42 (.07) 65 (.04) 6/14Controls +1.6 900 2350FU 50 2X3# -1.6 260 (.29) 1030 (.44)FO 16.5 2X3 -3.3 270 (.30) 646 (.27)FO 12.5 2X3 -1.5 198 (.22) 828 (.35)6-MP 25 2X3 +3.6 65 (.07) 70 (.03)

*FU = 5-fluorouracil.6-MP = 6-mercaptopurine.FO = 5-fluoroorotic acid.

t Number of mice that were tumor-free either at death or on the 18th day/total number of mice.ÎTwo weeks' treatment with five injections per week.§The numbers in parentheses are the fractions of the volumes of the untreated controls.# Two weeks' treatment with three injections per week.

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312 Cancer Research Vol. 18, April, 1958

into these mice and withdrawn and injected intorecipient mice. No tumors developed. It shouldbe emphasized that in these experiments treatment was continued for only 7 days by intraperi-toneal injection, starting 1 day after transplantation. There was no increase in survival time if thedrugs were injected subcutaneously or intramuscularly. It should be noted that OPSPA and6-mercaptopurine are ineffective against this tumor, and that nitrogen mustard, colchicine derivatives (7), Sarkomycin, Fumigillin, TEM (24),and thioguanine and azaserine, under proper conditions of dosage (16), have been reported to havesignificant activity against this tumor.

TABLE 5

EFFECTSOFDRUGSONTHEE OTTIMAMMARYADENOCARCINOMA

Drugs given daily by intraperitoneal injection for 10 daysstarting on the day of transplantation. Animals

were sacrificed on 15th day.

DrugControls

FUFU

6-MP

DosengAs/day)201010Bodywt.change(gm.)+0.6-0.8+0.5-2.5Tumorvolume(cu.

mm.)26506961910¿05RatioT/Cvol».0.260.720.078

The effects of 5-fluorouracil combined withother compounds on this tumor are given in Table8. There was no significant effect, on the survivalof mice, of an initial starvation to a loss of 7.4 gm.There was no suggestion of potentiation in thistumor of fluorouracil combined with 6-mercapto-

VOL.22,000"

15,000

10,000'

5,000-

FLEXNER-JOBLING

FU 25 MG/KG/7O7) FO 12 MG/K G/7 (16)

.65

.33

15 DAYS 22AWT.

+ 40+ 20

0-20

CHART 8.—Effects of 5-fluorouracil and 5-fluoroorotic acidon the Flexner-Jobling carcinoma. Tumor vol. (cu. mm.) andbody weight change (gm.) vs. days after transplantation.X = controls; O = treated.

.35

15 22

TABLE 6

COMBINATIONEXPERIMENTS,FLEXNER-JOBLINGCARCINOMA,MEASUREDONISTH DAY

Group

Controls5-Fluorouracil6-Mercaptopurine5-Fluorouracil

+6-Mercaptopurine

Controls5-FluorouracilAzaserine5-Fluorouracil

-(-Azaserine

Controls5-FluorouracilOPSPA5-Fluorouracil

+OPSPA

Controls5-FluorouracilUrethan5-Fluorouracil

+ Urethan

Controls5-FluorouracilA-methopterin5-Fluorouracil

+A-methopterin

Controls5-Fluorouracil5-Bromouracil5-Fluorouracil

+5-Bromouracil

Dose(mg/kg/day)18512S122.5122.5120.5120.5li30012300250.6250.610SO1030Bodywt.change(gm.)+31+28+33+22+26+25+20+44+21+

15+36+

15+44+32+34+24+40+35+37+

9+39+20+39+30Tumor

volume(cu.mm.)137021505753517801010908145030201020246035212,10015,3006,7503,83013,4006,5008,3507,14010,2007,65011,0003,070RatioT/Cvol».1

56.42.026.57.51.82.84.81.111.17.56.32.49.63.54.7«1.08.31

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HEIDELBERGER et al.—Studies on Fluorinated Pyrimidines. II 313

purine or OPSPA, in contrast to the findings withS-180 and Flexner-Jobling carcinoma. This resultmight have been anticipated, since the latter twocompounds have no effect on this tumor. A typicalexperiment is also cited to show that a completely5-fluorouracil-resistant line of the tumor has beenobtained.

L1210 leukemia.—The survival times of micebearing this tumor in ascites form, under a varietyof treatments, are shown in Chart 10. Here,

TABLE7

EFFECTOFDRUGSONTHEWALKER256CARCINOSARCOMADrugs given daily for 14 days by subcutaneous injection, start

ing on the day of transplantation. Sacrificed on the15th day. Ten rats per group.

GroupControlsFUFUFOFOFOTEM

Dose Body weight Tumor(mg/kg/day)20105025100.1change(gm.)+94+81+85+11+11+51+94wt.(gm.)37.68.518.19.115.813.10.6Ratio

T/C weight

0.230.480.240.420.350.016

5-fluorouracil and 5-fluoroorotic acid appear to beequally effective, and both more so than 6-mer-captopurine given at maximum tolerated dose.With the combination of 5-fluorouracil and |5-fluo-roorotic acid, a doubling in mean survival time wasobserved, with the earliest deaths delayed until

EHRLICH ASCITES CARCINOMA

1 CONTROLS 144o FU 25/7 63

FO 12/7 51

25

7 10 15 20 DAYS 30 50 55

CHABT9.—Percent survival of mice bearing Ehrlich ascitescarcinoma vs. days after transplantation.

TABLE 8

EFFECTSOFDRUGSONSURVIVALOFMICEBEARINGEHRLICHASCITESCARCINOMA

GROUPControlsStarved 5 days to —7.4gm.5-Fluorouracil

Controls5-Fluorouracil5-Fluoroorotic5-Fluorouracil

+5-Fluoroorotic

Controls5-Fluorouracil5-Bromouracil5-Fluorouracil

+5-Bromouracil

Control, susceptiblef5-Fluorouracil, susceptible5-Fluoroorotic, susceptible

Control, resistant5-Fluorouracil, resistant5-Fluoroorotic, resistant

Controls5-Fluorouracil6-Mercaptopurine5-f'luorouracil

+6-Mercaptopurine

Controls5-FluorouracilOPSPA5-Fluorouracil

+OPSPA

* The ratio T/C survival =

DOSE(mg/kg/day)

SuBVIVAL DAYS

25

10101010

15¿015¿0

2512.5

2512.5

1212I*12

127

12. 7

av. survival of treated mice

Av.11.614.235.2

9.619.220.222.8

9.223.611.021.4

12.622.421.8

16.416.425.411.420.715.021.1

11.421.412.422.2

Extremes7-22

10-2111-639-15

17-2013-2617-27

9-1019-338-16

15-28

10-1618-2614-82

13-1714-2014-84

7-2211-437-2610-47

10-1418-2411-1616-28

RATIO*T/C SURVIVAL

1.123.03

2.002.102.37

2.561.202.33

1.781.73

1.001.55

1.811.321.S5

1.881.101.95

av. survival of control mice 't The term "susceptible" refers to the usual Ehrlich ascites tumor used throughout these

experiments; the term "resistant" refers to the 5-fluorouracil-resistant Ehrlich ascites tumordefined under "Materials and Methods."

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314 Cancer Research Vol. 18, April, 1958

the 19th day. No suggestions of potentiation werefound in the combination of 6-mercaptopurinewith either 5-fluorouracil or 5-fluoroorotic acid.

Through the generosity of Dr. L. W. Law, wewere able to test our compounds on his 6-mercap-topurine-resistant line of the L1210 leukemia. Theexperiments in Chart 11 show that the tumor wasindeed resistant to 6-mercaptopurine, but thatboth 5-fluorouracil and 5-fluoroorotic acid pro-

L-1210 LEUKEMIA, ASCITES FORM

a>X CONTROLS 10

o FU 25/7

A FO 12.5/7

+ 6MP 50/7

«FU 12.5/7 t6MP 7.5/7

a FO 6.75/7+6MP 7.5/7

FU 12.5/7 +FO 6.75/7

CHART10.—Percent survival of mice bearing L1210 leukemia vs. days after transplantation.

L-I2IO LEUKEMIA,6MP RESISTANT LINE (LW.LAW)

X CONTROLS 10

O FU 25/7

A FO 12.5/7

4- 6MP50/7

FU I2.5/7+6MP 75/7

FO 6.75/7 + 6MP 75/7

FU 125/7 + FO 6.75/7

IO 15 DAYS 20 25

CHART11.—Percent survival of mice bearing the 6-MP-resistant line of L1210 leukemia vs. days after transplantation.

longed the life of these animals. Thus, we considerthat there is no cross-resistance. Although the prolongation of survival was not as great as we observed in the susceptible line, this is not consideredto be a partial resistance, because the differences inappearance, behavior, and growth rate of the twotumors do not permit a rigorous quantitative comparison. In this tumor, as well, the combination of5-fluorouracil and 5-fluoroorotic acid appeared tobe beneficial.

Yoshida osciles sarcoma.—In this tumor, asshown in Chart 12, 5-fluorouracil produced a very

significant prolongation of survival, considerablymore than 5-fluoroorotic acid. However, OPSPAproduced cures in the three rats tested, an effectreminiscent of that produced by Nitromin (9). Itis of interest that we obtained 60 per cent curesin rats treated for prolonged periods with the combination of 5-fluorouracil and 6-mercaptopurine,although the latter compound was ineffectiveagainst this tumor. Relatively little work was carried out with this tumor because of the survivalfor a considerable time by a few of the untreatedcontrols.

YOSHIDA ASCITES SARCOMA

X CONTROLS 18o FU 25/7 IIu FO 12.5/7 9«FU 12.5/55 •*•6MP 2.5/55 5

•FU 12.5/55 5v FU 20/7 + 6MP 5/7 4

CHART12.—Percent survival of rats bearing the Yoshidaascites sarcoma vs. days after transplantation.

NOVIKOFF HEPATOMA

100 X CONTROLS

° FU 25/7

FO 125/7

+ 6MP 25/7

OPSPA 4/7

9 FU IO/7+6MP 10/7 6

»FU 125/7* FO 6.75/7 6

A F06.75/7 + 6MP 10/7 6

37

25

2620

6

10 DAYS 25 64 80

CHART18.—Percent survival of rats bearing the Novikoffhepatoma vs. days after transplantation.

Novikoff hepatoma.—Incontrast to the findingin the Yoshida ascites tumor of complete cureswith OPSPA, in the Novikoff hepatoma that compound as well as 6-mercaptopurine was ineffective, as illustrated in Chart 13. In this tumor5-fluorouracil produced a considerable increase insurvival time, more so than fluoroorotic acid, withthree rats surviving for 64, 70, and 81 days. Therewere no advantages to any of the combinationstried.

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HEIDELBERGERet al.—Studies on Fluorinated Pyrimidines. II 315

Inactive compounds.—In Table 9 it is shownthat 2-thio-5-fluorouracil, 2-methyl- or ethyl-mercapto-5-fluorouracil, and 5-fluorocytosine werewithout significant activity against S-180, Flexner-Jobling carcinoma, and Ehrlich ascites carcinoma.

HlSTOLOGICAL STUDIES

Tissue studies to show the influence of 5-fluoro-uracil and 5-fluoroorotic acid on the morphologyof transplantable tumors were carried out in miceimplanted subcutaneously with tumor particles(S-180, Sarcoma A-l) by trocar, or with cell suspensions containing 100,000 cells (Ehrlich ascitestumor) or 20,000,000 cells (L1210). Subcutaneous(remote from the site of implantation) or intra-abdominal treatment with 5-fluorouracil (25 mg/kg) and 5-fluoroorotic acid (12.5 mg/kg) was

necrosis was also more marked than in the controls. In the experiments with S-180, sections oftumors excised after 12 or 4 days after the lasttreatment were also examined. In these tumorsthe differences between treated and control micewere much less conspicuous. Giant cells (6),which are frequently observed in S-180 undergoingtreatment with 6-mercaptopurine, were only occasionally found. Arrested growth, which is notrare after administration of 6-mercaptopurine(25-50 mg/kg), did not occur in the animals whichhad received 5-fluorouracil and 5-fluoroorotic acid.

The lymphoma-like masses which developedafter the subcutaneous implantation of cell suspensions of L-1210 responded in a similar manner tothe two fluoro compounds. In the control animalsinfiltrati ve growth of lymphoblast-like cells into

TABLE9EFFECTSOFINACTIVECOMPOUNDSONVARIOUSTUMORS

COMPOUND

2-Thio-5-fluorouracil2-Methylmercapto-S-fluorouracil5-Fluorocytosine

2-Thio-5-fluorouracil2-Ethylmercapto-5-fluorouracil5-Fluorocytosine

2-Thio-5-fluorouraciI2-Methylmercapto-5-fluorouracil5-Fluorocytosine

BODY WT. CHANGE

DOSE Controls Treated(mg/kg/day) (gm.) (gm.)

Sarcoma 180, measured on llth day100 +0.6 +1.230 -1.2 -4.935 -0.4 -0.7

Flexner-Jobling carcinoma, measured on 15th day

75 +45 +31+25 +12

VOLUME

10025 +26 + 18

Controls(cu. mm.)

309684234

206019501780

Treated(cu. mm.)

396332268

207014401380

Khrlich ascites carcinomaSURVIVALDAYS

DOSE Av. Extremes(mg/kg/day) Controls Treated Controls Treated

100 11.0 14.6 10-13 8-1960 11.0 13.6 10-13 12-1635 16.4 14.6 15-18 10-18

RATIOT/C VOLUME

1.280.491.14

1.030.740.78

RATIOT/C SURVIVAL

1.281.240.89

started shortly after the implantation and continued once daily for 3-8 days. On the 3d, 6th, and9th day, three animals of each treated group andof an untreated control group were sacrificed, andthe site of implantation or, in the later stages, thetumor growth was removed for microscopic examination. Hematoxylin-eosin staining was used inall instances.

The results were uniform. In the case of thesarcomas S-180 and A-l and the solid Ehrlich carcinoma, only small groups or plaques of tumorcells were found in the subcutaneous fat tissue onthe 3d day when the control animals exhibitedsmall, well circumscribed tumors. On the 6th daythe treated animals had developed larger tumorswith all signs of active growth in the periphery butmany more necrotic areas than were found in thecontrol tumors at that time. This observation waseven more marked in the tumors taken on the 9thday. Although the growth had further increased,

fat and muscle tissue was noticed on the 3d day.A comparable extent of growth was found in twoof the six treated animals on the 6th day, when thecontrol mice had already developed an extensiveproliferation of the pathological tissue. Even onthe 9th day lymphomas were present in one outof three mice treated with either 5-fluorouracil or5-fluoroorotic acid. Specimens taken at later dayswere not available. A similar experiment withA-methopterin (2.5 mg/kg every 2d day) showedcomplete absence of subcutaneous lymphomas onthe 9th day; well developed tumors were presentafter 15 days.

The examination of the spleens confirmed theresults obtained with the subcutaneous tumormasses. The enlarged spleens of untreated controlmice were filled with large numbers ot pathologicalcells which caused regressive changes of the normallymphatic structures. This high degree of splenicinvolvement which was present in the controls on

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316 Cancer Research Vol. 18, April, 1958

the 6th day was absent in all mice treated with thefluoro compounds at this time. Animals treatedwith 5-fluorouracil showed a slight degree oflymphoblastic proliferation on the 9th day, buteven that was absent in the mice which had received 5-fluoroorotic acid.

Morphological studies seem, therefore, to indicate that the fluoro derivatives of uracil and oroticacid cause a delay in the development of trans-plantable tumors and leukemia which is mostmarked during the first stages of the growth.

DISCUSSIONThe foregoing data show that the fluorinated

pyrimidines, 5-fluorouracil and 5-fluoroorotic acid,have appreciable tumor-inhibitory properties,while 5-fluorocytosine possesses very little. Because of the fact that results vary so greatly inscreening experiments from laboratory to laboratory and from tumor to tumor, it is impossible tocompare the activity of these compounds with thedozens of other substances that are active againsttransplanted tumors. In order to make some validcomparisons, we have compared these drugs inseveral tumors with 6-mercaptopurine, an activeantimetabolite, and OPSPA, a potent alkylatingagent with which we have had some previousexperience (12). These studies have shown that thefluoropyrimidines have greater activity againstsome tumors, and less against others, as comparedwith 6-mercaptopurine, TEM, and OPSPA. Anumber of combinations have been tried, and thedata suggest that there is potentiation in Sarcoma180 and Flexner-Jobling carcinoma when fluoro-uracil and 6-mercaptopurine are given together.Law has stated3 that 5-fluorouracil has a considerably greater activity against a large variety ofmouse reticular neoplasms than any other compound that he has tested.

It is clear from the experiments on solid established tumors, with respect both to screening andhistológica!findings, that these drugs are growth-inhibitory, but not carcinolytic. However, the experiments in which 5-fluorouracil and x-ray treatment were combined show that destruction of established S-180 and 755 tumors could be accomplished. The most appreciable activity in thisseries of compounds was against the Ehrlichascites carcinoma.

The fact that this series of compounds was devised with reasonable deductions based on theobservation (15, 22) of some degree of a metabolicdifference between tumors and normal tissues intheir utilization of uracil for nucleic acid biosynthesis should be a stimulus for continued efforts

*Private communication.

to approach the problem of tumor chemotherapyby intensification of fundamental research aimedat discovering new exploitable biochemical differences between normal and neoplastic tissues. Itwould be reasonable to suppose that ribosides of5-fluorouracil might have increased activityagainst certain tumors. The results of the screeningof 5-fluorouridine and 5-fluoro-2'-deoxyuridine

against several transplanted tumors will be reported separately. Studies on the distribution,excretion, metabolism, and biochemical mechanisms of action of these compounds are reportedin the accompanying papers (3, 5, 8).

SUMMARY1. The activity of a new series of fluorinated

pyrimidines has been screened against severaltransplanted tumors.

2. In general, the activity of 5-fluorouracil wasgreater than that of 5-fluoroorotic acid. 5-Fluoro-cytosine, 2-methylmercapto-5-fluorouracil, and2-thio-5-fluorouracil did not have appreciabletumor-inhibitory activity.

3. The toxicity of 5-fluorouracil was characterized by delayed deaths, hematopoietic depression, diarrhea, weight loss, and hemorrhages in thelungs and intestines.

4. These compounds acted as tumor growth-inhibitors rather than as carcinolytic substances.

5. The activity of 5-fluorouracil was considerably greater than that of 6-mercaptopurine andOPSPA on the Ehrlich ascites carcinoma, Novi-koff hepatoma, and L1210 leukemia, and less thaneither or both of those compounds on Sarcoma 180,755 and E 0771 mammary adenocarcinomas,Sarcoma A-l, Flexner-Jobling carcinoma, Walker256 carcinosarcoma, and Yoshida ascites sarcoma.

6. Some potentiation of activity against thesolid tumors has been obtained when 5-fluorouracil was combined with 6-mercaptopurine, 5-bromo-uracil, or x-ray treatment.

ACKNOWLEDGMENTSIt is a great pleasure to acknowledge the pleasant and pro

ductive collaboration with the scientists of Hoffmann-LaRoche, Inc. The synthesis of the new compounds has beencarried out by Dr. R. Duschinsky and Mr. E. Pleven (10).The bacteriological work has been done by Mr. J. Scheiner(23), and the preclinical toxicity studies in dogs and monkeyshave been carried out by Dr. T. Wong, W. Benson, and E. L.Severinghaus (25). The initial clinical studies have been donein the University of Wisconsin Hospitals by Drs. F. A.Mclver, A. R. Curreri, R. F. Schilling, and H. Waisman (18).

REFERENCES1. ARMSTRONG,M. D., and LEWIS, J. D. The Toxicity of

o and p-Fluoro-Phenyl-DL-alanine for the Rat. J. Biol.Chem., 188:91-95, 1951.

2. BOLLAG,W. Tierexperimentelle Untersuchungen mit dem

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HEIDELBERGERet al.—Studies on Fluorinated Pyrimidines. II 317

Cytostaticum 5-Fluoruracil, Schweiz Med. Wchnschr.,87:817-20, 1957.

3. BOSCH,L.; HARBERS,E.; and HEIDELBERGER,C. Studieson Fluorinated Pyrimidines. V. Effects on Nucleic AcidMetabolism in Vitro. Cancer Research, 18:335-43, 1958.

4. BUFFA,P., and PETERS,R. A. The in Vivo Formation ofCitrate by Fluoroacetate and Its Significance. J. Physiol.,110:488-500, 1949.

5. CHAUDHURI,N. K.; MONTAG,B. J.; and HEIDELBERGER,C. Studies on Fluorinated Pyrimidines. III. The Metabolism of 5-Fluorouracil-2-C14 and 5-Fluoroorotic-2-Cu Acidin Vim. Cancer Research, 18:318-28, 1958.

6. CLARKE,D. A.; PHILIPS, F. S.; STERNBERG,S. S.; andSTOCK,C. C. Effects of 6-Mercaptopurine and Analogs onExperimental Tumors. Ann. N.Y. Acad. Sc., 60:235-43,1954.

7. CREECH,H. J.; HAUSCHKA,T. S.; HANKWITZ,R. F., JR.;LITTLETON,B. J.; and ANDRE,J. Studies with Four Distinct Lines of the Ehrlich Ascites Tumor for the Evaluation of Twenty-Seven Potential Inhibitors. Cancer Research Suppl. 3, pp. 47-51, 1955.

8. DANNEBERG,P. B.; MONTAG,B. J.; and HEIDELBERGER,C. Studies on Fluorinated Pyrmidines. IV. Effects onNucleic Acid Metabolism in Vivo. Cancer Research, 18:829-34,1958.

9. DRUCKREY,H.; SCHMÄHL,D.; DANNEBERG,P.; KAISER,K.; NIEPEB, H. A.; Lo, H. W.; and MECKE, R., JR.Vergleichende Prüfungder chemotherapeutischen Wirkung von N-oxyd-Lost und anderen alkylierenden Substanzen auf Tumoren von Ratten. Arzneimittel-Forsch.,9:539-50, 1956.

10. DUSCHINSKY,R.; PLEVEN, E.; and HEIDELBERGER,C.The Synthesis of 5-Fluoropyrimidines. J. Am. Chem. Soc.,79:4559-60, 1957.

11. FRIED,J., and SABO,E. F. 9-o Fluoro Derivatives of Cortisone and Hydrocortisone, J. Am. Chem. Soc., 76:1455,1954.

12. HAKALA,M. T.; LAW,L. W.; and WELCH,A. D. InhibitoryActivity of 6-Azauracil, 6-Azauracil Methyl Sulfone andRelated Compounds on the Growth of Mouse Lymphomasand Sarcoma 180. Proc. Am. Assoc. Cancer Research,2(2): 113, 1956.

13. HEIDELBERGER,C., and BAUMANN,M. E. Studies onOPSPA. I. The Effect of Several Phosphoramides onTransplanted Tumors. Cancer Research, 17:277-83, 1957.

14. HEIDELBERGER,C.; CHATIDHURI,N. K.; DANNEBERO,P.;MOOREN,D. ; GRIESBACH,L. ;DUSCHINSKY,R. ; SCHNITZER,R. J.; PLEVEN,E.; and SCHEINER,J. Fluorinated Pyrimidines, a New Class of Tumor-Inhibitory Compounds.Nature, 179:663-66, 1957.

15. HEIDELBERGER,C.; LEIBMAN,K. C.; HARBERS,E.; andBHARGAVA,P. M. The Comparative Utilization of Uracil--2-C" by Liver, Intestinal Mucosa, and Flexner-JoblingCarcinoma in thÃR̈at. Cancer Research, 17:399-404,1957.

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17. McCoMBiE, H., and SAUNDERS,B. C. Alkyl Fluorophos-phonates, Preparation and Physiological Properties. Nature, 157:287-89, 1946.

18. MclvER, F. A.; CURHERI,A. R.; MEYER,O. O.; SCHILLING,R. F.; and WAISMAN,H. Clinical Studies with 5-Fluoro-uracil. Proc. Am. Assoc. Cancer Research, 2(3): 230, 1957.

19. MILLER,J. A.; MILLER,E. C.; and FINGER,G. C. On theEnhancement of the Carcinogenicity of 4-Dimethylamino-azobenzene by Fluoro Substitution. Cancer Research,13:93-97, 1953.

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1958;18:305-317. Cancer Res   Charles Heidelberger, Lois Griesbach, Betty Jo Montag, et al.   TumorsStudies on Fluorinated Pyrimidines: II. Effects on Transplanted

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