Structure-Activity Relationships among Purines …...structure with activity. Studies with...

Structure-Activity Relationships among PurinesRelated to 6-Mercaptopurine*

DONALDA. CLARKE,GERTRUDEB. ELION,GEORGEH. HITCHINGS,ANDC. CHESTERSTOCK

(Division of Experimental Chemotherapy, Sloan-Kettering Institute for Cancer Research, New York, N.Y.,and the Wellcome Research Laboratories, Tuckahoe, N. Y.)

The systematic investigation of purities asantagonists of nucleic acid synthesis in bacteria,viruses, and neoplasms has been under way inthese laboratories for a number of years (6, 29, 30,33, 47, 49). The purines and their analogs havebeen studied in a wide variety of biological systems in an attempt to correlate details of chemicalstructure with activity. Studies with Lactobacilluscasei (14, 23, 30) revealed certain structure-activity relationships which formed the basis forfurther syntheses and for the examination of thesecompounds against neoplastic growth. The findingthat 6-mercaptopurine (6-MP) is inhibitory toSarcoma 180 (8) and produces a high percentageof regressions of this tumor led to a more intensivestudy of its activity in other animal tumors (8,45, 48), its mechanism of action in microorganisms(20, 21, 36), and its metabolic fate in the mouse(12) and in man (28). Clinical investigation (35)showed it to be effective in producing remissions inhuman leukemias.

The studies presented here deal with the relationship between structure and activity amongsubstances related to 6-mercaptopurine. Studiesof this type are in order quite generally to determine the structural specificities of biologicalactivities. They possess both informational andpractical significance, since an improvement in thetherapeutic index of the drug under considerationis always a possible outcome. In the present study,no outstanding derivative other than thioguaninehas been discovered, but a number of derivativeshave been found to possess antitumor activity of

* This investigation was supported in part by a researchgrant (C-2026) from the National Cancer Institute of the National Institutes of Health, Public Health Service, in part by aninstitutional research grant (INSTR-10) and a grant-in-aid(CH-22) from the American Cancer Society, and in part by agrant from the Charles F. Kettering Foundation to the Wellcome Research Laboratories.

Preliminary accounts of this work have been given (10, 17).

sufficient interest to warrant further investigations.

MATERIALS AND METHODSTumor inhibition test.â€”The procedure for testing com

pounds for inhibition of S-180 has been described previously(8, 46). However, since there have been some refinements intechnic since that time, further details are given below.

Donor mice received bilateral subcutaneous implants ofS-180, and the tumors were permitted to grow for 7 days. Adonor mouse was sacrificed during the 7th day of tumorgrowth, and the two masses were dissected free into a pettidish. A specimen of the tumors was removed for a sterilitytest; the specimen was incubated at 37Â°C. for 72 hours in athioglycollate-ascitic fluid medium. Aseptic technic was employed throughout this procedure. The remaining tumor tissuewas drenched with Locke-Ringer solution (pH 7.0) containing100 units of penicillin/ml. Debridement of the tumor tissuewas done, and that tissue presenting, grossly, good appearancewas cut into small, uniform implant pieces weighing about5 mg. wet weight. Fifty such pieces were cut from the tumortissue from one donor. These pieces were randomized in thepetri dish into ten groups of five.

Female HalCR/Swiss mice,1 18-22 gm., were selectedfrom a common (as to source) population. The mice wereweighed, organized into ten groups of five mice per group, andcaged, two groups in each cage. The mice were supplied withwater and Purina Laboratory Chow supplemented with sunflower seeds and lettuce leaves ad libitum. At an appropriatetime, the animals used in the experiments were implanted, eachwith its previously designated tumor fragment. Implantationwas achieved by means of a loaded trocar which was introducedsubcutaneously in the groin region; the trocar was advancedto the right axillary region, where the fragment was discharged.

Nine of these experimental groups were designated to receive therapy with chemicals being examined for possible tumor-inhibitory capacity. The tenth group served as a control. Acommon diluent was used for the nine chemicals under study;the control mice received injections of this diluent alone.

Twenty-four hours after implantation of the tumors,therapy was initiated. A selected daily dose of a candidatecompound was divided into two equal parts and injected atapproximately 9:15 A.M.and 4:45 P.M.each day for 7 successive days. An exception was the 1st day of therapy when butone injection, that in the afternoon, was given. Thus, the micereceived actually 65 days of treatment with the chemicals.

The experiments were terminated the day following the lastinjection. The animals were weighed, and the average weightwas calculated for each experimental group both on the day of

Received for publication December 2, 1957. 1 Millerton Research Farm, Inc., and Taconic Farms.

445

on June 13, 2020. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

446 Cancer Research Vol. 18, May, 1958

implantation and the day following completion of therapy.Changes in weight which might be attributable to therapy werecomputed and compared with similar values obtained for thecontrol group. Any lethality attributable to the individualagents was noted; an experiment was considered inconclusiveif three or more of the five mice had died during therapy. Thetumors of surviving mice were measured in situ, through theskin, with vernier calipers. The tumor mass was measuredthrough a diameter which, on inspection, appeared to be thegreatest. A second measurement was made through an axisperpendicular to the first measurement. These measurementswere averaged for the group and results expressed in terms ofthe parameter "average tumor diameter." The average tumor

diameter of a treated group was compared with the same parameter for the control; if the average diameter of a treatedgroup was found to be between 0 and 76 per cent of the controlaverage diameter, the compound was considered effective. InTable 1 the ratio of treated :control average tumor diametersis given as "diameter index."

Isotonic NaCl solution was employed in these studies as asolvent for water-soluble agents. The usual volume of diluentinjected was 0.5 ml/mouse at each injection. Insoluble chemicals were suspended, in a state of fine subdivision, in 0.5 percent carboxymethycellulose, in isotonic NaCl solution. Theinjection volume was the same as that used for solutions.

Microbiologicalstudies.â€”Allthe compounds were tested fortheir activity on the growth of Lactobacillus casei according tothe screening procedure previously described (23, 82). By addition of supplements to the basal medium (0), the followingmedia were obtained: OFA, 0 + 0.05 m/jg of folie acid/ml;FA+, 0 + 0.62 mug of folie acid/ml.; FT, 0 + l /ig of thymine/ml +10 itg of adenine sulfate/mi; PFA, OFA + 10 Â¿tgofadenine sulfate/ml.

Syntheses.â€”Referencesfor syntheses previously publishedare included in Table 1. The syntheses of new compounds willbe reported elsewhere. All compounds were prepared in theWellcome Research Laboratories.

RESULTSThe effects of the purines on the growth of Sar

coma 180 are given in Table 1. Some typical results with 6-MP are included for comparison.The first eighteen compounds in Table 1 are thosein which the hydrogen of the 6-mercapto group of6-MP has been replaced by an alkyl, aralkyl, oraryl group. In general, such substitution leadsto decreased activity. The only compounds inthis group which show significant tumor inhibitory effects are 6-SCH3, 6-SCH2CeH6, and6-SCH2C6H.iN02(3') purine. Since some of these6-substituted mercaptopurines do show activityand because their metabolic fates in the animalmay be different from that of 6-MP (e.g., 6-SCH3purine is not oxidized by xanthine oxidase,2 theyare being studied further.

Since the physiological purines are either 6-monosubstituted or 2,6-disubstituted, it was ofinterest to test the effect of various substituentsat position 2 of 6-MP. Compounds with CH3, Cl,OH, or SH at position 2 are inactive, and 2-CH3S-6-SH purine is active only at high doses.

1D. C. Lorz, G. B. Elion, and G. H. Hitchings, unpublishedexperiments (cf. 38).

However, 2-amino-6-mercaptopurine (thiogua-nine), which had actually been synthesized priorto 6-MP itself, is about 20-fold as active, andtoxic, as 6-MP. Although the activity of thiscompound against Sarcoma 180 has been reportedpreviously in a preliminary fashion (7), a detailedreport has not been given.

Sarcoma 180 responds to thioguanine therapyin a nicely graded manner, as the dose is increased.Table 2 illustrates this dose-response relationshipfollowing therapy by the intraperitoneal route. Atthe higher dose levels, mortality during the testperiod is appreciable and becomes excessive whendelayed deaths are taken into account. Thioguanine was examined for tumor-inhibitory capacity by direct gastric intubation with dosesranging from 25 to 5 mg/kg/day. This was anexploratory type of experiment with small numbers of animals used for each dose; the tumorswere about equally inhibited irrespective of thedose, and the majority of animals died eitherduring therapy or shortly thereafter. The resultssuggested that the compound would prove at leastas potent an inhibitor by the oral as by the intraperitoneal route. Subsequent experiments wereconducted with the use of dietary supplementation(Table 3). If it may be assumed with fairness thatthese mice consumed 4 gm. of supplemented dieteach 24-hour period, the doses would be 10 mgand 20 mg/kg of body weight per day, respectively, for the 0.005 and 0.01 per cent supplements. Acomparison of the data in Tables 2 and 3 suggeststhat thioguanine administered in this way is lesspotent than by the intraperitoneal route by a factor of 5-10. In the preliminary experiments, inwhich single doses were given by direct gastricintubation, doses of 10 mg or 20 mg/kg producedinhibition of tumor growth to an extent substantially greater than that seen in these dietarysupplement experiments (64 and 61 per cent inhibition, respectively). It may, therefore, be possible that the preferred method of administrationof thioguanine by mouth is a single daily doserather than small increments administered at frequent intervals around the clock.

One unique property of 6-mercaptopurine wasthat its use in therapy rendered the tumor unableto produce viable transplants in a majority ofinstances (8). Tumors removed from animalstreated with thioguanine demonstrated a similarloss in viability when implanted into new hostmice. Occasionally, however, a tumor would develop from either 6-MP or thioguanine-treatedS-180. It seemed possible that the implants whichgrew could be considered to have viable cells innumbers sufficient to exceed the minimum-num-



TABLE 1

EFFECTSOFPURINESONSARCOMA180

CH,

a

OH

PUHINESUBSTITUENTÂ»6

SH

SCHÂ»

SC.H,

SCJIiOH

Other

SCH.COCH,

SCH.COOH

SCH,C,H4C1(2')

SCH,C,H4C1(3')

SCH,CJl4NOI(3')

SCH,C,H4OH(3')

SCH,C,H4CH,(4')

SC.H,

SC.H4CI(4')

SC,H4CH3(40

SC,H4CH,(3')

SC,H4CH3(2')

SB.

SH

SH

REF.13ISÃŽtttt18tÃŽttÃŽtÃŽÃŽtÃŽÃŽtt4DOSE(mgAg)2510066125125250125250400U.',500750125tuo50050012525050012612525025050012525025050012525050012525050050012525025050012550012525050012550012550012525012512525065125250500250250200500DIAMETERINDEX*0.60.50.80.60.70.950.551.050.70.90.750.91.20.950.750.750.550.70.90.71.00.70.750.750.91.050.70.950.750.750.51.150.950.850.90.951.01.01.150.850.900.850.851.01.81.80.851.00.85Av.WT.CHANGE

t-1.5/+1.0-2.0/+1.0-2.0/-0.5-1.0/-1.6-1.0/+0.50/+1.0-2.5/+1.00/+1.0+0.5/+2.5+/1.0/0-2.5/+0.50/+2.50/0+2.0/+0.5+0.5/+1.5+0.5/+0.5+1.0/+4.5-2.0/0+2.5/+1.5-1.0/+1.5-0.5/+0.5+0.5/+2.5+0.5/+1.0-2.0/+0.6-0.5/+1.0-2.0/+0.5-1.0/+2.5-0.5/0+1.0/+2.5+

1.5/+2.5-2.0/+2.5+1.0/+1.5-1.5/+0.5+0.5/00/+2.6+2.5/+1.5-1.0/+1.50/+0.5-1.0/+0.5-0.5/-0.5-1.5/00/-0.5-2.5/-0.5-1.5/0+0.5/+0.5+2.5/+3.S+2.0/+4.5-1.0/-1.0+2.0/+2.0DEATHSPEB

FIVEMICE01012S02S0050180105120141011018004211010100300111100100s4120SEFFECT

ON/..Â«un!-8400+47-11+16-87-310-22-1Â»-28-12-41-18-92-91-84-63-470-28

* Diameters of treated tumors/diameters of control tumors.

t Average weight change of treated animals/average weight change of control animals.Â§Per cent change in titer in the presence of 100 pg/ml of compound, OFA medium.t Synthesis to be published elsewhere.

447



TABLE Iâ€”Continued

SH

Pl'KI.NK SUBSTITUANTS

6

SHOther

SCH, SH

NH, SH

NHCH, SH

HNC.H, SH

NHCtHuM SH

N(CH,), SH

NHC.H, SH

piperidino SH

NHi

NH,

NH,

NH,

NH.

NHi

NH,

NH,

NH,

NH,

NH,

NH,

NH,

SCHi

SC.HI

SCÂ«H,(n-)

SCHiCOOH

SCHiCOCH3

SCH.CJ1,

SCH,C,H4C1(4')

SCH,CJH4CH3(2')

SCH,C,H4CHs(4')

SCH,C,H,C11(3',4')

RET.4ÃŽ151Â»19Ã®1Â»1919Ã•Ã®tÃ®tÃ®ttlttttDOBE(mgAg)5007507507501503005002.6456200200507512512512525025050012550012550012550050012512512525012540012512525050012512525012525050012525025062.5125125125250250125125250125500125500125500125500125250500DUKZTEBINDEX*1.00.80.90.850.851.00.70.750.60.50.350.550.61.160.750.751.01.051.01.051.20.850.850.90.91.00.450.750.70.41.00.950.60.850.00.650.850.750.90.90.850.850.70.30.60.60.50.70.70.551.00.90.850.90.90.751.051.01.11.21.0Av.WT.CHANGE

t+1.5/-0.5-0.5/+3.5-1.5/0+0.5/-1.5-1.0/-0.5+0.5/+3.5-1.0/+4.0-1.0/-1.0-1.5/-1.0-2.5/-1.0-2.5/-1.0-3.0/-0.5-3.0/+0.5+0.5/+4.5-1.5/-0.50/-0.5+0.5/+1.0-l.O/

0.5-0.5/1.5+1.0/-0.5+3.5/+3.0+1.0/+1.5+0.5/+1.0+1.5/+1.6+1.0/+1.0+0.5/-0.5-0.5/+3.0+3.

0/ +4.50/+2.00/+4.5-0.5/0+0.5/+1.5+

1.0/+2.0-1.0/0+0.5/+2.0+1.0/+2.0-1.5/00/+2.0+1.5/+3.00/+2.0-2.0/+0.50/+1.0-2.0/+1.5-1.5/+3.0+0.5/+2.0+0.5/+4.60/+4.5+0.5/+1.00/0-1.0/+2.0+1.5/0-1.5/+1.00/+1.0-1.5/+2.5+

1.0/+2.0-1.5/+1.0+2.0/+2.0-0.6/+1.0-0.5/+2.0-1.0/+0.5-1.0/0DEATHSPER

FIVE MilK010000000120101/103100240100002/10000100014100010003300002610000110111020EFFECTON/..

catti^-470-78-79-84-91-18-60-930-16-16-5600-76-50-40-84-71-39-45

448



TABLE Iâ€”Continued

2NH,NH,NH,NH,aSHOHNH,IVllINE

8CB8TITUENTS6

OtherSHDiacetylSH

DibenzoylSH

8-CH,SH

8-OHSH

8-SHSH

8-SHSH

7-CHjSH

7-CH,SH

8-OHSH

8-OHS

1-CH,S

1-CH,S

3-CH,SH

7-CH,SH

9-CH,SH

7-CH.C.H,SH9-CH,C,HfSO,CH,SOÂ»KSCNBrICNCSNH,REF.ÃŽtÃŽÃŽtti24tÃŽ11t112424ttttt16168089DOSE(mgAg)2.5510Â«5100125125eoo500eoo40060065651502502502501802005007505007501261252602505008080806012516530050012512550050012512525806512512650020SO6012512550020020050012525050030125250DIAMETEBINDEX*0.50.60.70.951.051.00.90.80.90.650.90.950.850.950.01.10.951.150.850.750.750.950.550.551.00.951.00.950.651.10.91.050.80.950.00.91.151.40.80.850.80.850.650.90.951.10.951.050.95Av.

WT.CHANGEt-0.5/+0.5-1.5/+2.5-3.0/0-1.0/+0.5-1.0/+0.5+1.0/+1.0+4.0/+4.6-0.5/+0.5+S.5/+4.5+0.5/+2.0-1.0/+1.5+3.0/+1.0-2.0/-0.5-1.0/-0.5-1.0/+2.5+0.5/-0.5+1.0/+1.5-1.5/-0.50/+1.0-1.0/-0.50/+0.5-0.5/-1.0-2.0/+1.0-1.0/0+0.5/+0.5+

1.5/+1.50/+0.5+2.0/+2.0+1.0/0-1.0/0-3.0/+1.5-1.5/0+2.5/+1.5+3.5/+1.5+2.0/+0.5+2.0/+1.0-1.0/0-2.0/-0.6+2.0/+0.5-1.0/+0.5+1.0/+1.0-1.5/+1.0+0.5/0-0.5/0+0.5/+0.5-1.5/+0.5+2.5/+1.0-0.5/-1.0+1.0/0DEATHSPER

FIVEHICK5040S1303000008022000000110182085010211000000840012460000111s125ErracT

ON/,.carni-89-87-8300-95-75000-15-6400000+

1800-20-15

449



TABLE \â€”Continued

PDBINEBUBSTITCENTB6 Other

CONH,

COOH

COOC.H,

NHCH,

NHCJH,

HNCJI.in)

NHC10H.,(n)

N(CH3),

NHCH,CÂ«Hi

NHCHiCJl4OCHt(4')

NHCH, furyl (a)

NHCtH,(HCl.H,0)

NHC,H4C1(4')

NHC,H4C1(3')

Ã‘ VcH,

OCHi

OC.H,

Unsubstituted (na)

RKF.39SOtIS13ISISIS5t4113ISttISIStIS26tSOso4,

37DOSE(mg/kg)6.2512.

S253012525030125250636375125Â£01253012Â«12520040060BO501001252505001252505001255001002005005001255001251503006512650065250600256012522S462050125125600760100500250500DlAMKTEBINDEX*0.90.850.91.00.90.70.950.80.91.10.90.90.80.90.80.91.20.850.950.951.250.751.150.850.80.951.00.951.050.851.10.951.10.851.20.950.751.050.91.051.051.10.951.250.850.91.21.150.920.45Av.

WT.CHANGE

t-2.5/+1.0-3.5/+1.0-3.5/+0.5+1.0/-1.0-3.0/0-2.5/0-6.0/-0.5-4.0/-1.5-0.5/+0.50/-0.5-2.0/0-0.5/+2.5-3.0/-1.5-2.0/-1.6-3.5/-1.5-1.0/0+1.0/+1.5-1.0/+0.5+2.0/+1.5+1.0/+1.5+

1.0/+1.60/-1.0+1.0/+1.0+1.5/+2.5-1.5/0â€”

0.5/â€”O.Ãâ€”2/00/-0.5+3.0/+2.5+1.5/-1.5-1.0/0+1.0/0+

1.5/+4.5+2.0/+2.0+2.0/-0.50/0-2.5/-0.5+O.Ã–/+1.50/+1.50/-1.5-2.5/-0.5+0.5/0-1.5/-0.5+0.5/+2.0+1.5/+2.0+0.5/+3.0+1.0/+1.5-0.5/+1.0-1.0/-1.00/-2.0DEATHSPEB

FIVEUICE00530402300130322S10001300S00S0600100200000600400S000600000010000EFFECT

ON/,.<Â»Â«Â«'Â§-16-780+7400-180-39-28000000-

70+22-97000-92

450



CLARKE et al.â€”Structure-Activity Relationships among Purines 451

ber-required threshold for growth. The proportionof implants which would be viable, if this weretrue, could be increased substantially by increasing the size of the original implant from treatedtumors. An experiment of this type was done withthioguanine used as the antitumor agent. This experiment is summarized in Table 4. From thesedata it may be seen that the growth of regular-sized implant pieces and implant pieces roughly 3times as large, cut from treated tumors, did notoccur during the first 7 days after implantation. Inthe ensuing weeks, however, growth occurred inabout half of those animals implanted with thelarger pieces, leading to death, whereas growth occurred in only about one-quarter of the mice receiving the smaller pieces with resulting death.

TABLE 2

that organisms resistant to 6-MP possessed cross-resistance to thioguanine (20, 21). Furthermore,the same purine derivatives which blocked the effects of 6-MP were also effective in blocking the effects of thioguanine in these microbial systems(22).

None of the purine derivatives (i.e., adenine,guanine, hypoxanthine, and xanthine) which hadshown moderate to good blockade of the inhibitions of the microbial systems by 6-MP or thioguanine was effective in blocking or otherwisealtering the in vivo antitumor effects. This approach, therefore, could not be utilized to examine

TABLE4Loss OFVIABILITYOFS-180FOLLOWING

THIOGUANINETHERAPY*

TUMQRINHIBITIONANDTOXICITYOFTHIOGUANINEINMICEBEARINGSARCOMA180*Dailydose(mg/kg)012

34

BAv.

tumordiametersÂ±standarddeviation(mm.)11.2

+0.39.4+ 1.2

6.9 + 1.36.4 + 1.15.2 + 0.34.5 + 0.5Av.wt.change(gm.)-1.5-1.5

-1.5-1.0-2.5-3.0Mortality0/200/20

0/200/201/202/20Cumu

lative mortalitythroughid

week3/201/20

2/207/20

13/2017/20Av.

wet wt.of implant

(mg.)Fromtreatedmice:4.815.2From

controlmice:5.615.7*

Twenty mice

hours later intrapedose of 3 mg/kg; tltumors fromsevenmerits

were imnln

Av. tumordiameter after

7 days of growth(mm.)

2.63.7

12.315.9

Ultimaterecovery(per cent)

76M

00

* Twenty mice per group. The compound was given by

the intraperitoneal route once daily for seven successive days.

TABLE 3

INHIBITIONOFSARCOMAiso BYDIETARYSUPPLEMENTATIONWITHTHIOGUANINE*

Per centthioguanine

0.0050

0.010

Av. tumor diameter(mm.iS.D.)

7.311.310.1 + 1.0

6.7 + 1.110.4Â±1.0

Av. wt.change(gm.)

0-1.5-3.0-1.0

* There were 30 mice in each group. The animals were

placed on a diet of ground Purina Laboratory Chow supplemented with thioguanine 24 hours after implantation andcontinued on this diet ad libitum for 7 successive days. Theexperimental groups consumed this diet at about the same rateas their respective controls, i.e., between 3 and 5 gm. of food/24-hour period.

This would seem to verify the assumption that alimited number of cells not adversely affected("killed") by thioguanine were present in treated

tumors. If enough of these cells are implanted intonew hosts, growth will occur.

Thioguanine is closely related to 6-MP structurally but is somewhat over 20 times more potent as an inhibitor of S-180 (7). Evidence hadaccrued indicating that the two agents possesseda similar type of activity in microbial systems in

ments were implanted into two groups of 25 mice. Twodifferent-sized implants were used: the size usually employed inthese laboratories for experiments of this type, ca. S mg. wetweight and a size approximately 3 times as great. The tumorswere palpated and measured at 7-day intervals thereafteruntil either all mice had died as a result of tumor growth ortumors were no longer palpable. Tumors from mice serving ascontrols for the original treatment group were handled similarly. Experiment was terminated after 9 weeks.

TABLE 5

CROSS-RESISTANCEOFAO-MP-RESISTANTS-180TOTHIOGUANINE*

Av. TI.'MUU DIAMETERS AT END Or THEHAPT

DAILYDOSE* (mm.Â¿standard deviation)(mg/kg) 6-MP-R-S-180 Normal S-180

0 8.1 + 0.9 11.6Â±1.23 7.5Â±1.S 5.8Â±1.0

* Twenty mice received subcutaneous implants of S-180/6-

MP on the right side, and the same animals received implantsof normal S-180 on the left side. Ten of these mice served ascontrols, and the remaining ten were treated with thioguanine.Therapy consisted of single daily intraperitoneal injectionsthrough 7 successive days. On the day after therapy wascompleted, all tumors were measured.

the similarity ot action of the two inhibitorsagainst S-180. A tumor line had been developed,however, which was resistant to the inhibitory andcell-damaging effects of 6-MP (S-180/6-MP) (8),and this line did demonstrate cross-resistance tothioguanine. The results of a typical experimentare detailed in Table 5. S-180/6-MP grows at a




slightly slower rate than normal S-180: this hasbeen a consistent finding and is evident in themeasurements in the table. Nevertheless, inhibition of this tumor is clear-cut if an appropriateagent is selected with which to treat it (e.g.,azaserine [9]). Common hosts for both tumorswere employed to rule out any possible host-mediated effect. It is evident from the table thatnormal S-180 responded in the usual manner tothe 3 mg. dose of thioguanine, whereas S-180/6-MP failed to respond.

The high activity of thioguanine led to anexploration of thioguanine derivatives in whichthe 2-amino group was replaced by alkyl, aryl,and heterocyclic amino groups (Table 1). Ofthese, none showed activity except the 2-methyl-amino derivative, which is about -^ as active asthioguanine. Substitutions of the sulfur of thioguanine were made in much the same way as on6-MP, with similar results. The 6-SCH3 and 6-SCH^CeHs derivatives were again active; in addition, the 6-SCH2CoH4Cl(2') compound showed

activity. In each case, however, the minimal effective dose was about 20 times that of thioguanine.

Three derivatives of 6-MP (i.e., CH3) OH, SH)and one of thioguanine (i.e., SH) with substituentson the 8-position were tested. All were inactive.Similarly, 2,6(SH)j-8-OH-purine was inactive and2,8(OH)s-6-SH-purine did not show activity at alevel at least 5 times greater than the minimallyactive level of 6-MP. This is of particular interest,since the latter compound, 6-thiouric acid, is oneof the metabolic products of 6-mercaptopurine inthe human (28).

It had been noted previously in microbiologicalstudies (14, 23) that N-alkylation of the purinering in general decreases or eliminates the growth-stimulatory or growth-inhibitory effects of purinesand that the compounds most closely simulatingthe unalkylated derivatives are the 1-methyl compounds. This was likewise shown to be true withthe antitumor activities. The 1-methyl derivativesof 6-MP and thioguanine are active against Sarcoma 180 but much less so than their parent compounds. The 3-, 7-, and 9-monomethyl derivativesof 6-MP are inactive, as are the 7- and 9-benzylcompounds.

Since 6-MP had originally been prepared because of its close structural similarity to adenineand hypoxanthine, a variety of other 6-mono-substituted purines was synthesized for the samereason. Among these were compounds containingoxidized sulfur, thiocyano, halogen, cyano, car-boxy, carboxamido, thiocarboxamido, alkylamino,arylamino, heterocyclic amino, hydrazino, alkyl,alkoxy, and phenoxy groups in the 6-position. As

can be seen from Table 1, none of these appearedto show any promise as antitumor agents. However, activity has been reported for the 6-chloroderivative (7). The high toxicity of 6-methylpurineis of some interest (44). This compound showed adelayed inhibitory effect on Sarcoma 180, whichwas not apparent in the usual assay time (7). Increasing the length of the alkyl chain to w-propylabolished the toxicity as well as the activity.

All the compounds reported in Table 1 werestudied in the L. casei screening test previouslydescribed (31-33). The microbiological activitiesof several of the compounds have been reportedelsewhere (23) but are included here for the sake ofcompleteness. Only the per cent inhibition in theOF A medium is given in Table 1. For compoundsshowing over 50 per cent inhibition at 100 Â¿ug/mlin this medium, further data are given in Table 6,except for 2-amino-6,8-dimercaptopurine and 2-amino-6-mercapto-7-methylpurine, which have already been reported (23). The results for 6-mercaptopurine and 2-amino-6-mercaptopurine,although also reported previously (21, 23), are repeated in abbreviated form in Table 6 to providea standard of comparison with related compounds.Certain structure-activity relationships are apparent in the microbiological results (cf. 30).Alkyl, benzyl, and phenyl substituents on the sulfur of 6-mercaptopurine reduced its microbiological activity, substituted aryl groups (e.g.,6-SC6H4Cl(4')-purine) did not. Substitution on the2-position of 6-mercaptopurine by CHa, CI, OH,SH, SCHa likewise reduced activity against L.casei, whereas those derivatives with a primary orsecondary amino group on the 2-position retainedstrong activity. Alkylation of the sulfur of thioguanine markedly reduced its inhibitory effect, asin the case of 6-mercaptopurine; however, S-benzyl derivatives were active. The diacetyl anddibenzoyl derivatives of thioguanine also were inhibitory; the position of the acyl groups have notyet been definitely established. Alkylation of thering nitrogens abolished microbiological activityin most cases, except for the 1-CH3 and 7-CH3derivatives of thioguanine. Among the 6-mono-substituted purines the only appreciably activecompounds were 6-SH, 6-COOH, and 6-CH,purine. Purine itself is also a good inhibitor.

The data in Table 6 indicate whether the inhibitory activities of the compounds are reversibleby folie acid (the FA+ medium contains 12.5 timesas much folie acid as the OF A) or by adenine (thePFA medium contains 10 /Â¿g/mlof adenine sulfate). In addition, an inhibitory effect in the PTmedium (where folie acid is replaced by thymineand adenine) suggests an ability of the compoundto interfere with the utilization of exogenous



CLARKEet al.â€”Structure-Activity Relationships among Purines 453

adenine (or thymine). In general, folie acid was nism toward the natural purines and whether thenot efficient in restoring the growth of the micro- antagonism was directed toward any specificorganism inhibited by the compounds in Table 6. purine (e.g. adenine, guanine, xanthine, or hy-In a few cases there was a partial reversal of the poxanthine), experiments were designed to obtaininhibition by folie acidâ€”e.g., 6-SCcH4CH3(3'), this information with representative compoundsS-NHj-e-SCÃHÃCHaCS'),l-CH3-2-NH,-6-S and 6- (Table 7). Each of the physiological purines wasCH3 purinesâ€”and in a few there was complete examined for its ability to reverse the inhibitoryreversal when the concentration of the compound effects of at least two levels of the compound in thewas very lowâ€”e.g., 6-SC6H4CHj(4'), 6-COOH. OFA medium. A similar study with 6-mercapto-Many of the compounds were inhibitory in purine and thioguanine (22, 23) had shown thatPT, with some exceptionsâ€”6-SCeH4CH3(2'), 2- both of these compounds were competitive withCÃHÃ¼NH-G-SH,l-CH3-2-NH2-6-S, 6-COOH and the four natural purines over at least a fourfold6-CHs purines. Reversal of inhibition by adenine range of concentration. On the other hand, none(i.e., in PFA medium) was good in most cases, of the compounds in Table 7 showed this sort ofwith the exception of several of the 6-phenyl- competition, although l-CH3-2-NH2-6-S purinemercaptopurines. came closer than any of the others to being a

Since the data in Table 6 do not show whether competitive antagonist for the natural purines.the compounds exhibited a competitive antago- It is of interest also that the inhibitions caused by

TABLE6MICROBIOLOGICALACTIVITIESOFPURINESSHOWINGSTRONGINHIBITORYEFFECTSONL.com'

MEDITI!COKCN. OFA FA+ PT PFA

IViiiNK (tig/ml) Per cent cbange in titer6-SH 100 -84 -85 -35 -12

6-scÂ«H4Ci(4') io o o -47 o50 -91 -78 -89 -68

100 -92 -92 -94 -80

6-SC,HÂ«CH,(4') 5 -91 0+25 0100 -85 -96 -54 -86

6-SCÂ«H<CHa(3') 50 -36 -21 -IS -10100 -84 -38 -51 -61

6-SC6H4CHa(20 100 -63 -12 0 -38

2-NH2-6-SH* 5 -48 -86 -17 0100 -78 -79 -35 -32

2-CH,NH-6-SH 100 -79 -69 -16 0

Ã-CÃ¼HÃNH-e-SH 100 -84 -78 0 -37

2-CiHâ€žNH-6-SH 100 -91 -94 -87 -S3

2-CoHsNH-6-SH 100 -60 -58 O -152-piperidino-6-SH 100 -93 -89 â€”79 -27

2-NHs-6-SCHjCeHÃ 100 -76 -42 -71 -332.NH2-6-SCH2C6H4a(2') 100 -60 -33 -71 -12

2-NHj-6-SCH2C<Jl,CH,(20 100 -84 -64 -50 -152-NHj-6-SCHsCJL,CH,(3') 100 -71 -37 -69 -38

Diacetyl-2-NH,-6-SH 5 -62 -35 +27 0100 -89 -90 -25 -29

Dibenzoyl-2-NHj-6-SH 5 â€”67 â€”41 0 â€”10100 -87 -84 -35 -45

l-CHÂ»-2-NH2-6-S 100 -64 -16 0 0

6-COOH 5 -32 000100 -78 -64 0 0

6-CH3 5 -67 -33 0 -25100 -95 -94 -19 -25

Unsubstituted l -58 -SO 0 05 -83 -67 0 0

100 -92 -65 0 -14

* The thioguanine used in these experiments was purer and more inhibitory than the batches previously

reported (22, 23). Slight contamination of the compound with guanine, such as was found in experimentswith the 6-mercaptopurine-resistant mutant of L. casei (21), reduces its inhibitory effect considerably.




both purine and 6-methylpurine were relievedpreferentially by adenine, although the reversalby adenine was noncompetitive in nature.

DISCUSSIONIt was realized at the outset of this work that,

in a series of biologically active materials, thetherapeutic index often depends on details of finechemical structure which can be revealed only bythe preparation and testing of a large number ofrelated compounds (34). Because of the specificityof toxic responses, it is often not possible to predictthe effects of chemotherapeutic agents in onespecies on the basis of results in another. Thus,high doses of 6-mercaptopurine produce a pleuraleffusion in the rat but not in any other animal

on eleven compounds which strongly inhibit thegrowth of L. casei but which do not affect thegrowth of Sarcoma 180 in mice. This discrepancyis not unexpected, since, in addition to differencesin the nucleic acid metabolism of the tumor andL. casei, one is dealing also with the difference between an in vivo and an in vitro system. In the invivo system the differential toxicity to host andparasitic tissue becomes of prime importance, aproblem which is not present in an in vitro system.In addition, such phenomena as detoxification,excretion, and lack of absorption may play significant roles in preventing the drug from reachingthe desired tissue in the necessary concentrations.

The differences between an in vivo and an in

l'i IlINt.

Unsubstituted

TABLE7REVERSALSOFINHIBITORYEFFECTSINL.caseiBYNATURALPURINES

OFA SUPPLEMENTED WITH PCRINE8 (jig/ml)Adenine sulfate Guanine HC1 Xanthine

3 10 3 10 3 10Per cent change in titer

6-CH3

6-COOH*

l-CH,-2-NH:-6-S

2-CH3NH-6-SH

2-NH2-6-SCH2C,H56-SCJL.CK4')

* Sterilized by filtration.

COTÃ•C.(Mg/ml)10so1002010030100so100so1000-91-91-92-81-89-55-77-67-80-18-51

Hjrpoxanthine3 10

-37-49-53-54-580-450-200-15-14-16-14-16-25-10-18000+

10-66-68-72-54-67-20-15-10-1600-56-71-75-34-66-10-270000-53-72-73-58-76-21-15-10-150-10-60-81-80-40-77-21-100-1000

100

1750

-68

-86-92

-18

-48-76

-13

-44-68

-18

-48-77

-14-46-71

-31

-48-70

-17

-43-72

-60-65-75

-46-48

-10-30

0-25

00

-19-52-70

-65-65-75

-18-45

-16-31

00

00

-17-43-68

studied (8). Thioguanine is 10 times as toxic as 6-mercaptopurine to mice, whereas both drugs appear to have equal toxicity in man (27, 42).

Thioguanine differs from 6-MP in an importantaspect of its biological activity. Mice which havebeen intoxicated with thioguanine show little detectable pathology other than splenic depletionand profound depression of bone marrow cellular-ity (43, 44). 6-MP produces pathology in othersystems as well as causing depression of marrowcellularity, but, on a mole for mole basis, thioguanine is strikingly more potent than 6-MP as amarrow depressant.

In an investigation such as this, microbiologicalstudies have a limited usefulness, since they donot supply any information on the metabolic fate,toxicity, or tissue distribution of the drugâ€”factorswhich may determine the therapeutic index of apotential tumor inhibitor. Table 6 contains data

vitro system may sometimes work to advantage aswell as to disadvantage. Thus, among the manyderivatives of 6-mercaptopurine there may be oneor more which can liberate the parent substancein vivo. Obviously, if such a reaction occurred at ahigher rate in tumor than in normal tissues, thedifferential effect of the drug might be greatly enhanced. Among the seventeen compounds reportedhere which showed inhibitory effects on the tumor, only nine are found in Table 6 among thestrong inhibitors of L. casei growth. The fact thatthe others are compounds all theoretically convertible to 6-mercaptopurine or thioguanine invivo may be an indication that such a mechanismmay exist and may yet yield a better tumor inhibitor than any of the parent compounds.

The value of microbiological studies in connection with the testing of tumor inhibitors lies intheir contributions to an understanding of mech-



CLARKE et al.â€”Structure-Activity Relationships among Purines 455

anisms of action of such inhibitors (2, 21, 31).With some compounds (e.g., 6-mercaptopurine,thioguanine), reversal of inhibition has beenachieved by all four physiological purines in acompetitive manner. With others, reversal by thepurines has been partial and not competitive. Ingeneral, it would seem that the better tumor-inhibitors in this series are among the compoundswhich behave as competitive antagonists for thenatural purines.

The greater antitumor activity of thioguanine,as compared with 6-mercaptopurine, and thecross-resistance of the S-180/6-MP to thioguanineare of considerable interest and consistent withthe view that the two purine analogs may bemetabolically interrelated. Presumably, these anti-metabolites are transformed into more complexsubstances, and the ultimate site of activity is acompetitive inhibition of the synthesis of one ormore nucleotides or polynucleotides. It is tempting to postulate that just as hypoxanthine, in theform of a nucleoside or nucleotide, is transformedto a similar product with a guanine moiety (1, 3,26, 40), 6-mercaptopurine may be transformedinto some metabolite of thioguanine. To demonstrate such a transformation convincingly, theisolation of such a product would be required.

SUMMARYThe specificity of action of 6-mercaptopurine

(6-MP) against Sarcoma 180 has been examinedthrough the synthesis and testing of 102 relatedsubstances. The substitution of a variety of othergroups, e.g., alkyl, halogen, cyano, and carboxyfor the mercapto group led in general to less activeor inactive compounds. Other changes on thepurine nucleus such as substitution at positions 2and 8 or alkylation of the ring nitrogens resulted,in general, in loss of activity. The exception to thiswas the 2-amino-derivative (thioguanine) which,like 6-MP, is a close analog of a natural purine.Thioguanine was active against Sarcoma 180 atabout ^Â¡ythe dosage of 6-MP but had about thesame therapeutic index. Replacement of the 2-amino group of thioguanine by alkylamino,arylamino, or heterocyclic amino groups loweredthe activity markedly.

The abilities of the substances to exert anti-metabolic effects in the growth of Lacfobacilluscasei also were examined. The two most activesubstances, 6-mercaptopurine and thioguanine,are clearly antagonists of the correspondingnatural oxygen-containing purines. A number ofderivatives exerted similar effects in both biological systems; however, several substances,which are essentially inactive microbiologically,

exerted antitumor effects. Since these are foundchiefly among S-substituted derivatives, the possibility was considered of metabolic cleavage of suchsubstances with formation of the parent mer-captan.

ACKNOWLEDGMENTS

We are indebted to Samuel Singer and Henry Nathan formicrobiological results, to William H. Lange for chemicalassistance, and to Irving R. Goodman for several of the compounds listed in Table 1.

REFERENCES1. AIIKAMS,R., and HKNTLKY,M. Biosynthesis of Nucleic

Acid Purines. II. Role of Hypoxanthine and XanthineCompounds. Arch. Biochem. & Biophys., 68:109â€”18,1955.

2. BALIS,M. E.; LEVIN,D. H.; BBOWN,G. B.; ELION,G. B.;NATHAN,H. C.; and HITCHINGS,G. H. The Effects of 6-Mercaptopurine on Lactobacittus casei. Arch. Biochem. &Biophys., 71:858-66, 1957.

3. BALIS,M. E.; LEVIN,D. H.; BROWN,G. B.; ELION,G. B.;VANDERWEBFF,H.; and HITCHINQS,G. H. Utilization ofSome Purine Riboside Derivatives by Laclobacittus casei.3. Biol. Chem., 199:227-34, 1952.

4. BEAMAN,A. G New Syntheses of Purine, J. Am. Chem.Soc., 76:5633-36, 1954.

5. BULLOCK,M. W.; HAND,J. J.; and STOKSTAD,E. L. R.Syntheses of 6-Substituted Purines. J. Am. Chem. Soc.,78:3693-96, 1956.

6. BUHCHENAL,J. H.; BENDICH,A.; BROWN,G. B.; ELION,G. B.; HITCHINQS,G. H.; RHOADS,C. P.; and STOCK,C. C.Preliminary Studies on the Effect of 2,6-Diaminopurine onTransplanted Mouse Leukemia. Cancer, 2:119-20, 1949.

7. CLABKE,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.

8. CLARKE,D. A.; PHILIPS,F. S.; STERNBERG,S. S.; STOCK,C. C.; ELION,G. B.; and HITCHINGS,G. H. 6-Mercaptopurine: Effects on Mouse Sarcoma 180 and in NormalAnimals. Cancer Research, 13:593-604, 1953.

9. CLARKE,D. A.; PHILLIPS,F. S. ; STOCK,C. C. ; ELION,G. B.;and HITCHINGS,G. H. Combined Chemotherapy of MouseSarcoma 180: Azaserine and 6-Mercaptopurine. AbstractsAm. Chem. Soc., Kansas City, 1954, p. 12M.

10. ELION, G. B. Structure-Activity Relationships. Proc.Royal Soc. Med., 60:7-8, 1957.

11. . Some New N-Methylpurines. Ciba FoundationSymposium on the Chemistry and Biology of Purines, p.39. London: J. & A. Churchill Ltd., 1957.

12. ELION,G. B.; BIBBER,S.; and HITCHINGS,G. H. The Fateof 6-Mercaptopurine in Mice. Ann. N.Y. Acad. Sc., 60:297-303, 1954.

13. ELION, G. B.; BURGI,E.; and HITCHINGS,G. H. Studieson Condensed Pyrimidine Systems. IX. The Synthesis ofSome 6-Substituted Purines. J. Am. Chem. Soc., 74:411-14, 1952.

14. ELION, G. B., and HITCHINGS,G. H. Antagonists ofNucleic Acid Derivatives. III. The Specificity of thePurine Requirement of Lactobacittus casei. J. Biol. Chem.,185:651-55, 1950.

15. . The Synthesis of Thioguanine. J. Am. Chem. Soc.,77:1676, 1955.

16. . Studies on Condensed Pyrimidine Systems. XVII.Some Halogenopurines. Ibid., 78:3508-10, 1956.

17. ELION, G. B.; HITCHINQS,G. H.; CLARKE,D. A.; andSTOCK,C. C. Structure Activity Relationships among




Purines Related to 6-Mercaptopurine. Proc. Am. Assoc.Cancer Research, 2:199, 1957.

18. ELION, G. B.; LANGE,W. H.; and HITCHINGS,G. H-Studies on Condensed Pyrimidine Systems. XVI. Purinesand Thiazolo(5,4-<Z)pyrimidines from 4-Amino-S-form-amido-6-mercaptopyrimidines. J. Am. Chem. Soc., 78:Â¡2858-65,1956.

19. . Studies on Condensed Pyrimidine Systems. XIII.Some Amino-substituted Derivatives of Guanine and 6-Thioguanine. Ibid., pp. 217-20.

20. ELION, G. B.; SINGER,S.; and HITCHINGS,G. H. Microbiological Effects of 6-Mercaptopurine. Ann. N.Y. Acad.Sc., 60:200-206, 1954.

21. . The Furine Metabolism of a 6-Mercaptopuiine-Resistant Lactobacillus casei. J. Biol. Chem., 204:35-11,1953.

22. ELION,G. B.; SINGER,S.; HITCHINGS,G. H.; BALIS,M. E.;and BROWN,G. B. Effects of Furine Antagonists on aDiaminopurine-Resistant Strain of Lactobacillus casei. J.Biol. Chem., 202:647-54, 1953.

23. ELION,G. B.; HITCHINGS,G. H.; and VANDERWERFF,H.Antagonists of Nucleic Acid Derivatives. VI. Purines. J.Biol. Chem., 192:505-18, 1951.

24. FISCHER,E. TJeber Thiopurine. Ber., 31:431-46, 1898.25. GABRIEL,S., and COLMAN,J. Synthesen in der Purinreihe.

Ber., 34:1234-57, 1901.26. GETLER,H.; ROLL, P. M.; TINKER, J. F.; and BROWN,

G. B. Study of the Metabolism of Dietary Hypoxanthiueand Xanthine in the Rat. J. Biol. Chem., 178:259-64,1949.

27. HALES,D. R.; JERNER,R. W.; HALL, B. E.; WILLETT,F. M.; FRANCO,J.; and FEICHTMEIR,T. V. Comparisonof Therapeutic Effect of 6-Methylmercaptopurine, 6-Chloropurine, 6-Thioguanine and 6-Mercaptopurine inHuman Acute Leukemia. Clin. Research Proc., 5:32, 1957.

28. HAMILTON,L., and ELION,G. B. The Fate of 6-Mercaptopurine in Man. Ann. N.Y. Acad. Sc., 60:304-14, 1954.

29. HITCHINGS,G. H., and ELION,G. B. The Chemistry andBiochemistry of Furine Analogs. Ann. N.Y. Acad. Sc.,60:195-99, 1954.

30. â€¢ . Chemistry and Biochemistry of AntimetabolitesRelated to the Purines. Proc. Third Internat. CongressBiochem., Brussels, pp. 55-61, 1955.

31. . The Lactobaciilus casei Screening Test. Cancer Research Suppl. 3:66-68, 1955.

32. HITCHINGS,G. H.; ELION,G. B.; FALCO,E. A.; RUSSELL,P. B.; SHERWOOD,M. B.; and VANDEBWERFF,H. Antagonists of Nucleic Acid Derivatives. I. The Lactobacilluscasei Model. J. Biol. Chem., 183:1-9, 1950.

33. HITCHINGS,G. H.; ELION,G. B.; FALCO,E. A.; RUSSELL,P. B.; and VANDERWERFF,H. Studies on Analogs of

Purines and Pyrimidines. Ann. N.Y. Acad. Sc., 52:1318-35, 1950.

34. HITCHINGS,G. H., and RHOADS,C. P. (ed.). Studies onAnalogs of Purines and Pyrimidines. Antimetabolites andCancer, pp. 231-41. Washington: A.A.A.S., 1955.

35. HITCHINGS,G. H., and RHOADS,C. P. (Co-Chairmen),6-Mercaptopurine. Ann. N.Y. Acad. Sc., 60:183-508, 1955.

36. HUTCHISON,D. Biological Activities of 6-Mercaptopurine:Effects on Streptococcus faecalis. Ann. N.Y. Acad. Sc.,60:212-19, 1954.

37. ISAT,O. Eine Synthese des Purins. Ber., 39:250-65, 1906.38. LoRz, D. C., and HITCHINGS,G. H. Specificity of Xanthine

Oxidase. Fed. Proc., 9:197, 1950.39. MACKAY,L. B., and HITCHINGS,G. H. Studies on Con

densed Pyrimidine Systems. XVHI. Substances Relatedto 6-Purine Carboxylic acid. J. Am. Chem. Soc., 78:3511-13, 1956.

40. MAGASANIK,B. ; MOTED,H. S. ; and KARIBIAN,D. Inter-conversion of Purines in the Biosynthesis of Nucleic AcidAllenine and Guanine and of Histidine. J. Am. Chem. Soc.,78:1510-11, 1956.

41. MILLER,C. O.; SKOOG,F. ; OKUMURA,F. S.; VONSALTZA,M. H.; and STRONG,F. M. Structure and Synthesis ofKinetin. J. Am. Chem. Soc., 77:2662-63, 1955.

42. MURPHY,M. L.; TAN,T. C.; ELLISON,R. R.; KARNOFSKY,D. A.; and BURCHENAL,J. H. Clinical Evaluation ofChloropurine and Thioguanine. Proc. Am. Assoc. CancerResearch, 2:36, 1955.

43. PHILIPS, F. S.; STERNBERG,S. S. ; CLARKE,D. A.; andHAMILTON,L. The Effects of Thioguanine in Mammals.Cancer, 9:1092-1101, 1956.

44. PHILIPS, F. S.; STERNBERG,S. S.; HAMILTON,L.; andCLARKE,D. A. The Toxic Effects of 6-Mercaptopurine andRelated Compounds. Ann. N.Y. Acad. Sc., 60:283-96,1954.

45. SKIPPER,H. E. Effects of 6-Mercaptopurine on Experimental Tumors. Ann. N.Y. Acad. Sc., 60:267-72, 1954.

46. STOCK,C. C. Aspects of Approaches in ExperimentalChemotherapy. Am. J. Med.. 8:658-74, 1950.

47. STOCK,C. C.; CAVALIERI,L. F.; HITCHINGS,G. H.; andBUCKLEY,S. M. A Test of Triazolopyrimidines on MouseSarcoma 180. Proc. Soc. Exper. Biol. & Med., 72:565-67,1949.

48. SUGIURA,K. Studies in a Spectrum of Mouse and RatTumors. Cancer Research SuppL, 3:18-27, 1955.

49. SUGIURA,K.; HITCHINGS,G. H.; CAVAIJERI,L. F.; andSTOCK,C. C. The Effect of 8-Azaguanine on the Growthof Carcinoma, Sarcoma, Osteogenic Sarcoma, Lympho-sarcoma and Melanoma in Animals. Cancer Research,10:178-85, 1950.

50. U.S. Patent 2,746,961.



1958;18:445-456. Cancer Res Donald A. Clarke, Gertrude B. Elion, George H. Hitchings, et al. 6-MercaptopurineStructure-Activity Relationships among Purines Related to

Updated version

http://cancerres.aacrjournals.org/content/18/4/445

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/18/4/445To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Structure-Activity Relationships among Purines …...structure with activity. Studies with...

Documents

Transcript of Structure-Activity Relationships among Purines …...structure with activity. Studies with...