The potential utility of thioguanine nucleosides in producing carcinostasis, with tumor cells resistant to thioguanine, has been suggested (9). The a- and @9-anomersof 2'-deoxythioguanosine (TGDR) have been shown tobe active against ascites tumors having either of two typesof resistance to thioguanine (10). The kinases necessaryfor conversion of these nucleosides to nucleotides wereapparently present in the tumor cell lines examined. However, the effectiveness of TGDR against the tumors wasfound to be limited because of an unfavorable ratio in thetwo activities : nucleoside cleavage and phosphorylation ofnucleoside to nucleotide. This problem is not limited tothe use of thioguanosines for carcinostasis, but is involvedin the use of other fraudulent nucleosides for tumortherapy. Birnie et o2. (1) studied the cleavage of 5-fluoro2'-deoxyuridine and attempted to find inhibitors of thereaction. Welch and Prusoff (15) observed the samelimitation in the use of 5-iodo-2'-deoxyuridine.

This report describes a number of experiments conductedto determine the effects of structural modifications on thecleavage of nucleosides. Some of the adenine nucleosidesincluded in the investigation were substrates for adenosinedeaminase. Studies concerning the activity and catabolism of these adenine nucleosides are included.

EXPERIMENTAL

Separation of the components studied was accomplished

1 This work was supported by Contract SA-43-ph-3068 from the

Cancer Chemotherapy National Service Center, National CancerInstitute, National Institutes of Health.

Received for publication August 31, 1964.

by gradient elution from columns of Dowex-1 formats orDowex-50-H, or by one of a variety of paper chromatographic systems. It was necessary in each instance to determine a procedure that would separate the componentsof the incubation mixture and the potential products.The separation system was judged satisfactory only ifhigh recovery was obtained (85—100%). The cleavage ofthioguanine and 6-mercaptopurine nucleosides was routinely measured by an isotope dilution method, using6-thioguanine-8-C'4 or 6-mercaptopurine-8-C'4 to dilute thefree base which was the cleavage product. The adeninenucleosides and their metabolites could, in most instances,be separated on Dowex-50-H columns by gradient elutionwith hydrochloric acid. Two of these adenine nucleosides, cordycepin and homoadenosine, had to be chromatographed at 2-4°C. to avoid chemical decomposition during the separations.

The methods previously described by LePage et at. (9,10) were used for the growth and manipulation of theascites tumor cell lines. Two types of C3H mammarytumors were used : (a) seventh to twelfth transplant generations of those described by LePage and Howard (8),and spontaneous C3H mammary tumors in 9- to 12-month-old C3H females obtained from the Roscoe B.Jackson Laboratories at 6 weeks of age. The latter wereheld in our facilities until such tur@iors arose.

Nucleoside cleavage.—Some difficulty was encountered inestablishing whether the nucleoside cleavage by mousetumor cells, or by extracts prepared from the cells, wasphosphorylytic or hydrolytic. The first experiments were

46

Metabolism of Purine Nucleoside Analog&

G. A. LEPAGE AND IRENE G. JUNGA(Life Sciences Research, Stanford Re8earch Institute, Menlo Park, California)

SUMMARY

A study was made of the cleavage of ribosides and 2'-deoxyribosides of thioguanine, adenine, and 6-mercaptopurine in mouse tissues. Evidence indicated that thiswas a phosphorylytic cleavage. Changes in the sugar moiety from ribose or 2'-deoxyribose to xylose, arabinose, 3'-deoxyribose, 5'-deoxyallose or 6'-deoxyallose prevented the cleavage. A shift in the ribosidic linkage from position 9 to 7 of the purineprevented cleavage, as did esterification of the ribose moiety with acetyl or propylgroups. The nucleoside phosphorylase was found to be active on both a- and @-anomers of 2'-deoxythioguanosine and 2'-deoxyribosyl-6-mercaptopurine. The relative rates of cleavage of these anomers varied with the tissue source. The adenosinedeaminase of mouse tissues was active on @-anomers,but not on a-anomers. Variouschanges in the sugar moiety of adenosine decreased or abolished the adenosine deaminase activity. The Km'S were determined for ribosyl, arabinosyl and xylosyladenine. The substrate affinity was of a higher order for ribosyl adenine. As a resuit, it was demonstrated that ribosyl adenine could be used in combination withxylosyl or arabinosyl adenine to protect the latter two analogs from the adenosinedeaminase of mouse blood, so that they were able to reach subcutaneous tumors viathe circulation and produce some inhibitory effects not otherwise possible.

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Tissuefl-Anomer°a-Anomer―Transplanted

tumorTA32623Mecca

lymphosarcoma97486C3HED11097Ca-755112104Ehrlich108i07Sarcoma

180112110L1210114113Mammary

tumorBC3HIlii72II607287III900886IV143149V283149VI138118Spontaneous

mammary tumorC3HI94138II29264III183124IV11379Normal

mousetissueLiver,BAF@―917886Kidney,BAF1932907Spleen,BAF,921900Liver,C3H900921Kidney,C3H880900Spleen,C3H865880Blood,―

BAF11.0

LEPAGE AND JUNGA—PUrine Nucleoside Analogs 47

conducted by incubating intact ascites tumor cells in vitrowith nucleosides (e.g., thioguanosine [see Addendum]) inRobinson's medium (14) with glucose and a bicarbonatebuffer at pH 7.4. The cells were extracted with perchloricor trichioroacetic acids. The extracts were analyzed forinorganic phosphate by the method of Lowry and Lopez(11), before and after acid hydrolysis, in order to detect anyribose-1-phosphate formed. Materials present in the cxtracts so inhibited color development that the tests wereinconclusive. L1210 ascites tumor cells were used to compare the rate of thioguanosine cleavage for whole cells incubated in Robinson's medium with that obtained for ahomogenate of L1210 cells. Cells used for homogenizationwere incubated 1 zulu. in 3 volumes of ice water, thenhomogenized in a Potter-Elvehjem homogenizer, andtonicity restored with Robinson's medium. The cleavageof thioguanine riboside by equal aliquots of the whole cellsand homogenate was identical (20 mg dry weight of cells,or the equivalent amount of homogenate, cleaved 2.1j@molesin 15 mm. at 38@C.) In another experiment, it wasdemonstrated that the cleavage activity of a wholehomogenate was equaled by an equivalent amount of thesupernatant fluid from centrifugation of the homogenatefor 1 hr. at 16,000 g in an International PR2 centrifuge at2°C. However, repeated experiments with such supernatant fractions showed that : (a) activity was reducedabout 38 % when Tris buffer at pH 7.4 was used instead ofRobinson's medium; (b) activity was almost entirely lostwhen the cell extracts were dialyzed at 2°C.for 5 hr. againstTris buffer at pH 7.4 and was not restored by the additionof inorganic phosphate; (c) freezing and storage overnightat —22°C. led to partial (50 %) loss of activity ; (d) thecleavage rate was linear for 15—20zulu., but dropped offappreciably by 30 mm.

For a period of several months, when the distilled watersupply was obtained from a gas-fired Barnstead still andthe feed water was of low quality, it was not possible to getreproducible assays of the nucleoside cleavage. Waterwhich did give satisfactory results was produced thereafterby feeding water deionized with a Barnstead Bantam Dcmineralizer to the pot of an all-glass still purchased fromCorning Glass Works.

Cleavage of thioguanine riboside and of 2'-deoxythioguanosine by homogenates of mouse tissues (e.g., liver) was

measured, with variations of pH, in Tris-phosphate buffers.The activity was maximal at pH 7.0—7.4,and all subsequent studies were then conducted at pH 7.4. In theroutine assay used for the subsequent studies, the cell

suspension or homogenate was added to chilled 50 mlErlenmyer flasks containing Robinson's medium at pH 7.4to a final volume of 10 ml and also containing 7.5 zmolesof the nucleoside. The flasks were flushed with an atmosphere of 95 % oxygen and 5 % carbon dioxide, closed,and incubated 15 min. at 38CC. The flasks were againchilled, deproteinized with perchioric acid, neutralizedwith potassium hydroxide and stored in a deep freeze at—22°C.until analyzed. Although a sufficient level oftissue cleaved up to 94 % of the nucleoside, the results ofassays in which there was 65 % cleavage or less were routinely used, since in these the nucleoside cleavage was proportional to tissue concentration and was not increased on

. TABLE1CLEAVAGE OF a- AND ft-ANOMERS OF 2'-DEOXYTHIOGUANOSINE BY

Mouse Tissuza

The first seven tumors were used as suspensions of ascites cells.The other tissues were used as homogenates.

a Each figure is the average of duplicate analyses, and is cx

pressed as Mmoles of nucleoside cleaved per gram wet weight perhour. Incubation was in 10 ml Robinson's medium (14) for 15mm. at 38°C. in an atmosphere of 95% oxygen and 5% carbondioxide.

b BAF1 refers to (A/Jax X C57BL/Jax)F,.

C Cells centrifuged, plasma discarded, and cells laked in 3

volumes of ice water immediately before the assay. Since plasmaadded to incubation mixtures had no effect, activity was presentonly in the cells.

doubling the amount of the nucleoside present. Thus substrate was not limiting to the reaction.

In order to resolve the issue of whether cleavage wasphosphorylytic or hydrolytic, an experiment was conductedwith an extract of L1210 cells. The incubation mixturecontained 22 ml of extract prepared as described earlier(1 :4 extract), plus 22 ml of Robinson's medium at pH 7.4and 45 @smolesof thioguanine nboside. After the reactionhad been incubated 15 mm. at 38°C.,the mixture was deproteinized with 3.6 ml of 2.6 M perchioric acid and neutralized to pH 7.0 with 2 MKOH. The KC1O4was removedin the cold. An aliquot of the solution was used to deter

mine, by isotope dilution with thioguanine-8-C'4, theamount of thioguanine formed. This analysis indicatedthat 18.6 @imolesof thioguanine had been formed. A portion

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

TissueChemical agentCleavage rate

48 Cancer Research

TABLE 2

Vol. 25, January 1965

INFLUENCE OF STRUCTURAL V@uuAnoNs ON NUCLEOSIDE CLEAVAGE

TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210L1210

@ L1210L1210

@ . TA3, 6C3HED, L12i0

TA3TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3,6C3HED, L1210TA3,6C3HEDTA3, 6C3HEDTA3, 6C3HEDTA3,6C3HEDTA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L12i0TA3, 6C3HED, L1210TA3, 6C3HED, L12i0TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L1210TA3, 6C3HED, L12i0TA3TA3TA3TA3TA3TA3TA3TA3TA3TA3TA3

7-@-Ribosyltheophylline (BG 43)Triacetyl-6-mercaptopurine ribosideTriacetyl-thioguanosineTripropyl-thioguanosineHomoadenosinePsicofuranineXylosyladenineArabinosyl adenineXylosyl-6-mercaptopurineArabinosyl-6-mercaptopurineThioadenosineArabinosyl thymine94-i@-ribofuranosyl)-6-aminopurineRibosyl-6-mercaptopurineCordycepin3'-Deoxy-3'-S-ethyl ribosyl-6-mercaptopurine5'-Methyl-ribosyl-6-mercaptopurine6-Methyl thioguanosine@9-2'-Deoxyribosyl-6-mercaptopurinea-2'-Deoxyribosyl-6-mercaptopurine2'-S-Ethyl adenosine3'-S-Ethyl adenosine5'-Deoxythioguanosine5'-Deoxy-5'-S-ethyl thioguanosineAllosyl adenineAllosyl-6-mercaptopurineThioguanosineThioguanosine + BG 43

@-2'-Deoxythioguanosine@-2'-Deoxythioguanosine + BG 43

ThioguanosineThioguanosine + allosyl-6-mercaptopurineRibosyl adenineRibosyl adenine + homoadenosine

@-2'-Deoxythioguanosine@-2'-Deoxythioguanosine + 9-@-hydroxyethyladenine

$-2'-Deoxythioguanosine + 9-@-cb1orethyl-6-benzylpurinep-2'-Deoxythioguanosine + 9-@-hydroxyethyl-6-benzylthiopurine$-2'-Deoxythioguanosine + 9-@-hydroxyethyl-6-thiopurine

@-2'-Deoxythioguanosine + 8-bromoadenosine@-2'-Deoxythioguanosine + 8-bromoguanosine@-2'-Deoxythioguanosine + 8-bromoxanthosine@-2'-Deoxythioguanosine+ 8-thioguanosine

fl-2'-Deoxythioguanosine + 8-methyitbioguanosine@-2'-Deoxythioguanosine+ 8-thioadenosine

00000

>60000000010,9.9,19002.8, 3.4, 4.719,23,1524, 21, 2417, 20, 97.2, 7.67.1, 5.35.5,180

<0.9<1.513,10,307.3, 7.3, 2619,10,3219, 9.4, 2610,10,2410, 10, 1913,0,2612, 0, 232639191839384943405150

Each figure is the average of 2—4analyses, conducted with cell suspensions as described for Table 1, expressed as micromoles ofnucleoside cleaved per gram wet weight per hour. Analyses listed as 0 for all three tumors assayed represent cleavage rates probablyless than 0.2 pmoles. Figures for the two allosyl nucleosides could not be scored 0 because of some chemical cleavage in controlsresulting from the analytical procedures. Where combinations of two substrates were used, they were at approximately equimolarlevels.

of the extract (10 ml) was passed through a small (5 X 35mm) column of Dowex-50-H to remove residual thioguanosine. Then an aliquot (4 ml) of this eluate waspassed through a small (5 X 35 mm) column of Dowex-1formate. This would retain all ribose esters, but wouldallow any free ribose to pass through. Analyses for pentose were negative. A second 4.0 ml aliquot was made to0.1 M with perchioric acid and heated 2.5 min. at 100°C.,then neutralized with KOH as before and passed through

a similar Dowex-1 formate column. The effluent wasfound to contain pentose, which was equivalent for thewhole extract to 10.7 @molesof ribose. This was calculated to be 58 % of the amount expected if ribose-1-phosphate had been the other product formed along with thioguaninc. Information on the acid-hydrolysis of ribose-1-phosphate (6) indicates that it should be approximately50 % hydrolyzed by the treatment used. The data therefore support the interpretation that the cleavage was

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

AgentDose,2

treatments!day(mg/kg)Duration

oftreatment

(days). ‘ays1.Saline614.0

±3.3Ribosyl-6-mercaptopurine3069.7±1.3Triacetyl

ribosyl-6-mercapto 40.569.9 ±1.1purineThioguanosine0

.9610.4 ±1.3Triacetylthioguanosine1 .3623 .7 ±1.0Tripropylthioguanosine1 .4616.4 ±3.5Saline

•313.7 ±3.3Ribosyl-6-mercaptopurine30312.3±3.8Triacetyl

ribosyl-6-mercapto 40.5312.8 ±3.1purineRibosyl-6-thioguanine2.737

.9 i0.7Triacetylthioguanosine4.3318.4 ±2.1Tripropylthioguanosine4.7317 .8 ±2.2

TissueNudeosideRate ofDeaminationTA3,

6C3HED, L1210Ribosyl adenine, @-anomer56, 6.6,>100TA3,L1210Arabinosyladenine,a-anomer0TA3Arabinosyl

adenine,@-anomer29TA3,L1210Xylosyl adenine,a-anomer0TA3,L1210Xylosyl adenine, @-anomer28,>76L1210Thioadenosine50TA3,

6C3HED, L1210Homoadenosine,@-anomer0L1210L-Adenosine0TA3,

6C3HED, L1210Allosyladenine0TA3Cordycepin(3'-deoxyadenosine)>100TA3,

6C3HED,L1210Psicofuranine0BAF1mouse bloodRibosyladenine76BAF1mouse bloodArabinosyl adenine,@-anomer20BAF1mouse bloodXylosyl adenine,j9-anomer20TA3Xylosyl

adenine,@9-anomer28TA3Xylosyladenine, j9-anomer + 4X

ofa-anomerlevel17TA3,6C3HED, L1210Ribosyl adenine56, 6.6,>100TA3,6C3HED, L1210Ribosyl adenine + homoadenosine53, 4.2, >100

LEPAGE AND JUNGA—Purine Nvdeoside Analogs 49

phosphorylytic. Recently Paterson and Sutherland (12)have found that the cleavage of 6-mercaptopurine ribosidein Ehrlich ascites tumor cells is a phosphorylytic reaction.

Assays were conducted, with a spectrum of mousetumors and normal tissues, on the cleavage of the a- andf@-anomersof 2'-deoxythioguanosine. For the normal tis

TABLE 3SURVIVAL TESTS OF PURINETHIOL NUCLEOSIDES AGAINST EHRLICH

TGR II Asci@s Timions

sues and for the mammary tumors, homogenates wereused. The others, used as ascites cell tumors, were assayed as cell suspensions. Data obtained from theseassays are presented in Table 1.

Attempts were made to influence cleavage by : (a) substitution on the nucleoside base; (b) changes in the structure of the carbohydrate moiety of the nueleoside, including variations in the sugar itself and esteriflcation of thehydroxyl groups; (c) change of the attachment of sugar tobase from position 9 to 7. Where such changes interferedwith nucleosidic cleavage, the influence of such nucleosideson cleavage of thioguanine nucleosides was determined.Data from such tests are presented in Table 2. Many ofthe changes in configuration of the sugar, but by no meansall of those tested, resulted in complete loss of nucleosidephosphorylase activity. However, addition of inactivenucleosides to the medium in most instances had no effecton the cleavage of active nucleosides. An example of apositive result, inhibition of thioguanosine cleavage byarabinosyl adenine, has been reported (9). The change ofthe ribosidic linkage on the base from position 9 to 7 ledto an inactive nucleoside. Addition of this compound toreaction mixtures containing thioguanosine, at an equimolar level, produced some reduction in the rate. But therate of cleavage of 2'-deoxythioguanosine appeared unaffected. This would seem to encourage further study withother nucleosides having the linkage at position 7. Pikeet at. (13) recently reported that tubercidin, a nucleosidewith an unnatural base, was not cleaved by nucleosidephosphorylase.

Substitution with acetyl or propyl groups on the sugarprevented cleavage. Since it seemed likely that such

TABLE 4

The data are presented as the average survival time, for groupsof 10 mice in each of two experiments ±the average deviation.Treatment (I.P.) was begun 24 hr. after the I.P. transplant of6 X 10' Erhlich TGR II cells per mouse in random-bred Swissfemales weighing 23—25gm.

DEAMINATION OF ADENINE NUCLEOSIDES BY MOUSE TISSUES

Assays were conducted by incubation of a suitable tissue aliquot in 10 ml of Robinson's medium(14) at pH 7.4, containing 7.5 pmoles of substrate, for 15 mm. at 38°C.under 95% O@and 5% CO2.Under these conditions, substrate level was optimal and the rate was linear for at least 20 mm. Ratesare expressed as micromoles per gram wet weight per hour. For blood, the cells contained the totalactivity; washed cells were, therefore, used. Laking the blood cells did not change the level of activity.Tumors were used as cell suspensions.

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ADDITIONS (p@toi.zs)PIODUCTS(@sMor.xs)IRibo@yI

adenineXylosyl!adenineArabinosyladenineInosineHypoxan thineAdeninenudeosides7.5

—

7.5—

7.5—

7.57.5—

——

—

—

7.57.54.87

2.625.082.204.351.65

01.8001.500.98

4.888.125.309.10

TumorAgentDose.2

treatments/day(mg/kg)Duration

oftreatment

(days).

Tumor wei@ts@@ IfliMaximumhost weight

loss(%)TA3Control277±

300TA3Xylosyladenine253205±317TA3Ribosyladenine503268±230TA3Xylosyladenine andribosyl

adenine25and 503175±149S-180Control401E

450S-180Ribosyladenine503409±430S-180Xylosyladenine253375±415S-180Xylosyladenine and ribosyl

adenine25and 503252±358TA3Control197±

230TA3Arabinosyladenine403197±190TA3Arabinosyladenine and ribo

syl adenine40and 803133±313Mammarytumor

IVGControl365±300Mammarytumor

IVXylosyl adenine253357±288Mammarytumor

IVXylosyl adenine and ribosyladenine25

and 503222± 1310

50 Cancer Research Vol. 25, January 1965

esters would be acted upon in vivo to give free nucleoside,tests were performed for carcinostatic activity against an

ascites tumor resistant to thioguanine because of lack ofthe guanosine-5'-phosphate pyrophosphorylase (EhrlichTGR II), where the protection of thioguanosine by esterification might lead to better levels of nucleotide formation.

The results are presented in Table 3. This tumor hasbeen described (5). It is resistant to both 6-mercaptopurineand 6-thioguanine. The esters were used at levels ap

TABLE 5

PRODUCTS OF INCUBATION OF ADENINE Nuci.EosIDE MIXTURESWITH TA3 CELLS

proximately equimolar with the free nucleosides, at thedoses of the latter found to give the best responses insensitive tumor lines. The esterified nucleosides of thioguanine appear to produce a significant inhibition of thesethioguanine-resistant cells, where thioguanosine does not.The latter actually decreases survival time, due probablyto suppression of the host's immune response, as discussedearlier(10).

We have no current explanation for the apparent stimulation of the cleavage of 2'-deoxythioguanosine producedby purines substituted in position 8. The supplies of thesewere very small and permitted only a single experimentwith duplicate samples.

Deamin.a@estudi€8with adenine nucleoside8.—Inthestudies on nucleoside cleavage, a number of adenine nucicosides with sugar moieties other than ribose were tested.Some of these were found to have carcinostatic activity.One, the arabinosyl adenine, was studied in some detailand produced markedly inhibitory effects in certain ascitestumors which were relatively low in adenosine deaminase(3). However, tumors high in adenosine deaminase andsubcutaneous tumors were relatively unresponsive, in thelatter case because of the high adenosine deaminaseactivity of mouse blood (4). The adenosine deaminase ofmouse tissues deaminates arabinosyl adenine, though not asrapidly as it does the natural substrate. Studies weremade of the activity of this enzyme on various adenine

TABLE 6

Incubations were for 15 mm. with 0.50 ggm wet weight of TA3cells in 10 ml of medium. This tissue level is somewhat high for avalid assay on ribosyl adenine, but was desired in order to havesufficient activity on the analogs of adenosine.

INFLUENCE OF ADENINE NUCLEOSIDES ON GROWTH OF SUBCUTANEOUS MOUSE TUMORS

TA3 implants were in (A/Jax d' X C57BL/Jax 9 )F, female mice; S-iSO in random-bred Swiss females. Treatment was started when tumors had become of palpable size and established. Mice weresacrificed 1 day after the last treatment and the tumors were weighed. There were 6 mice in each group.

Differences in tumor weights between groups treated with xylosyl adenine of arabinosyl adeninealone and controls were not significant. Differences between controls and groups treated with eitheranalog in combination with ribosyl adenine were significant at the 0.02 level.

a Twelfth generation transplants of a spontaneous mammary tumor in C3H mice, as described by

LePage and Howard (8).

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

LEPAGE AND JUNGA—PUrine Nucleoside Analogs 51

nucleosides, with both a- and $-anomers tested where theywere available. The relative activities were determinedin the same medium used for the cleavage studies—Robinson's medium with glucose and a bicarbonate buffer atpH 7.4 (14). The pH optimum for this enzyme is verybroad and pH 7.4 is within the area of maximum activity(7). Measurementson the rate of deaminationof various&lenine nucleosides are presented in Table 4. The rateswere found to be the same with suspensions of ascites cells

or homogenates prepared from them as described earlier.The data indicate that adenosine deaminase, unlike thenucleoside phosphorylase, is not active on the a-anomers,but does have activity on the @9-anomersof the xylosyland arabinosyl analogs. A number of other changes in thesugar structure prevented deaminase activity. Block andNichol (2) have recently reported that psicofuranine and arelated nucleoside, decoyinine, are unaffected by adenosinedeaminase from the intestine. A fourfold addition of theinactive a-anomer of xyiosyl adenine had a small butsignificant effect on the rate of deamination of the $-anomer. Homoadenosine, an inactive nucleoside whichwas not acted upon, had a significant effect on deamination of ribosyl adenine by 6C3HED cells at an equimolarlevel. But the other two tumors tested did not show asignificant inhibition.

When equimolar mixtures of ribosyl adenine with xylosyl adenine or arabinosyl adenine were incubated andcompared with the deaminase assays for the individualnucleosides, it was found that the natural substrate,ribosyl adenine, was apparently used in great preference tothe analogs. This is ifiustrated in Table 5 where, becauseof the cleavage which also occurred with ribosyl adenine,it was evident which nucleoside was being used. It appeared that ribosyl adenine could protect xylosyl adenine orarabinosyl adenine from deamination. The substratelevel giving half-maximum activity was determinedfor the adenosine deaminase of TA3 cells. For ribosyladenine, arabinosyl adenine, and xylosyl adenine respectively, these values were 4.25 X 10@, 2.36 X 10@, and3.94 x 10-@ M. It was evident that the ribosyl adeninehad a higher order of affinity for the enzyme.

The potentiality of ribosyl adenine, given in combinationwith the analogs, to protect the analog for passage throughmouse blood to a subcutaneous tumor site was tested.Table 6 presents some data from tests on several tumorswith xylosyl adenine or arabinosyl adenine, alone and incombination with ribosyl adenine. It is evident that theprotection of an analog in this manner is a feasible meansof allowing the analog to reach a tumor through the circulation. This particular example may have no clinical utility,since assays of adenosine deaminase on human blood showsomewhat different results. In human tissues ribosyl,xylosyl, and arabinosyl adenines were deaminated at approximately the same rate (G. A. LePage and I. G. Junga,unpublished data).

DISCUSSION

The initial objective of this study was to increase theutility of thioguanine nucleosides for the treatment ofthioguanine-resistant tumors by reducing the rate ofnucleoside cleavage. This objective was not achieved.

However, potential means of accomplishing this weresuggested. The use of nucleosides esterifled with fattyacid groups appears to have promise, as does the use ofnucleoside derivatives with the sugar moiety attached atposition 7. In addition, it was found that both a- and @9-

anomers of the nucleosides can be acted upon by nucleosidephosphorylase. Since the a-anomer of 2'-deoxythioguanosine is much less toxic to mice than the j9-anomer (10),it may be inferred that the nucleoside phosphorylase ofmouse bone marrow has a relatively low activity on thea-anOmer, or that the kinases are inactive on the a-anomer.

A number of changes made in the sugar moiety abolishednucleoside phosphorylase activity and resulted in newnucleosides with carcinostatic properties. The adenosinedeaminase of mouse tissues showed more substrate specificity than did the nucleoside phosphorylase, was notactive on a-anOmers, and was less active, or had no activity,when any of a number of structural changes in the sugarmoiety were made.

The nucleoside phosphorylase of mouse tissues appearedto be a relatively labile enzyme under the conditionsstudied.

ADDENDUM

Trivial names are used for most of the nucleosides mentioned.The trivial name or abbreviation used for each nucleoside is givenhere, followed by the full name. (Supplied by: (a) Dr. C. C. Chengof Midwest Research Institute; (b) Dr. Howard Bond of theCancer Chemotherapy National Service Center; (c) Dr. KarlFolkers of Stanford Research Institute ; (d) Dr. Leon Goodmanof Stanford Research Institute ; (e) Dr. Roland K. Robins ofArizona State University; (J) Dr. John Montgomery of SouthernResearch Institute.)

7-fl-ribosyl theophylline, BG 43, 7 (8-D-ribofuranosyl)-1 ,3-dimethylxanthine (e); triacetyl-6-mercaptopurine riboside, 2',3',5'-triacetyl-9-(fl-D-ribofuranosyl)-6-mercaptopurine (a); tnacetyl thioguanosine, 2',3' ,5'-triacetyl-9-(fl-D-nibofuranosyl)-2-amino-6-thiopurine (a) ; tnipropyl thioguanosine, 2',3',5'-tnipropyl-9-(fl-D-nibofuranosyl)-2-amino-6-thiopunine (a) ; homoadenosine , 9- (ft-5'-deoxy-D-allofuranosyl)-6-amino-punine (d);psicofuranine, 9-(ft-D-psicofuranosyl)-6-aminopunine (b) ; xylosyladenine , 9-(fl-D-xylofuranosyl)-6-aminopurine (d) (also a-anomer)(d) ; arabinosyl adenine, 9-(8-D-arabinofuranosyl)-6-aminopurine(d) ; arabinosyl adenine, 9-(a-D-arabinofuranosyl)-6-aminopunine(1); xylosyl-6-mercaptopunine,9-(fl-D-xylofuranosyl)-6-mercaptopunine (d) ; arabinosyl-6-mencaptopunine, 9- (8-D-arabinofunanosyl)-6-mercaptopunine (d) ; thioadenosine, 9-(fl-D-4'-thioribofuranosyl)-6-aniinopurine (d) ; arabinosyl thymine, 9- (fl-Darabinofuranosyl)-5-methyl pyrimidine (d) ; L-adeflosifle, 9-(flL-nibofuranosyl)-6-aminopunine (d) ; ribosyl-6-mercaptopurine,9-(fl-D-nibofuranosyl)-6-mercaptopunine (b); cordycepin, 9-(fl-D-3'-deoxy-nibofunanosyl)-6-aminopunine (c) ; 3'-deoxy-3'-S-ethylnibosyl-6-mercaptopunine, 9-(fl-D-3'-S-ethyl-3'-thioribofuranosyl)-6-mercaptopunine (d) ; 5'-methyl-ribosyl-6-mercaptopurine, 9-($-D-6'-deoxyallofuranosyl)-6-mercaptopunine (d) ; 6-methylthioguanosine, 9- (8-D-nibofuranosyl)-6-methylthiopunine (d); 2'-deoxynibosyl-6-mercaptopunine, 9-(ft-D-2'-deoxynibofunanosyl)-6-mercaptopunine (d) ; 2'-deoxynibosyl-6-mercaptopunine, 9-(a-D-2'-deoxynibofuranosyl)-6-mercaptopunine (e) ; 2'-S-ethyl adenosine, 9-(8-D-2'-S-ethyl-2'-thioribofuranosyl)-6-aminopunine (d);3'-S-ethyl adenosine, 9-(fl-D-3'-S-ethyl-3'-thionibofuranosyl)-6-aminopunine (d) ; 5'-deoxythioguanosine, 9-(fl-D-5'-deoxynibofuranosyl)-2-amino-6-thiopunine (d); 5'-deoxy-5'-S-ethyl thioguanosine, 9-(fl-D-5'-S-ethyl-5'-thioribofuranosyl)-2-amino-6-thiopunine (d) ; allosyl adenine, 9-(fl-D-6'-deoxyallofuranosyl)-6-aminopurine (d) ; allosyl-6-mencaptopunine, 9-(8-D-6'-deoxyallofunanosyl)-6-mercaptopunine (d) ; thioguanosine, 9-(fl-D-nibofuranosyl)-2-amino-6-thiopunine (b); 2'-deoxythioguanosine, 9-(fl-D-2'-deoxynibofuranosyl)-2-amino-6-thiopunine (d) ; (also a-

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

52 Cancer Research

anomer) (d) ; nibosyl adenine, adenosine, 9-$-D-nibofuranosyl)-6-aminopunine; 9-@-hydroxyethyladenine, 9-2-hydroxyethyl-6-aminopunine (e); 9-j9-chlorethyl-6-benzylpurine (e); 9-@-hydroxyethyl-6-benzylthiopurine (e); 9-j@-hydroxyethyl-6-benzythiopurine(e) ; 9-@-hydroxyethyl-6-thiopunine (e) ; 8-bromoadenosine, 9-(fl-D-nibofuranosyl)-6-ammo-8-bromopunine (e) ; 8-bromoguanosine, 9-(fl-D-ribofuranosyl)-2-amino-8-bromopurine (e); 8-bromoxanthosine, 94-D-nibofuranosyl)-2,6-dioxypurine (e); 8-thioguanosine, 9-(fl-D-nibofuranosyl)-2-amino-8-thiopunine (e); 8-methyithioguanosine, 9-(fl-D-nibofuranosyl)-2-amino-8-methylthiopunine (e); 8-thioadenosine, 9-(ft-D-nibofuranosyl)-6-amino-8-thiopurine (e); 9-(ft-L-nibofuranosyl)-6-aminopunine (d).

The authors very much appreciate the cooperation of the investigator8 cited, who supplied the many nucleosides and substituted punines needed in this investigation.

REFERENCES1. BIRNIE, G. D.; KROEGER,H.; ANDHEIDELBERGER,C. Studies

on Fluorinated Pynimidines. XVIII. The Degradation of 5-Fluoro-2'-deoxyuridine and Related Compounds by Nucleoside Phosphorylase. Biochemistry, 2th66—72, 1963.

2. BL0CX, A., ANDNICHOL,C. A. Inhibition of RibosephosphatePyrophosphokinase Activity by Decoyinine, an AdenineNucleoside. Biochim. Biophys. Rca. Commun., 16:400-03,1964.

3. BRINK, J. J., AND LEPAGE, G. A. Metabolic Effects of 9-D-Arabinosyl Punines in Ascites Tumor Cells. Cancer lies.,24312—18,1964.

4. - . Metabolism and Distribution of 9-$-D-Arabinofuranosyladenine in Mouse Tissues. Thid., 24:1042-49, 1964.

5. ELLIS, D. B., ANDLEPAGE, G. A. Biochemical Studies of Resistance to 6-Thioguanine. Cancer lies., 23:436-43, 1963.

6. KALCE.AR,H. M., ANDBESSMAN,A. N. The Determination ofAdenine Compounds. J. Biol. Chem., 167:445-59, 1947.

7. KALCE.AR,H. M., AND SHAFRAN,M. The Enzymic Synthesisof Punine Ribosides. J. Biol. Chem., 167:477—86, 1947.

8. LEPAGE, G. A., ANDHOWARD,N. Chemotherapy of Mammary Tumors of C3H Mice. Cancer lies., 23.622-27, 1963.

9. LEPAGE,G. A., ANDJUNGA,I. G. Use of Nucleosides in Resistance to 6-Thioguanine. Cancer lies., 23:739-43, 1963.

10. LEPAGE,G. A. ; JUNGA,I. G. ; ANDBOWMAN,B. Biochemicaland Carcinostatic Effects of 2'-Deoxythioguanosine. Cancerlies.,24835-40,1964.

11. LOWRY,0. H., AND LOPEZ, J. The Determination of Inorganic Phosphate in the Presence of Labile Phosphate Esters.J. Biol. Chem., 162:421-28,1946.

12. PATERSON,A. R. P., AND SUTHERLAND,A. Metabolism of6-Mercaptopunine Ribonucleoside by Ehrlich Ascites Carcinoma Cells. Can. J. Biochem., 42: 1415-24, 1964.

13. PIxz, J. E. ; SLECRTA,L. ; ANDWILEY, P. F. Tubercidin andRelated Compounds. J. Heterocyclic Chem., 1:159-61, 1964.

14. ROBINSON,J. R. Some Effects of Glucose and Calcium uponthe Metabolism of Kidney Slices from Adult and NewbornRats. Biochem. J., 45.68-74, 1949.

15. WELCH,A. D., ANDPRU5OFF,W. H. A Synopsis of Recent Investigations of 5-Iodo-2'-deoxyunidine. Cancer ChemotherapyRep., 6:29—36,1960.

Vol. 25, January 1965

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1965;25:46-52. Cancer Res   G. A. LePage and Irene G. Junga  Metabolism of Purine Nucleoside Analogs

  Updated version

  http://cancerres.aacrjournals.org/content/25/1_Part_1/46

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

  .pubs@aacr.orgDepartment 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/25/1_Part_1/46To request permission to re-use all or part of this article, use this link

on April 18, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from