THE JOURNAL OF BIOLOG~L CHEMISTRY Vol. 250, No. 18, Issue of September 25. pp. 7343-7351.1975

Printed in U.S.A.

On the “Activation” of Cytokinins” (Received for publication, January 20, 1975)

SIDNEY M. HECHT, R. BRUCE FRYE, DIETER WERNER, AND S. D. HAWRELAK

From the Massachusetts Institute of Technology, Department of Chemistry, Cambridge, Massachusetts 02139

FOLKE SKOOG AND RUTH Y. SCHMITZ

From the University of Wisconsin, Institute of Plant Development, Madison, Wisconsin 53706

A number of cytokinin analogs containing modifications in the heterocyclic moiety were prepared. These compounds were tested for activity as cytokinins and anticytokinins in the tobacco bioassay and the results were used to determine whether any position(s) of the heterocyclic nucleus of cytokinins may require derivatization as part of an over-all “activation” process. 3-Substituted 4- alkylaminopyrazolo[3,4-dlpyrimidines and 4-alkylaminopyrrolo[2,3-dlpyrimidines, for example, have (substituted) carbon rather than nitrogen atoms at positions 3 and 5, respectively (analogous to position 7 in purines) and would be predicted to be metabolically stable at these positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable position, and by extension at position 7 in cytokinin analogues which are purines, is not a prerequisite for the expression of cytokinin activity. Similar consideration of other heterocyclic analogs which have cytokinin activity suggests that the active form of a cytokinin can be the exogenous compound itself.

Certain structural analogs of cytokinins were found to inhibit the growth of tobacco callus promoted by 6-(3-methyl-2-butenylamino)purine. These compounds were studied as potential cytokinin antagonists, i.e. having activity analogous to the 7-alkylamino-3-methylpyrazolo[4,3-dlpyrimidines (Hecht, S. M., Bock, R. M., Schmitz, R. Y., Skoog, F., and Leonard, N. J. (1971) Proc. N&l. Ad. Sci. U. S. A. 68, 26082610; Skoog, F., Schmitz, R. Y., Hecht, S. M., and Bock, R. M. (1973) Phytochemistry 12, 25-37). The activity of these compounds is discussed and criteria are proposed to distinguish between those species which are specific anticytokinins and those which otherwise inhibit growth.

Cytokinins are hormones believed to be involved in the growth, cell division, and differentiation processes in all plants (1, 2). When added to cultures of excised auxotropic tissues of higher plants, the cytokinins promote rapid cell division and continuous growth of callus. In structural terms, the unit common to all highly active natural and synthetic cytokinins is the N’-alkyladenine moiety, although cytokinins have been shown to exist in nature at the purine, ribonucleoside, ribonu- cleotide, and tRNA levels (1-13). The occurrence of cytokinins at these several levels of structural modification and the additional finding that cytokinins can be metabolized to a more stable form, tentatively identified as the 7-glucosyl derivative, after addition to excised plant tissues (14-17), have led to the suggestion that cytokinins may mediate their effects in plant tissues only after conversion to an “active” form (17, 18). Moreover, the inability of tobacco tissue to metabolize a cytokinin analog to its corresponding glucosyl derivative was

reported (18) to confer “anticytokinin” activity on that molec- ular species.

Our concern with the mechanism of cytokinin action (21) and with the design of anticytokinins (19, 20) prompts us to report the results of experiments which suggest that exogenous cytokinins do not require activation before the expression of cytokinin activity and that certain cytokinin analogs may have a general inhibitory action rather than functioning as specific anticytokinins.

7343

The experimental procedures for synthesis of the test compounds are presented as a miniprint supplement immediately following this paper.’

* This investigation was supported at the Massachusetts Institute of Technology by Public Health Service Research Grant CA-14896 from the National Cancer Institute and at the University of Wisconsin by Research Grant BMS72-02226 (GB-35260X) from the National Science Foundation.

‘F. Skoog, R. Y. Schmitz, S. M. Hecht, and R. B. Frye, manuscript in preparation.

2Material published in miniprint form can be easily read with the aid of a large field reading glass of a type readily available at most opticians. For the convenience of those who prefer to obtain supple- mentary material in the form of full size photocopies, these same data are available as JBC Document No. 75M-89. Orders for supplementary material should specify the title, authors, and reference to this paper and the JBC Document Number, and the number of copies desired. Orders should be addressed to The Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Maryland 20014, and must be accom- panied by a remittance to the order of the Journal in the amount of $1.35.

EXPERIMENTAL PROCEDURE

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RESULTS 4-AMlNOPYRAZOLO[3,4-d]PYRIMIDINES

Compounds 6 and 7 were prepared by the condensation of 5-amino-3,4-dicyanopyrazole with N-(3-methyl-2-butenyl)for- mamide and N-(3-methylbutyl)formamide, respectively, ac- cording to a previously published procedure (22). The forma- mides were not dried rigorously so that the substituted pyrazolo [3,4-dlpyrimidine-3carboxamides were obtained di- rectly during the rearrangement by concomitant hydration of the 3-cyan0 moiety.

NO R R’ 1 R’ ACTlVlTY CYTOKININI7ZDLor lNHl6lTORY -EZf- . . . . . . ..A.... I I

5

6

NC CN

NH&H + 0

H

6

Compounds 18 to 20 were prepared by extension of the ex- change amination procedure of Whitehead and Traverso (30). Thus, treatment of formycin(7-amino-3-(/3-D-ribofuranosyl)- pyrazolo[4,3-dlpyrimidine) with n-pentyl-, n-hexyl- or cyclo- hexylamine and the respective amine hydrochloride at 165” in a sealed tube afforded the corresponding W-substituted 7-amino-3-(8-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidines (18 to 20) in yields of 16 to 26%. This compared favorably with an earlier procedure (21) based on the alkylation of the N-6 posi- tion of formycin with 3-methyl-2-butenyl bromide and sub- sequent Dimroth rearrangement (23) to afford 7-(3-methyl-2- butenylamino) - 3-(/3-D-ribofuranosyl)pyrazolo[4,3-dl-pyrimi- dine (17). It was also comparable to the over-all results ob- tained in four-step sequences subsequently reported for the synthesis of 17 and 7-benzylamino-3-(&D-ribofuranosyl)py- razolo [4,3-dlpyrimidine (26).

-NH2 +

HO OH HO OH

16 18

Compounds 1 to 20 were tested for cytokinin and an- ticytokinin activity in the tobacco bioassay. A summary of cytokinin activity (determined in the absence of additional cytokinins) and inhibitory activity (measured in the presence of 3 x lOWa pM 6-(3-methyl-2-butenylamino)purine) for the pyrazolo[3,4-dlpyrimidines (1 to 7) is given in Fig. 1; growth curves determined in individual experiments for each of these species and the highly cytokinin 6-(3-methyl-2-butenyl- amino)purine (21) are shown in Fig. 2. The parent member of this series, 4-(3-methyl-2-butenylamino)pyrazolo[3,4-d]pyrimi- dine (5), which is isomeric with 21, had only about 1% of the growth-promoting activity of the latter, but was nevertheless an effective cytokinin. Also weakly active as cytokinins were

CONCENTRATION (yM)

FIG. 1. Summary of the cytokinin and antagonist activities of the pyrazolo [3,4d]pyrimidine derivatives. The compounds are numbered as in the text. The base-lines represent the tested concentration ranges for each compound and the vertical arrowheads indicate the start and end points of the linear growth response as determined in individual assays, whereas the bars represent the mean activity range as determined for all assays of each compound. In the case of cytokinin activity, this is the range over which growth increases as a nearly linear function of the log of concentration; in the case of “antagonism” (growth inhibition), the bars extend from the lowest concentration at which inhibition can be detected to that at which growth is completely inhibited in the presence of 0.003 PM 6-(3-methyl-2-butenylamino)pu- rine. Bars representing cytokinin activity are placed above the base-lines and are striped. Bars representing antagonist activity (inhibition of growth) are below the base-lines and are stipled.

L

j:

1 10-4

t I L

t

I

II 0

5

1

i-3 IO-L 10-1 I CONCENTRATION (uM)

FIG. 2. Comparison of the effect on growth of tobacco callus of serial concentrations of the seven pyrazolo[3,4-dlpyrimidine derivatives (Compounds 1 to 7) and of the active cytokinin 6-(3-methyl-2- butenylamino)purine (21). The compounds are numbered as in the text and their structures are also indicated in Fig. 1. Tissues were cultured for a 5-week period. Results of four separate experiments, performed between August, 1966 and July, 1974.

the 4-alkylaminopyrazolo [3,4-dlpyrimidine-3carboxamides 1 and 2. Compounds 3, 6, and 7, on the other hand, had no cyto- kinin activity, but instead inhibited the growth of tobacco tis- sue cultured on a medium containing 3 x lo-’ MM 6-(3-methyl-

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CONCENTRATION (PM)

FIG. 3. Effect on growth of tobacco callus of serial concentrations of 4 in the presence of optimal (0.08 NM, Cl) or suboptimal (0.003 PM, A) concentrations or in the absence (0) of the cytokinin 6-(3-methyl-2- butenylamino)purine. Culture period: May 9 to June 12, 1974.

CONCENTRATION (,.,M)

FIG. 4. Summary of the cytokinin and inhibitory activities of the pyrrolo[2,3d]pyrimidine derivatives. The compounds are numbered as in the text. Procedural details as in Fig. 1.

2-butenylamino)purine.8 It should be noted, however, that the related compound 4-aminopyrazolo [3,4d]pyrimidine (4), which lacked substituents on the 2-, 3- or N’-positions, was equally as inhibitory and that a higher concentration of 6-(3-methyl-2-butenylamino)purine (0.08 PM) did not reverse this inhibition in tests with compounds 4 and 6, as shown for 4 in Fig. 3. In the pyrrolo[2,3d]pyrimidine series (Fig. 4), 4-(3-methyl-2-butenylamino)pyrrolo [2,3d]pyrimidine (9) was found to be about % as active as a cytokinin as its aza analog, 4-(3-methyl-2-butenylamino)pyrazolo[3,4]pyrimidine (5), but 10 times as active as 4-n-hexylaminopyrrolo[2,3-dlpyrimidine (10). In contrast to this, 4-amino-7-( &n-ribofuranosyl)pyrrolo- [2,3d]pyrimidine (tubercidin; 8) was found to be without cytokinin activity, but became toxic at a concentration of about 5 PM.

As shown in Fig. 5, several of the tested compounds in the pyrazolo [4,3-d lpyrimidine series were active as cytokinins, although not all of these were capable of eliciting a maxi- mal growth response over the concentration range tested (19). Among the ribosyl derivatives, 7-(3-methyl-2-butenyl- amino)-3-(B-n-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (17) was weakly active as a cytokinin, whereas the ‘I-n-pentyl, 7cyclopenty1, and 7-n-hexyl derivatives (18 to 20) were inac- tive as cytokinins and inhibited the growth of tobacco tissue grown in the presence of 3 x lo-’ pM 6-(3-methyl-2- butenylamino)purine. As in the previous series, inhibition was

‘Interestingly, compounds 1 and 6, which differ only by the a-methyl group on 1, have different qualitative activities in the tobacco bioassay, suggesting that the methyl group may be metabolically stable. This is in direct contrast to the results reported for 6-ben- zylaminopurine and 6-benzylamino-9-methylpurine (31-33).

l,R= A, R’. CONH2, R”z CH3

2,R= -, R’= CONH2. R”= CH3

R’= CN, R”: CH3

4,R= H,

5,R= /\/I\ , :;;; ;:::l

R’= CONH2, R”= H

R’= CONH2, R”= H

RNH

8, R=H, R’= p-D-ribofuranosyl

9,R= 6/k, R’=H

JO,R= -, R’= H

achieved at approximately the same concentration using the corresponding compound unsubstituted on the exocyclic nitro- gen atom, 7-amino-3-( /3-n-ribofuranosyl)pyrazolol4,3-dlpyrimi- dine (formycin; 16).

Also of interest were the results obtained in tests of 9-(5’-deoxy-8-Dribofuranosyl)-6-(3-methyl-2-butenylamino)pu- rine (23) (Figs. 6 and 7). This compound had essentially the same cytokinin activity as 6-(3-methyl-2-butenylamino)-9-B Dribofuranosylpurine (22) and the corresponding 2’-deoxy ana- log 24 (34). All three ribonucleosides were about one-tenth as active as the corresponding purine, 6-(3-methyl-2- butenylamino)purine (21).

DISCUSSION

Deleuze et al. (17) have recently reported that a number of plant tissues convert exogenously added 6-benzylaminopurine to a stable, long lived metabolite, tentatively identified as 6-benzylamino-7-glucofuranosylpurine. They speculated that this compound might be a “protected or active form of the cytokinin.” Parker et al. (14, 15) have reported the isolation of a cytokinin-active, glucosylated metabolite of zeatin after addition of the cytokinin to radish seedlings. The metabolite,

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RNH

15,R= - , RI= SCH3, R”=CH3

16,R= H, R’= H , R”= ri bose

17.R= R’= H, R”= ri bose

18.R= - I R’= H, R”= ri bose

19,R= D

R’. H. R “5 H

R’= H , R”=H

R’= H, R”=CH3

R’= SCH3, R”=CH3

R’= H, R”= ri bose

20,R= m , R’=H, R”= ribose

7-AMINOPYRAZOLO[4,3-d]PYRIMIDINES

IO 20

CONCENTRATION (PM)

FIG. 5. Summary of the cytokinin and inhibitory activities of the pyrazolo[4,3-dlpyrimidine derivatives. The compounds are numbered as in the text. Procedural details as in Fig. 1.

named raphanatin, was shown to be distinct from synthetic 9-P-D-glucopyranosylzeatin and was suggested to be 7-glucosyl- zeatin on the basis of ultraviolet spectral data. Parker and Letham (16) subsequently reported the presence of 9+-D- glucopyranosylzeatin as a major and 7-glucosylzeatin as a minor metabolite of zeatin in maize. In the belief that 7-glucosylation of exogenous cytokinins might be required for the expression of cytokinin activity, Iwamura et al. (18)

furylamino-7-( j3-Dribofuranosyl)pyrrolo[2,3d]pyrimidine) as a potential anticytokinin and found that this analog did cause inhibition of the growth of tobacco tissue cultured on kinetin (6-furfurylaminopurine).

We have previously studied the activity of cytokinin analogs in the substituted pyrazolo [4,3d]pyrimidine series (19, 21). Thus, 7-(3-methyl-2-butenylamino)pyrazolo[4,3-d]pyrimidine

(11) and 7-(3-methylbutylamino)pyrazolo[4,3-dlpyrimidine (12), as well as 3-methyl-7-(3-methyl-2-butenylamino)pyrazolo- [4,3-dlpyrimidine (13), were found to be moderately active cytokinins, whereas the corresponding ribosyl derivative 17 was

prepared the “7-deaza” analog of kinetin ribonucleoside (4-fur- found to be only weakly active. Because position 3 in these

l--f 21 HO ‘3’

22) R=OH, Rk0l-i

23 I R= H, R’=OH

24,R=OH,R’=H

6-(3-METHYL-Z-BUTENYLAMINO)PURINES

10-4 10-3 10-2 10-I I IO

CONCENTRATION (FM)

FIG. 6. Summary of the cytokinin activities of the 6-(3-methyl-2- butenylamino)purine nucleoside derivatives. The compounds are num- bered as in the text. Procedural details as in Fig. 1.

0 IO“ 10-a 10-c 10-1 I IO

CONCENTRATION (IrM)

FIG. 7. Comparison of the effect on growth of tobacco callus of the 6-(3-methyl-2-butenylamino)purine nucleoside derivatives. Com- pounds are numbered as in the text and structures are also indicated in Fig. 6. Culture period: March 1 to April 5, 1974.

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sites before the compounds can promote cell division and growth. Rogoziriska et al. (40) have studied the cytokinin activity of analogs of 6-(3-methyl-2-butenylamino)purine (21) and 6-furfurylaminopurine which contain carbon rather than nitrogen atoms in positions 1 or 3, i.e., compounds in the substituted imidazo[4,5-blpyridine and imidazo[4,5-clpyri- dine series. Although the latter were much less active than the corresponding analogs in the purine series, all of the com- pounds tested did have cytokinin activity. Although no deriva- tives of the l- or 3-deaza analogs additionally substituted at positions 1 or 3 have been reported, it seems unlikely that enzymic modification of the l- or 3-deaza analogs could occur in the same sense or to nearly the same extent as with the corresponding nitrogen analogs. A requirement for enzymic modification seems especially remote for compounds in the 1-deaza series because 7-(3-methyl-2-butenylamino)imidazo- [4,&b]pyridine has fully 50% of the cytokinin activity of 6- (3-methyl-2-butenylamino)purine (21) in the tobacco bioassay.6 Finally, it should be noted that disubstitution of the exocyclic nitrogen atom or replacement with another heteroatom still af- fords compounds which are active as cytokinins (35,41). On the assumption that the substituted and deaza analogs of the purine cytokinins which possess cytokinin activity are meta- bolically stable at the ring position containing the (substi- tuted) carbon atoms, the cumulative evidence from the cytoki- nin testing of these various analogs suggested that no common site need be modified before the analogs can promote cell division and growth in the tobacco bioassay, i.e. that the active form of the cytokinins can be the exogenous species themselves. ’

Because naturally occurring cytokinins have also been iso- lated at the nucleotide level (4, 7, 42), it also seemed of interest to consider whether cytokinin ribonucleosides require phospho- rylation before their utilization as growth-promoting species. The essentially equal cytokinin activities of 6-(3-methyl-2- butenylamino)-9-(P-n-ribofuranosyl)purine (22) and 9-(5’-de- oxy - p - D - ribofuranosyl) - 6 - (3 - methyl - 2 - butenylamino)purine (23), as well as the corresponding 3’-deoxy analog (24) (34), suggests that either phosphorylation is not required or that all three compounds are converted to a common species, e.g. by metabolic cleavage of the sugars to afford 6-(3-methyl-2- butenylamino)purine (21).

In view of considerable recent interest in the synthesis of additional classes of potential cytokinin antagonists (18, 26, 40) and the claim that 4-furfurylamino-7-( &n-ribofuranosyl)- pyrrolo [2,3-dlpyrimidine possesses anticytokinin activity (18), it seems worthwhile to outline criteria to distinguish between compounds which are specific anticytokinins and those which inhibit growth for other reasons. If a cytokinin antagonist is to function by competing for the same cellular “receptor sites” as the cytokinins, then three criteria can be specified.

1. The apparent anticytokinin activity of compounds in a given heterocyclic series should reside with those compounds

analogs, corresponding to position 9 in the purine series, consisted of a carbon rather than a nitrogen atom, intracellular ribosylation or deribosylation of the exogenous compounds was considered unlikely and it was concluded that cytokinins can probably function without ribosylation of the exogenous com- pounds at this position.

That the exogenous pyrazolo [4,3-dlpyrimidines functioned as cytokinins without separation of the 7-substituent and heterocyclic nucleus may be inferred from the presence of specific anticytokinin activity in the closely related analog 3 - methyl - 7 - (3 - methylbutylamino)pyrazolo [4,3 - dlpyrimidine (19). This compound, which has been postulated to function by competing with the structurally related cytokinins for cellular “receptor sites” (19), would afford the same component parts as those derived from cytokinins 12 and 13 if dissociation of the ‘I-substituent and heterocyclic nucleus occurred and the basis for its anticytokinin activity would be difficult to envision. The inactivity in the tobacco bioassay of heterocycles such as 7-hydroxy-3-methylpyrazolo[4,3-dlpyrimidine (20) and ade- nine (35) and of “substituents” such as 3-methyl-2-butenyla- mine and 3-methyl-2-butenyl alcohol (35) further suggested that such dissociation is not prerequisite to the development of cytokinin activity.

In the present work, we have extended the principles utilized previously to study additional positions in cytokinins which may require derivatization as part of an over-all “activation” process. In the pyrazolo [3,4-dlpyrimidine series, for example, 2-methyl-4-(3-methyl-2-butenylamino)pyrazolo[3,4-d]pyrimi- dine-3-carboxamide (l), 4-n-hexylamino-2-methylpyrazolo[3,4- dlpyrimidine-3-carboxamide (2) and 4-(3-methyl-2-butenyl- amino)pyrazolo[3,4-dlpyrimidine (5), all of which contain a carbon atom in position 3 of the heterocylic nucleus and two of which are additionally substituted, were active as cytokinins, suggesting that position 3, corresponding to position 7 in the purine series, need not be glucosylated or otherwise derivatized as a prerequisite to the development of cytokinin activity. In spite of the apparent “anticytokinin” activity reported to be associated with 4-furfurylamino-7-( &n-ribofuranosyl)pyrrolo- [2,3-dlpyrimidine (18), we have found that the related com- pounds 4-(3-methyl-2-butenylamino)pyrrolo[2,3-d]pyrimidine (9) and 4-n-hexylaminopyrrolo [2,3-dlpyrimidine (10) do pos- sess cytokinin activity. Because position 5 in these analogs, corresponding to position 7 in the purine series, also consists of a carbon rather than a nitrogen atom, it would seem that this position need not be modified to produce an “active” cytokinin. This conclusion is reinforced by the activity in the leaf senescence test of 2-hydroxy-4-(3-methyl-2-butenylamino)py- rimidine, which has no imidazole moiety (36). *

An examination of those substituted heterocycles known to possess cytokinin activity is of interest in defining additional molecular sites which may require modification for the expres- sion of cytokinin activity. B-Substituted purines, for example, have carbon atoms at positions 2 and 8. The formal addition of a variety of different substituents to these positions still affords compounds which are active as cytokinins (38, 39), so that there is presumably no common modification required at these

‘Fox et al. (37) have suggested that “if glycosidation at the ‘I-position is required for cytokinin activity, then a metabolically stable, g-sub- stituted cytokinin derivative should be either inactive or act as a cytokinin antagonist.” I f this principle is correct, the cytokinin activity associated with compounds 13 to 15 and 17, all of which are “metabolically stable, g-substituted cytokinin derivatives,” may be taken as evidence that glycosidation at position 7 is not required for the expression of cytokinin activity.

‘In the 3-deaza series, the analogs of 6.(3.methyl-2. butenylamino)purine and 6-furfurylaminopurine were considerably less active than the parent compounds (40), but the corresponding 8-aza-3-deaza analogs (i.e. 4-substituted-u-triazolo[4,5-clpyridines) were much closer in activity to their respective 6-substituted 8-azapu- rines.

‘We cannot exclude the remote possibility that there is a required mechanism for “activation” of the compounds which involves different modifications for individual analogs or that the cytokinins are “acti- vated” via a mechanism which involves extensive degradation of the exogenous compounds.

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which resemble cytokinins structurally. 2. The antagonist must diminish the utilization of cytoki-

nins in some system, the functioning of which is limited by the availability of cytokinins.

3. The inhibition of growth obtained at a given concentra- tion of cytokinin antagonist and cytokinin must be “reversi- ble,” i.e. less inhibition must be apparent in cultures contain- ing the same concentration of antagonist, but more cytokinin.

Although the second criterion can only be satisfied with certainty when the molecular basis of cytokinin action is known, the other two can be tested experimentally.

In the pyrazolo [3,4d]pyrimidine series, e.g. compounds 3, 6, and 7 were found to be inhibitory to the net growth of tobacco tissue grown on a medium containing 3 x lo-* PM 6-(3-methyl-2-butenylamino)purine (21). However, as shown in Fig. 1, the related heterocycle without the N’substituent, 4-aminopyrazolo[3,4d]pyrimidine (4), was also inhibitory to the growth of the tissue and at approximately the same concentration. The absence of a positive correlation between the structural relationship to the cytokinins and inhibitory activity, as well as the finding that the inhibition was not substantially reversed at higher cytokinin concentrations (Fig. 3), as has been shown previously for anticytokinins in the 7-alkylamino-3-methylpyrazolo[4,3d]pyridimine series (Figs. 1 and 2, Ref. 19), must be taken as evidence that inhibitors 3, 6, and 7 have little specific anticytokinin activity. In the 3-(P-nribofuranosyl)pyrazolo[4,3d]pyrimidine series, 7- amino-3-(p-n-ribofuranosyl)pyrazolo [4,3-dlpyrimidine (formy- tin; 16) strongly inhibited the growth of tobacco callus when supplied at concentrations of 6.6 and 20 PM and this in- hibition was not counteracted by 0.08 PM 6-(3-methyl-2- butenylamino)purine (21).’ The same concentrations of the three 7-alkylamino-3-(8-n-ribofuranosyl)pyrazolo[4,3-d]pyrimi- dines (18 to 20) also inhibited callus growth to about the same extent, but for each of these compounds the inhibition was at least partially relieved by 0.08 PM 6-(3-methyl-2- butenylamino)purine. Although there would seem to be a lack of correlation between structural relationship to the cytokinins and inhibitory activity for these compounds as well, the qualitative difference in reversal of inhibitory activity at higher cytokinin concentration obtained for 16 as compared with 18 to 20 suggests that the substituted species may have some specific anticytokinin activity. A similar relationship exists in the 7-(/I-n-ribofuranosyl)pyrrolo[2,3-dlpyrimidine series. The 4- amino derivative, tubercidin (8), was strongly inhibitory and at concentrations above 2.2 pM this inhibition was not coun- teracted by 6-(3-methyl-2-butenylamino)purine (21). 4-Fur- furylamino-7-(8-n-ribofuranosyl)pyrrolo[2,3-d]pyrimidine has been reported to be a “potent anticytokinin” (18), although the published data do not clearly indicate reversal over the entire tested concentration range and especially at higher concentrations.8 Our tests of a preparation of 4-furfuryl- amino-7-(8-n-ribofuranosyl)pyrrolo[2,3-d]pyrimidine kindly furnished by Professor Iwamura showed that kinetin 0.24 PM) or 6-(3-methyl-2-butenylamino)purine (0.08 PM) completely counteracted the inhibitory effect of the 2.2 pM concentration of this analog. Partial reversal was obtained at a 6.6 pM con- centration of the analog.

Certain observations can be made concerning the effect of formal alteration of the imidazole portion of the purine nucleus

‘F. Skoog and R. Y. Schmitz, unpublished data. BRever~al of cytokinin antagonist inhibition by a cytokinin can be

appreciated more fully from Figs. 1 and 2 and Ref. 19.

1. 2.

3. 4. 5. 6. 7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

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Burrows, W. J., Skoog, F., and Leonard, N. J. (1971) Biochemistry 10, 2189-2194

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on cytokinin activity. The 6-(3-methyl-2-butenylamino)purine analogs 5, 9, and 11, as well as the ribosylated analog 17, were all active as cytokinins. In the absence of the substituent on the exocyclic nitrogen atom, however, the analogs (4, 8, and 16) had no cytokinin activity and caused inhibition of growth which could not be reversed effectively by increasing the concentration of cytokinin. Apparently, the formal exchange of atoms in the adenine nucleus afforded inhibitory compounds which are not anticytokinins. When these inhibitors were formally substituted on the exocyclic nitrogen atom, they exhibited weak cytokinin or anticytokinin activity, depending on the nature of the substituent, and possibly more general inhibitory activity at higher concentrations. Although the compounds discussed here do not meet the criteria specified for anticytokinins to the same extent as the original series of such compounds (19), we will soon report on certain compounds in the substituted pyrrolo[2,3-dlpyrimidine series, the behavior of which in the tobacco bioassay is consistent with the criteria described above for anticytokinins.

Acknowledgment-We thank Professor Nelson J. Leonard for a synthetic sample of 4-(3-methyl-2-butenylamino)pyrazolo- [3,4-dlpyrimidine and Mrs. Anna Hilden for technical as- sistance with the bioassays.

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S M Hecht, R B Frye, D Werner, S D Hawrelak, F Skoog and R Y SchmitzOn the "activation" of cytokinins.

1975, 250:7343-7351.J. Biol. Chem. 

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