Action of pyrazolopyrimidine derivatives on Trypanosoma rangeli culture forms

Camp. Eiochem. Physiol. Vol. 83C, No. 2, pp. 291-294, Printed in Great Britain

1986

8

0306-4492/86 $3.00 + 0.00 1986 Pergamon Press Ltd

ACTION OF PYRAZOLOPYRIMIDINE DERIVATIVES ON TRYPANOSOMA RANGELI CULTURE FORMS Jo& lnstituto

LUIS AVILA, MARIA A. POLEGRE and ROLAND K. ROBINS

de Biomedicina,

Apartado

4043, Caracas IOIOA, Venezuela Provo. UT 84602. USA (Rewired

19 August

and Brigham

Young

University,

1985)

Abstract-l. The capacity of 54 different pyrazolo(3,4-d)or pyrazolo(4,3-d)pyrimidine derivatives to inhibit the multiplication of Tr~~punosomn range/i culture forms was evaluated. 2. Among pyrazolo(3,4-d)pyrlmidines. I4 derivatives showed trypanostatic activity, 4-aminopyrazolo(3,4-d)pyrimidine (APP) being the most active, with 4-hydroxypyrazolo(3,4-d)pyrimidine (HPP) lacking trypanostatic activity. 3. 7-Hydroxy-3-B-D-ribofuranosylpyrazolo(4,3-~)pyrimidine (FOB) was as active as 7-amino-3-/?-oribofuranosylpyrazolo(4,3-d)pyrimidine (FoA), both compounds being five-fold less inhibitory than APP. 4. It can be concluded that, regarding T. rangeli, the chemical analogy to hypoxanthine or inosine of pyrazolo(3,4-d)and pyrazolo(4,3-d)pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.

INTRODUCTION Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine, HPP] inhibits the growth of the cultured forms of both American (Trypunosomu crud) and African (T. hrucei and T. rhociesiense) trypanosomes (Marr et c/l., 1978; Berens et al., 1980; Avila and Avila, 1981) but not of T. rangrli, a non-pathogenic American trypanosome (Avila et al., 1981). Biochemical investigation of the metabolism of HPP using sensitive organisms has shown that this compound is metabolized by a sequential conversion to allopurinol mononucleotide and 4-aminopyrazolo(3,4-d)pyrimidine(APP) mono-, di- and trinucleotides. The latter are then incorporated into RNA (Marr et cd., 1978). In the case of T. cru~i it has been demonstrated that succino-AMP synthetase (EC 6.3.4.4) and succino-AMP lyase (EC 4.3.2.2) activities are capable of catalyzing the conversion of HPP-ribose 5’-phosphate (HPPRP) to APP-ribose-5’phosphate (APPRP) and that the rate-limiting step is the formation of succino-APP-ribose 5’-phosphate as catalyzed by succino-AMP synthetase. This intermediate is cleared rapidly by succino-AMP lyase (Spector rt al., 1982). In the case of T. runpli it has been demonstrated that HPP, even at high concentrations (up to 100 /(g/ml), lacks in vitro activity, this fact being due to a poor affinity for HPPRP of the T. rangeli succino-AMP synthetase activity. This interpretation is further supported by the fact that the addition of APP to an adeninedepleted culture medium strongly inhibited T. rungdi growth, suggesting the rapid conversion of this drug to APP-ribonucleotides (Avila et al.. 1981). The related inosine analog, Formycin B [7-hydroxy-3-8-D-ribofuranosylpyrazolo(4,3-~)pyrimidine, FOB] also has antitrypanosomal activity (Rainey et ul., I983), its metabolism being identical to that of HPPR (Rainey et al., 1983; Avila, 1983). This study compares the anti-T. rungeli effect of several pyrazolo(4.3-ci)and pyrazolo(3,4-d)pyrimidine de-

rivatives in order to determine possible relationships between structure and inhibitory activity. MATERIALS AND

METHODS

Culturc~ mrrhods Trypanosomu

rangc~li (San Juan de Los Morros strain) at 28‘C in a purine-depleted liver infusiontryptose (LIT) medium (Avila el ul., 1981). After dialvsis the concentration of total purines or pyrjmidines in -the culture medium was lower than 0.1 /‘g/ml (including 0.01 /‘g/ml adenine) as determined by high-pressure liquid chromatography (HPLC). Most culture medium components were obtained from GIBCO Laboratories (Grand Island. NY). Glucose. hemin and APP 2’-deoxyriboside were purchased from Sigma Chemical Co., St Louis, MO.

was grown

The 42 pyrazolo(3,4-d)pyrimidine and I2 pyrazolo(4,3-d)pyrimidine derivatives evaluated for antitrypanosomal activity are shown in Tables 1 and 2, respectively. These derivatives were synthesized as described previously (Robins, 1956; Cheng and Robins. 1956; Lewis and Townsend, 1980).

Various concentratrons of active drugs were added aseptically in alkaline saline solution. without causing changes in the pH of the culture medium. After 5-7 days of culture. parasites were counted in a hemocytometer as previously described (Avila e/ ~1.. 1979), and the counts were compared with those of controls grown in the absence of the drug.

RESULTS

Table I shows that of the 42 pyrazolo(3,4-d)pyrimidine derivatives tested on Trypunosomu range/i culture forms I4 showed a trypanostatic effect. all these compounds being APP derivatives. The most inhibitory compound was APP (compound 6); minor modifications of this compound slightly decreasing 291

292

Jo& LUIS AVIM ef ul

Percentage

CO”7pOU”d No.

R,

R?

R

I

H

H

I1

2

H

Hydroxyl

3

H

Hydroxyl

/~-1,.Rihofuranosyl

Other

contr0l

substitutions

growlh 91

H

96 100

4

H

Hydroxyl

/l-~Rihofuranosyl

3.Br

5

H

Hydroxyl

/j-I,-Rlhafilranosyl

3-CN

6 1

H Hydroxyl

85 Ill0

Amino

H

8

Amino

H

69

R

H

Amino

/,-Bromophenyl

71

Y

H

Amlno

~Chlorophenyl

92

IO

72

Ammo

H

II

H

Amino

El

I2

H

Amino

/I-wRlhofura”obyI

55

I3

H

Amino

/~-1,.D~oxyrihofuran~l~yl

IO0

14

H

Amino

/I-wRlhofureno\yl

I5

Cl

Amino

/I-wRihofurano\yl

I6

IH

Isopropylamino

H

72

I7

H

Dimethylammo

H

69

IX

H

Methylamino

Ii

60

IY

H

Dimethylamino

Phenyl

65

20

H

21

H

Methyhhlo

/~-Diethylaminoethylamino Dielhylamino

3.Br

3.Br

29

36 67

H

59

11

69

H

Anilino

22

H

23

H

,,I-Bromoamlino

H

24

H

Benzylamino

H

25

H

3.4.Dichlorohenzylamino

26

H

3.4-Dichlorobenzylamino

27

H

2.4-Dlchlorobenzylamino

2x

H

2.4-Dichlorobenzylamino

/~-i,-Rihofurano~ll

93

29

H

o-Fluorohenzylamino

/l-1,.Rihofuran~)\~I

94

30

H

p-Fluorobenzylamino

/~-1,.Ribofu~an[,\\I

84

31

Renzylamino

H /I-~,-Rlh(,ful-an(,s~l 11

H

Benrylamino

60 -.

56 6X 37 82 52

72

32

H

o-Chlorobenzylammo

Methyl

69

33

H

p-Chlorobenzylammo

Methyl

57

34

H

2.Elhylhexylamino

Vuhyl

35

H

Allylamino

Methyl

IO0

36

H

Phenylc~hylamino

Methyl

IO0

37

Cl

Methyl

IO0

ix

H

Mcihql

65

3’)

H

4n

H

41

H

42

H

Tested

colnpounds

Initial

lnoculum

wcrc

Ammo ~Butylamino Thio Dime~hylamino

present

in the culture

was 0 I x IO” parasites/ml

medium

of San Juan

63 72 82

IOpg/ml.

de Los

Morros

activity, as for example 3-bromination (compound I I). The importance of a free 4-amino group is evident as modifications of this group as in compounds 16-25, 27 decreased the cytostatic activity of these drugs on T. ran&i culture forms, the 3,4-dichlorobenzylamino-N-4-substituted derivative [4-(3,4-DCB) APP] (compound 25) being the most active derivative. It is of interest to note that in general I-ribosylation of the parent compound decreased the cytostatic effect, as for example compounds 12, 14, 26 and 28. lt is noteworthy that p-bromophenyl (compound 8), phenyl (compound 19) and some methyl-l derivatives (compounds 32-34 and 38-40) maintained discrete inhibitory activity on T. rangeli culture forms. trypanostatic

72

Methyl ,I-Chlo~~phenyl at

51

Mclhql II

Hydrarino 3-Morphohnopropylammo

of

blvhln

It is of interest to note that 6-hydroxy (compound 7), 6-chlorinated (compound 15) and 6-methylthio (compound IO) derivatives of APP showed littlc cytostatic activity as compared with the parent compound (compound 6). suggesting the importance of a free carbon atom at the 6-position for obtaining strong inhibitory activity. Finally. four 4-hydroxy derivatives (compounds 2-5) showed no cytostatic activity, as also occurred with the I-/I-D-deoxyribofuranosyl-4-amino derivative (compound 13). Figure I show that the IDA,, (the dose causing a 50% inhibition of cell growth) was about I /(g/ml for compound 6. about 4/lg/ml for compounds I1 and 25 and 6 /(g/ml for compound 14. Table 2 shows the strong cytostatic effect of FoA (compound 43), FOB (compound 45) and

Activity

of pyrazolopyrimidines

Fig. I, Effect of several pyrazolnpyrimidines on the multiplication of Tr?‘/)“““.s”“lLl rtrn~eli culture f9rms. (A) Pyrazolo(3.4-rl)pyrimidines: a. SBrAPP; 0. 3-BrAPPR:

7-thioformycin (compound 54) on T. run&i culture forms. It can be seen in Fig. I that the ID,,, was about 5 Llgjml. These results show that FoA, FOB and 7-thioformycin were about five-fold less active than APP. Additional experiments revealed that pyrazolo(3,4-tl)and pyrazolo(4,M)pyrimidines have only a trypanostatic effect. Finally, different chemical modifications of the pyrazolo(4,3-cf)pyrimidine ring blocked trypanostatic activity against T. run~c~li culture forms. This is the case for N-l-. N-2- and N-Gmethyl formycin B derivatives (compounds 46, 47 and 49), 7-methylformycin (compound 5 I). 7-selenoxoformycin (compound 52) and 7-methylselenoformycin (compound 53) however S-amino and 4-methyl substitutions maintained inhibitory activity.

The observations adenine and inosine

reported analogs

here indicate that are very reasonable

Table

tn

2.

vwo

cytotoxicity

on

A.

293

on T. rccn&i

APP; a. pyrimidines:

4-(3,4-DCB) APP. (B) Pyrazoio(4.3-d)0. FOB: m\ FoA; A. Thioformycin.

models for the design of agents with antiprotozoan activity, and suggest ways in which pyrazolopyrimidines may be structurally modified so as to alter their cytostatic activity on T. rungeli culture forms. Thus, of 54 pyrazolopyrimidine derivatives tested 14 pyrazolo(3,4-d)pyrimidines and six pyrazolo(4,3_d)pyrimidines showed strong to modcratc cytostatic activity. In contrast with results obtained for pyrazolo(3,4_d)pyrimidines on T. cruzi epimastigotes. which need only a free 4-amino group or just a ribosylatcd N-l group for full cytostatic activity (Avila (‘I rrl.. 1986a), T. range/i culture forms accepted several different N-l and N-4 substitutions in the APP molecule while maintaining inhibitory effects, however 4-hydroxy derivatives totally lacked cytostatic activity. Previous results present T. mngdi culture forms as similar to certain mammalian cells, as it has been reported that I -methyl or 4-methylamino APP dcrivi rr,vpanosoma

ran&i

p-I~-ribofuranosylpyrazolo(4,3-d)pyrimidlne

culture

forms

derivatives

Percentage

CompounJ

Other

No.

44

Methylamino

4s

Hydroxy

46 47

Hydroky

4x

HydIWy

49 50 51

-

42 28

N-2-Methyl -

100

Methyl

Hydroxy Hydroxy

-

100 24

Methyl

-

N-&Methyl 5-Amino

53

Methylseleno

wre

was I)

h?

100

-

100

-

100

Thio

inoculum

33

-

100

S&!IlOXO

compounds

growth

-

N-l-Methyl

52

Tested

substitutions

Hydroxy

54

Initial

R,

R, Amino

4.7

control

38

prexnt

m the culture

I x IOhparasites/ml

medium

at IOfigjml.

of San Juan de Los

Morros

strain.

of

of

294

Jo& LUIS

atives arc active inhibitors of the subcutaneous growth of L5178 leukemia in mice (Skipper PZ ul., 1957). Of particular interest was the fact, already found in T. crusi epimastigotes (Avila et al., 1986a) and American Leishmania promastigotes (Avila et al., 1986b), that 3-bromination of APP (compound I I) decreased trypanostatic activity, a situation also found for the ribosylated APP derivative (compound 14). Our results with compound I2 arc similar to those found on T. cruzi euimastigotes (Avila et al.. 1986a). but different to those found for American Leiskaniu (Avila et al., 1986b) and for other eukaryotic cells, indicating that APPR is a more effective inhibitor of both the purine de t7ow biosynthetic pathway and cell growth than APP per SC (Bennett et ~1.. 1966). The fact that APP is more active than APPR on both American trypanosomes could represent a characteristic of these kinetoplastidic cells. With regard to pyrazolo(4.3-d)pyrimidines, methylation of the 7-amino group (compound 44) did not abolish cytostatic activity, while N-l, N-2 or N-6-methylations in the FOB molecule (compounds 46. 47 and 49) blocked all inhibitory activity. Our results are again similar to those obtained on T. cruri epimastigotes (Avila et al., 1986a), but different to those found on American Leishmania promastigotes (Avila et al., 1986b) suggesting that T. rangeli culture forms. in the presence of certain pyrazolopyrimidines. behave similar to T. cruzi epimastigotes, this being due perhaps to its close taxonomic classification. In our hands. and this time in contrast with the results obtained on T. cruzi epimastigotes (Avila et al., 1986a) and on American Leishmania promastigotes (Avila et al., 1986b), the IDS,, for FoA and FOB was identical and about five-fold higher than that obtained for APP. We can conclude from previous considerations that, regarding pyrazolopyrimidines and their effects on T. rangeli culture forms, the chemical analogy to hypoxanthine and inosine of pyrazolo(3.4-d) and (4,3_d)pyrimidines, respectively, is not absolutely critical and also that T. rangeli, as previously found for American Leishnzunia, are less specific in their requirements on the pyrazolopyrimidine ring than T. cruzi epimastigotes concerned.

as

far

as

inhibitory

activity

is

Ackno~l,k~dXrmenrs-This work was supported by CONICIT and Fundacion Polar. We express our gratitude to Angela Avila for careful maintenance of the T. rungeli strain used in this paper and to Candelaria de Aranguren for excellent secretarial work.

AVILA

et ul

REFERENCES

Avila J. L. (1983) New rational approaches to Chagas disease chemotherapy. htercienciu 8, 4054 17. Avila J. L. and Avila A. (1981) Tr~vpanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease. E.vp. farusi/o/. 51, 204~208. Avila J. L., Bretaiia A.. Casanova M. A., Avila A. and Rodriguez F. (1979) Trypcmosomu cruzi: defined medium for continuous cultivation of virulent parasites. E.xp. Prrrrrsitol. 48, 27735. Avila J. L., Avila A. and Casanova M. A. (1981) Differential metabolism of allopurinol and derivatives in TrJpcmo.xomu ranpelr and T. cru:i culture forms. M&c. Bio&m. Parcr.vitoi 4, 265 272. Avila J. L.. Poleare M. A. and Robins R. K. (1986al Action of pyrazolopyrimidine derivatives on Trypunosomu cru;i epimastigotes and trypomastigotes. Antimicroh. Agenr.c Chemorhrr. In Press. Avila J. L.. Polegre M. A., Avila A. and Robins R. K. (1986b) Action of pyrazolopyrimidine derivatives on American Leishmania spp. promastigotes. Comp. Biochem. Ph_wiol. 83C, 285-289. Bennett L. L., Vail M. H., Chumley S. and Montgomery J. A. (1966) Activity of adenosine analogs against a cell culture line resistant to 2-Auoroadenine. Biochcm. Phnrmrrcol. 15, I7 I9 1128. Berens R. L., Marr J. J. and Brun R. (1980) Pyrazolopyrimidine metabolism in African trypanosomes: metabolic similarities to Trypanosoma cruzi and Lei.thmunio spp. M&c. Biochem. Purusirol. 1, 69. 13. Cheng C. C. and Robins R. K. (1956) Potential nurine antagonists. VI. Synthesis of I-alkyl and I&-4substituted pyrazolo(3,4-tl)pyrimidines. J. urx. Chem. 21, 1240-1256. Lewis A. F. and Townsend L. B. (1980) Pyrazolo(4.3-d)pyrimidine nucleosides. 9. Studies on the isomeric N-methylformycins. J. Am. chrm. SW. 102, 2817 2822. Marr J. J.. Berens R. L. and Nelson D. J. (1978) Antitrypanosomal effect of allopurinol: conversion in riw to aminopyrdzolopyrimidine nucleotides by Tr~puno.wmu crxi. Screnrr 201, 1018 1020. Rainey P.. Garrett C. E. and Santi D. V. (1983) The metabolism and cytotoxic effects of Formycin B in Trypano.romrz cruzi.. B&hem. Phurmacol. 32, 749 152. Robins R. K. (1956) Potential purine antagonists. I. Synthesis of some 4,6-substituted pyrazolo(j,4-rl)pyrimidines. J. Am. chrm. Sot. 78, 784-790. Skipper H. E.. Robins R. K.. Thomson J. R.. Cheng C. C., Brockman R. W. and Schabel F. M. Jr. (1957) Structureeactivity relationships observed on screening a series of pyrazolopyrimidines against experimental neoplasms. Cuncrr. Rex 17, 5799596. Spector T., Berens R. L. and Marr J. J. (1982) Adenylosuccinate synthetase and adenylossucinate lyase from Trypcrnosoma cruzi. Specificity studies with potential chemotherapeutic agents. Biodwm. Pharmucol. 31, 225- 229.