Cyanopyrazoles as analogs of purine precursors—1. Inhibitory effect on purine biosynthesis de novo

Inr. J. Biochem.Vol. 16, No. 10, PP. 1091-1094, 1984 Printedin Great Britain. All rights reserved

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0020-711X/84 $3.00 + 0.00 1984 PergamonPress Ltd

CYANOPYRAZOLES AS ANALOGS OF PURINE PRECURSORS-I. INHIBITORY EFFECT ON PURINE BIOSYNTHESIS DE i’VOV0 MARIA K.

Institute

SPASSOVA, KONSTANTIN CH. GRANCHAROV

of Molecular

Biology,

Bulgarian

Academy

and

EVCENY V. G~LOVINSKY

of Science,

1113 Sofia, Bulgaria

(Received 3 January 1984) Abstract-l. The in vitro inhibition of purine biosynthesis de nouo by a series of cyanopyrazoles was studied. 2. At concentration 1 mM trichloromethyl analogs (3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole and N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole) were found to inhibit IMP synthesis 80 and 30% respectively. 3. GAR synthesis was inhibited at a lower degree at the same range of concentrations.

4. The compounds demonstrated a similar pattern of inhibition formylation and cyclization as found on the whole pathway.

of the last steps, e.g. AICAR

INTRODUCTION

Many purine and pyrimidine analogs are known as effective agents in modern pharmacobiochemistry. There are numerous studies on their synthesis, mechanism of action and application. The interest in precursor analogs in purine and pyrimidine biosynthesis has recently increased after several metabolic inhibitors such as ribavirin (Sidwell et al., 1979), bredinin (Jap. Pat., 1974; Mizuno et al., 1974) and pyrazofurin (Williams and Hoelm, 1973; Westhed and Prince, 1974; Cadman et al., 1976; Cummings et al., 1979) were discovered. In a series of substituted diaminoand aminotrichloromethylpyrazoles (Fig. 1) synthesized as structural analogs of purine precursors in de nova biosynthesis, the trichloromethyl containing cyanopyrazoles (R, = H, CH,CH,OH; R, = CN; R, = Ccl,) exerted significant antibacterial activity in vitro (Spassova et al., 1980). The same compounds showed an expressed inhibitory effect against experimental tumors. They were found to inhibit the growth of Melanoma B 16 90% and to increase the life span of the animals above 50% at doses of 50 mg/kg b.w. To clear up the mode of action of these evidently active compounds as well as the metabolic level (molecular or cellular) of their action, we tested the analogs on different systems. This paper presents data about the activity of the compounds on de novo biosynthesis of purine nucleoside monophosphates. In previous works we have already found some effective inhibitors of this pathway (Spassova et al., 1976; Spassova et al., 1980) and have shown the influence of substituents at the nitrogen in the pyrazole ring as well as the substituents at 3rd and 4th position on the activity. The new analogs (Fig.

R, =H, CH,, R,=COOEt R,=

Fig.

CH,CH,OH ,COOH,CN,

I. General

structure of the diaminotrichloromethylpyrazoles.

R2

R,=H,

R,=R,=H

R,=CCl,

I , R,= CCC,

II

m

R, = CH,CH,OH,

R, =H

Fig. 2. The four compounds 1091

and

amino-

l-R, = H, CH,CH,OH; R, = CN; R, = Ccl,) contained substituents at 5th position. It was interesting to compare their activity with the activity of similar cyanoanalogs without substituent at the same position. So in the work presented, four compounds were tested with the main structure shown in Fig. 2. Three sets of experiments were performed which gave information about different steps of de nova pathway. Thus we studied the action of the compounds on IMP biosynthesis, on the first steps up to GAR where metabolic control of the pathway is taking place and on the last steps of AICAR formylation and cyclization.

R, = CH,CH,OH

Abbreviations: IMP-inosine 5’-monophosphate; GAR5’-phosphoribosylglycineamide, glycineamideribotide; AICAR-5’-phosphoribosyl-5-amino-4-imidazole carboxamide.

CONHNH2

NH,,CCl,

IT

tested.

MARIA K. SPAS~~VA et al.

1092 MATERIALS

AND METHODS

The inhibitors: 3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole(I), N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole (II), 3(5)-amino-4-cyanopyrazole (III) and N-hydroxyethyl-3(5)-amino-4-cyanopyrazole (IV) were synthesized as described previously (Spassova e/ al., 1980). [“‘C]Formate was obtained from Isocommerz (D.D.R.), glutamine, ribose-5-phosphate, ATP, 3-phosphoglycerate, glycine and 5’-phosphoribosyl-5-amino-4-imidazole carboxamide (AICAR) from Sigma, o,L-homocysteine from Fluka. All other substances were analytical grade reagents. (I) IMP

bioassay

The preparation of pigeon liver acetone powder as enzyme source was prepared according to Goldthwait and Greenberg (1955). The enzyme system was isolated before use by extraction of the powder with IO vol of veronal buffer, pH 7.5. The action of analogs on purine biosynthesis de nouo was tested by measuring incorporation of [‘4C]formate into IMP. The incubation mixture contained in a final volume of 2 ml the following compounds in mM; glutamine, 4.3; ATP, 2.2; ribose-5-phosphate, 2.2; 3-phosphoglycerate, 3.2; MgC&, 2.2; D,L-homocysteine, 2.2; inhibitors, 5, 2.5, 2, I, 0.5 resp.; veronal buffer, 22 ml (pH 7.6); boiled extract of pigeon liver, 0.20 ml; enzyme extract, 0.50; [i4C]formate, 0.5 FCi. The samples were incubated at 37°C for 30 min and then 1ml of IO% CCl,COOH was added to precipitate the proteins. After ether extractions hypoxantine 0.5mg was added to each sample as a carrier, and hydrolysis was carried out with 0.3 ml of 2% FeCl, in 2 N HCI in a boiling water bath for 40min. The samples were evaporated to dryness, dissolved in 0.5 ml of water and chromatographed on Whatman N3 paper. The chromatograms were developed in water-saturated n-butanol and dried. The hypoxantine spots were cut out, extracted with 2 ml of 0. I HCI, aliquots mixed with IO vol of toluene-Triton X-100 mixture and counted with a LKB Ultrobeta liquid-scintillation counter. (2) GAR synthesis

assay

The action of inhibitors on GAR synthesis was tested as described (Spassova et al., 1979) by measuring in uifro incorporation of [I-‘4C]glycine into GAR using the technique of Goldthwait et al. (1954). The enzyme system was isolated before use by extraction of acetone powder with 0.05 M K,HPO,. The extract was passed through a Dowex-1 (bicarbonate form, 4% cross linkage) column, dialyzed overnight against 0.05 M K,HPO, and lyophilyzed. The enzyme preparation gave about 1% incorporation of [I-r4C]glycine into GAR in the controls under the conditions as follows. The incubation mixture contained in a final volume of 0.7ml the following compounds in mM: glutamine, 14.3; ATP, 7; ribose-5-phosphate, 57; 3-phosphoglycerate, 20; MgCl,, 7; inhibitors, 5, 2.5, I resp.; [l-‘4C]glycine, 2pCi; K,HPO,, 0.05 M, O.lOml; lyophilized extract, 20 mg. The samples were incubated at 38°C for 30 min, then 0.5 ml of 20% CCI,COOH was added, the precipitates were removed by centrifugation at 4000 g for I5 min and aliquots of 0. I ml of I M K,HPO, were added to each sample. The residual [I-i4C]glycine was removed by decarboxylation with ninhydrin, 1 ml of 0.2 mM, in a boiling water bath for 30 min. The aliquots were aerated with CO, for 15 min and diluted with water to IO ml. Two milliliters of aliquots were mixed with IO ml of toluene-Triton X-100 scintillation liquid and counted with a LKB Ultrobeta liquid-scintillation counter. (3) AICAR

assay

For the last steps of IMP biosynthesis, e.g. AICAR formylation and cyclization a bioassay with enzyme extract (as described for IMP biosynthesis) and AICAR as sub-

strate was used. The effect of analogs was studied by measuring [“‘Clformate incorporation into IMP. The incubation mixture contained in a final volume of 2ml the following compounds in mM: AICAR, 2;

o,L-homocysteine, 2.3; MgCl,, 2.2; inhibitors, 5, 2.5, I respectively; phosphate buffer, 50 (pH); pigeon liver boiled extract, 0.20 ml; enzyme extract, 0.50; [‘4C]formate, 0.5 pCi. RESULTS

The inhibitory effects of pyrazole derivatives on IMP biosynthesis are presented on Fig. 3. The dosedependence is determined after 30 min incubation. The time-course of [‘4C]formate incorporation is

Concentration

100

(mM

)

(b)

5 60;

0

I

2 3 Concentrotlon

Concentrotlon

7mMl

5

I mM 1

Fig. 3. Inhibitory effect of analogs on: (a) IMP synthesis; (b) GAR synthesis; (c) AICAR formylation and cyclization. 1-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole; IIN-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylIV-NIII-3(5)-amino-4-cyanopyrazole; pyrazole; hydroxyethyl-3(5)-amino-4-cyanopyrazole.

Cyanopyrazoles

as analogs

of purine

1093

precursors-i

stronger inhibitors than the compounds without substituent at the 5th position. As close structural analogs of AICAR base-residue cyanopyrazoles were studied on the last steps of de nova pathway-formylation of AICAR and cyclization to IMP. The compounds demonstrated a similar pattern of inhibition as found under conditions of IMP assay (Fig. 3~). I, II and III at concentration of 1 mM inhibited about 3Cr50% the incorporation of [‘4C]formate, while IV was not active at all. DISCU!3SiON 0

5

IO

I5

Time

20

25

30

(mn)

Fig. 4. Time-course of [‘%]formate incorporation in the presence of I (2.5 mM).

into IMP

(Fig. 4). The introduction of CCl,-group at the 5th position increases the inhibitory effect of the compounds tested. This effect is strongly expressed at the compound I without substituent as pyrazole ring nitrogen (Fig. 3a). It can be seen that at concenlinear

tration

of

1 mM

the

inhibition

of IMP

synthesis

is

about SO”/,. When the same analog (I-O.5 mM) was preincubated without enzyme system under the conditions of IMP assay, no changes of the inhibitory effect was observed (Fig. 5a). On the contrary, preincubation of I in the presence of the enzymes resulted in increase of the inhibition of IMP synthesis indicating metabolic activation (Fig. 5b). To discount the possibility of spontaneous decomposition or hydrolysis eventually taking place, water solutions of the analogs were kept for 1,2 and 3 days at room temperature. There were no differences observed in UV spectra run. In order to assess more properly the effect of cyanopyrazoles on IMP biosynthesis they were tested as retroinhibitors on the steps up to GAR. As seen (Fig. 3b), GAR synthesis was inhibited at a lower degree (15525%) in comparison with that of IMP. Of the compounds tested I and II were proved to be 100

i

84

It has been found that some new cyanopyrazoles synthesized as analogs of purine nucleotide precursors are inhibitors of IMP biosynthesis de nova in pigeon liver cell free system. All experimental data presented show that the 5th position of pyrazole ring is important for the activity of the analogs. The introduction of GCl,-group significantly increases inhibitory action. The compounds are stable and no tendency to spontaneous hydrolysis or nonenzymatic conversion is found. The effect on IMP biosynthesis is stronger when there is no substituent at ring nitrogen. Moreover, there is an increase in the inhibitory effect of the unsubstituted analog up to 75% at 0.5 mM after preincubation with the enzyme system. This indicates metabolic activation (most probably phosphorjbosylation). There are data about similar transformations in literature (Brockman and Anderson, 1963). It is known that the first steps of purine biosynthesis de novo up to GAR are involved in the metabolic control of this pathway. A lot of metabolites (purine nucleotide monophosphates, purine nucleosides, free bases, some purine precursors) as well as some purine analogs inhibit these steps. From the compounds tested I, II and III inhibit to some extent GAR synthesis (Fig. 3b) but the effect is low to explain their activity on IMP biosynthesis by a feed back inhibition only. The differences are better seen in the three sets of experiments at low concentration (Fig. 3). The analogs more strongly inhibit the steps of AICAR formylation and cyclization to IMP than GAR synthesis. This means that these compounds act predominantly as AICAR analogs and their inhibitory effect on IMP biosynthesis is mainly due to the inhibition of the steps after AICAR synthesis. REFERENCES

Brockman R. W. and Anderson E. P. (1963) Biochemistry of cancer (metabolic aspects). A. Rev. Biochem. 32, 463. Cadman E. C., Dix D. E. and Handschumacher R. E. (1976) Pyrazofurin: biochemical and clinicopharmacological studies. Proe. Am. Ass. Cancer Res. 17, 208-214. Cummings F. J., Steller R. G.. Kaplan H. G. and Catabresi P. (1979) Clinical trial of pyrazofurin. Cancer ~re~r~e~r

20

Rep. 63, 1363- 1365.

/

I

I IO

0

I 20

Prelncubotlon Fig. 5. Inhibitory with-all substrates with the substrates

I 30 time

I 40

I mm1

effect of I (OSmM) preincubated: (a) (start of measuring with enzyme); (b) and the enzyme system (start of mea-

suring with [14Cjformate).

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(Toyc

Jozo

Co.,

Ltd)

(1974)

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1094

MARIA K. SPASSOVA et al.

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