Inhibition Of L1210 Cell Growth Research Articles

ChemInform Abstract: Synthesis and Antifolate Evaluation of 10-Ethyl-5-methyl-5,10- dideazaaminopterin and an Alternative Synthesis of 10-Ethyl-10- deazaaminopterin (Edatrexate).

Previous findings suggesting that 5,10-dialkyl-substituted derivatives of 5,10-dideazaaminopterin warranted study as potential antifolates prompted synthesis of 10-ethyl-5-methyl-5,10- dideazaaminopterin (12a). The key step in the synthetic route to 12a was Wittig condensation of the tributylphosphorane derived from 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (7a) with methyl 4-propionylbenzoate. Reaction conditions for the Wittig condensation were developed using the tributylphosphorane prepared from 6-(bromomethyl)-2,4-pteridinediamine (7b) as a model. Each of the respective Wittig products 8a and 8b was obtained in 75-80% yield. Hydrogenation of 8a and 8b at their 9,10-double bond afforded 4-amino-4-deoxy-10-ethyl-5-methyl-5,10-dideazapteroic acid methyl ester (9a) and 4-amino-4-deoxy-10-ethyl-10-deazapteroic acid methyl ester (9b). This route to 9b intersects reported synthetic approaches leading to 10-ethyl-10-deazaaminopterin (10-EDAM, edatrexate), an agent now in advanced clinical trials. Thus the Wittig approach affords an alternative synthetic route to 10-EDAM. Remaining steps were ester hydrolysis of 9a,b to give carboxylic acids 10a,b followed by standard peptide coupling with diethyl L-glutamate to produce diethyl esters 11a,b, which on hydrolysis gave 12a and 10-EDAM (12b), respectively. The relative influx of 12a was enhanced about 3.2-fold over MTX, but as an inhibitor of dihydrofolate reductase (DHFR) from L1210 cells and in the inhibition of L1210 cell growth in vitro, this compound was approximately 20-fold less effective than MTX (DHFR inhibition, Ki = 4.82 +/- 0.60 pM for MTX, 100 pM for 12a; cell growth, IC50 = 3.4 +/- 1.0 nM for MTX, 65 +/- 18 nM for 12a).

Synthesis, antiproliferative and antiviral activity of imidazo[4,5-d]isothiazole nucleosides as 5:5 fused analogs of nebularine and 6-methylpurine ribonucleoside.

A series of imidazo[4,5-d]isothiazole nucleosides related to the antibiotic nebularine and the highly cytotoxic 6-methyl-9-beta-D-ribofuranosylpurine have been synthesized from the corresponding heterocycles. The sodium salt glycosylation of the imidazo[4,5-d]isothiazoles proceeded smoothly, giving mixtures of N-4 and N-6 regioisomers in generally good yields. The protected derivatives were deblocked using standard conditions to afford the desired imidazo[4,5-d]-isothiazole nucleosides, usually as crystalline solids. None of the new nucleosides or heterocycles displayed selective activity against human cytomegalovirus (HCMV) or herpes simplex virus type 1 (HSV-1). The N-6 glycosylated imidazo[4,5-d]isothiazoles were completely inactive up to the highest concentration tested. The N-6 glycosylated imidazo[4,5-d]isothiazoles also were inactive in antiproliferative and cytotoxicity assays, except for 3-methyl-6-beta-D-ribofuranosylimidazo[4,5-d]isothiazole (15a) and 5-(benzylthio)-6-(2-deoxy-beta-D-ribofuranosyl)imidazo[4,5-d]isothiaz ole (5e), which showed moderate inhibition of L1210 cell growth. However, the heterocycles and several of the N-4 glycosylated derivatives were toxic to HFF, KB and L1210 cells; compounds with 5-benzylthio substituents were the most cytotoxic agents in this series.

Adozelesin, a potent new alkylating agent: cell-killing kinetics and cell-cycle effects.

Adozelesin (U-73975) was highly cytotoxic to V79 cells in culture and was more cytotoxic than several clinically active antitumor drugs as determined in a human tumor-cloning assay. Phase-specificity studies showed that cells in the M+early G1 phase were most resistant to adozelesin and those in the late G1 + early S phase were most sensitive. Adozelesin transiently slowed cell progression through the S phase and then blocked cells in G2. Some cells escaped the G2 block and either divided or commenced a second round of DNA synthesis (without undergoing cytokinesis) to become tetraploid. Adozelesin inhibited DNA synthesis more than it did RNA or protein synthesis. However, the dose needed for inhibition of DNA synthesis was 10-fold that required for inhibition of L1210 cell growth. The observation that cell growth was inhibited at doses that did not cause significant inhibition of DNA synthesis and that cells were ultimately capable of completing two rounds of DNA synthesis in the presence of the drug suggests that adozelesin did not exert its cytotoxicity by significant inhibition of DNA synthesis. It is likely that adozelesin alkylates DNA at specific sites, which leads to transient inhibition of DNA synthesis and subsequent G2 blockade followed by a succession of events (polyploidy and unbalanced growth) that result in cell death.

Synthesis and antifolate evaluation of 10-ethyl-5-methyl-5,10-dideazaaminopterin and an alternative synthesis of 10-ethyl-10-deazaaminopterin (edatrexate)

Previous findings suggesting that 5,10-dialkyl-substituted derivatives of 5,10-dideazaaminopterin warranted study as potential antifolates prompted synthesis of 10-ethyl-5-methyl-5,10- dideazaaminopterin (12a). The key step in the synthetic route to 12a was Wittig condensation of the tributylphosphorane derived from 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (7a) with methyl 4-propionylbenzoate. Reaction conditions for the Wittig condensation were developed using the tributylphosphorane prepared from 6-(bromomethyl)-2,4-pteridinediamine (7b) as a model. Each of the respective Wittig products 8a and 8b was obtained in 75-80% yield. Hydrogenation of 8a and 8b at their 9,10-double bond afforded 4-amino-4-deoxy-10-ethyl-5-methyl-5,10-dideazapteroic acid methyl ester (9a) and 4-amino-4-deoxy-10-ethyl-10-deazapteroic acid methyl ester (9b). This route to 9b intersects reported synthetic approaches leading to 10-ethyl-10-deazaaminopterin (10-EDAM, edatrexate), an agent now in advanced clinical trials. Thus the Wittig approach affords an alternative synthetic route to 10-EDAM. Remaining steps were ester hydrolysis of 9a,b to give carboxylic acids 10a,b followed by standard peptide coupling with diethyl L-glutamate to produce diethyl esters 11a,b, which on hydrolysis gave 12a and 10-EDAM (12b), respectively. The relative influx of 12a was enhanced about 3.2-fold over MTX, but as an inhibitor of dihydrofolate reductase (DHFR) from L1210 cells and in the inhibition of L1210 cell growth in vitro, this compound was approximately 20-fold less effective than MTX (DHFR inhibition, Ki = 4.82 +/- 0.60 pM for MTX, 100 pM for 12a; cell growth, IC50 = 3.4 +/- 1.0 nM for MTX, 65 +/- 18 nM for 12a).

Quinoline antifolate thymidylate synthase inhibitors: variation of the C2- and C4-substituents.

Modifications to the bicyclic ring system of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (1, CB3717) have led to the synthesis of a series of quinoline antifolates bearing a variety of substituents at the C2 and C4 positions. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with a disubstituted 6-(bromomethyl)quinoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 TS. As a measure of cytotoxicity, the compounds were tested for their inhibition of the growth of L1210 cells in culture. Good enzyme inhibition and cytotoxicity were found for compounds containing chloro, amino, or methyl substituents at the C2 position with chloro or bromo substituents at C4. The effect on enzyme inhibition of varying the N10 substituent of 2h was similar to that observed in the quinazolinone-containing antifolates, indicating that the quinoline compounds may be interacting with the enzyme in a similar way to the quinazolinones. Also, the introduction of a 2'-fluoro substituent into the benzoyl ring of several of the quinoline antifolates led to an increase in both TS inhibition and the inhibition of L1210 cell growth. These data demonstrate that the N3-H of the pyrimidine ring of the quinazolinone antifolates is not required for binding to TS if appropriate substituents are placed at the C2 and C4 positions of the bicyclic ring system.

Kinetic characteristics of ICI D1694: a quinazoline antifolate which inhibits thymidylate synthase

The thymidylate synthase (TS) inhibitor ICI D1694 ( N-(5-[ N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)- N-methylamino] -2-thenoyl)- S-glutamic acid) is a structural analogue of the substrate N 5, N 10-methylenetetrahydrofolate (5,10-CH 2FH 4) and is currently under clinical evaluation as a treatment for cancer. The compound is shown here to be a mixed non-competitive inhibitor of TS from murine leukemia (L1210) cells when 5,10-CH 2FH 4 is varied. This result suggests formation of an inactive complex between TS, 5,10-CH 2FH 4 and the inhibitor. Thus, binding to only one of the two active sites on the TS homodimer may be sufficient to prevent catalysis fully. Treatment of L1210 cells with ICI D1694 is known to cause intracellular accumulation of the tetraglutamate derivative which is shown here to have a 60-fold higher affinity for TS. The ic 50 for inhibition of L1210 cell growth is below the K i value of ICI D1694 for L1210 TS but above that of the tetraglutamate. The formation of polyglutamates and concentration of drug inside cells, therefore, seem to be responsible for biological activity.

Synthesis of alkoxy-substituted diaryl compounds and correlation of ring separation with inhibition of tubulin polymerization: differential enhancement of inhibitory effects under suboptimal polymerization reaction conditions.

A number of cytostatic compounds (2-4, 7, and 8), which can be described as "diaryl", inhibit tubulin polymerization, cause cells to accumulate in mitotic arrest, and competitively inhibit the binding of colchicine to tubulin. They differ, however, in the separation of the two aryl moieties. To attempt to understand this variability we prepared a series of analogues modeled on 3 and 4 ("benzodioxole series") and on 7 and 8 ("combretastatin series") which differed only in the number of methylene units (ranging from none to four) separating the aryl moieties. These compounds were evaluated for their effects on tubulin polymerization, colchicine binding, and the growth of L1210 murine leukemia cells. In terms of inhibitory effects on tubulin polymerization, for the combretastatin series there was an optimal separation of the two phenyl rings by a two-carbon bridge (compound 24), with progressively decreasing inhibitory activity when the separation was by one carbon (20), three carbons (25), or four carbons (28) (the biphenyl analogue 16 was inactive). The benzodioxole series, however, did not permit us to generalize this finding, because the least active agents prepared (39 and 40) had a two-carbon bridge, while those with one- (5 and 6) and three-carbon (46 and 47) bridges were nearly equivalent in potency. Submicromolar IC50 values for inhibition of L1210 cell growth were only obtained for compounds 20 (IC50, 0.2 microM), 24 (0.07 microM), and 25 (0.4 microM). While evaluating the effects of these agents on tubulin polymerization, we noted with the combretastatin series and with several standard agents that apparent potency (in terms of IC50 values) was always lower if the reaction was performed at 30 degrees C, with 0.25 mM MgCl2, than at 37 degrees C, with 1.0 mM MgCl2. This enhancement of IC50 values in the former system as compared with the latter was particularly dramatic for the less active agents (e.g., 28) as compared with the more active (e.g. 24).

Studies on the mechanisms of inhibition of L1210 cell growth by 3,4-dihydroxybenzohydroxamic acid and 3,4-dihydroxybenzamidoxime

Didox and Amidox inhibit L1210 cell growth in culture. At least one of the mechanisms in the mode(s) of action of the compounds is directed at the ribonucleotide reductase site. Partially purified preparations of ribonucleotide reductase activity are inhibited by Amidox and Didox. The formation of deoxycytidine nucleotides from [ 14C]cytidine in intact L1210 cells is also blocked. Didox and Amidox cause the decrease in the intracellular pools of the four dNTPs. Hydroxyurea-resistant L1210 cells are not cross-resistant to either Didox or Amidox. These data suggest that Didox and Amidox are not inhibiting ribonucleotide reductase through a mechanism similar to hydroxyurea.

Quinazoline antifolate thymidylate synthase inhibitors: 2'-Fluoro-N10-propargyl-5,8,-dideazafolic acid and derivatives with modifications in the C-2 position

The synthesis of 2'-fluoro-10-propargyl-5,8-dideazafolic acid and its 2-desamino, 2-desamino-2-hydroxymethyl, and 2-desamino-2-methoxy analogues is described. In general the synthetic route involved the coupling of diethyl N-[2-fluoro-4-(prop-2-ynylamino)benzoyl]-L-glutamate with the appropriate 6-(bromomethyl)quinazoline followed by deprotection with mild alkali. These four compounds together with the 2-desamino-2-methyl analogue were tested for their activity against L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth of the L1210 cell line and of two mutant L1210 cell lines, the L1210:R7A that overproduces dihydrofolate reductase (DHFR) and the L1210:1565 that has impaired uptake of reduced folates. Compared with their non-fluorinated parent compounds, the 2'-fluoro analogues were all approximately 2-fold more potent as TS inhibitors. Similarly, they also showed improved inhibition of L1210 cell growth (1.5-5-fold), and this activity was prevented by co-incubation with thymidine. All had retained or improved activity against both the L1210:R7A and L1210:1565 cell lines.

Synthesis, antiproliferative, and antiviral activity of certain 4-substituted and 4,5-disubstituted 7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyrimidines

The sodium salts of 4-chloro- and several 4-chloro-5-substituted-7H-pyrrolo[2,3-d]pyrimidines were treated with [1,3-bis(benzyloxy)-2-propoxy]methyl chloride (6) to provide the corresponding 4-chloro- and 4-chloro-5-substituted-7-[[1,3-bis(benzyloxy)-2-propoxy]methyl]pyrrolo [2,3-d]pyrimidines (7-11). Debenzylation with boron trichloride at -78 degrees C furnished 4-chloro- and several 4-chloro-5-substituted-7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3- d]pyrimidines (12.16). Subsequent amination of 12-16 yielded the 4-amino-5-substituted-7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3- d]pyrimidines (17-21). Treatment of 14 with methylamine and 13 and 14 with ethylamine yielded the 4-(alkylamino)-5-halo-7-[(1,3-dihydroxy-2- propoxy)methyl]pyrrolo[2,3-d]pyrimidines (22-24). Treatment of 12-15 with hydroxylamine in refluxing 2-propanol yielded the 5-substituted-4-(hydroxyamino)-7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrol o [2,3-d]pyrimidines (25-28). Treatment of compound 12 with Pd/C under a hydrogen atmosphere has furnished the nebularine analogue 31. The antiproliferative activity of compounds 17-28 and 31 was studied using L1210 cells in vitro. The 4-amino- and 4-(hydroxyamino)-5-halogenated derivatives (compounds 18-20, 26-28) inhibited cell growth. Although the effect of compounds 18-20 and 27 on final growth rate was pronounced (IC50 = 2.3, 0.7, 2.8, and 3.7 microM, respectively), cells underwent at least one doubling before cell division stopped. The remaining compounds were less cytotoxic, with IC50's greater than 30 microM for 21, 23, 26, and 28, whereas no inhibition of L1210 cell growth was observed with compounds 17, 22, 24, 25, and 31 at 100 microM. The antiviral activity of these compounds also was tested. Compounds 18-20 and 26-28 were active against human cytomegalovirus and herpes simplex type 1. The 4-amino derivatives (18-20) were more active than the 4-hydroxyamino derivatives (26-28), the 4-amino-5-bromo and 4-amino-5-iodo derivatives produced more than five log reductions in virus titer at concentrations of 10-100 microM. Although some cytotoxicity was observed at these concentrations, compound 19 was active against murine cytomegalovirus in vivo. At 5.6 mg/kg, 14/15 animals survived compared to 10/15 treated with 5.6 mg/kg of ganciclovir or 1/15 treated with placebo.

Potent inhibition of thymidylate synthase by two series of nonclassical quinazolines

The synthesis and biological activity of two series of nonclassical thymidylate synthase (TS) inhibitors are described. The first is a series of 10-propargyl-5,8-dideazafolic acid derivatives (10a-j) and the second is a series of the analogous 2-desamino derivatives (13a-c,k), both bearing a more lipophilic substituent on the phenyl ring than the CO-glutamate of classical antifolates. Compounds 10a-j were prepared in a straightforward manner, generally by treatment of N-[6-(bromomethyl)-3,4-dihydro-4-oxo-2-quinazolinyl]-2,2-dimethylprop anamide (6) with various phenyl-substituted N-propargylanilines (8), followed by deprotection. Compounds 13a-c,k were prepared similarly from [6-(bromomethyl)-4-oxo-3(4H)-quinazolinyl] methyl 2,2-dimethylpropanoate (11). The compounds were tested for inhibition of purified L1210 TS and for inhibition of L1210 cell growth in vitro. Several of these nonclassical analogues approached the TS inhibitory potency of 10-propargyl-5,8-dideazafolic acid (1, CB3717), a glutamate-containing TS inhibitor. 2-Amino target compounds 10a-j were generally potent inhibitors of L1210 TS, with IC50s within the range of 0.51-11.5 microM, compared to 0.05 microM for 1. The order of potency for phenyl substitution at the 4-position in this series was the following: COCF3 greater than or equal to NO2 greater than or equal to CONH2 greater than or equal to COCH3 greater than SO2NMe2 greater than CN much greater than OCF3 greater than or equal to F. The 2-desamino target compounds 13a-c,k also exhibited significant, although diminished, TS inhibition. Both series were growth inhibitory to cells in tissue culture and this inhibition could be reversed by thymidine alone, indicating that the primary target was TS. None of the compounds was a potent inhibitor of dihydrofolate reductase. These studies indicate that the presence of the glutamate moiety in folate analogues is not an absolute requirement for potent inhibition of TS.

In vitro and in vivo anti‐tumor activity of L‐glutamic acid γ‐monohydroxamate against L1210 leukemia and B16 melanoma

A glutamine analogue, L-glutamic acid gamma-monohydroxamate (GAH) demonstrated complete cytotoxicity against L1210 cells in culture and marked anti-tumoral activity in vivo against L1210 leukemia and B16 melanoma. In vitro, GAH caused concentration-dependent inhibition of L1210 cell growth, with complete cell death being reached at 72 hr and at a 500 microM concentration. A minimal incubation time of 38 hr with 500 microM GAH was necessary to obtain complete cell death at 72 hr. During incubation, GAH is metabolized to hydroxylamine. Hydroxylamine acts as the active form of GAH, since the concentration-dependent inhibition of cell growth caused by hydroxylamine is the same as that observed with GAH. The cytotoxic effects of GAH and hydroxylamine on L1210 cells were not reversed or prevented by L-glutamine or L-glutamic acid and purine nucleosides but were prevented or reversed by pyruvate, 2-oxaloacetate and 2-oxoglutarate. In vivo, GAH considerably increased survival of mice bearing L1210 leukemia or a solid tumor, the B16 melanoma. Antitumor activity of GAH against L1210 leukemia and B16 melanoma was schedule-dependent. The administration of GAH 3 times daily was more effective than a twice daily treatment and the maximum ILS was observed using split-dose schedules on days 1 through 3 and 7 through 9 without noticeable toxicity. Under these conditions hydroxylamine is highly toxic, suggesting that in vivo GAH might act as an hydroxylamine releaser in the tumor cells and is not significantly metabolized in the body.

Structure and activity relationship of several novel CC-1065 analogs.

CC-1065 was found to cause delayed toxicity at therapeutic doses, therefore, a large number of analogs have since been synthesized. A series of analogs with simplified but closely related structures were chosen for this investigation because some were found to be superior to CC-1065 in the treatment of several experimental tumors. The inhibition of L1210 cell growth by U-68,415 was comparable to that by CC-1065. A similar situation was true in terms of their in vivo potency; however, U-68,415 was superior to CC-1065 in terms of anti-P388 leukemia activity. At the optimal dosage, U-68,415 produced 4 out of 6 long-term (greater than 30 day) survivors; whereas CC-1065 produced a mere 62% increase of life span (ILS) and no long-term survivors. The order of antitumor potency and effectiveness of the CC-1065 analogs was U-68,415 greater than U-66,694 greater than U-68,819 greater than U-66,664, which was parallel to the inhibition of L1210 cell growth. CC-1065 and all the analogs tested here inhibited DNA synthesis approximately 10 times more than RNA synthesis. Protein synthesis was the least inhibited. On a molar basis, U-68,415 was about 6-9 times more inhibitory toward cellular DNA synthesis than CC-1065, yet the interaction and/or binding of CC-1065 to DNA determined by circular dichroism, DNA melting or differential cytotoxicity assay was much stronger than that of U-68,415.(ABSTRACT TRUNCATED AT 250 WORDS)

Didemnin B. The first marine compound entering clinical trials as an antineoplastic agent.

A new class of marine compounds, the didemnins, with potent antitumor activity has been identified. They share the novel structure of a cyclic depsipeptide. Among three structurally related compounds, didemnin B is by far the most potent in its in vitro cytotoxicity and in vivo antitumor activity (0.001 microgram/ml inhibits the growth of L1210 leukemia cells by 50%). It also demonstrates good antitumor activity against B16 melanoma and moderate activity against M5076 sarcoma and P388 leukemia. The compound also has good antiviral and potent immunosuppressive properties. Although the precise mechanism of action for the cytotoxicity remains unknown, the agent inhibits protein synthesis more than DNA synthesis and the inhibition of protein synthesis is closely correlated with inhibition of L1210 cell growth. Toxicology studies in CD2F1 mice, Fischer 344 rats and beagle dogs reveal that major target organs are the lymphatics, gastrointestinal tract, liver and kidney. Phase I trials are currently in progress under the auspices of the National Cancer Institute.

Lack of enhanced cytotoxicity of cultured L1210 cells using folinic acid in combination with sequential methotrexate and fluorouracil.

Previous studies from this laboratory have demonstrated that treatment of cultured cells with sequential methotrexate (MTX) and fluorouracil (FUra) leads to synergistic cell killing in several murine and human neoplasms in vitro. In this study leucovorin (folinic acid, LCV) was added to the MTX/FUra combination with the intention of generating elevated levels of methylenetetrahydrofolate to promote the formation of a stable fluorodeoxyuridylate-thymidylate synthetase ternary complex, thereby augmenting the cytotoxicity of the MTX-FUra sequence. The addition of 10 or 100 microM LCV concurrently with or after 10 microM FUra following MTX (1 microM) pretreatment did not augment the inhibition of L1210 cell growth or the clonigenicity compared with MTX prior to FUra without LCV. The effects of LCV scheduling on the sequential MTX and FUra-induced inhibition of thymidylate synthesis were measured by examining the rate of [6-3H] dUrd incorporation into the acid-precipitable cell fraction and by direct quantitation of the thymidylate synthetase ternary complex. Combination of 100 microM LCV with 10 microM FUra after 1 microM MTX resulted in significantly more ternary complex formation than did 1 microM MTX before 10 microM FUra alone. The inhibitory effects of FUra on thymidylate synthetase in the presence of MTX, however, could not be augmented by LCV as determined by [6-3H] incorporation into acid-precipitable material, nor did the addition of LCV result in increased cytotoxicity. Factors other than the inhibition of DNA synthesis may be critical to the cytotoxicity of sequential MTX and FUra in L1210 cells.

Inhibition of ribonucleotide reductase and L1210 cell growth by N-hydroxy- N′-aminoguanidine derivatives

A series of N-hydroxy- N′-aminoguanidine derivatives was studied for their effects on L1210 cell growth and ribonucleotide reductase activity. With the twelve compounds studied, there was a good correlation between the inhibition of L1210 cell growth and the inhibition of ribonucleotide reductase activity. The most potent compound required concentrations of only 1.4 and 2 μM for 50% inhibition of L1210 cell growth and ribonucleotide reductase activity respectively. These guanidine analogs specifically inhibited the conversion of [ 14C]cytidine and deoxycytidine nucleotides in the nucleotide pool and the incorporation of [ 14C]cytidine into DNA without altering the incorporation of [ 14C]cytidine into RNA. Ribonucleotide reductase activity in drug-treated cells was reduced markedly. Iron-chelating agents did not either increase or decrease the inhibition caused by the N-hydroxy- N′-aminoguanidine derivatives. No evidence was obtained that these derivatives selectively inactivated one of the subunits of ribonucleotide reductase. These compounds appear to inhibit ribonucleotide reductase by a mechanism different from hydroxyurea or the thiosemicarbazone derivatives.

Effect of the dialdehyde derivative of 5′-deoxyinosine on pyrimidine deoxyribonucleoside metabolism in L1210 cells

The effects of the dialdehyde derivatives of inosine (Inox) and 5′-deoxyinosine (5′-dInox) on L1210 cells were compared. The growth of L1210 cells was inhibited to a greater extent by 5′-dInox than by Inox. The increased inhibition of L1210 cell growth by 5′-dInox was also reflected by the increased inhibition of the incorporation of precursors into RNA, DNA and proteins. Even though 5′-dInox was a more potent inhibitor, Inox accumulated in the L1210 cells to levels 4- to 5-fold greater than 5′-dInox. The metabolism of [5- 3H]deoxycytidine and [5- 3H]deoxyuridine by L1210 cells in culture, in the presence of Inox or 5′-dInox, indicated that dCMP deaminase was an intracellular site of action for 5′-dInox. The dCMP deaminase activity in cell-free extracts prepared from 5′-dInox-treated cells was reduced markedly. This decrease in activity was not reversed by increased substrate concentrations nor was the activity subject to allosteric activation by dCTP. Deoxyuridine and deoxycytidine were able to reverse the effects of 5′-dInox on the inhibition of L1210 cell growth.

The utility of combinations of drugs directed at specific sites of the same target enzyme — ribonucleotide reductase as the model

Ribonucleotide reductase is a key enzyme in DNA replication and, as such, has been a target for antitumor agents. This enzyme is composed of two non-identical protein subunits which can be specifically and independently inhibited. Combinations of drugs directed at the effector-binding and non-heme iron subunits of ribonucleotide reductase resulted in the synergistic inhibition of L1210 cell growth and synergistic L1210 cell kill. These combinations included dAdo/EHNA/IMPY/Desferal; dAdo/EHNA/hydroxyurea/Desferal (the EHNA was required to protect dAdo from deamination while Desferal modulated the effects of IMPY or hydroxyurea); 2-F-araA/IMPY/Desferal and 2-F-2′-dAdo/IMPY/Desferal (EHNA was not required to protect 2-F-araA or 2-F-2′-dAdo from deamination); and dGuo/8-AGuo/IMPY/Desferal (8-AGuo was required to protect dGuo from phosphorolysis). Although thymidine alone inhibited L1210 cell growth, it was not possible to potentiate the effects of thymidine with the pyrimidine nucleoside phosphorylase inhibitors, acyclothymidine, 5-chlorouracil and 2,6-dihydroxypyridine. Combinations of drugs directed at the ribonucleotide reductase and DNA polymerase sites were studied for their effects on L1210 cell growth. With these combinations, no synergistic inhibition of L1210 cell growth was observed. The combinations of aphidicolin and IMPY/Desferal and aphidicolin and dAdo/EHNA inhibited L1210 cell growth in an additive manner; the combinations of IMPY/Desferal and BuAU or IMPY/Desferal and BuPdG resulted in antagonistic inhibition of L1210 cell growth. From these results it is clear that combination chemotherapy directed at independent sites of the same key target enzyme can result in strong synergistic inhibition of cell growth and cytotoxicity offering a clear therapeutic advantage. In contrast, the combinations directed at sequential key enzymes (e.g. ribonucleotide reductase and DNA polymerase) did not result in synergistic inhibition of cell growth. The utility of combinations of drugs directed at specific but independent sites of the target enzyme (e.g. ribonucleotide reductase) has been demonstrated in tumor cell systems in culture and now must be demonstrated in vivo.

Drug action on ribonucleotide reductase

Ribonucleotide reductase catalyzes the rate-limiting step in DNA synthesis. It represents a key metabolic site at which specific inhibitors have been directed as potential antitumor agents. Several different classes of ribonucleotide reductase inhibitors have been generated and studied. Because of the nature of the DNA polymerase reaction in which all four dNTPs are required, the initial velocity vs dNTP concentration curve gives sigmoidal rather than hyperbolic kinetics. As a result, a 50 per cent decrease in ribonucleotide reductase activity causes a decrease in DNA polymerase activity of 75 per cent or greater depending on the ratio of [dNTP] to its K m. This has been demonstrated with theoretical calculations, actual DNA polymerase determinations and precursor studies in intact tumor cells. The structural requirements for a compound to serve as a specific inhibitor of ribonucleotide reductase, either as the non-heme iron or effector-binding subunit, are stringent. Each protein subunit comprising the active enzyme can be specifically and independently inhibited. When combinations of agents, each directed at one of the subunits of ribonucleotide reductase, are used, strong synergistic inhibition of L1210 cell growth and synergistic cytotoxicity result.

An Evaluation of the Effects of Combination ChemotherapyIn VitroUsing DNA-Reactive Agents

The combined application of DNA unwinding and strand-scission agents is a novel and potentially important approach to cancer therapy, based in part on mechanistic considerations of drug action. In order to evaluate this hypothesis a number of experiments were performed in which the cellular cytotoxicity of DNA reactive agents (ethidium bromide, adriamycin or cis-platinum) were evaluated alone and in combination with bleomycin, a strand-scission agent, using a number of different tumor cell systems in vitro. The results of these studies indicated that combinations of these agents were found to be much more effective than treatment with single drugs alone. This conclusion was warranted for the action of ethidium bromide followed by bleomycin with murine L1210 leukemia, melanoma B16-BL6 and human HeLa cells, and cis-platinum followed by bleomycin with L1210 and B16-BL6 cells. These data support previous findings in which synergistic growth inhibition of L1210 cells by ethidium bromide, followed by bleomycin was explained by a two-step mechanism; first, ethidium bromide introduces changes in DNA-conformation resulting in the facilitation of a second step in which bleomycin cleaves DNA more efficiently. Therefore, the rational use of combinations of DNA reactive agents based on mechanistic considerations should result with improved therapeutic regimens for the treatment of cancer.