In vitro and in vivo properties of novel nucleoside transport inhibitors with improved pharmacological properties that potentiate antifolate activity.

The activity of antimetabolite inhibitors of de novo deoxyribonucleotide biosynthesis can be compromised by the salvage of extracellular preformed nucleosides and nucleobases. Dipyridamole (DP) is a nucleoside transport inhibitor that has been used clinically in an attempt to increase antimetabolite activity; however, DP binds tightly to the serum protein alpha1-acid glycoprotein (AGP) thereby rendering this therapeutic strategy largely ineffective. Four novel DP analogues (NU3076, NU3084, NU3108, and NU3121) have been developed with substitutions at the 2,6- and 4,8-positions of the pyrimidopyrimidine ring. The novel DP analogues inhibit thymidine (dThd) uptake into L1210 cells in vitro (NU3076 IC(50), 0.25 microM; NU3084 IC(50), 0.27 microM; NU3108 IC(50), 0.31 microM; NU3121 IC(50), 0.26 microM; and DP IC(50), 0.37 microM), but, unlike DP, their activity remains largely unaffected in the presence of 5 mg/ml AGP. The four DP analogues inhibit dThd and hypoxanthine rescue from Alimta (multitargeted antifolate)-induced growth inhibition in A549 and COR L23 human lung carcinoma cell lines in the presence of 2.5 mg/ml AGP, whereas the activity of DP is completely abolished. i.p. administration of 10 mg/kg NU3108, NU3121, and DP produced peak plasma concentrations of 4.4, 2.1, and 6.7 microM, respectively, and levels were sustained above 1 microM for approximately 45 min (DP) and 120 min (NU3108 and NU3121). [3H]thymidine incorporation into COR L23 xenografts grown in CD1 nude mice was reduced by 64% (NU3108), 44% (NU3121), and 65% (DP) 2 h after administration of the nucleoside transport inhibitors. In conclusion, two novel DP analogues (NU3108 and NU3121) have been identified that do not bind to AGP and that display superior pharmacokinetic profiles in comparison to DP and inhibit [3H]thymidine incorporation into human tumor xenografts in vivo.

Reaction-kinetic parameters of glycidamide as determinants of mutagenic potency.

Values for reaction-kinetic parameters of electrophiles can be used to predict mutagenic potency. One approach employs the Swain-Scott relationship for comparative kinetic studies of electrophilic agents reacting with nucleophiles. In this way glycidamide (GA), the putatively mutagenic/carcinogenic metabolite of acrylamide, was assessed by determining the rates of reaction with different nucleophiles. The rate constants (kNu) were determined using the "supernucleophile" cob(I)alamin [Cbl(I)] as an analytical tool. The Swain-Scott parameters for GA were compared with those of ethylene oxide (EO). The substrate constants, s values, for GA and for EO were found to be 1.0 and 0.93, respectively. The reaction rates at low values of nucleophilic strength (n=1-3), corresponding to oxygens in DNA, were determined to be 2-3.5 times higher for GA compared to EO. GA was also more reactive than EO towards other nucleophiles (n=0-6.4). The mutagenic potency of GA was determined in Chinese hamster ovary cells (hprt mutations in CHO-AA8 cells per dose unit with gamma-radiation as reference standard). The potency of GA was estimated to be about three mutations per 10(5) cells and mMh corresponding to about 40 rad-equ./mMh. A preliminary comparison of the mutagenic potency (per mMh and as rad-equivalents) of GA and EO shows an approximately seven times higher potency for GA. A higher mutagenic potency of GA compared to EO is compatible with expectation from reaction-kinetic data of the two compounds. The data confirmed that GA is not a strong mutagen, which is in line with what is expected for simple oxiranes. The present study shows the value of cob(I)alamin for the determination of reaction-kinetic parameters and their use for prediction of mutagenic potency.

The role of inhibitors of poly(ADP-ribose) polymerase as resistance-modifying agents in cancer therapy.

Poly(ADP-ribose) polymerase (PARP) plays an important role in a number of cellular processes including DNA repair. Since poly(ADP-ribosyl)ation occurs in response to radiation- or drug-induced DNA damage, inhibitors of the enzyme may enhance the antitumour activity of radiotherapy or cytotoxic drug treatment. In this review the development of PARP inhibitors is discussed, and structure-activity relationships amongst inhibitors of the enzyme are presented. Studies to date regarding the in vitro and in vivo activity of PARP inhibitors, as resistance modifying agents in cancer therapy, are also overviewed.

Resistance-modifying agents. 5. Synthesis and biological properties of quinazolinone inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP).

Clinical studies concerning the role of poly(ADP-ribose) polymerase (PARP) in the repair of drug- and radiation-induced DNA damage have been impeded by the poor solubility, lack of potency, and limited specificity of currently available inhibitors. A series of 2-alkyl- and 2-aryl-substituted 8-hydroxy-, 8-methoxy-, and 8-methylquinazolin-4(3H)-ones has been synthesized and evaluated for PARP inhibitory activity in permeabilized L1210 murine leukemia cells. 8-Methoxy- and 8-methylquinazolinones (14-34) were readily prepared by acylation of 3-substituted anthranilamides with the appropriate acid chloride, followed by base-catalyzed cyclization. The requisite 8-hydroxyquinazolinones (6, 35-39) were synthesized by demethylation of the corresponding 8-methoxyquinazolinones with BBr3. N-Methylation of 8-methoxy-2-methylquinazolinone (15) with MeI, followed by O-demethylation by BBr3, afforded the control N3-methylquinazolinones 42 and 43, respectively. In general, an 8-hydroxy or 8-methyl substituent enhanced inhibitory activity in comparison with an 8-methoxy group. 2-Phenylquinazolinones were marginally less potent than the corresponding 2-methylquinazolinones, but the introduction of an electron-withdrawing or electron-donating 4'-substituent on the 2-aryl ring invariably increased potency. This was particularly evident in the 8-methylquinazolinone series (IC50 values 0.13-0.27 microM), which are among the most potent PARP inhibitors reported to date. N3-Methylquinazolinones 42 and 43 were essentially devoid of activity (IC50 values > 100 microM). In studies with L1210 cells in vitro, a concentration of 200 microM 8-hydroxy-2-methylquinazolinone (6, NU1025) (IC50 value 0.40 microM) potentiated the cytotoxicity of the monomethylating agent 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide and gamma-radiation 3.5- and 1.4-fold, respectively, at the 10% survival level.

Resistance-modifying agents. 9. Synthesis and biological properties of benzimidazole inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents. Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-benzimidazole-4-carboxamide (23, K(i) = 15 nM), in which the phenyl ring contains substituents, have been synthesized. Many of these derivatives exhibit K(i) values for PARP inhibition < 10 nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, K(i) = 1.6 nM) being one of the most potent. Insight into structure-activity relationships (SAR) for 2-aryl-1H-benzimidazole-4-carboxamides has been enhanced by studying the complex formed between 2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, K(i) = 6 nM) and the catalytic domain of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide (45, K(i) = 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780 cells in vitro (by 2.8- and 2.9-fold, respectively).

Resistance-modifying agents. 8. Inhibition of O(6)-alkylguanine-DNA alkyltransferase by O(6)-alkenyl-, O(6)-cycloalkenyl-, and O(6)-(2-oxoalkyl)guanines and potentiation of temozolomide cytotoxicity in vitro by O(6)-(1-cyclopentenylmethyl)guanine.

A series of O(6)-allyl- and O(6)-(2-oxoalkyl)guanines were synthesized and evaluated, in comparison with the corresponding O(6)-alkylguanines, as potential inhibitors of the DNA-repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Simple O(6)-alkyl- and O(6)-cycloalkylguanines were weak AGT inactivators compared with O(6)-allylguanine (IC(50) = 8.5 +/- 0.6 microM) with IC(50) values ranging from 100 to 1000 microM. The introduction of substituents at C-2 of the allyl group of O(6)-allylguanine reduced activity compared with the parent compound, while analogous compounds in the O(6)-(2-oxoalkyl)guanine series exhibited very poor activity (150-1000 microM). O(6)-Cycloalkenylguanines proved to be excellent AGT inactivators, with 1-cyclobutenylmethylguanine (IC(50) = 0.55 +/- 0.02 microM) and 1-cyclopentenylmethylguanine (IC(50) = 0.39 +/- 0.04 microM) exhibiting potency approaching that of the benchmark AGT inhibitor O(6)-benzylguanine (IC(50) = 0.18 +/- 0.02 microM). 1-Cyclopentenylmethylguanine also inactivated AGT in intact HT29 human colorectal carcinoma cells (IC(50) = 0.20 +/- 0.07 microM) and potentiated the cytotoxicity of the monomethylating antitumor agent Temozolomide by approximately 3- and 10-fold, respectively, in the HT29 and Colo205 tumor cell lines. The observation that four mutant AGT enzymes resistant to O(6)-benzylguanine also proved strongly cross-resistant to 1-cyclopentenylmethylguanine indicates that the O(6)-substituent of each compound makes similar binding interactions within the active site of AGT.

Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles.

Substituted guanines and pyrimidines were tested as inhibitors of cyclin B1/CDK1 and cyclin A3/CDK2 and soaked into crystals of monomeric CDK2. O6-Cyclohexylmethylguanine (NU2058) was a competitive inhibitor of CDK1 and CDK2 with respect to ATP (Ki values: CDK1, 5 +/- 1 microM; CDK2, 12 +/- 3 microM) and formed a triplet of hydrogen bonds (i.e., NH-9 to Glu 81, N-3 to Leu 83, and 2-NH2 to Leu 83). The triplet of hydrogen bonding and CDK inhibition was reproduced by 2,6-diamino-4-cyclohexylmethyloxy-5-nitrosopyrimidine (NU6027, Ki values: CDK1, 2.5 +/- 0.4 microM; CDK2, 1.3 +/- 0.2 microM). Against human tumor cells, NU2058 and NU6027 were growth inhibitory in vitro (mean GI50 values of 13 +/- 7 microM and 10 +/- 6 microM, respectively), with a pattern of sensitivity distinct from flavopiridol and olomoucine. These CDK inhibition and chemosensitivity data indicate that the distinct mode of binding of NU2058 and NU6027 has direct consequences for enzyme and cell growth inhibition.

Molecular features necessary for the uptake of diamines and related compounds by the polyamine receptor of rat lung slices.

The influence of 17 putrescine analogues on the uptake of putrescine and/or paraquat by rat lung slices has been determined. Most of these compounds are competitive inhibitors of putrescine and/or paraquat uptake, but three show no inhibiting activity. Apparent Ki values of the putrescine derivatives increase, and thus the inhibitory effects decrease, with increasing N-methylation. Comparison of N-methyl-1,4-diaminobutane (Ki = 8 microM) with N,N'-bis-methyl-1,4-diaminobutane (Ki = 25.5 microM) shows that a single primary amino group is desirable for high inhibiting activity. Dimethylation at one amino function does not greatly decrease inhibitory potential (thus N,N-dimethyl-1,4-diaminobutane has Ki = 11.5 microM). Increasing the size of N-alkyl substituents in putrescine derivatives, decreased their inhibitory action on the uptake of putrescine. Investigation of the effect of conformationally-restricted analogues of putrescine shows that both (E) and (Z) isomers of 1,4-diaminobut-2-ene are poor inhibitors of putrescine uptake. Analogues of putrescine with bulky substituents on the butyl chain, i.e. the meso- and rac-isomers of 1,1-dichloro-2,3-diaminomethylcyclopropane, do not inhibit putrescine uptake. Inhibiting putrescine derivatives which contain aziridine groups are competitive inhibitors of putrescine and paraquat uptake. Surprisingly, N-(4-aminobutyl)aziridine is the most effective inhibitor of putrescine uptake studied, and is a better inhibitor of paraquat uptake than the endogenous polyamine, putrescine. N-(4-Aminobutyl)aziridine binds reversibly to the polyamine transporter and its inhibitory effects do not appear to be due to any cytotoxic activity of the aziridine. The parameter A (mM)-1 defined as 1000/Ki (where Ki units are microM) was taken as a measure of the affinity of a compound for the polyamine receptor in this paper.

Chemistry of muconaldehydes of possible relevance to the toxicology of benzene.

(Z,Z)-Muconaldehyde reacted with primary amines to give N-substituted-2(2'-oxoethyl)-pyrroles, which were reduced to N-substituted-2-(2'-hydroxyethyl)-pyrroles by sodium borohydride. The pyrrole-forming reaction is exhibited by valine and its methyl ester, and is being developed with terminal valine in hemoglobin as a means of dose monitoring (Z,Z)-muconaldehyde, a putative metabolite of benzene. Reactions in aqueous solution between (Z,Z)-muconaldehyde and adenosine, deoxyadenosine, guanosine, or deoxyguanosine leading to pyrrole-containing adducts are described. The elucidation of the structures of the adducts was assisted by the study of reactions between (Z,Z)-muconaldehyde and both nucleoside derivatives and a model compound for guanosine. Reactions of (Z,Z)-muconaldehyde are complicated by its isomerization to (E,Z)- and (E-E)-muconaldehyde. The kinetics of this process have been studied in benzene, acetonitrile, and dimethylsulfoxide.

Studies on the molecular toxicology of buta-1,3-diene and isoprene epoxides.

Reactions of ethenyloxirane with amino (RNH2) and thiol (R'SH) nucleophiles occur by an SN2 mechanism involving competing ring cleavage at C-2 and C-3. In contrast, 2-ethenyl-2-methyloxirane reacts with amino (RNH2) and thiolate (R'S-) nucleophiles in methanol by regioselective SN2 attack at C-3 ("neo-pentyl" position). However, in pure water or methanol SN1 reaction occurs mainly at C-2.

Metabolism and molecular toxicology of isoprene.

Isoprene (2-methylbuta-1,3-diene) is a large-scale petrochemical used principally in the manufacture of synthetic rubbers. It is also produced by plants and trees and is the major endogenous hydrocarbon formed by mammals, probably from mevalonic acid. Isoprene is metabolised by mammals in processes that involve epoxidation by cytochrome P450-dependent monooxygenases to the isomeric mono-epoxides, (1-methylethenyl)-oxirane and 2-ethenyl-2-methyloxirane. Further metabolism of the mono-epoxides to mutagenic isoprene di-epoxides, (2, 2')-2-methylbioxiranes, can also occur. The oxidations to the mono- and di-epoxides occur enantioselectively and diastereoselectively. The mono-epoxides are hydrolysed enantioselectively to vicinal diols under catalysis by epoxide hydrolase. 2-Ethenyl-2-methyloxirane is also readily hydrolysed non-enzymatically. Because of the stereochemical possibilities for metabolites, the metabolism of isoprene is complex. The metabolism of isoprene by liver microsomes in vitro from a range of species including rat, mouse and human shows significant differences between species, strains and gender in respect of the diastereoselectivity and enantioselectivity of the metabolic oxidation and hydrolysis reactions. The impact of the extra methyl in isoprene on di-epoxide reactivity also appears to be critically important for the resulting biological effects. Isoprene di-epoxides may exhibit a lower cross-linking potential in vivo compared to butadiene di-epoxides. Differences in metabolism and reactivity of metabolites may be factors contributing to the significant differences in toxicological response to isoprene observed between species.

Structure and mechanism of formation of a new adduct from formaldehyde and guanosine.

Reactions between formaldehyde and guanosine in the range pH 4-10 gave N2-hydroxymethylguanosine 1, bis-N2-guanosinylmethane 2 and 7-[7-(beta-D-ribofuranosyl)-purin-6(7H)- on-2-yl]-3-(beta-D-ribofuranosyl)-5,6,7,8-tetrahydro-sym-triazino[ 1,2-alpha] purin-10(3H)-one 3a. The latter is a new adduct, the formation of which occurs in neutral solution, is favoured at higher pH and can be rationalised by a sequence of condensations involving two molecules each of guanosine and formaldehyde.

Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026.

In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation.