Stabilities of 7-alkylguanosines and 7-deoxyguanosines formed by phosphoramide mustard and nitrogen mustard

7-Alkylguanosine and 7-alkyldeoxyguanosine were prepared from phosphoramide mustard and nitrogen mustard in nonaqueous conditions. The guanosine products were N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl] phosphorodiamidic acid, and N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl]methylamine, respectively. These were also formed in aqueous reactions but they rapidly underwent secondary reactions. The half-life of the phosphoramide mustard-guanosine adduct was 3.1 h (37 degrees C, pH 7.4) and that of the nitrogen mustard adduct 1 h (25 degrees C, pH 7.4), as determined by HPLC. The half-lives of the respective adducts for imidazole ring-opening were 9.5 h and 0.78 h (37 degrees C, pH 7.4). The respective deoxyguanosine derivatives depurinated with half-lives of 8.5 h and 1.6 h (25 degrees C, pH 4.2). The mustard adducts are notably more labile than simple alkyl substituted guanosines and deoxyguanosines.     

Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen mustard analogues in an SV40-based shuttle plasmid.

It was previously shown that the predominant mutations induced by melphalan (L-phenylalanine mustard) in the supF gene of shuttle plasmid pZ189 during replication in human cells are A.T----T.A transversions. In order to determine whether adenine adducts were formed at sequence positions corresponding to these mutations, melphalan-induced thermolabile adducts were mapped in the supF gene by selective depurination followed by strand cleavage in alkali. All A.T base pairs which were frequent sites for melphalan-induced A.T----T.A transversions were also prominent sites for formation of thermolabile adenine adducts. Although no mutations were detected at some prominent adduct sites, there was a significant correlation between adduct sites and mutation sites. While runs of two or more adenines were particularly prominent adduct sites, comparison of results obtained with 3'- and 5'-end-labeled DNA gave no evidence for intrastrand cross-links between adjacent adenines. Chlorambucil, another aromatic nitrogen mustard, showed sequence specificities for both mutagenesis and adenine adduct formation nearly identical to those seen with melphalan. The nonaromatic analogues mechlorethamine and phosphoramide mustard were much less efficient in inducing thermolabile adenine adducts, and mechlorethamine induced significantly fewer transversions at A.T base pairs than chlorambucil or melphalan. Formation of thermolabile adenine adducts by the aromatic nitrogen mustards was markedly reduced by blockage of the minor groove with distamycin, or by prior heat denaturation of the DNA. These results suggest that alkylation occurs primarily at the N-3 rather than N-7 position of adenine, probably as a consequence of the affinity of the aromatic rings of melphalan and chlorambucil for the minor groove.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of dietary carrot on cytostatic drug activity of cyclophosphamide and its main directly acting metabolite: induction of sister-chromatid exchanges in normal human lymphocytes, Chinese hamster ovary cells, and their DNA repair-deficient cell lines

We have utilized an in vivo drug metabolism technique (i.e. injecting the chemical into rat and isolating plasma with metabolites from blood) for detecting the genotoxicity of indirectly acting cyclophosphamide and its directly acting metabolite phosphoramide mustard in cultures of human peripheral blood lymphocytes of normal individuals, Fanconi's anaemia (FA) and aplastic anaemia (AA) patients, wild-type Chinese hamster ovary cells (CHO) and its DNA repair-deficient mutant 43-3B cells. In addition, the influence of dietary carrot on the clastogenic activity of these 2 chemicals in all the different cell types was studied. The genotoxicity was assessed by the ability of the metabolites of these agents to induce sister-chromatid exchanges in the treated cells. A dose-dependent increase in the frequencies of sister-chromatid exchanges was observed in all cell strains following treatment with activated metabolites of cyclophosphamide or phosphoramide mustard. The sensitivity of lymphocytes from normal donors, FA and AA patients to these 2 chemicals was similar. In CHO cell lines the induced frequency of sister-chromatid exchanges was slightly higher after treatment with the metabolites of cyclophosphamide than with phosphoramide mustard. The mutant 43-3B cells responded with higher frequencies of SCEs when compared to the wild-type CHO cells, about 1.5-2-fold, at low doses. Pretreating of rats with fresh carrot juice effectively inhibited the increase in the frequencies of sister-chromatid exchanges induced by cyclophosphamide in wild-type and mutant CHO cells (P less than 0.01), and to a lesser extent in human lymphocytes (p less than 0.05). In contrast, no inhibitory effect was observed in any of these cell types in combination of dietary carrot for direct acting phosphoramide mustard on the frequency of induced sister-chromatid exchanges. The possibility that dietary carrot exerts its antimutagenic effect by affecting the processes of enzymatic activation of cyclophosphamide is discussed.     

Effects of phosphoramide mustard and acrolein, cytotoxic metabolites of cyclophosphamide, on mouse limb development in vitro

Phosphoramide mustard and acrolein are toxic and reactive metabolites of the widely used anticancer drug and known teratogen cyclophosphamide. To study the mechanism(s) involved and to determine which of the active metabolites of cyclophosphamide is responsible for the production of limb malformations, the effects of exposure of cultured limb buds to phosphoramide mustard and acrolein were investigated. Fore- and hindlimbs were excised from ICR mice on day 12 of gestation and cultured in roller bottles for 6 days. Limbs were exposed to either phosphoramide mustard or acrolein (10 or 50 micrograms/ml) for the first 20 hours of the culture period. Exposure to phosphoramide mustard produced limb reduction malformations in both the fore- and hindlimbs; total limb bone area was greatly reduced, while the relative contribution of the paw to this area in forelimbs was increased. There was a fourfold reduction in both DNA and RNA; protein content was reduced only by one-half. Alkaline phosphatase activity was significantly decreased in fore- and hindlimbs exposed to phosphoramide mustard, whereas creatine phosphokinase activity was only reduced in hindlimbs in the limbs exposed to the higher concentration of phosphoramide mustard. Exposure to acrolein also produced malformed limbs with a mangled appearance; however, total limb bone area and the relative contribution of the long bones versus paw structures were not altered. Acrolein exposure had little effect on growth parameters such as DNA (decreased only in hindlimbs exposed to 50 micrograms/ml), RNA (increased in hindlimbs exposed to 50 micrograms/ml), or protein content. Alkaline phosphatase and creatine phosphokinase activities were not altered in acrolein-exposed fore- or hindlimbs. Thus, phosphoramide mustard and acrolein have dramatically different effects on developing limbs in vitro; this observation may indicate that they have different targets and/or mechanisms of action as teratogens in the limb. The effects of phosphoramide mustard are very similar to those of "activated" cyclophosphamide (4-hydroperoxycyclophosphamide).     

Mechanism of genotoxicity of diethylstilbestrol in vivo

Diethylstilbestrol (DES) is a carcinogen in humans and rodents which has eluded mechanistic clarification of its carcinogenic action. In vitro and in vivo, binding of DES to DNA has been found previously, but covalent DNA adducts could not be identified. In this study, the nature of binding was investigated by 32P-postlabeling, a rapid and highly sensitive assay for covalent DNA damage, to distinguish between a genotoxic or epigenetic mechanism of carcinogenesis by DES. A unique and distinct DNA adduct pattern was observed in kidney, liver, uterus (or testes) of female (or male, respectively) Syrian hamsters treated with a single injection of DES (200 mg/kg body weight). This set of DNA adducts closely matched patterns generated in vitro by reaction of diethylstilbestrol-4',4'-quinone with DNA or 2'-deoxyguanosine 3'-monophosphate. The major and several minor DES-DNA adducts in vivo had identical chromatographic mobilities in 11 different solvent systems with corresponding adducts obtained in vitro. The major adduct spot, generated in vitro by reaction of diethylstilbestrol-4',4'-quinone and DNA, was chemically unstable (half-life at 37 degrees C: 4-5 days). The persistence in vivo of these DNA modifications was low (biological half-life: 14 h) presumably because of chemical instability in concert with DNA repair. After injection of identical dosages of DES, adduct concentrations were 4-6-fold higher in females than in males. These results demonstrate that DES is capable of covalently modifying DNA. Moreover, diethylstilbestrol-4',4"-quinone is the major reactive metabolic intermediate responsible for the genotoxic activity of DES. Tumors are expected to arise only in rapidly dividing cells due to the short biological lifetimes of DES-DNA adducts.     

Peptide conjugates of 4-aminocyclophosphamide as prodrugs of phosphoramide mustard for selective activation by prostate-specific antigen (PSA)

In our continued effort to develop prodrugs of phosphoramide mustard, conjugates of 4-aminocyclophosphamide (4-NH₂-CPA) with three PSA-specific peptides were synthesized and evaluated as substrates of PSA. These include conjugates of cis-(2R,4R)-4-NH₂-CPA with a tetrapeptide Succinyl-Ser-Lys-Leu-Gln-OH, a hexapeptide Succinyl-His-Ser-Ser-Lys-Leu-Gln-OH, and a pentapeptide Glutaryl-Hyp-Ala-Ser-Chg-Gln-OH. These conjugates were cleaved by PSA efficiently and exclusively after the expected glutamine residue to release 4-NH₂-CPA, the activated prodrug form of phosphoramide mustard. The cleavage was most efficient for the pentapeptide conjugate 3 (Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-CPA), which showed a half-life of 55 min with PSA, followed by the hexapeptide conjugate 2 (Succinyl-His-Ser-Ser-Lys-Leu-Gln-NH-CPA) and the tertrapeptide conjugate 1 (Succinyl-Ser-Lys-Leu-Gln-NH-CPA) with half-lives of 6.5 and 12 h, respectively. These results indicate a potential of the conjugate 3 as an anticancer prodrug of phosphoramide mustard for selective PSA activation.     

Synthesis and analysis of DNA adducts of arylamines

Atylamines and nitroarenes are very important environmental and occupational pollutants. Genotoxic effects of arylamines are believed to be initiated by the formation of DNA adducts. DNA adducts of arylamines have been found in experimental animals and in exposed humans, and are predominantly formed with the carbon 8 of 2'-deoxyguanosine. Reference standards are necessary to develop methods for the quantification of DNA-adducts. Therefore, we have synthesized the 2'-deoxyguanosin-8-yI adducts of 2-methylaniline, 2-chloroaniline, 4-chloroaniline, 2,4dimethylaniline, and 2,6-dimethylaniline. The products were characterized by ¹H-NMR, ¹³C-NMR, MS and UV. The corresponding 2'-deoxyguanosine-3' -monophosphate adducts were synthesized for the quantification of DNA adducts by the ³²P-postlabelling technique. A GC-MS method was developed for the analysis of the new adducts as an alternative to the ³²P-postlabelling. DNA was spiked with the synthesized adducts and treated with 0.3 m NaOH overnight at 110 °C in the presence of a deuterated internal standard. We observed up to 80% recovery from about 1 adduct in 10⁸ to 1 in 10⁵ nucleotides.     

N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide as a potential bioreductively activated prodrug of phosphoramide mustard

N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide isomers (DMNA-NH-CPA, 4) were synthesized stereospecifically from Boc-l-Hse(OBn)-OH and the degradation of the corresponding reduced amine 5a was investigated by UV/vis spectroscopy and LC/MS. The rate of cyclization of 5a was found to increase with decreasing pH, with half-lives ranging from 3.2 to 54 min at pH 4–7.4, suggesting that the cyclization is catalyzed by the hydronium ions. LC/MS analysis of the degradation products of 5a indicates that 4-aminocyclophosphamide is rapidly released from 4 upon reductive activation under acidic conditions and further decomposes into the cytotoxic phosphoramide mustard. These results validated 4-aminocyclophosphamide as a prodrug form of phosphoramide mustard and suggest that compound 4 can potentially be used as a prodrug of phosphoramide mustard for bioreductive activation.     

32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.     

Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.     

Synthesis and stability of 2'-deoxyguanosine 3'-monophosphate adducts of dimethyl sulfate, ethylene oxide and styrene oxide

Deoxyguanosine 3'-monophosphate (dGMP) was alkylated at the 7-position by dimethyl sulfate, ethylene oxide and styrene oxide in aqueous media and glacial acetic acid, respectively, to yield reasonable quantities of the products, which were purified by HPLC. dGMP adducts are needed as standards for the 32P-postlabelling assay. The stability of the adducts was studied at 37 degrees and neutral pH. The half-lives of disappearance of 7-methyl-dGMP and the beta-isomers of the styrene oxide adducts were about 250 min; 7-hydroxy-ethyl-dGMP and the alpha-isomers of the styrene oxide adducts had respective half-lives of 340 and 440 min. In all cases the main degradation product was the corresponding guanine adduct. The results demonstrate considerable lability of the 7-alkylation products of dGMP which has to be taken into consideration in devising the 32P-postlabelling assay.     

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.     

Phenylalanyl-aminocyclophosphamides as model prodrugs for proteolytic activation: synthesis, stability, and stereochemical requirements for enzymatic cleavage

4-Aminocyclophosphamide (4-NH2-CPA, 7) was proposed as a prodrug moiety of phosphoramide mustard. Four diastereomers of phenylalanine-conjugates of 4-NH2-CPA were synthesized and their stereochemistry was assigned based on chromatographic and spectroscopic data. All diastereomers were stable in phosphate buffer but only the cis-(4R)-isomer of 15 was efficiently cleaved by alpha-chymotrypsin with a half-life of 20 min, which is much shorter than the 8.9h to >12h half-lives found for the other diastereomers. LC-MS analysis of the proteolytic products of cis-(4R)-15 indicated that 4-NH2-CPA was released upon proteolysis and further disintegrated to phosphoramide mustard. These results suggest the feasibility of using peptide-conjugated cis-(4R)-4-NH2-CPA as potential prodrugs for proteolytic activation in tumor tissues.     

32P-postlabeling of N-7, N2 and O6 2'-deoxyguanosine 3'-monophosphate adducts of styrene oxide

Adducts were prepared by reacting styrene oxide with 2-deoxyguanosine 3'-monophosphate (dGMP). Four isomeric N-7-, two diastereomeric N2- and three isomeric O6-adduct were isolated and characterized. The adducts were used as substrates in the 32P-postlabeling reaction. No phosphorylation products were seen with the N-7-alkylation products. One diastereomeric N2-adduct was labeled with 20% efficiency and the second with a markedly lower efficiency. Two of the three O6-adducts were labeled with 5% and the third with 10% labeling efficiency. The results suggest that large N-7-dGMP adducts are very poor substrates of T4 polynucleotide kinase. The diastereomeric products are labeled at different efficiencies indicating stereoselectivity in the kinase reaction.     

Improved detection limit for a direct determination of 8-hydroxy-2'-deoxyguanosine in untreated urine samples by capillary electrophoresis with optical detection

Method for a direct determination of 8-hydroxy-2'-deoxyguanosine (8OHdG) in untreated urine samples by capillary electrophoresis with optical detection was developed. Optimisation of conditions resulted in a significant lowering of the limit of detection (LOD) by a factor of 400 as compared to our previous study. Optimum separation of 8OHdG from other urine components was achieved using the separation electrolyte containing 80 mM 2-(cyclohexylamino)ethanesulfonic acid, 9 mM LiOH (pH 8.6), and 0.1 mM cetyltrimethylammonium bromide ensuring the electro-osmotic flow inversion. In the model aqueous samples, these conditions allow separating 8OHdG and 2'-deoxyguanosine (dG) from other nucleosides/nucleotides including 2'-deoxycitidine 5'-monophosphate (dCMP), thymidine 5'-monophosphate (TMP), adenosine (A), and thymidine (T). On the other hand, 2'-deoxyadenosine 5'-monophosphate (dAMP) and 2'-deoxyguanosine 5'-monophosphate (dGMP) migrate together, and guanosine (G), 2'-deoxyadenosine (dA), 2'-deoxycytidine (dC) are transported as neutral species with the electro-osmotic flow. In the spiked urine samples, 8OHdG and dG are well separated from each other and from other urine components and exhibit a linear calibration over the concentration range of 0.1-2.0 microM for 8OHdG (LOD = 42 nM) and 0.2-5.0 microM for dG (LOD = 86 nM), but urine metabolites interfere with the determination of dCMP, TMP, A and T. Method is applicable to untreated urine samples with slightly enhanced levels of 8OHdG compared to that found in healthy individuals.     

Specificity and kinetics of interstrand and intrastrand bifunctional alkylation by nitrogen mustards at a G-G-C sequence.

Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are potential sites for various bifunctional alkylations involving guanine N-7. To identify the DNA lesion(s) which may be responsible for these mutations, an end-labeled DNA duplex containing a frequent site of melphalan-induced mutation in the aprt gene was treated with melphalan, mechlorethamine or phosphoramide mustard. The sequence specificity and kinetics of formation of both interstrand and intrastrand crosslinks were determined. All mustards selectively formed two base-staggered interstrand crosslinks between the 5'G and the G opposite C in the 5'G-G-C sequence. Secondary alkylation was much slower for melphalan than for the other mustards and the resulting crosslink was more stable. Mechlorethamine and phosphoramide mustard induced intrastrand crosslinks between the two contiguous Gs in the G-G-C sequence in double-stranded DNA, but melphalan did not. Molecular dynamic simulations provided a structural explanation for this difference, in that the monofunctionally bound intermediates of mechlorethamine and phosphoramide mustard assumed thermodynamically stable conformations with the second arm in a position appropriate for intrastrand crosslink formation, while the corresponding melphalan monoadduct did not.     

DNA adduct formation, metabolism, and morphological transforming activity of aceanthrylene in C3H10T1/2CL8 cells

Aceanthrylene (ACE), a cyclopenta-fused polycyclic aromatic hydrocarbon (CP-PAH) related to anthracene, has been studied for its ability to be metabolized, to form DNA adducts, and to morphologically transform C3H10T1/2CL8 mouse embryo fibroblasts in culture. Although ACE has been previously shown to be a strong mutagen in Salmonella typhimurium strains TA89 and TA100, it did not transform C3H10T1/2 cells (0.4-16 micrograms/ml) under 2 treatment protocols: treatment (for 24 h) 1 day after seeding the cells; treatment (for 24 h) 5 days after seeding the cells. Both protocols are effective in detecting the morphological transforming activity of PAH and CP-PAH and the latter protocol has been shown to be effective in detecting chemicals which are active in the first protocol only with the additional treatment of the cells with a tumor promoter. ACE is metabolized by C3H10T1/2 cells to ACE-1,2-dihydrodiol (the cyclopenta-ring dihydrodiol) at a rate of 450 pmoles ACE-1,2-dihydrodiol formed/h/10(6) cells. ACE-7,8-dihydrodiol and ACE-9,10-dihydrodiol, identified as major Aroclor-1254-induced rat liver microsomal metabolites from their UV, NMR, and mass spectral data, were not identified in incubations of C3H10T1/2 cells with ACE. ACE-DNA adducts in C3H10T1/2 cells were isolated, separated, identified, and quantitated using the 32P-postlabeling method. ACE forms 4 major adducts and each was identified as an ACE-1,2-oxide/2'-deoxyguanosine adduct. The level of adduction was 2.18 pmoles ACE adducts/mg DNA after a 24-h incubation of ACE (16 micrograms/ml) with C3H10T1/2 cells. ACE-DNA adduct persistence and repair were evaluated in C3H10T1/2 cells using a hydroxyurea block after ACE treatment. ACE-DNA adducts were not repaired under the conditions used in the morphological transformation studies. Thus, ACE provides an interesting example of a mutagenic PAH which is metabolized by C3H10T1/2 cells to active intermediates, forms relatively stable and persistent 2'-deoxyguanosine adducts in C3H10T1/2 cells, and yet induces no detectable morphological transforming activity under the experimental conditions used.     

Reassignment of the guanine-binding mode of reduced mitomycin C

Mitomycin C (1) is a clinically used antitumor antibiotic that binds covalently to deoxyribonucleic acid under reductive or acidic catalysis. We have determined the structures of the adducts resulting from attack of reductively activated 1 on the dinucleoside phosphate d(GpC) to be N2-(2' beta, 7'-diaminomitosen-1'alpha-yl)-2'-deoxyguanosine (2) and its 1' beta-isomer (3). This represents a revision of the previously reported structures for these adducts in that the mitomycin residue is linked to the N2- rather than O6-position of 2'-deoxyguanosine. This revision is the result of applying to the mitomycin case a newly developed general method that leads to unambiguous assignment of the linkage position in complex alkylated guanosines. The method as described here takes advantage of the resolution enhancement gained by calculation of the second derivatives of absorbance Fourier transform infrared spectra. In addition, we present 1H NMR data that corroborate the assigned structures of 2 and 3 and that should serve as a useful reference for future investigations into the binding of mitomycin C to DNA. The convenient synthesis of adducts 2 and 3 from deoxyguanosine and mitomycin C reported here should facilitate such investigations as well. Furthermore, we demonstrate a useful acetylation procedure for adducts and metabolites of mitomycin C that furnishes spectroscopically superior chemical derivatives (e.g., triacetates 4 and 5, derived from acetylation of adducts 2 and 3).     

Synthesis and stereochemical preference of peptide 4-aminocyclophosphamide conjugates as potential prodrugs of phosphoramide mustard for activation by prostate-specific antigen (PSA)

In an effort to develop proteolytically activated prodrugs of phosphoramide mustard by prostate-specific antigen (PSA), a series of tetrapeptide (Cbz-Ser-Ser-Phe-Tyr)-conjugated 4-aminocyclophosphamide (4-NH₂-CPA) isomers were synthesized and evaluated as substrates of PSA. The cleavage of the conjugates by PSA were found to be stereoselective as only the two isomers with 4R-configuration were efficiently cleaved by PSA. The cis-(2R,4R)-isomer was the best substrate of PSA with a half-life of 12 min. LC/MS analysis of the incubation solution of this isomer with PSA suggests that 4-NH₂-CPA is released upon proteolysis and quickly degrades to cytotoxic phosphoramide mustard. These results clarified the stereochemical requirements of PSA on the peptide conjugates of 4-NH₂-CPA and demonstrated the potential of these conjugates as potential PSA-activated prodrugs targeting prostate cancer.     

Comparison of the mutagenic activity of the benzene metabolites, hydroquinone and para-benzoquinone in the supF forward mutation assay: a role for minor DNA adducts formed from hydroquinone in benzene mutagenicity

Benzene, a ubiquitous environmental pollutant and occupational hazardous chemical, is a recognised human leukaemogen and rodent carcinogen. The mechanism by which benzene exerts its carcinogenic effects is to date unknown but it is considered that mutations induced by benzene-DNA adducts may play a role. The benzene metabolite, para-benzoquinone (p-BQ) following reaction in vitro with DNA, forms four major adducts, which include two adducts on 2'-deoxyguanosine 3'-monophosphate (dGp). Reaction of DNA with the benzene metabolite hydroquinone (HQ) results in only one major DNA adduct, which corresponds to one of the dGp adducts formed following reaction with p-BQ. The mutagenicity of the adducts formed from these two benzene metabolites was investigated using the supF forward mutation assay. Metabolite-treated plasmid (pSP189) containing the supF gene was replicated in human Ad293 cells before being screened in indicator bacteria. Treatment with 5-20 mM p-BQ gave a 12 to 40-fold increase in mutation rate compared to 5-20 mM HQ treatment, a result reflected in the level of DNA modification observed (8 to 26-fold increase compared to HQ treatment). Treatment with p-BQ gave equal numbers of GC --> TA transversions and GC --> AT transitions, whereas treatment with HQ gave predominantly GC-->AT transitions. The spectra of mutations achieved for the two individual treatments were shown to be significantly different (P = 0.004). A combination of both treatments also resulted in a high level of GC --> AT transitions and a synergistic increase in the number of multiple mutations, which again predominated as GC --> AT transitions. Sites of mutational hotspots were observed for both individual treatments and one mutational hotspot was observed in the multiple mutations for the combined treatment. These results suggest that the dGp adducts formed from benzene metabolite treatment may play an important role in the mutagenicity and myelotoxicity of benzene.