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At least 19 recordsLinked to original sources
PubMed1993

Protonation of phosphoramide mustard and other phosphoramides.

The chemistry of the bifunctional alkylating agent phosphoramide mustard and model phosphoramides was probed by multinuclear NMR spectroscopy as a function of pH. Between pH 1 and 11, both the 31P and 15N resonances for phosphoramide mustard displayed a single monobasic titration curve with a pKa of 4.9. The protonation below pH 4.9 correlates with the loss in reactivity of the mustard. The 17O NMR spectrum of 17O-enriched phosphoramide mustard shows little change with pH. The data on the mustard was compared to 15N and 31P NMR data on 15N-enriched phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide. Contrary to the conclusions of previous studies, our combined 31P, 15N, and 17O NMR results are more consistent with N-protonation of phosphoramide mustard rather than an O-protonation. Theoretical calculations on the phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide show O-protonation to be more stable in the gas phase. For the latter two compounds, the calculations suggest that N-protonation may be the most stable protonated form in the aqueous phase. These findings influence our understanding of the structure-activity relationships of phosphoramide mustards.

Amides↗
PubMed1994

Role of apoptosis in mediating phosphoramide mustard-induced rat embryo malformations in vitro.

Phosphoramide mustard, an active metabolite of the anticancer drug cyclophosphamide, causes malformations in rat embryos undergoing organogenesis in vitro. The purpose of the present study was to investigate the hypothesis that apoptosis plays an important role in mediating the teratogenicity of phosphoramide mustard. Apoptosis is a process of active or programmed cell death which is characterized by internucleosomal DNA fragmentation and de novo RNA and protein synthesis. Sulphated glycoprotein-2 (SGP-2) or clusterin is induced in some models of apoptosis and is one of the proteins likely to be involved in the maintenance of cell integrity. In the present study, day 10 rat embryos were cultured for 6, 12, 24, and 45 hr, with or without the addition of 10 microM phosphoramide mustard. After culture for 24 or 45 hr with exposure to 10 microM phosphoramide mustard, the embryos were both growth-retarded and malformed. Exposure to phosphoramide mustard for 6 or 12 hr did not significantly alter the relative amounts of either the mRNA or protein for SGP-2; this treatment also had no effect on DNA fragmentation in embryos or their yolk sacs. After 24 hr in culture, the relative amounts of SGP-2 protein, but not mRNA, were increased 2-fold in the yolk sacs of the phosphoramide mustard-exposed embryos, but not in the embryos themselves. At this time, DNA fragmentation was detected in phosphoramide mustard-exposed embryos, but not in their yolk sacs or in control embryos. After 45 hr in culture, SGP-2 protein and mRNA levels were increased 2-4-fold above the controls in the phosphoramide mustard-exposed embryos and their yolk sacs. Immunohistochemical analysis revealed that in control embryos cultured for 45 hr, the SGP-2 reaction product was localized in the heart, hindgut, and yolk sac. In contrast, in phosphoramide mustard-treated embryos cultured for 45 hr, SGP-2 immunostaining was found throughout the embryo, with a strong immunoreaction in the mesenchyme and ectoplacental cone. DNA fragmentation in the embryos exposed to phosphoramide mustard for 45 hr was more extensive than that found after 24 hr, but fragmentation was still not detected in the yolk sac. Thus exposure in vitro to a teratogenic concentration of phosphoramide mustard resulted in DNA fragmentation and an increased expression of SGP-2 in the embryo. These data suggest that apoptosis is involved in mediating the teratogenicity of phosphoramide mustard.

Abnormalities, Drug-Induced↗
PubMed2007

Phosphoramidate pronucleotides: a comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins.

To facilitate the delivery of nucleotide-based therapeutics to cells and tissues, a variety of pronucleotide approaches have been developed. Our laboratory and others have demonstrated that nucleoside phosphoramidates can be activated intracellularly to the corresponding 5'-monophosphate nucleotide and that histidine triad nucleotide binding proteins (Hints) are potentially responsible for their bioactivation. Hints are conserved and ubiquitous enzymes that hydrolyze phosphoramidate bonds between nucleoside 5'-monophosphate and an amine leaving group. On the basis of the ability of nucleosides to quench the fluorescence of covalently linked amines containing indole, a sensitive, continuous fluorescence-based assay was developed. A series of substrates linking the naturally fluorogenic indole derivatives to nucleoside 5'-monophosphates were synthesized, and their steady state kinetic parameters of hydrolysis by human Hint1 and Escherichia coli hinT were evaluated. To characterize the elemental and stereochemical effect on the reaction, two P-diastereoisomers of adenosine or guanosine phosphoramidothioates were synthesized and studied to reveal a 15-200-fold decrease in the specificity constant (kcat/Km) when the phosphoryl oxygen is replaced with sulfur. While a stereochemical preference was not observed for E. coli hinT, hHint1 exhibited a 300-fold preference for d-tryptophan phosphoramidates over l-isomers. The most efficient substrates evaluated to date are those that contain the less sterically hindering amine leaving group, tryptamine, with kcat and Km values comparable to those found for adenosine kinase. The apparent second-order rate constants (kcat/Km) for adenosine tryptamine phosphoramidate monoester were found to be 107 M-1 s-1 for hHint1 and 106 M-1 s-1 for E. coli hinT. Both the human and E. coli enzymes preferred purine over pyrimidine analogues. Consistent with observed hydrogen bonding between the 2'-OH group of adenosine monophosphate and the active site residue, Asp43, the second-order rate constant (kcat/Km) for thymidine tryptamine phosphoramidate was found to be 3-4 orders of magnitude smaller than that for uridine tryptamine phosphoramidate for hHint1 and 2 orders of magnitude smaller than that for E. coli hinT. Ara-A tryptamine phosphoramidate was, however, shown to be a good substrate with a specificity constant (kcat/Km) only 10-fold lower than the value for adenosine tryptamine phosphoramidate. Consequently, nucleoside phosphoramidates containing unhindered primary amines and either an alpha or beta 2'-OH group should be easily bioactivated by Hints with efficiencies rivaling those for the 5'-monophosphorylation of nucleosides by nucleoside kinases. The differential substrate specificity observed for human and E. coli enzymes represents a potential therapeutic rationale for the development of selective antibiotic phosphoramidate pronucleotides.

Amides↗
PubMed1985

Kinetics of phosphoramide mustard hydrolysis in aqueous solution.

Hydrolysis of phosphoramide mustard was investigated using HPLC, 31P NMR, and GC-MS with specific deuterium labels. The hydrolysis of phosphoramide mustard in sodium phosphate buffers was found to follow apparent first-order kinetics. The rate of hydrolysis was temperature and pH dependent, being slower under acidic conditions. The hydrolysis was not catalyzed by hydroxyl ion, and its pH dependence appeared to be the result of a change in the mechanism of hydrolysis at different pH values. At a pH value approximately above the pKa of the phosphoramide mustard nitrogen, the major hydrolytic pathway of phosphoramide mustard was via the formation of the aziridinium ion, followed by nucleophilic attack. At pH values below its pKa, cleavage of the P-N bond predominated. At pH 7.4, the formation of an aziridinium ion was followed by a rapid hydrolysis to yield the monohydroxy and, subsequently, the dihydroxy products. The hydrolysis at this pH was adequately described by consecutive first-order kinetics. Seven species in the hydrolytic mixture have been identified as intact phosphoramide mustard, N-(2-chloroethyl)-N-(2-hydroxyethyl)phosphorodiamidic acid, N,N-bis-(2-hydroxyethyl)phosphorodiamidic acid, phosphoramidic acid, phosphoric acid, N,N-bis-(2-chloroethyl)amine, and N-(2-chloroethyl)-N-(2-hydroxyethyl)amine by GC-MS with the aid of deuterium labels. Phosphoramide mustard was found to be stabilized by chloride ion. The stabilization was linearly related to the chloride ion concentration, and the mechanism was found to be via the formation of phosphoramide mustard from the aziridinium and chloride ions. Phosphoramide mustard was significantly more stable in human plasma and in 5% human serum albumin as compared to aqueous buffers, an observation that may be important in vivo.

Bromides↗
PubMed1998

RNA mimetics: oligoribonucleotide N3'-->P5' phosphoramidates.

The synthesis and properties of novel RNA mimetics, oligoribonucleotide N3'-->P5' phosphoramidates, are described. These oligonucleotides contain 3'-aminoribonucleosides connected via N3'-->P5' phosphoramidate linkages, replacing the native RNA O3'-->P5' phosphodiester counterparts. The key monomers 2'-t-butyldimethylsilyl-3'-(monomethoxytrityl)-amino-5'-phospho ramidi tes were synthesized and used to prepare the oligonucleotide phosphoramidates using a solid phase methodology based on the phosphoramidite transfer reaction. Oligoribophosphoramidates are very resistant to enzymatic hydrolysis by snake venom phosphodiesterase. These compounds form stable duplexes with complementary natural phosphodiester DNA and RNA strands, as well as with 2'-deoxy N3'-->P5' phosphoramidates. The increase in melting temperature, Delta T m, was 5-14 degrees C relative to the 2'-deoxy phosphoramidates for decanucleotides. Also, the thermal stability of the ribophosphoramidatehomoduplex was noticeably higher (Delta T m +9.5 degrees C) than that for the isosequential 2'-deoxy phosphoramidate complex. Furthermore, the oligopyrimidine ribo N3'-->P5' phosphoramidate formed an extremely stable triplex with an oligopurine/oligopyrimidine DNA duplex with Delta T m +14.3 degrees C relative to the 2'-deoxy N3'-->P5' phosphoramidate counterpart. The properties of the oligoribonucleotide N3'-->P5' phosphoramidates indicate that these compounds can be used as hydrolytically stable structural and functional RNA mimetics.

Base Sequence↗
PubMed1998

alpha-Oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and duplex formation.

The synthesis and hybridization properties of novel nucleic acid analogs, alpha-anomeric oligodeoxyribonucleotide N3'-->P5' phosphoramidates, are described. The alpha-3'-aminonucleoside building blocks used for oligonucleotide synthesis were synthesized from 3'-azido-3'-deoxythymidine or 3'-azido-2',3'-dideoxyuridine via acid catalyzed anomerization or transglycosylation reactions. The base-protected alpha-5'-O-DMT-3'-aminonucleosides were assembled into dimers and oligonucleotides on a solid support using the oxidative phosphorylation method.1H NMR analysis of the alpha-N3'-->P5' phosphoramidate dimer structures indicates significant differences in the sugar puckering of these compounds relative to the beta-N3'-->P5' phosphoramidates and to the alpha-phosphodiester counterparts. Additionally, the ability of the alpha-oligonucleotide N3'-->P5' phosphoramidates to form duplexes was studied using thermal denaturation experiments. Thus the N3'-->P5' phosphoramidate decamer containing only alpha-thymidine residues did not bind to poly(A) and exhibited lower duplex thermal stability with poly(dA) than that for the corresponding beta-anomeric phosphoramidate counterpart. A mixed base decamer alpha-CTTCTTCCTT formed duplexes with the RNA and DNA complementary strands only in a parallel orientation. Melting temperatures of these complexes were significantly lower, by 34-47 or 15-25 degrees C, than for the duplexes formed by the isosequential beta-phosphoramidates in antiparallel and parallel orientations respectively. In contrast, the alpha-decaadenylic N3'-->P5' phosphoramidate formed duplexes with both RNA and DNA complementary strands with a stability similar to that of the corresponding beta-anomeric phosphoramidate. Moreover, the self-complementary oligonucleotide alpha-ATATATATAT did not form an alpha:alpha homoduplex. These results demonstrate the effects of 3'-aminonucleoside anomeric configuration on sugar puckering and consequently on stability of the duplexes.

Base Sequence↗
PubMed1989

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).

Acrolein↗
PubMed2002

Pharmacokinetics of amino acid phosphoramidate monoesters of zidovudine in rats.

In vitro studies have demonstrated that water-soluble, nontoxic phosphoramidates of azidothymidine (zidovudine [AZT]) have significant and specific anti-human immunodeficiency virus and anticancer activity. Although polar, these compounds are internalized and processed to the corresponding nucleoside monophosphates. Eight methyl amide and methyl ester phosphoramidate monoesters composed of D- or L-phenylalanine or tryptophan and AZT were synthesized. The plasma stability and protein binding studies were carried out in vitro. Then in vivo pharmacokinetic evaluations of six of the compounds were conducted. Sprague-Dawley rats received each compound by intravenous bolus dose, and serial blood and urine samples were collected. AZT and phosphoramidate concentrations in plasma and urine were quantitated by high-performance liquid chromatography with UV or fluorescence detection. Pharmacokinetic parameters were calculated by standard noncompartmental means. The plasma half-lives of the phosphoramidates were 10- to 20-fold longer than the half-life of AZT. Although the renal clearances of the phosphoramidates were similar to AZT, their total body clearances were significantly greater than that of AZT. The 3- to 15-fold-larger volume of distribution (Vss) for the phosphoramidates relative to AZT appeared to be dependent on the stereochemistry of the amino acid, with the largest values being associated with the L-amino acids. The increased Vss indicates a much greater tissue distribution of the phosphoramidate prodrugs than of AZT. Amino acid phosphoramidate monoesters of AZT have improved pharmacokinetic properties over AZT and significant potential as in vivo pronucleotides.

Amides↗
PubMed2004

Binding and conformational analysis of phosphoramidate-restriction enzyme interactions.

Phosphoramidates are modified deoxyoligonucleotides that feature nitrogen in place of the 3'-oxygen of a phosphodiester linkage. Noted for stability against nuclease activity, these linkages are of both mechanistic and therapeutic interest. While a number of studies characterizing the properties of oligonucleotides composed entirely of phosphoramidate linkages have been published, little is known about how singly substituted phosphoramidate substitutions affect the thermodynamics and structure of protein-oligonucleotide interactions. We chose to investigate these interactions with PvuII endonuclease, the DNA binding behavior of which is well-characterized. Oligonucleotide duplexes containing a phosphoramidate substitution at the scissile phosphates were resistant to cleavage by the enzyme, even after extended incubations. However, the enzyme was able to cleave the native strand in a native:phosphoramidate heteroduplex at a rate comparable to that observed with the native substrate. Ca(II)-stimulated PvuII binding for a phosphoramidate-substituted oligonucleotide is comparable to that of the native duplex (K(d) approximately 200 pM). K(d) values obtained in the presence of Mg(II) are somewhat weaker (K(d) approximately 10 nM). Under metal-free conditions, the enzyme exhibited a remarkable approximately 50-fold greater affinity for the modified oligonucleotide relative to the native substrate (5 vs 240 nM). While (31)P NMR spectra indicate increased chemical shift dispersion in the free phosphoramidate duplex, the spectrum of the enzyme-bound duplex is similar to that of the native duplex. (1)H-(15)N HSQC analysis indicates that enzyme conformations in the presence of these oligonucleotides are also comparable. The tight binding of the phosphoramidate duplex under metal-free conditions and its resistance to cleavage are attributed to local conformational adjustments propagating from the O-->N substitution.

Amides↗
PubMed1996

Synthesis and biological activity of aromatic amino acid phosphoramidates of 5-fluoro-2'-deoxyuridine and 1-beta-arabinofuranosylcytosine: evidence of phosphoramidase activity.

The amino acid phosphoramidate diesters of FUdR (2) and Ara-C (6), 5-fluoro-2'-deoxy-5'-uridyl N-(1-carbomethoxy-2-phenylethyl)phosphoramidate (5a), 5-fluoro-2'-deoxy-5'- uridyl N-(1-carbomethoxy-2-indolylethyl)phosphoramidate (5b), 1-beta-arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-phenylethyl) phosphoramidate (8a), and 1-beta-arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-indolylethyl)phosphoramidate (8b), were synthesized and tested for their antitumor activity against L1210 mouse lymphocytic leukemia cells and CCRF-CEM human T-cell lymphoblastic leukemia cells. Ara-C phosphoramidates 8a,b were found to be inactive at a concentration of 100 microM, while the FUdR conjugates 5a,b exhibited IC50 values within a range of 0.30-0.40 microM. Stability studies revealed that > 99% of the phosphoramidates remained intact after incubation for > 2 days in 20% calf or 20% human serum. Intracellular thymidylate synthase (TS) inhibition studies revealed that treatment of L1210 and CCRF-CEM cells with 5a or 5b resulted in significant inhibition of TS in intact and permeabilized cells, while treatment of L929 TK- cells with these compounds did not result in inhibition of TS activity in intact cells. However, permeabilization of L929 TK- cells enhanced the activity of 5a,b toward intracellular TS by 900- and 1500-fold, respectively. In addition, incubation of cell-free extracts of CEM cells with radiolabeled 5b resulted in the rapid production of FUdR 5'-monophosphate and a lag in the generation of FUdR. Consequently, it is proposed that the metabolism of the phosphoramidate diesters of FUdR in proliferating tissue proceeds through two separate enzymatic steps involving P-N bond cleavage by an unknown phosphoramidase followed by P-O bond cleavage by phosphatases such as 5'-nucleotidase.

Animals↗
PubMed1982

Analysis and excision of ring-opened phosphoramide mustard-deoxyguanine adducts in DNA.

The reaction products formed by reacting deoxyguanosine with phosphoramide mustard at pH 7.4 have been analyzed by high-performance liquid chromatography and Schiff's reaction. The adducts consisted of five fractions of phosphoramide mustard-imidazole ring-opened deoxyguanosine complexes and one fraction of each of intact phosphoramide mustard-deoxyguanosine and phosphoramide mustard-dideoxyguanosine complexes. Thus, contrary to views held previously, the imidazole ring of alkylated guanine can undergo fission at physiological pH. Schiff's reaction suggests that some fractions of phosphoramide mustard-ring-opened deoxyguanosine adducts contain formyl groups, while others do not. When DNA containing phosphoramide mustard-ring-opened guanine adducts was treated with formamidopyrimidine-DNA glycosylase, there was enzymatic removal of formylated ring-opened guanine adducts. The quantification of the full amount of ring-opened guanine released by formamidopyrimidine-DNA glycosylase was precluded by the limitations of our assay system, which requires that any two ring-opened guanines cross-linked by phosphoramide mustard be both excised in order to be detected.

DNA, Bacterial↗
PubMed2002

Oligonucleotide N3'-->P5' phosphoramidates as efficient telomerase inhibitors.

Human telomerase is a unique reverse transcriptase that is expressed in multiple cancers, but not in the vast majority of normal cells. The enzyme is responsible for telomere protection and maintenance, and supports the proliferative immortality of cancer cells. Thus, it has been proposed that the specific inhibition of telomerase activity in tumors might have significant and beneficial therapeutic effects. To this goal we have designed, synthesized, and evaluated several oligonucleotide N3'-->P5' phosphoramidates as telomerase inhibitors. These oligonucleotides are complementary to the template region of the RNA domain of telomerase (hTR). The prepared compounds were evaluated in HME50-5E breast epithelial cells, where their effects on telomerase activity were determined using a cell-based telomerase (TRAP) assay at 24 as well as 72 h after exposure to compounds. The oligo-N3'-->P5' phosphoramidate inhibited telomerase activity in cells in the presence of the cellular up-take enhancer (FuGENE6) in a dose- and sequence-dependent manner, with IC(50) values of approximately 1 nM. Inhibition of telomerase activity by this compound without the lipid carrier was not efficient. However, the isosequential oligonucleotide N3'-->P5' thio-phosphoramidate was able to inhibit telomerase activity with or without lipid carriers at nM, or low-microM concentrations, respectively. This inhibition of telomerase activity in HME50-5E cells by the oligonucleotide thio-phosphoramidates was also sequence specific. Long-term treatment of the cells with 0.5 microM of FuGENE6 formulated 13-mer thio-phosphoramidates, fully complementary to hTR, resulted in gradual telomere shortening, followed by cellular senescence and apoptosis, as would be predicted for a telomerase inhibitor. The mismatched control compound had no effect on cell proliferation. The results suggest that the oligonucleotide N3'-->P5' phosphoramidates, and particularly thio-phosphoramidates, might be further developed as selective anti-telomerase reagents.

Amides↗