Large-scale performance and design for construction activity erosion control best management practices.

The National Pollutant Discharge Elimination System (NPDES) Phase II requires construction activities to have erosion and sediment control best management practices (BMPs) designed and installed for site storm water management. Although BMPs are specified on storm water pollution prevention plans (SWPPPs) as part of the construction general permit (GP), there is little evidence in the research literature as to how BMPs perform or should be designed. The objectives of this study were to: (i) comparatively evaluate the performance of common construction activity erosion control BMPs under a standardized test method, (ii) evaluate the performance of compost erosion control blanket thickness, (iii) evaluate the performance of compost erosion control blankets (CECBs) on a variety of slope angles, and (iv) determine Universal Soil Loss Equation (USLE) cover management factors (C factors) for these BMPs to assist site designers and engineers. Twenty-three erosion control BMPs were evaluated using American Society of Testing and Materials (ASTM) D-6459, standard test method for determination of ECB performance in protecting hill slopes from rainfall induced erosion, on 4:1 (H:V), 3:1, and 2:1 slopes. Soil loss reduction for treatments exposed to 5 cm of rainfall on a 2:1 slope ranged from-7 to 99%. For rainfall exposure of 10 cm, treatment soil loss reduction ranged from 8 to 99%. The 2.5 and 5 cm CECBs significantly reduced erosion on slopes up to 2:1, while CECBs < 2.5 cm are not recommended on slopes >or= 4:1 when rainfall totals reach 5 cm. Based on the soil loss results, USLE C factors ranged from 0.01 to 0.9. These performance and design criteria should aid site planners and designers in decision-making processes.

Storm water pollutant removal performance of compost filter socks.

In 2005, the U.S. Environmental Protection Agency (USEPA) National Menu of Best Management Practices (BMPs) listed compost filter socks (FS) as an approved BMP for controlling sediment in storm runoff on construction sites. The objectives of this study were to determine if FS with or without the addition of a flocculation agent to the FS system can significantly remove (i) suspended clay and silt particulates, (ii) ammonium nitrogen (NH(4)-N) and nitrate-nitrite nitrogen (NO(3)-N), (iii) fecal bacteria, (iv) heavy metals, and (v) petroleum hydrocarbons from storm water runoff. Five separate (I-V) 30-min simulated rainfall-runoff events were applied to soil chambers packed with Hartboro silt loam (fine-loamy, mixed, active, nonacid, mesic fluvaquentic Endoaquepts) or a 6-mm concrete veneer on a 10% slope, and all runoff was collected and analyzed for hydraulic flow rate, volume, pollutant concentrations, pollutant loads, and removal efficiencies. In corresponding experiments, runoff was analyzed for (i) size of sediment particles, (ii) NH(4)-N and NO(3)-N, (iii) total coliforms (TC) and Escherichia coli, (iv) Cd, Cr, Cu, Ni, Pb and Zn, and (v) gasoline, diesel, and motor oil, respectively. Results showed that: (i) FS removed 65% and 66% of clay (<0.002 mm) and silt (0.002-0.05 mm), respectively; (ii) FS removed 17%, and 11% of NH(4)-N and NO(3)-N, respectively and when NitroLoxx was added to the FS, removal of NH(4)-N load increased to 27%; (iii) total coliform and E. coli removal efficiencies were 74 and 75%, respectively, however, when BactoLoxx was added, removal efficiency increased to 87 and 99% for TC and 89 and 99% for E. coli, respectively; (iv) FS removal efficiency for Cd, Cr, Cu, Ni, Pb, and Zn ranged from 37 to 72%, and, when MetalLoxx was added, removal efficiency ranged from 47 to 74%; and (v) FS removal efficiency for the three petroleum hydrocarbons ranged from 43 to 99% and the addition of PetroLoxx increased motor oil and gasoline removal efficiency in the FS system.

Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Evaluation of the in vivo antitumor activity and in vitro cytotoxic properties of auranofin, a coordinated gold compound, in murine tumor models.

The coordinated gold compound, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S-triethyl phosphine gold (auranofin; Ridaura), was evaluated for antitumor activity in a variety of mouse tumor models. Of the 15 tumor models evaluated, auranofin was found to be active only against i.p. P388 leukemia. A number of dose schedules was used to measure activity against P388 with optimal activity observed at 12 mg/kg given daily, i.p., on Days 1 to 5. Auranofin was active against i.p. P388 leukemia only when administered i.p.; the drug was completely inactive when administered i.v., s.c., or p.o. on Days 1 to 5. Evaluation of the effects of auranofin in vitro demonstrated that survival curves for B16 melanoma cells as measured by the clongenic and dye exclusion assays were exponential and monophasic; cell cycle distribution was not altered, and auranofin displayed no preferential cytotoxicity to logarithmic or plateau growth phase cell populations; auranofin inhibited DNA, RNA, and protein synthesis at cytotoxic concentrations but showed no selective effect; the cytotoxic activity and cellular association of gold from auranofin were dose, time, and temperature dependent; and binding of auranofin gold to serum proteins markedly decreased cellular uptake of gold and cytotoxicity of auranofin in vitro.

A murine model to evaluate the ability of in vitro clonogenic assays to predict the response to tumors in vivo.

The use of the human tumor cloning assay as a predictor of clinical response of human tumors to drugs is predicated on the hypothesis that the in vivo response of a tumor to a drug can be correlated with the in vitro response of cells derived from the tumor. To test this hypothesis, we utilized a murine tumor model in which the in vivo and in vitro responses of a tumor can be accurately and reproducibly compared. Drug activity was assessed in P388 leukemia with the standard in vivo antitumor assay (i.p. tumor/i.p. drug administration) and an in vitro assay wherein the ascites tumor cells are removed from mice, treated with a drug, and directly cloned in soft agar to measure clonogenic capacity. The response of P388 cells to analogues within four separate classes of antitumor agents, anthracyclines, anthraquinones, platinum(II) coordination complexes, and phosphinogold(I) complexes was evaluated. The clonogenic assay failed to discriminate between highly active in vivo antitumor agents and analogues with only marginal in vivo efficacy (i.e., doxorubicin and daunorubicin versus rhodomycins A and B, ametantrone versus NSC 276740, cisplatin versus transplatin, [Au(dppe)2]Cl versus [Au(depe)2]PF6. Furthermore, the in vitro clonogenic assay failed to detect carboplatin which was a highly active agent in vivo. The basis for these discrepancies was explored by a more detailed comparison of doxorubicin and rhodomycin B. In vivo or in vitro drug exposure with subsequent measurement of cell kill by the in vitro clonogenic and in vivo tumorigenic assay demonstrated that the in vitro assay overestimated the cytotoxic potency of the drugs relative to the tumorigenic assay. Treatment of tumors in vivo with doxorubicin at doses below the maximally tolerated dose in mice resulted in multiple log cell kill as measured in vitro or in vivo, whereas rhodomycin B was cytotoxic only at dose levels exceeding its maximally tolerated dose. The results indicate that a subset of tumor stem cells capable of forming colonies in soft agar are significantly more sensitive to the cytotoxic effects of anthracyclines than are in vivo tumorigenic stem cells. Cytotoxic potency as measured by an in vitro soft agar clonogenic assay is not an accurate predictor of in vivo antitumor efficacy even in a model in which ascites tumor cells are directly exposed to i.p. drug. The in vitro cytotoxicity assay is useful only as a nonselective prescreen and must be used in combination with other indicators of tumor cell selectivity and dose-limiting organ toxicity.

Tumorigenicity of the cyc- variant of the S49 murine lymphoma deficient in the Gs-alpha subunit of adenylate cyclase.

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.

Modulation of the antitumor and biochemical properties of bis(diphenylphosphine)ethane with metals.

Bis(diphenylphosphine)ethane (DPPE) and its bis[chlorogold(I)] [DPPE(Au2Cl2)], and bis[trichlorogold(III)] [DPPE(Au2Cl6)], complexes have in vivo antitumor activity. To determine if interaction with metals in situ can play a role in the antitumor activity of DPPE, we have studied the effects of DPPE, DPPE(Au2Cl2), DPPE(Au2Cl6) and mixtures of DPPE with metal salts on in vitro and in vivo biological systems. The in vitro cytotoxic potencies of the two DPPE-gold complexes were approximately 10-fold greater than that of DPPE. In addition, the cytotoxic potency of DPPE was increased when incubated with cells in the presence of Au(III) and Cu(II) salts, whereas Mg(II), Zn(II), Mn(II), Fe(II), Co(II), and Cd(II) had no effect. The effects of DPPE, DPPE(Au2Cl2) and mixtures of DPPE and metal salts on the activity of a model enzyme system, DNA polymerase alpha were measured. While DPPE did not inhibit the activity of DNA polymerase alpha, the DPPE(Au2Cl2) complex and mixtures of DPPE and Cu(II) salts inhibited the activity of the enzyme. Consistent with the effects observed in vitro, coadministration of Cu(II) or Au(III) increased the in vivo potency of DPPE in mice bearing i.p. P388 leukemia. Fifteen other DPPE analogues were evaluated for in vivo antitumor activity and for the effect of Cu(II) on their in vitro cytotoxic potency; there was a relationship between the ability of Cu(II) to potentiate the cytotoxic activities of DPPE analogues and their having in vivo antitumor activity.

In vivo antitumor activity and in vitro cytotoxic properties of bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride.

We have previously reported the cytotoxicity and antitumor activity of bis(diphenylphosphino)ethane (DPPE) and a variety of its transition metal complexes. During studies of the chemistry of a gold complex of this group [(AuCl)2(DPPE)], it was observed that this complex readily underwent ring closure on reaction with DPPE to form the tetrahedral complex [Au(DPPE)2]+. Various counterion forms (e.g., Cl-) of this cation were isolated and were found to exhibit a remarkably high stability in solution. Evaluation of [Au(DPPE)2]Cl in mice bearing i.p. P388 leukemia demonstrated that the compound produced an average of 87% increase in life span at its maximally tolerated dose (2-3 mumol/kg/day for 5 days). Activity was also seen in i.p. M5076 reticulum cell sarcoma (60% increase in life span) and s.c. mammary adenocarcinoma 16/c. Modest activity was evident in i.p. B16 melanoma and L1210 leukemia. A subline of P388 leukemia resistant to cisplatin was not cross-resistant to [Au(DPPE)2]Cl. In addition, combination therapy of [Au(DPPE)2]Cl and cisplatin against i.p. P388 demonstrated an advantage over single-agent therapy. In vitro studies of [Au(DPPE)2]Cl showed that the compound: is cytotoxic to tumor cell lines; is only minimally inhibited in its cytotoxic activity by the presence of serum; produces DNA protein cross-links and DNA strand breaks in cells; and inhibits macromolecular synthesis with a preferential inhibitory effect on protein synthesis relative to DNA and RNA synthesis. 31P nuclear magnetic resonance spectroscopy indicated that the compound is stable in the presence of serum proteins, thiols, or disulfides and that it reacts with Cu(II) resulting in the formation of a Cu(I)DPPE complex. The results of these in vivo and in vitro experiments suggest that the contrasting pharmacological profile of [Au(DPPE)2]Cl with respect to other gold(I) phosphine complexes may be related to both the kinetic stability of the complex and its stability in the presence of thiols.

Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.

The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth.

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

The tyrosine89 residue of yeast FKBP12 is required for rapamycin binding.

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent RmR rbp1 mutants and screened them for both RBP1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of the in vitro cytotoxic and in vivo antitumor activities of gold(I) coordination complexes.

A series of gold(I) coordination complexes including analogues of the antiarthritic agent auranofin 1 were evaluated for in vitro cytotoxic potency against both B16 melanoma cells and P388 leukemia cells and in vivo antitumor activity against P388 leukemia in mice. A number of the complexes showed potent cytotoxic activity in vitro and antitumor activity in vivo, with the phosphine-coordinated gold(I) thiosugar complexes demonstrating the greatest in vitro and in vivo activity. The data compiled for 63 complexes of the general structural formula LAuX provide the basis for the following observations: potent in vitro cytotoxic activity is observed for substituted (phosphine) gold complexes, lack of potency in vitro correlates well with lack of antitumor activity, potent cytotoxicity in vitro is not necessarily predictive of activity in vivo, in vivo antitumor activity is generally optimized by ligation of Au(I) with a substituted phosphine and a thiosugar.

Synthesis, tubulin binding, antineoplastic evaluation, and structure-activity relationship of oncodazole analogues.

In an attempt to identify a soluble oncodazole analogue that could be easily formulated, a series of substituted oncodazoles was synthesized and evaluated for tubulin binding affinity, in vitro cytotoxicity against cultured mouse B-16 cells, and ability to prolong lifespan at the maximally tolerated dose in the P388 mouse leukemia model. Biological evaluation of all the isomeric methyloncodazoles demonstrated the thiophene 4'-position to be the only site of significant bulk tolerance, although substitution of this position with polar or charged functional groups abolished biological activity. Simple esters of the 4'-carboxymethyloncodazole were shown to have enhanced antitumor activity and tubulin binding affinity relative to oncodazole. Despite a failure of this study to identify a water-soluble oncodazole with antitumor activity, the structure-activity relationship developed led to a derivative with enhanced activity in the P388 leukemia model and facilitated the preparation of a biologically active photolabile analogue.

Antitumor activity of bis(diphenylphosphino)alkanes, their gold(I) coordination complexes, and related compounds.

Bisphosphines related to bis(diphenylphosphino)ethane (dppe) and their gold complexes are described that are active in a spectrum of transplantable tumor models. When administered ip on days 1-5 at its maximally tolerated dose (MTD) of 40 mumol/kg, dppe reproducibly gives 100% increase in life span (ILS) in mice bearing ip P388 leukemia. Coordination of chlorogold(I) to each phosphine in dppe gave a complex that had similar activity but at a much lower dose level than dppe; the MTD for the gold(I) complex was 7 mumol/kg. Among other metal complexes of dppe, the Au(III) complex was active (greater than 50% ILS) whereas Ag(I), Ni(II), Pt(II), Pd(II), and Rh(I) complexes were inactive. Among dppe analogues, replacement of phenyl groups with ethyl or benzyl groups resulted in inactivity for both ligands and the corresponding gold complexes whereas substitution with cyclohexyl or heterocyclic ring systems yielded ligands and/or gold complexes with antitumor activity. Among substituted-phenyl dppe and dppe(AuCl)2 analogues, 3-fluoro, 4-fluoro, perdeuterio, 4-methylthio, and 2-methylthio analogues were active; 4-methyl, 3-methyl, 4-methoxy, 4-dimethylamino, and 4-trifluoromethyl analogues were marginal or inactive. Analogues in which the ethane bridge of dppe or dppe(AuCl)2 was varied between one and six carbons, unsaturated or substituted, revealed that activity was maximal with ethane or cis-ethylene. Compounds with good P388 activity were also active in other animal tumor models.

Cytotoxicity and antitumor activity of some tetrahedral bis(diphosphino)gold(I) chelates.

We report the cytotoxicity toward B16 cells and antitumor activity in three transplantable tumor models of a series of ionic, tetrahedral, bischelated gold diphosphine complexes of the type [Au1(R2PYPR2')2]X, where Y = (CH2)2, (CH2)3, or cis-CH = CH. The anion (X = Cl, Br, I, CH3SO3, NO3, PF6) had little effect upon activity. The R = R' = phenyl complexes 1, 7, and 8 [Y = (CH2)2, (CH2)3, cis-CH = CH, X = Cl] were the most active against P388 leukemia, with an increase in lifespan ranging from 83 to 92% and were also active against M5076 sarcoma and B16 melanoma. Complexes with pyridyl or fluorophenyl substituents had reduced activities. For the latter, 19F and 31P NMR were used to verify the formation of bischelated gold(I) complexes in solution. The reduced activity of the complex with R = Et and R' = Ph and inactivity with R = R' = Et are discussed in terms of their increased reactivity as reducing agents. 31P NMR studies show that [AuI(Et2P(CH2)2PPh2)2]Cl readily reacts with serum, albumin, and Cu2+ ions to give oxidized ligand.

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Antimicrobial and anticancer activity of tetrahedral, chelated, diphosphine silver(I) complexes: comparison with copper and gold.

Tetrahedral, bischelated Ag(I) diphosphine complexes [Ag(P-P)2]NO3, where P-P is Ph2P(CH2)2PPh2 (dppe), Et2P(CH2)2PPh2 (depe), and cis-Ph2P(CH = CH)PPh2 (dppey), are potently cytotoxic to B16 melanoma cells in vitro (IC50 4 microM) and exhibit good activity against ip P388 leukemia in mice. The complex [Ag(dppe)2]NO3 is active against M5076 reticulum cell sarcoma. The antibacterial and antifungal activities of Ag(I) diphosphine and related Cu(I) and Au(I) complexes were assessed. The complexes [Au(dppey)2]Cl, [Au(dppp)2]Cl and (CuCl)2(dppe)3 show modest activity against three of the 12 bacterial strains tested, but all complexes exhibit antifungal activity against three strains of C. albicans in a "defined" medium, [Ag(depe)2]NO3 and [Au(dppp)2]Cl having comparable activity to fungizone. Antifungal activity of the complexes is reduced in Sabouraud's broth medium, and lost altogether for the Ag(I) complexes. Reactions of some of the Ag(I) complexes with glutathione and blood plasma were studied by 31P NMR.

Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin.

The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are both sensitive to camptothecin, an inhibitor of DNA topoisomerase I. An S. cerevisiae DNA repair mutant, rad52, is hypersensitive to the drug. In both species, topoisomerase I mutants totally lacking the enzyme are completely resistant to the drug. A strain with a mutation leading to a temperature-sensitive topoisomerase I exhibits temperature dependence in its in vivo response to camptothecin. A strain carrying a plasmid that overproduces topoisomerase I is hypersensitive to the drug. The rad52 mutant is killed by overproduction of the enzyme, even in the absence of the drug. The response of several of these strains to camptothecin analogs, to DNA topoisomerase II inhibitors, and to other drugs is reported. The cytotoxic effects of camptothecin are discussed in terms of the drug extending the lifetime of a topoisomerase I-DNA covalent intermediate, which is recognized as DNA damage by a DNA repair system.