A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2'-deoxyuridine and their 5'-phosphates

2′-Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2′-deoxyuridine inhibited herpes simplex (type 1 and 2) virus-induced cytopathogenicity by 50 per cent at a dose (id₅₀) of 10–30 μg/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an id₅₀ of 30–70 μg/ml for herpes simplex virus, 20 μg/ml for vaccinia virus, and 280 μg/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2′-deoxyuridine 5′-phosphate inhibited vaccinia replication with an IC₅₀ of 20 μg/ml and L-1210 cell culture growth with an id₅₀ of 80 μg/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase (Lactobacillus casei) with the following $\text{K}{}_{\mathrm{i}}\text{K}{}_{\mathrm{m}}$ ratios: 3-nitrostyryl, 0.035; 4-nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.     

Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Antiherpes activity of [E]-5-(1-propenyl)-2′-deoxyuridine and 5-(1-propenyl)-1-β-d-arabinofuranosyluracil

5-(1-Propenyl)-1-β-d-arabinofuranosyluracil has been synthesized, and this compound and [E]-5-(1-propenyl)-2′-deoxyuridine have been tested for inhibition of herpes virus multiplication. Only [E]-5-(1-propenyl)-2′-deoxyuridine was found to be an active inhibitor reducing by 50% the plaque formation of herpes simplex virus type 1 (HSV-1) at about 1 μM. A comparison to the bromovinyl derivatives showed the following order of decreasing activity; $\text{[}\text{E}\text{]-5-(2-}\text{bromovinyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(2-}\text{bromovinyl}\text{)-1-β-}\text{d}\text{-}\text{arabinofuranosyluracil}\text{≥ [}\text{E}\text{]-5-(1-}\text{propenyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(1-}\text{propenyl}\text{)-1-β-}\text{arabinofuranosyluracil}$. HSV-1 mutants lacking thymidine kinase or resistant against acycloguanosine were resistant against [E]-5-(1-propenyl)-2′-deoxyuridine. All compounds seemed to be phosphorylated by HSV-1 thymidine kinase in a cell-free assay. The compounds were phosphorylated to a lower extent by cellular or HSV-2 thymidine kinase, and the HSV-2 strains tested were inhibited by less than 50% at 100 μM in plaque assays. A selective inhibition of HSV-1 DNA synthesis by [E]-5-(1-propenyl)-2′-deoxyuridine was observed in infected cells indicating an effect on viral DNA polymerase. [E]-5-(1-Propenyl)-2′-deoxyuridine had a low cellular toxicity and a therapeutic effect when applied topically to HSV-1-infected guinea pig skin.     

Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions

The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this enzyme. The substrate analog, 8-aminoguanine (8-AG), has the lowest dissociation constant (K_i = 0.2–1.2 μM) of any compound reported to date and V_max = 16 per cent (relative to guanine). Other C(8) substituents decreased the affinity for PNP and, with the exception of the methyl and sulfhydryl groups, abolished substrate activity. Halogens or a thiomethyl group at C(5') of inosine resulted in unchanged or improved affinities (K_i = 10–30 μM) and greatly decreased substrate activity (V_max < 1 per cent relative to inosine). The K_i of formycin B was reduced from 100 μM to 10 μM or less by substitution of a halogen at C(5'). Phosphorolysis of purine nucleosides was inhibited significantly by 8-AG in intact human erythrocytes and murine Sarcoma 180, L1210 and L5178Y cells. Although a K_i value of 17 μM was determined for 8-aminoguanosine, it was equally effective in inhibiting PNP activity in intact cells. The nucleoside was cleaved to 8-AG which was not a substrate for guanase or hypoxanthine-guanine phosphoribosyltransferase. Despite its low substrate activity (V_max < 0.2%). 5′-deoxy-5′-iodoinosine was cleaved by intact L1210 and L5178Y cells.     

Role of thymidine kinase in the inhibitory activity of 5-substituted-2'-deoxyuridines on the growth of human and murine tumor cell lines

Twenty-four 5-substituted 2'-deoxyuridines have been evaluated for their inhibitory effects on the growth of three human lymphoblast cell lines (Namalva, Raji and TK^? (thymidine kinase deficient) Raji) and these inhibitory effects were compared to those for two murine leukemia cell lines (L1210/0 and L1210/BdUrd). The latter was selected from the parental L1210/0 cell line by its ability to grow at high concentrations of 5-bromo-dUrd and could also be considered as TK^?. There was a close correlation between the inhibitory effects of the deoxyuridine analogs on Namalva, Raji and L1210 cells: the correlation coefficient (r) for log id₅₀ (median inhibitory dose) for L1210 cell growth, on the one hand, and log id₅₀ for Namalva or Raji cell growth, on the other hand, was 0.902 and 0.929, respectively. There was also a strong correlation (r = 0.936) between the log id₅₀ values for the two human lymphoblast cell lines. However, there was no significant correlation (r < 0.40) either between the log id₅₀ for the TK^? Raji cells and the parental TK⁺ Raji cells, or between the log id₅₀ for the TK^? L1210/BdUrd cells and the parental TK⁺ L1210/0 cells. We may conclude therefore, that (i) the murine leukemia L1210 cell system is predictive for the growth-inhibitory effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines, and (ii) the antitumor cell activity of the 5-substituted 2'-deoxyuridines is, to a large extent, dependent on the thymidine kinase activity of the tumor cells.     

Cytotoxicities of the l and d isomers of phenylalanine mustard in L1210 cells

The cytotoxicity of d-phenylalanine mustard (medphalan) to murine L1210 leukemia cells in culture was reduced by both the d and l isomers of leucine. l-Leucine only partially protected L1210 cells from medphalan cytotoxicity at drug concentration's above 10 μM, suggesting that medphalan uptake occured via both an amino acid carrier and an as yet undetermined agency, possibly passive diffusion. At equitoxic concentrations of l-phenylalanine mustard (melphalan) and medphalan, l-leucine reduced medphalan cytotoxicity by only one-sixth that obtained with melphalan. Analysis of melphalan and medphalan inhibition of the initial rate of l-leucine transport indicated a melphalan K_i of 0.085 mM, a value one-seventh that of melphalan (K_i, 0.635 mM).     

The effect of alkylating agents on adenosine 3',5'-monophosphate metabolism in Walker carcinoma.

The effect of some alkylating agents on the activity of the enzymes adenylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase has been studied using Walker carcinoma cells in tissue culture. The monofunctional agent 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), which has previously been shown to elevate the level of adenosine 3′,5′-monophosphate (cyclic AMP) in sensitive Walker cells, has been shown to have no effect on the activity of adenylate cyclase either in the presence or absence of the protecting agent 4-amino-2-phenylimidazole-5-carboxamide (2-phenyl-AlC). Chlorambucil (p-N,N(di-2-chloroethylamino)phenylbutyric acid) (5 μg/ml) while having no effect on either the basal or fluoride-stimulated adenylate cyclase activity caused an inhibition of the high affinity form of the cyclic AMP phosphodiesterase which reached a maximum after 1 hr. This was accompanied by an increase in the intracellular level of cAMP which was proportional to the dose of chlorambucil up to a maximal 2-fold increase at 6.4 μg/ml, a dose which caused complete inhibition of cell growth. Further increases in the concentration of chlorambucil up to 100 μg/ml caused no further increase in cAMP level. Merophan ($\text{dl}\text{-}\text{o}\text{-N,N(}\text{di}\text{-2-}\text{chloroethylamino}\text{)}\text{phenylalanine}$) (0.5 μg/ml) similarly caused an inhibition of the low K_m form of the phosphodiesterase, but the rate of inhibition was slower than that observed with chlorambucil. The molecular forms of the cAMP phosphodiesterase in Walker cells sensitive or resistant to chlorambucil have been resolved using Sepharose 6B gel chromatography. The resistant lines displayed a reduction in the specific activity of the high affinity form of the enzyme which was accompanied by a shift to lower molecular weight forms. This could explain the lack of effect of chlorambucil on cAMP levels in Walker cells with acquired resistance to this agent.     

2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline (tmq), a potent non-classical folate antagonist inhibitor--I effect on dihydrofolate reductase and growth of rodent tumors in vitro and in vivo.

Seventeen non-classical 2,4-diamino-6-[(anilino)methyl]quinazoline antifolates were tested as inhibitors of dihydrofolate reductase from L1210 leukemia cells and from human leukemia cells (acute lymphocytic leukemia). Several potent inhibitors of this enzyme were found, some with I₅₀ values of 10^?9 M, thus displaying activity comparable to that of methotrexate. In general, the potency of dihydrofolate reductase inhibition correlated with the inhibition of cell growth in vitro against L1210 cells. Two of these compounds, compound 14 (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline; TMQ, JB-11, NSC 249008) and compound 3 (2,4-diamino-5-chloro-6-[(3,4-dichloroanilino)methyl]quinazoline; NSC 208652), were further evaluated against murine tumors in vivo and both showed a broad spectrum of antitumor effects. The results of these studies encourage further evaluation of these compounds, in particular compound 14, as possible anti-neoplastic agents in the treatment of human disease.     

Stereospecificity at carbon 6 of fomyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

The unnatural diastereoisomer of l-5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [³H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K_i, were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K_i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

Inhibition of hypoxanthine-guanine phosphoribosyl transferase.

The affinities of eighteen purines or purine analogs for human erythrocytic hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase) were compared to assess the feasibility of obtaining active inhibitors of the enzyme. Three compounds appeared to inhibit the utilization of hypoxanthine by L5178Y cells in vitro due to inhibition of the enzyme rather than depletion of the intracellular 5-phosphoribosyl-1-pyrophosphate pool. The three competitive inhibitors and their affinity constants (K_i) using 6-mercaptopurine as substrate were: 6-mercapto-9-(tetrahydro-2-furyl)-purine, 37 μM; 2,6-bis-(hydroxyamino)-9-β-d-ribofuranosyl-purine, 12 μM; and 6-iodo-9-(tetrahydro-2-furyl)-purine, 108 μM. The KInm for 6-mercaptopurine was 9 μM. Thus, the enzyme tolerates bulky substitution at N⁹. 6-Mercapto-9-(tetrahydro-2-furyl)-purine also potentiated the chemotherapeutic effect of azaserine, an inhibitor of de novo purine biosynthesis, in L5178Y ascites tumor-bearing mice. Four 2-substituted, oxazolo-[5, 4-d]-pyrimidine-7-ones and 2-methylthiazolo-[5, 4-d]-pyrimidine-7-one had K_i values in the range of 84–173 μM. Consequently, isosteric substitution at N⁹ may also be a fruitful and logical course to pursue in the design and synthesis of more potent inhibitors of this important enzyme.     

beta-adrenergic receptors of human leukocytes: studies with intact mononuclear and polymorphonuclear cells and membranes comparing two radioligands in the presence and absence of chloroquine

Owing to the large differences in reported values for β-adrenergic receptor numbers and binding affinity in normal leukocytes, we undertook a systematic re-examination of the binding of two widely used beta antagonists, (-)-[³H]dihydroalprenolol (DHA) and $\text{(±)-}\text{[}{}^{125}\text{I]iodohydroxybenzylpindolol}$ (HYP), to intact normal mononuclear (MN) leukocytes and polymorphonuclear (PMN) leukocytes and membrane preparations. Assays were conducted in the presence and absence of chloroquine, which has been proposed recently to eliminate ligand uptake into a non-receptor cell compartment such as lysosomes. The binding curves relating radioligand concentration to specific sitesper intact cell were biphasic. At high (10–24 nM) (-)-DHA ligand concentration in the absence of chloroquine, a large number (20,000–60,000 sites/cell) of low affinity (K_d 12–15 nM) stereospecific binding sites were detected in both cell types. This class of binding sites was eliminated by 10 ,μM chloroquine not only in PMN cells but also in the lysome-poor MN cells (? 90% lymphocytes), leaving 2000–3000 specific high affinity (-)-DHA sites/cell. In the absence of chloroquine, comparably low numbers of specific high affinity binding sites/cell were also obtained by the use of appropriately low concentrations of (-)-DHA or (±)-HYP (800 pM or less). However, even at these low radioligand concentrations chosen to measure high affinity specific binding, the addition of 10 μM chloroquine produced a moderate reduction in the number of sites/cell, without a detectable change in the apparent K_d. Mean (± S.E.M.) site numbers obtained in the presence of chloroquine were: 1331 ± 100 sites/MN cell and 1135 ±129 sites/PMN cell (K_d 143–153 pM) using (-)-DHA; and 1487 ± 210 sites/MN cell and 1065 ± 69 sites/PMN cell [avg. K_d(±) 224–274 pM] using (±)-HYP. Chloroquine had no effect on agonist-stimulated cAMP production but produced an apparent increase in the effectiveness of (-)-propranolol as an inhibitor of DHA binding. Competition studies on the binding of DHA and HYP with zinterol and practolol confirmed that the receptor was of the β₂-subtype for both MN and PMN cells. The detection of a moderately larger number of high affinity binding sites at saturation (Scatchard analysis) by (±)-HYP than by (-)-DHA was a consistent finding with either intact cells or membranes, with or without chloroquine. The possible overestimation of receptor numbers by a racemic ligand such as (±)-HYP is discussed and leads us to favor the use of a pure stereoisomer such as (-)-DHA. A system employing 800 pM (-)-[³H]DHA, 1 ,μM (-)-propranolol and 10, μM chloroquine with intact MN and PMN cells yielded reproducible and plausible results. Our values for β-adrenergic receptor numbers of intact MN and PMN cells and membranes are compared to others in the literature.     

Mechanism of action of 5'-methylthioadenosine in S49 cells

5'-Deoxy-5'-methylthioadenosine, a naturally occurring co-product of polyamine biosyn-thesis, has been shown to inhibit a variety of biological processes. To investigate the mode of action of this nucleoside and to assess the involvement of cAMP in this action, the effect of methylthioadenosine on S49 wild type and two cAMP-related mutant cells was examined. The sulfur-containing nucleoside potently inhibited the growth of the parental strain ($\text{IC}{}_{50}\text{= 50 μ}\text{M}$), whereas nearly 10-fold greater resistance was demonstrated by S49 adenylate cyclase deficient ($\text{IC}{}_{50}\text{= 420 μ}\text{M}$) and S49 cAMP-dependent protein kinase deficient ($\text{IC}{}_{50}\text{= 520 μ}\text{M}$) mutant cells. Methylthioadenosine was shown to competitively inhibit the S49-derived high-affinity cAMP phosphodiesterase (K_i = 62 μM) in vitro, whereas methylthioadenosine phosphorylase activity was equivalent in all three cell types. The intracellular levels of the regulatory nucleotide, cAMP, increased dramatically in the wild type (17-fold) and protein kinase deficient (6-fold) strains in response to 100 μM concentrations of the drug. It is concluded that the growth arrest produced by 5'-methylthioadenosine in S49 cells is primarily due to the inhibition of cAMP phosphodiesterase and the subsequent increase in cAMP levels that result.     

Effect of a prostaglandin antagonist, N-0164, on cAMP generation and hydrolysis in the rat uterus

N-0164 (sodium-p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl] phenylphosphonate) (20–100 μM), an antagonist of the contractile effect of prostaglandins, reversed the prostaglandin E₂ (PGE₂) inhibition of isoproterenol-induced cAMP accumulation in rat uterus. N-0164, at the same concentrations, was a potent cAMP-phosphodiesterase inhibitor in broken cell preparations and potentiated the cAMP response to isoproterenol in intact tissue. The potency of N-0164 to inhibit cAMP-phosphodiesterase and to reverse the effect of PGE₂ on the cAMP response to isoproterenol were comparable (ec₅₀:50 and 60 μM respectively). In the presence of 10 mM theophylline, N-0164 did not affect the inhibitory effect of PGE₂. Furthermore, N-0164 produced similar proportional increases in the cAMP response to isoproterenol in the presence and absence of PGE₂. These results suggest that the apparent reversal by N-0164 of the PGE₂ effect on the cAMP response to isoproterenol is not due to its prostaglandin antagonistic action but to inhibition of cAMP-phosphodiesterase. N-0164, at concentrations lower than those inhibiting cAMP-phosphodiesterase, selectively inhibited the PGE₂-induced contractions of the rat uterus (ec₅₀, 4 μM), while at higher concentrations it also diminished carbachol-induced contractions. These results indicate that in the rat uterus N-0164 has at least two effects, prostaglandin antagonism and cAMP-phosphodiesterase inhibition, and suggest that the contractile effect of PGE₂ is independent of the effect of PGE₂ on the isoproterenol-induced rise in cAMP.     

A novel metabolic effect of the adenosine deaminase inhibitor coformycin,a potentiator of antiviral adenosine analogues

Coformycin [$\text{(R)-3-(β-}\text{d}\text{-erythropentofuranosyl}\text{)-3,6,7,8-}\text{tetrahydroimidazo}\text{[4,5}\text{d}\text{]-[1,3]}\text{diazepin}\text{-8-(R)-}\text{ol}$] is an inhibitor of adenosine deaminase and thus increases the antiviral potency of a series of adenosine analogues. The influence of chemically synthesized coformycin triphosphate on RNA-polymerizing enzymes (RNA polymerases from E. coli and from calf thymus, poly(A) polymerase) and on DNA-synthesizing enzymes (DNA polymerases and terminal deoxynucleotidyltransferase from calf thymus) was studied. Only the poly(A) polymerase was found to be sensitively inhibited by coformycin triphosphate. The inhibition is non-competitive with respect to ATP (K_i = 2.9 μM). Inhibition was reversed by simple dilution. The inhibitor does not act as chain terminator.     

Acetylpanaxydol und Panaxydolchlorhydrin,zwei neue,gegen L1210-Zellen cytotoxische Polyine aus Koreanischem Ginseng

Acetylpanaxydol and Panaxydolchlorohydrin, Two New Poly-ynes from Korean Ginseng with Cytotoxic Activity against L1210 Cells Two new polyines showing good cytotoxic activity against L1210 cells were isolated from Korean ginseng root. These were 3-acetyloxy-9,10-epoxy-heptadec-1-en-4,6-diyne (acetylpanaxydol, ED₅₀= 0.52 μg/nl) and 10-chloro-3,9-dihydroxyherptadec-1-en-4,6-diyne(Panaxydolchlorohydrin, ED₅₀= 0.50 μg/ml).     

Patent Evaluation: Novel Platinum Complexes as Antitumour Agents

Summary

Novelty: Novel platinum (II) complexes are claimed to be cytotoxic. They are potentially useful as antitumour agents.

Biology: Antitumour activity was assayed in vitro using murine leukaemia L1210 cells (10⁵); IC₅₀ values were in the range 0.06-2.36 μg/kg (cis-platin = 0.06). A similar test in vivo using male DBA/2 mice showed compounds have excellent life prolongation effect compared with carboplatin. Water solubility was 0.9- > 100 mg/ml at 20°C. Nephrotoxicity was very low. Of thirty-nine compounds assayed in an acute toxicity test, LD₅₀ = 34-304.8 mg/kg (cis-platin = 7.7 mg/kg).

Chemistry: Forty-five examples of preparation of the compounds are given by standard techniques. Characterization is by mp, IR, NMR, MS and elemental analysis. The dihalogendiamine platinum (II) complexes used as starting materials were synthesized as described in nineteen preparative examples. A preferred compund is (glycolato-O,O')[(4R,5R)-4,5-bis-(aminomethyl)-2,2-diethyl-1,3-dioxolane]platinum (II).     

The role of transcription factor Nrf2 in the toxicity of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in C57BL/6 mouse astrocytes

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are members of perfluoroalkyl substances (PFAS). This study aimed to determine the protective role of Nrf2 against the toxicity of these agents. Nrf2-/- and wild-type astrocytes were exposed to PFOS (75−600 μM) and PFOA (400−1000 μM) for 24 h. Lactate dehydrogenase (LDH) release was significantly higher in nrf2-/- than in the wild-type astrocytes. Exposure to 600 μM PFOS and 800 μM PFOA showed significant increases in reactive oxygen species, lipid peroxidation, and apoptosis in nrf2-/- astrocytes as compared to wild-type astrocytes. The GSH/GSSG ratio was significantly decreased in nrf2-/- astrocytes when compared to wild-type astrocytes. Additionally, PFOS and PFOS caused dramatic ultrastructural alterations to the mitochondria. BHT pretreatment in wild-type cells decreased ROS production with exposure to both agents. Results of the present study conclude that PFOS and PFOA are cytotoxic to astrocytes and that nrf2 -/- cells are more sensitive to toxicity by these agents.