Development of nitroxides for selective localization inside cells.

The use of nitroxides to measure intracellular phenomena, especially oxygen concentrations, is a new and potentially important approach to a number of physiological and pathophysiological studies. This study provides data indicating the feasibility of developing nitroxides that localize selectively in the intracellular compartment; it is based on the use of readily hydrolysed ester linkages, such that the nitroxides become converted intracellularly to ionic derivatives that do not cross cell membranes readily. Up to 120-fold increased concentrations of intracellular nitroxides (and their one electron reduction product, the hydroxylamines) were obtained. The ESR spectra of the intracellular nitroxides were consistent with their conversion to the ionic species. Preliminary studies indicate that these nitroxides have the properties needed for their use as probes of intracellular concentrations of oxygen and that it should be feasible to synthesize nitroxides that will be even more effective for this purpose.

In the search for new anticancer drugs. 19. A predictive design of N,N:N',N':N'',N''-tri-1,2-ethanediylphosphorothioic triamide (TEPA) analogues.

The nitroxyl-labeled analogues of N,N:N',N':N",N"-tri-1,2-ethanediylphosphoric triamide (TEPA), N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,6,6-tetramethyl-1-oxypiperidi n-4- yl)amino]carbonyl]phosphoric triamide (5a) and N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)amino]carbonyl]phosphoric triamide (11a), possess therapeutic indexes that are 8-12 times higher than those of thio-TEPA (1) and TEPA (2). The introduction of methyl groups into the aziridine ring, or the replacement of the nitroxyl moiety with hydroxylamine or amine derivatives, or with an adamantane moiety, results in compounds of lesser activity. An attempt is made to rationalize these results using a lipophilicity scale. A predictive design pattern is established.

In the search for new anticancer drugs. 17. Linear and cyclic polyether analogues of N,N:N',N':N'',N''-tri-1,2-ethanediylphosphoric triamide and N,N:N',N':N'',N''-tri-1,2-ethanediylphosphorothioic triamide.

Linear and cyclic polyether derivatives of N,N:N',N':N'',N''-tri-1, 2-ethanediylphosphoric triamide (TEPA) and N,N:N',N':N'',N''-tri-1,2-ethanediylphosphorothioic triamide (thio-TEPA) are synthesized and evaluated for their antineoplastic activity against the murine lymphocytic leukemia P388. All compounds, except for 7d, were active ranging from 42% to 287% increase in life span (% ILS). All CD2F1 male mice treated with the most active compound (7a) at 90 mg/kg per day for 9 days were alive after 30 days, whereas all mice treated with the clinical drug thio-TEPA were dead. The % ILS for compound 7a on day 60 was 525. A correlation is presented between the structural features of compounds and their lipophilicities and antineoplastic activities.

In the search for new anticancer drugs. XVIII. Various mono- and bis-anthraquinone hydrazones containing the N,N;N',N'-bis(1,2-ethanediyl)phosphoric diamide moiety.

The structure-anticancer activity and the activity-lipophilicity relationship of 8 mono- and bis-anthraquinone hydrazones containing the N,N;N',N'-bis(1,2-ethanediyl) phosphoric diamide moiety were evaluated. These compounds were tested in vivo, using murine lymphocytic leukemia P388. Seven of these compounds were active and one was marginal at optimum doses. The highest activity was exhibited by the bis[N,N;N',N'-bis(1,2-ethanediyl)-[N2-(1',4',5',8'-tetrahydroxy anthracenylidene)-N1-methyl hydrazin-1-yl]]-phosphoric triamide, bis[N,N;N',N'-bis(1,2-ethanediyl)-[N2-(1',4'-dihydroxy anthracenylidene)-N1-methylhydrazin-1-yl]]phosphoric triamide and bis-[N,N;N',N'-bis(1,2-ethanediyl)-[N2-(1',4',5',8'-tetrahydroxy anthracenylidene)-N1-phenylhydrazin-1-yl]]phosphoric triamide as is evidenced by their percent T/C of 183, 175 and 172, respectively. The correlation of the anticancer activities of these compounds with their lipophilicities leads to the general hypothesis that the intercalating and alkylating capabilities of a potential drug may play only a secondary role as compared to the selective permeability of the drug through normal and cancerous membranes.

In the search for new anticancer drugs. XV. Various aliphatic and aromatic hydrazones containing the N,N;N',N'-bis(1,2-ethanediyl)phosphoric diamide moiety.

The scope of the structure-anticancer activity relationship among various aliphatic and aromatic hydrazones containing the N,N,N',N'-bis(1,2-ethanediyl)-phosphoric diamide moiety was studied. A total of 19 compounds were tested in vivo, using murine lymphocytic leukemia P388. At optimum dose all these compounds were active. The highest activity was exhibited by the N,N;N',N'-bis(1,2-ethanediyl)-[N2-(p-chlorobenzylidene)-N1- hydrazin-1-yl]phosphoric triamide, N,N;N',N'-bis(1,2-ethanediyl)-N2-(p-chlorobenzylidene)-N1- methylhydrazin-1-yl]phosphoric triamide and N,N;N',N'-bis(1,2-ethanediyl)-[N2-(p-chlorobenzylidene)-N1- phenylhydrazin-1-yl]phosphoric triamide as is evidenced by the percent T/C of 212, 194 and 192, respectively. An attempt was made to correlate the anticancer activities with the corresponding lipophilicities. On the basis of the activity-lipophilicity results, it is concluded that, in general, the activity scale follows the lipophilicity scale in the reverse order, i.e. the more active compounds possess lower lipophilicity than the less active compounds.

In the search for new anticancer drugs. XX: A comparison of the in vitro growth inhibition of P388 cells by TEPA and N,N;N',N'-bis (1,2-ethanediyl)-N''-(1-oxyl-2,2,6,6-tetramethyl-4- piperidinylaminocarbonyl) phosphoric triamide and congeners.

We tested the in vitro growth inhibitory activity of TEPA, and three analogs against P388 murine lymphocytic leukemia cells in culture. The analogs consist of spin-labeled TEPA and two reduced forms containing the NH and NOH groups instead of the nitroxyl function. Spin label TEPA was obtained by replacing one of the aziridine groups in TEPA with spin-labeled urea. In a concentration range of 10(-6)-10(-5) M, only the reduced analog containing the NH group was active. That is, to achieve a 50% inhibition of cell growth, a five-fold excess in concentration of this analog (IC50 = 10 X 10(-6) M) was needed as compared to the parent compound TEPA (IC50 = 2 X 10(-6) M). These results are in contrast with those obtained in vivo against the same leukemia cell line, indicating inherent discrepancies between in vivo and in vitro evaluation of antitumor agents.

Cellular pharmacology of N,N',N''-triethylene thiophosphoramide.

N,N',N''-triethylene thiophosphoramide (Thio-TEPA) is an alkylating agent whose antineoplastic activity has been known for nearly 30 years. Human plasma pharmacokinetic studies revealed the presence of TEPA, a Thio-TEPA metabolite which after 4 h achieved plasma concentrations equal to those of the parent compound. We studied the activity of both Thio-TEPA and TEPA against murine leukemia P388 cells in culture. We found that Thio-TEPA is approximately two-fold more active than TEPA in arresting cell growth (IC50 = 2.8 microM for TEPA and 1.5 microM for Thio-TEPA). In inhibiting [3H]thymidine incorporation, Thio-TEPA and TEPA have the same activity (IC50 = 2 microM for both compounds). Experiments in which drug was removed from cell cultures which were further incubated in drug-free media, revealed that the bulk of the cell damage occurs during the first 4 h of incubation. Cell cultures exposed to 0.5 microM Thio-TEPA for 22 h fully recovered their [3H]thymidine incorporation ability after 24 h of drug-free incubation. Cells exposed to 2.5 microM Thio-TEPA for 22 h partially recovered their ability to incorporate [3H]thymidine. Cells exposed to 10 microM Thio-TEPA for 22 h did not recover their ability to incorporate [3H]thymidine. Gas liquid chromatographic analysis of the media from incubated cells showed that the concentration of Thio-TEPA remained unchanged during the incubations and that TEPA was not present. In Thio-TEPA doses ranging from 0.1 microM to 100 microM, [3H]uridine and [3H]-leucine incorporation were less affected than [3H]thymidine incorporation. This may indicate that a longer observation time may be needed to allow the DNA damage to be expressed in terms of protein or RNA synthesis.

In the search for new anticancer drugs. XXIII: Exploration of a predictive design for anticancer drugs of carbohydrates containing N-nitrosochloroethylamino, N-nitrosomethyl, and N-nitrosoaminoxyl components.

The spin-labeled glucose nitrosoureas 13-16, streptozotocin (18), chlorozotocin (31), streptozotocin analogues of galactosyl 24 and mannosyl 28, and their tetra-O-acetyl derivatives 25 and 29, MCNU (Cymerin, 34), and glucamine (21) were synthesized and evaluated in vivo for their anticancer activities against the murine lymphocytic leukemia P388. Compounds 13-16, 18, 24, 28, 31, and 34 possessed activities ranging from 33 to 603% increase in life span (%ILS), whereas 21, 25, and 29 were inactive (%ILS = 9 to 10). All CD2F1 male mice treated with the most active compounds (13, 14, and 34) at 20 mg/kg were alive after 30 days, whereas all mice treated with the clinical drug streptozotocin (18) and clinically tested chlorozotocin (31) succumbed. Compounds 13-16, 18, 31, and 34 were further evaluated for their antineoplastic activity against lymphoid leukemia L1210. Compounds 13 and 34 on day 60 exhibited %ILS values of 557 and 713, respectively, as compared with %ILS values of 646 and 713 for CCNU (1) and the spin-labeled SLCNU (3), respectively. The lipophilicities of 13-16, 18, 21, 24, 25, 28, 29, 31, and 34 were determined using EPR and/or UV methods. A predictive design pattern was observed, with the most active drug (34) possessing some hydrophobic property (log P = 1.24), followed by 13 (log P = 1.87) and 14 (log P = 1.81) as the more active drugs with higher hydrophobicity than 34. The clinical drugs streptozotocin (18) and chlorozotocin (31) were distinctly hydrophilic and less active. Finally, it was concluded that various scattered results of anticancer activity in the literature can be explained by a linear correlation of activities with lipophilicities.

In the search for new anticancer drugs. XXIV: Synthesis and anticancer activity of amino acids and dipeptides containing the 2-chloroethyl- and [N'-(2-chloroethyl)-N'-nitroso]-aminocarbonyl groups.

A series of L,L- (42, 44, 46, and 60) and D,D- (43, 45, 47, and 61) dipeptide derivatives composed of phenylglycine, phenylalanine, homophenylalanine, and valine and containing a 2-chloroethylamino group at the C-terminus and an N'-(2-chloroethyl)-N'-nitroso-aminocarbonyl group at the N-terminus of the dipeptides were prepared. The dipeptide derivatives (42-47, 60, and 61) were first evaluated in vivo for their anticancer activities against the murine lymphocytic leukemia P388. Compounds 42, 44, 46, and 60 possessed activities ranging from 46 to 111 percent increase in life span (%ILS), whereas 43 was marginal (%ILS = 31) and 45, 47, and 61 were inactive. In general, the L,L-series exhibited low to good activity (%ILS = 46-111), whereas the corresponding D, D-series, except for 43 (%ILS = 31), was devoid of activity. The analogously structured monoamino acid derivatives of L-alanine (74), L-phenylalanine (75), and L-aspartic acid (76) exhibited higher activity against P388 than the dipeptide derivatives (i.e., 481, 297, and 481 %ILS, respectively). The more active representatives of dipeptides (i.e., 42, 44, and 60) and the amino acids derivatives 74-76 were then tested in vivo against the murine lymphoid leukemia L1210. Compounds 42, 44, and 60 exhibited either low or marginal activity (i.e., the %ILS values were 46, 31, and 26, respectively). Compounds 74, 75, and 76 possessed low to moderate activity, as evidenced by the %ILS values of 56, 48, and 64, respectively. The %ILS parameters obtained against the P388 and L1210 tumor lines were correlated with the corresponding lipophilicities, and there is a trend towards higher activity with concomitant decrease in hydrophobicity.

Chemical and electrochemical reduction rates of cyclic nitroxides (nitroxyls).

The reduction rates of five-membered pyrrolidine and pyrroline, and six-membered piperidine nitroxides (alternatively termed nitroxyls) containing various substituents were determined under homogeneous conditions using ascorbate, and electrochemically under heterogeneous conditions. The results were compared with data from the literature. It was shown that the increased rates of reduction of six-membered nitroxides, compared with those of the five-membered nitroxides, cannot be explained on the basis of differences in electrochemical potentials but, rather, can be ascribed to differences in the accessibility of the nitroxide group. A double bond in the five-membered nitroxyls increases the reduction rate. Within any ring system, the reduction rates of nitroxides using ascorbate can be correlated with the inductive substituent constants. The half-way potentials for electrochemical reduction within a series of nitroxides based on the same ring correlate with logarithms of the rates using ascorbate and with the inductive constants. The potentials for one-electron oxidation of the nitroxides were related to the inductive constants.

In the search for new anticancer drugs, XXI. Spin labeled nitrosoureas.

The spin labeled nitrosoureas 7a-e and 12 were synthesized and evaluated in vivo for their anticancer activities against the murine lymphocytic leukemia P388. Compounds 7a-c, 7e and 12 possessed activities ranging from 31 to 542 percent increase in life span (%ILS), whereas compound 7d was marginal (%ILS = 21). All CD2F1 male mice treated with the most active compounds (7a and 12) at 35 mg/kg for 9 days were alive after 30 days, whereas all mice treated with the clinical drug CCNU (1c) succumbed. Compounds 7a-e and 12 were further evaluated for their antineoplastic activity against lymphoid leukemia L 1210. Compounds 7a and 12 exhibited, on day 60, a %ILS of 713 and 620, respectively. The lipophilicities of compounds 7a-e and 12 were determined using the EPR and UV methods. Compounds 7a and 12 which differ from CCNU and MeCCNU by the replacement of the cyclohexyl and methylcyclohexyl groups with six and five membered nitroxyl radical moieties were more hydrophilic than the clinical drugs.

Evaluation of spin labeled TEPA and CCNU analogs against osteosarcoma and MNU-induced mammary carcinoma of the SD-rat.

The effectiveness to reduce tumor growth by 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6-tetramethylpiperidinyl)- 1-nitrosourea (SLCNU) and N,N,N,'N'- bis(1,2-ethanediyl)-N"-(1-oxyl-2,2,6,6-tetramethyl- 2-piperidinylaminocarbonyl)-phosphoric triamide (SLDU) was studied in osteosarcoma and MNU-induced mammary carcinoma in the SD-rat. Both compounds elicited neither an inhibitory effect on these tumors nor an increase in the mean/median life span as compared to the control group.

Foodborne transmission of cholera in Micronesian households.

During a cholera epidemic in Truk (Micronesia), in a survey of 1 village, transmission of Vibrio cholerae O1 in the 28 households with illness appeared to be through food contaminated in the home. Households in which the index case was a foodhandler had significantly higher attack rates than households in which the index case was not a foodhandler. Members of households with illness were significantly more likely to become ill if they had eaten food prepared by a foodhandler who had recently been ill. A matched-pair case-control study on several adjacent islands confirmed this relation. Those who had tended an ill person were not at increased risk of cholera in either study. This study suggests that in this outbreak, foodborne transmission of V cholerae O1 was more important than contact spread.