George Sosnovsky

Factors affecting nitroxide reduction in ascorbate solution and tissue homogenates

Because of their paramagnetic properties, nitroxides are potentially useful as contrast agents in magnetic resonance imaging (MRI). They are reduced in vivo to their corresponding hydroxylamines which are nonparamagnetic and have no contrast enhancing property. Nitroxides with high resistance to reduction would be advantageous as pharmaceutical contrast enhancing agents. We show that in the presence of ascorbic acid and in tissue homogenates, the reduction is faster for piperidine than for pyrrolidine nitroxides and for positively-charged than for negatively-charged derivatives. The data also suggest that nitroxide reduction in tissue homogenates is mainly due to sulfhydryl groups on proteins and that endogenous ascorbic acid plays a relatively minor role.

The quest for a predictive design of anticancer drugs

A predictive design is explored with nitroxyl labeled derivatives, and other congeners of alkylating drugs TEPA/Thio-TEPA, CCNU/MeCCNU, and Streptozotocin/Chlorozotocin. The design is based on correlations of lipophilicities of these drugs with their antineoplastic activities in vivo against the murine lymphocytic leukemia P388. Some results are also included of evaluations using the nturine lymphoid leukemia L1210. therapeutic indices than those of the parent drugs which are in clinical use, and, hence, warrant further studies. Several of these compounds possess considerably higher

Simultaneous measurements of the intra- and extra-cellular oxygen concentration in viable cells

An EPR method that can measure the intra- and extra-cellular oxygen concentration [O2] simultaneously in vitro has been developed using specially designed nitroxides. In the presence of Fe(CN)6(3-) in the medium, intracellular [O2] is measured by a neutral 15N-nitroxide and extracellular [O2] is measured by a negatively charged 14N-nitroxide, since charged species do not enter cells and the EPR spectrum of a 15N-nitroxide does not overlap with that of a 14N-nitroxide. The method is based in part on the minimal broadening of negatively charged nitroxides by Fe(CN)6(3-) and the very effective broadening of neutral nitroxides by the same paramagnetic ions. Results with this method confirm the existence of gradients in [O2] between the extracellular and intracellular compartments in CHO cells and M5076 tumor cells, even without stimulation of cellular respiration by CCCP. The nature of the barrier that needs to be involved to account for the experimental results raises some significant questions.

In the search for new anticancer drugs XII. Synthesis and biological evaluation of spin labeled nitrosoureas

The spin labeled nitrosourea 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6- tetramethyl-piperidinyl)-1-nitrosourea (SLCNU, 4) and its analogues 5-7 were synthesized either by a regio-selective method or by a conventional route via the nitrosation of the spin labeled intermediates (11a-e). Nitrosation of the ureas 11a-e with dinitrogen tetraoxide resulted in better yields than those obtained with sodium nitrite. The nitrosoureas 4-8 were tested for their anticancer activity against the lymphocytic leukemia P388 in mice. Thus, either at the equal molar dose or at the dose of equal toxicity level, the SLCNU (4) was found to be more active than the clinically used CCNU (1). Unlike CCNU (1) whose LD50 is 56 mg/kg, the SLCNU (4) possesses a low toxicity (LD50 123 mg/kg). Therefore, SLCNU (4) is a promising new entry into the nitrosourea class of anticancer drugs.

Diradical nitroxyl spin label contrast agents for magnetic resonance imaging. A comparison of relaxation effectiveness.

The proton relaxation enhancement characteristics of seven potential MRI contrast agents containing two nitroxyl spin labels per molecule (diradicals) were compared with eight similar agents with only one spin label per molecule (monoradicals). Diradical nitroxyls were evaluated to test the hypothesis that multiple paramagnetic centers in one molecule will result in stronger proton relaxation enhancement characteristics, allowing effective contrast enhancement at lower molar concentrations and thus a reduced osmotic load and greater safety. The acute toxicity of these agents is believed to be largely related to osmotic load. Five of seven diradical nitroxyls tested had spin-lattice relaxivities that were substantially greater than all eight of the monoradicals tested. The spin-spin relaxation properties of these agents and other pertinent characteristics are favorable for contrast enhancement. The results indicate that diradical nitroxyl spin labels may be used advantageously for the design of safer, more effective MRI contrast agents.

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.

Influence of chemical structure on nitroxyl spin label magnetic relaxation characteristics

Abstract An attempt was made to develop guidelines for the design of new contrast agents using nitroxyl spin labels (NSL). The structural parameters of ring size and of substituents were correlated with the stability towards reduction and the relaxation effectiveness using 5 piperidine and 5 pyrrolidine nitroxyls containing the same substituents. The susceptibility of NSL to reduction was assessed by EPR spectroscopy. The relaxation effectiveness of NSL on protons in buffer and plasma solution was measured on a NMR spectrometer. The ring size and substituents has a decisive effect on the stability of NSL, whereby the ring size effect was dominant. In the case of spin—lattice relaxivities (R 1 ) and spin-spin relaxivities (R 2 ), the ring size and the substitution effect were marginal in buffer solution, while in plasma these effects were more pronounced. A number of guidelines were proposed for the design of suitable NSL contrast agents for MRI.

In the search for new anticancer drugs. X. N,N; N', N'-bis(1,2-ethynediyl)-N″-(1-oxyl-2,2,6,6-tetramethyl-4- piperidinylaminocarbonyl) phosphoric triamide — A new potential anticancer drug of high activity and low toxicity

A new nitroxyl labeled TEPA derivative 5 containing the urea bridge between the phosphorus and the nitroxyl moiety, and the congeners containing the NOH and NH groups instead of the nitroxyl function were synthesized, and tested in vivo on CD2F1 mice for anticancer activity against P388 and L1210. The nitroxyl compound is more active than the reduced forms. The nitroxyl compound 5 elicits 170% ILS at 90 mg/kg after 30 days and 439% ILSmax after 60 days against P388, and has a higher therapeutic ratio (26.4) than the clinically used Thio-TEPA (2.75). The LD50 of 5 is 270 mg/kg, while that of Thio-TEPA is 18 mg/kg. Consequently, the nitroxyl compound 5 is a promising new anticancer drug.

Gadolinium, neodymium, praseodymium, thulium and ytterbium complexes as potential contrast enhancing agents for NMR imaging

Abstract Complexes of ferric (3) and gadolinium (5) citrate, gadolinium tricarballylate (6) and complexes of gadolinium, neodymium, praseodymium, thulium and ytterbium with complexons EDTA (7), CDTA (8), EGTA (9), DTPA (10) and TTHA (11) were synthesized and evaluated as potential contrast enhancing agents for NMR imaging by measuring their stability constants and their effectiveness in eliciting spin-lattice (T1) and spin-spin (T2) relaxations of protons in water and plasma. High stability constants (log10 k = 22 or higher) and excellent relaxation properties were found for Gd-EGTA 19, Yb-EGTA 20, Gd-DTPA 23, Yb-DTPA 24, Gd-TTHA 28, Yb-TTHA 29 and Tm-TTHA 30. These compounds were considered as potential intravenous contrast agents for NMR imaging. Whereas, the stability constant of 28 was higher (log10 k = 28.4) than that (log10 k = 22.4) of the clinically used 23, the LD50 value, using Swiss male mice, for 28 was lower (2.88 mmol/kg) than that for 23 (3.74 mmol/kg). The somewhat higher toxicity of 28 than that of 23 was explained by a higher osmolality of 28 as compared to 23. The gadolinium citrate 5 and gadolinium tricarballylate 6 were proposed as potential oral contrast agents.

Spin Labeled Bovine Serum Albumin, Spin Labeled Bovine Serum Albumin Chelating Agents and Their Gadolinium Complexes. Potential Contrast Enhancing Agents for Magnetic Resonance Imaging

Abstract Bovine Serum Albumin (BSA) was spin labeled using the active esters 3 and 5 containing a nitroxyl radical to give the spin labeled BSA derivatives 4 and 6. Spin labeled C D TA , DTPA and TTHA chelating agents 10, 13 and 16 and the corresponding BSA derivatives 17, 18 and 19 and their gadolinium complexes 20, 21 and 22 were synthesized. All these BSA derivatives were evaluated as possible contrast enhancing agents for NMR imaging by measurement of spin-lattice (T1) and spin-spin (T2) relaxations and by calculations of relaxivities i.e., slopes of 1/T1 versus concentration plots. The spin labeled BSA-complexon-gadolinium conjugates 20, 21 and 22 were found to have better relaxation times and relaxivities than the BSA spin labeled derivatives 4, 6, 17, 18 and 19. The spin count for the BSA derivatives 4, 6, 17, 18 and 19 and for their gadolinium conjugates 20, 21 and 22 was found to be between 9 -1 0 spins/molecule of the protein. The gadolinium conjugates 20, 21 and 22 of spin labeled BSA possess high relaxivities combined with a nitroxyl marker that could be used with EPR to assess pharmacokinetic behaviors of these potential contrast agents