WO1999050244A1 - Nitroxide free radical dual indicator agents - Google Patents

Abstract

Dual indicator agents having synergistically enhanced fluorescent properties are provided that include a nitroxide moiety and a nitroxide free radical center. The indicator molecule may include any variety of chemical or biochemical marking agents, such as a fluorescent molecule. The dual agent may further include a spacer molecule or linker, that will function to attach the indicator molecule, such as a fluorescent moiety, to the nitroxide moiety. These may be used to measure the redox potential in living cells, as well as to determine redox potentials in diagnostics, as well as in many other applications.

Description

NITROXIDE FREE RADICAL DUAL INDICATOR AGENTS

The government may own rights in the present invention as financing of the development of the invention was from the Cooperation in Applied Science Technology

(CAST) grant from the National Research Office of International Affairs date 10, 1996 The present application is a continuating application of U.S. Provisional Serial No.: 60/079,848, filed March 28, 1998 to which priorities claimed.

FIELD OF THE INVENTION

The present invention relates generally to the fields of novel molecular probes and labels that combine the features of fluorescent probes with the paramagnetism of spin probes. They can be recognized by means of both fluorescent spectroscopy and electron paramagenetic (or spin) resonance (EPR on ESR) spectroscopy as well. The invention also relates to the fields of immunodiagnostics, protein and membrane analysis, spin oximetry, and studies of intracellular pH and surface potentials of cells, as the novel probes and labels may be used to render measurements by fluorescent spectroscopy and/or EPR.

BACKGROUND OF THE INVENTION

Nitroxides (nitroxyl radicals) are examples of stable organic free radicals (Berliner, L J., Spin Labeling. Theory and Applications. Vol. 1 and 2, Academic Press, New York, 1976, 1979). These N,N-disubstituted >NO radicals are stable enough to be prepared, handled and stored like conventional organic compounds (Keana, J.F. W., Chem. Rev., 78:37, 1978). They possess an open electron shell (an unpaired electron), and, like all free radicals, display paramagnetic properties. This characteristic allows their use as reporter molecules, recognizable by Electron Paramagnetic (or Spin) Resonance (EPR or ESR) spectroscopy (Keana, J.F.W., Chem. Rev., 78:37, 1978). Sensitivity of their EPR spectra to changes in their surroundings (Gaffhey, B.J., and Chen, S.C, in Method in Membrane Biology. Vol. 8, edited by E.D. Korn (Plenum Press, New York, US), Chapter 4, 1977), coupled with the high chemical stability of the oxide moiety (Berliner, L.J., Spin Labeling. Theory and Applications. Vol. 1 and 2, Academic Press, New York, 1976, 1979) makes them especially attractive as probes and labels, usually referred to as spin probes and labels. EPR spectra of nitroxide radicals are affected by characteristics of the probe's microenvironment such as mobility, micro viscosity, pH, concentrations of metals and molecular oxygen (Bacic, G., Nitges, M.J., Bennett, H.F., Magin, R.L., and Swartz, H.M., Magn. Resort. Med, 6:445,1988; Dobrucki, J.W., Demsar, T., Walczak, T., Woods, R.K., Bacic. G., and Swartz, H.M., Br. J Cancer, 61 :221, 1990; and Subczinski, W.K., Lukiewicz, S., and Hyde, S., Magn. Reson. Med., 3:747, 1986). Spin probes are already used in molecular biology for enzyme mechanism studies, immunonodiagnostics Rauckman, E.J., Rosen, G.M., and Griffeth, L.K., in Spin Labeling in Pharmacology, Holzman, J.L., Ed., Academic Press, New York, 1984, 1975), protein- and membrane research (Castle, J.D., and Hubbell, Biochemistry, 15:4818, 1976), spin oxymetry (Strzalka, K., Walczak, T., Sarna, T., and Swartz, H.M., Arch. Biochem. Biophys., 281:312,1990), and studies of intracellular pH and surface potentials (Mehlhorn, R.J., Candau, P., and Packer, L., Method EnzymoL, 88:751, 1982; Mehlhorn, R.J., and Packer, L., Method EnzymoL, 56:515, 1979). Recent developments in spin labeling techniques involve the expansion of their biological use from simple systems to more complex ones. The use of nitroxides in living systems has expanded rapidly recently (Zhdanov, R.I., Ed. Bioactive Spin Labels, Springer-Verlag, Berlin, 1992). At first, nitroxides were used in cells and subcellular fractions. Most recently, with the development of EPR imaging and in vivo EPR spectroscopy, their use has been extended to tissues and whole animals (Bacic, G., Walczac, T., Demsar, F., and Swartz, H.M., Magn. Reson. Med, 8:209, 1988). This demonstrates their potential as an investigative tool. In principle, all EPR techniques that have used nitroxides in nonbiological systems and model systems also can be used with functional cell fractions, intact cells, tissues, or isolated organs, as well as in vivo. These techniques include the measurement of local motion in membranes; the motion of macromolecules; viscosity, including intracellular volume; content of sulfhydryl groups (Khramtsov, V.V., Yelinova, V.I., Weiner, L.M., Berezina, T.A., Martin, V.V., and Volodarsky, L.B., Anal. Biochem., 182:58, 1989); concentration of oxygen (Sosnovsky, G., Rao, N.U.M., Li, S.W., and Swartz, H.M., J Org. Chem., 54:3667,1989); diffusion of nitroxides (Demsar, F., Swartz, H.M., and ScharA., M.., Magn. Reson. Med Boil., 1 : 17,

1988); viability of cells (Dobrucki, J.W., Sutherland, R.M., and Swartz, H.M., Magn. Reson. Med, 19:42,1991); and temperature (Chato, J.C., Swartz, H.M., and Robles, J.E., in Boiheat Transfer- Applications in Hyperthermia. Emarging Horizons in Instrumentation and Modeling, Romer, R.B., McGrath, J.J., and Bowman, H.F., Eds., American Society of Mechanical Engineers, New York, 1989, 12 1).

The recent development of in vivo EPR techniques has further increased the interest in and the need to understand the biological interactions of nitroxides. These techniques usually require adding nitroxides to the system because of the lack of sufficiently high concentrations of naturally occurring paramagnetic molecules in cells or tissues. There is also considerable potential for the use of in vivo EPR techniques to make measurements of classical biophysical parameters such as motion of macromolecules and the state of membranes. In addition, most of the new capabilities of EPR that have been developed in the last few years also could be studied by means of in vivo EPR techniques. These capabilities include the measurement of oxygen concentration (Chan, H.C., Glockner, J., and Swartz, H.M., Biochim. Biophys. Ada, 1014:141,1989), diffusion (Demsar, F., Walczak, T.,Morse, P.D., Bacic, G.,Zolnai, Z., and Swartz, H.M., J Magn. Reson., 6:114,X9%%), viability (Dubruckij.W., Demsar, F., Walczak, T., Woods, R.K., Bacic, G., and Swartz, H.M., Br. J Cancer, 61 :221, 1990), and temperature (Chato, J.C., Swartz, H.M., and Robles, J.E., in Boiheat Transfer-Applicahons in Hyperthermia. Emarging Horizons in Instrumentation and Modeling, Romer, R.B., McGrath, J.J., and Bowman, H.F., Eds., American Society of Mechanical Engineers, New York, 1989, 121).

Other developments include the growing use of nitroxide spin probes outside the field of molecular biology. The use of nitroxide spin probes is being investigated in such areas as chelating reagents for analytical chemistry, probes for polymer dynamics study, labeling of underground flows for oil production, and quality control of the surface of diamond jewelry, among others.

The nitroxide radical center is stable, e.g. paramagnetic properties of the spin probes are not time-dependent. However, in early studies with biological systems, it was observed that there was the potential for loss of the paramagnetism due to oxidation or reduction of the nitroxide group, resulting in the pairing of the unpaired electron of the nitroxide, and hence the loss of the EPR signal (Corcer, G.A. Klein, M.P., and Calvin, M., Proc. Natl. Acad Sci. USA, 56:1365,1966).

The process by which the EPR signal is lost in biological systems is through reduction of the nitroxide to the corresponding hydroxylamine (Scheme 1).

Scheme 1 More rarely, oxidation, often via interactions with unstable radicals, is possible, for example with hydroxyl radicals (Floyd, R., Biochem. Biophys. Ada, 756:204,1983; Nohl, H., Jordan, W., and Hegner, D., FEBS Lett., 123:241, 198 1). These reactions are responsible for the inhibition of radical polymerization processes as well as for the enhancement of tumor radiosensitivity by nitroxides. Acid-catalyzed dispropotrionation of nitroxides, which yields a mixture of hydroxylamino derivative H and nitrosoniurn salt N also resulted in the loss of paramagnetizm.

Scheme 2

H H

Another pathway for the loss of paramagnetism is through reaction with unstable carbon-centered radicals (R), which led to R-substituted diamagnetic hydroxylamine derivative. Scheme 3

\

N-O R s N-OR

These reactions affecting nitroxide radical centers impose additional experimental concerns and constraints on the use of nitroxides in functional biological systems due to the potential for the nitroxides to modify the biological system they are introduced into. These same characteristics make nitroxides important tools for investigating redox reactions and free radical processes in complex systems. Monitoring the decay of electron paramagnetic resonance (EPR) signal makes it possible to measure the redox potentials in living cells or to study the free radical processes in vivo.

To supply nitroxide radicals for spin labeling applications, the synthetic chemistry of nitroxides has been developed. It deals with the synthesis of organic molecules containing a nitroxide radical center and is based on the possibility of executing various reactions without affecting the unpaired electron. The stability of the nitroxide group in withstanding several synthetic manipulations has made it possible to synthesize a wide variety of spin-labeled molecules. The success and versatility of the spin labeling method, is attributable to the possibilities opened up by the discovery of reactions that do not effect the nitroxide moiety. Despite their outstanding stability, nitroxide groups are capable of losing their free radical character on interaction (inter- and intramolecular) with a variety of reagents and functional groups (Keana, J.F.W., Chem. Rev., 78, 37, 1978; Volodarsky L.B., ed. Synthetic Chemistry ofNitrroxide Radicals, CRC Press, Boca Raton, FI, 1995). This reactivity imposes limitations both on the scope of the reaction that can be employed for nitroxide synthesis and on the possible structures for functional deπvatives containing a radical center.

The present day attempts to synthesize nitroxides also recognizes that specific properties need to be incorporated in the nitroxide for use in particular fields of research. Some of these properties include development of nitroxides which are resistant to in vivo reduction (Keana, J.F.W., Pou, S., and Rosen, G.M., Magn Reson. Med, 5: 525, 1987), nitroxides which specifically localize in a biological system (Swatz, H.M., and Pals, M., Handbook of Biomedicine of Free Radicals and Antioxidants, Vol. 3, edited by J. Miguel, H Weber and A Wumtamlha (CRC Press), 19 89, 141), nitroxides with spectral features which respond to oxygen concentration (Swatz, H.M., and Pals, M., Handbook of Biomedicine of Free Radicals and Antioxidants, Vol. 3, edited by J. Miguel, H. Weber and A. Wumtamlha (CRC Press), 1989, 141), nitroxides which cause relaxation of water protons or other nuclei studied by NMR techniques (Swartz, H M., in Advances in Magnetic Resonance Imaging, edited by E. Feig (Ablex Publishing Company, Norwood, New Jersey, US), 1989, 49), and nitroxides with a combination of these properties appropriate for particular biological systems.

Syntheses of spin labels for their numerous biological applications are the subject of several books and reviews (Volodarsky L.B., ed. Synthetic Chemistry offitroxide Radicals, CRC Press, Boca Raton, FI, 1995; Imidazohne nitroxides. Synthesis and properties, V.l; Applications, V.2, Volodarsky, L.B., Ed., Boca Raton, Flonda: CRC press, 1988;

Likhtenstein G.I.; Kuhkov, A V , Kotelmkov, A.I., Levonenko, L.A Biochem Biophys. Methods, 12 1, 1986; Kochergmsky, N ; Swartz, H.M. Nitroxide Spin Labels Reactions in Biology and Chemistry, CRC Press, Boca Raton, FI. 1995; Khramtsov, V V , Vamer, L.M Russ Chem Revs., 57:824, 1988; Keana' J.F.W in Spin Labeling in Pharmacology, ed. Holtzman, J.L. Academic Press, Orlando, FI, 1984, p 1, and, Gaffhey, B.J. in Spin Labeling Theory and Apphcahons, ed. Berliner, L.J Academic Press, New York, 1976, p. 183)

Recent research in spm labeling techniques has helped define both its advantages and its limitations. The latter are associated with the limited stability of free radicals (even nitroxide free radicals) in the systems studied and with the limitations of EPR spectroscopy itself.

Bioreduction is the most pervasive and bothersome aspect of the use of nitroxides m living biological systems, principally because of the possibility of differential rates of reduction in the vaπous parts of a complex system and/or the potential biological consequences of the process of reduction (Kocherginsky, N.; Swartz, H.M. Nitroxide Spin Labels: Reactions in Biology and Chemistry. CRC Press, Boca Raton, FI, 1995).

There are a number of approaches that can be used to minimize the effects of bioreduction. These include the development and use of the chemical structures, particularly nitroxide moieties that are more resistant to bioreduction. Also important are methods of facilitating the oxidation of the hydroxylamines back to nitroxides and methods of physically shielding the nitroxides from metabolism by incorporating them into liposomes, microspheres, or plastic tubes (Keana, J.F.W. U.S. Patent 4,863,717). Another important approach is to try to compensate for bioreduction by establishing a temporary or pseudo-equilibrium.

Another important limitation is the attainment of sufficient sensitivity so that the desired measurements can be made on a time scale that is appropriate to the biological process being investigated, without introducing biological or spectroscopic complications from excessive concentrations of the nitroxides. This is extremely important because the strong oxidative properties of nitroxide radical centers can cause irreversible modification of the system under examination. Sensitivity is a major problem in EPR studies of living animals or organs because of the decreased sensitivity of the lower frequency EPR spectrometers which are used for such studies (Halpern, H.J.; Bowman, M.K. in EPR Imaging and In Vivo EPR. Eaton, G.R.; Eaton, S.S.; Ohno, K., eds. CRC Press, Boca Raton, FI, 1991). It should be emphasized that EPR spectroscopy itself is not among the user- friendliest of methods. Difficulties in experimental technique and in the interpretation of data must be added to the problems of low sensitivity and the high cost of equipment.

There are three general approaches to trying to increase sensitivity: improve the instrumentation, optimize the paramagnetic properties of the nitroxides, and increase the specificity of the reporting by the nitroxide. Improvement of the instrumentation is an area in which there is currently great activity and progress (Pou, S.; Halpern, H.J.; Tsai, P.; Rosen, G.M. Ace. Chem. Res., 1999, 32, 155). An approach to enhancing the paramagnetic properties of the nitroxides has been to use isotopic substitutions to decrease the number of lines and to decrease the line widths (Venkataramu, S.D.; Pearson, D.E.; Beth; A.H. U.S. Patent 4,332,946).

Despite several advantages of spin labeling technique, the use of the EPR spectroscopy for monitoring reporter molecules is not an easy task because of its limited sensitivity, complexity and the high cost of equipment. Furthermore, the interpretation of EPR data is difficult. Fluorescent spectroscopy is an extremely sensitive method, uses relatively simple equipment, and enjoys a central role in a large number of biological probe methodologies (Lakowicz, J.R., ed. Topics in Fluorescence Spectroscopy. Plenum Press, New York, 1991). However, a need continues to exist for molecular tools that are capable of utilizing the high sensitivity of fluorescence measurements. A number of experimental techniques and a variety of specially designed fluorescent probes have been developed (Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals. 6th Ed., Molecular Probes, Inc. Eugene, OR, 1996) that do at least in part, provide for this.

Although fluorescent and nitroxide probes are often used together, the combination of both nitroxide and fluorescent moieties in one molecule yields a non- fluorescent compound. This is because of the quenching of the excited singlet state of aromatic fluorescent compounds by a free radical moiety (Atik, S.S.; Singer, L.A., J Am. Chem. Soc, 100:3234,1978). Thus, a compound having both a fluorophore and nitroxide radical in the same molecule in close proximity (7-8 A) does not have fluorescent properties, since the excited singlet state of fluorophore is fully quenched by the nitroxide group.

The hybrid nitroxide-fluorescent probe was introduced by the N. Blough group (Blough N.V.; Simpson, O ., JAm. Chem. Soc, 110: 1915, 1988; Green, S.A.; Simpson, D.J.; Zhou, G.; Ho, P.S.; Though, N.V., JAm. Chem. Soc, 112:7337, 1990; Gerlock, J.L.; Zacmandis, P.J.; Bauer, D.R.; Simpson, D.J.; Though, N.V.; Salmeen, I.T., Free Radical Res. Commun., 10:119, 1990; Kieber, D.; Though N.V., Free Radical Res. Commun., 10: 109, 1990). Early studies of the fluorescence quenching phenomena in hybrid molecules were performed by the G.I.; Likhtenstein group at the Institute of Chemical Physics in Chemogolovka, USSR Bystrayk, I.M.; Likhtenstein, G.I.; Kotehiikov, A.I.; Hideg, K., J Chem.Phys. (Moscow), 61:2769, 1986). In addition to its study of the fluorescent properties of hybrid labels, the N. Blough group performed a theoretical analysis of possible contributions of different quenching mechanisms, including energy transfer (both Forster and Dexter therms), electron transfer, and electron exchange (Green, S.A.; Simpson, D.J.; Zhou, G.; Ho, P.S.; Though, N.Y., JAm. Chem. Soc, 112:7337, 1990). It was concluded that the contributions of energy transfer mechanisms as well as electron transfer are negligible and that the quenching results from a relaxation process induced by electron exchange.

The first of the hybrid nitroxide-fluorescent probes studied by N. Blough were naphthoic acid esters with nitroxide alcohol (Scheme 4). Their low solubility in water limited their value for biological research. A more convenient type of hybrid probe, discovered later by the same group, was an adduct of nitroxide-amine with fluorescarnine (Kieber, D.; Though N.V., Anal. Chem., 62:2275, 5 1990). A similar probe was used by the G. Rosen group, who employed it for detection of the biologically important hydroxyl radical and superoxide in the course of their study of free-radical processes in vivo using confocal fluorescent microscopy (Pou, S.; Huang, Y.I.; Bhan, A.; Bhadfi, V.S.; Hosmane, R.S.; Wu, S.Y.; Cao, G.L; Rosen, G.M. Analyt. Biochem., 212:65,1993; Pou, S.; Wu, S.Y.; Lederer, W.I.; Rosen G.M. Int. Congr. Ser. - Experta Med, 1992,988 (Oxygen Radicals), 20 179.

Scheme 4

The first few examples of the uses of hybrid molecules show their potential as research tools. However, the variety of hybrid molecules used in the reported research is limited, both in terms of their fluorescent properties and nitroxide triggers.

SUMMARY OF THE INVENTION

The present invention in a general and overall sense, provides for molecular probes having improved paramagnetic properties and high sensitivity. These probes may be more particularly described as hybrid probes or dual fluorophore-nitroxide probes comprising a first nitroxide moiety (NR) capable of emitting fluorescence and at least one nitroxide free radical N-O^* These probes may be further described as having a highly sensitive fluorescence indicator.

Transmitting fluorescence and at least one nitroxide free radical (N-O)^*. These probes may be further described as having a highly sensitive fluorescence indicator.

In some embodiments, the probes of the invention may be further defined as having a structure:

wherein FI is a fluorescent moiety, NR is a nitroxide moiety, and N-O^* is a nitroxide free radical center. Several FI and or NR moieties may be present in a single molecule of the invention.

The present invention, in yet another aspect, provides a method for the synthesis and use of hybrid molecules by combining moieties capable of emitting fluorescence with nitroxide free radical centers. These hybrid molecules display a strong interaction between the free radical center and the fluorophore which effectively quenches fluorescence. On transformation of the nitroxide free radical center to a diamagnetic functional group, the hybrid molecule becomes fluorescent and can easily be detected in extremely low concentrations. Therefore, these hybrid molecules are chemical compounds possessing internal "triggers" which can provide a dramatic molecular response when acted on by specific agents (reductants, free radicals, etc.). The use of these unique molecular probes, among other uses, provide utility for use in biological research and medical diagnostics.

A variety of hybrid molecules are described herein together with the characterization of their fluorescent properties. Several types of fluorophores and nitroxide "triggers", with different reduction potentials, were employed.

Transformation of the nitroxide radical moiety in the hybrid probe into a diamagnetic state, either by reduction or reaction with an active, unstable free radical R, will trigger an intense fluorescence (Scheme 5) (Blough N.V.; Simpson, D.J., J Am. Chem. Soc, 110: 1915, 1988). This feature dramatically improves the sensitivity over monitoring the EPR signal. A conventional fluorimeter can be used to make measurements instead of a complex and expensive EPR spectrometer. Scheme 5

Fl NR N-OR

Radical (spin) adduct, highly fluorescent

F! NR N-O

Dual Probe, non-fluorescent

Reduction product, fluorescent

In some aspects, the present invention provides methods to synthesize several series of hybrid molecules. These hybrid molecules are also characterized for their fluorescent properties.

The invention, in yet another aspect, provides for hybrid compounds. In some embodiments, these hybrid molecules can be defined as a compound depicted in Scheme 6. The hybrid compounds are depicted in Scheme 6 in a general drawing consisting of the fluorophore and the nitroxide moieties, separated by a spacer. Scheme 6

Fluorophore (FI) is the part of the hybrid molecule that makes detection by a spectrometer possible. In some embodiments, fluorophores for the hybrid molecules of the presently described probes may be further described as having the following characteristics: 1. Compatibility with the presence of a nitroxide moiety. The fluorophore is substantially without strong acidic, oxidative or reductive properties; 2. Comprising functionalities suitable for incorporation into a hybrid molecule without loss of their fluorescent properties;

3. Comprising spectral properties (by way of example, quantum yield, excitation and emission regions), suitable for this particular application;

Most of the fluorescent dyes, used as conventional fluorescent probes, including polycyclic aromatic fragment, acridine, coumarine, dansyl, fluorescein, rhodamine, Texas

Red, Cascade Blue, Lucifer Yellow and their derivatives comply with these requirements and may be used for the dual probe's preparations.

The nitroxide part (NR) of the hybrid molecule is responsible for triggering the fluorescence on reduction or reaction with a free radical. Reduction of nitroxide free radicals into diamagnetic hydroxylamino derivatives, being the major metabolic pathway in vivo, has been studied by several research groups. It has been reported that the reduction potential of the nitroxide group is determined by its structural features such as size of the cycle (for cyclic nitroxides), the presence of additional heteroatoms in the cycle and substitution (Briggs, S.P., Haug, A.R., and Scheffer, R.P., Plant Physiol, 70:668, 1982; Shchukin, G. , and Grigoriev, A., Oxidation-reduction properties, in Imidazoline Nitroxides, Vol. 1, Synthesis and Properties, Volodarsky, L.B., Ed.,CRC Press, Boca Raton, FL, 1988,172).

Several nitroxide moieties were employed in the synthesis of dual agents. They represented both ends of the nitroxide radicals' oxidation scale. This would permit the preparation of an assortment of hybrid molecules, suitable for studying the oxidation processes, involving both strong and weak reductants. Another important structural feature of some nitroxide moieties is its ability to provide the density of unpaired electron (spin density) into the system. The extended studies by Nuclear Magnetic Resonance (NNIR) spectroscopy, EPR spectroscopy and quantum-chemical calculations show that the amount of spin density, contributed by nitroxide fragment through spin-polarization mechanism into the molecule, is determined by the structure. Structurally different nitroxide fragments could differ by the orders of magnitude on their spin density donor properties (Grigor'ev, A., and Dikanov, S.A., in Imidazoline Nitroxides, Vol. 1, Synthesis and Properties, Volodarsky, L.B., Ed., CRC Press, Boca Raton, FL, 1988). Since the fluorescence quenching is determined by electron exchange between fluorophore and nitroxide, the ability of nitroxide fragment to donate density of unpaired electron is important for the molecular design of dual probes.

The spacer (SP) is the part of the molecule that connects the fluorophore (FI) and the nitroxide moiety (NR). It should provide maximum interaction between the nitroxide and fluorophore moieties and may optionally contain additional functionalities for addressing a hybrid probe to a specific environment. The actual design of the spacer is determined by the desired properties of the dual agents, that in turn reflect the intended application of the agent. Structurally, spacer could consist of the following:

A. Functional group, derived from the connection reaction. This single point short connection spacer is supposed to provide maximum interaction between nitroxide and fluorophore fragment due to its short length. Spacer of this type facilitates electronic exchange by "through space" formation of a FI - NR collision complex.

B. Chemical bond, derived from incorporation of the fluorophore fragment directly to the nitroxide moiety (or vice versa). Several possibilities exist to introduce a fluorescent substituent into a free nitroxide radical or a nitroxide substituent into a fluorescent molecule. The resulting zero spacer could have one connection point (single bond), or two connection points (spiro-substitution or ring annelation). Depending upon the method of incorporation, FL and NR could be in "through space" collision proximity, or forming a conjugated system, where the exchange is derived from "through bond" electron tranfer.

C. Longer conjugated spacer, capable of ensuring ensure electron exchange between the nitroxide and the fluorophore through the system of conjugate chemical bonds. Such spacers may comprise consist of ethyle ic- or heteratom double bonds, or acytylenic bonds, or aromatic (heteroaromatic) disubstituted fragment. A spacer of this type works as a "through bond" exchange provider. D. Flexible spacers may comprise a confomationally mobile fragment, providing the interaction between nitroxide and fluorophore upon "through space" formation of the collision complex. Such a spacer could contain a methylene group, separating a short functional group spacer (as in A. above), or a longer hydrocarbon chain. Because of a longer space, fluorescence in the dual agents of this type is not fully quenched, but depends upon the imminent distance between the FI and NR moieties.

Several types of connections between the nitroxide and fluorophore moieties are within the scope of the present invention. The above type of spacers (A-D) were examined in this concept including: A. A single point short connection, by formation of an ester, an amide or a sulfamide bond.

Fl-COOH + R-OH - Fl-COOR

Fl-OH + R-COOH -» Fl-OOCR

F1-NH₂ + R-COX -» Fl-NHCOR F Fll--CCOOXX + R-NH₂ -» Fl-CONHR

Fl-SO₂X + R-NH₂ ^■» F1-S02NHR

To provide these connections, the hydroxy, or amino derivatives were treated with acylation or sulfonylation reagents. The hydroxy, or amino group to be used in this connection could be placed on either the fluorophore or the nitroxide moiety. It could also be a part of a heterocycle, amidine or guanidine system. Since nitroxide acids are more readily available than amines and their active derivatives have already been studied in connection with spin labeling (Hideg, K., and Hankovszky, O.H., in Organic Magnetic Resonance: SpinLabeling VIII; Berliner, L.J., Reuben, J., Ed., Plenum Press, NY, 1989, Chapter 9) and MRI contrast media synthesis (Volodarsky L.B., ed. Synthetic Chemistry of Nitroxide Radicals, CRC Press, Boca Raton, FI, 1995; Keana, J.F.W., Martin, V.V., Ralston, W.H., U.S. Patent No: 5,567,411, 1996), the nitroxide acids were the main key intermediates in the preparations described in the Examples presented here. B Incorporation of fluorophore moieties. Another type of connection provided in the present invention is the incorporation of the fluorophore moiety into heterocyclic systems, containing a nitroxide free radical center. Two ways of incorporation were examined: 1. Synthesis of nitronyl-nitroxide radicals with fluorophore in position 2 of the heterocycle ring (Osiecki, J.H., and Uliman, E.F., J Amer. Chem. Soc, 1078 (1968) (Scheme 7). Scheme 7

o From the point of view of interaction between the moieties, nitronyl nitroxides are of particular interest due to their strong delocalization of the spin density of the aromatic substituent in position 2 (Uliman, E.F., Call, L., and Osiecki, J.H., J Org. Chem., 35:3623,1980). Such a delocalization should provide strong "through bond" electron exchange between the moieties resulting in strong quenching of fluorescence in the radical 5 form.

2. Incorporation of the fluorescent moiety into immediate proximity of a radical center using nucleophilic addition to a nitrone group followed by oxidation of the intermediate hydroxylamine derivative (Scheme 8). Scheme 8 0

5 The above organometallic addition-oxidation sequence is one of the most general methods of nitroxide synthesis (Keana, J.F.W., Chem. Rev., 78, 37, 1978; Volodarsky L.B., ed. Synthetic Chemistry of Nitroxide Radicals, CRC Press, Boca Raton, FI, 1995). However, most of the reported examples were limited to aliphatic Grignard nucleophiles (Keana, J.F.W. in Spin Labeling in Pharmacology, ed. Holtzman, J.L. Academic Press, 0 Orlando, FI, 1984, p. 1) with only a few of the aromatic reagents involved (Martin, V.V., Volodarsky, L.B., and Vishnivetskaya, L. A., Izv. Sib. Otd. Akad. Nauk SSSR, Ser. Khirn. Nauk, 4:94, 198 1). The aromatic nucleophiles are of particular interest, because of their structural similarity with polycyclic aromatic fluorophores. C. Incorporation of the nitroxide moiety. The nitroxide radical center could be incorporated into the fluorophore molecule as a substituent. One method of such preparation is a reaction of organometallic derivative with nitroso-t-butane, followed by oxidation of intermediate N,N-disubstituted hydroxylamine.

A similar reaction was used for the preparation of several simple aromatic nitroxides (Thesing, J.; Mayer, H. Chem. Ber 1956, 89, 2159), and later was employed in singlet oxygen nitroxide probe (Keana, J.F.W.; Prabhu, V.S.; Ohmiya, S.; Klopfenstein, C.E.. J. Amer. Chem. Soc, 1985, 107, 5020); however the fluorescent properties of the resulting aromatic nitroxide has not been investigated. Another way of preparing a similar compound is the reaction of organometallic reagents with aromatic nitro and nitroso compounds (Keana, J.F.W., Chem. Rev., 78:37, 1978)

D. Conjugated systems as spacers. Two conjugated systems examined were: 1). Synthesis of ethylenic spacer, using C-H acidic properties of methylene- nitrone group.

2). Using aromatic p-phenylenediarnine fragment as a spacer.

D. Flexible spacers.

Earlier studies of the fluorescence quenching mechanism in hybrid molecules showed that quenching is the result of the electron exchange which occurs at short distances (7-10 A), between nitroxide and fluorescent moieties. Similarity in the mechanisms of fluorescence quenching in hybrid molecules with spin exchange in nitroxide diradicals (Parmon, V. N ., Kokorin, A. I., and Zhidomirov, Stable Diradicals, Nauka, Moscow, 1980) allowed the use of the present inventor's experience in diradical synthesis (Martin, V.V., and Keana, J.W.F., J. Chem. Soc, Chem. Commun. 723, 1995) to hybrid-nitroxide probes. Based on this analogy, electron exchange between nitroxide and fluorophore moieties were concluded by the present inventors to happen by two mechanisms: a) indirect exchange (through bond interaction) or b) direct exchange by collision of nitroxide and fluorophore. The direct mechanism requires formation of the collision complex with close nitroxide- fluorophore distance (7- 1 0 A), similar to that of the spin exchange in diradicals (Parmon, V. N., Kokorin, A.I. , and Zhidomirov, Stable Diradicals, Nauka, Moscow, 1980). In flexible hybrid molecules, quenching of the fluorescence will depend on the conformational mobility of the molecule. The difference between diradical and hybrid molecules is a limited lifetime of the latter in their excited state. The characteristic lifetime of conformation is usually longer than that of fluorescence. This means that the excited molecule will undergo photon emission before formation of the collision complex. However, it is reasonable to expect a Gibbs distribution of the possible probe conformers in accordance with their energy levels. Changes in the probe's microenvironment will affect the conformer's distribution through energy changes. When favorable conformations have closer nitroxide-fluorophore distances, the net result will be quenching and decrease in the fluorescent signal. These assumptions are in agreement with earlier studies of the hybrid molecules.

Hybrid probes based on conformation mobility are yet further embodiments of the present invention. They will provide information on changes in the microenvironment that could result in conformational mobility, such as temperature, microviscosity, fluidity, etc. Accommodation of the amide nitrogen, capable of protonation in the bridge, could provide information on the ionic strength and pH of the environment (Martin, V.V., and Keana,

J.W.F., J Chem. Soc, Chem. Commun. 723, 1995). These molecules would be true nitroxide fluorescent probes, because their working principle is based on reversible changes and does not require the destruction of the nitroxide part of the probe.

Several hybrid probes having a flexible bridge between nitroxide and fluorophore moieties have also been prepared by the present inventors. The diradical structural analogs having a second nitroxide moiety instead of fluorophores were also synthesized and examined. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Following long-standing patent law convention, the terms 'a" and "an" mean "one or more" when used in this application, including the claims.

Melting points were obtained in an Electrothermal apparatus and are uncorrected. All the chemicals, unless noted otherwise, were purchased from Aldrich Chemical Co. and used without additional purification. Ethanol-free chloroform was prepared by passing commercial chloroform through an alumina column. Analytical TLC was performed on a Merck silica gel 60 F₂₅ , plates; preparative TLC - on Analtech Uniplate silica gel plates

(20x20 cm, I mm), preparative column chromatography was performed on Aldrich silica gel, Davisil, grade 643 (200-425 mesh). Analytical HPLC analyses were performed on the Rainin Microsorb C18 4.6 x 250 mm column using acetonitrile - water (with 0.2 % TFA) gradients. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES 1 - 4

SYNTHESIS OF STARTING MATERIALS

Nitroxide structural units used as key intermediates (Scheme 9) in the preparations are readily available, stable, and non-toxic compounds. Carboxylic acid derivatives of the five- membered heterocycles, containing nitroxide group, namely - 3-carboxy-2,2,5,5-tetramethyl- 3-pyrroline-l-oxyl (1), 4-carboxy-2,2,5,5-tetramethyl-3-imidazoline-l-oxyl (2), 4-carboxy- 2,2,2, 5,5-tetramethyl-3-imidazoline-3 oxide-1-oxyl (3), and bicyclic nitroxide 4,4,6,6- tetramethyl-5H-imidazo[l,5-b]-isoxazole (4) were used as starting compounds in dual probe's synthesis. Several active derivatives, capable of forming the amide bonds upon the reaction with amines were prepared from these key intermediates 1-4. Another class of nitroxide starting materials used in these preparations are nitroxides an with amino group, namely 4- phenylamino-2,2,2,5-tetrainethyl-3-imidazoline-l-oxyle (5) and 4-amino-2,2,6,6- tetramethylpiperidine-1-oxyl (6). Amine 6 is commercially available and was purchased from Aldrich Chemical Co. Most of the preparations listed below are adaptations of the procedures, published elsewhere (Hideg, K; Hankovszky, H.O. In Spin Labeling: Theory and Applications, Vol.8. Berliner, L.J.; Reuben, J., Eds. Plenum Press, New York, 1989, Chapter 9. Volodarsky L.B., ed. Synthetic Chemistry ofNitrroxide Radicals, CRC Press, Boca Raton, FI, 1995, and references therein; also: In Imidazoline nitroxides. Synthesis and properties, V.l; Volodarsky, L.B., Ed., Boca Raton, Florida: CRC press, 1988), however, certain modifications were made to simplify and improve the procedures. Also the inventors found that when alternative methods of preparation were involved, the fluorescent properties of the dual probes were not well-reproducible. These results were probably caused by unidentified micro-impurities in starting materials. The methods of preparations, listed in Examples 1-5 below afforded the materials 1 - 5, which gave reproducible properties of the resulting dual probes. Scheme 9

EXAMPLE 1. PYROLINE DERIVATIVES

The carboxylic acid 1 was prepared in a four-step reaction sequence from commercial 4-oxo-2,2,6,6-tetramethyl piperidine (triacetoneamine) 6a (Scheme 10). Bromination of the triacetonamine yielded dibromide 7 that was converted to amide 8. Oxidation of this compound with peroxide gave a nitroxide amide 9. Basic hydrolysis of the amide 9 completed the synthesis of the acid 1. Two activated derivatives - N- hydroxysuccinimide ester 10 and mixed anhydride 11 were prepared from the parent carboxylic acid 1. These active derivatives 10, 11 are somewhat less stable in comparison with the parent carboxylic acid 1; however, they can be stored in a freezer for several weeks without decomposition. Scheme 10

10 11

3.5-Dibromo-2.2.6.6-tetramethyl-piperidone hydrobromide (7).

A 17.5 g (0.11 mol) of triacetoneamine 6a was placed into a 500 ml flask equipped with a 100 ml pressure-equalizing dropping funnel and magnetic stirrer. The flask was placed in an ice-water cooling bath and 70 ml of glacial acetic acid was added. At this point, 11.6 ml (0.225 mol) of bromine in 50 ml of glacial acetic acid was added dropwise upon vigorous stirring. After the bromine color disappeared, the stirring was stopped, and reaction mixture was left to stand for a day at room temperature. The precipitated crystals of dibromide 7 were collected by filtration, washed on the filter successively with acetic acid (3x5 ml), water (2x5 ml), ether (5x 10 ml) and dried on air to give 29.5 g (68%) of yellow solid, used in the next step without purification.

3-Carbamoyl-2.2.5.5-tetramethylpyrrolin (8).

A 29 g (73 mmol) of 3,5-dibromo-2,2,6,6-tetramethyl-4-oxopiperidine hydrobromide 7 was added slowly to a 25% aqueous ammonia (25 ml) under vigorous stirring and the resulting solution was saturated with solid K₂C0 (about 150 g) for 3 hr. The light crystalline precipitate of the amide was filtered off and dried in vacuo, yielding 12 g (97%). The crude product was contaminated with some inorganic impurities, however it was used in the next step without purification. The attempts to remove the inorganic impurities either by washing of crude material with water or by recrystallization leads to significant decrease of yield.

3-Carbamoyl-2.2.5-tetramethylpyrroline-l-oxyl (9).

A 2-L, three-necked flask equipped with a magnetic stirrer, thermometer and a reflux condenser was charged with 12 g (70 mmol) of 3-carbamoyl-2,2,5,5-tetramethylpyrroline 8, 140 ml of water, 6g of sodium tungstate, 6g of ethylenediaminetetraacetic acid (EDTA) disodium salt, and 25 ml (0.22 mol) of 30% hydrogen peroxide. The mixture was briefly heated to 60°C under stirring, then heating was removed. The mixture was left overnight to cool to 10°C. The precipitated crystals of the product 9 were collected by filtration, washed with water (3 x 5 ml) and dried under reduced pressure to give 10.9 g (85%) of the amide 9 as an orange solid.

3-Carboxy-2.2.5.5-tetramethyIpyrroline-l-oxyl (l .

A suspension of 2.7 g (15 mmol) of 3 -carbomoyl-2,2,5,5 -tetramethylpyrroline- 1- oxyl 9 in 100 ml of 10% sodium hydroxide was stirred under reflux. A clear solution was formed after 1 hr. The mixture was heated for additional 2hr (control byTLC: eluent - mixture chloroform - methyl alcohol 9:1), cooled to room temperature and acidified under stirring by the dropwise addition of 15% hydrochloric acid to pH = 2. The reaction mixture was stirred for 30 min and a precipitated product was filtered off, washed on a filter with water (3 x 10 ml), and dried in vacua, yielding 2.5 g of the acid 1 as a yellow solid. The combined filtrate was extracted with chloroform (3 x 20 mL), dried over magnesium sulfate, and evaporated to give additional 0.2 g of the acid 1. The combined yield of the acid 1 is about 2.7 g ( about 100%). N-Hydroxysuccinimide ester of 3-carboxy-2.2.5.5-tetramethylpyrroline-l-oxyl (10).

A 250 ml flask equipped with a magnetic stirring bar and 100 ml pressure-equalizing addition funnel, was placed into a cold water bath, and was charged under an argon atmosphere with 2.26 g (14 mmol) of 3-carboxy-2,2,5,5-tetramethylpyrroline-l-oxyl 1, 1.4 g (14 mmol) of N-hydroxysuccinimide, and 25 ml of ethyl acetate. A solution containing 2.53 g (14 mmol) of 1,3-dicyclohexylcarbodiimide in 35 ml of ethyl acetate was added slowly to the cooled and rapidly stirred reaction mixture over a 10 min. period. After the addition was completed, the reaction mixture was stirred for an additional 16 hr. at room temperature (TLC control: eluent -chloroform). The white precipitate of dicyclohexylurea were removed by filtration and washed with ethyl acetate (3x15 ml). The combined organic filtrate was evaporated, yielding 3.42 g (90%) of the active ester 10 as a yellow solid. Mixed anhydride 11. To a stirred suspension of the acid 1 (1,84 g, 10 mmol) in ether (50 mL), triethylamine (1.38 ml, 10 mmol) was added. The solution was cooled to 3 °C, and the solution of ethyl chloroformate (1.29 ml, 10 mmol) in ether (50 ml) was introduced dropwise over 30 min. The mixture was allowed to heat to room temperature, stirred for additional 1 h. The precipitated triethylamine hydrochloride was filtered off, washed with ether (5 x 20 mL) on a filter. Combined organic filtrate was evaporated and the residue was treated with cold petroleum ether (20 ml) and cooled to 0° C. The product was filtered off, washed with petroleum ether (2 x 5 ml) and dried on air to yield anhydride 11, 2.43 g (95%) as an orange- yellow solid.

EXAMPLE 2. PREPARATION OF IMIDAZOLINE CARBOXYLIC ACID (2)

Imidazoline carboxylic acid 2 was prepared according to methods known to those of ordinary skill in the art. Readily available hindered amine, 2,2,4,5, 5-pentamethyl-3- imidazoline-3-oxide 12 was used as a starting material. Compound 12 was oxidized to a nitroxide 13, which was brominated with N-bromsuccinimide to give a mixture of mono-14 and dibromo-15 derivatives. Monobromide 14 reacted with t-butyl amine in nucleophilic substitution followed by intramolecular oxidation resulting in the loss of N-oxide oxygen and the formation of imine 16. Imine 16 was coverted to amide 17 in a one-pot procedure using hydroxylamine-O-sulphonic acid to give initial nitrile that was subjected to peroxide- catalyzed basic hydrolysis in situ. Amide 17 yield the targeted acid 2 upon heating with equimolar amount of sodium hydroxide at 90°C. Free acid 2 was not very stable at room temperatures and was isolated as sodium salt, more convenient for handling.

Scheme 11

2 17 _{1 β}

2.2.4.5.5-Pentamethyl-3-imidazoline-3-oxide-l-oxyl (13).

In a 1-L three necked flask equipped with a magnetic stirrer, thermometer, and reflux condenser, 6 g of sodium tunstate, 6g of disodium EDTA, 100 ml of water, and 15.6 g (0.1 mol) of 2,2,4,5, 5-pentamethyl-3-imidazoline-3-oxide 12 were dissolved under stirring. To this, 150 ml of 30% hydrogen peroxide was added. The mixture was heated to 60°C for 2 hr. The heating was removed and the reaction mixture was cooled to room temperature, and was extracted with chloroform (5 x 150 ml). The extract was dried over magnesium sulfate, filtered, and evaporated, yielding 14.5 g (85%) of the radical 13 as yellow-orange solid.

Bromination of 2.2.4.5.5-pentametbyl-3-imidazoline-3-oxide-l-oxyI (14).

To the solution of 11.9 g of 2,2,4,5, 5-pentamethyl-3-imidazoline-3-oxide-l-oxyl 13 in 150 ml of carbon tetrachloride a dry of N-bromosuccinimide (11.9 g) was added in single portion, followed by 1 ml of trifluoroacetic acid (catalyst). The mixture was stirred for 3 hr at room temperature (control by TLC: eluent-chloroform), and precipitated succinimide was filtered off and washed on a filter with carbon tetrachloride (5x10 ml). The combined organic filtrate was evaporated and the residue was flash chromatographed on a silica gel column (4x50 cm); eluent: mixture hexane - chloroform = 1:1, yielding 4.6 g (26%) of monobromo derivative 14 and 3.4 g (15%) of dibromo derivative 15. 4-Tert-buthyliminomethyl-2.2.5.5-tetramethvI-3-imidazoline-l-oxyl (16). A 25 ml flask, equipped with a magnetic stirring bar was charged with 5.8 ml (55 mmol) of tert-buthylamine and 4 ml of water. A solid 4.6 g (18 mmol) of 4-bromomethyl-2,2,5,5- tetrainethyl-3-imidazoline-3-oxide-l-oxyl 14 was added in portions over 2 hr. at 40°C (bath temperature). After the addition was completed, stirring was continued for another 2 hr. at 40°. The mixture was cooled to room temperature, and 3 ml of water was added. The precipitated solid was filtered, washed with water (3x10 ml), and dried under reduced pressure, yielding 4.09 g (99%) of the imine 16 as an orange solid.

4-Carbamoyl-2.2.5.5-tetramethyl-3-imidazoline-l-oxyl (17V To a suspension of 4-tert-butyliminomethyl-2,2,5,5-tetramethyl-3-imidazoline-l-oxyl 16 (4.09 g, 18 mmol) in 35 ml of water 2.47 g (22 mmol) of hydroxylamino-O-sulfonic acid was added over 10 min. The mixture was stirred for another 10 min, then 36.5 ml of 6% hydrogen peroxide was added in single portion, followed by the dropwise addition of 2N sodium hydroxide (1.46 g, 36 mmol, in 20 ml of water). The mixture was stirred for 1 hr. and the precipitated product was filtered off, washed with water (3x15 ml), dried in vacuo. The combine aqueous filtrate was extracted with chloroform (3x20 ml). The extract was dried over magnesium sulfate, filtered, and evaporated. The residual solid was combined with initial precipitate to yield 2.2 g (66%) of the amide 17 as a red solid.

4-Carboxy-2.2.5.5-tetramethyl-3-imidazoline-l-oxyl (sodium salt) (2). A suspension of the 4-carbamoyl-2,2,5,5-tetramethyl-3-imidazoline-l-oxyl 17 (2.2 g,

12 mmol) in 12 ml of aqueous IN sodium hydroxide was heated under stirring to 90°C for 3 hr. At that time the mixture, which became a clear solution, was evaporated to dryness. The residual water was removed by co-evaporation with benzene (3x20 ml). To the solid residue a dry ester (20 ml) was added. The precipitated solid was filtered and dried in vacuo, yielding 2.4 g (96%) of a sodium salt 2 as a thin yellow powder.

EXAMPLE 3. SYNTHESIS OF 4-CARBOXY-3-IMIDAZOLINE-3-OXIDE (3)

Synthesis of 3-imidazoline-N-oxide carboxylic acid 3 was from available 1 -hydroxy - 3-imidazoline-3-oxide 18. The starting compound 18 was brominated with excess sodium hypobromite to give tribromide 19. Basic hydrolysis of tribromide 19 resulted in carboxylic acid 3 (Scheme 12). Scheme 12

4-Tribromomethyl-2.2.5.5-tetramethyl-3-imidazoline-3-oxide-l-oxyl (19

A solution of potassium hypobromite was prepared by addition of bromine (8.4 ml, 0.16 mol) to the solution of potassium hydroxide (24.6 g, 0.44 mol) in water (60 ml) at -10°C. To this solution, a starting l-hydroxy-2,2,4,5,5-pentamethyl-3-imidazolin-3-oxide 18 (6.8 g, 0.04 mol) in water (60 ml) was added dropwise within 10 min at -5°C. The mixture was kept for 30 min at -5°C under stirring. The precipitated product was filtered off, washed with water, and dried in air, yielding 15 g (92%) of the tribromide 19 as an orange solid.

4-Carboxy-2.2.5.5-tetramethyl-3-imidazoline-3-oxide-l-oxyl (3). To a stirred solution of potassium hydroxide (8.2 g, 0.15 mol) in ethanol (50 ml) and water (10 ml) a solid 4-tribromomethyl-2,2,5,5-tetramethyl-3-imidazoline-3-oxide-l-oxyl 19 (15 g, 0.036 mol) was added in portions over 1 h. The resulting mixture was stirred at room temperature for 1 h and extracted with chloroform (2x20 ml). The extract was discarded. The aqueous layer was acidified with 2N hydrochloric acid to pH 2 under stirring, and was extracted with chloroform (4x20 ml). The extract was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give 4.05 g (55%) of the acid 3 as a yellow solid.

EXAMPLE 4. SYNTHESIS OF BICYCLIC ACID 4

Bicyclic carboxylic acid 4 was synthesized via cycloaddition of aldonitrone radical 21 with methyl acrylate followed by the hydrolysis of methyl ester 22 (Scheme 13). Key to this scheme aldonitrone-radical 21 was prepared by oxidation of the l-hydroxy-3-imidazoline-3- oxide 20. Aldonitron 21 is not a stable compound and was used immediately upon isolation (Volodarsky, L.B.; Martin, V.V.; Leluch, T.F. Tetrahedron Lett., 1985, 26(39), 4801).

Scheme 13

2.2.5.5-Tetramethyl-3-imidazoline-3-oxide-l-oxyl (21).

To a stirred solution of 3.0 g (19 mmol) of l-hydroxy-2,2,5,5-tetramethyl-3- imidazoline-3-oxide 20 in 50 ml of chloroform, 3 g of manganese dioxide were added in single portion. The mixture was stirred for 1 hr ( control by TLC: eluent-chloroform). The inorganic material was removed by filtration and washed on a filter with chloroform (10 x 5 ml). The combined organic filtrate was evaporated and the residue was flash- chromatographed on a silica gel column (4x10 cm) using chloroform as the eluent. Yield of the yellow-orange aldonitron 21 was 2.7 g (90%>). This compound was immediately used in the next step.

2-Metoxycarbonyl-4.4.6.6-tetramethyl-5-oxyl-2.3.4.6.7.8-hexahydro-5H-imidazo[15-b]- isoxazole (22).

To a solution of 2.7 g (17 mmol) of 2,2,5, 5-tetramethyl-3-imidazoline-3-oxide-l-oxyl 21 in 30 ml of chloroform was added 2.3 ml (26 mmol) of methylacrilate. The reaction mixture was left to stand for 16 hr at room temperature (control by TLC: eluent - chloroform). The reaction mixture was evaporated to dryness and cold hexane (20 ml) was added to the solid residue. The precipitated solid product was filtered, washed with hexane

(2x10 ml) to give 3.48 g (83%) of the methyl ester 22 as an orange solid.

2-Carboxy-4.4.6.6-tetramethyl-5-oxyl-2.3.4.6.7.8-hexahydro-5H-imidazo[1.5-b]- isoxazole (4 .

A mixture of 2-mehoxycarbonyl isoxazole 22 (3.48 g, 14 mmol) in methyl alcohol (40 ml) and IN aqueous sodium hydroxide (20 ml) was stirred for 60 min. at room temperature

(control by TLC: eluent chloroform). To the mixture 50 ml of water was added, and the reaction mixture was extracted with chloroform (3x10 ml). The extract was discarded. The mixture was slowly acidified with 2N hydrochloric to pH 2 and extracted with chloroform

(3x15 ml). The extract was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The solid residue was treated with hexane (10 mL) and filtered to give 2.65 g, (82%ι) of the acid 4 as bright yellow crystals.

EXAMPLE 5. SYNTHESIS OF AMINO DERIVATIVE 5.

The preparation of amidine-nitroxide 5 was also based on 1,3 dipolar cycloaddition of 2,2,5,5tetramethyl-3-imidazoline-3-oxide-l-oxyl 21, similar to what was used in Example 4. Cycloaddition of aldonitrone-radical 21 to phenylisocyanate gave oxadiazolidine 23. This cycloadduct was transformed to amidine 5 by the reaction with MeONa in aqueous alcohol (Scheme 14).

Scheme 14

21 2»

3-Phenyl-4.4.6.6-tetramethyl-5-oxyl-2-oxoperhydroimidazo[1.5-b1-oxadiazoles-1.2.4 (23).

A 2.27 g (14.5 mmol) of freshly prepared 2,2,5, 5-tetramethyl-3-imidazoline-3-oxide-l-oxyl 21 were dissolved in 5 ml of ethanol-free chloroform, and 1.73 ml (15.9 mmol) of phenylisocyanate were added. The reaction mixture was kept for 16 hr. at room temperature (control by TLC: eluent - chloroform) and evaporated to dryness. The residue was washed with hexanes (5x10 ml) and hexanes washings containing the excess phenylisocyanate were discarded. The solid residue was dissolved in chloroform (10 ml), loaded on a silica gel column (2.5x20 cm), and flash chromatographed in chloroform to yield 3.90 g (95%) of the cycloadduct 23 as an orange solid.

4-Phenylamino-2.2.5.5-tetramethyl-3-imidazoUne-l -oxyl (5).

A 50 ml flask was charged with 3.99 g (14.5 mmol) of 3-phenyl-4,4,6,6-tetramethyl- 5-oxyl-2-oxoperhydroimidazo[l,5-b]-oxodiazoles-l,2,4 23, 25 ml of methanol, and 5.4 ml (29 mmol) of 30% sodium methoxide. The reaction mixture was kept for 16 hr at room temperature and evaporated to dryness. Water (15 ml) was added to the solid residue and the resulting mixture was extracted with chloroform (3x15 ml). The chloroform was extracted with 2% hydrochloric acid (3x15 ml). The acidic solution was neutralized with solid sodium carbonate to pH 8 and extracted with chloroform (3x15 ml). The chloroform extract was dried over magnesium sulfate, filtered, evaporated and the residue was flash chromatographed over silica gel column (2.5x20 cm) using chloroform as eluent, yielding 1.35 g (40%) of the amidine 5 as an orange solid.

EXAMPLES 6-10. SYNTHESIS OF (AMINOMETHYL)PYRENE DERIVATIVES

Attachment of the nitroxide free radical moiety to the fluorescent moiety (methylenepyrene residue) was made in the examples below by means of acylation reactions of 1 -aminomethylpyrene, involving activated derivatives of the carboxylic acids 1-4 (Scheme 15). For the pyrroline acid 1 both N-hydroxysuccinimide ester 10, and mixed anhydride 11, were successfully employed. For heterocychc acids 2-4 correspondent hydroxysuccinimide esters 24b-d were found to be unstable and were not isolated in pure form.. However they were used without isolation and purification, in a way that was used previously for synthesis of nitroxide molecular amplifiers (Keana, J. F. W.; Martin, V. V.; Ralston, W. H. U.S. Patent 5,567,411).

The resulting dual probes 25 a-d have a spacer including methylene group. Their fluorescent properties depend upon the conditions, therefore they could be considered a dual agents with flexible spacers.

Scheme 15

EXAMPLE 6.

3-N-d'-Pyrenemethyl) aminocarbonyl-2.2.5.5-tetramethylpyrroline-l -oxyl (25a).

A 50 ml flask equipped with a magnetic stirring bar was charged with 0.4 g (1.4 mmol) of N-hydroxysuccinimide ester 10, 0.38 g (1.4 mmol) of 1-pyrenemethylamine hydrochloride, 5 ml of N,N-dimethylformamide, and 0.3 ml (2.2 mmol) of triethylamine, and. The reaction mixture was stirred for 16 hr at room temperature under argon (control by TLC: eluent - chloroform). The solvent was removed on a rotary evaporator. The resulting solid was treated with water (10 mL), filtered, washed with water (2x15 ml), and dried in vacuo. The crude product was purified by flash chromatography on a silica gel column (3.5x10 cm), eluent - chloroform to yield 0.48 g (84%) of compound 25a as yellow crystals: mp. 184.9 - 185.9°C (from hexane:ethylacetate = 1 :1). Anal. Calculated for C₂₆H₂₅,N₂0₂: C.78.55; H 6.34; N 7.05. Found: C 78.53; H 6.45; N 7.12. The same compound 25a was made with mixed anhydride 11 as an acylating reagent in similar conditions.

EXAMPLE 7.

4-N-( -Pyrenemethyl)aminocarbonyl-2.2.5.5-tetramethyl-3-imidazoline-l-oxyl (25b).

0.4g (1.9 mmol) of sodium salt of 4-carboxy-2,2,5,5-tetramethyl-3-imidazoline- 1- oxyl 2 was dissolved in 5 ml of water, acidified with 2N hydrochloric acid to pH 2, and extracted with chloroform (2x 10 ml). The extract was dried over magnesium sulfate, filtered, and evaporated to give a free carboxylic acid 2. A 50 ml flask containing a magnetic stirring bar was charged with acid 2, 0.23 g (2 mmol) of hydroxysuccinimide, and 15 ml of ethylacetate. A solution of 1,3-dicyclohexylcarbodiimide (0.41 g, 2 mmol) in 15 ml of ethyl acetate was added dropwise at 0°C for 10 min. upon stirring under argon. The mixture was allowed to warm to room temperature, and stirred for an additional 4 hr. To the resulting mixture 1-pyrenemethylamine hydrochloride (0.53 g , 2 mmol) was added followed by dropwise addition of triethylamine (0.4 ml, 3 mmol). The reaction mixture was stirred for 16 hr (control by TLC: eluent - chloroform). The mixture was filtered from precipitated 1,3- decyclohexylurea, which was washed with ethylacetate (5x10 ml). The combined filtrate was washed successively with water (20 ml), 2N hydrochloric acid (20 ml), water (20 ml), saturated sodium bicarbonate (20 ml), water (20 ml), and dried over magnesium sulfate, filtered, and evaporated. The residue was flash chromatographed over silica gel column

(2x10 cm), eluent - chloroform to yield 0.22 g (29%) of the compound 25b as yellow solid:, mp. 154.3 - 155.3°C (from hexane: ethylacetate = 2:1). Anal. Calculated for C₂₅H₂₄N₃0₂: C75.34; H 6.07; N 10.55. Found: C 75.31; H 6.06; N 10.36.

EXAMPLE 8.

4-N-( -Pyrenemethyl)aminocarbonyl-2.2.5.5-tetramethyl-3-imidazoline-3-oxide-l-

oxyl (25c).

A 50 ml flask was charged with 0.4 g (2 mmol) of 4-carboxy-2,2,5,5-tetramethyl-3— imidazoline-3-oxide- 1 -oxyl 3, 0.24 g (2 mmol) of hydroxy succinimide, and 15 ml of ethyl acetate. A solution of 1,3-decyclohexylcarbodiimide (0.42 g, 2 mmol) in 5 ml of ethyl acetate was added dropwise at 0°C upon stirring under argon . The reaction mixture was allowed to warm to room temperature, and stirred for an additional 3 hr. To the resulting mixture a solid 0.55 g (2 mmol) of 1-pyrenemethylamine hydrochloride was added, followed by dropwise addition of 0.42 ml (3 mmol) of triethylamine. The mixture was stirred for 16 hr (control by TLC: eluent - chloroform). The mixture was filtered from 1,3-decyclohexylurea, which was washed with ethyl acetate (5x10 ml). The combine filtrate was washed successively with water (25 ml), 2N hydrochloric acid (25 ml), water (25 ml), saturated sodium bicarbonate (25 ml), water (25 ml), dried over magnesium sulfate, filtered, evaporated. The residue was flash-chromatographed over silica gel column (3.5x10 cm), eluent - chloroform, yielding 0.36 g (44%) of the compound 25c as yellow crystals: mp. 156- 157°C (from hexanes:ethyl acetate = 1:1). Anal. Calculated for C₂₅H₂₄N₃0₃: C,72.43; H 5.84; N 10.14. Found: C 72.12; H 5.71; N 10.36.

EXAMPLE 9

2-N-( -PyrenemethyI)aminocarbonyl-4.4.6.6-tetramethyl-5-oxyl-2.3.4.6.7.8-hexahydro- 5H-imidazo [1.5-bl-isoxazole (25d).

A 50 ml flask was charged with 0.4 g (1.7 mmol) of 2-carboxy-4,4,6,6-tetramethyl-5- oxyl-2,3,4,6,7,8-hexahydro-5H-imidazo[l,5-b]isoxazole 4, 0.2 g (1.8 mmol) of hydroxy succinimide, and 15 ml of ethyl acetate. A solution of 1,3-decyclohexylcarbodiimide (0.37 g,

1.8 mmol) in 5 ml of ethylacetate was added dropwise at 0°C. The reaction mixture was allowed to warm to room temperature, and stirred for an additional 4 hr. To the resulting solution, a solid 1-pyrenemethylamine (0.48 g, 1.8 mmol) was introduced, followed by dropwise addition of 0.36 ml (2.6 mmol) of triethylamine. The mixture was stirred for 16 hr (control by by TLC: eluent - chloroform). The mixture was filtered from 1,3- decyclohexylurea, which was washed with ethyl acetate (5x10 ml). The combined organic filtrate was evaporated and the residue was purified by column chromatography on silica gel (2.5x10 cm), eluent - chloroform to give 0.4 g (55%) of the compound 25c: mp 174.2- 174.4°C (from hexanes: ethyl acetate = 1:2). Anal. Calculated for C₂₇H₂₈N₃O₃; C,73.27; H 6.38; N 9.50. Found: C 73.34; H 6.45; N 9.40.

EXAMPLES 10-11. SYNTHESIS OF DUAL AGENT WITH A SHORT SPACER

The examples listed below represent the synthesis of fluorophore-nitroxide agent 32 with a short amide spacer. To achieve this synthesis, the acid 1 was converted to a strong acylation agent, acyl chloride 31. Compound 31 was not isolated, but was used immediately without isolation and purification. The high reactivity of acyl chloride 32 allowed to form the amide bond with a weak nucleophile, 1-aminopyrene (Scheme 16). Same dual probe 32 was prepared also by employing mixed anhydride 11, whereas attempts to use weaker acylation agent, N-hydroxysuccinimide ester 10 failed.

Scheme 16

11

EXAMPLE 9. l-(2'.2'.5^,.5'-TetramethyI-r-oxyl-pyrroline-3'-carbonyl)-aminopyrene (32). (Method A)

To a stirred solution of carboxylic acid 1 (0.18 g, 1 mmol) in 2 ml of dichloromethane and 0.2 ml (2.5 mmol) of pyridine, a freshly distilled thionyl chloride (0.09 ml 1.2 mmol) was added. The reaction mixture was stirred for 30 min at room temperature under argon, then a mixture of 220 mg (1 mmol) of 1-aminopyrene in 2 ml of N,N-dimethylformamide and 0.28 ml (2 mmol) of triethylamine was introduced dropwise. The reaction mixture was stirred for additional for 60 min (control by TLC: eluent - chloroform:methanol = 9:1). The mixture was evaporated and the residue was purified by flash-chromatography over silica gel column (2x20 cm), eluent - chloroform to give 0.18 g (47%) of the compound 32 as a yellow solid.

EXAMPLE 9. l-(2'.2'.5'.5'-Tetramethyl-r-oxyl-pyrroline-3'-carbonyl)-aminopyrene (32). (Method B) To a stirred solution of carboxylic acid 1 in 2 ml of 1,2-dimethoxyethane and 0.19 ml (1.4 mmol) of triethylamine was added. 0.11 ml (1.2 mmol) of ethylchloro formate at -5°C under argon. The reaction mixture was stirred for 1 hr, then 0.22 g (1 mmol) of 1-aminopyrene in 1 ml of N,N-dimethylformamide and 0.19 ml (1.4 mmol) of triethylamine was introduced dropwise. The resulting mixture was allowed to heat to room temperature and stirred for additional 24 h (control by TLC: eluent - chloroforrmmethanol = 9:1). Water (10 ml) was added and the resulting mixture was extracted with ethyl acetate (5x 10 ml). The organic solution was washed with saturated sodium bicarbonate (15 ml) and saturated sodium chloride (15 ml), dried over a mixture of magnesium sulfate :potassium carbonate = 5:1, filtered, and evaporated. The residual crude product was purified by prep. TLC, eluent - chloroform, yielding 0. 1 6 g (42%) of the compound 32, identical to that prepared in the above Example 9.

EXAMPLES 11-12. SYNTHESIS OF DUAL AGENTS WITH FLEXIBLE SPACERS

The examples 11,12 below represent the synthesis of nitroxide-fluorophore dual probes 42a,b having longer hydrocarbon spacers. These compounds were prepared in similar way by acylation of the terminal amino group of monosubstituted diamines 41a,b. The key intermediates 41a,b were synthesized from parent diamines 38a,b in the reaction sequences that include protection, introduction of fluorescent unit (dansyl) followed by deprotection (Scheme 17).

Scheme 17

amtno proup

a. n=2; R = CBz. b. n=3; R = BOC.

EXAMPLE 11. N-Dansyl-N'-(3-carbonyl-2.2.5.5-tetramethyl-l-oxyi-pyrroline)- ethylenediamine (42a).

l-Benzyloxycarbonyl-2-dansyl-ethylenediamine (40a). A 25 ml flask equipped with a magnetic stirring bar and an argon inlet was charged with 0.14 g (0.75 mmol) of benzyloxycarbonyl-ethylenediamine 39a, 0. 13 g (0.5 mmol) of dansyl chloride, and 5 ml of pyridine. The reaction mixture was stirred at room temperature for 3 hr (control by TLC: eluent - chloroform : methyl alcohol 15:1) and evaporated to dryness. Chloroform (10 ml) was added to the residue, and the white solid was filtered off and washed on a filter with chloroform (2x5 ml). The combined organic filtrate was concentrated, and the residue was purified by flash chromatography over silica gel column (1.5x20 cm), eluent chloroform, yielding 0.13 g (61%>) of the compound 40a as off-white solid.

Dansyl-ethylenediamine (41a). A 300 ml bench-top high-pressure reactor was charged with 1.17 g (3 mmol) of 1 - dansyl-2-bezyloxycarbonyl-ethylenediamine 40a, 100 ml of dry methanol, and 60 mg Pd/C (catalyst). The mixture was hydrogeneted for 2 hr at 30 psi hydrogen pressure, for additional 2 hr at 40 psi, and 68 h at 50 psi. The pressure was released, and the catalyst was removed by filtration through Celite-aided filter washed with methanol (3x10 ml). The combined filtrate was evaporated and crude conpound 40a was dried under reduced pressure and used without further purification.

N-Dansyl-N'-(3-carbonyl-2.2.5.5-tetramethyI-l-oxyl-pyrroline)-ethylenediamine (42a).

A mixture of 0.4 g (1.4 mmol) of dansyl-ethylenediamine 41a and 0.38 g (1.4 mmol) of N-hydroxy-succinimide ester 10 in 5 ml of N,N-dimethylformamide was stirred under argon for 16 hr (control by TLC: eluent - chloroform). The mixture was evaporated, and the residue was flash-chromatographed over silica gel column (3.5x10 cm) using chloroform as eluent. The fractions containing the desired substance were combined and evaporated, yielding 0.26 g (41%) of the compound 42a as yellow solid: mp 167-168 C (from hexanes:ethyl acetate = 1:5). Anal. Calculated for C₂₃H₃ιN₄0₄S: C,60.10; H 6.80; N 12.20. Found: C 60.01; H 6.85; N 12.05. EXAMPLE 12. N-Dansyl-N^,-(3'-carbonyl-2'.2^,.5^,.5^,-tetramethyl-l'-oxyl- pyrroline)propane-l) diamine (42b).

l-Dansyl-3-tertbutyloxy-carbonylpropane-1.3-diamine (40b). To a stirred solution of 0.38 g (2.2 mmol) of tert-butyloxycarbonylpropane-1,3- diamine 39b in 2 ml of pyridine a 0.58 g (2 mmol) of dansyl chloride was added portionwise for 20 min under argon upon stirring. The reaction was completed (control by TLC: eluent - chloroform:methanol = 10:1) after stirring for additional 15 min. The mixture was evaporated, and the residue was flash chromatographed over silica gel column (2.5x20 cm) using chloroform as eluent to yield 0.55 g (73%) of the compound 40b as an off-white solid.

Dansyl-propane-1.3-diamine (41b).

A solution of 0.25 g (0.7 mmol) of l-dansyl-3-tert-butyloxicarbonylpropane-l,3- diamine 40b in 5 ml of dichloromethane was acidified with 0.77 ml (10 mmol) of trifluoroacetic acid. The progress of the reaction was followed by TLC (eluent - chloroform). The reaction was completed after stirring for 30 min. The reaction mixture was evaporated, and the crude amino compound 41b was used in the next step without additional purification.

N-Dansyl-N'-(3'-carbonyl-2'.2'.5'.5'-tetramethyl- -oxyi-pyrroline)propane-l) diamine (42h).

To a stirred solution of N-dansyl-propane- 1,3 -diamine 41b (0.18 g, 0.6 mmol) in 2 ml of N,N-dimethlyformamide and 0.25 ml (2 mmol) of triethylamine a solid N-hydroxysuccinimide ester 10 (0.18 g, 0.6 mmol) was added portionwise over 10 min. The reaction mixture was stirred for 16 hr (control by TLC: eluent - chloroform). The mixture was evaporated to dryness and 10 ml of chloroform was added to the residue. The mixture was washed with water (2 x 5 ml), dried over magnesium sulfate, filtered, evaporated, and flash- chromatographed over silica gel column (2.5x20 cm) using chloroform as eluent to yield 0.18 g (57%o) of the compound 42b as yellow solid: mp 140. -142°C (hexanes: ethyl acetate = 1:1). Analysis: Calculated for C₂ H₃₃N₄O₄S: C 60.86; H 7.03; N 11.84; S 6.76. Found: C 59.84; H 7.24; N 11.50; S 6.50. i EXAMPLE 13, 14. SYNTHESIS OF DUAL AGENTS WITH CONJUGATED ETYLENIC SPACERS

The examples 13 and 14 represent the synthesis of dual agents where the exchange between NR and FI moieties happen through the system of conjugated ethyl enic bonds. To ensure a high degree of exchange interaction a 3 -imidazoline-3 -oxide- 1 -oxide nitroxide moiety that provides the highest degree of spin density delocalization was employed in these preparations. Condensation of l-hydroxy-2,2,4,5,5-pentamethyl-3-imidazoline-3-oxide 18 with 9-antracenyl aldehyde in basic conditions yielded diamagnetic condensation product 63. This compound was oxidized in mild conditions into nitroxide radical 64. The compound 64 contains a rigid trans-ethylenic bridge between nitroxide and fluorescent moieties. In the following Example 14 this compound, having a nitrone group, was desoxygenated to get dual agent 66 with an imino group instead. Heating compound 64 with sodium dithionite resulted in simultaneous loss of N-oxide oxygen atom and reduction of the nitroxide center to give 3- imidazoline derivative 65. The regeneration of the radical center was achieved by mild oxidation into targeted compound 66 (Scheme 18). Scheme 18

Transl-⁽9-antracenyl⁾-2-⁽l"-hvdroxy-2".2".5".5"-tetramethvI-3"-imidazoline-3"-oxide- 4"-yl)ethylene (63).

A mixture of l-hydroxy-2,2,4,5,5-pentamethyl-3-imidazoline-3-oxide 18 (0.86 g, 5 mmol), 9-antracenyl aldehyde (0.52 g, 2.5 mmol) and NaOH (0.22 g, 5.5 mmol) in 5 ml of methanol was refluxed under stirring for 16 hr., then cooled to room temperature and poured into water (100 ml). The crude precipitate was filtered, washed with water (5x40 ml) and dried on air. The crude product was dissolved in ether - hexane (1 :3) mixture, loaded on silica gel column (2x15 cm) and chromatographed using ether: hexane mixture (1:3) as eluent to give 0.39 g (43%) of compound 63.

Trans-l-(9'-antracenyl)-2-(I"-oxyi-2".2".5".5"-tetramethyl-3"-imidazoline-3"-oxide-4"- yPethylene (64).

To a stirred solution of trans-l-(9'-antracenyl)-2-(l"-hydroxy-2",2",5",5"-tetramethyl-3"- imidazoline-3"-oxide-4"-yl)ethylene 63 (0.390 g , 1.1 mmol) in 20 ml of CHC1_3, 1 g of manganese dioxide was added. The mixture was stirred for 10 min, filtered from inorganic material, which was washed on a filter with chloroform (10 x 20 mL). Combined organic filtrate was evaporated and the residue was chromatographed over silica gel column (2x20 cm), eluent - chloroform to give 0.34 g (92%) of the compound 64.

EXAMPLE 14.

Trans-l-(9'-antracenyl)-2-(l"-oxy-2".2".5".5"-tetramethyl-3" - imidazol ine-4"- yPethylene (66).

A solution of 100 mg (0.3 mmol) of trans-l-(9'-antracenyl)-2-(l"-oxy-2",2",5",5"- tetramethyl-3"-imidazoline-3"-oxide-4"-yl)ethylene 64 in 1 ml of ethanol was added to 0.5 ml of sodium dithionite (50 mg, 0.3 mmol) in water. The mixture was refluxed for 21 hr and evaporated to dryness. The residue of the crude compound 65 was dissolved in 20 ml of chloroform and 0.2 g of manganese dioxide was added. The mixture was stirred for 40 min and the inorganic material was filtered off and washed on a filter with chloroform (10 x 5 ml). Combined organic extract was evaporated and the residue was purified by prep. TLC (eluent - chloroform) to yield 40 mg (42%) of the compound 66 as an orange solid.

EXAMPLE 15. SYNTHESIS OF DUAL AGENTS HAVING CONJUGATED PHENYLENEDIAMINE SPACER.

Example 15 below represents the synthesis of the hybrid fluorophore-nitroxide molecule 44 with a conjugated p-phenylenediamine spacer. The key step of this preparation is the acylation of phenylendiamine with acyl chloride 27, prepared in situ from 3- imidazoline-3-oxide carboxylic acid 3. An excess of p-phenylenediamide was employed to avoid possible diacylation. Treatment of the intermediate amine 43 with fluorescamine led to the hybrid molecule 44 (Scheme 19). Scheme 19

(2'.2'5'.5'-Tetramethyl-r-oxyl-3'-imidazoline-3'-oxide-4'-carbonyl)-phenylenediamine- 1,4 (43).

To a stirred solution of 4-carboxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide-l-oxyl 3 (0.2 g, 1 mmol) in 2 ml of dichloromethane and 0.2 ml (2.5 mmol) of pyridine was added

0.09 ml (1.2 mmol) of thionyl chloride at -20°C. The reaction mixture was stirred for 90 min at the same temperature and 130 mg (1.2 mmol) of p-phenylenediamine in 2 ml of N,N- dimethylformamide and 0.15 ml (2 mmol) of triethylamine was introduced. The resulting reaction mixture was stirred for 90 min at -20°C under argon and allowed to warm to room temperature for 2 h (control by TLC: eluent - chloroform:methanol 9:1). The mixture was evaporated to dryness and the residue was purified by flash-chromatography over silica gel column (2x20 cm), eluent - chloroform to give 0.02g (7%) of the aniline 43 as a brown solid.

The reaction of (2'.2'.5'.5'-tetramethyI- -oxyl-3'-imidazoline-3'-oxide-4'-carbonyl)- phenlylenediamine-1.4 (43) with fluorescamine. To a stirred solution of 20 mg (0.07 mmol) of compound 43 in 1 ml of acetonitrile was added 20 mg of fluorescamine. The reaction was completed after stirring for 20 min (control by TLC: eluent - chloroform:methanol 9:1). The mixture was evaporated to dryness and the solid residue was treated with water (2 ml). The solid material was filtered off, washed with water (2 x 1 ml) and dried in vacuo to yield 27 mg (aboutl00%>) of hybrid probe 44 as a yellow solid. EXAMPLE 16. SYNTHESIS OF THE DUAL AGENT (45) FROM NITROXIDE AMIDINE (5)

The Example 16 below represents the synthesis of dual probe 45 from nitroxide amidine 5. The starting material 5 was treated with dansyl chloride in pyridine in the presence of triethylamine. The resulting dual agent has a relatively short sulfamide spacer between the nitroxide moiety and the fluorophore (Scheme 20). Scheme 20

4-(N-DansyLN-phenyl)amino-2.2.5.5-tetramethyI-3-imidazoline-l-oxyI (45).

To the stirred solution of 4-phenyl-2,2,5,5-tetramethyl-3-imidazoline-l-oxyl 5 (80 mg, 0.3 mmol) in 1 ml of pyridine and 0.14 ml (1 mmol) of triethylamine, a solid dansyl chloride (110 mg, 0.4 mmol) was added in small portions. The reaction mixture was stirred for 100 h at room temperature under argon (control by TLC: eluent - benzene: ether = 9:1).

The mixture was evaporated under reduced pressure and the residue was purified by prep.

TLC, eluent - benzene:ethyl ester = 9:1 to give 70 mg (44%) of the compound 45 as a yellow solid: mp 189-191°C (from hexanes: ethyl acetatekl).

EXAMPLES 17-19. SYNTHESES OF NITRONYL NITROXIDE AND IMINO NITROXIDE DUAL AGENTS

The Examples 17-19 below represent the series of dual molecules were polycyclic aromatic fluorophores are incorporated into a conjugated system of nitronyl nitroxide 53a-d, or imino nitroxide 54a-c compounds. Compounds of both nitronyl nitroxide and imino nitroxide series were prepared from commercially available aromatic aldehydes and bis- hydroxylamine 51. The starting bis-hydroxylamine 51 was obtained by reduction of bis- nitroso compound, 2,3-dimethyl-2,3-dinitrobutane 50. This intermediate in turn was prepared from commercial 2-nitropropane 49 by treatment with bromine. Condensation of bis- hydroxylamine 51 with aldehydes givesl,2-dihydroxy-2-R-4,4,5,5-tetramethylimidazolidines 52a-d. These compounds were immediately oxidized without isolation and purification by air in the presence of catalyst. The transformation of the dihydroxyamino intermediate 52 proceeded in two competitive pathways: oxidation of both hydroxyamino groups results in the formation of nitronyl nitroxide series, 2-imidazolin-l-oxyl-3-oxides 53a-d. Alternative initial dehydration of one hydroxy group, followed by oxidation of another gave imino nitroxide compounds, 2-imidazolin-l-oxyles 54a-c (Scheme 21). Scheme 21

4? SO 51

Reno

M«-d &«••« E2ϋ-<l

R= a) 1 -pyryl; b) 9-phenantryl; c) 1-chrysyl;

EXAMPLE 17. SYNTHESIS OF IMINO- AND NITRONYL NITROXIDE DUAL AGENTS HAVING CONJUGATED PYRENE FLUOROPHORE

2.2-Dimetyl-2.3-dinitrobutane (50).

To a stirred mixture of 89 g (1 mol) of 2-nitropropane 49 and 170 ml of 6 N NaOH was added 25.8 ml (0.5 mol) of bromine, followed by 330 ml of ethanol. The stirred solution was boiled gently under a reflux condenser for 3 h. The the mixture was cooled to room temperature and the resulted slurry was poured into 500 ml of ice-water. Precipitated bis-nitro compound 50 was filtered, washed with water (3 x 100 mL) and dried on air to yield 65.58 g (75%).

The monosulfate salt of N.N'-dihydroxy-2.3-diamino-2.3-dimethylbutane (51).

65.5g of 2,3-dinitrobutane 50 was stirred with ammonium chloride (35.8 g) in a 50% aqueous ethanol solution (600 ml) and kept below 20°C while zinc dust (121 g) was added in small portions for 3 hours. The reaction mixture was filtered and the cake of zinc oxide was washed on a filter with 96% ethanol (425 ml), the combined filtrate and washings diluted with ethanol (1425 ml), and the solution was slowly titrated with 20% ethanolic sulfuric acid until final pH=2. The precipitated crystals were washed with 96%> ethanol (100 ml) and dried, yielding 65.9 g (72%) of bis-hydroxylamine 51 as a monosulfate salt.

4.4.5.5-Tetramethyl-2-pyryl-2-imidazoHne-l-oxyI (54a).

To a stirred solution of 2,3-bis(hydroxylamino)-2,3-dimethylbutane monosulfate salt 51 (800 mg, 3.3 mmol) in 10 ml of methanol a 30% solution of sodium methoxide (1.2 ml, 6.5 mmol) was added quickly under argon. The reaction mixture was stirred for 30 min, evaporated to dryness and diluted with 10 ml of benzene. To the resulting suspension, a solid pyrene aldehyde (0.5 g, 2.2 mmol) was added and the stirred mixture was boiled for 16 hours. Analytical TLC (eluent - CHC1 ) indicated complete consumption of the starting aldehyde and formation of two products. The mixture was evaporated to dryness. The residue was diluted with 10 ml of methanol and vigorously stirred in a 25-ml beaker with 1 ml of oxidation catalysts [20 mg of copper (II) acetate and 0.5 ml of cone, ammonium hydroxide diluted with methanol to 5 ml] for 2 hours. The reaction mixture was evaporated to dryness and the residue was flash-chromatographed over silica gel column (2x20 cm), eluent chloroform to give 70 mg (9%>) of 4,4,5, 5-tetramethyl-2-pyryl-2-imidazoline- 1 -oxyl (54a) as an orange solid (R_f= 0.6) and 80 mg (10%) of 4,4,5, 5-tetramethyl-2-pyryl-2-imidazoline-3- oxide-1-oxyl (53a) as a deep blue solid (Rf = 0.4). 4.4.5.5-Tetramethyl-2-pyryl-2- imidazoline-l-oxyl (54a): mp. 152-153°C. Anal. Calcd for C₂₃H₂ιN₂O: C.80.91; H 6.20; N 8.20. Found: C 80.69; H 6.09; N 8.13.

4.4.5.5-Tetrametbyl-2-pyryl-2-imidazoIine-3-oxide-l-oxyl 53a: mp. 179-180°C. Anal. Calcd for C₂₃H₂ιN₂θ₂: C,77.29; H 5.92; N 7.82. Found: C 76.99; H 5.54; N 7.70.

EXAMPLE 18. SYNTHESIS OF IMINO- AND NITRONYL NITROXIDE DUAL AGENTS HAVING CONJUGATED PHENANTRYL FLUOROPHORE

4.4.5.5-Tetramethyl-2-(9'-phenantryl)-2-imidazoline-l-oxyl (54b). 4.4.5.5-tetramethyl-2-(9'-phenantryl)-2-imidazoline-3-oxide-l-oxyl (53b). To a stirred suspension of 2,3-bis(hydroxylainino)-2,3-dimethylbutane monosulfate salt 51 (1.79g, 7.3 mmol) in 20 ml of methanol a 30% sodium methoxide solution (2.7 ml, 14.6 mmol) was added quickly under argon. The mixture was stirred for 30 min, then 1 g (4.9 mmol) of phenanthrene-9-carboxaldehyde was added in portions over 5 min. The stirred mixture was boiled for 16 hours. Analytical TLC (eluent -chroloform) indicated complete consumption and the formation of two products. The mixture was evaporated to dryness, diluted with 30 ml of methanol and stirred vigorously in 25-ml beaker with 2 ml of oxidation catalyst [similar to what used in Example 17 above] for 2 hours. The mixture was evaporated to dryness, and the residue was flash-chromatographed over silica gel column (2x30 cm), eluent - chloroform to yield 80 mg (5%) of 4,4,5,5- tetramethyl-2-(9'-phenantryl)- 2-imidazoline-l-oxyl 54b as an orange solid (Rf = 0.6), and 310 mg (20%) of 4,4,5,5- tetramethyl-2-(9'-phenantryl)-2-imidazoline-3-oxide-l-oxyl 53b as a deep blue solid (Rf = 0.4). 4.4.5.5-Tetramethyl-2-(9'-phenantryl)-2-imidazoline-l-oxyl 54b: mp. 135-136°C. Anal. Calcd for C₂ιH₂ιN₂O₂: C,79.47; H 6.67; N 8.83. Found: C 79.20; H 6.52; N 8.74. 4.4.5.5-TetramethyI-2-(9'-phenantryl)-2-imidazoline-3-oxide-l-oxyl 53b: mp 195-196°C. Anal. Calcd for C₂ιH₂₁N₂O₂: C,75.65; H 6.35; N 8.40. Found: C 75.36; H 6.31; N 8.43.

EXAMPLE 19. SYNTHESIS OF IMINO- AND NITRONYL NITROXIDE DUAL AGENTS HAVING CONJUGATED CHRYSENE FLUOROPHORE

4.4.5.5-Tetramethyl-2-(rchrysyl)-2-imidazoline-i-oxyl (54c).

To a stirred suspension of 2,3-bis(hydroxylainino)-2,3-dimethylbutane monosulfate salt 51 (l.Olg, 4.1 mmol) in 10 ml of methanol a 30%> sodium methoxide solution (1.5 ml, 8.2 mmol) was added quickly under argon. The mixture was stirred for 30 min, then 0.7 g (2.7 mmol) of phenanthrene-9-carboxaldehyde was added. The stirred mixture was boiled for 16 hours. Analytical TLC (eluent -chroloform) indicated complete consumption and the formation of two products. The mixture was evaporated to dryness, diluted with 20 ml of chloroform and stirred with 2 g of manganese (IV) oxide for 30 min. The inorganic material was filtered off, washed on a filter with chloroform (10 x 10 ml). Combined organic filtrate was evaporated and the residue was flash-chromatographed over silica gel column (20 x 1.5 cm), eluent - chloroform to yield 260 mg (40%) of 4,4,5,5-tetramethyl-2-(r-chrysyl)-2-imidazoline-l-oxyl 54c as an orange solid, and 50 mg (5%) of 4,4,5, 5-tetramethyl-2-(l'-chrysyl)-2-imidazoline- 3-oxide-l-oxyl 53c as a deep blue solid. 4.4.5.5-Tetramethyl-2-(l'-chrysyl)-2-imidazoline- 1-oxyl 54c: mp 184-185°C. Anal. Calcd for C₂5H₂₃N₂0: C.81.72; H 6.31; N 7.62. Found: C 82.04; H 6.37; N 7.27. 4.4.5.5-tetramethyl-2-(l'-chrysyl)-2-imidazoline-3-oxide-l-oxyl 53c: mp 192-193°C. Anal. Calcd for C2₅H₂₃N₂O₂: C 78.30; H 6.05; N 7.31. Found: C 77.67; H 5.81; N 7.17.

EXAMPLE 20. SYNTHESIS OF A NITRONYL NITROXIDE DUAL AGENT HAVING PYRENE FLUOROPHORE AND METHYLENE SPACER

The Example 20 below represents the synthesis of a double agent 62 with a pyrene fluorophore which is connected to the nitronyl nitroxide moiety through methylene spacer. The synthesis of compound 62 was accomplished via nucleophilic substitution of bromine in a bromomethyl group of 2-bromomethyl nitronyl nitroxide 61. Preparation of this key intermediate was performed by condensation of bis-hydroxylamine 51 with bromoacetaldehyde followed by the oxidation of intermediate dihydroxyimidazolidine 60 (Scheme 22).

62

2-Bromomethyl-4.4.5.5-tetramethylimidazoline-3-oxide-l-oxyI (61).

To a stirred suspension of 2,3-bis(hydroxylainino)-2,3-dimethylbutane monosulfate salt 51 (2.98g, 12 mmol) in 20 ml of methanol a 30% sodium methoxide solution (4.2 ml, 24 mmol) was added quickly under argon. The mixture was stirred for 30 min, then 1 ml (12 mmol) of bromoacetaldehyde dropwise. The mixture was stirred for additional 30 min and evaporated to dryness. The residue of intermediate 60 was diluted with 50 ml of methanol and stirred vigorously in 100-ml beaker with 1 ml of oxidation catalyst [similar to what used in Example 17,18 above] for 2 hours. The mixture was evaporated to dryness, and flash- chromatographed by SiO column (2x20 cm), eluent - chloroform to yield 0.4 g (13 %) of 2- bromomethyl-4,4,5,5-tetramethyl-2-imidazoline-3-oxide-l-oxyl 61 as a deep blue solid.

2-(r-Pyreneamino)methyl-4.4.5.5-tetramethyl-2-imidazoHne-3-oxide-l-oxyl (62)

A solution of 100 mg (0.4 mmol) of 2-bromomethyl-4,4,5,5-tetramethyl-2- imidazoline-3-oxide-l-oxyl 61 in 1 ml of acetonitrile was added to a solution of 210 mg (0.8 mmol) of 1-pyrenemethylamine hydrochloride in 0.13 ml (0.72 mmol) of 30%) sodium methoxide and 5 ml of acetonitrile. The mixture was heated under argon to 60°C with 10 mg of potassium iodide (catalyst) and stirred at this temperature for 15 min (control by TLC: eluent -chloroform-methanol = 10:1). The mixture was evaporated to dryness and the residue was purified by prep. TLC, eluent chloroform to yield 80 mg (50%>) of the compound 62 as a deep blue solid.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Berliner, L.J. Spin Labeling. Theory and Applications, Vol. 1 and 2, Academic Press, New York, 1976, 1979.

Keana, J.F.W., Chem. Rev., 78, 37, 1978.

Gaffhey, B.J., and Chen, S.C, in Method in Membrane Biology, Vol. 8, edited by E.D. Korn (Plenum Press, New York, US) 1977, chapter 4.

Bacic, G., Nitges, M.J., Bennett, H.F., Magin, R.L., and Swartz, H.M., Magn. Reson. Med, 6:445, 1988.

Dobrucki, J.W., Demsar, T., Walczak, T., Woods, R.K., Bacic, G., and Swartz, H.M., Br. J Cancer, 61 :221, 1990. Subczinski, W. K., Lukiewicz, S., and Hyde, S., Magn. Reson. Med., 3:747, 1986.

Riley, R.J., Hemingway, S.A., Grahan, M.A., and Workman, P., Biochem. Pharmacol., 45:1065, 1993. Rauckman, E.J., Rosen, G.M., and Griffeth, L.K., in Spin Labeling in Pharmacology, Holzman, J.L., Ed., Academic Press, New York, 1984, 1975.

Castle, J.D., and Hubbell, Biochemistry, 15:4818, 1976. Strzalka, K., Walczak, T., Sarna, T., and Swartz, H.M., Arch. Biochem. Biophys., 281:312, 1990.

Mehlhorn, R.J., Candau, P., and Packer, L., Method EnzymoL, 88:751, 1982. Mehlhorn, R.J., and Packer, L., Method EnzymoL, 56:515, 1979.

Zhdanov, R.I., Ed. Bioactive Spin Labels, Springer-Verlag, Berlin, 1992.

Bacic, G., Walczac, T., Demsar, F., and Swartz, H.M., Magn. Reson. Med, 8:209, 1988.

Khramtsov, V.V., Yelinova, V.I., Weiner, L.M., Berezina, T.A., Martin, V.V., and Volodarsky, L. ., Anal. Biochem., 182:58, 1989.

Sosnovsky, G., Rao, N.U.M., Li, S.W., and Swartz, H.M., J Org. Chem., 54:3667, 1989.

Demsar, F., Swartz, H.M., and Schar A, M., Magn. Reson. Med Boil., 1:17, 1988. Dobrucki, J.W., Sutherland, R.M., and Swartz, H.M., Magn. Reson. Med, 19:42, 1991.

Chato, J.C., Swartz, H.M., and Robles, J.E., in Boiheat Transfer-Applications in Hyperthermia. Emarging Horizons in Instrumentation and Modeling, Romer, R.B., McGrath, J.J., and Bowman, H.F., Eds., American Society of Mechanical Engeneers, New York, 1989, 121.

Chan. H.C., Glockner, J., and Swartz, H.M., Biochim. Biophys. Ada, 1014:141,1989.

Demsar, F., Walczak, T., Morse, P.D., Bacic, G.,Zolnai, Z., and Swartz, H.M., J Magn Reson., 76:224, 1988.

Dubrucki, J.W., Demsar, F., Walczak, T., Woods, R.K., Bacic, G., and Swartz, H.M., Br. J Cancer, 61 :221, 1990.

Corcer, G.A. Klein, M.P., and Calvin, M., Proc. Natl. Acad Sci USA, 56:1365,1966.

Floyd, R., Biochem. Biophys. Ada, 756:204, 1983.

Nohl, H., Jordan, W., and Hegner, D., FEBSLett., 123:241, 1981.

Volodarsky L.B., ed. Synthetic Chemistry of Nitroxide Radicals, CRC Press, Boca Raton, FI, 1995.

Keana, J.F.W., Pou, S., and Rosen, G.M., Magn. Reson. Med, 5: 525, 1987.

Swatz, H.M., and Pals, M., Handbook of Biomedicine of Free Radicals and Antioxidants, Vol. 3, edited by J. Miguel, H. Weber and A. Wuintanilha (CRC Press), 1989, 141.

Swartz, H.M., in Advances in MagneNc Resonance Imaging, edited by E. Feig (Ablex Publishing Company, Norwood, New Jersey, US), 1989, 49.

Imidazoline nitroxides. Synthesis and properties, V.l; Applications, V.2 / Volodarsky, L.B., Ed., Boca Raton, Florida: CRC press, 1988.

Likhtenstein G.L; Kulikov, AN.; Kotelnikov, A.I.; Levonenko, L.A. Biochem. Biophys. Methods, 12:1, 1986. Kochergmsky, Ν.; Swartz, H.M. Nitroxide Spin Labels: Reactions in Biology and Chemistry, CRC Press, Boca Raton, FI. 1995.

Khramtsov, V.V.; Vainer, L.M. Russ Chem. Revs., 57:824,1988. Keana, J.F.W. in Spin Labeling in Pharmacology, ed. Holtzman, J.L. Academic Press, Orlando, FI, 1984, p. 1.

Gaffhey, B.J. in Spin Labeling: Theory and Applications, ed. Berliner, L.J. Academic Press, New York, 1976, p. 183. Lakowicz, J.R., ed. Topics in Fluorescence Spectroscopy. Plenum Press, New York, 1991.

Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals. 6th Ed., Molecular Probes, Inc. Eugene, OR, 1996.

Likhtenstein G.L, Biophysical Labeling Methods in Molecular Biology. Cambridge University Press, New York, 1993.

Atik, S.S.; Singer, .A., JAm. Chem. Soc, 100:3234, 1978.

Though N.V.; Simpson, D.J., J Am. Chem. Soc, 110: 1915, 1988.

Green, S.A.; Simpson, D.J.; Zhou, G.; Ho, P.S.; Though, N.V., J Am. Chem. Soc, 112:7337, 1990.

Gerlock, J.L.; Zacmandis, P.J.; Bauer, D.R.; Simpson, D.J.; Though, N.V.; Salmeen, I.T., Free Radical Res. Commun., 10:119, 1990.

Kieber, D.; Though N.V., Free Radical Res. Commun., 10:109. 1990.

Kieber, D.; Though N.V., Anal. Chem., 62:2275, 1990.

Bystrayk, I.M.; Likhtenstein, G.L; Kotelnikov, A.I.; Hideg, K., J.Chem.Phys. (Moscow), 61:2769, 1986.

Pou, S.; Huang, Y.I.; Bhan, A.; Bhadti, V.S.; Hosmane, R.S.; Wu, S.Y.; Cao, G.L; Rosen, G.M. Analyt. Biochem., 212:65, 1993.

Pou, S.; Wu, S.Y.; Lederer, W.I.; Rosen G.M. Int. Congr. Ser. - Experta Med., 1992, 988 (Oxygen Radicals), 179.

Briggs, S.P., Haug, A.R., and Scheffer, R.P., Plant PhysioL, 70:668, 1982.

Shchukin, G.L, and Grigoriev, I. A., Oxidation-reduction properties, in Imidazoline Nitroxides, Vol. 1, Synthesis and Properties, Volodarsky, L.B., Ed.,CRC Press, Boca Raton, FL, 1988, 172.

Hideg, K., and Hankovszky, O.H., in Organic Magnetic Resonance: SpinLabeling VIII; Berliner, L.J., Reuben, J., Ed., Plenum Press, NY, 1989, Chapter 9.

Keana, J.F.W., Martin, V.V., Ralston, W.H., U.S. Patent No: 5,567,411, 1996.

Keana, et al., U.S. Patent No: 5,252,317, 1993. Keana, et al., U.S. Patent No: 4,099,918, 1978.

Osiecki, J.H., and Uliman, E.F., JAmer. Chem. Soc, 1078,1968.

Uliman, E.F., Call, L., and Osiecki, J.H., J Org. Chem., 35:3623, 1980. Martin, V.V., Volodarsky, L.B., and Vishnivetskaya, L. A., Izv. Sib. Otd. Akad Nauk SSSR, Ser. Khirn. Nauk, 4:94,1981.

Parmon, V. N., Kokorin, A. I., and Zhidomirov, Stable Diradicals, Nauka, Moscow, 1980.

Martin, V.V., and Keana, J.W.F., J Chem. Soc, Chem. Commun. 723, 1995.

Rezaikov, V.A., Volodarsky, L.B., Khim. Geterotsill. Soedin., 1990, N 6, 772. Grigor ev, A., Volodarsky, L.B., Starichenko, VF., and Kirilyuk, LA., Tetrahedron Lett., 26:5085, 1985.

Grigor ev, LA., Volodarsky, L.B., Starichenko, V.F., and Kirilyuk, LA., Tetrahedron Lett., 3 0:751, 1989.

Rezaikov, V.A., and Volodarsky, L.B., Izv. Akad Nauk S.S.S.R., Ser. Khim., 1990, N 2, 3 90.

Claims

WHAT IS CLAIMED IS:

1. A dual fluorophore-nitroxide composition comprising at lease one fluorescent moiety (F1 ), at least one nitroxide moiety (NR), and at least one nitroxide free radical center (N-O^*), said dual flurophore nitroxide composition having a structure:

2. The dual fluorophore-nitroxide composition of claim 1 wherein the fluorescent moiety comprises a polycyclic aromatic fragment, acridine, coumarine, dansyl, fluorescein, rhodamine, Texas Red, Cascade Blue, Lucifer Yellow, a derivative thereof, or a combination thereof.

3. The dual fluorophore-nitroxide composition of claim 1 wherein the nitroxide moiety (NR) is a stable free radical entity, comprising at least one nitroxide free radical center -N(O)-.

4. The dual fluorophore-nitroxide composition of claim 3 wherein the nitroxide moiety (NR) comprises a part of a heterocyclic system or an opened-chain fragment.

5. The dual fluorophore-nitroxide composition of claim 4 wherein the heterocyclic system comprises a partially or fully hydrogenated derivative of pyrrole, pyridine, imidazole, or oxazole.

6. A dual (hybrid) fluorescent-nitroxide composition having a structure:

wherein FI is a fluorescent moeity as defined in claim 2; NR is a nitroxide moiety comprising a heterocyctic system or an opened-chain fragment; and SP is a spacer that connects FI and NR.

7. The dual (hybrid) fluorescent-nitroxide composition of claim 6 wherein the NR is a heterocyclic system comprising a partially of fully hydrogenated derivative of pyrrole, pyridine, imidazole, or oxazole.

8. The dual fluorophore-nitroxide probe of claim 6 wherein the spacer is further defined as a functional group -COO-, -CONH-; -SO₂NH-; CSNH-; -NHNH; -NR-, -O-, or -S-.

9. The dual fluorophore-nitroxide probe of claim 6 wherein the spacer is further defined as providing a through-space electron interaction between FI and NR and is capable of quenching fluorescence of FI.

10. The dual fluorophore-nitroxide composition of claim 6 wherein the spacer is further defined as a conjugated system of double or triple bonds -(C=C)_n; (C=N)_n; (CΓëíC) _n wherein _n is a - 1 ,2,3,4,5,6,7,8,9 or 10 carbon chain.

11.The dual fluorophore-nitroxide composition of claim 9 wherein the spacer is a conjugated system further defined as comprising part of an aromatic system, a heterocyclic system, or an opened-chain fragment.

12. The dual fluorophore-nitroxide composition of claim 9 or 10, wherein the conjugated system is further defined as capable of providing a through-bond electron interaction between FI and NR, and is capable of quenching the fluorescence of FI.

13. A dual fluorophore-nitroxide composition comprising a radical center and a fluorophore fragment in immediate proximity of the radical center.

14. The dual fluorophore-nitroxide composition of claim 13 further defined as an opened-chain molecule or a part of a cyclic system with a general structure:

^ A N)"^ F.I

wherein FI is a fluorescent entity comprising a polycyclic aromatic fragment, acridine, coumarine, dansyle, fluorescein, rhodamine, Texas Red, Cascade Blue, Lucifer Yellow, their derivatives, or a combination thereof.

15. A dual fluorophore-nitroxide composition comprising a nitroxide moiety NO' directly connected to an aromatic fluorophore F1 , said dual fluorophore-nitroxide composition having a general structure:

16. A dual fluorescent-nitroxide molecule comprising a nitroxide moiety as a part of a conjugated nitronyl-nitroxide- or imino-nitroxide grouping, with a general structure:

where (O) means presence or absence of N-oxide oxygen atom; R is an C C₁₀ alkyl, aryl, or a substituted alkyl or aryl group; and Z is a fluorescent moiety comprising a polycyclic aromatic fragment, acridine, coumarine, dansyl, fluorescein, rhodamine, Texas Red, Cascade Blue, Lucifer Yellow, a derivative thereof, or a combination hereof.

17. The dual fluorescent-nitroxide molecule of claim 16 wherein the fluorescent moeity is attached through a spacer SP to the conjugated nitronyl-nitroxide or imino- nitroxide grouping.

18. The dual fluorophore-nitroxide molecule of claim 5,6,7,8 or 9 further defined as capable of regaining fluorescent properties upon transformation of the nitroxide moiety into a non-radical diamagnetic group.

19. The dual fluorophore-nitroxide molecule of claim 18 wherein the non-radical dramagnetic group is a hydroxyamine or an amino group.

20. The dual fluorophore-nitroxide molecule of claim 18 wherein the transformation of the nitroxide moiety into a non-radical diamagnetic group is further defined as a reduction or recombination with a reactive free radical.

21. The dual fluorophore-nitroxide molecule of claims 6 or 10 wherein the spacer SP is further defined as a flexible system capable of conformational mobility.

22. The dual fluorophore-nitroxide molecule of clam 21 wherein the spacer SP is a hydrocarbon, heteroatom chain, a functionality thereof, or a combination thereof.

23. The dual fluorophore-nitroxide molecule of claim 22 wherein the distance between FI and NR moieties will determine the fluorescence of the molecule.