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WO1999050244A1 - Agents indicateurs doubles comportant un radical libre nitroxyde - Google Patents

Agents indicateurs doubles comportant un radical libre nitroxyde Download PDF

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WO1999050244A1
WO1999050244A1 PCT/US1999/006688 US9906688W WO9950244A1 WO 1999050244 A1 WO1999050244 A1 WO 1999050244A1 US 9906688 W US9906688 W US 9906688W WO 9950244 A1 WO9950244 A1 WO 9950244A1
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nitroxide
dual
fluorophore
fluorescent
moiety
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PCT/US1999/006688
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English (en)
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WO1999050244A9 (fr
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Vladimir V. Martin
Alexander Weis
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Lipitek, Inc.
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Priority to AU31175/99A priority Critical patent/AU3117599A/en
Publication of WO1999050244A1 publication Critical patent/WO1999050244A1/fr
Publication of WO1999050244A9 publication Critical patent/WO1999050244A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
    • C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/88—Nitrogen atoms, e.g. allantoin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • 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.
  • Nitroxides 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.
  • EPR Electron Paramagnetic Resonance
  • 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.
  • in vivo EPR techniques have 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
  • 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).
  • 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.
  • 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 de ⁇ vatives containing a radical center.
  • 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.
  • 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 va ⁇ ous 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).
  • 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.
  • 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.
  • 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.
  • hybrid molecules show their potential as research tools.
  • variety of hybrid molecules used in the reported research is limited, both in terms of their fluorescent properties and nitroxide triggers.
  • 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 *
  • NR nitroxide moiety
  • N-O * nitroxide free radical
  • the probes of the invention may be further defined as having a structure:
  • FI is a fluorescent moiety
  • NR is a nitroxide moiety
  • N-O * is a nitroxide free radical center.
  • 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.
  • the hybrid molecule 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 provide utility for use in biological research and medical diagnostics.
  • the present invention provides methods to synthesize several series of hybrid molecules. These hybrid molecules are also characterized for their fluorescent properties.
  • hybrid compounds in yet another aspect, provides for hybrid compounds.
  • 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.
  • Fluorophore is the part of the hybrid molecule that makes detection by a spectrometer possible.
  • 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;
  • fluorescent dyes used as conventional fluorescent probes, including polycyclic aromatic fragment, acridine, coumarine, dansyl, fluorescein, rhodamine, Texas
  • 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.
  • 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.
  • 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 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:
  • connection reaction 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.
  • 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.
  • 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.
  • A-D A single point short connection, by formation of an ester, an amide or a sulfamide bond.
  • 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.
  • 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; Hopkins, 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.
  • 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.
  • diradical and hybrid molecules 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.
  • 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,
  • Nitroxide structural units used as key intermediates (Scheme 9) in the preparations are readily available, stable, and non-toxic compounds.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • bromine 8.4 ml, 0.16 mol
  • potassium hydroxide 24.6 g, 0.44 mol
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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 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).
  • 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-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 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.
  • 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.
  • 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).
  • Example 15 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).
  • Example 16 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).
  • 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.
  • R a) 1 -pyryl; b) 9-phenantryl; c) 1-chrysyl;
  • 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).
  • Example 20 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).

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Abstract

Agents indicateurs doubles à propriétés de fluorescence améliorées par synergie, qui comportent une fraction nitroxyde et un centre radical libre nitroxyde. La molécule indicatrice peut comprendre toute une série d'agents marqueurs chimiques ou biochimiques, tels qu'une molécule fluorescente. Ledit agent double peut comporter en outre une molécule d'espacement ou de liaison qui fonctionne pour attacher la molécule indicatrice, telle que la fraction fluorescente, à la fraction nitroxyde. Ces sondes peuvent être utilisées pour mesurer le potentiel d'oxydoréduction dans des cellules vivantes, ainsi que pour déterminer les potentiels d'oxydoréduction dans des diagnostics et dans de nombreuses autres applications.
PCT/US1999/006688 1998-03-28 1999-03-26 Agents indicateurs doubles comportant un radical libre nitroxyde WO1999050244A1 (fr)

Priority Applications (1)

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AU31175/99A AU3117599A (en) 1998-03-28 1999-03-26 Nitroxide free radical dual indicator agents

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US7984898P 1998-03-28 1998-03-28
US60/079,848 1998-03-28

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WO1999050244A1 true WO1999050244A1 (fr) 1999-10-07
WO1999050244A9 WO1999050244A9 (fr) 1999-12-09

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JP2009143855A (ja) * 2007-12-14 2009-07-02 Toyota Central R&D Labs Inc 有機ラジカル化合物、蓄電デバイス用電極及び蓄電デバイス
WO2009088801A1 (fr) * 2007-12-31 2009-07-16 Medical College Of Wisconsin Composés de piégeage de spin à base de nitrone bifonctionnel et trifonctionnel, et leurs utilisations
CN110483383A (zh) * 2018-05-14 2019-11-22 新发药业有限公司 一种吡啶衍生物的制备方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008414A1 (fr) * 2000-06-27 2002-01-31 National Institute Of Advanced Industrial Science And Technology Nouvelles sondes d'acides nucléiques et procédés d'essai d'acide nucléique par utilisation des mêmes
JP2009143855A (ja) * 2007-12-14 2009-07-02 Toyota Central R&D Labs Inc 有機ラジカル化合物、蓄電デバイス用電極及び蓄電デバイス
WO2009088801A1 (fr) * 2007-12-31 2009-07-16 Medical College Of Wisconsin Composés de piégeage de spin à base de nitrone bifonctionnel et trifonctionnel, et leurs utilisations
CN110483383A (zh) * 2018-05-14 2019-11-22 新发药业有限公司 一种吡啶衍生物的制备方法
CN110483383B (zh) * 2018-05-14 2021-04-16 新发药业有限公司 一种吡啶衍生物的制备方法

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AU3117599A (en) 1999-10-18
WO1999050244A9 (fr) 1999-12-09

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