Syntheses and Study of a Pyrroline Nitroxide Condensed Phospholene Oxide and a Pyrroline Nitroxide Attached Diphenylphosphine

The reaction of a diene nitroxide precursor with dichlorophenylphosphine in a McCormac procedure afforded 1,1,3,3-tetramethyl-5-phenyl-1,2,3,4,5,6-hexahydrophospholo[3,4-c]pyrrole-5-oxide-2-oxyl. Lithiation of the protected 3-iodo-pyrroline nitroxide followed by treatment with chlorodiphenylphosphine after deprotection afforded (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)diphenylphosphine oxide, and after reduction, (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)diphenylphosphine was realized, which was also supported by X-ray single crystal diffraction measurements. This pyrroline diphenylphosphine derivative was converted to hexadecylphosphonium salt, which is an analogue of antineoplastic agent, MITO-CP.


Introduction
Heterocyclic nitroxide derivatives of piperidine pyrrolidine, pyrroline and imidazoline have found various applications [1,2]. Stable nitroxide (aminoxyl) free radicals possess unique properties and reactivity, and despite decades of experience working with them, accessing new scaffolds without affecting the free valence is a challenge for every synthetic chemist active in this field [3]. Sometimes nitroxide moieties require temporary protection [4,5] to avoid irreversible destruction during various chemical transformations. Nitroxides have a wide range of applications; they are used as co-oxidants in organic chemistry, building blocks for magnetic materials, superoxide dismutase mimics, antiproliferative compounds, mediators of polymerization, redox active materials in batteries, magnetic resonance imaging (MRI), and electron paramagnetic resonance imaging (EPRI) contrast agents [1,2]. These various applications require numerous paramagnetic scaffolds adjusted to different utilizations. Our idea is to combine various phosphorus-containing functional groups with nitroxides, which may offer fascinating possibilities for synthetic, structural and biological studies and the utilization of these new compounds [6,7]. In continuation of our interest in the synthesis of nitroxide-based phosphorus compounds, we have promoted the synthesis of phosphorus-containing heterocycles condensed with pyrroline nitroxide and pyrroline nitroxide-diphenylphosphine and pyrroline nitroxidediphenylphosphine oxide compounds. Although similar nitroxides have been previously reported with nitronyl nitroxides [8] and a phosphorus-containing heterocycle [9], our approach described in the present work might open a new route for synthesizing such novel types of paramagnetic phosphorus-containing compounds.

Synthesis of Paramagnetic Phospholene Oxide
A standard procedure to form phospholene oxide is McCormac cycloaddition attempted from compound 1a [10] and dichlorophenylphosphine [11,12]. However, this addition did not give the expected, isolable product. Proposing the disruption of the nitroxide under the reaction conditions applied, we protected nitroxide as an O-acetyl derivative 1b [4]. Treatment of compound 1b with dichlorophenylphosphine in a three-week-long reaction time in pentane at 37 • C enabled us to obtain compound 2b after hydrolysis with a modest 36% yield. Deprotection of the O-acetyl group by a catalytic amount of NaOMe, followed by oxidation of the N-hydroxylamine with MnO 2 , offered 1,1,3,3tetramethyl-5-phenyl-1,2,3,4,5,6-hexahydrophospholo [3,4-c]pyrrole-5-oxide-2-oxyl 2a as the first pyrroline nitroxide condensed phospholene oxide (Scheme 1). Compound 2a was reduced to its hydroxylamine derivative in situ in the NMR tube (see Section 3.1.) and in the resulted 31 P-NMR we found a single peak at 61. 8  been previously reported with nitronyl nitroxides [8] and a phosphorus-containing heterocycle [9], our approach described in the present work might open a new route for synthesizing such novel types of paramagnetic phosphorus-containing compounds.

Synthesis of Paramagnetic Phospholene Oxide
A standard procedure to form phospholene oxide is McCormac cycloaddition attempted from compound 1a [10] and dichlorophenylphosphine [11,12]. However, this addition did not give the expected, isolable product. Proposing the disruption of the nitroxide under the reaction conditions applied, we protected nitroxide as an O-acetyl derivative 1b [4]. Treatment of compound 1b with dichlorophenylphosphine in a threeweek-long reaction time in pentane at 37 °C enabled us to obtain compound 2b after hydrolysis with a modest 36% yield. Deprotection of the O-acetyl group by a catalytic amount of NaOMe, followed by oxidation of the N-hydroxylamine with MnO2, offered 1,1,3,3-tetramethyl-5-phenyl-1,2,3,4,5,6-hexahydrophospholo [3,4-c]pyrrole-5-oxide-2oxyl 2a as the first pyrroline nitroxide condensed phospholene oxide (Scheme 1). Compound 2a was reduced to its hydroxylamine derivative in situ in the NMR tube (see Section 3.1.) and in the resulted 31 P-NMR we found a single peak at 61.8 ppm and methylene protons as multiplets 2.68-2.73 and 2.85-2.29 ppm with 2H-2H integrals, suggesting that compound 2a contains an endocyclic double bond.

Synthesis of Pyrroline Nitroxide Diphenylphosphine and Its Phosphonium Salt
As triphenylphosphine is an essential building block of mitochondria-targeted antioxidants and neoplastic agents [13,14], e.g., lipophilic triphenylphosphonium cations, we aimed to synthesize the paramagnetic analog of triphenylphosphine. We intended to determine whether or not we can synthesize different types of nitroxide-containing mitochondrially targeted molecules compared to MITO-CP ( Figure 1) [15]. In our case, nitroxide would function as a superoxide dismutase (SOD) mimic at the cationic "warhead". Compound 3 [16] was converted to the corresponding 4 O-methyl derivative in a coupled Fenton reaction, which generates methyl radicals. This protected pyrroline nitroxide 4 was treated with 1.1 eq. BuLi followed by the addition of diphenylchlorophosphine to furnish compound 5. Phosphine 5 proved to be rather stable during the flash

Synthesis of Pyrroline Nitroxide Diphenylphosphine and Its Phosphonium Salt
As triphenylphosphine is an essential building block of mitochondria-targeted antioxidants and neoplastic agents [13,14], e.g., lipophilic triphenylphosphonium cations, we aimed to synthesize the paramagnetic analog of triphenylphosphine. We intended to determine whether or not we can synthesize different types of nitroxide-containing mitochondrially targeted molecules compared to MITO-CP ( Figure 1) [15]. In our case, nitroxide would function as a superoxide dismutase (SOD) mimic at the cationic "warhead". been previously reported with nitronyl nitroxides [8] and a phosphorus-containing heterocycle [9], our approach described in the present work might open a new route for synthesizing such novel types of paramagnetic phosphorus-containing compounds.

Synthesis of Paramagnetic Phospholene Oxide
A standard procedure to form phospholene oxide is McCormac cycloaddition attempted from compound 1a [10] and dichlorophenylphosphine [11,12]. However, this addition did not give the expected, isolable product. Proposing the disruption of the nitroxide under the reaction conditions applied, we protected nitroxide as an O-acetyl derivative 1b [4]. Treatment of compound 1b with dichlorophenylphosphine in a threeweek-long reaction time in pentane at 37 °C enabled us to obtain compound 2b after hydrolysis with a modest 36% yield. Deprotection of the O-acetyl group by a catalytic amount of NaOMe, followed by oxidation of the N-hydroxylamine with MnO2, offered 1,1,3,3-tetramethyl-5-phenyl-1,2,3,4,5,6-hexahydrophospholo [3,4-c]pyrrole-5-oxide-2oxyl 2a as the first pyrroline nitroxide condensed phospholene oxide (Scheme 1). Compound 2a was reduced to its hydroxylamine derivative in situ in the NMR tube (see Section 3.1.) and in the resulted 31 P-NMR we found a single peak at 61.8 ppm and methylene protons as multiplets 2.68-2.73 and 2.85-2.29 ppm with 2H-2H integrals, suggesting that compound 2a contains an endocyclic double bond.

Synthesis of Pyrroline Nitroxide Diphenylphosphine and Its Phosphonium Salt
As triphenylphosphine is an essential building block of mitochondria-targeted antioxidants and neoplastic agents [13,14], e.g., lipophilic triphenylphosphonium cations, we aimed to synthesize the paramagnetic analog of triphenylphosphine. We intended to determine whether or not we can synthesize different types of nitroxide-containing mitochondrially targeted molecules compared to MITO-CP ( Figure 1) [15]. In our case, nitroxide would function as a superoxide dismutase (SOD) mimic at the cationic "warhead". Compound 3 [16] was converted to the corresponding 4 O-methyl derivative in a coupled Fenton reaction, which generates methyl radicals. This protected pyrroline nitroxide 4 was treated with 1.1 eq. BuLi followed by the addition of diphenylchlorophosphine to furnish compound 5. Phosphine 5 proved to be rather stable during the flash Compound 3 [16] was converted to the corresponding 4 O-methyl derivative in a coupled Fenton reaction, which generates methyl radicals. This protected pyrroline nitroxide 4 was treated with 1.1 eq. BuLi followed by the addition of diphenylchlorophosphine to furnish compound 5. Phosphine 5 proved to be rather stable during the flash chromatography purification process and could be stored for weeks under an Ar atmosphere at −18 • C without oxidation (e.g., appearance of compound 6). However, refluxing in toluene in air oxidized it to phosphine 6 oxides. Treatment of compounds 5 and 6 with meta-chloroperbenzoic acid (m-CPBA) to remove the protecting methyl group from the oxygen atom furnished paramagnetic phosphine oxide 7. Compound 7 could be reduced with paramagnetic pyrroline nitroxide diphenylphospine by heating it with 4 eq. trichlorosilane in toluene at 80 • C to reduce the phosphinoxide function [17,18] to phosphine and reduce nitroxide to hydroxylamine. The latter could be selectively oxidized to nitroxide 8 by PbO 2 (Scheme 2) without oxidation of phosphorus.
Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 chromatography purification process and could be stored for weeks under an Ar atmosphere at −18 °C without oxidation (e.g., appearance of compound 6). However, refluxing in toluene in air oxidized it to phosphine 6 oxides. Treatment of compounds 5 and 6 with meta-chloroperbenzoic acid (m-CPBA) to remove the protecting methyl group from the oxygen atom furnished paramagnetic phosphine oxide 7. Compound 7 could be reduced with paramagnetic pyrroline nitroxide diphenylphospine by heating it with 4 eq. trichlorosilane in toluene at 80 °C to reduce the phosphinoxide function [17,18] to phosphine and reduce nitroxide to hydroxylamine. The latter could be selectively oxidized to nitroxide 8 by PbO2 (Scheme 2) without oxidation of phosphorus.
Compound 8 was heated with hexadecylbromide for 5 days in acetonitrile at 90 °C in a closed vial to afford compound 9 in a low 5% yield in a sluggish reaction, presumably because of sterical hindrance due to the pyrroline nitroxide ring and because of side-reactions (Scheme 3). Scheme 2. Synthesis of pyrroline nitroxide diphenylphosphine and pyrroline nitroxide diphenylphosphin oxide.
Compound 8 was heated with hexadecylbromide for 5 days in acetonitrile at 90 • C in a closed vial to afford compound 9 in a low 5% yield in a sluggish reaction, presumably because of sterical hindrance due to the pyrroline nitroxide ring and because of sidereactions (Scheme 3).

Scheme 3.
Synthesis of a paramagnetic phosphonium salt.

X-ray Crystallographic Study of Pyrroline Nitroxide-Diphenylphosphine
X-ray-quality crystals of 8 were grown by slow crystallization from pentane/Et2O (2:1) solution by spontaneous evaporation of the solvent. A suitable crystal was fixed under a microscope onto a Mitegen loop using high-density oil. Diffraction intensity data were collected at 200 K using a Bruker-D8 Venture diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) equipped with INCOATEC IμS 3.0 (Incoatec GmbH, Geesthacht, Germany) dual (Cu and Mo) sealed tube micro sources and a Photon II Charge-Integrating Pixel Array detector (Bruker AXS GmbH, Karlsruhe, Germany) using Mo Kα (λ = 0.71073 Å) radiation. High multiplicity data collection and integration were performed using APEX3 (version 2017.3-0, Bruker AXS Inc., 2017, Madison, WI, USA) software. Data reduction and multiscan absorption correction were performed using SAINT (version 8.38A, Bruker AXS Inc., 2017, Madison, WI, USA). The structure was solved using direct methods and refined on F 2 using the SHELXL program [19] incorporated into the APEX3 suite. Refinement was performed anisotropically for all nonhydrogen atoms. Hydrogen atoms were placed into geometric positions. The CIF file was manually edited using Publcif software [20], while graphics were prepared using the Mercury program [21]. The results for the X-ray diffraction structure determinations were very good according to the Checkcif functionality of PLATON software (Utrecht University, Utrecht, The Netherlands) [22], and structural parameters such as bond length and angle data were in the expected range (for selected data, see the caption for Figure 2). The crystallographic and refinement details are given in Table 1    . The structure was solved using direct methods and refined on F 2 using the SHELXL program [19] incorporated into the APEX3 suite. Refinement was performed anisotropically for all nonhydrogen atoms. Hydrogen atoms were placed into geometric positions. The CIF file was manually edited using Publcif software [20], while graphics were prepared using the Mercury program [21]. The results for the X-ray diffraction structure determinations were very good according to the Checkcif functionality of PLATON software (Utrecht University, Utrecht, The Netherlands) [22], and structural parameters such as bond length and angle data were in the expected range (for selected data, see the caption for Figure 2). The crystallographic and refinement details are given in Table 1

X-ray Crystallographic Study of Pyrroline Nitroxide-Diphenylphosphine
X-ray-quality crystals of 8 were grown by slow crystallization from pentane/Et2O (2:1) solution by spontaneous evaporation of the solvent. A suitable crystal was fixed under a microscope onto a Mitegen loop using high-density oil. Diffraction intensity data were collected at 200 K using a Bruker-D8 Venture diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) equipped with INCOATEC IμS 3.0 (Incoatec GmbH, Geesthacht, Germany) dual (Cu and Mo) sealed tube micro sources and a Photon II Charge-Integrating Pixel Array detector (Bruker AXS GmbH, Karlsruhe, Germany) using Mo Kα (λ = 0.71073 Å) radiation. High multiplicity data collection and integration were performed using APEX3 (version 2017.3-0, Bruker AXS Inc., 2017, Madison, WI, USA) software. Data reduction and multiscan absorption correction were performed using SAINT (version 8.38A, Bruker AXS Inc., 2017, Madison, WI, USA). The structure was solved using direct methods and refined on F 2 using the SHELXL program [19] incorporated into the APEX3 suite. Refinement was performed anisotropically for all nonhydrogen atoms. Hydrogen atoms were placed into geometric positions. The CIF file was manually edited using Publcif software [20], while graphics were prepared using the Mercury program [21]. The results for the X-ray diffraction structure determinations were very good according to the Checkcif functionality of PLATON software (Utrecht University, Utrecht, The Netherlands) [22], and structural parameters such as bond length and angle data were in the expected range (for selected data, see the caption for Figure 2). The crystallographic and refinement details are given in Table 1     Absolute structure parameter -0.05 (3) The molecular structure of 8 as the targeted nitroxide derivative of the diphenylphosphinopyrrole derivative is fully supported by the X-ray diffraction study. Both the N-O distance and the double bond between C3 and C4 (see Figure 2) were proven. A search of the Cambridge Structural Database (version 5.41 Updates March 2020) [23] revealed 73 hits for similar 2,2,5,5 tetramethyl pyrrole nitroxide compounds, with an average N-O distance of 1.278(33) Å. We observed a similar value of 1.271(3) Å. However, no phosphorous derivative at C3 or C4 could be found, showing the uniqueness of our compound. The compound crystallized in the monoclinic space group P21, is chiral. Moreover, the Flack parameter is very close to 0 (Table 1) indicating that we have a chiral lattice for our achiral molecule. The packing diagram shows a very small portion of the unit cell as a void (Figure 3).

General Methods and Reagents
The mass spectra were recorded with a GCMS-2020 (Shimadzu, Tokyo, Japan) operated in EI mode (70 eV) and a ThermoScientific Q-Extractive HPLC/MS/MS with ESI(+) ionization (Thermo Scientific, Waltham, MA, USA). Elemental analyses were carried out with a Fisons EA 1110 CHNS elemental analyzer (Fisons Instruments, Milan, Italy). The melting points were determined with a Boetius micromelting point apparatus (Franz Küstner Nachf. K. G., Dresden, Germany). 1 H NMR spectra were recorded with a Bruker Avance 3 Ascend 500 system (Bruker BioSpin Corp., Karlsruhe, Germany) operated at 500 MHz, and 13 C NMR spectra were obtained at 125 MHz and 31 P NMR 202 MHz in CDCl3 or DMSO-d6 at 298 K. The "in situ" reduction of nitroxides was achieved by the addition of five equivalents of hydrazobenzene (DPPH/radical). The EPR spectra were recorded using a MiniScope MS 200 (Magnettech GMBH, Berlin, Germany) in CHCl3 solution, and the sample concentrations were 1.0 × 10 −4 M. All monoradicals gave a triplet line at aN= 14.4 G. IR spectra were obtained with a Bruker Alpha FT-IR instrument (Bruker Optics, Ettlingen, Germany) with ATR support on a diamond plate. Flash column chromatography was performed using a Merck Kieselgel 60 (0.040-0.063 mm). Qualitative TLC was carried out using commercially available plates (20 × 20 × 0.02 cm) coated with Merck Kieselgel (Darmstadt, Germany) GF254. Compounds 1a [9], 3 [15] were synthesized as previously reported. All the other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA), Molar Chemicals (Halásztelek, Hungary).

General Methods and Reagents
The mass spectra were recorded with a GCMS-2020 (Shimadzu, Tokyo, Japan) operated in EI mode (70 eV) and a ThermoScientific Q-Extractive HPLC/MS/MS with ESI(+) ionization (Thermo Scientific, Waltham, MA, USA). Elemental analyses were carried out with a Fisons EA 1110 CHNS elemental analyzer (Fisons Instruments, Milan, Italy). The melting points were determined with a Boetius micromelting point apparatus (Franz Küstner Nachf. K. G., Dresden, Germany). 1 H NMR spectra were recorded with a Bruker Avance 3 Ascend 500 system (Bruker BioSpin Corp., Karlsruhe, Germany) operated at 500 MHz, and 13 C NMR spectra were obtained at 125 MHz and 31 P NMR 202 MHz in CDCl 3 or DMSO-d 6 at 298 K. The "in situ" reduction of nitroxides was achieved by the addition of five equivalents of hydrazobenzene (DPPH/radical). The EPR spectra were recorded using a MiniScope MS 200 (Magnettech GMBH, Berlin, Germany) in CHCl 3 solution, and the sample concentrations were 1.0 × 10 −4 M. All monoradicals gave a triplet line at a N = 14.4 G. IR spectra were obtained with a Bruker Alpha FT-IR instrument (Bruker Optics, Ettlingen, Germany) with ATR support on a diamond plate. All spectra are shown in the Supplementary Material. Flash column chromatography was performed using a Merck Kieselgel 60 (0.040-0.063 mm). Qualitative TLC was carried out using commercially available plates (20 × 20 × 0.02 cm) coated with Merck Kieselgel (Darmstadt, Germany) GF 254 . Compounds 1a [9], 3 [15] were synthesized as previously reported. All the other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA), Molar Chemicals (Halásztelek, Hungary).

Conclusions
In this paper, we demonstrated that an O-acetyl nitroxide-protected paramagnetic diene can be used to synthesize phospholo [3,4-c] pyrrole scaffolds. Protected O-methyl pyrroline vinyl iodide can be converted to pyrroline nitroxide-diphenylphosphine oxide, for which reduction afforded pyrroline nitroxide-diphenylphosphine. A single-crystal X-ray diffraction study unambiguously supported the molecular structure. The pyrroline nitroxide diphenylphosphine can be converted into a hexadecyl phosphonium salt. In general, the proposed and adopted approaches could be used for a pyrroline nitroxidecontaining phosphine-and pyrroline nitroxide-condensed P-heterocycle. Further synthetic and biological study of these compounds are in progress in our laboratory.
Supplementary Materials: Supplementary materials are available online. The 1 H NMR and 13 C NMR, IR, MS spectra of novel compounds, and EPR spectra of compound 9 are available online. Table S1: Elememtal analysis of new compounds synthesized, Figure S1: Preliminary biological data of compound 9 and cetyltriphenylphosphonium bromide (compound 10) compared to MITO-CP.