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Unexpected one-pot formation of the 1H-6a,8a-epiminotri- cyclopenta[a,c,e][8]annulene system from cyclopentanone, ammonia and dimethyl fumarate. Synthesis of highly strained polycyclic nitroxide and EPR study

The unexpected formation of a highly strained polycyclic amine was observed in a one-pot synthesis from cyclopentanone, dimethyl fumarate and ammonium acetate. This multistep reaction includes 1,3-dipolar cycloaddition of dimethyl fumarate to the cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was easily converted into a sterically shielded polycyclic nitroxide. The EPR spectra and spin relaxation behavior of the nitroxide were studied in solution. The spin relaxation seems well suited for the use as a biological spin label and are comparable with those of cyclic nitroxides with two spirocyclic moieties adjacent to the N–O· group. Introduction Domino reactions have attracted much attention as an approach for the synthesis of complex molecules in a few steps [1]. The utility of multicomponent reactions involving amines, activated olefins and carbonyl compounds for the synthesis of heterocyclic compounds has been repeatedly demonstrated [2,3]. We recently used a domino reaction of amino acid, ketone and dimethyl fumarate for the one-pot synthesis of a substituted pyrrolidine, which then was converted into a reduction-resistant pyrrolidine nitroxide [4]. Here we report the unexpected formation of a highly strained polycyclic amine from cyclopenBeilstein J. Org. Chem. 2019, 15, 2664–2670. 2665 Table 1: NMR Assignment of 1 based on the HSQC, COSY 1H,1H and 1H,13C spectra. 13C (ppm) 1H (ppm) 13C (ppm) 1H (ppm) CH2 21.0 1.71; 2.00 3aCH 45.9 2.45 CH2 24.1 1.43; 1.68 CH3 51.5; 51.6 3.57; 3.66 CH2 25.3 1.51; 1.63 3bCH 55.1 2.39 CH2 30.1 1.93; 2.24 7CH 59.9 3.44 CH2 32.4 2.01; 2.15 8CH 60.4 3.19 CH2 33.9 1.41; 1.94 6aC, 8aC 76.08; 76.12 – CH2 34.4 1.11; 1.87 11aC 133.5 CH2 36.4 1.45; 1.63 11bC 137.5 CH2 40.9 1.96; 2.01 CO2 172.7; 175.3 tanone, dimethyl fumarate and ammonium acetate. This multistep reaction obviously includes the 1,3-dipolar cycloaddition of dimethyl fumarate with cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was then converted into a sterically shielded polycyclic nitroxide. Sterically hindered nitroxides have high chemical stability [5] and can be used as spin labels to study biopolymers in cells [6]. The introduction of spirocyclic moieties has a smaller effect on the reduction rates of nitroxides than does the introduction of linear alkyl substituents. However, spirocyclic nitroxides may have much longer spin relaxation times at 70–150 K which make them attractive agents for spin labeling [7-9]. Sterically hindered nitroxides can be used as spin labels for measurements at room temperature [10]. In this paper we examined the properties of this new nitroxide, in particular, the electron spin relaxation at different temperatures in water/glycerol solution. Results and Discussion Synthesis Polycyclic amine A mixture cyclopentanone, dimethyl fumarate and ammonium acetate was refluxed in benzene in a Dean–Stark apparatus. The reaction mixture underwent a strong tarring and after standard extraction with aqueous sulfuric acid, the main product 1 was isolated in a low yield (ca. 5%, see Scheme 1). In another experiment a much higher yield was obtained (ca. 15%), however, we failed to reproduce this result and in subsequent experiments the yields were close to the 5% limit. The 1H NMR spectra of 1 show signals of two ester groups and multiple methylene and methyne protons with an overall intensity of Scheme 1: Synthesis of compound 1. 22 protons. The 13C NMR spectrum showed signals from 21 carbon atoms, including an isolated, fully substituted C=C moiety at 133.5 and 137.5 ppm, methoxy groups at 51.5 and 51.6 ppm and carboxylate groups at 172.7 and 175.3 ppm, respectively. There are also four signals originating from CH groups at 45.9, 55.1, 59.9 and 60.4 ppm. The two downfield CH groups belong to isolated spin systems in the 1H NMR spectra at 3.19 and 3.44 ppm with hfs constants of 3 Hz. In the HMBC spectrum, the latter protons show cross peaks with the carboxylate carbons and with nodal carbons at 76.08 and 76.12 ppm. The above data imply the presence of a 2,2,5,5-tetrasubstituted3,4-bis(methoxycarbonyl)pyrrolidine ring. The 1H signal at 3.19 ppm shows a cross peak with an atom of a fully substituted C=C moiety, while another lower field signal of a CH group at 3.44 ppm interacts with a CH-group carbon at 55.1 ppm. The hydrogen of the latter group shows cross peaks with the carbon of the 3aCH group at 45.9 ppm and with the carbon of the 11bC=C moiety at 137.5 ppm. These interactions clearly indicate a 9-azabicyclo[4.2.1]non-2-ene system in the structure of 1. The remaining assignments were made on the basis of HSQC, COSY 1H,1H and 1H,13C NMR spectra (see Table 1). The structure of the compound was finally confirmed Beilstein J. Org. Chem. 2019, 15, 2664–2670. 2666 Scheme 2: Possible mechanism for the formation of 1. by X-ray analysis and elemental analysis data (Figure 1). A possible mechanism for the formation of 1 is presented in Scheme 2. Figure 1: X-ray structure of compound 1 (one of the two enantiomers present in the crystal). It is well-known that cyclopentanone is prone to self-condensation. In the presence of ammonia these reactions may lead to heterocycle formation [11]. Presumably, a prototropic shift in the enamine-imine intermediate 4 is followed by electrocyclization to the cyclic azomethine ylide, which then reacts with dimethyl fumarate in a 1,3-dipolar cycloaddition. The suggested mechanism accounts for the trans-position of the methyne hydrogens in the azepine ring: electrocyclization proceeds via a conrotatory mechanism due to the antisymmetry of the HOMO (Figure 2). Figure 2: A possible mechanism for the trans-position of the methyne hydrogens in the azepine ring: the electrocyclization proceeds via a conrotatory mechanism due to the antisymmetry of the HOMO. The selective formation of a single diastereomer in the 1,3dipolar cycloaddition reaction is likely a result from secondary interactions of orbitals of the π-systems of the dipole and dipolarophile (Figure 3). Figure 3: Selective formation of a single diastereomer in the 1,3dipolar cycloaddition reaction. Nitroxide Oxidation of 1 with m-CPBA afforded the nitroxide 6 with 48% yield (Scheme 3). It is noteworthy that the oxidation of the amino group is accompanied by the stereospecific hydroxylation at position 4 of the 2,3,4,7-tetrahydroazepine ring. The structure assignment was based on the single-crystal X-ray analysis (Figure 4) and a possible mechanism for this hydroxylation is shown in Scheme 4. Oxidation of amines with peracids is known to proceed through oxoammonium cation formation [12]. The close proximity of this reactive group to the allyl Beilstein J. Org. Chem. 2019, 15, 2664–2670. 2667 Scheme 4: A proposed mechanism for nitroxide 6 synthesis. Scheme 3: Synthesis of nitroxide 6. Figure 4: X-ray structure of compound 6 (one of the two enantiomers present in the crystal). hydrogen results in hydride abstraction with the formation of carbocation 9 and subsequent cyclization to the bicyclic alkoxyamine 10. The resulting isoxazolidine ring is then opened with m-CPBA in the usual way, retaining the configuration of the asymmetric center at 3aC-OH [13] (Scheme 4). The steric strain in molecules 1 and 6 is characterized by an elongation of the bonds C6A–C7 1.579(2), C8–C8A 1.573(2) Å in 1 and C3B–C6A 1.580(2), C8–C8A 1.573(2) Å in 6. The conformation of the azepane ring in 1 is close to a distorted boat with a kink at the C3A–N1 line, while in 6 the conformation of this ring is close to a distorted half-chair. Presumably, this difference is due to the formation of an intramolecular hydrogen bond O6–H...O5 (H...O 1.95(2) Å, O–H...O 154(2)°) in molecule 6. In the crystal of 1 the amino group participates in intermolecular hydrogen bonding N1–H...O3 (H...O 2.39(2) Å, N–H...O 167(1)°) forming chains of molecules along the a-axis. EPR measurements Figure 4 demonstrates the X-band CW EPR spectra of nitroxide 6 in water/glycerol solution at 180 K and at room temperature with simulations (red) using the parameters listed in the caption. The electron spin relaxation of nitroxides with different bulky substituents has been studied in water/glycerol solutions at low temperatures in numerous papers [9,14-16]. Previously, we have investigated the electron spin relaxation of a series of nitroxides with different bulky substituents in water solution and in trehalose [10]. Because 6 is a new class of a hindered nitroxide, we investigated its electron spin relaxation properties in water/glycerol solution which is the solvent of choice for biomolecular distance measurements by PELDOR/DEER. If the rotation of the radical is prevented, the primary relaxation mechanisms are (i) modulation of the 14N hyperfine interaction Beilstein J. Org. Chem. 2019, 15, 2664–2670. 2668 Figure 5: A, B) Temperature dependence of the electron spin relaxation times in water/glycerol at X-band frequency. The solid red dots are for nitroxide 6; the open black squares are for spirocyclohexane-substituted nitroxide. C, D) CW EPR spectra of nitroxide 6 at 180 K (C) and at room temperature (D) in water/glycerol. Black lines are experimental spectra and the red lines are simulations for hfi, g-factor and correlation times of A(N) = [0.71; 0.71; 3.71] mT, g = [2.0087; 2.0055; 2.0019], tcorr = 0.53 ns (at 298 K). (hfi) anisotropy of the NO group by librational motion, and (ii) modulation of the hfi with other nuclei in the radical by rotation of the groups containing those nuclei (e.g., rotation of methyl groups). The temperature dependence of spin relaxation reveals the relevant mechanism. For comparison, relaxation of a spirocyclohexane-substituted nitroxide [10] is also shown in Figure 5. The Tm vs T dependence generally shows consistent trends in frozen solutions. For 2,5-tetramethyl-substituted pyrrolidine and piperidine nitroxides, a local maximum appears in their ph


Introduction
Domino reactions have attracted much attention as an approach for the synthesis of complex molecules in a few steps [1]. The utility of multicomponent reactions involving amines, activated olefins and carbonyl compounds for the synthesis of heterocyclic compounds has been repeatedly demonstrated [2,3]. We recently used a domino reaction of amino acid, ketone and dimethyl fumarate for the one-pot synthesis of a substituted pyrrolidine, which then was converted into a reduction-resistant pyrrolidine nitroxide [4]. Here we report the unexpected formation of a highly strained polycyclic amine from cyclopen- tanone, dimethyl fumarate and ammonium acetate. This multistep reaction obviously includes the 1,3-dipolar cycloaddition of dimethyl fumarate with cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was then converted into a sterically shielded polycyclic nitroxide.
Sterically hindered nitroxides have high chemical stability [5] and can be used as spin labels to study biopolymers in cells [6]. The introduction of spirocyclic moieties has a smaller effect on the reduction rates of nitroxides than does the introduction of linear alkyl substituents. However, spirocyclic nitroxides may have much longer spin relaxation times at 70-150 K which make them attractive agents for spin labeling [7][8][9]. Sterically hindered nitroxides can be used as spin labels for measurements at room temperature [10]. In this paper we examined the properties of this new nitroxide, in particular, the electron spin relaxation at different temperatures in water/glycerol solution.

Results and Discussion
Synthesis Polycyclic amine A mixture cyclopentanone, dimethyl fumarate and ammonium acetate was refluxed in benzene in a Dean-Stark apparatus. The reaction mixture underwent a strong tarring and after standard extraction with aqueous sulfuric acid, the main product 1 was isolated in a low yield (ca. 5%, see Scheme 1). In another experiment a much higher yield was obtained (ca. 15%), however, we failed to reproduce this result and in subsequent experiments the yields were close to the 5% limit. The 1 H NMR spectra of 1 show signals of two ester groups and multiple methylene and methyne protons with an overall intensity of  13 C NMR spectra (see Table 1). The structure of the compound was finally confirmed Scheme 2: Possible mechanism for the formation of 1.
by X-ray analysis and elemental analysis data ( Figure 1). A possible mechanism for the formation of 1 is presented in Scheme 2. It is well-known that cyclopentanone is prone to self-condensation. In the presence of ammonia these reactions may lead to heterocycle formation [11]. Presumably, a prototropic shift in the enamine-imine intermediate 4 is followed by electrocyclization to the cyclic azomethine ylide, which then reacts with dimethyl fumarate in a 1,3-dipolar cycloaddition. The suggested mechanism accounts for the trans-position of the methyne hydrogens in the azepine ring: electrocyclization proceeds via a conrotatory mechanism due to the antisymmetry of the HOMO ( Figure 2). The selective formation of a single diastereomer in the 1,3dipolar cycloaddition reaction is likely a result from secondary interactions of orbitals of the π-systems of the dipole and dipolarophile ( Figure 3).

Nitroxide
Oxidation of 1 with m-CPBA afforded the nitroxide 6 with 48% yield (Scheme 3). It is noteworthy that the oxidation of the amino group is accompanied by the stereospecific hydroxylation at position 4 of the 2,3,4,7-tetrahydroazepine ring. The structure assignment was based on the single-crystal X-ray analysis ( Figure 4) and a possible mechanism for this hydroxylation is shown in Scheme 4. Oxidation of amines with peracids is known to proceed through oxoammonium cation formation [12]. The close proximity of this reactive group to the allyl Scheme 4: A proposed mechanism for nitroxide 6 synthesis.   Figure 4 demonstrates the X-band CW EPR spectra of nitroxide 6 in water/glycerol solution at 180 K and at room temperature with simulations (red) using the parameters listed in the caption. The electron spin relaxation of nitroxides with different bulky substituents has been studied in water/glycerol solutions at low temperatures in numerous papers [9,[14][15][16]. Previously, we have investigated the electron spin relaxation of a series of nitroxides with different bulky substituents in water solution and in trehalose [10]. Because 6 is a new class of a hindered nitroxide, we investigated its electron spin relaxation properties in water/glycerol solution which is the solvent of choice for biomolecular distance measurements by PELDOR/DEER. If the rotation of the radical is prevented, the primary relaxation mechanisms are (i) modulation of the 14 N hyperfine interaction (hfi) anisotropy of the NO group by librational motion, and (ii) modulation of the hfi with other nuclei in the radical by rotation of the groups containing those nuclei (e.g., rotation of methyl groups). The temperature dependence of spin relaxation reveals the relevant mechanism. For comparison, relaxation of a spirocyclohexane-substituted nitroxide [10] is also shown in Figure 5. The T m vs T dependence generally shows consistent trends in frozen solutions. For 2,5-tetramethyl-substituted pyrrolidine and piperidine nitroxides, a local maximum appears in their phase relaxation (1/T m ) at T > 100 K as thermally activated rotation of their methyl groups becomes rapid. Above 140 K this rotation is rapid enough to average the hfi anisotropy and to cause some decrease in the phase relaxation rates. Finally, at T > 220 K the librational motion of the NO group dominates phase relaxation and causes the relaxation rate to increase with temperature in soft matter [14,17]. Consequently, the common tetramethyl-substituted nitroxides are characterized by a local bell-shaped maximum in their phase relaxation at T > 100 K, that is absent in more hindered nitroxides. In contrast, no bell-like shape is observed in the phase relaxation of nitroxides with two spirocyclohexane moieties adjacent to the N-O • group [8][9][10] or for nitroxide 6. The temperature dependence of 1/T m is similar for both radicals in Figure 5 with some divergence above 180 K.

Conclusion
An unexpected formation of a highly strained polycyclic amine from cyclopentanone, dimethyl fumarate and ammonium acetate was observed, and this amine was converted to a sterically-shielded polycyclic nitroxide. The temperature dependence of the electron spin relaxation of the nitroxide in water/ glycerol solution is very similar to that of the previously studied nitroxides with two spirocyclohexane moieties adjacent to the N-O• group despite their very different structures.
Experimental 1 H NMR spectra were recorded at 400 or 600 MHz, and 13 C NMR spectra were recorded at 100 or 150 MHz, respectively. 1 H and 13 C NMR chemical shifts (δ) were internally referenced to the residual solvent peak. IR spectra were acquired on an FTIR spectrometer in KBr and are reported in wavenumbers (cm −1 ). Reactions were monitored by TLC using UV light 254 nm, 1% aqueous permanganate and Dragendorff reagent as visualizing agents. Column chromatography was performed on silica gel 60 (70-230 mesh). X-ray diffraction data were obtained with a Bruker KAPPA APEX II diffractometer using ϕ, ω scans with Mo Kα radiation (λ = 0.71073 Å) and a graphite monochromator. CCDC 1947797 (for 1) and 1947798 (for 6) contain the supplementary crystallographic data for this paper.

EPR measurements
Continuous wave (CW) EPR measurements were carried out using a commercial Bruker Elexsys E540 X-band spectrometer. The CW EPR spectra were simulated using EasySpin [18].
Pulse EPR experiments were carried out using a commercial Bruker Elexsys E580 X/Q-band spectrometer equipped with an Oxford helium flow cryostat and temperature control system. An ER 4118X-MD5W resonator was used for X-band measurements. T m was measured using a two-pulse electron spin echo (ESE) sequence; T 1 was measured using the inversion-recovery technique with a π-pulse for inversion and a two-pulse ESE sequence for detection. The π-pulse lengths were nominally 20 ns.

Supporting Information
Supporting Information File 1 Copies of the IR and 1 H, 13 C, HMBC, HSQC NMR spectra and X-ray analysis data.