Unexpected one-pot formation of the 1H-6a,8a-epiminotricyclopenta[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 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.