The ¹H NMR Spectroscopic Effect of Steric Compression Is Found in [3.3.1]Oxa- and Azabicycles and Their Analogues

Abstract

The through-space ¹H NMR effect of steric compression by the lone-pair electrons of O- and N-atoms is shown in synthetic [3.3.1]oxa- and azabicycles. The electrons of the compressed proton bond are pushed away by the repulsive force generated by the lone-pair electrons of the heteroatom. There is a corresponding significant increase in the chemical shift of the compressed proton. The intensity of this deshielding effect is related to the proximity and overlap of the lone-pair or compressing atom. The steric compression decreases when the lone-pair electrons of the heteroatom and the compressed proton are not directly overlapped, for example, in [4.3.1]- and [3.2.1]azabicycles. Steric compression is also caused by a proton, deuterium, or an ethyl group close in space to the compressed proton. The protonated [3.3.1]azabicycle adopts a true-boat/true-chair conformation in its crystal lattice, but in solution the conformation is true-chair/true-chair.

1. Introduction

The intramolecular through-space interaction that causes steric compression is detectable in ¹H NMR studies. The chemical shifts of the compressed proton and its neighbor on the methylene group shift significantly.¹ Some inflexible C-skeleta (Figure 1), for example, half-cage cyclopentyl (1), norbornenes (2 and 3), and imino[14]annulene (4), have been designed and synthesized to demonstrate elegantly that the compressed proton must be close in space to the source of the repulsive force, for example, H, OH,^1,2 ether oxygen,³ alkene π-cloud,⁴⁻⁶ and NH.⁷

Figure 1.

Designed reporter molecules (1–4) and the natural product methyllycaconitine (5, MLA).

For two protons attached to the same carbon of a cyclohexane ring in a chair conformation, it is known that the chemical shift of the equatorial proton (∼1.6 ppm) is larger than that of its geminal axial proton (1.1 ppm) by Δδ ∼ 0.5 ppm (at −103 °C in CS₂, 60 MHz),⁸ and this is the result of the magnetic anisotropic effect.⁹ For 4-H_a and 4-H_e of cyclohexanone, the difference between their chemical shifts (0.24 ppm, at <−185 °C in a 5:1 CHClF₂/CHCl₂F mixture, 251 MHz) is relatively small.¹⁰

C₁₉-Norditerpenoid alkaloid methyllycaconitine (5, MLA) is one of the most potent competitive antagonists of α7-nicotinic acetylcholine receptors (α7-nAChRs) with a highly selective targeting of the snake venom toxin α-bungarotoxin (α-BgTx) binding sites.¹¹ In terms of structure, norditerpenoid alkaloids are hexacyclic with bridged structures leading to well-defined conformations. The synthesis of cyclic analogues mimicking MLA (5)¹²⁻¹⁶ has been reported where the C2 axial and equatorial methylene group protons show a significant difference (∼1 ppm) in their chemical shifts.¹⁷⁻²²

In this study, the large ¹H NMR separations of the two signals on the methylene groups of different synthetic bridged [3.3.1]oxa- and azabicycles are reported. In order to explain this observation, comprehensive 1D/2D NMR spectroscopy and single-crystal X-ray analyses have been undertaken, which demonstrate that the effect of steric compression is found in these [3.3.1]bicycles and compared with the effect in analogues of various ring sizes.

2. Results and Discussion

2.1. Conformational Analysis of [3.3.1]Azabicycles

An A/E-bicyclic [3.3.1]analogue (8) of norditerpenoid alkaloids was prepared via a double-Mannich reaction (Scheme 1). ¹H, ¹³C, heteronuclear single-quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser enhancement spectroscopy (NOESY) were used to assign the product (8).¹²⁻²² One of the protons attached to C7 is significantly deshielded and resonates at 2.86 ppm; the geminal methylene proton resonates at 1.53 ppm. The chemical shifts of these 7-H are separated by 1.33 ppm. This deshielding must be through space, as there are no significantly electronegative functional groups nearby.

Scheme 1. Synthesis of [3.3.1]Azabicycle (8).

The tertiary amine nitrogen is assumed to be responsible for the observed shift as it should be close to 7-H_a, if the [3.3.1]azabicycle (8) adopts a chair/chair conformation. The deshielding was observed for 7-H_a. This is an example of steric compression, where the lone-pair electrons of the N-atom generate a repulsive force pushing the electron cloud surrounding 7-H_a away and thus decreasing its electron density, leading to 7-H_a resonating at a low field. This explanation holds if the [3.3.1]azabicycle (8) adopts a chair/chair conformation with the N-ethyl group in the equatorial position and therefore the N-atom lone-pair electrons are close to 7-H_a. Due to the intramolecular hindrance in the “half-cage” structure, it is certainly possible that the AE-[3.3.1]bicycle (8) can adopt a boat/chair or a chair/boat conformation rather than an “obvious” chair/chair conformation. NOESY data (Figure 2) of the [3.3.1]azabicycle (8) were obtained. 2-H_a is correlated with 4-H_a. 2-H_e and 4-H_e are correlated with 8-H_e and 6-H_e, respectively. Therefore, the piperidine ring of [3.3.1]azabicycle (8) has adopted a chair conformation.

Figure 2.

Key NOESY correlations and the coupling patterns of key ¹H NMR signals of [3.3.1]azabicycle (8).

The coupling pattern of 5-H of the compound (8) is shown in Figure 2, which is displayed as a dq peak (5.3, 3.0 Hz). The lack of a large coupling constant (caused by 6-H_e in the eclipsed position of a boat/chair conformation, Figure 3) for 5-H strongly suggests that the cyclohexane ring of this [3.3.1]azabicycle (8) is in a chair conformer. 5-H couples with 4-H_a, 4-H_e, 6-H_a, and 6-H_e. If the [3.3.1]azabicycle (8) adopts a chair/chair conformation, dihedral angles ∠(4-H_a)–C4–C5–(5-H), ∠(4-H_e)–C4–C5–(5-H), ∠(6-H_a)–C6–C5–(5-H), and ∠(6-H_e)–C6–C5–(5-H) are the same and equal to ∼60° (Figure 3). Therefore, two small ³J_ae (typically 5–7 Hz, 4-H_a, and 6-H_a) and two even smaller ³J_ee (typically 2–4 Hz, 4-H_e and 6-H_e) are supposed to be displayed in the coupling pattern of the equatorial 5-H, according to the Karplus relationship. If the bicycle (8) adopts a boat/chair conformation, dihedral angles ∠(4-H_a)–C4–C5–(5-H), ∠(4-H_e)–C4–C5–(5-H), and ∠(6-H_a)–C6–C5–(5-H) are still the same ∼60° (6-H_a in the cyclohexane ring adopting a boat conformation is 6-H_e in the cyclohexane ring adopting a chair conformation), but 6-H_e is now in the eclipsed position of the equatorial 5-H, so ∠(6-H_e)–C6–C5–(5-H) is ∼0° which generates a large coupling constant (∼10 Hz), so the signal of 5-H should be a “doublet” when the conformation is a boat/chair.

Figure 3.

[3.3.1]Azabicycles in two different conformations.

The coupling patterns of 7-H_a and 7-H_e are different (Figure 2), which were used to assign their orientations. 7-H_a couples with 7-H_e, 6-H_a, 8-H_a (²J_gem = ³J_aa = 12.4 Hz) and 6-H_e and 8-H_e (³J_ae = 6.1 Hz); therefore, it shows as a qt. 7-H_e couples with 7-H_a (²J_gem = 12.4 Hz); 6-H_a, 8-H_a (³J_ae = 6.1 Hz); and 6-H_e and 8-H_e (³J_ae = 3.0 Hz); therefore, it resonates as a dtt. These assignments confirmed that the 7-H_a is deshielded rather than shielded by the lone-pair electrons of the N-atom.

The coupling constants of 6-H_a and 8-H_a that are caused by 7-H_a are equal (12.4 Hz); therefore, the dihedral angles ∠(6-H_a)–C6–C7–(7-H_a) and ∠(8-H_a)–C8–C7–(7-H_a) are the same or highly similar, indicating this chair conformation of the cyclohexane ring is true (not twisted).^23,24

NMR spectra of azabicycle (8) are also obtained in CDCl₃, CD₃OD, and d₆-DMSO, and the chemical shifts for 7-H_a and 7-H_e are given in Table S8. In all three solvents, the 7-H_a of azabicycle (8) is significantly deshielded; therefore, this effect cannot be attributed to solvent effects. In addition, variable temperature ¹H NMR experiments of azabicycle (8) in d₆-DMSO were used to investigate the stability of the chair/chair conformation (Figure S1). It is clear that the chemical shifts of both 7-H_a and 7-H_e barely change (∼0.2 ppm) when the azabicycle (8) solution was heated, so 7-H_a still experiences steric compression; therefore, the molecule adopts a nearly inflexible chair/chair conformation at 25–125 °C.

The 7,7-dimethyl[3.3.1]azabicycle (10) was prepared (Scheme 2) as an example of adopting a boat/chair conformer. As two methyl groups are introduced at C7, the cyclohexane or piperidine ring flips into a boat conformation. The piperidine ring is in a chair conformation as shown by the NOESY correlations of 2-H_a/4-H_a, 2-H_e/8-H_e, and 4-H_e/6-H_e (Figure 4). In contrast to the coupling pattern of 5-H in [3.3.1]azabicycle (8, Figure 2), 5-H of 7,7-dimethyl[3.3.1]azabicycle (10) displayed a dq with a large coupling constant (³J_x–x = 10.5 Hz) that originates from eclipsed 6-H_e (Figure 4), confirming that the cyclohexane ring of 7,7-dimethyl[3.3.1]azabicycle (10) adopts a boat conformation.

Scheme 2. Synthesis of 7,7-Dimethyl[3.3.1]azabicycle (10).

Figure 4.

NOESY correlations and the coupling pattern of 5-H of boat/chair [3.3.1]azabicycle (10).

To support the ¹H NMR assignment of 7-H_a and 7-H_e of the azabicycle (8), 7-alkyl substituted [3.3.1]azabicycle (12), (14), and (17) were synthesized (Scheme 3). In these [3.3.1]azabicycles (12), (14), and (17), the bulky 7-alkyl groups will preferentially adopt the equatorial positions and therefore the δ (7-H_a) can be demonstrated unequivocally. The δ (7-H_a) of products (12), (14), and (17) resonate (in CDCl₃) at 3.02, 3.42, and 3.42 ppm, respectively, which are similar values to the δ (7-H_a) of azabicycle (8) as these 7-H_a are experiencing the same steric compression. Therefore, assignments of 7-H_a at 2.86 ppm and of 7-H_e at 1.53 ppm of the [3.3.1]azabicycle (8) are confirmed.

Scheme 3. Synthesis of 7-Alkyl-Substituted [3.3.1]Azabicycles.

2,4-Dinitrophenylhydrazine (2,4-DNPH) was used to derivatize these oily ketones (8), (12), and (17) in order to obtain crystalline derivatives (18–20, respectively Scheme 4) for single-crystal X-ray diffraction (SXRD). NMR data of dinitrophenylhydrazone (DNP) derivatives (18–20) also show that each 7-H_a resonates at a low field (Table S9), suggesting that both the ketone starting materials (8), (12), and (17) and their DNP derivatives (18–20) adopt the same solution conformations. SXRD data (18–20) were obtained (Figure 5). The [3.3.1]azabicyclic DNP derivatives (18), (19), (20a), and (20b) adopt true-chair/true-chair conformations with the N-ethyl groups in the equatorial positions and 9-imine hydrazinyl groups adopt E-configurations. 7-iPr and 7-Me groups of DNP derivatives (19), (20a), and (20b) are equatorial. These conclusions, based on the SXRD data, are consistent with the NMR studies of the synthetic [3.3.1]azabicycles (8), (12), and (17–20). The single crystals of 7-Me derivatives (20a) and (20b) are packed in the same unit cell; they are enantiomers.

Scheme 4. 2,4-DNP Derivatization of [3.3.1]Azabicycles (8), (12), and (17).

Figure 5.

SXRD data of 2,4-DNP derivatives (18–20), ORTEP presentations of the crystal structures show the atom position with a 50% probability for each ellipsoid.

2.2. Impacts of Change in Ring Size on Steric Compression

To investigate whether the ring size influences the ¹H NMR effect of steric compression, [4.3.1]- and [3.2.1]azabicyclic analogues (23) and (27) were prepared (Scheme 5). According to key NOESY correlations of [4.3.1]- and [3.2.1]azabicycles (23) and (27) (Figure 6), all the orientation assignments are confirmed and the piperidine rings of both analogues (23) and (27) are proven to adopt chair conformations. As δ (3-H_a′) and δ (4-H_a′) of [4.3.1]azabicycle (23) are larger than δ (3-H_e′) and δ (4-H_e′), thus these 3-H_a′ and 4-H_a′ are experiencing steric compression (Δδ_3-H = 0.68 ppm, Δδ_4-H = 0.45 ppm, Table S10), and the cycloheptane ring adopts a chair conformation.²⁴ Axial and equatorial are suitable for describing the orientations of protons attached to a six-membered ring, for example, cyclohexane in a true-chair conformation. For cyclopentane and cycloheptane rings, protons attached to the ring are better described as pseudo-axial (a′) and pseudo-axial (e′).²⁵ In this study, a (a′) and a (e′) are preferred rather than exo and endo for retaining consistency with labeling used in the monoring system. Δδ_6-H and Δδ_7-H of [3.2.1]azabicycle (27) are only small values (<0.2 ppm). The steric compression decreases if the N-atom and the compressed proton are in a staggered relationship.

Scheme 5. Synthesis of [4.3.1]- and [3.2.1]Azabicyclic Analogues.

Figure 6.

Key NOESY correlations of [4.3.1]- and [3.2.1]azabicyclic analogues (23) and (27).

Both [4.3.1]azabicycle (23) and [3.2.1]azabicycle (27) were converted into 2,4-DNP derivatives in order to produce crystalline products (24) and (28), and crystals of [4.3.1]azabicyclic derivative (24) suitable for X-ray analysis were obtained. Twin-packed crystal structures in one unit cell in this crystal are stereoisomers (24a) and (24b), and these stereoisomers are extracted from the original SXRD data and shown separately, as shown in Figure 7. Chair conformations of piperidine rings with equatorial N-ethyls are displayed in both isomers with hydrazone imines in the E-configuration, and these two stereoisomers (24a) and (24b) can be distinguished as 1R- and 1S-esters. Unlike the twin-packed crystal structures of enantiomers 7-Me [3.3.1]azabicyclic DNP derivative (20a) and (20b), the two crystal stereoisomers (24a) and (24b) are not mirror images on the basis of comparison between them in different view angles (Figure 8). A cycloheptane ring is more flexible and its conformations are more various than that of a cyclohexane ring.²⁶

Figure 7.

SXRD data of [4.3.1]azabicyclic DNP derivatives (24a) and (24b).

Figure 8.

Comparison between bicyclic carbon skeleta in different stereoisomers (24a, left and 24b, right).

The NMR spectroscopic study of the [4.3.1]azabicycles (23 and 24, Table S10) showed that the Δδ_3-H is slightly larger than Δδ_4-H, suggesting that 3-H_a′ is experiencing more steric compression than 4-H_a′, and thus, it may, on average, sit closer to the N-atom. This theoretically preferred conformation of [4.3.1]azabicycle revealed by NMR is similar to 1S,6S-isomer 24b (Figure 8, right).

Computer projections of the cycloheptane boat and chair conformations were reported by Bocian et al.(27) In their paper, they especially drew attention to the eclipsed hydrogens at the “stern”, the left side of the projection (Figure 9) of the boat, and the chair conformations. In the twist-chair conformation, these previously eclipsed hydrogen atoms are now shown on the basis of SXRD data and NMR analysis to be staggered.

Figure 9.

3D depictions of [4.3.1]azabicycle (24b).

2.3. No Steric Compression in a Mono-Mannich Product

To prove that 7-H_a of the synthetic [3.3.1]azabicycles is being sterically compressed by the lone-pair electrons of the N-atoms, a mono-Mannich reaction was designed and carried out to give the monocyclic product (29). β-Keto ester (6) was treated with 0.9 equiv. formaldehyde and 0.9 equiv. ethyl amine, and the reaction was heated at 40 °C, rather than under reflux, for 3 h giving the target product (29) (Scheme 6). In the mono-Mannich product (29), there is no piperidine ring; thus, the lone-pair electrons of the N-atom are away from 5-H that correlates to 7-H of the double-Mannich products (8).

Scheme 6. Synthesis of Mono-Mannich Product (29).

The ¹³C signal assignments of the mono-Mannich product (29) are assigned (Table S11) compared with those of β-keto ester (6, keto tautomer). Compared with Δ_7-H (1.33 ppm) of the double-Mannich product (8), Δ_5-H (0.09 ppm) of the mono-Mannich product (29) is significantly smaller, demonstrating that a significant ¹H NMR steric compression of 7-H_a of [3.3.1]azabicycles requires the N-atom to be close in space to 7-H_a.

2.4. Reducing 9-Ketone

An alternative explanation for the observed chemical shifts may possibly be attributed to the 9-ketone group of [3.3.1]azabicycle (8) displaying a long-range anisotropic effect on axial or equatorial protons. To investigate this, the ketone (8) was treated with lithium aluminum hydride to reduce both the 1-ester and the 9-ketone functional groups affording a diol (30, Scheme 7).

Scheme 7. Reduction of Azabicycle (8) Providing Diol (30).

In CDCl₃, CD₃OD, and d₆-DMSO, 9-ketone of azabicycle (8) has no obvious magnetic effect on 7-H_a or 7-H_e as chemical shifts of 7-H_a and 7-H_e of the diol (30) remain at similar values compared to those of 7-H_a and 7-H_e of ketone (8) when 9-ketone is reduced (Table S12). This is also consistent with diol (30) adopting true-chair/true-chair conformations in these three solvents. Interestingly, the relative difference in the shifts for 7-H_a of the diol (30) obtained in D₂O is reduced by ∼0.7 ppm to Δδ_7-H = 0.45 ppm. The intensity of the effect of steric compression in diol (30) decreases in D₂O, which is consistent with the solution conformation of the cyclohexane ring adopting a boat conformation. This makes the N-atom away from 7-H_a. The orientation of 8-H_e of diol (30) has been confirmed by NOESY correlation 2-H_e/8-H_e, and this signal is a dd peak (Figure 10) as it couples with 8-H_a (²J_gem = 13.5 Hz) and 7-H_a (³J_ae = 7.0 Hz). Due to the absence of a large ³J_aa ∼ 14 Hz (7-H_f, dihedral angle ∼ 180°, Figure 11), the diol (30) adopts a chair/chair conformation in D₂O. When the cyclohexane ring of the diol (30) adopts a chair conformation (Figure 11), the coupling pattern of 8-H_e, the proton that is close to 2-H_e, should contain only one large coupling constant of ²J_gem ∼ 14 Hz (8-H_a), and it also couples with 7-H_a and 7-H_e (typically ³J_ae ∼ 6 Hz and ³J_ee ∼ 3 Hz, both dihedral angles ∼ 60°). If the cyclohexane ring of this diol (30) adopts a boat conformation, 8-H_a, which is 8-H_e of the chair-like cyclohexane ring, the proton that is close to 2-H_e, then its coupling pattern should consist of two large coupling constants, ²J_gem ∼ 14 Hz (8-H_a) and ³J_aa ∼ 14 Hz (7-H_f, dihedral angle ∼ 180°), and it also couples with 7-H_b, which results in a small coupling constant (dihedral angle ∼ 60°).

Figure 10.

Key NOE correlations and the coupling pattern of 8-H_e of diol (30) in D₂O.

Figure 11.

Diol (30) in two different conformations.

2.5. Protonation

Norditerpenoid alkaloids and their synthetic analogues are bases. They are typically partly protonated in the aqueous components of body fluids even at neutral pH. Thus, it is valuable to understand the conformation of the protonated form of [3.3.1]azabicycle (8) that is synthesized for mimicking the A/E-rings of bioactive norditerpenoid alkaloids especially MLA (5). The [3.3.1]azabicycle (8) was separately dissolved in d₄-acetic acid and concd HCl aq solutions in order to obtain protonated compounds (31–33, Scheme 8), which are the ketone salts (31) and (32) and the ketal (hydrate) salt (33), respectively.

Scheme 8. Acidification of [3.3.1]Azabicycle (8).

The acetate salt (31) shows δ (7-H_a) = 2.42 ppm > δ (7-H_e) = 1.72 ppm in d₄-acetic acid, Δδ_7-H = 0.70 ppm (Table S13), which means the NH(D) is able to provide steric compression acting on 7-H_a. Therefore, the conformation of this salt (31) is determined to be true-chair/true-chair as significant compression is displayed. The compression is caused by the electrons in the new bond of NH/D, as there are no lone-pair electrons available. SXRD data of the chloride salt (32) were determined, which shows that this salt (32) adopts a true-boat/true-chair crystal conformation with the N-ethyl in the equatorial position (Figure 12).

Figure 12.

SXRD data of HCl salt (32) and the coupling pattern of 5-H of ketal salt (33).

NMR spectroscopic analyses of the crystalline chloride salt (32) in d₄-acetic acid gave similar results to those of the acetate salt (31) in d₄-acetic acid. NMR data of the chloride salt (32) show a significant Δδ_7-H = 0.87 ppm (Table S13), suggesting that the solution conformation of this chloride salt (32) in d₄-acetic acid is true-chair/true-chair. Therefore, the crystal conformation of the synthetic [3.3.1]azabicyclic chloride salt (32) is different from its solution conformation.

When the crystalline ketone chloride salt (32) was dissolved in D₂O (or wet solvents, e.g., CD₃OD and d₆-DMSO), the ketal (hydrate) salt (33) was obtained. To determine the conformation of this salt (33), the ¹H signal of 5-H was employed (Figure 12). On the basis of the shape (even though broad) of this 5-H, it does not contain a large coupling constant such as ³J_x–x (10.5 Hz) of the 5-H of 7,7-dimethyl azabicycle (10), cf.Figure 4, this ketal salt (33) adopts a chair/chair conformation.

2.6. Methylation

[3.3.1]Azabicycle (8) was methylated with MeI (5.0 equiv) heated under reflux for 24 h (Scheme 9). The key NOESY data of this methylated product (34) are given in Figure 13. The 2-H_a of the compound (34) has a NOESY correlation with 4-H_a, and 2-H_a is also NOESY correlated with 8-H_e, so the piperidine ring adopts a boat conformation. The 7-H_a is close to the 2-H_a in the space determined by NOESY correlation 2-H_a/7-H_a, thus the cyclohexane ring is in a chair conformation.

Scheme 9. N-Methylation of [3.3.1]Azabicycle (8).

Figure 13.

Key NOESY correlations and coupling patterns of key ¹H NMR signals methylated [3.3.1]azabicycle (34).

The N-2 has NOESY correlations with both 2-H_a and 2-H_e, but the N–Me only show NOESY correlation with the 2-H_e; therefore, the N–Me is in the flagpole position and the N–Et is determined to be in the bowsprit position.

The 7-H_a experiences the effect of steric compression presented by δ (7-H_a) = 2.84 ppm > δ (7-H_e) = 1.87 ppm, Δδ_7-H = 0.97 ppm. It is notable that the steric compression acting on the 7-H_a is caused by the methyl group of the N–Et rather than the lone-pair electrons of the N-atom, as the lone-pair electrons are no longer available.

If the piperidine ring adopts a true-boat conformation, the methyl group of the N–Et is far away from 7-H_a in space; therefore, the boat-like piperidine ring has to be mono-flattened (only the N-atom is flattened rather than both the N-atom and the C9 are flattened) allowing the methyl group of the N–Et to be close to the 7-H_a showing a significant steric effect on the 7-H_a.

Both 6-H_a and 8-H_a of methylated [3.3.1]azabicycle (34) (Figure 13) contribute equal coupling constants of ³J_aa = 13.8 Hz to the 7-H_a, which suggests that the cyclohexane ring adopts a true-chair conformation. The value of the ²J_gem between 7-H_a and 7-H_e of the methylated derivative (34) is 16.1 Hz, which is significantly larger than that of the ²J_gem (12.4 Hz) of the 7-H_a of azabicycle (8). This suggests that the ∠(7-H_a)–C7–(7-H_e) of the methylated derivative (34) becomes smaller than that of the azabicycle (8), as 7-H_a experiences a strong compression through space changing the geminal bond angle of ∠(7-H_a)–C7–(7-H_e).

2.7. Synthesis and Analysis of [3.3.1]Oxabicycle

To add further data about the effect of steric compression on ¹H NMR signals, a [3.3.1]oxabicyclic tetrahydropyranyl ether (36) was designed and then synthesized by intramolecular dehydration (Scheme 10).²⁸

Scheme 10. Synthesis of [3.3.1]Oxabicycle (36).

The product was recrystallized from EtOAc (∼14 h). SXRD data of this [3.3.1]bicyclic ether (36) show a true-chair/true-chair conformation with 9-OH in the equatorial position (Figure 14), supported by the related NOESY data.

Figure 14.

SXRD data, key NOESY correlations, and the coupling patterns of key ¹H NMR signals of [3.3.1]oxabicycle (36).

The signal of 6-H_e (8-H_e) resonates as a dd (Figure 14), coupled with 6-H_a (8-H_a, ²J_gem = 13.5 Hz) and 7-H_a (³J_ae = 6.5 Hz), determining that in solution the [3.3.1]oxabicycle (36) adopts a chair/chair conformation. The 7-H_a signal at 2.32 ppm is sterically compressed [δ (7-H_e) = 1.51 ppm, Δδ_7-H = 0.81 ppm] by the lone-pair electrons of the ether O-atom. Hence, the solution conformation of this ether (36) is true-chair/true-chair as found in its crystal lattice (Figure 14). The solution data are supported by the coupling pattern of 7-H_a resonating as a typical qt peak.

NMR data of [3.3.1]oxabicycle (36) in d₆-DMSO, d₆-acetone, and D₂O are also obtained, and key ¹H NMR data are given in Table S14. Compared with Δδ_7-H (0.81 ppm) of this ether (36) in CD₃OD, Δδ_7-H acquired from d₆-DMSO and d₆-acetone are similar, 0.80 and 0.92 ppm, respectively. However, Δδ_7-H measured in the D₂O solution is smaller (0.59 ppm). D₂O (H₂O) may form H-bonds with the ether O-atom, resulting in the oxygen lone-pair electrons perhaps being less available for compressing 7-H_a.

3. Conclusions

A through-space ¹H NMR effect of steric compression displayed in [3.3.1]azabicycles is demonstrated and fully discussed. It is caused by the lone-pair electrons of the N-atom generating an intramolecular repulsive force acting on 7-H_a leading to this proton being significantly deshielded. By comprehensive conformational analysis on these bicyclic compounds and their analogues via NOESY, coupling pattern analysis of key ¹H NMR signals, and SXRD the conformation of the bicycles and the configuration of protons of different methylene groups that experience steric compression are unambiguously assigned. The intensity of this compression is also proven to be related to the distance between the N-atom, especially its lone-pair electrons and the compressed proton: smaller distance and larger intensity. The key NMR data of several typical [3.3.1]bicycles in this work are summarized in Table 1 and shown compared to the literature data in Figure 15.

Table 1. ¹H NMR Data of Key Methylenes in [3.3.1]Bicycles (δ in ppm).

compound	solvent	δ (7-Ha)	δ (7-He)	Δδ7-H
[3.3.1]azabicycle (8)	CDCl3	2.86	1.53	1.33
	CD3OD	2.88	1.52	1.36
7-iPr [3.3.1]azabicycle (12)	CDCl3	3.02
[3.3.1]azabicyclic diol (30)	CDCl3	2.59	1.48	1.11
	CD3OD	2.58	1.42	1.16
	D2Oa	2.04	1.59	0.45
protonated [3.3.1]azabicycle ketone chloride salt (32)	d4-acetic acid	2.60	1.73	0.87
protonated [3.3.1]azabicycle ketal chloride salt (33)	CD3OD	1.78; 1.89b		0.11
	D2O	1.72; 1.83b		0.11
methylated [3.3.1]azabicycle (34)	CDCl3	2.84	1.87	0.97
[3.3.1]oxabicyclic ether (36)	CD3OD	2.32	1.51	0.81
	D2O	2.16	1.57	0.59

^aWith the addition of 2 drops of d₆-DMSO.

^bNo reliable evidence was obtained to identify the orientation of these protons.

Figure 15.

Intramolecular through-space interactions that cause steric compression are reported by the key ¹H NMR signals across a range of inflexible half-cage-type molecules where the chemical shifts of the compressed proton and its methylene group neighbor shift significantly compared with those in unsubstituted chair conformers of cyclohexane⁸ and cyclohexanone¹⁰ (δ in ppm).

Half-cage cyclopentyl (1), norbornenes (2 and 3), and imino[14]annulene (4) elegantly demonstrate that the compressed proton must be close in space to the source of the repulsive force, for example, H, OH, ether oxygen, alkene π-cloud, and the only literature example of a secondary amine. Winstein and colleagues reported (in 1965) a new kind of steric compression on one proton of a methylene pair when the other proton is strongly compressed, for example, by an oxygen functional group. This was found together with unusually large deshielding effects in their half-cage or endo,endo-fused skeleta.^1,2 Such rigid geometries and enormous H–H or H–O steric oppositions are ideally suited for the study of effects of steric compression on chemical shifts, where the inside protons are strongly deshielded. Cava and Scheel (in 1967) concluded that the ether oxygen bridge exerts considerable shielding and deshielding effects on the methylene bridge protons, which are separated from each other by Δδ = 1.76 ppm, where such a large difference in the chemical shifts of the methylene bridge protons of a norbornene or norbornane was then unprecedented.³ Marchand and Rose (in 1968) reported that of particular interest is the effect of the alkene electron π-cloud causing the unusually large value of Δδ = 1.49 ppm between the bridge norbornene protons.⁴ A comparable value had been noted only once before in the literature by Cava and Scheel.

The expected aromatic character was found in syn-1,6-imino-8,13-methano[14]annulene (4), first synthesized by Vogel and colleagues.⁷ This is the first, and outside of the [3.3.1]azabicycle of MLA (5) and related natural products and their analogues, and essentially the only (secondary) amine to show such a strong steric compression effect. The NH proton is exo-orientated, the bridge methylene protons are so magnetically different; they are an AX system, −1.52 (d, H_exo) and 2.08 (d, H_endo) (J_AX gem = 10.2 Hz). The chemical shifts of the two CH₂ protons therefore differ by 3.6 ppm! The chemical shifts of the exo-CH₂- and the NH-bridge protons are observed at a relatively high field, the endo-CH₂-bridge proton is strongly deshielded. Such a large Δδ might be due in a considerable part to its steric interaction with the spatially very close (H)N-group, possibly due to the van der Waals effect. The NH-bridge proton is assigned to the exo-position with a high degree of certainty on the basis of its resonance at a relatively high field, NH_exo [CCl₄, tetramethylsilane (TMS)] −2.07 (br s) ppm. If this proton were to be in the endo-position, then it would not only have to be markedly deshielded as a result of the H–H (steric) compression but also show a not-present nuclear Overhauser effect.⁷

This effect of steric compression can not only be caused by the lone-pair electrons of the N- or another heteroatom (e.g., O-atom) (Figure 16) but also by a proton (deuterium) or alkyl, for example, ethyl group that is close in space to the compressed proton. This conclusion can help in understanding the conformations of molecules related to [3.3.1]bicycles as a true-chair/true-chair conformation allows a significant steric compression to be demonstrated.

Figure 16.

Steric compression reported by the 7-methylene key ¹H NMR signals of [3.3.1]azabicycle (8) (upper) and [3.3.1]oxabicycle (36) (lower).

4. Experimental Section

4.1. Materials and General Methods

2,4-Dinitrophenylhydrazine (∼70%, wet with ∼30% water) was purchased from Fluorochem (U.K.). All other chemicals were purchased from Sigma-Aldrich (U.K.) and used as received. Deuterated solvents including d₄-acetic acid, d₆-acetone, d-chloroform, d₄-methanol, d₆-DMSO, and deuterium oxide (D₂O) were used for NMR experiments (99.8% D atom, Cambridge Isotope Laboratories, Inc., USA). All other solvents were of high-performance liquid chromatography grade, ≥99.9% purity (Fisher Scientific, U.K. and VWR, U.K.) including anhydrous solvents (Sigma-Aldrich, U.K. and Acros Organics, U.K.). Petroleum ether (Fisher Scientific, U.K.) specifically refers to the 40–60 °C distillate.

4.1.1. Instrumentation

¹H NMR spectra were recorded on Bruker Avance III spectrometers (¹H Larmor precession frequency, 400 and 500 MHz) at 25 °C. Chemical shifts are expressed in parts per million (ppm) downfield from TMS or 3-(trimethylsilyl)-propionic-2,2,3,3-d₄ acid sodium salt (TMSP) as internal or external standards, and residual (protio) solvent peaks were also used as internal standards if required. Chemical shifts (δ_H) are reported as the position (accurate δ_H of overlapping signals were extracted from 2D NMR spectra, e.g., HSQC, COSY, and NOESY), relative integral, multiplicity, and assignment. Multiplicity is abbreviated: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet; br = broad. Coupling constants (J) are line separations (absolute values expressed in hertz) rounded and rationalized to 0.1 Hz.

¹³C NMR spectra were recorded with complete proton decoupling on Bruker Avance III spectrometers (¹³C Larmor precession frequency 100 and 125 MHz) at 25 °C as well as 2D NMR experiments including HSQC and HMBC. Chemical shifts are expressed in a ppm downfield shift from TMS or TMSP as internal or external standards, and solvent peaks were also used as internal standards if required. Data are reported as the position (δ_C), number of attached protons (CH₃, CH₂, CH, quat = quaternary), and assignment.

Positive-ion [M + H]⁺ mode-mass spectrometry was performed on samples dissolved in methanol, using Bruker micrOTOF and Agilent Q-TOF mass spectrometers equipped with electrospray ionization (ESI) sources. Negative-ion [M – H]⁻ mode-mass spectrometry was performed on samples dissolved in methanol, on an Agilent ESI-Q-TOF mass spectrometer. High-resolution mass spectra were within 5 ppm error unless otherwise stated.

Intensity SXRD data were collected at 150 ± 2 K on a Rigaku SuperNova Dual, EosS2 system using monochromated Cu Kα radiation (λ = 1.54184 Å). Unit cell determination, data collection, and data reduction were performed using CrysAlisPro software CrysAlisPro 1.171.39.46 (Rigaku Oxford Diffraction, 2018). An empirical absorption correction using spherical harmonics was employed. The structures were solved with SHELXT and refined by a full-matrix least-squares procedure based on F² (SHELXL-2018/3).²⁹ All nonhydrogen atoms were refined anisotropically. Hydrogen atoms were placed onto calculated positions and refined using a riding model.

The removal of solvents by evaporation in the procedures specifically refers to the use of a Buchi R-114 rotary evaporator with warming samples to 40 °C on a Buchi B-480 water bath and in vacuo (50–500 mbar).

4.1.2. Chromatography

Flash chromatography³⁰ was performed using silica gel 60A 35–70 μm (Fluorochem Ltd., U.K. and Sigma-Aldrich, U.K.) with the indicated solvents. Thin-layer chromatography (TLC) was performed using 0.2 mm thick precoated silica gel plates (Merck KGaA 60 F₂₅₄). Compounds were visualized under ultraviolet light (UV, λ = 254 nm) and by staining with different reagents including iodine vapor, potassium permanganate aq solution (0.05 M), p-anisaldehyde solution (p-anisaldehyde/concd aq H₂SO₄/H₂O/acetic acid = 3:2:50:40, v/v), ninhydrin solution (0.2% w/v ninhydrin in ethanol), or Dragendorff’s reagent: bismuth subnitrate (1.7 g), acetic acid (20 mL), water (80 mL), and 50% w/v solution of potassium iodide in water (100 mL) were mixed and stored as a stock solution. The stock solution (10 mL) and acetic acid (20 mL) were mixed and made up to 100 mL with water to give Dragendorff’s reagent.

All the products after purification by chromatography were detected by TLC (UV, λ = 254 nm, and staining with at least two reagents) showing a single spot, and the residual solvents were removed under high vacuum for ∼14 h, then the NMR data of the products were recorded.

4.1.3. Synthesis and Structural Identification

4.1.3.1. Ethyl 2-Oxocyclohexane-1-carboxylate (6, Keto)

δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.28 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.68 (1H, m, 5-H_A), 1.84 (1H, m, 4-H_A), 1.87 (1H, m, 5-H_B), 1.97 (1H, m, 4-H_B), 2.11 (1H, m, 6-H_A), 2.16 (1H, m, 6-H_B), 2.37 (1H, ddd, J = 14.9, 10.3, 5.5 Hz, 3-H_A), 2.51 (1H, dt, J = 12.1, 5.5 Hz, 3-H_B), 3.37 (1H, dd, J = 9.7, 5.8 Hz, 1-H) and 4.21 (2H, m, OCH₂CH₃); δ_C (125 MHz; CDCl₃; calibrated with TMS): 14.17 (CH₃, OCH₂CH₃), 23.31 (CH₂, C5), 27.12 (CH₂, C4), 29.98 (CH₂, C6), 41.57 (CH₂, C3), 57.25 (CH, C1), 61.09 (CH₂, OCH₂CH₃), 170.02 (quat, COOEt) and 206.32 (quat, C2).

4.1.3.2. Ethyl 2-Hydroxycyclohex-1-ene-1-carboxylate (7, Enol)

δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.0 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.60 (2H, quin, J = 6.0 Hz, 5-H), 1.68 (2H, quin, J = 6.0 Hz, 4-H), 2.22 (2H, t, J = 6.3 Hz, 6-H), 2.27 (2H, t, J = 6.3 Hz, 3-H), 4.21 (2H, q, J = 7.1 Hz, OCH₂CH₃) and 12.25 (1H, s, OH); δ_C (125 MHz; CDCl₃; calibrated with TMS): 14.32 (CH₃, OCH₂CH₃), 21.97 (CH₂, C4), 22.42 (CH₂, C5 or C6), 22.44 (CH₂, C5 or C6), 29.11 (CH₂, C3), 60.15 (CH₂, OCH₂CH₃), 97.79 (quat, C1), 172.01 (quat, C2) and 172.80 (quat, COOEt).

4.1.3.3. Ethyl 3-Ethyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (8)¹²

Ethyl 2-oxocyclohexane-1-carboxylate (6, 50 μL, 0.30 mmol), ethylamine aq solution (66–72% w/w, 28 μL, 0.33 mmol), formaldehyde aq solution (37–40% w/w, 54 μL, 0.66 mmol), and EtOH (2 mL) were mixed with stirring at 20 °C. The solution was then heated under reflux for 2 h when TLC monitoring showed the reaction was complete (petroleum ether/ethyl acetate = 20:1 v/v, target compound R_f = 0.35, stained with p-anisaldehyde solution, or iodine vapor). After the solvent was removed by evaporation, the crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate = 20:1 v/v; mobile phase was basified concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase) giving pale yellow oil (8, 60 mg, 79%). MS (m/z): [M + H]⁺ found 240.1603, C₁₃H₂₂NO₃ requires 240.1600 and [M + Na]⁺ found 262.1410, C₁₃H₂₁NO₃Na requires 262.1419 and [2M + Na]⁺ found 501.3066, C₂₆H₄₂N₂O₆Na requires 501.2941. δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.10 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.29 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.53 (1H, dtt, J = 12.4, 6.1, 3.0 Hz, 7-H_e), 2.08 (1H, ddddd, J = 13.9, 12.4, 6.1, 5.3, 1.7 Hz, 6-H_a), 2.14 (1H, ddt, J = 13.9, 5.8, 3.0 Hz, 6-H_e), 2.24 (1H, ddt, J = 14.0, 6.1, 3.0 Hz, 8-H_e), 2.41 (2H, qd, J = 7.2, 2.4 Hz, NCH₂CH₃), 2.46 (1H, dq, J = 5.3, 3.0 Hz, 5-H), 2.53 (1H, dddd, J = 14.0, 12.4, 6.1, 1.7 Hz, 8-H_a), 2.57 (1H, br ddd, J = 11.1, 3.0, 1.3 Hz, 4-H_a), 2.86 (1H, qt, J = 12.4, 6.1 Hz, 7-H_a), 2.93 (1H, dd, J = 11.4, 1.7 Hz, 2-H_a), 3.15 (1H, dt, J = 11.1, 3.0 Hz, 4-H_e), 3.22 (1H, dd, J = 11.4, 2.4 Hz, 2-H_e) and 4.21 (2H, q, J = 7.2 Hz, OCH₂CH₃). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.73 (CH₃, NCH₂CH₃), 14.15 (CH₃, OCH₂CH₃), 20.56 (CH₂, C7), 34.19 (CH₂, C6), 36.84 (CH₂, C8), 47.23 (CH, C5), 51.12 (CH₂, NCH₂CH₃), 58.84 (quat, C1), 59.94 (CH₂, C4), 61.10 (CH₂, OCH₂CH₃), 61.67 (CH₂, C2), 171.24 (quat, COOEt) and 212.83 (quat, C9). δ_H (500 MHz; CD₃OD; calibrated with TMS): 1.11 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.26 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.52 (1H, dtt, J = 12.4, 6.1, 3.1 Hz, 7-H_e), 2.03 (1H, m, 6-H_a), 2.18 (1H, m, 6-H_e), 2.24 (1H, m, 8-H_e), 2.40 (1H, m, 5-H), 2.41 (2H, m, NCH₂CH₃), 2.48 (1H, m, 8-H_a), 2.52 (1H, m, 4-H_a), 2.88 (1H, m, 7-H_a), 2.90 (1H, m, 2-H_a), 3.23 (1H, m, 4-H_e), 3.23 (1H, m, 2-H_e) and 4.16 (2H, q, J = 7.2 Hz, OCH₂CH₃). δ_C (125 MHz; CD₃OD; calibrated with TMS): 13.05 (CH₃, NCH₂CH₃), 14.45 (CH₃, OCH₂CH₃), 21.52 (CH₂, C7), 35.32 (CH₂, C6), 37.99 (CH₂, C8), 48.71 (CH, C5), 52.20 (CH₂, NCH₂CH₃), 60.26 (quat, C1), 61.00 (CH₂, C4), 62.20 (CH₂, OCH₂CH₃), 62.88 (CH₂, C2), 172.66 (quat, COOEt) and 214.64 (quat, C9). δ_H [400 MHz; d₆-DMSO; calibrated with residual DMSO (2.50 ppm)]: 1.04 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.18 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.45 (1H, m, 1H, dtt, J = 12.1, 5.9, 2.7 Hz, 7-H_e), 1.95 (1H, m, 6-H_a), 2.10 (1H, ddq, J = 13.7, 5.5, 2.7 Hz, 6-H_e), 2.18 (1H, dq, J = 13.7, 2.7 Hz, 8-H_e), 2.35 (2H, m, NCH₂CH₃), 2.37 (1H, m, 8-H_a), 2.37 (1H, m, 5-H), 2.45 (1H, br dd, J = 11.1, 3.2 Hz, 4-H_a), 2.77 (1H, qt, J = 12.1, 5.9 Hz, 7-H_a), 2.81 (1H, dd, J = 11.5, 1.5 Hz, 2-H_a), 3.16 (1H, dt, J = 11.2, 2.2 Hz, 4-H_e), 3.22 (1H, dd, J = 2.2, 11.4 Hz, 2-H_e) and 4.21 (2H, qd, J = 7.2, 1.1 Hz, OCH₂CH₃). δ_C [100 MHz; d₆-DMSO; calibrated with d₆-DMSO (39.52 ppm)]: 12.51 (CH₃, NCH₂CH₃), 14.01 (CH₃, OCH₂CH₃), 19.97 (CH₂, C7), 33.57 (CH₂, C6), 36.12 (CH₂, C8), 46.46 (CH, C5), 50.49 (CH₂, NCH₂CH₃), 58.27 (quat, C1), 59.21 (CH₂, C4), 60.48 (CH₂, OCH₂CH₃), 61.10 (CH₂, C2), 170.46 (quat, COOEt) and 212.16 (quat, C9).

4.1.3.4. Ethyl 3-Ethyl-7,7-dimethyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (10)

Methyl 5,5-dimethyl-2-oxocyclohexane-1-carboxylate (9, 50 μL, 0.29 mmol), ethylamine aq solution (66–72% w/w, 27 μL, 0.32 mmol), formaldehyde aq solution (37–40% w/w, 52 μL, 0.64 mmol), and MeOH (2 mL) were mixed together successively with stirring. The round-bottomed flask with reactants was heated and stirred under reflux on a heating mantle for 2 h under an atmosphere of anhydrous nitrogen. TLC showed the reaction was complete (petroleum spirit/ethyl acetate = 2:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound R_f = 0.24, stained with potassium permanganate solution). After the solvents were removed by evaporation, the products were acidified with aq HCl (1 M) to pH = 2 followed by washing with dichloromethane (DCM) (3 × 5 mL), then the aqueous layer was basified with aq NaHCO₃ (pH = 8) and extracted with DCM (3 × 5 mL). The combined organic phase was dried (Na₂SO₄), filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate = 30:1 → petroleum ether/ethyl acetate = 20:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase), and finally pure colorless oil (10, 10 mg, 14%) was obtained. MS (m/z): [M + Na]⁺ found 276.1559, C₁₄H₂₃NO₃Na requires 276.1576. δ_H [500 MHz; CDCl₃; calibrated with residual CHCl₃ (7.26 ppm)]: 0.94 (3H, s, 2′-H), 1.00 (3H, s, 1′-H), 1.08 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.89 (1H, ddd, J = 13.3, 10.5, 2.8 Hz, 6-H_e), 2.12 (1H, dd, J = 13.3, 2.2 Hz, 6-H_a), 2.17 (1H, dd, J = 13.1, 2.8 Hz, 8-H_e), 2.37 (1H, m, 4-H_a), 2.40 (1H, m, 8-H_a), 2.45 (1H, dq, J = 10.5, 2.8 Hz, 5-H), 2.52 (2H, q, J = 7.2 Hz, NCH₂CH₃), 2.62 (1H, d, J = 11.0 Hz, 2-H_a), 2.93 (1H, dd, J = 10.5, 2.8 Hz, 4-H_e), 3.02 (1H, dd, J = 11.0, 2.8 Hz, 2-H_e) and 3.74 (3H, s, OCH₃). δ_C [125 MHz; CDCl₃; calibrated with CDCl₃ (77.16 ppm)]: 12.68 (CH₃, NCH₂CH₃), 27.83 (CH₃, C2′), 29.23 (quat, C7), 31.83 (CH₃, C2′), 44.53 (CH₂, C6), 44.80 (CH, C5), 47.26 (CH₂, C8), 50.31 (CH₂, NCH₂CH₃), 52.34 (CH₃, OCH₃), 57.84 (quat, C1), 62.59 (CH₂, C4), 63.74 (CH₂, C2), 172.75 (quat, COOMe) and 216.33 (quat, C9).

4.1.3.5. Methyl 3-Ethyl-7-isopropyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (12)

A solution of methyl 5-isopropyl-2-oxocyclohexane-1-carboxylate (11, 420 mg, 2.12 mmol), ethylamine aq solution (66–72% w/w, 0.20 mL, 2.33 mmol), and paraformaldehyde (140 mg, 4.66 mmol) in MeOH (16 mL) was stirred and heated under reflux for 2 h under an atmosphere of anhydrous nitrogen. TLC showed the reaction was complete (petroleum ether/ethyl acetate = 20:1 v/v, R_f = 0.3, stained with p-anisaldehyde solution). After the solvents were removed by evaporation, the crude products were acidified with aq HCl (1 M) to pH = 2 followed by washing with DCM (3 × 5 mL), then the aqueous layer was basified with a sat. NaHCO₃ aq solution (pH = 8) and extracted with DCM (3 × 5 mL). The combined organic phase was dried (Na₂SO₄), filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate = 20:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase) producing colorless oil (12, 63 mg, 11%). MS (m/z): [M + H]⁺ found 268.1913, C₁₅H₂₆NO₃ requires 268.1913 and [M + Na]⁺ found 290.1723, C₁₅H₂₅NO₃Na requires 290.1732. δ_H (500 MHz; CDCl₃; calibrated with TMS): 0.89 (3H, d, J = 6.1 Hz, 2′-H_A), 0.90 (3H, d, J = 6.1 Hz, 2′-H_B), 1.10 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.28 (1H, m, 1′-H), 1.72 (1H, td, J = 12.9, 6.0 Hz, 6-H_a), 2.13–2.29 (3H, m, 6-H_e, 8-H_a and 8-H_e), 2.41 (2H, q, J = 7.2 Hz, NCH₂CH₃), 2.45 (1H, br quin, J = 2.6 Hz, 5-H), 2.55 (1H, br dd, J = 11.2, 2.4 Hz, 4-H_a), 2.93 (1H, br dd, J = 11.2, 2.5 Hz, 2-H_a), 2.93 (1H, br dd, J = 11.4, 1.9 Hz, 2-H_a), 3.02 (1H, tq, J = 12.4, 6.0 Hz, 7-H_a), 3.14 (1H, br td, J = 11.2, 2.4 Hz, 4-H_e), 3.22 (1H, dd, J = 11.4, 2.5 Hz, 2-H_e) and 3.75 (3H, s, OCH₃). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.69 (CH₃, NCH₂CH₃), 20.24 (CH₃, C2_A^′), 22.38 (CH3, C2_B), 33.76 (CH, C1′), 37.01 (CH, C7), 38.35 (CH₂, C6), 41.17 (CH₂, C8), 47.08 (CH, C5), 51.04 (CH₂, NCH₂CH₃), 52.25 (CH₃, OCH₃), 58.66 (quat, C1), 59.75 (CH₂, C4), 61.59 (CH₂, C2), 171.67 (quat, COOMe) and 213.10 (quat, C9).

4.1.3.6. Ethyl 3-Ethyl-7-methyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (14)

A solution of methyl 5-isopropyl-2-oxocyclohexane-1-carboxylate (13, 200 mg, 1.09 mmol), ethylamine aq solution (66–72% w/w, 0.10 mL, 1.20 mmol), and paraformaldehyde (173 mg, 2.40 mmol) in EtOH (8 mL) was stirred and heated under reflux for 2 h under an atmosphere of anhydrous nitrogen. TLC showed the reaction was complete (petroleum ether/ethyl acetate = 20:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound R_f = 0.25, stained with potassium permanganate solution). After the solvents were removed by evaporation, the products were acidified with aq HCl (1 M) to pH = 2 followed by washing with DCM (3 × 20 mL), then aqueous layer was basified with a sat. NaHCO₃ aq solution (pH = 8) and extracted with DCM (3 × 20 mL). The combined organic phase was dried (Na₂SO₄), filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate, 20:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase) producing yellow oil (14, 11 mg, 4%). MS (m/z): [M + H]⁺ found 254.1752, C₁₄H₂₄NO₃ requires 254.1757 and [M + Na]⁺ found 276.1573, C₁₄H₂₃NO₃Na requires 276.1576. δ_H (500 MHz; CDCl₃; calibrated with TMS): 0.88 (3H, d, J = 6.7 Hz, 1′-H), 1.10 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.29 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.68 (1H, m, 6-H_a), 2.09–2.24 (3H, m, 6-H_e, 8-H_a and 8-H_e), 2.40 (2H, m, NCH₂CH₃), 2.42 (1H, m, 5-H), 2.54 (1H, d, J = 11.2 Hz, 4-H_a), 2.93 (1H, dd, J = 11.5, 2.0 Hz, 2-H_a), 3.16 (1H, dt, J = 11.2, 2.0 Hz, 4-H_e), 3.23 (1H, dt, J = 11.5, 2.0 Hz, 2-H_e), 3.42 (1H, tq, J = 12.3, 6.1 Hz, 7-H_a) and 4.21 (2H, q, J = 7.2 Hz, OCH₂CH₃). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.73 (CH₃, NCH₂CH₃), 14.15 (CH₃, OCH₂CH₃), 22.01 (CH₃, C1′), 26.28 (CH, C7), 42.56 (CH₂, C6), 44.99 (CH₂, C8), 47.30 (CH, C5), 51.13 (CH₂, NCH2CH3), 58.54 (quat, C1), 59.65 (CH₂, C4), 61.14 (CH₂, OCH₂CH₃), 61.46 (CH₂, C2), 171.03 (quat, COOEt) and 212.93 (quat, C9).

4.1.3.7. N,N-Bis(ethoxymethyl)ethanamine (16)¹⁹

Freshly distilled ethylamine (14.2 g, 315 mmol) was slowly added to a solution of pre-dried paraformaldehyde (18.9 g, 630 mmol) and oven-dried potassium carbonate (43.5 g, 315 mmol) in anhydrous ethanol (120 mL) at 0 °C with magnetic stirring, and the mixture was stirred vigorously for 2 days at 20 °C. The suspension was filtered, and the residue was rinsed with anhydrous ethanol (20 mL). The crude product solution was first purified by fractional vacuum distillation through a Vigreux column at 90–130 °C, 400 mbar, mainly in order to remove the excess of alcohol and then the Vigreux column was removed and the products were further purified by fractional vacuum distillation (130–150 °C, 100–110 mbar) followed by collecting the fraction with a boiling point of 100–105 °C, forming the clear liquid product amine (16, 21.3 g, 41%). δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.11 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.19 (3H × 2, t, J = 7.1 Hz, OCH₂CH₃ × 2), 2.89 (2H, q, J = 7.2 Hz, NCH₂CH₃), 3.44 (2H × 2, q, J = 7.1 Hz, OCH₂CH₃ × 2) and 4.30 (2H × 2, s, NCH₂O × 2). δ_C (125 MHz; CDCl₃; calibrated with TMS): 13.69 (CH₃, NCH₂CH₃), 15.25 (CH₃ × 2, OCH₂CH₃ × 2), 43.81 (CH₂, NCH₂CH₃), 62.58 (CH₂ × 2, OCH₂CH₃ × 2) and 84.27 (CH₂ × 2, NCH₂O × 2).

4.1.3.8. Methyl 3-Ethyl-7-methyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (17)¹⁹

To a solution of methyl 2-oxocyclopentane-1-carboxylate (15, 94 mg, 0.55 mmol) and N,N-bis(ethoxymethyl)ethanamine (16, 179 mg, 1.10 mmol) in acetonitrile (2 mL), trichloromethylsilane (217 mg, 1.11 mmol) was added followed by stirring for 20 h at 20 °C. The reaction was quenched with sat. aq NaHCO₃ (pH = 8) and extracted with ethyl acetate (3 × 10 mL). The combined organic extracts were washed with sat. aq brine (10 mL), dried (Na₂SO₄), and filtered, and then the solvents were removed by evaporation. The crude products were purified by chromatography over silica gel (petroleum ether/ethyl acetate = 20:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.25, stained with Dragendorff’s reagent and p-anisaldehyde solution) giving a yellow oil product (17, 26 mg, 73%). MS (m/z): [M + H]⁺ found 240.1618, C₁₃H₂₂NO₃ requires 240.1594 and [M + Na]⁺ found 262.1448, C₁₃H₂₁NO₃Na requires 262.1414. δ_H [500 MHz; CDCl₃; calibrated with residual CHCl₃ (7.26 ppm)]: 0.87 (3H, d, J = 6.7 Hz, 1′-H), 1.09 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.66 (1H, tdd, J = 13.8, 6.0, 1.5 Hz, 6-H_a), 2.09–2.23 (3H, m, 6-H_e, 8-H_a and 8-H_e), 2.39 (2H, qd, J = 7.2, 2.0 Hz, NCH₂CH), 2.42 (1H, m, 5-H), 2.54 (1H, m, 4-H_a), 2.92 (1H, dd, J = 11.4, 2.1 Hz, 2-H_a), 3.15 (1H, dt, J = 11.2, 2.1 Hz, 4-H_e), 3.23 (1H, dt, J = 11.4, 2.1 Hz, 2-H_e), 3.42 (1H, tt, J = 12.3, 6.0 Hz, 7-H_a) and 3.74 (3H, s, OCH₃). δ_C [125 MHz; CDCl₃; calibrated with CDCl₃ (77.16 ppm)]: 12.80 (CH₃, NCH₂CH₃), 22.11 (CH₃, C1′), 26.40 (CH, C7), 42.70 (CH₂, C6), 45.18 (CH₂, C8), 47.39 (CH, C5), 51.25 (CH₂, NCH2CH3), 52.34 (CH₃, OCH₃), 58.90 (quat, C1), 59.75 (CH₂, C4), 61.59 (CH₂, C2), 171.60 (quat, COOMe) and 212.93 (quat, C9).

4.1.3.9. Ethyl (E)-9-(2-(2,4-Dinitrophenyl)hydrazinylidene)-3-ethyl-3-azabicyclo[3.3.1]nonane-1-carboxylate (18)³¹

2,4-Dinitrophenylhydrazine (412 mg, 2.08 mmol) and trifluoroacetic acid (TFA) (119 mg, 1.04 mmol) were added to a solution of ethyl 3-ethyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (8, 415 mg, 1.73 mmol) in tetrahydrofuran (THF, 12 mL), and the resulting solution was stirred and heated under reflux for 3 h under an atmosphere of anhydrous nitrogen. After the solution was cooled to 20 °C, about 7 mL of the solvent was removed by evaporation. The residue was basified with sat. aq NaHCO₃ (pH = 8) and extracted with DCM (3 × 15 mL), and then the combined organic layers were washed with sat. aq brine (10 mL), dried (Na₂SO₄), and filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate 10:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.3, stained with iodine vapor or ninhydrin solution) giving an orange solid product (18, 472 mg, 65%). MS (m/z): [M + H]⁺ found 420.1934, C₁₉H₂₆N₅O₆ requires 420.1878 and [M + Na]⁺ found 442.1729, C₁₉H₂₅N₅O₆Na requires 442.1697. δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.11 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.36 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.58 (1H, m, 7-H_e), 1.93 (1H, tt, J = 12.2, 6.0 Hz, 6-H_a), 2.14 (1H, m, 6-H_e), 2.16 (1H, m, 8-H_e), 2.40 (2H, q, J = 7.2 Hz, NCH₂CH₃), 2.42 (1H, m, 4-H_a), 2.49 (1H, J = 12.9, 6.0, 1.1 Hz, 8-H_a), 2.89 (2H, m, 7-H_a and 2-H_a), 3.11 (1H, m, 5-H), 3.16 (1H, m, 4-H_e), 3.19 (1H, m, 2-H_e), 4.30 (2H, qd, J = 7.1, 1.9 Hz, OCH₂CH₃), 7.80 (1H, d, J = 9.5 Hz, 6′-H), 8.28 (1H, dd, J = 9.5, 2.6 Hz, 5′-H), 9.11 (1H, d, J = 2.6 Hz, 3′-H) and 11.17 (1H, s, NH). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.54 (CH₃, NCH₂CH₃), 14.37 (CH₃, OCH₂CH₃), 20.85 (CH₂, C7), 32.45 (CH₂, C6), 33.22 (CH, C5), 36.38 (CH₂, C8), 51.54 (CH₂, NCH₂CH₃), 52.34 (quat, C1), 58.42 (CH₂, C4), 61.13 (CH₂, OCH₂CH₃), 61.82 (CH₂, C2), 116.20 (CH, C6′), 123.48 (CH, C3′), 129.10 (quat, C2′), 129.97 (CH, C5′), 137.88 (quat, C4′), 145.40 (quat, C1′), 163.86 (quat, C9) and 172.12 (quat, COOEt).

4.1.3.10. Methyl (E)-9-(2-(2,4-Dinitrophenyl)hydrazinylidene)-3-ethyl-7-isopropyl-3-azabicyclo[3.3.1]nonane-1-carboxylate (19)

2,4-Dinitrophenylhydrazine (55 mg, 0.19 mmol) and TFA (11 mg, 0.10 mmol) were added to a solution of methyl 3-ethyl-7-isopropyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (12, 43 mg, 0.16 mmol) in THF (3 mL), and the resulting solution was stirred and heated under reflux for 3 h under an atmosphere of anhydrous nitrogen. The solution was basified with sat. aq NaHCO₃ (pH = 8) and extracted with DCM (3 × 10 mL), and then the combined organic layers were washed with sat. aq brine (5 mL), dried (Na₂SO₄), and filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate 10:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.2, stained with iodine vapor or ninhydrin solution) giving an orange solid product (19, 44 mg, 61%). MS (m/z): [M + H]⁺ found 448.2206, C₂₁H₃₀N₅O₆ requires 448.2191 and [M + Na]⁺ found 470.2026, C₂₁H₂₉N₅O₆Na requires 470.2010. δ_H (500 MHz; CDCl₃; calibrated with TMS): 0.89 (3H, d, J = 6.1 Hz, 2′-H_A), 0.91 (3H, d, J = 6.1 Hz, 2′-H_B), 1.10 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.26 (1H, m, 1′-H), 1.93 (1H, td, J = 13.4, 4.1 Hz, 6-H_a), 2.13 (1H, m, 8-H_a), 2.19 (1H, m, 6-H_e), 2.20 (1H, m, 8-H_e), 2.40 (3H, m, 4-H_a and NCH₂CH₃), 2.89 (1H, br d, J = 11.3 Hz, 2-H_a), 2.97 (1H, m, 7-H_a), 3.12 (1H, m, 5-H), 3.14 (1H, m, 4-H_e), 3.18 (1H, m, 2-H_e), 3.84 (3H, s, OCH₃), 7.77 (1H, d, J = 9.6 Hz, 6″-H), 8.29 (1H, dd, J = 9.6, 2.5 Hz, 5″-H), 9.12 (1H, d, J = 2.5 Hz, 3″-H) and 11.16 (1H, s, NH). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.48 (CH₃, NCH₂CH₃), 20.18 (CH₃, C2_A^′), 20.28 (CH₃, C2_B), 33.33 (CH, C5), 34.12 (CH, C1′), 36.67 (CH₂, C6), 37.43 (CH, C7), 40.68 (CH₂, C8), 51.35 (CH₂, NCH₂CH₃), 52.22 (CH₃, OCH₃), 52.54 (quat, C1), 58.23 (CH₂, C4), 61.65 (CH₂, C2), 116.16 (CH, C6″), 123.46 (CH, C3″), 129.14 (quat, C2″), 130.05 (CH, C5″), 137.92 (quat, C4″), 145.34 (quat, C1″), 163.83 (quat, C9) and 172.57 (quat, COOMe).

4.1.3.11. Methyl (E)-9-(2-(2,4-Dinitrophenyl)hydrazinylidene)-3-ethyl-7-methyl-3-azabicyclo[3.3.1]nonane-1-carboxylate (20)³²

2,4-Dinitrophenylhydrazine (91 mg, 0.46 mmol) and concd aq H₂SO₄ solution (15 μL, 0.10 mmol) were added to a solution of methyl 3-ethyl-7-methyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (17, 22 mg, 0.09 mmol) in MeOH (3 mL) at 0 °C, and the resulting solution was stirred and heated under reflux for 3 h under an atmosphere of anhydrous nitrogen. The solution was basified with sat. aq NaHCO₃ (pH = 8) and extracted with DCM (3 × 10 mL), and then the combined organic layers were washed with sat. aq brine (5 mL), dried (Na₂SO₄), and filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate = 20:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.32, stained with iodine vapor or ninhydrin solution) giving an orange solid product (20, 20 mg, 53%). MS (m/z): [M + H]⁺ found 420.1902, C₁₉H₂₆N₅O₆ requires 420.1878 and [M + Na]⁺ found 442.1720, C₁₉H₂₅N₅O₆Na requires 442.1697. δ_H (500 MHz; CDCl₃; calibrated with TMS): 0.81 (3H, d, J = 6.6 Hz, 1′-H), 1.13 (3H, t, J = 7.1 Hz, NCH₂CH₃), 1.42 (1H, m, 6-H_a), 2.01 (1H, m, 8-H_a), 2.08 (1H, m, 6-H_e), 2.10 (1H, m, 8-H_e), 2.32 (1H, m, 4-H_a), 2.33 (2H, m, NCH₂CH₃), 2.82 (1H, br d, J = 11.3 Hz, 2-H_a), 3.03 (1H, m, 5-H), 3.09 (1H, d, J = 11.2 Hz, 4-H_e), 3.13 (1H, d, J = 11.4 Hz, 2-H_e), 3.28 (1H, tq, J = 12.2, 6.1 Hz, 7-H_a), 3.76 (3H, s, OCH₃), 7.70 (1H, d, J = 9.5 Hz, 6″-H), 8.22 (1H, d, J = 9.5 Hz, 5″-H), 9.03 (1H, d, J = 2.5 Hz, 3″-H) and 11.08 (1H, s, NH). δ_C [125 MHz; CDCl₃; calibrated with CDCl₃ (77.16 ppm)]: 12.60 (CH₃, NCH₂CH₃), 22.49 (CH₃, C1′), 26.71 (CH, C7), 33.57 (CH, C5), 40.94 (CH₂, C6), 44.78 (CH₂, C8), 51.56 (CH₂, NCH₂CH₃), 52.32 (CH₃, OCH₃), 52.74 (quat, C1), 58.19 (CH₂, C4), 61.59 (CH₂, C2), 116.26 (CH, C6″), 123.56 (CH, C3″), 129.26 (quat, C2″), 130.17 (CH, C5″), 138.04 (quat, C4″), 145.45 (quat, C1″), 163.70 (quat, C9) and 172.54 (quat, COOMe).

An orange solid (42 mg in total) was obtained after purification, and there were three main components in both ¹H and ¹³C NMR spectra, which are the target product, the conformational isomer of the target product, and the byproduct, 1-(2,4-dinitrophenyl)-2-(propan-2-ylidene)hydrazine derived from acetone. The ratio of ¹H integrals of these components was 20:4:17; therefore, the yield is 53%. The total content of the isomer was too low to be analyzed in detail using NMR spectroscopy. These products were therefore not purified further after the purification by flash chromatography over silica gel.

4.1.3.12. 1-(2,4-Dinitrophenyl)-2-(propan-2-ylidene)hydrazine

MS (m/z): [M – H]⁻ found 237.0614, C₉H₉N₄O₄ requires 237.0623, and MS (m/z): [M + HCOO]⁻ found 283.0669, C₁₀H₁₁N₄O₆ requires 283.0679. δ_H (500 MHz; CDCl₃; calibrated with TMS): 2.02 (3H, s, 2′-H), 2.11 (3H, s, 3′-H), 7.89 (1H, d, J = 7.1 Hz, 6″-H), 8.22 (1H, m, 5″-H), 9.05 (1H, d, J = 2.4 Hz, 3″-H) and 10.95 (1H, br s, NH). δ_C [125 MHz; CDCl₃; calibrated with CDCl₃ (77.16 ppm)]: 17.17 (CH₂, C2′), 25.64 (CH₂, C3′), 116.49 (CH, C6), 123.69 (CH, C3), 129.07 (quat, C2), 130.12 (CH, C5), 137.76 (quat, C4), 145.27 (quat, C1) and 155.36 (quat, C1′).

4.1.3.13. Methyl 8-Ethyl-10-oxo-8-azabicyclo[4.3.1]decane-1-carboxylate (23)

A solution of methyl 5-isopropyl-2-oxocyclohexane-1-carboxylate (21, 511 mg, 3.01 mmol), aq ethylamine (66–72% w/w, 0.28 mL, 3.31 mmol), and formaldehyde aq solution (37–40% w/w, 0.54 mL, 6.65 mmol) in MeOH (20 mL) was stirred and heated under reflux for 4 h under an atmosphere of anhydrous nitrogen. TLC showed the reaction was complete (petroleum ether/ethyl acetate = 20:1 v/v, R_f = 0.2, stained with p-anisaldehyde solution). After the solvents were removed by evaporation, the crude products were acidified with aq HCl (1 M) to pH = 2 followed by washing with DCM (3 × 20 mL), then the aqueous layer was basified with a sat. NaHCO₃ aq solution (pH = 8) and extracted with DCM (3 × 20 mL). The combined organic phase was dried (Na₂SO₄), filtered, and then the solvents were removed by evaporation. The crude compound was purified by column chromatography over silica gel (petroleum ether/ethyl acetate = 20:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase) producing colorless oil (23, 459 mg, 64%). MS (m/z): [M + H]⁺ found 240.1624, C₁₃H₂₂NO₃ requires 240.1594 and [M + Na]⁺ found 262.1428, C₁₃H₂₁NO₃Na requires 262.1414. δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.11 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.36 (1H, dtdd, J = 15.0, 10.3, 3.6, 1.4 Hz, 3-H_e′), 1.51 (1H, m, 4-H_e′), 1.69 (1H, m, 5-H_e′), 1.75 (1H, m, 5-H_a′), 1.85 (1H, ddd, J = 13.5, 10.5, 3.1 Hz, 2-H_e′), 1.96 (1H, m, 4-H_a′), 2.04 (1H, m, 3-H_a′), 2.41 (1H, ddd, J = 13.5, 10.5, 3.1 Hz, 2-H_a′), 2.47 (2H, q, J = 7.2 Hz, NCH₂CH₃), 2.58 (1H, dd, J = 11.5, 4.4 Hz, 7-H_a), 2.70 (1H, quin, J = 3.6 Hz, 6-H), 2.83 (1H, d, J = 11.5 Hz, 9-H_a), 2.88 (1H, m, 9-H_e), 2.89 (1H, m, 7-H_e) and 3.74 (3H, s, OCH₃). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.62 (CH₃, NCH₂CH₃), 26.00 (CH₂, C4), 26.25 (CH₂, C3), 32.45 (CH₂, C5), 33.54 (CH₂, C2), 48.47 (CH, C6), 51.50 (CH₂, NCH₂CH₃), 52.34 (CH₃, OCH₃), 58.45 (CH₂, C7), 61.50 (CH₂, C9), 62.09 (quat, C1), 172.95 (quat, COOMe) and 208.75 (quat, C10).

4.1.3.14. Methyl (E)-10-(2-(2,4-Dinitrophenyl)hydrazinylidene)-8-ethyl-8-azabicyclo[4.3.1]decane-1-carboxylate (24)

2,4-Dinitrophenylhydrazine (425 mg, 1.46 mmol) and concd aq H₂SO₄ solution (40 μL, 0.73 mmol) were added to a solution of methyl 8-ethyl-10-oxo-8-azabicyclo[4.3.1]decane-1-carboxylate (23, 70 mg, 0.29 mmol) in MeOH (8 mL) at 0 °C, and the resulting solution was stirred and heated under reflux under an atmosphere of anhydrous nitrogen. After 4.5 days, TLC monitoring showed the reactant [4.3.1]azabicycle was fully reacted and the target compound was generated (petroleum ether/ethyl acetate = 3:1 v/v, target compound TLC R_f = 0.8, stained with p-anisaldehyde). After the solution was cooled to 20 °C, 4 mL of the solvent was removed by evaporation. The rest of the solution was basified with sat. aq NaHCO₃ (pH = 8) and extracted with DCM (3 × 15 mL), and the combined organic layers were washed with sat. aq brine (15 mL), dried (Na₂SO₄), and filtered, and then the solvents were removed by evaporation. The residue was purified by chromatography over silica gel (petroleum ether/ethyl acetate = 30:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.25, stained with iodine vapor or ninhydrin solution) giving an orange solid product (24, 16 mg, 13%). MS (m/z): [M + H]⁺ found 420.1903, C₁₉H₂₆N₅O₆ requires 420.1878 and [M + Na]⁺ found 442.1706, C₁₉H₂₅N₅O₆Na requires 442.1697. δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.11 (3H, t, J = 7.1 Hz, NCH₂CH₃), 1.39 (1H, m, 4-H_e′), 1.42 (1H, m, 3-H_e′), 1.79 (1H, ddd, J = 10.1, 7.8, 2.9 Hz, 5-H_e), 1.91 (1H, m, 2-H_e′), 1.95 (2H, m, 5-H_a′ and 4-H_a′), 2.05 (1H, m, 3-H_a′), 2.44 (1H, m, 7-H_a), 2.45 (2H, m, NCH₂CH₃), 2.51 (1H, ddd, J = 13.1, 8.2, 3.8 Hz, 2-H_a), 2.78 (1H, d, J = 11.0 Hz, 9-H_a), 2.86 (1H, dd, J = 11.0, 2.3 Hz, 9-H_e), 2.90 (1H, br dt, J = 11.4, 1.9 Hz, 7-H_e), 3.19 (1H, m, 6-H), 3.80 (3H, s, OCH₃), 7.75 (1H, d, J = 9.5 Hz, 6′-H), 8.29 (1H, dd, J = 9.5, 2.6 Hz, 5′-H), 9.12 (1H, d, J = 2.6 Hz, 3′-H) and 11.24 (1H, s, NH). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.41 (CH₃, NCH₂CH₃), 25.83 (CH₂, C3), 26.07 (CH₂, C4), 30.51 (CH₂, C5), 34.50 (CH, C6), 26.80 (CH₂, C2), 51.74 (CH₂, NCH₂CH₃), 52.32 (CH₃, OCH₃), 55.55 (quat, C1), 58.05 (CH₂, C7), 61.92 (CH₂, C9), 116.26 (CH, C6′), 123.42 (CH, C3′), 129.36 (quat, C2′), 130.11 (CH, C5′), 138.05 (quat, C4′), 145.20 (quat, C1′), 160.08 (quat, C10) and 173.98 (quat, COOMe).

4.1.3.15. Ethyl 3-Ethyl-8-oxo-3-azabicyclo[3.2.1]octane-1-carboxylate (27)

To a solution of ethyl 2-oxocyclopentane-1-carboxylate (25, 253 mg, 1.64 mmol) and N,N-bis(ethoxymethyl)ethanamine (533 mg, 3.28 mmol) in acetonitrile (5 mL) was added trichloromethylsilane (642 mg, 3.28 mmol) at 0 °C followed by stirring for 20 h at 20 °C. The reaction was quenched with sat. aq NaHCO₃ (pH = 8) and extracted with ethyl acetate (3 × 15 mL). The combined organic layers were washed with sat. aq brine (15 mL), dried (Na₂SO₄), and filtered, and then the solvents were removed by evaporation. The crude products were purified by chromatography over silica gel (petroleum ether/ethyl acetate = 7:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.25, stained with Dragendorff’s reagent and p-anisaldehyde solution) giving a pale yellow oil product (27, 183 mg, 50%). MS (m/z): [M + H]⁺ found 226.1448, C₁₂H₂₀NO₃ requires 226.1438 and [M + Na]⁺ found 248.1265, C₁₂H₁₉NO₃Na requires 248.1257. δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.09 (3H, t, J = 7.1 Hz, NCH₂CH₃), 1.28 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.95 (2H, m, 6-H_a′ and 6-H_e′), 2.25 (1H, ddd, J = 12.6, 10.1, 6.1 Hz, 7-H_a′), 2.36 (1H, m, 5-H), 2.38 (1H, m, 7-H_e′), 2.51 (1-H, m, 4-H_a), 2.55 (2H, m, NCH₂CH₃), 2.70 (1H, d, J = 10.9 Hz, 2-H_a), 3.00 (1H, ddd, J = 10.5, 4.0, 2.7 Hz, 4-H_e), 3.15 (1H, dd, J = 11.0, 2.7 Hz, 2-H_e) and 4.21 (2H, q, J = 7.2 Hz, OCH₂CH₃). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.66 (CH₃, NCH₂CH₃), 14.19 (CH₃, OCH₂CH₃), 21.86 (CH₂, C6), 27.61 (CH₂, C7), 46.42 (CH, C5), 49.78 (CH₂, NCH₂CH₃), 57.65 (quat, C1), 61.13 (CH₂, C4), 61.17 (CH₂, OCH₂CH₃), 62.24 (CH₂, C2), 170.45 (quat, COOEt) and 213.80 (quat, C8).

4.1.3.16. Ethyl (E)-8-(2-(2,4-Dinitrophenyl)hydrazinylidene)-3-ethyl-3-azabicyclo[3.2.1]octane-1-carboxylate (28)

2,4-Dinitrophenylhydrazine (212 mg, 0.75 mmol) and concd aq H₂SO₄ solution (22 μL, 0.10 mmol) were added to a solution of ethyl 3-ethyl-8-oxo-3-azabicyclo[3.2.1]octane-1-carboxylate (27, 33 mg, 0.15 mmol) in MeOH (5 mL) at 0 °C, and the resulting solution was stirred and heated under reflux for 3 h under an atmosphere of anhydrous nitrogen. After the solution was cooled to 20 °C, about 2 mL of the solvent was removed by evaporation. The solution was basified with sat. aq NaHCO₃ (pH = 8) and extracted with DCM (3 × 10 mL), and then the combined organic layers were washed with sat. aq brine (5 mL), dried (Na₂SO₄), and filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (petroleum ether/ethyl acetate = 5:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.35, stained with iodine vapor or ninhydrin solution) giving an orange solid product (22 mg, 37%). The product (28, 26 mg) was obtained after purification and the byproduct of 1-(2,4-dinitrophenyl)-2-(propan-2-ylidene)hydrazine was displayed. The ratio of ¹H integrals between the target product and the byproduct was 5:1; thus, the yield is 37%. MS (m/z): [M + H]⁺ found 406.1755, C₁₈H₂₄N₅O₆ requires 406.1721. δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.10 (3H, t, J = 7.1 Hz, NCH₂CH₃), 1.36 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.94 (1H, tdd, J = 12.0, 6.4, 4.7 Hz, 6-H_a′), 2.07 (1H, m, 6-H_e′), 2.19 (1H, m, 7-H_a′), 2.35 (1H, m, 4-H_a), 2.37 (1H, m, 7-H_e′), 2.55 (2H, q, J = 7.1 Hz, NCH₂CH₃), 2.64 (1H, d, J = 10.7 Hz, 2-H_a), 2.99 (1H, ddd, J = 10.3, 4.1, 1.6 Hz, 4-H_e), 3.18 (1H, m, 2-H_e), 3.19 (1H, m, 5-H), 4.31 (2H, qd, J = 7.1, 4.0 Hz, OCH₂CH₃), 7.84 (1H, d, J = 9.5 Hz, 6′-H), 8.29 (1H, dd, J = 9.5, 2.6 Hz, 5′-H), 9.11 (1H, d, J = 2.6 Hz, 3′-H) and 11.02 (1H, s, NH). δ_C (125 MHz; CDCl₃; calibrated with TMS): 12.44 (CH₃, NCH₂CH₃), 14.42 (CH₃, OCH₂CH₃), 25.40 (CH₂, C6), 29.81 (CH₂, C7), 36.62 (CH, C5), 50.18 (CH₂, NCH₂CH₃), 54.58 (quat, C1), 58.70 (CH₂, C4), 61.14 (CH₂, OCH₂CH₃), 62.64 (CH₂, C2), 116.20 (CH, C6′), 123.56 (CH, C3′), 129.07 (quat, C2′), 130.01 (CH, C5′), 137.86 (quat, C4′), 145.38 (quat, C1′), 168.39 (quat, C8) and 171.05 (quat, COOEt).

4.1.3.17. Ethyl 1-[(Ethylamino)methyl]-2-oxocyclohexane-1-carboxylate (29)

A solution of ethyl 2-oxocyclohexane-1-carboxylate (6, 532 mg, 3.13 mmol), ethylamine aq solution (0.24 mL, 2.82 mmol), and paraformaldehyde (85 mg, 2.82 mmol) in EtOH (10 mL) was stirred and heated for 3 h at 40 °C under an atmosphere of anhydrous nitrogen. TLC monitoring showed the reaction was complete (DCM/MeOH = 40:1 v/v, target compound R_f = 0.2, stained with p-anisaldehyde solution and iodine vapor). After the solvent was removed by evaporation, the crude product was purified by chromatography over silica gel (DCM/MeOH = 40:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase) producing the pale yellow oil product of the mono-Mannich reaction (29, 37 mg, 5%). MS (m/z): [M + H]⁺ found 228.1602, C₁₂H₂₂NO₃ requires 228.1594. δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.04 (3H, t, J = 7.1 Hz, NCH₂CH₃), 1.27 (3H, t, J = 6.8 Hz, OCH₂CH₃), 1.68 (3H, m, 4-H_A, 5-H_A and 6-H_A), 1.77 (1H, m, 5-H_B), 2.00 (1H, m, 4-H_B), 2.42 (1H, m, 6-H_B), 2.43 (1H, m, 3-H_A), 2.58 (1H, m, 3-H_B), 2.60 (2H, m, NCH₂CH₃), 2.73 (1H, d, J = 11.7 Hz, 1′-H_A), 2.92 (1H, d, J = 11.7 Hz, 1′-H_B), 4.23 (2H, m, OCH₂CH₃). δ_C (125 MHz; CDCl₃; calibrated with TMS): 14.15 (CH₃, OCH₂CH₃), 15.15 (CH₃, NCH₂CH₃), 22.38 (CH₂, C5), 27.26 (CH₂, C4), 34.77 (CH₂, C6), 41.12 (CH₂, C3), 44.60 (CH₂, NCH₂CH₃), 53.76 (CH₂, C1′), 61.23 (CH₂, OCH₂CH₃), 62.19 (quat, C1), 172.06 (quat, COOEt) and 209.24 (quat, C2).

4.1.3.18. 3-Ethyl-1-(hydroxymethyl)-3-azabicyclo[3.3.1]nonan-9-ol (30)

Lithium aluminum hydride (410 mg, 10.75 mmol, 1.6 equiv) was added to a solution of the ethyl 3-ethyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (8, 513 mg, 2.15 mmol) in anhydrous THF (50 mL) at 0 °C; then the mixture was warmed to 20 °C and stirred under an atmosphere of anhydrous nitrogen for 4 h. The reaction mixture was diluted with anhydrous THF (30 mL) and then quenched by the dropwise addition of water (20 mL) cooled in an ice-bath at 0 °C. The organic solvent (40 mL) was removed by evaporation and the remaining aq solution was extracted with DCM (3 × 30 mL). The combined organic layers were washed with sat. aq brine (50 mL), dried (Na₂SO₄), filtered, and then the solvents were removed by evaporation. The crude product was purified by chromatography over silica gel (DCM/MeOH = 11:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.25, stained with iodine vapor) to give the title compound as colorless oil (30, 78 mg, 18%). [M + Na]⁺ found 222.1450, C₁₁H₂₁NO₂Na requires 222.1470. δ_H (500 MHz; CDCl₃; calibrated with CDCl₃): 1.04 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.28 (1H, dd, J = 13.4, 6.2 Hz, 8-H_e), 1.48 (1H, m, 7-H_e), 1.52 (1H, m, 6-H_e), 1.83 (1H, br m, 2-H_a), 1.86 (1H, br m, 5-H), 1.91 (1H, m, 6-H_a), 2.01 (1H, dtd, J = 13.4, 6.2, 2.0 Hz, 8-H_a), 2.19 (1H, br m, 4-H_a), 2.25 (2H, br m, NCH₂CH₃), 2.59 (1H, m, 7-H_a), 2.64 (1H, d, J = 11.1 Hz, 2-H_e), 2.98 (1H, d, J = 10.8 Hz, 4-H_e), 3.37 (1H, d, J = 10.8 Hz, 1′-H_A), 3.43 (1H, d, J = 10.8 Hz, 1′-H_B) and 3.72 (1H, br d, J = 2.0 Hz, 9-H). δ_C [125 MHz; CDCl₃; calibrated with CDCl₃ (77.16 ppm)]: 12.83 (CH₃, NCH₂CH₃), 20.59 (CH₂, C7), 24.02 (CH₂, C6), 26.59 (CH₂, C8), 26.27 (CH, C5), 28.29 (quat, C1), 52.56 (CH₂, NCH₂CH₃), 58.43 (CH₂, C4), 60.52 (CH₂, C2), 71.30 (CH₂, C1′), and 75.52 (CH, C9). δ_H (500 MHz; CD₃OD; calibrated with TMS): 1.05 (3H, t, J = 7.1 Hz, NCH₂CH₃), 1.35 (1H, dd, J = 13.5, 6.5 Hz, 8-H_e), 1.42 (1H, m, 7-H_e), 1.48 (1H, dd, J = 13.5, 6.5 Hz, 6-H_e), 1.75 (1H, tdd, J = 13.5, 6.5, 2.4 Hz, 8-H_a), 1.81 (1H, br m, 5-H), 1.96 (1H, m, 2-H_a), 1.99 (1H, br m, 6-H_a), 2.19 (1H, m, 4-H_a), 2.25 (2H, br m, NCH₂CH₃), 2.58 (1H, br m, 7-H_a), 2.83 (1H, d, J = 9.5 Hz, 2-H_e), 3.00 (1H, d, J = 8.5 Hz, 4-H_e), 3.28 (1H, d, J = 11.0 Hz, 1′-H_A), 3.34 (1H, d, J = 11.0 Hz, 1′-H_B) and 3.56 (1H, br d, J = 3.6 Hz, 9-H). δ_C (125 MHz; CD₃OD; calibrated with TMS): 13.13 (CH₃, NCH₂CH₃), 21.77 (CH₂, C7), 25.35 (CH₂, C6), 28.03 (CH₂, C8), 37.69 (CH, C5), 39.85 (quat, C1), 53.76 (CH₂, NCH₂CH₃), 59.97 (CH₂, C4), 62.02 (CH₂, C2), 69.91 (CH₂, C1′) and 73.98 (CH, C9). δ_H [500 MHz; d₆-DMSO; calibrated with residual DMSO (2.50 ppm)]: 0.97 (3H, br t, J = 7.2 Hz, NCH₂CH₃), 1.23 (1H, dd, J = 13.0, 6.5 Hz, 8-H_e), 1.29 (1H, m, 7-H_e), 1.35 (1H, br dd, J = 12.6, 4.9 Hz, 6-H_e), 1.58 (1H, tdd, J = 13.0, 6.5, 2.2 Hz, 8-H_a), 1.69 (1H, br m, 5-H), 1.85 (1H, br m, 2-H_a), 1.89 (1H, m, 6-H_a), 2.04 (1H, br d, J = 10.0 Hz, 4-H_a), 2.14 (2H, br s, NCH₂CH₃), 2.47 (1H, br m, 7-H_a), 2.73 (1H, d, J = 10.1 Hz, 2-H_e), 2.88 (1H, d, J = 10.0 Hz, 4-H_e), 3.08 (1H, dd, J = 10.6, 5.5 Hz, 1′-H_A), 3.17 (1H, dd, J = 10.6, 5.5 Hz, 1′-H_B), 3.37 [1H, m (overlapped with HDO peak), 9-H], 4.29 (1H, br s, CH₂OH) and 4.43 (1H, br s, 9-OH). δ_C [125 MHz; d₆-DMSO; calibrated with d₆-DMSO (39.52 ppm)]: 12.28 (CH₃, NCH₂CH₃), 20.65 (CH₂, C7), 24.32 (CH₂, C6), 27.01 (CH₂, C8), 35.91 (CH, C5), 38.50 (quat, C1), 52.16 (CH₂, NCH₂CH₃), 58.62 (CH₂, C4), 60.83 (CH₂, C2), 67.64 (CH₂, C1′) and 70.92 (CH, C9). δ_H [500 MHz; D₂O/d₆-DMSO (2 drops); calibrated with residual DMSO (2.71 ppm)]: 1.12 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.45 (1H, dd, J = 13.0, 7.0 Hz, 8-H_e), 1.59 (1H, m, 7-H_e), 1.60 (1H, m, 6-H_e), 1.62 (1H, tdd, J = 13.0, 7.0, 2.5 Hz, 8-H_a), 1.91 (1H, tt, J = 13.5, 7.0 Hz, 6-H_a), 2.02 (1H, m, 5-H), 2.04 (1H, m, 7-H_a), 2.25 (1H, br d, J = 12.0 Hz, 2-H_a), 2.46 (3H, m, 4-H_a and NCH₂CH₃), 3.02 (1H, d, J = 12.0 Hz, 2-H_e), 3.17(1H, d, J = 12.0 Hz, 4-H_e), 3.32 (1H, d, J = 11.3 Hz, 1′-H_A), 3.46 (1H, d, J = 11.3 Hz, 1′-H_B) and 3.75 (1H, d, J = 2.5 Hz, 9-H). δ_C [125 MHz; D₂O/d₆-DMSO (2 drops); calibrated with d₆-DMSO (39.52 ppm)]: 12.46 (CH₃, NCH₂CH₃), 20.49 (CH₂, C7), 24.14 (CH₂, C6), 27.19 (CH₂, C8), 36.33 (CH, C5), 39.86 (quat, C1), 55.47 (CH₂, NCH₂CH₃), 59.65 (CH₂, C4), 61.46 (CH₂, C2), 68.81 (CH₂, C1′) and 72.29 (CH, C9).

4.1.3.19. 1-(Ethoxycarbonyl)-3-ethyl-9-oxo-3-azabicyclo[3.3.1]nonan-3-ium Acetate Salt (31)

Ethyl 3-ethyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (8, 20 mg) was dissolved in d₄-acetic acid (0.8 mL) and the title ketone (31) was produced. δ_H [500 MHz; d₄-acetic acid; calibrated with residual acetic acid (2.05 ppm)]: 1.29 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.38 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.72 (1H, m, 7-H_e), 2.12 (1H, tdd, J = 14.3, 5.8, 5.2 Hz, 6-H_a), 2.35 (1H, m, 6-H_e), 2.41 (1H, m, 8-H_e), 2.42 (1H, m, 7-H_a), 2.58 (1H, td, J = 14.3, 5.8 Hz, 8-H_a), 2.83 (1H, tt, J = 5.2, 2.4 Hz, 5-H), 3.35 (2H, qd, J = 7.2, 2.4 Hz, NCH₂CH₃), 3.66 (1H, dd, J = 13.2, 5.2 Hz, 4-H_a), 4.01 (1H, d, J = 13.4 Hz, 2-H_a), 4.12 (1H, m, 4-H_e), 4.15 (1H, m, 2-H_e) and 4.26 (2H, q, J = 7.2 Hz, OCH₂CH₃). δ_C [125 MHz; d₄-acetic acid; calibrated with d₄-acetic acid (20.00 ppm)]: 9.98 (CH₃, NCH₂CH₃), 14.30 (CH₃, OCH₂CH₃), 18.45 (CH₂, C7), 33.93 (CH₂, C6), 36.80 (CH₂, C8), 45.11 (CH, C5), 55.30 (CH₂, NCH₂CH₃), 57.03 (CH₂, C4), 58.06 (quat, C1), 58.20 (CH₂, C2), 63.24 (CH₂, OCH₂CH₃), 170.15 (quat, COOEt) and 208.66 (quat, C9).

4.1.3.20. 1-(Ethoxycarbonyl)-3-ethyl-9-oxo-3-azabicyclo[3.3.1]nonan-3-ium Chloride (32)

A solution of ethyl 3-ethyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (8, 225 mg) in MeOH (2 mL) was titrated with concd aq HCl solution (37%, w/w) to pH = 2, then all the solvents and the excess of HCl were removed by evaporation to give the ketone as its HCl salt (32). δ_H [500 MHz; d₄-acetic acid; calibrated with residual acetic acid (2.05 ppm)]: 1.30 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.47 (3H, t, J = 7.1 Hz, NCH₂CH₃), 1.74 (1H, m, 7-H_e), 2.15 (1H, m, 6-H_a), 2.48 (1H, br d, J = 13.3 Hz, 6-H_e), 2.55 (1H, m, 8-H_e), 2.60 (1H, m, 7-H_a), 2.61 (1H, m, 8-H_a), 2.82 (1H, m, 5-H), 3.44 (2H, q, J = 7.1 Hz, NCH₂CH₃), 3.64 (1H, br d, J = 9.7 Hz, 4-H_a), 4.01 (1H, br d, J = 13.5 Hz, 2-H_a), 4.16 (1H, m, 2-H_e), 4.19 (1H, m, 4-H_e) and 4.27 (2H, q, J = 7.2 Hz, OCH₂CH₃). δ_C [125 MHz; d₄-acetic acid; calibrated with d₄-acetic acid (20.00 ppm)]: 10.12 (CH₃, NCH₂CH₃), 14.31 (CH₃, OCH₂CH₃), 19.35 (CH₂, C7), 33.52 (CH₂, C6), 36.46 (CH₂, C8), 45.28 (CH, C5), 55.62 (CH₂, NCH₂CH₃), 57.47 (CH₂, C4), 57.98 (quat, C1), 58.55 (CH₂, C2), 63.34 (CH₂, OCH₂CH₃), 169.91 (quat, COOEt) and 208.26 (quat, C9).

4.1.3.21. 1-(Ethoxycarbonyl)-3-ethyl-9,9-dihydroxy-3-azabicyclo-[3.3.1]nonan-3-ium Chloride (33)

The ketone (32) HCl salt was dissolved in the indicated solvents and the title ketal (33, hydrate) salt was generated. δ_H (500 MHz; D₂O; calibrated with TMSP): 1.29 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.37 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.72 (1H, qt, J = 14.1, 7.0 Hz, 7-H_A), 1.83 (1H, m, 7-H_B), 1.85 (1H, m, 6-H_e), 2.00 (1H, dd, J = 14.1, 7.0 Hz, 8-H_e), 2.15 (1H, ddddd, J = 14.1, 13.1, 7.0, 4.2, 1.8 Hz, 6-H_a), 2.23 (1H, tt, J = 4.2, 2.2 Hz, 5-H), 2.39 (1H, tdd, J = 14.1, 7.0, 2.0 Hz, 8-H_a), 3.26 (2H, q, J = 7.2 Hz, NCH₂CH₃), 3.50 (1H, br ddd, J = 13.2, 4.2, 1.8 Hz, 4-H_a), 3.64 (2H, m, 4-H_e and 2-H_a), 3.82 (1H, d, J = 13.4 Hz, 2-H_e) and 4.26 (2H, qd, J = 7.2, 1.0 Hz, OCH₂CH₃). δ_C (125 MHz; D₂O; calibrated with TMSP): 11.90 (CH₃, NCH₂CH₃), 16.05 (CH₃, OCH₂CH₃), 20.73 (CH₂, C7), 27.97 (CH₂, C6), 32.66 (CH₂, C8), 41.39 (CH, C5), 51.96 (quat, C1), 57.47 (CH₂, C4), 57.93 (CH₂, C2), 58.73 (CH₂, NCH₂CH₃), 66.03 (CH₂, OCH₂CH₃), 95.55 (quat, C9) and 176.04 (quat, COOEt). δ_H (500 MHz; CD₃OD; calibrated with TMS): 1.30 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.10 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.78 (1H, m, 7-H_A), 1.88 (1H, m, 6-H_e), 1.89 (1H, m, 7-H_B), 1.94 (1H, m, 8-H_e), 2.20 (1H, m, 6-H_a), 2.40 (1H, m, 8-H_a), 2.45 (1H, m, 5-H), 3.23 (1H, m, 4-H_a), 3.24 (2H, m, NCH₂CH₃), 3.55 (1H, m, 4-H_e), 3.60 (1H, m, 2-H_a), 3.75 (1H, m, 2-H_e) and 4.24 (2H, qd, J = 7.2, 1.0 Hz, OCH₂CH₃). δ_C [125 MHz; CD₃OD; calibrated with CD₃OD (49.50 ppm)]: 10.53 (CH₃, NCH₂CH₃), 14.86 (CH₃, OCH₂CH₃), 19.95 (CH₂, C7), 26.92 (CH₂, C6), 32.93 (CH₂, C8), 34.69 (CH, C5), 50.69 (quat, C1), 55.85 (CH₂, C4), 56.50 (CH₂, C2), 57.73 (CH₂, NCH₂CH₃), 63.78 (CH₂, OCH₂CH₃), 96.99 (quat, C9) and 174.77 (quat, COOEt). δ_H [500 MHz; d₆-DMSO/D₂O (2 drops); calibrated with residual DMSO (2.50 ppm)]: 1.20 (3H, m, OCH₂CH₃), 1.24 (3H, m, NCH₂CH₃), 1.55 (1H, br m, 7-H_A), 1.68 (1H, br m, 6-H_e), 1.78 (1H, br dd, J = 13.7, 5.2 Hz, 8-H_e), 1.90 (1H, m, 7-H_B), 2.21 (1H, m, 6-H_a), 2.03 (1H, br m, 5-H), 2.25 (1H, m, 8-H_a), 3.11 (2H, m, NCH₂CH₃), 3.25 (1H, br d, J = 12.0 Hz, 4-H_a), 3.45 (1H, m, 2-H_a), 3.47 (1H, m, 4-H_e), 3.55 (1H, br d, J = 13.2 Hz, 2-H_e) and 4.14 (2H, m, OCH₂CH₃). δ_C [125 MHz; d₆-DMSO/D₂O (2 drops); calibrated with d₆-DMSO (39.52 ppm)]: 9.72 (CH₃, NCH₂CH₃), 14.25 (CH₃, OCH₂CH₃), 17.87 (CH₂, C7), 25.41 (CH₂, C6), 29.88 (CH₂, C8), 38.65 (CH, C5), 49.19 (quat, C1), 54.10 (CH₂, C4), 55.02 (CH₂, C2), 55.62 (CH₂, NCH₂CH₃), 61.75 (CH₂, OCH₂CH₃), 91.92 (quat, C9) and 172.29 (quat, COOEt).

4.1.3.22. 1-(Ethoxycarbonyl)-3-ethyl-3-methyl-9-oxo-3-azabicyclo-[3.3.1]nonan-3-ium Iodide (34)

A solution of ethyl 3-ethyl-9-oxo-3-azabicyclo[3.3.1]nonane-1-carboxylate (8, 101 mg, 0.42 mmol) and methyl iodide (0.13 mL, 2.09 mmol, 5 equiv) in THF (3 mL) was heated under reflux under an atmosphere of anhydrous nitrogen for 24 h. After the solvent and the excess of methyl iodide were removed by evaporation, and the residue was washed with Et₂O (5 × 2 mL) and EtOAc (5 × 2 mL). The crude product was purified by chromatography over silica gel (DCM/MeOH 10:1 v/v; the mobile phase was basified with concd aq 0.880 ammonia, 0.5% v/v of the prepared mobile phase; target compound TLC R_f = 0.2, stained with Dragendorff’s reagent) giving the title compound (34, 23 mg, 15%). [M]⁺ found 254.1733, C₁₄H₂₄NO₃⁺ requires 254.1751 (254.17562 – 0.00055 ≈ 254.1751 Da, difference = 7 ppm). δ_H (500 MHz; CDCl₃; calibrated with TMS): 1.35 (3H, t, J = 7.2 Hz, OCH₂CH₃), 1.54 (3H, t, J = 7.2 Hz, NCH₂CH₃), 1.87 (1H, dt, J = 16.1, 5.0 Hz, 7-H_e), 2.07 (1H, tt, J = 13.8, 5.0 Hz, 6-H_a), 2.35 (1H, m, 6-H_e), 2.38 (1H, m, 8-H_e), 2.47 (1H, td, J = 13.8, 5.0 Hz, 8-H_a), 2.84 (1H, dtt, J = 16.1, 13.8, 5.0 Hz, 7-H_a), 3.15 (4H, m, 5-H and NCH₃), 4.24 (1H, d, J = 13.8 Hz, 2-H_e), 4.31 (2H, m, OCH₂CH₃), 4.35 (2H, m, NCH₂CH₃), 4.43 (2H, d, J = 7.1 Hz, 4-H) and 4.56 (1H, d, J = 13.8 Hz, 2-H_a). δ_C (125 MHz; CDCl₃; calibrated with TMS): 8.96 (CH₃, NCH₂CH₃), 14.12 (CH₃, OCH₂CH₃), 16.68 (CH₂, C7), 36.08 (CH₂, C6), 38.50 (CH₂, C8), 42.21 (CH, C5), 47.90 (CH₃, NCH₃), 57.19 (quat, C1), 63.22 (CH₂, OCH₂CH₃), 63.98 (CH₂, C4), 65.33 (CH₂ × 2, C2 and NCH₂CH₃) and 169.55 (quat, COOEt) and 207.19 (quat, C9).

4.1.3.23. ((1R,5S,9s))-9-Hydroxy-3-oxabicyclo[3.3.1]nonane-1,5-diyl Dimethanol (36)²⁸

Tetramethylolcyclohexanol (35, 1.0 g) was heated to melting at 160 °C, and then the anhydrous gaseous HCl (concd aq HCl in a dropping funnel was added dropwise into a three-necked round-bottomed flask with concd aq H₂SO₄, and the generated gaseous HCl was piped into a Drechsel gas washing bottle containing concd aq H₂SO₄, forming the anhydrous gaseous HCl) was slowly pumped into the round-bottomed flask with the melted reactant for 10 min. Then, the reaction was heated at 160 °C for 15 min to remove residual HCl. After cooling to 20 °C, water (10 mL) was added to the crude products. Aqueous solution was extracted with chloroform (3 × 10 mL), then extracted with ethyl acetate (5 × 15 mL), and the combined organic extracts were concentrated by evaporation forming a white solid ether (36, 142 mg, 16%). MS (m/z): [M + H]⁺ found 203.1281, C₁₀H₁₉O₄ requires 203.1283 and [M + Na]⁺ found 225.1104, C₁₀H₁₈O₄Na requires 225.1103. δ_H (500 MHz; CD₃OD; calibrated with TMS): 1.44 (2H, tdd, J = 13.5, 6.5, 2.4 Hz, 6-H_a and 8-H_a), 1.51 (1H, dt, J = 13.5, 6.5 Hz, 7-H_e), 1.78 (2H, dd, J = 13.5, 6.5 Hz, 6-H_e and 8-H_e), 2.32 (1H, qt, J = 13.5, 6.5 Hz, 7-H_a), 3.33 (2H, d, J = 11.1 Hz, O-1_A and O-1_A^′), 3.37 (2H, d, J = 11.1 Hz, O-1_B and O-1_B), 3.52 (1H, s, 9-H), 3.53 (2H, d, J = 11.4 Hz, 2-H_e and 4-H_e) and 3.84 (2H, d, J = 11.4, 2.4 Hz, 2-H_a and 4-H_a). δ_C (125 MHz; CD₃OD; calibrated with TMS): 21.66 (CH₂, C7), 34.20 (CH₂ × 2, C6 and C8), 40.71 (quat × 2, C1 and C5), 67.84 (CH₂ × 2, O-1 and O-1′), 70.07 (CH₂ × 2, C2 and C4) and 74.39 (CH, C9). δ_H [500 MHz; d₆-DMSO; calibrated with residual DMSO (2.50 ppm)]: 1.35 (2H, m, 6-H_a and 8-H_a), 1.38 (1H, m, 7-H_e), 1.70 (2H, dd, J = 13.0, 5.7 Hz, 6-H_e and 8-H_e), 2.18 (1H, qt, J = 13.0, 5.7 Hz, 7-H_a), 3.15 (2H × 2, m, O-1 and O-1′), 3.29 (1H, d, J = 4.2 Hz, 9-H), 3.38 (2H, m, 2-H_e and 4-H_e) and 3.59 (2H, dd, J = 11.1, 2.1 Hz, 2-H_a and 4-H_a). δ_C [125 MHz; d₆-DMSO; calibrated with d₆-DMSO (39.52 ppm)]: 20.42 (CH₂, C7), 32.91 (CH₂ × 2, C6 and C8), 43.44 (quat × 2, C1 and C5), 65.48 (CH₂ × 2, O-1 and O-1′), 68.76 (CH₂ × 2, C2 and C4) and 71.15 (CH, C9). δ_H [500 MHz; d₆-acetone; calibrated with residual acetone (2.05 ppm)]: 1.37 (2H, tdd, J = 13.5, 5.8, 2.5 Hz, 6-H_a and 8-H_a), 1.43 (1H, dt, J = 13.5, 5.8 Hz, 7-H_e), 1.66 (2H, dd, J = 13.5, 5.8, 2.5 Hz, 6-H_e and 8-H_e), 2.35 (1H, qt, J = 13.5, 5.8 Hz, 7-H_a), 3.36 (2H, d, J = 10.9 Hz, O-1_A and O-1_A^′), 3.39 (2H, d, J = 10.9 Hz, O-1_B and O-1_B), 3.45 (2H, d, J = 11.2 Hz, 2-H_e and 4-H_e), 3.64 (1H, br s, 9-H) and 3.90 (2H, dd, J = 11.2, 2.5 Hz, 2-H_a and 4-H_a). δ_C [125 MHz; d₆-acetone; calibrated with d₆-acetone (29.84 ppm)]: 21.38 (CH₂, C7), 33.82 (CH₂ × 2, C6 and C8), 39.94 (quat × 2, C1 and C5), 68.27 (CH₂ × 2, O-1 and O-1′), 69.32 (CH₂ × 2, C2 and C4) and 75.83 (CH, C9). δ_H (500 MHz; D₂O; calibrated with TMSP): 1.52 (2H, m, 6-H_a and 8-H_a), 1.57 (1H, m, 7-H_e), 1.82 (2H, dd, J = 12.8, 5.8 Hz, 6-H_e and 8-H_e), 2.16 (1H, tq, J = 12.8, 5.8 Hz, 7-H_a), 3.37 (2H, d, J = 11.5 Hz, O-1_A and O-1_A^′), 3.42 (2H, d, J = 11.5 Hz, O-1_B and O-1_B), 3.58 (1H, s, 9-H), 3.63 (2H, d, J = 11.2 Hz, 2-H_e and 4-H_e) and 3.73 (2H, br d, J = 11.2 Hz, 2-H_a and 4-H_a). δ_C (125 MHz; D₂O; calibrated with TMSP): 22.69 (CH₂, C7), 35.21 (CH₂ × 2, C6 and C8), 42.03 (quat × 2, C1 and C5), 68.34 (CH₂ × 2, O-1 and O-1′), 71.13 (CH₂ × 2, C2 and C4) and 74.32 (CH, C9).

4.1.4. Recrystallization

The conditions of recrystallization obtaining single crystals for the X-ray diffraction studies are shown in Table 2. Generally, the selected hot solvent(s) were added dropwise to a glass vial (5–10 mL) containing the indicated sample (5–25 mg) heated on a water bath (40–60 °C) with slow shaking until the sample was fully dissolved, and then the glass vial was covered with foil, allowed to cool to 20 °C, and then partially evaporated at 20 °C for the required period. After the single crystals were formed (in solution), intensity data for compounds (18), (19), (20), (24), (32), and (36) were collected on a Rigaku Supernova Dual, EosS2 system using monochromated Cu Kα radiation (λ = 1.54184 Å) at 150 ± 2 K. The details of recrystallization and SXRD data are reported in Tables S1–S7.

Table 2. Conditions of Recrystallization.

compound	solvent(s)	time
18	MeOH	3 days
19	MeOH/Et2O	2 days
20	MeOH/Et2O	4 days
24	MeOH/Et2O	1 week
32	EtOAc/CHCl3/MeOH	3 weeks
36	EtOAc	14 h

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c01093.

• Key 2D NMR correlations (COSY, HMBC, and NOESY), ESI-MS spectra, and fully assigned 1D/2D NMR spectra (PDF)

• Crystallographic data from the structural analysis with CCDC no. 1975722 (18) (CIF)

• Crystallographic data from the structural analysis with CCDC no. 1975720 (19) (CIF)

• Crystallographic data from the structural analysis with CCDC no. 1975723 (20) (CIF)

• Crystallographic data from the structural analysis with CCDC no. 1975725 (24) (CIF)

• Crystallographic data from the structural analysis with CCDC no. 1975721 (32) (CIF)

• Crystallographic data from the structural analysis with CCDC no. 1975724 (36) (CIF)

Author Present Address

^§ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhang Jiang Hi-Tech Park, Shanghai 201203, P. R. China.

The authors declare no competing financial interest.