Multi-conformers caused by conformational change of A-ring in the C18- and C19-N-dealkyl diterpenoid alkaloids

Abstract This paper describes a rare phenomenon of multi-conformers caused by conformational change of A-ring in the C18- and C19- N-dealkyl diterpenoid alkaloids. The possible reasons for the generation of multiple conformational isomers are complex, which could be affected by the substituents at C-1, C-3, C-13, C-14, and C-15, pH, solvents, the intramolecular hydrogen bond between 1α-OCH3/1α-OH and N-H groups, acid-base treatment, preparation methods, and work-up procedures. Graphical Abstract


Introduction
In 2006, a cardioactive alkaloid, N-deethylaconine (1a, Figure 1), was isolated by Chao et al from the lateral roots of Aconitum carmichaelii ("Fu Zi") [1,2]. Later, we conducted the preparation of compound 1a via semi-synthesis, which proved to be more difficult than expected [3]. A three-step transformation including acylation, Ndeethylation, and hydrolysis from aconitine (2) yielded N-deethylaconine (1c), the 13 C NMR data of which were inconsistent with those of the natural 1a (Table 1).
More importantly, the synthetic 1c exhibited no cardioactive effect. On the other hand, N-deethylaconine (1d) generated by a sequence of N-oxidation/reductive demethylation followed by hydrolysis from mesaconitine (3) also revealed a different NMR data compared to the natural compound 1a ( Table 1). Adjustment of the reaction sequence by hydrolysis prior to N-demethylation gave compounds 1e and 1f, both did not match the natural sample 1a in NMR spectra (Table 1). Finally, we found that first hydrolysis of 3 with NaOH/MeOH followed by N-oxidation/reductive demethylation under mild neutral or slight basic work-up conditions allowed for the generation of synthetic 1 g with fully consistent data with 1a (Table 1) [3].
The above results indicated that the natural N-deethylaconine (1a) was unstable. Due to the conformational change of A-ring, multi-conformers could be formed during preparation or work-up procedures. N-Deethylaconine may contain at least 6 conformers judging from the 13 C NMR spectroscopic data (Table 1, 1a-1f). This represents a very rare phenomenon in the chemistry of diterpenoid alkaloids, even in the wide scope of alkaloid natural products.
Because of the rigid polycyclic cage-like structures of diterpenoid alkaloids, only the conformations of rings A and D are variable [4], which are dependent on the substituents at C1 and C15 and solvents [5][6][7][8]. In addition, the conformation of ring A could be influenced by pH compared to that of ring D [6,[9][10][11]. For the diterpenoid alkaloids containing N-alkyl and 1a-OMe groups such as mesaconine (4 , Table  1), as shown in Figure 2, their ring A generally adopts chair form a (H-1: d 3.15, dd, J ¼ 11.2, 6.8 Hz; H-3: d 3.71, dd, J ¼ 11.2, 5.2 Hz), which could be converted to boat or twisted-boat form b after protonation with acids. On the other hand, for the Table 1. 13 C NMR spectral data of conformers 1a-1g and mesaconine (4) (100 MHz, CD 3 OD).
1a [1,2] 1b [1] 1c 1d 1e 1f compounds possessing 1a-OH such as neoline (5) [5,6,8], the ring A exists in boat or twisted-boat conformers (Figure 2(c,d)) regardless of their form as free base or salt. Notably, the preparation method and acid-base workup do not affect the conformation of the above-mentioned diterpenoid alkaloids. However, by contrast, the conformation of ring A in the diterpenoid alkaloid N-deethylaconine (1), which structurally doesn't contain N-alkyl groups, is highly dependent on the preparation method and acid-base treatment. Generally, the A ring conformation could be determined by the multiplicity of the H-1 and H-3 signals in the 1 H NMR spectra. Specifically, the ring A adopts a chair form when both H-1 and H-3 signals are doublet of doublet (dd)-type peaks, while a broad singlet (brs) or triplet (t) peak for H-1 and a brs, broad doublet (brd), or dd/ddd peak for H-3 correspond to the twistedboat form of the ring A [7,[9][10][11]. In addition, A-ring adopts a boat conformation when both H-1 and H-3 are t-type peaks according to the Dreiding molecular model. In this paper, we wish to report our investigations on the multi-conformers caused by conformational change of ring A in the C 18 -and C 19 -N-dealkyl diterpenoid alkaloids.

The amino alcohol type alkaloids (10-15)
As shown in Table 1, compound 1 contains at least 6 conformers (1a-1f), indicating that the 3a-OH or 15a-OH groups significantly affect the number of conformers, compared to that of compounds 10 and 11. The absence of 3a-OH (e.g., in 10) or 15a-OH (e.g., in 11) led to a decrease of conformers. The conformation of A-ring after acid-base treatment remained unchanged in compounds without 15a-OH, such as 12a (with 3a-OH) and 13a (without 3a-OH). The above results implied that 15a-OH had a great impact on the A-ring conformation of the amino alcohol type diterpenoid alkaloids. In a comparison with 11a, after acid-base treatment of 14a (without 1a-OMe), the conformation of ring A in the resulting 14b did not change, probably demonstrating that 1a-OMe was also one of the factors in conformational change of A-ring. The A-ring adopted a boat conformation in compound 15a which possesses 1a-OH [11]. After acid-base treatment, the resulting 15b exhibited a different twisted-boat conformer of A-ring according to its NMR data (Tables 2 and 3), compared to 15a.

The diester or polyester type alkaloids (16-22)
Acid-base treatment of 16a bearing 3a-OH furnished 16b, similar operation converted 16b into 16c. The NMR data ( Tables 4 and 5) of 16a-c revealed that all three compounds possessed distinct A-ring conformations. Acid-base treatment of 16c yielded 16d, which displayed same conformation for A-ring. Similarly, the conversion of 18a into 18b (Tables 4 and 5), as compared with 17a (without 3a-OH), revealed that 3a-OH could also influence the conformation of A-ring. Interestingly, the difference of A-ring conformation in 19a and 19b which lack the 3a-OH seemingly indicated the irrelevance to the 3a-OH functionality. On the other hand, after acid-base treatment, both diester alkaloid 18a (without 15a-OH) and triester alkaloid 20a (without 15a-OH) led to their corresponding conformational isomers 18b and 20b, respectively, implying that the A-ring conformation in these compounds might be not related to 15a-OH. In addition, compared to 20a-c, compound 21a with a 15a-OAc unit was unique because the A-ring conformation remained unaltered in compound 21b resulting from acid-base treatment of 21a (Tables 4 and 5). By contrast, the generation of new conformational isomer 22b was observed in compound 22a without 3a,6a,13,15a-oxygenation groups after acid-base treatment (Tables 4 and 5).

The C 18 -diterpenoid alkaloid N-deethyllappaconitine (23a)
After exposure of 23a to acid-base, the NMR data (Table 6) of the resulting 23b indicated the presence of at least two conformational isomers. More importantly, the signals at aromatic areas were complicated in the NMR spectra of 23b (Table 6, Figures  S59 and S60). We suspected that the complication was not only caused by the amide rotamers.

The lycoctonine-type diterpenoid alkaloid N-deethyldelsoline (24a)
After treatment of 24a with acid-base, very complex NMR profiles (  Table 6). Apart from pH [6,[9][10][11], the A-ring conformation in the diterpenoid alkaloids was also dependent on solvent [6][7][8]. As shown in Table 7, different deuterated solvents such as CDCl 3 , CD 3   was shifted down-field by 4.1 ppm compared to that in 16a, indicating the presence of the intramolecular hydrogen bonding between 1a-OCH 3 and N-H (Figure 3(b)). The above observation was analogous to the chemical shift change of 15 N signal caused by protonation of diterpenoid alkaloids [8,11,18]. Collectively, the A-ring might adopt a twisted-chair conformation in 16a (Table 5, H-1: d 3.18, d, J ¼ 7.8 Hz;    Figure 3(b)). Based on the aforementioned evidence, the presence of an intramolecular hydrogen bonding between 1a-OCH 3 and N-H represented one of the key factors leading to conformational change of A-ring. Furthermore, the twistedboat conformation of A-ring in compounds 15a and 15b containing 1a-OH could be deduced because of the formation of two different intramolecular hydrogen bonds (Figure 3(c,d)).    To summarize, we have for the first time reported the multi-conformers caused by conformational change of A-ring in the C 18 -and C 19 -N-dealkyl diterpenoid alkaloids, which represents a very rare phenomenon in the chemistry of alkaloids. The Aring conformation of these alkaloids could be dependent on the substitution groups at different positions, pH, solvents, the possibility of forming intramolecular hydrogen bond between 1a-OCH 3 /OH and N-H groups, as well as preparation methods and work-up procedures. It is noteworthy that, after acid-base treatment of the C 18 -diterpenoid alkaloid 23a and the lycoctonine-type alkaloid 24a, the NMR profiles of the resulting 23b and 24b tend to be very complex (Table 6).

Preparation of compound 1d
To a solution of mesaconitine

Preparation of compound 1f
A solution of mesaconitine (3, 1.2 g, 1.902 mmol) in 5% NaOH methanol (40 ml) was stirred at room temperature for 1.5 h and general work-up and column chromatography (silica gel H, 25 g, CHCl 3 -MeOH ¼ 9:1, þ 1% NH 4 OH) gave the residue I (0.65 g), which was subjected to N-oxidation with mCPBA (465 mg) at room temperature for 2 h, and the usual work-up gave the residue II (0.66 g). To a solution of the residue II (0.66 g) in methanol (25 ml) on ice water bath, Fe 2 SO 4 Á7H 2 O (755 mg) was added under stirring condition and the reaction solution was stirred at room temperature for 3 h. A general work-up and column chromatography (silica gel H, CHCl 3 -MeOH ¼ 9:1, þ 1% NH 4 OH) gave the pure compound 1f (62 mg). 13 C NMR spectral data (100 MHz, CD 3 OD): See Table 1.
10a-15a: 13 C NMR: See Table 2; 1 H NMR: See Table 3. 21a, 22a: 13 C NMR: See Table 4; 1 H NMR: See Table 5. 23a: NMR: See Table 6 3.8. Preparation of compounds 16a-19a, and 24a: N-deethylation (with NBS) general procedure To a solution of the alkaloid in glacial HOAc, NBS was added and the solution was stirred at room temperature for the time shown in Section below. The reaction solution was poured into ice water, followed by extraction with CHCl 3 after basifying with conc. NH 4 OH (pH 11), evaporation, and column chromatography (silica gel H, CHCl 3 -MeOH ¼ 9:1) gave the pure compound.