Revised structure of deacetyl-1,10-didehydrosalvinorin G

Abstract

In comparison with the NMR data of salvinorin A and its 8-epimer, the published structure of deacetyl-1,10-didehydrosalvinorin G was revised to its 8-epimer. The stereochemistry of 8-epi-deacetyl-1,10-didehydrosalvinorin G was further confirmed by NOESY and chemical synthesis.

Salvinorin A (1a), a non-nitrogenous neoclerodane diterpenoid, was isolated from the Mexican medicinal plant Salvia divinorum.^1,2 1a was identified as a potent and selective kappa (κ) opioid receptor (KOR) agonist and the key ingredient for psychoactive effects.^3–5 During the course of structure-activity relationship (SAR) studies,^6–18 it was found that 1a and its derivatives readily underwent epimerization at C-8 under basic conditions. Using various inorganic bases, such as NaBH₄,^2,12,19 NaHCO₃,²⁰ Na₂CO₃,^7,12 K₂CO₃,⁸ LiSEt¹⁰ and LiI,¹¹ 1a produced corresponding natural (8-Hβ) and unnatural (8-Hα, also called 8-epi-) mixtures. Surprisingly, treatment of 1a with excess strong base KOH or Ba(OH)₂ gave a natural salvinorin derivative deacetyl-1,10-didehydrosalvinorin G that was claimed by different research groups to have structures 2a and 3.^13,8 In general, the affinity and potency of natural salvinorin derivatives show much higher KOR binding activities than those of unnatural 8-epimers.^8,12,15,21 Since the configuration of a molecule can significantly affect KOR binding, it is essential to establish a reliable method which would unambiguously determine the stereochemistry at C-8 of salvinorins.

This prompted us to conduct a comprehensive analysis of the ¹H and ¹³C NMR data of natural and unnatural salvinorin derivatives. The differences of 1a and its 8-epimer (1b) in their ¹H and ¹³C NMR spectra are discussed in this paper. Based on the summarized NMR data and chemical synthesis, the stereochemistry of 2a at C-8 is revised to its 8-epimer (2b) as shown in Figure 1.

Figure 1.

Conformational structures of 2a and 2b

In connection with our previous study related to determining the stereochemistry of betulinic acids,²² we recognized that the ¹H and ¹³C NMR data of salvinorins and their corresponding 8-epimers have significant differences. A pair of 8-epimers (1a and 1b), isolated⁹ and synthesized⁸ in our group, were selected as standard compounds for NMR analyses. Using 2D NMR techniques, including COSY, NOESY, HMQC and HMBC, permitted the full assignments of all ¹H and ¹³C NMR chemical shifts. The relative stereochemistry at the C-8 position of these compounds was unambiguously determined, based on their ¹H and ¹³C NMR data. For instance, in the ¹H NMR spectra, the C-8-H of 1a resonated at δ 2.07 (dd, J = 3.0 and 12.0 Hz), confirming axial orientation, while that of 1b resonated at δ 2.45 (d, J = 2.7 Hz) for equatorial orientation. The chemical shift change (Δδ) between 1a and 1b is about 0.38 ppm (Table 1). In addition, the C-12-H of 1b shifted upfield (Δδ 0.27 ppm) to δ 5.26 (dd, J = 1.8 and 12.0 Hz) in comparison with that of 1a at δ 5.53 (dd, J = 4.8 and 11.7 Hz). The coupling constants of H-12 revealed the axial orientation of H-12 in both 1a and 1b. The X-ray analyses showed that the lactone ring in 1a adopts a chair confirmation,^1,2,17 while the one in 1b is a boat conformation.²¹ The different conformation may explain the J values differences of H-12 between 1a and 1b. On the other hand, the H-8 configuration also strongly affects the ¹³C NMR data in B- and C-ring carbons. For instance, the methylene carbon (C-11), carbonyl carbon (C-17) and axial methyl carbon (C-20) of 1b showed lower-field chemical shifts in comparison with those of 1a (Table 2), while the ¹³C resonances of C-6, C-8, C-12, C-13 and C-19 of 1b shifted to upper field. In summary, the characteristic carbon peaks of C-12, C-17 and C-20 can be employed readily for identification of natural and unnatural 8-epimers. In the previous published papers,^10,13,21 8-epimers were mainly determined based on the coupling constants of H-8 and H-12, irradiation of H-12 for a NOE enhancement of H-8, and a general TLC Rf value. Our generalized NMR data should provide reliable information for identification of future salvinorin analogs including C-8 epimers.

Table 1.

¹H NMR Data (300 MHz) at H-8 and -12 for 1a, 1b and 2b in CDCl₃

Compound	1aa	1ba	Δδ1b-1a	2b
H-8 (δ)	2.07	2.45	+ 0.38	2.99
J value	dd, 3.0, 12.0	d, 2.7		dd, 5.1, 9.6
H-12 (δ)	5.53	5.26	− 0.27	5.45
J value	dd, 4.8, 11.7	dd, 1.8, 12.0		dd, 2.4, 12.3

^aSee references 10, 20 and 23 for full ¹H NMR assignments of 1a and 1b.

Table 2.

¹³C NMR Data (75 MHz) for 1a, 1b and 2b in CDCl₃

Carbon #	1a	1b	Δδ1b-1aa	2b	Carbon #	1a	1b	Δδ1b-1aa	2b
1	202.0	202.3		145.1	13	125.2	123.4	− 1.8	124.4
2	75.0	75.2		180.7	14	108.4	108.5		108.4
3	30.7	30.7		128.2	15	143.7	143.5		143.7
4	53.6	52.9		157.5	16	139.4	139.7		139.6
5	42.1	42.2		42.3	17	171.1	173.4	+ 2.3	173.2
6	38.1	34.0	− 4.1	28.3	18	171.5	171.8		165.4
7	18.1	17.6		21.9	19	16.4	15.2	− 1.2	30.3
8	51.4	45.2	− 6.2	44.8	20	15.2	24.6	+ 9.4	24.4
9	35.4	34.7		37.6	-COOMe	52.0	51.7		52.6
10	64.1	64.0		140.0	-COCH3	170.0	169.7		-
11	43.4	48.0	+ 4.6	36.8	-COCH3	20.6	20.5		-
12	72.0	70.0	− 2.0	70.8

^aData is given when Δδ1b-1a is more than 1.0 ppm.

We reported previously that treatment of 1a with Ba(OH)₂ in MeOH gave an unexpected oxidative-elimination product that was assigned as structure 3.⁸ In a very short time span, the structure of 3 was revised to deacetyl-1,10-didehydrosalvinorin G (2a), which was synthesized in 1 M KOH methanol solution.¹³ The structural revision was based on extensive NMR experiments including ¹H NMR, ¹³C NMR, DEPT, COSY, HMQC, HMBC and NOE, and HR-ESI-MS. Following the published procedure (Scheme 1),¹³ indeed, we isolated the same product.²⁴ The NMR data were in full agreement with previous report¹³ except that the assignments of H-7α (δ1.98) and H-7β (δ2.24) should be reversed as H-7α (δ 2.24) and H-7β (δ1.98). After careful comparison of our ¹H and ¹³C NMR data with those of 1a and 1b (Tables 1 and 2), we found that structure 2a assigned to the product was incorrect,¹³ and the correct structure should be 8-epi-deacetyl-1,10-didehydrosalvinorin G (2b, Figure 1). In the ¹H NMR spectrum, the C-8-H of 2b shifted much lower field to δ 2.99 (dd, J = 5.1 and 9.6 Hz), while the C-12-H shifted upfield slightly compared with that of 1a (Table 1). The H-12 coupling constants (dd, J = 2.4 and 12.3 Hz) of 2b were closer to those of 1b (dd, J = 1.8 and 12.0 Hz) than of 1a (dd, J = 4.8 and 11.7 Hz), but the H-8 of 2b showed the J values (5.1 and 9.6 Hz) for axial orientation.¹³ However, this perplexing configuration at C-8-H was soon resolved by comparing the C-ring ¹³C NMR data of 2b with those of 1a and 1b (Table 2). The ¹³C NMR chemical shifts at C-8, C-12, C-17 and C-20 are very similar to those of 1b, indicating the orientation of H-8 is α. The revised structure 2b was further confirmed by the NOE interactions shown in Figure 2. In the NOESY spectrum (see Supplementary Data), H-8 (δ2.99) showed cross peaks to H-7α (δ2.24, strong), H-7β (δ1.98, weak), H-19 (δ1.72) and H-20 (δ1.67), while H-12 (δ5.45) related to H-11α (δ3.11, strong), H-11β (δ2.02, weak) and H-20 (δ1.67). It should be noted that the crossed peak between H-8 and H-12 was very weak. Furthermore, when 1b, the 8-epimer of 1a, was treated with 1 M KOH in MeOH, it afforded the endione 2b in a 67% isolated yield (Scheme 1).²⁴ Valdes et al.² reduced 1a with NaBH₄ in i-PrOH and afforded equal amounts of 8-epimeric mixtures (diol 4a and 8-epi-diol 4b), while Munro et al.¹³ reduced 2a with NaBH₄ in EtOH/CH₂Cl₂ and obtained sole product - 8-epi-diol 4b. All of this evidence supports the structure 2b.

Scheme 1.

Figure 2.

Key NOE interactions of 2b

The coupling constants of H-8 in 2b were misleading in comparison with those of other 8-epi-salvinorins. This is the main reason that Munro et al.¹³ determined H-8 as β configuration. Because of the double bond between C-1 and C-10, the conformation of 2b is distorted. It is known that A/B/C rings in 1a are in chair/chair/chair conformation^1,2 and A/B/C rings in 1b are in chair/chair/boat conformation.²¹ Based on NOESY and molecular modeling analyses, A/B/C rings in 2b should be face-down boat /twist-chair/twist-chair conformation (Figure 1). Under this circumstance, both H-7β and H-8 resemble axial orientations and showed a closer diaxial coupling constant (9.6 Hz). Without sufficient NOE data,¹³ the incorrect assignments of H-7α and H-7β in 2b might depend on those of the known salvinorins C (5a), D (5b), E (5c) and F (5d), isolated from S. divinorum by Munro and Rizzacasa.²⁵ Because the chemical shifts of H-7 and H-8 in 5a, 5b, 5c and 5d overlapped,^{19, 25} the unambiguous assignments of H-7α and H-7β with NOESY spectra became very difficult. Unexpectedly, one year latter, Munro gave the revised assignments of H-7α and H-7β of 2b without any scientific explanation.²⁰ Based on our NOE data, all protons in 2b were fully assigned.²⁴

In conclusion, a concise and informative NMR method for the determination of the C-8-H configuration of salvinorins was established. Based on this method, the correct product obtained by the treatment of 1a with hydroxide in MeOH has been identified. The C-8 epimeric structure (2b) was confirmed by NOESY spectrum and chemical conversion.

Supplementary Material

01

Supplementary data

Copies of ¹H and ¹³C NMR spectra of 1a, 1b and 2b, and 2D NOESY spectrum of 2b.