Structural Elucidation of Presilphiperfolane-7α,8α-diol, a Bioactive

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Structural Elucidation of Presilphiperfolane-7α,8α-diol, a Bioactive Sesquiterpenoid from Pulicaria vulgaris: A Combined Approach of Solvent-Induced Chemical Shifts, GIAO Calculation of Chemical Shifts, and Full Spin Analysis Niko S. Radulovic,́ *,† Marko Z. Mladenovic,́ † Nikola M. Stojanovic,́ ‡ Pavle J. Randjelovic,́ ‡ and Polina D. Blagojevic†́ Downloaded via UNIV OF SOUTHERN INDIANA on July 22, 2019 at 12:33:10 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Chemistry, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia Faculty of Medicine, University of Niš, Bulevar dr Zorana Đinđića 81, 18000 Niš, Serbia

‡

S Supporting Information *

ABSTRACT: Structural elucidation of a new triquinane sesquiterpenoid, presilphiperfolane-7α,8α-diol, 1a, isolated from Pulicaria vulgaris, was accomplished by combining solvent-induced removal of chemical shift degeneracy and computational (DFT-GIAO) prediction of NMR spectra with the analysis of 1H NMR splitting patterns. In addition to extensive NMR experiments (in 10 different solvents), MS, and FTIR, the identity of 1a was also confirmed by chemical transformations. The applied approach can facilitate structural elucidation of organic molecules and decrease the probability of an erroneous identification, permitting an unambiguous stereochemical elucidation and full NMR assignment. The pharmacological/toxicological profile of 1a was evaluated in several different models.

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since they represent structural precursors of angular and propellane triquinane sesquiterpenoids.1,8−10 However, there is only one previous report regarding synthetic compounds with a 7,8-dioxygenated presilphiperfolane skeleton, only one known vicinal presilphiperfolane diol, and a handful of naturally occurring presilphiperfolanols.11 As the existence of such a compound may shed additional light on the biosynthetic pathways involved in the production of triquinane sesquiterpenes, we opted to unambiguously confirm the identification and determine the correct relative configuration of the new compound. To achieve this goal, three main tools were used: (1) solvent-induced chemical shifts, e.g., solvent-induced chemical shifts, SICS; aromatic solvent-induced shift, ASIS,12,13 (2) computational prediction of NMR parameters,14−17 and (3) full spin analysis.18−23 All of the aforementioned approaches may significantly aid structural elucidation when used as stand-alone tools, but in a multitude of cases they are inoperative, i.e., molecules without strongly complexing functional groups, solubility restrictions, and extensive conformational freedom.24 In addition to the structural elucidation, the pharmacological/toxicological profile of the new compound was evaluated. As there are only limited data regarding the biological activity of presilphiperfolanes, its acute toxicity was

wing to the absence or remoteness of functional groups with a strong (de)shielding effect, NMR spectra of many natural compounds often display accidental chemical shift degeneracy, resulting in a number of (overlapped) higherorder multiplets, and in that way, a straightforward structural determination and/or NMR assignment is obstructed. The small differences between 1H NMR chemical shifts, and also 13 C NMR resonances, can cause overlap of cross-peaks and render 2D NMR spectra equivocal, especially when it comes to the assessment of configuration.1−3 Similar problems were encountered during structural elucidation of a compound isolated from the Et2O extract of Pulicaria vulgaris Gaertn. (Asteraceae). This process was approached using standard methodologies (GC-MS, IR, and 1D/2D NMR data analyses). Additional structural data were collected by a study of the chemical behavior (BF3-induced rearrangement and reaction with dichlorodimethylsilane) of the compound. The results suggested it was a vicinal diol, with two mutually cis-oriented OH groups attached to C-7 and C-8 of the presilphiperfolane core, i.e., one of the eight possible presilphiperfolane-7,8-diol diastereomers, 1a−1h, Figure 1. Presilphiperfolanes are rare triquinane sesquiterpenoids that were previously found in a number of Asteraceae taxa, including Pulicaria,1−7 and are notorious for being NMRchallenging: for example, a 1H NMR spectrum of presilphiperfolanol displayed nine proton resonances (including three unresolved multiplets) in the narrow interval of ca. 0.1 ppm.1,2 It is no wonder the structures of some of them were initially wrongly assigned.3 Presilphiperfolanes were extensively studied © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 8, 2019

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DOI: 10.1021/acs.jnatprod.9b00120 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 1. Numbering Scheme1,11,28 and Chemical Transformations of 1a: The Reaction with Dichlorodimethylsilane (DCDMS) Gave a Single Silylation Product (3); Upon Treatment with BF3, 1a Quantitatively Rearranged to Cameroonanone (2a)a

Figure 1. Structures of eight diastereomeric presilphiperfolane-7,8diols (1a−1h) and the differences in their zero-point-corrected electronic energies (ΔE, kcal/mol); all energies shown are relative to 1f and calculated using (a) B3LYP/6-311++G(d,p) or (b) mPW1PW91/6-311++G(d,p)//B3LYP/6-311++G(d,p) (electronic energies were corrected for zero-point energies from B3LYP/6-311+ +G(d,p)).8 For 1a and 1b, two energy minima were found (conformers 1 and 2 for 1a and 1b are shown in Figure 5).

first evaluated. The Artemia salina model was chosen, as this assay is considered to be useful for the initial assessment of potential general toxicity and cytotoxicity of different compounds.25 The new compound was administered to mice, and the mice were tracked for behavioral changes as a sign of intoxication. Additionally, the stomach mucosa of the animals were inspected for the presence of lesions, of interest in the case of orally applied botanical drugs.26 As the extracts of some Pulicaria species seem to have anticonvulsant activity,27 the anticonvulsant potential of the title compound was also screened.

a

MW is molecular weight.

The first assumption was that 1 would stereochemically correspond to some of the known presilphiperfolanols. For example, the relative configuration of diastereomer 1a (Figure 1) would match that of presilphiperfolane-8α-ol.11 However, naturally occurring presilphiperfolanes are known to have different relative configurations; for example, those of C-4 and C-9 are different in presilphiperfolane-8α-ol and 9-epipresilphiperfolane-1β-ol.11 These stereochemical ambiguities could not be resolved by a simple inspection of the NOESY spectrum (Figure S4, Supporting Information) due to severe signal overlap. Values of vicinal H−H coupling constants for selected pairs of protons would allow differentiation between possible diastereomers 1a−1h. However, due to strong vicinal and/or germinal couplings and mutual overlap, resulting in higher-order multiplets, signals in the δ ranges 1.28−1.19, 1.54−1.34, and 1.96−1.76 were noninterpretable. Solvent-Induced Chemical Shifts. As polar molecules may have substantially different chemical shifts in aromatic and less magnetically active solvents, i.e., solvents with less pronounced anisotropic effects,12,13 the NMR spectra of 1 were recorded in several different deuterated and nondeuterated solvents. Four types of interactions are recognized as responsible for the solvent dependence of 1H NMR shifts:

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RESULTS AND DISCUSSION Chromatographic fractionation of the Et2O extract of dry aerial parts of P. vulgaris permitted the isolation of 240 mg (0.1% yield of the dry plant material, w/w) of compound 1, which has a molecular formula of C15H26O2. The sample was initially subjected to a series of 1D and 2D NMR experiments measured in CDCl3 that provided insight into the connectivity of the molecule, indicative of a presilphiperfolane skeleton. The results of IR analysis and the outcome of the reaction with dichlorodimethylsilane (DCDMS; Scheme 1) suggested that it is a vicinal cis-diol. Compound 1 was assigned the structure of presilphiperfolane-7,8-diol. The reasoning and spectroscopic data that led to the proposed 2D structure of 1 are shown in the Supporting Information file. The results of NMR analyses, e.g., 1H, selective homonuclear decoupling experiments, 13C, DEPT90/135, 1H−1H COSY, gHMQC, gHMBC, and NOESY, of the compound are summarized in Tables 1, 2, and S1−S3, while the numbering scheme is given in Scheme 1 as defined in Coates et al., 1996,1 Zhang et al., 2017,11 and the Dictionary of Natural Products.28 B

DOI: 10.1021/acs.jnatprod.9b00120 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. Parameters (Chemical Shifts, δ, and Line Widths) Used in the Final Step of the Simulation of 1H NMR Spectra (Recorded in 10 Different Solvents) of the New Presilphiperfolane Diol δa (ppm)/line width (Hz) nucleus

CDCl3

CCl4

CS2

benzene-d6

pyridine-d5

methanol-d4

CD3CN

acetone-d6

DMSO-d6

1,4-dioxane

H-1 H-2a H-2b H-3a H-3b H-5a H-5b H-9 H-10a H-10b H-11a H-11b H-12 H-13 H-14 H-15 OH-a OH-b

1.2575/0.8 2.3575/0.5 1.9060/0.8 1.8844/0.7 1.2330/1.0 1.9900/0.7 1.2525/0.5 1.4898/1.2 1.3900/0.5 1.4900/0.5 1.7110/0.5 1.8152/0.8 1.2850/0.5 1.1200/0.5 1.1520/0.5 0.8700/0.5 2.0700/4.5 2.6200/4.5

1.2100/0.8 2.3470/0.9 1.8795/0.7 1.8920/0.6 1.2420/0.7 1.9920/0.8 1.2050/0.5 1.4680/0.9 1.3875/0.9 1.4658/1.0 1.6850/0.5 1.7850/0.8 1.2865/0.5 1.1200/0.5 1.1520/0.5 0.8700/0.5 2.0150/20.0 2.4800/45.0

1.1890/0.6 2.3280/0.5 1.8730/0.6 1.8760/0.7 1.2030/0.7 1.9765/0.5 1.2050/0.5 1.4552/1.0 1.3710/0.5 1.4520/0.6 1.6650/0.5 1.7740/1.0 1.2580/0.5 1.0800/0.5 1.1200/0.5 0.8700/0.5 1.9810/2.5 2.4300/4.0

1.0478/1.2 2.2198/0.5 1.7200/0.5 2.0110/0.5 1.1320/0.5 2.1665/0.7 1.1870/0.5 1.2645/0.7 1.4365/0.7 1.3255/0.5 1.5950/0.5 1.6025/0.6 1.0848/0.5 1.1023/0.5 1.2360/0.3 0.8065/0.5 1.8900/2.5 2.1990/6.5

1.5890/1.2 2.4639/0.55 1.9243/1.1 2.3370/0.5 1.2650/0.5 2.4180/0.8 1.3125/0.5 1.4899/0.8 1.7500/1.0 1.4909/0.8 1.9338/0.7 1.8790/0.8 1.3060/0.5 1.4125/0.5 1.2480/0.5 0.8700/0.5 4.9399/1.5 5.3300/0.5

1.2430/0.7 2.3478/0.5 1.8904/0.7 1.9145/0.7 1.1700/0.7 2.0290/0.5 1.1720/0.5 1.5060/0.7 1.4050/0.6 1.4497/0.5 1.6800/0.7 1.8445/0.5 1.2900/0.5 1.0840/0.5 1.1385/0.5 0.8610/0.5 / /

1.1761/0.8 2.2970/0.6 1.8799/0.7 1.8756/1.1 1.1400/0.7 1.9700/0.5 1.1590/0.5 1.4860/1.0 1.3410/0.6 1.4293/1.0 1.6280/0.7 1.8170/0.6 1.2600/1.0 1.0495/0.5 1.1150/0.5 0.8394/0.5 2.9710/0.7 2.9835/0.5

1.2800/1.0 2.3450/0.5 1.8820/0.8 1.9960/0.6 1.1480/0.7 2.0700/0.8 1.1620/0.5 1.5100/0.9 1.4420/0.9 1.4200/0.9 1.7000/0.5 1.8385/1.0 1.2940/0.5 1.0985/0.5 1.1375/0.5 0.8480/0.5 3.5100/0.5 3.7030/0.5

1.1680/1.2 2.2380/0.8 1.7700/1.0 1.8690/1.0 1.0570/0.7 1.9500/0.7 1.0700/0.5 1.4055/1.5 1.3217/1.0 1.3599/1.0 1.5990/0.5 1.7075/1.2 1.2050/0.5 1.0170/0.5 1.0610/0.5 0.7980/0.5 3.8960/0.7 4.1270/0.8

1.1020/1.0 2.2205/0.9 1.7874/1.1 1.8420/1.1 1.0670/1.0 1.9390/0.7 1.0745/0.7 1.4080/1.5 1.3183/1.5 1.3480/0.9 1.5507/0.5 1.7303/1.2 1.1925/0.5 0.9960/0.5 1.0420/0.5 0.7780/0.5 3.1490/1.0 3.3000/4.5

Chemical shifts obtained after a manual refinement of δ values that correspond to the center of signals observed in the HSQC spectra of 1a.

a

Table 2. Parameters (1H−1H Coupling Constants, J) Used in the Final Step of the Simulation of 1H NMR Spectra (Recorded in 10 Different Solvents) of the New Presilphiperfolane Diol J (Hz)a JH‑1,H‑2ab JH‑1,H‑2b JH‑1,H‑3a JH‑1,H‑3b JH‑1,H‑9 JH‑2a,H‑2b JH‑2a,H‑3a JH‑2a,H‑3b JH‑2b,H‑3a JH‑2b,H‑3b JH‑3a,H‑3b JH‑3a,H‑5b JH‑3a,H‑12 JH‑3b,H‑12 JH‑5a,H‑5b JH‑5a,H‑12 JH‑9,H‑10a JH‑9,H‑10b JH‑9,H‑15 JH‑10a,H‑10b JH‑10a,H‑11a JH‑10a,H‑11b JH‑10b,H‑11a JH‑10b,H‑11b JH‑11a,H‑11b JH‑11b, OH‑a

CDCl3

CCl4

CS2

9.1 3.4 −0.2 −0.8 9.5 −13.7 8.8 1.0 9.5 8.0 −10.5 0.3 1.2 0.3 −11.6 0.85 12.4 4.0 6.3 −13.3 3.0 13.0 3.2 3.2 −14.3

8.8 3.6 −0.3

9.1 3.8 −0.3

benzene-d6 9.2 3.6

9.5 −13.7 8.9 0.9 9.2 8.2 −10.9 0.3 1.2 0.3 −11.6 0.75 13.1 3.5 6.3 −12.9 3.1 13.0 3.3 2.8 −14.2

10.0 −13.8 8.9 1.0 9.2 8.2 −11.5

10.5 −13.9 9.0 0.9 9.6 8.1 −10.7

1.1 0.3 −11.6 0.9 12.0 3.3 6.3 −13.3 3.4 13.0 3.3 3.3 −14.1

1.2 0.3 −11.6 1.0 12.0 3.3 6.3 −13.2 3.2 13.2 3.5 3.3 −14.4

pyridine-d5

methanol-d4

CD3CN

acetone-d6

9.1 3.5 −0.3 −0.3 10.3 −13.4 8.9 0.85 8.4 7.6 −10.5 0.3 1.2 0.3 −11.5 0.9 12.0 3.6 6.3 −12.9 3.2 13.0 3.3 3.3 −14.5

8.9 3.6 −0.5 −0.5 10.5 −13.4 8.4 1.0 9.2 7.9 −10.5 0.3 1.2

9.2 3.6 −0.5

9.0 3.7 −0.3

8.9 3.4

9.0 3.5

10.5 −13.7 8.8 0.9 9.1 8.0 −10.9 0.3 1.2 0.3 −11.6 0.85 12.0 3.5 6.3 −13.3 3.1 13.0 3.2 3.7 −14.3 1.5

9.5 −13.6 9.0 0.9 9.2 7.4 −10.5 0.3 1.0 0.3 −11.6 1.0 13.0 3.5 6.3 −12.9 3.2 11.5 3.3 4.0 −14.0 1.3

10.0 −13.7 8.8 0.85 9.5 8.0 −10.9 0.3 1.0 0.3 −11.6 0.85 12.4 4.2 6.3 −13.3 3.0 13.0 3.2 3.2 −14.0 1.0

10.0 −13.4 8.9 0.85 9.0 7.7 −10.4

−11.6 0.9 12.0 3.7 6.3 −13.1 3.2 13.1 3.3 3.3 −14.2

DMSO-d6

1,4-dioxane

1.0 −11.7 0.85 12.0 3.3 6.3 −13.3 3.0 13.0 3.3 3.3 −14.1 1.4

a1 H−1H coupling constants obtained after a manual refinement of the values directly obtained from the experimental spectra or calculated for 1a, conformer 1 (DFT level of theory). bJx,y refers to the 1H−1H coupling constant between x and y protons.

hydrogen bonding, the anisotropy of the solvent molecules, polar, and van der Waals effects.12 The following nine solvents were used to dissolve the isolated presilphilperfolane diol in further NMR experiments: CCl4, CS2 (CS2 and CCl4 solutions

contained 10% CDCl3, v/v), methanol-d4, DMSO-d6, acetoned6, CD3CN, 1,4-dioxane, benzene-d6, and pyridine-d5. The interchange of NMR solvents significantly influenced 1

C

H NMR shifts of some protons in 1 (Tables 1, 2, S1, and S3, DOI: 10.1021/acs.jnatprod.9b00120 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Simulated (A, blue/top) and experimental (B, black/bottom) 1H NMR spectra of the new presilphiperfolane diol in CDCl3, the residual spectrum obtained when the experimental and simulated spectra were subtracted (B−A, magnification 15×, red/middle; root-mean-square deviation, RMSD = 0.0007, normalized root-mean-square deviation, NRMSD = 0.039%), and appropriate expansions of the signals. Designation of the nuclei corresponds to that presented in Scheme 1. Simulation parameters are given in Tables 1 and 2.

The values of solvent-induced shifts, ΔδA, for nonpolar and polar H atoms ranged from −0.25 to 0.45 ppm and from −0.05 to 2.84 ppm, respectively (Figures 4 and S19). The highest downfield shift values of ΔδA were observed for the CDCl3/ pyridine-d5 interchange and for the following nuclei: OH protons, H-1, H-3 at 1.88, H-5 at 1.99, H-10 at 1.39, H-11 at 1.71, and C-6-CH3 at 1.12 ppm. For other 1H-nuclei, ΔδA was generally equal to or less than 0.1 ppm. It is reasonable to expect the basic pyridine-d5 to strongly interact with OH groups, bringing the strongly magnetically anisotropic pyridine aromatic core in close proximity to H atoms that are on the same side of the presilphiperfolane core as OH groups, an assumption supported by the results of calculations done on solvation complexes of a C5H5N molecule with the title compound; for details, please see the text that follows. The indicated association should result in more pronounced solvent shifts for proximal protons. By following this logic, it was possible to deduce which of the geminal H-3, H-5, H-10, or H11 was cis to the OH groups; the solvent-induced shifts of H3a, H-5a, H-10a, and H-11a were, in general, 5−10 times higher than the ones observed for their geminal counterparts. In addition, the high value of ΔδH‑1 showed that H-1 is, most probably, in the axial orientation with respect to the cyclohexane ring and cis to OH; diastereomers 1a, 1b, 1e, and 1f possess such a configuration at C-1. Contrary to that, from the low values of ΔδH‑12, one could assume that this methyl group was on the other side of the presilphiperfolane core, i.e.,

Figures 2, 3, and S6−S18, Supporting Information). For some signals, the solvent-induced changes in the chemical shifts abolished higher-order effects. This was the case with the H-2 signal (Figure 3). However, in almost all of the solvents utilized, H-9 and H-10 (a/b) signals partially overlapped, as well as the H-1 signal with those of H-3 and/or H-5 (a/b) (e.g., Figure S10, Supporting Information). Despite this, the SICS approach disclosed the relative configurations at C-1 and C-4 and enabled the assignment of signals corresponding to diastereotopic geminal protons. Figures 4, S19, and S20 show solvent-induced chemical shifts, calculated relative to 1H and 13C NMR spectra recorded in CDCl3, of H and C atoms of the title compound. The values of solvent-induced shifts ΔδA (ΔδA = δS − δ0; δS is the chemical shift of nucleus A recorded in solvent S; δ0 is the chemical shift of nucleus A recorded in CDCl3) for C atoms ranged from −2.0 to 1.4 ppm. As expected based on similar polarities and H-bonding abilities, the lowest absolute ΔδA values were observed in the cases of CDCl3/CS2 and CDCl3/CCl4 exchange. Analogously, within the group of Hacceptors, CD3CN, methanol-d4, and acetone-d6, and that of aromatic solvents, benzene-d 6 and pyridine-d 5, similar influences on 13C NMR resonances were noted. The highest absolute value of ΔδA for 13C NMR shifts (Figure S19) was seen in the cases of DMSO-d6 and 1,4-dioxane, which interacted particularly strongly with OH-8. D

DOI: 10.1021/acs.jnatprod.9b00120 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Appearance of the signal of H-2a in 10 different solvents used for NMR recordings. In the spectra recorded in benzene-d6 and pyridine-d5, the H-2a signal overlapped with OH and/or H-5a signals. Black (bottom): experimentally observed signals, blue (top): simulated signals (simulation parameters are given in Tables 1 and 2); CS2, CCl4, and 1,4-dioxane solutions contained 10% (v/v) CDCl3.

Figure 4. Solvent-induced NMR shifts (ΔδA) of the chemical shifts of protons of 1a; ΔδA = δS − δ0 (δS is the chemical shift of nucleus A recorded in solvent S, δ0 is the chemical shift of nucleus A recorded in CDCl3; values are given in parts per million, ppm); H atom designations as given in Scheme 1. Solvents: 1-CD3CN, 2-methanol-d4, 3-acetone-d6, 4−1,4-dioxane (+10% CDCl3), 5-DMSO-d6, 6-benzene-d6, 7-pyridine-d5, 8-CS2 (+10% CDCl3), 9-CCl4 (+10% CDCl3) (10% CDCl3 was added for 2H lock).

trans to the OH groups; diastereomers 1a−1d have the appropriate C-4 configuration. The low value of ΔδH‑9 might suggest that H-9 and two OH groups are trans. However, that would mean Me-9 (H3-15) has to be equatorial as in diastereomers 1a and 1e. Nonetheless, the opposite configuration at C-9 might also be possible, i.e., equatorial H-9 cis to OH, diastereomers 1b and 1f. By taking all of the above into account, the results of the (A)SICS experiments suggested that the structure of the title compound most probably corresponds to that of diastereomer 1a or 1b; these were the only two diastereomers with appropriate configurations at both C-1 and

C-4. Thus, these experiments provided convincing proof of the relative configuration of C-1 and C-4, but were not able to distinguish between C-9 epimers. Geometry Optimization and Prediction of Chemical Shifts and Coupling Constants for Diastereomers 1a− 1h. To acquire additional relevant data, we (a) calculated the chemical shifts for diastereomers 1a−1h and correlated them with the experimentally observed ones and (b) calculated the coupling constants for 1a−1h and used these values to try to interpret the observed complex, overlapped higher-order multiplets. The preliminary search of 1a−1h conformational E

DOI: 10.1021/acs.jnatprod.9b00120 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 5. Optimized geometries (B3LYP/6-311++G(d,p)) of eight diastereomeric presilphiperfolane-7,8-diols (1a−1h). For clarity, only exchangeable H atoms are shown; two conformers were found for 1a and 1b (conformers 1 and 2).

spaces was performed by combining a manual search based on the common knowledge on preferred conformers of substituted cyclopentane and cyclohexane rings and molecular dynamic simulations. Geometries with the lowest energies were further optimized using the B3LYP functional and 6-311+ +G(d,p) basis set.29 This permitted the location of two minima in the case of diastereomers 1a (conformers 1 and 2) and 1b (conformers 1 and 2) and one in the case of 1c−1h (Figure 5). The relative energies of all found conformers are given in Figure 1 and were calculated using B3LYP/6-311++G(d,p) or mPW1PW91/6-311++G(d,p)//B3LYP/6-311++G(d,p), previously employed for the calculation of energies of presilphiperfolane-related compounds.8 The results showed that diastereomers 1e−1h with Me-4 cis to OH-7 and OH-8, which displayed a chairlike geometry of the cyclohexane ring, were thermodynamically more stable than diastereomers 1a− 1d with Me-4 trans to OH groups, with a twist-boat and chairlike conformers of the cyclohexane moiety. The differences between the calculated energies of conformers 1 and 2 of 1a (ΔE = 11.13 and 8.44 kcal/mol for B3LYP/6-311++G(d,p) and mPW1PW91/6-311++G(d,p)// B3LYP/6-311++G(d,p), respectively) strongly suggested conformer 1 as the dominant species of 1a, with almost 100% Boltzmann population. Such a pronounced energy difference and the fact that considerable ring strain is built-up into this tricyclic ring system led to the conclusion that little or no conformational exchange would be possible for 1a or all other isomers. In the case of 1b, more than 93% of conformer 1 would exist in equilibrium, corresponding to ΔE = 1.54 and 1.60 kcal/mol, at the same levels of theory, respectively.

The optimized geometries of conformer 1 of both 1a and 1b are in accordance with the results of (A)SICS experiments: axial H-1, H-10a, and H-5a and equatorial H-11a are in close proximity to the OH groups and could be under strong influence of the anisotropic cone of a H-bonded pyridine molecule. Conformer 2 of 1a and 1b could be ruled out as the pseudoaxial 9-CH3 group of 1a or H-9 of 1b would also be close to the pyridine hydrogen-bonded OH groups, and, by analogy, with other syn-facial protons, higher ΔδA in pyridine than the observed ones should be expected. The opposite was true for H-10a. The distances of the selected protons/carbons from the oxygen atoms used only to assess the distance to the possibly H-bonded pyridine of 1a and 1b, which support conformer 1 of both 1a and 1b as the possible hit structures, are summarized in Table S1 (Supporting Information). Chemical shifts and coupling constants for all energy minima of 1a−1h were calculated using the gauge including atomic orbital (GIAO) method with the B3LYP density functional and 6-311++G(d,p) basis set (further on in the text, this method will be referred to as “method a”) or with the recently proposed WP04 functional, a version of the B3LYP that was reparametrized explicitly for calculating chemical shifts in CDCl3: WP04/aug-cc-pVDZ, “method b”.16 Correlation of the calculated values of the 1H NMR chemical shifts of the nonexchangeable protons shifts with the experimental values strongly implies that the structure of the title compound matches that of 1a (Figures S22−S26 and Table S2, Supporting Information). The correlation coefficients for H atoms of conformers 1 and 2 of 1a were R2 = 0.92 and 0.86, calculated by method b (Table S2, Supporting Information). In F

DOI: 10.1021/acs.jnatprod.9b00120 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 6. (A) Full spin analysis of 1H NMR spectra (DMSO-d6) of 1 with manual fitting performed by the spin simulation option in MestReNova. Step 1: simulated spectrum of 1a; initial GIAO-calculated chemical shifts, δ, and coupling constants, J, in the polarizable continuum model of DMSO-d6. Step 2: initial refinement; adjustment to available experimental δ and J values. Step 3: further (manual) refinement of δ and J values and the adjustment of line widths; this was repeated until the difference between the simulated and experimental spectra, expressed as normalized rootmean-square deviation, NRMSD, 0.91) for all diastereomers; this is not surprising, as the C-framework of the rigid, tricyclic presilphiperfolane core was relatively similar for 1a−1h (Figure 5). The values of rootmean-square errors (RMSEs) also support 1a as the correct structure. They ranged from 2.7 for conformer 1 of 1a to even 48.2 ppm for 1g (method b) for 13C NMR chemical shifts, i.e., from 0.14 for conformer 1 of 1a to even 1.30 ppm for 1h (method b) for 1H NMR chemical shifts. According to RMSE values calculated without any scaling, method b, although slightly overestimating δ values, gave somewhat better results than method a, which systematically underestimated δ values, although, R2 for conformer 1 of 1a was slightly higher in the case of method a. As previously shown,15 the calculation may be improved by taking into account solvent effects. Thus, the calculations of the chemical shifts were repeated for conformer 1 of 1a by representing the experimentally used solvent as a polarizable continuum, according to the method implemented in PCMSCRF.26 In the case of CDCl3, the calculations were performed using both methods a and b. However, despite the much lower computational cost of method a, it gave highly similar, if not better, results (Figure S26, Supporting Information) than that of method b. Thus, for all other solvents, only method a was employed. For the majority of solvents, the calculated δ values were in excellent agreement with the experiment (R2 > 0.94), with the low values of RMSE, that were ca. 0.1 ppm without any scaling, comparable to that of method b. The poorest G

DOI: 10.1021/acs.jnatprod.9b00120 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

similar values of geminal 1H−1H coupling constants for all studied structures. However, the values of some of the vicinal constants in different isomers/conformers were significantly different and could be used to easily discern between 1a− 1h;for example, JH‑1,H‑9 for conformers 1 and 2 of 1a were predicted to be 8.4 and 0.4 Hz, respectively. However, these constants, JH‑1,H‑9, JH‑9,H‑10a, JH‑9,H‑10b, etc., were not directly available from the spectra. Full Spin Analysis. Next, a slightly different approach to the one employed in the 1H iterative full spin analysis (HiFSA) was applied, an NMR methodology successfully used to discern regio- and diastereomers with highly similar NMR spectra, e.g., steviol glycosides or mixtures of natural compounds.21 In this study, manual iteration was used, whereas published HiFSA cases all employ computational iteration. To ascertain similarities between the simulated and experimental spectra, the commonly employed root-meansquare deviation, RMSD, and normalized root-mean-square deviation, NRMSD, were calculated; specifically, the simulation was considered successful when NRMSD was