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Complementarity of DFT Calculations, NMR Anisotropy, and ECD for the Configurational Analysis of Brevipolides K−O from Hyptis brevipes G. Alejandra Suárez-Ortiz,† Carlos M. Cerda-García-Rojas,‡ Mabel Fragoso-Serrano,† and Rogelio Pereda-Miranda*,† †

Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, Mexico City 04510, Mexico ‡ Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, A. P. 14-740, Mexico City 07000, Mexico S Supporting Information *

ABSTRACT: Brevipolides K−O (1−5), five new cytotoxic 6(6′-cinnamoyloxy-2′,5′-epoxy-1′-hydroxyheptyl)-5,6-dihydro2H-pyran-2-ones (IC50 values against six cancer cell lines, 1.7− 10 μM), were purified by recycling HPLC from Hyptis brevipes. The structures, containing a distinctive tetrahydrofuran ring, were established by comprehensive quantum mechanical calculations and experimental spectroscopic analysis of their NMR and ECD data. Detailed analysis of the experimental NMR 1H−1H vicinal coupling constants in comparison with the corresponding DFT-calculated values at the B3LYP/ DGDZVP level confirmed the absolute configuration of 3 and revealed its conformational preferences, which were further strengthened by NOESY correlations. NMR anisotropy experiments by the application of Mosher’s ester methodology and chemical correlations were also used to conclude that this novel brevipolide series (1−5) share the same absolute configuration corresponding to C-6(R), C-1′(S), C-2′(R), C-5′(S), and C-6′(S).

cytotoxic compounds, named brevipolides A−J (Figure S1, Supporting Information), all sharing a 6-heptyl-5,6-dihydro-2Hpyran-2-one framework with a cyclopropane ring moiety. These products were identified as inhibitors of the chemokine receptor CCR5 (the principal human immunodeficiency virus Type 1 co-receptor)11 and also found to be active in an enzyme-based ELISA NF-κB assay.9 Polyacylated-6-heptyl-5,6dihydro-2H-pyran-2-ones have been isolated from species of the genus Hyptis, which exhibit a wide range of biological activities.12 In particular, those containing the 5,6-dihydro-2Hpyran-2-one unit with an R-configuration at the C-6 stereogenic center of the α,β-unsaturated δ-lactone exhibit antimicrobial, antifungal, and cytotoxic properties.13 It is believed that this well-known Michael acceptor constitutes the pharmacophoric moiety responsible for the biological properties of these 5,6dihydro-2H-pyran-2-ones from the mint family,14,15 which resemble the antiproliferative activity against various cancer cell lines of (−)-pironetin, a potent inhibitor of tubulin assembly.14 As part of our continuing investigations on the isolation and structural elucidation of polyacylated-6-heptyl-5,6-dihydro-2Hpyran-2-ones,10 this report describes the absolute configuration

Hyptis brevipes Poit. is a member of the mint family (Lamiaceae). This species has been described as originating in Southern Mexico and is native to the Americas, but has been naturalized in Asia.1 It is a weed of cultivated and plant waste areas, plantation crops, orchards, and forest edges and is becoming abundant in fallow ground. As a weed of crops in a wide range of South American (e.g., banana plantations)2 and Asian (e.g., rice crops)1,3 countries, there is a significant risk of accidental introduction of H. brevipes in the southern United States with contaminated seeds or other agricultural products, where it could pose a threat to crops and natural vegetation in the warm, humid regions of all the Gulf States.4 This species has been reported to be used in folk medicine for the treatment of asthma, malaria, and different types of cancer,5,6 as well as a pest-repelling plant for cereal conservation and to repel mosquitoes.5 Biological studies on crude extracts of H. brevipes have shown inhibitory activities against bacterial and fungal growth, as well as DNA intercalation properties.7 In addition, the essential oil of this plant exhibited notable free-radical scavenging activities, potential antitumor activities, and in vitro antimicrobial activity against pathogenic human bacteria and plant fungi.8 Previous chemical studies of H. brevipes collected in Indonesia9 and Mexico10 resulted in the isolation of 10 © 2017 American Chemical Society and American Society of Pharmacognosy

Received: October 16, 2016 Published: January 18, 2017 181

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of five new compounds named brevipolides K−O (1−5) from H. brevipes. Determination of the configurational features of this class of compounds offered a substantial challenge due to their flexibility and the presence of multiple chiral centers.15 Various approaches exist to solve this problem, e.g., chiroptical methods,16 NMR anisotropy methods,17 and X-ray crystallography.18 In addition, the DFT-NMR methodology has been recently applied as a reliable tool to determine the configuration for this class of highly flexible natural products.15 The principle of DFT-NMR calculations is simple, although requiring extensive NMR analysis, including a synergistic relationship between experimental parameters and quantum mechanical calculations.19 In the DFT-NMR integrated approach, calculated 3JHH values are contrasted with the experimental 1H−1H NMR coupling constants, and when data for both sets are comparable, an accurate assignment is obtained among all possible isomers.15,19 Accordingly, the structural assignment of compounds 1−5 was accomplished by the application of DFT-NMR calculations, complemented with the Mosher’s ester method that induces anisotropy in NMR spectroscopy as well as the comparison between calculated and experimental electronic circular dichroism data.

que.20 The previously isolated major brevipolides G−J10 (Scheme S1, Supporting Information), all of which share a 6heptyl-5,6-dihydro-2H-pyran-2-one framework bearing a cyclopropane moiety, were used as HPLC standards to identify those fractions containing novel minor constituents. This procedure allowed the purification of five additional compounds (1−5), which displayed cytotoxicity against a variety of tumor cell lines (Table 1). The most significant values were obtained against nasopharyngeal (KB) and cervix (HeLa) cancer cells with IC50 values of 1.7−10 μM. The molecular formula of the new brevipolide K (1) was deduced as C22H26O7 by HRFABMS (m/z 403.1739 [M + H]+). The 6-heptyl-5,6-dihydro-2H-pyran-2-one framework was recognized through its characteristic 1H and 13C NMR signals (Tables 2 and 3, respectively; Figures S1 and S2, Supporting Information) by comparison with the already published data for the brevipolide series.10 The downfieldshifted methine signal at C-6′ (δ 4.97) in the heptenyl side chain and the singlet at δH 3.83 (3H; δC 55.5) corroborated the substitution of this stereogenic center by a p-methoxycinnamoyl ester, as previously reported.10 In addition, three downfield-shifted signals were observed corresponding to oxygenated methine groups at δH 4.36 (δC 78.0, C-2′), 3.96 (δC 82.2, C-5′), and 3.46 (δC 73.3, C-1′) in the HETCOR spectrum (Figure S4, Supporting Information). COSY experiments (Figure S3, Supporting Information) showed the correlations between the highfield-shielded methylene protons at δH 2.06 and 1.77 (1H each; δC 28.5, C-4′) and 1.97 (2H; δC 27.4, C-3′) and the methines at δH 3.96 and 4.36, respectively, confirming the presence of a tetrahydrofuran ring. Correlations of H-6 and H-2′ with H-1′ were also observed that indicated the lactone and the tetrahydrofuran ring to be connected through the C-1′ center. Figure 1 illustrates the observed correlations in both the 1H−1H COSY and HMBC spectra (Figure S5, Supporting Information) that permitted the assignment of the 6-heptyl-5,6-dihydro-2H-pyran-2-one framework with a tetrahydrofuran ring between C-2′ and C-5′. Three diagnostic 3JCH correlations supporting this connectivity were observed between C-1′ and the methylene protons at C-3′, C7′ and the methylene protons at C-4′, and C-5′ and the C-6′ terminal methyl group. The relative cis configuration around the tetrahydrofuran ring was determined by the NOESY correlation between protons H-2′ and H-5′ (Figure S6, Supporting Information). The HRFABMS of brevipolide L (2) showed the protonated molecule at m/z 389.1578 [M + H]+ consistent with the formula C21H24O7. The NMR spectra of 1 and 2 were quite similar (Tables 2 and 3). For compound 2, the only difference was the absence of the −OCH3 signal as observed in both 1H and 13C NMR spectra of 1 (Figures S7 and S8, Supporting Information). Thus, the p-methoxycinnamoyl substituent at C6′ in 1 was replaced by a coumaryl group in 2. Brevipolide M (3) gave the same molecular formula as 1, which was confirmed by HRFABMS analysis displaying the [M + H]+ ion at m/z 403.1738. The difference between this compound and brevipolide K (1) was observed in the chemical shifts and coupling constant for the double-bond protons in the coumaroyl moiety centered at δH 7.65 (H-7″; δC 145.4) and 6.30 (H-8″; δC 115.5) with a coupling constant value of J = 15.9 Hz, which indicated that compound 3 is the trans geometrical isomer of 1 (Figures S9 and S10, Supporting Information). In turn, the molecular formula of brevipolide N (4) was determined as C21H24O7, from the protonated molecule ion

■

RESULTS AND DISCUSSION The aerial parts of H. brevipes were powdered and macerated with CHCl3. The extract was fractionated by column chromatography on silica gel. 1H NMR analysis allowed the selection of the 5,6-dihydro-2H-pyran-2-one-containing fractions through the diagnostic signals for the α,β-unsaturated δlactone moiety. The selected fractions were submitted to preparative reversed-phase HPLC using the recycling techni182

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Table 1. Cytotoxicity for Brevipolides K−O (1−5)a IC50 (μM) compound

HCT-15

MCF-7

KB

PC-3

HeLa

Hep-2

1 2 3 4 5 vinblastine

>10 >10 >10 >10 >10 0.002

>10 9.3 >10 >10 >10 0.004

9.5 7.5 1.7 3.6 4.9 0.006

10.0 8.8 7.5 >10 >10 0.001

4.2 3.3 6.0 9.5 6.9 0.001

>10 7.7 5.7 >10 9.4 0.007

a

Abbreviations: HCT-15 = colon carcinoma; MCF-7 = breast carcinoma; HeLa = cervix carcinoma, PC-3 = prostate carcinoma, KB = nasopharyngeal carcinoma, Hep-2 = laryngeal epidermoid carcinoma, NT = not tested.

Table 2. 1H NMR Data of Compounds 1−5 (measured at 300 MHz in CDCl3, δ in ppm, J in Hz) position 3 4 5proR 5proS 6 1′ 2′ 3′proR 3′proS 4′proR 4′proS 5′ 6′ 7′ 2″ 3″ 5″ 6″ 7″ 8″ OCH3 OH

1 5.93 ddd (9.8, 2.6, 1.2) 6.78 ddd (9.8, 5.9, 2.6) 2.51 dddd (18.7, 5.9, 4.0, 1.2) 2.38 dddd (18.7, 11.5, 2.6, 2.6) 4.25 ddd (11.5, 8.6, 4.0) 3.51 ddd (9.4, 8.6, 1.3) 4.36 ddd (7.9, 6.8, 1.3) 1.96 dddd (13.6, 8.7, 7.9, 5.7) 2.04 dddd (13.6, 9.0, 7.9, 6.8) 1.78 dddd (12.6, 9.0, 5.7, 5.2) 2.05 dddd (12.6, 8.7, 8.0, 7.9) 3.96 ddd (8.0, 6.9, 5.2) 4.98 dq (6.9, 6.5) 1.25 d (6.5) 7.72 d (8.7) 6.87 d (8.7) 6.87 d (8.7) 7.72 d (8.7) 6.88 d (12.8) 5.82 d (12.8) 3.83 s 3.38 brd (9.4)

2 5.92 ddd (9.8, 2.6, 1.2) 6.77 ddd (9.8, 5.9, 2.6) 2.49 dddd (18.4, 5.9, 4.0, 1.2) 2.37 dddd (18.4, 11.0, 2.6, 2.6) 4.25 ddd (11.0, 8.6, 4.0) 3.51 dd (8.6, 1.0) 4.35 ddd (7.8, 6.8, 1.0) 1.98 m dddd (13.6, 8.6, 7.8, 5.7) 2.05 dddd (13.6, 9.0, 7.9, 6.8) 1.76 dddd (12.6, 9.0, 5.7, 5.2) 2.07 dddd (12.6, 8.6, 7.8, 7.9) 3.95 4.97 1.24 7.64 6.82 6.82 7.64 6.84 5.81

ddd (7.8, 6.7, 5.3) dq (6.7, 6.5) d (6.5) d (8.6) d (8.6) d (8.6) d (8.6) d (12.8) d (12.8)

4.75 brs

3 5.96 ddd (9.8, 2.6, 1.2) 6.88 ddd (9.8, 5.9, 2.6) 2.68 dddd (18.8, 5.9, 4.0, 1.2) 2.49 dddd (18.8, 11.5, 2.6, 2.6) 4.35 (11.5, 8.6, 4.0) 3.55 m 4.35 ddd (7.9, 6.8, 1.3) 1.96 dddd (13.6, 8.7, 7.9, 5.7) 2.05 dddd (13.6, 9.0, 7.9, 6.8) 1.77 dddd (12.6, 9.0, 5.7, 5.2) 2.06 dddd (12.6, 8.7, 8.0, 7.9) 4.00 ddd (8.0, 6.7, 5.2) 5.03 dq (6.7, 6.5) 1.28 d (6.5) 7.46 d (8.6) 6.89 d (8.6) 6.89 d (8.6) 7.46 d (8.6) 7.65 d (15.9) 6.30 d (15.9) 3.83 s 3.55 m

peak at m/z 389.1572 [M + H]+ in the HRFABMS. From its 1 H and 13C NMR spectra (Tables 2 and 3; Figures S11 and S12, Supporting Information), it was deduced that this natural product represents the trans isomer of compound 2. Finally, the molecular formula of brevipolide O (5) was confirmed from its HRFABMS (m/z 405.1551 [M + H]+) as C21H24O8. These data suggested that compound 5 has one additional oxygen atom when compared with compound 4. The 1H and 13C NMR spectra (Figures S13 and S14, Supporting Information) provided evidence for the presence of a 3,4-dihydroxycinnamoyl substituent at C-6′ in compound 5.10 The evidence for the C-6 and C-6′ absolute configurations in compounds 1−5 was obtained from electronic circular dichrosim (ECD) spectroscopic analysis. A positive n → π* Cotton effect for the α,β-unsaturated δ-lactones (1−5) was observed for the five compounds centered at λmax 256−260 nm (Figure 2), confirming the R-configuration for the stereogenic center C-6, as has been described for all 6-substituted 5,6dihydro-α-pyrones from the mint familiy.13 The ECD spectra for the n → π* transition of the cis-cinnamoyl derivative at

5b

4 5.96 brdd (9.8, 2.6) 6.89 ddd (9.8, 5.9, 2.6) 2.70 brddd (18.8, 5.9, 4.0)

5.88 brdd (9.6, 2.0) 6.94 ddd (9.6, 4.4, 2.0) 2.45−2.60 m

2.49 dddd (18.8, 11.0, 2.6, 2.6) 4.34 ddd (11.0, 8.6, 4.0)c 3.60 brdd (9.4, 8.6) 4.32 m 1.90−2.15 m

2.45−2.60 m

1.90−2.15 m

1.90−2.00 m

1.78 m

1.77 dddd (12.6, 9.0, 5.7, 5.2) 2.05 dddd (12.6, 8.7, 8.0, 7.9) 3.98 ddd (8.0, 6.8, 5.2) 4.97 dq (6.8, 6.5) 1.25 d (6.5) 7.14 d (2.2)

1.90−2.15 m 3.99 5.03 1.28 7.37 6.83 6.83 7.37 7.62 6.25

ddd (7.8, 6.7, 5.3) dq (6.7, 6.5) d (6.5) d (8.7) d (8.7) d (8.7) d (8.7) d (15.9) d (15.9)

3.80 brd (9.4)

4.36 ddd (11.0, 8.6, 4.0) 3.60 brs 4.10 ddd (7.0, 7.0, 1.3) 1.90−2.00 m

6.84 7.02 7.58 6.29

d (8.1) dd (8.1, 2.2) d (15.9) d (15.9)

3.50 brs

chiral C-6′ in compounds 1 and 2 both showed a negative Cotton effect at 312 nm in contrast with those of the trans compounds 3−5, which each exhibited a positive Cotton effect centered around 300 nm (Figure 2), confirming the C-6′(S)configuration for the new brevipolides 1−5. These effects were similar to those previously reported for the brevipolide series possessing the cyclopropane moiety with the C-6′(S)configuration10 (Scheme S1, Supporting Information). This Sconfiguration for the stereogenic center at C-6′ seems to be a unique biogenetic feature present in all natural 6-heptenyl-5,6dihydro-2H-pyran-2-ones from the Lamiaceae.15 For the brevipolide series, the C-6′(S)-configuration was also established by X-ray diffraction analysis10 of brevipolide I (Scheme S1, Supporting Information). The absolute configuration for the stereogenic center C-1′ substituted with a hydroxy group adjacent to the tetrahydrofuran ring was determined by Mosher’s ester methodology,21 for which the 1H NMR spectra for the (R)-MTPA and (S)-MTPA derivatives of compound 3 were recorded in the NMR tube.22 The chemical shift difference values (ΔδH = δS − δR; Table 183

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Table 3. 13C NMR Data of Compounds 1−5 (measured at 75 MHz in CDCl3, δ in ppm) position

1

2

3

4

5

2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ OCH3

163.9 121.1 145.7 26.6 77.9 73.3 78.1 27.4 28.5 82.3 72.6 17.2 127.3 132.7 113.5 160.7 113.5 132.7 144.6 116.9 166.8 55.5

164.3 120.9 146.1 26.6 77.8 73.3 78.0 27.3 28.5 82.5 72.9 17.2 127.0 132.9 115.1 157.5 115.1 132.9 144.6 116.8 167.3

163.9 121.2 145.7 26.6 78.1 73.1 78.3 27.4 28.5 82.4 72.8 17.3 127.0 130.0 114.5 161.7 114.5 130.0 145.4 115.5 168.0 55.5

164.3 121.1 146.1 26.6 78.2 73.1 78.3 27.5 28.5 82.5 72.9 17.3 126.7 130.3 116.1 158.7 116.1 130.3 145.8 115.0 168.3

164.8 121.0 146.5 25.6 78.4 73.4 78.5 27.7 28.3 82.5 72.4 17.5 127.1 114.7 144.4 147.3 122.3 115.5 146.1 115.1 168.0

not reflect in a qualitative way their vicinity to the MTPA moiety, and there was an unexpected positive low-magnitude value (δ +0.08) for this α-proton signal. The good correlation for the remaining Δδ values shown in Table 4 is noteworthy. Previous studies involving a large group of acetogenins, containing tetrahydrofuran rings adjacent to secondary hydroxy groups, have also shown an abnormal behavior for the α-proton signals (ΔδH ≅ 0) to the MTPA moiety when the hydroxy group and the adjacent C−O bond of the 2,5-disubstituted tetrahydrofurans have a threo relationship.23 Therefore, the absolute configuration for C-2′ was assigned as R, to sustain this relationship between the chiral centers C-1′(S)/C-2′(R), and for the C-5′ center as S, in accordance with the cis configuration for the tetrahydrofuran ring as supported by the observed NOESY interaction in all compounds 1−5. This unusual effect could be clearly understood for the brevipolides when molecular models for the minimum energy conformers of the MTPA-esters were DFT calculated at the B3LYP/DGDZVP level,23 with the oxygen atoms between C1′(S)/C-2′(R) centers in a threo configuration, which produced a synclinal relationship between H-1′ and H-2′. It was observed that the Mosher ester moiety is always located on the opposite side of the molecule with respect to H-2′, causing an insignificant anisotropic effect upon this atom (Figure 3). In the (R)-Mosher ester, conformers 3R-a and 3R-b are substantially stabilized by the π−π interaction between the aromatic ring and the α,β-unsaturated lactone ring, resulting in the most favored conformation. This situation is not equivalent in the (S)-Mosher ester since an equilibrium between conformers 3S-a and 3S-b is established by rotation around the C-6−C-1′ bond, due to the lack of the π−π stabilizing interaction, causing an imbalance in the anisotropic effects due to the predominance of conformation 3S-a over 3S-b (Figure 3). Consequently, the observed abnormal value for H-2′ (ΔδH = δS − δR = +0.08) arises from the threo configuration that nullifies the anisotropic effects of the substituents in the (S)MTPA derivative over this proton. This configuration precludes H-2′ from the strong shielding effect of the aromatic moiety in conformer 3S-a, in addition to a 1,3 deshielding effect caused by the O-1 atom that is parallel to H-2′ in the minimum energy conformation 3S-b (Figure 3). Additionally, in these four conformations and as expected for the classical MTPA-ester model,21 the strong protection for H-4′proR is evident with a ΔδH = δS − δR = −0.43 as well as the observed paramagnetic shift for the lactone CH2 group with a ΔδH = δS − δR = +0.28, as summarized in Table 4 (Figure S22, Supporting Information). A DFT molecular model for brevipolide M (3) was generated to correlate the 3D structure with its spectroscopic and chiroptical properties. To accomplish this task, a systematic conformational search was carried out by 120° rotations of the C-6−C-1′, C-1′−C-2′, C-5′−C-6′, and C-6′−O-6′ bonds to afford 81 conformers. Several conformational arrangements were discarded according to their high MMFF24 energy arising from steric hindrance. The most stable arrangements within a 3 kcal/mol range were optimized geometrically at the B3LYP/ DGDZVP level,25 affording 18 contributing conformers. In addition, the pseudorotation26 of the tetrahydrofuran ring and the orientation of the cinnamoyl moiety were explored by varying the pertinent dihedral angles. This procedure allowed supplementary local minima structures to be found, which were reoptimized using the same level of theory. Table 5 lists the relative free energies as well as the Boltzmann distribution for

Figure 1. COSY and key HMBC correlations for 1.

Figure 2. ECD spectra of brevipolides K (1 in black), L (2 in green), M (3 in blue), N (4 in orange), and O (5 in magenta).

4)21a obtained by comparing the relevant 1H NMR data of the (R)- and (S)-MTPA (Figure S19, Supporting Information) indicated the absolute configuration for the chiral center to be C-1′(S). Additionally, the Δδ value for the H-2′ proton does 184

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Table 4. 1H NMR Chemical Shift Data for Diagnostic Signals from the (S)- and (R)-Ester Derivatives of 3 to Support the C1′(S)-Configuration for the Brevipolide Series (Measured at 300 MHz in CDCl3, δ in ppm) proton chemical shifts (ΔδH = δS − δR) MTPA-ester

H-3

H-4

CH2-5

H-6

H-1′

H-2′

H-3′proR

H-3′proS

H-4′proR

H-4′proS

H-5′

H-6′

H-7′

R S ΔδH

5.88 5.94 +0.06

6.74 6.79 +0.05

2.22 2.50 +0.28

4.61 4.72 +0.11

5.57 5.44

4.10 4.18 +0.08

1.97 1.95 −0.02

1.72 1.63 −0.09

1.72 1.29 −0.43

1.97 1.72 −0.25

3.98 3.90 −0.08

5.02 4.96 −0.06

1.22 1.15 −0.07

DFT conformations were useful in rationalizing the observed NOESY correlations (Figure S6, Supporting Information). For example, conformer 3a is responsible for the observed key correlations between H-2′−H-5′, H-3′−H-6′, and H-6′-OH, while 3a′ produces the complementary interactions between H4′−H-6′ and H-5′−H-7′. The subsequent conformers also contributed by enhancing the above-mentioned effects. The magnetic shielding tensors of these minimum energy structures were calculated with the gauge-including atomic orbital method, followed by theoretical calculation of the NMR spin−spin coupling constants, also determined at the B3LYP/ DGDZVP level. These values were Boltzmann-averaged to yield DFT-calculated coupling constants in accordance with previously described protocols.15 However, in this case, since the tetrahydrofuran ring can be rapidly interconverted by pseudorotation26 in a large number of conformational states close in energy, the applied approach consisted of averaging the energies and populations of the conformational pairs obtained by tetrahydrofuran ring inversion (3a with 3a′, etc.) before obtaining the Boltzmann populations for the full conformational equilibrium. The whole set of calculated values showed a good correlation (RMSD value of 0.64 Hz) with the experimentally registered JH−H for 3 (Table 5), confirming the configuration for the 6-(6′-cinnamoyloxy-2′,5′-epoxy-1′hydroxyheptyl)-5,6-dihydro-2H-pyran-2-one core, and provided a complete picture for the conformational behavior of brevipolide M (3) in CHCl3 solution. The experimental 1H NMR spectra of 1 and 3 were well-reproduced by nonlinear fitting to their simulated parameters by an iterative processing of chemical shifts, JH,H, and line widths based on the original 1H NMR plots (Figure S20, Supporting Information), using MestReNova software.15c Furthermore, the ECD spectrum for the most stable conformations of brevipolide M (3) was also calculated at the B3LYP/DGDZVP level.10 The DFT-ECD calculations were in agreement with the sign and intensity of the positive broad shoulder around 300 nm (Table 6), which also reproduced the small negative values for both Rvelocity and Rlength responsible for the hyperfine splitting of the experimental ECD spectrum around 300−325 nm for the chiral transcinnamoyl derivatives 3−5, in contrast with the cis compounds 1 and 2, which showed a negative Cotton effect at 320 nm, as shown in Figure 2. To extend the absolute configuration assignment to the 6(6′-cinnamoyloxy-2′,5′-epoxy-1′-hydroxyheptyl)-5,6-dihydro2H-pyran-2-one series, compounds 1−4 were submitted individually to a catalytic hydrogenation procedure using Pd/ C. Brevipolides K (1) and M (3) yielded a convergent tetrahydro derivative that was identical spectroscopically (NMR) with compound 6 (Figures S15 and S16; Supporting Information), while brevipolides L (2) and N (4) afforded derivative 7 (Figures S17 and S18; Supporting Information). Methylation of 7 with CH2N2 afforded compound 6. These results supported the same absolute configuration for all stereogenic centers in both pairs of geometric isomers.

Figure 3. Minimum energy structures (kcal/mol) and population (%) of MTPA esters of compound 3.

the 10 most relevant conformers of 3 within a ΔG° range of between 0 and 2 kcal/mol, and Figure 4 shows the eight most representative conformers accounting for 98.8% of the population. All of these adopted a pseudo-chair conformation for the α,β-unsaturated δ-lactone and a bent U-shaped geometry for the 6-heptyl-5,6-dihydro-2H-pyran-2-one framework, which was stabilized by a hydrogen bond formed between the hydroxy group at C-1′ and either the carbonyl group (conformers 3a, 3a′, 3b, and 3b′) or the ether oxygen (3c, 3c′, 3d, and 3d′) of the cinnamoyl moiety at C-6′. Specifically, the presence of these hydrogen bonds was responsible for the stabilization of the structure in a limited number of conformers. In these minimum energy structures, an envelope conformation for the tetrahydrofuran moiety prevailed (Figure 4). Conformers 3a, 3b, 3c, and 3d showed the C-3′ atom as out of plane, while C-4′ was the atom out of plane in conformations 3a′, 3b′, 3c′, and 3d′. Also, all relevant 185

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ΔGa

18.43 18.43 14.19 14.19 8.82 8.82 7.95 7.95 0.69 0.55

Pb J4,5proS 2.23 2.28 2.27 2.28 2.24 2.26 2.23 2.26 2.24 2.23 2.26 2.6

J3,4

9.86 9.79 9.79 9.79 9.87 9.78 9.86 9.78 9.88 9.85 9.81 9.8

7.23 6.97 6.96 6.97 7.18 6.95 7.17 6.95 7.21 7.19 7.05 5.9

J4,5proR 12.58 12.96 13.04 12.98 12.53 13.03 12.55 13.02 12.52 12.54 12.84 11.5

J5proS,6

J6,1′ 9.72 9.61 9.63 9.62 9.63 9.56 9.63 9.56 9.40 9.37 9.63 8.6

J5proR,6 3.58 4.02 4.02 3.99 3.51 3.92 3.52 3.93 3.45 3.47 3.83 4.0

J1′,2′ 1.07 0.21 1.03 0.23 0.49 0.55 0.48 0.54 9.36 9.31 0.70 1.3

J2′,3′proS 11.73 0.82 10.93 0.89 11.74 1.13 11.71 1.14 −0.01 −0.02 6.15 6.8

J2′,3′proR 5.48 10.43 5.63 10.52 5.77 10.67 5.76 10.67 8.32 8.33 8.08 7.9

0.30 12.41 0.02 12.32 0.07 12.53 0.07 12.54 14.67 14.68 6.39 5.7

J3′proR,4′proR 7.72 9.46 7.92 9.56 9.02 9.07 9.04 9.06 7.79 7.78 8.78 8.7

J3′proR,4′proS 7.24 9.31 7.95 9.42 8.54 9.36 8.54 9.36 7.00 7.00 8.60 9.0

J3′proS,4′proR 14.97 0.01 13.63 0.02 14.59