Isolation and structural characterization of dihydrobenzofuran congeners of licochalcone A

Abstract

In an effort to explore the residual complexity of naturally occurring chalcones from the roots of Glycyrrhiza inflata (Fabaceae), two new licochalcone A (LicA) derivatives were isolated as trace metabolites from enriched fractions. Both constituents contain a dihydrofuran moiety linked to carbons C-4 and C-5 of the retrochalcone core. Compound 1 (LicAF1) represents a new chemical entity, whereas compound 2 (LicAF2) has previously been reported as a Lewis acid catalyzed rearrangement of LicA. Evaluation of chirality revealed that both dihydrofuran derivatives existed as a mixture of R and S enantiomers. Interestingly, when solutions were exposed to sunlight, both dihydrofuran retrochalcones, initially isolated as trans isomers, were found to rapidly isomerize yielding trans and cis isomers. Analysis of the 1D ¹H NMR spectra of the photolysis products revealed the presence of two sets of proton resonances ascribed to each of the geometric isomers. An up-field shift of all proton resonances arising from the cis isomer was observed, suggesting that anisotropic shielding effects were introduced through an overall perturbation of the 3-dimensional structure upon photoisomerization. Similar up-field shifts were observed in the ¹³C spectrum of the cis isomer, except for the CO, C-α, and C-6 carbons, which experienced downfield shifts. Analogous NMR results were observed for LicA. Hence, the results presented herein encompass the isolation and full characterization of LicAF analogs 1 and 2, and observations of their trans-to-cis photoisomerization through the systematic analysis of their NMR spectra.

Keywords: Cis/trans isomerization; Dihydrobenzofuran; Licochalcone A; Raw NMR data; Residual complexity; Retrochalcone.

Fig. 1

Echinatin, licochalcone A (LicA) isomers and its dihydrobenzofuran derivatives. A. Summary of the biosynthetic route leading to the production of echinatin, the first described retrochalcone [7][8][9]. During the biosynthesis, methoxylation occurs in position 2′, while the adjacent keto group undergoes a reduction and dehydration, thereby leading to an inversion of the identified A- and B-rings. Hence, retrochalcones are characterized by a lack of oxygen functionalities in C-2′/C-6′, and the presence of a methoxy group in position 2. B. Licochalcone C, E, F and compound 2 are all licochalcone A isomers, here represented in the trans geometry. Licochalcone A isomers as well as compounds 1 and 2 share the same retrochalcone core structure corresponding to echinatin, (black carbon edges), but differ structurally by the type of aliphatic side chain attached to C-5. For compounds 1 and 2, this chain forms a dihydrofuran ring with the hydroxyl in C-4.

Fig. 2

LC-MS chromatograms and MS/MS spectra of compounds 1 and 2. LC-MS Chromatograms (left side) and MS/MS spectra (right side) readily obtained after isolation of compounds 1 (A) and 2 (B). The MS/MS spectra were taken at 20eV in positive ionization mode. The extracted ion chromatograms (m/z 355 for compound 1, and m/z 339 for compound 2) displayed two peaks with the exact same MS/MS spectra, thereby suggesting the presence of two isomeric forms. The fragment ion at m/z 121 suggests a hydroxyphenyl moiety, whereas the loss of ketene (m/z 313 and 297, respectively) confirmed that both compounds and their isomeric forms are 2-methoxy or 2-hydroxychalcone, as previously described [25].

Fig. 3

Comparison of the 1D ¹H NMR spectra of compounds 1 and 2 (DMSO-d₆, 900 MHz) revealed that both compounds share the same pattern of aromatic protons associated with the retrochalcone core. The main differences are related to a set of signals corresponding to the methyl protons. Compound 1 (LicAF1) has two sets of methyl groups, whereas compound 2 (LicAF2) has three sets, including one doublet having the same coupling constant (J = 6.53 Hz) as the quartet proton at 4.45

Fig. 4

Overlay HMBC and HSQC spectra for structural determination of compound 1 (LicAF1). The structural assignment on the overlay HMBC and HSQC spectra of (trans) compound 1 (LicAF1) is represented by color coded arrows. All the correlations observable for H-6 and H-3 are in green and red, respectively. The correlations related to the α-β unsaturated ketone unit are in dashed lines, and finally the correlation associated with the dihydrofuran moiety and the methoxy group are in blue. The bold bonds of the structure represent the observable COSY correlations. The same type of observations was detected for compound 2 (raw data are deposited in the Harvard Dataverse repository at doi:http://dx.doi.org/10.7910/DVN/DFAVTE).

Fig. 5

¹H NMR spectrum of LicAF1, and chemical shift difference between trans & cis isomers. 3D representations of LicAF1 optimized inside the MMS module of the PERCH software, ¹H NMR spectrum (Exp., 900 MHz, DMSO-d₆) and HiFSA simulated (Sim.) spectra obtained for both trans (green) and cis (red) isomers and showing the up-field shifts of proton resonances corresponding to the cis isomer. The ratio of isomers was calculated as 10.6/89.4 cis/trans for a sample protected from light prior to NMR analysis.

Fig. 6

¹H NMR spectrum of LicAF2, kept in day light, and chemical shift difference between trans & cis isomers. (A) 3D representations of LicAF2 optimized inside the MMS module of the PERCH software, ¹H NMR spectrum (Exp., 900 MHz, DMSO- d₆) and HiFSA simulated (Sim.) spectra obtained for both trans (green) and cis (red) isomers and showing the up-field shifts of proton resonances corresponding to the cis isomer. The ratio of isomers was calculated as 52.5/47.5 cis/trans for a sample unprotected from light a week prior to NMR analysis. (B) Calculated ¹H and ¹³C chemical shifts (Δδ mean ± stdv) between the trans and cis isomers, for LicAFs and LicA. The results suggest a general anisotropic shielding effect for the ¹H occurring through the modification of the 3-dimensional structures. A de-shielding effect was measured for the C=O, C-α and -6, whereas C-2, C-5 and to a lesser extend C-4 were found to be more shielded in the cis isomer. The most important Δδ were measured for the C=O, CH-α, CH-β, and CH-6.