H and 13 C NMR Spectral Data Assignment for Two Dihydrobenzofuran Neolignans

In this work we present a complete proton (H) and carbon 13 (C) nuclear magnetic resonance (NMR) spectral analysis of two synthetic dihydrofuran neolignans (±)-trans-dehydrodicoumarate dimethyl ester and (±)-trans-dehydrodiferulate dimethyl ester. Unequivocal assignments were achieved by H NMR, proton decoupled C (C{H}) NMR spectra, gradient-selected correlation spectroscopy (gCOSY), J-resolved, gradient-selected heteronuclear multiple quantum coherence (gHMQC), gradient-selected heteronuclear multiple bond coherence (gHMBC) and nuclear Overhauser effect spectroscopy (NOESY) experiments. All hydrogen coupling constants were measured, clarifying all the hydrogen signals multiplicities. Computational methods were also used to simulate the H and C chemical shifts and showed good agreement with the trans configuration of the substituents at C7 and C8.


Introduction
Neolignans (NL) are a class of plant-derived natural products which are produced from shikimic acid pathway. 1 They differ from related lignans by the way the two C 6 C 3 units are joined by other bonds.According to the International Union of Pure and Applied Chemistry (IUPAC) recommendations, the term lignan refers to structures where the two C 6 C 3 units are β,β' (8-8') linked, whereas the term neolignan must be used for compounds that originate from coupling other than 8-8' coupling. 2mong NL, compounds exhibiting a dihydrobenzofuran moiety as structure feature have attracted special attention because their wide range of biological activities, such as antioxidant, 3 antitumor, 4 anti-inflammatory, 5 antileishmanial, 6 trypanocidal, 7,8 insecticidal 9 and cytotoxic. 3][12] However, the oxidative coupling of phenylpropanoids is so far the most commonly reported synthetic route to obtain DBNL, such as compounds (±)-trans-dehydrodicoumarate dimethyl ester (2a) and (±)-trans-dehydrodiferulate dimethyl ester (2b; Figure 1).Compound 2b is reported to have antileishmanial, 13 antiplasmodial, 13 cytotoxic, 13 antiangiogenic, 14 antitumor, 15 and antioxidant 16 activities.Despite of these biological activities, nuclear magnetic resonance (NMR) data found in literature for both compounds are generally incomplete and, in some cases, inaccurate. 14,15,17,18wing to our interest in the detailed NMR study of natural [19][20][21] and synthetic [22][23][24][25] compounds, in this study we have performed a thorough assignment of all proton ( 1 H) and carbon 13 ( 13 C) NMR data for the synthetic dihydrobenzofuran neolignans 2a and 2b using one-(1D) and two-dimensional (2D) NMR techniques.
The main 1 H and 13 C NMR data for (±)-transdehydrodicoumaroate dimethyl ester (2a) and (±)-transdehydrodiferulate dimethyl ester (2b) are presented in Tables 1 and 3. Two-dimensional NMR data (gradientselected correlation spectroscopy, gCOSY; gradientselected heteronuclear multiple quantum coherence, gHMQC; gradient-selected heteronuclear multiple bond coherence, gHMBC; and nuclear Overhauser effect spectroscopy, NOESY) for the same compounds are given in Tables 2 and 4, respectively.Firstly, the 1 H NMR spectra were analyzed in detail, which made it possible to verify all chemical shifts.Further analysis of 1 H NMR spectra led to the measurement of most homonuclear hydrogen coupling constants.Some J values were measured only in J-resolved spectrum and all couplings were confirmed by gCOSY experiments.Then, most signals of the proton decoupled 13 C ( 13 C{ 1 H}) NMR spectra were assigned through gHMQC and distortionless enhancement by polarization transfer (DEPT) 135 experiments.The assignment of nonhydrogenated carbons was carried out by the use of gHMBC information and by comparison with calculated spectra.
1 H and 13 C NMR data previously reported for compound 2a and 2b were obtained in CDCl 3 or acetone-d 6 .
Most of the signals in the 1 H NMR spectrum were between d H 6.0 and d H 8.0, but the hydrogen signal multiplicities are ambiguous.In this work, we found that for compound 2a in acetone-d 6 , the signals at d H 7.6-7.7 are referred to four hydrogen atoms and their overlapping precluded their correct assignment (Figure 2).Therefore, CDCl 3 provided much clearer spectra for 2a, but not for 2b, due to the solvent influence on chemical shifts.For compound 2b, three hydrogen atoms resonate at d H 6.91 in the 1 H HMR spectrum in CDCl 3 .On the other hand, the 1 H NMR signals of 2b were resolved by using acetone-d 6 as solvent, which allowed verification of the multiplicities, observation of the chemical shifts and measurement of the coupling constants.
The 1 H NMR (400 MHz, CDCl 3 ) of compound 2a (Table 1) showed resonances for one trans disubstituted  Detailed 1 H and 13  Assignments of the carbonyl C 9 and C 9' and methoxy groups C 10 and C 10' are directly performed and those groups can clearly be differentiated on the basis of the gHMBC spectrum (Table 2).The gHMBC correlations are observed between d C at 170.9 and the signals at d H 4.27 (H 8 ), 6.09 (H 7 ), and 3.83 (s, 3H); therefore, the d C at 170.9 is attributed to C 9 , and d H at 3.83 is assigned to H 10 .On the other hand, gHMBC correlations between d C at 167.9 and the signals at d H 7.66 (H 7' ), 7.55 (H 7' ) and 3.81 (s, 3H) allowed to assign the d C 167.9 to C 9' , and the d H 3.81 to H 10' .In addition, the d c 51.7 and d c 52.9 were assigned to C 10' and C 10 , respectively, on the basis of the correlations observed in the gHMQC spectrum with d H 3.81 (H 10' ) and d H 3.83 (H 10 ).Finally, the non-hydrogenated sp 2 -hybridized carbons C 1' (127.8),C 4' (161.2) and C 5' (125.1) were unambiguously assigned to C 1' , C 4' and C 5' on the basis of their long-range C−H correlations in the gHMBC spectrum with d H 6.32 (H 8' ), 7.43 (H 2' ) and 6.09 (H 7 ), respectively.Similarly, the assignment of C 1 and C 4 to d C 132.0 and 156.1 was established on the basis of the correlations with d H 7.27 (H 2 =H 6 ) and 6.84 (H 3 =H 5 ), respectively.Considering that C 4 is expected to be unshielded when compared to C 1 due to the inductive effect of the oxygen hydroxyl, this corroborates the assignment.
The 1 H NMR (400 MHz, acetone-d 6 ) data of compound 2b are shown in Table 3 and their 2D NMR data are compilated in Table 4.
The structure of compound 2b is related to the natural dimer 3',4-di-O-methylcedrusin, which is one of the active compounds in dragon's blood.This blood-red latex, produced by some Croton species growing in the South America, is employed in traditional medicine for wound-healing and anticancer properties. 27Lemière et al. 17 have previously reported the synthesis of 3',4-di-O-methylcedrusin and other related neolignans, including compound 2b, and assigned the 13 C NMR data of these compounds on the basis of DEPT experiments and long-range of heteronuclear correlation (HETCOR) correlations.In this work, we found that the 13 C NMR data assignment based on DEPT, gHMQC and gHMBC were similar to that reported by Lemière et al. 17 and therefore, will not be discussed in details here.On the other hand, the with H 7 has not been previously reported in the literature.In this work, we could measure the scalar coupling constants J 2,7 and 6,7 in the J-resolved spectrum as being 0.8 and 0.6 Hz, respectively.
The relative stereochemistry of the substituents at C 7 and C 8 in (±)-2a and (±)-2b, only the trans-(7R,8R) stereoisomers, are reported in Figure 1 and Scheme 1 was determined on the basis of the J 7,8 value and some theoretical calculations, all corroborated by nuclear Overhauser effect (NOE) data (Figure 3).
Firstly, a comparison of J 7,8 values for 2a and 2b with J values reported for other dihydrobenzofuran neolignans, 28 showed a clear agreement with the trans configuration.It is well-established in the literature that the coupling constant J 7,8 in the skeleton of neolignans is higher for cis isomers (8.2-8.4Hz) than for the trans isomer (6.5-7.3Hz). 28evertheless, it has also been reported that conclusions on the relative stereochemistry in five membered rings based on J values for vicinal hydrogens cannot be so reliable for some compounds, as these hydrogens are susceptible to a great variety of dihedral angles and that cis or trans H−H coupling constants can be exactly the same. 29On the other hand, Muñoz and Joseph-Nathan 30 suggested that different stereoisomers might show rather large differences in their 13 C chemical shifts, and that these differences can be used for the structural identification, reassignment and confirmation.Thus, we decided to use theoretical calculations of 1 H and 13 C chemical shifts as an extra effort to elucidate the relative stereochemistry of 2a and 2b.We hence calculated the 1 H and 13 C chemical shifts for trans-(7R,8R and 7S,8S) and cis-(7S,8R and 7R,8S) stereoisomers of compounds 2a and 2b and plotted these results in a cross-comparison to experimental values obtained for these compounds.In our case, each group of experimental data was compared to the group of cis and trans calculated data.The database that shows better agreement with experimental data should indicate which isomer we are dealing with.As an evaluation of this comparison, two main values were considered: the root mean square (rms) error and the coefficient of determination (R 2 ), as recently used to clarify conformation and configuration of several structures. 31This first one, the rms error, was obtained by the comparison of chemical shift values atom by atom, both hydrogen and carbon.In this case, rms value was always lower for trans compounds, regardless the comparison made: only 1 H chemical shifts (d H ), only 13 C chemical shifts (d C ) or d H plus d C .Table 5 shows the rms values obtained, which clearly indicates trans configuration for both compounds 2a and 2b.
The coefficients of determination were obtained from graphics where 1 H and 13 C experimental chemical shift values were plotted in one axis and the corresponding calculated values in the other one.Three different graphics were drawn for each structure: one with d H , one with d C and one with both d H and d C .Invariably, the values obtained for trans structures were closer to the experimental rather than the cis values.Figure 4 shows the example of R 2 obtained for compounds 2a and 2b versus 1 H and 13 C chemical shifts  for the trans-(7R,8R) structure.The R 2 values for 2a and 2b trans-(7R,8R) were 0.9984 and 0.9974, respectively; while the R 2 value for the diasteroisomers cis-(7S,8R) were 0.9977 and 0.9963, respectively.These data also indicate that the obtained compounds have a trans configuration.
Moreover, H 7 exhibited significant NOE correlation with H 2 and H 6 , in the NOESY spectrum (Table 4).However, NOE correlation of H 7 with H 8 is weak, indicating the relative trans configuration for compounds 2a and 2b.In addition, the trans stereochemistry is also consistent with the diastereoselectivity observed in previously reported syntheses of dihydrobenzofuran neolignans by oxidative coupling, in which the main product is normally a trans racemic mixture. 32In this work, the formation of a trans racemic mixture for both 2a and 2b was confirmed on the basis of their specific optical rotation values ([a] D 25 = 0°).

Conclusions
The complete and unequivocal assignments of 1 H and 13 C NMR data for two dihydrobenzofuran neolignans are achieved, leaving no ambiguities.This work included the measurement of all hydrogen homonuclear coupling constants values and all hydrogen signal multiplicities were clarified.Confirmation of the relative stereochemistry was also achieved by density functional theory (DFT) calculations and NOE experiments.This study provides an important 1 H and 13 C NMR database for these two substances and eliminates all previous ambiguities.The stereochemistry was also confirmed by means of J values comparison.This is the first complete assignment reported for each one of these two compounds.

Synthesis of compounds 2a and 2b
Dihydrobenzofuran neolignans 2a and 2b were synthesized as previously reported. 15,17,26Briefly, compounds 2a and 2b were obtained by oxidative coupling of methyl coumarate (1a) and methyl ferulate (1b) using Ag 2 O as oxidant.The reactions were carried out employing a mixture of acetone and benzene (5:8, v/v) in a two-necked flask with aluminum foil, equipped with a magnetic stirrer and a gas tube of N 2 for 20 h at room temperature.The product was purified by column chromatography (2.2 × 100 cm, silica gel 60, 0.040-0.063mm) with hexane and ethyl acetate (2:1, v/v) as eluent affording compounds 1 (36% yield) and 2 (43% yield) as mixture of trans-enantiomers.All structures were confirmed by NMR analysis.

NMR analyses
All 1 H and 13 C NMR experiments were performed on a Bruker Avance DRX400 spectrometer (Karlsruhe, Germany, 400.13 MHz for 1 H and 100.61MHz for 13 C).A direct 5-mm probe head (BBO) was used for 13 C{ 1 H} NMR experiments and an inverse 5-mm probe head (BBI) was used for other experiments.The 1 H NMR spectra were acquired with a solar water heating (SWH) of 8.28 kHz, a time domain (TD) of 64 K, and a number of scans (NS) of 16, which provided a digital resolution of ca.0.126 Hz ( 1 H 30° pulse width = 8.5 μs).As for the 13 C NMR spectra, an SWH of 23.98 kHz was employed, with TD of 32K and NS of 1024, giving a digital resolution of ca.0.732 Hz ( 13 C 30° pulse width = 14.25 μs).DEPT (512 scans), 1 H/ 1 H and 13 C/ 1 H 2D chemical shift correlation experiments were carried out using standard pulse sequences supplied by the spectrometer manufacturer.Long-range 13 C/ 1 H chemical shift correlations were obtained in experiments with delay values optimized for 2 J(C,H) = 8 Hz.Experiments were performed at 300 K and the concentrations for all samples were in the range 10-15 mg mL −1 , in CDCl 3 or acetone-d 6 , using tetramethylsilane (TMS) as internal reference.

Computational methods
Full geometry optimization and vibrational frequency calculations were carried out using the Gaussian09 program package, 33 employing the B3LYP hybrid functional 34 and 6-311+G(2d,p) basis set. 35The nature of the stationary point was determined by performing Hessian matrix analysis. 1H and 13 C NMR chemical shifts values are calculated within Gauge-Independent Atomic Orbital (GIAO) method, [36][37][38] using the TMS as the reference molecule.The mixed option was included to consider the Fermi contact contribution and improve the accuracy of spin-spin coupling constants. 39All NMR calculations were performed at the mPW1PW91/6-311+G(2d,p) level of theory, following the recommendations from Tantillo and co-workers [40][41][42] for 1 H and 13 C computed chemical shifts.In addition, the solvent effect in the NMR calculations was taken into account via the self-consistent reaction field (SCRF) approach. 43

Figure 2 .
Figure 2. Expansions of the 1 H NMR spectrum of compounds 2a and 2b obtained in CDCl 3 and acetone-d 6 .

Figure 3 .
Figure 3. Main nuclear Overhauser effect (NOE) correlations observed in the nuclear Overhauser effect spectroscopy (NOESY) spectra of compounds 2a and 2b.
C NMR Spectral Data Assignment for Two Dihydrobenzofuran Neolignans

Table 1 .
13 and13C NMR data assignments for compound 2a (400 MHz, CDCl 3 ) a Multiplicities assigned on the basis of distortionless enhancement by polarization transfer (DEPT) 135 experiments; b multiplicities and coupling constant values measured within 1 H NMR and J-resolved spectra with the help from 1 H-1 H correlation spectroscopy (COSY) results.

Table 2 .
2D NMR data for compound 2a (400 MHz, CDCl 3 ) 1 H NMR data of compound 2b available in the literature seems inaccurate.Multiplicities of the signals of the 1 H NMR spectrum of 2b are often reported as singlet (H 10 , H 10' , H 11 and H 11' ), doublet (H 2 , H 5 , H 7 , H 8 , H 7' and H 8' ), doublet of doublets (H 6 ) or broad singlet (H 2' and H 6' ) and have not been previously explored.In this work, 1 H-1 H COSY and 2D J-resolved spectra were used to understand the multiplicity and to measure the coupling constants.As reported for compound 2a, analysis of the 1 H-1 H COSY spectrum of 2b (Table 4) revealed a long-range coupling ( 4 J) of H 7' (ddd, 1H, d H 7.63) with H 2' (d H 7.33) and H 6' (d H 7.29).The coupling constant values J 7',6' and J 7',2' were measured in the J-resolved spectrum to be 0.8 Hz and 0.4 Hz, respectively.Similarly, the signal at H 2' (dd, d H 7.33) correlates with d H 7.29 (H 6' , J 2',6' 2.6 Hz) and d H 7.63 (H 7' ).A long-range coupling ( 4 J) between H 6' and H 8 was also deduced from the correlations between d H 7.29 (ddd, H 6' ) and 4.47 (dd, H 8' , J 7',8 1.4 Hz) in the 1 H-1 H COSY spectrum.It was possible to establish the spin systems corresponding to the C 2' /C 6' /C 7' /C 8 and C 2 /C 3 /C 5 /C 6 /C 7 portions of 2b.The long-range coupling ( 4 J) of both H 2 and H 6

Table 3 .
13 and13C NMR data assignments for compound 2b (400 MHz, acetone-d 6 ) a Multiplicities assigned on the basis of distortionless enhancement by polarization transfer (DEPT) 135 experiments; b multiplicities and coupling constant values measured within 1 H-NMR and J-resolved spectra with the help from 1 H-1 H correlation spectroscopy (COSY) results.

Table 5 .
13ot mean square (rms) values from the comparison of experimental 1 H and13C chemical shifts of compounds 2a and 2b with those calculated for their cis-(7S,8R) and trans-(7R,8R) diastereoisomers a Calculated in CDCl 3 ; b calculated in acetone-d 6 .