The structure proposed for apteniol D is different from that of the compound obtained by total synthesis

Abstract We describe the synthesis of 4,4′-oxyneolignan, the proposed structure for naturally occurring apteniol D. The diphenyl ether moiety in 4,4′-oxyneolignan was formed via classical Ullmann ether synthesis using excess copper powder in N,N-dimethylacetamide. The spectral data of synthesised apteniol D show differences compared to those of naturally occurring apteniol D.


Introduction
Apteniols A-G (1-7, Figure 1), isolated from Aptenia cordifolia by DellaGreca in 2005 and 2007, are oxyneolignans, and are reported to be secondary metabolites that inhibit the germination of lettuce seeds (DellaGreca et al. 2005(DellaGreca et al. , 2007. Compounds proposed to have the same structures as apteniols A, B, C and G were synthesised in our laboratories (Nishikawa et al. 2014;Noshita et al. 2015), and a compound with the proposed structure of apteniol C was synthesized by Jung and Bräse (2009); however, the spectral data of the naturally occurring apteniols were not consistent with the spectral data of the synthesised versions. To confirm the structures of the remaining apteniols, we now report the synthesis of the compound with the structure proposed for apteniol D (4).

Results and discussion
The synthetic route for the synthesis of apteniol D is shown in Figure 2. Formation of the diphenyl ether, the key step in this synthesis, was performed via the Ullmann ether synthesis that was previously used in the preparation of apteniols A, B, C and G (Nishikawa et al. 2014;Noshita et al. 2015). First, coupling of vanilline and 4-bromo-3,5-dimethylbenzaldehyde via either the Ullmann ether synthesis or the Buchwald-Hartwig reaction was examined (data not shown). However, the reaction did not proceed under the conditions used for the Buchwald-Hartwig reaction. The desired diphenyl ether was also not obtained using the Ullmann ether synthesis; instead, 3,5-dimethoxybenzaldehyde, the product formed from reductive elimination of bromine, was obtained. in contrast, Ullmann etherification using syringaldehyde and 4-bromo-3-methoxybenzadehyde in a sealed tube at 200 °C produced the desired compound 8 in 29% yield. Because about a half of aldehyde without reacting are recovered, the yield of this reaction is low. in this step, 8 was obtained using the classical conditions, heating at 200 °C with excessive copper powder in N,N-dimethylacetamide (Shioe et al. 2013). Coupling of the phenol and aryl halide to form 8 is supported by the presence of two aldehyde carbon peaks at 189.5 and 190.9 ppm in the 13 C NMR spectrum and detection of the pseudomolecular ion at m/z 317.1025 [M + H] + , consistent with the molecular formula of C 17 H 16 O 6 , using high-resolution fast atom bombardment mass spectrometry (HRFAB-MS). The formyl groups in 8 were then converted into the α,β-unsaturated diethyl ester 9 via the Horner-Wadsworth-emmons reaction. Catalytic hydrogenation and subsequent hydrolysis of the ester groups afforded the desired dicarboxylic acid that corresponds to the proposed structure of apteniol D (4). The chemical structures of all the synthesised compounds were determined by 1 H and 13 C NMR spectroscopy and HR-MS analyses. The 1 H and 13 C NMR spectral data for 4 synthesized in this work and the data previously reported for 4 are shown in Table S1 (supplementary material). The reported NMR data (see Table S1) for natural apteniol D (4) are similar to those of synthesised 4; however, differences exist between the data, even though they were obtained using the same solvent. Distinct differences are observed in the 1 H NMR chemical shifts of H-5′, H-7′ and H-8′, which differ by 0.45, 0.19 and 0.22 ppm, respectively. in addition, the 1 H NMR coupling patterns observed in the peaks for H-2′ and H-6′ for synthesised 4 (doublet and double-of-doublets, respectively) are different from naturally occurring 4 (singlet and doublet, respectively). in the 13 C NMR spectra, the C-3, C-5, C-3′ and C-4′ carbon signals show more than 6 ppm differences when comparing synthesised 4 to naturally occurring 4.  At this stage, it is unclear if the correct structure was determined for naturally occurring 4; however, given the synthetic route that was used and the NMR and HRFAB-MS data, the structure determined for synthesised 4 is correct. The differences in the NMR data between synthesised and naturally occurring 4 suggest the possibility of an error in the interpretation of the data used for the structural determination of naturally occurring 4. Or this difference may come from rotational isomer. As shown in Figure 3, rotational isomers may exist because the diphenyl ether has two rotation axes (C-4-O and C-4′-O). Therefore, the difference in both spectra may come from these isomers (Mazzocchi et al. 1978;Feigel 1996;Duong et al. 2015).

General experimental procedures
Melting points were measured using an MP-J3 (Yanaco, Kyoto, Japan), and are uncorrected. The iR spectra were obtained using a Nicolet iS10 FT-iR spectrometer (Thermo Fisher Scientific, Waltham, MA, U.S.A) with a diamond horizontal attenuated reflectance (ATR) accessory and co-addition of 16 interferograms. Calibration models were generated using OMNiC 9.2.98 software. The 1 H and 13 C NMR spectra were recorded using an Agilent 400-MR DD2 (Agilent, SantaClara CA, U.S.A) spectrometer with tetramethylsilane as the internal standard. Mass spectra were recorded using a JMS-700 (JeOl, Tokyo, Japan) mass spectrometer. Column chromatography was performed on silica gel 60 N (100-210 mesh, Kanto Chemical Co. Tokyo, Japan). All chemicals were reagent grade and used as received, without further purification.

Conclusion
Synthesis of apteniol D (4) was accomplished, based on the reported structure of naturally occurring 4. The 1 H and 13 C NMR data of synthesised 4 was similar but did not perfectly agree with the previously reported data from naturally occurring 4. The discrepancy in the NMR data has also been observed in the comparison of synthesised apteniols A, B, C and G in our previous works (Nishikawa et al. 2014;Noshita et al. 2015) to their reported natural product structures, suggesting that the actual structure of naturally obtained apteniols may be slightly different then their synthesised counterparts. However, it may be necessary to consider the presence of the rotational isomer. The biological activities of the synthesised apteniol D as well as compounds 8, 9 and 10 will be reported in the future.

Funding
This work was supported by Sasakawa Scientific Research Grant from the Japan Science Society.