Abietane-Type Diterpenoids from the Arils of Torreya grandis

A chemical investigation of the arils of Torreya grandis led to the isolation of seven abietane-type diterpenoids (compounds 1–7) including three previously undescribed compounds, one unreported natural product, and three known analogs. The structures of these compounds were determined by means of spectroscopy, single-crystal X-ray diffraction, and ECD spectra. An antibacterial activity assay showed that compounds 5 and 6 had significant inhibitory effects on methicillin-resistant Staphylococcus aureus, with MIC values of 100 μM. Moreover, compounds 1, 3, 4, and 7 exhibited anti-neuroinflammatory activity in LPS-stimulated BV-2 microglia cells, with the IC50 values ranging from 38.4 to 67.9 μM.


Introduction
Taxaceae plants are the economically and medicinally important coniferous evergreen trees that comprise the well-known genera Taxus, Pseudotaxus and Austrotaxus, whereas the genera Torreya and Amentotaxus attributed to this family have been controversial for a long time due to their close resemblance to the family of Cephalotaxaceae [1].Among them, Torreya is a primitive member of the gymnospermous yew family (Taxaceae), which consists of seven species and is distributed in the Northern Hemisphere, including North America (T.taxifolia and californica), Japan (T. nucifera), and China (T.fargesii, T. grandis, T. jackii, and T. yunnanensis) [2].However, species of the genus Torreya are similar in morphology, and the relationship within this genus is still vague [3].
No.  Compound 2 was shown to have a molecular formula of C19H27O2 based on its molecular negative-ion peak at m/z 287.2018 [M − H] − (calcd.for C19H27O2, 287.2017) in its HR-ESI-MS spectrum.The 1 H and 13 C NMR data (Table 1) of compound 2 were very similar to those of torregrandol A [20].Fortunately, compound 2 was crystallized from a methanol   13 C NMR data (Table 1) of compound 2 were very similar to those of torregrandol A [20].Fortunately, compound 2 was crystallized from a methanol solution, which led to the confirmation of its structure and the assignment of its absolute configuration as 4R,5R,10S, based on its single-crystal X-ray diffraction analysis (Cu Kα) (Figure 5).Compound 2 was shown to have a molecular formula of C19H27O2 based on its molecular negative-ion peak at m/z 287.2018 [M − H] − (calcd.for C19H27O2, 287.2017) in its HR-ESI-MS spectrum.The 1 H and 13 C NMR data (Table 1) of compound 2 were very similar to those of torregrandol A [20].Fortunately, compound 2 was crystallized from a methanol solution, which led to the confirmation of its structure and the assignment of its absolute configuration as 4R,5R,10S, based on its single-crystal X-ray diffraction analysis (Cu Kα) (Figure 5).Torregrandin B (compound 3) shared an identical molecular formula (C19H27O2) with compound 2, according to its molecular negative-ion peak at m/z 287.2021 [M − H] − (calcd.for C19H27O2, 287.2017) in its HR-ESI-MS spectrum.The 1 H and 13 C NMR data (Table 2) of this compound were very similar with those of compound 2 except for a downfield signal at δC 30.9, ascribable to the 4-CH3 in the 13 C NMR spectrum.To the best of our knowledge, the carbon resonances of an α-oriented CH3 group at C-4 in abietane-type diterpenoids can range from 25 to 35 ppm, whereas in a β-oriented CH3 group, they can range from 15 to 25 ppm [6,21].The downfield chemical shift of 4-CH3 (δC 30.9) in comparison with that of compound 2 (δC 22.9) revealed that compound 3 was an epimer of compound 2 at C-4. Next, the NOESY correlations of H-3α (δH 1.72) with H-5 (δH 1.42) and H3-19 (δH 1.25), of H-5 (δH 1.42) and H-1β (δH 2.21), and of H-1α (δH 1.42) with H3-20 (δH 1.30) established the relative configuration of compound 3, as shown (Figure 3).The absolute configuration was determined by means of ECD calculation, which unambiguously pinpointed the (4S,5R,10S)-configuration of compound 3 (Figure 6).2) of this compound were very similar with those of compound 2 except for a downfield signal at δ C 30.9, ascribable to the 4-CH 3 in the 13 C NMR spectrum.To the best of our knowledge, the carbon resonances of an α-oriented CH 3 group at C-4 in abietane-type diterpenoids can range from 25 to 35 ppm, whereas in a β-oriented CH 3 group, they can range from 15 to 25 ppm [6,21].The downfield chemical shift of 4-CH 3 (δ C 30.9) in comparison with that of compound 2 (δ C 22.9) revealed that compound 3 was an epimer of compound 2 at C-4. Next, the NOESY correlations of H-3α (δ H 1.72) with H-5 (δ H 1.42) and H 3 -19 (δ H 1.25), of H-5 (δ H 1.42) and H-1β (δ H 2.21), and of H-1α (δ H 1.42) with H 3 -20 (δ H 1.30) established the relative configuration of compound 3, as shown (Figure 3).The absolute configuration was determined by means of ECD calculation, which unambiguously pinpointed the (4S,5R,10S)-configuration of compound 3 (Figure 6).Compound 4, namely methyl 12-hydroxy-7-oxodehydroabietate, was recently isolated from Torreya grandis [20], which was previously synthesized by Hamulić and coworkers [22].However, its absolute configuration was not assigned.In the present work, the absolute configuration of compound 4 was firstly confirmed by the single-crystal X-ray diffraction analysis (Cu Kα) (Figure 7).The remaining known diterpenoids were characterized as dehydroabietinol (5) [23], dehydroabietic acid (6) [24], and torreyagrandate (7) [25] by comparing the 1 H and 13 C NMR data as well as mass spectrometric spectra to the published data.
All isolated compounds were tested for their antibacterial activitity against Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Salmonella.However, only compounds 5 and 6 had significant inhibitory effects on MRSA, with MIC values of 100 µM, which were comparable to those of the positive control, rifampicin (MIC = 0.625 µM).Other compounds displayed no obvious antibacterial activity at 100 µM.Moreover, the anti-neuroinflammatory activity in LPS-induced BV-2 cells was evaluated for these compounds.The results indicated that compounds 1, 3, 4, and 7 showed a weak inhibitory effect on NO production, with IC50 values of 49.4, 41.9, 38.4, and The remaining known diterpenoids were characterized as dehydroabietinol (5) [23], dehydroabietic acid (6) [24], and torreyagrandate (7) [25] by comparing the 1 H and 13 C NMR data as well as mass spectrometric spectra to the published data.
All isolated compounds were tested for their antibacterial activitity against Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Salmonella.However, only compounds 5 and 6 had significant inhibitory effects on MRSA, with MIC values of 100 µM, which were comparable to those of the positive control, rifampicin (MIC = 0.625 µM).Other compounds displayed no obvious antibacterial activity at 100 µM.Moreover, the anti-neuroinflammatory activity in LPS-induced BV-2 cells was evaluated for these compounds.The results indicated that compounds 1, 3, 4, and 7 showed a weak inhibitory effect on NO production, with IC 50 values of 49.4, 41.9, 38.4, and 52.6 µM (Table 3), respectively.Meanwhile, none of the remaining compounds exhibited an inhibitory effect on NO production at 100 µM.All the results are representative of three independent experiments.

Plant Materials
The arils of Torreya grandis Fort.ex Lindl.cv.Merrillii.(Taxaceae) were collected in November of 2022 in Yuexi, Anhui Province, People's Republic of China.The plant sample was identified by Prof. Zhen-Hai Wu at College of Life Sciences, Northwest A&F University.A voucher specimen has been deposited in our institute with the following accession number: TG-arils-2022-AH.

Extraction and Isolation
The air-dried powder of the arils of Torreya grandis (1.5 kg) was presoaked three times with 95% EtOH (3 × 2 L) at room temperature to produce an extract (338 g) which was first re-suspended in H 2 O and then partitioned with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol.The EtOAc extract (75 g) was eluted with PE-EtOAc (v/v, 50:1→1:1), which was then combined under the guidance of the thin-layer chromatography (TLC) analysis to obtain eight fractions (A-H).Fraction E (11.6 g) was subjected to a silica gel.

ECD Calculation
The ECD calculation was performed according to our previously reported methods [26].

Figure 4 .
Figure 4.The experimental and calculated ECD spectra of compound 1.

Compound 2
was shown to have a molecular formula of C 19 H 27 O 2 based on its molecular negative-ion peak at m/z 287.2018 [M − H] − (calcd.for C 19 H 27 O 2 , 287.2017) in its HR-ESI-MS spectrum.The 1 H and

Figure 4 .
Figure 4.The experimental and calculated ECD spectra of compound 1.

Figure 5 .
Figure 5. X-ray structure of compound 2. Torregrandin B (compound 3) shared an identical molecular formula (C 19 H 27 O 2 ) with compound 2, according to its molecular negative-ion peak at m/z 287.2021 [M − H] − (calcd.for C 19 H 27 O 2 , 287.2017) in its HR-ESI-MS spectrum.The 1 H and 13 C NMR data (Table2) of this compound were very similar with those of compound 2 except for a downfield signal at δ C 30.9, ascribable to the 4-CH 3 in the13 C NMR spectrum.To the best of our knowledge, the carbon resonances of an α-oriented CH 3 group at C-4 in abietane-type diterpenoids can range from 25 to 35 ppm, whereas in a β-oriented CH 3 group, they can range from 15 to 25 ppm[6,21].The downfield chemical shift of 4-CH 3 (δ C 30.9) in comparison with that of compound 2 (δ C 22.9) revealed that compound 3 was an epimer of compound 2 at C-4. Next, the NOESY correlations of H-3α (δ H 1.72) with H-5 (δ H 1.42) and H 3 -19 (δ H 1.25), of H-5 (δ H 1.42) and H-1β (δ H 2.21), and of H-1α (δ H 1.42) with H 3 -20 (δ H 1.30) established the relative configuration of compound 3, as shown (Figure3).The absolute configuration was determined by means of ECD calculation, which unambiguously pinpointed the (4S,5R,10S)-configuration of compound 3 (Figure6).

Figure 6 .
Figure 6.The experimental and calculated ECD spectra of compound 3.
a Recorded in CDCl 3 .b Recorded in CD 3 OD.Chemical shifts (δ) are expressed in ppm, and J values are expressed in Hz.Molecules 2024, 29, x FOR PEER REVIEW 3 of 10
a Recorded in CDCl 3 .b Recorded in DMSO-d 6 .Chemical shifts (δ) are expressed in ppm, and J values are expressed in Hz.