Two New Benzophenones From the Endohydric Moss Polytrichastrum formosum

Abstract

Two new benzophenone derivatives (1 and 2), were obtained from the endohydric moss Polytrichastrum formosum. Their structures were established by 1D and 2D NMR spectroscopic and HRESIMS methods. The cytotoxicity of 1 and 2 against HCT-116, A-549, HepG2 and HeLa cell lines was evaluated, but no obvious cytotoxic activities were observed (IC₅₀ >100 µM). Benzophenones may be characteristic components of Polytrichaceae.

Mosses (Musci, Bryophyta) have relatively simple anatomical structures ¹ and are currently represented by approximately 14 000 species that colonize diverse habitats. ² The Polytrichaceae family are comprised of 17 genera and approximately 200 species, ³ of which the genera Polytrichum and Polytrichastrum are among the most familiar of all mosses, especially in boreal and temperate regions, due to their abundance, distinctiveness, and large size. ⁴ Previous phytochemical investigation of Polytrichaceae mosses has resulted in the isolation of benzonaphthoxanthenones, ^{5 -11} benzophenones, ¹² cinnamoyl, ⁷ coumarin glucosides, ¹³ and flavonoids^10,11with broad biological activities such as cytotoxicity, ^{5 -7,10,12} antioxidant, ⁹ anti-inflammatory, ¹⁴ tyrosine phosphatase 1B (PTP1B) inhibition, ⁸ and anti-neuroinflammatory activities. ¹¹ During continuing chemical research of products from mosses, 2 new benzophenone derivatives (1 and 2) (Figure 1) were obtained from the endohydric moss species Polytrichastrum formosum. Herein, we report their chromatographic separation, structure elucidation, and bioactivity.

Figure 1

The structures of compounds 1 and 2.

Results and Discussion

Compound 1, obtained as a white powder, had a molecular formula of C₂₃H₂₁NO₄ as deduced from its HRESIMS ion at m/z 398.1363 (cacld for C₂₃H₂₁NNaO₄, 398.1363). The ¹H NMR spectrum displayed signals for a hydroxy group (δ _H 12.58), 2 monosubstituted aromatic rings (δ _H 7.63, 2 hours, d, J = 7.5 Hz, 7.51, 2 hours, t, J = 7.5 Hz, 7.59, 1 hour, t, J = 7.5 Hz; 7.18, 2 hours, d, J = 7.4 Hz, 7.30, 2 hours, t, J = 7.4 Hz, 7.23, 2 hours, t, J = 7.4 Hz), a 1, 2, 4-trisubstituted benzene ring (δ _H 7.54, 1 hour, d, J = 8.9 Hz; 6.49, 1 hour, d, J = 2.1 Hz; 6.38, 1 hour, dd, J = 8.9, 2.1 Hz), and an oxygenated methylene group (δ _H 4.51, 2 hours, s). The ¹³C NMR spectrum with the assistance of DEPT spectroscopic data revealed the presence of 23 carbon resonances (Table 1), attributed to 7 quaternary (one keto carbonyl), 3 methylene (one oxygenated), and 13 aromatic methine carbons. The above-mentioned information suggested that 1 might be a benzophenone derivative with 3 aromatic rings. ^12,15,16

Table 1

¹H NMR (500 MHz) and ¹³C NMR (125 MHz) Spectroscopic Data for Compounds 1 and 2 in CDCl₃ (δ in ppm, J in Hz).

Position	1		2
	δ H	δ C	δ H	δ C
1		114.4		114.4
2		166.1		166.1
3	6.49 (1 H, d, 2.1)	102.8	6.55 (1 H, d, 1.5)	102.9
4		163.3		163.3
5	6.38 (1 H, dd, 8.9, 2.1)	106.7	6.45 (1 H, dd, 8.8, 1.5)	106.7
6	7.54 (1 H, d, 8.9)	135.8	7.56 (1 H, d, 8.8)	135.8
7		200.3		200.3
1'		138.1		138.1
2', 6'	7.63 (2 H, d, 7.5)	128.8	7.63 (2 H, d, 7.5)	129.0
3', 5'	7.51 (2 H, t, 7.5)	128.5	7.50 (2 H, t, 7.5)	128.5
4'	7.59 (1 H, t, 7.5)	131.9	7.58 (1 H, t, 7.5)	131.9
1"		167.1		167.1
2"	4.51 (2 H, s)	67.3	4.54 (2 H, s)	67.3
1"'	3.62 (2 H, q)	40.3	3.32 (2 H, q)	41.0
2"'	2.86 (2 H, q)	35.7	1.58 (2 H, m)	22.9
3"'		138.5	0.94 (3 H, t，7.4)	11.4
4"', 8"'	7.18 (2 H, d, 7.4)	128.9
5"', 7"'	7.30 (2 H, t, 7.4)	126.8
6"'	7.23 (1 H, t, 7.4)
12-OH	12.58 (1 H, s)		12.57 (1 H, br s)
1"-NH	6.53 (1 H, br s)		6.52 (1 H, br s)

The hydroxy resonance at δ _H 12.58 showed HMBC correlations to C-1 (δ _C 114.4), C-2 (δ _C 166.1), C-3 (δ _C 102.8), and C-4 (δ _C 163.3), indicating that HO-2 was ortho to the quaternary C-1 and meta to the oxygenated quaternary. The HMBC correlations of H-6 (δ _H 7.55) to C-1, C-7 (δ _C 200.3), and C-4; and of H-2' (and H-6') (δ _H 7.63) to C-7 connected a monosubstituted and a 1, 2, 4-trisubstituted aromatic rings to the keto carbonyl group to give a benzophenone core. Except for the other one monosubstituted aromatic ring, the remaining NMR signals were assigned to an amide side chain based on the COSY correlations (Figure 2) of N-H (δ _H 6.53)/H₂-1"' (δ _H 3.62)/H₂-2"' (δ _H 2.86) and HMBC correlations of H₂-2" (δ _H 4.51) to C-1" (δ _C 167.1); of H₂-1"' to C-1" and C-2"' (δ _C 35.7); and of H₂-2"' to C-1"' (δ _C 40.3). The HMBC correlations of H-2" to C-4; of H₂-1"' to C-3"' (δ _C 138.5); and of H₂-2"' to C-3"' and C-4"' (and C-8"') (δ _C 128.9) suggested that the amide side chain was attached to C-4 via an oxygen atom, and a monosubstituted benzene ring was linked to C-2"' via a C-2"'/C-3"' single bond. Thus, the structure of compound 1 was proposed as shown in Figure 1, and named 2-(4-benzoyl-2-hydroxyphenoxy)-N-phenethylacetamide.

Figure 2

Key COSY and HMBC correlations of compounds 1 and 2.

Compound 2 showed a molecular ion peak at m/z 336.1207 (calcd. for C₁₈H₁₉NNaO₄, 336.1206) in its HREIMS, suggesting a molecular formula of C₁₈H₁₉NO₄. Comparison of the NMR data of 2 (Table 1) with those of 1 revealed that the main differences were the absence of one monosubstituted aromatic ring and the presence of a methyl group. The COSY correlations of H₂-1"' (δ _H 3.32)/H₂-2"' (δ _H 1.58)/H₃-3"' (δ _H 0.94) and HMBC correlations of this methyl group (δ _H 0.94, 3 hours, t, J = 7.4 Hz) with C-1"' (δ _C 41.0) and C-2"' (δ _C 22.9) (Figure 2) indicated that 2 possessed a similar benzophenone core as that of 1, and a methyl group instead of a benzene ring was linked to C-2"'. Accordingly, the structure of 2 was elucidated as shown and named 2-(4-benzoyl-3-hydroxyphenoxy)-N-propylacetamide.

The cytotoxicity of the 2 new compounds against HCT-116, A-549, HepG2 and HeLa human cancer cell lines were assessed, but no obvious cytotoxic activity was observed (IC₅₀ >100 µM).

Experimental

General Experimental Procedures

Optical rotations were measured on a JASCO P-1020 polarimeter in MeOH at room temperature. IR spectra were recorded on a Bruker Tensor 27 spectrometer using KBr pellets, 1D and 2D NMR spectra on a Bruker AVIII 500 MHz spectrometer (Bruker, Karlsruhe, Germany) in CDCl₃ with tetramethylsilane (TMS) as an internal standard, and high-resolution electrospray mass spectrometry (HRESIMS) on an Agilent 6529B Q-TOF instrument (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was performed on a Prep-HTGF ODS column (21.2 mm × 250 mm i.d., 7 µm, Agilent Technologies, Santa Clara, CA, USA) with a flow rate of 20.0 mL/min. Silica gel (200, 300 mesh, Qingdao Haiyang Chemical Co., Ltd, Qingdao China), Sephadex LH-20 gel (Amersham Biosciences), MCI (Mitsubishi, Japan) and RP-C18 silica (40, 63 mm, FuJi, Japan) were used for column chromatography.

Plant Material

The endohydric moss, Polytrichastrum formosum (Hedw.) G. L. Sm, was collected from Cape Mount, Dujun city, Guizhou Province (latitude 26°35′42.8″N, longitude 107°38′32.1″E, altitude 1250 m), People’s Republic of China in November, 2019. A specimen (HBSD2019110401) was deposited in the College of Life Sciences, Hebei Normal University.

Extraction and Isolation

The air-dried whole plant (1.3 kg) of P. formosum was powdered and extracted with 95% EtOH by infusion (5 × 5 L, 3 days each). The concentrated exact (39 g) was suspended in H₂O and partitioned with light petroleum, CH₂Cl₂, EtOAc, and n-BuOH, successively. The CH₂Cl₂ extract (7.5 g) was subjected to MCI gel column chromatography eluting with MeOH/H₂O mixtures (30%, 60%-90%), and the 90% MeOH/H₂O fraction (2.6 g) was chromatographed on a silica gel column eluting with light petroleum and acetone mixtures (10:1−5:1−3:1−3:2−1:1) to give 5 fractions, Fr.1−Fr.5. Fraction Fr.3 (650 mg) was separated on an ODS gel column (eluted with MeOH/H₂O, 60%‐100% in a step gradient manner) to afford 5 fractions, Fr.3.1−Fr.3.5. Fraction Fr.3.2 (0.92 g) was separated by RP-C₁₈ preparative HPLC with 60% ACN/H₂O and the chromatographic peak at t _R = 19 minutes was collected and purified by Sephadex LH-20 CC (eluted with MeOH) to afford compound 1 (9.3 mg). Compound 2 (6.1 mg) was obtained from the chromatographic peak at t _R = 31 minutes by recrystallization from CDCl₃.

2-(4-Benzoyl-2-Hydroxyphenoxy)-N-Phenethylacetamide (1)

White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{25}$ −1.9 (c 0.1, MeOH); UV (MeOH) λ _max (log ɛ) 205 (4.38), 243 (3.86), 284 (4.05), 324 (3.81) nm; IR (KBr) ν _max 3351, 1651, 1627, 1543, 1346, 1261, 1223, 1192, 1112, 1053, 700 cm⁻¹; ¹H and ¹³C NMR see Table 1; HRESIMS m/z 398.1363 [M + Na]⁺ (calcd. for C₂₃H₂₁NNaO₄, 398.1363).

2-(4-Benzoyl-3-Hydroxyphenoxy)-N-Propylacetamide (2)

Colorless crystals (CDCl₃); $[\alpha {]}_{\mathrm{D}}^{25}$ −5.7 (c 0.1, MeOH); UV (MeOH) λ _max (log ɛ) 204 (4.26), 243 (3.86), 284 (4.05), 327 (3.81) nm; IR (KBr) ν _max 3390, 1679, 1627, 1546, 1505, 1283, 1171, 1131, 1045, 702 cm⁻¹; ¹H and ¹³C NMR see Table 1; HRESIMS m/z 336.1207 [M + Na]⁺ (calcd. for C₁₈H₁₉NNaO₄, 336.1206).

Cytotoxicity

Cytotoxicity was measured using the sulforhodamine B method with human colon cancer (HCT-116), human lung carcinoma (A-549), liver hepatocellular carcinoma (HepG2), and human cervical carcinoma (HeLa)cell lines. The half-maximal inhibitory concentration value was analyzed using GraphPad Prism 5 software. Adriamycin was used as a positive control. ¹⁷

Conclusion and Discussion

Two new benzophenones, 2-(4-benzoyl-2-hydroxyphenoxy)-N-phenethylacetamide (1) and 2-(4-benzoyl-3-hydroxyphenoxy)-N-propylacetamide (2), were isolated from the endohydric moss P. formosum. The 2 compounds showed no activity against 4 hoursuman cancer cell lines. Our findings enrich the knowledge of the phytochemical content of Polytrichaceae. In addition, our previous research found that benzophenones were isolated from the genera Pogonatum and Polytrichum. ^12,18 From the perspective of phytochemical taxonomy, it shows that these 3 genera are closely related. Benzophenone compounds may be characteristic chemical constituents of the Polytrichaceae.

Supplemental Material

Supplementary material - Supplemental material for Two New Benzophenones From the Endohydric Moss Polytrichastrum formosum

Supplemental material, Supplementary material, for Two New Benzophenones From the Endohydric Moss Polytrichastrum formosum by Xu-Hong Duan, Xue-Wen Zhang, Meng Qin, Pei He, Lin Pei, Jian-Cheng Zhao and Yu-Ling Chen in Natural Product Communications