Three Sulfur-Containing Natural Products From Rubus Hirsutus Thunb

Abstract

Chemical investigation of the leaves and stems of Rubus hirsutus Thunb has resulted in the isolation and characterization of a rare new tricyclic thienocyclopentapyran (1) and 2 known thiophenes, 5-(3-buten-1-ynul)−2,2’-bithiophene (2) and α-terthienyl (3). The structure of compound 1 was unambiguously determined by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and time-dependent density functional theory electronic circular dichroism calculations. The new compound 1 exhibited impressive antibiotic properties against methicillin resistant Staphylococcus aureus (MRSA), with an MIC value of around 1 µg/mL.

Sulfur contributes significantly to the chemical diversity of natural compounds thanks to its particular features which allow fundamental biological reactions. ¹ Plant-derived sulfur-containing secondary metabolites constitute a small group of low-molecular weight natural products, which play a vital role in plant-pest interactions in numerous plant families and represent major defense molecules in the Asteraceae, Alliaceae, and Brassicaceae families. ² Both primary and secondary S-containing compounds have great effects on plant health and regulatory mechanisms when under stress. ³ In addition to antibacterial activity, some sulfur-containing natural products have shown promising antifungal activities against a wide range of plant pathogenic fungi. For instance, the phytoalexins sinalbins A and B were proved to have antifungal activity against the plant pathogenic fungus Leptosphaeria maculans. ⁴ Sulfur-containing monoterpenoids have been reported as potential antithrombotic drugs. ⁵ Sulfur-containing natural products have emerged as a new class of compounds that have the potential to exert strong anti-inflammatory effects and may, therefore, stand as a potential “goldmine” for the discovery of future anti-inflammatory drugs. ⁶ In this study, we investigated the chemical composition of the leaves and stems of Rubus hirsutus Thunb, leading to the isolation and characterization of a rare new tricyclic thienocyclopentapyran (1) and 2 known thiophenes, 5-(3-buten-1-ynul)−2,2’-bithiophene (2) and α-terthienyl (3). The antibacterial activities of these 3 sulfur-containing compounds were also evaluated.

The MeOH extracts of the leaves and stems of R. hirsutus were subjected to preparative HPLC (C18), affording a new sulfur-containing compound 1 and the known compounds 5-(3-buten-1-ynul)−2,2’-bithiophene (2) ⁷ and α-terthienyl (3) (Figure 1). ⁸

Figure 1

The structures of compounds 1‐3.

Results and Discussion

Compound 1 was isolated as a brown powder. The molecular formula was determined to be C₁₇H₁₆O₅S by analysis of the HR-TOF-MS ion peak at m/z 331.0639 [M - H]^- (calcd. 331.0640). The formula was supported by the ¹³C NMR data, which showed 10 degrees of unsaturation. The NMR data (Table 1) in the downfield region of 1 were similar to those of (+) and (-) xanthienopyran. ⁹ This type of molecule contains 2 distinct domains, a tricyclic thienocyclopentapyran and a conjugated diene chain, connected by an oxygenated methine carbon. ⁹ The ¹³C NMR spectrum showed the presence of 10 signals for the tricyclic thienocyclopentapyran domain, including a cyclopenta[b]thiophene basic skeleton and a hydroxylated pyran lactone moiety, with the remaining 7 resonances corresponding to a conjugated diene chain and a hydroxymethyl moiety. When compared with the NMR data of (-) xanthienopyran, isolated from the seeds of Xanthium strumarium, compound 1 showed similar chemical shifts for the main tricyclic thienocyclopentapyran skeleton. However, the chemical shifts for the substructures of the thiophene and attached methyl unit at the adjacent position of the sulfur atom were less similar to those of xanthienopyran.

Table 1

the NMR Data (600 MHz, MeOH-D ₄) of Compound 1.

Position	δC	δH (mult; J in Hz)	HMBC (1H→13C)
2	150.6
3	120.8	7.23 (1H, s)	C-2, C-3a, C-4, C-8b, C-15
3a	144.9
4	115.2	7.40 (1H, s)	C-3, 3a, C-5, C-8a, C-8b
4a	121.5
5	151.5
6	171.8
8	82.8	6.06 (1H, d, J = 6.0 Hz)	C-6, C-9, C-10
8a	136.7
8b	127.8
9	126.0	5.68 (1H, dd, J = 15.2, 6.0 Hz)	C-8, C-11
10	135.7	6.49 (1H, dd, J = 15.2, 10.4 Hz)	C-8, C-11, C-12
11	132.2	6.17 (1H, dd, J = 15.4, 10.4 Hz)	C-9, C-10, C-13
12	134.6	5.83 (1H, dt, J = 15.4, 7.0 Hz)	C-10, C-13, C-14
13	37.1	2.31 (2H, q, J = 6.8 Hz)	C-11, C-12, C-14
14	62.4	3.59 (2H, t, J = 6.8 Hz)	C-12, C-13
15	60.7	4.85 (2H, s)	C-2, C-3

The 10 signals for the tricyclic thienocyclopentapyran domain comprised 2 aromatic methines at δ_C 115.2 (C-4), and 120.8 (C-3), an oxygenated methine at δ_C 82.8 (C-8), six quaternary aromatic carbons at δ_C 151.5 (C-5), δ_C 121.5 (C-4a), δ_C 136.7 (C-8a), δ_C 127.8 (C-8b), δ_C 144.9 (C-3a), and δ_C 150.6 (C-2), corresponding to 3 sets of double bonds, and a lactone carbonyl carbon at δ_C 171.8 (C-6). Among the six quaternary aromatic carbons, an enol carbon at δ_C 151.5 (C-5) was observed. In the ¹H, ¹H COSY spectrum of 1 (Figure 2), the oxygenated methine at δ_H 6.06 (d, H-8) was coupled with the olefinic proton at δ_H 5.68 (dd, H-9), which further coupled with another olefinic proton at δ_H 6.49 (dd, H-10), revealing that the side chain was positioned at C-8 of the thienocyclopentapyran main skeleton. The ¹H, ¹H COSY spectrum of 1 also showed a diagnostic cross peak between 2 double bonds, demonstrating that a chain of 2 conjugated double bonds was attached at C-8. A sequence of HOCH₂-CH₂- was deduced by the observation of the ¹H, ¹H COSY correlations between the proton signals at δ_H 3.59 (t, H-14) and δ_H 2.31 (q, H-13). From the above analysis of the COSY correlations, a side chain of HOCH₂-CH₂-CH = CH-CH=CH was inferred, attached at C-8 of the main backbone. The stereochemistry of both C-9 and C-11 was deduced as E on the basis of J values (J _9,10= 15.2 Hz. J _11,12= 15.4 Hz).

Figure 2

Key ¹H, ¹H COSY and HMBC correlations of compound 1.

HMBC analyses showed the formation of the tricyclic thienocyclopentapyran skeleton with a characteristic (E)-hexa-3,5-dien-1-ol pedant (Figure 2). HMBC cross peaks between olefinic H-9,10 and the oxygenated methine C-8 confirmed that the side chain was located at C-8 of ring A. In the ¹H NMR spectrum of 1, 2 olefinic protons at δ_H 7.23 (s, H-3) and δ_H 7.40 (s, H-4) displayed 2 singlet signals, both of which showed HMBC correlations with 2 quaternary carbon, C-3a and C-8a, 2 fusing atoms between ring B and ring C. As such, these 2 singlet aromatic protons were assigned to C-4 of ring B and C-3 of ring C. A diagnostic long range correlation from H-8 to the carbonyl at C-6 was observed, showing that a pyran lactone ring was bridged between C-6 and C-8. The long range correlations from the hydroxylated methylene protons at δ_H 4.85 (H₂-15) to the double bond carbon signals at δ_C 150.6 (C-2) and δ_C 120.8 (C-3) positioned the hydroxymethyl group at C-2. The hydroxymethyl pendant attached at C-2 was obviously different from that of the known xanthienopyran. The enol carbon at δ_C 151.5 (C-5) was attributed to C-5 from the observation of long range correlations from the proton signals at δ_H 7.40 (H-4) to C-5. The fusing pattern of a pyran ring A, a cyclopentane ring B and a thiophene ring C was completed from the analysis of the HMBC cross peaks from H-4 of ring B to C-13 and C-8a of ring A and 8b of ring C. The absolute configuration of 1 was determined from the experimental electronic circular dichroism (ECD) spectrum in comparison with the Boltzmann-weighted time-dependent density functional theory (TDDFT) calculated spectrum (Figure 3), confirming the absolute configuration of (8R)−1. From the above analysis, the structure of 1 was established as a new thienocyclopentapyran. The new compound was thus given the trivial name hirsuthienopyran.

Figure 3

Experimental and calculated ECD spectra of (8R)−1.

The isolated new and known compounds were tested for their bacteriostatic as well as bactericidal abilities against 3 pathogenic bacteria namely methicillin resistant Staphylococcus aureus MRSA, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Hirsuthienopyran (1) exhibited impressive antibiotic abilities with an MIC value of around 1 µg/mL, whereas 5-(3-buten-1-ynul)−2,2’-bithiophene (2) and α-terthienyl (3) showed no obvious activities.

Experimental

General

The melting point (uncorrected): Reichert apparatus. Optical rotations: Perkin-Klmer-341 polarimeter. IR spectra: NicoletAvatar-360FT-IR spectrometer. ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) spectra: Agilent 600 NMR spectrometer with TMS as internal standard (at 25 °C). Mass spectra: AB Sciex 5500 Q-TRAP and LC/MS Q-Orbitrap (Q Exactive Focus, ThermoFisher Scientific). TLC: Merck precoated plates (silica gel 60 F254) of 0.25 mm thickness. Chromatography: Waters 600 Preparative HPLC, with a Shim-pack PREP-ODS (250 × 20 mm) column; Sephadex LH-20 (Amersham).

Plant Material

The leaves and stems of Rubus hirsutus were collected in Zhejiang Province, China, in September 2018 and identified by Prof. Changxi Zhang (Jinhua Medical College, Jinhua, China.). A voucher specimen (No. zju 7541) is kept in the College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China.

Extraction and Isolation

The shade-dried leaves and stems (5 kg) were extracted with methanol (20 L), and 134 g extract was obtained, which was partitioned with light petroleum (1200 ml), EtOAc (1500 ml) and n-BuOH (1000 ml), successively. The light petroleum extract (27 g) was subjected to column chromatography (4 × 40 cm, 700 g, 200‐300 mesh) over silica gel, eluting with light petroleum/EtOAc (10:0-0:10, gradients, 3000 ml) to afford 4 fractions. Fraction 3 was purified preparative HPLC (flow rate 8 mL/min, UV detector 250 nm), using CH₃OH-H₂O (75:25) as eluent, to afford compound 1 (5.3 mg, t_R 33.6 minutes). The EtOAc extract (30 g) was fractionated by column chromatography (5 × 45 cm, 850 g, 200‐300 mesh) over silica gel, eluting with light petroleum/EtOAc (8:1-0:10, gradients, 3000 ml) to afford six fractions. Fraction 4 (8.4 g) was applied to a Sephadex LH-20 column (4 × 150 cm, 100 g, Amersham), and eluted with acetone (1200 ml) at 15 °C for 1 day to give pure 2 (6.3 mg) and 3 (4.9 mg).

Computation Section

The geometry was optimized starting from initial conformations, with DFT calculations at the B3LYP/6‐31 + G(d) level using the Gaussian 09 program. Frequency analysis was made at the same level of theory to verify that these optimized structures are real minima on the potential energy surface. Time-dependent DFT calculations were performed on the lowest-energy conformations for each configuration using 30 excited states and in methanol solution. ECD spectra were generated using the program SpecDis by applying a Gaussian band shape with 0.2 eV width, from dipole-length rotational strengths. ^{10 -12}

Antibiotic Activity Screening of Stress Metabolites

The conventional broth dilution assay was used to evaluate the antibacterial activities. Three clinical pathogens namely MRSA, P. aeruginosa [CMCC(B)10104], and K. pneumoniae [CMCC(B)46117] were cultured and left overnight to grow. Each pathogenic culture was then diluted in 0.9% saline to an inoculum density of 5 × 10⁵ cfu by comparison with a McFarland standard. Tetracycline was dissolved in Mueller–Hinton broth with a starting concentration of 512 µg/mL and was used as positive control, whereas methanol was used as negative control. A total of 125 µL of MHB was distributed into the 96-well plates. A total of 125 µL of the different samples was dispensed into well 1 and serially diluted across the plate. Ultimately, addition of 125 µL of the bacterial inoculum was made and the plates were incubated at 37 °C for 18 hours. The bacteriostatic abilities of the compounds were noted as MICs, which were made in triplicate. Five mg per mL of a methanolic solution of 3-[4,5-dimethylthiazol-2-yl]−2,5-diphenyltetrazolium bromide (MTT; Lancaster) was used to detect bacterial growth by a change in color from yellow to blue. ¹³

Hirsuthienopyran (1)

Brown powder

$[\alpha {]}_{\mathrm{D}}^{20}$ −23 (c 0.1, MeOH).

Rf : 0.40 (CHCl₃-MeOH, 2:1).

UV/Vis λ_max (MeOH) nm (log ε): 210 (4.24), 265 (2.47), 324 (3.14).

ECD (c 50 ppm, MeOH) λ_max (Δε) 224 (-1.56), 215 (+0.01), 203 (−3.57) nm.

ESI MS m/z 331.0639 [M - H]^- (calcd. 331.0640).

Product yield: 0.02%.