Callistemonols A and B, Potent Antimicrobial ... - ACS Publications

Jul 5, 2019 - Copyright © 2019 American Chemical Society and American Society ... in conjunction with time-killing and biophysical studies, revealed ...
1 downloads 0 Views 2MB Size
Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Callistemonols A and B, Potent Antimicrobial Acylphloroglucinol Derivatives with Unusual Carbon Skeletons from Callistemon viminalis Jie-Wei Wu,†,‡,# Bai-Lin Li,†,# Chunping Tang,‡ Chang-Qiang Ke,‡ Nan-Lin Zhu,‡ Sheng-Xiang Qiu,*,§ and Yang Ye*,‡

Downloaded via NOTTINGHAM TRENT UNIV on July 17, 2019 at 13:57:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.



Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, People’s Republic of China ‡ State Key Laboratory of Drug Research, & Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China § Program for Natural Product Chemical Biology, Key Laboratory of Plant Resources Conservation and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China S Supporting Information *

ABSTRACT: A phytochemical investigation on the leaves of Callistemon viminalis resulted in the isolation of two unusual compounds, callistemonols A (1) and B (2). Callistemonol A (1) possesses a novel skeleton of a furan ring fusing both an α,βtriketone and a phloroglucinol unit, while callistemonol B (2) is an acylphloroglucinol derivative featuring two methyl substituents on a five-membered ring and an isovaleryl side chain. Their structures were fully characterized on the basis of extensive spectroscopic analysis, including 1D and 2D NMR parameters, as well as the IR and HRESIMS data. Callistemonol A (1) represents an example of a natural dibenzofuran with two phenyl moieties, and a plausible biogenetic pathway to generate this novel dibenzofuran through a C−C bond-forming radical SAM enzyme is proposed. Moreover, antimicrobial assays, in conjunction with time-killing and biophysical studies, revealed that 1 and 2 exert potent bactericidal activities against a panel of methicillin-resistant pathogenic microbes.

M

oglucinols with promising antimicrobial activities is therefore a prime focus of ongoing investigations. The genus Callistemon belongs to the plant family Myrtaceae and is well known for its attractive evergreen shrubs and small trees. Known as red bottlebrush, Callistemon viminalis (Sol. ex Gaertn.) G. Don was introduced from Australia to mainland China decades ago and is now cultivated widely throughout the country due to its ornamental and medicinal properties. This plant was reported to possess a variety of biological effects including antidiabetic,14 antimicrobial,15−17 antifungal,15 hemolytic,18 anthelmintic,19,20 and insecticidal activities.21−23 In Australia, C. viminalis has long been used by the aboriginals to treat gastroenteritis, diarrhea, and skin infections.24 Nowadays, it has been adopted as a folk medicine to treat a variety of

ethicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-acquired infections and is commonly associated with significant morbidity, mortality, increased length of stay, and enhanced cost burden.1,2 Presently, vancomycin remains the most important first-line therapy for severe MRSA infection. However, the emergence of MRSA with reduced susceptibility to vancomycin has been reported.3,4 Given that bacteria naturally evolve toward developing resistance to all antibiotics to which they are exposed, there is a critical need for research focusing on the search for novel antibacterial agents to combat MRSA. In the past decade, considerable attention has been paid to phloroglucinols of natural origin due to their biological functions, especially their significant antimicrobial activity.5−13 For instance, caespitate, a phloroglucinol compound isolated from Helichrysum caespititium, demonstrated antibacterial activity with an MIC value of 0.5−5 μg/mL against several Gram-positive bacteria species.5 Seeking novel natural phlor© XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 1, 2019

A

DOI: 10.1021/acs.jnatprod.9b00064 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for 1 and 2

symptoms such as colds and arthralgia in the People’s Republic of China.25 Dozens of phytochemical studies have been carried out on C. viminalis. The results showed that the plant biosynthesizes phloroglucinol derivatives, other phenolics, flavonoids, triterpenoids, saponins, steroids, and alkaloids.17,26−28 Our previous work on new antibacterial constituents from C. viminalis resulted in the identification of a series of phloroglucinols with potent antibacterial activities based on a variety of structural types.27,28 In a continuing effort to search for additional bioactive phloroglucinol derivatives, research was conducted on an ethanol extract of C. viminalis. As a result, two new potent antimicrobial acyphloroglucinol derivatives, callistemonols A and B (1 and 2, Figure 1), were afforded. Callistemonol A (1)

1a position 1 2 3 4 5 6 7 8a 8b 9 10 11 1′ 2′ 3′ 4′a 4′b 5′ 6′ 7′ 8′ 9′ 10′ CH3O-3 CH3O-3′ OH-5 OH-2′

Figure 1. Structures of compounds 1 and 2.

possesses an unique carbon skeleton that features a furan ring fused with both an α,β-triketone functionality and a phloroglucinol unit. In this study, the isolation, structural elucidation, antibacterial testing, and a possible mechanism of 1 and 2 are discussed. A plausible biosynthetic pathway for callistemonol A (1) is also proposed.



RESULTS AND DISCUSSION Compound 1 was purified as a yellow powder. The molecular formula, C23H28O9, was established by the 13C NMR data and an HRESIMS ion at m/z 471.1634 [M + Na]+ (calcd 471.1631), requiring 10 indices of hydrogen deficiency. Two characteristic absorptions at 3437 and 1718 cm−1 in the IR spectrum indicated the presence of hydroxy and carbonyl groups, respectively. The 13C NMR data and the HSQC spectrum of 1 exhibited 23 carbons, attributed to 12 quaternary, two methylene, three methine, and six methyl carbons. From these resonances, three carbonyls (δC 191.4, 204.2, 205.5) and two methoxy (δC 56.1, 53.2) groups could be clearly identified. The 1H NMR data (Table 1) showed characteristic resonance signals for four methyl groups at δH 1.00 (H3-10), 0.98 (H3-11), 1.16 (H3-9′), and 0.80 (H3-10′), as well as two methoxy groups at δH 3.82 (OMe-3) and 3.58 (OMe-3′). The 1H−1H COSY spectrum of 1 indicated the correlations from δH 2.91/2.74 (H28) to δH 2.12 (H-9), 1.00 (H3-10), and 0.98 (H3-11) and from δH 3.41 (H-8′) to δH 1.16 (H3-9′) and 0.80 (H3-10′), revealing the presence of two spin-coupled fragments (Figure 2). From the HMBC data, the proton resonance at δH 1.00 (H3-10) correlated with the carbon signals at δC 51.7 (C-8), 25.9 (C-9), and 22.9 (C-11), and that at δH 2.12 (H-9) correlated with signals at δC 204.2 (C-7). In turn, the signal at δH 1.16 (H3-9′) correlated with the signals at δC 205.5 (C-7′), 34.2 (C-8′), and 19.0 (C-10′). Therefore, the above data were indicative of the presence of an isovaleryl group and an isopropionyl group. Moreover, the HMBC correlations of OH-5 (δH 13.70) to C-4 (δC 94.3) and C-6 (δC 101.6), of H-4 (δH 6.02) to C-2 (δC 105.6) and C-6 (δC 101.6), and of OMe-3 (δH 3.82) to C-3 (δC

a

δH (mult, J Hz)

6.02 s

2.74 dd (7.1, 14.6) 2.91 dd (7.1, 14.6) 2.12 m 1.00 d (6.7) 0.98 d (6.7)

3.08 d (17.4) 3.23 d (17.4)

3.41 m 1.16 d (6.7) 0.80 d (6.7) 3.82 s 3.58 s 13.70 s 4.62 s

2b δC

position

160.5 105.6 162.2 94.3 168.7 101.6 204.2

1 2 3 4 5 6 7a

51.7

7b

25.9 22.8 22.9 191.2 82.9 108.5 42.2

δH (mult, J Hz)

5.37 s

8

2.43 dd (6.6, 18.3) 2.68 dd (6.6, 18.3) 2.06 m

9 10 11 12 CH3O-3

0.89 d (6.7) 0.89 d (6.7) 1.05 s 1.20 s 3.95 s

δC 203.1 93.0 197.9 49.7 100.9 211.5 49.4

24.7 22.9 22.9 18.8 26.4 60.0

191.4 109.2 205.5 34.2 18.2 19.0 56.1 53.2

Recorded in CDCl3. bRecorded in methanol-d4.

Figure 2. Key 1H−1H COSY and HMBC correlations (H→C) of 1 and 2.

162.2) indicated the presence of a pentasubstituted benzene moiety. Furthermore, the HMBC correlations from H2-8 (δH 2.74, 2.91) to C-6 (δC 101.6) suggested a connection of the isovaleryl group to C-6 of the benzene moiety. Thus, a typical phloroglucinol unit (I) could be constructed, consistent with an observation made in a previous investigation.27,28 Apart from the signals of unit I, a cyclic methylene unit (δH 3.08, d, J = 17.4 Hz, H-4′a; δH 3.23, d, J = 17.4 Hz, H-4′b) was observed in the 1H NMR spectrum. In addition, a carbonyl (δC 191.4, C-5′), a ketal (δC 108.5, C-3′), and an oxygenated quaternary carbon (δC 82.9, C-2′), along with two olefinic carbons (δC 191.2, C-1′; δC 109.2, C-6′), were identified from the 13C NMR spectrum. In the HMBC experiment, the key HMBC correlations of H2-4′ (δH 3.08, 3.23) to C-2′ (δC 82.9), C-3′ (δC 108.5), C-5′ (δC 191.4), and C-6′ (δC 109.2) suggested B

DOI: 10.1021/acs.jnatprod.9b00064 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

A plausible biosynthetic pathway of 1 was proposed in Scheme 1. Catalyzed by radical S-adenosylmethionine (SAM) enzymes, a new C−C bond (C-2−C-2′) was formed to link two starting monomers,29,30 which led to the putative key intermediate C. Subsequent deprotonation could occur at the phenols HO-1 and HO-1′, respectively, accompanied by the release of an electron and forming the precursor D. Further dearomatization of E would generate the intermediate F, resulting in the construction of the unique tertiary hydroxy carbon and the formation of the dibenzofuran architecture in callistemonol A (1) through a Michael addition. In this proposed pathway, an empirical transition state model could be used to explain the configurational outcomes. To avoid steric repulsions caused by the hydroxy group at C-2′ (intermediate F), the hydroxy group at C-1 would attack from the reverse side of the methoxy group, thus leading to the cofacial orientation of OH-2′ and OMe-3′. Compound 2, isolated as a yellow powder, was assigned a molecular formula of C13H20O4, with four indices of hydrogen deficiency according to the HRESIMS ion at m/z 241.1448 [M + H]+ (calcd 241.1440) and its 13C NMR spectrum. The IR absorption bands suggested the presence of hydroxy (3435 cm−1) and carbonyl (1715 cm−1) functionalities. Analysis of the 13 C NMR and HSQC data revealed the presence of 13 carbons, which were ascribed to five quaternary, two methylene, one methine, and five methyl carbons. From these resonances, two carbonyls (δC 203.1, 211.5), one olefinic quaternary carbon (δC 197.9), and one methoxy (δC 60.0) group could be identified clearly. The 1H NMR data (Table 1) showed signals characteristic for five methyl groups at δH 0.89 (6H, d, J = 6.7 Hz, H3-9, H3-10), 1.05 (3H, s, H3-11), and 1.20 (3H, s, H3-12), respectively, and one methoxy group at δH 3.95 (OMe-3). The 1H−1H COSY spectrum of 2 showed the correlations from δH 2.68/2.43 (H2-7) to δH 2.06 (H-8) and from δH 2.06

the presence of a cyclohexane ring moiety. In addition, the isopropionyl group was assigned at C-6′ from the HMBC correlation between H-8′ (δH 3.41) and C-6′ (δC 109.2). Thus, a second substructural unit (II) of 1 could be defined (Figure 2). The connection of structural units I and II could not be inferred from the HMBC experiment due to the absence of a suitable correlative proton. However, as units I and II accounted for nine indices of hydrogen deficiency, the remaining one thus implied the presence of another ring in the molecule. Therefore, the structure of 1 was proposed as shown, with a newly formed furan ring bridging units I and II through connections of C-1− O−C-3′ and C-2−C-2′. The relative configuration of 1 was deduced from analysis of the ROESY correlations (Figure 3). The key ROESY

Figure 3. Key ROESY correlations of 1.

correlations of H3-10/H-4′b and H-4/H3-10′, as well as of H310/H3-9′, indicated that OH-2′ and OMe-3′ are cofacial. Therefore, the structure of 1 could be proposed as shown, and this compound was given the trivial name callistemonol A, representing a type of phloroglucinol derivative with an unprecedented dearomatized dibenzofuran core combining an α,β-triketone with a phloroglucinol unit.

Scheme 1. Proposed Biosynthetic Pathway for Callistemonol A (1)

C

DOI: 10.1021/acs.jnatprod.9b00064 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 2. Antibacterial Activities of Compounds 1 and 2 against Bacteria Strains MIC/MBC (μg/mL) compound

MRSA JCSC4744

MRSA JCSC4469

S. aureus CMCC26003

B. cereus CMCC63302

E. coli ATCC8739

1 2 vancomycina

3.12/6.25 1.56/3.12 0.78/2.50

3.12/6.25 3.12/6.25 0.78/2.50

25/50 3.12/6.25 1.56/2.50

25/50 12.5/25 50/ND

>100/ND >100/ND 50/ND

a

Positive control. ND: not determined.

Figure 4. Time−kill assay of MRSA (JCSC 4744) by 1 and 2.

Figure 5. Cytoplasmic membrane depolarization assay of MRSA (JCSC 4744) with different concentrations of 1 (A) and 2 (B) by the DiSC3−5 probe; melittin MIC value: 1.25 μg/mL.

(H-8) to δH 0.89 (H3-9, H3-10), which revealed the presence of one spin-coupled fragment, H2-7/H-8/H3-9(H3-10). The HMBC data showed that the protons at δH 0.89 (H3-9, H310) correlated to the carbons at δC 24.7 (C-8) and 49.4 (C-7), and the proton at δH 2.06 (H-8) to the carbons at δC 49.4 (C-7) and 211.5 (C-6). All of the above-mentioned data were indicative of the occurrence of an isovaleryl group. In addition, one double bond and two carbonyls accounted for three of four indices of hydrogen deficiency so that the one remaining suggested 2 might possess one ring moiety in the molecule. The HMBC experiment showed that H-5 (δH 5.37) correlated to C-1 (δC 203.1), C-2 (δC 93.0), C-3 (δC 197.9), and C-4 (δC 49.7), indicating further the presence of a five-membered ring. Moreover, two methyl groups (CH3-11 and CH3-12) could be placed at C-4, as deduced from the HMBC correlations from H311 (δH 1.05) to C-3 (δC 197.9), from H3-12 (δH 1.20) to C-3 (δC 197.9), and from H-5 (δH 5.37) to C-12 (δC 26.4). Thus, the structure of 2, containing two methyl substituents on a fivemembered ring and an isovaleryl side-chain, was constructed, and this compound was named callistemonol B. The optical rotations of both compounds 1 and 2 were recorded as being zero, suggesting that they might be

enantiomeric. Subsequently, the analytical resolution of the respective enantiomers succeeded by HPLC on a chiral phase wherein two liquid chromatography (LC)−UV peaks could be observed (Figure S18, Supporting Information). That the two LC−UV peaks observed indeed corresponded to the respective enantiomers was verified by LC−circular dichroism (CD) analysis. As a result, the LC−CD spectra of 1a and 1b recorded online provided almost mirror-imaged CD curves (Figure S19, Supporting Information), indicating the existence of enantiomers of 1. Similarly, compound 2 was also verified as a pair of enantiomers (Figure S20, Supporting Information). The antibacterial activities of 1 and 2 were evaluated against a panel of bacteria including two methicillin-resistant strains of S. aureus MRSA JCSC 4744 and MRSA JCSC 4469 using a standard MIC method according to CLSI guidelines (Table 2).31 The result showed that both 1 and 2 possess significant anti-MRSA activities, with MICs and MBCs ranging from 1.56 to 6.25 μg/mL. Their potencies are 2−4-fold less than that of vancomycin, which was used as a positive control. However, both 1 and 2 were inactive toward the Gram-negative bacterium Escherichia coli (ATCC 8739). D

DOI: 10.1021/acs.jnatprod.9b00064 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 6. SYTOX green assay of MRSA by 1 and 2 (8 × MIC) and melittin (10 μg/mL) as positive control (A); SYTOX green assay of MRSA with different concentrations of 1 and 2 for 4 h (B).

Figure 7. Scanning electron microscopies: MRSA (JCSC 4744) cells treated for 4 h with negative control (DMSO) (A); 8 × MIC of 1 (B) and 2 (C).

the membrane was not as vigorous as melittin, 1 and 2 showed permeability effects that were also concentration-dependent (Figure 6B). Scanning electron micrographs of 1 and 2 against MRSA (JCSC 4744) cells are shown in Figure 7. After treatment with an 8-fold MIC dose of 1 and 2 for 4 h, cell lysis was distinctly observed with fissures and deformations on the bacterial membrane in each case. These results were in accord with the aforementioned studies showing that 1 and 2 can induce cell lysis.

Moreover, time−kill kinetic experiments of 1 and 2 against MRSA (JCSC 4744) were performed (Figure 4). At the dose of an 8-fold MIC value, MRSA (JCSC 4744) survivors were lessened significantly with increasing time and subsequently fell to a dramatically low point after 24 h when treated with 1. Similar results were found when treated with 2. In particular, a 2log decrease (99% reduction) in MRSA cell-survival curve was rapidly attained within 4 h at the dose of 1-fold MIC level and completely eliminated after a 24 h exposure. Vancomycin with the same fold MIC value, however, depressed fewer CFUs in the same period of time. These results indicated that 1 and 2 exhibited more rapid and complete in vitro bactericidal activities than vancomycin, under the test conditions used. Based on the fact that numerous species in the Myrtaceae family exploit an antibiotic advantage by targeting the bacterial membrane,32,33 comprehensive investigations were conducted to further understand the antimicrobial mechanism of 1 and 2 against clinical MRSA (JCSC 4744). First, 3,3-dipropylthiacarbocyanine (DiSC3−5), a fluorescence probe, was utilized to elucidate whether 1 and 2 may function as dissipaters for membrane potential.34,35 In the presence of 1 and 2 with serial concentrations, noticeable increments in fluorescence signal were detected (Figure 5), implying that the bactericidal ability of 1 and 2 could account for a significant membrane depolarization. Next, a SYTOX green assay was performed, during which the membrane-impermeable dye SYTOX green will bind to nucleic acids and give rise to an enhanced fluorescence signal once the tested membrane is disrupted. Distinct from the positive control, melittin, which disrupted the microbial membrane almost immediately within the first 2 min, treatment with the 8-fold MIC values of 1 and 2 resulted in only a slight increase in the fluorescence intensity (Figure 6A). After a 4 h treatment with sequential concentrations of 1 and 2, however, increases of florescence were detected. Although the destructive potency on



EXPERIMENTAL SECTION

General Experimental Procedures. The instruments and materials for the purification of the compounds from the title plant and for the spectroscopic measurements of the purified compounds are detailed in the Supporting Information. Plant Material. The leaves of C. viminalis were collected at the South China Botanical Garden, Chinese Academy of Sciences, in March 2016, and identified by Professor Fu-Wu Xing of the South China Botanical Garden, CAS. A voucher specimen (No. 201603) was deposited at the Laboratory of Natural Product Chemistry Biology, SCBG. Extraction and Isolation. The dried and powdered leaves of C. viminalis (20 kg) were extracted with 95% ethanol at room temperature (3 times, 3 days each), and the solvent was evaporated under vacuum to afford a crude extract (3.8 kg), which was further suspended in H2O (5 L) and extracted with EtOAc (5 L × 3) to afford an EtOAc-soluble fraction. The EtOAc fraction was subjected to column chromatography (CC) over silica gel eluted with an n-hexane/EtOAc mixtures of increasing polarity (from 20:1 to 0:1) to yield pooled fractions A−F. Fraction C (36.2 g) was chromatographed on a silica gel column with a gradient of CHCl3/MeOH (100:1 to 20:1) to yield three subfractions (C1−C3). Subfraction C2 (4.1 g) was further purified by CC over Sephadex LH-20 (MeOH) and then preparative HPLC (CH3CN/H2O from 50% to 90%), yielding 1 (12 mg). Fraction D (40.1 g) was subjected to CC over silica gel eluting with a gradient of CHCl3/MeOH (100:1 to 20:1) to yield six subfractions (D1−D6). Subsequently, subfraction D3 (8.2 g) was further purified by CC over Sephadex LH-20 E

DOI: 10.1021/acs.jnatprod.9b00064 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(7) Singh, I. P.; Sidana, J.; Bansal, P.; Foley, W. J. Expert Opin. Ther. Pat. 2009, 19, 847−866. (8) Pia Schiavone, B. I.; Verotta, L.; Rosato, A.; Marilena, M.; Gibbons, S.; Bombardelli, E.; Franchini, C.; Corbo, F. Anticancer Agents Med. Chem. 2014, 14, 1397−1401. (9) Casero, C.; Machín, F.; Méndez-Á lvarez, S.; Demo, M.; Ravelo, Á . G.; Pérez-Hernández, N.; Joseph-Nathan, P.; Estévez-Braun, A. J. Nat. Prod. 2015, 78, 93−102. (10) Zhang, Y. B.; Li, W.; Jiang, L.; Yang, L.; Chen, N. H.; Wu, Z. N.; Li, Y. L.; Wang, G. C. Phytochemistry 2018, 153, 111−119. (11) Su, C. J.; Wang, S.; Cheng, W.; Huang, X. J.; Li, M. M.; Jiang, R. W.; Li, Y. L.; Wang, L.; Ye, W. C.; Wang, Y. J. Org. Chem. 2018, 83, 8522−8532. (12) Cao, J. Q.; Wu, Y.; Zhong, Y. L.; Li, N. P.; Chen, M.; Li, M. M.; Ye, W. C.; Wang, L. Chem. Biodiversity 2018, 15, No. e1800172. (13) Pham, T. A.; Shair Mohammad, I.; Vu, V. T.; Hu, X. L.; Birendra, C.; Ulah, A.; Guo, C.; Lü, X. Y.; Ye, W. C.; Wang, H. Chem. Biodiversity 2018, 15, No. e1800052. (14) Nazreen, S.; Kaur, G.; Alam, M. M.; Shafi, S.; Hamid, H.; Ali, M.; Alam, M. S. Fitoterapia 2012, 83, 1623−1627. (15) Delahaye-Mckenzie, C.; Rainford, L.; Nicholson, A.; Mitchell, S.; Lindo, J.; Ahmad, M. J. Med. Biol. Sci. 2009, 3, 1−7. (16) Oyedeji, O. O.; Lawai, O. A.; Shade, F. O.; Oyedeji, A. O. Molecules 2009, 14, 1990−1998. (17) Ashmawy, N. A.; Behiry, S. I.; Ali, H. M.; Salem, M. Z. M. J. Pure Appl. Microbiol. 2014, 8, 667−673. (18) Saleem, A.; Nasir, S.; Rasool, N.; Bokhari, T. H.; Rizwan, K.; Shahid, M. Int. J. Chem. Biochem. Sci. 2015, 7, 29−34. (19) Garg, S. C.; Kasera, H. L. Fitoterapia 1982, 53, 179−181. (20) Veerakumari, L. Asian J. Sci. Technol. 2015, 6, 1881−1894. (21) Khambay, B. P. S.; Beddie, D. G.; Hooper, A. M.; Simmonds, M. S. J.; Green, P. W. C. J. Nat. Prod. 1999, 62, 1666−1667. (22) Ndomo, A. F.; Tapondjou, A. L.; Tendonkeng, F.; Tchouanguep, F. M. Tropicultura 2009, 27, 137−143. (23) Gohar, A. A.; Maatooq, G. T.; Gadara, S. R.; Aboelmaaty, W. S.; El-Shazly, A. M. Iran J. Pharm. Res. 2014, 13, 505−514. (24) Elliot, W. R.; Jones, D. L. Encyclopedia of Australian Plants, Vol. 2; Lothian Publishing Company: Melbourne, 1982. (25) State Administration of Traditional Chinese Medicine of the People’s Republic of China. Chinese Materia Medica; Shanghai Scientific and Technical Publishers: Shanghai, 1999; Vol. 15, p 4711. (26) Wollenweber, E.; Wehde, R.; Dorr, M.; Lang, G.; Stevens, J. F. Phytochemistry 2000, 55, 965−970. (27) Liu, H. X.; Chen, Y. C.; Liu, Y.; Zhang, W. M.; Wu, J. W.; Tan, H. B.; Qiu, S. X. Fitoterapia 2016, 114, 40−44. (28) Liu, H. X.; Chen, K.; Liu, Y.; Li, C.; Wu, J. W.; Xu, Z. F.; Tan, H. B.; Qiu, S. X. Fitoterapia 2016, 115, 142−147. (29) Landgraf, B. J.; McCarthy, E. L.; Booker, S. J. Annu. Rev. Biochem. 2016, 85, 485−514. (30) Yokoyama, K.; Lilla, E. A. Nat. Prod. Rep. 2018, 35, 660−694. (31) Wu, M.; Hancock, R. E. J. Biol. Chem. 1999, 274, 29−35. (32) Tan, H. B.; Liu, H. X.; Zhao, L. Y.; Yuan, Y.; Li, B. L.; Jiang, Y. M.; Gong, L.; Qiu, S. X. Eur. J. Med. Chem. 2017, 125, 492−499. (33) Xiang, Y. Q.; Liu, H. X.; Zhao, L. Y.; Xu, Z. F.; Tan, H. B.; Qiu, S. X. Sci. Rep. 2017, 7, 2363. (34) Wu, M. H.; Hancock, R. E. W. J. Biol. Chem. 1999, 274, 29−35. (35) Zhang, L. J.; Dhillon, P.; Yan, H.; Farmer, S.; Hancock, R. E. W. Antimicrob. Agents Chemother. 2000, 44, 3317−3321. (36) Van Bambeke, F. Curr. Opin. Investig. Drugs 2006, 7, 740−749. (37) Rathinakumar, R.; Walkenhorst, W. F.; Wimley, W. C. J. Am. Chem. Soc. 2009, 131, 7609−7617.

(MeOH) and then preparative HPLC (CH3CN/H2O from 30% to 60%) to afford 2 (6 mg). Callistemonol A (1): yellow powder, [α]20D 0 (c 0.1 in CH3OH); UV (MeOH) λmax (log ε) 238.5 (3.15), 282.1 (3.34) nm; IR (KBr) νmax 3437, 2958, 2924, 2852, 1718, 1680, 1629, 1585, 1460, 1444, 1367, 1261, 1167, 1099, 1026, 805 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 471.1634 [M + Na]+ (calcd for C23H28O9Na, 471.1631). 1a: ECD (c 2.2 × 10−3 mol/L, n-hexane/isopropanol 98:2, v/v) λmax (Δε) 220.0 (+18.39), 243.0 (−12.08), 272.0 (+3.14) nm. 1b: ECD (c 2.23 × 10−3 mol/L, n-hexane/isopropanol 98:2, v/v) λmax (Δε) 220.0 (−20.55), 243.0 (+14.48), 272.0 (−4.04) nm. Callistemonol B (2): yellow power; [α]20D 0 (c 0.1 in CH3OH); UV (MeOH) λmax (log ε) 241.1 (3.68) nm; IR (KBr) νmax 3435, 2959, 2872, 1715, 1682, 1585, 1531, 1456, 1366, 1342, 1288, 1234, 1171, 1124, 986, 835 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 241.1448 [M + H]+ (calcd for C13H21O4, 241.1440). 2a: ECD (c 2.1 × 10−3 mol/L, n-hexane/isopropanol, 94:6, v/v) λmax (Δε) 213.0 (+19.61), 253.0 (−15.51), 311.0 (+7.11) nm. 2b: ECD (c 2.08 × 10−3 mol/L, n-hexane/isopropanol, 94:6, v/v) λmax (Δε) 213.0 (−22.88), 253.0 (+21.12), 311.0 (−7.19) nm. In Vitro Antibacterial Activity Assay. The MIC and MBC evaluations, a time−killing study, a cytoplasmic membrane depolarization experiment, a SYTOX green assay, and scanning electron microscopy studies on compounds 1 and 2 were carried out according to the standard methods.31−37 Detailed procedures are described in the Supporting Information.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00064. IR, HRESIMS, 1H and 13C NMR, 1H−1H COSY, HSQC, HMBC, and ROESY spectra of compounds 1 and 2, detailed procedures of antimicrobial assays, and the time−killing and biophysical studies (PDF)



AUTHOR INFORMATION

Corresponding Authors

*Tel: 86-20-37252958. Fax: 86-20-37252958. E-mail: sxqiu@ scbg.ac.cn (S.-X. Qiu). *Tel: 86-21-50806726. Fax: 86-21-50806726. E-mail: yye@ mail.shcnc.ac.cn (Y. Ye). ORCID

Yang Ye: 0000-0003-1316-5915 Author Contributions #

J.-W. Wu and B.-L. Li contributed equally to this study.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was financially supported by the State Key Laboratory of Drug Research (SIMM1803KF-09) and China Postdoctoral Science Foundation (2015M582436).



REFERENCES

(1) Xiao, G.; Chen, Z.; Lv, X. Infect. Drug Resist. 2018, 11, 1473−1481. (2) Lewis, P. O.; Heil, E. L.; Covert, K. L.; Cluck, D. B. J. Clin. Pharm. Ther. 2018, 43, 614−625. (3) Ghahremani, M.; Jazani, N. H.; Sharifi, Y. J. Glob. Antimicrob. Resist. 2018, 14, 4−9. (4) Kemung, H. M.; Tan, L. T.; Khan, T. M.; Chan, K. G.; Pusparajah, P.; Goh, B. H.; Lee, L. H. Front Microbiol 2018, 9, 2221. (5) Meyer, J. J. M.; Mathegka, A. D. M. WO 0123342, 2001. (6) Singh, I. P.; Bharate, S. B. Nat. Prod. Rep. 2006, 23, 558−591. F

DOI: 10.1021/acs.jnatprod.9b00064 J. Nat. Prod. XXXX, XXX, XXX−XXX