Antibacterial Prenylated Acylphloroglucinols from Psorothamnus

Oct 15, 2015 - †National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, and ‡Department of Biomolecular Scie...
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Antibacterial Prenylated Acylphloroglucinols from Psorothamnus fremontii Qian Yu,†,§ Ranga Rao Ravu,† Qiong-Ming Xu,† Suresh Ganji,† Melissa R. Jacob,† Shabana I. Khan,†,‡ Bo-Yang Yu,§ and Xing-Cong Li*,†,‡ †

National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, and ‡Department of Biomolecular Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States § Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, 211198, People’s Republic of China S Supporting Information *

ABSTRACT: Psorothatins A−C (1−3), three antibacterial prenylated acylphloroglucinol derivatives, were isolated from the native American plant Psorothamnus f remontii. They feature an unusual α,β-epoxyketone functionality and a β-hydroxy-α,βunsaturated ketone structural moiety. The latter forms a pseudosix-membered heterocyclic ring due to strong intramolecular hydrogen bonding, as indicated by the long-range proton− carbon correlations in the NMR experiments. Psorothatin C (3) was the most active compound against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium, with IC50 values in the range 1.4−8.8 μg/mL. The first total synthesis of 3 described herein permits future access to structural analogues with potentially improved antibacterial activities.

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ntibiotic resistance has increasingly posed a profound threat to human health. The Centers for Disease Control and Prevention, Atlanta, GA, estimates that drug-resistant bacteria cause two million illnesses leading to approximately 23 000 people dying each year in the United States alone.1 The discovery of new lead compounds from natural products for antibacterial drug development remains an important approach to combat antibiotic resistance.2 In our search for antibacterial compounds from plants, the MeOH extract of the aerial parts of Psorothamnus f remontii (Torr.) Barneby (Fabaceae) was shown to have IC50 values of 56.7 and 69.7 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 and methicillin-sensitive S. aureus ATCC 29213, respectively. This plant is a native American desert shrub distributed in California and arid areas of Nevada3 and has been used to treat internal hemorrhage and stomach problems by native Americans.4 Two isoflavonoids were isolated from its roots and showed antimutagenic activity.5 In addition, previous phytochemical investigations of several Psorothamnus species revealed the isolation of chalcones, flavonoids, benzofurans, isoflavonoids, pterocarpans, and triterpenoids, of which some showed anticancer, antiparasitic, antiprotozoal, and cytotoxic activities.6−9 In the current study, we report the isolation, structure elucidation, and antibacterial activity of three new prenylated acylphloroglucinol derivatives, psorothatins A−C (1−3), from this plant, as well as the total synthesis of 3. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The dried twigs, leaves, and flowers of P. f remontii collected in Utah, USA, were extracted with MeOH. The MeOH extract was partitioned between hexanes and 90% MeOH in H2O. The hexanes portion that was active against MRSA 33591 (IC50, 40.4 μg/mL) was chromatographed on Sephadex LH-20 using CHCl3−MeOH as an eluting solvent system. The most active column fraction (IC50 < 8.0 μg/mL against MRSA 33591) was chromatographed further on normal-phase and reversed-phase silica gel and finally purified by HPLC to afford psorothatins A−C (1−3). All three compounds were isolated as yellow oils and were not optically active. The UV spectrum of psorothatin A (1) showed absorption maxima at 255 and 285 nm. Its HRESIMS gave an [M − H]− ion peak at m/z 291.1232, suggesting a molecular formula of C16H20O5 (calcd for C16H19O5−, 291.1238). The 13C NMR spectrum displayed 16 distinct resonances. The DEPT NMR Received: August 13, 2015

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spectrum permitted identification of five methyls, one methylene, two methines, and eight quaternary carbons (Table 2). The 1H NMR spectrum also showed corresponding

60.0 and C-9 at δC 51.5 suggested that C-1 is oxygenated. An isobutyryl group in the molecule was evident from the COSY correlations from H-12 at δH 3.97 (1H, m, J = 6.5 Hz) to Me13/Me-14 at δH 1.13/1.14 (3H each, d, J = 7.0 Hz), which was supported by the HMBC correlations from the two methyl protons and H-12 to the carbonyl C-11 at δC 207.9. Further analysis of HMBC correlations indicated that the methyl protons (Me-17) at δH 1.89 (s) correlated with three quaternary carbons at δC 114.3 (C-5), 191.1 (C-6), and 163.0 (C-10), while the hydroxy group proton at δH 19.37 (s) showed cross-peaks with four quaternary carbons at δC 114.3 (C-5), 191.1 (C-6), 106.7 (C-7), and 207.9 (C-11) and one methine carbon at δC 35.9 (C-12). The remaining quaternary carbon at δC 185.1 (C-8) was correlated with H-1 through a three-bond H−C correlation obtained from an HMBC experiment (nJCH = 5 Hz) that emphasized small long-range couplings.10 This experiment also generated a weak 4JCH correlation between Me17 and C-9. These correlations allowed construction of the carbon skeleton of 1, and its molecular composition requires the formation of an ether bond between C-10 and C-3 of the isopentyl group and the presence of an epoxy group between C-1 and C-9. The proposed planar structure reasonably explains the chemical shifts for each proton and carbon. For example, C-6 was shifted downfield to δC 191.1 due to the strong electron-withdrawing effects of the two carbonyl groups. In turn, C-9 resonated in the upfield region at δC 51.5 due to the strained oxirane ring and the shielding effects of the planar diene-dione system. The C-6 hydroxy group forms a strong hydrogen bonding with the C-11 carbonyl group, leading to a significant shift of the hydroxy group proton to the downfield region. The long-range correlations observed from this hydroxy group proton to C-11 and C-12 in both HMBC experiments (nJ CH optimized for 5 and 10 Hz) were thus considered to result from two-bond and three-bond couplings, respectively, via the hydrogen bond between the C-11 carbonyl group and the C-6 hydroxy group (Figure 1), as a 5JCH correlation from this

Table 1. 1H NMR Data for Compounds 1−3 in CDCl3a H

1

1 2α 2β 12 13 14 15 16 17 18 20 21 OH

4.19, 2.42, 2.10, 3.97, 1.13, 1.14, 1.56, 1.38, 1.89,

2

br s d (16) d (16) m (6.5) d (7)b d (7)b s s s

19.37, s

4.21, 2.45, 2.13, 3.85, 1.77, 1.14, 1.58, 1.40, 1.91, 0.92,

3

br s d (16) d (16) m (6.5) 1.48, m d (7) s s s t (7.6)

19.48, s

4.19, br s 2.42, d (16) 2.12, d (16) 3.95, m (6.5) 1.13, d (7)b 1.14, d (7)b 1.56, s 1.39, s 3.13, d (7) 5.12, t (8) 1.73, s 1.67, s 19.40, s

Recorded at 400 MHz. δH, ppm, mult (J in Hz). bInterchangeable assignment.

a

Table 2. 13C NMR Data for Compounds 1−3 in CDCl3 1a

2a

3b

C

δC

DEPT

δC

DEPT

δC

DEPT

1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

60.0 34.8 80.7 114.3 191.1 106.7 185.1 51.5 163.0 207.9 35.9 18.9c 19.1c 30.1 31.6 8.0

CH CH2 C C C C C C C C CH CH3 CH3 CH3 CH3 CH3

60.0 34.8 80.7 114.4 191.2 105.2 185.3 51.6 162.9 207.4 42.3 26.7 16.6 30.1 31.6 8.0 12.1

CH CH2 C C C C C C C C CH CH2 CH3 CH3 CH3 CH3 CH3

59.9 34.8 80.7 118.1 190.8 106.9 185.1 51.5 162.8 207.9 35.8 18.9d 19.1d 30.0 31.6 21.9 121.2 132.6 18.1 26.0

CH CH2 C C C C C C C C CH CH3 CH3 CH3 CH3 CH2 CH C CH3 CH3

Figure 1. 2D NMR key correlations of 1 and 3.

Recorded at 100 and 125 MHz, respectively. δc, ppm. c,d Interchangeable assignment. a,b

hydroxy group proton to C-12 is unlikely to occur through a five-bond coupling. This is of particular significance, since, to the best of our knowledge, this kind of HMBC correlation has not been described previously in the literature. The chemical shift of C-6 (δC 191.1), which may be considered to result from a carbonyl carbon resonance, also supports a pseudo-sixmembered heterocyclic ring involving an intramolecular hydrogen bond. The HRESIMS of psorothatin B (2) gave an [M − H]− ion peak at m/z 305.1397, which was consistent with a molecular formula of C17H22O5. The 13C NMR spectrum displayed 17 resonances, most of which were closely comparable to those of psorothatin A (Table 2). The difference between the two compounds is that psorothatin B bears a 2-methylbutyryl group replacing the isobutyryl group at C-7 in psorothatin A. The 1H

resonances, in addition to a characteristic resonance significantly shifted downfield at δH 19.37 (1H, s) arising from a hydroxy group proton (Table 1). Analysis of these 1H and 13C NMR chemical shifts enabled the conclusion to be made that this C16 molecule is unprecedented, and 2D NMR experiments were thus employed to establish structural moieties and C−C connectivity of the molecule. The presence of an oxygenated isopentyl unit in 1 was determined from a COSY correlation from H-1 at δH 4.19 (br s) to H-2 at δH 2.10 and 2.42 (1H each, d, J = 16.0 Hz) as well as HMBC correlations from both Me-15 at δH 1.56 (s) and Me-16 at δH 1.38 (s) to the oxygenated quaternary C-3 at δC 80.7 and the methylene C-2 at δC 34.8. Additional HMBC correlations from H-2 to C-1 at δC B

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and 13C NMR chemical shifts of the 2-methylbutyryl group observed were consistent with the reported data in the literature.11 Psorothatin C (3) gave a molecular formula of C20H26O5 as determined by HRESIMS at m/z 345.1710 [M − H]− (calcd for C20H25O5−, 345.1707). The 13C NMR spectrum showed 20 carbon resonances, which were classified into six methyl, two methylene, three methine, and nine quaternary carbons by DEPT NMR. Comparison of its 1H and 13C NMR spectra with those of psorothatin A (1) indicated that they are analogues, and a C5 isoprenyl unit was substituted at C-5 in 3 (Table 2). This was supported by detailed 2D NMR analysis as shown in Figure 1. In particular, a reasonable NOESY spectrum for this compound was obtained, which facilitated 1H NMR assignments and understanding of the relative configuration and conformation of the molecule. The C-2 methylene proton at δH 2.12 (H-2β) showed NOE correlations with both H-1 and Me16 (δH 1.39). Another methylene proton at δH 2.42 (H-2α) showed NOE correlations with H-1 as well as Me-15 (strong) and Me-16 (δH 1.39) (weak). This indicated that the tetrahydropyran ring with an epoxy functionality between C-1 and C-9 should adopt a half-chair conformation, as shown in Figure 2. In this geometry optimized by ChemBio 3D Ultra

Scheme 1. Hypothetical Biosynthesis of 3

ruled out for these compounds to possess alternative structures formed via possible keto−enol tautomerization. The optical inactivity of all three compounds indicates that they are racemates, suggesting that they were generated via a nonstereoselective oxidation mechanism. Similar phenomena were observed in naturally occurring racemized phenolic compounds with an oxygenated prenyl moiety.13,14 Although hundreds of structurally diverse acylphloroglucinols and their derivatives have been isolated from natural sources,11,15−17 this is the first report for this class of compounds with such an unprecedented oxidation pattern. Antibacterial testing showed that the isolated compounds 1− 3 had IC50 values of 11.1, 6.2, and 1.4 μg/mL, respectively, against MRSA 33591 and 10.0, 5.6, and 2.4 μg/mL, respectively, against S. aureus ATCC 29213. Since compound 3 was the most active one, we embarked on a chemical synthesis of this compound. Taking advantage of available synthetic routes for similar prenylated acylphlorogluconols,18−20 a synthetic strategy for 3 was designed as shown in Scheme 2. Friedel−Crafts reaction of anhydrous phloroglucinol (9) with isobutyryl chloride and AlCl3 in nitrobenzene gave

Figure 2. Optimized geometry of 3.

14.0, the calculated distances from H-2β to H-1 and equatorial Me-16 are 2.646 and 2.389 Å, respectively, while the distances from H-2α to H-1, Me-16, and axial Me-15 are 2.601, 2.614, and 2.471 Å, respectively, explaining the observed NOE correlations. The ring fusion of this molecule requires the expoxy group (α-oriented) and H-1 (β-oriented) to take opposite orientations, thereby defining the relative configuration of C-1 and C-9. Since psorothatins A (1) and B (2) have the same NMR behaviors as psorothatin C (3) for their bicyclic ring system (Tables 1 and 2), they should also possess the same relative configuration and conformation. Psorothatins A−C (1−3) are novel natural products featuring a unique α,β-epoxyketone functionality and a βhydroxy-α,β-unsaturated ketone structural moiety. They are likely biosynthesized from acylphloroglucinol.12 A possible biosynthetic pathway for 3 is proposed as shown in Scheme 1. Prenylation of isobutyrylphloroglucinol (4) would yield prenylated metabolite 5, which could undergo an intramolecular cyclization followed by dehydrogenation to afford the prenylated chromene 6. Tautomerization of 6 into the key intermediate 7 would make epoxidation of the exo-double bond possible to form 8, which may be tautomerized further to the final product 3 as a more stable tautomer. Compounds 1 and 2 would also be biosynthesized in a similar fashion. Since the 1H and 13C NMR spectra of compounds 1−3 indicated that only one compound was predominantly present in the NMR solvent at room temperature (see Supporting Information), it can be

Scheme 2. Total Synthesis of 3

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Table 3. In Vitro Antibacterial Activities and Cytotoxicity of Synthetic 3a antibacterial activity (IC50, μg/mL)b

3 methicillin vancomycin doxorubicin

cytotoxicity (IC50, μg/mL)c

SA 29213d

MRSA 33591e

MRSA 1696f

MRSA 1708f

MRSA 1717f

EF 29212g

VRE 51299h

VRE 700221i

HEPG2j

LLCPK1k

Verol

2.4 0.5 − −

1.4 >50 − −

8.8 3.0 − −

8.6 3.2 − −

1.6 26.9 − −

5.3 −m 1.4 −

2.8 − 3.3 −

1.6 − >20 −

31 − − 0.4

30 − − 0.5

21 − − 5.4

a Both antibacterial activity and cytotoxicity are expressed as mean values of three experimental data. bIC50: concentration responsible for 50% growth inhibition of bacterial cells. The highest test concentrations for compound 3, methicillin, and vancomycin were 20, 50, and 20 μg/mL, respectively. cIC50: concentration responsible for 50% growth inhibition of mammalian cells. The highest test concentrations for compound 3 and the positive control doxorubicin were 50 and 10 μg/mL, respectively. dMethicillin-sensitive Staphylococcus aureus ATCC 29213. eStandard methicillin-resistant S. aureus ATCC 33591. fMethicillin-resistant S. aureus ATCC strains with various degrees of resistance: 1696 is the communityacquired USA400 strain, 1708 is a mupirocin-resistant strain, and 1717 is the community-acquired USA300 strain. gVancomycin-sensitive Enterococcus faecalis ATCC 29212. hLow-level vancomycin-resistant E. faecalis ATCC 51299. iVancomycin-resistant E. faecium ATCC 700221. j Human hepatic carcinoma. kPig kidney epithelial. lAfrican green monkey kidney fibroblasts. mNot tested.

aluminum sheets (silica gel 60 F254, Merck, Darmstadt, Germany) and reversed-phase glass plates (RP-18 F254S, Merck) and visualized by UV 254 nm and spraying with 10% H2SO4, followed by heating. Flash column chromatography was performed on normal-phase silica gel (230 × 400 mesh, J. T. Baker, Center Valley, PA, USA) and reversed-phase silica gel (RP-18, 40 μm, J. T. Baker). HPLC was performed on a Waters 2795 series HPLC system composed of an LC20AT pump with an SPD-20A detector (Shimadzu Corp., Kyoto, Japan). A Phenomenex C18 semipreparative HPLC column (250 × 10.0 mm i.d., 5 μm, Palo Alto, CA, USA) was used. The mobile phase was composed of solvent A (5% H2O) and solvent B (95% acetonitrile). The flow rate, injection volume, column temperature, and UV wavelength were set at 1.8 mL/min, 25 μL, 25 °C, and 325 nm, respectively. Reactants or reagents for synthesis including anhydrous phloroglucinol, isobutyryl chloride, 3,3-dimethylallyl bromide, 3-methyl-2-butenal, aluminum chloride, 1,8-diazabicyclo[5.4.0]undec-7-ene, ethylenediamine diacetate, nitrobenzene, tetrahydrofuran, and sodium sulfate were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) in appropriate grades and were used without further purification. Plant Material. The twigs, leaves, and flowers of Psorothamnus f remontii were collected in Garfield, Utah, USA, with coordinates of 38°09′00″ N, 112°00′00″ W by Elray Nixon in June 2011, who also identified this plant (specimen no. NIX0200611). An authentic sample of the plant material is deposited at the National Center for Natural Products Research at The University of Mississippi (NIX20061-1-A). Extraction and Isolation. The dried twigs, leaves, and flowers (350 g) were extracted with MeOH (1 L × 3) at room temperature for 12 h. Removal of the solvent by a rotary evaporator under vacuum at 40 °C yielded an MeOH extract (56.5 g), which was dissolved in 90% aqueous MeOH (500 mL) and partitioned with hexanes (500 mL × 3). The hexanes extract (12.5 g) was active against MRSA ATCC 33591, with an IC50 value of 40.4 μg/mL. It was chromatographed on a Sephadex LH-20 column using a CHCl3−MeOH gradient eluting solvent system to yield pooled fractions A−F. Fraction D (5.09 g, IC50 < 8 μg/mL against MRSA ATCC 33591) was chromatographed by normal-phase silica gel using hexanes−CHCl3 (9:1−3:7, 5 L) as the gradient eluting solvent system, followed by MeOH (1 L) to give pooled fractions D1−D12. The combined active fractions D7−D9 (350 mg, IC50 ≈ 0.8 μg/mL against MRSA ATCC 33591) were separated by a reversed-phase silica gel column with aqueous CH3CN (55−100%) followed by semipreparative reversed-phase HPLC purification using 95% CH3CN in H2O, to give psorothatins A (1) (3.5 mg, tR 10.5 min), B (2) (2.3 mg, tR 11.7 min), and C (3) (2.2 mg, tR 13.5 min). Psorothatin A (1) [6-hydroxy-7-isobutyryl-3,3,5-trimethyl-1a,2dihydro-3H,8H-oxireno[2,3-d]chromen-8-one]: yellow oil, [α]20D 0 (c 1.0, CHCl3); UV (MeOH) λmax log (ε) 255 (4.04), 285 (3.22) nm; IR (CHCl3) 3750, 2975, 2929, 1662, 1526, 1466, 1124, 889, 810, 719 cm−1; NMR (CDCl3) data, see Tables 1 and 2; HRESIMS m/z 291.1232 [M − H]− (calcd for C16H19O5−, 291.1238).

acylphloroglucinol 4. Alkylation of 4 with prenyl bromide in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and dry tetrahydrofuran (THF) afforded prenylated acylphloroglucinol 10. Compound 10 reacted with 3-methyl-2-butenal in ethylenediamine diacetate (EDDA) and CH2Cl2 yielded a mixture of prenylated acylchromenes (6 and 11). Treatment of 6 with oxygen in EDDA and CH2Cl2 furnished the final product 3. However, similar treatment of 11 did not produce the corresponding epoxide. This can be explained by the strong intramolecular hydrogen bonding in 11 preventing tautomerization, whereas in 6 there is a free hydroxy group that facilitates tautomerization for oxidation (Scheme 1). Synthetic psorothatin C (3) showed equivalent antibacterial activity to that of the natural product in the initial testing against the aforementioned Staphylococcus strains. It was further tested against multiple drug-sensitive and -resistant S. aureus and Enterococcus strains. As shown in Table 3, compound 3 was active against methicillin-resistant S. aureus and vancomycinresistant E. faecium with IC50 values in the range 1.4−8.8 μg/ mL. In vitro cytotoxicity testing showed that 3 exhibited IC50 values in the range 21−31 μg/mL against three mammalian cell lines (HEPG2, LLC-PK1, and Vero), indicating favorable antibacterial selectivity for this compound. In conclusion, three novel antibacterial prenylated acylphloroglucinols, psorothatins A−C (1−3), have been isolated from P. f remontii. Compound 3 has been synthesized from phloroglucinol by a facile four-step reaction sequence. This study has provided a novel chemically tractable natural product template for the synthesis of analogues with potentially improved antibacterial properties against drug-resistant strains.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a SEPA-3000 automatic digital polarimeter. UV spectra were measured on a Hewlett-Packard 8453 spectrometer. IR spectra were run on an ATI Mattson Genesis Series FTIR spectrometer. The NMR spectra using standard pulse programs were recorded at room temperature on a Bruker Avance DPX-400 spectrometer operating at 400 (1H) and 100 (13C) MHz or a Bruker Avance DRX 500 FT spectrometer operating at 500 (1H) and 125 (13C) MHz. The chemical shift (δ, ppm) values were calibrated using the residual NMR solvent. 2D NMR spectra were measured with standard pulse programs and acquisition parameters, except for the HMBC experiments, which were set for the delay times of 50 and 100 ms, corresponding to nJCH coupling constants of 10 and 5 Hz, respectively. HRESIMS was conducted on an Agilent series 1100 SL spectrometer equipped with an ESI source. TLC was performed on silica gel D

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Compound 11: pale yellow oil; 1H NMR (400 MHz, CDCl3) δ 14.28 (s, OH), 6.55 (1H, d, J = 9.9 Hz, H-1), 6.35 (1H, br s, OH), 5.44 (1H, d, J = 9.9 Hz, H-2), 5.27 (1H, t, J = 7.0 Hz, H-18), 3.90 (1H, m, J = 7.8 Hz, H-12), 3.38 (2H, d, J = 7.0 Hz, H-17), 1.82 (3H, s, Me20), 1.77 (3H, s, Me-21), 1.48 (6H, s, Me-15,16), 1.18 (6H, d, J = 7.0 Hz, Me-13,14); 13C NMR (100 MHz, CDCl3) δ 210.9 (C-11), 163.6 (C-10), 157.3 (C-6), 154.8 (C-8), 136.6 (C-19), 124.9 (C-2), 122.0 (C-18), 116.8 (C-1), 105.5 (C-7), 105.0 (C-5), 102.0 (C-9), 77.9 (C3), 39.5 (C-12), 28.0 (C-15), 27.9 (C-16), 26.1 (C-20), 21.8 (C-17), 19.8 (C-13,14), 18.0 (C-21); HRESIMS m/z 331.1965 [M + H]+ (calcd for C20H27O4+, 331.1904). The NMR assignments of this new synthetic compound (with a numbering system similar to psorothatins, which is shown in the Supporting Information) were made by comparison with similar compounds in the literature.22 Synthesis of Psorothatin C (3). To a solution of compound 6 (62.7 mg, 0.189 mmol) in CH2Cl2 (5 mL) was added EDDA (0.009 g, 0.06 mmol). The reaction mixture was stirred at room temperature under oxygen for 24 h. After addition of H2O (5 mL), the reaction mixture was extracted with CH2Cl2 (8 mL × 3). The combined CH2Cl2 layers were dried over Na2SO4. Evaporation of the solvent gave a crude product, which was purified by column chromatography on silica gel using hexanes−acetone (95:5) to give 3 (15 mg, 23% yield): yellow oil; its 1H and 13C NMR spectra are identical with those of the natural product. In Vitro Antibacterial Assays. Microorganisms were obtained from the American Type Culture Collection (Manassas, VA, USA) including Staphylococcus aureus ATCC 29213 (methicillin-sensitive), Staphylococcus aureus ATCC 33591 (standard methicillin-resistant strain), Staphylococcus aureus ATCC BAA-1696 (the communityacquired USA400 strain), Staphylococcus aureus ATCC BAA-1708 (mupirocin-resistant strain), Staphylococcus aureus ATCC BAA-1717 (the community-acquired USA300 strain), vancomycin-sensitive Enterococcus faecalis ATCC 29212, low-level vancomycin-resistant Enterococcus faecalis ATCC 51299, and vancomycin-resistant Enterococcus faecium ATCC 700221. All organisms were tested using modified versions of the CLSI (formerly NCCLS) method,23 which has been described previously.24 Briefly, samples (dissolved in DMSO) were serially diluted in 20% DMSO−saline and transferred (10 μL) in duplicate to 96-well flat-bottom microplates. Inocula were prepared by adjusting the OD630 of microbe suspensions in the assay to afford the final desired CFU/mL. The positive control drugs included methicillin and vancomycin (from ICN Biomedicals, Aurora, OH, USA). The highest test concentrations of compounds and the control drugs are indicated in Table 3. IC50 values (concentration that affords 50% inhibition relative to controls) were calculated using XLfit software (IDBS, Alameda, CA, USA) with Fit Model 201. In Vitro Cytotoxicity Assays. A panel of mammalian cell lines obtained from ATCC (Manassas, VA, USA) included human hepatic carcinoma (HEPG2), pig kidney epithelial (LLC-PK1), and African green monkey kidney fibroblasts (Vero). The detailed assay procedure has been described in our previous papers.25,26

Psorothatin B (2) [6-hydroxy-3,3,5-trimethyl-7-(2-methylbutanoyl)-1a,2-dihydro-3H,8H-oxireno[2,3-d]chromen-8-one]: yellow oil, [α]20D 0 (c 1.0 CHCl3); UV (MeOH) λmax log (ε) 255 (4.06), 290 (3.28) nm; IR (CHCl3) 3726, 2971, 2932, 1662, 1622, 1525, 1467, 1120, 973, 879, 798, 668 cm−1; NMR (CDCl3) data, see Tables 1 and 2; HRESIMS m/z 305.1397 [M − H]− (calcd for C17H21O5−, 305.1394). Psorothatin C (3) [6-hydroxy-7-isobutyryl-3,3-dimethyl-5-(3methylbut-2-en-1-yl)-1a,2-dihydro-3H,8H-oxireno[2,3-d]chromen8-one]: yellow oil, [α]20D 0 (c 1.0, CHCl3); UV (MeOH) λmax log (ε) 230 (4.68), 290 (3.13) nm; IR (CHCl3) 3736, 2974, 2924, 1662, 1622, 1525, 1442, 1376, 1250, 889, 798, 749 cm−1; NMR (CDCl3) data, see Tables 1 and 2; HRESIMS m/z 345.1710 [M − H]− (calcd for C20H25O5−, 345.1707). Synthesis of Isobutylphloroglucinol (4). To a solution of anhydrous phloroglucinol (9, 5.0 g, 39.7 mmol) in nitrobenzene (40 mL) was added AlCl3 (21.2 g, 158.5 mmol). The reaction mixture was stirred at room temperature under nitrogen for 30 min. Isobutyryl chloride (4.57 mL, 43.6 mmol) was slowly added, and the reaction mixture was heated at 65 °C for 21 h. The reaction was quenched by addition of ice−water (20 mL). The reaction mixture was extracted with EtOAc (50 mL × 3). The combined EtOAc layers were treated with 2 M NaOH (80 mL × 2). The aqueous layers were neutralized with 2 M HCl and extracted with EtOAc (50 mL × 3). The combined EtOAc layers were washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford a crude product, which was purified by column chromatography on silica gel using CHCl3−MeOH (9:1) to give 4 (4.5 g, 58% yield): pale yellow solid. Identification of compound 4 was made by comparison of its NMR data with those reported in the literature.18 Synthesis of 2-Methyl-1-(2,4,6-trihydroxy-3-(3-methylbut-2en-1-yl)phenyl)propan-1-one (10). A mixture of 4 (2.21 g, 11.3 mmol), prenyl bromide (1.79 g, 12 mmol), and DBU (2.01 g, 13.2 mmol) in dry THF (50 mL) was stirred at room temperature under nitrogen for 48 h. After addition of a 2 N HCl solution (50 mL), the reaction mixture was extracted with EtOAc (50 mL × 3). The combined EtOAc layers were washed with brine and dried over Na2SO4. Removal of the solvent afforded a crude product, which was purified by column chromatography on silica gel using hexanes− EtOAc (7:3) to give 10 (1.3 g, 43% yield): pale yellow solid. Identification of compound 10 was made by comparison of its NMR data with those reported in the literature.21 Synthesis of 1-(5,7-Dihydroxy-2,2-dimethyl-8-(3-methylbut2-en-1-yl)-2H-chromen-6-yl)-2-methylpropan-1-one (6) and 1(5,7-Dihydroxy-2,2-dimethyl-6-(3-methylbut-2-en-1-yl)-2Hchromen-8-yl)-2-methylpropan-1-one (11). To a solution of compound 10 (649.8 mg, 2.46 mmol) and 3-methyl-2-butenal (0.3 mL, 5.1 mmol) in CH2Cl2 (30 mL) at room temperature under nitrogen was slowly added EDDA (0.09 g, 0.6 mmol). The reaction mixture was stirred at room temperature for 12 h. H2O (30 mL) was added, and the reaction mixture was extracted with CH2Cl2 (30 mL × 3). The combined CH2Cl2 layers were dried over Na2SO4. Evaporation of the solvent under reduced pressure afforded a residue, which was chromatographed on silica gel using hexanes−EtOAC (8:2) to give 6 (147 mg, 18% yield) and 11 (301 mg, 37% yield). Compound 6: pale yellow oil; 1H NMR (400 MHz, CDCl3) δ 13.27 (s, OH), 6.65 (1H, d, J = 9.9 Hz, H-1), 5.46 (1H, d, J = 9.9 Hz, H-2), 5.19 (1H, t, J = 6.5 Hz, H-18), 3.90 (1H, m, J = 7.8 Hz, H-12), 3.35 (2H, d, J = 6 Hz, H-17), 1.85 (3H, s, Me-20), 1.79 (3H, s, Me21), 1.42 (6H, s, Me-13,14), 1.16 (6H, d, J = 7.0 Hz, Me-15,16); 13C NMR (100 MHz, CDCl3) δ 211.0 (C-11), 163.6 (C-6), 158.9 (C-10), 157.0 (C-8), 136.2 (C-19), 125.6 (C-2), 121.7 (C-18), 116.5 (C-1), 105.6 (C-7), 104.3 (C-5), 102.8 (C-9), 77.9 (C-3), 39.6 (C-12), 28.4 (C-15), 27.9 (C-16), 26.1 (C-20), 21.9 (C-17), 19.7 (C-13,14), 18.1 (C-21); HRESIMS m/z 331.1982 [M + H]+ (calcd for C20H27O4+, 331.1904). The NMR assignments of this new synthetic compound (with a numbering system similar to psorothatins, which is shown in the Supporting Information) were made by comparison with similar compounds in the literature.22

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00721. NMR and ESIMS spectra of compounds 1−4, 6, 10, and 11 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 662-915-6742. Fax: 662-915-7989. E-mail: xcli7@olemiss. edu (X.-C. Li). Notes

The authors declare no competing financial interest. E

DOI: 10.1021/acs.jnatprod.5b00721 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Article

ACKNOWLEDGMENTS The authors thank Ms. M. Wright for in vitro antimicrobial testing, Mr. J. Trott for cytotoxicity testing, Dr. B. Avula for mass spectrometry analyses, and Mr. Frank T. Wiggers for 2D NMR data. This work was supported by the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-64082-0009 and China Scholarship Council.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00721 J. Nat. Prod. XXXX, XXX, XXX−XXX