Peniphenones AâD from the Mangrove Fungus Penicillium

Article pubs.acs.org/jnp

Peniphenones A−D from the Mangrove Fungus Penicillium dipodomyicola HN4-3A as Inhibitors of Mycobacterium tuberculosis Phosphatase MptpB Hanxiang Li,† Jieyi Jiang,† Zhaoming Liu,† Shaoe Lin,† Guoping Xia,† Xuekui Xia,† Bo Ding,† Lei He,‡ Yongjun Lu,*,‡ and Zhigang She*,† †

School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China School of Life Sciences and Biomedical Center, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: A pair of unusual benzannulated 6,6-spiroketal enantiomers [(−)-1 and (+)-1] and three new biogenetically related compounds (2−4), together with two known related analogues (5 and 6), have been isolated from a mangrove fungus, Penicillium dipodomyicola HN4-3A. Their structures were elucidated on the basis of spectroscopic analysis (1D and 2D NMR data) and X-ray crystallography. The absolute configurations of enantiomers (−)-1 and (+)-1 were determined using quantum chemical calculations of the electronic circular dichroic (ECD) spectra. Compounds 2 and 3 exhibited strong inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with IC50 values of 0.16 ± 0.02 and 1.37 ± 0.05 μM, respectively.

T

unusual benzannulated 6,6-spiroketal enantiomers [(−)-1 and (+)-1] and three new biogenetically related compounds (2−4), together with two known related analogues (5 and 6), were obtained from the fungal culture. Compounds 2 and 3 were tested and exhibited strong inhibitory activity against MptpB. Details of the isolation, structure elucidation, and biological activity of these compounds are reported herein.

uberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis. According to the latest report from the World Health Organization (WHO),1 there were 8.7 million cases of TB and 1.4 million TB-related deaths in 2011. The situation is further worsened by co-infection with HIV2−4 and the prevalence of multi-drug-resistant tuberculosis (MDRTB).5−7 There is an urgent requirement to develop new targets and innovative strategies to tackle TB infections. M. tuberculosis secretes two eukaryote-like protein tyrosine phosphatases, MptpA and MptpB, which selectively dephosphorylate human host proteins involved in the interferon-γ signaling pathways, thereby preventing the initiation of host defense mechanisms.8−12 Since MptpB has no direct human orthologues, the inhibition of MptpB might prevent the proliferation of M. tuberculosis in human host macrophages and therefore represents a promising strategy for the development of selective antibiotics against this severe pathogen. Recently, there has been increased interest in finding new inhibitors against MptpB.13−16 Filamentous fungi have been an important source of pharmacological metabolites.17−19 Among them, marine fungi are a particularly promising source of novel active natural products.20−22 Our research group has focused in the past decade on the exploration of bioactive metabolites from mangrove fungi collected from the South China Sea.23−28 Recently, a chemical investigation of mangrove fungal strain HN4-3A was carried out. This fungus was isolated from the stem of the mangrove plant Acanthus ilicifolius and was identified as a Penicillium dipodomyicola strain. A pair of © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound (±)-1 was isolated as pale yellow crystals. The molecular formula of (±)-1 was determined to be C19H24O5 on the basis of HREIMS (m/z 332.1621 [M]+, calcd 332.1618), indicating eight degrees of unsaturation. The 1H, 13C, and HSQC NMR data (Table 1) showed resonances of five methyls (Me-14, Me-16, Me-17, Me-18, and Me-19), two methylenes (C-7 and C-12), four methines (C-3, C-8, C-10, and C-13), and 10 quaternary carbons, whose chemical shift values indicated the presence of two carbonyl carbons (δC 206.4, C-11; δC 202.9, C-15), a ketal carbon (δC 105.0, C-9), one oxygenbearing methane carbon (δC/δH 67.5/3.93, C-13/H-13), and one pentasubstituted benzene ring (δC 155.6, C-1; δC 117.0, C2; δC/δH 129.3/7.32, C-3/H-3; δC 113.2, C-4; δC 160.0, C-5; and δC 111.2, C-6). The pentasubstituted benzene ring was assigned as a 2,4-dihydroxy-5-methylacetophenone moiety, according to HMBC correlations (Figure 1) from H3-14 (δH 2.04) to C-1, C-2, and C-3, from H-3 (δH 7.32) to C-1 and C-5, Received: October 17, 2013

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dx.doi.org/10.1021/np400880w | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. NMR Data for (±)-Peniphenone A [(±)-1] in CDCl3

a

no.

δC, type

1 2 3 4 5 6 7a 7b 8 9 10 11 12a 12b 13 14 15 16 17 18 19

155.6, C 117.0, C 129.5, CH 113.2, C 160.0, C 112.2, C 23.5, CH2 30.8, CH 105.0, C 49.2, CH 206.4, C 48.6, CH2 67.5, CH 15.1, CH3 202.9, C 26.2, CH3 15.2, CH3 7.5, CH3 21.7, CH3 OH-5

δH, mult. (J in Hz)

7.32, s

C-1, -5, -14, -15

2.53, dd (16.6, 3.1) 2.77, dd (16.6, 5.6) 2.09, m

C-6, -8, -9 C-1, -5, -6, -8, -9 C-7, -9, -17

2.87, qd (6.7, 0.8)

C-9, -11, -18

2.34, 2.50, 3.93, 2.04,

C-11, -13, -19 C-10, -11

ddd (13.8, 11.3, 0.8) dd (13.8, 3.1) m s

2.54, s 1.23, d (6.8) 1.14, d (6.7) 1.19, d (6.2) 12.81, brd

Figure 1. 1H−1H COSY (bonds) and selected HMBC (arrows) of (±)-1 and 2−4.

HMBCa

a 1,7-dioxaspiro[5.5]undecane moiety. On the basis of these data, the benzannulated 6,6-spiroketal structure of (±)-1 was established as shown and named (±)-peniphenone A. The structure of (±)-peniphenone A [(±)-1] was confirmed on the basis of a single-crystal X-ray diffraction analysis (Figure 2a). The relative configuration of (±)-1 was consequently established as 8R*,9R*,10R*,13R*. However, the crystal of (±)-1 had the centrosymmetric space group P21/c, which was indicative of its racemic nature. The deduction was also proved by the absence of any CD (circular dichroism) maximum and optical rotation. Subsequent chiral HPLC purification of (±)-1 led to the separation of the two enantiomers, (−)-1 and (+)-1 (Figure 2b), which displayed opposite Cotton effects in their CD spectra (Figure 3) and opposite optical rotations. According to the relative stereochemistry 8R*,9R*,10R*,13R* established by the single-crystal X-ray diffraction, the quantum chemical calculation on electronic circular dichroism (ECD) was utilized to determine their absolute stereochemistry. The structure with all R configurations was used in the computations. Its ECD was computed at the B3LYP/6-311+ +G(2d,p)//B3LYP/6-31+G(d) level using the reported effective methods which Zhu has summarized.29−31 As illustrated in Figure 3, the experimentally acquired CD spectrum for (−)-1 agreed well with the ECD curve computed for (8R,9R,10R,13R)-1. Thus, the most likely absolute configuration of (−)-1 was established as 8R,9R,10R,13R, and that of its enantiomer as 8S,9S,10S,13S. Compound 2 was obtained as yellow crystals. Its molecular formula was established as C21H18O8 by HREIMS data (m/z 398.0997 [M]+, calcd 398.0996). The NMR spectra (Table 2) showed resonances for two methyls (Me-19 and Me-21), one methylene (C-7), 16 aromatic/olefinic carbons, one α,βunsaturated carboxylic carbon (C-9), and one ketone carbon (C-20). With 13 degrees of unsaturation accounted for by the molecular formula, the structure of 2 was suggested to contain three rings. Analysis of the 1H−1H COSY and HMBC data of 2 defined the same moiety, 3,4-diol-5-methyl acetophenone (ring A), as found in (±)-1. HMBC cross-peaks from H2-7 (δH 3.66)

C-1, -2, -3 C-4, C-7, C-9, C-4, C-4,

-15 -8, -9 -10, -11 -5, -6 -5, -6

HMBC correlations are presented from proton to indicated carbon.

from H3-16 (δH 2.54) to C-4 and C-15, and from 5-OH (δH 12.82) to C-4, C-5, and C-6. Additional HMBC cross-peaks between H-7 (δH 2.77/2.53) and C-1, C-5, and C-6 linked C-7 (δC 23.5) to C-6 of the acetophenone moiety. The 1H−1H COSY spectrum (Figure 1) showed the presence of three spin systems (H2-7/H-8/H3-17, H-10/H3-18, and H2-12/H-13/H319). Both of the former units were linked to the ketal carbon C9 on the basis of HMBC correlations from Hb-7 (δH 2.77), H-8 (δH 2.09), H-10 (δH 2.87), Me-17 (δH 1.23), and Me-18 (δH 1.14) to C-9. Correlations of H2-12 (δH 2.34/2.50) to C-11 and C-10 (δC 49.2) and of Me-18 to C-11 enabled the connection of the latter two units. Considering the chemical shifts of C-1 (δC 155.6) and C-13 (δC 67.5) and the unsaturation requirement of (±)-1, the two carbons should attach to C-9 via two oxygen atoms, respectively, to form the substructure for B



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Figure 2. Single-crystal X-ray structure (a) and chiral HPLC chromatogram (b) of (±)-1.

Figure 3. Experimental CD spectra of (−)-1 and (+)-1 in MeCN and the calculated ECD spectrum of (8R,9R,10R,13R)-1.

same substituted acetophenone moiety as (±)-1. The presence of a 2,5-dimethyl-3-hydroxyl-p-benzoquinone moiety was deduced from the chemical shifts of C-8−C-13 (δC 143.4, 191.2, 116.8, 151.6, 183.7, and 139.8) in 3. This was confirmed by HMBC correlations of H2-7 (δH 3.75) to C-8, C-9, and C13, H3-17 (δH 1.92) to C-9, C-10, and C-11, and H3-18 (δH 2.37) to C-8, C-12, and C-13. Additional HMBC correlations from H2-7 to C-1, C-5, and C-6 established the connectivity of the acetophenone and p-benzoquinone moieties via the methylene C-7, completing the planar structure of 3. The above conclusion was supported by an X-ray crystal structure (Figure 4b). The new compound 3 was named peniphenone C. Compound 4 (named peniphenone D) was obtained as colorless crystals. Its HREIMS spectrum displayed a molecular ion peak at m/z [M]+ 292.0942 (calcd 292.0941), corresponding to the molecular formula C15H16O6. The 1H and 13C NMR spectra together with HMQC correlations for peniphenone D (4) showed three methyls (Me-12, Me-14, and Me-15), one methylene (C-7), one oxygenated sp3 methine (C-10), eight aromatic/olefinic carbons (one protonated), one α,β-unsaturated carboxylic carbon (C-9), and one ketone carbon (C-13). Comparison of the 1H and 13C NMR data (Table 2) with those of (±)-1 showed the presence of the same 2,4-dihydroxy-5methylacetophenone moiety. Further HMBC cross-peaks (Figure 1) from H2-7 (δH 3.42/3.48) to C-8 (δC 101.5), C-9 (δC 177.1), and C-11 (δC 175.7) and from H3-15 (δH 1.46) to C-10 (δC 76.1) and C-11 (δC 175.7) together with the

to C-8 (δC 100.0), C-9 (δC 167.0), and C-12 (δC 168.4) and from H-11 (δH 6.49) to C-8, C-10 (δC 158.0), and C-12 established a substituted α-pyrone moiety (ring B), which was attached to ring A through a methylene carbon C-7 (δC 17.1) on the basis of correlations from H2-7 to C-1 (δC 161.4), C-5 (δC 160.6), and C-6 (δC 113.2). The remaining three aromatic protons [δH 6.83 (d, J = 8.4 Hz), H-15; δH 7.09 (dd, J = 8.4, 2.3 Hz), H-14; δH 7.14 (d, J = 2.3 Hz), H-18], appearing as an ABX spin system, together with their relevant HMBC correlations (H-14/C-16, C-18; H-15/C-13, C-17; H-18/C-14, C-16), identified the structure of a 1,2,4-trisubstituted benzene ring (ring C). Further HMBC cross-peaks from H-11 (δH 6.49) to C-13 (δC 121.9) and from H-14 (δH 7.09) and H-18 (δH 7.14) to C-10 (δC 158.0) led to the connection of ring B and ring C through the C-10−C-13 bond. This structural assumption was confirmed on the basis of a single-crystal X-ray diffraction analysis (Figure 4a). Therefore, the structure of compound 2 was elucidated as shown and named peniphenone B. Compound 3 was isolated as a white solid and displayed a molecular formula of C18H18O6 (10 degrees of unsaturation), as determined by HREIMS (m/z 330.1096 [M]+, calcd 330.1098). The 13C and DEPT NMR spectra exhibited 18 carbon signals for four methyls (Me-14, Me-16, Me-17, and Me-18), one methylene (C-7), 10 aromatic/olefinic carbons, one ketone carbon (C-15), and two α,β-unsaturated carbonyl carbons (C-9 and C-12) (Table 2). Comparison of the 1H and 13C NMR spectroscopic data with those of (±)-1 revealed that it had the C



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(±)-1, 3, and 4, the intermediates 1i, 3i, and 4i arise from C2 or C3 units via an acetate pathway. For compound 2, the formation of intermediate 2i was also concerned with tyrosine from the shikimate pathway. The key steps were that the nucleophilic intermediates 1i, 2i, 3i, and 4i attacked at the terminal methylene group of 5i, providing the corresponding intermediates. The corresponding intermediates could finally be transformed into 1, 2, 3, and 4, respectively, via a series of reductions or oxidations (Scheme S1, Supporting Information). The co-isolated compounds 5 and 6 from the fungus in this study further supported the plausible biosynthetic pathway. As shown in Figure 5, compounds 2 and 3 exhibited strong inhibitory activity against MptpB with IC50 values of 0.16 ± 0.02 and 1.37 ± 0.05 μM, respectively. Unfortunately, due to the limited quantity of (±)-1 and 4, their biological activities were not tested. The promising inhibitory activities of 2 and 3, together with their unique structures, indicate that they could represent a new type of lead compounds for the development of new drugs tackling TB infections.

Table 2. NMR Data for Pheniphenones B−D (2−4) peniphenone B (2)a

peniphenone C (3)b

peniphenone D (4)b



δC, type


7.41, brs

160.2, C 119.5, C 130.7, CH

3.75, s

113.3, C 158.8, C 112.8, C 25.9, CH2

no.

δC, type

1 2 3

161.4, C 116.6, C 130.6, CH

4 5 6 7

112.5, C 160.6, C 113.2, C 17.2, CH2

8 9 10

100.0, C 167.0, C 158.0, C

11 12

97.1, CH 168.4, C

13 14

121.9, C 117.3, CH

15

116.1, CH

16 17 18

148.5, C 145.7, C 112.3, CH

19 20 21

16.0, CH3 202.6, C 26.6, CH3 OH OH

a

δC, type

7.52, s

161.1, C 118.4, C 131.1, CH

3.66, s

112.8, C 161.8, C 110.6, C 22.0, CH2

6.49, s

7.09, dd (8.4, 2.3) 6.83, d (8.4)

143.4, C 191.2, C 116.8, C

101.5, C 177.1, C 76.1, CH

151.6, C 183.7, C

175.7, C 15.8, CH3

139.8, C 16.1, CH3

203.4, C 25.9, CH3

2.22, d (0.4)

202.7, C 26.2, CH3 8.1, CH3 12.4, CH3

2.53, s 1.92, s 2.37, s

2.51, s 9.61, brs

OH-5

9.37, brs

OH-1

13.18, brs 9.55, brs

7.14, d (2.3) 2.10, s

7.41, d (0.4)

3.42, d (15.1) 3.28, d (15.1)

4.84, q (6.8)

1.46, d (6.8)

OH-5

13.76, brs 8.55, brs 8.22, brs

EXPERIMENTAL SECTION

General Experimental Methods. Melting points were determined on an X-4 micromelting point apparatus and are uncorrected. Optical rotations were determined with an MCP 300 (Anton Paar) polarimeter at 25 °C. UV data were measured on a UV-240 spectrophotometer (Shimadzu). CD data were recorded with a J720 spectropolarimeter (JASCO, Tokyo, Japan). The NMR data were recorded on a Bruker Avance 400 spectrometer at 400 MHz for 1H and 100 MHz for 13C in CDCl3 or DMSO-d6. All chemical shifts were referenced relative to the corresponding solvent signals (δH 7.26/δC 77.2 for CDCl3; δH 2.50/δC 49.5 for DMSO-d6), and coupling constants (J) are given in Hz. HREIMS data were measured on a MAT95XP high-resolution mass spectrometer (Thermo) and EIMS on a DSQ EI-mass spectrometer (Thermo). Single-crystal data were measured on an Oxford Gemini S Ultra diffractometer. Column chromatography (CC) was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Pharmacia). The chiral HPLC preparation of pure enantiomers was accomplished for (±)-1 over a Chiralpak AS-H (column size: 4.6 × 250 mm; Daicel Chemical Industries, Ltd.; mobile phase: hexane−2propanol, 80/20 (v/v); flow rate: 0.8 mL/min). Fungal Material. The fungal strain HN4-3A was isolated from the stem of the mangrove plant Acanthus ilicifolius collected from the South China Sea in Hainan Province, China. By classical microscopic analysis, the fungus was identified as a member of the genus Penicillium. It was further identified as Penicillium dipodomyicola according to a molecular biological protocol by DNA amplification and sequencing of the ITS region (see Supporting Information). The sequence data have been submitted to and deposited at GenBank (accession no. KF649207). This fungal strain has been preserved at the China Center for Type Culture Collection (CCTCC) under the patent depository number CCTCC M 2013233. Cultivation. Starter cultures were maintained on PDA medium (20 g of glucose, 20 g of agar, and 2 g of sea salt in 1 L of potato infusion). Plugs of agar supporting mycelial growth were cut and transferred aseptically to 250 mL Erlenmeyer flasks containing 100 mL of PDB medium (20 g of glucose and 2 g of sea salt in 1 L of potato infusion). The flasks were incubated at 28 °C on a rotary shaker for seven days, and then the mycelia were aseptically transferred to 500 mL Erlenmeyer flasks containing 200 mL of PDB medium. The flasks were then incubated statically at room temperature (25−30 °C) for one month. Extraction and Isolation. The fungal culture (10 L) was filtered through cheesecloth. The filtrate was concentrated to 5 L below 50 °C and then extracted with EtOAc (5 L × 3) to give 6.2 g of extract. The extract was subjected to silica gel CC using gradient elution with petroleum ether−EtOAc from 90:10 to 0:100 (v/v) to give nine

2.57, s

17.2, CH3

OH-11 OH-1

■

2.21, d (0.4)

In DMSO-d6. bIn CDCl3.

unsaturation requirement of 4 identified an α,β-unsaturated γlactone moiety. Finally, the two moieties mentioned above were linked to C-7 by the relevant HMBC correlations from H2-7. The structure of 4 was subsequently confirmed by a singlecrystal X-ray diffraction experiment using Cu Kα radiation [flack parameter −0.02(16)] (Figure 4c), and its absolute configuration was also established as 10R. The remaining two known compounds 5 and 6 were identified as 2,4-dihydroxy-3,5-dimethylacetophenone and 2,4dihydroxy-5-methylacetophenone, respectively, by comparison of their NMR and MS data with those reported.32,33 (±)-Peniphenone A [(±)-1], possessing a benzannulated 6,6-spiroketal, represents an unusual structural class. Spiroketals as substructures of miscellaneous natural products have been obtained from many sources such as plants, fungi, marine organisms, and insects,34 but having an aryl ring fused to the spiroketal moiety is relatively rare, especially as in the benzannulated 6,6-spiroketals. A probable biosynthetic pathway for compounds (±)-1 and 2−4 was proposed as shown in Scheme 1. On the basis of the structural similarity, the common biogenetic precursor of compounds (±)-1 and 2−4 was proposed to be the ortho-quinone methide 5i, which was derived from 5 (the methylated product of 6). For compounds D



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Figure 4. Single-crystal X-ray structures of 2 (a), 3 (b), and 4 (c).

Scheme 1. Proposed Biosynthetic Pathways for Compounds (±)-1 and 2−6

fractions (Fr.1−Fr.9). Fr.3 (142 mg) was applied to Sephadex LH-20 CC, eluted with petroleum ether−CHCl3−MeOH (2:1:1), to obtain compounds (±)-1 (1.4 mg) and 3 (3.1 mg). Fr.4 (235 mg) was rechromatographed on silica gel (gradient of CHCl3−MeOH from 1:0 to 1:1) to give eight fractions (Fr.4-1−Fr.4-8). Fr.4-3 (27 mg), Fr.4-5 (35 mg), and Fr.4-6 (42 mg) were applied to Sephadex LH-20 CC eluted with CHCl3−MeOH (1:1) to yield compounds 4 (1.2 mg), 5

(5.6 mg), and 2 (4.7 mg), respectively. Fr.6 was recrystallized from EtOAc to afford 6 (10.2 mg). (±)-Peniphenone A [(±)-1]: pale yellow crystals (EtOAc); mp 161−163 °C; [α]25 D ±0 (c 0.45, MeOH); UV (MeOH) (λmax) (log ε) 231 (3.14), 274 (4.11) nm; 1H and 13C NMR spectroscopic data, see Table 1; EIMS m/z 332 [M]+; HREIMS m/z 332.1621 [M]+ (calcd for C19H24O5, 332.1618). (−)-Peniphenone A [(−)-1]: [α]25 D −172 (c 0.2, MeOH). E



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Figure 5. IC50 determination of compounds 2 (a) and 3 (b). Mean values and standard deviations are represented. (+)-Peniphenone A [(+)-1]: [α]25 D +167 (c 0.2, MeOH). Peniphenone B (2): yellow crystals (acetone); mp 223−224 °C; UV (MeOH) (λmax) (log ε) 285 (3.06), 353 (3.92) nm; 1H and 13C NMR spectroscopic data, see Table 2; EIMS m/z 398 [M]+; HREIMS m/z 398.0997 [M]+ (calcd for C21H18O8, 398.0996). Peniphenone C (3): white, amorphous solid (MeOH); mp 197− 199 °C; UV (MeOH) (λmax) (log ε) 267 (3.96), 331 (2.84) nm; 1H and 13C NMR spectroscopic data, see Table 2; EIMS m/z 330 [M]+; HREIMS m/z 330.1096 [M]+ (calcd for C18H18O6, 330.1098). Peniphenone D (4): colorless crystals (EtOAc); mp 127−128 °C; [α]25 D −72 (c 0.5, MeOH); UV (MeOH) (λmax) (log ε) 239 (3.45), 292 (3.89), 325 (3.17) nm; 1H and 13C NMR spectroscopic data, see Table 2; EIMS m/z 292 [M]+; HREIMS m/z 292.0942 [M]+ (calcd for C15H16O6, 292.0941). X-ray Crystallographic Analysis of (±)-1 and 2−4. All singlecrystal X-ray diffraction data were collected at 20 °C on an Oxford Gemini S Ultra diffractometer with Cu Kα radiation (λ = 1.541 78 Å) or Mo Kα radiation (λ = 0.710 73 Å). The structures were solved by direct methods (SHELXS-97) and refined using full-matrix leastsquares difference Fourier techniques. Hydrogen atoms bonded to carbons were placed on the geometrically ideal positions by the “ride on” method. Hydrogen atoms bonded to oxygen were located by the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors. Crystallographic data for (±)-1 and 2−4 have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44-(0)1223-336033, or e-mail: [email protected]]. Crystal Data of (±)-1: C19H24O5, Mr = 332.16, monoclinic, a = 21.945(4) Å, b = 15.450(3) Å, c = 10.384(2) Å, α = 90°, β = 103.603(4)°, γ = 90°, V = 3422.0(11) Å3, space group P21/c, Z = 4, Dx = 1.290 mg/m3, μ(Mo Kα) = 0.093 mm−1, and F(000) = 1424. Crystal dimensions: 0.50 × 0.45 × 0.40 mm3. Independent reflections: 7496 (Rint = 0.0601). The final R1 values were 0.0560, wR2 = 0.1330 (I > 2σ(I)). CCDC number: CCDC 965576. Crystal Data of 2: C21H18O8, Mr = 398.10, monoclinic, a = 4.3655(6) Å, b = 49.452(5) Å, c = 8.9782(11) Å, α = 90°, β = 91.731(13)°, γ = 90°, V = 1937.4(4) Å3, space group Cc, Z = 4, Dx = 1.489 mg/m3, μ(Cu Kα) = 1.020 mm−1, and F(000) = 912. Crystal dimensions: 0.30 × 0.25 × 0.12 mm3. Independent reflections: 1884 (Rint = 0.0821). The final R1 values were 0.0699, wR2 = 0.1724 (I > 2σ(I)). CCDC number: CCDC 965577. Crystal Data of 3: C18H18O6, Mr = 330.32, orthorhombic, a = 8.3424(18) Å, b = 11.460(2) Å, c = 16.107(3) Å, α = 90°, β = 90°, γ = 90°, V = 1540.0(6) Å3, space group P212121, Z = 4, Dx = 1.425 mg/m3, μ(Mo Kα) = 0.107 mm−1, and F(000) = 696. Crystal dimensions: 0.42 × 0.27 × 0.25 mm3. Independent reflections: 3367 (Rint = 0.0369). The final R1 values were 0.0408, wR2 = 0.0814 (I > 2σ(I)). CCDC number: CCDC 965578.

Crystal Data of 4: C16H16O6, Mr = 292.09, orthorhombic, a = 4.52790(10) Å, b = 15.4658(3) Å, c = 19.9377(3) Å, α = 90°, β = 90°, γ = 90°, V = 1396.19(5) Å3, space group P212121, Z = 4, Dx = 1.390 mg/m3, μ(Cu Kα) = 0.912 mm−1, and F(000) = 616. Crystal dimensions: 0.43 × 0.32 × 0.24 mm3. Independent reflections: 2233 (Rint = 0.0364). The final R1 values were 0.0255, wR2 = 0.0714 (I > 2σ(I)). Flack parameter = −0.02(16). CCDC number: CCDC 794211. MptpB Inhibition Assay. The target enzyme MptpB was prepared according to a modified literature procedure (see Supporting Information).23 The inhibition assays were performed using the RediPlate 96 EnzChek tyrosine phosphatase assay kit (Invitrogen) by monitoring the hydrolysis of the fluorogenic phosphatase substrate 6,8-difluoromethylumbelliferyl phosphate according to the manufacturer’s instruction. The IC50 value was determined at different substrate concentrations by nonlinear regression fitting of the inhibitor concentration versus inhibition rate. Sodium orthovanadate was used as positive control (IC50 = 82 nM). All measurements were done in triplicate from two independent experiments. The reported IC50 was the average value of two independent experiments.

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ASSOCIATED CONTENT

* Supporting Information S

Supplementary experimental section, biosynthetic pathways, NMR spectra, and CIF files for compounds (±)-1 and 2−4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Y. Lu) E-mail: [email protected]. Phone: +86-2084110778. Fax: +86-20-84110778. *(Z. She) E-mail: [email protected]. Phone: +86-2084113356. Fax: +86-20-84113356. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (41376149, 41276146), the 863 Foundation of China (2011AA09070201), the Science & Technology Plan Project of Guangdong Province of China (2010B030600004, 2011A080403006), the Guangzhou Project of Science & Technology Planning (2010J1-E331), the China Postdoctoral Science Foundation (2013M542224), and the China’s Marine Commonweal Research Project (2010050222). We also thank Dr. L. Jiang (Sun Yat-sen University) for F



Article

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helpful instruction in crystallography and Dr. H. Zhu (Kunming Institute of Botany, Chinese Academy of Sciences) for helpful instruction in ECD calculation.

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