Antibacterial Polyketide Heterodimers from - ACS Publications

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Antibacterial Polyketide Heterodimers from Pyrenacantha kaurabassana Tubers Leslie Boudesocque-Delaye,† Daniel Agostinho,† Charles Bodet,‡ Isabelle Thery-Kone,† Hassan Allouchi,† Alain Gueiffier,† Jean-Marc Nuzillard,§ and Cécile Enguehard-Gueiffier*,† †

Equipe Recherche et Innovation en Chimie Médicinale, UMR INRA 1282 Infectiologie et Santé Publique, Université de Tours François Rabelais, 37200 Tours, France ‡ Laboratoire Inflammation, Tissus Epithéliaux et Cytokines EA4331, Université de Poitiers, 86022 Poitiers, France § Institut de Chimie Moléculaire de Reims UMR CNRS 7312, Université Reims Champagne-Ardenne, 51100 Reims, France S Supporting Information *

ABSTRACT: Two heterodimers comprising anthraquinone and methylbenzoisocoumarin moieties (1 and 2) were isolated, together with emodin and physcion from the tubers of Pyrenacantha kaurabassana. The structures of 1 and 2 were established by NMR spectroscopy, including the analysis of a 2D INADEQUATE spectrum. On the basis of the data obtained, the structures that were previously proposed in the literature for these compounds were revised. Compounds 1 and 2 showed antibacterial activity against three different strains of Staphylococcus aureus. Compound 2 also showed bactericidal activity against Helicobacter pylori.

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Reported herein is a phytochemical study of the tubers of P. kaurabassana as a potential source of anti-infectious agents, with the structures determined of two polyketide heterodimers (1 and 2) based on spectroscopic data interpretation, along with an evaluation of their antibacterial activity against Staphylococcus aureus and Helicobacter pylori.

n southeast Africa, traditional medicine still represents the first option to treat many diseases for most of the population. Many plants are routinely used by traditional healers. For example, the flora of Mozambique includes about 5500 different species known as medicinal plants.1,2 Some species are endangered due to forest fires, domestic use (lumber, firewood), or intensive medicinal use. Several traditional healers have organized themselves into associations such as the “Associação dos Médicos Tradicionais de Moçambique” (AMETRAMO) to regulate access to medicinal plants, in collaboration with local authorities.3 The genus Pyrenacantha (Icacinaceae) is composed of about 30 different species growing mainly in tropical regions of Asia and Africa and in Madagascar.1 Only three of these species are found in southern Africa, namely, P. grandif lora Baill., P. scandens Planch, and P. kaurabassana Baill. In local traditional medicine, Pyrenacantha species are used widely to treat infectious diseases such as ulcers, diarrhea, herpes, and AIDS.2−4 Of these, P. kaurabassana is a dioecious liana found mainly in Zimbabwe, Tanzania, and Mozambique. Few constituents have been isolated from P. kaurabassana. Thus, only two xanthones with weak anti-HIV activity and a chrysene derivative have been described, by Omolo and co-workers.4 © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A literature survey showed that only two xanthones (1* and 2*, see structures) and a triterpenoid have been isolated from P. kaurabassana tubers by Omolo et al.4 From the entire Pyrenacantha genus, flavonoids, triterpenoids, and saponins have been isolated, mainly from the leaves of P. staudtii.5−8 In the present study, two heterodimers comprising an anthraquinone moiety linked to a 3-methylbenzodihydroisocoumarin unit were isolated (125 mg of compound 1 and 55 mg of compound 2) from P. kaurabassana tubers (2 g of CH2Cl2 extract), together with emodin (5 mg) and physcion (6 mg). Compound 1 gave the molecular formula C31H24O10 on the basis of the 13C NMR data and an HRESIMS ion at m/z Received: April 14, 2014

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DOI: 10.1021/np5003252 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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nm, suggesting the presence of a quinone functionality. The IR spectrum exhibited an absorption band of a hydrogen-bonded hydroxy group (νmax 3380 cm−1), a hydroxy group (νmax 2850, 2917 cm−1), and a carbonyl function (νmax 1652, 1615, and 1585 cm−1). The NMR data of compound 1 (Figures S3 to S14, Supporting Information) were obtained initially in CDCl3 and then in a mixture of acetone-d6 and DMSO-d6 (as summarized in Table 1) in order to improve solubility. The 1H NMR spectrum of compound 1 showed five aromatic proton signals at δH 7.52 (1H, s), 7.51 (1H, s), 7.12 (1H, s), 6.54 (1H, d, J = 2.4 Hz), and 6.19 (1H, d, J = 2.4 Hz). These two last signals were typical of meta-substituted aromatic protons. Surprisingly, meta coupling between protons at δH 7.52 and 7.12 was not observed at 950 MHz even though it was observed at 300 MHz. In CDCl3, at 300 MHz, these protons appeared as broad doublets with a coupling constant estimated at 1.9 Hz. The small value of this coupling constant showed that it could not be resolved when the concentration of the sample was high, like in the 950 MHz experiment. The same reason could explain that Omolo and co-workers also did not notice meta coupling for this same compound.4 This is a common practice in the field of natural products research: NMR samples are

555.1298 [M − H]− (calcd 555.1291). The UV spectrum showed maximum absorption bands at 224, 264, 308, and 372 Table 1. 1H and 13C NMR Data of Compound 1 position

δC, typea,b

1 3 4

170.9, C 75.6, CH 32.2, CH2

5 6 7 8 9 10 11 12 13 14 Me-3 OMe-9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ OH-1′ OMe-3′ Me-6′ OH-8′

133.1, 116.8, 139.0, 98.2, 162.3, 100.2, 158.9, 108.0, 162.6, 99.1, 19.9, 55.0, 161.2, 118.9, 164.2, 103.2, 120.7, 148.7, 124.1, 161.6, 190.6, 181.1, 132.6, 113.3, 134.8, 110.8,

C C C CH C CH C C C C CH3 CH3 C C C CH CH C CH C C C C C C C

56.4, CH3 21.3, CH3

δHa (mult., J (Hz))

COSY

4.69, m a: 2.75, dd (16.6, 3.3) b: 2.63, dd (16.6, 10.6)

4, Me-3

HMBCa,c 5 3, 5, 6, 14, Me-3

6.19, d (2.4)

10

6, 9, 10, 12

6.54, d (2.4)

8

8, 9, 11, 12

1.35, d (6.4) 3.66, s

3

3, 4 9

7.51, s 7.52, s

7′, Me-6′

1′, 2′, 3′, 10′, 13′, 14′, 6 6′, 7′ 8′, 9′, 10′, 12′, Me-6′

7.12, s

5′, Me-6′

5′, 8′, 9′, 11′, 12′, Me-6′

12.24, s 3.89, s 2.62, s 11.80, s

1′, 2′ 14′ 3′ 5′, 6′, 7′ 7′, 8′, 12′

INADEQUATEa 14 Me-3, 4 3, 5 4, 6, 14 5, 7, 2′ 6, 8, 12 7, 9 8, 10 9, 11 10, 12 7, 11, 13 12, 14 1, 5, 13 3 2′, 14′ 1′, 3′, 6 2′, 4′ 3′, 13′ 6′, 11′ 5′, Me-6′, 7′ 6′, 8′ 7′, 12′ 12′, 14′ 11′, 13′ 5′, 10′, 12′ 8′, 9′, 11′ 4′, 10′, 14′ 1′, 9′, 13′

6′

Run at 950 MHz for the 1H NMR spectrum and 239 MHz for the 13C NMR spectrum, in DMSO-d6−acetone-d6 (80:20, v/v). bThe number of attached protons was determined by analysis of the 2D NMR spectra. cHMBC correlations are from proton(s) to the indicated carbon.

a

B



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usually highly concentrated to reduce the recording time of 13C and 2D NMR spectra, even though it decreases 1H NMR resolution. Two aromatic methoxy signals in 1 were observed at δH 3.89 (3H, s) and 3.66 (3H, s), as well as two methyl signals at δH 2.62 (3H, s) and 1.35 (3H, d, J = 6.4 Hz). Two coupled methylene protons were identified at δH 2.75 (1H, dd, J = 16.6, 3.3 Hz) and 2.63 (1H, dd, J = 16.6, 10.6 Hz). In addition, two phenolic signals were evident at δH 12.24 (1H, s) and 11.80 (1H, s). The proton at 4.59 (1H, m) was attached to C-3 based on HSQC spectrum analysis. The 13C NMR spectrum displayed signals for 31 carbons, including three carbonyl carbons at δC 190.6, 181.1, and 170.9, 11 quaternary sp2 carbons at δC 148.7, 139.0, 134.8, 133.1, 132.6, 118.9, 116.8, 113.3, 110.8, 108.0, and 99.1, six oxygenated tertiary carbons at δC 164.2, 162.6, 162.3, 161.6, 161.2, and 158.9, five tertiary sp2 carbons at δC 124.1, 120.7, 103.2, 100.2, and 98.2, a secondary oxygenated carbon at δC 75.6, a secondary sp3 carbon at δC 32.2, and four primary sp3 carbons, two of which are linked to an oxygen atom at δC 56.4 and 55.0. The large number of aromatic carbons (20) was considered reminiscent of a polyketide-type structure. The partial structure of the lactone moiety of 1 was deduced from COSY correlations. A correlation between H-3 (δH 4.69) and Me-3 (δH 1.35) was observed together with correlations between H-3 and H-4a (δH 2.75) and between H-3 and H-4b (δH 2.63). The large coupling constant of H-4b (J = 10.3 Hz) suggests that this proton is trans relative to H-3. The presence of the lactone unit was supported by the chemical shift of C-1 (δC 170.9) in addition to the chemical shift of C-3 (δC 75.6), typical of an oxygenated secondary carbon, and the long-range HMBC correlation between C-1 and H-3. In the COSY spectrum, correlations between H-5′ and H-7′ and of both H5′and H-7′ with the protons of Me-6′ were observed. The benzylic-type couplings explained why the signals of H-5′ and H-7′ were broadened in comparison with H-8 and H-10 signals. All these analytical observations are related closely to those published by Omolo et al. for xanthones 1* and 2*.4 Comparison of the 13C NMR shifts in CDCl3 of compounds 1 and 1* (Table S2, Supporting Information) revealed a 0.05 ppm difference in the means, providing justification to conclude that compound 1* proposed by Omolo and co-workers4 is identical to compound 1. However, some differences were evident based on MS and NMR data: (1) HRESIMS data of compound 1 (Figure S1, Supporting Information) corresponded to a molecular formula with one oxygen atom less than xanthone 1*; (2) the COSY spectrum of 1 revealed a correlation between H-8 and H-10, whereas these protons are not located on the same ring and are separated by five bonds in the proposed xanthone structure 1*.4 In view of the large number of nonprotonated aromatic carbons, the structure of compound 1 could not be established using 2D 1H−13C NMR chemical shift correlations. It was thus not possible at this stage to confirm the xanthone structure of compound 1. Thus, a 2D INADEQUATE experiment at natural 13C abundance was performed on compound 1. Only 13 C−13C NMR chemical shift correlations could provide reliable structural data here since only poor 1H−13C correlation information was available.9−11 Following the analysis of 2D INADEQUATE data (Table 1 and Figures 1 and S10 to S14, Supporting Information), compound 1 appeared to be a heterodimer comprising two tricyclic moieties, an anthraquinone and a 3,4-dihydro-3-methylbenzoisocoumarin, and not a

Figure 1. Key INADEQUATE correlation of compound 1 (bonds in bold).

combination of tetracyclic and bicyclic moieties as in xanthones 1* and 2*.4 The linkage of the two constituent units was deduced from the correlation between C-6 (δC 116.8) and C-2′ (δC 118.9). Even though this junction was also suggested by the HMBC correlation between H-4′ (δH 7.51) and C-6 (δC 116.8), it could not be reliably identified without the 2D INADEQUATE spectrum, due to the lack of available HMBC correlations and the presence of the neighboring nonprotonated carbon atoms. Correlations of C-9′ (δC 190.6) with C-12′ and C-14′ and of C-10′ (δC 181) with C-11′ and C-13′ ruled out the presence of an aromatic carboxylic acid moiety in compound 1. The C-9′ and C-10′ centers appeared to be part of the quinone ring, confirming the anthraquinone structure. The meta-relationship of H-8 and H-10 was confirmed from the correlations of both C-8 and C-10 with C-9. The presence of methoxy groups at C-9 and C-3′ was deduced from the HMBC spectra. The positions of two hydroxy groups were established from the HMBC spectra through the correlations of C-1′ with the proton of OH-1′ and of C-8′ with the proton of OH-8′. Two other hydroxy groups were deduced from the chemical shifts of C-11 (δC 158.9) and C-13 (δC 162.6), consistent with aromatic carbons linked to an oxygen atom. In addition to the C-3 stereogenic center, compound 1 possesses a chirality axis (C-2′/C-6) due to its biaryl-type structure (atropoisomerism). The absolute configuration of C-3 in structures 1* and 2* was not assigned.4 The rotational barrier about the biphenyl axis was established in the 100 kcal· mol−1 range using Macromodel software (Schrödinger LLC, Portland, OR, USA), which precludes interconversion between atropoisomers. This was confirmed by the 1H NMR spectrum, which showed a single set of proton signals reminiscent of the presence of a single atropoisomer (one diastereomer only). In order to determine the absolute configurations of the biphenyl axis and of the C-3 center, the electronic circular dichroism (ECD) spectrum was recorded (see Figure S22, Supporting Information). Derivative 1 displayed a clear positive exciton coupling bisignate signature,12 i.e., a strong negative ECD band at 270 nm (Δε ≈ −100 M−1 cm−1) and a strong positive ECD band at 285 nm (Δε ≈ +80 M−1 cm−1), together with additional weak bands at 310, 362, and 405 nm (respectively negative, positive, and negative). Only two publications have provided ECD data of closely related compounds in the literature, a dimer of the hydroxyanthracenone, peroxisomicine A1,15 and a dimer of anthraquinone, alterporriol G.14 The experimental ECD spectra of peroximicine A1 and alterporriol G showed a negative exciton coupling bisignate signature in the 250−280 nm range, which is opposite the data found for compound 1 and 10−20 nm ipsochromically shifted. This shift can be explained by the more C



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π-conjugated nature of compound 1 rings. For both A1 and alterporriol G, the axial stereochemistry was assigned as aS and confirmed by theoretical calculations.14,15 Since the chromophores of peroximicine A1 are closely related to those of compound 1, the axial stereochemistry of compound 1 can be assigned, by direct comparison, to aS, which corresponds to the opposite spatial arrangement of the two chromophores but with inverted priority orders.

Table 2. NMR Data of Compound 2 position

δC, typea,b

1 3

172.1, C 76.2, CH

4

5 6 7 8 9 10 11 12 13 14 Me-3 OMe-9 OH-11 OH-13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ OH-1′ OMe-3′ OMe-5′ Me-6′

Since the ECD spectrum is dominated by the strong exciton coupling bisignate Cotton effect originating from the biaryltype scaffold of compound 1, the determination of the C-3 stereochemistry could not be reliably determined. Indeed, as was already described by Proksch and co-workers,14 the ECD spectrum is expected to be marginally affected by the stereogenic center configuration. Compound 2 gave a molecular formula of C32H26O11 based on its 13C NMR spectroscopy data and the HRESIMS ion at m/z 585.1405 [M − H]− (calcd 585.1500) (Figure S2, Supporting Information). The NMR data of compound 2 (Table 2 and Figures S15 to S21, Supporting Information) were closely related to those of compound 1, supporting the hypothesis of an anthraquinone and methylbenzodihydroisocoumarin heterodimeric-type structure. When compared to compound 1, the 13C NMR spectrum of compound 2 showed an additional quaternary carbon at δC 146.5 and a third methoxy substituent at δC 62.4. These findings, combined with the disappearance of one aromatic proton signal in the 1H NMR spectrum, suggested that C-5′ is substituted by a methoxy group in compound 2. This assumption was confirmed by correlation between C-5′ and the OMe-5′ protons in the HMBC spectrum. These findings were not compatible with the xanthone-like template 2*, 4 but were in good agreement with a heterodimeric-type structure of anthraquinone and 3-methylbenzo-3,4-dihydroisocoumarin moieties. Comparison between the 13C NMR shifts in CDCl3 for compounds 2 and 2*4 (Table S3, Supporting Information) revealed a 0.025 ppm difference in the means, which supported the assumption that the structure of compound 2* proposed by Omolo and co-workers is identical to our compound 2. In the lactone moiety, the multiplicity of the H-3 signal (δH 4.73) was well-defined as a dqd (J = 10.2, 6.0, 3.6 Hz). Thus, the structure of compound 2 was defined as the 5′-methoxy derivative of compound 1. The ECD spectrum of compound 2 (Figure S23, Supporting Information) is closely related to the ECD spectrum of compound 1, indicating that its axial chirality is also aS. This heterodimer template is close to the structures of talaroderxines and viriditoxin, two binaphthopyrones isolated from Talaromyces derxii16 and Aspergillus viridi-nutans,17 respectively, and of xanthoradones, dimers of dihydronaphto-

OH-8′

33.1, CH2

132.6, 117.5, 139.3, 99.1, 163.1, 100.6, 159.6, 108.5, 163.3, 99.3, 20.9, 55.5,

162.0, 119.2, 164.0, 102.4, 146.6, 132.0, 127.3, 154.4, 187.5, 181.7, 126.3, 123.3, 134.9, 112.3,

C C C CH C CH C C C C CH3 CH3

C C C CH C C CH C C C C C C C

56.7, CH3 62.5, CH3 16.5, CH3

δH (mult., J (Hz))a

COSY

4.73, dqd (10.2, 6.0, 3.6) a: 2.68, dd (16.2, 3.6) b: 2.63, dd (16.2, 10.8)

4, Me-3

4, Me-3

3

Me-3

6.17, d (2.3)

10

6, 9, 10, 12

6.58, d (2.3)

8

8, 9, 11, 12

1.45, d (6.3) 3.71, s 9.78, s 14.19, s

3

1, 3, 4 9 10, 11, 12 12, 13, 14

OH-13 OH-11

7.52, s

8.08, s

13.17, s 3.88, s 4.02, s 2.44, s 6.82, s

HMBCa,c

2′, 3′, 13′, 14′

Me-6′

7′

8′, 12′, Me-6′

1′, 2′, 14′ 3′ 5′ 5′, 6′, 7′, 8′, 12′ 5′, 6′, 8′

a

Run at 600 MHz for the 1H NMR spectrum and 150 MHz for the C NMR spectrum, in CDCl3. bThe number of attached protons was determined by analysis of the 2D NMR spectra. cHMBC correlations are from proton(s) to the indicated carbon. 13

pyranone and naphtoquinone moieties, isolated from the culture broth of Penicillium radicum13,18 (see structures). D



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UK) and were uncorrected. Optical rotations were determined in CHCl3 with a PerkinElmer 241 polarimeter. UV spectra were obtained with a Secoman UV spectrometer. ECD spectra were measured on a JASCO J810 spectropolarimeter using spectroscopic grade solvent (CHCl3) and a 10 mm quartz cell with a 100 nm/min scan rate and 1 nm slit. IR spectra were recorded on a Bruker Alpha-T FT-IR with ATR crystal (Bruker Biospin, Wissenbourg, France). NMR experiments were performed at 600 MHz (1H) and 151 MHz (13C) on a Bruker Avance 600 MHz spectrometer or on a Bruker Avance III spectrometer operating at 950 MHz, in CDCl3 or in acetone-d6− DMSO-d6 (20:80, v/v). The INADEQUATE spectra were recorded on a Bruker Avance III spectrometer operating at 238.89 MHz for 13C NMR (950.03 MHz on 1H). The standard Bruker pulse sequence inadphsp was used. The sequence was tuned for an optimal detection of 13C−13C pairs with a coupling constant of 60 Hz, thus favoring the signals from pairs of sp2 carbons. The refocusing pulse in the middle of the delay for double quantum (DQ) state creation was the Crp80comp.4 composite adiabatic pulse of 2 ms length, thus ensuring a minimum signal loss at the edges of the 13C spectral window by an offset effect. A 200 ppm wide acquisition window was used, with the recording of 16 384 complex data points, resulting in a 341 ms acquisition time, thus taking advantage of most of the available free precession signal. A 400 ppm wide spectral window was used in the indirect dimension. A high number of t1 increments, 1024 for each cosine and sine modulation, ensured a sufficient resolution in the DQ evolution frequency domain, so that no ambiguity in the assignment of the observed responses occurred. The data set, recorded in StatesTPPI quadrature detection mode in F1, was processed to obtain a 16 384 × 2048 data matrix of real values. A 4 s relaxation delay and 32 scans per t1 increment resulted in an overall 3 days and 7 h recording time. Samples were prepared by dissolving 100 μmol of compound 1 in DMSO-d6−acetone-d6 (80:20, v/v). Experiments were recorded at 40 °C to avoid sample precipitation. HRMS spectra were recorded on an ExactivePlus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), equipped with a heated electrospray probe (H-ESI II). The instrument was operated in the negative ionization mode using the MS full-scan mode over a mass range of 100−1000 Da. The system was controlled by Xcalibur 2.2 (Thermo Fisher Scientific). ESI and MS parameters were set as follows: spray voltage −4.0 kV, sheath gas and auxiliary nitrogen pressures 40 and 5 arbitrary units, respectively, capillary and heater temperatures 260 and 350 °C, respectively, tube lens voltage 100 V. The automatic gain control target value was set at 1 × 106 charges, and maximum injection time was set to 200 ms. HPLC analysis was performed on a Dionex UHPLC U3000RS system (ThermoFisher SA, Voisins le Bretonneux, France). The system was fitted with an Accucore aQ (15 cm × 3 mm i.d., 2.6 μm particle size) column, itself protected by an Accucore aQ defender guard 13 × 3.0 mm cartridge (ThermoFisher SA, Voisins le Bretonneux, France). The mobile phases were solvent A (0.025% TFA in H2O) and solvent B (CH3CN). The gradient was set as follows: initial CH3CN content was 0%, it was raised to 100% in 15.25 min and maintained for 2 min. TLC analysis of the fractions was performed on Merck 60 F254 silica gel plates. The mobile phase was a mixture of toluene−ethyl formate−formic acid (50:20:10, v/v/v). After elution, the plates were visualized with H2SO4−anisaldehyde. Flash chromatography was performed on Flashsmart One Chain (AIT, Houilles, France) using an Interchim PF-30-SiHP silica gel column. Plant Material. Tubers of Pyrenacantha kaurabassana were collected in March 2010 in collaboration with traditional healers from the Gaza district (Mozambique) and identified taxonomically by botanist Dr. Silva Mulhovo of the Agriculture Research Institute of Mozambique, Maputo. A voucher specimen (number 4157) is deposited in the herbarium of the botanical sector of the same institution. Extraction and Isolation. Dried powdered tubers (452 g) of P. kaurabassana were extracted in a Soxhlet apparatus using cyclohexane (1.2 L), CH2Cl2 (1 L), and MeOH (1 L) successively. The extracts were concentrated to dryness under reduced pressure to yield 1.894 g of a cyclohexane extract, 2.336 g of a CH2Cl2 extract, and 17.029 g of a MeOH extract. The CH2Cl2 extract (2.0 g) was subjected to flash

However, no heterodimers comprising anthraquinone and 3methylbenzodihydroisocoumarin moieties were reported previously. Talaroderxines and viriditoxin have bactericidal activity against Bacillus subtillis,16 and xanthoradones are potentiators of imipenem activity against Staphylococcus aureus.16 Moreover, considering the extensive use of Pyrenacantha species in traditional medicine to cure infectious diseases, the crude extract of this plant and compounds 1 and 2 were tested for antibacterial activity against various bacterial strains. Table 3 illustrates that compounds 1 and 2 exhibited antibacterial activities against Staphylococcus aureus and Helicobacter pylori. Table 3. Antibacterial Activities of Compounds 1 and 2a S. aureus compound kanamycin amoxicillin

H. pylori

MICb (μM)

MICc (μM)

MICd (μM)

8.3

8.3

8.3

1

89.9

89.9

89.9

2

10.7

10.7

2.7

log10 (CFU/mL) of killinge concentration (μM) 0.51 ± 0.09 274 0.42 ± 0.13 179.8 6±0 170.6

a

Minimum inhibitory concentrations (MIC) of compounds and kanamycin against three strains of S. aureus. bATCC 29213. cA clinical strain isolated from tibial osteomyelitis. dA clinical strain isolated from a leg ulcer. eCompound and amoxicillin bactericidal activity for a starting inoculum of 106 CFU/mL of H. pylori J99 after 2 h incubation expressed as log10 (CFU/mL) of kill relative to the initial inoculum.

The crude dichloromethane P. kaurabassana tuber extract as well as compounds 1 and 2 gave MIC values against S. aureus ATCC 29213 of 50 μg·mL−1 and 50 μg·mL−1 (89.9 μM) and 6.25 μg·mL−1 (10.7 μM), respectively. Interestingly, although the chemical structures of compounds 1 and 2 were closely related, compound 2 was more effective against S. aureus. For the three strains of S. aureus tested, the MIC values for compounds 1 and 2 were 90 and 10.7 μM, respectively. The low MIC values exhibited by compound 2 suggest that this compound has potential for prophylactic or therapeutic treatment in S. aureus infections. Compound 2 also had bactericidal activity against H. pylori, reducing the viable counts by 6 log10 CFU/mL (>99.9% kill relative to the initial inoculum) after 2 h of incubation at a concentration of 170 μM. A smaller reduction in the viability of the initial bacterial inoculum was observed under the same conditions with compound 1. These findings validated the traditional use of P. kaurabassana tubers, to cure stomach ulcers. These results showed that the newly isolated compounds (1 and 2) from P. kaurabassana possess promising antibacterial properties against S. aureus and/or H. pylori, and they may possess potential for the development of new therapeutic agents for treatment of infections caused by these major pathogens.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a capillary apparatus (Stuart, Stone, Staffordshire, E



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chromatography using silica gel as stationary phase and a solvent gradient of heptane−CH2Cl2 and CH2Cl2−acetone successively. The heptane−CH2Cl2 (30:70, v/v) and heptane−CH2Cl2 (1:99, v/v) fractions afforded two orange powders, namely, physcion (6.0 mg) and compound 1 (125 mg), respectively. The pure CH2Cl2 and CH2Cl2− acetone (98:2, v/v) fractions afforded compound 2 (55 mg) and emodin (5.0 mg), respectively. 6-(9′,10′-Dihydro-1′,8′-dihydroxy-3′-methoxy-6′-methyl-9′,10′dioxoanthryl)-2′-yl-11,13-dihydroxy-9-methoxy-3-methylbenzo[1,2-g]isochroman-1-one (1): orange, microcrystalline powder, mp 288−292 °C; [α]25D −73 (c 0.1, CHCl3); UV (CH3CN) λmax (log ε) 224 (4.27), 264 (4.58), 308 (3.88), 372 (3.87) nm; ECD (c 2.2 × 10−5 mol/L, CHCl3) mol. ellip. −100 (270 nm), 0 (280 nm), +80 (285 nm), −20 (310 nm), +10 (362 nm), 0 (385 nm), −10 (405 nm); IR νmax 3380 (broad signal), 2917 and 2850, 1652, 1615, 1585 cm−1; 1H NMR [(CDCl3, 600 MHz); (DMSO-d6−acetone-d6 (80:20, v/v), 950 MHz)] and 13C NMR [(CDCl3, 151 MHz); (DMSO-d6−acetone-d6 (80:20, v/v), 249 MHz)], see Table 1; HRESIMS m/z 555.1298 [M − H]− (calcd for C31H23O10, 555.1291). 6-(9′,10′-Dihydro-1′,8′-dihydroxy-3′,5′-dimethoxy-6′-methyl9′,10′-dioxoanthryl)-2′-yl-11,13-dihydroxy-9-methoxy-3methylbenzo[1,2-g]isochroman-1-one (2): orange, microcrystalline powder, mp 224−225 °C; [α]25D −50 (c 0.1, CHCl3); UV (CH3CN) λmax (log ε) 228 (4.42), 266 (4.51), 286 (4.51), 381 (4.07) nm; ECD (c 1.2 × 10−5 mol/L, CHCl3) mol. ellip. −25 000 (265 nm), 0 (275 nm), +20 000 (287 nm), 0 (325 nm), −5000 (325 nm), 0 (345 nm), +7000 (362 nm), 0 (380 nm), −10 000 (405 nm); IR νmax 3392 (broad signal), 2917 and 2849, 1624, 1585, 1567 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 151 MHz) see Table 2; HRESIMS m/z 585.1405 [M − H]− (calcd for C32H25O11, 585.1500). 1,3,8-Trihydroxy-6-methylanthracene-9,10-dione (emodin): orange needles (CH2Cl2); physical data are in accordance with the literature.15 1,8-Dihydroxy-3-methoxy-3-methylanthracene-9,10-dione (physcion): orange needles (CH2Cl2); physical data are in accordance with the literature.15 Bacterial Culture. Three strains of Staphylococcus aureus were used: the reference strain ATCC 29213 as well as two clinical strains isolated from patients with tibial osteomyelitis and a leg ulcer at the Poitiers University Hospital. S. aureus were grown routinely on Muller Hinton agar plates (Oxoid, Dardilly, France) and incubated for 24 h at 37 °C. The Helicobacter pylori J99 strain was cultured routinely on Skirrow’s medium (Oxoid) and incubated at 37 °C in microaerobic conditions using CampyGen bags (Oxoid). Activity of Compounds against Staphylococcus aureus. MICs for compounds were determined by broth microdilution at final concentrations of 0.39 to 100 μg/mL. The adjusted bacterial inocula (50 μL/well, 2 × 105 CFU/mL) were added to each well of a sterile Ubased microtiter plate containing various concentrations of compounds (50 μL/well). Each condition was tested in triplicate. Consequently, the last inoculum concentration of 105 CFU/mL was obtained in each well, and this plate was incubated for 24 h at 37 °C. The lowest concentration of compounds that inhibited the visible bacterial growth was determined as the MIC value against each S. aureus strain. Each experiment was repeated in triplicate. Bactericidal Activity of Compounds against Helicobacter pylori. For bactericidal assays, suspensions of H. pylori J99 at 2 × 106 cells per mL were prepared in brain heart infusion (Oxoid) using 24 h bacterial cultures. Bacterial concentrations were determined by measuring the optical density of the culture at 600 nm. In addition, colony-forming unit (CFU) counts of H. pylori were performed. Compounds 1 and 2 were tested against H. pylori J99 (106 CFU/mL) at a final concentration of 100 μg/mL by thoroughly mixing 1 mL of a compound solution (200 μg/mL) and 1 mL of the bacterial suspension in a 5 mL round-bottomed plastic tube. The tubes were incubated for 2 h at 37 °C in a water bath. Bacterial cells mixed with BHI alone or containing amoxicillin at a final concentration of 10 μg/ mL were used as controls. Samples (100 μL) were removed and immediately diluted 10-fold in warm PBS for the determination of viable counts (numbers of CFU per milliliter). Volumes (20 μL) from

each dilution were spread in triplicate onto Skirrow’s medium. The inoculated plates were incubated at 37 °C under microaerophilic conditions for 72 h before the colonies were counted. Compound bactericidal activity was expressed as log10 CFU per milliliter (0 h) − log10 CFU per milliliter (2 h). Each experiment was repeated four times.

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ASSOCIATED CONTENT

* Supporting Information S

Full NMR and MS data and spectra of compounds 1 and 2 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +33 02 47 36 71 72. Fax: +33 02 47 36 71 44. E-mail: cecile.enguehard-gueiffi[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the TGIR-RMN-THC FR 3050 CNRS is gratefully acknowledged. D.A. thanks Egide for financial support. Financial support by CNRS, Conseil Regional Champagne Ardenne, Conseil General de la Marne, Ministry of Higher Education and Research (MESR), and EUProgramme FEDER to the PlAneT CPER project is gratefully acknowledged. The authors aknowledge Dr. J. Crassous (UMR CNRS 6226) for fruitful discussions about ECD data. The authors acknowledge F. Montigny from the PPF platform and D. Harakat, from UMR CNRS 7312, for MS analysis, and C. Sayagh and A. Martinez from UMR CNRS 7312 for polarimetry and ECD. The authors thank N. Bellin, L. Fréon, and C. Sérot for technical support.

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REFERENCES

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