Lovastatin Analogues from the Soil-Derived ... - ACS Publications

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Lovastatin Analogues from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178 Patima Phainuphong,† Vatcharin Rukachaisirikul,*,† Saowanit Saithong,† Souwalak Phongpaichit,‡ Kawitsara Bowornwiriyapan,‡ Chatchai Muanprasat,§ Chutima Srimaroeng,⊥ Acharaporn Duangjai,∥ and Jariya Sakayaroj△ †

Department of Chemistry and Center of Excellence for Innovation in Chemistry and ‡Natural Products Research Center of Excellence and Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand § Department of Physiology, Faculty of Science, Mahidol University, Rajathevi, Bangkok 10400, Thailand ⊥ Department of Physiology, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai 50200, Thailand ∥ Division of Physiology, School of Medical Sciences, University of Phayao, Muang, Phayao 56000, Thailand △ National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang, Pathumthani 12120, Thailand S Supporting Information *

ABSTRACT: Three new lovastatin analogues (1, 4, and 5) together with four known lovastatin derivatives, namely, lovastatin (2), α,β-dehydrolovastatin (3), α,β-dehydrodihydromonacolin K (6), and α,β-dehydro-4a,5-dihydromonacolin L (7), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Their structures were established using spectroscopic evidence. Compound 5 exhibited the most potent activity against HMG-CoA reductase, with an IC50 value of 387 μM. In addition, the present study indicated the direct interaction of compound 5 with HMG-CoA reductase. Compound 5 was considered to be noncytotoxic against noncancerous Vero cells, with an IC50 value of 40.0 μM, whereas compound 2 displayed much stronger activity, with an IC50 value of 2.2 μM.

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Princess Maha Chakri Sirindhorn at Ratchaprapa Dam in Suratthani Province, Thailand. All crude extracts from A. sclerotiorum PSU-RSPG178, including the broth and mycelial ethyl acetate extracts, were antimalarial (Plasmodium falciparum, K1 strain) and cytotoxic (KB oral cavity, MCF-7 breast cancer cells, and Vero-African green monkey kidney fibroblast cells), with IC50 values of 2.39, 6.70, 11.30, and 5.37 μg/mL for the broth ethyl acetate extract and 3.17, 12.05, 41.19, and 14.73 μg/ mL for the mycelial ethyl acetate extract, respectively. In addition, the broth ethyl acetate extract exhibited antimycobacterial (Mycobacterium tuberculosis, H37Ra strain) activity with an MIC value of 12.50 μg/mL. A chemical investigation of these crude extracts led to the isolation of one new lovastatin derivative (1) and two known compounds, lovastatin (2)10 and α,β-dehydrolovastatin (3),11 from the broth ethyl acetate extract, along with two new lovastatin derivatives (4 and 5),

icroorganisms have been recognized as an abundant source of new bioactive products with applications for the pharmaceutical and agricultural industries.1 More than 30,000 natural compounds have been isolated from microorganisms since the 1940s, over 10,000 of which are biologically active.2 Lovastatin, a fungal secondary metabolite and a competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase (HMGR), is a cholesterol-lowering agent in humans and is cytotoxic against breast (MCF-7), cervical (HeLa), liver (HepG2), and skin (melanoma) cell lines.3−5 Lovastatin analogues such as mevastatin, pravastatin, and simvastatin exhibit antihepatitis C virus,6 antioxidant,7 and anti-inflammatory8 activities, respectively. Several fungal species have been reported to produce lovastatin including Aspergillus terreus, Penicillium citrinum, Pleurotus spp., and Monascus ruber.9 In our ongoing search for new bioactive metabolites from soil fungi, we investigated secondary metabolites produced by the soil fungus Aspergillus sclerotiorum PSU-RSPG178 isolated from a soil sample collected from the Plant Genetic Conservation Project under the Royal Initiation of Her Royal Highness © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 29, 2015

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DOI: 10.1021/acs.jnatprod.5b00961 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of compounds 1−7 isolated from Aspergillus sclerotiorum PSU-RSPG178.

Figure 2. ORTEP drawings of compounds 2 and 6.

together with two known compounds, α,β-dehydrodihydromonacolin K (6)12 and α,β-dehydro-4a,5-dihydromonacolin L (7)11 from the mycelial ethyl acetate extract. The isolated compounds 2, 3, 5, and 6 were evaluated for antimycobacterial (M. tuberculosis, H37Ra strain), antimalarial (P. falciparum, K1 strain), and cytotoxic (KB, MCF-7, and Vero cells) activity. Because of the cholesterol-lowering activities of lovastatin derivatives, these compounds were evaluated for HMG-CoA reductase activity.

olefinic protons of a cis-disubstituted alkene [δH 5.60 (ddd, J = 9.5, 5.0, and 2.5 Hz, 1H)/5.31 (d, J = 9.5 Hz, 1H), two oxymethylene protons (δH 3.66, d, J = 7.5 Hz, 2H), five methine protons [δH 2.24, 1.95, 1.80, 1.51, and 1.07, each m, 1H)], five sets of nonequivalent methylene protons [δH 2.44 (ddd, J = 15.5, 10.0, and 5.0 Hz, 1H)/2.24 (ddd, J = 15.5, 9.5, and 6.5 Hz, 1H), 1.92 (m, 1H)/1.39 (m, 1H), 1.85 (m, 1H)/ 1.51 (m, 1H), 1.75 (m, 1H)/1.25 (m, 1H), and 1.67 (m, 1H)/ 1.02 (m, 1H)], and one methyl group (δH 0.85, d, J = 7.0 Hz, 3H). The 13C NMR spectrum (Table 1) displayed signals for one carbonyl carbon of a carboxylic acid (δC 177.5), seven methine carbons (δC 132.7, 131.0, 41.0, 39.7, 38.1, 36.0, and 31.9), one oxymethylene carbon (δC 64.2), five methylene carbons (δC 33.8, 31.5, 27.3, 24.4, and 23.8), and one methyl carbon (δC 14.9). These data were similar to those of 7.11 The obvious differences were the replacement of carbon signals for the δ-lactone unit and 6-Me group in 7 with signals for the carboxylic carbonyl carbon (δC 177.5, C-11) and a hydroxymethyl unit (δH 3.66, d, J = 7.5 Hz and δC 64.2, C-1′) in 1, respectively. This assignment was confirmed by HMBC correlations of Hab-9 (δH 1.92 and 1.39) with C-11 and the correlations of H2-1′ (δH 3.66) with C-5 (δC 33.8), C-6 (δC 36.0), and C-7 (δC 27.3) (Table 1). The hydronaphthalene moiety of 1 displayed similar NOEDIFF data (Figure 3) to those of 7, indicating their identical relative configuration. The hydroxymethyl unit was located at the axial position because irradiation of H2-1′ enhanced signal intensity of the axial H-4a (δH 1.80). The observed optical rotation of 1, [α]21 D = +119.0 (c 0.10, CHCl3), was similar to that of (1S,2S,4aR,6R,8aR)-

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RESULTS AND DISCUSSION All lovastatin analogues (1−7) (Figure 1) were purified using chromatographic techniques, and their structures were elucidated by using various spectroscopic techniques. The relative configuration was assigned according to the NOEDIFF data and was confirmed by the X-ray data for 23 and 6 (Figure 2). The X-ray data of 6 are reported for the first time. The absolute configuration of the isolated compounds was established by comparison of the optical rotations and CD data with those of known or structurally related compounds. In addition, Mosher’s method13,14 was employed for the assignment of the absolute configuration of the secondary alcohol in 5. Compound 1 was obtained as a colorless gum with the molecular formula C15H24O3 determined by the HRESIMS peak at m/z 275.1623 [M + Na]+. The IR spectrum showed characteristic absorption bands of a hydroxy group at 3394 cm−1 and a carbonyl group at 1717 cm−1. The 1H NMR spectroscopic data (Table 1) consisted of signals for two B



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Table 1. 1H and 13C NMR Spectroscopic Data of 1, 4, and 5 [CDCl3, δ (ppm)] 1 δC,a type

δH,b mult. (J, Hz)

1

41.0, CH

1.51, m

2

31.9, CH

2.24, m

position

2-Me 3

14.9, CH3 132.7, CH

4

131.0, CH

0.85, d (7.0) 5.60, ddd (9.5, 5.0, 2.5) 5.31, d (9.5)

4 HMBC

δC,a type

4a, 8, 8a, 9, 2-Me 3, 4, 8a, 2-Me 1, 2, 3 1, 2, 4a

42.1, CH 32.7, CH 15.3, CH3 134.3, CH

5 δH,d mult. (J, Hz)

HMBC

δC,c type

2.57, dt (9.3, 5.0) 2.73, m

2, 3, 4, 4a, 8a, 9, 2-Me 1, 3

41.8, CH

1.75, m

8a, 10

35.5, CH

1.98, m

3, 8a

1.08, d (7.4) 5.73, dd (9.5, 3.3) 6.36, dd (9.5, 2.5)

1, 2, 3 1, 2, 4a, 2-Me

11.8, CH3 69.5, CH

0.81, d (7.5) 3.90, t (4.0)

2, 3 4, 4a

2, 4a, 5, 6, 8, 8a

127.4, CH

5.94, dd (5.0, 1.5)

2, 3, 5, 8a

6.87, d (1.5)

4, 6-Me

140.1, C 77.4, CH

4.00, d (2.5)

4, 6, 7, 8a, 6-Me

2.32, s 6.95, dd (7.8, 1.5)

5, 7, 8 5, 8, 8a, 6-Me

34.3, CH 19.1, CH3 30.7, CH2

2.06, m 1.01, d (7.5) α: 1.69, m

5 5, 6, 7 5, 6, 8a 5, 6, 6-Me 4a, 6, 7, 8a, 1′

4a 5

38.1, CH 33.8, CH2

1.80, m a: 1.75, m

2, 4a, 8a, 2-Me 5, 6 1, 4, 4a, 6, 1′

6 6-Me 7

36.0, CH

b: 1.25, m 1.95, m

4a, 6, 8a, 1′ 4a, 5, 7, 8, 1′

27.3, CH2

α: 1.85, m

5, 6, 8, 8a

136.1, C 21.0, CH3 127.1, CH

8

24.4, CH2

β: 1.51, m α: 1.02, m

5, 8, 8a, 1′ 4a, 6, 7, 8a

127.5, CH

6.96, d (7.8)

1, 4a, 6

71.0, CH

β: 2.22, m 5.33, q (3.0)

8a 9

39.7, CH 23.8, CH2

β: 1.67, m 1.07, m a: 1.92, m

4a, 6, 7, 8a 1, 2, 5, 8 1, 2, 8a, 10, 11 1, 2, 8a, 10, 11 1, 9, 11

135.6, C 22.4, CH2

a: 1.82, m

1, 10, 11

38.0, CH 24.6, CH2

2.39, m 1.58, m

b: 1.60, m

1, 10, 11

a: 1.75, m

1, 9, 11, 12

33.0, CH2

a: 1.87, m

b: 1.50, m

1, 9, 11, 12

4.30, m 2.25, m

9 10, 11, 13, 14, 15

6.83, ddd (9.8, 5.3, 2.2) 5.99, td (9.8, 1.8)

11, 12, 15

144.9, CH

12, 15

121.5, CH

b: 1.39, m 10

11 12

31.5, CH2

a: 2.44, ddd (15.5, 10.0, 5.0) b: 2.24, ddd (15.5, 9.5, 6.5)

126.9, CH

δH,b mult. (J, Hz)

133.3, C 126.8, CH

33.1, CH2

1, 9, 11

177.5, C

78.6, CH 29.6, CH2

13

144.9, CH

14

121.5, CH

15 1′ 2′ 2′-Me 3′

164.5, C 64.2, CH2

3.66, d (7.5)

78.4, CH 29.6, CH2

C C CH CH3 CH2

4.38, m a: 2.39, m b: 2.29, m 6.87, ddd (9.5, 6.0, 2.5) 6.02, ddd (9.5, 2.5, 1.0)

2.30, m 1.12, d (7.0) a: 1.66, m b: 1.46, m

4′ a

11.8, CH3 b

c

11

b: 1.46, m

164.3, 176.0, 41.8, 16.7, 26.7,

5, 6, 7

HMBC

0.88, t (7.5)

13

11, 12, 15 12, 15

1′ 1′, 2′, 3′ 1′, 2′, 4′, 2′-Me 1′, 2′, 4′, 2′-Me 2′, 3′

d

Recorded at 125 MHz. Recorded at 500 MHz. Recorded at 75 MHz. Recorded at 300 MHz.

heptaketide,11 [α]20 D = +120.3 (c 0.10, CHCl3), indicating that they would have the same absolute configuration. Consequently, 1 was assigned as a new lovastatin derivative lacking the δ-lactone unit. Compound 4 was obtained as a colorless gum with the molecular formula C19H22O2 deduced from HREIMS, indicating the presence of 9 degrees of unsaturation. The UV spectrum showed absorption bands at 214 and 265 nm for conjugated carbonyl and aromatic chromophores.12,15 The IR spectrum showed an absorption band at 1718 cm−1 for an α,βunsaturated carbonyl lactone. The 1H NMR spectroscopic data (Table 1) consisted of signals for three aromatic protons of a 1,2,4-trisubstituted benzene [δH 6.96 (d, J = 7.8 Hz, 1H), 6.95

(dd, J = 7.8 and 1.5 Hz, 1H), and 6.87 (d, J = 1.5 Hz, 1H)], two sets of cis-disubstituted alkenes [δH 6.83 (ddd, J = 9.8, 5.3, and 2.2 Hz, 1H)/5.99 (td, J = 9.8 and 1.8 Hz, 1H) and 6.36 (dd, J = 9.5 and 2.5 Hz, 1H)/5.73 (dd, J = 9.5 and 3.3 Hz, 1H)], one oxymethine proton (δH 4.30, m, 1H), two methine protons [δH 2.57 (dt, J = 9.3 and 5.0 Hz, 1H) and 2.73 (m, 1H)], three sets of methylene protons [δH 2.25 (m, 2H), 1.82 (m, 1H)/1.60 (m, 1H), and 1.75 (m, 1H)/1.50 (m, 1H)], and two methyl groups [δH 2.32 (s, 3H) and 1.08 (d, J = 7.4 Hz, 3H)]. The 13C NMR spectrum (Table 1) displayed signals for one carbonyl carbon of the α,β-unsaturated lactone (δC 164.5), three quaternary carbons (δC 136.1, 135.6, and 133.3), one oxymethine carbon (δC 78.6), nine methine carbons (δC 144.9, 134.3, 127.5, 127.1, C



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Figure 3. Key NOEDIFF data of compounds 1 and 5.

Compound 5 was obtained as a colorless gum with the molecular formula C24H36O6 determined from the HRESIMS peak at m/z 443.2410 [M + Na]+. The UV spectrum showed an absorption band at 213 nm for a conjugated carbonyl chromophore.12 The IR spectrum displayed absorption bands at 3421 cm−1 for a hydroxy group, 1736 cm−1 for an ester carbonyl functional group, and 1716 cm−1 for a lactone carbonyl group. Comparison of the 1H and 13C NMR spectroscopic data of 5 (Table 1) with those of 612 indicated that they possessed identical substituents at C-1 (δC 41.8) and C-8 (δC 71.0), which are a 6-ethyl-5,6-dihydro-2H-pyran-2-one unit [δH 6.87 (ddd, J = 9.5, 6.0, and 2.5 Hz, 1H), 6.02 (ddd, J = 9.5, 2.5, and 1.0 Hz, 1H), 4.38 (m, 1H), 2.39 (m, 1H), 2.29 (m, 1H), 1.87 (m, 1H), 1.58 (m, 2H), and 1.46 (m, 1H)] and a 2methyl-1-butanoyl unit [δH 2.30 (m, 1H), 1.66 (m, 1H), 1.46 (m, 1H), 1.12 (d, J = 7.0 Hz, 3H), and 0.88 (t, J = 7.5 Hz, 3H)], respectively. The differences in their 1H NMR spectroscopic data were the presence of signals for one olefinic proton of a trisubstituted alkene (δH 5.94, dd, J = 5.0 and 1.5 Hz, 1H) and two oxymethine protons [δH 4.00 (d, J = 2.5 Hz, 1H) and δH 3.90 (t, J = 4.0 Hz, 1H)] in 5 instead of signals for the cis-disubstituted alkene (H-3 and H-4), the methine proton (H-4a), and the methylene protons (Hab-5) in 6. The oxymethine proton at δH 3.90 ppm was assigned as H-3 on the basis of its HMBC correlations with C-4 (δC 127.4) and C4a (δC 140.1) as well as its 1H−1H COSY correlation with H-2 (δH 1.98). Moreover, H-3 showed a 1H−1H COSY correlation with the olefinic proton at δH 5.94, which was consequently assigned as H-4. This assignment was confirmed by HMBC correlations of H-4 with C-2 (δC 35.5), C-3 (δC 69.5), C-5 (δC 77.4), and C-8a (δC 38.0) (Table 2). The other oxymethine proton at δH 4.00 ppm was then assigned as H-5 due to its 1 H−1H COSY correlations with H-4 and H-6 (δH 2.06) as well as its HMBC correlations with C-4, C-6 (δC 34.3), 6-Me (δC 19.1), C-7 (δC 30.7), and C-8a. The relative configuration was determined by NOEDIFF data (Figure 3) as well as coupling constants. In the NOEDIFF experiment, irradiation of H-8a enhanced signal intensities of H-8, H2-9, and 2-Me, supporting the location of H-8a and 2-Me at the pseudoaxial position and H-8 and H2-9 at the pseudoequatorial position. The signal intensity of H-3 was affected by irradiation of the pseudoaxial 2Me, indicating the location of H-3 at the pseudoequatorial

126.9, 126.8, 121.5, 42.1, and 32.7), three methylene carbons (δC 33.1, 29.6, and 22.4), and two methyl carbons (δC 21.0 and 15.3). These data resembled those of 7. However, the signals of three methine protons (H-4a, H-6, and H-8a) and three sets of nonequivalent methylene protons (Hab-5, Hab-7, and Hab-8) in the hydronaphthalene ring of 7 were replaced with signals of three aromatic protons of a 1,2,4-trisubstituted benzene (δH 6.96, 6.95, and 6.87) in 4. These aromatic protons were assigned as H-8, H-7, and H-5, respectively, on the basis of their multiplicity and coupling constants together with HMBC correlations from H-5 to C-4 (δC 126.9) and 6-Me (δC 21.0) and from H-8 to C-1 (δC 42.1), C-4a (δC 133.3), and C-6 (δC 136.1) (Table 2). In an NOEDIFF experiment, irradiation of 2Table 2. Antimycobacterial and Cytotoxic Activities for Compounds 2, 3, 5, 5OAc, and 6 antimycobacterial (MIC, μg/mL)

cytotoxic (IC50, μM)

compound

M. tuberculosis H37Ra strain

KB

MCF-7

Vero

2 3 5 5OAc 6 control

INa 12.50 INa INa INa 0.0063b 0.6250c 0.0469d 0.3910e 0.4690f

15.6 20.1 31.6 19.8 15.2 2.03g 1.19h

INa 57.3 INa 74.7 INa 6.75h 6.99i

2.2 8.1 40.0 8.4 14.5 1.00g

a f

IN = inactive. bRifampicin. cStreptomycin. dIsiniazid. eOfloxacin. Ethambutol. gEllipticine. hDoxorubicin. iTamoxifen.

Me (δH 1.08) enhanced signal intensity of Ha-9 (δH 1.82), indicating the cis-relationship of the 2-Me and the substituent at C-1. The absolute configuration at C-11 was assigned to be R, identical to that of 7, according to negative Cotton effects (Δε = −4.1 and −1.8, at 208 and 259 nm, respectively), similar to those of 2,3-dehydrosolistatinol (Δε = −6.0 and −2.0, at 207 and 258 nm, respectively).16 Since 1, 4, and 7 were cometabolites, the absolute configurations of both C-1 and C-2 were proposed to be S. Therefore, 4 was assigned as a dehydro derivative of 7. D



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position, which was consistent with a small coupling constant of 4.0 Hz between H-2 and H-3. Accordingly, 3-OH was then located at the pseudoaxial position. In addition, H-5 was assigned as pseudoequatorial on the basis of signal enhancement of H-5 and H-2′ upon irradiation of pseudoaxial 6-Me. The absolute configuration at C-3 in 5 was established to be S by Mosher’s method (Figure 4).13,14 Unfortunately, the

Figure 4. Δδ (=δS − δR) values for (S)- and (R)-MTPA esters of compound 5.

absolute configuration at C-5 could not be determined by Mosher’s method because there was no proton at C-4a.17 Consequently, the remaining absolute configurations were then assigned as 1S, 2R, 5S, 6R, 8S, and 8aR, of which the absolute configurations at C-1 and C-8 were identical to those in compounds 2, 3, and 6. The CD spectrum of 5 showed negative Cotton effects at 205 and 258 nm, indicating the absolute configuration at C-11 to be R. The absolute configuration at C-2′ was proposed to be identical to that of compounds 2, 3, and 6, as they were co-metabolites. Compounds 2, 3, 5, 6, and 5OAc (the diacetylated derivative of 5) were tested for antimycobacterial (Mycobacterium tuberculosis, H37Ra strain), antimalarial (P. falciparum, K1 strain), and cytotoxic (KB, MCF-7, and Vero cell lines) activities (Table 2). None of them displayed antimalarial activity, whereas only 3 exhibited mild antimycobacterial activity, with the MIC value of 12.50 μg/mL. For cytotoxic activity, all of the tested compounds were inactive against KB and MCF-7 cell lines, with IC50 values greater than 10 μM. In addition, compounds 2, 3, and 5OAc showed cytotoxic actvity toward Vero cells with the IC50 values in the range 2.2−8.4 μM. All of the isolated compounds showed no antimalarial activity and weaker cytotoxic activity when compared with those of the crude extracts. Further chemical investigation of the crude extracts is being performed in order to isolate compounds that are responsible for these activities. The effect of compounds 2, 3, 5, 6, and 5OAc on HMGR activity was evaluated using the HMG-CoA reductase assay kit with slight modification. Interestingly, lovastatin (2) and 5 (200 μM) significantly inhibited HMGR activity by ∼42% and 50%, respectively (Figure 5A). The remaining compounds (at 200 μM) were weakly active, with percent inhibition of 8.4%, 17.2%, and 20.4%, respectively. These results indicated that the potency of 5 is slightly higher than that of 2 but much lower than pravastatin, a positive control, which inhibited HMGR activity by ∼90.4% at 1 μM. According to these results, 2 and 5 were further evaluated for their IC50 values in comparison with pravastatin, a positive control. Unfortunately, only the IC50 value of 5 was obtained because 2 precipitated at concen-

Figure 5. Inhibitory effect of pravastatin and lovastatin derivatives from Aspergillus sclerotiorum PSU-RSPG178 on HMG-CoA reductase activity in a cell-free-based assay. (A) HMGR activity was determined in the absence (control) or presence of either pravastatin (1 μM) or lovastatin derivatives including compounds 2, 3, 5, 5OAc, and 6 (200 μM). Data are expressed as percent inhibition. *, p < 0.05 compared with control (n = 3−5). (B) IC50 value of compound 5 against HMGCoA reductase activity (n = 3).

trations higher than 400 μM. As shown in Figure 5B, the IC50 value of 5 was 387 ± 70 μM, while the IC50 value of pravastatin was 0.4 ± 0.1 μM (data not shown). This potency difference is in agreement with results from the previous study, showing that the IC50 value of pravastatin in HMGR inhibition was markedly lower than that of lovastatin.18 Additionally, it is known that lovastatin, simvastatin, and atorvastatin are lactone prodrugs requiring cellular metabolic activation via cytochrome P450 3A4 isoenzyme (CYP3A4),19 whereas pravastatin is readily active in hydroxy acid form.20 The IC50 value of 5 seems to be higher than that of lovastatin reported in previous studies,18 which may be explained by the differences in the assay protocols. It should also be noted that the HMGR inhibitory activity of 5 observed in the IC50 studies appeared to be less than that obtained from the screening experiments. This may be due to the variation of dose−response data at high doses (especially 800 μM) of 5. To determine the direct binding of 5 to the catalytic domain of HMGR, effects of 5 at the concentration of 50 μM on HMGR activity with various concentrations of HMG-CoA substrate (800, 1600, and 3200 μM) were determined. As shown in Figure 6, 5 displayed an HMG-CoA-concentration-independent effect, similar to pravastatin and 2. Pravastatin and 2 are known to directly bind to the active site of HMGR catalytic domain.18,21 Therefore, these results indicated that 5 directly interacted with HMGR.

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting points were determined on an Electrothermal 9100 melting point apparatus and E



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Na2SO4 and then evaporated to dryness to obtain a brown gum (4.34 g). The broth extract was separated by CC over Sephadex LH-20 using MeOH to give three fractions (A−C). Fraction A (1.11 g) was fractionated by CC over Sephadex LH-20 using MeOH−CH2Cl2 (1:1) to afford four fractions (A1−A4). Fraction A3 (368.7 mg) was purified by CC over silica gel using a gradient of MeOH−CH2Cl2 (2:98 to 100:0) to give 3 (23.5 mg). Fraction B (8.26 g) was separated by CC over silica gel using a gradient of EtOAc−petroleum ether (15:85 to 100:0) to afford nine fractions (B1−B9). Fraction B4 (4.48 g) was further separated by CC over silica gel using a gradient of acetone− CHCl3 (0:100 to 100:0) to give eight fractions (B4A−B4H). Fraction B4E (1.70 g) was rechromatographed on CC over silica gel using a gradient of acetone−petroleum ether (30:70 to 100:0) followed by CC over silica gel using a gradient of EtOAc−petroleum ether (15:85 to 100:0) to provide 2 (73.1 mg). Fraction B4H (72.6 mg) was further purified using the same procedure as fraction A3 to give 1 (1.8 mg). The mycelial ethyl acetate extract (4.34 g) was separated using the same procedure as the broth extract to afford four fractions (CE1− CE4). Fraction CE3 (2.47 g) was rechromatographed on CC over silica gel using a gradient of MeOH−CH2Cl2 (0:100 to 100:0) to give six fractions (CE3A−CE3F). Fraction CE3B (18.6 mg) was subjected to CC over silica gel using EtOAc−petroleum ether (5:95) to provide 4 (3.6 mg) and 7 (2.7 mg). Fraction CE3D (61.2 mg) was further purified by CC over silica gel using EtOAc−petroleum ether (15:85) to afford 6 (12.3 mg). Fraction CE3E (1.11 g) was subjected to CC over silica gel using the same solvent system as fraction A3 to give five fractions (CE3E1−CE3E5). Fraction CE3E5 (371.7 mg) was purified by CC over Sephadex LH-20 using MeOH, subsequent CC over silica gel using a gradient of MeOH−CH2Cl2 (3:97 to 100:0), followed by CC over silica gel using a gradient of MeOH−CH2Cl2 (2:98 to 100:0) to give three fractions. The second fraction (40.3 mg) was subjected to acetylation reaction followed by CC over silica gel using MeOH− CH2Cl2 (1:99) to afford 5OAc (13.7 mg). The last fraction (30.3 mg) was further purified by CC over silica gel using CH2Cl2−MeOH− EtOAc (92:4:4) and subsequent PTLC using CH2Cl2−MeOH− EtOAc (92:4:4) as a mobile phase (5 runs) to provide 5 (9.2 mg). Compound 1: colorless gum; [α]21 D +119.0 (c 0.1, CHCl3); IR (neat) νmax 3394, 2928, 1717 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+ 275.1623 (calcd for C15H24O3Na, 275.1623). Compound 4: colorless gum; [α]24 D +72.00 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 214 (3.99), 265 (3.38), 301 (2.54) nm; CD (MeOH, c 0.0024) λmax (log ε) 208 (−4.06), 259 (−1.75) nm; IR (neat) νmax 2924, 1718 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+ 305.1517 (calcd for C19H22O2Na, 305.1517). Compound 5: colorless gum; [α]24 D +44.00 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 213 (3.72) nm; CD (MeOH, c 0.0012) λmax (log ε) 205 (−42.88), 258 (−4.18) nm; IR (neat) νmax 3421, 2933, 1736, 1716 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+ 443.2410 (calcd for C24H36O6Na, 443.2410). Compound 5OAc: colorless gum; [α]24 D +57.33 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 211 (3.90) nm; IR (neat) νmax 2936, 1741, 1739, 1735, 1717 cm−1; 1H NMR (CDCl3, 300 MHz) δH 6.88 (ddd, J = 9.6, 5.7, and 3.0 Hz, 1H, H-13), 6.02 (ddd, J = 9.6, 2.4, and 0.9 Hz, 1H, H-14), 5.97 (dd, J = 5.4 and 1.5 Hz, H-4), 5.41 (q, J = 3.0 Hz, 1H, H-8), 5.11 (brs, 1H, H-5), 4.96 (dd, J = 5.1 and 3.0 Hz, 1H, H-3), 4.38 (m, 1H, H-11), 2.40 (m, 1H, Ha-12), 2.35 (m, 1H, H-2′), 2.30 (m, 1H, Hb-12), 2.26 (m, 1H, H-8a), 2.12 (m, 1H, Ha-7), 2.08 (m, 1H, H-6), 2.05 (m, 1H, H-2), 2.03 (s, 3H, 1″-Me), 2.02 (s, 3H, 1‴-Me), 1.84 (m, 1H, Ha-10), 1.77 (m, 1H, H-1), 1.75 (m, 1H, Hb-10), 1.74 (m, 1H, Hb7), 1.72 (m, 1H, Ha-3′), 1.57 (m, 1H, H-9), 1.49 (m, 1H, Hb-3′), 1.15 (d, J = 6.9 Hz, 3H, 2′-Me), 1.05 (d, J = 7.5 Hz, 3H, 6-Me), 0.90 (t, J = 7.2 Hz, H3-4′), 0.77 (d, J = 6.9 Hz, 3H, 2-Me); 13C NMR (CDCl3, 75 MHz) δC 175.8 (C, C-1′), 170.4 (C, C-1″), 169.8 (C, C-1‴), 164.3 (C, C-15), 144.9 (CH, C-13), 137.5 (C, C-4a), 125.9 (CH, C-4), 121.5 (CH, C-14), 78.4 (CH, C-5), 78.3 (CH, C-11), 71.4 (CH, C-3), 69.5 (CH, C-8), 41.8 (CH, C-2′), 38.9 (CH, C-8a), 32.8 (CH2, C-10), 32.3 (CH, C-2), 32.2 (CH, C-1, C-6), 31.2 (CH2, C-7), 29.6 (CH2, C-

Figure 6. Inhibitory effect of pravastatin and compounds 2 and 5 on the active site of the catalytic domain of HMG-CoA reductase in a cellfree-based assay. HMG-CoA reductase activity was determined in a cell-based assay containing HMG-CoA at concentrations of 800, 1600, and 3200 μM, NADPH, and HMGR in the absence (control) or presence of either 0.25 μM pravastatin or 50 μM compound 2 or 5. Data are expressed as percent inhibition. *, p < 0.05 compared with control (n = 3). reported without correction. Optical rotations were recorded on a JASCO P-1020 polarimeter. The ultraviolet (UV) absorption spectra were measured in MeOH on a PerkinElmer Lambda 45 spectrophotometer. The infrared (IR) spectra were recorded neat using a PerkinElmer 783 FTS165 FT-IR spectrometer. Mass spectra were obtained from a MAT 95 XL mass spectrometer (Thermo Finnigan), a Bruker MicrOTOF mass spectrometer, or a liquid chromatograph− mass spectrometer (2090, LCT, Waters, Micromass). 1H and 13C NMR spectra were recorded on a 300 or 500 MHz Bruker FTNMR Ultra Shield spectrometer. Chemical shifts are expressed in δ (parts per million, ppm) referring to the tetramethylsilane peak. Thin-layer chromatography (TLC) and preparative TLC (PTLC) were performed on silica gel 60 GF254 (Merck). Column chromatography (CC) was carried out on Sephadex LH-20 with MeOH, silica gel (Merck) type 60 (230−400 mesh ASTM) or type 100 (70−230 mesh ASTM), or reversed-phase C18 silica gel. Fungal Material. The soil fungus PSU-RSPG178 was isolated from a soil sample from Suratthani Province, Thailand, and deposited as BCC56851 at BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. PSURSPG178 was identified by morphological characteristics and the analysis of an internal transcribed spacer (ITS1-5.8S-ITS2) rDNA using universal fungal primers.22 Its colony was yellow, floccose, and rapid growing. The microscopic features showed hyaline and septate hyphae and unbranched conidiophores with a vesicle. Phialides were borne directly on metulae. Phylogenetic analysis using the maximum parsimony method revealed that the fungus RSPG178 (GenBank accession number KC478521) was placed in a subclade with several strains of Aspergillus sclerotiorum with A. sclerotiorum AY373866 being the most similar taxon (99% nucleotide identity). Therefore, the fungus RSPG178 could be identified as A. sclerotiorum. Fermentation, Extraction, and Purification. A. sclerotiorum PSU-RSPG178 was grown on potato dextrose agar at 25 °C for 5 days. Five pieces (0.5 × 0.5 cm2) of mycelial agar plugs were inoculated into 500 mL Erlenmeyer flasks containing 300 mL of potato dextrose broth, which were then incubated at room temperature for 3 weeks. The flask culture (16 L) was filtered to separate into the filtrate and wet mycelia. The filtrate was extracted twice with ethyl acetate (2 × 300 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness under reduced pressure to afford a dark brown gum (9.43 g). The mycelial cakes were extracted with MeOH (500 mL). The MeOH layer was concentrated under reduced pressure. To the MeOH extract was added H2O (50 mL), and the mixture was washed twice with hexane (2 × 300 mL). The hexane layer was dried over anhydrous Na2SO4 and evaporated to dryness under reduced pressure to obtain a crude extract as a dark brown gum (1.40 g). The aqueous residue was extracted three times with an equal amount of EtOAc (3 × 300 mL). The EtOAc layer was dried over anhydrous F



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12), 26.5 (CH2, C-3′), 24.6 (CH2, C-9), 21.2 (CH3, 1″-Me), 21.3 (CH3, 1‴-Me), 18.6 (CH3, 6-Me), 16.5 (CH3, 2′-Me), 11.8 (CH3, C4′), 10.9 (CH3, 2-Me); HRESIMS m/z [M + Na]+ 527.2621 (calcd for C28H40O8Na, 527.2621) Compound 6: colorless crystals; mp 125−127 °C; [α]24 D +50.33 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 215 (3.72) nm; IR (neat) νmax 2963, 1737, 1717 cm−1. Preparation of the (R)- and (S)-MTPA Ester Derivative of 5.13,14 Pyridine (100 μL) and (+)-(R)-MTPACl (40 μL) were added to a CH2Cl2 solution (300 μL) of 5 (1.5 mg). The reaction mixture was stirred at room temperature overnight. After removal of the solvent, the mixture was purified by CC over silica gel using MeOH− CH2Cl2 (5:95) to afford the (S)-MTPA ester (2.8 mg, 93.3% yield). Compound 5 (1.3 mg) was treated in a similar way with (−)-(S)MTPACl and, after purification by CC over silica gel, the (R)-MTPA ester (2.4 mg, 92.3% yield) was obtained. X-ray Crystallographic Analysis of 6. A suitable crystal of 6 (Figure 3) was selected for data collection, which was performed on a Bruker Smart Apex CCD diffractometer equipped with a graphitemonochromatic Mo Kα radiation (λ = 0.71073 Å) source at 293(2) K. Cell refinement, data reductions, and absorption correction were performed using SAINT and SADABS. The structure was solved using direct methods with SHELXS23 and refined with the full-matrix leastsquares methods based on F2 with the SHELXL program.24 Nonhydrogen atoms were allowed to vibrate anisotropically in cycles of refinement. All hydrogen atoms were placed in calculated, ideal positions and refined as riding model approximations on their respective parent atoms. The WinGX v2014.125 and Mercury26 programs were used to prepare the materials and molecular graphics for publication. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit no. CCDC 1431870). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax: +44-(0)223-336033 or e-mail: [email protected]. uk). Crystal Data of 6 (CCDC 1431870): C24H36O4, M = 388.53, T = 293(2) K, orthorhombic space group P212121, a = 5.7192(4) Å, b = 19.6161(13) Å, c = 19.9873(12) Å, V = 2242.3(3) Å3, α = β = γ = 90°, Z = 4, Dcalc = 1.151 Mg/m3, crystal dimensions 0.449 × 0.107 × 0.057 mm3, μ = 0.076 mm−1, F(000) = 848, 23 929 reflections measured, 3885 unique (Rint = 0.1370). The final refinement gave R1 = 0.0980 and wR2 = 0.1919 [I > 2σ(I)]. Flack parameter = 0.1(10). Antimycobacterial Assay. Antimycobacterial activity was determined against M. tuberculosis H37Ra using green fluorescent protein (GFP)-based fluorescent detection.27 The positive controls were rifampin, streptomycin, isoniazid, ofloxacin, and ethambutol. Antimalarial Assay. The activity was evaluated against the parasite P. falciparum (K1, multidrug-resistant strain), using the microculture radioisotope technique based on the method described.28 Dihydroartemisinine and mefloquine were used as standard compounds and exhibited IC50 values of 0.0023 and 0.0269 μM, respectively. Cytotoxicity Assays. The activity assay against African green monkey kidney fibroblast (Vero) cells was performed in triplicate employing the method described by Hunt and co-workers.29 The activities against KB and MCF-7 cell lines were evaluated using the resazurin microplate assay.30 The positive controls were ellipticine for both Vero and KB cells, doxorubicin for both KB and MCF-7 cells, and tamoxifen for MCF-7 cells. HMG-CoA Reductase Assay. Effects of lovastatin derivatives from Aspergillus sclerotiorum PSU-RSPG178 on HMG-CoA reductase were evaluated using the HMG-CoA reductase assay kit (Sigma, MO, USA) according to the manufacturer’s instructions with slight modifications. Briefly, 1× assay buffer, NADPH, and HMG-CoA (at a final concentration of 800 μM) were added into 96-well plates (Corning Inc., NY, USA) in the absence (control) or presence of test compound (at a final concentration of 200 μM for the screening and at the indicated concentrations for dose−response studies). Subsequently, a catalytic domain of human HMGR (at a final concentration of 0.6 μM) was added to initiate the reaction at 37 °C. Pravastatin at a final concentration of 0.89 μM was used as a positive control. The oxidation

rate of HMGR was analyzed by measuring absorbance at 340 nm for 10 min at 20 s intervals using a Synergy HT microplate reader (Biotek, VT, USA). The data were expressed as percent inhibition of control. In order to further examine the inhibitory effect of the test compounds on HMGR, effects of submaximal concentrations of pravastatin (0.225 μM) and lovastatin derivatives (50 μM) were tested with the use of increasing concentrations of HMG-CoA (800, 1600, and 3200 μM).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00961. 1 H and 13C NMR spectra for 1, 4, 5, and 5OAc (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +66 74 288 435. Fax: +66 74 558 841. E-mail: vatcharin. [email protected] (V. Rukachaisirikul). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS V.R. thanks the NSTDA Chair Professor grant of the Crown Property Bureau and the National Science and Technology Development Agency. P.P. is grateful to the TRF through the Royal Golden Jubilee Ph.D. program (grant number PHD/ 0031/2555) and Prince of Songkla University (PSU-Ph.D. scholarship) for a joint funding scholarship. The Center of Excellence for Innovation in Chemistry (PERCH−CIC) and Prince of Songkla University Graduate School are acknowledged for partial support. Finally, the National Center for Genetic Engineering and Biotechnology (BIOTEC) is acknowledged for antimycobacterial, antimalarial, anticancer, and cytotoxic assays.

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REFERENCES

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