Irpexolidal Represents a Class of Triterpenoid from the Fruiting Bodies

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Irpexolidal Represents a Class of Triterpenoid from the Fruiting Bodies of the Medicinal Fungus Irpex lacteus Yang Tang, Zhen-Zhu Zhao, Kun Hu, Tao Feng, Zheng-Hui Li, He-Ping Chen, and Ji-Kai Liu J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 23, 2019

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The Journal of Organic Chemistry

Irpexolidal Represents a Class of Triterpenoid from the Fruiting Bodies of the Medicinal Fungus Irpex lacteus Yang Tang,†,§ Zhen-Zhu Zhao,‡ Kun Hu,§ Tao Feng,† Zheng-Hui Li,† He-Ping Chen,†,§,* Ji-Kai Liu†,§,*

†School

of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, People’s Republic

of China §State

Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese

Academy of Sciences, Kunming 650201, People’s Republic of China ‡College

of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450008, People’s Republic of China

GRAPHICAL ABSTRACT

ACS Paragon Plus Environment

The Journal of Organic Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Irpexolidal (1), a triterpenoid with an unprecedented carbon skeleton, along with its biogenetic-related compound irpexolide A (2), were isolated from the fruiting bodies of the medicinal fungus Irpex lacteus. Irpexolidal features a 6/5/6/5/6/5-fused polycyclic skeletal system which arises from the eburicane-type triterpene by a 6,7-seco-6,8-cyclo pattern. The structures of 1 and 2 were established by means of extensive spectroscopic techniques, ECD calculation, and DP4+ probability based on GIAO NMR chemical shift calculations. The plausible biosynthetic pathways for compounds 1 and 2 were proposed. Their biological activities were evaluated.


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INTRODUCTION Triterpenoids from mushrooms are a large group of secondary metabolites but with less structural variety comparing to the higher fungal sesquiterpenoids. The medicinal fungi Ganoderma lucidum, Poria cocos, Antrodia cinnamomea, and Inonotus obliquus, especially their fruiting bodies, are the main origin of mushroom triterpenoids.1 The Ganoderma lanostanoids have long been hot topics for their isolation, structural diversity, and biological activity.2 To the best of our knowledge, more than five hundred of Ganoderma triterpenoids (GTs) have been reported from 23 species.1,2d Although there is a large GT library reported, the vast majority of the GTs are lanostane-type with usual modifications; the same is true for the Poria cocos-derived triterpenoids.1 The well-known medicinal fungus Antrodia cinnamomea is ascribable for the all-reported ergostane triterpenoids from fungi.1 Among those reported

higher

fungal

triterpenoids,

the

spiro

cyclic

system-harboring

compounds

spiroinonotsuoxodiol3 and officimalonic acid A4 which generated by a pinacol rearrangement between the positions C-8 and C-9, together with the 3,4-seco-hexanortriterpenoid ganolearic acid A5, are the three ones with pronounced skeletal changes apart from the C–C cleavage and degradation patterns. The fungus Irpex lacteus is a widely-accepted folk medicine across the mainland China. The crude polysaccharide fraction of I. lacteus was approved by the China Food and Drug Administration with the name Yishenkang for the treatment of chronic glomerulonephritis.6 However, little attention was paid on the secondary metabolites of this fungus, only several sesquiterpenoids7 and nematicidal substances8 were reported from the cultures of I. lacteus. In a project to discover promising natural products as drug templates from medicinal mushrooms, the EtOAc layer of crude extract of a Xishuangbanna rainforest-originated I. lacteus was chemically investigated. Five 1,10-seco-/1,10– 9,11-diseco- and ring B aromatic eburicane-type triterpenoids with significant inhibitory activity against NO production in LPS-activated RAW 264.7 were isolated.9 Herein, we intend to describe the isolation and structure elucidation of two unusual triterpenes, irpexolidal (1) and irpexolide A (2) (Figure 1), from the fruiting bodies of I. lacteus. Irpexolidal harbors an unprecedented 6,7-seco-6,8cyclo-eburicane skeleton and represents a new class of triterpenoid from nature.



21 20

18 11

HO

9 1 10

HO

15

8 6

5

3

17

13

19

H 23 O

O

O

26 25

OH

27

1

31

30

CHO 7

28 29

O

1

3

HO

13 9

5

17

H O 31

8 7

OH OH 2

Figure 1. Chemical structures of compounds 1 and 2.


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O 26

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RESULTS AND DISCUSSION Irpexolidal (1) was obtained as white powder. The molecular formula, C31H42O6, with 11 degrees of unsaturation was deduced from HRMS (ESI-TOF) spectrum m/z: [M+Na]+ calcd for C31H42O6Na, 533.2874; Found 533.2870. The 1H NMR of 1 measured in methanol-d4 displayed proton signals which ascribable to eight methyls (δH 0.78, 0.88, 1.04, 1.09, 1.17, 1.39, 1.77, and 1.96), three oxygenated methine protons [δH 3.91 (dd, J = 11.9, 4.5 Hz), 4.50 (ddd, J = 8.0, 8.0, 6.7 Hz), 4.82 (t-like, J = 3.0 Hz)], two olefinic protons [5.41, 6.31 (t, J = 4.0 Hz)], and an aldehyde proton at δH 9.85 (Table 1). Additionally, the

13C

NMR and DEPT spectra of 1 showed 31 carbon signals, including four

methylenes (δC 33.7, 38.3, 39.66, 39.73), eight methines (two olefinic ones at δC 121.3, 125.2, three oxygenated ones at δC 74.1, 76.6, 80.6, and an aldehyde group at δC 202.1), eleven quaternary carbons (five sp2 ones, six sp3 ones), and eight methyls. All the signals were assignable to three double bonds and two carbonyls, accounting for five of the eleven degrees of unsaturation, which implied the existence of six rings in the structure of 1. Comprehensive analysis of the 2D NMR (HSQC, 1H–1H COSY, HMBC) spectra of 1 allowed the completion of its planar structure and chemical shift assignments. The 1H–1H COSY spectrum revealed three isolated spin systems, which were identified as follows: H-1/H-2/H-3, H-11/H-12, and H-15/H-16/H-17/H-20/H-22 (H-21) (Figure 2). The HMBC correlations from the methyl H3-27 to C24, C-25, C-26, from H3-31 to C-23, C-24, and C-25, and from H-16 to C-23, in conjunction with the IR absorption band at 1763 cm1 indicated that the existence of a spiroketal moiety which were respectively assigned as rings E and F. The two rings shared the spiro carbon C-23, and ring F harbored an α,β-unsaturated-γ-lactone functionality. Moreover, HMBC correlations from the angular methyl H3-18 to C-12, C-13, C-14, and C-17, from H3-30 to C-8, C-13, C-14, C-15, and also from H-11 to C-8, C-9 enabled the 5/6 ring-fused moiety to be defined (rings C and D in Figure 2). Additionally, HMBC correlations from the angular methyl H3-19 to C-1, C-5, and C-10, from the geminal methyls H3-28, H3-29 to C-3, C-4, and C-5 allowed the establishment of a six-membered dihydroxy-substituted ring, i.e. ring A.



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Table 1. 1H (800 MHz) and 13C (200 MHz) NMR Spectroscopic Data of Compounds 1 and 2. 1a 2b No. δC, type δH, multi. δC, type δH, multi. 1 74.1, CH 3.91, dd (11.9, 4.5) 74.0, CH 4.72, ddd (7.2, 6.5, 6.5) 2 33.7, CH2 2.10, overlapped 36.1, CH2 2.50, ddd (13.3, 6.5, 2.0) 1.93, overlapped 2.48, td (13.3, 13.3, 2.0) 3 80.6, CH 4.82, t-like (3.0) 69.5, CH 4.88, br. d (13.3) 4 39.5, C 45.1, C 5 157.6, C 79.5, C 6 125.2, CH 5.41, s 65.5, CH 4.53, d (6.0) 7 202.1, CH 9.85, s 123.2, CH 5.83, d (6.0) 8 69.6, C 142.7, C 9 150.2, C 141.4, C 10 54.7, C 44.9, C 11 121.3, CH 6.31, t (4.0) 118.6, CH 5.62, d (6.5) c 12 39.66, CH2 2.18, dd (17.8, 4.0) 37.8, CH 2.28, d (17.5) 2.08, dd (17.8, 4.0) 2.01, dd (17.5, 7.0) 13 45.0, C 43.7, C 14 49.6, C 48.8, C c 15 39.73, CH2 2.32, dd (13.3, 6.7) 40.4, CH2 2.20, dd (12.5, 8.0) 2.11, overlapped 1.88, dd (13.0, 6.5) 16 76.6, CH 4.50, ddd (8.0, 8.0, 6.7) 75.5, CH 4.70, ddd (8.0, 8.0, 6.5) 17 55.3, CH 1.73, dd (9.7, 8.0) 53.3, CH 1.68, t-like (8.0) 18 23.2, CH3 0.88, s 18.7, CH3 0.87, s 19 22.6, CH3 1.39, s 28.3, CH3 1.93, s 20 27.4, CH 1.88, m 26.1, CH 1.81, overlapped 21 20.9, CH3 1.04, d (6.3) 20.6, CH3 0.97, d (7.4) 22 38.3, CH2 1.91, overlapped 37.4, CH2 1.81, overlapped 1.80, d (13.0) 23 109.5, C 107.9, C 24 160.5, C 158.5, C 25 124.8, C 124.0, C 26 174.7, C 172.5, C 27 8.2, CH3 1.77, d (1.0) 8.3, CH3 1.77, d (1.0) 28 27.2, CH3 1.17, s 19.1, CH3 1.16, s 29 25.7, CH3 1.11, s 22.7, CH3 1.60, s 30 21.3, CH3 0.78, s 25.4, CH3 0.99, s 31 11.1, CH3 1.97, (1.0) 10.7, CH3 1.91, d (1.0) 1-OH 6.42, d (7.2) 3-OH 6.15, br. s 5-OH 6.00, s 6-OH 6.62, br. s a Measured in CD OD; b Measured in C D N; c Assignments were rounded to two decimal places to tell apart the 3 5 5 close resonances.


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O

O F

E

HO

C

D

O

B

H

A

HO

1

H-1H COSY

O

HMBC

ROESY

Figure 2. Key 2D NMR correlations of compound 1. Regardless of the yet unassigned aldehyde group (C-7) and a double bond (C-5 and C-6), the above assignments accounted for eight degrees of unsaturation, suggesting that an additional ring remained to be settled. The key HMBC correlations from the methyl H3-19 to C-9, from the olefinic proton H-6 to C-4, C-5, C-8, C-9, and C-10, from the aldehyde proton H-7 to C-8, C-9, and C-14 indicated that the presence of a five-membered ring (ring B) constructed by C5–C6–C8–C9–C10 with the aldehyde group attached to C-8 (Figure 2). Thus, the planar structure of 1 was established as an unprecedented triterpene with a unique 6,7-seco-6,8-cyclo carbon skeleton as depicted in Figure 1. The rigidity of 1 facilitated the determination of the relative configuration of its chiral centers in rings A–D by analysis of ROESY spectrum and 3J coupling constants. The key ROESY correlations between H3-19/H-7/H3-18/H-20 suggested that the β orientation for CHO-7, Me-18, and Me-19, while α orientation for Me-21. Likewise, the key ROESY correlations between H3-30/H-16/H-17 indicated that Me-30, H-16, H-17 were α orientation (Figure 2). As regards the relative configuration of 1,3-diol group in A ring, 3J coupling constant analysis is helpful to unambiguously determined the relative stereochemistry. Significant ROESY cross peaks between H-2β/H3-19, and H-1/H-2α led to the projection of C1-C2 as shown in Figure 3, revealing H-1 was a double doublet with a large and a small coupling constants generated by H-1/H-2β and H-1/H-2α, respectively, which also in accordance with the experimental 1H NMR results (H-1, dd, J = 11.9, 4.5 Hz), i.e. 1-OH was β orientation. In contrast, ROESY correlations between H-3 and H-2α, H-2β, H3-28, H3-29 as well as the t-like peak type of H3 with a small coupling constant 3.0 Hz suggested that the 3-OH was α orientation. As for the spiro



Page 8 of 19

carbon C-23, the close chemical shifts for protons H3-31, H-22, H-20 hampered the establishment of the relative configuration of C-23 because of the possible correlations overlapped in diagonal peaks.

(A)

H2

CH3

HO H2

(B) H3 H2

H1

3

JH1H2

large

3

JH1H2

small

Exp. H-1,dd, J = 11.9, 4.5 Hz

OH

H2 CH3 CH3

3

JH1H2 small

3

JH1H2

small

Exp. H-3, t-like, J = 3.0 Hz

OH

Figure 3. Newman Projections, 3J coupling constants & ROESY ( ) analyses, and corresponding 1H NMR peaks of (A) C1-C2, and (B) C2-C3.

However, after failing to obtain suitable crystals for X-ray diffraction analysis, the shortage of sample (totally obtained 0.9 mg) of 1 presented challenges in attempting to obtain single crystals of its derivatives. Nonetheless, the absolute configuration of 1 was determined by ECD calculation in combination with the experimental CD data, which has proven to be a powerful and reliable method in establishing the absolute configurations of natural products.10 Concerning the conserved configuration and inexistence of enantiomers of natural triterpenoid skeleton, the ECD calculations were performed on two tentative C-23 epimers (23R)-1 and (23S)-1 (Supporting Information). Conformational analysis of the two possible epimers were carried out using the MMFF94s force field.11 The selected conformers with distribution higher than 1% were further optimized by Gaussian 09 program at the level of B3LYP/6-31G(d).12 These predominant conformers were subjected to theoretical calculation of ECD using density functional theory (DFT) at ωB97XD/Def2SVP level in MeOH with IEFPCM model.13 The theoretical ECD spectra of (23R)- and (23S)-1 were then compared to the experimental CD spectrum. As depicted in Figure 4, this comparison led to the experimental CD best matched to the curve calculated for the 23R isomer. Thus, the absolute configuration of 1 was unequivocally determined to be 1R,3R,8R,10S,13R,14R,16S,17R,20R,23R.


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8 6 4 2

Δε

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 -2

Calcd ECD for (23S)-1

-4

Calcd ECD for (23R)-1

-6

Exptl CD for 1

-8 200

225

250

275

300

325

Wavelength (nm)

350

375

400

Figure 4. Experimental CD and calculated ECD spectra of 1.

To further reinforced the structure of 1, calculations of NMR chemical shifts of 1 using GaugeIndependent Atomic Orbitals (GIAO) method were performed at B3LYP/6-31G(d,p) level with PCM model in methanol based on the optimized conformers. The results showed that the correlation coefficient (R2) between the calculated and experimental data from linear regression analysis was 0.9986 for 23R isomer of 1 (Figure 5A). There were three outliers higher than 3 ppm, and the largest one was Δδ = 5.1 (C-26), the mean absolute deviation (MAD) and the root-mean-square deviation (RMSD) were 1.68 and 2.03 ppm, respectively (Supporting Information, Page S19-S20). However, the 13C NMR calculation results of the 23S isomer also exhibited acceptable data with four outliers higher than 3 ppm (R2 = 0.9984, MAD = 1.72, RMSD = 2.13) (Supporting Information, S22-S24). Therefore, for this example, NMR chemical shift calculation on two feasible isomers were not effective strategies to differentiate the real isomer from the virtual one. The results became worse when the functionals were changed from B3LYP to mPW1PW91, which suggested that the absolute configuration of 1 to be 23S (23R: R2 = 0.9984, MAD = 1.49, RMSD = 1.82; 23S: R2 = 0.9988, MAD = 1.49, RMSD = 1.87, Supporting Information S21 and S26). But, to say the least, the NMR calculation results consolidated the planar structure of compound 1. The DP4+ probability is a method shows clear superiority than the regression analysis of the NMR data in discriminating isomers when only one set of experimental data is available,14 as occurred in the case of compound 1. To assign the absolute configuration of 1, the DP4+ probability of the two tentative isomers of 1 were calculated with two different functionals (B3LYP and mPW1PW91). Fortunately, the unscaled and scaled DP4+ data of the protons were capable of discriminating between the 23R and 23S isomers. As shown in Figures 5C and 5D, the 23R isomer showed striking



predominance than the 23S isomer. The results suggested that (23R)-1 was the most likely candidate, which echoed with the outcome of ECD calculation. In conclusion, all the evidences strengthened the structural correctness of 1. 200

A

δexp (ppm)

160 120 80 40 0 0

40

80

120

δcalc (ppm)

160

200

δexp (ppm)

200

B

160 120 80 40 0 0

40

80

120

δcalc (ppm)

160

PCM-mPW1PW91/6-31G(d,p)

200

C

100% 80% 60% 40% 20%

uD

uD

P4 +

(H P4 dat a) +( uD C da P4 ta +( ) al ld at a) D P4 + (H D da P4 ta + ) (C D P4 da ta + ) (a ll da ta )

a) at

ta ) P4

+(

al

ld

da C

+(

P4

sD

sD

sD

P4

+(

H

da

ta )

0%

(23R)-1

(23S)-1

PCM-B3LYP/6-31G(d,p) 100%

D

80% 60% 40% 20%

sD

sD

P4 +

(H

P4 dat a) +( C da P4 ta +( ) al ld at a) uD P4 + uD (H d at P4 a) + uD (C da P4 t a) +( al ld at a) D P4 + (H D da P4 ta + ) (C D da P4 t + a) (a ll da ta )

0%

sD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(23R)-1

(23S)-1


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Figure 5. Regression analysis of experimental vs. calculated 13C NMR chemical shifts of A) (23R)-1 and B) (23S)1 at PCM-B3LYP/6-31G(d,p) level (methanol); DP4+ probability for (23R)-1 and (23S)-1 at C) PCMmPW1PW91/6-31G(d,p) and D) PCM-B3LYP/6-31G(d,p) level.

The structure 2, dubbed “irpexolide A”, obtained as white powder, showed an ion peak in the positive HRMS (ESI-TOF) experiment m/z: [M+Na]+ calcd for C31H44O7Na, 551.2979; Found 551.2978, appropriate for a molecular formula of C31H44O7, indicative of ten double bond equivalence. The 1D NMR and HSQC spectra recorded in pyridine-d5 displayed signals for eight methyls, four methylenes, seven methines (four oxygenated, two olefinic), eleven quaternary carbons (five sp2 ones, six sp3 ones) (Table 1), and hydroxy groups at δH 6.42 (1-OH), 6.15 (3-OH), 6.00 (5-OH), and 6.62 (6-OH). Comparison of these data with those of 1 revealed that the two compounds harbored similar structure fragments. Exhaustive analysis of the 2D NMR spectra allowed the completion of the planar structure as well as relative configuration. From the 1H-1H COSY spectrum, four independent spin systems were characterized as follows: (a) 1-OH/H-1/H-2/H-3/3-OH, (b) 6-OH/H-6/H-7, (c) H-11/H12, (d) H-15/H-16/H-17/H-20/H3-21 (H-22) (Figure 6). The HMBC correlations from H3-28/H3-29 to C-3, C-4, and C-5, and from H3-19 to C-1, C-5, C-9, and C-10, from the hydroxy proton 5-OH to C-4, C5, C-6, and C-10 as well as from H-6 to the olefinic carbon C-8 suggested that the structural discrepancies between 1 and 2 occurred in B ring, which was a normal six-membered ring with a C7/C-8 double bond and 5,6-diol substitutional patterns. Other assignments are same with those of 1. All the evidence led to the accomplishment of the planar structure of 2 as a highly oxygenated triterpene as described in Figure 6. The relative configuration of 2 was established via thorough analysis of the ROESY spectrum. Significant ROESY correlations between H3-18 (δH 0.87) and H-20, H3-30 and H-16, H-16 andH-17 as well as the instrumental correlation between H3-31 (δH 1.91) and H3-18 (Figure S13, Supporting Information) demonstrated that the same configuration of rings C, D, E, and F between 1 and 2 (23R* for 2). Fortunately, the presence of all the hydroxy proton signals in 1H NMR expedited the determination of relative stereochemistry of rings A and B. As illustrated in Figure 6, characteristic ROESY correlations between 5-OH/H3-19, H-6/H3-28, H-6/H3-29 indicated that the β orientation of 5-OH and 6-OH. Moreover, the ROESY correlations between H-3/1-OH, 5-OH, H3-29 suggested the β orientation of 1-OH, and α orientation of 3-OH. Therefore, the relative configuration of 2 was 1R*,3R*,5S*,6R*,10S*,13R*,14R*,16S*,17R*,20R*,23R*.



O

O OH

HO

1

O

OH OH

H-1H COSY

ROESY

HMBC

Figure 6. Key 2D NMR correlations of compound 2. Given that the structure of 2 holds similarity with those of 1, especially for the spiro-lactone moiety. But unlike compound 1, the crucial ROESY correlation between H3-31 and H3-18 was observable when pyridine-d5 was used as the NMR solvent, making it possible to assign the relative configuration of C-23 as R. To substantiate the assignment, the 23S isomers of 2 was also taken into consideration in the establishment of the absolute configuration by computational methods. The absolute configuration of 2 was established by ECD calculation. As depicted in Figure 7, the calculated ECD curve of (1R,3R,5S,6R,10S,13R,14R,16S,17R,20R,23R)-2 showed consistent trends with

the

experimental

one,

suggesting

the

absolute

configuration

1R,3R,5S,6R,10S,13R,14R,16S,17R,20R,23R.

3

Calcd ECD for (23S)-2 Calcd ECD for (23R)-2 Exptl CD for 2

1

Δε

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 19

-1

-3

-5 195

220

245

270

295

320

Wavelength (nm)

345

370

395

Figure 7. Experimental CD and calculated ECD spectra of 2.


of

2

as

Page 13 of 19

The absolute configuration of 2 was further confirmed by NMR calculation and DP4+ probability analysis. As described in Figure 8, the regression analysis of the calculating NMR data were also incapable of distinguishing (23R)-2 from (23S)-2 [(23R)-2: R2= 0.9983, MAD = 1.47, RMSD = 1.93, Figure 8A; (23R)-2: R2= 0.9980, MAD = 1.54, RMSD = 2.07, Figure 8B]. But the DP4+ probability analysis of the unscaled DP4+, scaled DP4+, and all DP4+ data of both the protons and the carbons unambiguously indicated the absolute configuration of 2 to be 23R (Figure 8C). 200

A

δexp (ppm)

160 120 80 40 0 0

40

80 120 δcalc (ppm)

160

200

200

B

δexp (ppm)

160 120 80 40 0 0

40

80 120 δcalc (ppm)

160

200

C

PCM-mPW1PW91/6-31G(d,p) 100% 80% 60% 40% 20%

) ta

ta )

da

ta )

P4 +

(a

ll

da

da

C D

H +(

P4

P4

D

D

+(

)

a)

ld al

+(

P4

uD

(23R)-1

at

ta

) da

C

H

+(

+(

P4

P4

uD

uD

da

ta

a)

al +(

P4

sD

at

ta )

ld

da C

+( P4

sD

P4

+(

H

da

ta )

0%

sD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23S)-1

Figure 8. Regression analysis of experimental vs. calculated 13C NMR chemical shifts of A) (23R)-2 and B) (23S)-2 at PCM- mPW1PW91/6-31G(d,p) level (pyridine); DP4+ probability for (23R)-1 and (23S)-1 at C) PCMmPW1PW91/6-31G(d,p) level.



Page 14 of 19

Although a variety of triterpenoids have been reported from higher fungi, irpexolidal (1) is a structurally unique triterpene possessing an unprecedented 6/5/6/5 hexacyclic scaffold which was named as “irpexonane”. The plausible biosynthetic origin of compounds 1 and 2 were discussed (Scheme 1). It was postulated that they might be traced back to A, an eburicane skeleton (sometimes named 24-methyl lanostane) which is widely distributed in higher fungi. In brief, A via repeatedly oxidation processes and ketalization to gives the key intermediate C. Compound C undergoes an oxidative cleavage between C6–C7 to yield compound D with two aldehyde groups. The intramolecular aldol condensation of compound D and E1 elimination of the resultant compound E yields compound 1. On the other hand, the intermediate C adopts cascade electrophilic addition and elimination procedures to give compound 2.

Scheme 1. Proposed Biosynthetic Pathway of Compounds 1 and 2. HO H

[O]

OH

[O] Oxidation HO

H

HO OH

OH

O

O

H

OH

HO

COOH

HO

OH

H

OH O

HO

C

H

OH H

2

H

O

OH

HO

CHO CHO

O

O

OH Aldol condensation HO

D

O

H O

HO O

O

H

OH

O

O

[O]

O

O

H

HO

O

Oxidative cleavage

2H2O O

H+ +H2O

H

OH

O

OH

H

B O

H

3H2O

H

A

HO

COOH

OH

[O] [O]

HO OH

OH

OH H E

O

H+ H2O 1

Both the two compounds were evaluated for their cytotoxicity, anti-NO activity, and vascularrelaxing activity. However, none of them displayed remarkable biological activity in the bioassays. In conclusion, chemical investigation on the secondary metabolites of the EtOAc layer of the medicinal mushroom I. lacteus led to the encounter of two unusual triterpenoids with a spiro lactone moiety, of which irpexolidal (1) possesses a unique 6,7-seco-6,8-cyclo-eburicane skeleton. Their


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structures were unequivocally determined via extensive spectroscopic analysis and computational investigation. More and more attention should be paid on the natural products of the fungus I. lacteus. EXPERIMENTAL SECTION General experimental procedures Optical rotations were obtained on a JASCO P-1020 digital polarimeter (Horiba, Kyoto, Japan). UV spectra were recorded on a Shimadzu UV-2401PC (Shimadzu, Kyoto, Japan). CD spectra were measured on an Applied Photophysics Chirascan Circular Dichroism Spectrometer (Applied Photophysics Limited, Leatherhead, Surrey, UK). 1D and 2D NMR spectra were obtained on Bruker Ascend™ 800 MHz and Bruker Avance III 600 MHz spectrometers (Bruker Corporation, Karlsruhe, Germany). HRMS (TOF) data were measured on an Agilent 6200 Q-TOF MS system (Agilent Technologies, Santa Clara, CA, USA). Medium Pressure Liquid Chromatography (MPLC) was performed on a Büchi Sepacore System equipping with pump manager C-615, pump modules C-605 and fraction collector C-660 (Büchi Labortechnik AG, Flawil, Switzerland), and columns packed with Chromatorex C-18 (40–75 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan). Preparative high performance liquid chromatography (prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with a Zorbax SB-C18 column (5 μm, 9.4 mm × 150 mm, 7 mL·min1) and a DAD detector. Fungal Material The fungus Irpex lacteus was collected from the Wangtianshu Scenic Area, Xishuangbanna, Yunnan Province in July 2014, and authenticated by Prof. Yu-cheng Dai (Beijing Forestry University), who is a mushroom specialist. A voucher specimen of Irpex lacteus was deposited at the Mushroom Bioactive Natural Products Research Group in Kunming Institute of Botany (No. HFG 201407). Extraction and isolation The dry fruiting bodies of I. lacteus (1.47 kg) were pulverized and macerated five times with 95% EtOH (total 5 L) at room temperature. The extract was evaporated under reduced pressure and partitioned between EtOAc and water for three times to give an EtOAc layer (53 g). The crude extract was eluted on MPLC with a stepwise gradient of MeOH–H2O (20–100%) to afford eight fractions (A– H). Fraction B (2 g) was subjected to Sephadex LH-20 column chromatography (CC) to afford three subfractions (B1–B3). Subfraction B2 was purified on prep-HPLC (MeCN–H2O: 38–43 %, 20 min, 7



Page 16 of 19

mL/min) to yield compound 1 (0.9 mg, tR = 16.0 min). Subfraction B1 was purified on prep-HPLC (MeCN–H2O: 35–40 %, 20 min, 7 mL/min) to yield compound 2 (1.5 mg, tR = 15.9 min).

24

Irpexolidal (1): Amorphous white powder; [α] D +14.4 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 204.5 (4.03), 308.0 (3.00) nm; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C31H42O6Na, 533.2874; Found 533.2870. Irpexolide A (2): Amorphous white powder; [α] D 25.5 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 197 24

(3.00), 211 sh (2.92) 249 (2.98) nm; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C31H44O7Na 551.2979; Found 551.2978. ECD calculation The

ECD

calculations

for

compounds

(1R,3R,8R,10S,13R,14R,16S,17R,20R,23S)-1

(1R,3R,8R,10S,13R,14R,16S,17R,20R,23R)-1 (1b),

(1R,3R,5S,6R,10S,13R,14R,16S,17R,20R,23R)-2

(1a), and

(2a),

and

(1R,3R,5S,6R,10S,13R,14R,16S,17R,20R,23S)-2 (2b) were performed using Gaussian 09 software package.12 Conformation searches based on molecular mechanics with MMFF94s force field were performed for 1a, 1b, 2a, and 2b gave 9, 11, 15, and 7 conformers with populations higher than 1%. All these conformers were further optimized by the density functional theory method at the B3LYP/631G(d) level in gas phase in Gaussian 09 program package, led to six conformers of (23R)-1 (1aa– 1af), six conformers of (23S)-1 (1ba–1bf), seven conformers of (23R)-2 (2aa–2ag), and seven conformers of (23S)-2 (2ba-2bg) within 3 kcal/mol energy threshold from global minimum, respectively (Supporting Information). These conformers were subjected to theoretical calculation of ECD using time-dependent density functional theory (TDDFT) at ωB97XD/Def2SVP level in MeOH with IEFPCM model. The weighted ECD for 1a, 1b, 2a, and 2b were all generated using SpecDis 1.71, respectively.15 13C

NMR calculation

Gauge-Independent Atomic Orbital (GIAO) calculations of 13C NMR of the conformers 1aa-1af and 1ba-1bf were accomplished by density functional theory (DFT) at the B3LYP/6-31G(d,p) and mPW1PW91/6-31G(d,p) level with PCM model in methanol in Gaussian 09 software package (Supporting Information).12 Besides, the same calculation procedures were also applied to the conformers of 2aa-2ag and 2ba-2bg except for using the B3LYP/6-31G(d,p) level. The isotropic


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values of TMS was calculated in the same level and used as references. The calculated NMR data of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. Compared to the experimental data, linear correlation coefficients (R2), mean absolute deviation (MAD), and root-mean-square deviation (RMSD) were calculated for evaluation of the results.

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website https:// at DOI: . Spectroscopic data including 1D & 2D NMR, HRMS, UV and CD spectra, and calculation details of 1 and 2 (PDF).

AUTHOR INFORMATION Corresponding authors E-mail: [email protected] (H.-P. Chen) E-mail: [email protected]; [email protected] (J.-K. Liu)

ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (No. 81561148013, 81773590), National Key Technologies R&D Program of China (2017YFC1704007), and the State Key Laboratory of Phytochemistry and Plant Resources in West China (No. P2017KF01). We thank Analytical & Measuring Center, School of Pharmaceutical Sciences, South-Central University for Nationalities for MS and NMR spectra tests. The computational work was supported by the Supercomputing Center of the Chinese Academy of Sciences in Beijing.

REFERENCES (1) Chen, H. P.; Liu, J. K. Secondary metabolites from higher fungi. Prog. Chem. Org. Nat. Prod. 2017, 106, 1−201. (2) (a) Bishop, K. S.; Kao, C. H. J.; Xu, Y. Y.; Glucina, M. P.; Paterson, R. R. M.; Ferguson, L. R. From 2000 years of Ganoderma lucidum to recent developments in nutraceuticals.



Page 18 of 19

Phytochemistry 2015, 114, 56–65. (b) Ahmad, M. F. Ganoderma lucidum: Persuasive biologically active constituents and their health endorsement. Biomed. Pharmacother. 2018, 107, 507–519. (c) Cor, D.; Knez, Z.; Hrncic, M. K. Antitumour, antimicrobial, antioxidant and antiacetylcholinesterase effect of Ganoderma lucidum terpenoids and polysaccharides: A Review. Molecules 2018, 23, 649. (d) Baby, S.; Johnson, A. J., Govindan, B. Secondary metabolites from Ganoderma. Phytochemistry 2015, 114, 66–101. (3) Handa,

N.;

Yamada,

T.;

Tanaka,

T.

An

unusual

lanostane-type

triterpenoid,

spiroinonotsuoxodiol, and other triterpenoids from Inonotus obliquus. Phytochemistry 2010, 71, 1774–1779. (4) Han, J.; Li, L.; Zhong, J.; Tohtaton, Z.; Ren, Q.; Han, L.; Huang, X.; Yuan, T. Officimalonic acids A–H, lanostane triterpenes from the fruiting bodies of Fomes officinalis. Phytochemistry 2016, 130, 193–200. (5) Peng, X. R.; Huang, Y. J.; Lu, S. Y.; Yang, J.; Qiu, M. H. Ganolearic Acid A, a hexanorlanostane triterpenoid with a 3/5/6/5-fused tetracyclic skeleton from Ganoderma cochlear. J. Org. Chem. 2018 DOI: 10.1021/acs.joc.8b01906. (6) Dong, X. M.; Song, X. H.; Liu, K. B.; Dong, C. H. Prospect and current research status of medicinal fungus Irpex lacteus. Mycosystema 2017, 36, 28−34. (7) Ding, J. H.; Feng, T.; Cui, B. K.; Wei, K.; Li, Z. H.; Liu, J. K. Novel sesquiterpenoids from cultures of the basidiomycete Irpex lacteus. Tetrahedron Lett. 2013, 54, 2651−2654. (8) Hayashi, M.; Wada, K.; Munakata, K. New nematicidal metabolites from fungus, Irpex lacteus. Agric. Biol. Chem. 1981, 45, 1527−1529. (9) Tang, Y.; Zhao, Z. Z.; Yao, J. N.; Feng, T.; Li, Z. H.; Chen, H. P.; Liu, J. K. Irpeksins A–E, 1,10-seco-eburicane-type triterpenoids from the medicinal fungus Irpex lacteus and their antiNO activity. J. Nat. Prod. 2018, 81, 2163–2168 (10)Superchi, S.; Scafato, P.; Gorecki, M.; Pescitelli, G. Absolute configuration determination by quantum mechanical calculation of chiroptical spectra: basics and applications to fungal metabolites. Curr. Med. Chem. 2018, 25, 287–320. (11)(a) Gotō, H.; Ōsawa, E. Corner flapping: a simple and fast algorithm for exhaustive generation of ring conformations. J. Am. Chem. Soc., 1989, 111, 8950–8951; (b) Gotō, H.; Ōsawa, E. An efficient algorithm for searching low-energy conformers of cyclic and acyclic molecules. J.


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Chem. Soc., Perkin Trans. 2, 1993, 187–198 (12)Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Ogliaro, F.; Bearpark,M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.;Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J.C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene,M.; Knox, J. E. J.; Cross, B.; Bakken, V.; Adamo, C.; Jaramillo, J.;Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.;Cioslowski, J.; Fox, D. J. Gaussian 09, revision C.01; Gaussian, Inc.: Wallingford CT, 2010. (13)Pescitelli, G; Bruhn T. Good computational practice in the assignment of absolute configurations by TDDFT calculations of ECD spectra. Chirality 2016, 28, 466–474. (14)(a) Smith, S. G.; Goodman, J. M. Assigning stereochemistry to single diastereoisomers by GIAO NMR calculation: the DP4 probability. J. Am. Chem. Soc. 2010, 132, 12946–12959. (b) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. Beyond DP4: an improved probability for the stereochemical assignment of isomeric compounds using quantum chemical calculations of NMR shifts. J. Org. Chem. 2015, 80, 12526–12534. (15) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Pescitelli, G. SpecDis version 1.71, Berlin, Germany, 2017, http:/specdis-software.jimdo.com.


Irpexolidal Represents a Class of Triterpenoid from the Fruiting Bodies

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