Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Cytochalasans from the Endophytic Fungus Xylaria cf. curta with Resistance Reversal Activity against Fluconazole-Resistant Candida albicans Wen-Xuan Wang,† Xinxiang Lei,† Hong-Lian Ai,*,† Xue Bai,‡ Jing Li,† Juan He,† Zheng-Hui Li,† Yong-Sheng Zheng,† Tao Feng,*,† and Ji-Kai Liu*,† †
School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, Hubei 430074, P.R. China Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, P.R. China
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‡
S Supporting Information *
ABSTRACT: Curtachalasins C−E (1−3), which have an unprecedent bridged 6/6/6/ 6 ring system, were identified from the endophytic fungus Xylaria cf. curta. Residual dipolar coupling analyses associating with density functional theory calculations were utilized to determine the relative configuration of noncrystallizable compound 2. The absolute configurations of 1−3 were determined by X-ray diffraction and electronic circular dichroism calculations. Remarkably, curtachalasin C (1) showed significant resistance reversal activity against fluconazole-resistant Candida albicans.
Candida albicans is the most life-threatening fungus, infecting millions of people with high rates of mortality.1 Unfortunately, the frontline anticandidal drugs are limited, and consequent drug resistance occurs frequently.2 However, the urgent demand for safe and effective treatment options can hardly be fulfilled by current development of new antifungal drugs.3 Therefore, we have been dedicated to discovering structurally novel natural products with anticandidal activity from fungal origins.4 Cytochalasans are a group of fungal products with high structural diversity and multiple bioactivities.5 In the previous study, we reported a new type of cytochalasans from the endophytic fungus Xylaria cf. curta E10.6 Considering the potential of Xylariales species to produce novel secondary metabolites,7 we were inspired to further investigate its traceable cytochalasans produced in different fermentation conditions. From the ethyl acetate extract of 100 L of liquid medium fermented by X. cf. curta E10, three cytochalasans (curtachalasins C−E, 1−3) with an unprecedented bicyclo[3.3.1] lactam core structure were isolated (Figure 1). They were identified by NMR analyses, DFT calculations, and X-ray diffraction. Due to the lack of attached protons on some key carbons, their relative configuration represents a challenging case for conventional structural elucidation technologies, if X-ray structures are unavailable. Curtachalasin C (1) was obtained as a yellowish powder with its molecular formula determined to be C29H33NO4 by HRESIMS ([M + H]+, m/z 460.2481, calcd for 460.2482). © XXXX American Chemical Society
Figure 1. Structures of 1−3.
The 1H, 13C NMR and HSQC spectra (Table S1 and S2) of 1 revealed a single substituted phenyl group (δC = 136.4, 129.0, 129.2, and 127.4 ppm), four other carbon−carbon double bonds (δC = 135.1, 128.8, 128.6, 134.5, 132.2, 129.0, 110.0, and 182.4 ppm), one α,β-unsaturated ketone carbonyl group (δC = 203.0 ppm), one amide carbonyl group (δC = 175.7 ppm), one sp3 quaternary carbon (δC = 47.7 ppm), five sp3 tertiary carbons (δC = 58.4, 45.8, 37.7, 83.1, and 39.0 ppm), two secondary carbons (δC = 40.5 and 35.7 ppm), one methoxy group (δC = 62.5 ppm), and four other methyl groups Received: January 2, 2019
A
DOI: 10.1021/acs.orglett.9b00015 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters (δC = 16.1, 18.3, 23.2, and 14.6 ppm). Moreover, the 1H−1H COSY spectrum showed 1 has three spin−spin coupling systems, namely H-25/H-26/H-27/H-28/H-29, H2-10/H-3/ H-4/H-5/H3-11, and H2-15/H-16/H3-23. By detailed analysis on the key HMBC correlations, the planar structure of 1 can be determined as shown in Figure 2. The ROESY spectrum of
configuration of C-7 could not be assigned by their NOE correlations either. Residual dipolar couplings (RDCs) are produced by the relatively oriented dipolar pairs, coding the global structural information on the molecule, which allow us to assign stereocenters difficult to be determined by NOE or 3J coupling values.8 With the established method in our previous work,9 we measured the 1DCH RDCs of compounds 1 and 2. The results revealed that they have different sets of RDC values (Table S3). Presuming the absolute configuration of C-3 is also S in 2, there are three possible relative configurations other than compound 1 (7S,16S), namely 7S,16R, 7R,16S, and 7R,16R. We sampled the conformational spaces of the structures with these four relative configurations by Frog2.10 We performed optimization and frequency calculations on the B3PW91-D3/ 6-311+G(d) level of theory as well as single-point energy calculations on the M06-2X-D3/ma-def2-TZVP level of theory for the obtained stable conformers. The Gibbs free energy of each conformer was calculated by summing Gibbs free energy thermal correction and single-point energy. The conformers with Boltzmann population over 1% were imported into MSpin (version 2.3.3),11 with all heavy atoms superimposed. The theoretic RDCs of the four configurations were fitted with the experimental RDCs of 1 and 2, with fitting population (Figures 4 and 5 and Table S4), following the reported protocol.11
Figure 2. 1H−1H COSY, key HMBC, and key ROESY correlations of 1 and 2.
1 showed a cross peak between H-3 and H-5, which suggested the C-4/C-5/C-6 bridge is on the same side with H-3. Because of the rigidity of the bridged structure, correspondingly, the relative configurations of C-4, C-5, and C-6 can be deduced to be as shown in Figure 2. Fortunately, the single crystal of 1 was successfully analyzed by X-ray diffraction, and the absolute configuration of 1 can be determined to be 3S,4R,5R,6S,7S,16S (Flack parameter x value was −0.11 ± 0.006) (Figure 3).
Figure 4. Comparison of the Q factors of four possible configurations fitted with the experimental RDCs of 1 or 2.
Calculated with the experimental RDCs of 1, the 7S,16S configuration presented the lowest Q factor among the four structures, which matches the structure determined by the Xray diffraction experiment. The 7S,16R configuration, furthermore, showed the lowest Q factor (0.081) with the experimental RDCs of 2. For comparison, when the 7S,16R configuration was fitted with the experimental RDCs of 1, the Q factor was a higher value of 0.176. By contrast, when the configuration of C-7 is R, the Q factors are much higher with both the experimental RDCs of 1 and 2. Therefore, the configuration of 2 was unambiguously determined to be 3S,4R,5R,6S,7S,16R or its enantiomer. ECD calculation of 2 was performed, and the result of the 3S,4R,5R,6S,7S,16R structure matched the experimental ECD curve well. Hence, the absolute configuration of 2 can be fully assigned (Figure 6). Curtachalasin E (3) was obtained as a colorless powder with a molecular formula C28H33NO5 determined by HRESIMS ([M + H]+, m/z 464.2432, calcd for 464.2431). 1H and 13C NMR spectra (Table S1 and S2) of 3 showed similar signals to
Figure 3. X-ray structure of 1.
Curtachalasin D (2) was purified as a yellowish powder with a close retention time to 1 in analytical HPLC conditions (Figure S1). Compared to 1, HRESIMS and 1D/2D NMR analyses showed 2 has the same molecular formula with slightly different 1H NMR and 13C NMR chemical shifts (Tables S1 and S2), as well as the same HSQC, 1H−1H COSY, HMBC, and ROESY correlation patterns. Thus, the planar structure and the relative configuration of C-3, C-4, C5, and C6 of 2 can be deduced to be the same as 1. Because the distances between the protons on the C-15/C-16/C-23 fragment and other chiral references are beyond the maximum range (around 5 Å) for NOE correlation, the relative configuration of C-16 could not be determined by ROESY/ NOESY experiments. Moreover, as a reason for the short distance, NOE correlations were observed between both H-7/ H3-12 and 7-OMe/H3-12, which suggested the relative B
DOI: 10.1021/acs.orglett.9b00015 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
Figure 7. 1H−1H COSY, key HMBC, and key ROESY correlations of 3.
Figure 5. Correlation between calculated RDCs (y-axis) of 7S,16S and 7S,16R configurations and experimental RDCs (x-axis) of 1 or 2. Figure 8. X-ray structure of 3.
Figure 6. Calculated ECD curves and the experimental ECD of 2. Figure 9. Inhibitory ratio against C. albicans with different concentrations of 1 (combined with 10 g/mL of fluconazole) and fluconazole alone.
compounds 1 and 2, except that 3 has one less double bond of enol and two more oxygenated carbons (C-17 and CH-19; δC = 83.0 and 71.5 ppm, respectively). The 1H−1H COSY spectrum of 3 showed three spin−spin coupling systems, namely H-25/H-26/H-27/H-28/H-29, H2-10/H-3/H-4/H-5/ H3-11, and H2-15/H-16/H3-23. The planar structure was deduced by the key HMBC correlations as shown in Figure 7. A single crystal of 3 was successfully measured by X-ray diffraction, and the stereochemistry of 3 was then fully assigned (Flack parameter x value was 0.06 ± 0.012) (Figure 8). Fluconazole is a commonly used drug for C. albicans infection, owing to its efficacy and low toxicity, which in turn causes the frequent occurrence of fluconazole resistance of C. albicans strains.3 In the resistance reversal assay for 1 and 3, we used the C. albicans strain with resistant genes Cdr1, Cdr2, and Mdr1, against which the MIC50 value of fluconazole is higher than 500 μg/mL (Figure 9). When this resistant strain was treated by 1 combined with 10 μg/mL of fluconazole, 1 showed dose-dependent resistant reversal activity (Figure 9, Table S5). Upon treatment with 16 μg/mL of 1 combined with 10 μg/mL of fluconazole, the inhibitory ratio against this strain was close to 50%, which was
a significant improvement compared to fluconazole alone. Markedly, 1 alone showed no inhibitory activity even at a concentration of 128 μg/mL (inhibitory ratio of −4.188 ± 1.047%), implying that 1 may be nontoxic to eukaryotes. However, compound 3 showed only weak resistance reversal activity at a concentration of 128 μg/mL (Table S5). Compound 2 was not tested in this assay because of the low amount. The biosynthesis of 1−3 is proposed to involve cyclization, aromatization, and rearrangement from the known compound 19,20-epoxycytochalasin C (the major cytochalasan produced by X. cf. curta E10) as shown in Scheme 1. Ring C is supposed to be formed by decarboxylation and aromatization. Furthermore, the intermediate with a formyl group on the residual amino group is proposed to be the key precursor of the bridged lactam product. The chiral difference at position 16 of 2 probably results from the epimerization of an intermediate, which could be induced by the keto−enol tautomerism of the 17-ketone group. C
DOI: 10.1021/acs.orglett.9b00015 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters *E-mail:
[email protected].
Scheme 1. Proposed Biosynthesis of 1−3
ORCID
Hong-Lian Ai: 0000-0002-6832-0970 Tao Feng: 0000-0002-1977-9857 Ji-Kai Liu: 0000-0001-6279-7893 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 31801789, 31560010, 31870513, 81872762, 81773590, 81561148013, 21502239, and 81803395) and the National Key R&D Plan (No. 2017YFC1704007). We are grateful for the convenience for the HRMS and NMR measurement provided by Analytical & Measuring Center, School of Pharmaceutical Sciences, South-Central University for Nationalities.
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In conclusion, compounds 1−3 represent a new type of cytochalasan with a unique bridged lactam ring system. Notably, the difference in the single chiral center between 1 and 2 does not cause any characterizable variation of 1H and 13 C chemical shifts or ECD spectra but can be designated by RDCs. Combining RDC analysis and DFT calculations, the stereochemical structure of noncrystallizable compound 2 (0.8 mg) was fully determined. The results of resistant reversal assay against C. albicans implied these compounds are worthwhile for further resistant reversal agent research.
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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.orglett.9b00015. Experimental procedures, 1D and 2D NMR, MS, IR, and ECD spectra for compounds 1−3 (PDF) Accession Codes
CCDC 1884667−1884668 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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DOI: 10.1021/acs.orglett.9b00015 Org. Lett. XXXX, XXX, XXX−XXX
