Phloroglucinol Derivatives with Unusual Skeletons from Cleistocalyx

Jul 2, 2018 - (2−4) In just the past year, over 100 new natural phloroglucinol ... s), 1.34 (3H, s), and 1.34 (3H, s); δC 26.4, 23.8, 21.0, and 8.3...
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Article Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX

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Phloroglucinol Derivatives with Unusual Skeletons from Cleistocalyx operculatus and Their in Vitro Antiviral Activity Jun-Cheng Su,†,§,‡ Shan Wang,†,§,‡ Wen Cheng,† Xiao-Jun Huang,†,§ Man-Mei Li,† Ren-Wang Jiang,† Yao-Lan Li,† Lei Wang,†,§ Wen-Cai Ye,*,†,§ and Ying Wang*,†,§ †

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Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China § Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM & New Drugs Research, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *

ABSTRACT: Four novel phloroglucinol derivatives (1−4) featuring a 2,4-dimethyl-cinnamyl-phloroglucinol moiety, along with their putative biosynthetic precursors 5 and 6, were isolated from the leaves of Cleistocalyx operculatus. Compounds 1 and 2 are two pairs of new enantiomeric phloroglucinol dimers possessing an unprecedented polycyclic skeleton with a highly functionalized dihydropyrano[3,2-d]xanthene tetracyclic core. Compounds 3 and 4 are two new phloroglucinol-terpene adducts (PTAs) with a novel carbon skeleton. The structures of 1−4 including their absolute configurations were unambiguously accomplished by combination of extensive spectroscopic analyses, X-ray crystallography, and quantum chemical ECD calculations. A hypothetical biosynthetic pathway for 1−4 was also proposed. Compound 1 exhibited a promising in vitro antiherpes simplex virus type-1 (HSV-1) effect.

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INTRODUCTION Phloroglucinols and their derivatives are an important class of secondary metabolites widely distributed in plants, marine organisms, and microorganisms. As one of the most important sources of natural phloroglucinols, Myrtaceous phloroglucinols discovered from the plants of family Myrtaceae are characterized by a polymethylated or formyl-substituted acylphloroglucinol moiety incorporating various kinds of biogenetic precursors via different coupling patterns. Such incorporations not only resulted in fascinating architectures and ring systems but also provided broader bioactivities than their parent compounds.1 On account of their intriguing chemical scaffolds and the biological significances, Myrtaceous phloroglucinols have attracted extensive attention from both chemical and biological communities in recent years.2−4 In just the past year, over 100 new natural phloroglucinol derivatives with various skeletons have been reported from family Myrtaceae. As an ongoing project of our group in recent years, we had carried out phytochemical investigations on several medicinal plants of family Myrtaceae. As a result, a series of novel phloroglucinol derivatives with significant bioactivities were © XXXX American Chemical Society

discovered from the genera Psidium, Callistemon, Myrtus, Eucalyptus, and Rhodomyrtus.5 In our continuing study, Cleistocalyx operculatus (Roxb.) Merr. & Perry, another member of family Myrtaceae which has been used as a folk medicine in the southern areas of China for the treatment of cold, fever, and inflammation, was selected as a subject due to the potential medicinal importance of this plant.6 An LC-MS-UV-guided isolation of the ethanol extract of the leaves of the title plant resulted in the discovery of four novel phloroglucinol derivatives (1−4), together with their proposed biosynthetic precursors, 2′,4′-dihydroxy-3′,5′-dimethyl-6′-methoxychalcone (5) and champanone B (6). (±)-Cleistoperlones A and B [(±)-1 and (±)-2] are two pairs of new dimeric phloroglucinol enantiomers, which possess an unprecedented polycyclic carbon backbone featuring a highly functionalized dihydropyrano[3,2-d]xanthene tetracyclic core (Figure 1, rings A/B/C/D). In particular, their central tetracyclic core contains seven continuous quaternary carbons (including two adjacent stereogenic centers) and a fully substituted benzene ring (ring Received: May 1, 2018

A

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of compounds 1−4.

degrees of unsaturation. The UV spectrum showed the absorption maxima at 210, 302, and 338 nm. The IR spectrum revealed the presence of a hydroxyl (3479 cm−1), conjugated carbonyl (1606 cm−1), as well as benzene ring (1560 and 1460 cm−1). The 1H and 13C NMR spectra of 1 implied the presence of two chelated hydroxyls [δH 15.77 (1H, s) and 13.49 (1H, s)], three ketone carbonyls (δC 209.1, 198.0, and 193.6), one oxygenated tetrasubstituted double bond (δC 184.6 and 105.2), one trans-disubstituted double bond [δH 7.94 (1H, d, J = 15.7 Hz) and 7.83 (1H, d, J = 15.7 Hz); δC = 143.2 and 126.8], a hexasubstituted benzene ring (δC 161.1, 158.7, 155.5, 110.7, 110.0, and 106.7), two monosubstituted benzene rings [δH 7.63 (2H, m), 7.40 (3H, overlapped), 7.31 (3H, overlapped), and 7.22 (2H, m); δC 139.4, 135.5, 130.3, 129.0 (2C), 128.8 (2C), 128.6 (2C), 128.4, and 125.6 (2C)], one methoxyl [δH 3.62 (3H, s); δC 62.5], and four methyls [δH 2.04 (3H, s), 1.44 (3H, s), 1.34 (3H, s), and 1.34 (3H, s); δC 26.4, 23.8, 21.0, and 8.3]. Analyses of 1D and 2D NMR data resulted in the full assignment of the 1H and 13C NMR signals of 1 (Table 1). Interpretation of the 1H−1H COSY correlations led to the establishment of four isolated spin systems (Figure 2). In the HMBC spectrum, the correlations between H-8 and C-10, between H-9 and C-7/C-11/C-15, between Me-16 and C-1/ C-3, between H2-17 and C-3/C-5, as well as between 5-OH and C-4/C-6 indicated the presence of a cinnamylphloroglucinol unit (1a). In addition, the HMBC correlations between H-8′ and C-10′, between H-9′ and C-1′/C-11′/C15′, between 7′-OH and C-4′/C-5′/C-6′/C-8′, between Me17′/ Me-18′ and C-3′/C-5′, as well as between Me-16′ and C-

A), which brought challenge for their structural elucidation only by NMR experiments. With the aid of X-ray crystallography analyses and quantum chemical calculations of ECD spectra, the structures with absolute configurations of all optically pure enantiomers, (+)-1, (−)-1, (+)-2, and (−)-2, were unambiguously assigned. Operculatols A (3) and B (4) are two new optically pure phloroglucinol-terpene adducts (PTAs) with a novel carbon skeleton, which represent the first examples of PTAs bearing a 2,4-dimethyl-cinnamyl-phloroglucinol moiety isolated from family Myrtaceae. Herein, we report the isolation and structural elucidation of these novel phloroglucinol derivatives. Moreover, a hypothetical biogenetic pathway for 1−4 and their in vitro antiherpes simplex virus type-1 (HSV-1) activities are also presented.

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RESULTS AND DISCUSSION The air-dried and powdered leaves of C. operculatus (20 kg) were extracted with 95% EtOH three times at room temperature. The combined EtOH residue (3.2 kg) was suspended in water and successively partitioned with petroleum ether, chloroform, and n-butanol. The obtained fractions were subsequently analyzed by UPLC-QTOF/MS, and the detection of characteristic UV profiles and ion peaks in the petroleum ether soluble fraction suggested the existence of phloroglucinol derivatives in this fraction. The petroleum ether soluble fraction (1200 g) was separated by a series of chromatographic procedures to afford compounds 1−6. The molecular formula of cleistoperlone A (1) was assigned as C36H34O8 by its HR-ESI-MS (m/z 595.2325 [M + H]+, calcd for C36H35O8 595.2326), indicating the presence of 20 B

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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The Journal of Organic Chemistry Table 1. NMR Data of 1 and 2 (in CDCl3, J in Hz)a 1 δH

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′ 1-OMe 5-OH 7′-OH

7.94 (d, 15.7, 1H) 7.83 (d, 15.7, 1H) 7.63 7.40 7.40 7.40 7.63 2.04

(m, 1H) (1H) (1H) (1H) (m, 1H) (s, 3H)

a 3.57 (d, 16.6, 1H) b 2.64 (d, 16.6, 1H)

2.86 (2H) 5.29 (dd, 10.5, 4.7, 1H) 7.22 (m, 1H) 7.31 (1H) 7.31 (1H) 7.31 (1H) 7.22 (m, 1H) 1.34 (s, 3H) 1.34 (s, 3H) 1.44 (s, 3H) 3.62 (s, 3H) 13.49 (s, 1H) 15.77 (s, 1H)

2 δC

δH

158.7 110.7 155.5 106.7 161.1 110.0 193.6 126.8 143.2 135.5 128.6 129.0 130.3 129.0 128.6 8.3 24.0 99.4 50.3 209.1 51.9 198.0 105.2 184.6 38.3 68.9 139.4 125.6 128.8 128.4 128.8 125.6 21.0 26.4 23.8 62.5

7.97 (d, 15.7, 1H) 7.84 (d, 15.7, 1H) 7.64 (m, 1H) 7.40 (1H) 7.40 (1H) 7.40 (1H) 7.64 (m, 1H) a 3.48 (d, 16.1, 1H) b 2.71 (d, 16.1, 1H) 2.04 (s, 3H)

2.85 (2H) 5.31 (dd, 10.2, 5.1, 1H) 7.23 (m, 1H) 7.32 (1H) 7.32 (1H) 7.32 (1H) 7.23 (m, 1H) 1.34 (s, 3H) 1.33 (s, 3H) 1.43 (s, 3H) 3.77 (s, 3H) 13.23 (s, 1H) 15.72 (s, 1H)

δC 158.0 107.6 155.4 109.6 161.7 110.3 193.6 126.6 143.4 135.4 128.6 129.1 130.4 129.1 128.6 24.3 7.8 99.1 50.3 209.5 52.0 198.1 105.2 184.4 38.1 69.0 139.3 125.8 128.8 128.5 128.8 125.8 21.0 26.4 23.7 62.7

a

Overlapped signals are reported without designating multiplicity.

Figure 2. 1H−1H COSY and key HMBC correlations of 1 and 2.

1′/C-3′ suggested the existence of a highly modified cyclic polyketide-like fragment (1b). The connection of 1a and 1b via C-17−C-2′ bond could be defined by the HMBC

correlations between H2-17 and C-1′/C-3′. Furthermore, the remaining one degree of unsaturation as well as the downfield sp3-hybridized quaternary carbon C-1′ (δC 99.4) and upfield C

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Figure 3. X-ray ORTEP drawings of 1, 2, and 3.

Figure 4. Experimental and calculated ECD spectra of 1 and 2.

sp2-hybridized ones C-3 (δC 155.5) suggested the closure mode of ring B (Figure 2). Thus, based on the above spectroscopic information, the planar structure of 1 could be established as a novel phloroglucinol dimer with a complex C35 polycyclic framework. However, due to the extreme proton deficient property of the gross structure of 1 (16 quaternary carbons, including seven continuous nonaromatic quaternary carbons and a hexasubstituted benzene ring), it was not surprising that the NOE experiment did not provide any valuable information for the establishment of the stereochemistry of 1. In order to verify the planar structure as well as define the stereochemistry of 1, an X-ray crystallography experiment seemed to be necessary. Fortunately, a qualified crystal suitable for an X-ray diffraction experiment was obtained from an acetone solution. As a result, the structure with a relative configuration of 1 was determined as shown in Figure 3. On the basis of the presence of the symmetric space group C2/c in its crystal structure as well as the optical rotation data close to zero, we proposed that 1 was a racemic mixture. Subsequently, the racemic mixture of 1 was separated into a pair of enantiomers, (+)-1 and (−)-1, with a ratio of 1:1, by HPLC on a chiral stationary phase. The absolute configurations of (+)-1 and (−)-1 were further determined by quantum chemical ECD calculation. The time-dependent density functional theory (TDDFT) calculated ECD spectra of the two enantiomers were compared with the experimental ones. As shown in Figure 4, the predicted ECD curves of (1′R,2′R,9′S)-1 and (1′S,2′S,9′R)-1 revealed a good agreement with the experimental ones of (+)-1 and (−)-1, respectively, which led to the establishment of the absolute configurations of (+)-1 and (−)-1 (Figure 1). The molecular formula of cleistoperlone B (2) was determined to be identical to that of 1 by its HR-ESI-MS data at m/z 617.2149 [M + Na]+ (calcd for C36H34NaO8 617.2146). Similar to 1, the 1H and 13C NMR spectra of 2 also revealed the presence of typical signals due to a dimeric

phloroglucinol skeleton. With the aid of 1H−1H COSY, HSQC, and HMBC experiments, all of the 1H and 13C NMR signals of 2 were assigned as shown in Table 1. The NMR spectral data of 2 highly resembled those of 1, except for the differences of the resonances assigned to C-2, C-4, C-16, and C-17 (Table 1), suggesting the presence of a different linkage position between the two phloroglucinol monomers in 2. The HMBC spectrum of 2 showed the correlations between H2-16 and C-1/C-3 as well as between Me-17 and C-3/C-5 (Figure 2). Furthermore, in the NOESY spectrum, the NOE correlation between 1-OMe and H-16b was also observed, suggesting that 2a and 2b were connected through the C-16− C-2′ bond in 2 instead of the C-17−C-2′ bond in 1. Similarly, based on the molecular formula information, the C-3 and C-1′ were bridged through an oxygen atom to form ring B. Moreover, the planar structure and the relative configuration of 2 were completely defined by single-crystal X-ray crystallographic analysis (Figure 3). Interestingly, the tested crystal structure of 2 was found to present a space group of R3 with a Flack parameter of 0.011(4), suggesting that 2 seemed to be an optically pure compound. However, with the optical rotation value close to zero as well as the absence of a Cotton effect in its ECD spectrum, 2 was also assumed to be a racemic mixture. Similarly, 2 was further resolved by chiral HPLC to afford two optically pure enantiomers (+)-2 and (−)-2. Using a similar quantum chemical ECD calculation method as described for 1, the absolute configurations of (+)-2 and (−)-2 were established as 1′S,2′S,9′R and 1′R,2′R,9′S, respectively (Figure 4). Operculatol A (3) was obtain as yellow blocks. The molecular formula of 3 was deduced to be C28H32O4 by its HR-ESI-MS [M + H]+ ion at m/z 433.2371 (calcd for C28H33O4 433.2373). The UV spectrum of 3 showed absorption maxima at 214 and 346 nm. The IR spectrum revealed the presence of a hydroxyl (3236 cm−1), conjugated carbonyl (1622 cm−1), benzene ring (1560 and 1449 cm−1), as well as methyl (1352 cm−1). Similar to 1, the 1H and 13C NMR D

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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The Journal of Organic Chemistry Table 2. NMR Data of 3 and 4 (in CDCl3, J in Hz)a 3 δH

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 1-OMe 5-OH

8.02 (d, 15.7, 1H) 7.84 (d, 15.7, 1H) 7.65 7.41 7.41 7.41 7.65 2.67 2.03

(m, 1H) (1H) (1H) (1H) (m, 1H) (2H) (s, 3H)

2.25 (t, 5.7, 1H) 2.63 (m, 1H) a 2.12 (m, 1H) b 1.37 (m, 1H) 1.84 (m, 1H) a 2.06 (m, 1H) b 0.86 (d, 10.7, 1H) 1.43 (s, 3H) 1.29 (s, 3H) 1.08 (s, 3H) 3.68 (s, 3H) 13.65 (s, 1H)

4 δC

δH

158.2 109.1 162.4 109.7 162.7 108.7 193.2 127.1 142.6 135.7 128.5 129.1 130.2 129.1 128.5 23.1 7.7

8.04 (d, 15.7, 1H) 7.83 (d, 15.7, 1H) 7.65 (m, 1H) 7.40 (1H) 7.40 (1H) 7.40 (1H) 7.65 (m, 1H) 2.03 (s, 3H) a 2.80 (dd, 15.1, 3.0, 1H) b 2.55 (dd, 15.2, 6.1, 1H) 2.24 (t, 5.7, 1H)

55.4 85.9 33.4 33.8

2.67 (m, 1H) a 2.12 (m, 1H) b 1.32 (ddd,13.6, 8.2, 1.9, 1H) 1.83 (m, 1H)

40.5 40.4 26.9

a 2.07 (m, 1H) b 0.79 (d, 10.5, 1H) 1.44 (s, 3H) 1.29 (s, 3H) 1.09 (s, 3H) 3.66 (s, 3H) 13.74 (s, 1H)

29.2 28.1 23.1 62.8

δC 159.1 111.0 162.0 106.5 162.6 108.6 193.2 127.2 142.5 135.7 128.5 129.0 130.2 129.0 128.5 8.3 21.5 55.5 86.1 32.9 34.1 40.6 40.4 27.3 29.2 28.3 23.0 62.3

a

Overlapped signals are reported without designating multiplicity.

Figure 5. 1H−1H COSY and key HMBC correlations of 3 and 4.

The 1H−1H COSY spectrum of 3 revealed the presence of three spin systems drawn in bold (Figure 5). In the HMBC spectrum, correlations between H2-4′ and C-2′, between Me-8′ and C-1′/C-3′, as well as between Me-9′/Me-10′ and C-1′/C5′ allowed the establishment of a monoterpene moiety in 3 (3b), which was further confirmed by comparison of its NMR data with those of the known compounds.5f,7 The connection between fragments 3a and 3b through C-16−C-3′ could be deduced by the HMBC correlations between H2-16 and C-1/ C-3/C-2′/C-4′ as well as between H-3′ and C-2. Moreover, the typical upfield chemical resonance of C-3 (δC 162.4) and downfield shift of C-2′ (δC 85.9), as well as the molecular

spectra of 3 showed characteristic signals due to a 2,4dimethyl-cinnamyl-phloroglucinol unit (3a). Besides, the remaining NMR signals could be attributed to two quaternary carbons (δC 85.9 and 40.4), three methines [δH 2.63 (1H, m), 2.25 (1H, t, J = 5.7 Hz), and 1.84 (1H, m); δC 55.4, 40.5, and 33.4 ], two methylenes [δH 2.12 (1H, m), 2.06 (1H, m), 1.37 (1H, m), and 0.86 (1H, d, J = 10.7 Hz); δC 33.8 and 26.9], as well as three methyls (δH 1.43, 1.29, and 1.08; δC 29.2, 28.1, and 23.1). The above spectral data indicated that 3 was a monoterpene-based PTA. On the basis of the detailed analysis of 1D and 2D NMR spectra, all proton and carbon resonances of 3 could be fully assigned (Table 2). E

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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Figure 6. Key NOE correlations of 3 and 4.

Figure 7. Experimental and calculated ECD spectra of 3 and 4.

2′ based on their characteristic chemical shift values as well as the molecular formula information. The relative configuration of 4 could be determined by the NOESY spectrum. In the NOESY spectrum, the correlations between H-3′ and Me-8′/Me-10′ as well as between Me-10′ and H-4′a indicated that these protons were cofacial (Figure 6). Meanwhile, the NOE correlations between H-7′a and Me9′ as well as between H-7′b and H-4′b suggested that these protons possessed the same orientation (Figure 6). Finally, the calculated ECD curve of 1′S,2′R,3′S,5′R-4 displayed a good agreement with the experimental one (Figure 7), which permitted the establishment of absolute configuration of 4 as 1′S,2′R,3′S,5′R. Compounds 1−4 are four novel phloroglucinol derivatives with a characteristic 2,4-dimethyl-cinnamyl-phloroglucinol moiety. Structurally, compounds 1 and 2 shared an unprecedented polycyclic skeleton featuring a densely functionalized dihydropyrano[3,2-d]xanthene tetracyclic core. To our knowledge, it has never been reported in natural products before. Compounds 3 and 4 represent the first examples of 2,4-dimethyl-cinnamyl-phloroglucinol-based PTAs disclosed from family Myrtaceae. Based on the findings of two phloroglucinol precursors [2′,4′-dihydroxy-3′,5′-dimethyl-6′methoxychalcone (5) and champanone B (6)] from the same plant material, a detailed description of the hypothetical biogenetic pathway for 1−4 is presented in Scheme 1. The generation of the two putative phloroglucinol precursors involved a polyketide pathway utilizing cinnamoyl-CoA as a starting unit. A cascade sequence of carbon chain extension, cyclization, methylation, and enolization modification led to the formation of achiral phloroglucinol precursors 5 and 6. Oxidation of 5 afforded highly reactive intermediates i and ii, which subsequently coupled with 6 via a nonstereoselective hetero-Diels−Alder cycloaddition to produce intermediates iiia, iiib, iva, and ivb, respectively. An intramolecular Michael

formula information allowed the construction of a dihydropyran ring between 3a and 3b. Therefore, the gross structure of 3 was assigned to be a PTA composing of a 2,4-dimethylcinnamyl-phloroglucinol unit and a α-pinene moiety. In the NOESY spectrum of 3, the correlations between H-3′ and Me-8′/Me-10′ as well as between Me-10′ and H-4′a indicated that these protons were located in the same orientation of the molecule. In addition, the NOE correlations between H-7′a and Me-9′ as well as between H-7′b and H-4′b suggested that these protons were cofacial (Figure 6). Furthermore, the structure and stereochemistry of 3 were fully assigned by an X-ray diffraction experiment (Figure 3). With a final refinement of the Flack parameter −0.08(15), the absolute configuration of 3 was defined as 1′S,2′R,3′S,5′R, which was further confirmed by quantum chemical ECD calculations (Figure 7). Operculatol B (4) was isolated as a yellow oil with the identical molecular formula to 3 by its HR-ESI-MS data at m/z 433.2374 ([M + H]+ calcd for C28H33O4 433.2373). The UV and IR spectra of 4 showed the similar characteristic absorptions to those of 3. Similar to 3, the 1H and 13C NMR spectra of 4 (Table 2) showed feature signals due to a 2,4-dimethyl-cinnamyl-phloroglucinol unit (4b) and a monoterpene moiety, indicating that 4 was also a monoterpenebased PTA. A C-17 to C-1′ spin−spin system (Figure 5) could be deduced by the 1H−1H COSY correlations. The HMBC correlations between H2-4′ and C-2′, between Me-8′ and C1′/C-3′, as well as between Me-9′/Me-10′ and C-1′/C-5′ allowed the establishment of a α-pinene moiety in 4 (4b), which is identical to 3b. Furthermore, in the HMBC spectrum of 4, the correlations between H2-17 and C-3/C-5/C-2′/C-4′ (Figure 5) were observed, indicating that the phloroglucinol unit (4a) and monoterpene moiety (4b) were connected via C-17−C-3′ bond instead of C-16−C-3′ bond in 3. Similar to 3, the remaining oxygen atom was assigned to bridge C-3 and CF

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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The Journal of Organic Chemistry Scheme 1. Hypothetical Biosynthetic Pathway of 1−4

addition enabled the construction of the final dihydropyran ring, which resulted in the generation of (−)-1, (+)-1, (+)-2, and (−)-2, respectively. The last step Michael addition reaction provided a high stereoselectivity owing to the steric bulk of the nearby phloroglucinol moiety (blue one). On the other hand, compounds 3 and 4 could be generated from intermediates i and ii coupling with α-pinene (which has been previously identified from the leaves of the title plant8) via a hetero-Diels−Alder cycloaddition reaction, respectively.9 In order to exclude the possibility that 1−4 may be artifacts generated during the isolation procedure, the three putative precursors 5, 6, and α-pinene (obtained from commercial approach) were mixed and resolved in methanol. The solution was exposed to sunlight for 10 days and subsequently analyzed by UPLC-QTOF/MS. As a result, neither an oligomeric nor hybridized product was detected, demonstrating that compounds 1−4 could not be spontaneously generated. Combined with our initial UPLC-QTOF/MS analysis result of the crude extract of the title plant, the above result indicated that 1−4

could not be yielded during the extraction and isolation procedures, and they should be natural products generated by the plant. Furthermore, considering the racemic nature of 1 and 2, the crucial hetero-Diels−Alder reaction between intermediates i/ii and 6 seemed to be a nonenzymatic process.10 However, the generations of the highly reactive intermediates i and ii from 5 should be an enzyme catalytic procedure in the biosynthesis of 1−4. Compounds 3 and 4 were obtained as optically pure compounds maybe due to the stereoselectivity of the monoterpene moiety. All of the isolated compounds were evaluated for their in vitro antivirus activity against HSV-1 and GFP-HSV-1 by using a cytopathic effect (CPE) reduction assay and fluorescence assay, respectively. Notably, as shown in Table 3 and Figure 8, compounds 1 and 6 exhibited obvious anti-HSV-1 activity in a dose-dependent manner with an IC50 value of 7.50 ± 1.25 μM and 5.63 ± 0.78 μM, respectively, while compounds 2−5 did not show anti-HSV-1 activity at the tested concentrations. Meanwhile, both monomeric phloroglucinol precursors 5 and G

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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The Journal of Organic Chemistry Table 3. In Vitro Anti-HSV-1 Activities of Compounds 1−6 compound

CC50 (μM)a

IC50 (μM)b

SIc

1 2 3 4 5 6

>100 >100 >100 >100 14.21 ± 1.76 12.00 ± 0.38

7.50 ± 1.25 >100 >100 >100 NAd 5.63 ± 0.78

>13.33

of 1−4, the discovery of these novel compounds would definitely enlarge the structural diversity of Myrtaceous phloroglucinols.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation values were measured on a Jasco P-1020 polarimeter (Jasco, Tokyo, Japan) at room temperature. UV spectra were determined on a Jasco V-550 UV/vis spectrophotometer (Jasco, Tokyo, Japan). IR spectra were recorded on a Jasco FT/IR-480 plus Fourier Transform infrared spectrometer (Jasco, Tokyo, Japan) using KBr pellets. Melting points were obtained on an X-5 micromelting apparatus (Fukai Instrument, Beijing, China) without correction. ECD spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) at room temperature. HR-ESI-MS spectra were demonstrated on an Agilent 6210 ESI/TOF mass spectrometer (Agilent, Pala Alto, CA, USA). UPLC-QTOF/MS analysis was carried out by using an Xevo G2 QTOF mass spectrometer using an electrospray ionization (ESI) source (Waters, Milford, MA, USA) equipped with a BEH-C18 column (2.1 mm × 100 mm, 1.7 μm; Waters, Milford, MA, USA). Xray crystallographic analysis was carried out on a Rigaku Oxford Diffraction Supernova diffractometer with Cu Kα radiation. NMR data were determined on Bruker AV-500 and AV-600 spectrometers (Bruker, Karlsruhe, Germany) with TMS as an internal standard. Column chromatographies were performed on silica gel (200−300 mesh, Qingdao Marine Chemical Plant, Qingdao, China), reversedphase C18 silica gel (Merck, Darmstadt, Germany), and Sephadex LH20 (Pharmacia Biotech AB, Uppsala, Sweden). The analytical and preparative HPLC was performed on an Agilent 1260 instrument equipped with a multiple wavelength diode array detector (DAD), accompanied by Cosmosil C18 (4.6 mm × 250 mm, 5.0 μm) and Cosmosil 5C18-MS-II (10 mm × 250 mm, 5.0 μm) columns, respectively. The chiral separation of the enantiomers was carried out using Phenomenex Lux Cellulose-3 (4.6 mm × 250 mm, 5 μm) and Cellulose-4 (4.6 mm × 250 mm, 5 μm) chiral HPLC columns. All solvents used in column chromatography and HPLC were of

2.13

a

CC50 is the concentration of compound with half maximal inhibition on the growth and survival of Vero cells. bIC50 is the concentration of compound that reduced 50% CPE as compared to control cells infected with HSV-1. cSelectivity index value equaled CC50/IC50. d Not active at the tested concentration.

6 showed strong cytotoxicity against the host cells. The above results suggested that the 2,4-dimethyl-cinnamyl-phloroglucinol moiety is not essential for the anti-HSV-1 activity; however, coupling with this unit could significantly reduce the cytotoxicity.

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CONCLUSION Four novel phloroglucinol derivatives (1−4) representing two unusual carbon frameworks, together with their hypothetical phloroglucinol precursors, were discovered from the leaves of Cleistocalyx operculatus. Compounds 1 and 2 are two pairs of new enantiomeric phloroglucinol dimers with an unprecedented polycyclic architecture possessing a highly functionalized dihydropyrano[3,2-d]xanthene tetracyclic core. Compounds 3 and 4 are two novel PTAs featuring a 2,4-dimethylcinnamyl-phloroglucinol motif, which is unprecedented in family Myrtaceae. Compound 1 exhibited obvious in vitro antiHSV-1 activity. With the rare phloroglucinol precursors and unusual coupling pattern involved in the biosynthetic pathway

Figure 8. (A) Antivirus activities of 1 and 6 against GFP-HSV-1 by using a fluorescence assay. Scale bar, 50 μm. Fluorescence intensity of (B) 1 and (C) 6 on GFP-HSV-1. Effects of (D) 1 and (E) 6 on the viability of Vero cells. All data represent the means ± SD of three different experiments. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, different from the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, different from the virus group. H

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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CDCl3, 25 °C) and 13C NMR (125 MHz, CDCl3, 25 °C) data see Table 2; HR-ESI-MS m/z 433.2371 ([M + H]+ calcd for C28H33O4 433.2373). Operculatol B (4): yellow oil; [α]25 D +69.0° (c 0.10, MeOH); UV (CHCl3) λmax (log ε) 215 (4.29), 346 (3.98) nm; CD (MeOH) λmax (Δε) 219 (2.32), 248 (−7.39), 280 (−2.84), 338 (6.54); IR (KBr) νmax 3351, 2921, 1620, 1560, 1449, 1349, 1174, 1106, 989 cm−1; 1H (500 MHz, CDCl3, 25 °C) and 13C NMR (125 MHz, CDCl3, 25 °C) data see Table 2; HR-ESI-MS m/z 433.2374 ([M + H]+ calcd for C28H33O4 433.2373). 2′,4′-Dihydroxy-3′,5′-dimethyl-6′-methoxychalcone (5): orange blocks (MeOH), mp 168−169 °C; UV (MeOH) λmax (log ε) 212 (4.36), 344 (4.22) nm; IR (KBr) νmax 3399, 1626, 1604, 1542, 1352, 1222, 1167, 1107, 986 cm−1; 1H NMR (500 MHz, CDCl3, 25 °C) δ 13.67 (s, 1H), 8.00 (d, J = 15.7 Hz, 1H), 7.84 (d, J = 15.7 Hz, 1H), 7.66−7.61 (m, 2H), 7.44−7.37 (m, 3H), 5.83 (s, 1H), 3.66 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H); 13C NMR (125 MHz, CDCl3, 25 °C) δ = 193.5, 162.1, 159.6, 158.9, 143.0, 135.4, 130.3, 129.0 (×2), 128.5 (×2), 126.8, 109.2, 109.1, 106.9, 62.4, 8.4, 7.7; HR-ESI-MS m/z 321.1101 ([M + Na]+ calcd for C18H18NaO4 321.1097). Champanone B (6): yellow oil; UV (MeOH) λmax (log ε) 234 (3.49), 297 (3.30), 373 (3.65) nm; IR (KBr) νmax 3295, 2980, 2927, 2860, 1655, 1620, 1512, 1449, 1387, 1314, 1197, 1148, 1115, 1011, 876 cm−1; 1H NMR (600 MHz, DMSO-d6, 25 °C) δ 8.23 (d, J = 15.9 Hz, 1H), 7.83 (d, J = 15.9 Hz, 1H), 7.71−7.67 (m, 2H), 7.48−7.45 (m, 3H), 1.81 (s, 3H), 1.34 (s, 6H, 2 × CH3) ppm; 13C NMR (150 MHz, DMSO-d6, 25 °C) δ 196.8, 190.3, 185.7, 176.2, 143.0, 134.9, 130.6, 129.2 (×2), 128.5 (×2), 123.6, 104.8, 103.4, 48.6, 24.3, 7.6 ppm; HR-ESI-MS m/z 321.1100 ([M + Na]+ calcd for C18H18NaO4 321.1097). X-ray Crystallographic Analyses. Compounds 1, 2, 3, and 5 were crystallized by using the solvent vapor diffusion method. The crystal data of compounds 1, 2, 3, and 5 were collected on a Rigaku Oxford Diffraction Supernova diffractometer with Cu Kα radiation (λ = 1.54184 Å). The crystal structures were solved by direct methods using the SHELXS-2015 software package and refined by the fullmatrix least-squares method. In the structure refinements, nonhydrogen atoms were refined anisotropically, and the hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. Crystal data of compounds 1, 2, 3, and 5 in the standard CIF format were deposited with the Cambridge Crystallographic Data Centre with CCDC numbers of 1573199, 1573202, 1837527, and 1837526, respectively. The crystallographic data of compounds 1, 2, 3, and 5 were shown in Tables S1−S4. Quantum Chemical ECD Calculations. The systematic random conformational analysis of 1−4 were performed in the SYBYL X 2.1 program using MMFF94s molecular force field, which afforded 46, 44, 41, and 34 conformers for 1−4, respectively, with an energy cutoff of 10 kcal mol−1 to the global minima. All of the obtained conformers were further optimized using DFT at the B3LYP/6-311G* level in the gas phase by using Turbomole 7.0 (1 and 2) or Gaussion09 (3 and 4) software. The number of stable conformers optimized for 1−4 were 7, 5, 5, and 10, respectively. These conformers were selected and used for TDDFT [B3LYP/6-311G*] computations in Turbomole 7.0 (1 and 2) or Gaussion09 (3 and 4), with the consideration of the first 100 (1 and 2) or 50 (3 and 4) excitations. The overall ECD curves of 1−4 were all weighted by Boltzmann distribution. The calculated ECD spectra of 1−4 were subsequently compared with the experimental ones. The ECD spectra were produced by SpecDis 1.70.1 software. Cell and Virus. African green monkey kidney cells (Vero cells, ATCC CCL-81) were purchased from the ATCC. Herpes simplex virus type 1 (HSV-1 wild strains, ATCC VR-733) was kindly provided by Medicinal Virology Institute of Wuhan University, China. Vero cells were grown in Dulbecco’s modified eagle medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 1% penicillin−streptomycin solution. The medium containing DMEM with 2% FBS and 1% penicillin− streptomycin solution were used for the cytotoxicity assay and antiviral assay, respectively. HSV-1 propagated on Vero cells and was

analytical grade (Tianjin Damao Chemical Plant, Tianjin, China) or chromatographic grade (Merck, Darmstadt, Germany), respectively. Plant Material. The leaves of Cleistocalyx operculatus (Roxb.) Merr. & Perry were collected from Haikou, Hainan province of China in July of 2015 and authenticated by Professor Guang-Xiong Zhou (Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, China). A voucher specimen (no. 20150731) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University. Extraction and Isolation. The air-dried and powdered leaves of C. operculatus (20.0 kg) were extracted with 95% EtOH three times at room temperature. The combined EtOH residue (3.2 kg) was suspended in water and extracted successively with petroleum ether, chloroform, and n-butanol. The petroleum ether soluble fraction (1200 g) was therefore subjected to silica gel column eluted with a gradient mixture of petroleum ether/ethyl acetate (100:0 to 0:100, v/ v) to afford 13 major fractions (Fr. A−Fr. M). Fr. H (50.6 g) was subsequently separated by an ODS column using MeOH−H2O (60:0 to 100:0, v/v) as an eluent to afford six subfractions (Fr. H1−Fr. H6). Then, Fr. H4 was separated by a Sephadex LH-20 column (MeOH− CHCl3, 50:50, v/v) to afford Fr.H4a−Fr. H 4f. Fr. H4c was further subjected to an ODS column using MeCN−H2O (90:10, v/v) as an eluent to afford compounds 1 (21 mg) and 2 (22 mg). Fr. E (49.2 g) was separated by a silica gel column using a gradient mixture of petroleum ether−acetone (100:0 to 0:100, v/v) as the eluent to afford Fr. E1−E7. Subsequently, Fr. E3 was subjected to an ODS column using MeCN−H2O (70:0 to 100:0, v/v) as an eluent to yield Fr. E3a−Fr. E3d. Then, Fr. E3c was separated by a Sephadex LH-20 column (MeOH−CHCl 3, 70:30, v/v) followed by repeated preparative HPLC to afford compound 3 (7 mg) and 4 (18 mg). Fr. K was subjected to a silica gel column eluted with a gradient mixture of cyclohexane−ethyl acetate (100:5 to 0:100, v/v) to obtain four subfractions (Fr. K1−Fr. K4). Fr. K4 was further separated by a Sephadex LH-20 column with methanol as the mobile phase followed by preparative HPLC to afford compounds 5 (80 mg) and 6 (9 mg). Chiral Separation of (±)-1 and (±)-2. The racemic mixture of (±)-1 and (±)-2 were subjected to a chiral HPLC column, respectively, to afford the optical pure enantiomers (+)-1, (−)-1, (+)-2, and (−)-2. For (±)-1, a Phenomenex Lux Cellulose-4 column (4.6 mm × 250 mm, 5 μm) was used as the stationary phase and MeCN−H2O (85:15, v/v) was used as the mobile phase, while (±)-2 was subjected to a Phenomenex Lux Cellulose-3 column (4.6 mm × 250 mm, 5 μm) eluted with MeCN−H2O (67:33, v/v). (±)-Cleistoperlone A (1): yellow blocks (acetone), mp 186−187 °C; [α]25 D ± 0° (c 0.10, MeOH); UV (CHCl3) λmax (log ε) 210 (3.69), 302 (3.49), 338 (3.45) nm; IR (KBr) νmax 3479, 1606, 1560, 1460, 1409, 1361, 1162, 1107, 908, 698, 621 cm−1; 1H (500 MHz, CDCl3, 25 °C) and 13C NMR (125 MHz, CDCl3, 25 °C) data see Table 1; HR-ESI-MS m/z 595.2325 ([M + H]+, calcd for C36H35O8 595.2326). (+)-1: [α]25 D +25.0° (c 0.10, MeOH), CD (MeOH) λmax (Δε) 217 (16.83), 240 (−1.90), 277 (−0.74), 295 (1.14), 334 (−7.46) (−)-1: [α]25 D −24.0° (c 0.10, MeOH), CD (MeOH) λmax (Δε) 220 (−7.73), 240 (3.21), 277 (1.74), 303 (−0.91), 334 (5.50) (±)-Cleistoperlone B (2): yellow blocks (CHCl3−MeOH, 1:1), mp 180−181 °C; [α]25 D ± 0° (c 0.10, MeOH); UV (CHCl3) λmax (log ε) 209 (3.68), 302 (3.47), 339 (3.44) nm; IR (KBr) νmax 3459, 1616, 1565, 1449, 1413, 1358, 1162, 1106, 902, 689, 619 cm−1; 1H (500 MHz, CDCl3, 25 °C) and 13C NMR (125 MHz, CDCl3, 25 °C) data see Table 1; HR-ESI-MS m/z 617.2149 ([M + Na]+ calcd for C36H34NaO8 617.2146). (+)-2: [α]25 D +19.7° (c 0.10, MeOH), CD (MeOH) λmax (Δε) 237 (8.94), 252 (2.21), 270 (9.71), 299 (17.72), 325 (−10.10). (−)-2: [α]25 D −19.5° (c 0.10, MeOH), CD (MeOH) λmax (Δε) 237 (−9.55), 252 (−1.82), 270 (−7.74), 299 (−16.42), 325 (6.41). Operculatol A (3): yellow blocks (MeOH), mp 175−176 °C; [α]25 D −33.0°( c 0.10, MeOH); UV (CHCl3) λmax (log ε) 214 (4.28), 346 (4.16) nm; CD (MeOH): λmax (Δε) 206 (−14.00), 227 (−4.79), 249 (6.06), 301 (3.16), 347 (−1.80); IR (KBr) νmax 3236, 2989, 2921, 2857, 1622, 1560, 1449, 1352, 1181, 1106, 998 cm−1; 1H (500 MHz, I

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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harvested when 100% cytopathic effect of the virus was observed. Virus titers were determined by the 50% tissue culture infective dose (TCID50) method and the HSV-1 strains were stored at −80 °C until use. Cytotoxicity Assay. The cytotoxicity of compounds was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in 96-well plates. In brief, Vero cells were seeded at a density of 1.2 × 104 cells per well. Subsequently, 2-fold serial dilutions of compounds (100, 50, 25, 12.5, 6.25, and 3.125 μM) were added to confluent Vero cell monolayers in triplicate, and the medium without sample was used as a cell control. After incubation for 48 h, the cells were treated with MTT solution for another 4 h. Subsequently, the supernatant was removed, and dimethyl sulfoxide (DMSO) was added to dissolve the formazan. The optical density (OD) values were measured with an enzyme immunoassay reader (Thermo Labsystems Multiskan MK3) at 570 nm. The 50% cytotoxic concentration (CC50) of the sample was calculated by regression analysis of the dose− response curve generated from the OD values. The maximal noncytotoxic concentration (MNCC) of the sample on the cells was also calculated through the OD values. Antiviral Assay. The anti-HSV-1 activity of the compounds was determined by the cytopathic effect (CPE) reduction assay. The antiviral activity of each compound was tested using the MNCC as the initial concentration. Briefly, Vero cell monolayers (1.2 × 104 cells per well) were inoculated with the virus suspension (100 TCID50) and mixture of a serial 2-fold dilution of sample. The virus suspension and medium without sample were added as virus and cell controls, respectively. Acyclovir was used as a reference compound. After 48 h of incubation at 37 °C, the CPE of each well was examined using an inverted microscope (CKX41, Olympus Corporation, Tokyo, Japan). The antiviral effects of compounds were expressed by the 50% inhibitory concentration (IC50) that defined as the concentration required for reducing 50% of CPE in respect to the virus control. Fluorescence Assay. Vero cells were seeded at a concentration of 1.2 × 104 cells/well in 96-well plates and incubated overnight to form a monolayer. Afterward, the cell monolayer was treated with 1 or 6, and infected with GFP-HSV-1 at MOI = 0.5. After infection for 24 h in the 37 °C, 5% CO2 incubator, the green fluorescence was photographed by the High-Content Cellular Analysis System (INCELL 6000 imaging system, GE, USA).

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‡

J.-C.S. and S.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (81622045, U1401225, and 81630095) and the Science and Technology Planning Project of Guangdong Province (2016B030301004). This work was also supported by the high-performance computing platform of Jinan University.

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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.joc.8b01050. UPLC-QTOF/MS analysis of petroleum ether soluble fraction, chiral HPLC separation of (±)-1 and (±)-2, UV, IR, HR-ESI-MS, and NMR spectra data of 1−6, detailed quantum chemical ECD calculations for 1−4, and crystallographic data of 1−3 and 5 (PDF) X-ray data for 1 (CIF) X-ray data for 2 (CIF) X-ray data for 3 (CIF) X-ray data for 5 (CIF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jun-Cheng Su: 0000-0002-1458-5383 Xiao-Jun Huang: 0000-0002-3636-4813 Lei Wang: 0000-0001-9242-1109 Wen-Cai Ye: 0000-0002-2810-1001 Ying Wang: 0000-0003-4524-1812 J

DOI: 10.1021/acs.joc.8b01050 J. Org. Chem. XXXX, XXX, XXX−XXX

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