Cnidimonins A–C, Three Types of Hybrid Dimer from Cnidium

Letter pubs.acs.org/OrgLett

Cnidimonins A−C, Three Types of Hybrid Dimer from Cnidium monnieri: Structural Elucidation and Semisynthesis Fangyi Su,† Zheng Zhao,† Shuanggang Ma,† Rubing Wang,† Yong Li,† Yunbao Liu,† Yuhuan Li,‡ Li Li,† Jing Qu,*,† and Shishan Yu*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China ‡ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China S Supporting Information *

ABSTRACT: Three pairs of racemic dimers, (±)-cnidimonins A−C (1−3), were isolated from the fruits of Cnidium monnieri. They represent novel hybrid-dimerization patterns of coumarin skeleton with structurally diverse units (flavonol, benzofuran, and chromone) via an unprecedented terminal chiral carbon of prenyl. The absolute configurations of the enantiomers were determined by electronic circular dichroism (ECD). To investigate their bioactivities in depth, (±)-cnidimonins A−C (1−3) were synthesized. The racemic mixture (±)-1 exhibited stronger antiviral activity against HSV-1 (IC50: 1.23 μM) than its corresponding optically pure enantiomers.

Herein, we describe the isolation, structural elucidation, chiral separation, semisynthesis, and antiviral activity of (±)-cnidimonins A−C (1−3, Figure 1). Cnidimonin A (1) was obtained as a yellow powder. The HRESIMS of 1 ([M + H]+, m/z 529.1499) combined with the 1 H NMR and 13C NMR data indicated a molecular formula of C30H24O9 (19 degrees of unsaturation). The IR spectrum showed the presence of hydroxyl (3262 cm−1), carbonyl (1717, 1649 cm−1), and aromatic groups (1601, 1512 cm−1). The 1H NMR spectrum (Table S1) exhibited signals of two pairs of ABtype doublets δH 7.94, 6.21 (each 1H, J = 9.6 Hz, H-4″, H-3″); δH 7.51, 6.97 (each 1H, J = 8.4 Hz, H-5″, H-6″) as well as one methoxyl (δH 3.62, 3H, s), suggesting the presence of a 7methoxy-8-substituted coumarin nuclei.13 The presence of the prenyl chain was determined on the basis of the 1H NMR signals at δH 6.01, 6.10 (each 1H, d, J = 9.6 Hz, H-11″, 12″) and two methyls (δH 1.63, 1.57, each 3H, s), corresponding to the 13 C NMR signals at δC 31.0, 125.3, 129.3, 17.5, 25.4, which was verified by the HMBC correlations of H3-15″/C-12″, C-13″; H3-14″/C-13″; H-11″/C-12″, and C-13″. In addition, the remaining aromatic singlet at δH 6.12 (1H, s) as well as four

Cnidium monnieri (L.) Cusson (Umbelliferae) is an annual gregarious herb widely grown in Vietnam, China, Japan, and Russia. Its fruits have been used as a well-known traditional Chinese medicine for the treatment of asthma, cough, fungal infection, and arrhythmia.1−5 Recent studies have shown that the isolated constituents from this plant exhibit diverse biological activities.2−8 For example, osthol, a coumarin compound isolated from the fruits of C. monnieri, was found to have antihepatitis B virus,9 anti-inflammatory,10 antidiabetic,11 and antitumor activities.12 To find new bioactive constituents from this plant, three pairs of enantiomeric natural dimers, (±)-cnidimonins A−C (1−3), which represent three novel types of hybrid dimers, coumarin−flavonol, coumarin−benzofuran, and coumarin− chromone, featuring an unprecedented terminal chiral-carbon linkage of prenyl, were isolated. Structurally, cnidimonins A−C possess the same coumarin moiety and only one chiral center at (C-11″), indicating that they were derived from a common biosynthesis precursor of osthol. Although their unprecedented structure piqued our interest, it was difficult to investigate their bioactivities in depth due to the scarcity of material. Thus, it was necessary to develop a synthetic strategy for the cnidimonins. © 2017 American Chemical Society

Received: August 2, 2017 Published: August 31, 2017 4920

DOI: 10.1021/acs.orglett.7b02290 Org. Lett. 2017, 19, 4920−4923

Letter

Organic Letters

Figure 1. Structures of (±)-cnidimonins A−C.

protons belonging to a para-substituted phenyl were assigned to 4′,5,7-trihydroxyflavonol (kaempferol)14,15 substituted at C-6 or C-8. In the HMBC experiment (Figure 2), the attachment of

Figure 3. Experimental ECD spectra of (+)-1/(−)-1, (+)-2/(−)-2,and (+)-3/(−)-3 with calculated ECD spectra of 1a-(11″S)/1b-(11″R), 2a-(11″R)/2b-(11″S), and 3a-(11″S)/3b-(11″R).

showed a total of 27 carbon resonances. Except for an osthol moiety, the 13C NMR data showed the presence of one carbonyl carbon, six aromatic carbons from a benzene ring, two olefinic carbons, two methylenes, and one methoxy. The above data, combined with the degrees of unsaturation, indicated the presence of a benzofuran moiety, which was confirmed by the HMBC correlations of H-1′/C-4, C-5; H-6/C-1′. Additionally, the coupling system detected on the COSY and HSQC spectra consisted of two methylenes at δH 2.87 (2H, t, J = 7.5 Hz, 15.0 Hz, H-7) and δH 2.57 (2H, t, J = 7.5 Hz, 15.0 Hz, H-8). The presence of the C7−C9 unit was verified by the HMBC correlations of H2-7/C-9. Moreover, the C7−C9 unit was determined to be attached to C-1 through C-7 by the HMBC correlations from H-7 to C-1 (δC 123.8), C-2 (δC 153.2), and C-6 (δC 121.2) (Figure 4). The linkage of the osthol moiety

Figure 2. Selected 1H−1H COSY and HMBC correlations for 1.

the prenyl group to the kaempferol moiety was deduced to be via C-11″−C-8 based on HMBC correlations of H-11″with C-7 (δC 161.7), C-8 (δC 107.3), and C-9 (δC 154.5). Additionally, the linkage of the prenyl group to coumarin moiety through C11″−C-8″ was further supported by the observed HMBC correlations of H-11″ to C-7″ (δC 160.1), C-8″ (δC 119.4), and C-9″ (δC 152.5). Thus, the planar structure of 1 was determined as depicted (Figure 1). Despite the presence of a stereogenic center at C-11″, measurement of the optical rotations of 1 gave a [α]25D value near zero, and no Cotton effects were observed in its ECD spectra, indicating that 1 was a racemic mixture. Subsequently, chiral resolution of 1 was performed on chiral column to yield (+)-cnidimonin A [(+)-1] and (−)-cnidimonin A [(−)-1] in equal amounts (see Figure S57). The CD spectra of (+)-1 and (−)-1 revealed opposite Cotton effects, confirming their enantiomeric relationship. The absolute configurations of (+)-1 and (−)-1 were determined by comparison of the experimental and calculated ECD spectra. As shown in Figure 3, the calculated ECD spectra for (11″R)-1b and (11″S)-1a were consistent with the experimental ECD spectra for (−)-1 and (+)-1, respectively. Accordingly, the absolute configuration of the enantiomers were determined as (+)-(11″S)-1 and (−)-(11″R)-1. Cnidimonin B (2) was isolated as a white, amorphous powder. Its molecular formula was established as C27H26O7 by HRESIMS ([M + H]+, m/z 463.1751), corresponding to 15 degrees of unsaturation. The IR spectrum of 2 showed the presence of hydroxyl (3329 cm−1), carbonyl (1732 cm−1), aromatic groups (1605, 1460 cm−1). The comparison of NMR data (see Table S1) of 1 and 2 suggested that the structure of 2 should possess an osthol moiety. The 13C NMR spectrum of 2

Figure 4. Selected 1H−1H COSY and HMBC correlations for 2.

and the benzofuran moiety via C-11″-C-2′ was established by the HMBC correlations of a methine proton at δH 5.73 (H11″) with C-1′ (δC 102.7), C-2′ (δC 158.0), and C-8″ (δC 117.3). Thus, the planar structure of 2 was established as shown in Figure 1. In contrast to 1, 2 also possessed a stereogenic center at C-11″. (+)-Cnidimonin B [(+)-2] and (−)-cnidimonin B [(−)-2] were successfully obtained in a ratio of approximately 1:1 by using HPLC on a chiral column (see Figure S57). The absolute configuration of each enantiomer was determined by calculation of the ECD spectrum. As shown in Figure 3, the absolute configuration of (−)-2 was assigned as 11″R and that of (+)-2 as 11″S. Cnidimonin C (3) was purified as a white, amorphous powder. It possessed a molecular formula of C30H30O8 with 16 degrees of unsaturation, as deduced from HRESIMS ([M + H]+, m/z 519.2016). The IR spectrum of 3 showed the presence of hydroxyl (3401 cm−1), carbonyl (1729 cm−1), 4921

DOI: 10.1021/acs.orglett.7b02290 Org. Lett. 2017, 19, 4920−4923

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Organic Letters aromatic groups (1604, 1495 cm−1). Analysis of its NMR data (see Table S1) suggested 3 possessed the same osthol moiety as 1 and 2. Except for the osthol moiety, the 13C NMR spectrum revealed 15 carbon resonances ascribed to one carbonyl carbon, six aromatic carbons from a benzene ring, four olefinic carbons, one methylene, two methyls, and one hydroxymethyl. The NMR data of 3 exhibited signals corresponding to a 6-substituted 5,7-dihydroxy-2-methylchromone ring,16 which was confirmed by comparison with literature data of related structures. Additionally, the HMBC correlations of H3-5′/C-2′, C-3′, C-4′; H2-4′/C-2′, and H2-1′/ C-5, C-6, C-7 verified the presence of the prenyl chain and its location at C-6 (Figure 5). The key HMBC correlations of a

Scheme 1. Synthetic Route of 7

Scheme 2. Synthetic Route to 11

Alkylation of 12 with prenyl bromide led to the known compound 13,20 which was oxidized with selenium dioxide in 95% ethanol solution to afford 14. The key intermediate 15 can be constructed from 14 over two steps via acetylation with acetic anhydride in pyridine and aldol reaction by NaH (Scheme 3).21

Figure 5. Selected 1H−1H COSY, HMBC, and NOE correlations for 3.

methine proton at δH 6.05 (H-11″) with C-7 (δC 162.1), C-8 (δC 108.3), and C-9 (δC 155.7) suggested that the osthol moiety was linked to chromone moiety via C-11″−C-8. Meanwhile, the C-2′/C-3′ olefin was assigned Z-geometry based on NOE correlations of H-2′ with H3-5′ and H-1′a, and H2-4′ with H-1b (Figure 5). Thus, the planar structure of 3 was established as shown in Figure 1. Three was a racemate based on the lack of optical activity. Subsequently, 3 was separated into two equal amounts of optically pure enantiomers, (+)-cnidimonin C [(+)-3] and (−)-cnidimonin C [(−)-3] using HPLC on a chiral column (see Figure S57). The absolute configurations of (+)-3 and (−)-3 were established by comparing of the calculated ECD spectra with experimental spectra. From the above evidence, the absolute stereochemistry for (+)-3 (11″R) and (−)-3 (11″S) were unambiguously determined as shown in Figure 3. The novel structures of cnidimonins A−C were intriguing. However, it is difficult to further explore their bioactivities due to the scarcity of material, which makes the synthesis of cnidimonins extraordinarily meaningful. In this paper, the synthesis of cnidimonins A−C was efficiently achieved. One common characteristic of the structures is that each cnidimonin derivative possesses the same moiety 7, which simplifies the retrosynthetic analysis (see Figure S56) and makes it reasonable to propose the synthetic strategy of connecting 7 with 8, 11, and 15, especially since 8 (kaempferol) is commercially available. Osthol 4 was isolated from C. monnieri. Following the Sharpless dihydroxylation of 4 with an AD-mix-β,17 diol 5 was obtained in high yield at room temperature for 12 h. Subsequent reaction of 5 with N,N′-carbonyldiimidazole (CDI) afforded the cyclic carbonate 6, which was converted into the key intermediate 7 by reaction with DBU in methylbenzene at 120 °C for 12 h (Scheme 1).18 Starting from commercially available psoralen 9, 11 can be accomplished in 41% yield over two steps by sequences of hydrolyzation with MeONa in dry methanol and reduction by magnesium powder (Scheme 2).19

Scheme 3. Synthetic Route to 15

With 7, 8, 11, and 15 in hand, we next focused on their coupling. Condensation of 7 with 8, 11, and 15 in the presence of Nafion 50 was accomplished in methanol solution at room temperature for 12 h to afford the racemic dimers (±)-1−3 in 15%, 12%, and 10% yield, respectively (Scheme 4).22 Spectral data for 1−3 were identical in all respects to the natural Scheme 4. Synthesis of (±)-cnidimonins A-C (1-3)

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DOI: 10.1021/acs.orglett.7b02290 Org. Lett. 2017, 19, 4920−4923

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Organic Letters *E-mail: [email protected].

cnidimonins A−C, respectively (see Figures S47−S55). Synthetic racemates 1−3 were successfully separated by HPLC using a CHIRALPAK AD-H column to obtain the optically pure enantiomers. The synthetic racemic mixtures of cnidimonins A−C as well as their enantiomers were tested for antiviral (HSV-1) activity (see Table 1). Each enantiomer was obtained with enantio-

ORCID

Yunbao Liu: 0000-0002-1338-0271 Shishan Yu: 0000-0003-4608-1486 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the CAMS Innovation Fund for Medical Science (CIFMS: 2016-I2M-1-010) and the National Natural Science Foundation of China (No. 81573321). We are grateful to the Department of Instrumental Analysis of Peking Union Medical College for the spectroscopic measurements.

Table 1. Antiviral Activity against HSV-1 and Cytotoxicity of Compounds in Vero Cellsa compd

TC50b (μM)

IC50 (μM)

(±)-1 (+)-1 (−)-1 (±)-2 (+)-2 (−)-2 (±)-3 (+)-3 (−)-3 acyclovird

2.14 19.25 16.02 23.11 23.11 23.11 100 >100 >100 >100

1.23 6.41 3.70 >11.11 11.11 >11.11 >33.33 >33.33 >100 0.41

SIc 1.7 3.0 4.3

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2.1

>243.9

a

Data represent mean values for three independent determinations. Cytotoxic concentration required to inhibit Vero cell growth by 50%. c Selectivity index value equaled TC50/IC50; dPositive control. b

meric excess (ee) ≥99%. Acyclovir was used as the positive control, with IC50 values of 0.41 μM. The racemic mixture (±)-1 possessed strong activity against HSV-1 with IC50 values of 1.23 μM. The pure enantiomers, (+)-1 and (−)-1, exhibited weaker activity against HSV-1 (IC 50 : 6.41, 3.70 μM, respectively) than their corresponding racemic mixture (±)-1. (+)-2 showed moderate activity, with IC50 values of 11.11 μM. The bioassay data indicated that synergistic effects between (+)-1 and (−)-1 against HSV-1 may exist. In this contribution, cnidimonins A−C represent a new carbon skeleton formed through unique hybrid-dimerization patterns of coumarin with structurally diverse units (flavonol, benzofuran, and chromone) via an unprecedented terminal chiral carbon of prenyl. To investigate their bioactivity in depth, the synthesis of cnidimonins A−C was efficiently achieved. Coupling two different moieties through convergent synthesis was used as the key step to construct dimers. Remarkably, the racemic mixture, (±)-1, showed stronger antiviral activity than the corresponding pure enantiomers, suggesting that a synergistic effect occurred, which might give some insight into new lead ligands for the development of antiviral drugs. Meanwhile, further investigations such as the synthesis of analogues with more potent bioactivity and in depth biological testing, are still required.

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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.7b02290. Detailed experimental procedures; 1D and 2D NMR, MS, and IR spectra for novel compounds (PDF)

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*E-mail: [email protected]. 4923

DOI: 10.1021/acs.orglett.7b02290 Org. Lett. 2017, 19, 4920−4923