Fitoterapia 95 (2014) 234–239
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Antimalarial diterpene alkaloids from the seeds of Caesalpinia minax Guoxu Ma a, Zhaocui Sun a, Zhonghao Sun a, Jingquan Yuan b, Hua Wei c, Junshan Yang a, Haifeng Wu a,⁎, Xudong Xu a,⁎ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, PR China b National Engineering Laboratory of Southwest Endangered Medicinal Resources Development, National Development and Reform Commission, Guangxi Botanical Garden of Medicinal Plant, Nanning 530023, PR China c Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, PR China
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Article history: Received 27 February 2014 Accepted in revised form 30 March 2014 Available online 13 April 2014 Keywords: Caesalpinia Diterpene alkaloid Antimalarial activity Biosynthetic pathway
a b s t r a c t Two new diterpene alkaloids, caesalminines A (1) and B (2), possessing a tetracyclic cassane-type furanoditerpenoid skeleton with γ-lactam ring, were isolated from the seeds of Caesalpinia minax. Their structures were determined by different spectroscopic methods and ECD calculation. The plausible biosynthetic pathway of caesalminines A and B was proposed. The anti-malarial activity of compounds 1 and 2 is presented with IC50 values of 0.42 and 0.79 μM, respectively. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The search for new antimalarial drug is still urgent because malaria parasites have developed resistance to almost every drug human has thrown at them over the decades. The isolation of artemisinin has inspired us to pay close attention to traditional medicinal plants. Based on its activity against the malaria strain Plasmodium falciparum, a methanol extract of the seeds of Caesalpinia minax was selected for evaluation as a possible source of new antimalarial agents. C. minax, whose seeds have been known as Kushilian in Guangxi folk medicine long used for the treatment of anemopyretic colds, dysentery, skin itching and sores, is a rich source of cassane-type diterpenes [1]. The cassane-type diterpenoids, mainly found ⁎ Corresponding authors at: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing, China. Tel./fax: + 86 10 5783 3296. E-mail addresses: [email protected] (H. Wu), [email protected] (X. Xu).
http://dx.doi.org/10.1016/j.fitote.2014.04.001 0367-326X/© 2014 Elsevier B.V. All rights reserved.
to be distributing in various genera of Fabaceae family (especially Caesalpinia genus), are a group of structurally diverse natural products exhibiting a wide range of pharmacological activities such as antimalarial, antitumor, antiinflammatory, antiviral, antimicrobial and antitrypanosomal properties [2,3]. In recent years, successive discoveries of new cassane diterpenoids, some of which possess novel frameworks, have made great contribution to structural diversities of this family of diterpenes [4–17]. To date, more than 400 cassane diterpenoids have been obtained. Among them, caesanines A–D, four new diterpene alkaloids isolated from Caesalpinia sappan, possess unprecedented N bridge in cassane skeleton [17]. Our previous studies on C. minax have resulted in the isolation of a series of new diterpenes [18–25]. In our further search for structurally unique diterpenes with antimalarial activity, two new diterpene alkaloids, caesalminines A (1) and B (2) (Fig. 1), possessing a tetracyclic cassane-type furanoditerpenoid skeleton with a nitrogen atom incorporated in lactone ring, were isolated from the MeOH extracts of the seeds of C. minax. Compounds 1 and 2 showed significant antimalarial activities against the
G. Ma et al. / Fitoterapia 95 (2014) 234–239
O
R N
16 15
12
OAc 11 AcO
13
20
2 1 3 4
10 5
14 9 8 6 7
17
OH 19
18
1 R=H 2 R = C2H4OH Fig. 1. Caesalminines A (1) and B (2) from Caesalpinia minax.
multidrug-resistant K1 strain of P. falciparum. Herein, we describe the isolation and structure elucidation of the new bioactive compounds. 2. Experimental section 2.1. General Optical rotations were obtained on a PerkinElmer 341 digital polarimeter. UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometer, respectively. ECD spectra were obtained using a JASCO J-815 spectropolarimeter. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HR-ESI-MS spectra were performed on a LTQ-Obitrap XL spectrometer. C18 reversed-phase silica gel (40–63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel (CHP 20P, 75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan) and silica gel (100–200 and 300–400 mesh, Qingdao Marine Chemical plant, Qingdao, People's Republic of China) were used for column chromatography. And pre-coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works). All isolated compounds were purified to 95% purity or better, as judged by HPLC and by NMR before determining bioactivity. The NMR spectra can be found in the Supplementary data. 2.2. Plant material The seeds of C. minax were collected in October 2011 from Nanning, Guangxi Province, China and identified by Prof. Jing-Quan Yuan, Department of Pharmaceutical Chemistry, Guangxi Botanical Garden of Medical Plant. A voucher specimen (NO. 21782) was deposited at the Guangxi Botanical Garden of Medical Plant. 2.3. Extraction and isolation The air-dried and powered seeds of C. minax Hance (750 g), were extracted three times with methanol. Removal of the
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methanol under reduced pressure yielded a methanol extract (190 g). The residue was subjected to column chromatography on silica gel eluting with hexane, chloroform, ethyl acetate, acetone and methanol, respectively. The chloroform fraction (28 g) was found to be antimalarial with an IC50 value of 4.8 μg/mL and was subjected to silica gel (100–200 mesh) column chromatography using a petroleum ether–EtOAc gradient (from 1:0 to 1:1) as eluent, to yield twelve fractions (Fr. A–L). Fr. J (920 mg) was separated over the sephadex LH-20 column and MCI and four fractions (Fr. J1–J3) were collected. Then fractions J1 (230 mg) were chromatographed on a silica gel (300–400 mesh) column using petroleum ether–CHCl3 gradient (20:1; 15:1; 6:1; 2:1), followed with CHCl3–MeOH gradient (80:1; 50:1; 30:1; 10:1) as eluent, then purified by semipreparative liquid chromatography with a YMC RP-18 column using a MeOH–H2O (70:30) system yielded 1 (2.3 mg, Rt 27.6 min). Fr. J2 (190 mg) was chromatographed on a silica gel (300–400 mesh) column using petroleum ether–EtOAc (50:1; 20:1; 8:1; 4:1; 1:1; 0:1), then purified by semi-preparative liquid chromatography using a MeOH–H2O (73:27) system yield 2 (1.5 mg, Rt 30.1 min). 2.3.1. 5α-hydroxy-1α,2α-diacetoxycass-8,11,13(15)-trien-16,12lactam (caesalminine A, 1) White amorphous powder (2.3 mg); [a]20 D −24.2 (c 0.05, MeOH); UV (MeOH) λmax(log ε) 212 (3.86), 254 (3.04) nm; ECD (MeOH) 299 (Δε −0.08), 252 (Δε −0.24) nm; IR (KBr) νmax 3190, 2935, 1735 cm−1; For 1H and 13C APT spectroscopic data, see Table 1; HR-ESI-MS m/z 452.2045 (calcd for C24H31O6NNa, 452.2049). 2.3.2. 5α,2′-dihydroxy-1α,2α-diacetoxycass-8,11,13(15)-trienN-ethylidene-16,12-lactam (caesalminine B, 2) White amorphous powder (1.5 mg); [a]20 D − 22.9 (c 0.05, MeOH); UV (MeOH)λmax (log ε): 210 (3.92), 254 (3.13) nm; ECD (MeOH) 298 (Δε − 0.07) nm; IR (KBr) νmax 3194, 2940, 1738 cm−1; For 1H and 13C APT spectroscopic data, see Table 1; HR-ESI-MS m/z 496.2332 (calcd for C26H35O7NNa, 496.2311). 2.4. Rh-complex of 1 Compound 1 (0.5 mg) was dissolved in a dry solution of [Rh2(OCOCF3)4] (1.0 mg) in CDCl3 (300 μL). The first ECD spectrum was recorded immediately after mixing, and the time was monitored until stabilization of the spectrum. The inherent ECD was then subtracted, and the observed sign of the E band at 350 nm in the induced ECD spectrum was correlated with the absolute configuration of the C-5 tertiary alcohol moiety. 2.5. Antimalarial bioassay Antimalarial activity in vitro was determined by means of the microculture radioisotope technique based on the method described by Desjardins et al. [26]. The parasite P. falciparum (K1, multidrug-resistant strain) was cultured continuously according to the method of Trager and Jensen [27]. Three preparations were used for each experiment. Data are presented as means ± SEM. Statistical analyses were done by means of the
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G. Ma et al. / Fitoterapia 95 (2014) 234–239
Table 1 1 H and 13C NMR spectroscopic data of 1 and 2 (600 and 150 MHz). Position
1a δC
δH (J in Hz)
δC
δH (J in Hz)
1 2
73.1 d 67.5 d
5.71, d (2.4) 5.34, d (11.4,2.4)
74.9 d 68.0 d
3
35.3 t
2.18, m 1.36, m
36.1 t
5.96, d (2.4) 5.49, dt (11.4, 2.4) 2.20, m 1.47, m
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OCOCH3
40.0 s 74.4 s 23.4 t 23.1 t 126.6 s 141.9 s 47.4 s 101.9 d 141.0 s 122.5 s 132.0 s 35.0 t 176.3 s 15.6 q 27.7 q 25.1 q 30.0 q 169.7 s 20.6 q 169.8 s 20.7 q
2-OCOCH3 5-OH \NH \NCH2CH2OH
2b
40.1 s 75.6 s 23.9 t 23.7 t 128.7 s 142.6 s 48.4 s 102.0 d 142.0 s 122.0 s 133.2 s 35.3 t 176.5 s 16.2 q 28.1 q 25.7 q 30.3 q 170.7 s 21.1 q 170.9 s 21.4 q
1.95, m 2.61, m
6.26, s
3.32, s 2.02, 1.09, 1.14, 1.31,
s s s s
1.93, s 1.94, s 3.08, br s 10.18, s
43.4 t
\NCH2CH2OH a b
61.0 t
1.97, m 2.74, m
6.48, s
3.42, s 2.13 s 1.19, s 1.22, s 1.45, s 2.00, s 2.05, s 2.84, br s 3.67, m 3.91, m 3.78, dd (10.8, 5.4)
Recorded in DMSO-d6. Recorded in CDCl3.
Student's t-test. A P value of less than 0.05 was considered significant difference. 3. Results and discussion Compound 1 was isolated as a white amorphous powder with [a]20 D −24.2 (c 0.05, MeOH). The UV spectrum is consistent with an oxindole chromophore (λmax 212 and 254 nm) and IR spectrum shows absorption bands from amidogen (3190 cm−1) and carbonyl function (1735 cm−1) [28]. Compound 1 showed a pseudomolecular ion peak at m/z 452 [M + Na]+ in the positive-ion mode ESI-MS. The odd numbered molecular weight
suggested a nitrogen atom in the molecular formula of 1. The HR-ESI-MS spectrum showed a pseudomolecular ion at m/z 452.2025 [M + Na]+, from which in conjunction with NMR data the molecular formula was established as C24H31O6N, compatible with 10° of unsaturation. The 1H NMR spectrum of 1 (Table 1), exhibited six methyl signals including three characteristic tertiary at δH 1.09 (H3-18), 1.14 (H3-19), and 1.31 (H3-20), two acetyl at δH 1.93, 1.94, and a methyl group at δH 2.02 (H3-17) attached to aromatic ring. One proton signal on sp2 carbon at δH 6.26 (s, H-11) and two oxygenated protons signals at δH 5.71 (d, J = 2.4 Hz, H-1) and 5.34 (dd, J = 11.4, 2.4 Hz, H-2), were also observed. In the 1H–1H COSY spectrum (Fig. 2), the correlation of proton signals at δH 5.71 and 5.34 suggested the presence of two oxygenated methines. A singlet at δH 10.18 was assigned to the N–H proton of the amidogen ring, its chemical shift being influenced by the amidogen [29]. The 13C APT spectrum (Table 1) showed 24 carbon resonances including three carbonyl carbons at δC 169.7, 169.8, and 176.3, six olefinic carbons at δC 101.9, 122.5, 126.6, 132.0, 141.0, and 141.9, three quaternary carbons at δC 40.0, 47.4, and 74.4, two oxymethines at δC 67.5 and 73.1, four methylenes at δC 23.1, 23.4, 35.0, and 35.3, and six methyls at δC 15.6, 20.6, 20.7, 25.1, 27.7, and 30.0. All the proton and protonated carbon resonances in the NMR of 1 were unambiguously assigned by interpretation of 2D NMR spectra. The overall 1H and 13C NMR spectroscopic data for 1 confirmed that the compound was a dehydrogenated tetracyclic cassane-type diterpene by comparison with those of previously reported compounds [30,31]. In the HMBC spectrum, the correlations of H3-OAc (δH 1.93) and H-1 with δC carbonyl signal at δC 169.7 and of H3-OAc (δH 1.94) and H-2 with carbonyl signal at δC 169.8 suggested that the acetoxy groups were attached to C-1 and C-2. Furthermore, the HMBC correlations of H-11 with C-9 (δC 141.9), C-12 (δC 141.9), C-8 (δC 126.6), and C-13 (δC 122.5) and of H3-17 with C-8 (δC 126.6), C-13 (δC 122.5), and C-14 (δC 132.0) implied the presence of benzene ring and the methyl group at C-14. In the HSQC spectrum, the singlet at δH 3.32 (s, H2-15) had direct correlation with carbon signal δC 35.0 (C-15) which indicated the presence of the non-coupled methylene. This methylene was located at C-15 by the HMBC correlations of H-15 with C-12 (δC 141.0), C-13 (δC 122.5), C-14 (δC 132.0), and C-16 (δC 176.3). The proton signal of N–H exhibited a downfield chemical shift at δH 10.18 due to the presence of the carbonyl at C-16 (δC 176.3), as
O
H N H3COCO H3COCO OH
Fig. 2. Key 1H–1H COSY (bands) and HMBC (arrows) correlations for 1.
Fig. 3. Selected NOESY correlations of 1 (↔).
G. Ma et al. / Fitoterapia 95 (2014) 234–239
237
Fig. 4. Comparison of CD spectra of 1 (blue) and 2 (black).
supported by the HMBC spectrum. In the HMBC spectrum, the correlations of N–H (δH 10.18, s) with C-12 (δC 141.9), C-13 (δC 122.5), and C-16 (δC 176.3) confirmed the presence of lactam ring. The relative configuration was determined from the NOESY spectrum. The NOEs from H3-20 to H-1, H-2, and H3-19; from H3-18 to 5-OH (δH 3.08, br s) indicated that rings A and B are in chair conformations with a trans-fused ring junction, thus, confirming the relative configurations at C-1, C-2, C-5, and C-10 (Fig. 3). The ECD spectrum of compound 1 exhibited two negative Cotton effects at 252 (Δε − 0.24) and 299 (Δε −0.08) nm due to the n → π* and π → π* transitions of the chromophores (Fig. 4). However, the absence of proper model compounds to use as references made the assignment of the absolute configuration at C-10 unreliable. Therefore, an ECD analysis was conducted for the Rh-complex of 1 (Fig. 5), which displayed a significant negative cotton effect at 350 (Δε −0.48) suggesting an R absolute configuration for C-5 [32,33]. Thus, the structure of 1 was established as
5α-hydroxy-1α,2α-diacetoxycass-8,11,13(15)-trien-16,12lactam and named caesalminine A, with unusual cassane diterpene skeleton possessing a lactam ring, representing a new class of diterpenoid alkaloids. Compound 2, also obtained as a white amorphous powder, was assigned as C26H35O7N by evaluation of HR-ESI-MS (m/z 496.2332 [M + Na]+) and NMR spectroscopic data. IR absorption bands revealed the presence of hydroxyl (3194 cm−1) and carbonyl (1738 cm−1) functionalities in 2. The NMR (Table 1) and UV spectroscopic data for this compound were analogous to those of 1, except for the appearance of two downfield methylene signals and the absence of N–H proton signal. In the 1H–1H COSY spectrum, two multiplets corresponding to the protons (δH 3.67, m; 3.91, m) had the correlation with the protons (δH 3.78, dd, J = 10.8, 5.4 Hz), suggesting the presence of oxygenated ethylidene side-chain. In the HMBC spectrum, the correlations of H-2′ (δH 3.67, m; 3.91, m) with carbon signal at δC 142.0 (C-9) and 176.5 (C-16) indicated that
Fig. 5. CD spectrum of the Rh-complex of 1 with the inherent CD spectrum subtracted, recorded in CHCl3.
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O
O H2N
O Geranylgeranyl pyrophosphate
aminolysis
O
O
HN
HN 2
aromatization 1
OAc
[O]
AcO OH
Scheme 1. A proposed biosynthetic route of 1 and 2.
the ethylidene side-chain was located at nitrogen atom. The similar NOESY (Supporting information) and ECD spectroscopic data of 2 and 1 (Fig. 4) suggested that their absolute configurations were identical. Accordingly, the structure of 2 was identified as 5α,2′-dihydroxy-1α,2α-diacetoxycass8,11,13(15)-trien-N-ethylidene-16,12-lactam and named caesalminine B. The natural occurrence of 1 and 2 is further supported by its extraction and isolation procedures, which were under mild conditions without the use of any nitrogen-containing solvents and chromatographic materials. The biogenesis of 1 and 2 is of great interest given its unique structure. To account for the biogenetic origin of compound 1 and 2, a plausible biosynthetic pathway was proposed as illustrated in Scheme 1. Compounds 1 and 2 were produced from the precursor of geranylgeranyl pyrophosphate (GGPP) through aminolysis, oxidation, and aromatization reactions [34]. To the best of our knowledge, no ethanolamine adducts of terpenoid alkaloids have been reported so far. However, N-hydroxyethyl derivative of natural alkaloids has been isolated [35]. The in vitro antimalarial activities (IC50 values) of compounds 1 and 2 were tested against the multidrug-resistant K1 strain of P. falciparum. In this experiment, compounds 1 and 2 exhibited significant activity with IC50 values of 0.42 and 0.79 μM, respectively, compared to IC50 value of 0.48 μM for chloroquine used as a control. Considering the seeds of C. minax as therapeutical agents for the treatment of malaria in the Chinese traditional medicine, it can be concluded that the cassane diterpenoid alkaloids may be responsible for the biological activity of C. minax [36].
Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as
influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This work was financially supported from the technological large platform for comprehensive research and development of new drugs in the Twelfth Five-Year “Significant New Drugs Created” Science and Technology Major Projects (No. 2012ZX09301-002-001-026), National Natural Science Foundation of China (No. 30973626), the Science and Technology Grant of Guangxi Province (No. 0639039) and special purpose of basic scientific research operation grant for Commonweal Academy and Institute of Central Authorities (No. YZ-1-24). Innovation capacity-building in Guangxi Science and Technology Agency (0443002-2). References [1] Jiangsu New Medical College. Dictionary of Chinese traditional medicine. Shanghai People's Publishing House; 1986. p. 1289–90. [2] Maurya R, Ravi M, Singh S, Yadav PP. A review on cassane and norcassane diterpenes and their pharmacological studies. Fitoterapia 2012;83:272–80. [3] Liu YX, Harinantenaina L, Brodie PJ, Bowman JD, Cassera MB, Slebodnick C, et al. Bioactive compounds from Stuhlmannia moavi from the Madagascar dry forest. Bioorg Med Chem 2013;21:7591–4. [4] Kalauni SK, Awale S, Tezuka Y, Banskota AH, Linn TZ, Kadota S. Methyl migrated cassane-type furanoditerpenes of Caesalpinia crista from Myanmar. Chem Pharm Bull 2005;53:1300–4. [5] Cheenpracha S, Srisuwan R, Karalai C, Ponglimanont C, Chantrapromma S, Chantrapromma K, et al. New diterpenoids from stems and roots of Caesalpinia crista. Tetrahedron 2005;61:8656–62. [6] Pudhom K, Sommit D, Suwankitti N, Petsom A. Cassane furanoditerpenoids from the seed kernels of Caesalpinia bonduc from Thailand. J Nat Prod 2007;70:1542–4. [7] Wu ZH, Wang YY, Huang J, Sun BH, Wu L. A new cassane diterpene from Caesalpinia bonduc (Fabaceae). J Asian J Trad Med 2007;2:135–9. [8] Dickson RA, Houghton PJ, Hylands PJ. Antibacterial and antioxidant cassane diterpenoids from Caesalpinia benthamiana. Phytochemistry 2007;68:1436–41. [9] Hou Y, Cao S, Brodie P, Miller JS, Birkinshaw C, Ratovoson F, et al. Antiproliferative cassane diterpenoids of Cordyla madagascariensis ssp. madagascariensis from the Madagascar rainforest. J Nat Prod 2008;71:150–2.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Antimalarial diterpene alkaloids from the seeds of Caesalpinia minax Guoxu Ma a, Zhaocui Sun a, Zhonghao Sun a, Jingquan Yuan b, Hua Wei c, Junshan Yang a, Haifeng Wu a,⁎, Xudong Xu a,⁎ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, PR China b National Engineering Laboratory of Southwest Endangered Medicinal Resources Development, National Development and Reform Commission, Guangxi Botanical Garden of Medicinal Plant, Nanning 530023, PR China c Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, PR China
a r t i c l e
i n f o
Article history: Received 27 February 2014 Accepted in revised form 30 March 2014 Available online 13 April 2014 Keywords: Caesalpinia Diterpene alkaloid Antimalarial activity Biosynthetic pathway
a b s t r a c t Two new diterpene alkaloids, caesalminines A (1) and B (2), possessing a tetracyclic cassane-type furanoditerpenoid skeleton with γ-lactam ring, were isolated from the seeds of Caesalpinia minax. Their structures were determined by different spectroscopic methods and ECD calculation. The plausible biosynthetic pathway of caesalminines A and B was proposed. The anti-malarial activity of compounds 1 and 2 is presented with IC50 values of 0.42 and 0.79 μM, respectively. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The search for new antimalarial drug is still urgent because malaria parasites have developed resistance to almost every drug human has thrown at them over the decades. The isolation of artemisinin has inspired us to pay close attention to traditional medicinal plants. Based on its activity against the malaria strain Plasmodium falciparum, a methanol extract of the seeds of Caesalpinia minax was selected for evaluation as a possible source of new antimalarial agents. C. minax, whose seeds have been known as Kushilian in Guangxi folk medicine long used for the treatment of anemopyretic colds, dysentery, skin itching and sores, is a rich source of cassane-type diterpenes [1]. The cassane-type diterpenoids, mainly found ⁎ Corresponding authors at: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing, China. Tel./fax: + 86 10 5783 3296. E-mail addresses: [email protected] (H. Wu), [email protected] (X. Xu).
http://dx.doi.org/10.1016/j.fitote.2014.04.001 0367-326X/© 2014 Elsevier B.V. All rights reserved.
to be distributing in various genera of Fabaceae family (especially Caesalpinia genus), are a group of structurally diverse natural products exhibiting a wide range of pharmacological activities such as antimalarial, antitumor, antiinflammatory, antiviral, antimicrobial and antitrypanosomal properties [2,3]. In recent years, successive discoveries of new cassane diterpenoids, some of which possess novel frameworks, have made great contribution to structural diversities of this family of diterpenes [4–17]. To date, more than 400 cassane diterpenoids have been obtained. Among them, caesanines A–D, four new diterpene alkaloids isolated from Caesalpinia sappan, possess unprecedented N bridge in cassane skeleton [17]. Our previous studies on C. minax have resulted in the isolation of a series of new diterpenes [18–25]. In our further search for structurally unique diterpenes with antimalarial activity, two new diterpene alkaloids, caesalminines A (1) and B (2) (Fig. 1), possessing a tetracyclic cassane-type furanoditerpenoid skeleton with a nitrogen atom incorporated in lactone ring, were isolated from the MeOH extracts of the seeds of C. minax. Compounds 1 and 2 showed significant antimalarial activities against the
G. Ma et al. / Fitoterapia 95 (2014) 234–239
O
R N
16 15
12
OAc 11 AcO
13
20
2 1 3 4
10 5
14 9 8 6 7
17
OH 19
18
1 R=H 2 R = C2H4OH Fig. 1. Caesalminines A (1) and B (2) from Caesalpinia minax.
multidrug-resistant K1 strain of P. falciparum. Herein, we describe the isolation and structure elucidation of the new bioactive compounds. 2. Experimental section 2.1. General Optical rotations were obtained on a PerkinElmer 341 digital polarimeter. UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometer, respectively. ECD spectra were obtained using a JASCO J-815 spectropolarimeter. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HR-ESI-MS spectra were performed on a LTQ-Obitrap XL spectrometer. C18 reversed-phase silica gel (40–63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel (CHP 20P, 75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan) and silica gel (100–200 and 300–400 mesh, Qingdao Marine Chemical plant, Qingdao, People's Republic of China) were used for column chromatography. And pre-coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works). All isolated compounds were purified to 95% purity or better, as judged by HPLC and by NMR before determining bioactivity. The NMR spectra can be found in the Supplementary data. 2.2. Plant material The seeds of C. minax were collected in October 2011 from Nanning, Guangxi Province, China and identified by Prof. Jing-Quan Yuan, Department of Pharmaceutical Chemistry, Guangxi Botanical Garden of Medical Plant. A voucher specimen (NO. 21782) was deposited at the Guangxi Botanical Garden of Medical Plant. 2.3. Extraction and isolation The air-dried and powered seeds of C. minax Hance (750 g), were extracted three times with methanol. Removal of the
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methanol under reduced pressure yielded a methanol extract (190 g). The residue was subjected to column chromatography on silica gel eluting with hexane, chloroform, ethyl acetate, acetone and methanol, respectively. The chloroform fraction (28 g) was found to be antimalarial with an IC50 value of 4.8 μg/mL and was subjected to silica gel (100–200 mesh) column chromatography using a petroleum ether–EtOAc gradient (from 1:0 to 1:1) as eluent, to yield twelve fractions (Fr. A–L). Fr. J (920 mg) was separated over the sephadex LH-20 column and MCI and four fractions (Fr. J1–J3) were collected. Then fractions J1 (230 mg) were chromatographed on a silica gel (300–400 mesh) column using petroleum ether–CHCl3 gradient (20:1; 15:1; 6:1; 2:1), followed with CHCl3–MeOH gradient (80:1; 50:1; 30:1; 10:1) as eluent, then purified by semipreparative liquid chromatography with a YMC RP-18 column using a MeOH–H2O (70:30) system yielded 1 (2.3 mg, Rt 27.6 min). Fr. J2 (190 mg) was chromatographed on a silica gel (300–400 mesh) column using petroleum ether–EtOAc (50:1; 20:1; 8:1; 4:1; 1:1; 0:1), then purified by semi-preparative liquid chromatography using a MeOH–H2O (73:27) system yield 2 (1.5 mg, Rt 30.1 min). 2.3.1. 5α-hydroxy-1α,2α-diacetoxycass-8,11,13(15)-trien-16,12lactam (caesalminine A, 1) White amorphous powder (2.3 mg); [a]20 D −24.2 (c 0.05, MeOH); UV (MeOH) λmax(log ε) 212 (3.86), 254 (3.04) nm; ECD (MeOH) 299 (Δε −0.08), 252 (Δε −0.24) nm; IR (KBr) νmax 3190, 2935, 1735 cm−1; For 1H and 13C APT spectroscopic data, see Table 1; HR-ESI-MS m/z 452.2045 (calcd for C24H31O6NNa, 452.2049). 2.3.2. 5α,2′-dihydroxy-1α,2α-diacetoxycass-8,11,13(15)-trienN-ethylidene-16,12-lactam (caesalminine B, 2) White amorphous powder (1.5 mg); [a]20 D − 22.9 (c 0.05, MeOH); UV (MeOH)λmax (log ε): 210 (3.92), 254 (3.13) nm; ECD (MeOH) 298 (Δε − 0.07) nm; IR (KBr) νmax 3194, 2940, 1738 cm−1; For 1H and 13C APT spectroscopic data, see Table 1; HR-ESI-MS m/z 496.2332 (calcd for C26H35O7NNa, 496.2311). 2.4. Rh-complex of 1 Compound 1 (0.5 mg) was dissolved in a dry solution of [Rh2(OCOCF3)4] (1.0 mg) in CDCl3 (300 μL). The first ECD spectrum was recorded immediately after mixing, and the time was monitored until stabilization of the spectrum. The inherent ECD was then subtracted, and the observed sign of the E band at 350 nm in the induced ECD spectrum was correlated with the absolute configuration of the C-5 tertiary alcohol moiety. 2.5. Antimalarial bioassay Antimalarial activity in vitro was determined by means of the microculture radioisotope technique based on the method described by Desjardins et al. [26]. The parasite P. falciparum (K1, multidrug-resistant strain) was cultured continuously according to the method of Trager and Jensen [27]. Three preparations were used for each experiment. Data are presented as means ± SEM. Statistical analyses were done by means of the
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Table 1 1 H and 13C NMR spectroscopic data of 1 and 2 (600 and 150 MHz). Position
1a δC
δH (J in Hz)
δC
δH (J in Hz)
1 2
73.1 d 67.5 d
5.71, d (2.4) 5.34, d (11.4,2.4)
74.9 d 68.0 d
3
35.3 t
2.18, m 1.36, m
36.1 t
5.96, d (2.4) 5.49, dt (11.4, 2.4) 2.20, m 1.47, m
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OCOCH3
40.0 s 74.4 s 23.4 t 23.1 t 126.6 s 141.9 s 47.4 s 101.9 d 141.0 s 122.5 s 132.0 s 35.0 t 176.3 s 15.6 q 27.7 q 25.1 q 30.0 q 169.7 s 20.6 q 169.8 s 20.7 q
2-OCOCH3 5-OH \NH \NCH2CH2OH
2b
40.1 s 75.6 s 23.9 t 23.7 t 128.7 s 142.6 s 48.4 s 102.0 d 142.0 s 122.0 s 133.2 s 35.3 t 176.5 s 16.2 q 28.1 q 25.7 q 30.3 q 170.7 s 21.1 q 170.9 s 21.4 q
1.95, m 2.61, m
6.26, s
3.32, s 2.02, 1.09, 1.14, 1.31,
s s s s
1.93, s 1.94, s 3.08, br s 10.18, s
43.4 t
\NCH2CH2OH a b
61.0 t
1.97, m 2.74, m
6.48, s
3.42, s 2.13 s 1.19, s 1.22, s 1.45, s 2.00, s 2.05, s 2.84, br s 3.67, m 3.91, m 3.78, dd (10.8, 5.4)
Recorded in DMSO-d6. Recorded in CDCl3.
Student's t-test. A P value of less than 0.05 was considered significant difference. 3. Results and discussion Compound 1 was isolated as a white amorphous powder with [a]20 D −24.2 (c 0.05, MeOH). The UV spectrum is consistent with an oxindole chromophore (λmax 212 and 254 nm) and IR spectrum shows absorption bands from amidogen (3190 cm−1) and carbonyl function (1735 cm−1) [28]. Compound 1 showed a pseudomolecular ion peak at m/z 452 [M + Na]+ in the positive-ion mode ESI-MS. The odd numbered molecular weight
suggested a nitrogen atom in the molecular formula of 1. The HR-ESI-MS spectrum showed a pseudomolecular ion at m/z 452.2025 [M + Na]+, from which in conjunction with NMR data the molecular formula was established as C24H31O6N, compatible with 10° of unsaturation. The 1H NMR spectrum of 1 (Table 1), exhibited six methyl signals including three characteristic tertiary at δH 1.09 (H3-18), 1.14 (H3-19), and 1.31 (H3-20), two acetyl at δH 1.93, 1.94, and a methyl group at δH 2.02 (H3-17) attached to aromatic ring. One proton signal on sp2 carbon at δH 6.26 (s, H-11) and two oxygenated protons signals at δH 5.71 (d, J = 2.4 Hz, H-1) and 5.34 (dd, J = 11.4, 2.4 Hz, H-2), were also observed. In the 1H–1H COSY spectrum (Fig. 2), the correlation of proton signals at δH 5.71 and 5.34 suggested the presence of two oxygenated methines. A singlet at δH 10.18 was assigned to the N–H proton of the amidogen ring, its chemical shift being influenced by the amidogen [29]. The 13C APT spectrum (Table 1) showed 24 carbon resonances including three carbonyl carbons at δC 169.7, 169.8, and 176.3, six olefinic carbons at δC 101.9, 122.5, 126.6, 132.0, 141.0, and 141.9, three quaternary carbons at δC 40.0, 47.4, and 74.4, two oxymethines at δC 67.5 and 73.1, four methylenes at δC 23.1, 23.4, 35.0, and 35.3, and six methyls at δC 15.6, 20.6, 20.7, 25.1, 27.7, and 30.0. All the proton and protonated carbon resonances in the NMR of 1 were unambiguously assigned by interpretation of 2D NMR spectra. The overall 1H and 13C NMR spectroscopic data for 1 confirmed that the compound was a dehydrogenated tetracyclic cassane-type diterpene by comparison with those of previously reported compounds [30,31]. In the HMBC spectrum, the correlations of H3-OAc (δH 1.93) and H-1 with δC carbonyl signal at δC 169.7 and of H3-OAc (δH 1.94) and H-2 with carbonyl signal at δC 169.8 suggested that the acetoxy groups were attached to C-1 and C-2. Furthermore, the HMBC correlations of H-11 with C-9 (δC 141.9), C-12 (δC 141.9), C-8 (δC 126.6), and C-13 (δC 122.5) and of H3-17 with C-8 (δC 126.6), C-13 (δC 122.5), and C-14 (δC 132.0) implied the presence of benzene ring and the methyl group at C-14. In the HSQC spectrum, the singlet at δH 3.32 (s, H2-15) had direct correlation with carbon signal δC 35.0 (C-15) which indicated the presence of the non-coupled methylene. This methylene was located at C-15 by the HMBC correlations of H-15 with C-12 (δC 141.0), C-13 (δC 122.5), C-14 (δC 132.0), and C-16 (δC 176.3). The proton signal of N–H exhibited a downfield chemical shift at δH 10.18 due to the presence of the carbonyl at C-16 (δC 176.3), as
O
H N H3COCO H3COCO OH
Fig. 2. Key 1H–1H COSY (bands) and HMBC (arrows) correlations for 1.
Fig. 3. Selected NOESY correlations of 1 (↔).
G. Ma et al. / Fitoterapia 95 (2014) 234–239
237
Fig. 4. Comparison of CD spectra of 1 (blue) and 2 (black).
supported by the HMBC spectrum. In the HMBC spectrum, the correlations of N–H (δH 10.18, s) with C-12 (δC 141.9), C-13 (δC 122.5), and C-16 (δC 176.3) confirmed the presence of lactam ring. The relative configuration was determined from the NOESY spectrum. The NOEs from H3-20 to H-1, H-2, and H3-19; from H3-18 to 5-OH (δH 3.08, br s) indicated that rings A and B are in chair conformations with a trans-fused ring junction, thus, confirming the relative configurations at C-1, C-2, C-5, and C-10 (Fig. 3). The ECD spectrum of compound 1 exhibited two negative Cotton effects at 252 (Δε − 0.24) and 299 (Δε −0.08) nm due to the n → π* and π → π* transitions of the chromophores (Fig. 4). However, the absence of proper model compounds to use as references made the assignment of the absolute configuration at C-10 unreliable. Therefore, an ECD analysis was conducted for the Rh-complex of 1 (Fig. 5), which displayed a significant negative cotton effect at 350 (Δε −0.48) suggesting an R absolute configuration for C-5 [32,33]. Thus, the structure of 1 was established as
5α-hydroxy-1α,2α-diacetoxycass-8,11,13(15)-trien-16,12lactam and named caesalminine A, with unusual cassane diterpene skeleton possessing a lactam ring, representing a new class of diterpenoid alkaloids. Compound 2, also obtained as a white amorphous powder, was assigned as C26H35O7N by evaluation of HR-ESI-MS (m/z 496.2332 [M + Na]+) and NMR spectroscopic data. IR absorption bands revealed the presence of hydroxyl (3194 cm−1) and carbonyl (1738 cm−1) functionalities in 2. The NMR (Table 1) and UV spectroscopic data for this compound were analogous to those of 1, except for the appearance of two downfield methylene signals and the absence of N–H proton signal. In the 1H–1H COSY spectrum, two multiplets corresponding to the protons (δH 3.67, m; 3.91, m) had the correlation with the protons (δH 3.78, dd, J = 10.8, 5.4 Hz), suggesting the presence of oxygenated ethylidene side-chain. In the HMBC spectrum, the correlations of H-2′ (δH 3.67, m; 3.91, m) with carbon signal at δC 142.0 (C-9) and 176.5 (C-16) indicated that
Fig. 5. CD spectrum of the Rh-complex of 1 with the inherent CD spectrum subtracted, recorded in CHCl3.
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O
O H2N
O Geranylgeranyl pyrophosphate
aminolysis
O
O
HN
HN 2
aromatization 1
OAc
[O]
AcO OH
Scheme 1. A proposed biosynthetic route of 1 and 2.
the ethylidene side-chain was located at nitrogen atom. The similar NOESY (Supporting information) and ECD spectroscopic data of 2 and 1 (Fig. 4) suggested that their absolute configurations were identical. Accordingly, the structure of 2 was identified as 5α,2′-dihydroxy-1α,2α-diacetoxycass8,11,13(15)-trien-N-ethylidene-16,12-lactam and named caesalminine B. The natural occurrence of 1 and 2 is further supported by its extraction and isolation procedures, which were under mild conditions without the use of any nitrogen-containing solvents and chromatographic materials. The biogenesis of 1 and 2 is of great interest given its unique structure. To account for the biogenetic origin of compound 1 and 2, a plausible biosynthetic pathway was proposed as illustrated in Scheme 1. Compounds 1 and 2 were produced from the precursor of geranylgeranyl pyrophosphate (GGPP) through aminolysis, oxidation, and aromatization reactions [34]. To the best of our knowledge, no ethanolamine adducts of terpenoid alkaloids have been reported so far. However, N-hydroxyethyl derivative of natural alkaloids has been isolated [35]. The in vitro antimalarial activities (IC50 values) of compounds 1 and 2 were tested against the multidrug-resistant K1 strain of P. falciparum. In this experiment, compounds 1 and 2 exhibited significant activity with IC50 values of 0.42 and 0.79 μM, respectively, compared to IC50 value of 0.48 μM for chloroquine used as a control. Considering the seeds of C. minax as therapeutical agents for the treatment of malaria in the Chinese traditional medicine, it can be concluded that the cassane diterpenoid alkaloids may be responsible for the biological activity of C. minax [36].
Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as
influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This work was financially supported from the technological large platform for comprehensive research and development of new drugs in the Twelfth Five-Year “Significant New Drugs Created” Science and Technology Major Projects (No. 2012ZX09301-002-001-026), National Natural Science Foundation of China (No. 30973626), the Science and Technology Grant of Guangxi Province (No. 0639039) and special purpose of basic scientific research operation grant for Commonweal Academy and Institute of Central Authorities (No. YZ-1-24). Innovation capacity-building in Guangxi Science and Technology Agency (0443002-2). References [1] Jiangsu New Medical College. Dictionary of Chinese traditional medicine. Shanghai People's Publishing House; 1986. p. 1289–90. [2] Maurya R, Ravi M, Singh S, Yadav PP. A review on cassane and norcassane diterpenes and their pharmacological studies. Fitoterapia 2012;83:272–80. [3] Liu YX, Harinantenaina L, Brodie PJ, Bowman JD, Cassera MB, Slebodnick C, et al. Bioactive compounds from Stuhlmannia moavi from the Madagascar dry forest. Bioorg Med Chem 2013;21:7591–4. [4] Kalauni SK, Awale S, Tezuka Y, Banskota AH, Linn TZ, Kadota S. Methyl migrated cassane-type furanoditerpenes of Caesalpinia crista from Myanmar. Chem Pharm Bull 2005;53:1300–4. [5] Cheenpracha S, Srisuwan R, Karalai C, Ponglimanont C, Chantrapromma S, Chantrapromma K, et al. New diterpenoids from stems and roots of Caesalpinia crista. Tetrahedron 2005;61:8656–62. [6] Pudhom K, Sommit D, Suwankitti N, Petsom A. Cassane furanoditerpenoids from the seed kernels of Caesalpinia bonduc from Thailand. J Nat Prod 2007;70:1542–4. [7] Wu ZH, Wang YY, Huang J, Sun BH, Wu L. A new cassane diterpene from Caesalpinia bonduc (Fabaceae). J Asian J Trad Med 2007;2:135–9. [8] Dickson RA, Houghton PJ, Hylands PJ. Antibacterial and antioxidant cassane diterpenoids from Caesalpinia benthamiana. Phytochemistry 2007;68:1436–41. [9] Hou Y, Cao S, Brodie P, Miller JS, Birkinshaw C, Ratovoson F, et al. Antiproliferative cassane diterpenoids of Cordyla madagascariensis ssp. madagascariensis from the Madagascar rainforest. J Nat Prod 2008;71:150–2.
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