Curcumanes A and B, Two Bicyclic Sesquiterpenoids with Significant

Jan 31, 2019 - †School of Pharmacy and ‡Institute of Innovative Medicine ... Academy of Medical Sciences and Peking Union Medical College, Beijing...
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Curcumanes A and B, Two Bicyclic Sesquiterpenoids with Significant Vasorelaxant Activity from Curcuma longa Yu Liu,†,‡,∥ Fei Liu,†,‡,∥ Ming-Ming Qiao,†,‡ Li Guo,†,‡ Ming-Hua Chen,§ Cheng Peng,*,†,‡ and Liang Xiong*,†,‡ School of Pharmacy and ‡Institute of Innovative Medicine Ingredients of Southwest Specialty Medicinal Materials, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China § Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China Downloaded via UNIV DE BARCELONA on February 5, 2019 at 01:00:24 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: Two novel sesquiterpenoids, curcumanes A (1) and B (2), possessing unprecedented skeletons with a dicyclo[3.2.1]octane and a dicyclo[3.3.1]nonane moiety, respectively, were isolated from Curcuma longa. Both of them had remarkable vasorelaxant activity on rat aorta via blocking extracellular Ca2+ influx through VDCCs and ROCCs. The activity of 1 was endothelium-independent, while that of 2 was endothelium-dependent. Compound 2 also prolonged APTT and TT to inhibit blood coagulation.

Curcuma longa L. (Zingiberaceae) plays an important role in people’s daily life as a food and phytomedicine. As a widely used traditional Chinese medicine for promoting blood circulation, the rhizomes of C. longa have been recorded in many Chinese medical reports.1 Modern studies have revealed that it possesses relevant bioactivities, including antiplatelet aggregation,2 anticoagulation,3 cardiovascular protection,4 and antiatherosclerosis.5 The prior studies showed that curcumins and sesquiterpenoids were the main material basis for the above activities in C. longa, but with the most focus on curcumins.6 In recent phytochemical studies, more than 380 sesquiterpenoids belonging to 11 subtypes were isolated from the genus Curcuma, and several novel sesquiterpenoid skeletons have been discovered.1,7 As part of a program to explore active natural compounds from traditional Chinese medicines that can promote blood circulation, an ethanolic extract of the rhizomes of C. longa was investigated. After separation by column chromatography over polyamide, the ethanolic extract was mainly divided into two parts (sesquiterpenoids and curcumins). Interestingly, the vasorelaxant activity assay showed that the sesquiterpenoid portion had a significant effect with an EC50 value of 31.19 μg/mL, while the curcumin portion was inactive even at 500 μg/mL. Thus, our further phytochemical investigation focused on sesquiterpenoids and their ability to promote blood circulation. Two structurally unique sesquiterpenoids, curcumanes A (1) and B (2), were isolated (Figure 1). The ability of these two compounds to promote blood circulation was evaluated from vasorelaxant activity, effects on blood coagulation functions, © XXXX American Chemical Society

Figure 1. Structures of curcumanes A (1) and B (2).

and antiplatelet activity. The preliminary mechanisms for vasodilation were also investigated. Compound 1 was obtained as a colorless oil. Its molecular formula was established as C15H22O2 by HR-ESI-MS (m/z 235.1696 [M + H]+; calcd 235.1698), corresponding to five degrees of unsaturation. Its UV absorption maximum at 238 nm showed the possibility of existence of an α,β-unsaturated ketone unit.8 The IR spectrum displayed the characteristic absorptions for hydroxy and carbonyl functionalities (3425 and 1621 cm−1). The 1H NMR spectrum of 1 (Table 1) showed signals of two tertiary methyls (δH 1.83 and 2.04), one secondary methyl (δH 1.01), one oxymethine (δH 4.23), a Received: January 14, 2019

A

DOI: 10.1021/acs.orglett.9b00149 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of 1 and 2 in Acetone-d6 (δ in ppm, J in Hz) 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

2

δH

no.

3.05 t (4.8) 2.17 1.96 1.73 2.65 2.61 4.23

m, 1.94 m m, 1.35 m m m t (6.0) dd (9.6, 4.8)

6.14 brs 1.83 2.04 4.61 1.01

s s t (2.4), 4.52 t (2.4) d (6.6)

δC

δH

55.0 146.7 28.0 25.9 45.9 32.5 61.9 73.9 199.1 124.9 153.4 27.4 20.6 111.3 23.1

2.10 2.29 2.97 1.58 1.88 3.93 5.53

δC

m ddd (9.0, 9.0, 3.6) m m m m brs

42.1 48.4 75.0 43.0 44.1 64.1 127.5 139.5 31.0 129.1 132.7 18.2 26.0 16.5 25.5

1.86 m, 1.50 dt (12.0, 2.4) 5.06 brd (9.0) 1.65 1.69 1.10 1.69

s s d (7.2) s

carbon skeleton containing a dicyclo[3.2.1]octane moiety. As shown in Figure 3, the relative configuration of 1 was determined on the basis of ROESY correlations of H-8 with H-1, H-5, H-7, and H3-15 and of H-5 and H-7 with H3-15.

terminal double bond (δH 4.52 and 4.61), a conjugated trisubstituted double bond (δH 6.14), and several aliphatic methylenes and methines between δH 1.30 and 3.10 ppm. The 13 C NMR and DEPT data (Table 1) revealed the presence of 15 carbons, including three methyls, three methylenes (one olefinic), six methines (one oxygen-bearing and one olefinic), two olefinic quaternary carbons, and one carbonyl carbon. The above functionalities accounted for three degrees of unsaturation and suggested a bicyclic sesquiterpenoid structure. A comprehensive analysis of the 2D NMR spectra was conducted. The 1H−1H COSY correlations of H-1/H-8/H5/H2-4/H2-3 and H-8/OH-8 as well as HMBC correlations of OH-8 with C-1, C-5, and C-8, of H-8 with C-1, C-2, C-4, and C-5, and of H2-14 with C-1, C-2, and C-3 established the existence of ring A with a hydroxy group at C-8 and an

Figure 3. Key ROESY correlations of 1 and 2.

Compound 2 was also obtained as a colorless oil. The positive HR-ESI-MS data (m/z 259.1689 [M + Na]+; calcd 259.1674) suggested that the molecular formula of 2 was C15H24O2 with four indices of hydrogen deficiency. The 1H NMR spectrum of 2 displayed resonances attributable to four methyl groups [δH 1.10 (d, J = 7.2 Hz), 1.65 (s), 1.69 (s), and 1.69 (s)], two olefinic methines [δH 5.06 (brd, J = 9.0 Hz) and 5.53 (brs)], and two oxymethines [δH 3.93 and 2.97] (Table 1). The 13C NMR and DEPT data of 2 exhibited four olefinic and two oxygenated carbon signals [δC 139.5 (C), 132.7 (C), 129.1 (CH), 127.5 (CH), 75.0 (CH), and 64.1 (CH)] corresponding to two trisubstituted double bonds and two oxymethines. Thus, the remaining two degrees of unsaturation suggested that 2 was a bicyclic sesquiterpenoid. The 1H−1H COSY correlations of H-1/H-2/H-3/H-4/H-5/H2-9/H-1, H2/H-10, and H-4/H3-14 established the six-membered ring A with an isobutenyl unit at C-2 and a methyl group at C-4, which was confirmed by the HMBC correlations from H2-9 to C-1, C-2, C-4, and C-5, from H-3 to C-1, C-4, C-10, and C-14, from H-10 to C-2, C-3, C-12, and C-13, from H3-12 and H3-13 to C-10 and C-11, and from H3-14 to C-3, C-4, and C-5 (Figure 2). The other six-membered ring B possessing OH-6 and Me-8 substituents was elucidated by the HMBC correlations of OH-6 with C-5, C-6, and C-7, of H3-15 with C-1, C-7, and C-8, of H2-9 to C-6 and C-8, of H-7 with C-1, C-

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

exocyclic double bond at C-2 in 1 (Figure 2). Additionally, the H−1H COSY correlations of H-5/H-8/H-1/H-7/H-6/H3-15, combined with the HMBC correlations from H-1 to C-5, C-6, and C-7, from H-6 to C-4, C-5, C-7, C-8, and C-15, and from H3-15 to C-5, C-6, and C-7, undoubtedly revealed the substructure of ring B with a methyl group attached to C-6, although the 1H−1H COSY correlation of H-5/H-6 was absent because the dihedral angle between them was approximately 90°. Finally, the fragment C (the side chain connected to C-7) was determined by the HMBC correlations of H3-12 and H313 with C-10 and C-11 and of H-10 with C-7 and C-9, together with their chemical shifts. Thus, compound 1 was determined to be a bicyclic sesquiterpenoid with a novel 1

B

DOI: 10.1021/acs.orglett.9b00149 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

of enzymes in the plant, curcumanes A (1) and B (2) were generated. In our ongoing endeavor to discover bioactive secondary metabolites from traditional Chinese medicines that can promote blood circulation, a series of compounds have been found to relax aortic rings,10 inhibit platelet aggregation,11 or affect blood coagulation functions.12 In particular, most of the active isolates were terpenoids. C. longa is a well-known traditional Chinese medicine for promoting blood circulation, and thus, curcumanes A (1) and B (2) from C. longa were tested for their vasorelaxant activity, influences on blood coagulation functions, and antiplatelet activity. Both compounds 1 and 2 exhibited significant dosedependent vasorelaxant activity against KCl- and PHE-induced contractions of rat aorta rings (Figure 5). The EC50 of 1, 2, and

5, and C-15, and of H-2 with C-8. Thus, the planar structure of 2 was determined. In the NOE difference spectrum of 2, H-1, H-2, and H-5 were enhanced when H-9b was irradiated, and H-1, H-4, and H-9b were enhanced when H-2 was irradiated (Figure 3), indicating that H-2 and H-4 were oriented on the same side with the methano bridge (C-1−C-9−C-5). In addition, correlations of H3-14 with H-3 and H-6, of H-6 with H-3 and H3-14, and of H-3 with H-10 and H3-14, together with the coupling constant of J5,6 (≈0 Hz), revealed that these protons were oriented on the opposite side with the methano bridge. The absolute configurations of 1 and 2 were determined by comparing the experimental electronic circular dichroism (ECD) spectrum with the calculated ECD data.9 The detailed ECD calculative process is shown in the Supporting Information. As shown in Figure 4, the calculated ECD

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

spectra of (1S,5S,6S,7R,8S)-1 and (1R,2R,3R,4S,5S,6S)-2 were matched with the experimental ECD spectra of 1 and 2, respectively. Thus, the absolute configurations of 1 (1S,5S,6S,7R,8S) and 2 (1R,2R,3R,4S,5S,6S) were established. A possible biosynthesis pathway for 1 and 2 is shown in Scheme 1. They shared the same mevalonic acid pathway

Figure 5. Vasorelaxant activity of (a) 1, 2, and methoxyverapamil against KCl-induced contractions and (b) 1, 2, and phentolamine mesylate against PHE-induced contractions.

positive control (methoxyverapamil) against KCl-induced contractions were 2.40, 0.91, and 0.69 μM, respectively, while the EC50 of 1, 2, and positive control (phentolamine mesylate) against PHE-induced contractions were 3.37, 0.83, and 0.05 μM. It is well-known that the vascular tension intensity is modulated by Ca2+, which is supplied from intracellular Ca2+ release and extracellular Ca2+ influx, and Ca2+ influx results from the opening of voltage-dependent calcium channels (VDCCs) and receptor-operated calcium channels (ROCCs).13 K+-depolarization (KCl)-activated Ca2+ entry permitting sustained force maintenance in vascular smooth muscle is attributed to activation of VDCCs,14 while the PHEinduced contraction results from activation of ROCCs via stimulating the α-1 adrenoceptor.15 Thus, the results indicated that 1 and 2 possibly block extracellular Ca2+ influx by both VDCCs and ROCCs. To confirm the above deduction, the effect of 1 on intracellular Ca2+ release and extracellular Ca2+ influx was studied. The aorta rings were precontracted with high K+ in normal K−H solution to ensure rich Ca2+ storage in the sarcoplasmic reticulum, and then the aorta rings were subsequently allowed to rest in Ca2+-free solution for 30 min. After the rings were exposed to 1 (6.25 μM) or the solvent control for 20 min, PHE was added to induce contraction. There was no significant difference for Emax (maximum tension rate) between 1 and the control (Figure 6a). Then 3 mM CaCl2 solution was added when the PHEinduced contraction was stable. The results showed that the Emax of the control was 23.38 ± 1.93%, while the Emax of 1 was 12.14 ± 1.41% (p < 0.05, n = 5; Figure 6b). Thus, compound 1

Scheme 1. Possible Biosynthetic Pathways for 1 and 2

(MVA). First, one γ,γ-dimethyl allyl pyrophosphate (DMAPP) unit and two isopentenyl pyrophosphate (IPP) units connected end to end to form the farnesyl pyrophosphate (FPP) chain. In this process, isoprenoid units (DMAPP and IPP) were elongated by prenyltransferases. Second, the FPP cycled from C-4′ to C-5′′ to form an intermediate with a sixmembered ring. Third, the basic skeletons of compounds 1 and 2 were formed by the cyclization from C-1′′ to C-1′ and C-1′′ to C-5 of the intermediate, respectively. There may be some new cyclases in the plant leading to new biochemical modifications. Finally, through the reduction and oxidation C

DOI: 10.1021/acs.orglett.9b00149 Org. Lett. XXXX, XXX, XXX−XXX

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Figure 6. Inhibitory effect of compound 1 (6.25 μM) on extracellular Ca2+ influx. *p < 0.05 compared with control (n = 5).

Figure 8. Effects of 1 and 2 on (a) APTT and (b) TT.

significantly reduced the extracellular Ca2+ influx to relax the aorta. Vascular endothelial cells usually synthesize and secrete multiple bioactive materials, especially some important vascular endothelium-derived relaxing factors, such as nitric oxide,16 prostacyclin,17 and endothelium-derived hyperpolarizing factor,18 and these materials can regulate the vasorelaxant effect via impacting the Ca2+.19 Therefore, the effects of 1 and 2 on the PHE-contracted rat aortic rings with intact endothelium (E+) or without endothelium (E−) were investigated. As shown in Figure 7, compound 1 exhibited

and B (2) are components of C. longa for activating blood circulation, and their mechanism of action works through vasodilation and prolongation of APTT and TT, not via antiplatelet aggregation.

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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.9b00149. Experimental procedures as well as physical−chemical properties, experimental and calculated ECD spectra, 1D and 2D NMR, HR-ESI-MS, IR, and UV spectra for 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID Figure 7. Vasorelaxant effects of 1 and 2 on rat aortic rings with intact endothelium (E+) or without endothelium (E−).

Ming-Hua Chen: 0000-0001-5634-4599 Liang Xiong: 0000-0001-6222-8340 Author Contributions

almost the same relaxant activity on E+ and E−, while the effect of 2 on E+ was significantly stronger than that on E−. This result indicated that vasodilation by 1 occurred in an endothelium-independent fashion but in an endotheliumdependent fashion for 2. In addition, to evaluate the effects of 1 and 2 on blood coagulation functions, the coagulation four indices test [activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (Fib)] was performed using rabbit blood plasma. Compound 2 significantly prolonged APTT and TT in a dose-dependent manner (p < 0.05 vs control at 25 μM, p < 0.01 vs control at 50 and 100 μM; Figure 8). However, compound 1 showed a weak effect on APTT and no effect on TT. In the platelet aggregation activity assay, both 1 and 2 were inactive. In conclusion, two sesquiterpenoids (1 and 2) with novel carbon skeletons were isolated from the rhizomes of C. longa, a widely used traditional Chinese medicine for promoting blood circulation. Interestingly, both 1 and 2 exhibited significant relaxant activity on rat aorta rings contracted by KCl or PHE, and the effect was exerted via blocking extracellular Ca2+ influx through both VDCCs and ROCCs. Additionally, the vasorelaxant activity of 1 was endothelium-independent and was endothelium-dependent for 2. In addition, compound 2 also significantly prolonged APTT and TT to inhibit blood coagulation. Thus, it is concluded that curcumanes A (1)

∥

Y.L. and F.L. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Sciences Foundation of China (NNSFC, Grant No. 81891012) and the Youth Science and Technology Innovation Research Team Program of Sichuan (Grant Nos. 2017TD0001 and 2016TD0006).

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DOI: 10.1021/acs.orglett.9b00149 Org. Lett. XXXX, XXX, XXX−XXX