Diterpenoids from the aerial parts of Leonurus macranthus

Yunfang Zhao,Zheng Huang,Jing Sun,Daoran Pang,Xiaonan Chen,Huina Yao,Zhixiang Zhu,Jiao Zheng,Pengfei Tu,Jun Li*

Modern Research Center for Traditional Chinese Medicine,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China

Key words:Lamiaceae Labdane diterpenoid cis-Clerodane diterpenoid Anti-neuroin fl ammation

ABSTRACT Four new diterpenoids including one cis clerodane-type(1)and three highly oxygenated labdane-type diterpenes(2–4)were isolated from the aerial parts of Leonurus macranthus.Their structures were elucidated on the basis of spectroscopic data(NMR,UV,IR,and MS).Compound 1 represents the fi rst example of cis clerodane-type diterpene in the plants of Leonurus genus.Compounds 1 and 4 exhibited weak inhibition of nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells with IC50 values of 35.8?3.6 m mol/L and 48.6?4.8 m mol/L,respectively

Leonurus macranthus Maxim.,family Lamiaceae(syn.Labiatae),is a perennial herbaceous plant mainly distributed in northeast Asia.The dried aerial parts of L.macranthus are used asmotherw ort locally for the treatment of menoxenia,dysmenorrhea,amenorrhea,lochia,edema,oliguresis,and ulcerations[1,2].Our previously phytochemical investigations of this plant revealed the presence of diterpenoids,lignans,fl avonoids,coumarin,and phenolic acids[2,3].As a continued program to search for bioactive diterpenoids from medicinal plants[2,4,5],four new diterpenoids including one cis clerodane-type(1)and three highly oxygenated labdane-type diterpenes(2–4)(Fig.1)were isolated from the acetone extract of the aerial parts of L.macranthus.Herein,the isolation and structural elucidation of the new compounds are described as well as their inhibitory effects on nitric oxide(NO)production in lipopolysaccharide(LPS)-stimulated BV-2 microglial cells.

The acetone extract of the aerial parts of L.macranthus was processed by solvent partitioning with n-hexane and CH2Cl2,successively.The CH2Cl2-soluble fraction was repeatedly subjected to silica gel,Sephadex LH-20,and Lichroprep RP-C18 gel column chromatography(CC)and semipreparative RP-C18 HPLCto afford one new cis clerodane-type(1)and three new labdane-type diterpenes(2–4).Detailed experimental procedures and full spectroscopic data for compounds 1–4 can be found in Supporting information.

Macranthin H(1)was obtained as a colorless gum,?30.Its13C NMR and negative-ion HRESIMS data,exhibiting a deprotonated molecular ion peak at m/z 321.2425,corresponded to a molecular formula of C20H34O3(calcd.for C20H33O3:321.2435),with four indicesof hydrogen de fi ciency.The IRabsorption band at 3419 cm?1was indicative of the presence of hydroxy group.The1H NMR data(Table 1)displayed characteristic resonances of fi ve methyls[dH1.07,1.28,1.67,and 1.68(s,each 3H);dH0.99(d,3H,J=6.5 Hz)],one oxygenated methylene[dH4.07(2H,m)],tw o oxygenated methines[dH3.57(dt,1H,J=11.0,4.0 Hz);dH4.07(m,1H)],and tw o ole fi nic protons[dH5.34(m,1H);dH5.35(m,1H)].The13C NMR data(Table 1)showed 20 carbon resonances comprising fi ve methyls,fi ve sp3methylenes,four sp3methines,tw o sp3quaternary carbons,and four sp2carbons.These functionalities accounted for tw o out of the four indices of hydrogen de fi ciency,requiring the presence of tw o rings in 1.The aforementioned data suggested that 1 is a clerodane diterpenoid[8].The1H NMR and13C NMR spectroscopic data(Table 1)of 1 were comparable to those of kolavenol[8],which was previously isolated from the root bark of Entada abyssinica.The significantly deshielded C-2(dC69.6)and C-7(dC70.5)resonances as well asthe HMBC correlations from H2-1/H-10 to C-2,from H-2 to C-3/C-4,from H2-6/H-8/H3-17 to C-7,and from H-7 to C-17(Fig.2)indicated that C-2 and C-7 are oxygenated.The planar structure of 1 was further supported by HMBC correlations from H-3 to C-5/C-18,from H2-12 to C-11/C-14,from H2-15 to C-13/C-14,from H3-16 to C-12/C-13/C-14,from H3-18 to C-3/C-5,H3-19 to C-4/C-6/C-10,and from H3-20 to C-8/C-10/C-11,as well as1H-1H COSYcorrelations of H-10/H2-1,H2-1/H-2,H-2/H-3,H2-6/H-7,H-7/H-8,and H-8/H3-17(Fig.2).In the NOESY spectrum(Fig.3),the NOE correlation between H3-19 and H-10 supported the cis-fusion of A/Brings in 1.The NOE correlations of H-7/H2-11,H-7/H3-17,H-7/H3-19,H-10/H2-11,H-10/H3-19,H2-11/H3-19,and H3-19/6a indicated that these hydrogens are cofacial and a-oriented,while NOEs of H-8/H3-20,H-8/H-6b,and H-1b/H-6b suggested that these protons are b-cofacially oriented.The presence of NOE correlation of H-2/H-10 supported 2-OH to be b-oriented.NOEs of H2-15/H3-16,H2-12/H-14 indicated E con fi guration of the double bond between C-13 and C-14. Therefore,the structure of 1(macranthin H)was characterized as 2b,7bdihydroxy-(5a,8a,9b,10a)-cleroda-3,13E-dien-15-ol.

Fig.1.Structures of compounds 1–4 from Leonurus macranthus.

It is w orth to note that the cis-fusion of rings A/B is rarely encountered in clerodane-type diterpenoids[9].Compound 1 represents the fi rst example of cis clerodane-type diterpene in the plants of Leonurus genus.

Macranthin I(2)gave a molecular formula of C20H32O4based on the13C NMR data and a deprotonated ion peak at m/z 335.2235[M?H]?(calcd.for C20H31O4:335.2228)in the negative-ion HRESIMS.The IR spectrum indicated the presence of hydroxy(3432 cm?1)and carboxyl carbonyl(1693 cm?1)functionalities.The1H NMRdata(Table 1)displayed resonances of three methyls[dH0.88,1.21,1.67(s,each 3H)],tw o oxygenated methylenes[dH3.95(d,1H,J=12.0 Hz),3.91(d,1H,J=12.0 Hz);dH4.07(d,2H,J=6.5 Hz)],and tw o ole fi nic protons[dH5.36(t,1H,J=6.5 Hz);dH5.57(br s,1H)].The13CNMRdata(Table 1)of 2 showed 20 carbon resonances due to three methyls,eight methylenes(tw o oxygenated),four methines(tw o ole fi nic),and fi ve quaternary carbons(one carbonyl,tw o ole fi nic).Detailed analysis of the1H NMRand13CNMRspectroscopic data(Table 1)of 2 showed a close structural resemblance to leojaponin B[10],which was previously isolated from Leonurusjaponicus.The major difference wasthe replacementof the C-17 methyl group in leojaponin Bby a hydroxymethyl group in 2.This deduction was supported by the significantly deshielded H2-17[dH3.95(d,1H,J=12.0 Hz),3.91(d,1H,J=12.0 Hz);D dH+2.31,2.27]and C-17(dC66.3;D dC+43.0)resonances as well as the HMBC correlations from H2-17 to C-7/C-8/C-9(Fig.2).The 2D structure of 2 was also supported by1H-1H COSY correlations of H2-1/H2-2,H2-2/H2-3,H-5/H2-6,H2-6/H-7,H-9/H2-11,H2-11/H2-12,and H-14/H2-15(Fig.2).The relative con fi guration of 2 was established on the basis of NOESY data(Fig.3).The NOE correlations of H3-20/H-6b and H-5/H-6a supported the trans A/B junction in 2.NOEs of H-5/H-6a,H-5/H2-11,and H-5/H3-18 indicated that these protons are on the same face and a-oriented,while the correlations of H3-20/H-6b and H3-20/H-9 suggested that these protons are b-oriented.The NOE correlations of H2-15/H3-16,H2-12/H-14 indicated that the double bond between C-13 and C-14 is Econ figuration.Thus,the structure of 2(macranthin I)was elucidated as 15,17-dihydroxylabd-7,13E-dien-19-oic acid.

Table 1 1H NMR(500 MHz)and 13C NMR(125 MHz)data of compounds 1–4(d in ppm,in methanol-d4).a

Fig.2.1H-1H COSY and selected HMBC(arrow s point from protons to carbons)correlations of compounds 1–4.

Fig.3.Selected NOE correlations of compounds 1–4.

Compound 3 was obtained as colorless gum,a?2D1–30.Its molecular formula,C24H38O8,was deduced from the HRESIMS(m/z 455.2645[M+H]+,calcd.for C24H39O8:455.2639)and13C NMR spectroscopic data,indicating six indices of hydrogen de fi ciency.The IRspectrum showed absorption bands for hydroxy(3545 cm?1)and carbonyl(1731 cm?1)functionalities.The1H NMR data(Table 1)displayed characteristic resonances for three tertiary methyl[dH1.05,1.26,and 1.45(s,each 3H)],one secondary methyl[dH1.06(d,3H,J=6.5 Hz)],tw o oxygenated methylene[dH3.62(m,1H),3.67(m,1H);dH4.42(d,1H,J=11.5 Hz),4.46(d,1H,J=11.5 Hz)],tw o oxygenated methine[dH3.80(br s,1H);dH5.15(d,1H,J=2.5 Hz)],and tw o O-acetyl[dH2.04,2.12(s,each 3H)]groups.The13C NMR data(Table 1)of 3 showed 24 carbon resonances due to six methyl(dC10.0,20.7,21.1,21.2,22.4,27.5),seven sp3methylene(dC26.2,28.5,30.4,40.1,46.2,60.0,68.7),four sp3methine(dC46.4,47.9,70.7,78.7),four quaternary sp3(dC44.4,44.7,85.6,98.4),tw o ester carbonyl(dC171.3 and 172.8),and one keto carbonyl(dC206.9)carbons.These functionalities accounted for three out of the six indices of hydrogen de fi ciency,requiring the presence of a tricyclic system in 3.The aforementioned data suggested that 3 is a highly oxygenated tricyclic spirolabdane diterpenoid[2,11].The1H NMR and13C NMR spectroscopic data(Table 1)of 3 were comparable to those of macranthin A[2],except for the absence of resonances of an acetoxy group in 3.The deshielded resonances of H-6(dH5.15)and H2-19(dH4.42,4.46)indicated that tw o O-acetyl groups are located at C-6 and C-19,respectively.Thiswascon fi rmed by the HMBCcorrelationsfrom H-6 and H2-19 to the acetoxy carbonyl carbons at dC171.3 and 172.8,respectively(Fig.2).The planar structure of 3 was further supported by1H ?1H COSY correlations of H2-1/H2-2,H2-2/H-3,H-5/H2-6,H-8/H3-17,H2-11/H2-12,and H2-14/H2-15(Fig.2).The relative con fi gurations at C-3,C-4,C-5,C-6,C-8,C-9,C-10,and C-13 were the same asthose of macranthin Aon the basis of NOESYdata(Fig.3).Thus,the structure of 3 was de fined as 6b,19-diacetoxy-9,13-epoxy-3a,15-dihydroxylabdan-7-one,and this compound was named 15-O-deacetylmacranthin A.

Compound 4 was isolated as a colorless gum,a?2D1?22,and its13CNMRand positive-ion HRESIMSdata show ing a protonated ion peak at m/z 455.2641[M+H]+established a molecular formula of C24H38O8,indicating six indices of hydrogen de fi ciency.The IR spectrum indicated the presence of hydroxy(3421 cm?1),ester carbonyl(1737 cm?1),and a,b-unsaturated carbonyl(1655 cm?1)functionalities.The1HNMRdata(Table 1)exhibited resonancesfor four tertiary methyl[dH1.10,1.25,1.47,and 1.83(s,each 3H)],tw o oxygenated methylene[dH3.73(m,1H),dH3.76(m,1H);dH4.69(d,1H,J=11.5 Hz),4.82(d,1H,J=11.5 Hz)],tw o oxygenated methine[dH4.19(d,1H,J=3.5 Hz);5.08(br s,1H)],and tw o O-acetyl(dH2.05 and 2.07,s,each 3H)groups.The13C NMR spectrum showed 24 carbon resonances including six methyls,seven methylenes(tw o oxygenated),three methines(tw o oxygenated),and eight quaternary carbons(one ketocarbonyl,tw o ester carbonyl,tw o ole fi nic,and one oxygenated).The aforementioned data suggested that compound 4 is a highly oxygenated bicyclic labdane diterpenoid similar to macranthin F[2].The1H NMR and13C NMR spectroscopic data(Table 1)were highly similar to those of macranthin F[2],with the exception of the deshielded H-3 resonance(dH5.08;D dH+1.28)and the presence of an additional acetyl group(dH2.05;dC172.3,20.7),indicating 4 to be the 3-O-acetyl derivative of macranthin F.Thisdeduction wascon fi rmed by the HMBCcorrelation from H-3 to the carbonyl carbon(dC172.3)of the acetyl group(Fig.2).The 2D structure of 4 was further supported by1H-1H COSY correlations of H2-1/H2-2,H2-2/H-3,H-5/H2-6,H-8/H3-17,H2-11/H2-12,and H2-14/H2-15(Fig.2).The relative con fi guration of 4 was established on the basis of the NOESYdata(Fig.3).The NOEcorrelations from H3-20 to H2-19 and from H-5 to H3-18 indicated that the A/Brings are trans-fused.NOE correlations of H-6/H-5,H-5/H-1a,and H-6/H3-18 indicated these protons are cofacial and a-oriented,while NOEs of H-3/H2-19,H2-19/H3-20 were consistent with these protons being b-cofacially oriented(Fig.3).However,the con fi guration at C-13 could not be assigned.Accordingly,the structure of 4 was de fined as 3a,19-diacetoxy-6b,13,15-trihydroxylabd-8(9)-en-7-one,named 3-O-acetylmacranthin F.

Compounds 1–4 were evaluated for their inhibitory effects on the NO production in LPS-stimulated BV-2 microglial cells using Griess assay[2,6,7].Compounds 1 and 4 showed weak inhibition of NO production with IC50values of 35.8?3.6 m mol/Land 48.6?4.8 m mol/L,respectively.Indomethacin(IC50=30.6?2.2 m mol/L)was used as a positive control.Compounds 2 and 3 were inactive(<50%inhibition at 100 m mol/L,the highest concentration tested).In order to investigate w hether the inhibitory activities of compounds 1 and 4 were due to cytotoxicity,their effects on cell proliferation/viability using the MTT method were measured.These tw o compounds(up to 100 m mol/L)did not show any cytotoxicity with LPStreatment for 24 h.

In summary,one cis clerodane-type(1)and three highly oxygenated labdane-type diterpenes(2–4)were isolated from the aerial parts of Leonurus macranthus.The cis-fusion of rings A/B is rarely encountered in clerodane-type diterpenoids.Compound 1 represents the fi rst example of cis clerodane-type diterpene in the plants of Leonurus genus.Compounds 1 and 4 exhibited weak inhibition of NO in LPS-stimulated BV-2 microglial cells with IC50values of 35.8?3.6 m mol/L and 48.6?4.8 m mol/L,respectively.

Acknow ledgm ents

This work was financially supported by the National Natural Science Foundation of China(Nos.81573572,81530097)and New Century Excellent Talents in University(No.NCET-13-0693).

Appendix A.Supplem entary data

Supplementary data associated with thisarticle can be found,in the online version,at https://doi.org/10.1016/j.cclet.2017.11.033.