Simultaneous determination of seven major diterpenoids in Siegesbeckia pubescens Makino by high‐performance liquid chromatography coupled with evaporative light scattering detection

A novel HPLC method with evaporative light scattering detection was developed for the simultaneous quantification of seven major diterpenoids of two types, including ent‐pimarane type: Kirenol, Hythiemoside B, Darutigenol, and ent‐kaurane type: ent‐16β,17,18‐trihydroxy‐kauran‐19‐oic acid, ent‐17,18‐dihydroxy‐kauran‐19‐oic acid, ent‐16β,17‐dihydroxy‐kauran‐19‐oic acid, 16α‐hydro‐ent‐kauran‐17,19‐dioic acid in the aerial parts of Siegesbeckia pubescens Makino, an important traditional Chinese medicinal herb. Chromatographic separation was achieved on a Waters Symmetry Shield^TM RP18 column (250 mm× 4.6 mm id, 5 μm) with a gradient mobile phase (A: 0.3% v/v aqueous formic acid and B: acetonitrile) at a flow rate of 1.0 mL/min. The drift tube temperature of evaporative light scattering detection was set at 103°C, and nitrogen flow rate was 3.0 L/min. The method was validated for accuracy, precision, LOD, and LOQ. All calibration curves showed a good linear relationship (r > 0.999) in test range. Precision was evaluated by intra‐ and interday tests that showed RSDs were less than 3.5%. Accuracy validation showed that the recovery was between 96.5 and 102.0% with RSDs below 2.8%. The validated method was successfully applied to determine the contents of seven diterpenoids in the different parts of Siegesbeckia pubescens Makino from two sources and to determine the contents of ent‐pimarane, ent‐kaurane, and total diterpenoids.     

Two New Diterpenoids and other Constituents from Isodon Nervosus

Two new ent‐kaurane diterpenoids, 15β‐hydroxy‐6,7‐seco‐6,11β:6,20‐diepoxy‐1α,7‐olide‐ent‐kaur‐16‐ene ( 1 ), 11α,15α‐dihydroxy‐6β‐methoxy‐6,7‐seco‐6,20‐epoxy‐1α,7‐olide‐ent‐kaur‐16‐ene ( 2 ), together with four known diterpenoids, nodosin ( 3 ), isodocarpin ( 4 ), odonicin ( 5 ) and maoyecrystal F ( 6 ) were isolated from the aerial parts of Isodon nervosus. The structures of the new compounds were elucidated on the basis of their spectral evidence, especially on 2D NMR.     

Diterpenes from Xylopia langsdorffiana

The phytochemical investigation of Xylopia langsdorffiana A.St.‐Hil. & Tul . led to the isolation of eight diterpenes, i.e., of the four new compounds (5β,7β,8α,9β,10α,12α)‐atisane‐7,16‐diol 7‐acetate ( 1 ), named xylodiol 7‐acetate, (5β,8α,9β,10α,12α)‐16‐hydroxyatisan‐7‐one ( 2 ), named xylopinone, (3α,12Z)‐3‐hydroxy‐ent‐labda‐8(20),12,14‐trien‐18‐oic acid ( 3 ), named labdorffianic acid A, and 8,20‐epoxy‐13‐hydroxy‐ent‐labd‐14‐en‐18‐oic acid ( 4 ), named labdorffianic acid B, and of the four known compounds 5 – 8 , i.e., ent‐kauran‐16‐ol, ent‐kaur‐16‐en‐19‐oic acid, ent‐kaur‐16‐en‐19‐ol, and ent‐trachyloban‐18‐oic acid. The structures were established by IR, HR‐ESI‐MS, and NMR data analysis with the aid of 2D techniques.     

Diterpenoids from Isodon Eriocalyx

Two new diterpenoids, Maoecrystal U and epi‐maoecrystal P , together with nine known diterpenoids and eight other known compounds were isolated from Isodon eriocalyx ( ). The structures of two new diterpenoids were elucidated as 6β‐acetoxy‐15β‐hydroxy‐3α,20‐epoxy‐ent‐kaur‐16‐ene‐1,7‐dione and 16(S)‐methoxymethy‐6β,7β‐dihydroxy‐7α,20‐epoxy‐ent‐kaur‐2,3‐ethenylene‐1,15‐dione on the basis of spectroscopic evidence, especially by 2D NMR techniques.     

Diterpenes and Disulfides from the Marine Mangrove Plant Bruguiera sexangula var. rhynchopetala

Phytochemical investigation of the stems of Bruguiera sexangula var. rhynchopetala resulted in the isolation and characterization of four new and seven known secondary metabolites. The new compounds were spectroscopically identified as 17‐hydroxy‐16‐oxobeyer‐9(11)‐en‐19‐al ( 1 ), 16,17‐dihydroxy‐19‐nor‐ent‐kaur‐9(11)‐en‐3‐one ( 2 ), (16R)‐13,17‐epoxy‐16‐hydroxy‐ent‐kaur‐9(11)‐en‐19‐al ( 3 ), and (−)‐3,4‐dihydro‐3‐hydroxy‐7‐methoxy‐2H‐1,5‐benzodithiepine‐6,9‐dione ( 10 ). The configurations of the known compounds brugierol ( 11 ) and isobrugierol ( 12 ) were re‐investigated. Compounds 11 and 12 , together with 2,6‐dimethoxy‐1,4‐benzoquinone ( 13 ), are proposed to be degradation products of the novel, unusual disulfide 10 (Scheme).     

Cytotoxic Diterpenoids from the Stem Bark of Annona squamosa L.

Three new ent‐kaurane diterpenoids, (4α)‐19‐nor‐ent‐kaurane‐4,16,17‐triol ( 1 ), (4α,16α)‐17‐(acetyloxy)‐19‐nor‐ent‐kaurane‐4,16‐diol ( 2 ), and 17‐hydroxy‐ent‐kaur‐15‐en‐19‐al ( 3 ), together with 11 known compounds, were isolated from the stem bark of Annona squamosa L. The structures of 1 – 3 were identified by analysis of their spectroscopic data. All compounds were evaluated for cytotoxic activity against human lung cancer (95‐D) and ovarian cancer (A2780) cell lines, and compounds 3, 5, 7, 11 – 14 exhibited promising antiproliferative activities with IC₅₀ values ranging from 0.38 to 34.66 μM .     

The Chemical Constituents from the Callus Culture of Trewia nudiflora

Five ent‐atisane diterpenoids, including three new ones, i.e., (16α)‐17‐hydroxy‐ent‐atisan‐19‐oic acid methyl ester ( 1 ), (16α)‐17‐dihydroxy‐ent‐atisan‐19‐oic acid methyl ester ( 2 ), (16α)‐ent‐atisan‐16,17,19‐triol ( 3 ), and two known compounds, i.e., 17‐hydroxy‐ent‐atisan‐19‐oic acid ( 4 ), (16α)‐16,17‐dihydroxy‐ent‐atisan‐19‐al ( 5 ), together with one known diterpene glycoside, i.e., sumogaside, two known triterpenes, i.e., germanicone and (3β)‐β‐amyrin, three known phenolic compounds, i.e., (+)‐gallocatechin, (+)‐catechin, and gallic acid, and two known sterols, i.e., β‐sitosterol and daucosterol, were isolated from the callus cultures of Trewia nudiflora. Their structures were elucidated by spectroscopic analysis including 1D‐ and 2D‐NMR experiments. No maytansinoids were isolated or detected by LC‐ESI‐MS in the extracts of the calli, which suggests that the callus cultures can not produce maytansinoids under these conditions.     

Chemical Constituents from the Pericarp of Trewia nudiflora

Three new ent‐atisane diterpenes, i.e., 17‐hydroxy‐ent‐atisan‐19‐oic acid ( 1 ), 17‐hydroxy‐ent‐atisan‐19‐oic acid methyl ester ( 2 ), and 16α,17‐dihydroxy‐ent‐atisan‐19‐al ( 3 ), together with five known phenolic compounds, i.e., gallic acid, ethyl gallate, protocatechuic acid, 3,4,4′‐tri‐O‐methylellagic acid, and α‐tocopherol, and two other known compounds, i.e., trans‐cinnamic acid and taraxerone, were isolated from the pericarp of Trewia nudiflora collected in Xishuangbanna, Yunnan Province, China. Their structures were elucidated by spectroscopic analysis including 1D‐ and 2D‐NMR. Trace amounts of maytansinoids were isolated by antifungal‐activity‐guided fractionation and determined by LC‐ESI‐MS analysis; they were prominent antifungal constituents in the pericarp of Trewia nudiflora.     

New Sesquiterpenes from Ligulariopsis Shichuana

A thorough investigation of Ligulariopsis shichuana afforded five new sesquiterpenes, including 1β,10β‐epoxy‐3α‐angeloyloxy‐9β‐acetoxy‐8α,11β‐dihydroxybakkenolide ( 1 ), 1β,7‐dihydroxy‐3β‐acetoxy‐noreremophil‐6(7),9(10)‐dien‐8‐one ( 2 ), 8α‐hydroxy‐3‐oxoeremophil‐1(2),7(11),9(10)‐trien‐8β(12)‐olide ( 3 ), 1β,10β‐dihydroxy‐3β‐acetoxyeremophil‐7(11),8(9)‐dien‐8(12)‐olide ( 4 ) and 1β,10β‐epoxy‐8,12‐dihydroxy‐3β‐acetoxy‐9β‐angeloyloxyeremophil‐7(11)‐en‐8,12‐disemiketal ( 5 ). Their structures were established by spectroscopic methods and 2D NMR techniques. In addition, bakkenolide ( 1 ) and eremophilenolides ( 5 ) showed antibacterial and cytotoxic activities.     

Synthesis of Natural Atisane‐Type Diterpenoids by retro‐Biomimetic Transformations

An efficient one step, retro‐biomimetic procedure for the synthesis of natural products having the atisane structure is described (Scheme 2), natural products which are components of medicinal plants and possess relevant biological activity. Their structures were confirmed by chemical transformations and spectral data. The starting materials were the known ent‐kaur‐16‐en‐19‐oic acid ( 1 ) and ent‐trachyloban‐19‐oic acid ( 2 ), diterpenoids readily available from the waste of sunflower.     

Three New ent‐Trachylobane Diterpenoids from Co‐cultures of the Calli of Trewia nudiflora and Fusarium sp. WXE

Five diterpenoids, including three new ent‐trachylobane diterpenoids, i.e., (3α)‐3‐hydroxy‐ent‐trachylobane‐17,19‐dioic acid 19‐methyl ester ( 1 ), ent‐trachylobane‐17,19‐dioic acid 19‐methyl ester ( 2 ), ent‐trachylobane‐17,19‐dioic acid ( 3 ), and two known atisane‐type ones, i.e., (16α)‐16,17‐dihydroxy‐ent‐atisan‐19‐oic acid methyl ester ( 4 ), and 17‐hydroxy‐ent‐atisan‐19‐oic acid methyl ester ( 5 ), were isolated from the co‐culture extract of the calli of Trewia nudiflora and its endophytic fungus Fusarium sp. WXE. Their structures were elucidated by spectroscopic analyses, including 1D‐ and 2D‐NMR experiments, and HR‐Q‐TOF mass spectrometry. The antitumor and antibacterial properties of the new compounds were evaluated.     

Two New, 1‐Oxygenated ent‐Kaurane‐Type Diterpenes from Croton kongensis

One new ent‐8,9‐secokaurane diterpene, kongensin D ( 1 ), and one new ent‐kaurane diterpene, kongensin E ( 2 ), along with one known compound, (7α)‐7,18‐dihydroxy‐ent‐kaur‐16‐en‐15‐one 18‐acetate ( 3 ), were isolated from the aerial parts of Croton kongensis. The structures of the new compounds were elucidated by means of HR‐MS and in‐depth NMR experiments, and by comparison with literature data. Compounds 1 and 2 showed an unusual oxygenation pattern with an OH or AcO group at C(1).     

New ent‐Pimarane Diterpenes from the Roots of Aralia dumetorum

Four new ent‐pimarane diterpenes were isolated from the EtOH extract of Aralia dumetorum, together with three known compounds involving ent‐pimar‐8(14),15‐dien‐19‐oic acid ( 5 ), ent‐pimar‐8(14),15‐dien‐19‐ol ( 6 ), and ent‐kaur‐16‐en‐19‐oic acid ( 7 ). By detailed analyses of the MS, IR, and NMR data, the structures of four new diterpenes were characterized as (5β,9β,10α,13α)‐pimara‐6,8(14),15‐trien‐18‐oic acid ( 1 ), (5β,7β,9β,10α,13α)‐7‐methoxypimara‐8(14),15‐dien‐18‐oic acid ( 2 ), (5β,9β,10α,13α,14β)‐14‐methoxypimara‐7,15‐dien‐18‐oic acid ( 3 ), and (5β,10α,13α,14α)‐14‐hydroxypimara‐7,9(11),15‐trien‐18‐oic acid ( 4 ). The cytotoxic activities of compounds 1  –  7 were assayed in vitro through MTT method.     

Novel ent‐Abietane Diterpenoids from Isodon eriocalyx var. laxiflora

Two novel ent‐abietane diterpenoids, 3α,20‐epoxy‐6β‐hydroxy‐1,7‐dioxo‐ent‐abiet‐15(17)‐en‐16‐oic acid ( 1 ) and ent‐abieta‐7,15(17)‐diene‐3β,16,18‐triol ( 2 ) were isolated from Isodon eriocalyx var. laxiflora. Their structures were determined by extensive spectroscopic analysis and confirmed by X‐ray crystallography. Compound 1 is an unprecedented example that establishes that a naturally occurring ent‐abietane diterpenoid can have an oxygenation pattern almost identical to those of 3α,20‐epoxy‐ent‐kaurane diterpenoids.     

Rubescensins S and T: Seco‐ent‐Kaurane Diterpenoids from Isodon rubescens var. taihangensis

Two new diterpenoids, rubescensin S (=(1α,6β,14β)‐7α,20‐epoxy‐1,7,14‐trihydroxy‐16‐oxo‐15,16‐seco‐ent‐kauran‐6,15‐olide; 1 ) and rubescensin T (=(1α,6β,11β,20S)‐7α,20‐epoxy‐1,6,7‐trihydroxy‐20‐methoxy‐8,15‐seco‐ent‐kaur‐16‐en‐11,15‐olide; 2 ) were isolated from the Chinese medicinal herb Isodon rubescens var. taihangensis. Compound 1 possesses a unique, unprecedented 15,16‐seco‐ent‐kaurane skeleton. Both compounds exhibited cytotoxic activities against K562 human leukemia cells.     

A Diterpenoid with a New Skeleton and Cytotoxic Terpenoids Isolated from Amentotaxus formosana

The new diterpenoid 6α‐hydroxy‐ent‐kaur‐16‐en‐15‐one ( 1 ) and a diterpenoid with a new skeleton, i.e., amentotaxin BB ( 5 ), as well as three new lanostanoids, i.e., (3β,23R)‐3‐methoxy‐24‐methylenelanost‐8‐en‐23‐ol ( 6 ), (23S)‐23‐methoxy‐24‐methylenelanost‐8‐en‐3‐one ( 7 ), and (3β,24ξ)‐3‐methoxy‐24‐methyl‐9,19‐cyclolanostan‐25‐ol ( 8 ), were isolated from the bark and leaves of Amentotaxus formosana. Their structures, including the relative configurations, were elucidated from spectroscopic data. Compound 1 and a known compound, ent‐kaur‐16‐en‐15‐one ( 2 ), showed potent cytotoxic effects against a number of cancer cells in vitro.     

Three New Cytotoxic ent‐Kaurane Diterpenoids from Isodon weisiensis C. Y. Wu

Three new cytotoxic ent‐kaurane diterpenoids, (1α,7α,14β)‐1,7,14‐trihydroxy‐ent‐kaur‐16‐en‐15,18‐dione ( 1 ), (1α,7α,14β)‐1,7,14,18,20‐pentahydroxy‐ent‐kaur‐16‐en‐15‐one ( 2 ), and (3β,7α,14β)‐3,7,14‐tris(acetyloxy)‐ent‐kaur‐16‐en‐15‐one ( 3 ), were isolated from Isodon weisiensis C. Y. Wu. Their structures were elucidated by spectroscopic methods, including 2D‐NMR techniques, and the crystal structure of 1 was determined by single‐crystal X‐ray‐diffraction analysis. The chosen crystal of 1 was orthorhombic, space group P2₁2₁2₁, and there were two molecules with little difference in bond length and bond angle in the least‐asymmetry unit. Compounds 1–3 showed significant cytotoxic activities against human‐cancer cell lines Bel‐7402 and HO‐8910.     

Chemical Transformations of Phyllocladane (=13β‐Kaurane) Diterpenoids

Earlier phytochemical work on Plectranthus ambiguus (Lamiaceae) afforded a series of tetracyclic phyllocladane‐type (=13β‐kaurane) diterpenoids (see 1a – f ). In the course of investigations concerning the reaction behavior of this rare natural‐products, a new constituent of P. ambiguus was isolated, (2S,3R,16R)‐phyllocladane‐2,3,16,17‐tetrol 2,3‐diacetate ( 1g ), and another eighteen new phyllocladanes were prepared by chemical transformations and characterized. The main constituent 1b of P. ambiguus was chemically transformed to the known natural diterpenoid calliterpenone (=(16R)‐16,17‐dihydroxyphyllocladan‐3‐one; 2 ) thus unambiguously establishing its structure (Scheme 1). Epimerization at C(16) via the epoxy derivative 20 yielded 16‐epicalliterpenone ( 21 ), 17‐hydroxyphylloclad‐15‐ene‐3‐one ( 22 ), and (16R)‐3‐oxophyllocladan‐17‐al ( 23 ) (Scheme 6). Comparing this reaction sequence with the corresponding one starting from diastereoisomeric (16R)‐16,17‐dihydroxy‐ent‐kauran‐3‐one (=abbeokutone; 27 ) showed basically the same outcome (Scheme 7). Furthermore, three new C(16)‐substituted ent‐kauran‐3‐ones were characterized. Reliable spectroscopic arguments for the determination of the configuration at C(16) in phyllocladanes and kauranes as well as for the differentiation of the diastereoisomeric skeletons are presented.     

Two New C(20)‐Oxygenated ent‐Kaurene Diterpenoids from Isodon henryi

Two new C(20)‐oxygenated ent‐kaurene diterpenoids, taibaihenryiins A ( 1 ) and B ( 2 ), along with five known compounds, shikokianin ( 3 ), longikaurin D, 2α‐hydroxyursolic acid, longikaurin F, and lasiodin, were isolated from the EtOH extract of the leaves and tender branches of Isodon henryi (Hemsl .) Kudo . The structures of the new compounds were determined as 11α‐acetoxy‐7,20‐epoxy‐1α,6β,7β‐trihydroxy‐ent‐kaur‐16‐en‐15‐one ( 1 ) and 7,20‐epoxy‐3β,6β,7β,11α‐tetrahydroxy‐ent‐kaur‐16‐en‐15‐one ( 2 ) on the basis of detailed spectroscopic analyses.     

Three New 24‐Noroleanane Triterpenoids from Quercus aliena var. acuteserrata

Three new 24‐noroleanane triterpenoids, i.e., 2α,19α‐dihydroxy‐3‐oxo‐24‐norolean‐12‐en‐28‐oic acid ( 1 ) and 19α‐hydroxy‐3‐oxo‐24‐norolean‐12‐en‐28‐oic acid ( 2 ), and 2α,3β,19α‐trihydroxy‐24‐norolean‐12‐en‐28‐oic acid ( 3 ) were isolated from Quercus aliena var. acuteserrata, together with three known compounds, bartogenic acid ( 4 ), ilexgenin A ( 5 ), and aophitolic acid ( 6 ). Their structures were established by spectroscopic methods, especially 2D‐NMR and MS analyses.