Tetrahedron report number 863

Germacrenes A–E and related compounds: thermal, photochemical and acid induced transannular cyclizations

Germacrane sesquiterpenes are widespread in nature; in higher plants such as Solidago species (Solidago canadensis, Solidago altissima L.),2, 20, 20(a), 20(b) Ageratum conyzoides L.,²¹ Chloranthus spicatus (Thunb.) Makino,²² caraway herb and root,²³ fresh costus roots (Saussurea lappa),²⁴ Hedycarya angustifolia A. Cunn.,²⁵ Smyrnium olusatrum,²⁶ Acorus calamus L.,27, 27(a), 27(b), 27(c) and Meum athamanticum;²⁸ in liverworts such as Scapania undulata,²⁹ Lepidozia fauriana, Lepidozia vitrea,³⁰ and for more liverwort species read the review by Asakawa;³¹ in mosses such as Mnium hornum, Mnium marginatum, Mnium stellare, Plagiothecium undulatum and Taxiphyllum wisgrillii;³² in arthropods such as termite (Amitermes wheeleri,33, 33(a) Reticulitermes),^33b American cockroaches (Periplaneta Americana)34, 34(a), 34(b), 34(c) and aphid (Therioaphis maculala);35, 35(a), 35(b) in marine invertebrates such as Eunicea mimosa,³⁶ Sinularia mayi,³⁷ Sinularia polydactyla, Lithophyton arboretum, Strereonephthea cundabiluensis,³⁸ Pacifigorgia pulchra exilis, Pacifigorgia media and Tubipora musica,³⁹ and in microorganisms such as Helminthosporium sativum fungus⁴⁰ and Streptomyces citreus bacteria.41, 41(a), 41(b)

Structurally, the cyclodecadiene system of germacranes consists of two endocyclic double bonds at 1(10)- and 4(5)- positions and methyl groups at C₄ and C₁₀ with an isopropyl group at C₇. This cyclodecadiene system consists of an isoprene units linked in head-to-tail fashion consistent with the Isoprene rule42, 42(a), 42(b) (Scheme 2). With respect to the endocyclic double bonds in the 1(10)- and 4(5)- positions of the cyclodecadiene system, germacranes are classified into four possible geometric isomers namely E,E-germacranes, (Z,E)-germacrenes (melampolides), (E,Z)-germacrenes (heliangolides) and (Z,Z)-germacrenes,6, 11(l), 43 (Scheme 2). The trend of natural occurrences of geometric isomers is as follows: (E,E)>(Z,E)∼(E,Z)>(Z,Z), suggesting that of all these four isomers, E,E-germacranes are the most common in nature with few (Z,Z)-germacranes.

Recently, a regioisomer of (E,E)-germacrane with the isopropyl group at C₆ was reported18, 44 and it has been proposed to be biogenetic precursor of the rare gorgonanes, zieranes and iso-humulane sesquiterpenoids skeleton,¹⁸ Scheme 1.

Germacrane sesquiterpenes have been reported to have various biological purposes and effects in nature.¹⁰ These purposes range from biosynthetic intermediates of several sesquiterpenes16(a), 16(b), 13(a), 13(b), 14(a), 14(b), 14(c), 15, 45 to specific biological activities such as sex pheromones,34(a), 34(b), 34(c), 46 alarm pheromone,35(a), 35(b) anti-inflammatory activities,47, 47(a), 47(b) anti-cancer agents,48, 48(a), 48(b), 48(c) antiplasmodial activity,⁴⁹ antibacterial and antifungal activities,50, 50(a), 50(b), 50(c) antifeedant,⁵¹ tubulin inhibitor,⁵² phytotoxic activity,⁵³ inhibitor of tumour necrosis and interleukin-6,⁵⁴ PTP1B inhibitor,⁵⁵ NF-κB inhibitors,⁵⁶ inhibitors of preadipocyte differentiation,⁵⁷ inhibitors of germination and seedling growth in plant species,⁵⁸ moth antennal receptor neuron activator,⁵⁹ nematicidal activity,⁶⁰ cytotoxic activity,⁶¹ tyrosinase inhibitory activities⁶² and other various biological activities.

Interestingly, germacrene artefacts (elemenes) or the Cope rearrangement products isolated from the traditional Chinese medicinal herbs Rhizoma zedoariae, Curcuma Wenyujin Y. H. Chen et C. Ling and other higher plants have been reported to be effective in the treatment of leukemia and carcinomas of the brain, breast, liver and other tissues in China.63, 63(a), 63(b), 63(c), 63(d), 63(e), 63(f), 63(g), 63(h), 63(i)

The identification of germacranes is usually accomplished by gas chromatography coupled with mass spectrometry (GC–MS) in electron ionization mode (EI). Several general mass spectral libraries such as the Wiley and the NIST library⁶⁴ are available, but specialized libraries for volatile compounds (e.g., MassFinder), which contains a large collection of mono- and sesquiterpenoids,⁶⁵ are sometimes more useful. These libraries offer tremendous identification opportunity, but they can also fool an inexperienced user, because a closest hit within the library might be taken as positive identification. In cases where germacrane related compounds generate similar mass spectra, for example, different positional and geometrical isomers, compound identification cannot be performed on the basis of the mass spectrum alone. Therefore, inclusion of additional data, such as gas chromatographic retention indices, is critical for structure determination66, 66(a), 66(b), 66(c), 66(d), 66(e) (Table 1).⁶⁵

Generally, gas chromatographic (GC) analyses of extracts or essential oils commonly show the co-existence of germacrenes and its thermal artefact (elemane). And sometimes, presence of a particular elemene-type serves as a reliable indicator of its precursor germacrane. Thus, in cases where germacrane is present in trace amounts the corresponding elemane allows accurate prediction because the elemane-type is usually more abundant or higher.26, 67, 67(a), 67(b), 67(c), 67(d), 67(e), 67(f), 67(g), 67(h)

Often, higher plants have sesquiterpenes of opposite enantiomer to that of lower plants or microorganisms,66(d), 68, 68(a), 68(b), 68(c), 68(d), 68(e), 68(f), 68(g), 68(h) for example, (−)-germacrene A (2) was first isolated from the gorgonian Eunicea mammosa³⁶ while its (+)-enantiomer is commonly widespread in the higher plants such as caraway herb and root,²³ and fresh costus roots (S. lappa).²⁴

Only a few germacranes are readily available in their pure form. This is due to instability associated with their isolation during the sample handling procedures from their natural sources. The isolation difficulties originate from the sensitivity of their endocyclic double bonds, which sometimes undergo closures that give other bicyclic sesquiterpenes in slightly acidic conditions2, 24, 69, 69(a), 69(b), 69(c), 69(d), 69(e) and elevated temperatures.1, 11(e), 11(m), 14(b), 14(c), 24, 29, 36, 69(e)

Therefore, the isolation of germacrane requires mild conditions and methods used also depend on complexity of the sample. These methods include column chromatography on silica gel or Al₂O₃ or prep-TLC or prep-GC or HPLC or a combination of two to three of these methods. Isolations have been achieved on column chromatography using silica gel with eluent such as n-pentane or n-pentane–ethyl acetate at room temperature or at −25 °C,2, 11(m), 69(e), 70, 71, 71(a), 71(b), 71(c), 71(d) AgNO₃-impregnated silica gel with Et₂O and Et₂O–MeOH as eluents,⁷⁰²⁴ AgNO₃-impregnated Al₂O₃ with hexane–Et₂O as eluent,1, 20(b) deactivated-Al₂O₃ with n-pentane–Et₂O as eluent,¹⁸ 30% powdered sucrose and 70% florisil mixed as dry solids with n-hexane³⁶ and by prep-TLC (silica gel) at room temperature for further purification^11m or at −25 °C.^69e Other methods include prep-GC with SE-30 column,2, 18, 29, 72, 72(a) and prep-GC with enantioselective cyclodextrin columns such as heptakis(6-O-tert-butyldimethyl-silyl-2,3-di-O-methyl)-β-cyclodextrin and octakis(2,6-di-O-methyl-3-O-pentyl)-γ-cyclodextrin.2, 29, 72(b) For germacranolides, which are less volatile and thermolabile, HPLC on reverse phase or sometimes normal phase may be preferable,⁷³ or at times a combination of silica gel column chromatography, Sephadex LH-20 and HPLC.⁷⁴ For more information on analytical techniques readers are forwarded to a review by Merfort.⁷⁵

Of all the four germacrane geometric isomers, the conformational behaviour of (E,E)-germacranes has been studied intensively.11(d), 11(e), 11(f), 11(g), 11(i), 11(j), 11(k), 11(l), 76, 76(a), 76(b), 76(c) These studies have shown that cyclodecadiene ring of germacranes in this case hedycaryol (3) can adopt four distinct conformations, which has been denoted as UU, DU, DU, and DD. U (up) and D (down) refer to the orientation of the methyl groups at C₄ and C₁₀ (Scheme 3). The UU and DD conformations have a crossed endocyclic double bonds while UU and DD have a parallel endocyclic double bonds. The inversion of the C₇–C₈ unit in the cyclodecadiene ring is highly dependent of substituent at C₇, which preferably occupy an equatorial or pseudoequatorial position.11(d), 11(e), 11(f), 11(g), 11(j), 11(m)

Samek and Harmantha (1978) introduced an extensive notation to denote the cyclodecadiene conformers formed from E,E-, E,Z-, Z,E- and Z,Z-germacranes. ^11g The corresponding notations for the case of E,E-cyclodecadiene are as follows: UU=(₁D¹⁴, ¹⁵D₅), UD=(₁D¹⁴, ₁₅D⁵), DD=(¹D₁₄, ₁₅D⁵), and DU=(¹D₁₄, ¹⁵D₅).^11g

In solution, most (E,E)-cyclodecadiene system have been reported to show a temperature dependent NMR spectra indicative of several interconvertible conformational isomers in equilibria.11(e), 11(f), 11(j), 11(m), 14(b), 14(c) This interconversion usually results in broadened or multiplet sets of NMR signals.11(d), 11(f), 11(m), 14(b), 14(c), 36, 77

Reliable predictions of most stable conformations of the germacranes have been achieved by molecular mechanics calculations (MMC), NMR (NOE) and X-ray studies or sometimes a combination of any of these methods. The molecular mechanics calculations (MMC) have been applied to study many hydrocarbons including cyclodecadiene rings and reliable predictions of conformations, heats of formation, steric energies, and in some cases estimates of energy barriers between conformers have been achieved.11(b), 11(c), 11(k), 11(l), 11(m), 78, 78(a), 78(b), 78(c)

Before MMC, the most favourable transition state of (E,E)-cyclodecadiene system reported is the chair–chair (CC).11(a), 11(f), 69(b) This observation was later confirmed when MMC was applied to the thermal isomerization products of (E,E)-1,5-cyclodecadiene.11(h), 11(i) Terada and Yamamura (1979) have evaluated the relative stabilities of each conformation of germacrene A (2), hedycaryol (3) and germacrene B (4) in their ground states and transition states. In these experiments they confirmed that elemenes are formed from the corresponding germacrenes through the most stable transition states (CC), regardless of the most stable conformers in each ground state. They also indicated that the two elemanes shyobunone (5) and epi-shyobunone (6), resulting from acoragermacrone (7), are formed from CC, and CC and CT, respectively.11(h), 11(i) Molecular mechanics calculations (MMC) also revealed that germacrene B (4) has two comparably stable conformations.^76b

Ground state conformation of all possible geometrical isomers of hedycaryol (3) (E,E-, E,Z-, Z,E-, Z,Z-) have also been estimated by MMC and all the four isomers are indicated to exist in more than two conformations in which the parallel conformation (TC or TT) is reported to be the most stable for all isomers.76(c), 78(c)

Using MMC, Tashkhodzhaev and Abduazimov (1997)^11l have suggested a detailed probable structural types of cis/trans-guaianes and cis/trans-eudesmanes formed from the four conformers of E,E-germacranes via a transannular cyclization. They have also suggested the possibility of similar structural guaianes and eudesmanes type of compounds from (E,Z)- and (Z,E)-germacranes.^11l

Intramolecular nuclear Overhauser effect (NOE) measurements have been used to deduce the stable conformation of germacrene-type sesquiterpenes in solution.^11e In a few cases, NOE experiments, in combination with variable-temperature ¹H NMR spectra and/or molecular mechanic calculations, have been applied to establish the preferred conformation germacranes.11(a), 79, 79(a), 79(b), 79(c), 79(d) Wharton et al. (1973) concluded that hedycaryol (3) consists mainly of one crossed and two parallel conformations and that the conformational changes is rapid at 90 °C and slow at −30 °C, with the parallel conformations predominating to 75% at −30 °C and that they are favoured to the parallel forms at higher temperatures.^11d

Recently, using variable-temperature 500 MHz ¹H NMR spectra in CDCI₃, the three NMR-distinguishable conformational isomers of (+)-germacrene A (2) were established to occur in the ratio 5:3:2 at or below ordinary probe temperature (25 °C) by Coate's group.^11m They assigned 52% UU, 29% UD and 19% DU to the conformer structures based on ¹H NMR data comparison, NOE experiments and vicinal couplings.^11m

Using a variable-temperature ¹H NMR spectroscopy, Allemann's group (2007) showed that 5-fluorogermacrene A (8) indicates the existence of two conformers in the ratio 3.22:1 that were in slow exchange at −60 °C, while at 90 °C the two isomers gave rise to averaged NMR signals.^14b Consistent with 8, ¹H NMR and ⁹F NMR spectra of (−)-1-fluorogermacrene (9) at 25 °C also revealed the existence of a 7:3 mixture of UU and UD conformers in solution.^14c

X-ray analysis of silver nitrate adducts of pregeijerene A (10),⁸⁰ costunolide (11),81, 81(a) germacrene B (4)^81b and germacrone (1)⁸² have shown that the most stable chair–chair (CC) conformation is also the one that is preferred in the solid state. Recently, attempts to prepare X-ray quality crystals of the AgNO₃ adduct of (+)-germacrene A (2) using the literature methods have been reported to be unsuccessful.^11m

Cope rearrangement in germacranes is a stereospecific [3,3]-sigmatropic rearrangement that proceeds via the most stable chair-like transition state of 1,5-cyclodecadiene to give 1,2-divinylcyclohexane system. The configuration of 1,2-divinylcyclohexane formed depends on most stable chair-like transition state conformation of the corresponding 1,5-cyclodecadiene.11(f), 11(h), 11(i), 15, 83 The mechanism of Cope rearrangement has been studied84, 84(a), 84(b), 84(c), 84(d) and various groups have contributed to the understanding of this rearrangement.85, 85(a), 85(b), 85(c), 85(d), 85(e), 85(f), 85(g), 85(h), 85(i)

It is also well known that Cope rearrangement is a reversible process86, 86(a), 86(b), 86(c), 86(d) and that equilibrium between 1,5-cyclodecadiene and 1,2-divinylcyclohexane system depends on the nature and configuration of substituents.85(i), 86(d), 87, 87(a), 87(b) While E,E-cyclodeca-l,5-dienes are known to undergo Cope rearrangement to give trans-l,2-divinylcyclohexane,11(m), 14(b), 14(c), 36, 69(e) the Z,E-cyclodeca-1,5-dienes give cis-1,2-divinyl derivative29, 67(a), 76(c), 88 and it has been established that the presence of a fused five-membered ring at positions C₆ and C₇ in the Z,E-cyclodeca-1,5-diene derivatives influences the stereostructure of the 10-membered ring transition state, which has an important effect on the rearrangement.⁸⁸

Recent investigation by Sezter (2008) on energetics of Cope rearrangement product of 17 germacrane sesquiterpenoids by density functional theory (B3LYP/6-31G^∗) and post Hartree–Fock (MP2/6-31G^∗∗) showed that different substitution patterns affect the relative energetics of the germacrene–elemene Cope rearrangement.⁸⁹ The results of these two calculations are in qualitative agreement with the experimentally observed Cope rearrangements, eventhough the two methods gave slightly different results.⁸⁹

Generally, germacranes undergo Cope rearrangements to elemanes, and it is possible to detect variation in the amount of elemane produced from their germacrane precursor by variation of GC injector port temperature. The increase in temperature of GC injector port leads to an increase in the amount of elemane produced with a corresponding decrease in the precursor germacrane.11(m), 17, 24, 29, 69(e), 90 In few cases, small amount of diastereomeric elemanes could also be formed in addition to the major elemane produced at slightly high temperature.²⁹

The existence of different germacrane conformations have been postulated to explain the formation of a small amount of diastereomeric elemanes during the thermal Cope rearrangement of a few cyclodecadienes such as acetyl caulesol (12), which rearranges into two elemanes 13 and 14 in ratio 5:3, respectively.⁹¹ Thermal reactions conducted at higher temperatures (ca. 500 °C) with synthesized hedycaryol isomers E,E-(3), Z,E-(3), E,Z-(3) and Z,Z-(3) led to the formation of diastereomeric elemols (15, 16, 17, 18) in addition to the low temperature formed elemols from corresponding hedycaryol isomer.^76c They suggested that although the diastereomeric elemols can be explained as being formed from respective conformation of hedycaryols, and they also noted a few features against the explanation and as a result, an additional pathway involving the retro-Cope rearrangements of the preformed low temperature elemols was considered.^76c

Occurrence of similar diastereomeric elemanes have also been reported for germacrene A (2),24, 69(e) and it derivatives (alcohol, aldehyde and carboxylic acid),²⁴ and helminthogermacrene (19).²⁹ In 5-fluorogermacrene A (8) and 1-fluoro-germacrene A (9), which appear more stable than germacrene A (2), only the corresponding major fluoro-elemane were observed.14(b), 14(c)

Germacranes undergo transformation to a large variety of products upon acid treatment. Acid induced cyclization of (E,E)-germacranes has been studied extensively,14(c), 24, 25, 69(a), 69(b), 69(d), 69(e), 76(c), 92, 92(a), 92(b), 92(c), 92(d), 92(e) whereas the induced transannular cyclizations of its geometric isomers such as (Z,E)-germacranes (melampolides),76(c), 93, 93(a), 93(b) (E,Z)-germacranes (heliangolides)2, 69(e), 76(c), 92(d) and (Z,Z)-germacranes appear to be limited to a few studies.^11l

Commonly, induced transannular cyclization of (E,E)-germacranes system produced corresponding trans-selinane-type, while (Z,E)- and (E,Z)-germacranes system produced cadinane-type and trans- or cis-selinane-type or guaiane-type sesquiterpenes.2, 69(e), 92(d)

From molecular mechanics calculations (MMC), a detailed probable cis/trans-guaianes and cis/trans-eudesmanes structural types that could result from transannular cyclization of (E,E)-germacranes have been reported by Tashkhodzhaev and Abduazimov (1997) and they have also suggested the possibility of having similar structural types of compounds from (E,Z)- and (Z,E)-germacranes.^11l

Several kinds of acidic reagents and solvents that have been used to induce cyclization include 80% aq AcOH, 80% aq HCOOH, AlCl₃ in absolute ether, AcOH in PhSH, 100% HCOOH in PhSH and concd H₂SO₄ in PhSH;^92d 80% aq AcOH at 0 °C for 1 h;^93c adsorption on SiO₂;69(e), 92(a) adsorption on SiO₂, acetic acid, p-toluene sulphonic acid, or Amberlyst^® 15, BF₃, AICI₃;² p-toluenesulfonic acid;²⁴ storage at 4 °C in CDCl₃ for at least one week, treated with BF₃ in diethyl ether, and acidic ion exchange resin Amberlyst^® 15;^69e treated with CF₃CO₂H in CDCl₃;^14c and when subjected to basic treatment such as basic alumina.93(d), 93(e)

The rearrangements products obtain from acid induced cyclizations depend on acid reagents or solvents use, reaction time and temperature,2, 69(e), 92(d) while the percentage (%) yield of products is dependent of the overall reaction conditions.² With increasing reaction time, secondary rearrangement products could also be formed, which cannot be related to the starting material.²

The studies on biotransformation of germacranes have led to the discovery of bio-activation and/or bio-inactivation metabolites with unique structures. Biotransformation of germacrone (1) by Aspergillus niger⁹⁴ and cultured plant cells of Curcuma zedoaria⁹⁵ produced similar as well as new sesquiterpenoid metabolites of hydroxylated guaiane-type (20, 21), eudesmane-type (22, 23, 24, 25), together with allylic alcohols (26) and glucosylated eudesmane (27) (Scheme 4). The configurations of some of the derivatives were opposite to those of the sesquiterpenes isolated from the Curcuma species, such as Curcuma aromatica, Curcuma longa and C. zedoaria.⁹⁵ Biotransformation of germacrone (1) examined by suspension cultured cells of Lonicera japonica, Bulpleurum falcatum, Polygonumn tinctorium and S. altissima afforded several types of sesquiterpenes, such as guaiane, eudesmane and seco-guaianes (28, 29).⁹⁶

Incubation of (+)-hedycaryol (3)⁹⁷ administered to a mortared root suspension of fresh chicory for four days gave cryptomeridiol⁹⁸ as its sole product.^69d Biotransformation of germacrane epoxides by a suspension of fresh chicory root (Cichorium intybus) gave hydroxylated guaianes and eudesmane derivatives.⁹⁹

Biotransformation have also been reported in other germacrane-type compounds such as curdione (30),100, 100(a), 100(b) costunolide (11),100(c), 100(d), 100(e) (1α,10β),(4β,5α)-diepoxygermacrane (31)¹⁰¹ and shiromodiol diacetate (32).¹⁰² Commonly, biotransformation increases the polarity and water solubility of germacrane metabolites via hydroxylation, oxidation and glucosylation.

Germacrene A synthases have been isolated, purified and characterized from chicory roots.15, 103 For detailed recent review on germacrene A synthases and biosynthesis read a recent publication by Faraldos et al. (2007).^11m In addition, the intermediacy of germacrene A (2) in the mechanism involving conversion of (E,E)-farnesyl diphosphate (FPP) to aristolochene^14b and epi-aristolochene^14c have been indirectly confirmed with fluoro-FPP derivatives.14(b), 14(c)

Biosynthesis of germacrene C (33) was confirmed by a preliminary radioactive labelling experiment involving an enzymatic conversion of 2-¹⁴C-mevalonic acid lactone via trans,trans-farnesyl pyrophosphate (FPP).¹⁰⁴ The enzymatic solvolysis of farnesyl pyrophosphate (FPP) by prenyltranferase yielded 33 as part of the products.¹⁰⁵ The isolation, purification and characterization of germacrene C synthases from Lycopersicon esculentum cv. VFNT Cherry have been achieved.¹⁰⁶

The biosynthetic pathway of germacrene D (34) was elucidated by incubation of isolated enzymes with isotopically labelled FDP¹⁰⁷ and mass spectrometric analysis of the products,¹⁰⁸ which indicated different steps for both enantiomers as already suspected by Niwa et al., 1980.^20b The labelling experiments carried out on S. canadensis with 1-[5,5-D₂]deoxy-d-xylulose-5-phosphate (D₂-DOXP), [5-¹³C]mevalonolactone (¹³C-MVL) and [1-¹³C]-d-glucose,¹⁰⁹ and Tanacetum vulgare (Asteraceae)¹¹⁰ revealed that the biosynthesis of germacrene D (34) proceeds predominantly via the methylerythritol phosphate pathway. Enantiospecific (+)- and (−)-germacrene D synthases, cloned from ‘goldenrod’ S. canadensis showed a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.¹¹¹

Generally, the synthesis of germacranes have met tremendous set backs because of their thermal instability1, 11(e), 24, 35(a), 36, 112, 112(a), 112(b) and their sensitivity towards slight acidic conditions,24, 69(a), 69(b), 69(d), 69(e), 92(d) and UV radiations.² Despite these set backs many reports on germacrane syntheses have been published in the last 50 years. In 1999, an extensive review on synthesis of germacrane sesquiterpenes and related compounds has been reported by Minnaard et al.⁶ therefore, only related chemical synthesis of compounds reported on or after this review will be mentioned briefly. Germacrene B (4) and (±)-9-methylgermacrene B (35) have been synthesized.113, 113(a), 113(b) 9-Methylgermacrene B (35) has also been synthesized enantiospecifically.¹¹⁴ Recently, Mehta and Kumaran (2005) reported a short, flexible and enantioselective approach towards 10-membered germacratrienones from the commercially available monoterpene (−)-carvone.¹¹⁵