Macrolides and Alcohols as Scent Gland Constituents of theMadagascan Frog Mantidactylus femoralis and Their IntraspecificDiversityDennis Poth,†,‡ Pardha Saradhi Peram,† Miguel Vences,*,§ and Stefan Schulz*,†

†Institut fur Organische Chemie, Technische Universitat Braunschweig, Hagenring 30, 38106 Braunschweig, Germany§Zoologisches Institut, Technische Universitat Braunschweig, Mendelssohnstraße 4, 38106 Braunschweig, Germany

*S Supporting Information

ABSTRACT: Acoustic and, to a lesser degree, visual signalsare the predominant means of signaling in frogs. Nevertheless,certain lineages such as the mantelline frogs from Madagascaruse the chemical communication channel as well. Malespossess femoral glands on the hind legs, which recently havebeen shown to contain volatile compounds used incommunication as pheromones. Many mantelline speciesoccur in sympatry, and so far species recognition is regardedto occur mainly by acoustic signals. The analysis of the glandconstituents of Mantidactylus femoralis by GC/MS revealed the presence of volatile macrolides and secondary alcohols. The newnatural products mantidactolides A (4) and B (6), as well as several methyl carbinols, were identified, and their structures wereconfirmed by synthesis. The analysis of individuals from different locations of Madagascar revealed the presence of two groupscharacterized by specific patterns of compounds. While one group contained the alcohols and mantidactolide B, the othershowed specific presence of the macrolides phoracantholide I (1) and mantidactolide A (4). Genetic analysis of some individualsshowed no congruence between genetic relatedness and gland constituents. Several other individuals from related species haddifferent gland compositions. This suggests that a basic set of biosynthetic machinery might be available to a broader group ofrelated species.

Amphibians are an animal class in which the knowledge onchemical communication is relatively scarce. The pre-

dominant means of signaling in anurans (frogs) is acoustic and,to a lesser degree, visual.1 Variations in the advertisement callswithin species of frogs are typically restricted to the effects oftemperature, individual body size, or hormone-related sexualmotivation.2 Qualitative differences in call structure are, on thecontrary, indicative of species-level differentiation and usuallycorrelate with strong genetic differences. Despite theundoubted importance of bioacoustic communication infrogs, a considerable proportion of these animals arecharacterized by sexually dimorphic macroglands at differentparts of their bodies,3 suggesting that the secretions of theseglands may be of importance in their mating or territorialbehavior. Although it is well established that amphibians usepheromones, until recently only water-soluble compounds suchas peptides or prostaglandins have been identified aspheromones.4 In a recent study we have demonstrated thatvolatile alcohols or macrolides are also used by frogs aspheromones.5

In Madagascar and the Comoros, one endemic group ofmore than 200 species of frogs, the Mantellidae, containsvarious genera that are characterized by distinct glands on theunderside of male thighs. These so-called femoral glands6 areparticularly distinct in the genus Mantidactylus, where rudi-ments are also visible in females. In a previous study of

Mantidactylus multiplicatus, M. betsileanus, and several mantellidspecies of the genus Gephyromantis, it was shown that thesescent-emitting femoral glands of the males contain species-specific mixtures or compounds, many of them with unknownstructure.5 These compounds likely are used as pheromones, ashas been shown for M. multiplicatus. Although a highinterspecific variability in gland composition is obvious, theintraspecific diversity of the composition is as yet unstudied.The present study focuses on analyzing the volatile

compounds from the femoral glands of Mantidactylus femoralisas well as the variation of these substances within populationsof M. femoralis and closely related species in the subgenusOchthomantis of Madagascan river bank frogs. Individualgenetic analysis of the frogs determined their geneticrelatedness, and the characterization of new compounds fromM. femoralis glands demonstrated a considerable geographicvariation of femoral skin secretions in this species.

■ RESULTS AND DISCUSSION

Identification of Volatile Femoral Gland Constituentsof Mantidactylus femoralis. During the analysis of CH2Cl2extracts of femoral glands from various Madagascan mantelline

Received: February 11, 2013Published: September 4, 2013

Article

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frogs we encountered an individual of M. femoralis from theFotsialanana river collection site (in the Makira Reserve,eastern Madagascar) that contained two volatile compounds inhigh concentrations (Figure 1).Compound A was identified as phoracantholide I (1) due to

its characteristic mass spectrum by comparison with data fromthe literature.7 It was first described as a constituent of thedefense secretion of the Australian beetle Phoracanthasynonyma, where it occurs as the R-enantiomer. Syntheticreference material of both enantiomers was obtained byhydrogenation of its unsaturated analogue phoracantholide J(2), which was previously synthesized.5 Gas chromatographyon a chiral phase revealed that natural phoracantholide I (1)found in M. femoralis has the S-configuration (Figure S1), whilebeetles produce the opposite enantiomer.7

The other major component was an unknown naturalproduct with a mass spectrum similar to that of 1, pointing to amacrolide structure. High-resolution mass spectrometryrevealed an m/z of 184.1483, corresponding to a molecularformula of C11H20O2 and indicating the presence of anadditional CH2 compared to phoracantholide I. Phoracantho-lide I exhibits a gas chromatographic retention index (I) of1268, while compound B showed I = 1336. The difference (ΔI= 68) ruled out the possibility of an extended chain, as, forexample, in 9- or 10-undecanolide, because this would require aΔI of about 100.8 Therefore, compound B was likely abranched 9-decanolide in which the additional CH2 is locatedas a methyl group somewhere along the chain. Macrolides such

as phoracantholides I and J are likely derived from fatty acidbiosynthesis. A common motif for the formation of methylbranches in fatty acid biosynthesis is the incorporation ofmethylmalonate instead of malonate during biosynthesis.9 Thiswill result in the location of methyl groups at even-numberedcarbon atoms. The resulting target structures are shown inScheme 1, and the building blocks are highlighted. Because the

mass spectra of cyclic compounds do not allow easy location ofmethyl groups in cyclic compounds, all four structures weresynthesized, and their mass spectra were compared with that ofthe natural compound. An approach using ring-closingmetathesis10 with C6F6 activation

10c followed by hydrogenationwas used in all cases.2-Methyl-9-decanolide (3) was synthesized from available

phoracantholide J (2) by α-methylation of 2 with NaHMDS/MeI, followed by hydrogenation of lactone 7 (Scheme 2).A short synthesis was developed for 4-methyl-9-decanolide

(4, Scheme 3). Ethyl 3-bromopropionate (8) was coupled withisopropenylmagnesium bromide under Li2CuCl4 catalysis toform ethyl 4-methylpent-4-enoate (9). Saponification withKOH yielded 4-methylpent-4-enoic acid (10). Acid 10 wasthen coupled with hept-6-en-2-ol (12), which was obtained bythe addition of 3-butenylmagnesium bromide (11) to

Figure 1. Gas chromatogram of the femoral gland extract of a single male Mantidactylus femoralis (individual number ZCMV 11251) collected at theFotsialanana River. The main constituents A and B occur in high concentrations and are accompanied by a mixture of fatty acid ethyl esters andglycerides. X: artifact.

Scheme 1. Possible Structures of Compound B According toFatty Acid Biosynthesis

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propylene oxide. After formation of unsaturated ester 13 a ring-closing metathesis using the Hoveyda−Grubbs II catalystformed the unsaturated lactone 14. The target compound 4 wasobtained via hydrogenation.The synthesis of 6-methyl-9-decanolide (5) was carried out

in six steps starting from 6-methyltetrahydro-2H-pyran-2-one(15) (Scheme 4). Introduction of a methyl group in the α-

position with LDA/MeI and the subsequent partial reductionwith LAH furnished 3,6-dimethyltetrahydro-2H-pyran-2-ol(16), which was transformed by a Wittig reaction into alcohol17. Coupling with 4-pentenoic acid furnished the ester 18, theprecursor for the following ring-closing metathesis using theHoveyda−Grubbs II catalyst. Finally, macrolide 5 was obtained

by hydrogenation of the double bond of unsaturated macrolide19.The synthesis of 8-methyl-9-decanolide (6) is shown in

Scheme 5. 4-Pentenoic acid (20) was converted into thecorresponding acid chloride, coupled with (S)-4-benzyloxazo-lidin-2-one and stereoselectively methylated to form (S)-4-benzyl-3-((S)-2-methylpent-4-enoyl)oxazolidin-2-one (21). Re-duction with LAH led to alcohol 22. The oxidation of 22 to thecorresponding aldehyde was followed by a Grignard reactionwith methylmagnesium bromide to furnish (S)-3-methylhex-5-en-2-ol (23). Next 23 was coupled with 5-hexenoic acid to yieldthe unsaturated ester 24. Cyclization with Grubbs-II catalystformed the unsaturated lactone 25, which was hydrogenated inthe last step to yield 8-methyl-9-decanolide (6).After the completion of the syntheses, the mass spectra and

gas chromatographic retention times of the synthesizedmacrolides were compared (Figure 2). All compounds showeddifferent mass spectra, and only that of 4-methyl-9-decanolidematched that of the natural compound B. We then set out toestablish the absolute configuration of the natural compound bysynthesizing an enantiomerically enriched stereoisomer(Scheme 6).Commercially available (+)-(β)-citronellene (26) was

selectively epoxidized with mCPBA at the trisubstituted doublebond.11 The resulting epoxide 27 was converted into thealdehyde 28 by a Lemieux−Johnson oxidation12 and furtheroxidized by Jones oxidation to yield the chiral acid 29. (S)-Hex-5-en-2-ol (33) was obtained via a copper-mediated coupling ofallyl bromide (32) and (S)-propylene oxide.13 Esterification ofacid 29 with alcohol 33 led to the unsaturated ester 30, whichwas converted into the unsaturated lactone 31 via ring-closingmetathesis. A final hydrogenation of the double bond formedthe desired (4R,9S)-4-methyl-9-decanolide (4R,9S-4). Theother three enantiomers were synthesized via the same routeusing appropriately configured starting materials. The buildingblock (R)-4-methylhex-5-enoic acid (R-29) was synthesizedfrom (−)-(β)-citronellene and (R)-hex-5-en-2-ol (R-33)obtained by the addition of allyl bromide to (R)-propyleneoxide. The combination of the different enantiomers of 29 and33 gives access to all four enantiomers of 4 via the respectiveester 30. Using gas chromatography on a chiral Lipodex-Gphase it could then be demonstrated that the natural compoundfrom M. femoralis has the shown (4R,9S)-configuration (FigureS2). For this new natural product we propose the namemantidactolide A. In conclusion, the femoral gland constituentsof this frog consisted of the two major componentsphoracantholide I (1) and mantidactolide A (4).

Chemical Diversity of Femoral Gland Contents ofIndividuals. In addition to the specimen from the FotsialananaRiver containing the two macrolides described above, femoralgland extracts of 11 individuals of M. femoralis collected at fivedifferent locations throughout Madagascar were analyzed.These samples contained a varying mixture of volatilecompounds, some of which have not been described beforeas natural products. Figure 3 shows a total ion chromatogram ofthe femoral gland extract of an individual collected inBemanevika containing the volatile compounds C−F.Surprisingly, neither macrolide 2 nor 4 was present in this

sample. Instead a new macrolide with I = 1342 (F) was found.Compound F’s mass spectrum (panel F; Figure 2) was identicalto that of macrolide 6 prepared previously. The retention indexmatched that of the later eluting diastereomer. Comparison ofthe H−H coupling constant J8,9 of the minor, first-eluting

Scheme 2. Synthesis of 2-Methyl-9-decanolide (3)a

aConditions: (a) NaHMDS, MeI, CH2Cl2, 2 h, 72%; (b) H2, 10% Pd/C, MeOH, 5 h, 62%, CH2Cl2.

Scheme 3. Synthesis of 4-Methyl-9-decanolide (4)a

a(a) Li2CuCl4, isopropenylmagnesium bromide, THF, 0 °C, 12 h,77%; (b) KOH, EtOH, H2O, 4 h, 80%; (c) (i) CuCN, Et2O, (ii)propylene oxide, 0 °C, 12 h, 81%; (d) 10 + 12, EDC·HCl, DMAP,CH2Cl2, 0 °C, 3 h, 75%; (e) Hoveyda−Grubbs II, C6F6, toluene, 80°C, 3 h, 80%; (f) H2, 10% Pd/C, MeOH, 5 h, 59%.

Scheme 4. Synthesis of 6-Methyl-9-decanolide (5)a

aConditions: (a) LDA, MeI, THF, 93%; (b) LAH, Et2O, 56%; (c)MePPh3Br, nBuLi, NaHMDS, THF, −78 °C to rt, 31%; (d) 4-pentenoic acid, EDC·HCl, DMAP, CH2Cl2, 0 °C, 3 h, 82%; (e)Hoveyda−Grubbs II, C6F6, toluene, 80 °C, 6 h, 48%; (f) H2, 10% Pd/C, MeOH, 5 h, 31%.

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diastereomer (J8,9 = 3.4 Hz) and the major later elutingdiastereomer (J8,9 = 10.4 Hz) with those predicted by softwaresimulation14 (2.8 and 8.2 Hz) led to the tentative assignment ofrelative configurations. The first-eluting diastereomer (I =1329) shows an (8R*,9S*)-configuration, while the later elutingnatural diastereomer (I = 1346) shows an (8R*,9R*)-configuration. Therefore, compound F was identified as thenew natural compound 8-methyl-9-decanolide (6), which wepropose calling mantidactolide B, most likely exhibiting an(8R*,9R*)-configuration. In only one of the samples we foundalso trace amounts of the (8R*,9S*)-diastereomer besides themajor (8R*,9R*)-diastereomer.Compound D was readily identified as 8-methylnonan-2-ol,

which is a known femoral gland constituent from M.multiplicatus.5 Gas chromatography on a chiral phase revealedthat the natural 8-methylnonan-2-ol from M. femoralis also hasthe R-configuration (Figure S3), identical to the configurationoccurring in M. multiplicatus.

The other two unknown volatile compounds C and E hadmass spectra (Figure 4) similar to that of 8-methylnonan-2-ol(D), suggesting these to be secondary alcohols as well. Theretention indices indicated these alcohols to contain a methylbranch either at the ω-1, as in D, or at the ω-2 position. Thevariation of the retention index depending on the position ofthe methyl branch along the chain can be estimated using anempirical gas chromatographic retention index system,developed by us.15 Both compounds 6-methyl- and 7-methyloctan-2-ol were then synthesized.3-Methylpentanol (34) was converted into the correspond-

ing bromide 35, which was then coupled with propylene oxideunder copper catalysis, forming 6-methyloctan-2-ol (36)(Scheme 7). 7-Methyloctan-2-ol was synthesized by a similarroute starting from 1-bromo-4-methylpentane (see SI). Acomparison of the mass spectra revealed slight differences(Figure 4), and the retention index I = 1070 for 7-methyloctan-2-ol was higher than for 6-methyloctan-2-ol (36), I = 1065. The

Scheme 5. Synthesis of 8-Methyl-9-decanolide (6)a

aConditions: (a) oxalyl chloride, Et2O, rt, 67%; (b) (S)-4-benzyloxazolidin-2-one, THF, rt, 84%; (c) NaHMDS/MeI, THF, rt, 12 h, 74%; (d) LAH,Et2O, rt, 3 h, 68%; (e) oxalyl chloride, DMSO, Et3N, CH2Cl2, 0 °C; CH3MgBr, rt, 1 h, 40%, 2 steps; (f) 5-hexenoic acid, EDC·HCl, DMAP, CH2Cl2,rt, 5 h, 73%; (g) Grubbs II, toluene, 80 °C, 3 h, 45%; (h) H2, 10% Pd/C, MeOH, 5 h, 76%.

Figure 2. Mass spectra of natural compounds B and F and of the synthesized macrolides 3−6.

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Scheme 6. Synthesis of (4R,9S)-4-Methyl-9-decanolide (4R,9S-4)a

aConditions: (a) mCPBA, NaOAc, CH2Cl2, 2 h, 90%; (b) NaIO4, dioxane, H2O, 24 h, 94%; (c) CrO3, H2SO4, acetone, 10 min, 49%; (d) (i) Mg,Et2O, (ii) CuCN, (iii) (S)-propylene oxide, 0 °C, 12 h, 51%; (e) 29 + 33, EDC·HCl, DMAP, CH2Cl2, 0 °C, 3 h, 92%; (f) Grubbs II, C6F6, toluene,80 °C, 3 h, 69%; (g) H2, 10% Pd/C, MeOH, 5 h, 74%.

Figure 3. Gas chromatogram of a femoral gland extract of an individual male Mantidactylus femoralis collected at Bemanevika. In addition to thealready known compounds D (8-methylnonan-2-ol), G (squalene), and H (cholesterol), several other volatile alcohols, C and E, as well as themacrolide compounds F were present. X: artifact.

Figure 4. Mass spectra of (a) 7-methyloctan-2-ol, (b) 6-methyloctan-2-ol (C, 36), and (c) 8-methyldecan-2-ol (E, 39) and characteristic massspectrometric fragmentation.

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latter matched the value found for natural compound C, I =1066.In the mass spectra of both C and synthetic 36 a fragment

with m/z = 97 was generated, which is absent in the massspectrum of 7-methyloctan-2-ol (Figure 4). This fragment isformed by the loss of water and a C2H5 fragment, as indicatedin Figure 4. Compound C was identified to be 6-methyloctan-2-ol (36). The typical fragmentation of a ω-2-branchedmethylcarbinol was also present in the mass spectrum ofcompound E, exhibiting the characteristic ion at m/z 125.Thus 8-methyldecan-2-ol (39) was proposed for alcohol E. A

short synthesis starting from 2-methylbutan-1-ol (37) verifiedthe proposed structure (Scheme 7).

Alcohol 37 was converted into the corresponding tosylate,which was then coupled with hex-5-enylmagnesium bromideunder copper catalysis to form alkene 38. The double bond wasoxidized via a Wacker oxidation, and the resulting ketone wassubsequently reduced using LAH to yield 8-methyldecan-2-ol(39). The mass spectrum as well as the retention index of thesynthetic compound matched those of the natural compound(C). The minor component 7-methyl-2-nonanol (40),occurring in some individuals, was tentatively identified by itsmass spectrum and I value.M. femoralis is found along the eastern portion of

Madagascar.16 Although some geographic variation can beexpected among populations of such a widespread species, thediversity of compounds encountered in their femoral glandextracts was unsuspected. All the data refer to the glands ofadult males, as samples from the rudimentary glands of femalefrogs and from belly skin did not contain any volatilecompounds and are not shown. Only eight of the 12 individualsanalyzed contained volatile compounds in their femoral glands.The absolute concentration of the samples varied widely. Someindividuals contained up to 500 μg of volatile material, whileothers contained nothing or very small amounts, only. Thisdivergence can be explained be either recent usage of the glandor lack of femoral gland constituent accumulation due tophysiological conditions. Among individuals, varying mixturesof the identified alcohols and the three macrolides werepresent, which was dependent on their collection site (Figure5).

Scheme 7. Synthesis of Natural Alcohols 6-Methyloctan-2-ol(36) and 8-Methyldecan-2-ol (39)a

aConditions: (a) PBr3, pyridine, Et2O, 2 d, 80%; (b) (i) Mg, Et2O; (ii)CuCN, THF; (iii) propylene oxide, 12 h, 0 °C, 64%; (c) pyridine,TsCl, CH2Cl2, 1 h, 95%; (d) hex-5-enylmagnesium bromide, Li2CuCl4,0 °C, 24 h, 53%; (e) PdCl2, Cu(OAc)2, O2, DMF, H2O, 72 h, 85%; (f)LAH, Et2O, rt, 2 h, 88%.

Figure 5. Occurrence of volatile compounds in femoral gland extracts of Mantidactylus femoralis from different localities in Madagascar. The differentmarks in the table indicate the amount of the compounds compared to the largest peak. ×××: 30−100%, ××: 10−30%, ×: 1−10%, ○: below 1%.The samples varied in absolute amount of the volatile compounds. The samples from Analabe contained only low amounts, while the samples fromAngozongahy and Vohiparara and one sample from Bemanevika showed higher concentrations of gland constituents. The largest amounts werepresent in the sample from Fotsialanana-Makira and one sample from Bemanevika. The color code is used to show collection sites on the map: (1)Angozongahy (yellow); (2) Analabe (red, 3 individuals), including individual ZCMV 12228; (3) Bemanevika (green, 2 individuals); (4) Vohiparara(blue), including individual ZCMV 8029; (5) Fotsialanana-Makira (gray), including individual ZCMV 11251.

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The results showed that the individuals can be classified intotwo groups with similar chemistry. While the samples fromAngozongahy, Analabe, and Bemanevika (group 1) containedsecondary alcohols, these were lacking in extracts fromVohiparara and Fotsialanana (group 2). In contrast, phor-acantholide I (1) and mantidactolide A (4) occurred only inindividuals of group 2. Furthermore, mantidactolide B (6) isthe major component of most individuals of group 1, but canbe found only in traces in group 2. Nevertheless, these groupsare not uniform in composition and differences can be seenbetween individuals.The methyl-branched alcohols D, 36, and 39 were present in

all femoral gland extracts of group 1. (R)-8-Methylnonan-2-ol(D) was always the major secondary alcohol except in onesample from Bemanevika, in which 6-methyl-2-octanol (36)dominated. Alcohols 39 and 40 were mostly minor metabolites.Mantidactolide A (4) was the major component in the extractfrom Fotsialanana, but only a minor component in theVohiparara individual, while both contained 1 in major and 6in trace amounts. In one sample from Bemanevika macrolide 6was only a minor component besides the major secondaryalcohol D.Most surprising are the strong differences among the samples

from Fotsialanana and Angozongahy, as these two sites are veryclose to each other; both are located in the Makira Reserve. Infact, various specimens from the Makira Reserve weregenetically heterogeneous, as exemplified by those fromHevirina (which yielded only empty femoral gland extracts)and Fotsialanana (Figure 6). According to 16S rRNA sequencedata (not shown; all sequences deposited in GenBank), theAngozongahy specimen was also somewhat geneticallydivergent despite the small geographic distances betweenthese three sites (less than 5 km). Together with the strong

differences in the femoral gland compounds, this might suggestthat M. femoralis as currently understood contains cryptic orincipient species. Individuals from Bemanevika were geneticallysimilar to those from Analabe and also showed the greatestqualitative similarity in femoral gland compounds to samplesfrom Analabe. While evolutionary diversification is one possibleexplanation for the differing femoral gland composition, also afood dependence might exist, although direct uptake of thecompounds by feeding and storage in the femoral glands isunlikely to be the only mechanism involved. In laboratoryraised M. betsileanus macrolide 2 is produced even when fruitflies were used as sole food source, which did not contain themacrolide (D. Poth, M. Vences, S. Schulz, unpublished results).This differs from poison frogs, which sequester dietary alkaloidsin their skin and lack these toxins when captive-raised onalkaloid-free food.17

In individual samples of high concentration several tracecomponents were detected that are prominent constituents ofother individuals. The Fotsialanana sample contains traces ofcompound 6 and methyl 2-octenoate. One sample fromBemanevika contained the respective methyl ketones of thesecondary alcohols as trace components. These findings mightsuggest that a relatively broad biosynthetic flexibility is presentin the frogs, allowing an easy evolutionary alteration of majorgland constituents, depending on the species or even theindividual.In order to understand the geographic and species-specific

nature of the differences in the femoral gland compositions, amolecular phylogenetic analysis based on the DNA sequencesof mitochondrial genes was performed on samples fromdifferent locations, which also confirmed the taxonomy of thecollected samples (Figure 6). This analysis shows that theanalyzed M. femoralis indeed group together despite the genetic

Figure 6. Phylogeny of the subgenus Ochthomantis in the genus Mantidactylus based on DNA sequences (3071 bp) of 5 mitochondrial genes. Thephylogram is a Bayesian 50% majority-rule consensus tree with other compatible groupings also shown. Bayesian posterior probabilities (BPP) >0.95are shown by circles at nodes. The specimens colored in green contained volatile compounds in their femoral glands. No such compounds werefound in the single analyzed gland of M. mocquardi (red); no data are available for the other species. Undescribed (candidate) species are numberedaccording to previous work.16

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differences found among some of them (see above) and thatthe lineage of all M. femoralis samples is genetically distinctfrom all related species.Diversity between Species. To better understand

patterns of intra- and interspecific variability of the femoralgland chemistry in mantellid frogs, extracts of various additionalspecies related to M. femoralis, all belonging to the subgenusOchthomantis (Figure 6), were analyzed. No volatile com-pounds were identified in the single sample available for M.mocquardi, but a variety of such compounds, which remain tobe studied in detail, were found in M. majori and theundescribed candidate species Mantidactylus sp. 47 andMantidactylus sp. 63. As revealed by the phylogenetic analysis,these species with M. femoralis represent all the major cladeswithin Ochthomantis (Figure 6), suggesting that volatilecompounds in femoral glands are probably universal withinthis subgenus. Although the amount of data is rather limited,the chemical composition of the gland secretions appears to beparticularly different where species occur in sympatry.For instance, in the Ranomafana region, where M. majori and

Mantidactylus sp. 47 occur together with M. femoralis (samplefrom Vohiparara), these three species do not share major orminor components of their glands. Phoracantholide I (1) was amajor compound in M. femoralis from the Ranomafana area(Vohiparara) but at the same locality was not detected in M.majori or Mantidactylus sp. 47. The major compounds in threeof four of the individuals of M. majori were identified as 8-methylnonan-2-one and 10-methylundecan-2-one, while allfour specimens of Mantidactylus sp. 47 contained primarilymethyl 2-octenoate and, in lower concentrations, 9-methyl-decan-2-one. Nevertheless, the close biosynthetic similarity tothe alcohols from M. femoralis is evident for the ketones, andtraces of methyl 2-octenoate can be found in some of the M.femoralis individuals.Of the two samples of Mantidactylus sp. 63 studied, both

from the Analabe forest, one contained no volatile compounds,but the other shared compounds with various specimens of M.femoralis. For instance, it contained phoracantholide I (1), butremarkably this compound was not observed in the syntopicspecimens of M. femoralis from Analabe. However, macrolide 1was observed in other, geographically distant populations of M.femoralis from Fotsialanana and Vohiparara. In addition, thesample of Mantidactylus sp. 63 also contained phoracantholide J(2), which was previously identified from other species ofMantidactylus.5 Furthermore, several unidentified compoundsoccurred in these species.

■ CONCLUSIONIn this study the structures of all the volatile compoundsidentified in the femoral gland extracts of M. femoralis collectedat different Madagascar localities were confirmed by synthesis.Furthermore, the absolute configuration of the new macrolidenatural product mantidactolide A (4), as well as theconfiguration of the already described compounds (R)-8-methylnonan-2-ol (D) and (S)-phoracantholide I (1), wasdetermined using gas chromatographic methods with chiralphases. In addition, mantidactolide B (6) was described as anew natural compound.The data herein demonstrate that, although the femoral

gland constituents are mostly different among related speciesand especially in sympatry, macrolides are in general frequentlyoccurring in the femoral glands of mantellid frogs. Species ofthe subgenus Ochthomantis are morphologically cryptic and not

very vocal.16 When occurring in sympatry, they appear to differin the pheromone cocktail of their gland secretions. On theother hand, at least the most widespread species M. femoralispresents a remarkable variation in these compounds across itsdistribution, even in neighboring populations such as those ofMakira, and the possibility of incipient speciation processesinvolving chemical communication as a premating isolationmechanism should be researched further.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Specific rotations were

obtained with a Propol digital automatic polarimeter (Dr. Kernchen)with a 1 cm cuvette at a wavelength of 578 nm using the solventsreported. NMR spectra were obtained with the following instruments:Bruker DPX-200 (1H 200 MHz, 13C 50.5 MHz), DRX-400 (1H 400MHz, 13C 101 MHz), or AV II-600 (1H 600 MHz, 13C 151 MHz).Chemical shifts are reported in ppm relative to tetramethylsilane as aninternal standard (δ = 0). High-resolution MS data were obtained witha gas chromatograph (GC 6890, Agilent Technologies) equipped witha Phenomenex ZB5-MS column (30 m × 0.25 mm i.d. × 0.25 μm)coupled to a time-of-flight mass spectrometer (JMS-T100GC,GCAccuTOF, JEOL, Japan) in EI mode (70 eV). JEOL MassCenterworkstation software was used. The system was tuned with PFK toachieve a resolution of 5000 (fwhm) at m/z 292.9824. GC-MS wasperformed on a HP 6890 gas chromatograph coupled to an MSD 5973(EI 70 eV) (Hewlett-Packard) and on a GC 7890A coupled to anMSD 5975C (Agilent Technologies). Separation was performed on afused-silica capillary column BPX-5 (SGE Inc., 25 m × 0.22 mm i.d. ×0.25 μm) and an HP5-MS (Agilent Technologies, 30 m × 0.25 mm i.d.× 0.25 μm). Chiral phase gas chromatography was performed using aHydrodex-6-TBDMS phase (Macherey-Nagel, 25 m × 0.25 mm i.d.)or a Lipodex-G phase (Macherey-Nagel, 50 m × 0.25 mm i.d.).Commercially available starting material and solvents were purchasedfrom Sigma-Aldrich and used without further purification. Technicalsolvents were distilled before use. All reactions involving water-sensitive chemicals were performed in heat gun-dried glass equipmentwith magnetic stirring under a nitrogen atmosphere. TLC wasperformed on Polygram SIL G/UV254 plates (Macherey-Nagel) withdetection by UV (254 nm) or by immersion in a 10% ethanolicsolution of phosphomolybdic acid, followed by heating. Flashchromatography was performed on silica gel M60 (0.04−0.063 mm,230−400 mesh ASTM) (Macherey-Nagel) under pressure or on aflash chromatograph (Combi Flash Companion, Teledyne Isco) withthe eluent mentioned.

Sample Preparation and Analysis. A total of 25 samples ofdifferent species were collected from sites within north and northeastMadagascar during the rainy periods of the years 2008−2012 andanalyzed to investigate the gland constituents of individuals of thesubgenus Ochthomantis. The femoral glands were excised and stored invials containing dichloromethane. The samples were filtered, and thesolution was analyzed using GC/MS. The detected volatilecompounds were identified by comparison of their mass spectra,fragmentation patterns, and gas chromatographic retention indiceswith those of reference compounds, synthesized as described above.Gas chromatographic co-injection experiments verified their identity.Control samples of the belly skin and the femoral skin of females werealso analyzed, but they did not contain any volatile compounds. It wasnot possible to obtain NMR data of the natural compounds due to thediverse sample compositions and low concentrations in the biologicalmaterial.

Molecular Genetics. In order to verify the species identity ofsampled specimens, and due to the high morphological similarity ofmost species of Ochthomantis, a fragment of the mitochondrial 16SrRNA gene of all the relevant specimens was sequenced, followingestablished protocols.18 These sequences were compared with acomprehensive sequence data set from a previous study.16 Tocomprehend the phylogeny of these frogs, sequences of additionalmitochondrial genes were determined using standard methods andprimers commonly used in Madagascan frogs.18,19,20 The final

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alignment contained fragments of the genes cytochrome b,cytochrome oxidase subunit I, 12S rRNA, 16S rRNA, and ND1. Thesequences were resolved on an ABI 3130XL automated sequencer(Applied Biosystems). All newly determined sequences weresubmitted to GenBank (accession numbers KF426665-KF426724).The sequences were checked, and reading errors were corrected

manually in CodonCode Aligner (CodonCode Corp.). The align-ments were done in MEGA 521 and were unambiguous (only a fewgaps required in the rRNA genes, mostly to accommodate theoutgroup sequences). MrModeltest version 2.322 was used to selectthe best fitting nucleotide model of evolution under the Akaikeinformation criterion (a GTR+I+G model). Phylogenetic analysisbased on Bayesian inference was computed with MrBayes v3.0b423

using Markov chain Monte Carlo (MCMC) sets for 20 × 106

generations and sampled every 1000 generations. The treescorresponding to the first 10 × 106 generations were conservativelydiscarded as burn-in after empirically assessing the log-likelihoodvalues of the sampled trees.Synthesis. (S)-Hex-5-en-2-yl (S)-4-methylhex-5-enoate (30). (S)-

4-Methylhex-5-enoic acid (29, 40 mg, 0.31 mmol), (S)-hex-5-en-2-ol(33, 36 mg, 0.36 mmol), and DMAP (4 mg, 0.03 mmol) weredissolved in 10 mL of absolute CH2Cl2 and cooled to 0 °C. N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 76mg, 0.4 mmol) was added in one portion, and the reaction wasstirred for 1 h at 0 °C and for 2 h at room temperature (rt), similar tothe procedure described by Patel et al.24 Then the reaction mixturewas diluted with tert-butyl methyl ether, washed with saturatedNaHCO3, and dried with MgSO4. After solvent removal underreduced pressure the crude product was purified by columnchromatography on silica gel, yielding pure (S)-hex-5-en-2-yl (S)-4-methylhex-5-enoate (30, 34 mg, 0.29 mmol, 92%).Rf = 0.53 (pentane/tert-butyl methyl ether, 40:1); 1H NMR (200

MHz, CDCl3) δ 5.52−5.96 (2H, m), 4.81−5.12 (5H, m), 2.20−2.33(2H, m), 1.96−2.19 (3H, m), 1.45−1.80 (4H, m), 1.15−1.25 (3H, d, J= 6.1 Hz), 0.97−1.04 (3H, d, J = 6.8 Hz); 13C NMR (50 MHz,CDCl3) δ 173.4, 143.5, 137.8, 114.9, 113.5, 70.2, 37.5, 35.1, 32.5, 31.5,29.7, 20.1, 19.9; EIMS (70 eV) m/z (%) 153 (1), 128 (40), 111 (36),82 (57), 67 (72), 55 (100), 41 (49).(4S,5Z,9S)-4-Methyl-5-decen-9-olide (31). A solution of (S)-hex-5-

en-2-yl (S)-4-methylhex-5-enoate (30, 20 mg, 0.095 mmol) andhexafluorobenzene (1.8 mL, 6 mmol) was prepared in 150 mL of drytoluene according to the procedure of Rost et al.25 The Hoveyda−Grubbs-II catalyst dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](2-isopropoxyphenylmethylene)ruthenium(II)(12 mg, 0.019 mmol) was added to this solution, and the reaction wasstirred for 3 h at 80 °C. The reaction was quenched by the addition ofsaturated NaHCO3 solution after cooling to rt, and the phases wereseparated. The organic layer was dried with MgSO4, and the solventwas removed under reduced pressure. After column chromatographicpurification on silica gel (4S,5Z,9S)-4-methyl-5-decen-9-olide (31, 12mg, 0.066 mmol, 69%) was obtained.Rf = 0.55 (pentane/tert-butyl methyl ether, 19:1); 1H NMR (400

MHz, CDCl3) δ 5.28−5.46 (1H, m), 4.98−5.15 (1H, m), 4.61 (1H,m), 2.16−2.25 (2H, m), 2.10−2.16 (1H, m), 1.94−2.05 (2H, m),1.46−1.58 (4 H, m), 1.09 (3H, d, J = 6.5 Hz), 1.01 (3H, d, J = 7.0 Hz);13C NMR (100 MHz, CDCl3) δ 175.8, 132.6, 129.0, 71.1, 34.5, 33.1,32.5, 32.0, 29.8, 21.7, 15.2; EIMS (70 eV) m/z (%) 182 (5) [M]+, 140(5), 125 (7), 109 (15), 85 (100), 67 (51), 55 (31).(4R,9S)-4-Methyldecan-9-olide (4R,9S-4). A solution of

(4S,5Z,9S)-4-methyl-5-decen-9-olide (31) (12 mg, 0.066 mmol) in 1mL of absolute MeOH was prepared in a 1 mL vial, and 2 mg of 10%palladium on activated charcoal was added to hydrogenate the doublebond according to the procedure of Kitahara et al.7 Hydrogen wasbubbled through this solution with a pressure of 1 bar for 5 h. Thenthe catalyst was filtered off, and methanol was carefully evaporated in agentle stream of nitrogen. Pure (4R,9S)-4-methyldecan-9-olide (4, 9mg, 0.049 mmol, 74%) was obtained.[α]D

26.7 +10.3 (c 0.74, CH2Cl2);1H NMR (400 MHz, CDCl3) δ

4.88−5.00 (1H, m), 2.07−2.21 (2H, m), 1.77−1.96 (2H, m), 1.63−1.75 (2H, m), 1.51−1.62 (3H, m), 1.25−1.43 (4H, m), 1.20 (3H, d, J

= 6.3 Hz), 0.81 (3H, d, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ174.3, 72.8, 34.6, 32.5, 31.5, 30.2, 28.1, 23.5, 21.0, 20.7, 19.3; EIMS(70 eV) m/z (%) 184 (1) [M]+, 169 (1), 124 (8), 112 (50), 96 (20),83 (61), 69 (44), 55 (100); HREIMS m/z 184.1458 (calcd forC11H20O2, 184.1463); I = 1336.

(3S)-3-Methylhex-5-en-2-yl hex-5-enoate (24). (3S)-3-Methylhex-5-en-2-ol (23) (0.19 g, 1.7 mmol), 5-hexenoic acid (0.18 g, 1.57mmol), and DMAP (21 mg, 0.17 mmol) were added to absoluteCH2Cl2 (10 mL) at 0 °C. Then EDC·HCl (0.33 g, 1.75 mmol) wasadded to the above solution.24 The reaction mixture was stirred at 0°C for 1 h and at rt for 5 h until the complete consumption of thealcohol (23) was observed by TLC. The reaction mixture was dilutedwith Et2O (40 mL) and washed with saturated NaHCO3 (2 × 30 mL).The organic phase was dried using MgSO4, and the crude product waspurified by column chromatography to yield (3S)-3-methylhex-5-en-2-yl hex-5-enoate (24) (0.26 g, 1.23 mmol, 73%).

Rf = 0.4 (pentane/Et2O, 30:1);1H NMR (300 MHz, CDCl3) δ 5.76

(2H, m), 5.01 (4H, m), 4.85 (1H, m), 2.29 (2H, t, J = 7.6 Hz), 2.19(1H, m), 2.09 (2H, m), 1.89 (1H, m), 1.73 (3H, m), 1.16 (3H, d, J =7.0 Hz), 0.91 (3H, d, J = 8.1 Hz); 13C NMR (75 MHz, CDCl3) δ173.1, 137.7, 136.7, 116.2, 115.3, 73.2, 37.6, 37.2, 34.0, 33.1, 24.2, 17.0,16.3; EIMS (70 eV) m/z (%) 166 (1), 128 (1), 114 (4), 99 (2), 97(42), 81 (27), 72 (2), 69 (47), 68 (8), 55 (87), 43 (22), 41 (100), 39(39).

(5Z,8S)-8-Methyl-5-decen-9-olide (25). (3S)-3-Methylhex-5-en-2-ylhex-5-enoate (24) (77 mg, 0.36 mmol) was dissolved in dry toluene(250 mL), and hexafluorobenzene (4.4 g, 23 mmol) was added to theabove solution. Then Grubbs II catalyst (1,3-bis(2,4,6-trimethylphen-y l ) - 2 - im i d a zo l i d i ny l i d ene )d i ch l o r o (pheny lme thy l en e )(tricyclohexylphosphine)ruthenium (40 mg, 0.076 mmol) was added,and the reaction mixture was heated to 80 °C for 3 h. The reaction wascooled to rt and washed with saturated NaHCO3. The organic phasewas dried using MgSO4. The organic solvents were evaporated, andthe Grubbs catalyst was filtered off on a silica gel filled microcolumn.The column was washed (four column volumes) with pentane/Et2O(40:1). The column fractions were evaporated, and the crude prodcutwas purified by column chromatography to yield (5Z,8S)-8-methyl-5-decen-9-olide (25) (30 mg, 0.16 mmol, 45%).

Rf = 0.52 (pentane/Et2O, 10:1);1H NMR (300 MHz, CDCl3) δ

5.36 (2H, m), 4.71 (1H, qd, J = 6.5, 4.1 Hz), 2.27 (3H, m), 2.17 (2H,m), 1.91 (2H, m), 1.73 (2H, m), 1.20 (3H, d, J = 6.4 Hz), 0.98 (3H, d,J = 6.6 Hz); 13C NMR (75 MHz, CDCl3) δ 175.3, 133.9, 131.0, 75.0,38.3, 33.7, 32.2, 26.5, 26.0, 17.3, 14.1; EIMS (70 eV) m/z (%) 182(12) [M]+, 164 (4), 149 (5), 138 (14), 126 (12), 110 (25), 99 (89),95 (32), 93 (17), 84 (37), 81 (71), 79 (40), 68 (26), 67 (81), 55 (76),53 (41), 41 (100), 39 (80).

(8S,9RS)-8-Methyl-9-decanolide (6). (5Z,8S)-8-Methyl-5-decen-9-olide (25) (30 mg, 0.16 mmol) was dissolved in MeOH (30 mL,HPLC grade), and 10 mg of 10% palladium on activated carbon wasadded. Then hydrogen gas was passed into the reaction solution at apressure of 2 bar for 5 h. Next the catalyst was filtered off on a Celite-filled microcolumn, and the column was washed with MeOH. TheMeOH fractions were collected and evaporated to yield (8S,9RS)-8-methyl-9-decanolide (6) (23 mg, 0.12 mmol, 76%). The ratio (8S,9S):(8S,9R) was 1:0.7.

[α]D21.5 −8.3 (c 0.7, CHCl3);

1H NMR (600 MHz, CDCl3) δ 4.8(1H, qd, J = 6.8, 3.4 Hz), 4.4 (1H, dq, J = 10.4, 6.2 Hz) 2.4 (2H, m)2.2 (1H, ddt, J = 10.8, 7.2, 3.6 Hz), 2.1 (1H, m), 2.0 (2H, m), 1.8 (1H,m), 1.7 (1H, m), 1.6 (2H, m), 1.4 (8H, m), 1.2 (4H, m), 1.1 (3H, d, J= 6.2 Hz), 1.1 (3H, d, J = 6.8 Hz), 0.8 (3H, d, J = 7.0 Hz), 0.7 (3H, d, J= 7.0 Hz); 13C NMR (151 MHz, CDCl3) (8S,9S)-6 δ 173.3, 75.7, 35.7,35.1, 30.6, 27.7, 24.2, 22.5, 20.4, 19.8, 12.8; (8S,9R)-6 δ 174.0, 77.7,40.0, 34.7, 29.7, 27.0, 25.9, 22.6, 20.6, 18.9, 17.8; EIMS (70 eV) m/z(%) 184 (1) [M]+, 166 (2), 148 (2), 140 (11), 123 (2), 112 (17), 109(3), 98 (62), 94 (6), 83 (19), 81 (12), 69 (24), 67 (14), 56 (26), 55(71), 53 (13), 45 (14), 42 (46), 41 (100), 39 (62); HREIMS m/z184.1466 (calcd for C11H20O2 184.1463); I(8S,9S)‑6 = 1346, I(8S,9R)‑6 =1329.

6-Methyloctan-2-ol (36). 3-Methylmagnesium bromide wasprepared by dropwise addition of 1-bromo-3-methylpentane (35,

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500 mg, 3.05 mmol) to a solution of activated magnesium turnings(0.9 g, 4 mmol) in 5 mL of absolute Et2O following the procedure ofYu et al.26 The solution was stirred for 3 h and then transferred into acooled solution (−40 °C) of copper cyanide (0.3 g, 0.37 mmol) in 3mL of absolute THF. After 20 min propylene oxide (177 mg, 3.05mmol) was added slowly. The reaction mixture was stirred for 12 h at0 °C and then quenched with saturated NH4Cl solution. The aqueouslayer was extracted three times with ethyl acetate, and the combinedorganic layers were washed with brine and dried with MgSO4. Thecrude product was purified by column chromatography on silica gel(pentane/tert-butyl methyl ether, 9:1) after evaporation of the solventto yield 6-methyloctan-2-ol (36, 280 mg, 1.94 mmol, 64%).Rf = 0.6 (pentane/tert-butyl methyl ether, 2:1); 1H NMR (400

MHz, CDCl3) δ 3.79 (1H, sxt, J = 6.0 Hz), 1.8 (1H, brs) 1.28−1.51(7H, m), 1.17−1.21 (3H, d, J = 6.0 Hz), 1.06−1.16 (2H, m), 0.86(3H, t, J = 7.6 Hz), 0.85 (3H, d, J = 6.8 Hz); 13C NMR (100 MHz,CDCl3) δ 68.0, 39.6, 36.5, 34.3, 29.4, 23.4, 23.2, 19.1, 11.3.; EIMS (70eV) m/z (%) 129 (3), 111 (5), 97 (24), 83 (9), 69 (42), 57 (24), 55(44), 45 (100), 41 (34), 39 (11); HREIMS m/z 129.1283 (calcd forC8H17O1 (M-15), 129.1279); I = 10658-Methyldecan-2-ol (39). 8-Methyldecan-2-one (162 mg, 0.96

mmol) was dissolved in dry Et2O (30 mL) under an inert atmosphere,and the flask was cooled to 0 °C. Following the procedure describedby Hansen et al.,27 LiAlH4 (36.5 mg, 0.96 mmol) was added to thesolution and the reaction was stirred at rt until the completeconsumption of starting material was observed by TLC. The reactionwas quenched using saturated NH4Cl, and the aqueous phase wasseparated after dissolution of the formed aluminum with concentratedHCl. After extraction with Et2O (3 × 30 mL) the combined organicphases were dried with MgSO4, the solvent was removed, and thecrude product was purified by column chromatography to yield pure 8-methyldecan-2-ol (39, 145 mg, 0.84 mmol, 88%).Rf = 0.5 (pentane/tert-butyl methyl ether, 5:1); 1H NMR (400

MHz, CDCl3) δ 3.79 (1H, sext, J = 6.0 Hz), 1.34−1.51 (3H, m),1.24−1.34 (9H, m), 1.19 (3H, d, J = 6.1 Hz), 1.06−1.16 (1H, m), 0.85(3H, t, J = 7.1 Hz), 0.84 (3H, t, J = 6.3 Hz); 13C NMR (100 MHz,CDCl3) δ 68.2, 39.4, 36.6, 34.4, 30.0, 29.5, 27.1, 25.8, 23.5, 19.2, 11.4;EIMS (70 eV) m/z (%) 157 (1), 125 (10), 97 (9), 83 (22), 70 (27),55 (38), 45 (100), 41 (53); HREIMS m/z 157.1592 (calcd forC10H21O1 (M-15), 157.1589); I = 1268.

■ ASSOCIATED CONTENT*S Supporting InformationSynthetic procedures, chromatography on chiral phases, andNMR spectra are available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: stefan.schulz@tu-bs.de. Tel: (+49)-531-3915271 Fax:(+49)-531-3915272. Home page: http://www.oc.tu-bs.de/schulz/index.html.Present Address‡Center for Marine Biotechnology and Biomedicine, ScrippsInstitution of Oceanography, UCSD, 8655 Kennel Way, LaJolla, CA 92037, USA.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe are grateful to F. Glaw, James and C. Patton, E.Rajeriarison, T. Rajoafiarison, R. D. Randrianiaina, F.Ratsoavina, and D. R. Vieites for their help in the field, andto J. Glos, S. Ndriantsoa, A. Rakotoarison, J. Riemann, and M.-O. Rodel for providing samples. The fieldwork was supportedby the Volkswagen Foundation and carried out in the

framework of collaborations with the Departement de BiologieAnimale, Universite d’Antananarivo. We are grateful to theMadagascar Institute for the Conservation of Tropical Environ-ments MICET and the Valbio biological station for logisticsupport, and to the Madagascan authorities for grantingresearch and export permits. We thank the DeutscheForschungsgemeinschaft for research grant SCHU 984/10-1.

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