Malbrancheamide, a new calmodulin inhibitorfrom the fungus Malbranchea aurantiaca*

Sergio Martınez-Luis,a Rogelio Rodrıguez,b Laura Acevedo,a Marıa C. Gonzalez,c

Alfonso Lira-Rochaa and Rachel Mataa,*

aDepartamento de Farmacia, Facultad de Quımica, Universidad Nacional Autonoma de Mexico, DF 04510 Mexico, MexicobDepartamento de Bioquımica, Facultad de Quımica, Universidad Nacional Autonoma de Mexico, DF 04510 Mexico, Mexico

cInstituto de Biologıa, Universidad Nacional Autonoma de Mexico, DF 04510 Mexico, Mexico

Received 5 October 2005; revised 17 November 2005; accepted 18 November 2005

Available online 20 December 2005

Abstract—Bioassay-directed fractionation of an ethyl acetate extract (mycelia and broth) of the fungus Malbranchea aurantiaca led to theisolation of the novel phytotoxic alkaloid (5aS,12aS,13aS)-8,9-dichloro-12,12-dimethyl-2,3,11,12,12a,13-hexahydro-1H,6H-5a,13a(epiminomethano)indolizino[7,6b]carbazol-14-one (1) of the brevianamide series. The phytotoxin was given the trivial name ofmalbrancheamide (1). The structure of 1 was unequivocally established by UV, NMR, MS and X-ray studies. The absolute configuration wasestablished by X-ray analysis according to the method of Flack. According to the conformational studies using molecular mechanicsanalyses, 1 exists in one preferred conformation, which was optimized by DFT calculations. Compound 1 caused moderate inhibition ofradicle growth of Amaranthus hypochondriacus (IC50Z0.37 mM) and inhibited the activation of the calmodulin-dependent enzyme PDE1(IC50Z3.65G0.74 mM). This effect was comparable to that of chlorpromazine (IC50Z2.75G0.87 mM) a well characterized CaM antagonist.The inhibition mechanism of 1 was competitive with respect to CaM according to a kinetic analysis.q 2005 Elsevier Ltd. All rights reserved.

N

H

Cl 9 10a11

12

16

H

1. Introduction

Continuing with our search of fungal phytotoxins withcalmodulin-inhibitor properties,1–4 we have investigated anew culture of the ascomycete Malbranchea aurantiaca Siglerand Carmich (Myxotrichaceae). Previously we described theisolation, structure elucidation and the phytotoxic effect of1-hydroxy-2-oxoeremophil-1(10),7(11),8(9)-trien-12(8)-olide and penicillic acid from this species.4 Herein, we reportthe isolation, structure, absolute configuration and calmodulininhibitors properties of (5aS,12aS,13aS)-8,9-dichloro-12,12-dimethyl-2,3,11,12,12a,13-hexahydro-1H,6H-5a,13a-(epi-minomethano)indolizino[7,6b]carbazol-14-one (1) from thisfungi. Compound 1 is a new member of the brevianamidestype of alkaloids and was given the trivial name ofmalbrancheamide (1) (Fig. 1). The brevianamides as well asthe allied aspergamides, macfortines, paraherquamides,

0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.tet.2005.11.047

* Taken in part from the PhD thesis of Sergio Martınez-Luis.

Keywords: Malbranchea aurantiaca; Amaranthus hypochondriacus;Calmodulin; cAMP phosphodiesterase; Brevianamides; Malbrancheamide.* Corresponding author at present address: Colonia Parque San Andres,

Inglaterra 102, 4040 Coyoacan, Mexico.Tel.: C525 55 622 5289; fax: C525 55 622 5329;e-mail: rachel@servidor.unam.mx

sclerotamides and stephacidins belongs to a rare type ofindol alkaloids possessing an unusual bicyclo [2.2.2]diazaoctane ring system.5–19 These alkaloids are biosynthe-sized from tryptophan, proline or lysine, and at least oneisoprene unit. It has been proposed that the bicyclo [2.2.2]diazaoctane ring arises via an intramolecular Diels–Alderreaction of a suitable intermediate.20–21 Since their discoveryin 1969, these natural products have been isolated periodicallyto the present from different strains of fungi of the generaAspergillus and Penicillium. Their pharmacological action,synthesis and biosynthesis have been the subject of severalelegant investigations.5–21

Tetrahedron 62 (2006) 1817–1822

N

O

H

N

Cl

1

3

5a

67

136b

14

Figure 1. Structure of malbrancheamide (1).

S. Martınez-Luis et al. / Tetrahedron 62 (2006) 1817–18221818

2. Results and discussion

2.1. Isolation and structure elucidation

Column chromatography of a phytotoxic (againstAmaranthus hypochondriacus seedlings, IC50Z195.0 mg/mL) extract (mycelium and broth) of M. aurantiaca led tothe isolation of a novel indole alkaloid, which was given thetrivial name of malbrancheamide (1). The novel compound1 was isolated as a colorless crystalline solid. Its molecularformula was determined as C21H23ON3Cl2 by HRMS.The compound showed a positive color reaction withDragendorff’s and Erlich’s reagents. This information aswell as the UV absorption maxima at 233 and 293 nmsuggested that 1 was an indole alkaloid. The presence of twochlorine atoms in the molecule was consistent with therelative abundance of the [MC2] and [MC4] peaks withrespect to the molecular ion [M] in the mass spectrum(approximately two-third and one-tenth, respectively, of theintensity of the molecular ion peak). The IR spectrumshowed typical absorption bands for lactams at 3299 and1659 cmK1. In the 1H NMR spectrum (Table 1) of 1, twosinglets signals showed the existence of a tetrasubstitutedaromatic ring. Also apparent in the 1H NMR spectrum wereresonances for two methyl, one aliphatic methine and sixmethylene groups. The 13C NMR spectrum (Table 1)revealed 21 carbon-resonances, interpreted from multi-plicity-edited HSQC data as ten quaternary, three methine,six methylene, and two methyl carbons; this spectrum alsosupported the presence of a lactam functionality and anindole group in 1. The chemical shift of the resonances at dC

125.4 and 128.2 were consistent with the placement of the

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR data for compound 1 inMeOD

Position d 13C d 1H HMBC

1 28.1 A 2.52m H2, H3, H13

B 1.46m2 23.6 1.87m H1, H3

3 55.3 A 3.05m H1, H2, H5

B 2.15q 2, 55 59.4 A 2.25dd 2, 10 H3, H6, H12a

B 3.42d5a 57.5 H5, H6, H12a,

H13, H16

6 30.0 A 2.86mB 2.85m

6a 104.8 H6, H7

6b 123.3 H7, H10

7 119.6 7.47s H6, H10

8 125.4 H7, H10

9 128.210 113.1 7.39s H7

10a 137.3 H7, H10

11a 145.2 H6, H16, H17

12 35.5 H12a, H13, H16,H17

12a 48.5 2.14m H5, H6, H13,H16, H17

13 32.5 A 1.99m H1, H2

B 1.94m13a 66.1 H1, H3, H5, H13

14 176.7 H1, H13

16 30.6 1.32s H12a, H17

17 24.2 1.42s H12a, H16

chlorines at C-8 and C-9. The above-mentioned structuralfragments accounted for seven of the 11 degrees ofunsaturation required for the molecular formula, thereforethere must be four additional rings in the structure ofcompound 1. Detailed 2D-NMR spectra analyses (COSY,HETCOR, HMBC, and NOESY) led to the establishment ofthe connectivity of functional groups and, in turn, of themolecular structure. The position of the functional groupsalong the pentacyclic moiety was corroborated by anHMBC experiment (Table 1). Thus, the correlationsC-10a/H-7, C-6b/H-10, C-6a/H-6, H-7, C-5a/H-12a,C-12a/H-6, H-16, H-17 and C-11a/H-6, H-16, H-17corroborated the position of the chlorine atoms and thefusion of the indole nucleus to the dimethyl cyclohexanering throughout C-6a and C-11a. On the other hand, thecross peaks C-5a/H-6, H-12a, H-13, H-5; C-12a/H-5and C-13a/H-13, H-1, H-3, H-5 indicated that the bicyclo[2.2.2] diazaoctane ring system has the same arrangementthan the one observed in alkaloids (K) VM55599 17 andstephacidin A.19

On the basis of the above data, the structure of 1 waselucidated as depicted and corroborated by X-ray analysis(Fig. 2). In the bicyclo [2.2.2] diazaoctane system, ring C(carbocyclic) adopts a boat conformation whereas ring F(lactam) an envelope conformation. Ring D was alsoobserved as an envelope while ring E adopts a twistedenvelope conformation. The atoms C13a–C1–C2–C3 arelocated in the ring plane while N4 is out-of the plane.Finally, the absolute configuration was determined applyingthe Flack’s method.22

In order to gain a better understanding about theconformational behavior, a high level density functionaltheory (DFT) investigation was performed. The initialstructure was built from standard fragments and minimizedusing molecular mechanics analysis as implemented in theSPARTAN’04 program. This initial study revealed a minimumenergy conformer (EZ137.77 kcal/mol) which was fullyoptimized by DFT (B3LYP/G31G*).23 In the DFToptimized structure (EZK1973.98 kcal/mol) ring E dis-plays an envelope conformation and the atoms N4–C13a–C1–C2 are located in the plane while the C3 is out-of thering-plane (Fig. 3).

As previously proposed for the brevianamides, it is highlyprobable that the biogenesis of malbrancheamide (1)proceed from L-tryptophan, L-proline and one isopreneunit,6,7,21 being the bicyclo [2.2.2] ring system arisingthrough an intramolecular Diels–Alder reaction.21,22

From the structural point of view, it is important to point outthat malbrancheamide is the first chlorinated indole alkaloidpossessing a bicyclo [2.2.2] ring. The brevianamides,paraherquamides, aspergamides and macfortines type ofcompounds possess a spiro-j-indoxyl system, whilemalbracheamide does not. On the other hand, the maindifference between malbrancheamide and (K) VM5559919

and stephacidin A17 seems to be the relative configuration atC12a in the bicyclo [2.2.2] diazaoctane ring system.Altogether, these features make malbrancheamide (1)unique among these of complex indole alkaloid derivatives.

Figure 2. ORTEP view of malbrancheamide (1).

Figure 3. Optimized structure of compound 1 obtained by DFT analysis.

Figure 4. CaM-dependent PDE activity with constant concentrations ofcAMP and CaCl2. CaM concentration was held at 12.5 (;), 25 (:), 50(C) and 100 (&) nM, and the concentration of 1 was varied. Error barsrepresent the standard error. Each point was repeated 6 to 12 times. Thelines are the result of fitting all individual readings globally to a competitiveinhibition model with CaM as essential activator and 1 as the inhibitor.

S. Martınez-Luis et al. / Tetrahedron 62 (2006) 1817–1822 1819

2.2. Biological testing

Compound 1 showed phytotoxic effects when tested againstseedlings of A. hypochondriacus using a Petri dishbioassay.1 Compound 1 (IC50Z0.37 mM) inhibited radiclegrowth of this species with a similar potency to 2,2-dichlorophenoxyacetic acid [2,4-D; IC50Z0.18 mM], whichwas used as a positive control.

The in vitro effect of the phytotoxin 1 on calmodulin (CaM)-sensitive PDE1 activity was investigated using a coupledenzymatic reaction. The PDE1 assay is well characterizedand commonly used to detect CaM antagonists. The activityof PDE1 is correlated with the amount of inorganicphosphorous (Pi) released by the hydrolysis of cAMP inthe presence of CaM and a nucleotidase. In turn, Pi wasmeasured spectrophotocolorimetrically at 595 nm.2–4,24

Compound 1 inhibited the activation of PDE1 in aconcentration-dependent manner. The IC50 (concentrationof the testing material inhibiting the activity of the enzymeby 50%) value calculated was 3.65G0.74 mM. This effectwas comparable to that of chlorpromazine (IC50Z2.75G0.87 mM) a well characterized CaM antagonist. During thecourse of these measurements 1 was found to have a mildbut reproducible effect on the basal PDE1 activity in thepresence of bovine-serum albumin (BSA), however, this lasteffect is unlikely to have physiological significance becauseits half-amplitude was above 200 mM, and was not pursued.

In order to obtain further evidence of the involvement ofCaM in the inhibition of CaM-PDE1, a kinetic analysis ofthe inhibition of the activity of PDE1 was assessed usingdifferent amounts of CaM in the presence of differentconcentrations of 1 and in the absence of BSA. The BSAwas eliminated in order to reduce, though not completelyeliminate, the effect of the compound 1 on PDE1 itself. Theresults were analyzed by means of Dixon plots.25 In thisanalysis, the vertical axes are the reciprocal of the PDE1activity in the presence of each Ca2C-CaM and 1concentrations, and the horizontal axes are the 1 concen-trations. Figure 4 shows the kinetic analysis of 1 inducedinhibition of CaM-activated PDE1 by means of Dixon plots.These results suggested that 1 acts as competitive antagonistof CaM, thus competing with the formation of the CAM-PDE1 active complex. The estimated Ki (inhibitionconstant) value was 47.4G5.63 mM. The differencebetween the IC50 and the Ki values can be explained bythe difference in experimental conditions. In fact, ananalysis of the impact of 1 on the assay indicated that the

S. Martınez-Luis et al. / Tetrahedron 62 (2006) 1817–18221820

Ki value might be as much as three times lower than theIC50, but never higher.

In conclusion, malbrancheamide (1) represents a novel typeof CaM inhibitor although other natural indole alkaloidssuch as vincristine, vinblastine26 and K-252a27 as well as thesynthetic compound DY-9760e28 have been reported asCaM antagonists. The CaM antagonist effect of 1 might berelated with its phytotoxic action and other pharmacologicalproperties yet to be discovered. M. aurantiaca is verysensitive to light and temperature conditions. In dark andcolder (25 8C) environments the fungi produce sesqui-terpenes and polyketides but in warmer surroundings(32 8C) and under natural day-light cycles, malbranchea-mide (1) is produced. The ecological significance of thesevariations remains an open question.

3. Experimental

3.1. General experimental procedures

Melting point determinations were carried out on a Fisher-Johns apparatus and are uncorrected. The optical rotationwas recorded on a JASCO DIP 360 digital polarimeter. TheCD spectrum was registered on a JASCO 720 spectro-polarimeter at 25 8C in MeOH solution. The IR spectrumwas obtained using KBr disk on a Perkin-Elmer 599Bspectrophotometer. The UV spectrum was recorded on aShimadzu 160 UV spectrometer in MeOH solution. NMRspectra including COSY, NOESY, HMBC and HMQCexperiments were recorded in CDCl3 on a Varian Unity Plus500 spectrometer or on a Bruker DMX500 spectrometer at500 MHz (1H) or 125 MHz (13C) NMR, using tetramethyl-silane (TMS) as an internal standard. HREIMS was obtainedon a JEOL JMS-AX505HA mass spectrometer. Columnchromatography: silica gel 60 (70–230 mesh, Merck). TLC(analytical and preparative) was performed on precoatedsilica gel 60 F254 plates (Merck).

3.2. Fungal material and fermentation

The isolate of Malbranchea aurantiaca was obtained frombat guano collected at the Juxtlahuaca cave located inRamal del Infierno, State of Guerrero, Mexico, in 1998. Avoucher specimen (24428) is deposited in the NationalHerbarium (MEXU), Instituto de Biologıa, UniversidadNacional Autonoma de Mexico, Mexico City. Twelve 2-LErlenmeyer flasks, each containing 1 L of PDB (Difco),were individually inoculated with one 1-cm2 agar plug takenfrom a stock culture of M. aurantiaca maintained at 4 8C onpotato dextrose agar (PDA). Flask cultures were incubatedat environmental temperature and aerated by agitation on anorbital shaker at 200 rpm for 15 days.

3.3. Extraction and isolation of compound

After incubation, all flask contents were combined andfiltered. The combined culture filtrate (10 L) was extractedexhaustively with AcOEt (3!10 L). The combined organicphase was filtered over anhydrous Na2SO4 and concentratedin vacuo to give a dark brown solid (3.5 g). The myceliumwas macerated with AcOEt (3!2 L). After evaporating the

solvent in vacuo, the combined mycelial and culture extract(4.0 g) was subjected to silica gel (400 g) open columnchromatography eluting with a gradient of hexane–CH2Cl2(10:0/0:10) and CH2Cl2:MeOH (9.9:0.1/1:1) to yield 10major primary fractions (FI-FX). Bioactivity in thebioautographic bioassay showed one active pool: FVI(120 mg), eluted with CH2Cl2–AcOEt (7:3). From theactive fraction crystallized a colorless solid, which waspurified by re-crystallization from a mixture ofMeOH–CH2Cl2 to yield 20 mg of 1. Fraction FII, elutedwith CH2Cl2–AcOEt (9:1), afforded 5 mg of each linoleicand oleic acids.29

3.3.1. Malbrancheamide (1). Crystalline colorless solid,mp 321–324 8C; [a]D C428 (MeOH; c, 1). IR nmax (KBr)cmK1: 3299, 2959, 2924, 1739, 1659, 1460, 1317, 1241. UVlmax (MeOH) nm (log 3): 233 (3.94), 293 (4.74) nm: CD(MeOH) lmax nm (D3): 2.03!106 (245), 1.34!106 (218),1.18!106 (227), 1.51!105 (296); 1H and 13C NMR, seeTable 1; EIMS m/z (rel int.) 407 (1), 405 (3), 404 (1), 403[MC(5)], 363 (10.6), 361 (53.6), 359 (87.9), 345 (17.2), 325(12.2), 264 (15.7), 262 (21.4), 240 (5), 228 (5), 180 (7), 164(100), 163 (25), 135 (15.7), 120 (10) 96 (7); HREIMS m/z403.1106 [M]C (calcd for C21H23Cl2N3O, 403.332).

3.4. X-ray crystal structure determination of 1

Single crystals suitable for X-ray analysis were obtained byrecystallization from CH2Cl2–MeOH (8:2). A colorlessprism crystal having approximate dimensions of 0.398!0.232!0.166 mm was mounted on a glass fiber. Allmeasurements were made on a Bruker Smart Apex CCDdiffractometer equipped with graphite-monochromatedMo Ka radiation (lZ1.54178 A) at 293 K. Crystal data:C21H23Cl2N3O, 0.25*(C4H8O2), MW 426.35,tetragonal, space group P43212, with unit cell parametersaZ18.8877(7) A, bZ18.8877(7) A, cZ13.1336(9) A, bZ908, VZ4685.3 (4) A3, ZZ8, F(000)Z1792 and DcalcZ1.209 g/cmK3. Intensity data were collected in the range of1.89!q!25.038 using a u scan. Of the 37704 independentreflections collected, 4149 with IO2s(I) were consideredobserved and used in the calculations. The structure wassolved by direct methods and refined by full matrix least-squares methods on F2 using SHELXTL V 6.1232 to give afinal R-factor of 0.0751 (wR2Z0.2101), confirming thereported configuration, with a data-restrains-parametersratio of 4149/2/254. The absolute configuration wasdetermined from the refined value of the Flack x parameter(0.05(9)).22 Crystallographic data for the structure reportedin this paper have been deposited with the CambridgeCrystallograpic Data Center (CCDC 285932). Copies of thedata can be obtained, free of charge, on application to theDirector, CCDC, 12 Union Road, Cambridge CB21EZ, UK.

3.5. Molecular modeling calculations

Minimum energy structures were generated using theMMF94 (Monte Carlo protocol) as implemented in theSPARTAN’04 Program (Wavefunction, Inc. Irvine, CA). Theconformational search was carried out by exploringtorsional internal degrees of freedom of dihedral anglesselected by the automatic set up procedure. The torsional

S. Martınez-Luis et al. / Tetrahedron 62 (2006) 1817–1822 1821

angles considered were: C12a–C13–C13a, C13a–C1–C2,C1–C2C–3, C2–C3–N4, N4–C5–C5a. The rotation anglewas 308. The minimum energy conformer was fullyoptimized by DFT. DFT calculations were carried out atthe B3LYP/G31G* level.

3.6. Phytogrowth-inhibitory bioassays

The phytogrowth-inhibitory activity of the crude extract,fractions and pure compounds was evaluated on seeds ofAmaranthus hypochondriacus using a Petri dish bioas-say.1–4 In addition, a bioautographic phytogrowth inhibitorybioassay was employed to guide secondary fractionation.1–4

Seeds of A. hypochondriacus were purchased from Mercadode Tulyehualco, Mexico City. The results were analyzed byANOVA (p!0.05) and IC50 values were calculated byprobit analysis based on percent of radicle growth orgermination inhibition. Samples were evaluated at 10, 100and 1000 mg ml-1. 2,4-D was used as the positive control.The bioassays were performed at 28 8C.

3.7. Cyclic nucleotide phosphodiesterase assay

Phosphodiesterase activity was measured according to themethod described by Sharma and Wang30 with somemodifications. Bovine brain CaM (44 ng) was incubatedwith 0.015 units of CaM-deficient-CaM-dependent cAMPfrom bovine brain during 40 min in 40 mL of assay solutioncontaining 0.3 units of 5 0-nucleotidase, 45 mM Tris–HCl,5.6 mM magnesium acetate, 45 mM imidazole, 2.5 mMcalcium chloride, and 10 mM bovine serum albumin (BSA),pH 7.0. Compounds were then added to the assay medium at1, 2, 4, 7, 13, 20, 32, 50 and 65 mM in ACN, and the sampleswere incubated during 30 min. Then, 10 mL of 10.8 mMcAMP was added to start the assay. After 15 min, the assaywas stopped by the addition of 190 mL of malachite greensolution. All the above steps were carried out at 30 8C. Thephosphodiesterase reaction was coupled to the 5 0-nucleo-tidase (Crotalus atrox venom from Sigma) reaction; theamount of inorganic phosphate released, measured spectro-photometrically at 595 nm, correlated with the activity ofthe PDE1. The experiments to determine Ki values wereperformed as described above but in the presence of fourdifferent concentrations of CaM (10, 20, 40 and 80 ng/mL),and in the absence of BSA. All the results are expressed asthe mean of at least six experiments GSEM. The IC50

(concentration inhibiting by 50% the activity of the enzyme)values were determined by non-linear regression analysis byfitting to hyperbolic inhibition. The Ki was calculated froma global fit of data against inhibitor and CaM concentrationusing the simple competitive inhibition equation:

n ZVMAX S½ �

KMA 1 C I½ �KI

� �C S½ �

where VMAXZActivity at saturating CaM concentration;KMAZDissociation constant of CaM-PDE1 complex; IZConcentration of the inhibitor and SZConcentration ofCaM.

Non-linear regression was performed with the programOrigin 3.0 (Microcal) or with the SigmaStat 2-0 statisticalpackage (Jendel Scientific). Both programs rendered very

similar estimates. Chlorpromazine was used as a positivecontrol.

Acknowledgements

This work was supported by Facultad de Quımica andPAEP-2005, Posgrado en Ciencias Quımicas, UNAM andCONACYT 45814-Q. We also are grateful to the followingpeople: Marisela Gutierrez and Georgina Duarte forrecording UV, IR and mass spectra; Isabel Rivero-Cruzfor technical assistance; and Blanca Rivero-Cruz for hervaluable help in the CaM assays.

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