4 PUBLISHED RESULTS

4 PUBLISHED RESULTS

1.

J. H. C. Ii, R. C. Vieira, V. Eccard, J. Skerry, V. Montgomery, Y. Campbell, V.

Roxas-duncan, W. Leister, C. A. Leclair, D. J. Maloney, D. Padula, G. Pescitelli,

I. Khavrutskii, X. Hu, A. Wallqvist, and L. A. Smith,

“Separation of Betti Reaction Product Enantiomers : Absolute Configuration and

Inhibition of Botulinum Neurotoxin A”

ACS Medicinal Chemistry Letters, 2011, 2, 396–401.

2.

I. Ahmed, H. Hussain, B. Schulz, S. Draeger, D. Padula, G. Pescitelli, T. van

Ree, and K. Krohn

“Three New Antimicrobial Metabolites from the Endophytic Fungus Phomopsis

sp.”

European Journal of Organic Chemistry, 2011, 2867–2873.

3.

I. N. Siddiqui, A. Zahoor, H. Hussain, I. Ahmed, V. U. Ahmad, D. Padula, S.

Draeger, B. Schulz, K. Meier, M. Steinert, T. Kurtán, U. Flörke, G. Pescitelli,

and K. Krohn

“Diversonol and blennolide derivatives from the endophytic fungus

Microdiplodia sp.: absolute configuration of diversonol.”

Journal of Natural Products, 2011, 74, 365–73.

4.

M. Barbero, S. Bazzi, S. Cadamuro, L. Di Bari, S. Dughera, G. Ghigo, D.

Padula, and S. Tabasso,

“Synthesis of 3-aryl-4-methyl-1,2-benzenedisulfonimides, new chiral Brønsted

acids. A combined experimental and theoretical study”

Tetrahedron, 2011, 67, 5789–5797.

5.

D. Padula, L. Di Bari, F. Santoro, H. Gerlach, and A. Rizzo

“Analysis of the Electronic Circular Dichroism Spectrum of (–)-[9](2,5)

Pyridinophane”

Chirality, 2012, 24, 994–1004.

6. G. Pescitelli, D. Padula, and F. Santoro

“Intermolecular Exciton Coupling and Vibronic Effects in Solid-State Circular

Dichroism: a Case Study”

Physical Chemistry Chemical Physics, 2013, 15, 795-802.

7. H. –W. Liu, X. –Z. Yu, D. Padula, G. Pescitelli, Z. –W. Lin, F. Wang, K. Ding,

M. Lei, and J. –M. Gao

“Lignans from Schisandra sphenathera Rehd. et Wils. and semisynthetic

schisantherin A analogues: Absolute configuration, and their estrogenic and

anti-proliferative activity”

Published:

March 11, 2011

pubs.acs.org/acsmedchemlett

Separation of Betti Reaction Product Enantiomers: Absolute

Configuration and Inhibition of Botulinum Neurotoxin A

John H. Cardellina II,

Rebecca C. Vieira,

Vanessa Eccard,

Janet Skerry,

Vicki Montgomery,

Yvette Campbell,

Virginia Roxas-Duncan,

William Leister,

Christopher A. LeClair,

David J. Maloney,

Daniele Padula,

Gennaro Pescitelli,

Ilja Khavrutskii,

Xin Hu,

Anders Wallqvist,

and Leonard A. Smith*

†

_{Division of Integrated Toxicology, U.S. Army Medical Research Institute for Infectious Diseases, Frederick, Maryland, United States}

_{NIH Chemical Genomics Center, National Human Genome Research Institute, NIH, 9800 Medical Center Drive, MSC 3370,}

Bethesda, Maryland, United States

§

_{Department of Chemistry, University of Pisa, Pisa, Italy}

)

Biotechnology High Performance Computer Software Application Institute, Telemedicine and Advanced Technology Research Center,

U.S. Army Medical Research and Materiel Command, Frederick, Maryland, United States

^

_{Office of Chief Scientist, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, United States}

b

W

e recently reported the identification of 7-substituted

8-hydroxyquinolines (e.g., 1), products of the Betti

reac-tion, as leads to potential chemotherapies for botulinum

poison-ing.

The Betti reaction

originated as a condensation of Schiff

bases with 2-naphthol,

but it was later extended to other

nucleophilic aromatic substrates, e.g., 8-hydroxyquinoline.

While a chiral center is formed in this reaction, the products

are typically racemic mixtures, as the reagents are usually achiral

(Scheme 1). Over the years, there have been several efforts to

resolve Betti product racemates, most of which involve preparing

diastereomeric salts with chiral acids.

Chiral chromatography seemed an obvious alternative for

resolution of these racemates, but we examined a total of six

chiral columns and 18 different methods before observing

sufficient resolution of the two enantiomers on a Chiralcel OD

column to permit semipreparative purification of adequate

quantities of (þ)-1 and (
)-1 for evaluation in our bioassays

and assignment of absolute configuration (Figures S1 and S2 of

the Supporting Information).

Initial comparison of the botulinum neurotoxin serotype A

(BoNT/A) inhibitory activity of the (þ) and (
) enantiomers of

1

was accomplished via an HPLC-based assay using a full-length

recombinant BoNT/A protease light chain (LC).

To our

surprise, both compounds displayed similar IC

values, 1.0

and 1.1

μM for (þ)-1 and (
)-1, respectively. We subsequently

evaluated their potential to inhibit the biological activity of

BoNT/A holotoxin in murine neuroblastoma N2a cells.

No

difference was observed in percent inhibition (P > 0.05) of

SNAP-25 cleavage for both enantiomers and the racemate (()-1

at the four concentrations tested (60, 45, 30, and 15

μM). We

then examined the efficacy of these compounds in mouse phrenic

nerve hemidiaphragm preparations (MPNHDA).

Similar to

observations in HPLC and cell-based assays, both (þ)-1 and

(
)-1 were equipotent (P = 0.94) in the tissue-based assay. At 2

μM concentrations, both enantiomers dramatically delayed (P =

1.58

 10

and 2.30

 10

for (þ)-1 and (
)-1, respectively)

the BoNT/A-induced paralytic half-time 3-fold. The

compara-tive testing of (()-1 and the two enantiomers is summarized in

Table 1.

Received:

February 3, 2011

Accepted:

March 6, 2011

ABSTRACT:

The racemic product of the Betti reaction of

5-chloro-8-hydroxyquinoline, benzaldehyde, and

2-aminopyri-dine was separated by chiral HPLC to determine which

enantiomer inhibited botulinum neurotoxin serotype A. When

the enantiomers unexpectedly proved to have comparable

activity, the absolute structures of (þ)-(R)-1 and (
)-(S)-1

were determined by comparison of calculated and observed

circular dichroism spectra. Molecular modeling studies were

undertaken in an effort to understand the observed bioactivity

and revealed different ensembles of binding modes, with

roughly equal binding energies, for the two enantiomers.

KEYWORDS:

Chiral resolution, Betti reaction products, TDDFT CD calculations, molecular docking, inhibition of botulinum

neurotoxin

397 dx.doi.org/10.1021/ml200028z |ACS Med. Chem. Lett. 2011, 2, 396–401

ACS Medicinal Chemistry Letters

We then turned to assigning the absolute configuration of

(þ)-1 and (
)-1 via comparison of calculated and experimental

electronic dichroism (CD) spectra. As a prelude, we determined

the 3-dimensional conformation of 1 through a series of NMR

experiments. Proton and carbon resonances were assigned from a

combination of COSY, HSQC, and HMBC experiments (Table

S1, Supporting Information). Numerous NOE interactions were

observed in the NOESY experiment (Scheme 2), with those of

NH, OH, H6, and H9 providing the greatest insight into the

conformation of the compound. While all NMR data were

obtained for the racemic mixture, the (S)-enantiomer is shown

for ease of portraying the structural analysis. The observed NOE

between H6 and H18a,b and lack of NOE between the C8

hydroxyl proton and H18a,b indicate that the phenyl ring is

rotated away from the C8 OH. The NOE between the OH and

the NH, as well as the OH and H9 interaction, supports this

conformation. The phenyl ring assumes a position perpendicular

to the quinoline ring system to minimize steric interactions. This

is supported by NOEs of H18a,b with H6, H9, and the NH.

Interestingly, H15 and H16 both have an NOE with the NH, but

not with H9, suggesting that the pyridine ring is oriented so as to

place the ring nitrogen toward H9, while H15 and H16 are

projected away. Also, this allows the pyridine ring to be

ortho-gonal to the quinoline and pseudoparallel to the phenyl ring,

further minimizing steric interactions. A locked conformation of

the compound due to hydrogen bonding between the lone pair of

the NH and the proton of the C8 hydroxyl is highly probable.

However, the high number of NOEs for the NH (OH, H6, H9,

H18a,b, and H19a,b) implies that the structure may not be

entirely rigid.

To assign the absolute configuration of (þ)- and (
)-1, their

electronic CD spectra

were recorded in solution and

com-pared with those calculated using the time-dependent DFT

(TDDFT) method.

The enantiomers of 1 show almost

mirror image CD spectra in methanol, as expected (Figure 1).

Due to the presence of three different aromatic chromophores,

these spectra feature many bands between 200 and 350 nm. For

the (þ)-enantiomer, the first band appearing in the 280
340 nm

region is broad and positive, followed by a moderately intense

negative band centered around 260 nm and two stronger bands, a

positive one at 245 nm and a negative one around 220 nm.

Several rotatable bonds in the structure of 1, most directly

affecting the relative orientation of the chromophores, made

obtaining a reliable set of input structures crucial for CD

calculations. A preliminary molecular-mechanics conformational

search was run with the MMFF force

field, using a starting

geometry with (R) absolute configuration (see Supporting

Information for details). All low-energy structures obtained were

optimized with the DFT method at the B3LYP/6-31G(d) level,

converging to a set of nine distinct conformers within 10 kJ/mol.

Their energies and populations at 298.15 K were estimated with

B3LYP/6-311þþG(d,p) in methanol (PCM solvent model).

The low-energy DFT structures were then checked against the

conformational picture provided by NMR experiments, bearing

in mind that the coexistence of several low-energy minima

rendered interpretation of NOEs in terms of a single

conforma-tion quesconforma-tionable. The calculated structures may be divided into

Scheme 1

Table 1. Comparative Testing of (()-1, (þ)-1, and (
)-1

HPLC assay N2a cell culture assay MPNHDAb

% inhibition IC50 % inhibition of SNAP-25 cleavage minutesc,d

sample 20μM 5μM (μM) 60μM 45μM 30μM 15μM 2μM

(()-1 92 88 1.5 91 90 74 41 NDe

(þ)-1 94 90 1.1 96 94 89 56 191f

(
)-1 91 90 1.0 96 93 69 54 188f

a

_{Assays conducted as described in ref 1 and the Supporting Information.}

_{Mouse phrenic nerve hemidiaphragm assay.}

_{Average time to 50% loss of}

twitch tension (min).

Average value for BoNT/A toxin control was 63 min.

Not determined.

P value < 0.001 (highly significant) for comparison with

values recorded for the BoNT/A control; statistical analyses performed using SigmaPlot 10 (Systat Software, San Jose, CA).

two subsets according to the rotation around the C7
C9 bond.

In one (major) subset, composed of four lowest-energy

con-formers, H9 is directed toward the OH. Within this subset, two

conformers are especially stable (absolute minimum, 30.8%

population at 298.15 K; second lowest minimum,

þ0.37 kJ/

mol, 26.5%; Figure 2); the structures and relative energies for all

minima are reported in the Supporting Information. In the

second (minor) subset, composed of the remaining

five

con-formers accounting for 25% overall population, H9 is again in the

plane of the quinoline ring but directed toward C6. These

findings corroborate the observed NOEs that NH and H9 each

have with both the C8 OH and H6.

Taking the lowest-energy structure as a test molecule,

TDDFT calculations were run using different combinations of

hybrid DFT functionals (B3LYP, PBE0, CAM-B3LYP, BH&

HLYP) and basis sets (SVP, TZVP, aug-TZVP),

in vacuo or in

methanol. The three basis sets led to very similar results (using

B3LYP), except for a small red shift observed for all computed

transitions on increasing the basis size. It appears that the two

“standard” functionals B3LYP and PBE0 led to calculated

transition energies underestimated with respect to the

experi-ment, while the opposite was true for Coulomb-attenuated

B3LYP (CAM-B3LYP) and the half a d-half functional

BH&HLYP. Looking, for example, at the absorption band

measured at 250 nm, the calculated transition wavelength was

270 and 230 nm with B3LYP/aug-TZVP and CAM-B3LYP/

SVP, respectively. Apart from a systematic wavelength shift, the

shape of the calculated CD spectrum was similar in all cases.

Finally, including the solvent model in CAM-B3LYP/SVP

calculations did not appreciably change calculated frequencies

and spectra.

TDDFT calculations were then run on all low-energy

struc-tures using the two combinations B3LYP/aug-TZVP and

CAM-B3LYP/SVP in vacuo. The resulting CD spectra were weighted

with the respective Boltzmann factors (estimated from B3LYP/

6-311þG(d,p) internal energies in methanol) at 298.15 K

and summed to afford weighted average spectra. In all cases,

input structures had (R) absolute configuration. Figure 3 displays

spectra computed with the two methods for the lowest-energy

structure and the weighted averages over nine structures. Apart

from the already discussed wavelength shift (taken into account

by a frequency correction in Figure 3), the overall shapes of the

four spectra are quite similar, especially in the low-energy region

(where TDDFT calculations are intrinsically more accurate).

CAM-B3LYP/SVP results agree especially well with the

experi-mental spectrum for (þ)-1 in sign, position, and intensity of

bands (Figure 3b). Therefore, the absolute configuration of the

enantiomers of compound 1 may be assigned as (þ)-(R)-1 and

(
)-(S)-1. It must be noted that the calculated average CD

spectrum is the superposition of very heterogeneous component

spectra; thus, the apparent bands are actually the convolution

from several transitions (Supporting Information).

The unexpected observation of virtually equivalent BoNT/

A-inhibitory activity for the enantiomers in three different

bioassays prompted us to examine the bound state of the ligands

to rationalize the apparent lack of discrimination. Given the

observed pharmacological activity, we hypothesized that both

enantiomers bind BoNT/A, presumably in the Zn

-containing

active site. Nevertheless, experimental observations suggest that

binding of a similar ligand is not exclusively due to Zn

chelation.

To test this hypothesis computationally, we

as-sessed relative binding free energies for the enantiomers by

performing Thermodynamic Integration with Molecular

Dy-namics simulations

(see the Supporting Information for

details). Because the protonation state of the bound ligand is

unknown, we assumed that both enantiomers bind the active site

Zn

without ionization of the OH group. Using 10 lowest stable

conformations of each ligand, we generated corresponding

complex conformations by superposing the quinoline moiety

onto a template complex derived with AutoDock4.0.

The

resulting calculations indicated that the (
)-S-enantiomer would

Figure 2.

DFT-optimized structures for the absolute lowest energy (left) and second lowest energy (right) conformers of (R)-1.

Figure 1.

CD spectra of the enantiomers of 1 in methanol;

399 dx.doi.org/10.1021/ml200028z |ACS Med. Chem. Lett. 2011, 2, 396–401

ACS Medicinal Chemistry Letters

bind BoNT/A by 3.8

( 2.4 kcal/mol more favorably than its

(þ)-R-counterpart.

The large standard deviation in the free energy calculations

indicates significant variability in the conformational ensembles

of the bound ligands. To analyze the variety of binding modes of

both (þ)-R-1 and (
)-S-1 to BoNT/A, along with the

corre-sponding binding features, we performed GROMOS-style

clustering

of the sparsely saved simulation trajectories in the

physical state for each of the 10 starting configurations of the

(R)-and (S)- complexes. (Table S3 of the Supporting Information

summarizes the 16 most prominent features describing the two

ensembles and comprising H-bonding interactions, coordinating

bonds to the Zn

ion and

π
π interactions.) The most robust

features include coordinate bonds of atoms O, N1 (quinoline),

and N12 (pyridine) to the Zn

ion. Depending on the number

of bonds, the ligand can be monodentate or bidentate. When

both O and N12 chelate the Zn

, the N10 (amine) atom is

brought sufficiently close to the Zn

_{that this coordination}

mode could be considered as tridentate. The most common

H-bonding interaction is that of the coordinated OH group with

the carboxyl of E224.

Clustering analysis of the trajectories of the protein
ligand

complexes captured the diversity of the binding modes that can

be described by a few features. Most of these features are

common to both enantiomers and pertain to specific interactions

of the ligand with the binding site residues. We found that the

(
)-S-1 ensemble, on average, has more interaction features per

cluster, 3.6 versus 3.0 for that of the (þ)-R-1. In addition, the

(
)-S-1 ensemble has a larger number of unique interactions

with the active site compared to that of the (þ)-R-1. These

observations are consistent with the computed binding free

energy difference. Figure 4 illustrates examples of the likely

binding mode for each enantiomer.

Only a few interactions exist that can differentiate between the

two enantiomers. In particular, the N10H
E224 interaction is

realized only by the (R)-enantiomer, whereas the interactions

OH-Y366, N12
F163(NH), and Ar:Phe-Y366 are only realized

by the (S)-enantiomer. Although it is possible that different

realizations of common interaction features by the enantiomers

can further contribute to binding disparity, quantifying those

differences could be daunting.

The fact that our calculations favor BoNT/A binding by the

(
)-S-1 suggests that, in reality, the ligands might bind

differ-ently. Unlike experimental measurements, our calculated values

provide a more limited assessment of the relative binding

affinities of the two enantiomers under specified assumptions.

It is also possible that the ligand can undergo epimerization,

Figure 3.

CD spectra calculated for (R)-1 with two TDDFT methods: (A) B3LYP/aug-TZVP; (B) CAM-B3LYP/SVP. Blue lines: spectra calculated

for the lowest-energy DFT structure (divided by 2 for better comparison). Black lines: averages of spectra calculated for nine low-energy DFT structures

weighted with Boltzmann factors at 298.15 K using populations estimated with B3LYP/6-311Gþþ(d,p) in methanol. Red dashed line: experimental

spectrum for (þ)-1. Frequency corrections of þ2500 cm

_and

_{2000 cm}

_{have been applied to calculated spectra in panels A and B, respectively.}

Figure 4.

Examples of binding modes for the (R)-enantiomer (A) and

(S)-enantiomer (B). Active site residues (yellow) of the protein (green)

participate in binding of the Zn

(magenta) and the ligand (gray). Blue

and red colors correspond to oxygen and nitrogen atoms. Certain active

site residues and hydrogen atoms are omitted for clarity. The red dashed

lines show some coordinating and hydrogen bonds.

similar to that of ibuprofen

in the presence of certain

enzymes.

In conclusion, we separated the enantiomers of 1 (chiral

HPLC), established their chemical structure (NMR) and

abso-lute configuration (CD, molecular modeling), evaluated their

BoNT/A inhibitory activity, and explored their docking motifs

with BoNT/A LC. To our knowledge, this is the

first use of CD

calculations to assign the absolute configuration of Betti

reac-tion product enantiomers. While chiral chromatography has

recently been applied to the separation of naphthol-based Betti

products,

this is the

first such separation of Betti products

comprised of 8-hydroxyquinoline, an aryl aldehyde and an

aryl amine.

While, in a vast majority of cases, one enantiomer of a racemic

drug or drug candidate has significantly greater pharmacological

activity than the other, as has recently been demonstrated for one

BoNT/A inhibitor,

we found essentially equivalent BoNT/

A-inhibitory activity in (þ)-(R)-1 and (
)-(S)-1. However, this

unexpected

finding can be explained by the proposed docking

motifs for the two enantiomers—different ensembles, but nearly

equivalent in energy. We are currently seeking to confirm those

binding models by crystallizing each enantiomer in the active site

of BoNT/A.

’ ASSOCIATED CONTENT

b

Supporting Information.

Experimental protocols for

synthesis, chiral separation, and biological testing of (()-1,

H

and

C NMR assignments and NOE interactions, CD

calcula-tions leading to assignment of the absolute configuracalcula-tions of

1 and (
)-1, and molecular modeling and docking of

(þ)-and (
)- 1 into the BoNT/A light chain. This material is

available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone:

301-619-4238.

’ ACKNOWLEDGMENT

This work was supported by a grant to L.A.S. from the Defense

Threat Reduction Agency, JSTO-CBD Project Number

3.10037_07_RD_B. R.C.V. was funded by a National Research

Council Research Associateship Award. This work was

spon-sored by the U.S. Department of Defense High Performance

Computing Modernization Program (HPCMP), under the High

Performance Computing Software Applications Institutes

(HSAI) initiative. We thank James Bougie for help with the

chiral HPLC separation. Opinions or assertions contained herein

are the private views of the authors and are not to be construed as

reflecting the official views of the United States Department of

Defense.

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ar, P.; Hixon, M. S.; Silvaggi, N. R.; Allen,

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DOI: 10.1002/ejoc.201100158

Three New Antimicrobial Metabolites from the Endophytic Fungus

Phomopsis sp.

[‡]

Ishtiaq Ahmed,

_{Hidayat Hussain,}

_{Barbara Schulz,}

_{Siegfried Draeger,}

Daniele Padula,

_{Gennaro Pescitelli,*}

_{Teunis van Ree,}

_{and Karsten Krohn*}

Keywords: Natural products / Biological activity / Oxygen heterocycles / Circular dichroism / Density functional

calculations

Two new chromones, phomochromone A and B (1 and 2), and one new natural cyclopentenone derivative, phomotenone (3), together with six known compounds, phomosines A–D (4–7), (1S,2S,4S)-trihydroxy-p-menthane (8), and 5-meth-ylmellein (9), have been isolated from the endophytic fungus Phomopsis sp. through a bioassay-guided procedure. The structures of the new compounds were assigned on the basis of the 1_{H and} 13_{C NMR spectra, DEPT, and 2D COSY,} HMQC, HMBC, and NOESY experiments, supported by

mo-Introduction

As part of a program to isolate biologically active

com-pounds as leads for new products for pharmacy and plant

protection, we have been investigating the secondary

metab-olites produced by endophytic fungi. This relatively

unex-plored fungal group has proved to be a rich source of novel

secondary metabolites.

_{For example, the palmarumycines,}

a new class of naphthalene spiroketals, have been isolated

from the endophytic fungus Coniothyrium palmarum.

_We

now report on the investigation of constituents of the

asco-mycete Phomopsis sp., an endophyte isolated from Cistus

monspeliensis

. The crude fermentation extracts showed

algi-cidal (Chlorella fusca), antibacterial (Bacillus megaterium),

and antifungal (Microbotryum violaceum, Septoria tritici,

Botrytis cinerea

, and Phytophthora infestans) activity

[‡] Biologically Active Secondary Metabolites from Fungi, 49. Part 48: M. Saleem, H. Hussain, I. Ahmed, S. Draeger, B. Schulz, K. Meier, M. Steinert, G. Pescitelli, T. Kurtán, U. Flörke, K. Krohn, Eur. J. Org. Chem. 2011, 808–812.

[a] Department Chemie, Universität Paderborn, Warburger Straβe 100, 33098 Paderborn, Germany Fax: +49-5251-60-3245

E-mail: [email protected]

[b] Institut für Mikrobiologie, Technische Universität Braunschweig,

Spielmannstraße 7, 31806 Braunschweig, Germany [c] Dipartimento di Chimica e Chimica Industriale, Università

degli Studi di Pisa,

Via Risorgimento 35, 56126 Pisa, Italy E-mail: [email protected]

[d] Department of Chemistry, University of Venda, Thohoyandou 0950, South Africa

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201100158.

lecular modelling. The absolute configurations of compounds

1–3 were assigned by TD-DFT CD calculations and, surpris-ingly, the two related phomochromones A and B have an op-posite configuration at C-2. Compounds 1–3 show good anti-fungal (Microbotryum violaceum), antibacterial (Escherichia coli, Bacillus megaterium), and antialgal (Chlorella fusca) ac-tivities. Compound 8 shows good antialgal and antibacterial activity.

against selected fungi and bacteria. Further studies on the

active constituents of this fungus led to the isolation of two

new chromanone derivatives (1 and 2), one new natural

cy-clopentenone derivative (3), and six known compounds,

phomosines A–D (4–7), (1S,2S,4S)-trihydroxy-p-menthane

(8), and 5-methylmellein (9).

Results and Discussion

The fungus Phomopsis sp. was cultivated on a biomalt

agar medium for 4 weeks at 21 °C and subsequently

ex-tracted with ethyl acetate. The crude extract was

fraction-ated on a silica gel column using gradients of

dichlorometh-ane/ethyl acetate to yield a crude mixture containing 1–9

(Scheme 1). The less polar fraction contained mainly fatty

G. Pescitelli, K. Krohn et al.

FULL PAPER

acids and lipids. The polar fraction was further purified by

preparative TLC with n-hexane/ethyl acetate as eluent to

give the pure compounds.

The high-resolution electron-impact mass spectrum

(HREIMS) of phomochromone A (1), which was obtained

as a white solid, shows an [M]

_{peak at m/z = 206.0943}

corresponding to the molecular formula C

H

O

. The IR

spectrum of 1 displays a carbonyl group (1660 cm

_{) and a}

hydroxy group (3380 cm

_{). The}

_{H NMR spectrum of 1}

shows the signals of two tertiary methyl groups [δ

= 2.20

(s, 6-Me), 2.12 ppm (s, 8-Me)], one secondary methyl [δ

=

1.50 ppm (d, J = 6.0 Hz, 2-Me)], one methylene [δ

=

2.60 ppm (m, 2 H, 3-H)], and one oxymethine signal [δ

=

4.54 ppm (m, 2-H)]. The

_{H NMR spectrum of 1 also}

shows the presence of one aromatic proton at δ

= 7.57 (s,

5-H).

_{H, broadband and DEPT}

_{C NMR, and HMQC}

spectra indicate the presence of three methyl groups, one

methylene, one sp

_{methine adjacent to oxygen, one sp}

methine, five quaternary sp

_{carbon atoms, and one}

carbonyl. The

_{C NMR signals at δ}

C

= 191.9 (s, C-4), 159.7

(s, C-8a), 74.5 (d, C-2), and 44.3 ppm (t, C-3) suggest the

presence of a chromanone skeleton.

The HMBC correlations (Scheme 2) between 7-OH and

C-6, C-7, and C-8 indicate that the hydroxy group is

at-tached at 7. The correlations between 6-Me and 5,

C-6, and C-7, and between 8-Me and C-7, C-8, and C-8a

indi-cate the positions of the two methyls at C-6 and C-8,

respec-tively. The

_H–

_{H COSY and HMBC correlations}

(Scheme 2) establish the structure as

7-hydroxy-2,6,8-tri-methyl-2,3-dihydrochromen-4-one (1).

Scheme 2. Key 1_H–1_{H COSY and HMBC correlations of} com-pound 1.

Phomochromone B (2) was found to have a molecular

formula of C

H

O

due to its molecular ion peak at m/z

= 222.0889 [M]

_{in the HREIMS spectrum. The structure}

of 2 was determined by comparison of its NMR

spectro-scopic data with those of 1. However, one main difference

was apparent in the spectrum of compound 2: a methine

proton appeared at δ

= 4.68 ppm (d, J = 6.0 Hz, 1 H)

instead of a two-proton multiplet for the methylene group

at C-3 [δ

= 2.60 ppm (m, 2 H)], as found in compound 1.

The

_{C NMR spectrum shows the disappearance of the}

methylene carbon atom [δ

= 44.3 ppm (C-3) in 1] and the

appearance of a methine carbon atom (δ

= 71.7 ppm) at

C-3. This was further confirmed by the molecular ion peak

of compound 2, which had 16 a.m.u. more than compound

1. The structure of 2 was definitively determined by 2D

NMR experiments, COSY and HMBC giving pertinent

correlations. The two 2-H and 3-H cis hydrogen atoms of

www.eurjoc.org © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem.2011, 2867–2873

2868

compound 2 show the expected coupling constant of J =

6.0 Hz; usually a trans relationship in these systems leads to

a larger coupling constant (9.0 Hz).

_{The cis relationship}

between the CH

and OH substituents on ring B (see

Scheme 1) implies that they cannot occupy equatorial

posi-tions at the same time. To establish the preferential

confor-mation assumed by phomochromone B in solution, a

com-bination of NOESY experiments and molecular modeling

was employed. All conformers obtained by a MMFF

(Merck molecular force field) conformational search were

optimized by the DFT method at the

B3LYP/6-311++G(d,p) level including the PCM solvent model for

acetonitrile; zero-point-corrected (ZPC) free energies were

estimated thereafter (structures and energies are shown in

the Supporting Information). The two conformers with an

equatorial 3-OH group were found to be much more stable

(73 % relative Boltzmann population at 300 K) than the

four conformers with an equatorial 2-CH

group. The main

reason for this preference lies in the possibility of

establish-ing an intramolecular hydrogen bond between 4-C=O and

3-OH when this latter is equatorial (see structures in the

Supporting Information). Accordingly, the NOESY spectra

recorded in CD

CN show a strong NOE between the

equa-torial 2-H and the axial 3-H and only a weak NOE between

3-H and axial 2-CH

, which confirms the preferential anti

arrangement between these two latter groups.

The absolute configurations of phomochromones A and

B were assigned by electronic circular dichroism (CD)

spec-troscopy. The CD spectra of (+)-1 and (–)-2 in acetonitrile

(see Figures 1 and 2, a) show several bands in the 190–

380 nm region allied with the transitions of the substituted

chroman-4-one ring.

_{Apparently, however, the spectrum}

of compound 2 is richer in bands than that of 1. A helicity

rule exists for analyzing the CD spectra of chromanones

based on the sign of the first two bands (from the red)

as-signed to the n–π* and π–π* transitions. In the CD

spec-trum of (+)-1, the positive n–π* band at 335 nm and the

negative π–π* band at 311 nm can be correlated to the P

helicity of ring B, that is, a positive value for the C8a–O–

C2–C-3 dihedral angle (see inset in Figure 1).

_{On the}

as-sumption that the methyl substituent at C-2 tends to

prefer-entially occupy an equatorial position, P helicity

corre-sponds to a 2R absolute configuration (see molecular

mod-els in the Supporting Information). This assignment is

sub-stantiated by TD-DFT CD calculations

_using

B3LYP-SCRF/6-311++G(d,p)-optimized structures as input. An

MMFF conformational search followed by DFT

optimiza-tion (same level as for 2) revealed for phomochromone A

(1) a strong preference for two conformers with equatorial

2-CH

(97 % overall population at 300 K, estimated from

ZPC free energies). TD-DFT calculations were first run

with different combinations of functionals (B3LYP,

CAM-B3LYP, BH&HLYP) and basis sets (SVP, TZVP,

aug-TZVP) on the lowest-energy DFT structure with 2R

config-uration. Thereafter, the B3LYP/TZVP combination was

chosen for final calculations that included the PCM for

ace-tonitrile. The Boltzmann-weighted average of the calculated

CD spectra (Figure 1) reproduces very well the

experimen-tal CD spectrum. In particular, the expectations based on

the helicity rule

_{are confirmed. Thus, the first CD band}

calculated at 321 nm is of n–π* nature and it is positive for

a P helicity of ring B (positive C8a–O–C2–C3 dihedral

angle as shown in the inset in Figure 1). The absolute

con-figuration of phomochromone A is therefore definitively

es-tablished as (+)-(2R)-1.

Figure 1. CD spectra of (+)-(2R)-phomochromone A (1). Solid line: Experimental spectrum in acetonitrile. Dotted line: Boltz-mann-weighted average spectrum calculated at the B3LYP-SCRF/ TZVP//B3LYP/6-311++(d,p) level with PCM in acetonitrile. Gaussian band-width 0.2 eV; the calculated spectrum was scaled by a factor 0.2. Inset: Portion of the molecular structure of (2R)-1 showing the preferred conformation of the B ring. P helicity corre-sponds to a positive C8a–O–C2–C3 dihedral angle.

Application of the chromanone helicity rule discussed

above to phomochromone B (2) is hampered by the fact

that the observed spectrum is actually the convolution of

spectra of different conformers, as highlighted by CD

calcu-lations. After a preliminary test of various functionals and

basis sets as above, the B3LYP-SCRF/TZVP method with

PCM in acetonitrile was employed. The two sets of spectra

calculated for the two families of conformers with

equato-rial and axial 3-OH are very different (see average spectra

in Figure 2, b). In particular, the position of the CD band

allied with the n–π* transition, which is considered in the

helicity rule,

_{varies a lot depending on the presence of the}

intramolecular hydrogen bond, which causes a blueshift of

40 nm. Thus, the n–π* transition is the first calculated band

at 340 nm for conformers with axial 3-OH, but it is the

second calculated transition at 300 nm for conformers with

equatorial 3-OH. Interestingly enough, the predicted n–π*

CD band is negative in both cases, which contradicts the

helicity rule.

_{In fact, the two families of conformers have}

opposite helicity of the B ring (absolute C8a–O–C2–C3

di-hedral, see molecular models in the Supporting

Infor-mation). However, for conformers with axial 3-OH the n–

π* CD band is negative for M helicity and 2S

configura-tion, in keeping with the rule. The overall

Boltzmann-weighted average spectrum (thick line in Figure 2, b)

calcu-lated for the (2S,3R) configuration reproduces well the main

aspects of the experimental spectrum in terms of the shape,

sign, and relative intensity of the sequence of bands (with

the exception of the negative band predicted at 235 nm,

which appears as a trough in the experimental spectrum,

probably submerged by flanking bands). Therefore the

ab-solute configuration of phomochromone B is established as

(–)-(2S,3R)-2.

Figure 2. CD spectra of (–)-(2S,3R)-phomochromone B (2). (a) Ex-perimental spectrum in acetonitrile. (b) Boltzmann-weighted average spectra calculated at the B3LYP-SCRF/TZVP//B3LYP/ 6-311++(d,p) level with PCM in acetonitrile on the first six low-energy structures. Gaussian band-width 0.15 eV; the calculated spectrum has been shifted 10 nm to the right.

It has to be noted that somewhat unexpectedly the two

related phomochromones A and B have opposite

configura-tions of the common chirality center at C-2. In fact, they

also have opposite specific rotatory powers, [α]

= +42.1

for 1 and –62.2 for 2. This discrepancy was also reproduced

by optical rotation calculations

_{at the D line for the two}

compounds at the B3LYP/TZVP//B3LYP/6-311++G(d,p)

level. Boltzmann-weighted calculated averages over all

iso-lated DFT energy minima were +83.6 for (2R)-1 and –56.3

for (2S,3R)-2. The agreement with experimental values

fur-ther confirms the configurational assignments for both

compounds.

The molecular formula of phomotenone (3) was

deter-mined to be C

H

O

(three degrees of unsaturation) on

the basis of HREIMS and NMR spectroscopic data.

Analysis of

_{H and broadband and DEPT}

_{C NMR}

spec-troscopic data (see Exp. Sect.) revealed the presence of three

methyl groups, three methylene units, two sp

_{methines (one}

G. Pescitelli, K. Krohn et al.

FULL PAPER

of which is oxygenated), a tetrasubstituted olefin, and a

ketone carbonyl. These signals accounted for the molecular

formula and required phomotenone (3) to be monocyclic.

Analysis of COSY data (Scheme 3, bottom) led to the

iden-tification of a continuous spin system incorporating half of

the protons present in 3 to confirm the presence of a

1-hydroxybutyl group. HMBC correlations of 10-H and 5-H

to the ketone carbonyl at δ

= 212.7 ppm indicate that C-1

is connected to C-5. HMBC correlations of the vinylic

methyl signal (10-H

) to C-1 as well as to the two olefinic

carbon atoms (C-2 and C-3) indicate that the olefin unit

must be conjugated to the ketone C atom with the methyl

group located in α position to the ketone.

Scheme 3. Key1_H–1_{H COSY, HMBC, and NOESY correlations of} compound 3.

Because the remaining linkages in the molecule had

al-ready been established on the basis of COSY data, a

cyclo-pentenone ring was deduced. The chemical shifts of the

ketone and olefinic carbons are fully consistent with such a

structure.

_{HMBC correlations of 6-H to C-3, C-2, and}

C-4 confirmed the attachment of the hydroxybutyl group

to C-3. Thus, the structure of 3, a new cyclopentenone

de-rivative, was established as

3-(1-hydroxybutyl)-2,4-dimeth-ylcyclopent-2-enone.

Because of the flexibility of the chain attached to C-3,

the relative configuration of the two chirality centers of

compound 3 could be assigned only by careful

interpret-ation of experimental NMR spectroscopic data by using

molecular modeling. This is not uncommon for compounds

having multiple chirality centers on distinct moieties linked

by flexible bonds.

_{Two starting geometries were}

con-structed with the 4R*,6S* and 4R*,6R* configurations and

used for MMFF conformational searches followed by DFT

geometry optimizations at the B3LYP/6-31G(d) level. They

resulted in several low-energy minima for both

dia-stereomers (16 and 12 structures within 2 kcal/mol for

4R,6S and 4R,6R, respectively). In both cases the most

stable conformers have 6-H oriented towards 10-CH

. In

the case of the 4R,6R isomer, the C-6–H bond is practically

eclipsed by the C-2–C-10 bond. Thus, the n-propyl chain is

oriented towards the 4-H and the 6-OH group towards the

www.eurjoc.org © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem.2011, 2867–2873

2870

11-CH

. For the 4R,6S isomer, the n-propyl chain is instead

roughly perpendicular to the ring on the side of 11-CH

.

Thus, a different network of NOEs is expected for the two

structures. In particular, for the 4R,6S isomer, significant

NOEs are expected between 7-CH

and both 10-H and

11-CH

but not 4-H (see the structures in the Supporting

In-formation). In contrast, for the 4R,6R isomer, a strong

NOE is expected between 7-H

and 4-H. The pattern of

experimental NOEs extracted from NOESY experiments in

CD

CN (Scheme 3, top) is in agreement with the 4R*,6S*

relative configuration.

The same CD protocol described above for

phomochro-mones was applied to establish the absolute configuration

of phomotenone. The CD spectrum of (–)-3 in acetonitrile

displays two positive bands centered at 230 and 320 nm

al-lied with π–π* and n–π* transitions of the enone

chromo-phore (Figure 3).

_{Because the five-membered ring is}

practically planar, the CD does not arise from an

intrin-sically chiral enone moiety. To calculate the CD spectrum,

the first 13 low-energy DFT structures were further

opti-mized at the B3LYP/6-311++G(d,p) level in acetonitrile

(PCM) and their zero-point-corrected free energies were

evaluated (see structures and energies in the Supporting

In-formation). The first six low-energy conformers thus

ob-tained, amounting to an overall 82 % Boltzmann

popula-tion at 300 K, were considered for CD calculapopula-tions with a

4R,6S configuration. After screening DFT functionals and

basis sets as described above, the B3LYP/TZVP

combina-tion was again used. The Boltzmann-weighted average

spec-trum (Figure 3) is in keeping with the experimental one and

allows assignment of the absolute configuration of

phomot-enone as (–)-(4R,6S)-3.

Figure 3. CD spectra of (–)-(4R,6S)-phomotenone (3). Solid line: Experimental spectrum in acetonitrile. Dotted line: Average spec-trum calculated for the first six low-energy structures at the B3LYP/ TZVP level, Boltzmann weighted at 300 K using zero-point-cor-rected free energies for B3LYP/6-311++(d,p) optimized structures. Gaussian band-width 0.4 eV.

Phomotenone (3) bears some resemblance to

cyclo-pentenone-containing

prostaglandins

from

various

sources.

_{However, the fungal metabolites that are}

in-Table 1. Biological activities of pure metabolites 1–3 and 8 against microbial test organisms in an agar diffusion assay. Pure substances were used, concentration 0.05 mg (50 μL of 1 mg/mL). The radius of the zone of inhibition was measured in mm. P.I. denotes partial inhibition, that is, there was some growth within the zone of inhibition.

Compound Antibacterial Antibacterial Antialgal Antifungal Antifungal Antifungal Antifungal Antifungal

E.c. B.m. C.f. M.v. B.c. P.o. S.t. P.i.

1 8 P.I. 8 5 8 P.I. 7 0 6 0 2 7 P.I. 8 5 5 P.I. 10 0 9 0 3 6 P.I. 8 8 8 0 0 0 0 8 8 P.I. 8 7 0 0 0 0 0 Penicillin 14 18 0 0 Tetracycline 18 18 P.I. 10 0 Nystatin 0 0 0 20 Actidione 0 0 35 50 Acetone 0 0 0 0

clude phomapentenone A,

_{jasmonic acid derivatives}

from Botryodiplodia theobromae,

_{wasabienone B}

, a

cy-clopentenone derivative from Phoma wasabiae,

_and

terr-ein, originally isolated from Aspergillus terreus.

_The

simi-larity of the structure of 3 to the phytohormone jasmonic

acid allows one to speculate that this fungal metabolite may

play a role in modulating the fungal interaction with its

plant host.

The known compounds were readily identified as

pho-mosines A–D (4–7).

_{(1S,2S,4S)-trihydroxy-p-menthane}

(8),

_{and 5-methylmellein (9)}

_{by analysis of their NMR}

spectra and comparison with data reported in the literature.

Biological Activity

The isolated compounds 1–3 and 8 were tested in an agar

diffusion assay for their antifungal, antibacterial, and

algi-cidal properties towards Botrytis cinerea (B.c.), Pyricularia

oryzae

(P.o.), Septoria tritici (S.t.), Phytophthora infestans

(P.i.), Microbotryum violaceum (M.v.), Escherichia coli

(E.c.), Bacillus megaterium (B.m.), and Chlorella fusca (C.f.)

(Table 1). Interestingly, compounds 1 and 2 showed good

antifungal, antibacterial, and algicidal properties towards

S.t.

, M.v., B.c., E.c., B.m., and C.f. Similarly, compound 3

exhibited good antifungal, antibacterial, and algicidal

ac-tivity towards M.v., E.c., B.m., and C.f., whereas 8 displayed

only antialgal and antibacterial properties.

Conclusion

Two new chromones, phomochromone A and B (1 and

2), and one new natural cyclopentenone derivative,

phomot-enone (3), together with six known compounds,

phomos-ines A–D (4–7), (1S,2S,4S)-trihydroxy-p-menthane (8), and

5-methylmellein (9), have been isolated from the endophytic

fungus Phomopsis sp. through a bioassay-guided procedure

and structurally characterized. Compounds 1–3 showed

good antifungal (Microbotryum violaceum), antibacterial

(Escherichia coli, Bacillus megaterium), and antialgal

(Chlo-rella fusca

) activities. Compound 8 showed good antialgal

and antibacterial activity.

Experimental Section

General: Column chromatography: commercial silica gel (Merck,

0.040–0.063 mm) and Sephadex LH-20 (Amersham Biosciences). Analytical and preparative thin-layer chromatography (TLC): Pre-coated silica gel plates (Merck, G60 F-254 or G50 UV-254, respec-tively). Optical rotation: Perkin–Elmer 241 MC polarimeter at the sodiumd line. IR spectra: Nicolet-510P spectrophotometer; ν˜max= in cm–1_.1_{H and}13_{C NMR spectra: Bruker Avance 500 (500 MHz} for 1_{H and 125 MHz for}13_{C) spectrometer; chemical shifts δ in} ppm, coupling constants J in Hz. NOESY spectra: Varian INOVA 600 spectrometer, using a standard pulse sequence with 1 s mixing time. CD spectra: Jasco J715 spectropolarimeter using a 0.01 cm cell and 8.5 (2) and 10.4 m_{m (3) concentrated solutions in} acetoni-trile and with the following conditions: speed 50 nm/min, response 1 s, bandwidth 2.0 nm, four accumulations. EI-MS and HREI-MS: MAT 8200 and Micromass LCT mass spectrometers, measured in m/z. Microbiological methods and culture conditions were as de-scribed previously.[22,23]

Culture, Extraction, and Isolation: The endophytic fungus

Phom-opsis sp. was isolated from the plant Cistus monspeliensis. It was cultivated at room temperature for 28 d[22,23]_{on biomalt solid agar} medium. The culture media were then extracted with ethyl acetate to afford 67 g of a residue after removal of the solvent under re-duced pressure. The extract was separated into two fractions by column chromatography (CC) on silica gel using gradients of dichloromethane/ethyl acetate (85:15, 50:50, 0:100). The less polar fraction 1 (5 g) contained mainly fatty acids and lipids. The remain-ing fraction was further purified by silica gel column chromatog-raphy (CC) and preparative TLC with n-hexane/ethyl acetate (10:1, 1000 mL, 5:1, 1000 mL) to give pure compounds 1 (10 mg), 2 (6 mg), 3 (4 mg), 4 (3 g), 5 (7 mg), 6 (8 mg), 7 (5 mg), 8 (10 mg), and 9 (8 mg).

Phomochromone A (1): Colorless solid; m.p. 188 °C. [α]25D= +42.1 (c = 0.8, CH2Cl2). UV (CH2Cl2): λmax[log (ε/m–1cm–1)] = 214 [3.1], 280 [2.0] nm. IR (CH2Cl2): ν˜ = 3390, 1660, 1605, 1460, 1360, 1285, 1215, 1190 cm–1_.1_{H NMR (CDCl}

3, 500 MHz): δH= 1.50 (d, J = 6.0 Hz, 3 H, 2-Me), 2.12 (s, 3 H, 8-Me), 2.20 (s, 3 H, 6-Me), 2.60 (m, 2 H, 3-H), 4.54 (m, 1 H, 2-H), 5.16 (s, 1 H, 7-OH), 7.57 (s, 1 H, 5-H) ppm.13_{C NMR (CDCl}

3, 125 MHz): δC= 191.9 (s, C-4), 159.7 (s, C-8a), 158.5 (s, C-7), 125.8 (d, C-5), 117.0 (s, C-6), 114.3 (s, C-4a), 110.4 (s, C-8), 74.5 (d, C-2), 44.3 (t, C-3), 21.0 (q, 2-Me), 15.3 (q, 6-Me), 7.9 (q, 8-Me) ppm. MS (EI, 230 °C): m/z (%) = 206.0 (22) [M]+_{. HREIMS: calcd. for C}

12H14O3206.0943; found 206.0938.

Phomochromone B (2): Colorless solid; m.p. 196 °C. [α]D25= –62.2 (c = 0.9, CH2Cl2). UV (CH2Cl2): λmax[log (ε/m–1cm–1)] = 215 [3.2],

G. Pescitelli, K. Krohn et al.

FULL PAPER

285 [2.3] nm. IR (CH2Cl2): ν˜ = 3380, 1660, 1600, 1460, 1360, 1285, 1215, 1190 cm–1_.1_{H NMR (CDCl}

3, 500 MHz): δH= 1.25 (d, J = 6.5 Hz, 3 H, 2-Me), 2.20 (s, 3 H, 8-Me), 2.21 (s, 3 H, 6-Me), 4.68 (d, J = 6.0 Hz, 1 H, 3-H), 4.94 (dq, J = 6.5, 6.0 Hz, 1 H, 2-H), 7.50 (s, 1 H, 5-H) ppm.13_{C NMR (CDCl}

3, 125 MHz): δC= 192.6 (s, C-4), 159.4 (s, C-8a), 157.5 (s, C-7), 125.4 (d, C-5), 117.3 (s, C-6), 112.0 (s, C-4a), 111.3 (s, C-8), 76.7 (d, C-2), 71.7 (t, C-3), 15.2 (q, 2-Me), 11.2 (q, 6-Me), 7.8 (q, 8-Me) ppm. MS (EI, 230 °C): m/z (%) = 206.0 (22) [M]+_{. HREIMS: calcd. for C}

12H14O4222.0889; found 222.0892.

Phomotenone (3): Colorless oil. [α]D25= –32.2 (c = 0.9, CH2Cl2). UV (CH2Cl2): λmax[log (ε/m–1cm–1)] = 266 [3.7] nm. IR (CH2Cl2): ν˜ = 3320, 1715, 1700, 1630, 1340 cm–1_.1_{H NMR (CDCl} 3, 500 MHz): δH= 0.98 (t, J = 6.5 Hz, 3 H, 9-H), 1.17 (t, J = 5.5 Hz, 3 H, 11-H), 1.34 (m, 2 H, 8-11-H), 1.45 (m, 1 H, 7b-11-H), 1.54 (m, 1 H, 7a-11-H), 1.72 (s, 3 H, 10-H), 2.09 (m, 1 H, 5b-H), 2.38 (m, 1 H, 4-H), 2.98 (m, 1 H, 5a-H), 4.78 (dd, J = 5.3, 8.3 Hz, 1 H, 3-H) ppm. 13_C NMR (CDCl3, 125 MHz): δC= 212.7 (s, C-1), 171.1 (s, C-3), 134.6 (s, C-2), 69.3 (d, C-6), 39.1 (d, C-4), 37.6 (t, C-7), 33.8 (t, C-5), 18.7 (t, C-8), 16.6 (q, C-11), 13.9 (q, C-9), 8.3 (q, C-10) ppm. MS (EI, 230 °C): m/z (%) = 181.1 (22) [M]+_{. HREIMS: calcd. for} C11H18O2182.1307; found 182.1300.

Agar Diffusion Test for Biological Activity: Compounds 1–3 and 8

were dissolved in acetone at a concentration of 1 mg/mL. The solu-tions (50 μg. 50 μL) were pipetted onto a sterile filter disk (Schleicher & Schuell, 9 mm), which was placed onto an appropri-ate agar growth medium for the corresponding test organism and subsequently sprayed with a suspension of the test organism.[23] The test organisms were the Gram-negative bacterium Escherichia coli, the Gram-positive bacterium Bacillus megaterium (both grown on NB medium), the fungi Microbotryum violaceum, Septoria tritici, Botrytis cinerea, Pyricularia oryzae, the fungal-like Phytoph-thora infestans, and the alga Chlorella fusca (all grown on MPY medium). Reference substances were penicillin, nystatin, actidione, and tetracycline. Commencing at the middle of the filter disk, the radius of the zone of inhibition was measured in millimeters. These micro-organsims were chosen because (a) they are nonpathogenic and (b) they have in the past proved to be accurate initial test or-ganisms for antibacterial, antifungal, and antialgal/herbicidal ac-tivities.

Computational Section: Molecular mechanics and preliminary DFT

calculations were run with Spartan 08[28]_{with standard parameters} and convergence criteria. DFT and TD-DFT calculations were run with Gaussian 09[24]_{with default grids and convergence criteria.} Conformational searches were run by employing the “systematic” procedure implemented in Spartan 08 using MMFF (Merck molec-ular force field). All MMFF minima were reoptimized with DFT at the 31G(d) level and then at the B3LYP/6-311++G(d,p) level using the integral equation formalism variant of the polarizable continuum model (IEF-PCM)[25] _{for acetonitrile} with default parameters. Zero-point-corrected free energies were evaluated from frequency calculations at the same level of theory. Calculated low-energy structures and free energies are shown in the Supporting Information. TD-DFT calculations were run by using various combinations of the hybrid DFT functionals B3LYP, CAM-B3LYP, and BH&HLYP, and the basis sets SVP, TZVP, and aug-TZVP,[26]_{including at least 36 excited states in all cases and} by using IEF-PCM for acetonitrile. The aug-TZVP set was con-structed by augmenting TZVP with a (1s1p1d/1s1p) set of primitive functions taken from the most diffuse functions of aug-cc-pVDZ. All transitions computed with B3LYP/TZVP responsible for the CD bands observed above 200 nm had energies below the estimated

www.eurjoc.org © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem.2011, 2867–2873

2872

ionization potentials. CD spectra were generated by using the pro-gram SpecDis[27]_{by applying a Gaussian band-shape with a width} of 0.15–0.4 eV from dipole-length rotational strengths; the differ-ence with dipole-velocity values was negligible (⬍10%) for most transitions.

Supporting Information (see footnote on the first page of this

arti-cle): DFT-optimized structures for compounds 1–3 with relative free energies and TD-DFT-calculated CD spectra for all relevant structures.

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Received: February 3, 2011 Published Online: April 5, 2011