Previous PageTable Of ContentsNext Page

Structure-Activity Studies (SAR) on phytotoxic breviane derivatives.

Francisco A. Macías, Ceferino Carrera, María D. García-Díaz, and Juan C. G. Galindo

Cadiz Allelopathy Group (GAC), Department of Organic Chemistry, Faculty of Sciences University of Cadiz. Apdo. 40, 11510 – Puerto Real, Cádiz, Spain. Email


Breviones are new phytotoxic diterpenes isolated from the endemic New Zealand fungi Penicillium brevicompactum cv. Dierkx. Their chemical structure constitutes a unique challenge for synthetic organic chemists and is an attractive target for chemical modifications and structure-activity relationship studies. From a biogenetic point of view they can be described as resulting from a mixed biogenetic pathway: the tricyclic system formed by rings A, B, and C presents a diterpenic nature, while ring E can be considered as formed from the poliketide pathway. This vision can be translated to their retrosynthetic analysis, as a convergent synthetic design for each part that merges in the last steps to give rise to the breviane skeletons. Herein, we present the preliminary results of our ongoing project on the total synthesis and bioactivity evaluation of breviones and brevione-like analogues. A SAR study has been carried out using the etiolated wheat coleoptiles and the Standard Target Species (STS) bioassays.

Media summary

The diterpenic part of Breviones A-D has been synthesized and the phytotoxic activity of these and other structurally related compounds have been tested.

Key Words

Breviones, SAR, Penicillium brevicompactum, etiolated coleoptiles bioassay, synthesis


The discovery of new allelochemicals from plants and microorganisms has attracted much interest in recent years because of their ecological implications and potential applications. Allelochemicals are bioactive natural products, produced by living organisms with a precise role and meaning within the specific territory of the producers, which profoundly influence their associated biosphere. Their applications as templates for development of new environmentally compatible agrochemicals have been extensively reviewed (Duke 2000, 2000b, 2002; Vyvyan, 2002) and include herbicides, antimicrobials, pesticides and plant growth regulators, which presents the possibility for discovery of new target sites and modes of action. Partial and/or total synthesis are usual methods for mass production of these compounds. Also, biotransformation is also a very attractive production method, especially for those compounds coming from fungi and microorganisms that can be mass-produced by culture.

The genus Penicillium is noted for producing a variety of bioactive metabolites possessing diverse biological activities including plant growth regulators (Matsuzaki 1998; Tomoda, 1998; Kimura 2000). In particular, P. brevicompactum produces polyketides, peptides and a collection of diverse heterocyclic compounds with pyrrole, pyrroline, and oxazine structures with anti-juvenile-hormonal, insecticidal, and fungicidal activities (Canonica 1971; Moya, 1998; Cantin, 1999). Recently, our group isolated a new class of compounds of mixed biogenesis named brevianes, obtained from strains of Penicillium brevicompactum Dierckx from leaf litter collected in the Waipoua Forest, New Zealand. Breviones A-D (Figure 1) were isolated from the ethyl acetate fraction of the culture broth and mycelia acetone/aqueous extract following a etiolated wheat coleoptile (Parker 1995; Cutler 1999) bioassay-guided search protocol (Macias, 2000b). Breviones C and E presented excellent activities, producing a total inhibition of growth in wheat coleoptiles at 100μM. These activities together with their unique structure make them an attractive goal for total synthesis. Consequently, the total synthesis of breviones A and B has been recently reported (Takikawa 2004). Herein we present the results of our synthetic approach to the diterpenic A,B,C rings of Breviones A-D and some preliminary Structure-Activity Relationship (SAR) studies based on the activities of the derivatives obtained.


The total synthesis of breviones present three key points: the configuration of their many stereogenic centers, the spiranic carbon placed between rings C and D, and the polyketide lactone ring E. The retrosynthetic analysis of Breviones A and B is shown in Scheme 1. In the case of Breviones C and D, we envisioned a retrosynthetic methodology involving an expansion of the A ring to build up the cycloheptanone system from the common intermediate R-3 (Scheme 2).

Figure 1. Brevianes isolated from P. brevicompactum (1-5) and from Lygodium flexuosum (6). The different breviane backbones are depicted on the right side of the figure.

Scheme 1. Retrosynthetic analysis for rings A-C of Breviones A and B.

Scheme 2. Retrosynthetic analysis for rings A-C of Breviones C and D.


We used a wheat coleoptile bioassay in test tubes, and germination and growth bioassay with monocots and dicots in Petri dishes. The general procedure for the coleoptile bioassay has been previously described (Macías et al., 2000b).

Products were purified (+99%) by HPLC and tested at concentrations of 1000 µM to 10 µM in a buffered nutritive aqueous solution (citric acid-sodium hydrogenphosphate buffer, pH 5.6; 2% sucrose). Mother solutions of pure compounds were prepared in DMSO and diluted to the proper concentration with the buffer to a 0.5% v/v DMSO final maximum concentration. Subsequent dilutions were prepared maintaining the same buffer and DMSO concentrations. Bioassays were performed in 10 mL test tubes as follows: five coleoptiles were placed per tube containing 2 mL of test solution each; three replicates were prepared for each test solution and the experiments were run in duplicate. Test tubes were placed in a roller tube apparatus and rotated at 6 rpm for 24 h at 22 °C in the dark. Increments of coleoptile elongation were measured by digitalization of their photographic images and data were statistically analyzed.

Petri dish bioassay. The general procedure has been previously described (Macías et al., 2000b). Mother test solutions (10-3or10-4M) were prepared using MES (2-[N-morpholino]-ethanesulfonic acid) 10 mM and the rest were obtained by dilution. Parallel controls were performed. All pH values were adjusted to 6.0 before bioassay with MES. All products were purified prior to the bioassay using HPLC equipped with a refractive index detector. Minimum degree of purity was of 99% as extracted from the chromatograms. Germination index, root length and shoot length were measured and recorded at the end of the experiment using Phytomed®.


According to the retrosynthetic analysis depicted in Scheme 1, we gained access to the A-D ring system of Breviones A and B with the proper stereochemistry at each stereogenic carbon (Scheme 3). We are in process of preparing synthon A (Scheme 1) that will allow for a coupling to yield the spiranic D ring.

Scheme 3. Key: a) 1) H2O, 50 ºC, 24 h, pent-1-en-3-one (EVK) 2) S-phenylalanine, 7 days, (+)-camphorsulfonic acid, DMF, 30→70ºC; 3) NH4Cl, aq. sat.; b) NaBH4, MeOH; c) KOH/MeOH 10%, EVK, 36h; d) MeI, Li/NH3; e) H2, Pd/C, 2atm., 5 days; f) HOCH2CH2OH, p-TSOH; g) PDC, CH2Cl2

Scheme 4. Synthetic scheme for cycloheptane ring preparation.

Using compound 3 as starting material, we gained access to the silyl enol-ether preparing the way for the proposed cyclopropanation and ring expansion reactions (Scheme 4).


We have synthesized the ABC ring-system of Breviones A-D in an enantioselective way. Bioassays with all the intermediates and related compounds have been performed using the etiolated wheat coleoptiles and Petri dishes bioassays.


Canonica, L; Kroszczynski, W; Ranzi, BM; Rindone, B; Scolastico, C. (1971). Biosynthesis of mycophenolic acid. Journal of the Chemical Society [Section] D: Chemical Communications 6, 257.

Cantin, A; Moya, P; Castillo, MA; Primo, J; Miranda, MA; Primo-Yufera, E. (1999) Isolation and synthesis of N-(2-methyl-3-oxodec-8-enoyl)-2-pyrroline and 2-(hept-5-enyl)-3-methyl-4-oxo-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b]-1,3-oxazine. Two new fungal metabolites with in-vivo anti-juvenile-hormone and insecticidal activity. European Journal of Organic Chemistry 1, 221-226.

Cutler, HG; Cutler, SJ; Ross, SA; El Sayed, K; Dugan, FM; Bartlett, MG; Hill, AA; Hill, RA; Parker, SR (1999). Koninginin G, a New Metabolite from Trichoderma aureoviride. Journal of Natural Products 62, 137-139.

Duke, SO; Dayan, FE; Rimando, AM (2000). Natural products and herbicide discovery. En ‘Herbicides and Their Mechanisms of Action’ (Eds.: Cobb, AH; Kirkwood, RC). Sheffield Academic Press, Sheffield, UK. 105-133.

Duke, SO; Dayan, FE; Rimando, AM; Schrader, KK; Aliotta, G; Oliva, A; Romagni, JG (2002). Chemicals from nature for weed management. Weed Science 50, 138-151.

Duke, SO; Rimando, A; Scheffler, B; Dayan, FE. (2000b). Strategies for research in applied aspects of allelopathy. In ‘Allelopathy’ (Eds.: Reigosa, MJ and Pedrol, N). Science Publishers, Inc., Enfield, N.H. 139-152.

Kimura Y; Mizuno T; Kawano T; Okada K; Shimada A (2000). Peniamidienone and penidilamine, plant growth regulators produced by the fungus Penicillium sp. No. 13. Phytochemistry 53, 829-31.

Macías, FA, Varela, RM, Simonet, AM, Cutler, HG, Cutler, SJ, Ross, SA, Dunbar, DC, Dugan, FM, Hill, RA (2000). Allelochemicals from New Zealand fungi. (+)-Brevione A. The first member of a novel family of bioactive spiroditerpenoids isolated from Penicillium brevicompactum Dierckx. Tetrahedron Letters 41, 2683-2686

Macías, FA, Varela, RM, Simonet, AM, Cutler, HG, Cutler, SJ, Dugan, FM, Hill, RA (2000b). Novel Bioactive Breviane Spiroditerpenoids from Penicillium brevicompactum Dierckx. Journal of Organic Chemistry 65, 9039-9046

Macías, FA, Castellano, D, Molinillo, JMG, (2000). Search for a Standard Phyotoxic Bioassay for Allelochemicals. Selection of Standard Target Species. Journal of Agricultural and Food Chemistry 48, 2512-2521.

Matsuzaki, K; Tahara, H; Inokoshi, J; Tanaka, H; Masuma, R; Omura, S. (1998). New brominated and halogen-less derivatives and structure-activity relationship of azaphilones inhibiting gp120-CD4 binding. Journal of Antibiotics 51, 1004-1011.

Moya, P; Cantin, A; Castillo, MA; Primo, J; Miranda, MA; Primo-Yufera, E. (1998). Isolation, Structural Assignment, and Synthesis of N-(2-Methyl-3-oxodecanoyl)-2-pyrroline, a New Natural Product from Penicillium brevicompactum with in Vivo Anti-Juvenile Hormone Activity. Journal of Organic Chemistry 63, 8530-8535.

Parker, SR; Cutler, HG; Schreiner, PR (1995). Koninginin E: isolation of a biologically active natural product from Trichoderma koningii. Bioscience, Biotechnology, and Biochemistry 59, 1747-9.

Takikawa, H; Hirooka, M; Sasaki, M (2003). The first synthesis of (±)-brevione B, an allelopathic agent isolated from Penicillium sp. Tetrahedron Letters 44, 5235-5238.

Tomoda H; Tabata N; Masuma R; Si, SY; Omura S (1998). Erabulenols, inhibitors of cholesteryl ester transfer protein produced by Penicillium sp. FO-5637. I.Production, isolation and biological properties. Journal of Antibiotics 51, 618-23.

Vyvyan, JR (2002). Allelochemicals as leads for new herbicides and agrochemicals. Tetrahedron 58, 1631-1646.

Previous PageTop Of PageNext Page