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A novel sesquiterpene furan compound CJ-01 was isolated from the methanol extract of the whole plant of Chloranthus japonicus SIEB. by monitoring the inhibitory activity of chitin synthase 2 from Saccharomyces cerevisiae. Based on spectroscopic analysis, the structure of compound CJ-01 was determined as 3,4,8a-trimethyl-4a,7,8,8a-tetrahydro-4a-naphto[2,3-b]furan-9-one. The compound inhibited chitin synthase 2 of Saccharomyces cerevisiae in a dose-dependent manner with an IC50 of 39.6 microg/ml, whereas it exhibited no inhibitory activities against chitin synthase 1 and 3 of S. cerevisiae up to 280 microg/ml. CJ-01 has 1.7-fold stronger inhibitory activity than polyoxin D (IC50=70 microg/ml), a well-known chitin synthase inhibitor. These results indicate that the compound is a specific inhibitor of chitin synthase 2 from S. cerevisiae. In addition, CJ-01 showed antifungal activities against various human and phytopathogenic fungi. Therefore, the compound might be an interesting lead to develop effective antifungal agents.

Potent chitin synthase 2 inhibitors, methyllinderone (1), linderone (2) and kanakugiol (3) were isolated from the stem bark of L. erythrocarpa Makino (Lauraceae). These compounds inhibited chitin synthase 2 with IC(50) values of 23.3, 21.4 and 23.8 microg/mL, respectively. Methyllinderone (1) and linderone (2) exhibited no inhibitory activities for chitin synthases 1 and 3 from S. cerevisiae, and chitin synthase 1 from Candida albicans up to the concentration of 280 microg/mL, while kanakugiol (3) exhibited very weak activity against chitin synthase 1 of C. albicans with an IC(50) of 160 microg/mL. All of the compounds showed moderate to weak antifungal activities against various pathogenic fungi (MIC: 8 - >128 microg/mL) including Cryptococcus neoformans, Aspergillus fumigatus, and Colletotrichum lagenarium. The results indicate that these compounds are specific inhibitors of chitin synthase 2 and can potentially serve as antifungal agents.

In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases inhibitor, 2'-benzoyloxycinnamaldehyde (2'-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliinervis NAKAI. 2'-BCA inhibited chitin synthase 1 and 2 of Saccharomyces cerevisiae with the IC50s of 54.9 and 70.8 microg/ml, respectively, whereas it exhibited no inhibitory activity for chitin synthase 3 up to 280 microg/ml. Its derivatives, 2'-chloro- (V) and 2(-bromo-cinnamaldehyde (VI), each showed 1.9 and 2.7-fold stronger inhibitory activities than 2'-BCA, with the IC50s of 37.2 and 26.6 microg/ml, respectively. Especially, the IC50 of compound VI against chitin synthase 2 represented 1.7-fold more potent inhibitory activity than polyoxin D, a well-known chitin synthase inhibitor. Furthermore, compounds V and VI showed potent antifungal activities against various fungi including human pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC = 16 microg/ml). Although the chemical synthesis of this compound has been reported, the present study is the first report to describe the isolation of 2'-BCA from natural resources and chitin synthases inhibitory activities of its derivatives. These results suggested that 2'-BCA and its derivatives can potentially serve as useful lead compounds for development of antifungal agents.

Phellinsin A (1a) was prepared in a concise way, thereby elucidating the relative stereochemistry of the aryl and carboxylic acid groups in 1a. The synthesis employed selective monohydrolysis of the dilactones derived from oxidative dimerization of cinnamic acid derivatives. This approach provided a practical synthetic route to α‐arylidene‐γ‐lactones.

In the course of search for potent inhibitors of chitin synthase II from natural resources, seven tannins and related compounds were isolated from the aerial part of Euphorbia pekinensis and identified as gallic acid (1), methyl gallate (2), 3-O-galloyl-(-)-shikimic acid (3), corilagin (4), geraniin (5), quercetin-3-O-(2"-O-galloyl)-beta-D-glucoside (6), and kaempferol-3-O-(2"-O-galloyl)-beta-D-glucoside (7). These and nine related compounds, (-)-quinic acid (8), (-)-shikimic acid (9), ellagic acid (10), kaempferol (11), quercetin (12), quercitrin (13), rutin (14), quercetin-3-O-(2"-O-galloyl)-beta-D-rutinoside (15) and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (16), were evaluated for the inhibitory activity against chitin synthase II and III. They inhibited chitin synthase II with IC(50) values of 18-206 microM, except for two organic acids, (-)-quinic acid (8) and (-)-shikimic acid (9). Among them, 3-O-galloyl-(-)-shikimic acid (3) was the most potent inhibitor against chitin synthase II of Saccharomyces cerevisiae with an IC(50) value of 18 microM. The inhibition appears to be selective for chitin synthase II, as they did not appreciably inhibit chitin synthase III.

In the course of the search for inhibitors of ScCHS2 from natural sources, we have isolated a new type of chitin synthase 2 inhibitor, obovatol, which has a biphenol skeleton, from Magnolia obovata. Obovatol inhibited chitin synthase 2 activity of Saccharomyces cerevisiae with an IC(50) of 38 microM. Its derivative, tetrahydroobovatol, inhibited chitin synthase 2 activity under the same conditions with an IC(50) of 59 microM. These compounds exhibited no inhibitory activity for ScCHS3, and showed less inhibitory activity for chitin synthase 1 than for chitin synthase 2 (IC(50) > 1 mM). These results indicated that obovatol and tetrahydroobovatol are specific inhibitors of ScCHS2. They also inhibited CaCHS1, which is structurally and functionally analogous to ScCHS2, with similar IC(50)s to ScCHS2 (IC(50) 28 and 51 microM, respectively). The compounds exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate [inhibition constant (K(i)) 21.8 microM for obovatol and 23.1 microM for tetrahydroobovatol]. Furthermore, they showed antifungal activities against various pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC 7.8 mg/L). The results indicate that obovatol and tetrahydroobovatol can potentially serve as antifungal agents.

Chaetoatrosin A, a novel chitin synthase II inhibitor, was isolated from the culture broth of fungus F449, which was identified as Chaetomium atrobrunneum F449. Chaetoatrosin A was purified by solvent partition, silica gel, ODS, preparative TLC, and Sephadex LH-20 column chromatographies, consecutively. The structure of chaetoatrosin A was assigned as 1,8-dihydroxy-3(2-hydroxypropionyl)-6-methoxynaphthalene on the basis of various spectroscopic analyses including UV, IR, mass spectral, and NMR. Its molecular weight and formula were found to be 262 and C 14 H 14 O 5 , respectively. Chaetoatrosin A inhibited chitin synthase II by 50% at the concentration of 104 μg/ml in an enzyme assay system. This compound showed antifungal activities against Rhizoctonia solani, Pyricularia oryzae, Botrytis cinerea. Cryptococcus neoformans and Trichophyton mentagrophytes.

Phellinsin A, a novel chitin synthases inhibitor was isolated from the cultured broth of fungus PL3, which was identified as Phellinus sp. PL3. Phellinsin A was purified by solvent partition, silica gel, ODS column chromatographies, and preparative HPLC, consecutively. The structure of phellinsin A was assigned as a phenolic compound on the basis of various spectroscopic analyses including UV, IR, Mass, and NMR. Its molecular weight and formula were found to be 358 and C18H14O8, respectively. Phellinsin A selectively inhibited chitin synthase I and II of Saccharomyces cerevisiae with an IC50 value of 76 and 28 microg/ml, respectively, in our cell free assay system. This compound showed antifungal activity against Colletotrichum lagenarium, Pyricularia oryzae, Rhizoctonia solani, Aspergillus fumigatus, and Trichophyton mentagrophytes.

Chitin is the β-1,4-linked homopolymer of N-acetyl-Dglucosamine (GlcNAc) that constitutes an important structural component of the cell walls of nearly all zoopathogenic and phytopathogenic fungi, and plays a crucial role in the determination of cell morphology. Chitin have been known to be synthesized by chitin synthase I, II, and III in Saccharomyces cerevisiae. Chitin synthase II is an essential enzyme for the formation of primary septum between mother and daughter cells, and chitin synthase III is responsible for the formation of chitin ring at bud emergence, whereas chitin synthase I is a repair enzyme of the damaged cells on cell division. Accordingly, specific inhibitors of chitin synthase II and III are expected to be an attractive target for the development of antifungal agents. In the course of exploring a chitin synthase II inhibitor from microbial sources, we isolated a new phenolic lignan bearing a γ-lactone ring from the cultured broth of Phellinus sp. PL3. The lignan compound named phellinsin A (3a) exhibited inhibition of chitin synthase II with an IC50 value of 27 μg/mL and showed 2.5 times stronger inhibitory activity than polyoxin D, and its structure was established by NMR analysis and synthesis. Our efforts toward the development of a potent antifungal agent have focused on examining structure-activity relationships for phellinsin A. We have modified the aryl group in phellinsin A by changing the number of phenolic OH groups in order to investigate the effect of the number of free phenolic OH groups in γ-lactone analogues of phellinsin A (3a) on chitin synthase II inhibitory activity. Herein, we describe the biological evaluation of phellinsin A analogues 3. The γ-lactone analogues 3c-f of phellinsin A were prepared by oxidative dimerization of cinnamic acid derivatives 1 into the corresponding dilactones 2, followed by monohydrolysis of the dilactones as shown in Scheme 1. The γlactones 3a and 3b were prepared by demethylation of compounds 3d and 3e using BBr3, respectively. The γ-lactone analogues 3 of phellinsin A were examined for structure-activity relationships of phellinsin A (3a). Inhibitory activities of the compounds against chitin synthase II were evaluated by measurement of the formation of chitin with UDP-[C]-N-acetyl-D-glucosamine. Table 1 showed the inhibitory activities of phellinsin A analogues at 140 and 280 μg/mL concentrations. The activities were largely dependent on the presence of free phenolic OH substituents in γ-lactone analogues 3. Generally, the compound 3f with non-phenolic OH groups did not show

Chaetoatrosin A, a novel chitin synthase II inhibitor, was isolated from the culture broth of fungus F449, which was identified as Chaetomium atrobrunneum F449. Chaetoatrosin A was purified by solvent partition, silica gel, ODS, preparative TLC, and Sephadex LH-20 column chromatographies, consecutively. The structure of chaetoatrosin A was assigned as 1,8-dihydroxy-3(2-hydroxypropionyl)-6-methoxynaphthalene on the basis of various spectroscopic analyses including UV, IR, mass spectral, and NMR. Its molecular weight and formula were found to be 262 and C 14 H 14 O 5 , respectively. Chaetoatrosin A inhibited chitin synthase II by 50% at the concentration of 104 μg/ml in an enzyme assay system. This compound showed antifungal activities against Rhizoctonia solani, Pyricularia oryzae, Botrytis cinerea. Cryptococcus neoformans and Trichophyton mentagrophytes.

In the course of search for potent chitin synthase inhibitors from plant extracts, the chitin synthase 2 inhibitors, O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one which have diterpene skeleton, were isolated from the leaves of Chamaecyparis pisifera. These compounds inhibited chitin synthase 2 of Saccharomyces cerevisiae with the IC50 values of 5.8 and 226.4 microM, respectively. Especially, O-methyl pisiferic acid showed 15.3-fold stronger inhibitory activity than polyoxin D (IC50=88.6 microM), a well-known chitin synthase inhibitor. These compounds exhibited weaker inhibitory activities against chitin synthase 1 than chitin synthase 2, whereas it showed no inhibitory activity for chitin synthase 3. The compound exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate (Ki=5 microM). These results indicated that O-methyl pisiferic acid is a specific inhibitor of chitin synthase 2. The compound also inhibited chitin synthase 1 of Candida albicans, which represents analogues to chitin synthase 2 of S. cerevisiae, with an IC50 of 75.6 microM, which represents 1.8-fold weaker activity than that of polyoxin D. Although O-methyl pisiferic acid has been reported for antibacterial and insecticidal activities, the present study is the first report on its inhibitory activity against chitin synthase 2.

Chitin is the β-1,4-linked homopolymer of N-acetyl-Dglucosamine (GlcNAc) that constitutes an important structural component of the cell walls of nearly all zoopathogenic and phytopathogenic fungi, and plays a crucial role in the determination of cell morphology. Chitin have been known to be synthesized by chitin synthase I, II, and III in Saccharomyces cerevisiae. Chitin synthase II is an essential enzyme for the formation of primary septum between mother and daughter cells, and chitin synthase III is responsible for the formation of chitin ring at bud emergence, whereas chitin synthase I is a repair enzyme of the damaged cells on cell division. Accordingly, specific inhibitors of chitin synthase II and III are expected to be an attractive target for the development of antifungal agents. In the course of exploring a chitin synthase II inhibitor from microbial sources, we isolated a new phenolic lignan bearing a γ-lactone ring from the cultured broth of Phellinus sp. PL3. The lignan compound named phellinsin A (3a) exhibited inhibition of chitin synthase II with an IC50 value of 27 μg/mL and showed 2.5 times stronger inhibitory activity than polyoxin D, and its structure was established by NMR analysis and synthesis. Our efforts toward the development of a potent antifungal agent have focused on examining structure-activity relationships for phellinsin A. We have modified the aryl group in phellinsin A by changing the number of phenolic OH groups in order to investigate the effect of the number of free phenolic OH groups in γ-lactone analogues of phellinsin A (3a) on chitin synthase II inhibitory activity. Herein, we describe the biological evaluation of phellinsin A analogues 3. The γ-lactone analogues 3c-f of phellinsin A were prepared by oxidative dimerization of cinnamic acid derivatives 1 into the corresponding dilactones 2, followed by monohydrolysis of the dilactones as shown in Scheme 1. The γlactones 3a and 3b were prepared by demethylation of compounds 3d and 3e using BBr3, respectively. The γ-lactone analogues 3 of phellinsin A were examined for structure-activity relationships of phellinsin A (3a). Inhibitory activities of the compounds against chitin synthase II were evaluated by measurement of the formation of chitin with UDP-[C]-N-acetyl-D-glucosamine. Table 1 showed the inhibitory activities of phellinsin A analogues at 140 and 280 μg/mL concentrations. The activities were largely dependent on the presence of free phenolic OH substituents in γ-lactone analogues 3. Generally, the compound 3f with non-phenolic OH groups did not show

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli , three sets of an artificial gene cluster which contained three genes of heterologous origins— PAL from the yeast Rhodotorula rubra , 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata— were constructed. The constructions of the three sets were done as follows: (i) PAL , 4CL , and CHS were placed in that order under the control of the T7 promoter (P T7 ) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P T7 in front of PAL , and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P T7 and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.

Chalcones, the central precursor of flavonoids, are synthesized exclusively in plants from tyrosine and phenylalanine via the sequential reaction of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL) and chalcone synthase (CHS). Chalcones are converted into the corresponding flavanones by the action of chalcone isomerase (CHI), or non-enzymatically under alkaline conditions. PAL from the yeast Rhodotorula rubra, 4CL from an actinomycete Streptomyces coelicolor A3(2), and CHS from a licorice plant Glycyrrhiza echinata, assembled as artificial gene clusters in different organizations, were used for fermentation production of flavanones in Escherichia coli. Because the bacterial 4CL enzyme attaches CoA to both cinnamic acid and 4-coumaric acid, the designed biosynthetic pathway bypassed the C4H step. E. coli carrying one of the designed gene clusters produced about 450 microg naringenin/l from tyrosine and 750 microg pinocembrin/l from phenylalanine. The successful production of plant-specific flavanones in bacteria demonstrates the usefulness of combinatorial biosynthesis approaches not only for the production of various compounds of plant and animal origin but also for the construction of libraries of "unnatural" natural compounds.

Two triterpenoid compounds, ursolic acid and uvaol, were isolated from Crataegus pinnatifida Bunge leaves. Ursolic acid inhibits chitin synthase II from S. cerevisiae with an IC50 value of 0.84 microgram/ml and the inhibition appears to be selective for chitin synthase II, whereas uvaol has no inhibitory activity up to 280 micrograms/ml. Oleanolic acid, alpha-hederin hydrate, and betulic acid inhibited the chitin synthase II activity under the same conditions with an IC50 of 5.6, 64.3, and 98.7 micrograms/ml, respectively.

Two flavonoids, (+/-)-catechin and (-)-epicatechin, were isolated from the stem bark of Taxus cuspidata by monitoring chitin synthase II inhibitory activity. The compounds inhibit chitin synthase II with an IC50 of 15 and 29 micrograms/ml, respectively and appear to be selective for chitin synthase II. They did not inhibit chitin synthase III.