Three New Amides from Streptomyces sp . H 7372

As amidas fenatato de metila (1), actifenamida (2) e 1-b-D-glucopiranosídeo-actinofenol (3), juntamente com outros 13 compostos de estruturas conhecidas, foram isolados do extrato de uma cultura de Streptomyces sp H7372. As estruturas destes compostos foram determinadas por análises de RMN uni e bidimensionais e por espectrometria de massas. Cicloheximida (6) e ciclo (ΔAla-L-Val) (8) apresentaram forte atividade inibitória na interação entre Ras-Raf-1 pela técnica de duplo híbrido em leveduras, com halos de inibição de 10 e 25 mm em SD His, respectivamente, enquanto foram inativos em SD His, na concentração de 25 μg/disco.


Introduction
Protein kinases constitute an important group of enzymes that are essential in many physiological processes.Protein phosphorylation is one of the major regulatory mechanisms involved in signal transduction pathways including apoptosis, cell proliferation, cell differentiation, and metabolism. 1 Abnormalities in protein phosphorylation are often associated with human diseases.Thus, the inhibitors of both protein kinases and phosphatases appear to be very promising drug targets in the chemotherapeutic treatment of cancer and have received wide attention. 1The Ras family GTPases play a central role in the growth factor signaling controlling cell proliferation, differentiation, and survival. 2The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties.Recently, the approval of NEXAVAR ® (sorafenib) by the FDA for advanced renal cell carcinoma provides justification for the development of Ras-Raf-1 kinase inhibitors.Radicicol, is a macrocyclic antibiotic isolated from the fungus Monosporium bonorden that inhibits the Ras-Raf-1 protein interaction at concentrations of 0.1 ca. 1 mg mL -1 . 3To identify such inhibitors, we employed the yeast two-hybrid system in our screening. 3he actinomycete family continues to be a rich reservoir of microorganisms that produce a diverse array of biologically active metabolites effective against a broad spectrum of health problems.For example, staurosporine from Streptomyces staurosporeus exhibited potent inhibition

General experimental procedures
The optical rotation [a] D values were determined with an Autopol ® IV Automatic polarimeter.UV spectra were measured on a Shimadzu PharmaSpec-1700 UV-Visible spectrophotometer.IR spectra were recorded on a Shimadzu-8400S FT-IR spectrometer.Mass spectra and high-resolution MS spectra were taken with a BioTOF II ESI mass spectrometer.1D and 2D NMR spectra were recorded in chloroform-d and methanol-d 4 on an INOVA Unity (500 MHz) Varian spectrometer equipped with an xyz-shielded gradient triple resonance probe.Reversed-phase HPLC was carried out on a Beckman Coulter Gold-168 system equipped with a photodiode array detector using an Alltech semipreparative Econosil C 18 column (10 μm, 10 × 250 mm) run with a flow rate of 2.0 mL min -1 .Column chromatography (CC) was carried out on Merck silica gel 60 (70-230 mesh).Precoated plates of silica gel 60 F 254 were used for analytical purposes.

Microbial material and sample collection
The organism was isolated from mud sample (pH 7.30) under the root of Bruguiera sp., located at sea shore near mouth of river into mangrove swamp, Kampung Termunong, Tuaran, Sabah, Malaysia in 2003. 5The strain was isolated by suspending 0.1 g of soil into 10 mL of sterile distilled water and later diluting this up to 10 -3 .One hundred-mL of the suspension is spread on humic-acid (HV)-vitamins B plus cycloheximide, 6 with pH 7.2.Isolated strains were transferred from HV medium onto similar pH oatmeal agar plates and incubated at 28 °C for 14 days until sporulation.The strain H7372 was characterized by microscopy and determination of cell wall DAP (meso or LL).It was further characterized by 16S rRNA gene analysis.A pure culture of strain H7372 was deposited in National Measurement of Australia and was given an accession number V07/019103.Frozen spore stock of Streptomyces sp.H7372 was stored in 20% glycerol at -78 °C.The vial was thawed and spores spread on an ISP4 plate for complete sporulation.

Seed medium
The composition of the seed medium (20 g L -1 of D-mannitol, 20 g L -1 of peptone, and 10 g L -1 of dextrose) was prepared with distilled water, and the pH was adjusted to 7.0 prior to sterilization.The medium was dispensed at 50 mL per 250 mL in Belco baffled shaker flasks.A single colony from the agar plate was used as inoculum into each flask of mannitol-peptone media, cultured at 30 °C at 250 rpm for two days.

Production medium
An aliquot (1%) from the seed medium was inoculated into the production medium.The composition of the production medium was similar to that of the seed medium.The production cultures were incubated at 30 °C at 250 rpm for seven days and were then harvested.The supernatants were filtered and partitioned with n-BuOH three times.Both the n-BuOH-soluble and aqueous layers were tested against the yeast two-hybrid assay.

Yeast two-hybrid assay
The inhibition of yeast two-hybrid assay in Saccharomyces cerevisiae was performed as previously described. 2The LZ strain, H10014 of the genotype MATa trp1 leu2 his3 LYS: lexA-HIS3 URA3: lexA-lacZ [pLexA-RAS V12 and pVP16-RAF] was grown on the SD medium agar plate lacking histidine for the generation of stock plate.These cells were grown in the SD medium lacking histidine at 30 °C, 220 rpm, for 2 days.Four hundred microliters of this culture were inoculated into 100 mL of medium containing 1% agar supplemented with (His + ) or without (His -) 130 mmol L -1 histidine.Compounds of known concentration dissolved in MeOH were dispensed onto disks in 20 mL aliquots.The air-dried disks were applied directly onto the plates.The plates were incubated at 30 °C for 48 h, and the zones of inhibition were measured and compared between those on histidine-minus and histidine plates.The yeast strain carrying both pLexA-RAS V12 and pVP16-RAF (strain LZ) will be to grown on the SD-(His -) plate and allowed to express b-galactosidase.Ilimaquinone and geldanamycin were employed as positive controls.Active compounds were then tested at lower concentrations (20-2.5 mg per disk).The assays were performed in triplicate.
b-Galactosidase assay b-Galactosidase activity was assayed according to the literature with modifications. 7One mL culture strains was taken at A 600 between 0.50-1.00and mixed with 20 μL test compounds.The mixture was kept at 30 °C for 2 h and was harvested by centrifugation in a microfuge tube.Yeast pellets were resuspended in 0.7 mL of Z buffer (60 mmol L -1 Na 2 HPO 4 • 7H 2 O, 40 mmol L -1 NaH 2 PO 4 • H 2 O, 10 mmol L -1 KCl, 1 mmol L -1 MgSO 4 • 7H 2 O, 2.7 μL mL -1 b-mercaptoethanol), and followed by adding 20 μL of freshly prepared 0.1% SDS and 50 μL of chloroform into a microfuge tube with vortexing for 15 seconds.At time zero, the assay is initiated by adding 160 μL of 4 mg mL -1 2-nitrophenyl-b-D-galactopyranoside to each microfuge tube.After 20-60 min at 30 °C, the reactions were terminated by the addition of 0.4 mL of 1mol L -1 Na 2 CO 3 and the liberated p-nitrophenol was measured at A 420 nm.The b-galactosidase activity expressed in Miller Units (MU) which is calculated with formula below: Miller Unit = (A 420 × 1000)/(A 600 × t × V) t = reaction time (minutes); V = reaction volume in mL, equals to 1 if there is no dilution of cells.
Cells treated with extracts with less than ½ MU of the control cells are scored as positive. 7
All isolates were evaluated for their inhibitory activities of Ras-Raf-1 interaction in the yeast two-hybrid assay (Table 2) according to a literature method. 3In brief, we looked for a compound giving a clear zone of inhibition (ZOI) that inhibited growth of LZ cells in the SD His -plates and not in the SD His + plates, together with inhibition of b-galactosidase activity.This would indicate the compound is an inhibitor of Ras-Raf-1 interaction.Compounds 6 and 8 showed high potency with 10 and 25 mm clear ZOIs on SD His -and inactive on SD His + at 2.5 mg per disk, respectively, whereas all other compounds 1-5, 7, 9-14 were inactive.In addition, both compounds gave a clear ZOI that inhibited growth of LZ cells in the SD His - plates and not in the SD His + plates, and together with inhibition of b-galactosidase activity, indicated 6 and 8 were inhibitors of Ras-Raf-1 interaction.The potency of these compounds was comparable to that of doxorubicin and geldanamycin but higher than those of the positive controls.Cycloheximide (6) showed the strongest activity against b-galactosidase with 79.8% inhibition of b-galactosidase activity whereas cyclo(DAla-L-Val) (8) exhibited a weaker effect (62.7%).Geldanamycin, a heat shock protein 90 (HSP90) inhibitor and ilimaquinone were employed as positive controls and gave 82.3%, and 96.1% inhibition of b-galactosidase activity, respectively.Ilimaquinone, previously isolated from the marine sponge Hippospongia metachromia, inhibited tyrosine kinase activity by 87% at a concentration of 1 mg mL -1 . 18Cycloheximide (6) was isolated from the cultures of Streptomyces griseus and has been demonstrated to inhibit protein synthesis in mammalian systems. 3,19AH-135Y (5) has been reported from Streptomyces albovinaceus is reported to have antiherpetic, cytotoxic and antifungal activities. 9any terrestrial yeast, lichens, and fungi are known to produce diketopiperazines by condensation of two amino acids.Natural diketopiperazines show a remarkable preference for proline as a building block. 20They consist mostly of two L-amino acids, but DL-, LD-, and DD-isomers can be generated by non-enzymatic epimerization, possibly accelerated by steric effects. 21In the literature there are two report of DD-enantiomers of diketopiperazines as natural compounds. 22,23The DD-enantiomers exhibited

Table 1 .
13 (500 MHz) and13C (125 MHz) NMR Assignments of compounds 1-3 in CD 3 OD (d, mult, J in Hz) J 17.8, 5.3 Hz, H-8b) from the COSY experiments.The remaining resonances observed in the 1 H and13C NMR spectra of 3 were attributable to a glycosyl unit.The 1 H and13C NMR spectroscopic data (Table1) displayed resonances characteristic for a glycosyl moiety containing five oxymethine groups at d H /d C 4.35/105.8

Table 2 .
Yeast two-hybrid screening of compounds 6 and 8 a Ilimaquinone gave 11 mm zone of inhibition on SD His -but not on SD His + at 80 mg per disk.na indicates not active.