A New Geldanamycin Analogue from Streptomyces hygroscopicus

A new geldanamycin analogue was isolated from Streptomyces hygroscopicus A070101. The structure was elucidated as 11-methoxy-17-formyl-17-demethoxy-18-O-21-O-dihydrogeldanamycin (1) on the basis of extensive 1D and 2D NMR as well as HRESI-MS spectroscopic data analysis. Compound 1 showed considerable cytotoxicity (SRB) against human cancer cell lines (breast cancer MCF-7, skin melanoma SK-MEL-2 and lung carcinoma COR-L23).


Introduction
Steroid hormone receptors are generally intracellular receptors and initiate signal transduction for steroid which lead to changes in gene expression over a time period ranging from hours to days [1]. During the translation, steroid receptors are assembled into a multi-protein complex containing hsp90 (one of the most abundant proteins expressed in cells) [2], p23, an immunophilin, and often some hsp70 [3]. Geldanamycin analogues were found to bind to hsp90 and disrupt its function, impedes dexamethasone-dependent trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus, led many of them are protooncogenic and play a prominent role in cancer [4]. For example, geldanamycin and its analog 17-AAG showed significant cytotoxicity against human cancer cell line SKBr3 (IC 50 = 41 and 33 nM, respectively) [5]. During our continued work on bioactive bacteria and fungi, a Streptomyces hygroscopicus strain was isolated from the soil of Chang-Bai Mountain. A new geldanamycin analogue was isolated from a 20 L fermentation of this strain. Its structure was elucidated as 11-methoxy-17-formyl-17-demethoxy-18-O-21-O-dihydrogeldanamycin (1, Figure 1) on the basis of extensive 1D and 2D NMR as well as HRESI-MS spectroscopic data analysis. Compound 1 was tested with five-day in vitro SRB cytotoxicity against human tumors cell lines and showed considerable cytotoxicity against human cancer cell lines (breast cancer MCF-7, skin melanoma SK-MEL-2 and lung carcinoma COR-L23).

Characterization of compound 1
The molecular formula of compound 1 was determined as C 30 H 42 N 2 O 9 on the basis of its HR-ESI-MS (m/z 575.2981 [M+H] + , calcd. 575.2969) and NMR data ( Table 1). Analysis of the 13 C-NMR spectrum and DEPT experiments, allowed the identification of seven methyl groups, two methylenes, eleven methines and ten quaternary carbons. Analysis of the 1 H-1 H COSY ( Figure 2) and HMQC spectra suggested the presence of two 1 H-1 H spin systems: H 3 -H 4 -H 5 -H 6 -H 7 , H 9 -H 10 -H 11 -H 12 -H 13 -H 14 -H 15 . The chemical shifts of the protons and carbons (Table 1) were similar to those of the previously reported compound, 17-formyl-17-demethoxy-18-O-21-O-dihydrogeldanamycin, a geldanamycin analogue isolated from recombinant S. hygroscopicus strain [6]. The main differences between the two metabolites concerned a newly appeared methoxyl group [δ C 57.2, δ H 3.37 (s)] in compound 1. The location of the methoxyl group was established taking into account the correlation observed between 11-OCH 3 (δ 3.37) and C-11 (δ 156.8) in the HMBC experiment of 1 ( Figure 2). The coupling constant between the H5 and H6 (11.2 Hz), H9 and H10 (8.8 Hz), were similar to the literature values 10.4 Hz and 9.6 Hz [6], respectively, led to determine the relative stereochemistry same as reported compound KOSN 1645.  The biosyntheses of geldanamycin analogues were reported to be involved in the assembly of 3-amino-5-hydroxybenzoic acid (AHBA) as a starter unit, following elongation with the acyl-Coenzyme A substrates malonyl-CoA, methylmalonyl-CoA, and 2-methoxymalonyl-ACP, the polyketide intermediate undergoes intra-molecular lactamization by gdmF to form progeldanamycin [5,7] (Figure 3). The compound 1 isolated in this study and previously reported herbimycin A [6], proposed that an O-methylation step exist after the formation of polyketide backbone which may lead to identify a new O-methyltransferase. To prove this hypothesis, mutant lines could be established for screening of this 11-O-methyltransferase.

General
The 1 H-and 13 C-NMR spectra were measured on a Bruker Avance DRX 500 NMR spectrometer in DMSO-d 6 , using TMS as an internal standard. Chemical shifts (δ) are expressed in parts per million (ppm), with the coupling constants (J) reported in Hertz (Hz). The HR-ESI mass spectrum was obtained from a MDS SCIEX API QSTAR-MS instrument. TLC was performed with silica gel plates (Macherey -Nagel, SilG / UV254, 0.20mm); Semi-preparative HPLC was carried out with Agilent 1100 on a Zorbax C 18 column (250 x 10 mm, Phenomenex, Torrance, CA), UV absorption data (λ 280 ) were analyzed with Agilent Chemstation Ver 8.01. All solvents used in this study were HPLC grade, purchased from the Chinese Chemical Group, Beijing, China.

Extraction and isolation of compound 1
The lyophilized culture broth was extracted with 80% EtOH at room temperature. The extract was concentrated under reduced pressure to give the pale brown residue (625 mg) that was fractionated by reverse phase (C-8) chromatography using H 2 O, aqueous MeOH (30%, 60%, 90%) and MeOH to give four fractions: the 90% MeOH fraction was further fractionated on a Sephadex LH-20 column (CHCl 3 : MeOH=1:1) and then purify by semi-preparative HPLC to yield compound 1 (3.6 mg, 0.18 mg/L, whereas yield of geldanamycin was reported as ~10 mg/liter) [6].

In vitro cytotoxicity assays
Five-day in vitro SRB cytotoxicity tests against human tumors cell lines were carried out at the Cell Culture Laboratory, Pharmaceutical College, Jilin University, using modified protocols for MCF-7 (breast cancer), SK-MEL-2 (skin melanoma) and COR-L23 (lung carcinoma), the normal cells were used as control [12]. Generally, 5x10 3 /mL cells were placed in a 24-well plate and treated with compound 1. The plate was incubated at 37 ºC for 5 days. Then the medium was removed from the 24-well plate, and 10% ice-cold TCA (trichloroacetic acid, 1 mL) was added. The plate was kept at 4 °C for two hours after which was washed four times with cold water, then stained with SRB (Sulforhodamine B, Sigma St. Louis, MO, USA). After washing with 1% acetic acid, the bound dye was solubilized with Tris base A (Sigma) and 100 μL of each sample were transferred into a 96-well plate, and then read at 492 nm.