JBIR-14, a highly oxygenated ergostane, from Isaria sp. NBRC 104353

Many proteins linked with accelerated proliferation of human cancer cells repress the growth of yeast.^{1, 2, 3} The inhibitors of these human proteins can restore the inhibition of yeast proliferation with these human proteins, such as Cdk4, PTEN and poly(ADP-ribose) polymerase;⁴ therefore, these observations have led to the development of a cell-based high-throughput screening system for anticancer drugs. Our group has revealed that a previously uncharacterized protein, termed dynAP (dynactin-associating protein), inhibits the growth of a mutant budding yeast lacking Mad2, a principal component of mitotic checkpoint, but does not affect the growth of wild-type yeast.⁵ DynAP is localized in Golgi and plasma membrane and interacts with dynactin components; it thus forms a complex and acts as a minus end-directed microtubule motor. Furthermore, dynAP expresses in approximately 50% of human cancer cell lines, contrary to its low-level expression in normal cells. Thus, dynAP could be a new target of anticancer drug discovery.

Inhibitors of dynAP could be discovered by monitoring the restoration of dynAP-induced growth inhibition of the mutant yeast lacking Mad2. In the course of our screening program for inhibitors of dynAP, the culture extract of an entomopathogenic fungus, Isaria sp. NBRC 104353, exhibited the restoration of dynAP-induced growth inhibition. We isolated a new steroidal compound, named JBIR-14 (1), by activity-guided isolation from the culture extract of Isaria sp. NBRC 104353 (Figure 1a). We report herein the fermentation, isolation and structure elucidation of 1.

Figure 1

(a) Structure of 1. (b) Key correlations of ¹H-¹H double-quantum-filtered-COSY (bold line) and heteronuclear multiple bond correlation (arrow, proton to carbon) of 1. (c) Rotating-frame Overhauser enhancement spectroscopy correlations of 1.

Isaria sp. NBRC 104353, purchased from the National Institute of Technology and Evaluation (Tokyo, Japan), was cultivated in 50 ml test tubes containing 15 ml of potato dextrose broth (24 g l^–1 potato dextrose; BD Biosciences, San Jose, CA, USA). The test tubes were shaken in a reciprocal shaker (355 r.p.m.) at 27 °C for 3 days. Aliquots (1 ml) of the culture were transferred to 100 ml Erlenmeyer flasks containing a medium consisting of 3 g oatmeal (Quaker, Chicago, IL, USA) and 10 ml V8 Mix Juice (Campbell Soup Company, Camden, NJ, USA) and were incubated in static culture at 27 °C for 14 days.

The culture (20 flasks) was extracted with 80% aq. Me₂CO. After concentration in vacuo, the aqueous concentrate was extracted with EtOAc (100 ml × 3). The organic layer was dried over Na₂SO₄ and then evaporated to dryness. The dried residue (0.58 g) was subjected to normal-phase medium-pressure liquid chromatography (MPLC; Purif-Pack SI 60 μm, size: 60 (26.5 i.d. × 100 mm), Moritex, Tokyo, Japan) and eluted with a stepwise system of n-hexane–EtOAc and CHCl₃–MeOH, successively, to yield an active fraction (73.0 mg) in CHCl₃–MeOH (19:1) eluate. The active fraction was rechromatographed on the normal-phase column (Purif-Pack SI 60 μm, size: 20 (20 i.d. × 60 mm) with CHCl₃–MeOH (99:1, 49:1, successively). Finally, the active fraction (9.5 mg) was purified by preparative reverse-phase HPLC using a Senshu Pak PEGASIL ODS column (20 i.d. × 150 mm; Senshu Scientific, Tokyo, Japan) developed with 80% MeOH–H₂O including 0.1% formic acid (flow rate: 10 ml min^–1) to yield 1 (3.4 mg, retention time 19.7 min).

Compound 1 was obtained as a colorless amorphous solid ([α]²⁴_D+4.0°, c 0.12, in MeOH; UV λ_max 240 nm, sh, in MeOH) and its molecular formula was determined to be C₂₈H₄₂O₆ by HR-electrospray ionization-MS (m/z 475.3078 [M+H]⁺, calcd for C₂₈H₄₃O₆, 475.3060). The IR (ν_max 1714 cm¹) spectra of 1 suggested the presence of a carbonyl group. The direct connectivity between each proton and carbon was established by a heteronuclear single-quantum coherence spectrum. The ¹³C and ¹H NMR spectral data for 1 are shown in Table 1. NMR spectra show 28 signals, including six methyl signals (C-18, δ_C 20.13, δ_H 1.37; C-19, δ_C 18.2, δ_H 0.89; C-21, δ_C 17.6, δ_H 1.18; C-26, δ_C 20.07, δ_H 0.98; C-27, δ_C 18.1, δ_H 0.94; C-28, δ_C 12.7, δ_H 0.96). Four partial structures were established by a double-quantum-filtered-COSY spectrum, together with a constant time-heteronuclear multiple bond correlation⁶ spectrum as follows:

Table 1 ¹³C and ¹H NMR data for 1

The sequence from methylene protons 1-H (δ_H 2.03, 1.55) to a methine proton 9-H (δ_H 3.06) through methylene protons 2-H (δ_H 1.93, 1.52), an oxymethine proton 3-H (δ_H 3.70), methylene protons 4-H (δ_H 2.52, 2.27), two olefinic protons 6-H (δ_H 6.33) and 7-H (δ_H 5.77) including allylic couplings between 4-H and 6-H and between 7-H and 9-H was established as shown in Figure 1b. The ¹H–¹³C correlations from a methyl proton 19-H (δ_H 0.89) to a methylene carbon C-1 (δ_C 37.9), an olefinic carbon C-5 (δ_C 140.1), a methine carbon C-9 (δ_C 56.8) and a quaternary carbon C-10 (δ_C 38.4) observed in the heteronuclear multiple bond correlation spectrum of 1 revealed a 9-methyldecalin-2,4(10)-dien-6-ol structure (Figure 1b). The sequence from a methyl proton 21-H (δ_H 1.18) to a methyl proton 26-H (δ_H 0.98) through a methine proton 20-H (δ_H 1.67), two epoxy protons 22-H (δ_C 62.0, δ_H 2.80) and 23-H (δ_C 61.4, δ_H 2.90), a methine proton 24-H (δ_H 1.34) and a methine proton 25-H (δ_H 1.83), together with spin couplings between 24-H and a methyl proton 28-H (δ_H 0.96) and between 25-H and a methyl proton 27-H (δ_H 0.94), established a 3,4-epoxy-5,6-dimethylhept-2-yl substructure (Figure 1b). The presence of an oxacyclopropane ring at the positions of C-22 and C-23 was determined by their characteristic ¹³C shifts, and its stereochemistry was determined to be cis by their coupling constant (J=4.4 Hz). In a similar manner, ¹H–¹H spin couplings for two fragment structures—a hydroxymethine at C-12 (δ_H 4.10; OH: δ_H 3.49) and a 2-hydroxypropane composed of 15-H (δ_H 1.73, 1.62) through 16-H (δ_H 4.38; OH: δ_H 4.11) to 17-H (δ_H 1.53)—were observed as shown in Figure 1b. Long-range couplings from the two methine protons 9-H and 12-H and from the hydroxyl proton 12-OH to a carbonyl carbon C-11 (δ_C 214.7); from a methyl proton 18-H (δ_H 1.37) to two methine carbons C-12 (δ_C 84.3) and C-17 (δ_C 53.6) and to two quaternary carbons C-13 (δ_C 58.1) and C-14 (δ_C 83.7); and from a hydroxyl proton 14-OH (δ_H 4.48) to C-8 (δ_C 132.5), C-14 and a methylene carbon C-15 (δ_C 44.2) elucidated the connectivity among the three substructures, decalin, hydroxymethine and hydroxypropane, in the steroid nucleus. Finally, the remaining 3,4-epoxy-5,6-dimethylhept-2-yl substructure was determined to be attached to C-17 as revealed by ¹H–¹³C long-range couplings from 20-H, 21-H and 22-H to C-17. Thus, the planar structure of 1 was determined as shown in Figure 1a.

Relative configuration was assigned on the basis of coupling constants and the analysis of a rotating-frame Overhauser enhancement spectroscopy experiment. The large coupling constants for J_2βH,3H (11.0 Hz) and J_3H,4βH (11.0 Hz) and for the rotating-frame Overhauser enhancement spectroscopy correlations (Figure 1c) between 1-αH and 3-H; between 2-βH and 19-H; and between 4-βH and 19-H indicated that ring-A should be in a chair conformation with the hydroxyl groups at C-3 in β-equatorial orientation and with the methyl group (C-19) at C-10 in β-axial orientation. On the other hand, the rotating-frame Overhauser enhancement spectroscopy correlations between 1-αH and 9-H; between 9-H and 15-αH; and between 9-H and 17-αH revealed that the ring junction proton 9-H is located in α-axial orientation. Further, the rotating-frame Overhauser enhancement spectroscopy correlations between 12-H and 18-H; between 12-OH and 14-OH; between 14-OH and 18-H; between 14-OH and 15-βH; between 14-OH and 16-OH; between 18-H and 20-H; and between 18-H and 21-H revealed that the three hydroxyl groups at C-12, C-14 and C-16, the methyl group (C-18) at C-13 and the side chain at C-17 are in β-orientation, but that of C-28 was not determined. Thus, the structure of 1 was established as (3β,12β,14β,16β)-22,23-epoxy-3,12,14,16-tetrahydroxyergosta-5,7-dien- 11-one (Figure 1).

Compound 1 restored the dynAP-induced growth inhibition of Mad2-lacking mutant yeast at a concentration of 25 μM (Figure 2). Furthermore, compounds related to 1 induced dynAP-mediated Golgi fragmentation and apoptosis in human cancer cells.⁵ Thus, 1 may serve as an important compound for developing anticancer drugs and also as a valuable tool for conducting studies on the action mechanism of dynAP. The detailed biological activity and the structure–activity relationship of 1 will be reported elsewhere.⁵

Figure 2

Recovery of the dynAP-induced growth inhibition of Mad2-lacking mutant yeast by 1. Exponentially growing Mad2-lacking mutant cells harboring the empty vector or dynAP expression vector were washed and adjusted for their cell density, followed by incubation in galactose medium containing 1 in DMSO or DMSO alone. After 48 h, growth recoveries were calculated by measuring the OD of cell cultures. OD_vec is OD of mutant cells harboring the empty vector treated with DMSO. OD_dynAP is OD of dynAP-expressed mutant treated with 1.