Bioorganic & Medicinal Chemistry 22 (2014) 2912–2918

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Anti-inflammatory drimane sesquiterpene lactones from an Aspergillus species Silke Felix a, Louis P. Sandjo b, Till Opatz b,⇑, Gerhard Erkel c,⇑ a

Institute of Biotechnology and Drug Research (IBWF), Erwin-Schrödinger-Strasse 56, D-67663 Kaiserslautern, Germany Institute of Organic Chemistry, University of Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany c Department of Molecular Biotechnology and Systems Biology, University of Kaiserslautern, Paul-Ehrlich-Strasse 23, D-67663 Kaiserslautern, Germany b

a r t i c l e

i n f o

Article history: Received 18 February 2014 Revised 7 April 2014 Accepted 8 April 2014 Available online 18 April 2014 Keywords: CXCL10 Inhibitor Inflammation Fungal metabolites

a b s t r a c t IFN-c inducible protein 10 (IP-10, CXCL10) is a 10 kDa chemokine, which is secreted from various cell types after exposure to pro-inflammatory stimuli. This chemokine is a ligand for the CXCR3 receptor and regulates immune responses by activating and recruiting leukocytes such as T cells, eosinophils, monocytes, and NK cells to sites of inflammation. Altered expression of CXCL10 has been associated with chronic inflammatory and infectious diseases and therefore CXCL10 represents a promising target for the development of new anti-inflammatory drugs. In a search for inhibitors of CXCL10 promoter activity, three structurally related drimane sesquiterpene lactones (compounds 1–3) were isolated from fermentations of an Aspergillus species. Compounds 1 and 2 inhibited the IFN-c/TNF-a/IL-1b induced CXCL10 promoter activity in transiently transfected human DLD-1 colon carcinoma cells in a dose-dependent manner with IC50 values of 12.4 lM for 1 and 55 lM for 2, whereas 3 was devoid of any biological activity. Moreover, compounds 1 and 2 reduced CXCL10 mRNA levels and synthesis in IFN-c/TNF-a/IL-1b stimulated DLD-1 cells. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction CXCL10/IFN-c-inducible protein 10 (IP-10), a member of the CXC chemokine subfamily, is a small molecule that is secreted mainly by monocytes, dendritic cells, fibroblasts, and endothelial cells in response to diverse stimuli, such as IFN-c, lipopolysaccharide (LPS), interleukin-1b (IL-1b), and viral infection.1,2 CXCL10 exerts its function as ligand for the CXCR3 receptor and regulates immune responses by activating and recruiting leukocytes such as T cells, eosinophils, monocytes, and NK cells to the sites of inflammation.3 Local CXCL10 expression in inflammation sites leads to a potent chemotaxis for IFN-c producing Th1 cells. This in turn results in a positive feed-back loop with resident cells that further release CXCL10 upon IFN-c stimulation which promotes Th1-type cytokine production while down-regulating Th2 cytokines.4 The expression of CXCL10 is mainly regulated at the transcriptional level by the activation of the NF-jB, STAT1 and IRF-3 transcription factors after cytokine induction and engagement of the Toll-like receptors (TLRs) by LPS.5,6 Although CXCL10 plays an essential role in host defense against bacterial and some viral infections, increased serum and/or tissue

⇑ Corresponding authors. Tel.: +49 631 205 2881; fax: +49 631 205 2999 (G.E.). E-mail addresses: [email protected] (T. Opatz), [email protected] (G. Erkel). http://dx.doi.org/10.1016/j.bmc.2014.04.015 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

expression of CXCL10 have been observed in different autoimmune diseases such as asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel diseases (IBD), rheumatoid arthritis (RA) as well as reactions associated with allograft rejection.7,8 Blocking CXCL10 with neutralizing antibodies in a murine model of colitis reduced colonic Th1 recruitment and downregulated the mRNA levels of important pro-inflammatory cytokines, such as TNF-a and IFN-c, and chemokines, such as CXCL9 and CXCL10.9 In addition, phase II clinical trials with a anti-CXCL10 antibody in patients with rheumatoid arthritis or moderately-toseverely active ulcerative colitis showed significant clinical activities.10,11 Therefore, inhibition of the CXCL10/CXCR3 axis represents an attractive target for the development of new therapeutics against various chronic inflammatory and autoimmune diseases.12 In order to search for new anti-inflammatory compounds, we used a human CXCL10 promoter dependent transcriptional reporter that used the CXCL10 gene regulatory sequence driving a luciferase reporter gene. In a screening of fungal extracts inhibiting the inducible CXCL10 promoter activity, we found that cultures of Aspergillus sp. produced two drimane sesquiterpene lactones with inhibitory activity on CXCL10 promoter dependent luciferase reporter gene expression in transiently transfected DLD-1 human colon carcinoma cells together with a structurally related drimane sesquiterpene lactone devoid of anti-inflammatory activities (see Fig. 1). In this paper, the fermentation, structure elucidation, and

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some biological properties of the three fungi-derived sesquiterpenoids are described.

showed high similarity to an uncultured soil fungus (100% in 554 bp, Genbank accession no. GQ921753.1) and to Aspergillus janus (98% in 575 bp, Genbank accession no. EU021598). For maintenance, the fungus was grown on HMG agar slants consisting of: 1% malt extract, 1% glucose, 0.4% yeast extract, pH 5.5 and 2% agar for solid media. Fermentation was performed in a Biolafitte C-6 fermenter containing 20 L of HMG medium with aeration (3 L air/min) and agitation (120 rpm) at 22 °C. The compounds were isolated from the culture fluid by bioactivity-guided fractionation using the CXCL10 transcriptional reporter assay in TNF-a/IL-1b/IFN-c stimulated DLD-1 cells as described below. The fermentation was stopped after 14 days, when the glucose in the medium was depleted and the inhibition of the CXCL10 promoter activity reached a maximum. The culture fluid was separated from the mycelium by filtration, extracted twice with an equal volume of ethyl acetate (EtOAc) and dried over Na2SO4. The solvent was evaporated in vacuo and the crude extract (3.8 g) was separated by chromatography on silica gel (Merck 60). Elution with cyclohexane/EtOAc (50:50 v/v) resulted in 510 mg of an enriched fraction which was further purified by preparative HPLC (Macherey–Nagel, Düren, Germany; Nucleosil RP18; column 21  250 mm, flow 20 mL/min) with MeCN/H2O (60:40) as eluent to yield compound 1 (10 mg, tR: 15 min), compound 2 (40 mg, tR: 14 min) and compound 3 (4 mg, tR: 12 min).

2. Materials and methods 2.1. General procedures 1D and 2D NMR data were recorded with a Bruker AVANCE III-600 MHz spectrometer equipped with a 5 mm inverse TCI cryoprobe using standard pulse sequences. APCI-MS spectra were measured from a solution of the analyte in MeCN/H2O with a Hewlett Packard MSD 1100 using an evaporator temperature of 400 °C, a drying gas temperature of 350 °C at a flow of 6 L/h (N2). In positive ionization mode, the capillary voltage amounted to 3.5 kV, the corona discharge current was 4 lA. In negative ionization mode, the capillary voltage amounted to 2.2 kV, the corona discharge current was 12 lA. HR-ESI-MS data were measured from a solution of the analyte in acetonitrile with a Waters Q-TOF-Ultima 3 equipped with a LockSpray interface (tri-n-octylamine as external reference). IR and UV spectra were measured with a Bruker IFS48 FTIR spectrometer and a Perkin-Elmer Lambda-16 spectrophotometer, respectively. The optical rotation was measured on a Perkin-Elmer 241 polarimeter at 578 nm and 546 nm and extrapolated to 589 nm using Drude’s equation.13

2.2.1. (5R,5aS,9aS)-9b-Hydroxy-6,6,9a-trimethyl-1-oxo1,3,5,5a,6,7,8,9,9a,9b-decahydro-naphtho[1,2-c]furan-5-yl hexanoate (1) 1 Yellowish oil, [a]20 ) 3433, D 209.8 (c 0.37, CD3OD); IR (m cm 2953, 1778, 1732, 1464, 1339, 1294; HR-ESI-MS: m/z 387.2140 [M+Na]+, calcd for [C21H32O5+Na]+, 387.2142; APCI-MS neg. mode m/z 364.2 [M]–, 248 [MH3C(CH2)4CO2H]–, pos. mode m/z 347.3 [MH2O+H]+, 249.1 [MH3C(CH2)4CO2H+H]+, 231.1 [MH3C(CH2)4CO2HH2O+H]+. NMR data (see Table 1).

2.2. Producing organism, fermentation and isolation of compounds 1–3 Aspergillus sp. strain IBWF002-96 was obtained from the culture collection of the Institute of Biotechnology and Drug Research (IBWF e.V.), Kaiserslautern, Germany. The strain IBWF002-96 showed all characteristics of the genus Aspergillus, the species however could not be unequivocally determined. ITS sequence analysis of the ITS1-5.8S rDNA-ITS2 region of nuclear DNA14

Table 1 H (600 MHz) and

1

13

C NMR (150 MHz) data of compounds 1–3

Position

Compound 1, CD3OD dH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 60 OH-1 OH-9 OH-11

1.97 2.01 1.55 1.72 1.32 1.42 — 2.04 5.63 5.84 — — — — 4.74 4.93 1.12 1.18 0.99 — 2.33 1.63 1.32 1.33 0.91 — — —

(1H, (1H, (1H, (1H, (1H, (1H,

m) dd, 4.2, 13.6) m) qt, 3.6, 13.6) m) m)

(1H, d, 4.8) (1H, m) (1H, m)

(1H, (1H, (3H, (3H, (3H,

dt, 1.3, 12.6) dt, 2.4, 12.6) s) s) s)

(2H, (2H, (2H, (2H, (3H,

m) m) m) m) t, 7.0)

Compound 2, CD3CN

Compound 3, CD3CN

dC

dH

dC

dH

dC

31.1

4.18 (1H, m)

70.1

4.02 (1H, m)

70.9

19.0

1.66 (2H, m)

27.0

27.2

46.02

1.34 (2H, m)

42.5

1.58 (1H, m) 1.64 (1H, m) 1.35 (2H, m)

34.8 46.04 67.9 123.3 138.0 75.2 39.0 176.6 69.9

— 1.86 5.55 5.91 — — — — 4.87 5.04 1.03 1.13 0.96 — 2.31 1.60 1.29 1.30 0.88 4.78 4.41 —

34.3 45.3 67.1 124.1 135.9 77.3 43.9 179.4 71.5

— 1.90 5.53 5.61 — — — 5.32 4.15 4.49 1.08 1.14 0.95 — 2.29 1.59 1.29 1.30 0.88 3.26 3.59 4.96

34.0 46.3 67.9 119.5 142.9 77.7 44.7 99.3 67.3

18.9 25.2 33.0 174.5 35.7 25.6 32.4 23.4 14.3 — — —

(1H, d, 4.9) (1H, m) (1H, m)

(1H, (1H, (3H, (3H, (3H,

dt, 1.3, 12.8) dt, 2.5, 12.8) s) s) s)

(2H, (2H, (2H, (2H, (3H, (1H, (1H,

m) m) m) m) t, 7.0) d, 1.1) s)

12.9 24.8 32.2 173.7 35.3 25.2 31.9 23.0 14.2 — — —

(1H, d, 4.7) (1H, m) (1H, m)

(1H, (1H, (1H, (3H, (3H, (3H,

br s) dt, 1.6, 13.3) dt, 2.3, 13.3) s) s) s)

(2H, (2H, (2H, (2H, (3H, (1H, (1H, (1H,

m) m) m) m) t, 7.1) d, 3.7) br s) d, 7.6)

42.9

11.9 25.0 32.7 173.7 35.4 25.2 31.9 23.0 14.2 — — —

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2.2.2. (5R,5aS,9R,9aR,9bR)-9,9b-Dihydroxy-6,6,9a-trimethyl-1oxo-1,3,5,5a,6,7,8,9,9a,9b-decahydronaphtho[1,2-c]furan-5-yl hexanoate (2) 1 Colourless oil, [a]20 ) 3339, D 225.2 (c 0.25, CD3CN); IR (m cm 2953, 1733, 1464, 1372, 1239; HR-ESI-MS: m/z 403.2107 [M+Na]+, calcd for [C21H32O6+Na]+, 403.2102; APCI-MS neg. mode m/z 380.2 [M]–, 263 [MH3C(CH2)4CO2HH], pos. mode m/z 381.2 [M+H]+, 345.2 [M2  H2O+H]+, 247.1 [MH3C(CH2)4CO2HH2O+H]+. NMR data (see Table 1). 2.2.3. (1R,5R,5aS,9R,9aR,9bR)-1,9,9b-Trihydroxy-6,6,9atrimethyl-1,3,5,5a,6,7,8,9,9a,9b-decahydronaphtho[1,2-c]furan5-yl hexanoate (3) 1 Colourless oil, [a]20 ) 3420, D 218.2 (c 0.11, CD3CN); IR (m cm 2956, 1730, 1464, 1287; HR-ESI-MS: m/z 405.2256 [M+Na]+, calcd for [C21H34O6+Na]+, 405.2251; NMR data (see Table 1).

cxcl10 (Genbank Accession NM001565) forward: 50 -TGAGCCTACAGCAGAGGAA-30 and reverse: 50 -TACTCCTTGAATGCCACTTAGA30 (size of the PCR product is 102 bp), gapdh (Genbank Accession M33197) forward: 50 -CCTCCGGGAAACTGTGG-30 and reverse: 50 AGTGGGGACACGGAAG-30 (size of the PCR product is 140 bp), inos (Genbank Accession NM00625.4) forward: 50 -GCAGGTCACTTATGTCACTTATC-30 and reverse: 50 -GTTCTCAAGGCACAGGTCTC-30 (size of the PCR product is 127 bp), il-1b (Genbank Accession NM00576) forward: 50 -AAGCTGAGGAAGATGCTG-30 and reverse: 50 -ATCTACACTCTCCAGCTG-30 (size of the PCR product is 390 bp), tnf-a (Genbank Accession M10988) forward: 50 -TCTTCTGCCTGCTGCACTTTGG-30 and reverse: 50 -ATCTCTCAGCTCCACGCCATTG-30 (size of the PCR product is 224 bp). Relative mRNA amounts were determined using the mathematical model for relative quantification in real-time PCR as proposed by Pfaffl.17 2.7. Proteome profiler

2.3. Cell culture DLD-1 (DSMZ ACC278) cells were maintained in RPMI 1640 medium with 25 mM HEPES buffer and 2 mM L-glutamine, supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 lg/mL streptomycin at 37 °C and 5% CO2. 2.4. Reporter gene assays The NF-jB driven reporter plasmid pNF-jB-Luc and the Stat1 driven plasmids pGAS-TA-Luc, which contains two copies of the Stat1 enhancer element, and pISRE-Luc, which contains five copies of the ISRE enhancer element, were obtained from Clontech (SaintGermain-en-Laye, France). The plasmid pRL-CMV for normalizing transfection efficiency was obtained from Promega (Dual-Luciferase-Reporter-Assay). The 972 bp human CXCL10 promoter driven reporter plasmid has been previously reported.15 Transient transfections of DLD-1 cells were performed using the liposomal formulation ‘Lipofectamine 2000’ purchased from Invitrogen. DLD-1 cells were seeded at 1  105 cells/mL in RPMI 1640 medium containing 10% FCS into 24-well plates to attain 90–95% confluence at time of transfection. One day before transfection the medium was replaced by RPMI 1640 medium containing 0.5% FCS without antibiotics. The lipofection was performed according to the manufacturer´s protocol with 0.8 lg of the indicated reporter plasmids. For induction of luciferase expression the cells were treated with 10 ng/mL TNF-a, 10 ng/mL IFN-c and 5 ng/mL IL-1b (CM), for 24 h. Luciferase activity was measured with a luminometer, using the Dual-Glo Luciferase assay system (Promega, Mannheim, Germany) according to the manufacturer’s instructions. 2.5. Cell viability testing The cytotoxicity of the compounds was determined after 48 h using a XTT-based cell viability assay as previously described by Roehm et al.16 2.6. Quantitative real-time polymerase chain reaction analysis (qRT-PCR analysis) DLD-1 cells were seeded into 6-well plates at a cell density of 5  105 cells/mL in RPMI 1640 medium with 10% FCS and allowed to grow for 24 h. After starving the cells for 16 h in RPMI 1640 medium containing 0.5% FCS, cells were pretreated with or without the test compounds for 1 h and induced with 10 ng/mL TNF-a, 10 ng/mL IFN-c and 5 ng/mL IL-1b for additional 5 h. Untreated samples and samples without stimulation served as controls. The mRNA expression in human DLD-1 cells was analyzed by two-step RT-PCR as described before15 with gene-specific primers for human

DLD-1 cells were starved for 16 h in RPMI 1640 medium with 0.5% fetal calf serum. The cells were seeded at 5  106 cells/mL in 6-well plates, pretreated with or without test compound for 1 h and induced with 10 ng/mL TNF-a, 10 ng/mL IFN-c and 5 ng/mL IL-1b (CM) for additional 16 h. For analyses of cytokine and chemokine production in culture supernatants of DLD-1 cells the ‘Human Cytokine Array Panel A’ array system (R&D Systems, Wiesbaden, Germany) was used according to the manufacturer´s instructions. The ImageJ software was used for the quantification of the protein spots (http://rsb.info.nih.gov/ij). 3. Results and discussion 3.1. Identification and structure elucidation The active compounds 1 and 2 were isolated from the culture fluid extract by bioactivity-guided fractionation as described in the experimental section together with an inactive congener; compound 3 (see Fig. 1). Compound 1 was obtained as a yellowish oil, gave a peak at m/z 387.2140 (calcd 387.2147) in its HR ESI MS spectrum from which the molecular formula [C21H32O5+Na]+ was deduced. The elemental composition was consistent with six double bond equivalents. The IR spectrum showed the presence of OH groups (3433 cm1), and carbonyl groups (1778 and 1732 cm1). The NMR spectra of 1 (Table 1) revealed signals of an alkanoate chain, namely a terminal CH3 group (d 0.91/14.3), four CH2 groups (d 1.32/32.4, 1.33/ 23.4, 1.63/25.6, 2.33/35.7) and a carbonyl (d 174.5). In addition, fifteen resonances including three CH3 groups (d 0.99/33.0, 1.12/18.9, 1.18/25.2), four CH2 groups (d 1.32, 1.42/46.02; 1.55, 1.72/19.0; 1.97, 2.01/31.1; 4.74, 4.93/69.9), three CH groups (d 2.04/46.04, 5.63/67.9, 5.84/123.3) and five quaternary carbons (d 34.8, 39.0, 75.2, 138.0, 176.6) were observed suggesting a hexanoic acid ester of a sesquiterpene. Correlations displayed in the COSY spectrum (Fig. 2) between CH2 protons [(d 1.97, 2.01), (d 1.55, 1.72) and (d 1.32, 1.42)] as well as those observed between CH protons (d 2.04, 5.63 and 5.84) suggested a drimane skeleton. The decalin portion was deduced from the HMBC spectrum (Fig. 2) which revealed correlations of CH3 protons at d 1.12 (H-13) to carbons at d 31.1 (C1), 39.0 (C-10), 46.04 (C-5) and 75.2 (C-9) as well as between CH3 protons at d 1.18 (H-14) and 0.99 (H-15) and carbon atoms at d 46.02 (C-3), 46.04 (C-5) and 34.8 (C-4). Moreover, oxidized carbons C-11 and C-12 formed together with C-8 (d 138.0) and C-9 (d 75.2) a c-lactone ring since HMBC correlations were revealed between H-12 (d 4.74 and 4.93) and C-7 (d 123.3), C-8 (d 138.0), C-9 (d 75.2), and C-11 (d 176.6). The carbonyl of the lactone ring was located at C-11 since weak allylic COSY contacts were observed between protons H-12 (d 4.74 and 4.93) and H-7 (d 5.84). The side

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O 1

2

10

3

4

O 11 OH O OH

11

O OH

13

9 5

8

6

12

1

3

7

H

2'

14 15 O 1'

9

4' 3'

O

5'

12

8

6

H

6'

HO 11 OH O OH 1

7

H

6'

O 1'

12

9 6

7

O

1

6'

O 1' O

2

3

Figure 1. Structures of compounds 1–3.

O

H

O OH

O

O

H

O OH

O

O

O O

H O

O OH

O O

O

2

3

1

Figure 2. COSY (bold) and HMBC (arrow) correlations of compounds 1–3.

chain was attached to C-6 based on further long-range correlations of H-6 (d 5.63) to the carbonyl at d 174.5 (C-10 ). The relative configuration was assigned based on NOESY data (Fig. 3). Thus, spatial correlations were observed between H-15 (d 0.99), H-3 (d 1.32), H-5 (d 2.04), and H-6 (d 5.63). Besides, H-14 (d 1.18) showed similar interactions to protons at d 1.42 (H-3) and 1.72 (H-2) which in turn correlated with H-13 (d 1.12). The aforementioned data suggested compound 1 to contain a trans-decalin moiety in which the relative stereochemical assignment for C-5, C-6 and C-10 was S, R and S, respectively. The stereochemistry at C-9 could not be determined using NMR data. The absolute configuration was tentatively assigned based on the stereochemistry reported for similar scaffolds.18,19 The foregoing data led to identify compound 1 as (5R,5aS,9aS)-9b-hydroxy-6,6,9a-trimethyl-1-oxo-1,3,5,5a,6,7, 8,9,9a,9b-decahydronaphtho[1,2-c]furan-5-yl hexanoate. Compound 2 was obtained as a colourless oil. The molecular formula C21H32O6 was deduced from its HR ESI MS analysis which gave a peak at m/z 403.2107 (calcd for [M+Na]+, 403.2097). The elemental composition required six double bond equivalents as found for compound 1. Both metabolites differed in mass by 16 amu. The IR spectrum of compound 2 showed absorption bands for OH (3339 cm1) and C@O (1733 cm1). NMR data of 2 (Table 1) revealed features similar to those of compound 1 including resonances of an alkanoate chain (d 0.88/14.2, 1.30/23.0, 1.29/31.9, 1.60/25.2, 2.31/35.3, and 173.7). In addition, signals of five quaternary carbon (d 34.3, 43.9, 135.9, 77.3, 179.4), four CH groups (d 4.18/70.1, 1.86/45.3, 5.55/67.1, 5.91/124.1), three CH2 groups (d 1.66/27.0; 1.34/42.5; 4.87, 5.04/71.5) and three CH3 groups (d 1.03/12.9, 1.13/24.8, 0.96/32.2) were observed. Thus, compound

O

H

O OH

H

H

H H

H

O

O

H O O

HO OH

H

O

H O

O

O

O 1

H H

H

O

2 turned to be another drimane sesquiterpene lactone containing an additional OH group compared to its congener 1. The presence of a signal (d 4.78) in the 1H NMR spectrum corresponding to an exchangeable proton supported this assumption. The novel OH group was located at C-1 based on the HMBC correlations (Fig. 2) of H-13 (d 1.03) to C-1 (d 70.1) and C-9 (d 77.3). Moreover, the relative configuration at C-1 was assigned by NOESY data (Fig. 3) which disclosed a correlation between the exchangeable proton (d 4.78) of OH-1 and H-13 (d 1.03). Similarly, the OH group at C9 was anti to Me-13 since its exchangeable proton at d 4.41 showed correlations with H-1 (d 4.18), and H-5 (d 1.86). A detailed analysis of NOESY data in conjunction to those reported in the literature for similar secondary metabolites18,19 led to assign the relative and tentative absolute configuration at C-1, C-5, C-6, C-9, and C-10 to be R, S, R, R, and R, respectively. From the complete assignment, compound 2 was identified as (5R,5aS,9R,9aR,9bR)-9,9b-dihydroxy-6,6,9a-trimethyl-1-oxo-1,3,5, 5a,6,7,8,9,9a,9b-decahydronaphtho[1,2-c]furan-5-yl hexanoate. Compound 3 was obtained as a colourless oil. The molecular formula C21H34O6 was deduced from its HR ESI MS and NMR data (Table 1). The composition corresponded to five double bond equivalents and differed from that of 2 by 2 amu. This assumption was supported by the absence in the 13C NMR spectrum of 3 of a carbonyl at d 179.4 as found for 2 and the presence of an acetalic carbon resonating at d 99.3. Apart from that, the other signals were similar to those of compound 2 including resonances of the alkanoate chain (d 2.29/35.4, 1.59/25.2, 1.29/31.9, 1.30/23.0, 0.88/14.2 and 173.7) and the decalin moiety bearing three CH3 groups (d 1.08/11.9, 1.14/25.0, and 0.95/32.7) and formed by two CH2

2 Figure 3. NOESY correlations of compounds 1–3.

O 3

O

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groups (d 1.58, 1.64/27.2, and 1.35/42.9), four CH groups (d 4.02/ 70.9, 1.90/46.3, 5.53/67.9, and 5.61/119.5) and four quaternary carbons (d 34.0, 44.7, 77.7, and 142.9). Furthermore, a five membered heterocyclic ring was deduced from the HMBC correlations to be formed by the acetal (d 5.32/ 99.3), one of the olefinic carbon (d 142.9), the carbinol (d 77.7) and the oxymethylene (d 4.15, 4.49/67.3). The latter was located in position 12 since allylic coupling constants were observed in the 1H NMR data for the signals at 4.15 and 4.49 ppm (J = 1.6 and 2.3 Hz, respectively). This conclusion was supported by COSY correlations (Fig. 2) of both geminal OCH2-protons to the olefinic proton (d 5.61). As in compounds 1 and 2, compound 3 contained a trans-decalin portion since hydrogens of Me-13 (d 1.08) and Me14 (d 1.14) showed NOESY correlations (Fig. 3) with H-2 at d 1.64 while H-5 (d 1.90) unveiled similar interactions with H-15 (d 0.95). Besides, a strong NOESY contact was observed between H1 (d 4.02) and H-5 (d 1.90) and a weak one with the exchangeable proton of OH-9 (d 3.59). While H-14 correlated with the acetal proton (d 5.32), H-15 showed a NOE contact with H-6 (d 5.53). The aforementioned spatial correlations along with the stereochemistry reported for similar drimane sesquiterpenes18,19 allowed to assign the relative and tentative absolute configuration at C-1, C5, C-6, C-9, C-10, and C-11 to be R, S, R, R, R, and R. Thus, compound 3 was identified as (1R,5R,5aS,9R,9aR,9bR)-1,9,9b-trihydroxy-6,6, 9a-trimethyl-1,3,5,5a,6,7,8,9,9a,9b-decahydronaphtho[1,2-c]furan5-yl hexanoate. A search for the identified structures in the SciFinder database revealed structural matches for compounds 3 and 2 in catalogues of commercial suppliers but a different relative stereochemistry has been assigned in both cases. To the best of our knowledge, no scientific publication exists on any of the three compounds. 3.2. Biological activity For the identification of active compounds and in order to characterize their influence on CXCL10 expression, we transfected human colon carcinoma DLD-1 cells with a full length human CXCL10-promoter dependent transcriptional reporter which reflects the activation of transcription factors binding to recognition sequences in the cxcl10 gene promoter. Treatment of the cells with 10 ng/mL TNF-a, 10 ng/mL IFN-c and 5 ng/mL IL-1b increased luciferase activity 8–10 fold compared to non-stimulated cells. As shown in Figure 4 AB, the inducible CXCL10-promoter activity was dose-dependently inhibited by compounds 1 and 2 with IC50 values of 12.4 lM and 55 lM, respectively. The constitutive activity of the CMV-promoter was not affected up to the highest concentration tested for both compounds indicating that the compounds do not interfere with transcription in a general manner. Interestingly, the closely related compound 3 which differs from the most active compound 1 by the occurrence of an acetalgroup at C-11 on the furan-ring and a hydroxyl-group at C-1 on the drimane scaffold did not show any inhibitory activity on the inducible CXCL10 promoter activity up to 260 lM (data not shown). The response of the 972 bp CXCL10 promoter construct largely depends on the synergistic interaction between transcription factors activated by IFN-c (e.g., Stat1) and TNF-a/IL-1b (e.g., NFjB).5,6,20,21 The NF-jB pathway has been shown to be an essential modulator of the transcription of chemokine genes such as cxcl10.22 We therefore investigated the effect of compounds 1 and 2 on NF-jB driven expression of the reporter gene luciferase in TNF-a/IFN-c/IL-1b (CM) stimulated DLD-1 cells. As shown in Figure 4 AB, they, strongly inhibited the inducible NF-jB-dependent luciferase expression in DLD-1 cells with IC50-values of 10 lM (1) and 68.4 lM (2).

Figure 4. Effect of Compounds 1 (A) and 2 (B) on CXCL10-, CMV-promoter activity, NF-jB-, Stat1- and ISRE-driven reporter gene expression in DLD-1 cells. DLD-1 cells were transiently transfected with the reporter gene constructs pNF-jB-Luc, pGASTA-Luc, pISRE-Luc, pRL-CMV and a human CXCL10-promoter dependent reporter plasmid by using the liposomal formulation ‘Lipofectamine 2000’ and stimulated with TNF-a/IL-1b/IFN-c (CM) for 24 h with and without test compounds. Control (100%): stimulation only. The expression of the reporter genes was determined as described in the experimental section. Data represent the mean ± SEM of at least three independent experiments.

In addition to NF-jB, the response of the 972 bp CXCL10 promoter construct largely depends on three distal interferon stimulated response elements (ISRE) and two Stat recognition sites.5 We therefore investigated the effect of 1 and 2 on Stat-1 and ISRE-driven expression of the reporter gene luciferase in TNF-a/ IFN-c/IL-1b stimulated DLD-1 cells. As shown in Figure 4AB, the ISRE-dependent expression of the reporter gene was inhibited by both compounds with IC50-values of 14.3 lM (1) and 49 lM (2). The Stat1-dependent transcriptional reporter was inhibited in a dose dependent manner with IC50-values of 11.8 lM compound 1 and 50.7 lM compound 2, respectively. In accordance with the CXCL10-promoter assay, the structurally related drimane 3 did not affect NF-jB-, ISRE- and Stat1-dependent reporter gene expression in DLD-1 cells up to 260 lM. To investigate the effect of compounds 1 and 2 on the inducible transcription of the cxcl10 gene, quantitative real-time PCR experiments were performed with total RNA isolated from cells treated with different concentrations of test compounds for one hour and stimulated with TNF-a/IFN-c/IL-1b (CM) for 5 h as described in the materials and methods section. Values are expressed as relative mRNA content of induced versus uninduced cells (100%), and induced and compound treated versus induced and untreated cells, each corrected for the constitutive expressed housekeeping gene

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gapdh as reference determined in the same sample in parallel. As shown in Figure 5, stimulation of DLD-1 cells with TNF-a/IFN-c/ IL-1b resulted in a strong up-regulation (1200-fold; 100%) of mRNA levels for cxcl10. Uninduced cells did not produce detectable amounts of CXCL10 mRNA. Compound 1 at a concentration of 27.5 lM, completely down-regulated the mRNA level of cxcl10 in induced DLD-1 cells, whereas compound 2 was 4-fold less potent significantly reducing CXCL10 mRNA levels at 131.5 lM. The measured mRNA values for gapdh did not vary significantly upon treatment of the cells with TNF-a/IFN-c/IL-1b or test compounds (data not shown). We also investigated the effect of the two active compounds on the expression of selected pro-inflammatory marker genes, which are regulated at the transcriptional level by NF-jB- and Stat transcription factors by real-time PCR experiments.23,24 DLD-1 cells were preincubated for 1 h with test compounds, stimulated with TNF-a/IFN-c/IL-1b (CM) for 5 h and relative mRNA-levels of selected induced genes were determined as described above. Compared to control cells, the mRNA expression of IL-1b, TNF-a and iNOS was strongly up-regulated upon cytokine stimulation and significantly reduced by treatment of the cells with 13.75 lM of compound 1 or 67.75 lM of compound 2 (Fig. 6). Cytotoxic properties of the compounds 1 and 2 were evaluated against DLD-1 cells by measuring the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium (XTT) into a coloured formazan. No significant cytotoxic activities against DLD-1 cells could be observed up to 50 lM for 1 and 158 lM for 2 during a 48 h incubation period (data not shown). We further investigated the influence of compounds 1 and 2 on the inducible synthesis and excretion of pro-inflammatory cytokines and chemokines in DLD-1 cells with an antibody array. As shown in Figure 7, DLD-1 cells stimulated with CM for 16 h mainly express chemokines, such as CXCL10, MIF, IL-8 and CXCL1, and cytokines, such as IL-1b, IL-27, and TNF-a. Treatment of CM stimulated DLD-1 cells with 27.5 lM of compound 1 and 131.5 lM of compound 2 significantly inhibited the synthesis of most of the pro-inflammatory mediators investigated. Drimane sesquiterpenes are frequently occurring metabolites in plants, marine organisms and fungi which exhibit a variety of biological activities.25 The production of drimane sesquiterpenoids has been described in various genera of fungi such as Aspergillus

Figure 5. Effect of compounds 1 and 2 on mRNA levels of cxcl10 in TNF-a/IL-1b/IFNc (CM) stimulated DLD-cells. Values are expressed as ratios (log2) of relative mRNA levels of stimulated (5 h CM) versus unstimulated cells as control and compound pretreated and stimulated versus untreated, stimulated cells, corrected for gapdh as reference determined in the same sample in parallel. Data are shown as mean values ± SEM of three independent experiments.

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Figure 6. Effect of compounds 1 and 2 on mRNA levels of tnf-a, il-1b and inos in TNF-a/IL-1b/IFN-c (CM) stimulated DLD-1 cells. Values represent the relative mRNA levels of stimulated (5 h CM) versus unstimulated cells as control (100%) and compound pretreated and stimulated versus untreated, stimulated cells, corrected for gapdh as reference determined in the same sample in parallel. Data are shown as mean values ± SEM of three independent experiments.

exhibiting cytotoxic or endothelin-receptor antagonistic activities,18,19,26 Marasmius with spore-germination inhibitory activities against plant-pathogenic fungi27 as well as Kuehneromyces and Mniopetalum producing inhibitors of reverse transcriptases.28,29 We identified the fungal drimane sesquiterpene lactones 1 and 2 as inhibitors of CXCL10 expression on the transcriptional and protein level. We demonstrated that they interfere with signal transduction pathways leading to the activation of transcription factors, which participate in the transcriptional activation of the cxcl10 gene, such as NF-jB and Stat1. In addition, the two drimane sesquiterpene lactones 1 and 2 inhibited the expression of proinflammatory marker genes, such as tnf-a, il-1b, cxcl1 and inos in TNF-a/IL-1b/IFN-c stimulated DLD-1 cells. Although compound 1 showed 5-fold higher anti-inflammatory activity than compound 2, compound 3 was completely devoid of any biological activity indicating that the c-lactone ring as well as the lack of the hydroxyl-group on C-1 account for the inflammatory activities of the isolated drimanes. Interestingly, the closely related drimane sesquiterpene lactone SF 002-96-1 which we have recently isolated from fermentations of the same Aspergillus strain as a transcriptional inhibitor of the anti-apoptotic protein survivin did not show any inhibitory activity on CXCL10 promoter activity.30

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spec. strain IBWF002-96, and P. Stark for skillful technical assistance. Supplementary data Supplementary (NMR spectra) data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bmc.2014.04.015. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. Figure 7. Effect of compounds 1 and 2 on cytokine and chemokine production in TNF-a/IL-1b/IFN-c (CM) stimulated DLD-1 cells. DLD-1 cells were pretreated with 27.5 lM 1 and 131.5 lM 2 for 1 h prior to stimulation with CM for 16 h. The supernatant was used to determine the cytokine and chemokine production with a human cytokine array (Proteome Profiler-Human Cytokine Array Panel A, R&D Systems) according to manufacturer´s instructions. A summary of two quantitative analyses (ImageJ) is shown. The data (mean ± SEM) represent relative protein amounts of significantly (>2-fold) induced chemokines and cytokines.

11. 12. 13. 14.

15. 16.

4. Conclusions We identified two new structurally related drimane sesquiterpene lactones, compounds 1 and 2, which reduce the inducible expression of pro-inflammatory genes in human colon carcinoma DLD-1 cells. These compounds were obtained along with an inactive congener, compound 3. Although the exact molecular target remains to be elucidated, the metabolites may serve as leadstructures for the development of new therapeutics for the treatment of chronic inflammatory diseases. Acknowledgements This work was supported by a Grant from the Stiftung Rheinland-Pfalz für Innovation as well as by the Zeiss foundation. We thank Prof. Dr. T. Anke and Prof. Dr. H. Anke for providing Aspergillus

17. 18. 19. 20.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Groom, J. R.; Luster, A. D. Immunol. Cell Biol. 2011, 89, 207. Lee, E. Y.; Lee, Z. H.; Song, Y. W. Autoimmun. Rev. 2009, 8, 379. Groom, J. R.; Luster, A. D. Exp. Cell Res. 2011, 317, 620. Campbell, J. D.; Gangur, V.; Simons, F. E.; HayGlass, K. T. FASEB J. 2004, 18, 329. Spurrell, J. C.; Wiehler, S.; Zaheer, R. S.; Sanders, S. P.; Proud, D. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005, 289, L85. Yeruva, S.; Ramadori, G.; Raddatz, D. Int. J. Colorectal Dis. 2008, 23, 305. Ramana, D.; Sobolik-Delmairea, T.; Richmond, A. Exp. Cell Res. 2011, 317, 575. Romagnani, P.; Crescioli, C. Clin. Chim. Acta 2012, 413, 1364. Hyun, J. G.; Lee, G.; Brown, J. B.; Grimm, G. R.; Tang, Y.; Mittal, N.; Dirisina, R.; Zhang, Z.; Fryer, J. P.; Weinstock, J. V.; Luster, A. D.; Barrett, T. A. Inflamm. Bowel Dis. 2005, 11, 799. Yellin, M.; Pliienko, I.; Balanescu, A.; Ter-Vartanian, S.; Tseluyko, V.; Xu, L.-A.; Tao, X.; Cardarelli, P. M.; LeBlanc, H.; Nichol, G.; Ancuta, C.; Chirieac, R.; Luo, A. Arthritis Rheum. 2012, 64, 1730. Mayer, L.; Sandborn, W. J.; Stepanov, Y.; Geboes, K.; Hardi, R.; Yellin, M.; Tao, X.; Xu, L.-A.; Salter-Cid, L.; Gujrathi, S.; Aranda, R.; Luo, A. Y. Gut 2014, 63, 442. Garin, A.; Proudfoot, A. E. I. Exp. Cell Res. 2011, 317, 602. Lippke, G.; Thaler, H. Starch 1970, 22, 344. White, T. J.; Bruns, T.; Lee, S.; Taylor, J. In PCR Protocols: A Guide to Methods and Applications; Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J., Eds.; Academic Press: San Diego, 1990; p 315. Jung, M.; Triebel, S.; Anke, T.; Richling, E.; Erkel, G. Mol. Nutr. Food Res. 2009, 53, 1263. Roehm, N. W.; Rodgers, H.; Hatfield, S. M.; Glasebrook, A. L. J. Immunol. Methods 1991, 142, 257. Pfaffl, M.-W. Nucleic Acid Res. 2001, 29, e45. Lu, Z.; Wang, Y.; Miao, C.; Liu, P.; Hong, K.; Zhu, W. J. Nat. Prod. 2009, 72, 1761. Liu, H.; Edrada-Ebel, R.; Ebel, R.; Wang, Y.; Schulz, B.; Draeger, S.; Müller, W. E. G.; Wray, V.; Lin, W.; Proksch, P. J. Nat. Prod. 2009, 72, 1585. Hardaker, E. L.; Bacon, A. M.; Carlson, K.; Roshak, A. K.; Foley, J. J.; Schmidt, D. B.; Buckley, P. T.; Comegys, M.; Panettieri, R. A.; Sarau, H. M.; Belmonte, K. E. FASEB J. 2004, 18, 191. Clarke, D. L.; Clifford, R. L.; Jindarat, S.; Proud, D.; Pang, L.; Belvisi, M.; Knox, A. J. J. Biol. Chem. 2010, 285, 29101. Richmond, A. Nat. Rev. Immunol. 2002, 2, 664. Gaestel, M.; Kotlyarov, A.; Kracht, M. Nat. Rev. Drug Disc. 2009, 8, 480. Pautz, A.; Art, J.; Hahn, S.; Nowag, S.; Voss, C.; Kleinert, H. Nitric Oxide 2010, 23, 75. Jansen, B. J. M.; de Groot, Ae Nat. Prod. Rep. 2004, 21, 449. Ogawa, T.; Uosaki, Y.; Tanaka, T.; Tsuka, E.; Mihara, A.; Matsuda, E. J. Antibiot. 1996, 49, 168. Liermann, J. C.; Thines, E.; Opatz, T.; Anke, H. J. Nat. Prod. 2012, 75, 1983. Erkel, G.; Lorenzen, K.; Anke, T.; Velten, R.; Giminez, A.; Steglich, W. Z. Naturforsch. 1995, 50c, 1. Kuschel, A.; Anke, T.; Velten, R.; Klostermeyer, D.; Steglich, W.; König, B. J. Antibiot. 1994, 47, 733. Felix, S.; Sandjo, L. P.; Opatz, T.; Erkel, G. Beilstein J. Org. Chem. 2013, 9, 2866.