Clindanones A and B and cladosporols F and G, polyketides from the deep-sea derived fungus Cladosporium cladosporioides HDN14-342

Abstract

Four new tetralone derivatives, clindanones A and B (1 and 2) and cladosporols F and G (3 and 4), together with three known biogenetically related polyketides (5–7), were isolated from the deep-sea derived fungus Cladosporium cladosporioides HDN14-342. The structures of 1–4, including absolute configurations, were deduced based on MS, NMR and TD-DFT calculations of specific ECD spectra. The absolute configurations of the known cladosporols C (5) and E (6) were also revised. Compounds 1 and 2 possessed new dimeric forms of the skeleton composed by coupling of indanone and 1-tetralone units, and 4 showed the best cytotoxic activity against HeLa cells with an IC₅₀ value of 3.9 μM.

---

Introduction

The 1-tetralone homology dimers linked by a C–C bond are widely distributing polyketides produced by fungi and plants.^1–7 The structure diversity is arisen from both the various substitution groups and dimeric patterns including the naturally occurred 2–2′,⁷ 7–3′,^5,6 and 8–4′ (ref. 1–4) linkages, and the chemically synthesized 4–4′, 5–5′, 6–2′, 7–7′ and 8–8′ linkages^8–12 (Fig. S38 in ESI†). Cladosporols are naturally occurring 8–4′ 1-tetralone dimeric derivatives. Up to now, less than 10 natural cladosporol analogues (cladosporols A–E, cladosporone A, alterfungin and its derivates) have been discovered from the fungi cladosporium spp. and the bacterium Altemaria alternate var. monosporus.^1–4

In our on-going search for bioactive molecules from deep-sea derived microorganisms,^13–16 a fungal strain Cladosporium cladosporioides HDN14-342, isolated from a sediment sample collected in the Indian Ocean (depth 3471 m), was selected for its interesting HPLC-UV profile (Fig. S39, ESI†) and cytotoxic activity (inhibitory rate 66% on P388 cells at the concentration of 100 μg mL⁻¹ of the EtOAc extract). A chemical investigation of the EtOAc extract led to the isolation of seven polyketides including four new ones including the heterodimeric clindanones A and B (1 and 2) and the 1-tetralone homology dimers cladosporols F and G (3 and 4), together with three known compounds, cladosporol C (5),² cladosporol E (6)² and isosclerone (7)¹⁷ (Fig. 1). Among them, 1 and 2 possessed a new dimeric forms of the skeleton composed by coupling of indanone and 1-tetralone units, and 4 is the first halogenated cladosporol derivatives. The cytotoxicity of new compounds was tested and 4 showed the best activity with an IC₅₀ value of 3.9 μM. Herein, we reported the details of the isolation, structure elucidation and biological activities of these new compounds. The absolute configurations of the known cladosporols C (5) and E (6) were also revised.

Fig. 1 The chemical structures of compounds 1–7.

Results and discussion

The fungus C. cladosporioides HDN14-342 was cultured under static conditions (60 L). The EtOAc extract (5 g) of fermentation was fractionated by Sephadex LH-20 column chromatography, MPLC (ODS) and semi-preparative HPLC to afford the mixture of 1 and 2 and compounds 3–7.

The ¹H and ¹³C NMR spectra of the initial isolated mixture of compounds 1 and 2 showed two sets of almost identical signals with the ratio of nearly 1 : 1 (Fig. S40 and S41, ESI†). Further fractionation of the mixture on a chiral phase HPLC column achieved the isolation of compounds 1 and 2 (Fig. S42, ESI†).

Clindanones A and B (1 and 2) were both obtained as brown powders with molecular formula C₂₂H₁₈O₇ according to the protonated HRESIMS peaks at m/z 395.1121 and 395.1120, respectively, indicating fourteen degrees of unsaturation. The 1D NMR data (Tables 1 and 2) of 1 and 2 were highly similar. Each of them suggested the presence of one methyl, three methylenes, six methines with five aromatic ones, and twelve non-protonated carbons including four carbonyls. The planar structures of 1 and 2 were determined to be the same by interpretation of 1D and 2D NMR spectroscopic data. The 1-tetralone moiety was suggested by the comparison of chemical shifts with those of the known compounds 5–7,^2,17 and was further confirmed by the COSY and HMBC correlations (Fig. 2). The 1,3-indandione moiety was indicated by the COSY correlation (H-5/H-6) and the HMBC correlations from H-5 to C-4, C-4a and C-7, from H-6 to C-4 and C-7a, and from H₂-8 to C-1, C-2 and C-3, as well as the chemical shifts.¹⁸ The substituents on C-2 were suggested to be an acetonyl and a hydroxyl groups based on the HMBC correlations from H₃-10 to C-8 and C-9, together with the chemical shifts of C-2 (δ_C 72.7) and C-9 (δ_C 205.9). Finally, the planar structure was constructed by connecting C-7 and C-4′ based on the key HMBC correlation from H₂-3′ to C-7.

Table 1 ¹H NMR (500 MHz) data for compounds 1–5

No.	1^a	2^a	3^b	4^c	5^d
	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)
a In acetone-d6.b In DMSO-d6.c In CD3OD.d In CDCl3.e Data were recorded in DMSO-d6.
2	—	—	2.70, m; 2.54, m	4.94, d (9.9)	2.91, m; 2.60, m
3	—	—	2.34, m; 1.95, m	3.97, dd (3.2, 9.9)	2.38, m; 2.25, m
4	—	—	4.86, dd (2.7, 4.8)	5.43, d (3.2)	5.25, t (5.9)
5	7.27, d (8.5)	7.28, d (8.5)	—	—	—
6	7.42, d (8.5)	7.47, d (8.5)	6.99, d (8.6)	6.99, d (8.5)	6.92, (8.3)
7	—	—	6.85, d (8.6)	6.85, d (8.5)	6.75, (8.3)
8	3.44, s	3.44, s	—	—	—
9	—	—	3.25, s	—	—
10	2.13, s	2.13, s	—	—	—
2′	2.77, m; 2.72, m	2.82, m; 2.71, m	2.70, m; 2.57, m	2.65, m	2.62, m; 2.56, m
3′	2.39, m	2.39, m	2.11, m	2.33, m; 2.21, m	2.36, m; 2.12, m
4′	5.46, s	5.49, s	5.27, t (6.0)	5.34, t (5.0)	5.47, t (5.9)
5′	6.43, d (8.0)	6.38, d (8.0)	6.24, d (7.9)	6.36, d (8.0)	6.32, d (7.8)
6′	7.40, t (8.0)	7.39, t (8.0)	7.36, t (7.9)	7.31, t (8.0)	7.25, t (7.8)
7′	6.82, d (8.0)	6.80, d (8.0)	6.76, d (7.9)	6.75, d (8.0)	6.73, d (7.8)
OH-3	—	—	—	5.83, d (6.5)^e	—
OH-4	9.44, s	9.49, s	—	5.55, d (4.8)^e	—
OH-5	—	—	9.91, s	10.04, s^e	9.35, s
OH-8′	12.65, s	12.64, s	12.56, s	12.55, s^e	12.55, s

---

Table 2 ¹³C NMR (125 MHz) data for compounds 1–5

No.	1^a	2^a	3^b	4^c	5^d
	δC, type	δC, type	δC, type	δC, type	δC, type
a In acetone-d6.b In DMSO-d6.c In CD3OD.d In CDCl3.
1	199.4, C	196.9, C	200.9, C	193.2, C	200.4, C
2	72.7, C	72.7, C	34.3, CH2	64.6, CH	36.6, CH2
3	200.8, C	200.8, C	25.8, CH2	73.5, CH	30.5, CH2
4	155.8, C	155.8, C	68.7, CH	64.9, CH	66.0, CH
4a	125.8, C	125.8, C	129.8, C	128.4, C	131.0, C
5	123.1, CH	123.2, CH	153.7, C	154.3, C	154.5, C
6	138.8, CH	138.8, CH	119.8, CH	120.0, CH	120.8, CH
7	135.2, C	135.2, C	130.8, CH	131.5, CH	131.0, CH
7a	137.6, C	137.7, C	—	—	—
8	48.0, CH2	48.0, CH2	135.0, C	136.4, C	136.4, C
8a	—	—	132.8, C	129.9, C	130.6, C
9	205.9, C	205.9, C	56.3, CH3	—	—
10	28.4, CH3	28.4, CH3	—	—	—
1′	204.9, C	204.9, C	206.0, C	205.4, C	205.6, C
2′	36.3, CH2	36.7, CH2	37.5, CH2	36.2, CH2	37.3, CH2
3′	29.4, CH2	29.4, CH2	30.7, CH2	30.3, CH2	30.7, CH2
4′	38.8, CH	38.8, CH	39.4, CH	40.0, CH	40.4, CH
4′a	146.9, C	147.1, C	149.7, C	148.5, C	148.7, C
5′	119.5, CH	119.5, CH	120.4, CH	119.8, CH	120.1, CH
6′	136.5, CH	136.5, CH	137.0, CH	136.1, CH	136.4, CH
7′	115.7, CH	115.6, CH	115.3, CH	115.0, CH	115.5, CH
8′	162.6, C	162.6, C	162.2, C	162.4, C	162.6, C
8′a	117.4, C	117.2, C	117.5, C	117.3, C	117.7, C

---

Fig. 2 Key 2D NMR correlations for compounds 1–4.

After failing to make crystals, the absolute configurations of compounds 1 and 2 were proposed by comparison of the experimental to the calculated ECD spectra. Although experimental ECD curves of 1 and 2 are similar, slight differences around 330 nm were found, which indicates that they were not enantiomers (Fig. 3). Considering that the relative stereo-relationship of the two chiral centers (C-2 and C-4′) were not established due to the lack of valid signals, the computational ECD spectra of the two epimers (2S,4′S)-1 and (2R,4′S)-1, covering all the possible relative configurations, were calculated. The molecular mechanics conformational analysis of them were performed using Spartan software,¹⁹ followed by re-optimization using DFT at the B3LYP/6-31+g(d) level with Gaussian 09 software.²⁰ The Boltzmann-weighted ECD curves (Fig. 4) of (2S,4′S)-1 and (2R,4′S)-1 were also similar. Most of the Cotton effects in the experimental and computational ECD spectra were agreed with each other, indicating the 4′S absolute configurations of 1 and 2. The major differences of the computational ECD spectra lies also around 330 nm in which the (2R,4′S)-1 showed positive Cotton effect while the (2S,4′S)-1 showed negative. Although the calculated curves were not in agreement with the experimental ones perfectly, the absolute configurations for C-2 were proposed tentatively as S in 1 and R in 2 based on the negative and positive Cotton effects around 330 nm in their experimental ECD spectra (Fig. 3 and 4). The results also suggested that the ECD curves of this kind of compounds were dominated by the configuration of C-4′.

Fig. 3 Experimental ECD spectra of 1 (red) and 2 (black).

Fig. 4 B3LYP/6-31+G(d) calculated spectra of (2S,4′S)-1 (blue) and (2R,4′S)-1 (black) (σ = 0.18 eV).

Cladosporol F (3) was isolated as a brown powder. The molecular formula of 3 was determined to be C₂₁H₂₀O₅ based on the HRESIMS ion peak. The 1D NMR data (Tables 1 and 2) of 3 suggested the presence of one methoxy (δ_C 56.3 and δ_H 3.25), four methylenes, seven methines, and nine non-protonated carbons. Comparison of the ¹H and ¹³C NMR spectra of 3 with those of cladosporol C (5)² revealed that the 5-OH in 5 was replaced by a methoxy in 3 which was further supported by the HMBC correlation from H-4 to C-9.

The negative LRESIMS spectrum of cladosporol G (4) exhibited a characteristic chlorinated molecule ion peak cluster at m/z 387/389 (3 : 1) ([M − H]⁻), and the molecular formula was determined as C₁₀H₁₇ClO₆ according to the HRESIMS deprotonated peak at m/z 387.0627. The similar 1D NMR data of 4 (Tables 1 and 2) and cladosporol E (6) suggested that they should share the same 1-tetralone dimeric skeleton.² The differences of them were the replacement of the 2-OH in 6 by a chlorine in 4 based on the COSY correlations of H-2/H-3/H-4, OH-3/H-3 and OH-4/H-4 and the key HMBC correlations from H-4 to C-5, C-4a and C-8a, and from H-2 to C-1 as well as the chemical shift of C-2 (δ_C 64.6). In addition, the coupling constants of ³J_H-2,H-3 (9.9 Hz) and the ³J_H-3,H-4 (3.2 Hz) indicated that H-2 and H-3 was trans and H-3 and H-4 was cis. Cladosporol G (4) is the first halogenated cladosporol derivative, up to our knowledge.

Compounds 5 and 6 were proved to be identical to the reported cladosporols C (5) and E (6) by the NMR data (Tables 1 and 2), the optical rotation and the ECD behaviour.² The absolute configurations of cladosporols C and E had been determined as 4S,4′R and 2S,3R,4R,4′R based on the comparison of ECD data with cladosporol,² whose absolute configuration had been deduced by ECD exciton chirality method and comparing with those of the model compounds (+)-epoxydon and (+)-isoepoxydon.¹ Due to that the major cotton effects of 1–6 were similar but the absolute configuration of C-4′ were disagree, the ECD spectra of (4S,4′R)-5 and (4S,4′S)-5 were both calculated for a double check (Fig. 5). The well agreement of calculated ECD spectra of (4S,4′S)-5 with the experimental one suggested the revision of absolute configuration of cladosporol C as 4S,4′S. Accordingly, the absolute configuration of cladosporol E (6) was also revised as 2S,3R,4R,4′S, and the absolute configurations of 3 and 4 were deduced as 4S,4′S and 2S,3R,4R,4′S, according to the ECD spectra respectively (Fig. 6).

Fig. 5 B3LYP/6-31+G(d) calculated spectra of (4S,4′S)-5 (red, σ = 0.20 eV), (4S,4′R)-5 (green, σ = 0.20 eV) and the experimental one of 5 (black).

Fig. 6 The experimental ECD spectra of 3 (black), 4 (red), 5 (blue) and 6 (green).

The cytotoxicity of compounds 1–6 were evaluated against HeLa, A549 and HCT-116 cell lines by SRB method²³ and K562 and HL-60 cell lines by the MTT method.²⁴ Compounds 3 and 4 were active against HeLa, K562 and HCT-116 cell lines with IC₅₀ values ranging from 3.9 to 23.0 μM while other compounds were not active (IC₅₀ > 50.0 μM) (Table 3).

Table 3 Cytotoxicity of 1–6 (IC₅₀ μM)

Compd.	HeLa	K562	HCT-116	HL-60	A549
a ADM = doxorubicin (positive control). NT = not test.
1	>50.0	>50.0	>50.0	>50.0	>50.0
2	>50.0	>50.0	>50.0	>50.0	>50.0
3	13.8	23.0	23.0	>50.0	>50.0
4	3.9	8.8	19.4	>50.0	>50.0
5	>50.0	>50.0	NT	NT	NT
6	>50.0	>50.0	NT	NT	NT
ADM^a	0.5	0.6	0.2	0.2	0.8

---

As a relatively rare natural products, cladosporols showed various biological activities such as antifungi activity (cladosporol A, alterfungin and its derivates),^1,3 inhibitory activity against a panel of cancer cells (cladosporol A, cladosporone A),^4,29,30 inhibiting the urediniospore germination of the bean rust agent Uromyces appendiculatus (cladosporols B–E),² and inhibitory activity of COX-2 (cladosporone A).⁴ The differences of structures and cytotoxic bioactivities between compounds 4 and 6 indicate that the halogen on C-2 could enhance the cytotoxicity, while those between compounds 3 and 5 imply the importance of 4-OCH₃ to cytotoxicity.

Comparing to the common 1-tetralone homology dimers, the heterodimers are rare, with only two related cases reported as synthetic products (Fig. S43, ESI†).^21,22 Clindanones A and B (1 and 2) are the first naturally occurring heterodimeric products with a new dimeric forms of the skeleton composed by coupling of indanone and 1-tetralone units via a 7–4′ linkage. Biogenetically, compounds 1 and 2 are proposed to be formed from two suitable phenolic precursors which could be derived from a polyketide pathway involving one acetyl-CoA and four (or five) malonyl-CoA units.²⁵ Followed by dehydration involving the alcoholic hydroxy group and an aromatic proton,^26,27 the indandione moiety is generated through further oxidation and ring-open (Fig. 7).

Fig. 7 Proposed biosynthetic pathway of compounds 1 and 2.

Experimental section

General

Optical rotations were obtained on a JASCOP-1020 digital polarimeter. UV spectra were recorded on Waters 2487, while the ECD spectrum was measured on JASCO J-815 spectropolarimeter. ¹H NMR, ¹³C NMR, DEPT and 2D NMR spectra were recorded on an Agilent 500 MHz DD2 spectrometer. HRESIMS and ESIMS data were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Column chromatography (CC) was performed with Sephadex LH-20 (Amersham Biosciences). MPLC was performed using a C18 column (Agela Technologies, YMC-Pack ODS-A, 3 × 40 cm, 5 μm, 20 mL min⁻¹). Preparative HPLC collection used a C18 column (Waters, YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 3 mL min⁻¹). Chiral-phase HPLC used a Daicel Chiralpack IC column (4.6 × 250 mm, 5 μm, 1 mL min⁻¹).

Strains and culture conditions

The fungal strain was isolated from the deep-sea sediment from Indian Ocean (depth 3471 m, E 89.27°, N 8.76°, collected in April, 2013) and identified as Cladosporium cladosporioides based on sequencing of the ITS region (GenBank no. KT936390) with 100% similarity. The strain was deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, People's Republic of China.

Erlenmeyer flasks (1 L) containing 300 mL fermentation media were directly inoculated with spores. The media contained starch (10 g) and peptone (1 g) dissolved in 1 L naturally-collected seawater (Huiquan Bay, Yellow Sea). The flasks were static cultured for 60 days at 15 °C.

Extraction and purification

The whole fermentation broth (60 L) was filtered through cheese cloth to separate the supernatant from the mycelia. The supernatant was extracted with EtOAc (3 × 60 L), and the mycelia were macerated and extracted with acetone (3 × 15 L). All extracts were evaporated under reduced pressure to give a crude gum (5 g). The supernatant and mycelia extracts were combined after evaporation to dryness based on their similar HPLC-UV profiles. The extract was separated on a Sephadex LH-20 column with MeOH to provide six subfractions (Fr.1 to Fr.6). Fr.3 was further separated by MPLC (ODS) using a stepped gradient elution of MeOH–H₂O (40 : 60 to 100 : 0) to yield twelve subfractions (Fr.3-1 to Fr.3-12). Fr.3-4 was then separated by semi-preparative HPLC eluted with MeCN–H₂O (40 : 60) to obtain the mixture of 1 and 2 (5.0 mg, t_R = 18 min). Compounds 1 and 2 were further achieved by HPLC on a chiral phase column eluted with hexane–isopropanol (70 : 30), affording 1 (2.0 mg, t_R = 7 min) and 2 (2.1 mg, t_R = 10 min) (Fig. S42, ESI†). Fr.3-6 was then separated by semi-preparative HPLC eluted with MeCN–H₂O (35 : 65) to obtain 5 (27.0 mg, t_R = 26 min). Fr.3-8 was then separated by semi-preparative HPLC eluted with MeCN–H₂O (42 : 58) to obtain compound 3 (8.0 mg, t_R = 27 min). Fr.4 was further separated on a Sephadex LH-20 column with MeOH to provide four subfractions (Fr.4-1 to Fr.4-4). Fr.4-2 was then fractionated by semi-preparative HPLC eluted with MeOH–H₂O (45 : 55) to obtain compounds 7 (7.0 mg, t_R = 9 min) and 6 (5.0 mg, t_R = 21 min). Fr.5 was further separated by MPLC (C-18 ODS) using a stepped gradient elution of MeOH–H₂O (40 : 60 to 100 : 0) to yield five subfractions (Fr.5-1 to Fr.5-5). Fr.5-3 was future separated by semi-preparative HPLC eluted with MeOH–H₂O (55 : 45) to obtain compound 4 (30.0 mg, t_R = 25 min).

Clindanone A (1). Brown powder, [α]²⁰_D +23 (c 0.10, MeOH); UV (MeOH) λ_max (log ε): 234 (4.23), 343 (3.52) nm; CD (2.5 mM MeOH) λ_max (Δε) 216 (−1.89), 230 (+0.99), 245 (−1.84), 263 (+0.99) and 352 (+0.61) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 395.1121 [M + H]⁺ (calcd for C₂₂H₁₉O₇, 395.1125).

Clindanone B (2). Brown powder, [α]²⁰_D +15 (c 0.10, MeOH); UV (MeOH) λ_max (log ε): 234 (4.23), 343 (3.52) nm; CD (2.5 mM MeOH) λ_max (Δε) 216 (−2.44), 230 (+0.56), 245 (−1.30), 263 (+0.97) and 352 (+0.50) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 395.1120 [M + H]⁺ (calcd for C₂₂H₁₉O₇, 395.1125).

Cladosporol F (3). Brown powder, [α]²⁰_D +56 (c 0.10, MeOH); UV (MeOH) λ_max (log ε): 221 (4.01), 250 (3.88), 330 (3.64) nm; CD (2.8 mM MeOH) λ_max (Δε) 210 (+7.66), 233 (−12.92), 257 (+2.73) and 352 (+1.02) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 351.1234 [M − H]⁻ (calcd for C₂₁H₁₉O₅, 351.1227).

Cladosporol G (4). Brown powder, [α]²⁰_D +73 (c 0.10, MeOH); UV (MeOH) λ_max (log ε): 221 (4.32), 250 (4.02), 330 (3.84) nm; CD (2.6 mM MeOH) λ_max (Δε) 210 (+3.20), 233 (−5.19), 257 (+1.06) and 352 (+0.22) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 387.0627 [M − H]⁻ (calcd for C₂₀H₁₆ClO₆, 387.0630).

Cladosporol C (5). Brown powder; [α]²⁰_D +85 (c 0.10, MeOH); UV (MeOH) λ_max (log ε): 221 (3.98), 250 (4.14), 330 (3.90) nm; CD (3.0 mM MeOH) λ_max (Δε) 210 (+6.62), 233 (−12.02), 257 (+2.45) and 352 (+0.91) nm; ¹H and ¹³C NMR data see Tables 1 and 2.

Bioassays

Cytotoxic activities of 1–6 were evaluated using the HeLa cell line (human cervical cancer), the A549 cell line (human lung cancer) and the HCT-116 cell line (human colon cancer) by the SRB method,²³ and the K562 cell line (human acute myelocytic leukemia) and the HL-60 cell line (human promyelocytic leukemia) by the MTT method,²⁴ with ADM (0.2–0.8 μM) as positive control. The five human cancer cell lines above were purchased from America Type Culture Collection, ATCC (Rockville, MD, USA), with the K562 and HL-60 cultured in RPMI-1640, the HCT-116 in DEME, the A549 in F12k and HeLa in MEM. The four culture media above were purchased from Gibco (New York, USA) and supplemented with 100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin, and 10% heat-inactivated fetal bovine serum (Gibco) at 37 °C in humidified atmosphere with 5% CO₂.

In the MTT assay, cell lines were grown in RPMI-1640. Cell suspensions, 90 μL, at a density of 8 × 10⁵ cells per mL were plated in 96-well microtiter plates. Then, 10 μL of the test solution was added to each well and further incubated for 72 h. Then, 20 μL of the MTT solution (in PBS) was added to each well and incubated for 4 h. Then, 100 μL of the three linked dissolved solution was added to each well and incubated for 10 h. Absorbance was then determined on a Spectra Max Plus plates reader at 570 nm.

In the sulforhodanine B (SRB) assay, 90 μL of the suspensions were plated in 96-well plates at a density of 6 × 10⁵ cells per mL. Then, 10 μL of the test solution was added to each well, and the culture was further incubated for 72 h. The cells were fixed with 12% trichloroacetic acid, and the cell layer was strained with 0.4% SRB. The absorbance of SRB solution was measured at 515 nm. Dose–response curves were generated, and the IC₅₀ values, the concentration of compound required to inhibit cell proliferation by 50%, were calculated from the linear portion of log dose–response curves.

Computation section

Conformational searches were run by employing the “systematic” procedure implemented in Spartan'14 (ref. 19) using MMFF (Merck molecular force field). All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian 09 program.²⁰ The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were performed on three lowest-energy conformations for (2S,4′S)-1, three lowest-energy conformations for (2R,4′S)-1, two lowest-energy conformations for (4S,4′R)-5 and two lowest-energy conformations for (4S,4′S)-5 (>5% population) using 30 excited states, and using a polarizable continuum model (PCM) for MeOH. ECD spectra were generated using the program SpecDis²⁸ by applying a Gaussian band shape with 0.18 eV width for 1 and 0.20 eV width for 5, from dipolelength rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for about 97% of the weights. The calculated spectra were shifted by 5 nm to facilitate comparison to the experimental data.

Conclusions

In summary, four new tetralone derivatives, clindanones A and B (1 and 2) and cladosporols F and G (3 and 4), together with three known biogenetically related polyketides (5–7), were isolated from the deep-sea derived fungus Cladosporium cladosporioides HDN14-342. Clindanones A and B (1 and 2) possessed a new dimeric forms of the skeleton composed by coupling of indanone and 1-tetralone units, and 4 showed the best cytotoxic activity against the HeLa cells with an IC50 value of 3.9 μM. Deep-sea fungi still an important source of active natural products.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (41176120 and 21372208), the Shandong Provincial Natural Science Fund for Distinguished Young Scholars (JQ201422), the Program for New Century Excellent Talents in University (NCET-12-0499), and NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402).

Notes and references

1. Y. Sakagami, A. Sano, O. Hara, T. Mikawa and S. Marumo, Tetrahedron Lett., 1995, 36, 1469–1472.
2. G. Nasini, A. Arnone, G. Assante, A. Bava, S. Moricca and A. Ragazzi, Phytochemistry, 2004, 65, 2107–2111.
3. J. P. Chen, S. J. Deng, L. L. Duan, R. H. Wang, S. L. Zhao, X. L. Qiu, S. M. Wei, P. Z. Chen, S. X. Cheng and Z. J. Zheng, PCT Int. Appl., WO 2006036131 A1 20060406, 2006.
4. W. Ai, X. Lin, Z. Wang, X. Lu, F. Mangaladoss, X. Yang, X. Zhou, Z. Tu and Y. Liu, J. Antibiot., 2015, 68, 213–215.
5. O. Weigenand, A. A. Hussein, N. Lall and J. J. Meyer, J. Nat. Prod., 2004, 67, 1936–1938.
6. N. Lall, O. Weiganand, A. A. Hussein and J. J. M. Meyer, S. Afr. J. Bot., 2006, 72, 579–583.
7. J. T. Fan, B. Kuang, G. Z. Zeng, S. M. Zhao, C. J. Ji, Y. M. Zhang and N. H. Tan, J. Nat. Prod., 2011, 74, 2069–2080.
8. K. M. Baker, K. H. Bell and B. R. Davis, Aust. J. Chem., 1968, 21, 2945–2950.
9. H. Akimoto and S. Yamada, Tetrahedron, 1971, 27, 5999–6009.
10. H. H. Huang, Aust. J. Chem., 1976, 29, 2415–2422.
11. A. R. Katritzky and C. M. Marson, J. Chem. Soc., 1983, 9, 1455–1461.
12. M. Y. Chang, T. W. Lee and M. H. Wu, Org. Lett., 2012, 14, 2198–2201.
13. W. Q. Guo, Z. Z. Zhang, T. J. Zhu, Q. Q. Gu and D. H. Li, J. Nat. Prod., 2015, 78, 2699–2703.
14. G. W. Wu, X. H. Sun, G. H. Yu, W. Wang, T. J. Zhu, Q. Q. Gu and D. H. Li, J. Nat. Prod., 2014, 77, 270–275.
15. W. Q. Guo, J. X. Peng, T. J. Zhu, Q. Q. Gu, R. A. Keyzers and D. H. Li, J. Nat. Prod., 2013, 76, 2106–2112.
16. G. W. Wu, H. Y. Ma, T. J. Zhu, J. Li, Q. Q. Gu and D. H. Li, Tetrahedron, 2012, 68, 9745–9749.
17. T. Morita and H. Aoki, Agric. Biol. Chem., 1974, 38, 1501–1505.
18. Y. Hayakawa, T. Kobayashi and M. Izawa, J. Antibiot., 2013, 66, 731–733.
19. Spartan 14, Wavefunction Inc., Irvine, CA, 2013.
20. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Revision B.01, Gaussian, Inc., Wallingford CT, 2010.
21. S. Kneubuehler, V. Carta, C. Altomare, A. Carotti and B. Testa, Helv. Chim. Acta, 1993, 76, 1956–1963.
22. J. P. Poupelin and G. Narcisse, Fr. Demande., FR 2382424 A1 19780929, 1978.
23. P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney and M. R. Boyd, J. Natl. Cancer Inst., 1990, 82, 1107–1112.
24. T. J. Mosmann, Immunol. Methods, 1983, 65, 55–63.
25. T. J. Shan, J. Tian, X. H. Wang, M. Mou, Z. L. Mao, D. W. Lai, J. G. Dai, Y. L. Peng, L. G. Zhou and M. G. Wang, J. Nat. Prod., 2014, 77, 2151–2160.
26. C. Li, Y. C. Fang, T. J. Zhu, Q. Q. Gu and W. M. Zhu, J. Nat. Prod., 2008, 71, 66–70.
27. M. Colon, P. Guevara and W. H. Gerwick, J. Nat. Prod., 1987, 50, 368–374.
28. T. Bruhn, Y. Hemberger, A. Schaumloffel and G. Bringmann, SpecDis, Version 1.53, University of Wuerzburg, Germany, 2011.
29. D. Zurlo, C. Leone, G. Assante, S. Salzano, G. Renzone, A. Scaloni, C. Foresta, V. Colantuoni and A. Lupo, Mol. Carcinog., 2013, 52, 1–17.
30. D. Zurlo, G. Assante, S. Moricca, V. Colantuoni and A. Lupo, Biochim. Biophys. Acta, 2014, 1840, 2361–2372.

---

Footnote

† Electronic supplementary information (ESI) available: The HPLC-UV profile of crude extract, chiral HPLC separation chromatogram of 1/2, MS, 1D and 2D NMR spectra for compounds 1–5 and details for ECD calculations. See DOI: 10.1039/c6ra14640f

---