Identification and Biological Activity of NFAT-133 Congeners from Streptomyces pactum

Abstract

The soil bacterium Streptomyces pactum ATCC 27456 produces a number of polyketide natural products. Among them is NFAT-133, an inhibitor of the nuclear factor of activated T cells (NFAT) that suppresses interleukin-2 (IL-2) expression and T cell proliferation. Biosynthetic gene inactivation in the ATCC 27456 strain revealed the ability of this strain to produce other polyketide compounds including analogues of NFAT-133. Consequently, seven new derivatives of NFAT-133, TM-129 – TM-135, together with a known compound, panowamycin A, were isolated from the culture broth of S. pactum ATCC 27456 ΔptmTDQ. Their chemical structures were elucidated on the basis of their HRESIMS, 1D and 2D NMR spectroscopy, and ECD calculation and spectral data. NFAT-133, TM-132, TM-135 and panowamycin A showed no antibacterial activity or cytotoxicity, but weakly reduced the production of LPS-induced nitric oxide (NO) in RAW264.7 cells in a dose-dependent manner. A revised chemical structure of panowamycin A and proposed modes of formation of the new NFAT-133 analogues are also presented.

Graphical Abstract

Soil bacteria (actinomycetes), especially those from the genus Streptomyces, are known to be a prolific source of bioactive secondary metabolites.¹ They produce a wide variety of products including polyketides, peptides, terpenes, glycosides, and aminocyclitols.^2–5 Genome sequences of many strains of actinomycetes reveal the presence of biosynthetic gene clusters whose number far exceeds the number of known secondary metabolites produced by those bacteria. Evidently, only a small number of the biosynthetic gene clusters are expressed under typical laboratory culture conditions to produce detectable amounts of compounds.⁶ This information further underscores the enormous potential of actinomycetes as a source of bioactive natural products.

Streptomyces pactum is known to produce a number of bioactive secondary metabolites, e.g., pactamycin, piericidins, and actinopyrones.^7–9 Some strains of S. pactum, such as the ATCC 27456, also produce a high amount of NFAT-133 (1).^10,11 This polyketide-derived aromatic compound has been reported to have multiple biological activities, such as immunosuppressive, antidiabetic, and antitrypanosomal properties.^12–15 It blocks nuclear factor of activated T cells (NFAT)-dependent transcription and increases insulin-stimulated glucose uptake in L6 myotubes by activating the AMPK pathway.^12,15 NFAT-133 has also been reported in Streptomyces sp. strain AB 2184C-502, Streptomyces sp. PM0324667, Streptomyces sp. K07–0010, and Streptomyces karnatakensis NBRC 13051.^12–14,16 While the absolute stereoconfiguration of 1 was initially reported to be 10R, 11R, 12S,¹⁶ a more recent study by Ogura and co-workers concluded that the absolute stereoconfiguration is 10S, 11R, 12S.¹⁷

In addition to NFAT-133, a number of its analogues have also been isolated from several species of Streptomyces. Those include benwamycins from Streptomyces sp. KIB-H1471, panowamycins from Streptomyces sp. K07–0010, and NFAT-133/conglobatin hybrid compounds, TM-127 and TM-128, from S. pactum ATCC 27456.^14,18,19 Benwamycins B and F showed anti-proliferation activity on human T-cell activated with anti-CD3/anti-CD28 antibodies without appreciable cytotoxicity against naïve human T cells,¹⁸ whereas panowamycins A and B showed moderate antitrypanosomal activity.¹⁴

As part of an effort to mine new natural products from soil bacteria, we investigated minor metabolites produced by a mutant strain of S. pactum ATCC 27456. Here, we report the isolation and structure characterization of seven new NFAT-133 analogues, TM-129 – TM-135, as well as a known compound, panowamycin A. A revised chemical structure of panowamycin A, the proposed modes of formation of TM-129 – TM-135, and the biological activities of some of the new compounds are also presented.

Results and Discussion

S. pactum ATCC 27456 has been used as a model organism for the biosynthetic studies of the antitumor antibiotic pactamycin.^10,20–27 During the course of those studies, the potential of the strain to produce other bioactive natural products was recognized. Furthermore, inactivation of the NFAT-133 biosynthetic gene cluster in S. pactum ATCC 27456 ΔptmTDQ, which does not produce pactamycin,^11,19,24 resulted in a mutant that also lacks the ability to produce NFAT-133 and a number of unknown minor metabolites.¹¹ To investigate the identity of the minor metabolites, the ATCC 27456 ΔptmTDQ strain was cultivated in BTT liquid medium and the culture broth was subjected to column chromatography and HPLC (Fig. S1). Consequently, seven new derivatives of NFAT-133, TM-129 – TM-135 (2 – 8), together with a known compound, panowamycin A, were isolated. Inspection of the ATCC 27456 wild-type strain showed that the compounds were also produced by the wild-type, albeit in very low yields.

TM-129 (2) was isolated as a colorless viscous oil, and its high-resolution ESI-MS suggested a molecular formula of C₁₇H₂₄O₃. The ¹H NMR spectrum of TM-129 showed the presence of four methyl groups, a methylene, three methines, and five olefinic/aromatic protons. The ¹³C NMR spectra of TM-129 also showed the presence of four methyl carbons, an oxygenated methylene carbon, three methine carbons (one of which is oxygenated), eight olefinic/aromatic carbons, and an ester/lactone carbon (δ_C 212.6). The COSY and HMBC spectra of TM-129 showed proton-proton and proton-carbon correlations that are consistent with the cinnamyl alcohol moiety and the linear polyketide side chain of NFAT-133 (Fig. 1). However, direct comparisons of the ¹H and ¹³C NMR data for TM-129 with those for NFAT-133 revealed a significant change in the chemical shifts of H-1 and C-1. In addition, the coupling constant between H-2 and H-3 (11 Hz) in TM-129 is much smaller than that of NFAT-133 (16 Hz) indicating that TM-129 has a cis double bond, instead of the trans double bond found in NFAT-133.

Figure 1.

COSY and key HMBC correlations of compounds 2, 4, and 6.

TM-130 (3) was isolated as a colorless viscous oil, and its high-resolution ESI-MS suggested a molecular formula of C₁₇H₂₄O₃. The ¹H and ¹³C NMR spectra of TM-130 showed resonances that are similar to those of NFAT-133, particularly those for the primary allyl alcohol and the aromatic moieties (Tables 1 and 2). Differences in the chemical shifts and coupling constants were observed for H-10, H-11, and H-12. The COSY, HSQC, and HMBC spectra showed cross-peaks that connect most of the proton and carbon resonances to form a backbone structure identical to NFAT-133 (Fig. S2). The changes in the chemical shifts and coupling constants of H-10, H-11, and H-12 indicate that TM-130 is a diastereomer of NFAT-133 where one or more of the stereoconfigurations at C-10, C-11, or C-12 are different from NFAT-133.

Table 1.

¹H NMR data for NFAT-133 analogues (CD₃OD, 700 MHz)

	1	2	3	4	5	6	7	8	Pano-A
position	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)	δH (J in Hz)
1	4.27, dd (2, 6)	4.12, dd (7, 13), 4.17, dd (7, 13)	4.27, d (5)	5.24, dt (2, 10), 5.38, dt (2, 17)	5.15, dt, (1, 10), 5.25, dt (1, 17)	1.92, dd (2, 7)	1.69, dd (2, 7)	3.72, m, 3.84, dt (7, 11)	3.79, ddd (4, 7, 11), 3.89, ddd (7, 9, 11)
2	6.17, dt (6, 16)	5.93, dt (7, 11)	6.18, dt (5, 16)	6.13, ddd (5, 10, 17)	5.98, ddd (6, 10, 17)	6.05, dd (7, 15)	5.88, dd (7, 11)	1.90, 2.29, m	1.90, 2.13, m
3	6.97, d (16)	6.71, d (11)	6.94, d (16)	5.46, d (5)	5.52, d (6)	6.74, d (15)	6.57, d (11)	4.87, dd (2, 9)	5.00, dd (2, 11)
4	-	-	-	-	-	-	-	-	-
5	7.27, s	6.96, s	7.27, s	7.24, s	7.34, s	7.2, s	6.98, s	6.96, s	6.88, s
6	-	-	-	-	-	-	-	-	-
7	7.09, d (8)	7.13, d (8)	7.07, d (8)	7.11, d (8)	7.10, d (8)	7.05, d (8)	7.10, d (8)	7.00, d (8)	6.99, dd (1, 8)
8	7.16, d (8)	7.21, d (8)	7.16, d (8)	7.21, d (8)	7.19, d (8)	7.13, d (8)	7.20, d (8)	6.98, d (8)	6.97, d (8)
9	-	-	-	-	-	-	-	-	-
10	3.11, m	2.99–2.95, m	3.25, m	3.24–3.18, m	3.17–3.05, m	3.14–3.04, m	2.99, m	2.69, m	2.68, m
11	4.22, dd (3, 9)	4.23, dd (3, 9)	3.75, dd (7, 13)	4.25, dd (4, 8)	4.24, dd (4, 9)	4.23, dd (3, 9)	4.24, dd (3, 9)	3.71, dd (2, 10)	3.94, dd (2, 10)
12	2.39, m	2.36, m	2.72, m	2.52, m	2.49, m	2.36, m	2.35, m	2.92, m	2.92, m
13	-	-	-	-	-	-	-		-
14	2.08, s	2.09, s	2.10, s	2.10, s	2.09, s	2.09, s	2.08, s	2.25, s	2.27, s
15	2.32, s	2.32, s	2.32, s	2.32, s	2.33, s	2.30, s	2.32, s	2.31, s	2.30, s
16	1.31, d (7)	1.29, d (7)	1.27, d (7)	1.34, d (7)	1.26, d (7)	1.31, d (7)	1.28, d (7)	1.14, d (7)	1.10, d (7)
17	0.97, d (7)	0.96, d (7)	1.11, d (7)	1.01, d (7)	1.07, d (7)	0.95, d (7)	0.95, d (7)	1.31, d (7)	1.31, d (7)

Table 2.

¹³C NMR data for NFAT-133 analogues (CD₃OD, 175 MHz)

	1	2	3	4	5	6	7	8	Pano-A
position	δC	δC	δC	δC	δC	δC	δC	δC	δC
1	62.4, CH2	58.3, CH2	62.4, CH2	113.8, CH2	113.6, CH2	17.5, CH3	13.1, CH3	58.4, CH2	58.4, CH2
2	131.4, CH	131.8, CH	131.1, CH	141.5, CH	141.0, CH	127.7, CH	127.0, CH	38.9, CH2	37.9, CH2
3	128.4, CH	129.4, CH	127.8, CH	70.3, CH	70.1, CH	128.9, CH	129.0, CH	74.9, CH	72.0, CH
4	135.7, C	134.9, C	135.4, C	139.9, C	140.0, C	136.6, C	135.7, C	136.7, C	136.6, C
5	127.8, CH	130.0, CH	126.9, CH	128.0, CH	126.7, CH	127.2, CH	130.2, CH	124.1, CH	125.3, CH
6	135.6, C	135.2, C	135.5, C	135.4, C	135.5, C	135.5, C	135.0, C	135.5, C	135.3, C
7	127.2, CH	128.4, CH	127.9, CH	128.3, CH	128.0, CH	127.8, CH	127.8, CH	127.0, CH	127.1, CH
8	126.5, CH	126.4, CH	127.1, CH	126.8, CH	126.5, CH	126.3, CH	126.2, CH	128.5, CH	128.3, CH
9	138.8, C	139.6, C	138.9, C	139.5, C	138.5, C	138.2, C	139.6, C	137.6, C	137.0, C
10	38.2, CH	39.0, CH	36.4, CH	37.6, CH	37.1, CH	38.3, CH	39.0, CH	34.3, CH	34.0, CH
11	75.2, CH	75.1, CH	76.6, CH	74.9, CH	75.2, CH	75.2, CH	75.2, CH	77.7, CH	71.3, CH
12	49.8, CH	49.8, CH	50.2, CH	49.8, CH	49.7, CH	49.8, CH	49.8, CH	49.2, CH	49.0, CH
13	212.7, C	212.6, C	212.8, C	212.8, C	212.8, C	212.6, C	212.6, C	212.1, C	212.1, C
14	27.1, CH3	27.0, CH3	28.3, CH3	27.2, CH3	27.1, CH3	27.0, CH3	27.0, CH3	27.7, CH3	27.8, CH3
15	19.7, CH3	19.6, CH3	19.7, CH3	19.7, CH3	19.8, CH3	19.7, CH3	19.6, CH3	19.9, CH3	19.8, CH3
16	18.0, CH3	17.8, CH3	13.8, CH3	18.4, CH3	18.1, CH3	18.0, CH3	18.0, CH3	16.4, CH3	16.1, CH3
17	8.2, CH3	8.1, CH3	13.1, CH3	9.0, CH3	8.7, CH3	8.0, CH3	7.6, CH3	13.9, CH3	14.4, CH3

To determine the absolute configuration of TM-130, all eight possible diastereoisomers of TM-130, except NFAT-133, were subjected to ECD calculation (Figs. 2 and S3). The experimental ECD spectrum of TM-130 (Fig. 2B, black solid line) was in good agreement with the theoretically calculated ECD spectrum for 10R, 11R, 12R (Fig. 2B, red solid line). Thus, the chemical structure of TM-130 was suggested as shown.

Figure 2.

ECD spectra of TM-130 (3), TM-131 (4), and TM-132 (5). (A) The lowest energy conformers of 10R, 11R, 12R and 10S, 11S, 12S of 3; (B) Calculated and experimental ECD spectra of 3; (C) The lowest energy conformers of 4 and 5; (D) Calculated and experimental ECD spectra of 4 and 5.

TM-131 (4) was isolated as a colorless viscous oil, and its high-resolution ESI-MS suggested a molecular formula of C₁₇H₂₄O₃, which is identical to NFAT-133. While the ¹H and ¹³C NMR spectra of TM-131 showed resonances that are similar to those of NFAT-133 (Tables 1 and 2), significant shifts in resonances in the downfield region were observed. In particular, the presence of two doublets of triplet at 5.24 ppm (J = 2, 10 Hz) and 5.38 (J = 2, 17 Hz), a doublet at 5.46 ppm (J = 5 Hz), and a doublet of doublet of doublets at 6.13 ppm (J = 5, 10, 17 Hz) indicated the presence of a secondary allyl alcohol moiety in TM-131 (instead of a primary allyl alcohol in NFAT-133). This was supported by the lack of signals for a hydroxymethylene group in TM-131. HMBC correlation between H-3 (δ_H 5.46, d, J = 5 Hz) and the aromatic carbon C-5 (δ_C 128.0) suggested that the secondary allyl alcohol moiety is attached to the aromatic ring, indicating that TM-131 contains a vinyl benzyl alcohol moiety instead of a cinnamyl alcohol moiety. The stereoconfiguration of this hydroxy group was suggested to be R, as the experimental ECD spectrum of TM-131 was in good agreement with the theoretically calculated ECD spectrum for 3R, 10S, 11R, 12S (Fig. 2C–D, blue dotted line).

TM-132 (5) was isolated as a colorless viscous oil with a molecular mass of 276 (m/z 299.1617 [M+Na]⁺), which is also identical to NFAT-133. The ¹H and ¹³C NMR spectra of TM-132 also showed resonances for a secondary allyl alcohol moiety, but the chemical shifts are slightly different from those of TM-131. In addition, the methyl protons H₃-16 and the aromatic proton H-5 in TM-132 were observed at δ_H 1.26 and 7.34 ppm, respectively; both of them are about 0.1 ppm shifted downfield and upfield, respectively, from those in TM-131. Detailed analysis of the COSY, HSQC, and HMBC spectra of TM-132 resulted in a planar chemical structure identical to TM-131. Therefore, TM-131 and TM-132 were determined to be diastereomers differing only in the stereoconfiguration of the hydroxy group at C-3. The stereoconfiguration of the C-3 hydroxy group in TM-132 was suggested to be S, as the experimental ECD spectrum of TM-132 was in good agreement with the calculated ECD spectrum for 3S, 10S, 11R, 12S (Fig. 2C–D, red dotted line).

TM-133 (6) and TM-134 (7) were isolated as colorless solids, and their high-resolution ESI-MS suggested a molecular formula of C₁₇H₂₄O₂ for both compounds. While the ¹H and ¹³C NMR spectra of TM-133 and TM-134 showed resonances that are similar to those of NFAT-133 (Tables 1 and 2), they lack a hydroxy methylene resonance seen in NFAT-133. Instead, both TM-133 and TM-134 have an additional methyl group, which indicates that the hydroxy methylene moiety in NFAT-133 has been reduced to a methyl group in TM-133 and TM-134. HMBC correlations between the methyl group and the olefinic carbon C-2 in both compounds confirmed this assignment. The coupling constant between H-2 and H-3 in TM-133 is 15 Hz, indicating that TM-133 contains an olefin moiety with an E configuration, whereas the coupling constant of the olefinic protons H-2 and H-3 in TM-134 is 11 Hz, indicating that TM-134 contains an olefin moiety with a Z configuration.

TM-135 (8) and panowamycin A were isolated as colorless solids, and their high-resolution ESI-MS suggested a molecular formula of C₁₇H₂₄O₃ for both. The ¹H NMR data of panowamycin A in CDCl₃ are almost identical to those reported in the literature,¹⁴ whereas the ¹H NMR spectra of TM-135 either in CDCl₃ or CD₃OD showed resonances that are similar to those of panowamycin A, except for protons H-1, H-2, H-3, H-5, and H-11 (Table 1 and Supporting Information). Similarly, the ¹³C NMR spectrum of TM-135 is also almost identical to that of panowamycin A, except that the resonances for C-2 and C-3 of TM-135 are shifted slightly compared to those of panowamycin A. The planar chemical structure of TM-135 was further confirmed by COSY, HSQC, and HMBC spectral analysis.

The relative stereoconfiguration of TM-135 was determined to be 3R*, 10S*, 11R*, 12S* on the basis of the NOESY correlations between H-11 and H-3, H-10, H-17 (Fig. 3A–B). Surprisingly, this assignment is the same as the reported relative stereoconfiguration of panowamycin A, which is 3S*, 10R*, 11S*, 12R* (Fig. 3C).¹⁴ The reported stereoconfiguration of panowamycin A was determined based on the ROESY data (measured in CDCl₃), particularly on the correlation between H-3 and H-11.¹⁴ However, we discovered that when measured in CDCl₃ the H-11 resonance overlaps with that of H-1, making it difficult to determine whether the observed cross peak is indeed between H-3 and H-11 or between H-3 and H-1. Re-evaluation of the NOESY spectrum of panowamycin A in CD₃OD showed a clear correlation between H-11 and H-2, H-10, H-17, and no correlation between H-11 and H-3, suggesting that its relative stereoconfiguration is 3R*, 10R*, 11S*, 12R* (Fig. 3D). Considering that both TM-135 and panowamycin A are derived from NFAT-133, and the absolute configuration of NFAT-133 is 10S, 11R, 12S (Fig. 3F),¹⁷ the absolute configuration of TM-135 and panowamycin A are postulated to be 3R, 10S, 11R, 12S and 3S, 10S, 11R, 12S, respectively (Figs. 3B and 3E).

Figure 3.

Partial NOESY correlations and proposed stereoconfigurations of TM-135 and panowamycin A. (A) Partial NOESY correlations of TM-135; (B) proposed absolute configuration of TM-135; (C) reported relative stereoconfiguration of panowamycin A; (D) Partial NOESY correlations of panowamycin A; (E) proposed absolute stereoconfiguration of panowamycin A; (F) absolute stereoconfiguration of NFAT-133.

Several of the compounds reported above are likely derived from the major metabolite NFAT-133. An inspection of the biosynthetic gene cluster of NFAT-133 in S. pactum did not provide any clues as to which genes, if any, in the cluster that are involved in the conversions (Fig. 4A).¹¹ Also, it is possible that the conversions are catalyzed by enzymes that are coded by genes located outside of the cluster. As all of the compounds were isolated in low yields, it may be postulated that their formations are not very efficient. Nevertheless, TM-129 (2) may be formed from NFAT-133 (1) nonenzymatically through light-induced isomerization of the double bond.²⁸ Likewise, TM-131 (4) and TM-132 (5) may be derived from NFAT-133 through a 1,3-rearrangement (transposition) of the allylic alcohol, presumably under an acidic condition of the late log/stationary phase cultures (Fig. 4B). This type of rearrangement has been reported in the literature,²⁹ but how often it occurs in nature is unclear. It is also possible that 4 and 5 are intermediates or early shunt products of the NFAT-133 pathway. TM-130 (3), which adopts a 10R, 11R, 12R configuration may be produced as a minor product by the NFAT-133 polyketide synthases. Although the chirality of the hydroxy and methyl groups in polyketides is usually determined by the ketoreductase (KR) domains of the polyketide synthases,³⁰ the stereoselectivity and stereospecificity of the KR domains are not always perfect. A small amount of diastereomers may be produced.³¹ Although possible, it is less likely that 3 is formed from 1 by epimerizations at C-10 and C-12, particularly under the mild culture and isolation conditions. The mechanism underlying the formation of TM-133 (6) and TM-134 (7) is unclear at this point. However, TM-133 (6) and TM-134 (7) are more likely formed from TM-131 (4) and/or TM-132 (5) or other intermediates than directly from NFAT-133 (1) and/or TM-129 (2). TM-135 (8) and panowamycin A may be formed from 1 through an intramolecular oxy-Michael reaction involving the 11-OH and the C-2/3 olefin (Fig. 4C). While this cyclization reaction appears to be non stereospecific, the involvement of two different enzymes that give either 8 or panowamycin A cannot be ruled out.

Figure 4.

Proposed formation of NFAT-133 analogues. (A) Biosynthetic gene cluster of NFAT-133 (1); (B) Proposed 1,3-rearrangement of 1 to give TM-131 (4) and TM-132 (5); (C) Proposed cyclization of 1 to give TM-135 and panowamycin A.

NFAT-133, TM-132, TM-135 and panowamycin A were inactive against Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6051, Escherichia coli ATCC 11775, and Pseudomonas aeruginosa ATCC 9721. Other NFAT-133 analogues were not tested due to insufficient amounts of the samples. Cytotoxicity assays against RAW264.7, HeLa, NCI-H460 and MCF-7 cancer cell lines also indicated that NFAT-133, TM-132, TM-135 and panowamycin A are not cytotoxic up to 50 μM (Fig. S4 and S5A). All of the tested compounds weakly reduced the production of NO in RAW264.7 cells in a dose-dependent manner (Fig. S5B).

Experimental Section

General experimental procedures.

Optical rotations were measured on a Jasco P1010 polarimeter. UV spectra were obtained on an Eppendorf BioSpectrometer. Circular Dichroism (CD) spectra were recorded on a Jasco J-815 Circular Dichroism spectropolarimeter. The ¹H and ¹³C NMR spectra were measured on a Bruker Avance III 700 MHz spectrometers (Bruker Bio Spin AG, Industriestrasse 26, Fällanden, Switzerland). Low-resolution mass spectra were obtained from an AB SCIEX 3200 QTRAP mass spectrometer and high-resolution ESI mass spectra were obtained using an Agilent 1260 HPLC upstream of an Agilent 6545 Q-ToF. HPLC was performed using a Shimadzu dual LC-20AD solvent delivery system with a Shimadzu SPD-M20A UV/vis photodiode array detector. Column chromatography was performed over silica gel (SiO₂, 200–300 mesh).

Bacterial strains.

S. pactum ATCC 27456 was purchased from American Type Culture Collection (ATCC). S. pactum ΔptmTDQ mutant was constructed according to procedure described in our previous papers.^20,24

Isolation of NFAT-133 derivatives from S. pactum ΔptmTDQ.

The S. pactum ΔptmTDQ triple mutant strain was cultured in BTT liquid medium [glucose (10 g/L), beef extract (1 g/L), yeast extract (1 g/L), soytone (2 g/L), trace elements (1 mL/L)] (5 L) at 30 °C for 7 days with shaking at 200 rpm. The fermentation broth was collected by centrifugation and subjected to resin HP20 column chromatography. The metabolites were gradually eluted with 1 bed volume of different concentrations of EtOH (20%, 40%, 80% and 100%). Based on TLC analysis using CHCl₃-MeOH (95:5) as a solvent system and p-anisaldehyde as a staining reagent, the 80% and 100% EtOH fractions were pooled. The combined fraction (2.25 g) was subjected to SiO₂ flash column chromatography using step gradient elution with CHCl₃–MeOH (95:5–0:100) to give five fractions. The five fractions were then analyzed by HPLC. Fraction 1 (0.54 g), which contained most of the metabolites of S. pactum ΔptmTDQ, was subjected to SiO₂ column chromatography eluted with a gradient of CHCl₃–MeOH (100:0 – 90:10) to give fractions 1–1, 1–2, and 1–3. Fraction 1–2 (0.325 g) was then separated by semipreparative HPLC (ACE C₁₈ column 10 × 250 mm, 5 μm) to yield TM-129 (1 mg), TM-130 (0.5 mg), TM-131 (0.8 mg), TM-132 (1.5 mg), TM-133 (0.5 mg), TM-134 (0.3 mg), TM-135 (2.4 mg), panowamycin (1.6 mg) and NFAT-133 (98 mg) (Fig. S1). The elution condition was as follows: solvent A, H₂O; solvent B, MeOH; flow rate, 2.5 mL/min; 0−5 min, 45% B; 6−15 min, linear gradient to 50% B; 16−20 min, 50% B; 21−30 min linear gradient to 80% B; 31−35 min, 80% B. The elution was monitored at 216 nm.

TM-129 (2):

a colorless viscous oil, ${\left[\alpha \right]}_{\text{D}}^{25}=+9.6°$ (c = 0.5, MeOH). UV(MeOH), λ_max (log ε) 208 (4.45), 233 (4.00) nm. IR (neat) ν_max 3383, 2926, 1682, 1436, 1204, 1184, 1135, 802, 724 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 299.1614 [M+Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

TM-130 (3):

a white solid, ${\left[\alpha \right]}_{\text{D}}^{25}=+67.6°$ (c = 0.05, MeOH). UV(MeOH), λ_max (log ε) 214 (4.39), 247 (4.00) nm. IR (neat) ν_max 3397, 2924, 1700, 1457, 1375, 1359, 1173, 1012, 971 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 299.1615 [M+Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

TM-131 (4):

a colorless viscous oil, ${\left[\alpha \right]}_{\text{D}}^{25}=+19.9°$ (c = 0.40, MeOH). UV(MeOH), λ_max (log ε) 203 (4.43), 218 (3.82) nm. IR (neat) ν_max 3389, 2921, 1702, 1562, 1458, 1425, 1165, 1124 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 299.1616 [M+Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

TM-132 (5):

a colorless viscous oil, ${\left[\alpha \right]}_{\text{D}}^{25}=+27.2°$ (c = 0.15, MeOH). UV(MeOH), λ_max (log ε) 204 (4.47), 218 (3.95) nm. IR (neat) ν_max 3397, 2957, 2921, 1696, 1559, 1452, 1418, 1259, 1085, 1026, 796, 669 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 299.1617 [M+Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

TM-133 (6):

a colorless solid, ${\left[\alpha \right]}_{\text{D}}^{25}=+19.7°$ (c = 0.25, MeOH). UV(MeOH), λ_max (log ε) 210 (4.40), 245 (3.87) nm. IR (neat) ν_max 3397, 2971, 2926, 1701, 1589, 1453, 1354, 1175, 968 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 283.1671 [M+Na]⁺ (calcd for C₁₇H₂₄O₂Na, 283.1674).

TM-134 (7):

a colorless solid, ${\left[\alpha \right]}_{\text{D}}^{25}=+29.1°$ (c = 0.15, MeOH). UV(MeOH), λ_max (log ε) 206 (4.33), 233 (3.85) nm. IR (neat) ν_max 3453, 2972, 2930, 1701, 1593, 1457, 1356, 1177, 1082, 976, 821, 712 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 283.1672 [M+Na]⁺ (calcd for C₁₇H₂₄O₂Na, 283.1674).

TM-135 (8):

a colorless solid, ${\left[\alpha \right]}_{\text{D}}^{25}=+39.0°$ (c = 0.16, MeOH). UV(MeOH), λ_max (log ε) 203 (4.35), 215 (3.89) nm. IR (neat) ν_max 3388, 2967, 2931, 2875, 1708, 1500, 1456, 1374, 1102, 1054, 820 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 299.1618 [M+Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

Panowamycin A:

a colorless solid, ${\left[\alpha \right]}_{\text{D}}^{25}=-61.6°$ (c = 0.24, MeOH). UV(MeOH), λ_max (log ε) 203 (4.34), 215 (3.95) nm. IR (neat) ν_max 3411, 2964, 2931, 2876, 1707, 1502, 1454, 1373, 1101, 1053, 949, 819 cm⁻¹. ¹H NMR (700 MHz, CD₃OD): Table 1. ¹³C NMR (175 MHz, CD₃OD): Table 2. HRESIMS: m/z 299.1618 [M+Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1623).

ECD calculation.

The absolute configurations of TM-130, TM-131 and TM-132 were determined by quantum chemical calculation of ECD spectra using Gaussian 09 software.³² The stereoisomer was geometrically optimized using the DFT method at the B3LYP/6–31+G(d) level in acetonitrile to afford a preferred conformer.³³ The ECD spectrum was calculated using the TDDFT method at the PBE1PBE/6–311++G(d,p) level in acetonitrile.^34,35 The ECD spectra were generated by the program SpecDis³⁶ using a Gaussian band shape with a 0.3eV exponential half-width from dipole-length dipolar and rotational strengths.

Antibacterial Activity Assay.

A disk diffusion assay was used to test the antibacterial activity of the isolated compounds. The test bacteria, Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6051, Escherichia coli ATCC 11775, and Pseudomonas aeruginosa ATCC 9721 were inoculated into liquid LB medium (3 mL) and grown overnight at 200 rpm, 30 °C. The cultures (100 μL each) were mixed with warm LB agar (0.7%, 10 mL) and plated out in a Petri dish. Whatman paper disks were impregnated with the compound solution (10 mM, 10 μL) and transferred onto the agar plate. The plate was then incubated at 37 °C. Antibacterial activity was determined based on the formation of an inhibition zone and the diameter of the zone. Ampicillin (2 mg/mL) was used as a positive control.

In vitro cytotoxicity assay.

RAW264.7 murine macrophage, HeLa, NCI-H460, and MCF-7 cancer cell lines were cultured at 37 °C in a 5% CO₂ humidified incubator and maintained in high glucose Dulbecco’s Modified Eagle Medium containing streptomycin (100 mg/mL), amphotericin B (2.5 mg/L), and heat-inactivated fetal bovine serum (FBS) (10%). Suspensions of tested cell lines (cal. 1.0 × 10⁴ cells/well) were seeded in a 96-well culture plates and cultured for 12 h followed by treatment with various diluted concentrations of compounds. Control cultures were treated with culture medium alone. Cell viabilities were evaluated using MTT assay. Absorbance was read using a SpectraMax 190 micro plate reader at a wavelength of 490 nm. Cells in the exponential phase were used for all experiments.

Nitric oxide production inhibitory assay.

RAW264.7 macrophages (cal. 1 × 10⁴ cells/well) were seeded in a 96-well culture plate and cultured for 12 h. Cells were pre-treated with various concentrations of the drugs for 1 h and then co-incubated with LPS (25 ng/mL) for 24 h. NO concentrations in the medium were determined using Griess assay. Griess reagent (80 μL) was added to the media supernatants (80 μL) and then incubated at 37 °C for 15 min in the dark. Absorbance was measured at 520 nm using a SpectraMax 190 micro plate reader. NO concentrations were calculated using 0–100 μM sodium nitrite standards. The significance between the groups was determined by T-test. Results are expressed as the mean ± SD of the indicated numbers of independent experiments. Values of p < 0.05 were considered statistically significant.