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May 27, 2017 - Isolation, Synthesis, and Biological Activity of Chlorinated. Alkylresorcinols from Dictyostelium Cellular Slime Molds. Haruhisa Kikuch...
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Article Cite This: J. Nat. Prod. 2017, 80, 2716-2722

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Isolation, Synthesis, and Biological Activity of Chlorinated Alkylresorcinols from Dictyostelium Cellular Slime Molds Haruhisa Kikuchi,*,† Ikuko Ito,† Katsunori Takahashi,‡ Hirotaka Ishigaki,‡ Kyoichi Iizumi,§ Yuzuru Kubohara,§ and Yoshiteru Oshima† †

Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aza-Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan Department of Medical Technology, Faculty of Health Science, Gunma Paz University, 1-7-1, Tonyamachi, Takasaki 370-0006, Japan § Graduate School of Health and Sports Science, Juntendo University, 1-1 Hiraga-gakuendai, Inzai, Chiba 270-1695, Japan ‡

S Supporting Information *

ABSTRACT: Eight chlorinated alkylresorcinols, monochasiol A−H (1−8), were isolated from the fruiting bodies of Dictyostelium monochasioides. Compounds 1−8 were synthesized to confirm their structures and to obtain sufficient material for performing biological tests. Monochasiol A (1) selectively inhibited the concanavalin A-induced interleukin-2 production in Jurkat cells, a human T lymphocyte cell line. Monochasiols were biogenetically synthesized by the combination of biosynthetic enzymes relating to the principal polyketides, MPBD and DIF-1, produced by Dictyostelium discoideum.

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he cellular slime mold Dictyostelium discoideum is thought to be an excellent model organism for studying cell and developmental biology because of its simple developmental pattern.1 Vegetative cells of D. discoideum grow as a single amoeba by eating bacteria. When these cells are starved, they initiate a developmental program of morphogenesis, forming a slug-shaped multicellular aggregate. This aggregate differentiates into two cell types, prespore and prestalk cells, which are precursors to spores and stalk cells, respectively. At the end of its development, the aggregate forms a fruiting body consisting of spores and a multicellular stalk.2 In 2005, the genome sequencing of D. discoideum clearly revealed the presence of approximately 45 polyketide synthase (PKS) gene clusters.3 Another cellular slime mold species, D. purpureum, has also been reported to contain 50 predicted PKS genes.4 These numbers are even greater than those observed in Streptomyces avermitilis, which is known to contain abundant secondary metabolites. Thus, we have focused on the utility of cellular slime molds as a source of natural compounds. Recently, our group has isolated α-pyronoids,5a,d,g amino sugar derivatives,5b and aromatics5c,e,f with unique structures and various biological activities. Brefelamide exhibits an inhibitory effect on osteopontin expression.5c,6 Ppc-1 is a prenylated quinolone derivative isolated from Polysphondylium pseudocandidum; it serves as a mitochondrial uncoupler and is a candidate for new antiobesity drugs.5e,7 The above results indicate that cellular slime molds are an important source of lead compounds for drug discovery. This paper reports the isolation, structure elucidation, and synthesis of new chlorinated alkylresorcinols, monochasiols A− H (1−8), from the cellular slime mold Dictyostelium monochasioides (Figure 1). The selective inhibitory activities © 2017 American Chemical Society and American Society of Pharmacognosy

of these compounds against the production of interleukin-2 (IL-2) in Jurkat cells, a human T lymphocyte cell line, are also described.

Figure 1. Structures of monochasiols A−H (1−8).

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RESULTS AND DISCUSSION Isolation and Structural Elucidation. From the fruiting bodies (85 g) of D. monochasioides, monochasiols A (1, 1.6 mg), B (2, 3.4 mg), C (3, 3.6 mg), D (4, 3.4 mg), E (5, 8.2 mg), F (6, 3.8 mg), G (7, 5.2 mg), and H (8, 2.9 mg) were isolated. The aromatic regions of their 1H and 13C NMR data showed signals at δH 6.45 (2H, s) and 5.29 (2H, br s) in the 1H NMR data and δC 151.4, 143.8, 108.2, and 104.3 in the 13C NMR data. These indicate that the compounds contain a common aromatic skeleton. Received: May 27, 2017 Published: September 18, 2017 2716

DOI: 10.1021/acs.jnatprod.7b00456 J. Nat. Prod. 2017, 80, 2716−2722

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Two peaks observed in the EIMS of monochasiol A (1) at m/z 326 and 328 for the molecular ion peaks at a ratio of 3:1 suggest that it contains one chlorine atom. Its HREIMS (m/z 326.2012 [M]+) indicated that its molecular formula is C19H31O2Cl. Except for those in the common aromatic skeleton, protons associated with 12 methylene groups (δ 2.49 (2H, t, J = 7.8 Hz), 1.56 (2H, quint, J = 7.8 Hz), and 1.22−1.32 (20H, m)) and one methyl group (δ 0.88 (3H, t, J = 6.9 Hz)) were observed in the 1H NMR data. These indicate the presence of a tridecyl group (C13H27). Thus, the remaining molecular formula for the common aromatic skeleton is C6H4O2Cl, which corresponds to a symmetrical tetrasubstituted benzene ring (δC 151.4, 143.8, 108.2, and 104.3) containing a chlorine atom and two phenolic hydroxy groups (δH 5.29 (2H, br s)). In the HMBC spectrum of 1, the correlation between two phenyl protons (δH 6.45 (2H, s)) and a benzylic methylene carbon (δC 35.7, Figure 2) suggests that

Scheme 1. Determination of Position of the Double Bond of Monochasiols D (4) and E (5)

data.10 The structure of monochasiol G (7), the HREIMS (m/z 366.2320 [M]+) of which showed the molecular formula C22H35O2Cl, also contained a cis-cyclopropane ring in its C15 side chain. The opening of a cyclopropane ring into a methylbranched alkyl chain has been widely exploited for determining the position of a cyclopropane ring in fatty acid methyl esters.11 This procedure was utilized for the hydrogenation of 6 catalyzed by platinum(IV) oxide; however, several products were obtained by the partial hydrogenation of the benzene ring of 6. Thus, we planned to synthesize plausible structures of 6 and 7 to elucidate these structures. Typically, a cyclopropane ring in an alkyl chain is biogenetically synthesized by the methylenylation of a double bond.12 Therefore, the structures of 6 and 7 are hypothesized to contain a cyclopropane ring between C-6′ and C-7′ and between C-8′ and C-9′, respectively, which correspond to those of 4 and 5, respectively. These plausible structures should be confirmed by the comparison of the NMR and mass spectra of the natural and synthesized compounds. In addition, the absolute configurations of the cyclopropane moieties of 6 and 7 were not determined because of the extremely low optical activities observed for their quasisymmetric structures ([α]D −1.4 (c 0.38, CHCl3) for 6; [α]D +0.9 (c 0.48, CHCl3) for 7). It is also possible that these compounds are racemic. Although a method for forming α-oxocyclopropane has been reported to determine such absolute configurations,13 small amounts of isolated natural monochasiols F (6) and G (7) restricted applying this method due to very low yield of the oxidation. The ω-7 long alkyl chains of 6 and 7 suggest that the biogenetic origin of 6 and 7 is lactobacillic acid (discussed later) (Figure 3), which is widespread in several bacteria, e.g., Klebsiella aerogenes,14 fed with D. monochasioides under the culture conditions employed herein. Thus, the absolute configurations of 6 and 7 are assumed to be 6′R, 7′S and 8′R, 9′S, respectively, which are the same as those in lactobacillic acid.15 The molecular formula of monochasiol H (8) as suggested from its HREIMS (m/z 350.2000 [M]+) was C21H31O2Cl. This formula differs from that of 3 by four hydrogen atoms. Olefinic protons (δ 5.38 (4H, m)) and allylic protons (δ 2.05 (8H, m)) in the 1H NMR data indicate that 8 contains two double bonds in its C15 side chain. The DMDS adduct method was applied; however, no products affording mass fragments that reveal the

Figure 2. Structural elucidation of a common aromatic skeleton of monochasiols.

two phenyl protons are located on C-2 and C-6. Thus, two phenolic hydroxy groups are located on C-3 and C-5, and a chlorine atom is located on C-4 because of the symmetry of substitutions on a benzene ring, confirming the structure of 1. The molecular formulas of monochasiols B (2) and C (3) were C20H33O2Cl and C21H35O2Cl, as revealed by the HREIMS data (m/z 340.2162 [M]+ and 354.2333 [M]+ for 2 and 3, respectively). The formulas differ from that of 1 by one and two methylene units, respectively. In addition, the signals of methylene protons (δ 1.20−1.34 (22H, m) and 1.21−1.36 (24H, m)) were observed in the 1H NMR data of 2 and 3, respectively, instead of the signal of δ 1.22−1.32 (20H, m) in 1. Thus, the structures of 2 and 3 contain a tetradecyl and pentadecyl side chain, respectively. From the HREIMS data (m/z 324.1873 [M] + ) of monochasiol D (4), its molecular formula was determined to be C19H29O2Cl, which differs from that of 1 by two hydrogen atoms. Olefinic protons (δ 5.34 (2H, m)) and allylic protons (δ 2.01 (4H, m)) in the 1H NMR data indicate that 4 contains one double bond in its C13 side chain. The double-bond positions were determined through the dimethyl disulfide (DMDS) adduct method.8 In the EIMS of the DMDS adduct of 4, we observed two strong peaks at m/z 319 and 145, respectively, which indicate that the double bond is located between C-6′ and C-7′ (Scheme 1A). In the 13C NMR data of 4, the signals corresponding to allylic carbons (C-5′ and C-8′) at 27.2 and 27.1 ppm, respectively, are indicative of the cis double-bond configuration.9 Similarly, monochasiol E (5) contained a C15 alkenyl chain with a cis double bond between C-8′ and C-9′ (Scheme 1B). The molecular formula of monochasiol F (6) as revealed by its HREIMS (m/z 338.2035 [M]+) was C20H31O2Cl. This formula differs from that of 1 by one carbon atom. The structure of 6 contains a cis-cyclopropane ring in its C13 side chain, due to the higher-field signals (δ 0.64 (2H, m), 0.56 (1H, dt, J = 8.3, 4.9 Hz), −0.34 (1H, q, J = 4.9 Hz)) in its 1H NMR 2717

DOI: 10.1021/acs.jnatprod.7b00456 J. Nat. Prod. 2017, 80, 2716−2722

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sorcinol was directly chlorinated, a mixture of the favored 2chloro compound and the disfavored 4-chloro compound was obtained. Thus, after the selective chlorination of 3,5dimethoxyaniline, the Sandmeyer iodination of 8 provided 9, the methoxy groups of which were converted into the methoxymethyl (MOM) groups to give 10 (Scheme 4). Scheme 4. Synthesis of the Aromatic Part of Monochasiols F (6) and G (7)

Figure 3. Structural comparison between plausible strucutures of monochasiols F (6) and G (7) and lactobacillic acid.

location of double bonds were obtained. Instead, the molecular masses of the product obtained from the cross-metathesis16 of 8 with styrene were 234, indicating the presence of two methylene units between two double bonds. In addition, 1 H−1H COSY showed the linkage of benzylic methylene (δ 2.51 (2H, t, J = 7.8 Hz), H-1′)−homoallylic methylene (1.64 (2H, quint, J = 7.8 Hz), H-2′)−allylic methylene (2.04−2.11 (2H, m), H-3′). Therefore, the double bonds are located between C-4′ and C-5′ and between C-8′ and C-9′, respectively (Scheme 2). Scheme 2. Determination of Position of the Double Bonds of Monochasiol H (8) Sonogashira coupling of 10 with 3-butyn-1-ol afforded phenylalkyne 11f. Its triple bond was hydrogenated using a palladium-activated carbon ethylenediamine complex [Pd/ C(en)]19 instead of ordinary Pd/C to avoid dechlorination to give 12f; next, 12f was subjected to thioetherification and subsequent oxidation to form phenyltetrazole sulfone 13f, which corresponds to the aromatic part of 6. ((1R,2S)-2-Hexylcyclopropyl)methanol, the precursor of the chiral cyclopropane part of 6 and 7, has been already synthesized.20 Oxidation into an aldehyde and the subsequent Julia−Kocienski olefination with 13f afforded 14f as a mixture of cis/trans isomers (E/Z = 7:1) (Scheme 5). Then, the diimide

Synthesis of Monochasiols. To confirm the structures and to obtain a sufficient amount of material for performing several biological tests, monochasiols were synthesized. In particular, the structures of 6 and 7 had to be reconfirmed. Although the synthesis of alkylresorcinols has been reported in some studies,17 cyclopropane-containing and halogenated alkylresorcinols have not been synthesized thus far. Our strategy to synthesize plausible structures of 6 and 7 involved the connection of an aromatic part and a cyclopropane part via the Julia−Kocienski olefination18 (Scheme 3). A tetrasubstituted benzene ring of the aromatic part had to be constructed regioselectively. Nevertheless, when 5-alkylre-

Scheme 5. Synthesis of Monochasiols F (6) and G (7)

Scheme 3. Retrosynthetic Pathway of Monochasiols F (6) and G (7) reduction of 14f and deprotection of the MOM groups produced monochasiol F (6). Similarly, monochasiol G (7) was also synthesized by the connection of ((1R,2S)-2hexylcyclopropyl)methanol and 13g. The spectral data of synthetic 6 and 7 were identical to those of natural 6 and 7, respectively. Particularly, all fragment ion peaks from the EIMS of synthetic and natural compounds were completely consistent (Supporting Information). Thus, the proposed planar structures of 6 and 7 are confirmed, although their absolute 2718

DOI: 10.1021/acs.jnatprod.7b00456 J. Nat. Prod. 2017, 80, 2716−2722

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configurations are not elucidated because of low optical activities ([α]D +0.2 (c 0.54, CHCl3) for synthetic 6; [α]D −0.2 (c 0.90, CHCl3) for synthetic 7). Monochasiols A−C (1−3) were synthesized by the Sonogashira coupling of 10 with terminal alkynes and subsequent saturation of triple bonds catalyzed by Pd/C(en) (Scheme 6). Scheme 6. Synthesis of Monochasiols A−C (1−3)

For the synthesis of monochasiol D (4), acetylenic alcohol 16d was obtained by a similar synthetic pathway to that for 11f and 11g (Scheme 7). After the hydrogenation and bromination Scheme 7. Synthesis of Monochasiols D (4), E (5), and H (8)

Figure 4. (A) Antiproliferative activities of monochasiols A−H (1−8) on HeLa cells. Cells were incubated with 20 μM of each compound for 4 days, and the relative cell number was estimated. (B) Effects of monochasiol A−H (1−8) on ConA-induced IL-2 production in Jurkat cells. (C) Selective immunosuppressive effect of monochasiol A (1). Cells were preincubated for 30 min with 0.1% DMSO (control) or indicated concentrations of each compound. After addition of ConA, cells were further incubated for 12 h and assayed for IL-2 protein production and cell viability. Results are presented as mean values of three independent experiments (n = 3). The bars indicate the standard errors.

of 16d, the extension of the chain with 1-octyne and subsequent deprotection of the MOM groups provided 18d. Finally, partial hydrogenation of triple bonds catalyzed by palladium-polyethylenimine (Pd/PEI)21 afforded monochasiol D (4). Similarly, the chain extension of bromides 17e,h with 1octyne and dodec-5-en-1-yne (19) afforded monochasiols E (5) and H (8), respectively. Biological Evaluation. The biological activity of monochasiols A−H (1−8) was investigated. Some monochasiols (20 μM) only exhibited weak antiproliferative activity against HeLa cells (Figure 4A). On the other hand, monochasiols exhibited inhibitory activities against concanavalin A (ConA)-induced IL2 production22 in Jurkat cells. Monochasiols (20 μM) inhibited the production of IL-2 to less than 50% of the control (Figure 4B). In particular, monochasiol A (1) exhibited inhibitory activity (IC50 16 μM) in a concentration-dependent manner without suppressing cell proliferation (Figure 4C). Thus, this effect of monochasiol A (1) is selective for IL-2 production, suggesting that compound 1 is a promising candidate for novel immunosuppressive drugs.

Plausible Biosynthesis. Although some studies have reported chlorinated alkylresorcinols from nature,23 monochasiols are the first examples of chlorinated resorcinols bearing a long alkyl chain (particularly, those greater than 10 carbons). Monochasiols are assumed to be biogenetically synthesized by analogous enzymes to essential biosynthetic enzymes for principal polyketides, 4-methyl-5-pentylbenzene-1,3-diol (MPBD),24 and differentiation-inducing factor-1 (DIF-1)25 (Scheme 8) produced by D. discoideum. The plausible biosynthetic pathways for 6 and 7 could involve one or two β-oxidations of lactobacillic acid followed by elongation of the polyketide chain three times by an analogous enzyme of Steely A,26 an essential polyketide synthase for MPBD, affording tetraketides 23f and 23g. They were cyclized into resorcinols 24f and 24g, respectively. Then, chlorination of 24f and 24g was done using an analogous enzyme of flavin-dependent halogenase,27 an essential biosynthetic enzyme for DIF-1, to afford 6 and 7. These plausible biosynthetic pathways support 2719

DOI: 10.1021/acs.jnatprod.7b00456 J. Nat. Prod. 2017, 80, 2716−2722

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with increasing polarity to afford hexane−EtOAc (9:1) eluent (fraction A, 163 mg). Fraction A was further separated by ODS column using a water−acetonitrile solvent system to give a water−acetonitrile (1:4) elutant (fraction B, 86.1 mg). Fraction B was subjected to recycle preparative HPLC (column, JAIGEL-GS310 (ϕ 20 mm × 500 mm, Japan Analytical Industry Co., Ltd.); solvent, ethyl acetate) to give three fractions, C-1 (20.9 mg), C-2 (36.1 mg), and C-3 (6.5 mg). Fraction C-1 was subjected to preparative HPLC (column, Mightysil RP-18GP (ϕ 20 mm × 250 mm, Kanto Chemical Co., Inc.); solvent, water−acetonitrile (1:9)) to give monochasiol D (4) (3.4 mg), monochasiol F (6) (3.8 mg), and monochasiol H (8) (2.9 mg). Fraction C-2 was subjected to preparative HPLC (column, Mightysil RP-18GP (ϕ 20 mm × 250 mm, Kanto Chemical Co., Inc.); solvent, water−acetonitrile (2:23)) to give monochasiol B (2) (3.4 mg), monochasiol C (3) (3.6 mg), and monochasiol G (7) (5.2 mg). Fraction C-3 was subjected to preparative HPLC (column, Wakopak Navi C30-5 (ϕ 20 mm × 250 mm, Wako Pure Chemical Industries, Ltd.); solvent, water−acetonitrile (13:87)) to give monochasiol A (1) (1.6 mg) and monochasiol E (5) (8.2 mg). Monochasiol A (1): colorless, amorphous solid; 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.29 (2H, br s), 2.49 (2H, t, J = 7.8 Hz), 1.50−1.60 (2H, m), 1.22−1.34 (20H, m), 0.88 (3H, t, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3) δ 151.4 (2C), 143.8, 108.2 (2C), 104.3, 35.7, 32.0, 31.0, 29.74, 29.72, 29.71, 29.70, 29.6, 29.5, 29.4, 29.2, 22.8, 14.2; EIMS m/z 328 [M + 2]+ (11%), 326 [M]+ (31%), 160 (34%), 158 (100%); HREIMS m/z 326.2012 [M]+ (326.2013 calcd for C19H31O235Cl). Monochasiol B (2): colorless, amorphous solid; 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.28 (2H, br s), 2.49 (2H, t, J = 7.8 Hz), 1.52−1.61 (2H, m), 1.20−1.34 (22H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.2 (2C), 104.3, 35.7, 31.9, 31.0, 29.71, 29.69, 29.68, 29.67 (2C), 29.57, 29.5, 29.4, 29.2, 22.7, 14.1; EIMS m/z 342 [M + 2]+ (8%), 340 [M]+ (22%), 160 (35%), 158 (100%); HREIMS m/z 340.2162 [M]+ (340.2169 calcd for C20H33O235Cl). Monochasiol C (3): colorless, amorphous solid; 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.28 (2H, br s), 2.49 (2H, t, J = 7.8 Hz), 1.51−1.61 (2H, m), 1.21−1.36 (24H, m), 0.88 (3H, t, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.3 (2C), 104.3, 35.7, 31.9, 31.0, 29.71, 29.70, 29.69, 29.68, 29.67 (2C), 29.57, 29.48, 29.37, 29.2, 22.7, 14.1; EIMS m/z 356 [M + 2]+ (8%), 354 [M]+ (23%), 160 (34%), 158 (100%); HREIMS m/z 354.2333 [M]+ (354.2326 calcd for C21H35O235Cl). Monochasiol D (4): colorless, amorphous solid; 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.30−5.40 (4H, m), 2.49 (2H, t, J = 7.4 Hz), 1.97−2.09 (4H, m), 1.57 (2H, quint, J = 7.2 Hz), 1.21−1.39 (12H, m), 0.88 (3H, t, J = 6.7 Hz); 13C NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.8, 130.1, 129.6, 108.2 (2C), 104.3, 35.7, 31.8, 30.9, 29.7, 29.6, 29.0, 28.8, 27.2, 27.1, 22.7, 14.1; EIMS m/z 326 [M + 2]+ (7%), 324 [M]+ (20%), 230 (6%), 228 (20%), 160 (38%), 158 (100%); HREIMS m/z 324.1873 [M] + (324.1856 calcd for C19H29O235Cl). Monochasiol E (5): colorless, amorphous solid; 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.32−5.37 (2H, m), 5.29 (2H, br s), 2.49 (2H, t, J = 7.8 Hz), 1.96−2.08 (4H, m), 1.52−1.62 (2H, m), 1.22−1.39 (16H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.8, 130.0, 129.8, 108.2 (2C), 104.3, 35.7, 31.8, 30.9, 29.7 (2C), 29.3, 29.2, 29.1, 29.0, 27.2, 27.1, 22.7, 14.1; EIMS m/z 354 [M + 2]+ (6%), 352 [M]+ (16%), 258 (5%), 256 (15%), 160 (35%), 158 (100%); HREIMS m/z 352.2153 [M]+ (352.2169 calcd for C21H33O235Cl). Monochasiol F (6): colorless, amorphous solid; [α]D −1.4 (c 0.38, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.20−5.40 (2H, br s), 2.50 (2H, t, J = 7.6 Hz), 1.58 (2H, quint., J = 7.6 Hz), 1.20−1.42 (14H, m), 1.08−1.17 (2H, m), 0.88 (3H, t, J = 7.1 Hz), 0.60−0.69 (2H, m), 0.56 (1H, dt, J = 8.3, 4.9 Hz), −0.34 (1H, q, J = 4.9 Hz); 13C NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.3 (2C), 104.3, 35.7, 32.0, 31.0, 30.2, 30.0, 29.4, 29.2, 28.7, 28.6, 22.7, 15.8, 15.7, 14.1,

Scheme 8. (A) Biosynthetic Pathway of MPBD and DIF-1; (B) Plausible Biosynthetic Pathway of Monochasiols F (6) and G (7)

our assumption of the absolute configurations of 6 and 7, which are described earlier. Other monochasiols can also be biosynthesized from their corresponding fatty acids. The biosynthesis of monochasiols via the combination of the biosynthetic enzymes for principal polyketides, MPBD and DIF-1, suggests that cellular slime molds produce considerably diverse secondary metabolites; thus, cellular slime molds are a promising source for novel metabolites.

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EXPERIMENTAL SECTION

General Experimental Procedures. Analytical TLC was performed on silica gel 60 F254 (Merck). Column chromatography was carried out on silica gel 60 (70−230 mesh, Merck). NMR spectra were recorded on JEOL ECA-600 and AL-400. Chemical shifts for 1H and 13C NMR are given in parts per million (δ) relative to tetramethylsilane (δ H 0.00) and residual solvent signals (δC 77.0) as internal standards. Mass spectra were measured on JEOL JMS-700 and JMS-DX303. Organism and Culture Conditions. Dictyostelium monochasioides JKS728 was kindly supplied by Dr. Hagiwara, National Science Museum, Tokyo, Japan. Spores were cultured at 22 °C with Klebsiella aerogenes on A-medium consisting of 0.5% glucose, 0.5% polypeptone, 0.05% yeast extract, 0.225% KH2PO4, 0.137% Na2HPO4·12H2O, 0.05% MgSO4·7H2O, and 1.5% agar. When fruiting bodies had formed after 4 days, they were harvested and stored in a refrigerator at −18 °C. Collected fruiting bodies were lyophilized for extraction. Isolation of Monochasiols. The lyophilized fruiting bodies (84.6 g) of D. monochasioides were extracted three times with methanol at room temperature to give an extract (19.4 g), which was then partitioned with ethyl acetate and water to yield ethyl acetate solubles (3.04 g). The ethyl acetate solubles were chromatographed over silica gel, and the column was eluted with hexane−ethyl acetate solutions 2720

DOI: 10.1021/acs.jnatprod.7b00456 J. Nat. Prod. 2017, 80, 2716−2722

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10.9; EIMS m/z 340 [M + 2]+ (4%), 338 [M]+ (13%), 227 (19%), 225 (55%), 160 (35%), 158 (100%); HREIMS m/z 338.2035 [M]+ (338.2013 calcd for C20H31O235Cl). Monochasiol G (7): colorless, amorphous solid; [α]D +0.9 (c 0.48, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.30 (2H, br s), 2.49 (2H, t, J = 7.8 Hz), 1.57 (2H, quint., J = 7.8 Hz), 1.21−1.42 (18H, m), 1.08−1.17 (2H, m), 0.88 (3H, t, J = 7.0 Hz), 0.60−0.69 (2H, m), 0.56 (1H, dt, J = 8.3, 4.9 Hz), −0.34 (1H, q, J = 4.9 Hz); 13C NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.3 (2C), 104.3, 35.7, 32.0, 31.0, 30.2 (2C), 29.55, 29.52, 29.4, 29.2, 28.73, 28.69, 22.7, 15.8, 15.7, 14.1, 10.9; EIMS m/z 368 [M + 2]+ (5%), 366 [M]+ (14%), 330 (6%), 270 (8%), 197 (10%), 160 (35%), 158 (100%); HREIMS m/z 366.2320 [M]+ (366.2326 calcd for C22H35O235Cl). Monochasiol H (8): colorless, amorphous solid; 1H NMR (600 MHz, CDCl3) δ 6.45 (2H, s), 5.33−5.39 (4H, m), 5.31 (2H, br s), 2.51 (2H, t, J = 7.8 Hz), 2.04−2.11 (6H, m), 2.00 (2H, q, J = 6.9 Hz), 1.64 (2H, quint, J = 7.8 Hz), 1.22−1.35 (8H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (150 MHz, CDCl3) δ 151.5 (2C), 143.5, 130.5, 130.0, 129.4, 129.1, 108.3 (2C), 104.4, 35.2, 31.8, 30.9, 29.7, 29.0, 27.5, 27.34, 27.29, 26.7, 22.7, 14.1; EIMS m/z 352 [M + 2]+ (9%), 350 [M]+ (27%), 314 (10%), 253 (4%), 251 (11%), 225 (20%), 199 (10%), 197 (29%), 160 (35%), 158 (100%); HREIMS m/z 350.2000 [M]+ (350.2013 calcd for C21H31O235Cl). Dimethyl Disulfide Adduction of Monochasiols D and E. To a solution of monochasiol D (4) (0.23 mg) in diethyl ether (0.1 mL) were added dimethyl disulfide (20 μL) and iodine (1.2 mg). After being stirred for 5 h at 50 °C, the reaction mixture was cooled to room temperature, poured into 5% sodium thiosulfate solution (0.5 mL), and extracted with diethyl ether (0.3 mL) three times. The combined organic layer was concentrated in vacuo to give a crude DMDS adduct of 4 for MS analysis. EIMS m/z 466 (25%), 464 (52%), 420 (24%), 418 (54%), 370 (28%), 321 (37%), 319 (79%), 275 (25%), 273 (69%), 227 (14%), 225 (40%), 145 (100%). In a similar way, monochasiol E (5) was converted into a DMDS adduct for MS analysis. EIMS m/z 494 (25%), 492 (50%), 448 (14%), 446 (35%), 398 (23%), 349 (48%), 347 (100%), 303 (19%), 301 (54%), 145 (78%). Cross-Metathesis of Monochasiol H (8) with Styrene. To a solution of monochasiol H (8) (0.85 mg) in dichloromethane (1 mL) were added Grubbs catalyst second generation (0.2 mg) and styrene (3.5 mg). After being stirred for 1.5 h at 40 °C under an argon atmosphere, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was chromatographed over silica gel eluted by hexane−ethyl acetate (49:1) to give styrene-adducted fragment of 8 for MS analysis. EIMS m/z 234 (43%), 117 (100%). Assay for Cell Growth in HeLa Cells. Cells were maintained at 37 °C (5% CO2) in tissue culture dishes filled with an appropriate medium. HeLa cells were in DMEM-HG (Dulbecco’s modified Eagle’s medium containing a high concentration (4500 mg/L) of glucose supplemented with the antibiotics and 10% fetal bovine serum). HeLa (5 × 103 cells/well) cells were allowed to grow for 3 days in 12-well plates; each well was then filled with 1 mL of DMEM-HG containing DMSO (0.2%) or the test compound. The relative cell number was assessed using Alamar blue (cell number indicator) as described previously.29 Assay for IL-2 Production by Jurkat Cells and Determination of Cell Viability. Jurkat cells were preincubated for 30 min in 12-well culture plates filled with 1 mL of RPMI (at 1 × 106 cells/mL) in the presence of the test compounds or 0.1% DMSO (vehicle). After the preincubation, ConA (final concentration, 25 μg/mL) was added to each culture, and the cells were further incubated for 12 h. Aliquots of the culture media were collected, and the levels of IL-2 were assessed by using immunoassay kits (ENDOGEN, Rockford, IL, USA). Briefly, 50 μL aliquots (in duplicate) of the culture media or standards for IL-2 from the kits were added to the wells of 96-well plates precoated with antihuman IL-2 antibody. After incubation with biotinylated antibodies to human IL-2 and then with streptavidin-horseradish peroxidase, color was developed and the levels of IL-2 were quantified by measuring the absorbance of each sample at 450 and 550 nm. The readings at 550 nm were subtracted from the readings at 450 nm. To

determine cell viability, cells were incubated under the same conditions, and the percentage of viable cells compared with controls was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay according to standard procedures.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00456. Experimental methods for synthesis of monochasiols and NMR spectra of the new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail (H. Kikuchi): [email protected]. Phone: +81-22-795-6824. Fax: +81-22-795-6821. ORCID

Haruhisa Kikuchi: 0000-0001-6938-0185 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the Grants-in-Aid for Scientific Research (no. 16H03279) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; the Platform Project for Supporting Drug Discovery and Life Science Research funded by Japan Agency for Medical Research and Development (AMED); the Shorai Foundation for Science and Technology; Kobayashi International Scholarship Foundation; and the Takeda Science Foundation.

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REFERENCES

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