Note pubs.acs.org/jnp
Identification of Pyridinium with Three Indole Moieties as an Antimicrobial Agent Masahiro Okada,*,† Tomotoshi Sugita,† Chin Piow Wong, Toshiyuki Wakimoto,‡ and Ikuro Abe* Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan S Supporting Information *
ABSTRACT: A novel pyridinium with three indole moieties, tricepyridinium, was obtained from the culture of an Escherichia coli clone incorporating metagenomic libraries from the marine sponge Discodermia calyx. For the important structural elements of tricepyridinium to be investigated for antibacterial activity, tricepyridinium and its analogues were chemically synthesized. Tricepyridinium had antimicrobial activity, but not against E. coli, and cytotoxicity against P388 cells. Additional bioassays with its synthetic analogues revealed that the intriguing combination of the indole moieties, most likely derived from three tryptamines, as well as the pyridinium moiety were chiefly responsible for its potent biological activities.
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any natural products, especially secondary metabolites, are attractive as drugs and drug leads due to their broad biological activities. The indole ring, derived from tryptophan, or its derivatives such as tryptamine and indole itself, is one of the most common moieties in many biologically active alkaloids.1−4 Hence, numerous biologically active natural products containing an indole ring have been isolated from various natural sources including marine sponges using bioassay-guided screening. Marine sponges including their symbiotic microorganisms are attractive sources for the discovery of biologically active secondary metabolites. For instance, the potent protein phosphatase inhibitor calyculin A and its derivatives were isolated from the marine sponge Discodermia calyx collected in relatively shallow waters from the ocean around Japan.5 We also previously reported several cytotoxic secondary metabolites, calyxamides6 and sulfolipodiscamides,7 from D. calyx and D. kiiensis, respectively. The metagenome mining approach is a powerful tool not only to identify biosynthetic gene clusters of secondary metabolites but also to directly isolate bioactive compounds likely derived from genes of symbiotic microorganisms.8,9 Indeed, the biosynthetic gene cluster of calyculin A was recently identified by using metagenomic fosmid libraries of the spongemicrobe consortium of D. calyx.10 The huge NRPS-PKS hybrid genes corresponding to calyculin A synthase are encoded by an uncultured symbiotic bacterium, Candidatus “Entotheonella”.11 On the other hand, the clones transformed with metagenomic libraries serve as a rich source of various biologically active compounds. Therefore, we screened the Escherichia coli EPI300 clones transformed with metagenomic libraries of D. calyx to search for new bioactive molecules and identified several antibacterial compounds.12−15 In particular, several antibacterial indole metabolites have been isolated, such as indole-porphyrin © 2017 American Chemical Society and American Society of Pharmacognosy
hybrid molecules,14 2,2-di(3-indolyl)-3-indolone,15 and turbomycins.15,16 In this study, a novel pyridinium with three indole moieties, tricepyridinium (1), originally detected from the culture extracts of the E. coli clone incorporating metagenomic libraries of D. calyx, and its analogues were chemically synthesized. The biological activities of 1 and its synthetic analogues are also reported herein. In our previous research, 3-hydroxypalmitic acid was isolated as a moderate antibacterial compound against Bacillus cereus from the culture extracts of the E. coli EPI300 clone (pDC113) incorporating metagenomic libraries of D. calyx.12 After purification of the culture extracts by column chromatography with Diaion HP-20, a fraction containing compound 1 exhibited significant antibacterial activity. Compound 1 was separated by HPLC with a MeCN-aqueous acetic acid solvent system to give 1 probably as an acetate salt (1a). NMR analysis indicated the presence of two symmetrical 3-substituted indole rings and one 3-ethylindole moiety. In addition, two equivalent aromatic protons and one additional aromatic proton were Received: December 15, 2016 Published: March 14, 2017 1205
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observed as a singlet in the 1H NMR spectrum (Table 1, Figures S1 and S2). These findings together with MS analysis
palladium(II) dichloride [PdCl2(dppf)] and potassium acetate resulted in the synthesis of diboronate 3.17 Successive Suzuki− Miyaura cross-coupling of 1-Boc-3-bromoindole with catalytic PdCl2(dppf) and potassium carbonate gave bisindolyl compound 4.18 After deprotection of the Boc group, the resulting 5 was treated with 3-(2-bromoethyl)indole at 90 °C for 42 h to give the desired 1-[2-(3-indolyl)ethyl]-3,5-di(3-indolyl)-pyridinium (1) as a bromide salt (1b). As expected, all the spectroscopic data for synthetic 1 were identical to those of the bacterial product. Thus, the chemical structure of 1 was confirmed to be 1-[2-(3-indolyl)ethyl]-3,5-di(3-indolyl)-pyridinium, which we named tricepyridinium. In addition, we synthesized pyridinium derivatives with two indole moieties (6 and 7), a monoindolylpyridine derivative (8), and pyridinium derivatives with one indole moiety (9 and 10) to investigate structure−activity relationships (Figure 1 and Supporting Information).
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data of Tricepyridinium in CD3OD tricepyridinium (1) position 1 2, 6 3, 5 4 1′ (NH) 2′ 3′ 3a′ 4′ 5′ 6′ 7′ 7a′ 1″ (NH) 2″ 3″ 3a″ 4″ 5″ 6″ 7″ 7a″ 8″ 9″
δC, type 136.2, CH 137.0, C 135.7, CH
δH (J in Hz)
HMBC
8.49, 2H, s
2 (6), 3′, 9′
8.78, s
2 (6), 3′ 3 (5), 3′, 3a′, 7a′
126.0, 109.3, 124.1, 118.0, 121.1, 122.5, 112.0, 137.6,
CH C C CH CH CH CH C
7.70, 2H, s
124.0, 108.4, 126.7, 117.4, 119.3, 121.9, 111.7, 136.9, 27.4, 62.5,
CH C C CH CH CH CH C CH2 CH2
7.01, s
7.39, 7.14, 7.22, 7.47,
7.53, 7.06, 7.18, 7.34,
2H, 2H, 2H, 2H,
d (7.6) dd (7.4, 7.6) dd (7.4, 7.9) d (7.9)
d (7.6) dd (7.4, 7.6) dd (7.4, 7.9) d (7.9)
3a′, 3a′, 4′, 3a′,
6′, 7a′ 7′ 7a′ 5′
3″, 3a″, 7a″
3a″, 3a″, 4″, 3a″,
7a″ 7″ 7a″ 5″
Figure 1. Chemical structures of synthetic tricepyridinium analogues. 3.58, 2H, t (4.8) 5.00, 2H, t, (4.8)
2″, 3″, 3a″ 2 (6), 3″
The antimicrobial activity of 1 was investigated by measuring the minimum inhibitory concentration (MIC) according to the Clinical & Laboratory Standards Institute (CLSI) protocol.19 Tricepyridinium bromide (1b) displays significant antibacterial activity against Bacillus cereus with an MIC value of 0.78 μg/mL and methicillin-sensitive Staphylococcus aureus (MSSA) with an MIC value of 1.56 μg/mL as well as moderate antifungal activity against Candida albicans with an MIC value of 12.5 μg/ mL with no activity against E. coli at 100 μg/mL, as shown in Table 2.
indicated that 1 was 1-[2-(3-indolyl)ethyl]-3,5-di(3-indolyl)pyridinium, which has never been isolated previously. To confirm the structure of 1 and evaluate its biologically activities, we chemically synthesized 1. Three indole moieties were introduced in two steps via Suzuki−Miyaura crosscoupling and an SN2 reaction of an alkyl bromide, as shown in Scheme 1. Using 3,5-dibromopyridine 2 as a starting material, a diborylation reaction by cross-coupling of bis(pinacolato)diboron with catalytic [1,1′-bis(diphenylphosphino)ferrocene]Scheme 1. Synthesis of Tricepyridinium Bromide (1b)
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in the fosmid containing the metagenomics gene cluster (pDC113) based on our previous research,12 the genes responsible for the biosynthesis of 1 remain elusive. Unfortunately, it is difficult to completely eliminate the possibility at this time that tricepyridinium is nonenzymatically synthesized.24 We propose that the introduction of the fosmid stimulated an endogenous biosynthetic pathway to tryptamine in the host E. coli cells. Notably, tryptamine and formaldehyde, which are toxic to the E. coli host, are consumed to produce tricepyridinium (1) that can be exported as a possible detoxification mechanism. Intriguingly, the released tricepyridinium (1) exhibits significant toxicity to other microorganisms but not to the E. coli producer. The discovery of novel compound 1 demonstrates that transformation of an environmentally derived DNA library is a useful approach to induce serendipitous synthesis of small molecules in a host organism. In this study, we isolated a novel pyridinium with three indole moieties, tricepyridinium (1), from E. coli transformed with metagenomic libraries of D. calyx, based on functional screening. Tricepyridinium (1) possessed three unsubstituted indole moieties and exhibited antibacterial activity, but not against E. coli, antifungal activity, and cytotoxicity against P388 cells. All three indoles contributed to these activities. In contrast to antimicrobial activity, elimination of the indole group at the 8″-position (6b) resulted in more potent cytotoxicity against P388 cells than that of tricepyridinium bromide (1b). It was demonstrated in this study that the synthesis of tricepyridinium (1) is feasible and that three indole moieties are easily substituted with similar derivatives using our synthetic scheme. The biological activity of 1 can be expected to significantly improve through future structure−activity relationship studies.
Table 2. Antimicrobial Activities of Tricepyridinium Bromide and Its Analogues MIC [μg/mL] compound
B. cereus
S. aureus (MSSA)
C. albicans
E. coli (W3110)
1b 5 6b 7b 8 9b 10b ampicillin
0.78 50.0 1.56 25.0 >100 >100 >100 0.063
1.56 50.0 1.56 50.0 100 >100 >100 0.50
12.5 50.0 25.0 100 100 100 100 100
>100 >100 100 >100 >100 >100 >100 4.00
The synthetic analogues with two indole moieties (5, 6b, and 7b) exhibited antimicrobial activity against B. cereus, MSSA, and C. albicans, but the intensities of 5 and 7b were much weaker than that of 1b. Intriguingly, the 1-ethylpyridinium analogue with two indole moieties (6b) exhibited antimicrobial activity not only superior to all synthetic analogues, comparable to that of 1b, but also weak activity against E. coli with an MIC value of 100 μg/mL. In contrast, the synthetic analogues with one indole moiety (8, 9b, and 10b) exhibited no or only weak antimicrobial activity with the MIC value of 100 μg/mL. Subsequently, we tested the toxicity of these compounds against the murine leukemic cell line P388. Compound 1b had cytotoxicity to P388 cells with an IC50 value of 0.53 ± 0.07 μg/ mL (1.0 ± 0.1 μM). Interestingly, the 1-ethylpyridinium analogue with two indole moieties (6b) exhibited the most potent cytotoxicity to P388 cells with the IC50 value of 0.093 ± 0.029 μg/mL (0.22 ± 0.07 μM), whereas the other analogues showed weaker cytotoxicity than that of 1b (Table S1). These results indicated that the three indole moieties of 1 and the charged pyridinium were chiefly responsible for the high cytotoxicity toward Gram-positive bacteria and fungi. In particular, the indole group at the 3- or 5-position was more crucial than an indole group at the 8″-position. Previous structure−activity relationship studies on pyridinium compounds demonstrated that 1-alkylpyridinium compounds generally exhibited antimicrobial activity.20−23 However, previous studies also revealed that the hydrophobic alkyl chain could improve antimicrobial activity in most cases up to an alkyl chain having 18 carbon atoms, and the 1alkylpyridinium compounds exhibited broad-spectrum antimicrobial activity including against E. coli. Actually, the 1ethylpyridinium analogue (6b) exhibited weaker antimicrobial activity against B. cereus and C. albicans but stronger activity against E. coli compared to that of 1b. Therefore, tricepyridinium (1) is likely to be a unique antimicrobial agent with a different mechanism of action from typical 1-alkylpyridinium compounds. In contrast to antimicrobial activity, the presence of the indole group at the 8″-position does not enhance cytotoxicity against P388 cells. This structure−activity relationship assessment indicated that the two indole moieties played a concerted role with the 1-ethylpyridinium moiety to induce cytotoxicity of P388 cells. It is remarkable that a molecule with three simple indole moieties displays high bioactivities, and the synthetic route we established is highly applicable to preparation of its derivatives. The biosynthesis of 1 is predicted to involve tryptamine and formaldehyde precursors (Scheme S1), and the three indole rings are likely derived from three tryptamines. Because an ORF involved in the biosynthesis of tryptamine is not present
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EXPERIMENTAL SECTION
General Experimental Procedures. The UV spectrum was recorded on a Shimadzu UV-1280 spectrophotometer. The IR spectrum was recorded on a JASCO FT/IR-4100 spectrometer with CaF2 plates. NMR spectra were recorded on a JEOL ECX-500 or ECA-500 spectrometer (1H NMR: 500 MHz; 13C NMR: 125 MHz) with either TMS (at 0.0 ppm) or residual solvent peaks with specific shift values as an internal standard (CDCl3: δH 7.26, δC 77.1; CD3OD: δH 3.31, δC 49.0). ESI-HRMS analysis was performed with a Bruker Compact QqTOF mass spectrometer. HPLC was performed on a Shimadzu LC-20A system using an ODS column (4.6 × 250 mm, COSMOSIL 5C18-PAQ, NACALAI TESQUE, Inc.). Solvents and chemicals were purchased from Wako Pure Chemical Industry, Kanto Chemical Co., Inc., or Sigma-Aldrich JAPAN unless otherwise noted. Preparation of E. coli EPI300 Clone pDC113. The preparation of the metagenomic DNA library from the marine sponge was previously reported.13,14 Briefly, metagenomic DNA was prepared from the marine sponge D. calyx collected at Shikine-jima Island in Japan, and then the DNA fragments larger than 35 kbp were separated from the total DNA by agarose gel electrophoresis. The DNA was blunt-ended, ligated into the pCC1FOS fosmid vector, and packaged to transfect E. coli according to the manufacturer’s instructions (Epicenter). The packaged vector was transformed into E. coli EPI300T1R, and the cells were plated on LB agar containing chloramphenicol (12.5 mg/L) as a selection marker according to the manufacturer’s instructions (Epicenter). Purification of Tricepyridinium Acetate (1a). After incubation of the clone pDC113 in LB medium supplemented with chloramphenicol (12.5 mg/L) at 30 °C and 120 rpm for 3 days, the broth was subjected to solid phase extraction with Diaion HP-20 resin (Mitsubishi Chemical Corp.) and then eluted with MeOH. The eluent was purified by HPLC on an ODS column (Cosmosil COSMOSIL 1207
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7.4, 7.6 Hz), 7.06 (1H, dd, J = 7.4, 7.6 Hz), 5.00 (2H, t, J = 4.8 Hz), 3.58 (2H, t, J = 4.8 Hz); 13C NMR (125 MHz, CD3OD) δC 137.6, 137.0, 136.9, 136.2, 135.7, 126.7, 126.0, 124.1, 124.0, 122.5, 121.9, 121.1, 119.3, 118.0, 117.4, 112.0, 111.7, 109.3, 108.4, 62.5, 27.4; HRESIMS m/z 453.2075 [M]+ (calcd for C31H25N4, 453.2074). Antimicrobial Activity. According to the CLSI protocol,19 the turbidity of suspensions of E. coli W3110, MSSA1, B. cereus, and C. albicans was adjusted to 0.5 McFarland, respectively, in PBS(−) and then diluted 300-fold with growth medium (Mueller Hinton Broth, Sigma-Aldrich) for E. coli, MSSA, and B. cereus, Roswell Park Memorial Institute 1640 medium (RPMI 1640, Sigma-Aldrich) with glutamine, without sodium bicarbonate, with 2% (w/v) glucose, and with phenol red, buffered to pH 7.0 with MOPS at 165 mM for C. albicans) in 96well plates. Each well contained serially diluted solutions of the compounds. Final culture volume for each well was adjusted to 100 μL, and after 20 h (24 h for C. albicans) incubation at 37 °C, MICs were scored visually. Ampicillin was used as a positive control. Cell Culture and Cytotoxic Activity. The murine leukemic cell line P388 was maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (MP Biomedicals) and 1.0% penicillin (10,000 U/mL)-streptomycin (10 mg/L). Cytotoxicity of the compounds was evaluated via MTT assay based on mitochondrial succinate dehydrogenase activity and confirmed via microscopic observation. At the end of the incubation, 15 μL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 5.0 mg/mL was added to each of the wells. The cultures were incubated for another 3 h before the cell supernatants were removed. After the removal of the cell supernatants, 100 μL of dimethyl sulfoxide was added to each well to dissolve the formed formazan. The optical density was measured using a microplate reader (Bio-Rad) at 550 nm with reference wavelength at 700 nm. Cisplatin and doxorubicin were used as positive controls.
5C18-PAQ, Nacalai Tesque), eluting with a gradient system from H2O to MeOH containing 0.1% acetic acid to give compound 1a as a white powder (0.76 mg/L broth). UV (MeOH) λmax (log ε) 224 (5.47), 261 (5.13), 279 (5.13), 324 (5.19), 394 (sh) (4.60) nm; IR (CaF2) νmax 3181, 1589, 1537, 1455, 1430, 1338, 1245 cm−1; 1H NMR (500 MHz, CD3OD) δH 8.77 (1H, s), 8.47 (2H, s), 7.68 (2H, s), 7.51 (1H, d, J = 7.6 Hz), 7.46 (2H, d, J = 7.9 Hz), 7.40 (2H, d, J = 7.9 Hz), 7.37 (1H, d, J = 7.9 Hz), 7.22 (2H, dd, J = 7.4, 7.9 Hz), 7.13 (2H, dd, J = 7.4, 7.9 Hz), 7.05 (1H, dd, J = 7.4, 7.6 Hz), 4.99 (2H, t, J = 4.8 Hz), 3.57 (2H, t, J = 4.8 Hz), 1.87 (3H, s); 13C NMR (125 MHz, CD3OD) δC 174.7, 137.9, 137.54, 137.49, 136.8, 136.5, 127.1, 126.3, 124.6, 124.4, 122.9, 122.3, 121.4, 119.7, 118.3, 117.8, 112.4, 112.4, 109.8, 108.8, 62.9, 27.8, 23.1; HRESIMS m/z 453.2072 [M]+ (calcd for C31H25N4, 453.2074). Synthetic Protocols for Tricepyridinium (1). Synthesis of 3,5Bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (3). To a solution of 3,5-dibromopyridine (2) (1.39 g, 5.87 mmol) in 1,4dioxane (50 mL) were added bis(pinacolato)diboron (3.42 g, 13.5 mmol), KOAc (3.45 g, 35.2 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) [PdCl2(dppf)2] (341 mg, 0.418 mmol). After the reaction mixture was stirred at 85 °C for 19 h, it was concentrated on a rotary evaporator. After the residue was suspended in CH2Cl2, the suspension was filtered. The filtrate was concentrated on the rotary evaporator to give a black solid. After the solid residue was dissolved in H2O containing formic acid (pH 1), the aqueous solution was washed with EtOAc. The aqueous solution was evaporated to give 3 (1.38 g, 4.17 mmol, 71%) as a brown powder, which was used without any further purification. 1H NMR (500 MHz, CDCl3) δH 8.87 (3H, s), 1.20 (24H, s); 13C NMR (125 MHz, CDCl3) δC 156.2, 149.5, 128.2, 84.3, 24.7. Unfortunately, HRESIMS could not be obtained. Synthesis of 3,5-Bis(1-Boc-indol-3-yl)pyridine (4). To a solution of 3 (231 mg, 0.698 mmol) in DMSO (7.0 mL) were added 1-Boc-3bromoindole (496 mg, 1.67 mmol), K2CO3 (964 mg, 6.98 mmol), and PdCl2(dppf)2 (57.0 mg, 69.8 μmol). After the mixture was stirred at 90 °C for 2 h, it was quenched with H2O and filtered through Celite to remove the palladium catalyst. The filtrate was extracted with EtOAc, washed with saturated aqueous NaCl, dried over Na2SO4, and evaporated. The residue was purified by silica gel column chromatography (hexane/acetone = 15:1 to 10:1) to give a mixture of dindolyl/monoindolyl compounds in a 5:1 molar ratio as a white powder. The two compounds were completely separated by HPLC to give 4 (261 mg, 0.512 mmol, 73%) as a white powder. 1H NMR (500 MHz, CDCl3) δH 8.89 (2H, brs), 8.25 (3H, d, J = 1.7 Hz), 7.91−7.76 (4H, m), 7.49−7.29 (4H, m), 1.72 (18H, s); 13C NMR (125 MHz, CDCl3) δC 149.6, 146.4, 136.0, 134.8, 130.6, 128.5, 125.2, 123.9, 123.5, 119.5, 118.2, 115.8, 84.5, 28.3; HRESIMS m/z 510.2389 [M + H]+ (calcd for C31H32N3O4, 510.2387). Synthesis of 3,5-Di(indol-3-yl)pyridine (5). To a solution of 4 (171 mg, 0.336 mmol) in CH2Cl2 (1.0 mL) was slowly added TFA (3.0 mL) in CH2Cl2 (1.0 mL) at 0 °C, and the mixture was stirred at 0 °C for 1 h. The reaction mixture was quenched and basified with 1 M aqueous KOH. The mixture was extracted with EtOAc, washed with saturated aqueous NaCl, dried over Na2SO4, and evaporated to give 5 (101 mg, 0.326 mmol, 97%) as a brown powder, which was used without any further purification. 1H NMR (500 MHz, CDCl3) δH 8.93 (1H, s), 8.53 (2H, s), 7.94 (2H, m), 7.50−7.35 (4H, m), 7.30−7.20 (4H, m); 13C NMR (125 MHz, CDCl3) δC 148.1, 136.8, 134.8, 131.9, 130.4, 125.5, 122.9, 122.4, 120.9, 119.4, 111.7; HRESIMS m/z 310.1332 [M + H]+ (calcd for C21H16N3, 310.1339). Synthesis of Tricepyridinium Bromide (1b). To a solution of 5 (90.0 mg, 0.291 mmol) in 1,4-dioxane (0.60 mL) was added 3-(2bromoethyl)indole (98.0 mg, 0.437 mmol), and the mixture was stirred at 90 °C for 42 h. After the mixture was quenched with H2O, EtOAc was added, and the mixture was extracted with H2O. The aqueous layer was evaporated to give 1b (97.0 mg, 0.182 mmol, 63%) as an orange solid. For further analysis, the compound was purified by HPLC to give analytically pure 1b as a yellow powder. 1H NMR (500 MHz, CD3OD) δH 8.78 (1H, s), 8.49 (2H, s), 7.70 (2H, s), 7.53 (1H, d, J = 7.6 Hz), 7.47 (2H, d, J = 7.9 Hz), 7.39 (2H, d, J = 7.6 Hz), 7.34 (1H, d, J = 7.9 Hz), 7.22 (2H, dd, J = 7.4, 7.9 Hz), 7.14 (2H, dd, J =
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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.6b01152. Synthetic protocols for analogues, NMR spectra, table of cytotoxicity, and plausible biosynthetic pathway (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Toshiyuki Wakimoto: 0000-0003-2917-1797 Ikuro Abe: 0000-0002-3640-888X Present Address ‡
T.W.: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060−0812, Japan Author Contributions †
M.O. and T.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant Number JP15H01836, JP16H06443, and JP24688011). Support was also provided by Takeda Science Foundation, Kobayashi International Scholarship Foundation, and a Grantin-Aid for the Cooperative Research Project from Institute of 1208
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Natural Medicine, University of Toyama in 2016 (to M.O.). We thank Dr. Rui He and Dr. Yuya Takeshige for their technical assistance.
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REFERENCES
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