Synthesis and Antimicrobial Evaluation of Fire Ant Venom Alkaloid

Oct 12, 2017 - The first synthesis of 2-methyl-6-pentadecyl-Δ1,6-piperideine (1), ... faecium with an IC50 value of 19.4 μg/mL, while compounds 2 an...
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Synthesis and Antimicrobial Evaluation of Fire Ant Venom Alkaloid Based 2‑Methyl-6-alkyl‑Δ1,6-piperideines Yujie Yan,† Yu An,† Xiaozhong Wang,† Yingqi Chen,† Melissa R. Jacob,‡ Babu L. Tekwani,‡ Liyan Dai,*,† and Xing-Cong Li*,‡ †

Zhejiang Provincial Key Laboratory of Advanced Chemical Engineering Manufacture Technology, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China ‡ National Centers for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of Mississippi, University, Mississippi 38677, United States S Supporting Information *

ABSTRACT: The first synthesis of 2-methyl-6-pentadecyl-Δ1,6-piperideine (1), a major alkaloid of the piperideine chemotype in fire ant venoms, and its analogues, 2-methyl-6-tetradecyl-Δ1,6-piperideine (2) and 2-methyl-6-hexadecyl-Δ1,6-piperideine (3), was achieved by a facile synthetic method starting with glutaric acid (4) and urea (5). Compound 1 showed in vitro antifungal activity against Cryptococcus neoformans and Candida albicans with IC50 values of 6.6 and 12.4 μg/mL, respectively, and antibacterial activity against vancomycin-resistant Enterococcus faecium with an IC50 value of 19.4 μg/mL, while compounds 2 and 3 were less active against these pathogens. All three compounds strongly inhibited the parasites Leishmania donovani promastigotes and Trypanosoma brucei with IC50 values in the range of 5.0−6.7 and 2.7−4.0 μg/mL, respectively. Boc-aminoketones 9−11. Finally, treatment of 9−11 with HCl furnished the desired product.10 It should be pointed out that tetrafluoroacetic acid (TFA), which is generally used to cyclize similar N-Boc-aminoketones,5,11 did not work well in this case. Previous studies have shown that the antifungal activities of compounds with a long aliphatic chain are strongly associated with the chain length.5,12,13 For example, both 6-alkyl-Δ1,6piperideines5 and phloeodictine derivatives with a C14−C16 aliphatic chain13 demonstrated strong in vitro antifungal activities. In addition, caspofungin, the first antifungal drug in the echinocandin class, was derived from the natural product pneumocandin B0, in which the aliphatic chain length was C14,14 while micafungin, the second antifungal drug in this class, was developed from FR901379 with a C16 aliphatic tail.15 We thus designed and synthesized the fire ant venom piperideine alkaloid 1 with a C15 aliphatic chain, along with analogues 2 and 3 with aliphatic chain lengths of C14 and C16, respectively. Compounds 1−3 were evaluated for in vitro antifungal activity against the clinically important opportunistic fungal pathogens Cryptococcus neoformans, Candida albicans, and Aspergillus f umigatus as well as antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), Escherichia coli, and Pseudomonas aeruginosa. All three compounds were inactive against A. f umigatus, MRSA, E. coli, and P. aeruginosa at the highest test concentration of 20 μg/mL. Compounds 1 and 2 showed antifungal activity against C. neoformans and C. albicans, while compound 3 was only active against C. neoformans. For the antibacterial activity, only compound 1 was active against

2-Methyl-6-alkylpiperidines represented by the solenopsins are characteristic alkaloids in the venoms of the imported fire ants Solenopsis invicta and Solenopsis richteri.1 In recent years, a series of 2,6-dialkyl-Δ1,6-piperideines were also identified by GC-MS from the venoms of these two imported fire ants and their hybrid.2−4 It was speculated that 2,6-dialkylpiperideines were precursors of the corresponding 2,6-dialkylpiperidines in their biosynthetic pathways.3 2-Methyl-6-pentadecyl-Δ1,6-piperideine (1) was proven to be a major constituent in the venom of S. invicta.4 Inspired by the potent antifungal activity of steroidal alkaloids containing a Δ1,6-piperideine structural moiety, 6alkyl-Δ1,6-piperideines, simple versions of 1, were synthesized and exhibited strong in vitro antifungal activity against human fungal pathogens.5 Another study showed that the total piperideines from the venom of S. invicta demonstrated potent in vitro activity against the agricultural fungal pathogen Pythium ultimum and protected cucumber seedlings from infection.6 Thus, 2,6-dialkylpiperideines are a class of compounds that may possess interesting biological activities. So far there have been no purified naturally occurring or synthetic 2,6-dialkylpiperideines available for biological studies. In the present study, we have developed a facile synthetic method to synthesize racemic 2-methyl-6-alkyl-Δ1,6-piperideines and evaluated their antifungal, antibacterial, and antiprotozoal activities. The synthetic methodology is shown in Scheme 1. First, glutaric acid (4) and urea (5) were used as starting materials to generate glutarimide (6) under melting conditions at 175 °C.7 Next, a Grignard reaction of 6 with CH3MgBr followed by reduction with NaBH3CN introduced a methyl group on the piperidinone ring of compound 7.8 Nitrogen protection of 7 with (Boc)2O catalyzed by 4-DMAP9 afforded compound 8, which was reacted with selected Grignard reagents to obtain N© XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 21, 2017

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DOI: 10.1021/acs.jnatprod.7b00625 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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4.0 μg/mL, comparable to the control drug alpha-difluoromethylornithine, with an IC50 value of 6.1 μg/mL. However, they did not inhibit THP-1 cells at the highest test concentration of 10 μg/mL, indicating low cytotoxicity. In conclusion, the first synthesis of the naturally occurring fire ant venom alkaloid 2-methyl-6-pentadecyl-Δ1,6-piperideine (1) and its analogues (2 and 3) was achieved by a five-step synthetic sequence. The facile synthetic method makes it possible to access other piperideine alkaloids. The demonstrated in vitro antifungal, antibacterial, and antiprotozoal activities of compounds 1−3 warrant further studies of this chemotype of compounds on additional biological activities, e.g., chemical defense of fire ants against insects and pests.16,17

Scheme 1. Synthesis of 2-Methyl-6-alkyl-Δ1,6-piperideines

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General Experimental Procedures. Melting points were determined on a WRS-18 digital melting-point apparatus. IR spectra were recorded on an Agilent Technologies Cary 630 FTIR spectrometer. NMR spectra were recorded in CDCl3 on a Bruker Avance DMX-500 MHz instrument, and chemical shifts were expressed in ppm relative to tetramethylsilane as an internal standard (δ = 0). The high-resolution mass spectra were obtained on a GCT Premier GC-TOFMA with an EI source. Thin-layer chromatography analyses were conducted on GF254 plates. The Grignard reagent nC16H33MgBr was prepared according to a reported procedure.18 Anhydrous tetrahydrofuran (THF) used in the Grignard reactions was prepared by distillation over sodium, with diphenyl ketone indicator. The solvents petroleum ether (PE), CH2Cl2, EtOAc, and EtOH were dried over anhydrous sodium sulfate. All other reagents were purchased from commercial sources and were used without further purification unless otherwise indicated. Glutarimide (6). Glutaric acid (20.02 g) and urea (13.64 g) were mixed without any solvent. The mixture was heated for 40 min at 140 °C, then for an additional 2 h at 175 °C. Upon cooling, the crude product mixture was recrystallized from EtOH, then purified by column chromatography on silica gel using PE−EtOAc (1:3) as eluent to afford compound 6 as a white solid (12.96 g, yield, 76%): HREIMS m/z 113.0475 (calcd for C5H7NO2, 113.0477). 6-Methyl-2-piperidinone (7). The Grignard reagent CH3MgBr in 2-methyltetrahydrofuran (3 M, 4.42 mL) was added dropwise to a magnetically stirred solution of glutarimide (6, 0.50 g) in CH2Cl2 (15 mL) at −78 °C under a nitrogen atmosphere. Then the temperature was raised to 25 °C. After the reaction mixture was stirred at 25 °C for 18 h, NaBH3CN (0.33 g) was added, followed by the slow addition of a 6 M HCl solution to keep the pH at 3−4. The reaction was continued at 25 °C for 5 h, then neutralized with 10% NaOH solution and extracted with CH2Cl2 (5 × 20 mL). The organic phase was combined and dried over anhydrous Na2SO4. After workup, the product was purified by column chromatography on silica gel using PE−EtOH (15:1) as eluent to obtain compound 7 as a white solid (0.41 g, yield, 82%): 1H NMR (500 MHz, CDCl3) δ 1.20 (d, 3H, CH3), 1.33 (m, 1H), 1.68 (m, 1H), 1.89 (m, 2H), 2.29 (m, 1H), 2.35 (m, 1H), 3.51 (m, 1H), 6.77 (s, br, 1H); HREIMS m/z 113.0839 (calcd for C6H11NO, 113.0841). 6-Methyl-2-oxo-piperidine-1-carboxylic acid tert-butyl ester (8). In a three-necked flask equipped with a magnetic stirrer, Et3N (0.20 g), DMAP (0.03 g) and (BOC)2O (1.10 g) were added, in order, to a solution of compound 7 (0.23 g) in CH2Cl2 (30 mL). Then the mixture was heated to reflux. After 12 h the solvent was evaporated, and the organic mixture was separated with column chromatography on silica gel using PE−EtOH (20:1) as eluent to get compound 8 as a yellow oil (0.34 g, yield, 78%): 1H NMR (500 MHz, CDCl3) δ 1.20 (d, 3H, CH3), 1.45 (s, 9H), 1.62 (m, 2H), 1.88 (m, 2H), 2.39 (m, 2H), 4.20 (m, 1H). General Procedure for Preparation of N-Boc-aminoketones (9−11). The Grignard reagent RMgX (X = Br for 9 and 11; X = Cl for 10) (1.33 equiv) and tetramethylethylenediamine (TMEDA) (1.33 equiv) were added dropwise to a magnetically stirred solution of N-

VRE. The 50% growth inhibition (IC50) and minimum inhibitory concentration (MIC) values of the three compounds are shown in Table 1. Compound 1 appears to be the most active among the three synthetic products against these pathogens. Table 1. In Vitro Antifungal and Antibacterial Activities of Compounds 1−3a IC50b (MICc), μg/mL compound 1 2 3 amphotericin B

C. neoformans 6.6 ± 1.0 (10.0 ± 0) 8.3 ± 0.7 (13.3 ± 5.8) 9.9 ± 0.8 (20.0 ± 0) 10) >10 (>10) >10 (>10) 0.9 ± 0.05 (1.4 ± 0.07) ntd

3.1 ± 0.2 (>10) 3.4 ± 0.1 (8.8 ± 0.4) >10 (>10) 0.2 ± 0.02 (0.4 ± 0.04) ntd

0.6 (7.9 ± 0.8) 0.7 (9.0 ± 1.1) 0.3 (8.3 ± 0.7) 0.03 (0.5 ± 0.04)

T. brucei 2.7 ± 4.0 ± 3.5 ± ntd 6.1 ±

THP-1

0.2 (3.6 ± 0.03) 0.3 (6.8 ± 0.03) 0.2 (6.5 ± 0.04) 0.7 (12.1 ± 1.3)

>10 (>10) >10 (>10) >10 (>10) >2 (>2) ntd

a

Data are mean values with standard deviations from two independent experiments, each with two replicates. The highest test concentration for 1− 3, amphotericin B (AMB), and α-difluoromethylornithine (DFMO) are 10, 2, and 20 μg/mL, respectively. b50% inhibitory concentration. c90% inhibitory concentration. dNot tested. Boc-piperidinone (8, 1 equiv) in THF at −30 °C for 9 and 10 or at 0 °C for 11, under a nitrogen atmosphere. After stirring at 25 °C for 10 h, 2 M HCl was added to quench the reaction, and the mixture was extracted with CH2Cl2 (3 × 5 mL). The organic phase was dried over anhydrous Na2SO4. After workup, the products were separated by column chromatography on silica gel using PE−EtOAc (15:1) as eluent to give the corresponding N-Boc-aminoketone as a white solid. tert-Butyl 6-oxohenicosyl-2-carbamate (9): yield 82% from nC15H31MgBr, white solid; 1H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.11 (d, 3H, CH3), 1.25 (m, 26H, CH2), 1.44 (s, 9H, CH3 × 3), 1.54−1.61 (m, 4H, CH2), 2.36−2.49 (m, 4H, CH2), 3.64 (m, 1H, CH), 4.34(s, br, 1H, NH); 13C NMR (125 MHz, CDCl3) δC 14.4 (CH3), 20.4 (CH3), 21.5 (CH2), 22.9 (CH2), 24.1 (CH2), 28.7 (CH3 × 3), 29.5 (CH2), 29.6 (CH2), 29.67 (CH2), 29.73 (CH2), 29.86 (CH2), 29.90 (CH2), 29.93 (CH2), 32.2 (CH2), 36.9 (CH2), 42.5 (CH2), 43.1 (CH2), 46.4 (CH), 79.2 (C), 155.7 (NHCO), 211.5 (CO). tert-Butyl 6-oxoicosyl-2-carbamate (10): yield 86% from nC14H29MgCl, white solid; 1H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.11 (d, 3H, CH3), 1.25 (m, 24H, CH2), 1.44 (s, 9H, CH3 × 3), 1.55−1.61 (m, 4H, CH2), 2.36−2.42 (m, 4H, CH2), 3.63 (m, 1H, CH), 4.33 (s, br, 1H, NH); 13C NMR (125 MHz, CDCl3) δC 14.4 (CH3), 20.3 (CH3), 21.4 (CH2), 22.9 (CH2), 24.1 (CH2), 28.6 (CH3 × 3), 29.5 (CH2), 29.58 (CH2), 29.65 (CH2), 29.70 (CH2), 29.84 (CH2), 29.88 (CH2), 29.89 (CH2), 29.91 (CH2), 32.2 (CH2), 36.8 (CH2), 42.5 (CH2), 43.1 (CH2), 46.3 (CH), 79.2 (C), 155.6 (NHCO), 211.5 (CO). tert-Butyl 6-oxodocosyl-2-carbamate (11): yield 56% from nC16H33MgBr, white solid; 1H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.11 (d, 3H, CH3), 1.25 (m, 28H, CH2), 1.44 (s, 9H, CH3 × 3), 1.55−1.61 (m, 4H, CH2), 2.36−2.42 (m, 4H, CH2), 3.64 (m, 1H, CH), 4.33 (s, br, 1H, NH); 13C NMR (125 MHz, CDCl3) δC 14.4 (CH3), 20.3 (CH3), 21.5 (CH2), 22.9 (CH2), 24.1 (CH2), 28.7 (CH3 × 3), 29.5 (CH2), 29.61 (C CH2), 29.67 (CH2), 29.73 (CH2), 29.86 (CH2), 29.90 (CH2), 29.92 (CH2), 29.94 (CH2), 32.2 (CH2), 36.9 (CH2), 42.5 (CH2), 43.1 (C CH2), 46.4 (CH), 79.3 (C), 155.7 (NHCO), 211.5 (CO). General Procedure for Preparation of 2-Methyl-6-alkyl-Δ1,6piperideines (1−3). To a solution of N-Boc-aminoketone (9−11) (100 mg) in CH2Cl2 (2 mL) at 0 °C was added 12 M HCl (1 mL) dropwise over 5 min. After stirring for 5 h at 0 °C, the reaction mixture was basified with 2 M NaOH and then extracted with CH2Cl2 (3 × 5 mL). The organic phase was dried over anhydrous Na2SO4 to get the 2-methyl-6-alkyl-Δ1,6-piperideines as white solids. 2-Methyl-6-pentadecyl-Δ1,6-piperideine (1): yield 97% from 9, white solid; IR (neat) 2917, 2850, 1705, 1508, 1470, 1379, 1261, 1220, 1069, 1019, 803, 721 cm−1; 1H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.25 (m, 24H, CH2), 1.33 (d, 3H, CH3), 1.43−1.99 (m, 6H, CH2), 2.54−2.68 (m, 4H, CH2), 3.90 (m, 1H, CH); 13C NMR (125 MHz, CDCl3) δC 14.4 (CH3), 18.9 (CH3), 19.7 (CH2), 22.9 (CH2), 24.1 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2), 29.89 (CH2), 29.92 (CH2), 29.93 (CH2), 32.2 (CH2), 34.4 (CH2), 42.1 (CH2), 43.2 (CH2), 48.6 (CH2), 63.3 (CH), 172.3 (CN); HREIMS m/z 307.3237 (calcd for C21H41N, 307.3239).

2-Methyl-6-tetradecyl-Δ1,6-piperideine (2): yield 98% from 10, white solid; IR (neat) 2917, 2850, 1692, 1463, 1262, 1064, 1018, 780, 729 cm−1; 1H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.30 (m, 22H, CH2), 1.42 (d, 3H, CH3), 1.53−1.80 (m, 6H, CH2), 2.37− 2.48 (m, 4H, CH2), 3.75 (m, 1H, CH); 13C NMR (125 MHz, CDCl3) δC 14.4 (CH3), 18.9 (CH3), 19.7 (CH2), 22.9 (CH2), 24.1 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2), 29.89 (CH2), 29.92 (CH2), 32.2 (CH2), 34.2 (CH2), 42.1 (CH2), 43.2 (CH2), 48.5 (CH2), 63.3 (CH), 172.3 (CN); HREIMS m/z 293.3087 (calcd for C20H39N, 293.3083). 2-Methyl-6-hexadecyl-Δ1,6-piperideine (3): yield 96% from 11, white solid; IR (neat) 2917, 2850, 1705, 1467, 1259, 1068, 1019, 800, 721 cm−1; 1H NMR (500 MHz, CDCl3) δH 0.88 (t, 3H, CH3), 1.25 (m, 26H, CH2), 1.34 (d, 3H, CH3), 1.54−1.95 (m, 6H, CH2), 2.32− 2.45 (m, 4H, CH2), 3.68 (m, 1H, CH); 13C NMR (125 MHz, CDCl3) δC 14.4 (CH3), 18.8 (CH3), 19.7 (CH2), 22.9 (CH2), 24.1 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 29.75 (CH2), 29.79 (CH2), 29.85 (CH2), 29.88 (CH2), 29.91 (CH2), 32.1 (CH2), 34.4 (CH2), 42.1 (CH2), 43.2 (CH2), 48.5 (CH2), 63.3 (CH), 172.3 (CN); HREIMS m/z 321.3396 (calcd for C22H43N, 321.3396). In Vitro Antifungal Assay. A modified version of the CLSI (formerly NCCLS) method was used for susceptibility testing. Organisms (C. neoformans ATCC 90113, C. albicans ATCC 90028, and A. f umigatus ATCC 204305) were obtained from the American Type Culture Collection (ATCC). Amphotericin B (ICN Biomedicals) was used as a positive control. The detailed procedure has been described previously.12 In Vitro Antibacterial Assay. Microorganisms were obtained from ATCC including methicillin-resistant S. aureus ATCC 1708, vancomycin-resistant E. faecium ATCC 700221, E. coli ATCC 2452, and P. aeruginosa ATCC 2108. The positive control drugs included methicillin and vancomycin (from ICN Biomedicals). All organisms were tested using a modified version of the CLSI method, which has been described previously.19 Antiprotozoal Assay. The in vitro antileishmanial and antitrypanosomal assays were done on cell cultures of L. donovani promastigotes, axenic amastigotes, THP1-amastigotes, and T. brucei trypomastigotes by Alamar Blue assays as described earlier.20 Compounds with appropriate dilution were added to the test organism (2 × 106 cells/mL) in clear flat-bottom 384-well microplates, which were incubated at 26 °C for 72 h. IC50 and IC90 values were calculated from the growth inhibition curve using XLFit. Amphotericin B and alpha-difluoromethylornithine were used as positive controls.

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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.7b00625. 1

H and 13CNMR, MS, and IR spectra of intermediates and products 1−3 (PDF) C

DOI: 10.1021/acs.jnatprod.7b00625 J. Nat. Prod. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Authors

*(L. Dai) Tel: +86-576-87952693. E-mail: [email protected]. *(X.-C. Li) Tel: 662-915-6742. Fax: 662-915-7989. E-mail: [email protected]. ORCID

Liyan Dai: 0000-0001-6141-6278 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank the National Center for Natural Products Research at the University of Mississippi for biological testing (supported by the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-6408-1-603) and Zhejiang University Analysis and Testing Center for obtaining NMR and HREIMS spectra.

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DOI: 10.1021/acs.jnatprod.7b00625 J. Nat. Prod. XXXX, XXX, XXX−XXX