A new sulfoxide analog of 1,2,3,6-tetrahydrophenylpyridine and antimicrobial activity

Abstract

Bioactivities of thiotetrahydropyridines were previously described. Herein, a novel bioactive sulfoxide analog; N-acetyl-2-(1-adamantylsulfoxo)-3-acetoxy-4-phenyl-6-hydroxy-1,2,3,6-tetrahydropyridine (3) from the deoxydative substitution of 4-phenylpyridine 1-oxide is reported. Its structure was elucidated using spectral data including 2D-NMR, MS, IR and UV. The sulfoxide 3 exhibited antibacterial activity against Moraxella catarrhalis and Streptococcus pyogenes with minimum inhibitory concentration of 128 and 256 µg/mL, respectively.

Introduction

Tetrahydropyridines are a class of compounds that possess a vast array of bioacti-vities. In particular, synthesis and bioactivities of thiotetrahydropyridines were previously reported (Prachayasittikul et al., 1985[9], 1991[8], 2009[11]). The tetrahydropyridines are N-acetyl-1,2,3,4- and 1,2,3,6- isomers (1 and 2, respectively) containing 1-adamantylthio and hydroxyl (acetoxyl) groups (Figure 1(Fig. 1)) (Egan et al., 1969[1]; Kokosa et al., 1975[5]; Hershenson and Bauer, 1969[2]; Prachayasittikul et al., 1985[9], 1991[8], 2009[11]). These tetrahydropyridines were isolated from the deoxydative substitution reaction of pyridine 1-oxides by thiols in the presence or absence of triethylamine (Egan et al., 1969[1]; Kokosa et al., 1975[5]; Hershenson and Bauer, 1969[2]; Prachayasittikul et al., 1985[9], 1991[8], 2009[11]). According to our experience working on such reactions, it occurred in mind that more and new interesting tetrahydropyridines remained to be explored. As part of our continuing study, isolation and characterization of new tetrahydropyridines from 4-phenylpyridine 1-oxide and its antimicrobial activity have been reported.

Figure 1. Chemical structures of 1,2,3,4- and 1,2,3,6-tetrahydropyridines.

Material and Methods

General

Melting points were determined on an Electrothermal melting point apparatus (Electrothermal 9100) and are uncorrected. ¹H- and ¹³C-NMR spectra were recorded on a Bruker AVANCE 300 NMR spectrometer (operating at 300 MHz for ¹H and 75 MHz for ¹³C, respectively). Infrared spectra (IR) were obtained on a Perkin Elmer System 2000 FTIR. Ultraviolet (UV) spectra were recorded on a Milton Roy Spectronic 300 Arrays. Mass spectra were recorded on a Finnigan INCOS 50 and a Bruker Daltonics (micro TOF). Column chromatography was carried out using silica gel 60 (0.063-0.200 mm). Analytical thin layer chromatography (TLC) was performed on silica gel 60 PF₂₅₄ aluminium sheets (cat. No. 7747 E., Merck). Solvents were distilled prior to use. Reagents for cell culture and assays were of analytical grade. All chemicals for reaction were used as supplied. 1-Adamantanethiol (1-AdmSH) was prepared by the literature method (Khullar and Bauer, 1971[4]).

Synthesis of N-acetyl-2-(1-adamantyl-sulfoxo)-3-acetoxy-4-phenyl-6-hydroxy-1,2,3,6-tetrahydropyridine (3)

A solution of 4-phenylpyridine 1-oxide (17.1 g, 0.1 mol) in acetic anhydride (75 mL) containing triethylamine (0.35 mol, 50 mL) was added dropwise (30 min) a solution of 1-AdmSH (16.8 g, 0.1 mol) in acetic anhydride (25 mL). The reaction mixture was heated at 95°C for 3 h, then evaporated in vacuo, neutralized with 50 % Na₂CO₃ and extracted with toluene (3 × 100 mL). After washing with water (50 mL), dried (anh. Na₂SO₄), the solvent was removed in vacuo to give a dark-brown solid (55 g). The solid was chromatographed on a silica gel (500 g) column. Elution with petroleum ether : toluene (1:1, 1.5 L) then toluene: CHCl₃ (4:1, 11 L) gave a mixture of 1-AdmSCOCH₃ (14.3 g) and pyridyl sulfides (10.6 g). Further elution with CHCl₃ (7 L) gave a mixture of tetrahydropyridines which was rechromatographed on the silica gel (120 g) to yield 3.2 g solid of tetrahydropyridines from petroleum ether: CHCl₃ (1:1, 1.1 L). The 3.2 g solid was re-isolated on the silica gel (120 g) column to afford tetrahydropyridine 2.3 g from CHCl₃ (4.5 L). The product was recrystallized from ether to provide 0.55 g of N-acetyl-2-(1-adamantylsulfoxo)-3-acetoxy-4-phenyl-6- hydroxy-1,2,3,6-tetrahydropyridine (3); mp 158-159 °C, UV (95 % ethanol) λ_max (log ε) 205 (4.31) and 245 (4.02) nm, IR (UATR) 3257, 2909, 2852, 1741, 1672, 1498, 1448, 1390, 1370, 1299, 1217, 1067, 1013, 987 cm^-1 , ¹H-NMR (300 MHz, CDC1₃): δ = 1.55, 1.62, 1.82, 1.85, 2.07, (5s, 15H, 1-AdmH), 1.85 (s, 3H, NCOCH₃), 2.33 (s, 3H, OCOCH₃), 5.50-5.56 (m, 2H, H-3, H-6), 5.63 (d, J = 11.4 Hz, 1H, OH), 6.28 (d, J = 2.1 Hz, 1H, H-2), 6.35 (d, J = 3.0 Hz, 1H, H-5), 7.25 (s, 5H, PhH). ¹³C-NMR (75 MHz, CDC1₃) : δ = 20.8 (NCOCH₃), 21.7 (OCOCH₃), 29.3 (3 × CH-1-Adm), 35.0, 36.0 (6 × CH₂-1-Adm), 57.8 (Cqua-1-Adm), 61.8 (C-2), 67.7 (C-3), 73.7 (C-6), 125.6, 129.0, 136.6 (Ph-C), 132.1 (C-5), 136.6 (C-4), 170.5 (NCOCH₃), 172.7 (OCOCH₃). LRMS (EI):m/z (%) = 457 (6.30) [M - H]⁺, 456 (30.56), 427 (17.50), 426 (18.03), 348 (14.67), 256 (25.52), 215 (24.24), 214 (100.00), 172 (12.92), 156 (13.75), 135 (3.03). HRMS (TOF): m/z [M+Na]⁺ calcd for C₂₅H₃₁NSO₅Na: 480.1815; found : 480.1815.

Antimicrobial assay

Antimicrobial activity of the tested compound was performed using agar dilution method as previously described (Prachayasittikul et al., 2009[10]). Briefly, the tested compound dissolved in DMSO was individually mixed with 1 mL Müller Hinton (MH) broth. The solution was then transferred to the MH agar solution to yield the final concentrations of 64-256 μg/mL. Twenty-one strains of microorganisms, cultured in MH broth at 37 °C for 18-24 h, were diluted with 0.9 % normal saline solution to adjust the cell density to 1×10⁸ cells/mL compared with 0.5 McFarland. The organisms were inoculated onto each plate and further incubated at 37 °C for 24- 48 h. Compounds which possessed high efficacy to inhibit bacterial cell growth were analyzed.

Results and Discussions

Chemistry

Reaction of 4-phenylpyridine 1-oxide with 1-AdmSH in acetic anhydride containing triethylamine was performed. The products were isolated by repeated silica gel column chromatography to give first 1-AdmSCOCH₃ (Prachayasittikul and Bauer, 1985[7]) and a mixture of α- and β-pyridyl sulfides from toluene : CHCl₃ (4:1 elution) (Prachayasittikul et al., 1991[8]). From more polar CHCl₃ elution afforded a mixture of tetrahydropyridines (based on ¹H-NMR spectra). Upon extensive rechromatographic separation, a new tetrahydropyridine (3) was found as a crystalline solid of m.p. 158-159°C. Structure of compound 3 was elucidated using 2D-NMR; ¹H- and ¹³C-NMR, LRMS, HRMS, IR and UV spectra. Its IR spectra showed the presence of OH group (3257 cm^-1), strong ester and amide CO at 1741 and 1672 cm^-1, respectively. The very strong S = O moiety was observed at 1013 cm^-1. The UV spectra displayed λ_max at 245 nm indicating an unconjugated alkene amide of 1,2,3,6-tetrahydropyridine (Prachayasittikul et al., 1991[8], 2009[11]).

¹H-NMR spectra showed δ 6.28 of H-2 as a doublet with a coupling constant (J) of 2.1 Hz. In addition, H-3 and H-6 were a multiplet at δ 5.50-5.56, H-5 at δ 6.35 was noted as a doublet with the J value of 3.0 Hz. The signal at δ 5.63 as a doublet was collapsed by D₂O exchange, suggesting the presence of hydroxyl group at C-6 with the large J value of 11.4 Hz. The stereochemistry at C-2 and C-3 was assigned using the J value between H-2 and H-3 and the Karplus relationship. The coupling constant of 2.1 Hz suggested that the H-2 and H-3 are trans-quasidiequatorial. Hence, the sulfoxide at C-2 and acetate at C-3 are trans-quasidiaxial. Comparision with the well established tetrahydropyridine (Prachayasittikul et al., 1985[9], 1991[8], 2009[11]; Egan et al., 1969[1]), the OH group at C-6 is assigned to have the same stereochemistry as C-2. The two substituents at C-2 and C-6 are cis-quasidiaxial in twist chair form. Furthermore, the ¹H-detected heteronuclear multiple bond correlation (HMBC) experiment was also employed to confirm the assignments of protons in the sulfoxide 3 through a long-range coupling of protons and carbons (Table 1(Tab. 1)). The results showed the correlations between H2 with C-3, C-6, C-4, NCOCH₃ and quaternary carbon of 1-Adm; H3 with Ph-C-1' and OCOCH₃; H5 with C-3 and Ph-C-1' and H6 with C-4. Mass spectra showed a low intensity fragment of [M-H]⁺ with m/z 457 including a base peak at m/z 214 resulting from the loss of 1-AdmSO, CH₃CO and H₂O from the molecule of 3. The presence of amide and ester carbonyls was confirmed by ¹³C-NMR showing δ 170.5 and 172.7 ppm, respectively. Based on the spectral data, thus, compound 3 was identified to be the new sulfoxide analog of tetrahydropyridine; N-acetyl-2-(1-adamantylsulfoxo)-3-acetoxy-4-phenyl-6-hydroxy-1,2,3,6-tetrahydropyridine. Its molecular formula was confirmed by HRMS as C₂₅H₃₁NSO₅.

Table 1. HMBC spectroscopic data of the sulfoxide 3 in CDCl₃.

Analogously, such 1,2,3,6-tetrahydropyridine from 4-phenylpyridine 1-oxide was reported previously (Prachayasittikul et al., 1991[8]), but as a sulfide derivative (4). When the reaction was studied without inclusion of triethylamine, 1,2,3,6-tetrahydropyridine (4a) was found in which the sulfide group was deposited at C-3 (Prachayasittikul et al., 2009[11]). The isolated sulfoxide of 1,2,3,6-tetrahydropyridine (3) has been proposed to be formed via episulfonium ion intermediate (a) (Prachayasittikul et al., 1985[9], 1991[8], 2009[11]) to give first sulfide 4. Finally, oxidation of the sulfide function by acetic anhydride in triethylamine furnished the sulfoxide product 3 (Figure 2(Fig. 2)).

Figure 2. Chemical structure of the sulfoxide 3 and related compounds.

Conversion of the sulfide 4 to sulfoxide 3 is likely to occur through S-acylation of 4 to give acetylsulfonium salt (b) which was attacked by an acetate anion to produce acetoxythiol acetate (c). Further intramolecular nucleophilic attack by carbonyl oxygen to afford three membered ring intermediate (d) with subsequent cleavage of C-S and C-O bonds to generate the sulfoxide 3 (Figure 3(Fig. 3)). Oxidation of sulfides to sulfoxides was reviewed in the literatures using a variety of oxidants (Kowalski et al., 2005[6]; Kaczorowska et al., 2005[3]) such as halogen derivatives e.g. molecular halogens and hypervalent iodine (III and IV) reagents (Kowalski et al., 2005[6]) as well as hydrogen peroxide in various solvents and in the presence of catalyst e.g. TiCl₃, SeO₂ and TeO₂ (Kaczorowska et al., 2005[3]). This study illustrates the use of Ac₂O/Et₃N as an oxidant converting the sulfide 4 to sulfoxide 3.

Figure 3. Proposed transformation of sulfide 4 to sulfoxide 3.

Antimicrobial activity

The sulfoxide 3 was tested for anti-microbial action using the agar dilution method against twenty-one microorganisms (gram-positive and gram-negative bacteria and diploid fungus). It was found that the tetrahydropyridine 3 inhibited the growth of Moraxella catarrhalis and Streptococcus pyogenes with minimum inhibitory concentration (MIC) of 128 and 256 µg/mL, respectively (Table 2(Tab. 2)). In addition, the growth of Corynebacterium diphtheriae NCTC 10356 was partially inhibited (80 %) at 256 µg/mL. However, the sulfides of 1,2,3,6-tetrahydropyridines (4 and 4a) were reported to be an inactive antimicrobial agents (Prachayasittikul et al., 2009[11]). The result showed that the sulfoxide function provide the compound with bioactivity. This oxidation could possibly be extended to other sulfides in order to improve their bioactivities.

Table 2. Antimicrobial activity^a of tetrahydropyridines.

Conclusion

The deoxydative substitution of 4-phenylpyridine 1-oxide by 1-AdmSH in acetic anhydride containing triethylamine was reinvestigated to afford the new sulfoxide analog of 1,2,3,6-tetrahydropyridine (3) as a unique by-product. The sulfoxide 3 exerted its antibacterial activity against M. catarrhalis and S. pyogenes with MIC of 128 and 256 µg/mL, respectively. Partial growth inhibition (80 %) of 3 was noted against C. diphtheriae NCTC 10356 at 256 µg/mL. This study leads to the discovery of new bioactive sulfoxide of 1,2,3,6-tetrahydropyridine from 4-phenylpyridine 1-oxide.

Notes

Supaluk Prachayasittikul and Virapong Prachayasittikul (Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand; Telephone: 662-441-4376, Fax: 662-441-4380, E-mail: mtvpr@mahidol.ac.th) contributed equally as corresponding authors.

Acknowledgements

This project was in part supported by the research grant of Mahidol University (B.E. 2551-2555). R.P. gratefully acknowledges support from the Young Scholars Research Fellowship from the Thailand Research Fund (TRF, Grant No. MRG5280092). A.W. is supported by the TRF Royal Golden Jubilee (Ph.D.) scholarship under supervision of V.P.