Synthesis and Biological Activity Studies of Methyl-5-(Hydroxymethyl)-2-Furan Carboxylate and Derivatives

Introduction

Infectious diseases have been serious public health problems in previous decades.^1-2  As drug resistance has evolved against antimicrobial and antineoplastic agents, the search for novel and potent antibacterial agents is necessary.³ Furans are an important heterocyclic class that possess variety of bioactivities.^4-8 The furan moiety is considered to be a common structural motif in many natural products, and many research groups have reported the synthesis of substituted furans for biological assay. Methyl-5-(hydroxymethyl) – 2-furan carboxylate (1) was first discovered in 2009 from Curvularia lunata and reported as a toxin from fungal caused curvularia leaf spot of maize.^9-10 In 2014, Yu-Tang Tung and co-workers isolated methyl-5-(hydroxymethyl)-2-furan carboxylate (1) from Antrodia camphorate¹¹ and found it to possess anti-inflammatory activities.¹² Recently the extraction of bacteria Streptomyces sp.¹³ from Zingiber zerumbet (L.) Smith¹⁴ in Chantaburi province, Thailand was reported to contain methyl-5-(hydroxymethyl)-2-furan carboxylate (1) together with many other biological compounds.¹⁵ This compound has interesting antibacterial activity with MIC 1.00 µg/mL against Staphylococcus aureus ATCC25923. This observation prompted us to synthesize new chemical entities incorporating the active furan pharmacophores to evaluate their biological activities. We report the synthesis of 1-(hydroxymethyl) furan derivatives (2-3) (Figure 1) and in vitro biological activities to identify viable leads for further studies.

Figure 1: Structure of Methyl–5-(hydroxymethyl)-2–furan carboxylate 1 and derivatives. Click here to view figure

 

Materials and Methods

General Methods

Unless otherwise stated, nuclear magnetic resonance (NMR) was acquired in CDCl₃ and on a Bruker Avance 300 spectrometer. Stuart Scientific SMP 2 was an apparatus used to measure the melting points and are uncorrected. Mass spectra were detected with a HEWLETT PACKARD 5973 or POLARIS Q mass spectrometer. To monitor the reactions, Merck silica plate 60 F254 was used for thin layer chromatography (TLC). Products were purified using Merck Kieselgel 60 via column chromatography.

Synthesis Method

General Procedure for the Synthesis of Amines Via Reductive Amination

A mixture of primary amine (1.0 equi), aldehyde (1.1 equi) and acetic acid (3 drops) in MeOH (10 mL) was heated under reflux for 1 h. After cooling to RT, sodium borohydride (0.6 equi) was added and stirred at 0°C for 1 h under Argon. The saturated NaHCO₃ was added and extracted with EtOAc (3×20 mL). The organic phase was dried (Na₂SO₄), filtered and concentrated to give the dark brown residue. The residue was purified by flash column chromatography.

 (5-(((2-(1H–indol–3–yl)ethyl)amino)methyl)furan–2–yl)methanol (8a) 

Compound (8a) was obtained as yellow oil (30.40 mg, 10.5%) from tryptamine (0.10 g, 0.62 mmol) and 5-(hydroxymethyl)furan-2-carbaldehyde (5) (0.10 g, 0.62 mmol).; IR (ATR, cm^-1) 3277, 3045, 2923, 1727, 1618, 1456, 1339, 1288, 1096, 1009, 795, 740, 583; UV (λ_max, nm )  203, 223, 281, 290; ¹H NMR δ 2.94 (4H, t, J = 3.3 Hz, 2CH₂), 3.70 (2H, s, NCH₂), 4.46 (2H, s, OCH₂), 6.06 (1H, d, J = 3.1 Hz, furan), 6.13 (1H, d, J = 3.1 Hz, furan), 6.93 (1H, d, J = 2.0 Hz, indole), 7.08 (1H, t, J = 7.5 Hz, CH-Ar), 7.17 (1H, t, J = 7.5 Hz, CH-Ar), 7.33 (1H, d, J = 8.0 Hz, CH-Ar), 7.57 (1H, d, J = 8.0 Hz, CH-Ar), 8.35 (1H, br, NH); ¹³C NMR δ 24.52, 45.14, 47.83, 56.41, 107.45, 107.48, 110.57, 118.03, 118.46, 121.22, 121.67, 126.59, 135.76, 152.27, 153.09, 153.51; HR-ESI MS calculated for C₁₆H₁₉N₂O₂ [M+H]⁺ 271.1447, found 271.1439.

(5-(((3–aminopropyl)amino)methyl)furan–2–yl)methanol (8b)

Compound (8b) was obtained as the unstable yellow oil (0.19 g, 25.6%) from the reaction of 1,3-diaminopropane (0.35 mL, 4.12 mmol and 5-(hydroxymethyl)furan-2-carbaldehyde (5) (0.49 g, 3.89 mmol).  However, the colour of compound (8b) was changed from yellow to brown after left in the refrigerator without solvent for few days.);IR (ART, cm^-1) 3270, 3050, 2925, 2851, 1628, 1595, 1555, 1456, 1339, 1227, 1096, 1009, 795, 740, 583; UV (λ_max, nm )  202, 223, 281, 290; ¹H NMR δ 1.61 (2H, qui, J = 6.9 Hz, CH₂-2′), 2.65 (2H, t, J = 6.9 Hz, CH₂-1′), 2.73 (2H, t, J = 6.8 Hz, CH₂-3′), 3.72 (2H, s, NCH₂), 4.50 (2H, s, OCH₂), 6.10 (1H, d, J = 3.0 Hz, furan), 6.16 (1H, d, J = 3.0 Hz, furan); ¹³C NMR δ 30.77, 37.87, 43.96, 44.49, 54.74, 105.44, 105.75, 151.36, 152.01; HR-ESI MS calculated for C₉H₁₇N₂O₂ [M+H]⁺ 185.1290, found 185.1283.

(5-(((2-(1H–indol–3–yl)ethyl)amino)methyl)furan–2–yl)methyl acetate (8c)

Compound (8c) was obtained as  the yellow oil (50.57 mg, 13.5%)from (5-formylfuran-2-yl)methyl acetate (6)  (0.20 g, 1.20 mmol) and tryptamine (0.29 g, 1.80 mmol).; IR (ATR, cm^-1) 3275, 3056, 2920, 2850, 1734, 1619, 1456, 1339, 1230, 1095, 1009, 795, 740, 584; UV (λ_max, nm ) 202, 222, 282, 290; ¹H NMR  δ 2.05 (3H, s, CH₃), 2.98 (4H, s, 2CH₂), 3.78 (2H, s, NCH₂), 4.97 (2H, s, OCH₂), 6.11 (1H, d, J = 3.2 Hz, furan), 6.30 (1H, d, J = 3.2 Hz, furan), 7.00 (1H, d, J = 2.2 Hz, indole), 7.10 (1H, t, J = 7.5 Hz, CH-Ar), 7.19 (1H, t, J = 7.5 Hz, CH-Ar), 7.36 (1H, d, J = 8.0 Hz, CH-Ar), 7.60 (1H, d, J = 7.8 Hz, CH-Ar); ¹³C NMR δ 19.12, 23.33, 43.83, 46.83, 56.45, 106.92, 109.72, 109.75, 110.75, 116.85, 117.33, 120.09, 120.81, 125.49, 134.85, 147.12, 151.76, 169.22; HR-ESI MS calculated for C₁₈H₂₁N₂O₃ [M+H]⁺ 313.1552, found 313.1543.

(5-(((3–aminopropyl)amino)methyl)furan–2–yl)methyl acetate (8d)

Compound (8d) (63.3 mg, 63.6%) was prepared from 1,3-diaminopropane (0.04 mL, 0.47 mmol) and (5-formylfuran-2-yl)methyl acetate (6) (73.8 mg, 0.44 mmol); (ATR, cm^-1) 3273, 3050, 2924, 2850, 1627, 1593, 1554, 1456, 1339, 1227, 1095, 1009, 795, 740, 583; UV (λ_max, nm ) 203, 222, 280, 290; ¹H NMR δ 1.68 (2H, qui, J = 6.4 Hz, CH_2–2′),  1.95 (3H, s, CH₃), 2.69 (2H, t, J = 6.3 Hz, CH₂-3′), 3.30-3.35 (2H, m, CH₂-1′), 3.76 (2H, s, NCH₂), 4.56 (2H, s, OCH₂), 6.13 (1H, d, J = 3.0 Hz, furan), 6.21 (1H, d, J = 3.1 Hz, furan); ¹³C NMR δ 22.30, 28.14, 37.08, 45.21, 45.55, 56.34, 107.72, 108.15, 151.58, 153.75, 171.07; HR-ESI MS calculated for C₁₁H₁₉N₂O₃ [M+H]⁺ 227.1396, found 227.1394.

General procedure for the synthesis of amide derivatives (9a and 9b)

A mixture of methyl 5-(hydroxymethyl)-2-furan carboxylate (1) (1.0 equi), amine (1.0 equi) and triethylamine (1.0 equiv) in MeOH (10 mL) was refluxed overnight.The solvent was concentrated and purified by flash column chromatography.

N-(2-(1H–indol–3–yl)ethyl)-5-(hydroxymethyl)furan–2–carboxamide (9a)

Compound (9a) was obtained as the yellow oil of (88.80 mg, 28.1%) from methyl 5-(hydroxymethyl)-2-furan carboxylate (1) (0.20 g ,1.11 mmol) and tryptamine (0.18 g, 1.12 mmol); ¹H NMR (CDCl₃: MeOD, 9:1) δ 2.99 (2H, t, J = 6.9 Hz, CH₂), 3.60-3.68 (2H, m, CH₂), 4.48 (2H, s, OCH₂), 6.26 (1H, d, J = 3.3 Hz, furan), 6.96 (1H, d, J = 3.3 Hz, furan), 6.98 (1H, s, indole), 7.08 (1H, t, J = 7.4 Hz, CH-Ar), 7.16 (1H, t, J = 7.4 Hz, CH-Ar), 7.35 (1H, d, J = 8.1 Hz, CH-Ar), 7.60 (1H, d, J = 7.7 Hz, CH-Ar); ¹³C NMR (CDCl₃: MeOD, 9:1) δ 23.76, 38.28, 55.34, 108.14, 110.05, 113.53, 117.06, 117.69, 120.41, 121.04, 125.80, 135.08, 145.53, 155.05, 157.62, 157.69; HR-ESI MS calculated for C₁₆H₁₇N₂O₃ [M+H]⁺ 285.1239, found 285.1244.

N-(3–aminopropyl)-5-(hydroxymethyl)furan–2–carboxamide (9b)

Compound (9b) was obtained as the yellow oil of (0.14 g, 33.8%) from 1,3-diaminopropane (0.17 mL, 2.00 mmol) and methyl 5-(hydroxymethyl)-2-furan carboxylate (1) (0.32 g, 2.07 mmol); ¹H NMR (MeOD) δ 1.77 (2H, qui, J = 6.8 Hz, CH₂-2′), 2.72 (2H, t, J = 6.9 Hz, CH₂-3′), 3.43 (2H, t, J = 6.9 Hz, CH₂-1′), 4.59 (2H, s, OCH₂), 6.47 (1H, d, J = 3.4 Hz, furan), 7.06 (1H, d, J = 3.4 Hz, furan); ¹³C NMR (MeOD) δ 31.76, 36.04, 38.21, 56.05, 108.96, 114.58, 146.94, 157.42, 159.65; HR-ESI MS calculated for C₉H₁₅N₂O₃ [M+H]⁺ 199.1083, found 199.1072.

(5-((2-(1H–indol–3–yl)ethyl)carbamoyl)furan–2–yl)methyl acetate (9c)

A mixture of 5-(Acetoxymethyl)furan-2-carboxylic acid (7) (0.13 g, 0.71 mmol) in DCM (5 mL), tryptamine (0.11 g, 0.67 mmol), dicyclohexyl carbodiimide (DCC) (0.21 g, 1.02 mmol) and 4-dimethylaminopyridine (DMAP) (8.26 mg, 0.07 mmol) was stirred for overnight. After that, the mixture was filtered and the filtrate was evaporated. The residue was purified by PLC (hexane: EtOAc, 2:1) to give yellow oil of (5-((2-(1H-indol-3-yl)ethyl)carbamoyl)furan-2-yl)methyl acetate (9c) 76.10 mg (34.6%).; ¹H NMR (CDCl₃: MeOD, 9:1) δ 2.06 (3H, s, CH₃), 3.04 (2H, t, J = 6.9 Hz, CH₂), 3.68-3.74 (2H, m, CH₂), 4.98 (2H, s, OCH₂), 6.44 (1H, d, J = 3.4 Hz, furan), 7.02 (1H, d, J = 3.6 Hz, furan), 7.03 (1H, s, indole), 7.09 (1H, t, J = 7.7 Hz, CH-Ar), 7.17 (1H, t, J = 7.7 Hz, CH-Ar), 7.38 (1H, d, J = 8.0 Hz, CH-Ar), 7.62 (1H, d, J = 7.8 Hz, CH-Ar); ¹³C NMR (CDCl₃: MeOD, 9:1) δ 18.84, 23.45, 37.73, 55.85, 109.50, 110.85, 113.00, 116.65, 117.28, 120.02, 120.41, 125.38, 134.55, 134.70, 146.20, 149.22, 156.64, 168.85; HR-ESI MS calculated for C₁₈H₁₈N₂NaO₄ [M+Na]⁺ 349.1164, found 349.1158.

Biological Activity

Determination of Anticancer Activity

All the synthesised compounds were tested for cytotoxicity and anticancer activity by MTT assay on Vero cells (kidney of African green monkey cell line), HepG2 (human liver carcinoma cell line), and HeLa (human cervical carcinoma cell line).¹⁶ All cell lines were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum about 80% confluence. About 1 × 10⁵cells/mL were plated on a 96-well microliter plates and incubated at 37°C under 5% CO₂ for 24 h. The medium containing difference concentration of tested compounds was replaced. After 24 h of inoculation, 100 μL of the MTT solution (400 µg/mL) was added to each well and incubated at 37°C for 4 h, then the media were removed and added 100% DMSO 100 µL/well. Formazan was solubilized and the concentration was determined by optical density at 540 nm. The absorbance reading from each well was calculated the percentage of survival cells for each compound with different concentration.

Determination of Antibacterial Activity

Antibacterial activity was tested against four bacterial strains: B. subtilis ATCC 6633, B. cereus ATCC 7064, S. aureus ATCC 25932 and Escherichia coli ATCC10536 by disc diffusion method.¹⁷ The 500 μg of each compound were added in to a sterile paper disk. The disks were placed on nutrient agar plates that it containing of bacterial strains and were incubated at 37 °C for 24 h. The diameter zone of inhibition was measured in mm.

The minimal inhibitory concentration (MIC) was evaluated by micro broth dilution method (Taechowisan et al., 2018). The agents were dissolved in methanol (10,000 µg/mL) was serially diluted with nutrient broth supplemented with 10% glucose (NBG) and 0.5% phenol red as a pH indicator to get the varying concentration and 100 μL of each concentration was placed in the well.  Then, 10 μL of the bacterial suspension (10⁵ cells/mL) was inoculated into each well of a 96-well microplate. The drug-free and 5% methanol was used as a positive control and tetracycline was used as a reference.  Microtiter plates were incubated for 24 h at 37 °C. MIC was defined as the lowest concentrations which no change of pH indicator.

Results and Discussion

Chemistry of Synthesis

The furfuryl alcohol (4) was converted to 5-(hydroxyethyl)-furaldehyde (5) by formylation using the Vilsmeier–Haack reaction according to Miyazawa et al.¹⁸ The o-acylation¹⁹ followed by Pinnick oxidation to afford carboxylic acid (7).²⁰ Esterification of the carboxylic acid (7) under acidic conditions afforded the desired methyl-5-(hydroxymethyl)-2-furan carboxylate (1)²¹ (Scheme 1).

Scheme 1: Synthetic route to methyl–5-(hydroxymethyl)-2–furan carboxylate.Click here to view scheme

 

The 5-(hydroxymethyl) furan derivatives were prepared by amination of aldehydes (5) and (6) with tryptamine and 1,3-diaminopropane. The amine derivatives (8a–8d) were obtained in low yields due to the solubility of the mixture in aqueous solution after worked up. The aminopropane derivative (8b) was unstable, the colour changed from yellow to brown after left in the refrigerator without solvent for few days. Thus, the fresh prepared compound (8b) was determined the structure by NMR spectroscopy and mass spectrum analysis prior to evaluate for the biological activities (Scheme 2).

Scheme 2: Amine derivatives of methyl–5-(hydroxymethyl)-2–furan carboxylate.Click here to view scheme

 

The acyl protection of alcohol compound (8c), compound (8d) was stable and the structure was compared with compound (8b) via NMR and mass spectrometry techniques and. To study the bio-activity of the amide derivatives of (9a-c), the reaction of methyl ester (1) with tryptamine and diaminopropane in the presence of the base triethylamine produced secondary amides (9a and 9b) in 28% and 34% yield respectively. Derivative (9c) was synthesized from the carboxylic acid (7) reacted with tryptamine via amide formation using dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) to give 35% yield (Scheme 3).

Scheme 3: Amide derivatives of methyl–5-(hydroxymethyl)-2–furan carboxylate. Click here to view scheme

 

Biological Activity Assay

The cytotoxicity of the methyl-5-(hydroxymethyl)-2-furan carboxylate and derivatives was evaluated against HeLa, HepG2 and Vero cell lines using MTT assay. The IC₅₀ are shown in Table 1. Compounds (1, 8c and 9c) showed weak cytotoxicity against the normal cell lines but significant anticancer activities against the HeLa cell lines. The presence of tryptamine and acyl ether in the structures led to an increase in anticancer activities, while the amide linkage reduced the activities in comparison with the amine linkage. In contrast, compound 8a showed selective anticancer activities against HepG2.

Table 1: IC₅₀ of furan derivatives against the HeLa, HepG2 and Vero cell lines.

Compounds			IC50(µg/mL)
	HeLa		HepG2		Vero
tryptamine		275.26		149.08		193.64
1		64.00		154.60		247.19
8a		143.64		96.98		137.45
8b*		215.01		150.03		307.39
8c		62.37		120.06		124.46
8d		200.73		135.90		241.67
9a		150.00		110.32		287.14
9b		270.03		157.00		204.76
9c		82.27		121.11		171.69

 

* This compound was unstable and cytotoxicity was evaluated after preparation.

The synthesized compounds were also evaluated for antibacterial activities against pathogenic bacteria. The zones of inhibitions (mm) and MIC values are shown in Table 2. It was found that the synthetic compound (1) inhibited the Gram (+) bacteria; Staphylococcus aureus and Bacillus cereus with MIC 500.00 µg/mL, while all of the derivatives of (1) lost these activities. Thus, the methyl ester of compound (1) is necessary for inhibition. Most of the derivatives showed the ability to inhibit photogenic bacteria; B. subtilis and E. coli except the substituted aminopropanes (8b and 9b), which had no activities against any bacteria strains. Compound (8c) showed the most potent antibacterial activities against B. subtilis and E. coli (MIC 250.00 µg/mL) compared to all tested compounds. The amide derivative (9c) showed lower activity compared to (8c). This agrees with the importance of acyl and tryptamine substitution of the furan ring for anticancer activity.

Table 2: Antibacterial activity of furan derivatives against different bacteria.

Compounds	B. cereus			S. aureus		B. subtilis			E. coli
	ATCC7064			ATCC25923		ATCC6633			ATCC10536
	IZ	MIC	IZ	MIC		IZ	MIC	IZ		MIC
tryptamine	–	NT	7.3	500		9.8	500	8.5		500
1	7.3	500	8.0	500		9.1	500	8.3		500
8a	–	NT	–	NT		8.1	500	7.0		>500
8b	–	NT	–	NT		–	NT	–		NT
8c	–	NT	–	NT		19.0	250	10.0		250
8d	–	NT	–	NT		6.9	>500	–		NT
9a	–	NT	–	NT		13.3	500	8.0		500
9b	–	NT	–	NT		–	NT	–		NT
9c	–	NT	–	NT		9.0	500	8.0		500
tetracycline	24.5	0.98	29.0	<0.49		27.3	0.98	25.3		3.91

 

IZ= zones of inhibitions (in mm), MIC= minimum inhibition (µg/mL)

(-) indicates = No zone of inhibition, NT = Not tested

All results are the average of triplicates.

Conclusion

Methyl-5-(hydroxymethyl)-2-furan carboxylate (5) and derivatives (8a-d), (9a-c) were prepared and tested for their biological activities. The core molecule (5) displayed both anticancer and antibacterial activity. The amine derivative, (8c) is more potent than the other compounds in this series, but it is not active against the bacteria S. aureus and B. cereus. Because of its carbonyl group, the amide derivative, (9c) is significantly less potent than (8c). Based on these results, the tryptamine and acyl protection of the primary alcohol are important for the furan ring to exhibit anticancer activity and antibacterial activity. These furan analogues could be served as important leads for further synthesis and optimization of the novel drug syntheses.

Acknowledgements

The authors are grateful to the Department of Chemistry and Department of Microbiology, Faculty of Science, Silpakorn University for the research support. We also gratefully acknowledged The Chulabhorn Research Institute for HRMS spectroscopy.

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