2-(Cyclopropylamino)-5-(4-methoxybenzylidene)thiazol-4(5H)-one

Abstract

Multicomponent reactions effectively contribute to modern organic and medicinal chemistry. 4-Thiazolidinone core and cyclopropyl moiety are important structural motifs for design of potential biologically active molecules. In the present paper, the convenient step-economy and cost-effective synthesis of 2-(cyclopropylamino)-5-(4-methoxybenzylidene)thiazol-4(5H)-one (2) is described based on the application of the MCR methodology. The proposed approach includes direct one-pot interaction of 2-thioxothiazolidin-4-one (rhodanine), 4-methoxybenzaldehyde with cyclopropylamine which was used in 10% excess compare to other reagents. The structure of synthesized compound 2 was confirmed using ¹H, ¹³C, 2D NMR, LC-MS, IR and UV spectra. The presence of prototropic amino/imino tautomerism for synthesized compound 2 was observed based on spectral analysis data. Screening of antimicrobial activity against 12 strains of Gram-positive and Gram-negative bacteria, as well as yeasts, was performed for synthesized derivative 2.

1. Introduction

Multicomponent reactions (MCRs) are a highly effective tool for the synthesis of polyfunctionalized heterocyclic small molecules [1,2,3]. This approach has numerous advantages and benefits as compared to sequential multistep synthesis, namely, atom economy, efficient yields, a high bond-forming index, avoiding the use of toxic reagents, and low solvent consumption [1,2,3].

The 4-thiazolidinone core represents an important building block for the construction of multiple biologically active molecules, as well as approved drugs [4,5,6,7]. Over the past few decades, among biologically active 4-thiazolidinones scholars have identified various agents with anticancer, antibacterial, antifungal, antiviral, and antiparasitic activities. Taking into account the wide use of 4-thiazolidinone derivatives in modern drug design, further functionalization of the mentioned heterocycles with other pharmacophore fragments is an attractive area for the development of their pharmacological potential and of the search for new active agents.

The cyclopropane ring has also been the target of considerable focus in modern medicinal chemistry [8,9,10]. Due to the unique steric and electronic properties, cyclopropane fragments provide the means to obtain various potent biologically active synthetic molecules. Besides, there are numerous cyclopropyl-moiety-containing drugs approved by the FDA (Figure 1). Additionally, the cyclopropane ring can be found in bioactive natural products like terpenoids, steroids, and alkaloids.

Taking into account all the data mentioned above and our permanent interest in the field of the new MCR protocols development for the design of novel biologically active 4-thiazolidinones [11,12,13], herein we describe the synthesis of 2-(cyclopropylamino)-5-(4-methoxybenzylidene)thiazol-4(5H)-one (2) based on the application of the MCR methodology. The results of antimicrobial activity screening against 12 strains of Gram-positive and Gram-negative bacteria, as well as yeasts for synthesized derivative 2, are presented as well.

2. Results and Discussion

2.1. Synthesis of the Title Compound 2

Two possible pathways could be proposed using the retrosynthetic approach for synthesis of title 2-(cyclopropylamino)-5-(4-methoxybenzylidene)thiazol-4(5H)-one (2) (routes A and B, Scheme 1) via two-stage protocols. However, these schemes need the application of base catalysts at least in one of the stages, and additional difficulties could be met with the separation and purification of product 1a.

That is why we developed a convenient step-economy and cost-effective multicomponent protocol for the synthesis of title compound 2. The proposed approach is based on direct one-pot interaction of 2-thioxothiazolidin-4-one (rhodanine) (1), 4-methoxybenzaldehyde (both reagents used in the equimolar amounts) with cyclopropylamine which was used in 10% excess compared to 1 and to aromatic aldehyde (Scheme 2). Application of cyclopropylamine excess allows for avoiding the additional use of base catalysts. The mixture of the above-mentioned reagents was refluxed for 4 h in the dry dioxane medium, and after cooling to room temperature, the target derivative 2 was obtained with a yield 71%. Synthesized in such a way, compound 2 possesses a good level of purity; however, it could be recrystallized from glacial acetic acid.

The structure of synthesized compound 2 was confirmed using ¹H, ¹³C, 2D NMR, LC-MS, IR and UV spectra (copies of spectra are presented in the Supplementary Materials).

The presence of prototropic amino/imino tautomerism for synthesized compound 2 was observed based on spectral analysis data, which is in correspondence with early-reported data for this type of heterocycles [11,14,15,16]. The possible tautomeric forms for 2 are presented on the on Figure 2.

The pattern of signals is complicated in the ¹H NMR spectrum of compound 2 due to the amino/imino tautomerism. Protons of cyclopropyl moiety give a set of doubled signals at 0.67–0.62 (m), 0.72 (p), 0.80 (dt), 0.85 (dt), 2.78 (tt) and 3.07 (tt) ppm. The signal of methoxy group protons is also doubled and appeared as two singlets at 3.78 and 3.80 ppm. Aromatic protons resonate as a three multiplets at 7.04–7.11, 7.47–7.51 and 7.56–7.61 ppm. Proton of methylidene moiety gives a pair of singlets at 7.54 and 7.55 ppm. The broad signal at 9.95 ppm belongs to the proton of NH-amino-group. In the ¹³C NMR spectrum of compound 2, there are presented signals of all carbon atoms in the relevant areas. Carbons of cyclopropyl moiety resonate at 6.6, 7.5 (C-17, C-18), 27.7 (C-16), and 55.8, 55.9 (C-15) ppm. Carbons of 4-thiazolidinone ring give a set of signals at 126.8 (C-5), 174.5 (C-2) and 180.4 (C-4, C=O) ppm. Carbon of methylidene moiety gives a pair of singlets at 129.5 and 130.1 ppm. The molecular ion peak observed at the m/z value of 275.0 [M + H]⁺ in the positive ionization mode in the mass spectrum confirmed the formation of the title compound 2.

2.2. Antimicrobial Activity Evaluation In Vitro of Compound 2

The antibacterial activity of compound 2 was prescreened against 12 strains of Gram-positive and Gram-negative bacteria, as well as yeasts (

Table 1. In vitro antimicrobial activity of compound 2 (zone of growth inhibition at conc. 1 mg/mL after 24–48 h).

Type of Species		Species of Bacteria or Fungi	Zone of Growth Inhibition (mm ± SE)
			2	DMSO	Vancomycin	Ciprofloxacin	Clotrimazole
Gram-negative bacteria	Reference strains	Pseudomonas aeruginosa ATCC 10145	-	-	-	35.2 ± 0.2	-
		Raoultella terrigena ATCC 33257	-	-	-	37.3 ± 0.4	-
		Raoultella ornithinolytica DSM 7464	21.3 ± 0.4	9.2 ± 0.2	-	36.4 ± 0.2	-
	Clinical strains	Klebsiella pneumoniae 189	-	-	-	21.3 ± 0.2	-
		Aeromonas hydrophila 196	-	-	-	20.4 ± 0.2	-
Gram-positive bacteria	Reference strains	Streptococcus agalactiae ATCC 13813	-	-	24.3 ± 0.3	32.4 ± 0.2	-
		Staphylococcus aureus subsp. aureus ATCC 25923	-	-	25.4 ± 0.2	31.4 ± 0.2	-
		Staphylococcus epidermidis ATCC 12228	-	-	25.3 ± 0.2	39.5 ± 0.2	-
	Clinical strains	Enterococcus faecalis 191	-	-	23.2 ± 0.2	12.4 ± 0.2	-
		Staphylococcus aureus N 23	-	-	11.4 ± 0.3	15.2 ± 0.4	-
Fungi	Reference strain	Candidaalbicans ATCC 885–653	19.4 ± 0.2	-	-	-	19.3 ± 0.2
	Clinical strain	Candida albicans 67	28.3 ± 0.4	17.2 ± 0.3	-	-	8.4 ± 0.4

“-“—no inhibition was observed in the experiment; Vancomycin 30 μg (inhibition zone 17–21 mm for S. aureus), ciprofloxacin 5 μg, (inhibition zone 30–40 mm for E. coli, 22–30 for S. aureus, 22–33 for P. aeruginosa), clotrimazole 10 μg, (inhibition zone 12–17 mm for Candida).

). The diameter of the microbial growth inhibition zone and the minimal inhibitory concentrations (MICs) were used to assess the antibacterial activity.

Compound 2 indicated differential antimicrobial property against both (clinical and reference) strains of Candida albicans and the reference strain of Raoultella ornithinolytica (Table 1).

MIC to reference Raoultella ornithinolytica and clinical Candida albicans 67 was in range 0.25 mg/mL (752.1285 μM) (

Table 2. MIC value (μM) of compound against bacterial species.

	μM
	Compound 2	Vancomycin	Ciprofloxacin	Clotrimazole
Klebsiella pneumoniae 189	1504.3	-	2.2635	-
Enterococcus faecalis 191	3008.5	3.4499	12.0722	-
Candida albicans 67	752.1285	-	-	5.8
Raoultella ornithinolytica DSM 7464	752.1285	-	0.754512	-

‘-’ not tested.

).

Based on EUCAST recommendation, MIC determination and zone diameter breakpoints for ciprofloxacin and clotrimazole sensitivity tests could be used equally justified. In our case, compound 2 MIC determination showed less efficiency (752.1285 μM) than ciprofloxacin (0.754512 μM) and clotrimazole (5.8 μM) but, using zone diameter breakpoints (disc diffusion technique), showed better results than ciprofloxacin and clotrimazole (28.3 ± 0.4 mm vs. 8.4 ± 0.4 mm for Candida albicans 67).

3. Materials and Methods

3.1. General Information and Compound 2 Synthesis

Melting points were measured in open capillary tubes on a Cole-Parmer IA9200 melting point apparatus (Antylia Scientific Ltd., Stone, UK) and are uncorrected. The elemental analyses (C, H, N) were performed using the Thermo Scientific FlashSmart Elemental Analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and were within ±0.4% of the theoretical values. The 600 MHz-¹H and 150 MHz-¹³C spectra were recorded on Varian Unity Plus 600 (600 MHz) spectrometer (Varian Inc., Paulo Alto, CA, USA). All spectra were recorded at room temperature, except where indicated otherwise, and were referenced internally to solvent reference frequencies. Chemical shifts (δ) are quoted in ppm and coupling constants (J) are reported in Hz. LC–MS spectra were obtained on a Finnigan MAT INCOS-50 (Thermo Finnigan LLC, San Jose, CA, USA). The reaction mixture was monitored by thin layer chromatography (TLC) using commercial glass-backed TLC plates (Merck Kieselgel 60 F₂₅₄). Solvents and reagent (Cyclopropylamine, CAS number: 765-30-0), which is commercially available, was used without further purification. The 2-thioxothiazolidin-4-one (CAS number: 141-84-4) (1) was prepared according to the protocol described in [17].

2-(Cyclopropylamino)-5-(4-methoxybenzylidene)thiazol-4(5H)-one (2)

A mixture of 10 mmol of 2-thioxothiazolidin-4-one (1), 10 mmol of 4-methoxybenzaldehyde and 11 mmol of cyclopropylamine was refluxed for 4 h in 10 mL of dioxane. After cooling to room temperature, the yellow powder was filtered off and recrystallized from acetic acid.

Yellow crystals, yield 71%, Rf = 0.80 (ethyl acetate/benzene: 1/2), mp 238–240 °C (AcOH). ¹H NMR (600 MHz, DMSO-d₆, δ): 0.67–0.62 (m, 1H, cyclopropyl), 0.72 (p, J = 5.0, 4.5 Hz, 1H, cyclopropyl), 0.80 (dt, J = 7.1, 3.5 Hz, 1H, cyclopropyl), 0.85 (dt, J = 7.0, 3.4 Hz, 1H, cyclopropyl), 2.78 & 3.07 (tt, J = 7.0, 3.5 Hz, 1H, cyclopropyl), 3.78 & 3.80 (s, 3H, OCH₃), 7.04–7.11 (m, 2H, arom), 7.47–7.51 (m, 1H, arom), 7.54 & 7.55 (s, 1H, =CH), 7.56–7.61 (m, 1H, arom), 9.95 (brs, 1H, NH, amino). ¹³C NMR (150 MHz, DMSO-d₆, δ): 6.6 & 7.5 (C-17, C-18), 27.7 (C-16), 55.8 & 55.9 (C-15), 115.2 & 115.3 (C-11, C-13), 126.1 (C-9), 126.8 (C-5), 129.5 & 130.1 (C-8), 131.7 & 131.8 (C-10, C-14), 160.74 (C-12), 174.5 (C-2), 180.4 (C-4). IR (KBr): 3156, 3015 (N-H), 2768 (C-H), 1678 (C=O), 1618 (C=C), 1595 (C=C) cm⁻¹. UV–Vis (acetone) λ_max (lgε, L·mol⁻¹·cm⁻¹): 351 (2.03), 250 (0.88) nm. LCMS (ESI+) m/z 275.0 (98.7%, [M+H]+). Anal. calc. for C₁₄H₁₄N₂O₂S: C, 61.29%; H, 5.14%; N, 10.21%. Found: C, 61.10%; H, 5.30%; N, 10.37%.

3.2. Antimicrobial Activity

Applying the agar diffusion and resazurin-based microdilution assays, the synthesized compound 2 was evaluated in vitro for its antibacterial and antifungal properties [18,19]. Vancomycin, ciprofloxacin, clotrimazole, and dimethyl sulfoxide (DMSO) were employed as a control. Reference and clinical microbial strains that had been previously identified using the 16S rRNA gene and the MALDI TOF method (Bruker, Bremen, Germany) were applied. All clinical strains were multidrug resistant with various antibiotic resistance patterns. Clinical strains were isolated from a patient at one of the nearby hospitals who had healthcare-associated infections. Testing was done three times in total.

4. Conclusions

In the present paper the multicomponent approach to the synthesis of cyclopropyl-containing 4-thiazolidinone derivative is proposed. The method is based on one-pot interaction of rhodanine, 4-methoxybenzaldehyde and cyclopropylamine and lead to the obtaining of target compound with a high yield. The synthesized title molecule is characterized by prototropic amino/imino-tautomerism. Additionally, the studied compound showed selective antimicrobial effects against reference Raoultella ornithinolytica, and a significant antifungal effect against clinical clotrimazole resistance strain Candida albicans, both of which are prominent for further studies. Obtained data contribute to the organic and medicinal chemistry of this type of heterocycle.