Methyl 9-(2-Iminothiazol-3(2H)-yl)-9-oxononanoate

Abstract

Methyl 9-(2-iminothiazol-3(2H)-yl)-9-oxononanoate was synthesized through Schotten–Baumann type reaction between 2–aminothiazole and methyl 9-chloro-9-oxononanoate. The structure of the newly synthesized compound was elucidated based on ¹H-NMR, ¹³C-NMR, DEPT, NOE, ESI-MS, FT-IR and UV-Vis spectroscopy.

1. Introduction

2-Aminothiazole derivatives represent very important scaffolds in many fields of applied chemistry, such as dyes [1], pharmaceuticals [2], and medicinal chemistry [3,4,5,6,7,8,9,10]. For a long time, our interests have lain in thiazole chemistry both from the synthetic and mechanistic point of view and in the study of biological effects of 9-hydroxystearic acid (9-HSA, Figure 1a) and its derivatives. 9-HSA is an endogenous cellular product with antiproliferative activity against different human cancer cell lines, and, interestingly, it is not toxic for normal cells. It acts as a histone deacetylase inhibitor (HDACi) [11,12]. Looking at the structure of Vorinostat, also known as SAHA (suberoylanilide hydroxamic acid or N-Hydroxy-N′-phenyloctane diamide, Figure 1b), and 9-HSA, and considering that both act as inhibitors of HDAC by interacting, respectively, through hydroxamate- or carboxylate-zinc coordination modes, we realized that a structural modification of 9-HSA on the C-9, preserving the methylene chain as a spacer between the carboxylic group and an amide functionality, might produce promising candidates as inhibitors of HDAC. Thus, we incorporated a long aliphatic chain with ester or the carboxylic acid ending group to a series of amino aza-heterocycles via an amide connection (Figure 1c,d), and the compounds obtained showed antiproliferative ability against a series of human cancer cell lines [13,14].

Based on the above and considering that the 2-aminothiazolyl moiety is present in a lot of hybrid compounds of interest, such as anticancer drugs [15], we planned, and herein report, together with the preliminary results on its biological activity, the synthesis of a novel hybrid bearing both the 2-aminothiazolyl and azelayl moieties.

2. Results

The synthesis of methyl 9-(2-iminothiazol-3(2H)-yl)-9-oxononanoate (3) (Scheme 1) was performed through a Schotten–Baumann-type reaction from 2-aminothiazole (1) and methyl 9-chloro-9-oxononanoate (2) in dichloromethane at room temperature. At the end of the reaction, product 3 was purified by column chromatography on silica gel using a mixture of light petroleum and diethyl ether, 2/8 v/v ratio, as eluent (yield 43%); no presence of other isomers was detected. The newly synthesized compound was characterized by ¹H-NMR, ¹³C-NMR, ESI-MS, FT-IR, and UV-Vis spectroscopy.

In principle, 2-aminothiazole has three nucleophilic sites, namely the C-5, the endo–cyclic nitrogen atom, and the exo–cyclic nitrogen atom. The ¹H NMR spectrum of the unique product that was recovered from the current reaction shows, in addition to the aliphatic chain and methoxy signals, two doublets at δ = 7.42 and 7.00 ppm due to C-4 and C-5 hydrogen atoms belonged to the thiazole ring. This information, however, does not permit discrimination between the two possible structures 3 and 3′ derived from the attack to the endo- or the exo-cyclic nitrogen atom, respectively (Scheme 2).

Thus, a NOESY-1D experiment was carried out, and the result provided an unequivocal elucidation of the structure. In particular, the irradiation of the signal at 2.55 ppm (CH₂ in α position with respect to the amide group) produced a Nuclear Overhauser Effect on the signal at 7.42 ppm belonging to the C-4 hydrogen atom (Figure S2 in Supplementary Materials), indicating that the group irradiated was near to the H-4 and this was possible when the chain was bound to the endocyclic nitrogen atom of the thiazole ring.

This observation that resulted was crucial for assigning the structure of the reaction product to 3, as derived from the attack on the endo–cyclic nitrogen atom.

We chose to test the effects of compound 3 on two cell lines since they came from the neoplastic transformation of two different human tissues: HT29 was isolated from a primary tumor obtained from a patient with colorectal adenocarcinoma, while U2OS was derived from a moderately differentiated sarcoma of an osteosarcoma patient.

The effects on cell viability were evaluated using the MTT assay, and the results are shown in

Table 1. IC₅₀ values of compound 3.

Cell Line	IC50
HT29	≥100 μM
U2OS	50 μM

. Interestingly, compound 3 exhibits inhibitory activity only on U2OS, indicating a cell-specific activity.

HT-29 cells have a mutation in the p53 gene at position 273, while U2OS are wt for this protein. This difference is important as it explains the different biological responses to therapeutic agents. Indeed, it has been demonstrated that the administration of 9-HSA to HT29 inhibits HDAC1-inducing histone hyperacetylation, which is associated with a cytostatic effect [11]. On the contrary, the treatment of U2OS produces a hyperacetylation of p53 with consequent cytotoxic effects [16]. Moreover, 9-HSA is a selective inhibitor of class I HDACs [12], while other agents, in particular, SAHA (non-selective for HDACi), affect the hyperacetylation of all histones and cause massive cell death [17].

3. Materials and Methods

The ¹H, ¹³C, and DEPT (Distortionless Enhancement by Polarization Transfer), NOE (Nuclear Overhauser Effect) spectra were recorded on an Inova 600 (Varian, Palo Alto, CA, USA) spectrometer operating at 600 MHz (for ¹H NMR) and at 150 MHz (for ¹³C NMR). Chemical shifts were referenced to the solvent for ¹H and ¹³C NMR (δ = 7.26 ppm and δ = 77.0 ppm, respectively, for CDCl₃). Signal multiplicities were established by DEPT experiments. Chemical shifts are measured in δ (ppm). J values are given in Hertz. Electron spray ionization mass spectra (ESI-MS) were recorded with a WATERS 2Q 4000 instrument (Waters Corporation, Milford, MA, USA). IR spectra were recorded on a Perkin Elmer FT-IR Mod. 1600 spectrophotometer (Perkin Elmer, Waltham, MA, USA). UV/Vis spectra were recorded on a PerkinElmer Lambda 12 spectrophotometer (Perkin Elmer, Waltham, MA, USA) at 20 °C in CHCl₃ and in quartz cells (path length cell: 1 cm). Melting points (m.p.) were measured on a Büchi 535 apparatus (Büchi, Flawil, Switzerland). Chromatographic purifications (FC) were carried out on glass columns packed with silica gel (Merck grade 9385, 230−400 mesh particle size, 60 Å pore size) at medium pressure (Merck & Co. Readington, NJ, USA). Thin layer chromatography (TLC) was performed on silica gel 60 F₂₅₄ coated aluminum foils (Fluka, Buchs, Switzerland). Methyl 9-chloro-9-oxononanoate was prepared by us as previously reported [13]; 2-aminothiazole was purchased by Sigma-Aldrich (Milan, Italy), as well as all the solvents used.

The human colon cancer cell line HT29 and the human bone osteosarcoma U2OS cell line were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in an RPMI 1640 medium (Labtek Eurobio, Milan, Italy), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) or fetal calf serum (Euroclone, Milan, Italy) 100 U/mL penicillin, 100 g/mL streptomycin (GE Healthcare, Chicago, IL, USA) and 2mM L-glutamine (Sigma-Aldrich, Milan, Italy), at 37 °C in a 5% CO₂ atmosphere. Compound 3 was dissolved in DMSO (Sigma-Aldrich, Milan, Italy) in a stock solution.

3.1. Methyl 9-(2-Iminothiazol-3(2H)-yl)-9-oxononanoate (3)

A solution of 2-aminothiazole (1) (150 mg, 1 mmol, in 1 mL of anhydrous CH₂Cl₂) was added to a solution of 9-chloro-9-oxononanoate (2) (110 mg, 0.5 mmol), prepared from azelaic acid mono-methyl ester and oxalyl chloride, in 5 mL of anhydrous CH₂Cl₂, under a nitrogen atmosphere, as reported in ref. [13]. The mixture was magnetically stirred for 3 h. The reaction course was monitored by TLC (eluent diethyl ether/petroleum ether 9/1). The product was purified by chromatography on silica gel (eluent diethyl ether/petroleum ether 8/2). The product yield was 61 mg, 43%.

Pale yellow solid, m.p.: 105.5–105.8 °C; ¹H-NMR (600 MHz, CDCl₃, 25 °C) δ, ppm: 12.25 (s, 1H, NH), 7.42 (d, J = 3.6 Hz, 1H, CH=), 7.00 (d, J = 3.6 Hz, 1H, CH=), 3.66 (s, 3H, COOCH₃), 2.54 (t, J = 7.5 Hz, 2H, CH₂CON), 2.29 (t, J = 7.5 Hz, 2H, CH₂COOCH₃), 1.80–1.74 (m, 2H, CH₂CH₂CON), 1.64–1.58 (m, 2H, CH₂CH₂COOCH₃), 1.43–1.30 (m, 6H, aliphatic CH₂); ¹³C NMR: (150 MHz, CDCl₃, 25 °C) δ ppm: 174.2 (C), 171.2 (C), 160.1 (C), 136.1 (CH), 113.5 (CH), 51.4 (CH₃), 36.1 (CH₂), 34.0 (CH₂), 29.0 (CH₂), 28.95 (CH₂), 28.87 (CH₂), 25.0 (CH₂), 24.8 (CH₂); FT-IR (CHCl₃), ν (cm⁻¹): 3019, 2930, 2860, 2397, 1731, 1691, 1552, 1532, 1217; UV-Vis λmax = 266 nm; ESI-MS (m/z): 285 [M+H]⁺, 307 [M + Na]⁺, 323 [M + K]⁺; Elemental Analysis for C₁₃H₂₀N₂O₃S, Calculated C, 54.91; H, 7.09; N, 9.85; found C, 54.97; H, 7.12; N, 9.81.

3.2. Cell Viability Assays

To evaluate the compound activity, the cells were treated for 24 h with a vehicle (DMSO, as control) or with the test sample at concentrations between 0.01 μM and 250 μM. Cell growth was assessed by the colorimetric 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay (MTT, Sigma-Aldrich, Milan, Italy). As for MTT assays, the culture medium was removed, and cells were incubated with 0.1 mL of MTT dissolved in PBS at a concentration of 0.2 mg/mL following Micheletti et.al. [18]. The absorbance at 570 nm was measured using a multiwell plate reader (Tecan, Männedorf, Switzerland). The IC₅₀ was determined from the dose–response curve by using Prism Graph Pad Software v.6.0 (GraphPad Software, San Diego, CA, USA).