2-((6,7-Dimethoxy-4-oxo-3-(4-(trifluoromethyl)phenethyl)-3,4-dihydroquinazolin-2-yl)thio)-N-(4-ethylphenyl)butanamide

Abstract

2-({3-[2-(1-cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111; K284), the representative CHI3L1 inhibitor, has interesting biological activities, including anti-cancer and anti-inflammatory effects on neuroinflammation. Following our hit-to-lead program, we report the most active novel derivative, named CBJL-025, 2-((6,7-dimethoxy-4-oxo-3-(4-(trifluoromethyl)phenethyl)-3,4-dihydroquinazolin-2-yl)thio)-N-(4-ethylphenyl)butanamide. The title compound, CBJL-025, was successfully synthesized by S-alkylation of the p-trifluoromethyl phenethyl group possessing quinazoline and the corresponding bromide. The structure of CBJL-025 was confirmed by ¹H and ¹³C nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS).

1. Introduction

Chitinase-3-like protein 1 (CHI3L1), encoded by the CHI3L1 gene, is a secreted glycoprotein in humans. CHI3L1, also known as YKL-40, has an approximate molecular weight of 40 kDa. An increasing number of studies [1,2] have reported that the expression of CHI3L1 is markedly upregulated in various inflammatory conditions [3,4], Alzheimer’s disease [5], and several types of cancer [6]. Accordingly, numerous reports have demonstrated a close association between CHI3L1 and these diseases. In particular, a recent lead optimization study based on the compound K284-611 for the treatment of glioblastoma has been reported [7] (Figure 1).

We also conducted a medicinal chemistry program based on K284-611 and herein report the title compound 1, CBJL-025 (2-((6,7-dimethoxy-4-oxo-3-(4-(trifluoromethyl)phenethyl)-3,4-dihydroquinazolin-2-yl)thio)-N-(4-ethylphenyl)butanamide), which exhibited the most potent cytotoxic activity among the synthesized compounds. The title compound was designed to incorporate a p-trifluoromethylphenyl group, motivated by the well-documented success of fluorinated substituents in medicinal chemistry [8].

2. Results

To synthesize CBJL-025, quinazoline 2 and bromide 3 were first prepared from commercially available starting materials according to a reported procedure [7]. With key intermediates 2 and 3 in hand, they were coupled under reflux conditions using potassium carbonate in acetone, as depicted in Scheme 1. The structure of the synthesized compound 1 was confirmed by ¹H NMR (Figure S1), ¹³C NMR (Figure S2), ¹⁹F NMR (Figure S3), and high-resolution mass spectrometry (HRMS, Figure S4). Notably, this strategy provided an overall yield exceeding 70% on the gram scale. The synthesized compound 1, CBJL-025, exhibited a fourfold increase in cytotoxic activity compared with the reference compound K284-611.

Encouraged by these promising results, we investigated the structural modeling of CBJL-025 in complex with the CHI3L1 protein. Two distinct hydrogen-bonding interactions between the trifluoromethyl group and CHI3L1 were identified (Figure 2). These strong intermolecular interactions are expected to significantly enhance the binding affinity of the designed molecule toward the CHI3L1 protein.

3. Materials and Methods

3.1. General Information

All reactions performed under air were conducted with a reflux condenser, leaving the vent open to prevent solvent evaporation. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), TCI (Tokyo, Japan), Alfa Aesar (Ward Hill, MA, USA), and Acros Organics (Geel, Belgium) and were used directly without further purification unless otherwise indicated. Thin-layer chromatography (TLC) was performed on silica gel 60 F₂₅₄ glass plates (Merck, Darmstadt, Germany), visualized by UV light at 254 or 365 nm, using anisaldehyde, ninhydrin, and KMnO₄. ¹H, ¹³C, and ¹⁹F NMR spectra were recorded with a Jeol ECZL 400S FT NMR (Tokyo, Japan) (400 MHz for ¹H NMR) using chloroform-d as the solvent. Chemical shifts (δ) were reported in parts per million (ppm), referenced to the residual solvent signals as follows: ¹H-7.26 ppm for chloroform-d and ¹³C-77.16 ppm for chloroform-d. The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad. Coupling constants, J, were reported in Hertz (Hz). ¹³C NMR spectra were recorded with broad-band proton decoupling. High-resolution mass spectrometry was performed with a SCIEX X500B Q-TOF (Framingham, MA, USA), a Bruker MaXis 4G Q-TOF (Billerica, MA, USA), and a Waters Xevo G2-XS Q-Tof (Milford, MA, USA) in positive ion mode using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) as an ionization method. Silica gel column chromatography was performed using silica gel with a particle size of 40–63 μm (230–400 mesh).

3.2. 2-((6,7-Dimethoxy-4-oxo-3-(4-(trifluoromethyl)phenethyl)-3,4-dihydroquinazolin-2-yl)thio)-N-(4-ethylphenyl)butanamide (CBJL-025)

To a solution of quinazoline 2 (410 mg, 1.0 mmol) and bromide 3 (256 mg, 1.0 mmol) in acetone, potassium carbonate was added (276 mg, 2.0 mmol). The mixture was heated to reflux for 5 h. After completion, acetone was evaporated under reduced pressure, and the residue was extracted with DCM (2 × 150 mL). The combined organic layers were washed with water, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by flash chromatography using hexane/ethyl acetate (3:2 to 1:1) as the eluent to afford final compound 1 (491 mg) in 82% yield.

¹H NMR (400 MHz, CDCl₃,) δ 9.71 (s, 1H), 7.56–7.58 (m, 3H), 7.42 (d, 2H, J = 8.0 Hz), 7.36 (d, 2H, J = 8.0 Hz), 7.09 (d, 2H, J = 8.0 Hz), 7.04 (s, 1H), 4.49 (t, 1H, J = 7.6 Hz), 4.21 (t, 2H, J = 8.0 Hz), 4.03 (s, 3H), 4.02 (s, 3H), 3.05–3.19 (m, 2H), 2.58 (q, J = 7.6 Hz, 2H), 2.31 (m, 1H), 1.92 (m, 1H), 1.15–1.19 (m, 6H); ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 168.8, 160.6, 155.8, 155.7, 149.1, 143.0, 141.7, 140.4, 135.9, 129.4 (q, ²J_C–F = 32.6 Hz), 129.4, 128.5, 125.8 (q, ³J_C–F = 3.8 Hz), 124.3 (q, ¹J_C–F = 270.2 Hz), 119.4, 112.7, 106.4, 105.8, 56.6, 56.5, 50.1, 46.1, 34.0, 28.4, 23.1, 15.8, 12.4; ¹⁹F NMR (CDCl₃, 376 MHz) δ (ppm)–62.35 (s); HRMS-Q-TOF m/z calcd for C₃₁H₃₂F₃N₃O₄S [M + H]⁺; and 600.2138, found: 600.2139.

4. Conclusions

In summary, 2-((6,7-dimethoxy-4-oxo-3-(4-(trifluoromethyl)phenethyl)-3,4-dihydroquinazolin-2-yl)thio)-N-(4-ethylphenyl)butanamide (1, CBJL-025) was synthesized by a simple S-alkylation reaction under mild conditions in a satisfactory yield. The pure compound was obtained as a white solid. Its structure was confirmed by HRMS, as well as ¹H NMR, ¹³C NMR, and ¹⁹F NMR spectroscopy. Together with this compound, a comprehensive medicinal chemistry study of related derivatives, including their diverse biological activities, is currently underway and will be reported in future publications.