Synthesis,crystal structure and biological activity of 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline

Abstract

The title compound, 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline, was synthesized by selective nucleophilic attack at C-1 of 1-bromo-3-chloropropane by the potassium salt of 4-(2-fluorophenylamino)-7-methoxyquinazolin-6-ol, which was prepared from 7-methoxy-4-oxo-3,4-dihydroquinazolin-6-yl acetate in three steps. The compound crystallized as an ethyl acetate complex (C₂₀H₂₁ClFN₃O₃, M_r = 405.85), and X-ray crystallography showed that the crystal belongs to the orthorhombic system, space group Pbca with a = 12.7407(4) Å, b = 14.0058(5) Å, c = 21.7726(7) Å, α = 90°, β = 90° and γ = 90°. The whole molecule is stacked into a three-dimensional structure via weak N–H…N hydrogen bonding between molecules. The compound acts as an effective inhibitor on the proliferation of a lung cancer cell line.

Introduction

A number of quinazoline derivatives have been reported to demonstrate good pharmacological activity as anticancer,^1–4 antimicrobial,^5–8 antiviral ⁹ and anti-inflammatory ¹⁰ agents. In view of this, in order to discover novel bioactive compounds, several new types of quinazolines were synthesized in our laboratory. ¹¹ As part of our ongoing research programme on quinazolines that may serve as leads for designing novel anticancer agents, we now report a four-step synthesis of 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline. In order to confirm its structure and investigate its stereoconfiguration, a single crystal of the title compound was obtained and the molecular structure was determined by X-ray diffraction. The compound was tested for inhibition of the proliferation of lung cancer cell lines.

Results and discussion

The synthetic route of the title compound is summarized in Scheme 1 and follows closely to that used by us for the synthesis of a series of six 4-arylamino-7-methoxyquinazolin-6-yl tosylates. ¹¹ The starting material, 7-methoxy-4-oxo-3,4-dihydroquinazolin-6-yl acetate (1), which was prepared as described previously,^11–15 was reacted with phosphoryl chloride (POCl₃) in the presence of toluene at 78 °C for 6 h to give a chloro compound 2, which was treated in situ with 2-fluoroaniline to afford compound 3 in high yield. Compound 3 was converted into compound 4 by reaction with ammonium hydroxide (28%) in methanol solution at 25 °C for 2 h. Finally, the title compound 5 was prepared by the etherification reaction of the hydroxy group of 4 with 1-bromo-3-chloropropane in dimethylformamide (DMF) at 70 °C for 10 h. The title compound 5 was characterized by spectroscopic and analytical methods. All data were consistent with structure 5. The infrared radiation (IR) showed peaks at 1287 and 1254 cm⁻¹ due to the stretching vibration of the aromatic ether. ¹H NMR data were consistent with structure 5. For example, the ¹H NMR spectrum exhibited three multiplets at 2.29, 3.85 and 3.94 ppm corresponding to the methylene protons and one singlet at 4.26 ppm due to the methoxy protons. In the mass spectrum, a peak appeared at m/z 362.1 ((M + H)⁺, 100%), which is in accordance with its molecular formula.

Scheme 1.

The synthetic route of title compound.

The IR and ¹H NMR spectra (Figures S1 and S2 in the supplemental material), mass spectroscopy (MS) spectra, elemental analysis and X-ray diffraction data for the product are in good agreement with the structure of the title compound. The molecular structure of the title compound is shown in Figure 1. The crystal data are shown in Table 1. Figure 2 shows the crystal structure and the molecular packing and hydrogen bonds in a unit cell. The molecular structure of the compound is noteworthy, since the title compound and ethyl acetate form a complex structure. In the crystal, the Cl(1)–C(18), F(1)–C(2), O(1)–C(13), N(1)–C(7) and C(1)–C(2) bond lengths are 1.842(6), 1.334(4), 1.350(3), 1.363(3) and 1.372(4) Å, and the C(13)–O(1)–C(15) and C(17)–C(18)–Cl(1) bond angles are 116.9(2)° and 111.0(3)°, respectively. From these results, it is shown that all the bond lengths and bond angles are in normal ranges. ¹⁶ The torsion angles C(14)–C(9)–C(10)–C(7) and N(3)–C(9)–C(10)–C(11) are −175.4(2)° and −178.0(2)°, respectively, which indicate that the quinazoline nucleus is almost coplanar. Intermolecular hydrogen bonds N(1)–H(1)…N(3)#1 are also present (Table 2), which contribute to the formation of a stable molecule in the unit cell.

Figure 1.

Structure of C₂₀H₂₁ClFN₃O₃ with all non-H atom-labelling scheme and ellipsoids drawn at the 30% probability level.

Table 1.

Crystal data for the title compound.

Crystal size	0.320 × 0.160 × 0.060 mm3
Formula	C20H21ClFN3O3
Molecular weight, Mr	405.85
T (K)	150(2) K
Crystal system	Orthorhombic
Space group	Pbca
a (Å)	12.7407 (4)
b (Å)	14.0058 (5)
c (Å)	21.7726 (7)
α (°)	90
β (°)	90
γ (°)	90
V (Å)3	3885.2 (2)
Z	8
Dc (g cm−3)	1.388
F(000)	1696
Goodness-of-fit on F2	1.060
Reflection/unique	3420/2861
R1, wR2 (I > 2σ(I))	0.0600, 0.1832
R1, wR2 (all data)	0.0702, 0.1946

R₁=∑(||F_o|–|F_c||)/∑|F_o| … wR₂ = (∑w(F_o²–F_c²)²/∑w(F_o²)²)^1/2

Figure 2.

Packing diagram of C₂₀H₂₁ClFN₃O₃.

Table 2.

Hydrogen bond lengths (Å) and bond angles (°) for the title compound.

D–H···A	d (D–H)	d (H···A)	d (D···A)	∠DHA
N(1)–H(1)…N(3)#1	0.88	2.07	2.871(3)	151.1

Symmetry transformations used to generate equivalent atoms: #1 x–1/2, y, –z+1/2.

Anticancer activity was determined by a methyl thiazolyl tetrazolium (MTT) bioassay in two lung cancer cell lines A431 and H1299, and the results are shown in Table 3. Gefitinib was employed as a control. The results indicated that the title compound inhibited A431 cell lines with almost similar IC₅₀ values of 23.1 µg mL⁻¹ (21.5 µg mL⁻¹ for gefitinib). In addition, in H1299 cells lines, the title compound possessed IC₅₀ values of 36.7 µg mL⁻¹ compared with 84.2 µg mL⁻¹ for gefitinib. These results suggested that the title compound is more sensitive than the control gefitinib on anticancer activity. Further structure optimization may result in more active anticancer compounds.

Table 3.

In vitro anticancer activity of 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline on A431 and H1299 lung cancer cell lines.

Compound	IC50 (µg mL−1) a
	A431	H1299
6-(3-Chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline	23.1	36.7
Gefitinib	21.5	84.2

MTT: methyl thiazolyl tetrazolium.

a

Test MTT colorimetric assay in A431 and H1299 lung cancer cell lines.

In conclusion, a novel title compound was prepared. X-ray single crystal and powder diffraction, ¹H NMR, MS and IR spectroscopy were used for the compound characterization. The results obtained from these techniques are consistent. The product obtained by crystallization from the ethyl acetate solution was the single-phase substance. A preliminary bioassay indicated that the title compound possessed some anticancer activity. Further studies on structural optimization and biological activities of quinazolines are underway in our laboratory and will be reported in the future.

Experimental analysis

All melting points were taken on a Beijing Taike X-4 microscopy melting point apparatus and are uncorrected. Thin-layer chromatography (TLC) was performed on aluminium sheets precoated with silica gel (Merck Kieselgel 60 F254).¹H NMR spectra were obtained on a Bruker BioSpin 400 MHz spectrometer in dimethyl sulfoxide-d₆ (DMSO-d₆) using tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) are given in ppm and coupling constants (J) are given in hertz (Hz). IR spectra were recorded on a Bruker Platinum ART Tensor II FTIR spectrometer. MS spectra were obtained on an Agilent 6460 QQQ mass spectrometer analysis system. Elemental analysis of the newly synthesized compounds was carried out on a Carlo Erba 1108 analyzer. Crystal data were obtained on a Bruker D8 VENTURE X-ray diffractometer. All solvents and reagents were obtained from commercial sources and were used without purification.

Synthesis of 4-((2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl acetate (3)

A suspension of 7-methoxy-4-oxo-3,4-dihydroquinazolin-6-yl acetate (1; 2.0 g, 8.54 mmol), triethylamine (2.4 mL, 17.32 mmol) and phosphorus oxychloride (2.4 mL, 26.22 mmol) in toluene (25 mL) was heated to 78 °C for 6 h. Then, a mixture of 2-fluoroaniline (1.04 g, 9.40 mmol) in toluene (40 mL) was added followed by stirring for 5 h. Upon completion of the reaction (TLC), the resulting mixture was cooled to 0 °C. The solid thus obtained was filtered off under reduced pressure and washed with toluene (30 mL). Isopropanol (40 mL) was added to the solid, which was then stirred for 2 h. The resulting solid was filtered off and washed with cold isopropanol (20 mL). The solid was dried at 60 °C to afford 3 as a white powder: Yield 2.34 g (83.7%, two steps); m.p. 196.5–198.2 °C; IR (KBr, cm⁻¹): 1770, 1644, 1578, 1474, 1433, 1274, 1255, 1105, 1066, 760, 521; ¹H NMR (DMSO-d₆, 400 MHz): δ 10.31 (s, 1H, NH), 8.90 (s, 1H, ArH), 8.79 (d, J = 6.4 Hz, 1H, ArH), 7.59–7.52 (m, 2H, ArH), 7.48–7.39 (m, 2H, ArH), 7.36–7.31 (m, 1H, ArH), 4.02 (s, 3H, OCH₃), 2.40 (s, 3H, CH₃). Anal. calcd for [C₁₇H₁₄FN₃O₃]: C, 62.38; H, 4.31; N, 12.84; found: C, 62.45; H, 4.37; N, 12.77%.

Synthesis of 4-((2-fluorophenyl)amino)-7-methoxyquinazolin-6-ol (4)

4-((2-Fluorophenyl)amino)-7-methoxyquinazolin-6-yl acetate (1.0 g, 3.1 mmol; 3) was admixed with methanol (15 mL). The mixture was cooled to 0 °C, treated with ammonia solution (5 mL) and stirred for 2 h at 25 °C. The solid thus obtained was filtered off and washed with a mixture of cold methanol (10 mL) and water (10 mL). The resulting solid was dried at 60 °C in an oven to afford 4 as a white powder: Yield 0.78 g (89.5%); m.p. 247.2–249.3 °C; IR (KBr, cm⁻¹): 3334, 1623, 1582, 1449, 1426, 1272, 1256, 1106, 1060, 751, 525; ¹H NMR (DMSO-d₆, 400 MHz): δ 9.70 (s, 1H, NH), 9.34 (s, 1H, OH), 8.31 (s, 1H, ArH), 7.61 (d, J = 6.0 Hz, 2H, ArH), 7.24 (d, J = 3.6 Hz, 4H, ArH), 3.97 (s, 3H, OCH₃). Anal. calcd for [C₁₅H₁₂FN₃O₂]: C, 63.15; H, 4.24; N, 14.73; found: C, 63.20; H, 4.28; N, 14.79%.

The title compound (5)

A mixture of 4-((2-fluorophenyl)amino)-7-methoxyquinazolin-6-ol (0.50 g, 1.8 mmol; 4), K₂CO₃ (1.21 g, 8.8 mmol) and 1-bromo-3-chloropropane (0.30 g, 1.9 mmol) in dried N, N-DMF (30 mL) was stirred at 70 °C for 10 h. The reaction mixture was diluted with water (50 mL), extracted with ethyl acetate (3 × 30 mL), dried over anhydrous sodium sulphate and concentrated in vacuo to give a crude product, which was purified by column chromatography to afford 5 as a white powder: Yield 0.54 g (85.2%); m.p. 155.3–157.1 °C; IR (KBr, cm⁻¹): 2935, 1619, 1578, 1472, 1452, 1287, 1254, 1101, 1063, 756, 552; ¹H NMR (DMSO-d₆, 400 MHz): δ 9.53 (s, 1H, NH), 8.35 (d, J = 13.6 Hz, 1H, ArH), 7.87 (d, J = 13.2 Hz, 1H, ArH), 7.54 (s, 1H, ArH), 7.39–7.19 (m, 4H, ArH), 4.26 (s, 3H, OCH₃), 3.94 (m, 2H, CH₂), 3.85 (m, 2H, CH₂), 2.29 (m, 2H, CH₂); MS (ESI) m/z (%): (M + H)⁺: 362.1 (100). Anal. calcd for [C₁₈H₁₇ClFN₃O₂]: C, 59.76; H, 4.74; N, 11.61; found: C, 59.81; H, 4.79; N, 11.66%.

Crystal data structure determination

The white powder of the title compound was dissolved in ethyl acetate. After slowly evaporating the solvent for two days, some single crystals suitable for X-ray analysis were obtained. A white crystal (C₂₀H₂₁ClFN₃O₃) with dimensions of 0.320 × 0.160 × 0.060 mm³ was selected for data collection which was performed on a Bruker D8 VENTURE diffractometer equipped with graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å) using an ω scan mode at 150(2) K. A total of 21,205 reflections were collected in the range of 3.0° < θ < 25.0° (index ranges: –15 < h < 14, –13 < k < 16 and −25 < l < 25) and 3420 were independent (R_int = 0.0286), of which 294 observed reflections with I > 2σ (I) were used in the structure determination and refinements. The structure was solved by intrinsic phasing methods with the SHELXT 2014 program ¹⁷ and expanded by the Fourier technique. The non-hydrogen atoms were refined anisotropically. The hydrogen atoms bound to carbon were determined by theoretical calculations. The structure was refined by the full-matrix least-squares techniques on F² with SHELXL-2017. ¹⁷ The final refinement gave R = 0.0600 and wR = 0.1832 (w = 1/(σ²(F_o²) + (0.1161P)² + 4.0254P), where P = (F_o² + 2F_c²) /3), S = 1.06, (Δ/σ) max = 0.0044, (Δρ) max = 1.229 and (Δρ) min = –0.604 e Å⁻³. All calculations were performed using the Crystal Structure crystallographic software package except for the refinement. Crystallographic data and experimental details of structural analyses for the title compound are summarized in Table 1. The hydrogen bond data of the title compound are listed in Table 2.

CCDC1879449 contains the supplementary data for this paper. The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via https://summary.ccdc.cam.ac.uk/structure-summary-form.

In vitro anticancer activity test of the title compound on A431 and H1299 lung cancer cell lines

The title compound was evaluated for its in vitro against two lung cancer cell lines (A431 and H1299) by the MTT-based assay using gefitinib as a positive control (Table 3). Cells were placed in 96-multiwell plates (104 cells per well) for 24 h before treatment with the test compounds to allow attachment of the cells on the surface of the plate. The test compound and gefitinib were dissolved in DMSO and diluted with saline to the appropriate volume. Different concentrations of the test compound (0, 5, 12.5, 25 and 50 μg mL⁻¹) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the test compound for 48 h at 37 °C in an atmosphere of 5% CO₂. The relation between surviving fraction and compound concentration was plotted, and IC₅₀ (the concentration required for 50% inhibition of cell viability) was calculated for the title compound.

Supplemental Material

esi-ir – Supplemental material for Synthesis, crystal structure and biological activity of 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline

Supplemental material, esi-ir for Synthesis, crystal structure and biological activity of 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline by Zhi-Qiang Cai, Chen-kang Zhao, Meng-Yao Li, Xiao-Min Shuai, Hai-Guan Ding, Qing-Lin Wang, Jia Fu, Zheng-Sheng Jin, Shuai Li and Le-Jing Zhao in Journal of Chemical Research

Supplemental Material

esi-nmr – Supplemental material for Synthesis, crystal structure and biological activity of 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline

Supplemental material, esi-nmr for Synthesis, crystal structure and biological activity of 6-(3-chloropropoxy)-4-(2-fluorophenylamino)-7-methoxyquinazoline by Zhi-Qiang Cai, Chen-kang Zhao, Meng-Yao Li, Xiao-Min Shuai, Hai-Guan Ding, Qing-Lin Wang, Jia Fu, Zheng-Sheng Jin, Shuai Li and Le-Jing Zhao in Journal of Chemical Research