Quinazoline-urea, new protein kinase inhibitors in treatment of prostate cancer

Abstract

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2), two protein tyrosine kinases, are involved in pathological disorders and the progression of different types of carcinomas. Concomitant inhibition of both tyrosine kinase activities appears to be an attractive target for cancer chemotherapy. A series of new quinazoline derivatives substituted by amide, urea, or carbamic acid ester groups have been synthesized. The biological activities of these new compounds have been evaluated for their enzyme inhibition and antiproliferative activities.

Keywords::

• Quinazoline
• EGFR
• VEGFR
• inhibitors
• cancer
• prostate

Introduction

The prostate gland is the most common site for cancer in males within the developed world. Increasingly, amongst the elderly male population, prostate disease is having a significant effect on morbidity, mortality, and health care resources¹. Although endocrine and cytotoxic treatments remain therapies of choice to induce tumor growth arrest, resistance and recurrence often appear with toxicity side effects². Protein kinases play important roles in regulating most of the cellular functions (proliferation, cell cycle, cell metabolism, survival, apoptosis, DNA damage/repair, etc.), and their overexpression is involved in many cancer cells^3,4. Most signal transduction pathways are mediated by protein kinases, and aberrant kinase signaling leads to proliferation of cancer cells and also angiogenesis and growth of solid tumors such as prostatic, colon, breast, and gastric cancers^5,6.

Among protein kinases, the epidermal growth factor (EGF)/ErbB family of receptor tyrosine kinases, notably EGFR, is activated following binding with peptide growth factor, inducing the formation of receptor homo- or heterodimers and activation of the intrinsic tyrosine kinase domain. Signal transduction pathways are initiated, such as Ras/MAPK, PI3K/Akt, or JAK/STAT, leading to tumor initiation in the case of overactivation^7–9.

The vascular endothelial growth factor (VEGF) is a potent angiogenic factor produced by tumor cells. This peptide stimulates the formation of new blood vessels, a fundamental event in normal and pathologic angiogenesis. Activation of the VEGF receptor tyrosine kinase (TK) family, notably VEGFR-2, by this growth factor results in endothelial cell survival, mitogenesis, migration, and differentiation^10–12.

Tumor angiogenesis is critically important for cancerous growth, supplying nutrients and oxygen to tumors¹³. It is well-established that tumor cells express EGFR and VEGFR-2, in particular in tumor endothelial cells¹⁴. These two glycoproteins have been identified in many cancers, and a relationship is established between them: a targeting EGFR strategy inhibits tumor growth, decreasing the product of VEGF, and inhibition of VEGFR-2 increases the antitumoral effect of inhibitors of EGFR^15,16. According to the knowledge of tumor biology and mechanisms of oncogenesis activation, simultaneous blockade of the intracellular domain of EGFR and VEGFR-2 by small competitive molecules at the adenosine phosphate (ATP) site offers new opportunities for the development of anticancer drugs with a synergic effect¹⁷. A number of compounds representing a generation of ATP-mimics (Figure 1), such as the 4-anilinoquinazolines gefitinib (ZD1839/Iressa) and erlotinib (OSI-774/Tarceva)^18,19, have been approved by the Food and Drug Administration (FDA).

Figure 1.  Structures of ATP-mimic inhibitors for treatment of solid tumors.

Some 4-anilinoquinazolines inhibit EGFR and VEGFR-2 TK activities and are described in the literature^20,21. Vandetanib (ZD6474), an orally bioavailable drug in phase III, is considered to be a dual tyrosine kinase inhibitor targeting EGFR and VEGFR-2²². This quinazoline substituted with a halogen at the 2- and 4-positions on the phenyl group showed potent inhibitory activities (IC₅₀ = 500 nM for EGFR and 40 nM for VEGFR-2) in enzyme-linked immunosorbent assays (ELISAs) with recombinant enzymes^22,23. The heterocycle employed appears to be a potent linker to interact with the ATP site of these two proteins. The effects of substituents on the arylamino group and ether linker at the 6- or 7-position of quinazoline influence the inhibitory activities, and it could be interesting to investigate this aryl group. Many compounds which target the VEGFR-2 kinase domain are designed with amide or urea groups^24–26. The most potent VEGFR inhibitor is represented by the urea derivative sorafenib (BAY 93-4006/Nexavar). Based on the structure–activity relationships established for the described protein tyrosine kinase inhibitors^20–26 and previous work carried out in our laboratory^27–29, we have undertaken the synthesis of a series of amide, carbamic acid ester, and urea derivatives, coupled with a 4-anilinoquinazoline skeleton, leading to new dual EGFR and VEGFR inhibitors (Figure 2).

Figure 2.  Structures of synthesized compounds.

Three series of compounds, differentiating the ether linker (methoxy or diethylaminoethoxy) at the C-6 and C-7 positions of the quinazoline core, were synthesized. Introduction of the diethylamino side chain may improve physicochemical and pharmacological properties such as water solubility, bioavailability, and cell penetration. According to the position, the inhibitory activity of the 4-anilinoquinazoline might change. Then, the final new compounds reported in this article were evaluated for anticancer activity on hormone-independent PC3 prostate cancer cells and the inhibition of various kinase activities, notably EGFR and VEGFR-2; the results were compared with the activity of the reference compounds, semaxanib (SU5416) and gefitinib. To evaluate the selectivity of these compounds, they were also studied on Aurora-A, a serine–threonine kinase whose inhibition activity disrupts the cell cycle and blocks proliferation. Vertex³⁰, a pyrimidine derivative, was used as reference for this test.

Materials and methods

Chemistry

Experimental protocols

Melting points were determined in open capillary tubes on a Büchi reference B-530 digital melting point apparatus and are uncorrected. Kieselgel 60 F-254 commercial plates were used for analytical thin layer chromatography (TLC) with ultraviolet (UV) light and/or iodine to follow the course of the reaction. Silica gel Kieselgel Si 60, 0.063–0.200 mm (Merck), was used for column chromatography. The structures of all compounds were supported by infrared (IR) spectrometry (using a Bruker Vector 22 instrument) ¹H-nuclear magnetic resonance (NMR) (300 MHz) spectra were recorded on a Bruker AC300P NMR spectrometer in dimethylsulfoxide (DMSO)-[D₆] or in CDCl₃ at room temperature. Chemical shifts (δ) are reported in ppm downfield from tetramethylsilane (TMS). J values are in Hertz and the splitting patterns are designed as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. APCI+ (atmospheric pressure chemical ionization) mass spectra were obtained on a Thermo Electron Surveyor MSQ liquid chromatography-mass spectrometry (LC/MS) system. Gefitinib was synthesized according to described procedures³¹. Semaxanib (SU5416) is a commercial product of Sigma-Aldrich, reference S8442-5MG, lot number 085K4618. These two compounds were references for tests on cellular EGFR and VEGFR-2 activities. Vertex, a product of Huskerchem, reference MK-0457, was the commercial reference for tests on Aurora-A activity.

General procedure for compound 2–6 (series A)

4-(2-Diethylaminoethoxy)-3-methoxybenzoic acid methyl ester hydrochloride 2 Potassium carbonate (0.44 mol) was added to a solution of methyl vanillate 1 (0.11 mol) in acetone (300 mL) and was stirred for 5 min. 2-Diethylaminoethyl chloride hydrochloride (0.16 mol) was added and the mixture was refluxed for 16 h. The inorganic solid was filtered off and the filtrate was concentrated in vacuo. The oily residue was dissolved in 2-propanol (10 mL), and 2-propanol saturated with HCl was added (15 mL). The resulting white precipitate was filtered, washed with diethyl ether, and dried in vacuo to afford 2. Mp = 90–92°C (toluene); yield = 76%. IR (ν, cm⁻¹): 2579 and 2467 (NH⁺), 1695 (C=O). ¹H-NMR (DMSO), δ, ppm: 1.25 (m, 6H, NCH₂CH₃), 3.20 (m, 4H, NCH₂CH₃), 3.50 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 3.80 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 7.15 (d, 1H, ArH, J = 8.70 Hz), 7.50 (d, 1H, ArH, J = 2.00 Hz), 7.60 (dd, 1H, ArH, J = 8.70 Hz and J = 2.00 Hz), 12.20 (s, 1H, NH⁺). LC/MS (APCI+), m/z: 282.3 (M – HCl + H)⁺.

4-(2-Diethylaminoethoxy)-3-methoxy-6-nitrobenzoic acid methyl ester hydrochloride 3 A solution of tin(IV) chloride (28 mmol) and nitric fuming acid (28 mmol) in CH₂Cl₂ (20 mL) was added dropwise to a solution of 2 (9.40 mmol) in CH₂Cl₂ (150 mL) cooled at–70°C. After stirring for 8 h at–70°C and warming to room temperature, the residue was filtered off and dissolved in saturated potassium carbonate solution (100 mL). The aqueous solution was then extracted with ethyl acetate (3 × 70 mL), dried over magnesium sulfate, and concentrated in vacuo. The oily residue was dissolved in 2-propanol (3 mL), and 2-propanol saturated with HCl was added (7 mL). The resulting white precipitate was filtered, washed with diethyl ether, and dried in vacuo to afford 3. Mp = 128–131°C (cyclohexane); yield = 88%. IR (ν, cm⁻¹): 2562 (NH⁺), 1717 (C=O), 1510 (NO₂). ¹H-NMR (DMSO), δ, ppm: 1.10 (m, 6H, NCH₂CH₃), 2.60 (m, 4H, NCH₂CH₃), 3.00 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 3.90 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 4.20 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 7.05 (s, 1H, ArH), 7.50 (s, 1H, ArH), 11.90 (s, 1H, NH⁺). LC/MS (APCI+), m/z: 327.3 (M – HCl + H)⁺.

2-Amino-4-(2-diethylaminoethoxy)-3-methoxybenzoic acid methyl ester hydrochloride 4 The compound 3 (6 mmol) was dissolved in methanol (50 mL), and Raney nickel (0.4 g) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 16 h. The product was passed through a plug of Celite before being concentrated and purified by column chromatography on silica gel eluting with CH₂Cl₂/MeOH (9/1). Petroleum ether provided the title compound as a brown solid. Mp = 176–178°C; yield = 66%. IR (ν, cm⁻¹): 3480 and 3385 (NH₂), 1707 (C=O). ¹H-NMR (DMSO), δ, ppm: 1.25 (m, 6H, NCH₂CH₃), 3.20 (m, 4H, NCH₂CH₃), 3.00 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 3.90 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 4.30 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 6.45 (s, 1H, ArH), 6.50 (s, 2H, NH₂), 7.20 (s, 1H, ArH). LC/MS (APCI+), m/z: 297.4 (M – HCl + H)⁺.

7-(2-Diethylaminoethoxy)-6-methoxyquinazolin-4-one 5 A mixture of 4 (3 mmol) and ammonium formate (9 mmol) in formamide (1 mL) was heated at 140°C for 16 h. The reaction was hydrolyzed with potassium carbonate solution (1 N, 40 mL) and extracted with ethyl acetate (5 × 30 mL). The combined organic layers were dried over magnesium sulfate and concentrated under vacuum. The residue was purified by column chromatography on silica gel eluting with CH₂Cl₂/MeOH (9/1) to give a brown solid. Mp = 137–139°C; yield = 68%. IR (ν, cm⁻¹): 1697 (C=O), 1611 (NH). ¹H-NMR (DMSO), δ, ppm: 1.05 (m, 6H, NCH₂CH₃), 2.70 (m, 4H, NCH₂CH₃), 3.00 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 3.95 (s, 3H, OCH₃), 4.20 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 7.10 (s, 1H, ArH), 7.50 (s, 1H, ArH), 8.00 (s, 1H, ArH), 12.20 (s, 1H, OH). LC/MS (APCI+), m/z: 292.3 (M + H)⁺.

4-Chloro-7-(2-diethylaminoethoxy)-6-methoxyquinazoline 6 A mixture of 5 (3 mmol) and phosphorous oxychloride (20 mL) was refluxed for 2 h. After evaporation under vacuum, ice water (50 mL) was added and the mixture was neutralized by ammonium hydroxide. The aqueous layer was extracted with CH₂Cl₂ (3 × 50 mL) and the extract was washed with saturated aqueous sodium hydrogen carbonate solution and dried over calcium chloride. The solvent was removed by evaporation and the residue was purified by column chromatography on silica gel eluting with CH₂Cl₂/MeOH (9/1) to provide a white solid. Mp = 189–191°C (cyclohexane/toluene); yield = 95%. ¹H-NMR (DMSO), δ, ppm: 1.50 (m, 6H, NCH₂CH₃), 3.20 (m, 4H, NCH₂CH₃), 3.45 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 4.05 (s, 3H, OCH₃), 4.60 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 7.45 (m, 2H, ArH), 8.90 (s, 1H, ArH). LC/MS (APCI+), m/z: 310.8 (M)⁺ and 312.8 (M + 2)⁺.

General procedure for compound 14–18 (series C)

3-(2-Diethylaminoethoxy)-4-methoxybenzoic acid methyl ester hydrochloride 14 Compound 14 was obtained by the same procedure as used for 2. Starting from 13 (0.08 mol), a white solid was synthesized. Mp = 103–105°C (toluene); yield = 77%. IR (ν, cm⁻¹): 2580 and 2465 (NH⁺), 1697 (C=O). ¹H-NMR (DMSO), δ, ppm: 1.25 (m, 6H, NCH₂CH₃), 3.20 (m, 4H, NCH₂CH₃), 3.50 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 3.80 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 7.10 (d, 1H, ArH, J = 8.70 Hz), 7.55 (d, 1H, ArH, J = 2.00 Hz), 7.60 (dd, 1H, ArH, J = 8.70 Hz and J = 2.00 Hz), 10.90 (s, 1H, NH⁺). LC/MS (APCI+), m/z: 282.3 (M – HCl + H)⁺.

3-(2-Diethylaminoethoxy)-4-methoxy-6-nitrobenzoic acid methyl ester hydrochloride 15 A solution of tin(IV) chloride (28 mmol) and nitric fuming acid (28 mmol) in CH₂Cl₂ (20 mL) was added dropwise to a solution of 14 (9.40 mmol) in CH₂Cl₂ (150 mL) cooled at–70°C. After stirring for 8 h at–70°C and warming to room temperature, water was added (100 mL). The aqueous layer was basified with 6 N NaOH and extracted with CH₂Cl₂ (3 × 70 mL). The combined organic layers were washed with saturated potassium carbonate solution and saturated sodium chloride solution and dried over calcium chloride. After concentration in vacuo, the oily residue was dissolved in 2-propanol (3 mL), and 2-propanol saturated with HCl was added (7 mL). The resulting white precipitate was filtered, washed with diethyl ether, and dried in vacuo to afford 15. Mp = 134–136°C (cyclohexane); yield = 91%. IR (ν, cm⁻¹): 2560 (NH⁺), 1697 (CGENZ 417121O), 1515 (NO₂). ¹H-NMR (DMSO), δ, ppm: 0.95 (m, 6H, NCH₂CH₃), 2.60 (m, 4H, NCH₂CH₃), 2.85 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 3.80 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 4.20 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 7.40 (s, 1H, ArH), 7.60 (s, 1H, ArH), 11.90 (s, 1H, NH⁺). LC/MS (APCI+), m/z: 327.3 (M – HCl + H)⁺.

2-Amino-3-(2-diethylaminoethoxy)-4-methoxybenzoic acid methyl ester hydrochloride 16 The compound 16 was obtained by using the same procedure as that for 4. Starting from 15 (6 mmol), a brown solid was obtained after purification by column chromatography on silica gel, eluting with CH₂Cl₂/CH₃OH (9/1). Mp = 142–146°C; yield = 77%. IR (ν, cm⁻¹): 3480 and 3385 (NH₂), 1709 (C=O). ¹H-NMR (DMSO), δ, ppm: 1.20 (m, 6H, NCH₂CH₃), 3.20 (m, 4H, NCH₂CH₃), 3.40 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 3.70 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 4.20 (t, 2H, NCH₂CH₂O, J = 4.60 Hz), 6.40 (s, 1H, ArH), 6.50 (s, 2H, NH₂), 7.20 (s, 1H, ArH), 10.60 (s, 1H, NH⁺). LC/MS (APCI+), m/z: 297.4 (M – HCl + H)⁺.

6-(2-Diethylaminoethoxy)-7-methoxyquinazolin-4-one 17 A mixture of 16 (3 mmol) and ammonium formate (9 mmol) in formamide (1 mL) was heated at 140°C for 16 h. The reaction was treated with water (50 mL) and extracted with CH₂Cl₂ (2 × 30 mL). The aqueous layer was neutralized by a saturated potassium carbonate solution. The precipitated solid was collected by filtration and washed with water and diethyl ether to provide a white solid after crystallization. Mp = 233–236°C (ethanol/water); yield = 71%. IR (ν, cm⁻¹): 1688 (C=O), 1610 (NH). ¹H-NMR (DMSO), δ, ppm: 1.00 (m, 6H, NCH₂CH₃), 2.65 (m, 4H, NCH₂CH₃), 2.80 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 3.95 (s, 3H, OCH₃), 4.10 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 7.05 (s, 1H, ArH), 7.45 (s, 1H, ArH), 8.00 (s, 1H, ArH), 12.00 (s, 1H, OH). LC/MS (APCI+), m/z: 292.3 (M + H)⁺.

4-Chloro-6-(2-diethylaminoethoxy)-7-methoxyquinazoline 18 The compound 18 was obtained by using the same procedure as that for 6. Starting from 17 (3 mmol), a white solid was prepared after purification by column chromatography on silica gel eluting with CH₂Cl₂/CH₃OH (95/5) Mp = 200–203°C (heptane); yield = 92%. ¹H-NMR (DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.55 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 4.00 (s, 3H, OCH₃), 4.60 (t, 2H, NCH₂CH₂O, J = 4.50 Hz), 7.50 (m, 2H, ArH), 8.90 (s, 1H, ArH). LC/MS (APCI+), m/z: 310.8 (M)⁺ and 312.8 (M + 2)⁺.

General procedure for compounds b–c (intermediates)

4-Nitrophenyl isocyanate 20 (6 mmol) was dissolved in a mixture of dichloromethane/methanol (b) or ethanol (c) (30 mL) and Raney nickel (0.4 g) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 16 h. The product was filtered through a plug of Celite before being concentrated and purified by column chromatography on silica gel eluting with CH₂Cl₂/MeOH (9/1).

N-(4-Aminophenyl)carbamic acid methyl ester b Mp = 88–90°C; yield = 80%. IR (ν, cm⁻¹): 3480 and 3385 (NH₂), 2467 (NH–CO), 1695 (C=O). ¹H-NMR (DMSO), δ, ppm: 3.70 (s, 3H, OCH₃), 5.80 (s, 2H, NH₂), 7.15 (d, 2H, ArH, J = 8.10 Hz), 7.60 (d, 2H, ArH, J = 8.10 Hz), 9.40 (s, 1H, NH). LC/MS (APCI+), m/z: 167.2 (M + H)⁺.

N-(4-Aminophenyl)carbamic acid ethyl ester c Mp = 72–74°C (73–74°C, lit.³²); yield = 85%. IR (ν, cm⁻¹): 3480 and 3375 (NH₂), 2465 (NH–CO), 1695 (C=O). ¹H-NMR (DMSO), δ, ppm: 1.70 (m, 3H, CH₂CH₃), 2.85 (m, 2H, CH₂CH₃), 5.90 (s, 2H, NH₂), 7.25 (d, 2H, ArH, J = 8.20 Hz), 7.65 (d, 2H, ArH, J = 8.20 Hz), 9.35 (s, 1H, NH). LC/MS (APCI+), m/z: 181.2 (M + H)⁺.

General procedure for compounds d–j (intermediates)

A solution of appropriate isocyanate (10 mmol) in anhydrous chloroform (15 mL) was added dropwise to a solution of 1,4-phenylenediamine 21 (9 mmol) in anhydrous chloroform (25 mL). After stirring for 2 h at room temperature, the precipitated solid was filtered off, washed with water and diethyl ether, with the exception of i which was extracted with ethyl acetate (3 × 15 mL), and obtained without further purification.

N-(4-Aminophenyl)-N’-phenylurea d Mp = >250°C; yield = 91%. IR (ν, cm⁻¹): 3480 and 3385 (NH₂), 1663 (C=O urea). ¹H-NMR (DMSO), δ, ppm: 4.75 (s, 2H, NH₂), 6.50 (d, 2H, ArH, J = 8.50 Hz), 6.90 (t, 1H, ArH), 7.10 (d, 2H, ArH, J = 8.50 Hz), 7.25 (m, 2H, ArH), 7.45 (m, 2H, ArH), 8.10 (s, 1H, NH), 8.45 (s, 1H, NH). LC/MS (APCI+), m/z: 228.3 (M + H)⁺.

N-(4-Aminophenyl)-N’-(4-methoxyphenyl)urea e Mp = >250°C; yield = 74%. IR (ν, cm⁻¹): 3480 and 3385 (NH₂), 1665 (C=O urea). ¹H-NMR (DMSO), δ, ppm: 3.70 (s, 3H, OCH₃), 4.80 (s, 2H, NH₂), 6.50 (d, 2H, ArH, J = 8.60 Hz), 6.80 (d, 2H, ArH, J = 9.10 Hz), 7.15 (d, 2H, ArH, J = 8.60 Hz), 7.30 (d, 2H, ArH, J = 9.10 Hz), 8.10 (s, 1H, NH), 8.25 (s, 1H, NH). LC/MS (APCI+), m/z: 258.3 (M + H)⁺.

N-(4-Aminophenyl)-N’-(2,4-difluorophenyl)urea f Mp = >250°C; yield = 83%. IR (ν, cm⁻¹): 3480 and 3390 (NH₂), 1655 (C=O urea), 1220 (C-F). ¹HNMR (DMSO), δ, ppm: 5.85 (s, 2H, NH₂), 6.50 (d, 2H, ArH, J = 8.30 Hz), 6.65–6.80 (m, 2H, ArH), 7.25 (d, 2H, ArH, J = 8.30 Hz), 7.55 (m, 1H, ArH), 8.15 (s, 1H, NH), 8.25 (s, 1H, NH). LC/MS (APCI+), m/z: 264.3 (M + H)⁺.

N-(4-Aminophenyl)-N’-(3-chloro-4-fluorophenyl)urea g Mp = >250°C; yield = 82%. IR (ν, cm⁻¹): 3480 and 3390 (NH₂), 1657 (C=O urea), 1221 (C-F), 1069 (C-Cl). ¹H-NMR (DMSO), δ, ppm: 4.85 (s, 2H, NH₂), 6.55 (d, 2H, ArH, J = 8.50 Hz), 7.05 (d, 2H, ArH, J = 8.50 Hz), 7.25–7.40 (m, 2H, ArH), 7.75 (m, 1H, ArH), 8.20 (s, 1H, NH), 8.70 (s, 1H, NH). LC/MS (APCI+), m/z: 280.5 (M)⁺ and 282.5 (M + 2)⁺.

N-(4-Aminophenyl)-N’-butylurea h Mp = 176–178°C; yield = 95%. IR (ν, cm⁻¹): 3470 and 3380 (NH₂), 1650 (C=O urea). ¹H-NMR (DMSO), δ, ppm: 1.00 (m, 3H, CH₂CH₃), 1.20–1.40 (m, 6H, CH₂CH₂CH₂CH₃), 6.00 (s, 2H, NH₂), 6.20 (s, 1H, NH), 7.50 (d, 2H, ArH, J = 8.80 Hz), 8.10 (d, 2H, ArH, J = 8.80 Hz), 9.70 (s, 1H, NH). LC/MS (APCI+), m/z: 208.3 (M + H)⁺.

N-(4-Aminophenyl)-N’-ethylurea i Mp = 149–151°C; yield = 70%. IR (ν, cm⁻¹): 3480 and 3385 (NH₂), 1655 (C=O urea). ¹H-NMR (DMSO), δ, ppm: 1.60 (m, 3H, CH₂CH₃), 2.80 (m, 2H, CH₂CH₃), 5.80 (s, 1H, NH), 6.10 (s, 2H, NH₂), 6.50 (d, 2H, ArH, J = 8.20 Hz), 7.50 (d, 2H, ArH, J = 8.20 Hz), 8.90 (s, 1H, NH). LC/MS (APCI+), m/z: 180.2 (M + H)⁺.

N-(4-Aminophenyl)-N’-cyclohexylurea j Mp = 199–202°C; yield = 85%. IR (ν, cm⁻¹): 3470 and 3380 (NH₂), 1664 (C=O urea). ¹H-NMR (DMSO), δ, ppm: 1.20–1.50 (m, 10H, CH(CH₂)₅), 2.55 (m, 1H, CH(CH₂)₅), 5.90 (s, 2H, NH₂), 6.15 (s, 1H, NH), 7.45 (d, 2H, ArH, J = 8.60 Hz), 7.80 (d, 2H, ArH, J = 8.60 Hz), 8.90 (s, 1H, NH). LC/MS (APCI+), m/z: 234.2 (M + H)⁺.

General procedure for compounds 7a–j, 11a–j, and 19a–j

The chloride derivate 6, 10, or 18 (0.32 mmol) was dissolved in 2-propanol during refluxing (5 mL) and the synthesized b–j or commercial aniline a (1.2 eq.) was added. The mixture was refluxed for 3–6 h. Final products were obtained either by filtration and washing with 2-propanol and diethyl ether or by purification by column chromatography on silica gel eluting with CH₂Cl₂/MeOH (9/1).

Series A

N-{4-[7-(2-Diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}acetamide hydrochloride 7a Mp = 221–223°C; yield = 56%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 2463 (NH⁺), 1680 (C=O). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 2.05 (s, 3H, COCH₃), 3.20 (m, 4H, NCH₂CH₃), 3.40 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 4.00 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 7.15 (s, 1H, ArH), 7.50 (d, 2H, ArH, J = 8.10 Hz), 7.55 (d, 2H, ArH, J = 8.10 Hz), 7.90 (s, 1H, ArH), 8.40 (s, 1H, ArH), 9.55 (s, 1H, NH), 9.85 (s, 1H, NH). LC/MS (APCI+), m/z: 424.5 (M – HCl + H)⁺.

Methyl {4-[7-(2-diethylaminoethoxy)-6-methoxy- quinazolin-4-ylamino]phenyl}carbamate hydrochloride 7b Mp = 189–191°C; yield = 48%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 2463 (NH⁺), 1682 (C=O). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.20 (m, 4H, NCH₂CH₃), 3.55 (m, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.70 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 4.55 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 7.30 (s, 1H, ArH), 7.50 (d, 2H, ArH, J = 8.10 Hz), 7.65 (d, 2H, ArH, J = 8.10 Hz), 8.00 (s, 1H, ArH), 8.50 (s, 1H, ArH), 8.70 (s, 1H, NH), 9.85 (s, 1H, NH). LC/MS (APCI+), m/z: 440.5 (M – HCl + H)⁺.

Ethyl {4-[7-(2-diethylaminoethoxy)-6-methoxy- quinazolin-4-ylamino]phenyl}carbamate hydrochloride 7c Mp = 198–201°C; yield = 52%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 2465 (NH⁺), 1675 (C=O). (1H-NMR DMSO), δ, ppm: 1.30–1.50 (m, 9H, NCH₂CH₃ and OCH₂CH₃), 3.25 (m, 4H, NCH₂CH₃), 3.60 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 4.00 (s, 3H, OCH₃), 4.15 (m, 2H, OCH₂CH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 7.25 (s, 1H, ArH), 7.55 (d, 2H, ArH, J = 8.30 Hz), 7.70 (d, 2H, ArH, J = 8.30 Hz), 8.10 (s, 1H, ArH), 8.55 (s, 1H, ArH), 8.80 (s, 1H, NH), 9.85 (s, 1H, NH). LC/MS (APCI+), m/z: 454.5 (M – HCl + H)⁺.

N-{4-[7-(2-Diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}-N’-phenylurea hydrochloride 7d Mp = 215–217°C; yield = 65%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1657 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.65 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 4.00 (s, 3H, OCH₃), 4.55 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 6.90 (t, 1H, ArH), 7.25 (m, 3H, ArH), 7.40–7.55 (m, 4H, ArH), 7.65 (d, 2H, ArH), 8.25 (s, 1H, ArH), 8.70 (s, 1H, ArH), 9.24 (s, 1H, NH), 9.25 (s, 1H, NH), 10.50 (s, 1H, NH). LC/MS (APCI+), m/z: 501.3 (M – HCl + H)⁺.

N-{4-[7-(2-Diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}-N’-(4-methoxy phenyl)urea hydrochloride 7e Mp = 207–209°C; yield = 73%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1661 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.55 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.85 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 4.55 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 6.90 (d, 2H, ArH, J = 8.70 Hz), 7.20 (s, 1H, ArH), 7.40 (d, 2H, ArH, J = 8.70 Hz), 7.50 (d, 2H, ArH, J = 8.80 Hz), 7.60 (d, 2H, ArH, J = 8.80 Hz), 8.10 (s, 1H, ArH), 8.55 (s, 1H, ArH), 8.70 (s, 1H, NH), 8.80 (s, 1H, NH), 10.55 (s, 1H, NH). LC/MS (APCI+), m/z: 531.6 (M – HCl + H)⁺.

N-{4-[7-(2-Diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}-N’-(2,4-difluoro phenyl)urea hydrochloride 7f Mp = 202–204°C; yield = 89%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1657 (C=O urea), 1221 (C–F). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.60 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 4.00 (s, 3H, OCH₃), 4.55 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 7.00 (d, 1H, ArH), 7.20 (s, 1H, ArH), 7.30 (t, 1H, ArH), 7.50 (d, 2H, ArH, J = 8.80 Hz), 7.65 (d, 2H, ArH, J = 8.80 Hz), 7.95 (s, 1H, ArH), 8.10 (d, 1H, ArH), 8.50 (s, 1H, ArH), 8.60 (s, 1H, NH), 9.15 (s, 1H, NH), 10.50 (s, 1H, NH). LC/MS (APCI+), m/z: 537.6 (M – HCl + H)⁺.

N-{4-[7-(2-Diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}-N’-(3-chloro-4-fluorophenyl)urea hydrochloride 7g Mp = 218–220°C; yield = 64%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1664 (C=O urea), 1219 (C–F), 1069 (C–Cl). (1H-NMR DMSO), δ, ppm: 1.25 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.55 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.95 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 7.10–7.30 (m, 3H, ArH), 7.50 (d, 2H, ArH, J = 8.70 Hz), 7.65 (d, 2H, ArH, J = 8.70 Hz), 7.95 (s, 1H, ArH), 8.10 (d, 1H, ArH), 8.45 (s, 1H, ArH), 9.15 (s, 1H, NH), 9.35 (s, 1H, NH), 10.40 (s, 1H, NH). LC/MS (APCI+), m/z: 553.6 (M – HCl)⁺ and 555.6 (M – HCl + 2)⁺.

N-{4-[7-(2-Diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}-N’-butylurea hydrochloride 7h Mp = 222–225°C; yield = 59%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1651 (C=O urea). (1H-NMR DMSO), δ, ppm: 0.95 (m, 3H, CH₂CH₃), 1.20 (m, 6H, NCH₂CH₃), 1.30–1.50 (m, 6H, CH₂CH₂CH₂CH₃), 3.20 (m, 4H, NCH₂CH₃), 3.35 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 3.95 (s, 3H, OCH₃), 4.45 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 6.20 (s, 1H, NH), 7.20 (s, 1H, ArH), 7.40 (d, 2H, ArH, J = 8.90 Hz), 7.55 (d, 2H, ArH, J = 8.90 Hz), 7.95 (s, 1H, NH), 8.35 (s, 1H, ArH), 8.55 (s, 1H, ArH), 9.55 (s, 1H, NH). LC/MS (APCI+), m/z: 481.6 (M – HCl + H)⁺.

N-{4-[7-(2-Diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}-N’-ethylurea hydrochloride 7i Mp = 207–209°C; yield = 40%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1659 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.05 (m, 3H, CH₂CH₃), 1.30 (m, 6H, NCH₂CH₃), 3.10 (m, 2H, CH₂CH₃), 3.35 (m, 4H, NCH₂CH₃), 3.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 4.00 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 6.30 (s, 1H, NH), 7.30 (s, 1H, ArH), 7.45 (d, 2H, ArH, J = 8.10 Hz), 7.55 (d, 2H, ArH, J = 8.10 Hz), 8.10 (s, 1H, ArH), 8.55 (s, 1H, ArH), 8.80 (s, 1H, NH), 10.45 (s, 1H, NH). LC/MS (APCI+), m/z: 453.6 (M – HCl + H)⁺.

N-Cyclohexyl-N’-{4-[7-(2-diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}urea hydrochloride 7j Mp = 191–194°C; yield = 68%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1657 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.20–1.70 (m, 17H, NCH₂CH₃ and CH–(CH₂)₅), 3.25 (m, 4H, NCH₂CH₃), 3.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 4.00 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 6.40 (s, 1H, NH), 7.25 (s, 1H, ArH), 7.50 (d, 2H, ArH, J = 8.80 Hz), 7.65 (d, 2H, ArH, J = 8.80 Hz), 8.00 (s, 1H, ArH), 8.60 (s, 1H, ArH), 8.70 (s, 1H, NH), 10.90 (s, 1H, NH). LC/MS (APCI+), m/z: 507.7 (M – HCl + H)⁺.

Series B

N-{4-[6,7-Dimethoxy-quinazolin-4-ylamino]phenyl}acetamide 11a Mp = 211–213°C; yield = 44%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 1668 (C=O). (1H-NMR DMSO), δ, ppm: 2.05 (s, 3H, COCH₃), 4.00 (s, 3H, OCH₃), 4.05 (s, 3H, OCH₃), 7.40 (s, 1H, ArH), 7.55–7.75 (m, 4H, ArH), 8.30 (s, 1H, ArH), 8.70 (s, 1H, ArH), 10.20 (s, 1H, NH), 11.30 (s, 1H, NH). LC/MS (APCI+), m/z: 339.4 (M + H)⁺.

Methyl {4-[6,7-dimethoxy-quinazolin-4-ylamino]phenyl}carbamate 11b Mp = >250°C; yield = 72%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 1690 (C=O). (1H-NMR DMSO), δ, ppm: 3.65 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 4.05 (s, 3H, OCH₃), 7.30 (s, 1H, ArH), 7.45 (d, 2H, ArH, J = 8.10 Hz), 7.60 (d, 2H, ArH, J = 8.10 Hz), 8.20 (s, 1H, ArH), 8.80 (s, 1H, ArH), 9.80 (s, 1H, NH), 11.30 (s, 1H, NH). LC/MS (APCI+), m/z: 355.4 (M + H)⁺.

Ethyl {4-[6,7-dimethoxy-quinazolin-4-ylamino]phenyl}carbamate 11c Mp = 198–201°C; yield = 52%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 1669 (C=O). (1H-NMR DMSO), δ, ppm: 1.30 (t, 3H, OCH₂CH₃), 3.94 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 4.20 (m, 2H, OCH₂CH₃), 7.20 (d, 2H, ArH, J = 9.00 Hz), 7.35 (s, 1H, ArH), 7.55 (d, 2H, ArH, J = 9.00 Hz), 7.65 (s, 1H, ArH), 8.50 (s, 1H, ArH), 8.80 (s, 1H, NH), 9.75 (s, 1H, NH). LC/MS (APCI+), m/z: 369.4 (M + H)⁺.

N-{4-[6,7-Dimethoxy-quinazolin-4-ylamino]phenyl}-N’-phenylurea 11d Mp = >250°C; yield = 56%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1662 (C=O urea). (1H-NMR DMSO), δ, ppm: 3.93 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 6.90 (m, 1H, ArH), 7.30 (m, 3H, ArH), 7.45–7.60 (m, 4H, ArH), 7.65 (d, 2H, ArH), 8.25 (s, 1H, ArH), 8.60 (s, 1H, ArH), 9.25 (d, 2H, 2NH), 10.40 (s, 1H, NH). LC/MS (APCI+), m/z: 416.6 (M + H)⁺.

N-{4-[6,7-Dimethoxy-quinazolin-4-ylamino]phenyl}-N’-(4-methoxyphenyl)urea 11e Mp = 193–196°C; yield = 54%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1655 (C=O urea). (1H-NMR DMSO), δ, ppm: 3.70 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 6.85 (d, 2H, ArH, J = 8.80 Hz), 7.20 (s, 1H, ArH), 7.30 (d, 2H, ArH, J = 8.80 Hz), 7.50 (d, 2H, ArH, J = 9.00 Hz), 7.60 (d, 2H, ArH, J = 9.00 Hz), 8.00 (s, 1H, ArH), 8.55 (s, 1H, ArH), 8.65 (s, 1H, NH), 8.80 (s, 1H, NH), 10.10 (s, 1H, NH). LC/MS (APCI+), m/z: 446.6 (M + H)⁺.

N-{4-[6,7-Dimethoxy-quinazolin-4-ylamino]phenyl}-N’-(2,4-difluorophenyl)urea 11f Mp = 168–171°C; yield = 58%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1657 (C=O urea), 1220 (C-F). (1H-NMR DMSO), δ, ppm: 3.95 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 7.00 (m, 1H, ArH), 7.20 (s, 1H, ArH), 7.30 (m, 1H, ArH), 7.50 (d, 2H, ArH, J = 9.00 Hz), 7.60 (d, 2H, ArH, J = 9.00 Hz), 7.95 (s, 1H, ArH), 8.10 (m, 1H, ArH), 8.55 (s, 1H, ArH), 8.60 (s, 1H, NH), 9.20 (s, 1H, NH), 10.10 (s, 1H, NH). LC/MS (APCI+), m/z: 452.5 (M + H)⁺.

N-{4-[6,7-Dimethoxy-quinazolin-4-ylamino]phenyl}-N’-(3-chloro-4-fluorophenyl)urea 11g Mp = 241–243°C; yield = 71%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1658 (C=O urea), 1220 (C-F), 1069 (C-Cl). (1H-NMR DMSO), δ, ppm: 3.93 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 7.20–7.40 (m, 3H, ArH), 7.55 (s, 4H, ArH), 7.80 (m, 1H, ArH), 8.15 (s, 1H, ArH), 8.80 (s, 1H, ArH), 9.35 (s, 1H, NH), 9.45 (s, 1H, NH), 11.20 (s, 1H, NH). LC/MS (APCI+), m/z: 468.9 (M)⁺and 470.9 (M + 2)⁺.

N-{4-[6,7-Dimethoxy-quinazolin-4-ylamino]phenyl}-N’-butylurea 11h Mp = 196–198°C; yield = 50%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1662 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.00 (t, 3H, CH₂CH₃), 1.20–1.40 (m, 6H, CH₂CH₂CH₂CH₃), 4.00 (s, 3H, OCH₃), 4.05 (s, 3H, OCH₃), 6.30 (s, 1H, NH), 7.25 (s, 1H, ArH), 7.40 (d, 2H, ArH, J = 8.70 Hz), 7.60 (d, 2H, ArH, J = 8.70 Hz), 8.20 (s, 1H, ArH), 8.60 (s, 1H, ArH), 8.90 (s, 1H, NH), 10.60 (s, 1H, NH). LC/MS (APCI+), m/z: 396.5 (M + H)⁺.

N-{4-[6,7-Dimethoxy-quinazolin-4-ylamino]phenyl}-N’-ethylurea 11i Mp = 189–192°C; yield = 40%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1657 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.15 (t, 3H, CH₂CH₃), 3.20 (m, 2H, CH₂CH₃), 3.94 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 6.35 (s, 1H, NH), 7.20 (s, 1H, ArH), 7.45 (d, 2H, ArH, J = 8.80 Hz), 7.55 (d, 2H, ArH, J = 8.80 Hz), 8.00 (s, 1H, ArH), 8.40 (s, 1H, ArH), 8.90 (s, 1H, NH), 10.30 (s, 1H, NH). LC/MS (APCI+), m/z: 368.4 (M + H)⁺.

N-Cyclohexyl-N’-{4-[6,7-dimethoxy-quinazolin-4-ylamino]phenyl}urea 11j Mp = 151–153°C; yield = 53%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1659 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.20 (m, 5H, (CH₂)₅), 1.40 (m, 1H, CH–(CH₂)₅), 1.65 (m, 5H, (CH₂)₅), 3.95 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 6.30 (s, 1H, NH), 7.25 (s, 1H, ArH), 7.50 (m, 4H, ArH), 8.10 (s, 1H, ArH), 8.70 (s, 1H, ArH), 8.70 (s, 1H, NH), 11.00 (s, 1H, NH). LC/MS (APCI+), m/z: 422.5 (M + H)⁺.

Series C

N-{4-[6-(2-Diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}acetamide hydrochloride 19a Mp = 239–241°C; yield = 60%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 2461 (NH⁺), 1671 (C=O). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 2.05 (s, 3H, COCH₃), 3.25 (m, 4H, NCH₂CH₃), 3.40 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.95 (s, 3H, OCH₃), 4.45 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 7.20 (s, 1H, ArH), 7.45 (d, 2H, ArH, J = 8.10 Hz), 7.55 (d, 2H, ArH, J = 8.10 Hz), 7.90 (s, 1H, ArH), 8.35 (s, 1H, ArH), 9.75 (s, 1H, NH), 9.95 (s, 1H, NH). LC/MS (APCI+), m/z: 424.5 (M – HCl + H)⁺.

Methyl {4-[6-(2-diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}carbamate hydrochloride 19b Mp = 239–241°C; yield = 56%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 2461 (NH⁺), 1680 (C=O). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.25 (m, 4H, NCH₂CH₃), 3.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.65 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 7.20 (s, 1H, ArH), 7.45 (d, 2H, ArH, J = 8.10 Hz), 7.65 (d, 2H, ArH, J = 8.10 Hz), 8.00 (s, 1H, ArH), 8.40 (s, 1H, ArH), 8.65 (s, 1H, NH), 9.85 (s, 1H, NH). LC/MS (APCI+), m/z: 440.5 (M – HCl + H)⁺.

Ethyl {4-[7-(2-diethylaminoethoxy)-6-methoxy-quinazolin-4-ylamino]phenyl}carbamate hydrochloride 19c Mp = 201–203°C; yield = 49%. IR (ν, cm⁻¹): 3480 and 3385 (NH), 2462 (NH⁺), 1679 (C=O). (1H-NMR DMSO), δ, ppm: 1.30–1.50 (m, 9H, NCH₂CH₃ and OCH₂CH₃), 3.25 (m, 4H, NCH₂CH₃), 3.55 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.95 (s, 3H, OCH₃), 4.15 (m, 2H, OCH₂CH₃), 4.45 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 7.25 (s, 1H, ArH), 7.50 (d, 2H, ArH, J = 8.50 Hz), 7.70 (d, 2H, ArH, J = 8.50 Hz), 8.15 (s, 1H, ArH), 8.55 (s, 1H, ArH), 8.80 (s, 1H, NH), 9.85 (s, 1H, NH). LC/MS (APCI+), m/z: 454.5 (M – HCl + H)⁺.

N-{4-[6-(2-Diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}-N’-phenylurea hydrochloride 19d Mp = 177–179°C; yield = 81%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1665 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.60 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 3.95 (s, 3H, OCH₃), 4.65 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 7.00 (t, 1H, ArH), 7.20 (m, 3H, ArH), 7.40–7.55 (m, 4H, ArH), 7.70 (m, 2H, ArH), 8.30 (s, 1H, ArH), 8.60 (s, 1H, ArH), 9.10 (s, 1H, NH), 9.12 (s, 1H, NH), 10.45 (s, 1H, NH). LC/MS (APCI+), m/z: 501.6 (M – HCl + H)⁺.

N-{4-[6-(2-Diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}-N’-(4-methoxy phenyl)urea hydrochloride 19e Mp = 145–147°C; yield = 82%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1661 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.20 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.60 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.80 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 4.60 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 6.90 (d, 2H, ArH, J = 8.70 Hz), 7.20 (s, 1H, ArH), 7.35 (d, 2H, ArH, J = 8.70 Hz), 7.55 (d, 2H, ArH, J = 8.80 Hz), 7.65 (d, 2H, ArH, J = 8.80 Hz), 8.00 (s, 1H, ArH), 8.50 (s, 1H, ArH), 8.70 (s, 1H, NH), 8.80 (s, 1H, NH), 10.50 (s, 1H, NH). LC/MS (APCI+), m/z: 531.6 (M – HCl + H)⁺.

N-{4-[6-(2-Diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}-N’-(2,4-difluoro phenyl)urea hydrochloride 19f Mp = 143–146°C; yield = 66%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1660 (C=O urea), 1221 (C–F). (1H-NMR DMSO), δ, ppm: 1.25 (m, 6H, NCH₂CH₃), 3.30 (m, 4H, NCH₂CH₃), 3.55 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 3.85 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 7.00 (d, 1H, ArH), 7.20 (s, 1H, ArH), 7.30 (m, 1H, ArH), 7.50 (d, 2H, ArH, J = 8.80 Hz), 7.60 (d, 2H, ArH, J = 8.80 Hz), 8.00 (s, 1H, ArH), 8.15 (m, 1H, ArH), 8.50 (s, 1H, ArH), 8.60 (s, 1H, NH), 9.20 (s, 1H, NH), 10.50 (s, 1H, NH). LC/MS (APCI+), m/z: 537.6 (M – HCl + H)⁺.

N-{4-[6-(2-Diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}-N’-(3-chloro-4-fluorophenyl)urea hydrochloride 19g Mp = 215–217°C; yield = 59%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1658 (C=O urea), 1220 (C–F), 1067 (C–Cl). (1H-NMR DMSO), δ, ppm: 1.30 (m, 6H, NCH₂CH₃), 3.25 (m, 4H, NCH₂CH₃), 3.55 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 3.95 (s, 3H, OCH₃), 4.55 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 7.10–7.30 (m, 3H, ArH), 7.55 (d, 2H, ArH, J = 8.70 Hz), 7.65 (d, 2H, ArH, J = 8.70 Hz), 7.95 (s, 1H, ArH), 8.10 (m, 1H, ArH), 8.45 (s, 1H, ArH), 9.25 (s, 1H, NH), 9.45 (s, 1H, NH), 10.45 (s, 1H, NH). LC/MS (APCI+), m/z: 553.6 (M – HCl)⁺and 555.6 (M – HCl + 2)⁺.

N-{4-[6-(2-Diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}-N’-butylurea hydrochloride 19h Mp = 102–105°C; yield = 49%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1661 (C=O urea). (1H-NMR DMSO), δ, ppm: 0.95 (m, 3H, CH₂CH₃), 1.15 (m, 6H, NCH₂CH₃), 1.25–1.45 (m, 6H, CH₂CH₂CH₂CH₃), 3.10 (m, 4H, NCH₂CH₃), 3.30 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 3.90 (s, 3H, OCH₃), 4.30 (t, 2H, NCH₂CH₂O, J = 4.40 Hz), 6.10 (s, 1H, NH), 7.15 (s, 1H, ArH), 7.40 (d, 2H, ArH, J = 9.05 Hz), 7.60 (d, 2H, ArH, J = 9.05 Hz), 7.90 (s, 1H, NH), 8.40 (s, 1H, ArH), 8.50 (s, 1H, ArH), 9.50 (s, 1H, NH). LC/MS (APCI+), m/z: 481.6 (M – HCl + H)⁺.

N-{4-[6-(2-Diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}-N’-ethylurea hydrochloride 19i Mp = 194–196°C; yield = 33%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1660 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.10 (m, 3H, CH₂CH₃), 1.20 (m, 6H, NCH₂CH₃), 2.90 (m, 4H, NCH₂CH₃), 3.05 (m, 2H, CH₂CH₃), 3.40 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 3.90 (s, 3H, OCH₃), 4.30 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 6.20 (s, 1H, NH), 7.20 (s, 1H, ArH), 7.40 (d, 2H, ArH, J = 8.80 Hz), 7.60 (d, 2H, ArH, J = 8.80 Hz), 7.90 (s, 1H, ArH), 8.40 (s, 1H, ArH), 8.50 (s, 1H, NH), 9.50 (s, 1H, NH). LC/MS (APCI+), m/z: 453.6 (M – HCl + H)⁺.

N-Cyclohexyl-N’-{4-[6-(2-diethylaminoethoxy)-7-methoxy-quinazolin-4-ylamino]phenyl}urea hydrochloride 19j Mp = 184–186°C; yield = 64%. IR (ν, cm⁻¹): 3200 and 2800 (NH), 1661 (C=O urea). (1H-NMR DMSO), δ, ppm: 1.20–1.70 (m, 17H, NCH₂CH₃ and CH–(CH₂)₅), 3.25 (m, 4H, NCH₂CH₃), 3.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 4.00 (s, 3H, OCH₃), 4.50 (t, 2H, NCH₂CH₂O, J = 4.30 Hz), 6.40 (s, 1H, NH), 7.25 (s, 1H, ArH), 7.50 (d, 2H, ArH, J = 8.80 Hz), 7.65 (d, 2H, ArH, J = 8.80 Hz), 8.00 (s, 1H, ArH), 8.60 (s, 1H, ArH), 8.70 (s, 1H, NH), 10.90 (s, 1H, NH). LC/MS (APCI+), m/z: 507.7 (M – HCl + H)⁺.

Pharmacology

Cell culture and cell proliferation assay

Human prostate cancer cells PC3 were grown at 37°C in a humidified atmosphere containing 5% CO₂ in RPMI-1640 medium supplemented with 10% fetal bovine serum, glutamine (2 mM), penicillin (100 IU/mL), and streptomycin (100 μg/mL). In the cell proliferation assay, cells were plated in triplicate on 96-well plates (3 × 10³ cells per well) and incubated for 72 h. The cell medium was changed to serum-free medium, and the cells were starved for 24 h for culture synchronization. Cells were then incubated in culture medium that contained various concentrations of tested compounds, each dissolved in less than 0.1% DMSO. After 72 h, cell growth was estimated by the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test.

In vitro kinase assays

Kinase assays were performed in 96-well plates (Multiscreen Durapore, Millipore) using [γ-³²P]ATP (Amersham Biosciences) and the synthetic polymer poly(Glu₄/Tyr) (Sigma Chemicals) as a phosphoacceptor substrate. Tested compounds were dissolved in 1H-NMR DMSO, and the final concentration of 1H-NMR DMSO in assay solutions was 0.1% (total kinase activity) or 0.01% (EGFR kinase activity), which was shown to have no effect on kinase activity.

EGFR tyrosine kinase activity 20 ng of EGFR (purified from human carcinoma A431 cells; Sigma Chemicals) was incubated for 1 h at 28°C using various concentrations of tested compounds in kinase buffer (HEPES 50 mM pH 7.5, bovine serum albumin (BSA) 0.1 mg/mL, MnCl₂ 10 mM, MgCl₂ 5 mM, Na₃VO₄ 100 μM, dithiothreitol (DTT) 0.5 mM, poly(Glu₄/Tyr) 250 μg/mL, ATP 5 μM, [γ-³²P]ATP 0.5 μCi).

VEGFR-2 tyrosine kinase activity 10 ng of VEGFR-2 (recombinant human protein; Invitrogen) was incubated for 1 h at 28°C using various concentrations of tested compounds in kinase buffer (Tris 50mM pH 7.5, BSA 25 μg/mL, MnCl₂ 1.5 mM, MgCl₂ 10 mM, DTT 2.5 mM, Na₃VO₄ 100 μM, β-glycerophosphate 5 mM, poly(Glu₄/Tyr) 250 μg/mL, ATP 5 μM, [γ-³²P]ATP 0.5 μCi).

The reaction was stopped by adding 20 μL of trichloroacetic acid 100%. Wells were screened out and washed 10 times with trichloroacetic acid 10%. Plates were counted in a Top Count for 1 min per well.

Aurora-A kinase activity 2 μg of Aurora-A(His)₆ was incubated in 15 μL of kinase buffer (50 mM Tris-HCl pH 7.5, 25 mM NaCl, 1 mM DTT, 10 mM MgCl₂) in the presence of 5 μCi [γ-³²P]ATP at 3000 Ci/mmol and 10 μg histone H3 with or without the compounds. The control was performed with a solution of 1H-NMR DMSO in water corresponding to the dilution used to prepare the chemical compounds. After 20 min incubation at 30°C, the incubations were spotted into 2.5 cm × 3 cm pieces of Whatman P81 phosphocellulose paper, and, after 20 s, the filters were washed five times (for at least 10 min each time) in a solution of phosphoric acid in water (1.2%). The wet filters were transferred into 6 mL plastic scintillation vials, and 5 mL ACS scintillation fluid (Amersham) was added prior to measuring the radioactivity in a Packard counter. The kinase activity was expressed as percent of the control assay.

Molecular modeling

All the calculations were carried out using the Sybyl 6.9.1 molecular modeling package³³ running on Silicon Graphics Octane 2 workstations. The ligands were built from the internal fragments library of Sybyl and their geometry was optimized by the Powell method available in the Maximin2 procedure to a gradient of 0.001 kcal/mol/å. The dielectric constant was set to 4 to implicitly represent a biological middle, the atomic charges were attributed following the Gasteiger–Hückel method, and energy minimization was run using the Tripos force field³⁴. The structure of the protein co-crystallized with an inhibitor was obtained from the Protein Data Bank (http://www.pdb.org)³⁵ under the entries 1M17³⁶ and 1YWN³⁷. The co-crystallized inhibitor and water molecules were removed and hydrogens were added to the protein, taking care to be as close as possible to the biological protonation state of the residues. The binding mode of the compounds was investigated by a two-step process. They were first docked into the two binding sites of EGFR and VEGFR-2 by Gold 3.2³⁸. The multiple conformations generated were then ranked by a consensus scoring based on Goldscore³⁸ and X-Score³⁹. The consistency of the best ranked conformation was visually assessed to insure it was the most stable binding mode prediction.

Results and discussion

Chemistry

The synthesis of the three series (A, B, C) of quinazolines 7a–j, 11a–j, and 19a–j is presented in Schemes 1, 2, and 3, respectively, according to a parallel synthesis route. We have described the synthesis of 4-chloroquinazoline derivates 6, 10, and 18, differentiating the ether linker at the C-6 and C-7 positions (Scheme 1).

Scheme 1.  Synthesis of 4-chloroquinazoline derivatives 6, 10, and 18. Reagents and conditions: (i) ClCH₂CH₂N(CH₂CH₃)₂.HCl, K₂CO₃, acetone, reflux, 3 h; (ii) SnCl₄, HNO₃, CH₂Cl₂,–25°C, 4 h; (iii) Raney Ni, H₂, MeOH, rt, 16 h; (iv) HCOONH₄, HCONH₂, 140°C, 16 h; (v) POCl₃, 120°C, 2 h; (vi) NaOMe, formamide, DMF/MeOH, 110°C, 4 h; (vii) SOCl₂, MeOH, 0°C at refluxing, 3 h.

These compounds were synthesized with high yields using an optimized and potent synthesis route. For series A, we used a synthesis route according to previously described procedures⁴⁰ to synthesize compounds 2 and 3. Catalytic hydrogenation of the nitro group led to amino derivative 4 (67% yield), which was converted, by heating at 140°C in formamide in the presence of ammonium formate, to the corresponding substituted quinazolinone derivative 5. Treatment of quinazolinone 5 in refluxing phosphorus oxychloride afforded the chloroquinazoline derivative in good yield (>90%). In series B, compound 10 was prepared according to procedures previously described by Furuta et al.⁴¹ using methyl 2-amino-4,5-dimethoxybenzoate 8 as starting material. To prepare series C, the same synthesis route was employed as for series A. After a step in which 3-hydroxy-4-methoxybenzoic acid 12 was converted into its methyl ester 13⁴⁰, we synthesized intermediates 14–18 with high yields. These chloride derivatives 6, 10, and 18 were then engaged in a nucleophilic substitution reaction in the presence of aniline (a–j).

The synthesis of non-commercially available aniline derivatives b–j is depicted in Scheme 2. During these preparations, we also report a novel beneficial (80–85%) approach to synthesizing (4-aminophenyl)carbamic acid methyl and ethyl ester (b, c). For this, we used 4- nitrophenylisocyanate 20 in the presence of a dichloromethane/alcohol mixture under a hydrogen atmosphere and Raney nickel as catalyst. Condensation of 1,4-phenylenediamine 21 and the corresponding isocyanate in CHCl₃⁴² afforded the desired ureas d–j in high yields and with short reaction times.

Scheme 2.  Synthesis of aniline intermediates b–j. Reagents and conditions: (i) Raney Ni, H₂, CH₂Cl₂/MeOH or EtOH, rt, 10 h; (ii) isocyanate (R2–N=C=O) diluted in CHCl₃, CHCl₃, rt, 2 h.

Obtaining of the final 4-anilinoquinazoline derivatives 7, 11, and 19 was performed by the same synthesis strategy: nucleophilic displacement of the chlorine atom with various arylamino groups in 2-propanol. The final products were crystallized in various solvents (Scheme 3).

Scheme 3.  Synthesis of targeted derivatives 7a–j, 11a–j, and 19a–j.

Molecular modeling

Molecular modeling of many compounds described in the literature was realized in order to understand their binding modes with the ATP site of EGFR and VEGFR-2. The models used the reported crystal structure of the EGFR domain in complex with erlotinib³⁶ and the VEGFR-2 domain with a compound from the literature, a 4-amino-furo[2,3-d]pyrimidine derivative³⁷.

Preliminary docking simulations were carried out in order to predict the binding mode of the first synthesized compound 11a into the ATP active site of EGFR and VEGFR occupied by vandetanib, a dual EGFR/VEGFR kinase inhibitor. The most stable docking model in EGFR (Figure 3a) shows a binding mode very similar to the orientation of vandetanib. Compound 11a interacts via a H-bonding interaction with Met769 (between a methionine NH and the N-1 nitrogen of quinazoline from 11a), Thr766 (via a bridge between a water molecule, the OH threonine side chain, and the N-3 nitrogen of quinazoline from 11a), and Thr830 (between a threonine OH and the sp2 oxygen from the amide group of 11a).

Figure 3.  Docking mode of ZD6474 (vandetanib) and compound 11a to the ATP site of EGFR and VEGFR-2. In each case, hydrophobic regions are represented in brown. (a) Active site of EGFR with docked vandetanib (magenta) and compound 11a (green). (b) Active site of VEGFR-2 with docked vandetanib (magenta) and compound 11a (green).

We also compared the volume occupied by vandetanib and 11a in the ATP active site of VEGFR-2. Both compounds interact differently with this active site. The quinazoline cores adopt essentially volumes and conformations in opposite directions. We observed new interactions between, respectively, the CO and NH amide groups from 11a and the backbone of Asp1044 and the Lys866–Glu883 salt bridge (Figure 3b). This observation led us to introduce different bulky substituents, such as amide, carbamate, or urea, which are a common moiety in EGFR and VEGFR inhibitors, in order to strengthen the ligand–receptor interactions and by the way probably increase the affinity.

Biological evaluation

The substituted anilinoquinazoline derivatives 7a–j (series A), 11a–j (series B), and 19a–j (series C) were evaluated for both their antiproliferative activity toward the hormone-independent PC3 prostate cancer cell and their in vitro EGFR and VEGFR-2 kinase inhibition. The results are reported in Table 1. Urea derivatives were slightly less active in the cell proliferation assays than the reference compound gefitinib, while other quinazolines were found to be poorly cytotoxic with IC₅₀ >10 μM. Substitution of urea with a phenyl or 2,4-difluorophenyl group seemed to be most tolerant of the steric bulk of substituents for in vitro antiproliferative activities. Replacement of one of the methoxy groups at the C-6 or C-7 position of the quinazoline core with a diethylaminoethyl resulted in a clear decrease in activity. Also, the antiproliferative activities of quinazoline substituted by carbamic acid ester (b and c) and aliphatic-urea (h, i, and j) were decreased.

Table 1.  Antiproliferative activity toward hormone-independent PC3 prostate cancer cells and in vitro EGFR and VEGFR-2 kinase inhibition.

Compounds			% Inhibition^a (IC50, μM)^b
			PC3 cancer cells^c	EGFR^d	VEGFR-2^e
Gefitinib		/	81.9 (7.31)	92.3 (0.072)	10.8
Semaxanib		/	35.2	16.6	80.2 (3.60)
R
a		7a	7.2	0	1.3
		11a	13.4	31.3	14.7
		19a	26.1	0	2.3
b		7b	2.2	1.7	45.2
		11b	15.9	72.4 (6.87)	79.8 (5.79)
		19b	16.1	11.1	26.7
c		7c	10.2	9.4	34.2
		11c	22.7	47.9	81.2 (5.62)
		19c	21.2	18.6	29.2
d		7d	0	9.1	44.1
		11d	78.1 (8.80)	28.7	77.8 (5.12)
		19d	19.1	4.3	26.4
e		7e	30.2	17.1	33.2
		11e	29.1	34.5	75.6 (6.18)
		19e	28.7	21.5	28.3
f		7f	27.9	8.3	30.7
		11f	69.1 (9.45)	2.1	22.2
		19f	31.2	1.7	20.9
g		7g	10.4	43.2	35.4
		11g	33.2	15.2	81.9 (4.31)
		19g	27.8	29.2	29.7
h		7h	17.4	5.2	72.1 (6.62)
		11h	28.2	30.6	80.3 (4.71)
		19h	0.8	3.4	24.5
i		7i	17.3	29.1	40.3
		11i	0	36.3	78.3 (5.82)
		19i	0	0	19.2
j		7j	11.3	28.2	1.9
		11j	35.1	51.3 (9.81)	30.4
		19j	6.2	32.3	8.2

^aCompounds tested at a concentration of 10 μM.

^bValues correspond to n = 3 (SD <10%).

^cCell proliferation was realized by MTT assay at 10 μM from at least three independent determinations.

^dInhibition of EGFR (purified from human carcinoma A431 cells) tyrosine kinase activity.

^eInhibition of VEGFR-2 (recombinant human protein) tyrosine kinase activity.

All the compounds tested exhibited weak to moderate in vitro EGFR and VEGFR kinase inhibitions (Table 1). The best results were obtained when the quinazoline derivatives were substituted on the aniline moiety by an alkyl or arylurea group, excepted for compounds 11f and 11j, leading to selective VEGFR inhibitors with IC₅₀ values from 4.31 to 6.62 μM (7h, 11d, 11e, 11g, 11h, 11i). This suggests that the oxygen of urea and the NH moiety interact with the binding site of VEGFR-2 as described in previous investigations. Surprisingly, amide derivative 11a was found to be inactive in both in vitro antiproliferative and enzymatic assays. Replacement of the urea with a carbamic acid methyl ester derivative led to dual EGFR/VEGFR active compound 11b, while its ethyl (11c) counterpart was found to be inactive on EGFR enzymatic activity. These observations demonstrated that the introduction of bulky substituents suh as ethyl carbamate (compared to methyl) or urea derivatives is the key modification to increase VEGFR-2 kinase inhibition, while those changes in the aniline substitution resulted in a total loss of EGFR activity.

We investigated the inhibitory activity of many compounds toward Aurora-A to demonstrate selectivity against this serine–threonine kinase. These compounds did not significantly impact on the enzyme-inhibitory activity (IC₅₀ >10 μM) compared to the reference Vertex³⁰ (80% inhibition at 1 μM). The quinazoline core employed is not a favorable structure to provide a potent inhibitory activity on Aurora-A, caused by the conformational restriction.

Conclusion

Three series of new quinazolines, differentiating ether linkers at the 6- and 7-positions of the core, incorporating a donor/acceptor group such as acetamide, carbamic acid ester, or urea substituted on an arylamino moiety, have been designed using structure–activity relationships. Suppression of the basic side chain on the quinazoline core confers an increase of cellular and enzymatic inhibitory activity. Our results show that some derivates of series B (6,7- dimethoxyquinazoline) have highly selective activity on EGFR and VEGFR-2 compared to series A and C. The introduction of a urea group was demonstrated by the increase of VEGFR-2 enzymatic activity but not EGFR. Also, replacement of the urea entity by a carbamic acid methyl ester group such as in 11b presented a dual EGFR/VEGFR-2 activity. According to the investigations, substitution by halogens on the middle phenyl group of carbamic acid ester and urea derivatives (b–j) appears to be an opportunity to develop a new dual inhibitor for EGFR/VEGFR-2. This suggests that this type of compound could bind in the ATP pocket of these two enzymes with better affinity leading to a synergic growth inhibition of the tumors.

Declaration of interest

The authors are grateful to the “Ligue Contre le Cancer” for its financial support.