Patent Application
20150246891
1. Technical Field
The disclosure relates to pyrimidine and quinazoline derivatives and their use as anticancer drugs.
2. Description of Related Art
Overexpression of cancerous inhibitor of protein phosphatase 2A (abbreviated as CIP2A) has been found in several common human cancers including acute leukemia, prostate cancer, non-small cell lung cancer, gastric cancer, head and neck cancer, colon cancer and breast cancer and has been linked to clinical aggressiveness in tumors and promotion of the malignant growth of cancer cells. CIP2A interacts directly with the transcription factor c-Myc and inhibits the PP2A dephosphorylation of c-Myc, thereby stabilizing the oncogenic c-Myc from degradation.
Protein phosphatase 2A (abbreviated as PP2A) is a crucial regulator of cell proliferation by dephosphorylation of protein kinases on serine or threonine residues. PP2A is composed of three subunits which regulate substrate specificity, cellular localization and enzymatic activity. For example, PP2A dephosphorylates p-Akt at serine 473 and reduces the cell growth. Hence, the CIP2A-PP2A-Akt signaling cascade is thought to be an important survival regulator in cancers. Accordingly, downregulation of c-Myc and p-Akt by CIP2A ablation is a promising anticancer strategy.
Some compounds have been found to be capable of repressing repress CIP2A expression and subsequently reducing p-Akt level and induce apoptosis in hepatocellular carcinoma (HCC). For example, the above phenomenon had been observed for bortezomib, a proteasome inhibitor.
The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
In one aspect, the present invention is directed to an aryl amine substituted pyrimidine having a chemical structure (I) or (II) below:
The R1 and R2 above are same or different substituted phenyl groups, and the substituted phenyl group each is
In another aspect, the present invention is directed to an aryl amine substituted quinazoline having a chemical structure (III) or (IV) below:
The R3 above is an aliphatic-substituted phenyl group, a halo-substituted phenyl group, a hydroxyl-substituted phenyl group, or an aryloxy-substituted phenyl group. The R4 above is H, an aliphatic group with carbon number of 1-5, an amino-substituted aliphatic group, or a benzyl group. The R5 above aliphatic substituted phenyl group, a halo-substituted phenyl group, an aryloxy-substituted phenyl group, a benzyl group, a halo substituted benzyl group, an alkoxy substituted phenyl group, an arylamino-substituted phenyl group, an amidyl-substituted phenyl group, an ArO(CO)NH-substituted phenyl group, or Ph-SO2—NH-substituted phenyl group, with the proviso that when R3 is
and R4 is H, the R5 does not include
According to an embodiment, the R3 is
According to another embodiment, the R4 is H, Me
According to yet another embodiment, the R5 is
In yet another aspect, the present invention directs to a pharmaceutical composition. The pharmaceutical composition comprises an effective amount of a compound having a chemical structurer (VII) below and a pharmaceutically acceptable carrier.
In yet another aspect, the present invention directs to a method of inhibiting the expression of cancerous inhibitor of PP2A. The method comprises contacting a cell with an effective amount of a compound having a chemical structure (VII) above. The R5 above is
In yet another aspect, the present invention directs to a method of treating cancer. The method comprises administrating an effective amount of a compound having a chemical structure (VII) above by a needed subject. The R5 above is
Many of the attendant features will be more readily appreciated as the same becomes better understood by reference to the following detailed description considered in connection with the accompanying drawings.
In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.
The following abbreviations are used: CDCl3, deuterated chloroform; DMSO-d6, dimethyl sulfoxide-d6; i-PrOH, isopropyl alcohol; EtOAc, ethyl acetate; DMF, N,N-dimethylformamide; MeOH, methanol; THF, tetrahydrofuran; EtOH, ethanol; DMSO, dimethyl sulfoxide; DIPEA, diisopropylethylamine; DCM, dichloromethane.
In the synthesis scheme I above, R1 and R2 can be the same or different substituted phenyl group, such as a mono-substituted phenyl group or a di-substituted phenyl group. The mono-substituted phenyl group can be
The di-substituted phenyl group can be
One or both of the R1 and R2 also can be a benzyl group,
The general synthesis procedure of the pyrimidine derivatives is described as follow.
A solution of 2,4-dichloropyrimidine (1.0 mmol) and N,N-diisopropylethylamine (DIPEA) (100 μl) in isopropyl alcohol was added with 0.7 mmol of a substituted phenyl amine, and the mixtures was stirred in ice-bath for 30 minutes. The resulting mixture was stirred at room temperature for 8 hours. After the reaction was completed, the reaction mixture was washed with water, extracted with EtOAc, and the organic layer was dried over MgSO4. After removal of MgSO4 by filtration and evaporation of solvents, the crude residue was purified by chromatography on a silica gel column (silica gel columns 60, 0.063-0.200 mm or 0.040-0.063 mm, Merck; basic silica gel) using MeOH/CH2Cl2 as eluent (0% to 2%) to give compounds 1-7 (yield: 3-27%) below.
The spectral data of the above compounds are listed below.
1H NMR (400 MHz, MeOH-d4): δ 3.48 (s, 1H), 6.66 (d, J=6.0 Hz, 1H), 7.18 (d, J=7.6 Hz, 1H), 7.30 (t, J=7.6 Hz, 1H), 7.61 (d, J=8.4 Hz, 1H), 7.72 (s, 1H), 8.05 (d, J=6.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 78.6, 82.8, 102.9, 123.7, 123.9, 126.5, 129.8, 130.0, 137.5, 158.5, 161.1, 162.6; HRMS calculated for C12H8ClN3 (M+H): 230.0485. Found: 230.0478. Yield: 5%.
1H NMR (400 MHz, CDCl3): δ 6.59 (d, J=5.6 Hz, 1H), 7.18 (d, J=8.0 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H), 7.31 (t, J=8.0 Hz, 1H), 7.36 (s, 1H), 8.15 (d, J=5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 102.9, 120.6, 122.6, 125.8, 130.6, 135.2, 138.4, 158.2, 160.8, 162.0; HRMS calculated for C10H7Cl2N3 (M+H): 240.0095. Found: 240.0101. Yield: 6%.
1H NMR (400 MHz, CDCl3): δ 6.55 (d, J=5.6 Hz, 1H), 7.11 (s, 1H), 7.51 (d, J=8.4 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.72 (s, 1H), 8.20 (d, J=5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 103.6, 120.8 (q), 123.7, 125.8, 128.1, 129.2, 129.5, 132.5, 136.2, 158.3, 160.9, 161.4; HRMS calculated for C11H6Cl2F3N3 (M+H): 307.9969. Found: 307.9969. Yield: 3%.
1H NMR (400 MHz, MeOH-d4): δ 6.63 (d, J=6.0 Hz, 1H), 6.96-7.00 (m, 4H), 7.08 (t, J=7.2 Hz, 1H), 7.33 (t, J=8.0 Hz, 2H), 7.54 (d, J=8.0 Hz, 2H), 8.01 (d, J=6.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 102.1, 119.0, 119.6, 123.7, 125.5, 129.8, 131.7, 155.6, 156.7, 158.0, 160.8, 163.0; HRMS calculated for C16H12ClN3O (M−H): 296.0591. Found: 296.0583. Yield: 14%.
1H NMR (400 MHz, MeOH-d4): δ 4.55 (s, 2H), 6.60 (d, J=5.2 Hz, 1H), 7.19-7.22 (m, 1H), 7.26-7.31 (m, 4H), 8.12 (d, J=5.2 Hz, 1H); 13C NMR (100 MHz, MeOH-d4): δ 43.8, 104.4, 126.9, 127.4, 128.2, 138.3, 154.3, 160.2, 163.7; HRMS calculated for C11H10ClN3 (M+H): 220.0642. Found: 220.0640. Yield: 27%.
The spectral data of the above compounds are listed below.
1H NMR (400 MHz, CDCl3): δ 3.04 (s, 1H), 3.10 (s, 1H), 6.16 (d, J=6.0 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 7.19-7.31 (m, 4H), 7.37 (d, J=8.0 Hz, 1H), 7.45 (s, 1H), 7.54 (d, J=8.4 Hz, 1H), 7.72 (s, 1H), 7.92 (brs, 1H), 8.06 (d, J=6.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 77.0, 77.8, 83.0, 83.8, 97.2, 120.5, 122.4, 122.6, 123.0, 123.1, 125.2, 126.1, 128.0, 128.8, 129.3, 138.5, 139.7, 157.2, 159.8, 161.0; HRMS calculated for C20H14N4 (M+H): 311.1297. Found: 311.1291. Yield: 10%.
1H NMR (400 MHz, CDCl3): δ 6.16 (d, J=5.6 Hz, 1H), 6.60 (s, 1H), 7.10 (s, 1H), 7.38 (d, J=8.4 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H), 7.60 (d, J=8.4 Hz, 1H), 7.68 (d, J=8.8 Hz, 1H), 7.72 (s, 1H), 7.92 (s, 1H), 8.12 (d, J=5.6 Hz, 1H); 13C NMR (100 MHz, MeOH-d4): δ 99.6, 118.0 (q), 118.4 (q), 118.7, 118.9, 121.4, 121.6, 123.0, 123.3, 123.9, 124.1, 124.2, 124.3, 126.91-128.30 (m), 131.2, 131.4, 139.0, 139.6, 155.8, 159.1, 160.6; HRMS calculated for C18H10Cl2F6N4 (M+H): 467.0265. Found: 467.0254. Yield: 5%.
In the synthesis scheme II, R3 and R5 can be the same or different substituted phenyl groups, such as a mono-substituted phenyl group or a di-substituted phenyl group. The mono-substituted phenyl group can be
The di-substituted phenyl group can be
One or both of R3 and R5 also can be a benzyl group,
R4 can be H or methyl group.
A series of quinazoline derivatives were designed and synthesized by the general procedure illustrated in scheme II above. Based on the core quinazoline structure of these quinazoline derivatives, a commercially available dichloro-quinazoline was chosen as a starting material. A series of mono-quinazoline derivatives (compounds 8-17) were generated with various substituted phenylamines by replacement of the chloride in the quinazoline (Embodiment 3). Then, the other chloride from the mono-substitute quinazolines was replaced with various substituted phenylamines to yield compounds 18-24 (Embodiment 4).
The general synthesis procedure of the mono-substituted quinazoline derivatives are stated as follow. Substituted phenyl amine (0.8 mmol) was added to a solution of 2,4-dichloro-6,7-dimethoxyquinazoline (1.0 mmol) in isopropyl alcohol (5 ml), followed by the addition of a drop of concentrated HCl (100 μl). The resulting mixture was stirred at 60° C. for 2 hours. The mixture was filtered, and the solid was washed with isopropyl alcohol then dried under vacuum to give compounds 8, and 10-17. This procedure afforded the expected coupling product as a white or yellow solid (yield: 21%-95%).
Compound 9 was synthesized from compound 8, and the further synthesis procedure is as follow. Methyl iodide (56 μl, 0.90 mmol) was added to a solution of compound 8 (61.0 mg, 0.18 mmol) and sodium hydride (60% oil suspension, 8.63 mg, 0.36 mmol) in 2 ml of DMF cooled to 0° C. The mixture was stirred at 0° C. for 1 hour, then allowed to warm to room temperature and stirred for another 1 hour. The reaction mixture was washed with water, and then extracted with EtOAc. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by chromatography on a silica gel column (silica gel columns 60, 0.063-0.200 mm or 0.040-0.063 mm, Merck; basic silica gel) using EtOAc/hexane as eluent (0% to 40%) to give compound 9.
The spectral data of the above compounds are listed below.
1H NMR (400 MHz, DMSO-d6): δ 3.93 (s, 3H), 3.99 (s, 3H), 4.21 (s, 1H), 7.18 (s, 1H), 7.26 (d, J=8.0 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.89 (s, 1H), 7.94 (s, 1H), 10.00 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 56.4, 57.2, 81.1, 83.8, 103.9, 106.6, 107.9, 122.2, 123.8, 126.0, 127.6, 129.2, 139.5, 148.1, 149.5, 154.1, 155.5, 158.4; HRMS calculated for C18H14ClN3O2 (M+H): 340.0853. Found: 340.0850. Yield: 94%.
1H NMR (400 MHz, CDCl3): δ 3.07 (s, 1H), 3.28 (s, 3H), 3.57 (s, 3H), 3.88 (s, 3H), 6.21 (s, 1H), 7.05 (s, 1H), 7.15 (d, J=7.2 Hz, 1H), 7.31-7.37 (m, 3H); 13C NMR (100 MHz, CDCl3): δ 42.2, 55.2, 56.1, 78.8, 82.0, 104.8, 106.7, 108.6, 124.2, 126.7, 129.5, 130.0, 130.2, 147.5, 147.8, 150.4, 154.4, 155.0, 161.3; HRMS calculated for C19H16ClN3O2 (M+H): 354.1009. Found: 354.1016. Yield: 60%.
1H NMR (400 MHz, DMSO-d6): δ 3.90 (s, 3H), 3.95 (s, 3H), 7.18 (s, 1H), 7.20 (d, 1H, J=8.0 Hz), 7.44 (t, J=8.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.95 (s, 1H), 7.97 (s, 1H), 10.08 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 56.5, 57.1, 103.3, 106.6, 107.8, 121.3, 122.4, 124.1, 130.5, 133.1, 140.7, 148.2, 149.6, 154.1, 155.6, 158.2; HRMS calculated for C16H13Cl2N3O2 (M+H): 350.0463. Found: 350.0466. Yield: 75%.
1H NMR (400 MHz, DMSO-d6): δ 3.15 (s, 1H), 3.91 (s, 3H), 3.94 (s, 3H), 6.59 (d, J=6.8 Hz, 1H), 7.13-7.21 (m, 4H), 7.98 (s, 1H), 9.96 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 56.4, 56.9, 58.5, 103.3, 106.4, 107.7, 110.6, 112.1, 114.2, 129.5, 139.8, 147.6, 149.5, 154.2, 155.4, 158.0; HRMS calculated for C16H14ClN3O3 (M+H): 332.0802. Found: 332.0810. Yield: 60%.
1H NMR (400 MHz, DMSO-d6): δ 2.32 (s, 3H), 3.87 (s, 3H), 3.92 (s, 3H), 7.12-7.16 (m, 2H), 7.22 (d, J=10.4 Hz, 1H), 7.29 (d, J=7.6 Hz, 1H), 7.90 (s, 1H), 10.05 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 20.1, 56.0, 56.3, 102.8, 105.7, 106.8, 115.61, 115.8, 124.9, 125.0, 128.1, 128.2, 128.5, 133.6, 133.7, 146.9, 149.1, 153.9, 153.9, 155.1, 156.3, 159.1; HRMS calculated for C17H15ClFN3O2 (M+H): 348.0915. Found: 348.0911. Yield: 60%.
1H NMR (400 MHz, DMSO-d6): δ 3.92 (s, 3H), 3.96 (s, 3H), 7.19 (s, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.99 (s, 1H), 8.19 (d, J=8.8 Hz, 1H), 8.42 (s, 1H), 10.32 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 56.0, 56.8, 102.8, 105.9, 107.3, 118.7, 120.9, 120.9, 121.0, 121.0, 121.5, 124.2, 124.5, 125.9, 126.2, 126.5, 126.9, 131.6, 138.3, 147.5, 149.2, 153.1, 155.2, 157.2; HRMS calculated for C17H12Cl2F3N3O2 (M+H): 418.0337. Found: 418.0340. Yield: 86%.
1H NMR (400 MHz, DMSO-d6): δ 3.91 (s, 3H), 3.94 (s, 3H), 7.03 (d, J=8.6 Hz, 2H), 7.07 (d, J=8.8 Hz, 2H), 7.11-7.16 (m, 2H), 7.40 (t, J=6.0 Hz, 2H), 7.71 (d, J=8.8 Hz, 2H), 7.90 (s, 1H), 9.93 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 56.5, 57.0, 103.4, 105.9, 107.5, 118.8, 119.5, 123.8, 125.3, 130.5, 134.3, 146.9, 149.5, 153.6, 153.9, 155.5, 157.4, 158.4; HRMS calculated for C22H18ClN3O3 (M+H): 408.1115. Found: 408.1121. Yield: 55%.
1H NMR (400 MHz, DMSO-d6): δ 2.70 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H), 6.64 (d, J=8.4 Hz, 1H), 6.71 (s, 1H), 7.04 (d, J=8.4 Hz, 1H), 7.11 (s, 1H), 7.83 (s, 1H), 9.67 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 18.5, 56.4, 56.5, 102.8, 107.0, 107.3, 113.5, 117.3, 128.0, 129.2, 136.7, 148.1, 149.2, 155.1, 155.5, 156.5, 160.1; HRMS calculated for C17H16ClN3O3 (M+H): 346.0958. Found: 346.0951. Yield: 23%.
1H NMR (400 MHz, DMSO-d6): δ 3.91 (s, 3H), 3.94 (s, 3H), 6.94 (d, J=8.0 Hz, 1H), 7.17 (s, 1H), 7.21 (d, J=8.8 Hz, 2H), 7.50 (t, J=8.0 Hz, 1H), 7.65 (m, 2H), 7.86 (d, J=8.8 Hz, 2H), 7.90 (s, 1H), 10.00 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 56.0, 56.2, 102.1, 105.3, 106.6, 107.2, 113.8, 115.4, 118.4, 118.6, 118.7, 130.2, 134.6, 140.4, 148.2, 149.0, 153.9, 154.6, 155.0, 157.6, 160.7; HRMS calculated for C23H17ClN4O3 (M−H): 431.0911. Found: 431.0909. Yield: 74%.
1H NMR (400 MHz, MeOH-d4): δ 3.90 (s, 3H), 3.92 (s, 3H), 4.79 (s, 2H), 6.96 (s, 1H), 7.23 (t, J=7.2 Hz, 1H), 7.31 (t, J=7.2 Hz, 2H), 7.39 (d, J=7.2 Hz, 2H), 7.47 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 43.4, 55.8, 56.0, 102.2, 106.5, 106.8, 126.9, 127.4, 128.3, 138.9, 147.2, 148.5, 154.4, 155.0, 159.9; HRMS calculated for C17H16ClN3O2 (M+H): 330.1009. Found: 330.1007. Yield: 21%.
The general synthesis procedure of the di-substituted quinazoline derivatives are stated as follow. Substituted phenyl amine (0.5 mmol) were added to a solution of compound 8 (0.2 mmol) in isopropyl alcohol (3 ml), followed by the addition of a drop of concentrated HCl (100 μl). The resulting solution was heated by using microwave irradiation to 150° C. for 30 min. After cooling, the mixture was filtered, and the solid was washed with isopropyl alcohol. The crude solid was dissolved in CH2Cl2 and washed with saturated NaHCO3 solution. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by chromatography on a silica gel column (silica gel columns 60, 0.063-0.200 mm or 0.040-0.063 mm, Merck; basic silica gel) using MeOH/CH2Cl2 as eluent (0% to 5%) to give compounds 18-24 (yield: 20-80%).
The spectral data of the above compounds are listed below.
1H NMR (400 MHz, MeOH-d4): δ 3.47 (s, 1H), 3.91 (s, 3H), 3.93 (s, 3H), 6.88 (s, 1H), 7.22 (d, J=7.6 Hz, 2H), 7.31 (t, J=8.0 Hz, 1H), 7.34 (d, J=9.2 Hz, 1H), 7.53 (s, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.83 (s, 1H), 8.01-8.04 (m, 2H); 13C NMR (100 MHz, MeOH-d4): δ 54.9, 55.4, 77.1, 77.2, 83.1, 101.9, 105.0, 105.1, 117.1-117.2 (q), 121.5, 121.7, 121.9 (d), 122.5, 122.7, 122.9, 123.3, 124.4, 125.5, 126.9, 127.3, 127.6, 128.3, 128.5, 131.17, 139.5, 140.3, 147.0, 148.3, 155.1, 155.5, 157.8; HRMS calculated for C25H18ClF3N4O2 (M+H): 499.1149. Found: 499.1142. Yield: 33%.
1H NMR (400 MHz, DMSO-d6): δ 3.93 (s, 3H), 3.94 (s, 3H), 4.18 (s, 1H), 6.98 (d, 4H, J=8.8 Hz), 7.13 (s, 1H), 7.15 (d, 1H, J=7.6 Hz), 7.33-7.45 (m, 6H), 7.69 (d, 1H, J=7.6 Hz), 7.75 (s, 1H), 8.10 (s, 1H), 10.33 (s, 1H), 10.90 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 56.1, 56.2, 77.6, 83.2, 100.0, 104.4, 106.3, 117.9, 120.0, 120.8, 122.4, 122.5, 122.7, 125.2, 127.7, 128.8, 129.5, 135.9, 138.6, 146.7, 149.2, 151.3, 155.1, 155.9, 156.9, 158.2; HRMS calculated for C30H24N4O3 (M+H): 489.1927. Found: 489.1925. Yield: 40%.
1H NMR (400 MHz, MeOH-d4): δ 3.47 (s, 1H), 3.93 (s, 3H), 3.94 (s, 3H), 6.88 (d, J=8.0 Hz, 1H), 6.92 (s, 1H), 7.17 (t, J=8.0 Hz, 1H), 7.22 (d, J=7.6 Hz, 1H), 7.34 (t, J=8.0 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.58 (s, 1H), 7.80 (s, 2H), 7.87 (d, J=8.4 Hz, 1H); 13C NMR (100 MHz, MeOH-d4): δ 54.9, 55.4, 77.1, 83.1, 102.0, 104.9, 104.9, 117.0, 118.4, 120.5, 122.6, 123.0, 125.5, 126.9, 128.5, 129.3, 133.7, 139.6, 142.3, 146.9, 148.5, 155.1, 155.8, 157.9; HRMS calculated for C24H19ClN4O2 (M+H): 431.1275. Found: 431.1280. Yield: 34%.
1H NMR (400 MHz, MeOH-d4): δ 2.18 (s, 3H), 3.45 (s, 1H), 3.95 (s, 3H), 3.96 (s, 3H), 6.75 (brs, 1H), 6.91-6.98 (m, 2H), 7.23 (d, J=7.6 Hz, 1H), 7.31 (t, J=8.0 Hz, 1H), 7.62 (s, 1H), 7.75-7.77 (m, 2H), 7.91 (d, J=7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 21.2, 56.1, 56.1, 75.5, 100.3, 104.4, 105.5, 114.0, 114.2, 121.4, 122.2, 122.3, 122.7, 122.7, 125.3, 127.6, 127.7, 127.8, 128.9, 133.7, 133.7, 138.6, 146.9, 148.1, 149.7, 152.1, 155.2, 155.2, 157.1; HRMS calculated for C25H21FN4O2 (M+H): 429.1727. Found: 429.1721. Yield: 20%.
1H NMR (400 MHz, DMSO-d6): δ 3.84 (s, 3H), 3.85 (s, 3H), 4.15 (s, 1H), 4.53 (d, J=6.4 Hz, 2H), 6.75 (s, 1H), 7.10-7.19 (m, 3H), 7.27 (t, J=8.0 Hz, 1H), 7.28 (d, J=7.2 Hz, 2H), 7.33 (d, J=7.2 Hz, 2H), 7.63 (s, 1H), 7.90 (s, 1H), 9.14 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 29.3, 45.2, 55.7, 55.9, 76.8, 83.0, 100.1, 103.4, 105.2, 121.6, 122.2, 124.2, 126.7, 126.9, 127.1, 128.1, 128.4, 138.5, 139.3, 145.7, 154.7, 156.5, 158.0; HRMS calculated for C25H22N4O2 (M+H): 411.1821. Found: 411.1826. Yield: 15%.
1H NMR (400 MHz, CDCl3): δ 3.05 (s, 1H), 3.93 (s, 3H), 3.95 (s, 3H), 6.61 (d, J=7.6 Hz, 1H), 6.92 (s, 1H), 6.94 (s, 1H), 7.01 (d, J=9.2 Hz, 2H), 7.20 (d, J=7.6 Hz, 1H), 7.26 (t, J=8.0 Hz, 2H), 7.35 (d, J=7.2 Hz, 2H), 7.54 (d, J=9.2 Hz, 2H), 7.64 (s, 1H), 7.66 (d, J=8.0 Hz, 1H), 7.73 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 56.1, 56.2, 77.6, 83.2, 100.1, 104.6, 105.3, 106.3, 110.5, 112.8, 115.2, 118.0, 119.0, 122.7, 122.8, 125.4, 127.9, 128.8, 130.1, 133.9, 138.5, 142.1, 147.0, 148.9, 155.1, 155.1, 155.3, 157.0, 161.6; HRMS calculated for C31H23N5O3 (M+H): 514.1879. Found: 514.1888. Yield: 80%.
1H NMR (400 MHz, DMSO-d6): δ 3.92 (s, 3H), 3.94 (s, 3H), 4.22 (s, 1H), 7.06 (d, J=8.4 Hz, 2H), 7.10-7.15 (m, 3H), 7.36 (d, J=7.6 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H), 7.53 (d, J=7.6 Hz, 2H), 7.68 (d, J=7.6 Hz, 1H), 7.74 (s, 1H), 7.85 (d, J=8.4 Hz, 2H), 8.03 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 56.1, 56.2, 77.6, 83.2, 100.2, 104.6, 105.0, 106.3, 117.3, 119.0, 120.6, 121.0, 122.6, 122.7, 125.5, 127.8, 128.8, 134.0, 137.6, 138.6, 146.8, 148.6, 149.2, 155.1, 155.7, 157.0, 162.4; HRMS calculated for C31H23N5O3 (M+H): 514.1879. Found: 514.1876. Yield: 75%.
1H NMR (400 MHz, DMSO-d6) δ 3.92 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 4.22 (s, 1H), 4.24 (s, 1H), 7.13 (s, 1H, ArH), 7.25 (d, J=8.0 Hz, 1H, ArH), 7.32-7.36 (m, 2H, ArH), 7.44 (t, J=8.0 Hz, 1H, ArH), 7.53 (s, 2H, ArH), 7.69 (s, 1H, ArH), 7.73 (d, J=8.0 Hz, 1H, ArH), 8.05 (s, 1H, ArH); 13C NMR (100 MHz, CDCl3) δ 56.0, 56.1, 77.0, 77.54, 83.3, 83.9, 100.1, 104.5, 106.2, 119.7, 122.2, 122.2, 122.5, 122.6, 125.0, 125.4, 127.6, 128.7, 128.9, 138.6, 140.1, 146.7, 148.9, 154.9, 155.4, 156.8; HRMS calculated for C26H20N4O2 [M++H] 421.1665. found 421.1671.
1H NMR (400 MHz, DMSO-d6) δ 1.09 (t, J=7.6 Hz, 3H, CH3), 2.52 (q, J=7.6 Hz, 2H, CH2), 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.23 (s, 1H), 7.00 (d, J=6.8 Hz, 1H, ArH), 7.11 (s, 1H, ArH), 7.23-7.27 (m, 3H, ArH), 7.37-7.43 (m, 2H, ArH), 7.71 (d, J=8.0 Hz, 1H, ArH), 7.74 (s, 1H, ArH), 8.06 (s, 1H, ArH); 13C NMR (100 MHz, DMSO-d6) δ 15.3, 28.0, 56.2, 56.3, 81.1, 82.8, 98.5, 102.9, 105.0, 119.1, 120.8, 122.1, 124.0, 125.5, 127.8, 128.7, 128.9, 129.1, 135.8, 136.6, 137.4, 144.6, 147.2, 150.6, 155.9, 158.5; HRMS calculated for C26H24N4O2 [M++H] 425.1978. found 425.1978.
1H NMR (400 MHz, MeOH-d4) δ 3.43 (s, 1H), 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.62 (s, 2H, CH2), 6.84 (s, 1H, ArH), 7.17 (d, J=8.0 Hz, 1H, ArH), 7.22 (t, J=8.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H, ArH), 7.50 (d, J=7.6 Hz, 1H, ArH), 7.56 (s, 1H, ArH), 7.62 (d, J=8.0 Hz, 1H, ArH), 7.70 (s, 1H, ArH), 7.86 (s, 1H, ArH); 13C NMR (100 MHz, CDCl3) δ 44.5, 56.0, 56.2, 78.2, 83.2, 100.3, 103.9, 105.6, 121.4, 121.9, 122.6, 124.1, 124.9, 126.44-126.48 (t), 127.5, 127.6, 127.9, 128.6, 130.4, 131.3, 131.6, 138.6, 139.3, 146.3, 155.1, 157.0, 158.1; HRMS calculated for C26H20ClF3N4O2 [M++H] 513.1305. found 513.1309.
1H NMR (400 MHz, MeOH-d4) δ 3.42 (s, 1H), 3.88 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.61 (s, 2H, CH2), 6.81 (s, 1H, ArH), 7.05 (d, J=7.6 Hz, 1H, ArH), 7.14 (d, J=7.6 Hz, 1H, ArH), 7.17-7.21 (m, 2H, ArH), 7.30 (s, 1H, ArH), 7.32 (t, J=7.6 Hz, 1H, ArH), 7.51 (s, 1H, ArH), 7.66 (d, J=7.6 Hz, 1H, ArH), 7.87 (s, 1H, ArH); 13C NMR (100 MHz, MeOH-d4) δ 45.3, 56.2, 56.8, 78.1, 84.3, 103.6, 105.1, 105.3, 119.8, 120.4, 120.4, 122.9, 123.6, 123.7, 126.5, 126.6, 127.9, 129.3, 130.6, 140.7, 144.6, 147.2, 149.9, 150.4, 150.4, 156.2, 159.1, 159.8; HRMS calculated for C26H21F3N4O3 [M++H] 549.1644. found 549.1639.
1H NMR (400 MHz, MeOH-d4) δ 3.45 (s, 1H), 3.69 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.52 (s, 2H, CH2), 6.83-6.88 (m, 3H, ArH), 6.95 (s, 1H, ArH), 7.17 (dt, J=8.0 Hz, 1H, ArH), 7.25 (t, J=8.0 Hz, 1H, ArH), 7.56 (s, 1H, ArH), 7.74 (d, 1H, ArH), 7.96 (s, 1H, ArH); 13C NMR (100 MHz, CDCl3) δ 14.1, 29.6, 44.7, 55.8, 56.2, 61.6, 77.3, 83.0, 102.4, 102.8, 110.7, 111.0, 119.4, 122.6, 122.9, 125.1, 128.3, 128.7, 130.8, 138.2, 146.7, 148.2, 149.0, 155.7, 157.6, 165.2, 165.7; HRMS calculated for C27H26N4O4 [M++H] 471.2032. found 471.2031.
1H NMR (400 MHz, MeOH-d4) δ 3.47 (s, 1H), 3.77 (s, 3H), 3.86 (s, 3H), 6.66 (d, J=8.0 Hz, 1H), 6.82 (s, 1H), 7.04 (t, J=8.0 Hz, 1H), 7.19 (d, J=8.0 Hz, 1H), 7.26 (t, J=8.0 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 7.40 (t, J=8.0 Hz, 3H), 7.46-7.48 (m, 2H), 7.53 (s, 1H), 7.75 (m, 4H); 13C NMR (100 MHz, MeOH-d4) δ 56.3, 56.8, 78.8, 84.4, 103.4, 104.7, 105.7, 113.4, 115.6, 117.5, 123.9, 124.6, 127.0, 128.2, 128.7, 129.8, 129.9, 130.3, 133.8, 139.3, 140.4, 141.0, 141.8, 146.5, 148.3, 155.7, 156.6, 159.13; HRMS calcd for C30H25N5O4S [M++H] 552.1627. found 552.1707. Yield: 45%.
1H NMR (400 MHz, DMSO-d6) δ 3.94 (s, 6H), 4.23 (s, 1H), 6.88 (d, 1H NMR (400 MHz, MeOH-d4) 6, 1H), 7.13 (s, 2H), 7.19 (t, J=8.0 Hz, 1H), 7.35-7.43 (m, 3H), 7.70-7.73 (m, 2H), 7.78 (t, J=8.0 Hz, 1H), 8.01 (d, J=8.0 Hz, 1H), 8.05-8.07 (m, 2H), 8.11 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 56.7, 57.1, 81.6, 83.3, 99.3, 103.6, 105.4, 114.2, 115.0, 116.8, 118.9, 122.3, 122.4, 122.8, 123.6, 124.0, 125.1, 125.8, 126.3, 127.8, 128.0, 129.3, 129.8, 129.9, 130.1, 130.4, 130.8, 131.2, 131.4, 136.8, 137.9, 138.2, 141.0, 147.8, 151.1, 156.3, 158.8, 158.9; HRMS calcd for C31H24F3N6O4S [M++H] 620.1501. found 620.1546. Yield: 30%.
1H NMR (400 MHz, MeOH-d4) δ 3.46 (s, 1H), 3.94 (s, 6H), 6.73 (d, J=8.0 Hz, 1H), 7.00 (s, 1H), 7.06 (t, J=8.0 Hz, 1H), 7.20 (d, J=8.0 Hz, 1H), 7.29-7.35 (m, 2H), 7.59 (s, 1H), 7.69 (d, J=8.4 Hz, 1H), 7.74 (s, 1H), 7.80 (s, 1H), 7.87 (d J=8.4 Hz, 1H), 8.01 (s, 1H), 8.26 (d, J=8.4 Hz, 1H); 13C NMR (100 MHz, MeOH-d4) δ 56.4, 56.8, 78.6, 84.4, 103.4, 106.3, 106.4, 112.6, 114.6, 117.0, 121.8, 122.5, 123.9, 124.3, 125.2, 125.6, 125.7, 126.7, 128.2, 129.8, 130.2, 133.2, 133.3, 134.3, 135.9, 136.2, 138.0, 141.0, 143.1, 143.7, 148.2, 149.6, 156.4, 157.1, 159.2; HRMS calcd for C31H23BrF3N6O4S [M++H] 698.0606. found 698.0682. Yield: 40%.
1H NMR (400 MHz, DMSO-d6) δ 3.49 (s, 6H), 4.24 (s, 1H), 7.14 (s, 1H), 7.29-7.39 (m, 3H), 7.51-7.62 (m, 5H), 7.79 (s, 2H), 7.88 (s, 1H), 7.94 (d, J=8.0 Hz, 2H), 8.13 (s, 1H); 13C NMR (100 MHz, MeOH-d4) δ 55.7, 56.1, 80.6, 82.4, 98.3, 102.5, 104.4, 113.7, 116.5, 117.4, 121.4, 124.8, 127.0, 127.1 (2C), 127.8 (2C), 128.3, 128.5, 131.1, 134.2, 135.5, 136.2, 136.9, 139.2, 139.2, 146.7, 150.3, 155.4, 157.8, 165.0; HRMS calcd for C31H26N6O3 [M++H] 516.1957. found 516.2025. Yield: 70%.
1H NMR (400 MHz, CDCl3) δ 3.02 (s, 1H), 3.78 (s, 3H), 3.96 (s, 3H), 4.23 (s, 2H), 6.24 (d, J=8.0 Hz, 1H), 6.72-6.78 (m, 2H), 6.92-7.12 (m, 5H), 7.18-7.45 (m, 8H), 7.72 (d, J=8.0 Hz, 1H), 7.85 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 29.4, 55.9, 56.0, 76.1, 83.0, 102.0, 104.0, 104.6, 106.6, 109.2, 121.0, 122.3, 123.9, 126.4, 126.9, 127.4, 127.9, 128.3, 128.5, 129.3, 137.3, 138.5, 139.2, 139.3, 139.7, 146.7, 148.7, 155.3, 157.8; HRMS calcd for C31H27N5O2 [M++H] 502.2243. found 502.2245. Yield: 45%.
1H NMR (400 MHz, CDCl3) δ 2.94 (s, 1H), 3.66 (s, 3H), 3.77 (s, 3H), 6.66 (s, 1H), 6.93 (d, J=8.0 Hz, 1H), 6.97 (d, J=8.0 Hz, 1H), 7.08 (t, J=8.0 Hz, 2H), 7.12-7.20 (m, 2H), 7.54 (d, J=8.0 Hz, 1H), 7.78 (s, 1H), 7.95 (brs, 1H), 8.01-8.05 (m, 2H), 8.28 (s, 2H), 8.39 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 55.9, 56.1, 77.6, 83.1, 100.8, 104, 3, 104.8 (q), 112.2, 115.0, 116.4, 121.5, 122.3, 122.6, 124.2, 125.2 (q), 125.3, 126.9, 127.5 (q), 128.6, 129.3, 131.7, 131.7, 132.1, 132.4, 132.7, 137.0, 137.1, 138.6, 140.3, 146.9, 154.3, 155.2, 156.8, 163.2; HRMS calcd for C33H23F6N5O3 [M++H] 652.1783. found 652.1777. Yield: 30%.
1H NMR (400 MHz, MeOH-d4) δ 3.41 (s, 1H), 3.78 (s, 3H), 3.84 (s, 3H), 6.80 (s, 1H), 7.01 (d, J=8.0 Hz, 1H), 7.14-27 (m, 3H), 7.35 (d, J=8.0 Hz, 1H), 7.45 (s, 1H), 7.66 (t, J=8.0 Hz, 1H), 7.81-7.88 (m, 3H), 8.05 (s, 1H), 8.11 (d, J=8.0 Hz, 1H), 8.21 (s, 1H); 13C NMR (100 MHz, MeOH-d4) δ 56.2, 56.7, 78.5, 84.5, 103.2, 106.1, 113.4, 113.8, 113.9, 115.8, 117.3, 123.7, 124.0, 124.1, 125.6 (q), 126.5, 126.6, 126.7, 128.0, 129.1, 129.4, 129.5, 129.8, 130.5, 131.4, 131.73, 132.0, 132.2, 132.3, 137.3, 137.4, 139.6, 140.9, 142.4, 147.9, 149.5, 156.2, 157.1, 158.7, 166.9; HRMS calcd for C32H24F3N5O3 584.1909. found 584.1918.
The general synthesis procedure of the di-substituted quinazoline derivatives are stated as follow.
A solution of 2,4-dichloro-6,7-dimethoxyquinazoline in isopropyl alcohol (IPA) was treated with 3-ethynylaniline and a drop of concentrated HCl and then stirred at 60° C. for 2.5 h to provide TD-1. A series of phenoxyanilines was added to a solution of TD-1 in IPA, followed by the addition of concentrated HCl. The mixture was heated under microwave radiation at 150° C. for 30 min to obtain compounds 87-90, 95 and 96.
The spectral data of the above compounds are listed below.
1H NMR (400 MHz, DMSO-d6) δ 3.92 (s, 3H), 3.94 (s, 3H), 4.22 (s, 1H), 7.00 (d, J=9.2 Hz, 2H), 7.09 (s, 1H), 7.27 (d, J=7.6 Hz, 1H), 7.34 (t, J=8.4 Hz, 1H), 7.45-7.50 (m, 2H), 7.55 (d, J=8.8 Hz, 1H), 7.68 (s, 2H), 7.82 (dt, J=9.2, 2.4 Hz, 2H), 8.05 (s, 1H), 10.50 (s, 1H), 10.85 (s, 1H).
1H NMR (400 MHz, DMSO-d6) δ 3.92 (s, 3H), 3.93 (s, 3H), 4.20 (s, 1H), 6.93 (dd, J=8.8, 2.4 Hz, 1H), 7.08 (s, 1H), 7.23 (s, 1H), 7.25 (d, J=2.4 Hz, 1H), 7.34 (t, J=8.0 Hz, 1H), 7.43 (dd, J=8.8, 2.0 Hz, 1H), 7.56 (d, J=8.8 Hz, 2H), 7.66 (d, J=1.2 Hz, 2H), 7.92 (d, J=8.8 Hz, 1H), 8.11 (s, 1H), 10.70 (s, 1H), 11.05 (s, 1H).
1H NMR (400 MHz, DMSO-d6) δ 2.43 (s, 3H), 3.92 (s, 3H), 3.93 (s, 3H), 4.22 (s, 1H), 6.77 (dd, J=8.4, 2.4 Hz, 1H), 6.94 (d, J=2.4 Hz, 1H), 7.08 (s, 1H), 7.26 (d, J=7.6 Hz, 1H), 7.33 (t, J=7.6 Hz, 1H), 7.41-7.45 (m, 2H), 7.53 (d, J=8.4 Hz, 1H), 7.66 (s, 1H), 7.67 (s, 1H), 7.72 (d, J=8.8 Hz, 1H), 8.12 (s, 1H), 10.67 (s, 1H), 11.04 (s, 1H).
1H NMR (400 MHz, DMSO-d6) δ 3.36 (s, 3H), 3.92 (s, 3H), 3.93 (s, 3H), 4.20 (s, 1H), 6.90 (d, J=8.8 Hz, 2H), 6.97 (s, 4H), 7.09 (s, 1H), 7.34-7.40 (m, 4H), 7.69 (d, J=7.2 Hz, 1H), 7.77 (s, 1H), 8.15 (s, 1H), 10.36 (s, 1H), 11.00 (s, 1H).
1H NMR (400 MHz, DMSO-d6) δ 3.94 (s, 6H), 4.20 (s, 1H), 6.97 (dd, J=8.8, 2.8 Hz, 1H), 7.07 (d, J=8.8 Hz, 2H), 7.13 (s, 1H), 7.23 (d, J=2.8 Hz, 1H), 7.35 (dt, J=7.6, 1.2 Hz, 1H), 7.42 (t, J=7.6 Hz, 1H), 7.49 (d, J=8.8 Hz, 2H), 7.63 (d, J=8.8 Hz, 1H), 7.69 (d, J=7.6 Hz, 1H), 7.76 (s, 1H), 8.10 (s, 1H), 10.38 (s, 1H), 10.92 (s, 1H).
1H NMR (400 MHz, DMSO-d6) δ 3.93 (s, 3H), 3.94 (s, 3H), 4.20 (s, 1H), 6.98-7.03 (m, 4H), 7.14 (s, 1H), 7.37 (t, J=7.6 Hz, 1H), 7.40-7.48 (m, 5H), 7.69 (d, J=8.0 Hz, 1H), 7.76 (s, 1H), 8.08 (s, 1H), 10.33 (s, 1H), 10.88 (s, 1H).
The R4 and R5 listed in the Table below can be paired arbitrarily.
In one aspect, the present invention directs to a pharmaceutical composition. The pharmaceutical composition comprises an effective amount of a compound having a chemical structure (I), (II), (Ill), or (VII) below and a pharmaceutically acceptable carrier.
The R3 above is
and R4 is H or methyl group. The R5 above is
In another aspect, the present invention directs to a method of inhibiting the expression of cancerous inhibitor of PP2A. The method comprises contacting a cell with an effective amount of a compound having the chemical structure (I), (II), (III), or (VII) above.
In yet another aspect, the present invention directs to a method of treating cancer. The method comprises administrating an effective amount of a compound having a chemical structure (I), (II), (Ill), or (VII) above by a needed subject. The cancer above can be a hepatocellular carcinoma or a lung cancer.
Quinazolines have been used as a scaffold for synthesizing a variety of pharmacological compounds. For example, antagonists of human adenosine A3 receptor, inhibitors of histone lysine methyltransferase G9a, inhibitors of poly(ADP-ribose)polymerase, an inhibitor of protein kinase c isotypes, agonists of histamine H4 receptor and inhibitors of thymidylate synthase inhibitors. Some quinazoline derivatives having amino substitutes at position 4 of the quinazoline structure have been demonstrated to be inhibitors of epidermal growth factor receptor (EGFR) kinase. EGFR kinase is a receptor tyrosine kinase, which regulates cell proliferation. The numbering of quinazoline is shown below.
Some quinazoline derivatives having amino substitutes at position 4, such as erlotinb, gefitinib, and lapatinib, have been approved for clinical use in cancer patients. The chemical structure of compound 8 is similar to erlotinb (
PC9 cells (3×105 cells) were treated with erlotinb or compound 8 at 0.5, 1, 2, 4, and 8 μM in 60 mm dishes for 24 hours. 40 μg/per lane of cell lysates were analyzed by western blot. The antibodies of actin, EGFR, and p-EGFR were from Cell Signaling (Danvers, Mass.). The results of western blot are shown in
H358 cells were exposed to erlotinib or compound 8 at 1, and 5 μM for 24 hours and cell lysates were analyzed for EGFR phosphorylation. The result is shown in
The results above suggested that functional group connected to 2-position of quinazoline ring impeded nitrogen atom of quinazoline to act as a hydrogen acceptor and break the binding with EGFR. Therefore, a series of quinazoline derivatives (compounds 8-17) having a substituent at the 2 position of the quinazoline skeleton were synthesized. Another series of quinazoline derivatives (compounds 18-33) having various phenyl amine substituents at the position 2 of quinazoline were further synthesized. Moreover, the quinazoline skeleton was further simplified by using pyrimidine skeleton instead (compounds 1-7). The bioactivity of these compounds were analyzed and described below.
Pyrimidine derivatives (compounds 1-7) and quinazoline derivatives (compounds 8-33) were screened against a panel of SK-Hep-1 cell lines (a hepatocellular carcinoma (HCC) cell) for growth-inhibitory activities. MTT assay was used to measure growth inhibition. The compound concentrations causing 50% cell growth inhibition (IC50 values) were determined by interpolation from dose-response curves. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed as follow.
The effect of individual test compounds on cell viability was assessed by using the MTT in 6 replicates. Sk-Hep-1 cells were seeded and incubated in 96-well, flat-bottomed plates for 24 hours, and were exposed to various concentrations of test compounds dissolved in DMSO (final concentration, 0.1%) for 48 hours. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. The medium was removed, replaced by 200 μL of 0.5 mg/mL MTT in 10% fetal bovine serum containing DMEM (Dulbecco's Modified Eagle Medium), and cells were incubated in the carbon dioxide incubator at 37° C. for 2 hours. Supernatants were removed from the wells and the reduced MTT dye was solubilized in 100 μL/well DMSO. Absorbance at 570 nm was determined on a plate reader.
The result of MTT assay showed that IC50 values of compounds 6, 8, 13, 18-23, and 25-33 were smaller than 10 μM. Especially, compounds 31 had the smallest IC50 value (1.95 μM) among these compounds. Next, IC50 values of compounds 7, 9, 10, 14, 17, and 24 were within 10-20 μM. As for compounds 1-5, 11, 12, 15, 16, the IC50 values were all greater than 40 μM.
For compounds 8 and 9, the IC50 values were very close (7.5±0.5 v. 12.3±0.6). Compound 9 was synthesized by methylation of the substituted phenyl amine group at the position 4 of the quinazoline. This implies that hydrogen donor ability of the substituted phenyl amine group is not necessary for the induction of cell death. In addition, as methylation at the substituted phenyl amine group is known to dramatically reduce the binding ability of quinazoline with EGFR, compound 9 further provides a proof that induction of cell death of quinazoline derivatives is independent of EGFR inhibition.
For compounds 11 and 15, a hydroxyl group was introduced into the phenyl ring. The IC50 greater than 40 μM shows that no activity against SK-Hep1 cells. This suggests that hydrophobic interaction is required in this area.
For compounds 14 and 16, a phenyloxy group and a 4-cyano-phenyloxy was introduced into the phenyl ring. Compound 14 exhibited higher activity than compound 16 (15.3±0.6 μM v. >40 μM). This reveals that an electron-withdrawing group on the benzene ring is not favored for inducing cell death.
For the mono-substituted quinazoline derivatives (Embodiment 3), compound 8 exhibited the most potent growth inhibitory activity (7.5±0.5 μM). Interestingly, when the quinazoline skeleton of the mono-substituted quinazoline derivatives was simplified to the pyrimidine skeleton (Embodiment 1), no inhibitions were detected in cell growth assays. However, the compounds 6 and 7 with phenylamine di-substituents at positions 2, 4 in pyrimidine (Embodiment 2) showed more potent anti-tumor activity than the mono-substituted pyrimidine derivatives (Embodiment 1) in cell growth assays. This result suggests that a phenylamine group connected to position 2 of pyrimidine plays a crucial role in cancer-cell growth inhibitory activity.
Accordingly, a second substituted phenyl amine group was further introduced at the position 2 of the quinazoline (Embodiment 4). These derivatives showed more potent activity than mono-substituted derivatives against HCC cells. This result suggests that the second substituted phenyl amine group at the position 2 of the quinazoline plays a significant role in the cancer-cell growth inhibitory activity.
In compounds 18-33, compounds 19, 22, 27, 28, 31 and 33 exhibited higher potency with low IC50 values (2.8, 2.8, 2.60, 2.55, 1.95 and 2.65 μM, respectively) against HCC cells whereas compound 24 only showed moderate activity (14.5 μM), indicating that substitutions with hydrophobic properties, such as phenyloxy (compound 19) and benzyl groups (compound 22) exhibited higher CIP2A inhibitory activity than the hydrophilic cyanophenyl groups (compound 24). In addition, compound 23 showed much better inhibition than compound 24 (3.9 μM v. 14.5 μM), suggesting that the connection position of cyanophenyl group to phenyl ring plays an important role in CIP2A inhibition.
Next, compounds 19, and 34-36 were screened against a panel of Hep3B cell lines for growth-inhibitory activities. The result of MTT assay showed that IC50 values of compounds 19, and 34-36 were 1.28, 3.35, 3.27 and 3.38 μM, respectively. Compounds 87-90, 95 and 96 were screened against a panel of PLC5 cell lines for growth-inhibitory activities. The result of MTT assay showed that IC50 values of compounds 87-90, 95 and 96 were 8.2, 12.74, 20.2, 0.77, 0.95 and 0.61 μM.
Quinazoline derivatives have previously been evaluated as EGFR inhibitors. However, the quinazoline derivatives above had very low potency against EGFR because of the second substituted group at the position 2 of the quinazoline. However, the quinazoline derivatives above were found to be capable of repressing oncoprotein CIP2A expression and induced cell death as shown above. Therefore, it was hypothesized that quinazoline derivatives downregulate CIP2A and p-Akt, and consequently enhance cell apoptosis.
Accordingly, the pyrimidine and quinazoline derivatives above were screened by using western blot analysis for expression of CIP2A in SK-Hep1 cells. Before the western blot analysis, the SK-Hep-1 cells were maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate and 25 μg/mL amphotericin B in a 37° C. humidified incubator in an atmosphere of 5% CO2 in air. In this western blot analysis, SK-Hep1 cells were respectively treated with compounds 1-24 at a concentration of 20 μM for 24 hours. The results of inhibition effect on CIP2A expression are shown in
PLC/PRF/5 cells were exposed to compounds 88, 95 and 96 at various doses (5 and 10 μM) for 48 hours. Total lysates were subjected to CIP2A protein analysis by western blot. Actin was used as a loading control. The results of inhibition effect on CIP2A expression in a dose-dependent manner are shown in
Quantitative Polymerase Chain Reaction (qPCR) Assay
In addition, a quantitative polymerase chain reaction (qPCR) assay was used to find the correlation coefficient (R2) between the IC50 of CIP2A inhibition and the IC50 of cell growth. The correlation coefficient (R2) between the IC50 of CIP2A inhibition and the IC50 of cell growth was found to be 0.9519. This indicates that the decreased level of CIP2A induced by these derivatives is well correlated with cell toxicity.
The qPCR was performed as follow. Total RNA was isolated from SK-Hep1 cell line with TRIzol (Invitrogen). An aliquot of 2.5 μg/12.1 μL of total RNA was used as the template in the synthesis of first-strand cDNA using an oligo(dT) primer and the AMV reverse transcriptase system (Roche Diagnostics) by Thermal Cycler (RTC-200, MJ Reaserch). The method of qPCR was followed according to the method described by Ponchel et al (Ponchel, F. et al. BMC Biotechnol 2003, 3, 18). qPCR was performed using a Roche Light Cycler 480 sequence detection system (Roche Applied Science,). Thermocycling was performed in a final volume of 20 μl containing 2.5 μl of cDNA sample, 200 nM of each of the primers, and 6.5 μL of SYBR Green I master mix (Roche).
The relative differences in expression levels between genes were expressed using cycle time (Ct) values as follows: the Ct value of the gene of CIP2A was first normalized to that for GAPDH in the same sample, then the difference between the treatment and control group was calculated and expressed as an increase or decrease in cycle numbers compared with the control. Oligonucleotide sequences were as follows: CIP2A, 5′-TGG CAA GAT TGA CCT GGG ATT TGG A-3′(sense) and 5′-AGG AGT AAT CAA ACG TGG GTC CTG A-3′(antisense); GAPDH, 5′-CGA CCA CTT TGT CAA GCT CA-3′(sense) and 5′-AGG GGT CTA CAT GGC AAC TG-3′ (antisense). The following PCR conditions were used: denaturation at 95° C. for 10 min followed by 40 cycles of 94° C. for 1 min, annealing for 1 min at 60° C., and elongation for 1 min at 72° C., and a final elongation step at 72° C. for 10 min.
Next, the most potent compounds 19 and 22 were used to study whether down-regulation CIP2A and p-Akt is correlated to EGFR phosphorylation.
Cell viability, measured by MTT assay, and CIP2A expressions, analyzed by western blot, in response to compound 19 or erlotinib treatment in SK-Hep1 cell line were analyzed. SK-Hep-1 cells were treated with erlotinib and compound 19 at 2.5 and 5 μM for 24 h, respectively. The SK-Hep1 cells were then analyzed with western blot assay and MTT assay. The results are shown in
Similarly, CIP2A expressions analyzed by western blot were also performed for lung cancer cells, H358, H460, and H322 cell lines. Moreover, drug-induced apoptotic cell death was also assessed by western blot analysis of activated caspases cleaved poly(ADP-ribose) polymerase (PARP). The cleavage of PARP is explained as follow. The compound induces apoptotic signal which cleave the procaspase 3 to the active caspase 3. The activation of caspase 3 further cleaves PARP and inactivate PAPR function. The events are thought to be required in late apoptosis. Therefore, the PARP cleavage is an indicator of apoptosis.
The results are shown in
Correlation Between Down-regulating CIP2A and Suppressing p-Akt
Next, compounds 11, 19, and 22 were used to explore whether downregulation of CIP2A lead to suppression of p-Akt.
The results of
In order to know whether CIP2A is a key regulator of cell survival, we have used genetic knockdown CIP2A and then determine the cell survival with colongenic assay.
For colony formation, SK-Hep1 cells transfected with scramble siRNA or CIP2A-specific siRNA for 48 hours were seeded in triplicate onto 6 cm plates (10,000 cells per plate). After 7 days of culturing, cells were stained with crystal violet, and colonies containing more than 50 cells were counted. The obtained results are shown in
The effect of okadaic acid (OA) in compound 19 induced CIP2A inhibition is shown in
In vivo efficacy was determined in nude mice with PLC5 and Huh-7 xenog rafts.
Male NCr athymic nude mice (5-7 weeks of age) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The mice were housed in groups and maintained under standard laboratory conditions on a 12-hour light-dark cycle. They were given access to sterilized food and water ad libitum. All experimental procedures using these mice were performed in accordance with protocols approved by the Institutional Laboratory Animal Care and Use Committee of National Taiwan University. Each mouse was inoculated s.c. in the dorsal flank with 1×106 HCC cells suspended in 0.1 ml of serum-free medium containing 50% Matrigel (BD Biosciences, Bedford, Mass.). When tumors reached 150-200 mm3, mice received erlotinib at 50 mg/kg/day or compounds 8 and 19 at 20 mg/kg/day daily by oral gavage for 3 weeks. Controls received vehicle.
Tumor growth data points are reported as mean tumor volume±SE. Comparisons of mean values were performed using the independent samples t test in SPSS for Windows 11.5 software (SPSS, Inc., Chicago, Ill.).
PLC5 HCC cell line was obtained from American Type Culture Collection (ATCC; Manassas, Va.). The Huh-7 HCC cell line was obtained from the Health Science Research Resources Bank (HSRRB; Osaka, Japan; JCRB0403).
All cells were immediately expanded and frozen down such that all cell lines could be restarted every 3 months from a frozen vial of the same batch of cells. No further authentication was conducted in our lab. Cells were maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate, and 25 μg/mL amphotericin B in a 37° C. humidified incubator and an atmosphere of 5% CO2 in air. Lysates of HCC cells treated with drugs at the indicated concentrations for various periods of time were prepared for immunoblotting of PARP, P-Akt, Akt, etc.
The in vivo effects of erlotinib, compound 8, and compound 19 on HCC xenograft tumors were analyzed. Tumor-bearing mice were treated with vehicle, erlotinib (50 mg/kg/day), compound 8 (20 mg/kg/day), or compound 19 (20 mg/kg/day) p.o. daily for 3 weeks. All animals tolerated the treatments well without observable signs of toxicity and had stable body weights throughout the course of study. No gross pathologic abnormalities were noted at necropsy.
The results of xengraft study above were listed in Table below. The data listed in the Table below were the data after 21 days of treatment.
In light of foregoing, a series of pyrimidine and quinazoline-derived compounds were synthesized and their cytotoxicity was explored with interesting SAR results. Structural modifications indicated that di-phenylamine derivatives with quinazoline and pyrimidine skeletons are required for activity.
According to MTT assay, most of these derivatives had micromolar level potency against SK-Hep-1 cells. Compounds 19 and 22 showed the most potent inhibition of CIP2A expression and cell survival activity, whereas compound 4 had no activity in either assay. Furthermore, compounds 19 and 22 reduced Akt phosphorylation after repressing CIP2A, whereas compound 4 had no activity against p-Akt and CIP2A.
These results suggest selective sensitivity in response to the different substituted functional groups in quinazoline. Moreover inhibition of CIP2A expression correlated with cytotoxicity in SK-Hep-1 cells upon drug treatment. Testing of compounds 19 and 22 in an in vivo HCC model shows that compounds 19 and 22 are capable of significantly reducing the growth of tumor in sensitive PLC5 tumors.
All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, each feature disclosed is one example only of a generic series of equivalent or similar features.
| Number | Date | Country | |
|---|---|---|---|
| 61700923 | Sep 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14026545 | Sep 2013 | US |
| Child | 14631248 | US |
