Patent Grant
7767707
This invention is related to novel fused pyrazolyl compounds, and the use thereof in inhibiting cancer cell growth.
U.S. Pat. No. 7,378,532 B2 discloses novel fused pyrazolyl compounds, and the use thereof in inhibiting cancer cell growth, details of which are incorporated herein by reference.
There is a need for searching derivatives of the novel fused pyrazolyl compounds disclosed in U.S. Pat. No. 7,378,532 B2, which possess pharmaceutically useful properties.
An objective of the present invention is to provide novel fused pyrazolyl compounds having an aminoalkylcarbonyl group, and the use thereof in inhibiting cancer cell growth.
Another objective of the present invention is to provide a pharmaceutical composition comprising an effective amount of a novel fused pyrazolyl compound having an aminoalkylcarbonyl group.
In order to accomplish the aforesaid objectives of the present invention a fused pyrazolyl compound synthesized according to the present invention has the following formula:
wherein A is
in which n is 0, 1, 2, or 3;
Ar1 is benzene, thiophene or furan;
Ar2 is furyl; and
Ar3 is phenyl; and
R1 and R2 independently are hydrogen, halogen or —(CH2)mORe;
R3 is hydrogen or alkyl;
R4 is —(CH2)r-A1, wherein r is an integer of 1-5, and A1 has a formula of —O—C(O)—(CRcH)q—NRc′Rd′;
R5 and R6 independently are hydrogen, halogen, or alkyl, or R5 and R6 together are —O(CH2)mO—;
Rc is H, halogen, nitro, cyano, alkyl, or aryl;
Re is H, alkyl, or aryl;
Rc′ and Rd′ independently are H, alkyl, or aryl;
m is 0, 1, 2, 3, 4, 5, or 6; and
q is 1, 2, 3, 4, 5, or 6; or
a salt thereof.
The present invention also provide a pharmaceutical composition comprising an effective amount of the novel fused pyrazolyl compound having the above formula.
Preferably, Ar2 is 2′-furyl.
Preferably, R3 is H and R4 is bonded to position 5 of furyl.
Preferably, Rc′ and Rd′ independently are H or alkyl. More preferably, Rc′ and Rd′ are both H.
Preferably, Rc is H.
Preferably, q is 1 or 2.
Preferably, r is 1.
Preferably, Ar1 is benzene.
Preferably, A is —CH2-phenyl.
Preferably, the compound has the following structure:
wherein R is
Abbreviation:
Glycine (Gly) (0.5 g, 6.66 mmole) was dissolved in 10% NaCO3 (14 ml) under stirring in a 50-ml flask. To the resulting solution, which had been put into an ice bath, 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) (1.72 g, 6.66 mmol) in dioxane (12 ml) was gradually added. The reaction mixture was stirred at room temperature for 4 hours, and water (150 ml) was then added. The aqueous phase layer was separated from the reaction mixture and stripped with ether three times (75 ml×3). The stripped aqueous layer was acidified with 2N HCl aqueous solution to a pH value of 2, followed by extraction with ethyl acetate three times (75 ml×3). The organic phase layer was recovered and concentrated to obtain crude product of 1.70 g. The crude product was recrystallized in a mixed solvent of ethyl acetate hexane=1:2 (30 ml), and white solid denoted as Fmoc-Gly was obtained after filtration at a reduced pressure. Yield: 1.69 g (85%).
NMR (CD3OD) δ 3.84 (s, 2H, CH2COOH), 4.24 (t, 1H, J=7.3 Hz, Fmoc-CH), 4.35 (d, 2H, J=7.0 Hz, Fmoc-CH2), 7.31 (t, 2H, J=7.0 Hz, Ar—H), 7.40 (t, 2H, J=7.3 Hz, Ar—H), 7.68 (d, 2H, J=7.5 Hz, Ar—H), 7.80 (d, 2H, J=8.0 Hz, Ar—H).
In a 50 ml flask Fmoc-Gly (488 mg, 1.64 mmole), HBTU (621 mg, 1.64 mmole), DMAP (200 mg, 1.64 mmole) and ACN (10 ml) were added and stirred until a solution was formed. To the solution YC-1 (500.0 mg, 1.64 mmole) was added and stirred at room temperature for two days. Fmoc-Gly (244 mg, 0.82 mmole), HBTU (311 mg, 0.82 mmole), DMAP (100 mg, 0.82 mmole) and ACN (10 ml) were added as supplements, and then stirred at room temperature for another three days. The reaction mixture was filtered to obtain a white crude product (1.21 g). The crude product was dissolved thoroughly in a mixed solvent of methanol: CH2Cl2=1:9 (10 ml), and CAN (10 ml) was added to form a precipitate. White solid 3 was obtained after filtration. Yield: 662 mg (69%).
NMR (CD3OD) δ 4.04 (d, 2H, J=5.5 Hz, CH2NH), 4.21 (t, 1H, J=7.0 Hz, Fmoc-CH), 4.38 (d, 2H, J=3.5 Hz, Fmoc-CH2), 5.27 (s, 2H, CH2-furan), 5.63, (s,2H, CH2-Ph), 6.59 (d, 1H, J=3.5 Hz, furan-H), 6.87 (d, 1H, J=3.5 Hz, furan-H), 7.18-7.38 (m,12H, Ar—H), 7.57 (d, 2H, J=8.0 Hz, Ar—H), 7.75 (d, 2H, J=8.0 Hz, Ar—H), 8.05(d, 1H, J=8.0 Hz, Ar—H)
Compound 3 (413 mg, 0.71 mmole) was dissolved in CH2Cl2 (10 ml) in a 50 ml flask. To the solution DBU (0.1 ml, 0.71 mmole) was added dropwise, and then stirred at room temperature for 20 minutes. Water (30 ml) was added, and the aqueous phase layer was separated and extracted with CH2Cl2 (30 ml). The organic phase layer was recovered and stripped with H2O (50 ml). The stripped organic phase layer was concentrated and dissolved in ethyl acetate (5 ml) thoroughly. To the solution, H3PO4/ethyl acetate=1/10 (2 ml) was added dropwise and stirred for 30 minutes. Yellowish solid (Compound 5) was obtained after filtration at a reduced pressure.
NMR (CD3OD) δ 3.88 (s, 2H, CH2NH), 5.41 (s, 2H,CH2-furan), 5.70 (s, 2H, CH2-Ph), 6.75 (d, 1H, J=3.5 Hz, furan-H), 7.00 (d, 1H, J=3.5 Hz, furan-H), 7.23-7.33(m,6H, Ar—H), 7.46 (t, 1H, J=7.8 Hz, Ar—H), 7.56 (d, 2H, J=8.5 Hz, Ar—H), 8.15(d, 1H, J=8.0 Hz, Ar—H)
β-Alanine (β-Ala) (0.5 g, 6.61 mmole) was dissolved in 10% NaCO3 (12 ml) under stirring in a 50 ml flask. To the resulting solution, which had been put into an ice bath, Fmoc-Cl (1.45 g, 5.61 mmol) in dioxane (10 ml) was gradually added. The reaction mixture was stirred at room temperature for 4 hours, and water (80 ml) was then added. The aqueous phase layer was separated from the reaction mixture and stripped with ether three times (75 ml×3). The stripped aqueous layer was acidified with 2N HCl aqueous solution to a pH value of 2, followed by extraction with ethyl acetate three times (75 ml×3). The organic phase layer was recovered and concentrated to obtain crude product of 1.50 g. The crude product was recrystallized in a mixed solvent of ethyl acetate:hexane=1:2 (30 ml), and white solid denoted as Fmoc-β-Ala was obtained after filtration at a reduced pressure. Yield: 1.41 g (81%).
NMR (CDCl3) δ 2.60 (t, 2H, NHCH2CH2COOH), 3.47 (d, 2H, J=7.0 Hz, NHCH2CH2COOH), 4.19 (t, 1H, J=6.0 Hz, Fmoc-CH), 4.39 (d, 2H, J=7.0 Hz, Fmoc-CH2), 7.29 (t, 2H, J=7.3 Hz, Ar—H), 7.38 (t, 2H, J=7.3 Hz, Ar—H), 7.56 (d, 2H, J=7.0 Hz, Ar—H), 7.74 (d, 2H, J=7.5 Hz, Ar—H).
In a 50 ml flask Fmoc-β-Ala (488 mg, 1.64 mmole), HBTU (621 mg, 1.64 mmole), DMAP (200 mg, 1.64 mmole) and ACN (10 ml) were added and stirred until a solution was formed. To the solution YC-1 (500.0 mg, 1.64 mmole) was added and stirred at room temperature for two days. Fmoc-β-Ala (244 mg, 0.82 mmole), HBTU (311 mg, 0.82 mmole), DMAP (100 mg, 0.82 mmole) and ACN (10 ml) were added as supplements, and then stirred at room temperature for another three days. The reaction mixture was filtered to obtain a yellowish crude product (1.05 g). The crude product was dissolved thoroughly in a mixed solvent of methanol:CH2Cl2=1:9 (5 ml), and ACN (10 ml) was added to form a precipitate. White solid 4 was obtained after filtration. Yield: 690 mg (70%).
NMR (CDCl3) δ 2.60 (t, 2H, NHCH2CH2), 3.48 (d, 2H, J=2.8 Hz, NHCH2CH2), 4.21 (t, 1H, J=7.0 Hz, Fmoc-CH), 4.33 (d, 2H, J=3.5 Hz, Fmoc-CH2), 5.23 (s, 2H, CH2-furan), 5.61, (s,2H, CH2-Ph), 6.58 (d, 1H, J=3.0 Hz, furan-H), 6.88 (d, 1H, J=3.5 Hz, furan-H),7.17-7.37 (m, 12H, Ar—H), 7.53 (d, 2H, J=7.5 Hz, Ar—H), 7.72 (d, 2H, J=7.5 Hz, Ar—H), 8.05(d, 1H, J=8.0 Hz, Ar—H).
Compound 4 (363 mg, 0.61 mmole) was dissolved in CH2Cl2 (10 ml) in a 50 ml flask. To the solution DBU (0.08 ml, 0.61 mmole) was added dropwise, and then stirred at room temperature for 20 minutes. Water (30 ml) was added, and the aqueous phase layer was separated and extracted with CH2Cl2 (30 ml). The organic phase layer was recovered and stripped with H2O (50 ml). The stripped organic phase layer was concentrated and dissolved in ethyl acetate (5 ml) thoroughly. To the solution, H3PO4/ethyl acetate=1/10 (2 ml) was added dropwise and stirred for 30 minutes. Yellowish solid (Compound 6) was obtained after filtration at a reduced pressure.
NMR (CD3OD) δ 2.79 (t, 2H, J=6.5 Hz, CH2CH2NH), 3.20 (t, 2H, J=6.5 Hz, CH2CH2NH), Ar—H), 5.32 (s, 2H, CH2-furan), 5.70, (s,2H, CH2-Ph), 6.70 (d, 1H, J=3.5 Hz, furan-H), 6.99 (d, 1H, J=3.0 Hz, furan-H), 7.23-7.33(m,6H, Ar—H), 7.46 (t, 1H, J=3.5 Hz, Ar—H), 7.56 (d, 2H, J=8.5 Hz, Ar—H), 8.14(d, 1H, J=8.5 Hz, Ar—H).
The target compound 5, 6 and related succinate (A), glutamate (B) derivatives were evaluated in a MTT assay against NCI H226 cells, and the results are shown in Table 1. All of the tested compounds exhibited significant cytotoxicity.
1. Cell Culture and Treatment
The NCI H-226 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBCO/BRL), penicillin (100 unit/mL)/streptomycin (100 μg/mL) (GIBCO/BRL) and 1% L-glutamine (GIBCO/BRL) at 37° C. in a humidified atmosphere containing 5% CO2. Logarithmically growing tumor cells were used for all experiments.
2. Cytotoxicity Assay
The cytotoxicity was assessed using MTT assay.1 NCI H-226 cells (4×104) were treated separately with compounds A, B, 5, and 6 for 72 h. After treatment, the cells were collected, washed with cold PBS, and then added 10 μL of MTT solution (5 mg/mL)(Sigma) with 50 μL of cells suspension in HBSS into 96-well plate and incubated them at 37° C. in the dark for 2 h. Treatment of living cells with MTT produces a dark blue formazan product, whereas no such staining is observed in dead cells. The formazan product was dissolved by adding 140 μL DMSO and then the absorbance was measured on an ELISA reader at a best wavelength of 570 nm.
3. Statistic Evaluation
Values are expression as the mean ±S.D. of three independent experiments. Student's t tests were used to assess the statistical significance of the differences, with “p” values of less than 0.05 being considered statistically significant.
This patent application claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 60/996,515, filed Nov. 21, 2007.
| Number | Name | Date | Kind |
|---|---|---|---|
| 7378532 | Kuo et al. | May 2008 | B2 |
| Number | Date | Country | |
|---|---|---|---|
| 20090131681 A1 | May 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60996515 | Nov 2007 | US |
