Substituted 2-phenyl-4-quinolone-3-carboxylic acid compounds and their use as antitumor agents

Substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives and their salts were synthesized. The results of preliminary screening revealed that these compounds are potent in killing solid tumor cancers.

FIELD OF THE INVENTION

The present invention relates to a series of substituted 2-phenyl-4-quinolone-3-carboxylic acid compounds; and in particular to their uses in treating human cancers.

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

The present invention synthesizes a substituted 2-phenyl-4-quinolone-3-carboxylic acid compound having the formula (I) and its salt:
wherein

R2′, R3′, R4′, R5′ and R6′ independently are H, (CH2)nCH3, (CH2)nYH, Y(CH2)nCH3, Y(CH2)nYH, Y(CH2)nNR8R9, X, or NR8R9, wherein n is an integer of 0˜4, Y is O or S, X is F, Cl, or Br, and R8and R9independently are H, (CH2)nYH, (CH2)nN(CnH2n+1) or (CH2)nCH3, wherein n and Y are defined as above, and m is an integer of 0˜4;

R is H.

Preferably, R2′, R4′, R5′ and R6′ are H; and R3′ is H, Y(CH2)nCH3, NH(CH2)nCH3or X, wherein n, Y and X are defined as above; R3is H; and R4is Y(CH2)nCH3, or X, wherein n and X are defined as above. More preferably, R3′ and R4independently are methoxy, F or Cl. Most preferably, R3′ is F and R4is methoxy.

Preferalby, the salt of the compound (I) is a quaternary ammonium salt or pharmaceutically acceptable inorganic salt.

The present invention also discloses a pharmaceutical composition for the killing of solid tumor cells, which comprises a therapeutically effective amount of a substituted 2-phenyl-4-quinolone-3-carboxylic acid compound of the formula (I) as set forth in any one of claim1to claim4or a pharmaceutically acceptable salt thereof, as an active ingredient, in admixture with a pharmaceutically acceptable carrier or diluent for the active ingredient, wherein the solid tumor cells comprise human breast cancer, CNS cancer, colon cancer, lung cancer, melanoma, ovarian cancer, renal cancer, stomach cancer, prostate cancer, ileocecal carcinoma, glioblastoma, bone cancer, or epidermoid carcinoma of the nasopharynx.

The present invention further discloses a method of killing solid tumor cancers comprising administering a therapeutically effective amount of a substituted 2-phenyl-4-quinolone-3-carboxylic acid compound of the formula (I) as set forth in any one of claim1to claim8to a subject having solid tumor cells, wherein the solid tumor cells comprise human breast cancer, CNS cancer, colon cancer, lung cancer, melanoma, ovarian cancer, renal cancer, stomach cancer, prostate cancer, ileocecal carcinoma, glioblastoma, bone cancer, or epidermoid carcinoma of the nasopharynx.

DETAILED DESCRIPTION OF THE INVENTION

The synthesis of key intermediates, 3-ethoxy carbonyl derivatives of substituted 2-phenyl-4-quinolones (VII) was illustrated in the following Schemes 1 and 2. At first, the reaction of substituted anilines (I) with substituted benzyl chlorides (II) in toluene, at room temperature, yielded the corresponding N-(substituted phenyl)-substituted benzamides (III), subsequent chlorination of compounds III with PCl5afforded the carboximidoyl chlorides (IV) that, without further purification, where then treated with sodium diethylmalonate in toluene to give their corresponding N-[1-(substituted phenyl)-2-diethoxycarbonylvinyl]-N-(4-substituted phenyl)amines (V).

In the next step, these intermediates V were used without purification and were thermally cyclized into their corresponding ethyl substituted 2-phenyl-4-quinolone-3-carboxylates (VII). At the same time, substituted 2-phenyl-4-quinolones (VIII-1˜4) were obtained, during the purification of products VII-1˜3 and VII-6, as their de-ethoxycarboxylate derivatives. This indicated that mono-ethoxycarbonyl vinyl derivatives (VI) might have also been produced in side reaction during the preparation of intermediates V-1˜3 and V-6. Indeed, the mono-ethoxycarbonyl vinyl derivative (VI-1) was isolated. Thermal cyclization of the mono-ethoxycarbonyl vinyl intermediates (VI-1) also led to the formation of 3′-chloro-6-fluoro-2-phenyl-4-quinolone (VIII-1).

The synthetic routes from key intermediates VII to the target compounds IX, X were illustrated in the following Scheme 3. As shown, compounds VII were first hydrolyzed into carboxylic acids (IX) which were subsequently treated with tromethamine in mixed butyl chloride/methanol solvent to afford the corresponding salts (X).
Experimental Section

Melting points were determined on a Yanaco MP-500D melting point apparatus and are uncorrected. IR spectra were recorded on Shimadzu IR-440 and Nicolet Impact 400 FT-Irspecrophotometers as KBr pellets, NMR spectra were obtained on a Bruker Advance DPX-200 FT-NMR spectrometer in DMSO-d6. The following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad MS spectra were measured with an HP 5995 GC-MS instrument. The UV spectra were recorded on a Shimadzu UV-160A UV-vis recording spectrophotometer as methanolic solutions. Elemental analyses (C, H, N) were performed at China Medical College, Taiwan, and the results were within ±0.4% of the calculated values.

PCl5(2.24 g, 0.01 mol) was added into compound III-1 (2.50 g, 0.01 mol) and the mixture was heated to 110° C., stirred for additional 1 h, and then evaporated under vacuum to yield carboximidoyl chloride (IV-1) as viscous liquids.

Meanwhile, diethyl malonate (4.8 g, 0.03 mol) was introduced dropwise into the solution of Na0(0.72 g, 0.03 mol) in absolute ethanol (50 ml), at 20±2° C. The mixture was stirred for 1 h at 50±2° C. Subsequent removal of ethanol under vacuum afforded sodium diethyl moloante as gel.

Then compound IV-1 was dissolved in toluene (20 ml) and added into the suspension of sodium diethyl moloante in toluene (20 ml). The resulted mixture was allowed to react 4 hr at 110° C. After filtration the filtrate was concentrated and extracted with ether, washed with water, dried over MgSO4and concentrated under vacuum to yield V-1 and VI-1 as yellowish, viscous liquid.

Without further purification, the viscous liquid was heated for 4 h at 170° C. to give yellowish solid that was purified further by column chromatography (silica gel, 100:1 CHCl3-EtOH) and recrystallized from CHCl3-EtOH to produce compounds VII-1 (1.21 g, 35%) and VII-1 (0.41 g, 15%).

Following the same synthetic route of method A for preparating compounds V-1 and VI-1. The yellowish viscous liquid was chromatographed on silica gel with CHCl3as eluant to give N-[1-(3-chlorophenyl)-2,2-diethoxycarbonylvinyl]-N-(4-fluoro phenyl) amine (V-1) (1.84 g, 47%) and N-[1-(3-chloropheny)-2-ethoxycarbonylvinyl]-N-(4-fluorophenyl)amine (VI-1) (1.09 g, 34%). Compound V-1 (1.17 g, 3 mmol) was heated for 4 hr at 170° C. to give yellowish solid, that was purified by column chromatography (silica gel, CHCl3-EtOH) to product compound VII-1 (0.84 g, 81%). From compound VI-1 (0.96 g, 3 mmol), the same procedure used for the synthesis of compound VII-1 gave product VIII-1 (0.60 g, 73%).

Evaluation Against Human Cancer Cell Line Panel Cell Lines.

Measurements of Cell Growth Inhibition and Data Analysis.

Results

Preliminary Screening of Antitumor Activity

Preliminary screening of antitumor activity was based on the cytotoxicity tested in human long cancer (A-549) cell lines. As shown in Table 1, 3-ethoxycarbonyl derivatives of substituted 2-phenyl-4-quinolones (VII-5, VII-7˜10) did not show any significant activity. Among 3-carboxylic acid derivatives (IX), the cytotoxicity was not remarkable when the m-position of 2-phenyl group was substituted by OCH3group (IX-3, 7, 11). ED50values were greater than 20 μg/mL. On the contrary, the cytotoxicity increased greatly when the m-position was substituted by F atom (IX-2, 5, 8), 3′-fluoro-6-methoxy-2-phenyl-4-quinolone-3-carboxylic acid (IX-8) was the most potent compound with an ED50of 0.19 μg/mL. The replacement of F atom with Cl atom on the m-position (IX-1, 6, 9) caused the activity to decrease, which nevertheless were more active then the m-methoxy derivatives (IX-3, 7, 10). The antitumor activity was not good, if there was no substitution on the m-position of 2-phenyl group (IX-11).

The cytotoxicity of the tromethamine salts of compounds IX was presented in Table 2, the SARs were similar to the carboxylic acids (IX), namely, 3′-fluoro-derivatives (X-2, 5, 8) had the best activity followed by 3′-chloro-derivatives (X-1, 6, 9), and 3′-methoxy derivatives (X-3, 7, 10) had the weakest activity. Among these salts, the corresponding salt (X-8) of compound IX-8 again showed the best antitumor activity with an ED50of 2.4 μg/mL.

The SARs of the described above compounds IX and X revealed that the size of the substituents on the m-position of 2-phenyl group affected its activity greatly. We conjectured that the coplanarity between 2-phenyl group and quinolone ring had a strong influence in the activity. Meanwhile the results from preliminary screening indicated that compound IX-8 and X-8 had the best cytotoxicity against A-549. These two compounds were further tested on other 8 human tumor cell lines (HTCL) to evaluate their activities on the replication of these cell lines.

TABLE 2The Cytotoxicity of ethyl 3′,6-disubstituted2-phenyl-4-quinolone-3-carboxylic acid tromethamine salts(X-1˜3, X-4˜11) against lung cancer cell line (A-549).CompoundR1R2ED50(μg/mL)aX-1FCl17.6X-2FF4.3X-3FOCH3NAX-5ClF10.9X-6ClCl>20X-7ClOCH3>20X-8OCH3F2.4X-9OCH3Cl17.6X-10OCH3OCH3NAX-11OCH3H>20aCytotoxicity expressed as ED50against A-549 (the concentration of compounds that causes a 50% reduction in adsorbance at 562 nm relative to untreated cells using the SRB assay).
The Activity of Compounds IX-8 and X-8 Against HTLC Replication

Antitumor activity of compounds IX-8 and X-8 was further tested on the following 8 cancer cell lines: CAKI, HOS, KB, KB-VIN, SK-MEL-2, U87-MG, HCT-8 and IA9. As shown in Table 3, these two compounds demonstrated marked inhibition against almost all of these 8 cancer cell lines. It is worth mentioning that these two compounds were quite active against human ovarian cancer cells (IA9), which were known to have a high level of drug resistant P-glycoprotein. IC50of compound IX-8 against these cancer cells is only 0.03 μg/mL. Thus, this compound is really worth developing, particular against drug resistant ovarian cancer.

On the other hand, compound IX-8 was equally active against vincristine sensitive and resistant KB cells. It is also worth developing, against vincristine-resistant epidermal carcinoma of the nasopharynx.

Evaluation of Compound IX-8 Against Human Cancer Cell Line Panel

Our previous screening of cytotoxicity indicated that compound IX-8 possessed excellent antitumor activity. It was further evaluated against human caner cell line panel which includes 5 breast cancer, 6 CNS cancer, 5 colon cancer, 7 lung cancer, 1 melanoma, 5 ovarian cancer, 2 renal cancer, 6 stomach cancer, and 2 prostate cancer cell lines. These various cancer cells were cultured in 96 wells plate. Compound IX-8 was employed at 5 different dosages between 10−4and 10−8M, and cultured with the cells for 48 hr. Cells were stained with Sulforhodamine B for the assay of cell number. Dose response curves were obtained from computer analysis. GI50was obtained from such curves.

Compound IX-8 was active against most cancer cell lines. The MG-MID (mean growth midpoint) of log GI50is −6.22M. It was most effective against HGC2998 and the log GI50is −6.86M; next came against OVCAR-4 with a log GI50of −6.76 M.

TABLE 3The activity of compounds IX-8 and X-8 against HTCL replicationED50(μg/mL)aCompoundCAKlbHOSbKBbKB-VINbSK-MEL-2bU87-MGbHCT-8bIA9bIX-810.00.140.130.130.161.500.140.03X-8NT5.0NTNT2.05.01.200.88aCell line/mean ED5gin mcg/mL. (Huplieated varied no more than 5%)Note:if inhibition is less than 50% at 10 mcg/mL then inhibition values observed are the bracketed values.NT = Not testedbRenal cancer (CAKI), bone (HOS), epidermoid carcinoma of the nasopharynx (KB), Vincristine-resBtant epidermoid carcinoma of the nasopharynx (KB-VIN), melanoma (SK-MEL-2), glioblastoma (C187-MG), ileocecal Carcinoma (HCT-8) and human ovarian cancer (IA9) cell lines.