Conjugate of benzofuranone and indole or azaindole, and preparation and uses thereof

Abstract

The present invention relates to an oxo indirubin or isoindigo derivative, an oxo aza indirubin or isoindigo derivative, and their optical isomers, racemes, cis/trans isomers and pharmaceutically acceptable salts, which can be used for preparing a drug for treating or preventing diseases such as glucose metabolic disorder, inflammatory or autoimmune disease, neurodegenerative disease, a mental illness, tissue proliferation disease or tumors.

Claims

1. A compound of formula I-1 or formula II-1, or an enantiomer, a racemate, a cis-trans isomer or any combination thereof, or a pharmaceutically acceptable salt thereof: ##STR00019## wherein, R.sup.1 is selected from the group consisting of H, D, or the following groups that may be unsubstituted or substituted by 1 to 3 substituents: C.sub.1C.sub.6 alkyl, aryl, aralkyl, acyl, glycosyl protected by acyl, or glycosyl; wherein said substituents are selected from the group consisting of halogen, hydroxyl, C.sub.1C.sub.3 alkyl, nitro or amino; R.sup.2, R.sup.2, R.sup.4 and R.sup.5 are H; R.sup.3 is selected from the group consisting of H, halo, and C.sub.1C.sub.4 alkyl; R.sup.4 is selected from the group consisting of H, halo, and C.sub.1C.sub.4 alkyl; R.sup.5 is selected from the group consisting of H, halo, and C.sub.1C.sub.4 alkyl; R.sup.3 is selected from the group consisting of H, halo, C.sub.1C.sub.4 alkoxyl and C.sub.1C.sub.4 alkyl; and R is selected from the group consisting of oxygen, sulfur, selenium, or NOR.sup.6 group, wherein R.sup.6 is H or C.sub.1C.sub.6 straight-chain or branched-chain alkyl.

2. The compound of claim 1, wherein in formula I-1, R.sup.1 is selected from the group consisting of H, D, C.sub.1C.sub.6 alkyl, phenyl, phenyl-CH.sub.2, arabinosyl, xylosyl, ribosyl, mannosyl, and glucosyl; R.sup.3 is selected from the group consisting of H, halo, and C.sub.1C.sub.4 alkyl; R.sup.4 is selected from the group consisting of H and halo; R.sup.5 is selected from the group consisting of H and halo; R.sup.3 is selected from the group consisting of H and halo; and R is selected from the group consisting of oxygen and NOR.sup.6 group, wherein R.sup.6 is H or C.sub.1C.sub.6 straight-chain or branched-chain.

3. The compound of claim 1, wherein the compound is selected from the group consisting of the following Compound ID Nos. 1-60: TABLE-US-00009 Compound ID No. R.sup.1 R.sup.3 R.sup.4 R.sup.5 R.sup.3 R 1 H H H H H O 2 H H H F H O 3 CH.sub.2CH.sub.3 H H H H O 4 CH.sub.2CH.sub.3 H H F H O 5 CH.sub.2CH.sub.3 Cl H H H O 6 CH.sub.2CH.sub.3 H Cl H H O 7 CH.sub.2CH.sub.3 CH.sub.3 H H H O 8 i-C.sub.3H.sub.7 H H H H O 9 i-C.sub.3H.sub.7 F H H H O 10 i-C.sub.3H.sub.7 H H F H O 11 i-C.sub.3H.sub.7 Cl H H H O 12 i-C.sub.3H.sub.7 H Cl H H O 13 i-C.sub.3H.sub.7 Me H H H O 14 CH.sub.2Ph H H H H O 15 CH.sub.2Ph F H H H O 16 CH.sub.2Ph Cl H H H O 17 CH.sub.2Ph Me H H H O 18 CH.sub.2CH.sub.3 F H H H O 19 CH.sub.2CH.sub.3 H H H Cl O 20 CH.sub.2CH.sub.3 H H F Cl O 21 CH.sub.2CH.sub.3 Cl H H Cl O 22 CH.sub.2CH.sub.3 H Cl H Cl O 23 i-C.sub.3H.sub.7 Cl H H Cl O 24 i-C.sub.3H.sub.7 H Cl H Cl O 25 H H H H H NOH 26 H H H F H NOH 27 CH.sub.2CH.sub.3 H H H H NOH 28 CH.sub.2CH.sub.3 H H F H NOH 29 CH.sub.2CH.sub.3 Cl H H H NOH 30 CH.sub.2CH.sub.3 H Cl H H NOH 31 CH.sub.2CH.sub.3 CH.sub.3 H H H NOH 32 i-C.sub.3H.sub.7 H H H H NOH 33 Ribosyl H H H H NOH 34 i-C.sub.3H.sub.7 H H F H NOH 35 i-C.sub.3H.sub.7 Cl H H H NOH 36 i-C.sub.3H.sub.7 H Cl H H NOH 37 i-C.sub.3H.sub.7 Me H H H NOH 38 CH.sub.2Ph H H H H NOH 39 CH.sub.2Ph F H H H NOH 40 CH.sub.2Ph Cl H H H NOH 41 CH.sub.2Ph Me H H H NOH 42 CH.sub.2CH.sub.3 F H H H NOH 43 CH.sub.2CH.sub.3 H H H Cl NOH 44 CH.sub.2CH.sub.3 H H F Cl NOH 45 CH.sub.2CH.sub.3 Cl H H Cl NOH 46 CH.sub.2CH.sub.3 H Cl H Cl NOH 47 i-C.sub.3H.sub.7 Cl H H H NOH 48 i-C.sub.3H.sub.7 H Cl H Cl NOH 49 H H H H H NOCH.sub.3 50 H H H F H NOCH.sub.3 51 CH.sub.2CH.sub.3 H H H H NOCH.sub.3 52 CH.sub.2CH.sub.3 H H F H NOCH.sub.3 53 Glucosyl Cl H H H NOCH.sub.3 54 i-C.sub.3H.sub.7 H Cl H H NOCH.sub.3 55 i-C.sub.3H.sub.7 Me H H H NOCH.sub.3 56 CH.sub.2Ph H H H H NOCH.sub.3 57 CH.sub.2Ph F H H H NOCH.sub.3 58 CH.sub.2Ph Cl H H H NOCH.sub.3 59 CH.sub.2CH.sub.3 F H H H NOCH.sub.3 60 i-C.sub.3H.sub.7 H Cl H Cl NOCH.sub.3.

4. The compound of claim 1, wherein the compound is selected from the group consisting of the following Compound ID Nos. 91-120: TABLE-US-00010 Compound ID No. R.sup.1 R.sup.3 R.sup.4 R.sup.5 R.sup.3 91 H H H H H 92 H F H H H 93 CH.sub.3 H H H H 94 CH.sub.3 H H Cl H 95 CH.sub.3 Cl H H H 96 CH.sub.3 H Cl H H 97 CH.sub.3 CH.sub.3 H H H 98 CH.sub.3 H H F H 99 CH.sub.3 F H H H 100 CH.sub.2CH.sub.3 H H H H 101 CH.sub.2CH.sub.3 H H H OCH.sub.3 102 CH.sub.2CH.sub.3 Cl H H H 103 CH.sub.2CH.sub.3 H Cl H H 104 CH.sub.2CH.sub.3 F H H H 105 CH.sub.2CH.sub.3 H H F H 106 i-C.sub.3H.sub.7 H H H H 107 i-C.sub.3H.sub.7 Me H H H 108 i-C.sub.3H.sub.7 F H H H 109 i-C.sub.3H.sub.7 H H F H 110 i-C.sub.3H.sub.7 Cl H H H 111 Triacetylribosyl Cl H H H 112 Glucosyl H H H H 113 i-C.sub.3H.sub.7 Cl H H Cl 114 i-C.sub.3H.sub.7 H Cl H Cl 115 CH.sub.2Ph H H H H 116 CH.sub.2Ph H H H OCH.sub.3 117 CH.sub.2Ph F H H H 118 CH.sub.2Ph H H F H 119 CH.sub.2Ph Cl H H H 120 CH.sub.2Ph H Cl H H.

5. The compound of claim 1, wherein the pharmaceutically acceptable salt is a salt formed with an inorganic acid or an organic acid; the inorganic acid includes hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid; the organic acid includes methanoic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1, 5), asiatic acid, carbenoxolone, glycyrrhetinic acid, tartaric acid, oleanolic acid, crataegolic acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, lactic acid, salicylic acid, benzoic acid, butylcarboxylic acid, diethylacetic acid, malonic acid, amber acid, fumaric acid, pimelic acid, hexanedioic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, para-toluenesulfonic acid, citric acid and amino acid.

6. A pharmaceutical composition comprising (a) the compound of claim 1, or enantiomer, racemate, cis-trans isomer or any combination thereof, or pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers.

7. The pharmaceutical composition of claim 6, wherein dosage form of said composition is low capacity injection, medium capacity injection, high capacity injection, dried powder injection, emulsion for injection, tablet, pill, capsule, paste, cream, patch, liniment, powder, spray, implant, drop, suppository, ointment, various nano preparations, or liposomes.

8. The compound of claim 1, wherein in formula II-1, R.sup.1 is selected from the group consisting of H, D, C.sub.1C.sub.6 alkyl, phenyl, phenyl-CH.sub.2, arabinosyl, xylosyl, ribosyl, mannosyl, and glucosyl; R.sup.3 is selected from the group consisting of H, halo, and C.sub.1C.sub.4 alkyl; R.sup.4 is selected from the group consisting of H and halo; R.sup.5 is selected from the group consisting of H and halo; and R.sup.3 is selected from the group consisting of H, halo and C.sub.1C.sub.4 alkoxyl.

9. The pharmaceutical composition of claim 6 comprising (a) the compound of claim 2, or enantiomer, racemate, cis-trans isomer or any combination thereof, or pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers.

10. The pharmaceutical composition of claim 6 comprising (a) the compound of claim 3, or enantiomer, racemate, cis-trans isomer or any combination thereof, or pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers.

11. The pharmaceutical composition of claim 6 comprising (a) the compound of claim 4, or enantiomer, racemate, cis-trans isomer or any combination thereof, or pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers.

12. The pharmaceutical composition of claim 6 comprising (a) the compound of claim 5, or enantiomer, racemate, cis-trans isomer or any combination thereof, or pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers.

13. The pharmaceutical composition of claim 6 comprising (a) the compound of claim 8, or enantiomer, racemate, cis-trans isomer or any combination thereof, or pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers.

Description

FIGURE LEGENDS

(1) FIG. 1. The docking results of the ATP-binding domain of CDK2 and the compound 75 or 7-azaindirubin.

(2) The dotted line in green represents the hydrogen bonding interaction. Oxygen (0) is labeled as red; nitrogen (N), as blue; hydrogen (H), as white; and carbon (C), as grey. (a) The H at position N1 and the O at position 2 in both compounds form hydrogen bonding with their respective amino acid residues in the ATP binding pocket of CDK2. The docking score is 99.294 for the compound 75 (right); 102.251, for 7-azaindirubin (left). (b) Similarity of interactions between the ATP-binding pocket with the compound 75, and with 7-azaindirubin. The docking software used was Discovery Studio. The docking method was LibDock. The three dimensional structure of CDK2 was from CDK2/cyclinA co-crystallized with sulfonyl-indirubin.

(3) FIG. 2. The docking results of the SH2 domain of STAT3 and the compound-93 or N-methyl-isoindigo.

(4) The dotted lines in green in the left of the figure represent the hydrogen bonding interaction. Oxygen (O) is labeled as red; nitrogen (N), as blue; hydrogen (H), as white; and carbon (C), as yellow. (d1): the H at position N-1 and the O at position 2 of N-methyl-isoindigo form hydrogen bonds with respective residues of amino acids in the binding pocket in the SH2 domain of the human STAT3 protein. LibDockScore: 93.9797; (d2, d3)): the O at position 1 and the O at position 2 of N-methyl-1-oxo-isoindigo form hydrogen bonds with respective residues of amino acids in the binding pocket in the SH2 domain of human STAT3 protein. LibDockScore is 91.0665 for N-methyl-1-oxo-isoindigo positioned at the same binding format as N-methyl-isoindigo (d2); LibDockScore is 94.5181 for N-methyl-1-oxo-isoindigo positioned at the 180 degree-reversed structural plane as that of N-methyl-isoindigo (d3), a higher score suggesting a more favorable docking position. The dockings shown in the right of the figures (d1d3) indicate a clear similarity between the interactions of STAT3-SH2 with N-methyl-isoindigo, and with N-methyl-1-oxo-isoindigo. The three dimensional structure of STAT3-SH2 domain was from a crystallized form of STAT3 with AAPpYL. The docking software was Discovery Studio. The docking method was LibDock.

DETAILED PREPARATION METHODS

(5) The present invention is further demonstrated as followings, with individual examples together with the figures. It should be understood that the described examples are used only to illustrate the present invention, not to limit the scope of the present invention.

I. Method for Chemical Syntheses

I-1. Syntheses of Intermediates and Target Compounds of 1-oxo-indirubins (I-13)

I-1-1. Syntheses of the Intermediates N1-substituted Indole-2,3-dione (A1) and N1-substituted-5/7-azaindole-2,3-dione (A2/A3)

(6) N1-alkyl-indole-2,3-dione (A1) was synthesized by alkylating the N-1 position of isatins that have desired substituents at different positions;

(7) N1-alkyl-5-azaindole-2,3-dione (A2) and N1-alkyl-7-azaindole-2,3-dione (A3) were synthesized by alkylating the N-1 position of 5-azaindole and 7-azaindole, respectively, that have desired substituents at different positions, then oxidizing the intermediates in the presence of CrO.sub.3 and CH.sub.3COOH.

(8) ##STR00004##

(9) Wherein, R.sup.1 represents CH.sub.3, C.sub.2H.sub.5, n-C.sub.3H.sub.7, n-C.sub.4H.sub.9, PhCH.sub.2, glycosyl protected by acyl, etc.; R.sup.2, R.sup.3, R.sup.4, R.sup.5 independently represent H, D, halogen, hydroxyl, sulfhydryl, C.sub.1C.sub.4 alkyl, nitro, amino, amido, amide, C.sub.1C.sub.4 alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonyl, isocyanate;

I-1-2. Syntheses of the Intermediates, Benzofurane-3-one (B)

(10) Benzofurane-3-ones (B) were synthesized by esterification of salicylic acid that has desired substituents at different positions, substitution reaction with ethyl chloroacetate, hydrolytic reaction of the esters, cyclization of anhydride acid, and then hydrolytic reaction under acidic condition.

(11) ##STR00005##

(12) Wherein, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently represent H, D, halogen, hydroxyl, sulfhydryl, C.sub.1C.sub.4 alkyl, nitro, amino, amido, amide, C.sub.1C.sub.4 alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonyl, isocyanate.

I-1-3. Syntheses of 1-oxo-(aza)indirubins (I-13)

(13) The target compounds were synthesized through reactions of benzofurane-3-ones (B) and N1-alkyl-indole-2,3-diones (A1) or N1-alkyl-5-azaindole-2,3-diones (A2) or N1-alkyl-7-azaindole-2,3-diones (A3) using acetic acid as solvent in the presence of sodium acetate anhydrous under condition of 85 C. for 8 hours with stirring. The reactions were terminated by pouring the reaction mixture into ice water in which the products crystallized into solids. The target products (I-1, I-2, and I-3) were obtained by filtration, desiccation, and purification through column chromatography.

(14) ##STR00006##

(15) Wherein, R.sup.1 represents H or D, C.sub.1C.sub.6 alkyl, aryl, aralkyl, acyl, aroyl, glycosyl or biosyl protected by acyl, glycosyl or biosyl; R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently represent H, halogen, hydroxyl, sulfhydryl, C.sub.1C.sub.4 alkyl, nitro, amino, amido, amide, C.sub.1C.sub.4 alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonyl, sulfamoyl, isocyanate, or alkyl isocyanate;

(16) Wherein R represents oxygen, sulfur, selenium, or a NR.sup.6 or NOR.sup.6 group, wherein R.sup.6 is H, C.sub.1C.sub.6 straight-chain or branched-chain alkyl, aryl, aralkyl, C.sub.3C.sub.6 alicyclic group, acyl, aroyl, sulfonyl or phosphoryl.

I-1-4. Syntheses of the Target Compounds, 3-oxime-1-oxo-indirubins

(17) 3-oxime-1-oxo-indirubins were synthesized through heating reflux of hydroxylamine hydrochloride with N1-alkyl-(aza)indirubins in pyridine.

(18) ##STR00007##

(19) Wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 represent the same as described in 1-3.

I-1-5. Syntheses of Target Compounds, 1-oxo-indirubins-3-oxime Ester

(20) 1-oxo-indirubins-3-oxime ester were synthesized by reacting 3-oxime-1-oxo-indirubins with halocarbon in alkaline alcohol solution.

(21) ##STR00008##

(22) Wherein R represents CH.sub.3ON, EtON; R.sup.1 represents CH.sub.3, C.sub.2H.sub.5, n-C.sub.3H.sub.7, n-C.sub.4H.sub.9, PhCH.sub.2, etc.; R.sup.2, R.sup.4 represent H, CH.sub.3, C.sub.2H, n-C.sub.3H.sub.7, n-C.sub.4H.sub.9, PhCH.sub.2, etc.

I-2. Syntheses of Intermediates and Target Compounds of 1-oxo-isoindigos (II-13)

(23) Syntheses of the key intermediates: Benzofuran-2-ones (C) are actually the lactones of salicylic acid derivatives. They are commercially available. The target compounds were synthesized through reactions of benzofurane-2-ones (C) and N1-alkyl-indole-2,3-diones (A1) or N1-alkyl-5-azaindole-2,3-diones (A2) or N1-alkyl-7-azaindole-2,3-diones (A3) using acetic acid as solvent in the presence of sodium acetate anhydrous under condition of 85 C. for 8 hours with stirring. The reactions were terminated by pouring the reaction mixture into ice water in which the products became crystallizing solid. The target products (II-1, II-2, and II-3) were obtained by filtration, desiccation, and purification through column chromatography.

(24) ##STR00009##

(25) Wherein, R.sup.1 represents H or D, C.sub.1C.sub.6 alkyl, aryl, aralkyl, acyl, aroyl, glycosyl or biosyl protected by acyl, glycosyl or biosyl; R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 independently represent H, halogen, hydroxyl, sulfhydryl, C.sub.1C.sub.4 alkyl, nitro, amino, amido, amide, C.sub.1C.sub.4 alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonyl, sulfamoyl, isocyanate, or alkyl isocyanate.

II. Exemplification of Chemical Synthesis

(26) The present invention is further illustrated through the following examples. It should be appreciated that the following examples are provided merely for the purposes of illustration and not intended to limit the scope of the invention. In examples, the experimental methods usually follow conventional methods or recommendations from manufacturers, unless specifically indicated. Unless otherwise indicated, the portion and percentage are mass portion and weight percentage.

II-1. Instruments and Reagents

(27) The melting points of 1-oxo-(aza)indirubins and 1-oxo-(aza)isoindigos presented in this example were measured by the Mel-TEMP melting point instrument without calibration. Infra-red (IR) spectrum was measured by the Nicolet Avatar 370 DTGS IR spectrometer. The electrospray ionization mass spectrum (ESI-MS) was determined using the HP1100LC/MSD mass spectrometer; the high resolution MS was determined by the Agilent Q-TOF 6520 high resolution mass spectrometer. The thin layer chromatographic (TLC) plates were made from silica gel GF254 (Qingdao Haiyang Chemical Co., Ltd) and 0.8% CMC-Na solution in distilled water, activated at 100-110 C. for 1 hour, preserved in dryers, and developed under ultraviolet light (at 254 nm and 365 nm). The chromatographic columns were packed by the dry method with silica gel (100-200 mesh or 200-300 mesh) (Qingdao Haiyang Chemical Co., Ltd). The hydrogen spectrum (.sup.1H-NMR) was determined by the Bruck AV-300 nuclear magnetic resonance spectrometer using tetramethylsilane (TMS) as an internal standard. The elemental analysis was performed using the Elementar Vario EL. III elemental analyzer.

(28) The reagents were commercially available chemically pure (CP) or analytical reagents (AR). Unless otherwise indicated, the reagents were used directly without any prior treatment.

II-2. Example: Preparation of the Compounds

II-2-1. Preparation of the Intermediates

(29) Example 1: Preparation of the Intermediate, N1-benzyl-indole-2,3-dione (A1)

(30) To a solution of indole-2,3-dione (1.0 g, or 0.007 mol) in 10 ml DMF (N,N-Dimethylformamide) incubated in ice water, NaH (0.24 g, or 0.01 mol) was slowly added; 10 minutes later, benzyl chloride (0.88 g, 0.007 mol) was added; and the reaction mixture was incubated under room temperature while stirring for 9 hours. The reaction was terminated by pouring the reaction mixture into ice water in which the products became crystallized red solids. TLC showed that the reaction mixture still contained a small amount substrate. The target product was obtained by filtration, desiccation, and purification through column chromatography (petroleum ether: ethyl acetate=4: 1). A solid red product of 0.85 g was obtained, with a yield of 59%; ESI-MS m/z: 238.2 [M+H].sup.+, C.sub.15H.sub.11NO.sub.2 (237.3).

(31) Example 2: Preparation of the Intermediate, N1-methyl-5-azaindole-2,3-dione (A2)

(32) To a solution of 1-methyl-5-azaindole (2.0 g, or 15 mmol) in 70 ml acetic acid, a suspension of 3.2 g CrO.sub.3 in 20 ml water was added. The reaction mixture was stirred for 0.5 h at room temperature (the reaction was monitored by TLC), and then diluted with water. The product was extracted using trichloromethane three times; the combined organic phases were washed with water, dried and evaporated. An orange product (1-methyl-5-azaindole-2,3-dione) of 1.5 g was obtained, with a yield of 62%; mp: 140-142 C.

(33) Example 3: Preparation of the Intermediate, N1-methyl-7-azaindole-2,3-dione (A3)

(34) To a solution of N1-methyl-7-azaindole (1.01 g, or 7.65 mmol) in 30 ml acetic acid (AcOH), a suspension of 1.5 g CrO.sub.3 in 10 ml water was added. The reaction mixture was stirred for 0.5 h at room temperature (the reaction was kept tracked by TLC), and then diluted with water. The product was extracted using trichloromethane three times; the combined organic phases were washed with water, dried and evaporated. A yellow solid product (1-methyl-5-azaindole-2,3-dione) of 1.0 g was obtained, with a yield of 84%; mp: 159-160 C. (mp reported in literature: 160-161 C.) (Tatsugi, et al., An improved preparation of isatins from indoles. ARKIVOC 2001; (i) 67-73).

(35) Example 4: Preparation of the Intermediate, benzofuran-3-one (B)

(36) (1) Methyl Salicylate

(37) To a solution of salicylate (30 g, or 0.217 mol) in 150 ml absolute methanol in a 500 ml-three-necked bottle, 90 ml thionyl chloride was slowly added with a dropping funnel. A lot of heat was released during this procedure. The reaction mixture was then heated and refluxed for 5 hours, during which the reaction was monitored by TLC. The mixture was cooled down and then poured into a 250 ml eggplant-shaped bottle, and 200 ml ethyl acetate was added after the methanol was removed by rotary evaporation under reduced pressure. The reaction mixture was washed 3 or more times until the fluorescence disappeared from the water and the ethyl acetate phase became pH-neutral. The product was desiccated with anhydrous sodium sulfate, filtered to remove the desiccant, and dried by decompression evaporation. A colorless liquid of 32.15 g, a yield of 98%, was obtained after purification through column chromatography (petroleum ether: ethyl acetate=5:1) and was ready for the next reaction.

(38) (2) 2-Ethoxycarbonyl Methoxyl-Methyl Benzoate

(39) The methyl salicylate (32.15 g, or 0.213 mol) obtained from previous reaction was dissolved and mixed well in 150 ml acetone, added with K.sub.2CO.sub.3 (72 g, or 0.727 mol) and ethyl chloroacetate (22.5 ml, or 0.211 mol). The mixture was heated and refluxed for 11 hours, after which the reaction was terminated and the acetone was removed by rotary evaporation under reduced pressure. The reaction mixture was dissolved in water, extracted multiple times with ethyl acetate, desiccated with anhydrous sodium sulfate, filtered to remove the desiccant, and then dried by decompression evaporation. A milk-white solid of 48 g, a yield of 94%, was obtained after purification through column chromatography (petroleum ether: ethyl acetate=5: 1) and was ready for the next reaction.

(40) (3) 2-Carboxymethyloxy Benzoic Acid

(41) The 2-ethoxycarbonyl methoxyl-methyl benzoate (48 g, or 0.201 mol) obtained from the previous reaction was dissolved in 60 ml methanol and added with 300 ml of 10% K.sub.2CO.sub.3 solution. The mixture was stirred at room temperature for 3 hours; the reaction was then terminated and the methanol was removed by rotary evaporation under reduced pressure. The reaction mixture was added with concentrated HCl and stirred until a white solid separated out with an acidic pH. The product was filtered, washed with water and desiccated under infrared lamp. A white solid of 18.15 g, a yield of 46%, was obtained and was ready for the next reaction.

(42) (4) 3-Acetoxyl-Benzofuran

(43) The 2-carboxymethyloxy benzoic acid (18.15 g) obtained from previous reaction was divided into 2 parts, 9 g and 9.15 g for two separate reactions. The 2-carboxymethyloxy benzoic acid (9 g, or 0.046 mol) together with the anhydrous sodium acetate (11.88 g) were dissolved in 300 ml acetic anhydride and 47.25 ml acetic acid, stirred and heat-refluxed for 4 hours. The reaction mixture was cooled down and added with 400 ml water, thoroughly mixed, and extracted with dichloromethane. The dichloromethane phase was repeatedly washed with saturated sodium bicarbonate solution, and then with saturated sodium chloride solution. For the remaining 2-carboxymethyloxy benzoic acid (9.15 g, or 0.047 mol), the same molar ratios of raw materials and procedures as described above were used in the same reactions. A combined product of 11.06 g, a final yield of 68%, was obtained, and was ready for the next reaction.

(44) (5) Benzofuran-3-One

(45) The 3-acetoxyl-benzofuran (11.06 g, or 0.058 mol) obtained from the previous reaction was dissolved in 80 ml methanol, added with 2.5 ml concentrated HCl, and then with 25 ml water, stirred, heat-refluxed for 1 hour. The reaction mixture was then cooled down in ice water. The light-yellow product was crystallized, filtered, desiccated under infrared lamp. The final product obtained was 6.63 g, with a yield of 79%; ESI-MS m/z: 135.1[M+H].sup.+, C.sub.8H.sub.6O.sub.2 (134.1).

II-2-2. Preparation of the Representative Target Compounds (I-13)

(46) Example 5: N1-benzyl-1-oxo-indirubin (14)

(47) N1-benzyl-indole-2,3-dione (0.35 g, or 1.5 mmol) was dissolved in 15 ml acetic acid, added with anhydrous sodium acetate (0.37 g, or 4.5 mmol) solid, stirred until dissolved and then added with benzofuran-3-one (0.2 g, or 1.5 mmol). The reaction was maintained at 85 C. for 8 hours and then terminated. The cooled reaction mixture was poured into 200 ml ice water and thoroughly mixed. A maroon solid was crystallized, filtered, desiccated, and purified through column chromatography (dichloromethane: ethyl acetate=120: 1, then, petroleum ether: ethyl acetate=6:1, v/v). The product was re-crystallized with dichloromethane and petroleum ether. 0.26 g of the purified red solid product was obtained, with a yield of 48%, m.p. 235237 C.; IR (KBr, v, cm.sup.1): 3435, 3326, 3318, 1706, 1654, 1595, 1465, 1400, 1361, 1305, 1162, 1081, 954;

(48) ESI-MS m/z: 392.2[M+K].sup.+, C.sub.23H.sub.15NO.sub.3(353.4);

(49) .sup.1H-NMR (DMSO-d6, 300 MHz) : 8.92 (d, J=7.60 Hz, 1H, ArH), 7.86 (t, J=7.00 Hz, 2H, ArH), 7.56 (d, J=7.80 Hz, 1H, ArH), 7.357.32 (m, 7H, ArHs), 7.10 (t, J=7.60 Hz, 1H, ArH), 6.71 (d, J=7.80 Hz, 1H, ArH), 4.99 (s, 2H, ArCH2);

(50) Anal. for C.sub.23H.sub.15NO.sub.3 Calcd (%): C, 78.17; H, 4.28; N, 3.96.

(51) Found (%): C, 78.09; H, 4.40; N, 3.68.

(52) Example 6: N1-Benzyl-1-Oxo-Indirubin-3-Oxime (38)

(53) N1-benzyl-1-oxo-indirubin (0.2 g, or 0.57 mmol) was dissolved in 12 ml methanol, added with 4 ml anhydrous pyridine and 0.1 g (1.4 mmol) hydroxylamine hydrochloride and heat-refluxed for 1 hour. The reaction mixture was cooled down and concentrated by removing most of the solvent. The remaining mixture was poured into 60 ml ice water and stirred vigorously. An orange solid was crystallized, filtered and purified through silica gel column chromatography (petroleum ether: ethyl acetate=3:1, v/v). 0.16 g orange solid product, N1-benzyl-1-oxo-indirubin-3-oxime (38), was obtained, with a yield of 75%; m.p.: 253-255 C.; IR (KBr, v, cm.sup.1): 3240, 3060, 3028, 2804, 2775, 1668, 1614, 1589, 1463, 1332, 1213, 1170, 1018, 997, 696;

(54) ESI-MS: 369.1[M+H].sup.+, C.sub.23H.sub.16N.sub.2O.sub.3(368.4);

(55) .sup.1H NMR (AV-300, D6-DMSO, ppm) : 13.7 (s, 1H, NOH), 8.90 (d, J=7.60 Hz, 1H, ArH), 7.82 (t, J=7.00 Hz, 2H, ArH), 7.53 (d, J=8.55 Hz, 1H, ArH), 7.32-7.30 (m, 7H, ArHs), 7.11 (t, J=7.60 Hz, 1H, ArH), 6.67 (d, J=7.80 Hz, 1H, ArH), 4.94 (s, 2H, ArCH.sub.2);

(56) Anal. for C.sub.23H.sub.16N.sub.2O.sub.3 Calcd (%): C, 74.99; H, 4.38; N, 7.60.

(57) Found (%): C, 74.51; H, 4.60; N, 7.42.

(58) Example 7: N1-benzyl-1-oxo-indirubin-3-oxime ether (56)

(59) N1-benzyl-1-oxo-indirubin-3-oxime (1.5 g, or 4.1 mmol) was added to 50 ml anhydrous ethanol containing 5% KOH, dissolved in mild heating condition, filtered, added drop wise with 3 ml CH.sub.3I while being stirred. Heat was released at this step and dark red precipitate was formed. After stirring for 0.5 hours, the reaction mixture was filtered, water-washed until pH became neutral, and then desiccated. The dark red crude was re-crystallized in acetone. 1.22 g dark red crystal, N1-benzyl-1-oxo-indirubin-3-oxime ether (56), was obtained, with a yield of 78%; mp: 201-200 C.;

(60) ESI-MS: 383.0 [M+H].sup.+, C.sub.24H.sub.18N.sub.2O.sub.3(382.4);

(61) .sup.1H-NMR (AV-300, D6-DMSO, ppm) : 4.26 (s, 3H, OCH.sub.3), 4.88 (s, 2H, NCH.sub.2), 8.92 (d, J=7.60 Hz, 1H, ArH), 7.76 (t, J=7.00 Hz, 2H, ArH), 7.52 (d, J=8.55 Hz, 1H, ArH), 7.30-7.27 (m, 7H, ArHs), 7.11 (t, J=7.60 Hz, 1H, ArH), 6.69 (d, J=7.80 Hz, 1H, ArH);

(62) Anal For C.sub.24H.sub.18N.sub.2O.sub.3 Calcd (%): C, 75.38; H, 4.74; N, 7.33.

(63) Found (%): C, 75.19; H, 4.59; N, 7.48.

II-2-3. Preparation of all Other Target Compounds (I-13)

II-2-3-1. Syntheses of 1-oxo-indirubin Derivatives (Formula I-1, No. 1No. 60)

(64) Example 8: Twenty-Four Derivatives of 1-oxo-indirubin (No. 1No. 24) were Synthesized by the Method Described Above for the Synthesis of N1-benzyl-1-oxo-indirubin (14) in Example 5

(65) Example 9: Twenty-Four Derivatives of 1-oxo-indirubin-3-oxime (No. 25No. 48) were Synthesized by the Method Described Above for the Synthesis of N1-benzyl-1-oxo-indirubin-3-oxime (38) in Example 6

(66) Example 10: Twelve Derivatives of 1-oxo-indirubin-3-oxime ether (No. 49No. 60) were Synthesized by the Method Described Above for the Synthesis of N1-benzyl-1-oxo-indirubin-3-oxime ether (56) in Example 7

(67) Molecular structures of the compounds 160 are listed in Table 1. All these novel compounds were structurally characterized by IR, ESI-MS, .sup.1H-NMR and elemental analysis.

(68) ##STR00010##

(69) In the formula I-1, R.sup.2, R.sup.2, R.sup.4 and R.sup.5 represent H. Rest of the substituents are described in Table 1:

(70) TABLE-US-00002 TABLE 1 Structures of 1-oxo-indirubins (I-1: 1~60) ID R.sup.1 R.sup.3 R.sup.4 R.sup.5 R.sup.3 R 1 H H H H H O 2 H H H F H O 3 CH.sub.2CH.sub.3 H H H H O 4 CH.sub.2CH.sub.3 H H F H O 5 CH.sub.2CH.sub.3 Cl H H H O 6 CH.sub.2CH.sub.3 H Cl H H O 7 CH.sub.2CH.sub.3 CH.sub.3 H H H O 8 i-C.sub.3H.sub.7 H H H H O 9 i-C.sub.3H.sub.7 F H H H O 10 i-C.sub.3H.sub.7 H H F H O 11 i-C.sub.3H.sub.7 Cl H H H O 12 i-C.sub.3H.sub.7 H Cl H H O 13 i-C.sub.3H.sub.7 Me H H H O 14 CH.sub.2Ph H H H H O 15 CH.sub.2Ph F H H H O 16 CH.sub.2Ph Cl H H H O 17 CH.sub.2Ph Me H H H O 18 CH.sub.2CH.sub.3 F H H H O 19 CH.sub.2CH.sub.3 H H H Cl O 20 CH.sub.2CH.sub.3 H H F Cl O 21 CH.sub.2CH.sub.3 Cl H H Cl O 22 CH.sub.2CH.sub.3 H Cl H Cl O 23 i-C.sub.3H.sub.7 Cl H H Cl O 24 i-C.sub.3H.sub.7 H Cl H Cl O 25 H H H H H NOH 26 H H H F H NOH 27 CH.sub.2CH.sub.3 H H H H NOH 28 CH.sub.2CH.sub.3 H H F H NOH 29 CH.sub.2CH.sub.3 Cl H H H NOH 30 CH.sub.2CH.sub.3 H Cl H H NOH 31 CH.sub.2CH.sub.3 CH.sub.3 H H H NOH 32 i-C.sub.3H.sub.7 H H H H NOH 33 Ribosyl H H H H NOH 34 i-C.sub.3H.sub.7 H H F H NOH 35 i-C.sub.3H.sub.7 Cl H H H NOH 36 i-C.sub.3H.sub.7 H Cl H H NOH 37 i-C.sub.3H.sub.7 Me H H H NOH 38 CH.sub.2Ph H H H H NOH 39 CH.sub.2Ph F H H H NOH 40 CH.sub.2Ph Cl H H H NOH 41 CH.sub.2Ph Me H H H NOH 42 CH.sub.2CH.sub.3 F H H H NOH 43 CH.sub.2CH.sub.3 H H H Cl NOH 44 CH.sub.2CH.sub.3 H H F Cl NOH 45 CH.sub.2CH.sub.3 Cl H H Cl NOH 46 CH.sub.2CH.sub.3 H Cl H Cl NOH 47 i-C.sub.3H.sub.7 Cl H H H NOH 48 i-C.sub.3H.sub.7 H Cl H Cl NOH 49 H H H H H NOCH.sub.3 50 H H H F H NOCH.sub.3 51 CH.sub.2CH.sub.3 H H H H NOCH.sub.3 52 CH.sub.2CH.sub.3 H H F H NOCH.sub.3 53 Glucosyl Cl H H H NOCH.sub.3 54 i-C.sub.3H.sub.7 H Cl H H NOCH.sub.3 55 i-C.sub.3H.sub.7 Me H H H NOCH.sub.3 56 CH.sub.2Ph H H H H NOCH.sub.3 57 CH.sub.2Ph F H H H NOCH.sub.3 58 CH.sub.2Ph Cl H H H NOCH.sub.3 59 CH.sub.2CH.sub.3 F H H H NOCH.sub.3 60 i-C.sub.3H.sub.7 H Cl H Cl NOCH.sub.3

II-2-3-2. Syntheses of 1-oxo-5-azaindirubin Derivatives (Formula I-2, No. 61No. 74)

(71) Example 11: Synthesis of N-butyl-1-oxo-5-azaindirubin (69)

(72) N1-butyl-5-azaindole-2,3-dione (0.31 g, or 1.5 mmol) was dissolved in 15 ml acetic acid, added with 0.37 g anhydrous sodium acetate (4.5 mmol) solid, stirred until dissolved and then added with 0.2 g benzofuran-3-one (1.5 mmol). The reaction was maintained at 85 C. for 8 hours and then terminated. The cooled reaction mixture was poured into 200 ml ice water and thoroughly mixed. A maroon solid was crystallized, filtered, desiccated, and purified through column chromatography (dichloromethane: ethyl acetate=5: 1, v/v). The crude product was re-crystallized with dichloromethane and petroleum ether. 0.26 g of the purified maroon solid product, N-butyl-1-oxo-5-azaindirubin (69), was obtained, with a yield of 52%, m.p. 141143 C.; IR (KBr, v, cm.sup.1): 3428, 3290, 3133, 2946, 1675, 1627, 1612, 1594, 1455, 1402, 1384, 1197, 1116, 981, 748; .sup.1H-NMR(CDCl.sub.3, 300 MHz) : 8.91 (dd, J=0.90, 7.80 Hz, 1H, ArH), 8.43 (d, J=7.60 Hz, 1H, ArH), 8.02 (dd, J=0.90, 7.80 Hz, 1H, ArH), 7.33 (t, J=7.72 Hz, 1H, ArH), 7.10 (t, J=7.72 Hz, 1H, ArH), 6.98 (m, 1H, ArH), 6.86 (d, J=7.60 Hz, 1H, ArH), 167-176 (m, 4H, NCH.sub.2CH.sub.2), 1.41-1.45 (m, 2H, CH.sub.2), 0.99 (s, 3H, CH.sub.3);

(73) ESI-MS m/z: 359.2[M+K].sup.+, C.sub.19H.sub.16N.sub.2O.sub.3(320.3);

(74) Anal. for C.sub.19H.sub.16N.sub.2O.sub.3 Calcd (%): C, 71.24; H, 5.03; N, 8.78.

(75) Found (%): C, 71.05; H, 5.10; N, 8.69.

(76) Example 12: Fourteen Derivatives of 1-oxo-5-azaindirubin (Formula I-2, No. 61No. 74) were Synthesized by the Methods Described Above for the Syntheses of N-butyl-1-oxo-5-azaindirubin (69) in Example 11, 1-oxo-indirubin-3-oxime (38) in Example 6, and 1-oxo-indirubin-3-oxime ether (56) in Example 7

(77) Molecular structures of the compounds 61-74 are listed in Table 2. All these novel compounds were structurally characterized by IR, ESI-MS, .sup.1H-NMR and elemental analysis.

(78) ##STR00011##

(79) In the formula I-2, R2, R.sup.4 and R.sup.5 represent H. Rest of the substituents are described in Table 2.

(80) TABLE-US-00003 TABLE 2 Structures of 1-oxo-5-azaindirubins (I-2: 61-74) ID R.sup.1 R.sup.2 R.sup.4 R.sup.5 R.sup.3 R 61 H H H H H O 62 CH.sub.3 H H H H O 63 CH.sub.2CH.sub.3 H H H H O 64 CH.sub.2CH.sub.3 H H F H O 65 CH.sub.2CH.sub.3 CH.sub.3 H H H O 66 i-C.sub.3H.sub.7 H H H H O 67 i-C.sub.3H.sub.7 H H F H O 68 i-C.sub.3H.sub.7 Me H H H O 69 n-C.sub.4H.sub.9 H H H H O 70 n-C.sub.4H.sub.9 H H F H O 71 CH.sub.2CH.sub.3 H H H Cl O 72 CH.sub.2Ph H H H H NOH 73 CH.sub.2CH.sub.3 H H H Cl NOH 74 CH.sub.2Ph H H H H NOCH.sub.3

II-2-3-3. Syntheses of 1-oxo-7-azaindirubin Derivatives (Formula I-3, No. 75No. 90)

(81) Example 13: Synthesis of N-isopropyl-1-oxo-7-azaindirubin (80)

(82) N-isopropyl-7-azaindole-2,3-dione (0.29 g, or 1.5 mmol) was dissolved in 15 ml acetic acid, added with 0.37 g anhydrous sodium acetate (4.5 mmol) solid, stirred until dissolved and then added with 0.2 g benzofuran-3-one (1.5 mmol). The reaction was maintained at 85 C. for 8 hours and then terminated. The cooled reaction mixture was poured into 200 ml ice water and thoroughly mixed. A red solid was crystallized, filtered, desiccated and purified through column chromatography (dichloromethane: ethyl acetate=5: 1, v/v). The crude product was re-crystallized with dichloromethane and petroleum ether. 0.20 g of the purified red solid product, N-isopropyl-1-oxo-7-azaindirubin (80), was obtained, with a yield of 43%, m.p. 215216 C.; IR (KBr, v, cm.sup.1): 3411, 3307, 3101, 2981, 1700, 1658, 1592, 1467, 1429, 1371, 1315, 1253, 1180, 1097, 1054, 781, 744;

(83) .sup.1H-NMR (CDCl.sub.3, 300 MHz) : 8.92 (dd, J=1.15, 7.80 Hz, 1H, ArH), 8.44 (d, J=7.80 Hz, 1H, ArH), 7.99-8.03 (m, 1H, ArH), 7.31-7.35 (m, 1H, ArH), 7.10 (d, J=7.80 Hz, 1H, ArH), 7.04 (d, J=7.80 Hz, 1H, ArH), 6.97 (dd, J=1.15, 7.80 Hz, 1H, ArH), 4.70 (m, 1H, NCH), 1.55 (d, 6H, 2CH.sub.3);

(84) ESI-MS m/z: 339.1[M+Na].sup.+, C.sub.18H.sub.14N.sub.2O.sub.3(306.3)

(85) Anal. for C.sub.18H.sub.14N.sub.2O.sub.3 Calcd (%): C, 70.58; H, 4.61; N, 9.15.

(86) Found (%): C, 70.46; H, 4.52; N, 9.04.

(87) Example 14: Sixteen Derivatives of 1-oxo-7-azaindirubin (Formula I-3, No. 75No. 90) were Synthesized by the Methods Described Above for the Syntheses of N-isopropyl-1-oxo-7-azaindirubin (80) in Example 13, 1-oxo-indirubin-3-oxime (38) in Example 6, and 1-oxo-indirubin-3-oxime ether (56) in Example 7

(88) Molecular structures of the compounds 75-90 are listed in Table 3. All these novel compounds were structurally characterized by IR, ESI-MS, .sup.1H-NMR and elemental analysis.

(89) ##STR00012##

(90) In the formula I-3, R2, R.sup.2 and R.sup.4 represent H. Rest of the substituents are described in Table 3.

(91) TABLE-US-00004 TABLE 3 Structures of 1-oxo-7-azaindirubins (I-3: 75-90) ID R.sup.1 R.sup.3 R.sup.4 R.sup.3 R.sup.5 R 75 H H H H H O 76 CH.sub.3 H H H H O 77 CH.sub.2CH.sub.3 H H H H O 78 CH.sub.2CH.sub.3 F H H H O 79 CH.sub.2CH.sub.3 H H H Cl O 80 i-C.sub.3H.sub.7 H H H H O 81 i-C.sub.3H.sub.7 F H H H O 82 i-C.sub.3H.sub.7 Cl H H H O 83 i-C.sub.3H.sub.7 H Cl H H O 84 i-C.sub.3H.sub.7 Me H H H O 85 CH.sub.2CH.sub.3 H H H Cl O 86 CH.sub.2Ph H H H H NOH 87 CH.sub.2CH.sub.3 H H H Cl NOH 88 i-C.sub.3H.sub.7 Cl H H H NOH 89 i-C.sub.3H.sub.7 Cl H H H NOCH.sub.3 90 CH.sub.2Ph H H H H NOCH.sub.3

II-2-4. Preparation of the Target Compounds (II-13)

II-2-4-1. Syntheses of 1-oxo-isoindigo Derivatives (Formula II-1, No. 91No. 120)

(92) Example 15: Synthesis of N-methyl-1-oxo-indirubin (93)

(93) 0.24 g N-methyl-indole-2,3-dione (1.5 mmol) was dissolved in 15 ml acetic acid, added with 0.37 g anhydrous sodium acetate (4.5 mmol) solid, stirred until dissolved and then added with 0.2 g benzofuran-2-one (1.5 mmol). The reaction was maintained at 85 C. for 8 hours and then terminated. The cooled reaction mixture was poured into 200 ml ice water and thoroughly mixed. A purple solid was crystallized, filtered, desiccated and purified through column chromatography (dichloromethane: ethyl acetate=6: 1, v/v). The product was re-crystallized with dichloromethane and petroleum ether. 0.21 g of the purified dark purple solid product, N-methyl-1-oxo-isoindigo (93), was obtained, with a yield of 62%, m.p. 232233 C.; IR (KBr, v, cm.sup.1): 3442,3238, 3131, 3022, 1702, 1618, 1591, 1485, 1463, 1398, 1324, 1282, 1214, 1106, 1047, 868 594;

(94) .sup.1H-NMR(CDCl.sub.3, 300 MHz) : 9.31 (d, J=8.00 Hz, 1H, ArH), 9.05 (d, J=8.00 Hz, 1H, ArH), 7.49-7.43 (m, 2H, ArH), 7.28-7.24 (m, 1H, ArHs), 7.16-7.12 (m, 2H, ArH), 6.82 (d, J=7.50 Hz, 1H, ArH), 3.32 (s, 3H, NCH.sub.3);

(95) ESI-MS m/z: 278.1 [M+H].sup.+, 300.1 [M+Na].sup.+, C.sub.17H.sub.11NO.sub.3(277.3)

(96) Anal. for C.sub.17H.sub.11NO.sub.3 Calcd (%): C, 73.64; H, 4.00; N, 5.05.

(97) Found (%): C, 73.51; H, 4.09; N, 5.14.

(98) Example 16: Thirty Derivatives of 1-oxo-isoindigo (Formula II-1, No. 91No. 120) were Synthesized by the Methods Described Above for the Syntheses of N-methyl-1-oxo-isoindigo (93) in Example 15

(99) Molecular structures of the compounds 91-120 are listed in Table 4. All these novel compounds were structurally characterized by IR, ESI-MS, .sup.1H-NMR and elemental analysis.

(100) ##STR00013##

(101) In the formula II-1, R2, R.sup.2, R.sup.4 and R.sup.5 represent H. Rest of the substituents are described in Table 4.

(102) TABLE-US-00005 TABLE 4 Structures of 1-oxo-7-azaindirubins (II-1: 91-120) ID R.sup.1 R.sup.3 R.sup.4 R.sup.5 R.sup.3 91 H H H H H 92 H F H H H 93 CH.sub.3 H H H H 94 CH.sub.3 H H Cl H 95 CH.sub.3 Cl H H H 96 CH.sub.3 H Cl H H 97 CH.sub.3 CH.sub.3 H H H 98 CH.sub.3 H H F H 99 CH.sub.3 F H H H 100 CH.sub.2CH.sub.3 H H H H 101 CH.sub.2CH.sub.3 H H H OCH.sub.3 102 CH.sub.2CH.sub.3 Cl H H H 103 CH.sub.2CH.sub.3 H Cl H H 104 CH.sub.2CH.sub.3 F H H H 105 CH.sub.2CH.sub.3 H H F H 106 i-C.sub.3H.sub.7 H H H H 107 i-C.sub.3H.sub.7 Me H H H 108 i-C.sub.3H.sub.7 F H H H 109 i-C.sub.3H.sub.7 H H F H 110 i-C.sub.3H.sub.7 Cl H H H 111 Triacetylribosyl Cl H H H 112 Glucosyl H H H H 113 i-C.sub.3H.sub.7 Cl H H Cl 114 i-C.sub.3H.sub.7 H Cl H Cl 115 CH.sub.2Ph H H H H 116 CH.sub.2Ph H H H OCH.sub.3 117 CH.sub.2Ph F H H H 118 CH.sub.2Ph H H F H 119 CH.sub.2Ph Cl H H H 120 CH.sub.2Ph H Cl H H

II-2-4-2. Syntheses of 1-oxo-5-azaisoindigo Derivatives (Formula II-2, No. 121No. 135)

(103) Example 17: Synthesis of N-ethyl-1-oxo-5-azaisoindigo (127)

(104) N-ethyl-5-azaindole-2,3-dione (0.26 g, or 1.5 mmol) was dissolved in 15 ml acetic acid, added with 0.37 g anhydrous sodium acetate (4.5 mmol) solid, stirred until dissolved and then added with 0.2 g benzofuran-2-one (1.5 mmol). The reaction was maintained at 85 C. for 8 hours and then terminated. The cooled reaction mixture was poured into 200 ml ice water and thoroughly mixed. A purple solid was crystallized, filtered, desiccated and purified through column chromatography (dichloromethane: ethyl acetate=6: 1, v/v). The product was re-crystallized with dichloromethane and petroleum ether. 0.28 g of the purified dark purple solid product, N-ethyl-1-oxo-5-azaisoindigo (127), was obtained, with a yield of 65%, m.p. 212214 C.; IR (KBr, v, cm.sup.1): 3434, 3121, 2985, 1718, 1695, 1606, 1479, 1456, 1398, 1384, 1159, 1105, 983, 762, 638;

(105) .sup.1H-NMR(CDCl.sub.3, 300 MHz) : 9.29 (d, J=8.40 Hz, 1H, ArH), 9.01 (d, J=8.40 Hz, 1H, ArH), 7.52-7.47 (m, 2H, ArH), 7.23-7.20 (m, 1H, ArHs), 7.05-7.02 (m, 1H, ArH), 6.72 (s, 1H, ArH), 3.86 (q, 2H, NCH.sub.2), 1.32 (t, 3H, CH.sub.3);

(106) ESI-MS m/z: 293.1 [M+H].sup.+, 315.1[M+Na].sup.+, C.sub.17H.sub.12N.sub.2O.sub.3(292.3)

(107) Anal. for C.sub.17H.sub.12N.sub.2O.sub.3 Calcd (%): C, 69.86; H, 4.14; N, 9.58.

(108) Found (%): C, 69.99; H, 4.05; N, 9.46.

(109) Example 18: Fifteen Derivatives of 1-oxo-5-azaisoindigo (Formula II-2, No. 121No. 135) were Synthesized by the Methods Described Above for the Syntheses of N-ethyl-1-oxo-5-azaisoindigo (127) in Example 17

(110) Molecular structures of the compounds 121-135 are listed in Table 5. All these novel compounds were structurally characterized by IR, ESI-MS, .sup.1H-NMR and elemental analysis.

(111) ##STR00014##

(112) In the formula II-2, R2, R.sup.2 and R.sup.4 represent H. Rest of the substituents are described in Table 5.

(113) TABLE-US-00006 TABLE 5 Structures of 1-oxo-5-azaisoindigos (II-2: 21-135) ID R.sup.1 R.sup.4 R.sup.5 R.sup.3 R.sup.5 121 H H H H H 122 H H F H H 123 CH.sub.3 H H H H 124 CH.sub.3 H Cl H H 125 CH.sub.3 H F H H 126 CH.sub.3 H H CH.sub.3 H 127 CH.sub.2CH.sub.3 H H H H 128 CH.sub.2CH.sub.3 Cl H H H 129 CH.sub.2CH.sub.3 H H H OCH.sub.3 130 i-C.sub.3H.sub.7 F H H H 131 i-C.sub.3H.sub.7 H H F H 132 i-C.sub.3H.sub.7 Cl H H H 133 i-C.sub.3H.sub.7 F H H H 134 CH.sub.2Ph H H H H 135 CH.sub.2Ph H H H OCH.sub.3

II-2-4-3. Syntheses of 1-oxo-5-azaisoindigo Derivatives (Formula II-3, No. 136No. 153)

(114) Example 17: Synthesis of N-methyl-1-oxo-7-azaisoindigo (139)

(115) N-methyl-7-azaindole-2,3-dione (0.24 g, or 1.5 mmol) was Dissolved in 15 ml acetic Acid, added with 0.37 g anhydrous sodium acetate (4.5 mmol) solid, stirred until dissolved and then added with 0.2 g benzofuran-2-one (1.5 mmol). The reaction was maintained at 85 C. for 8 hours and then terminated. The cooled reaction mixture was poured into 200 ml ice water and thoroughly mixed. A purple solid was crystallized, filtered, desiccated and purified through column chromatography (dichloromethane: ethyl acetate=6: 1, v/v). The product was re-crystallized with dichloromethane and petroleum ether. 0.25 g of the purified dark purple solid product, N-methyl-1-oxo-7-azaisoindigo (139), was obtained, with a yield of 61%, m.p. 261263 C.; IR (KBr, v, cm.sup.1): 3438, 3175, 3126, 1697, 1618, 1598, 1457, 1403, 1384, 1347, 1101, 984;

(116) .sup.1H-NMR (DMSO-d6, 300 MHz) : 9.32 (d, J=8.20 Hz, 1H, ArH), 9.04 (d, J=8.20 Hz, 1H, ArH), 7.50-7.44 (m, 2H, ArH), 7.29-7.25 (m, 1H, ArHs), 7.17-7.14 (m, 1H, ArH), 6.83 (d, J=7.80 Hz, 1H, ArH), 3.35 (s, 3H, NCH.sub.3);

(117) ESI-MS m/z: 279.1 [M+H].sup.+, 301.1 [M+Na].sup.+, C.sub.16H.sub.10N.sub.2O.sub.3(278.3)

(118) Anal. for C.sub.16H.sub.10N.sub.2O.sub.3 Calcd (%): C, 69.06; H, 3.62; N, 10.07.

(119) Found (%): C, 69.11; H, 3.51; N, 10.16.

(120) Example 20: Eighteen Derivatives of 1-oxo-7-azaisoindigo (Formula II-3, No. 136No. 153) were Synthesized by the Methods Described Above for the Syntheses of N-methyl-1-oxo-7-azaisoindigo (139) in Example 19

(121) Molecular structures of the compounds 136-153 are listed in Table 6. All these novel compounds were structurally characterized by IR, ESI-MS, .sup.1H-NMR and elemental analysis.

(122) ##STR00015##

(123) In the formula II-3, R.sup.2, R.sup.2 and R.sup.4 represent H. Rest of the substituents are described in Table 6

(124) TABLE-US-00007 TABLE 6 Structures of 1-oxo-7-azaisoindigos (II-3: 136-153) ID R.sup.1 R.sup.3 R.sup.4 R.sup.3 R.sup.5 136 H H H H H 137 H F H H H 138 H H Cl H H 139 CH.sub.3 H H H H 140 CH.sub.3 H H H CH.sub.3 141 CH.sub.3 H H CH.sub.3 H 142 CH.sub.3 CH.sub.3 H H H 143 CH.sub.3 Cl H H H 144 CH.sub.3 F H H H 145 CH.sub.2CH.sub.3 H H H H 146 CH.sub.2CH.sub.3 F H H H 147 CH.sub.2CH.sub.3 Cl H H H 148 CH.sub.2CH.sub.3 H H H OCH.sub.3 149 i-C.sub.3H.sub.7 F H H H 150 i-C.sub.3H.sub.7 H H F H 151 i-C.sub.3H.sub.7 Cl H H H 152 CH.sub.2Ph H H H H 153 CH.sub.2Ph F H H H

III. Exemplification of Antitumor Activities in Cell-Based Assays

(125) Example 21: Inhibitory Effects of the Compounds (I-13 and II-13) on Tumor Cell Growth

III-1. Materials and Instruments

III-1-1. Tumor Cell Lines

(126) human hepatocellular carcinoma cell line HepG-2, human lung adenocarcinoma epithelial cell line A549

III-1-2. Reagents

(127) RPMI Medium 1640 (GIBCOBRL, Inc. U.S.A.), calf serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.), MTT (Sigma), HEPES (Shanghai Livzon Pharmaceutical Co., Ltd.), L-glutamine (imported from Japan), dimethyl sulfoxide (DMSO, analytical reagent);

(128) Measured samples: 90 compounds from 1-oxo-indirubins (I) and 1-oxo-isoindigos (II) (prepared in-house, listed in Table 7);

(129) Reference substances: 1-ethyl-indirubin (154) and 1-ethyl-indirubin-3-oxime (155) (prepared in-house, structures characterized);

III-1-3. Preparation of Reagents

III-1-3-1. Cell Culture Medium

(130) 10.4 g of 1640 medium powder, 2.1 g of sodium bicarbonate, 0.3 g of glutamine, 5.95 g of HEPES, 100,000 units of penicillin and 100,000 units of streptomycin were added to 1000 ml double distilled water. The mixture was sterilized by filtration using Millipore filter, and aliquots were stored at 20 C. The inactivated calf serum was added to the medium prior to use;

III-1-3-2. Calf Serum

(131) It was inactivated for 30 minutes in 56 C. water bath, and the aliquots were stored at 20 C.;

III-1-3-3. MTT

(132) It was diluted to 5 mg/ml with PBS, stored at 4 C. and kept away from light. It was used within two weeks;

III-1-3-4. PBS

(133) 8.00 g of sodium chloride, 0.20 g of potassium chloride, 3.4 g disodium phosphate dodecahydrate and 0.20 g of potassium phosphate dibasic were fully dissolved in double distilled water at 37 C., diluted to 1000 ml, and aliquots were stored at 4 C.;

III-1-3-5

(134) 90 measured samples, the reference substances (154 and 155) were dissolved in DMSO, and stored at 20 C.

III-1-4. Main Equipment

(135) CO.sub.2 incubator (GB16, Heraeus company, German); clean bench (SW-CJ-1F, Suzhou Antai Air Tech Co., Ltd.); horizontal centrifuge (LXJ-II, Shanghai Third Medical Instruments Factory); enzyme-linked immune detector (BIO RAD Model 550, USA); inverted biological microscope (XSZ-D2, Chongqing Optical Instrument Factory); rapid mixer (SK-1 type, Changzhou Guohua Electric Appliance Co., Ltd.); electrical heating water thermostat system (DK-8D, Shanghai Medical Constant Temperature Equipment Factory): flow cytometry (FACSCalibur, American BD Company); plate oscillator (752-A, Shanghai Medical Analysis Instrument Factory); electronic balance (BS110S, Sartorius Company, German).

III-2. Methods

III-2-1. Cell Culture

(136) Tumor cells were seeded in RPM11640 medium containing 10% calf serum, incubated at 37 C., 5% CO.sub.2 in CO.sub.2 incubator, and passaged every 2-3 days. Cells at logarithmic growth phase were used for this experiment.

III-2-2. Grouping

(137) The cells at logarithmic growth phase were formulated into suspension. The cell viability by trypan blue staining was more than 98%. The cell suspension was divided into several groups: 1 as blank control group (cell suspension plus solvent DMSO); and 2 as experimental groups (cell suspension plus compounds to be tested).

III-2-3. Determination of IC50 Values by MTT (the 50% Inhibitory Concentration)

(138) The drugs were formulated into a stock solution in DMSO, and the concentration of the stock solution was 20 mmol (used within 4 hours). In the experiment, the work solution of the drugs was diluted with RPMI1640 medium containing 10% calf serum under aseptic condition, to a final concentration of 80 M. The drug concentrations were increased by 2 times (1.25-20 M).

(139) The cells in logarithmic growth phase were selected, centrifuged, counted, and formulated into a cell suspension (2.5104/ml) with RPMI1640 medium containing 10% calf serum. The cell suspension were inoculated into 96-well plates at a density of 5000 cells/200 l per well, incubated for 24 hours at 37 C., 5% CO.sub.2. According to the above concentration of the drug, the cells were inoculated to 6 groups (including one control group), with 8 wells per group. After incubating for 72 hours, the viability of cells was measured by MTT assay. The absorbance value (A) was measured with detection wavelength at 540 nm, reference wavelength at 630 nm. The inhibitory rate (I) was calculated by the following equation, where T was the absorbance value of the experimental groups, and C was the absorbance value of the blank control group:
I=(1T/C)100%

(140) Equation of regression line was drawn from concentration-inhibitory rate curve, from which the IC.sub.50 of tested sample was calculated.

III-3. Results

(141) Inhibitory potencies (IC.sub.50, M) of the 90 compounds described above on the proliferation of the tumor cell lines A549 and HepG2 are listed in Table 7.

(142) TABLE-US-00008 TABLE 7 IC.sub.50 (M) of 1-oxo-indirubins (I) and 1-oxo-isoindigos (II) on the tumor cells A549 and HepG2 ID A549 HepG2 1 40.0 1.4 >100 2 >100 ND 3 21.9 0.9 26.4 1.1 4 9.5 2.1 31.5 2.9 5 11.5 1.0 87.0 2.1 6 11.9 1.4 60.0 1.7 7 8.0 1.2 19.0 1.6 8 10.0 1.4 32.2 2.4 9 18.5 1.8 25.0 1.0 10 8.1 0.6 28.5 1.3 11 8.5 1.6 45.0 1.9 12 14.0 1.7 26.0 2.7 13 11.0 1.2 42.0 1.8 14 7.0 1.6 24.0 1.5 15 7.4 1.7 34.0 0.9 16 16.0 2.0 61.0 2.9 17 8.0 2.3 25.0 2.7 18 7.05 1.1 17.0 1.9 19 16.0 1.5 8.0 1.5 20 1.8 1.0 12.0 1.4 21 4.0 2.4 12.0 1.9 22 2.7 1.2 7.1 1.1 23 3.1 0.8 6.9 0.8 24 3.8 1.5 12.1 0.9 25 3.7 0.9 5.0 0.9 26 5.4 0.6 29.0 1.8 27 7.1 0.7 19.0 1.1 28 7.0 1.3 30.0 1.5 29 3.6 0.2 7.7 1.2 30 3.8 0.5 3.0 0.5 31 1.2 0.8 2.8 0.4 32 10.5 1.3 12.8 1.6 33 14.8 1.2 21.0 0.9 34 14.1 1.5 51.1 2.6 35 17.5 1.8 29.2 1.4 36 10.0 0.6 31.5 1.8 37 9.0 2.1 41.0 1.7 38 9.05 1.4 32.2 1.5 39 33.0 2.9 19.0 2.1 40 14.0 1.2 >100 41 8.3 1.7 45.0 3.0 42 15.0 1.2 28.0 2.5 43 14.0 1.7 42.0 4.2 44 >100 >100 45 4.8 0.6 3.6 0.2 46 3.4 0.7 3.8 0.5 47 1.7 0.8 1.6 0.8 48 1.9 0.4 2.6 0.4 49 9.5 0.6 4.8 0.7 50 7.7 1.2 2.9 0.5 51 3.9 0.5 1.7 0.6 52 2.8 0.4 2.1 0.2 53 2.1 0.5 16.0 1.5 54 12.0 1.4 1.8 1.9 55 12.0 1.9 4.0 2.4 56 7.1 1.1 2.7 1.2 57 6.9 0.8 3.1 0.8 58 12.1 0.9 3.8 1.5 59 5.0 0.9 3.7 0.9 60 29.0 1.8 5.4 0.6 62 9.5 2.1 11.5 1.0 65 18.5 1.8 40.0 3.1 66 7.4 1.7 1.8 0.6 68 14.0 1.7 18.5 1.8 70 4.5 0.6 3.6 0.4 74 9.4 0.6 19.0 1.1 75 8.0 1.2 10 1.4 79 17.4 1.7 31.5 2.5 82 16 2.0 18.0 2.3 85 25.0 2.7 18.5 1.5 88 87.0 2.1 80.1 1.5 90 29.0 1.8 30.2 1.5 92 31.7 1.5 15.2 1.2 96 17.2 1.6 14.2 1.3 100 7.0 1.4 9.2 2.0 106 35.5 2.9 3.8 1.5 112 90.1 4.5 >100 116 3.7 0.6 7.4 1.7 119 39.6 1.7 27.5 1.3 120 49.1 2.6 43 2.2 121 11.6 1.1 15.6 1.2 123 5.8 0.8 9.1 1.0 127 4.7 0.9 8.4 1.3 130 28.5 1.6 19.6 1.3 134 11.5 1.2 26.0 2.1 138 12.1 1.4 25.8 1.7 140 1.9 0.8 7.3 0.8 146 26.3 2.1 35.2 2.3 150 7.9 1.3 5.6 0.6 152 18.2 1.2 42.3 1.8 154 19.0 1.1 7.1 0.7 155 30.3 1.5 7.0 1.3 Note: Molecular structures of the reference samples, 154 and 155, are described below: 1-ethyl-indirubin (154), 1-ethyl-indirubin-3-oxime (155)

(143) ##STR00016##

IV. Exemplification of Computational Modeling

(144) Example 22: Computer-Aided Drug Design (CADD) Indicates that 1-oxo-7-azaindirubin can Bind CDK2 at the Same Domain as 7-azaindirubin Binds, the Binding Affinities are Similar for 1-oxo-7-azaindirubin and 7-azaindirubin (FIG. 1)

(145) Although 1-oxo-7-azaindirubin and 7-azaindirubin are chemically distinct, their three dimensional structures are similar. This similarity may confer them with similar abilities to interact with molecular targets and to therefore have similar biological activity. Based on this hypothesis, we have performed a docking study to demonstrate the interaction between CDK2 and 1-oxo-7-azaindirubin (75), or 7-azaindirubin (molecular structure as below). FIG. 1 shows that the two compounds bind to the same ATP-binding domain of CDK2 with similar affinities. This result suggests that the replacement of the NH in the scaffold of 7-azaindirubin with an oxygen atom will not result in significant changes to the molecular mechanism of action of this type of chemical compound. It also suggests that computational modeling, through, for example, docking studies, may be a useful means for further structural modification of 1-oxo-(aza)indirubins (I) and 1-oxo-(aza)isoindigos (II).

1-oxo-7-azaindirubin (75) and 7-azaindirubin

(146) ##STR00017##

(147) Example 23: Computer-Aided Drug Design (CADD) Indicates that N-methyl-1-oxo-isoindigo can Bind STAT3-SH2 Domain in the Same Way as N-methylisoindigo, with Similar Binding Affinities (FIG. 2).

(148) N-methyl-isoindigo (molecular structure as below) is a known STAT3 allosteric inhibitor. It can fit in the binding pocket of human STAT3 SH2 domain and form hydrogen bonds with specific amino acid residues of the protein. The docking score is 93.9797. N-methyl-1-oxoisoindigo shares a similar three dimensional structure with N-methyl-isoindigo, and therefore potentially has functions similar to N-methyl-isoindigo, i.e., inhibiting STAT3 function through SH2 domain binding. Based on this hypothesis, we performed a docking study of N-methyl-1-oxoisoindigo with human STAT3-SH2. The results indicate that the bindings of N-methyl-1-oxoisoindigo and N-methyl-isoindigo with human STAT3-SH2 are indeed similar, suggesting that the replacement of the nitrogen atom at position 1 of N-methyl-isoindigo with an oxygen atom does not significantly change the mode-of-action for these types of molecules.

N-methylisoindigo and N-methyl-1-oxoisoindigo (93)

(149) ##STR00018##

(150) What has been described above illustrates and demonstrates the basic principles, the main features and the potential advantages of the present invention. Technical professionals in this field should understand that the present invention is not limited to the examples listed in the text. These examples as well as other descriptions in the present invention serve to illustrate the scientific fundamentals of the present invention; various changes and improvements can also be made within the scope and the spirit of the present invention, and all these changes and improvements fall into the category of the claims of the present invention. The protection category of the present invention is defined by the attached Claims and their equivalents.