HK1036452B - Substituted bisindolymaleimides for the inhibition of cell proliferation - Google Patents

Description

Substituted bisindolylmaleimides for inhibiting cell proliferation

Technical Field

The present invention relates to substituted pyrroles of the formula

Wherein R is¹Is hydrogen and R²Is methyl, or

R¹Is methyl and R²Is hydrogen, or

R¹Is hydroxymethyl and R²Is methyl

It also relates to pharmaceutically acceptable prodrugs or pharmaceutically acceptable salts thereof. Background of the inventioncompounds of formula I have antiproliferative activity, in particular, they inhibit cell division in the G2/M phase of the cell cycle and are therefore commonly referred to as "G2/M phase cell cycle" inhibitors.

The compounds of formula I are covered by formula I of u.s.p.5,057,614 (but not specifically disclosed as such or alone). Furthermore, the above-mentioned activity of the compounds of the invention is not disclosed or indicated in U.S. Pat. No. 5,057,614 and is therefore surprising.

Summary of the inventionthe formula I above includes the following three compounds:

the term "pharmaceutically acceptable prodrug" denotes a compound that: it may be converted to any compound of formula I or to a pharmaceutically acceptable salt of said compound under physiological conditions or by solvolysis.

The compounds of formula I and pharmaceutically acceptable salts of said compounds are prepared by the reactions represented by the following schemes. The synthesis of each of these compounds is also described in examples 1-3.

Compound I-1 can be prepared by: (1-methyl-6-nitro-1H-indol-3-yl) -oxo-acetyl chloride (3) is reacted with [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl ] -3-ethylidene amino acid 1-methylethyl ester hydrochloride (7), and the reaction product is treated with a base.

Compound I-2 can be prepared by: (1-methyl-1H-indol-3-yl) -oxo-acetyl chloride (17) is reacted with [1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl ] -3-ethylidene amino acid 1-methylethyl ester hydrochloride (19), and the reaction product is treated with a base.

Compound I-3 can be prepared by: (1-methoxymethyl-1H-indol-3-yl) -oxo-acetyl chloride (10) is reacted with (1-methyl-6-nitro-1H-indol-3-yl) -3-ethylideneamino acid 1-methylethylidene hydrochloride (15), and the reaction product is treated with an acid.

FIG. 1 schematically shows

FIG. 2 is a schematic view of

The antiproliferative activity of the compounds of the invention is illustrated below. These effects suggest that: the compounds are useful in the treatment of cancer, particularly solid tumors.

The estrogen receptor negative epithelial breast cancer line (MDA-MB-435) was purchased from the American type cell culture Collection (ATCC; Rockville, Md.) and grown in the media recommended by the ATCC. To analyze the effect of test compounds on the growth of these cells, cells were plated at 2000 cells per well in 96-well tissue culture plates ("assay plates") and incubated at 37 ℃ and 5% CO₂Incubate overnight in the presence. The next day, test compounds were dissolved in 100% Dimethylsulfoxide (DMSO) to give a 10mM stock solution. Each compound was diluted to 1mM with sterile distilled water and then added to triplicate wells of a 96 well "master plate" (the wells contained sufficient medium to give a final concentration of 40. mu.M). The compounds in the "master plate" were diluted one by one with medium. One quarter of the final volume of the diluted compound was transferred to two "test plates". DMSO was added to a row of "control cells" to give a final DMSO concentration of 0.1% per well. The "test plate" was returned to the incubator and one "test plate" was analyzed 3 days after the addition of the test compound as follows. Similarly, 5 days after the addition of test compound, the second "test panel" was paneled as follows.

3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyl-2H-tetrazolium bromide (thiazolyl blue); MTT) was added to each well to a final concentration of 1 mg/ml. Then at 37 deg.CThe plates were incubated for 3 hours. Then, the medium containing MTT was removed and 50. mu.l of 100% ethanol was added to each well to dissolve the formed formazan * metabolite. To ensure complete dissolution, the plates were shaken for 15 minutes at room temperature. The absorbance at a wavelength of 570nm (650 nm as reference) was read in a microtiter plate reader (molecular dynamics). Percent inhibition was calculated by subtracting the blank from all wells and subtracting the quotient of the mean absorbance of triplicates divided by the mean of the controls for each trial from 1.00. Inhibitory Concentration (IC) was determined from linear regression of a plot of log of concentration versus percent inhibition₅₀And IC₉₀)。

Colon adenocarcinoma line SW480 and colon carcinoma line HCT-116 were also obtained from ATCC and tested according to the same protocol as previously provided (modified as follows). Cell line SW480 was plated at 1000 cells per well and analyzed 6 days after addition of test compound. Cell line HCT-116 was plated at 750 cells per well and analyzed 4 days after addition of test compound. For the MTT assay, the plates were centrifuged at 1000rpm for 5 minutes and 100. mu.l of 100% ethanol was used to dissolve the formazan * before aspirating the MTT-containing medium.

The results of the previous in vitro tests are set forth in tables I-III.

TABLE I

Antiproliferative activity in the cell line MDA-MB-435

Compound (I)	IC50(μM)
		Compound I-1	0.03*
TransformingCompound I-3	0.05*
		Compound I-2	0.6*

^*Average of at least three independent tests

TABLE II

Antiproliferative activity in the cell line HCT-116

Compound (I)	IC50(μM)
		Compound I-1	0.17*
Compound I-3	0.23*
		Compound I-2	1.66*

^*Average of at least three independent tests

TABLE III

Antiproliferative activity in the cell line SW480

Compound (I)	IC50(μM)
		Compound I-1	0.20*
Compound I-3	0.22*
		Compound I-2	1.86*

^*Average of at least three independent tests

To analyze the effect of the compounds on cell cycle progression, MDA-MB-435 cells (ATCC; Rockville, Md.) were plated at 1X 10 cells per 10cm dish⁶Individual cells/10 ml were plated in growth medium as follows: RPMI 1640+ 10% heat-inactivated fetal bovine serum, 2mM L-glutamine and 5U/ml pen-strep (both from GIBCO/BRL, Gaithersburg, Md.). At 37 ℃ and 5% CO₂Cells were incubated overnight in the presence. The next day, 10 μ l of each test compound in 100% DMSO was added to each dish to give 1/1000 × final concentration stock solution. In addition, 10. mu.l of 100% DMSO was added to the control dish. The final concentration of DMSO in all plates, including the control plate, was 0.1%. The plates were returned to the incubator.

Then, at each time period, the medium in each plate was transferred to a 50ml centrifuge tube. The cell layer remaining in the dish was washed with 5ml of phosphate buffered saline (PBS; GIBCO/BRL). PBS was removed and combined with the medium in the respective tubes. Cells were trypsinized at 37 ℃ for 5 minutes, and the solution was collected and combined with the medium and PBS in the respective tubes. The tubes were then centrifuged at 1200rpm for 5 minutes. The cells were thus fixed: the supernatant was removed, the pellet was dispersed by tapping the centrifuge tube, and 5ml of cold 70% ethanol was added while gently spinning. The cells were then stored at-20 ℃ for more than 24 hours.

And taking the tube containing the cells out of the refrigerator, and standing at room temperature for 20-30 minutes. The tubes were centrifuged at 3000rpm for 5 minutes. The supernatant wave was removed and the pellet washed with 5ml PBS and the tube centrifuged as above. Subsequently, the supernatant was removed and the pellet was resuspended in 0.5ml PBS. Thereafter, 0.5ml RNase A (RNAseA) (1mg/ml in PBS) was added to each tube and the tubes were incubated at 37 ℃ for 15 minutes. Mu.l of propidium iodide (Sigma, St. Louis, Mo) (1mg/ml in PBS) was added to each tube, and the tubes were incubated at room temperature for 2-3 minutes. Each of the resulting solutions was filtered through a filter cap tube (Becton Dickinson, San Jose, Calif., # 2235).

The samples were read in a FACStort instrument (Becton-Dickinson) using the manufacturer's CellQUEST program and analyzed using the manufacturer's ModFIT software. This assay provides an indication of the following percentage of cells in each phase: stage G0/G1, stage DNA synthesis (S) and stage G2/M.

The results of the cell cycle progression experiments were analyzed one day after addition of test compounds I-1, I-2 and I-3 and are summarized in Table IV below.

TABLE IV

Effect of test Compounds on cell cycle

Compound (I)	Concentration of	Cell in each cell cycle%
					G1	S	G2/M
		DMSO	0.1％	43.93％				41.08％	14.99％
Compound I	0.1μM				8.27％	25.21％	66.52％
		Compound I	0.03μM	45.30％				34.67％	20.03％
Compound I	0.01μM				44.95％	41.04％	14.00％
		Compound I-3	0.3μM	1.11％				24.99％	73.90％
Compound I-3	0.1μM				15.54％	24.06％	60.40％
		Compound I-3	0.03μM	45.45％				38.06％	16.50％
Compound I-2	10μM				10.41％	35.25％	54.34％
		Compound I-2	3μM	3.26％				48.75％	47.99％
Compound I-2	1μM				27.21％	30.19％	42.60％

The results summarized in the foregoing tables I-IV indicate that: compounds I-1, I-2 and I-3 have antiproliferative activity; specifically, they cause the accumulation of cells in the G2/m phase of the cell cycle.

The aforementioned azoles of the formula I and the aforementioned salts thereof can be used as medicaments (for example in the form of pharmaceutical preparations) which can be administered orally, for example in the form of tablets, coated tablets, dragees, hard or soft gelatine capsules, solutions, emulsions or suspensions. They may also be administered rectally, for example, in the form of suppositories; or parenterally, e.g., in the form of injection solutions.

To produce pharmaceutical preparations, these compounds can be formulated with therapeutically inert inorganic or organic carriers. Lactose, corn starch or derivatives thereof, talc, stearic acid (stearic acid) or its salts can be used as such carriers in tablets, coated tablets, dragees and hard gelatine capsules. Suitable carriers for soft gelatin capsules are: vegetable oils, waxes, fats, semi-solid or liquid polyols. Depending on the nature of the active substance, however, no carriers are generally required in the case of soft gelatin capsules. Suitable carriers for the production of solutions and syrups are: water, polyols, sucrose, invert sugar and glucose. Suitable carriers for injection solutions are: water, alcohol, polyols, glycerol, vegetable oils, phospholipids and surfactants, suitable carriers for suppositories are: natural or hardened oils, waxes, fats and semi-liquid polyols.

The pharmaceutical preparations may also contain preserving agents, solubilizers, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They may even contain other therapeutically useful substances.

As previously mentioned, pyrroles of formula I and their aforementioned salts may be used in the treatment or control of neoplastic diseases. The dosage can vary within wide limits and, of course, should be adjusted to the individual requirements in each particular case. Generally, for oral or parenteral administration to an adult human weighing about 70kg, a daily dosage of about 10mg to about 10,000mg, preferably about 200mg to about 5,000mg, more preferably about 200mg to about 1000mg, should be suitable, although this upper limit can be exceeded if necessary. The daily dose may be administered as a single or divided dose, or, in the case of parenteral administration, as a continuous infusion.

The following examples illustrate the invention.

Example 1 3- (1H-indol-3-yl) -4- (1-methyl-6-nitro-1H-indol-3- Preparation of yl) -pyrrole-2, 5-dione (I-1)A. 1-methyl-6-nitro-1H-indole (2)

To a slurry of 0.33g (8.3mmol) NaH (60% dispersion in oil) in 30ml dry dimethylformamide ("DMF") was added 0.973g (6.00mmol) of commercially available 6-nitro-1H-indole (1) over a period of 10 minutes at 0-5 deg.C. After stirring at the same temperature for 1 hour, 0.75ml (12.1mmol) of methyl iodide was added, and the mixture was stirred at the same temperature for 30 minutes, further stirred at room temperature for 1 hour, poured into ice water, and extracted with ethyl acetate. The organic phase was washed with brine over MgSO₄Dried and concentrated to give 0.814g (77.5%) of 1-methyl-6-nitro-1H-indole (2) as a yellow solid. The material was used directly without purification. B.(1-methyl-6-nitro-1H-indole)-3-yl) -oxo-acetyl chloride (3)

To a solution of 1.33g (7.55mmol) of 1-methyl-6-nitro-1H-indole (2) in 40ml of diethyl ether was added 1.5ml (17.2mmol) of oxalyl chloride under an argon atmosphere at 0 to 5 ℃. A precipitate formed. After stirring for 3 hours, the solid formed is filtered, washed with a little ether and dried to yield 1.9g (95%) (1-methyl-6-nitro-1H-indol-3-yl) -oxo-acetyl chloride (3) as a yellow solid. The material was used directly without purification. C.[1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl]-acetonitrile (6)

Using the procedure of subsection A above, an N-alkylation reaction of 10.2g (65mmol) of commercially available (1H-indol-3-yl) -acetonitrile (5) with 8.7ml (71mmol) of trimethylacetyl chloride and 3.4g (85mmol) of NaH (60% dispersion in oil) as base in 115ml of DMF gave, after purification by chromatography, 6.6g (38.7%) [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl ] -acetonitrile]Acetonitrile (6) yellow oil. D.[1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl]-3-ethylideneamino acid 1- Ethyl methyl ester hydrochloride (7)

To 6.6g (27.5mmol) of [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl obtained as above in step C during 20 min at 0-5 deg.C]Acetonitrile (6) to a slurry in 105ml of 2-propanol 40ml (0.563mol) of acetyl chloride are added dropwise. The reaction mixture was stirred overnight at room temperature, concentrated, and the residue diluted with approximately 75ml of ethyl acetate, heated on a steam bath for 15 minutes, cooled and placed in a freezer. The precipitate was filtered and dried to give 6.0g (65.0%) of [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl]-3-ethylideneamino acid 1-methylethyl ester hydrochloride (7) as a white solid. E.3- [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl]-4- (1-methyl-6) -nitro-1H-indol-3-yl) -pyrazine-2, 5-dione (4)

Add 1.25g (4.69mmol) of (1-methyl-6-nitro-1H-indol-3-yl) -oxo-acetyl chloride (3) obtained as in step B above and 1.6g (4.75mmol) of [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl obtained as in step D above under an argon atmosphere at 0 deg.C]To a solution of (7) 3-ethylideneamino acid 1-methylethyl ester in 80ml of methylene chloride was added 2.6ml (18.65 m)mol) of triethylamine. After stirring at the same temperature for 30 minutes, the reaction mixture was stirred at room temperature for 31/2 hours and diluted with more dichloromethane. The organic phase was washed with water, 0.5N HCl solution, brine, over MgSO₄Dried and concentrated to give 3.01g of a foam. This material was dissolved in 50ml of toluene and treated with 987.9mg (5.19mmol) of p-toluenesulfonic acid at 0 ℃. After stirring at room temperature for 3 hours, the reaction mixture was extracted with dichloromethane. With saturated NaHCO₃The organic phase was washed with brine over MgSO₄Dried and concentrated to give 3.9g of crude material. Purification by chromatography on a silica gel column gives 1.7g (77%) of 3- [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl]-4- (1-methyl-6-nitro-1H-indol-3-yl) -pyrrole-2, 5-dione (4) as an orange solid. mp > 146 deg.C (decomposition). MS: (M)⁺)，m/z 470。3- (1H-indol-3-yl) -4- (1-methyl-6-nitro-1H-indole-3 -yl) -pyrrole-2, 5-dione (I-1)

With 5.6ml (8.96mmol) of 1.6 mol NaOCH₃Treatment of 1.7g (3.61mmol) of 3- [1- (2, 2-dimethyl-propionyl) -1H-indol-3-yl radical from step E above with methanolic solution]-4- (1-methyl-6-nitro-1H-indol-3-yl) -pyrrole-2, 5-dione (4) in 60ml methanol. The reaction was stirred at room temperature for 1 hour, poured onto 2N HCl/ice and extracted with ethyl acetate. Over anhydrous MgSO₄The organic extracts were dried, concentrated and purified by chromatography to give 394.7mg (28%) 3- (1H-indol-3-yl) -4- (1-methyl-6-nitro-1H-indol-3-yl) -pyrrole-2, 5-dione (I-1) as a red solid, mp > 280 ℃. MS: (M)⁺)m/z386。

Example 2 3- (1-hydroxymethyl-1H-indol-3-yl) -4- (1-methyl-6-nitro-1H Preparation of (I) -indol-3-yl) -pyrrole-2, 5-dione (I-3)A. 1-methoxymethyl-1H-indole (9)

Using the procedure of step A in example 1, 1.17g (10mmol) of commercially available indole (8) were N-alkylated with 1ml (13.1mmol) of chloromethyl methyl ether and 0.48g (12mmol) of NaH (60% dispersion in oil) as base in 22ml of DMF and purified by chromatography to give 1.4g (86.9%) of 1-methoxymethyl-1H-indole (9) as colorlessAnd (3) oil. B.(1-methoxymethyl-1H-indol-3-yl) -oxo-acetyl chloride (10)

Using the procedure of step B of example 1, 0.23g (1.43mmol) of 1-methoxymethyl-1H-indole (9) from step A above was reacted with 0.25ml (2.86mmol) of oxalyl chloride in 3.5ml of diethyl ether to give 0.174g (48.5%) (1-methoxymethyl-1H-indol-3-yl) -oxo-acetyl chloride (10) as a yellow solid. The material was used directly without purification. C.(6-Nitro-1H-indol-3-yl) -acetonitrile (13)

To 44.27g (0.204mol) of 6-nitrograminine (12) [ Jackson B.Hester, J.Org.Chem ] was added with stirring during one hour at 0-5 deg.C29：1158(1964)]44.59g (0.31mol) of methyl iodide were added to 450ml of the acetonitrile solution. The reaction mixture was stirred at room temperature for three hours, after which 26.6g (0.543mol) of aqueous solution of sodium cyanide (225 ml) were added in one portion. The reaction mixture was heated at 32 ℃ overnight, cooled to room temperature and the product was extracted 3 times with a total of 800ml of ethyl acetate and 300ml of water. The combined extracts were washed with water, 1N HCl solution, saturated sodium bicarbonate solution over MgSO₄Dried and the solvent evaporated in vacuo. The orange-brown residue (41.3g) was dissolved in 200ml of warm ethyl acetate and passed through a small pad of silica gel to yield 28.9g (70.4%) of (6-nitro-1H-indol-3-yl) -acetonitrile (13) as a yellow solid after evaporation of the solvent. D.(1-methyl-6-nitro-1H-indol-3-yl) -acetonitrile (14)

65.5g (0.474mol) of powdered potassium carbonate are added at room temperature to a solution of 28.9g (0.143mol) of (6-nitro-1H-indol-3-yl) -acetonitrile (13) from step C above in 230ml of dimethylformamide. The suspension is stirred for 40 minutes and then 25.48g (0.179mol) of methyl iodide are added dropwise over 65 minutes. After stirring overnight at room temperature, the reaction mixture was cooled and poured into a total of 600ml of water. The precipitate was filtered, washed with a small amount of water and dried over phosphoric anhydride (phosphoric anhydride) until constant weight. This procedure yielded 30.4g (95.4%) (1-methyl-6-nitro-1H-indol-3-yl) -acetonitrile (14), which was used directly without further purification. E.(1-methyl-6-nitro-1H-indol-3-yl)) -3-ethylideneamino acid 1-A Ethyl ester hydrochloride (15)

A stream of HCl gas was bubbled through a suspension of 82g (0.382mol) of (1-methyl-6-nitro-1H-indol-3-yl) -acetonitrile (14) from step D above in 1000ml of 2-propanol with stirring at 0-10 ℃. After addition of about 350g of HCl, diethyl ether was added to the reaction mixture until a precipitate formed. The solid was collected, washed with ether and dried in vacuo to give 102g (85.7%) (1-methyl-6-nitro-1H-indol-3-yl) -3-ethylideneamino acid 1-methylethyl ester hydrochloride (15). F.3- (1-methoxymethyl-1H-indol-3-yl) -4- (1-methyl-6-nitro-n-methyl-n-propyl-p-nitrophenyl sulfate yl-1H-indol-3-yl) -pyrrole-2, 5-dione (11)

Using the procedure of step E in example 1, 1.3g (5.17mmol) of oxoacetyl chloride (10) obtained as in step B above was condensed with 1.7g (5.45mmol) of (1-methyl-6-nitro-1H-indol) -3-ethylideneamino acid 1-methylethylidene hydrochloride (15) obtained as in step E above in 95ml of dichloromethane to give 1.08g (48.5%) of 3- (1-methoxymethyl-1H-indol-3-yl) -4- (1-methyl-6-nitro-1H-indol-3-yl) -pyrrole-2, 5-dione (11) as an orange solid, mp > 250 deg.C (decomposition). MS: (M)⁺)，m/z430。

G. 3- (1-hydroxymethyl-1H-indol-3-yl) -4- (1-methyl-6-nitro-compound yl-1H-indol-3-yl) -pyrrole-2, 5-dione (I-3)

727.5mg of 3- [1- (methoxymethyl) -1H-indol-3-yl group obtained from step F above were treated with about 40ml of 2N HCl]-a solution of 4- (1-methyl-6-nitro-1H-indol-3-yl) -pyrrole-2, 5-dione (11) in 65ml THF. The reaction mixture was refluxed for 5 hours, cooled and the product extracted with ethyl acetate. Over MgSO₄The organic phase was dried and the solvent evaporated to give an orange solid. This material was purified by chromatography to give 123.3mg of 3- (1-hydroxymethyl-1H-indol-3-yl) -4- (1-methyl-6-nitro-1H-indol-3-yl) -pyrrole-2, 5-dione (I-3) as a red solid, mp 210-213 ℃. MS: (M)⁺)，m/z416。

Example 3 3- (1-methyl-1H-indol-3-yl) -4- (6-nitro-1H-indoleIndole-3- Preparation of Yl) -Pyri-p-2, 5-dione (I-2)A. (1-methyl-1H-indol-3-yl) -oxo-acetyl chloride (17)

Using the procedure of step B in example 1, 6ml (47mmol) of commercially available 1-methyl-1H-indole (16) were reacted with 8ml (92mmol) of oxalyl chloride in 120ml of diethyl ether to yield 7.6g (73.2%) (1-methyl-1H-indol-3-yl) -oxo-acetyl chloride (17) as a yellow solid. The material was used directly without purification. B.[1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl]-acetonitrile (18)

Using the procedure of step A in example 1, 346.6mg (1.72mmol) of 6-nitro-1H-indolyl-3-acetonitrile (13) from step C in example 2 were N-alkylated in 8ml of DMF using 0.3ml (2.44mmol) of trimethylacetyl chloride and 70.8mg (1.77mmol) of NaH (60% dispersion in oil) as base to yield 287.7mg (43.2%) of [1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl after purification by chromatography]Acetonitrile (18) yellow oil. C.[1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl]-3-ethylene Amino acid 1-methylethyl hydrochloride (19)

Bubbling HCl gas stream into 1.45g (5.08mmol) of 1[ - (2, 2-dimethyl-propionyl) -6-nitro-1H-indolyl radical obtained from step B above at 0-5 deg.C under constant stirring]-3-acetonitrile (18) in a suspension of 90ml 2-propanol. The reaction mixture was stirred at room temperature for 21 hours. The solvent was evaporated in vacuo to give 1.95g (100%) of a yellow solid (19). The material was used without further purification. D.3- [1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl]-4-(1 -methyl-1H-indol-3-yl) -pyrrole-2, 5-dione (20)

Using the procedure of step E in example 1, 1.1g (4.96mmol) of oxoacetyl chloride (17) obtained as in step A above was reacted with 1.95g (5.08mmol) of [1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl obtained as in step C above]Reaction of (19) 3-ethylideneamino acid 1-methylethyl ester hydrochloride and 2.1ml (17.94mmol) of triethylamine in 120ml of dichloromethane, treatment with 1.1g (5.78mmol) of p-toluenesulfonic acid monohydrate in 80ml of tolueneThe resulting product gave 1.3g (62.1%) of 3- [1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl]-4- (1-methyl-1H-indol-3-yl) -pyrrole-2, 5-dione (20) as an orange solid; mp > 245 deg.C (decomposition). MS: (M)⁺)，m/z470。E. 3- (1-methyl-1H-indol-3-yl) -4- (6-nitro-1H-indol-3-yl) -yl) -pyrrole-2, 5-dione (I-2)

Using the procedure of step F in example 1, 4.3ml (6.88mmol) of 1.6 mol NaOCH are used₃A solution in 65ml of methanol versus 1.3g (2.76mmol) of 3- [1- (2, 2-dimethyl-propionyl) -6-nitro-1H-indol-3-yl obtained as above in step D]-4- (1-methyl-1H-indol-3-yl) -pyrrole-2, 5-dione (20) was N-deprotected to give after crystallization from ethyl acetate and hexane 300.6mg (28.1%) 3- (1-methyl-1H-indol-3-yl) -4- (6-nitro-1H-indol-3-yl) -pyrrole-2, 5-dione (I-2) as a red solid; mp > 260 ℃. MS: (M)⁺)，m/z386。

Example 4

Tablet formulation

Item	Components	mg/tablet
								1	Compound A*	5	25	100	250	500	750
2	Anhydrous lactose	103	83	35	19	38	57
								3	Croscarmellose Sodium (Croscarmellose Sodium)	6	6	8	16	32	48
4	Povidone K30(Povidone K30)	5	5	6	12	24	36
								5	Magnesium stearate	1	1	1	3	6	9
	Total weight of	120	120	150	300	600	900

^*Compound a represents a compound of the present invention.

The preparation operation comprises the following steps:

1. items 1, 2 and 3 were mixed in a suitable mixer for 15 minutes.

2. The powdered mixture of step 1 was granulated with 20% povidone K30 solution (item 4).

3. The granulate of step 2 was dried at 50 ℃.

4. The granulation of step 3 is passed through a suitable milling apparatus.

5. Add item 5 to the milled granulation of step 4 and mix for 3 minutes.

6. The granulation of step 5 is pressed on a suitable press.

Example 5

Capsule formulation

Item	Components	mg/tablet
							1	Compound A*	5	25	100	250	500
2	Hydrous lactose	159	123	148	--	--
							3	Corn starch	25	35	40	35	70
4	Talcum powder	10	15	10	12	24
							5	Magnesium stearate	1	2	2	3	6
	Total filling weight	200	200	300	300	600

^*Compound a represents a compound of the present invention.

The preparation operation comprises the following steps:

1. items 1, 2 and 3 were mixed in a suitable mixer for 15 minutes.

2. Add items 4&5 and mix for 3 minutes.

3. Filling into suitable capsules.

Example 6

Injectable solution/emulsion formulations

Item	Components	mg/ml
			1	Compound A*	1mg
2	PEG 400	10-50mg
			3	Lecithin	20-50mg
4	Soybean oil	1-5mg
			5	Glycerol	8-12mg
6	Proper amount of water	1ml

^*Compound a represents a compound of the present invention.

The preparation operation comprises the following steps:

1. dissolve item 1 in item 2.

2. Items 3, 4 and 5 were added to item 6 and mixed until dispersed, then homogenized.

3. The solution of step 1 was added to the mixture of step 2 and homogenized until the dispersion became translucent.

4. Sterile filtration was performed through a 0.2 μm filter and filled into vials.

Example 7

Injectable solution/emulsion formulations

Item	Components	mg/ml
			1	Compound A*	1mg
2	Glycofurol	10-50mg
			3	Lecithin	20-50mg
4	Soybean oil	1-5mg
			5	Glycerol	8-12mg
6	Water (W)	q.s.1ml

^*Compound a represents a compound of the present invention.

The preparation operation comprises the following steps:

1. dissolve item 1 in item 2.

2. Items 3, 4 and 5 were added to item 6 and mixed until dispersed, then homogenized.

3. The solution of step 1 was added to the mixture of step 2 and homogenized until the dispersion became translucent.

4. Sterile filtration was performed through a 0.2 μm filter and filled into vials.

Claims (5)

1. A compound of the formula

Wherein R is¹Is hydrogen and R²Is methyl, or

R¹Is hydroxymethyl and R²Is methyl and pharmaceutically acceptable salts thereof.

2. The compound of claim 1 of the formulaAnd pharmaceutically acceptable salts of said compounds.

3. A compound of the formulaAnd pharmaceutically acceptable salts of said compounds.

4. A pharmaceutical composition comprising a compound of the formula:

wherein R is¹Is hydrogen and R²Is methyl, or

R¹Is hydroxymethyl and R²Is methyl or a pharmaceutically acceptable salt of said compound and a pharmaceutically acceptable carrier.

5. Use of a compound of claim 1 for the preparation of a pharmaceutical composition for the treatment of solid tumors.