KR101132599B1 - Indole derivatives with apoptosis-inducing effect - Google Patents

Abstract

The present invention relates to indole derivatives which are used as drugs for the treatment of neoplastic diseases, especially in the case of resistance of drugs to other active ingredients and metastatic carcinomas.

Neoplastic disease, drug resistance, metastatic carcinoma, indole derivatives

Description

Indole derivatives with apoptosis-inducing effect

The present invention has excellent biological effects, is excellent in tolerability and oral bioavailability, and is used as a drug for treating tumor diseases, especially in the presence of drug resistance to other active compounds and metastasis of carcinoma. It relates to a novel indole derivative.

Treatment of cancer diseases is very important in the medical field. There is a worldwide need for effective cancer therapies to achieve appropriate and targeted oriented therapies for patients. This can be seen from a number of scientific studies emerging in the field of basic research related to applied oncology and cancer treatment in recent years.

The effect of tumor suppressors is due to a very wide range of mechanisms, only some of which are known. Known tumor drugs found to have novel mechanisms of action are not particular. This is also expected in the case of the compounds according to the invention. Many tumor drugs block cell division mechanisms in cells, inhibit the supply of nutrients and oxygen to the tumor (antiangiogenesis), inhibit metastasis, inhibit the receipt of growth signals and subsequent transmission to tumor cells, or , By mechanisms such as leading tumor cells to planned cell death (apoptosis).

Because they have different mechanisms of action, including interaction with different intracellular targets, clinically relevant cytostatic inhibitors are often administered together to achieve a synergistic therapeutic effect.

Indole derivatives are used as pharmacodynamic active compounds in a wide variety of ways and as structural units for synthesis in pharmaceutical chemistry.

International publications WO 99/51224 A1 and WO 01/22954 A1 have anti-tumor effects and have 2-, 3-, 4- and 8-quinoline radicals or 2-, 3-, Indol-3-yl derivatives are described which may be substituted by a number of groups including 4-, 5- and 6-pyridine radicals. It is mentioned in Example 60 that the 2-methyl-8-quinolinyl group is a substituent on the amide group. However, no biological properties are mentioned at all.

WO 99/55696 A1 describes hydroxyindoles substituted as inhibitors of phosphodiesterase 4. However, the compounds according to the invention are not described as having antitumor activity and do not suggest that they can have such activity.

WO 02/08225 A1 describes 2- (1H-indol-3-yl) -2-oxo-acetamide derivatives having antitumor effect in connection with solid tumors. However, specific examples containing quinoline, pyridopyrazine or indazolyl radicals are not mentioned.

Patent specification WO 00/67802 describes indole-3-glyoxylamides substituted with relatively long chain fatty acids as potent antitumor agents. However, specific examples containing quinoline, pyridopyrazine or indazolyl radicals are not mentioned. There is no biological data at all associated with this example.

Publications [W.-T. Li et al., J. Med. Chem. 2003, 46, 1706 ff. Describe N-heterocyclic indolylglyoxylamide as an oral active compound with antitumor activity. However, no information is provided about their mechanism of action.

WO 03/022280 A2 describes 3-glyoxylamide indole and their use as anti-tumor therapeutic drugs. Their formula also includes 6-quinoline derivatives. In addition, two examples containing 6-quinoline radicals are mentioned as examples and demonstrated as biological results. However, there is no mention of specific examples containing pyridopyrazine or indazolyl radicals.

No. 03/0181482 A1 describes a novel indolylglyoxylamide. In this case, the compounds according to the invention are described as antitumor agents and angiogenesis inhibitors having cytotoxic activity. In addition, 6-quinoline derivatives are shown as examples (Compound 3: p. 10) and antiproliferative data [p. 19; Tables 1a and 1b] and antiangiogenic properties (p. 20). However, there is no mention of specific examples containing pyridopyrazine or indazolyl radicals.

Applicant has already described a second group of indole derivatives for treating tumors in WO 02/10152 A2. In this patent document, in particular, the anti-proliferative effect of the active compound N- (2-methyl-6-quinolyl)-[1- (4-chlorobenzyl) indol-3-yl] glyoxylamide in various tumor cell lines Tested about.

Clinically tested compounds that bind to microtubules (paclitaxel and vincristine) or inhibit topoisomerase II (doxorubicin, etoposide and mitoxantrone) are currently present, particularly in breast, ovarian, gastric and lung cancers. It has been successfully used for cancer therapy, Kaposi's sarcoma and leukemia. However, their use is limited by the emergence of drug resistance and severe neurological, gastrointestinal, cardiovascular and liver side effects.

It is a primary object of the present invention to produce available cytotoxic substances which comprise a complex mechanism of action and which are particularly suitable for treating a large number of tumors when active compound resistance is present against other drugs and the carcinoma metastases.

This object is achieved by indole derivatives of formula I, their tautomers, their stereoisomers, including diastereomers and enantiomers, and their physiologically acceptable salts.

In Formula I,

R is a substituted or unsubstituted (C ₂ -C ₁₄ ) -heterocycle containing one or more hetero atoms selected from groups N, O and S and which is saturated, unsaturated or aromatic bonded directly to the amide nitrogen, wherein the heterocycle is Preferably (i) unsubstituted or substituted 5-, 6- and 7-quinolyl, (ii) unsubstituted or substituted 2-, 3-, 6-, 7- and 8-pyridopyrazinyl, ( iii) unsubstituted or substituted 3-, 4-, 5-, 6- and 7-indazolyl, (iv) unsubstituted or substituted 2-, 3-, 4-, 5- and 6-pyridyl, ( v) unsubstituted or substituted 3-, 4- and 5-isoxazolyl or (vi) unsubstituted or substituted 3-, 4- and 5-isothiazolyl,

R 1 is unsubstituted or substituted alkyl-aryl,

R 2 is (i) hydrogen or (ii) unsubstituted or substituted (C ₁ -C ₆ ) -alkyl,

R3 to R6 are (i) hydrogen, (ii) unsubstituted or substituted (C ₁ -C ₆ ) -alkyl, (iii) unsubstituted or substituted (C ₃ -C ₇ ) -cycloalkyl, (iv) amino , Mono- (C ₁ -C ₄ ) -alkylamino, di- (C ₁ -C ₄ ) -alkylamino, (v) halogen, (vi) (C ₁ -C ₄ ) substituted by one or more fluorine atoms -alkyl, preferably methyl group, (vii) trifluoromethyl, cyano, straight-chain or branched-cyano - (C ₁ -C ₆₎ - _{alkyl, (viii) (C 1 -C} 6) - alkylcarbonyl, ( ix) carboxyl, (C ₁ -C ₄ ) -alkoxycarbonyl, carboxy- (C ₁ -C ₆ ) -alkyl or (C ₁ -C ₆ ) -alkoxycarbonyl- (C ₁ -C ₆ ) -alkyl, (x) hydroxyl, (xi)-(C ₁ -C ₆ ) -alkoxy, (xii) aryl- (C ₁ -C ₄ ) -alkoxy, preferably benzyloxy or (xiii) (C ₁ -C ₆ ) -Alkoxycarbonylamino, (C ₁ -C ₆ ) -alkoxycarbonylamino- (C ₁ -C ₆ ) -alkyl,

R7 is (C ₁ -C ₆ ) -alkylcarbonyl, preferably acetyl or propionyl, (C ₁ -C ₆ ) -alkoxycarbonyl, preferably methoxycarbonyl, ethoxycarbonyl or propoxycarbon Neal,

X and Y are oxygen or sulfur.

When R is an unsubstituted or substituted 2-, 3-, 4-, 5- or 6-pyridyl group and R1 to R6 are as described above, R7 is in this case an acetyl radical or tertiary butyloxycarbonyl group Should not be this.

상기 화학식 I에서, The invention also relates to indole derivatives of formula (I), to tautomers thereof, to diastereomers thereof and to stereoisomers thereof, including physiologically acceptable salts thereof:
Formula I

In Formula I,

R is (i) substituted 6-quinolyl, unsubstituted or substituted 7-quinolyl, directly bonded to the amide nitrogen, except for 2-methyl-6-quinolyl, and if X is a sulfur atom, R is also substituted Unsubstituted 6-quinolyl, or (ii) unsubstituted or substituted 2-, 3-, 6-, 7- and 8-pyridopyrazinyl or (iii) unsubstituted or substituted 3-, 4 -, 5-, 6- and 7-indazolyl,

R 1 is unsubstituted or substituted alkyl-aryl,

R2 is hydrogen,

R3 to R6 are (xiv) hydrogen, (xv) unsubstituted or substituted (C ₁ -C ₆ ) -alkyl, (xvi) unsubstituted or substituted (C ₃ -C ₇ ) -cycloalkyl, (xvii) amino , Mono- (C ₁ -C ₄ ) -alkylamino, di- (C ₁ -C ₄ ) -alkylamino, (xviii) halogen, (xix) (C ₁ -C ₄ ) substituted by one or more fluorine atoms -alkyl, preferably methyl group, (xx) trifluoromethyl, cyano, straight-chain or branched-cyano - (C ₁ -C ₆₎ - _{alkyl, (xxi) (C 1 -C} 6) - alkylcarbonyl, ( xxii) carboxyl, (C ₁ -C ₄ ) -alkoxycarbonyl, carboxy- (C ₁ -C ₆ ) -alkyl or (C ₁ -C ₆ ) -alkoxycarbonyl- (C ₁ -C ₆ ) -alkyl _{, (xxiii) - (C 1} -C 6) - alkoxy, (xxiv) aryl - (C ₁ -C ₄₎ - alkoxy, preferably benzyloxy or _{(xxv) (C 1 -C 6} ) - alkoxycarbonyl Amino, (C ₁ -C ₆ ) -alkoxycarbonylamino- (C ₁ -C ₆ ) -alkyl,

R7 is hydrogen,

X and Y are oxygen or sulfur.

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The present invention is a further development of the invention described in WO 02/10152. Unsubstituted or substituted 2-, 3-, 6-, 7- and 8-pyridopyrazinyl or unsubstituted 3-, 4-, 5-, 6- And it has been observed that indole derivatives obtained by replacing with 7-indazolyl show good antiproliferative effects against many tumor cell lines.

It has also been observed that the compounds according to the invention exert potent cytotoxic effects which can be attributed to a wide variety of different mechanisms. One mechanism of the compounds according to the invention, demonstrated in the present invention, is based on inhibiting tubulin polymerization and inhibiting topoisomerase II. It inhibits tumorigenic cells in the G2M stage. In addition, the compounds according to the invention induce apoptosis.

It has also been observed that the compounds according to the invention have good solubility in water and consequently good oral bioavailability.

It has also been demonstrated that the introduction of acetyl radicals as R7 radicals leads to compounds according to the invention with good in vivo activity and good tolerance.

The class of substances described in the present invention should open up the possibility of obtaining lower tumors, longer lasting, and more tolerant anti-tumor drugs than can be achieved using conventional cell proliferation inhibitors. In particular, it should be possible to avoid the development of adverse resistances known to occur in the case of many anti-tumor agents. The increased effect achieved with the indole derivatives according to the invention should make the drug use more efficient. In addition, treatment should be prolonged if the treatment is resistant.

In a preferred embodiment, in the indole derivative of formula (I) R 1 is 4-chlorobenzyl, R 2 to R 6 are hydrogen, R is heterocycle and R 7 is alkylcarbonyl or alkoxycarbonyl.

In another preferred embodiment, R in the indole derivative of formula I is unsubstituted 5-quinolyl, unsubstituted 6-quinolyl or unsubstituted 7-quinolyl, and R7 is acetyl or propionyl.

In another preferred embodiment, R in the indole derivative of formula I is unsubstituted 5-quinolyl, unsubstituted 6-quinolyl or unsubstituted 7-quinolyl, and R7 is methoxycarbonyl, ethoxycarbon Nil or propionoxycarbonyl.

Some terms used in the description and the claims are defined below.

In the context of “heterocycle”, the term is used to describe pyrrole, furan, thiophene, pyrazole, thiazole, indole, oxazole, imidazole, isothiazole, isoxazole, 1,2, unless explicitly stated above. 3-triazole, 1,2,4-triazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, 1,2,5-thiadiazole, 1,3,4- Thiadiazole, tetrazole, pyridine, pyrimidine, pyridazine, pyrazine, benzofuran, indazole, carbazole, benzoxazole, benzimidazole, benzothiazole, benzotriazole, quinoline, cinnoline, quinoxaline, quina Sleepy, phthalazine, pyridopyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, purine, putridine, acridine and phenanthridine It is understood to mean.

Within the meaning of the present invention, the expression "alkyl" includes saturated or unsaturated acyclic hydrocarbons which may be straight or branched. In the context of "alkyl", the term "substituted" means hydrogen radicals F, Cl, Br, I, CN, NH ₂ , NH-alkyl, NH-cycloalkyl, unless explicitly defined above within the scope of the present invention. , OH or O-alkyl, wherein the polysubstituted radical is at least once, for example twice or three times at different atoms or at the same atom, eg, -CF ₃ and -CH ₂ It is understood to mean those substituted three times at the same C atom as in the case of CF ₃ or at different positions as in the case of —CH (OH) —CH ₂ —CH ₂ —CHCl ₂ . Polysubstitution may be performed using the same substituents or different substituents.

The expression "alkyl-aryl" means (C ₁ -C ₆ ) -alkyl- (C ₆ -C ₁₄ ) -aryl, preferably (C ₁ -C ₆ ) -alkyl-C ₆ -aryl.

With respect to "alkyl-aryl" and "cycloalkyl", "one or more times substituted" means one or more hydrogen atoms in the ring system unless explicitly stated as above within the meaning of the present invention. Atom at or, optionally, at different atoms (substituents may optionally be substituted as part thereof), F, Cl, Br, I, CN, NH ₂ , NH-alkyl, OH, O-alkyl, CF Single, several times, for example twice, with ₃ , alkyl, (C ₆ -C ₁₀ ) -aryl, (C ₆ -C ₁₀ ) -aryl- (C ₁ -C ₆ ) -alkyl and / or heterocyclyl, It is understood that it is substituted three or four times. In this regard, multiple substitutions are carried out using the same substituents or different substituents.

In the context of "heterocycle", "one or more times substituted" means within one meaning of one or more hydrogen atoms in the ring system, unless explicitly stated as above, Thus, at different atoms (substituents may optionally be substituted as part thereof), F, Cl, Br, I, nitro, amino, C ₁ -C ₆ -alkyl, preferably methyl, mono- (C ₁ -C ₆ ) -alkylamino, di- (C ₁ -C ₆ ) -alkylamino, hydroxyl, C ₁ -C ₆ -alkoxy, benzyloxy, carboxyl, (C ₁ -C ₆ ) -alkoxycarbonyl, ( C ₁ -C ₆ ) -alkoxycarbonylamino, or (C ₁ -C ₆ ) -alkyl substituted one or more times with fluorine, preferably trifluoromethyl, (C ₁ -C ₆ ) -aryl and / or It is understood that it is substituted once or several times, for example twice, three times or four times with (C ₆ -C ₁₀ ) -aryl- (C ₁ -C ₆ ) -alkyl. In this regard, multiple substitutions are carried out using the same substituents or different substituents.

As long as the compounds of the formula (I) according to the invention comprise at least one asymmetric center, they are in racemic form, pure enantiomers and / or diastereomer forms or in the form of mixtures of these enantiomers and / or diastereomers. May exist. Stereoisomers may be present in any proportion of mixtures. As far as this is possible, the compounds according to the invention may exist in tautomeric forms.

Thus, using known methods, for example, compounds of formula (I) according to the invention comprising at least one chiral center and produced as racemates can be separated into their optical isomers, ie enantiomers or diastereomers. Can be. Separation can be carried out by column separation on chiral or from the optically active solvent or by recrystallization with an optically active acid or base or by derivatization with an optically active reagent such as an optically active alcohol and then cutting off the residue.

If they contain sufficiently acidic groups, for example carboxyl groups, the compounds of formula I according to the invention can be converted into their physiologically acceptable salts using inorganic bases and / or organic bases. Examples of suitable inorganic bases include sodium hydroxide, potassium hydroxide and calcium hydroxide, and examples of suitable organic bases include ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dibenzylethylenediamine and lysine. In this regard, the stoichiometry of the salts of the compounds according to the invention formed may be one complete or incomplete majority.

If they comprise sufficiently basic groups, for example secondary or tertiary amines, the compounds of formula (I) according to the invention can be converted into salts using inorganic or organic acids. Pharmaceutically acceptable salts of formula (I) according to the invention are preferably hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, carbonic acid, formic acid, acetic acid, trifluoroacetic acid, oxalic acid, It is formed using malonic acid, maleic acid, succinic acid, tartaric acid, pyruvic acid, malic acid, embonic acid, mandelic acid, fumaric acid, lactic acid, citric acid, glutamic acid or aspartic acid. Salts formed are, in particular, hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, sulfoacetic acid, tosylate, carbonate, hydrogen carbonate, formate, acetate, triflate, oxalate, malonate, maleate, succinate , Tartrate, maleate, emcarbonate, mandelate, fumarate, lactate, citrate and glutamate. In this regard, the stoichiometry of the salts of the compounds according to the invention formed may be one complete or incomplete majority.

Preference is also given to solvates, in particular hydrates of the compounds of the formula (I) according to the invention, which can be obtained, for example, by crystallization from solvents or aqueous solutions. In this regard, one, two, three or any number of solvates or water molecules can be mixed with the compounds according to the invention to form solvates and hydrates.

Chemicals are known to form solids that exist in different forms, to a degree called polymorphic forms or variants. Different variants of the polymorphic materials may differ greatly in their physical properties. The compounds of formula (I) according to the invention may exist in different polymorphic forms and certain variants may be metastable.

Both compounds of formula (I) and salts thereof are biologically active. The compounds of formula (I) can be administered in free form or as salts with physiologically acceptable acids or bases.

The compounds of formula (I) can be administered orally, rectally, oral route (eg sublingual), parenteral (eg subcutaneous, intramuscular, intradermal or intravenous), topical or transdermal.

The invention also relates to a drug comprising at least one compound of formula (I) or a salt with a physiologically acceptable inorganic or organic acid and, optionally, a pharmaceutically acceptable carrier material and / or diluent or auxiliary substance.

These drugs are used for the treatment of tumor diseases, in particular for the treatment of tumor diseases including tumor diseases and / or metastatic carcinomas with drug resistance to other active compounds.

Examples of suitable dosage forms are tablets, dragees, capsules, solutions for infusion or ampoule, suppositories, plasters, powder preparations that can be used for inhalation, suspensions, creams and ointments.

The compounds according to the invention may also be dispersed in particulates, for example nanoparticle compositions.

Specifically, the properties found to be therapeutically useful are related to the following benefits:

? The compounds according to the invention are characterized by potent antiproliferative properties,

? The compounds according to the invention inhibit tubulin polymerization,

? The compounds according to the invention inhibit topoisomerase II,

? Compounds according to the invention arrest cell division at the G2 / M stage,

? Compounds according to the invention induce apoptosis,

? The compounds according to the invention are characterized by potent antitumor activity and also good tolerance in vivo,

? The compounds according to the invention are in vitro active on mdr-resistant cell lines, in contrast to paclitaxel, vincristine, doxorubicin or etoposide.

Most preferred are compounds of formula I according to the invention, which are included in the following options:

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-pyrido [2,3-b] pyrazin-7-ylacetamide (Compound 1),

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N- (1H-indazol-5-yl) -2-oxoacetamide (Compound 4),

N- {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} -N-quinolin-6-ylacetamide (Compound 2),

Methyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} quinoline-6-ylcarbamate (compound 3),

Ethyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} quinolin-6-ylcarbamate (compound 5),

Propyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} quinoline-6-ylcarbamate (Compound 6),

N- {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} -N-quinolin-6-ylpropionamide (Compound 7),

Ethyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} pyridin-4-ylcarbamate (compound 8) or

2- [1- (4-Chlorobenzyl) -1H-indol-3-yl] -N-quinolin-6-yl-2-thioxoacetamide (Compound 11).

Compounds (1), (4) and (11) are compounds wherein the radical R7 is hydrogen. Compounds (2), (3), (5) and (6) to (8) contain alkylcarbonyl groups of alkoxycarbonyl groups as group R7.

The following compounds (9), (10), (12), (13), (14) and (15) are also compounds studied for comparison. Compounds (9), (10), (14) and (15) are known from the prior art. Compound (9) is described in WO 02/10152, Compound (10) is described in WO 03/022280, and Compound (13) is disclosed in WO 02/152. 08225 A1, the compounds (12), (14) and (15) are included in the claims of WO 99/51224 A1 and WO 01/22954.

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N- (2-methylquinolin-6-yl) -2-oxoacetamide (Compound 9),

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-quinolin-6-ylacetamide (Compound 10),

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-quinolin-8-ylacetamide (Compound 12),

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N-isoquinolin-5-yl-2-oxoacetamide (Compound 13),

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-pyridin-4-ylacetamide (Compound 14),

2- [1- (4-Fluorobenzyl) -1H-indol-3-yl] -N- (2-methylquinolin-8-yl) -2-oxoacetamide (Compound 15).

Compounds of formulas (la) and (lb) can be obtained according to the following scheme 1:

Compounds of Formula (Ic) wherein X is S can be prepared according to Scheme 2:

Compounds of formula (Ic) wherein Y is S are described in W.-D. Malmberg et al. Liebigs Ann. Chem. 10, 1983; 1649-1711 can be obtained using known methods.

Starting compounds of formula (II), (III) and (IV) can be prepared using methods which are commercially available or known per se. Starting compounds of formula (II), (III) and (IV) are useful intermediates for preparing indole derivatives of formula (I) according to the invention.

To prepare starting compounds and target compounds, reference may be made, for example, to the following standard literature on organic synthesis, which is incorporated by reference in its entirety herein:

? Houben-Weyl, Volume E 7a (Part 1) pp. 290-492, pp. 571-740

? Houben-Weyl, Volume E 7a (Part 2) pp. 119-156, pp. 205-686, pp. 157- 204

? The monograph "Heterocyclic Compounds" (Elderfield),

       Volume 1, pp. 119-207, pp. 397-616

       Volume 3, pp. 1-274

       Volume 6, pp. 101-135, pp. 234-323

 ? The monograph "Comprehensive Organic Chemistry"

   (S. D. Barton, W. D. Ollis)

       Volume 4, pp. 155-204, pp. 205-232, pp. 493-564

The skilled person is, due to their expertise, well aware of the solvents and auxiliaries and, in some cases, reaction parameters to be used, for example reaction temperatures and reaction durations.

The following compounds, included in the tests below, apparent from their respective chemical naming, are synthesized according to the general guidelines for steps 1, 2 and 3, based on the synthesis schemes 1 and 2. The compounds according to the invention are characterized analytically by their melting point and / or 1 H NMR spectroscopy and / or mass spectroscopy.

Chemicals and solvents used are conventional sources (Acros, Avocado, Aldrich, Fluka, Lancaster, Maybridge, Merck). ), Sigma, TCI, etc.) or are synthesized.

The invention is described in more detail with the aid of the following examples, but is not limited thereto.

Example

Example 1 (Reaction according to Step 1 of Scheme 1)

Preparation of 1- (4-chlorobenzyl) indole

A solution of 5.86 g (0.05 mol) of indole in 25 ml of DMSO is added to a mixture of 1.32 g (0.055 mol, mineral oil suspension) of sodium hydride in 50 ml of dimethyl sulfoxide. The resulting mixture is heated at 60 ° C. for 1.3 h, then cooled and 17.7 g (0.11 mol) of 4-chlorobenzyl chloride are added dropwise. The solution is allowed to warm to 60 ° C. and left overnight, then poured into 200 ml of water with stirring. The mixture is extracted several times with a total of 75 ml of CH ₂ Cl ₂ , then the organic phase is dried over anhydrous sodium sulfate, filtered and the filtrate is evaporated in vacuo.

Yield: 11.5 g (95% of theory)

Example 2 (reaction according to step 2 of Scheme 1):

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-pyrido [2,3-b] pyrazin-7-ylacetamide (Compound 1)

A solution of 10.2 g (10.7 mmol) of 1- (4-chlorobenzyl) -indole in 200 ml of ether is added dropwise to a solution of 1.12 ml of oxalyl chloride in 50 ml of ether at 0 ° C. under nitrogen. The mixture is heated to reflux for 2 hours and then the solvent is evaporated. Then 30 ml of DMF was added to the residue, followed by 1.93 g (13.9 mmol) of potassium carbonate, the suspension was cooled to 0 ° C., and then a solution of 1.57 g (10.7 mmol) of the amino component in 10 ml of DMF was added dropwise. The reaction mixture is left to stir overnight at room temperature. Final stirring in ice water and the resulting precipitate is suction filtered. The crude product obtained is chromatographed on 100 g of silica gel using n-heptane / ethyl acetate = 4: 1.

Yield: 3.23 g (68.0%)

Melting Point: 250 ℃

¹ H-NMR (DMSO-D6) δ = 11.56 (s, 1H), 9.53 (d, 1H), 9.12 (s, 1H), 9.09 (d, 1H), 9.04 (s, 1H), 8.32 (d, 1H), 7.6 (d, 1H), 7.40 (d, 2H), 7.35 (m, 3H), 7.32 (m, 2H), 5.64 (s, 2H) ppm

Example 3 (Reaction according to Step 3 (a) of Scheme 1)

N- {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} -N-choline-6-ylacetamide (2)

0.833 g (6.82 mmol) of DMAP, 1.38 g (13.6 mmol) of triethylamine and 13.9 g (136 mmol) of acetic anhydride were added to a 2- [1- (4-chlorobenzyl) -1H-indole- in 60 ml of DMF under nitrogen. To a stirred solution of 6.0 g (13.6 mmol) of 3-yl] -2-oxo-N-quinolin-6-ylacetamide. The reaction mixture is stirred at room temperature for 10 minutes and then poured into 200 ml of ethyl acetate. After adding 300 ml of water, the mixture is shaken in a separatory funnel and the two phases are separated. It begins to settle after 20 minutes. The pale yellow crystals are filtered and dried under vacuum at 60 ° C.

Yield: 4.04 g (61.5%)

Melting point: 122.9 ℃

¹ H NMR (600 MHz, DMSO-d6) δ = 9.02 (d, 1H), 8.54 (s, 1H), 8.44 (d, 1H), 8.21 (d, 1H), 8.17 (d, 1H), 8.10 ( m, 1H), 7.88 (m, 1H), 7.65 (m, 1H), 7.58 (m, 1H), 7.44 (d, 2H), 7.33 (d, 2H), 7.28 (m, 2H), 5.60 (s , 2H), 2.15 (s, 3H).

MS (ESI) m / z 482.1 (MH ⁺ ), (theoretical: 481.94)

Example 4 (Reaction according to Step 3 (a) of Scheme 1):

Methyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} quinoline-6-ylcarbamate (3)

930.2 mg (27.3 mmol) of NaH (60% concentration dispersion in mineral oil) of 10.0 g (22.7 mmol) 2- [1- (4-chlorobenzyl) -1H-indol-3-yl] in 500 ml of dry THF under nitrogen To a cooled and stirred solution of 2-oxo-N-quinolin-6-ylacetamide is added. The solution is stirred at 0 ° C. until the yellow precipitate is separated and then for an additional 15 minutes. 2.58 g (27.3 mmol) of methyl chloroformate are then added dropwise at a temperature below + 5 ° C. The reaction is monitored by thin layer chromatography (eluent: n-heptane / ethyl acetate 1/1 RF = 0.11). The reaction mixture is poured into water and the resulting mixture is extracted with ethyl acetate and the organic phase is washed with saturated sodium chloride solution and dried over anhydrous MgSO ₄ . The solvent is evaporated to give the crude product which is purified by column chromatography (n-heptane / acetone 2/1) to give compound (3). Thin layer chromatography shows that compound (3) still contains some impurities, which impurities can be removed by stirring the crude compound (3) with acetone for 1 hour. Filtration affords compound 3 as pale yellow crystals.

Yield: 3.0 g (26.5%)

Melting Point: 178.5 ℃

¹ H NMR (600 MHz, DMSO-d6) δ = 9.02 (d, 1H), 8.58 (s, 1H), 8.47 (d, 1H), 8.17 (m, 3H), 7.84 (m, 1H), 7.63 ( m, 2H), 7.44 (d, 2H), 7.34 (m, 4H), 5.60 (s, 2H), 3.65 (s, 3H).

MS (ESI) m / z 498.2 (MH ⁺ ), (Theoretical: 497.94)

Example 5 (Reaction according to Step 3 (b) of Scheme 2 :)

Preparation of 2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N-quinolin-6-yl-2-thioxoacetamide (11)

4.68 g (9.1 mmol) of 2,4-bis (4-methoxyphenyl) -1,3-dithia-2,4-diphosphetane-2,4-disulfide in 200 ml of toluene under nitrogen (9.1) mmol) was added to a suspension of 2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-quinolin-6-ylacetamide, and the mixture was then stirred at 75 ° C. for 3 hours. Heat during. The residue formed is filtered hot from the reaction solution and then washed with 100 ml of methylene chloride. The filtrate is concentrated in vacuo and the residue is chromatographed on flash silica gel (eluant: methylene chloride / methanol 99: 1). After the solvent is removed once more, the product fractions are filtered over flash silica gel (eluant: n-heptane / ethyl acetate 1: 1).

Yield: 0.46 g (11% of theory)

ESI-MS: m / e = 456.1 (MH ⁺ ), (theoretical: 455.97)

¹ H-NMR (DMSO-D6) δ = 10.89 (s, 1H), 8.8 (s, 1H), 8.75 (s, 1H), 8.55 (s, 1H), 8.12 (d, 1H), 8.35 (d, 1H), 8.0 (d, 1H), 7.93 (d, 1H), 7.63 (d, 1H), 7.50 (m, 1H), 7.4 (m, 3H), 7.3 (m, 3H), 5.6 (s, 2H ) ppm.

The following compounds of formula (I) are simplified according to Examples 2 and 3, similar to the synthetic route of Scheme 1.

Example 6:

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N- (1H-indazol-5-yl) -2-oxoacetamide (Compound 4)

Melting Point: 203 ℃

¹ H-NMR (DMSO-D6) δ = 13.02 (s, 1H), 10.7 (s, 1H), 9.04 (s, 1H), 8.48 (s, 1H), 8.42 (d, 1H), 8.06 (s, 1H), 7.73 (d, 1H), 7.6 (d, 1H), 7.55 (d, 1H), 7.40 (d, 2H), 7.28-7.35 (m, 4H), 5.63 (s, 2H) ppm

Example 7 :

Ethyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} -quinolin-6-ylcarbamate (compound 5)

Melting point: 199 ℃

¹ H NMR (600 MHz, DMSO-d6) δ = 9.02 (m, 1H), 8.60 (s, 1H), 8.48 (d, 1H), 8.15 (m, 3H), 7.83 (m, 1H), 7.63 ( m, sH), 7.43 (d, 2H), 7.32 (m, 4H), 5.60 (s, 2H), 4.15 (q, 2H), 0.95 (t, 3H).

MS (ESI) m / z 514.2, 512.1 (MH ⁺ ), (Theoretical: 511.97)

Example 8:

Propyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} quinoline-6-ylcarbamate (Compound 6)

Melting point: 164 ℃

¹ H NMR (600 MHz, DMSO-d6) δ = 9.02 (m, 1H), 8.60 (s, 1H), 8.48 (d, 1H), 8.17 (m, 3H), 7.84 (m, 1H), 7.63 ( m, 2H), 7.43 (d, 2H), 7.33 (m, 4H), 5.61 (s, 2H), 4.03 (t, 2H), 1.32 (m, 2H), 0.56 (t, 3H).

Example 9:

N- {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} -N-quinolin-6-ylpropionamide (Compound 7)

¹ H NMR (600 MHz, DMSO-d6) δ = 9.03 (m, 1H), 8.52 (s, 1H), 8.45 (d, 1H), 8.23 (d, 2H), 8.18 (d, 1H), 8.13 ( m, 1H), 7.88 (m, 1H), 7.65 (m, 1H), 7.58 (m, 1H), 7.45, (d, 2H), 7.30 (m, 4H), 5.59 (s, 2H), 2.61 ( q, 3H), 0.88 (t, 3H).

Example 10:

Ethyl {2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxoacetyl} -pyridin-4-ylcarbamate (compound 8)

Melting Point: 62 ℃

¹ H NMR (500 MHz, DMSO-d6) δ = 8.74 (m, 2H), 8.52 (s, 1H), 8.12 (m, 1H), 7.60 (m, 1H), 7.55 (m, 2H), 7.40 ( m, 2H), 7.30 (m, 4H), 5.57 (s, 2H), 4.10 (q, 2H), 0.95 (t, 3H).

Example 11 (comparative material):

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N- (2-methylquinolin-6-yl) -2-oxoacetamide (Compound 9)

Yield: 14.8 g (77.3% of theory)

Melting point: 182-185 ℃

¹ H-NMR (CDCl ₃ ) δ = 9.58 (s, 1H), 9.12 (s, 1H), 8.5 (s, 1H), 8.41 (s, 1H), 8.05 (t, 2H), 7.78 (d, 1H ), 7.4 (dd, 1H), 7.32 (m, 4H), 7.26 (s, 1H), 7.15 (d, 1H), 5.38 (s, 2H), 2.73 (s, 3H) ppm

Example 12 (comparative material)

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-quinolin-6-ylacetamide (Compound 10)

Melting point: 200 ℃

¹ H-NMR (DMSO-D ₆ ) δ = 11.5 (s, 1H), 9.05 (s, 1H), 8.85 (s, 1H), 8.66 (s, 1H), 8.32 (d, 2H), 8.12 (d , 1H), 8.03 (d, 1H), 7.63 (d, 1H), 7.53 (dd, 1H), 7.42 (d, 2H), 7.30-7.38 (m, 4H), 5.63 (s, 2H) ppm

Example 13 (comparative material)

2- [1- (4-Chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-quinolin-8-ylacetamide (Compound 12)

Melting point: 178 ℃

Example 14 (comparative material)

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N-isoquinolin-5-yl-2-oxoacetamide (Compound 13)

Melting Point: 239-241 ° C

Example 15 (comparative material)

2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-pyridin-4-ylacetamide (Compound 14)

Melting Point: 264 ℃

Example 16 (comparative material):

2- [1- (4-fluorobenzyl) -1H-indol-3-yl] -N- (2-methylquinolin-8-yl) -2-oxoacetamide (Compound 15)

Melting point: 200-202 ° C

Biological Effects of the Compounds According to the Invention

In vitro and in vivo tests on selected tumor models are performed to show the presence of the following pharmacological activity.

Example 17 Antiproliferative Effects on Various Tumor Cell Lines

Antiproliferative activity of Substances 1, 2, 4, 9, 11, 12, 13 and 15 was investigated in proliferative tests performed on established tumor cell lines (DA Scuderio et al. Cancer Res. 1988, 48, 4827-4833). The test used allows measuring the dehydrogenase activity of the cell and indirectly determining cell viability and cell number. Cell lines used are human cervical carcinoma cell line KB / HeLa (ATCC CCL17), ovarian adenocarcinoma cell line SKOV-3 (ATCC HTB77), human glioblastoma cell line SF-268 (NCI 503138) and lung carcinoma cell line NCI-H460 (NCI 503473) to be.

The results show that Examples 1, 2, 4 and 11 are very potent inhibitors for the proliferation of selected tumor cell lines.

Example 18 Antiproliferative Effects in MDR Tumor Cell Lines

For further characterization, substances 1, 2, 4 and 11 were examined for their effect on multidrug resistant cell lines compared to effects on non-resistant wild type cell lines.

The cell lines investigated were mouse cell line L1210, acute myeloid leukemia cell line LT12 and resistant lines L1210 / mdr and LT12 / mdr. Murine cell lines P388 (methylcholaterene-induced lymphoid tumor) and doxorubicin resistant P388 were also included as test systems.

The results are summarized in Table 2 below:

Substances 1, 2, 4 and 11 show very potent inhibitory effects on all cell lines tested, but typical substances with tubulin inhibitory effects, such as pacyclictaxel or vincristine, and topoisomerase II inhibitors It can be seen that (doxorubicin, mitoxantrone and etoposide) has at least a greatly reduced effect in MDR1 resistant cell lines.

Example 19: Tubulin Polymerization Inhibition

Substances 1, 4, 9, 11, 12, 13 and 15 were tested for their ability to inhibit bovine tubulin polymerization in an in vitro test (DM Bollag et al. Cancer Res. 1995, 55, 2325-2333). The test uses tubulin which can be purified in polymerization and depolymerization cycles and polymerized by adding GTP and heating it. Table 3 provides EC ₅₀ values that can inhibit the polymerization of tubulin containing 30% related protein (MPA).

The results (see Table 3) show that substances 1, 4, 9 and 11 have a very strong inhibitory effect on tubulin polymerization, while compounds 12, 13 and 15 do not exert any effect.

Example 20 Inhibition of Topoisomerase II

The topoisomerase II inhibition of substance 1 was tested in two different in vitro tests.

? KDNA assays for testing topoisomerase II activity:

In this assay, described in P. Arimondo, Anti-Cancer Drug Design 2000, 15 (6), 413-421, kDNA was expressed with human DNA topoisomerase II in the absence or presence of a test compound. Treated. In this assay, Compound 1 according to the invention was tested at three different concentrations (100, 31.6 and 10 μΜ). Concentrations of 100 μM each of the positive control and reference compounds m-amsacrine (m-amsa), paclitaxel (taxol) and vincristine were used for comparison.

Perform analysis:

2 μl of 10 × Assay Buffer, 1 μl of kDNA (200 ng), 0.5 μl of human topoisomerase II (1 unit) and 15.5 μl of H ₂ O using a pipette, in test material first introduced (100% DMSO) 20 folds) was added and the reagents were mixed.

Reaction analysis samples were placed in a heating block preheated to 37 ° C. and incubated at 37 ° C. for 10 minutes. Incubation was stopped after adding 4 μl of 5 × Stop buffer and the material was extracted with CIA. 20 μl of the supernatant was then added onto a 1% agarose gel containing 0.25 μg / ml of ethidium bromide and separated at 100 V for 1 hour. Finally, the gel was photographed under ultraviolet excitation (see FIG. 1). Inhibition of decatenation of kDNA was quantified using GelPro ^R Analyzer Software (see FIG. 2).

? PRYG relaxation assay to test topoisomerase II activity

This relaxation assay was used to further demonstrate the inhibition of topoisomerase II of the compounds according to the invention. In this assay, Compound 1 according to the present invention was tested at three different concentrations (100, 31.6 and 10 μΜ). Reference compounds m-amsacrine, paclitaxel (taxol) and vincristine were used at concentrations of 316 μM and 100 μM for comparison.

The analysis is performed as follows:

A test substance first introduced (100% DMSO) using a pipette of ₂ μl of 10 × assay buffer, 0.5 μl of pRYG DNA (125 ng), 0.5 μl of human topoisomerase II (1 unit) and 16 μl of H ₂ O Concentrated to 20 times) and the reagents were mixed. Reaction analysis samples were placed in a heating block preheated to 37 ° C. and incubated at 37 ° C. for 30 minutes. 4 μl of 5 × stop buffer is added and incubation is stopped. 10 μl of assay sample was then added onto a 1.2% agarose gel containing 0.25 μg / ml of ethidium bromide and separated at 100 V for 2.5 h. Finally, the gel was photographed under ultraviolet excitation (see FIG. 3). Inhibition of pRYG relaxation was quantified using GelPro ^R Analyzer Software (see FIG. 4).

Overall, it can be seen that Compound 1 according to the present invention significantly inhibits topoisomerase II in both assays. The results obtained with compound 1 are comparable to the inhibition values obtained with m-amsacrine, a topoisomerase II inhibitor. As expected, paclicktaxel and vincristine were observed to have no inhibitory effect in both assays.

Example 21 Cell Cycle Analysis

The cell cycle includes cell progression from one cell generation to the next.

During the resting (G0) and presynthetic (G1) cells contain a diploid chromosome set (2c). In the synthesizer (S), the amount of DNA is increased by replication. The S stage is terminated when the cells reach a preliminary mitosis stage (G2M) comprising a replicated complement of the chromosome (4c) and twice the DNA. In subsequent mitosis (M) of short duration, the cloned chromosome is evenly distributed between the two daughter cells, each containing a double fold amount of DNA once again and present at stage G01, which causes the cell cycle to resume It means you can start.

For cell cycle analysis, KB / HeLa cells were treated with different concentrations of test substance (0.1-1000 nM) at 37 ° C. for 24 hours.

After treatment with reference material or selected test material, cell inhibition rate at the G2 / M stage of the cell cycle is shown in Table 4 below. Results were analyzed using professional analysis software (ModFit ^™ ).

Compounds 1, 2, 4 and 11 according to the present invention show comparable activity to that of the reference compounds paclitaxel and mitoxantrone.

Example 22: Demonstration of Apoptosis

CDD ^plus nucleosome ELISA test:

Nuclear fission is the end result of the apoptosis process. Changes that can be observed in this regard can be attributed to the DNA chain cleaved by the endonuclease and the fragments into nucleosome particles produced therefrom.

The CDD ^plus nucleosome ELISA test described in Roche Molecular Biochemicals was used for nucleosome particle identification. To this end, the effects of compounds 1 and 2 in the U-937 cell line were investigated at different concentrations (1 nM to 10 μM; treatment for 24 hours) (see FIGS. 5 and 6).

In this test, one can observe a concentration dependent increase in nucleosomes in cell lysates for compounds 1 and 2. Significant increases in cell culture supernatants could not be demonstrated, which is evidence supporting apoptotic cell death that occurs after treatment with Compounds 1 and 2.

Example 23 Demonstration of Saturation Solubility in Water of Compounds According to the Invention

Saturated solubility in water of compounds 1, 2, 10 and 14 was measured as described below. Up to 1% DMSO was added for the purpose of solubilizing the material and improving the wettability of the sample. HPLC-UV method was used to check the content. The results are summarized in Table 5 below:

Compounds 1 and 2 according to the invention differ from compounds 10 and 14 which are more soluble in water.

Example 24 In Vivo Activity

In vivo activity and tolerability of Compound 2 according to the present invention compared to the cases of Materials 10 and 14 were tested with a human xenograft model (melanoma, MEXF-462). The results are summarized in Table 6 below:

In this xenograft model, Compound 2 was observed to induce complete remission of tumors in treated animals and also very well tolerant (no death). Comparable antitumor effects were observed for compounds 10 and 14, but their tolerance was much less.

Example 25 In Vivo Activity

In vivo activity and tolerability of Compound 2 according to the invention compared to the case of substance 10 was tested by another xenograft model (mammal gland, MAXF857).

The results are shown in the following table:

Compounds 2 and 10 were observed to have comparable antitumor effects, but substance 10 (2 of 6 rats died) was far less tolerant than compound 2.

Claims (13)

delete delete delete delete delete 2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -2-oxo-N-pyrido [2,3-b] pyrazin-7-ylacetamide (Compound 1) and Indole selected from the group consisting of 2- [1- (4-chlorobenzyl) -1H-indol-3-yl] -N- (1H-indazol-5-yl) -2-oxoacetamide (compound 4) derivative. delete A pharmaceutical composition for treating a tumor disease comprising at least one indole derivative according to claim 6. The pharmaceutical composition of claim 8, comprising an indole derivative in the particulate or nanoparticle composition. A liquid according to claim 8, which may be used for tablets, dragees, capsules, infusions or ampoules, suppositories, plasters, inhalations, including indole derivatives and pharmaceutically acceptable carriers and / or diluents and auxiliary substances. A pharmaceutical composition in the form selected from the group consisting of powder preparations, suspensions, creams and ointments. delete The pharmaceutical composition of claim 8 for treating a tumor disease having drug resistance to other active compounds. The pharmaceutical composition of claim 8 for treating a tumor disease comprising metastatic carcinoma.

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