HK1163674A - Indole derivatives as anticancer agents - Google Patents

Description

Indole derivatives as anticancer agents

Technical Field

The present invention relates to indole compounds and compositions comprising the compounds as anti-cancer agents. In addition, the invention provides methods of making the disclosed compounds, compositions containing them, and methods of using them, for example, as pharmaceuticals, particularly for the treatment of cancer.

This application claims priority from european patent application No. 09152089.0, which is incorporated herein by reference. Without being bound by any theory, it is presently believed that, although MDM2 and p53 represent key elements in tumor cell biology and play a major role in cellular responses to cell injury or stress, and although the present compounds are capable of increasing the expression of p53, the proven preclinical antitumor activity elicited by the compounds of the present invention, they do not appear to represent a major molecular target. Initial observations were directed to direct or indirect effects on DNA synthesis and/or replication-stress responses as a support for the preclinical antitumor activity observed with the compounds. The compounds of the invention also exhibit antiproliferative effects in tumor cells lacking p53, lacking functional p53, or having mutant p53, and in addition, they may sensitize tumorigenic cells to chemotherapy and radiation therapy.

p53 is a tumor suppressor protein that plays a key role in the regulation of cell proliferation and the cytostatic/apoptotic balance. Under normal conditions, the half-life of p53 is very short, so the concentration of p53 in cells is low. However, in response to cellular DNA damage or cellular stress (e.g., oncogene activation, telomere erosion, hypoxia), the concentration of p53 increases. This increase in p53 concentration results in activation of transcription of many genes that drive the cell into growth inhibition or apoptosis processes. Thus, an important function of p53 is to prevent uncontrolled proliferation of damaged cells, thereby protecting tissues from cancer growth. The term "MDM 2" (Murine Double Minute 2: Murine Double Minute 2) as used herein refers to a protein expressed via MDM2 gene. Within the meaning of this term, MDM2 encompasses all proteins encoded by MDM2, mutants thereof, or sheet proteins (slice proteins) thereof, as well as phosphorylated proteins thereof. Furthermore, as used herein, the term "MDM 2" encompasses MDM2 isoforms, such as MDMX (also known as MDM4) and MDM2 homologs and other animal isoforms, such as the human homolog HDM2 or the human isoform HDMX. MDM2 is a key negative regulator of p53 function. It forms a negative self-regulating loop by binding to the amino-terminal activation region of p53, so that MDM2 can inhibit both the ability of p53 to activate transcription and target p53 for proteolytic degradation. Under normal conditions, this regulatory loop is responsible for maintaining low concentrations of p 53.

Background

JP 11130750 describes substituted phenylaminocarbonylindolyl derivatives and the like as 5-HT receptor antagonists.

EP1129074 describes anthranilic acid amides as inhibitors of Vascular Endothelial Growth Factor Receptors (VEGFR) and for the treatment of angiogenic diseases. WO01/42224 provides carboxyamide-based derivatives for the treatment of Alzheimer's disease. EP1317443 discloses tricyclic tertiary amine derivatives useful as modulators of chemokine receptors CXCR4 or CCR5 for the treatment of human immunodeficiency virus and feline immunodeficiency virus.

EP1379239 discloses N- (2-arylethyl) benzylamines as 5-HT₆A receptor antagonist. WO00/15357 provides piperazine-4-phenyl derivatives as inhibitors of the interaction between MDM2 and p 53. EP1137418 provides tricyclic compounds for restoring the conformational stability of proteins of the p53 family. WO03/040402 provides compounds that inhibit protein interactions, such as between MDM2 and p 53. EP1443937 describes substituted 1, 4-benzodiazepinesAnd their use as inhibitors of the MDM2-p53 interaction. EP1458380 provides cis-2, 4, 5-triphenyl-imidazolones which inhibit the interaction between MDM2 protein and p 53-like peptides and have antiproliferative activity.

EP1519932 discloses bisarylsulfonamide compounds which bind to MDM2 and are useful in cancer therapy.

WO2006/032631, WO2007/107543, WO2007/107545, WO2009/019274, WO2009/037308 and WO2009/037343 disclose inhibitors of the interaction between MDM2 and p 53.

There is a need for effective small molecules with effective tumor cell growth inhibition, broad safety profiles, and fewer undesirable side effects.

The compounds of the present invention show excellent in vitro activity and excellent in vivo antitumor effect. They have low affinity for P450 enzymes, which reduces the risk of adverse drug-drug interactions, allowing broader safety margins. Furthermore, the compounds of the present invention have low drug-induced neurological effects and have improved cardiovascular profile, which may advantageously alter the dose limiting toxicity (dose limiting toxicity) of the compounds.

Description of the invention

The present invention provides compounds, compositions and methods for treating cancer. In addition, the compounds and compositions of the present invention are useful for enhancing the efficacy of chemotherapy and radiotherapy.

The invention relates to compounds of formula (I)

The compounds include any stereochemically isomeric form thereof, wherein

R¹Is a hydroxy group C_1-6Alkyl or C_2-6An alkenyl group; provided that R is¹The substituent is located at the 6 or 7 position of the indole moiety;

R²is hydrogen or C_1-4An alkyl group;

z is a group selected from:

R³is hydrogen or hydroxy C_1-4An alkyl group;

R⁴is hydroxy or C_1-4An alkoxy group;

R⁵is hydrogen or C_1-4An alkyl group; or

R⁴And R⁵Together form an oxo group;

a pharmaceutically acceptable salt thereof or a solvate thereof.

The compounds of formula (I) may also exist in their tautomeric form. Although these forms are not explicitly indicated in the above formula, they are intended to be included within the scope of the present invention.

The invention also relates to the use of the compounds of formula (I) for the preparation of a medicament for the treatment of cancer and to the use of the compounds of formula (I) in the treatment of cancer.

The following explains the above definitions and many terms used hereinafter. These terms are sometimes used alone or in combination terms.

C as a radical or part of a radical_1-4Alkyl is defined as a straight or branched chain saturated hydrocarbon group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 1-methylethyl, butyl; c as a radical or part of a radical_1-6Alkyl is defined as a straight or branched chain saturated hydrocarbon radical having from 1 to 6 carbon atoms, e.g. para-C_1-4Alkyl groups and pentyl, hexyl, 2-methylbutyl and the like. C_2-6Alkenyl is defined as a straight or branched hydrocarbon group containing one double bond and having 2 to 6 carbon atoms, such as vinyl, 2-propenyl, 3-butenyl, 2-pentenyl, 3-methyl-2-butenyl, and the like.

The line drawn from the substituent to the ring system indicates that the bond may be attached to any suitable ring atom.

For therapeutic use, salts of the compounds of formula (I) are those wherein the counterion is pharmaceutically acceptable. However, salts of acids and bases which are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of pharmaceutically acceptable compounds. All salts, whether pharmaceutically acceptable or not, are included within the scope of the invention.

The pharmaceutically acceptable salts mentioned hereinbefore and hereinafter are meant to comprise the therapeutically active non-toxic acid addition salt forms which the compounds of formula (I) are able to form. The latter is conveniently obtained by treating the base form with an appropriate acid such as: such as inorganic acids, e.g., hydrohalic acids, such as hydrochloric acid, hydrobromic acid, and the like; sulfuric acid; nitric acid; phosphoric acid, and the like; or organic acids such as acetic acid, propionic acid, glycolic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1, 2, 3-propanetricarboxylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, cyclohexanesulfonic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid and the like. Conversely, the salt form can be converted to the free base form by treatment with a base.

The compounds of formula (I) containing acidic protons may be converted into their therapeutically active non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. The pharmaceutically acceptable salts mentioned hereinbefore and hereinafter are also meant to comprise the therapeutically active non-toxic metal or amine addition salt forms which the compounds of formula (I) are able to form. Suitable base addition salt forms comprise, for example, the ammonium salt, alkali metal or alkaline earth metal salts, for example the lithium, sodium, potassium, magnesium, calcium salts and the like, salts of organic bases, for example the primary, secondary and tertiary aliphatic and aromatic amines, such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-N-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline, benzathine, N-methyl-D-glucamine, 2-amino-2- (hydroxymethyl) -1, 3-propanediol, hydrabamine, and salts with amino acids, such as arginine, lysine and the like. Conversely, the salt form can be converted to the free acid form by treatment with an acid.

The term salt also encompasses quaternary ammonium salts (quaternary amines) which can be formed from compounds of formula (I) by reaction between the basic nitrogen of the compound of formula (I) and a suitable quaternizing agent such as, for example, optionally substituted C_1-6Alkyl/aryl halides, C_1-6Alkyl/aryl carbonyl halides or aryl C_1-6Alkyl halides (e.g. methyl iodide or benzyl iodide), wherein aryl represents unsubstituted or substituted phenyl. Other reactants with good leaving groups, such as trifluoromethanesulfonic acid, may also be usedAcid C_1-6Alkyl esters, methanesulfonic acid C_1-6Alkyl esters and p-toluenesulfonic acid C_1-6An alkyl ester. Quaternary amines have a positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate, acetate, triflate, sulfate, sulfonate. Ion exchange resins may be used to introduce selected counterions.

The term salt preferably refers to pharmaceutically acceptable acid addition salt forms and pharmaceutically acceptable metal or amine addition salt forms.

The term solvate includes hydrates and solvent addition forms which the compounds of formula (I) are able to form, and salts thereof. Examples of such forms are, for example, hydrates, alcoholates and the like.

It will be appreciated that certain compounds of formula (I) and salts and solvates thereof may contain one or more chiral centers and exist in stereochemically isomeric forms.

The term "stereochemically isomeric forms of the compounds of formula (I)" as used hereinbefore is defined as: all possible compounds consisting of the same atoms bonded by the same sequence of bonds but with different three-dimensional structures which are not interchangeable, the compounds of formula (I) and their pharmaceutically acceptable salts or physiologically functional derivatives may have this form.

Unless otherwise mentioned or indicated, the chemical designation of a compound refers to the mixture of all possible stereochemically isomeric forms, as well as to each of the individual isomeric forms of formula (I) and their salts or solvates, which are substantially free of other isomers, i.e. are accompanied by less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, more preferably less than 2%, and most preferably less than 1% of other isomers. Thus, for example, when a compound of formula (I) is designated as R, it means that the compound is substantially free of the S isomer. Or if, for example, a compound of formula (I) is designated as E, it means that the compound is substantially free of the Z isomer.

In particular, the stereogenic center may have either the R-or S-configuration; the substituents on the divalent cyclic (partially) saturated groups may have either the cis (cis-) or trans (trans-) configuration. The double bond-containing compounds may have E (hetero) or Z (homo) -stereochemistry at the double bond. The terms cis, trans, R, S, E and Z are well known to those skilled in the art.

The stereochemically isomeric forms of the compounds of formula (I) are expressly intended to be embraced within the scope of the present invention.

Of most interest are those compounds of formula (I) which are stereochemically pure.

According to CAS-nomenclature conventions, when two stereocenters of known absolute configuration are present in a molecule, the R or S descriptor is assigned (based on Cahn-Ingold-Prelog order rule) to the lowest numbered chiral center, the reference center. The configuration of the second stereocenter uses a relative descriptor [ R ]^*，R^*]Or [ R ]^*，S^*]Is represented by the formula (I) in which R^*Always designated as the reference center, and [ R ]^*，R^*]Refers to centers of the same chirality, [ R ]^*，S^*]Refers to centers of different chirality. For example, if the lowest numbered stereocenter in the molecule has the S configuration and the second center is R, the stereodescriptor will be designated S- [ R ]^*，S^*]. If "α" and "β" are used: in the ring system with the lowest ring number, the position of the highest priority substituent on the asymmetric carbon atom is always exclusively in the "α" position of the mean plane (mean plane) defined by the ring system. The position of the highest priority substituent on the other asymmetric carbon atoms in the ring system relative to the position of the highest priority substituent on the reference atom is designated "α" if it is on the same side of the mean plane defined by the ring system or "β" if it is on the other side of the mean plane defined by the ring system.

The terms cis and trans as used herein are consistent with the Chemical Abstracts nomenclature (J.org.chem.1970, 35(9), 2849-2867) and refer to the position of a substituent on a ring moiety.

The compounds of formula (I) may be synthesized as racemic mixtures of enantiomers which can be separated from each other according to art-known resolution methods. The racemic compounds of formula (I) can be converted into the corresponding non-stereoisomeric salt forms by reaction with an appropriate chiral acid. The non-stereoisomeric salt forms are then separated, for example by selective or fractional crystallization, and the enantiomers are then liberated therefrom by base. Another method for separating the enantiomeric forms of the compounds of formula (I) involves liquid chromatography using a chiral stationary phase. The pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically. Preferably, if a particular stereoisomer is desired, the compound will be synthesized by stereospecific methods of preparation. These processes will advantageously use enantiomerically pure starting materials.

The present invention is also intended to include any isotopes of atoms present in the compounds of the present invention. For example, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include C-13 and C-14.

Whenever used hereinafter, the term "compound of formula (I)" or "compound of the invention" is intended to also include pharmaceutically acceptable salts, in particular acid or base (metal or amine) addition salts, all stereoisomeric forms, solvates and all polymorphic crystalline forms or amorphous forms.

Whenever used in context, substituents may each independently be selected from a list of numerous definitions, all possible chemical combinations are contemplated as being chemically possible.

One embodiment of interest of the present invention are those compounds of formula (I) wherein R is¹The substituent is located at the 7-position of the indole moiety. Thus, the compound has the formula.

One embodiment of interest of the present invention are those compounds of formula (I), whichIn R¹The substituent is located at the 6-position of the indole moiety. Thus, the compound has the formula.

One embodiment of interest of the present invention are those compounds of formula (I), (I-a) or (I-b), wherein R¹Is a hydroxy group C_1-6Alkyl, especially-CH₂-OH、-C(CH₃)₂-OH、-CH(CH₃)-OH、-CH₂-CH₂-OH、-CH(CH(CH₃)₂)-OH、-CH₂-CH₂-CHOH-CH₃More particularly-C (CH)₃)₂-OH。

One embodiment of interest of the present invention are those compounds of formula (I), (I-a) or (I-b), wherein R¹Is C_2-6Alkenyl, especially-CH ═ CH₂、-C(CH₃)＝CH₂。

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as indicated above, wherein R is²Is hydrogen.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is²Is C_1-4Alkyl, especially methyl.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is³Represents hydrogen.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is³Represents a hydroxyl group C_1-4Alkyl, especially-CH₂-OH. Preferably the R is³The substituent is located at the 2-position of the pyridine moiety.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is⁴Represents a hydroxyl group.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is⁴Represents C_1-4Alkoxy, especially methoxy.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is⁵Represents hydrogen.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is⁵Represents C_1-4Alkyl, especially methyl.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is⁴And R⁵Together form an oxo group.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is⁴Is hydroxy and R⁵Is hydrogen.

One interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein R is⁴Is hydroxy and R⁵Is C_1-4Alkyl is especially methyl.

An embodiment of interest of the present invention are those compounds of the formula (I), (I-a) or (I-b) or which may be indicated above as relevantEmbodiments of note wherein R⁴Is C_1-4Alkoxy and R⁵Is hydrogen.

An interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein Z is a group of formula (Z-1).

An interesting embodiment of the present invention are those compounds of formula (I), (I-a) or (I-b) or interesting embodiments thereof as may be indicated above, wherein Z is a group of formula (Z-2).

All possible combinations of the above-identified interesting embodiments are considered to be within the scope of the present invention.

One embodiment of interest of the present invention are those compounds of formula (I), (I-a) or (I-b), wherein R¹is-CH₂-OH、-C(CH₃)₂-OH、-CH(CH₃)-OH、-CH₂-CH₂-OH、-CH(CH(CH₃)₂)-OH、-CH₂-CH₂-CHOH-CH₃、-CH＝CH₂or-C (CH)₃)＝CH₂；R²Is hydrogen or methyl; r³Is hydrogen or-CH₂-OH；R⁴Is hydroxy or methoxy; r⁵Is hydrogen or methyl, or R⁴And R⁵Together form an oxo group.

The compounds of the interesting embodiments indicated above are preferably stereochemically pure.

Preferred compounds of the invention are selected from the following compounds, including any stereochemically isomeric form thereof; a pharmaceutically acceptable salt thereof or a solvate thereof:

(^*relative stereochemistry).

Preferred compounds of the invention are selected from the following compounds, including any stereochemically isomeric form thereof; a pharmaceutically acceptable salt thereof or a solvate thereof:

a preferred compound of the invention is a compound having the formula:

a preferred compound of the invention is a compound having the formula including any stereochemically isomeric form thereof; a pharmaceutically acceptable salt thereof or a solvate thereof:

a preferred compound of the invention is an enantiomer having the formula,

and said enantiomer has levorotatory activity, measured at a temperature of 20 ℃ and a cell pathlength of 1dm, in chloroform at a concentration of 8.59mg/ml, measured with light of the wavelength (589nm) of the sodium D-line (D-line); or

A pharmaceutically acceptable salt thereof or a solvate thereof.

A preferred compound of the invention is an enantiomer having the formula,

and said enantiomer having a dextrorotatory character, measured at a temperature of 20 ℃ and at a concentration of 10.33mg/ml in methanol, at a wavelength of the D-line of sodium (589nm) in a cell path length of 1 dm; or a pharmaceutically acceptable salt or solvate thereof.

A preferred compound of the invention is an enantiomer having the formula,

and said enantiomer having levorotatory activity, measured at a temperature of 20 ℃ and a cell path length of 1dm, in methanol at a concentration of 10.74mg/ml, measured with light of the wavelength of the D-line of sodium (589 nm); or a pharmaceutically acceptable salt or solvate thereof.

The compounds of formula (I), their pharmaceutically acceptable salts and stereochemically isomeric forms may be prepared by conventional methods. The starting materials and certain intermediates are known compounds and may be provided commercially or may be prepared according to conventional reaction procedures as are generally known in the art. Reference is also made in this respect to the synthetic methods described in WO 2006/032631.

Many such methods of preparation are described in more detail below. Other methods of obtaining the final compound of formula (I) are described in the examples.

In general, the compounds of formula (I) may be prepared by reacting an intermediate of formula (II) with an intermediate of formula (III), wherein W is₁Is a suitable leaving group, such as halogen, e.g. fluorine, chlorine, bromine or iodine, or sulfonyloxy, e.g. methylsulfonyloxy, 4-methylphenylsulfonyloxy, and the like. The reaction may be carried out in a reaction-inert solvent such as, for example: alcohols such as methanol, ethanol, 2-methoxy-ethanol, propanol, butanol, etc.; ethers, such as 1, 4-dioxane (which is preferably in the presence of a suitable acid, such as HCl), 1' -oxydiprane, and the like; ketones such as 4-methyl-2-pentanone; or N, N-dimethylformamide, nitrobenzene, acetonitrile, and the like. To neutralize the acid liberated during the reaction, it is possible to use the addition of suitable bases, such as carbonates or bicarbonates of alkali metal or alkaline earth metal salts, or organic bases, such as N, N-diisopropylethylamine, triethylamine, sodium carbonate or potassium carbonate. A small amount of an appropriate metal iodide (e.g., sodium or potassium iodide) may be added to facilitate the reaction. Stirring can increase the reaction rate. The reaction can be conveniently carried out at a temperature ranging from room temperature to the reflux temperature of the reaction mixture, and if desired, the reaction can be carried out under pressure.

In the above reaction, wherein R¹is-C (OH) (CH)₃)₂To the corresponding intermediate of formula (II) wherein R¹is-C (CH)₃)＝CH₂The final compound of formula (I).

The above reaction of the intermediate of formula (III) with the intermediate of formula (II) may also be carried out in the presence of a suitable catalyst, such as Pd (dba)₂(bis [ (1, 2, 4, 5-. eta.) -1, 5-diphenyl-1, 4-pentadien-3-one]Palladium), a suitable ligand, such as BINAP (2, 2'Bis (diphenylphosphino) -1, 1' -binaphthyl), a suitable base, such as sodium tert-butoxide, and a suitable solvent, such as toluene.

Wherein R is¹Represents a hydroxyl group C_1-4Alkyl compounds of formula (I), said compounds being represented by formula (I-1), also by reaction with a suitable reducing agent (e.g. NaBH)₄) And a suitable solvent (such as an alcohol, e.g., methanol) to reduce the corresponding carbonyl derivative of formula (IV).

The compound of formula (I-1) can also be prepared by reacting with a suitable reducing agent (e.g., LiAlH)₄) And in the presence of a suitable solvent (e.g., tetrahydrofuran), reducing the corresponding compound wherein R^xrepresents-C_1-3Alkyl C (═ O) OC_1-4An ester derivative of the formula (V) of an alkyl group.

A compound of the formula (I-1), wherein R¹Represents a hydroxyl group C_1-4Alkyl and said hydroxy group C_1-4The alkyl group is located at the 7-position of the indole moiety, and the compounds represented by formula (I-1-a) may be prepared by hydrolysis of an intermediate of formula (VI) with a suitable base, such as sodium hydroxide, and a suitable solvent, such as tetrahydrofuran or an alcohol, such as ethanol and the like.

The compounds of formula (I) may also be converted to each other by reactions or functional group transformations known in the art.

The compounds of formula (I) may also be converted into pharmaceutically acceptable acid addition salts by reaction with a suitable acid, such as hydrochloric acid, in a suitable solvent, such as an alcohol, e.g. 2-propanol, diethyl ether, diisopropyl ether.

Certain intermediates and starting materials are known compounds and are commercially available or can be prepared according to procedures known in the art.

In general, intermediates of formula (II) may be prepared by hydrogenation of intermediates of formula (VII), the reaction being carried out in the presence of a suitable metal catalyst such as: raney nickel in the presence of a suitable solvent, such as an alcohol, e.g. methanol or ethanol and the like; or platinum on carbon in the presence of a suitable catalyst poison, such as a thiophene solution; and vanadium pentoxide in the presence of a suitable solvent, such as tetrahydrofuran.

Intermediates of formula (VII) can be prepared by reacting an intermediate of formula (VIII) with an intermediate of formula (IX) in the presence of a suitable solvent such as N, N-dimethyl sulfoxide, wherein W is₂Represents a suitable leaving group such as a halogen atom (e.g., chlorine, bromine, fluorine, etc.). To neutralize the acid liberated during the reaction, it is possible to use the addition of suitable bases, such as alkali metal or alkaline earth metal carbonates or bicarbonates or organic bases, such as N, N-diisopropylethylamine, triethylamine, sodium carbonate, sodium bicarbonate or potassium carbonate.

Intermediates of formula (VIII) can be prepared by reaction between Raney nickel and NH₃Prepared from the corresponding intermediate of formula (X) in the presence of a suitable solvent, such as an alcohol, e.g. methanol, etc.

An intermediate of formula (X), wherein R¹Represents a hydroxyl group C_1-4Alkyl radicals, e.g. -CH (OH) (CH)₃) Said intermediate is represented by formula (X-a), which can be prepared by reacting an intermediate of formula (XI) with CH in the presence of a suitable solvent such as tetrahydrofuran₃And (3) MgCl reaction preparation.

The same reaction can be used to prepare hydroxy C_1-4Other alternatives to alkyl. For example, wherein R¹represents-C (OH) (CH)₃)₂The intermediate of (a) may be prepared from the corresponding compound having a group-C (═ O) -CH₃The intermediate preparation of (1).

Intermediates of formula (XI) can be prepared by reacting an intermediate of formula (XII) with sodium cyanide in the presence of a suitable solvent, such as N, N-dimethylformamide.

Alternatively, intermediates of formula (XI) may also be prepared by oxidation of the corresponding hydroxy analogue in the presence of Dess-martinperiodine (Dess-martinperiodine) and a suitable solvent such as dichloromethane.

Intermediates of formula (XII) may be prepared by reacting an intermediate of formula (XIII) with CH in the presence of a suitable solvent, such as an alcohol, e.g. ethanol₃I, reaction preparation.

Intermediates of formula (XIII) can be prepared by reacting an intermediate of formula (XIV) with an Eschenmosser salt in the presence of acetic acid.

The intermediate of formula (XIV) can be prepared by reacting the intermediate in a suitable oxidizing agent (e.g., MnO)₂) In the presence of a suitable solvent, such as methylene chloride, to oxidize the corresponding hydroxy analog.

An intermediate of formula (III) wherein Z is a group of formula (Z-2) and R⁴Is a hydroxyl group, said intermediate being represented by formula (III-a), by reacting an intermediate of formula (III-b) with a suitable reducing agent (e.g., NaBH) in the presence of a suitable solvent (e.g., an alcohol such as methanol)₄) Reducing and preparing.

The intermediate of formula (III-a) can be prepared by reaction with C in the presence of a suitable base (e.g., sodium hydride) and a suitable solvent (e.g., tetrahydrofuran)_1-4The alkyl iodide reacts and is converted into the intermediate of formula (III-c). Intermediates of formula (III-b) can be prepared by reacting an intermediate of formula (III-a) with a suitable oxidizing agent (e.g., MnO) in the presence of a suitable solvent (e.g., dichloromethane)₂) Prepared by reaction and oxidation. The intermediate of formula (III-b) can be prepared by reaction with C in the presence of a suitable solvent, such as tetrahydrofuran_1-4The alkyl MgCl reacts and is converted into the intermediate of the formula (III-d).

The intermediates of formula (III-a) can also be prepared by reaction of the intermediates in methanol/NH₃Stirring the intermediate of formula (XV) in the mixture. If desired, the intermediate of formula (III-a) may be prepared by stirring in a mixture of acetic anhydride and pyridineAnd converted into an intermediate of formula (XV).

The S-enantiomer of intermediate (III-a) (referred to herein as the intermediate of formula (III-a-1)) and the R-enantiomer of the intermediate of formula (XV) (referred to herein as the intermediate of formula (XV-b)) may be prepared by adding Candida antarctica lipase B (Lipase Candida antartia B) to a racemic mixture of the intermediate of formula (III-a) in a suitable solvent such as vinyl acetate (see scheme 1). When required, the reaction mixture can be prepared by reaction in MeOH/NH₃In a reaction to convert the intermediate of formula (XV-b) to the R enantiomer of intermediate (III-a) (referred to herein as intermediate of formula (III-a-2)).

Starting from the racemic mixture, this process provides a conversion of the acetate salt of one enantiomer with ee > 99% and a yield of 58%, while the second enantiomer is isolated with ee > 99% in a yield of 42%.

The term enantiomeric excess (ee) is well known to those skilled in the art of stereochemistry. With compositions given as molar or weight fractions F (+) and F (-) for the (+) and (-) enantiomer mixtures, where F (+) + F (-) is 1, and F: (for F^*) The enantiomeric excess of (A) is defined as F (+) -F (-), the percentage enantiomeric excess is 100x [ F (+) -F (-)]。

Scheme 1

Alternatively, the R-enantiomer of intermediate (III-a), referred to herein as the intermediate of formula (III-a-2), and the S-enantiomer of the intermediate of formula (XV), referred to herein as the intermediate of formula (XV-a), may be prepared by adding Candida antarctica lipase B to a racemic mixture of the intermediate of formula (XV) in water (see scheme 2). When required, canBy reaction in MeOH/NH₃Converting the intermediate (XV-a) into the intermediate (III-a-1).

Scheme 2

Alternatively, intermediates of formula (III-a) may be separated into their enantiomers by chiral column chromatography.

The intermediate of formula (XV) may be prepared by stirring the intermediate of formula (XVI) in acetic anhydride.

Wherein W₁The intermediate of formula (XVI), which is chlorine, referred to herein as the intermediate of formula (XVI-a), can be prepared by adding benzyltriethylammonium chloride and sodium chloride to a solution of the intermediate of formula (XVII) in acetonitrile, followed by the addition of concentrated hydrochloric acid.

The intermediate of formula (XVII) may be prepared by addition of fuming nitric acid to sulfuric acid, followed by the fractional addition of 6, 7-dihydro-5H-cyclopenta [ b ] pyridine 1-oxide.

The intermediate of formula (IV) may be prepared according to the reaction scheme above for the intermediate of formula (II).

Intermediates of formula (V) can be prepared by deprotecting an intermediate of formula (XVIII), wherein P represents a suitable protecting group, such as-C (═ O) -O-C (CH), in the presence of a suitable acid, such as hydrochloric acid, and a suitable solvent, such as acetonitrile₃)₃。

The intermediate of formula (XVIII) can be prepared by reacting an intermediate of formula (XIX) with an intermediate of formula (III), in the presence of a reaction-inert solvent such as: alcohols such as methanol, ethanol, 2-methoxy-ethanol, propanol, isopropanol butanol, etc.; ethers, such as 1, 4-dioxane (which is preferably in the presence of a suitable acid, such as HCl), 1' -oxydiprane, and the like; ketones such as 4-methyl-2-pentanone; or N, N-dimethylformamide, nitrobenzene, acetonitrile, and the like. To neutralize the acid liberated during the reaction, it is possible to use the addition of suitable bases, such as carbonates or bicarbonates of alkali metal or alkaline earth metal salts, or organic bases, such as N, N-diisopropylethylamine, triethylamine, sodium carbonate or potassium carbonate. A small amount of an appropriate metal iodide (such as sodium or potassium iodide) may be added to facilitate the reaction. Stirring can increase the reaction rate. The reaction can conveniently be carried out at a temperature in the range of from room temperature to the reflux temperature of the reaction mixture and, if desired, under elevated pressure.

Intermediates of formula (XIX) may be prepared according to the reaction scheme above for intermediates of formula (II).

Intermediates of formula (VI) can be prepared by reacting an intermediate of formula (XX) wherein P represents a suitable protecting group as defined above, and R represents_yrepresents-C_1-4alkyl-O-Si (CH)₃)₂C(CH₃)₃。

Intermediates of formula (XX) can be prepared by reacting an intermediate of formula (XXI) with an intermediate of formula (III), in the presence of a reaction-inert solvent such as: alcohols such as methanol, ethanol, 2-methoxy-ethanol, propanol, isopropanol butanol, etc.; ethers, such as 1, 4-dioxane (preferably in the presence of a suitable acid, such as HCl), 1' -oxydiprane, etc.; ketones such as 4-methyl-2-pentanone; or N, N-dimethylformamide, nitrobenzene, acetonitrile, and the like. To neutralize the acid liberated during the reaction, it is possible to use the addition of suitable bases, such as carbonates or bicarbonates of alkali metal or alkaline earth metal salts, or organic bases, such as N, N-diisopropylethylamine, triethylamine, sodium carbonate or potassium carbonate. A small amount of an appropriate metal iodide (such as sodium or potassium iodide) may be added to facilitate the reaction. Stirring can increase the reaction rate. The reaction can be conveniently carried out at a temperature ranging from room temperature to the reflux temperature of the reaction mixture, and if desired, the reaction can be carried out under pressure.

Intermediates of formula (XXI) can be prepared by hydrogenation of the corresponding nitro intermediates of formula (XXII) in the presence of a suitable catalyst, such as raney nickel, and in the presence of a suitable solvent, such as an alcohol, e.g. methanol, etc.

Wherein P represents-C (═ O) -O-C_1-4Alkyl intermediates of formula (XXII), herein designated as formula (XXII-a), can be prepared by reacting an intermediate of formula (XXIII) with di-tert-butyl dicarbonate in the presence of a suitable base, such as triethylamine, and a suitable solvent, such as 4- (dimethyl) aminopyridine.

Intermediates of formula (XXIII) can be prepared by reacting an intermediate of formula (XXIV) with an intermediate of formula (IX) in the presence of a suitable base, such as sodium bicarbonate, and a suitable solvent, such as N, N-dimethyl sulfoxide.

Intermediates of formula (XXIV) can be prepared by reacting an intermediate of formula (XXV) with NH in the presence of a suitable catalyst (e.g., Raney nickel) in the presence of a suitable solvent (e.g., an alcohol such as methanol, etc.)₃And (3) reaction preparation.

Intermediates of formula (XXV) can be prepared by reacting an intermediate of formula (XXVI) with tert-butyldimethylsilyl chloride in the presence of a suitable base, such as imidazole, and a suitable solvent, such as tetrahydrofuran.

Intermediates of formula (XXVI) can be prepared as described above for intermediates of formula (XI).

The compounds of formula (I) and some of the intermediates of the present invention may contain asymmetric carbon atoms. Pure stereochemically isomeric forms of said compounds and of said intermediates may be obtained by applying the methods known in the art. For example, diastereomers can be separated by physical methods such as selective crystallization or chromatographic techniques such as reflux distribution, liquid chromatography, and the like. Enantiomers can be obtained from the racemic mixture as follows: first converting the racemic mixture into a mixture of diastereomeric salts or compounds by means of a suitable resolving agent, such as a chiral acid; then, physically separating the mixture of diastereomeric salts or compounds by methods such as selective crystallization, supercritical fluid chromatography or chromatographic techniques such as liquid chromatography; and finally converting the separated diastereomeric salt or compound into the corresponding enantiomer. Pure stereochemically isomeric forms may also be obtained by reacting the appropriate intermediates and starting materials in pure stereochemically isomeric forms, provided that the intermediate reaction occurs stereoselectively.

The compounds of formula (I), including any stereochemically isomeric form thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, possess valuable pharmacological properties for their increased expression of p53, exhibit potent antiproliferative activity, and exhibit potent antitumor activity.

As indicated previously, the compounds of the invention increased the expression of p53 in the assay described in C.1. This increase may be due to, but is not limited to, one or more of the following mechanisms of action:

interaction with an upstream or downstream target, such target for example comprising a kinase or an enzymatic activity in ubiquitination or SUMO modification,

direct or indirect stabilization of the p53 protein, for example by keeping it in functional structural form, or by preventing misfolding,

enhancement of the expression of p53 or of members of the p53 family (such as p63 and p73),

-increasing p53 activity, such as by (but not limited to) enhancing its transcriptional activity, and/or

Increasing the expression of genes and proteins of the p 53-signalling pathway (signalling pathway), such as, but not limited to, p21waf1, cip1, MIC-1(GDF-15), PIG-3, Bax, Puma, Noxa and ATF-3.

Accordingly, the present invention discloses compounds of formula (I) for use as medicaments, in particular for the treatment of cancer or related diseases, for inhibiting tumor growth, for increasing the expression of p 53.

Furthermore, the present invention relates to the use of a compound for the manufacture of a medicament for the treatment of a disease mediated by a reduction in p53 expression, in particular for the treatment of cancer, wherein the compound is a compound of formula (I).

The term "treatment" as used herein encompasses any treatment of a disease and/or disorder in an animal (particularly a human) and includes: (i) preventing the development of a disease and/or disorder in a subject who may be susceptible to the disease and/or disorder but has not yet been diagnosed as diseased; (ii) inhibiting the disease and/or disorder, i.e., arresting its development; (iii) relieving the disease and/or condition, i.e., causing regression of the disease and/or condition.

By the term "disease mediated by a reduction in p53 expression" is meant any undesirable or deleterious condition that can be inhibited, prevented from developing, alleviated, or caused to regress by apoptosis, cell death induction, or cell cycle regulation.

The present invention also provides methods of treating diseases mediated by a decrease in p53 expression, in particular, methods of treating cancer, by administering to a subject, such as a mammal (and more particularly a human) in need of such treatment an effective amount of a compound of the present invention.

The compounds of the invention may have an antiproliferative effect on tumour cells, even if the cells lack functional p 53. More particularly, the compounds of the invention have antiproliferative effects on tumors with wild-type or mutant p53 and/or on tumors overexpressing MDM 2. The compounds may affect angiogenesis, tumor cell migration, invasion or metastasis.

Accordingly, the present invention also provides a method of inhibiting tumor growth by administering to a subject, such as a mammal (and more particularly a human) in need of such treatment an effective amount of a compound of the present invention.

Examples of tumors include adult and pediatric malignancies that can be inhibited by the compounds of the present invention, including but not limited to: lung cancer including small cell lung cancer and non-small cell lung cancer (e.g., adenocarcinoma), pancreatic cancer, colon cancer (e.g., colorectal cancer, e.g., colon adenocarcinoma and colon adenoma), esophageal cancer, oral squamous carcinoma, tongue cancer, gastric cancer, liver cancer, nasopharyngeal cancer, hematopoietic tumors of the lymphatic system (e.g., acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), non-Hodgkin's lymphoma (e.g., mantle cell lymphoma), Hodgkin's disease, myeloid leukemia (e.g., Acute Myeloid Leukemia (AML) or Chronic Myeloid Leukemia (CML)), acute lymphoblastic leukemia, Chronic Lymphocytic Leukemia (CLL), thyroid follicular cancer, myelodysplastic syndrome (MDS), mesenchymal neoplasia, soft tissue sarcoma, liposarcoma, gastrointestinal stromal sarcoma, Malignant Peripheral Nerve Sheath Tumor (MPNST) Ewing's sarcoma, leiomyosarcoma, interstitial chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, melanoma, teratocarcinoma, neuroblastoma, brain tumor, medulloblastoma, glioma, benign skin tumor (e.g., keratoacanthoma), breast cancer (e.g., late breast cancer), kidney cancer, wilms' tumor, ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, prostate cancer (including late stage disease and hormone refractory prostate cancer), testicular cancer, osteosarcoma, head and neck cancer, epidermal cell carcinoma, multiple myeloma (e.g., refractory multiple myeloma), mesothelioma. The characteristic cancers that can be treated with the compounds of the present invention are breast cancer, colorectal cancer, non-small cell lung cancer, Acute Myeloid Leukemia (AML).

The compounds of the invention are also useful in the treatment and prevention of inflammatory diseases.

Accordingly, the present invention also provides a method of treating and preventing inflammatory diseases by administering an effective amount of a compound of the present invention to a subject, such as a mammal (and more particularly a human) in need of such treatment.

The compounds of the invention are also useful in the treatment of autoimmune diseases and disorders. The term "autoimmune disease" refers to any disease in which the immune system of an animal reacts adversely to self-antigens. The term "autoantigen" refers to any antigen normally found in an animal. Representative autoimmune diseases include, but are not limited to: hashimoto's thyroiditis, Grave's disease, multiple sclerosis, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), dermatomyositis, Crohn's disease, Wegener's granulomatosis, anti-glomerular basement membrane disease, anti-phospholipid syndrome, 25 herpetiform dermatitis, allergic encephalomyelitis, glomerulonephritis, membranous glomerulonephritis, Goodpasture's syndrome, Lambert-Eaton's syndrome, myasthenia gravis, pemphigus, polyendocrinopathy (polynicorinopathy), Reiter's (Reiter) disease, and Stiff person's (Stiff-Man) syndrome.

Accordingly, the present invention also provides a method of treating autoimmune diseases and disorders by administering to a subject, such as a mammal (and more particularly a human) in need of such treatment an effective amount of a compound of the present invention.

The compounds of the present invention are also useful in the treatment of diseases associated with structurally unstable or misfolded proteins.

Examples of diseases associated with structurally unstable or misfolded proteins include, but are not limited to: cystic Fibrosis (CFTR), Marfan's (Marfan) syndrome (fibrillin), amyotrophic lateral sclerosis (superoxide dismutase), scurvy (collagen), maple syrup urine disease (alpha-keto acid dehydrogenase complex), osteogenesis imperfecta (type 1 collagenolytic pro-alpha), Creutzfeldt-Jakob's disease (prion), Alzheimer's (β -amyloid), familial amyloidosis (lysozyme), cataracts (crystallin), familial hypercholesterolemia (LDL receptor), α I-antitrypsin deficiency, Tay-Sachs (β -hexosaminidase), retinitis pigmentosa (rhodopsin), and leprosy (insulin receptor).

Accordingly, the present invention also provides a method of treating a disease associated with structurally unstable or misfolded proteins by administering an effective amount of a compound of the invention to a subject, such as a mammal (and more particularly a human), in need of such treatment.

Depending on their useful pharmacological properties, the compounds of interest may be formulated into a variety of pharmaceutical forms for administration purposes.

To prepare the pharmaceutical compositions of the present invention, an effective amount of a compound of the present invention as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may vary widely depending upon the form of preparation desired for administration. These pharmaceutical compositions are desirably in suitable unit dosage form, preferably for oral, rectal, transdermal, or by parenteral injection. For example, when the compositions are prepared in oral dosage forms, in the case of oral liquid preparations (e.g., suspensions, syrups, elixirs and solutions), any of the usual pharmaceutical media may be employed, such as water, glycols, oils, alcohols and the like; or in the case of powders, pills, capsules and tablets, solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like may be employed.

Because of their ease in administration, tablets and capsules represent the most advantageous unit dosage form for oral administration, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will generally comprise sterile water (at least in large part), although other ingredients may be included, for example to aid solubility. For example, injectable solutions may be prepared in which the carrier comprises saline solution, glucose solution or a mixed solution of saline and glucose. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In compositions suitable for transdermal administration, the carrier thereof optionally comprises a penetration enhancer and/or a suitable wetting agent, optionally in minor proportions, in combination with suitable additives of any nature which do not cause a significant deleterious effect on the skin. The additives may facilitate dermal administration and/or may aid in the preparation of the desired composition. These compositions may be administered in different ways, e.g. as transdermal patches, drops (spot-on), ointments. It is particularly advantageous to formulate the above pharmaceutical compositions in unit dosage form for ease of administration and uniform dosage. A unit dosage form as used herein the specification and claims refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls, and the like, and segregated multiples thereof.

It is particularly advantageous to formulate the above pharmaceutical compositions in unit dosage form for ease of administration and uniform dosage. A unit dosage form as used herein the specification and claims refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls, and the like, and segregated multiples thereof.

The compounds of the invention are administered in an amount sufficient to cause apoptosis, cause cell death, or regulate the cell cycle.

Thus, the compounds of the present invention are administered in an amount sufficient to increase the expression of p53, or to exert its anti-tumor activity.

The effective amount can be readily determined by one skilled in the art from the test results given below. In general, it is contemplated that a therapeutically effective amount will be from 0.005mg/kg to 100mg/kg body weight, and in particular from 0.005mg/kg to 10mg/kg body weight. It may be appropriate to administer the required dose, e.g. one, two, three, four or more doses, at appropriate intervals throughout the day. The sub-doses may be presented in unit dosage form, e.g. containing from 0.5 to 500mg, especially from 1mg to 500mg, more especially from 10mg to 500mg of active ingredient per unit dosage form.

Depending on the mode of administration, the pharmaceutical composition will preferably comprise from 0.05 to 99% by weight, more preferably from 0.1 to 70% by weight, even more preferably from 0.1 to 50% by weight of a compound of the invention, and from 1 to 99.95% by weight, more preferably from 30 to 99.9% by weight, even more preferably from 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

As a further aspect of the invention, combinations of the compounds of the invention with other anti-cancer agents are contemplated, especially for use as medicaments, more particularly in the treatment of cancer or related diseases.

For the treatment of the above-mentioned conditions, the compounds of the invention can be advantageously used in combination with one or more other pharmaceutical agents, more specifically in combination with other anti-cancer agents or adjuvants in the treatment of cancer. Examples of anti-cancer agents or adjuvants (supporting agents in therapy) include, but are not limited to:

-platinum coordination compounds, such as cisplatin optionally in combination with amifostine, carboplatin or oxaliplatin;

taxane compounds, e.g. paclitaxel, paclitaxel protein-binding particles (Abraxane)^TM) Or docetaxel;

topoisomerase I inhibitors, such as camptothecin compounds, for example irinotecan, SN-38, topotecan hydrochloride;

topoisomerase II inhibitors, e.g. anti-tumour epipodophyllotoxins or podophyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide (teniposide);

anti-tumour vinca alkaloids, such as vinblastine, vincristine or vinorelbine;

antineoplastic nucleoside derivatives, such as 5-fluorouracil, leucovorin, gemcitabine hydrochloride, capecitabine, cladribine, fludarabine, nelarabine;

alkylating agents, such as nitrogen mustards or nitrosoureas, for example cyclophosphamide, chlorambucil, carmustine, thiotepa, melphalan, lomustine, hexamethylmelamine, busulfan, dacarbazine, estramustine, ifosfamide (ifosfamide) optionally in combination with mesna (mesna), pipobroman, procarbazine, streptozotocin (streptazocin), temozolomide (telozolomide), uracil;

-antineoplastic anthracycline (anthracycline) derivatives, such as daunorubicin, doxorubicin optionally in combination with dexrazoxane, doxorubicin hydrochloride liposome, idarubicin (idarubicin), mitoxantrone, epirubicin hydrochloride, valrubicin;

molecules targeting the IGF-1 receptor, such as podophyllotoxin;

-a tetrocardin derivative, such as tetrocardin a;

-glucocorticoidsSuch as prednisone;

antibodies, such as trastuzumab (HER2 antibody), rituximab monomer (CD20 antibody), gemumab ozolomicin, cetuximab, pertuzumab (pertuzumab), bevacizumab, alemtuzumab, seculizumab (eculizumab), ibritumomab, nymphamab (nofetummab), panitumumab (panitumumab), tositumomab, CNTO 328;

-an estrogen receptor antagonist or a selective estrogen receptor modulator or an estrogen synthesis inhibitor, such as tamoxifen, fulvestrant, toremifene, droloxifene, faslodex, raloxifene or letrozole;

aromatase inhibitors, such as exemestane, anastrozole, letrozole (letrozole), testolactone and vorozole;

differentiating agents (differentiating agents), such as retinoids, vitamin D or retinoic acid and Retinoic Acid Metabolism Blockers (RAMBA), for example isotretinoin;

-DNA methyltransferase inhibitors, such as azacytidine or decitabine;

antifolates, such as pemetrexed disodium;

antibiotics, such as actinomycin D (antinomycin D), bleomycin, mitomycin C, dactinomycin, carminomycin, daunorubicin, levamisole, plicamycin (plicamycin), mithramycin;

antimetabolites, such as clofarabine (clofarabine), aminopterin, cytarabine (cytisine) or methotrexate, azacitidine, cytarabine (cytarabine), fluorouracil, pentostatin, thioguanine;

apoptosis inducers and anti-angiogenic agents, such as Bcl-2 inhibitors, e.g. YC 137, BH 312, ABT 737, gossypol, HA 14-1, TW 37 or decanoic acid;

-tubulin-binding agents, such as, for example, bepotastine (combretatin), colchicine or nocodazole;

kinase inhibitors (e.g. EGFR (epidermal growth factor receptor) inhibitors, MTKI (multi-target kinase inhibitors), mTOR inhibitors) such as flazidoid (flavoperidol), imatinib mesylate, erlotinib (erlotinib), gefitinib (gefitinib), dasatinib (dasatinib), lapatinib (lapatinib), lapatinib ditosylate, sorafenib (sorafenib), sunitinib (sunitinib), sunitinib maleate, temsirolimus (temsirolimus);

farnesyl transferase inhibitors, such as tipifarnib (tipifarnib);

-Histone Deacetylase (HDAC) inhibitors, such as sodium butyrate, suberoylanilide hydroxamic acid (SAHA), depsipeptide (FR901228), NVP-LAQ824, R306465, JNJ-26481585, trichostatin (trichostatin) a, vorinostat;

ubiquitin-proteasome pathway inhibitors, such as PS-341, mln.41 or bortezomib;

-Yondelis；

inhibitors of telomerase, such as, for example, telomestatin;

matrix metalloproteinase inhibitors, such as batimastat, marimastat (marimastat), prinostat (prinostat) or metamastat (metastat);

recombinant interleukins, such as aldesleukin, dinierkin 2, interferon alpha 2a, interferon alpha 2b, pegylated interferon (peginteferon) alpha 2 b;

-a MAPK inhibitor;

retinoids, such as alitretinoin, bexarotene, tretinoin;

-arsenic trioxide;

-asparaginase;

steroids, such as drotasandrosterone propionate, megestrol acetate, nandrolone (caprate, phenylpropionate), dexamethasone;

gonadotropin releasing hormone agonists or antagonists, such as abarelix, goserelin acetate (goserelin acetate), histrelin acetate, leuprorelin acetate;

-thalidomide, lenalidomide;

mercaptopurine, mitotane, pamidronate (pamidronate), pegase, pemetrase (pegaspargegase), labyrinase;

-BH3 mimetics, such as ABT-737;

MEK inhibitors, such as PD98059, AZD6244, CI-1040;

colony stimulating factor analogues, such as filgrastim, pegfilgrastim (pegfilgrastim), sargrastim; erythropoietin or an analog thereof (e.g., dabigatran α); interleukin 11; ompreil interleukin, zoledronate, zoledronic acid; fentanyl; bisphosphonates (bisphosphatate); palifermin (palifermin).

-inhibitors of steroid cytochrome P45017 α -hydroxylase-17, 20-lyase (CYP17), such as abiraterone, abiraterone acetate.

As mentioned above, the compounds of the present invention also have therapeutic applications in sensitizing tumor cells to radiation therapy and chemotherapy.

Thus, the compounds of the invention may be administered as "radiosensitizers" and/or "chemosensitizers" or may be administered in combination with other "radiosensitizers" and/or "chemosensitizers".

The term "radiosensitizer," as used herein, refers to a molecule, preferably a low molecular weight molecule, which is administered to an animal in a therapeutically effective amount to increase the sensitivity of cells to ionizing radiation and/or to facilitate the treatment of diseases that can be treated with ionizing radiation.

The term "chemosensitizer" as used herein refers to a molecule, preferably a low molecular weight molecule, which is administered to an animal in a therapeutically effective amount to increase the sensitivity of cells to chemotherapy and/or to facilitate the treatment of a disease that can be treated by chemotherapy.

Several mechanisms for the mode of action of radiosensitizers have been proposed in the literature, including: hypoxic cell radiosensitizers (e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds) that mimic oxygen or behave like a bioreductive agent under hypoxia; non-hypoxic cell radiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNA bases and preferentially incorporate into the DNA of cancer cells, thereby promoting radiation-induced DNA molecule destruction, and/or avoiding normal DNA repair mechanisms; and in the treatment of disease, various other mechanisms of action on radiosensitizers have been hypothesized.

Radiosensitizers are now used in combination with x-ray radiation in many cancer treatment protocols. Examples of x-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmetholonidazole, pimonidazole, etanidazole, nimozole, mitomycin C, RSU1069, SR 4233, EO9, RB 6145, niacinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives thereof.

Photodynamic therapy (PDT) of cancer uses visible light as a radiation activator of sensitizers. Examples of photodynamic radiosensitizers include, but are not limited to, the following: hematoporphyrin derivatives, photoporphyrins (Photofrin), benzoporphyrin derivatives, protoporphyrins, pheophorbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and therapeutically effective analogs and derivatives thereof.

The radiosensitizer can be administered in combination with a therapeutically effective amount of one or more other compounds, including, but not limited to: a compound that promotes binding of the radiosensitizer to the target cell; compounds that control the therapeutic regimen, nutrients and/or oxygen to the target cells; chemotherapeutic agents acting on tumors with or without additional radiation; or other therapeutically effective compounds for the treatment of cancer or other diseases.

The chemosensitizer may be administered in combination with a therapeutically effective amount of one or more other compounds, including but not limited to: a compound that promotes the binding of the chemosensitizer to the target cell; compounds that control the therapeutic regimen, nutrients and/or oxygen to the target cells; chemotherapeutic agents that act on tumors, or other therapeutically effective compounds for the treatment of cancer or other diseases. Calcium antagonists such as verapamil have been found to be effective in combination with anti-tumor agents to establish chemosensitivity of tumor cells resistant to the chemotherapeutic agents received and to achieve efficacy of such compounds in agent-sensitive malignancies.

In view of their useful pharmacological properties, the ingredients of the compositions of the present invention, i.e., one or more other pharmaceutical agents and the compounds of the present invention, may be formulated into a variety of pharmaceutical forms for administration purposes. The ingredients may be separately formulated into individual pharmaceutical compositions or into a single pharmaceutical composition containing all the ingredients.

The invention therefore also relates to a pharmaceutical composition comprising one or more further pharmaceutical agents and a compound of the invention together with a pharmaceutically acceptable carrier.

The invention also relates to the use of a composition of the invention in the manufacture of a pharmaceutical composition for inhibiting the growth of tumor cells.

The invention also relates to a product comprising as a first active ingredient a compound according to the invention and as further active ingredients one or more anticancer agents, for simultaneous, separate or sequential use as a combined preparation in the treatment of a patient suffering from cancer.

One or more additional agents and a compound of the invention may be administered simultaneously (e.g., separately or in a single composition), or sequentially in any order. In the latter case, the two or more compounds are administered over a period of time and in amounts and in methods sufficient to ensure that a beneficial or synergistic effect is achieved. It will be understood that the preferred method and sequence of administration, and the individual dosages and schedules of the individual components of the compositions, will depend upon the particular other agent being administered and the compound of the invention, its route of administration, the particular tumor being treated, and the particular host being treated. The optimal method and sequence of administration, as well as the dosage and administration regimen, can be readily determined by one of skill in the art using routine methods and in view of the information set forth herein.

When administered as a composition, the weight ratio of the compound of the present invention to the one or more other anti-cancer agents can be determined by one skilled in the art. As is well known to those skilled in the art, the proportion and the exact dose and frequency of administration will depend upon the particular compound of the invention and other anti-cancer agents used, the particular condition being treated, the severity of the condition in the particular patient being treated, the age, body weight, sex, diet, time and general physical condition of administration, the mode of administration and other drugs which the individual may be taking. Furthermore, it is clear that the daily effective amount may be reduced or increased depending on the subject's response and/or as assessed by the physician prescribing the compounds of the instant invention. The specific weight ratio of the compound of formula (I) and the other anticancer agent of the present invention may be in the range of 1/10 to 10/1, more particularly 1/5 to 5/1, even more particularly 1/3 to 3/1.

The platinum coordination compound is advantageously present in an amount of 1 to 500mg per square meter (mg/m)²) Dosage of body surface area, e.g. 50 to 400mg/m²In particular, the dose of cisplatin per course of treatment is about 75mg/m²And about 300mg/m for carboplatin²。

The taxane compound is advantageously present in an amount of 50-400mg per square meter (mg/m)²) Dosing of body surface area, e.g. 75-250mg/m²In particular, the dose of paclitaxel per treatment course is about 175-250mg/m²And about 75-150mg/m for docetaxel²。

The camptothecin compound is advantageously present in an amount of 0.1-400mg per square meter (mg/m)²) Dosage of body surface area, e.g. 1-300mg/m²In particular, the dose of irinotecan is about 100-350mg/m per course of treatment²And topotecan is about 1-2mg/m²。

The antitumor podophyllotoxin derivative is advantageously administered in an amount of 30-300mg per square meter (mg/m)²) Dosage of body surface area, e.g. 50-250mg/m²In particular, the dose of etoposide per course of treatment is about 35-100mg/m²And about 50-250mg/m for tenipop²。

The antitumor alkaloid is advantageously 2-30mg per square meter (mg/m)²) The dosage of the body surface area is administered, in particular, about 3-12mg/m for the dose of vinblastine per treatment period²About 1-2mg/m for vincristine dosage²And about 10-30mg/m for vinorelbine²。

The antitumor nucleoside derivative is advantageously present at 200-²) Dosage of body surface area, e.g. 700-1500mg/m²In particular, the dose for 5-FU is 200-500mg/m per treatment course²About 800-1200mg/m for gemcitabine²And about 1000-²。

Alkylating agents, such as nitrogen mustards or nitrosoureas, are advantageously used in amounts of 100-500mg per square meter (mg/m)²) Dosage of body surface area, e.g. 120-200mg/m²In particular, the dose of cyclophosphamide per treatment course is about 100-500mg/m²About 0.1-0.2mg/kg for chlorambucil and about 150mg/m for carmustine²And about 100-150mg/m for the dose of lomustine²。

The antitumor anthracycline derivative is advantageously present in an amount of 10-75mg per square meter (mg/m)²) Dosage of body surface area, e.g. 15-60mg/m²In particular, the dose of doxorubicin per course of treatment is about 40-75mg/m²About 25-45mg/m for daunorubicin dose²And about 10-15mg/m for an idarubicin dose²。

Depending on the particular agent and condition being treated, the antiestrogen is advantageously administered at a dosage of about 1-100mg per day. Tamoxifen is advantageously administered orally at a dose of 5-50mg, preferably 10-20mg, twice daily, for a sufficient time to achieve and maintain the therapeutic effect. Toremifene is advantageously administered orally at about 60mg once daily for a sufficient time to achieve and maintain the therapeutic effect. Anastrozole is advantageously administered orally at about 1mg once daily. Droloxifene is advantageously administered orally at about 20-100mg once daily. Raloxifene is advantageously administered orally at 60mg once daily. Exemestane is advantageously administered orally at about 25mg once daily.

The antibody is advantageously present at about 1-5mg per square meter (mg/m)²) The dosage of body surface area is administered, or if different, as is known in the art. Trastuzumab is advantageously administered at 1-5mg per square meter (mg/m) per course of treatment²) Dosing of body surface area, in particular, 2-4mg/m²。

These doses may be administered, for example, once, twice or more per course of treatment, for example, the course of treatment may be repeated every 7, 14, 21 or 28 days.

The compounds of formula (I), pharmaceutically acceptable addition salts, in particular the pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof, may have valuable diagnostic properties, since they may be used to detect or identify the formation of complexes between the labeled compound and other molecules, peptides, proteins, enzymes or receptors.

The detection or identification method may use a compound labeled with a labeling substance such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like. Examples of radioactive isotopes include¹²⁵I、¹³¹I、³H and¹⁴C. enzymes are typically made detectable by conjugation to an appropriate substrate, followed by catalysis of a detectable reaction. Examples thereof include, for example, β -galactosidase, β -glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase, preferably horseradish peroxidase. Luminescent substances include, for example, luminol derivatives, luciferin, jellyfish, and luciferase.

A biological sample may be defined as a body tissue or a body fluid. Examples of body fluids are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva, etc.

The following examples illustrate the invention.

Experimental part

Hereinafter, "DMF" is defined as N, N-dimethylformamide, "DCM" as dichloromethane, "DIPE" as diisopropyl ether, "DMSO" as dimethyl sulfoxide, "EtOAc" as ethyl acetate, "EtOH" as ethanol, "MeOH" as methanol and "THF" as tetrahydrofuran.

A. Preparation of intermediate compounds

Example A1

a) Preparation of intermediate 1

Manganese oxide (33.31g, 13Eq, 0.3832mol) was added portionwise to a solution of 4-chloro-6, 7-dihydro-5H-cyclopenta [ b ] pyridin-7-ol (intermediate 3) [ CAS 126053-15-4] (5g, 1Eq, 0.029mol) in DCM (50 ml). The mixture was stirred at room temperature for 24 hours, then filtered through celite (celite). Evaporation of the solvent gave 3.25g (66%) of intermediate 1.

b) Preparation of intermediate 2

Methylmagnesium chloride (1.47ml, 3.2Eq, 0.0.004mol) was added dropwise to a solution of intermediate 1(0.220g, 1Eq, 0.0013mol) in THF (4ml) at-5-0 ℃ under a stream of nitrogen. The reaction was stirred at room temperature for 1.5 hours. Ammonium chloride (10% aqueous) and EtOAc were added at 0-10 ℃. The mixture was extracted 3 times with EtOAc, then the organic layer was separated over MgSO₄Drying, filtering and evaporating the solvent. The residue (0.215g) was purified by column chromatography over silica gel (eluent: cyclohexane/EtOAc 50/50). The pure fractions were collected and the solvent was evaporated, yielding 0.120g (50%) of intermediate 2.

Example A2

a) Preparation of intermediate 3

Sodium tetrahydroborate (1.92g, 50.67mmol) was added portionwise to a solution of intermediate 1(7.72g, 46.06mmol) in MeOH (80ml) at 5 ℃. The reaction mixture was stirred at room temperature for 1 night and then poured inIn water. The mixture was extracted 3 times with EtOAc. The organic layer was washed with water and NaCl solution, separated and MgSO₄Drying, filtration and evaporation of the solvent gave 6.38g (81.7%) of intermediate 3(50/50 mixture RS). The product was used in the next step without further purification.

b) Preparation of intermediate 4

At 0 ℃ and N₂Sodium hydride (1.06g, 44.22mmol) was added portionwise to a solution of intermediate 3(3.00g, 17.70mmol) in THF (anhydrous, 30ml) under atmosphere. The reaction was stirred at 0 ℃ for 15 minutes. Methyl iodide (1.65ml, 26.53mmol) was then added dropwise to the mixture at 0 ℃. The reaction was stirred at room temperature for 1 night. The mixture was cooled to room temperature and cold water was added dropwise. The mixture was stirred for 1 hour, then extracted 3 times with EtOAc. The organic layer was separated over MgSO₄Drying, filtration and evaporation of the solvent gave 3.10g (95.4%) of intermediate 4.

The product was used directly in the next step without further purification.

Example A3

a) Preparation of intermediate 5

Acetic acid (300ml) containing methyl 1H-indole-7-carboxylate (0.091mol), Eschenmoser salt (0.1mol) was heated at 65 ℃ for 2 hours. The precipitate was filtered and dissolved in DCM and potassium carbonate 10%. Potassium carbonate (solid) was added, and the mixture was stirred at room temperature for 1 hour and then extracted. The organic layer was separated over MgSO₄Drying, filtration and evaporation of the solvent gave 10g of intermediate 5.

b) Preparation of intermediate 6

Intermediate 5(0.043mol), methyl iodide (0.047mol) were stirred in EtOH (300ml) overnight at room temperature. The precipitate was filtered and dried to yield 8.3g of intermediate 6.

c) Preparation of intermediate 7

Intermediate 6(0.023mol), sodium cyanide (0.03mol) in DMF (100ml) was heated at 110 ℃ for 2 h. The reaction mixture was poured into ice water and stirred for 1 hour. The precipitate was filtered and then taken up in DCM. Extracting the solution, separating the organic layer, and removing the organic layer with MgSO₄Drying, filtration and evaporation gave 5.1g of intermediate 7.

d) Preparation of intermediate 8

Methylmagnesium chloride (0.058mol) was added dropwise to a solution of intermediate 7(0.018mol) in THF (50ml) at 5 ℃ under a stream of nitrogen. The reaction mixture was stirred at 5 ℃ for 1 hour. Ammonium chloride 10% was added dropwise at 5 ℃. The reaction mixture was extracted with EtOAc. The organic layer was separated over MgSO₄Drying, filtration and evaporation gave 4g of intermediate 8.

e) Preparation of intermediate 9

Intermediate 8(0.019mol), Raney nickel (4g) in MeOH/NH at room temperature at 3 bar₃Hydrogenation (50ml) for 2 hours. The reaction mixture was filtered through celite, washed with DCM and the filtrate evaporated to give 3.5g of intermediate 9.

f) Preparation of intermediate 10

A mixture of intermediate 9(0.016mol), 2-fluoro-5-nitrotoluene (0.018mol), sodium bicarbonate anion (0.019mol) in DMSO (100ml) was heated overnight at 60 ℃. The mixture was cooled to room temperature. Ice water was added and DCM was added. Extracting the reaction mixture, separating the organic layer, and purifying with MgSO 2₄Dried, filtered and concentrated. The residue was purified by column chromatography over silica gel (eluent: cyclohexane/EtOAc 60/40). The pure fractions were collected and the solvent was evaporated, yielding 2.8g of intermediate 10.

g) Preparation of intermediate 11

Intermediate 10(0.0079mol), vanadium oxide (0.05g), DIPE (1ml) in thiophene (4%) solution, Pt/C5% (1.3g) in THF (50ml) were hydrogenated at room temperature under atmospheric pressure for 1 night. The reaction was filtered and the solvent evaporated to afford intermediate 11. The product was used in the next step without further purification.

The following intermediates were prepared according to method a 3.

Example A4

Intermediate 7 may alternatively be prepared as follows.

a) Preparation of intermediate 12

1- (Triphenylphosphinene) -2-propanone (34.4mmol) was added portionwise to a solution of 1H-indole-7-carbaldehyde (34.44mmol) in methylbenzene (60ml) at room temperature. The reaction mixture was heated at 100 ℃ for 3 hours, then cooled to room temperature and evaporated to dryness. The residue (17.4g) was purified by HPLC over silica gel: 20-45 μm (450g) purified (eluent: 99.5/0.5 DCM/MeOH). The pure fractions were collected and the solvent was evaporated, yielding 4.3g of intermediate 12.

b) Preparation of intermediate 13

Acetic acid (10ml) containing intermediate 12(3.4mmol) and Eschenmoser salt (3.8mmol) was heated at 65 ℃ for 2 h. The precipitate was filtered and dissolved in DCM and potassium carbonate 10%. Potassium carbonate (solid) was added and the mixture was stirred at room temperature for 1 hour. Extracting the reaction mixture, separating the organic layer, and purifying with MgSO 2₄Drying, filtration and evaporation of the solvent gave 0.7g of intermediate 13.

c) Preparation of intermediate 14

EtOH (300ml) containing intermediate 13(0.13mol), methyl iodide (0.14mol) was stirred for 2 days at room temperature. The precipitate was filtered and dried to yield 47g of intermediate 14.

d) Preparation of intermediate 15

Intermediate 14(136.5mmol), sodium cyanide (177.5mmol) in DMF (400ml) was stirred at room temperature for 2 h. Water was added and the reaction mixture was extracted with EtOAc. The organic layer was separated over MgSO₄Drying, filtering and evaporating. The residue was purified by high performance liquid chromatography (Irregular SiOH 20-45 μm 1000g MATREX, mobile phase: cyclohexane 70%/EtOAc 30%). The pure fractions were collected and the solvent was evaporated, yielding 11.8g of intermediate 15.

e) Preparation of intermediate 16

Methylmagnesium chloride (0.07mol) was added dropwise to a solution of intermediate 15(0.022mol) in THF (50 ml). Ammonium chloride 10% and EOAc were added. Extracting the reaction mixture, separating the organic layer, and purifying with MgSO 2₄Drying, filtering and evaporating the solvent. The residue (2.9g) was purified by high performance liquid chromatography (Irregular SiOH 20-45 μm 450g MATREX, mobile phase: cyclohexane 60%/EtOAc 40%). The pure fractions were collected and the solvent was evaporated to yield 2g of intermediate 16.

f) Preparation of intermediate 7

Dess-Martin periodinane (24.9ml) was added dropwise to a solution of intermediate 16(10mmol) in DCM (20ml) at room temperature. The reaction mixture was stirred at room temperature for 1 hour, then poured into ice water and filtered through celiteThe filtrate was extracted with DCM. The organic layer was separated over MgSO₄Drying, filtering and concentrating the solvent. The residue (2.8g) was purified by silica gel column chromatography (eluent: cyclohexane/EtOAc 60/40). The pure fractions were collected and the solvent was evaporated, yielding 0.8g of intermediate 7.

Example A5

Intermediate 15 may alternatively be prepared as follows.

a) Preparation of intermediate 17

Lithium tetrahydroaluminate (0.018mol, 0.69g) was added portionwise to a solution of methyl 3- (cyanomethyl) -1H-indole-7-carboxylate (0.012mol, 2.6g) in THF (50ml) at 5 ℃ under a stream of nitrogen. The reaction mixture was stirred at room temperature for 30 minutes. Water was added dropwise at 5 ℃, and the reaction mixture was filtered through celite, washed with EtOAc and extracted. The organic layer was separated over MgSO₄Drying, filtration and concentration gave 2.4g of intermediate 17.

b) Preparation of intermediate 15

Dess-martin periodinane (0.016mol) was added dropwise to a solution of intermediate 17(0.0081mol) in DCM (15ml) at 5 ℃. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered through celite and the filtrate was evaporated. The residue was purified by column chromatography over silica gel (eluent: cyclohexane/EtOAc 70/30). The pure fractions were collected and the solvent was evaporated, yielding 0.5g of intermediate 15.

Example A6

Preparation of intermediates 18, 19 and 20

4-chloro-6, 7-dihydro-5H-cyclopenta [ b ] at room temperature]A mixture of pyridin-7-ol (5g, 0.029mol) and Candida antarctica lipase B (2.5g) in vinyl acetate (50ml) was stirred for 4 hours. The reaction mixture was filtered through celite. Celite was washed with DCM. The solvent of the filtrate was evaporated. The residue (6.3g) was purified by silica gel column chromatography (eluent: DCM/MeOH, 100/0-98/2; 15-40 μm). Two different product fractions were collected and the solvent of each product fraction was evaporated to yield 2.1g of intermediate 18 (42%; S-enantiomer) and 3.6g of intermediate 19 (58%; R-enantiomer). When required, the reaction mixture can be prepared by reaction in MeOH/NH₃Intermediate 19 is converted to the R enantiomer of intermediate 18 to give intermediate 20.

Intermediates 18 and 20 may alternatively be prepared as follows.

Sodium tetrahydroborate (1.37g, 36.10mmol) was added portionwise to a solution of intermediate 1(5.50g, 32.82mmol) in MeOH (50ml) at 5 deg.C. The reaction mixture was stirred at room temperature for 1 night, then poured into water and the mixture was extracted 3 times with EtOAc. The organic layer was washed with water and aqueous NaCl, separated, and MgSO₄Drying, filtering and evaporating the solvent. The residue (4.42g) was purified by manual supercritical fluid chromatography on CHIRALPAK AD-H5 μm250X20 mm. Mobile phase: 85% CO₂，15％MeOH。

The pure fractions were collected and the solvent was evaporated, yielding 1.625g (29.2%) of compound 18 (S-enantiomer) (DMF, 20 ℃, concentration 0.33 w/v%, 589 nm: -77.58 °) and 1.620g (29.1%) of compound 20 (R-enantiomer) (DMF, 20 ℃, concentration 0.33 w/v%, 589 nm: +76.74 °).

Example A7

Preparation of intermediate 21

Oxalyl chloride (0.0079mol) was added dropwise to a solution containing 7- [2- [ [ (1, 1-dimethylethyl) dimethylsilyl group at 5 deg.C]Oxy radical]Ethyl radical]-1H-indole (0.0047mol) in diethyl ether (20 ml). The reaction mixture was stirred at 5 ℃ for 2 hours. To this solution NH was added dropwise at 5 deg.C₄OH (concentrated, 20 ml). The reaction mixture was stirred at room temperature overnight. Water and EtOAc were added. The organic layer was separated over MgSO₄Drying, filtration and concentration gave 1.8g of intermediate 21.

Preparation of intermediate 22

Lithium tetrahydroaluminate (0.021mol) was added dropwise to a solution of intermediate 21(0.0052mol) in THF (50ml) at 5 ℃. The mixture was stirred at reflux overnight and then cooled to 5 ℃ with an ice bath. The excess lithium tetrahydroaluminate is carefully hydrolyzed by dropwise addition of water. The mixture was filtered through celite, washed with DCM and extracted. The organic layer was separated over MgSO₄Drying, filtration and concentration gave 0.6g of intermediate 22.

Example A8

a) Preparation of intermediate 23

Oxalyl chloride (2.9mmol) was added dropwise to a solution of 4- (1H-indol-7-yl) -3-butan-2-one (2.7mmol) in diethyl ether (10ml) at 5 ℃. The cold bath was removed and the reaction was allowed to warm to room temperature (1.5 hours). The precipitate was filtered and dried to yield 0.49g of intermediate 23.

b) Preparation of intermediate 24

Ammonium hydroxide (10ml) was added dropwise to a solution of intermediate 23(0.725mmol) in diethyl ether (10ml) at 5 ℃. The reaction mixture was stirred at room temperature overnight. The precipitate was filtered and dried to yield 0.11g of intermediate 24.

c) Preparation of intermediate 25

Lithium tetrahydroaluminate (39.5mmol) was added dropwise to a solution of intermediate 24(7.9mmol) in THF (20ml) at 5 deg.C. The mixture was stirred at 80 ℃ for 2 hours and then cooled to 5 ℃ with an ice bath. The excess lithium tetrahydroaluminate is carefully hydrolyzed by dropwise addition of water. The mixture was filtered through celite, washed with DCM and extracted. The organic layer was separated over MgSO₄Drying, filtration and concentration gave 1g of intermediate 25.

d) Preparation of intermediate 26

A mixture of intermediate 25(4.3mmol), 2-fluoro-5-nitrotoluene (4.7mmol), sodium bicarbonate anion (5.1mmol) in DMSO (10ml) was heated at 60 ℃ overnight. The mixture was cooled to room temperature. Ice water was added and DCM was added. Extracting the reaction mixture, separating the organic layer, and purifying with MgSO 2₄Dried, filtered and evaporated. The residue was purified by column chromatography (eluent: DCM/MeOH 97/3)). The pure fractions were collected and the solvent was evaporated. The residue was purified by high performance liquid chromatography (Irregular SiOH 15-40 μm 300g MERCK). Mobile phase (NH)₄OH 0.1% -dichloroMethane 99% -MeOH 1%). The pure fractions were collected and the solvent was evaporated, yielding 200mg of intermediate 26.

e) Preparation of intermediate 27

A mixture of intermediate 26(0.35mmol), Raney nickel (200mg) in MeOH (5ml) was hydrogenated at room temperature under atmospheric pressure. The residue was filtered through celite, washed with DCM and the residue was evaporated to give 0.14g of intermediate 27.

Example A9

a) Preparation of intermediate 28

Tert-butyldimethylsilyl chloride (195mg, 0.91mmol) was added to a solution of intermediate 16(162mg, 0.81mmol), imidazole (143mg, 2.1mmol) in THF (5ml) at room temperature. The reaction mixture was stirred at room temperature overnight. The precipitate was filtered and washed with DCM. The filtrate was evaporated. The residue (365mg) was purified by flash chromatography on silica gel (15-40 μm, 30g, DCM/MeOH: 100/0-99/1). The pure fractions were collected and evaporated to dryness to yield 190mg of intermediate 28.

b) Preparation of intermediate 29

Room temperature, 3 bar H₂Next, intermediate 28(190mg, 0.6mmol), Raney nickel (0.7g) in NH-containing solution was added₃Stirred for 4 hours in MeOH 7N (10 ml). The reaction was filtered through celite, and the celite was washed 3 times with DCM/MeOH (90/10). Evaporating the solvent to obtainTo 147mg of intermediate 29.

c) Preparation of intermediate 30

A mixture of intermediate 29(147mg, 0.46mmol), 1-fluoro-2-methyl-4-nitro-benzene (78mg, 0.51mmol), sodium salt of carbonic acid (1: 1) (85mg, 1mmol) in DMSO (2ml) was heated at 65 ℃ for 1 night. The mixture was poured into ice and stirred for 10 minutes. DCM was added and the mixture was extracted 2 times with DCM. The organic layer was washed with water and then MgSO₄Drying, filtering and evaporating the solvent. The residue was purified by flash chromatography on silica gel (15-40 μm, 30g, cyclohexane/EtOAc 85/15). The pure fractions were collected and evaporated to dryness to yield 200mg of intermediate 30.

d) Preparation of intermediate 31

Intermediate 30(2.8g, 6.17mmol), di-tert-butyl dicarbonate (8g, 37mmol), 4-dimethylaminopyridine (0.15g, 1.2mmol) and triethylamine (1.89ml, 13.6mmol) were stirred in THF (50ml) at 60 ℃ for 24 h. The mixture was evaporated to dryness. The residue (7.4g) was purified by high performance liquid chromatography on silica gel (Cartidge 15-40 μm, 90 g). Mobile phase (cyclohexane 50%: dichloromethane 50%). The pure fractions were collected and the solvent was evaporated, yielding 3.3g of intermediate 31.

e) Preparation of intermediate 32

A mixture of intermediate 31(3.3g, 5mmol), Raney nickel (3g) in MeOH (100ml) was hydrogenated at 2.5 bar for 2 h. The residue was filtered through celite, washed with DCM and the residue was evaporated to give 2.7g of intermediate 32.

f) Preparation of intermediate 33

Intermediate 32(2.6g, 4.2mmol), 4-chloro-6, 7-dihydro-5H-cyclopenta [ b ] are reacted]Pyridin-7-ol (0.78g, 4.6mmol), dioxane containing HCl 4M (0.21ml, 0.8mmol) was heated in acetonitrile (28ml) and EtOH (7ml) at 65 ℃ for 72 h. After cooling to room temperature, water and potassium carbonate powder were added and the mixture was extracted 2 times with EtOAc. The organic layer was washed with water and MgSO₄Dried, filtered and evaporated. The residue was purified by high performance liquid chromatography (Irregular SiOH 20-45 μm 450g MATREX). Mobile phase (NH)₄0.5 percent of OH; 95% of dichloromethane; MeOH 5%). The pure fractions were collected and the solvent was evaporated, yielding 2.35g of intermediate 33.

g) Preparation of intermediate 34

Manganese (IV) oxide (4.3g) was added portionwise to a solution of intermediate 33(2.2g, 2.9mmol) and tris (2- (2-methoxyethoxy) ethyl) amine (47mg, 0.145mmol) in DCM (50ml) at room temperature. The mixture was stirred at room temperature for 18 hours. The mixture was filtered through celite. Celite was washed with DCM and the solvent was evaporated to give 1.84g of intermediate 34.

h) Preparation of intermediate 35

Trifluoroacetic acid (1.2ml, 15.9mmol) containing intermediate 34(1.2g, 1.6mmol) and DCM (10ml) were stirred for 2 days at room temperature. Potassium carbonate 10% was added and the mixture was extracted 2 times with DCM. The organic layer was dried over MgSO4, filtered and evaporated to give 0.8g of intermediate 35, which was used in the next step without further purification.

Example A10

a) Preparation of intermediate 36

Methyl iodide (0.024mol) was added to a solution of methyl 3- [ (dimethylamino) methyl ] -1H-indole-7-carboxylate (0.022mol) in EtOH (50 ml). The mixture was stirred at rt overnight and then concentrated to dryness in vacuo to give 5.6g (68%) of intermediate 36.

b) Preparation of intermediate 37

A mixture of intermediate 36(0.015mol) and sodium cyanide (0.019mol) in DMF (60ml) was stirred at 100 ℃ for 2 hours. Water was added. The precipitate was filtered. The filtrate was evaporated to yield 3.5g (100%) of intermediate 37.

c) Preparation of intermediate 38

Intermediate 37(0.016mol) and Raney nickel (3g) in MeOH/NH at room temperature under 3 bar pressure₃The mixture in (50ml) was hydrogenated and then filtered through celite. The filtrate was evaporated to give 3.2g (89%) of intermediate 38.

d) Preparation of intermediate 39

A mixture of intermediate 38(0.0092mol), 1-fluoro-4-nitro-benzene (0.01mol) and diisopropylethylamine (0.023mol) was stirred at 180 ℃ for 2 hours. Water was added. The mixture was extracted with DCM. The organic layer was separated and dried (MgSO)₄) Filtered and the solvent evaporated. The residue (2.6g) was purified by column chromatography from kromasil (eluent: cyclohexane/EtOAc 60/40). The pure fractions were collected and the solvent was evaporated, yielding 1.4g (45%) of intermediate 39.

e) Preparation of intermediate 40

Tert-butoxycarbonyl anhydride (0.015mol) was added to a mixture of intermediate 39(0.05mol) and 4- (dimethylamino) pyridine (small amount) in THF (50ml) at room temperature. The mixture was stirred at room temperature overnight. The mixture was extracted with EtOAc. The organic layer was washed with water and dried (MgSO)₄) The solvent was filtered and evaporated. The residue (4g) was purified by column chromatography over silica gel (eluent: cyclohexane/EtOAc 60/40). The pure fractions were collected and the solvent was evaporated, yielding 3.3g (82%) of intermediate 40.

f) Preparation of intermediate 41

A mixture of intermediate 40(0.0061mol) and raney nickel (3.3g) in MeOH (50ml) was hydrogenated at room temperature under 3 bar pressure and then filtered through celite. The celite was washed with DCM/MeOH (98/2). The filtrate was evaporated. The residue (2.8g) was purified by silica gel column chromatography (eluent: cyclohexane/EtOAc 70/30). The pure fractions were collected and the solvent was evaporated, yielding 1.3g (42%) of intermediate 41.

g) Preparation of intermediate 42

Intermediate 41(0.0018mol), 4-chloro-6, 7-dihydro-5H-cyclopenta [ b ] at 65 deg.C]A mixture of pyridin-7-ol (0.33g) and HCl/isopropanol (5 drops) in acetonitrile (20ml) was stirred for 24 hours. Potassium carbonate 10% was added. The mixture was extracted with EtOAc. The organic layer was separated and dried (MgSO)₄) The solvent was filtered and evaporated. The residue (1.1g) was purified by column chromatography over silica gel (eluent: DCM/MeOH/NH)₄OH 95/5/0.1). The pure fractions were collected and the solvent was evaporated, yielding 0.054g of intermediate 42.

h) Preparation of intermediate 43

A mixture of intermediate 42(0.0009mol) and HCl 3N (0.0039mol) in acetonitrile (10ml) was stirred at 65 ℃ for 3 h. Potassium carbonate 10% was added. The mixture was extracted with EtOAc. The organic layer was separated and dried (MgSO)₄) The solvent was filtered and evaporated. The residue (0.4g, 100%) was crystallized from diethyl ether/acetonitrile. The precipitate was filtered and dried to yield 0.3g of intermediate 43, mp 164 ℃.

B. Preparation of the end product

Example B1

Preparation of Compound 1

Intermediate 11(1.00g, 3.092mmol), intermediate 2(0.437g, 2.381mmol), HCl (0.155ml, 0.618mmol) were stirred in acetonitrile (10ml) at 65 ℃ for 5 h. The reaction was cooled to room temperature, then basified with potassium carbonate (10% aqueous) and extracted 3 times with DCM. The organic layer was washed with water and MgSO₄Dry, filter and evaporate the solvent. The residue (1.37g) was purified normally by silica gel (5 μm 150X30.0 mm). The mobile phase is as follows: gradient from 0% NH₄OH, 100% DCM, 0% MeOH to 0.8% NH₄OH, 92% DCM, 8% MeOH. The pure fractions were collected and the solvent was evaporated. The residue (0.350g) was taken up into acetonitrile for 1 week at room temperature. The product crystallizes, is filtered and dried in vacuo to yield 300mg (20.8%) of compound 1, m.p. 209 ℃.

Example B2

Preparation of Compounds 2 and 3

A solution of intermediate 11(0.0031mol), N-diisopropylethylamine (0.0028mol), 4-bromopyridine hydrochloride (0.0034mol) in acetonitrile (20ml) was heated at 65 ℃ for 6 hours. Potassium carbonate 10% and EtOAc were added. Extracting the reaction mixture, separating the organic layer, and purifying with MgSO 2₄Dried, filtered and concentrated. The residue (1.3g) was purified by column chromatography over silica gel (eluent 95/5/0.5 DCM/MeOH/NH)₄OH). The two fractions were collected and the solvent was evaporated to give 60mg of compound 3 and 80mg of F2. The residue F2(80mg) was purified by supercritical fluid chromatography (AMINO column 150X21.2mmm, eluent: MeOH/CO)₂30/70). The pure fractions were collected and the solvent was evaporated, yielding 43mg of compound 2.

Example B3

Preparation of Compound 4

Intermediate 35(0.8g, 1.7mmol) and sodium hydroxide 3N (2.57ml, 2.57mmol) were stirred in EtOH (22ml) and THF (10ml) for 30 min at room temperature. Addition of NH₄Cl 10% and DCM, the mixture was filtered through celite. The organic layer was decanted and MgSO₄Dried, filtered and evaporated. The residue was purified over the normal phase of Spherical SiOH (10 μm 60g PharmPrep MERCK). The mobile phase is NH₄OH 0.1%, 95% DCM, 5% MeOH. The pure fractions were collected and the solvent was evaporated, yielding 250mg of compound 4.

Example B4

Preparation of Compound 5

Sodium tetrahydroborate (0.69mmol) was added to a solution of intermediate 49(0.46mmol) in MeOH (5ml) at 5 deg.C. The reaction mixture was stirred at room temperature overnight. Adding water, extracting the reaction mixture with EtOAc, separating the organic layer, and removing MgSO₄Dried, filtered and evaporated. The residue (0.14g) was crystallized from acetonitrile, and the precipitate was filtered and dried to give 0.1g of compound 5.

Example B5

Preparation of Compound 6

In N₂Lithium tetrahydroaluminate (0.0003mol) was added dropwise to a solution of intermediate 43(0.0002mol) in THF (4ml) at 5 ℃ under a stream of air. The mixture was stirred at room temperature for 2 hours. Water was added. The mixture was extracted with EtOAc. Separating the organic layer, drying (MgSO₄) The solvent was filtered and evaporated. The residue (0.18g) was purified by column chromatography over kromasil (eluent: DCM/MeOH/NH)₄OH 98/2/0.2-85/15/1; 3.5 μm). The pure fractions were collected and the solvent was evaporated, yielding 0.066g (70%) of compound 6.

Example B6

Preparation of Compounds 7 and 8

Intermediate 47((1.00g, 3.23mmol), intermediate 4(0.653g, 3.55mmol), HCl/dioxane (0.242ml, 0.97mmol) in acetonitrile (15ml) was stirred at 70 ℃ overnight the reaction mixture was cooled to room temperature water and potassium carbonate (10% aq) were added in sequence and the mixture was extracted 3 times with DCM, the organic layer was separated, MgSO₄Dry, filter and evaporate the solvent. The residue was purified forward on silica gel (cartidge 15-40 μm 30g) with 96% DCM, 4% MeOH as mobile phase. The pure fractions were collected and the solvent was evaporated, yielding 0.092g (6.5%) of compound 7 and 0.030g (2.0%) of compound 8.

Example B7

Preparation of Compound 9

Intermediate 27(0.41mmol) and 4-chloro-6, 7-dihydro-5H-cyclopenta [ b ]]Pyridin-7-ol (0.46mmol) was heated at 125 ℃ for 2 h. The residue was purified by column chromatography over silica gel (eluent 90/10/0.1 DCM/MeOH/NH)₄OH). The pure fractions were collected and the solvent was evaporated, yielding 58mg of compound 9.

Example B8

Preparation of Compounds 24 and 25

Compound No. 24:^*S

compound No. 25:^*R

^*refers to the relative stereochemistry (the absolute stereochemistry is unknown). Thus, if compound 24 is the S enantiomer, then compound 25 is the R enantiomer, or if compound 24 is the R enantiomer, then compound 25 is the S enantiomer.

The reaction is carried out in N₂The reaction is carried out under an atmosphere.

Under reflux, intermediate 47(11.31mmol), intermediate 4(12.44mmol), Pd (dba)₂A mixture of (1.13mmol), BINAP (1.13mmol) and tBuONa (22.62mmol) in toluene (100ml) was stirred overnight. The mixture was filtered, washed with MeOH and purified by high performance liquid chromatography (column Synergi 50x 250mm, 10 μm; eluent: CH)₃CN/H₂O (0.1% trifluoroacetic acid) gradient 15% CH at time 0 min₃CN to 40% CH at time 25 min₃CN). The desired fractions were collected and the solvent was evaporated in vacuo. The residue was extracted with DCM and NaHCO₃Washing with aqueous solution over Na₂SO₄Drying, filtration and evaporation gave compound 8(3.8g, 98% purity, 65% yield) which was subjected to supercritical fluid chromatography (Chiralcel OJ, 20 μm, 250mm by 20 mm; supercritical CO)₂Isopropanol (0.05% diethylamine), 40: 60v/v, 70ml/min) to give two fractions:

1000.79mg (19%) of Compound 24, mp 110.5-111.6 ℃.

(ee％＝99％，[α]_D ²⁰+5.034 ° (wavelength 589, 20 ℃, concentration 10.33mg/ml in methanol), and

939.19mg (18%) of Compound 25, mp 111.4-113.3 ℃.

(ee％＝99％，[α]_D ²⁰3.443 ° (wavelength 589, 20 ℃, 10.74mg/ml in methanol).

Example B9

Preparation of the Compounds

Compound No. 26:^*S

compound No. 27:^*R

^*refers to the relative stereochemistry (the absolute stereochemistry is unknown). Thus, if compound 26 is the S enantiomer, compound 27 is the R enantiomer, or compound 26 is the R enantiomer, compound 27 is the S enantiomer.

The reaction is carried out in N₂The reaction is carried out under an atmosphere.

Under reflux, intermediate 11(9.25mmol), intermediate 2(9.25mmol), tBuONa (18.5mmol), Pd (dba)₂A mixture of (0.93mmol) and BINAP (0.93mmol) in toluene (40ml) was stirred for 3 hours. The resulting mixture was cooled to room temperature and filtered. The filtrate was evaporated in vacuo and the residue was purified by high performance liquid chromatography (column Synergi 50x 250mm, 10 μm; eluent: CH)₃CN/H₂O (0.1% trifluoroacetic acid), gradient: 10% CH at time 0 min₃CN to 40% CH at time 25 min₃CN). The desired fractions were collected and adjusted to pH > 7. The solvent was concentrated, extracted with ethyl acetate (3 × 100ml), and the desired organic layer was washed with brine, over Na₂SO₄Drying, filtering and evaporation of the solvent in vacuo gave 2.5g (57%) of Compound 1, which was isolated by supercritical fluid chromatography (AD 250mm 20mm, 20 μm; supercritical CO)₂Isopropanol (0.05% diethylamine) 40: 60v/v, 70ml/min) to give two fractions.

Fraction 1(1g) was subjected to high performance liquid chromatographyOne-step purification (column Synergi 50x 250mm, 10 μm; eluent: CH)₃CN/H₂O (0.1% trifluoroacetic acid), gradient: 10% CH at time 0 min₃CN to 40% CH at time 25 min₃CN) to yield 0.88g (20%) of compound 26, mp 122.3-124 ℃, (ee 100%).

Fraction 2(1g) was further purified by HPLC (column Synergi 50x 250mm, 10 μm; eluent: CH)₃CN/H₂O (0.1% trifluoroacetic acid), gradient: 10% CH at time 0 min₃CN to 40% CH at time 25 min₃CN) to yield 0.96g (22%) of compound 27, mp 121.4-122.6 ℃, (ee 99%).

Table 1 lists compounds prepared according to one of the above examples.

TABLE 1

^*Relative stereochemistry

Compound characterization

Melting point:

the values are either peak or melting range, and the resulting values have experimental uncertainties typically associated with analytical methods.

Kofler

The melting point of compound 4 was determined using a Kofler hot plate (hot bench) consisting of a hot plate with a linear temperature gradient, a sliding pointer and a temperature scale in degrees celsius.

WRS-2A

For compounds 11, 24, 25, 26, 27, 28, 29, 30, 31, their melting points were determined using a WRS-2A melting point apparatus, available from Shanghai precision scientific instruments, Inc. The linear heating rate for melting point determination is 0.2-5.0 deg.C/min. The reported values are melting ranges. The maximum temperature was 300 ℃.

DSC

The melting point of compound 1 was determined by DSC (differential scanning calorimetry). The melting point was determined using a temperature gradient of 10 ℃/min. The maximum temperature was 350 ℃. The value is the peak value.

LCMS

LCMS-characterization of the compounds of the invention was determined using the following procedure.

General procedure A

The HPLC measurements were performed using an Alliance HT 2795(Waters) system comprising a quaternary pump with degasser, an autosampler, a Diode Array Detector (DAD) and a column specified in the methods below, which was maintained at a temperature of 30 ℃. The split stream from the column enters the MS spectrometer. The MS detector was equipped with an electrospray ionization source. Capillary needle Voltage (capillary needle Voltage)3kV, LCT (Waters time of flight Zspray)^TMMass spectrometer) the ion source temperature (source temperature) was maintained at 100 ℃. Nitrogen was used as the atomizing gas. Data acquisition was performed using a Waters-Micromass MassLynx-Openlynx data System.

General procedure B

LC assay employs a UPLC (ultra performance liquid chromatography) acquity (waters) system comprising a binary pump with degasser, an autosampler, a Diode Array Detector (DAD) and a column specified in the following methods, which is maintained at a temperature of 40 ℃. Liquid from the column enters the MS detector. The MS detector was equipped with an electrospray ionization source. The capillary needle voltage 3kV, ion source temperature on Quattro (triple quadrupole mass spectrometer from Waters) was maintained at 130 ℃. Nitrogen was used as the atomizing gas. Data acquisition was performed using a Waters-MicromassMassLynx-Openlynx data System.

General procedure C

HPLC assays were performed using an Agilent 1100 module, which contained a pump, a Diode Array Detector (DAD) (using a wavelength of 220nm), a column heater, and columns as specified in the methods below. The split stream from the column entered the Agilent MSD series G1946C and G1956A. The MS detector was configured with API-ES (atmospheric pressure electrospray ionization). Mass spectra were obtained by 100-. Capillary needle voltage: the positive ionization mode is 2500V, and the negative ionization mode is 3000V. Fragmentation voltage 50V. The drying gas temperature was maintained at 350 ℃ and the flow rate was 10 l/min.

Method 1

In addition to general procedure a: reverse phase HPLC was performed using a Xterra-MS C18 column (5 μm, 4.6X150mm) at a flow rate of 1.0 ml/min. Gradient conditions were carried out using two mobile phases (mobile phase A: 100% 7mM ammonium acetate; mobile phase B: 100% acetonitrile): 85% A, 15% B (3 min hold), 20% A, 80% B in 5 min, 6 min hold at 20% A and 80% B, and equilibrate for 3 min with initial conditions. A20. mu.l injection volume was used. A positive ionization mode cone voltage (cone voltage) of 20V and a negative ionization mode of 20V. Mass spectra were acquired by 100-900 scans over 0.8 seconds with an inter-scan delay (Interercn delay) of 0.08 seconds.

Method 2

In addition to general procedure B: reverse phase UPLC was performed on a Waters Acquity BEH (bridged ethylsiloxane/silica gel hybrid) C18 column (1.7 μm, 2.1X100mm) at a flow rate of 0.35 ml/min. Gradient conditions were carried out using two mobile phases (mobile phase A: 95% 7mM ammonium acetate/5% acetonitrile; mobile phase B: 100% acetonitrile): 90% A and 10% B (hold 0.5 min), changing to 8% A and 92% B in 3.5 min, hold 2min, return to initial conditions in 0.5 min, hold 1.5 min. A sample volume of 2. mu.l was used. The cone hole voltage of positive ionization mode and negative ionization mode is 20V. Mass spectra were acquired by 100-1000 scans within 0.2 seconds with a 0.1 second delay between scans.

Method 3

In addition to general procedure C: reverse phase HPLC was performed on YMC-Pack ODS-AQ, 50X2.0mm5 μm column at a flow rate of 0.8 ml/min. Two mobile phases (mobile phase A: water with 0.1% TFA; mobile phase B: acetonitrile with 0.05% TFA) were used. First 90% A and 10% B were held for 0.8 min. Then gradient was performed to 20% a and 80% B in 3.7 min and held for 3 min. Typical sample injection volumes were used: 2 μ l. The temperature of the incubator is 50 ℃. (MS polarity: Positive).

Table 2: LCMS data and melting Point

(Retention time R_t(min)，(MH)⁺Peak, LCMS procedure refers to method for LCMS)

Optical rotation

The optical rotation was measured using a polarimeter. [ alpha ] to]_D ²⁰The optical rotation measured at 20 ℃ using light having a D-line wavelength (589nm) of sodium is shown. The sample cell optical path was 1 dm. After the actual values, the concentrations and solvents of the solutions used for the determination of the optical rotation are noted.

Table 3: optical rotation

Compound numbering	[α]D 20	Concentration of	Solvent(s)
				18	-37.4°	0.516w/v％	DMF
20	+36.87°	0.575w/v％	DMF
				21	-39.68°	0.378w/v％	DMF
24	+5.03°	10.30mg/ml	MeOH
				25	-3.44°	10.70mg/ml	MeOH
26	-13.74	8.59mg/ml	Chloroform
				27	+11.89	9.00mg/ml	Chloroform
30	-5.25	4.00mg/ml	MeOH
				31	+7.99	4.00mg/ml	MeOH
28	-4.90	10.00mg/ml	MeOH
				29	+5.69	9.89mg/ml	MeOH

C. Pharmacological agentsExample of study

The ability of the compounds of the invention to protect p53 in a2780 cells was determined using the p53 enzyme-linked immunosorbent assay. The p53 assay is a "sandwich" enzyme immunoassay employing two polyclonal antibodies. A polyclonal antibody specific for p53 protein has been immobilized on the surface of a plastic well. Any p53 present in the sample to be tested will bind to the capture antibody (capture antibody). The biotinylated detector polyclonal antibody also recognizes the p53 protein and will bind to any p53 that has been immobilized by the capture antibody. The detector antibody is then bound by horseradish peroxidase conjugated streptavidin. Horseradish peroxidase catalyzes the conversion of the chromogenic substrate o-phenylenediamine with a catalytic strength proportional to the amount of p53 protein bound to the plate. And quantifying the chromogenic reaction product by using a spectrophotometer. Quantification was achieved by constructing a standard curve with known concentrations of purified recombinant HIS-tagged p53 protein (see example c.1.).

C.1.p53 ELISA

At 37 deg.C, with 5% CO₂A2780 cells (ATCC) were cultured in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS), 2mM L-glutamine and gentamicin.

A2780 cells were seeded at 20000 cells per well in 96-well plates, cultured in a humidified incubator at 37 ℃ for 24 hours and treated with compounds for 16 hours. After incubation, the cells were washed once with phosphate buffered saline, and then 30. mu.l of low-salt RIPA buffer (20mM tris, pH7.0, 0.5mM EDTA, 1% Nonidet P40, 0.5% DOC, 0.05% SDS, 1mM PMSF, 1. mu.g/ml aprotinin and 0.5. mu.g/ml leupeptin) was added to each well. The plate was placed on ice for 30 minutes to complete lysis. The p53 protein was detected in split lysates (de lysate) using a sandwich ELISA as described below.

High binding polystyrene EIA/RIA 96 well plates (Costar 9018) were used in coating buffer (0.1M NaHCO)₃pH8.2) at a concentration of 1. mu.g/ml, and 50. mu.l per well, of capture antibody pAb1801(Abcamab 28-100). The antibody was allowed to adhere overnight at 4 deg.C. The coated plates were washed once with Phosphate Buffered Saline (PBS)/0.05% Tween 20, and 300 μ l blocking buffer (PBS, 1% Bovine Serum Albumin (BSA)) was added for an incubation period of 2 hours at room temperature. Dilutions of purified recombinant HIS-tagged p53 protein were prepared in blocking buffer in the range of 3-200ng/ml and used as standards.

Each plate was washed 2 times with PBS/0.05% Tween 20 and blocking buffer or standard was added at 80. mu.l/well. To the standard was added 20. mu.l lysis buffer. Samples were added to the other wells in an amount of 20 μ l lysate/well. After overnight incubation at 4 ℃, each plate was washed 2 times with PBS/0.05% Tween 20. An aliquot of 100. mu.l of secondary polyclonal antibody p53(FL-393) (Tebubio, sc-6243) was added to each well at a concentration of 1. mu.g/ml in blocking buffer and allowed to adhere for 2 hours at room temperature. Plates were washed 3 times with PBS/0.05% Tween 20. The detection antibody anti-rabbit HRP (sc-2004, Tebubio) was added at a concentration of 0.04. mu.g/ml in PBS/1% BSA and incubated at room temperature for 1 hour. Each plate was washed 3 times with PBS/0.05% Tween 20, 100. mu.l substrate buffer (substrate buffer ready for use: 1 piece of 10mg o-phenylenediamine (OPD) (from Sigma) and 125. mu.l 3% H₂O₂Add to 25ml OPD buffer: 35mM citric acid, 66mM Na₂HPO₄At ph 5.6). After 5-10 minutes, 50. mu.l stop buffer (1M H) was added through each well₂SO₄) The color reaction was stopped. The absorbance was measured at a dual wavelength of 490/655nm using a Biorad microplate reader and the results were then analyzed.

For each experiment, a control (no drug) and a blank culture (no cells or drug) were run in parallel. Blank values were subtracted from all control and sample values. For each sample, the p53 values (absorbance units) are expressed as a percentage of the p53 values in the control. Percentage maintenance above 140% is defined as significant. Thus, the effect of the test compound is expressed as the lowest dose (LAD) giving at least 140% of the value of p53 present in the control (see table 4).

In some experiments, the assay was adapted in 384-well plates.

Table 4: results for compounds tested in the above p53 ELISA protocol (A2780 cells)

C.2 proliferation assay

Human a2780 ovarian cancer cells were given by dr.t.c.hamilton (Fox Chase cancer centre, Pennsylvania, u.s.a.). The cells were cultured in RPMI 1640 medium supplemented with 2mM L-glutamine, 50. mu.g/ml gentamicin and 10% fetal bovine serum.

Reagents used in Alamar Blue (Alamar Blue) assay

Resazurin was purchased from Aldrich (Prod.No. 199303). Potassium ferrocyanide, potassium ferricyanide, KH₂PO₄And K₂HPO₄Purchased from Sigma (prod. No. P9387, P8131, P5655 and P8281, respectively).

Potassium phosphate buffer 0.1M (PPB) was prepared as follows: mixing 2.72g KH₂PO₄And 13.86gK₂HPO₄Dissolving in 500ml milli-Q H₂In O, the pH was adjusted to pH 7.4 and then adjusted with milli-Q H₂O add volume to 1 liter; the buffer was filter sterilized and then stored at room temperature. Resazurin stock solution (PPB-A) was freshly prepared by dissolving 45mg of Resazurin in 15ml of PBS. 30mM potassium ferricyanide (PPB-B) was prepared by dissolving 0.987g potassium ferricyanide in 100ml PPB. 1.266g of potassium ferrocyanide was dissolved in 100ml of PPB to prepare 30mM potassium ferrocyanide (PPB-C).

Mixtures of PPB-A, PPB-B and PPB-C were made by mixing equal volumes of each solution. A resazurin working solution (referred to herein as "alamar blue" solution) was prepared by diluting the mixture 20 × (vol/vol) in PPB followed by filter sterilization; the alamar blue solution can be stored at 4 ℃ for up to 2 weeks.

Alma blue analysis program

For experiments in 384-well plates, cells were plated at a density of 5 × 10³Cells/ml were seeded in 45. mu.l of medium in black Falcon 384-well plates (Life Technologies, Merelbeke, Belgium) with a clear bottom. Cells were allowed to adhere to the plastic for 24 hr. Test compounds were pre-diluted (1/50 in medium) and 5 μ l of the pre-diluted compound was then added to each well. After 4 days of culture, 10. mu.l of Amanian blue solution was added to each well, and the cells were further cultured at 37 ℃ for 5 hours (A2780). The fluorescence intensity for each well was determined using a fluorescent plate reader (Fluorskan, Labsystems, 540nm excitation and 590nm emission).

Antiproliferative activity was calculated as the percentage of viable cells retained under the treated conditions compared to the control (untreated cells) conditions. In the experiment, the results under each experimental condition are the average of 3 parallel test wells. Where appropriate, the experiment was repeated to determine a complete concentration-response curve. IC calculation using probability analysis (Finney, d.j., Probit analytics, second edition, chapter 10, Graded Responses, Cambridge University Press, Cambridge 1962) on hierarchical data, where appropriate₅₀Value (concentration of drug required to reduce cell growth to 50% of control). Herein, the effect of the test compound is expressed as pIC₅₀(IC₅₀Negative log of values (M)) (see Table 5A).

Table 5A: results of Compounds assayed in the above cell proliferation protocol (A2780 cells)

Compound numbering	A2780 cell proliferation inhibition pIC50
		6	6.67
17	7.40
		12	7.20
13	6.90
		19	7.34
2	7.38
		5	7.28
22	6.26
		15	7.27
20	7.29
		18	7.47
21	6.85
		9	6.85
11	6.55
		14	6.43
4	6.74
		10	6.70
7	6.96
		8	7.06
16	6.87
		23	6.52
1	6.94
		27	6.85
24	7.18
		31	6.84
26	7.11
		30	7.04
29	7.14
		28	6.89

Compounds can also be tested according to the above protocol, but using other cell lines: colorectal cancer cell line HCT116, non-small cell lung cancer cell line H1299 and human prostate cancer cell line DU 145. The results are reported in table 5B.

TABLE 5B

C.3. P450 analysis

CYP P450 (e.coli) expressed protein (3a4, 2D6, 2C9, 1a2&2C19) Converting its specific substrate into a fluorescent molecule^{1 2}. The fluorescent molecules are measured using a fluorescent plate reader. Compounds that inhibit this enzymatic reaction will result in a reduction of the fluorescent signal.

Transformation mediated by cytochrome P450 isozymes expressed by each cDN4

Abbreviations:

CEC: 7-ethoxy-3-cyanocoumarin; CHC: 3-cyano-7-hydroxycoumarin;

MFC: 7-methoxy-4-trifluoromethylcoumarin; 7-HFC: 7-hydroxy-trifluoromethyl coumarin is used as the active component,

CEC: 7-ethoxy-3-cyanocoumarin; CHC: 3-cyano-7-hydroxycoumarin;

AMMC: 3- [2- (N, N-diethyl-N-methylamino) ethyl ] -7-methoxy-4-methylcoumarin;

AHMC: 3- [2- (N, N-diethylamino) ethyl ] -7-hydroxy-4-methylcoumarin hydrochloride,

BFC: 7-benzyloxy-trifluoromethylcoumarin;

DBF: dibenzylfluorescein, 7-BQ: a benzyloxyquinoline.

Cofactor mixture: (for all CYP enzymes except CYP2D6)

Abbreviations:

G-6-P: glucose-6-phosphate; G-6-P-DH: glucose-6-phosphate-dehydrogenase

Cofactor mixture: (for CYP2D6)

CYP P450 enzyme solution:

all these CYP enzymes were dissolved in 0.01M Na-K-phosphate buffer + 1.15% KCl and stored on ice until use. CYP P450 enzymes were stored at-80 ℃.

Dilution of compound and reference inhibitor：

The compound and the reference inhibitor were delivered to the department as 5mM DMSO solutions. Preparation of 5.10 via serial dilution with acetonitrile^-4M, as a working solution. The final compound concentration for the primary screen was 10^-5M, and a final solvent concentration of 2%. At a concentration of 10^-5After primary screening under M, at 3.10^-9-10^-5Concentration range of M determination of IC of selected effective inhibitors₅₀The value is obtained. In the primary screen, all compounds were tested in triplicate.

At 10^-9-10^-4Concentration range of M, test reference inhibitor.

Reference inhibitors

Substrate solution:

the substrate stock was dissolved in acetonitrile and stored at-20 ℃. The final working solution was dissolved in 0.1M Na-K-phosphate buffer pH 7.4 and this solution was always prepared fresh before starting the assay.

Data analysis

The preparation of the board, data transmission, data analysis, result verification and approval and data uploading are all performed semi-automatically by using Lexis-Laplace software (Laplace-DLM-RVAM).

The equation used for the calculation is:

% activity ═ x (100/(positive control mean-negative control mean)) x (sample mean-negative control mean)

% inhibition of 100 [% activity

When calculated, the IC is automatically generated based on the intersection with the 50% control axis via extrapolation of the plot in RVAM₅₀The value is obtained.

Method of producing a composite material

The assay was performed in black 96-well Costar plates. The assay contained per well: mu.l CYPP450 enzyme solution (in the negative control sample, 40. mu.l 0.1M Na-K-phosphate buffer pH 7.4 without enzyme was added); 40 μ l cofactor cocktail; 2 μ l of compound or reference inhibitor for negative control samples or solvent for positive control samples. After pre-incubation for 5 minutes in a shake incubator at 37 ℃, 20 μ l of substrate solution was added. Each plate was incubated at 37 ℃ for 10 minutes (CYP3A4/DBF), 15 minutes (CYP1A2), 30 minutes (CYP2C9, CYP3A4/BFC, and CYP3A4/7-BQ & CYP2C19), and 45 minutes (CYP2D 6). The reaction was stopped by adding 200. mu.l acetonitrile. For CYP3A4/DBF, the reaction was stopped by adding 200. mu.l 2M NaOH. For CYP3A4/7-BQ, the reaction was terminated by the addition of 40. mu.l Tris/acetonitrile (1: 5) (V: V) followed by centrifugation at 2000rpm for 10 minutes. The fluorescence signal is detected by a fluorescence Victor2(Wallac) or Fluoroskan (Labsystems) reader. The excitation and emission wavelengths for the different enzymes and their specific substrates are described in table 6.

Table 6: excitation and emission wavelengths

Enzyme	Substrate	Excitation wavelength	Emission wavelength
				CYP1A2	CEC	410nm	460nm
CYP3A4	BFC	405nm	535nm
				CYP3A4	DBF	485nm	538nm
CYP3A4	7-BQ	405nm	535nm
				CYP2C9	MFC	405nm	535nm
CYP2C19	CEC	410nm	460nm
				CYP2D6	AMMC	390nm	460nm

Reference to the literature

[1] Microtiter Plate Assays for Inhibition of Human, Drug-metabolising cytochromes P450 (for Microtiter Plate analysis of Human Drug-metabolising cytochromes P450) Charles L.Crespi, Vaughn P.Miller, Bruce W.Penman (Gentest) Analytical Biochemistry 248, 188-charge 190(1997) particle n.AB 972145

[2] Novel High Throughput fluorescent P450 assays v.p.miller, j.ackermann, d.m. stressors, c.l.crespigent Internet site.

Table 7 provides data for the compounds of the invention tested in 7 trials (compound numbers 18, 20, 9, 14 and 4) when compared to prior art compounds. As described above, five P450 enzymes were tested, one of which was tested on three different substrates (hence 7 trials in total). In Table 7 it is indicated how many P450 tests the compounds showed to have IC₅₀< 1.00E-06.

Table 7: comparative data on CYP inhibition

C.4. antagonism of Ro-4-1284 in mice^3，4，5

The test is Colpaert et al (1975)³A variant of said procedure. Male NMRI mice (22 ± 3g) were housed in macrolon observation cages (LxWxH: 11x12x17 cm; n-3 mice/cage). At the beginning of the experiment, immediately prior to administration of the test compound, the initial body temperature of the mice was measured with an accuracy of 0.1 ℃ by inserting a thermosensitive probe (1.0 mm diameter) of an electronic thermometer (combar) to a fixed depth of 3 cm in the esophagus until a stable reading was obtained. The right eye pupil diameter was measured with a scale microscope and expressed in units of 1/24 mm. 15 minutes after administration of the test compound, mice were challenged with Ro-4-1284(10mg/kg, s.c.). Ro-4-1284 is a serpentinoid vesicular monoamine transporter (VMAT-2) inhibitor that rapidly depletes secretory vesicles^3，4. Ro-4-1284 mice were evaluated for eyelid openness (0, 1, 2, 3, 4, 5) and motor ability (-1, 0, 1, 2, 3) 15, 30 and 60 minutes after challenge. At 60 minute intervals, immediately after the overt behavior score, the right eye pupil diameter and esophageal temperature were measured again. Abnormal phenomena such as dense sniffing, chewing, uprighting, engorgement, piloerection, salivation, tremors, convulsions, and death were recorded (subsequent phenomena were also recorded when they occurred prior to administration of Ro-4-1284). Criteria for drug-induced action: repeated ptosis: eyelid openness score > 1 at 15, 30, or 60 minutes (2.7, 0.5, and 0% false positive controls, respectively; n > 350); induction of collapse: exercise score-1 at 15, 30 or 60 minutes (not seen in control); the repetitive motion is weakened: motor scores > 0 at 15, 30 and 60 min (2.2, 0.8 and 0% false positive controls, respectively); repeated miosis: pupil diameter > 5 units (0.8% false positive) at 60 min; enhancement of body temperature reduction: temperature drop (over 1h time interval) > 9.0 ℃ (1.4% false positive); repeated hypothermia: the temperature drop was < 3.0 deg.C (1.8% false positive).

R0-4-1284 was injected 15 minutes after subcutaneous or oral administration and observation was initiated for 15 minutes according to standard procedures. The dose is initially administered to3 animals, and the compound is considered active when at least 2 of the 3 animals exhibit activity for at least one observation. In other cases, the compound is considered inactive under specific time-path-dose therapy and is classified as complete.

Compounds No. 20, 18, 1, 27, 24, 25, 31, 30, 7, 29, 28, 26 and 17, which were tested on 3 animals each after oral administration at a concentration of 80mg/kg, showed no activity on repeated miosis, repeated ptosis, repeated hypothermia. Compound 4 was tested only at 40mg/kg and showed no activity on repeated miosis, repeated ptosis, repeated hypothermia.

The compounds of EP 1809622 were also tested in the same test and the results are shown in table 8 below.

Table 8: comparing data

Reference to the literature

[3] Colpaert, f.c., Lenaerts, f.m., nieegees, c.j.e., Janssen, p.a.j.: "clinical study of Ro-4-1284 antagonism in mice," Arch. int. Pharmacodyn.21540-90 (1975).

[4] Colzi, a., D' Agostini, r., Cesura, a.m., Borroni, e., Da Prada, m.: "Monoamine oxidases-A inhibitors and dopamine metabolism inrata caudatus: evidence of a monoamine oxidase A inhibitor in rat tail and dopamine metabolism: in vivo evidence of dopamine-substituted reversible monoamine oxidase A inhibitor with increased levels of cytosol) was used, J.Pharmacol.Exp.Ther.265103-111 (1993).

[5] Filiger, e.j.: "Effect of a reserpine-like agent on the release and metabolism of [3H ] NA in cell bodies and terminals", Gen.Pharmacol.251039-1043(1994).

C.5 Patch-clamp test (patch-clamp test) -HERG-mediated K in HEK293 cells ⁺ Electric power Flow suppression

Experiments were performed using HEK293 cells stably expressing the HERG potassium channel. At 37 ℃ and 5% CO₂The cells were grown in MEM medium supplemented with 10% heat-inactivated fetal calf serum, 1% L-glutamine-penicillin-streptomycin-solution, 1% non-essential amino acids (100X), 1% sodium pyruvate (100mM), and 0.8% Geneticin (Geneticin) (50 mg/ml). Before use, the cells were sub-cultured in MEM medium without 5ml L-glutamine-penicillin-streptomycin. For use in the automated patch clamp system PatchXpress7000A (Axoinstruments), cells were collected to obtain a cell suspension of single cells.

The extracellular solution contained (mM): 150NaCl, 4KCl, 1MgCl₂、1.8CaCl₂10HEPES, 5 glucose (adjusted to pH 7.4 with NaOH). Pipette solutions contained (mM): 120KCl, 10HEPES, 5EGTA, 4ATP-Mg2, 2MgCl₂、0.5CaCl₂(adjusted to pH7.2 with KOH).

Patch clamp experiments were performed in voltage-clamp mode and whole cell currents were recorded by automated patch clamp testing using the patch xpress7000A system (Axon Instruments). The current signal is amplified and digitized by multicamp amplifiers, stored and analyzed by PatchXpress, DataXpress software, and Igor 5.0 (Wavemetrics).

The control potential (holding potential) was-80 mV. HERG current (K) determined as the maximum tail current after 2 seconds to +60mV of depolarization⁺-selective outward current) at-40 mV. The pulse cycle rate was 15 seconds. Before each test pulse, a brief pulse (0.5 sec) from the control potential to-60 mV was given to determine the (linear) leakage current. After establishing a whole-cell pattern (whole-cell configuration) and a stabilization period, the medium was applied for 5 minutes, followed by 10 minutes^-7M、3x10^-7M and 3x10^-6M increasing concentrations of test substance were applied. Each concentration of test substance was applied twice. The effect of each concentration after 5 minutes was measured as the average current of 3 consecutive voltage pulses. To determine the extent of the blockage, the residual current is compared to the pre-treated medium. Data (table 9) are presented as the mean of the percent inhibition compared to the control (control 100%) (standard error of the mean (SEM), if available).

Table 9: for HERG mediated K in HEK293 cells⁺Suppression of electric current

Surprisingly, the compounds of the present invention show excellent in vitro activity, which combines low affinity for P450 enzymes, no in vivo drug-induced neurological effects, and low cardiovascular effects.

C.6. In vivo antitumor effect

In vivo antitumor activity can be detected as follows:

NCI standard reference:

biservy, M-c, and chat, g.g.history and new development methods of anti-cancer drug use in vivo and invitro (history and new advances in methods of screening and evaluating anti-cancer drugs used in vivo and in vitro). 587-602.

Animal model:

these studies used immunodeficient (athymic) male NMRI nude (Nu/Nu) mice (20-25 g, obtained from Ja)nvier, France). The initial body weight was approximately 23-34 grams. All animals were kept in "complete isolation" conditions of SPF with free access to food and water. Mice were housed in groups in Techniplast type 3 IVC cages under conditions: 12 hours day night cycle (giving light at 06: 00), temperature 19-22 ℃ and humidity 35-40%. Mice were fed standard laboratory food. All experiments were performed according to the guidelines of the European Commission (86/609/EEC) and had to be approved by the local ethical Commission. For the established tumor allograft model (tumor volume-200 mm)³) Mice were randomly grouped, 5 mice per treatment group, according to tumor volume.

The test system comprises:

the human U87 glioma tumor cell line originates from a 44-year-old female Caucasian patient. At 37 deg.C in a humid atmosphere (5% CO)₂95% air), the cells were cultured in DMEM medium supplemented with 2mM L-glutamine, 2.0mM sodium pyruvate, 25 units/ml penicillin/25 μ g/ml streptomycin, and 10% fetal bovine serum. Cells were maintained as monolayer cell cultures using the following procedure at 3X10 per T175 flask⁶Individual cells, passaged twice a week. Briefly, cells were treated with PBS (without Mg) prior to addition of trypsin-EDTA to the flask²⁺、Ca²⁺) And (6) washing. After cell detachment, trypsin-EDTA was inactivated by adding complete medium. The cell suspension was then transferred to a 50ml Falcon tube and centrifuged at 1200rpm for 3 minutes. The medium was aspirated and the cells were resuspended in the appropriate volume of complete medium. Cells were counted in a hemocytometer and their viability was assessed by exclusion with 0.25% trypan blue (trypan blue). The appropriate volume of cell suspension is then added to a new T175 flask or roller bottle containing fresh medium. For large-scale growth of U87 tumor cells, an appropriate number of roller bottles were used at 0.5X 10 weeks prior to inoculation into mice⁷-1×10⁷And (4) inoculating the cells. The medium was changed twice during this period, and the latter change was performed the day before cell injection. Except that after centrifugation the cells were resuspended in cold (4)C.) serum free media, cells were harvested as described above. Mice were injected in the groin area with 1X10 total volumes of 200. mu.l⁷And (4) cells.

Research and design:

on day 0 (D0), human U87 glioma cells were injected directly into the inguinal region (1 × 10) of male NMRI nude mice⁷Individual cells/200 microliters/animal). On days 7-10 (which may vary with tumor survival/growth between cell batches), when the tumor volume has reached an average of about 200mm³At that time, mice were randomly divided into 5 mice per treatment group according to tumor volume. Mice were then treated 1 time daily (QD) with either vehicle (10% HP- β -CD) or vehicle containing test compound (20mg/kg) administered by gavage (p.o.) for 5 days in a volume of 10ml/kg body weight. On the sixth day (24 hours after the 5 th dose), the specific tumors were measured and again on day 10. Tumor regrowth was monitored (after dosing stopped) so that a log of 2000mm could be reached³Time of volume (TTR 2000). This gives additional information about the duration of drug action on tumor growth. Typically, tumor size and body weight were measured 2 times per week and mice were monitored daily for clinical signs of toxicity during the treatment period. Clinical signs of toxicity include, but are not limited to, sustained loss of appetite or dehydration, prone position (posture), moribund, lethargy, hypothermia, and/or respiratory effort (according to the ukccr (UK coordinated committee on Cancer Research) guidelines for animal welfare in experimental neoplasia) (July1997), and Workman, p. et al.

And (3) data analysis:

for each individual animal, its body weight and tumor size were monitored 2 times per week throughout the study period [ using commonly recognized equations: tumor volume (mm)³)＝(a×b²2); wherein "a" represents tumor length and "b" represents tumor width, as measured by caliper]. A sustained weight loss of more than 15% of the initial body weight is considered clinically toxic,and the animals were removed from the study and sacrificed. The time course of tumor growth is expressed as a median value, or can be normalized to the starting tumor volume on the day of treatment initiation and expressed as mean ± standard error of the mean (SEM) (5 animals per group). For pre-established tumors, the relative tumor volume (treated tumor volume/tumor volume on day 0) can be calculated for each mouse and expressed as mean ± SEM for each treatment group. The unilateral p-value analyzed by Wilcoxon-Mann-Whitney (Wilcoxon rank sum test; Wilcoxon rank test) indicates statistical significance, and p < 0.05 is considered statistically significant. On days 6 and 10 (day 1-treatment start, mice were randomized to the appropriate treatment group), treatment/control (T/C) ratios were calculated from the final relative tumor volumes using NCI criteria. An effective criterion for the T/C ratio is 42%.

Table 10: % T/C on days 6 and 10 after initiation of dosing

Compound numbering	Day 6% T/C	Day 10% T/C
			6	105	99
Compound numbering	Day 6% T/C	Day 10% T/C
			12	64	71
19	57	40
			2	14	-0.23
5	103	67
			22	40	55
15	53	88
			18	9	-3
21	102	95
			9	145	113
11	72	79
			4	77	129
10	96	71
			16	63	64
23	72	95
			1	17	8
27	193	22
			24	60	1
31	179	1
			26	354	-11
30	-61	-10
			29	-196	-13
28	-280	-10
			25	330	-3

D. Composition examples: film coated tablet

Preparation of the tablet core

A mixture of 100g of the compound of formula (I), 570g of lactose and 200g of starch is mixed well and then moistened with a solution of 5g of sodium lauryl sulfate and 10g of polyvinylpyrrolidone in about 200ml of water. The wet powder mixture was sieved, dried, and sieved again. Then, 100g of microcrystalline cellulose and 15g of hydrogenated vegetable oil were added. All mixed well and compressed into tablets to obtain 10000 tablets, each containing 10mg of the compound of formula (I).

Coating film

To a solution of 10g of methylcellulose in 75ml of denatured ethanol was added a solution of 5g of ethylcellulose in 150ml of dichloromethane. Then 75ml of methylene chloride and 2.5ml of 1, 2, 3-propanetriol were added, and 10g of polyethylene glycol was melted and then dissolved in 75ml of methylene chloride. The latter solution was added to the former solution, then 2.5g magnesium stearate, 5g polyvinyl-pyrrolidone and 30ml concentrated pigment suspension were added and the whole mixture was homogenized. The tablet cores are coated with the thus obtained mixture in a coating apparatus.

Claims (15)

1. A compound of formula (I):

the compounds include any stereochemically isomeric form thereof, wherein

R¹Is a hydroxy group C_1-6Alkyl or C_2-6An alkenyl group; provided that R is¹The substituent is located at the 6 or 7 position of the indole moiety;

R²is hydrogen or C_1-4An alkyl group;

z is a group selected from:

R³is hydrogen or hydroxy C_1-4An alkyl group;

R⁴is hydroxy or C_1-4An alkoxy group;

R⁵is hydrogen or C_1-4An alkyl group; or

R⁴And R⁵Together form an oxo group;

a pharmaceutically acceptable salt thereof or a solvate thereof.

2. The compound of claim 1, wherein the compound has the formula:

3. the compound of claim 1 or2, wherein R¹Is hydroxy C_1-6An alkyl group.

4. The compound of any one of claims 1-3, wherein R²Is C_1-4An alkyl group.

5. The compound of any one of claims 1-4, wherein Z is a group of formula (Z-1).

6. The compound of any one of claims 1-4, wherein Z is a group of formula (Z-2).

7. The compound of claim 6, wherein R⁴Is hydroxy and R⁵Is hydrogen.

8. The compound of claim 6, wherein R⁴Is hydroxy and R⁵Is C_1-4An alkyl group.

9. The compound of claim 1, wherein the compound is an enantiomer having the formula,

and said enantiomer having levorotatory activity, measured at a temperature of 20 ℃ and at a concentration of 8.59mg/ml in chloroform and at a wavelength of the D-line of sodium (589nm) in a cell path length of 1 dm; or a pharmaceutically acceptable salt or solvate thereof.

10. The compound of claim 1, wherein the compound is an enantiomer having the formula,

and said enantiomer having a dextrorotatory character, measured at a temperature of 20 ℃ and at a concentration of 10.33mg/ml in methanol, at a wavelength of the D-line of sodium (589nm) in a cell path length of 1 dm; or

A pharmaceutically acceptable salt thereof or a solvate thereof.

11. A compound according to any one of claims 1 to 10 for use as a medicament.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound according to any one of claims 1 to 10.

13. Use of a compound according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of cancer.

14. A combination of one or more anti-cancer agents and a compound of any one of claims 1-10.

15. A process for the preparation of a compound according to claim 1, characterized in that:

a) reacting an intermediate of formula (II) with an intermediate of formula (III) in a reaction-inert solvent, optionally in the presence of a suitable base, wherein W₁For a suitable leaving group, or by reacting an intermediate of formula (II) with an intermediate of formula (III) in the presence of a suitable catalyst, a suitable ligand, a suitable base and a suitable solvent, wherein W is₁In the case of a suitable leaving group,

wherein R is¹、R²And Z is as defined in claim 1;

b) reducing the corresponding carbonyl derivative of formula (IV) in the presence of a suitable reducing agent and a suitable solvent,

wherein R is²And Z is as defined in claim 1;

c) reducing the corresponding ester derivative of formula (V) wherein R is^xrepresents-C_1-3Alkyl C (═ O) OC_1-4An alkyl group, a carboxyl group,

wherein R is²And Z is as defined in claim 1;

d) hydrolyzing the intermediate of formula (VI) with a suitable base in the presence of a suitable solvent,

wherein R is²And Z is as defined in claim 1;

or, if desired, converting the compounds of formula (I) into each other according to conversion methods known from the literature and, if desired, further converting the compounds of formula (I) into therapeutically active, non-toxic acid addition salts by treatment with acids or into therapeutically active, non-toxic base addition salts by treatment with bases or, conversely, converting the acid addition salt form into the free base by treatment with bases or converting the base addition salts into the free acids by treatment with acids; or, if desired, preparing a stereochemically isomeric form thereof or a solvate thereof.