Benzo[b]thiophene derivatives and their use for the inhibition of Fibroblast Growth Factor Receptor kinases (FGFRs) for the use of neo- and hyperplasia therapies

Field of the invention

The present invention relates to benzo[b]thiophene derivatives of general formula (I) and pharmaceutically acceptable salts, solvates, hydrates, stereoisomeric and polymorphic forms thereof, procedures for manufacturing them, the use of them as of medication, as well as pharmaceutical compositions containing at least one of them as pharmaceutically active agent(s) together with pharmaceutically acceptable carrier, excipient and/or diluent, especially for the inhibition Fibroblast Growth Factor Receptor kinases (FGFR's), e.g. for the treatment of cancer.

Background of the invention

The FGFR receptor family and its involvement in disease

FGFRs regulate numerous principal cellular processes, such as proliferation, cell growth, differentiation, migration and survival; therefore they are fundamental to embryonic and after birth development, regulation of angiogenesis and wound healing in adults.

Dysregulation of the FGF/FGFR signaling pathway has been associated with multiple developmental disorders and with cancer. The causes of oncogenic behavior are mainly amplifications, activating mutations, overexpression and constitutively active fusions. FGFRs are trans-membrane proteins and receptor tyrosine kinases (RTKs). Four members of the family (FGFR1 , FGFR2, FGFR3 and FGFR4) are enzymatically active tyrosine kinases of great interest in drug discovery namely, whereas the gene for FGFR5 lacks a kinase domain. Hence the FGFR5 protein and the gene are also known as Fibroblast growth factor receptorlike 1 (FGFRL1). FGFRL1 has not been targeted in any kind of disease yet.

FGFRs are receptors of Fibroblast Growth Factors (FGFs). FGF family consists of 18 secreted protein ligands, which can be separated into 2 subfamilies: the hormone-like FGFs (FGF19, 21 and 23) and the canonical FGFs (FGF1-10, 16-18 and 20). FGFR family members differ from one another in their ligand affinities and tissue distribution. The combination and concentration and receptor affinity of those present at a given tissue and the pattern of FGFR expression determines the cellular behavior of the given tissue. Besides, FGFR signaling is strictly regulated by several feedback mechanisms in distinct points in the signaling network.

Based on these findings it is clear that FGFR signaling pathway is multifactorial and complex. It has evolved in a way that regulates numerous different biological functions in a well-controlled temporal and spatial manner throughout human development and adult life. This implicates that dysregulation of this fine-tuned system will lead to developmental dysfunctions and via regulation of cell proliferation it will lead to cancer formation.

There are multiple possible mechanisms that can affect FGFR signaling and lead to various diseases. These mechanisms are FGFR mutations, FGFR overexpression via gene amplifications and chromosomal translocations, constitutively active fusion proteins via other types of chromosomal translocations, altered FGFR splicing or the absence of signal transductional negative feedback.

Gene amplification of FGFR2 was found in approximately 10% of gastrointestinal cancer. Gastrointestinal cancer has especially high prevalence in Asia which represents an important proportion of patients and in many cases the wild type FGFR2 protein expressed in exceptionally high levels.

We interpret here another example of unequivocal FGFR overexpression in multiple myeloma (MM). MM is a cancer of white blood cells that is characterized by multiple genetic abnormalities. About 15% to 20% of MM patients harbor a chromosomal translocation, t(4; 14), which brings FGFR3 under the influence of a strong IgH enhancer region, leading to FGFR3 overexpression. The t(4; 14) translocation is associated with poor prognosis, and FGFR3 has been recognized as a potent oncogene in MM and an attractive target for novel drug development. Several studies have reported antitumor activity of small-molecule FGFR3 inhibitors as well as inhibitory anti-FGFR3 antibodies in MM cell lines carrying the t(4; 14) translocation and in MM xenograft mouse models.

There are numerous further cases where FGFR2 or FGFR3 overexpression, mutation or translocation is crucial for cancer formation and development. Targeting FGFRs with small molecule inhibitors is an excellent basis for cancer therapy development. Known FGFR inhibitors and the need for selective agents

Given the crucial involvement as cancer driver genes in many cancer types, FGFRs are commonly accepted, validated targets in terms of cancer therapy development. Several families of compounds have been investigated in preclinical and clinical studies or currently are in clinical trials. We currently know 3 FDA approved kinase inhibitor drugs which amongst many other targets also affect FGFRs. These are Ponatinib, Lenvatinib and Nintedanib and there is no evidence for a strictly FGFR-based mechanism of action for anyone of these 3 drugs. Another compound, ENMD-2076 received orphan drug certification for the treatment of hepatocellular carcinoma, but the FGFR family is not the main target group of the compound. 3 compounds reached Phase III in clinical trials, one of them are already suspended. 6 compounds are examined in Phase II clinical trials. Another 6 compounds started phase I studies now. The Fibroblast Growth Factor Receptor kinase family has a special place in cancer treatment as a target, since non-selective inhibition of the whole family would produce adverse effects - despite fighting cancer successfully - because FGFR1 in most cancer cases shouldn't be targeted, since inhibition of FGF23/FGFR1 signaling leads to hyperphosphataemia (bone mineralization defects) in clinical settings, which has been observed in lung cancer trials using a non-selective FGFR inhibitor.

Pyrido[2,3-d]pyrimidines are FGFR inhibitors of great interest because of their selectivity. Development started to focus on FGFRs as targets when a lead compound PD089828, a multitarget RTK inhibitor which showed inhibition of tumor growth was found to be also an FGFR inhibitor. PD173074 is the most active derivative of this compound family. We utilized this compound as a reference in our in vitro studies because of the well defined IC ₅₀ proportion within the FGFR family. As another reference we used LY-2874455 which is under investigation in clinical trials and is a very potent novel agent against the FGFR family.

Estimation of the number of patients

In order to estimate the size of the market of a selective FGFR2/3 inhibitor we used COSMIC database (Catalogue Of Somatic Mutations In Cancer v75) which contains detailed information on the ratio of given mutations and amplifications broken down by type of cancer. We assessed that how many patient samples carry FGFR2/3 mutation which may result in increased activity or how many patients have amplified FGFR2/3 which results in overexpression. In the case of FGFR2 the ratio of the samples which carried FGFR2 mutation was 1.47%, 419 samples were mutant out of 28 587. The same ratio in the case of FGFR3 was 9.35%(3552 mutant out of 37 980 samples). Table 1 contains the two gene's mutation frequency broken down in the most potent target cancer types. The therapeutical importance of a potent FGFR2/3 inhibitor are the highest in these cancer types.

Table 1. FGFR2 and FGFR3 mutants in the most important cancer types

According to the latest available data (Cancer.org was queried on 2016-01-21) in 2012 the total global incidence of all cancer types was 14 100 000. Using the ratios of FGFR2 and FGFR3 mutated patient samples obtained from the COSMIC database we concluded that in 2012 and throughout the following years at least 206 664 new FGFR2 mutant patients and at least 1 318 673 new FGFR3 mutant patients are diagnosed in each year who may benefit from a selective FGFR2/3 inhibitor.

We recognized the need for a selective FGFR2/3 inhibitor and developed a compound group that is new in FGFR inhibitor development.

Summary of the invention

1. The present invention relates to compounds of the general formula (I) and pharmaceutically acceptable salts, solvates, hydrates, and polymorphic forms thereof:

wherein

R1 is selected from the group of hydrogen; hydroxyl; substituted or unsubstituted heterocyclyl; amino, optionally substituted with 1 or 2 group(s) selected independently from the following group: C1-8 alkyi, C1-8 alkyi substituted with heterocycloalkyi, C1-8 hydroxyalkyi, C1- 8 hydroxyalkyi esterified with carboxylic acid, C3-8 cycloalkyl, C3-8 hydroxycycloalkyl, alkylcarbonyl, and alkoxyalkyl;

X, Y and Z are selected independently from the followings: CH (methine), nitrogen;

R2, R3, R4 and R5 are independently selected from the group of:

a) hydrogen;

b) halogen;

c) hydroxyl, optionally esterified with alkylcarboxylic acid;

d) alkyi, alkenyl, alkynyl,

e) alkoxy, and

f) amino, optionally substituted with 1 or 2 alkyi, alkylcarbonyl or alkoxycarbonyl group(s); preferably amino, optionally substituted with an alkoxycarbonyl group; 2. A compound according to above point 1 , wherein the substituent of the heterocyclyl of R1 is selected from the following group:

a) hydroxy I;

b) carboxyl, optionally esterified with hydroxyalkyl;

c) alkyl, optionally substituted with hydroxyl, cyano, amino or acetylamino;

d) alkoxycarbonyl

e) alkoxyalkyl;

f) carbamoyl;

g) alkylsulfonyl

h) amino, optionally substituted with 1 or 2 alkyl or alkylcarbonyl.

3. A compound according to above point 1 or 2, wherein the optionally substituted amine of R1 is selected from the following group:

a) di(alkyl)-amino

b) di(hydroxyalkyl)-amino

c) alkyl-hydroxyalkyl-amino

d) alkyl-amino

e) cycloalkyl-amino

f) alkoxyalkyl-amino

g) hydroxyalkyl-amino

h) dihydroxyalkyl-amino

i) (alkylsulfonyl-heterocycloalkyl-alkyl)-amino

j) alkylcarbonyl-alkyl-amino

k) alkylcarbonyl-cycloalkyl-amino

I) alkylcarbonyl-(hydroxyalkyl esterified with carboxylic acid)-amino

m) amino.

4. A compound according to above point 1 or 2, wherein R1 is substituted or unsubstituted heterocyclyl.

5. A compound according to above point 1 or 3, wherein R1 is amino, optionally substituted with 1 or 2 group(s).

6. A compound according to above point 1 to 3, wherein R1 is selected from the following group:

3-hydroxy-piperidin-1-yl

3- hydroxy-pyrrolidin-1-yl

4- carboxylic-piperidin-1-yl

N,N-Dimethylamino 2- hydroxymethyl-piperidin-1-yl

3- hydroxymethyl-piperidin-1-yl

piperazine-1-yl

4- hydroxymethyl-piperidin-1-yl

hydrogen

4-methyl-piperazin-1-yl

morpholin-4-yl

N,N-diethylamino

pyrrolidin-1-yl

N,N-bis-(2-hydroxyethyl)-amino

N-ethyl, N-(2-Hydroxyethyl)-amino

N-methyl, N-(2-Hydroxyethyl)-amino

2-hydroxymethyl-pyrrolidin-1-yl

azetidin-1-yl

piperidin-1-yl

N,N-dipropylamino

4-tertbutoxycarbonyl-piperazin-1-yl

4-hydroxy-piperidin-1-yl

2-Methoxymethyl-pyrrolidin-1-yl

thiazolidin-3-yl

2-carboxamido-pyrrolidin-1-yl

2- (carboxylic acid tert-butyl ester)-pyrrolidin-1-yl

3- amino-pyrrolidin-1-yl

hydroxyl

2-(carboxylic acid)-pyrrolidin-1-yl

2-(carboxylic acid methyl ester)-pyrrolidin-1-yl

Isopropylamino

cyclopropylamino

cyclopentylamino

2-methoxy-1-methyl-ethylamino

sec-butylamino

tert-Butylamino

1-ethyl-propylamino

1 -hydroxymethyl-propylamino

Cyclohexylamino

1 ,2,2-trimethyl-propylamino

1 , 1-dimethyl-propylamino bis-(2-Hydroxyethyl)-amino

4-ethyl-piperazin-1-yl

4-(2-cyano-ethyl)-piperazin-1-yl

3-aminomethyl-piperazin-1-yl

(2-hydroxy-ethyl)-amino

3-(acetylamino-methyl)-piperidine-1-yl

(2-hydroxy-1-hydroxymethyl-ethyl)-amino

1-methanesulfonyl-piperidin-4-yl-methylamino

N-lsopropyl-acetamido

N-Cyclopentyl-acetamido

N-sec-Butyl-acetamido

N-(1 -Ethyl-propyl)-acetamido

N-cyclopropyl-acetamido

tert-Butylamino

N-(0-Acetyl-butanol-2-yl)-acetamido

N-tert-Butyl-acetamido

N-cyclohexyl-acetamido

N-(1 , 1-Dimethyl-propyl)-acetamido

N-(1 ,2-Dimethyl-propyl)-acetamido

Phthalimido

N-Methyl-acetamido

3-(Dimethylamino)pyrrolidin-1-yl.

7. A compound according to any of the above points 1 to 6, wherein X, Y and Z are CH (methine).

8. A compound according to any of the above points 1 to 7, wherein the phenyl moiety of formula (I) is substituted with -CH ₂ -R1 in position 3 or 4.

9. A compound according to any of the above points 1 to 8, wherein

R2 is H;

R3 is H, amino optionally substituted with an alkoxycarbonyl group; alkoxy, halogen; R4 is H, OH, halogen or hydroxyl, optionally esterified with alkylcarboxylic acid; and R5 is H, alkyl or halogen.

10. A compound according to any of the above points 1 to 9, wherein

R2 is H;

R3 is H, -NH-CO-OMe, OMe, CI or Br;

R4 is H, OH, Br, F -OAc; and R5 is H, alkyl or CI.

1 1. A compound according to any of the above points 1 to 10, wherein

R2 is H;

R3 is CI or Br;

R4 is OH; and

R5 is H or CI.

The invention also relates to procedures for manufacturing of the above compounds, the use of them as of medication, as well as pharmaceutical compositions containing at least one of them as pharmaceutically active agent(s) together with pharmaceutically acceptable carrier, excipient and/or diluent, especially for the inhibition Fibroblast Growth Factor Receptor kinases (FGFR's), e.g. for the treatment of cancer, especially in the case of cancer of gastrointestinal, skin, brain, head and neck, lung, breast, stomach, bladder, kidney, endometrial and prostate cancer and in sarcomas and multiple myeloma.

Detailed description of the invention

As used herein in the meaning of R1 , the term "heterocyclyl" alone or in combination, means a group derived from a saturated, partially unsaturated or aromatic ring system with 4 to 9 carbon atoms and 1 to 4 heteroatom(s) selected from the group of N, O and S [i.e. group of N (nitrogen), O (oxygen) or S (sulfur) atoms]. The "heterocyclyl" preferably means a group derived from a saturated or partially unsaturated group, even more preferably a saturated group. Examples for "heterocyclyl" are piperidinyl, piperazinyl, morpholinyl, azetidinyl, pyrrolidinyl, thiazolidinyl, indolyl, indazolyl, 1 ,3-benzodioxolyl, dihydro-1 ,4-benzodioxinyl, furanyl, pyrrolyl, pyridinyl, quinolinyl, isoquinolinyl, pyranyl, oxazinyl, imidazolyl, benzoimidazolyl, pyrazolyl, purinyl, phthalimidyl where piperidinyl, piperazinyl, morpholinyl, azetidinyl, pyrrolidinyl, thiazolidinyl and phthalimidyl are preferred.

Those substituted heterocyclyl groups are also within the scope which contain one or more substituent(s) usually applied in the organic chemistry for substitution of heterocyclyl groups. So, the substituted heterocyclyl groups carry one or more, preferably 1 to 4 substituent(s), e.g. 1 to 3 or 1 to 2 substituent(s), independently selected from the group of alkyl, hydroxyl, halogen, hydroxyalkyl, cyanoalkyl, carboxyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, sulphate, amino, aminoalkylcarbonyl, acylamino, carboxylate, carbamoyl, optionally substituted amide, monoalkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl and cyano, where hydroxyl, carboxyl, optionally esterified with hidroxyalkyl; alkyl, optionally substituted with hydroxyl, cyano, amino or acetylamino; alkoxycarbonyl, alkoxyalkyl, carbamoyl, alkylsulfonyl and amino, optionally substituted with 1 or 2 alkyl or alkylcarbonyl are preferred groups. As used herein, the term "halogen" means fluorine, chlorine, bromine or iodine.

As used herein, the term "alkyl" alone or in combinations means, if it is not signed otherwise, a straight or branched-chain alkyl group containing from 1 to 6, preferably 1 to 5 carbon atom(s) (i.e. "CW or "Ci_ ₅ " alkyl groups), such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, f-butyl and pentyl. In special cases this phrase can relate to alkyl groups containing from 1 to 4, or 1 to 3 or 1 to 2 carbon atom(s) (i.e. "CW or "Ci. ₃ " or "Ci_ ₂ " alkyl groups). Those substituted alkyl groups are also within the scope which contain one or more substituent(s) usually applied in the organic chemistry for substitution of alkyl groups. So, the substituted alkyl groups carry one or more, preferably one or two substituent(s), independently selected from the group of halogen, aryl, hydroxyl, carboxyl, benzyloxy, alkoxy, nitro, sulphate, amino, acylamino, monoalkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl and cyano.

As used herein, the term "alkoxy" means an alkyl-O- group in which the alkyl group is as previously described. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond to the parent moiety is through the ether oxygen. If the alkoxy group is substituted with halogen then it is named as haloalkoxy group. If an alkyl group is substituted with an alkoxy then it is named as alkoxyalkyl group.

As used herein, the term "alkylcarbonyl" means an alkyl-CO- group in which the alkyl group is as previously described.

The term "alkenyl" means a linear or branched aliphatic hydrocarbon group containing a carbon-carbon double bond having a single radical and 2 to 6 carbon atoms.

The term "alkynyl" means a linear or branched aliphatic hydrocarbon group containing a carbon-carbon triple bond having a single radical and 2 to 6 carbon atoms.

The term "salt" means any ionic compound formed between one of the embodiments of the present invention and an acidic or basic molecule that can donate or accept ionic particle to/from its partner. The quaternary amine salts are also included.

As used herein, the term "cycloalkyl" means a cyclised alkyl group, in line with the general meaning of cycloalkyl applied in organic chemistry.

Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Salts of the compounds of the formula (I) may be formed, for example, by reacting a compound of formula (I) with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

Exemplary acid addition salts include acetates, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2- hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates (such as those mentioned herein), tartarates, thiocyanates, toluenesulfonates (also known as tosylates,) undecanoates, and similar to these. Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are known.

The term "solvate" means a compound formed by the combination of solvent molecules with molecules or ions of the solute (solvation). Solute can be any of the embodiments of the present invention and the solvent can be water (forming hydrates) or any organic solvent.

The invention also relates to all polymorphic forms of compounds of general formula (I). In the context of the present invention, general formula (I) includes all stereoisomeric forms of the compounds of the present invention. The term "stereoisomer" as used herein includes all possible stereoisomeric forms, including all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure, unless the specific stereochemistry or isomer form is specifically indicated. Where the compounds of the present invention contain one or more chiral centers, all possible enantiomeric and diastereomeric forms, as well as the racemate, are included. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All processes used to prepare compounds of the present invention and intermediates made therein are considered to be part of the present invention.

Another subject of the invention is providing pharmaceutical composition containing as active ingredient one or more compound(s) of general formula (I) together with one or more usual pharmaceutical auxiliary material(s). Formally another subject is the use of the compounds of general formula (I) in preparing such compositions. The applicable auxiliary materials are those which are generally applied in the preparation of pharmaceutical compositions, e.g. carriers, diluents, vehicles, coloring agents, flavoring agents, stabilizers, surfactants, carriers for the preparation of sustained release compositions etc. Further details can be found in the following book: Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990, Volume 5., Chapter 25.2). The specific embodiments of the present invention can be used for the preparation of a pharmaceutical composition for prophylaxis and/or treatment of a disease where the inhibition Fibroblast Growth Factor Receptor kinases (FGFR's) affects the patient's condition positively, e.g. in the treatment of cancer, for example cancer of gastrointestinal, skin, brain, head and neck, lung, breast, stomach, bladder, kidney, endometrial and prostate cancer and in sarcomas and multiple myeloma, especially cancer of gastrointestinal, skin, endometrial and prostate cancer and multiple myeloma. The invention also relates to procedures for manufacturing of compounds of general formula (I), in line with the following methods.

Route 3 Materials and methods:

General route 1

Step A: Preparation of 2-Amino-6,6-ethylenedioxy-4,5,6,7-tetrahydro- benzo[b]thiophene-3-carboxylic acid amide:

3.12 g (20 mmol) cyclohexane-1 ,4-dion monoethylene ketal, 1.68 g (20 mmol) cyano- acetamide, and 0.64 g (20 mmol) sulphur were dissolved in the mixture of 17 mL ethanol and 1.2 mL water. 1.2 mL (13.87 mmol) morpholine was added to the solution, and the mixture was stirred at reflux temperature overnight. The reaction mixture was cooled down to room temperature, and was diluted with 5 mL ethanol and 5 mL water. The solid product was filtered off, washed with 15 mL n-hexane and dried.

Yield: 3.12 g (62 %)

Step B: Preparation of (3-Carbamoyl-6,6-ethylenedioxy-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)-carbamic acid 2,2,2-trichloro-ethyl ester:

4.7 mL (34.9 mmol) chloroformic acid 2,2,2-trichloro-ethyl ester was added at 0 °C to the solution of 8 g (31.5 mmol) 2-Amino-6,6-ethylenedioxy-4,5,6,7-tetrahydro- benzo[b]thiophene-3-carboxylic acid amide in 95 mL abs. pyridine. The reaction mixture was stirred at room temperature for one day; then the solvent was removed under vacuum, the residue was stirred with 20 mL water. The solid product was filtered off, dried, and recrystallized from 10 mL acetonitrile.

Yield: 81 %. Step C: Preparation of (3-Carbamoyl-6,6-ethylenedioxy-benzo[b]thiophen-2- carbamic aci -trichloro-ethyl ester

430 mg (1.00 mmol) (3-Carbamoyl-6,6-ethylenedioxy-4,5,6,7-tetrahydro- enzo[b]thiophen-2-yl)-carbamic acid 2,2,2-trichloro-ethyl ester and 0.34 g (2.00 mmol) cupper(ll) chloride dihydrate was stirred in 25 cm ³ acetonitrile for three hours at room temperature. Then the solvent was evaporated under reduced pressure, the residue was triturated with 20 mL 1 N hydrochloride acid solution, and extracted three times with 20 mL ethyl-acetate. The combined organics were washed with 20 mL brine and dried over magnesium-sulphate. Evaporation of the solvent gave the crude product, which was recrystallized from 10 cm ³ acetonitrile afforded the pure product.

Yield: 68 %.

Step D: (3-Carbamoyl-5,7-dichloro-6-hydroxy-benzo[b]thiophen-2-yl)-c arbamic acid 2,2,2-trichlo

384 mg (0.10 mmol) (3-Carbamoyl-6,6-ethylenedioxy-benzo[b]thio phen-2-yl)-carbamic acid 2,2,2-trichloro-ethyl ester and 0.40 g (3.00 mmol) N-chloro-succinimide were stirred at room temperature for 24 hours in 15 mL acetonitrile. The reaction mixture was diluted with 15 mL water, the solid was filtered off, and was washed three times with 5 mL water. The crude product was recrystallized from 40 mL acetonitrile to give pure product.

Yield: 80 %.

Step E: Acetic acid 3-carbamoyl-5,7-dichloro-2-(2,2,2-trichloro-ethoxycarbonylam ino)- benzo[b]thiophen-6-yl ester

1.81 g (4.00 mmol) (3-Carbamoyl-5,7-dichloro-6-hydroxy-benzo[b]thiophen-2-yl)- carbamic acid 2,2,2-trichloro-ethyl ester was dissolved in 20 mL pyridine and 0.39 g (0.36 mL, 1.10 mmol) acetyl chloride was added dropwise into the reaction mixture at 0 °C. After stirring at this temperature for four hours the solid was filtered off, was washed with 20 mL 1 N HCI and two times with 20 mL water. The crude product was refluxed in 25 mL acetonitrile for a half an hour, was cooled to room temperature, and was filtered off to give pure product.

Yield: 80 %.

0.25 g (0.50 mmol) acetic acid 3-carbamoyl-5,7-dichloro-2-(2,2,2-trichloro- ethoxycarbonylamino)-benzo[b]thiophen-6-yl ester was dissolved in 20 mL tetrahydrofuran, and was treated first with 2.00 mL (2.00 mmol) 1 M NaH ₂ P0 ₄ solution, and 1.96 g (30.00 mmol) freshly prepared activated Zn. The reaction mixture was stirred at room temperature for 24 hours, and then the solid was filtered off, washed with 5 mL tetrahydrofuran two times. The collected solution was evaporated. The residue was stirred at room temperature for one hour in 20 mL water, the solid was filtered off, and washed three times with 5 mL water. The crude product was refluxed in 10 mL acetonitrile for a half an hour, was cooled to room temperature, and filtered off to give pure product.

Yield: 65 %.

Step G: Acetic acid 3-carbamoyl-5,7-dichloro-2-(4-chloromethyl-benzoylamino)- benzo[b]thiophen-6-yl ester

198 mg (1.05 mmol) 4-chloromethyl-benzoylchloride was added to the solution of 319 mg (1 mmol) acetic acid 2-amino-3-carbamoyl-5,7-dichloro-benzo[b]thiophen-6-yl ester in 10 ml_ tetrahydrofuran. The reaction mixture was stirred at reflux temperature for 24 hours, and cooled down to room temperature. The solvent was removed under vacuum, the residue was stirred with 10 mL acetonitrile at reflux temperature for 30 minutes. The mixture was cooled down to room temperature, the product was filtered off, washed with a small amount of acetonitrile and dried.

Yield: 85 %.

Step H: Reaction of acetic acid 3-carbamoyl-5,7-dichloro-2-(4-chloromethyl- benz

The solution of 142 mg (0.30 mmol) acetic acid 3-carbamoyl-5,7-dichloro-2-(4- chloromethyl-benzoylamino)-benzo[b]thiophen-6-yl ester and 3.00 mmol appropriate amine compound were stirred in 20 mL abs. tetrahydrofuran at room temperature until the starting benzo[b]thiophene compound disappeared by TLC. The solvent was removed under vacuum, and the crude product was purified with HPLC.

Yield: 10 - 90 %.

Step I: Acetylation of 6-hydroxyl products

0.50 mmol 6-hydroxyl derivative was solved in 10 mL abs. pyridine. 470 (5 mmol) acetic acid anhydride was added to the solution, and the mixture was stirred at room temperature for 6 hours. The solvent was removed under vacuum, and the residue was stirred with 10 mL water for 15 minutes. The solid was filtered off, and recrystallized from 15 mL acetonitrile.

Yield: 73 %.

70 (0.60 mmol) p-toluolyl chloride was added to the mixture of 160 mg (0.50 mmol) acetic acid 2-amino-3-carbamoyl-5,7-dichloro-benzo[b]thiophen-6-yl ester and 80 mg potassium carbonate in 25 mL tetrahydrofuran. The reaction mixture was stirred at reflux temperature for 24 hours, and cooled down to room temperature. The solvent was removed under vacuum, the residue was solved in the mixture of 30 mL methanol and 2 mL 2N NaOH/aq, and stirred for 15 minutes. The pH was set to acidic with 1 N HCI/aq, the product was filtered off and recrystallized from 10 mL acetonitrile.

Yield: 55 %.

Step K: 5,7-Dichloro-6-hydroxy-2-(4-hydroxymethyl-benzoylamino)-benz o[b]thiophene- 3-carboxylic acid amide

The mixture of 190 mg (0.40 mmol) acetic acid 3-carbamoyl-5,7-dichloro-2-(4- chloromethyl-benzoylamino)-benzo[b]thiophen-6-yl ester, 660 mg (8.00 mmol) sodium acetate in 30 mL tetrahydrofuran was stirred at reflux temperature for 3 hours. The solvent was removed under vacuum, the residue was stirred with 15 mL water. The solid was filtered off, solved in the mixture of 30 mL methanol and 2 mL 2N NaOH/aq, and stirred for one hour. The solvent was removed under vacuum, the residue was treated with 1 N HCI/aq. After 15 minutes stirring the solid was filtered off, and recrystallized from 10 mL acetonitrile.

Yield: 78 %.

General route 2

Step A: Acetic acid 3-carbamoyl-5,7-dichloro-2-(4-chloromethyl-benzoylamino)- benzo[b]thi

210 (4 mmol) bromine was added dropwise to the solution of 430 mg (1 mmol) (3- carbamoyl-6,6-ethylenedioxy-4,5,6,7-tetrahydro-benzo[b]thiop hen-2-yl)-carbamic acid 2,2,2- trichloro-ethyl ester and 330 mg (4 mmol) sodium acetate in 20 mL acetic acid. The reaction mixture was stirred at room temperature for 24 hours. The solution was cooled down with ice, the solid was filtered off, washed two times with 10 mL cold water, and stirred at reflux temperature for 30 minutes in 10 mL acetonitrile. After cooling to room temperature the product was filtered off and dried.

Yield: 39 %. Step B: Acetic acid 5,7-dibromo-3-carbamoyl-2-(2,2,2-trichloro-ethoxycarbonylami no)- benzo[b]t

The solution of 0.21 g (0.39 mmol) acetic acid 3-carbamoyl-5,7-dichloro-2-(4- chloromethyl-benzoylamino)-benzo[b]thiophen-6-yl ester and one drop pyridine in 2 mL acetic acid anhydride was stirred at room temperature for 5 hours. The solvent was removed under vacuum, the residue was dissolved in 20 mL ethanol, and after a few minutes stirring the ethanol was removed under vacuum. 5 mL acetonitrile was added to the residue, and the mixture was stirred at reflux temperature for 30 minutes. The solution was cooled down to 0 °C, the solid product was filtered off and dried.

Yield: 74 %.

Step C: Acetic acid 2-amino-5-bromo-3-carbamoyl-benzo[b]thiophen-6-yl ester

0.29 g (0.50 mmol) acetic acid 5,7-dibromo-3-carbamoyl-2-(2,2,2-trichloro- ethoxycarbonylamino)-benzo[b]thiophen-6-yl ester was dissolved in 20 mL tetrahydrofuran, and was treated with first 2.00 mL (2. 00 mmol) 1 M NaH ₂ P0 ₄ solution, then 1.96 g (30 mmol) freshly prepared activated Zn. The reaction mixture was stirred at room temperature for 24 hours, and then the solid was filtered off, and washed two times with 5 mL tetrahydrofuran. The collected solutions were evaporated under vacuum. The residue was stirred at room temperature for one hour in 20 mL water; the solid was filtered off, and washed three times with 5 mL water. The crude product was recrystallized from 10 mL acetonitrile.

Yield: 61 %. Step D: Acetic acid 5-bromo-3-carbamoyl-2-(4-chloromethyl-benzoyl benzo[b

The solution of 0.17 g (0.5 mmol) acetic acid 2-amino-5-bromo-3-carbamoyl- benzo[b]thiophen-6-yl ester, and 0.1 1 g (0.6 mmol) 4-chloromethylbenzoyl-chloride in 25 mL tetrahydrofuran was stirred at reflux temperature for 7 hours. The solvent was removed under vacuum, and the residue was stirred in 5 mL acetonitrile at reflux temperature for 30 minutes. The mixture was cooled down to room temperature; the solid product was filtered off and dried.

Yield: 65 %.

Step E: Reaction of Acetic acid 5-bromo-3-carbamoyl-2-(4-chloromethyl- benzoylamino)-benzo[b]thiophen-6-yl ester with amines

The solution of 0.24 g (0.5 mmol) acetic acid 5-bromo-3-carbamoyl-2-(4-chloromethyl- benzoylamino)-benzo[b]thiophen-6-yl ester, and 1.5 mmol appropriate amine in 50 mL tetrahydrofuran was stirred at reflux temperature for 24 hours. After cooling down to room temperature, the solvent was removed under vacuum, the residue was solved in 25 mL methanol, and 5 mL saturated HCI in isopropanol was added. The mixture was stirred at room temperature for 8 hours, the solvent was removed under vacuum, and the crude product was recrystallized from acetonitrile.

Step F: Acetylating of 6-hydroxyl products

0.50 mmol 6-hydroxyl derivative was solved in 10 mL abs. pyridine. 470 μΙ_ (5 mmol) acetic acid anhydride was added to the solution, and the mixture was stirred at room temperature for 6 hours. The solvent was removed under vacuum, and the residue was stirred with 10 mL water for 15 minutes. The solid was filtered off, and recrystallized from 15 mL acetonitrile.

Yield: 73 %.

Step G: 6-Hydroxy-2-(4-piperidin-1-ylmethyl-benzoylamino)-5-vinyl-be nzo[b]thiophene- -carboxylic acid amide

244 mg (0.5 mmol) 5-Bromo-6-hydroxy-2-(4-piperidin-1-ylmethyl-benzoylamino)- benzo[b]thiophene-3-carboxylic acid amide was solved in 6 mL abs. toluene. 25 mg tetrakis(triphenylphosphine)palladium(0), and 180 mg (0.55 mmol) hexamethylditin was added to this solution, and the mixture was stirred at reflux temperature for 2 hours. The reaction mixture was allowed to cool down, the solid was filtered off, and washed twice with 5 mL toluene. The filtrate was evaporated under vacuum. The oily residue was solved in 8 mL abs DMF, then 25 mg palladium(ll)acetate, 100 triethylamine and 1 mL vinyl bromide (1.0 M solution in THF) was added to it. The reaction mixture was stirred at 95 °C for 2 hours. The mixture was cooled down to room temperature, filtrated through Cellite, the solvent was removed under vacuum, and the crude product was purified with flash chromatography.

Yield: 4 mg pale-yellow oil. General route 3

Step A: 3-Amino-5-methyl-cyclohex-2-enone

The solution of 50.00 g (396 mmol) 5-methyl-cyclohexane-1 ,3-dione and 31.13 g (404 mmol) ammonium acetate in 1000 mL toluene was stirred at reflux temperature for 5 hours in a flask equipped with Marcusson separator. After cooling down to room temperature the toluene was removed under vacuum, the remaining solid was washed with saturated NaHC03/aq, water and diisopropyl ether.

Yield: 83 %.

Ste B: (5-Methyl-3-oxo-cyclohex-1-enyl)-carbamic acid tert-butyl ester

15.02 g (120 mmol) 3-amino-5-methyl-cyclohex-2-enone was solved in the mixture of 600 mL abs. tetrahydrofuran and 100 mL abs. Ν,Ν-dimethylformamide, and this solution was added to the mixture of 9.60 g (240 mmol) sodium hydride (60 w/w% in paraffin) in 120 mL abs. tetrahydrofuran at 0 °C. After stirring for one hour at +5 °C, 31.40 g (144 mmol) carbonic acid di-tert-butyl ester was added in small portions. The mixture was stirred for 4 hours at +5 °C, and was allowed to warm up to room temperature. The solution was diluted with 200 mL water and 400 mL ethyl acetate, the solid was filtered off. The organic phase was washed with saturated NH ₄ CI solution, saturated NaHC0 ₃ solution, water and brine, dried over MgS04, and evaporated.

Yield: 60 %.

Step C: [3-(Carbamoyl-cyano-methylene)-5-methyl-cyclohex-1-enyl]-car bamic acid tert- butyl ester

15.89 g (70.52 mmol) (5-Methyl-3-oxo-cyclohex-1-enyl)-carbamic acid tert-butyl ester and 5.924 g (70.52 mmol) was solved in 500 mL abs. tetrahydrofuran. 17 mL titanium- tetrachloride was added to this solution dropwise, at 0 °C. The mixture was stirred at 0 °C for 1.5 hours, and 28 mL abs pyridine was added dropwise, at 0 °C

The reaction mixture was allowed to warm up to room temperature, and was stirred overnight. 200 mL water and 200 mL ethyl acetate were added to the mixture; the organic phase was separated, washed three times with saturated NaHC0 ₃ solution, dried over MgS0 ₄ and evaporated under vacuum. The oily residue was crystallized from diisopropyl ether.

Yield: 72 %.

Step D: (2-Amino-3-carbamoyl-7-methyl-benzo[b]thiophen-5-yl)-carbami c acid tert-butyl este

The mixture of 14.10 g (48.4 mmol) [3-(Carbamoyl-cyano-methylene)-5-methyl- cyclohex-1-enyl]-carbamic acid tert-butyl ester, 9.20 g (288 mmol) sulfur and 40.00 g NaHC0 ₃ in 450 mL ethanol was stirred at reflux temperature overnight. The solid compounds were filtered off; the filtrate was concentrated under vacuum. The crude product was purified with column chromatography.

Yield: 27 %,

Step E: [3-Carbamoyl-2-(4-chloromethyl-benzoylamino)-7-methyl-benzo[ b]thiophen-5- yl]

1.60 g (5 mmol) (2-Amino-3-carbamoyl-7-methyl-benzo[b]thiophen-5-yl)-carbami c acid tert-butyl ester, 604 (7.5 mmol) abs. pyridine, and 1.134 g (6 mmol) 4-chloromethyl- benzoylchloride were stirred in 60 mL abs. tetrahydrofuran at room temperature for 3 days. The solvent was removed under vacuum; the residue was solved in the mixture of 300 mL ethyl acetate and 100 mL water. The organic phase was separated, washed with water, saturated NaHC0 ₃ solution and brine, dried over MgS0 ₄ and evaporated under vacuum.

Yield: 94 %. Step F: [3-Carbamoyl-7-methyl-2-(4-pyrrolidin-1-ylmethyl-benzoylamin o)- benzo[b]thiophen-5-yl]-carbamic acid tert-butyl ester

600 mg (1.27 mmol) [3-Carbamoyl-2-(4-chloromethyl-benzoylamino)-7-methyl- benzo[b]thiophen-5-yl]-carbamic acid tert-butyl ester and 12 mmol R1-H (e.g. pyrrolidine, which is the specific compound of next step G ) were stirred in 15 mL abs. tetrahydrofuran at room temperature for 20 hours. The solvent was removed under vacuum; the residue was recrystallized from 20 mL acetonitrile.

Yield: 34 %.

Step G: 5-Amino-7-methyl-2-(4-pyrrolidin-1-ylmethyl-benzoylamino)-be nzo[b]thiophene- 3-carboxylic acid amide

180 mg (0.354 mmol) [3-Carbamoyl-7-methyl-2-(4-pyrrolidin-1-ylmethyl-benzoylamin o)- benzo[b]thiophen-5-yl]-carbamic acid tert-butyl ester was dissolved and stirred in 6 mL trifluoro acetic acid at room temperature for 4 hours. The solvent was removed under vacuum, the residue was mixed with 5 mL diisopropyl-ether, and the solid product was filtered off, washed with diisopropyl ether and dried.

Yield: 67 %. General route 4

Step A: 3-metho

10.00 g (89.18 mmol) cyclohexane-1 ,3-dione, and 50 mg p-Toluenesulfonic acid were dissolved in 200 ml methanol and the solution was stirred at reflux temperature for 4 hours in a flask equipped with Marcusson separator. The reaction mixture was evaporated under vacuum. The residual oil was taken up with 50 ml saturated aq. NaHC0 ₃ and extracted twice with 100 ml ethyl-acetate. The combined organic layer was dried over MgS0 ₄ . The desiccant was filtered off and the solvent was evaporated under reduced pressure affording the product as yellowish oil. -cyano-2-(3-methoxycyclohex-2-en-1-ylidene)acetamide

10.00 g (79.26 mmol) 3-methoxycyclohex-2-en-1 -one and 6.66 g (79.26 mmol) were solved in 500 ml_ abs. tetrahydrofuran. 18 mL titanium-tetrachloride was added to this solution dropwise at 0 °C. The mixture was stirred at 0 °C for 1.5 hours, and 32 mL abs pyridine was added dropwise at 0 °C. The reaction mixture was allowed to warm up to room temperature, and was stirred overnight. 200 mL water and 200 mL ethyl acetate were added to the mixture; the organic phase was separated, washed three times with saturated NaHC0 ₃ solution, dried over MgS0 ₄ and evaporated under vacuum. The oily residue was crystallized from diisopropyl ether.

Yield: 71 %.

Step C: 6-Fluoro-5-methoxy-2-(4-piperidin-1-ylmethyl-benzoylamino)- benzo[b]thiophene-3-carboxylic acid amide (Example 104)

120 mg (0.28 mmol) 5-Methoxy-2-(3-piperidin-1-ylmethyl-benzoylamino)- benzo[b]thiophene-3-carboxylic acid amide, and 110 mg (0.31 mmol, 1.1 eq.) Selectfluor® (1- chloromethyl-4-fluoro-1 ,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)) were solved in 15 mL acetoritrile. The reaction mixture was stirred at reflux temperature for 6 hours, then the solvent was removed under vacuum, and the crude product was purified with flash chromatography.

Yield: 4 mg yellow solid.

Route 5

Step A: 2-Amino- -hydroxy-benzo[b]thiophene-3-carboxylic acid amide

110 mg (0.5 mmol) 2-Amino-5-methoxy-benzo[b]thiophene-3-carboxylic acid amide was solved in 15 mL abs. chloroform. 1 mL boron tribromide solution (1.0 M in methylene chloride) was added, and the mixture was stirred 80 °C for 4 hours. The reaction mixture was allowed to cool down to room temperature, and the solvent was removed under vacuum. The oily residue was stirred with 30 mL 0.5N NaHC0 ₃ solution, the crude product was filtered off, washed with water and n-hexane, and purified with flash chromatography.

Yield: 37 mg white solid.

Analytical characterization

All of the prepared compounds were characterized by the following analytical methods:

NMR

The 300 MHz ¹ H-NMR analysis was performed with an apparatus of type Bruker AVANCE-300 at 25 °C, exact frequency was 300.14 MHz. Generally DMSO-d ₆ was used as solvent, exceptions given. The 600 MHz ¹ H-NMR and ¹³ C-NMR spectra were recorded on a Varian lnova-600 MHz device at 25 °C, the solvent was DMSO-d ₆ (5 _C = 39.50 and δ _Η = 2.50).

LCMS

The LCMS analysis was performed with a liquid chromatography mass-spectrometer Waters chromatograph with the following parameters:

Waters HPLC/MS:

MS detector:

Method "A": MicroMass ZMD

Method "B": Waters SQD

UV detector: Waters 996 DAD

Separation module: Waters Alliance 2795

HPLC:

Column:

Waters XBridge C18, 50 mm x 4.6 mm, 3.5 μηι

Solvent I: Water/ 0.1 % HCOOH

Solvent II: AcCN

Acetonitrile: Riedel-deHaen; G Chromasolv (34998)

Water: Mili-Q Academic

Formic acid: Riedel-deHaen; extra pure (27001)

Flow rate: 2 ml/min

Injection: 5 μg

Gradient:

Ionization: ES ⁺ /ES ^"

Source block temperature: 1 10 °C

Desolvation temperature: 250 °C

Desolvation gas: 500 L/h

Cone gas: 80 L/h Capillary voltage: 3000 V

Cone voltage: 30 V

Extractor voltage: 6 V

Rf lens voltage: 0.1 V

Scan: 80 to 1000 m/z in 1 sec.

Inter-scan delay: 0.1 s

Table 2. Identification of the prepared compounds.

All the compounds prepared by the examples are important embodiments of the invention.

Table 3. The analytical data of the prepared compounds

13.16(s,1 H); 10.37(s,1 H);

10.02(br. s,1 H); 8.22(dd,J~1.5

and 1.5Hz,1 H); 8.01(s,1 H);

7.90-8.00(ovl. m,4 H); 2.64 497.0579 496.03 498.06 97 7.74(dd,J~7.5 and 7.5Hz, 1 H);

5.33(br,2 H); 4.58(s,2 H);

3.82(m,4 H); 3.20(m,4 H)

13.15(br. s,1 H); 8.14(s,1 H);

8.03(br. s,2 H);

7.97(dm, =8.1 Hz,0.7 H) and

7.89(dm,J=8.1 Hz,1.3 H);

7.52(dm, =8.1 Hz,0.7 H) and

7.46(dm,J=8.1 Hz,1.3 H);

4.68(m,0.35 H) and 4.20(m,0.65

H); 4.64(s,0.7 H) and 4.54(s,1.3 4.41 535.0735 534.15 536.12 99 H); 2.46(s,3 H); 2.18(s,1.95 H)

and 1.91(s,1.05 H);

1.10(d,J=6.7Hz,3.9 H) and

1.04(d, =6.7Hz,2.1 H); Partly two

signal sets in ratio of 65:35 due

to restricted rotation of the

tertiary amide

13.16(br. s,1 H); 8.14(s,1 H);

8.03(br. s,2 H);

7.97(dm,J=8.0Hz,0.7 H) and

7.89(dm,J=8.0Hz,1.3 H);

7.48(dm,J=8.0Hz,0.7 H) and

7.42(dm,J=8.0Hz,1.3 H);

4.71 (m, 0.35 H) and 4.30(m,0.65

4.06 519.0786 518.20 520.15 99 H); 4.64(s,0.7 H) and 4.54(s,1.3

H); 2.46(s,3 H); 2.21 (s,1.95 H)

and 1.91(s,1.05 H); 1.33- 1.85(ovl,m,8H); Partly two signal

sets in ratio of 65 : 35 due to

restricted rotation of the tertiary

amide

13.16(br. s,1 H); 8.14(s,1 H);

8.03(br. s,2 H);7.97(dm, =8.1 Hz,

0.7 H) and 7.87(dm,J=8.1 Hz,1.3

H); 7.52(dm, =8.1 Hz,0.7 H) and

7.48(dm,J=8.1 Hz,1.3 H);

4.66(d, =17.5Hz,0.35 H) and

4.62(d,J=16.5Hz,0.65 H);

4.57(d, =17.5Hz,0.35 H) and

4.39(d,J=16.5Hz,0.65 H);

4.47(m,0.35 H) and 3.91(m,0.65 4.59 549.0892 548.21 550.16 97 H); 2.46(s,3 H); 2.17(s,1.95 H)

and 1.93(s,1.05 H); 1.33- 1.60(ovl. m,2 H);

1.07(d, =6.8Hz,1.95 H) and

1.00(d,J=6.8Hz,0.95 H);

0.79(t, J=7.3Hz,3 H); Partly two

signal sets in ratio of 65:35 due

to restricted rotation of the

tertiary amide

13.11(br. s,1 H); 10.32(s,1 H);

7.99(s,1 H); 7.94(dm, J=8.0Hz,0.7

H) and 7.87(dm, =8.0Hz,1.3 H);

7.92(br. s,2 H);

7.46(dm,J=8.0Hz,0.7 H) and

7.41 (dm, =8.0Hz,1.3

H);4.72(m,0.35 H) and

4.06 519.0786 518.20 520.15 99 4.29(m,0.65 H); 4.64(s,0.7 H)

and 4.53(s,1.3 H); 2.20(s,1.95 H)

and 1.90(s,1.05 H); 1.32- 1.85(ovl,m,8 H); Partly two signal

sets in ratio of 65:35 due to

restricted rotation of the tertiary

amide

13.11(br. s,1 H); 10.32(s,1 H);

7.99(s,1 H); 7.94(dm,J=8.1 Hz,0.7

H) and 7.85(dm,J=8.1 Hz,1.3 H);

7.92(br. s,2 H);

7.52(dm,J=8.1 Hz,2 H); 4.58(s,0.7

H) and 4.46(s,1.3 H);

4.30(m,0.35 H) and 3.65(m,0.65

4.15 521.0943 520.20 522.18 98 H); 2.15(s,1.95 H) and

1.98(s,1.05 H); 1.35-1.54(ovl,m,4

H); 0.77(t,J=7.0Hz,2.1 H) and

0.76(t, J=7.0Hz,3.9 H); Partly two

signal sets in ratio of 65:35 due

to restricted rotation of the

tertiary amide 13.11(br. s,1 H); 10.32(s,1 H);

7.99(s,1 H); 7.95(dm,J=8.1 Hz,0.7

H) and 7.86(dm,J=8.1 Hz,1.3 H);

7.92(br. s,2 H);

7.50(dm,J=8.1 Hz,0.7 H) and

7.46(dm,J=8.1 Hz,1.3 H);

4.66(d,J=17.5Hz,0.35 H) and

4.61 (d,J=16.4Hz,0.65 H);

4.56(d,J=17.5Hz,0.35 H) and

4.39(d,J=16.4Hz,0.65 H); 3.96 507.0786 506.13 508.13 99 4.47(m,0.35 H) and 3.91(m,0.65

H); 2.17(s,1.95 H) and

1.93(s,1.05 H); 1.33-1.60(ovl.

m,2 H); 1.07(d,J=6.5Hz,1.95 H)

and 1.00(d,J=6.5Hz,0.95 H);

0.79(t, J=7.3Hz,3 H); Partly two

signal sets in ratio of 65:35 due

to restricted rotation of the

tertiary amide

13.12(br. s,1 H); 10.32(s,1 H);

7.99(s,1 H);7.92(br. s,2 H);

7.90(dm,J=8.0Hz,2 H);

7.44(dm,J=8.0Hz,2 H); 4.60(s,2

H); 2.73(m,1 H); 2.22(s,3 H); 3.76 491.0473 490.13 492.08 96 0.74-0.82(ovl,m,4 H); Partly two

signal sets in ratio of 65:35 due

to restricted rotation of the

tertiary amide

13.11(br. s,1 H); 10.32(s,1 H);

7.99(s,1 H); 7.95(dm,J=8.1 Hz,0.7

H) and 7.86(dm,J=8.1 Hz,1.3 H);

7.93(br. s,2 H);

7.50(dm,J=8.1 Hz,0.7 H) and

7.45(dm,J=8.1 Hz,1.3 H);

4.67(m,0.35 H) and 4.20(m,0.65

H); 4.63(s,0.7 H) and 4.54(s,1.3 3.58 493.0630 492.07 494.08

97 H); 2.18(s,1.95 H) and

1.91 (s,1.05 H);

1.10(d,J=6.7Hz,3.9 H) and

1.03(d,J=6.7Hz,2.1 H); Partly two

signal sets in ratio of 65:35 due

to restricted rotation of the

tertiary amide

13.17(br. s,1 H); 8.14(s,1 H);

8.03(br. s,2 H);

7.92(dm, =8.0Hz,2 H);

4.36 533.0579 532.12 534.10 99 7.45(dm,J=8.0Hz,2 H); 4.61(s,2

H); 2.73(m,1 H); 2.46(s,3 H);

2.23(s,3 H); 0.74-0.82(ovl,m,4 H) 13.18(s,1 H); 8.24(s,1 H);

8.00(s,1 H); 7.98(br. s,2 H);

7.92(dm, =7.8Hz,2 H);

7.47(dm, =7.8Hz,2 H); 4.68(s,0.6

H) and 4.60(s,1.4 H);

2.97(s,2.1 H) and 2.83(s,0.9 H); 3.60 517.0307 516.08 518.02 98 2.37(s,3H); 2.10(s,2.1 H) and

2.05(s,0.9 H); Partly two signal

sets in ratio of 7 : 3 due to

restricted rotation of the tertiary

amide

13.30(br.s,1 H); 7.75-8.00(br. ovl.

m,5H); 7.42-7.67(br. ovl. m,3H);

6.98(br, 1 H); 3.87(s, 3H); 3.59 (s,

2H); 2.41(m, 4H); 1.55(m, 4H); 2.93 423.1617 422.4 424.3 96 1.42(m, 2H). The signals at 30

°C are broad, heating up to 120

°C, the signals sharpen.

not determined 2.89 548.1052 547.10 549.11 94 not determined 2.78 506.0946 505.09 507.10 92 not determined 2.75 478.0633 477.06 479.07 94 not determined 2.73 464.0477 463.04 465.06 95 not determined 2.67 493.0742 492.07 494.08 96 not determined 2.65 479.0586 478.05 480.07 98 not determined 2.58 465.0429 464.04 466.05 97 not determined 2.67 423.1617 422.15 424.17 97 not determined 2.48 501.0722 500.06 502.08 98 not determined 2.75 441.1523 440.14 442.16 97 not determined 2.79 427.1366 426.13 428.14 95 not determined 2.83 445.1272 444.12 446.14 93 not determined 2.62 435.1617 434.15 436.17 96 not determined 2.63 435.1617 434.15 436.17 92 not determined 2.65 433.1460 432.14 434.15 94 not determined 2.65 433.1460 432.14 434.15 94 Biological results

Materials and methods

Description of the FGFR family in vitro biochemical assays

The FGFR1 , FGFR2, FGFR3 and FGFR4 kinase assays were performed in low protein binding, black, round bottom 384-well plates (Corning Inc., Corning, NY). Potent kinase inhibitor compounds were dissolved in 100 % DMSO to 5 mM and then we prepared a serial dilution in H ₂ 0 from 30 μΜ to 0.014 μΜ concentrations with the divisor of three. In all cases we used Transcreener® ADP Assay FP method (BellBrook Labs LLC, Fitchburg, Wl). The reaction conditions were different in each enzyme's case.

In the FGFR1 assays we used the following materials in the following final concentrations for the reaction: 15 nM FGFR1 (ProQinase GmbH, Freiburg, Germany), 0.01 mg/mL Poly Glu-Tyr (Sigma-Aldrich) as a substrate, 20 mM HEPES pH 7.5 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich, St. Louis, MO), 10 mM MgCI ₂ (Sigma-Aldrich), 0.4 mM MnCI ₂ (Sigma-Aldrich), and 0.01 V/V% NP40 (Sigma-Aldrich) detergent. In each assay the identical materials used were the same stocks from the vendors described above.

In the FGFR2 assays the reaction conditions were the following: 1.5 nM FGFR2

(SignalChem Corp., Richmond, Canada), 0.01 mg/mL Poly Glu-Tyr, 20 mM HEPES pH 8, 1 mM DTT, 10 mM MgCI ₂ , 0.4 mM MnCI ₂ , and 0.01 V/V% Tween20 (Sigma-Aldrich) detergent. In the FGFR3 assays we used the following materials and concentrations for the reaction: 15 nM FGFR3 (ProQuinase), 0.01 mg/mL Poly Glu-Tyr, 20 mM HEPES pH 7.5, 1 mM DTT, 10 mM MgCI ₂ , 0.4 mM MnCI ₂ , and 0.01 V%V Brij35 detergent (Sigma-Aldrich).

In the FGFR4 assays the reaction conditions were the following: 5 nM FGFR4 (ProQuinase), 0.01 mg/mL Poly Glu-Tyr, 20 mM HEPES pH 8, 1 mM DTT, 10 mM MgCI ₂ , 10 mM MnCI ₂ , and 0.01 V%V Brij35 detergent (Sigma-Aldrich).

In each assay the ATP (Sigma-Aldrich) concentrations were consistent with the given KM _ap p concentration. The kinase reaction had started by the addition of 2 5X enzyme, and the reaction had been progressing in the volume of 10 for 1 hour at room temperature. The reaction was stopped by adding 10 μί Transcreener® Stop and Detection Solution and had been incubated for additional 1 hour. The solution contained in every case 20 mM HEPES pH 7.5, 40 mM EDTA, 0.02 V/V% Brij35 and 3 nM ADP Alexa633 Tracer. The ADP antibody concentration was 10.61 μg/mL for the FGFR1 assay, 2.29 μg/mL for the FGFR2 assay, 5.24 μg/mL for the FGFR3 assay and 4.78 μg/mL for the FGFR4 assay. Then the fluorescence polarization and fluorescence intensity was measured was using Analyst GT (Molecular devices) and by Infinite M1000 Pro Multimode Reader (Tecan Group Ltd., Mannedorf, Switzerland). IC ₅₀ curves were fitted with XLfit curve fitting add-in software (IDBS) for Microsoft Office Excel.

Description of the cell viability assay

H716 and HCT-1 16 cell lines were purchased from ATCC (via LGC Standards,

Teddington, UK), RKO cell line was obtained from Semmelweis Unversity, U266 and LP1 cell lines were obtained from Max Planck Institute. All cells were cultured in RPMI-1640 medium (Sigma-Aldrich); supplemented with 10 % heat inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1 % Ab/Am (Sigma-Aldrich, St. Louis, MO), except LP1 which line was cultured in IMDM, supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1 % Ab/Am (Sigma-Aldrich, St. Louis, MO) at 37 °C in a 5 % C02 containing, humidified incubator.

Cell viability was determined with CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega Co., Madison, Wl). The luminescence signal was detected by Infinite M1000 Pro Multimode Reader (Tecan). All compounds were dissolved in DMSO. 1000 cells per well were plated in 384-well flat-bottom plates (PerkinElmer Inc., Waltham, MA) in 30 μί. One day after seeding, compounds were added to each cell line. Experimental data were gained from the 10 point serial dilution of the compounds (dilution range varied between 30-0.00152 μΜ). Cell viability was measured after 72 hours. The ratio of the number of survived cells and the number of cells in the untreated samples (positive control) was determined. The negative control was medium without cells containing 10 μΜ DMSO.

Biochemical assay and cell viability assay results

We tested the inhibitory effect of the compounds in four in vitro biochemical assays, each optimized for the given enzyme. The four enzymes were wild type FGFR1 , FGFR2, FGFR3 and FGFR4 proteins. We also measured selected compounds' effect on cell viability. The cellular model of choice was H716, which is an FGFR2 amplificated cell line with increased FGFR2 expression and activity which makes it a good and common model for the characterization of FGFR-targeted compounds. The IC ₅₀ values on each target are shown in the table below. ND indicates that there is no available data. Ta E _l bam _p exle 4. FGFR inhibitory data and H716 cell viability data of selected compounds

FGFR1 IC ₅₀ FGFR2 IC ₅₀ FGFR3 IC ₅₀ FGFR4 IC ₅₀

H716 IC ₅₀ [μΜ] [μΜ] [μΜ] [μΜ] [μΜ]

1 >16 0.00701 0.15341 1.00434 0.50839

2 2.29837 0.00771 1.58704 10.87673 0.9625

3 0.04058 0.0019 0.02488 2.8684 >30

4 0.27491 0.00966 0.05824 10.75192 0.39522

5 0.10144 0.00763 0.07315 5.32417 0.78702

6 0.21897 0.00861 0.16197 5.27249 0.31552

7 0.24818 0.00636 0.11208 3.02077 0.68197

8 0.38042 0.00834 0.0448 2.17721 10.30119

9 0.16102 0.00721 0.0014 1.77341 0.58907

1 1 0.31324 0.0193 0.20624 >16 1.72934

12 >16 0.02501 0.35075 4.59914 0.52749

14 0.23651 0.01369 0.21333 5.92525 0.41198

16 0.81886 0.01142 0.17696 >16 0.60755

19 0.24925 >16 0.38569 8.17909 ND

20 0.08398 0.01149 0.01672 3.07034 0.19971

23 ND ND 0.03053 ND 0.44693

24 ND ND 0.0467 ND 0.62573

25 0.62905 0.02715 0.49713 >16 0.56577

27 0.63996 0.01025 0.28668 >16 2.02947

28 ND ND 4.09665 ND ND

29 0.31 109 0.011 13 0.18517 2.88856 4.35881

30 2.17351 0.03883 0.62046 8.13277 ND

31 0.22606 0.01447 0.01865 >16 16.41891

33 >16 0.05604 0.26475 >16 ND

35 ND ND 2.87519 ND ND

36 ND ND 3.08024 ND ND

37 ND ND >28.8 ND ND

38 ND ND 0.10176 ND ND

39 ND ND 0.13526 ND ND

40 ND ND 0.09837 ND ND

41 ND ND 0.22301 ND ND

42 ND ND 0.08726 ND 0.47897

43 ND ND 0.08109 ND 0.821 13

44 ND ND 0.08925 ND ND

45 ND ND 0.14927 ND ND ND ND 0.21415 ND ND

ND ND 0.09977 ND ND

ND ND 0.09668 ND ND

ND ND 0.0778 ND 1.27503

0.23386 0.01546 0.05315 >30 0.37275

0.2104 0.01434 0.11033 5.30369 0.55507

0.35009 0.03781 0.04501 1.74163 0.8741

0.25894 0.01484 0.3059 2.47931 0.68504

0.67947 0.01896 0.0692 >30 0.7611

0.62484 0.02712 0.16696 3.47732 1.64524

ND ND 0.05534 ND 1.18589

0.89562 0.04006 0.23295 30 ND

ND ND 0.03491 ND 3.26909

ND ND 0.0771 ND 3.2864

ND ND 0.07495 ND 9.23112

ND ND 0.07276 ND 1.45985

ND ND 3.56 ND ND

ND ND 0.30788 ND ND

ND ND 0.09089 ND ND

ND ND 0.16698 ND ND

ND ND 0.12205 ND ND

ND ND 0.15713 ND ND

ND ND 0.05009 ND 0.42995

ND ND 0.1181 ND ND

ND ND 0.13123 ND ND

ND ND 0.08342 ND 1.40143

ND ND >28.8 ND ND

ND ND >28.5 ND ND

ND ND 3.14136 ND ND

ND ND 1.37388 ND ND

ND ND 0.83079 ND ND

ND ND 2.47889 ND ND

ND ND >28.5 ND ND

ND ND 0.0879 ND ND

ND ND 0.10988 ND ND

ND ND 0.09229 ND ND

ND ND 3.24579 ND ND

ND ND 4.68793 ND ND

ND ND >28.5 ND ND Besides H716, we had been seeking for other human cell lines which may underline compound efficacy based on the compounds' inhibitory effects on FGFR family members. We were searching for FGFR mutated cell lines in order to investigate efficacy in tumor cells where the FGFRs may be particularly important. We had carried out COSMIC database searches to identify cell lines which harboured FGFR mutations and thus may help to demonstrate efficacy. HCT-1 16 is a KRAS mutation driven colon tumor, but the searches also revealed several FGFR2 mutations in addition to the mutations in FGFR1 . The FGFR mutational status of RKO colon cell line includes several non-silent FGFR1 mutations. The U266 and the LP1 multiple myeloma cell lines have mutations in FGFR3 which may fairly contribute to their malignancy. Based on these data, we tested commercially available FGFR inhibitors on HCT-1 16, RKO, U266 and LP1 cell lines and found that these compounds inhibited the growth of these cell lines, therefore FGFRs are potent targets in these systems. In a subsequent step, we tested our own compounds on HCT-1 16, RKO, U266 and LP1 cell lines and several of these compounds proved to be effective. Results from these experiments are shown in Table 5.

Table 5.

HCT-116 ICso RKO ICso U266 ICso OPM266 ICso

Example

[μΜ] [μΜ] [μΜ] [μΜ]

1 3.09880 ND ND ND

2 5.71906 ND ND ND

3 30 >30 >30 >30

4 2.65743 2.32724 3.46631 3.09561

5 3.72815 2.73543 2.78012 2.35581

6 1 .81439 2.39598 ND ND

7 0.93423 ND ND ND

8 30 >30 >30 23.09733

9 2.06919 1 .48604 1 .16328 0.90589

10 26.55316 ND ND ND

1 1 8.13630 >30 ND ND

12 2.12452 ND ND ND

13 3.94047 ND ND ND

14 2.38773 ND ND ND

15 23.03024 ND ND ND 16 3.46303 3.04127 ND ND

17 6.43289 ND ND ND

18 17.23210 ND ND ND

19 ND 3.32688 ND ND

20 0.77273 1 .08012 2.82975 2.41935

21 3.09867 ND ND ND

22 1 1 .26905 ND ND ND

23 ND ND ND ND

24 ND ND ND ND

25 5.1 1991 3.59578 ND ND

26 4.20780 ND ND ND

27 5.17085 5.77025 ND ND

28 ND ND ND ND

29 17.27935 8.88540 ND ND

30 9.0271 1 ND ND ND

31 30 >30 >30 >30

33 8.02934 ND ND ND

35 ND ND ND ND

36 ND ND ND ND

37 ND ND ND ND

38 ND 9.13673 ND ND

39 ND ND ND ND

40 ND ND ND ND

41 ND ND ND ND

42 ND ND ND ND

43 ND ND ND ND

44 ND ND ND ND

45 ND ND ND ND

46 ND ND ND ND

47 ND ND ND ND

48 ND ND ND ND

49 ND ND ND ND

50 0.81241 1 .51768 1 .13476 0.86216

51 2.18962 1 .58109 ND ND

52 3.98671 4.34140 4.31561 2.46750

53 30 ND ND ND

54 2.54784 1 .64149 ND ND

55 2.97322 1 .65762 2.401 14 1 .56823

56 7.46859 6.48388 ND ND 57 7.49755 13.36340 ND ND

58 ND 1 .51073 ND ND

59 30 >30 ND ND

60 ND 5.30200 ND ND

61 ND ND ND ND

62 ND ND ND ND

64 ND ND ND ND

65 ND ND ND ND

66 ND ND ND ND

67 5.90948 ND ND ND

68 6.62813 ND ND ND

69 3.44749 ND ND ND

70 7.09668 ND ND ND

71 30 ND ND ND

72 ND ND ND ND

73 30 ND ND ND

77 ND ND ND ND

78 ND ND ND ND

79 ND ND ND ND

80 ND ND ND ND

81 ND ND ND ND

88 ND ND ND ND

89 ND ND ND ND

90 ND ND ND ND

91 ND ND ND ND

92 ND ND ND ND

94 ND ND ND ND