WO2016096631A1

WO2016096631A1 - New antibacterial compounds

Info

Publication number: WO2016096631A1
Application number: PCT/EP2015/079352
Authority: WO
Inventors: Rosella Ombrato; Barbara Garofalo; Giorgina Mangano; Alessandra CAPEZZONE DE JOANNON; Gaia CORSO; Gabriele Magaro'; Guido Furlotti; Tommaso Iacoangeli
Original assignee: Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.P.A.
Priority date: 2014-12-17
Filing date: 2015-12-11
Publication date: 2016-06-23
Also published as: US20170368017A1; US10369130B2; EP3233826A1; MA41168A; AR103028A1; JP2017537938A

Abstract

The present invention relates to novel antibacterial compounds, pharmaceutical compositions containing them and their use as antimicrobials.

Description

TITLE

"New antibacterial compounds"

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

DNA topoisomerases are enzymes involved in the modification of the DNA-supercoiling during replication or transcription. These enzymes bind to single-stranded or double-stranded DNA and cut the phosphate backbone of the DNA such that the DNA strand is untangled or unwound. At the end of the replication or transcription processes, the enzymes themselves reseal the DNA backbone.

DNA topoisomerases are classified as type I when cut a single strand of a DNA double helix and as type II when cut both strands of a DNA double helix.

Bacterial type II topoisomerases comprise DNA gyrase and topoisomerase IV (TopolV), which are heterotetrameric enzymes concurrently present in almost all the prokaryotic cells. Both the enzymes are necessary for DNA replication and, hence, for bacterial cell growth and division.

Bacterial type II topoisomerases are a proven antibacterial target, in particular of compounds belonging to fluoroquinolone class.

Fluoroquinolones are broad-spectrum antibacterial drugs that play an important role in treatment of bacterial infections, especially hospital-acquired infections and infections in which resistance to other antibacterial classes is suspected. Fluoroquinolones act by inhibiting the DNA gyrase in Gram negative bacteria and the topoisomerase IV in Gram positive bacteria.

However, resistance to fluoroquinolones emerged in recent years due to mutations that altered either the active site of the drug targets DNA gyrase and topoisomerase IV or the drug accumulation. In addition, resistance to quinolones can be mediated by plasmids that produce the Qnr protein, which protects the quinolone targets from inhibition (G.A. Jacoby, CID, 2005:41 , Suppl. 2, SD1 20-S1 26). According to the World Health Organization, the antimicrobial resistance (AMR) is the resistance of a microorganism to an antimicrobial drug to which it was originally sensitive. Resistant bacteria are able to withstand attack by antibiotics and antibacterial drugs, so that standard treatments become ineffective and infections persist increasing risk of spread to others.

WO 02/072572, WO 2006/021448, WO 2008/139288, WO 201 0/081874, WO 2010/084152, WO 201 3/068948 and WO 2013/080156 disclose heterocyclic compounds endowed with antimicrobial activity.

WO 96/10568 and WO 2012/003418 disclose heterocyclic compounds endowed with other therapeutic activity.

SUMMARY OF THE INVENTION

The Applicant recognized that there is a strong and continuous need for antibacterial drugs that overcome the problem of resistant bacteria.

Thus, the Applicant faced the problem to develop new antibacterial compounds.

In particular, the Applicant faced the problem to develop new antibacterial compounds having broad spectrum of activity, i.e. useful against Gram positive and/or Gram negative bacteria.

Thus, in a first embodiment, the present invention relates to a compound of formula (I):

A-L B-L₂-Y-L₃-C (I)

wherein

A is a 10-membered fused bicyclic ring;

Li is σ bond, -O- or -N(R')-, wherein R' is H or (Ci-₃)alkyl;

B is a divalent residue of a piperazine or piperidine ring;

L₂ is σ bond, -CH₂-, -O-, or -N(R')-, wherein R' is H or (d-₃)alkyl;

Y is a (Ci-₆)alkylenyl, (C₂-₆)alkenylenyl, (C₂-₆)alkynylenyl, or (C₃- 6)cycloalkylenyl group, said group being optionally substituted with one or more groups selected from -OH, (Ci-₃)alkyl, (C₃-₆)cycloalkyl, 3- to 5-membered oxacycloalkyl, -COOR', -NR'R" wherein R' and

R", identical or different each other, are hydrogen atom or (Ci- 3)alkyl; L₃ is -0-, -(CH₂)₂-, -N(R')-, -N(R')-C(=0)-, -N(R')-C(=S)-, -N(R')-(d- 6)alkylenyl-, -C(=0)-N(R')-, -C(=O)-N(R')-(Ci-₆)alkylenyl-, -SO₂- N(R')-, -N(R')-SO₂-, wherein R' is hydrogen atom or (Ci_-3)alkyl;

C is a group having a 5- or 6-membered saturated or unsaturated ring, or a 9- or 10-membered fused bicyclic ring;

and pharmaceutically acceptable salts, enantiomers, N-oxides and quaternary ammonium salts thereof.

In a second embodiment, the present invention relates to a pharmaceutical composition comprising at least one compound of formula (I).

In a third embodiment, the present invention relates to the compounds of formula (I) for use in medicine.

In a fourth embodiment, the present invention relates to the compounds of formula (I) for use in the treatment of bacterial infections.

In a fifth embodiment, the present invention relates to a method for treating a bacterial infection, comprising the administration of a compound of formula (I) to a patient in need thereof.

According to a preferred aspect of the present invention, A is a fused bicyclic ring having the following formula (II):

wherein

G1 and G2, identical or different each other, are CH or N;

Ri is H, halogen atom, CN or CF₃;

R₂ is H, halogen atom, OH, CN, CF₃, (Ci-₆)alkyl or (Ci-₆)alkoxy;

D is a divalent group selected from

wherein R₃ is H, (Ci-₆)alkyl or (Ci-₆)alkoxy.

According to a preferred aspect of the present invention, A is a fused bic clic rin havin one of the following fo

Preferably, Ri is H or halogen atom. More preferably, Ri is H or F.

Preferably, R₂ is H, halogen atom, OH, (d-₃)alkyl or (Ci-₃)alkoxy. More preferably, R₂ is H, F, CI, OH, CH₃ or OCH₃.

Preferably, R₃ is H, (Ci-₃)alkyl or (Ci-₃)alkoxy. More preferably, R₃ is H, CH₃ or OCH₃.

Preferably, Li is a σ bond or -N(R')- wherein R' is H or methyl. More preferably, U is a σ bond or -NH-. According to a preferred aspect of the present invention, B is a group having the following formula (II I)

— G3 G4—

\ G5

wherein

G3 and G4, identical or different each other, are C(R₄) or N, provided that at least one of G3 and G4 is N,

R₄ is hydrogen atom, -OH, -CN, -COOH, -NR'R" wherein R' and R", identical or different each other, are hydrogen atom or (Ci-₃)alkyl;

G5 is C(=0) or C(H)(R₅);

R₅ is hydrogen atom, CF₃, -(Ci-₃)alkyl-CF₃, -COOR' and -CONR'R", wherein R' and R", identical or different each other, are hydrogen atom or (Ci- 3) alky I.

Preferably, R₄ is hydrogen atom.

Preferably, R₅ is H, -CH₂CF₃, -COOR' or -CONR'R", where R' and R" are H or CH₃.

According to a preferred aspect of the present invention, B is a ring residue having one of the following formulas:

Preferably, L₂ is σ bond, or -NH-.

More preferably, L₂ is σ bond.

Preferably, Y is a (Ci-₄)alkylenyl, (C₂-₄)alkynylenyl, or (C₅-₆)cycloalkylenyl group, said group being optionally substituted with one or more groups selected from -OH, methyl, (C₃-₄)cycloalkyl, 3- or 4-membered oxacycloalkyl, -COOH, -NR'R" wherein R' and R", identical or different each other, are hydrogen atom or methyl.

More preferably, Y is a (Ci-₄)alkylenyl, (C₂-₄)alkynylenyl or cyclohexylenyl group, said group being optionally substituted with one or more hydroxy groups.

Preferably, L₃ is -O-, -(CH₂)₂-, -N(R')- wherein R' is H or (Ci-₃)alkyl, -NH- C(=O)-, -NH-(Ci-₃)alkylenyl-, -C(=O)-NH- or

More preferably, L₃ is -O-, -(CH₂)₂-, -N(R')- wherein R' is H or methyl, -NH- C(=O)-, -NH-CH₂-, -C(=O)-NH-, -C(=O)-NH-CH₂-.

According to a preferred aspect of the present invention, C is a group having one of the following formulae (IV), (V), (VI), (VII) or (VIII):

(IV) (V) (VI)

(VII) (VI II) wherein

n is an integer from 0 to 3

Pi and P₂, equal or different each other, are CH or N

P₃ is O, S or NH;

R₆ and R₇ together form a 5- or 6-membered aliphatic or aromatic ring, optionally comprising at least one heteroatom selected from N and O,

R₈ and R₉, equal or different each other, are a hydrogen atom or an aryl group, or together form a 5- or 6-membered aliphatic or aromatic ring, optionally comprising at least one heteroatom selected from N and O, Rio and R , equal or different each other, are a hydrogen atom, or a (d- ₃)alkyl group, or together form a 5- or 6-membered aliphatic or aromatic ring, and wherein each hydrogen atom linked to a carbon or nitrogen atom forming a ring of formulae (IV), (V), (VI), (VII) or (VIII) is optionally substituted with at least one substituent selected from the group consisting of halogen atom, OH, (Ci-₃)alkyl or (Ci-₃)alkoxy.

Preferably, n is an integer from 0 to 2, more preferably n is 0 or 1 .

Preferably, R₆ and R₇ together form a ring selected from the group consisting of benzene, furan, tetrahydrofuran, dioxolane, piperidine and piperazine.

Preferably, R₈ and R₉, equal or different each other, are a hydrogen atom or an aryl group, or together form a ring selected from the group consisting of benzene, pyridine, and piperidine.

Preferably, R₁₀ and R , equal or different each other, are a hydrogen atom, or a (Ci-₃)alkyl group, or together form a cyclohexane or an aryl ring.

Preferably, each hydrogen atom linked to a carbon or nitrogen atom forming the ring of formulae (IV), (V), (VI), (VII) or (VIII) is optionally substituted with at least one substituent selected from the group consisting of chlorine atom, fluorine atom, OH, methyl or methoxy.

According to a preferred aspect of the present invention, C is a group having one of the following formulas:

In the present description and in the following claims, the term "(Ci- 6)alkyl" means a linear or branched alkyl chain comprising from 1 to 6 carbon atoms, such as for example methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, sec-pentyl, 3- pentyl, n-hexyl, isohexyl, neo-hexyl, 3-methyl-pentyl, 2,3-dimethylbutyl.

In the present description and in the following claims, the term "(Ci- 3)alkyl" means a linear or branched alkyl chain comprising from 1 to 3 carbon atoms, such as for example methyl, ethyl, propyl, isopropyl.

In the present description and in the following claims, the term "(Ci- ₆)alkoxy" means a linear or branched alkoxy chain comprising from 1 to 6 carbon atoms, such as for example methoxy, ethoxy, propoxy, isopropoxy, n- butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentoxy, tert-pentoxy, sec-pentoxy, 3-pentoxy, n-hexoxy, isohexoxy, neo- hexoxy, 3-methyl-pentoxy, 2,3-dimethy-lbutoxy.

In the present description and in the following claims, the term "(Ci- ₃)alkoxy" means a linear or branched alkoxy chain comprising from 1 to 3 carbon atoms, such as for example methoxy, ethoxy, propoxy, isopropoxy.

In the present description and in the following claims, the term "(Ci- 6)alkylenyl" means a divalent linear or branched alkyl chain comprising from 1 to 6 carbon atoms, such as for example methylenyl (-CH₂-), ethylenyl (- CH₂CH₂-), propylenyl (-CH₂CH₂CH₂-), butylenyl (-CH₂CH₂CH₂CH₂-), pentylenyl (-CH₂CH₂CH₂CH₂CH₂-) or hexylenyl (-CH₂CH₂CH₂CH₂CH₂-). ln the present description and in the following claims, the term "(Ci- ₄)alkylenyl" means a divalent linear or branched alkyl chain comprising from 1 to 4 carbon atoms, such as for example methylenyl (-CH₂-), ethylenyl (- CH₂CH₂-), propylenyl (-CH₂CH₂CH₂-), butylenyl (-CH₂CH₂CH₂CH₂-).

In the present description and in the following claims, the term "(C₂-

₆)alkenylenyl" means a divalent linear or branched alkylen chain comprising from 2 to 6 carbon atoms, such as for example ethenylenyl (-CH=CH-), propenylenyl (-CH=CH-CH₂- or -C(CH₃)=CH-) or butenylenyl (-CH=CH- CH₂CH₂- or -CH₂CH=CH-CH₂- or -C(CH₃)=CH-CH₂-).

In the present description and in the following claims, the term (C₂-

₆)alkynylenyl means a divalent linear or branched alkynyl chain comprising from 2 to 6 carbon atoms, such as for example ethynylenyl (-C≡C-), propynylenyl (-C≡C-CH₂- or -CH₂-C≡C-), butynylenyl (-C≡C-CH₂CH₂- or - CH₂-C≡C-CH₂- or -C(CH₃)-C≡C-), pentynylenyl (-C≡C-CH₂CH₂CH₂- or - CH₂CH₂-C≡C-CH₂- or -C(CH₂CH₃)-C≡C-), or hexynylenyl (-C≡C- CH₂CH₂CH₂CH₂- or -CH₂CH₂C≡C-CH₂CH₂- or - CH₂CH₂C(CH₃)-C≡C-).

In the present description and in the following claims, the term (C₂- ₄)alkynylenyl means a divalent linear or branched alkynyl chain comprising from 2 to 4 carbon atoms, such as for example ethynylenyl (-C≡C-), propynylenyl (-C≡C-CH₂- or -CH₂-C≡C-), butynylenyl (-C≡C-CH₂CH₂- or - CH₂-C≡C-CH₂- or -C(CH₃)-C≡C-).

In the present description and in the following claims, the term "(C₃- ₆)cycloalkylenyl" means a divalent cycloalkyl group comprising from 3 to 6 carbon atoms, such as cyclopropylenyl, cyclobutylenyl, cyclopentylenyl and cyclohexylenyl

In the present description and in the following claims, the term "(C₅- ₆)cycloalkylenyl" means a divalent cycloalkyl group comprising from 4 to 6 carbon atoms, such as cyclopentylenyl and cyclohexylenyl.

In the present description and in the following claims, the term "(C₃- ₆)cycloalkyl" means a cycloalkyl group comprising from 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In the present description and in the following claims, the term "(C₃- ₄)cycloalkyl" means a cycloalkyl group comprising 3 or 4 carbon atoms, such as cyclopropyl and cyclobutyl. ln the present description and in the following claims, the expression "3- to 5-membered oxacycloalkyl" means a cycloalkyl group comprising at least one O atom and from 2 to 4 carbon atoms.

In the present description and in the following claims, the expression "3- or 4-membered oxacycloalkyl" means a cycloalkyl group comprising at least one O atom and from 2 to 3 carbon atoms.

Certain compounds of this invention may exist in tautomeric forms, and this invention includes all such tautomeric forms of those compounds unless otherwise specified.

Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Thus, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention. Thus, this invention encompasses each diastereomer or enantiomer substantially free of other isomers (>90%, and preferably >95%, free from other stereoisomers on a molar basis) as well as a mixture of such isomers.

Particular optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, e.g., by formation of diastereomeric salts, by treatment with an optically active acid or base. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid and then separation of the mixture of diastereomers by crystallization followed by liberation of the optically active bases from these salts. A different process for separation of optical isomers involves the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers. Still another method involves synthesis of covalent diastereomers by reacting compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure compound. Optically active compounds of the invention can be obtained by using active starting materials. These isomers may be in the form of a free acid, a free base, an ester or a salt.

The compounds of this invention can exist in radiolabeled form, i.e., said compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number ordinarily found in nature. Radioisotopes of hydrogen, carbon, phosphorous, fluorine and chlorine include ³H, ¹⁴C, ³²P, ³⁵S, ¹⁸F and ³⁶CI, respectively. Compounds of this invention which contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, radioisotopes are particularly preferred for their ease of preparation and detectability.

Radiolabeled compounds of this invention can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed herein except substituting a readily available radiolabeled reagent for a non-radiolabelled reagent.

In a second embodiment, the present invention relates to a pharmaceutical composition comprising at least one compound of formula (I) as described above, a salt thereof with a pharmaceutically acceptable organic or inorganic acid or base, or an enantiomer thereof, or a quaternary ammonium salt thereof, or a N-oxide thereof, and at least one inert pharmaceutically acceptable excipient.

Preferably, the pharmaceutical composition of the present invention is prepared in suitable dosage forms.

Examples of suitable dosage forms are tablets, capsules, coated tablets, granules, solutions and syrups for oral administration; solutions, pomade and ointment for topical administration; medicated patches for transdermal administration; suppositories for rectal administration and injectable sterile solutions. Other suitable dosage forms are those with sustained release and those based on liposomes for oral, injectable or transdermal administration. The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation or delivered by implantation (e.g., surgically), such as with an implantable or indwelling device like a stent. Other suitable dosage forms are those with sustained release and those based on liposomes for oral, injectable or transdermal administration.

The dosage forms of the pharmaceutical composition of the present invention can be prepared by techniques that are familiar to a pharmaceutical chemist, and comprise mixing, granulation, compression, dissolution, sterilization and the like.

Typically, the amount of compound of formula (I) or of the pharmaceutically acceptable quaternary ammonium salt, N-oxide and salt thereof in the pharmaceutical composition of the present invention will be between 0.01 mg to 1 ,500 mg, preferably between 0.1 mg and 500 mg and more preferably between 1 mg and 200 mg.

Typically, the amount of compound of formula (I) in the pharmaceutical composition of the present invention will be such to ensure a level of administration from 0.001 to 20 mg/kg/day. Preferably, the level of administration is from 0.01 to 7.5 mg/kg/day, more preferably from 0.1 to 5 mg/kg/day, and most preferably from 0.5 to 2.5 mg/kg/day.

As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

As mentioned above, depending on the nature of the substituents, the compound of formula (I) may form addition salts with a pharmaceutically acceptable organic or inorganic acid or base. Typical examples of suitable physiologically acceptable inorganic acids are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid.

Typical examples of suitable physiologically acceptable organic acids are acetic acid, ascorbic acid, benzoic acid, citric acid, fumaric acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-toluenesulfonic acid, benzenesulfonic acid, succinic acid, tannic acid and tartaric acid.

Typical examples of suitable physiologically acceptable inorganic bases are hydroxides, carbonates and hydrogen carbonates of ammonium, calcium, magnesium, sodium and potassium, for instance ammonium hydroxide, calcium hydroxide, magnesium carbonate, sodium hydrogen carbonate and potassium hydrogen carbonate.

Typical examples of suitable physiologically acceptable organic bases are: arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, N-methylglucamine, glucamine, glucosamine, histidine, N-(2-hydroxyethyl)-piperidine, N-(2- hydroxyethyl)pyrrolidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, theobromine, triethylamine, trimethylamine, tripropylamine and tromethamine.

As described herein, the pharmaceutical composition of the present invention comprises a compound of the invention together with a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, diluents, or other vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.

Some examples of materials which can serve as pharmaceutically acceptable excipient include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants.

The terms "pharmaceutically acceptable" and "physiologically acceptable" are intended to define, without any particular limitation, any material suitable for preparing a pharmaceutical composition to be administered to a living being.

Preferably, said bacterial infection is a skin infection, a mucosal infection, a gynaecological infection, a respiratory tract infection (RTI), a CNS infections, a gastro-intestinal infection, a bone infection, a cardiovascular infection, a sexually transmitted infection, or a urinary tract infection.

More in particular, said bacterial infection is a acute exacerbation of chronic bronchitis (ACEB), an acute otitis media, an acute sinusitis, an infection caused by drug resistant bacteria, a catheter-related sepsis, a chancroid, a chlamydia, a community-acquired pneumonia (CAP), a complicated skin and skin structure infection, an uncomplicated skin and skin structure infection, an endocarditis, a febrile neutropenia, a gonococcal cervicitis, a gonococcal urethritis, a hospital-acquired pneumonia (HAP), a osteomyelitis, a sepsis, a syphilis, a ventilator-associated pneumonia, an intraabdominal infections, a gonorrhoeae, a meningitis, a tetanus, or a tuberculosis.

Even more, said bacterial infection can be an atherosclerosis or cardiovascular disease related to infection by Helicobacter pylori or Chlamydia pneumonia; a blood and tissue infections, including endocarditis and osteomyelitis, caused by S. aureus, S. haemolyticus, E. faecalis, E. faecium, E. durans, including strains resistant to known antibacterials such as, but not limited to, beta-lactams, vancomycin, aminoglycosides, quinolones, chloramphenicol, tetracyclines and macrolides; bronchitis; catheter-related sepsis; chancroid; chlamydia; community-acquired pneumonia; disseminated Mycobacterium avium complex (MAC) disease related to infection by Mycobacterium avium, or Mycobacterium intracellular; endocarditis; febrile neutropenia; gas gangrene related to infection by Clostridium perfringens or Bacteroides spp; gastroenteritis infection; glomerulonephritis related to infection by Streptococcus pyogenes, Groups C and G streptococci, Corynebacterium diphtheriae, or Actinobacillus haemolyticum; gonococcal cervicitis; gonococcal urethritis; gynaecological infection; hospital-acquired pneumonia (HAP); infection caused by drug resistant bacteria; infections caused by Mycobacterium tuberculosis, M. leprae, M. paratuberculosis, M. kansasii, or M. chelonei; intestinal protozoa related to infection by Cryptosporidium spp; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H. injluenzae, or Listeria spp. ; mastoiditis related to infection by Streptococcus pneumoniae, Haemophilus injluenzae, Moraxella catarrhalis , Staphylococcus aureus, Enterococcus faecalis, E. faecium, E. casseliflavus, S. epidermidis, S. haemolyticus, or Peptostreptococcus spp; odontogenic infection related to infection by viridans streptococci; osteomyelitis; otitis media; persistent cough related to infection by Bordetella pertussis; pharyngitis; puerperal fever related to infection by Staphylococcus aureus, coagulase-negative staphylococci Streptococcus pyogenes, Streptococcus agalactiae, Streptococcal groups C-F (minute colony streptococci), viridans streptococci Corynebacterium minutissimum, Clostridium spp., or Bartonella henselae; respiratory tract infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus injluenzae, or Chlamydia pneumoniae; rheumatic fever; sepsis; sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasma urealyticum, or Neiseria gonorrhoeae; sinusitis,; syphilis; systemic febrile syndromes related to infection by Borrelia recurrentis; tonsillitis; toxin diseases related to infection by S. aureus (food poisoning and toxic shock syndrome), or Groups A, B, and C streptococci; ulcers related to infection by Helicobacter pylori; uncomplicated acute urinary tract infections related to infection by Staphylococcus aureus coagulase- negative staphylococcal species, or Enterococcus spp.; uncomplicated skin and soft tissue infections and abscesses ; urethritis and cervicitis; urinary tract infection; central nervous system infections; device related infections caused by staphylococci; muscoleskeletal infection caused by staphylococci; Shiga toxin-producing E. coli; Haemophilus influenzae (invasive disease); legionellosis; psittacosis/ornithosis clamydia psittaci; salmonellosis caused by salmonella spp.; shigellosis by shigella spp.; streptococcal toxic shock syndrome; staphylococcal toxic shock syndrome; and typhoid fever caused by Salmonella typhi.

The bacterial infection can be an infection caused by Acinetobacter spp,

Bacteroides spp, Burkholderia spp, Campylobacter spp, Chlamydia spp, Chlamydophila spp, Clostridium spp, Enterobacter spp, Enterococcus spp, Escherichia spp, Gardnerella spp, Haemophilus spp, Helicobacter spp, Klebsiella spp, Legionella spp, Moraxella spp, Morganella spp, Mycoplasma spp, Neisseria spp, Peptostreptococcus spp, Proteus spp, Pseudomonas spp, Salmonella spp, Serratia spp, Staphylococcus spp, Streptoccocus spp, Stenotrophomonas spp, Ureaplasma spp, aerobes, obligate anaerobes, facultative anaerobes, gram-positive bacteria, gram-negative bacteria, gram- variable bacteria, and atypical respiratory pathogens.

More in particular, the bacterial infection can be an infection caused by

Acinetobacter baumanii, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter johnsonii, Acinetobacter Iwoffi, Bacteroides bivius, Bacteroides fragilis, Burkholderia cepacia, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia urealyticus, Chlamydophila pneumoniae, Clostridium difficile, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Gardnerella vaginalis, Haemophilus parainfluenzae, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Methicillin-resistant Staphylococcus aureus, Methicillin-susceptible Staphylococcus aureus, Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Penicillin-resistant Streptococcus pneumoniae, Penicillin-susceptible Streptococcus pneumoniae, Peptostreptococcus magnus, Peptostreptococcus micros, Peptostreptococcus anaerobius, Peptostreptococcus asaccharolyticus, Peptostreptococcus prevotii, Peptostreptococcus tetradius, Peptostreptococcus vaginalis, Proteus mirabilis, Pseudomonas aeruginosa, Quinolone-Resistant Staphylococcus aureus, Quinolone-Resistant Staphylococcus epidermis, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella typhimurium, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Stenotrophomonas maltophilia, Ureaplasma urealyticum, Vancomycin-Resistant Enterococcus faecium, Vancomycin-Resistant Enterococcus faecalis, Vancomycin-Resistant Staphylococcus aureus, and Vancomycin-Resistant Staphylococcus epidermis.

Examples of compounds according to the present invention are provided in the following Table 1 .

Table 1

The above compounds can be prepared as explained in the synthetic examples below.

The man skilled in the art has a well-established literature of heterocyclic and other relevant chemical transformations, recovery and purification technologies to draw upon, in combination with the information contained in the examples which follow, for guidance on synthetic strategies, protecting groups, and other materials and methods useful for the synthesis, recovery and characterization of the compounds of this invention, including compounds containing the various choices for A L_{1 ;} B, L₂, Y, L₃ and C.

Various synthetic approaches may be used to produce the compounds described herein, including those approaches depicted schematically below. The man skilled in the art will appreciate that protecting groups may be used in these approaches. "Protecting groups", are moieties that are used to temporarily block chemical reaction at a potentially reactive site (e.g., an amine, hydroxy, thiol, aldehyde, etc.) so that a reaction can be carried out selectively at another site in a multifunctional compound. In preferred embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is suitable for the planned reactions; the protecting group should be selectively removable in good yield by readily available, preferably nontoxic reagents that do not unduly attack the other functional groups present; the protecting group preferably forms an readily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group preferably has a minimum of additional functionality to avoid the complication of further sites of reaction. A wide variety of protecting groups and strategies, reagents and conditions for deploying and removing them are known in the art.

Also, one may chose reagents enriched for a desired isotope, e.g. tritium in place of hydrogen, to create compounds of this invention containing such isotope(s). Compounds containing tritium in place of hydrogen in one or more locations, or containing various isotopes of C, N, P and O, are encompassed by this invention and may be used, for instance, for studying metabolism and/or tissue distribution of the compounds or to alter the rate or path of metabolism or other aspects of biological functioning. The compounds of the this invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or by a variation thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to those described below. The reactions are performed in a solvent appropriate to the reagents and materials employed and suitable for the transformation being effected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule should be consistent the transformations proposed. This will sometimes require some judgment to modify the order of the synthetic steps or to select one particular process scheme over another in order to obtain a desired compound of the invention.

A compound of the present invention could be prepared as outlined in the synthetic pathways described hereinafter and via standard methods known to those skilled in the art.

EXAMPLES

List of the abbreviations used in the synthetic pathways described hereinafter:

Boc - ferf-butyl carbamate

cHex - cyclohexane

CV - column volume

DCM - dichloromethane

DIPEA - A/,A/-diisopropylethylamine

DMA - Λ/JV-dimethylacetamide

DMF - N,N-dimethylformamide

DMSO - dimethyl sulfoxide

Et₂0 - diethyl ether

EtOAc - ethylacetate

EtOH - ethanol

HATU - 1 -[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate

HBTU - A/,A/,A/',A/'-tetramethyl-0-(1 H-benzotriazol-1 -yl)uronium

hexafluorophosphate LC - liquid chromatography

MeCN - acetonitrile

MeOH - methanol

MS - mass spectroscopy

TEA - triethylamine

TFA - trifluoroacetic acid

THF - tetrahydrofuran

Pd/C - palladium on activated charcoal

Pd(dba)₂ - bis(dibenzylideneacetone)palladium(0)

Pd(OAc)₂ - palladium(ll) acetate

UPLC - ultra high performance liquid chromatography

Preparation of compound 2 : N-{3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}pyridine-3-carboxamide hydrochloride

Compound 2 was prepared as described hereinbelow.

Step 1 : A/-(3,3-diethoxypropyl)pyridine-3-carboxamide (2a)

Pyridine-3-carbonyl chloride (10 g, 56.2 mmoles, 1 eq.) was dissolved in DMF (50 mL) and CH₃CN (200 mL) with TEA (19.5 mL, 140.4 mmoles, 2.5 eq.). 3,3-diethoxypropan-1 -amine (10.35 g, 70.2 mmoles, 1 .25 eq.) in CH₃CN (20 mL) was added at 0°C. The solution was stirred overnight then was concentrated, diluted with DCM and washed with sat. NaHC0₃ and brine. The organic phase was then separated, dried over sodium sulphate and evaporated in vacuum to obtain N-(3,3-diethoxypropyl)pyridine-3- carboxamide 2a (6.8 g, Y= 45%). LC-MS (M-H+): 253.0.

Step 2: A/-(3-oxopropyl)pyridine-3-carboxamide (2b)

Intermediate 2a (500 mg, 1 .98 mmoles, 1 eq.) was dissolved in DCM (5 ml_). TFA (2 ml_) was added, and after 3 h the solution was concentrated and washed several times with toluene, diethyl ether and ethyl acetate to afford 900 mg of N-(3-oxopropyl)pyridine-3-carboxamide 2b (Y= quant.). LC-MS (M- H⁺): 1 79.0.

Step 3: 1 -(piperazin-1 -yl)isoquinoline (2c)

1 -Chloroisoquinoline (3g, 1 8.3 mmoles, 1 eq.) was dissolved in CH₃CN

(1 50 ml_) with piperazine (23.7g, 274.5 mmoles, 15 eq.) and potassium carbonate (3.8 g, 27.5 mmoles, 1 .5 eq). The solution was heated to reflux for 48 h then was concentrated, diluted with DCM (250 ml_) and washed with sat. NaHC0₃ and brine. The organic phase was separated, dried over sodium sulfate and evaporated in vacuum to obtain 1 -(piperazin-1 - yl)isoquinoline 2c (3.9 g, Y= 94%). LC-MS (M-H⁺): 214.1 .

Step 4: A/-{3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}pyridine-3- carboxamide hydrochloride (2)

Intermediate 2b (1 50 mg, 0.51 mmoles, 1 eq.) was dissolved in DCM (5 mL). DIPEA (0.078 mL, 0.45 mmoles, 0.9 eq.), acetic acid (one drop) and intermediate 2c (87 mg, 0.41 mmoles, 0.8 eq.) were added subsequently at room temperature. After ten minutes sodium triacetoxyborohydride (141 mg, 0.66 mmoles, 1 .3 eq.) was added and the solution was left stirring overnight. The mixture was diluted with DCM, washed with sat. NaHC0₃ and sat. NaCI, dried over sodium sulphate, filtered and evaporated in vacuum. The crude material was purified with a NH-Column eluting with ethyl acetate to obtain the desired amine (78 mg, 0.21 mmoles). The product was dissolved in DCM (5 mL) and 1 M HCI in Et₂0 (0.84 mL, 4 eq.) was added dropwise at 0 ^<C. The solution was left stirring for 2 h then concentrated in vacuum and triturated with diethyl ether to obtain title compound N-{3-[4-(isoquinolin-1 -yl)piperazin- 1 -yl]propyl}pyridine-3-carboxamide hydrochloride 2 (79 mg, Y= 39%). LC-MS (M-H⁺): 376.2.

¹ H NMR (300MHz, DMSO-d₆) δ = 1 1 .37 (br. s., 1 H), 9.29 - 9.14 (m, 2 H), 8.88 (dd, J = 1 .5, 5.5 Hz, 1 H), 8.60 (td, J = 1 .8, 8.1 Hz, 1 H), 8.21 (d, J = 8.4 Hz, 1 H), 8.06 (d, J = 6.2 Hz, 1 H), 8.02 {6, J = 1.1 Hz, 1 H), 7.93 - 7.78 (m, 2 H), 7.72 (ddd, J = 1 .4, 7.0, 8.3 Hz, 1 H), 7.59 (d, J = 6.2 Hz, 1 H), 6.71 (br. s., 2 H), 4.06 (d, J = 13.5 Hz, 2 H), 3.88 - 3.56 (m, 4 H), 3.44 (q, J = 6.5 Hz, 4 H), 3.34 - 3.20 (m, 2 H), 2.09 (quin, J = 6.1 Hz, 2 H).

Preparation of compound 3 : /V-{3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}benzamide hydrochloride

Compound 3 was prepared according to the procedure described for compound 2, using in step 1 benzoyl chloride. LC-MS (M-H⁺): 375.2.

¹ H NMR (300 MHz, DMSO-d₆) δ = 1 1 .68 (br. s., 1 H), 8.79 (t, J = 5.4 Hz, 1 H), 8.25 (d, J = 8.6 Hz, 1 H), 8.06 (d, J = 8.6 Hz, 1 H), 8.02 (dd, J = 1 .0, 6.3 Hz, 1 H), 7.98 - 7.86 (m, 3 H), 7.75 (ddd, «7 = 1 .1 , 7.0, 8.5 Hz, 1 H), 7.63 (d, J = 6.3 Hz, 1 H), 7.58 - 7.41 (m, 3 H), 4.16 (d, J = 13.5 Hz, 2 H), 3.90 (t, J = 1 2.4 Hz, 2 H), 3.66 (d, J = 1 3.1 Hz, 2 H), 3.56 - 3.34 (m, 4 H), 3.25 (quin, J = 5.0 Hz, 2 H), 2.08 (quin, J = 7.2 Hz, 2 H).

Preparation of compound 4 : /V-{2-[4-(isoquinolin-1-yl)piperazin-1- yl]ethyl}benzamide hydrochloride

Compound 4 was prepared according to the procedure described for compound 2, using in step 1 benzoyl chloride and 2,2-diethoxyethanamine. LC-MS (M-H⁺): 361 .2.

¹ H NMR (300MHz, DMSO-d₆) δ = 8.71 (t, J = 5.5 Hz, 1 H), 8.1 5 (d, J = 5.1 Hz, 1 H), 8.14 (d, J = 8.6 Hz, 1 H), 7.93 (d, J = 7.5 Hz, 1 H), 7.91 - 7.87 (m, 2 H), 7.75 (ddd, J = 1 .5, 7.0, 8.3 Hz, 1 H), 7.64 (ddd, J = 1 .7, 7.0, 8.3 Hz, 1 H), 7.61 - 7.45 (m, 4 H), 6.06 (s, 2 H), 3.68 (q, J = 6.2 Hz, 2 H), 3.63 - 3.19 (m, 1 0 H).

Preparation of compound 14

Compound 14 was prepared according to the procedure described for the synthesis of compound 2, using 1 ,6-dichloroisoquinoline in step 3 (Y=78%). LC-MS (M-H⁺) = 410.2

¹H NMR (300 MHz, DMSO-d₆) δ = 11.36 (br. s., 1 H), 9.30 (t, J= 5.3 Hz, 1 H), 9.25 (d, J = 1.7 Hz, 1 H), 8.90 (dd, J = 1.3, 5.3 Hz, 1 H), 8.67 (d, J = 7.9 Hz, 1 H), 8.19 (d, J= 8.9 Hz, 1 H), 8.14 (d, J= 3.3 Hz, 1 H), 8.13 (s, 1 H), 7.88 (dd, J= 5.4, 8.1 Hz, 1 H), 7.67 (dd, J = 2.1 , 9.1 Hz, 1 H), 7.51 (d, J=5.9 Hz, 1 H), 3.96 (d, J= 13.9 Hz, 2 H), 3.76 - 3.54 (m, 4 H), 3.50 - 3.34 (m, 4 H), 3.32 - 3.19 (m, J= 4.6 Hz, 2 H), 2.19 - 1.99 (m, 2 H).

Preparation of compound 21 : /V-{3-[4-(3-methylisoquinolin-1- yl)piperazin-1 -yl]propyl}pyridine-3-carboxamide hydrochloride

Compound 21 was prepared according to the procedure described for compound 2, using in step 3 1-chloro-3-methylisoquinoline. LC-MS (M-H⁺): 390.2.

1H NMR (400MHz, DMSO-d₆) δ = 11.37 (br. s., 1H), 9.30 (t, J=5.4 Hz, 1H),

9.25 (d, J=1.5 Hz, 1H), 8.90 (dd, J=1.4, 5.1 Hz, 1H), 8.65 (d, J=8.0 Hz, 1H), 8.12 (d, J=8.5 Hz, 1H), 7.92 - 7.83 (m, 2H), 7.78 (t, J=7.5 Hz, 1H), 7.60 (t, J=7.5 Hz, 1 H), 7.37 (s, 1 H), 7.30 - 5.98 (m, 3H), 5.76 (s, 1 H), 3.98 (d, J=13.3 Hz, 2H), 3.78 - 3.54 (m, 4H), 3.51 - 3.34 (m, 4H), 3.27 (td, J=4.6, 9.6 Hz, 2H), 2.54 (s, 3H), 2.19 - 1.99 (m, 1H)

Preparation of compound 28 : 4-({1-[3-(isoquinolin-1-yl)prop-2-yn-1- yl]piperidin-4-yl}amino)-2H-chromen-2-one

Compound 28 was prepared as described hereinbelow.

Step 1 : 2-oxo-2H-chromen-4-yl trifluoromethanesulfonate (28a)

4-Hydroxycoumarine (1 g, 6.17 mmoles, 1 eq.) was dissolved in DCM (30 mL), triethylamine (1 .72 mL, 1 2.34 mmoles, 2 eq.) was added and the resulting mixture was cooled to -I CC. Trifluoromethanesulfonic anhydride (1 .25 mL, 7.4 mmoles, 1 .2 eq.) in DCM (5 mL) was added dropwise and the solution was left stirring at -10°C for 2 h. The resulting reddish brown solution was warmed to room temperature, diluted with cHex/Et₂0 1 /1 (50 mL) and filtered through a pad of silica gel using cHex/Et₂0 1 /1 as eluent. The solvent was removed in vacuum to obtain 2-oxo-2H-chromen-4-yl trifluoromethanesulfonate 28a (1 .76 g, Y= 97%). LC-MS (M-H⁺): 295.0.

Step 2: ieri-butyl 4-[(2-oxo-2H-chromen-4-yl)amino]piperidine-1 - carboxylate (28b)

Triethylamine (1 mL, 7.2 mmoles, 1 .2 eq.) in acetonitrile (2 mL) was added dropwise to a stirred solution of intermediate 28a (1 .76 g, 5.99 mmoles, 1 eq.) and 1 -Boc-4-aminopiperidine (1 .2 g, 5.99 mmoles, 1 eq.) in dry acetonitrile (20 mL). The mixture was heated at reflux for 2 h then was cooled to room temperature, diluted with DCM (100 mL) and washed with saturated NaHC0₃ (50 mL) and water (50 mL). The organic phase was separated, dried over sodium sulfate and evaporated in vacuum. The crude material was purified by trituration with methanol to obtain ieri-butyl 4-[(2-oxo-2H-chromen- 4-yl)amino]piperidine-1 -carboxylate 28b (1 .51 g, Y= 73%). LC-MS (M-H⁺): 345.2.

Step 3: 4-(piperidin-4-ylamino)-2H-chromen-2-one trifluoroacetic acid salt (28c)

Intermediate 28b (1 g, 2.90 mmoles, 1 eq.) was dissolved in DCM (10 ml) and TFA (3 mL) was added dropwise at 0°C. The solution was left stirring for 2 h then was concentrated in vacuum and washed with toluene (25 ml) and diethyl ether (25 ml) to obtain 4-(piperidin-4-ylamino)-2H-chromen-2-one trifluoroacetic acid salt 28c, which was used without any further purification (1 .22 g, Y= quant.). LC-MS (M-H⁺): 245.1 .

Step 4: 4-{[1 -(prop-2-yn-1 -yl)piperidin-4-yl]amino}-2H-chromen-2-one (28d)

To a solution of intermediate 28c (50 mg, 0.14 mmoles, 1 eq.) and potassium carbonate (38.7 mg, 0.28 mmoles, 2 eq.) in DMF (2 mL) 3-bromo- 1 -propyne (20.8 mg, 0.14 mmoles, 1 eq.) was added at room temperature. The reaction was left stirring overnight then was filtered and the filtrate concentrated. The residue was purified by Si-column eluting with eluting with EtOAc to EtOAc/Methanol 8:2 to give 4-{[1 -(prop-2-yn-1 -yl)piperidin-4- yl]amino}-2H-chromen-2-one 28d (25.6 mg, Y= 64%). LC-MS (M-H⁺): 283.1 . Step 5: 4-({1 -[3-(isoquinolin-1 -yl)prop-2-yn-1 -yl]piperidin-4-yl}amino)-2H- chromen-2-one (28)

Intermediate 28d (186 mg, 0.66 mmoles, 1 eq.), 1 -chloroisoquinoline (120 mg, 0.72 mmoles, 1 .1 eq.) and Cul (12 mg, 0.066 mmoles, 0.1 eq.) were dissolved in dry DMF (3.6 ml). DIPEA (0.473 ml, 2.64 mmoles, 4 eq.) was added. The mixture was degassed by alternatively applying vacuum and nitrogen, then bis(triphenylphosphine)palladium(ll) dichloride (45 mg, 0.066 mmoles, 0.1 eq.) was added and the mixture was heated at 60 °C. After 3 h water (10 mL) was added and the mixture was extracted with ethyl acetate (3x 10 mL). The organic phase was washed with brine, dried over Na₂S0₄, filtered and concentrated in vacuum to give 283 mg of crude material. After purification by Si-column eluting with EtOAc to EtOAc/MeOH 8:2 compound 4-({1 -[3-(isoquinolin-1 -yl)prop-2-yn-1 -yl]piperidin-4-yl}amino)-2H-chromen-2- one 28 (87mg, Y=32%) was obtained. LC-MS (M-H+): 410.2. ¹ H NMR (400MHz, METHANOL-d₄) δ = 8.42 - 8.34 (m, 2 H), 7.97 - 7.93 (m, 1 H), 7.89 (d, J = 7.8 Hz, 1 H), 7.79 - 7.66 (m, 3 H), 7.58 - 7.49 (m, 1 H), 7.28 - 7.20 (m, 2 H), 5.30 (s, 1 H), 3.78 (s, 2 H), 3.60 - 3.47 (m, 1 H), 3.13 (d, J = 1 2.0 Hz, 2 H), 2.59 (dt, J = 1 .8, 1 1 .8 Hz, 2 H), 2.1 1 (d, J = 1 1 .3 Hz, 2 H), 1 .79 (dq, J = 3.0, 12.0 Hz, 2 H).

Preparation of compound 31 : /V-{3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}furo[3,2-b]pyridine-6-carboxamide hydrochloride

Compound 31 was prepared as described hereinbelow.

Step 1 : ieri-butyl {3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl} carbamate

(31 a)

A mixture of 1 -(piperazin-1 -yl)isoquinoline 2c (1 .40 g, 6.54 mmoles. 1 eq.), ieri-butyl N-(3-bromopropyl)carbamate (1 .48 g, 6.22 mmoles, 0.95 eq.), potassium iodide (0.54 g, 3.27 mmoles, 0.50 eq.) and potassium carbonate (1 .81 g, 13.1 mmoles, 2 eq.) in DMF (20 ml_) was stirred at room temperature for 2 days. The reaction was partitioned between EtOAc (200 ml_) and brine (250 ml_). The organic phase was separated then was washed with brine (3x 200 ml_), dried over sodium sulfate and evaporated under reduced pressure. The residue was chromatographed on silica gel (SNAP 100) eluting with a gradient of 20-100% of A in cyclohexane, where A is MeOH/EtOAc (5:95), to give 2.10 g (Y=87%) of ieri-butyl {3-[4-(isoquinolin-1 -yl)piperazin-1 - yl]propyl}carbamate 31 a. LC-MS (M-H+) = 371 .2.

Step 2: 3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propan-1 -amine TFA salt (31 b)

Intermediate 31 a (6.35 g, 8.74 mmoles) was dissolved in DCM (25 ml_) and TFA (1 ml_) was added dropwise at 0°C. The solution was stirred for 2 h then was concentrated in vacuum, washed with toluene and diethyl ether to obtain 3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propan-1 -amine TFA salt 31 b (6.35 g, Y= 93%). LC-MS (M-H⁺) = 271 .2.

Step 3: A/-{3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}furo[3,2-b]pyridine-6- carboxamide hydrochloride (31 )

Intermediate 31 b (250 mg, 0.34 mmoles, 1 eq.) and commercially available furo[3,2-b]pyridine-6-carboxylic acid (56 mg, 0.34 mmoles, 1 eq.) were dissolved in dry DMF (2 mL). TEA (0.25 mL, 1 .7 mmoles, 5 eq.) was added, the mixture was cooled to 0°C and HATU (136 mg, 0.36 mmoles, 1 .05 eq.) was added. After stirring at room temperature for 2 h DCM was added, the organic phase was washed with sat. NaHC0₃ and brine and evaporated in vacuum. The crude material was purified by Si column eluting with EtOAc to EtOAc/MeOH 8:2 to obtain 100 mg of the desired compound. The product was dissolved in DCM (5 mL) and HCI (1 M solution in diethyl ether, 0.70 mL, 4 eq.) was added dropwise at 0°C. The solution was left stirring for 30 min then concentrated in vacuum and triturated with diethyl ether to obtain N-{3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}furo[3,2- b]pyridine-6-carboxamide hydrochloride 31 (120 mg, 0.23 mmoles, Y= 68%). LC-MS (M-H⁺) = 416.2.

1 H NMR (300 MHz, DMSO-d₆) δ = 1 1 .42 (br. s., 1 H), 9.09 (d, J = 1 .7 Hz, 1

H), 9.05 (t, J = 5.6 Hz, 1 H), 8.57 (s, 1 H), 8.51 (d, J = 2.4 Hz, 1 H), 8.23 (d, J = 8.7 Hz, 1 H), 8.10 - 8.00 (m, 2 H), 7.91 (t, J = 7.5 Hz, 1 H), 7.74 (ddd, J = 1 .0, 7.0, 8.4 Hz, 1 H), 7.61 (d, J = 6.3 Hz, 1 H), 7.25 (dd, J = 1 .0, 2.4 Hz, 1 H), 4.1 1 (d, J = 13.2 Hz, 2 H), 3.81 (t, J = 12.7 Hz, 2 H), 3.67 (d, J = 1 1 .5 Hz, 2 H), 3.55 - 3.36 (m, 4 H), 3.35 - 3.20 (m, 2 H), 2.1 1 (quin, J = 7.0 Hz, 2 H).

Preparation of compound 35: /V-{3-[4-(6-methylisoquinolin-1- yl)piperazin-1-yl]propyl}pyridine-3-carboxamide

Compound 35 was prepared according to the procedure described for compound 2, using in step 3, 1 -chloro-6-methylisoquinoline. LC-MS (M-H⁺): 390.2.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .44 (br. s., 1 H), 9.26 (br. s., 1 H), 9.22 (s, 1 H), 8.88 (d, J=4.8 Hz, 1 H), 8.59 (br. s., 1 H), 8.14 (d, J=8.5 Hz, 1 H), 7.98 (d, J=6.3 Hz, 1 H), 7.84 (s, 2H), 7.59 (d, J=8.5 Hz, 1 H), 7.53 (d, J=6.3 Hz, 1 H), 4.12 (d, J=14.3 Hz, 2H), 3.82 (t, J=1 1 .7 Hz, 2H), 3.66 (d, J=12.3 Hz, 2H), 3.50 - 3.35 (m, 4H), 3.32 - 3.21 (m, 2H), 2.54 (s, 3H), 2.08 (quin, J=7.3 Hz, 2H)

Preparation of compound 36: A/-{3-[4-(6-methoxyisoquinolin-1- yl)piperazin-1 -yl]propyl}pyridine-3-carboxamide hydrochloride

Compound 36 was prepared according to the procedure described for compound 2, using in step 3 1 -chloro-6-methoxyisoquinoline. LC-MS (M-H⁺): 406.2.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .03 (br. s., 1 H), 9.13 (d, J=1 .3 Hz, 1 H), 9.07 (t, J=5.0 Hz, 1 H), 8.80 (dd, J=1 .0, 5.0 Hz, 1 H), 8.40 (d, J=7.5 Hz, 1 H), 8.14 (d, J=9.3 Hz, 1 H), 7.98 (d, J=6.3 Hz, 1 H), 7.72 - 7.64 (m, 1 H), 7.52 (d, J=6.5 Hz, 1 H), 7.48 (s, 1 H), 7.32 (dd, J=2.3, 9.3 Hz, 1 H), 4.39 - 3.53 (m, 8H), 3.49 - 3.33 (m, 4H), 3.31 - 3.19 (m, 2H), 2.1 6 - 1 .95 (m, 2H)

Preparation of compound 38: /V-(2,5-difluorobenzyl)-3-[4-(isoquinolin- 1-yl)piperazin-1-yl]propan-1 -amine hydrochloride

Intermediate 31 b (0.21 mmoles, 1 eq.) was dissolved in dry DCE (3 ml_) and 2,5-difluorobenzaldehyde (32.8 mg, 0.23 mmoles, 1 .1 eq), DIPEA (0.146 ml_, 0.84, 4 eq.) and acetic acid (one drop) were added subsequently at room temperature. The mixture was stirred for 4 h at 60 °C. Sodium borohydride (1 2 mg, 0.315 mmoles, 1 .5 eq.) was added. The mixture was stirred at room temperature for 12 h then DCM was added and the mixture was washed with sat. NaHC0₃. The collected organic phases were evaporated in vacuum and the crude material was purified by Si-column eluting with ethyl acetate to ethyl acetate/MeOH 6:4 to afford the desired compound as free base. The product was dissolved in DCM then HCI (1 M solution in diethyl ether, 0.1 56 ml_, 3 eq.) was added dropwise at 0°C. The solution was left stirring for 2 h then was concentrated in vacuum and triturated with diethyl ether to obtain N-(2,5-difluorobenzyl)-3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propan-1 -amine hydrochloride 38 (26.6 mg, Y= 24%). LC-MS (M-H+): 397.2.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .00 (br. s., 1 H), 9.47 (br. s., 2H), 8.21 - 8.08 (m, 2H), 7.95 (d, J=8.0 Hz, 1 H), 7.77 (t, J=7.0 Hz, 1 H), 7.70 - 7.59 (m, 2H), 7.51 (d, J=5.8 Hz, 1 H), 7.45 - 7.32 (m, 2H), 5.75 (s, 1 H), 4.23 (t, J=5.4 Hz, 2H), 3.90 (d, J=12.8 Hz, 2H), 3.58 (br. s., 4H), 3.43 - 3.22 (m, 4H), 3.18 - 3.07 (m, 2H), 2.22 (quin, J=7.4 Hz, 2H) Preparation of compound 39: 2,5-difluoro-N-{3-[4-(isoquinolin-1- yl)piperazin-1 -yl]propyl}benzamide hydrochloride

Compound 39 was prepared according to the procedure described for compound 31 , using in step 3, 2,5-difluorobenzoic acid. LC-MS (M-H⁺):

41 1 .2.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 0.04 (br. s., 1 H), 8.63 - 8.53 (m, 1 H), 8.15 (d, J=5.8 Hz, 2H), 7.95 (d, J=8.0 Hz, 1 H), 7.76 (t, J=7.0 Hz, 1 H), 7.69 - 7.60 (m, 1 H), 7.55 - 7.45 (m, 2H), 7.44 - 7.32 (m, 2H), 3.88 (d, J=10.0 Hz, 2H), 3.63 (br. s., 3H), 3.46 - 3.32 (m, 6H), 3.26 (td, J=5.3, 1 0.7 Hz, 2H), 2.00 (quin, J=7.2 Hz, 2H)

Preparation of compound 42: /V-{3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}furo[2,3-c]pyridine-5-carboxamide hydrochloride

The compound was prepared according to the procedure described for compound 31 , using in step 3 furo[2,3-c]pyridine-5-carboxylic acid. LC-MS (M-H⁺): 489.2.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 0.24 (br. s., 1 H), 9.07 (t, J=5.9 Hz, 1 H), 9.03 (s, 1 H), 8.43 (d, J=0.8 Hz, 1 H), 8.36 (d, J=2.3 Hz, 1 H), 8.15 (d, J=8.3 Hz, 1 H), 8.12 (d, J=5.8 Hz, 1 H), 7.96 (d, J=8.3 Hz, 1 H), 7.79 (t, J=7.4 Hz, 1 H), 7.66 (t, J=7.4 Hz, 1 H), 7.52 (d, J=5.8 Hz, 1 H), 7.23 (d, J=1 .5 Hz, 1 H), 3.91 (d, J=1 1 .5 Hz, 2H), 3.60 (br. s., 4H), 3.50 - 3.33 (m, 5H), 3.31 - 3.16 (m, 2H), 2.16 - 1 .95 (m, 2H)

Preparation of compound 43: /V-{3-[4-(1 ,6-naphthyridin-5-yl)piperazin- 1 -yl]propyl}pyridine-3-carboxamide hydrochloride

Compound 43 was prepared according to the procedure described for compound 2, using in step 3 5-chloro-1 ,6-naphthyridine. LC-MS (M-H⁺): 377.2.

¹ H NMR (400MHz, DMSO-d₆) δ = (CHLOROFORM-d) 9.04 (d, J=2.0 Hz, 1 H), 9.00 (dd, J=1 .6, 4.1 Hz, 1 H), 8.71 (dd, J=1 .8, 4.8 Hz, 1 H), 8.39 - 8.33 (m, 2H), 8.22 (br. s., 1 H), 8.1 7 (td, J=1 .9, 8.0 Hz, 1 H), 7.52 (d, J=5.8 Hz, 1 H), 7.43 (dd, J=4.3, 8.5 Hz, 1 H), 7.39 (dd, J=4.9, 7.9 Hz, 1 H), 3.65 (q, J=5.8 Hz, 2H), 3.56 - 3.41 (m, 4H), 2.81 (br. s., 4H), 2.73 (t, J=5.8 Hz, 2H), 1 .91 (quin, J=6.0 Hz, 2H)

Preparation of compound 45: A/-{3-[4-(6-fluoroisoquinolin-1- yl)piperazin-1 -yl]propyl}pyridine-3-carboxamide hydrochloride Compound 45 was prepared according to the procedure described for compound 2, using in step 3 1 -chloro-6-fluoroisoquinoline. LC-MS (M-H⁺): 394.2.

¹ H NMR (300 MHz, DMSO-d₆) δ = 9.40 - 9.32 (m, 1 H), 9.28 (s, 1 H), 8.93 (d, J = 5.3 Hz, 1 H), 8.72 (d, J = 7.6 Hz, 1 H), 8.30 (dd, J = 5.6, 9.1 Hz, 1 H), 8.09 (d, J = 6.1 Hz, 1 H), 7.99 - 7.89 (m, 1 H), 7.84 (dd, J = 2.2, 9.5 Hz, 1 H),

7.63 - 7.52 (m, 2 H), 4.02 (d, J = 13.4 Hz, 2 H), 3.75 (t, J = 12.6 Hz, 2 H),

3.64 (d, J = 12.0 Hz, 2 H), 3.43 (ddd, J = 5.7, 5.8, 1 1 .8 Hz, 4 H), 3.34 - 3.20 (m, 2 H), 2.20 - 1 .97 (m, 2 H).

Preparation of compound 50

Compound 50 was prepared as described herein below.

Step 1 - Synthesis of ferf-butyl N-{3-[4-(naphthalen-1 -yl)piperazin-1 - yl]propyl}carbamate:

A mixture of commercial 1 -(naphthalen-1 -yl)piperazine hydrochloride (200 mg, 0.80 mmol), ieri-butyl N-(3-bromopropyl)carbamate (182 mg, 0.76 mmol), potassium iodide (66 mg, 016 mmol) and potassium carbonate (221 mg, 1 .6 mmol) in DMF (3 mL) was stirred 18 h at room temperature. The reaction mixture was partitioned between EtOAc (50 mL) and brine (50 mL), the organic phase was washed with brine (50 mL), dried over sodium sulfate and evaporated under reduced pressure. The residue was chromatographed on silica gel (SNAP 25) eluting with a gradient of 30-100% EtOAc in cyclohexane to give 263 mg (0.71 mmol, 94% yield) of the title compound. LC-MS (M-H⁺) = 370.4

Step 2 - Synthesis of 3-[4-(naphthalen-1 -yl)piperazin-1 -yl]propan-1 -amine:

TFA (2 mL) was added to a solution of ferf-butyl N-{3-[4-(naphthalen-1 - yl)piperazin-1 -yl]propyl}carbamate (261 mg, 071 mmol) in dichloromethane (6 mL). The resulting mixture was stirred 1 h at room temperature then the volatiles were evaporated under reduced pressure. The residue was dissolved in dichloromethane (10 mL) and evaporated under reduced pressure twice. The resulting residue was dissolved in MeOH (4 mL) and loaded onto a preconditioned SCX cartridge (5g) eluting with MeOH and with a 2M solution of ammonia in methanol. The basic fractions were dried under reduced pressure to give 193 mg (0.71 mmol, quant.) of the title compound as a white solid. LC-MS (M-H⁺) = 270.3

Step 3 - Synthesis of 4-({3-[4-(naphthalen-1 -yl)piperazin-1 - yl]propyl}amino)-2H-chromen-2-one hydrochloride (compound 50):

Compound 50 was prepared according to the procedure described for the synthesis of 53 (step 3, Y=21 %). LC-MS (M-H+) = 414.2

¹ H NMR (400 MHz, DMSO-d₆) δ = 10.79 (br. s., 1 H), 8.19 - 8.08 (m, 2 H), 7.98 - 7.87 (m, 2 H), 7.67 (d, J = 8.0 Hz, 1 H), 7.61 (t, J = 7.3 Hz, 1 H), 7.57 - 7.50 (m, 2 H), 7.46 (t, J = 7.9 Hz, 1 H), 7.34 (t, J = 7.7 Hz, 1 H), 7.32 (d, J = 8.3 Hz, 1 H), 7.1 8 (d, J = 7.3 Hz, 1 H), 5.28 (s, 1 H), 3.64 (d, J = 1 0.0 Hz, 2 H), 3.53 - 3.1 9 (m, 10 H), 2.1 6 (quin, J = 7.0 Hz, 2 H).

Preparation of compound 53: 4-[{3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}(methyl)amino]-2H-chromen-2-one hydrochloride

Compound 53 was prepared as described hereinbelow.

Step 1 : ieri-butyl {3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}

methylcarbamate (53a)

53a 53b 53 Sodium hydride (24 mg, 60% dispersion in mineral oil, 0.61 mmoles, 1 .5 eq.) was added to a stirred solution of intermediate 31 a (150 mg, 0.41 mmoles, 1 eq.) in DMF (2 mL) at room temperature. The mixture was stirred for 10 minutes then iodomethane (38 μί, 0.61 mmoles, 1 .5 eq.) was added. Stirring was continued for 2 hours then the reaction was quenched with water (50 mL) and extracted with EtOAc (50 mL). The organic phase was washed with brine (50 mL), dried over sodium sulfate and evaporated under reduced pressure. The residue was chromatographed on NH-modified silica gel (2x SNAP 1 1 in series) eluting with a gradient of 10-50% EtOAc in cyclohexane to give 78 mg of ferf-butyl {3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}methyl carbamate 53a (Y=50%). LC-MS (M-H+) = 385.4.

Step 2: 3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]-N-methylpropan-1 -amine (53b)

TFA (1 mL) was added to a solution of intermediate 53a (78 mg, 0.2 mmoles, 1 eq.) in dichloromethane (3 mL) at room temperature and the resulting mixture was stirred for 60 minutes. The volatiles were evaporated under reduced pressure then the residue was dissolved in MeOH (2 mL) and loaded onto a preconditioned SCX cartridge (1 g). The SCX was eluted with MeOH and then a 2M solution of ammonia in methanol. The basic fractions were evaporated under reduced pressure to give 56 mg of 3-[4-(isoquinolin- 1 -yl)piperazin-1 -yl]-N-methylpropan-1 -amine 53b (Y=quant.). LC-MS (M-H+) = 285.3.

Step 3: 4-[{3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}(methyl)amino]-2H- chromen-2-one hydrochloride (53)

A solution of intermediate 53b (56 mg, 0.2 mmoles, 1 .1 eq.), triethylamine

(37 μί, 0.27 mmoles, 1 .5 eq.) and 2-oxo-2H-chromen-4-yl tnfluoromethanesulfonate 28a (53 mg, 0.18 mmoles, 1 eq.) in acetonitrile (2 mL) was heated to 70⁵C for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between dichloromethane (10 mL) and a brine/sodium bicarbonate mixture (1 :1 , 10 mL). The mixture was filtered through a hydrophobic frit (Phase Separator) washing with dichloromethane (1 0 mL). The organic phase was evaporated under reduced pressure and the residue was chromatographed on NH- modified silica gel (2x SNAP 1 1 in series) eluting with a gradient of 20-100% EtOAc in cyclohexane to give 79 mg of a colorless sticky gum. The product was dissolved in dichloromethane (3 mL) and treated with 1 M HCI solution in diethyl ether (0.46 mL) causing precipitation. The resulting mixture was evaporated under reduced pressure and the residue was triturated with diethyl ether. The solids were dried to give 85 mg of 4-[{3-[4-(isoquinolin-1 - yl)piperazin-1 -yl]propyl}(methyl)amino]-2H-chromen-2-one hydrochloride 53 as a white solid (Y = 95%). LC-MS (M-H+) = 429.2.

¹ H NMR (400MHz, DMSO-d₆) δ = 8.19 (d, J = 8.3 Hz, 1 H), 8.12 (d, J = 5.8 Hz, 1 H), 8.00 (d, J = 8.0 Hz, 1 H), 7.88 - 7.80 (m, 1 H), 7.71 (t, J = 7.5 Hz, 1 H), 7.63 (t, J = 7.5 Hz, 1 H), 7.57 (d, J = 5.8 Hz, 1 H), 7.43 - 7.34 (m, 1 H), 5.66 (s, 1 H), 3.98 (d, J = 13.8 Hz, 2 H), 3.73 - 3.53 (m, 4 H), 3.51 - 3.35 (m, 4 H), 3.31 - 3.14 (m, 2 H), 3.08 (s, 3 H), 2.24 (br. quin, J = 6.5 Hz, 2 H).

Preparation of compound 57: 4-({3-[4-(isoquinolin-1-yl)piperazin-1-yl]- 3-oxopropyl}amino)-2H-chromen-2-one hydrochloride

Compound 57 was prepared as described hereinbelow.

57a 57b Step 1 : ferf-butyl {3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]-3- oxopropyl}carbamate (57a)

1 -(piperazin-1 -yl)isoquinoline 2c (100 mg, 0.46 mmoles, 1 eq.) was added to a stirred solution of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (104 mg, 0.54 mmoles, 1 .2 eq.), 1 -hydroxybenzotriazole hydrate (72 mg, 0.54 mmoles, 1 .2 eq.), 3-{[(tert- butoxy)carbonyl]amino}propanoic acid (86 mg, 0.46 mmoles, 1 eq.) and TEA (1 90 μΙ, 0.1 38 mmoles, 3 eq.) in DCM (5 mL) at room temperature. The resulting mixture was stirred overnight then the organic phase was washed with sodium bicarbonate solution, filtered through a hydrophobic frit (Phase Separator) and evaporated under reduce pressure. The residue was chromatographed on silica gel (SNAP 10) eluting with a gradient of 1 0-100% EtOAc in cyclohexane to give 148 mg of tert-butyl {3-[4-(isoquinolin-1 - yl)piperazin-1 -yl]-3-oxopropyl} carbamate 57a (Y=85%). LC-MS (M-H+) = 385.4.

Step 2: 3-amino-1 -[4-(isoquinolin-1 -yl)piperazin-1 -yl]propan-1 -one (57b)

Intermediate 57b was prepared according to the procedure described for the synthesis of intermediate 53b in the step 2 (Y=92%). LC-MS (M-H+) = 285.3.

Step 3: 4-({3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]-3-oxopropyl}amino)-2H- chromen-2-one hydrochloride (57)

Compound 57 was prepared according to the procedure described for the synthesis of 53 in step 3 (Y=87%). LC-MS (M-H+) = 429.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 8.26 (d, J=8.5 Hz, 1 H), 8.1 1 - 8.00 (m, 2H), 7.98 - 7.87 (m, 2H), 7.80 - 7.68 (m, 2H), 7.63 - 7.56 (m, 1 H), 7.54 (d, J=6.5 Hz, 1 H), 7.38 - 7.25 (m, 2H), 5.19 (s, 1 H), 4.1 5 (br. s., 1 H), 3.87 - 3.62 (m, 8H), 3.55 (q, J=6.4 Hz, 2H), 2.82 (t, J=6.8 Hz, 2H)

Preparation of compound 58: 4,4-({3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}amino)quinolin-2(1 H)-one hydrochloride

Compound 58 was prepared as described hereinbelow.

58

Step 1 : 2-OXO-1 ,2-dihydroquinolin-4-yl trifluoromethanesulfonate (58a)

1 ,1 , 1 -Trifluoro-N-phenyl-N-(trifluoromethane)sulfonylmethane

sulfonamide (1 .06 g, 2.98 mmoles, 1 .2 eq.) was added to a stirred suspension of 4-hydroxy-1 ,2-dihydroquinolin-2-one (0.4 g, 2.48 mmoles, 1 eq.) and triethylamine (1 .04 mL, 7.44 mmoles, 3 eq.) in DMF (15 mL) at room temperature. The reaction was stirred for 2 hours then was poured into water (1 00 mL) and extracted with EtOAc (60 mL). The organic phase was washed with brine (50 mL), dried over sodium sulphate and evaporated under reduced pressure. The residue was absorbed onto silica gel (10 g) and placed on top of a silica gel column (SNAP 50) which was eluted with 50- 1 00% EtOAc in cyclohexane to give 537 mg (1 .83 mmoles, Y=74%) of 2-oxo- 1 ,2-dihydroquinolin-4-yl trifluoromethanesulfonate 58a as a white solid. LC- MS (M-H+) = 294.1 .

Step 2: 4,4-({3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}amino) quinolin- 2(1 H)-one hydrochloride (58)

Compound 58 was prepared according to the procedure described for the synthesis of compound 53 (Y=28%), using in step 3 intermediates 58a and 31 b. LC-MS (M-H+) = 414.4.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 2.1 6 (br. s., 1 H), 1 1 .06 (br. s., 1 H), 8.27 (br. s., 1 H), 8.18 (d, J=8.3 Hz, 1 H), 8.1 0 (d, J=5.3 Hz, 1 H), 7.99 (d, J=7.8 Hz, 1 H), 7.89 - 7.78 (m, 1 H), 7.74 - 7.61 (m, 2H), 7.60 - 7.45 (m, 2H), 7.35 (d, J=5.8 Hz, 1 H), 5.82 (br. s., 1 H), 5.02 (br. s., 2H), 3.97 (d, J=1 1 .5 Hz, 2H), 3.79 - 3.55 (m, 4H), 3.53 - 3.23 (m, 6H), 2.1 9 (br. s., 2H)

Preparation of compound 60: 4-({3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}amino)-6-phenyl-2H-pyran-2-one hydrochloride

Compound 60 was prepared as described hereinbelow.

60 Step 1 : 4-hydroxy-6-phenyl-2H-pyran-2-one (60a)

Malonyl dichloride (0.25 mL, 2.6 mmoles, 1 eq.) was added dropwise to a stirred solution of trimethyl[(1 -phenylethenyl)oxy]silane (1 .07 mL, 5.2 mmoles, 2 eq.) in diethyl ether (16 mL) at -78 ⁵C. The reaction mixture was stirred overnight allowing the reaction to gradually warm to room temperature. The precipitated solid was collected, washed with diethyl ether (20 mL) and dried under vacuum to give 0.34 g of 4-hydroxy-6-phenyl-2H- pyran-2-one 60a (1 .8 mmoles, Y=70%), which was progressed without any further purification. LC-MS (M-H+) = 189.1 .

Step 2: 2-oxo-6-phenyl-2H-pyran-4-yl trifluoromethanesulfonate (60b)

Intermediate 60b was prepared according to the procedure described for the synthesis of 28a, using in step 1 intermediate 60a (Y=77%). LC-MS (M- H+) = 321 .1 .

Step 3: 4-({3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)-6-phenyl-2H- pyran-2-one hydrochloride (60)

Compound 60 was prepared according to the procedure described for the synthesis of 53, using in step 3 intermediates 60b and 31 b (Y=37%). LC-MS (M-H+) = 441 .4.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .05 (br. s., 1 H), 8.19 (d, J=8.3 Hz, 1 H), 8.10 (d, J=5.8 Hz, 1 H), 7.99 (d, J=7.8 Hz, 1 H), 7.83 (t, J=7.2 Hz, 1 H), 7.70 (dd, J=6.7, 14.7 Hz, 4H), 7.61 - 7.46 (m, 4H), 6.60 (br. s., 1 H), 5.62 (br. s., 1 H), 5.03 (d, J=1 .3 Hz, 1 H), 3.98 (d, J=1 1 .8 Hz, 2H), 3.64 (d, J=1 1 .5 Hz, 4H), 3.49 - 3.35 (m, 2H), 3.28 (br. s., 4H), 2.15 - 2.00 (m, 2H)

Preparation of compound 68: 4-(4-{3-[(2-oxo-2H-chromen-4- yl)amino]propyl} piperazin-1 -yl)quinolin-2(1 H)-one hydrochloride

Compound 68 was prepared as described hereinbelow.

Step 1 : ieri-butyl 4-[3-(1 ,3-dioxo-1 ,3-dihydro-2H-isoindol-2- yl)propyl]piperazine-1 -carboxylate (68a)

N-(3-Bromopropyl)phthalimide (2.1 g, 7.66 mmoles, 1 eq.), 1 -Boc- piperazine (1 .5 g, 8 mmoles, 1 eq.), potassium iodide (2.54 g, 15.3 mmoles, 2 eq.) and potassium carbonate (1 .76g, 12.7 mmoles, 1 .7 eq.) were dissolved in DMA (15 mL) and stirred at room temperature for 16h. The solvent was evaporated and the residue was dissolved in DCM. The salts were removed by filtration and the filtrate, after solvent evaporation, was purified by silica column (cHex: EtOAc 8:2 to cHex: EtOAc 1 :1 ). The desired product ieri-butyl 4-[3-(1 ,3-dioxo-1 ,3-dihydro-2H-isoindol-2- yl)propyl]piperazine-1 -carboxylate 68a was obtained as a pale yellow crystalline solid (2.8 g, Y=quant.). LC-MS (M-H+) = 374.1 .

Step 2: ieri-butyl 4-(3-aminopropyl)piperazine-1 -carboxylate (68b)

Intermediate 68a (1 .9 g, 5.08 mmoles) was dissolved in methylamine (33% in absolute ethanol, 20 mL) and heated at 40 °C for 4 h. The solvent was evaporated, the residue was dissolved in diethyl ether and filtered and the filtrate was evaporated to give ieri-butyl 4-(3-aminopropyl)piperazine-1 - carboxylate (68b) as a colourless oil (1 .1 g, Y=89%). LC-MS (M-H⁺) = 244.3.

Step 3: ieri-butyl 4-{3-[(2-oxo-2H-chromen-4-yl)amino]propyl}

piperazine-1 -carboxylate (68c).

A solution of intermediate 68b (1 .1 g, 4.5 mmoles, 1 .1 eq.), triethylamine (0.674 mL, 4.8 mmoles, 1 .2 eq.) and 2-oxochromen-4-yl trifluoromethanesulfonate 28a (1 .2 g, 4.1 mmoles, 1 eq.) in acetonitrile (20 mL) was heated to 70⁵C for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between dichloromethane and a brine/sodium bicarbonate mixture (1 :1 ). The mixture was filtered through a hydrophobic frit (Phase Separator) washing with dichloromethane. The organic phase was evaporated under reduced pressure and the residue was chromatographed on silica gel (SNAP50) eluting with a gradient of EtOAc in cyclohexane to give ferf-butyl 4-{3-[(2-oxo- 2H-chromen-4-yl)amino]propyl}piperazine-1 -carboxylate 68c (700 mg, Y=44%). LC-MS (M-H⁺) = 388.3.

Step 4: 4-{[3-(piperazin-1 -yl)propyl]amino}chromen-2-one (68d)

TFA (2 mL) was added to a solution of intermediate 68c (700 mg, 1 .8 mmoles) in dichloromethane (6 mL) at room temperature and the resulting mixture stirred for 20 minutes. The residue was evaporated under reduced pressure, dissolved in MeOH and loaded onto a preconditioned SCX cartridge. The SCX was eluted with MeOH and then a 2M solution of ammonia in methanol. The basic fractions were evaporated under reduced pressure to give 4-{[3-(piperazin-1 -yl)propyl]amino}chromen-2-one 68d (456 mg, Y=88%). LC-MS (M-H⁺) = 288.3.

Step 5: 4-(4-{3-[(2-oxo-2H-chromen-4-yl)amino]propyl}piperazin-1 - yl)quinolin-2(1 H)-one hydrochloride (68)

A solution of intermediate 68d (70 mg, 024 mmoles, 1 .1 eq.), triethylamine (35 uL, 0.26 mmoles, 1 .2 eq.) and intermediate 58a (71 mg, 0.22 mmoles, 1 eq.) in DMSO (5 mL) was heated to 80 ⁵C for 1 hour. The mixture was treated with water, the resulting solid was filtered, dissolved in dichloromethane (5 mL) and treated with HCI (1 M solution in diethyl ether, 0.630 mL) causing precipitation. The resulting mixture was evaporated under reduced pressure and the residue was triturated with diethyl ether. The solid was dried to give 1 13 mg of 4-(4-{3-[(2-oxo-2H-chromen-4- yl)amino]propyl}piperazin-1 -yl)quinolin-2(1 H)-one hydrochloride 68 as a white solid (Y=quant.). LC-MS (M-H⁺) = 431 .3.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .46 (br. s., 1 H), 1 1 .20 (br. s., 1 H), 8.18 (d, J=7.5 Hz, 1 H), 7.98 (br. s., 1 H), 7.69 (d, J=8.0 Hz, 1 H), 7.60 (t, J=7.3 Hz, 1 H), 7.49 (t, J=7.4 Hz, 1 H), 7.32 (d, J=7.5 Hz, 3H), 7.1 6 (t, J=7.3 Hz, 1 H), 5.95 (s, 1 H), 5.26 (s, 1 H), 3.78 - 3.1 9 (m, 1 3H), 2.1 5 (br. s., 2H)

Preparation of compound 70: 4-{3-[4-(isoquinolin-1 -yl)piperazin-1- yl]propoxy}-2H-chromen-2-one hydrochloride Com ound 70 was prepared as described hereinbelow.

Step 1 : 3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propan-1 -ol (70a):

A mixture of potassium carbonate (634 mg, 4.6 mmoles, 1 .5 eq.), 1 - chloroisoquinoline (500 mg, 3.1 mmoles, 1 eq.) and 3-(piperazin-1 -yl)propan- 1 -ol (881 mg, 6.1 mmoles, 2 eq.) in DMSO (4 mL) was heated to 120 °C under microwave irradiation for 5 hours. The reaction was allowed to cool to room temperature. The solid was filtered, washed with water then dried under reduced pressure to give 690 mg of 3-[4-(isoquinolin-1 -yl)piperazin-1 - yl]propan-1 -ol 70a (Y=81 %). LC-MS (M-H⁺) = 272.2.

Step 2: 4-{3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propoxy}-2H-chromen-2-one hydrochloride (70)

Methanesulfonyl chloride (57 μΙ_, 0.74 mmoles, 2 eq.) was added to a stirred solution of intermediate 70a (100 mg, 0.37 mmoles, 1 eq.) and TEA (206 μΙ_, 1 .48 mmoles, 4 eq.) in dichloromethane (10 mL) at 0 °C. The reaction was stirred for 30 minutes then was left to warm to room temperature. The reaction mixture was washed with saturated aqueous NaHC0₃ solution and water. The organic phase was dried over Na₂S0₄, filtered and concentrated under reduced pressure. The intermediate was used without any further purification for the following reaction.

Sodium hydride (60% dispersion in mineral oil, 15 mg, 0.37 mmoles, 1 eq.) was added to a stirred solution of 4-hydroxy-2H-chromen-2-one (60 mg, 0.37 mmoles, 1 eq.) in acetonitrile (4 mL). The suspension was stirred at room temperature for 30 minutes then was added to a solution of the previously obtained intermediate in acetonitrile (4 mL). K₂C0₃ (76 mg, 0.55 mmoles, 1 .5 eq.) was added and the mixture was heated to 80 °C for 2h. The volatiles were removed under reduced pressure, the residue was dissolved in dichloromethane and washed with water and brine. The organic phase was dried over Na₂S0₄, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with a gradient of mixture A in dichloromethane, where A is MeOH/NH₄OH 95/5, to give 72 mg of a white foam. The product was dissolved in dichloromethane (4 mL) and the minimum amount of MeOH then was treated with HCI (1 M solution in diethyl ether, 0.44 mL) causing precipitation. The solvents were evaporated under reduced pressure and the residue was triturated with diethyl ether. The resulting solid was dried to give 92 mg of 4-{3-[4-(isoquinolin-1 -yl)piperazin-1 - yl]propoxy}-2H-chromen-2-one hydrochloride 70 (Y=51 %). LC-MS (M-H⁺) = 416.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .41 (br. s., 1 H), 8.20 (d, J=8.5 Hz, 1 H), 8.10 (d, J=6.0 Hz, 1 H), 8.00 (d, J=8.3 Hz, 1 H), 7.95 (dd, J=1 .4, 7.9 Hz, 1 H), 7.85 (t, J=7.4 Hz, 1 H), 7.74 - 7.66 (m, 2H), 7.57 (d, J=6.0 Hz, 1 H), 7.45 - 7.37 (m, 2H), 5.95 (s, 1 H), 5.36 (br. s., 1 H), 4.38 (t, J=5.6 Hz, 2H), 4.01 (d, J=13.1 Hz, 2H), 3.79 - 3.59 (m, 4H), 3.55 - 3.36 (m, 4H), 2.45 - 2.35 (m, 2H)

Preparation of compound 71 : 4-(isoquinolin-1-yl)-1-{3-[(2-oxo-2H- chromen-4-yl)amino]propyl}piperazin-2-one hydrochloride

Com ound 71 was prepared as described hereinbelow.

Step 1 : 4-(isoquinolin-1 -yl)piperazin-2-one (71 a)

Intermediate 71 a was prepared according to the procedure described in the step 1 of the synthesis of compound 70, using piperazin-2-one (Y=77%). LC-MS (M-H⁺) = 228.1 . Step 2: ieri-butyl {3-[4-(isoquinolin-1 -yl)-2-oxopiperazin-1 -yl]

propyl}carbamate (71 b)

NaH (60% dispersion in mineral oil, 81 mg, 2 mmoles, 1 .1 eq.) and ieri- butyl N-(3-bromopropyl)carbamate (330 mg, 1 .4 mmoles, 0.75 eq.) were added to a stirred solution of intermediate 71 a (420 mg, 1 .8 mmoles, 1 eq.) in anhydrous DMF (6 ml_). The mixture was stirred for 4 hours then was quenched with water and extracted with ethyl acetate. The organic phase was dried over sodium sulphate and chromatographed on silica eluting with a gradient of 50-100% EtOAc in petroleum ether to give 273 mg of ieri-butyl {3- [4-(isoquinolin-1 -yl)-2-oxopiperazin-1 -yl]propyl}carbamate 71 b (Y=51 %). LC- MS (M-H+) = 385.2.

Step 3: 1 -(3-aminopropyl)-4-(isoquinolin-1 -yl)piperazin-2-one (71 c)

Intermediate 71 c was prepared according to the procedure described in step 2 of the synthesis of compound 53 (Y=quant.). LC-MS (M-H⁺) = 285.3. Step 4: 4-(isoquinolin-1 -yl)-1 -{3-[(2-oxo-2H-chromen-4- yl)amino]propyl}piperazin-2-one hydrochloride (71 )

Compound 71 was prepared according to the procedure described in step 3 of the synthesis of compound 53 (Y=80%). LC-MS (M-H⁺) = 429.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 8.23 (d, J=8.0 Hz, 1 H), 8.05 (d, J=8.0 Hz, 1 H), 8.02 - 7.93 (m, 2H), 7.87 (t, J=6.9 Hz, 1 H), 7.76 - 7.63 (m, 2H), 7.61

- 7.53 (m, 1 H), 7.50 (d, J=6.3 Hz, 1 H), 7.35 - 7.23 (m, 2H), 5.1 9 (s, 1 H), 5.04

- 4.38 (m, 1 H), 4.24 (br. s., 2H), 3.92 (br. s., 2H), 3.60 (br. s., 2H), 3.51 (t, J=7.0 Hz, 2H), 3.30 (q, J=7.0 Hz, 2H), 1 .93 (quin, J=7.0 Hz, 2H)

Preparation of compound 74: 4-({3-[4-(isoquinolin-1 -yl)piperazin-1- yl]cyclohexyl}amino)-2H-chromen-2-one hydrochloride

Compound 74 was prepared as described hereinbelow.

Step 1 : ieri-butyl {3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]cyclohexyl} carbamate (74a)

Sodium triacetoxyborohydride (591 mg, 2.8 mmoles,3 eq.) was added to a stirred solution of 1 -(piperazin-1 -yl)isoquinoline 2c (200 mg, 0.93 mmoles, 1 eq.) and ieri-butyl N-(3-oxocyclohexyl)carbamate (200 mg, 0.93 mmoles, 1 eq.) in dichloromethane (5 ml_). The reaction was stirred at room temperature for 2 hours then was diluted with DCM and quenched with water. The mixture was filtered through a hydrophobic frit (Phase Separator). The organic phase was washed with a mixture of brine and sodium bicarbonate solution (1 :1 ) and filtered through a hydrophobic frit (Phase Separator). The organic phase was evaporated under reduced pressure. The residue was chromatographed on silica gel ( SNAP 1 0) eluting with a gradient of EtOAc in cyclohexane to give 245 mg of ieri-butyl {3-[4-(isoquinolin-1 -yl)piperazin-1 - yl]cyclohexyl}carbamate 74a (Y=63%). LC-MS (M-H+) = 41 1 .4.

Step 2: 3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]cyclohexanamine (74b)

Intermediate 74b was prepared according to the procedure described in step 2 of the synthesis of compound 53 (Y=quant.). LC-MS (M-H⁺) = 31 1 .3.

Step 3: 4-({3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]cyclohexyl}amino)-2H- chromen-2-one hydrochloride (74)

Compound 74 was prepared according to the procedure described in step

3 of the synthesis of compound 53 (Y=87%). LC-MS (M-H+) = 455.4.

¹ H NMR (500 MHz, DMSO-d₆) δ ppm 1 .60 - 1 .76 (m, 2 H), 1 .76 - 1 .89 (m, 3 H), 1 .92 - 2.01 (m, 1 H), 2.13 - 2.22 (m, 1 H), 2.42 (d, J=12.72 Hz, 1 H), 3.36 - 4.06 (m, 8 H), 3.76 - 3.84 (m, 1 H), 4.24 (br. s., 1 H), 5.31 (s, 1 H), 6.97 (d, J=6.36 Hz, 1 H), 7.28 - 7.38 (m, 2 H), 7.55 (d, J=5.90 Hz, 1 H), 7.58 - 7.64 (m, 1 H), 7.69 (t, J=7.58 Hz, 1 H), 7.83 (t, J=7.60 Hz, 1 H), 7.99 (d, J=7.60 Hz, 1 H), 8.08 (d, J=5.87 Hz, 1 H), 8.21 (d, J=7.60 Hz, 1 H), 8.31 (d, J=7.34 Hz, 1 H), 10.88 (br. s., 1 H)

Preparation of compound 75: N-{2-[4-(isoquinolin-1-yl)piperazin-1- yl]ethyl}-2-oxo-2H-chromene-4-carboxamide hydrochloride

Compound 75 was prepared as described hereinbelow.

75b

Step 1 : 2-oxo-2H-chromene-4-carboxylic acid (75a)

Palladium dichloride (80 mg, 0.45 mmoles, 0.1 eq.) was added to a stirred mixture of 2-iodophenol (1 g, 4.5 mmoles, 1 eq.), dimethyl maleate (1 .71 mL, 1 3.6 mmoles, 3 eq.) and triethylamine (1 .90 mL, 1 3.6 mmoles, 3 eq.) in water (40 mL). The reaction mixture was heated to 85⁵C for 24 hours. The reaction mixture was acidified with 1 M HCI solution and extracted repeatedly with dichloromethane. The combined organic phases were filtered through a hydrophobic frit (Phase Separator) and evaporated under reduced pressure. The residue was chromatographed on RP-18 (SNAP 60) eluting with a gradient of 2-100% MeCN in water. The MeCN was removed under reduced pressure then the aqueous phase was basified withl M NaOH solution and washed with diethyl ether (2x 50 mL). The aqueous phase was acidified with 1 M HCI solution and extracted repeatedly with dichloromethane. The combined organic phases were filtered through a hydrophobic frit (Phase Separator) and evaporated under reduced pressure to give 228 mg of 2-oxo- 2H-chromene-4-carboxylic acid 75a as a pale yellow solid (Y=26%). LC-MS (M-H+) = 1 91 .0.

Step 2: 2-[4-(isoquinolin-1 -yl)piperazin-1 -yl]ethanamine (75b)

Intermediate 75b was prepared as described in step 2 of the synthesis of compound 68 using A/-(2-bromoethyl)phthalimide (Y=47%). LC-MS (M-H⁺) = 257.2.

Step 3: N-{2-[4-(isoquinolin-1 -yl)piperazin-1 -yl]ethyl}-2-oxo-2H-chromene- 4-carboxamide hydrochloride (75):

Compound 75 (59 mg, Y=35%) was prepared following the conditions described in step 1 of the synthesis of compound 57. LC-MS (M-H⁺) = 429.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .25 (br. s., 1 H), 9.33 (br. s., 1 H), 8.20 (d, J=8.3 Hz, 1 H), 8.1 1 (d, J=5.8 Hz, 1 H), 8.00 (d, J=8.0 Hz, 1 H), 7.90 - 7.79 (m, 2H), 7.69 (q, J=8.1 Hz, 2H), 7.56 (d, J=5.8 Hz, 1 H), 7.47 (d, J=8.3 Hz, 1 H), 7.39 (t, J=7.5 Hz, 1 H), 6.86 (s, 1 H), 4.61 - 3.04 (m, 13H)

Preparation of compound 81 : N-{2-[4-(isoquinolin-1-yl)piperazin-1- yl]ethyl}-2-oxo-2H-chromene-4-carboxamide hydrochloride

Compound 81 was prepared as described hereinbelow.

Compound 81 was prepared as described for compound 71 starting from methyl piperazine-2-carboxylate. LC-MS (M-H⁺) = 473.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 8.14 (d, J=5.8 Hz, 1 H), 8.08 (d, J=6.8 Hz, 2H), 7.85 - 7.75 (m, 2H), 7.71 - 7.64 (m, 1 H), 7.63 - 7.57 (m, 1 H), 7.54 (d, J=5.8 Hz, 1 H), 7.38 - 7.28 (m, 2H), 5.27 (s, 1 H), 4.64 (br. s., 7H), 4.12 - 3.56 (m, 6H), 3.38 (d, J=6.8 Hz, 4H), 2.24 - 1 .92 (m, 2H) Preparation of compound 82: 4-(isoquinolin-1 -yl)-1 -{3-[(2-oxo-2H- chromen-4-yl)amino]propyl}piperazine-2-carboxylic acid hydrochloride

To a solution of methyl 4-(isoquinolin-1 -yl)-1 -{3-[(2-oxo-2H-chromen-4- yl)amino]propyl}piperazine-2-carboxylate hydrochloride 81 (40 mg, 0.085 mmoles, 1 eq.) in THF/water 9:1 (1 ml_) LiOH (4 mg, 0.093 mmoles, 1 .1 eq.) was added. The mixture was stirred at 60 °C for 5 hours then was concentrated under reduced pressure. The residue was dissolved in water and 1 M HCI was added (6 eq.). The solvent was evaporated and the crude product was purified by C18 reversed chromatography (acetonitrile/water from 5:95 to 1 00:0) to obtain 4-(isoquinolin-1 -yl)-1 -{3-[(2-oxo-2H-chromen-4- yl)amino]propyl}piperazine-2-carboxylic acid hydrochloride 82 (38 mg, Y=84%). LC-MS (M-H+) = 459.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 8.1 8 - 8.10 (m, 2H), 8.05 (d, J=7.5 Hz, 1 H), 7.90 (d, J=8.0 Hz, 1 H), 7.77 - 7.66 (m, 2H), 7.59 (t, J=7.8 Hz, 2H), 7.42 (d, J=5.8 Hz, 1 H), 7.36 - 7.28 (m, 2H), 5.21 (s, 1 H), 3.71 - 3.62 (m, 1 H), 3.61 - 3.54 (m, 1 H), 3.52 - 3.08 (m, 9H), 2.96 (td, J=6.7, 13.2 Hz, 1 H), 2.82 - 2.70 (m, 2H), 1 .86 (quin, J=6.7 Hz, 2H)

Preparation of compound 83: 4-({3-[4-(isoquinolin-1-yl)-2-(2,2,2- trifluoroethyl)piperazin-1-yl]propyl}amino)-2H-chromen-2-one

hydrochloride

Compound 83 was prepared as described hereinbelow.

Compound 83 was prepared as described for compound 71 starting from 2-(2,2,2-trifluoroethyl)piperazine. LC-MS (M-H⁺) = 497.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .51 (br. s., 1 H), 8.25 - 8.08 (m, 3H), 7.97 (d, J=8.3 Hz, 1 H), 7.88 (br. s., 1 H), 7.79 (t, J=7.4 Hz, 1 H), 7.70 - 7.63 (m, 1 H), 7.63 - 7.57 (m, 1 H), 7.54 (d, J=5.5 Hz, 1 H), 7.40 - 7.26 (m, 1 H), 5.30 (s, 1 H), 5.42 (br. s., 2H), 4.46 - 2.74 (m, 13H), 2.14 (quin, J=7.0 Hz, 2H) Preparation of compound 85: 4-({3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}amino)-7-methoxy-2H-chromen-2-one hydrochloride

Compound 85 was prepared as described hereinbelow.

85

Step 1 : 7-methoxy-2-oxo-2H-chromen-4-yl trifluoromethanesulfonate (85a)

Intermediate 85a was prepared as described in step 1 of the synthesis of compound 28, starting from 4-hydroxy-7-methoxy-2H-chromen-2-one (Y=quant.). LC-MS (M-H⁺) = 325.1 .

Step 2: 4-({3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)-7-methoxy- 2H-chromen-2-one hydrochloride (85)

Compound 85 was prepared as described in step 4 of the synthesis of compound 71 , using intermediates 85a and 31 b (Y=22%). LC-MS (M-H⁺) = 445.4.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .07 (br. s., 1 H), 8.18 (d, J=8.3 Hz, 1 H), 8.09 (d, J=6.0 Hz, 1 H), 8.06 (d, J=8.8 Hz, 1 H), 7.99 (d, J=8.0 Hz, 1 H), 7.83 (t, J=7.2 Hz, 2H), 7.69 (t, J=7.5 Hz, 1 H), 7.55 (d, J=6.0 Hz, 1 H), 6.93 (dd, J=2.3, 8.8 Hz, 1 H), 6.88 (d, J=2.5 Hz, 1 H), 5.14 (s, 1 H), 4.10 (br. s., 1 H), 3.97 (d, J=12.5 Hz, 2H), 3.89 - 3.77 (m, 3H), 3.71 - 3.52 (m, 4H), 3.39 (d, J=5.8 Hz, 4H), 3.29 (br. s., 2H), 2.14 (quin, J=7.0 Hz, 2H)

Preparation of compound 87: 3-({3-[4-(isoquinolin-1-yl)piperazin-1- yl]propyl}amino)-2H-chromen-2-one hydrochloride

87

Compound 87 was prepared as described hereinbelow.

Step 1 : 3-bromo-2H-chromen-2-one (87a)

Bromine (0.35 ml_, 6.84 mmoles, 1 eq.) was added dropwise to a stirred solution of coumarin (1 g, 6.84 mmoles, 1 eq.) in chloroform (1 0 ml_) at 0 °C. The reaction mixture was allowed to gradually warm to room temperature overnight. Triethylamine (2 ml_) was added and the reaction mixture was washed with water (2 x 1 0 ml_). The organic phase was dried over Na₂S0₄ and evaporated under reduced pressure. The residue was chromatographed on silica gel (SNAP 50) eluting with 5% EtOAc in petroleum ether to give 504 mg of 3-bromo-2H-chromen-2-one 87a as a white solid (Y=33%). LC-MS (M- H+) = 225.0.

Step 2: 3-({3-[4-(isoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)-2H-chromen- 2-one hydrochloride (87)

A mixture of intermediates 31 b (50 mg, 0.185 mmoles, 1 eq.) and 87a (50 mg, 0.222 mmoles, 1 .2 eq.), Pd(OAc)₂ (5 mg, 0.019 mmoles, 0.1 eq.), Xantphos (1 1 mg, 0.019 mmoles, 0.1 eq.) and Cs₂C0₃ (90 mg, 0.28 mmoles, 1 .5 eq.) in 1 ,4-dioxane (2.5 ml_) was heated to 130°C in a sealed tube under microwave irradiation for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue was chromatographed on silica gel (SNAP25) eluting with a gradient of 0-2% mixture A in dichloromethane, where mixture A is MeOH/NH₄OH 10:1 . The partially pure product was further purified by preparative LC-MS to give 20 mg of a yellow gum. The product was dissolved in dichloromethane (4 mL) and treated with 1 M HCI solution in diethyl ether (0.16 mL) causing precipitation. The resulting mixture was evaporated under reduced pressure and the residue was triturated with diethyl ether. The solids were dried to give 24 mg of 3-({3-[4-(isoquinolin-1 - yl)piperazin-1 -yl]propyl}amino)-2H-chromen-2-one hydrochloride 87 (Y=27%). LC-MS (M-H+) = 415.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 1 .1 6 (br. s., 1 H), 8.19 (d, J=7.5 Hz, 1 H), 8.08 (d, J=5.8 Hz, 1 H), 8.00 (d, J=7.5 Hz, 1 H), 7.85 (t, J=6.5 Hz, 1 H), 7.70 (t, J=7.3 Hz, 1 H), 7.57 (d, J=5.3 Hz, 1 H), 7.47 (br. s., 1 H), 7.37 - 7.10 (m, 3H), 6.68 (s, 1 H), 6.27 (br. s., 1 H), 4.00 (d, J=12.3 Hz, 2H), 4.37 - 3.81 (m, 1 H), 3.64 (d, J=1 1 .0 Hz, 4H), 3.40 (d, J=9.3 Hz, 2H), 3.28 (d, J=5.8 Hz, 4H), 2.1 1 (br. quin, J=7.0 Hz, 2H)

Preparation of compound 96: 4-({3-[4-(7-fluoroisoquinolin-1- yl)piperazin-1-yl]propyl}amino)-5-methyl-2H-chromen-2-one

hydrochloride

Compound 96 was prepared as described hereinbelow.

96a 96b

96d Step 1 : 1 -(2-hydroxy-6-methylphenyl)ethanone (96a)

A solution of 2-hydroxy-6-methylbenzoic acid (2 g, 12.7 mmoles, 1 eq.) in THF (1 3 ml_) was cooled to -78°C. MeLi (1 .6 M in Et₂0, 38.1 mmoles, 3 eq.) was added dropwise. The mixture was stirred at room temperature overnight then was cooled to 0 °C and quenched with sat. NH₄CI. Ethyl acetate was added and the organic phase was washed with brine. The solvent was removed in vacuum and the crude material was purified by Si-column eluting with cHex to cHex/ethyl acetate 7:3 to obtain 980 mg of 1 -(2-hydroxy-6- methylphenyl)ethanone 96a (Y=51 %). LC-MS (M-H+) = 151 .0.

Step 2: 4-hydroxy-5-methyl-2H-chromen-2-one (96b)

A solution of intermediate 96a (0.98 g, 6.5 mmoles, 1 eq.) in toluene (10 ml_) was added dropwise to a stirred suspension of NaH (60% dispersion in mineral oil, 0.784 g, 19.6 mmoles, 3.0) in toluene (20 ml_)

at room temperature. Stirring was continued for 1 0 minutes, then a solution of diethyl carbonate (1 .58 ml_, 13.1 mmoles, 2 eq.) in toluene (10 ml_) was added dropwise and the mixture was heated to 1 1 5°C for 48 h. The solvent was evaporated under reduced pressure, the solid residue was added portion-wise to ice-cold water (100 ml_) to give a turbid solution. The aqueous phase was washed with diethyl ether (50 ml_) then acidified with 1 M HCI solution (60 ml_) causing precipitation of a white solid. The solid was collected by filtration and washed with water. The wet filter cake was suspended in dichloromethane (100 ml_) and evaporated under reduced pressure to give 949 mg of 4-hydroxy-5-methyl-2H-chromen-2-one 96b (Y=82%). LC-MS (M-H+) = 177.0.

Step 3: 5-methyl-2-oxo-2H-chromen-4-yl trifluoromethanesulfonate (96c)

Intermediate 96c was prepared as described in step 1 of the synthesis of compound 28, using intermediate 96b (Y=58%). LC-MS (M-H⁺) = 309.0.

Step 4: 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)-5- methyl-2H-chromen-2-one hydrochloride (96)

Compound 96 was prepared as described in step 4 of the synthesis of compound 71 , using intermediate 96d. Intermediate 96d was prepared according to the procedure described in step 2 of the synthesis of compound 31 , starting from 1 -chloro-7-fluoroisoquinoline (Y=31 %). LC-MS (M-H⁺) = 447.3. ¹H NMR (400MHz, DMSO-d₆) δ = 11.17 (br. s., 1H), 8.13 (d, J=5.8 Hz, 1H), 8.09 (dd, J=5.5, 8.8 Hz, 1H), 7.87 (d, J=9.8 Hz, 1H), 7.74 (t, J=8.5 Hz, 1H), 7.59 (d, J=5.8 Hz, 1H), 7.44 (t, J=7.9 Hz, 1H), 7.16 (d, J=8.0 Hz, 1H), 7.12 (d, J=7.5 Hz, 1H), 6.65 (br. s., 1H), 5.59 (br. s., 1H), 5.27 (s, 1H), 3.87 (d, J=12.8 Hz, 2H), 3.63 (d, J=11.3 Hz, 2H), 3.55 (t, J=12.8 Hz, 2H), 3.44 (d, J=5.5 Hz, 4H), 3.28 (br. s., 2H), 2.84 (s, 3H), 2.27 - 2.06 (m, 2H)

Preparation of compound 97: 4-({3-[4-(7-fluoroisoquinolin-1- yl)piperazin-1-yl]propyl}amino)-3-methyl-2H-chromen-2-one

hydrochloride

Compound 97 was prepared as described hereinbelow.

96d

Step 1 : 4-hydroxy-3-methyl-2H-chromen-2-one (97a) Intermediate 97a was prepared according to the procedure described in step 2 of the synthesis of compound 96, using 1 -(2-hydroxyphenyl)propan-1 - one (2.1 g, Y=20%). LC-MS (M-H⁺) = 1 77.0.

Step 2: 3-methyl-2-oxo-2H-chromen-4-yl trifluoromethanesulfonate (97b) Intermediate 97b was prepared according to the procedure described in step 1 of the synthesis of compound 28, using intermediate 97a (Y=58%). LC-MS (M-H⁺) = 309.0.

Step 3: 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)-3- methyl-2H-chromen-2-one hydrochloride (97)

Compound 97 was prepared as described in step 4 of the synthesis of compound 71 , using intermediate 96d. Intermediate 96d was prepared according to the procedure described in step 2 of the synthesis of compound 31 , starting from 1 -chloro-7-fluoroisoquinoline (Y=38%). LC-MS (M-H⁺) = 447.3.

1 H NMR (400MHz, DMSO-d₆) δ = 10.84 (br. s., 1 H), 8.20 - 8.1 1 (m, 2H),

8.07 (dd, J=5.5, 9.0 Hz, 1 H), 7.84 (dd, J=2.4, 1 0.2 Hz, 1 H), 7.71 (dt, J=2.5,

8.8 Hz, 1 H), 7.61 - 7.48 (m, 2H), 7.38 - 7.24 (m, 2H), 6.72 (br. s., 1 H), 4.48 (br. s., 1 H), 3.81 (d, J=1 1 .8 Hz, 2H), 3.65 (t, J=6.7 Hz, 2H), 3.59 (d, J=1 1 .0 Hz, 2H), 3.53 - 3.32 (m, 4H), 3.22 (br. s., 2H), 2.16 (s, 3H), 2.14 - 2.04 (m, 2H)

Preparation of compound 101 : 4-({3-[4-(7-fluoroisoquinolin-1- yl)piperazin-1-yl]propyl}amino)-3-methyl-2H-chromen-2-one

hydrochloride

Compound 101 was prepared as described hereinbelow.

101a 101 b 101c

101

Step 1 : ieri-butyl 4-(3-methoxy-3-oxopropyl)piperazine-1 -carboxylate (1 01 a)

Methyl 3-bromopropanoate (0.607 mL, 5.56 mmoles, 1 .05 eq.) was added to a solution of N-Boc-piperazine (1 g, 5.3 mmoles, 1 eq.) and TEA (0.870 mL, 6.36 mmoles, 1 .2 eq.) in anhydrous THF (8 mL), and the reaction was stirred overnight at room temperature. The mixture was diluted with EtOAc, the organic phase was washed with sodium bicarbonate solution and brine, dried over sodium sulphate and evaporated under reduced pressure. The residue was chromatographed on silica gel (SNAP 25) eluting with a gradient of 20-1 00% of EtOAc in ciclohexane to give 1 .3 g of ieri-butyl 4-(3-methoxy- 3-oxopropyl)piperazine-1 -carboxylate 101 a (Y=90%). LC-MS (M-H+) = 273.2.

Step 2: 3-[4-(tert-butoxycarbonyl)piperazin-1 -yl]propanoic acid (101 b)

To a solution of intermediate 101 a (1 .3 g, 4.7 mmoles, 1 eq.) in THF/water (9:1 ) LiOH (1 23 mg, 5.17 mmoles, 1 .1 eq.) was added. The mixture was stirred at 60 °C for 1 h then was concentrated under reduced pressure. The crude 3-[4-(tert-butoxycarbonyl)piperazin-1 -yl]propanoic acid 101 b (607 mg) was progressed without any further purification. LC-MS (M-H⁺) = 259.2.

Step 3: ieri-butyl 4-(3-amino-3-oxopropyl)piperazine-1 -carboxylate (101 c) Hexamethyldisilazane (0.488 mL, 2.35 mmoles, 1 eq.) was added to a stirred solution of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (540 mg, 2.82 mmoles, 1 .2 eq.), 1 -hydroxybenzotriazole hydrate (381 mg, 2.82 mmoles, 1 .2 eq.), intermediate 101 b (607 mg, 2.35 mmoles, 1 eq.) and TEA (977 μΙ, 7.05 mmoles, 3 eq.) in DCM (8 mL). The resulting mixture was stirred overnight at room temperature. The organic phase was washed with sodium bicarbonate solution, filtered through a hydrophobic frit (Phase Separator) and evaporated under reduce pressure. The residue was chromatographed on modified NH silica gel (SNAP 26) eluting with a gradient of 10-1 00% A in EtOAc, where A is MeOH/AcOEt (5:95), to give 454 mg of ferf-butyl 4-(3-amino-3-oxopropyl)piperazine-1 - carboxylate 101 c as a white solid (Y=75%).

LC-MS (M-H+) = 258.2.

Step 4: 3-(piperazin-1 -yl)propanamide (1 01 d)

Intermediate 101 d was prepared according to the procedure described in step 4 of the synthesis of compound 68 (Y=quant.). LC-MS (M-H⁺) = 158.1 . Step 5: 3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]propanamide (101 e)

Intermediate 101 e was prepared as described in step 1 of the synthesis of compound 73, using 1 -chloro-7-fluoroisoquinoline (Y=84%). LC-MS (M-H⁺) = 303.2.

Step 6: 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)-3- methyl-2H-chromen-2-one hydrochloride (1 01 )

4-(trifluoromethane)sulfonylchromen-2-one 28a (149 mg, 0.51 mmoles, 1 .3 eq.), 3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]propanamide 1 01 e (1 17 mg, 0.39 mmoles, 1 eq.), K₃P0₄ (165 mg, 0.78 mmoles, 2 eq.), Pd(dba)₂ (1 1 mg, 0.019 mmoles, 0.05 eq.) and Xantphos (1 1 mg, 0.019 mmoles, 0.05 eq.) were loaded into a Schlenk tube, which was filled with N₂. Dry dioxane (4 mL) was added and the resulting mixture was stirred at ^ 00 °C for 30 min. The organic phase was washed with H₂0, filtered through a hydrophobic frit (Phase Separator) and evaporated under reduced pressure. The residue was chromatographed on silica gel (SNAP 10) eluting with a gradient of 50-100% of EtOAc in ciclohexane. The resultant solid was dissolved in DCM and cooled to 0°C. 3 eq. of 1 M HCI in diethyl ether was added, after 1 h the mixture was evaporated in vacuo and the residue was triturated with diethyl ether to obtain 58 mg of 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 - yl]propyl}amino)-3-methyl-2H-chromen-2-one hydrochloride 1 01 (Y=29%). LC-MS (M-H+) = 447.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 10.81 (br. s., 1 H), 1 0.44 (s, 1 H), 8.34 (dd, J=1 .4, 8.4 Hz, 1 H), 8.16 (d, J=5.8 Hz, 1 H), 8.08 (dd, J=5.8, 9.0 Hz, 1 H), 7.86 (dd, J=2.5, 1 0.3 Hz, 1 H), 7.76 - 7.66 (m, 2H), 7.58 (d, J=5.8 Hz, 1 H), 7.48 - 7.41 (m, 2H), 7.24 (s, 1 H), 4.99 (br. s., 1 H), 3.85 (d, J=10.5 Hz, 2H), 3.66 - 3.59 (m, 2H), 3.59 - 3.52 (m, 2H), 3.50 - 3.41 (m, 4H), 3.31 (t, J=7.0 Hz, 2H)

Preparation of compound 107: 4-({3-[4-(7-fluoroisoquinolin-1- yl)piperazin-1-yl]propyl}amino)-7-hydroxy-2H-chromen-2-one

hydrochloride

Compound 107 was prepared as described hereinbelow.

96d

Step 1 : 7-(benzyloxy)-4-hydroxy-2H-chromen-2-one (107a)

Intermediate 107a was prepared according to the procedure described in step 2 of the synthesis of compound 96, using 1 -[4-(benzyloxy)-2- hydroxyphenyl]ethanone (Y=86%). LC-MS (M-H⁺) = 269.2.

Step 2: 7-(benzyloxy)-2-oxo-2H-chromen-4-yl trifluoromethanesulfonate (1 07b)

Intermediate 107b was prepared according to the procedure described in step 1 of the synthesis of compound 28 (Y=quant.). LC-MS (M-H⁺) = 401 .1 . Step 3: 7-(benzyloxy)-4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 - yl]propyl}amino)-2H-chromen-2-one (107c)

Intermediate 107c was prepared as described in step 4 of the synthesis of compound 71 , using intermediate 96d. Intermediate 96d was prepared according to the procedure described in step 2 of the synthesis of compound 31 , starting from 1 -chloro-7-fluoroisoquinoline (Y=37%). LC-MS (M-H⁺) = 539.4.

Step 4: 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)-7- hydroxy-2H-chromen-2-one hydrochloride (107)

To a suspension of intermediate 107c (49 mg, 0.09 mmoles, 1 eq.) in

EtOH (4 ml_) Pd/C 1 0% (0.05 eq.) was added and the mixture was stirred at room temperature under hydrogen (1 atm). After 30 h the mixture was filtered and concentrated in vacuum. The residue was loaded onto a SCX cartridge (1 g), which was eluted with MeOH and with a 1 M NH₃ solution in MeOH. The basic solution was concentrated in vacuum to give a colorless oil (34 mg). This was purified by flash chromatography (Biotage KP-Sil 10g SNAP cartridge, eluent A: EtOAc, B: EtOAc/MeOH 80/20, gradient A/B from 90:10 to 0:100) to give 1 8.2 mg of a white solid. The product was dissolved in MeOH and a 1 M HCI solution in diethyl ether (0.1 ml_) was added. The mixture was concentrated under nitrogen and the residue was triturated with diethyl ether (2x) to give 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 - yl]propyl}amino)-7-hydroxy-2H-chromen-2-one hydrochloride 107 as a white solid (18.7 mg, Y=40%). LC-MS (M-H+) = 449.3.

¹ H NMR (400MHz, DMSO-d₆) δ = 1 0.51 (br. s., 1 H), 1 0.42 (br. s., 1 H), 8.16 (d, J=5.8 Hz, 1 H), 8.07 (dd, J=5.5, 9.0 Hz, 1 H), 7.90 (d, J=8.8 Hz, 1 H), 7.85 (dd, J=2.3, 1 0.3 Hz, 1 H), 7.75 - 7.66 (m, 2H), 7.57 (d, J=5.8 Hz, 1 H), 6.75 (dd, J=2.3, 8.8 Hz, 1 H), 6.63 (d, J=2.3 Hz, 1 H), 5.09 (s, 1 H), 3.82 (d, J=8.8 Hz, 2H), 3.73 (br. s., 1 H), 3.61 (d, J=6.3 Hz, 2H), 3.47 - 3.33 (m, 6H), 3.28 (td, J=5.0, 10.0 Hz, 2H), 2.10 (quin, J=7.2 Hz, 2H)

Preparation of compound 111 : 4-({1-[(isoquinolin-1-yloxy)acetyl] piperidin-4-yl}amino)-2H-chromen-2-one

Compound 1 1 1 was prepared as described hereinbelow.

Step 1 : Ethyl (isoquinolin-l -yloxy)acetate (1 1 1 a)

To a suspension of ethyl hydroxyacetate (3.45 mL, 36.4 mmoles, 3 eq.) in THF (1 5 mL) NaH (60% dispersion in mineral oil, 1 .47 g, 36.6 mmoles, 3 eq.) was added portion-wise. The mixture was heated to 70 °C and stirred for 1 h then a solution of 1 -chloroisoquinoline (2 g, 1 2.3 mmoles, 1 eq.) in THF (15 mL) was added. The reaction mixture was stirred at 70 °C overnight then was cooled to room temperature, poured into a saturated solution of NaCI (15 mL) and extracted with EtOAc (3 x 10 mL). The collected organic phases were dried over Na₂S0₄ and concentrated under vacuum. The residue was purified via flash chromatography on silica gel eluting with hexane/EtOAc 9:1 to give 1 .85 g of ethyl (isoquinolin-l -yloxy)acetate 1 1 1 a (Y=65%). GC/MS: 231 .

Step 2: (isoquinolin-1 -yloxy)acetic acid (1 1 1 b)

To a solution of intermediate 1 1 1 a (1 .2 g, 5.2 mmoles) in a mixture of THF/EtOH 1 :1 (10 mL) NaOH (1 M solution, 20.4 mL, 4 eq.) was added. The solution was stirred for 3 h at room temperature then the solvents were removed under vacuum, water (10 mL) was added to the residue followed by 1 M HCI (neutralization). The mixture was stirred for 15 min. then the resulting solid was filtered, washed with water and dried to give 0.83 g of (isoquinolin-1 -yloxy)acetic acid 1 1 1 b (Y=78%). LC-MS (M-H⁺) = 204.1 .

Step 3: 4-({1 -[(isoquinolin-1 -yloxy)acetyl]piperidin-4-yl}amino)-2H- chromen-2-one (1 1 1 ) To a solution of intermediate 111b (0.3 g, 1.48 mmoles, 1 eq.) in DMF (10 ml_) TEA (0.46 ml_, 3.25 mmoles, 2.2 eq.), intermediate 28c (0.5 g, 1.63 mmoles, 1.1 eq.) and HBTU (616 mg, 1.63 mmoles, 1.1 eq.) were added. The mixture was stirred at room temperature overnight then was diluted with EtOAc and filtered. The solution was washed with water and concentrated. The residue was diluted with EtOAc, a white solid separated that was filtered and dried to give 182 mg of 4-({1 -[(isoquinolin-1 -yloxy)acetyl]piperidin-4- yl}amino)-2H-chromen-2-one 111 (Y=29%). LC-MS (M-H⁺) = 430.2.

¹H NMR (300MHz, DMSO-d₆) δ = 8.25 (dd, J= 0.7, 7.7 Hz, 1 H), 8.15 (d, J = 7.9 Hz, 1 H), 7.97 (d, J= 5.8 Hz, 1 H), 7.92 (d, J= 8.2 Hz, 1 H), 7.79 (ddd, J= 1.5, 7.0, 8.3 Hz, 1 H), 7.71 - 7.55 (m, 2 H), 7.41 (d, J= 5.3 Hz, 1 H), 7.38 - 7.25 (m, 3 H), 5.40 (s, 1 H), 5.34 (s, 2 H), 4.35 (d, J = 12.9 Hz, 1 H), 4.07 - 3.94 (m, 1 H), 3.92 - 3.70 (m, 1 H), 3.27 (d, J= 11.8 Hz, 1 H), 2.81 (tdd, J = 11.9, 12.1, 12.3 Hz, 1 H), 2.19 - 1.81 (m, 2 H), 1.66 (dq, J= 3.0, 11.8 Hz, 1 H), 1.49 (dq, J= 3.0, 11.8 Hz, 1 H).

Preparation of compound 117: N-(isoquinolin-1-yl)-2-{4-[(2-oxo-2H- chromen-4-yl)amino]piperidin-1-yl}acetamide

Compound 117 was prepared as described hereinbelow.

Step 1: Ethyl {4-[(2-oxo-2H-chromen-4-yl)amino]piperidin-1-yl}acetate (117a)

To a solution of intermediate 28c (0.64 g, 1.79 mmoles, 1 eq.) in MeCN (10 ml_) K₂CO₃ (0.74 g, 5.34 mmoles, 3 eq.) was added. The mixture was stirred under reflux for 1 h then a solution of ethyl bromoacetate (0.2 ml_, 1.78 mmoles, 1 eq.) in MeCN (5 ml_) was added dropwise. After stirring 2 h under reflux the mixture was cooled to room temperature, diluted with EtOAc, filtered and concentrated under vacuum. The residue was purified via flash chromatography on silica gel eluting with CHCI₃/MeOH 9:1 to give 0.45 g of ethyl {4-[(2-oxo-2H-chromen-4-yl)amino]piperidin-1 -yl}acetate 1 17a (Y=76%). LC-MS (M-H⁺) = 331 .2.

Step 2: {4-[(2-oxo-2H-chromen-4-yl)amino]piperidin-1 -yl}acetic acid (1 17b) Intermediate 1 1 7b was prepared according to the procedure described in step 2 of the synthesis of compound 1 1 1 , starting from intermediate 1 1 7a (Y=quant.). LC-MS (M-H⁺) = 303.1 .

Step 3: A/-(isoquinolin-1 -yl)-2-{4-[(2-oxo-2H-chromen-4-yl)amino]piperidin- 1 -yl}acetamide (1 17)

Compound 1 17 was prepared according to the procedure described in step 3 of the synthesis of compound 1 1 1 , using isoquinolin-1 -amine (Y=43%). LC-MS (M-H⁺) = 429.2. ¹ H NMR (300MHz ,DMSO-d₆) δ = 1 1 .47 - 1 0.96 (m, 1 H), 10.12 - 9.65 (m, 1 H), 8.34 (br. s., 1 H), 8.1 7 (d, J = 1.1 Hz, 2 H), 8.08 - 7.76 (m, 3 H), 7.72 (t, J = 1.8 Hz, 1 H), 7.66 - 7.54 (m, 1 H), 7.48 - 7.23 (m, 3 H), 5.38 (s, 1 H), 4.81 - 4.09 (m, 2 H), 4.05 - 3.41 (m, 4 H), 2.33 - 1 .83 (m, 4 H).

Preparation of compound 118: N-(isoquinolin-1-ylmethyl)-2-{4-[(2- oxo-2H-chromen-4-yl)amino]piperidin-1-yl}acetamide

Compound 1 18 was prepared according to the procedure described in step 3 of the synthesis of compound 1 1 1 , using intermediate 1 17b and 1 - (isoquinolin-l -yl)methanamine (Y=26%). LC-MS (M-H⁺) = 443.2.

¹ H NMR (300 MHz, DMSO-d₆) δ = 8.53 (t, J = 5.1 Hz, 1 H), 8.47 (d, J = 5.8 Hz, 1 H), 8.26 (qd, J = 1 .0, 8.3 Hz, 1 H), 8.18 (dd, J = 1 .3, 8.0 Hz, 1 H), 8.01 (d, J = 7.9 Hz, 1 H), 7.86 - 7.76 (m, 2 H), 7.72 (ddd, J = 1 .5, 7.0, 8.0 Hz, 1 H), 7.59 (dt, J = 1 .5, 7.7 Hz, 1 H), 7.39 - 7.15 (m, 3 H), 5.25 (s, 1 H), 5.01 (d, J = 5.3 Hz, 2 H), 3.63 - 3.39 (m, 1 H), 3.07 (s, 2 H), 2.95 (d, J = 1 1 .7 Hz, 2 H), 2.38 - 2.22 (m, 2 H), 2.02 - 1 .88 (m, 2 H), 1 .76 (dq, J = 3.6, 1 1 .7 Hz, 2 H).

Preparation of compound 119: /V-(1-benzothiophen-2-ylmethyl)-3-[4- (isoquinolin-1-yl)piperazin-1-yl]propan-1-amine

Compound 1 19 was prepared according to the procedure described in step 4 of the synthesis of compound 2 using intermediate 31 b and 1 - benzothiophene-2-carbaldehyde (Y=20%). LC-MS (M-H⁺) = 417.2.

¹ H NMR (300MHz, CHLOROFORM-d) δ = 8.13 (d, J = 5.8 Hz, 1 H), 8.07 (d, J = 8.6 Hz, 1 H), 7.83 - 7.66 (m, 3 H), 7.60 (ddd, J = 1 .3, 7.0, 8.3 Hz, 1 H), 7.49 (ddd, J = 1 .5, 7.0, 8.3 Hz, 1 H), 7.36 - 7.27 (m, 2 H), 7.24 (d, J = 5.8 Hz, 1 H), 7.20 (d, J = 0.7 Hz, 1 H), 4.14 (d, J = 0.9 Hz, 2 H), 3.47 (t, J = 5.0 Hz, 4 H), 2.85 (t, J = 6.5 Hz, 3 H), 2.77 (t, J = 5.0 Hz, 4 H), 2.59 (t, J = 6.9 Hz, 2 H), 1 .84 (quin, J = 6.7 Hz, 2 H).

Preparation of compound 120: Λ/-(1 ,3-benzodioxol-5-ylmethyl)-3-[4-

(isoquinolin-1-yl)piperazin-1-yl]propan-1-amine

Compound 1 20 was prepared according to the procedure described in step 4 of the synthesis of compound 2 using intermediate 31 b and 1 ,3- benzodioxole-5-carbaldehyde (Y=1 0%). LC-MS (M-H⁺) = 405.2.

1 H NMR (300 MHz, CHLOROFORM-d) δ = 8.13 (d, J = 5.8 Hz, 1 H), 8.02

(d, J = 8.5 Hz, 1 H), 7.75 (d, J = 8.0 Hz, 1 H), 7.61 (ddd, J = 1 .1 , 7.0, 8.3 Hz, 1 H), 7.50 (ddd, J = 1 .7, 7.0, 8.3 Hz, 1 H), 7.26 (dd, J = 0.6, 5.8 Hz, 1 H), 6.93 - 6.85 (m, 2 H), 6.81 (d, J = 7.8 Hz, 1 H), 5.94 (s, 2 H), 3.99 (s, 2 H), 3.47 - 3.23 (m, 4 H), 3.1 1 (t, J = 5.9 Hz, 2 H), 2.87 - 2.72 (m, 4 H), 2.65 (t, J = 5.8 Hz, 2 H), 1 .96 (quin, J = 5.8 Hz, 2 H).

Preparation of compound 122: 4-({3-[4-(7-fluoroisoquinolin-1- yl)piperazin-1 -yl]propyl}amino)-5,6,7,8-tetrahydro-2H-pyrano[3,2- c]pyridin-2-one hydrochloride

Compound 122 was prepared as described hereinbelow.

22a 122b 122c

122 122e

Step 1 : ieri-butyl 4-(pyrrolidin-1 -yl)-3,6-dihydropyridine-1 (2H)-carboxylate (1 22a)

Pyrrolidine (3.5 g, 50.2 mmoles, 2 eq.) was added to a solution of 1 -Boc-4- piperidone (5 g, 25 mmoles, 1 eq.) in dry toluene (30 mL). The mixture was heated at 90 °C with a Dean-Stark apparatus. After 3 h the mixture was concentrated in vacuum to give ieri-butyl 4-(pyrrolidin-1 -yl)-3,6- dihydropyridine-1 (2H)-carboxylate 122a as yellow oil (6.3 g, Y=quant.). The compound was used without any further purification and characterization. Step 2: ieri-butyl 3-acetyl-4-oxopiperidine-1 -carboxylate (122b)

Intermediate 122a (6.3 g, 25 mmoles, 1 eq.) was dissolved in 1 ,4-dioxane (25 mL). Acetic anhydride (5.21 mL, 55.2 mmol, 2.2 eq.) was added and the resulting mixture was allowed to stand at room temperature under nitrogen for 2 days. Water (6 mL) was added and the resulting mixture was refluxed for 1 hour then cooled to room temperature and concentrated in vacuum. Water (20 mL) was added and the aqueous phase was extracted twice with EtOAc (20 ml_). The combined organics extracts were washed with a 5% w/w HCI aqueous solution (20 ml_), dried over MgS0₄ and concentrated in vacuum. The resultant orange oil was purified by Si-column eluting with cyclohexane to cyclohexane/ethyl acetate 9:1 to afford 2,98 g of ieri-butyl 3- acetyl-4-oxopiperidine-1 -carboxylate 122b (Y=49%). LC-MS (M-H+) = 242.2.

Step 3: ieri-butyl 4-hydroxy-2-oxo-7,8-dihydro-2H-pyrano[3,2-c]pyridine- 6(5H)-carboxylate (122c)

A solution of intermediate 1 22b (1 g, 4.14 mmoles, 1 eq.) in THF (20 ml_) was cooled to -78 °C. Lithium bis(trimethylsilyl)amide (1 M solution in THF, 1 2.4 ml_, 3 eq.) was added dropwise. After stirring for 1 h at -78 °C, dimethylcarbonate (0.4 ml_, 4.6 mmoles, 1 .1 eq.) was added. The resulting mixture was allowed to warm slowly to -1 0°C. After 1 h the reaction was cooled to 0 °C and quenched with 1 M HCI until pH = 6. Ethyl acetate was added, the organic phase was separated, washed with brine and evaporated in vacuum to obtain crude ieri-butyl 3-(3-methoxy-3-oxopropanoyl)-4- oxopiperidine-1 -carboxylate, that was used without any further purification.

The crude material was dissolved in toluene (8 ml_), 1 ,8- diazabicyclo[5.4.0]undec-7-ene (0.62 ml_, 4.14 mmoles, 1 eq.) was added and the mixture was stirred under reflux. After 3 hours the reaction was cooled to 0°C and quenched with 1 M HCI. Ethyl acetate was added, the organic phase was separated, washed with brine and evaporated in vacuum. The crude material was purified by Si-column eluting with EtOAc to EtOAc/MeOH 7:3 to obtain 600 mg of ieri-butyl 4-hydroxy-2-oxo-7,8-dihydro- 2H-pyrano[3,2-c]pyridine-6(5H)-carboxylate 122c (Y=54%). LC-MS (M-H+) = 268.2.

Step 4: ieri-butyl 2-oxo-4-{[(trifluoromethyl)sulfonyl]oxy}-7,8-dihydro-2H- pyrano[3,2-c]pyridine-6(5H)-carboxylate (1 22d)

Intermediate 122d was prepared according to the procedure described in step 1 of the synthesis of compound 28 (Y=quant.). LC-MS (M-H⁺) = 400.1 . Step 5: ieri-butyl 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 - yl]propyl}amino)-2-oxo-7,8-dihydro-2H-pyrano[3,2-c]pyridine-6(5H)- carboxylate (122e)

Intermediate 122e (Y=17%) was prepared as described in step 4 of the synthesis of compound 71 , using intermediate 96d. Intermediate 96d was prepared according to the procedure described in step 2 of the synthesis of compound 31 , starting from 1 -chloro-7-fluoroisoquinoline. LC-MS (M-H⁺) = 538.4.

Step 6: 4-({3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]propyl}amino)- 5,6,7,8-tetrahydro-2H-pyrano[3,2-c]pyridin-2-one hydrochloride (122)

Compound 122 was prepared as described in step 4 of the synthesis of compound 68 (Y=72%). LC-MS (M-H⁺) = 438.2.

¹ H NMR (400 MHz, DMSO-d₆) δ = 1 0.74 (br. s., 1 H), 9.61 (br. s., 2 H), 8.16 (d, J = 5.8 Hz, 1 H), 8.07 (dd, J = 5.8, 9.0 Hz, 1 H), 7.84 (dd, J = 2.5, 1 0.3 Hz, 1 H), 7.71 (dt, J = 2.5, 8.8 Hz, 1 H), 7.57 (d, J = 5.8 Hz, 1 H), 7.1 6 (t, J = 5.3 Hz, 1 H), 5.06 (s, 1 H), 4.16 - 3.86 (m, 3 H), 3.81 (d, J = 10.8 Hz, 2 H), 3.59 (d, J = 9.0 Hz, 2 H), 3.50 - 3.32 (m, 6 H), 3.31 - 3.16 (m, 4 H), 2.73 (t, J = 5.8 Hz, 2 H), 2.14 - 1 .93 (m, J = 7.0, 7.0, 7.0, 7.0 Hz, 2 H).

Preparation of compound 123

Compound 123 was prepared as described herein below.

123a 123b 123c

Step ₄

123 Step 1 : 2-oxo-2H-chromen-4-yl trifluoromethanesulfonate (123a)

4-Hydroxycoumarine (1 g, 6.17 mmoles, 1 eq.) was dissolved in DCM (30 mL), triethylamine (1 .72 mL, 1 2.34 mmoles, 2 eq.) was added and the resulting mixture was cooled to -I CC. Trifluoromethanesulfonic anhydride (1 .25 mL, 7.4 mmoles, 1 .2 eq.) in DCM (5 mL) was added dropwise and the solution was left stirring at -10°C for 2 h. The resulting reddish brown solution was warmed to room temperature, diluted with cHex/Et20 1 /1 (50 mL) and filtered through a pad of silica gel using cHex/Et20 1 /1 as eluent. The solvent was removed in vacuum to obtain 2-oxo-2H-chromen-4-yl trifluoromethanesulfonate 1 23a (1 .76 g, Y= 97%). LC-MS (M-H+): 295.0.

Step 2: tert-butyl 4-[(2-oxo-2H-chromen-4-yl)amino]piperidine-1 - carboxylate (123b)

Triethylamine (1 mL, 7.2 mmoles, 1 .2 eq.) in acetonitrile (2 mL) was added dropwise to a stirred solution of intermediate 123a (1 .76 g, 5.99 mmoles, 1 eq.) and 1 -Boc-4-aminopiperidine (1 .2 g, 5.99 mmoles, 1 eq.) in dry acetonitrile (20 mL). The mixture was heated at reflux for 2 h then was cooled to room temperature, diluted with DCM (100 mL) and washed with saturated NaHC03 (50 mL) and water (50 mL). The organic phase was separated, dried over sodium sulfate and evaporated in vacuum. The crude material was purified by trituration with methanol to obtain tert-butyl 4-[(2-oxo-2H-chromen- 4-yl)amino]piperidine-1 -carboxylate 123b (1 .51 g, Y= 73%). LC-MS (M-H+): 345.2.

Step 3: 4-(piperidin-4-ylamino)-2H-chromen-2-one trifluoroacetic acid salt (1 23c)

Intermediate 123b (1 g, 2.90 mmoles, 1 eq.) was dissolved in DCM (10 ml) and TFA (3 mL) was added dropwise at 0°C. The solution was left stirring for 2 h then was concentrated in vacuum and washed with toluene (25 ml) and diethyl ether (25 ml) to obtain 4-(piperidin-4-ylamino)-2H-chromen-2-one trifluoroacetic acid salt 123c, which was used without any further purification (1 .22 g, Y= quant.). LC-MS (M-H+): 245.1 .

Step 4: 4-{[1 -(prop-2-yn-1 -yl)piperidin-4-yl]amino}-2H-chromen-2-one (1 23d)

To a solution of intermediate 123c (50 mg, 0.14 mmoles, 1 eq.) and potassium carbonate (38.7 mg, 0.28 mmoles, 2 eq.) in DMF (2 mL) 3-bromo- 1 -propyne (20.8 mg, 0.14 mmoles, 1 eq.) was added at room temperature. The reaction was left stirring overnight then was filtered and the filtrate concentrated. The residue was purified by Si-column eluting with eluting with EtOAc to EtOAc/Methanol 8:2 to give 4-{[1 -(prop-2-yn-1 -yl)piperidin-4- yl]amino}-2H-chromen-2-one 123d (25.6 mg, Y= 64%). LC-MS (M-H+): 283.1 .

Step 5: 4-({1 -[3-(isoquinolin-1 -yl)prop-2-yn-1 -yl]piperidin-4-yl}amino)-2H- chromen-2-one (123e)

Intermediate 1 23d (186 mg, 0.66 mmoles, 1 eq.), 1 -chloroisoquinoline (1 20 mg, 0.72 mmoles, 1 .1 eq.) and Cul (12 mg, 0.066 mmoles, 0.1 eq.) were dissolved in dry DMF (3.6 ml). DIPEA (0.473 ml, 2.64 mmoles, 4 eq.) was added. The mixture was degassed by alternatively applying vacuum and nitrogen, then bis(triphenylphosphine)palladium(ll) dichloride (45 mg, 0.066 mmoles, 0.1 eq.) was added and the mixture was heated at 60 °C. After 3 h water (10 mL) was added and the mixture was extracted with ethyl acetate (3x 10 mL). The organic phase was washed with brine, dried over Na2S04, filtered and concentrated in vacuum to give 283 mg of crude material. After purification by Si-column eluting with EtOAc to EtOAc/MeOH 8:2 compound 4-({1 -[3-(isoquinolin-1 -yl)prop-2-yn-1 -yl]piperidin-4-yl}amino)-2H-chromen-2- one 1 23e (87mg, Y=32%) was obtained. LC-MS (M-H+): 410.2.

Step 6 - Synthesis of 4-({1 -[3-(isoquinolin-1 -yl)propyl]piperidin-4-yl}amino)- 2H-chromen-2-one (123f):

To a solution of intermediate 123e 4-({1 -[3-(isoquinolin-1 -yl)prop-2-yn-1 - yl]piperidin-4-yl}amino)-2H-chromen-2-one (82 mg, 0.2 mmoles, 1 eq.) in EtOAc (5 ml) Pd/C 10% (0.05 eq.) was added and the mixture was stirred under hydrogen (3.5 atm) at room temperature. After 3 hours DCM was added, the mixture was then filtered and concentrated in vacuum to give crude material, which was purified by Si-column eluting with DCM to DCM/Methanol 8:2 to give 30 mg (0.072 mmol, 36% yield) of intermediate 1 23f. LC-MS (M-H⁺): 414.1

Step 7 - Synthesis of 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl]amino}ethyl)amino]propan-1 -ol (compound 1 23):

To a solution of 4-({1 -[3-(isoquinolin-1 -yl)propyl]piperidin-4-yl}amino)-2H- chromen-2-one (123f, 25 mg, 0.06 mmol) in acetone (5 mL) K2C03 (43 mg, 0.31 mmol) was added. After stirring 1 h the mixture was cooled to 0 °C then methyl iodide (3.7 μΙ_, 0.06 mmol) was added. The mixture was left to return to room temperature then was stirred overnight, filtered and concentrated in vacuo. The residue was purified via preparative HPLC (C18, acetonitrile from 1 0% to 30% in water) to give 1 3.3 mg (0.024 mmol, 40% yield) of the title compound. LC-MS (M-H⁺): 428.2

¹ H NMR (300 MHz, DMSO-d₆) δ = 8.44 (d, J = 5.6 Hz, 1 H), 8.32 (d, J = 8.5 Hz, 1 H), 8.16 - 8.09 (m, 1 H), 7.99 (d, J = 7.6 Hz, 1 H), 7.83 - 7.76 (m, 1 H), 7.76 - 7.68 (m, 2 H), 7.67 - 7.58 (m, 1 H), 7.54 (d, J = 7.3 Hz, 1 H), 7.40 - 7.30 (m, 2 H), 5.36 (s, 1 H), 3.96 - 3.79 (m, 1 H), 3.75 - 3.59 (m, 4 H), 3.57 - 3.36 (m, 4 H), 3.1 3 (s, 3 H), 2.39 - 2.05 (m, 6 H).

Preparation of compound 128: (2S,3S) and (2R,3R) 4-({3-[1-(7- fluoroisoquinolin-1-yl)piperidin-4-yl]-2,3-dihydroxypropyl}amino)-2H- chromen-2-one hydrochloride

Compound 128 was prepared as described hereinbelow.

128a 128b 128c 128d

128

Step 1 : [1 -(7-fluoroisoquinolin-1 -yl)piperidin-4-yl]methanol (1 28a)

Intermediate 128a was prepared according to the procedure described in step 1 of the synthesis of compound 70, using piperidin-4-ylmethanol (Y=82%). LC-MS (M-H⁺) = 261 .2. Step 2: 1 -(7-fluoroisoquinolin-1 -yl)piperidine-4-carbaldehyde (128b)

Intermediate 128a (675 mg, 2.6 mmoles, 1 eq.), sodium bicarbonate (1 .09 g, 13 mmoles, 5 eq.) and Dess-Martin periodinane (1 .65 g, 3.9 mmoles, 1 .5 eq.) were suspended in DCM (4 ml_). The reaction mixture was stirred at r.t. for 3 hours then was quenched with sat. NaHC0₃ / 2N Na₂S₂0₃ in a 1 :1 ratio. After 15 minutes water and DCM were added, the organic phase was separated, washed with brine, treated with Na₂S0₄ and concentrated under reduced pressure to give 1 -(7-fluoroisoquinolin-1 -yl)piperidine-4- carbaldehyde 128b, which was progressed without any further purification (671 mg, Y=quant.). LC-MS (M-H+) = 259.1 .

Step 3: methyl (2E)-3-[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4-yl]prop-2- enoate (128c)

Methyl 2-(dimethoxyphosphoryl)acetate (224 mg, 1 .23 mmoles, 1 .6 eq.) was added to a solution of potassium ieri-butoxide (1 38 mg, 1 .23 mmoles, 1 .6 eq.) in THF (3 ml_) at 0°C. The mixture was stirred at room temperature for 30 min. then a solution of intermediate 128b (199 mg, 0.77 mmoles, 1 eq.) in THF (2 ml_) was added dropwise at 0°C. The reaction was stirred at room temperature overnight. The reaction was quenched with water and extracted with EtOAc (2x). The organic phase was dried over sodium sulphate and evaporated under reduced pressure. The residue was chromatographed on silica gel (SNAP 10) eluting with a gradient of 0-30% EtOAc in cyclohexane to give 1 25 mg of methyl (2E)-3-[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4- yl]prop-2-enoate 1 28c (Y=52%). LC-MS (M-H+) = 315.1 .

Step 4: (2E)-3-[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4-yl]prop-2-en-1 -ol (1 28d)

Diisobutylaluminum hydride (1 M solution in cyclohexane, 13 ml_, 3.7 eq.) was added dropwise to a stirred solution of 1 28d (1 .1 g, 3.5 mmoles, 1 eq.) in THF (25 ml_) at -78°C. The mixture was stirred for 1 hour at the same temperature then was quenched with MeOH (4 ml_) and was allowed to warm to room temperature. EtOAc (100 ml_) and Rochelle saturated solution (1 50 ml_) were added, the resulting mixture was stirred vigorously for 15 minutes, the organic phase was separated and dried over sodium sulphate. The volatiles were removed under reduced pressure to give 0.9 g of (2E)-3-[1 -(7- fluoroisoquinolin-1 -yl)piperidin-4-yl]prop-2-en-1 -ol 128d (Y=90%). LC-MS (M- H+) = 287.4.

Step 5: di-ferf-butyl {(2E)-3-[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4-yl]prop- 2-en-1 -yl}imidodicarbonate (128e)

To a solution of intermediate 128d (800 mg, 2.8 mmoles, 1 eq.) in DCM

(4.5 ml_) TEA (0.97 ml_, 7 mmoles, 2.5 eq.) was added. The mixture was cooled to 0°C then methanesulfanonyl chloride (0.26 ml_, 3.35 mmoles, 1 .2 eq.) was added under N₂ atmosphere. The mixture was allowed to warm to room temperature and stirred for 1 h. DCM was added and the solution was washed with sat. NaHC0₃. The organic phase was separated and evaporated in vacuum to give (2E)-3-[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4- yl]prop-2-en-1 -yl methanesulfonate (Y=quant.), that was used without any further purification.

The intermediate (2E)-3-[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4-yl]prop-2- en-1 -yl methanesulfonate (2.8 mmoles, 1 eq.) was added dropwise to a stirred mixture of di-ieri-butyl-iminodicarboxylate (1 g, 4.75 mmoles, 1 .7 eq.), K₂C0₃ (0.7 g, 5 mmoles, 1 .8 eq.) and Lil (12 mg, 0.092 mmoles, 0.03 eq.) in acetonitrile (20 ml_). The reaction was refluxed overnight then sat. NaHC0₃ and EtOAc were added. The organic phase was washed with brine and dried over sodium sulphate. The residue was chromatographed on silica gel (SNAP 50) eluting with a gradient of 0-15 % solution A in cHex, where A is 20% MeOH in EtOAc, to give 580 mg of di-ieri-butyl {(2E)-3-[1 -(7- fluoroisoquinolin-1 -yl)piperidin-4-yl]prop-2-en-1 -yl}imidodicarbonate 128e (Y=48%). LC-MS (M-H+) = 486.4.

Step 6: (2S,3S) and (2R,3R) 3-amino-1 -[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl]propane-1 ,2-diol hydrochloride (1 28f)

To a stirred solution of intermediate 128e (580 mg, 1 .2 mmoles, 1 eq.) in t- butanol/water 1 :1 (8 ml_) 4-methylmorpholine-N-oxide (167 mg, 1 .4 mmoles, 1 .2 eq.) and one drop of osmium tetroxide (4% water solution) were added. After stirring 1 8 hours at room temperature, EtOAc was added and the organic phase was washed with water and brine, separated and evaporated in vacuum. 300 mg of the residue was dissolved in DCM/MeOH (5 ml_) and cooled to 0°C. 3 eq. of 1 M HCI in diethyl ether were added and the mixture was stirred at room temperature overnight and then at 60 °C for 5 h. The solvent was evaporated in vacuum to obtain 199 mg of (1 S,2S)-3-amino-1 -[1 - (7-fluoroisoquinolin-1 -yl)piperidin-4-yl]propane-1 ,2-diol hydrochloride 128f as a racemic mixture (Y=95%). LC-MS (M-H+) = 320.1 .

Step 7: (2S,3S) and (2R,3R) 4-({3-[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4- yl]-2,3-dihydroxypropyl}amino)-2H-chromen-2-one hydrochloride (128)

Compound 128, as a racemic mixture, was prepared as described in step 4 of the synthesis of compound 71 , using intermediates 128f and 28a (Y=86%). LC-MS (M-H⁺) = 464.3.

¹ H NMR (500 MHz, DMSO-d₆) δ ppm 1 .46 - 1 .61 (m, 2 H), 1 .73 - 1 .84 (m, 2 H), 2.06 - 2.13 (m, 1 H), 2.77 - 2.88 (m, 1 H), 3.25 (t, J=7.83 Hz, 1 H), 3.29 - 3.36 (m, 1 H), 3.40 - 3.48 (m, 1 H), 3.64 - 3.77 (m, 2 H), 3.84 - 3.98 (m, 1 H), 4.45 (d, J=7.83 Hz, 1 H), 4.83 (d, J=6.36 Hz, 1 H), 5.24 (s, 1 H), 7.29 - 7.36 (m, 2 H), 7.40 (d, J=5.87 Hz, 1 H), 7.56 - 7.71 (m, 4 H), 7.98 (dd, J=8.80, 5.87 Hz, 1 H), 8.07 (d, J=7.83 Hz, 1 H), 8.09 (d, J=5.87 Hz, 1 H).

Preparation of compound 129: (2R,3S) and (2S,3R) 4-({3-[1-(7- fluoroisoquinolin-1-yl)piperidin-4-yl]-2,3-dihydroxypropyl}amino)-2H- chromen-2-one hydrochloride

Compound 129 was prepared as described hereinbelow.

129f 129e Compound 129, as a racemic mixture, was prepared as described for the synthesis of compound 128, using methyl (2Z)-3-[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl]prop-2-enoate as key intermediate. LC-MS (M-H⁺) = 464.3.

1 H NMR (500 MHz, DMSO-de) δ ppm 1 .58 - 1 .96 (m, 4 H), 2.05 (br. s., 1

H), 3.1 1 - 3.27 (m, 3 H), 3.33 (dd, J=7.83, 2.93 Hz, 1 H), 3.61 - 3.69 (m, 1 H), 3.71 - 3.79 (m, 1 H), 4.00 (br. s., 2 H), 5.23 (s, 1 H), 7.25 - 7.38 (m, 2 H), 7.53 (d, J=5.87 Hz, 1 H), 7.59 (t, J=7.58 Hz, 1 H), 7.72 - 7.77 (m, 1 H), 7.82 (d, J=9.78 Hz, 2 H), 7.93 (d, J=4.40 Hz, 1 H), 8.13 (d, J=7.83 Hz, 2 H)

Preparation of compound 130: Λ/-(1 ,3-benzodioxol-5-ylmethyl)-2-[4-

(isoquinolin-1-yl)piperazin-1-yl]ethanamine

Compound 1 30 was prepared according to the procedure described in step 4 of the synthesis of compound 2, using intermediate 75b and 1 ,3- benzodioxole-5-carbaldehyde (Y=25%). LC-MS (M-H⁺) = 391 .2.

1 H NMR (300 MHz, DMSO-d₆) δ = 8.09 (d, J = 5.7 Hz, 2 H), 8.08 (d, J =

8.5 Hz, 1 H), 7.89 (dd, J = 0.9, 8.3 Hz, 1 H), 7.71 (ddd, J = 1 .2, 6.8, 8.0 Hz, 1 H), 7.61 (ddd, J = 1 .2, 6.8, 8.4 Hz, 1 H), 7.40 (d, J = 5.4 Hz, 1 H), 7.05 (t, J = 1 .0 Hz, 1 H), 6.95 (d, J = 1 .0 Hz, 2 H), 6.02 (s, 2 H), 3.97 (s, 2 H), 3.34 (br. s., 4 H), 2.94 (t, J = 6.2 Hz, 2 H), 2.76 - 2.58 (m, 6 H).

Preparation of compound 136

Compound 136 was prepared as described herein below.

Step 1 - Synthesis of 3-cyclohexyl-3-({(2R)-3-[4-(7-fluoroisoquinolin-1 - yl)piperazin-1 -yl]-2-hydroxypropyl}amino)propanoic acid (formate salt, compound 1 36):

Compound 3-cyclohexyl-3-({(2R)-3-[4-(7-fluoroisoquinolin-1 -yl) piperazin- 1 -yl]-2-hydroxypropyl}amino)propanoic acid (formate salt) as a mixture of diastereoisomers was prepared according to the synthesis described for compound 139 using racemic 3-amino-3-cyclohexylpropanoic acid (Y=44%). LC-MS (M-H⁺) = 459.5. ¹ H NMR (500 MHz, DMSO-d6) δ ppm 0.86 - 1 .29 (m, 5 H), 1 .49 - 1 .79 (m, 6 H), 2.05 - 2.17 (m, 1 H), 2.25 (dt, J=16.14, 4.16 Hz, 1 H), 2.48 (d, J=7.34 Hz, 2 H), 2.62 - 2.79 (m, 5 H), 2.80 - 2.95 (m, 2 H), 3.28 (br. s., 4 H), 3.84 - 3.94 (m, 1 H), 7.45 (d, J=5.38 Hz, 1 H), 7.65 (td, J=8.68, 2.69 Hz, 1 H), 7.69 (d, J=9.78 Hz, 1 H), 8.01 (dd, J=9.05, 5.62 Hz, 1 H), 8.12 (d, J=5.87 Hz, 1 H), 8.15 (s, 1 H).

Preparation of compound 137

Compound 137 was prepared as described herein below.

Step 1 - Synthesis of 3-cyclohexyl-3-({(2S)-3-[4-(7-fluoroisoquinolin-1 - yl)piperazin-1 -yl]-2-hydroxypropyl}amino)propanoic acid (formate salt, compound 1 37):

Compound 3-cyclohexyl-3-({(2S)-3-[4-(7-fluoroisoquinolin-1 -yl) piperazin- 1 -yl]-2-hydroxypropyl}amino)propanoic acid (formate salt) as a mixture of diastereoisomers was prepared according to the synthesis described for compound 138 using racemic 3-amino-3-cyclohexylpropanoic acid. LC-MS (M-H⁺) = 459.5. ¹ H NMR (500 MHz, DMSO-d6) δ 1 H NMR (500 MHz, DMSO- d6) d ppm 0.82 - 1 .32 (m, 5 H), 1 .47 - 1 .80 (m, 6 H), 2.04 - 2.1 6 (m, 1 H), 2.20 - 2.28 (m, 1 H), 2.48 (d, J=6.85 Hz, 2 H), 2.72 (br. s., 5 H), 2.80 - 2.94 (m, 2 H), 3.29 (br. s., 4 H), 3.88 (d, J=5.87 Hz, 1 H), 5.02 (br. s., 1 H), 7.45 (d, J=5.38 Hz, 1 H), 7.65 (td, J=8.56, 2.45 Hz, 1 H), 7.69 (d, J=9.78 Hz, 1 H), 8.01 (dd, J=9.05, 5.62 Hz, 1 H), 8.12 (d, J=5.87 Hz, 1 H), 8.1 5 (s, 1 H).

Preparation of compound 138

Compound 138 was prepared as described herein below.

Step 1 - Synthesis of 7-fluoro-1 -(4-{[(2R)-oxiran-2-yl]methyl}piperazin-1 - yl)isoquinoline:

The title intermediate was prepared according to the procedure described for the synthesis of 7-fluoro-1 -(4-{[(2R)-oxiran-2-yl]methyl}piperazin-1 - yl)isoquinoline (see compound 1 39) using (R)-(+)-epichlorohydrin (Y=92%). LC-MS (M-H⁺) = 288.3

Step 2 - Synthesis of (2S)-cyclohexyl({(2S)-3-[4-(7-fluoroisoquinolin-1 - yl)piperazin-1 -yl]-2-hydroxypropyl}amino)acetic acid (formate salt, compound 1

Compound 138 was prepared according to the procedure described for compound 139 using 7-fluoro-1 -(4-{[(2R)-oxiran-2-yl]methyl}piperazin-1 - yl)isoquinoline and (2S)-amino(cyclohexyl)acetic acid (Y=27%). LC-MS (M- H⁺) = 445.2. ¹ H NMR (500 MHz, DMSO-d6) δ ppm 1 .09 - 1 .37 (m, 5 H), 1 .62 - 1 .85 (m, 6 H), 2.57 - 2.60 (m, 4 H), 2.73 - 2.89 (m, 5 H), 2.94 (dd, J=1 1 .98, 4.16 Hz, 1 H), 3.1 1 (d, J=4.40 Hz, 1 H), 3.34 (d, J=7.34 Hz, 4 H), 3.96 - 4.09 (m, 1 H), 7.52 (d, J=5.38 Hz, 1 H), 7.71 (td, J=8.68, 2.69 Hz, 1 H), 7.76 (dd, J=10.03, 2.20 Hz, 1 H), 8.07 (dd, J=9.29, 5.87 Hz, 1 H), 8.18 (d, J=5.87 Hz, 1 H), 8.21 (s, 1 H).

Preparation of compound 139

Compound 139 was prepared as described herein below.

Step 1 - Synthesis of ferf-butyl 4-(7-fluoroisoquinolin-1 -yl)piperazine-1 - carboxylate:

The title intermediate was prepared as described for the synthesis of intermediate 2c using 1 -chloro-7-fluoroisoquinoline and /V-Boc-piperazine (Y=94%). LC-MS (M-H⁺) = 332.3

Step 2 - Synthesis of 7-fluoro-1 -(piperazin-1 -yl)isoquinoline (trifluoroacetic acid salt):

The title intermediate was prepared as described for the synthesis of intermediate 28c using ieri-butyl 4-(7-fluoroisoquinolin-1 -yl)piperazine-1 - carboxylate (Y = quant.). LC-MS (M-H⁺) = 232.2 Step 3 - Synthesis of 7-fluoro-1 -(4-{[(2S)-oxiran-2-yl]methyl} piperazin-1 - yl)isoquinoline:

7-fluoro-1 -(piperazin-1 -yl)isoquinoline (trifluoroacetic acid salt, 448 mg, 1 .3 mmol) was dissolved in MeOH (1 2 ml_), NaOH (1 56 mg, 3.9 mmol) was added followed by (S)-(+)-epichlorohydrin (0.92 ml_, 1 1 .7 mmol) at -10 ^<Ό. The reaction mixture was stirred at the same temperature for 30 h then was placed at 4°C overnight. The mixture was filtered, the solid was washed with acetone and the organic phase was evaporated in vacuum. The residue was chromatographed on silica gel (SNAP 50, Cy:EtOAc:MeOH 6:3:1 ) to give 370 mg (98% yield) of 7-fluoro-1 -(4-{[(2S)-oxiran-2-yl]methyl}piperazin-1 - yl)isoquinoline as a yellow oil. LC-MS (M-H⁺) = 288.3

Step 4 - Synthesis of (2R)-cyclohexyl({(2R)-3-[4-(7-fluoroisoquinolin-1 - yl)piperazin-1 -yl]-2-hydroxypropyl}amino)acetic acid (formate salt, compound 1 39):

7-fluoro-1 -(4-{[(2S)-oxiran-2-yl]methyl}piperazin-1 -yl)isoquinoline (60 mg, 0.21 mmol), (2R)-amino(cyclohexyl)acetic acid (98 mg, 0.63 mmol) and DIPEA (1 50 μΙ_, 0.84 mmol) were dissolved in a mixture of isopropanol/water 1 :1 and stirred at 90 °C for 3 h. The solvent was evaporated in vacuo, the residue was treated with EtOAc and chromatographed on C18 (SNAP 30, gradient: acetonitrile + 0.1 % formic acid 0 - 23% in water + 0.1 % formic acid) to give 35 mg (0.07 mmol, 33% yield) of (2R)-cyclohexyl({(2R)-3-[4-(7- fluoroisoquinolin-1 -yl)piperazin-1 -yl]-2-hydroxypropyl}amino)acetic acid (formate salt). LC-MS (M-H⁺) = 445.2.

¹ H NMR (500 MHz, DMSO-d6) δ ppm 0.99 - 1 .36 (m, 5 H), 1 .53 - 1 .78 (m, 6 H), 2.47 (d, J=6.36 Hz, 2 H), 2.65 - 2.81 (m, 5 H), 2.86 (dd, J=1 1 .98, 4.16 Hz, 1 H), 3.02 (d, J=4.40 Hz, 1 H), 3.28 (br. s., 6 H), 3.88 - 4.00 (m, 1 H), 7.45 (d, J=5.87 Hz, 1 H), 7.65 (td, J=8.68, 2.69 Hz, 1 H), 7.70 (dd, J=10.27, 1 .96 Hz, 1 H), 8.01 (dd, J=9.05, 5.62 Hz, 1 H), 8.12 (d, J=5.87 Hz, 1 H), 8.17 (s, 1 H).

Preparation of compound 140

Compound 140 was prepared as described herein below.

Step 1 - Synthesis of (2S)-cyclohexyl({(2R)-3-[4-(7-fluoroisoquinolin-1 - yl)piperazin-1 -yl]-2-hydroxypropyl}amino)acetic acid (formate salt, compound 140):

Compound (2S)-cyclohexyl({(2R)-3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 - yl]-2-hydroxypropyl}amino)acetic acid (formate salt) was prepared according to the synthesis described for compound 139 using (2S)- amino(cyclohexyl)acetic acid (Y=1 5%). LC-MS (M-H⁺) = 445.3.

¹ H NMR (500 MHz, DMSO-d6) δ ppm 0.99 - 1 .39 (m, 5 H), 1 .54 - 1 .78 (m, 6 H), 2.52 - 2.54 (m, 2 H), 2.67 - 2.90 (m, 6 H), 3.06 (d, J=4.40 Hz, 1 H), 3.27 (br. s., 4 H), 3.88 - 3.99 (m, 1 H), 7.45 (d, J=5.38 Hz, 1 H), 7.65 (td, J=8.80, 2.45 Hz, 1 H), 7.70 (dd, J=1 0.03, 2.20 Hz, 1 H), 8.01 (dd, J=9.05, 5.62 Hz, 1 H), 8.1 1 (d, J=5.38 Hz, 1 H), 8.1 6 (s, 1 H).

Preparation of compound 141

Compound 141 was prepared as described herein below.

Step 1 - Synthesis of (3R)-3-({(2R)-3-[4-(7-fluoroisoquinolin-1 -yl) piperazin- 1 -yl]-2-hydroxypropyl}amino)-3-phenylpropanoic acid (formate salt, compound 141 ):

Compound (3R)-3-({(2R)-3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]-2- hydroxypropyl}amino)-3-phenylpropanoic acid (formate salt) was prepared according to the synthesis described for compound 1 39 using (3R)-3-amino- 3-phenylpropanoic acid (Y=19%). LC-MS (M-H+) = 453.4.

¹ H NMR (500 MHz, DMSO-d6) δ ppm 2.33 - 2.46 (m, 4 H), 2.55 - 2.73 (m, 6 H), 3.23 (br. s., 4 H), 3.78 - 3.85 (m, 1 H), 4.1 1 (dd, J=9.78, 4.89 Hz, 1 H), 7.25 - 7.31 (m, 1 H), 7.36 (t, J=7.58 Hz, 2 H), 7.39 - 7.47 (m, 3 H), 7.61 - 7.70 (m, 2 H), 8.00 (dd, J=8.80, 5.87 Hz, 1 H), 8.1 1 (d, J=5.87 Hz, 1 H).

Preparation of compound 142

Compound 142 was prepared as described herein below.

Step 1 - Synthesis of (3R)-3-({(2S)-3-[4-(7-fluoroisoquinolin-1 -yl) piperazin- 1 -yl]-2-hydroxypropyl}amino)-3-phenylpropanoic acid (formate salt, compound 142):

Compound (3R)-3-({(2S)-3-[4-(7-fluoroisoquinolin-1 -yl)piperazin-1 -yl]-2- hydroxypropyl}amino)-3-phenylpropanoic acid (formate salt) was prepared according to the synthesis described for compound 1 38 using (3R)-3-amino- 3-phenylpropanoic acid (Y=19%). LC-MS (M-H+) = 453.4.

¹ H NMR (500 MHz, DMSO-d6) δ ppm 2.33 - 2.56 (m, 5 H), 2.57 - 2.72 (m, 5 H), 3.23 (br. s., 4 H), 3.74 - 3.81 (m, 1 H), 4.08 (dd, J=1 0.03, 4.65 Hz, 1 H), 7.26 - 7.31 (m, 1 H), 7.36 (t, J=7.58 Hz, 2 H), 7.40 - 7.46 (m, 3 H), 7.61 - 7.70 (m, 2 H), 8.00 (dd, J=9.05, 5.62 Hz, 1 H), 8.1 1 (d, J=5.87 Hz, 1 H).

Preparation of compound 167

Compound 167 was prepared as described herein below.

Step 1 - Synthesis of 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 -yl) piperidin-4-yl]amino}ethyl)amino]propan-1 -ol (compound 167):

To a solution of methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl]amino}ethyl)amino]propanoate (compound 1 89, 45 mg, 0.1 mmol) in THF (10 mL) NaBH₄ (19 mg, 0.5 mmol) was added. The mixture refluxed for 1 h then MeOH (0.13 mL, 3.3 mmol) was added. After stirring 18 h at reflux the mixture was cooled to rt, concentrated under vacuum and partitioned between water and DCM. The organic phase was dried over Na₂S0₄ and concentrated under vacuum. The residue was purified by preparative HPLC (C18, CH₃CN from 30% to 65% in H₂0) to afford 3- cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4- yl]amino}ethyl)amino]propan-1 -ol (14 mg, 0.03 mmol, 30% yield over two steps). LC-MS (M-H⁺) = 429.3.

¹ H NMR (300MHz, CHLOROFORM-d) δ = 8.12 (d, J = 5.9 Hz, 1 H), 7.76 (dd, J = 5.6, 8.9 Hz, 1 H), 7.67 (dd, J = 2.6, 10.2 Hz, 1 H), 7.40 (dt, J = 2.6, 8.6 Hz, 1 H), 7.27 (d, J = 5.5 Hz, 1 H), 4.01 (ddd, J = 3.8, 5.8, 1 1 .6 Hz, 1 H), 3.86 - 3.63 (m, 2 H), 3.49 - 2.72 (m, 13 H), 2.20 (d, J = 1 1 .2 Hz, 2 H), 2.09 - 1 .54 (m, 8 H), 1 .45 - 0.87 (m, 4 H).

Preparation of compound 189

Compound 189 was prepared as described herein below.

Step 1 - Synthesis of ferf-butyl [1 -(7-fluoroisoquinolin-1 -yl)piperidin-4- yl]carbamate:

Potassium carbonate (4.6 g, 33 mmol) was added to a stirred solution of 1 -chloro-7-fluoroisoquinoline (5 g, 27.5 mmol) and ferf-butyl piperidin-4- ylcarbamate (13.8 g, 68.8 mmol) in methyl isopropyl ketone (50 mL) at room temperature. The resulting mixture was heated to reflux overnight. UPLC check showed the reaction was complete. The mixture was then allowed to cool to room temperature then water was added. The mixture was extracted with EtOAc, the combined organic phases were washed with brine and dried over sodium sulfate. The solvents were evaporated under reduced pressure and the resulting residue was purified by flash chromatography on silica gel (from 100% hexane to hexane/Ethyl acetate 8:2) to obtain ieri-butyl [1 -(7- fluoroisoquinolin-1 -yl)piperidin-4-yl]carbamate (7.1 g, 20.6 mmol, 75% yield). LC-MS (M-H⁺) = 346.2

Step 2 - Synthesis of 1 -(7-fluoroisoquinolin-1 -yl)-A-[(4- methoxyphenyl)methyl]piperidin-4-amine hydrochloride:

To a solution of ferf-butyl [1 -(7-fluoroisoquinolin-1 -yl)piperidin-4- yl]carbamate (5.5 g, 15.9 mmol) in dry DMF (50 mL) NaH (60% mineral oil, 1 .5 g, 31 .8 mmol) was added. After stirring 1 h at room temperature p- methoxybenzyl chloride (5 mL, 31 .8 mmol) was added. The reaction mixture was stirred at room temperature overnight then was poured into cold water (300 mL). The solution was extracted with EtOAc, the combined organic layers were washed with brine, dried over sodium sulfate and concentrated to dryness. The dried residue was dissolved in a solution of HCI in MeOH (1 .25 M, 200 mL) and the mixture was refluxed for 2 h. The solvent was removed under vacuum and the resulting residue was treated with isopropanol and dried to give 1 -(7-fluoroisoquinolin-1 -yl)-A-[(4- methoxyphenyl)methyl]piperidin-4-amine hydrochloride (5.6 g, 15.3 mmol, 96% yield). LC-MS (M-H⁺) = 366.2

Step 3 - Synthesis of A/-(2-chloroethyl)-1 -(7-fluoroisoquinolin-1 -yl)-A/-[(4- methoxyphenyl)methyl]piperidin-4-amine:

To a solution of 1 -(7-fluoroisoquinolin-1 -yl)-A-[(4- methoxyphenyl)methyl]piperidin-4-amine hydrochloride (2 g, 5 mmol) and DIPEA (0.9 ml_, 5 mmol) in acetonitrile (10 ml_) chloroacetaldehyde (50 wt % solution in H₂0, 2.5 ml_, 19.9 mmol) was added. After stirring 15' at rt, acetic acid (0.57 ml) and NaBH(OAc)₃ (2.1 g, 10 mmol) were added. The mixture was stirred 3 h at rt adjusting pH to 5-6 by addition of 0.1 M HCI. After addition of NaHC0₃ the solution was extracted with DCM, the organic phase was washed with water, treated with Na₂S0₄ and concentrated under vacuum. EtOAc was added to the residue, the resulting solid was filtered off and the solvent was evaporated under reduced pressure. A purification by flash chromatography (silica gel, CHCI₃/MeOH 9:1 ) afforded N-{2- chloroethyl)-1 -(7-fluoroisoquinolin-1 -yl)-N-[(4- methoxyphenyl)methyl]piperidin-4-amine (650 mg, 1 .5 mmol, 30% yield). LC- MS (M-H⁺) = 428.1

Step 4 - Synthesis of methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl][(4-methoxyphenyl)methyl] amino}ethyl) aminojpropanoate:

A solution of sodium iodide (39 mg, 0.26 mmol) and A/-(2-chloroethyl)-1 -(7- fluoroisoquinolin-1 -yl)-A/-[(4-methoxyphenyl)methyl] piperidin-4-amine (1 10 mg, 0.26 mmol) in DMF (5 mL) was stirred 1 5' then was cooled to 0 °C. Potassium carbonate (1 07 mg, 0.77 mmol) was added followed by one-pot addition of methyl 3-amino-3-cyclohexylpropanoate hydrochloride (1 14 mg, 0.51 mmol) in DMF (5 mL). The mixture was stirred at 90°C overnight then was cooled and poured into cold water. The solution was extracted with DCM, the organic phase was dried over Na₂S0₄ and concentrated under vacuum to give methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl][(4-methoxyphenyl)methyl]amino}ethyl)amino]propanoate (1 10 mg, 0.20 mmol, 77% yield), that was progressed without further purification. LC-MS (M-H⁺) = 577.3

Step 5 - Synthesis of methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl]amino}ethyl)amino]propanoate (compound 189):

A mixture of methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl][(4-methoxyphenyl)methyl]amino}ethyl)amino]propanoate (1 10 mg, 0.20 mmol) and TFA (1 ml_) in DCM (10 ml_) was refluxed overnight. After cooling to rt sat. NaHC0₃ was added, the resulting solution was extracted with EtOAc, the organic phase was dried over Na₂S0₄ and concentrated under vacuum. Purification by preparative HPLC (C-1 8, CH₃CN from 1 0% to 45% in H₂0) provided 58 mg (0.13 mmol, 65% yield) of methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 -yl)piperidin-4- yl]amino}ethyl)amino]propanoate. LC-MS (M-H⁺) = 457.3.

¹ H NMR (500 MHz, DMSO-d6/D₂0) δ ppm 0.94 - 1 .33 (m, 5 H), 1 .53 - 2.03 (m, 8 H), 2.22 (d, J=1 1 .1 1 Hz, 2 H), 2.77 - 2.90 (m, 2 H), 3.04 - 3.18 (m, 2 H), 3.32 - 3.52 (m, 6 H), 3.64 - 3.71 (m, 3 H), 3.72 - 4.22 (m, 2 H), 7.53 (d, J=5.90 Hz, 1 H), 7.66 - 7.80 (m, 2 H), 7.98 - 8.12 (m, 2 H).

Preparation of compound 190

Compound 190 was prepared as described herein below.

Step 1 - Synthesis of lithium 3-cyclohexyl-3-[(2-{[1 -(7-fluoro isoquinolin-1 - yl)piperidin-4-yl]amino}ethyl)amino]propanoate (compound 190):

To a solution of methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)piperidin-4-yl]amino}ethyl)amino]propanoate (compound 189, 60 mg, 0.13 mmol) in a mixture of THF (0.4 ml_), MeOH (0.2 ml_) and water (0.2 ml_) LiOHxH20 (1 1 mg, 0.26 mmol) was added. The reaction mixture was stirred at 55 °C for 20 minutes then the solvent was evaporated and the crude was purified by reverse cromatography (C-1 8, from 100% water to 7/3 water/acetonitrile) to obtain lithium 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin- 1 -yl)piperidin-4-yl]amino}ethyl)amino]propanoate (32 mg, 0.07 mmol, 56% yield). LC-MS (M-H⁺) = 443.3.

¹ H NMR (500 MHz, DMSO-d6) δ ppm 0.82 - 1 .29 (m, 5 H), 1 .41 - 1 .80 (m, 8 H), 1 .86 - 2.04 (m, 3 H), 2.09 (dd, J=15.30, 3.91 Hz, 1 H), 2.58 - 2.79 (m, 6 H), 2.93 (t, J=12.50 Hz, 2 H), 3.64 (d, J=12.49 Hz, 2 H), 7.41 (d, J=5.76 Hz, 1 H), 7.59 - 7.69 (m, 2 H), 7.99 (dd, J=8.71 , 5.69 Hz, 1 H), 8.09 (d, J=5.80 Hz, 1 H).

Preparation of compound 191

Compound 191 was prepared as described herein below.

Step 1 - Synthesis of 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)pipehdin-4-yl]amino}ethyl)amino]-/V-hydroxypropanamide (compound 191 ):

To a stirred solution of hydroxylamine hydrochloride (2.6 g, 40 mmol) in MeOH (12 ml_) a solution of potassium hydroxide (3.3 g, 59 mmol) in MeOH (7 ml_) was added slowly at 0°C. After addition, the mixture was stirred for 30 minutes at 0°C then was allowed to reach room temperature. The resulting precipitate was filtered and the solution of free hydroxylamine was placed in 1 00 ml_ flask. Methyl 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 -yl)piperidin- 4-yl]amino}ethyl)amino]propanoate (compound 189, 87 mg, 0.19 mmol) in MeOH (5 ml_) was added to this solution. The resulting solution was degassed at 0 °C for 30 minutes then was concentrated to dryness. The residue was dissolved in water, the aqueous phase was acidified to pH 5 by adding 1 M HCI and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate and concentrated. The residue was purified by reverse chromatography (C-18, from 100% water to 8:2 water/acetonitrile) to give 3-cyclohexyl-3-[(2-{[1 -(7-fluoroisoquinolin-1 - yl)pipehdin-4-yl]amino}ethyl)amino]-/V-hydroxypropanamide (13 mg, 0.028 mmol, 1 5% yield). LC-MS (M-H⁺) = 458.3.

¹ H NMR (400 MHz, DMSO-d6) δ ppm 0.92 - 1 .81 (m, 12 H), 1 .31 - 1 .47 (m, 1 H), 1 .90 - 2.1 6 (m, 4 H), 2.65 - 2.72 (m, 3 H), 2.74 - 2.81 (m, 2 H), 2.81 - 2.89 (m, 1 H), 2.88 - 2.99 (m, 2 H), 3.69 (d, J=12.72 Hz, 2 H), 7.43 (d, J=5.26 Hz, 1 H), 7.60 - 7.70 (m, 2 H), 8.00 (dd, J=9.87, 5.92 Hz, 1 H), 8.07 - 8.12 (m, 1 H).

EXAMPLES

Example 1 Inhibition of DNA gyrase or Topo IV in E.coli and S. aureus

The above compounds were tested for the inhibition of the enzyme DNA gyrase in a gyrase supercoiling assay and for the inhibition of the enzyme topoisomerase IV in a decatenation assay, in both Gram positive and Gram negative bacteria, according to the following methods.

Both the assays were carried out according to a set-up method modified from the article to Blanche F, et al. "Differential Behaviors of Staphylococcus aureus and Escherichia coli Type II DNA Topoisomerases", Antimicrob. Agents Chemother., 1996, Vol. 40, No. 12 p. 2714-2720.

The compounds were screened at single concentration (200, 100 or 50 μΜ), in duplicate.

Ciprofloxacin and novobiocin were used as reference compounds, at single concentration of 200 and 50 μΜ, respectively.

DNA gyrase supercoiling assay.

Reagents from S.aureus and E.coli Gyrase Supercoiling Assay kits

(Inspiralis, UK) were used. A master mix with a total volume sufficient for the number of reactions to perform was prepared with the following reagents: 5x assay buffer, relaxed pBR322 substrate (0.5 μg/reaction), RNase-DNase free water. Aliquotes of this mix were dispensed in each tube, then 10x compound stock solutions or vehicle control (DMSO), were added to each reaction tube.

Reaction was started with E. Coli (2 U/reaction) or S.aureus (1 U/reaction) gyrase enzyme addition.

A sample added with an equal volume of dilution buffer was used as negative control (without enzyme).

The reaction tubes were gentle vortexed and incubated 30 minutes at

37 °C. Each reaction was stopped by adding 30 μΙ of Stop Buffer and 30 μΙ chloroform/isoamyl alcohol (24/1 ), briefly vortexed for 5-10 seconds and centrifuged at 20000xg for 2 minutes. Samples were loaded onto 1 % agarose gel and subjected to electrophoresis for 1 hour at 80V constant voltage in TAE (40 mM Tris-acetate, 2 mM EDTA).

Data acquisition and analysis. Treatment of relaxed pBR322 with DNA gyrase converted the relaxed topoisomers (DNAs of different linking number) to the supercoiled form of the plasmid, which migrates faster on an agarose gel. An upper band might also be visible, which consists of open-circular (nicked) DNA which is present in the relaxed substrate but co-migrates with some of the relaxed topoisomers.

Bands were visualized by ethidium bromide staining (dil 1 :20000) for 30 minutes followed by destaining in distilled water for 10 minutes.

In order to evaluate the compounds activity on the enzyme, the bands of supercoiled DNAs in the gel were photographed by a digital imaging system ImageQuant LAS 4000 (GE Healthcare) according to manufacturer's instructions.

The fluorescent intensity of each band was analysed by ImageQuant TL software and it was expressed as volume (volume of the uncalibrated quantity of material in the image feature after subtraction of the background intensity by using rolling ball method).

Each band intensity was compared, as percentage, to vehicle sample band intensity, which served as positive control, on the same gel.

Inhibitory activity was expressed as percent of inhibition versus the positive control.

The results are summarized in the following Table 2.

Topoisomerase IV decatenation assay

S.aureus and E.coli Topoisomerase IV decatenation kits (Inspiralis, UK) were used. A master mix with a total volume sufficient for the number of reactions to perform was prepared with the following reagents: 5x assay buffer (50 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate,10 mM magnesium acetate, 10 mM DTT, 1 mM ATP, 50 μg/ml albumin), kDNA substrate (200 ng/reaction), RNase-DNase free water. Aliquots of this mix were dispensed in each tube, then 1 0x compound stock solutions or vehicle control (DMSO), were added in each reaction tube.

Reaction was started with Topoisomerase IV enzyme (0.5 U/reaction) addition.

The reaction tubes were gentle vortexed and incubated 30 minutes at 37 °C. Each reaction was stopped by adding 30 μΙ of Stop Buffer and 30 μΙ of chloroform/isoamyl alcohol (24/1 ), briefly vortexed for 5-10 seconds and centrifuged at 20000xg for 2 minutes. Samples taken from the upper phase were loaded into 1 % agarose gel and subjected to electrophoresis for 1 hour at 80V constant voltage in TAE (40 mM Tris-acetate, 2 mM EDTA).

Data acquisition and analysis. Due to the high molecular mass, kDNA could not enter an agarose gel under normal electrophesis conditions, but remained in the wells. In the presence of Topo IV topoisomerase mini-circles (2.5 Kb) were released from kDNA by decatenation and were quickly and easily resolved in the gel at relatively high voltages.

For single concentration screening assay, in order to evaluate the compounds activity on the enzymes, the bands of decatenated DNAs in the gel were photographed by a digital imaging system ImageQuant LAS 4000 (GE Healthcare) according to manufacturer's instructions.

The fluorescent intensity of each band was analyzed by ImageQuant TL software and it was expressed as volume (volume of the uncalibrated quantity of material in the image feature after subtraction of the background intensity by using rolling ball method).

The results are summarized in the following Table 2.

Table 2

E.coli S. aureus

Compound

% inhibition % inhibition % inhibition % inhibition No

DNA gyrase Topo IV DNA gyrase Topo IV

2 n/a 65 n/a n/a

3 59 n/a n/a n/a

4 n/a 53 84 n/a

21 n/a n/a 67 n/a

31 n/a n/a 65 n/a E.coli S. aureus

Compound

% inhibition % inhibition % inhibition % inhibition No

DNA gyrase Topo IV DNA gyrase Topo IV

35 n/a n/a 78 n/a

36 n/a n/a 90 n/a

38 n/a n/a 63 n/a

39 50 n/a 74 n/a

42 n/a n/a 84 n/a

43 n/a n/a 100 n/a

45 n/a n/a 81 n/a

50 92 n/a n/a n/a

53 64 n/a 57 n/a

58 100 n/a n/a n/a

60 100 n/a 89 n/a

68 100 n/a n/a n/a

70 86 n/a 75 n/a

71 56 n/a n/a n/a

74 65 n/a 61 n/a

75 69 n/a 61 n/a

81 80 n/a n/a n/a

82 85 n/a 82 n/a

83 69 n/a n/a n/a

85 78 n/a 86 n/a

87 79 n/a 96 n/a

96 88 n/a 91 n/a

97 85 n/a 86 n/a

1 01 100 n/a 100 n/a

1 07 92 n/a 74 n/a

1 17 75 n/a n/a n/a

1 18 78 n/a n/a n/a E.coli S. aureus

Compound

% inhibition % inhibition % inhibition % inhibition No

DNA gyrase Topo IV DNA gyrase Topo IV

119 n/a n/a 60 n/a

120 n/a n/a 76 n/a

122 58 n/a n/a n/a

123 52 n/a n/a n/a

128 n/a n/a 66 n/a

129 69 n/a n/a n/a

130 n/a n/a 74 n/a

132 n/a n/a 53 n/a

133 53 n/a 58 n/a

136 57 n/a 75 n/a

137 57 n/a 75 n/a

138 n/a n/a 89 n/a

139 n/a n/a 54 n/a

140 68 n/a 92 n/a

141 n/a n/a 65 n/a

142 n/a n/a 52 n/a

167 n/a n/a 70 n/a

189 n/a n/a 84 n/a

190 61 n/a 73 n/a

191 n/a n/a 81 n/a n/a = not active

The above results showed that the exemplified compounds effectively inhibited DNA gyrase or topoisomerase IV in E.coli, which is a Gram positive bacterium, and/or S.aureus, which is a Gram negative bacterium.

Claims

1 . A compound of formula (I)

wherein

A is a 1 0-membered fused bicyclic ring;

Li is σ bond, -O- or -N(R')-, wherein FT is H or (Ci_-3)alkyl;

B is a divalent residue of a piperazine or piperidine ring;

L₂ is σ bond, -CH₂-, -0-, or -N(R')-, wherein R' is H or

s)alkyl;

Y is a (Ci-₆)alkylenyl, (C₂-₆)alkenylenyl, (C₂-₆)alkynylenyl, or (C₃- 6)cycloalkylenyl group, said group being optionally substituted with one or more groups selected from -OH, (Ci-₃)alkyl, (C₃-₆)cycloalkyl, 3- to 5-membered oxacycloalkyl, -COOR', -NR'R" wherein R' and R", identical or different each other, are hydrogen atom or (Ci-₃)alkyl;

L₃ is -O-, -(CH₂)₂-, -N(R')-, -N(R')-C(=O)-, -N(R')-C(=S)-, -N(R')-(Ci- 6)alkylenyl-, -C(=O)-N(R')-, -C(=O)-N(R')-(Ci-₆)alkylenyl-, -SO₂- N(R')-, -N(R')-SO₂-, wherein R' is hydrogen atom or (Ci-₃)alkyl;

and addition salts with pharmaceutically acceptable organic or inorganic acids or basis, enantiomers, N-oxides and quaternary ammonium salts of said compound of formula (I).

2. The compound according to claim 1 , wherein A is a fused bicyclic ring having the following formula (II):

wherein

G1 and G2, identical or different each other, are CH or N; Ri is H, halogen atom, CN or CF₃;

R₂ is H, halogen atom, OH, CN, CF₃, (Ci-₆)alkyl or (Ci-₆)alkoxy;

D is a divalent grou selected from

wherein R₃ is H, (Ci-₆)alkyl or (Ci-₆)alkoxy.

3. The compound according to claim 1 , wherein B is a group having the following formula (III)

— G3

/ wherein

G3 and G4, identical or different each other, are C(R₄) or N, provided that that at least one of G3 and G4 is N,

R is hydrogen atom, -OH, -CN, -COOH, -NR'R" wherein R' and R", identical or different each other, are hydrogen atom or (Ci_-3)alkyl;

G5 is C(=0) or C(H)(R₅);

wherein R₅ is hydrogen atom, CF₃, -(Ci_-3)alkyl-CF₃, -COOR' and - CONR'R", wherein R' and R", identical or different each other, are hydrogen atom or (Ci_-3)alkyl.

4. The compound according to claim 1 , wherein C has one of the following formulae (I (VI), (VII) or

(V) (VI)

(VII) (VI II)

wherein

n is an integer from 0 to 3

Pi and P₂, equal or different each other, are CH or N

P₃ is O, S or NH;

R₈ and R₉, equal or different each other, are a hydrogen atom or an aryl group, or together form a 5- or 6-membered aliphatic or aromatic ring, optionally comprising at least one heteroatom selected from N and O,

Rio and R , equal or different each other, are a hydrogen atom, or a (Ci- ₃)alkyl group, or together form a 5- or 6-membered aliphatic or aromatic ring, and wherein each hydrogen atom linked to a carbon or nitrogen atom forming the ring of formulae (IV), (V), (VI), (VII) or (VIII) is optionally substituted with at least one substituent selected from the group consisting of halogen atom, OH, (Ci-₃)alkyl or (Ci-₃)alkoxy.

5. The compound according to claim 4, wherein R₆ and R₇ together form a ring selected from the group consisting of benzene, furan, tetrahydrofuran, dioxolane, piperidine and piperazine.

6. The compound according to claim 4, wherein R₈ and R₉, equal or different each other, are a hydrogen atom or an aryl group, or together form a ring selected from the group consisting of benzene, pyridine, and piperidine.

7. The compound according to claim 1 , wherein C is a group having one of the following formulas:

8. The compound according to claim 1 , wherein said Y is a (Ci-₄)alkylenyl, (C₂-₄)alkynylenyl, or (C₅-₆)cycloalkylenyl group, said group being optionally substituted with one or more groups selected from -OH, methyl, (C_3- )cycloalkyl, 3- or 4-membered oxacycloalkyl, -COOH, -NR'R" wherein R' and R", identical or different each other, are hydrogen atom or methyl.

9. The compound according to claim 8, wherein said Y is a (Ci-₄)alkylenyl, (C₂-₄)alkynylenyl, or cyclohexylenyl group, said group being optionally substituted with one or more hydroxy groups.

10. The compound according to claim 1 , wherein said L₃ is -O-, -(CH₂)2-, - N(R')- wherein R' is H or (d-₃)alkyl, -NH-C(=O)-, -NH-(Ci-₃)alkylenyl-, -

C(=0)-NH- or -C(=O)-NH-(Ci-₃)alkylenyl-.

1 1 . The compound according to claim 10, wherein said L₃ is -O-, -(CH₂)2-, -N(R')- wherein R' is H or methyl, -NH-C(=O)-, -NH-CH₂-, -C(=O)-NH-, - C(=O)-NH-CH₂-.

12. A pharmaceutical composition comprising at least one compound of formula (I) according to any of the preceding claims 1 to 1 1 , a salt thereof with a pharmaceutically acceptable organic or inorganic acid or base, or an enantiomer thereof, or a quaternary ammonium salt thereof, or a N-oxide thereof, and at least one inert pharmaceutically acceptable excipient.

13. The compound of formula (I) according to any of the preceding claims 1 to 1 1 , for use in medicine.

14. The compound of formula (I) according to any of the preceding claims 1 to 1 1 , for use in the treatment of bacterial infections.

15. The compound of formula (I) according to any of the preceding claims 1 to 1 1 for use according to claim 14, wherein said bacterial infections is selected from the group consisting of a skin infection, a mucosal infection, a gynaecological infection, a respiratory tract infection (RTI), a CNS infections, a gastro-intestinal infection, a bone infection, a cardiovascular infection, a sexually transmitted infection, or a urinary tract infection.

16. A method for treating a bacterial infection, comprising the administration of the compound of formula (I) according to any of the preceding claims 1 to 1 1 to a patient in need thereof.