Particularly preferred in this group of compounds is at least

3-{1-[4-Amino-3-(5- hydroxypyridin-3-yl)-1H- pyrazolo[3,4-d]pyrimidin-1- yl]ethyl}-4-{3-[(2R)-1- methylpyrrolidin-2-yl]phenyl}-1H- isochromen-1-one;

[Image Omitted]

A further preferred group of compounds is that of formula (I) wherein:

- R₁and R₂are (C₁-C₆) alkyl which is methyl,
- R₃and R₄are independently H or (C₁-C₆) alkyl which is methyl; and
- R, R₅, R₆and p are as defined above;
- or pharmaceutically acceptable salts thereof.

Particularly preferred in this group of compounds is at least one of the compounds selected from those listed in the following table:

3-(1-(4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4- (3-((dimethylamino)methyl)phenyl)-1H- isochromen-1-one;

[Image Omitted]

3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4- {4-chloro-3- [(dimethylamino)methyl]phenyl}-1H- isochromen-1-one;

[Image Omitted]

3-(1-(4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4- (3-(1-(dimethylamino)ethyl)phenyl)-1H- isochromen-1-one;

[Image Omitted]

A further preferred group of compounds is that of formula (I) wherein:

- R is —H;
- R₅is —OH;
- R₆is H;
- R₁, R₂, R₃and R₄are as defined above;
  - or pharmaceutically acceptable salts thereof.

According to preferred embodiments, the present invention relates to at least one of the compound selected from those listed in the following table:

3-{1[4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4- {3-[(4-methylpiperazin-1- yl)methyl]phenyl}-1H-isochromen-1-one;

[Image Omitted]

3-(1-(4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4- (3-((dimethylamino)methyl)phenyl)-1H- isochromen-1-one;

[Image Omitted]

3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4- {4-chloro-3- [(dimethylamino)methyl]phenyl}-1H- isochromen-1-one;

[Image Omitted]

3-{1-[4-Amino-3-(5- hydroxypyridin-3-yl)-1H- pyrazolo[3,4-d]pyrimidin-1- yl]ethyl}-4-{3-[(2R)-1- methylpyrrolidin-2-yl]phenyl}-1H- isochromen-1-one;

[Image Omitted]

3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4- {4-fluoro-3-[(4-methylpiperazin-1- yl)methyl]phenyl}-1H-isochromen-1-one;

[Image Omitted]

3-(1-(4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4- (3-fluoro-5-((4-methylpiperazin-1- yl)methyl)phenyl)-1H-isochromen-1-one;

[Image Omitted]

3-(1-(4-Amino-3-(5-hydroxypyridin-3-yl)- 1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4- (3-(1-(dimethylamino)ethyl)phenyl)-1H- isochromen-1-one;

[Image Omitted]

and enantiomer or diastereoisomer mixtures in any proportion or single enantiomers or single diastereoisomers and pharmaceutically acceptable salt and solvates thereof.

Preferred salts according to the invention are selected from

- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Hydrobromide;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Hydrochloride;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Hemi 1,5-Naphthalenedisulfonate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Hemi sulfate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Tosylate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Mesylate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one 2-Naphthalene sulfonate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Isethionate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Maleate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Esylate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Hemipamoate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Xinafoate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Salycilate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one Benzoate.

Other preferred salts according to the invention are selected from

- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethylamino)methyl)phenyl)-1H-isochromen-1-one Hydrobromide;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethylamino)methyl)phenyl)-1H-isochromen-1-one Hydrochloride;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethyl amino)methyl)phenyl)-1H-isochromen-1-one Mesylate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethylamino)methyl)phenyl)-1H-isochromen-1-one 2-Naphthalene sulfonate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethyl amino)methyl)phenyl)-1H-isochromen-1-one Maleate;
- (R) and/or (S)-3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethylamino)methyl)phenyl)-1H-isochromen-1-one Esylates.

In one embodiment the present invention provides a method for treating respiratory disorders comprising administering an effective amount of a compound according to the invention or a pharmaceutically acceptable salt thereof to a patient in need thereof.

In one aspect, the present invention refers to a compound of formula (I) for use as a medicament.

In one aspect the present invention provides the use of a compound of the invention for the manufacture of a medicament.

The compounds of formula (I) including all the compounds here above listed can be generally prepared according to the procedure outlined in detail in the Schemes shown below using generally known methods.

- Schemes

[Image Omitted]

Scheme 1 provides a synthetic route for the preparation of Examples 1 and 2. Intermediate compounds of formula (VII), can be converted into Intermediates (VIII) by cross-coupling reactions, such as a Suzuki cross-coupling. Intermediates (VIII), wherein R′ is a formyl group, can be converted into corresponding amines by reductive amination with suitable amines, such as 1-methylpiperazine or dimethylamine, in presence of reducing agents such as for example sodium triacetoxyborohydride. Intermediates (IV), where X is a suitable leaving group (Lg) such as a halide atom, can be prepared from Intermediates (III) through substitution of the hydroxyl group promoted by suitable halogenating agents such as for example PBr₃. Intermediates (VI) can be prepared by reaction of Intermediates (IV) with a suitable nucleophile, such as a nitrogen based nucleophile of formula (V) wherein R″ is a protected hydroxypyridine e.g. methoxypyridine. Compounds (VI) can be deprotected in acidic conditions to give compound of formula (I) wherein R₃, R₄are H, R₆is H, R₅is —OH and R is H. Compounds of general structure (Ia) can be prepared from the corresponding racemic compound of formula (I) by means of chiral separation.

[Image Omitted]

Examples 3-7 and 4a can be synthesized as outlined in Scheme 2, by using a methodology similar to the one described in Scheme 1. Intermediates of general formula (IIa), wherein (R)p is at least one suitable substituent as defined above, can be prepared from Intermediate (VII) and proper synthesized boronic acids or esters (e.g. pinacol esters) of formula (IX) where R′″ is a formyl or protected formyl group. Intermediates (IIa) can be converted in Intermediates (X), where X represents a suitable Lg such as halide atom, by reaction with PBr₃, and subsequently reacted with commercially available 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4-amine to give Intermediates (XI). Intermediates (XII), wherein (R)p is a suitable substituent as above and R₄=R₃=H, can be prepared from Intermediates (XI) by de-protection of the aldehydic function through well-known procedures, followed by reductive amination with suitable amines such as for example 1-methylpiperazine or dimethylamine, in the presence of a reducing agent such as for example sodium triacetoxyborohydride (Scheme 2, step 4a,b). In another embodiment of the present invention Intermediates (XII), wherein R₁=R₂=R₄=H and R₃is a suitable (C₁-C₆) alkyl substituent, can be prepared from Intermediate (VII) by means of a Suzuki coupling with suitable commercially available boronic acid or synthesized boronate esters, followed by the conversion of OH into a Lg such as a halide atom by reaction with PBr₃and finally nucleophilic substitution with commercially available 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4-amine, according to Scheme 2, step 5a-c. Some compounds obtained after step 5 may contain a protected amino group which can be de-protected under well-known procedures and methylated through reductive amination to give Intermediates (XII) (Scheme 2, step 6a,b). Intermediates (XII) can be converted into compounds of general formula (I), wherein R₆is H, R₅is OH, by means of a Suzuki coupling with commercially available 5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)pyridin-3-ol. Compounds of general structure (Ia) can be prepared from the corresponding racemic compound of formula (I) by means of chiral separation. E.g. enantiomerically pure Example 4a was prepared from the corresponding diasteromeric mixture by means of chromatographic separation.

In a specific aspect the present invention relates to compounds of formula (II)

[Image Omitted]

wherein

when R=H, R₃and R₄=H, K=R″=metoxypyridine; p, R₁and R₂are as defined above (corresponding to compound (VI) in scheme 1)

In a further aspect the present invention relates to the use of compounds of formula (II)

[Image Omitted]

- wherein
- when R=H, R₃and R₄=H, K=R″=metoxypyridine; p, R₁and R₂are as defined above (corresponding to compound (VI) in scheme 1)
- or
- when K=I; p, R, R₁, R₂, R₃, R₄are as defined above (corresponding to compound compound (XII) in scheme 2)
- as intermediate in the preparation of compounds of formula (I) as above described.

The use of compounds of formula (II) as intermediate in the preparation of compounds of formula (I) is particularly useful in a process wherein compounds of formula (II) undergo subsequent steps of deprotection or coupling; followed by a final step of chiral separation.

The compounds of the invention are inhibitors of kinase activity, in particular PI3-kinase activity. Generally speaking, compounds which are PI3K inhibitors may be useful in the treatment of many disorders.

In one embodiment, the disorders that can be treated by the compounds of the present invention are respiratory diseases selected from cough such as idiopathic chronic cough, cough-variant asthma, cough associated with thoracic tumour or lung cancer, viral or post-viral cough, upper airways cough syndrome (UACS), post nasal drip cough, cough associated gastro-oesophageal reflux disease (both acid and non acid reflux), asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and interstitial lung disease (such as idiopathic pulmonary fibrosis (IPF)).

In a further embodiment, the disorder is selected from asthma, chronic obstructive pulmonary disease (COPD).

In a further embodiment, the disorder is selected from idiopathic pulmonary fibrosis (IPF), cough and chronic cough.

The methods of treatment of the invention comprise administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a patient in need thereof. As used herein, “effective amount” in reference to a compound of formula (I) or a pharmaceutically acceptable salt thereof or other pharmaceutically-active agent means an amount of the compound sufficient to treat the patient's condition but low enough to avoid serious side effects and it can nevertheless be routinely determined by the skilled artisan. The compounds of formula (I) or pharmaceutically acceptable salts thereof may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. Typical daily dosages may vary depending upon the particular route of administration chosen.

The invention also provides pharmaceutical compositions of compounds of formula (I) in admixture with one or more pharmaceutically acceptable carrier or excipient, for example those described in Remington's Pharmaceutical Sciences Handbook, XVII Ed., Mack Pub., N.Y., U.S.A.

Administration of the compounds of the present invention and their pharmaceutical compositions may be accomplished according to patient needs, for example, orally, nasally, parenterally (subcutaneously, intravenously, intramuscularly, intrasternally and by infusion), by inhalation, rectally, vaginally, topically, locally, transdermally, and by ocular administration.

Various solid oral dosage forms can be used for administering compounds of the invention including such solid forms as tablets, gelcaps, capsules, caplets, granules, lozenges and bulk powders. The compounds of the present invention can be administered alone or combined with various pharmaceutically acceptable carriers, diluents (such as sucrose, mannitol, lactose, starches) and known excipients, including suspending agents, solubilizers, buffering agents, binders, disintegrants, preservatives, colorants, flavorants, lubricants and the like. Time release capsules, tablets and gels are also advantageous in administering the compounds of the present invention.

Various liquid oral dosage forms can also be used for administering compounds of the invention, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable known inert diluents such as water and suitable known excipients such as preservatives, wetting agents, sweeteners, flavorants, as well as agents for emulsifying and/or suspending the compounds of the invention. The compounds of the present invention may be injected, for example, intravenously, in the form of an isotonic sterile solution. Other preparations are also possible.

For the treatment of the diseases of the respiratory tract, the compounds according to the invention are preferably administered by inhalation.

Inhalable preparations include inhalable powders, propellant-containing metering aerosols or propellant-free inhalable formulations and may be administered through a suitable inhalation device which may be respectively selected from dry powder inhaler, pressurized metered dosed inhaler, or a nebulizer.

For administration as a dry powder, single- or multi-dose inhalers known from the prior art may be utilized. In that case the powder may be filled in gelatine, plastic or other capsules, cartridges or blister packs or in a reservoir. A diluent or carrier, e.g. lactose or any other additive suitable for improving the respirable fraction may be added to the powdered compounds of the invention.

Inhalation aerosols containing propellant gas such as hydrofluoroalkanes may contain the compounds of the invention either in solution or in dispersed form. The propellant-driven formulations may also contain other ingredients such as co-solvents, stabilizers and optionally other excipients.

The propellant-free inhalable formulations comprising the compounds of the invention may be in form of solutions or suspensions in an aqueous, alcoholic or hydroalcoholic medium and they may be delivered by jet or ultrasonic nebulizers known from the prior art or by soft-mist nebulizers such as Respimat®, a registered trademark of Boehringer Ingelheim Pharmaceuticals (Wachtel, H., Kattenbeck, S., Dunne, S. et al. Pulm Ther (2017) 3:19. doi. org/10.1007/s41030-017-0040-8).

According to a particularly preferred embodiment the compositions of the invention are in the form of a dry powder formulation, wherein a carrier is present comprising fine and/or coarse particles of one or more pharmaceutically acceptable excipient, and further optionally an additive is present with lubricant or anti-adherent properties.

Preferably the carrier coarse particles have a mass diameter comprised between 30 and 500 micron.

The carrier is generally a crystalline sugar selected from the group consisting of glucose, arabinose, maltose, saccharose, dextrose and lactose or a polyalcohol selected from the group consisting of mannitol, maltitol, lactitol and sorbitol; and the additive materials are selected form the group consisting of amino acids, water soluble surface active agents, lubricants and glidants.

Preferably the carrier particles are particles of of alpha-lactose or beta-lactose, even more preferably are particles of alpha-lactose monohydrate.

Another preferred embodiment of the invention is a dry powder inhaler device filled with a pharmaceutical formulation in the form of a dry powder formulation as above defined.

A further preferred embodiment is a kit comprising a pharmaceutical formulation as defined above and a dry powder inhaler device.

The dry powder formulation according to the invention may be prepared according to known methods such as those described in WO0053157A1 and WO 96/23485

According to an alternative preferred embodiment the composition of the invention are in the form of a propellant-free pharmaceutical formulation for administration to the lung via nebulization wherein, the said compound or salt thereof, is dissolved or suspended in an aqueous vehicle, optionally comprising further pharmaceutically acceptable excipients.

Thus, a further preferred embodiment is a kit comprising a pharmaceutical formulation, that is a solution or a suspension of the compounds of the invention as above indicated, and a nebulizer.

The compounds of the invention can be administered as the sole active agent or in combination with other pharmaceutical active ingredients including those currently used in the treatment of respiratory disorders, and known to the skilled person.

The dosages of the compounds of the invention depend upon a variety of factors including the particular disease to be treated, the severity of the symptoms, the route of administration, the frequency of the dosage interval, the particular compound utilized, the efficacy, toxicology profile, and pharmacokinetic profile of the compound.

The following examples illustrate the invention without limiting its scope.

PREPARATIONS OF INTERMEDIATES AND EXAMPLES

Chemical Names of the compounds were generated with Structure To Name Enterprise 10.0 Cambridge Software.

Abbreviations

Et₂O=diethyl ether; Et₃N=triethyl amine; DCE=1,2-dichloroethane; TEA=triethyl amine; DCC=N,N′-Dicyclohexylcarbodiimide; HOBt=Hydroxybenzotriazole; HATU=(Dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminium hexafluorophosphate; HBTU=N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; EDC=1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; DMAP=4-dimethylaminopyridine; DMF=dimethylformamide; EtOAc=Ethyl acetate; RT=room temperature; THF=tetrahydrofuran; DCM=dichloromethane; MeOH=methyl alcohol; EtOH=ethylic alcohol; LHMDS=Lithium bis(trimethylsilyl)amide; m-CPBA=meta-Chloroperoxybenzoic acid; TFA=Trifluoroacetic acid; LC-MS=Liquid Chromatography/Mass Spectrometry; HPLC=high pressure liquid chromatography; MPLC=medium pressure liquid chromatography; SFC=Supercritical Fluid Chromatography; dppf=1,1′-Bis(diphenylphosphino) ferrocene; X-Phos-Pd-G2=Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II); S-Phos-Pd-G2=Chloro(2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II); DIEA or DIPEA=N,N-Diisopropylethylamine; MeCN=Acetonitrile; MTBE=tert-Butyl methyl ether; Ac2O=acetic anhydride; AcCl=acetyl chloride; HBTU=N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; TBDMSCl=tert-Butyl(chloro)dimethylsilane; DMSO=Dimethylsulfoxide; BoC2O=di-tert-butyl dicarbonate; UPLC=Ultra Performance Liquid Chromatography.

General Experimental Details

¹H-NMR spectra were performed on a Varian MR-400 spectrometer operating at 400 MHZ (proton frequency), equipped with: a self-shielded Z-gradient coil 5 mm 1H/nX broadband probe head for reverse detection, deuterium digital lock channel unit, quadrature digital detection unit with transmitter offset frequency shift, or on AgilentVNMRS-500 or on a Bruker Avance 400 spectrometers. Chemical shift are reported as δ values in ppm relative to trimethylsilane (TMS) as an internal standard. Coupling constants (J values) are given in hertz (Hz) and multiplicities are reported using the following abbreviation (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br. s=broad, nd=not determined).

LC/UV/MS Analytical Methods

LC/MS retention times are estimated to be affected by an experimental error of +0.5 min. LCMS may be recorded under the following conditions: diode array DAD chromatographic traces, mass chromatograms and mass spectra may be taken on UPLC/PDA/MS Acquity™ system coupled with Micromass ZQ™ or Waters SQD single quadrupole mass spectrometer operated in positive and/or negative electron spray ES ionization mode and/or Fractionlynx system used in analytical mode coupled with ZQ™ single quadrupole operated in positive and/or negative ES ionisation mode. Quality Control methods used operated under low pH conditions or under high pH conditions.

Method 1. Aquity UPLC-QDa Mass Spectrometer with a C18-reverse-phase column (50×2.1 mm Acquity CSH with 1.7 μm particle size) maintained at 40° C., elution with A: 95/5 water/acetonitrile+0.05% formic acid; B: 95/5 acetonitrile/water+0.05% formic acid.

- Gradient:

Gradient-Time
flow (mL/min)
% A
% B

0.00
1
99.0
1.0

1.50
1
0.1
99.9

1.90
1
0.1
99.9

2.00
1
99.0
1.0

- Detection-MS, UV PDA
- MS ionisation method-Electrospray (positive/negative ion).

Method 2, low pH conditions. Column: Acquity UPLC BEH C18, 1.7 μm, 2.1×50 mm, the column temperature was 40° C.; mobile phase solvent A was milliQ water+0.1% HCOOH, mobile phase solvent B MeCN+0.1% HCOOH. The flow rate was 1 mL/min. The gradient table was t=0 min 97% A-3% B, t=1.5 min 0.1% A-99.9% B, t=1.9 min 0.1% 0.1% A-99.9% B and t=2 min 97% A-3% B. The UV detection range was 210-350 nm and ES+/ES− range was 100=1000 amu.

Method 3, low pH conditions. Column: Acquity CSH C18, 1.7 μm, 2.1×50 mm, the column temperature was 40° C.; mobile phase solvent A was milliQ water+0.1% HCOOH, mobile phase solvent B MeCN+0.1% HCOOH. The flow rate was 1 mL/min. The gradient table was t=0 min 97% A-3% B, t=1.5 min 0.1% A-99.9% B, t=1.9 min 0.1% 0.1% A-99.9% B and t=2 min 97% A-3% B. The UV detection range was 210-350 nm and ES+/ES− range was 100=1000 amu.

Method 4. Aquity UPLC-QDa Mass Spectrometer with a C18-reverse-phase column (50×2.1 mm Acquity CSH with 1.7 μm particle size) maintained at 40° C., elution with A: 95/5 water/acetonitrile+0.05% formic acid; B: 95/5 acetonitrile/water+0.05% formic acid.

- Gradient:

Gradient-Time
flow (mL/min)
% A
% B

0.00
1
99.0
1.0

3.50
1
0.1
99.9

3.90
1
0.1
99.9

4.00
1
99.0
1.0

- Detection-MS, UV PDA
- MS ionisation method-Electrospray (positive/negative ion).

Method 5, high pH conditions. Column: Acquity UPLC BEH C18, 1.7 μm, 2.1×50 mm, the column temperature was 40° C.; mobile phase solvent A was 10 mM aqueous solution of NH₄HCO₃adjusted to pH=10 with ammonia, mobile phase solvent B MeCN. The flow rate was 1 mL/min. The gradient table was t=0 min 97% A-3% B, t=1.5 min 0.1% A-99.9% B, t=1.9 min 0.1% 0.1% A-99.9% B and t=2 min 97% A-3% B. The UV detection range was 210-350 nm and ES+/ES− range was 100=1000 amu.

Analytical Chiral for Chiral Compounds

The enantiomeric access of chiral compounds was determined by chiral HPLC analysis on a HPLC Agilent 1100 equipped with 6-position switching valve, DAD, and CD detectors. The following methods were used:

Method A1, Column: Chiralpak IC (25×0.46 cm), 5 μm; Mobile phase: n-Hexane/(Ethanol/Dichloromethane 90/10% v/v+0.1% isopropylamine) 80/10% v/v; Flow rate: 1.0 mL/min; DAD: 220 nm.

Method A2, Column: Whelk 0-1 (R,R) (25×0.46 cm), 10 μm; Mobile phase: n-Hexane/(2-Propanol/Methanol 1/1+0.1% isopropylamine) 30/70% v/v; Flow rate: 1.0 mL/min; DAD: 220 nm.

Method A3, Column: Chiralpak AD-H (25×0.46 cm), 5 μm; Mobile phase: n-Hexane/(Ethanol+0.1% isopropylamine) 75/25% v/v; Flow rate: 1.0 mL/min; DAD: 220 nm.

Method A4, Column: Chiralpak AD-H (25×0.46 cm), 5 μm; Mobile phase: (2-Propanol+0.1% isopropylamine) 32%; Flow rate: 2.5 mL/min; DAD: 220 nm.

Flash chromatography is carried out using an Isolera MPLC system (manufactured by Biotage) using pre-packed silica gel or reverse-phase cartridges (Supplied by Biotage). Many of the compounds described in the following Examples have been prepared from Stereochemically pure starting materials, for example 95% ee. The stereochemistry of the compounds in the Examples, where indicated, has been assigned on the assumption that absolute configuration at resolved stereogenic centers of starting materials is maintained throughout any Subsequent reaction conditions. In the procedures that follow, after each starting material, reference to a compound number is sometimes provided. This is provided merely for assistance to the skilled chemist. The starting material may not necessarily have been prepared from the batch referred to. When reference is made to the use of a “similar” or “analogous” procedure, as will be appreciated by those skilled in the art. Such a procedure may involve minor variations, for example reaction temperature, reagent/solvent amount, reaction time, work-up conditions or chromatographic purification conditions.

PREPARATION OF INTERMEDIATES
Intermediate A1: 3-(3-(1-Hydroxyethyl)-1-oxo-1H-isochromen-4-yl)benzaldehyde

[Image Omitted]

In a 500 mL 3-necked round bottomed flask (3-formylphenyl)boronic acid (1 g, 6.67 mmol) 4-bromo-3-(1-hydroxyethyl)-1H-isochromen-1-one (Intermediate A2 in WO 2015091685) (2.15 g, 8.00 mmol) and potassium phosphate hydrate (4.61 g, 20.01 mmol) were loaded then THF (50 mL) and Water (50 mL) were added. Argon was bubbled for 10 min then XPhos-Pd-G2 (0.367 g, 0.467 mmol) was added. Bubbling was continued for further 10 min and then brown turbid mixture was stirred under Argon atmosphere overnight. The mixture was poured into 300 mL of water then extracted twice with 200 mL of EtOAc. The organic phase was dried over sodium sulfate, filtered and concentrated. The crude was purified by chromatography (SNAP 340 g) eluting with HexaneEtOAc mixtures to leave 3-(3-(1-hydroxyethyl)-1-oxo-1H-isochromen-4-yl)benzaldehyde as a gummy solid (0.110 g, 0.374 mmol, 6% yield).

UPLC-MS: 1.45 min, 295.1.06 [M+H]+, method 4.

Intermediate A2: 4-[4-Chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-3-(1-hydroxyethyl)-1H-isochromen-1-one

[Image Omitted]

Step 1. 2-Chloro-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (Intermediate A2.1)

[Image Omitted]

A mixture of 5-bromo-2-chlorobenzaldehyde (1.50 g, 6.83 mmol), bis(pinacolato)diboron (2.08 g, 8.20 mmol), potassium acetate (1.34 g, 13.66 mmol) and Pd(dppf)Cl₂(0.150 g, 0.205 mmol) in anhydrous 1,4-dioxane (22.5 mL) was heated at 100° C. under N₂overnight. After cooling to r.t., the reaction mixture was diluted with AcOEt, filtered through a Celite pad and the solvent was removed under vacuum. The residue, 2-chloro-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (6.83 mmol theoric), was not further purified, but used as such in the subsequent step.

UPLC-MS: 0.86 min, 267.06 [M+H]+, method 2.

Step 2. 2-[4-Chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate A2.2)

[Image Omitted]

A mixture of 2-chloro-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (Intermediate A2.1) (crude, 6.83 mmol theoric) and pinacol (3.23 g, 27.32 mmol) was dissolved in 25.0 mL toluene. Then p-toluensulfonic acid monohydrate (0.065 g, 0.342 mmol) was added and the mixture was heated at 90° C. for 5 h. Then p-toluensulfonic acid monohydrate (0.100 g, 0.526 mmol) was added and the mixture was heated at 90° C. overnight. The mixture was concentrated in vacuum and the residue was solubilized in 20 mL of isopropanol. Then 30 mL of water were slowly added. Upon addition a solid precipitated and it was collected by filtration to give 2-[4-chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a brown solid residue (2.24 g, 6.11 mmol, 89%).

UPLC-MS: 1.53 min, 367.11 [M+H]+, method 2.

Step 3. 4-[4-Chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-3-(1-hydroxyethyl)-1H-isochromen-1-one

The title compound was made in a similar way as that of Intermediate A1 using 4-bromo-3-(1-hydroxyethyl)-1H-isochromen-1-one (Intermediate A2 in WO 2015091685) (1 g, 3.72 mmol), 2-[4-chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate A2.2) (1.24 g, 3.38 mmol), K₃PO₄(2.153 g, 10.14 mmol) and XPhos-Pd-G2 (0.133 g, 0.169 mmol). After work-up the residue was purified by flash chromatography on a 100 g silica gel Biotage SNAP column (eluent: gradient of AcOEt in cyclohexane from 5 to 50%). Evaporation of opportune fractions provided 4-[4-chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-3-(1-hydroxyethyl)-1H-isochromen-1-one as an off-white solid (0.458 g, 1.067 mmol, 32% yield).

UPLC-MS: 1.19 min, 429.3 [M+H]+, method 3.

Intermediate A3: (2R)-2-{3-[3-(1-hydroxyethyl)-1-oxo-1H-isochromen-4-yl]phenyl}pyrrolidine-1-carboxylate

[Image Omitted]

Step 1. Tert-Butyl (2R)-2-(3-bromophenyl)pyrrolidine-1-carboxylate (Intermediate A3.1)

[Image Omitted]

To a stirred solution of (2S)-2-(3-bromophenyl)pyrrolidine hydrochloride (1.00 g, 3.81 mmol) in DCM (12 mL) at 0° C. and under a nitrogen atmosphere, TEA (0.58 mL, 4.19 mmol) was added followed by a solution of Boc₂O (1.08 g, 4.95 mmol) in DCM (4 mL) portion-wise. The ice-bath was then removed and the resulting reaction mixture was stirred overnight at r.t. The mixture was diluted with DCM, washed with saturated water solution of NaHCO₃, dried over Na₂SO₄and concentrated under reduced pressure. The crude material was purified by FC on silica (Snap 25, eluting with Cy/EA from 100/0 to 90/10) to give tert-butyl (2R)-2-(3-bromophenyl)pyrrolidine-1-carboxylate (1.15 g, 3.53 mmol, 92%).

UPLC-MS: 1.32 min, 327.1 [M+H]+, method 3.

Step 2. Tert-Butyl (2R)-2-[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pyrrolidine-1-carboxylate (Intermediate A3.2)

[Image Omitted]

A mixture of tert-butyl (2R)-2-(3-bromophenyl)pyrrolidine-1-carboxylate (Intermediate A3.1) (1.15 g, 3.53 mmol), bis(pinacolato)diboron (1.34 g, 5.28 mmol), potassium acetate (0.70 g, 7.10 mmol) and Pd(dppf)Cl₂(0.080 g, 0.11 mmol) in anhydrous 1,4-dioxane (13 mL) was shaken in a capped vial at 100° C. for 6 h. Further bis(pinacolato)diboron (0.32 g, 1.26 mmol), AcOK (0.21 g, 2.14 mmol) and Pd(dppf)Cl₂(0.024 g, 0.033 mmol) were added and the mixture was shaken at 100° C. for additional 5 h. The reaction mixture was diluted with EtOAc, filtered and the solvent removed under reduced pressure. The crude material was purified by FC on silica (Snap 50, eluting with Cy/EA from 100/0 to 90/10) affording tert-butyl (2R)-2-[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pyrrolidine-1-carboxylate as whitish waxy solid (1.23 g, 3.29 mmol, 93%).

UPLC-MS: 1.41 min, 374.3 [M+H]+, method 3.

Step 3. 4 Tert-Butyl (2R)-2-{3-[3-(1-hydroxyethyl)-1-oxo-1H-isochromen-4-yl]phenyl}pyrrolidine-1-carboxylate

The title compound was made in a similar way as that of Intermediate A1 using 4-bromo-3-(1-hydroxyethyl)-1H-isochromen-1-one (Intermediate A2 in WO 2015091685) (1.33 g, 4.94 mmol), (2R)-2-[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pyrrolidine-1-carboxylate (Intermediate A3.2) (1.23 g, 3.29 mmol), K₃PO₄(2.10 g, 9.87 mmol) and X-Phos-Pd(crotyl)Cl (0.11 g, 0.16 mmol). After work-up the residue was purified by flash chromatography on a 55 g silica-NH Biotage SNAP column (eluent: gradient of AcOEt in cyclohexane from 0 to 35%). Evaporation of opportune fractions provided 4 tert-butyl (2R)-2-{3-[3-(1-hydroxyethyl)-1-oxo-1H-isochromen-4-yl]phenyl}pyrrolidine-1-carboxylate as dark oil (0.89 g, 2.04 mmol, 62%).

UPLC-MS: 1.19 min, 436.3 [M+H]+, method 3.

Intermediate A4: 4-[4-Fluoro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-3-(1-hydroxyethyl)-1H-isochromen-1-one

[Image Omitted]

Step 1. 2-[4-Fluoro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate A4.1)

[Image Omitted]

A mixture of 4-fluoro-3-formylphenylboronic acid (1.5 g, 8.93 mmol) and pinacol (4.22 g, 35.73 mmol) were dissolved in 30 mL toluene. Then p-toluensulfonic acid monohydrate (0.085 g, 0.45 mmol) was added and the mixture was heated at 90° C. for 4 h. The mixture was allowed to cool to room temperature. The solid formed was filtered off and the mother liquors were concentrated in vacuum to leave an oily residue that crystallizes upon standing. This solid was suspended in 30 mL of ⁱPrOH and heated at 70° C. to help dissolution. After cooling, 12 mL of water were added dropwise, to crystallize the compound. After addition, the thick slurry was filtered on a Buchner funnel. The cake was washed with water and dried under reduced pressure. From the mother liquors, a second solid precipitated and it was recovered by filtration on a Buchner funnel and the cake was washed with water. Both solids were dried together under reduced pressure to give 2-[4-fluoro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as an off-white solid (3.07 g, 8.76 mmol, 98% yield).

UPLC-MS: 1.44 min, 351.2 [M+H]+, method 3.

Step 2. 4-[4-Fluoro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-3-(1-hydroxyethyl)-1H-isochromen-1-one

[Image Omitted]

The title compound was made in a similar way as that of Intermediate A1 using 4-bromo-3-(1-hydroxyethyl)-1H-isochromen-1-one (Intermediate A2 in WO 2015091685) (2.83 g, 10.51 mmol), 2-[4-fluoro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate A4.1) (3.07 g, 8.76 mmol), K₃PO₄(5.58 g, 26.28 mmol) and XPhos-Pd-G2 (0.345 g, 0.438 mmol). After work-up the residue was purified by flash chromatography on a 100 g silica gel Biotage SNAP column (eluent: gradient of AcOEt in cyclohexane from 0 to 35%). Evaporation of opportune fractions provided 4-[4-fluoro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-3-(1-hydroxyethyl)-1H-isochromen-1-one as light yellow solid (1.79 g, 4.34 mmol, 50% yield).

UPLC-MS: 1.23 min, 413.2 [M+H]+, method 3.

Intermediate A5: 4-(3-Fluoro-5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl)-3-(1-hydroxyethyl)-1H-isochromen-1-one

[Image Omitted]

Step 1. 2-[3-Fluoro-5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate A5.1)

[Image Omitted]

A mixture of 3-fluoro-5-formylphenylboronic acid (1 g, 5.95 mmol) and pinacol 76-09-5 (2.8 g, 23.8 mmol) were dissolved in Toluene (20 mL). Then p-toluensulfonic acid monohydrate (0.057 g, 0.3 mmol) was added and the mixture was heated at 90° C. for 4 h. The mixture was allowed to cool to room temperature. It was concentrated under vacuum to give 2-[3-fluoro-5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as light yellow oil (crude material). Used as such in the next step.

UPLC-MS: 1.48 min, 351.3 [M+H]+, method 3.

Step 2. 4-(3-Fluoro-5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl)-3-(1-hydroxyethyl)-1H-isochromen-1-one

The title compound was made in a similar way as that of Intermediate A1 using 4-bromo-3-(1-hydroxyethyl)-1H-isochromen-1-one (Intermediate A2 in WO 2015091685) (1.9 g, 7.14 mmol), 2-[3-fluoro-5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate A5.1) (5.95 theoretical mmol), K₃PO₄(3.8 g, 17.85 mmol) and XPhos-Pd-G2 (0.236 g, 0.3 mmol). After work-up the residue was purified by flash chromatography on 110 g Silica-NH SNAP cartridge (eluent from Cy to EtOAc 30%). It was purified again by 25 g Silica gel SNAP cartridge (eluent from Cy to EtOAc 30%) to afford 4-(3-fluoro-5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl)-3-(1-hydroxyethyl)-1H-isochromen-1-one as white foam (0.250 g, 0.61 mmol, 9%).

UPLC-MS: 1.22 min, 413.3 [M+H]+, method 3.

Intermediate A6: 4-(3-(1-(Dimethylamino)ethyl)phenyl)-3-(1-hydroxyethyl)-1H-isochromen-1-one

[Image Omitted]

The title compound was made in a similar way as that of Intermediate A1 using 4-bromo-3-(1-hydroxyethyl)-1H-isochromen-1-one (Intermediate A2 in WO 2015091685) (1.62 g, 6.03 mmol), (3-(1-(dimethylamino)ethyl)phenyl)boronic acid (970 mg, 5.02 mmol), K₃PO₄(3.47 g, 15.1 mmol) and XPhos-Pd-G2 (0.277 g, 0.35 mmol). After work-up the residue was purified by chromatography with H₂OMeCNHCOOH 95:5:0.1% and MeCNH₂OHCOOH 95:5:0.1% to afford 4-(3-(1-(dimethylamino)ethyl)phenyl)-3-(1-hydroxyethyl)-1H-isochromen-1-one (0.356 g, crude material).

UPLC-MS: 0.45 min, 338.2 [M+H]+, method 1.

Intermediate B1: 3-(1-Hydroxyethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one

[Image Omitted]

In a 250 mL round bottomed flask 3-(3-(1-hydroxyethyl)-1-oxo-1H-isochromen-4-yl)benzaldehyde (Intermediate A1) (0.500 g, 1.699 mmol) was dissolved in DCM (20 mL), 1-methylpiperazine (0.582 mL, 5.10 mmol) was added followed by acetic acid (0.292 mL, 5.10 mmol). Sodium triacetoxyhydroborate (1.800 g, 8.49 mmol) was then added portion wise. The mixture was poured into a mixture of DCM and saturated water solution of NaHCO₃(100 mL/100 mL) and effervescence was observed. Organic phase was concentrated under vacuum and the resulting crude material was purified by RP chromatography (Biotage Isolera, 60 g C18 cartridge, gradient elution from 0 to 50% B in A in 10 CV; A: 95:5 water/acetonitrile+0.1% HCOOH, B: 5:95 water/acetonitrile+0.1% HCOOH; flow 30 ml/min) to give 3-(1-hydroxyethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one (0.482 g, 1.274 mmol, 75% yield).

UPLC-MS: 0.73 min, 379.1 [M+H]+, method 1.

Intermediates B2 found in the table below may be prepared starting from suitable reagents reported below following similar procedures as for compound B1.

UPLC-MS or

Intermediate & Name
Molecular Structure
Reagents
¹H-NMR

Intermediate B2 4-(3- ((Dimethylamino)methyl)phenyl)- 3-(1-hydroxyethyl)-1H- isochromen-1-one

[Image Omitted]

A1 and dimethylamine
Rt = 0.87 min, 324.0 [M + H]+, method 4.

Intermediate C1: 3-(1-Bromoethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one

[Image Omitted]

3-(1-Hydroxyethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one (Intermediate B1) (0.482 g, 1.274 mmol) was dissolved in dry DCM (20 mL) then 1M tribromophosphine in DCM (2.55 mL, 2.55 mmol) was slowly added. The mixture was stirred at r.t. The reaction was quenched by addition of 60 mL of a saturated water solution of NaHCO₃(gas evolution) and extracted with 60 mL of DCM, the organic phase was concentrated under vacuum to give 3-(1-bromoethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one as a red oil (crude material).

UPLC-MS: 1.07 min, 441.0 [M]+ and 442.9 [M+2]+, method 4.

Intermediates C2-C7 found in the table below may be prepared starting from suitable intermediate (Int.) reported below following similar procedures as for compound C1.

Intermediate & Name
Molecular Structure
Int.
UPLC-MS or ¹H-NMR

Intermediate C2 3-(1-Bromo ethyl)-4-(3- ((dimethylamino)methyl)phenyl)- 1H-isochromen-1-one

[Image Omitted]

B2
Crude material Rt = 1.27 min, 385.9 [M]+ and 387.9 [M + 2]+, method 4

Intermediate C3 3-(1-Bromo ethyl)-4-[4-chloro-3- (4,4,5,5-tetramethyl-1,3- dioxolan-2-yl)phenyl]-1H- isochromen-1-one

[Image Omitted]

A2
Rt = 1.43 min, 493.2 [M + H]+, method 3

Intermediate C4 tert-Butyl (2R)-2-{3-[3-(1- bromoethyl)-1-oxo-1H- isochromen-4- yl]phenyl}pyrrolidine-1- carboxylate

[Image Omitted]

A3
Rt = 1.46 min, 499.2 [M + H]+, method 3

Intermediate C5 3-(1-Bromoethyl)-4-[4-fluoro-3- (4,4,5,5-tetramethyl-1,3- dioxolan-2-yl)phenyl]-1H- isochromen-1-one

[Image Omitted]

A4
Rt = 1.44 min, 476.0 [M + H]+, method 3

Intermediate C6 3-(1-Bromoethyl)-4-(3-fluoro-5- (4,4,5,5-tetramethyl-1,3- dioxolan-2-yl)phenyl)-1H- isochromen-1-one

[Image Omitted]

A5
Rt = 1.48 min, 476.2 [M + H]+, method 3

Intermediate C7 3-(1-Bromoethyl)-4-(3-(1- (dimethylamino)ethyl)phenyl)- 1H-isochromen-1-one

[Image Omitted]

A6
Crude material Rt = 0.62 min, 400.0 [M]+ and 402.0 [M + 2]+, method 1

Intermediate D1: 3-(1-(4-Amino-3-(5-methoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one

[Image Omitted]

In a 250 mL round bottomed flask, 3-(5-methoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.463 g, 1.912 mmol) and potassium carbonate (0.528 g, 3.82 mmol) were suspended in dry DMF (15 mL) and the mixture heated at 60° C. for 15 min prior addition of the 3-(1-bromoethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one (Intermediate C1) dissolved in 5 mL of dry DMF. After two hours, 0.200 g of 3-(5-methoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine and an extra amount of K₂CO₃were added and heating was continued at 40° C. for 3 h. The reaction was quenched with a saturated water solution of NaHCO₃and extracted with 40 mL of DCM, then concentrated to leave a brown oil that was purified by RP chromatography (Biotage Isolera, 60 g C18 cartridge, gradient elution from 0 to 50% B in A in 10 CV; A: 95:5 water/acetonitrile+0.1% HCOOH, B: 5:95 water/acetonitrile+0.1% HCOOH) to leave 3-(1-(4-amino-3-(5-methoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3 methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one as a yellow oil (0.342 g, 0.567 mmol, 45% yield).

UPLC-MS: 0.60 min, 603.2 [M+H]+, method 1.

Intermediates D2 found in the table below may be prepared starting from suitable intermediate (Int.) reported below following similar procedures as for compound D1.

UPLC-

MS or

Intermediate & Name
Molecular Structure
Int.
¹H-NMR

Intermediate D2 3-(1-(4-Amino-3-(5- methoxypyridin-3-yl)-1H- pyrazolo[3,4-d]pyrimidin-1- yl)ethyl)-4-(3- ((dimethylamino)methyl)phenyl)- 1H-isochromen-1-one

[Image Omitted]

C2
Rt = 0.89 min, 548.0 [M + H]+, method 4.

Intermediate E1a: 3-(1-{4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-[4-chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-1H-isochromen-1-one

[Image Omitted]

3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.311 g, 1.19 mmol) and K₂CO₃(0.47 g, 3.4 mmol) were suspended in DMF (10 mL) and the mixture was stirred at 60° C. for 30 min. Then 3-(1-bromoethyl)-4-[4-chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-1H-isochromen-1-one (Intermediate C3) (0.557 g, 1.13 mmol) dissolved in DMF (5 mL) was slowly added and the mixture was maintained at 60° C. for 90 min. The reaction was cooled to r.t. and then AcOEt and water were added. The phases were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with water, dried over anhydrous Na₂SO₄and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on a 100 g Biotage SNAP KP-Sil cartridge, eluting with a gradient of AcOEt in cyclohexane from 50 to 100%, to give title compound 3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-[4-chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-1H-isochromen-1-one (0.379 g, 0.564 mmol, 50% yield).

UPLC-MS: 1.3 min, 671.2 [M+H]+, method 3.

Intermediates E2a-E5a found in the table below may be prepared starting from suitable intermediate (Int.) reported below following similar procedures as for compound E1a.

UPLC-MS or

Intermediate & Name
Molecular Structure
Int.
¹H-NMR

Intermediate E2a tert-Butyl (2R)-2-{3-[3-(1-{4- amino-3-iodo-1H- pyrazolo[3,4-d]pyrimidin-1- yl}ethyl)-1-oxo-1H- isochromen-4- yl]phenyl}pyrrolidine-1- carboxylate

[Image Omitted]

C4
Rt = 1.26 min, 679.38 [M + H]+, method 3.

Intermediate E3a 3-(1-{4-Amino-3-iodo-1H- pyrazolo[3,4-d]pyrimidin-1- yl}ethyl)-4-[4-fluoro-3- (4,4,5,5-tetramethyl-1,3- dioxolan-2-yl)phenyl]-1H- isochromen-1-one

[Image Omitted]

C5
Rt = 1.23 min, 656.2 [M + H]+, method 3.

Intermediate E4a 3-(1-(4-Amino-3-iodo-1H- pyrazolo[3,4-d]pyrimidin-1- yl)ethyl)-4-(3-fluoro-5- (4,4,5,5-tetramethyl-1,3- dioxolan-2-yl)lphenyl)-1H- isochromen-1-one

[Image Omitted]

C6
Rt = 1.26 min, 656.3 [M + H]+, method 3.

Intermediate E5a 3-(1-(4-Amino-3-iodo-1H- pyrazolo[3,4-d]pyrimidin-1- yl)ethyl)-4-(3-(1- (dimethylamino)ethyl)phenyl)- 1H-isochromen-1-one

[Image Omitted]

C7
Rt = 0.57 min, 581.0 [M + H]+, method 1.

Intermediate E1b: 5-[3-(1-{4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-1-oxo-1H-isochromen-4-yl]-2-chlorobenzaldehyde

[Image Omitted]

3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-[4-chloro-3-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl]-1H-isochromen-1-one (0.379 g, 0.565 mmol) was dissolved in CH₃CN (5 mL) and then 1N HCl_aqueous(5 mL) was added. The resulting suspension was stirred at room temperature overnight. The solid in suspension was recovered by filtration on a Buchner funnel. The resulting cake was washed several times with diethyl ether till the pH resulted neutral and it was dried under high vacuum. Compound 5-[3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-1-oxo-1H-isochromen-4-yl]-2-chlorobenzaldehyde (0.32 g, 0.56 mmol, 99% yield) was obtained as a white solid and used in the next step without further purification.

UPLC-MS: 1.06 min, 572.1 [M+H]+, method 3.

Intermediates E3b-E4b found in the table below may be prepared starting from suitable intermediate (Int.) reported below following similar procedures as for compound E1b.

Intermediate & Name
Molecular Structure
Int.
UPLC-MS or ¹H-NMR

Intermediate E3b 5-[3-(1-{4-Amino-3- iodo-1H-pyrazolo[3,4- d]pyrimidin-1- yl}ethyl)-1-oxo-1H- isochromen-4-yl]-2- fluorobenzaldehyde

[Image Omitted]

E3a
Rt = 0.98 min, 556.08 [M + H]+, method 3

Intermediate E4b 3-(3-(1-(4-Amino-3- iodo-1H-pyrazolo[3,4- d]pyrimidin-1- yl)ethyl)-1-oxo-1H- isochromen-4-yl)-5- fluorobenzaldehyde

[Image Omitted]

E4a
Rt = 1.01 min, 556.2 [M + H]+, method 3

Intermediate E1c: 3-(1-{4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-{4-chloro-3-[(dimethylamino)methyl]phenyl}-1H-isochromen-1-one

[Image Omitted]

To a stirred suspension of 5-[3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-1-oxo-1H-isochromen-4-yl]-2-chlorobenzaldehyde (Intermediate F1) (0.150 g, 0.26 mmol) in DCM/dioxane/CH₃CN (10 mL/2 mL/2 mL), 2M dimethylamine in THF (0.212 mL, 0.39 mmol) was added. This suspension was stirred at room temperature for 30 min. Then Na(OAc)₃BH (0.110 g, 0.52 mmol) was added and the mixture was stirred at r.t. overnight. Additional 1.5 eq 2M dimethylamine in THF (0.212 mL, 0.39 mmol) was added followed by 2 eq. of Na(OAc)₃BH (0.110 g, 0.52 mmol). The mixture was left to react at r.t. for 2 h. The mixture was quenched with a saturated water solution of NaHCO₃, the product extracted with DCM (3×) and washed with water (1×). The combined organic layers were passed through a phase separator and the solvent was removed under vacuum. The residue was purified by column chromatography on a 25 g Biotage silica gel column, using as eluent a gradient of MeOH in DCM from 0 to 10%. Desired compound 3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-{4-chloro-3-[(dimethylamino)methyl]phenyl}-1H-isochromen-1-one (0.105.2 g, 0.175 mmol, 67% yield) was obtained as a white solid.

UPLC-MS: 0.48-0.53 min, 568.3 [M+H]+, method 3.

Intermediates E3c-E4c found in the table below may be prepared starting from suitable reagents reported below following similar procedures as for compound E1c.

Intermediate & Name
Molecular Structure
Reagents
UPLC-MS or ¹H-NMR

Intermediate E3c 3-(1-{4-Amino-3- iodo-1H- pyrazolo[3,4- d]pyrimidin-1- yl}ethyl)-4-{4-fluoro- 3-[(4- methylpiperazin-1- yl)methyl]phenyl}- 1H-isochromen-1-one

[Image Omitted]

E3b and 1- methylpiperazine
Rt = 0.64-0.66 min, 640.1 [M + H]+, method 3

Intermediate E4c 3-(1-(4-Amino-3- iodo-1H- pyrazolo[3,4- d]pyrimidin-1- yl)ethyl)-4-(3-fluoro- 5-((4- methylpiperazin-1- yl)methyl)phenyl)- 1H-isochromen-1-one

[Image Omitted]

E4b and 1- methylpiperazine
Rt = 0.65-0.66 min, 640.2 [M + H]+, method 3

Intermediate E7: 3-(1-{4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-{3-[(2R)-pyrrolidin-2-yl]phenyl}-1H-isochromen-1-one hydrochloride

[Image Omitted]

tert-Butyl (2R)-2-{3-[3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-1-oxo-1H-isochromen-4-yl]phenyl}pyrrolidine-1-carboxylate (Intermediate E2a) (0.224 g, 0.330 mmol) was dissolved in 1.25M HCl in MeOH (5 mL) and the mixture was shaken at 38° C. for 4 h. The solvent was removed at reduced pressure to provide 3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-13-[(2R)-pyrrolidin-2-yl]phenyl-1H-isochromen-1-one hydrochloride as a white solid (0.212 g, crude). The material was used in the next step without further purification.

UPLC-MS: 1.03 min, 579.2 [M+H]+, method 5.

Intermediate E8: 3-(1-{4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-{3-[(2R)-1-methylpyrrolidin-2-yl]phenyl}-1H-isochromen-1-one

[Image Omitted]

To a stirred suspension of 3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-{3-[(2R)-pyrrolidin-2-yl]phenyl}-1H-isochromen-1-one hydrochloride (Intermediate E6) (0.212 g, crude) in DCM (4 mL), formaldehyde 37% Wt. aqueous solution (61 μL, 0.825 mmol) was added, followed by DIPEA (63 μL, 0.363 mmol) and acetic acid (1 drop). The mixture was stirred at room temperature for 30 minutes. Then Na(OAc)₃BH (0.084 g, 0.396 mmol) was added and the mixture was stirred at r.t. overnight. The mixture was quenched with saturated water solution of NaHCO₃and extracted with DCM. The organic phases were filtered through a hydrophobic phase separator and the solvent was removed at reduced pressure. The crude was purified by flash chromatography (Biotage SP1, 25 g KP-Sil-column, DCM/MeOH from 100:0 to 90:10 as eluent). The evaporation of opportune fractions provided 3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-{3-[(2R)-1-methylpyrrolidin-2-yl]phenyl}-1H-isochromen-1-one as an off-white solid (0.142 g, 0.24 mmol, 73% yield).

UPLC-MS: 1.16 min, 593.3 [M+H]+, method 5.

PREPARATION OF COMPOUNDS
Comparative Example 1: 3-(1-(4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one, Second Eluting Enantiomer

[Image Omitted]

Racemate 3-(1-(4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one (Example 142 in WO 2015091685) (0.110 g, 0.16 mmol) was dissolved in MeOH/DCM 1/1 (10 mL) and submitted to chiral resolution by Chiral preparative chromatography. Conditions: Column: Chiralpak IC (25×2.0 cm), 5 μm; Mobil phase: n-Hexane/(Ethanol/Dichloromethane 9/1+0.1% isopropyl amine) 82/18% v/v; Flow rate 18 mL/min; DAD detection: 220 nm; Loop: 500 μl; Injection: 6.5 mg/injection. The fractions containing the second eluted enantiomer were evaporated to dryness to afford title 3-(1-(4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one (0.031 g, 0.056 mmol).

Chiral HPLC (Method A1),

Rt 13.8 min (second eluting enantiomer), e.e.>99%.

UPLC-MS: 0.63-0.66, 606.4 [M+H]+, method 3.

¹H NMR (400 MHz, DMSO-d6) δ ppm 10.15-10.24 (m, 1H), 8.19-8.28 (m, 1H), 8.07 (s, 1H), 7.73-7.80 (m, 1H), 7.58-7.67 (m, 1H), 7.25-7.50 (m, 3H), 6.85-7.00 (m, 3H), 6.78-6.84 (m, 1H), 6.63-6.70 (m, 1H), 5.66-5.81 (m, 1H), 3.23-3.60 (m, 2H), 1.99-2.48 (m, 11H), 1.74-1.89 (m, 3H).

- Analytical Data of First Eluting Enantiomer

Chiral HPLC (Method A1),

Rt 12.0 min (first eluted enantiomer), e.e.>99%,

UPLC and ¹H NMR analyses of pure enantiomers were superimposable in all respect.

Example 1: 3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-isochromen-1-one hydrochloride

[Image Omitted]

In a 500 mL round bottomed flask equipped with a magnetic stirrer 3-(1-(4-amino-3-(5-methoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-isochromen-1-one (Intermediate D1) (0.342 g, 0.567 mmol) was suspended in DCM (10 mL) then 1M BBr₃in DCM (5 mL, 5.00 mmol) was added. The mixture was left stirring 24 h, then MeOH (20 mL) and a little amount of 2M HCl was added. The solution was then concentrated under vacuum and purified by RP chromatography (Biotage Isolera, 60 g C18 cartridge, gradient elution from 0 to 50% B in A in 10 CV; A: 95:5 water/acetonitrile+0.1% HCOOH, B: 5:95 water/acetonitrile+0.1% HCOOH, flow 20 ml/min) to give the desired product 3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-isochromen-1-one hydrochloride (0.093 g, 0.149 mmol, 26% yield).

UPLC-MS: 0.52 min, 589.0 [M+H]+, method 1.

¹H NMR (400 MHz, DMSO-d6) δ ppm 11.39 (br. s., 1H), 9.87-10.49 (m, 1H), 8.05-8.32 (m, 5H), 7.72-7.81 (m, 1H), 7.59-7.67 (m, 1H), 6.67-7.53 (m, 8H), 5.66-5.84 (m, 1H), 3.34-3.63 (m, 2H), 2.24-2.92 (s, 11H), 1.75-1.92 (m, 3H).

Example 3: 3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{4-chloro-3-[(dimethylamino)methyl]phenyl}-1H-isochromen-1-one

[Image Omitted]

A mixture of 3-(1-{4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl}ethyl)-4-{4-chloro-3-[(dimethylamino)methyl]phenyl}-1H-isochromen-1-one (Intermediate E1c) (0.105 g, 0.175 mmol), 5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)pyridin-3-ol (0.077 g, 0.35 mmol) and K₃PO₄(0.113 g, 0.53 mmol) in THF/water 3:1 (volume: 8 mL) was deoxygenated by N₂bubbling for 5 min, then SPhos-Pd-G2 (0.025 g, 0.035 mmol) was added and the mixture was heated at 85° C. for 3 h. The mixture was cooled to room temperature and portioned between water and DCM. The aqueous phase was extracted with DCM (3×), the combined organic layers were washed with water (1×), passed through a phase separator and the solvent was removed under reduced pressure. The obtained crude was purified by flash chromatography on a 11 g Biotage silica-NH column using as eluent a gradient of MeOH in DCM from 0 to 25%. The desired product 3-{1-[4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{4-chloro-3-[(dimethylamino)methyl]phenyl}-1H-isochromen-1-one was obtained as a yellowish solid (0.068 g, 0.12 mmol, 68% yield).

UPLC-MS: 0.61 min, 601.2 [M+H]+, method 3.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.18-8.25 (m, 3H), 8.05-8.10 (m, 1H), 7.73-7.79 (m, 1H), 7.58-7.66 (m, 1H), 7.30-7.58 (m, 3H), 6.85-7.03 (m, 2H), 5.71-5.79 (m, 1H), 3.13-3.22 (m, 2H), 2.01-2.25 (m, 6H), 1.80-1.87 (m, 3H).

Example 2 found in the table below was prepared starting from suitable intermediate (Int.) reported below following similar procedures as for compound 1. Examples 4-7 found in the table below were prepared starting from suitable intermediate (Int.) reported below following similar procedures as for compound 3.

Ex.
Name
Molecular Structure
Int.
UPLC-MS or ¹H-NMR

2
3-(1-(4-Amino-3-(5- hydroxypyridin-3-yl)- 1H-pyrazolo[3,4- d]pyrimidin-1- yl)ethyl)-4-(3- ((dimethylamino) methyl)phenyl)-1H- isochromen-1-one hydrochloride

[Image Omitted]

D2
Rt = 0.75 min, 534.0 [M + H]+, method 4. ¹H NMR (400 MHz, DMSO-d6) δ ppm 12.14 (br. s, 1H), 10.18 (br. s., 1H), 8.05-8.31 (m, 4H), 7.73- 7.81 (m, 1H), 7.59-7.67 (m, 1H), 7.45-7.57 (m, 1H), 7.28-7.42 (m, 3H), 6.84- 7.02 (m, 2H), 5.63-5.86 (m, 1H), 3.36-3.71 (m, 2H), 2.04-2.40 (m, 6H), 1.77- 1.87 (m, 3H).

4
3-{1-[4-Amino-3-(5- hydroxypyridin-3-yl)- 1H-pyrazolo[3,4- d]pyrimidin-1- yl]ethyl}-4-{3-[(2R)- 1-methylpyrrolidin-2- yl]phenyl}-1H- isochromen-1-one

[Image Omitted]

E7
Rt = 0.69 min, 560.4 [M + H]+, method 5 ¹H NMR (400 MHz, MeOD) δ ppm 8.31-8.38 (m, 1H), 8.24-8.29 (m, 1H), 8.17-8.23 (m, 1H), 8.04- 8.14 (m, 1H), 7.67-7.76 (m, 1H), 7.58-7.66 (m, 1H), 7.18-7.56 (m, 4H), 6.74- 7.08 (m, 2H), 5.86-6.04 (m, 1H), 2.82-3.32 (m, 2H), 1.56-2.48 (m, 11H).

5
3-{1-[4-Amino-3-(5- hydroxypyridin-3-yl)- 1H-pyrazolo[3,4- d]pyrimidin-1- yl]ethyl}-4-{4-fluoro- 3-[(4- methylpiperazin-1- yl)methyl]phenyl}- 1H-isochromen-1-one

[Image Omitted]

E3
Rt =: 0.61-0.63 min, 607.1 [M + H]+, method 5 ¹H NMR (400 MHz, DMSO) δ ppm 10.34 (br. s., 1H), 8.23 (s, 3H), 8.09 (s, 1H), 7.73-7.80 (m, 1H), 7.59-7.67 (m, 1H), 7.11- 7.42 (m, 3H), 6.97-7.06 (m, 1H), 6.86-6.93 (m, 1H), 5.70-5.80 (m, 1H), 3.22- 3.59 (m, 2H), 1.96-2.47 (m, 11H), 1.78-1.88 (m, 3H).

6
3-(1-(4-Amino-3-(5- hydroxypyridin-3-yl)- 1H-pyrazolo[3,4- d]pyrimidin-1- yl)ethyl)-4-(3-fluoro- 5-((4- methylpiperazin-1- yl)methyl)phenyl)- 1H-isochromen-1-one

[Image Omitted]

E4c
Rt =: 0.52-0.59 min, 607.3 [M + H]+, method 3 ¹H NMR (400 MHz, DMSO) δ ppm 10.22 (br. s., 1H), 8.17-8.28 (m, 3H), 8.04-8.11 (m, 1H), 7.72- 7.82 (m, 1H), 7.58-7.68 (m, 1H), 7.31-7.39 (m, 1H), 7.06-7.20 (m, 2H), 6.88- 6.93 (m, 1H), 6.64-6.75 (m, 1H), 5.74-5.86 (m, 1H), 3.31 (s, 2H), 2.01-2.47 (m, 11H), 1.76-1.88 (m, 3H).

7
3-(1-(4-Amino-3-(5- hydroxypyridin-3-yl)- 1H-pyrazolo[3,4- d]pyrimidin-1- yl)ethyl)-4-(3-(1- (dimethylamino)ethyl) phenyl)-1H- isochromen-1-one

[Image Omitted]

E5c
Rt = 0.50 min, 548.1 [M + H]+, method 1. ¹H NMR (400 MHz, DMSO) δ ppm 10.16 (br. s., 1H), 8.18-8.33 (m, 3H), 8.03-8.16 (m, 2H), 7.74- 7.82 (m, 1H), 7.58-7.67 (m, 1H), 7.27-7.54 (m, 4H), 6.85-7.09 (m, 3H), 5.66- 5.82 (m, 1H), 3.11-3.27 (m, 1H), 1.93-2.21 (m, 6H), 1.77-1.88 (m, 3H), 1.08- 1.20 (m, 3H).

Example 1a: 3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-isochromen-1-one, First Eluted Enantiomer

[Image Omitted]

Racemate 3-{1-[4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-isochromen-1-one hydrochloride (Example 1) (0.122 g, 0.195 mmol) was diluted with MeOH and was passed through a PL-HCO3 MP resin cartridge (5 g) to obtain the free base (0.080 g), which was dissolved in MeOH (5 mL) and submitted to chiral resolution by Chiral preparative chromatography. Conditions: Column: Whelk 01 (R,R) (25×2.1 cm), 10 μm; Mobil phase: n-Hexane/(2-Propanol/Methanol 1/1+0.1% isopropylamine) 30/70% v/v; Flow rate 17 mL/min; DAD detection: 220 nm; Loop: 300 μl; Injection: 4.8 mg/injection. The fractions containing the first eluted enantiomer were evaporated to dryness to afford title compound 3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-isochromen-1-one (0.021 g, 0.036 mmol).

Chiral HPLC (Method A2),

Rt 8.7 min (first eluted enantiomer), e.e.>99%

UPLC-MS: 0.48-0.54, 589.4 [M+H]+, method 3.

¹H NMR (400 MHz, DMSO-d6) δ ppm 9.84-10.55 (m, 1H), 8.02-8.36 (m, 4H), 7.72-7.81 (m, 1H), 7.57-7.67 (m, 1H), 7.25-7.52 (m, 4H), 6.87-7.01 (m, 2H), 6.53-7.88 (m, 2H), 5.64-5.83 (m, 1H), 3.13-3.62 (m, 2H), 2.00-2.47 (m, 11H), 1.76-1.88 (m, 3H).

Analytical Data of Second Eluting Enantiomer

Chiral HPLC (Method A2),

Rt 10.0 min (second eluted enantiomer), e.e.=97.4%

UPLC and ¹H NMR analyses of pure enantiomers were superimposable in all respect.

Single Crystal X-Ray Diffraction analysis (D. de Sanctis, A. Beteva, H. Caserotto, F. Dobias, J. Gabadinho, T. Giraud, A. Gobbo, M. Guijarro, M. Lentini, B. Lavault, T. Mairs, S. McSweeney, S. Petitdemange, V. Rey-Bakaikoa, J. Surr, P. Theveneau, G. A. Leonard, C. Mueller-Dieckmann, J. Sync. Rad. 2012, 19, 455-461) was performed on the crystallography beamline ID29 of European Synchrotron Radiation Facility (ESRF) following the method and conditions herewith specified: Single crystal was obtained with vapor diffusion experiments in EtOH/H2O 6% (internal solvent) and EtOAc (outside solvent). Diffraction data were recorded with a fast readout Pilatus 6M-F pixel detector (Dectris LTD). Data collection was performed at 100 K using an Oxford cryosystem applying rotating-crystal method, with monochromatic radiation at wavelength of 1.000 Å. The structure was solved by dual space algorithm implemented in the program ShelxT, and was refined with the program SHELXL-2016/6. The Flack parameter (S. Parsons, H. D. Flack, T. Wagner, Acta Cryst. 2013, B69, 249-259) was obtained from data collected at 1 Å as Flack x=0.07(18); and the absolute configuration was assigned as S to the first eluted enantiomer of example 1a. The determination was used to confirm absolute configuration of the structure as here reported:

(S)-3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-isochromen-1-one, first eluted enantiomer (the title compound of example 1a).

(R)-3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-isochromen-1-one, second eluted enantiomer.

Example 2a: 3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethylamino)methyl)phenyl)-1H-isochromen-1-one, First Eluted Enantiomer

[Image Omitted]

Racemate 3-{1-[4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(dimethylamino)methyl]phenyl}-1H-isochromen-1-one hydrochloride (Example 2) (0.228 g, 0.400 mmol) was diluted with MeOH and was passed through a PL-HCO3 MP resin cartridge (5 g) to obtain the free base (0.110 g), which was dissolved in Ethanol/Methanol/n-Hexane 1/1/1 (10 mL) and submitted to chiral resolution by Chiral preparative chromatography. Conditions: Column: Chiralpak AD-H (25×2.0 cm), 5 μm; Mobil phase: n-Hexane/(Ethanol+0.1% isopropylamine) 75/25% v/v; Flow rate 20 mL/min; DAD detection: 220 nm; Loop: 2500 μl; Injection: 27 mg/injection. The fractions containing the first eluted enantiomer were evaporated to dryness to afford title compound 3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((dimethylamino)methyl)phenyl)-1H-isochromen-1-one (0.040 g, 0.075 mmol).

Chiral HPLC (Method A3),

Rt 6.4 min (first eluted enantiomer), e.e.>99%.

UPLC-MS: 0.46-0.50, 534.3 [M+H]+, method 3.

¹H NMR (400 MHz, DMSO-d6) δ ppm 10.16 (bs, 1H), 8.18-8.30 (m, 3H), 8.04-8.12 (m, 1H), 7.72-7.81 (m, 1H), 7.57-7.68 (m, 1H), 7.43-7.53 (m, 1H), 7.25-7.40 (m, 4H), 6.82-7.04 (m, 3H), 5.67-5.83 (m, 1H), 3.16-3.49 (m, 2H), 1.97-2.25 (m, 6H), 1.78-1.87 (m, 3H).

Analytical Data of Second Eluting Enantiomer

Chiral HPLC (Method A3),

Rt 14.6 min (second eluted enantiomer), e.e.>99%

UPLC and ¹H NMR analyses of pure enantiomers were superimposable in all respect.

Example 4a: 3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((R)-1-methylpyrrolidin-2-yl)phenyl)-1H-isochromen-1-one, First Eluted Diastereomer

[Image Omitted]

3-{1-[4-Amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]ethyl}-4-{3-[(2R)-1-methylpyrrolidin-2-yl]phenyl}-1H-isochromen-1-one (Example 4) (0.050 g, 0.089 mmol) was dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol (2.5 mL) and submitted to chiral resolution by Chiral preparative chromatography. Conditions: Column: Chiralpak AD-H (25×2.0 cm), 5 μm; Mobile phase: (Ethanol+0.1% isopropylamine) 15%; Flow rate 46 mL/min; DAD detection: 220 nm; Loop: 600 μl; Injection: 12 mg/injection. The fractions containing the first eluted diastereomer were evaporated to dryness to afford title compound 3-(1-(4-amino-3-(5-hydroxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4-(3-((R)-1-methylpyrrolidin-2-yl)phenyl)-1H-isochromen-1-one (0.017 g, 0.03 mmol).

Chiral HPLC (Method A4),

Rt 3.3 min (first eluted diastereomer), d.e.=98.5%,

UPLC-MS: 0.70 min, 560.5 [M+H]+, method 5.

¹H NMR (400 MHz, MeOD) δ ppm 8.29-8.35 (m, 1H), 8.22-8.28 (m, 1H), 8.15-8.21 (m, 1H), 8.03-8.12 (m, 1H), 7.65-7.74 (m, 1H), 7.56-7.63 (m, 1H), 7.18-7.55 (m, 4H), 6.76-6.99 (m, 2H), 5.88-6.00 (m, 1H), 2.85-3.28 (m, 2H), 1.54-2.46 (m, 11H).

Analytical Data of Second Eluting Diastereomer

Chiral HPLC (Method A4),

Rt 7.0 min (second eluted diastereomer), d.e.=96.2%.

UPLC-MS: 0.70 min, 560.5 [M+H]+, method 5.

¹H NMR (400 MHz, MeOD) δ ppm 8.30-8.35 (m, 1H), 8.24-8.29 (m, 1H), 8.17-8.21 (m, 1H), 8.04-8.13 (m, 1H), 7.66-7.75 (m, 1H), 7.56-7.64 (m, 1H), 7.17-7.55 (m, 4H), 6.74-7.06 (m, 2H), 5.83-6.03 (m, 1H), 2.94-3.29 (m, 2H), 1.70-2.45 (m, 11H).

Salt Screening Examples

Particularly preferred salts of the invention are prepared following known methods of salification described for example in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, 2011 (P. Heinrich Stahl, Camille G. Wermuth (Eds.)°

The Salts reported in the table here below were prepared from the (S) enantiomer compound of example 1a and the salts from the (R) enantiomer are equally obtained using the said generally known methods of salification.

As an example the maleate salt was obtained dissolving 300 mg of free base in acetone (9 mL) and stirring at ˜20° C. for ˜2 hrs and then at 50° C. for ˜30 min to gain complete free base dissolution. Maleic acid (1.02 eq) was then added at 20° C. Slurry was left stirring at 650 rpm˜20° C. o/n. Sample was filtered on syringe filter (porosity 20 μL, PTFE frit) and dried under vacuum at room temperature until reaching constant weight (3 hrs) (yield=86%).

Equivalent

Acid Name
of acid
Salt name
salts form example 1a

HBr
1
Hydrobromide
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Hydrobromide

HCl
1
Hydrochloride
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Hydrochloride

Naphtalene-
0.5
Hemi 1,5-
(R) and/or (S)-3-(1-(4-amino-3-(5-

1,5-disulfonic

Naphthalene
hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

acid

disulfonate
d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Hemi 1,5-

Naphthalenedisulfonate

Sulfuric acid
0.5
Hemisulfate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Hemisulfate

P-toluensulfonic
1
Tosylate
(R) and/or (S)-3-(1-(4-amino-3-(5-

acid

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Tosylate

Methansulfonic
1
Mesylate
(R) and/or (S)-3-(1-(4-amino-3-(5-

acid

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Mesylate

Naphtalen-2-
1
2-Naphthalene
(R) and/or (S)-3-(1-(4-amino-3-(5-

sulfonic acid

sulfonate
hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one-2-Naphthalene

sulfonate

Isethionic acid
1
Isethionate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Isethionate

Maleic acid
1
Maleate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Maleate

Ethansulfonic
1
Esylate
(R) and/or (S)-3-(1-(4-amino-3-(5-

acid

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Esylate

Pamoic acid
0.5
Hemipamoate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Hemipamoate

1-hydroxy
1
Xinafoate
(R) and/or (S)-3-(1-(4-amino-3-(5-

naphthalene-2-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

carboxylic acid

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Xinafoate

Salycilic acid
1
Salycilate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Salycilate

Benzoic acid
1
Benzoate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-((4-

methylpiperazin-1-yl)methyl)phenyl)-

1H-isochromen-1-one Benzoate

Some further preferred salts are reported in the table here below prepared from the (S) enantiomer compound of example 2a and the salts from the (R) enantiomer are equally obtained using the said generally known methods of salification.

Equivalent

Acid Name
of acid
Salt name
salts form example 2a

HBr
1
Hydrobromide
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-

((dimethylamino)methyl)phenyl)-1H-

isochromen-1-one Hydrobromide

HCl
1
Hydrochloride
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-

((dimethylamino)methyl)phenyl)-1H-

isochromen-1-one Hydrochloride

Methansulfonic
1
Mesylate
(R) and/or (S)-3-(1-(4-amino-3-(5-

acid

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-

((dimethylamino)methyl)phenyl)-1H-

isochromen-1-one Mesylate

Isethionic acid
1
Isethionate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-

((dimethylamino)methyl)phenyl)-1H-

isochromen-1-one 2-Naphthalene

sulfonate

Maleic acid
1
Maleate
(R) and/or (S)-3-(1-(4-amino-3-(5-

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-

((dimethylamino)methyl)phenyl)-1H-

isochromen-1-one Maleate

Ethansulfonic
1
Esylate
(R) and/or (S)-3-(1-(4-amino-3-(5-

acid

hydroxypyridin-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-1-yl)ethyl)-4-(3-

((dimethylamino)methyl)phenyl)-1H-

isochromen-1-one Esylate

The salts listed above are obtained in solid form, most of them in substantially amorphous solid state; some particularly preferred were exhibiting a crystalline solid state.

Pharmacological Activity of the Compounds of the Invention.

In Vitro Determination of the PI3K Enzyme Inhibitory Activity in the Cell Free Assay

Human recombinant proteins PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ were purchased from Millipore Ltd (Billerica, MA). Compounds were dissolved at 0.5 mM in DMSO and were tested at different concentrations for their activity against PI3Ks using the ADP-Glo™ Kinase Assay (Promega, Madison WI) according to the manufacturer's instructions.

Briefly, the kinase reactions were performed in 384-well white plates (Greiner Bio-One GmbH, Frickenhausen). Each well was loaded with 0.1 μl of test compounds and 2.5 μl of 2× reaction buffer (40 mM Tris pH7.5, 0.5 mM EGTA, 0.5 mM Na₃VO₄, 5 mM β-glycerophosphate, 0.1 mg/ml BSA, 1 mM DTT), containing 50 μM PI and PS substrates (L-α-phosphatidylinositol sodium salt and L-α-phosphatidyl-L-serine, Sigma-Aldrich, St. Louis MO) and the PI3K recombinant proteins (PI3Kγ 0.25 ng/μl, PI3Kδ 1 ng/μl, PI3Kα 0.125 ng/μl, PI3Kβ 1 ng/μl).

The reactions were started by adding 2.5 μl of 2×ATP solution to each well (final concentrations: PI3Kγ ATP 30 μM; PI3Kδ ATP 80 μM; PI3Kα ATP 50 μM; PI3Kβ ATP 100 μM) and incubated for 60 min at room temperature. Subsequently, each kinase reaction was incubated for 40 min with 5 μl ADP-Glo™ Reagent, allowing depletion of unconsumed ATP. Then, the Kinase Detection Reagent (10 μl) was added in each well to convert ADP to ATP and to allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. Following 60 min incubation, the luminescence signal was measured using a Wallac EnVision® multilabel reader (PerkinElmer, Waltham MA).

Curve fitting and IC50 calculation were carried out using a four-parameter logistic model in XLfit (IDBS, Guilford, UK) for Microsoft Excel (Microsoft, Redmont, WA).

The results are provided below

Table 1a: Results of the in vitro determination of the PI3K enzyme inhibitory activity in the cell free assay for comparative examples; reproduced from WO2015091685.

Compound of
PI3K alpha
PI3K beta
PI3K delta
PI3K gamma

Example N.
inhibition
inhibition
inhibition
inhibition

142
+
+
+++
+

Comparative

example 1

168a
++
++
+++
++

Comparative

example 2

67a
++
++
+++
++

Comparative

example 3

wherein the compounds were classified in term of potency with respect to their inhibitory activity on PI3K -alfa, -beta, -gamma and -delta according to the following:

+++ : IC50 < 10 Nm

++ : IC50 in the range 10-1000 nM

+: IC50 > 1000 nM

Compounds of the present invention are potent delta inhibitors having Delta IC50<2 nM in the enzymatic assay as reported in the following table 1b.

In Vitro Determination of the PI3Kδ Enzyme Inhibitory Activity in the THP-1 Assay

The inhibitory activity of compounds on PI3Kinase in living cells was determined by evaluating the inhibition of the PI3Kδ-AKT pathway endogenously expressed on THP-1 cells. THP-1 cell suspension from T75 flask (0.4-1.0×10⁶/mL) was centrifuged and the cell pellet re-suspended in starvation medium at 1.5×10⁶cells/mL. Cell plate was prepared by dispensing 3×10⁵cells/well and incubated for 24 hours at 37° C. before the assay. Test compounds were serially diluted 1:3 in DMSO and then further diluted 1:100 in compound medium. Starved THP-1 cells were pre-incubated at 37° C. for 60 minutes with test compound solutions or vehicle. Cells were then stimulated for 10 minutes by Macrophage Colony-Stimulating Factor (M-CSF) 2.5 ng/mL or stimulus medium (control of basal pAKT levels). Cells were then lysed and the amount of phosphorylated AKT measured by using a Cisbio p-Ser473 AKT HTRF assay kit. Stimulation was ended by the addition of supplemented lysis buffer. Cell plate was shaken for 30 minutes at room temperature to complete cell lysis, followed by the addition of HTRF conjugates and incubated for further 4 hours at room temperature. Conjugates react with pAKT causing an increase in HTRF signal that is measured with the Envision plate reader with a HRTF reading protocol. Ratio data were fitted using a logistical four-parameter equation to determine IC₅₀. IPI-145 compound was used as pharmacological standard.

The compounds of the invention showed cellular IC50 (THP-1 IC₅₀nM) values lower than 4 nM with respect to the PI3K-delta subunit as reported in the following table 1b.

Enzymatic
THP-1

Delta
Delta
IC₅₀
Delta cell

Ki
IC₅₀
nM
drop-

Comments

nM
nM
Cellular
off**

Comparative example 1 Second eluting enantiomer by chiral separation from compound mixture of ex 142 in WO 2015/091685

[Image Omitted]

1.1
2.2
1.8
1.6

Comparative example 2 ex 168a in WO 2015/091685

[Image Omitted]

0.6
1.1
1.6
2.7

Comparative example 3 ex 67a in WO 2015/091685

[Image Omitted]

0.3
0.7
1.5
5.0

Example 1a

[Image Omitted]

0.9
1.7
2.8
3.1

Example 2a

[Image Omitted]

0.5
1.0
0.6
1.2

**Cellular/enzymatic activity = THP-1 IC₅₀nM/Delta Ki nM

enzymatic
THP-1

Delta
Delta
IC50

Ki
IC50
nM
Delta cell

Comments

nM
nM
cellular
drop-off

Example 3

[Image Omitted]

0.4
0.7
3.9
9.7

Example 4a

[Image Omitted]

0.1
0.3
1.0
10

Example 5

[Image Omitted]

0.5
1.0
1.8
3.6

Example 6

[Image Omitted]

0.4
0.8
3.0
7.5

Example 7

[Image Omitted]

0.8
1.6
3.9
4.9

** Cellular/enzymatic activity = THP-1 IC₅₀nM/Delta Ki nM

Protocol of Rat Model of Ovalbumin (OVA) Induced Lung Eosinophilia in Rat (WET Administration—Saline Solution)

- Animals

Male Brown Norway rats (6 weeks old at arrival) were purchased from Charles River Laboratories Italy (Calco, Lecco). Prior to use, animals were acclimated for at least 5 days to the local vivarium conditions (room temperature: 20-24° C.; relative humidity: 40-70%), having free access to standard rat chow and tap water. All animal protocols described herein were carried out approved by the intramural animal-welfare committee for animal experimentation of Chiesi Farmaceutici and Ministry of Health and comply with the European Directive 2010/63 UE and Italian D Lgs 26/2014 (authorization number 198-PR).

Sensitization and Allergen Exposure

Male Brown-Norway rats were sensitized by intraperitoneal injection of a suspension containing OVA (1 mg/rat) and Al(OH)₃(100 mg/rat) in 1 mL of saline for 3 consecutive days; two weeks later, the airway inflammation was induced by aerosol administration of the allergen. Specifically, the animals were exposed to an aerosol of OVA solution (1% in saline) that induced a massive influx of inflammatory cells in the airways, mainly represented by eosinophils and neutrophils. Briefly, rats were restrained in plexiglass tubes, introduced in the inhalation chamber and exposed to the aerosolised solution by nose-only inhalation for 30 min. The inhalation chamber was connected to a Medel Pro nebulizer that generated a nebulised solution in the chamber at a flow rate of approximately 3.3 l/min. Control animals were exposed to aerosolized saline.