EA041006B1 - CRYSTAL SALT, METHOD FOR ITS PRODUCTION, PHARMACEUTICAL COMPOSITION CONTAINING IT AND METHODS FOR INHIBITION AND TREATMENT WITH ITS USE - Google Patents

Abstract

The invention provides a crystalline hydrochloride salt of (K)-4-(3-((8)-1-(4-amino-3methyl-1H-pyrazolo[3,4-D]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2one, which is suitable as an inhibitor of phosphoinositide 3-kinase delta (PI3K6), a pharmaceutical composition containing it, as well as methods for using and preparing it.

Description

This application claims priority over U.S. Provisional Application No. 62/121697, filed Feb. 27, 2015, which is hereby incorporated by reference in its entirety.

Technical field

The invention provides a crystalline hydrochloric acid salt of (R)-4-(3-((S)-1-(4-amino-3-methyl1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5- chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, which is useful as a phosphoinositide-3-kinase delta (PI3Kδ) inhibitor, as well as methods for its use and preparation.

State of the art

Phosphoinositide 3-kinases (PI3Ks) belong to a large family of lipid signaling kinases that phosphorylate phosphoinositides at the D3 position of the inositol ring (Cantley, Science, 2002, 296(5573):1655-7). PI3Ks are classified into three classes (class I, II, and III) according to their structure, regulation, and substrate specificity. Class I PI3K, which includes PI3Ka, PI3Ke, PI3Ky, and PI3Kδ, is a dual-specific family of lipid kinases and protein kinases that catalyze the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP ₂ ) to give phosphatidylinositol-3,4,5-triphosphate ( _PIP3 ). PIP3 functions as a second messenger regulating a number of cellular processes, including growth, survival, adhesion, and migration. All four PI3K class I isoforms exist as heterodimers consisting of a catalytic subunit (p110) and a tightly coupled regulatory subunit that regulates their expression, activation, and intracellular location. PI3Ka, PI3Ke and PI3Kδ are associated with a regulatory subunit known as p85 and are activated by growth factors and cytokines through a tyrosine kinase dependent mechanism (Jimenez, et al., J. Biol. Chem., 2002, 277(44):41556-62 ), while PI3Ky is bound to two regulatory subunits (p101 and p84) and its activation is triggered by activation of G-protein coupled receptors (Brock, et al., J. Cell. Biol., 2003, 160(1):89 -99). PI3Ka and PI3Ke are ubiquitously expressed. In contrast, PI3Ky and PI3Kδ are predominantly expressed in leukocytes (Vanhaesebroeck, et al., Trends Biochem Sci., 2005, 30(4):194-204).

Different distribution of PI3K isoforms in tissues leads to their different biological functions. The genetic absence of both PI3Ka and PI3Ke results in embryonic death, indicating that PI3Ka and PI3Ke have essential and non-redundant functions, at least during development (Vanhaesebroeck, et al., 2005). In contrast, mice lacking PI3Ky and PI3Kδ are viable, reproductive, and have a normal lifespan, although their immune system is altered. PI3Ky deficiency results in impaired recruitment of macrophages and neutrophils to inflammatory sites, as well as impaired T cell activation (Sasaki, et al., Science, 2000, 287(5455):1040-6). PI3Kδ mutant mice have specific disturbances in B cell signaling that result in impaired B cell development and attenuated antibody responses after antigen challenge (Clayton, et al., J. Exp. Med. 2002, 196(6):753-63; Jou, et al., Mol Cell Biol 2002, 22(24):8580-91; Okkenhaug, et al., Science, 2002, 297(5583):1031-4).

The phenotypes of mice with PI3Ky and PI3Kδ mutants suggest that these enzymes may play a role in inflammation and other immune-related diseases, and this is supported in preclinical models. PI3Ky mutant mice are significantly protected from disease in mouse models of rheumatoid arthritis (RA) and asthma (Camps, et al., Nat. Med. 2005, 11(9):936-43; Thomas, et al., Eur. J Immunol 2005, 35(4):1283-91). Additionally, treatment of wild-type mice with a selective PI3Ky inhibitor has been shown to attenuate glomerulonephritis and improve survival in the MRL-lpr mouse model of systemic lupus erythematosus (SLE) and suppress inflammation and joint damage in RA models (Barber, et al., Nat. Med. 2005, 11(9):933-5; Camps, et al., 2005). Similarly, both PI3Kδ mutant mice and wild-type mice treated with a selective PI3Kδ inhibitor have been shown to exhibit attenuated allergic airway inflammation and hyperresponsiveness in a mouse model of asthma (Ali, et al., Nature. 2004, 431 (7011):1007-11; Lee, et al., FASEB J. 2006, 20(3):455-65), and an attenuated form of the disease is observed in the RA model (Randis, et al., Eur. J. Immunol. , 2008, 38(5):1215-24).

B cell proliferation has been shown to play a key role in the development of inflammatory autoimmune diseases (Puri, Frontiers in Immunology (2012), 3(256), 1-16; Walsh, Kidney International (2007) 72, 676-682). For example, B cells maintain T cell autoreactivity, an important component of inflammatory autoimmune diseases. After activation and maturation, B cells can move to sites of inflammation and recruit cells in the area of inflammation or differentiate into plasmablasts. Thus, the activity of B cells can be influenced by acting on cytokines that stimulate B cells, receptors on the surface of B cells, or by suppressing B cells. Rituximab, a chimeric mouse/human IgG1K monoclonal antibody directed against the CD20 receptor on the surface of B cells, has been shown to suppress CD20+ B cells. The use of rituximab has been shown to be effective in the treatment of idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia

- 1 041006 mii or vasculitis. For example, treatment with rituximab resulted in disease remission in patients suffering from antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis (AACV) with demonstrated B-cell suppression in peripheral blood (Walsh, 2007; Lovric, Nephrol Dial Transplant (2009) 24: 179-185). Similarly, a complete response has been reported in one-third to two-thirds of patients suffering from mixed cryoglobulinemic vasculitis after treatment with rituximab, including patients suffering from severe vasculitis that is resistant to or intolerant to other treatments (Cacoub, Ann Rheum Dis 2008 ; 67:283-287). Similarly, rituximab has been shown to be effective in the treatment of patients with idiopathic thrombocytopenic purpura (or immune thrombocytopenic purpura) (Garvey, British Journal of Haematology, (2008) 141, 149-169; Godeau, Blood (2008), 112(4), 999 -1004; Medeo, European Journal of Haematology, (2008) 81, 165-169) and autoimmune hemolytic anemia (Garvey, British Journal of Haematology, (2008) 141, 149-169).

PI3Kδ signaling is associated with B cell survival, migration, and activation (Puri, Frontiers in Immunology, 2012, 3(256), 1-16, at pages 1-5; and Clayton, J. Exp. Med., 2002 , 196(6):753-63). For example, PI3Kδ is required for antigen dependent B cell activation triggered by the B cell receptor. By blocking B cell adhesion, survival, activation, and proliferation, inhibition of PI3Kδ can interfere with the ability of B cells to activate T cells, preventing their activation and decreasing the secretion of autoantibodies and pro-inflammatory cytokines. Therefore, due to their ability to inhibit B cell activation, PI3Kδ inhibitors are expected to treat B cell mediated diseases that are treatable by similar methods such as B cell suppression with rituximab. Indeed, PI3Kδ inhibitors have been shown to be effective in mouse models of various autoimmune diseases that are also amenable to rituximab treatment, such as arthritis (Puri (2012)). Additionally, innate-like B cells, which are associated with autoimmunity, are sensitive to PI3Kδ activity, as MZ and B-1 cells are virtually absent in mice lacking the p110δ gene (Puri (2012). PI3Kδ inhibitors can reduce the directional migration and activation of MZ cells and B-1, which are involved in autoimmune diseases.

In addition to their possible role in inflammatory diseases, all four class I PI3K isoforms may play a role in cancer. The p110a gene is frequently mutated in common types of cancer, including breast, prostate, colon, and endometrial cancers (Samuels, et al., Science, 2004, 304(5670):554; Samuels, et al., Curr Opin Oncol 2006, 18(1):77-82). Eighty percent of these mutations are represented by one of three amino acid substitutions in the helical or kinase domains of the enzyme and result in significant upregulation of kinase activity leading to oncogenic transformation in cell culture and animal models (Kang, et al., Proc. Natl. Acad. Sci Bader, et al., Proc. Natl Acad. Sci. USA 2006, 103(5):1475-9). None of these mutations have been found in other PI3K isoforms, although there is evidence that they may contribute to the development and progression of malignant neoplasms. Corresponding overexpression of PI3Kδ is observed in acute myeloblastic leukemia (Sujobert, et al., Blood, 2005, 106(3):1063-6), and PI3Kδ inhibitors can prevent the growth of leukemic cells (Billottet, et al., Oncogene. 2006, 25( 50):6648-59). Increased overexpression of PI3Ky has been observed in chronic myeloid leukemia (Hickey, et al., J. Biol. Chem. 2006, 281(5):2441-50). Altered expression of PI3Ke, PI3Ky, and PI3Kδ has also been observed in brain, colon, and bladder cancers (Benistant, et al., Oncogene, 2000, 19(44):5083-90; Mizoguchi, et al., Brain. Pathol. 2004, 14(4):372-7; Knobbe, et al., Neuropathol Appl Neurobiol 2005, 31(5):486-90).

Additionally, all these isoforms have been shown to be oncogenic in cell culture (Kang, et al., 2006).

For these reasons, there is a need to develop new PI3K inhibitors that can be used in inflammatory disorders, autoimmune diseases and cancer. The present invention addresses this and other needs.

Brief description of the essence of the invention

The invention provides a hydrochloric acid salt of a compound of formula I

- 2 041006

which is crystalline.

The invention also provides pharmaceutical compositions having the ability to inhibit PI3Kδ kinase activity, comprising a crystalline hydrochloric acid salt as described herein, and a pharmaceutically acceptable carrier.

The invention further provides methods for inhibiting PI3K kinase activity, comprising contacting the kinase with a crystalline hydrochloride salt of a compound of formula I.

The invention also provides methods for treating a disease in a patient, said disease being associated with abnormal expression or activity of PI3K kinase, comprising administering to said patient a therapeutically effective amount of a crystalline hydrochloric acid salt of a compound of Formula I.

The invention also provides a process for the preparation of a crystalline hydrochloric acid salt of a compound of formula I, comprising bringing the compound of formula I

in contact with hydrochloric acid to form a suspension; heating the suspension to form a solution; and cooling the solution to crystallize the salt.

Brief description of graphic materials

In FIG. 1 shows a DSC thermogram typical of the salt of Example 3, FIG. 2 shows TGA data representative of the salt of Example 3, FIG. 3 is a XRD pattern typical of the salt of Example 3.

Detailed Description of the Invention

Salt.

The invention provides a salt of a compound of formula I

where Et is ethyl, which is crystalline.

More specifically, the invention provides the hydrochloric salt of (R)-4-(3-((S)-1-(4-amino-3-methyl1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)- 5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, which is crystalline. In some embodiments, the salt is a 1:1 stoichiometric ratio of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine- 1yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one to hydrochloric acid.

Different forms of the same substance have different bulk properties, which refers,

- 3 041006 for example, to hygroscopicity, solubility, stability, etc. Formulations with high melting points often have good thermodynamic stability, which is useful for extending the shelf life of drug formulations containing the solid form. Forms with lower melting points are often less thermodynamically stable, but convenient in that they have increased water solubility corresponding to increased drug bioavailability. Forms that are slightly hygroscopic are useful because of their stability to heat and moisture, and they are resistant to degradation on long-term storage.

The hydrochloride salt of the compound of formula I as provided herein is crystalline. As used herein, crystalline or crystalline form refers to a particular lattice configuration of a crystalline substance. Different crystalline forms of the same substance, as a rule, have different crystal lattices (for example, unit cells), corresponding to different physical properties, which are a characteristic feature of each of the crystalline forms. In some cases, different lattice configurations have different water or solvent contents.

Various salt forms can be identified using solid state techniques such as X-ray powder diffraction (XRD). Other testing methods such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DSP) and the like further assist in establishing the shape, as well as assist in determining the stability and solvent/water content.

The X-ray powder diffraction (X-ray powder diffraction) set of reflections (peaks) is generally considered to be characteristic of a particular crystalline form. It is well known that the relative intensities of XRD peaks can vary greatly depending on, among other things, the sample preparation method, the crystal size distribution, the various filters used, the sample setup method, and the specific instrument used. In some cases, new peaks appear or existing peaks disappear, depending on instrument type or parameters. As used herein, the term peak refers to a reflection having a relative height/intensity of at least about 4% of the peak's maximum height/intensity. In addition, equipment changes and other factors may affect the 2θ values. Thus, given peak values, such as those presented herein, can vary by plus or minus about 0.2° (2θ), and the term practically or about, as used in connection with VTP, herein encompasses the above variations.

In some embodiments, the hydrochloride salt of a compound of formula I has at least one peak in 2θ units selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28, 1°, about 31.2° and about 32.8°. In some embodiments, the hydrochloride salt of a compound of formula I has at least two peaks in 2θ units selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28, 1°, about 31.2° and about 32.8°. In some embodiments, the hydrochloride salt of a compound of formula I has at least three peaks in 2θ units selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28, 1°, about 31.2° and about 32.8°. In some embodiments, the hydrochloride salt of a compound of formula I has at least four peaks in 2θ units selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28, 1°, about 31.2° and about 32.8°. In some embodiments, the hydrochloride salt of a compound of formula I has at least five peaks in 2θ units selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28, 1°, about 31.2° and about 32.8°. In some embodiments, the hydrochloride salt of a compound of Formula I has substantially the same XRD pattern as shown in FIG. 3.

Similarly, temperature readings in connection with DSC, TGA, or other thermal experiments may vary by about ±3°C depending on equipment, specific parameters, sample preparation, etc. Therefore, the crystalline form described herein has a DSC thermogram substantially the same as shown in any of the figures, or the term about is considered to compensate for this difference. In some embodiments, the hydrochloride salt of a compound of Formula I has a DSC thermogram showing an endothermic peak at about 207°C. In some embodiments, the hydrochloride salt of a compound of formula I has a DSC thermogram substantially the same as shown in FIG. 1. In some embodiments, the hydrochloride salt of a compound of formula I has a TGA thermogram substantially the same as shown in FIG. 2.

In some embodiments, the salt described herein (the hydrochloric acid salt of the compound of Formula I) is substantially isolated. By isolated, it is generally understood that the salt is at least partially or substantially separated from the environment in which it was produced or found. Partial separation may include, for example, a composition enriched with the salt described herein. The separation to a large extent may include compositions containing at least about 50%, at least about 60%, at least

- 4 041006 at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the salt described herein . Methods for isolating salts are known in the art.

Ways.

The invention further provides a process for preparing a crystalline salt of a compound of formula I

In some embodiments, the method comprises bringing a compound of formula I

in contact with hydrochloric acid to obtain said salt.

In some embodiments of the invention, the specified hydrochloric acid is a 1M aqueous solution of hydrochloric acid.

In some embodiments of the invention, 1 eq. compounds of formula I use from about 3.3 to about 3.7 eq. of hydrochloric acid.

In some embodiments of the invention, the specified bringing into contact is carried out at a temperature of from about 45 to about 55°C.

In some embodiments of the invention, the method includes the steps in which hydrochloric acid is added to the compound of formula I at room temperature to form a slurry;

heating said slurry to a temperature of about 45 to about 55°C to form a solution; and cooling the solution to a temperature of about 0 to about 5°C to crystallize said salt.

It is understood that some features of the invention that are described for clarity in various embodiments of the invention may also be combined into one embodiment of the invention (although it is intended that the embodiments be combined as if written in a multi-dependent form). On the other hand, various features of the invention, which are described for brevity in one embodiment of the invention, may also be presented separately or in any suitable subcombination.

Separation of racemic salt mixtures can be carried out by any of the numerous methods known in the art. An exemplary method involves fractional recrystallization using a chiral resolving acid, which is an optically active salt-forming organic acid. Suitable resolving agents for fractional recrystallization processes are, for example, optically active acids such as the D- and L-forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acids such as βcamphorsulfonic acid . Other separating agents useful in fractional recrystallization methods include stereoisomerically pure forms of α-methylbenzylamine (e.g., S- and R-forms or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2- diaminocyclohexane and the like.

Separation of racemic mixtures can also be carried out by elution on a column containing an optically active resolving agent (eg dinitrobenzoylphenylglycine). Specialist in

- 5 041006 of the art can determine the appropriate composition of solvents for elution.

The salt of this invention may also contain all isotopes of atoms that occur in intermediate or final salts or compounds. Isotopes include those atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

In some embodiments, the salt may be present with other substances such as water and solvents (eg, hydrates and solvates), or may be isolated.

In some embodiments, the salt described herein (the hydrochloric acid salt of the compound of Formula I) is substantially isolated. By isolated, it is generally understood that the salt is at least partially or substantially separated from the environment in which it was produced or found. Partial separation may include, for example, a composition enriched with the compounds of this invention. The separation may substantially include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% , at least about 97%, at least about 99% by weight of the compounds of this invention or their salts. Methods for isolating compounds and their salts are known in the art.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions and/or dosage forms that, based on careful medical evaluation, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications. commensurate with a reasonable benefit/risk ratio.

It is to be understood that the salt provided herein may be prepared using known organic synthesis techniques and may be synthesized according to any of the various possible synthetic approaches. The methods described herein can be controlled in accordance with any suitable method known in the art. For example, product formation can be followed by spectroscopy, such as nuclear magnetic resonance spectroscopy (eg 1H or ¹³ C), infrared spectroscopy, or spectrophotometry (eg UV and visible region); or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC), or other related methods.

As used herein, the term bringing into contact is used as is known in the art and generally refers to the combination of chemical reactants in such a way that they are allowed to interact at the molecular level to achieve a chemical or physical transformation. In some embodiments of the invention, bringing into contact includes two reagents, and use one or more equivalents of the second reagent compared to the first reagent. The reaction steps of the methods described herein can be carried out at times and under conditions suitable to obtain the specified product.

The reactions of the methods described herein can be carried out in suitable solvents, which can be easily selected by a person skilled in the field of organic synthesis. Suitable solvents may be solvents that are substantially unreactive with the starting materials (reagents), intermediates or products at the reaction temperatures, for example at temperatures that may vary from the freezing point of the solvent to the boiling point of the solvent. This reaction can be carried out in one solvent or in a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for the particular reaction step may be selected.

Suitable solvents may include halogenated solvents such as carbon tetrachloride, bromodichloromethane, dibromochloromethane, bromoform, chloroform, bromochloromethane, dibromomethane, butyl chloride, dichloromethane, tetrachloroethylene, trichlorethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1, 1-dichloroethane, 2-chloropropane, 1,2-dichloroethane, 1,2-dibromoethane, hexafluorobenzene, 1,2,4-trichlorobenzene, 1,2-dichlorobenzene, chlorobenzene, fluorobenzene, mixtures thereof, and the like.

Suitable ether solvents include: dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether, anisole, methyl tert- butyl ether, mixtures thereof, and the like.

Suitable protic solvents may include, by way of example and without limitation, water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol, 2 -butanol, isobutyl alcohol, t-butyl alcohol, 2ethoxyethanol, diethylene glycol, 1-, 2- or 3-pentanol, neopentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenol or glycerin.

Suitable aprotic solvents may include, by way of example and without limitation, tetrahydrofuran (THF), K,K-dimethylformamide (DMF), K,K-dimethylacetamide (DMA), 1,3-dimethyl-3,4,5,6-6 041006 tetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP), formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethyl sulfoxide, propionitrile, ethyl formate, methyl acetate, hexachloroacetone, acetone, methyl ethyl ketone, ethyl acetate, sulfolane, N,N-dimethylpropionamide, tetramethylurea, nitromethane, nitrobenzene, or hexamethylphosphoramide.

Suitable hydrocarbon solvents include benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o- or p-xylene, octane, indane, nonane or naphthalene.

The reactions of the methods described herein can be carried out at suitable temperatures, which can be easily determined by a person skilled in the art. The reaction temperatures depend, for example, on the melting and boiling points of the reactants and the solvent, if present; reaction thermodynamics (for example, violent exothermic reactions may require lower temperatures); and reaction kinetics (for example, high activation energy barriers may require elevated temperatures).

The expressions ambient temperature and room temperature or rt, as used herein, are understood in the art and generally refer to a temperature, such as a reaction temperature, which is approximately equal to the temperature of the room in which the reaction is carried out, such as a temperature of about 20 up to about 30°C.

The reactions of the methods described herein can be carried out in air or in an inert atmosphere. In general, reactions involving reactants or products that are highly reactive with air can be carried out using air-sensitive synthetic techniques well known to those skilled in the art.

Application methods.

The salt of this invention can modulate the activity of one or more of various kinases, including, for example, phosphoinositide-3-kinases (PI3K). The term modulate refers to the ability to increase or decrease the activity of one or more members of the PI3K family. Therefore, the salt of this invention can be used in methods for modulating PI3K by bringing PI3K into contact with one or more of the salts, compounds, or compositions described herein. In some embodiments, the salt of the present application may act as an inhibitor of one or more PI3Ks. In further embodiments, a salt of the invention may be used to modulate PI3K activity in an individual in need of receptor modulation by administering a modulating amount of the salt of the invention. In some embodiments of the invention, the modulation is inhibition.

Because multiple signaling pathways influence the growth and survival of cancer cells, the present invention is suitable for the treatment of disease states characterized by drug-resistant kinase mutants. Additionally, various kinase inhibitors that modulate the activity of various kinases can be used in combination. This approach may be highly effective in treating disease states by targeting multiple signaling pathways, reducing the likelihood of drug resistance in the cell, and reducing the toxicity of disease treatments.

The kinases that the salt of the present invention binds and/or modulates (eg, inhibits) include any member of the PI3K family. In some embodiments, the PI3K is PI3Ka, PI3Ke, PI3Kδ, or PI3Ky. In some embodiments, the PI3K is PI3Kδ or PI3Ky. In some embodiments, the PI3K is PI3Kδ. In some embodiments, the PI3K is PI3Ky. In some embodiments of the invention PI3K contains a mutation. A mutation may be a substitution of one amino acid for another, or a deletion of one or more amino acids. In such embodiments, the mutation may be present in the kinase domain of PI3K.

In some embodiments, the salt is used in combination with another kinase inhibitor to inhibit the activity of one kinase (eg, PI3Kδ or PI3Ky).

In some embodiments, the salt is used in combination with another kinase inhibitor to inhibit the activity of more than one kinase (eg, PI3Kδ or PI3Ky), such as at least two kinases.

The salt of this invention may be selective. Selective means that a compound binds to or inhibits a kinase with greater affinity or potency, respectively, than at least one different kinase. In some embodiments, the salt of this invention is a selective inhibitor of PI3Kδ or PI3Ky over PI3Ka and/or PI3Ke. In some embodiments of the invention, the salt of this invention is a selective inhibitor of PI3Kδ (for example, compared to PI3Ka, PI3Ke and PI3Ky). In some embodiments of the invention, the salt of this invention is a selective inhibitor of PI3Kδ (for example, compared to PI3Ka, PI3Ke and PI3Ky). In some embodiments, the selectivity may be at least about 2-fold, 5-fold, 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold. times, at least about 200 times, at least about 500 times, or at least about 1000 times. Selectivity can be measured by methods known in the art. In some embodiments of the invention, selectivity can be studied at the concentration of K _m ATP for each enzyme. In some embodiments of the invention, the selectivity of the salt of this invention can be determined using cellular assays associated with the activity of a particular PI3K kinase.

Another aspect of this application relates to methods of treating a disease or disorder associated with a kinase (such as PI3K) in an individual (for example, a patient) by administering to an individual in need of such treatment, a therapeutically effective amount or dose of the salt of this application or a pharmaceutical composition for its basis. A disease associated with PI3K may include any disease, disorder or pathological condition that is directly or indirectly associated with the expression or activity of PI3K, including overexpression and/or abnormal activity. In some embodiments, the disease may be associated with Akt (protein kinase B), the mammalian target of rapamycin (mTOR), or phosphoinositide-dependent kinase 1 (PDK1). In some embodiments, the mTOR-associated disease may be inflammation, atherosclerosis, psoriasis, restenosis, benign prostatic hypertrophy, bone lesions, pancreatitis, angiogenesis, diabetic retinopathy, atherosclerosis, arthritis, immunological disorders, kidney disease, or cancer. A disease associated with PI3K may also include any disease, disorder, or condition that can be prevented, ameliorated, or treated by modulating PI3K activity. In some embodiments of the invention, the disease is characterized by abnormal PI3K activity. In some embodiments of the invention, the disease is characterized by a mutant PI3K. In such embodiments, the mutation may be present in the kinase domain of PI3K.

Examples of diseases associated with PI3K include immune diseases, including systemic ones, including, for example, rheumatoid arthritis, allergies, asthma, glomerulonephritis, lupus, or inflammation associated with any of the above.

Additional examples of diseases associated with PI3K include cancer, such as cancer of the breast, prostate, colon, endometrium, brain, bladder, skin, uterus, ovary, lung, pancreas, kidney, stomach, or hemoblastosis.

Additional examples of diseases associated with PI3K include lung diseases such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in adults.

Additional examples of PI3K-associated diseases include osteoarthritis, restenosis, atherosclerosis, bone lesions, arthritis, diabetic retinopathy, psoriasis, benign prostatic hypertrophy, inflammation, angiogenesis, pancreatitis, kidney disease, inflammatory bowel disease, myasthenia gravis, multiple sclerosis, or Sjögren's syndrome. and so on.

Additional examples of diseases associated with PI3K include idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AHA), vasculitis, systemic lupus erythematosus, lupus nephritis, pemphigus, membranous nephropathy, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), hairy cell leukemia, mantle cell lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone cell lymphoma, diffuse large B-cell lymphoma of activated B-cell-like cells (ABC), or diffuse B- large cell lymphoma from germinal center B-cells (GCC).

In some embodiments, the disease is selected from idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, vasculitis, systemic lupus erythematosus, lupus nephritis, pemphigus, autoimmune hemolytic anemia (AHA), membranous nephropathy, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma ( NHL), hairy cell leukemia, mantle cell lymphoma, Burkitt's lymphoma, small cell lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone cell lymphoma, Hodgkin's lymphoma, Waldenström's macroglobulinemia, prolymphocytic leukemia, acute lymphoblastic leukemia, myelofibrosis, lymphoma arising from mucosal-associated lymphoid tissue (MALT), B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, Hodgkin's disease, splenic marginal zone lymphoma, primary effusion lymphoma, intravascular B-large Ocellular lymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoldering myeloma (also called asymptomatic myeloma), monoclonal gammopathy of uncertain significance (MGUS), and B-cell lymphoma.

In some embodiments, the method is a method for treating idiopathic thrombocytopenic purpura (ITP) selected from recurrent ITP and refractory ITP.

- 8 041006

In some embodiments, the method is a method of treating a vasculitis selected from Behcet's disease, Cogan's syndrome, giant cell arteritis, polymyalgia rheumatica (RP), Takayasu's arteritis, Buerger's disease (thromboangiitis obliterans), central nervous system vasculitis, Kawasaki disease, polyarteritis nodosa, Churg-Strauss syndrome, mixed cryoglobulinemic vasculitis (essential or caused by hepatitis C virus (HCV)), Henoch-Schonlein purpura (HSP), leukocytoclastic vasculitis, microscopic polyangiitis, Wegener's granulomatosis, and antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis (ASV) .

In some embodiments, the method is a method for treating non-Hodgkin's lymphoma (NHL) selected from relapsed NHL, refractory NHL, and secondary follicular NHL.

In some embodiments, the method is a method of treating B-cell lymphoma, wherein said B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL).

In some embodiments, the method is a method of treating B-cell lymphoma, wherein said B-cell lymphoma is diffuse activated B-cell-like cell B-large cell lymphoma (AVC) or diffuse germ-center B-cell-like B-cell lymphoma (GCC).

In some embodiments, said disease is osteoarthritis, restenosis, atherosclerosis, bone lesions, arthritis, diabetic retinopathy, psoriasis, benign prostatic hypertrophy, inflammation, angiogenesis, pancreatitis, kidney disease, inflammatory bowel disease, myasthenia gravis, multiple sclerosis, or syndrome Shegren.

In some embodiments of the invention, the specified disease is rheumatoid arthritis, allergies, asthma, glomerulonephritis, lupus, or inflammation associated with any of the above.

In some embodiments, said lupus is systemic lupus erythematosus or lupus nephritis.

In some embodiments, said disease is breast cancer, prostate cancer, colon cancer, endometrial cancer, brain cancer, bladder cancer, skin cancer, uterine cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer or hemoblastosis.

In some embodiments of the invention, said hemoblastosis is acute myeloid leukemia or chronic myeloid leukemia.

In some embodiments, said hemoblastosis is B-cell lymphocytic leukemias, including indolent/aggressive B-cell non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL).

In some embodiments of the invention, the specified disease is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in adults.

As used herein, the term bringing into contact refers to the association of the indicated fragments in an in vitro system or in an in vivo system. For example, contacting a PI3K with a compound of this invention includes administering the salt of the present application to an individual or patient such as a human having PI3K, as well as, for example, administering the salt of this invention to a sample containing a cell or purified preparation containing PI3K.

As used herein, the terms individual or patient, used interchangeably, refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, and most preferably people.

As used herein, the phrase "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, individual, or human as required by a researcher, veterinarian, physician, or other clinician. In some embodiments, the dosage of salt administered to a patient or individual is about 1 mg to about 2 g, about 1 to about 1000 mg, about 1 to about 500 mg, about 1 to about 100 mg, about 1 up to 50 mg, or from about 50 to about 500 mg.

As used herein, the term treat or treatment refers to one or more of (1) preventing a disease; for example, preventing a disease, condition, or disorder in an individual who may be predisposed to the disease, condition, or disorder but does not yet experience or exhibit the pathology or symptomatology of the disease; (2) disease inhibition; for example, inhibiting a disease, condition, or disorder in an individual who experiences or exhibits the pathology or symptomatology of the disease, condition, or disorder (ie, arresting the further development of the pathology and/or symptomatology); and (3) ameliorating the disease; for example, amelioration of a disease, condition, or disorder in an individual who experiences or exhibits the pathology or symptomatology of the disease, condition, or disorder (i.e.,

reversal of pathology and/or symptomatology), such as a reduction in the severity of the disease.

Pharmaceutical compositions and dosage forms.

When the salt of this invention is used as a pharmaceutical preparation, it can be administered in the form of pharmaceutical compositions. These compositions can be prepared using methods well known in the art of pharmacy and can be administered in a variety of ways depending on whether topical or systemic treatment is needed and the area being treated. Administration may be topical (including transdermal, epidermal, ocular, and mucosal, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, particularly by nebulizer; intratracheal or intranasal) , oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; or intracranial, eg intrathecal or intraventricular, administration. Parenteral administration can be carried out in the form of a single bolus dose or, for example, continuously using a perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oil bases, thickeners, and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain, as an active ingredient, a salt of this invention (namely the hydrochloric acid salt of a compound of formula I) in combination with one or more pharmaceutically acceptable carriers (auxiliaries). In some embodiments of the invention, the composition is suitable for topical administration. To prepare the compositions of this invention, the active ingredient is generally mixed with an excipient, diluted with an excipient, or included in a carrier in the form of, for example, a capsule, sachet, paper bag, or other container. When the excipient serves as a diluent, it may be a solid, semi-solid or liquid substance which serves as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, dragees, powders, lozenges, sachets, starch capsules, infusions, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid), ointments containing, for example, up to 10 wt. % active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

To obtain a formulation, the salt may be milled to provide a suitable particle size prior to being combined with other ingredients. If the salt is practically insoluble, it can be ground to a particle size of less than 200 mesh. If the salt is highly soluble in water, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, eg about 40 mesh.

The salt of this invention can be milled using known milling methods such as wet milling to obtain particle sizes suitable for tablet formation and other types of formulations. Finely divided (in the form of nanoparticles) salt preparations according to this invention can be obtained using methods known in the art, see, for example, international application No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, molasses, and methylcellulose. The compositions may also additionally contain lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoate; sweeteners; and fragrances. The compositions of this invention can be designed to provide rapid, delayed or delayed release of the active ingredient after administration to a patient using methods known in the art.

Compositions may be formulated as unit dosage forms, with each dose containing from about 5 to about 1000 mg (1 g), typically from about 100 to about 500 mg, of the active ingredient. The term unit dosage forms refers to physically discrete entities suitable as single dosages for humans and other mammals, each unit dose containing a predetermined amount of the active substance, calculated to produce the desired therapeutic effect, in combination with a suitable pharmaceutical excipient.

In some embodiments of the invention, the compositions of this invention contain from about 5 to about 50 mg of the active ingredient. One skilled in the art should understand that this encompasses compositions containing about 5 to about 10, about 10 to about 15, about

- 10 041006 up to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 mg active ingredient.

In some embodiments, the compositions of this invention contain about 2.5, about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, or about 25 mg active ingredient. In some embodiments of the invention, the compositions of this invention contain about 5 mg of the active ingredient. In some embodiments of the invention, the compositions of this invention contain about 10 mg of the active ingredient.

Similar dosages can be used for the salt described herein in the methods and uses of the invention.

The salt can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. However, it will be understood that the amount of salt actually administered is usually determined by the physician in accordance with the circumstances, including the condition being treated, the route of administration chosen, the particular salt administered, the age, weight and response of the particular patient, the severity of the patient's symptoms, and etc.

To obtain solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to obtain a solid preformulation composition containing a homogeneous salt mixture of the present application. When these preformulation compositions are said to be homogeneous, it is understood that the active ingredient is generally dispersed evenly throughout the composition so that it can be readily subdivided into equally effective unit dosage forms such as tablets, dragees and capsules. This solid preformulation is then subdivided into unit dosage forms of the above types, containing, for example, from about 0.1 to about 1000 mg of the active ingredient of the present application.

Tablets or dragees according to the present application may be coated or modified in another way to obtain a dosage form that has the advantage of prolonged action. For example, a tablet or dragee may contain an internal dosage component and an external dosage component, the latter in the form of a shell for the former. The two components can be separated by an enteric layer which prevents decomposition in the stomach and allows the internal component to enter the duodenum unchanged or be released in a delayed manner. Various substances can be used for such enteric layers or coatings, such substances include a number of polymeric acids and mixtures of polymeric acids with substances such as shellac, cetyl alcohol or cellulose acetate.

Liquid forms in which the salt and compositions of this application may be incorporated for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil , coconut oil or peanut oil, as well as infusions and similar pharmaceutical carriers.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments of the invention, the compositions are administered orally or into the respiratory tract through the nose for a local or systemic effect. The compositions can be sprayed using inert gases. Nebulized solutions can be inhaled directly from the nebulizer, or the nebulizer can be attached to a face mask, oxygen tent, or intermittent positive pressure breathing apparatus. Solutions, suspensions or powder compositions can be administered orally or through the nose using devices that deliver the formulation in an appropriate manner.

Topical formulations may contain one or more conventional carriers. In some embodiments, the ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petrolatum, and the like. Carriers for cream compositions may be water based in combination with glycerol and one or more other components, for example glyceryl monostearate, PEGglyceryl monostearate and cetyl stearyl alcohol. Gels can be prepared using isopropyl alcohol and water in combination with other suitable ingredients such as, for example, glycerol, hydroxyethyl cellulose, and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 wt.% salt according to this invention. Topical formulations may be packaged in suitable tubes, for example 100 g, which may optionally contain instructions for the treatment of a selected symptom, such as psoriasis or other skin condition.

The amount of salt or composition administered to a patient will vary depending on what is being administered, the purpose of administration such as prophylaxis or therapy, the condition of the patient, the route of administration, and the like. For therapeutic purposes, the compositions may be administered to a patient already suffering from a disease in an amount sufficient to treat or at least partially alleviate the symptoms of the disease or its complications. Effective dosages will depend on the condition being treated as well as the judgment of the attending physician based on such factors as the severity of the disease, the age, weight and general condition of the patient, and the like.

The compositions administered to the patient may be in the form of the pharmaceutical compositions described above. These compositions can be sterilized using conventional sterilization methods or using sterile filtration. Aqueous solutions can be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH values of formulations with compounds should generally be between 3 and 11, more preferably between 5 and 9, and most preferably between 7 and 8. It is understood that the use of some of the aforementioned excipients, carriers, or stabilizers will result in the formation of pharmaceutically acceptable salts. .

The therapeutic dosage of the salt of the present invention may vary, for example, in accordance with the specific purpose for which the treatment is carried out, the method of administration of salt, the health and pathological condition of the patient and the opinion of the attending physician. The proportion or concentration of the salt of this invention in a pharmaceutical composition may vary depending on a number of factors, including dosage, chemical characteristics (eg hydrophobicity) and route of administration. For example, the salt of this invention may be in an aqueous physiological buffer solution containing from about 0.1 to about 10% (w/v) salt for parenteral administration. Some typical doses are in the range of about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments of the invention, the dose is in the range from about 0.01 to about 100 mg/kg of body weight per day. The dosage may depend on such factors as the type and extent of progression of the disease or disorder, the general health of the individual patient, the relative biological potency of the compound chosen, the composition of the excipients, and the route of administration. Effective doses can be extrapolated from dose-response curves obtained in vitro or from test model systems in animals.

The invention will be described in more detail with specific examples. The following examples are for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily find many non-critical parameters that can be changed or modified to give exactly the same results. The hydrochloride salt of a compound of formula I and a compound of formula I have been found to be PI3K inhibitors according to at least one assay described herein.

Examples

The invention will be described in more detail with specific examples. The following examples are for illustrative purposes and are not intended to limit the present invention in any way. The Example compounds or salts thereof containing one or more chiral centers were prepared in racemic form or as mixtures of isomers, unless otherwise indicated.

General methods.

Purification of some of the resulting compounds by preparative LC/MS was performed on Waters fractionation systems with a mass spectrometric detector. The basic hardware, protocols, and control software for operating these systems are detailed in the literature. See, for example, Two-Pump At Column Dilution Configuration for Preparative LC-MS, K. Blom, J. Combi. Chem. 4, 295 (2002); Optimizing Preparative LC-MS Configurations and Methods for Parallel Synthesis Purification, K. Blom, R. Sparks, J. Doughty, G. Everlof, T. Haque, A. Combs, J. Combi. Chem., 5, 670 (2003); and Preparative LC-MS Purification: Improved Compound Specific Method Optimization, K. Blom, B. Glass, R. Sparks, A. Combs, J. Combi. Chem., 6, 874-883 (2004). Isolated compounds were typically analyzed using analytical liquid chromatography with a mass spectrometric detector (LC/MS) to determine purity under the following conditions: instrument; Agilent 1100 Series LC/MSD, column: Waters Sunfire™ C ₁₈ 5 µm, 2.1x50 mm, buffers: mobile phase A: 0.025% TFA in water, and mobile phase B: acetonitrile; gradient from 2 to 80% B over 3 min with a flow rate of 2.0 ml/min.

Some of the resulting compounds were also separated at the preparative scale using reverse phase high performance liquid chromatography (RP-HPLC) with a mass spectrometric detector or flash chromatography (silica gel) as indicated in the examples. Typical preparative column reverse phase high performance liquid chromatography (RP-HPLC) conditions are shown below.

Purification at pH 2: Waters Sunfire™ C ₁₈ 5 µm, 19x100 mm column, eluting with mobile phase A: 0.1% TFA in water, and mobile phase B: acetonitrile; the flow rate was 30 ml/min, the separating gradient was optimized for each compound using a compound-specific technique optimization protocol as described in the literature [cf. Preparative LCMS Purification: Improved Compound Specific Method Optimization, K. Blom, B. Glass, R. Sparks, A.

- 12 041006

Combs, J. Comb. Chem., 6, 874-883 (2004)]. Typically, the flow rate used for the column

30x100 mm, was 60 ml/min.

Purification at pH 10: Waters XBridge C ₁₈ 5 µm column, 19x100 mm, eluting with mobile phase A: 0.15% NH ₄ OH in water, and mobile phase B: acetonitrile; the flow rate was 30 ml/min, the separating gradient was optimized for each compound using a compound-specific technique optimization protocol as described in the literature [cf. Preparative LCMS Purification: Improved Compound Specific Method Optimization, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)]. Typically, the flow rate used for a 30x100 mm column was 60 ml/min.

Some of the obtained compounds were also analyzed using differential scanning calorimetry (DSC). Typical equipment parameters for DSC are as follows:

TA Instrument Model Q200 DSC with autosampler: 30-350°C at 10°C/min; aluminum sample crucible with Tzero lid; 50 ml/min nitrogen gas flow, Mettler Toledo 822 Differential Scanning Calorimetry (DSC) instrument: 40-340°C with 10°C/min heating rate.

Some of the resulting compounds were also analyzed by thermogravimetric analysis (TGA). Typical equipment parameters for TGA are as follows:

thermogravimetric analyzer TA Instrument, model Pyris: temperature range from 25 to 300°C at 10°C/min; flow of nitrogen gas for purge at a rate of 60 ml/min; ceramic crucible for TGA samples, TA Instruments Q500: temperature range 20 to 300°C at 10°C/min.

Some of the resulting compounds were also analyzed using powder x-ray diffraction (XRD). Typical parameters of equipment for PDP are as follows:

Bruker D2 PHASER X-ray powder diffractometer: X-ray wavelength: 1.05406 A CuKAI; x-ray power: 30 kV, 10 mA; powder sample:

dispersed in a sample holder with zero background; general measurement conditions: initial angle 5°, limit angle 60°, step 0.015°, scanning speed 2°/min.

Rigaku Miniflex Powder Diffractometer: Cu at 1.054056 A with q-filter; general measurement conditions: initial angle 3°, limit angle 45°, step 0.02°, scanning speed 2°/min.

Example 1 Synthesis of (R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one

Step 1. 1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone (ii)

Into a 5 L flask at room temperature were placed 4-chloro-3-fluorophenol (i, 166 g, 1.11 mol) and acetyl chloride (107 ml, 1.50 mol). The reaction mixture was stirred, and it turned into a clear solution, while it was recorded that the temperature of the mixture decreased to 6°C. Then the reaction mixture was heated to 60° C. for 2 hours. Nitrobenzene (187.5 ml, 1.82 mol) was added to the reaction mixture, which was then cooled to room temperature. Aluminum chloride (160 g, 1.2 mmol) was then added to the mixture in three portions (50 g, 50 g and 60 g at 5 min intervals). After completion of the addition, the temperature of the mixture increased to 78°C. Then the reaction mixture was heated at 100-120°C

- 13 041006 for 3 hours, at which point HPLC analysis indicated that the reaction was complete. Then the reaction mixture was cooled to 0°C and was added a mixture of isomers of hexane (0.45 l), ethyl acetate (0.55 l), and then slowly at room temperature was added 1.0N. aqueous solution of hydrochloric acid (1.0 l). The addition of aqueous hydrochloric acid was exothermic and the temperature of the mixture increased from 26 to 60°C. The resulting mixture was stirred at room temperature for 20 minutes. The layers were separated and the organic layer was washed successively with 1.0N. an aqueous solution of hydrochloric acid (2x600 ml) and water (400 ml). Then the organic layer was extracted with 1.0N. an aqueous solution of sodium hydroxide (2x1.4 l). The combined stock solution was acidified to pH 2 by adding 12N. hydrochloric acid solution until no more precipitates form. The resulting solid was filtered off, washed with water and dried on a filter funnel under vacuum to give compound ii) as a yellow solid (187.4 g, 89.5%).

1H NMR (400 MHz, CDCl ₃ ) δ 12.44 (d, J=1.4 Hz, 1H), 7.78 (d, J=8.1 Hz, 1H), 6.77 (d, J= 10.2 Hz, 1H), 2.61 (s, 3H).

Stage 2. 1-(5-Chloro-4-fluoro-2-hydroxy-3-iodophenyl)ethanone (iii)

I k ,OH t iii O

1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone (ii, 100.0 g, 530.3 mmol) was dissolved in acetic acid (302 ml) and N-iodosuccinimide (179.2 g, 796.5 mmol). The reaction mixture was stirred at about 61 to about 71° C. for 2 hours, at which point HPLC analysis indicated that the reaction was complete. The reaction mixture was then cooled to room temperature, water (613 ml) was added and the resulting slurry was stirred at room temperature for 30 minutes. The product was collected by filtration and washed with water to give a brown solid. The wet product was dissolved in acetic acid (400 ml) at 60°C. Water (800 ml) was added to the solution (over 15 minutes) to precipitate the pure product. The product was collected by filtration and washed with water (100 ml). The product was dried on a filter funnel under vacuum for 18 hours to give compound iii) as a brown solid (164.8 g, 95.0% yield).

1H NMR (300 MHz, DMSO-de) δ 13.34 (s, 1H), 8.26 (d, J=8.4 Hz, 1H), 2.68 (s, 3H).

Stage 3. 1-(5-Chloro-2-ethoxy-4-fluoro-3-iodophenyl) ethanone (iv)

In a 5 L three neck round bottom flask equipped with a condenser and thermometer, 1-(5-chloro-4-fluoro-2-hydroxy-3-iodophenyl)ethanone (iii, 280 g, 840 mmol) was dissolved in N,N-dimethylformamide (600 ml ). During dissolution, the internal temperature dropped from 19.3 to 17.0°C. Iodoethane (81.2 ml, 1020 mmol) was added to the resulting mixture. Potassium carbonate (234 g, 1690 mmol) was then added to the reaction mixture over 2 minutes, no change was observed in the temperature of the mixture. The reaction mixture was heated to 60° C. for 3 hours, at which point HPLC analysis indicated that the reaction was complete. The reaction mixture was allowed to cool to room temperature and the product was collected by filtration. The solids were dissolved in a mixture of DCM (1.0 L), hexane (500 ml) and water (2.1 L). The biphasic system was stirred at 20°C for 20 min. The layers were separated and the aqueous layer was extracted with DCM (1.0 L). The combined organic layer was washed with water (2x250 ml) and saturated aqueous sodium chloride solution (60 ml). The organic phase was separated, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to dryness in vacuo to give compound iv as a yellow solid (292 g, 94% yield).

1H NMR (400 MHz, DMSO-de) δ 7.69 (d, J=8.4 Hz, 1H), 3.95 (q, J=7.0 Hz, 2H), 2.62 (s, 3H ), 1.49 (t, J=7.0 Hz, 3H). LC/MS for C ₁₀ H ₁₀ ClFIO ₂ (M+H)+: m/z=342.9.

Stage 4. 2-(5-Chloro-2-ethoxy-4-fluoro-3-iodophenyl)-2-methyl-1,3-dioxolane (v)

Solution of 1-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (iv, 250.0 g, 693.4 mmol) and 1,2-ethanediol (58.0 ml, 1040 mmol) in toluene (1.5 L) was treated with p-toluenesulfonic acid monohydrate (10.6 g, 55.5 mmol). A Dean-Stark trap was placed on the reaction flask and the mixture was heated with reverse

- 14 041006 refrigerator for 7 hours LC/MS analysis showed that the reaction mixture contained 8.3% of the starting material and 91.7% of the product. The reaction mixture was cooled to 106° C. and additional 1,2-ethanediol (11.6 mL, 208 mmol) was added via syringe. The reaction mixture was then heated at reflux for another 8 hours. LC/MS analysis indicated that the reaction mixture contained 3.6% starting material and 96.4% product. The reaction mixture was cooled to 106° C. and additional 1,2-ethanediol (7.73 mL, 139 mmol) was added via syringe. The reaction mixture was heated at reflux for an additional 15.5 hours. LC/MS analysis indicated that the reaction mixture contained 2.2% starting material and 97.8% product.

The reaction mixture was then cooled to 0°C and water (200 ml) and aqueous saturated NaHCO ₃ solution (300 ml) were added to adjust the pH of the mixture to 9. DCM (200 ml) was added and the mixture was stirred for 10 min. The layers were separated and the aqueous layer was extracted with toluene (300 ml). The combined organic layer was washed successively with a mixture of water (200 ml) and saturated aqueous NaHCO ₃ (200 ml), water (300 ml), saturated aqueous sodium chloride (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuum to dryness to give crude v as a light brown solid (268 g, 100% yield).

1H NMR (400 MHz, CDCl ₃ ) δ 7.59 (d, J=8.6 Hz, 1H), 4.26-3.96 (m, 4H), 3.92-3.72 (m, 2H ), 1.74 (s, 3H), 1.50 (t, J=7.0 Hz, 3H). LC/MS for SpNmSHYUz (M+H)+: m/z=387.0.

Step 5. 3-Chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde (vi)

To a solution of 2-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)-2-methyl-1,3-dioxolane (v, 135.0 g, 349.2 mmol) (purity determined using HPLC, 86.8% with 5.5% ketone) in anhydrous tetrahydrofuran (300 ml) at about 0 to about 3° C., with stirring, a complex of isopropyl magnesium chloride and lithium chloride in THF at a concentration of 1.3 M (322.3 ml , 419.0 mmol) for 1 hour.

The reaction mixture was stirred at about 0 to about 5° C. for 30 minutes, at which point HPLC analysis indicated that the iodine to magnesium replacement reaction was complete. N-formylmorpholine (71.1 mL, 700 mmol) was then added to the reaction mixture over 1 hour at about 0 to about 8°C. The reaction mixture was stirred at about 0 to about 8° C. for another 1 hour, at which point LC/MS and HPLC analysis indicated that the starting material had been consumed and a significant amount of deiodination by-product, 2-(5- chloro-2-ethoxy-4-fluorophenyl)-2-methyl-1,3-dioxolane. The reaction was quenched with an aqueous solution of citric acid (120.8 g, 628.6 mmol) in water (1.20 L) at 0°C. The quenched reaction mixture was then extracted with EtOAc (2x600 ml). The phases were easily separated. The combined organic layer was washed successively with water (300 ml) and saturated aqueous sodium chloride solution (500 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel flash column chromatography using 0-10% EtOAc/hexane to give the crude product vi) as a pale yellow solid which was a mixture containing the desired product, 3-chloro-6-ethoxy -2-fluoro-5-(2-methyl-1,3dioxolan-2-yl)benzaldehyde (vi, 80 g, 80%), and 36 mol. % by-product of deiodination, 2-(5-chloro2-ethoxy-4-fluorophenyl)-2-methyl-1,3-dioxolane, as determined by NMR analysis. The crude product vi) was further purified by formation of the appropriate adduct with sodium hydrosulfite.

Sodium hydrogen sulfite (36.91 g, 354.7 mmol) was dissolved in water (74.3 ml, 4121 mmol). To a solution of crude 3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde (vi, 80.00 g, 177.3 mmol) in ethyl acetate (256 0 ml) with stirring, a freshly prepared solution of sodium hydrosulfite was added in one portion. The solution was stirred for about 10 minutes and the formation of a precipitate was observed. The slurry was then stirred for another 1 h. The aldehyde-hydrosulfite adduct was collected by filtration, washed with EtOAc and dried under vacuum and nitrogen for 20 h to give a white solid (58.2 g, 83.6% yield) . To the aldehyde-hydrosulfite adduct (58.2 g, 148 mmol) which was stirred in 1.0 M aqueous sodium hydroxide solution (296 ml, 296 mmol) was added methyl tert-butyl ether (600 ml) (MTBE). The reaction mixture was stirred at room temperature for 6 minutes, which gave a clear biphasic mixture, and stirring was continued for another 5 minutes. The organic phase was collected and the aqueous layer was extracted with MTBE (2x300 ml). The combined organic layers were washed with saturated aqueous sodium chloride (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to dryness in vacuo to give pure compound vi) as a white crystalline solid (31.4 g, 73.4 yield %).

1H NMR (400 MHz, CDCl ₃ ) δ 10.27 (s, 1H), 7.78 (d, J=8.5 Hz, 1H), 4.10-3.96 (m, 4H), 3, 87-3.76 (m, 2H), 1.72 (s, 3H), 1.44 (t, J=7.0 Hz, 3H). LC/MS for C13H15QFO4 (M+H) ⁺ : m/z=289.0.

Step 6. (E)-2-(5-Chloro-2-ethoxy-4-fluoro-3-(2-nitrovinyl)phenyl)-2-methyl-1,3-dioxolane (vii)

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3-Chloro-6ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl ) benzaldehyde (vi, 566.2 g, 1961 mmol), nitromethane (1060 ml, 19600 mmol) and glacial acetic acid (1120 ml). Then benzylamine (53.6 ml, 490 mmol) was added to the reaction mixture and the resulting mixture was heated to 60°C, the reaction was followed by LC/MS for 5.5 hours. Initial baseline analysis was performed at t=0. The reaction was monitored after 2 hours and 5 hours. After 2 hours, about 20% of unreacted starting aldehyde remained. After 5 hours, the reaction profile was as follows: starting compound vi (<2%), intermediate imine (<4%), resulting compound vii (>93%) and Michael benzylamine adduct (not detected). After 5.5 h, the reaction was considered complete. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (3.0 L). For processing, the mixture was divided in half due to the large involved volume.

Each half was treated according to the following procedure. First, the reaction mixture was washed with 1.5 M NaCl in water (2x1500 ml; after each washing, the output volume of the aqueous solution increased compared to the input, indicating the removal of acetic acid and/or nitromethane). The mixture was then cooled to about 15°C and washed with 4M aqueous NaOH solution (4x300 ml) until the aqueous extract reached a pH of 8-9. The aqueous layer remained acidic on initial washes, but as the aqueous layer became slightly basic on subsequent washes, the mixture warmed up and the extract turned dark. The layers were separated. The organic layer was then washed with 1.5 M sodium chloride in water (1000 ml) and water (500 ml) after adjusting the pH. During these final washes, emulsion formation and slow separation were observed. The organic phase was dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The resulting amber viscous liquid was placed under high vacuum overnight. The viscous liquid solidified, 740 g of crude product was obtained.

Heptane (1.3 L) was added to the crude product to form a slurry which was heated in a 60° C. water bath until all solids had dissolved. The resulting solution was filtered on a fine-pored filter into a clean four-necked round-bottom flask with a capacity of 3 L, equipped with an overhead stirrer and a nitrogen inlet. The filter was washed with heptane (40 ml). The filtered solution was cooled to room temperature and stirred for 5 hours. Precipitation was observed and the slurry was cooled to 0°C in an ice bath for 1 hour. The product was collected by filtration and the resulting wet filter cake was washed with 500 ml of ice-cold heptane. The product was partially dried on the filter under vacuum and further dried under high vacuum overnight. The filtrate and washing solution were concentrated under reduced pressure, and the resulting residue was subjected to column purification.

The solids from the heptane crystallization were dissolved in a small volume of DCM and 20% EtOAc/hexane and loaded onto a column containing about 1 kg of silica gel. The column was eluted with 20% EtOAc/hexane. The desired fractions were combined and concentrated under reduced pressure to give a yellow solid. The solid was dried under high vacuum overnight to give 497 g of product vii) as a pale yellow crystalline solid.

The concentrated filtrate and the heptane crystallization wash were loaded onto the same column using 20% EtOAc/hexane. The column was eluted using the same solvent system to remove initial impurities and residual acetic acid. The desired fractions were combined and concentrated under reduced pressure to give a reddish amber oil. The oil was placed under high vacuum to give about 220 g of crude product. This crude product was dissolved in heptane (500 ml) and seeded with a small amount of the first portion of the solid product. The suspension was stirred at room temperature for 16 hours and then cooled in an ice bath for 3 hours. A second crop of product was collected by filtration. The product was dried under high vacuum to give 110 g of product as a yellow solid. The total amount of product vii) was 607 g (yield 93.3%).

¹ H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 2H), 7.68 (d, J=8.9 Hz, 1H), 4.07-3.95 (m, 4H), 3.82-3.73 (m, 2H), 1.65 (s, 3H), 1.39 (t, J=7.0 Hz, 3H). LC/MS for C14H16CIFNO5 (M+H)+: m/z=332.0.

Step 7. (R)-Dimethyl-2-(1-(3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl)-2nitroethyl)malonate (viii)

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Into a 2 L round-bottom flask with magnetic stirrer and nitrogen inlet, containing (E)-2-(5-chloro-2-ethoxy-4-fluoro-3-(2-nitrovinyl)phenyl)-2-methyl-1 ,3-dioxolane (vii, 352.8 g, 1064 mmol), anhydrous tetrahydrofuran (1.06 L) and dimethylmalonate (146 ml, 1280 mmol) were added. (1S,2S)-N,N'-dibenzylcyclohexane-1,2-diaminodibromonickel (Evans catalyst, 21.4 g, 26.6 mmol) was added to the reaction mixture. The reaction mixture turned brown and a homogeneous solution was observed. The reaction mixture was stirred at room temperature for 18.5 hours. After 18.5 hours, the reaction mixture was analyzed by HPLC. Unreacted starting material, compound vii) was present at 2%. The reaction mixture was concentrated under reduced pressure to remove THF and the resulting residue was purified by flash chromatography (DCM/hexane isomer mixture was used for loading, 1422 g of silica gel was used for this column, and the column was eluted with 10%-20% EtOAc/hexane; for fractions from columns were monitored by TLC using 30% EtOAc/hexane as eluent and visualized by UV). The desired fractions were combined and concentrated under reduced pressure. The residue was dried under high vacuum. Over time, the viscous liquid turned into light yellow solids (503.3 g), a sample was taken for chiral HPLC analysis. The chiral purity was 95.7% of the desired (R)-enantiomer and 4.3% of the unwanted (S)-enαantiomerα. Ethanol (1.0 L) was added to the solids and the mixture was heated in a 60° C. water bath until all solids had dissolved. The solution was filtered on a fine filter using Whatman No. 1 filter paper into a clean 3 L four neck round bottom flask. The filtered solution was cooled to room temperature with stirring. After 30 minutes of stirring, crystal formation was observed and the slurry was stirred at room temperature for 16 hours. The slurry was cooled in an ice bath for 1 hour. The product was collected by filtration and the resulting filter cake was washed with ice-cold ethanol (500 ml) and partially dried on the filter . The solids were dried under high vacuum to give the desired product viii) (377.5 g) as white crystalline solids. The chiral purity, determined by chiral HPLC, was 100% of the desired (R)-enantiomer and 0% of the unwanted (S)-enantiomer.

The filtrate and washings were combined and concentrated under reduced pressure to give an oil (118.9 g). The oil was dissolved in ethanol (475.0 ml) (4 ml/g) and seeded with the first portion of the crystals. The resulting suspension was stirred at room temperature for 16 hours and then cooled in an ice bath for 5.5 hours. The second portion of the product was isolated by filtration and partially dried on the filter. It was dried under high vacuum to give a second crop of product (31.0 g). The chiral purity, determined by chiral HPLC, was 98.3% of the desired (R)-α enantiomer and 1.7% of the unwanted (S)-enantiomer α. The combined yield of the first and second portions of the product was 408.5 g (82.8% yield).

¹ H NMR (500 MHz, DMSO-d6) δ 7.51 (d, J=9.0 Hz, 1H), 5.20-4.81 (m, 2H), 4.62 (m, 1H), 4.14-4.03(m, 2H), 4.03-3.97(m, 2H), 3.95-3.88(m, 1H), 3.84-3.72(m, 2H ), 3.70 (s, 3H), 3.38 (s, 3H), 1.61 (s, 3H), 1.39 (t, J=6.9 Hz, 3H). LC/MS for C19H24ClFO9 (M+H)+: m/z=463.9.

Step 8 (R)-Dimethyl-2-(1-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-nitroethyl)malonate (ix)

To a solution of (R)-dimethyl-2-(1-(3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl)-2nitroethyl)malonate ( viii, 244.0 g, 526.0 mmol) in acetone (1.2 L) in a 5 L three neck round bottom flask equipped with a mechanical stirrer, iodine (13.4 g, 52.6 mmol) was added with stirring at room temperature . The resulting brown solution was heated at 50° C. in a water bath for 30 minutes, at which point LC/MS analysis indicated completion of the reaction. The reaction mixture was cooled to room temperature and then quenched with a solution of sodium thiosulfate (17.0 g, 108 mmol) in water (160 mL) to give a pale yellow clear solution. At this point, additional water (1.2 L) was added to the quenched solution and the resulting white slurry was stirred at room temperature for 1 hour. The solid was then collected by filtration and redissolved in acetone (1.4 L) at 40°C. C. The solution was cooled to room temperature and then additional water (1.4 L) was added. The resulting white slurry was stirred at room temperature for 1 hour. The solid was collected by filtration and washed with water (3x100 ml). The solid was dried on a filter funnel under vacuum under a stream of nitrogen for 46 h to give compound ix) as a white solid (212 g, 96% yield).

1H NMR (300 MHz, CDC13) δ 7.54 (d, J=8.6 Hz, 1H), 5.09-4.67 (m, 3H), 4.10-3.83 (m, 3H) , 3.90 (s, 3H), 3.57 (s, 3H), 2.57 (s, 3H), 1.46 (t, J=7.0 Hz, 3H). LC/MS for C^HoClFNOg (M+H)+: m/z=420.1.

Step 9 Dimethyl 2-((R)-1-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)-2-nitroethyl)malonate (x)

x OH

To a solution of (3aS)-1-methyl-3,3-diphenyltetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaborol, ((S)MeCBS, 16.39 g, 59.12 mmol , 0.1 eq.) in anhydrous THF (100 ml) in a 5 L round bottom flask with stirring at room temperature was added a 1.0 M solution of the borane-THF complex in THF (591 ml, 591 mmol, 1 eq.), and then boron trifluoride etherate (3.75 ml, 29.6 mmol, 0.05 eq.). The resulting solution was stirred at room temperature for 30 minutes. A solution of dimethyl-[(1R)-1-(3-acetyl-5-chloro-2-ethoxy-6fluorophenyl)-2-nitroethyl]malonate (ix, 253.0 g , 591.2 mmol) in anhydrous THF (1.7 L). The flask that contained ketone ix) was washed with anhydrous THF (135 ml) and the solution was added dropwise via an addition funnel to the reaction mixture. The resulting solution was stirred at room temperature for another 10 minutes, at which point LC/MS analysis showed complete conversion of the ketone to alcohol. The reaction mixture was quenched by adding dropwise methanol (71.8 ml, 1770 mmol) at 0°C. The quenched reaction mixture was stirred at room temperature for 15 minutes before being concentrated in vacuo to give the crude product. The crude products from this batch and a similar batch (using 200 g starting material) were combined and purified by silica gel column chromatography using 0-5% MeOH/DCM as eluent to give compound x) as a white solid ( 437 g, 97.9% yield.

1H NMR (300 MHz, CDC13) δ 7.50 (d, J=8.8 Hz, 1H), 5.13 (q, J=6.3 Hz, 1H), 5.01-4.65 (m , 3H), 4.14-3.89 (m, 3H), 3.79 (s, 3H), 3.57 (s, 3H), 1.57-1.42 (m, 6H). LC/MS for C ₁₇ H ₂₁ C1FNNaO ₈ (M+Na)+: m/z=444.0.

Step 10 (4R)-Methyl-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)-2-oxopyrrolidine-3-carboxylate (xi)

A 3-neck Morton round bottom flask containing dimethyl-((1R)-1-{3-chloro-6-ethoxy-2-fluoro-5[(1R)-1-hydroxyethyl]phenyl}-2-nitroethyl)malonate (x , 100.0 g, 237.1 mmol) in tetrahydrofuran (800.0 ml) and Raney nickel (120 g after removal of water with a pipette), equipped with a condenser, mechanical stirrer (glass stirring rod and Teflon support) and two balls filled with gaseous hydrogen (purging after evacuation). The flask was placed in an oil bath at 65°C. The mixture was vigorously stirred for 16 hours, and the balls were periodically removed and refilled with hydrogen. A sample was taken and analyzed by HPLC. The product, compound xi), was present at 83%. The reaction mixture contained 7.8% uncirculated amine and 5.5% hydroxylamine as by-products. The catalyst was filtered off (it is necessary to ensure that the Raney nickel does not dry out and is not exposed to air). The filtrate was evaporated to dryness to give 91 g of crude product as a white foam. The crude product (91 g, 82.6% purity by area) was combined with the same portion of the crude product (93 g, 72.8% purity) for purification. The combined crude product (184 g) was purified by silica gel column chromatography (EtOAc/hexane as eluent) to give compound xi) (101.1 g, HPLC purity 93%, crude yield 59.3 %). The crude material was used in the next step without further purification. LC/MS for C ₁₆ H ₂₀ C1FNO ₅ (M+H)+: m/z=360.0.

Step 11 (4R)-4-(3-Chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)-2-oxopyrrolidine-3carboxylic acid (xii)

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A solution of (4R)-methyl-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1hydroxyethyl)phenyl )-2-oxopyrrolidine-3-carboxylate (xi, 268 g, 581 mmol) in tetrahydrofuran (2150 ml, 26500 mmol). A 1.0 M solution of sodium hydroxide in water (1420 ml, 1420 mmol) was added to the solution. The resulting cloudy solution became clear within 1 min. The reaction mixture was stirred overnight at room temperature. The reaction mixture was analyzed by LC/MS after 15 hours and the reaction was considered complete as no starting material was observed. The reaction mixture was cooled in an ice bath to an internal temperature of 9.5° C., and the mixture was acidified to pH 1-2 by adding 6.0M aqueous hydrochloric acid (237.0 mL, 1422 mmol) with an addition funnel over 30 min. The reaction mixture was divided in half and each half was extracted with ethyl acetate (2x1 l). The combined aqueous layers were further extracted with ethyl acetate (500 ml). The two organic layers were washed with saturated aqueous sodium chloride solution (20 wt.% NaCl/water, 2x1000 ml), dried over anhydrous MgSO ₄ , filtered and concentrated to give the crude intermediate acid xii) as a yellowish foam, which was used directly in the next step reactions.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.68 (br s, 1H), 8.26 (s, 1H), 7.52 (d, J=8.0 Hz, 1H), 5 .27 (br s, 1H), 4.90 (k, J=6.3 Hz, 1H), 4.28 (k, J=8.8 Hz, 1H), 3.92-3.81 ( m, 1H), 3.76-3.65 (m, 1H), 3.57 (t, J=9.6 Hz, 1H), 3.46 (d, J=9.4 Hz, 1H), 3.23 (q, J=9.5 Hz, 1H), 1.33 (t, J=6.9 Hz, 3H), 1.28 (d, J=6.4 Hz, 3H). LC/MS for C ₁₅ H ₁₇ ClFNNaO ₅ (M+Na)+: m/z=368.0.

Step 12 (R)-4-(3-Chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one (xiii)

XIII he

The crude compound xii) was dissolved in 1,4-dioxane (976 ml) and toluene (976 ml) and the resulting yellow solution was heated at 100°C. As the reaction progressed, the color of the solution turned brown. Samples were taken at 1h, 2h and 2.5h. After 2.5h, HPLC analysis showed that the acid, compound 12, was present at 0.38% and the desired product, compound xiii), at 78.8%. The reaction mixture was cooled to room temperature and filtered on a fine filter into a clean 3 L round bottom flask. The solution was then concentrated under reduced pressure and the resulting residue was placed under high vacuum to give a brown foam (254 g).

Acetonitrile (350 ml) was added to the brown viscous liquid and heated in a water bath at 65° C. until dissolved. The solution was cooled to room temperature and stirred for 16 hours. Solids were separated from the solution. The resulting slurry was cooled in an ice bath for 1 hour. The product was collected by filtration and the resulting filter cake was washed with 400 ml of ice-cold acetonitrile. Solids appear to be gyroscopic. The solid was dissolved in DCM (2.0 L) and concentrated to a viscous liquid which was placed under high vacuum to give compound xiii) as a white foam (106.4 g).

The filtrate was concentrated to a dark viscous liquid (ca. 120 g) which was purified by flash chromatography (4 x 330 g silica gel columns, loaded with DCM, eluted with 50 to 100% EtOAc/hexane, monitored by TLC using 100% EtOAc as eluent). Fractions from chromatography were concentrated under reduced pressure and placed under high vacuum to give a light brown foam (54.4 g). MTBE (400 ml) and MeOH (10 ml) were added to the foam and heated in a water bath at 56°C for 15 min, some solids remained. The suspension was cooled to room temperature with stirring. The slurry was filtered to remove insolubles. The filtrate was concentrated to a viscous liquid and placed under high vacuum to give a foam. Acetonitrile (72 ml, 1.5 ml/g) was added to the foam and heated in a water bath at 60° C. until the solution became homogeneous. The solution was cooled to room temperature with stirring, solids precipitated from the solution and it became very thick. Additional acetonitrile (24 ml) was added so that the dilution was 2 ml/g. The suspension was stirred at room temperature for 16 hours and then cooled in an ice bath for 1 hour. The product was collected by filtration and washed with acetonitrile. Compound xiii) (25 g) was obtained as a second portion. A total of 131.4 g of compound xiii) was obtained with a yield of 74.9% from compound xi).

¹ H NMR (400 MHz, DMSOCH) δ 7.83 (s, 1H), 7.47 (d, J=8.7 Hz, 1H), 5.24 (d, J=4.5 Hz, 1H) , 4.96-4.85 (m, 1H), 4.08-3.92 (m, 1H), 3.80 (kt, J=6.9, 3.5 Hz, 2H), 3.61 -3.51(m, 1H), 3.25(t, J=9.1Hz, 1H), 2.61-2.50(m, 1H), 2.35-2.26(m, 1H ), 1.33 (t, J=7.0 Hz, 3H), 1.27 (d, J=6.4 Hz, 3H). LC/MS for C14H18ClFNO3 (M+H)+: m/z=302.0.

Example 2 Alternative pyrrolidin-2-one synthesis of (R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)

-ohm

Etl K ₂ SS,

LE1

CFt

LDA DMF with io

DMF o

v-a oh

MeNO.

r| gyu.

i.leo about me

NJ EC Mii oo 1.0

J acetone

NO

OO[.c

I

AO-Ce jEt

CBS

F.

NO G . X ,OEt

VII

LI

VIII

fl

Ci'

IX

UN

IIN-----1

I

UN Raney nickel >H' ^{J Ph} rWF

L r Et ------heating toluene

JEt ci xii

XIII

Step 1. 1-(5-Chloro-2-ethoxy-4-fluorophenyl)ethanone (iv-a)

1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone (compound ii from example 1, step 1, 1350 g, 7160 mmol), N,N-dimethylformamide (3.32 L), iodoethane (1340 g, 8590 mmol) and potassium carbonate (1980 g, 14300 mmol) were mixed together and stirred at room temperature for 45 minutes. The temperature of the mixture increased from 22 to 55°C. The reaction mixture was heated to 60°C for 1 h (the temperature of the mixture reached 67°C after 30 min and then dropped to 60°C). HPLC analysis showed that all of the starting material had been consumed. Water (10 L) was added in one portion (agitation will stop if water is added in portions). The resulting slurry was stirred at room temperature for 30 minutes. The product was collected by filtration and washed with water (3 L). The product was dried on the filter under vacuum for 5 days to give compound iv-a as a tan solid (1418 g).

¹ H NMR (400 MHz, DMSO-J6) δ 7.69 (d, J=8.9 Hz, 1H), 7.30 (d, J=11.6 Hz, 1H), 4.15 (q, J=7.0 Hz, 2H), 2.51 (s, 3H), 1.37 (t, J=7.0 Hz, 3H).

Step 2. 2-(5-Chloro-2-ethoxy-4-fluorophenyl)-2-methyl-1,3-dioxolane (v-a)

A solution of 1-(5-chloro-2-ethoxy-4-fluorophenyl)ethanone (iv-a, 1481.0 g, 6836.3 mmol) was dissolved in toluene (6 L). 1,2-ethanediol (953 ml, 17100 mmol) and p-toluenesulfonic acid monohydrate (104 g, 547 mmol) were added to the solution. The reaction mixture was refluxed at 104-110° C. for 17.4 hours using a Dean-Stark trap to remove water. HPLC analysis showed that 37% of unreacted starting material remained. Approximately 600 ml of distillate was distilled off and the reaction mixture was heated at reflux for another 5 hours (22 hours total). HPLC analysis showed that the reaction no longer proceeded.

It was assumed that the residual amount of K ₂ CO ₃ in the original connection iv-a can stop the reaction. Therefore, the reaction mixture was cooled to room temperature and washed with 1N. an aqueous solution of hydrochloric acid (3x6.66 l). After washing with an aqueous acid solution, the organic layer was transferred back to the reaction vessel. 1,2-ethanediol (381 ml, 6840 mmol) and p-toluenesulfonic acid monohydrate (104 g, 547 mmol) were added and the reaction mixture was heated under reflux.

- 20 041006 Nick for 16 hours HPLC analysis showed that about 20% of unreacted starting material remained. Distilled off about 100 ml of distillate. 1,2-ethanediol (380 mL, 6800 mmol) was added and refluxed for 6 hours (22 hours total). HPLC indicated that 7% of unreacted starting material remained. Distilled off about 125 ml of distillate. The reaction mixture was heated at reflux for 6 hours (28 hours total). HPLC indicated that 5.4% of unreacted starting material remained. Distilled off about 125 ml of distillate. The reaction mixture was heated at reflux for another 7 hours. HPLC analysis indicated that 3.5% of unreacted starting material remained. Distilled off about 80 ml of distillate. At this point in time, the reaction was considered complete.

The reaction mixture was washed with 1N. an aqueous solution of sodium chloride (2x5.5 l). The first basic wash solution was extracted with toluene (2.1 L). The combined toluene solution was washed with water (7 L) and concentrated to give 2153 g of a dark oil. HPLC analysis indicated that the purity of the product was 93.8% with 1.90% starting material and 0.79% deiodinated product. 1H NMR analysis showed that about 0.5 eq. toluene (about 256 g). The corrected yield of compound v-a was 88.0%.

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.51 (d, J=8.8 Hz, 1H), 6.70 (d, J=11.0 Hz, 1H), 4.17-3.92 (m, 4H), 3.913.80 (m, 2H), 1.75 (s, 3H), 1.46 (t, J=7.0 Hz, 3H).

Stage 3. 3-Chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde (vi)

N,N-diisopropylamine (87.8 ml, 626 mmol) and anhydrous tetrahydrofuran (1090 ml, 13500 mmol). This solution was cooled to -72° C. and 2.5M n-butyllithium in a mixture of hexane isomers (261 ml, 652 mmol) was added thereto. The n-butyllithium solution was added over 18 minutes. The maximum internal temperature during the addition was -65°. The bath containing dry ice and acetone was changed to a bath containing ice and water, and the reaction mixture was heated to about -5 to about 0° C. and held for 15 minutes. Then the reaction mixture was cooled to -74.5°C.

Anhydrous tetrahydrofuran ( 456 ml) to dissolve solids. The resulting solution was cooled in an ice bath to about 0°C. The solution containing compound va was transferred to a solution of LDA (lithium diisopropylamide) for 40 min using a needle while maintaining the reaction temperature between -70 and -72.5°C. The reaction mixture became a yellow slurry and was stirred for 37 min at -74°C. N,N-dimethylformamide (60.6 ml, 783 mmol) was added in one portion via syringe, this addition resulted in heat evolution and temperature increase from -74.5 to -66.5°C. The reaction was monitored by HPLC 90 minutes after the addition. The starting material was present in an amount of 2.9%. The cooling bath was removed and the reaction mixture was warmed to ambient temperature. After 3 hours, a sample of the reaction mixture was taken and analyzed, unreacted starting material was present in an amount of 1.5%. The reaction was considered complete and quenched by adding the reaction solution to ice water (1.4 L), diluted with ethyl acetate (1.5 L). The aqueous layer was extracted with ethyl acetate (1.5 L) and the organic layers were combined and washed with saturated sodium chloride solution (20 wt.% aq. NaCl, 2x600 ml) and dried over anhydrous MgSO ₄ . MgSO ₄ was removed by filtration and the filtrate was concentrated to an oil in which some solids were present. The residue was dissolved in methylene chloride and added to a layer of silica gel (586 g). The silica gel layer was eluted with 2% EtOAc/DCM (monitored by TLC using 100% DCM as eluent). The desired fractions were combined and concentrated under reduced pressure to give a light amber oil. The oil was placed under high vacuum to give compound vi as a yellow solid (146.5 g, 95.1% yield).

1H NMR (400 MHz, CDCl ₃ ) δ 10.27 (s, 1H), 7.78 (d, J=8.5 Hz, 1H), 4.10-3.96 (m, 4H), 3, 87-3.76 (m, 2H), 1.72 (s, 3H), 1.44 (t, J=7.0 Hz, 3H). LC/MS for C ₁₃ H ₁₅ ClFO ₄ (M+H)+: m/z=289.1.

Stages 4-10. (R)-4-(3-Chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one (xiii).

The target compound was obtained using procedures similar to those described in example 1, steps 6-12.

Example 3 (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro -2ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride

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Step 1: (R)-1-(5-Chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl methanesulfonate ^(x iv)

(R)-4-(3-Chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one (xiii, 172.0 g, 570.0 mmol) (consists of 147 g, 99.83%: chiral purity 0.09%, chemical purity 99.33%; and 25 g, 87.46%: chiral purity 12.54%, chemical purity 86.74%) were dissolved in methylene chloride (860 ml). N,N-diisopropylethylamine (149 mL, 855 mmol) was added to the solution at about -7 to about 2°C. Methansulfonyl chloride (57.4 ml, 741 mmol) was added dropwise to the reaction mixture over 25 minutes. The suspension turned into a clear solution. After 30 min of the reaction, HPLC showed the completion of the reaction. The reaction mixture containing compound xiv was used directly in the next step.

Step 2. (R)-4-(3-Chloro-6-ethoxy-2-fluoro-5-((S)-1-hydrazinylethyl)phenyl)pyrrolidin-2-one (xv)

Hydrazine (178.9 mL, 5.7 mol) was added in one portion at 0°C followed by N-methylpyrrolidinone (860 mL) to the reaction mixture containing compound xiv from step 1. The reaction mixture became cloudy and some precipitate formed. . The mixture was heated to 40-57° C. under nitrogen for 90 minutes. HPLC showed that all of the mesylate had been consumed.

The reaction mixture was cooled to room temperature and a saturated solution of sodium hydrogen carbonate (28.3 g) in water (300 ml) was added. The mixture was stirred for 20 minutes after which dichloromethane (300 ml) was added. The organic layer was separated and stirred with a solution of sodium hydrogen carbonate (14.2 g) in water (150 ml). The aqueous layer was extracted with dichloromethane (200 ml x 2). The combined organic layers were washed with saturated aqueous sodium chloride (80 ml), dried over anhydrous Na ₂ SO ₄ (311 g), concentrated and azeotropically distilled with toluene (250 ml) to give a colorless solution containing compound xv in N -methylpyrrolidinone, which was used directly for the next reaction. The sample was purified for NMR analysis.

¹ H NMR (400 MHz, DMSOCH), δ 7.88 (s, 1H), 7.66 (d, J=8.5 Hz, 1H), 4.42 (q, J=6.7 Hz, 1H ), 4.06-3.88 (m, 2H), 3.79-3.66 (m, 1H), 3.65-3.51 (m, 1H), 3.24 (t, J=8 .8 Hz, 1H), 2.60-2.46 (m, 1H), 2.36-2.25 (m, 1H), 1.37 (t, J=6.9 Hz, 3H), 1 .26 (d, J=6.8 Hz, 3H). LC/MS for C ₁₄ H ₁₉ ClFN ₃ O ₂ (M+H)+: m/z=316.1.

Stage 3. 5-Amino-1-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)-3methyl -1H-pyrazole-4-carbonitrile (xvi)

- 22 041006

(1-ethoxyethylidene)malononitrile (101 g, 741 mmol) was added portionwise to a solution of compound xv from step 2 in N-methylpyrrolidinone with stirring, and the mixture was stirred at room temperature under nitrogen atmosphere. After 15 minutes, HPLC analysis indicated 11% starting hydrazine compound xv relative to product compound xvi. N,N-diisopropylethylamine (15 ml, 86 mmol) was added and the reaction mixture was stirred at room temperature for 15 hours. HPLC analysis indicated that 5.6% of unreacted starting material remained. N,N-diisopropylethylamine (5 ml, 30 mmol) was added and the reaction mixture was stirred at room temperature for 5 hours. HPLC indicated that 5.6% of starting material remained. The reaction mixture was stirred for 2.5 days and combined with two similar portions, processed together.

The reaction mixtures of three portions of compound xvi were combined. A 0.5M aqueous sodium hydroxide solution (3.8 L) was added at 10-20°C and stirred for 5 minutes. HPLC indicated that all of the starting material, (1-ethoxyethylidene)malononitrile, had been consumed. Ethyl acetate (4.0 L) was added and the mixture was stirred for 15 minutes. The layers were separated. The organic layer was washed with 0.5 M sodium hydroxide in water (2.38 L). The layers were separated. The combined organic layer was extracted with ethyl acetate (2x2 L). The combined organic layers were washed with 1.0M aqueous hydrochloric acid (3.56 L) and the pH of the resulting aqueous layer was 2-3. The organic layer was washed with saturated aqueous sodium chloride (5 L), dried over anhydrous Na ₂ SO ₄ , concentrated and dried under high vacuum for 40 h, which gave compound xvi as a light brown foamy solid (702.7 g) .

1H NMR (500 MHz, DMSO-H6) δ 7.78 (s, 1H), 7.44 (d, J=8.4 Hz, 1H), 6.53 (s, 2H), 5.64 (q , J=6.7Hz, 1H), 3.96(m, 1H), 3.74(m, 1H), 3.34(m, 1H), 3.58(m, 2H), 2.59 -2.50 (m, 1H), 2.29 (m, 1H), 2.04 (s, 3H), 1.57 (d, J=6.8 Hz, 3H), 1.37 (t, J=6.9 Hz, 3H). LC/MS for C19H22ClFN5O2 (M+H)+: m/z=406.1.

The total yield of compound xvi over the three steps (mesylation, hydrazinolysis, and pyrazole formation) was calculated to be 72.8% of the total amount of compound xiii administered. The purity determined by HPLC was about 80%. HPLC analysis indicated product in the main aqueous layer, which was successively extracted with EtOAc (2 L), washed with 1.0 M aqueous hydrochloric acid and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, concentrated and dried using a high vacuum pump over 40 hours to give compound xvi as a brown oil (134 g, 13.9%).

Step 4. (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5- chloro-2ethoxy-6-fluorophenyl)pyrrolidin-2-one (xvii)

To a reaction vessel with formamidine acetate (1802 g, 17.31 mol) and 1,2-ethanediol (3.51 L) was added 5-amino-1-((S)-1-(5-chloro-2-ethoxy- 4-fluoro-3-((R)-5-oxon-pyrrolidin-3-yl)-phenyl)-ethyl)-3-methyl1H-pyrazole-4-carbonitrile (xvi, 702.7 g, 1731 mmol). The reaction mixture was heated at 102-103°C with stirring for 18 hours. The reaction mixture was cooled to room temperature and ethyl acetate (7 L) and water (6 L) were added, the biphasic mixture was stirred for 15 minutes. The organic layer was separated and the aqueous layer was diluted with more water (4.5 L) and ethyl acetate (3 L), stirred for 10 minutes. The organic layer was separated. The aqueous layer was further extracted with ethyl acetate (2 L). The organic layers were combined and mixed with water (4.5 L). The aqueous layer was separated and the organic layer was filtered through a pad of celite (about 1 kg). The organic layer was extracted with 1.0M

- 23 041006 aqueous hydrochloric acid solution (7 l) by stirring the mixture for 10 minutes. The aqueous layer was separated. The clear brown organic layer was stirred with additional 1.0M aqueous hydrochloric acid (3 L) for 10 minutes. The aqueous layer was separated. The aqueous acidic layers were combined and washed with toluene (500 ml). The aqueous acidic solution was cooled in an ice-water bath and methylene chloride (4 L) was added. A solution of sodium hydroxide (530 g) in water (530 ml) (50% NaOH solution) was added slowly at 5-15° C. until the pH of the solution was 11-12. Precipitation was observed. Additional methylene chloride (3.5 L) and methanol (300 ml) were added and the mixture was stirred for 10-15 minutes. The solid was collected by filtration and dried on the filter under vacuum for 16 hours to give compound xvii (289.7 g) as a brown solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.11 (s, 1H), 7.82 (s, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.30 ( br s, 2H), 6.23 (k, J=7.0 Hz, 1H), 3.97 (n, J=9.2 Hz, 1H), 3.90-3.73 (m, 2H ), 3.57 (t, J=9.9 Hz, 1H), 3.25 (dd, J=9.2, 8.7 Hz, 1H), 2.48 (s, 3H), 2.60 -2.50 (m, 1H), 2.36-2.20 (m, 1H), 1.69 (d, J=7.1 Hz, 3H), 1.39 (t, J=6.9 Hz, 3H). LC/MS for C2oH2 ₃ C1FN602 (M+H)+: m/z=433.3.

The filtrate was transferred to a separating funnel and the organic layer was separated. The aqueous layer was stirred with methylene chloride (5 L) and methanol (200 ml). The combined organic layer was dried over anhydrous sodium sulfate, concentrated, dried using a high vacuum pump for 16 hours, which gave an additional amount of 259.3 g as a brown solid. The total yield xvii was 548.3 g (73.2% yield).

Step 5. (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5- chloro-2ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride (xviii)

1.0M aqueous hydrochloric acid (HCl, 5.0 L, 5.0 mol) was added to (R)-4-(3-((S)-1-(4-amino-3methyl-1H-pyrazolo[3 ,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (xvii, 609.8 g, 1.409 mol) at room temperature. Then the resulting thick suspension was heated to 50°C to obtain a clear solution. An additional 1.82 L of 1.0M aqueous hydrochloric acid (HCl, 1.82 L, 1.82 mol; total 6.82 L, 6.82 mol, 4.84 eq.) was added to the clear solution at 50°C. and then the solution was filtered through a fine filter at approximately 50°C. Filtered on a fine filter, the reaction mixture was gradually cooled to room temperature over 2 h before it was further cooled to 0-5°C. The reaction mixture was stirred at 05°C for at least 20 min to initiate the formation of a precipitate. The resulting solids were collected by filtration, washed with a portion of the cold mother liquor, then with 1.0 M aqueous hydrochloric acid (HCl, 200 ml) and dried on a filter funnel at room temperature under vacuum to constant weight (for about 39 h) to obtain hydrochloric acid salt of the compound of formula I: (R)-4-(3-((S)-1-(4-amino-3-methyl-1 H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl) -5-Chloro-2ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride (xviii, 348.7 g, theoretically 661.2 g, 52.7%) as a white crystalline powder.

1H NMR (400 MHz, DMSO-d6) δ 9.39 (br s, 1H), 9.05 (br s, 1H), 8.50 (s, 1H), 7.84 (s, 1H) , 7.59 (d, J=8.4 Hz, 1H), 6.28 (q, J=6.9 Hz, 1H), 3.95 (m, 1H), 3.79 (m, 2H) , 3.55 (m, 1H), 3.22 (m, 1H), 2.59 (s, 3H), 2.55 (ddd, J=16.8, 10.3, 2.3 Hz, 1H ), 2.28 (ddd, J=16.8, 8.6, 1.5 Hz, 1H), 1.73 (d, J=7.0 Hz, 3H), 1.38 (t, J= 6.9 Hz, 3H) ppm ¹³ C NMR (100 MHz, DMSO-d6) δ 175.3, 156.4 (Jcf=249.8 Hz), 153.8 (Jcf=7.0 Hz), 152.4, 150.8, 147, 3, 144.3, 131.4 (Jcf=3.5Hz), 127.3, 126.4 (Jcf=12.6Hz), 116.1 (Jcf=18.4Hz), 98.0, 72.1, 49.1, 46.6, 36.0, 29.4, 21.0, 15.4, 14.6 ppm ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ - 113.6 (d, J _FH =7.7 Hz) ppm. C ₂₀ H ₂₃ Cl ₂ FN ₆ O ₂ (mol. wt. 469.34); LC/MS (EI) m/e 433.2 (M++H; exact mass xvii: 432.15). Water content according to the KF method (Karl Fischer titration): 3.63% by weight; chloride content (Cl ^- ) determined by titration: 7.56% by weight (theoretical 7.56%).

Melting/decomposition range of the crystalline form of the salt (R)-4-(3-((S)-1-(4-amino-3-methyl-1Hpyrazolo[3,4-b]pyrimidin-1-yl)ethyl)-5 -chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride was determined by DSC from an initial temperature of 30°C to a final temperature of 350°C using a heating rate of 10°C/min. A single endothermic process was observed on the DSC thermogram, beginning at 194.37°C and peaking at 206.55°C, as shown in FIG. 1.

The TGA thermogram showed an overall weight loss of 4.3% up to 210°C. Above 210°C, the salt begins to decompose, as shown in FIG. 2.

- 24 041006

A typical X-ray powder diffraction (XRD) diffraction pattern is shown in FIG. 3, and the table shows the corresponding peaks and intensities.______

2-theta Relative intensity 2-theta Relative intensity 2-theta Relative intensity 5.739 2.20% 25.098 2.20% 42.916 2.40% 7.133 1.20% 25, 66 7.00% 43.373 0.50% 7.736 0.10% 25.895 4.00% 44.148 0.40% 10.225 2.30% 27.168 3.10% 45.29 0.30% 11.283 99.00% 27.792 8.50% 46.089 1.40% 11.303 94.10% 28.1 10.00% 47.572 0.40% 13.666 2.90% 28.464 5.50% 48.897 0.70% 14.166 0.90% 30.134 3.20% 49.647 0.50% 14.833 0.10% 31.239 13.70% 50.589 0.30% 15.364 3.80% 31.918 1.30% 51.042 0.10% 16.354 9.70% 32.827 9.50% 51.687 0.40% 17.136 0.50% 33.818 0.70% 52.624 0.40% 16.866 2.70% 34.198 2.80% 53.287 0.50% 17.435 5.50% 35.033 2.10% 54.104 0.20% 17.635 3.30% 35.423 2.10% 54.127 0.10% 18.811 5.10% 36, 226 0.30% 54.159 0.20% 18.898 6.60% 36,676 0.90% 55.42 0.30% 19.603 1.50% 37.47 0.90% 56, 821 0.10% 20.157 1.80% 37.951 0.50% 20.593 0.50% 38.457 1.70% 21.039 11.10% 39.055 0.20% 21.308 3.80% 39.968 0.20% 22.169 7.50% 40.184 0.30% 23.002 11.50% 40.962 0.20% 24.628 6.60% 42 1.30%

Example 4 Alternative Synthesis of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin1-yl)ethyl)-5-chloro -2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride

nm—A nm—A _L . reagent | _d> Dessa-Martin X F Jdati ___. I J A G CH _; C1 _; ] Г ^α ” · ' f ^с| - g xiii a OH Xi* C O HN---y' A EYU CN SK -Λ.,- DIPEA Eton xv-a NiiNH, TsOH HN---y'° 1. AQOD HCI Η Cl WJ .Ν Μ * V VA χνιϊι A ¹ NK---γ ° ΜΗ---y/ ^s JEt _F Г* ⁿ pp catalyst _F UH-LAA МВОН [[ MpGH ] J 1 Kilt ; Cl Г _C K^.-^·- ^J ** Ahboc “'Anvos PH ⁰ CM--A Г ПН IK F. J % „ ⁴ NK-GCDs p |' _R P - “ m/jn Г CN Jw HN xvii

- 25 041006

Step 1. (R)-4-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (xix)

(4R)-4-[3-Chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]pyrrolidin-2-one (as a mixture of two R-configuration diastereomers on pyrrolidinone and R- or S -configurations on secondary alcohol) (xiii, 16.7 g, 55.3 mmol) was dissolved in dichloromethane (167 ml). The solution was cooled in an ice-water bath and Dess-Martin periodinan (35.2 g, 83.0 mmol) was added in small portions. The reaction mixture was stirred at room temperature for 2 hours, at which point HPLC analysis indicated that the reaction was complete. A solution of sodium sulfite (28 g, 220 mmol) in water (70 ml) was added to the reaction mixture, and the mixture was stirred for 20 minutes. A 1.0 M sodium hydroxide solution was added to the mixture and stirred for 10 minutes. The layers were allowed to settle and the organic layer was separated and washed successively with 1M aqueous sodium hydroxide solution (66 ml) and water (60 ml). The organic layer was dried over anhydrous sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated to give (R)-4-[3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl]pyrrolidin-2-one as an oil which was used for the next reaction without further purification.

Step 2. (R,E)-tert-Butyl-2-(1-(5-chloro-2-ethoxy-4-fluoro-3-(5-oxopyrrolidin-3-yl)phenyl)ethylidene)hydrazinecarboxylate (xx)

The crude (R)-4-[3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl]pyrrolidin-2-one (compound xix from step 1) was dissolved in methanol (60 ml) and tert-butylcarbazate was added to the solution (8.04 g, 60.8 mmol). The reaction mixture was stirred at 65° C. for 3.5 days, at which point HPLC analysis indicated that the reaction was complete. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography eluting with 0-5% methanol in ethyl acetate to give (R,E)-tert-butyl-2-(1-(5-chloro-2-ethoxy-4- fluoro-3-(5-oxopyrrolidin-3-yl)phenyl)ethylidene)hydrazinecarboxylate (xx, 19.5 g, 85%).

1H NMR (500 MHz, DMSO-a ₆ ) δ 9.83 (s, 1H), 7.78 (s, 1H), 7.36 (d, J=8.6 Hz, 1H), 4.07 ( n, J=9.1 Hz, 1H), 3.84-3.69 (m, 2H), 3.59 (t, J=9.5 Hz, 1H), 3.28 (t, J=9 .5 Hz, 1H), 2.54 (m, 1H), 2.33 (m, 1H), 2.14 (s, 3H), 1.46 (s, 9H), 1.25 (t, J =7.0 Hz, 3H). LC/MS for C19H25ClFNsNaO4 (M+Na) ⁺ : m/z=436.1.

Stage 3. tert-Butyl-2-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl) hydrazinecarboxylate ( xxi)

(R,E)-tert-Butyl-2-(1-(5-chloro-2-ethoxy-4-fluoro-3-(5-oxopyrrolidin-3-yl)phenyl)ethylidene)hydrazinecarboxylate (xx, 0.5 g, 1.2 mmol) was dissolved in methanol (25 ml) and nitrogen gas was bubbled through the solution for 5 minutes. Bis(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate (35 mg, 0.086 mmol) and (R)-(-)-1-{(S)-2-[bis(4-trifluoromethylphenyl)phosphine] were added to the solution ferrocenyl}ethyl-di-tert-butylphosphine (64 mg, 0.094 mmol) and nitrogen gas was bubbled through the resulting reaction mixture for 30 minutes. The reaction mixture was then shaken under hydrogen pressure (386 kPa (56 psi)) for 2.5 days. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography, eluting with methanol (0-10%) in ethyl acetate. The desired fractions were concentrated to give tert-butyl-2-((S)-1(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl) hydrazine carboxylate (xxi, 428 mg, 85% yield).

1H NMR (500 MHz, DMSO-06) δ 8.18 (s, 1H), 7.78 (s, 1H), 7.53 (d, J=8.2 Hz, 1H), 4.73 (s , 1H), 4.41 (br s, 1H), 3.98 (m, 1H), 3.75 (m, 2H), 3.61 (m, 1H), 3.26 (m, 1H) , 2.53 (m, 1H), 2.29 (dd, J=17.6, 8.6 Hz, 1H), 1.32

- 26 041006 (s, 12H), 1.10 (d, J=6.5 Hz, 1H). LC/MS for C ₁₉ H ₂₇ ClFN ₃ NaO ₄ (M+Na)+: m/z=437.9.

Chiral HPLC analysis indicated that the product contained the desired diastereomer tert-butyl-2((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl )phenyl)ethyl)hydrazinecarboxylate (xxi) in an amount of 85.6% and an undesired diastereomer tert-butyl-2-((R)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R) -5oxopyrrolidin-3-yl)phenyl)ethyl)hydrazinecarboxylate in the amount of 14.3%.

Stage 4. 5-Amino-1 -((S)-1 -(5-chloro-2-ethoxy-4-fluoro-3 -((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)-3methyl -1 H-pyrazole-4-carbonitrile (xvi)

tert-Butyl-2-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)hydrazinecarboxylate (xxi, 130 mg, 0.31 mmol) and p-toluenesulfonic acid monohydrate (86 mg, 0.45 mmol) were added to ethanol (3 ml) and the reaction mixture was heated at 50°C for 20 h. HPLC analysis showed that about 88% unreacted starting material. Added additional amount of p-toluenesulfonic acid (86 mg, 0.45 mmol) and the reaction mixture was heated to 60°C for 24 hours HPLC analysis showed complete removal of Boc protection. To this reaction mixture were added (1ethoxyethylidene)malononitrile (61 mg, 0.45 mmol) and N,N-diisopropylethylamine (260 μl, 1.5 mmol). The reaction mixture was stirred at room temperature for 2 hours. HPLC showed completion of pyrazole ring formation. A 1.0M sodium hydroxide aqueous solution was added to the reaction mixture and stirred for 20 minutes. Ethyl acetate (20 ml) was added to the mixture and stirred. The biphasic mixture was allowed to settle. The ethyl acetate layer was collected and the aqueous layer was extracted with ethyl acetate (10 ml). To the combined ethyl acetate solution was added 1M aqueous hydrochloric acid (5 ml) and stirred for 15 minutes. The biphasic mixture was allowed to settle and the organic layer was collected and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration and the filtrate was concentrated to give 5-amino-1-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3yl)phenyl )ethyl)-3-methyl-1H-pyrazole-4-carbonitrile (xvi, 126 mg, quantitative yield of crude product), which was used in the next step without further purification.

Step 5. (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5- chloro-2ethoxy-6-fluorophenyl)pyrrolidin-2-one (xvii)

HN--cXe

A m / A ^N xvii ^h 2 ⁿ

K 5-amino-1-{(1S)-1-[5-chloro-2-ethoxy-4-fluoro-3-(5-oxopyrrolidin-3-yl)phenyl]ethyl}-3-methyl-1Hpyrazole-4 -carbonitrile (xvi, 126 mg, 0.31 mmol) was added formamidine acetate (323 mg, 3.1 mmol) and 1,2-ethanediol (2 ml). The reaction mixture was heated at 104-105°C with stirring. After 18 hours, HPLC analysis indicated that about 44% of the starting compound xvi remained. The reaction mixture was heated to 115° C. for 24 hours. HPLC analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature and ethyl acetate (10 ml) and water (5 ml) were added. The biphasic mixture was stirred. The layers were allowed to separate. The organic layer was collected and the aqueous layer was extracted with ethyl acetate (5 ml). The combined ethyl acetate solution was washed with water (5 ml), dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration and the filtrate was concentrated to a residue. The residue was purified by silica gel chromatography. The column was eluted with a mixture of methanol (0-5%) in methylene chloride. The desired fractions were combined and evaporated to give (R)-4-(3-((S)-1-(4-amino-3methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)- 5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (xvii, 94 mg, 69.9% yield).

¹ H NMR (400 MHz, DMSO-a ₆ ) δ 8.11 (s, 1H), 7.82 (s, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.30 (br s, 2H), 6.23 (k, J=7.0 Hz, 1H), 3.97 (n, J=9.2 Hz, 1H), 3.90-3.73 (m, 2H), 3.57 (t, J=9.9 Hz, 1H), 3.25 (dd, J=9.2, 8.7 Hz, 1H), 2.48 (s, 3H), 2, 60-2.50 (m, 1H), 2.36-2.20 (m, 1H), 1.69 (d, J=7.1 Hz, 3H), 1.39 (t, J=6, 9 Hz, 3H). LC/MS

- 27 041006 for C20H23CIFN6O2 (M+H)+: m/z=433.3.

Chiral HPLC analysis of the product showed that it contained the desired diastereomer (R)-4-(3((S)-1-(4-amino-3-methyl-1 H-pyrazolo[3,4-d]pyrimidine-1 -yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (xvii) in an amount of 87% and an undesired diastereomer (R)-4-(3-((R)-1-( 4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one in the amount of 13%.

Step 6 (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro -2ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride.

The desired product was obtained according to the procedure described in Example 3, Step 5. The resulting hydrochloride salt closely matches the material obtained using the synthesis method described in Example 3 in every comparable aspect, including chemical purity, chiral purity, and solid state characteristics.

Example A1. Analysis of PI3K enzymes.

Kit for luminescent analysis of PI3-kinase, including a lipid substrate for the kinase, Dmyophosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), D (+)-sn-1,2-du-O-octαnoylglucerul, 3U- phospho-bound (PIP2), biotinylated I(1,3,4,5)P4, PI(3,4,5)P3 detection protein, purchased from Echelon Biosciences (Salt Lake City, UT). TST AlphaScreen™ detection kit including donor and acceptor beads purchased from PerkinElmer Life Sciences (Waltham, MA). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, MA). ATP, MgCl ₂ , DTT, EDTA, HEPES, and CHAPS were purchased from Sigma-Aldrich (St. Louis, MO).

AlphaScreen™ analysis for PI3Kδ.

The kinase reaction was carried out in a Thermo Fisher Scientific 384-well REMP plate, the final volume was 40 µl. The inhibitors are first serially diluted with DMSO and added to the wells of the plate before the addition of other reaction components. The final concentration of DMSO for analysis is 2%. The PI3K assay is carried out at room temperature in 50 mM HEPES, pH 7.4, 5 mM MgCl ₂ , 50 mM NaCl, 5 mM DTT and 0.04% CHAPS. Reactions are initiated by the addition of ATP and the final reaction mixture, which consists of 20 μM PIP2, 20 μM ATP, 1.2 nM PI3Kδ, is incubated for 20 min. 10 µl of the reaction mixture is then transferred to 5 µl of 50 nM biotinylated I(1,3,4,5)P4 in quench buffer: 50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM EDTA, 5 mM DTT , 0.1% Tween-20, then add 10 µl of AlphaScreen™ donor and acceptor beads suspended in quench buffer containing 25 nM PI(3,4,5)P3 detection protein. The final concentration of both donor and acceptor beads is 20 mg/ml. After the plates are sealed, they are incubated in the dark at room temperature for 2 hours. Product activity is determined on a Fusion-alpha microplate reader (Perkin-Elmer). The determination of IC ₅₀ is performed by fitting the proportion of the activity of the control sample (in percent) against the logarithm of the concentration of the inhibitor, using the GraphPad Prism 3.0 software.

Example A2. Analysis of PI3K enzymes.

Substances.

The lipid substrate for the kinase, phosphoinositol 4,5-bisphosphate (PIP2), was purchased from Echelon Biosciences (Salt Lake City, UT). PI3K isoforms α, β, δ, and γ were purchased from Millipore (Bedford, Massachusetts). ATP, MgCl ₂ , DTT, EDTA, MOPS, and CHAPS were purchased from Sigma-Aldrich (St. Louis, MO).

Kinase reactions were carried out in a 96-well Thermo Fisher Scientific clear bottom plate with a final volume of 24 μl. The inhibitors are first serially diluted with DMSO and added to the wells of the plate before the addition of other reaction components. The final concentration of DMSO for analysis is 0.5%. PI3K analysis is performed at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl ₂ , 5 mM DTT, and 0.03% CHAPS. The reaction mixture was prepared to contain 50 μM PIP2, kinase, and various concentrations of inhibitors. Reactions are initiated by adding ATP containing 2.2 μCi of [y- ³³ P]ATP so that the final concentration is 1000 μM. The final concentrations of PI3K isoforms α, β, δ and γ for analysis are 1.3, 9.4, 2.9 and 10.8 nM, respectively. The reactions are incubated for 180 min and the reactions are terminated by adding 100 μl of quench buffer containing 1 M potassium phosphate, pH 8.0, 30 mM EDTA. An aliquot of 100 µl of the reaction solution is then transferred to a 96-well Millipore Multiscreen IP 0.45 µm PVDF filter plate (the filter plate is pre-wetted in turn with 200 µl of 100% ethanol, distilled water and 1M potassium phosphate solution pH 8.0 ). The liquid from the filter plate is aspirated under vacuum using a Millipore Manifold and the plate is washed with 18x200 μl of wash buffer containing 1 M potassium phosphate pH 8.0 and 1 mM ATP. After drying by aspiration and absorption, the plate is dried in air in a thermostat at 37°C overnight. The Packard TopCount adapter (Millipore) was then attached to the plate after adding 120 µl of Microscint 20 scintillation cocktail (Perkin Elmer) to each well. After the plate is sealed, the radioactivity of the product is determined by counting scintillations on a Topcount instrument (Perkin-Elmer). The determination of the IC ₅₀ is performed by curve fitting

- 28 041006 shares of the activity of the control sample (in percent) of the logarithm of the concentration of the inhibitor, using the GraphPad Prism 3.0 software.

In the assay of Example A2, (R)-4-(3-((S)-1-(4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5 hydrochloride salt was tested -chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one was found to be a selective inhibitor of PI3Kδ.

In the assay of Example A2, (R)-4-(3-((S)-1-(4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5 hydrochloride salt was tested -chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one was found to be 100 times more selective inhibitor of PI3Kδ than each of PI3Ka, PI3Ke and PI3Ky.

Example A3. Scintillation proximal PI3Kδ analysis.

Substances.

[Y- ^33P ]ATP (10 mCi/ml) was purchased from Perkin-Elmer (Waltham, MA). Kinase lipid substrate, D-myo-phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), D(+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho-linked (PIP2 ), CAS 204858-53-7, purchased from Echelon Biosciences (Salt Lake City, UT). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, MA). ATP, MgCl ₂ , DTT, EDTA, MOPS, and CHAPS were purchased from Sigma-Aldrich (St. Louis, MO). Scintillation beads for SPA analysis with immobilized wheat germ agglutinin (WGA) and YSi were purchased from GE Healthcare Life Sciences (Piscataway, NJ).

The kinase reaction was carried out in a 384-well white polystyrene array plate manufactured by Thermo Fisher Scientific, the final volume was 25 μl. The inhibitors were first serially diluted with DMSO and added to the wells of the plate before the addition of other reaction components. The final concentration of DMSO for analysis was 0.5%. PI3K analysis was performed at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl ₂ , 5 mM DTT, and 0.03% CHAPS. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [y- ³³ P] ATP, 4 nM PI3Kδ. The reactions were incubated for 210 min and the reactions were terminated by adding 40 µl of SPA beads suspended in quench buffer: 150 mM potassium phosphate, pH 8.0, 20% glycerol, 25 mM EDTA, 400 µM ATP. The final bead concentration for the SPA analysis was 1.0 mg/mL. After the plates were sealed, they were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by counting scintillations on a Topcount instrument (Perkin-Elmer). The determination of the IC ₅₀ was performed by fitting the curve of the proportion of activity of the control sample (in percent) from the logarithm of the concentration of the inhibitor, using the software GraphPad Prism 3.0. The compound of formula I in the assay of example A3 was found to have an IC ₅₀ of <10 nM.

Example B1. B-cell proliferation assay.

To obtain B cells, human PBMCs are isolated from the peripheral blood of healthy, drug-free donors using standard Ficoll-Hypague density gradient centrifugation (GE Healthcare, Piscataway, NJ) and incubated with anti-CD19 immobilized microbeads ( Miltenyi Biotech, Auburn, California). The B cells are then purified by positive immunosorting using autoMacs (Miltenyi Biotech) according to the manufacturer's instructions.

Purified B cells (2x105/well/200 μl) were cultured in 96-well ultra-low binding plates (Corning, Corning, NY) in RPMI1640 medium containing 10% PBS and goat F(ab') ₂ against human IgM ( 10 μg/ml) (Invitrogen, Carlsbad, CA) in the presence of various amounts of test compounds for three days. [ ³ H]-thymidine (1 μCi/well) (PerkinElmer, Boston, Massachusetts) in PBS was then added to B cell cultures for an additional 12 hours, after which the introduced radioactivity was separated by filtration with water through GF/B filters (Packard Bioscience, Meriden, Conn.) and determined by liquid phase scintillation counting using TopCount (Packard Bioscience).

Example B2. Pfeiffer cell proliferation assay.

The Pfeiffer cell line (diffuse large B cell lymphoma) was purchased from ATCC (Manassas, VA) and cultured in the recommended culture medium (RPMI and 10% FBS). To measure the antiproliferative activity of compounds, Pfeiffer cells were seeded in growth media (2x103 cells/well/200 μl) in 96-well ultra-low binding plates (Corning, Corning, NY) in the presence or absence of test compounds at various concentrations. After 3-4 days, [ ³ H]-thymidine (1 μCi/well) (PerkinElmer, Boston, Massachusetts) in PBS is added to the cell culture for another 12 hours, after which the introduced radioactivity is separated by filtration with water through GF/B filters (Packard Bioscience, Meriden, Conn.) and determined by liquid phase scintillation counting using TopCount (Packard Bioscience).

Example B3. SUDHL-6 cell proliferation assay.

The cell line SUDHL-6 (diffuse large B cell lymphoma) was purchased from ATCC (Manassas, VA) and cultured in the recommended culture medium (RPMI and 10% PBS). For

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Claims (31)

measurements of the antiproliferative activity of the compounds by ATP quantification, SUDHL-6 cells were seeded in nutrient medium (5000 cells/well/200 μl) in a 96-well polystyrene transparent black plate treated for tissue culture (Greiner-bio-one by VWR, New Jersey), in the presence or absence of test compounds at various concentrations. After 3 days, cell culture agent Cell Titer-GLO (luminescent) (Promega, Madison, Wisconsin) was added to each well for 10 minutes at room temperature to stabilize the luminescent signal. This assay determines the number of viable cells in culture based on the quantification of ATP present, which indicates the presence of metabolically active cells. Luminescence was measured with a TopCount 384 (Packard Bioscience via Perkin Elmer, Boston, MA). Example C Akt Phosphorylation Assay. Ramos cells (B-lymphocytes derived from Burkitt's lymphoma) were obtained from ATCC (Manassas, VA) and cultured in RPMI1640 medium and 10% PBS. Cells (3x10 ⁷ cells/tube/3 ml in RPMI) are incubated with various amounts of test compounds for 2 h at 37°C and then stimulated with goat F(ab') ₂ against human IgM (5 μg/ml) (Invitrogen) for 17 minutes in a water bath at 37°C. The stimulated cells are centrifuged at 4° C. and a whole cell extract is prepared using 300 μl of lysis buffer (Cell Signaling Technology, Danvers, MA). The resulting lysates are sonicated and the supernatants are collected. The level of Akt phosphorylation in supernatants was determined using PathScan phospho-Akt1 (Ser473) sandwich-type ELISA kits (Cell Signaling Technology) according to the manufacturer's instructions. For those skilled in the art, various modifications of the invention in addition to those described herein will be apparent from the foregoing description. Such modifications are also intended to be within the scope of the appended claims. Each source, including all patents, patent applications and publications cited in this application, is incorporated into this document in its entirety by reference. CLAIM 1. Salt which is the hydrochloric acid salt of (R)-4-(3-((S)-1-(4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1-yl)ethyl) -5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, which is crystalline. 2. A salt according to claim 1 which is a 1:1 stoichiometric ratio of (R)-4-(3-((S)-1-(4amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2one to hydrochloric acid. 3. Salt according to claim 1 or 2, which is characterized by a DSC thermogram showing an endothermic peak at 207±3°C. 4. Salt according to any one of claims 1 to 3, having a DSC thermogram substantially the same as shown in FIG. 1. 5. A salt according to any one of claims 1 to 4, having a TGA thermogram substantially the same as shown in FIG. 2. 6. Salt according to any one of claims 1 to 5, characterized by at least one peak in 2θ units selected from 11.3±0.2°, 16.4±0.2°, 21.0±0.2 °, 23.0±0.2°, 28.1±0.2°, 31.2±0.2° and 32.8±0.2°. 7. Salt according to any one of claims 1 to 5, characterized by at least two peaks in 2θ units selected from 11.3±0.2°, 16.4±0.2°, 21.0±0.2 °, 23.0±0.2°, 28.1±0.2°, 31.2±0.2° and 32.8±0.2°. 8. Salt according to any one of claims 1 to 5, characterized by at least three peaks in 2θ units selected from 11.3±0.2°, 16.4±0.2°, 21.0±0.2 °, 23.0±0.2°, 28.1±0.2°, 31.2±0.2° and 32.8±0.2°. 9. Salt according to any one of claims 1 to 5, characterized by at least four peaks in 2θ units selected from 11.3±0.2°, 16.4±0.2°, 21.0±0.2 °, 23.0±0.2°, 28.1±0.2°, 31.2±0.2° and 32.8±0.2°. 10. Salt according to any one of claims 1 to 5, characterized by at least five peaks in 2θ units selected from 11.3±0.2°, 16.4±0.2°, 21.0±0.2 °, 23.0±0.2°, 28.1±0.2°, 31.2±0.2° and 32.8±0.2°. 11. A salt according to any one of claims 1 to 5, having a XRD pattern substantially the same as shown in FIG. 3. 12. A pharmaceutical composition having the ability to inhibit the activity of PI3Kδ kinase, containing a therapeutically effective amount of a crystalline salt according to any one of claims 111 and a pharmaceutically acceptable carrier. 13. A method for inhibiting PI3Kδ kinase activity, comprising contacting the kinase with a crystalline salt according to any one of claims 1-11. 14. The method of claim 13 wherein said salt is a selective inhibitor of PI3Kδ over one or more of PI3Ka, PI3Ke or PI3Ky. 15. A method for treating a disease in a patient in which said disease is associated with abnormal expression or activity of PI3Kδ kinase, comprising administering to said patient a therapeutic - 30 041006 an effective amount of a crystalline salt according to any one of claims 1-11. 16. The method according to claim 15, characterized in that the disease is selected from idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, vasculitis, systemic lupus erythematosus, lupus nephritis, pemphigus, autoimmune hemolytic anemia (AHA), membranous nephropathy, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), hairy cell leukemia, mantle cell lymphoma, Burkitt's lymphoma, small cell lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone cell lymphoma, Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, prolymphocytic leukemia, acute lymphoblastic leukemia, myelofibrosis, mucosal-associated lymphoid tissue (MALT) lymphoma, B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, Hodgkin's disease, splenic marginal zone lymphoma, primary effusion lymphoma, intravascular B-large cell cellular lymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoldering myeloma (also called asymptomatic myeloma), monoclonal gammopathy of uncertain significance (MGUS), and B-cell lymphoma. 17. The method of claim 16, characterized in that it is a method of treating idiopathic thrombocytopenic purpura (ITP) selected from recurrent ITP and refractory ITP. 18. The method according to claim 16, characterized in that it is a method for treating vasculitis selected from Behçet's disease, Cogan's syndrome, giant cell arteritis, polymyalgia rheumatica (RP), Takayasu's arteritis, Buerger's disease (thromboangiitis obliterans), vasculitis of the central nervous system, Kawasaki disease, polyarteritis nodosa, Churg-Strauss syndrome, mixed cryoglobulinemic vasculitis (essential or caused by hepatitis C virus (HCV)), Henoch-Schonlein purpura (HSP), leukocytoclastic vasculitis, microscopic polyangiitis, Wegener's granulomatosis, and associated with antineutrophil cytoplasmic antibodies (ANCA) ) systemic vasculitis (ASV). 19. The method according to claim 16, characterized in that it is a method of treating non-Hodgkin's lymphoma (NHL) selected from relapsed NHL, refractory NHL and secondary follicular NHL. 20. The method of claim 16, wherein said B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL). 21. The method according to claim 16, characterized in that it is a method of treating B-cell lymphoma, wherein said B-cell lymphoma is diffuse B-large cell lymphoma from cells like activated B-cells (AVK), or diffuse B- large cell lymphoma from germinal center B-cells (GCC). 22. The method of claim 15, wherein said disease is osteoarthritis, restenosis, atherosclerosis, bone lesions, arthritis, diabetic retinopathy, psoriasis, benign prostatic hypertrophy, inflammation, angiogenesis, pancreatitis, kidney disease, inflammatory bowel disease, myasthenia gravis, multiple sclerosis or Sjögren's syndrome. 23. The method of claim 15, wherein said disease is rheumatoid arthritis, allergy, asthma, glomerulonephritis, lupus, or inflammation associated with any of the above. 24. The method of claim 23 wherein the lupus is systemic lupus erythematosus or lupus nephritis. 25. The method of claim 15, wherein said disease is breast cancer, prostate cancer, colon cancer, endometrial cancer, brain cancer, bladder cancer, skin cancer, uterine cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer or hemoblastosis. 26. The method of claim 25, wherein said hemoblastosis is acute myeloid leukemia or chronic myeloid leukemia. 27. The method of claim 15, wherein said disease is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in adults. 28. The method of claim 15, wherein said disease is follicular lymphoma. 29. The method of claim 15 wherein said disease is extranodal marginal zone cell lymphoma or marginal zone cell lymphoma of the spleen. 30. The method of claim 15, wherein said disease is mantle cell lymphoma. 31. A method for producing a crystalline salt according to any one of claims 1 to 11, including the interaction of a compound of formula I -