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WO2024256568A1 - Salt and crystal forms of a macrocyclic btk inhibitor - Google Patents

Salt and crystal forms of a macrocyclic btk inhibitor Download PDF

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Publication number
WO2024256568A1
WO2024256568A1 PCT/EP2024/066423 EP2024066423W WO2024256568A1 WO 2024256568 A1 WO2024256568 A1 WO 2024256568A1 EP 2024066423 W EP2024066423 W EP 2024066423W WO 2024256568 A1 WO2024256568 A1 WO 2024256568A1
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acid salt
compound
crystalline
ray powder
powder diffraction
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French (fr)
Inventor
Adrianus Petrus Antonius De Man
Rogier Christian Buijsman
Freek VAN CAUTER
Petrus Jozef Cornelis Maria VAN HOOF
Irina Andreevna NIKOLAEVA
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Crossfire Oncology Holding BV
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Crossfire Oncology Holding BV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present disclosure relates to novel salts and crystalline forms of a macrocyclic Bruton’s Tyrosine Kinase (BTK) inhibitor, to pharmaceutical compositions comprising the crystalline forms, to methods of using the crystalline forms to treat conditions treatable by the inhibition of (mutant) BTK, such as B-cell malignancies, in particular B-cell lymphomas, even more particular marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), non-Hodgkin lymphoma, Burkitt lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), hairy cell leukemia, B- cell non-Hodgkin lymphoma, B-cell prolymphocytic leukemia, Waldenstrom’s macroglobulinemia (WM), multiple myeloma (MM), arthritis, in particular rheuma
  • Kinases are enzymes that transfer a phosphate group from ATP to a protein. Kinases play an important role in regulating cellular functions such as cell proliferation, subcellular translocation, apoptosis, inflammation and metabolism (Attwood M.M. et al (2021) Nat Rev Drug Discov).
  • the human kinome is composed of over 500 kinases. The development of smallmolecule kinase inhibitors for the treatment of diverse types of cancer has proven successful in clinical therapy.
  • BTK Bruton's tyrosine kinase
  • BCR B-cell receptor
  • BTK small molecule inhibitors
  • small molecule inhibitors such as the FDA approved covalent BTK inhibitors ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib
  • CLL Chronic Lymphocytic Leukemia
  • MCL Mantle Cell Lymphoma
  • WM Macroglobulinemia
  • SLL Small Lymphocytic Lymphoma
  • BTK is also expressed and plays also pro-tumorigenic roles in several solid tumors (Xianhui Wang et al. 2021).
  • BTK inhibition with ibrutinib or acalabrutinib inhibited cell growth (Kokabee et al 2015).
  • BTK inhibitors have also showed inhibition of cellular proliferation and migration, and induced apoptosis and autophagy in glioblastoma cell lines (Wei et al., 2016; Wang et al., 2017).
  • a drawback of the first generation BTK inhibitor, ibrutinib is that drug resistance in B- cell malignancies can develop when BTK acquires mutations at the cysteine at position C481 of the kinase domain. This mutation abrogates the covalent binding of ibrutinib hampering its efficacy.
  • Quinquenel et al. performed a ‘snapshot’ screening to determine the prevalence of resistance mutations and found that the presence of the BTK mutation was significantly associated with subsequent CLL progression. The correlation between disease progression, and the emergence and temporal dynamics of the most common resistance inducing C481 S BTK mutation have been determined for CLL patients receiving single-agent Ibrutinib treatment (Bodor et al.
  • Second-generation BTK inhibitors which include acalabrutinib, zanubrutinib, and tirabrutinib, offer greater BTK selectivity and therefore limited off-target toxicity. These inhibitors, however, do not overcome resistance by C481 mutation.
  • non-covalent BTK inhibitors have been developed including LOXO- 305 (pirtobrutinib) and ARQ-531 (nemtabrutinib) and vecabrutinib. These agents do not require covalent binding to the BTK C481 residue and effectively inhibit both wild type and mutant BTK with C481 substitutions.
  • noncovalent BTK inhibitors including pirtobrutinib (LOXO-305), ARQ-351 and vecabrutinib, inhibited B-cell-receptor signaling in BTK C481- mutant cell and animal models.
  • phase 1 -2 clinical trial of pirtobrutinib showed promising efficacy for patients with B-cell cancer who had previously been treated with covalent BTK inhibitors (with 62% of patients with CLL having a response), including patients with or without BTK C481 mutations (with a response occurring in 71 % and 66% of the patients, respectively) (Wang et al. (2022) N. Engl. J. Med. 386, 735-43).
  • Pirtobrutinib was first disclosed in WO2017/103611 , ARQ-531 was disclosed in WO2017/111787 and vecabrutinib is disclosed in WO2013/185084. Further reversible BTK inhibitors are disclosed in WO2017/046604, , WQ2020/015735, WQ2020/239124, WQ2021/093839, WQ2020/043638, WO2013/067274, WO2018097234, WO2013/010380, W02016/161570, WO2016/161571 , WO2016/106624, WO2016/106625, WO2016/106626, WO2016106623, WO2016/106628 and WO2016/109222.
  • PCT Patent Application No. PCT/EP2022/085765 discloses macrocyclic inhibitors of Bruton’s tyrosine kinase (BTK).
  • Said macrocyclic BTK inhibitors can, for example, be used to treat various cancer, such as CLL.
  • the compound (I) is one of the BTK inhibitors disclosed in PCT Patent Application No. PCT/EP2022/085765, also referred to as having subformula 184.
  • the present disclosure is directed to i) novel pharmaceutically acceptable salts of compound (I) (e.g., compound (I) hydrochloric acid, compound (I) maleate, compound (I) semisulfate, compound (I) sulfate, compound (I) semi-edisylate, compound (I) hydrobromic acid) including the corresponding solid forms; and ii) a novel solid form of the free base of compound (I) (hereinafter collectively referred to as “Salt or Solid Forms of the Disclosure”).
  • the present disclosure provides a hydrochloric acid addition salt of compound (I).
  • the molar ratio between compound (I) and hydrochloric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1.
  • this salt is also referred herein as “compound (I) hydrochloric acid” or as “hydrochloric acid addition salt of compound (I)”.
  • the present disclosure provides a maleic acid addition salt of compound (I).
  • the molar ratio between compound (I) and maleic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1.
  • this salt is also referred herein as “compound (I) maleate” or as “maleic acid addition salt of compound (I)”.
  • the present disclosure provides a semi-sulfate addition salt of compound (I).
  • the molar ratio between compound (I) and sulfuric acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5.
  • this salt is also referred herein as “compound (I) semi-sulfate” or as “1 :0.5 sulphuric acid addition salt of compound (I)”.
  • the present disclosure provides a sulfate addition salt of compound (I).
  • the molar ratio between compound (I) and sulfuric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1 .
  • this salt is also referred herein as “compound (I) sulfate” or as “1 :1 sulphuric acid addition salt of compound (I)”.
  • the present disclosure provides a semi-edisylate addition salt of compound (I).
  • the molar ratio between compound (I) and ethane 1 ,2 sulfonic acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5.
  • this salt is also referred herein as “compound (I) semi- edisylate” or as “1 :0.5 ethane 1 ,2 sulfonic acid addition salt of compound (I)”.
  • the present disclosure provides a hydrobromic acid addition salt of compound (I).
  • the molar ratio between compound (I) and hydrobromic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1.
  • this salt is also referred herein as “compound (I) hydrobromic acid” or as “hydrobromic acid addition salt of compound (I)”.
  • the present disclosure provides a crystalline form of the free base of compound (I), having a crystalline Form G.
  • This crystalline form is also referred herein as compound (I) free base Form G.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention, in particular embodiments comprising at least one of a compound (I) hydrochloric acid salt, compound (I) maleate, compound (I) semisulfate, compound (I) sulfate, compound (I) semi-edisylate, compound (I) hydrobromic acid, compound (I) free base Form G or compound (I) and a pharmaceutically acceptable carrier or diluent.
  • BTK Tyrosine Kinase
  • an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention or a pharmaceutical composition according to the invention for use in the treatment of Bruton’s Tyrosine Kinase (BTK) mediated disorders wherein the Bruton’s Tyrosine Kinase (BTK) mediated disorder is selected from the group consisting of an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, a bone-related disease, and cancer.
  • a method for treating of cancer in a subject in need thereof comprising administering to the subject the compound according to the invention in an amount effective to treat cancer.
  • a method for treating a subject suffering with a Bruton's Tyrosine Kinase (BTK) mediated disorder comprising administering to the subject the compound the invention or pharmaceutical composition according to the invention in an amount effective to treat the BTK mediated disorder.
  • BTK Bruton's Tyrosine Kinase
  • composition as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.
  • pharmaceutically acceptable has its conventional meaning and refers to compounds, material, compositions and/or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.
  • an effective amount refers to an amount of the compound of the invention, and/or an additional therapeutic agent, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventive effect when administered to a subject suffering from a BTK-mediated disease or disorder.
  • a “subject” is a human or non-human mammal. In one embodiment, a subject is a human.
  • controlling is intended to refer to all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of the diseases and conditions affecting the mammal. However, “controlling” does not necessarily indicate a total elimination of all disease and condition symptoms, and is intended to include prophylactic treatment.
  • excipient as used herein has its conventional meaning and refers to a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing a granulate, solid or liquid oral dosage formulation.
  • salt as used herein has its conventional meaning and includes the acid addition and base salts of the compound of the invention.
  • solvate as used herein has its conventional meaning.
  • One or more compounds of the invention or the pharmaceutically acceptable salts thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • “Solvate” means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
  • Solvate encompasses both solution-phase and isolatable solvates. Examples of suitable solvates include ethanolates, methanolates, and the like.
  • “Hydrate” is a solvate wherein the solvent molecule is H2O and includes any hydrate of the compound or the salt of said compound.
  • treatment has its conventional meaning and refers to curative, disease controlling, palliative and prophylactic treatment.
  • unit dosage form has its conventional meaning and refers to a dosage form which has the capacity of being administered to a subject, preferably a human, to be effective, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising the therapeutic agent, i.e. the compound of the invention.
  • BTK Bruton's Tyrosine Kinase
  • BTK Bruton's tyrosine kinase
  • Src-related Tec family of protein kinases which are a large subset of kinases which play a central role in the regulation of a wide variety of cellular signaling processes.
  • BTK plays a key role in the B-cell receptor signaling and a critical role in the regulation of survival, proliferation, activation and differentiation of B- lineage cells.
  • BTK inhibitor as used herein has its conventional meaning and refers to an inhibitor for BTK.
  • a BTK inhibitor may be a small molecule inhibitor.
  • Inhibitors may be irreversible inhibitors, such as by forming a covalent bond, and may be reversible inhibitors, which may form a reversible interaction with BTK.
  • covalent BTK inhibitor has its conventional meaning and refers to a BTK inhibitor that reacts with its target protein (BTK) to form a covalent complex in which the protein has lost its function.
  • BTK target protein
  • Covalent inhibitors can be reversible or irreversible, depending on the rate of the reverse reaction.
  • 'covalent inhibitor' and 'irreversible inhibitor' are often used interchangeably.
  • irreversible BTK inhibitor has its conventional meaning and refers to a BTK inhibitor that possesses an off-rate that is slow relative to the rate of re-synthesis of the target protein (BTK) in vivo, so that once the target protein is inhibited, it does not regain activity.
  • rate of re-synthesis has its conventional meaning and refers to the rate at which a cell and/or organism replaces a protein target with freshly synthesized functional protein.
  • the re-synthesis rate defines the rate at which an irreversibly inhibited protein target will recover activity in vivo, once the inhibitor is no longer present.
  • reversible BTK inhibitor has its conventional meaning and refers to a BTK inhibitor that inactivates the BTK enzyme through non-covalent, transcient, interactions. Unlike an irreversible inhibitor, a reversible inhibitor can dissociate from the enzyme.
  • mutant-BTK has its conventional meaning and refers to mutations of BTK. Mutations of BTK may be referred to by an altered or modified amino acid target (such as C as single-letter amino acid code for cysteine) at a certain position of the BTK structure (such as 481). Additionally, the amino acid substitution at the modification position may be referred to by an additional amino acid single-letter amino acid code, such as C481 S for serine substitution and C481T for threonine substitution of cysteine at the 481 position.
  • an altered or modified amino acid target such as C as single-letter amino acid code for cysteine
  • an additional amino acid single-letter amino acid code such as C481 S for serine substitution and C481T for threonine substitution of cysteine at the 481 position.
  • modification has its conventional meaning and refers to modification of a sequence of amino acids of a polypeptide, protein or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively.
  • the term "resistance to BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor, which shows a reduction in effectiveness, after being effective initially in treating a hyperproliferative disease, in particular a B-cell hematological malignancy.
  • a drawback of the currently approved irreversible inhibitors is that patients treated with these inhibitors can develop drug resistance when BTK proteins with variations at the catalytic site are not able to bind efficiently to irreversible inhibitors in patients. This is a rather common event in patients treated with irreversible inhibitors and who experience relapse.
  • a major mechanism for the acquired resistance is the emergence of BTK cysteine 481 (C481) mutations. These mutations hamper binding of irreversible inhibitors such as ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib which form a covalent bond with this amino acid.
  • valine 416 V416)
  • A428 alanine 428
  • M437 methionine 437
  • T474 threonine 474
  • L528 leucine 528
  • drug resistance is meant the major cause of cancer treatment failure. While a treatment may be effective initially, the heterogeneity of cancer and its ability to adapt can allow the cancer to become resistant to the treatment and regrow.
  • relapse as used herein has the conventional meaning and refers to evidence of disease progression in a patient who has previously achieved criteria of a complete response or partial remission.
  • disease progression has the conventional meaning and refers to a measured increase in tumor size or tumor burden.
  • recurrent or refractory form of a hyperproliferative disease has the conventional meaning and refers to a particular cancer that is resistant, or non-responsive to therapy with a particular therapeutic agent.
  • a cancer can be refractory to therapy with a particular therapeutic agent either from the onset of treatment (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.
  • the term ‘’acquired drug resistance” as used herein has the conventional meaning and refers to resistance of a drug caused by mutations in the target protein.
  • the mutations in the target protein hamper the binding of drug resulting in a regrowth of the tumor
  • clinical drug resistance refers to growth of a tumour while the patient is on treatment, that develops following after an initial clinical benefit (a clinical response or prolonged stable disease).
  • wt-BTK or “WT-BTK” or “BTK WT ” as used herein has its conventional meaning and refers to wild-type Bruton’s Tyrosine Kinase.
  • a wild-type BTK has the regular meaning of a phenotype of the typical form of BTK as it occurs in nature. Originally, the wild-type was conceptualized as a product of the standard "normal” allele at a locus, in contrast to that produced by a non-standard, "mutant” allele.
  • microcycle as used herein has its conventional meaning and refers to a part of a molecule containing a ring consisting of 12 or more ring atoms forming said ring. In an example, a twelve membered ring consist of 12 atoms forming said ring.
  • compositions comprising components A and B
  • the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element or component are present, unless the context clearly requires that there is one and only one of the elements or components.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • Figure 1 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) hydrochloric acid Form A.
  • FIG. 2 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) hydrochloric acid Form A.
  • FIG. 3 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) hydrochloric acid Form A.
  • Figure 4 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) maleate Form B.
  • FIG. 5 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) maleate Form B.
  • FIG. 6 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) maleate Form B.
  • Figure 7 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :0.5 compound (I) semisulfate Form C.
  • Figure 8 shows the Thermogravimetric Analysis (TGA) of 1 :0.5 compound (I) semisulfate Form C.
  • FIG. 9 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :0.5 compound (I) semi-sulfate Form C.
  • Figure 10 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) sulfate Form D.
  • FIG 11 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) sulfate Form D.
  • Figure 12 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) sulfate Form D.
  • Figure 13 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :0.5 compound (I) semi-edisylate Form E.
  • FIG 14 shows the Thermogravimetric Analysis (TGA) of 1 :0.5 compound (I) semi- edisylate Form E.
  • FIG. 15 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :0.5 compound (I) semi-edisylate Form E.
  • Figure 16 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) hydrobromic acid Form F.
  • Figure 17 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) hydrobromic acid Form F.
  • Figure 18 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) hydrobromic acid Form F.
  • Figure 19 shows the X-ray Powder Diffraction (XRPD) pattern of compound (I) free base Form G.
  • FIG. 20 shows the Thermogravimetric Analysis (TGA) of compound (I) free base Form G.
  • FIG. 21 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of compound (I) free base Form G.
  • Figure 22 shows the X-ray Powder Diffraction (XRPD) pattern of compound (I) free base Form O.
  • FIG. 23 shows the Thermogravimetric Analysis (TGA) of compound (I) free base Form O.
  • FIG. 24 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of compound (I) free base Form O.
  • the present disclosure provides a hydrochloric acid addition salt of compound (I).
  • the molar ratio between compound (I) and hydrochloric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1.
  • this salt is also referred herein as “compound (I) hydrochloric acid”.
  • the present disclosure provides a maleic acid addition salt of compound (I).
  • the molar ratio between compound (I) and maleic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1 .
  • this salt is also referred herein as “compound (I) maleate”.
  • the present disclosure provides a semi-sulfate addition salt of compound (I).
  • the molar ratio between compound (I) and sulfuric acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5.
  • this salt is also referred herein as “1 :1 compound (I) semi-sulfate”.
  • the present disclosure provides a sulfate addition salt of compound (I).
  • the molar ratio between compound (I) and sulfuric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1 .
  • this salt is also referred herein as “compound (I) sulfate”.
  • the present disclosure provides a semi-edisylate addition salt of compound (I).
  • the molar ratio between compound (I) and ethane 1 ,2 sulfonic acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5.
  • this salt is also referred herein as “compound (I) semi- edisylate”.
  • the present disclosure provides a hydrobromic acid addition salt of compound (I).
  • the molar ratio between compound (I) and hydrobromic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1.
  • this salt is also referred herein as “compound (I) hydrobromic acid”.
  • the present disclosure provides a crystalline form of the free base of compound (I), having a crystalline Form G.
  • This crystalline form is also referred herein as compound (I) free base Form G.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention, in particular embodiments comprising at least one of a compound (I) hydrochloric acid salt, compound (I) maleate, compound (I) semisulfate, compound (I) sulfate, compound (I) semi-edisylate, compound (I) hydrobromic acid, or compound (I) free base Form G, and a pharmaceutically acceptable carrier or diluent.
  • the hydrochloric acid salt is at least partly crystalline.
  • said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven or eight peaks chosen from 4.2°, 8.4°, 9.0°, 14.9°, 18.0°, 23.0°, 23.8° and 25.4° ⁇ 0.2 in 20.
  • said crystalline hydrochloric acid salt is has Form A, having an X-ray powder diffraction pattern substantially similar to Figure 1 .
  • hydrochloric acid addition salt of compound (I) said hydrochloric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 3.
  • At least 90% by weight of the hydrochloric acid salt is crystalline Form A.
  • the X- ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
  • the hydrochloride acid addition is obtained or obtainable by a process comprising: combining compound (I) and hydrochloric acid in acetone; collecting said hydrochloric acid salt of compound (I).
  • the molar ratio between compound (I) and maleic acid is 1 :1.
  • the maleic acid salt is at least partly crystalline.
  • said crystalline maleic acid salt has crystalline Form B, having an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 9.7°, 10.7°, 12.2°, 16.5°, 23.5 and 25.4° ⁇ 0.2 in 20.
  • said crystalline maleic acid salt has Form B, having an X-ray powder diffraction pattern which comprises peaks at 9.7°, 10.7°, 12.2°, 16.5°, 23.5 and 25.4° ⁇ 0.2 in 20.
  • said crystalline maleic acid salt has Form B, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine, or ten peaks chosen from 8.1 °, 9.7°, 10.7°, 12.2°, 15.1 °, 16.5°, 17.7°, 19.6°, 20.5°, 21.2°, 22.4°, 23.5°, 23.9°, 25.4° and 30.0° ⁇ 0.2 in 20.
  • said crystalline maleic acid salt is Form B, having an X-ray powder diffraction pattern which comprises peaks at 8.1 °, 9.7°, 10.7°, 12.2°, 13.1 °, 15.1 °, 16.5°, 17.2°, 17.7°, 18.8°, 19.0°, 19.6°, 20.5°, 21.2°, 22.4°, 23.5°, 23.9°, 24.4°, 25.4°, 26.3°, 28.7°, 29.0° and 30.0° ⁇ 0.2 in 20.
  • said crystalline maleic acid salt is Form 1 , having an X-ray powder diffraction pattern substantially similar to Figure 4.
  • said maleic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 6.
  • said maleic acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 5.
  • the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
  • the maleic acid salt is obtained or obtainable by a process comprising: combining compound (I) and maleic acid in dioxane; collecting said maleic acid salt of compound (I).
  • the molar ratio between compound (I) and sulphuric acid is 1 :0.5.
  • the sulphuric acid salt is at least partly crystalline.
  • said crystalline sulphuric acid salt has crystalline Form C, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.8°, 9.6° and 20.8° ⁇ 0.2 in 20.
  • said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises peaks at 4.8°, 9.6° and 20.8° ⁇ 0.2 in 20.
  • said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises peaks at 4.8°, 6.0°, 9.6°, 10.3, 11.6°, 15.7°, 17.9°, 19.1 °, 19.9°, 20.8°, 21.6°, 21.8°, 22.4°, 23.4°, 24.0° and 25.7° ⁇ 0.2 in 20.
  • said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern substantially similar to Figure 7.
  • said sulphuric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 9.
  • thermogravimetric analysis (TGA) thermogram substantially similar to Figure 8.
  • At least 90% by weight of the sulphuric acid salt is crystalline Form C.
  • the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
  • the sulphuric acid salt is obtained or obtainable by a process comprising: combining compound (I) and sulphuric acid in ethanol; collecting said sulphuric acid salt of compound (I).
  • the sulphuric acid salt is at least partly crystalline.
  • said crystalline sulphuric acid salt has crystalline Form D, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.2°, 23.7°, and 25.4° ⁇ 0.2 in 20.
  • said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 23.7°, and 25.4° ⁇ 0.2 in 20.
  • said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises at least three, four, five, six or seven peaks chosen from 4.2°, 14.8°, 15.2°, 18.0°, 21 .0°, 23.7°, and 25.4° ⁇ 0.2 in 20.
  • said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern substantially similar to Figure 10.
  • said sulphuric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 12.
  • thermogravimetric analysis (TGA) thermogram substantially similar to Figure 11 .
  • At least 90% by weight of the sulphuric acid salt is crystalline Form D.
  • the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
  • the sulphuric acid salt is obtained or obtainable by a process comprising: combining compound (I) and sulphuric acid in acetone; collecting said sulphuric acid salt of compound (I).
  • Ethane 1 ,2-disulfonic acid addition salt of compound (I) In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), the molar ratio between compound (I) and ethane 1 ,2-disulfonic acid is 1 :0.5.
  • the ethane 1 ,2-disulfonic acid salt is at least partly crystalline.
  • said crystalline ethane 1 ,2-disulfonic acid salt has crystalline Form E, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.2°, 17.4° and 24.7° ⁇ 0.2 in 20.
  • said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 17.4° and 24.7° ⁇ 0.2 in 20.
  • said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises at least three, four or five peaks chosen from 4.2°, 14.9°, 17.4°, 21.3° and 24.7° ⁇ 0.2 in 20.
  • said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.9°, 15.5°, 17.4°, 21 .3° and 24.7° ⁇ 0.2 in 20.
  • said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern substantially similar to Figure 13.
  • said ethane 1 ,2-disulfonic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 15.
  • thermogravimetric analysis (TGA) thermogram substantially similar to Figure 14.
  • At least 90% by weight of the ethane 1 ,2-disulfonic acid salt is crystalline Form E.
  • the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
  • the ethane 1 ,2-disulfonic acid salt is obtained or obtainable by a process comprising: combining compound (I) and ethane 1 ,2-disulfonic acid in ethanol; collecting said ethane 1 ,2- disulfonic acid salt of compound (I).
  • Hydrobromic acid addition salt of compound (I) In preferred embodiments of the hydrobromic acid addition salt of compound (I), the molar ratio between compound (I) and hydrobromic acid is 1 :1 .
  • the hydrobromic acid salt is at least partly crystalline.
  • said crystalline hydrobromic acid salt has crystalline Form F, having an X-ray powder diffraction pattern which comprises at least three, four or five peaks chosen from 4.2°, 14.9°, 17.9°, 21.5°, 23.9°, 24.3° and 25.4° ⁇ 0.2 in 20.
  • said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.9°, 17.9°, 21 .5°, 23.9°, 24.3° and 25.5° ⁇ 0.2 in 20.
  • said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine, or ten peaks chosen from 4.2°, 8.4°, 9.0°, 9.8°, 12.6°, 14.9°, 15.4°, 16.3°, 17.3°, 17.9°, 19.9°, 20.7°, 21.5°, 23.0°, 23.9°, 24.3°, 25.5°, 26.4°, 27.1 ° and 27.9° ⁇ 0.2 in 20.
  • said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 8.4°, 9.0°, 9.8°, 12.6°, 14.9°, 15.4°, 16.3°, 17.3°, 17.9°, 18.6°, 19.9°, 20.7°, 21.5°, 23.0°, 23.9°, 24.3°, 25.5°, 26.4°, 27.1 °, 27.9°, 29.6° and 30.0° ⁇ 0.2 in 20.
  • said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern substantially similar to Figure 16.
  • said hydrobromic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 18.
  • thermogravimetric analysis (TGA) thermogram substantially similar to Figure 17.
  • At least 90% by weight of the hydrobromic acid salt is crystalline Form F.
  • the X- ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
  • the hydrobromic acid salt is obtained or obtainable by a process comprising: combining compound (I) and hydrobromic acid in acetone; collecting said hydrobromic acid salt of compound (I).
  • Freebase crystalline Form G of compound (I) In preferred embodiments ofthe freebase crystalline Form G of compound (I), said Form G has an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 4.2°, 9.3°, 22.0° and 25.2° ⁇ 0.2 in 20.
  • said Form G having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 9.3°, 22.0° and 25.2° ⁇ 0.2 in 20.
  • said Form G has an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven or eight peaks chosen from 4.2°, 9.3°, 13.0°, 13.5°, 16.4°, 22.0°, 24.7° and 25.2° ⁇ 0.2 in 20.
  • said Form G has an X-ray powder diffraction pattern which comprises peaks at 4.2°, 9.3°, 13.0°, 13.5°, 16.4°, 22.0°, 24.7° and 25.2° ⁇ 0.2 in 20.
  • said Form G has an X-ray powder diffraction pattern substantially similar to Figure 19.
  • said crystalline freebase form has a differential scanning calorimeter thermogram substantially similar to Figure 21 .
  • said Form G has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 20.
  • the freebase crystalline Form G of compound (I) at least 90% by weight of the freebase is crystalline Form G.
  • the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
  • the freebase crystalline Form G of compound (I) is obtained or obtainable by a process comprising: combining compound (I) to dioxane to form a mixture; collecting said freebase crystalline Form G of compound (I).
  • said Form O has an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from at least three or four peaks chosen from 5.9°, 6.9°, 9.9° and 11 .7° ⁇ 0.2 in 20.
  • said Form O has an X-ray powder diffraction pattern which comprises peaks at 5.9°, 6.9°, 9.9° and 11 .7° ⁇ 0.2 in 20.] In preferred embodiments ofthe freebase crystalline Form O of compound (I), said Form O has an X-ray powder diffraction pattern which comprises at least three, four, five or six peaks chosen from 5.9°, 6.2°, 6.9°, 9.9°, 11 .7° and 17.6° ⁇ 0.2 in 20.
  • said Form O having an X-ray powder diffraction pattern which comprises peaks at 5.9°, 6.2°, 6.9°, 9.9°, 11.7° and 17.6° ⁇ 0.2 in 20.
  • said Form O has an X-ray powder diffraction pattern substantially similar to Figure 22.
  • said crystalline freebase form has a differential scanning calorimeter thermogram substantially similar to Figure 24.
  • said Form O has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 23.
  • the compounds and the pharmaceutical compositions of the present invention are useful as inhibitors of tyrosine kinases, in particular of BTK.
  • compounds of this invention are useful as inhibitors of tyrosine kinases that are important in hyper-proliferative diseases, especially in cancer, such as a B-cell hematological malignancy.
  • a compound according to the invention or pharmaceutical composition according to the invention for use in therapy.
  • a compound according to the invention or pharmaceutical composition according to the invention for use in the treatment of Bruton’s Tyrosine Kinase (BTK) mediated disorders.
  • the Bruton’s Tyrosine Kinase (BTK) mediated disorder is selected from the group consisting of an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, a bone-related disease, and a hyperproliferative disease, such as cancer.
  • a compound according to the invention or pharmaceutical composition according to the invention for use in the treatment of cancer, lymphoma or leukemia.
  • a compound according to the invention or pharmaceutical composition according to the invention for use in the treatment of a disease selected from the group consisting of B-cell malignancy, B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocyte leukemia, non-Hodgkin lymphoma for example ABC-DLBCL, mantle cell lymphoma, follicular lymphoma, hairy cell leukemia B-cell non-Hodgkin lymphoma, Waldenstrom’s macroglobulinemia, Richter transformation, multiple myeloma, bone cancer, bone metastasis, chronic lymphocytic lymphomas, B-cell prolymphocyte leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma,
  • a compound according to the invention or pharmaceutical composition according to the invention for use in the treatment of a disease selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, deforming arthritis, osteoarthritis, traumatic arthritis, gouty arthritis, Reiter’s syndrome, polychondritis, acute synovitis and spondylitis, glomerulonephritis (with or without 40 nephrotic syndrome), autoimmune hematologic disorders, hemolytic anemia, aplasic anemia, idiopathic thrombocytopenia, and neutropenia, autoimmune gastritis, and autoimmune inflammatory bowel diseases, ulcerative colitis, Crohn’s disease, host versus graft disease, allograft rejection, chronic thyroiditis, Graves’ disease, schleroderma, diabetes (type I and type II), active hepatitis (acute and chronic), pancreatitis, primary billiary cirrhos
  • Behcet’s disease chronic renal insufficiency, Stevens-Johnson syndrome, inflammatory pain, idiopathic sprue, cachexia, sarcoidosis, Guillain-Barre syndrome, uveitis, conjunctivitis, kerato conjunctivitis, otitis media, periodontal disease, pulmonary interstitial fibrosis, asthma, bronchitis, rhinitis, sinusitis, pneumoconiosis, pulmonary insufficiency syndrome, pulmonary emphysema, pulmonary fibrosis, silicosis, chronic inflammatory pulmonary disease, and chronic obstructive pulmonary disease.
  • the hyperproliferative disease is a B-cell hematological malignancy.
  • the B-cell hematological malignancy is any one of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCB DLBCL), primary mediastinal B-cell lymphoma (PMBL), nonHodgkin lymphoma, Burkitt’s lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B-cell prolymphocytic leukemia, lym- phoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma
  • CLL
  • the B-cell hematological malignancy is mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B- cell lymphoma (DLBCL), Waldenstrom macroglobulinemia (WM), follicular lymphoma (FL) and marginal zone lymphoma (MZL).
  • MCL mantle cell lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • DLBCL diffuse large B- cell lymphoma
  • WM Waldenstrom macroglobulinemia
  • FL follicular lymphoma
  • MZL marginal zone lymphoma
  • the B-cell malignancy is Mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL).
  • composition/Formulation (Pharmaceutical composition)
  • a pharmaceutical composition comprising a salt of compound (I) according to the invention, or a freebase of compound (I) according to the invention, and a pharmaceutically acceptable carrier or a diluent.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection).
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be the VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD cosolvent system (VPD:5W) consists of VPD diluted 1 :1 with a 5% dextrose in water solution.
  • This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein stabilization may be employed.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • the invention is illustrated by the following examples.
  • Methyl (1 R,3/?)-3-aminocyclohexanecarboxylate hydrochloride (1.06 g, 5.47 mmol) was suspended in 10 mL water.
  • Sodium bicarbonate (1.38 g, 16.4 mmol) in 10 mL water was added followed by a drop-wise addition of a solution A/-(benzyloxycarbonyloxy)succinimide (1 .50 g, 6.01 mmol) in dioxane (30 mL).
  • the reaction mixture was stirred at room temperature o/n.
  • the mixture was diluted with ethyl acetate (50 mL) and water (50 mL) and the bi-phasic system was stirred 30 minutes at room temperature.
  • Triethylamine (10.4 mL, 74.62 mmol), 4-dimethylaminopyridine (605 mg, 4.95 mmol) and di-te/Y-butyl dicarbonate (13.5 g, 61.86 mmol) were added sequentially to a solution of 4- nitrobenzene sulfonamide (10 g, 49.46 mmol) in dichloromethane (100 mL).
  • the reaction mixture was stirred for 30 minutes at room temperature.
  • hydrochloric acid (1 N aqueous solution) until it becomes acidic.
  • the organic layer was separated and washed with saturated sodium chloride aqueous solution, dried over sodium sulfate, filtered and then concentrated under reduced pressure.
  • Lithium (E)-8-[3-[(1 R,3/?)-3-aminocyclohexyl]-1 -bromo-8-[(2,4-dimethoxyphenyl)- methylamino]imidazo[1 ,5-a]pyrazin-5-yl]oct-7-enoate (9.14 g, 15.07 mmol) was suspended dissolved in DMF (520 mL) and A/-ethylmorpholine (3.83 mL, 30.14 mmol) was added.
  • HT-XRPD patterns were obtained using the Ardena T2 high-throughput XRPD set-up.
  • the plates were mounted on a Bruker General Area Detector Diffraction System (GADDS) equipped with a VANTEC-500 gas area detector corrected for intensity and geometric variations.
  • GADDS General Area Detector Diffraction System
  • the calibration of the measurement accuracy (peaks position) was performed using NIST SRM1976 standard (Corundum).
  • Data collection was carried out at room temperature using monochromatic Cu Ka radiation in the 20 region between 1 .5° and 41 .5°, which is the most distinctive part of the XRPD pattern.
  • the diffraction pattern of each well was collected in two 20 ranges (1 .5° ⁇ 20 ⁇ 21 .5° for the first frame, and 19.5° ⁇ 20 ⁇ 41.5° for the second) with an exposure time of 90s for each frame. No background subtraction or curve smoothing was applied to the XRPD patterns.
  • the polarized light microscopy pictures were collected with a Leica DM 2500M optical microscope.
  • Mass loss due to solvent or water loss from the crystals was determined by TGA/DSC. Monitoring the sample weight, during heating in a TGA/DSC 3+ STARe system (Mettler-Toledo GmbH, Switzerland), resulted in a weight vs. Temperature curve and a heat flow signal.
  • the TGA/DSC 3+ was calibrated for temperature with samples of indium and aluminum. Samples (circa 2 mg) were weighed into 100 pL aluminum crucibles and sealed. The seals were pin- holed, and the crucibles heated in the TGA from 25 to 300 °C at a heating rate of 10 °C/min. Dry N2 gas was used for purging.
  • the gases coming from the TGA samples were analyzed by a mass spectrometer Omnistar GSD 301 T2 (Pfeiffer Vacuum GmbH, Germany).
  • the latter is a quadrupole mass spectrometer, which analyzes masses in the temperature range of 0-200 amu.
  • Detector 1 UV detector set at 240 nm
  • the compound integrity is expressed as a peak-area percentage, calculated from the area of each peak in the chromatogram, except the ‘injection peak’, and the total peak-area, as follows:
  • peak area peak area (%) - — - — - - — ⁇ 100% total area of all peaks
  • the peak area percentage of the compound of interest is employed as an indication of the purity of the component in the sample.
  • a UPLC-CAD method was used for the determination of the ratio of compound (I) and bromide, chloride and sulphate counterions in the respectively salts.
  • a calibration line was measured for bromide, chloride and sulphate, using a stock solution with a concentration of 0.1 mg/mL and 10 different injection volumes (from 1 pL to 5.5 pL in 0.5 pL steps).
  • the amount of compound (l)-salts used for the analysis was calculated, so that the concentration of the counterion would be about 0.05 mg/mL.
  • the ratio was then calculated using the expected area and the actual measured area considering the amount of water/solvent observed in TGMS.
  • the ratios determined using the UPLC-CAD method were all close to the expected ratios. The slight deviation that was observed was likely due to the hygroscopicity of the samples.
  • the salt screen experiments were carried out in 1 ,4-dioxane, acetone or ethanol.
  • the salt formation experiments were performed according to the “saturated solution” methodology for 1 ,4-dioxane and the solvent equilibrium method for acetone and ethanol.
  • the solids were separated from the liquid phases by centrifugation and analysed by HT-XRPD as vacuum-dried solids. Solvents from mother liquors were evaporated at ambient conditions and further dried under vacuum at 50°C. The obtained solids were also harvested and analysed by HT-XRPD.
  • PCT/EP2022/085765 was poorly crystalline and comprises a crystalline Form O, which is different from the freebase crystalline Form G, as obtained according to the invention.
  • Compound (l)-semi-sulfate salt (Form C) was physically stable after 2 days at 40°C/75% RH.
  • Compound (l)-sulfate salt (Form D) was physically stable for at least 2 days at 40°C/75% RH. Table 5. Peak list for solids of Compound (l)-sulfate salt (Form D).
  • Compound (l)-semi-edisylate salt (Form E) was physically stable for at least 2 days at 40°C/75% RH.
  • the HT-XRPD pattern of freebase crystalline Form G is shown in Figure 19 and the peak list is in Table 8.
  • Compound (I) freebase crystalline Form G was physically stable for at least 2 days at 40°C/75% RH.
  • the HT-XRPD pattern of freebase crystalline Form O is shown in Figure 22 and the peak list is in Table 9.
  • IMAP® assay (Molecular Devices) was used. Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4% DMSO in IMAP reaction buffer, which consists of 10 mM Tris- HCI, pH 15 7.5, 10 mM MgCL, 0.01 % Tween-20, 0.1 % NaNs and 1 mM freshly prepared dithiotreitol (DTT). Compound solution was mixed with an equal volume of full-length wt-BTK enzyme (Carna Biosciences, cat. no. 08-180) in IMAP reaction buffer.
  • DMSO dimethylsulfoxide
  • IMAP reaction buffer which consists of 10 mM Tris- HCI, pH 15 7.5, 10 mM MgCL, 0.01 % Tween-20, 0.1 % NaNs and 1 mM freshly prepared dithiotreitol (DTT).
  • DTT dithiotreitol
  • fluorescein-labeled MBP-derived substrate peptide (Molecular Devices, cat. no. RP 7123) was added, followed by ATP to start the reaction.
  • Final enzyme concentration was 1.2 nM, final substrate concentration 50 nM, and final ATP concentration was 4 pM.
  • the reaction was allowed to proceed for 2 hours at room temperature in the dark.
  • the reaction was stopped by quenching with IMAP progressive binding solution according to the protocol of the manufacturer (Molecular Devices). Fluorescence polarization was measured on an Envision multimode reader (Perkin Elmer, Waltham, MA, U.S.A.). ICso were calculated using XLfitTM5 software (ID Business Solutions, Ltd., Surrey, U.K.).
  • Compound (I) showed an IC50 value of ⁇ 5 nM.
  • IMAP® assay (Molecular Devices) was used. Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4 % DMSO in IMAP reaction buffer, which consists of 10 mM Tris-HCI, pH 15 7.5, 10 mM MgCh, 0.01 % Tween-20, 0.1 % NaNs and 1 mM freshly prepared dithiotreitol (DTT). Compound solution was mixed with an equal volume of full- length BTK C481 S enzyme (Carna Biosciences, cat. no. 08-547) in IMAP reaction buffer.
  • DMSO dimethylsulfoxide
  • IMAP reaction buffer which consists of 10 mM Tris-HCI, pH 15 7.5, 10 mM MgCh, 0.01 % Tween-20, 0.1 % NaNs and 1 mM freshly prepared dithiotreitol (DTT).
  • DTT dithiotreitol
  • REC-1 mantle cell lymphoma cells were purchased from American Type Culture Collection via Synthego Corporation (cat. no. CRL-3004, ATCC). Frozen stocks were thawed and cells were diluted in RPMI-1640 cell culture medium (cat. no. 61870036, Life Technologies), supplemented with 10% (v/v) fetal calf serum and 1 % penicillin/streptomycin. 3200 cells per well (in 45 pl) were seeded in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for 24 hours at 37 °C, 95 % humidity, and 5 % CO2.
  • Compound (I) showed an IC50 value of ⁇ 100 nM.
  • REC-1 cell lines expressing mutant BTK were created at Synthego Corporation.
  • a cell line expressing BTK T474I was generated via CRISPR/Cas9.
  • a clonal REC-1 BTK T474I cell line was obtained by single cell cloning.
  • the mutation status of BTK was confirmed via sequencing.
  • frozen cell stocks were thawed and cells were diluted in RPMI-1640 cell culture medium (cat. no. 61870036, Life Technologies), supplemented with 10% (v/v) fetal calf serum and 1 % penicillin/streptomycin. 3200 cells per well (in 45 pl) were seeded in a white 384-well culture plate (cat. no.
  • Compound (I) showed an IC50 value of ⁇ 100 nM.
  • Streptavidin-coated chips (Cat. No. BR100531), disposables and maintenance kits for Biacore were purchased from Cytiva (Eindhoven, The Netherlands).
  • Biotinylated wt-BTK enzym (Carna Biosciences, cat. no. 08-480-20N), BTK C481 S (Carna Biosciences, cat. no. 08-417- 20N), BTK T474I (Carna Biosciences, cat. no. 08-419-20N) or BTK T474S (Carna Biosciences, cat. no.
  • the kinetic constants of the compounds were determined with single cycle kinetics with five consecutive injections with an increasing compound concentration with ranges of 3.16 - 316 nM. Experiments were performed with an association time of 100 s per concentration and a dissociation time of 1200 s, except for compounds with a long target residence time, such as irreversible inhibitors, where dissociation time was increased. To circumvent problems of mass transport limitation, a flow rate of 30 pl/min was used. A blank run with the same conditions was performed before the compound was injected. The SPR sensorgrams were analyzed with Biacore Evaluation Software by using a method of double referencing. First the reference channel was subtracted from the channel containing immobilized protein. Subsequently, the reference curve obtained with buffer injections was subtracted.
  • the resulting curve was fitted with a 1 :1 binding model.
  • Compounds that bound according to an induced fit model were fitted with a two-state reaction model.
  • the kinetic constants (k a , kd, KD) of duplicates were geometrically averaged.
  • Compound (I) showed a KD (wt-BTK) value of ⁇ 5 nM.
  • Compound (I) showed a KD (BTK C481 S) value of ⁇ 5 nM.
  • Compound (I) showed a KD (BTK T474I) value of ⁇ 10 nM.
  • Compound (I) showed a KD (BTK T474S) value of ⁇ 5 nM.
  • wt-TMD8 diffuse large B-cell lymphoma cells were purchased from Tokyo Medical and Dental University and cultured in RPMI-1640 cell culture medium (cat. no. 61870036, Life Technologies), supplemented with 10% (v/v) heat-inactivated fetal bovine calf serum and 1 % penicillin/streptomycin. 1600 cells per well (in 45 pl) were seeded in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for at least 5 hours at 37 °C, 95 % humidity, and 5 % CO2.

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Abstract

Various salts and crystalline forms of macrocyclic compound (I) represented by the following structural formula, compound (I), and their corresponding pharmaceutical compositions, are disclosed. Particular crystalline forms of compound (I) hydrochloric acid, compound (I) maleate, compound (I) semi-sulfate, compound (I) sulfate, compound (I) semi-edisylate, and compound (I) hydrobromic acid, as well as a free base crystalline form are characterized by a variety of properties and physical measurements. Methods of preparing specific crystalline forms are also disclosed. The present disclosure also provides pharmaceutical compositions and methods of treating cancer via BTK inhibition, comprising said compounds.

Description

Salt and crystal forms of a macrocyclic BTK inhibitor
Field of the invention
The present disclosure relates to novel salts and crystalline forms of a macrocyclic Bruton’s Tyrosine Kinase (BTK) inhibitor, to pharmaceutical compositions comprising the crystalline forms, to methods of using the crystalline forms to treat conditions treatable by the inhibition of (mutant) BTK, such as B-cell malignancies, in particular B-cell lymphomas, even more particular marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), non-Hodgkin lymphoma, Burkitt lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), hairy cell leukemia, B- cell non-Hodgkin lymphoma, B-cell prolymphocytic leukemia, Waldenstrom’s macroglobulinemia (WM), multiple myeloma (MM), arthritis, in particular rheumatoid arthritis (RA), and multiple sclerosis (MS) and to processes useful in the preparation of the crystalline forms.
Background of the invention
Kinases are enzymes that transfer a phosphate group from ATP to a protein. Kinases play an important role in regulating cellular functions such as cell proliferation, subcellular translocation, apoptosis, inflammation and metabolism (Attwood M.M. et al (2021) Nat Rev Drug Discov). The human kinome is composed of over 500 kinases. The development of smallmolecule kinase inhibitors for the treatment of diverse types of cancer has proven successful in clinical therapy.
Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein kinases constituting a large subset of kinases, which play a central role in the regulation of a wide variety of cellular signaling processes. BTK plays a key role in B-cell receptor (BCR) signaling and a critical role in the regulation of survival, proliferation, activation and differentiation of B-lineage cells. Targeting of BTK with small molecule inhibitors such as the FDA approved covalent BTK inhibitors ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib has proven to be efficacious in several B cell malignancies including Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), Waldenstrom’s Macroglobulinemia (WM) and Small Lymphocytic Lymphoma SLL.
BTK is also expressed and plays also pro-tumorigenic roles in several solid tumors (Xianhui Wang et al. 2021). In prostate cancer cells BTK inhibition with ibrutinib or acalabrutinib inhibited cell growth (Kokabee et al 2015). Ibrutinib has also been shown to inhibit in vivo (xenograft) breast cancer cell growth (Wang et al., 2016) and inhibition of BTK with ibrutinib blocked gastric cancer cell growth (Wang et al., 2016). BTK inhibitors have also showed inhibition of cellular proliferation and migration, and induced apoptosis and autophagy in glioblastoma cell lines (Wei et al., 2016; Wang et al., 2017).
Where tumors have a strong oncogenic drive from one particular kinase, specific amino acid mutations have been observed as acquired resistance mechanisms of targeted drug molecules. These mutations may naturally occur in very low levels but become more prevalent upon drug treatment (pre-existing mutations), or these can also be produced by random mutation within tumors (acquired mutations) (Barouch-Bentov et al. 2011). Where such mutations have a clear advantage upon drug treatment (/.e. a mutation where the drug is no longer able to bind to the target) then resistance may rapidly develop and patients can relapse quickly. In these cases, the understanding of the impact and mechanism of these mutations has enabled additional drug discovery efforts to develop subsequent “generations” of drug molecules to target such resistance forms.
A drawback of the first generation BTK inhibitor, ibrutinib, is that drug resistance in B- cell malignancies can develop when BTK acquires mutations at the cysteine at position C481 of the kinase domain. This mutation abrogates the covalent binding of ibrutinib hampering its efficacy. Quinquenel et al. performed a ‘snapshot’ screening to determine the prevalence of resistance mutations and found that the presence of the BTK mutation was significantly associated with subsequent CLL progression. The correlation between disease progression, and the emergence and temporal dynamics of the most common resistance inducing C481 S BTK mutation have been determined for CLL patients receiving single-agent Ibrutinib treatment (Bodor et al. British Journal of Haematology 2021), Using digital droplet PCR the authors have been able to show that in 72.7% of the patients emergence of the BTK C481 S mutation preceded the symptoms of clinical relapse with a median of nine months. Woyach et al. showed that that detection of the C481 mutation in patients being treated with covalent BTK inhibitors is an indication for imminent relapse, usually within 12-18 months after detection (Woyach et al. (2017) J. Clin. Oncol. 35, 1437-1443). Since ibrutinib resistance confers poor survival, early detection of resistance provides clinically relevant information for the transition of affected patients to alternative treatment strategies.
Second-generation BTK inhibitors, which include acalabrutinib, zanubrutinib, and tirabrutinib, offer greater BTK selectivity and therefore limited off-target toxicity. These inhibitors, however, do not overcome resistance by C481 mutation. To treat patients with relapsed CLL having C481 BTK mutations non-covalent BTK inhibitors have been developed including LOXO- 305 (pirtobrutinib) and ARQ-531 (nemtabrutinib) and vecabrutinib. These agents do not require covalent binding to the BTK C481 residue and effectively inhibit both wild type and mutant BTK with C481 substitutions. In preclinical studies, noncovalent BTK inhibitors including pirtobrutinib (LOXO-305), ARQ-351 and vecabrutinib, inhibited B-cell-receptor signaling in BTK C481- mutant cell and animal models. Moreover, the phase 1 -2 clinical trial of pirtobrutinib showed promising efficacy for patients with B-cell cancer who had previously been treated with covalent BTK inhibitors (with 62% of patients with CLL having a response), including patients with or without BTK C481 mutations (with a response occurring in 71 % and 66% of the patients, respectively) (Wang et al. (2022) N. Engl. J. Med. 386, 735-43).
Pirtobrutinib was first disclosed in WO2017/103611 , ARQ-531 was disclosed in WO2017/111787 and vecabrutinib is disclosed in WO2013/185084. Further reversible BTK inhibitors are disclosed in WO2017/046604, , WQ2020/015735, WQ2020/239124, WQ2021/093839, WQ2020/043638, WO2013/067274, WO2018097234, WO2013/010380, W02016/161570, WO2016/161571 , WO2016/106624, WO2016/106625, WO2016/106626, WO2016106623, WO2016/106628 and WO2016/109222.
Despite the effective treatment of patients with a C481 mutation with pirtobrutinib new acquired mutations have emerged after treatment with this non-covalent inhibitor. Mutations were identified in these patients that were clustered in the kinase domain of BTK (V416L, A428D, M437R, T474I, and L528W) and that conferred resistance to pirtobrutinib and other non-covalent inhibitors.
The most well-known clinical documented BTK mutations for ibrutinib, acalabrutinib, zanubrutinib and pirtobrutinib are given in Figure 25.
PCT Patent Application No. PCT/EP2022/085765, the entire teachings of which are incorporated herein by reference, discloses macrocyclic inhibitors of Bruton’s tyrosine kinase (BTK). Said macrocyclic BTK inhibitors can, for example, be used to treat various cancer, such as CLL.
The structure of one of the BTK inhibitors disclosed in PCT Patent Application No. PCT/EP2022/085765, referred to herein as “Compound (I)” is shown below:
Figure imgf000004_0001
compound (I)
The compound (I) is one of the BTK inhibitors disclosed in PCT Patent Application No. PCT/EP2022/085765, also referred to as having subformula 184.
There is a need to develop new salt forms and/or solid crystalline forms of Compound (I) that are suitable for large scale manufacture and commercialization.
Summary of the invention
The present disclosure is directed to i) novel pharmaceutically acceptable salts of compound (I) (e.g., compound (I) hydrochloric acid, compound (I) maleate, compound (I) semisulfate, compound (I) sulfate, compound (I) semi-edisylate, compound (I) hydrobromic acid) including the corresponding solid forms; and ii) a novel solid form of the free base of compound (I) (hereinafter collectively referred to as “Salt or Solid Forms of the Disclosure”). In one aspect, the present disclosure provides a hydrochloric acid addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and hydrochloric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1. As noted above, this salt is also referred herein as “compound (I) hydrochloric acid” or as “hydrochloric acid addition salt of compound (I)”.
In another aspect, the present disclosure provides a maleic acid addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and maleic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1. As noted above, this salt is also referred herein as “compound (I) maleate” or as “maleic acid addition salt of compound (I)”.
In another aspect, the present disclosure provides a semi-sulfate addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and sulfuric acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5. As noted above, this salt is also referred herein as “compound (I) semi-sulfate” or as “1 :0.5 sulphuric acid addition salt of compound (I)”.
In another aspect, the present disclosure provides a sulfate addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and sulfuric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1 . As noted above, this salt is also referred herein as “compound (I) sulfate” or as “1 :1 sulphuric acid addition salt of compound (I)”.
In another aspect, the present disclosure provides a semi-edisylate addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and ethane 1 ,2 sulfonic acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5. As noted above, this salt is also referred herein as “compound (I) semi- edisylate” or as “1 :0.5 ethane 1 ,2 sulfonic acid addition salt of compound (I)”.
In another aspect, the present disclosure provides a hydrobromic acid addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and hydrobromic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1. As noted above, this salt is also referred herein as “compound (I) hydrobromic acid” or as “hydrobromic acid addition salt of compound (I)”.
In another aspect, the present disclosure provides a crystalline form of the free base of compound (I), having a crystalline Form G. This crystalline form is also referred herein as compound (I) free base Form G.
In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one of an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention, in particular embodiments comprising at least one of a compound (I) hydrochloric acid salt, compound (I) maleate, compound (I) semisulfate, compound (I) sulfate, compound (I) semi-edisylate, compound (I) hydrobromic acid, compound (I) free base Form G or compound (I) and a pharmaceutically acceptable carrier or diluent.
In another aspect is provided an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention or a pharmaceutical composition according to the invention for use as a medicament.
In another aspect is provided an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention or a pharmaceutical composition according to the invention for use in the treatment of Bruton’s Tyrosine Kinase (BTK) mediated disorders.
In another aspect is provided an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention or a pharmaceutical composition according to the invention for use in the treatment of Bruton’s Tyrosine Kinase (BTK) mediated disorders, wherein the Bruton’s Tyrosine Kinase (BTK) mediated disorder is selected from the group consisting of an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, a bone-related disease, and cancer.
In another aspect is provided an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention or a pharmaceutical composition according to the invention for use in treatment of cancer, lymphoma or leukemia.
In another aspect is provided a use of the compound according to the invention, for the manufacture of a medicament.
In another aspect is provided a method for treating of cancer in a subject in need thereof comprising administering to the subject the compound according to the invention in an amount effective to treat cancer.
In another aspect is provided a method for treating a subject suffering with a Bruton's Tyrosine Kinase (BTK) mediated disorder comprising administering to the subject the compound the invention or pharmaceutical composition according to the invention in an amount effective to treat the BTK mediated disorder.
Definitions
The term "pharmaceutical composition” as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.
The term "pharmaceutically acceptable” as used herein has its conventional meaning and refers to compounds, material, compositions and/or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.
The term "effective amount" as used herein, refers to an amount of the compound of the invention, and/or an additional therapeutic agent, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventive effect when administered to a subject suffering from a BTK-mediated disease or disorder.
A "subject" is a human or non-human mammal. In one embodiment, a subject is a human.
The term "controlling” is intended to refer to all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of the diseases and conditions affecting the mammal. However, "controlling” does not necessarily indicate a total elimination of all disease and condition symptoms, and is intended to include prophylactic treatment.
The term "excipient” as used herein has its conventional meaning and refers to a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing a granulate, solid or liquid oral dosage formulation.
The term "salt” as used herein has its conventional meaning and includes the acid addition and base salts of the compound of the invention.
The term "solvate” as used herein has its conventional meaning. One or more compounds of the invention or the pharmaceutically acceptable salts thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. "Solvate" means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Examples of suitable solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate wherein the solvent molecule is H2O and includes any hydrate of the compound or the salt of said compound.
The term "treatment” as used herein has its conventional meaning and refers to curative, disease controlling, palliative and prophylactic treatment.
The term "unit dosage form” has its conventional meaning and refers to a dosage form which has the capacity of being administered to a subject, preferably a human, to be effective, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising the therapeutic agent, i.e. the compound of the invention.
The term "BTK” as used herein has its conventional meaning and refers to Bruton's Tyrosine Kinase. Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein kinases which are a large subset of kinases which play a central role in the regulation of a wide variety of cellular signaling processes. BTK plays a key role in the B-cell receptor signaling and a critical role in the regulation of survival, proliferation, activation and differentiation of B- lineage cells. Targeting of BTK with small molecule inhibitors such as the FDA approved irreversible BTK inhibitors ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib has proven to be efficacious in several B-cell malignancies including Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), Waldenstrom’s Macroglobulinemia (WM) and Small Lymphocytic Lymphoma SLL. The term "BTK inhibitor” as used herein has its conventional meaning and refers to an inhibitor for BTK. A BTK inhibitor may be a small molecule inhibitor. Inhibitors may be irreversible inhibitors, such as by forming a covalent bond, and may be reversible inhibitors, which may form a reversible interaction with BTK.
The term “covalent BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor that reacts with its target protein (BTK) to form a covalent complex in which the protein has lost its function. Covalent inhibitors can be reversible or irreversible, depending on the rate of the reverse reaction. The terms 'covalent inhibitor' and 'irreversible inhibitor' are often used interchangeably.
The term "irreversible BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor that possesses an off-rate that is slow relative to the rate of re-synthesis of the target protein (BTK) in vivo, so that once the target protein is inhibited, it does not regain activity.
The term “rate of re-synthesis” as used herein has its conventional meaning and refers to the rate at which a cell and/or organism replaces a protein target with freshly synthesized functional protein. The re-synthesis rate defines the rate at which an irreversibly inhibited protein target will recover activity in vivo, once the inhibitor is no longer present.
The term "reversible BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor that inactivates the BTK enzyme through non-covalent, transcient, interactions. Unlike an irreversible inhibitor, a reversible inhibitor can dissociate from the enzyme.
The term "mutant-BTK” as used herein has its conventional meaning and refers to mutations of BTK. Mutations of BTK may be referred to by an altered or modified amino acid target (such as C as single-letter amino acid code for cysteine) at a certain position of the BTK structure (such as 481). Additionally, the amino acid substitution at the modification position may be referred to by an additional amino acid single-letter amino acid code, such as C481 S for serine substitution and C481T for threonine substitution of cysteine at the 481 position.
The term “modification" as used herein has its conventional meaning and refers to modification of a sequence of amino acids of a polypeptide, protein or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively.
The term "resistance to BTK inhibitor” as used herein has its conventional meaning and refers to a BTK inhibitor, which shows a reduction in effectiveness, after being effective initially in treating a hyperproliferative disease, in particular a B-cell hematological malignancy.
A drawback of the currently approved irreversible inhibitors is that patients treated with these inhibitors can develop drug resistance when BTK proteins with variations at the catalytic site are not able to bind efficiently to irreversible inhibitors in patients. This is a rather common event in patients treated with irreversible inhibitors and who experience relapse. A major mechanism for the acquired resistance is the emergence of BTK cysteine 481 (C481) mutations. These mutations hamper binding of irreversible inhibitors such as ibrutinib, acalabrutinib, zanubrutinib and tirabrutinib which form a covalent bond with this amino acid. Other mutations / modifications that can result in acquired resistance of irreversible covalent and/or reversible non- covalent BTK inhibitors are valine 416 (V416), alanine 428 (A428), methionine 437 (M437), threonine 474 (T474) and leucine 528 (L528) modifications, which can reduce BTK inhibitor binding to BTK.
By the term “drug resistance" as used herein is meant the major cause of cancer treatment failure. While a treatment may be effective initially, the heterogeneity of cancer and its ability to adapt can allow the cancer to become resistant to the treatment and regrow.
The term "relapse” as used herein has the conventional meaning and refers to evidence of disease progression in a patient who has previously achieved criteria of a complete response or partial remission.
The term ‘’disease progression” as used herein has the conventional meaning and refers to a measured increase in tumor size or tumor burden.
The term “ recurrent or refractory form of a hyperproliferative disease” as used herein has the conventional meaning and refers to a particular cancer that is resistant, or non-responsive to therapy with a particular therapeutic agent. A cancer can be refractory to therapy with a particular therapeutic agent either from the onset of treatment (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.
The term ‘’acquired drug resistance" as used herein has the conventional meaning and refers to resistance of a drug caused by mutations in the target protein. The mutations in the target protein hamper the binding of drug resulting in a regrowth of the tumor
The term ‘’clinical drug resistance” as used herein has the conventional meaning and refers to growth of a tumour while the patient is on treatment, that develops following after an initial clinical benefit (a clinical response or prolonged stable disease).
The term "wt-BTK” or “WT-BTK” or “BTKWT” as used herein has its conventional meaning and refers to wild-type Bruton’s Tyrosine Kinase. A wild-type BTK has the regular meaning of a phenotype of the typical form of BTK as it occurs in nature. Originally, the wild-type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele.
The term "macrocycle” as used herein has its conventional meaning and refers to a part of a molecule containing a ring consisting of 12 or more ring atoms forming said ring. In an example, a twelve membered ring consist of 12 atoms forming said ring.
The terms first, second, third and the like in the description and in the claims, are used for distinguishing between for example similar elements, compositions, constituents in a composition, or separate method steps, and not necessarily for describing a sequential or chronological order. The terms are interchangeable under appropriate circumstances and the embodiments of the invention can operate in other sequences than described or illustrated herein, unless specified otherwise.
Furthermore, the various embodiments, although referred to as “preferred” or “e.g.” or “for example” or “in particular” and the like are to be construed as exemplary manners in which the invention may be implemented rather than as limiting the scope of the invention.
The term “comprising”, used in the claims, should not be interpreted as being restricted to for example the elements or the method steps or the constituents of a compositions listed thereafter; it does not exclude other elements or method steps or constituents in a certain composition. It needs to be interpreted as specifying the presence of the stated features, integers, (method) steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a method comprising steps A and B” should not be limited to a method consisting only of steps A and B, rather with respect to the present invention, the only enumerated steps of the method are A and B, and further the claim should be interpreted as including equivalents of those method steps. Thus, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting only of components A and B, rather with respect to the present invention, the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components.
In addition, reference to an element or a component by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element or component are present, unless the context clearly requires that there is one and only one of the elements or components. The indefinite article "a" or "an" thus usually means "at least one".
Legend to the figures
Figure 1 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) hydrochloric acid Form A.
Figure 2 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) hydrochloric acid Form A.
Figure 3 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) hydrochloric acid Form A.
Figure 4 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) maleate Form B.
Figure 5 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) maleate Form B.
Figure 6 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) maleate Form B.
Figure 7 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :0.5 compound (I) semisulfate Form C. Figure 8 shows the Thermogravimetric Analysis (TGA) of 1 :0.5 compound (I) semisulfate Form C.
Figure 9 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :0.5 compound (I) semi-sulfate Form C.
Figure 10 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) sulfate Form D.
Figure 11 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) sulfate Form D.
Figure 12 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) sulfate Form D.
Figure 13 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :0.5 compound (I) semi-edisylate Form E.
Figure 14 shows the Thermogravimetric Analysis (TGA) of 1 :0.5 compound (I) semi- edisylate Form E.
Figure 15 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :0.5 compound (I) semi-edisylate Form E.
Figure 16 shows the X-ray Powder Diffraction (XRPD) pattern of 1 :1 compound (I) hydrobromic acid Form F.
Figure 17 shows the Thermogravimetric Analysis (TGA) of 1 :1 compound (I) hydrobromic acid Form F.
Figure 18 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of 1 :1 compound (I) hydrobromic acid Form F.
Figure 19 shows the X-ray Powder Diffraction (XRPD) pattern of compound (I) free base Form G.
Figure 20 shows the Thermogravimetric Analysis (TGA) of compound (I) free base Form G.
Figure 21 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of compound (I) free base Form G.
Figure 22 shows the X-ray Powder Diffraction (XRPD) pattern of compound (I) free base Form O.
Figure 23 shows the Thermogravimetric Analysis (TGA) of compound (I) free base Form O.
Figure 24 shows the Differential Scanning Calorimetry Analysis (DSC) thermogram of compound (I) free base Form O.
Figure 25 Map of clinically documented BTK mutants. Detailed description of the invention
In one aspect, the present disclosure provides a hydrochloric acid addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and hydrochloric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1. As noted above, this salt is also referred herein as “compound (I) hydrochloric acid”.
In another aspect, the present disclosure provides a maleic acid addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and maleic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1 . As noted above, this salt is also referred herein as “compound (I) maleate”.
In another aspect, the present disclosure provides a semi-sulfate addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and sulfuric acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5. As noted above, this salt is also referred herein as “1 :1 compound (I) semi-sulfate”.
In another aspect, the present disclosure provides a sulfate addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and sulfuric acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1 . As noted above, this salt is also referred herein as “compound (I) sulfate”.
In another aspect, the present disclosure provides a semi-edisylate addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and ethane 1 ,2 sulfonic acid is about 1 :0.5, in particular between 0.95 : 0.55 and 1.05 : 0.45, more preferably the molar ratio is 1 :0.5. As noted above, this salt is also referred herein as “compound (I) semi- edisylate”.
In another aspect, the present disclosure provides a hydrobromic acid addition salt of compound (I). In a preferred embodiment, the molar ratio between compound (I) and hydrobromic acid is about 1 :1 , in particular between 0.95 : 1 .05 and 1 .05 : 0.95, more preferably the molar ratio is 1 :1. As noted above, this salt is also referred herein as “compound (I) hydrobromic acid”.
In another aspect, the present disclosure provides a crystalline form of the free base of compound (I), having a crystalline Form G. This crystalline form is also referred herein as compound (I) free base Form G.
In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one of an addition salt of compound (I) according to the invention or a freebase of compound (I) according to the invention, in particular embodiments comprising at least one of a compound (I) hydrochloric acid salt, compound (I) maleate, compound (I) semisulfate, compound (I) sulfate, compound (I) semi-edisylate, compound (I) hydrobromic acid, or compound (I) free base Form G, and a pharmaceutically acceptable carrier or diluent.
Effective use of the compound (I) as BTK inhibitor has been experimentally demonstrated in several relevant assays, both for wt-BTK, as well as for mutant BTKs, such as BTK C481 S, BTK T474I and BTK T474S. Additionally, the exemplified addition salts of the compound of formula (I) according to the invention and other freebase forms of the compound of formula (I) have the same or similar effect.
Embodiments
Hydrochloric acid addition salt of compound (I)
In preferred embodiments of the hydrochloric acid addition salt of compound (I), the molar ratio between compound (I) and hydrochloric acid is 1 :1.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), the hydrochloric acid salt is at least partly crystalline.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), said crystalline hydrochloric acid salt has crystalline Form A, having an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 4.2°, 9.0°, 23.8° and 25.4° ± 0.2 in 20.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 9.0°, 23.8° and 25.4° ± 0.2 in 20.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven or eight peaks chosen from 4.2°, 8.4°, 9.0°, 14.9°, 18.0°, 23.0°, 23.8° and 25.4° ± 0.2 in 20.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 8.4°, 9.0°, 14.9°, 15.4°, 16.3°, 17.2°, 18.0°, 21.5°, 23.0°, 23.8° and 25.4° ± 0.2 in 20.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), said crystalline hydrochloric acid salt is has Form A, having an X-ray powder diffraction pattern substantially similar to Figure 1 .
In preferred embodiments of the hydrochloric acid addition salt of compound (I), said hydrochloric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 3.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), said hydrochloric acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 2.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), at least 90% by weight of the hydrochloric acid salt is crystalline Form A.
In preferred embodiments of the hydrochloric acid addition salt of compound (I), the X- ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH. In preferred embodiments of the hydrochloric acid addition salt of compound (I), the hydrochloride acid addition is obtained or obtainable by a process comprising: combining compound (I) and hydrochloric acid in acetone; collecting said hydrochloric acid salt of compound (I).
Maleic acid addition salt of compound (I)
In preferred embodiments of the maleic acid addition salt of compound (I), the molar ratio between compound (I) and maleic acid is 1 :1.
In preferred embodiments of the maleic acid addition salt of compound (I), the maleic acid salt is at least partly crystalline.
In preferred embodiments of the maleic acid addition salt of compound (I), said crystalline maleic acid salt has crystalline Form B, having an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 9.7°, 10.7°, 12.2°, 16.5°, 23.5 and 25.4° ± 0.2 in 20.
In preferred embodiments of the maleic acid addition salt of compound (I), said crystalline maleic acid salt has Form B, having an X-ray powder diffraction pattern which comprises peaks at 9.7°, 10.7°, 12.2°, 16.5°, 23.5 and 25.4° ± 0.2 in 20.
In preferred embodiments of the maleic acid addition salt of compound (I), said crystalline maleic acid salt has Form B, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine, or ten peaks chosen from 8.1 °, 9.7°, 10.7°, 12.2°, 15.1 °, 16.5°, 17.7°, 19.6°, 20.5°, 21.2°, 22.4°, 23.5°, 23.9°, 25.4° and 30.0° ± 0.2 in 20.
In preferred embodiments of the maleic acid addition salt of compound (I), said crystalline maleic acid salt is Form B, having an X-ray powder diffraction pattern which comprises peaks at 8.1 °, 9.7°, 10.7°, 12.2°, 13.1 °, 15.1 °, 16.5°, 17.2°, 17.7°, 18.8°, 19.0°, 19.6°, 20.5°, 21.2°, 22.4°, 23.5°, 23.9°, 24.4°, 25.4°, 26.3°, 28.7°, 29.0° and 30.0° ± 0.2 in 20.
In preferred embodiments of the maleic acid addition salt of compound (I), said crystalline maleic acid salt is Form 1 , having an X-ray powder diffraction pattern substantially similar to Figure 4.
In preferred embodiments of the maleic acid addition salt of compound (I), said maleic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 6.
In preferred embodiments of the maleic acid addition salt of compound (I), said maleic acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 5.
In preferred embodiments of the maleic acid addition salt of compound (I), at least 90% by weight of the maleic acid salt is crystalline Form B.
In preferred embodiments of the maleic acid addition salt of compound (I), the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH. In preferred embodiments of the maleic acid addition salt of compound (I), the maleic acid salt is obtained or obtainable by a process comprising: combining compound (I) and maleic acid in dioxane; collecting said maleic acid salt of compound (I).
Sulphuric acid addition salt of compound (I) - semi-sulfate
In preferred embodiments of the sulphuric acid addition salt of compound (I), the molar ratio between compound (I) and sulphuric acid is 1 :0.5.
In preferred embodiments of the sulphuric acid addition salt of compound (I), the sulphuric acid salt is at least partly crystalline.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has crystalline Form C, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.8°, 9.6° and 20.8° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises peaks at 4.8°, 9.6° and 20.8° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine, or ten peaks chosen from 4.8°, 6.0°, 9.6°, 10.3, 11.6°, 15.7°, 17.9°, 19.1 °, 19.9°, 20.8°, 21.6°, 21.8°, 22.4°, 23.4° and 24.0° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises peaks at 4.8°, 6.0°, 9.6°, 10.3, 11.6°, 15.7°, 17.9°, 19.1 °, 19.9°, 20.8°, 21.6°, 21.8°, 22.4°, 23.4°, 24.0° and 25.7° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern substantially similar to Figure 7.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said sulphuric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 9.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 8.
In preferred embodiments of the sulphuric acid addition salt of compound (I), at least 90% by weight of the sulphuric acid salt is crystalline Form C.
In preferred embodiments of the sulphuric acid addition salt of compound (I), the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH. In preferred embodiments of the sulphuric acid addition salt of compound (I), the sulphuric acid salt is obtained or obtainable by a process comprising: combining compound (I) and sulphuric acid in ethanol; collecting said sulphuric acid salt of compound (I).
Sulphuric acid addition salt of compound (I) -sulfate
In preferred embodiments of the sulphuric acid addition salt of compound (I), the sulphuric acid salt is at least partly crystalline.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has crystalline Form D, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.2°, 23.7°, and 25.4° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 23.7°, and 25.4° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises at least three, four, five, six or seven peaks chosen from 4.2°, 14.8°, 15.2°, 18.0°, 21 .0°, 23.7°, and 25.4° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.8°, 15.2°, 18.0°, 21.0°, 23.7°, and 25.4° ± 0.2 in 20.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern substantially similar to Figure 10.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said sulphuric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 12.
In preferred embodiments of the sulphuric acid addition salt of compound (I), said sulphuric acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 11 .
In preferred embodiments of the sulphuric acid addition salt of compound (I), at least 90% by weight of the sulphuric acid salt is crystalline Form D.
In preferred embodiments of the sulphuric acid addition salt of compound (I), the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
In preferred embodiments of the sulphuric acid addition salt of compound (I), the sulphuric acid salt is obtained or obtainable by a process comprising: combining compound (I) and sulphuric acid in acetone; collecting said sulphuric acid salt of compound (I).
Ethane 1 ,2-disulfonic acid addition salt of compound (I) In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), the molar ratio between compound (I) and ethane 1 ,2-disulfonic acid is 1 :0.5.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), the ethane 1 ,2-disulfonic acid salt is at least partly crystalline.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), said crystalline ethane 1 ,2-disulfonic acid salt has crystalline Form E, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.2°, 17.4° and 24.7° ± 0.2 in 20.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 17.4° and 24.7° ± 0.2 in 20.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises at least three, four or five peaks chosen from 4.2°, 14.9°, 17.4°, 21.3° and 24.7° ± 0.2 in 20.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.9°, 15.5°, 17.4°, 21 .3° and 24.7° ± 0.2 in 20.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern substantially similar to Figure 13.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), said ethane 1 ,2-disulfonic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 15.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), said ethane 1 ,2-disulfonic acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 14.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), at least 90% by weight of the ethane 1 ,2-disulfonic acid salt is crystalline Form E.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
In preferred embodiments of the ethane 1 ,2-disulfonic acid addition salt of compound (I), the ethane 1 ,2-disulfonic acid salt is obtained or obtainable by a process comprising: combining compound (I) and ethane 1 ,2-disulfonic acid in ethanol; collecting said ethane 1 ,2- disulfonic acid salt of compound (I).
Hydrobromic acid addition salt of compound (I) In preferred embodiments of the hydrobromic acid addition salt of compound (I), the molar ratio between compound (I) and hydrobromic acid is 1 :1 .
In preferred embodiments of the hydrobromic acid addition salt of compound (I), the hydrobromic acid salt is at least partly crystalline.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), said crystalline hydrobromic acid salt has crystalline Form F, having an X-ray powder diffraction pattern which comprises at least three, four or five peaks chosen from 4.2°, 14.9°, 17.9°, 21.5°, 23.9°, 24.3° and 25.4° ± 0.2 in 20.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.9°, 17.9°, 21 .5°, 23.9°, 24.3° and 25.5° ± 0.2 in 20.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine, or ten peaks chosen from 4.2°, 8.4°, 9.0°, 9.8°, 12.6°, 14.9°, 15.4°, 16.3°, 17.3°, 17.9°, 19.9°, 20.7°, 21.5°, 23.0°, 23.9°, 24.3°, 25.5°, 26.4°, 27.1 ° and 27.9° ± 0.2 in 20.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 8.4°, 9.0°, 9.8°, 12.6°, 14.9°, 15.4°, 16.3°, 17.3°, 17.9°, 18.6°, 19.9°, 20.7°, 21.5°, 23.0°, 23.9°, 24.3°, 25.5°, 26.4°, 27.1 °, 27.9°, 29.6° and 30.0° ± 0.2 in 20.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern substantially similar to Figure 16.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), said hydrobromic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 18.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), said hydrobromic acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 17.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), at least 90% by weight of the hydrobromic acid salt is crystalline Form F.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), the X- ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
In preferred embodiments of the hydrobromic acid addition salt of compound (I), the hydrobromic acid salt is obtained or obtainable by a process comprising: combining compound (I) and hydrobromic acid in acetone; collecting said hydrobromic acid salt of compound (I).
Freebase crystalline Form G of compound (I) In preferred embodiments ofthe freebase crystalline Form G of compound (I), said Form G has an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 4.2°, 9.3°, 22.0° and 25.2° ± 0.2 in 20.
In preferred embodiments ofthe freebase crystalline Form G of compound (I), said Form G, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 9.3°, 22.0° and 25.2° ± 0.2 in 20.]
In preferred embodiments ofthe freebase crystalline Form G of compound (I), said Form G has an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven or eight peaks chosen from 4.2°, 9.3°, 13.0°, 13.5°, 16.4°, 22.0°, 24.7° and 25.2° ± 0.2 in 20.
In preferred embodiments ofthe freebase crystalline Form G of compound (I), said Form G has an X-ray powder diffraction pattern which comprises peaks at 4.2°, 9.3°, 13.0°, 13.5°, 16.4°, 22.0°, 24.7° and 25.2° ± 0.2 in 20.
In preferred embodiments ofthe freebase crystalline Form G of compound (I), said Form G has an X-ray powder diffraction pattern substantially similar to Figure 19.
In preferred embodiments of the freebase crystalline Form G of compound (I), said crystalline freebase form has a differential scanning calorimeter thermogram substantially similar to Figure 21 .
In preferred embodiments ofthe freebase crystalline Form G of compound (I), said Form G has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 20.
In preferred embodiments of the freebase crystalline Form G of compound (I), at least 90% by weight of the freebase is crystalline Form G.
In preferred embodiments of the freebase crystalline Form G of compound (I), the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
In preferred embodiments of the freebase crystalline Form G of compound (I), the freebase crystalline Form G is obtained or obtainable by a process comprising: combining compound (I) to dioxane to form a mixture; collecting said freebase crystalline Form G of compound (I).
Freebase crystalline Form O of compound (I)
Properties of the original Freebase crystalline Form O of compound (I) are shown in Figures 22 - 24.
In preferred embodiments ofthe freebase crystalline Form O of compound (I), said Form O has an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from at least three or four peaks chosen from 5.9°, 6.9°, 9.9° and 11 .7° ± 0.2 in 20.
In preferred embodiments ofthe freebase crystalline Form O of compound (I), said Form O has an X-ray powder diffraction pattern which comprises peaks at 5.9°, 6.9°, 9.9° and 11 .7° ± 0.2 in 20.] In preferred embodiments ofthe freebase crystalline Form O of compound (I), said Form O has an X-ray powder diffraction pattern which comprises at least three, four, five or six peaks chosen from 5.9°, 6.2°, 6.9°, 9.9°, 11 .7° and 17.6° ± 0.2 in 20.
In preferred embodiments ofthe freebase crystalline Form O of compound (I), said Form O, having an X-ray powder diffraction pattern which comprises peaks at 5.9°, 6.2°, 6.9°, 9.9°, 11.7° and 17.6° ± 0.2 in 20.
In preferred embodiments ofthe freebase crystalline Form O of compound (I), said Form O has an X-ray powder diffraction pattern substantially similar to Figure 22.
In preferred embodiments of the freebase crystalline Form O of compound (I), said crystalline freebase form has a differential scanning calorimeter thermogram substantially similar to Figure 24.
In preferred embodiments ofthe freebase crystalline Form O of compound (I), said Form O has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 23.
Medical Use (methods of treatment)
The compounds and the pharmaceutical compositions of the present invention are useful as inhibitors of tyrosine kinases, in particular of BTK. In particular, compounds of this invention are useful as inhibitors of tyrosine kinases that are important in hyper-proliferative diseases, especially in cancer, such as a B-cell hematological malignancy.
In preferred embodiments, a compound according to the invention or pharmaceutical composition according to the invention, for use in therapy.
In preferred embodiments, a compound according to the invention or pharmaceutical composition according to the invention, for use in the treatment of Bruton’s Tyrosine Kinase (BTK) mediated disorders.
In preferred embodiments, the Bruton’s Tyrosine Kinase (BTK) mediated disorder is selected from the group consisting of an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, a bone-related disease, and a hyperproliferative disease, such as cancer.
In preferred embodiments, a compound according to the invention or pharmaceutical composition according to the invention, for use in the treatment of cancer, lymphoma or leukemia.
In preferred embodiments, a compound according to the invention or pharmaceutical composition according to the invention, for use in the treatment of a disease selected from the group consisting of B-cell malignancy, B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocyte leukemia, non-Hodgkin lymphoma for example ABC-DLBCL, mantle cell lymphoma, follicular lymphoma, hairy cell leukemia B-cell non-Hodgkin lymphoma, Waldenstrom’s macroglobulinemia, Richter transformation, multiple myeloma, bone cancer, bone metastasis, chronic lymphocytic lymphomas, B-cell prolymphocyte leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, lymphomatoid granulomatosis.
In preferred embodiments, a compound according to the invention or pharmaceutical composition according to the invention, for use in the treatment of a disease selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, deforming arthritis, osteoarthritis, traumatic arthritis, gouty arthritis, Reiter’s syndrome, polychondritis, acute synovitis and spondylitis, glomerulonephritis (with or without 40 nephrotic syndrome), autoimmune hematologic disorders, hemolytic anemia, aplasic anemia, idiopathic thrombocytopenia, and neutropenia, autoimmune gastritis, and autoimmune inflammatory bowel diseases, ulcerative colitis, Crohn’s disease, host versus graft disease, allograft rejection, chronic thyroiditis, Graves’ disease, schleroderma, diabetes (type I and type II), active hepatitis (acute and chronic), pancreatitis, primary billiary cirrhosis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosis, psoriasis, atopic dermatitis, contact dermatitis, eczema, skin sunburns, vasculitis (e.g. Behcet’s disease) chronic renal insufficiency, Stevens-Johnson syndrome, inflammatory pain, idiopathic sprue, cachexia, sarcoidosis, Guillain-Barre syndrome, uveitis, conjunctivitis, kerato conjunctivitis, otitis media, periodontal disease, pulmonary interstitial fibrosis, asthma, bronchitis, rhinitis, sinusitis, pneumoconiosis, pulmonary insufficiency syndrome, pulmonary emphysema, pulmonary fibrosis, silicosis, chronic inflammatory pulmonary disease, and chronic obstructive pulmonary disease.
In preferred embodiments, the hyperproliferative disease is a B-cell hematological malignancy.
In particular embodiments, the B-cell hematological malignancy is any one of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCB DLBCL), primary mediastinal B-cell lymphoma (PMBL), nonHodgkin lymphoma, Burkitt’s lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B-cell prolymphocytic leukemia, lym- phoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.
In preferred embodiments, the B-cell hematological malignancy is mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B- cell lymphoma (DLBCL), Waldenstrom macroglobulinemia (WM), follicular lymphoma (FL) and marginal zone lymphoma (MZL). In preferred embodiments, the B-cell malignancy is Mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL).
Composition/Formulation (Pharmaceutical composition)
In preferred embodiments, a pharmaceutical composition comprising a salt of compound (I) according to the invention, or a freebase of compound (I) according to the invention, and a pharmaceutically acceptable carrier or a diluent.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds can be formulated for parenteral administration by injection, e.g. bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD cosolvent system (VPD:5W) consists of VPD diluted 1 :1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. The invention is illustrated by the following examples.
ABBREVIATIONS
AAC Accelerated Aging Conditions (40°C/75% RH)
API Active Pharmaceutical Ingredient
Cl Counterion
Deliq Deliquescent
DSC Differential Scanning Calorimetry
DVS Dynamic Vapor Sorption
1H-NMR Proton Nuclear Magnetic Resonance
HT-XRPD High Throughput X-Ray Powder Diffraction
HR-XRPD High Resolution X-Ray Powder Diffraction
ML Mother liquor (liquid phases)
MS Mass Spectroscopy
Pc Poorly crystalline
RH Relative Humidity rT Room Temperature
TGMS Thermogravimetric Analysis coupled with Mass Spectroscopy
UPLC Ultra-Performance Liquid chromatography
CAD Charged Aerosol Detection
ACN Acetonitrile
DCM Dichloromethane
EtOH Ethanol
THF Tetra hydrofuran
EDY Ethane 1 ,2-disulfonic acid
HBr Hydrobromic acid
HCI Hydrochloric acid
MAE Maleic acid
SUL Sulfuric acid
SGF Simulated Gastric Fluid pH 1 .2
SIF Simulated Intestinal Fluid pH 6.8 Intermediate 1
Figure imgf000026_0001
enzy rom e
Benzyl (1 /?,5/?)-5-hydroxycyclohex-3-ene-1 -carboxylate
(a) (1 F?,4F?,5F?)-4-iodo-6-oxabicyclo[3.2.11octan-7-one
(R)-(+)-3-Cyclohexenecarboxylic acid (50.7 g, 402 mmol) was suspended in H2O (400 mL) under nitrogen. The mixture was cooled to 4 °C and sodium bicarbonate (101 .3 g, 1 .21 mol) was added, followed by a solution of potassium iodide (333 g, 2.01 mol) and iodine (107 g, 422 mmol) in H2O (400 mL). The reaction was allowed to come to room temperature and stirred o/n and then extracted with dichloromethane (4x100 mL). The combined organic layers were washed with sat. NaHSOs-solution (2x50 mL). The organic layer was protected from light, dried over Na2SO4, filtered and concentrated (20 mbar) to afford (1 R,4/?,5/?)-4-iodo-6- oxabicyclo[3.2.1]octan-7-one (90.1 g, 89.0 %) as an off-white solid. -6-oxabicyclo[3.2.11oct-3-en-7-one
Figure imgf000026_0002
(1 R,4/?,5/?)-4-iodo-6-oxabicyclo[3.2.1]octan-7-one (90.1 g, 358 mmol) was dissolved in dry THF (650 mL). Then, DBU (77 mL, 515 mmol) was added and the mixture was refluxed for 6 h. After cooling to room temperature, the suspension was filtered through Celite™, and concentrated in vacuo to -250 mL. This was used directly in the next step. -5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 1)
Figure imgf000026_0003
To the THF solution of (1 R,5/?)-6-oxabicyclo[3.2.1]oct-3-en-7-one (0.4 mol) in methanol (300 mL) was added 2M NaOH-solution (300 mL) and the mixture was stirred for 15 min at room temperature. The reaction was quenched by addition of 3M HCI-solution (300 mL) and the water layer was saturated by addition of sodium chloride. The mixture was extracted with ethyl acetate (3x100 mL). The combined organic phases were washed with brine, dried over sodium sulfate, filtered and the solvent removed in vacuo. The residue was dissolved in DMF (800 ml), cesium carbonate (129 g, 0.4 mol) and benzyl bromide (57 mL, 0.48 mol) were added subsequently. The mixture was stirred at room temperature for 30 min. The precipitate formed was filtered and the precipitate was washed with diethylether. The filtrate was washed with water, brine, dried over sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography (heptane/ethyl acetate = 95/5 to 45/55 v/v%) to give benzyl (1 R,5/?)-5- hydroxycyclohex-3-ene-1-carboxylate (57.1 g, 61.5% over 3 steps) as a cream-coloured oil. Intermediate 2
Figure imgf000027_0001
Ethyl (1 /?,5/?)-5-hydroxycyclohex-3-ene-1 -carboxylate
(a) (1 -4-iodo-6-oxabicyclo[3.2.11octan-7-one
Figure imgf000027_0002
(R)-(+)-3-Cyclohexenecarboxylic acid (50.7 g, 0.4 mol) was suspended in H2O (400 mL) under nitrogen. The reaction mixture was cooled to 4 °C and sodium bicarbonate (101 g, 1.2 mol) was added, followed by a solution of potassium iodide (333 g, 2 mol) and iodine (107 g, 0.42 mol) in H2O (400 mL). The reaction was allowed to come to room temperature and stirred o/n and then extracted with DCM (4x150 mL). The combined organic layers were washed with a solution of Na2S20s (120 g) in H2O (600 mL). The aqueous layer was extracted with DCM (2x150 mL). The combined organic layers were protected from light, dried over Na2SO4, filtered and concentrated (20 mbar) to afford (1 R,4/?,5/?)-4-iodo-6-oxabicyclo[3.2.1]octan-7-one (95.22 g, 94.5 %) as an off-white solid.
(b) (1 -6-oxabicyclo[3.2.11oct-3-en-7-one
(1 R,4/?,5/?)-4-iodo-6-oxabicyclo[3.2.1]octan-7-one (95.22 g, 377.9 mmol) was dissolved in dry THF (700 mL). Then, DBU (86.3 g, 566.9 mmol) was added and the mixture was refluxed o/n. The reaction mixture was cooled to room temperature, diluted with diethylether (500 mL) and extracted with aq. HCI (1 L, 1 M) and brine (250 mL). The aqueous layers were extracted with diethylether (2 x 480 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated (350 mbar to afford (1 R,5/?)-6-oxabicyclo[3.2.1]oct-3-en-7-one quantitatively as a yellowish oil which was used directly in the next step. -5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 2)
Figure imgf000027_0003
red solution of (1 R,5/?)-6-oxabicyclo[3.2.1]oct-3-en-7-one (377.9 mmol, theor.) in ethanol (750 mL) was added potassium carbonate (10.45 g, 75.6 mmol) at room temperature and the mixture stirred o/n. The reaction mixture was filtered through a Celite pad. Removal of ethanol under reduced pressure afforded the crude product that was purified by column chromatography plug filtration (eluent 40% EtOAc/heptane) to afford the title compound (41.38 g, 60.8% over 3 steps and column) as a yellow liquid. Intermediate 3
Figure imgf000028_0001
Methyl (1 /?,5/?)-5-hydroxycyclohex-3-ene-1 -carboxylate
(a) (1 -4-iodo-6-oxabicyclo[3.2.11octan-7-one
Figure imgf000028_0002
(R)-(+)-3-Cyclohexenecarboxylic acid (20.2 g, 160 mmol) was suspended in H2O (430 mL) under nitrogen. The reaction mixture was cooled to 0 °C and sodium bicarbonate (40.3 g, 480.3 mmol) was added, followed by a solution of potassium iodide (159.5 g, 961 mmol) and iodine (39.6 g, 168 mmol) in H2O (360 mL). The reaction was allowed to come to room temperature and stirred o/n and then extracted with DCM (3x150 mL). The combined organic layers were washed with a solution of Na2S20s (120 g) in H2O (600 mL). The aqueous layer was extracted with DCM (2x150 mL). The combined organic layers were protected from light, dried over Na2SO4, filtered and concentrated (20 mbar) to afford (1 R,4/?,5/?)-4-iodo-6- oxabicyclo[3.2.1]octan-7-one (37.88 g, 93.9 %) as an off-white solid.
(b) (1 -6-oxabicyclo[3.2.11oct-3-en-7-one
(1 R,4/?,5/?)-4-iodo-6-oxabicyclo[3.2.1]octan-7-one (37.88 g, 150.3 mmol) was dissolved in dry THF (750 mL). Then, DBU (34.3 g, 225.2 mmol) was added and the mixture was refluxed o/n. The reaction mixture was cooled to room temperature, diluted with diethylether (480 mL) and extracted with aq. HCI (1 L, 0.5 M) and brine (1 L). The aqueous layers were extracted with diethylether (2 x 480 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated (350 mbar to afford (1 R,5/?)-6-oxabicyclo[3.2.1]oct-3-en-7-one quantitatively as a yellowish oil which was used directly in the next step. -5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 3)
Figure imgf000028_0003
icarbonate (37.88 g, 0.451 mol) was added to a solution (1 R,5/?)-6- oxabicyclo[3.2.1]oct-3-en-7-one (150.3 mmol theor.) in anhydrous MeOH (300 mL). After stirring for 1 week at room temperature the solvent was removed in vacuo (40 °C/300 mbar). The residue was diluted with water (500 mL) and extracted with dichloromethane (3x250 mL). The combined extracts were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (21 .2 g, 90.3%) as a slightly coloured liquid. Intermediate 4 (Route A)
Figure imgf000029_0002
Figure imgf000029_0001
(1 /?,3/?)-3-(Benzyloxycarbonylamino)cvclohexanecarboxylic acid
(a) Methyl (1 /?,5S)-5-[bis(te/Y-butoxycarbonyl)amino1cyclohex-3-ene-1 -carboxylate
To an ice-cold (4 °C) solution of di-te/Y-butyl iminodicarboxylate (4.6 g, 21 .2 mmol), methyl (1 R,5/?)-5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 3) (3.31 g, 21.2 mmol) and triphenylphosphine (6.67 g, 25.4 mmol) in 2-MeTHF (180 mL) was added dropwise a solution of diisopropyl azodicarboxylate (6.26 mL, 31 .8 mmol) in 2-MeTHF (30 mL). The mixture was stirred for 30 min at 4 °C and then allowed to warm to room temperature and stirred for 3 h. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography using SiO2 and heptane/ethyl acetate = 3/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 5.68 g of methyl (1 R,5S)-5- [bis(te/Y-butoxycarbonyl)amino]cyclohex-3-ene-1 -carboxylate (yield 75.4 %).
(b) Methyl (1 F?,3F?)-3-[bis(te/Y-butoxycarbonyl)amino1cvclohexanecarboxylate
To a solution of methyl (1 /?,5S)-5-[bis(te/Y-butoxycarbonyl)amino]cyclohex-3-ene-1- carboxylate (2.48 g, 6.98 mmol) in ethanol (140 mL) was added 248 mg of 10% Pd/C. Catalytic hydrogenation was performed at room temperature for 16 h. The palladium-catalyst was filtered and the filtrate was concentrated in vacuo to give 2.6 g of the title compound (Yield: quantitative).
(c) Methyl (1 F?,3F?)-3-aminocvclohexanecarboxylate hydrochloride
To methyl (1 /?,3/?)-3-[bis(te/Y-butoxycarbonyl)amino]cyclohexanecarboxylate (2.6 g, 7.2 mmol) was added 4M HCI/dioxane solution (18 mL) and the mixture was stirred at room temperature o/n. The mixture was concentrated in vacuo to give 1 .06 g the title compound (yield: 76%).
(d) Methyl (1 F?,3F?)-3-(benzyloxycarbonylamino)cvclohexanecarboxylate
Methyl (1 R,3/?)-3-aminocyclohexanecarboxylate hydrochloride (1.06 g, 5.47 mmol) was suspended in 10 mL water. Sodium bicarbonate (1.38 g, 16.4 mmol) in 10 mL water was added followed by a drop-wise addition of a solution A/-(benzyloxycarbonyloxy)succinimide (1 .50 g, 6.01 mmol) in dioxane (30 mL). The reaction mixture was stirred at room temperature o/n. The mixture was diluted with ethyl acetate (50 mL) and water (50 mL) and the bi-phasic system was stirred 30 minutes at room temperature. The layers were separated and the water layer was extracted with ethyl acetate (2x20 mL). The combined organic layers were washed with water (50 mL), 0.5N aq. HCI-solution (50 mL), water (50 mL), 5% aq. NaHCOs-solution (50 mL), water (50 mL) and brine (25 mL), dried (Na2SO4), filtered and concentrated in vacuo to give 1.78 g of the title compound (yield: quantitative, crude).
(e) (1 f?,3f?)-3-(Benzyloxycarbonylamino)cvclohexanecarboxylic acid (Intermediate 4)
The crude product methyl (1 R,3/?)-3-(benzyloxycarbonylamino)cyclohexanecarboxy- late (1.45 g, 4.98 mmol) was dissolved in THF/dioxane/water = 4/1/1 v/v% (74 mL) and subsequently lithium hydroxide (358 mg, 14.9 mmol) was added. The mixture was stirred at room temperature o/n. Ethyl acetate (50 mL) and water (were added) and the pH of the mixture was adjusted to pH < 3 by addition of 2M HCI-solution. The organic phase was separated, washed with water, brine, dried over sodium sulfate , filtered and concentrated under reduced pressure to give 800 mg of (1 R,3/?)-3-(benzyloxycarbonylamino)cyclohexanecarboxylic acid (Intermediate 4) (yield:57.9%).
Intermediate 4 (Route B)
Figure imgf000030_0001
(1 -3-(Benzyloxycarbonylamino)cvclohexanecarboxylic acid
(a) tert-Butyl A/-(4-nitrophenyl)sulfonylcarbamate
Triethylamine (10.4 mL, 74.62 mmol), 4-dimethylaminopyridine (605 mg, 4.95 mmol) and di-te/Y-butyl dicarbonate (13.5 g, 61.86 mmol) were added sequentially to a solution of 4- nitrobenzene sulfonamide (10 g, 49.46 mmol) in dichloromethane (100 mL). The reaction mixture was stirred for 30 minutes at room temperature. To the reaction mixture was added hydrochloric acid (1 N aqueous solution) until it becomes acidic. The organic layer was separated and washed with saturated sodium chloride aqueous solution, dried over sodium sulfate, filtered and then concentrated under reduced pressure. The residue was dissolved in dichloromethane and purified by column chormatography over silica (heptane to ethyl acetate = 10/0 to 0/10) to give 13.09 g of the title compound (yield: 87.5%).
(b) Methyl (1 f?,5S)-5-[te/Y-butoxycarbonyl-(4-nitrophenyl)sulfonyl-amino1-cvclohex-3-ene-1 - carboxylate
To a cold (-20 °C) solution of methyl (1 R,5/?)-5-hydroxycyclohex-3-ene-1 -carboxylate (15 g, 96.0 mmol), te/Y-butyl A/-(4-nitrophenyl)sulfonylcarbamate (29.0 g, 96.0 mmol) and triphenylphosphine (27.7 g, 105.6 mmol) in THF (300 mL) was added dropwise a solution of diisopropyl azodicarboxylate (20.8 mL, 105.6 mmol) in THF (100 mL). The reaction mixture was concentrated under reduced pressure to give a residue which was purified by column chromatography (hexane/ethyl acetate = 85/15 v/v%) to give 36 g of the title compound, (yield: 85.1 %).
(c) Methyl (1 F?,5S)-5-(tert-butoxycarbonylamino)cyclohex-3-ene-1 -carboxylate
To a stirred solution of methyl (1 R,5S)-5-[te/Y-butoxycarbonyl-(4-nitrophenyl)sulfonyl- amino]-cyclohex-3-ene-1 -carboxylate (35.15 g, 79.8 mmol) in acetone (300 mL) was added DBU (23.85 mL, 159.6 mmol) and 2-mercaptoethanol (11.23 mL, 159.6 mmol). The reaction mixture was stirred for 3 h at room temperature. Acetone was removed under reduced pressure and the resulting residue was purified by column chromatography (hexane/ethyl acetate = 90/1 O to 88/12 v/v%) to give 14.1 g of the title compound (Yield: 69.2%) as a crystalline white solid.
(d) Methyl (1 F?,3F?)-3-(te/Y-butoxycarbonylamino)cvclohexanecarboxylate
To a solution of methyl (1 R,5S)-5-(tert-butoxycarbonylamino)cyclohex-3-ene-1- carboxylate (14.9 g, 58.36 mmol) in methanol (300 mL) was added 1 .5 g of 10% Pd/C. Catalytic hydrogenation was performed for 3 h at room temperature. The palladium-catalyst was filtered and the filtrate was concentrated in vacuo to afford the title compound in quantitative crude yield.
(e) Methyl (1 F?,3F?)-3-aminocvclohexanecarboxylate hydrochloride
To a cooled (4 °C) solution of methyl (1 /?,3R)-3-(te/Y-butoxycarbonylamino)cyclo- hexanecarboxylate (15.2 g, 58.36 mmol) in methanol (300 mL) was added drop-wise acetyl chloride (42 mL, 583.6 mmol). The reaction mixture was stirred for 1 h. The mixture was concentrated under reduced pressure and dried in vacuo to give the title compound in quantitative crude yield.
(f) Methyl (1 F?,3F?)-3-(benzyloxycarbonylamino)cvclohexanecarboxylate
To a cooled (4 °C) solution of methyl (1 R,3/?)-3-aminocyclohexanecarboxylate hydrochloride (58.36 mmol) in dioxane/water = 1/1 v/v% (200 mL) was added portion-wise sodium bicarbonate (14.7 g, 175 mmol). To the resulting suspension was added drop-wise a solution of A/-(benzyloxycarbonyloxy)succinimide (14.8 g, 59.02 mmol) in dioxane (150 mL) and the resulting mixture was stirred at room temperature o/w. LC-MS showed some starting material present. Additionally, 1 .5 g of Z-ONSu was added as a solution in dioxane. The mixture was stirred at room temperature o/n. Ethyl acetate was added and the resulting mixture was washed with 0.5M HCI solution, water and brine. The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure and dried in vacuo to give the title compound in quantitative crude yield.
(g) (1 R,3R)-3-(benzyloxycarbonylamino)cvclohexanecarboxylic acid (Intermediate 4)
To a solution of methyl (1 R,3R)-3-(benzyloxycarbonylamino)cyclohexanecarboxylate (58.36 mmol) in THF/water = 4/1 v/v% (375 mL) was added lithium hydroxide (4.21 g, 175 mmol) and the reaction mixture was stirred at room temperature o/n. Ethyl acetate (300 mL) and water (300 mL) were added to the mixture and the aqueous phase was separated. The ethyl acetate layer washed extracted with water (100 mL). The combined aqueous phases were washed with dichloromethane (100 mL) and acidified (pH < 2) by addition of 2M HCI-solution (90 mL). The water layer was extracted with ethyl acetate (3x250 mL). The combined ethyl acetate layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure and dried in vacuo to give 15.72 g of the title compound (Yield: 96.7% over 4
Figure imgf000032_0001
-3-(te/Y-butoxycarbonylamino)cvclohexanecarboxylic acid
Figure imgf000032_0002
(a) Ethyl (1 R,5S)-5-(1 ,3-dioxoisoindolin-2-yl)cyclohex-3-ene-1 -carboxylate
To an ice-cold (0 °C) solution of ethyl (1 R,5R)-5-hydroxycyclohex-3-ene-1 -carboxylate (Intermediate 2, 15.0 g, 88.13 mmol), phthalimide (14.26 g, 96.94 mmol) and triphenylphosphine (34.67 g, 132.2 mmol) in toluene (264 mL) was added dropwise diisopropyl azodicarboxylate (26.02 mL, 132.2 mmol) in 10 min. The reaction mixture was stirred at 0 °C for 30 min and then allowed to come to room temperature and stirred for 3 h. The mixture was evaporated under reduced pressure to give a yellow oil. Heptane/ethyl acetate = 7/3 v/v% (500 mL) was added and the mixture was heated to 70 °C. After cooling, the mixture was stirred for 72 h at room temperature. The solids were filtered, washed with heptane/ethyl acetate = 9/1 v/v% (2x50 mL) and the filtrate was evaporated under reduced pressure. The resulting residue was purified by column chromatography (heptane/ethyl acetate = 9/1 to 6/4 v/v%) to give 21 .96 g of the title compound (Yield: 83.0%) as an off-white solid.
(b) Ethyl (1 F?,3F?)-3-(1 ,3-dioxoisoindolin-2-yl)cvclohexanecarboxylate
To a solution of ethyl (1 R,5S)-5-(1 ,3-dioxoisoindolin-2-yl)cyclohex-3-ene-1 -carboxylate (27.73 g, 97.19 mmol) in methanol (975 mL) was added 2.7 g of 10% Pd/C. Catalytic hydrogenation was performed for 3 h at room temperature. The palladium-catalyst was filtered and the filtrate was concentrated in vacuo to afford 27.52 g the title compound (Yield: 94%).
(c) Ethyl (1 /?,3/?)-3-aminocvclohexanecarboxylafe
To a solution of ethyl (1 R,3/?)-3-(1 ,3-dioxoisoindolin-2-yl)cyclohexanecarboxylate (26.5 g, 87.95 mmol) in ethanol (440 mL) was added drop-wise hydrazine hydrate (64% in water, 4.68 mL, 96.74 mmol). The reaction mixture was stirred for 30 min. at room temperature and then refluxed for 3 h. Additional hydrazine hydrate (425 pL) was added and stirring under reflux was continued for 2 h. The mixture was concentrated under reduced pressure and dried in vacuo to give the title compound in quantitative crude yield.
(d) Ethyl (1 /?,3/?)-3-(te/Y-bufoxycarbonylamino)cvclohexanecarboxylafe
To a cold (0 °C) stirred suspension of ethyl (1 /?,3/?)-3-aminocyclohexanecarboxylate (91.27mmol, theoretical) in dichloromethane (456ml) was added portion-wise di-tert-butyl dicarbonate (21 .91 g, 100.4 mmol). The reaction mixture was stirred for 15 min at 0 °C, then allowed to come to room temperature. The mixture was washed with cold 0.5N NaOH-solution, water and brine. The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography (heptane/ethyl acetate = 8/2 to 6/4 v/v%) to give 20.5 g of the title compound (Yield: 83.0% over two steps) as an off-white solid.
(e) (1 /?,3/?)-3-(te/Y-bufoxycarbonylamino)cvclohexanecarboxylic acid
To a solution of ethyl (1 /?,3R)-3-(te/Y-butoxycarbonylamino)cyclohexanecarboxylate (20.5 g, 75.56 mmol) in THF (300 mL) was added a solution of lithium hydroxide (1.8 g, 75.56 mmol) in water (150 mL) and the reaction mixture was stirred at room temperature o/n. Additional lithium hydroxide (0.9 g) was added and stirring was continued for 24 h at room temperature. The mixture was acidified (pH < 2) by addition of 1 M HCI-solution (131 mL). The water layer was separated and extracted with dichloromethane (2x100 mL). The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure and dried in vacuo to give 16.81 g (91 %) of (1 /?,3R)-3-(te/Y-butoxycarbonylamino)cyclo- hexanecarboxylic acid (Intermediate 5).
Figure imgf000033_0001
(6-Bromo-3-chloro-pyrazin-2-yl)methanamine hydrochloride
(a) 3-Amino-6-bromo-pyrazine-2-carbonitrile
To a solution of commercially available 2-amino-3,5-dibromo pyrazine (25.12 g, 99.3 mmol) in DMF (160 mL) was added sodium cyanide (4.97 g, 101.3 mmol) and copper(l)cyanide (9.07 g, 101 .3 mmol) at room temperature. The mixture was stirred at 95 °C for 32 h. After cooling the mixture was and poured in a thoroughly stirred mixture of 5% sodium bicabonate solution (1 L) and ethyl acetate (500 mL) to give a black suspension. The mixture was filtered over Decalite®, flushed with warm ethyl acetate (2 x 150 mL). The organic layer from the biphasic filtrate was separated. The waterlayer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give 15.6 g of the crude title compound (yield: 78.9%)
(b) 6-Bromo-3-chloro-pyrazine-2-carbonitrile
To a solution of 3-amino-6-bromo-pyrazine-2-carbonitrile (15.6 g, 78.4 mmol) in acetonitrile (350 mL) was added copper(ll)chloride (12.65 g, 94.1 mmol) and the mixture was heated to 60 °C and stirred for 15 min. A solution of tert-butyl nitrite (1 1.75 mL, 98 mmol) in acetonitrile (50 mL) was added dropwise at 60 °C to the reaction mixture. The mixture was stirred at 60 °C o/n. After cooling, the mixture was filtered over Decalite®, washed with acetonitrile. The filtrate was added dropwise to a 1 N HCI-solution (250 mL) and ethyl acetate (250 mL) and stirred for 10 min. The layers were separated and the waterlayer was extracted with ethyl acetate (2x 100 mL). The combined organic layers were washed with 1 N-HCl-solution, water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography (plug filtration) using SiO2 and heptane/ethyl acetate = 95/5 to 9/1 to 8/2 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 10.4 g of the title compound (yield 60.7%).
(c) (6-Bromo-3-chloro-pyrazin-2-yl)methanamine hydrochloride
To a solution of 6-bromo-3-chloro-pyrazine-2-carbonitrile (10.4 g, 47.62 mmol) in acetic acid (160 mL) was added under nitrogen of Raney-Nickel (50% slurry in water, 1 .96 mL, -16.7 mmol). The resulting mixture was shaken in a Parr-vessel under 4 bar hydrogen at room temperature o/n. Raney-Nickel was removed by filtration over Decalite® and the filtrate was concentrated under reduced pressure and co-evaporated with toluene. The remaining green solid (12.6 g) was suspended in ethyl acetate at 50 °C and filtered. The filtrate was cooled on an ice-bath, 2N HCIgas in diethyl ether (48 mL) was added dropwise and the resulting suspension was stirred at room temperature o/n. The solids formed were collected by filtration, washed with diethyl ether and dried under reduced pressure at 40 °C to give 6.21 g (50%) of (6-bromo-3- chloro-pyrazin-2-yl)methanamine hydrochloride (Intermediate 6).
Intermediate 7
Figure imgf000035_0001
,
Ethyl (E)-8-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)oct-7-enoate (Intermediate 7)
(a) Ethyl oct-7-ynoate
To a solution of oct-7-ynoic acid (7.92g, 56.53 mmol) in ethanol (70 ml) was added conc.- H2SO4 (0.2 mL). The reaction mixture was stirred for 5 h. at 70 °C. After cooling of the mixture, MTBE (150 mL) was added and the organic phase was washed with sat. aq. NaHCOs-solution, water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give 8.84 g of ethyl oct-7-ynoate (yield: 93%).
(b) Ethyl (E)-8-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)oct-7-enoate (Intermediate 7)
To a cold (5 °C) oven-dried round bottom flask equipped with a magnetic stirring bar were added ethyl oct-7-ynoate (9.11 g, 54.16 mmol), Schwartz’s reagent (1.39 g, 5.41 mmol), EtaN (753 pL, 5.41 mmol) and pinacolborane (8.24 mL, 56.86 mmol), under an inert nitrogen atmosphere. The bottom flask was then sealed and the mixture was stirred at 60 °C for 5 hours. The reaction was allowed to cool to room temperature, diluted with dichloromethane, passed through a pad of silica gel and concentrated under reduced pressure at room temperature. The crude mixture was purified by column chromatography using SiO2 and hexane/ethyl acetate = 99/1 to 9/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 6.42 g of the title compound (yield: 40%).
Intermediate 8
Figure imgf000035_0002
A/-(4-Cyano-2-pyridyl)-4-(4,4,5,5-tetramethyl-1 , 3, 2-dioxaborolan-2-yl) benzamide (I ntermed iate 8)
(a) 4-(4,4,5,5-Tetramethyl-1 , 3, 2-dioxaborolan-2-yl) benzoyl chloride
To a cold (0 °C) solution of 4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzoic acid (24.8 g, 100 mmol) in dichloromethane (300 mL) was added a catalytic amount of DMF. A solution of oxalyl chloride (12.9 mL, 150 mmol) was added dropwise. After stirring for 60 min at 0 °C, the reaction mixture was allowed to warm to room temperature and the mixture was stirred o/n. The reaction mixture was concentrated to give 26.33 g of crude 4-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)benzoyl chloride (yield: 99%).
(b) A/-(4-Cyano-2-pyridyl)-4-(4,4,5,5-tetramethyl-1 , 3, 2-dioxaborolan-2-yl) benzamide (Intermediate 8)
To a cold (0 °C) solution of 4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzoyl chloride (26.0 g, 100 mmol) in acetonitrile (300 mL) was subsequently added 2-aminopyridine- 4-carbonitrile (14.29 g, 120 mmol) and 4-DMAP (14.66 g, 120 mmol) The mixture was stirred under nitrogen atmosphere at 0°C and allowed to warm to room temperature overnight. The reaction mixture was concentrated in vacuo. The crude oily solids were then dissolved in dichloromethane (300 mL) and washed with 5% citric acid (3x, 300 mL), 5% NaHCOs (2x300 mL) and brine (200 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue triturated in acetonitrile/water = 1/1 v/v% (300 mL). The solids were filtrated and dried under reduced pressure to give 20.5 g of 4-(4,4,5,5-tetramethyl- 1 ,3,2-dioxaborolan-2-yl)-A/-[4-(trifluoromethyl)-2-pyridyl]benzamide (Intermediate 8) (yield: 69%) as off-white solids.
Example A
Figure imgf000036_0001
Example A (compound (I))
(a) te/Y-Butyl A/-[(1 /?,3/?)-3-[(6-bromo-3-chloro-pyrazin-2-yl)methylcarbamoyl1cvclohexyl1- carbamate
To a cold (4 °C) suspension of (1 /?,3R)-3-(te/Y-butoxycarbonylamino)cyclohexane- carboxylic acid (Intermediate 5, 10.36 g, 42.62 mmol) and (6-bromo-3-chloro-pyrazin-2- yl)methanamine hydrochloride (intermediate 6, 10.51 g, 40.59 mmol) in dichloromethane (406 mL) was added subsequently, DiPEA (21 .21 mL, 121 .77 mmol) and HATU (16.2 g, 42.62 mmol). The resulting mixture was stirred at room temperature o/n. The reaction mixture was filtered over a Buechner filter. The filtrate was washed with 5% aq. NaHCOs-solution (100 mL), 5% aq. citric acid solution (3x100 mL), brine (100 mL), dried over sodium sulfate filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate = 8/2 to 3/7 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 17.4 g of the title compound (Yield: 94%).
(b) Ethyl (E)-8-[6-[[[(1 f?,3f?)-3-(te/Y-butoxycarbonylamino)cvclohexanecarbonyl1amino1methyl1- 5-chloro-pyrazin-2-yl1oct-7-enoate
A mixture of te/Y-butyl A/-[(1 R,3R)-3-[(6-bromo-3-chloro-pyrazin-2-yl)methylcarbamoyl]- cyclohexyl]carbamate (17.4 g, 38.86 mmol), ethyl (E)-8-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan- 2-yl)oct-7-enoate (Intermediate 7, 1 1.51 g, 38.86 mmol) and potassium carbonate (16.11 g, 116.6 mmol) in dioxane/water = 4/1 v/v% (200 mL) was degassed with nitrogen for 5 min at 30 °C. Pd(dppf)Cl2.CH2Cl2 (1.58 g, 1 .94 mmol) was added and the mixture was again degassed with nitrogen for 5 min at 30 °C. The reaction mixture was stirred at 80 °C for 4 h. After cooling ethyl acetate was added and the mixture was stirred for 15 min. The mixture was filtered over Decalite™ and the filtrate was washed with water and brine. The organic layer was separated, dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate = 7/3 to 3/7 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 20.9 g of the title compound (Yield: quantitative).
(c) Ethyl (E)-8-[3-[(1 -3-(te/Y-butoxycarbonylamino)cvclohexyl1-8-chloro-imidazo[1 ,5-
Figure imgf000037_0001
al py razi n -5-v II o ct-7-e n o ate
To a cold (-10 °C) solution of ethyl (E)-8-[6-[[[(1 R,3R)-3-(te/Y-butoxycarbonylamino)- cyclohexanecarbonyl]amino]methyl]-5-chloro-pyrazin-2-yl]oct-7-enoate (20.9 g, 38.9 mmol) in acetonitrile/DMF = 9/1 v/v% (117 mL) was added subsequently pyridine (12.58 mL, 155.7 mmol) and phosphorus oxychloride (7.25 mL, 77.84 mmol) and the mixture was stirred for 30 min. at 5 °C. The mixture was added carefully to 25% ammonia (200 mL) and crushed ice (400 mL) keeping the temperature below 0 °C. Ethyl acetate (200 mL) was added and the resulting mixture was stirred for 30 min. The water layer was separated and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate and concentrated in vacuo to give 19.4 g of the title compound (yield: 96%). (d) Ethyl (E)-8-[1-bromo-3-[(1 /?,3/?)-3-(te/Y-butoxycarbonylamino)cvclohexyl1-8-chloro-imidazo- [1 ,5-a1pyrazin-5-yl1oct-7-enoate
/V-Bromosuccinimide (7.31 g, 41.1 mmol) was added to a cold (0 °C) solution of ethyl (E)-8-[3-[(1 /?,3/?)-3-(te/Y-butoxycarbonylamino)cyclohexyl]-8-chloro-imidazo[1 ,5-a]pyrazin-5- yl]oct-7-enoate (19.4 g, 37.37 mmol) in acetonitrile (149 mL). The reaction mixture was stirred for 3 h. allowing the temperature to come to room temperature. The mixture was quenched with 10% aq. Na2S2O4-solution/5% aq. NaHCOs-solution/brine and ethyl acetate. The phases were separated and the water layer was extracted with ethyl acetate. The combined organic phases were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate = 9/1 to 6/4 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 17.07 g of the title compound (Yield: 74%).
(e) Ethyl (E)-8-[1-bromo-3-[(1 -3-(tert-butoxycarbonylamino)cvclohexyl1-8-[(2,4-
Figure imgf000038_0001
dimethoxyphenyl)methylamino1imidazo[1 ,5-a1pyrazin-5-yl1oct-7-enoate
To a suspension of ethyl (E)-8-[1-bromo-3-[(1 /?,3R)-3-(te/Y-butoxycarbonylamino)- cyclohexyl]-8-chloro-imidazo-[1 ,5-a]pyrazin-5-yl]oct-7-enoate (17.07 g, 28.55 mmol) in 1 -butanol (114 mL) was added 2,4-dimethoxybenzylamine (12.86 mL, 85.65 mmol) and the mixture was stirred at 110 °C for 2 h. The mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate/water 5/2 v/v% (700 mL). The organic layers was separated and washed with 5% NaHCOs-solution (100 mL) and brine (2x50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate = 85/15 to 6/4 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 18.9 g of the title compound (Yield: 91 %).
(f) Ethyl (E)-8-[3-[(1 /?,3/?)-3-aminocvclohexyl1-1-bromo-8-[(2,4-dimethoxyphenyl)methylamino1- imidazo[1 ,5-a1pyrazin-5-yl1oct-7-enoate
Ethyl (E)-8-[1 -bromo-3-[(1 /?,3/?)-3-(tert-butoxycarbonylamino)cyclohexyl]-8-[(2,4-dime- thoxyphenyl)methylamino]imidazo[1 ,5-a]pyrazin-5-yl]oct-7-enoate (18.85 g, 25.86 mmol) was dissolved in dichloromethane (129 mL). Trifluoroacetic acid (19.2 mL) was added and the mixture was stirred at room temperature for 2 h. The mixture was poured into dichloromethane/water = 1/4 v/v% (750 mL), the water layer was separated and extracted with dichloromethane (100 mL). The combined organic layers were washed with sat. aq. NaHCOs- solution (100 mL), brine (50 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give 16.68 g of the title compound (Yield: 102%).
(g) Lithium (E)-8-[3-[(1 f?,3f?)-3-aminocvclohexyl1-1 -bromo-8-[(2,4-dimethoxyphenyl)methyl- amino1imidazo[1 ,5-a1pyrazin-5-yl1oct-7-enoate
To a solution ethyl (E)-8-[3-[(1 /?,3/?)-3-aminocyclohexyl]-1-bromo-8-[(2,4-dimethoxy- phenyl)methylamino]-imidazo[1 ,5-a]pyrazin-5-yl]oct-7-enoate (16.68 g, 25.86 mmol) in THF/water=4/1 v/v% (129 mL) was lithium hydroxide (648 mg, 27.15 mmol) and the mixture was stirred at room temperature for 72 h. Water (300 mL) was added to the mixture and after evaporation of THF, the mixture was lyophilized to give 15.9 g of the title compound (yield: quantitative crude).
(h) Macrocyclic Intermediate 9
Lithium (E)-8-[3-[(1 R,3/?)-3-aminocyclohexyl]-1 -bromo-8-[(2,4-dimethoxyphenyl)- methylamino]imidazo[1 ,5-a]pyrazin-5-yl]oct-7-enoate (9.14 g, 15.07 mmol) was suspended dissolved in DMF (520 mL) and A/-ethylmorpholine (3.83 mL, 30.14 mmol) was added. This solution was added slowly to a heavily stirred suspension of HATU (17.18 g, 45.21 mmol) and A/-ethylmorpholine (5.74 mL, 45.21 mmol) in ethyl acetate (1 .0 L) over a 11 h period to obtain a clear solution. The mixture was poured into stirred cold water (2.0 L) and stirring was continued for 15 min. The water layer was separated and extracted with ethyl acetate/heptane = 95/5 v/v% (3x300 mL). The combined organic layer was washed with washed with 2.5% aq. citric acid solution (300 mL), sat. aq. NaHCOs-solution (300 mL), water (300 mL) and brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using SiO2 and heptane/ethyl acetate/methanol = 50/50/0 to 0/92.5/7.5 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 7.39 g of the DMB-protected Macrocyclic Intermediate 9 (Yield: 84%).
(i) Example A : compound (I)
DMB-protected Macrocyclic Intermediate 9 (9.83 g, 16.87 mmol) was dissolved in TFA (67 mL) and the mixture was stirred at 60 °C for 4 h. The reaction mixture was added slowly to a mixture of dichloromethane/water = 1/1 v/v% (1.0 L). The bi-phasic mixture was stirred for 10 min. The layers were separated and the water layer was extracted with dichloromethane (200 mL). To the combined organic layer was added slowly under stirring 12.5% NH4OH (300 mL). The layers were separated and the organic layer was washed with 12.5% NH4OH (150 mL), water (250 mL) and brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was triturated with hot (90 °C) acetonitrile (100 mL), filtered and dried to give 5.9 g (yield: 80%) of the deprotected Macrocyclic Intermediate 9 which was used directly in the next step.
Deprotected Macrocyclic Intermediate 9 (2.25 g, 5.2 mmol) was dissolved in dioxane/water = 9/1 v/v% (100 mL) and potassium carbonate (2.15 g, 15.6 mmol) was added. The solution was purged with nitrogen for 5 min and A/-(4-cyano-2-pyridyl)-4-(4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzamide (Intermediate 8) (2.18 g, 6.24 mmol) and Pd(dppf)Cl2.CH2Cl2 (424 mg, 0.52 mmol) were added. The reaction mixture was stirred for 4 h at 90 °C. The reaction mixture was diluted with dichloromethane/methanol = 9/1 v/v% (400 mL) after cooling to room temperature and filtered over Decalite™. The filtrate was washed with water, and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using SiO2 and dichloromethane/methanol = 98/2 to 94/6 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 1 .6 g of the title compound (Yield: 53%). Data: LCMS (B) Rt : 6.99 min; m/z 575.4 [M+H]+.
ANALYTICAL METHODS
High Throughput X-Ray Powder Diffraction
HT-XRPD patterns were obtained using the Ardena T2 high-throughput XRPD set-up. The plates were mounted on a Bruker General Area Detector Diffraction System (GADDS) equipped with a VANTEC-500 gas area detector corrected for intensity and geometric variations. The calibration of the measurement accuracy (peaks position) was performed using NIST SRM1976 standard (Corundum).
Data collection was carried out at room temperature using monochromatic Cu Ka radiation in the 20 region between 1 .5° and 41 .5°, which is the most distinctive part of the XRPD pattern. The diffraction pattern of each well was collected in two 20 ranges (1 .5°< 20 < 21 .5° for the first frame, and 19.5°< 20 < 41.5° for the second) with an exposure time of 90s for each frame. No background subtraction or curve smoothing was applied to the XRPD patterns.
High Resolution X-Ray Powder Diffraction
Before transferring into the capillary, the analysed solids were grounded in agate mortar till all of the visible crystals disappeared and material became a fine powder. The HR-XRPD data were collected on D8 Advance diffractometer using Cu Ka1 radiation (1.54056 A) with germanium monochromator at RT. Diffraction data were collected in the 2D range 2.15 - 41.5°. Detector scan on solid state LynxEye detector was performed using 0.0157° per step with 6 sec/step scan speed. The samples were measured in 8 mm long glass capillary with 0.7 mm outer diameter.
Calculation
Cell parameters as well as crystal system were obtained using LSI-lndex (Coelho, 2003; Coelho & Kern, 2005) indexing program.
For Rietveld analyses cell parameters, crystal system as well as atom positions were taken from the single crystal data (cif file). During the refinement the following parameters were refined:
- cell constants;
- background;
- instrument geometry;
- zero shift;
- absorption
Neither atoms positions nor thermal motion parameters were refined during whole process.
The following criteria of fit were used:
• Yo m and Yc,m are the observed and calculated data, respectively at data point m, • /W the number of data points,
• P the number of parameters,
• wm the weighting given to data point m which for counting statistics is given by wm=1/o(Yo,m)2 where o(Yo,m) is the error in Yo,m,
Figure imgf000041_0001
Polarized Light Microscopy
The polarized light microscopy pictures were collected with a Leica DM 2500M optical microscope.
Thermal analysis
TGA/SDTA and TGMS analysis
Mass loss due to solvent or water loss from the crystals was determined by TGA/DSC. Monitoring the sample weight, during heating in a TGA/DSC 3+ STARe system (Mettler-Toledo GmbH, Switzerland), resulted in a weight vs. Temperature curve and a heat flow signal. The TGA/DSC 3+ was calibrated for temperature with samples of indium and aluminum. Samples (circa 2 mg) were weighed into 100 pL aluminum crucibles and sealed. The seals were pin- holed, and the crucibles heated in the TGA from 25 to 300 °C at a heating rate of 10 °C/min. Dry N2 gas was used for purging.
The gases coming from the TGA samples were analyzed by a mass spectrometer Omnistar GSD 301 T2 (Pfeiffer Vacuum GmbH, Germany). The latter is a quadrupole mass spectrometer, which analyzes masses in the temperature range of 0-200 amu.
DSC analysis
Thermal events were obtained from DSC thermograms, recorded with a heat flux DSC3+ STARe system (Mettler-Toledo GmbH, Switzerland). The DSC3+ was calibrated for temperature and enthalpy with a small piece of indium (m.p. = 156.6 °C; 8Hf = 28.45 J/g) and zinc (m.p. = 419.6 °C; 8Hf = 107.5 J/g). Samples (circa 2 mg) were sealed in standard 40 pL aluminum pans, pin-holed and heated in the DSC from 25 °C to 300 °C, at a heating rate of 10 °C/min. Dry N2 gas, at a flow rate of 50 mL/min was used to purge the DSC equipment during measurement.
UPLC analytical methods
Purity (UPLC-MS)
Method name: S23018_01_LCMS
UPLC System: UPLC: Agilent 1290
Detector 1 : UV detector set at 240 nm
Detector 2: MSD XT in Negative Scan Mode
UPLC Conditions:
Auto sampler temp.: RT
Column: Agilent Eclipse Plus C18 HD (50 x 2.1 mm; 1.8pm)
Column temp: 40 °C
Mobile phase A: 10 mM ammonium acetate in water
Mobile phase B: Acetonitrile
Flow: 0.6 mL/min
Gradient: Time [min]: Eluent A: Eluent B:
0 95% 5%
0.1 95% 5%
2.5 5% 95%
2.55 5% 95%
2.56 95% 5%
3.5 95% 5%
Run time: 3.5 minutes
Sample:
Concentration: ca. 0.35 mg/mL
Solvent: Water:Acetonitrile (25:75 v/v) + 0.1% formic acid
Injection volume: 1 pL
Retention time: 1.78 min. S23018 - V1 m/z 573.4 [M-H]-
The compound integrity is expressed as a peak-area percentage, calculated from the area of each peak in the chromatogram, except the ‘injection peak’, and the total peak-area, as follows:
. peak area peak area (%) = - — - — - - — ■ 100% total area of all peaks
The peak area percentage of the compound of interest is employed as an indication of the purity of the component in the sample.
UPLC-CAD
A UPLC-CAD method was used for the determination of the ratio of compound (I) and bromide, chloride and sulphate counterions in the respectively salts.
A calibration line was measured for bromide, chloride and sulphate, using a stock solution with a concentration of 0.1 mg/mL and 10 different injection volumes (from 1 pL to 5.5 pL in 0.5 pL steps). The amount of compound (l)-salts used for the analysis was calculated, so that the concentration of the counterion would be about 0.05 mg/mL. The ratio was then calculated using the expected area and the actual measured area considering the amount of water/solvent observed in TGMS. The ratios determined using the UPLC-CAD method were all close to the expected ratios. The slight deviation that was observed was likely due to the hygroscopicity of the samples.
1H-NMR spectroscopy
1H-NMR spectroscopy in DMSO-de was used for compound integrity characterization. The spectra were recorded at room temperature on a 400 MHz instrument (Bruker BioSpin GmbH) using standard pulse sequences. The data was processed with ACD Labs software Spectrus Processor 2021 .2.2 (Advanced Chemistry Development Inc. Canada).
1H-NMR spectroscopy was used for the determination of the ratio of compound (I) and maleate and edisylate counterions in the respectively salts.
Example B: Salt screen
The salt screen experiments were carried out in 1 ,4-dioxane, acetone or ethanol. The salt formation experiments were performed according to the “saturated solution” methodology for 1 ,4-dioxane and the solvent equilibrium method for acetone and ethanol.
For the saturated solution method, compound (I) saturated solutions were prepared at room temperature in 1 ,4-dioxane (421 mg in 17 mL) and counterions (Cl) were added (from a 1 M stock solution of the Cl) in an compound (l):CI ratio of 1 :1 for acids with two ionization sites. The mixtures were subjected to a temperature profile including 3 heating-cooling cycles between 5 - 50°C and final aging at 5°C for 3 days.
For the solvent equilibrium method compound (I) suspensions with counterions in an compound (l):CI ratio of 1 :1 and 1 :0.5 (added from a 1 M stock solution of the Cl) were prepared in acetone and ethanol. The mixtures were subjected to thermocycling where the temperature profile includes 3 heating-cooling cycles between 5 - 50°C and final aging at 25°C for 3 days.
After the temperature profile, the solids were separated from the liquid phases by centrifugation and analysed by HT-XRPD as vacuum-dried solids. Solvents from mother liquors were evaporated at ambient conditions and further dried under vacuum at 50°C. The obtained solids were also harvested and analysed by HT-XRPD.
All solids were exposed to accelerated aging conditions (AAC, 40°C/75% RH) for 2 days and re-measured by HT-XRPD to test their physical stability.
RESULTS SELECTION STABLE SALTS AND SOLID CRYSTALLINE FREEBASE FORMS
Table 1. Overview and classification of the physical properties in relation to the developability of salts identified in this study. Analytical characterization was performed by TGMS (mass loss, % / solvation state), DSC (thermal events), UPLC-CAD (API:CI ratio) and XH-NMR (API:CI ratio).
Figure imgf000045_0001
The compound (I) as originally obtained from synthesis as described in
PCT/EP2022/085765 was poorly crystalline and comprises a crystalline Form O, which is different from the freebase crystalline Form G, as obtained according to the invention.
Characterization Compound (l)-HCI salt Form A
The HT-XRPD pattern of Compound (l)-hydrochloric acid salt (Form A) is shown in Figure 1 and the peak list is in Table 2. Compound (l)-hydrochloric acid salt (Form A) was physically stable for at least 2 days at 40°C/75% RH.
Table 2. Peak list for solids of Compound (l)-HCI salt (Form A).
Figure imgf000045_0002
Figure imgf000046_0001
The UPLC-CAD analysis was performed to assess the salt stoichiometric ratio. An Compound (I) : HCI ratio of 1 :1 was determined.
The TGA analysis of Compound (I)- hydrochloric acid salt (Form A) is shown in Figure 2 and showed a mass loss of 6.9%) between 25 and 170°C due to water (corresponding to 2.5 molecules of water per API molecule. Compound (I)- hydrochloric acid salt (Form A) appeared to be a hydrated phase.
The DSC trace of Compound (I)- hydrochloric acid salt (Form A) is shown in Figure 3 and showed a very broad endothermic event at Tpeak 110.4°C.
Characterization Compound (l)-maleate salt Form B
The HT-XRPD pattern of Compound (l)-maleate salt (Form B) is shown in Figure 4 and the peak list is in Table 3. Compound (l)-maleate salt (Form B) was physically stable for at least 2 days at
40°C/75% RH.
Table 3. Peak list for solids of Compound (l)-maleate salt (Form B).
Figure imgf000046_0002
Figure imgf000047_0001
The 1H-NMR spectrum of Compound (l)-maleate salt (Form B) showed Compound (I)- maleate salt (Form B) resonances were shifted compared to those observed in the spectrum of the free base and therefore, salt formation was confirmed. The chemical shift of the maleate anion appeared at 6.15 ppm, overlapping with the signal of a Compound (I) proton. The ratio Compound (I) : maleic acid was 1 :1.
The TGA analysis of Compound (l)-maleate salt (Form B) (Figure 5) showed a mass loss of 4.9% between 25 and 160°C due to water (corresponding to 1.7 molecules of water per API molecule). Thermal decomposition probably started after 160°C. Compound (l)-maleate salt (Form B) appeared to be a hydrated phase.
The DSC trace of Compound (l)-maleate salt (Form B) (Figure 6) showed two broad overlapping endothermic events at Tpeak 62.0 and 105.0°C. Small endo/exothermic events were recorded at Tpeak 176.8 and 180.1 °C. Characterization Compound (l)-semi-sulfate salt Form C
The HT-XRPD pattern of Compound (l)-semi-sulfate salt (Form C) is shown in Figure 7 and the peak list is in Table 4.
Compound (l)-semi-sulfate salt (Form C) was physically stable after 2 days at 40°C/75% RH.
Table 4. Peak list for solids of Compound (l)-semi-sulfate salt (Form C).
Figure imgf000048_0001
The UPLC-CAD analysis was performed to assess the salt stoichiometric ratio. An API:SUL ratio of 1 :0.4 was determined. The TGA analysis of Compound (l)-semi-sulfate salt (Form C) (Figure 8) showed a mass loss of 4.5% of water and/or ethanol between 25 and 180°C. According to 1H-NMR 0.08 molecules of ethanol was present (corresponding to a mass loss of 0.6% in TGMS). This means that 3.9% of the total mass loss was due to water (corresponding to 1 .3 molecules of water per API molecule). No thermal decomposition was observed before 200°C. Compound (l)-semi- sulfate salt (Form C) appeared to be a hydrated phase.
The DSC trace of Compound (l)-semi-sulfate salt (Form C) (Figure 9) showed a broad endothermic event at Tpeak 75.5°C, a small endothermic event at Tpeak 210.1 °C and a large endothermic event at Tpeak 314.7°C. Characterization Compound (l)-sulfate salt Form D
The HT-XRPD pattern of Compound (l)-sulfate salt (Form D) is shown in Figure 10 and the peak list is in Table 5.
Compound (l)-sulfate salt (Form D) was physically stable for at least 2 days at 40°C/75% RH. Table 5. Peak list for solids of Compound (l)-sulfate salt (Form D).
Figure imgf000049_0001
The UPLC-CAD analysis was performed to assess the salt stoichiometric ratio. An Compound (I) : sulfate ratio of 1 :0.9 was determined. The TGA analysis of Compound (l)-sulfate salt (Form D) (Figure 11) showed a mass loss of 4.9% between 25 and 170°C due to water (corresponding to 1.9 molecules of water per API molecule). No thermal decomposition was observed before 200°C. Compound (l)-sulfate salt (Form D) appeared to be a hydrated phase.
The DSC trace of Compound (l)-sulfate salt (Form D) (Figure 12) showed a very broad endothermic event at Tpeak 102.8°C, and very small endo/exothermic events at Tpeak 214.6 and 222.6°C. After 250°C decomposition occurred.
Characterization Compound (l)-semi-edisylate salt (Form E)
The HT-XRPD pattern of Compound (l)-semi-edisylate salt (Form E) is shown in Figure 13 and the peak list is in Table 6.
Compound (l)-semi-edisylate salt (Form E) was physically stable for at least 2 days at 40°C/75% RH.
Table 6. Peak list for solids of Compound (l)-semi-edisylate salt (Form E).
Figure imgf000050_0001
The 1 H-NMR spectrum of Compound (l)-semi-edisylate salt (Form E) showed Compound (I) resonances were shifted compared to those observed in the spectrum of the free base, suggesting that salt formation occurred. The chemical shift of the ethane-1 ,2-disulfonate anion appeared at 2.65 ppm. The ratio Compound (I) : edisylate was 1 :0.5. A trace of 1 ,4-dioxane was detected at chemical shift 3.57 ppm.
The TGA analysis of Compound (l)-semi-edisylate (Figure 14) showed a mass loss of 1.4% between 25 and 190°C due to water (corresponding to 0.6 molecules of water per Compound (I) molecule). Compound (l)-semi-edisylate (Form E) appeared to be a hydrated phase.
The DSC trace of Compound (l)-semi-edisylate (Form E) (Figure 15) showed broad endothermic events at Tpeak 89.3 and 132.2°C, in correspondence of the mass loss observed in the TGMS. A final endothermic event was recorded at Tpeak 261 .1 °C.
Characterization Compound (I)- hydrobromic acid salt Form F The HT-XRPD pattern of Compound (I)- hydrobromic acid salt (Form F) is shown in Figure 16 and the peak list is in Table 7.
Compound (I)- hydrobromic acid salt (Form F) was physically stable for at least 2 days at 40°C/75% RH. Table 7. Peak list for solids of Compound (l)-HBr salt (Form F).
Figure imgf000051_0001
I
The UPLC-CAD analysis was performed to assess the salt stoichiometric ratio. An Compound (I) : HBr salt ratio of 1 :1 was determined.
The TGA analysis of Compound (I)- hydrobromic acid salt (Form F) (Figure 17) showed a mass loss of 19.0% between 25 and 170°C due to water (corresponding to 8.5 molecules of water per Compound (I) molecule). No thermal decomposition was observed before 200°C. Compound (I)- hydrobromic acid salt (Form F) appeared to be a hydrated phase.
The DSC trace of Compound (I)- hydrobromic acid salt (Form F) (Figure 18) showed a single very broad endothermic event at Tpeak 1 12.9°C. Characterization Compound (I) freebase crystalline Form G
The HT-XRPD pattern of freebase crystalline Form G is shown in Figure 19 and the peak list is in Table 8.
Compound (I) freebase crystalline Form G was physically stable for at least 2 days at 40°C/75% RH.
Table 8. Peak list for solids of Compound (I) freebase crystalline Form G.
Figure imgf000052_0001
The TGA analysis of Compound (I) freebase crystalline Form G (Figure 20) showed a mass loss of 4.6% between 30 and 200°C, due to the loss of 1 ,4-dioxane and/or water based on the MS signal. According to 1H-NMR, about 0.06 molecules of 1 ,4-dioxane per API molecule were present (corresponding to a weight loss of 0.9% in TGMS). This means that 3.7% of the total mass loss was due to the water (corresponding to 1.2 molecules of water per Compound (I) molecule). No thermal decomposition was observed before 200°C. Compound (I) freebase crystalline Form G appeared to be a hydrated phase. The DSC trace of Compound (I) freebase crystalline Form G (Figure 21) showed two broad overlapping endothermic events at Tpeak 64.8 and 135.6 °C, and showed a endothermic point at Tpeak 263.9°C. No decomposition was observed.
Characterization of reference Compound (I) freebase crystalline Form O
The HT-XRPD pattern of freebase crystalline Form O is shown in Figure 22 and the peak list is in Table 9.
Table 9. Peak list for solids of Compound (I) freebase crystalline Form O.
Figure imgf000053_0001
The TGA analysis of Compound (I) freebase crystalline Form O (Figure 23) showed a mass loss of 3.0% between 25 and 200°C which could be attributed to the loss of water and/or residual process solvents, such as DCM (3.0% corresponds to 1 molecule of water per API molecule and to 0.2 molecules of DCM per Compound (I) molecule). NMR detected only traces of DCM. Compound (I) freebase crystalline Form O could be a monohydrate.
The DSC trace of Compound (I) freebase crystalline Form O (Figure 24) showed a very broad endothermic peak at Tpeak 86.1 °C (Tonset 30°C) and a endothermic point at Tpeak 182.4°C.
Example C
Biochemical kinase assay wt-BTK
To determine the inhibitory activity of compounds on wt-BTK enzyme activity, the IMAP® assay (Molecular Devices) was used. Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4% DMSO in IMAP reaction buffer, which consists of 10 mM Tris- HCI, pH 15 7.5, 10 mM MgCL, 0.01 % Tween-20, 0.1 % NaNs and 1 mM freshly prepared dithiotreitol (DTT). Compound solution was mixed with an equal volume of full-length wt-BTK enzyme (Carna Biosciences, cat. no. 08-180) in IMAP reaction buffer. After pre-incubation of 1 hour in the dark at room temperature, fluorescein-labeled MBP-derived substrate peptide (Molecular Devices, cat. no. RP 7123) was added, followed by ATP to start the reaction. Final enzyme concentration was 1.2 nM, final substrate concentration 50 nM, and final ATP concentration was 4 pM. The reaction was allowed to proceed for 2 hours at room temperature in the dark. The reaction was stopped by quenching with IMAP progressive binding solution according to the protocol of the manufacturer (Molecular Devices). Fluorescence polarization was measured on an Envision multimode reader (Perkin Elmer, Waltham, MA, U.S.A.). ICso were calculated using XLfit™5 software (ID Business Solutions, Ltd., Surrey, U.K.).
Compound (I) showed an IC50 value of < 5 nM.
Example D
Biochemical kinase assay BTK C481 S
To determine the inhibitory activity of compounds on BTK C481 S enzyme activity, the IMAP® assay (Molecular Devices) was used. Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4 % DMSO in IMAP reaction buffer, which consists of 10 mM Tris-HCI, pH 15 7.5, 10 mM MgCh, 0.01 % Tween-20, 0.1 % NaNs and 1 mM freshly prepared dithiotreitol (DTT). Compound solution was mixed with an equal volume of full- length BTK C481 S enzyme (Carna Biosciences, cat. no. 08-547) in IMAP reaction buffer. After pre-incubation of 1 hour in the dark at room temperature, fluorescein-labeled MBP-derived substrate peptide (Molecular Devices, cat. no. RP 7123) was added, followed by ATP to start the reaction. Final enzyme concentration was 1 .2 nM final substrate concentration 50 nM, and final ATP concentration was 7 pM. The reaction was allowed to proceed for 2 hours at room temperature in the dark. The reaction was stopped by quenching with IMAP progressive binding solution according to the protocol of the manufacturer (Molecular Devices). Fluorescence polarization was measured on an Envision multimode reader (Perkin Elmer, Waltham, MA, U.S.A.). IC50 were calculated using XLfit ™ 5 software (ID Business Solutions, Ltd., Surrey, U.K.). Compound (I) showed an IC50 value of < 5 nM.
Example E
Cell proliferation assay
REC-1 mantle cell lymphoma cells were purchased from American Type Culture Collection via Synthego Corporation (cat. no. CRL-3004, ATCC). Frozen stocks were thawed and cells were diluted in RPMI-1640 cell culture medium (cat. no. 61870036, Life Technologies), supplemented with 10% (v/v) fetal calf serum and 1 % penicillin/streptomycin. 3200 cells per well (in 45 pl) were seeded in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for 24 hours at 37 °C, 95 % humidity, and 5 % CO2. 5 pl compound solution was added to the cells and incubation was continued for 72 hours (3 days), followed by addition of 24 pl ATPIite 1 Step™ (PerkinElmer, Groningen, The Netherlands) solution was added to each well. Luminescence was recorded on an Envision multimode reader. The cell signal at the start of incubation was recorded separately in order to distinguish between cell population growth and cytotoxicity. In addition, maximum growth was determined by incubation of a duplicate without compound in the presence of 0.3 % DMSO. Percentage growth was used as the main y-axis signal. ICsos were fitted by non-linear regression using IDBS XLfit™5 using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50.
Compound (I) showed an IC50 value of < 100 nM.
Example F
Generation of a BTK T474I knock-in cell line and subsequent proliferation assay
REC-1 cell lines expressing mutant BTK were created at Synthego Corporation. A cell line expressing BTK T474I was generated via CRISPR/Cas9. A clonal REC-1 BTK T474I cell line was obtained by single cell cloning. The mutation status of BTK was confirmed via sequencing. For the proliferation assay, frozen cell stocks were thawed and cells were diluted in RPMI-1640 cell culture medium (cat. no. 61870036, Life Technologies), supplemented with 10% (v/v) fetal calf serum and 1 % penicillin/streptomycin. 3200 cells per well (in 45 pl) were seeded in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for 24 hours at 37 °C, 95 % humidity, and 5 % CO2. 5 pl compound solution was added to the cells and incubation was continued for 72 hours (3 days), followed by addition of 24 pl ATPIite 1 Step™ (PerkinElmer, Groningen, The Netherlands) solution was added to each well. Luminescence was recorded on an Envision multimode reader. The cell signal at the start of incubation was recorded separately in order to distinguish between cell population growth and cytotoxicity. In addition, maximum growth was determined by incubation of a duplicate without compound in the presence of 0.3 % DMSO. Percentage growth was used as the main y-axis signal. IC50S were fitted by nonlinear regression using IDBS XLfit™5 using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50.
Compound (I) showed an IC50 value of < 100 nM.
Example G
Binding kinetics measurements on wt-BTK, BTK C481 S, BTK T474I and BTK T474S (Surface Plasmon Resonance)
Streptavidin-coated chips (Cat. No. BR100531), disposables and maintenance kits for Biacore were purchased from Cytiva (Eindhoven, The Netherlands). Biotinylated wt-BTK enzym (Carna Biosciences, cat. no. 08-480-20N), BTK C481 S (Carna Biosciences, cat. no. 08-417- 20N), BTK T474I (Carna Biosciences, cat. no. 08-419-20N) or BTK T474S (Carna Biosciences, cat. no. 08-420-20N) were immobilized on a streptavidin-coated chip to a level of about 8000 response units (RU) using Biacore buffer (50 mM Tris pH 7.5, 0.05 % (v/v) Tween-20, 150 mM NaCI and 5 mM MgCh) + 1 mM TCEP. Remaining streptavidin was blocked with biocytin. Immobilization was performed at 4°C. Subsequent assay steps were conducted at 22°C. After changing buffer to Biacore buffer with 1 % (v/v) dimethylsulfoxide (DMSO), a pre-run was performed for a period of at least 30 min at a flow rate of 30 pl/min to obtain a stable surface. The kinetic constants of the compounds were determined with single cycle kinetics with five consecutive injections with an increasing compound concentration with ranges of 3.16 - 316 nM. Experiments were performed with an association time of 100 s per concentration and a dissociation time of 1200 s, except for compounds with a long target residence time, such as irreversible inhibitors, where dissociation time was increased. To circumvent problems of mass transport limitation, a flow rate of 30 pl/min was used. A blank run with the same conditions was performed before the compound was injected. The SPR sensorgrams were analyzed with Biacore Evaluation Software by using a method of double referencing. First the reference channel was subtracted from the channel containing immobilized protein. Subsequently, the reference curve obtained with buffer injections was subtracted. The resulting curve was fitted with a 1 :1 binding model. Compounds that bound according to an induced fit model were fitted with a two-state reaction model. The kinetic constants (ka, kd, KD) of duplicates were geometrically averaged.
Compound (I) showed a KD (wt-BTK) value of < 5 nM.
Compound (I) showed a KD (BTK C481 S) value of < 5 nM. Compound (I) showed a KD (BTK T474I) value of < 10 nM. Compound (I) showed a KD (BTK T474S) value of < 5 nM.
Example H
Cell proliferation assay wt-TMD8 diffuse large B-cell lymphoma cells were purchased from Tokyo Medical and Dental University and cultured in RPMI-1640 cell culture medium (cat. no. 61870036, Life Technologies), supplemented with 10% (v/v) heat-inactivated fetal bovine calf serum and 1 % penicillin/streptomycin. 1600 cells per well (in 45 pl) were seeded in a white 384-well culture plate (cat. no. 781080, Greiner Bio-One) and allowed to rest for at least 5 hours at 37 °C, 95 % humidity, and 5 % CO2. 5 pl compound solution was added to the cells and incubation was continued for 120 hours (5 days), followed by addition of 24 pl ATPIite 1 Step™ (PerkinElmer, Groningen, The Netherlands) solution was added to each well. Luminescence was recorded on an Envision multimode reader. The cell signal at the start of incubation was recorded separately in order to distinguish between cell population growth and cytotoxicity. In addition, maximum growth was determined by incubation of a duplicate without compound in the presence of 0.3 % DMSO. Percentage growth was used as the main y-axis signal. IC50S were fitted by non-linear regression using IDBS XLfit™5 using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50.
Compound (I) showed an IC50 value of < 100 nM. REFERENCES
Attwood et al. (2021) “Trends in kinase drug discovery targets, indications and inhibitor design.” Nat Rev Drug Discov 20, pages 839-861 (2021).
• Xianhui Wang et al. (2021) Bruton’s Tyrosine Kinase and Its Isoforms in Cancer. Front. Cell Dev. Biol. 9:668996
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• Wei, L., Su, Y„ Lin, C. M., Chao, T. Y., Huang, S., Huynh, T. D„ Jan, H. J., Whang- Peng, J., Chiou, J. F., Wu, A. T., & Hsiao, M. (2016). Preclinical investigation of ibrutinib, a Bruton’s kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes. Oncotarget, 7(43), 69961 -69975.

Claims

Claims
1. A hydrochloric acid addition salt of compound (I), represented by the following structural formula:
Figure imgf000058_0001
compound (I).
2. The hydrochloric acid addition salt of claim 1 , wherein the molar ratio between compound (I) and hydrochloric acid is 1 :1 .
3. The hydrochloric acid salt of any one of claims 1 - 2, wherein the hydrochloric acid salt is at least partly crystalline.
4. The hydrochloric acid salt of any one of claims 1 - 3, wherein said crystalline hydrochloric acid salt has crystalline Form A, having an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 4.2°, 9.0°, 23.8° and 25.4° ± 0.2 in 20, or wherein said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 9.0°, 23.8° and 25.4° ± 0.2 in 20, or wherein said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven or eight peaks chosen from 4.2°, 8.4°, 9.0°, 14.9°, 18.0°, 23.0°, 23.8° and 25.4° ± 0.2 in 20, or wherein said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 8.4°, 9.0°, 14.9°, 15.4°, 16.3°, 17.2°, 18.0°, 21.5°, 23.0°, 23.8° and 25.4° ± 0.2 in 20.
5. The hydrochloric acid salt of any one of preceding claims, wherein said crystalline hydrochloric acid salt has Form A, having an X-ray powder diffraction pattern substantially similar to Figure 1 , and/or wherein said hydrochloric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 3, and /or wherein said hydrochloric acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 2.
6. The hydrochloric acid salt of any one of preceding claims, wherein at least 90% by weight of the hydrochloric acid salt is crystalline Form A.
7. The hydrochloric acid salt of any one of preceding claims, wherein the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
8. The hydrochloride acid salt of any one of preceding claims, obtained or obtainable by a process comprising: combining compound (I) and hydrochloric acid in acetone; collecting said hydrochloric acid salt of compound (I).
9. A maleic acid addition salt of compound (I), represented by the following structural formula:
Figure imgf000059_0001
compound (I).
10. The maleic acid salt of claim 9, wherein the molar ratio between compound (I) and maleic acid is 1 :1 .
11 . The maleic acid salt of any one of claims 9 - 10, wherein the maleic acid salt is at least partly crystalline.
12. The maleic acid salt of claim 11 , wherein said crystalline maleic acid salt has crystalline Form B, having an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 9.7°, 10.7°, 12.2°, 16.5°, 23.5 and 25.4° ± 0.2 in 20, or wherein said crystalline maleic acid salt has Form B, having an X-ray powder diffraction pattern which comprises peaks at 9.7°, 10.7°, 12.2°, 16.5°, 23.5 and 25.4° ± 0.2 in 20, or wherein said crystalline maleic acid salt has Form B, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine, or ten peaks chosen from 8.1 °, 9.7°, 10.7°, 12.2°, 15.1 °, 16.5°, 17.7°, 19.6°, 20.5°, 21 .2°, 22.4°, 23.5°, 23.9°, 25.4° and 30.0° ± 0.2 in 20, or wherein said crystalline maleic acid salt has Form B, having an X- ray powder diffraction pattern which comprises peaks at 8.1 °, 9.7°, 10.7°, 12.2°, 13.1 °, 15.1 °, 16.5°, 17.2°, 17.7°, 18.8°, 19.0°, 19.6°, 20.5°, 21.2°, 22.4°, 23.5°, 23.9°, 24.4°, 25.4°, 26.3°, 28.7°, 29.0° and 30.0° ± 0.2 in 20.
13. The maleic acid salt of claim 9-12, wherein said crystalline maleic acid salt has Form B, having an X-ray powder diffraction pattern substantially similar to Figure 4, and/or wherein said maleic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 6, and/or wherein said maleic acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 5.
14. The maleic acid salt of any one of claims 9-13, wherein at least 90% by weight of the maleic acid salt is crystalline Form B.
15. The maleic acid salt of any one of claims 9-14, wherein the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C 1 75 % RH.
16. The maleic acid salt of any one of claims 9-15, obtained or obtainable by a process comprising: combining compound (I) and maleic acid in dioxane; collecting said maleic acid salt of compound (I).
17. A sulphuric acid addition salt of compound (I), represented by the following structural
Figure imgf000060_0001
compound (I).
18. The sulphuric acid salt of claim 17, wherein the molar ratio between compound (I) and sulphuric acid is 1 :0.5.
19. The sulphuric acid salt of claim 17 or 18, wherein the sulphuric acid salt is at least partly crystalline.
20. The sulphuric acid salt of claim 19, wherein said crystalline sulphuric acid salt has crystalline Form C, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.8°, 9.6° and 20.8° ± 0.2 in 20, or wherein said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises peaks at 4.8°, 9.6° and 20.8° ± 0.2 in 20, or wherein said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine. or ten peaks chosen from 4.8°, 6.0°, 9.6°, 10.3, 11.6°, 15.7°, 17.9°, 19.1 °, 19.9°, 20.8°, 21 .6°, 21 .8°, 22.4°, 23.4° and 24.0° ± 0.2 in 20, or wherein said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern which comprises peaks at 4.8°, 6.0°, 9.6°, 10.3, 11.6°, 15.7°, 17.9°, 19.1 °, 19.9°, 20.8°, 21 .6°, 21 .8°, 22.4°, 23.4°, 24.0° and 25.7° ± 0.2 in 20.
21. The sulphuric acid salt of any one of claims 17 - 20, wherein said crystalline sulphuric acid salt has Form C, having an X-ray powder diffraction pattern substantially similar to Figure 7, and/or wherein said sulphuric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 9, and/or wherein said crystalline sulphuric acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 8.
22. The sulphuric acid salt of any one of claims 17 - 21 , wherein at least 90% by weight of the sulphuric acid salt is crystalline Form C, and/or wherein the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C 1 75 % RH.
23. The sulphuric acid salt of any one of claims 17 - 22, obtained or obtainable by a process comprising: combining compound (I) and sulphuric acid in ethanol; collecting said sulphuric acid salt of compound (I).
24. A sulphuric acid addition salt of compound (I), represented by the following structural formula:
Figure imgf000062_0001
compound (I), and wherein the molar ratio between compound (I) and sulphuric acid is 1 :1 .
25. The sulphuric acid salt of claim 24, wherein the sulphuric acid salt is at least partly crystalline.
26. The sulphuric acid salt of claim 25, wherein said crystalline sulphuric acid salt has crystalline Form D, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.2°, 23.7°, and 25.4° ± 0.2 in 20, or wherein said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 23.7°, and 25.4° ± 0.2 in 20, or wherein said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises at least three, four, five, six or seven peaks chosen from 4.2°, 14.8°, 15.2°, 18.0°, 21.0°, 23.7°, and 25.4° ± 0.2 in 20, or wherein said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.8°, 15.2°, 18.0°, 21 .0°, 23.7°, and 25.4° ± 0.2 in 20.
27. The sulphuric acid salt of any one of claims 24 - 26, wherein said crystalline sulphuric acid salt has Form D, having an X-ray powder diffraction pattern substantially similar to Figure 10, and/or wherein said sulphuric acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 12, and/or wherein said sulphuric acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 1 1 .
28. The sulphuric acid salt of any one of claims 25, wherein at least 90% by weight of the sulphuric acid salt is crystalline Form D.
29. The sulphuric acid salt of any one of claims 25 - 28, wherein the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
30. The sulphuric acid salt of any one of claims 24 - 29, obtained or obtainable by a process comprising: combining compound (I) and sulphuric acid in acetone; collecting said sulphuric acid salt of compound (I).
31. An ethane 1 ,2-disulfonic acid addition salt of compound (I), represented by the following structural formula:
Figure imgf000063_0001
32. The ethane 1 ,2-disulfonic acid salt of claim 31 , wherein the molar ratio between compound (I) and ethane 1 ,2-disulfonic acid is 1 :0.5.
33. The ethane 1 ,2-disulfonic acid salt of claim 31 or 32, wherein the ethane 1 ,2-disulfonic acid salt is at least partly crystalline.
34. The ethane 1 ,2-disulfonic acid salt of claim 33, wherein said crystalline ethane 1 ,2-disulfonic acid salt has crystalline Form E, having an X-ray powder diffraction pattern which comprises at least two peaks chosen from 4.2°, 17.4° and 24.7° ± 0.2 in 20, or wherein said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 17.4° and 24.7° ± 0.2 in 20, or wherein said crystalline ethane 1 ,2- disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises at least three, four or five peaks chosen from 4.2°, 14.9°, 17.4°, 21 .3° and 24.7° ± 0.2 in 20, or wherein said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.9°, 15.5°, 17.4°, 21.3° and 24.7° ± 0.2 in 20.
35. The ethane 1 ,2-disulfonic acid salt of any one of claims 29 - 34, wherein said crystalline ethane 1 ,2-disulfonic acid salt has Form E, having an X-ray powder diffraction pattern substantially similar to Figure 13, and/or wherein said ethane 1 ,2-disulfonic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 15, and/or wherein said ethane 1 ,2-disulfonic acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 14.
36. The ethane 1 ,2-disulfonic acid salt of any one of claims 33 - 35, wherein at least 90% by weight of the ethane 1 ,2-disulfonic acid salt is crystalline Form E.
37. The ethane 1 ,2-disulfonic acid salt of any one of claims 33 - 36, wherein the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
38. The ethane 1 ,2-disulfonic acid salt of any one of claims 31 - 37, obtained or obtainable by a process comprising: combining compound (I) and ethane 1 ,2-disulfonic acid in ethanol; collecting said ethane 1 ,2-disulfonic acid salt of compound (I).
39. A hydrobromic acid addition salt of compound (I), represented by the following structural
Figure imgf000064_0001
40. The hydrobromic acid salt of claim 39, wherein the molar ratio between compound (I) and hydrobromic acid is 1 :1.
41. The hydrobromic acid salt of claim 39 or 40, wherein the hydrobromic acid salt is at least partly crystalline.
42. The hydrobromic acid salt of claim 41 , wherein said crystalline hydrobromic acid salt has crystalline Form F, having an X-ray powder diffraction pattern which comprises at least three, four or five peaks chosen from 4.2°, 14.9°, 17.9°, 21 .5°, 23.9°, 24.3° and 25.4° ± 0.2 in 20, or wherein said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 14.9°, 17.9°, 21 .5°, 23.9°, 24.3° and 25.5° ± 0.2 in 20, or wherein said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven, eight, nine, or ten peaks chosen from 4.2°, 8.4°, 9.0°, 9.8°, 12.6°, 14.9°, 15.4°, 16.3°, 17.3°, 17.9°, 19.9°, 20.7°, 21.5°, 23.0°, 23.9°, 24.3°, 25.5°, 26.4°, 27.1 ° and 27.9° ± 0.2 in 20, or wherein said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern which comprises peaks at 4.2°, 8.4°, 9.0°, 9.8°, 12.6°, 14.9°, 15.4°, 16.3°, 17.3°, 17.9°, 18.6°, 19.9°, 20.7°, 21.5°, 23.0°, 23.9°, 24.3°, 25.5°, 26.4°, 27.1 °, 27.9°, 29.6° and 30.0° ± 0.2 in 20.
43. The hydrobromic acid salt of any one of claims 39 - 42, wherein said crystalline hydrobromic acid salt has Form F, having an X-ray powder diffraction pattern substantially similar to Figure 16, and/or wherein said hydrobromic acid salt has a differential scanning calorimeter thermogram substantially similar to Figure 18, and/or wherein said hydrobromic acid salt has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 17.
44. The hydrobromic acid salt of any one of claims 41 - 43, wherein at least 90% by weight of the hydrobromic acid salt is crystalline Form F, and/or wherein the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
45. The hydrobromic acid salt of any one of claims 39 - 44, obtained or obtainable by a process comprising: combining compound (I) and hydrobromic acid in acetone; collecting said hydrobromic acid salt of compound (I).
46. A freebase crystalline Form G of compound (I), represented by the following structural formula:
Figure imgf000065_0001
compound (I), wherein said Form G, having an X-ray powder diffraction pattern which comprises at least three, four, five, six, seven or eight peaks chosen from 4.2°, 9.3°, 13.0°, 13.5°, 16.4°, 22.0°, 24.7° and 25.2° ± 0.2 in 20.
47. The freebase crystalline Form G of claim 46, wherein said Form G has an X-ray powder diffraction pattern which comprises at least three or four peaks chosen from 4.2°, 9.3°, 22.0° and 25.2° ± 0.2 in 20, or wherein said Form G has an X-ray powder diffraction pattern which comprises peaks at 4.2°, 9.3°, 13.0°, 13.5°, 16.4°, 22.0°, 24.7° and 25.2° ± 0.2 in 20.
48. The freebase crystalline Form G of claim 46 or 47, wherein said Form G has an X-ray powder diffraction pattern substantially similar to Figure 19, and/or wherein said crystalline freebase form has a differential scanning calorimeter thermogram substantially similar to Figure 21 , and/or wherein said Form G has a thermogravimetric analysis (TGA) thermogram substantially similar to Figure 20, and/or wherein at least 90% by weight of the freebase is crystalline Form G, and/or wherein the X-ray powder diffraction pattern is substantially stable during at least 2 days at 40°C / 75 % RH.
49. The freebase crystalline Form G of any one of claims 46 - 48, obtained or obtainable by a process comprising: combining compound (I) to dioxane to form a mixture; collecting said freebase crystalline Form G of compound (I).
50. A pharmaceutical composition comprising said salt of any one of claims 1 - 45, or said freebase of any one of claims 46 - 49, and a pharmaceutically acceptable carrier or a diluent.
51 . Compound or pharmaceutical composition according to any of the preceding claims, for use as a medicament.
52. Compound according to any of the claims 1 - 49 or pharmaceutical composition according to claim 50, for use in therapy.
53. Compound according to any of the claims 1 - 49 or pharmaceutical composition according to claim 50, for use in the treatment of Bruton’s Tyrosine Kinase (BTK) mediated disorders.
54. Compound according to claim 53, wherein the Bruton’s Tyrosine Kinase (BTK) mediated disorder is selected from the group consisting of an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, a bone-related disease, and cancer.
55. Compound according to any of the claims 1 - 49 or pharmaceutical composition according to claim 50, for use in the treatment of cancer, lymphoma or leukemia.
56. Compound according to any of the claims 1 - 49 or pharmaceutical composition according to claim 50, for use in the treatment of a disease selected from the group consisting of B-cell malignancy, B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocyte leukemia, non-Hodgkin lymphoma for example ABC-DLBCL, mantle cell lymphoma, follicular lymphoma, hairy cell leukemia B-cell non-Hodgkin lymphoma, Waldenstrom’s macroglobulinemia, Richter transformation, multiple myeloma, bone cancer, bone metastasis, chronic lymphocytic lymphomas, B-cell prolymphocyte leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, lymphomatoid granulomatosis.
57. Compound according to any of the claims 1 - 49 or pharmaceutical composition according to claim 50, for use in the treatment of a disease selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, deforming arthritis, osteoarthritis, traumatic arthritis, gouty arthritis, Reiter’s syndrome, polychondritis, acute synovitis and spondylitis, glomerulonephritis (with or without nephrotic syndrome), autoimmune hematologic disorders, hemolytic anemia, aplasic anemia, idiopathic thrombocytopenia, and neutropenia, autoimmune gastritis, and autoimmune inflammatory bowel diseases, ulcerative colitis, Crohn’s disease, host versus graft disease, allograft rejection, chronic thyroiditis, Graves’ disease, schleroderma, diabetes (type I and type II), active hepatitis (acute and chronic), pancreatitis, primary billiary cirrhosis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosis, psoriasis, atopic dermatitis, contact dermatitis, eczema, skin sunburns, vasculitis (e.g. Behcet’s disease) chronic renal insufficiency, Stevens-Johnson syndrome, inflammatory pain, idiopathic sprue, cachexia, sarcoidosis, Guillain-Barre syndrome, uveitis, conjunctivitis, kerato conjunctivitis, otitis media, periodontal disease, pulmonary interstitial fibrosis, asthma, bronchitis, rhinitis, sinusitis, pneumoconiosis, pulmonary insufficiency syndrome, pulmonary emphysema, pulmonary fibrosis, silicosis, chronic inflammatory pulmonary disease, and chronic obstructive pulmonary disease.
58. Use of the compound according to any of the claims 1 - 49, for the manufacture of a medicament.
59. The pharmaceutical composition of claim 49, which further comprises at least one additional therapeutically active agent.
60. A method for treating of cancer in a subject in need thereof comprising administering to the subject the compound according to any of the claims 1 - 49 in an amount effective to treat cancer.
61 . A method for treating a subject suffering with a Bruton's Tyrosine Kinase (BTK) mediated disorder comprising administering to the subject the compound of any of the claims 1 - 49 or pharmaceutical composition according to claim 50 in an amount effective to treat the BTK mediated disorder.
62. A method for treating a subject according to claim 61 , wherein the Bruton’s Tyrosine Kinase (BTK) mediated disorder is selected from the group consisting of an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, a bone-related disease, and cancer.
PCT/EP2024/066423 2023-06-13 2024-06-13 Salt and crystal forms of a macrocyclic btk inhibitor Pending WO2024256568A1 (en)

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