Pyrazolopyrrolidine derivatives and their use in the treatment of disease

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof;a method for manufacturing the compounds of the invention, and its therapeutic uses. The present invention further provides a combination of pharmacologically active agents and a pharmaceutical composition.

FIELD OF THE INVENTION

The invention provides pyrazolopyrrolidine derivatives and their use as BET inhibitors, for the treatment of conditions or diseases such as cancer.

BACKGROUND OF THE INVENTION

BET proteins are proteins encoded by either of the genes BRD2, BRD3, BRD4, or BRDT. Each of these proteins bears two N-terminal bromodomains. Bromodomains comprise of a conserved ˜110 amino acid segment found in at least 42 diverse proteins that specifically interact with acetylated lysines that occur for example on histone tails (Filippakopoulos and Knapp, FEBS Letters, 586 (2012), 2692-2704). Histones are a constituent part of chromatin and their covalent modifications including lysine acetylation regulate gene transcription. Bromodomains are thus believed to regulate transcription by recruiting proteins to genes that are marked with specific patterns of lysine acetylation.

Several published reports have linked the BET protein family to diseases including cancer, metabolic disease and inflammation. Oncogenic fusions of BRD4 or BRD3 and the Nuclear protein in Testis (NUT) gene caused by chromosomal translocations are underlying an aggressive cancer named NUT midline carcinoma (French et al., J Clin Oncol, 22 (2004), 4135-9; French et al., J Clin Pathol, 63 (2008), 492-6). The BRD3/4 bromodomains are preserved in these fusion proteins, and their inhibition either by knockdown or with the selective BET bromodomain inhibitor JQ1 leads to death and/or differentiation of these cancer cells both in vitro and in animal tumour models (Filippakopoulos et al., Nature, 468 (2010), 1067-73). JQ1 and several other selective BET inhibitors have been shown to bind to BET bromodomains and thereby prevent acetyl-lysine binding, which prevents BET proteins from interacting with chromatin and thereby regulating transcription. BRD4 was also identified from an RNAi screen as a target in acute myeloid leukemia (AML) (Zuber et al., Nature, 478 (2011), 524-8). This finding was validated in vitro and in vivo using the BET inhibitor JQ1 and another selective BET inhibitor named I-BET151 that is chemically unrelated to JQ1 (Dawson et al., Nature, 478 (2011), 529-33). These and other studies showed that BET inhibitors have broad anti-cancer activity in acute leukemias, multiple myeloma and other hematological malignancies. In several cancer models an acute downregulation of the oncogenic transcription factor Myc upon BET inhibition has been observed (Delmore et al., Cell, 146 (2011), 904-17; Mertz et al., Proc Natl Acad Sci USA, 108 (2011), 16669-74). More recent studies suggest that the therapeutic potential of BET inhibitors extends to other cancer indications, for example lung and brain cancer.

Another BET inhibitor named I-BET762 that is closely related to JQ1 in chemical structure and the manner in which it binds to BET bromodomains, was reported to modulate expression of key inflammatory genes and thereby protect against endotoxic shock and bacteria-induced sepsis in mouse models (Nicodeme et al., Nature, 468 (2010), 1119-23). This body of data has been used to support the clinical evaluation of the BET inhibitor RVX-208 in clinical trials in patients suffering from atherosclerosis, coronary artery disease, dyslipidemia, diabetes, and other cardiovascular diseases (McNeill, Curr Opin Investig Drugs, 3 (2010), 357-64 and www.clinicaltrials.gov), Both RVX-208 and I-BET762 have been shown to upregulate Apolipoprotein A-I, which is critically involved in reducing the tissue levels of cholesterol. Finally, BET proteins have been linked to propagation and transcription regulation of several viruses, and therefore it is believed that BET inhibitors could have anti-viral activity (Weidner-Glunde, Frontiers in Bioscience 15 (2010), 537-549).

In summary, inhibitors of BET bromodomains have therapeutic potential in several human diseases.

There remains a need for new treatments and therapies for the treatment of cancer. The invention provides compounds as BET inhibitors, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof. The invention further provides methods of treating, preventing or ameliorating cancer, comprising administering to a subject in need thereof an effective amount of a BET inhibitor.

Various embodiments of the invention are described herein. Particularly interesting compounds of the invention have good potency in the biological assays described herein. In another aspect they should have a favourable safety profile. In another aspect, they should possess favourable pharmacokinetic properties.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein
ring C is selected from

A is selected from

B is selected from

A is:

R1is selected from methyl, chloro and fluoro,
B is:

In another embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the definition of formula (I), or a pharmaceutically acceptable salt thereof, or subformulae thereof and one or more pharmaceutically acceptable carriers.

In another embodiment, the invention provides a combination, in particular a pharmaceutical combination, comprising a therapeutically effective amount of the compound according to the definition of formula (I), or a pharmaceutically acceptable salt thereof, or subformulae thereof and one or more therapeutically active agent.

Therefore according to the first aspect of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, of the following formulae:

The data disclosed in WO2013/08014A1 show that one enantiomer of the pyrazolopyrrolidine compounds possesses greater p53-MDM2 and p53-MDM4 activity. For examples 139, 140, 141, 142, 143 and 144, the (S)-enantiomer shows significantly greater activity. Surprisingly, for the compounds of the present invention, the optical antipode to the preferred p53-MDM2 inhibitors was found to possess significantly greater activity as BRD4 inhibitors.

DETAILED DESCRIPTION

Described below are a number of embodiments (E) of the first aspect of the invention, where for convenience E1 is identical thereto.

E1 A compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof.

E1.1 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1

wherein
ring C is selected from

A is selected from

B is selected from

A is:

R1is selected from methyl, chloro and fluoro,
B is:

E1.2 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1

wherein
ring C is selected from

A is selected from

B is selected from

A is:

R1is selected from methyl, chloro and fluoro,
B is:

R2is chloro or —CF3,
and the remaining substituents are as defined herein,
then R3is selected from H, methyl, ethyl, —CF3, —OH, ethoxy and methoxy.

E2 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1, E1.1 or E1.2, wherein A is selected from

or A is selected from

E3 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1 or E1.1 or E1.2, wherein A is

E4 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1 or E1.1 or E1.2, wherein A is

E5 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1 or E1.1 or E1.2, wherein A is

E6 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1 or E1.1 or E1.2, wherein A is selected from

E7 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E3, E4, E5 or E6, wherein B is

E8 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E3, E4, E5 or E6, wherein B is selected from

or B is selected from

E9 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E8, wherein R3is selected from H, methyl, ethyl, —CF3, isopropyl, —OH, ethoxy, methoxy and cyclopropyl, or R3is selected from methyl, ethyl, isopropyl and methoxy, or R3is selected from H, methyl, ethyl, —CF3, —OH, ethoxy and methoxy.

E10 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E9, wherein R3is methyl.

E11 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E10, wherein R4ais selected from methyl, isopropyl, cyclopropyl and

E12 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E11, wherein R4ais selected from cyclopropyl and

E13 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E10, wherein R4ais selected from H, methyl, ethyl, cyclopropyl, isopropyl, —(CH2)2—OH, —(CH2)2—O—CH3,

E14 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E10, wherein R4bis selected from ethyl, isopropyl, cyclopropyl, —(CH2)2—OH, —(CH2)2—O—CH3,

E15 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2 to E10 and E14, wherein R4bis cyclopropyl.

E17 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E3 and E7 to E16, wherein R1is methyl or chloro.

E18 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E3 and E7 to E17, wherein R1is methyl.

E19 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E3 and E7 to E18, wherein R2is chloro.

E20 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2 to E13 and E17 to E19, wherein ring C is i:

E21 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2 to DO and E14 to E19, wherein ring C is ii:

E22 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E21, wherein the stereochemistry is as shown in formula (Ia):

E23 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E21, wherein the compound is present as the racemate of the 2 enantiomeric forms (Ia) and (Ib) disclosed herein.

E24 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2 to E3 and E6 to E23, with the proviso that when ring C is selected from

and A is:

R1is selected from methyl, chloro and fluoro,
B is:

E25 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E3, E7, E11 to E20, E22 and E23,

with the proviso that

when ring C is i:

A is:

R1is selected from methyl, chloro and fluoro,
B is:

R2is chloro or —CF3,
and the remaining substituents are as defined herein,
then R3is selected from H, methyl, ethyl, —CF3, —OH, ethoxy and methoxy.

E26 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E10, wherein R4ais selected from H, (C1-C4)alkyl, (C3-C6)cycloalkyl, —(CH2)2—OH and —(CH2)2—O—CH3;

or R4ais selected from

E27 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2 and E2 to E10, wherein R4bis selected from H, (C1-C4)alkyl, (C3-C6)cycloalkyl, —(CH2)2—OH, —(CH2)2—O—CH3, and —(CH2)2—O—CH2—CF3;

or R4bis selected from

E28 a compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein
ring C is selected from

A is selected from

B is selected from

R5is H;
R2is selected from chloro and fluoro;
R2ais fluoro;
and
* indicates the point of attachment to the remainder of the molecule;
with the proviso that
when ring C is i:

A is:

R2is chloro or fluoro,
and the remaining substituents are as defined herein,
then R3is selected from methyl, ethyl, methoxy, —CH2OCH3and —CH2OH.

E29 A compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein
ring C is selected from

A is selected from

R4bis selected from H and (C3-C6)cycloalkyl, preferably R4bis H or cyclopropyl;
R5is H;
and
* indicates the point of attachment to the remainder of the molecule;
with the proviso that
when ring C is i:

A is:

and the remaining substituents are as defined herein,
then R3is selected from methyl, ethyl, —CH2OCH3and —CH2OH.

E30 A compound of formula (I), or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E4, E7, E9, E10, E15 and E21, wherein

C is ii:

E31 A compound of formula (I), or a pharmaceutically acceptable salt thereof, according to any one of E1, E1.1, E1.2, E2, E3, E7, E9, E10, E15 and E21, wherein

C is ii:

E32 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1, selected from:

E33 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1 or E32, selected from

E34 A compound of formula (I) or a pharmaceutically acceptable salt thereof, according to E1, E32 or E33, selected from

The present disclosure includes compounds of stereochemistry is as shown in formula (Ib):

Unless specified otherwise, the term “compounds of the present invention” refers to compounds of formula (I) and subformulae thereof, and salts thereof, as well as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties.

Various embodiments of the invention are described herein. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.

Depending on the choice of the starting materials and procedures, the compounds can be present in the form of one of the possible isomers or as mixtures thereof, for example as pure optical isomers, or as isomer mixtures, such as racemates and diastereoisomer mixtures, depending on the number of asymmetric carbon atoms. The present invention is meant to include all such possible isomers, including racemic mixtures, diasteriomeric mixtures and optically pure forms. Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have a cis- or trans-configuration. All tautomeric forms are also intended to be included.

As used herein, the terms “salt” or “salts” refers to an acid addition or base addition salt of a compound of the invention. “Salts” include in particular “pharmaceutical acceptable salts”. The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.

Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as2H,3H,11C,13C,14C,15N,18F,31P,32P,35S,36Cl,123I,124I,125I respectively. The invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as3H and14C, or those into which non-radioactive isotopes, such as2H and13C are present. Such isotopically labelled compounds are useful in metabolic studies (with14C), reaction kinetic studies (with, for example2H or3H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an18F or labeled compound may be particularly desirable for PET or SPECT studies. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.

Compounds of the invention, i.e. compounds of formula (I) that contain groups capable of acting as donors and/or acceptors for hydrogen bonds may be capable of forming co-crystals with suitable co-crystal formers. These co-crystals may be prepared from compounds of formula (I) by known co-crystal forming procedures. Such procedures include grinding, heating, co-subliming, co-melting, or contacting in solution compounds of formula (I) with the co-crystal former under crystallization conditions and isolating co-crystals thereby formed. Suitable co-crystal formers include those described in WO 2004/078163. Hence the invention further provides co-crystals comprising a compound of formula (I).

The term “a therapeutically effective amount” of a compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by BET proteins, or (ii) associated with BET protein activity, or (iii) characterized by activity (normal or abnormal) of BET proteins; or (2) reduce or inhibit the activity of BET proteins; or (3) reduce or inhibit the expression of BET. In another non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of BET proteins; or at least partially reducing or inhibiting the expression of BET proteins.

A “BET protein” is a protein encoded by either of the genes BRD2, BRD3, BRD4, or BRDT″. Unless indicated otherwise “BET proteins” or “BET protein” are used herein in the singular and plural forms interchangeably, and the use of either is not limiting. Unless indicated otherwise “BET proteins” includes all, or any combination of, such encoded proteins.

As used herein, the term “(C3-C6)cycloalkyl” refers to saturated monocyclic hydrocarbon groups of 3-6 carbon atoms. Exemplary C3-6cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, the term “(C1-C4)alkyl” refers to a fully saturated branched or unbranched hydrocarbon moiety having 1 to 4 carbon atoms. Representative examples of C1-4alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl and tert-butyl.

Any asymmetric atom (e.g., carbon or the like) of the compound(s) of the present invention can be present in racemic or enantiomerically enriched, for example the (R)-, (S)- or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)-configuration. Substituents at atoms with unsaturated double bonds may, if possible, be present in cis-(Z)- or trans-(E)-form.

Accordingly, as used herein a compound of the present invention can be in the form of one of the possible isomers, rotamers, atropisomers, tautomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (antipodes), racemates or mixtures thereof.

Furthermore, the compounds of the present invention, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization. The compounds of the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, it is intended that the invention embrace both solvated and unsolvated forms. The term “solvate” refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g., water, ethanol, and the like. The term “hydrate” refers to the complex where the solvent molecule is water.

The compounds of the present invention, including salts, hydrates and solvates thereof, may inherently or by design form polymorphs.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. For purposes of the present invention, unless designated otherwise, solvates and hydrates are generally considered compositions. Preferably, pharmaceutically acceptable carriers are sterile. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, etc. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. Typically, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of:

Tablets may be either film coated or enteric coated according to methods known in the art.

Suitable compositions for oral administration include an effective amount of a compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-75%, or contain about 1-50%, of the active ingredient.

Suitable compositions for topical application, e.g., to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like. Such topical delivery systems will in particular be appropriate for dermal application, e.g., for the treatment of skin cancer, e.g., for prophylactic use in sun creams, lotions, sprays and the like. They are thus particularly suited for use in topical, including cosmetic, formulations well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein a topical application may also pertain to an inhalation or to an intranasal application. They may be conveniently delivered in the form of a dry powder (either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids) from a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray, atomizer or nebuliser, with or without the use of a suitable propellant.

The compounds of formula I in free form or in pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, e.g. BET protein modulating properties, e.g. as indicated in tests as provided in the next sections, and are therefore indicated for therapy or for use as research chemicals, e.g. as tool compounds.

Having regard to their activity as BET inhibitors, compounds of the formula (I) in free or pharmaceutically acceptable salt form, are useful in the treatment of conditions which are mediated by the activity of BET proteins, such as cancer, and/or that are responsive (meaning especially in a therapeutically beneficial way) to inhibition of a BET protein, most especially a disease or disorder as mentioned herein below.

Compounds of the invention are believed to be useful in the treatment of diseases or disorders such as cancer. In particular, such cancers include benign or malignant tumours, a soft tissue sarcoma or a sarcoma such as liposarcoma, rhabdomyosarcoma or bone cancer, e.g. osteosarcoma, a carcinoma, such as of the brain, kidney, liver, adrenal gland, bladder, breast, gastric, ovary, colon, rectum, prostate, pancreas, lung (including small cell lung cancer), vagina or thyroid, a glioblastoma, meningioma, glioma, mesothelioma, a neuroendocrine tumor such as neuroblastoma, a multiple myeloma, a gastrointestinal cancer, especially colon carcinoma or colorectal adenoma, a tumor of the head and neck, a melanoma, a prostate hyperplasia, a neoplasia, a neoplasia of epithelial character, a neoplasia originating from blood or bone marrow, a leukemia such as acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) or B-cell chronic lymphocytic leukemia, a lymphoma, such as of B- or T-cell origin, such as diffuse large B cell lymphoma (DLBCL), NUT midline carcinoma or any other neoplasia with chromosomal rearrangements of the BET genes, and metastases in other organs.

In particular, the compounds of the invention may be useful in the treatment of a disease or disorder selected from a neoplasia originating from blood or bone marrow; a leukemia such as acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) or B-cell chronic lymphocytic leukemia; a lymphoma, such as of B- or T-cell origin, such as diffuse large B cell lymphoma (DLBCL); NUT midline carcinoma or any other neoplasia with chromosomal rearrangements of the BET genes; a neuroendocrine tumor such as neuroblastoma; a multiple myeloma; a lung cancer (including small cell lung cancer); and a colon cancer.

Compounds of the invention may also be of use in the treatment of atherosclerosis, coronary artery disease, dyslipidemia, diabetes, and other cardiovascular diseases, and/or as antiviral agents.

Thus, as a further embodiment, the present invention provides the use of a compound of formula (I) or a salt thereof, in therapy. In a further embodiment, the therapy is selected from a disease which may be treated by inhibition of BET proteins. In another embodiment, the disease is a cancer disease selected from the afore-mentioned list.

Thus, as a further embodiment, the present invention provides a compound of formula (I) or a salt thereof, for use in therapy. In a further embodiment, the therapy is selected from a disease which may be treated by inhibition of a BET protein. In another embodiment, the disease is a cancer disease selected from the afore-mentioned list.

In another embodiment, the invention provides a method of treating a disease which is treated by inhibition of a BET protein, comprising administration of a therapeutically acceptable amount of a compound of formula (I) or salt thereof. In a further embodiment, the disease is a cancer disease selected from the afore-mentioned list.

Thus, as a further embodiment, the present invention provides the use of a compound of formula (I) or salt thereof, for the manufacture of a medicament. In a further embodiment, the medicament is for treatment of a disease which may be treated by inhibition of a BET protein. In another embodiment, the disease is a cancer disease selected from the afore-mentioned list.

The pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50 mg of active ingredients. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

The above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof. The compounds of the present invention can be applied in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either enterally, parenterally, advantageously intravenously, e.g., as a suspension or in aqueous solution. The dosage in vitro may range between about 10−3molar and 10−9molar concentrations. A therapeutically effective amount in vivo may range depending on the route of administration, between about 0.1-500 mg/kg, or between about 1-100 mg/kg.

The compound of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agent. The compound of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents. A therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment or nucleic acid, which is therapeutically active or enhances the therapeutic activity when administered to a patient in combination with a compound of the invention.

Assays

The activity of a compound according to the present invention can be assessed by the following methods.

All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 μL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 μL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.

In order to assess bromodomain selectivity, we set up a binding assay using the bromodomain encoded by the CREBBP gene. Compounds were tested in the CREBBP assay with a similar protocol, however using AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay, Perkin Elmer) as detection readout instead of TR-FRET. The assay was started by stepwise addition of 4.5 μL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 324 nM His-CREBBP (1081-1197) (custom production at Viva Biotech Ltd.)) and 4.5 μL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 120 nM acetyl-histone H4 (AcK 5, 8, 12) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 45 μg/ml Ni-chelate acceptor beads, 45 μg/mL streptavidin-donor beads) (Perkin Elmer)) were added. After 60 minutes incubation at room temperature, plates were measured in a Perkin Elmer EnVision multilabel reader. IC50 values were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.

For further bromodomain selectivity profiling, additional panel assays were performed using analog protocols with minor modifications specific for the individual assay, using either TR-FRET or AlphaScreen for detection.

Preparation of Compound Dilutions

Test compounds were dissolved in DMSO (10 mM) and transferred into 1.4 mL flat bottom or V-shaped Matrix tubes carrying a unique 2D matrix. The stock solutions were stored at +2° C. if not used immediately. For the test procedure the vials were defrosted and identified by a scanner whereby a working sheet was generated that guided the subsequent working steps. Compound dilutions were made in 96 well plates. This format enabled the assay of maximally individual test compounds at 8 concentrations (single points) including 4 reference compounds, if desired (known BET inhibitors from the prior art, for this and other assays of the type disclosed herein). The dilution protocol included the production of “pre-dilution plates”, “master plates” and “assay plates”.

Polypropylene 96-well plates were used as pre-dilution plates. A total of 4 pre-dilution plates were prepared including 10 test compounds each on the plate positions A1-A10, one standard compound at A11 and one DMSO control at A12. All dilution steps were done on a HamiltonSTAR robot.

30 μL of individual compound dilutions including standard compound and controls of the 4 “pre-dilution plates” were transferred into a 384-well “master plate” including the following concentrations 10000, 3003, 1000, 300, 100, 30, 10 and 3 μM, respectively in 90% of DMSO.

Identical “assay plates” were then prepared by pipetting 50 nL each of compound dilutions of the “master plates” into 384-well “assay plates” by means of a HummingBird 384-channel dispenser. These plates were used directly for the assay which was performed in a total volume of 13.55 μL. This led to a final compound concentration of 37, 11, 3.7, 1.1, 0.37, 0.11, 0.037 and 0.011 μM and a final DMSO concentration of 0.37% in the assay.

Cell Growth Inhibition Assay

The human leukemia cell lines MV-4-11, THP-1 and K-562 were employed to characterize the effect of BET inhibitors on cellular proliferation and viability. Cells were obtained from the American Type Culture Collection (ATCC) and cultured at 37° C. in a humidified 5% CO2incubator in the following media: MV-4-11: DMEM high glucose (Animed #1-26F01-I), 10% FCS (Animed #2-01F26-I), 4 mM L-Glutamine (Animed #5-10K50), 1 mM Sodium Pyruvate (Animed # G03625P), 1× Penicillin-Streptomycin (Animed # F12478P); K-562: Iscove's MEM (Animed #1-28F16-I), 10% FCS (Animed #2-01F26-I), 4 mM L-Glutamine (Animed #5-10K50), 1× Penicillin-Streptomycin (Animed # F12478P); THP-1: RPMI-1640 (Animed #1-41F01-I), 10% FCS (Animed #2-01F26-I), 2 mM L-Glutamine (Animed #5-10K50), 10 mM HEPES (Animed #5-31F100), 1 mM Sodium Pyruvate (Animed # G03625P), 1× Penicillin-Streptomycin (Animed # F12478P). The AML lines MV-4-11 and THP-1 are very sensitive to BET inhibitors and show massive cell death upon BET inhibition (Zuber et al., Nature, 478 (2011), 524-8). Compound-mediated suppression of cell proliferation/viability was assessed by quantification of cellular ATP levels using the CellTiter-Glo (CTG) reagent (Promega). Briefly, cells were seeded in 20 μl fresh medium into 384-well plates, followed by addition of 5 μL medium containing compound dilutions at 5-fold their final intended concentration. Dose-response effects were assessed by 3-fold serial dilutions of the test compound, starting at 10 μM. Following incubation of the cells for days at 37° C. and 5% CO2, the effect of inhibitors on cell viability was quantified following addition of 20 μl CTG and luminescence quantification (integration time: 100 ms) as per vendor manual, using a correspondingly equipped Tecan M200 multi-mode platereader (TECAN, Switzerland). For data analysis, the assay background value determined in wells containing medium, but no cells, was subtracted from all data points. To enable differentiation of cytotoxic from cytostatic compounds, the number of viable cells is assessed relative to that observed at the time of compound addition using a separate cell plate (day 0). The effect of a particular test compound concentration on cell proliferation/viability is expressed as percentage of the background- and day 0-corrected luminescence reading obtained for cells treated with vehicle only (DMSO, 0.1% final concentration), which is set as 100%, whereas that luminescence reading for wells containing medium is set as −100%. Compound concentrations leading to half-maximal (IC50) and total growth inhibition (TGI) were determined using standard four parameter curve fitting.

HCC2494 NUT midline carcinoma cells (expressing BRD4-NUT-fusion) were obtained from the University of Texas Southwestern and cultured in RPMI-1640 medium containing 10% Foetal Calf Serum at 37° C. in a humidified 5% CO2incubator.

Compound-mediated inhibition of BRD4 activity was monitored by quantification of the number and intensity of nuclear BRD4-NUT foci using automated immunofluorescence microscopy. Briefly, 5000 cells in 20 μL fresh medium were seeded into Poly-D-Lysine-precoated 384-well plates and incubated overnight at 37° C. and 5% CO2, followed by addition of 5 μl medium containing compound dilutions at 5-fold their final intended concentration. Dose-response effects were assessed by 3-fold serial dilutions of the test compound, starting at 10 μM. Following incubation of the cells for 24 hours at 37° C. and 5% CO2, the cells were fixed by incubation with 3.7% formaldehyde for 10 min, followed by immunofluorescence staining using rabbit anti-NUT (Cell Signaling Technologies, Cat#3625) as primary, and AlexaFluor488-labeled goat anti-rabbit (Invitrogen, Cat#A11008) as secondary antibody (latter complemented with 1 μg/mL Hoechst33342 as DNA dye). Assay plates were imaged using the appropriate filter sets on the Cellomics VTi automated fluorescence microscopy platform (ThermoFisher Scientific) and the population average of the number of NUT-foci per nucleus is quantified using the Cellomics Spot Detection BioApplication image analysis algorithm (ThermoFisher Scientific). The effect of a particular test compound concentration on NUT-foci number and intensity is expressed as percentage of the value obtained for cells treated with vehicle only (DMSO, 0.1% final concentration), which was set as 100. Compound concentrations leading to half-maximal (IC50) inhibition of the aforementioned readout parameters were determined using standard four parameter curve fitting.

Using the biochemical and cellular assays as described hereinabove, compounds of the invention exhibit inhibitory efficacy as shown in Table 1 and Table 2.

In one embodiment, the invention provides a product comprising a compound of formula (I) and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a disease or condition mediated by a BET protein. Products provided as a combined preparation include a composition comprising the compound of formula (I) and the other therapeutic agent(s) together in the same pharmaceutical composition, or the compound of formula (I) and the other therapeutic agent(s) in separate form, e.g. in the form of a kit.

In one embodiment, the invention provides a pharmaceutical composition comprising a compound of formula (I) and another therapeutic agent(s). Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, as described above.

In one embodiment, the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of formula (I). In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like.

The kit of the invention may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the invention typically comprises directions for administration.

In the combination therapies of the invention, the compound of the invention and the other therapeutic agent may be manufactured and/or formulated by the same or different manufacturers. Moreover, the compound of the invention and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the comtripotassiupound of the invention and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the compound of the invention and the other therapeutic agent.

Accordingly, the invention provides the use of a compound of formula (I) for treating a disease or condition mediated by a BET protein, wherein the medicament is prepared for administration with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition mediated by a BET protein, wherein the medicament is administered with a compound of formula (I).

The invention also provides a compound of formula (I) for use in a method of treating a disease or condition mediated by a BET protein, wherein the compound of formula (I) is prepared for administration with another therapeutic agent. The invention also provides another therapeutic agent for use in a method of treating a disease or condition mediated by a BET protein, wherein the other therapeutic agent is prepared for administration with a compound of formula (I). The invention also provides a compound of formula (I) for use in a method of treating a disease or condition mediated by a BET protein, wherein the compound of formula (I) is administered with another therapeutic agent. The invention also provides another therapeutic agent for use in a method of treating a disease or condition mediated by a BET protein, wherein the other therapeutic agent is administered with a compound of formula (I).

The invention also provides the use of a compound of formula (I) for treating a disease or condition mediated by a BET protein, wherein the patient has previously (e.g. within 24 hours) been treated with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition mediated by a BET protein, wherein the patient has previously (e.g. within 24 hours) been treated with a compound of formula (I).

In one embodiment, the other therapeutic agent is an anticancer agent.

In a further embodiment, the other therapeutic agent is a modulator of a target in the field of epigenetics, such as an inhibitor of histone deacetylase (HDAC), or an inhibitor of histone methyltransferase (HMT).

Generic Schemes

Typically, the compounds of formula (I) can be prepared according to the Schemes provided infra.

Scheme 1 illustrates one method for preparing compounds of the invention (e.g. Example 1). An ethyl 5-alkyl-1H-pyrazole-3-carboxylate is reacted with N-iodosuccinimide (NIS) to provide a 4-iodo-pyrazole derivative whose pyrazole NH is protected by deprotonation with sodium hydride (NaH) and alkylation with 4-methoxy-benzyl chloride. The resulting iodo derivative can be converted in the corresponding magnesium chloride by reaction with isopropylmagnesium chloride lithium chloride complex solution 1.3 M in THF (TurboGrignard). This freshly made organometallic reagent is reacted with an aldehyde to generate the corresponding secondary alcohol adduct. Conversion of the alcohol into a leaving group, for example mesylate, is accomplished by reaction with methanesulfonic anhydride in the presence of an organic base such as pyridine (together with a catalytic amount of 4-dimethylaminopyridine) or triethylamine. The mesylate can be displaced by reaction with an amine. Cyclization to the lactam can be effected in two steps by initial saponification of the ester group on treatment with a base such as an alkali metal hydroxide (e.g. lithium hydroxide or sodium hydroxide) in a solvent such as wet cycloalkylether or alcohol (e.g. dioxane/water or methanol/water), at room temperature. The freed amino-acid intermediate obtained after neutralization of the reaction mixture with an acid (such as a mineral acid, e.g. hydrochloric acid), extraction and evaporation to dryness is then cyclized intramolecularly by treatment with 1-chloro-N,N,2-trimethylpropenylamine at 0° C.

Deprotection of the pyrazole moiety is achieved by treatment with an organic acid such as trifluoroacetic acid (TFA), at 100° C. under microwave irradiation (e.g. Example 2). Introduction of R4 can be effected by treatment with NaH in DMF followed by alkylation (e.g. Example 3). Specific alkyl (e.g. cyclopropyl) and aryl R4 can also be introduced by coupling reaction with a boronic acid in the presence of either copper(II) acetate and pyridine in dichloroethane (e.g. Example 81) or copper(II) acetate, sodium carbonate and 2,2′-bipyridine in acetonitrile, at 65-70° C. (e.g. Example 85).

Scheme 2 illustrates an alternative synthetic route for preparing compounds of the invention (e.g. Example 57). Amine, aldehyde and diketoester are reacted in acetic acid at 125° C. For R3=H, (E)-ethyl 4-(dimethylamino)-2-oxobut-3-enoate is used instead of the diketoester (e.g. Example 34). The resulting 3-hydroxy-1H-pyrrol-2(5H)-one intermediate undergoes condensation with the desired hydrazine usually in acetic acid or in a mixture of ethanol and toluene under heating. Modified experimental conditions for the condensation step are described in Examples 60 and 69.

Scheme 3 illustrates an alternative method to introduce A (e.g. Example 25). 5-(4-Methoxybenzyl)-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)- or 6(1H)-one intermediates, prepared according to the method described in Scheme 1 (e.g. Step 23.8) or 2 (e.g. Step 71.3), are deprotected by treatment with TFA, at 140° C. under microwave irradiation. The resulting compounds are reacted with the halide A-X in the presence of either 1) tripotassium phosphate, copper(I) iodide, and N,N′-dimethylethylenediamine in dioxane, at a temperature in the range of 100-120° C. (e.g. Examples 23, 24), or 2) Pd2(dba)3, Xantphos, and Cs2CO3, in dioxane at 100° C.

Scheme 4 illustrates one method for preparing compounds of the invention (e.g. Example 73, 76-77). Reaction of the aldehyde BCHO and the amine ANH2in ethanol at 85° C. produces the corresponding hydrazone which is reacted with the sodium salt of diethyl oxaloacetate in acetic acid at 110° C. The resulting intermediate is treated with methylhydrazine according to a protocol involving: 1) heating the two reactants at 110° C. in a solvent mixture of toluene and ethanol, 2) removal of the solvent by concentration, 3) dilution of the residue in acetic acid and heating of the resulting mixture at 100° C. to convert the intermediate obtained from the first step in a 3-hydroxy-2-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one. The hydroxy group can be alkylated by reaction with NaH in DMF, followed by addition of an organic halide (e.g. Examples 76, 77). The hydroxy group can also be converted in the corresponding triflate by treatment with trifluoromethanesulfonic anhydride and triethylamine in dichloromethane. Coupling reaction of the triflate with boronic acids in the presence of a palladium catalyst and a base (e.g. K3PO4) in dioxane at 110° C. allows for the introduction various R3groups.

The invention further includes any variant of the present processes, in which an intermediate product obtainable at any stage thereof is used as starting material and the remaining steps are carried out, or in which the starting materials are formed in situ under the reaction conditions, or in which the reaction components are used in the form of their salts or optically pure material. Compounds of the invention and intermediates can also be converted into each other according to methods generally known to those skilled in the art.

Synthetic Methods

The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon. Temperatures are given in degrees Celsius. If not mentioned otherwise, all evaporations are performed under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (=20-133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR. Abbreviations used are those conventional in the art.

All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesis the compounds of the present invention are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art. Further, the compounds of the present invention can be produced by organic synthesis methods known to one of ordinary skill in the art as shown in the following examples.

ABBREVIATIONS

To a stirred solution of 4-((4-chlorophenyl)(1-methyl-6-oxo-1,6-dihydropyridin-3-ylamino)methyl)-1-(4-methoxybenzyl)-5-methyl-1H-pyrazole-3-carboxylic acid (Step 1.5) (125 mg, 0.254 mmol) in CH2Cl2(2 mL) under Ar was added 1-chloro-N,N,2-trimethyl-1-propenylamine (0.047 mL, 0.355 mmol) at 0° C. The reaction mixture was stirred for 1 hr at 0° C., quenched with a saturated aqueous solution of NaHCO3(75 mL), and extracted with CH2Cl2. The combined organic layers were washed with a saturated aqueous solution of NaHCO3(100 mL), dried over Na2SO4the solvent was and evaporated off under reduced pressure. The crude residue was purified by silica gel column chromatography (CH2Cl2/MeOH 0.5-3.5%) to afford the title product (92 mg, 0.194 mmol, 76% yield) as a greenish solid. tR: 4.32 min (HPLC 1); tR: 0.97 min (LC-MS 2); ESI-MS: 475 [M+H]+(LC-MS 2); Rf=0.65 (CH2Cl2/MeOH 9:1).

To a stirred solution of ethyl 3-methylpyrazole-5-carboxylate (3.11 g, 20.17 mmol) in DMF (50 mL) under Ar at 0° C. was added NIS (5.90 g, 26.2 mmol). The reaction mixture was stirred 20 hr at rt and quenched with 500 mL of water. The resulting precipitate was collected to afford the title product (5.61 g, 20.03 mmol, 99% yield) as a white solid. tR: 3.75 min (HPLC 1); tR: 0.78 min (LC-MS 2); ESI-MS: 281 [M+H]+(LC-MS 2)

To a stirred solution of ethyl 4-iodo-5-methyl-1H-pyrazole-3-carboxylate (Step 1.1) (7.60 g, 27.1 mmol) in DMF (50 mL) under Ar was added NaH (1.302 g, 32.6 mmol) at 0° C. After 15 min, 4-methoxyphenyl chloride (3.70 mL, 27.1 mmol) was added. The reaction mixture was stirred for 1 hr at rt, quenched with a saturated aqueous solution of NaHCO3(100 mL) and extracted with EtOAc (100 mL). The combined organic layers were washed with a saturated aqueous solution of NaHCO3(100 mL), dried over Na2SO4and the solvent was evaporated off under reduced pressure. The crude material was purified by silica gel column chromatography (hexane/EtOAc 5-40%) to afford the title product (6.23 g, 15.57 mmol, 57% yield) as a colorless oil. tR: 5.24 min (HPLC 1); tR: 1.12 min (LC-MS 2); ESI-MS: 401 [M+H]+(LC-MS 2); Rf=0.66 (hexane/EtOAc 1:1).

To a stirred suspension of 3-chloro-2-hydroxy-5-nitropyridine (10 g, 57.3 mmol) and K2CO3(15.84 g, 115 mmol) in DMF (100 mL) under Ar was added MeI (5.37 mL, 86 mmol) at 0° C. The reaction mixture was stirred for 1 hr at rt, concentrated, diluted with water, and extracted with EtOAc. The combined organic layers were washed with water, dried over Na2SO4, and evaporated to afford the title product (10.38 g, 55.0 mmol, 96% yield) as a yellow solid. tR: 2.90 min (HPLC 1).

Reference Example 8

NIS (8.83 g, 39.2 mmol) and TFA (0.825 mL, 10.70 mmol) were added to a stirred solution of ethyl 1H-pyrazole-5-carboxylate (5 g, 35.7 mmol) in CH3CN (120 mL) under Ar. The reaction mixture was stirred for 6 hr at rt, concentrated, diluted with a saturated aqueous solution of NaHCO3, and extracted with EtOAc. The combined organic layers were washed with a saturated aq. NaHCO3solution, dried over Na2SO4and evaporated. The crude material was purified by silica gel column chromatography (hexane/EtOAc 10-50%) to afford the title product (8.31 g, 31.2 mmol, 88% yield) as a white solid. tR: 3.47 min (HPLC 1); tR: 0.72 min (LC-MS 2); ESI-MS: 267 [M+H]+(LC-MS 2); Rf=0.55 (hexane/EtOAc 1:1).

Reference Example 12

Reference Example 15

To a stirred solution of cyclopropyl magnesium bromide in THF (104 mL, 52.1 mmol) under Ar was added di-tert-butyl azodicarboxylate (12 g, 52.1 mmol) in THF (50 mL) at −78° C. The reaction mixture was stirred for 30 min at this temperature, quenched with a saturated aq. NH4Cl solution, and extracted with EtOAc. The combined organic layers were washed with saturated aq. NH4Cl solution, dried over Na2SO4and evaporated. The crude material was purified by silica gel column chromatography (hexane/EtOAc 2-10%) to afford the title product (11.84 g, 43.5 mmol, 83% yield) as a white solid. Rf=0.12 (CPS stain) (hexane/EtOAc 9:1)

A mixture of di-tert-butyl 1-cyclopropylhydrazine-1,2-dicarboxylate (Step 17.1) (11.84 g, 43.5 mmol) and HCl 4N in dioxane (109 mL, 435 mmol) was stirred for 24 hr at rt. The resulting precipitate was collected by filtration to afford the title product (4.90 g, 43.5 mmol, 100% yield) as a white solid.

Reference Example 18

The title compound was prepared in analogy to the procedure described in Step 5.1 using 3-methyl-5-nitropyridin-2-ol. The reaction mixture was filtered and dried, then diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to afford the title product as a yellow powder. tR: 0.59 min (LC-MS 2); ESI-MS: 169 [M+H]+(LC-MS 2).

A mixture of 1,3-dimethyl-5-nitropyridin-2(1H)-one (Step 20.1) (16.4 g, 98 mmol), Pd/C 10% (2.0 g), THF (200 mL) and MeOH (200 mL) was stirred for 3 hr at rt under a hydrogen atmosphere (0.1 bar). The reaction mixture was filtered over celite and concentrated. The crude material was purified by chromatography (CH2Cl2/MeOH/NH398/1/1) to afford the title product (10.3 g, 70.8 mmol, 73% yield) as a green oil. The green oil was triturated in diethyl ether to afford a powder. tR: 0.21 min (LC-MS 2); ESI-MS: 139 [M+H]+(LC-MS 2); Rf=0.35 (CH2Cl2/MeOH 9:1).

Reference Example 22

Sodium nitrite (0.708 g, 10.25 mmol) was added to a mixture of 5-iodo-3-methyl-pyridin-2-ylamine (2 g, 8.55 mmol) and H2SO4(12 mL) at 0° C. The reaction mixture was stirred 15 min at 60° C., allowed to cool down, and poured onto crushed ice. Boric acid (1.057 g, 17.09 mmol) was added and the solution was quickly heated to 100° C. The reaction mixture was cooled down and neutralized with a saturated aq. NH4OH solution. The suspension was filtered to afford the crude title product (1.67 g, 7.11 mmol, 83% yield) as a brown solid. tR: 2.85 min (HPLC 1); tR: 0.62 min (LC-MS 2); ESI-MS: 236 [M+H]+(LC-MS 2).

To a stirred solution of ethyl 1-cyclopropyl-4-iodo-5-methyl-1H-pyrazole-3-carboxylate (Step 23.4) (4.33 g, 13.53 mmol)) in THF (100 mL) under Ar was added TurboGrignard (14.88 mmol) at −10° C. After 15 min, 4-chlorobenzaldehyde (1.901 g, 13.53 mmol) was added. The reaction mixture was stirred for 30 min at this temperature, quenched with a saturated aqueous solution of NH4Cl (150 mL), extracted with EtOAc (2×150 mL). The combined organic layers were washed with a saturated aqueous solution of NH4Cl (75 mL), dried (Na2SO4), and evaporated. The crude material was purified by silica gel column chromatography (hexane/EtOAc 5-25%) to afford the title product (2.12 g, 6.33 mmol, 46.8% yield) as a colorless oil. tR: 1.16 min (LC-MS 2); ESI-MS: 335 [M+H]+(LC-MS 2); Rf=0.62 (hexane/EtOAc 1:1).

To a stirred solution of ethyl 4-((4-chlorophenyl)(hydroxy)methyl)-1-cyclopropyl-3-methyl-1H-pyrazole-5-carboxylate (Step 23.5) (2.12 g, 6.33 mmol) and triethylamine (4.41 mL, 31.7 mmol) in CH2Cl2(4 mL) under Ar was added Ms2O (2.206 g, 12.66 mmol) at −10° C. The reaction mixture was stirred for 30 min at this temperature. 4-Methoxybenzylamine (0.827 ml, 6.33 mmol) was added at 30° C. After 15 h at rt, the reaction mixture was quenched with a saturated aqueous solution of NaHCO3(100 mL), and extracted with CH2Cl2(2×100 mL). The combined organic layers were washed with a saturated aqueous solution of NaHCO3(100 mL), dried (Na2SO4) and evaporated. The crude material was purified by silica gel column chromatography (Hex/EtOAc 5-20%) to afford the title product (2.22 g, 4.89 mmol, 77% yield) as a colorless oil. tR: 1.12 min (LC-MS 2); ESI-MS: 454 [M+H]+(LC-MS 2); Rf=0.85 (hexane/EtOAc 1:1).

To a stirred solution of 4-((4-chlorophenyl)((4-methoxybenzyl)amino)methyl)-1-cyclopropyl-3-methyl-1H-pyrazole-5-carboxylic acid (Step 23.7) (2.30 g, 4.59 mmol) in CH2Cl2(30 mL) under Ar was added 1-chloro-N,N,2-trimethyl-1-propenylamine (0.846 mL, 6.43 mmol) at 0° C. The reaction mixture was stirred for 1 hr at rt, quenched with a saturated aqueous solution of NaHCO3(75 mL), and extracted with CH2Cl2(2×100 mL) The combined organic layers were washed with a saturated aqueous solution of NaHCO3(100 mL), dried (Na2SO4) and evaporated. The crude material was purified by silica gel column chromatography (Hex/EtOAc 5-30%) to afford the title product (1.82 g, 4.24 mmol, 92% yield) as a yellow oil. tR: 5.76 min (HPLC 1); tR: 1.27 min (LC-MS 2); ESI-MS: 408 [M+H]+(LC-MS 2); Rf=0.66 (hexane/EtOAc 1:1).

In a 20-mL MW vial was introduced (Step 23.8) (1.82 g, 4.46 mmol) and TFA (10.31 mL, 134 mmol). The reaction mixture was stirred for 2 hr at 100° C. and additional 2 hr at 120° C. MW irradiation. The reaction mixture was quenched with a saturated aq. NaHCO3solution and extracted with CH2Cl2. The combined organic layers were washed with a saturated aq. NaHCO3solution, dried over Na2SO4and evaporated. The crude material was purified by silica gel column chromatography (hexane/EtOAc 20-55%) to afford the title product (713 mg, 2.478 mmol, 55% yield) as a yellow solid. tR: 4.20 min (HPLC 1); tR: 0.93 min (LC-MS 2); ESI-MS: 288 [M+H]+(LC-MS 2); Rf=0.32 (hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described in Step 1.5 using ethyl 4-((4-chlorophenyl)((4-methoxybenzyl)amino)methyl)-1,5-dimethyl-1H-pyrazole-3-carboxylate (Step 25.3). The reaction mixture was quenched with 0.5N HCl, diluted with EtOAc, and cooled down to 0° C. The suspension was filtered to afford crude product as a white solid. tR: 3.38 min (HPLC 1); tR: 0.73 min (LC-MS 2); ESI-MS: 400 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 1.5 using ethyl 4-((4-chlorophenyl)(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-ylamino)methyl)-1-(4-methoxy-benzyl)-5-methyl-1H-pyrazole-3-carboxylate (Step 27.1). The reaction mixture was quenched with 0.5N HCl and diluted with EtOAc. The suspension was filtered to afford crude product as a white solid. tR: 4.07 min (HPLC 1); tR: 0.96 min (LC-MS 2); ESI-MS: 507 [M+H]+(LC-MS 2).

Reference Example 29

To a stirred solution of 3-methyl-5-nitrobenzo[d]isoxazole (Step 33.2) (1.8 g, 10.10 mmol) in AcOH (40 mL) was added a solution of tin(II) chloride dihydrate (6.84 g, 30.3 mmol) in HCl (15 mL, 494 mmol). The reaction mixture was stirred for 1 hr at 100° C., quenched with a saturated aq. NaHCO3solution, diluted with water, and extracted with CH2Cl2. The combined organic layers were washed once with a saturated aq. NH4Cl solution, dried over Na2SO4and evaporated. The crude material was purified by silica gel column chromatography (hexane/EtOAc 20-50) to afford the title product (458 mg, 3.09 mmol, 31% yield) as a pink solid. tR: 0.50 min (LC-MS 2); ESI-MS: 149 [M+H]+(LC-MS 2); Rf=0.45 (hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described in Step 10.1 using ethyl 1-cyclopropyl-1H-pyrazole-5-carboxylate (Step 34.1). The crude product was purified by silica gel column chromatography (hexane/EtOAc 2-10%). tR: 5.32 min (HPLC 1); tR: 1.12 min (LC-MS 2); ESI-MS: 307 [M+H]+(LC-MS 2); Rf=0.37 (hexane/EtOAc 9:1).

The title compound was prepared in analogy to the procedure described in Step 17.3 using isopropylhydrazine hydrochloride and ethyl 4-(dimethylamino)-2-oxobut-3-enoate at 120° C. for hr. The crude product was purified by silica gel column chromatography (hexane/EtOAc 2.5-40%). tR: 4.74 min (HPLC 1); tR: 1.00 min (LC-MS 2); ESI-MS: 183 [M+H]+(LC-MS 2); Rf=0.95 (hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described in Step 10.1 using ethyl 1-isopropyl-1H-pyrazole-5-carboxylate (Step 36.1). The crude product was purified by silica gel column chromatography (hexane/EtOAc 2-10%). tR: 5.64 min (HPLC 1); tR: 1.20 min (LC-MS 2); ESI-MS: 309 [M+H]+(LC-MS 2); Rf=0.41 (hexane/EtOAc 9:1).

The title compound was prepared in analogy to the procedure described in Step 10.1 using ethyl 1-cyclopropyl-1H-pyrazole-3-carboxylate (Step 37.1). The crude product was purified by silica gel column chromatography (hexane/EtOAc 5-20%). tR: 4.43 min (HPLC 1); tR: 0.95 min (LC-MS 2); ESI-MS: 307 [M+H]+(LC-MS 2); Rf=0.70 (hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described in Step 17.3 using cyclopropylhydrazine (Step 17.2) and ethyl 5,5,5-trifluoro-2,4-dioxopentanoate at 100° C. for 2 hr. The crude product was purified by silica gel column chromatography (Hexane/EtOAc 9:1). tR: 1.10 min (LC-MS 2); ESI-MS: 249 [M+H]+(LC-MS 2); Rf=0.17 (hexane/EtOAc 9:1).

LDA (3.70 mL, 6.66 mmol) was added dropwise to a stirred solution of ethyl 1-cyclopropyl-5-(trifluoromethyl)-1H-pyrazole-3-carboxylate (Step 40.1) (1.18 g, 4.75 mmol) in THF (30 mL). After 15 minutes at −78° C., a solution of 4-chlorobenzaldehyde (668 mg, 4.75 mmol) in THF (5 mL) was slowly added. The reaction was stirred at −78° C. for 15 min, quenched with 1 mL of saturated NH4Cl solution, partitioned between EtOAc and water and both phases separated. The aq. phase was extracted with EtOAc and the combined organic phases were washed with brine, dried over Na2SO4and concentrated. The crude was purified by silica gel column chromatography (hexane/20% EtOAc) to afford the title product (1.5 g, 3.40 mmol, 71% yield) as a yellow solid. tR: 1.27 min (LC-MS 2); ESI-MS: 389 [M+H]+(LC-MS 2); Rf=0.22 (hexane/EtOAc 8:2).

To a solution of ethyl 4-(((5-chloro-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)amino)(4-chlorophenyl)methyl)-1-cyclopropyl-5-(trifluoromethyl)-1H-pyrazole-3-carboxylate (Step 40.3) (350 mg, 0.569 mmol) in THF (3 mL) and MeOH (3 mL) was added 2M NaOH (2.84 mL, 5.69 mmol). The resulting mixture was stirred at RT for 1 hr. Volatiles were evaporated and the resulting aq. phase was adjusted to pH 5 with 2N HCl, extracted twice with EtOAc and the combined organic phases were washed with brine, dried over Na2SO4and concentrated under reduced pressure. The crude was triturated in EtOAc to afford the title product (222 mg, 0.443 mmol, 78% yield) as an off-white solid. tR: 1.00 min (LC-MS 2); ESI-MS: 501/503 [M+H]+, ESI-MS: 499/501 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 17.3 using isopropylhydrazine hydrochloride and ethyl 4-(Dimethylamino)-2-oxobut-3-enoate at 120° C. for hr. The crude product was purified by silica gel column chromatography (hexane/EtOAc 2.5-40%). tR: 3.73 min (HPLC 1); tR: 0.80 min (LC-MS 2); ESI-MS: 183 [M+H]+(LC-MS 2); Rf=0.69 (hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described in Step 10.1 using ethyl 1-isopropyl-1H-pyrazole-3-carboxylate (Step 41.1). The crude product was purified by silica gel column chromatography (hexane/EtOAc 5-25%). tR: 4.62 min (HPLC 1); tR: 0.98 min (LC-MS 2); ESI-MS: 309 [M+H]+(LC-MS 2); Rf=0.75 (hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described in Step 17.3 using cyclopropylhydrazine (Step 17.2) and ethyl 5,5,5-trifluoro-2,4-dioxopentanoate at 100° C. for 2 hr. The crude product was purified by silica gel column chromatography (hexane/EtOAc 9:1). tR: 1.23 min (LC-MS 2); ESI-MS: 249.2 [M+H]+(LC-MS 2); Rf=0.34 (hexane/EtOAc 9:1).

The title compound was prepared in analogy to the procedure described in Step 10.3 using ethyl 4-((4-chlorophenyl)(hydroxy)methyl)-1-cyclopropyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxylate

The title compound was prepared in analogy to the procedure described in Step 10.3 using ethyl 4-((4-chlorophenyl)(hydroxy)methyl)-1-cyclopropyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxylate

To a colorless solution of 3-methyl-salicylic acid (1 g, 6.57 mmol) in Et2O (50 mL) under argon and cooled down to 0° C. was added dropwise methyllithium 1.6M in Et2O (12.32 mL, 19.72 mmol) over 20 min. The resulting mixture was stirred at this temperature for 30 min then allowed to warm up and stir overnight at RT. The reaction mixture was slowly poured into a stirred mixture of 100 g of ice and HCl 4N (50 mL) and extracted twice with EtOAc. The combined organic layers were washed with 10% NaHCO3solution and brine, dried over MgSO4, filtered and concentrated under reduced pressure to afford the title product (920 mg, 5.94 mmol, 90% yield) as a pale yellow oil. tR: 0.91 min (LC-MS 2); ESI-MS: 481/483 [M+H]+, ESI-MS: 479/501 [M−H]−(LC-MS 2); Rf=0.55 (hexane/EtOAc 3:1).

(E)-1-(2-hydroxy-3-methylphenyl)ethanone oxime (Step 46.2) (5.5 g, 30 mmol) was added into Ac2O (48.1 mL, 509 mmol) under argon and the resulting mixture was stirred at RT for 1.5 hr. The reaction was concentrated to 10 mL volume under reduced pressure; the resulting suspension was diluted with cold water and stirred at RT until a fine precipitate occurred. The resulting solid was filtrated off, washed with water and dried to afford the title product (6.03 g, 28.8 mmol, 96% yield) as colorless solid. tR: 1.08 min (LC-MS 2); ESI-MS: 208 [M+H]+; Rf=0.60 (hexane/EtOAc 1:1).

3,7-dimethylbenzo[d]isoxazole (Step 46.4) (2 g, 12.77 mmol) was dissolved in H2SO4(5 mL), cooled down and stirred at 0° C. HNO3(0.878 mL, 12.77 mmol) was slowly added and the resulting mixture was stirred at 0° C. for 1 hr. The reaction was diluted with water (60 mL) and extracted three times with CH2Cl2. The combined organic layers were washed with NaHCO3solution and brine, dried over MgSO4, filtered and concentrated under reduced pressure. The resulting yellow solid was triturated with Et2O, filtrated off, washed with Et2O and dried to afford the title product (1.86 g, 9.29 mmol, 73% yield) as yellow solid. tR: 0.98 min (LC-MS 2); ESI-MS: 193 [M+H]+.

To a suspension of 3,7-dimethyl-5-nitrobenzo[d]isoxazole (Step 46.5) (50 mg, 0.260 mmol) in acetic acid (1.5 mL) was added dropwise a solution of SnCl2.2H2O (176 mg, 0.781 mmol) in HCl conc (0.316 mL, 10.41 mmol) and the resulting mixture was stirred at RT overnight. The reaction was poured into a cold 4N NaOH solution and extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane/EtOAc 0-55%) to afford the title product (25 mg, 0.153 mmol, 59% yield) as beige solid. tR: 0.57 min (LC-MS 2); ESI-MS: 163 [M+H]+; Rf=0.23 (hexane/EtOAc 1:1).

To a stirred solution of 5-bromo-2,4-dimethoxypyrimidine (400 g, 1.826 mol) in anhydrous THF (3 L) under argon and cooled down to 0° C. was added dropwise TurboGrignard (1.821 L, 2.367 mol). The resulting mixture was stirred at 0° C. until exothermic ceased then, allowed to warm up and stir at RT for 30 min. A solution of di-tert-butyl azodicarboxylate in anhydrous THF (1 L) was added dropwise to the mixture and the reaction was stirred at RT for 1 hr. The reaction was slowly quenched with a saturated aq. NH4Cl solution (2 L), diluted with EtOAc (2 L) and water (2 L) and both phases separated. The aq. phase was extracted with EtOAc (3 L), combined organic layers were washed with brine (3 L), dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting yellow oil was dissolved in Hexane (3 L) and the suspension was stirred at 0° C. for 3 hr, filtrated off and dried to afford a first batch of white crystals. The mother liquor was concentrated under reduced pressure and purified to afford a second batch of white crystals. The two batches were combined to afford the title product (507 g, 1.369 mol, 75% yield) as white crystals. tR: 1.03 min (LC-MS 1); ESI-MS: 371 [M+H]+, ESI-MS: 369 [M−H]−(LC-MS 1).

Di-tert-butyl 1-(2,4-dimethoxypyrimidin-5-yl)hydrazine-1,2-dicarboxylate (Step 46.7) (453 g, 1.223 mol) was dissolved in MeOH (2.5 L) and cooled down to 0° C. HCl 4N in gioxane (2.5 L, mol) was added and the resulting mixture was stirred at RT overnight. The reaction was concentrated under reduced pressure, NH34 N (2 L) was added, the resulting mixture was stirred for 1 hr and concentrated under reduced pressure. CH2Cl2(2 L) was added, the suspension was filtrated off and the filtrate was concentrated under reduced pressure. The crude product was stirred with Et2O (2 L) at 0° C. for 30 min. The resulting suspension was filtrated off and dried to afford the title product (150 g, 864 mmol, 70% yield) as light beige solid. tR: 0.32 min (LC-MS 1); ESI-MS: 171 [M+H]+(LC-MS 1).

5-hydrazinyl-2,4-dimethoxypyrimidine (Step 46.8) (140 g, 823 mmol) was dissolved in toluene (3 L) under nitrogen atmosphere and ethyl-2,4-dioxo-5-methylhexanoate (230 g, 1234 mmol) was added dropwise over 15 min. The reaction mixture was heated up and stirred at 110° C. for 1 hr. The brown solution was quenched with saturated aq. NaHCO3solution (2 L) and extracted with EtOAc (2×2 L). The combined organic layers were washed with brine (2 L), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was stirred in hexane (1 L) at RT and the resulting suspension was filtrated off and dried to afford the title product (193 g, 596 mmol, 72% yield) as white crystals. tR: 1.01 min (LC-MS 1); ESI-MS: 321 [M+H]+(LC-MS 1).

The title compound was prepared in analogy to the procedure described in Step 10.1 using ethyl 1-(2,4-dimethoxypyrimidin-5-yl)-5-isopropyl-1H-pyrazole-3-carboxylate (Step 46.9) at 80° C. for 6 hr. Purification by silica gel column chromatography (heptane/EtOAc 0-100%) afforded the title product as white crystals. tR: 1.14 min (LC-MS 1); ESI-MS: 4477 [M+H]+(LC-MS 1).

To a stirred solution of ethyl 4-((4-chlorophenyl)(hydroxy)methyl)-1-(2,4-dimethoxypyrimidin-5-yl)-5-isopropyl-1H-pyrazole-3-carboxylate (Step 46.11) (500 mg, 1.085 mmol) and triethylamine (0.756 mL, 5.42 mmol) in CH2Cl2(10 mL) cooled down to 0° C. was added Ms2O (378 mg, 2.170 mmol) and the reaction was stirred for 1 hr at this temperature. 3,7-dimethylbenzo[d]isoxazol-5-amine (Step 46.6) (194 mg, 1.193 mmol) was added and the reaction mixture was allowed to warm up to RT and stir for 1 hr. The reaction was quenched with aq. NaH2PO4solution and extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane/EtOAc 10-100%) to afford the title product (460 mg, 0.684 mmol, 63% yield) as yellow amorphous solid. tR: 1.38 min (LC-MS 2); ESI-MS: 605 [M+H]+(LC-MS 2); Rf=0.25 (hexane/EtOAc 1:1).

To a stirred solution of ethyl 4-((4-chlorophenyl)((3,7-dimethylbenzo[d]isoxazol-5-yl)amino)-methyl)-1-(2,4-dimethoxypyrimidin-5-yl)-5-isopropyl-1H-pyrazole-3-carboxylate (Step 46.12) (455 mg, 0.677 mmol) in MeOH (10 mL) cooled down to 0° C. was added dropwise 4M NaOH (2.54 mL, 10.15 mmol). The reaction mixture was allowed to warm up and stir at RT for 45 min, acidified with 4N HCl and MeOH was removed under reduced pressure. The aq. layer was extracted with EtOAc, combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure to afford the title product (450 mg, 0.663 mmol, 98% yield) as a beige amorphous solid. tR: 1.20 min (LC-MS 2); ESI-MS: 577 [M+H]+, ESI-MS: 575 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 17.3 using 5-hydrazinyl-2,4-dimethoxypyrimidine (Step 46.8) and ethyl 5,5,5-trifluoro-2,4-dioxopentanoate at 100° C. for 2 hr. The crude product was purified by silica gel column chromatography (hexane/30% EtOAc). tR: 1.09 min (LC-MS 2); ESI-MS: 347 [M+H]+(LC-MS 2); Rf=0.31 (hexane/30% EtOAc).

The title compound was prepared in analogy to the procedure described in Step 40.2 using ethyl 1-(2,4-dimethoxypyrimidin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-3-carboxylate (Step 47.1). The crude product was purified by silica gel column chromatography (hexane/35% EtOAc). tR: 1.24 min (LC-MS 2); ESI-MS: 487 [M+H]+(LC-MS 2); Rf=0.28 (hexane/35% EtOAc).

The title compound was prepared in analogy to the procedure described in Step 40.3 using ethyl 4-((4-chlorophenyl)(hydroxy)methyl)-1-(2,4-dimethoxypyrimidin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-3-carboxylate (Step 47.2) and 5-amino-3-chloro-1-methylpyridin-2(1H)-one (Step 5.2). The crude product was purified by silica gel chromatography (50% EtOAc/CH2Cl2). tR: 1.21 min (LC-MS 2); ESI-MS: 627 [M+H]+(LC-MS 2); Rf=0.20 (50% EtOAc/CH2Cl2).

Reference Example 48

Reference Example 50

A round-bottomed flask was charged 5-amino-1,3-dimethylpyridin-2(1H)-one (Step 20.2) (3 g, 21.71 mmol), 4-chlorobenzaldehyde (2.348 g, 16.70 mmol) and ethyl 2,4-dioxovalerate (3.17 g, 20.04 mmol) in AcOH (50 mL) and the reaction mixture was heated up and stirred at 125° C. for 1 hr. The reaction mixture was concentrated under reduced pressure, quenched with a saturated aq. NaHCO3solution, extracted with EtOAc. The organic layer was discarded and the aq. phase was adjusted to pH 1-2, extracted EtOAc. The combined organic layers were dried over Na2SO4and concentrated under reduced pressure to afford the title product (3.82 g, 8.71 mmol, 52% yield) as a black solid. tR: 3.54 min (HPLC 1); tR: 0.75 min (LC-MS 2); ESI-MS: 373 [M+H]+(LC-MS 2).

To a stirred solution of 4-chloro-2-methoxypyrimidine (5.7 g, 39.4 mmol) in EtOH (100 mL) under Ar was added hydrazine hydrate (3.83 mL, 79 mmol) and the reaction mixture was heated up and stirred at 85° C. for 1 hr. Volatiles were removed under reduced pressure and the resulting crude material was purified by silica gel column chromatography (CH2Cl2/MeOH/1-5%/NH31%) to afford the title product (4.40 g, 31.4 mmol, 80% yield) as white solid. ESI-MS: 141 [M+H]+(LC-MS 2); Rf=0.47 (CH2Cl2/MeOH 9:1).

The title compound was prepared in analogy to the procedure described in Step 1.5 using ethyl 4-((4-chlorophenyl)((1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)amino)methyl)-1-(2-methoxypyrimidin-4-yl)-3-methyl-1H-pyrazole-5-carboxylate (Step 59.5) for 2 hr at RT. tR: 0.92 min (LC-MS 2); ESI-MS: 493 [M+H]+, ESI-MS: 491 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 57.1 using 5-amino-1,3-dimethylpyridin-2(1H)-one (Step 20.2), 4-chlorobenzaldehyde and ethyl 2,4-dioxohexanoate under reflux for 7 hr. The crude product was purified by silica gel chromatography (hexane/CH2Cl2/MeOH 40:60:10 to 0:60:10). tR: 0.82 min (LC-MS 2); ESI-MS: 387 [M+H]+(LC-MS 2).

Reference Example 61

To a solution of 2-chloro-3-methyl-5-nitropyridine (35 g, 200 mmol) in EtOH (400 mL) was added hydrazine hydrate (30.0 g, 600 mmol) and the resulting reaction mixture was stirred at 60° C. for 1 hr. The reaction mixture was cooled down with an ice bath, the resulting precipitate was filtrated off, washed with cold H2O and Et2O and dried at 50° C. under reduced pressure to afford the title product (25.40 g, 113 mmol, 98%) as a yellow solid. tR: 0.43 min (LC-MS 2); ESI-MS: 169 [M+H]+; ESI-MS: 167 [M−H]−(LC-MS 2).

To a suspension 2-hydrazinyl-3-methyl-5-nitropyridine (Step 67.1) (33.2 g, 198 mmol) in dioxane (175 mL) was added Ac2O (20.5 mL, 217 mmol) and the reaction was stirred at RT for min. The reaction mixture was poured onto ice-water and stirred for 1 hr at 0° C. The precipitate was collected by filtration, washed with H2O and Et2O, and dried under reduced pressure at 50° C. to afford the title product (41.5 g, 198 mmol, 99.5% yield) as light beige solid. tR: 0.45 min (LC-MS 2); ESI-MS: 211 [M+H]+; ESI-MS: 209 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 10.3 using ethyl 4-((4-chlorophenyl)(hydroxy)methyl)-1-(2,4-dimethoxypyrimidin-5-yl)-5-isopropyl-1H-pyrazole-3-carboxylate (Step 46.11) and 3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-amine (Step 67.4) overnight at RT. tR: 1.02 min (LC-MS 2); ESI-MS: 605 [M+H]+(LC-MS 2).

To a stirred solution of 3-amino-2-methoxypyridine (5 g, 40.3 mmol) in 6N HCl (80 mL) was added dropwise a solution of NaNO2(2.78 g, 40.3 mmol) in water (50 mL) at 0° C. After 30 min at this temperature, a solution of SnCl2.2H2O (22.72 g, 101 mmol) in 6N HCl (80 mL) was added dropwise at 0° C. The reaction mixture was stirred 90 min at 0° C. The reaction mixture was adjusted to pH˜10-11 with a solution of KOH 40% in water and extracted with EtOAc. The combined organic layers were washed with water, dried over Na2SO4and evaporated under reduced pressure to afford the title product (4.97 g, 33.9 mmol, 84% yield) as red oil. tR: 0.31 min (LC-MS 2); ESI-MS: 140 [M+H]+(LC-MS 2).

To a stirred solution of 4-(4-chlorophenyl)-5-(4-methoxybenzyl)-1-(2-methoxypyridin-3-yl)-3-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one (Step 71.3) (2.8 g, 5.90 mmol) in CH3CN (80 mL) and H2O (20 mL) was added CAN (9.70 g, 17.69 mmol) and the reaction mixture was stirred at RT for 16 hr. The reaction mixture was quenched with brine and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4and evaporated under reduced pressure. The crude material was purified by silica gel column chromatography (CH2Cl2/MeOH 1-2.5%). The residue was triturated in CH2Cl2to afford the title product (1.06 g, 2.93 mmol, 50% yield) as white solid. tR: 4.36 min (HPLC 1); tR: 0.98 min (LC-MS 2); ESI-MS: 355 [M+H]+(LC-MS 2); Rf=0.39 (CH2Cl2/MeOH 9:1).

To a stirred solution of 5-amino-1,3-dimethylpyridin-2(1H)-one (Step 20.2) (5.2 g, 37.6 mmol) in EtOH (100 mL) were added 4-chlorobenzaldehyde (5.04 g, 35.8 mmol) and AcOH (0.410 mL, 7.17 mmol). The resulting mixture was heated up and stirred at 85° C. for 1 hr. The reaction was concentrated under reduced pressure and the resulting crude mixture was triturated in Et2O to afford the title product (7.6 g, 29.2 mmol) as beige solid. tR: 0.92 min (LC-MS 2).

To a stirred solution of (E)-5-((4-chlorobenzylidene)amino)-1,3-dimethylpyridin-2(1H)-one (Step 73.1) (4 g, 15.34 mmol) in AcOH (40 mL) was added diethyl oxaloacetate sodium salt (6.45 g, 30.7 mmol) and the resulting mixture was heated up and stirred at 110° C. for 1 hr. The reaction was concentrated under reduced pressure, diluted with CH2Cl2and water and both phases separated. The aqueous layer was extracted twice with CH2Cl2, combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was triturated in Et2O to afford the title product (4.42 g, 8.34 mmol, 54% yield) as beige solid. tR: 0.86 min (LC-MS 2); ESI-MS: 403 [M+H]+, ESI-MS: 401 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described for Step 46.7 using 1-methyl-5-bromopyrazole. The crude product was purified by silica gel column chromatography (hexane/EtOAc 5-30%). tR: 4.46 min (HPLC 1); tR: 1.04 min (LC-MS 2); ESI-MS: 496 [M+H]+(LC-MS 2); Rf=0.51 (Hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described for Step 17.2 using di-tert-butyl 1-(1-methyl-1H-pyrazol-5-yl)hydrazine-1,2-dicarboxylate (Step 74.1). ESI-MS: 113 [M+H]+(LC-MS 2).

Blank

The title compound was prepared in analogy to the procedure described in Example 78 using 5-(4-chlorophenyl)-1-(3,7-dimethylbenzo[d]isoxazol-5-yl)-4-propionylpyrrolidine-2,3-dione (Step 79.1) and 2-hydrazinylethanol. The crude material was purified by silica gel chromatography (hexane/EtOAc/MeOH 90:10:1 to 50:50:5 to 0:10:1). tR: 1.10 min (LC-MS 2); ESI-MS: 451 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 60.1 using 3,7-dimethylbenzo[d]isoxazol-5-amine (Step 46.6) at 110° C. for 5 hr. The crude material was purified by silica gel chromatography (hexane/CH2Cl2/MeOH 20:80:8 to 0: 100:10) followed by recrystallization in EtOAc/hexane. tR: 1.08 min (LC-MS 2); ESI-MS: 411 [M+H]+(LC-MS 2); Rf=0.18 (EtOAc).

The title compound was prepared in analogy to the procedure described in Example 79 using 5-(4-chlorophenyl)-1-(3,7-dimethylbenzo[d]isoxazol-5-yl)-4-propionylpyrrolidine-2,3-dione (Step 9.1) and 2-hydrazinylethanol. tR: 1.04 min (LC-MS 2); ESI-MS: 451 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Example 57 using 5-(3-acetyl-2-(4-chlorophenyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-1,3-dimethylpyridin-2(1H)-one (Step 57.1). tR: 3.36 min (HPLC 1).

The title compound was prepared in analogy to the procedure described for Step 46.7 using 5-bromo-1-methylimidazole. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 1-4%). tR: 3.29 min (HPLC 1); tR: 0.72 min (LC-MS 2); ESI-MS: 313 [M+H]+(LC-MS 2); Rf=0.49 (CH2Cl2/MeOH 9:1).

The title compound was prepared in analogy to the procedure described for Step 17.2 using di-tert-butyl 1-(1-methyl-1H-imidazol-5-yl)hydrazine-1,2-dicarboxylate (Step 83.1) at RT for 2 hr. ESI-MS: 113 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 67.1 using 2-chloro-3-methoxy-5-nitropyridine. tR: 0.46 min (LC-MS 2); ESI-MS: 185.0 [M+H]+(LC-MS 2); ESI-MS: 183 [M−H]−(LC-MS 2).

To a suspension of 2-hydrazinyl-3-methoxy-5-nitropyridine (Step 84.1) (20 g, 106 mmol) in dioxane (170 mL) was added at RT Ac2O (13.1 mL, 138 mmol) and the reaction mixture was stirred for 1 hr at RT. The reaction mixture was poured onto ice-water (700 mL) and stirred for 1 hr at 0° C. The precipitate was collected by filtration, washed with H2O and Et2O, and dried under reduced pressure at 50° C. to afford the title product (23.3 g, 101 mmol, 95% yield) as a yellow solid. tR: 0.45 min (LC-MS 2); ESI-MS: 227 [M+H]+(LC-MS 2); ESI-MS: 225 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 60.1 using 8-methoxy-3-methyl-[1,2,4]triazolo[4,3-a]pyridin-6-amine (Step 84.4) at 110° C. for 2 hr. The reaction mixture was concentrated under reduced pressure; the residue was diluted with 0.5N NaOH and extracted with EtOAc. The combined organic layers were washed with 0.1N NaOH. The combined aqueous layers were acidified with 4N HCl, saturated with NaCl and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. tR: 0.77 min (LC-MS 2); ESI-MS: 427 [M+H]+(LC-MS 2); ESI-MS: 425 [M−H]−(LC-MS 2).

A MW vial was charged with 5-bromo-1-fluoro-3-methyl-2-nitrobenzene (500 mg, 2.137 mmol) and methylamine 2M in THF (5 mL, 10.0 mmol). The MW vial was sealed and the reaction mixture was submitted to MW irradiation for 30 min at 100° C. The reaction was cooled down to RT and concentrated under reduced pressure to afford the title product (520 mg, 2.122 mmol, 99% yield) as yellow solid. tR: 1.19 min (LC-MS 2); ESI-MS: no ionisation (LC-MS 2).

To a solution of 5-bromo-N,3-dimethyl-2-nitroaniline (Step 85.1) (2.7 g, 11.02 mmol) in THF (100 mL) and MeOH (100 mL) was added Raney Nickel (189 mg, 2.203 mmol) and the resulting mixture was stirred under hydrogen atmosphere at RT for 16 hr. The reaction was filtered through a pad of Celite and the resulting filtrate was concentrated under reduced pressure to afford the title product (2.5 g, 10.56 mmol, 96% yield) as off-white solid. tR: 0.94 min (LC-MS 2); ESI-MS: 214 [M+H]+(LC-MS 2).

To a solution of 5-bromo-N1,3-dimethylbenzene-1,2-diamine (Step 85.2) (2.5 g, 11.62 mmol) in HCl cc (15 mL, 494 mmol) cooled down to 0° C. was slowly added a solution of NaNO2(0.962 g, 13.95 mmol) in water (25 mL). The resulting mixture was allowed to warm up and stir at RT for 2 hr. NaOH was added until basic pH, a precipitate occurred. The resulting solid was filtrated off, washed with water and dried under reduced pressure to afford the title product (2.5 g, 9.95 mmol, 86% yield) as beige solid. tR: 0.93 min (LC-MS 2); ESI-MS: 228 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Example 87 using 5-(4-chlorophenyl)-1-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-4-propionylpyrrolidine-2,3-dione (Step 70.1). tR: 0.90 min (LC-MS 2); ESI-MS: 465 [M+H]+(LC-MS 2).

To a stirred solution of 4-(4-chlorophenyl)-5-(4-methoxybenzyl)-3-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one (Step 85.4) (1 g, 2.72 mmol), PPh3(0.792 g, 3.02 mmol) and cyclobutanol (0.321 mL, 4.11 mmol) in THF (25 mL) under Ar was added dropwise DEAD in toluene (1.424 ml, 3.13 mmol) and the reaction mixture was stirred 16 h at RT. PPh3(0.792 g, 3.02 mmol) and DEAD in toluene (1.424 ml, 3.13 mmol) were further added and the reaction mixture was stirred for 24 hr at RT. The reaction was quenched with a saturated aq. NaHCO3solution and extracted with CH2Cl2. The combined organic layers were washed with a saturated aq. NaHCO3solution, dried over Na2SO4and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography (hexane/EtOAc 10-30%) to afford the title product (899 mg, 2.024 mmol, 75% yield) as white solid. tR: 6.24 min (HPLC 1); tR: 1.37 min (LC-MS 2); ESI-MS: 422 [M+H]+(LC-MS 2); Rf=0.83 (hexane/EtOAc 1:1).

The title compound was prepared in analogy to the procedure described in Example 91 using 4-acetyl-1-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-5-(4-(trifluoromethoxy)phenyl)pyrrolidine-2,3-dione (Step 95.1). tR: 1.03 min (LC-MS 2); ESI-MS: 459 [M+H]+(LC-MS 2).

Reference Example 99

Methylamine 2M in THF (67 mL, 134 mmol) was added to a stirred solution of 2,6-dichloro-3-nitropyridine (12, 93 g, 67 mmol) in THF (100 mL) at 0° C. and the resulting mixture was stirred at RT for 16 hr. The reaction was concentrated under reduced pressure; the residue was partitioned between water and EtOAc and both phases separated. The aq. layer was extracted with EtOAc, the combined organic layer were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography (25% EtOAc/hexane) to afford the title product (10.54 g, 53.4 mmol, 80% yield) as yellow solid. tR: 0.96 min (LC-MS 2); ESI-MS: 187 [M+H]+(LC-MS 2); Rf=0.72 (25% EtOAc/hexane).

The title compound was prepared in analogy to the procedure described in Step 85.2 using 6-chloro-N-methyl-3-nitropyridin-2-amine (Step 102.1) at RT for 23 hr. The crude material was purified by silica gel column chromatography (25% EtOAc/hexane) to afford a purple solid. tR: 0.64 min (LC-MS 2); ESI-MS: 158 [M+H]+(LC-MS 2); Rf=0.12 (25% EtOAc/Hexane).

Reference Example 103

The tile compound was prepared in analogy to the procedure described in Example 68 using 5-(4-chlorophenyl)-1-(8-methoxy-3-methyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-4-propionylpyrrolidine-2,3-dione (Step 84.5) and methyl hydrazine in MeOH. The crude material was first purified by silica gel column chromatography (hexane/EtOAc/MeOH 75:25:5 to 5:1) followed by preparative achiral SFC (Propyl-pyridyl-urea, gradient 13-18% in 6 min_total 11 min). tR: 0.89 min (LC-MS 2); ESI-MS: 437 [M+H]+(LC-MS 2).

Reference Example 106

The tile compound was prepared in analogy to the procedure described in Example 68 using 5-(4-chlorophenyl)-1-(8-methoxy-3-methyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-4-propionylpyrrolidine-2,3-dione (Step 84.5) and (2-(2,2,2-trifluoroethoxy)ethyl)hydrazine hydrochloride in MeOH and TEA. The crude material was first purified by silica gel chromatography (hexane/EtOAc/MeOH 75:25:5 to 5:1) followed by preparative achiral SFC (Propyl-pyridyl-urea, gradient 13-18% in 6 min_total 11 min). tR: 1.04 min (LC-MS 2); ESI-MS: 549 [M+H]+(LC-MS 2).

To a suspension of 2,6-dichloro-4-methylpyridine (1 g, 6.17 mmol) in trifluoroacetic acid anhydride (5 mL, 35.4 mmol) cooled down to 0° C. was added dropwise nitric acid (0.579 mL, 12.96 mmol) into it. The resulting solution was stirred at RT for 18 hr. The reaction mixture was added slowly to a chilled solution of sodium metabisulfite (1.183 g, 6.17 mmol) in water (8 mL) and stirred at RT for 2 hr. The reaction mixture was neutralized to pH 7 using 8N NaOH solution and extracted twice with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to afford the title product (1.187 g, 5.73 mmol, 93% yield) as white solid. tR: 1.07 min (LC-MS 2); ESI-MS: 208 [M+H]+(LC-MS 2);1H NMR (400 MHz, DMSO-d6) δ ppm 2.39 (s, 3H) 7.90 (s, 1H).

A mixture of 3,6-dichloro-4-methylpyridazine (5 g, 30.7 mmol) in hydrazine hydrate (30.4 mL, 153 mmol) was heated up and stirred at 80° C. for 24 hr. The reaction was cooled down to RT; the resulting solid was filtrated off, washed with water and dried under reduced pressure. Recrystallization in hot EtOH followed by trituration in EtOH and ultra sounds afforded the title product (721 mg, 4.55 mmol, 14.8% yield) as white solid. tR: 0.34 min (LC-MS 2); ESI-MS: 159 [M+H]+(LC-MS 2).

6-chloro-3-hydrazinyl-4-methylpyridazine (Step 111.1) (721 mg, 4.55 mmol) was dissolved in AcOH (15 mL, 262 mmol) and the resulting mixture was heated up and stirred at 115° C. for 1 hr. The reaction was cooled down to RT, diluted with CH2Cl2and poured into a saturated aq. NaHCO3solution. The aq. layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to afford the title product (752 mg, 3.91 mmol, 84% yield) as grey solid. tR: 0.59 min (LC-MS 2); ESI-MS: 183 [M+H]+(LC-MS 2).

Reference Example 112

A MW vial was charged with 3,4,6-trichloropyridazine (5 g, 27.3 mmol) and 7N NH3in MeOH (19.47 mL, 136 mmol). The MW vial was sealed and the resulting mixture was submitted to MW irradiation at 100° C. for 30 min. The reaction was cooled down to RT and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane/EtOAc 35-60%) to afford the title product (1.49 g, 8.63 mmol, 32% yield) as yellow solid. tR: 1.61 min (HPLC 1); tR: 0.45 min (LC-MS 2); ESI-MS: 163 [M+H]+(LC-MS 2); Rf=0.40 (hexane/EtOAc 1:1).

To a stirred suspension of 3,6-dichloropyridazin-4-amine (Step 112.1) (1.49 g, 9.09 mmol) in EtOH (15 mL) was added hydrazine hydrate (11.04 mL, 227 mmol) and the resulting mixture was heated up and stirred at 100° C. for 3 hr. The reaction was cooled down to RT and concentrated under reduced pressure. The crude product was triturated with water (25 mL) to afford the title product (478 mg, 3 mmol, 33% yield) as yellow solid. tR: 0.24 min (LC-MS 2); ESI-MS: 160 [M+H]+(LC-MS 2); ESI-MS: 158 [M−H]−(LC-MS 2).

To a stirred solution of tert-butyl (6-chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)carbamate (Step 112.4) (202 mg, 0.712 mmol) in DMF (4 mL) under Ar was added NaH (34.2 mg, 0.854 mmol) and the resulting mixture was stirred at RT for 30 min. MeI (0.053 mL, 0.854 mmol) was added to the mixture and stirred for further 30 min. The reaction was quenched with a saturated aq. NaHCO3solution and extracted with EtOAc. The combined organic layers were washed with a saturated aq. NaHCO3solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane/EtOAc 25-40%) to afford the title product (208 mg, 0.699 mmol, 98% yield) as white solid. tR: 4.45 min (HPLC 1); tR: 1.00 min (LC-MS 2); ESI-MS: 298 [M+H]+(LC-MS 2); Rf=0.45 (hexane/EtOAc 1:1).

To a stirred solution of 4-acetyl-5-(4-chlorophenyl)-3-hydroxy-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one (Step 71.2) (3 g, 8.07 mmol) in EtOH (50 mL) and toluene (50 mL) was added 5-hydrazinyl-1-methyl-1H-pyrazole (Step 74.2) (1.941 g, 10.49 mmol) and the resulting mixture was heated up and stirred at 115° C. for 20 hr. The reaction was quenched with a saturated aq. NaHCO3solution and extracted with EtOAc. The combined organic layers were dried over Na2SO4and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane/EtOAc 40-50%) to afford the title product (3.15 g, 6.89 mmol, 85% yield) as yellow solid. tR: 5.60 min (HPLC 1); tR: 1.24 min (LC-MS 2); ESI-MS: 448 [M+H]+(LC-MS 2); Rf=0.41 (hexane/EtOAc 1:1).

Reference Example 118

Reference Example 121

Reference Example 123

Reference Example 124

Reference Example 127

Methoxyacetone (5.22 mL, 56.8 mmol) was added dropwise to a cold (0° C.), stirred solution of sodium ethoxide (21% in EtOH, 20.2 g, 62.4 mmol) and EtOH (50 mL), under an argon atmosphere. The reaction mixture was stirred for 30 min at 0° C. Then, diethyl oxalate (7.71 mL, 56.8 mmol) was added. The reaction mixture was allowed to warm to rt, stirred for 16 h, quenched by addition of 1N HCl (75 mL), and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4and the solvent was evaporated off under reduced pressure. The crude material was purified by silica gel column chromatography (hexane/EtOAc 5-10%) to afford the title compound (3.21 g) as a yellow oil. tR: 0.70 min (LC-MS 2); ESI-MS: 189.1 [M+H]+(LC-MS 2); Rf=0.25 (hexane/EtOAc 1:1, CPS staining).

The title compound was prepared using an analogous procedure to that described in Step 57.1 but using equimolar amounts of ethyl 5-methoxy-2,4-dioxopentanoate (Step 130.1), 4-chlorobenzaldehyde, and 4-methoxybenzylamine. The reaction mixture was stirred for 2 h at 120° C. and allowed to cool to rt. The precipitate was collected by filtration to provide the title compound (5.21 g) as a colorless solid. tR: 0.94 min (LC-MS 2); ESI-MS: 402.1 [M+H]+(LC-MS 2).

Cyclopropylhydrazine (2.71 g) (Step 17.2) was added to a solution of 5-(4-chlorophenyl)-3-hydroxy-4-(2-methoxyacetyl)-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one (5 g, 12.4 mmol) (Step 130.2) in a mixture of EtOH and toluene (60 mL, 1:1, v/v). The reaction mixture was stirred 16 h at 115° C., concentrated, quenched with a saturated aq. NaHCO3solution, and extracted with EtOAc (2×100 mL). The combined organic layers were dried over Na2SO4and the solvent was evaporated off under reduced pressure. The crude material was purified by silica gel column chromatography (hexane/EtOAc 20-50%) to afford the title compound (2.62 g) as a yellow solid. tR: 1.24 min (LC-MS 2); ESI-MS: 438.1 [M+H]+(LC-MS 2); Rf=0.50 (hexane/EtOAc 1:1).

The title compound was prepared using an analogous procedure to that described in Step 23.9 but using 4-(4-chlorophenyl)-1-cyclopropyl-5-(4-methoxybenzyl)-3-(methoxymethyl)-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one (Step 130.3) and stirring the reaction mixture for 3 h at 110° C. under MW irradiation. The crude material was purified by silica gel column chromatography (hexane/EtOAc 40-65%) to afford the title compound (933 mg) as a colorless solid. tR: 0.91 min (LC-MS 2); ESI-MS: 318.0 [M+H]+(LC-MS 2); Rf=0.19 (hexane/EtOAc 1:1).

Hydrazine hydrate (119 mL, 2422 mmol) was added to a suspension of 5-bromo-2-chloro-3-methylpyridine (50 g, 242 mmol) in 2-methoxyethanol (250 mL). The resulting solution was stirred 30 h at 120° C. and concentrated. The yellow residue was purified by trituration with H2O to provide the title compound (24 g) as a colorless solid. tR: 0.39 min (LC-MS 2); ESI-MS: 201.9 [M+H]+(LC-MS 2)

A solution of acetic anhydride (12.22 mL, 129 mmol) in THF (10 mL) was added over a 10 min period to a suspension of 5-bromo-2-hydrazinyl-3-methylpyridine (25 g, 118 mmol) (Step 130.5) in dioxane (125 mL) and acetic acid (25 mL), at rt. The reaction mixture was stirred for 10 min at rt, heated to 100° C., stirred for 7 h at this temperature, and concentrated. The solid residue was purified by trituration with TBME to provide the title compound (25.7 g) as a colorless solid. tR: 0.39 min (LC-MS 2); ESI-MS: 225.9 [M+H]+(LC-MS 2).

The title compound was prepared using an analogous procedure to that described in Example stirring the reaction mixture for 30 min at 80° C. after addition of tert-butyl 3-iodoazetidine-1-carboxylate. The crude material was purified by silica gel column chromatography (CH2Cl2/MeOH 1-4.5%) to afford:

A mixture of 5-amino-1,3-dimethylpyridin-2(1H)-one (Step 20.2) (1.77 g, 12.78 mmol), 4-chlorobenzaldehyde (1.63 g, 11.62 mmol) and ethyl 4-cyclopropyl-2,4-dioxobutanoate (2 g, 11.62 mmol) in acetic acid (10 mL) was stirred for 2 h at 100° C. The reaction mixture was concentrated, diluted with CH2Cl2/1 N NaOH, and extracted with CH2Cl2. The combined organic extracts were discarded. The aqueous layer was acidified to pH 3 with 6 N HCl and extracted twice with CH2Cl2. The combined organic extracts were dried (Na2SO4) and concentrated to afford 3.06 g of the title compound as a beige foam. tR. 0.83 min (LC-MS 2); ESI-MS: 387.0 [M+H]+(LC-MS 2).

The preparation of the title compound is described in Example 149.

The title compound was prepared using an analogous procedure to that described in Example 134 using 4-(4-chlorophenyl)-5-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one (Example 98) (750 mg, 1.909 mmol) and stirring the reaction mixture for 30 min at 80° C. after addition of tert-butyl 3-iodoazetidine-1-carboxylate. The crude material was purified by silica gel column chromatography (CH2Cl2/MeOH 1-4%) followed by preparative HPLC (Gilson gx-281. Column: Sunfire C18, 30×100 mm, 5 μm. Flow: 30 mL/min. Gradient: 5% to 60% B in 20 min; A=0.1% TFA in H2O, B=CH3CN. Detection: UV) to afford:

4-Bromo-7-methoxy-1H-pyrrolo[2,3-c]pyridine (63 g, 0.27 mol) [Zhang, Zhongxing; Yang, Zhong; Meanwell, Nicholas A.; Kadow, John F.; Wang, Tao Journal of Organic Chemistry (2002), 67(7), 2345-2347] was dissolved in acetonitrile/water (1:1, 1260 mL) and potassium iodide (36.84 g, 0.22 mol) was added. The reaction mixture was stirred for 15 min at room temperature. Trimethylchlorosilane (52.7 mL, 0.41 mol) was added drop wise at room temperature. After completion of addition, the reaction mixture was heated to 70° C. and stirred for 20 h. The completion of the reaction was monitored by TLC using DCM: MeOH (9.3:0.7) as a mobile phase. After completion of the reaction, organic solvent was removed under reduced pressure. The resulting solid was filtered out and washed with water (1000 mL), hexane (1000 mL) and dried in vacuum to afford pure 50 g of the title compound.1H NMR (400 MHz, DMSO-d6) δ ppm 3.40 (s, 3H) 6.21 (dd, J=2.6, J=2.0, 1 H) 7.37 (t, J=2.8, 1 H) 11.18 (s, 1H) 12.36 (s, 1H).

The preparation of the title compound is described in Example 168. tR: 0.98 min (LC-MS 2); ESI-MS: 452.2 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 57.1 except using 2-fluoro,4-chloro benzaldehyde and 5-amino-1,3-dimethylpyridin-2(1H)-one (step 20.2) as starting materials. tR: 0.74 min (LC-MS 2); ESI-MS: 391.2[M+H]+/389.2[M−H] (LC-MS 2).

The title compound was prepared in analogy to the procedure described in Example 57 using 5-(3-acetyl-2-(4-chloro-2-fluorophenyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-1,3-dimethylpyridin-2(1H)-one (Step 170.1) and ethyl hydrazine as starting materials. Purification of the crude product afforded 18 mg of 4-(4-chloro-2-fluorophenyl)-5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-2-ethyl-3-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one (Example 171) and 45 mg of the title compound tR: 0.97 min (LC-MS 2); ESI-MS: 415.2 [M+H] (LC-MS 2).

The preparation of the title compound is described in Example 170. tR: 0.92 min (LC-MS 2); ESI-MS: 415.2 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Step 147.2 using 4-acetyl-5-(2,4-difluorophenyl)-1-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-hydroxy-1H-pyrrol-2(5H)-one (step 172.1) and hydrazine monohydrate as starting materials. Purification of the crude product by flash chromatography afforded 147 mg of the title compound tR: 0.70 min (LC-MS 2); ESI-MS: 395.3 [M+H]/393.2 [M−H] (LC-MS 2).

The title compound was prepared in analogy to the procedure described in Example 57 using 4-acetyl-5-(2,4-difluorophenyl)-1-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-hydroxy-1H-pyrrol-2(5H)-one (Step 172.1) and isopropyl hydrazine as starting materials. Purification of the crude product afforded 12 mg of 4-(2,4-difluorophenyl)-5-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-2-isopropyl-3-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one (Example 175) and 104 mg of the title compound tR: 0.96 min (LC-MS 2); ESI-MS: 437.3 [M+H] (LC-MS 2).

The preparation of the title compound is described in Example 174. tR: 0.91 min (LC-MS 2); ESI-MS: 437.3 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Example 57 using 5-(3-acetyl-2-(4-chloro-2-fluorophenyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-1,3-dimethylpyridin-2(1H)-one (Step 170.1) and cyclopropyl hydrazine as starting materials. Purification of the crude product afforded 87 mg of the title compound tR: 1.00 min (LC-MS 2); ESI-MS: 427.2 [M+H]+/425.2 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described in Example 57 using 5-(3-acetyl-2-(4-chloro-2-fluorophenyl)-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-1,3-dimethylpyridin-2(1H)-one (Step 170.1) and isopropyl hydrazine as starting materials. Purification of the crude product afforded 82 mg of the title compound tR:1.05 min (LC-MS 2); ESI-MS: 429.2 [M+H]+/427.3 [M−H]−(LC-MS 2).

The preparation of the title compound is described in Example 181. tR: 0.84 min (LC-MS 2); ESI-MS: 423.3 [M+H]+(LC-MS 2).

The title compound was prepared in analogy to the procedure described for Step 172.2 except for using 4-acetyl-5-(4-chloro-2-fluorophenyl)-1-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-hydroxy-1H-pyrrol-2(5H)-one (Step 168.1) as a starting material. Purification of the crude material afforded the title compound as a yellow solid. tR: 0.76 min (LC-MS 2); ESI-MS: 411.3 [M+H]+/ESI-MS: 409.2 [M−H]−(LC-MS 2).

The title compound was prepared in analogy to the procedure described for Step 172.3 except for using 4-(4-chloro-2-fluorophenyl)-5-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one (Example 183) as a starting material. Purification of the crude product afforded 7 mg of 4-(2,4-difluorophenyl)-5-(3,8-dimethyl-[1,2,4]triazolo[4,3-a]pyridin-6-yl)-2-ethyl-3-methyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one (Example 194) and 9 mg of the title compound tR: 1.01 min (LC-MS 2); ESI-MS: 518.2 [M+H]+, ESI-MS: 516.2 [M−H]−(LC-MS 2).

The preparation of the title compound is described in Example 185. tR: 0.90 min (LC-MS 2); ESI-MS: 439.3 [M+H]+(LC-MS 2).

The preparation of the title compound is described in Example 173. tR: 0.92 min (LC-MS 2); ESI-MS: 503.2 [M+H]+(LC-MS 2).

The preparation of the title compound is described in Example 183. tR: 0.98 min (LC-MS 2); ESI-MS: 518.2 [M+H]+(LC-MS 2).

3,6-Dichloro-4-methylpyridazine (Combi-Blocks) (60 g, 361 mmol) was dissolved in hydrazine monohydrate (Aldrich) (335 mL, 5411 mmol) and the solution was stirred at 80° C. for 1 h, forming a white precipitate. The reaction mixture is dilutes with water and the precipitated products isolated by filtration. The solid crude product is suspended in EtOH and left in an ultra sound bath for 1 h. The desired product (22.4 g) was obtained after filtration and drying under vacuum as a beige solid. tR: 0.31 min (LC-MS 2); ESI-MS: 160.0 [M+H]+(LC-MS 2).1H NMR (400 MHz; DMSO-d6) δ ppm 7.83 (br.s, 1H) 7.32 (s, 1H) 4.49 (br.s, 2H) 2.05 (s, 3H).

To a beige suspension of 6-chloro-3-hydrazinyl-4-methylpyridazine (step 195.1) (22.44 g, 127 mmol) in dioxane (250 mL) was added difluoroacetic acid (Aldrich) (9.40 mL, 146 mmol) and the reaction mixture was stirred at rt for 5 min, then heated-up to 120° C. for 2.5 hr. With heating the suspension turned into a red-orange solution. The reaction mixture was cooled to rt. Et2O (80 mL) was added and the suspension was stirred for 2 hours at 0° C. Precipitated solids were isolated by filtration, suspended in hexanes and filtered again. After repeated washings with hexanes the tittle compound was obtained as an orange solid.