CA3095019C - SUBSTITUTED PYRIDINE DERIVATIVES AND COMPOSITIONS THEREOF USEFUL AS INHIBITORS OF HISTONE DEMETHYLASES

8 claims: 3 independent, 5 dependent

1
CLAIMS:1. A compound of formula HaC^O^O Λ H Î V* CH 3 , or a pharmaceutically acceptable salt thereof.
7
The compound:Date Reçue/Date Received 2020-10-01 87210600 217
8
A pharmaceutically acceptable salt of the compound:

Independent claims3

1,764 paragraphs in 597 sections, as filed

SUBSTITUTED PYRIDINE DERIVATIVES AND COMPOSITIONS THEREOF USEFUL AS INHIBITORS OF HISTONE DEMETHYLASES

This application is a divisional of application no. 2885969, filed on October 1,2013.

FIELD OF THE INVENTION

The present invention relates to compounds capable of modulating the activity of histone demethylases (HDMEs), which compounds are useful for the prevention and/or the treatment of diseases in which genomic dysrégulation is involved in the pathogenesis, such as e.g. cancer.

BACKGROUND OF THE INVENTION

The DNA of eukaryotic cells is packaged into chromatin by winding of the DNA around histone proteins to form nucleosomes, the basic unit of chromatin. One of the important functions of chromatin is to determine regions of active and silenced transcription by changing the ordered chromatin structure. Such changes have profound effects on cellular function since they affect fundamental processes as differentiation, proliferation and apoptosis, and are often referred collectively to as epigenetic?' since they can lead to heritable changes that do not involve changes in gene sequences (Quina, A.S. et al. (2006), Biochem. Pharmacol. 72; 1563-1569)

These highly controlled chromatin changes are mediated by alterations histone proteins associated with DNA In the nucleosome. Most notably, the N-termlnal histone tall of Histone H3 and histone H4 are subject to such covalent changes, which include changes in methylation, acetylation, phosphorylation and ubiquitination. The addition or removal of these groups on histones is mediated by specific enzymes, e.g. histone methyl transferases and histone demethylases for methyl groups, histone acetyltransferases and histone deacetylases for acetyl groups, etc. In the event that the activity or expression of these epigenetic enzymes Is not correctly controlled and regulated it may lead to disease. Cancer, in particular, is an area of high importance in relation to dysregulated epigenetic enzyme activity due to the role of epigenetics in cell differentiation, proliferation and apoptosis, but epigenetics may also play a role in other diseases like metabolic, Inflammatory, neurodegeneratlve and cardiovascular diseases. Therefore the selective modulation of aberrant action of epigenetic enzymes may hold great promise for the treatment of human disease (Kelly, T.K. et al. (2010), Nat. Biotechnol. 28; 1069-1078, and Cloos, P.a.C. et al. (2008), Genes. Dev. 22; 115-1140).

Methylation and demethylation of lysine residues on the histone H3 tail constitute important epigenetic marks delineating transcriptionally active and inactive chromatin. For example, methylation of lysine 9 on histone H3 (H3K9) Is usually associated with epigenetlcally silenced chromatin (Fischle, W., et. al. (2003), Curr. Opinion Cell Biol. 15,172-83; Margueron, R., et al. (2005), Curr. Opinion Genet Dev. 15, 163-76) while methylation of lysine 4 on histone 3 is associated with transcriptionally active chromatin. Similarly, the lysine 27 histone H3 (H3K27) mark is repressive in its di- and tri-methylated states whereas the lysine 36 histone H3 mark is found in association with gene activation (Barski, A. et al. (2007), Cell, 129, 823-37; Vakoc, C. et al. (2006) Mol. Cell. Biol. 26, 9185-95; Wagner, EJ. & Carpenter, P.B. (2012) Nature Mol. Cell

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

Biol 13,115-26). There are, however, many exemptions from these general rules of association between methylation states of epigenetic marks and the effect they have on transcription.

As documented by studies of the SUV39H1 knockout mouse, loss of the tri-methyl variant of the H3K9 mark results in chromosomal aberrations and predisposes to cancer (Peters, A.H. et al., Cell 107, 323-37, 2001). The JMJD2C protein (KDM4C, GASC1) has been identified as an eraser of the H3K9 mark (a histone demethylase) and may therefore promote cancer if its expression and activity is not tightly controlled (Cloos, P. et al. (2006), Nature 442, 307-11; Klose, RJ. et al. (2006), Nature 442, 312-16; Liu, G. et al. (2009), Oncogene 28, 4491-500). For example, JMJD2C has been shown to induce transformed phenotypes like growth factor independent growth, anchorage independent growth and mammosphere formation, if it is overexpressed in cells (Liu, G. et al. (2009), Oncogene 28, 4491-500). These findings are supported by the overexpression of JMJD2C in a range of human tumours like squamous cell carcinoma, metastatic lung carcinoma, prostate cancer, breast cancer and several others (Yang, Z.Q. et al. (2000) Cancer Res. 60,4735-39; Yang, Z.Q. et al. (2001) Jpn. J. Cancer Res. 92, 423-28; Hu, N. et al. (2005) Cancer Res. 65, 2542-46; Liu, G. et al. (2009) Oncogene 28, 4491-500;

Wissmann, M. et al. (2007) Nat. Cell Biol. 9, 347-53), indicating the potential importance of JMJD2C as an oncogene.

The JMJD2A protein (KDM4A, JHDM3A) shows similar properties to JMJD2C. JMJD2A shows high sequence identity to JMJD2C in its JmjC catalytic domain, Is an eraser of the H3K9 mark and has also been shown to be overexpressed in prostate cancer (Cloos, P. Et al., Nature 442, 307-11, 2006). JMJD2A has been shown to interact with the estrogen receptor alpha (ER-alpha) and overexpression of JMJD2A enhances estrogen-dependent transcription and the down-regulation of JMJD2A reduced transcription of a seminal ER-alpha target gene, cydin DI (Kawazu et al., (2011) PLoS One 6; Berry et al., (2012) Int J Oncol 41). Additionally, it has been shown that catalytically inactive JMJD2A is compromised in its ability to stimulate ER-alpha mediated transcription, suggesting that inhibitors of JMJD2A may be beneficial for the treatment of ERalpha positive breast tumours (Berry et al., (2012) Int J Oncol 41).

Likewise, an eraser of the tri-methyl variant of the H3K4 mark, JARID1B (KDM5B, PLU1) has also been identified as potential oncogene. In cancer JARID1B most likely acts as a repressor of tumour repressor genes via removal of the H3K4 tri-methylation leading to decreased transcriptional activation in the affected chromatin regions. The oncogenic potential of JARID1B is demonstrated by its stimulation of proliferation in cell lines and further validated by shRNA knockdown studies of JARID1B expression showing Inhibition of proliferation In MCF7 human breast cancer cells, in SW780 and RT4 bladder cancer cells, in A549 and LC319 lung cancer cells and in 4T1 mouse tumour cells in vitro and/or in mouse xenograft experiments (Yamane K. et al. (2007), Mol. Cell 25, 801-12; Hayami S. et al. (2010) Mol. Cancer 9, 59; Catchpole S et al. (2011), Int. J. Oncol. 38, 1267-77). Finally, JARID1B is overexpressed in prostate cancer and is associated with malignancy and poor prognosis (Xiang Y. et al. (2007) PNAS 104).

□ate Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

JARID1A (KDM5A, RBP2) is also an eraser of the tri- and di-methyl variant of the H3K4 mark. JARID1A is overexpressed in gastric cancer (Zeng et al., (2010) Gastroenterology 138) and its gene is amplified in cervix carcinoma (Hidalgo et al, (2005) BMC Cancer 5). It has been suggested that JARID1A is fine-tuning progesterone receptor expression control by estrogens (Stratmann and Haendler (2011) FEBS J 278). Together with JARID1B, JARID1A has been Implicated In the maintenance of a slow-growing population of cancer cells that are required for continuous tumor growth and that are resistant to cytotoxic and targeted therapy (Roesch, et al, (2010) Cell 141; Sharma, et al., (2010) Cell 141). JARID1A is required for the tumor initiation and progression in Rb+/- and Menl-defective mice (Lin, et al., (2011) PNAS 108). Data from Pasini show that JARID1A binds to Polycomb group protein target genes which are involved in regulating important cellular processes such as embryogenesis, cell proliferation, and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions (Pasini et al., (2008) Genes & Dev 22). Additionally, JARID1A were also shown to binds the PRC2 complex and being regulator of PRC2 target genes (Pasini et al., (2008) Genes & Dev 22).

Another potential oncogene, an eraser of the di-methyl variant of the H3K36 mark, JHDM1B (KDM2B, FBXL10) has been shown to be highly expressed In human cancers (Tzatsos A et al. (2009), PNAS 106 (8), 2641-6; He, J. et al. (2011), Blood 117 (14), 3869-80). Knock-down of FBXL10 causes senescence in mouse embryonic fibroblasts (MEFs), which can be rescued by expression of catalytic active (but not catalytic inactive) JHDM1B (Pfau R et al. (2008), PNAS 105(6), 1907-12; He J et al. (2008), Nat Struct Mol Biol 15, 1169-75). JHDM1B demethylates H3K36me2 on the tumor-suppressor gene Ink4b (pl5'k4’), and thereby silences the expression of this senescence-mediating gene in MEFs and in leukemic cells (He, J. et al. (2008), Nat Struct Mol Biol 15, 1169-75; He, J. et al. (2011), Blood 117 (14), 3869-80). The catalytic dependency of JHDM1B is further shown by He et al. as catalytic activity is required for development of leukemia in a mouse AML model.

Inhibitors of the histone demethylase class of epigenetic enzymes, and in particular the potential oncogenes JARID1B, JARID1A, JMJD2C, JMJD2A, and JHDM1B, would present a novel approach for intervention in cancers and other proliferative diseases. Being one of the most devastating diseases, affecting millions of people worldwide, there remains a high need for efficacious and specific compounds against cancer.

Embodiments of the invention provide novel series of compounds capable of modulating the activity of histone demethylases (HDMEs), at least some of which compounds are useful for the prevention and/or the treatment of diseases in which genomic disregulation is involved in the pathogenesis, such as e.g. cancer. By way of further example, malnutrition or poor nutritiion is thought to have an adverse epigenetic effect and the compounds of the Invention may therefore be expected to have beneficial effect in treating such effects of poor nutrition. Furthermore, epigenetic changes have been found to be linked to behaviour. Accordingly, compounds according to the invention may be useful in behaviour modification. Alternatively or additionally

Date Reçue/Date Received 2020-10-01

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23 such compounds may be useful for exploring the extent to which different demethylases are inhibited by similar compounds as an investigation of the structure and functionality and mechanism of action of the demethylases.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a compound of the general Formula (I)

wherein

A is selected from -CHR2C(O)-, Cv8 alkylene, C2.8 alkenylene, C2.8 alkynylene, C3.10 cydoalkylene, heterocyclylene, heteroarylene and arylene, which alkylene, alkenylene, alkynylene, cydoalkylene, heterocyclylene, heteroarylene and arylene may optionally be substituted with one or more R3;

Y Is selected from -H, -NR&R7, -OR7, Cm alkyl, C2.8 alkenyl, C2.8 alkynyl, C3-10 cycloalkyl, heterocydyl, heteroaryl and aryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocydyl, heteroaryl and aryl may optionally be substituted with one or more R3 and may form a cyclic structure with R2;

R1 is selected from -H, Cm alkyl, C2 8 alkenyl, C2.8 alkynyl, C3-10 cydoalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more selected from -OH, aryl, C1-6alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and C3-6cycloalkyl; or more preferably is selected from -H and C1.4 alkyl; or with -A-Y forms a nitrogen containing optionally substituted heterocyclic group where the optional substitution may be Cl 8 alkyl, C2S alkenyl, C2.8 alkynyl, or C3-io cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more selected from -OH, aryl, Cm alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and Cm cycloalkyl;

R2 is selected from -H, Cm alkyl, C28 alkenyl, C28alkynyl, and C3-10 cycloalkyl, which alkyl, alkenyl, alkynyl and cydoalkyl may be optionally substituted with one or more selected from OH, aryl, Cm alkoxy, heteroaryl, aryloxy, heteraaryloxy, F, and C3-e cydoalkyl, and may form a cyclic structure with Y;

each R3 is independently selected from Cm alkyl, C1.4 fluoroalkyl, C1.4 hydroxyalkyl, C2.6 alkenyl, C2.6 alkynyl, C3.10 cydoalkyl, -Z-heterocyclyl, -Z-aryl, -Z- heteroaryl, -Z-NR6R7, -Z-C(=O)-NR6R7, -z-nr6-C(=O)-R7, -z-C(=O)-R7, -z-OR7, halogen, -z-SR7, -Z-SOR7, -z-S02R7, -z-S02nr6r7 and

Date Reçue/Date Received 2020-10-01

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

-Z-COOR7, wherein any heterocyclyl may be substituted with one or more R4, and wherein any heteroaryl and any aryl may be substituted with one or more R5;

Z is selected from a single bond, Ci-4 alkylene, heterocydylene and C3.6 cydoalkylene;

each R4 is independently selected from Ct-β alkyl, C1.4 fluoroalkyl, C1.4 hydroxyalkyl, C1.4 alkoxy, C3.10cycloalkyl, -N(RX)2, carbamoyl, and -OH;

each R5 is independently selected from Ci-& alkyl, Ci.4 fluoroalkyl, Ci.4 hydroxyalkyl, Ci.4 alkoxy, C3-6 cydoalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

each of R6 and R7 is independently selected from -H, Ci-aalkyl, Ci-4 fluoroalkyl, C1-4 perfluoroalkyl, Cj-4 hydroxyalkyl, C2 §alkenyl, C2.9 alkynyl, C3.ic cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl and -Zaryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl may optionally be substituted with one or more independently selected R8; or, alternatively, R6 and R7 may together with the N-atom to which they are attached form an N-heterocydic ring optionally substituted with one or more independently selected R8;

each R® is independently selected from Ci-6 alkyl, Ci-4fluoroalkyl, Ci-4 hydroxyalkyl, C2.6 alkenyl, C2-6 alkynyl, C3-10 cydoalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR1ORU, -Z-C(=O)NR10Rn, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9, which alkyl, alkenyl, alkynyl, cydoalkyl, heterocydics, heteroaryl and aryl may optionally be substituted with one or more selected from Ci-4 alkyl, Ci.4 fluoroalkyl, Ci.4 hydroxyalkyl, C3.6 cydoalkyl, -Z-heterocydyl, Z-heteroaryl, -Z-aryl, -Z-NR10R11, -Z-C(=O)-NR10Rn, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9; wherein any heterocyclyl may be further substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be further substituted with one or more Rs as defined above, and each R9 is independently selected from -H, Ci-aalkyl, C1-4fluoroalkyl, C1.4 hydroxyalkyl, C2-e alkenyl, C2.8 alkynyl, C3-10 cydoalkyl, -Z-heterocyclyl, -Z-aryl, and -Z- heteroaryl, wherein any heterocyclyl may be substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be substituted with one or more R5 as defined above;

each of R10 and R11 is independently selected from -H, Ci-6 alkyl, C1.4 fluoroalkyl, Ci-4 hydroxyalkyl, C2-8 alkenyl, C2-8 alkynyl, C3.10 cycloalkyl, heterocyclyl, heteroaryl, and aryl, wherein any heterocyclyl may be substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be substituted with one or more R5 as defined above, or, alternatively, R10 and R11 may together with the N-atom to which they are attached form an Nheterocyclic ring optionally substituted with one or more R4as defined above;

with the proviso that Y is not H when A is -CH2-;

or an isomer or a mixture of isomers thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Date Reçue/Date Received 2020-10-01

84515062

5a

In one aspect, there is provided a compound of the general Formula (I)

ΗΟχ^Ο ii < .N—A—Y

N / wherein

A is -CHR2C(O)-;

Y is -NR6R7;

R1 is selected from the group consisting of -H, Ci-β alkyl, Cz-β alkenyl, Cz-s alkynyl, and C3-10 cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl is unsubstituted or substituted with one or more substituents selected from the group consisting of -OH, aryl, C1-6 alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and C3-6 cycloalkyl;

R2 is selected from the group consisting of-H, Ci-s alkyl, Cz-s alkenyl, Cz-s alkynyl, and C3-io cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more substituents selected from the group consisting of -OH, aryl, Ci-s alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and C3-6 cycloalkyl, and R2 may form a cyclic structure with Y;

Z is selected from the group consisting of a single bond, Ci-4 alkylene, heterocyclylene and C3-s cycloalkylene;

each R4 is independently selected from the group consisting of Ci-β alkyl, C1-4 fluoroalkyl, Ci-4 hydroxyalkyl, Ci-4 alkoxy, C3-10 cycloalkyl, -N(R12)z, carbamoyl, and -OH;

each R5 is independently selected from the group consisting of Ci-β alkyl, C1-4 fluoroalkyl, C1-4 hydroxyalkyl, C1-4 alkoxy, C3-6 cycloalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

each of R6 and R7 is independently selected from the group consisting of -H, C1-8 alkyl, C1-4fluoroalkyl, C1-4 perfluoroalkyl, C1-4 hydroxyalkyl, C2-8 alkenyl, C2-8 alkynyl, C3-10 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl and -Z-aryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl may optionally be substituted with one or more independently selected R8;

Date Reçue/Date Received 2020-10-01

84515062

5b each R8 is independently selected from the group consisting of Ci-6 alkyl, Ci-4 fluoroalkyl, Ci-4 hydroxyalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 cycloalkyl, -Zheterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR1ORU, -Z-C(=O)-NR10Ru, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl is unsubstituted or substituted with one or more substituents selected from the group consisting of Ci-4 alkyl, Ci-4 fluoroalkyl, Ci-4 hydroxyalkyl, C3-6 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR10Rn, -Z-C(=O)-NR10Rn, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9; wherein any heterocyclyl is unsubstituted or substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl is unsubstituted or substituted with one or more R5 as defined above;

each R9 is independently selected from the group consisting of -H, Ci-s alkyl, Ci-4 fluoroalkyl, C1-4 hydroxyalkyl, C2-8 alkenyl, C2-8 alkynyl, C3-10 cycloalkyl, -Zheterocyclyl, -Z-aryl, and -Z- heteroaryl, wherein any heterocyclyl is unsubstituted or substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl is unsubstituted or substituted with one or more R5 as defined above;

each of R10 and R11 is independently selected from the group consisting of -H, C1-6 alkyl, C1-4 fluoroalkyl, C1-4 hydroxyalkyl, C2-8 alkenyl, C2-8 alkynyl, C3-10 cycloalkyl, heterocyclyl, heteroaryl, and aryl, wherein any heterocyclyl is unsubstituted or substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl is unsubstituted or substituted with one or more R5 as defined above; and each R12 is -H, C1-6 alkyl, C1-4 fluoroalkyl, C1-4 hydroxyalkyl, C2-8 alkenyl, C2-8 alkynyl, C3-10 cycloalkyl, heterocyclyl, heteroaryl, and aryl, wherein any heterocyclyl is unsubstituted or substituted with one or more substituent selected from the group consisting of C1-6 alkyl, C1-4 fluoroalkyl, C1-4 hydroxyalkyl, C1-4 alkoxy, C3-10 cycloalkyl, carbamoyl, and -OH, and wherein any heteroaryl and any aryl is unsubstituted or substituted with one or more R5 as defined above;

or a pharmaceutically acceptable salt thereof.

Date Reçue/Date Received 2020-10-01

84515062

5c

In another aspect, there is provided a compound of formula

or a pharmaceutically acceptable salt thereof.

In another aspect, there is provided a compound

or a pharmaceutically acceptable salt thereof.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

Where R1 forms with -A-Y an optionally substituted heterocyclic group, it is preferably an optionally substituted C3-7 for instance C3-6 or CS-6 heterocyclic group.

Where R2 forms a cyclic structure with Y, R2 is preferably C1-C2 alkylene. The cyclic structure is preferably an optionally substituted (with one or more R3) 5 or 6 membered, optionally heterocyclic, ring.

A preferred aspect of the present invention relates to a compound of the Formula (I)

wherein

A is selected from -CHR2C(O)-, Ci-8 alkylene, C2.8 alkenylene, C2.8 alkynyl ene, C3.10 cycloalkylene, heterocyclylene, heteroarylene and arylene, which alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, heteroarylene and arylene may optionally be substituted with one or more R5;

Y is selected from -H, -NR6R7, -OR7, Ci.8 alkyl, C2.8 alkenyl, C28 alkynyl, C3-10 cycloalkyl, heterocyclyl, heteroaryl and aryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl may optionally be substituted with one or more R3;

R1 is selected from -H and C1.4 alkyl;

R2 is selected from -H, Ci-a alkyl, C2s alkenyl, C2-8alkynyl, C3.10 cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more selected from -OH, aryl, C1-6alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and C3-6cycloalkyl;

each R3 is independently selected from C.-6 alkyl, CV4fluoroalkyl, Ci-4 hydroxyalkyl, C2.6 alkenyl, C2-6 alkynyl, C3-10 cycloalkyl, -Z-heterocyclyl, -Z-aryl, -Z- heteroaryl, -Z-NR6R7, -Z-C(=O)-NR6R7, -Z-NR6-C(=O)-R7, -Z-C(=O)-R7, -Z-OR7, halogen, -Z-SR7, -Z-SOR7, -Z-SO2R7, -Z-SO2NR6R7 and -Z-COOR7, wherein any heterocyclyl may be substituted with one or more R4, and wherein any heteroaryl and any aryl may be substituted with one or more R5;

Z is selected from a single bond, Ci-4 alkylene, heterocyclylene and C3.6 cycloalkylene;

each R4 is independently selected from C1.6 alkyl, Ci.4 fluoroalkyl, C1.4 hydroxyalkyl, Ci.4 alkoxy, C3.10cycloalkyl, -N(R1)2, carbamoyl, and -OH;

each R5 is independently selected from C:.8 alkyl, Ci.4fluoroalkyl, Ci.4hydroxyalkyl, Cj.4alkoxy, Cs e cycloalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457 each of R6 and R7 is independently selected from -H, Ci-3 alkyl, Ci-4 fluoroalkyl, Ci-4 perfluoroalkyl, Ci-4 hydroxyalkyl, C2-8alkenyl, C2-8 alkynyl, C3.ic cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl and -Zaryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl may optionally be substituted with one or more independently selected R8; or, alternatively, R6 and R7 may together with the N-atom to which they are attached form an N-heterocyclic ring optionally substituted with one or more independently selected R8;

each Rs is independently selected from Ci 6 alkyl, C1.4fluoroalkyl, C1.4 hydroxyalkyl, C2-6 alkenyl, C2.6 alkynyl, C3-i0 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR10Rn, -Z-C(=O)NR10Rn, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocydics, heteroaryl and aryl may optionally be substituted with one or more selected from Ci-4 alkyl, cb4 fluoroalkyl, cb4 hydroxyalkyl, c3 6 cycloalkyl, -z-heterocyclyl, Z-heteroaryl, -Z-aryl, -Z-NR1ORU, -Z-C(=O)-NRloRn, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9; wherein any heterocyclyl may be further substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be further substituted with one or more R5 as defined above, and each R9 is independently selected from -H, Ci.8alkyl, Cb4 fluoroalkyl, Ci.4 hydroxyalkyl, C2.8 alkenyl, C2.8 alkynyl, C3.10 cycloalkyl, -Z-heterocyclyl, -Z-aryl, and -Z- heteroaryl, wherein any heterocyclyl may be substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be substituted with one or more R5 as defined above;

each of R10 and R11 is independently selected from -H, Cb6 alkyl, Cb4 fluoroalkyl, Ci.4 hydroxyalkyl, C2.8 alkenyl, C2.8 alkynyl, C3.io cycloalkyl, heterocyclyl, heteroaryl, and aryl, wherein any heterocyclyl may be substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be substituted with one or more Rs as defined above, or, alternatively, R10 and R11 may together with the N-atom to which they are attached form an Nheterocyclic ring optionally substituted with one or more R4as defined above;

with the proviso that Y Is not H when A Is -CH2-;

or an isomer or a mixture of isomers thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In an alternative aspect, the invention relates to a compound of the Formual 1 wherein

A Is selected from -CHR2C(O)-, Cb8 alkylene, C2.e alkenylene, C2.e alkynylene, C3.i0 cydoalkylene, heterocydylene, heteroarylene and arylene, which alkylene, alkenylen, alkynylene, cydoalkylene, heterocydylene, heteroarylene and arylene may optionally be substituted with one or more R3;

Y Is selected from -H, -NR6R7, -OR7, Cb8 alkyl, C2.8 alkenyl, C2.8 alkynyl, C3-i0 cycloalkyl, heterocyclyl, heteroaryl and aryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl may optionally be substituted with one or more R3;

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

R1 is selected from -H and Ci-4 alkyl;

R2 is selected from -H, Ci.4 alkyl and Ci-4 hydroxyalkyl;

each R3 is independently selected from Ci^ alkyl, Ci.4 fluoroalkyl, Ci.4 hydroxyalkyl, C2.6 alkenyl, Cs-e alkynyl, C3.10 cydoalkyl, -Z-heterocyclyl, -Z-aryl, -Z- heteroaryl, -Z-NR6R2, -Z-C(=O)-NR6R7, -Z-OR7, halogen, -Z-SR7, -Z-SOR7, -Z-SO2R7 and -Z-COOR7, wherein any heterocydyl may be substituted with one or more R4, and wherein any heteroaryl and any aryl may be substituted with one or more R5;

Z is selected from a single bond, Cj.4 alkylene, heterocydylene and C3.6 cycloalkylene, each R4 is independently selected from alkyl, C14 fluoroalkyl, C14 hydroxyalkyl, C14 alkoxy,

C3-10cycloalkyl, -NCR1)?, carbamoyl, and -OH;

each R5 Is independently selected from Ci-6 alkyl, Ci-4 fluoroalkyl, Ci-4 hydroxyalkyl, CV4 alkoxy, C3.6 cydoalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

each of R6 and R7 is independently selected from -H, Ci-8alkyl, Ci-4 fluoroalkyl, Ci-4 hydroxyalkyl, C2-8 alkenyl, C2-8 alkynyl, C310 cydoalkyl, -z-heterocyclyl, -z-heteroaryl and -z-aryl, which alkyl, alkenyl, alkynyl, cydoalkyl, heterocydyl, heteroaryl and aryl may optionally be substituted with one or more independently selected R8; or, alternatively, R6 and R2 may together with the Natom to which they are attached form an N-heterocyclic ring optionally substituted with one or more independently selected R8;

each R6 is independently selected from Ci.6alkyl, Ci.4fluoroalkyl, Ci.4 hydroxyalkyl, C2.6alkenyl, C2.6 alkynyl, C3.10 cydoalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR10Rn, -Z-C(=O)NR10R11, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9, which alkyl, alkenyl, alkynyl, cydoalkyl, heterocydics, heteroaryl and aryl may optionally be substituted with one or more selected from Ci4alkyl, Ci 4 fluoroalkyl, Ci-4 hydroxyalkyl, C3.6 cydoalkyl, -Z-heterocydyl, · Z-heteroaryl, -Z-aryl, -Z-NR1ORU, -Z-C(=O)-NR10Ru, -Z-OR9, halogen, -CN, -Z-SR9, -Z-SOR9, -Z-SO2R9 and -Z-COOR9; wherein any heterocydyl may be further substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be further substituted with one or more Rs as defined above, and each R9 is independently selected from -H, CV6alkyl, Cw fluoroalkyl, Ci-4 hydroxyalkyl, C2-8 alkenyl, C2 8 alkynyl, C3-10 cycloalkyl, -Y-heterocyclyl, -Z-aryl, and -Z- heteroaryl, wherein any heterocydyl may be substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be substituted with one or more R5 as defined above;

each of R10 and R11 is independently selected from -H, Ci-8 alkyl, Ci-4 fluoroalkyl, Ci-4 hydroxyalkyl, C2.8 alkenyl, C2.8 alkynyl, C3-10 cycloalkyl, heterocydyl, heteroaryl, and aryl, wherein any heterocydyl may be substituted with one or more R4 as defined above, and wherein any heteroaryl and any aryl may be substituted with one or more R5 as defined above, or,

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457 alternatively, R10 and R11 may together with a N-atom to which they are attached form an Nheterocyclic ring optionally substituted with one or more R4 as defined above;

with the proviso that Y is not H when A i$ -CH2-; or an isomer or a mixture of isomers thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

A further aspect of the present invention relates to pharmaceutical compositions comprising at least one compound of Formula (I) as defined herein, and optionally one or more pharmaceutically acceptable excipients, diluents and/or carriers.

A further aspect of the present invention relates to a compound of Formula (I) as defined herein for use as a medicament.

A further aspect of the present invention relates to a compound of Formula (I) as defined herein for use in the treatment of a HDME dependent disease, such as for the treatment of cancer.

A further aspect of the present invention relates to a compound of Formula (I) as defined herein for use in the preparation of a pharmaceutical composition for the treatment of a HDME dependent disease, such as cancer.

A further aspect of the invention relates to a method of treating a HDME dependent disease in a subject, said method comprises administering to said subject a therapeutically effective amount of at least one compound of Formula (I) as defined herein.

Compounds of Formula (I) as defined herein can be used in the treatment of HDME dependent diseases by inhibiting HDMEs. Inhibiting HDMEs would provide a novel approach to the prevention and treatment of cancer and other proliferative diseases. Administered alone or optionally in combination with anti-neoplastic compounds, the compounds of the invention Increase the efficacy of the treatment of HDME dependent diseases. As will be shown below, compounds of the invention have a cytostatic or anti-proliferative action against cancer cells.

DETAILED DISCLOSURE OF THE INVENTION

Compounds of Formule (I)

As mentioned above, the present invention relates to compounds of Formula (I)

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

The above definition of the compounds of Formula (I) is referred to herein by the expressions compounds of Formula (1) as defined herein, compound of Formula (I) as defined herein, or simply compounds of Formula (I), etc. It should be understood, that such references are intended to encompass not only the above general formula, but also each and and every one of the embodiments, etc. discussed above or in the following. It should also be understood, that unless the opposite is stated, such references also encompass isomers, mixtures of isomers, pharmaceutically acceptable salts, solvates and prodrugs of the compounds of Formula (I).

Without being bound by any particular theory, the current results and X-ray crystallography studies give reasons to believe that the HOOC-pyridine-CH2-N< motif, cf. Formula (I), plays an important role when designing compounds capable of modulating the activity of histone demethylases (HDMEs). Furthermore, it is believed that the substituent combination -A-Y plays a role in establishing affinity for said histone demethylases. Histone demethylases contain an iron atom on which their activity depends. It is furthermore believed that the pyridine nitrogen and the nitrogen atom of Formula (I) also play a role in the binding of a particular cavity of the histone demethylases where the iron atom lies, probably by chelation of the iron itself.

It is also believed that the A-Y chain, itself and through its substituents, interacts with the area of the demethylase known to accommodate the lysine chain of the substrate. As the experimental results presented below in the examples clearly demonstrate, there is a wide range of possibilities for the -A-Y group that modulate activity at various demethylases, imparting selective inhibition, Some demethylases have the lysine chain area lined with acidic residues and compounds with basic groups in Y show enhanced potency on these enzymes. Similarly, some demethylases are better inhibited by compounds with apolar groups in the Y chain. Yet other demethylases are better Inhibited by compounds with non-charged polar groups in the Y chain. Yet other demethylases are better inhibited by compounds with non-charged polar groups in the Y chain.

More particularly, It Is believed that the carboxylic acid substituent of the pyridine ring plays a role in binding to a particular cavity of histone demethylases.

A is typically selected from -CHR2C(O)-, Ci e alkylene, Cj-s alkenylene, Cj-salkynylene, C3.10 cycloalkylene, heterocydylene, heteroarylene and arylene.

The alkylene, alkenylene, alkynylene, cycloalkylene, heterocydylene, heteroarylene and arylene as A may optionally be substituted with one or more R3 (see further below).

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

In one embodiment, A is selected from -CHR2C(O)·, Ci-8 alkylene, C3-i0 cydoalkylene, heterocyclylene, heteroarylene and arylene, in particular from -CHR2C(O)-, Ci.8 alkylene and heterocyclylene, such as -CHR2C(O)-, or Ci-8 alkylene, or heterocyclylene. In this context, R2 may take any of the sets of values given for it above or below.

In particular, A may be -CH2C(O)- or -CH2-CH2-.

Y is typically selected from -H, -NR6R7, -OR7, Ci-8 alkyl, C2 8 alkenyl, C2.8 alkynyl, C3.io cycloalkyl, heterocydyl, heteroaryl and aryl. Preferred values for R6 and R7 are defined further below.

The alkyl, alkenyl, alkynyl, cycloalkyl, heterocydyl, heteroaryl and aryl as Y may optionally be substituted with one or more R3 (see further below);

In one embodiment, Y is -NR6R7. In one variant, A is -CHR2C(O)-, especially -CH2C(O)-, and Y is -NR6R7.

PCT/EP2013/070457 preferred that -A-Y should contain at least one hetero-atom, preferably at least one nitrogen atom or at least one oxygen.

Options independently adoptable include that:

Y is

wherein n is from 1 to 3 and each of Rio and Ru independently is as defined above.

Y is

wherein n is from 1 to 3 and each m independently is from 0 to 2.

Y is selected from heterocyclyl, heteroaryl and aryl, which may be optionally substituted with one or more R3.

Based on the studies conducted and the results obtained so far, it is believed that the following compounds (numbered 1 to 45), including isomers, mixtures of isomers, as well as pharmaceutically acceptable salts, solvates and prodrugs thereof, are particularly interesting:

2-({[3-(lH-lmidazol-l-yl)propyl]amlno}methyl)pyrldine-4-carboxylic acid

2-({[2-(dimethylamino)ethyl]amino}methyl)pyridine-4-carboxylic acid

2-({[(2R)-2,3-dihydroxypropyl]amino}methyl)pyridine-4-carboxylic acid

2-{[(cyclopropylmethyl)amino]methyl}pyridine-4-carboxylic acid

2-({[2-(dlmethylamlno)ethyl](methyl)amlno}methyl)pyrldlne-4-carboxyllc acid

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

2-{[methyl(prop-2-yn-l-yl)amino]methyl}pyridine-4-carboxylic acid

2-{[(2-fluoroethyl)amino]methyl}pyridine-4-carboxylic acid

2-{[(furan-2-ylmethyl)amino]methyl}pyridine-4-carboxylic acid

2-({[(5-phenylfuran-2-yl)methyl]amino}methyl)pyridine-4-carboxylic add

2-({[(2,4-dimethoxyphenyl)methyl]amino}methyl)pyridine-4-carboxylic acid

2-({[2-(methylsulfanyl)ethyl]amino}methyl)pyridine-4-carboxylic acid

2-({[2-oxo-2-(pyrrolidin-l-yl)ethyl]amino}-methyl)pyridine-4-carboxylic add 14 2-[({[butyl(methyl)carbamoyl]methyl}amino)methyl]pyridine-4-carboxylic add

2-({[(l-methyl-lH-l,3-benzodiazol-2-yl)rnethyl]amino}methyl)pyridine-4-carboxylic acid

2-[({2-[4-(2-methoxyethyl)piperazin-l-yl]-2-oxoethyl}amino)methyl]pyridine-4-carboxylic add 17 2-[({[bis(prop-2-en-l-yl)carbamoyl]methyl}amino)methyl]pyridine-4-carboxylic add

2-[({2-oxo-2-[(2R)-2-(pyrrolldln-l-ylmethyl)pyrrolldln-l-yl]ethyl}amlno)methyl]-pyrldine4-carboxylic acid

2-({[(3R)-l-[(tert-butoxy)carbonyl]pyrrolidin-3-yl]amino}methyl)pyridine-4-carboxylic acid

2-({[(3R)-l-[(tert-butoxy)carbonyl]pyrrolidin-3-yl]amino}rnethyl)pyridine-4-carboxylic add

2-{[(3-{[3-(pyrrolidin-l-yl)propyl]amino}propyl)amino]methyl}pyridine-4-carboxylic add

2-{[(3-methylbutyl)amino]methyl}pyridine-4-carboxylic acid

2-[({[(2-carbamoylethyl)(methyl)carbamoyl]methyl}amino)methyl]pyridine-4-carboxylic add

2-[({2-[2-(hydroxymethyl)piperidin-l-yl]-2-oxoethyl}amino)methyl]pyridine-4-carboxylic acid

2-{[({methyl[3-(l-methyl·lH-imidazol·2-yl)propyl]carbamoyl}methyl)amino]methyl}pyridine«4«carboxylic add

2-{[({[(l-ethylpyrrolidin-2-yl)methyl]carbamoyl}methyl)amino]methyl}pyridine-4carboxylic add

2-{[({methyl[(l-methyl·lH-ρyrazol·5yl)methyl]¢arbamôyl}methyl)aminô]· methyl} py rid i ne-4-carboxy I ic acid

2-({[(3R)-l-(3-phenylpropyl)pyrrolidin-3-yl]amino}methyl)pyridine-4-carboxylic acid

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

2-({[({l-[(2-methoxyphenyl)methyl]piperidin-4-yl}carbamoyl)methyl]amino}· methyl)pyridine-4-carboxylic add

2-{[({[l-(3-phenylpropyl)piperidin-4-yl]carbamoyl}methyl)amino]methyl}pyridine-4carboxylic acid

2-{[({[l-(furan-2-ylmethyl)piperidin-4-yl]carbamoyl}methyl)amino]methyl}pyridine-4· carboxylic add

2-({[({l-[(5-phenylfuran-2-yl)methyl]piperidin-4-yl}carbamoyl)methyl]amino}~ methyl)pyridine-4-carboxylic add

2-[({[(2-cyanoethyl)(ethyl)carbamoyl]methyl}amino)methyl]pyridine-4-carboxylic add

2-({[2-(l-butylpyrrolidin-2-yl)ethyl]amino}methyl)pyridine-4-carboxylic acid

2-{[({[l-(3,7-dimethyloct-6-en-l-yl)pyrrolidin-3-yl]carbamoyl}methyl)amino]methyl} py rid i ne-4-carboxy I ic acid

2-{[(3-{[(2-fluorophenyl)methyl](methyl)amino}propyl)amino]methyl}pyridine-4carboxylic acid

2({[(lR)2·hydroxy·l·{methyl[3·(lmethyllH·imidazol·2·yl)propyl]carbamoyl} ethyl]amino}methyl)pyridine-4-carboxylic acid

2-[({2-[3-(lH-l,3-benzodiazol-2-ylmethyl)piperidin-l-yl]-2-oxoethyl}amino)methyl]pyridine-4-carboxylic add

2-{[({[l-(2-phenylethyl)pyrrolidin-3-yl]carbamoyl}methyl)amino]methyl}pyridine-4carboxylic acid

2-({[3-(4-benzylpiperidin-l-yl)propyl]amino}methyl)pyridine-4-carboxylic add

2-[({3-[(2-phenoxyethyl)amino]propyl}amino)methyl]pyridine-4-carboxylic acid

2-[({[methyl({4-[(4-methylpiperazin-l-yl)methyl]phenyl}methyl)carbamoyl]methyl}amino)methyl]pyridine-4-carboxylic acid

2-({[2-(2-benzylpyrrolidin-l-yl)-2-oxoethyl]amino}methyl)pyridine-4-carboxylic acid

2-({[({4-[benzyl(cydopropyl)amino]butyl}(methyl)carbamoyl)methyl]amino}methyl)pyridine-4-carboxylic acid

2-[({2-[(2S)-l-benzylpyrrolidin-2-yl]ethyl}amino)methyl]pyridine-4-carboxylic add

2-({[3-(pyrrolidin-l-yl)propyl]amino}methyl)pyridine-4-carboxylic acid methyl 2-({[3-(pyrrolidin-l-yl)propyl]amino}methyl)pyridine-4-carboxylate

2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4-carboxylic acid

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4carboxylic acid (5-methyl-2-oxo-2H-l,3-diOXOl-4-yl)methyl 2-({[4(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

4-methoxyphenyl 2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

2-(ethoxycarbonyl)phenyl 2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4carboxylate

2-(dimethylamino)ethyl 2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

3-(dimethylamino)propyl 2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate {4-{[(ethoxycarbonyl)amino]phenyl}methyl 2-({[4(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

2,6-dimethoxyphenyl 2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

2,6-dimethylphenyl 2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

4-methoxyphenyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

2-(ethoxycarbonyl)phenyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate {4-[(ethoxycarbonyl)(methyl)amino]phenyl}-methyl 2-({[4(diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

4-tert-butylphenyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl>pyridine-4-carboxylate

4-oxopentan-2-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

4-(trifluoroacetamido)butan-2-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

4-(2,2,2-trifluoro-N-methylacetamido)butan-2-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate ethyl 2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4carboxylate

5-(trifluoroacetamido)pent-l-en-3-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

5-(2,2,2-trifluoro-N-methylacetamido)pent-l-en-3-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

2-(2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4· carbonyloxy)-3-(hexadecanoyloxy)propyl hexadecanoate i-(2-{[({[2-(dlmethylamlno)ethyl](ethyl)carbamoyl}methyl)amlno]methyl}pyridine-4carbonyloxy)-3-(hexadecanoyloxy)propan-2-yl hexadecanoate methyl 2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine4-carboxylate

2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl)-Nmethanesulfonyl-N-methylpyridine-4-carboxamide

N-[2-(dimethylamino)ethyl]-N-ethyl-2-({[4-(2-oxo-l/3-oxazolidine-3-carbonyl)pyridin-2yl]methyl}amino)acetamide propan-2-yl 3-(2-<[«[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carbonyloxy)-4(trifluoroacetamido)butanoate propan-2-yl 3-(2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carbonyloxy)-5(trifluoroacetamido)pentanoate

2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}-N-(pyridin-4yl)pyridine-4’carboxamide

2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}-N-(5-methyl· l,3,4·oxadiazol·2·yl)pyridine·4·carboxamide

2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}-N-(l-methyl-lHpyrazol-5-yl)pyridine-4-carboxamide

2-(piperidin-l-ylmethyl)pyridine-4-carboxylic acid

2’(azetidin«l«ylmethyl)pyridine’4-carboxylic acid

2,2,2-trifluoroethyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

2-({ethyl[2-oxo-2-(piperidin-l-yl)ethyl]amino}methyl)pyridine-4-carboxylic acid

2-({butyl[2-oxo-2-(piperidin-l-yl)ethyl]amino}methyl)pyridine-4-carboxylic acid

2-({benzyl[2-oxo-2-(piperidin-l-yl)ethyl]amino}methyl)pyridine-4-carboxylic acid θ4 2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}-N-(l,3'Oxazol-2yl)pyridine-4-carboxamide

2,6-bis(propan-2-yloxy)phenyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

2-{[(2-methylpropyl)[2-oxo-2-(piperidin-l-yl)ethyl]amino]methyl}pyridine-4-carboxylic add

2-({[2-oxo-2-(plperldln-i-yl)ethyl](propyl)amino}methyl)pyrldlne-4-carboxyllc add

2-({[2-oxo-2-(piperidin-l-yl)ethyl](propan-2-yl)amino}methyl)pyridine-4-carboxylic add

2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}-N-(l-methyl-lHimidazol-2-yl)pyridine-4-carboxamide

2-fluoroethyl 2-{[«[2(dimethylamino)ethyl](ethyl)carbamoyl)methyl)amino]methyl}pyridine-4-carboxylate

2,2-difluoroethyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate

2-({[(lS)-l-(tert-butylcarbamoyl)-3-methylbutyl]amino}methyl)pyridine-4-carboxylic add

2-({methyl[(2S)-4-methyl-l-oxo-l-(piperidin-l-yl)pentan-2-yl]amino)methyl)pyridine-4carboxylic add

Further compounds of particular interest are illustrated in Table 1 below and in the other examples.

Definitions

The term alkyl as used herein refers to a saturated, straight or branched hydrocarbon chain. The hydrocarbon chain preferably contains from one to 8 carbon atoms (Ci.8-alkyl), more preferred from one to six carbon atoms (Ci.6-alkyl), in particular from one to four carbon atoms (Ci-4-alkyl), induding methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, tertiary butyl, pentyl, isopentyl, neopentyl, tertiary pentyl, hexyl, isohexyl, heptyl and octyl. In a preferred embodiment alkyl represents a Ci.4-alkyl group, which may in particular include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, and tertiary butyl. Correspondingly, the term alkylene means the corresponding biradical (-alkyl-).

The term cycloalkyl as used herein refers to a cyclic alkyl group, preferably containing from three to ten carbon atoms (C3.10-cydoalkyl), such as from three to eight carbon atoms (C3 8cydoalkyl), preferably from three to six carbon atoms (C3.6-cydoalkyl), induding cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cydooctyl. Furthermore, the term cycloalkyl as used herein may also include polycyclic groups such as for example bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptanyl, decalinyl and adamantyl. Correspondingly, the term cydoalkylene means the corresponding biradical (-cycloalkyl-).

The term alkenyl as used herein refers to a straight or branched hydrocarbon chain or cyclic hydrocarbons containing one or more double bonds, including di-enes, tri-enes and poly-enes. Typically, the alkenyl group comprises from two to eight carbon atoms (Cj.g-alkenyl), such as

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457 from two to six carbon atoms (C2.6-alkenyl), in particular from two to four carbon atoms (C2-4alkenyl), including at least one double bond. Examples of alkenyl groups include ethenyl; 1- or 2-propenyl; 1-, 2- or 3-butenyl, or 1,3-but-dienyl; 1-, 2-, 3-, 4- or 5-hexenyl, or 1,3-hex-dienyl, or 1,3,5-hex-trienyl; 1-, 2-, 3-, 4-, 5-, 6-, or 7-octenyl, or 1,3-octadienyl, or 1,3,5-octatrienyl, or 1,3,5,7-octatetraenyl, or cyclohexenyl. Correspondingly, the term alkenylene means the corresponding biradical (-alkenyl-).

The term alkynyl as used herein refers to a straight or branched hydrocarbon chain containing one or more triple bonds, including di-ynes, tri-ynes and poly-ynes. Typically, the alkynyl group comprises of from two to eight carbon atoms (C2.e-alkynyl), such as from two to six carbon atoms (C2.6-alkynyl), in particular from two to four carbon atoms (C2.4-alkynyl), including at least one triple bond. Examples of preferred alkynyl groups include ethynyl; 1- or 2-propynyl; 1-, 2or 3-butynyl, or 1,3-but-diynyl; 1-, 2-, 3-, 4- or 5-hexynyl, or 1,3-hex-diynyl, or 1,3,5-hextriynyl; 1-, 2-, 3-, 4-, 5-, 6-, or 7-octynyl, or 1,3-oct-diynyl, or 1,3,5-oct-triynyl, or 1,3,5,7-octtetraynyl. Correspondingly, the term alkynylene means the corresponding biradical (-alkynyl-).

The terms halo and halogen as used herein refer to fluoro, chloro, bromo or iodo. Thus a trihalomethyl group represents e.g. a trifluoromethyl group, or a trichloromethyl group. Preferably, the terms halo and halogen designate fluoro or chloro.

The term fluoroalkyl as used herein refers to an alkyl group as defined herein which is substituted one or more times with one or more fluorohalo, preferably perfluorated. The term perfluoroalkyl as used herein refers to an alkyl group as defined herein wherein all hydrogen atoms are replaced by fluoro atoms. Preferred fluoroalkyl groups include trifluoromethyl, pentafluoroethyl, etc.

The term alkoxy as used herein refers to an alkyl-O- group, wherein alkyl is as defined above.

The term hydroxyalkyl as used herein refers to an alkyl group (as defined hereinabove), which alkyl group is substituted one or more times with hydroxy. Examples of hydroxyalkyl groups include HO-CH2·, HO-CH2-CH2- and CH3-CH(OH)-.

The term oxy as used herein refers to an *·Ο· group.

The term ”0x0 as used herein refers to an ”=O group.

The term amine as used herein refers to primary (R-NH2, R * H), secondary (R2-NH, R2 * H) and tertiary (Rj-N, R * H) amines. A substituted amine is intended to mean an amine where at least one of the hydrogen atoms has been replaced by the substituent.

The term carbamoyl as used herein refers to a H2N(C=O)- group.

The term aryl”, as used herein, unless otherwise Indicated, includes carbocyclic aromatic ring systems derived from an aromatic hydrocarbon by removal of a hydrogen atom. Aryl furthermore includes bi-, tri- and polycyclic ring systems. Examples of preferred aryl moieties

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457 include phenyl, naphthyl, indenyl, indanyl, fluorenyl, biphenyl, indenyl, naphthyl, anthracenyl, phenanthrenyl, pentalenyl, azulenyl, and biphenylenyl. Preferred aryl is phenyl, naphthyl or indanyl, in particular phenyl, unless otherwise stated. Any aryl used may be optionally substituted. Correspondingly, the term arylene means the corresponding biradical (-aryl-).

The term heteroaryl, as used herein, refers to aromatic groups containing one or more heteroatoms selected from 0, S, and N, preferably from one to four heteroatoms, and more preferably from one to three heteroatoms. Heteroaryl furthermore includes bi-, tri- and polycyclic groups, wherein at least one ring of the group is aromatic, and at least one of the rings contains a heteroatom selected from 0, S, and N. Heteroaryl also Include ring systems substituted with one or more oxo moieties. Examples of preferred heteroaryl moieties include Nhydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, furanyl, triazolyl, pyranyl, thiadiazinyl, benzothiophenyl, dihydro-benzo[b]thiophenyl, xanthenyl, isoindanyl, acridinyl, benzisoxazolyl, quinolinyl, isoquinolinyl, phteridinyl, azepinyl, diazepinyl, imidazolyl, thiazolyl, carbazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, isothiazolyl, pyrrolyl, indolyl, benzimidazolyl, benzofuranyl, dnnolinyl, indazolyl, indolizinyl, phthalazinyl, triazinyl, isoindolyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl, tetrahydroisoquinolyl, benzofuryl, furopyridinyl, pyrolopyrimidinyl, azaindolyl, pyrazolinyl, and pyrazolidinyl. Non-limiting examples of partially hydrogenated derivatives are 1,2,3,4tetrahydronaphthyl, 1,4-dihydronaphthyl, and 1-octalin. Correspondingly, the term heteroarylene means the corresponding biradical (-heteroaryl-).

The term heterocyclyl as used herein, refers to cyclic non-aromatic groups containing one or more heteroatoms selected from 0, S, and N, preferably from one to four heteroatoms, and more preferably from one to three heteroatoms. Heterocyclyl furthermore Includes bi-, tri- and polycyclic non-aromatic groups, and at least one of the rings contains a heteroatom selected from 0, S, and N. Heterocyclyl also include ring systems substituted with one or more oxo moieties. Examples of heterocyclic groups are oxetane, pyrrolidinyl, pyrrolyl, 3H-pyrrolyl, oxolanyl, furanyl, thiolanyl, thiophenyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolidinyl, 3Hpyrazolyl, 1,2-oxazolyl, 1,3-oxazolyl, 1,2-thiazolyl, 1,3-thiazolyl, 1,2,5-oxadiazolyl, piperidinyl, pyridinyl, oxanyl, 2-H-pyranyl, 4-H-pyranyl, thianyl, 2H-thiopyranyl, pyridazinyl, 1,2-diazinanyl, pyrimidinyl, 1,3-diazinanyl, pyrazinyl, piperazinyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-diazinanyl, 1,4oxazinyl, morpholinyl, thiomorpholinyl, 1,4-oxathianyl, benzofuranyl, isobenzofuranyl, indazolyl, benzimidazolyl, quinolinyl, isoquinolinyl, chromayl, isochromanyl, 4H-chromenyl, 1Hisochromenyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, purinyl, naphthyridinyl, pteridinyl, indolizinyl, IH-pyrrolizinyl, 4H-quinolizinyl and aza-8-bicydo[3.2.1]octane. Correspondingly, the term heterocydylene means the corresponding biradical (-heterocyclyl-).

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

The term N-heterocyclic ring as used herein, refers to a heterocyclyl or a heteroaryl as defined hereinabove having at least one nitrogen atom, and being bound via a nitrogen atom. Examples of such N-heterocyclic rings are pyrrolidinyl, pyrrolyl, 3H-pyrrolyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolidinyl, 3H-pyrazolyl, 1,2-oxazolyl, 1,2-thiazolyl, 1,3-thiazolyl, piperidinyl, pyridinyl, pyridazinyl, pyrazinyl, piperazinyl, morpholinyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazolyl, pyrazinyl, tetrazolyl, etc.

Isomers

The compounds of Formula (I) may exist as geometric isomers (i.e. cis-trans isomers), optical isomers or stereoisomers, such as diastereomers, as well as tautomers. Accordingly, it should be understood that the definition of compounds of Formula (I) includes each and every individual isomers corresponding to the structural formula: Formula (I), including cis-trans isomers, stereoisomers and tautomers, as well as racemic mixtures of these and pharmaceutically acceptable salts thereof. Hence, the definition of compounds of Formula (I) is also intended to encompass all R- and S-isomers of a chemical structure in any ratio, e.g. with enrichment (i.e. enantiomeric excess or diastereomeric excess) of one of the possible isomers and corresponding smaller ratios of other isomers.

Diastereoisomers, i.e. non-superimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active acid or base. Examples of appropriate adds include, without limitation, tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. The mixture of diastereomers can be separated by crystallization followed by liberation of the optically active bases from these salts. An alternative process for separation of optical isomers includes the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules by reacting compounds of Formula (I) with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to obtain the enantiomerically pure compound. The optically active compounds of Formula (I) can likewise be obtained by utilizing optically active starting materials and/or by utilizing a chiral catalyst. These isomers may be in the form of a free acid, a free base, an ester or a salt. Examples of chiral separation techniques are given in Chiral Separation Techniques, A Practical Approach, 2nd ed. by G. Subramanian, Wiley-VCH, 2001.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Pharmaceutically acceptable salts

The compound of Formula (I) may be provided in any form suitable for the intended administration, In particular including pharmaceutically acceptable salts, solvates and prodrugs of the compound of Formula (I).

Pharmaceutically acceptable salts refer to salts of the compounds of Formula (I), which are considered to be acceptable for clinical and/or veterinary use. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of Formula (I) a mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition salts and base addition salts, respectively. It will be recognized that the particular counter-ion forming a part of any salt is not of a critical nature, so long as the salt as a whole is pharmaceutically acceptable and as long as the counter-ion does not contribute undesired qualities to the salt as a whole. These salts may be prepared by methods known to the skilled person. Pharmaceutically acceptable salts are, e.g., those described and discussed in Remington's Pharmaceutical Sciences, 17. Ed. Alfonso R. Gennaro (Ed.), Mack Publishing Company, Easton, PA, U.S.A., 1985 and more recent editions and in Encyclopedia of Pharmaceutical Technology.

Examples of pharmaceutically acceptable addition salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric, or phosphoric add; and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, trifluoroacetic, malic, lactic, formic, propionic, glycolic, gluconic, camphorsulfuric, isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), ethanesulfonic, pantothenic, stearic, sulfinilic, alginic and galacturonic acid; and arylsulfonic, for example benzenesulfonic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid; and base addition salts formed with alkali metals and alkaline earth metals and organic bases such as N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine and procaine; and internally formed salts.

Solvates

The compound of Formula (I) may be provided in dissoluble or Indissoluble forms together with a pharmaceutically acceptable solvent such as water, ethanol, and the like. Dissoluble forms may also include hydrated forms such as the mono-hydrate, the dihydrate, the hemihydrate, the trihydrate, the tetrahydrate, and the like.

Prodruqs

The compound of Formula (I) may be provided as a prodrug. The term prodrug used herein is intended to mean a compound which - upon exposure to certain physiological conditions - will liberate the compound of Formula (I) which then will be able to exhibit the desired biological action. A typical example is a labile ester of a carboxylic acid, in particular the pyridine carboxylic

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457 add group of the compound of formula (I), which e.g. is capable of liberating the latent carboxylic add group.

Illustrative examples of esters of a carboxylic acid group (in particular the pyridine carboxylic acid) are Cl-6 alkyl esters, e.g. methyl esters, ethyl esters, 2-propyl esters, phenyl esters, 2aminoethyl esters, etc., including (5-methyl-2-oxo-2H-l,3-dioxol-4-yl)methyl esters, 4methoxyphenyl esters, 2-(ethoxycarbonyl)phenyl esters, {4[(ethoxycartonyl)(methyl)amino]phenyl}methyl esters, 2-(dimethylamino)ethyl esters, 3(dlmethylamlno)propyl esters, [(ethoxycarbonyl)amlno]phenylmethyl esters, 2,6dimethoxyphenyl esters, 2,6-dimethylphenyl esters, 4-tert-butylphenyl esters, 4-oxopentan-2-yl esters, 4-(trifluoroacetamido)butan-2-yl esters, 4-(2,2,2-trifluoro-N-methylacetamido)butan-2-yl esters, 5-(trifluoroacetamido)pent-l-en-3-yl esters, 5-(2,2,2-trifluoro-N-methylacetamido)pentl-en-3-yl esters, l,3-bis(hexadecanoyloxy)propan-2-yl esters, 2,3-bis(hexadecanoyloxy)propyl esters, 4-oxo-4-(propan-2-yloxy)-l-(trifluoroacetamido)butan-2-yl esters, l-oxo-l-(propan-2yloxy)-5(trifluoroacetamido)pentan-3-yl esters 2,2,2-trifluoethyl esters, 2,6-bis(propan-2-yloxy)phenyl esters, 2-fluoroethyl esters, 2,2-difluoroethyl esters, etc.

In particular, a prodrug according to the invention may be of the form

wherin R12 is of the form (R13)2N- or of the form R13O-, where each R13 independently may be consistent with any of the examples of prodrugs given above or shown below. In particular, each R13 independently may be selected from Ci-s alkyl, C2-8 alkenyl, C2-s alkynyl, C3.10 cycloalkyl, and aryloxy which alkyl, alkenyl, alkynyl, cycloalkyl and aryloxy may be optionally substituted with one or more selected from -OH, aryl, Cm alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, a sulphonamide moiety, and C3.6cycloalkyl; and one R13 in (R13)2N- may be, and preferably Is, -H.

Date Reçue/Date Received 2020-10-01

WO 2014/053491

CA 02885969 2015-03-28

PCT/EP2013/070457

Methods for the preparation of compounds of Formula (I)

The compounds of Formula (I) as defined herein may be prepared by conventional methods of chemical synthesis, e.g. those described in the working examples, and starting from readily available starting materials. The starting materials for the processes described in the present application are known or may readily be prepared by conventional methods from commercially available chemicals.

The final products of the reactions described herein may be isolated by conventional techniques, e.g. by extraction, crystallisation, distillation, chromatography, etc.

In the following are described some of the possible routes to the compounds of Formula (I).

The compounds of the Formula (I) may be prepared as described in the following. Useful steps that may be used in the preparation steps of the compounds will be known to the skilled person. The methods below are given as non-limiting examples on how the compounds may be prepared.

Method A through C show the preparation of the compounds of this invention. Useful methods for the generation of intermediates are described afterwards.

Preparation of Compounds of Formula (I)

Scheme 1

Method A

The compound of Formula (I) may be prepared according to Scheme 1 at room temperature, or by heating for up to several hours by use of a solvent such as DMSO, an alcohol, or tetrahydrofuran, and a base such as LiOH, KOH, or NaOH. A purification method such as silica gel chromatography is employed if needed.

Method B

The compound of Formula (I) may be prepared according to Scheme 1 at room temperature, or by heating for up to several hours by use of a solvent such as water, DMSO, an alcohol, or tetrahydrofuran, and an aqueous acid. A purification method such as silica gel chromatography is employed if needed.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

Intermediates (x) may be prepared from (ix) according to Scheme 8 analogously to Method H.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Scheme 9

Method J

Intermediates (xii) may be prepared from aldehydes and intermediates (xi) according to Scheme 9 in one-pot or by a stepwise procedure by mixing with the amine (R': RI or a suitable protecting group) and a reducing agent such as NaBH4, NaBH(OAc)3, NaCNBH3, or Et3SiH, either at room temperature or by heating for up to several hours by use of a solvent such as an alcohol, DCE, DCM, water, or toluene, optionally adding a catalyst such as an acid or a Lewis add· Optionally, protecting groups may be removed and a purification method such as silica gel chromatography is employed if needed.

Method K

Intermediates (xii) may be prepared from (xi) according to Scheme 9 by use of a solvent such as DMF or THF, optionally a base, and a suitable electrophilic species such as an epoxide, an aliphatic, allylic or benzylic bromide, chloride, or sulfonate. A purification method such as silica gel chromatography is employed if needed.

Scheme 10

X

(xiv)

Method L

Intermediates (xiv) may be prepared from (xiii) according to Scheme 10 analogously to Method

J.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Method M

Intermediates (xiv) may be prepared from (xiii) according to Scheme 10 analogously to Method K.

Scheme 11

(xv)

(xvi)

Method N

Intermediates (xvi) may be prepared from intermediates (xv) according to Scheme 11 in one-pot or by a stepwise procedure by mixing with an amine, optionally containing orthogonal protected reactive sites, and a reducing agent such as NaBH4, NaBH(OAc)3, NaCNBH3, or Et3SiH, either at room temperature or by heating for up to several hours by use of a solvent such as an alcohol, DCE, DCM, water, or toluene, optionally adding a catalyst such as an acid or a Lewis add. Optionally, protecting groups may be removed and a purification method such as silica gel chromatography is employed if needed.

Scheme 12

(xvii) x

(xviii)

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Method 0

Intermediates (xviii) may be prepared from intermediates (xvii) according to Scheme 12 analogously to Method N.

Inhibitory effect

The inventors have surprisingly found that compounds of Formula (I) as defined herein have an inhibitory effect on the activity of one or more HDMEs. In this respect said one or more HDMEs may be any HDME, however preferably the one or more HDMEs are selected from the JmjC (Jumonji) family, more preferably said one or more HDME(s) are HDME of the human JmjC family and even more preferably are HDME belonging to the KDM6, KDM5, KDM4 or KDM2 families. The present invention also relates to a compound of Formula (I) as defined herein in a method for inhibiting HDMEs. The method includes contacting a cell with a compound of Formula (I). In a related embodiment, the method further provides that the compound is present in an amount effective to produce a concentration sufficient to inhibit the demethylation of a histone in the cell.

Thus, preferably in an assay for demethylation of a histone substrate by said HDME, then preferred compounds of Formula (I) are compounds capable of reducing or preferably inhibiting said demethylation by said HDME. Said histone substrate may be any histone, but preferably is histone H3 or a fragment thereof, even more preferred: a fragment comprising K4, K9, K27, or K36 of H3. Preferably, said inhibition is determined as the ICso of said compound of Formula (I) in respect of the said demethylation assay.

Preferred compounds of Formula (I) which have an IC5o at or below 1 pM, more preferably less than 300 nM, for example less than 100 nM, such as less than 50 nM in respect of demethylation of any of said histone substrates by any of said HDME. Thus very preferred compounds of Formula (I) which have an ICsc at or below 1 μΜ, more preferably less than 500 nM, for example less than 100 nM, such as less than 50 nM in respect of demethylation of histone H3 methylated at least on one lysine.

In a preferred embodiment ICS0 is determined as described in Example 2 herein below. Thus, particularly preferred are compounds of Formula (I) which have an ICso at or below 1 pM, more preferably less than 500 nM, for example less than 100 nM, such as less than 50 nM when said ICS0 >s determined as described in and one of the Examples herein below.

Particularly preferred compounds of Formula (I) are compounds that lead to a decreased tumour size and/or decreased number of metastases when tested in a xenograft model (Morton and Houghton, Nature Protocols, 2 (2) 247-250, 2007).

Pharmaceutical compositions

In one aspect of this invention, there is provided a pharmaceutical composition comprising at, as an active ingredient, at least one compound of Formula (I) as defined herein and optionally one

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457 or more pharmaceutically acceptable excipients, diluents and/or carriers. The compounds of Formula (I) may be administered alone or in combination with pharmaceutically acceptable carriers, diluents or excipients, in either single or multiple doses. Suitable pharmaceutically acceptable carriers, diluents and excipients include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.

The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 21st Edition, 2000, Lippincott Williams & Wilkins.

The pharmaceutical compositions formed by combining a compound of Formula (I) as defined herein with pharmaceutically acceptable carriers, diluents or excipients can be readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, suppositories, injectable solutions and the like, in powders, the carrier is a finely divided solid such as talc or starch which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

The pharmaceutical compositions may be specifically prepared for administration by any suitable route such as the oral and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.

Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings or they can be prepared so as to provide controlled release of the active ingredient such as sustained or prolonged release according to methods well known in the art.

For oral administration in the form of a tablet or capsule, a compound of Formula (I) as defined herein may suitably be combined with an oral, non-toxic, pharmaceutically acceptable carrier such as ethanol, glycerol, water or the like. Furthermore, suitable binders, lubricants, disintegrating agents, flavouring agents and colourants may be added to the mixture, as appropriate. Suitable binders indude, e.g., lactose, glucose, starch, gelatin, acacia gum, tragacanth gum, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes or the like. Lubricants include, e.g., sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride or the like. Disintegrating agents include, e.g., starch, methyl cellulose, agar, bentonite, xanthan gum, sodium starch glycolate, crospovidone, croscarmellose sodium or the like. Additional excipients for capsules include macrogols or lipids.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

For the preparation of solid compositions such as tablets, the active compound of Formula (I) is mixed with one or more excipients, such as the ones described above, and other pharmaceutical diluents such as water to make a solid pre-formulation composition containing a homogenous mixture of a compound of Formula (I). The term homogenous is understood to mean that the compound of Formula (I) is dispersed evenly throughout the composition so that the composition may readily be subdivided Into equally effective unit dosage forms such as tablets or capsules.

Liquid compositions for either oral or parenteral administration of the compound of Formula (I) include, e.g., aqueous solutions, syrups, elixirs, aqueous or oil suspensions and emulsion with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil. Suitable dispersing or suspending agents for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose or polyvinylpyrolidone.

Pharmaceutical compositions for parenteral administration Include sterile aqueous and nonaqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. For parenteral administration, solutions containing a compound of Formula (I) in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The oily solutions are suitable for intra-articular, intra-muscular and subcutaneous injection purposes.

The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

Depot injectable compositions are also contemplated as being within the scope of the present invention.

In addition to the aforementioned ingredients, the compositions of a compound of Formula (I) may include one or more additional ingredients such as diluents, buffers, flavouring agents, colourant, surface active agents, thickeners, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like.

A suitable dosage of the compound of Formula (I) will depend on the age and condition of the patient, the severity of the disease to be treated and other factors well known to the practicing physician. The compound may be administered for example either orally, parenterally or topically according to different dosing schedules, e.g. daily or with intervals, such as weekly intervals. In general a single dose will be in the range from 0.01 to 100 mg/kg body weight, preferably from about 0.05 to 75 mg/kg body weight, more preferably between 0.1 to 50 mg/kg body weight, and most preferably between 0.1 to 25 mg/kg body weight. The compound may be administered as a bolus (i.e. the entire daily dose is administered at once) or in divided doses two or more

Date Reçue/Date Received 2020-10-01

84515062 times a day. Variations based on the aforementioned dosage ranges may be made by a physician of ordinary skill taking into account known considerations such as weight, age, and condition of the person being treated, the severity of the affliction, and the particular route of administration.

The compounds of Formula (I) may also be prepared in a pharmaceutical composition comprising one or more further active substances alone, or in combination with pharmaceutically acceptable carriers, diluents, or excipients in either single or multiple doses. The suitable pharmaceutically acceptable carriers, diluents and excipients are as described herein above, and the one or more further active substances may be any active substances, or preferably an active substance as described in the section combination treatment herein below.

Clinical conditions and other uses of compounds

The compounds according to Formula (I) as defined herein are useful for treatment of a HDME dependent disease, disorder or condition. The treatment may include administering to a mammal, preferably a human, more preferably a human suffering from a HDME dependent disease, a therapeutically effective amount of a compound according to Formula (I) as defined herein.

Said HDME may be any HDME, however preferably the HDME of the present method is selected from the JmjC (Jumonji) family, as described in Cloos et. al., Genes & Development 22,11151140, 2008. More preferably said HDME is a HDME of the human JmjC family.

The present invention also relates to a compound of Formula (I) as defined herein for use in the treatment of a HDME dependent disease, such as for the treatment of cancer.

By the term HDME dependent disease is meant any disease characterized by elevated HDME expression and/or activity in at least in some instances of the disease, or a disease which is ameliorated by lowering the activity of HDMEs. Thus, the disease to be treated with the inhibitors of HDME, i.e. compounds of Formula (I), may be a proliferative or hyperproliferative disease, which includes benign or malignant tumors, for example a proliferative or hyperproliferative disease selected from the group consisting of a carcinoma of the brain, kidney, liver, adrenal gland, bladder, breast, stomach (for example gastric tumors), ovaries, esophagus, colon, rectum, prostate, pancreas, lung, vagina, thyroid, sarcoma, glioblastomas, multiple myeloma or gastrointestinal cancer, for example, colon carcinoma or colorectal adenoma, or a tumor of the neck and head, an epidermal hyperproliferation, for example, psoriasis, prostate hyperplasia, a neoplasia, including a neoplasia of epithelial character, including mammary carcinoma, and a leukemia.

In one embodiment, compounds of Formula (I) as defined herein are useful in the treatment of one or more cancers. The term cancer refers to any cancer caused by the proliferation of neoplastic cells, such as solid tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457 and the like. In particular, cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma, (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wllm's tumor, nephroblastoma, lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcfnoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholang iocarci noma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcorna, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic: blood (acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.

WO 2014/053491

PCT/EP2013/070457 or any combination thereof.

The phrase, aromatase inhibitor as used herein relates to a compound which inhibits the estrogen production, i.e., the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to steroids, especially atamestane, exemestane and formestane and, in particular, non-sterolds, especially aminoglutethimide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane can be administered, e.g., in the form as it is marketed, e.g., under the trademark AROMASIN. Formestane can be administered, e.g., in the form as it is marketed, e.g., under the trademark LENTARON. Fadrozole can be administered, e.g., in the form as it is marketed, e.g., under the trademark AFEMA. Anastrozole can be administered, e.g., In the form as It Is marketed, e.g., under the trademark ARIMIDEX. Letrozole can be administered, e.g., in the form as it is marketed, e.g., under the trademark FEMARA or FEMAR. Aminoglutethimide can be administered, e.g., in the form as it is marketed, e.g., under the trademark ORIMETEN. A combination of the invention comprising a chemotherapeutic agent which is an aromatase inhibitor is particularly useful for the treatment of hormone receptor positive tumors, e.g., breast tumors.

The term antiestrogen as used herein relates to a compound that antagonizes the effect of estrogens at the estrogen receptor level. The term includes, but is not limited to tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen can be administered, e.g., in the form as it is marketed, e.g., under the trademark NOLVADEX. Raloxifene hydrochloride can be administered, e.g., in the form as it is marketed, e.g., under the trademark EVISTA. Fulvestrant can be formulated as disclosed in US 4,659,516 or it can be administered, e.g., in the form as it is marketed, e.g., under the trademark FASLODEX. A combination of the invention comprising a chemotherapeutic agent which is an antiestrogen is particularly useful for the treatment of estrogen receptor positive tumors, e.g., breast tumors.

The term anti-androgen as used herein relates to any substance which is capable of inhibiting the biological effects of androgenic hormones and includes, but is not limited to, bicalutamide (CASODEX), which can be formulated, e.g., as disclosed In US 4,636,505.

<td rowspan="5"> Table 1</td><td> NMR</td><td>λΗ NMR (300 MHz, CD3OD), δ ppm: 8.10 (s, 1H), 7.58 (dd, 1H), 7.80 (t, 1H), 7.65 (t, 1H), 4.58 (s, 2H), 4.50 (t, 2H).</td><td>λΗ NMR (300 MHz, CD3OD), 8 ppm: 8.03 (s, 1H), 4.58 (s, 2H),3.63 (m, 4H),3.0(s, 6H).</td><td> *H NMR (300 MHz, CDjOD), 8 ppm: 8.12 (s, 1H), 4.04 (m, 1H), 3.62 (m, 2H), 3.35 (m, 1H), 3.18 (dd, 1H).</td><td> *H NMR (300 MHz, CD,OD), 8 ppm: 4.56 (s, 2H), 3.08 (d, 2H), 1.21 (m, 1H), 0.75 (m, 2H), 0.47 (m, 2H).</td>

PCT/EP2013/070457

<td> 5-(2,2,2-t rifl uoro-N methy la ceta mi do)pent-len-3-yl 2-{[({[2(dimethy lam ino)ethyl) (eth yl )ca rbamoyl}methyl)amin ojmethy 1}pyridi n e -4carboxylat</td><td> 2-(2-{[({[2(dimethyla m ino)ethy 1] (eth yl )ca rbamoyl}methyl)amin ojmethy 1 Jpyridi n e -4carbonyloxy)-3(hexadecanoyloxy)propyl hexadecanoate</td>

<td> CH, O CH, F L o o x:h3 ^ch2</td><td> Below</td>

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

9. X

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CH3

H

Synthetic Route E

Synthetic Route B

General Procedure D

----------► (p)

General Procedure E (d) ----------►

General Procedure B

(o)

General Procedure A (q) -----------► Title Compound

Prepared by General Procedure A from ethyl 2-({2,2,2-trifluoro-N-[({l-[(2methoxyphenyl)methyl]piperidin-4-yl}carbamoyl)methyl]acetamido}methyl)pyridine-4-carboxylate (q) using 2 equivalents of LiOH. Isolated as lithium salt after work up by dissolving residue in minimum of water and extract with DCM. The aqueous phase was evaporated to dryness yielding the title compound as white solid.

^-NMR (300MHz, CD3OD): δ 8.54(d,lH), 7.82(s, 1H), 7.65(d, 1H), 7.26(m, 2H), 6.94(m, 2H), 3.90(5, 2H), 3.85(S, 3H), 3.72(m, 3H), 3.62(s, 2H), 3.24(5, 2H), 2.94(m, 2H), 2.24(m, 2H), 1.90(m, 3H), 1.60(m, 2H).

ES-MS: 411 [M-H],

Date Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

2-({[({4[benzyl(cyclopropyl)amino]butyl}(methyl)carbamoyl)methyl]amino}methyl)pyridine-4carboxylic acid (#44)

Synthetic Route F

General Procedure B -----------► (t)

General Procedure B General Procedure A

-----------(u) ------------>- Title Compound

Prepared by General Procedure A from ethyl 2-«N-[({4benzyl(cyclopropyl)amino]butyl}(methyl)carbamoyl)methyl]-2,2,2trifluoroacetamido}methyl)pyridine-4-carboxylate (u) using 2.5 equivalents of LiOH. Isolated as lithium salt after work up by dissolving residue in minimum of water and extract with DCM. The aqueous phase was evaporated to dryness yielding the title compound as white solid.

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457 1H-NMR (300MHz, CDCh): δ 8-50 (d, 1H), 7.80 (S, 1H), 7.70 (d, 1H), 7.25 (m, 5H), 3-90 (S, 2H), 3.70 s, 2H), 3.50-3.40 (m, 3H), 2.25 (m, 1H), 2.95 (s, 3H), 2.55 (m, 2H), 1.75 (m, 1H), 1.50 (m, 4H), 0.50 (m, 2H), 0.40 (m, 2H).

ES-MS: 425 [M+l].

2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}-N-(pyridin-4yl)pyridine-4-carboxamide (#75)

Synthetic Route G □ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

100

WO 2014/053491

PCT/EP2013/070457

General Procedure G General Procedure A (#65) -----------► (v) -----------► (X)

General Procedure E or General Procedure H (y)

General Procedure D -----------D» Title Compound

Prepared by general procedure D from tert-butyl N-«[2(dimethylamino)ethyl](ethyl)carbamoyl)methyl)-N-({4-[(pyridin-4-yl)carbamoyl]pyridin-2yl}methyl)carbamate (y) to get the title compound as yellow oil.

^-NMR (300MHz, CD3OD): $ 8.73 (d, 2H), 8.44 (d, 2H), 8-02 (d, 2H), 6.85 (d, 1H), 4.62 ($, 2H),

4.30 (s, 2H), 3.83 (t, 2H), 3.33-3.31 (m, 4H), 2.99 (s, 6H), 1.26 (t, 3H).

ES-MS: 486 [M+l]

4-oxopentan-2-yl 2-<[«[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4-carboxylate (#62) □ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

101

WO 2014/053491

PCT/EP2013/070457

Synthetic Route H

Synthetic Route G

-----► w

General Procedure E or General Procedure H (2)

General Procedure F (aa)

General Procedure D -----------»» Title Compound

Prepared by General Procedure D from 4-oxopentan-2-yl 2-({[(tert-butoxy)carbonyl]({[2(dlmethylamlno)ethyl](ethyl)carbamoyl}methyl)amlno}methyl)pyrldine-4-carboxylate (aa) to get the title compound as a colorless oil.

^-NMR (300MHz, CD3OD): δ 8.75 (d, 1H ), 7.90-8.10 (m, 2H), 5.50 (m, 1H), 4.50 (s, 2H), 4.25 (s, 2H), 3.75 (m, 2H), 1.20-1.50 (m, 6H).

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

102

ES-MS: 393 [M+l]

2-({ethyl[2-oxo-2-(piperidin-l-yl)ethyl]amino}methyl)pyridine-4-carboxylic acid (#81)

Synthetic Route I

General Procedure B (ab) ----------->

(ac)

General Procedure A

----------Title Compound

Prepared by general procedure A from ethyl 2-({ethyl[2-oxo-2-(piperidin-l-yl)ethyl]amino}methyl) pyridine-4-carboxylate to get the title compound as yellow solid.

□ate Reçue/Date Received 2020-10-01

GA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

103 XH NMR (300 MHz, CD3OD) δ 8.48 (d, 1H), 7.94 ($, 1H), 7.70 (d, 1H), 3-80 (S, 2H), 3.47 (m, 4H), 3.36 (s, 2H), 2.63 (q, 2H), 1.54 (m, 6H), 1.10 (t, 3H).

ES-MS: 306 [M+l]

Intermediates

PCT/EP2013/070457

106

Ethyl 2-({N-[({4-[benzyl(cyclOpropyl)amino]butyl}(methyl)carbamoyl)methyl]-2l2i2trifluoroacetamido}methyl)pyridine-4-carboxylate (u)

PCT/EP2013/070457

112

Ethyl 2-({[(tert-butoxy)carbonyl]({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino}methyl)pyridine-4-carboxylate (v)

General Procedure G (Boc protection of amines)

The amine (ethyl 2-{[({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine4-carboxylate (#65)) (1 eq) and Boc2O (1.2 eq) were dissolved in THF/H2O. NaHCO3 solid (4 eq) was added. The reaction mixture was stirred at room temperature overnight. After solvent removal, the residue was purified by column chromatography with a gradient of 0-10% MeOH in DCM to give the title product as yellow oil.

’H-NMR (300MHz, CDCI3): δ 8.52 (m, 1H), 7.84 (m, 1H), 7.74 (m, 1H), 4.65 (m, 2H), 4.23 (m, 2H), 3.45 (m, 4H), 2.50 (m, 2H), 2.25 (m, 6H), 1.43 (m, 9H), 1.19 (m, 3H).

tert-butyl N-«[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)-N-({4-[(pyridin-4yl)carbamoyl]pyridin-2-yl}methyl)carbamate (y)

General Procedure H (Formation of esters, amides, and sulfonamides)

Et3N (3.0 eq) and propane phosphonlc add anhydride (2.0 eq) were added to a solution of an amine (pyrldin-4-amine) (or an alcohol or a sulfonamide) (1.5 eq) and an acid (2-({[(tertbutoxy)carbonyl](([2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl) amino}methyl)pyridine-4carboxylic acid (x)) (1.0 eq) in DMF. The reaction mixture was stirred for 12 h at r.t., DMF was removed in vacuum, diluted with DCM and washed with water to get the title compound as a brown oil. Purified by column chromatography or used without further purification.

lH-NMR (300MHz, D2O): δ 8.59 (d, 2H ), 8.21 (d, 2H), 7.88 (d, 2H), 6.75 (d, 1H), 4.35-4.32 (m, 2H), 3.71-3.66 (m, 2H), 3.38-3.24 (m, 2H), 3.13-3.06 (m, 4H), 2.90 (s, 6H), 1.20-1.15 (m, 12H).

Ethyl 2-{[2,2,2-trifluoro-N-(3-hydroxypropyl)acetamidO]methyl}pyridine-4-carboxylate (h) □ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

113

Prepared by General Procedure A from ethyl 2-[(2,2,2-trifluoro-N-{3[(trifluoroacetyl)oxy]propyl}acetamido)methyl]pyridine-4-carboxylate (g). Purified by flash chromatography to yield the title compound.

‘H NMR (300 MHz, CDCI3), (rotamers): δ 8.74 (two d, 1H), 7.80 (m, 2H), 4.85(s, 2H), 4.45 (m, 2H), 3.65 (m, 4H), 1.90 (m, 2H), 1.45 (m, 3H).

2-({[(tert-butoxy)carbonyl]({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino}methyl)pyridine-4-carboxylic acid (x)

Prepared by General procedure A from ethyl 2-({[(tert-butoxy)carbonyl]({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino}methyl)pyridine-4-carboxylate to get the title compound as yellow solid.

ΧΗ NMR (300 MHz, CD3OD) δ 8.67 (m, 1H), 7.67 (m, 1H), 7.73 (m, 1H), 4.70 (m, 2H), 4.42 (m, 2H), 4.14 (m, 2H), 3.36 (m, 4H), 2.44 (m, 2H), 2.24 (m, 6H), 1.43 (m, 12H), 1.15 (m, 3H).

Amine intermediates

Benzyl N-[(lR)-2-hydroxy-l-{methyl[3-(l-methyl-lH-imidazol-2yl)propyl]carbamoyl}ethyl]carbamate

The reaction mixture comprising of methyl[3-(l-inethyl-lH-imidazol-2-yl)propyl]amine (1.1 equiv), (2R)-2-{[(benzyloxy)carbonyl]amino}-3-hydroxypropanoic acid (1.0 equiv), HATU (1.2 equiv), and DIPEA (1.4 equiv), in DMF was stirred at room temperature until TLC showed a complete reaction. Then the reaction mixture was partitioned between EtOAc/satd NaHCOj. The EtOAc extract was concentrated to yield the title compound.

□ate Reçue/Date Received 2020-10-01

84515062

114 (2R)-2-amino-3-hydroxy-N-methyl-N-[3-(l-methyl-lH-imidazol-2y I ) propyl ] propanamide

A mixture of the Benzyl N-[(lR)-2-hydroxy-l-{methyl[3-(l-methyl-lH-imidazol-2yl)propyl]carbamoyl}ethyl]carbamate and 10% Pd/C (5% by weight) in MeOH was stirred under a hydrogen atmosphere (40 PSI) until no starting material was detected by TLC. Then the mixture was filtered through a pad of Celit™ and concentrated to yield the title compound.

2-amino-N-[2-(dimethylamino)ethyl]-N-ethylacetamide

Prepared by General Procedure E from Boc-Glycine and [2-(dimethylamino)ethyl](ethyl)amine. Subsequent treatment with HCI In MeOH gives the title product as hydrochloride .

* H NMR (300 MHz, CDCI3) δ 3.41 (m, 4H), 3.23 (m, 2H), 2.39 (m, 2H), 2.22 (s, 6H), 1.52 (s(br), 2H), 1.11 (m, 3H)

Alcohol intermediates

2,2,2-trifluoro-N-(3-hydroxybutyl)acetamide

General Procedure I (formation of trifluoro acetamides)

Ethyl 2,2,2-trifluoroacetate was added at 0 °C to a solution of 4-aminobutan-2-ol in MTBE . The mixture was brought to RT and stirring under nitrogen overnight. The solvent was evaporated in vacuum to yield The title compound as colorless oil.

* H NMR (300 MHz, CDCI3), δ 8.0 (brs, 1H), 3.95 (m, 1H), 3.6 (m, 1H), 3.3 (m, 2H), 1.75 (m, 1H), 1.6 (m, 1H), 1.2 (d, 6H).

Tert-butyl N-(3-hydroxybutyl)carbamate

Prepared by General Procedure G from 4-aminobutan-2-ol to give the title product as colorless gum.

* H NMR (300 MHZ, CDCl3), δ ppm: 5.0 (brs, 1H), 3.7 (m, 1H), 3.4 brs, 1H), 3.2 (m, 2H), 1.6 (m, 2H), 1.4 (S, 9H), 1.2 (d, 3H).

ES-MS: 224 [M+l].

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

115

4-(methylamino)butan-2-ol

LAH was added at 0 °C to a solution of tert-butyl 3-hydroxybutylcarbamate in THF. The mixture was brought to RT and heated at 70 °C with stirring under nitrogen for 3h. The reaction was quenched with 10% NaOH solution. Aqueous work up gave the title product as colorless oil.

‘H NMR (300 MHz, CDCI3), δ ppm: 3.9 (m, 1H), 3.6 (brs, 2H), 2.9 (m, 1H), 2.6 (m, 1H), 2.4 (s, 3H), 1.5 (m, 2H), 1.2 (d, 3H).

2,2,2-trifluoro-N-(3-hydroxybutyl)-N-methylacetamide

Prepared by General Procedure I from 4-(methylamino)butan-2-ol to give the title product as colorless color less oil.

* H NMR (300 MHz, CDO3), δ 3.8 (m, 1H), 3.7 (m, 1H), 3.2 (m, 1H), 3.2 (s, 3H), 1.7 (m, 1H), 1.6 (m, 1H), 1.2 (d, 3H).

ES-MS: 200 [M+l].

2,2,2-trifluoro-N-(3-hydroxypent-4-en-l-yl)acetamide

Prepared by General Procedure I from 5-Aminopent-l-en-3-ol to give the title compound as a brown foam.

^-NMR (300MHz, CDCI3): δ 7.50 (br s, 1H), 5.90 (m, 1H), 5.28 (d, 1H), 5.20 (d, 1H), 4.40 (m, 1H), 3.75 (m, 1H), 3.40 (m, 1H), 2.00-1.60 (m, 2H).

2,2,2-trifl uoro-N-( 3-oxopropyl )acetamide

Prepared by General Procedure I from 3,3-diethoxypropan-l-amine and ethyl trifluroacetate to get N-(3,3-diethoxypropyl)-2,2,2-trifluoroacetamide, which was treated with 2M HCI to get the title compound.

lH-NMR (300MHz, CDCI3): δ 9.81 (s, 1H), 7.15 (br S, 1H), 3.64 (q, 2H), 2.83 (t, 2H).

trimethyl({[l-(propan-2-yloxy)ethenyl]oxy})silane

To a solution of propan-2-yl acetate (1 eq) in THF was added LDA (1.1 eq) at -78 °C. The reaction mixture was stirred for 30 min and TMSCI (1 eq) added, allowed to warm at rt and stirred for 1 h.

Extracted with hexane, washed with water, brine and concentrated to get the title compound.

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

116 propan-2-yl 3-hydroxy-5-(trifluoroacetamido)pentanoate

To a solution of Z^^-trifluoro-N-CS-oxopropynacetamide (1 eq) in DCM was added trimethyl({[l(propan-2-yloxy)ethenyl]oxy})silane (leq) andTiCh (1 eq) at -78 °C. The reaction mixture was allowed to warm at rt and stirred for 1 h. Extracted with DCM, washed with water, brine and concentrated to get the title compound.

lH-NMR (300MHz, CDCh): δ 7.65 (br $, 1H), 5-05 (m, 1H), 4.19 (m, 1H), 3-35 (m, 1H), 2.46 (d, 2H), 1.85 - 1.61 (m, 2H), 1.25 (d, 6H).

Propan-2-yl 3-hydroxy-4-(trifluoroacetamido)butanoate

Prepared by General Procedure I from propan-2-yl 4-amino-3-hydroxybutanoate.

^-NMR (300MHz, CDCh): δ 6.94 (br s, 1H), 5.06 (m, 1H), 4.18 (m, 1H), 3.65 (d, 1H), 3.60 (m, 1H), 3.29 (m, 1H), 2.57 - 2.40 (m, 2H), 1.27 (d, 6H).

Example 2: Histone lysine demethylase AlphaLlSA assays for IC50 value determination.

This example demonstrates the ability of compounds of the invention to inhibit the activity in vitro of tested enzymes (Table 2a).

Assays are performed analogously to the protocol described by PerkinElmer (Roy et al. PerkinElmer Technical Note: AlphaLlSA #12, Apr. 2011). Results are seen in Table 3.

General method

Enzymes are dissolved in enzyme buffer and incubated for 10 min before 5 pL is added to 5 pL 3% DMSO solutions of compounds in enzyme buffer. Incubated for another 10 minutes, before 5 pL substrate solution is added and the reaction mixture is incubated at room temperature for the given period. 10 pL acceptor beads, suspended at given dilutions in Epigenetic Buffer (Perkin Elmer AL008) from stock, are added and the suspension is incubated 60 minutes in the dark at room temperature, before 10 pL suspension (at the given dilutions in Epigenetic Buffer) of streptavidin donor beads (Perkin Elmer 6760002) in Epigenetic Buffer is added- After incubation at room temperature in the dark the plates are read. Reaction conditions are seen in Table 2b.

Enzymes:

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

117

WO 2014/053491

PCT/EP2013/070457

<td colspan="4"> Table 2a</td>

<td> Protein name</td><td> Vendor/source</td><td> Sequence</td><td> Expression organism</td>

<td> KDM2A (FBXL10)</td><td> BPS, Bioscience, US</td><td> 1-650</td><td> Bac</td>

<td> KDM4A (JMJD2A)</td><td> BPS, Bioscience, US</td><td> 1-350</td><td> E.coli</td>

<td> KDM4C (JMJD2C)</td><td> BRIC, Denmark</td><td> 1-349</td><td> E.coli</td>

<td> KDM5 (JARID1C)</td><td> BPS</td><td> 2-1560</td><td> Bac</td>

<td> KDM3A (JMJD1A)</td><td> BPS, Bioscience, US</td><td> 2-end</td><td> Bac</td>

Substrates:

BK9M3: Biotin-ARTKQTAR(KMe3)STGGKAPRKQ-NH2 (Casio, Denmark)

BK9M2 : Biotin-ARTKQTAR(KMe2)STGGKAPRKQ-NH2 (AnaSpec 64359)

BK9M1: BiOtin-ARTKQTAR(KMe1)STGGKAPRKQ-NH2 (AnaSpec 64358)

H3K4M3B: H-ART(Kme3)QTARKSTGGKAPRKQLA-NH-Biotin (Casio, Denmark)

BK27M3; Biotin-ATKAAR(Kme3)SAPATGGVKKPHRY-NH2? (Casio, Denmark)

BH3K36M2: RKAAPATGGVK(Me2)KPHRYRPGTVK-(BIOTIN)? (AnaSpec) □ate Reçue/Date Received 2020-10-01

84515062

118

TM

Enzyme Buffer: 50 mM Hepes (pH see table 2b), 0.003% Tween-20, 0.1% BSA; 5 pM (NH4)2Fe(SO4)2

Buffer A: 50 mM Hepes (pH see table 2b), 0.003% Tween-20, 0.1% BSA

Reaction conditions

<td colspan="6"> Table 2b</td>

<td> Enzyme</td><td> Enzyme</td><td> Substrate solution</td><td> Acceptor Beads</td><td> Donor Beads</td><td> Incubation Time</td>

<td> KDM2B</td><td> Buffer A, pH 8.0 Final enzyme concentration: 2 nM</td><td> Buffer A, pH 8.0 + 25 pM LAsc, 10 μΜ oKG. Final substrate (BH3K36M2) conc: 50 nM</td><td> H3K36M1 (PerkinElmer Custom, 10120327RS) Diluted 1:200 from stock</td><td> Diluted 1:200 from stock</td><td> 60 min</td>

<td> KDM3A</td><td> Buffer A, pH 7.4 Final enzyme concentration: 1.6 nM</td><td> Buffer A, pH 7.4 + 25 pM LASC, 10 μΜ 0KG. Final substrate (BK9M2) conc: 100 nM</td><td> H3K9Mel (selfconjugated) Diluted 1:200 from stock</td><td> Diluted 1:200 from stock</td><td> 60 min</td>

<td> KDM3B</td><td> Buffer A, pH 7.4</td><td> Buffer A, pH 7.4 + 25 pM LASC, 10 pM 0-</td><td> H3K9Mel (self-</td><td> Diluted 1:200 from stock</td><td> 60 min</td>

Date Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

119

WO 2014/053491

PCT/EP2013/070457

<td></td><td> Anal enzyme concentration: 0.1 nM</td><td> KG. Final substrate (BK9M2) cone: 100 nM</td><td> conjugated) Diluted 1:200 from stock</td><td></td><td></td>

<td> KDM4A</td><td> Buffer A, pH 7.4 Anal enzyme concentration: 0.2 nM</td><td> Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK9M3) cone: 100 nM</td><td> H3K9Me2 (Cat #AL117, Perkin Elmer) Diluted 1:400 from stock</td><td> Diluted 1:400 from stock</td><td> 60 min</td>

<td> KDM4B</td><td> Buffer A, pH 7.4 Anal enzyme concentration: 1 nM</td><td> Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK9M3) cone: 100 nM</td><td> H3K9Me2 (Cat #AL117,Perkin Elmer) Diluted 1:400 from stock</td><td> Diluted 1:400 from stock</td><td> 60 min</td>

<td> KDM4C</td><td> Buffer A, pH 7.4 Anal enzyme concentration: 1 nM</td><td> Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK9M3) cone: 200 nM</td><td> H3K9Me2 (Cat #AL117,Perkin Elmer) Diluted 1:400 from stock</td><td> Diluted 1:400 from stock</td><td> 60 min</td>

<td> KDM5B</td><td> Buffer A, pH</td><td> Buffer A, pH 7.4 + 25 μΜ L-</td><td> H3K4Me2-l (Perkin Elmer</td><td> Diluted 1:200</td><td> 20 min</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

120

WO 2014/053491

PCT/EP2013/070457

<td></td><td> 7.4 Final enzyme concentration: 2 nM</td><td> Asc, 10 μΜ αKG. Final substrate (H3K4M3B) cone: 200 nM</td><td> AL116) Diluted 1:200 from stock</td><td> from stock</td><td></td>

<td> KDM5C</td><td> Buffer A, pH 7.4 Final enzyme concentration: 1 nM</td><td> Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (H3K4M3B) cone: 100 nM</td><td> H3K4Me2-l (Perkin Elmer A LI 16) Diluted 1:200 from stock</td><td> Diluted 1:200 from stock</td><td> 30 min</td>

<td> KDM6A</td><td> Buffer A, pH 7.4 Final enzyme concentration: 2 nM</td><td> Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK27M3) cone: 100 nM</td><td> H3K27me2-l (Perkin Elmer AL121) Diluted 1:200 from stock</td><td> Diluted 1:200 from stock</td><td> 60 min</td>

<td> KDM6B</td><td> Buffer A, pH 7.4 Final enzyme concentration: 1 nM</td><td> Buffer A, pH 7.4 + 25 μΜ LASC, 10 μΜ OKG. Final substrate (BK27M3) cone: 50 nM</td><td> H3K27me2 (Perkin Elmer AL121) Diluted 1:200 from stock</td><td> Diluted 1:200 from stock</td><td> 60 min</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

121

WO 2014/053491

PCT/EP2013/070457

<td> KDM7</td><td> Buffer A, pH 7.4 Final enzyme concentration: 2.5nM</td><td> Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK9M2) cone: 100 nM</td><td> H3K9Mel (selfconjugated) Diluted 1:200 from stock</td><td> Diluted 1:200 from stock</td><td> 60 min</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

122

HDME INHIBITION

<td></td><td> JARID1 C</td><td colspan="5"> 4· 4· 4·</td><td colspan="3"></td>

<td> Table Ξ</td><td> FBXL10</td><td colspan="5"> 4-</td><td colspan="3"></td>

<td></td><td> GASCI</td><td colspan="5"> 4· 4·</td><td colspan="3"> 4· + 4*</td>

<td></td><td></td><td> 1 I</td><td> i 1</td><td> E o c</td><td> 1 fl· 1 Φ C</td><td> 5 Q œ</td><td> nino) et nethyl)</td><td> fl· 1</td><td> : acid</td>

<td></td><td> E IQ z</td><td> 1 L——J 1 ΓΜ</td><td> imidazol</td><td> t IB CL g CL</td><td> u Q. »—s w 0)</td><td> u X e jS L. IB U</td><td> 2-({[2 (dimethylari hyl Jami no}r</td><td> pyridine</td><td> carboxylic</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

123

<td> 2-({[(2R)-2,3dihydroxypropyl]a mine} methyl )py ri di ne-4-carboxy lie acid</td><td> 2{[ (cyclopropyl meth yl )ami no] methyl }p yridine-4carboxylic acid</td><td> 2{[ (cyclopropyl meth yl )ami no] methyl}p yridine-4carboxylic acid</td><td> 2-({[2(d I meth ylam ino) et hyl ] (methyl )amino } methyl ) py r idi ne- 4carboxylic acid</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

124

138

WO 2014/053491

<td> 3( dimethylam i no) pr opyl 2-«[4(diethylamino)buty 1 ] ami n 0} meth y 1 )py ridin e-4carboxylate</td><td rowspan="2"> {4[(ethoxycarbonyl)amino]phenyl}m ethyl 2-«[4( d iethyl amino) butyl ] a mi no} meth y 1 ) py ri dine-4-ca rboxyl a te</td>

<td> Below</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

141

WO 2014/053491

PCT/EP2013/070457

Dato Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

142

WO 2014/053491

PCT/EP2013/070457

<td> 2,6 -d I methyip h e nyl 2-({[4(diethylamino)buty l]amino}methyl)py ridin e-4carboxylate</td><td rowspan="2"> 4-methoxyphenyl 2-{[({[2(dimethylamino)ethyl](ethyl)carba m oy 1 Jmethyl )ami no] m ethy 1 Jpy rl din e-4-car boxy late</td>

PCT/EP2013/070457

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

147

<td rowspan="2"> 4-(trifluoroacetamido)butan-2-yl 2<[«[2- (d imeth y laml no) eth y 1 ] (eth y 1 )carba mo yl} methyl )am I no] methyl} p y r idi ne-4carboxylate</td><td> 4-(2,2,2-trifluoro-Nmethylacetamido)butan-2-yl 2{[«[2(dlmethylamino) ethy 1] (ethy 1 )carba</td>

<td rowspan="2"> Below</td>

<td> Below</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

148

<td rowspan="2"> moyl}methyl)amino]nr)ettiyl}pyridin e-4-car boxy late</td><td> ethyl 2-{[«[2(d I meth ylam ino) et hyl ] (ethyl )carbamo yl}methyl )amino] methyl }pyridine-4carboxylate</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

149

WO 2014/053491

PCT/EP2013/070457

<td></td><td rowspan="2"> 5- (trifl u oroaceta m i do) pent-1 -en - 3 - y 1 2-<[«[2- ( di methyl a mi no )ethy 1] (ethyl )ca rbam oy 1} meth yl )ami no ] methyl} py ridine4-carboxylate</td>

<td> Below</td>

Dato Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

150

WO 2014/053491

PCT/EP2013/070457

□ate Reçue/Data Received 2020-10-01

CA 02385969 2015-03-23

151

WO 2014/053491

□ate Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

154

WO 2014/053491

PCT/EP2013/070457

<td> methyl 2-{[«[2(dimethylamino)et hyl](ethyl)carbamo yl}methyl)amino] methyl }pyridine-4carboxylate</td><td rowspan="2"> propan-2-yl 3-(2-{[({[2(dimethylamino)ethyl] (ethyl )carbam oyl }meth y I Jamino ] methyl} py ridine4-carbon yloxy ) -4(trifl uoroaceta mido) buta noate</td>

<td> Below</td>

Date Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

155

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

156

WO 2014/053491

PCT/EP2013/070457

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

157

WO 2014/053491

PCT/EP2013/070457

<td> 2,2,2-trifluoroethyl 2-<[«[2(d i meth ylam ino) et hy 1 ] (ethyl )carbamo yl>methyl )amino] methyl }pyridlne-4carboxylate</td><td> 2-« ethy 1 [2-OXO-2(piperidin-lyl )ethyl] a m inoj-me thyl)pyridine-4carboxylic acid</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

158

WO 2014/053491

PCT/EP2013/070457

□ate Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

159

PCT/EP2013/070457

<td> 2-<[«[2(d i meth ylam ino) et hyl ] (ethyl )carbamo y 1} methyl )ami n o] methyl}-N-(5methyl -1,3,4 oxa diazol-2 yl)pyridine-4carboxamide</td><td> 2-<[({[2(d I meth ylam ino) et hy 1 ] (ethyl )carbamo y 1} methyl )amino] methyl}-N-(lmethyl-lH-pyrazol5-yl)pyridine-4carboxamide</td><td rowspan="2"> 2-{[({[2(dimethylamino)ethyl](ethyl)carba moy 1} meth yl )ami no] methyl} - N( 1,3-oxazol -2 -y l)pyridine-4-</td>

<td> Below</td>

168

Example 3: Histone Lysine Demethvlase immunofluorescence Assays for IC50 value Determination in Cell

Histone Lysine Demethylase Immunofluorescence Assays for IC50 value Determination, nontransfected cells

This example demonstrates the ability of compounds of the invention to inhibit demethylation of H3K4 in a human osteosarcoma cancer cell line.

General method

U2OS cells were harvested and seeded into multi well plates into media containing compound. The media used was DMEM containing 5 % FBS and pen/strep. 20 hours after incubation of cells with compounds, the cells were washed once in PBS, harvested by fixation with formaldehyde 4 % aqueous solution, and washed 2 times in PBS. Subsequently, the cells were permeabilized in PBS with 0.2 % Triton X-100 for 10 min at room temperature. Blocking was performed in PBS with 0.2 % Triton X-100 and 5 % FBS for 45 min at room temperature. The cells were incubated with oH3K4me3 primary antibody (Cell Signaling, #97515) diluted 1:1000 in blocking solution over night at 4°C. After incubation with primary antibody, the cells were washed 3 times with PBS, incubated with secondary antibody diluted 1:1000 (Alexa fluor 594 goat anti rabbit IgG, Invitrogen, A11012) and Hoechst, 20 pg / ml (Sigma, 33342) in blocking solution, and washed again 3 times with PBS. Finally, PBS was added and high throughput imaging and analysis were performed by an IN Cell Analyzer 1000 (GE Healthcare). The IC50 values seen in Table 4 were based on an average measure of the staining of the H3K4me3 mark in cells.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

169

WO 2014/053491

PCT/EP2013/070457

Histone Lysine Demethylase Inhibition

<td> F |^XN 0 CH) 0 A 0 I H2C c-lj</td><td> 5-(trifluoroacetamido)pent-l-en-3-yl2«({[2(dimethylamino)ethyl](ethyl)carbamoyl} methyl)amino]methyl}pyridine-4carboxylate</td><td> +++</td>

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

175

WO 2014/053491

PCT/EP2013/070457

<td> F CH, Tt 0 CH, HjC ^CHj</td><td> 5-(2,2,2-trifluoro-Nmethylacetamido)pent-l-en-3-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl} methyl)amino]methyl}pyridine-4carboxylate</td><td></td>

<td> O^O A η Η ? X -ί\ Jx /x N x< N X CH, ^CHj</td><td> 2-(2-([({|2(dimethylamino)ethyl](ethyl)carbamoyl} methyl)amino|methyl}pyridine-4carbonyloxy)-3-(hexadecanoyk>xy)propyl hexadecanoate</td><td> +++</td>

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

179

WO 2014/053491

PCT/EP2013/070457

Date Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

180

WO 2014/053491

PCT/EP2013/070457

Example 4: Cell proliferation Assays for EC50 value Determination

This example demonstrates the ability of the compounds of the invention to inhibit the proliferation of a human breast cancer cell line.

General method

MCF7 cells were seeded at 1250 cells/well in 50pl medium/well in black 96 well plates. Cells were incubated for 24 hours before addition of compound. Compounds were diluted in complete medium (50pl/well) and added to the plates in duplicates. The total volume of medium in the wells was 100 pl, and the final concentration of DMSO 0.5 %. Complete medium used was DMEM with GlutaMAX containing 10 % FBS and pen/strep.

120 hours after addition of compounds, the plates were harvested and analyzed by ATPIite 1 Step (Perkin Elmer, cat no 6016739) according to the manufactures recommendation. Briefly, 100 pl ATP lite solution was added to each well, plates were vortexed at 700 rpm 2 minutes, followed by 20 minutes Incubation In the dark, and then analyzed for luminescence on EnSpire 2300 MuliOlabel reader (Perkin Elmer). EC50 values were calculated using GraphPad Prism 6. Results are seen in

Table 5.

□ate Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

181

WO 2014/053491

PCT/EP2013/070457

<td></td><td></td><td></td><td> iyl}pyridine-</td><td></td><td></td><td> )pyridine-4-</td><td></td>

<td></td><td> Name</td><td></td><td> 2 -{[ (cycl op ropyl methyljami no] m eth 4-carboxylic acid</td><td></td><td> <s CM</td><td> (methylsulfanyl)ethyl]amino}methyl</td><td> carboxylic acid</td>

<td> Table 5</td><td></td><td> r 0</td><td></td><td></td><td></td><td></td><td> Λ 5 \ co</td>

<td></td><td> Structure</td><td> 0</td><td> J HN --</td><td> E O</td><td></td><td></td><td> xz</td>

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

201 (a) +++: EC50 <lpM; ++: ΙμΜ < EC50 < 50μΜ; +: EC50 > 50μΜ

Example 5: Histone Lysine Demethvlase Immunofluorescence Assays for IC50 value Determination in Cell

This example demonstrates the ability of the compounds of the invention to inhibit demethylation of a specific H3 lysine in a human osteosarcoma cell line transfected to express a specific histone lysine demethylase.

General method

U2OS cells were seeded 24 hours before transfection. Transfection was performed with Fugene HD transfection reagent as recommended by the manufacturer. 6 hours after transfection, the cells were harvested and seeded into multi well plates into media containing compound. The media used was DMEM containing 5 % FBS and pen/strep. 20 hours after incubation of cells with compounds, the cells were washed once in PBS, harvested by fixation with formaldehyde 4 % aqueous solution, and washed 2 times in PBS. Subsequently, the cells were permeabilized in PBS with 0.2 % Triton X-100 for 10 min at room temperature. Blocking was performed in PBS with 0.2 % Triton X-100 and 5 % FBS for 45 min at room temperature. The cells were incubated with primary antibodies diluted 1 pg / ml in blocking solution over night at 4°C. The primary antibodies used in the assays were HA.ll (Covance, MMS-101P) and the antibody detecting the mark specified in the Table 6 below. After incubation with primary antibodies, the cells were washed 3 times with PBS, incubated with secondary antibodies diluted 1:1000 (Alexa fluor 594 goat anti rabbit IgG, Invitrogen, A11012; Alexa flour 488 donkey anti mouse IgG, Invitrogen, A21202) and Hoechst, 20 pg / ml (Sigma, 33342) in blocking solution, and washed again 3 times with PBS. Finally, PBS was added and high throughput imaging and analysis were performed by an IN Cell Analyzer 1000 (GE Healthcare). The robot software analyzed individual cells and divided these into HA+ (transfected cells) and HA' (non-transfected cells). The IC50 values in Table 7 below were based on an average measure of the staining of the mark specified In the Table 6 below in the transfected cells.

Date Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

202

WO 2014/053491

PCT/EP2013/070457

<td></td><td> Plasmid NCBI ID</td><td> NM_014663</td><td> NM_015061</td><td> 00 ΙΟ to Ο <3 1 Σ Z</td>

Date Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

203

HDME INHIBITION

<td rowspan="5"> Table 7</td><td> PL UI (KDM5B)</td><td> + +</td><td> + +</td><td> +</td>

<td> GASCI <KDM4C)</td><td> +</td><td> +</td><td> +</td>

<td> Name</td><td> 2 -({[3-( 1 H-im idazol-1 -yl )propyl]am ino}m ethy l)py r id ine4-carboxylic acid</td><td> 2-({[2-(d imethylam ino)ethy l]am inojmethyl )pyrid ine-4carboxylic acid</td><td> 2-({[(2R)-2,3-d ihydroxy propyl] am ino}methyl)pyridine-4carboxylic acid</td>

<td> Structure</td><td> 0 IZ X / O fl--V λ / z O \=/</td><td> n δ \ £ z—o IZ I / O r--/ λ U 'z Q</td><td> r o IZ O r--/ y U xz o \^=/ z</td>

□ate Reçue/Date Received 2020-10-01

CA 02385969 2015-03-23

204

WO 2014/053491

PCT/EP2013/070457

<td> 2-{[(cyclopropy 1 methyl Jam ino] methy l}pyridine-4 carboxylic acid</td><td> 2-({[4-(d imethylam ino Jbuty l]am inojmethyl Jpyrid ine-4car boxy lie acid</td><td> 2-«[2- (dimet hy lam ino Jethyl] (methy IJam inojmethyl Jpyrid ine-4 carboxylic acid</td>

□ate Reçue/Date Received 2020-10-01

CA 02885969 2015-03-23

WO 2014/053491

WO 2014/053491

PCT/EP2013/070457

<td> -butylpyrrolidin-2nyl )py rid ine-4-ca rboxyl ic</td><td> 2-{[(3-{[(2nethyl)am ino}propyl)am i e-4-carboxylic acid</td><td> 2-({[({4i ino]butyl}( methyl)carbai l)pyridine-4-carboxylic a</td><td> -1 -benzy Ipy rrolid in-2riyl] pyridine-4-carboxy I ic</td>

PCT/EP2013/070457

<td> {4-[(ethoxycartx>nylXmethyl)am ino] phenyljm ethyl 2({[44diethylamino)butyl]amino}methyl)pyridine-4carboxylate</td><td> 2-(2-(((((2- (d im ethy la m ino) ethyl] (ethyl)c a rbam oyljmethyl Jam ino] methyl) pyrid i ne-4-carbon y loxy )-3 -(hexadecanoyloxy (propyl hexa decanoate</td>

(a) +++: IC50 <1μΜ; ++: ΙμΜ < IC50 < 50μΜ; +: IC50 > 50μΜ

Dato Reçue/Date Received 2020-10-01

84515062

215

In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions Is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of.

No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

LIST OF REFERENCES

Catchpole S et al., Int. J. Oncol. 38, 1267-77, 2011

Cloos, P.a.C. et al. (2008), Genes. Dev. 22; 115-1140

Cloos, P. Et al., Nature 442, 307-11, 2006

Fischle, W., et. Al., Curr. Opinion Cell Biol. 15,172-83, 2003

Hayami S. et al. (2010) Mol. Cancer 9

He J et al., Blood 117 (14), 3869-80, 2011

He J et al. Nat Struct Mol Biol 15(11), 2008

Kelly, T.K. et al. (2010), Epigenetic modifications as therapeutic targets, Nat. Biotechnol. 28; 1069-1078

Klose, R.J. et al., Nature 442,312-16, 2006

Liu, G. Et al., Oncogene 28, 4491-500, 2009

Margueron, R., et al., Curr. Opinion Genet. Dev. 15, 163-76, 2005

Morton and Houghton, Establishment of human tumor xenografts in immunodeficient mice,

Nature Protocols, 2 (2) 247-250, 2007

Pfau Ret al., PNAS 105(6), 1907-12, 2008

Queguiner, G. and Pastour, P., Comptes Rendus des Séances de l’Académie des Sciences, Série

C: Sciences Chimiques, 268(2) 182-5, 1969.

Quina, A.S. et al. (2006), Chromatin structure and epigenetics, Biochem. Pharmacol. 72; 15631569

Roy et al. PerkinElmer Technical Note: AlphaLISA #12, Apr. 2011

Tzatsos A et al., PNAS 106 (8), 2641-6, 2009

Yamane K. et al., Mol. Cell 25, 801-12, 2007

Xiang Y. et al. (2007) PNAS 104

Date Reçue/Date Received 2020-10-01

Contents597

125 sheets

Sheet 1
Sheet 2
Sheet 3
Sheet 4
Sheet 5
Sheet 6
Sheet 7
Sheet 8
Sheet 9
Sheet 10
Sheet 11
Sheet 12
Sheet 13
Sheet 14
Sheet 15
Sheet 16
Sheet 17
Sheet 18
Sheet 19
Sheet 20
Sheet 21
Sheet 22
Sheet 23
Sheet 24
Sheet 25
Sheet 26
Sheet 27
Sheet 28
Sheet 29
Sheet 30
Sheet 31
Sheet 32
Sheet 33
Sheet 34
Sheet 35
Sheet 36
Sheet 37
Sheet 38
Sheet 39
Sheet 40
Sheet 41
Sheet 42
Sheet 43
Sheet 44
Sheet 45
Sheet 46
Sheet 47
Sheet 48
Sheet 49
Sheet 50
Sheet 51
Sheet 52
Sheet 53
Sheet 54
Sheet 55
Sheet 56
Sheet 57
Sheet 58
Sheet 59
Sheet 60
Sheet 61
Sheet 62
Sheet 63
Sheet 64
Sheet 65
Sheet 66
Sheet 67
Sheet 68
Sheet 69
Sheet 70
Sheet 71
Sheet 72
Sheet 73
Sheet 74
Sheet 75
Sheet 76
Sheet 77
Sheet 78
Sheet 79
Sheet 80
Sheet 81
Sheet 82
Sheet 83
Sheet 84
Sheet 85
Sheet 86
Sheet 87
Sheet 88
Sheet 89
Sheet 90
Sheet 91
Sheet 92
Sheet 93
Sheet 94
Sheet 95
Sheet 96
Sheet 97
Sheet 98
Sheet 99
Sheet 100
Sheet 101
Sheet 102
Sheet 103
Sheet 104
Sheet 105
Sheet 106
Sheet 107
Sheet 108
Sheet 109
Sheet 110
Sheet 111
Sheet 112
Sheet 113
Sheet 114
Sheet 115
Sheet 116
Sheet 117
Sheet 118
Sheet 119
Sheet 120
Sheet 121
Sheet 122
Sheet 123
Sheet 124
Sheet 125

81 members in 36 offices

This recordMember, by publication datePriority claim26 more offices are in the list below

Priority claims24

Priority claims
Document	Office	Kind	Date
201261708806	United States of America	P	2012-10-02
201261708806	United States of America	P	2012-10-02
61708806	United States of America	–	2012-10-02
PA201200599	Denmark	–	2012-10-02
PA201200599	Denmark	A	2012-10-02
PA201200599	Denmark	A	2012-10-02
201361770050	United States of America	P	2013-02-27
201361770050	United States of America	P	2013-02-27
61770050	United States of America	–	2013-02-27
PA201370112	Denmark	–	2013-02-27
PA201370112	Denmark	A	2013-02-27
PA201370112	Denmark	A	2013-02-27
2885969	Canada	A	2013-10-01
2885969	Canada	A	2013-10-01
2885969	–	–	–
61708806	–	–	–
61770050	–	–	–
CA20132885969	–	–	–
DK2012PA00599	–	–	–
DK2013PA70112	–	–	–
PA201200599	–	–	–
PA201370112	–	–	–
US201261708806P	–	–	–
US201361770050P	–	–	–

Members81

Family members, oldest first
Document	Office	Kind
CA2885969A1	Canada	A1	2014-04-10
CA3095019A1	Canada	A1	2014-04-10
WO2014053491A1	World Intellectual Property Organization (WIPO)	A1	2014-04-10
US2014371195A1	United States of America	A1	2014-12-18
AP2015008389A0	African Regional Intellectual Property Organization (ARIPO)	A0	2015-04-30
SG11201502452RA	Singapore	A	2015-05-28
PH12015500730A1	Philippines	A1	2015-06-01
KR20150063517A	Republic of Korea	A	2015-06-09
CR20150217A	Costa Rica	A	2015-06-11
PE20150998A1	Peru	A1	2015-06-29
US2015203453A1	United States of America	A1	2015-07-23
EP2903968A1	European Patent Office (EPO)	A1	2015-08-12
MD20150043A2	Republic of Moldova	A2	2015-08-31
CN104981458A	China	A	2015-10-14
JP2015532295A	Japan	A	2015-11-09
MX2015003959A	Mexico	A	2015-11-16
EA201500394A1	Eurasian Patent Organization (EAPO)	A1	2015-11-30
CL2015000829A1	Chile	A1	2015-12-04
ECSP15017165A	Ecuador	A	2016-01-29
HK1207373A	Hong Kong, China	A	2016-01-29
HK1207373A1	Hong Kong, China	A1	2016-01-29
EP3023415A1	European Patent Office (EPO)	A1	2016-05-25
HK1215710A	Hong Kong, China	A	2016-09-09
HK1215710A1	Hong Kong, China	A1	2016-09-09
EP2903968B1	European Patent Office (EPO)	B1	2016-12-07
DK2903968T3	Denmark	T3	2017-01-30
LT2903968T	Lithuania	T	2017-02-27
SMT201700109B	San Marino	B	2017-03-08
SMT201700109T1	San Marino	T1	2017-03-08
PT2903968T	Portugal	T	2017-03-13
SI2903968T1	Slovenia	T1	2017-03-31
EP3150582A1	European Patent Office (EPO)	A1	2017-04-05
HRP20170358T1	Croatia	T1	2017-04-21
PL2903968T3	Poland	T3	2017-05-31
ES2617905T3	Spain	T3	2017-06-20
ME02597B	Montenegro	B	2017-06-20
SG10201703682YA	Singapore	A	2017-06-29
BR112015007083A2	Brazil	A2	2017-07-04
CY1118695T1	Cyprus	T1	2017-07-12
CN104981458B	China	B	2017-07-21
RS55764B1	Serbia	B1	2017-07-31
HK1223612A	Hong Kong, China	A	2017-08-04
HK1223612A1	Hong Kong, China	A1	2017-08-04
IL253259A0	Israel	A0	2017-08-31
IL253259D0	Israel	D0	2017-08-31
IL237831A	Israel	A	2017-10-31
US2017320827A1	United States of America	A1	2017-11-09
SA515360233B1	Saudi Arabia	B1	2017-11-12
SA5601B1	Saudi Arabia	B1	2017-11-12
HUE033380T2	Hungary	T2	2017-11-28
CN107434803A	China	A	2017-12-05
EP3023415B1	European Patent Office (EPO)	B1	2017-12-06
SI3023415T1	Slovenia	T1	2018-01-31
JP2018016661A	Japan	A	2018-02-01
ES2654143T3	Spain	T3	2018-02-12
AU2018200876A1	Australia	A1	2018-02-22
AU2018200876A1	Australia	A1	2018-02-22
PT3023415T	Portugal	T	2018-02-27
MA38057A1	Morocco	A1	2018-02-28
MA38057A2	Morocco	A2	2018-02-28
IL253259A	Israel	A	2018-04-30
IL253259B	Israel	B	2018-04-30
PL3023415T3	Poland	T3	2018-06-29
NZ706635A	New Zealand	A	2018-08-31
EA030698B1	Eurasian Patent Organization (EAPO)	B1	2018-09-28
HK1247920A	Hong Kong, China	A	2018-10-05
HK1247920A1	Hong Kong, China	A1	2018-10-05
MA38057A3	Morocco	A3	2018-11-30
US10189787B2	United States of America	B2	2019-01-29
PH12017501306A1	Philippines	A1	2019-02-27
US10221139B2	United States of America	B2	2019-03-05
JP6530313B2	Japan	B2	2019-06-12
AU2018200876B2	Australia	B2	2019-09-19
AU2018200876B2	Australia	B2	2019-09-19
AU2018200876C1	Australia	C1	2020-02-06
AU2018200876C1	Australia	C1	2020-02-06
CN107434803B	China	B	2020-05-05
MA38057B1	Morocco	B1	2020-05-29
KR102160320B1	Republic of Korea	B1	2020-09-28
CA2885969C	Canada	C	2021-04-06
CA3095019CThis record	Canada	C	2022-06-14

6 legal events, as the office reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code
2025-09-02	Maintenance fee for patent paidMPN	MPN
2025-09-02	Fee paidST27 STATUS EVENT CODE: A-4-4-U10-U00-U101 (AS PROVIDED BY THE NATIONAL OFFICE); EVENT TEXT: MAINTENANCE REQUEST RECEIVEDU00	U00
2025-09-02	Full renewal or maintenance fee paidST27 STATUS EVENT CODE: A-4-4-U10-U11-U102 (AS PROVIDED BY THE NATIONAL OFFICE); EVENT TEXT: MAINTENANCE FEE PAYMENT PAID IN FULLU11	U11
2022-05-26	Examination requestEEER	EEER
2022-05-06	Examination requestEEER	EEER
2020-10-14	Examination requestEEER	EEER

Numbers

Publication: 3095019
Publication, DOCDB: 3095019
Publication, EPODOC: CA3095019
Application: 3095019
Application, DOCDB: 3095019
Application, EPODOC: CA20133095019

Titles2

English: SUBSTITUTED PYRIDINE DERIVATIVES AND COMPOSITIONS THEREOF USEFUL AS INHIBITORS OF HISTONE DEMETHYLASES
French: DERIVES DE PYRIDINE SUBSTITUES ET LEURS COMPOSITIONS UTILES COMME INHIBITEURS D'HISTONE DEMETHYLASES

Classification

CPC, 15: C07D213/79
C07D401/12
C07D405/12
C07D413/12
C07D405/14
C07D413/06
A61K31/44
A61K31/443
A61K31/4439
A61K31/4545
A61K31/496
C07D401/06
A61K31/444
A61P35/00
A61P43/00
Filled: the office’s first classification
IPC, 2: C07D213 79
A61K31 44

Abstract

Term

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

WO 2014/053491

PCT/EP2013/070457

SUMMARY OF THE INVENTION

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

CA 02885969 2015-03-23

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

CA 02885969 2015-03-23

DETAILED DISCLOSURE OF THE INVENTION

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

CA 02885969 2015-03-23

CA 02885969 2015-03-23

WO 2014/053491

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

CA 02885969 2015-03-23

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

CA 02885969 2015-03-28

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

X

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

WO 2014/053491

PCT/EP2013/070457

CA 02885969 2015-03-23

CA 02885969 2015-03-23

WO 2014/053491 PCT/EP2013/070457

CA 02885969 2015-03-23