OA17265A

OA17265A - Inhibitors of histone demethylases.

Info

Publication number: OA17265A
Application number: OA1201500120
Authority: OA
Inventors: Marc Labelle; Qasim Khan; Thomas Boesen; Mukund Mehrotra; Farman ULLAH
Original assignee: Epitherapeutics Aps
Priority date: 2012-10-02
Filing date: 2013-10-01
Publication date: 2016-04-20

Abstract

The present application discloses compounds capable of modulating the activity of histone demethylases (HDMEs), which are useful for prevention and/or treatment of diseases in which genomic dysregulation is involved in the pathogenesis, such as e.g. cancer. The present application also discloses pharmaceutical compositions comprising said compounds and the use of such compounds as a medicament. The compounds take the form (I).

Description

The présent invention relates to compounds capable of modulating the activity of histone demethylases (HDMEs), which compounds are useful for the prévention 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 nucléosomes, the basic unit of chromatin. One of the important functions of chromatin is to détermine régions of active and silenced transcription by changing the ordered chromatin structure. Such changes hâve profound effects on cellular function since they affect fondamental processes as différentiation, prolifération 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 nucléosome. Most notably, the N-terminal histone tail of Histone H3 and histone H4 are subject to such covalent changes, which include changes in méthylation, acétylation, phosphorylation and ubiquitination. The addition or removal of these groups on liistones is mediated by spécifie 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 rôle of epigenetics in cell différentiation, prolifération and apoptosis, but epigenetics may also play a rôle in other diseases like metabolic, infiammatory, neurodegenerative and cardiovascular diseases. Therefore the sélective 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).

Méthylation and déméthylation of lysine residues on the histone H3 tail constitute important epigenetic marks delineating transcriptionally active and inactive chromatin. For example, méthylation of lysine 9 on histone H3 (H3K9) is usually associated with epigenetically 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 méthylation of lysine 4 on histone 3 is associated with transcriptionally active chromatin. Similarly, the lysine 27 histone H3 (H3K27) mark is répressive 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, E.J. & Carpenter, P.B. (2012) Nature Mol. Cell Biol 13, 115-26). There are, however, many exemptions from these general rules of association between méthylation states of epigenetic marks and the effect they bave on transcription.

As documented by studies of the SUV39II1 knockout mouse, loss of the tri-methyl variant of the H3K.9 mark results in chromosomal aberrations and prédisposés to cancer (Peters, A.H. et al., Cell 107, 323-37, 2001). The JMJD2C protein (KDM4C, GASC1) has been identifîed as an eraserofthe 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, R.J. 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 înteract 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 séminal ER-alpha target gene, cyclin 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 bénéficiai for the treatment of ER-alpha 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 identifîed 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 régions. The oncogenic potential of JARID1B is demonstrated by its stimulation of prolifération in cell lines and further validated by shRNA knockdown studies of JARID1B expression showing inhibition of prolifération in MCF7 human breast cancer cells, in SW78O 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).

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 progestérone receptor expression control by estrogens (Stratmann and Haendler (2011) FEBS J 278). Together with JARID1B, JARID1A has been implîcated in the maintenance of a slow-growing population of cancer cells that are required for continuous tumor growth and that are résistant 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 Men 1-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 prolifération, 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^lnk4h), 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, JARID1 A, JMJD2C, JMJD2A, and JHDM1B, would présent a novel approach for intervention in cancers and other proliférative diseases. Being one of the most devastating diseases, affecting millions of people worldwide, there remains a high need for efTicacious and spécifie compounds against cancer.

Embodiments of the invention provide novel sériés of compounds capable of modulating the activity of histone demethylases (HDMEs), at least some of which compounds are useful for the prévention and/or the treatment of diseases in which genomic dysrégulation is involved in the pathogenesis, such as e.g.

cancer. By way of further example, malnutrition or poor nutrition is thought to hâve an adverse epigenetic effect and the compounds of the invention may therefore be expected to hâve bénéficiai effect in treating such effects of poor nutrition. Furthermore, epigenetic changes hâve been found to be linked to behaviour. Accordingly, compounds according to the invention may be usefiil in behaviour modification. Altematively or additionally such compounds may be usefiil for exploring the extent to which different demethylases are inhibited by similar compounds as an investigation ofthe 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)-, Cj.g alkylene, C2-8 alkenylene, C2-8 alkynylene, C3.10 cycloalkylene, heterocyclylene, heteroarylene and arylene, which alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, heteroarylene and arylene may optionally be substituted with one or more R³;

Y is selected from -H, -NR⁶R⁷, -OR⁷, C[.g alkyl, C2.8 alkenyl, C₂.8alkynyl, 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 and may form a cyclic structure with R²;

R¹ is selected from -H, C^g alkyl, C₂-8 alkenyl, C₂.8 alkynyl, C3._l0 cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more selected from -OH, aryl, C|.₆alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and C3.6 cycloalkyl; or more preferably is selected from -H and Cm alkyl; or with -A-Y forms a nitrogen containing optionally substituted heterocyclic group where the optional substitution may be Ci,₈ alkyl, C₂-8 alkenyl, C2.8 alkynyl, or C3.10cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more selected from -OH, aryl, C|.₆alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and C3.6 cycloalkyl;

R² is selected from -H, C|.g alkyl, C₂.s alkenyl, C₂-8 alkynyl, and C3.10 cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more selected from -OH, aryl, C].₆ alkoxy, heteroaryi, aryloxy, heteroaryloxy, F, and C3.6 cycloalkyl, and may form a cyclic structure with

Y;

each R³ is independently selected from alkyl, CM fluoroalkyl, CM hydroxyalkyl, C2.6 alkenyl, C2.6 alkynyl, C3.10 cycloalkyl, -Z-heterocyclyl, -Z-aryl, -Z- heteroaryi, -Z-NR^ftR⁷, -Z-C(=O)-NR⁶R⁷, -Z-NR⁶C(=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 heteroaryi and any aryl may be substituted with one or more R^s;

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

each R⁴ is independently selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, alkoxy, C3.10 cycloalkyl, -N(R')₂, carbamoyl, and -OH;

each R⁵ is independently selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, Cm alkoxy, C3.6 cycloalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

each of R⁶ and R⁷ is independently selected from -H, C|.₈alkyl, C_M fluoroalkyl, Cm perfluoroalkyl, Cm hydroxyalkyl, C₂.g alkenyl, C₂.g alkynyl, C3.10 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl and -Z-aryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryi and aryl may optionally be substituted with one or more independently selected R⁸; or, altematively, 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 R⁸ is independently selected from Ci_6alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C2.6 alkenyl, C2.6 alkynyl, C3.10 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR^IUR¹¹, -Z-C(=O)-NR¹⁰R^u, Z-OR⁹, halogen, -CN, -Z-SR⁹, -Z-SOR⁹, -Z-SO2R⁹ and -Z-COOR⁹, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclics, heteroaryi and aryl may optionally be substituted with one or more selected from C1 m alkyl, CM fluoroalkyl, Cm hydroxyalkyl, C3.6 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Zaryl, -Z-NR^IOR, -Z-C(=O)-NR^l0R, -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 heteroaryi and any aryl may be further substituted with one or more R⁵ as defined above, and each R⁹ is independently selected from -H, Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C₂.g alkenyl, C₂.g alkynyl, C3.10cycloalkyl, -Z-heterocyclyl, -Z-aryl, and -Z- heteroaryi, wherein any heterocyclyl may be substituted with one or more R⁴ as defined above, and wherein any heteroaryi and any aryl may be substituted with one or more R^s as defined above;

each of R¹⁰ and R¹¹ is independently selected from -H, Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C₂.g alkenyl, C₂.₈ alkynyl, C3.10 cycloalkyl, heterocyclyl, heteroaryi, and aryl, wherein any heterocyclyl may be substituted with one or more R⁴ as defined above, and wherein any heteroaryi and any aryl may be substituted with one or more R^s as defined above, or, altematively, R¹⁰ and R¹¹ may together with the Natom to which they are attached form an N-heterocyclic 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 sait, solvaté or prodrug thereof.

Where R¹ forms with -A-Y an optionally substituted heterocyclic group, it is preferably an optionally substituted C₃.₇ for instance C3.6 or C5.6heterocyclic 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 présent invention relates to a compound of the Formula (I) wherein

A is selected from -CHR²C(O)-, Cj-g alkylene, C₂._fi alkenylene, C2-8alkynylene, Cj.iocycloalkylene, heterocyclylene, heteroarylene and arylene, which alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, heteroarylene and arylene may optionally be substituted with one or more R³;

Y is selected from -H, -NR⁶R⁷, -OR⁷, Cj-g alkyl, C2.8 alkenyl, C₂-8 alkynyl, C₃._l0 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 Cm alkyl;

R² is selected from -H, Ci._g alkyl, C₂.e alkenyl, Ο₂-8 alkynyl, C₃.|₀ cycloalkyl, which alkyl, alkenyl, alkynyl and cycloalkyl may be optionally substituted with one or more selected from -OH, aryl, Cj.6 alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, and C₃.₆ cycloalkyl;

each R³ is independently selected from Cj.6 alkyl, C_:J fluoroalkyl, C_Mhydroxyalkyl, C₂.6 alkenyl, C₂.₆ alkynyl, C3.10 cycloalkyl, -Z-heterocyclyl, -Z-aryl, -Z- heteroaryl, -Z-NR⁶R⁷, -Z-C(=O)-NR⁶R⁷, -Z-NR⁶C(=O)-R⁷, -Z-C(=0)-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 R^s;

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

each R⁴ is independentiy selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, alkoxy, C3.10 cycloalkyl, -NiR¹)!, carbamoyl, and -OH;

each R^s is independentiy selected from Crî alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C_w alkoxy, Cj.₆cycloalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

each of R⁶ and R⁷ is independentiy selected from -H, C|.g alkyl, Cm fluoroalkyl, Cu perfluoroalkyl, Cu hydroxyalkyl, C₂-8 alkenyl, C₂.₈ alkynyl, Cj.₎₀ 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 independentiy selected R⁸; or, altematively, R⁶ and R⁷ may together with the N-atom to which they are attached form an N-heterocyciic ring optionally substituted with one or more independentiy selected R⁸;

each R⁸ is independentiy selected from Cm alkyl, Cu fluoroalkyl, Cm hydroxyalkyl, C2.6 alkenyl, C2.6 alkynyl, C3.|0 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NRæR¹¹, -Z-C(=O)-NR^1<)R¹¹, Z-OR⁹, haiogen, -CN, -Z-SR⁹, -Z-SOR⁹, -Z-SO2R⁹ and -Z-COOR⁹, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclics, heteroaryl and aryl may optionally be substituted with one or more selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C3.6 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Zaryl, -Z-NR^,ÜR, -Z-C(=O)-NR¹⁰R¹¹, -Z-OR⁹, haiogen, -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 R^s as defined above, and each R⁹ is independentiy selected from -H, C|.g alkyl, C_M fluoroalkyl, Cm hydroxyalkyl, C₂.₈ alkenyl, C₂._flalkynyl, 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 R^s as defined above;

each of R¹⁰ and R¹¹ is independentiy selected from -H, Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C₂.g alkenyl, C₂.g alkynyl, C3.₁₀cycloalkyl, 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, altematively, R^t0 and R¹¹ may together with the Natom to which they are attached form an N-heterocyclic ring optionally substituted with one or more R⁴as defined above;

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

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

A is selected from -CHR²C(O)-, Ci_₈ alkylene, C₂.₈ alkenylene, C₂.₈alkynylene, C₃.|₀ cycloalkylene, heterocyclylene, heteroarylene and arylene, which alkylene, alkenylen, alkynylene, cycloalkylene, heterocyclylene, heteroarylene and arylene may optionaily be substituted with one or more R³;

Y is selected from -H, -NR⁶R⁷, -OR⁷, C_b8 alkyl, C₂.₈ alkenyl, C₂.₈alkynyl, C₃.iocycloalkyl, heterocyclyl, heteroaryl and aryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl may optionaily be substituted with one or more R³;

R¹ is selected from -H and Ομ alkyl;

R² is selected from -H, Cm alkyl and Cm hydroxyalkyl;

each R³ is independently selected from Cm alkyl, CM fluoroalkyl, Cm hydroxyalkyl, C2.6 alkenyl, C2.6 alkynyl, C3.io cycloalkyl, -Z-heterocyclyl, -Z-aryl, -Z- heteroaryl, -Z-NR⁶R⁷, -Z-C(=O)-NR⁶R⁷, -Z-OR⁷, halogen, -Z-SR⁷, -Z-SOR⁷, -Z-SO2R⁷ 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 R^s;

Z is selected from a single bond, Cm alkylene, heterocyclylene and Cm cycloalkylene, each R⁴ is independently selected from Cm alkyl, C_M fluoroalkyl, Cm hydroxyalkyl, Cm alkoxy, C₃_i₀cycloalkyl, -NtR^, carbamoyl, and -OH;

each R^J is independently selected from C_t.₆ alkyl, C_M fluoroalkyl, C_M hydroxyalkyl, Cm alkoxy, C₃.₆cycloalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

each of R⁶ and R⁷ is independently selected from -H, Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C₂.₈alkenyl, C₂.₈ alkynyl, C₃.i₀ cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl and -Z-aryl, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl and aryl may optionaily be substituted with one or more independently selected R⁸; or, altemativeiy, R⁶ and R⁷ may together with the N-atom to which they are attached form an N-heterocyclic ring optionaily substituted with one or more independently selected R⁸;

each R⁸ is independently selected from CMalkyl, CM fluoroalkyl, Cm hydroxyalkyl, C2.6alkenyl, C2.6 alkynyl, C3.10 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR^R¹¹, -Z-C(=O)-NR^l0Rⁿ, Z-OR⁹, halogen, -CN, -Z-SR⁹, -Z-SOR⁹, -Z-SO2R⁹ and -Z-COOR⁹, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclics, heteroaryl and aryl may optionaily be substituted with one or more selected from CMalkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C3.6cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Zaryl, -Z-NR^IOR^U, -Z-C(=O)-NR^,0R, -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 R³ as defined above, and each R⁹ is independently selected from -H, C|._galkyl, C_M fluoroalkyl, C_M hydroxyalkyl, C₂.₈ alkenyl, C₂.₈alkynyl, C₃.|₀ cycloalkyl, -Y-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 R^s as defined above;

each of R¹⁰ and R¹¹ is independently selected from -H, Cj^alkyl, C_w fluoroalkyl, hydroxyalkyl, C₂.₈alkenyl, C₂.₈ alkynyl, Cj.jocycloalkyl, 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 R^s as defined above, or, altematively, R¹⁰ and R¹¹ may together with a Natom to which they are attached form an N-heterocyclic ring optionally substituted with one or more R⁴as defined above;

with the provîso that Y is not H when A is -CH₂-; or an isomer or a mixture of isomers thereof, or a pharmaceutically acceptable sait, solvaté or prodrug thereof,

A further aspect ofthe présent invention relates to pharmaceutical compositions comprising at least one compound of Formula (1) as defined herein, and optionally one or more pharmaceutically acceptable excipients, diluents and/or carriers.

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

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

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

A further aspect of the invention relates to a method of treating a HDME dépendent 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 dépendent diseases by inhîbiting HDMEs. Inhîbiting HDMEs would provide a novel approach to the prévention and treatment of cancer and other proliférative diseases. Administered alone or optionally in combination with anti-neoplastic compounds, the compounds of the invention increase the effîcacy of the treatment of

HDME dépendent diseases. As will be shown below, compounds of the invention hâve a cytostatïc or anti-proliferative action against cancer cells,

DETAILED DISCLOSURE OF THE INVENTION

Compounds of Formula (I)

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

HO

N

The above définition of the compounds of Formula (1) 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. Il should also be understood, that unless the opposite is stated, such references also encompass isomers, mixtures of isomers, pharmaceutically acceptable salts, solvatés and prodrugs ofthe compounds of Formula (I).

Without being bound by any particular theory, the cun-ent results and X-ray crystallography studies give reasons to believe that the HOOC-pyridine-CH2-N< motif, cf. Formula (I), plays an important rôle when designing compounds capable of modulating the activity of histone demethylases (HDMEs). Furthermore, it is believed that the substituent combination-A-Y plays a rôle in establishing affinity for said histone demethylases. Histone demethylases contain an iron atom on which their activity dépends. It is furthermore believed that the pyridine nitrogen and the nitrogen atom of Formula (1) also play a rôle in the binding of a particular cavity of the histone demethylases where the iron atom lies, probably by chélation 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 sélective inhibition. Some demethylases hâve 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 non10 charged polar groups in the Y chain. Yet other demethylases are belter inhibited by compounds with non-charged polar groups in the Y chain.

More particularly, it is believed that the carboxylic acid substituent of the pyridîne ring plays a rôle in binding to a particular cavity of histone demethylases.

A is typically selected from -CHR²C(O)-, Cm alkylene, C2.8 alkenylene, C2.8 alkynylene, C3.10 cycloalkylene, heterocyclylene, heteroarylene and arylene.

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

In one embodiment, A is selected from -CHR²C(O)-, Cm alkylene, C₃.10 cycloalkylene, heterocyclylene, heteroarylene and arylene, in particular from -CHR²C(O)-, Cm alkylene and heterocyclylene, such as CHR²C(O)-, or Cm 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, Cm alkyl, C2.8 alkenyl, C2.8 alkynyl, Cî-iocycloalkyl, heterocyclyl, heteroaryl and aryl. Preferred values for R⁶ and R⁷ are defined further below.

The alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, 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.

In particular, -A-Y may take any of the values illustrated in the compounds of Table 1 below and any value of-Y shown there may be combined with any illustrated value for -A.

In another variant, A is Cm alkyl and Y is -NR⁶R⁷. In one scénario within this embodiment and these variants, -NR⁶R⁷ represents an N-heterocyclic ring optionally substituted with one or more independently selected R⁸, preferably substituted with one to two independently selected R⁸. In another scénario within this embodiment and these variants wherein Y is -NR⁶R⁷, one of R⁶ and R⁷ represents -H or Cm alkyl. In still another scénario within this embodiment and these variants wherein Y is -NR⁶R⁷, R⁶ and R⁷ are independently selected from C|_s alkyl, C_M fluoroalkyl, C_M hydroxyalkyl, C₂.8 alkenyl, and Cm alkynyl, e.g. such that R⁶ and R⁷ are the same. In still another scénario within this embodiment and these variants wherein Y is -NR⁶R⁷, one of R⁶ and R⁷ is selected from heterocyclyl, heteroaryl and aryl.

In another embodiment, Y is -H. In one variant, A is selected from Cm alkylene, C₂.e alkenylene, C₂.₈ alkynylene, and C3.10 cycloalkylene. In another variant, A is selected from heterocyclyl.

In still another embodiment, Y is selected from heterocyclyl, heteroaryl and aryl. In one variant hereof, A is selected from Ci._g alkylene, Cj.giilkenylene, C₂.8 alkynylene, in particular from C|._g alkylene, such as from Cm alkylene, in particular from Cm alkylene.

R¹ is typically selected from -H and Cm alkyl (such as methyl, ethyl, propyl and butyl), in particular from -H and methyl, H being preferred.

R² is typically selected from -H, C|,₈ alkyl, C₂.8alkenyl, C₂._g alkynyl, C3.10cycloalkyl, 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 C3.6 cycloalkyl. In some embodiments, R² is selected from -H, Cm alkyl (such as methyl, ethyl, propyl and butyl) and Cm hydroxyalkyl (such as hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl), in particular from -H, methyl and hydroxymethyl, with H being preferred.

The R³ (possible substituents to some of the meanings of A and Y) is typically independently selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C2.G alkenyl, Cm alkynyl, C3.10 cycloalkyl, -Zheterocyclyl, -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 R^s. In another embodiment, R³ is independently selected from Cm alkyl, Cm fluoroalkyl, C). 4 hydroxyalkyl, Cm alkenyl, C2.₆ alkynyl, C3.₁₀ cycloalkyl, -Z-aryl, -Z- heteroaryl, -Z-NR⁶R⁷, -Z-C(=O)NR⁶R⁷, -Z-OR⁷, halogen, -Z-SR⁷, -Z-SOR⁷, -Z-SO₂R⁷ 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 R^s.

Z is typically selected from a single bond, Cm alkylene, heterocyclylene and C3.6 cycloalkylene. In one embodiment, Z is selected from Cm alkylene. In another embodiment, Z is selected from a single bond. It should be understood that the group Z may appear several times in Formula (I) and that such Z’s are independently selected.

Each R⁴ (possible substituents of heterocyclyl) is independently selected from Cm alkyl, Cm fluoroalkyl, Cmhydroxyalkyl, CMalkoxy, C3.₁₍₎ cycloalkyl, -N(R')₂, carbamoyl, and-OH,

Each R^s (possible substituents of heteroaryl and aryl) is independently selected from Cm alkyl, C_Mfluoroalkyl, C_M hydroxyalkyl, Cm alkoxy, cycloalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH.

Each of R⁶ and R⁷ (e.g. of the moiety -NR⁶R⁷) is independently selected from -H, Cm alkyl, Cm fluoroalkyl, C_M perfluoroalkyl, hydroxyalkyl, C₂._g alkenyl, C₂._g alkynyl, C3.₁₀cycloalkyl, -Zheterocyclyl, -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; or, altematively, R⁶ and R⁷ may together with the N-atom to which they are attached form an N-heterocyciic ring optionally substituted with one or more independently selected R⁸.

Each R⁸ is independently selected from C|.6alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C2.6 alkenyl, C2.5 alkynyl, C3.10 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR^IOR¹¹, -Z-C(=O)-NR^l0R, Z-OR⁹, halogen, -CN, -Z-SR⁹, -Z-SOR⁹, -Z-SO2R⁹ and -Z-COOR⁹, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclics, heteroaryl and aryl may optionally be substituted with one or more selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C3.6 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Zaryl, -Z-NR^I0R, -Z-C(=O)-NR^l0R^u, -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 R^J as defined above.

Each R⁹ is independently selected from -H, C|.g alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C2.8 alkenyl, C2g alkynyl, Cg.io 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. In one embodiment, each R⁹ is independently selected from -H, Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C2.8 alkenyl, C2-g alkynyl, C3.10 cycloalkyl, -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 (of the moiety -NR^1OR^U) is independently selected from -H, Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C₂.g alkenyl, C2.8 alkynyl, C3.1Qcycloalkyl, 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, altematively, R¹⁰and R¹¹ may together with the N-atom to which they are attached form an N-heterocyclic ring optionally substituted with one or more R⁴ as defined above.

It is to be understood that in the Formula (I), Y is not H when A is -CH₂-. Generally speaking, it is believed to be advantageous if the moiety -A-Y has a certain “size” with respect to the number of atom (disregarding hydrogen atoms) and/or the molecular weight. Also a limited flexibility of the moiety -A-Y appears to play a certain rôle.

Hence, it is believed that the moiety -A-Y should preferably consist of at the most 40 heavy atoms, such as at the most 30 heavy atoms, or at the most 25 heavy atoms, or at the most 20 heavy atoms. Preferably, the moiety -A-Y will consist of at least 3, or at least 4, or at least 8 or at least 10 heavy atoms. In some embodiments, the moiety -A-Y preferably consists of 3-40 heavy atoms, such as 4-30 heavy atoms, or 425 heavy atoms, or 4-20, or 8-30, or 8-20, or 8-15 heavy atoms. By the term “heavy atom” is meant ail atoms in the moiety except the hydrogen atom(s).

Moreover, it is believed that the compounds of Formula (I) should preferably hâve a molecular weight of at least 130, or at least 150, or at least 180, or at least 250, but not more than 1000, or not more than 800, or not more than 500, or not more than 400 and may be within any range constructable from these preferred upperand Iower limits, such as 130-1,000 g/mol, or 150-1,000 g/mol, such as 180-800 g/mol, e.g. 225-600 g/mol or 250-500 g/mol, or 250 to 400,

In some embodiments, and in order to introduce a limited flexibility of the moiety -A-Y, the moiety includes 1-4 rings, i.e. rings derived from cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl and/or aryl. In some variant, the moiety -A-Y includes 1-3 cyclic moieties selected from monocylic cycloalkyl, monocyclic heterocyclyl, monocylic heteroaryl, dicyclic heteroaryl and monocyclic aryl. Small substituents such as alkyls groups or hydroxyl on alkyl chains also reduce flexibility and favor certain conformations.

It may be préférable that if-A-Y does not include a ring, it includes at least one, for instance from 1 to 3, branches, each of which independently may be of from one heavy atom to six heavy atoms, for instance from one to three heavy atoms, or from one to two heavy atoms. It is 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 R|q and Rh independently is as defined above.

Yis

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 substîtuted 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), induding isomers, mixtures of isomers, as well as pharmaceutically acceptable salts, solvatés and prodrugs thereof, are particularly interesting:

2-( {[3 -( 1 H-i midazol-1 -yl)propyl]amino} methyl)pyridine-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- {[(cyclopropy lmethyl)amino] methyl} pyridi ne-4-carboxyl ic acid

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

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-ylmcthyl)amino]methyl}pyridine-4-carboxylic acid

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

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-(pynOlidm-l-yl)ethyl]amino}methyl)pyridine-4-carboxylic acid

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

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

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

2-[( {[bis(prop-2-en-1 -y l)carbamoy l]methy 1} amino)met hyl]pyridine-4 -carboxy 1 ic acid

2-[({2-oxo-2-[(2R)-2-(pyrroiidin-l-ylmethyl)pyrrolidin-l-yl]ethyl}amino)methyl]-pyridine-4carboxylic acid

2-( {[(3R)-1 -[(tert-butoxy)carbonyl]pyrrolidin-3-yl]amino}methyl)pyridine-4-carboxylic acid

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

2- {[(3- {[3-(pyrrolidin-1 -yl)propyl] amino} propyl)amino] methyl} pyridine-4-carboxylic acid

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

2-[( {[(2-carbamoylethyl)(methyl)carbamoyl]methyl)amino)methyl]pyridme-4-carboxylic acid

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

2-{ [( (methyl[3-( 1 -methyl-1 H-imidazol-2-yl)propyl]carbamoyl] methyl)amino]methyl }-pyridine-4carboxylic acid

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

2-{[({methyl[(l-methyl-lH-pyrazol-5-yl)methyl]carbamoyl}methyl)amino]-methyl}pyridine-4carboxylic acid

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

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

2-{[( {[1 -(3-phenylpropyl)piperidin-4-yl]carbamoyl}methyl)amino]methyl}pyridine-4-carboxylic acid

2- {[( {[ 1 -( furan-2-y 1 methy l)pip eridin-4 -y l]carbamoy 1} methyl)amino]methyl} pyridine-4-carboxy 1 ic acid

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

4-carboxylic acid

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

2-({[2-(1 -butylpyrrolidin-2-yl)ethy 1]amino} methyl)pyridine-4-carboxylie acid

2- {[( {[ 1 -(3,7-d i methy loct-6-en-1 -y l)pyrrol id in-3 -yljcarbamoy 1} methyl)amino] -methyl} pyridine-4carboxylic acid

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

2-( {[( 1 R)-2-hydroxy-1 - {methyl [3-( 1 -methyl-1 H-imidazol-2-yl)propyl]carbamoyl} - ethyl] amino} methy l)pyridine-4-carboxy 1 ic acid

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

2- {[({[ 1 -(2-phenylethyl)pyrrolidin-3-yl]carbamoyl} methyl)amino]methyl}pyridine-4-carboxylic acid

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

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

2-[( {[methyl( {4-[(4-methylpiperazin-1 -yl)methyl]pheny I} methyl)carbamoyl]methyl} - ami no)methy 1] pyridine-4-carboxyl ic acid

2-( {[2-(2-benzylpyrrolidin-1 -yl)-2-oxoethyl]amino} methyl)pyridine-4-carboxylic acid

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

2-[({2-[(2S)-l -benzylpyrrolidin-2-yl]ethyl} amino)methy l]pyridine-4-carboxy 1 ic acid

2-( {[3-(pyrro 1 idin-1 -y l)propy 1 ] ami no} methy l)pyrid i ne-4-carboxyl ic acid methyl 2-({[3-(pyrrolidin-l-yl)propyl]amino}methyl)pyridine-4-carboxylate

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

2- {[ ( {[ 2-(d i methylamino) ethyl] (ethyl) carbamoyl} methy l)ami nojmet hyl} pyridi ne-4-carboxyl ic acid (5-methyl-2-oxo-2H-l,3-dioxol-4-yl)methyl 2-({[4-(diethylamino)butyl]amino}methyl)pyridine-4carboxylate

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

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

2-(dimethylamino)ethyl 2-({[4-(diethylamino)butyl]aniino}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-4carboxylate

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

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

4-methoxyphenyl 2- {[( {[2- (d imethylamino)ethy l] (ethyl)carbamoyl} methyl)amino] methyl} pyridine-4-carboxylate

2-(ethoxycarbonyl)phenyl 2-{[({[2- (dimethylamino)ethy 1] ( ethyl )carbamoyl} methy l)ami no] methyl} pyrid ine-4-çarboxylate {4-[(ethoxycarbonyl) (methyl )amino] phenyl} methyl 2-({[4- (diethylamino)butyl]amino}methyl)pyridine-4-carboxylate

4-tert-butylphenyl 2- {[( {[2- e

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

4-(trifluoroacetamido)butan-2-yl 2-{[( {[2- (dimethylamino)ethyi](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-(tri fluoroacetamîdo)pent- 1 -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- (d imethylaniino)ethyl] (ethy l)carbamoyl} methyl)amino] methyl} pyridine-4-carboxylate

2-(2- {[( {[2-(dimethylamino)ethyl](ethyl)carbamoyl} methyl)amino]methyl} pyridine-4carbonyloxy )-3 -(hexadecanoy loxy)propy I hexadecanoate

1 -(2- {[( {[2-(di methyl a mi no)ethy 1 ] (ethy 1) c arbamoy 1} methy l)amino] methyl} pyridine-4carbony loxy)-3 -( hexadecanoy loxy)propan-2-y 1 hexadecanoate methyl 2- {[( {[2-(dimethylamino)elhyl](ethyl)carbamoyl} methyl)amino]methyl} pyridine-4carboxylate

2- {[( {[2-(dimethylamino)ethyl](ethyl)carbamoyl} methyl)amïno] methyl} -N-methanesulfonyl-Nmethylpyridine-4-carboxami de

N-[2-(dimethylamino)ethyl]-N-ethyl-2-( {[4-(2-oxo-l ,3-oxazolidine-3-carbonyl)pyridin-2- yl] methyl} ami no)acetami de 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} met hyl)amino] methyl} -N-(pyridin-4-yl)pyridine4-carboxamide

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

2- {[( {[2 -(dimethylami no)ethy 1] (ethy l)carbamoyl} methyl)amino] methyl} -N-( 1 -methyl-1 H-pyrazol-

5-yl)pyridine-4-carboxamide

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

2-(azetidin-1 -ylmethyl)pyridine-4-carboxylic acid

2,2,2-trifluoroethyl2-{[({[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}inethyl)pyridine-4-carboxylic acid

2-{[({[2-(di methylaminojethy 1] (et hy ljcarbamoy 1} methy l)ami no] methyl} -N-( 1,3 -oxazol-2yl)pyridine-4-carboxamide

2,6-bis(propan-2-yloxy)phenyl 2-{[({[2- (dimethyiamino)ethy 1] (ethy l)carbamoyl} methy l)amino] methyl} pyridine-4-carboxy late

2- {[(2-methylpropyl) [2-oxo-2-(piperid i η-1 -yi) ethyl]ami no] methyl} pyrid i ne-4-carboxylie acid

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

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

2- {[( {[2-(dimethylamino)ethyl](ethyl)carbamoyl} methyl) amino] methyl} -N-( 1 -methyl-1Himidazol-2-yl)pyridine-4-carboxamide

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

2,2-difluoroethyl 2-{[({[2- (d i methylamino)ethy l] (ethyl)carbamoy 1} met hyl)amino] methy 1} pyridine-4-carboxy late

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

2-( {methyl[(2S)-4-methyl- 1-oxo-l -(piperidin-1 -yl)pentan-2-yl]amino) methyl)pyridine-4carboxylic acid

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

Définitions

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 (C|.g-alkyl), more preferred from one to six carbon atoms (C|_6-alkyl), in particular from one to four carbon atoms (C)^-alkyl), including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, tertiary butyl, pentyl, isopentyl, neopentyl, tertiary pentyl, hexyl, isohexyl, heptyi and octyl. In a preferred embodiment “alkyl” represents a Cj.4-alkyl group, which may in particular include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, and tertiary butyl. Correspondingly, the term “alkylene” means lhe corresponding biradical (-alkyl-).

The term “cycloalkyl” as used herein refers to a cyclic alkyl group, preferably containing from three to ten carbon atoms (Cj.io-cycloalkyl), such as from three to eight carbon atoms (Cj.g-cycloalkyl), preferably from three to six carbon atoms (Cî-s-cycloalkyl), including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. 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 “cycloalkylene” 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 dî-enes, tri-enes and poly-enes. Typically, the alkenyl group comprises from two to eight carbon atoms (C₂._g-alkenyl), such as from two to six carbon atoms (C₂.6-alkenyl), in particular from two to four carbon atoms (Cj.4-alkenyl), 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.g-alkynyl), such as from two to six carbon atoms (C₂_6-alkynyl), in particular from two to four carbon atoms (C₂^-alkynyl), including at least one triple bond. Examples of preferred alkynyl groups include ethynyl; 1- or 2-propynyl; 1-, 2- or 3-butynyl, or 1,3-but-diynyl; 1-, 2-, 3-, 4- or 5-hexynyl, or 1,3-hex-diynyl, or 1,3,5-hex-triynyl; 1-, 2-, 3-, 4-, 5-, 6-, or 7-octynyl, or 1,3-octdiynyl, or 1,3,5-oct-triynyl, or 1,3,5,7-oct-tetraynyl. 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 perfiuorated. The term perfluoroalkyl as used herein refers to an alkyl group as defined herein wherein ail 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-CHj, HO-CH₂-CH₂- and CH₃-CH(OH)-.

The term “oxy” as used herein refers to an “-O-” group.

The term “oxo” 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 (R₃-N, R * H) amines. A substituted amine is intended to mean an amine where at least one ofthe 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-, triand polycyclic ring Systems. Examples of preferred aryl moieties 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 O, 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 O, S, and N. Heteroaryl also include ring Systems substituted with one or more oxo moieties. Examples of preferred heteroaryl moieties include N-hydroxytetrazolyl, N-hydroxytriazolyf, N-hydroxyimidazolyl, furanyl, triazolyl, pyranyl, thiadiazinyl, benzothiophenyl, dihydro-benzo[b]thiophenyl, xanthenyl, isoîndanyl, acridinyl, benzisoxazolyl, quinolinyl, isoquinolïnyl, phteridinyl, azepinyl, diazepinyl, imidazolyl, thiazolyl, carbazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, isothiazolyl, pyrrolyl, îndolyl, benzimidazolyl, benzofuranyl, cinnolinyl, 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 exemples of partially hydrogenated dérivatives are 1,2,3,4tetrahydronaphthyl, 1,4-dihydronaphthyl, and 1-octalin, Correspondingly, the term “heteroarylene” means the corresponding biradical (-heteroaryi-).

The term “heterocyclyl” as used herein, refers to cyclic non-aromatic groups containing one or more heteroatoms selected from O, 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 O, S, and N. Heterocyclyl also 10 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, 3H-pyrazolyl, 1,2-oxazolyl, 1,3-oxazolyl, 1,2-thiazolyl, 1,3thiazolyl, 1,2,5-oxadiazolyl, piperidinyl, pyridinyl, oxanyl, 2-H-pyranyl, 4-H-pyranyl, thianyl, 2Hthiopyranyl, pyrîdazinyl, 1,2-diazinanyl, pyrimidinyl, 1,3-diazinanyl, pyrazinyl, piperazinyl, 1,4-dioxinyl, 15 1,4-dioxanyl, 1,3-diazinanyl, 1,4-oxazinyl, morpholinyl, thiomorpholinyl, 1,4-oxathianyl, benzofuranyl, isobenzofuranyl, indazolyl, benzimidazolyl, quînolinyl, isoquinolinyl, chromayl, isochromanyl, 4Hchromenyl, IH-isochromenyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, purinyl, naphthyridinyl, pteridinyl, indolizinyl, 111-pyrrolizinyl, 4H-quinolizinyl and aza-8-bicyclo[3.2.1]octane. Correspondingly, the term “heterocyclylene” means the corresponding biradical (-heterocyclyl-).

The term “N-heterocyclic ring” as used herein, refers to a heterocyclyl or a heteroaryi as defined hereïnabove 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, pyrîdazinyl, pyrazinyl, piperazinyl, morpholinyl, pyridinyl, pyrîdazinyl, pyrimidinyl, pyrazolyl, pyrazinyl, tetrazolyl, etc.

Isomers

The compounds of Formula (I) may exist as géométrie isomers (i.e. cis-trans isomers), optical isomers or stereoisomers, such as diastereomers, as well as tautomers. Accordingly, it should be understood that the définition of compounds of Formula (I) includes each and every individual isomers corresponding to the 30 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 définition of compounds of Formula (I) is also intended to encompass ail 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 acids include, without limitation, tartane, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. The mixture of diastereomers can be separated by crystallization followed by libération of the optically active bases from these salts. An alternative process for séparation of optical isomers includes the use of a chiral chromatography column optimally chosen to maximize the séparation of the enantiomers. Still another avaiiable method involves synthesïs of covalent diastereoisomeric molécules 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 sait. Examples of chiral séparation techniques are given in Chïral Séparation Techniques, A Practical Approach, 2^nd ed. by G. Subramanian, WileyVCH, 2001.

Pharmaceutically acceptable salts

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

Pharmaceutically acceptable salts refer to salts of the compounds of Formula (1), which are considered to be acceptable for clînical and/or veterinary use. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of Formula (1) a minerai 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 sait is nol of a critical nature, so long as the sait as a whole is pharmaceutically acceptable and as long as the counter-ion does not contribute undesired qualifies to the sait 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 éditions and in Encyclopedia of Pharmaceutical Teclinology.

Examples of pharmaceutically acceptable addition salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric, or phosphoric acid;

and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, trifluoroacetic, malic, lactic, formic, propionic, glycolic, gluconic, camphorsulfurie, 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,Ndibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (Nmethylglucamine), lysine and procaine; and intemally formed salts.

Solvatés

The compound of Formula (I) may be provided in dissoluble or indissoluble forms together with a pharmaceutically acceptable solvent such as water, éthanol, 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.

Prodrugs

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 acîd group of the compound of formula (I), which e.g. is capable of liberating the latent carboxylic acid 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, 2-aminoethyl esters, etc., including (5-methyl-2-oxo-2H-l,3-dioxol-4-yl)methyl esters, 4-methoxyphenyl esters, 2(ethoxycarbonyl)phenyl esters, (4-[(ethoxycarbonyl)(methyl)amino]phenyl}methyl esters, 2(dimethylamino)ethyl esters, 3-(dimethylamîno)propyl esters, [(ethoxycarbonyl)amino]phenylmethyl esters, 2,6-dimethoxyphenyl esters, 2,6-dimethylphenyl esters, 4-tert-butylphenyl esters, 4-oxopentan-2yl 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)pent-l -en-3-yl esters, l,3-bis(hexadecanoyloxy)propan-2-yl esters, 2,3-bis(hexadecanoyloxy)propyl esters, 4-oxo-4-(propan-2yloxy)-1 -(trifluoroacetamido)butan-2-yl esters, 1 -oxo-1 -(propan-2-yloxy)-5-(trifluoroacetamido)pentan3-yl esters 2,2,2-trifluoethyl esters, 2,6-bis(propan-2-yloxy)phenyl esters, 2-fluoroethyl esters, 2,2difluoroethyl esters, etc.

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

A---Y wherin R¹² is of the form (R^I3)2N- or of the form R^I3O-, 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 C(.g alkyl, C2.8 alkenyl, C₂.g 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, C_t.₆ alkoxy, heteroaryl, aryloxy, heteroaryloxy, F, a sulphonamide moiety, and Cj.6 cycloalkyl; and one R¹³ in (R^I3)₂N- may be, and preferably is, -H.

Methods for the préparation 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 présent 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 préparation steps of the compounds will be known to the skilled person. The methods below are given as non-lîmiting examples on how the compounds may be prepared.

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

Préparation of Compounds of Formula fl)

Scheme 1

Method A

The compound of Formula (I) may be prepared according to Scheme 1 at room température, or by heating for up to several hours by use of a solvent such as DMSO, an alcohol, or tetrahydrofuran, and a 5 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 température, or by heating for up to several hours by use of a solvent such as water, DMSO, an alcohol, or tetrahydrofuran, 10 and an aqueous acid. A purification method such as silica gel chromatography is employed if needed.

Scheme 2

Method C

The compound of Formula (I) may be prepared from halides or triflates (X= halogen, OTf) according to 15 Scheme 2 either at room température or by heating for up to several hours by use of a solvent such as toluene or tetrahydrofuran, a base such as césium carbonate or potassium t-butoxide, a catalyst such as Pd complex optionally a sait such as lithium chloride and carbon monoxide. A purification method such as silica gel chromatography is employed if needed.

Préparation of Intermediates for Compounds of Formula (1)

Scheme 3

Method D

Intermediates (i) may be prepared from 2-formyl pyridines according to Scheme 3 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 NaBHi, NaBH(OAc)j, NaCNBHî, or EtjSiH, either at room température 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 acid. Optionally, protecting groups may be removed and a purification method such as silica gel chromatography is employed if needed.

Scheme 4

Method E

Intermediates (ii) may be prepared from 2-formyl pyridines according to Scheme 4 analogously to

Method D.

Scheme 5

Method F

Intermediates (iv) be prepared from (iii) according to scheme 5, where R’ is a suitable protecting group or RI, by use of a solvent such as DMF or THF, a base such as a hindered tertiary amine, a dehydrating agent such as EDCI or DCC and an amine, and by mixing at or above room température for a period up 5 to several hours. Optionally, the said protecting group may be removed, and a purification method such as silica gel chromatography is employed if needed.

Scheme 6

Method G

Intermediates (vi) may be prepared from (v) according to Scheme 6 analogously to Method F.

Scheme 7

Method H

Intermediates (viii) be prepared according to scheme 7 from (vii), where R’ is a suitable protecting group 15 or RI and R” is an orthogonal protecting group, which may be selectively removed, such as removal of

R”: 'Bu in presence of R’: CF3CO by treating with trifluoroacetic acid in a solvent such as dichlorométhane at room température for several hours. A purification method such as silica gel chromatography is employed if needed.

Scheme 8

(ix) *

(x)

Method I

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

Scheme 9

Method J

Intermediates (xii) may be prepared from aldéhydes and intermediates (xi) according to Scheme 9 in onepot or by a stepwise procedure by mixing with the amine (R*: RI or a suitable protecting group) and a 10 reducing agent such as NaBH4, NaBH(OAc)₃, NaCNBHj, or Et₃SiH, either at room température 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 acid. 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

Method L

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

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, NaCNBHj, or Et₃SiH, either at room température or by heating for up to several hours by use of a solvent such as an alcohol, DCE, DCM, water, or toluene, 15 optionally adding a catalyst such as an acid or a Lewis acid. Optionally, protecting groups may be removed and a purification method such as silica gel chromatography is employed if needed.

Scheme 12

(xvil)

(xvili)

Method O

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

Inhibitory effect

The inventors hâve surprisingly found that compounds of Formula (I) as defined herein hâve 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 présent 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 présent in an amount effective to produce a concentration sufficient to inhibit the déméthylation of a histone in the cell.

Thus, preferably in an assay for déméthylation of a histone substrate by said HDME, then preferred compounds of Formula (I) are compounds capable of reducing or preferably inhibiting said déméthylation 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 K.4, K9, K27, or K.36 of H3. Preferably, said inhibition is determined as the IC₅o of said compound of Formula (I) in respect of the said déméthylation assay.

Preferred compounds of Formula (I) which hâve an ICjo at or below 1 μΜ, more preferably less than 300 nM, for example less than 100 nM, such as less than 50 nM in respect of déméthylation of any of said histone substrates by any of said HDME. Thus very preferred compounds of Formula (I) which hâve an IC50 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 déméthylation of histone H3 methylated at least on one lysine.

In a preferred embodiment IC50 is determined as described in Example 2 herein below. Thus, particularly preferred are compounds of Formula (I) which hâve an IC_J0 at or below 1 μΜ, more preferably Iess than 500 nM, for example Iess than 100 nM, such as Iess than 50 nM when said IC₅₀ is 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 métastasés 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 ingrédient, at least one compound of Formula (I) as defined herein and optionally one 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 fïllers, stérile 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, 21 st 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 readiiy 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 parentéral (inciuding subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route. It will be appreciated that the preferred route will dépend on the general condition and âge of the subject to be treated, the nature of the condition to be treated and the active ingrédient 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 ingrédient 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 éthanol, 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 include, 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 stéarate, magnésium stéarate, sodium benzoate, sodium acetate, sodium chloride or the like. Disintegrating agents include, e.g., starch, methyi cellulose, agar, bentonite, xanthan gum, sodium starch glycolate, crospovidone, croscarmellose sodium or the like. Additional excipients for capsules include macrogols or lipids.

For the préparation 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 readiiy be subdivided into equally effective unit dosage forms such as tablets or capsules.

Liquid compositions for either oral or parentéral administration of the compound of Formula (I) include, e.g., aqueous solutions, syrups, élixirs, aqueous or oil suspensions and émulsion 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 parentéral administration include stérile aqueous and non-aqueous injectable solutions, dispersions, suspensions or émulsions as well as stérile powders to be reconstituted in stérile injectable solutions or dispersions prior to use. For parentéral administration, solutions containing a compound of Formula (I) in sesame or peanut oil, aqueous propylene glycol, or in stérile aqueous solution may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonie with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subeutaneous and intraperitoneal administration. The oily solutions are suitable for intra-articular, intra-muscular and subeutaneous injection purposes.

The préparation of ail these solutions under stérile conditions is readiiy 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 présent invention.

In addition to the aforementioned ingrédients, the compositions of a compound of Formula (I) may include one or more additional ingrédients 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 dépend on the âge 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 dosîng schedules, e.g. daîly 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 times a day. Variations based on the aforementioned dosage ranges may be made by a physician of ordinary skill taking into account known considérations such as weight, âge, 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 forther 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 dépendent disease, disorder or condition. The treatment may include administering to a mammal, preferably a human, more preferably a human suffering from a HDME dépendent disease, a therapeuticaily effective amount of a compound according to Formula (I) as defined herein.

Said HDME may be any HDME, however preferably the HDME of the présent method is selected from the JmjC (Jumonji) family, as described in Cloos et. al., Genes & Development 22,1115-1140, 2008, which is incorporated herein by reference in its entirety. More preferably said HDME îs a HDME of the human JmjC family.

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

By the term “HDME dépendent 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 proliférative or hyperproliferative disease, which includes benign or malignant tumors, for example a proliférative 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, pancréas, 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 epidennal hyperproliferatîon, for example, psoriasis, prostate hyperplasia, a neoplasia, including a neoplasia of épithélial 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 prolifération of neoplastic cells, such as solid tumors, neoplasms, carcinomes, sarcomas, leukemias, lymphomas 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), pancréas (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, Wilm's tumor, nephroblastoma, lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitîonal cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcfhoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: ostéogénie sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (réticulum 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), méningés (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congénital tumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma); Gynecological: utérus (endométrial 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, intraepithélial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcome), fallopian tubes (carcinoma); Hématologie: blood (acute myeloid leukemia, chrome 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: neuroblastome.

In one embodiment, the compounds of Formula (I) as defined herein are useful in the treatment of one or more cancers selected from the group consisting of: leukemias including acute leukemias and chronic leukemias such as acute lymphocytic leukemia (ALL), Acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and Hairy Cell Leukemia; lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T- cell lymphotrophic virus (HTLV) such as adult T-cell leukemia/Iymphoma (ATLL), Hodgkin's disease and non-Hodgkin's lymphomas, large-cell lymphomas, diffuse large B-cell lymphoma (DLBCL); Burkitt's lymphoma; mesothelioma, primary central nervous System (CNS) lymphoma; multiple myeloma; childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilm's tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal and esophageal), genito urinary cancers (e.g., prostate, bladder, rénal, uterine, ovarian, testicular, rectal and colon), lung cancer, breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, liver cancer and thyroid cancer.

In another very preferred embodiment, the compound of Formula (I) as defined herein are useful for the treatment of squamous cell carcinomas. Preferably said squamous cell carcinomas are cancers of the carcinoma type of squamous epithelium that may occur in many different organs, including the skin, lips, mouth, esophagus, urinary bladder, prostate, lungs, vagina, and cervix; brain cancer, that is neuroblastoma, glioblastoma and other malignant and benign brain tumors; breast cancer, pancreatic cancer, and multiple myeloma.

In yet another embodiment, the compounds of Formula (I) as defined herein are useful for treatment of brain cancer, tumors of adults such as head and neck cancers (e.g., oral, laryngeal and esophageal), genito urinary cancers (e.g., prostate, bladder, renai, uterine, ovarian, testicular, rectal and colon), and breast cancer.

Other cancer forms for which the compounds of Formula (I) are useful as treatment can be found in Stedman’s Medical Dictionary (Lippincott Williams & Wilkins, 28'¹' Ed., 2005), which is incorporated herein by reference in its entirety.

In still another related embodiment, the disease to be treated by compounds of Formula (I) as defined herein is selected from persistent proliférative or hyperproliferative conditions such as angiogenesis, such as psoriasis; Kaposi's sarcoma; restenosis, e.g., stent-induced restenosis; endometriosis; Hodgkin's disease; leukemia; hemangioma; angiofîbroma; eye diseases, such as neovascular glaucoma; rénal diseases, such as glomerulonephritis; malignant nephrosclerosis; thrombotic microangiopathic syndromes; transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver; mesangial cell-proliferative diseases; injuries of the nerve tissue; and inhibiting the re-occlusion of vessels after balloon cathéter treatment, for use in vascular prosthetics or after inserting mechanical devices for holding vessels open, such as, e.g., stents, as immune-suppressants, as an aid in scar-free wound healing, and treating âge spots and contact dennatitis.

The compounds of Formula (I) are suitable as active agents in pharmaceutical compositions that are efftcacious particularly for treating cellular proliférative or hyperproliferative ailments and/or aliments associated with dysregulated gene expression. Such pharmaceutical compositions hâve a therapeutically effective amount of the compound of Formula (I) along with other pharmaceutically acceptable excipients, carriers, and diluents and. The phrase, “therapeutically effective amount” as used herein indicates an amount necessary to administer to a host, or to a cell, tissue, or organ of a host, to achieve a therapeutic effect, such as an ameliorating or altematively a curative effect, for example an anti-tumor effect, e.g. réduction of or preferably inhibition of prolifération of malignant cancer cells, benign tumor cells or other proliférative cells, or of any other HDME dépendent disease.

Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of Formula (I) as defined herein, or a pharmaceutically acceptable sait, solvaté or prodrug thereof, in combination with at least one further anti-neoplastic compound, and a pharmaceutically acceptable excipient, carrier or diluent.

Method of treatment

In a further aspect the présent invention relates to a method of treating a diseases 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. The disease may be any disease or disorder as mentioned herein, such as for example mentioned in the section “HDME dépendent diseases”, and the compound may be administered alone or in a pharmaceutical composition, such as for example mentioned in the section “Pharmaceutical compositions”.

Hence, the invention also relates to a compound of Formula (I) as defined herein for use as a médicament.

The term “treating” and “treatment”, as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting the process of, or preventing the disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of a compound of Formula (I) to prevent the onset of the symptoms or the complications, or alleviating the symptoms or the complications, or eliminating the disease, condition, or disorder. Preferably treatment is curative or ameliorating.

In a preferred embodiment of this aspect of the invention the method is a method of treating a HDME dépendent disease in a subject, said method comprises administering to said subject a therapeutically effective amount of a compound of Formula (I) as defined herein to a subject in need of such treatment. The HDME dépendent disease may be any HDME dépendent disease as described herein above. Preferably the HDME dépendent disease is squamous cell carcinomas or any other of the cancer conditions mentioned above.

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

Further, the invention relates to the use of a compound of Formula (I) as defined herein for the préparation of a pharmaceutical composition for the treatment of a HDME dépendent disease.

In one embodiment of the method of treatment of a HDME dépendent disease, the compound of Formula (I) as defined herein is administered in combination with one or more further active substances. The active substances may be any active substances, and preferably an active substance as described herein above in the section “combination treatment”. More preferably the one or more additional active substances are selected from the group consisting of anti-proliferative or antî-neoplastic agents, Combination treatment

A compound of Formula (I) may also be used to advantage in combination with one or more other antiproliferative or antî-neoplastic agents. Such anti-proliferative agents include, but are not Iimited to other

HDME inhibitors, protéasome inhibitors, including bortezomib (Valcade) and Carfilzomib, aromatase inhibitors; antiestrogens; topoisomerase 1 inhibitors; topoisomerase II inhibitors; mïcrotubule active agents; alkylating agents; histone deacetylase inhibitors; compounds which induce cell différentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity and further antiangiogenic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonist s; anti-androgens; angiostatic steroids; méthionine aminopeptidase inhibitors; bisphosphonates; biological response modifiers; antiproliférative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; protéasome inhibitors; agents used in the treatment of hématologie malignancies; compounds which target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors; temozolomide (TEMOD AL(R)); leucovorin; immune stimulâting agents, such as BCG, IL-2 or IFN-α, antibodies, such as rituximab or herceptin and cancer vaccines.

A compound of Formula (I) as defined herein may also be used to advantage in combination with known therapeutic processes, e.g., the administration of hormones or tumor cell damaging approaches, especially ionizing radiation.

A compound of Formula (1) as defined herein may also be used as a radiosensitizer, including, for example, the treatment of tumors which exhibit poor sensitivity to radiotherapy.

By the term combination, is meant either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound of Formula (I) and a combination partner may be administered independently at the same time or separately within time intervals that especially allow that the combination partners show a cooperative, e.g., synergistic, effect, 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-steroids, especially aminoglutéthimide, 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. Aminoglutéthimide 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 usefui 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 fonnulated 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 androgénie hormones and includes, but is not limited to, bicalutamide (CASODEX), which can be formulated, e.g., as disclosed in US 4,636,505.

The phrase, gonadorelin agonist as used herein includes, but is not limited to abarelix, goserelin and goserelin acetate. Goserelin is disclosed in US 4,100,274 and can be administered, e.g., in the form as it îs marketed, e.g., under the trademark ZOLADEX. Abarelix can be formulated, e.g., as disclosed in US 5,843,901.

The phrase, topoisomerase I inhibitor as used herein includes, but is not limited to topotecan, gimatecan, irinotecan, camptothecan and its analogues, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148 (compound Al in WO99/ 17804). Irinotecan can be administered, e.g., in the form as it is marketed, e.g., under the trademark CAMPTOSAR. Topotecan can be administered, e.g., in the form as it is marketed, e.g., under the trademark HYCAMTIN.

The phrase, topoisomerase II inhibitor as used herein includes, but is not limited to the anthracyclines such as doxorubicin (inciuding liposomal formulation, e.g., CAELYX), daunorubicin, epîrubicin, idarubicin and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophyllotoxins etoposide and teniposide. Etoposide can be administered, e.g., in the form as it is marketed, e.g., under the trademark ETOPOPHOS. Teniposide can be administered, e.g., in the form as it is marketed, e.g., under the trademark VM 26-BRISTOL. Doxorubicin can be administered, e.g., in the form as it is marketed, e.g,, under the trademark ADRIBLASTIN or ADRLAMYCIN. Epîrubicin can be administered, e.g., in the form as it is marketed, e.g., under the trademark FARMORUBICIN. Idarubicin can be administered, e.g., in the form as it is marketed, e.g., under the trademark ZAVEDOS, Mitoxantrone can be administered, e.g., in the form as it is marketed, e.g., under the trademark NOVANTRON.

The phrase, microtubule active agent relates to microtubule stabilizing, microtubule destabilizing agents and microtublin polymerization inhibitors including, but not limited to taxanes, e.g., paclitaxel and docetaxel, vinca alkaloids, e.g., vinblastine, including vinblastine sulfate, vincristine including vincristine sulfate, and vinorelbine, discodermolides, cochicine and epothilones and dérivatives thereof, e.g., epothilone B or D or dérivatives thereof. Paclitaxel may be administered e.g., in the fo[pi]n as it is marketed, e.g., TAXOL. Docetaxel can be administered, e.g., in the form as it is marketed, e.g., under the trademark TAXOTERE. Vinblastine sulfate can be administered, e.g., in the form as it is marketed, e.g., under the trademark VINBLASTIN R.P. Vincristine sulfate can be administered, e.g., in the form as it is marketed, e.g., under the trademark FARMISTIN. Discodermolide can be obtained, e.g., as disclosed in US 5,010,099. Also included are Epothilone dérivatives which are disclosed in WO 98/10121, US 6,194,181, WO 98/25929, WO 98/08849, WO 99/43653, WO 98/22461 and WO 00/31247. Included are Epothilone A and/or B.

The phrase, alkylating agent as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or Gliadel). Cyclophosphamide can be administered, e.g., in the form as it is marketed, e.g,, under the trademark CYCLOSTIN. Ifosfamide can be administered, e.g., in the form as it is marketed, e.g., under the trademark HOLOXAN.

The phrase, histone deacetylase inhibitors or HDAC inhibitors relates to compounds which inhibit at least one example of the class of enzymes known as a histone deacetylase, and which compounds generally possess antiproliférative activity. Previously disclosed IIDAC inhibitors include compounds disclosed in, e.g., WO 02/22577, including N-hydroxy-3-[4-{[(2-hydroxyethyl)[2-(lH-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2- propenamide, N-hydroxy-3-[4-[[[2-(2-methyl-lH-indol-3-yl)-ethylJamino]methyl]phenyl]-2E-2- propenamide and pharmaceutically acceptable salts thereof. It further includes Suberoylanilide hydroxamic acid (SAHA). Other publicly disclosed HDAC inhibitors include butyric acid and its dérivatives, including sodium phenylbutyrate, thalidomide, trichostatin A and trapoxin.

The term antineoplastic antimetabolite includes, but is not limited to, 5-Fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating agents, such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folie acid antagonists such as pemetrexed. Capecitabine can be administered, e.g., in the form as it is marketed, e.g., under the trademark XELODA. Gemcitabine can be administered, e.g., in the form as it is marketed, e.g., under the trademark GEMZAR. Also included is the monoclonal antibody trastuzumab which can be administered, e.g., in the form as it is marketed, e.g., under the trademark HERCEPTIN.

The phrase, platin compound as used herein includes, but is not limited to, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatin can be administered, e.g., in the form as it is marketed, e.g., under the trademark CARBOPLAT. Oxaliplatin can be administered, e.g., in the form as it is marketed, e.g., under the trademark ELOXATIN.

Tumor cell damaging approaches refer to approaches such as ionizing radiation. The phrase, ionizîng radiation referred to above and hereinafler means ionizing radiation that occurs as either electromagnetic rays (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. See, e.g., Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., Eds., 4th Edition, Vol. l,pp. 248-275(1993).

The phrase, angiostatic steroids as used herein refers to agents which block or inhibit angiogenesis, such as, e.g., anecortave, triamcinolone, hydrocortisone, 1 l-[alpha]-epihydrocotisol, cortexolone, 17[alpha]-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone and dexamethasone,

Other chemotherapeutic agents include, but are not limited to, plant alkaloids, hormonal agents and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide dérivatives; or miscellaneous agents or agents with other or unknown mechanism of action.

The structure of the active agents identified by code numbers, generic or trade names may be taken from the actual édition of the standard compendium The Merck Index or from databases, e.g., Patents International (e.g., IMS World Publications).

The above-mentioned compounds, which can be used in combination with a compound of Formula (I), can be prepared and administered as described in the art such as in the documents cited above.

Furthermore, the compounds of the invention may be used in a method of profiling the functional and structural similarity of histone demethylases comprising taking a panel of at least two histone demethylases and a panel of at least two compounds of formula 1 and determining the extent to which each said compound of formula 1 inhibits the activity of each of said histone demethylases, and generating a similarity index reflecting the degree of similarity between the histone demethylases in respect of their inhibition by said compounds.

Examples

Example 1: Examples of Compounds of Formula (I)

General Methods and Materials

Table 1 below shows examples of compounds according to the invention and indicates routes that may be employed in their synthesis. Ail chemicais were purchased front Sigma-Aldrich, Alfa Aesar, Matrix, Combiblock, Oakwood, and Chembridge. Anhydrous solvents were Aldrich Sure/Seal™ brand. Ail reactions were carried out under a dry nitrogen atmosphère using dry solvents. Reactions were monitored by thin-layer chromatography carried out on Sigma-Aldrich 0.25 mm silica gel plates (60 A, fluorescent indicator). Spots were visualized under UV light (254 nm). Flash column chromatography was performed on Biotage SNAP Flash System, or silica gel 60 (particle size 0.032-0.063 mm) obtained from Silicycle, Inc. Low-resolution ES (electrospray) mass spectra were obtained using a Micromass Quattro Ultima mass spectrometer in the electrospray positive (ES+) or négative (ES-) ion mode. 1H-NMR spectra were recorded on a Broker AM-300 spectrometer and were calibrated using residual nondeuterated solvent as internai reference. Spectra were processed using Spinworks version 2.5 (developed by Dr. Kirk Marat, Department ofChemistry, Universîty of Manitoba). Préparative IIPLC was performed on Waters 2996 with Photodiode Array Detector, Waters 600 Controller, Waters 100 pump, and Waters 717 auto sampler, with UV détection at 254 and 280 nm. Flow rate: 15 mL/minute, ron time 30 minutes. Solvents: 0-100% (H₂O-MeOH), with and without added TFA (0.1%). Column used was Supelco Cl 8, 25 cm x 21.2 mm, particle size 10 micrometer.

Ethyl 2-formylpyridine-4-carboxylate was prepared analogously to Queguiner, G. and Pastour, P. ( Comptes Rendus des Séances de l'Academie des Sciences, Sérié C: Sciences Chimiques (1969), 268(2), 182-5).

Table 1
Structure	#	Name	Synthetic Route	NMR
0 OH X. N .N	1	2-({[3-(lH-imidazol-lyl)propyl]amino} methyl)pyri dine-4-carboxylic acid	A	^lH NMR (300 MHz, CDjOD), δ ppm: 8.10 (s, IH), 7.98 (dd, IH), 7.80 (t, IH), 7.65 (t, IH), 4.58 (s, 2H), 4.50 (t, 2H).
°γ^0Η il ^H ch₃	2	2< {[2- (dimethylamino)ethyljamino} methyl)pyridîne-4carboxylic acid	A	’H NMR (300 MHz, CD₃OD), δ ppm: 8.03 (s, IH), 4.58 (s, 2H), 3.63 (m, 4H), 3.0 (s, 6H).
^ΗΟγ° il ^H ?^H	3	2-({[(2R)-2,3dihydroxypropyl]amino} meth yl)pyridine-4-carboxylic acid	A	'H NMR (300 MHz, CDjOD), δ ppm: 8.12 (s, IH), 4.04 (m, IH), 3.62 (m, 2H), 3.35 (m, IH), 3.18 (dd, IH).

°γ^0Ητ Γ	4	2- {[(cyclopropylmethyl )amino] methyl} pyridine-4carboxylic acid	A	^lH NMR (300 MHz, CD₃OD), δ ppm: 4.56 (s, 2H), 3.08 (d, 2H), 1.21 (m, IH), 0.75 (m, 2H), 0.47 (m, 2H).
^ΗΟγ° [ί] ^Η ch₃	5	2- {[(cyclopropylmethyl)amino] methyl} pyridine-4carboxylic acid	A	Ή-NMR (300MHz, CDjOD), S ppm: 8.0 (s, IH), 4.42(s, 2H), 2.80(s, 6H).
°γ^0Η Γί ch₃	6	2<{[2- (dimethylamino)ethylj(methyl )amino} methyl)pyridine-4carboxylic acid	A	'H NMR (300 MHz, CD₃OD), δ ppm: 8.16(s, IH), 4.76 (s, 2H), 3.80 (s, 4H).
^ΗΟγ° 6j>^h	7	2- {[methyl(prop-2-yn-1 yljamino] methyl} pyridine-4carboxylic acid	A	'H NMR (300 MHz, CDjOD), δ ppm: 7.93 (s, IH), 3.77 (s, 2H), 3.39 (s, 2H).

^ΗΟγ° (X		8	2-{[(2fluoroethyl)amino] methyi }pyr idine-4carboxylic acid	A	‘H NMR (300 MHz, CD₃OD), δ ppm: 7.85 (s, IH), 4.65 (t, IH), 4.49 (t, IH), 4.02 (s, 2H).
HO -Ο
η			2-{[(fiiran-2-		'H NMR (300 MHz, CD₃OD),
		Q	yl methyl)amino]methyl} pyri d	A	δ ppm: 7.72 (s, IH), 6.30 (d,
ΗΝ_ν			ine-4-		IH), 6.22 (d, IH), 3.78 (s, 2H).
Ό'			carboxylic acid
^ΗΟγ°
Ο-					'H NMR (300 MHz, CD₃OD),
	Ί		2-( {[(5-phenyIfiiran-2-
ΗΝ.				δ ppm: 7.75 (s, IH), 6.68 (s,
		10	vDmethyl lamino} methyi )pyri	A
					IH), 6.38 (s, IH), 3.85 (s, 2H).
			dine-4-carboxylic acid
V

ο γ^0Ηά. <^ΝΗοη, V H₃C'°	11	2-({[(2,4dimethoxyphenyl)methyl] ami no} methyl)pyridine-4carboxylic acid	A	'H NMR (300 MHz, CD₃OD), δ ppm: 7.84 (s, IH),7.15(s, IH), 3.85 (s, 2H), 3.70 (s, 2H).
°γ^ΟΗ (ι ^Η < .^Ν-, ,CH, Ν -'' S ³	12	2-( {[2- (methyl sulfanyl)ethyl]amino} methyl)pyridine-4carboxylic acid	A	'H NMR (300 MHz, CD₃OD), δ ppm: 7.85 (s, IH), 3.96 (s, 2H), 2.82 (t, 2H), 2.67 (t, 2H).
°γ^ΟΗ	13	2-( {[2-oxo-2-(pyrrolidin-1 yl )ethyl]amino] methyl)pyri d i ne-4-carboxylic acid	B	’H-NMR (300MHz, MeOHda): δ ppm: 7.70 (s, IH), 3.70 (s, 2H), 4.0 (s, 2H), 3.40 (m, 6H).
^ΗΟγ° ÇVA^_CH3 ch₃	14	2-[({[butyl (methyl )carbamoyl]methyl} amino)methyl] pyridine-4carboxylic acid	B	Ή-NMR (300MHz, MeOHdq), δ ppm: 7.7 (s, IH), 3.7 (s, 2H), 3.4 (s, 2H) 3.2 (m, 2H).

^HO _V° K₃C	15	2-( {[(1 -methyl-1 H-1,3benzodiazol-2yl)methyl]amino}methyl)pyri dine-4-carboxylic acid	A	'H NMR (300 MHz, CD₃OD), δ ppm: 7.84 (s, IH), 4.16 (s, 2H), 4.02 (s, 2H), 3.90 (s, 3H).
^ΗΟγ° ’ k/^N\^_Q'^CH3	16	2-[({2-[4-(2- methoxyethyl)piperazin-1 -yl]- 2- oxoethyl} amino)methyl]pyrid ine-4-carboxylic acid	B	’H-NMR (300MHz, MeOHd4), δ ppm: 7.7 (s, IH), 3.7 (s, 2H), 3.3 (s, 3H), 1.8 (m, 8H).
^H° Ο x_n^^^ch2s CHj	17	2-[( {[bi s(prop-2-en-1 yl)carbamoyl] methyl} amino) methylJpyridine-4carboxylic acid	B	‘H-NMR (300MHz, MeOHd₄), δ ppm: 7.7 (s, IH), 5.6 (m, 2H), 4.9 (m, 4H), 3.5 (s, 2H).
Π° °	18	2-[({2-oxo-2-[(2R)-2(pyrrolidin-1ylmethyl)pyrTol i din-1 yl]ethyl}ammo)methyl]pyridi ne-4-carboxylic acid	B	‘H-NMR (300MHz, MeOH- dh), δ ppm: 7.7 (s, IH), 4.2 (m, IH), 3.4 (m, 6H), 3.0 (m, IH).

Ογ™ (S Η ο^η₃	19	2-({[(3R)-l-[(tertbutoxy)carbonyl]pyrrolidin-3yl] amino} methyl)pyridine-4carboxylic acid	A	'H-NMR (300MHz, MeOHdt), δ ppm: 7.75 (s, IH), 3.85 (s, IH), 1.50 (s, 9H).
^ΗΟγ° j L? h₃cT	20	2-({[(3R)-l-[(tertbutoxy)carbonyl]pyrrolidin-3yljamino} methyl)pyridine-4carboxylic acid	B	'H NMR (300 MHz, CDjOD), S ppm: 7.72 (s, IH), 6.32 (d, IH), 6.20 (d, IH), 4.00 (s, 2H), 3.84(s, 2H).
ΗΟ °	21	2- {[(3- {[3-(pyrrolidin-l yl)propyl]amino}propyl)amin ojmethyl } pyridine-4carboxylic acid	C	^lH NMR (300 MHz, D₂O), δ ppm: 7.96 (s, IH), 4.48 (s, 2H), 2.92-3.25 (m, 8H).
°γ^0Η ch₃	22	2-{[(3methylbutyl)amino]methyl }p yridine-4carboxylic acid	A	*H-NMR (300MHz, CDjOD), δ ppm: 7.80 (s, IH), 3.90(s, 2H), 2.60(m, 2H), 0.92 (d, 6H).

HO .Ο 0=:/ ch₃	23	2-[({[(2carbamoylethyl)(methyl)carba moyl ] methyl} ami no)methyl]p yridine-4carboxylic acid	B	‘H-NMR (300MHz, MeOHdLi), δ ppm: 7.9 (s, IH), 3.8 (s, 2H), 3.5 (m, 4H), 2.3 (m, 2H).
^ΗΟγ°	24	2-[({2-[2- (hydroxymethyl)piperidin-1 yl]-2oxoethyl} amino)methyl]pyrid ine-4-carboxylic acid	B	'H-NMR (300MHz, MeOH- da), δ ppm: 7.9 (s, IH), 3.9 (s, 2H), 3.5 (m, 2H).
^ΗΟγ° H,c-^Ny^N ch₃	25	2- {[( {methyl [3 -(1 -methyl- 1Himidazol-2yl)propyl]carbamoyl}methyl) amino]methyl}pyridine-4carboxylic acid	B	‘H-NMR (300MHz, MeOH<L): δ 7.7 (s, IH), 6.7 (d, IH), 6.6 (d, IH), 3.7 (s, 2H), 3.5 (s, 3H).
HO O ^H N J	26	2- {[({[( 1 -ethylpyrrolidin-2yl)methyl]carbamoyl} methyl) aminojmethyl} pyridine-4carboxylic acid	B	'H-NMR (300MHz, MeOHdj), δ ppm: 7.7 (s, IH), 3.7 (s, 2H), 3.4 (m, 2H), 3.3 (m, 3H), 1.0 (t, 3H).

HO Ο CH, N.*/ h₃ ³c'	27	2- {[( {methyl [( 1 -methyl-1HpyrazoI-5yl)methyl Jcarbamoyl} methyl) aminojmethyl }pyridine-4carboxylic acid	B	’H-NMR (300MHz, MeOHdj,Ô ppm: 7.7 (s, 1 H), 7.6 (d, IH), 7.3 (d, IH), 6.3 (d, IH), 4.6 (s, 2H), 4.0 (s, 2H).
0 OH M ^H	28	2<{[(3R)-l-(3- phenylpropyl)pyrrolidin-3yljamino} methyl)pyridine-4carboxylic acid	D	—’H-NMR (300MHz, MeOH- dj, δ ppm: 7.80 (s, 1 H), 7.20 (m, 5H), 3.85 (s, 2H).
°γ^ΟΗ ₀-^CH3 ίΧ^.Ό'τΐ H	29	2-{{[({l-[(2methoxyphenyl)methyl]piperi din-4yl}carbamoyl)methyl]amino} methyl)pyridi ne-4carboxylic acid	E	’H-NMR (300MH2, CDjOD), δ ppm: 7.82(s, IH), 7.26(m, 2H), 6.94(m, 2H), 3.90(s, 2H), 3.85(s, 3H), 3.62(s, 2H), 3.24(s, 2H).
°y^OH oaUFO H	30	2-{[({[l-(3- phenyl propyl)piperi din-4yl]carbamoyl} methyljamino] methyl} pyri d ine-4carboxylic acid	E	’H-NMR (300MHz, CDjOD), δ ppm: 7.84(s, IH), 3.92(s, 2H), 3.78(m, 2H), 3.24(s, 2H), 2.96(m, 2H).

°γ^0Η Η	31	2-{[{{[l-(furan-2ylmethyl)piperidin-4yljcarbamoyl }methyl)amino] methyl} pyridine-4carboxylic acid	E	'H-NMR (300MHz, CD₃OD), δ ppm: 7.82(s, IH), 7.64(d, IH), 7.42(s, IH), 6.38(d, IH), 6.30(d, IH), 3.92(s, 2H), 3.56(s, 2H).
°γ^ΟΗ Η	32	2-( {[( {1 -[(5 -phenyl furan-2yl)methyl]piperidin-4yl} carbamoyl )methyl] amino} methyl)pyridine-4carboxylic acid	E	’Η-NMR (300MHz,CD₃OD), δ ppm: 7.82 (s, 1 H),6.70(s, 1 H),6.40(d, 1 H),3 .92(s,2H).
^κογ° cxaZ h₃c^	33	2-[({[(2cyanoethyl)(ethyl )carbamoyl ] methyl }amino)methyl]pyridin e-4carboxylic acid	B	'H-NMR (300MHz, MeOHd4), δ ppm: 7.7 (s, IH), 4.0 (s, 2H), 3.6 (m, 4H), 1.2(t,3H).
°_V ^OH Λ _Η r^^CHs	34	2-( {[2-( 1 -butylpyrroi idin-2yl)ethyl]ammo} methyl)pyridi ne-4-carboxylîc acid	D	Ή NMR (300 MHz, Methanol-dj), δ ppm: 8.00 (s, IH), 4.53 (s, 2H), 3.58-3.13 (m, 6H), 1.01 (t, 3H).

h₃c °γ^0Η O^Jt_N£> H	35	2-{[( {[l-(3,7-dimethyloct-6en-I -yl)pyrrolidin-3yl ]carbamoyl} methyl)amîno] methyl} pyri dine-4carboxylic acid	E	'H-NMR (300MHz, CD₃OD), δ ppm: 7.72(s, IH), 5.10(m, IH), 4.36(m, IH), 3.90(s, 2H), 3.28{s, 2H).
°y^0H F	36	2-{[(3-{[(2fluorophenyl)methyl](methyl) amino} propyl )amino] methyl} pyridine-4carboxylic acid	C	'H-NMR (300MHz, CDjOD), δ ppm: 7.85 (s, IH), 3.90(s, 2H), 2.62(t, 2H), 2.48 (t, 2H).
°γ^ΟΗ UjÎ L CH, n OH H₃C^z	37	2-({[(lR)-2-hydroxy-l{methyl[3-(l -methyl- 1Himidazol-2yl)propyl]carbamoyl} ethyl]a mino} methyl)pyridine-4carboxylic acid	A	'H-NMR (300MHz, CDjOD), δ ppm: 8.32 (s, IH), 7.75(d, IH), 7.35(d, IH), 4.20(s, 2H).
^H0 n Λ P ^s,a-ⁿvAV^h	38	2-[({2-[3-(lH-l,3benzodiazol-2ylmethyl)piperi dîn-1 -yl]-2oxoethyl} amino)methyl]pyrid ine-4-carboxylic acid	B	'H-NMR (300MHz, MeOHd»), δ ppm: 7.9 (s, IH), 7.5 (m, 2H), 7.2 (m, 2H).

Q °γ^0Η ÇuA-û H	39	2-{[({[l-(2- phenylethyl)pyrrolidin-3yl] carbamoyl} methyl)amino] methyl} pyri dine-4carboxylic acid	E	Ή-NMR (300MHz, CD₃OD), S ppm: 8.52(d, IH), 7.80(s, IH), 4.36(m, IH), 3.92(m, IH), 3.28(m, 2H).
Η°γ° O	40	2-( {[3-(4-benzylpiperidin-lyl)propyl]amino}methyl)pyri dine-4-carboxylic acid	C	'H-NMR (300MHz, CD₃OD), δ ppm: 7.85 (s, IH), 7.20(m, 5H), 3.92(s, 2H), 3.35 (s, 2H).
^HOy°	41	2-[({3-[(2phenoxyethyl)amino]propyl} a mino)methyl ]pyridîne-4carboxylic acid	C	‘H-NMR (300MHz, CD₃OD), δ ppm: 7.82 (s, lH),4.18(t, 2H), 2.96(t, 2H), 2.70 (m, 4H).
HO X) Λ . ^έκ3	42	2-[({[methyl({4-[(4methylpiperazin-1 yl)methyl]phenyl }methyl)car bamoyl]methyl} amino)methyl ]pyrîdine-4carboxylic acid	B	'H-NMR (300MHz, MeOHd»), δ ppm: 7.7 (s, IH), 4.4 (s, 2H), 4.0 (s, 2H), 3.6 (s, 2H).

^{H0 0} UOJU/	43	2-( {[2-(2-benzy!pyrrolidm-1 yi)-2oxoethyljamino} methyl )pyrid ine-4-carboxylic acid	B	’H-NMR (300MHz, MeOHd»), δ ppm: 7.7 (s, 1H),7.2 (m, 4H), 4.4 (m, IH), 3.5 (m, 4H), 3.4 (s, 2H).
X » O ch₃ V	44	2-({[({4- [benzyl (cyclopropyl)ami no]b utyl} (methyl )carbamoyl)meth yl]amino} methyl)pyridine-4carboxylic acid	F	‘H-NMR (300MHz, MeOHdi), δ ppm: 7.80 (s, 1 H), 3.90 (s, 2H), 3.70 (s, 2H), 0.50 (m, 2H), 0.40 (m, 2H).
<V^OHίο	45	2-[({2-[(2S)-lbenzylpyrrolidin-2yl]ethyl} amîno)methyl]pyridi ne-4-carboxyIic acid	D	‘H-NMR (300MHz, MeOHdt), δ ppm: 7.74 (s, 1 H), 7.50 (m, 2H), 7.42 (m, 3H), 4.38 (s, 2H).

0	46	2-( {[3-(pyrrolid in-1 - yl)propyl]amino}methyl)pyri dine-4-carboxylic acid	A	‘H-NMR (300MHz, CD₃OD): 5 8.54 (d, IH), 7.86 (s, IH), 7.72 (dd, IH), 3.92 (s, 2H), 3.36 (s, 2H), 2.66 (t, 2H), 2.58-2.50 (m, 4H), 1.90 (s, 2H), 1.83-1.73 (m, 4H).
ο Άοο—ο	47	methyl 2-({[3-{pynOlidin-lyl)propyl]amino} methyl)pyri dine-4-carboxylate	G	’H-NMR (300MHz, CDjOD): 5 8.82 (d, 1 H), 8.03 (s, IH), 7.90 (dd, IH), 4.43 (s, 2H), 3.98 (s, 3H), 3.46-3.36 (m, 6H), 3.22 (t, 2H), 2.29-2.39 (m, 2H), 2.14-2.09 (m, 4H).

Η _Ί C N		48	2-({[4- (dîethylamino)butyl]amino} m	A	^lH NMR (300 MHz,) δ CDjOD 8.54 (dd, IH), 7.85 (s, IH), 7.72 (dd, IH), 3.91 (s,
1	T		ethyl)pyri dine-4-carboxylic		2H), 2.65-2.43 (m, 8H), 1.55 -
HaC^			acid		1.48 (m, 4H), 1.03 (t, 6H).

>h₃ f OH CH₃ o

49

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

B

‘H-NMR (300MHz, CD₃OD): δ 8.55(d, IH), 7.88 (s, IH), 7.73 (d, IH), 3.96 (s, 2H), 3.54 (s,2H), 3.52-3.31 (m,4H), 2.45-2.51 (m, 2H), 2.27 (m ,6H), 1.11-1.19 (m, 3H).

Λ ^H’^cx „ X \ ^ν J γ Hjtr

50

(5-methyl-2-oxo-2H-l ,3dioxol-4-yl)methyl 2-({[4(diethylamino)butyl]amino}m ethyl)pyridine-4-carboxylate

G

’H-NMR (300MHz, CD₃OD): 5 8.80 (m, IH), 7.98 (m, IH), 7.82 (m, 2H), 5.20 (s, 2H), 4.48 (s, 2H), 3.10 - 3.40 (m, 8H), 1.70-1.98 (m, 4H), 1.25- 1.40 (m,6H).

4-methoxyphenyl 2-( {[4(diethylammo)butyl]amino} m ethyl)pyridine-4-carboxylate ‘H-NMR (300MHz, CD₃OD): δ 8.88 (d, IH ),8.13 (s, IH), 8.05 (dd, IH), 7.20-7.15 (m, 2H), 7.01 - 6.96 (m, 2H), 4.53 (s, 2H), 3.81 (s, 3H), 3.27 3.16 (m, 8H), 1.90 -1.85(m, 4H), 1.33 (t, 6H).

2-(ethoxycarbonyl)phenyI 2¢{[4(diethylamino)butyl Jamino} m ethyl )pyridine-4-carboxylate 'H NMR (300 MHz, CD₃OD) δ 8.93 (dd, IH), 8.18 (s IH), 8.15-8.07 (m, 2H), 7.837.62 (m, IH), 7.49 (dd, IH), 7.33 (dd, IH), 4.57 (s, 2H),

4.22 (q, 2H), 3.47 - 2.99 (m, 10H), 2.01-1.73 (m, 2H), 1.34 (td, 9H).

2-(dimethylamino)ethyl 253

G

HjC

({[4(diethylamino)butyl]amino} m ethyl)pyridine-4-carboxylate 'H NMR (300 MHz, CD₃OD) δ 8.83 (d, IH), 8.09 (s, IH),

7.95 (d, IH), 4.76 (t, 2H), 4.10 (s, 2H), 3.65 (ζ 2H), 2.25 (m,

8H), 3.02(s, 6H), 1.85 (m, 4H), 1.36 (t, 6H).

^xch₃

3-(dimethylamino)propyl 2({[4(diethylamino)butyl]amino} m ethyl )pyridine-4-carboxylate 'H NMR (300 MHz, CDjOD) Ô 8.81 (d, IH), 8.02 (s, IH), 7.95 (d, IH), 4.50 (m, 4H), 3.38 (m, 2H), 3.26 (m, 8H), 2.98 (s, 6H), 2.25 (m, 2H), 1.87 (m, 4H), 1.38 (t,6H).

r^a’ ’ X—N \_ch₃

55

{4- {[(ethoxycarbonyl)aminojphe nyl} methyl 2-({[4- (diethylamino)butyl]amino] m ethyl )pyridine-4-carboxylate

G

’H-NMR (300MHz, CD30D): δ 8.70 (d, IH ), 7.90 (s, IH), 7.80 (d, IH), 7.30-7.50 (m, 4H), 5.35 (s, 2H), 4.50 (s, 2H), 3.20 (m, 9H), 1.30 (m, 9H).

2,6-dimethoxyphenyl 2-( {[4(dïethylamino)butyl]amino} m ethyl)pyridine-4-carboxylate

G

^lH NMR (300 MHz, CD3OD): 5 8.89(dd, IH), 8.15 (d, IH), 8.05 (dd, IH), 7.24 (t, IH),

6.77 (d, 2H), 4.53 (s, 2H), 3.79 (s, 6H), 3.29-3.13 (d, 8H), 1.92-1.80 (m, 4H), 1.33 (t, 6H).

2,6-dimethylphenyl 2-( {[4(diethylamino)butyl]amino} m ethyl)pyridine-4-carboxyl ate ’H-NMR (300MHz, CD3OD): δ 8.90 (d, IH ), 8.20 (s, IH), 8.00 (d, IH), 7.2 (s, 3H), 4.50 (s, 2H), 2.20 (s, 6H), 1.80 (m, 4H), 1.30 (m, 6H).

CH, 0 k t CHj 0

58

4-methoxyphenyl 2-{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl} methyl)amino]meth yl }pyridine-4-carboxy!ate

G

’H-NMR (300MHz, CD3OD): S 8.90(d, IH), 8.10(s, IH), 8.05 (d, IH), 7.10 (m,2H), 7.00 (m, 2H), 4.50 (s, 2H), 4.30 (s, 2H), 3.80 (s, 3H), 3.30 (m, 6H), 2.95 (s, 6H), 1.20 (t, 3H)

CH,

2-(ethoxycarbonyl)phenyI 2{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl} methyl )amino]meth yl} pyridine-4-carboxylate ‘H-NMR (300MHz, CD3OD): δ 8.90(d, IH), 8.20 (s, IH), 8.05 (d, IH), 7.75 (m, IH), 7.45 (m, IH), 7.30 (m, IH), 4.60 (s, 2H), 4.30 (s, 2H), 3.80 (m, 2H), 3.00 (s, 6H), 1.25 (m, 6H)

\^ch₃

60

{4[(ethoxycarbonyl)(methyl)ami no]phenyl} methyl 2-( {[4(d iethylamino)butyl Jamino} m ethyl)pyridine-4-carboxylate

G

’H-NMR (300MHz, CD3OD): δ 8.80 (d, IH ), 8.00 (s, IH), 7.90 (d, IH), 7.50 (d, 2H), 7.30 (d, 2H), 5.40 (s, 2H), 4.50 (s, 2H), 4.20 (m, 2H), 1.80 (m, 4H), 1.30 (m,6H), 1.20 (t, 3H)

H₃C

61

4-rert-butylphenyl 2-{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl} methyl)amino]meth yl} pyridine-4-carboxylate

G

’H-NMR (300MHz, CD3OD): 5 8.80 (d, IH ), 8.10 (s, IH), 8.00 (d, IH), 7.50 (d, 2H), 7.20 (d, 2H), 4.55 (s, 2H), 4.30 (s, 2H), 3.80 (m, 3H), 2.99 (s, 6H), 1.40 (s, 9H), 1.20 (t, 3H)

CH-, 0 Ν^ζΧΧ> Χ·^_ΝΛ>Χυγ°γγ“> 1 0 CHj 0 ^xch₃

62

4-oxopentan-2-yl 2-{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl} methyl)amino]meth yl }pyridine-4-carboxy!ate

H

’H-NMR (300MHz, CD3OD): 5 8.75 (d, IH), 7.90-8.10 (m, 2H), 5.50 (m, IH), 4.50 (s, 2H), 4.25 (s, 2H), 3.75 (m, 2H), 1.20-1.50 (m, 6H).

4-( tri fluoroacetamido)butan2-yl 2-{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl }methyl)amino]meth yl }pyridine-4-carboxylate 'H-NMR (300MHz, CDjOD): 5 8.80 (d, IH), 7.98 (brs, IH), 7.9 (d, 2H), 5.20 (m, IH), 4.9 (s, 2H), 4.5 (s, 2H), 4.3 (s,

2H), 3.8 (m, 2H), 3.5-3.3 (m,

6H), 3.0 (s, 3H), 2.0 (m, 2H),

1.3 (d, 3H), 1.2 (t, 3H).

F F F Z» 0^ ^CH3 ⁰

64

4-(2,2,2-trifluoro-Nmethylacetamido)butan-2-yI 2-{[({[2(dimethyl amino)ethyl ](ethyl)c arbamoyl} methyl)ami no] meth yl} pyri dine-4-carboxylate

G

‘H-NMR (300MHz, CD₃OD): δ 8.80 (d, IH), 8.0 (m, 2H), 5.20 (m, IH), 4.9 (s, 2H), 4.5 (s, 2H), 4.3 (s, 2H), 3.8 (m, 2H), 3.5-3.3 (m, 6H), 3.2 (s, 3H), 3.0 (s, 6H), 2.0 (m, 2H), 1.3 (d, 3H), 1.2(t,3H).

ethyl 2-{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl} methyl)ami no]meth yl} pyridîne-4-carboxylate

General

Procedure

B

’H-NMR (300MHz, CDC1₃): δ

8.7 (d, IH), 7.9 (s, IH), 7.8 (d,

IH), 4.4 (q, 2H), 4.0 (s, 2H),

3.5-3.3 (m, 4H), 3.2 (m, 2H),

2.5 (m,4H), 2.3 (s, 6H), 1.3 (t, 3H), 1.1 (t, 3H).

CH₃ 0 _F 'ch, 'xh,

66

5-(trifluoroacetamido)pent-len-3-yl 2-{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl} methyl)amino]meth yl}pyridine-4-carboxylate

G

'H-NMR (300MHz, CD,OD): Ô 8.84(d, IH), 8.02 (s, IH), 7.96 (d, IH), 6.04-5.93 (m, IH), 5.38 (d, IH), 5.29 (d, IH), 2.98 (s, 6H), 1.25 (t,3H).

CH₃ 0 CHj _F Vh, ⁰

67

5-(2,2,2-trifluoro-Nmethyl acetamido)pent-1 -en-3yl2-{[({[2(dimethylamino)ethyl](ethyl)c arbamoyl} methyl)amino] meth yl }pyridine-4-carboxylat

G

’H-NMR (300MHz, CD₃OD): δ 8.85 (d, IH), 8.02 (s, IH), 7.96 (d, IH), 6.05-5.93 (m, IH), 5.39 (d, IH), 5.29 (d, IH), 3.40 (m, 3H), 1.25 (t, 3H).

Below

68

2-(2-{[({[2- (dimethylamino)ethyl](etliyl) carbamoyl} methyl)amino]me thyl}pyridine-4carbonyloxy)-3(hexadecanoyloxy)propyl hexadecanoate

G

‘H-NMR (300MHz, CD3OD): δ 8.85 (d, IH), 8.05 (s, IH), 7.90 (d, IH), 5.50 (m, IH), 4.50 (s, 2H), 3.80 (m, 2H), 2.98 (s, 6H), 1.30 (m, 57H), 0.90 (m, 6H)

--4 oo

l-(2-{[({[2(dimethylamino)ethyl]( ethyl)carbamoyl} methy I)amino]methyl} pyridi ne-4-carbonyloxy)-3 (hexadecanoyloxy)prop an-2-yl hexadecanoate 'H-NMR (300MHz, CD3OD): Ô 8.80 (d, IH ), 8.05 (s, IH),

7.90 (d, 1 H), 4.50 (s, 2H),

4.40 (τη, 2H), 4.20 (s, 3H),

3.00 (s, 6H), 1.20-1.50 (m,

73H).

CHj 0 N '''CH, ^CH,

70

methyl 2-{[({[2(dimethylamino)ethyl]( ethyl )carbamoyl }methy l)amino]methyl} pyridi ne-4-carboxylate

G

’H-NMR (300MHz, CD3OD): δ 8.80 (d, IH ),8.10 (s, IH), 8.00 (d, IH), 4.70 (s, 2H), 4.40 (s, 2H), 4.00 (s, 3H), 3.00 (s, 6H), 1.25 (t, 3H).

CH. 0 k Λ/ C'S H,C^ 'p' //''CH,

71

2-((( {[2- (di methylamino)ethyl]( ethyl )carbamoyl} methy l)amî no] methyl} -Nmethanesulfonyl-Nmethylpyridine-4carboxamide

G

’H-NMR (300MHz, D₂O): δ 8.71 (d, IH),7.61 (s, IH), 7.58 (m, IH), 4.48 (s, 2H ), 4.20 (s, 2H), 3.70 (t, 2H), 3.41 (s, 3H), 3.30-3.24 (m, 4H), 3.20 (s, 3H), 2.86 (s, 6H), 1.10 (t, 3H).

CH₃ 0 „_ίο'''·^..Α/^ίίΧΧγ^ο S», X'J/'

72

N-[2(dimethylamino)ethyl]N-ethyl-2-( {[4-(2-oxol,3-oxazolidine-3carbonyl)pyridin-2yljmethyl) amino)aceta mide

G

‘H-NMR (300MHz, D₂O): δ 8.66 (d, IH ), 7.58-7.46 (m, 2H), 4.46 (s, 2H), 4.19 (s, 2H), 3.79 (t, 2H), 3.73-3.67 (m, 2H), 3.31-3.25 (m, 2H), 3.17-3.11 (m,2H), 3.03-2.94 (m, 2H), 2.86 (s, 6H), 1.121.07 (m, 3H).

H₃C CHj CHj 0 N H₃C ^NH A, F

73

propan-2-yl 3-(2{[({[2(dimethylamino)ethyl]( ethyl)carbamoyl} methy l)amino]methyl}pyridi ne-4-carbonyloxy)-4(trifluoroacetamido)but anoate

G

^!H-NMR (300MHz, CD₃OD): 6 8.83 (d, IH), 7.96 (s, IH), 7.90 (d, IH), 5.65 (m, IH), 4.56 (s, 2H), 4.26 (s, 2H), 3.00 (s, 6H), 1.30—1.16 (m, 9H).

propan-2-yl 3-(2{[({[2(dimethylamino)ethyl]( ethyl )carbamoyl} methy l)amino] methyl} pyri di ne-4-carbonyloxy)-5(trifluoroacetamido)pen tanoate 'H-NMR (300MHz, CDjOD):

8.83 (m, IH), 7.99 (s, IH),

7.93 (d, IH), 4.95 (m, IH),

4.56 (d, 2H), 4.38(d, 2H), 4.27 (s, 2H), 3.80 - 3.69 (m, 4H),

3.00 (s, 6H), 1.40-1.36 (m,

9H).

CHj O k ^T U

75

2-{[({[2(dimethylamino)ethyl]( ethyl )carbamoyl} methy l)amino] methyl} -N(pyridin-4-yl)pyridine4-carboxamide

G

'H-NMR (300MHz, CDjOD): 6 8.73 (d, 2H), 8.44 (d, 2H), 8.02 (d, 2H), 6.85 (d, IH), 4.62 (s, 2H), 4.30 (s, 2H), 3.83 (t, 2H), 3.33-3.31 (m, 4H), 2.99 (s, 6H), 1.26 (t, 3H).

/^CHî N _HC f i Χλ-» ch₃ o

76

2-{[<{[2- (dîmethylamino)ethyl]( ethyl)carbamoyl }methy l)amino]methyl}-N-(5methyl-1,3,4oxadiazol-2yl)pyridine-4carboxamide

G

’Η-NMR (300MHz, CD₃OD): 5 8.84 (d, IH), 7.99 (s, IH), 7.94 (d, IH), 4.58 (s, 2H), 4.28 (s, 2H), 3.84-3.79 (m, 2H), 3.43-3.36 (m, 2H), 3.32-3.28 (m, 2H), 2.98 (s, 6H), 2.52 (s, 3H), 1.27-1.22 (m, 3H).

/^CH3 ^Χ,/ Ç ΗΝ N ^H3^C\ ^CH3 N >< N TT ^O i il ^H 1 ch₃ 0

77

2-{[({[2(dimethylamino)ethyl]( ethyl)carbamoyl }tnethy l)amino]methyl} -N-( 1 methyl-1 H-pyrazol-5yl)pyridine-4carboxamide

G

'H-NMR (300MHz, CDjOD): δ 8.84 (d, IH), 7.96 (s, IH), 7.91 (d, IH), 7.56 (d, IH), 6.40 (d, IH), 4.59 (s, 2H), 4.28 (s, 2H), 3.84-3.80 (m, 2H), 3.41-3.35 (m, 4H), 3.82 (s, 3H), 2.98 (s, 6H), 1.26-1.21 (m, 3H).

^HO ° o	78	2-(piperidin-lyl methyl)pyri dine -4carboxylic acid	A	'H-NMR (300MHz, CD3OD): δ 8.50 (d, IH ), 7.40 (s, IH), 7.65 (d, IH), 3.65 (s, 2H), 2.50 (m, 4H), 1.60 (m, 4H), 1.40 (m,2H)
HO Λ	?
C N	N Q	79	2-(azetidin-lylmethyl )pyridine-4carboxylic acid	A	'H-NMR (300MHz, CD3OD): δ 8.25 (d, IH ), 7.50 (s, IH), 7.40 (d, IH), 3.50 (s, 2H), 3.10 (m, 4H), 1.90 (m, 2H) ppm.

CHj Ο N'Xi _F k ⁰

80

2,2,2-trifluorœthyl 2{[({[2(dimethylamino)ethyl]( ethyl )carbamoyl} methy l)amino] methyl Jpyridi ne-4-carboxylate

G

'H NMR (300 MHz, DMSO06)8 8.89(0, IH), 8.04 (s, IH), 7.91 (d, IH), 5.09 (q, 2H), 4.49 (s, 2H), 4.16 (s, 2H), 3.68 (t, 2H), 3.27 (q, 2H), 2.85 (s, 6H), 1.13 (t, 3H).

OH ^YV'rA ° <ch₃ ^N^

81

2 A {ethyl [2-oxo-2(piperidin-1yl)ethyl]amino} methyl) pyridine-4-carboxylic acid

I

‘H NMR (300 MHz, CD,OD) δ 8.48 (d, IH), 7.94 (s, IH), 7.70 (d, IH), 3.80 (s, 2H), 3.47 (m, 4H), 3.36 (s, 2H), 2.63 (q, 2H), 1.54 (m,6H), 1.10 (t, 3H).

r Ο Ν ?'

82

2-( {butyl[2-oxo-2(piperidin-1yl)ethyl] amino} methyl) pyri dine-4-carboxylic acid

I

'H NMR (300 MHz, CD₃OD) Ô 8.50 (d, IH), 7.94 (s, IH), 7.71 (d, IH), 3.80 (s, 2H), 3.46 (m, 4H), 3.35 (s, 2H), 2.54 (m, 2H), 1.51 (m, 8H), 1.28 (m, 2H), 0.88 (t, 3H).

À

83

2-( {benzyl [2-oxo-2(piperidtn-1yl)ethyl]amino} methyl) pyridine-4-carboxylïc acid

I

'H NMR (300 MHz, CD₃OD) δ 8.49 (d, IH), 8.05 (s, IH), 7.72 (d, IH), 7.48-7.08 (m, 5H), 3.85 (s, 2H), 3.71 (s, 2H), 3.43 (m, 2H), 3.31 (m, 4H), 1.73-1.33 (m, 6H).

,, x> f HN 0 ch₃ o

84

2-{[({[2(dimethylamino)ethyl]( ethyl)carbamoyl} methy l)amino]methyl}-N(l,3-oxazol-2yl)pyridîne-4carboxamide

G

‘H-NMR (300MHz, CD₃OD): 8 8.76 (d, IH), 7.94 (s, IH), 7.89 (d, IH), 7.65 (s, IH), 7.50 (s, IH), 4.51 (s, 2H), 4.21 (s, 2H), 3.77-3.73(m, 2H), 3.37- 3.23 (m, 4H), 2.92 (s, 6H), 1.21-1.16 (m, 3H).

CHj CH, 0 < Il H II I j< JL x JL ^JL -.o,. JL h,c^ \/ΧίΧ\/ \zJ\ L ° ^CH, Cr ----- CH,	85	2,6-bis(propan-2yloxy)phenyl 2-{[({[2(dimethylamino)ethyl]( ethyl)carbamoyl} methy l)ami nojmethyl} pyridi ne-4-carboxylate	G	¹ H-NMR (300MHz, CD,OD): δ 8.94 (m, IH), 8.16 (s, IH), 8.11 (d, IH), 7.18 (t, IH), 6.75 (d, 2H), 4.60 (m, 2H), 4.30 (s, 2H), 3.00 (s, 6H), 1.30-1.00 (m, 15H).
,---N O H₃C--/ \-- CHq PJ \ O	86	2-{[(2- methylpropyl)[2-oxo-2(piperidin-1yl ) ethyl ] aminojmethyl} pyri dine-4-carboxylic acid	I	‘H NMR (300 MHz, CD₃OD) 5 8.48 (d, 1 H), 7.99 (s, IH), 7.71 (d, IH), 3.79 (s, 2H), 3.55 - 3.37 (m, 4H), 3.32 (s, 2H), 2.34 (d, 2H), 1.82 (m, IH), 1.56 (m, 6H), 0.88 (d, 6H).

H.C 0 /-~---\	87	2-({[2-oxo-2(piperidin-1yl)ethyl](propyl)amino } methyi)pyridine-4carboxylic acid	I	'H NMR (300 MHz, CD₃OD) 5 8.48 (d, IH), 7.97 (s, IH), 7.71 (d, 1 H), 3.80 (s, 2H), 3.47 (m, 4H), 3.35 (s, 2H), 2.692.26 (m,2H), 1.75- 1.45 (m, 8H), 0.87 (t, 3H).
° /k h₃c ^ch₃	88	2-({[2-oxo-2(piperidin-1yl)ethyl](propan-2yl)amino} methyîjpyrid ine-4-carboxylic acid	I	'H NMR (300 MHz, CDjOD) δ 8.45 (d, IH), 8.00 (s, IH), 7.69 (d, IH), 3.76 (s, 2H), 3.45 - 3.37 (m, 4H), 3.34 (s, 2H), 3.12-2.93 (m, IH), 1.73 1.36 (m, 6H), 1.13 (d, 6H).

a) < HN N ΊΟ+ΥηΡ ch₃ ο ^Ν<^χ^

89

2-{[({[2- (di methylami no)ethyl]( ethyl)carbamoyl} methy l)amino]methyl}-N-(lmethyl-1 H-imidazol-2yl)pyridine-4carboxamide

G

¹ H-NMR (300MHz, CD₃OD): δ 8.82 (s, IH ),7.93 (s, IH), 7.34 (s, IH), 7.29 (s, IH), 6.76 (s, IH), 3.82-3.73 (m, 4H), 3.46-3.43 (m, 2H), 3.38-3.30 (m, 4H), 3.26 (s, 3H), 2.93 (s, 6H), 1.27-1.12 (m,3H).

2-fluoroethyl 2- {[( {[2(dimethylamino)ethyl]( ethyl)carbamoyl} methy l)amino]methyl} pyridi ne-4-carboxylate 'H-NMR (300MHz, CDC1₃): δ

8.72 (dd, IH ), 7.96 (d, IH),

7.73 (td, IH), 4.45 (td, IH), 4.04-4.03 (m, 2H), 3.95 (s,

2H), 3.52-3.49 (m, 4H), 3.523.49 (m, 4H), 3.47-3.38 (m, 2H), 2.52-2.39 (m, 3H), 2.30 (s, 6H), 1.17-1.11 (m, 3H).

^^CH3 ^CH3 ^{0 F}

91

2,2-difluoroethyl 2{[({[2(dimethylamino)ethyl]( ethyljcarbamoyl} methy l)amino]methyl} pyri di ne-4-carboxyIate

G

^lH-NMR (300MHz, CDClj): 5 8.72(d, IH),7.98 (s, IH), 7.76 (dd, IH), 6.09 (m, IH), 4.82 (m, IH), 4.68M.63 (m, 2H), 4.55 (m, IH), 4.04 (s, 2H), 3.50 (s, 2H), 3.49-3.38 (m, 2H), 3.31-3.24 (m, 2H), 2.29 (s, 6H), 1.18-1.11 (m, 3H).

H q C N ——\ K H₃c Y.uiiNH \----( ^hn—Z λ—^oh ^: \> F h,A^CH’

92

2-({[(lS)-l-(tert- butylcarbamoyl)-3methylbutyl]amino}me thyl)pyridine-4carboxylic acid

B

'H NMR (300 MHz, CD₃OD) 8 8.51 (d, IH), 7.87 (s, IH), 7.71 (d, IH), 3.84 (d, 2H), 3.31 (s, IH), 3.11 (dd, IH), 1.82-1.64 (m, IH), 1.53- 1.40 (m,2H), 1.32 (s, 9H), 0.90 (dd, 6H).

	2-{ {methyl[(2S)-4methyl-1-oxo-1-
93	(piperidin-1 -yl)pentan-
2-
	yl]amino}methyl)pyrid
	ine-4-carboxylic acid

‘H NMR (300 MHz, CD₃OD) δ 8.46 (d, IH), 7.96 (s, IH), 7.70 (d, IH), 3.98-3.65 (m,

3H), 3.57 (m, 3H), 2.81 (s, IH), 2.28 (s, 3H), 1.95-1.79 (m, IH), 1.76-1.41 (m, 6H),

0.93 (dd, 6H).

100

Illustrative préparations of compounds within Table 1 are as follows.

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

Synthctic Route A

(a)

General Procedure A

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

General Procedure A (Ester hydrolysis)

The ester (Ethyl 2-({[2-(dimethylamino)ethyl]amino}methyl)pyridine-4-carboxylate (a)) was dissolved in MeOH-THF-H₂O (1:1:1) and LiOH (1.0 equiv) was added. The reaction mixture was stirred at room température and monitored by TLC. Solvents were removed in vacuo. Worked up by dissolving in water 10 and extract with Et₂O. The aqueous basic layer was acidified with IN HCl to pH 1, and the solution was concentrated to dryness to afford the hydrochloric acid sait of title compound as a colorless solid.

'H NMR (300 MHz, Methanol-d₄), δ ppm: 8.82 (dd, IH), 8.03 (s, IH), 7.94 (dd, IH), 4.58 (s, 2H), 3.63 (m, 4H), 3.0 (s, 6H).

ES-MS: 224 [M+l].

101

2-({[2-oxo-2-(pyrrolidin-l-yl)ethyl]amino}methyl)pyridine-4-carboxylic acid (#13)

Synthetic Route B

General Procedure B

----------► (b)

General Procedure C (C)

General Procedure E (d) -----------►

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

Prepared by General Procedure A from ethyl 2-({2,2,2-ΐπί1υοΓθ-Ν-[2-οχο-2-(ργττο1ϊόιη-1yl)ethyl]acetamido}methyl)pyridine-4-carboxylate (e), Worked up by trituration of the solid residue with EtîO to give hydrochloric acid sait of the title compound as white powder.

'H-NMR(300MHz, MeOH-d₄): δ 8.4 (d, IH), 7.7 (s, IH), 7.6 (d, IH), 3.7 (s, 2H), 4.0 (s, 2H), 3.4 (m, 6H), 10 1.5 (m, 4H),

2-{[(3-{|3-(pyrrolidin-l-yl)propyl]aniino}propyl)amino|methyl}pyridine-4-carboxylic acid (#21)

102

Synthetic Route C

General Procedure C

-----►

General Procedure A (g) -----------►

General Procedure F (h) --------------► (j)

General Procedure B

---------► G)

General Procedure A

-----------►- Title Compound

Prepared by General Procedure A from ethyl 2-{[2,2,2-trifluoro-N-(3-{[3-(pyrrolidin-lyl)propyl]amino}propyl)acetamido] methyl}pyridine-4-carboxylate (j). Work up yielded hydrochloric acid sait of the title compound white sticky solid.

IH NMR(300 MHz, D2O): δ 8.72 (d, IH), 7.96 (s, IH), 7.90 (d, IH), 4.48 (s, 2H), 3.60 (m, 4H), 2.92-3.25 (m, 8H), 1.84-2.25 (m, 8H).

ES-MS: 321.40 [M+H].

2-({[(3R)-l-(3-phenylpropyl)pyrrolidin-3-yl]amino}methyl)pyridine-4-carboxylic acid (#28)

103

Synthetic Route D

ΙΙΊ	0	General Procedure B	General Procedure C
(k)	---------► (D

General Procedure D -----►	General Procedure B (m) ---------►	General Procedure A (n) ------------Title Compound

Prepared by General Procedure A from ethyl 2-({2,2,2-trifluoro-N-[(3R)-l-(3-phenylpropyl)pyrrolidin-3yi]acetamido)methyl)pyridine-4-carboxylate (n). Isolated as lithium sait 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.

'H-NMR (300MHz, CDClj): δ 8.50 (d, l H), 7.80 (s, 1 H), 7.65 (d, 1 H), 7.20 (m, 5H), 3.85 (s, 2H), 3.1 -2.4 (m, 8H), 2.25 (m, IH), 2.20 (m, IH), 1.90-1.55 (m, 3H).

ES-MS: 340 [M+1J.

2-({[({l-[(2-methoxyphenyl)tnethyl]piperidin-4-yl}carbamoyl)methyl]amino}methyl)pyridine-415 carboxylic acid (#29)

104

Synthetic Route E

General Procedure E

----------► (o)

General Procedure D

----------► (P)

General Procedure B

-----►

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

Prepared by General Procedure A from ethyl 2-({2,2,2-trifluoro-N-[({l-[(2methoxyphenyl)methy 1 ] piperidi n-4 -yl} carbamoyl)methyl ] acetami do} methyl)pyridine-4-carboxylate (q) using 2 équivalents of LiOH. Isolated as lithium sait 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.

'H-NMR (300MHz, CD₃OD): δ 8.54(d, I H), 7.82(s, 1 H), 7.65(d, 1 H), 7.26(m, 2H), 6.94(m, 2H), 3.90(s, 2H), 3.85(s, 3H), 3.72(m, 3H), 3.62(s, 2H), 3.24(s, 2H), 2.94(m, 2H), 2.24(m, 2H), 1.90(m, 3H), 1.60(m, 10 2H).

ES-MS: 411 [M-H],

2-({[({4-|benzyI(cyclopropyl)amino]butyl}(mcthyi)carbamoyl)mcthyl]amino}mcthyl)pyridinc-4carboxylic acid (#44)

105

Synthetic Route F

(d)

General Procedure E

-----►

General Procedure F (r) -----------► (s)

General Procedure B

------------► (t)

General Procedure 8

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

Prepared by General Procedure A from ethyl 2-({N-[( {4benzyl(cyclopropyl)amino]butyl}(methyl)carbamoyl)mcthyl]-2,2,2-trifluoroacetamido}methyl)pyridine-4carboxylate (u) using 2.5 équivalents of LiOH. Isolated as lithium sait 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.

'H-NMR (300MHz, CDClj): δ 8.50 (d, IH), 7.80 (s, IH), 7.70 (d, IH), 7.25 (m, 5H), 3.90 (s, 2H), 3.70 s, 2H), 3.50-3.40 (m, 3H), 2.25 (m, IH), 2.95 (s, 3H), 2.55 (m, 2H), 1.75 (m, IH), 1.50 (m, 4H), 0.50 (m, 2H), 0.40 (m, 2H).

ES-MS: 425 [M+l].

2-{[({[2-(diniethylamino)ethyl|(ethyl)carbamoyl}methyl)amino]methyl}-N-(pyridin-4-yl)pyridinc-415 carboxamide (#75)

106

General Procedure G

-----►

General Procedure A (V) -----------► (x)

General Procedure E or General Procedure H

-----► (y)

General Procedure D

-----------►· Title Compound

Prepared by general procedure D from tert-butyl N-({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl)5 N-({4-[(pyridin-4-yl)carbamoyl]pyridin-2-yl}methyl)carbamate (y) to get the title compound as yellow oil.

'H-NMR(300MHz, CD₃OD): δ 8.73 (d, 2H), 8.44 (d, 2H), 8.02 (d, 2H), 6.85 (d, IH), 4.62 (s, 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]

107

4-oxopentan-2-yl 2-{[({[2-(dimcthylaniino)ethyl](ethyl)carbamoyl}methyl)amino]methyl}pyridine-4 carboxylate (#62)

Synthetic Route H

CK Ο. OH,
a	Synthetic Route G -	(X)	General Procedure E or General Procedure H	(z)
		-
0	General Procedure F -----►	(aa)	General Procedure D	Title Compound
5		-----------►-

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

'H-NMR (300MHz, CD3OD): δ 8.75 (d, IH ), 7.90-8.10 (m, 2H), 5.50 (m, IH), 4.50 (s, 2H), 4.25 (s, 2H),

3.75 (m,2H), 1.20-1.50 (m, 6H).

108

ES-MS: 393 [M+l]

2-({ethyl[2-oxo-2-(piperidin-l-yl)ethyI]amino}methy])pyridine-4-carboxy)ic acid (#81)

General Procedure B

General Procedure B (a b) ---------------(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.

'H NMR (300 MHz, CD₂OD) δ 8.48 (d, 1 H), 7.94 (s, 1 H), 7.70 (d, 1 H), 3.80 (s, 2H), 3.47 (m, 4H), 3.36 (s, 10 2H), 2.63 (q, 2H), 1.54 (m,6H), 1.10 (t, 3H).

109

ES-MS: 306 [M+l]

Intermediates

Ethyl 2-({[2-(dimethylamÎno)ethyI]amino}niethyI)pyridine-4-carboxylate (a)

General Procedure B (Reductive amination)

To a mixture of aldéhyde (ethyl 2-formylpyridine-4-carboxylate) (1.0 equiv), and amine (N1,N1dimethylethane-1,2-diamine) (1.0 equiv), in 1,2-dichloroethane was added AcOH (1.0 equiv), followed by NaBH(OAc)j (2.5 equiv), and the mixture was stirred at room température, ovemight. Aqueous work up (EtOAc/NaHCOj) and purification by column chromatography (CH2C12/MeOH/NH(OH, 90:10:1) to yield the title compound as a colorless glue.

IHNMR(300 MHz, Methanol-dq): δ 8.68 (dd, IH), 7.87 (s, IH), 7.67 (dd, IH), 4.34 (q, 2H), 3.87 (s, 2H), 2.58 (t, 2H), 2.33 (t, 2H), 2.11 (s, 6H), 1.32 (t, 3H).

Ethyl 2-({[2-(tert-butoxy)-2-oxoetliyllamino}methyl)pyridine-4-carboxylate (b)

Prepared by General Procedure B from ethyl 2-formylpyridine-4-carboxylate and tert-butyl 2aminoacetate. Title compound isolated as yellow oil by column chromatography (EtOAc/hexanes).

‘H-NMR (300MHz, MeOH-d₄): δ 8.7 (d, 1 H), 7.8 (s, 1 H), 7.7 (d, 1 H), 4.4 (s, 2H), 4.3 (q, 2), 3.8 (s, 2H), 3.3 (s, 2H), 1.4 (s, (H), 1.3 (t, 3H)

ES-MS: 295 [M+l].

Ethyl 2-{[(3-hydroxypropyl)amino|methyl}pyridine-4-carboxylate (f)

Prepared by General Procedure B from ethyl 2-formylpyridine-4-carboxylate and 3-aminopropan-l-ol.

Ή NMR (300 MHz, CDClj), δ ppm: 8.72 (d, IH), 7.98 (s, IH), 7.75 (d, IH), 4.45(q, 2H), 3.95 (s, 2H), 3.70 (t, 2H), 2.80 (t, 2H), 1.75 (m, 2H), 1.40 (t, 3H).

110

Ethyl 2-({[(3R)-l-[(tcrt-butoxy)carbonyl]pyrrolidin-3-yl]amino}methyl)pyridÎne-4-carboxylate (k)

Prepared by General Procedure B from ethyl 2-formylpyridine-4-carboxylate and tert-butyl (3R)-3aminopyrrolidine-1-carboxylate. Coiumn chromatography (MeOH/DCM) gave the title compound as greenish oil.

'H-NMR(300MHz, CDC1₃): δ 8.70 (d, 1 H), 7.86 (s, 1 H), 7.74 (d, IH), 4.50 (q, 2H), 4.00 (s, 2H), 3.60-3.33 (m 4H), 3.25 (m, IH), 2.09 (m, IH), 1.90-1.72 (m, 2H), 1.46 (s, 9H), 1.41 (t, 3H).

ES-MS: 350 [M+l]

Ethyl 2-{[2,2,2-trinuoro-N-(3-{[3-(pyrrolidin-l-yl)propyl]amino}propyl)acetamido|methyl}pyridine4-carboxylate (j)

Prepared by General Procedure B from ethyl 2-{[2,2,2-trifluoro-N-(3oxopropyl)acetamido]methyl}pyridine-4-carboxylate (i) and 3-(pyrrolidin-l-yl)propan-l-amine. Coiumn chromatography (MeOH/DCM) gave the title compound.

'H NMR (300 MHz, CD₃OD): δ 8.66 (dd, 1 H), 7.95 (s, 1 H), 7.78 (dd, 1 H), 4.42 (q, 2H), 3.98 (s, 2H), 3.56 (m, 4H), 2.42-2.75 (m, 8H), 1.86 (m, 8H), 1,40 (t, 3H).

Ethyl 2-{|N-({|4-(cyclopropylamino)butyl](methyl)carbamoyi}mcthyl)-2,2,2trifluoroacetamido]methyl}pyridine-4-carboxylatc (t)

Prepared by General Procedure B from ethyl 2-[(2,2,2-trifluoro-N-{[methyl(4oxobutyl)carbamoyl]methyl}acetamido)methyl]pyridine-4-carboxylate (s) and cyclopropylamine. Coiumn chromatography (EtOAc/hexanes) gave the title compound as yellow oil.

'H-NMR (300MHz, CDC1₃): δ 8.70 (dd, IH), 7.85 (ss, IH), 7.74 (dd, IH), 4.95 (dd, 2H), 4.40 (q, 2H), 4.32 (ss, 2H), 3.31 (m, IH), 3.25 (m, IH), 2.90 (s, 3H), 2.73 (m, IH), 2.50 (m, IH), 2.01 (m, IH), 1.85-1.30 (m, 8H), 0.53.034 (m, 4H).

111

ES-MS: 459 [M+l]

Ethyl 2-({2,2,2-triiluoro-N-[(3R)-l-(3-phenylpropyl)pyrrolidîn-3-yl)acetamido}methyl)pyridine-4carboxylate (n)

Prepared by General Procedure B from 3-phenylpropanal ethyl 2-((2,2,2“trifluoro-N-[(3R)-pyrrolidin-3yl]acetamido] methyl)pyridine-4-carboxylate (m).

’H-NMR (300MHz, CDClj): 8 8.70 (d, IH), 7.80 (s, IH), 7.72 (dd, IH), 7.24 (m, 5H), 4.95 (q, 2H), 4.37 (m, 2H), 3.78 (m, IH), 2.60 (m, 3H), 2.46 (m, 2H), 2.32 (m, 2H), 2.21 (m, IH), 2.04 (m, 2H), 1.88 (m, IH), 1.73 (m, IH), 1.43 (t,3H).

Ethyl 2-((2,2,2-tnfluoro-N-[({l-[(2-methoxyphenyl)methyl|piperidin-4yl}carbamoyl)methyI|acetami(lo}methyl)pyridine-4-carboxylate (q)

Prepared by General Procedure B from 2-methoxybenzaldehyde and ethyl 2-[(2,2,2-trifluoro-N{[(piperidi n-4-y 1 Jcarbamoy 1 ] methyi} acetamido)methy ljpyrid ine-4-carboxylate (p).

'H-NMR (300MHz, CDC1₃): 8 8.75 & 8.71 (2d, 1 H; rotamer ), 7.91 - 7.78 (m, 2H), 7.36 (m, IH), 7.25 (m, IH), 7.99 - 7.86 (m, 2H), 4.95 & 4.72 (2s, 2H, rotamer), 4.45 (q, 2H), 4.30 & 4.08 (2s, 2H; rotamer), 3.83 (m, 4H), 3.60 (m, 2H) 2.95 (m, 2H), 2.22 (m, 2H), 1.90 (m, 2H), 1.60 (m, 2H), 1.40 (t, 3H).

Ethyl 2-({N-[({4-[benzyl(cyclopropyl)amino]butyl}(methyl)carbamoyl)mcthyI]-2,2,2trifluoroacetamido}methyl)pyridine-4-carboxylate (u)

Prepared by General Procedure B from benzaldehyde and ethyl 2-{[N-(([4(cyclopropylamino)butyl](methyl)carbamoyl}methyl)-2,2,2-trifluoroacetamido]methyl)pyridine-4carboxylate (t).

*H NMR (300 MHz, CD₃OD): 8 8.70 (m, 1 H), 7.83 (m, 1 H), 7.77 (m, 1 H), 7.30 (m, 5H), 4.83 (dd, 2H), 4.40 (m, 3H), 4.23 (m, IH), 3.65 (m, IH), 3.31 (m, IH), 3.19 (m, IH), 3.01 (m, IH), 2.91 (m, 3H), 2.49 (m, 2H), 1.70 (m, IH), 1.40 (m, 7H), 0.50 (m, 2H), 0.45 (m, 2H).

112

Ethyl 2-({[2-oxo-2-(piperidin-l-yl)ethyl]amino}mcthyI)pyrïdine-4-carboxylate (ac)

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

*H NMR (300 MHz, CDClj) δ 8.67 (m, IH), 8.04 (m, IH), 7.71 (m, IH), 4.40 (q, 2H), 3.89 (s, 2H), 3.46 (m, 4H), 2.68 (q, 2H), 1.77 - 1.33 (m, 6H), 1.40 (t, 3H), 1.08 (t, 3H).

Ethyl 2-({ethyl[2-oxo-2-(piperidin-l-yl)ethyi]amino}methyl)pyridine-4-carboxylate (ab) 'H NMR (300 MHz, CDClj) δ 8.68 (d, IH), 7.93 (s, IH), 7.71 (d, IH), 4.40 (q, 2H), 4.02 (s, 2H), 3.56 (t, 2H), 3.47 (s, 2H), 3.28 (t, 2H), 1.58 (m, 6H), 1.40 (t, 3H).

Ethyl 2-({N-|2-(tert-butoxy)-2-oxoethyl]-2,2,2-trifluoroacetamido}niethyl)pyridine-4-carboxylate (c)

General Procedure C (Formation of triflouroacetamide and/or trïfluoroacctate)

DIPEA (1.5 equiv.) was added to a solution of the amine (or alcohol) (Ethyl 2-({[2-(tert-butoxy)-2oxoethyl]amino}methyl)pyridine-4-carboxylate (b)) in anhydrous DCM. The mixture was stirred at 0°C and trifluoroacetic anhydride (1.5 equiv.) was added drop wise. After addition, the mixture was allowed to warm to room température and stirring was continued for 2h. The reaction was quenched with aqueous NaHCOj. Aqueous work up gave the title compound.

'H-NMR (300MHz, CDClj), (rotamers): δ 8.7 (dd, IH), 7.8 (ss, IH), 7.7 (dd, IH), 4.8 (ss, 2H), 4.3 (q, 2), 4.2 (ss, 2H), 1.4 (s, 9H), 1.3 (t, 3H).

Ethyl 2-[(2,2,2-trifluoro-N-{3“|(trifluoroacetyl)oxy]propyI}acetamido)methyl]pyridine-4-carboxylate (g)

Prepared by General Procedure C from ethyl 2-{[(3-hydroxypropyl)amino]methyl}pyridine-4-carboxylate (f) using 7 equîv. of DIPEA and 5 equiv. of trifluoroacetic anhydride.

113 'H NMR (300 MHz, CDC13), (rotamers) δ: 8.75 (two doublets, IH), 7.80 (m, 2H), 4.80(two singlets, 2H), 4.40 (m, 4H), 3.70 (two t, 2H), 2.20 (m, 2H), 1.45 (m, 3H).

Ethyl 2-({N-[(3R)-l-[(tert-butoxy)carbonyl]pyrrolidin-3-yl]-2,2,2trifluoroacetamido}methyl)pyridine-4-carboxyiate (1)

Prepared by General Procedure C from ethyl 2-({[(3R)-l-[(tert-butoxy)carbonyl]pyrrolidin-3yI]amino}methyl)pyridine-4-carboxylate (k). Reaction time 12 hours. Title compound was isolated as yellow oil.

’H-NMR (300MHz, CDClj): δ 8.70 (d, IH), 7.76 (m, 2H), 4.74-4.71 (m, 3H), 4.43 (q, 2H), 3.69-3.27 (m, 4H), 2.19-2.01 (m, 2H), 1.46 (s, 9H), 1,44 (t, 3H).

ES-MS: 446 [M+l].

2-(N-{[4-(ethoxycarbonyl)pyridin-2-yl]methyl}-2,2,2-trifluoroacetamido)acetic acid (d)

General Procedure D (Acids from tert butyl esters or amines from tert butoxy carbamates)

The ester (or carbamate) (Ethyl 2-({N-[2-(tert-butoxy)-2-oxoethyl]-2,2,2trifluoroacetamido}methyl)pyridine-4-carboxylate (c)) was dissolved in DCM and before trifluoroacetic acid (0.1 -1 equiv. volume of DCM) was added. Tire mixture was stirred at room température for ovemight. The solvent was evaporated in vacuum to get the title compound.

’H-NMR (300MHz, CDjOD): Ô 8.75 (m, 1 H), 7.8 8.00 (m, 2H), 5.45,4.99 (2s, 2H; rotamer), 4.20 - 4.40 (m, 4H), 1.40 (t,3H).

Ethyl 2-({2^,2-trifluoro-N-[(3R)-pyrrolidin-3-yl]acetamido}methyl)pyridine-4-carboxylate (m)

Prepared by General Procedure D from ethyl 2-({N-[(3R)-l-[(tert-butoxy)carbonyl]pyrrolidin-3-yl]-2,2,2trifluoroacetamido) methyl)pyridine-4-carboxylate (1),

114 ’H-NMR (300MHz, CDClj): δ 8.67 (d, 1 H), 7.81 (s, IH), 7.72 (d, 1 H), 4.39 (q, 2H), 3.97 (d, 2H), 3.84-3.64 (m, 3H), 3.51-3.45 (m, 2H), 2.11 (m, IH), 1.97 (m, IH), 1.39 (t, 3H).

ES-MS: 446 [M+l]

Ethyl 2-[(2,2,2-trifluoro-N-{[(piperidin-4-yl)carbamoyl]methyl}acetamido)methyl]pyridine-4carboxylate (p)

Prepared by General Procedure D from ethyl 2-({N-[({l-[(tert-butoxy)carbonyl]piperidin-4yl}carbamoyl)methyl]-2,2,2-trifluoroacetamido}methyl)pyridine-4-carboxylate (o). Purification by column chromatography (MeOH/DCM and 1% NH4OH) gave the title compound as a brown foam.

'H-NMR (300MHz, CDjOD): Ô 8.75 (m, 1H ), 7.90 (m, 2H), 5.00 & 4.90 (2s, 2H, rotamer), 4.42 (q, 2H), 4.32 & 4.12 (2s, 2H; rotamer), 3.95 (m, IH), 3.40 (m, 2H) 3.10 (m, 2H), 2.10 (m, 2H), 1.80 (m, 2H), 1.38 (t, 3H).

Ethyl 2-({2,2,2-trifluoro-N-[2-oxo-2-(pyrrolidin-l-yl)etliyl]acctamido]mcthyl)pyridinc-4-carboxylate (e)

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

An amine (pyrrolidine) (or an alcohol or a sulfonamîde) (2 equiv.) was added to a solution of an acid (2-(N{[4-(ethoxycarbonyl)pyridin-2-yl]niethyl}-2,2,2-trifluoroacetamido)acetic acid (d)) (1 equiv.) in DMF. Cooled to 0 °C before EDC HCl (1.5 équivalent) and ethyl(hydroxyl iminocyanoaectate (oxyma; 1.5 équivalent) were added. The reaction mixture was allowed to warm slowly to room température and stirred ovemight. Aqueous work up and purification by column chromatography gave the title compound.

‘H-NMR(300MHz, CDjOD): δ 8.75 (m, IH), 7.90 (s, IH), 7.85(m, IH), 4.90 =1.30 (two set of singlet & q , 6H, rotamer), 3.45 (m, 4H), 2.00-1.80 (m, 4H), 1.38 (t,3H).

Ethyl 2-({N-[({l-[(tert-butoxy)carbonyl]piperidin-4-yl}carbamoyl)methyl]-2,2,2trifluoroacetamido}methyl)pyridine-4-carboxylate (o)

115

Prepared by General Procedure E from tert-butyl 4-aminopiperidine-l-carboxylate and 2-(N-{[4(ethoxycarbonyl)pyridin-2-yl]methyl}-2,2,2-trifluoroacetamido)acetic acid (d) to give the title compound as a brown foam.

‘H-NMR (300MHz, CDClj): δ 8.70 & 8.60 (2d, IH; rotamer ), 7.80 (m, 2H), 4.90 & 4.78 (2s, 2H, rotamer), 4.42 (q, 2H), 4.30 & 4.10 (2s, 2H; rotamer), 4,10 (m, IH), 2.80 (m, 2H) 2.0 (m, 2H), 1.48 (s, 9H), 1.40 (t, 3H).

Ethyl 2-[(2,2,2-trifluoro-N-{((4hydroxybutyl)(methyl)carbamoyl]mcthyl}acetamido)methyl]pyridine-4-carboxylate (r)

Prepared by General Procedure E from 4-(methylamino)butan-l-ol and 2-(N-{[4-(ethoxycarbonyl)pyridin2-yl]methyl}-2,2,2-trifluoroacetamido)acetic acid (d).

'H-NMR(300MHz, CDC1₃): δ 8.70 (d, IH), 7.90 (s IH), 7.8 (m, IH), 4.95 (d, IH), 4.85 (d, IH), 4.41 (m, 3H), 4.34 (s IH), 3.67 (q, 2H), 3.40 (m, IH), 3.29 (m, IH), 2.97 (s, 3H), 1.74-1.52 (m, 5H), 1.43 (t, 3H).

ES-MS: 420 [M+l],

4-hydroxypentan-2-yl 2-({[(tert-butoxy)carbonyl|({[2-(dimcthylamino)cthyl](cthyl)carbamoyl} mcthyl)amino]mcthyl)pyridine-4-carboxylate (z)

General Procedure E from 2-({[(tert-butoxy)carbonyl]({[2 (dimethylamino)etliyl] (ethyl)carbamoyl}methyl)amino}methyl)pyridine-4-carboxylic acid (x) and pentane-2,4-diol. Purification by column chromatography (10-15% MeOH/DCM ) gave the title compound as a brown oil.

'H-NMR(300MHz, CDC13): δ 8.50 (d, IH ), 7.90 (m, IH), 7.50 (m, IH), 5.30 (m, IH), 4.55 (m, 2H), 4.20,4.10 (2s, 2H), 2.25 (2s, 6H), 1.40 (m, 12H)., 1.20 (m, 6H).

ES-MS: 495 [M+l]

116

Ethyl 2-{[2,2,2-trifluoro-N-(3-oxopropyl)acctamido]methyl}pyridine-4-carboxyIate (i)

General Procedure F (Swern oxidation of alcohols to aldéhydes and ketones)

DMSO (4.0 equiv) was diluted with DCM and cooled to -78 °C, oxalyl chloride (2.0 equiv) was added and the mixture was stirred for 30 minutes. Then a solution of the alcohol (ethyl 2-{[2,2,2-trifluoro-N-(3hydroxypropyl)acetamido]methyl}pyridine-4-carboxylate (h)) in DCM was added and the mixture was stirred for another 1 h. Then EtjN (5.0 equiv) was added and the mixtures was slowly warmed to room température. Aqueous work up and coiumn chromatography gave the title compound.

^lH NMR (300 MHz, CDC1₃), (rotamers): δ 9.80 (two singlets, IH), 8.70 (two doublets, IH), 7.80 (m, 2H), 4.90/4.75 (two singlets, 2H), 4.45 (m, 2H), 3.95/3.75 (m, 2H), 2.90 (two t, 2H), 1.45 (m, 3H).

Ethyl 2-[(2,2,2-trifluoro-N-{[mcthyl(4-oxobutyl)carbamoyl|mcthyl}acetamido)methyl]pyridine-4carboxylate (s)

Prepared by General Procedure F from ethyl 2-[(2,2,2-trifluoro-N-{[(4hydroxybutyl)(methyl)carbamoyl]methyl}acetamido)methyl]pyridine-4-carboxylate (r). Purified by coiumn chromatography (EtOAc/hexanes).

'H-NMR (300MHz, CDCIj): δ 9.85 (m, IH), 8.70 (dd, IH), 7.9 (ss, IH), 7.80 (dd, IH), 4.93 (ss, 2H), 4.43 (ss, 2H), 4.35 (q, 2), 3.01 (ss, 3H), 1.44 (t, 3H).

ES-MS: 418 [M+l].

4-oxopentan-2-yl 2-({|(tert-butoxy)carbonyl]({[2-(dimethylamino)ethyl](ethyl)carbamoyl}methyl) amino}mcthyl)pyridine-4-carboxylate (aa)

Prepared by General Procedure F from 4-hydroxypentan-2-yl 2-( {[(tert-butoxy)carbonyl]({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino}methyl)pyridine-4- carboxylate (z). Purification by coiumn chromatography ( 10% MeOH/DCM) gave the title compound as brown oil.

117 ’H-NMR (300MHz, CDCB): S 8.60 (d, IH ), 7.70 (m, IH), 7.60 (m, IH), 5.00 (m, IH), 4.60 (m, 2H), 4.15, 4.00 (2s, 2H), 2.15 (s, 6H), 1.40 (m, 14H)„ 1.10 (m, 6H)

ES-MS: 493 [M+l]

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

General Procedure G (Boc protection of amines)

The amine (ethyl 2-{[({[2-(dimethylamino)ethyl](ethyi)carbamoyl}methyl)amino]methyl}pyridine-4carboxylate (#65)) (1 eq) and Boc₂O (1.2 eq) were dissolved in THF/H₂O. NaHCOj solid (4 eq) was added. The reaction mixture was stirred at room température ovemight. After solvent removal, lhe 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, CDClj): δ 8.52 (m, IH), 7.84 (m, I H), 7.74 (m, IH), 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 phosphonic acid anhydride (2.0 eq) were added to a solution of an amine (pyridin-4-amine) (or an alcohol or a sulfonamide) (1.5 eq) and an acid (2-(((( tert-butoxy)carbonyl]( {[2(dimethylamino)ethyl](ethyl)carbamoyl)methyl) amino}methyl)pyridine-4-carboxylic 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.

‘H-NMR (300MHz, D₂O): δ 8.59 (d, 2H ), 8.21 (d, 2H), 7.88 (d, 2H), 6.75 (d, IH), 4.35A32 (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).

118

Ethyl 2-{[2,2,2-trifluoro-N-(3-hydroxypropyl)acetaniido]inethyl}pyridme-4-carboxylate (h)

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, CDC13), (rotamers): δ 8.74 (two d, IH), 7.80 (m, 2H), 4.85(5, 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}niethyl)amino}methyl)pyridine-4-carboxylic acid (x)

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

'H NMR (300 MHz, CD₃OD) δ 8.67 (m, 1 H), 7.67 (m, 1 H), 7.73 (m, IH), 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-{niethyl[3-(l-methyHH-imidazol-2yl)propyl] carbamoyl} ethyl] carbamate

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

(2R)-2-amino-3-hydroxy-N-methyl-N-i3-(l-methyl-lH-imidazol-2-yl)propyl]propanamîde

119

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 atmosphère (40 PSI) until no starting material was detected by TLC. Then the mixture was filtered through a pad of celite and concentrated to yield the title compound.

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

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

^lH NMR (300 MHz, CDCfi) 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 ovemight. The solvent was evaporated in vacuum to yield The title compound as colorless oil.

'H NMR (300 MHz, CDClj), δ 8.0 (brs, 1H),3.95 (m, IH), 3.6(m, IH), 3.3 (m,2H), 1.75 (m, IH), 1.6(m, IH), 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, CDClj), δ ppm: 5.0 (brs, IH), 3.7 (m, IH), 3.4 brs, IH), 3.2 (m, 2H), 1.6 (m, 2H), 1.4 (s,9H), 1.2 (d, 3H).

ES-MS: 224 [M+l],

4-(methylamino)butan-2-ol

120

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.

^lH NMR (300 MHz, CDC1₃), δ ppm: 3.9 (m, 1 H), 3.6 (brs, 2H), 2.9 (m, 1 H), 2.6 (m, 1 H), 2.4 (s, 3H), 1.5 (m, 2H), 1.2 (d, 3H).

2.2.2- trifluoro-N-(3-hydroxybutyl)-N-methyIacetamide

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

'H NMR (300 MHz, CDC1₃), 5 3.8 (m, 1 H), 3.7 (m, 1 H), 3.2 (m, 1 H), 3.2 (s, 3H), 1.7 (m, 1 H), 1.6 (m, 1 H),

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.

'H-NMR (300MHz, CDC1₃): δ 7.50 (br s, 1 H), 5.90 (m, 1 H), 5.28 (d, 1 H), 5.20 (d, 1 H), 4.40 (m, 1 H), 3.75 (m, IH), 3.40 (m, IH), 2.00-1.60 (m, 2H).

2.2.2- trifluoro-N-(3-oxopropyl)acctamide

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

^lH-NMR (300MHz, CDC1₃): δ 9.81 (s, IH), 7.15 (br s, IH), 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 LD A (1.1 eq) at -78 °C. The reaction mixture was stirTed for 30 min and TMSCI (l 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.

propan-2-yI 3-hydroxy-5-(trifluoroacetamîdo)pentanoate

121

To a solution of 2,2,2-trifluoro-N-(3-oxopropyl)acetamide (1 eq) in DCM was added trimethyl({[l(propan-2-yloxy)ethenyl]oxy})silane (leq) andTiCU (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.

‘H-NMR (300MHz, CDClj): δ 7.65 (br s, IH), 5.05 (m, IH), 4.19 (m, IH), 3.35 (m, IH), 2.46 (d, 2H), 1.85 - 1.61 (m,2H), 1.25 (d, 6H).

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

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

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

Example 2: Histone lysine demethvlase AlphaLISA assays for IC50 value détermination.

This example demonstrates the ability of compounds of the invention to inhîbit 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: AlphaLISA #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 réaction mixture is incubated at room température 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 température, 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 température in the dark the plates are read. Reaction conditions are seen in Table 2b.

Enzymes:

122

Table 2a
Protein name	Vendor/source	Sequence	Expression organism
KDM2A (FBXL10)	BPS, Bioscience, US	1-650	Bac
KDM3B (JMJDI B)	BRIC	842-1761	Bac
KDM4A (JMJD2A)	BPS, Bioscience, US	1-350	E.coli
KDM4B (JMJD2B)	BPS	2-500	Bac
KDM4C (JMJD2C)	BRIC, Denmark	1-349	E.coli
KDM5 (JARID1C)	BPS	2-1560	Bac
KDM5B (PLU-1)	BRIC	1-809	E.coli
KDM6A (UTX)	BRIC	919-1401	E.coli
KDM6B (JMJD3)	BPS	1043-end	Bac
KDM7 (PHF8)	BRIC	1-1322	Bac
KDM3A (JMJD1A)	BPS, Bioscience, US	2-end	Bac

Substrates:

BK.9M3:	Biotin-ARTKQTAR(KMe₃)STGGKAPRKQ-NH₂ (Caslo, Denmark)
BK9M2:	Biotin-ARTKQTAR(KMe₂)STGGKAPRKQ-NH₂ (AnaSpec 64359)

123

BK9M1:

Biotin-ARTKQTAR(KMe,)STGGKAPRKQ-NH₂ (AnaSpec 64358)

H3K4M3B:	H-ART(Kme3)QTARKSTGGKAPRKQLA-NH-Biotin (Caslo, Denmark)
BK27M3;	Biotin-ATKAAR(Kme3)SAPATGGVKKPHRY-NH2? (Caslo, Denmark)
BH3K36M2:	RKAAPATGGVK(Me2)KPHRYRPGTVK-(BIOTIN)? (Anaspec)

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

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

Reaction conditions

Table 2b
Enzyme	Enzyme	Substrate solution	Accepter Beads	Donor Beads	Incubation Time
KDM2B	Buffer A, pH 8.0 Final enzyme concentration: 2 nM	Buffer A, pH 8.0 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BH3K36M2) conc: 50 nM	H3K36M1 (PerldnElmer Custom, 10120327RS) Diluted 1:200 from stock	Diluted 1:200 from stock	60 min
KDM3A	Buffer A, pH 7.4 Final enzyme concentration:	Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG.	H3K9Mel (selfconjugated) Diluted 1:200	Diluted 1:200 from stock	60 min

124

	1.6 nM	Final substrate (BK9M2) conc: 100 nM	from stock
KDM3B	Buffer A, pH 7.4 Final enzyme concentration: 0.1 nM	Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK9M2) conc: 100 nM	H3K9Mel (selfconjugated) Diluted 1:200 from stock	Diluted 1:200 from stock	60 min
KDM4A	Buffer A, pH 7.4 Final enzyme concentration: 0.2 nM	Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ aKG. Final substrate (BK9M3) conc: 100 nM	H3K9Me2 (Cat #AL117,Perkin Elmer) Diluted 1:400 from stock	Diluted 1:400 from stock	60 min
KDM4B	Buffer A, pH 7.4 Final enzyme concentration: 1 nM	Buffer A, pH 7.4 + 25 μΜ L- Asc, 10 μΜ αKG. Final substrate (BK9M3) conc: 100 nM	H3K9Me2 (Cat #AL117,Perkin Elmer) Diluted 1:400 from stock	Diluted 1:400 from stock	60 min
KDM4C	Buffer A, pH	Buffer A, pH 7.4 + 25 μΜ L-	H3K9Me2 (Cat #AL117,Perkin	Diluted 1:400	60 min

125

	7.4 Final enzyme concentration: 1 nM	Asc, 10 μΜ α- KG. Final substrate (BK.9M3) conc: 200 nM	Elmer) Diluted 1:400 from stock	from stock
KDM5B	Buffer A, pH 7.4 Final enzyme concentration: 2 nM	Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ aKG. Final substrate (H3K4M3B) conc: 200 nM	H3K4Me2-l (Perkin Elmer ALI 16) Diluted 1:200 from stock	Diluted 1:200 from stock	20 min
KDM5C	Buffer A, pH 7.4 Final enzyme concentration: 1 nM	Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ ctKG. Final substrate (H3K4M3B) conc: 100 nM	H3K4Me2-l (Perkin Elmer ALI 16) Diluted 1:200 from stock	Diluted 1:200 from stock	30 min
KDM6A	Buffer A, pH 7,4 Final enzyme concentration: 2 nM	Buffer A, pH 7.4 + 25 μΜ L- Asc, 10 μΜ αKG. Final substrate (BK27M3)	H3K27me2-l (Perkin Elmer AL121) Diluted 1:200 from stock	Diluted 1:200 from stock	60 min

126

		conc: 100 riM
KDM6B	Buffer A, pH 7.4 Final enzyme concentration: 1 nM	Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK27M3) conc: 50 nM	H3K27me2 (Perkin El mer AL121) Diluted 1:200 from stock	Diluted 1:200 from stock	60 min
KDM7	Buffer A, pH 7.4 Final enzyme concentration: 2.5nM	Buffer A, pH 7.4 + 25 μΜ LAsc, 10 μΜ αKG. Final substrate (BK9M2) conc: 100 nM	H3K9Mel (selfconjugated) Diluted 1:200 from stock	Diluted 1:200 from stock	60 min

127

HDME INHIBITION

Table 3
Structure	Name	GA SCI	FBX L10	JARI D1C	JMJ D1B	JMJ D2A	JMJ D2B	JM JD3	PH F8	UT X	PL U1
CL OH X - - *j \/\z	2-({[3-(lHimidazol-1yl)propyl ]am ino} m ethyl)pyridine-4carboxylic acid	-H-	+	+++		+-H-		+		+
°y^0H [ίη h ί λ. ,ch, ch₃	2-({[2(dimethylami no)et hyljamino] methyl) pyridine-4carboxylic acid	+++			4-	+-H-	-H-+	+		+

128

^ΗΟγ° ίι ^Η ?^Η	2-<{[(2R)-2,3dihydroxypropylja mino} methyl)pyri d ine-4-carboxylic acid	++				+	+++
0 ΟΗ ΝΗ Δ	2{[(cyclopropylmet hyl)amino]methyl} pyridine-4carboxylic acid	++				+	+++
^ΗΟγ° ίι ^η CHj	2- {[(cyclopropylmet hyl)amino] methyl} pyridi ne-4carboxylic acid	+-H-	+++	+++	+++	+	+++
°γ^0Η Λ ?^η3 ch₃	2-({[2-(dimethylamino)ethyl](meth yl)amino } methyl )p yridine-4carboxylîc acid					+	++

129

^ΗΟγ° CH₃ [ 1 1 ³ ,CH	2- {[methyl(prop-2yn-1yi)amino]methyl} p yridine^lcarboxylic acid	+4-	+	-H-
^HOy° ^V-_N-^X\x'	2-{[(2fluoroethyl)amino] methyl} pyridine-4carboxylic acid	++	+	++
^HOy° i	2- {[(furan-2ylmethyl)amino]m ethyl }pyridine-4carboxylic acid	+-H-	+

130

0¼ HN ° / vy	2-({[(5phenylfuran-2yl)methyl]amino } methyl )pyri dîne -4-carboxylic acid	+					+	-H-
ΟγΟΗ άς ξτ° H_aC'°	2-({[(2,4dîmethoxyphenyl )methyl]amino} methyOpyridine4carboxylic acid	+	+	+	+	+	+	+++
°γ^0Η il ^H < -,Ν ,CH,	2-({[2- (methylsulfanyl) ethyl]amino}met hyl)pyridine-4carboxylic acid	+++					+	+++

131

°γ^0Η	2-{{[2-oxo-2(pyrrolidin-1yl)ethyl]amino} methyi)pyridine4-carboxylic acid	+++	+++	+++	+	+++	+++	+	++	+	+++
HO 0 ch₃	2- [( {[butyl(methyl) carbamoyl]meth yl}amino)methyl ]pyridine-4carboxylic acid	+++	-H-+	-H-+	+		+++	+	++	4-	+++
^HO _V° h₃C	2-({[(l-methylIH-1,3benzodiazol-2yl)methyl]amino }methyl)pyridine -4-carboxylic acid	-H-		+++		+++	+++	+		+	+++

132

ÇOJ._N^	2-[({2-[4-(2methoxyethyl)pi perazin-l-yl]-2oxoethyl} amino) methyl]pyridine4-carboxyIic acid	+++	+	+++
^ΗΟγ° ---^Ν '^X^_N-^V^^CH2 S CHj	2-[({[bis(prop-2en-1yl)carbamoyl]me thyl} amino)meth yl]pyridine-4carboxylic acid	+++	+	+-H-
Y o	2-[({2-oxo-2[(2R)-2(pyrrolidin-lylmethyl)pyTToli din-1yl]ethyl} amino) methyljpyridine4-carboxyIic acid	+++	+	+++

133

°γ^0Η CX N 0 -^^CH3	2-({[(3R)-l[(tertbutoxy)carbonyl] pyrrolidin-3yljamino} methyl )pyridine-4carboxylic acid	+	+	+
^ΗΟγ°	2-({[(3R)-l[(tertbutoxy)carbonyl] pyrrolidin-3yl]amino} methyl )pyridine-4carboxylic acid	+	+	+
^ΗΟγ°	2-{[(3-{[3(pyrrolidin-1yl)propyl]amino} propyl)amino]me thyl }pyridine-4carboxylic acid	+++	+	-H-+

134

°γ^0Η ch₃	2-{[(3methylbutyl)ami no]methyl}pyridi ne-4carboxylic acid	-H-	+	++
HO O ch₃	2-[({[(2carbamoylethyl)( methyl)carbamo yl]methyl}amino )methyl]pyridine -4carboxylic acid	+++	+	+++
H° ° “VU	2-[({2-[2(hydroxymethyl) piperidin-1 -yl]2oxoethyl}amino) methyljpyridine4-carboxylic acid	+++	+	+++

135

HO^O Η,Ο-'Ο Coxj CH,	2-{[({methyl[3(1 -methyl- 1Himidazol-2yl)propyl]carbam oyl} methyl)amin o]methyl}pyridin e-4carboxylic acid	+++	+	+++
«Y h, JO	2-{[({[(lethylpyrrolidin2yl)methyl]carba moyl} methyljam ino]methyl}pyrid îne-4carboxylic acid	+-H-	+	+++
^HOy° V-	2-{[({methyl [(1methyl-lHpyrazol-5yl)methyl]carba	+++	+	+++

136

	moyl} methyl )am ino]methyl}pyrid ine-4carboxylic acid
^ΟγΟΗ	2-{{[(3R)-l-(3phenylpropyl)pyr rolidîn-3yl]amino}methyl )pyridine-4carboxylic acid	+			+	++	++	+	+	+
°γ^0Η _q,ch, Η	2-( {[( {1-[(2methoxyphenyl) methyljpiperidin -4yl}carbamoyl)me thyl]amino}meth yl)pyridine-4carboxylic acid	+++	+++	+++	++	+++	+++		+++		+++

137

°γ^0Η Η	2-{[({[1-(3phenylpropyl)pip eridin-4yl]carbamoyl}me thyl)amino]meth yl}pyridine-4carboxylic acid	+++								4-	+++
°γ^ΟΗ Η	2-{[({[l-(furan- 2ylmethyl)piperidi n-4yl]carbamoyl }me thyl)amino]meth yl}pyridine-4carboxylîc acid	+++								++	+++

138

°γ°^Η Η	2-({[({1-[(5phenylfiiran-2yl)methyl]piperi din-4yljcarbamoyl) methyl]amino}m ethyl)pyridine-4carboxylic acid	-H-+								+	+++
^Η°γ° ζοχ./ h₃c^	2-[({[(2cyanoethyl) (ethyl)carbamoyl Jmethyl} amino) methyljpyridine4carboxylic acid	+++								+	-HH-

139

°_V ^OH JL x>\x'^CH3	2<{[2-{lbutylpyrrolidin2yl)ethyl]amino} methyl)pyridine4-carboxylic acid	+++		-H-+	+	+++	+++	+	+	+	+++
s	2-{[({[l-(3,7dimethyloct-6en-1yl)pyrrolidin-3yl]carbamoyl}me thyl)amino]meth yl}pyridine-4carboxylic acid	+++	++	-H-+	+	+++	+++	++	++	+	+-H-

140

0 °^H F	2-{[(3-{[(2fluoro phenyl)methyl]( methyl)amino}pr opyl)amino]meth yl}pyridine-4carboxylic acid	+++								+	+++
°γ°π [ίη h y L CH, ^OH HjC'	2-({[(lR)-2hydroxy-1{methyl[3-(lmethyl-lHimidazol-2yl)propyl]carbam oyl}ethyl]amino }methyl)pyridine -4carboxylîc acid	+44-	+	++	+	+++	+++	+	+	+

141

ΗΟ _Λ A 9	2-[( {2-(3-( 1 ΗΙ^-ύοηζοάΐβζοΙ2ylmethyl)piperidi n-1- yi]-2oxoethyl} amino) methyl]pyridine4-carboxylic acid	-H-+								+	-H-+
o » H	2-{[({[H2phenylethyl)pyrr olidin-3yl]carbamoyl)me thyl)amino]meth yl}pyridine-4carboxylic acid	+++								+	+++

142

ΟγΟ θ	2-(((3-(4benzylpiperidin1yl)propyl]amino} methyl)pyridine4-carboxylic acid	+++	+	+++
^Η°γ°	2-(((3-((2phenoxyethyl)am ino]propyl}amin o)methyl]pyridin e-4carboxylic acid	+++	+	+++
HO 0 ^,CH1	2-[({[methyl({4((4methylpiperazin1yl)methyl]phenyl }methyl)carbamo yl] methyl} amino )methyl]pyridine	4-H-	-H-	+++

143

	-4- carboxylic acid
Ύ r\	2-({[2-(2benzylpyrrolidinl-yl)-2oxoethyl]amino} methyl)pyridine4-carboxylic acid	+++							+	+-H-
4¼ V	2-({[({4- [benzyl(cyclopro pyl)amino]butyl} (methyl)carbamo yl)methyl]amino }methyl)pyridine -4carboxylic acid	+++	+++	+++	+	+++	+-H-	-H-

144

°_V ^OH l! ^h rO	2-[({2-[(2S)-lbenzylpyrrolidin2- yljethyl} amino) methyl ]pyridine4-carboxylic acid	+++								+	+++
H° °	2-({[3(pyrrolidin-1yl)propyl]amino} methyl)pyridine4-carboxylic acid	++		+++		-H-	+++	+	+	+	+++

145

Ch* V Ùz_O	methyi 2-({[3(pyrrolidin-lyl)propyl]amino} methyl)pyridine4-carboxylate	Ή									-H-
y° ^*hr ^CH,	2-( {[4- (dîethylamino)bu tyl]amino}methy I)pyridine-4carboxylic acid	-H-+		+++		+	+++	+	+	+	+++

146

⁰ r*	2-{[({[2(dimethylamino) ethyl](ethyl)carb amoyl }methyl)a mino]methyl}pyr idine-4carboxylic acid	+++	++	-H-+	+	+-H-	+++	+	++	+	+++
Below	4-methoxyphenyl 2-{ {[4- (diethylamino)butyl ]ami no} methyl )pyridîne-4-carboxylate	++								+	+++

147

148

Below

2-(ethoxycarbonyl )phenyl

2-({[4(diethylamino)butyl]amÎno} methyl)pyridine-4carboxylate ++

H ,N •N

149

^ογ°\ζ^_Ν/^Η1 [fjl H	2(dimethylamin o)ethyl 2-({[4(diethylamino) butyl] amino} methyl )pyridîn e-4carboxylate	4-H-								+	ΗΊ
CH, 1 >ch₃	3(dimethylamin o)propyl 2({[4(diethylamino) butyl]amino} methyljpyridin e-4-	++								+	+++

150 carboxyiate

Below {4-[(ethoxycarbonyl) amino]phenyl} methyl 2-{{[4(diethylamino)butyl]ami no} meth yl)pyridine-4-carboxylate ++

151

jOl Y	2,6dimethoxyphenyl 2({[4(diethylamino)butyl] amino }methyl)pyridi ne-4-carboxylate	++								+	+++
.,¼	2,6-dimethylphenyl 2< {[4(diethylamino)butyl] amino}methyl)pyridi ne-4-carboxylate	+								+	+++

152

Below

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

o

153

Below

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

154

Below {4[(ethoxycarbonyl)(methyl )ami nojphen yl}methyl 2-({[4(diethylamino)butyl ] amino} mcthyljpy ridine-4-carboxyl ate

H,C

155

Below

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

+++

HjC

156

ΥΥΎ“· JL ch, ο ίι^ ⁰ CH,

4-oxopentan-2-yl 2{[({[2(dimethylamino)ethy l](ethyl)carbamoyi} methyl )amino] methy l}pyridine-4carboxylate

+++

4-H-

157

Below

4-(trifluoroacetamido) butan-2-yl 2-{[({[2(dimethylamino)ethyl](ethy l)carbamoyl}methyl)amino] methyl }pyridîne-4carboxylate ++

158

Below	4-(2,2,2-trifluoro-Nmethylacetamido)butan2-yl 2-{[({[2- (dimethylamino)ethyl](e thyljcarbamoyl} methyl) ami no]methyl }pyri dine4-carboxylate	-H-+								+	++
F CHj r°Y° ° ^CH ° ^CH= CH,

159

^CH, Ύ ⁰

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

-H-

+++

++

160

Below

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

161

Below	5-(2,2,2-trifluoro-Nmethylacetamido)pent-l-en-3-yi 2{[¢{[2- (dimethylamino)ethyl](ethyl)carbain oyl} methyl)ami nojmethyl} pyridine4-carboxylate	++								4-H-	++
F CHj O CH₃ H,C^ '^CHj

162

Below

2<2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)amino]methyl} pyridine4-carbonyloxy)-3(hexadecanoyloxy)propyl hexadecanoate

163

Below

l-(2-{[({[2- (dimethyiamino)ethyl](ethyl)carbam oyl} methyl)amino] methyl} pyridi ne4-carbonyloxy)-3- (hexadecanoyloxy)propan-2-yI hexadecanoate

++

+++

++

164

165

CH, 1 v ^{0 CH}3

methyi 2-{[({[2(dimethylamino)ethy l](ethyl)carbamoyl} methyl)amino]methy l}pyridine-4carboxylate

++ +

+

-H-+

+-H-

166

Below	propan-2-yl 3-(2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl }methyl)amino]methyl }pyridine-4carbonyloxy)-4- (trifluoroacetamido)butanoate	-H-									++
/ÎX. ^H3^C\^^CH5 CHj 0 N y ^NH Xi. F

167

ρ. Y Q Γ*	propan-2-yl 3-(2{[({[2(dimethylamî no)ethy l](ethyl)carbamoyl} methyl)ammo]methy l}pyridine-4carbonyloxy)-5(trifluoroacetamido) pentanoate	++									+-H-
° °^H Γί	2-(piperidin-lylmethyl)pyridine-4carboxylic acid	+									+

168

^{0 0H} X N O	2-(azetidin-l ylmethyl)pyridine-4carboxylic acid	+	+								+
X ⁰ ch, <	2,2,2-tri fluoroethyl 2-{[({[2(dimethylamino)ethy l](ethyl)carbamoyl} methyl)amino]methy l}pyridine-4carboxylate	-H-+	+								+++

169

ά^α₀	2-({ethyl[2-oxo-2(pîperidin-1 yl )ethyi]amino} meth yl)pyridine-4carboxylic acid	++	-H-								-H-
X X	2-( (butyl[2-oxo-2(piperidin-1yl)ethyl]amino} meth yl)pyridine-4carboxylic acid	+	++								+

170

X P

2-( {benzyl[2-oxo-2(piperidîn-1yl)ethyl]ami no} meth yI)pyridine-4carboxylic acid

+

++

+

171

Below	2,6-bis(propan-2yloxy)phenyl 2{[({[2(dimethylamino)ethy l](ethyl)carbamoyl} methyl)amino]methy l}pyridine-4carboxylate	++									-H-
CH, O CH, 1 II H II 1 ° ^CHj HjC^^CH,

172

V F όυϊθ	2-{[(2methylpropyl)[2oxo-2-(piperidm-1 yl)ethyl]amino]meth yljpyridine^lcarboxylic acid	+	++							+++	+
°ν°^Η Γ	2-({[2-oxo-2(piperidin-1yl)ethyl](propyl)ami no} methyl)pyridine4-carboxylic acid	+	-H-							-H-+	++

173

OH 1 ^HîC\X'^CH3 pji 1 S	2-{ {[2-OXO-2(piperidin-1yl)ethyl](propan-2yl)amino}methyl)py ridine-4-carboxyiic acid	+	++							Ί· ί	+
r'' ί^ύ^υίΥ	2-fluoroethyl 2{[({[2(dimethylamino)ethy l](ethyl)carbamoyl} methyl)amino]methy I}pyridine-4carboxylate	++	+							+++	+++

174

	2,2-difluoroethyl 2(K{[2(dimethylamino)ethy l](ethyl)carbamoyl} methyl )amïno]methy l}pyridine-4carboxylatc	+++	+							4-H-	+++
°<γ^0Η ÎXsA.J<' Ξ H CH₃ Ύ L»rlj	2-({[(lS)-l-(tertbutylcarbamoyl )-3methylbutyljamino} methyl )pyri dine-4carboxylic acid	+	-H-							++	+

175

°<γ^0Η ^ηΜ kk CH 3	2-( {methyl[(2S)-4methyl-1 -oxo-1 (piperidin-1 yl)pentan-2yl Jamino} methyl)py ridine-4-carboxylic acid	+									+
Γ’ 1 °^ *° ifY\| ^{0 CM}J k^k^^A. /\γΧ ^CH, k ^CH,	2-{[(«2- (dimethylamino)ethy l](ethyl)carbamoyl} methyl)amino]methy 1}-N- methanesu 1 fonyl -Nmethylpyri dine-4carboxamide	+++	++							+	+-H-

176

ο Υϊ ^CH,	N-[2- (dimethylamino)ethy l]-N-ethyl-2-({[4-(2oxo-1,3-oxazolidine3-carbonyl)pyridin2- yl]methyl} amino)ac etamide	+++	+								+++
9 ^{0 Ν}» ο ch, ^CH,	2-{[({[2- (dimethylamino)ethy l](ethyl)carbamoyl} methyl)amino]methy l}-N-(pyridm-4yl)pyridine-4carboxamide	++									+++

177

	2-(1( {[2(dimethylamino)ethy I](ethyl)carbamoyl} methyl)amino]methy 1} -N-(5-methyl1,3,4-oxadiazol-2yl)pyridine-4carboxamide	+++									+-H-
h.c 6^* ° °s	2-(((((2(dimethylamino)ethy l](ethyl)carbamoyl} methyl)amino]methy 1} -N-( 1 -methyl -1Hpyrazol-5yl)pyridine-4carboxamide	+++									+++

178

Below	2-{[({[2(dimethylamino)ethyl](ethyl)carbamo yl} methyl)amino]methyl }-N -(1,3oxazol -2-yl )pyridme-4-carboxamide	+-H-									+++
° ch,

179

Below	2-{[({[2(dimethylanuno)ethyl](ethyl)carbamo yl} methyl)amino]methyl} -N-( 1 methyl-lH-imidazol-2-yl)pyridine-4carboxamide	++								+++	+++
H,C ° ^CH3 <jT “

(a) +++; IC50 <100nM; ++: ΙΟΟηΜ < IC5O < ΙΟΟΟηΜ; +: IC5O>1 OOOnM

180

Example 3: Histone Lysine Demethvlase Immunofluorescence Assays for IC50 value Détermination in

Cell

Histone Lysine Demethylase Immunofluorescence Assays for IC50 value Détermination, non-transfected cells

This example demonstrates the ability of compounds of the invention to inhibit déméthylation 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, 10 the cells were washed once in PBS, harvested by fixation with formaldéhyde 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 température. Blocking was performed in PBS with 0.2 % Triton X-100 and 5 % FBS for 45 min at room température. The cells were incubated with aH3K4me3 primary antibody (Cell Signaling, #97518) 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, Al 1012) 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.

181

Histone Lysine Demethylase Inhibition

Table 4
Structure	Name	IC50
H	2- {[( {[ 1 -(3-phenylpropyl)piperi din- 4- yl]carbamoy 1} methyl)amino] methyi }pyridine-4-carboxylic acid	+
CH, γ	methyi 2-f{[3-(pyrrolidin-l- y l)propyl]amino} methyl)pyridi ne-4carboxylate	++
HO ,0 x,„ n ir Χη,	2-( {[4- (diethylamino)bu ty 1] ami no} methy l)p yridine-4-carboxylic acid	++
H° ° r^Si ⁰ Î^H) TH,	2-{[({[2- (dimethylamino)ethyl](ethyl)carbam oyl} methyl)amino]methyl} pyridine4-carboxylic acid	++

182

o'^a’ ό Y X L,	4-methoxyphenyl 2-( {[4- (diethylamino)butyl]amino}methyl)p yridine-4-carboxyl ate	+++
X γ ¹ ^·Χ Ύ·τ ^CH,	2-(ethoxycarbonyl)phenyl 2-( {[4- (di ethylami no)butyl ] amino} methyl)p yridine-4-carboxyiate	+++
'V⁰'^^³ 1 CH₃ ^XX^Z\^/ZSsN^zZSxCH₃ L CH₃	2-(dimethylamino)ethyl 2-({[4- (diethylamino)butyl]amino}methyl)p yridine-4-carboxyiate	-H-

183

CH, 1 X/k/ ^xch₃ ς	3-(dimethylamino)propyl 2-( {[4(diethylamino)butyl]amino}methyl)p yridine-4-carboxylate	++
ΥΎΊ ⁰ Wy° M H LhA/^N^^\_h/\_ch L,	{4- [(ethoxycarbonyl )amino]phenyl} met hyl2-({[4- (diethylamino)butyl]amino}methyl)p yridine-4-carboxylate	1 1 1
Γγ ^h^%A>x₀/^ch> Ôu^.^ ς	2,6-dimethoxyphenyl 2-({[4(diethylami no)bu tyl]amino} methyl)p yridine-4-carboxylate	+++
iy°	2,6-dimethylphenyl 2-( {[4(diethylamino)butyl] amino} methyl)p yridine-4-carboxy 1 ate	+++

184

HjC^q ψ Y ^{0 CH}J	4-methoxyphenyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)amîno]methyl} pyridine4-carboxylate	•H4-
Cl ° A o (Χ»Α/χΛ	2-(ethoxycarbonyl)phenyl 2- {[( {[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl )ami nojmethyl} pyridi ne4-carboxylate	+++
HjC Ί <p Vn Y° il ^H W\z\yx/x, <	{4- [(ethoxycarbonyl)(methyl)amino]phe nyl) methyl 2-({[4- (diethylamino)butyl]amino}methyl)p yridine-4-carboxy 1 ate	+++

185

CH, HsC^KCH, v (Γί ⁰ UsAAz'xA.,, ΎΗ1	4-tert-butyl phenyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl )ami no] methyl} pyridine4-carboxylate	+++
θ^γ'-'γ'™³ 1. CH₃ 0 H θ CH₃ ^hr ^t>r ch, k,	4-oxopentan-2-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)amino]methyl }pyridine4-carboxylate	+-H-
'VoY ⁰ Λ ⁰ Γ Χχ/χΑχ^Αί.	4-(trifluoroacetamido)butan-2-yl 2([({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)amino]methyl} pyrid i ne4-carboxylate	Ί·· l·

186

F CH₃ ° ^‘rii ⁰	4-(2,2,2-trifluoro-Nmethylacetamido)butan-2-yl 2{[({[2- (dimethylamino)ethyl](ethyl)carbam oyl} methyl )amino]methy 1} pyri dine4-carboxylate	ΊΙΙ·
Ύ îXaA^xA N ^XN » CH,	ethyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl )amino] methyl} pyridine4-carboxylate	+++
'Â^N^s^yOL-⁸^-^ η,γ ^ch₃	5-(trifluoroacetamido)pent-l-en-3-yl 2-{[({[2- (dimethylamino)ethyl](ethyl)carbam oyl} methyl )amino] methyl} pyri dine4-carboxylate	+++

187

F CHj \|ÎÎ⁵'n 0 C HjCr ^chj	5-(2,2,2-tri fluoro-Nmethylacetamido)pent-l-en-3-yI 2{[({[2- (dimethylamino)ethyl](ethyl)carbam oyl} me thyl)amino] methyl} pyridine4-carboxylate	+44-
% /’ η 'CHj CT '0 0^0^/Ζ\^/^ f/Aï) 0 CH₃ C^mL/xA A	2-(2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)amino]methy 1} pyridine4-carbonyloxy)-3(hexadecanoyloxy)propyl hexadecanoate	Ή· 1·

188

X (ΛαΛ~Λ e.	H2-{[({[2- (dimethylammo)ethyl](ethyl)carbam oyl} methy l)amino]methy 1} pyri dine4-carbonyloxy)-3- (hexadecanoyloxy)propan-2-yl hexadecanoate	+++
CH, 1 Y ^{0 CH}3 k ^xch₃	methyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)amino] methyl} pyridine4-carboxylate	4-H-
CH, 1 Ύ ₀AA₀ Zn» o ch, ^H II ¹ X >< >*x x\ CH, k CH₃	2-{[({[2- (dimethyl amino)ethyl ] (ethyl)carbam oyl }methyl)ami no] methyl }-Nmethanesulfonyl-N-methylpyridine4-carboxamide	++
r\ ΥΎ Coy__A ^CH,	N-[2-(dimethylamino)ethyl]-Nethy 1-2-( {[4-(2-oxo-1,3-oxazol idine3-carbonyl)pyridin-2yljmethyl} amino)acetamîde	++

189

propan-2-yl 3-(2-{[( {[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)ami no]methyl} pyrid i ne4-carbonyloxy)-4(tri fluoroacetamido)butanoate +++ propan-2-yl 3-(2-{[({[2(dîmethylamino)ethyl](ethyl)carbam oyl} methyl)amï nojmethyl} pyridine4-carbonyloxy)-5(trifluoroacetamido)pentanoate

2,2,2-trifluoroethyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl)amino]methyl} pyridine4-carboxylate

190

AAn 0 CH, ''CH,	2-{[({[2- (dimethy 1 ami no)ethyl](ethyl)carbam oyl} methyl)amino]methyl} -N-( 1,3oxazol -2-yl)pyridine-4-carboxamide	++
CH, Ï O^THj (^\ 0 CHj _ίΐΑγ^Ογ^Ζ^Ζ^/^Ν^^Ν^^^Ν\ΟΗ_ίCu L 1 H₃c^x THj	2,6-bis(propan-2-yloxy)phenyl 2{[({[2- (dimethyl amino)ethyl ](ethyl)carbam oy1}methyl)amino]methyl}pyridine4-carboxylate	++
H,C f/^¹? S Ç^Hi 1 H H 1 A/A -n jî n XHj	2-{[({[2- ( dimethylamino)ethy 1] (ethyl)carbam oyl} methyl )amino] methyl} -N-( 1 methyl-lH-imidazol-2-yl)pyridine-4carboxamide	++
_r J CH, 0	2-fluoroethyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbam oyl} methyl )ami no] methyl} pyridine4-carboxylate	+++

191

	^.CH,	y O'— \|	2,2-difluoroethyl 2-{[({[2- (dimethylamino)ethyl](ethyl)carbam	1 \| \|
HjC \|			oyl} methyl )amino] methyl} pyridine-
CH,			4-carboxylate

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

Exampie 4: Cell prolifération Assays for EC50 value Détermination

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

General method

MCF7 cells were seeded at 1250 cells/well in 50μ1 medium/well in black 96 well plates. Cells were incubated for 24 hours before addition of compound. Compounds were diluted in complété medium (50pl/well) and added to the plates in duplicates. The total volume of medium in the wells was 100 μΙ, and the final concentration of DMSO 0.5 %. Complété 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 ATPlite 1 Step (Perkin Elmer, cat no 6016739) according to the manufactures recommendation. Briefly, 100 μΙ ATP lite solution was added to each well, plates were vortexed at 700 rpm 2 minutes, followed by 20 minutes incubation in 15 the dark, and then analyzed for luminescence on EnSpire 2300 Mulitilabel reader (Perkin Elmer). EC50 values were calculated using GraphPad Prism 6. Results are seen in Table 5.

192

Table 5
Structure	Name	EC 50
°<γ^ΟΗ Λ) NH I	2-{[(cyclopropylmethyl)amino]methyl}pyridine-4carboxylic acid	-H-
°·γ^0Η ίΐ ^H X aK /^CHî X/ x/ s	2-( {[2-(methylsulfanyl)ethyl]ainino}methyl)pyridine-4carboxylic acid	-H-

193

H° 0	2-( {[( 1 -methyl-1 H-l ,3-benzodiazol-2- yl )methyl]amino} methyl)pyri dine-4-carboxylic acid	-H-
s ,	2-[( {[bîs(prop-2-en-1 - yl)carbamoyl]methyl} amino)methyl ] pyri dine-4carboxylic acid	++
Y V-Pf CO^ÀJ CH _s	2- {[( {methyl[3-(l -methyl-1 H-imidazol-2yl)propyl]carbamoyl} methyl )amino]methyl} pyridine-4carboxylic acid	++

194

’Υ'™ ο⁰¹ 0-^-A.XYÔ Η	2-( {[( {1 -[(2-methoxyphenyl)methyl]piperidin-4yl}carbamoyl)methyl]amino}methyl)pyridine-4carboxylic acid	++
^η°Ύ>°	2-[({[(2- cyanoethyl)(ethyl)carbamoy l]methyl} amino)methyl]pyr idine-4-carboxylic acid	++

195

F	2-{[(3-{[(2- fluorophenyl )methyl ](methyl)amino} propyl)amino] met hyl}pyridine-4-carboxylic acid	-H-
ï£ σ^?	2-({[3-{4-benzylpiperidin-l- yl)propyl]ammo} methyl)pyridine-4-carboxyl ic acid	-H-

196

197

2-( {[({4[benzyl(cyclopropyl)ami nojbutyl} (methyl)carbamoyl)m ethyljamino} methyl)pyridine-4-carboxylic acid

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

198

CH ₃0 C Y ^cHj ^CHj	4-methoxyphenyl 2-({[4- (die±ylamino)butyl]amino}methyl)pyridine-4carboxylate	+++
(Y) Y ⁰ >r ch₃ ^CHj	2-(ethoxycarbonyI)phenyl 2-({[4(diethylamino)butyl]amino} methyl )pyri dine-4carboxylate	+++

199

200

201

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

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

202

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

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

-H++

203

204

CH₃ H₃C^^CH₃ 9 Y O ch₃ ^ch₃	4-tert-butylphenyl 2-{[({[2- (dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino] methyl }pyridine-4-carboxylate	+++
Tl Y n Y o ch, ^hT ^FK ^CHj ^CH_a	4-oxopentan-2-yl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl} methyl)amino] methyl} pyridine-4-carboxylate	+++

205

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

206

207

208

209

210

9 /	2-(2-{[({[2-
	(dimethylammo)ethyl](ethyl)carbanioyl}methyl)aniino]	{ \| \|
	methyl} pyridine-4-carbonyl oxy)-3-
1 Y	(hexadecanoyloxy)propyl hexadecanoate
ÎiY ° ^CHs N N CH, ί
^ch₃

211

212

CH₃ u 0 CH₃ IAzUaa», xaij	methyi 2-{[({[2- (dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino] methyi }pyridine-4-carboxylate	+++
CH, 1 ^O^^N^/^CH3 \| cXX fA'A'i O CH, fi 1 » n i A -A/A A n x^ — x^ ^ch₃ ^Α:η₃	2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl}methyl)amino] methyi} -N-methanesuIfonyl-N-methylpyridine-4carboxamide	+++

213

	%	ΓΆ 0 O CH₃ ,A^^K Ak =:«, ^ch₃	N-[2-(dimethylamino)ethyl]-N-ethyl-2-({[4-(2-oxo-l,3oxazolidine-3-carbonyl)pyridm-2- yl]methyl} amino)acetamide	++
oJ oA	:h, k CH₃ /CH₃	propan-2-yl 3-(2-{[({[2-
%	r y^NH	^ü Γ Wmk l^^N 0 A_n/^CH3	(dimethylamino)ethyl](ethyl)carbamoyl} methyl)ami no] methyl }pyridine-4-carbonyloxy)-4(trifluoroacetamido)butanoate	++
	F	1 ch₃

214

propan-2-yl 3-(2-{[({[2(di methylamino)ethyl](ethyl)carbamoyl} methyl)amino] methyl }pyridine-4-carbonyloxy)-5 (trifluoroacetamido)pentanoate

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

215

216

ch, ο ^ν<_5>Χ	2-fluoroethyl 2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl} methyl)amîno] methyl} pyridine-4-carboxylate	+++
’γ Q—J -- C*S ο	2,2-difluoroethyl 2-{[({[2- (dimethylamino)ethyl] (ethyl )carbamoyl} methyl)amino] methyl} pyridi ne-4-carboxylate	+++

(a) +++: EC50 <1 μΜ; ++: 1 μΜ < EC50 < 50μΜ; +: EC50 > 50μΜ

217

Example 5: Histone Lysine Demethylase Immunofluorescence Assays for IC50 value Détermination in Cell

This example demonstrates the ability of the compounds of the invention to inhibit déméthylation of a spécifie H3 lysine in a human osteosarcoma cell line transfected to express a spécifie 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 formaldéhyde 4 % aqueous solution, and washed 2 times în PBS. Subsequently, the cells were permeabilized in PBS with 0.2 % Triton X-100 for 10 min at room température. Blocking was performed in PBS with 0.2 % Triton X-100 and 5 % FBS for 45 min at room température. 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. 11 (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, Al 1012; 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 (nontransfected 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.

218

Table 6
Construct name	Vendor/ source	Sequence	Mark detected	Primary antibody used for détection of mark	Plasmid NCBI ID
pCMVHA JMJD2C	BRIC	Full length	H3K9me3	Abcam Ab8898	NM_014663
pCMVHA JMJD2A	BRIC	Full length	H3K9me3	Abcam Ab8898	NM_015061
pCMVHA PLU1	BRIC	Fragment (1-752)	H3K4me2	Millipore 07-030	NM_006618

219

HDME INHIBITION

Table 7
Structure	Name	GASC1 (KDM4C)	JMJD2A (KDM4A)	PLU1 (KDM5B)
° on	2-({[3-(lH-imidazol-l- yl)propyl]aniino}niethyl)pyridme-4-carboxylic acid	+		++
^{0 OH} C /^cHs 1 ch₃	2-({[2- (dimethylamino)ethyljamino} methyl)pyridine-4carboxylic acid	++		-H-

220

Ύ CVJL·-	2-({[(2R)-2,3- dihydroxypropyl]amino} methyl)pyridine-4carboxylic acid	+	+
° °Η A f	2- {[(cyclopropylmethyl)amino] methyl} pyridine-4carboxylic acid	+	++
'V Λ . ï <*3	2-({[4- (dimethyl amino)butyl]amino} methyl)pyridine-4carboxylic acid	+	++

221

γ ΙΊ	Η CH, 1 \ /^ΝΧ Ζ\ /^CH3 1 ch₃	2-({[2- (dimethylamino)ethyl](methyl)amnio}methyl)pyridin e-4-carboxylic acid	+		+
^ΗΟχ X	/0 k çh₃ ΑΧ*	2-{[methyl(prop-2-yn-l-yl)amino]methyl}pyridine- 4-carboxylic acid	+		+
	^HOy° ά ^Ν^\| UN °ό	2- {[(furan-2-ylmethyl)amino] methyl} pyridine-4carboxylic acid	++	++	++

222

^{0 0H} xk H X /^CH3	2-({[2- (methylsulfanyl)ethyl]amino}methyl)pyridine-4carboxylic acid		++
%γ^0Η fil ⁰	2-( {[2-oxo-2-(pyrrolidin-l - yl)ethyl]amino} methyl)pyridine-4-carboxylic acid	-H-		+
Y UOJL/^x_Hj 1 ch₃	2- [( {[butyl(methyl)carbamoyl] methyl} amino)methyl]p yridine-4-carboxylic acid	-H-		+

223

Ύ Λ γΥ η/	2-( {[( 1 -methyl-1 Η-1,3-benzodiazol-2yl)methyl]amino}methyl)pyridine-4-carboxylic acid	+	-H-
	2-[({2-[4-(2-methoxyethyl)piperazin-l-yl]-2oxoethyl} amino)methyl]pyridine-4-carboxylic acid	+	+
^ΗΟγ° 0A^KxA_n/^^w·	2- [( {[bis(prop-2-en-1 - yl)carbamoyl]methyl} amino)methyl]pyridine-4caiboxylic acid	-H-	++

224

^ΗΟγ°	2- {[(3- {[3-(pyrrolidin-1 - yl)propyl]amino} propyl)amino] methyl} pyridine-4carboxylic acid	+		+
HO J*	Y° o 1 ch₃	nh₂	2-[({[(2- carbamoylethyl)(methyl)carbamoyl]methyl}amino)m ethyl]pyridine-4-carboxylic acid	+
H	°γ° HaC'* UOJJ 1 ch₃	/=\ r	2-{[( {methyl[3-(l -methyl-1 H-imidazol-2yl)propyl]carbamoyl} methyl)amino] methyl} pyridine -4-carboxylic acid	++	++

225

^H0-y° ό _K JÎ ^H3^C-—^/^N /	2- {[({[( 1 -ethylpyrrolidin-2- yl)methyl]carbamoyl}methyl)amino]methyl} pyridine -4-carboxylic acid	+	++
^ΗΟγ° CH, Ν J x ^-N h₃c	2- {[( {methyl[( 1 -methyl-1 H-pyrazol-5yl)methyl]carbamoyl} methyl)amino]methyl} pyridine -4-carboxylic acid	+	+
0 0« 0-^SY\ Ly-V-, O	2-( {[(3R)-I -(3-phenylpropyl)pyrrolidin-3yl]amino}methyl)pyridme-4-carboxylic acid	+	++

226

° œ C/XO'+Î H	2-( {[( {1 -[(2-methoxyphenyl )methyl]piperidin-4yl}carbamoyl)methyl]amino}methyl)pyridîne-4carboxylic acid		-H-
Y H	2- {[( {[ 1 -(3-phenylpropyl)piperidin-4yljcarbamoyl} methyl)amino] methyl} pyridine-4carboxylic acid	+	+
CuroO·»	2- {[( {[ 1 -( fiiran-2-ylmethyl)piperidi n-4- yl] carbamoyl} methyl)amino] methyl} pyridine-4carboxylic acid	+	+

227

Ύ Η	2-( {[( {1 -[(5 -phenylfuran-2-yl)methyl]piperidiii-4yl} carbamoyl)methyl]ammo} methyl)pyridine-4carboxylic acid	+	+
HO 0 _HJCP	2-[({[(2- cyanoethyl)(ethyl)carbamoy I] methyl} amino)methyl] pyridine-4-carboxylic acid		++
° on Λ «	2-( {[2-( 1 -butylpyrrolidm-2- yl)ethyl]amino} methyl)pyridine-4-carboxylic acid		-w-
° °H ό & r n F	2-{[(3-{[(2fluorophenyl)methyl](methyl)amino}propyl)amino] methyl }pyridine-4-carboxylic acid	+	++

228

OH _Λ λ · r° (Ααλ^Χ, k V	2-({[({4- [benzyl(cyclopropyl)amino] butyl} (methyl)carbamoyl )methyl]amino} methyl)pyri dine-4-carboxylic acid		+
0 OH	2-[( {2-[(2S)-l -benzylpyrrolidin-2- yl] ethy 1} amino)methyl] pyridine-4-carboxylic acid	+	++ 1
^ΗΟγ° όυ^ο	2-( {[3-(pyiTolidin-1 - yl)propyl ] ami no} methyl)pyridine-4-carboxylic acid	++	-H-
CH, 1 Y (XU)	methyl 2-({[3-(pyrrolidin-l- yl)propyl]amino} methyl)pyridine-4-carboxylate	++	++

229

Ύ	2-( {[4-(diethylamino)butyI]amino} methyl)pyridine4-carboxylic acid	++		-H-
⁰ \X/“xjL_N^-k	2-{[({[2- (dimethylamino)ethyl](ethyl)carbamoyl} methyl)ami no]methyl}pyridine-4-carboxylic acid	+	++	++
O-⁰¹· Φ Y	4-methoxyphenyl 2-({[4(diethylamino)butyl] amino} methyl)pyridine-4carboxylate	-H-		+++

230

W γ » ^CH,	2-(ethoxycarbonyl)phenyl 2-({[4- (diethylamino)butyl]amino}methyl)pyridine-4carboxylate	++	+++
x CH, <	2-(dimethylamino)ethyl 2-({[4- (diethylamino)butyl]amino}methyl)pyridine-4carboxylate	++	++
î¹’ Ol ^h ^CH,	3-(dimethylamino)propyl 2-( {[4- (diethylamino)butyl] amino} methyl)pyridine-4carboxylate	++	++

231

H,C Ί “ νη ίΐ ^Η ''''CH,	{4-[(ethoxycarbonyl)(methyl)amino]phenyl} methyl 2-(([4-(diethylamîno)butyl] amino} methyl)pyridine- 4-carboxylate		+++
ΛΑ λ 9	2-(2-{[({[2(dimethylamino)ethyl](ethyl)carbamoyl} methyl)ami no]methyl} pyridine-4-carbonyloxy)-3 (hexadecanoyloxy)propyl hexadecanoate		++

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

232

In this spécification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that retums 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, Ail prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or représentation that the teaching thereof was common general knowledge in Austral ia 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; 10691078

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 R et al., PNAS 105(6), 1907-12, 2008

Queguiner, G. and Pastour, P., Comptes Rendus des Séances de ['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; 1563-1569 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

233

Claims

1, A compound of the general Formula (I)

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

Y is selected from -H, -NR⁶R⁷, -OR⁷, Cm alkyl, C₂.8 alkenyl, C₂._g 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 and may form a cyclic structure with R²;

R¹ is selected from -H, Cm alkyl, C₂-₈ alkenyl, C₂-₈ alkynyl, C₃.i₀ 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; or more preferably is selected from H and Cm alkyl; or with -A-Y forms a nitrogen containing optionally substituted heterocyclic group where the optional substitution may be Cm alkyl, C₂._g alkenyl, C₂._g 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 C_M cycloalkyl;

R² is selected from -H, Cm alkyl, C₂._g alkenyl, C₂.₈ alkynyl, and C₃.₎₀cycloalkyl, 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 C₃.₆cycloalkyl, and may form a cyclic structure with Y;

each R³ is independentiy selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyl, C₂.6 alkenyl, C₂.6 alkynyl, C₃.₁₀ cycloalkyl, -Z-heterocyclyl, -Z-aryl, -Z- heteroaryl, -Z-NR⁶R⁷, -Z-C(=O)-NR⁶R⁷, -Z-NR⁶C(=O)-R⁷, -Z-C(=O)-R⁷, -Z-OR⁷, haiogen, -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 R^s;

234

Z is selected from a single bond, Cm alkylene, heterocyclylene and C₃._h cycloalkylene;

each R⁴ is independently selected from Cmalkyl, C_M fluoroalkyl, Cm hydroxyalkyi, C_M alkoxy, C₃.i₀cycloalkyl, -N(R')₂, carbamoyl, and -OH;

each R^J is independently selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyi, Cm alkoxy, C₃.₆cycloalkyl, -CN, -F, -Cl, -Br, carbamoyl and -OH;

each of R⁶ and R⁷ is independently selected from -H, Cm alkyl, C_M fluoroalkyl, Cm perfluoroalkyl, Cm hydroxyalkyi, C₂._s alkenyl, C₂.₈ alkynyl, C₃_io 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; or, altematively, 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 R⁸ is independently selected from Cm alkyl, CM fluoroalkyl, Cm hydroxyalkyi, C2.6 alkenyl, Cm alkynyl, C3_i0 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Z-aryl, -Z-NR'^R¹¹, -Z-C(=0)-NR^ioR¹’, Z-OR⁹, halogen, -CN, -Z-SR⁹, -Z-SOR⁹, -Z-SO2R⁹ and -Z-COOR⁹, which alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclics, heteroaryl and aryl may optionally be substituted with one or more selected from Cm alkyl, Cm fluoroalkyl, Cm hydroxyalkyi, C3.6 cycloalkyl, -Z-heterocyclyl, -Z-heteroaryl, -Zaryl, -Z-NR^IOR, -Z-C(=O)-NR^l0R, -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 R^s as defined above, and each R⁹ is independently selected from -H, Cm alkyl, Cm fluoroalkyl, C_M hydroxyalkyi, C₂.galkenyl, C₂.g alkynyl, C₃._[0 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 R^s as defined above;

each of R¹⁰ and R is independently selected from -H, Cmalkyl, Cm fluoroalkyl, Cm hydroxyalkyi, C₂.

₈ alkenyl, C₂.8 alkynyl, C₃.₁₍₎ 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, altematively, R¹⁰ and R¹¹ may together with the

N-atom to which they are attached form an N-heterocyclic ring optionally substituted with one or more

R⁴ as defined above;

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

235 or an isomer or a mixture of isomers thereof, or a pharmaceutically acceptable sait, solvaté or prodrug thereof.

2. A compound according to claim 1, wherein A is selected from -CHR²C(O)-, or Ci._g alkylene, or heterocyclylene.

5

3. A compound according to claim 1 or claim 2, wherein Y is -NR⁶R⁷.

4. A compound according to claim 3, wherein A is -CHR²C(O)-.

5. A compound according to claim 4, wherein A is -CH₂-C(O)-.

6. A compound according to any one of claims 2 to 5, wherein Y is

Rio

Rio wherein n is from 1 to 3 and each of Rio and Rn independently is as defined in claim 1.

7. A compound according to claim 6, wherein Y is ;CH₂)_m----CH₃ wherein n is from 1 to 3 and each m independently is from 0 to 2.

8, A compound according to claim 1 or claim 2, wherein Y is selected from heterocyclyl,

15 heteroaryl and aryl, which may be optionaily substituted with one or more R³.

236

9. A compound according to any one of the preceding claims, which has a molecular weight of 130-1,000 g/mol, such as 180-800 g/mol, e.g. 225-600 g/mol or 250-500 g/mol.

10. A compound according to any one of the preceding claims, wherein the moiety -A-Y inciudes

1-3 cyclic moietîes selected from monocylic cycloalkyl, monocyclic heterocyclyl, monocylic

5 heteroaryl, dicyclic heteroaryl and monocyclic aryl.

11. A pharmaceutical composition comprising at least one compound of Formula (I) as defined in any one of the claims 1-10 and optionally one or more pharmaceuticaily acceptable excipients, diluents or carriers.

12. A pharmaceutical composition according to claim 7, which comprises one or more further

10 active substances.

13. A compound according to any one of the claims 1 -10 for use as a médicament.

14. A compound according to any one of the claims 1 -10 for use in the treatment of a HDME dépendent disease.

15. Use of a compound according to any one of the claims 1-10 for the préparation of a

15 pharmaceutical composition for the treatment of a HDME dépendent disease.

16. A method of treating a HDME dépendent 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 in any one of the claims 1-10.