RU2851631C2 - Quinoline derivatives for treatment of inflammatory diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the use of miR-124 in vitro or ex vivo as a biomarker for screening a compound of formula (I) for efficacy in the treatment and/or prevention of an inflammatory disease selected from the group consisting of inflammatory joint disease, inflammatory digestive tract disease, inflammatory skin disease, bronchitis, bronchial asthma, or any of its pharmaceutically acceptable salts, as well as to a method for screening in vitro or ex vivo a compound of formula (I) for efficacy in the treatment and/or prevention of an inflammatory disease selected from the group consisting of inflammatory joint disease, inflammatory digestive tract disease, inflammatory skin disease , bronchitis, bronchial asthma, and a method for in vitro or ex vivo evaluation and monitoring of the efficacy of of compound (I) in the treatment of an inflammatory disease selected from the group consisting of inflammatory joint disease, inflammatory digestive tract disease, inflammatory skin disease, bronchitis, bronchial asthma , comprising determining the presence or expression level of miR-124 in an isolated biological sample from an individual, wherein the expression level of miR-124 is compared to a control reference value, wherein an increase relative to the control reference value indicates efficacy.

EFFECT: use of miR-124 in vitro or ex vivo as a biomarker for screening compound of formula (I) for efficacy in the treatment and/or prevention of an inflammatory disease selected from the group consisting of inflammatory joint disease, inflammatory digestive tract disease, inflammatory skin disease, bronchitis, and bronchial asthma.

19 cl, 6 dwg, 3 tbl, 9 ex

Description

FIELD OF INVENTION

The invention relates to the identification of new quinoline derivatives effective for the treatment and/or prevention of inflammatory diseases, as well as to new types of therapeutic use of a quinoline derivative against inflammatory diseases.

The invention also relates to the field of biomarkers in combination with such inflammatory diseases.

PRIOR ART

Inflammation is the immune system's protective response to tissue damage and infection. However, in some circumstances, the inflammatory response can be damaging. In the acute phase, inflammation is characterized by pain, fever, redness, swelling, and loss of function. There is a wide range of inflammatory conditions that affect millions of people worldwide.

Indeed, inflammatory diseases encompass a wide range of conditions, including autoimmune-related inflammatory disease, inflammatory disease of the central nervous system (CNS), inflammatory joint disease, inflammatory gastrointestinal disease, and inflammatory skin disease. Of particular interest are inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis.

Inflammatory bowel disease (IBD) is a complex, multifactorial disorder It generally refers to two chronic conditions: ulcerative colitis (UC) and Crohn's disease (CD), which involve intestinal inflammation. IBD is common in developed countries, affecting up to 1 in 200 individuals in the Northern European region. Patients with IBD present several clinically significant challenges for physicians. Dextran sulfate sodium-induced colitis (DSS-induced colitis) is caused by upregulation of various proinflammatory cytokines, including tumor necrosis factor (TNF) alpha and interferon (IFN) gamma. Recently, the microRNA-124 gene has been shown to be dysregulated, particularly in pediatric patients with active UC, resulting in elevated levels of signal transducer and activator of transcription 3 (STAT3) expression and transcriptional activation of downstream targets, including proinflammatory cytokines (Koukos et al.; Gastroenterology; 145(4):842-52: 2013). However, despite recent advances, there remains a need for safe, well-tolerated therapies with rapid onset of action and improved ability to maintain long-term remission.

Rheumatoid arthritis (RA) is the most common autoimmune disease, affecting approximately 0.3 to 1% of the population worldwide. It is often associated with limited mobility, increased social dependence, and disability. RA is a systemic inflammatory disease affecting the tissue lining the joint, called synovium. Rheumatoid synovial tissue is characterized by hyperproliferation of fibroblast-like synoviocytes (FLS) in the intimal layer of the lining and infiltration of the subsynovial layer by macrophages, T and B cells, and other inflammatory cells, which contribute to inflammation and destruction of bone and cartilage. Intra-articular and systemic expression of proinflammatory cytokines, particularly tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1), and interleukin-6 (IL-6), produced primarily by synovial macrophages, plays a critical role in the pathogenesis of RA, for example, by contributing to the hyperproliferation of RA FPS. Patients with RA are typically treated with a group of small molecule drugs called disease-modifying rheumatoid arthritis drugs (DMARDs). DMARDs suppress the body's overactive immune and/or inflammatory systems in several ways, thereby slowing disease progression. RA patients who are unresponsive to DMARDs are treated with biologic agents such as tumor necrosis factor (TNF) antagonists. However, even though TNF antagonists are effective in approximately two-thirds of patients, they often become ineffective within 5 years. Therefore, alternative treatments are needed. Of particular interest are new therapeutic approaches developed for patients with RA in its early stages, before the disease becomes chronic.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disorder of the central nervous system, resulting in the destruction of myelin, oligodendrocytes, and axons. MS is characterized by multiple foci of inflammation and infiltration of macrophages and encephalitogenic T cells throughout the central nervous system. Microglia are present throughout the central nervous system and are involved in the onset and progression of inflammatory diseases of the central nervous system. Microglia, when activated, significantly damage CNS function through the production of neurotoxins, inflammatory cells (inflammatory protein-10, macrophage inflammatory protein-1, macrophage inflammatory protein-2, C-C chemokine ligand 19, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2), and antibody-producing immune cells. Microglia direct inflammatory responses that can result in the infiltration of the brain and spinal cord by immune cells directed against foreign organisms, as well as T cells that destroy myelin proteins. Peripheral macrophages appear in the CNS during inflammation, and these cells have a highly activated phenotype, effectively stimulate the proliferation of encephalitogenic T cells, and are believed to be involved in the destruction of nervous tissue.

MicroRNAs (miRNAs), which constitute the largest group of noncoding RNAs, are a class of noncoding RNAs approximately 22 nucleotides (nt) in length that inhibit gene expression by binding to the untranslated region (UTR) of target transcript mRNAs (Lai et al., Nature Genetics, vol. 30, no. 4, pp. 363–364, 2002; Bartel et al., Cell, vol. 136, no. 2, pp. 215–233, 2009). miRNA genes constitute approximately 1–2% of known eukaryotic genomes. It is predicted that each miRNA can target more than 200 transcripts, and that a single miRNA can be regulated by multiple miRNAs (LINDOW, DNA Cell Biol., vol. 26(5), pp. 339–351, 2007). miRNAs are formed from endogenous hairpin-shaped transcripts and act through base pairing with target mRNAs, resulting in mRNA cleavage or translational repression depending on the degree of base pairing. Two processing events lead to the formation of miRNAs: first, nascent miRNA transcripts (pri-miRNAs) are processed to 70-nucleotide precursors (pre-miRNAs), which are exported from the nucleus and cleaved in the cytoplasm to form short (approximately 22 nucleotides long) mature miRNAs (LEE, EMBO J., vol. 21, pp. 4663–4670, 2002). miRNAs can have intergenic or intragenic localization. When intergenic, their expression is coordinated with other miRNAs in a cluster (Altuvia et al., Nucleic Acids Research, vol. 33, no. 8, pp. 2697–2706, 2005; Ozsolak et al., Genes and Development, vol. 22, no. 22, pp. 3172–3183, 2008). When intragenic, i.e. When located within a protein-coding gene (almost exclusively in introns), they are often expressed from the same strand as their host gene (Liu et al., Cell Research, vol. 18, no. 10, pp. 985–996, 2008; Kim et al., EMBO Journal, vol. 26, no. 3, pp. 775–783, 2007), and at levels that correlate with it (Baskerville et al., RNA, vol. 11, no. 3, pp. 241–247, 2005).

Overexpression of miRNAs, particularly miRNA-124, has been shown to inactivate inflammatory macrophages and convert them into microglial-like cells. miRNA-124 is believed to inhibit macrophage activation by targeting CEBPα, a transcription factor responsible for myeloid cell differentiation. Intravenous injection of liposomes containing miRNA-124 significantly suppresses the clinical symptoms of experimental allergic encephalomyelitis (EAE) and inhibits the infiltration of encephalitogenic T cells and inflammatory macrophages into the central nervous system.

Indeed, Ponomarev et al. (“microRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α-PU.1 pathway”; Nature Medicine (2011); 17:1: 64–71) suggest that miRNA-124 may play a role as a key regulator of microglia quiescence and as a modulator of monocyte and macrophage activation. Based on an experimental allergic encephalomyelitis (EAE) model, this study suggests that the miRNA-124 expression pattern is modulated (either up-regulated or down-regulated depending on the cell type) in EAE mice.

WO 2010/151755 also discloses the administration of miRNA-124 for the treatment of an inflammatory disease of the central nervous system (CNS).

Sun et al.'s paper ("microRNA-124 mediates the cholinergic anti-inflammatory action through inhibiting the production of pro-inflammatory cytokines"; Cell Research (2013); 23:1270–1283) also suggests that miRNA-124 may mediate cholinergic anti-inflammatory effects by targeting STAT3 and TNF-alpha-converting enzyme (TACE).

On the other hand, new compounds have been identified, which are also referred to in this document as “quinoline derivatives”, but for specific indications.

As a reference, quinoline is a heterocyclic aromatic organic compound of the formula:

Thus, "quinoline derivatives" include substituted quinolines, such as mono- or polysubstituted quinolines.

WO 2010/143170 relates to the use of compounds for the treatment of conditions associated with premature aging.

From WO 2010/143169 and WO 2012/080953 follows the use of compounds for the treatment of acquired immunodeficiency syndrome (AIDS).

WO 2010/143168 discloses the use of compounds for the treatment of certain types of cancer. These compounds have been shown to be capable of correcting alternative splicing disorders.

SUMMARY OF THE INVENTION

It has now been discovered that compounds as hereinafter defined in formula (I) are useful in the treatment and/or prevention of inflammatory diseases.

Thus, the present invention relates to compounds of formula (I), as defined below, for use in the treatment and/or prevention of inflammatory diseases.

In particular, the invention relates to compounds of formula (I), as defined below, for use in the treatment and/or prevention of inflammation and/or inflammation that may occur in conjunction with such inflammatory diseases.

The invention also relates to the use of at least one miRNA in vitro or ex vivo, wherein at least one miRNA is miRNA-124, as a biomarker for screening a quinoline derivative and in particular a compound of formula (I), suspected of being effective in the treatment and/or prevention of an inflammatory disease.

The invention further relates to a compound of formula (Id) or (Ie), as defined herein below, as such.

The invention further relates to a pharmaceutical composition comprising at least one compound of formula (Id) or (Ie).

It also refers to a compound as such, selected from the list consisting of:

- (8) 8-chloro-5-(3-(piperidin-1-yl)propoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (9) 8-chloro-N ⁴ -(3-(piperidin-1-yl)propyl)-N ² -(4-(trifluoromethyl)pyridin-2-yl)quinoline-2,4-diamine;

- (10) 8-chloro-N-methyl-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (11) 8-chloro-N ⁴ -(2-morpholinoethyl)-N ² -(4-(trifluoromethyl)pyridin-2-yl)quinoline-2,4-diamine;

- (13) 4,8-dichloro-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (14) 8-chloro-N-(3-morpholinopropyl)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (15) 8-chloro-6-(2-morpholinoethoxy)-N-(3-morpholinopropyl)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (16) 8-chloro-5-(2-morpholinoethoxy)-N-(3-morpholinopropyl)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (17) 8-chloro-6-(2-(4-methylpiperazin-1-yl)ethoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (18) 8-chloro-6-(2-(piperidin-1-yl)ethoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (19) 8-chloro-6-(3-(piperidin-1-yl)propoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (20) 8-chloro-N-(3-fluoro-4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (21) N-(5-bromo-4-(trifluoromethyl)pyridin-2-yl)-8-chloroquinolin-2-amine;

- (22) N ² -(8-chloroquinolin-2-yl)-N ⁵ -(3-(4-methylpiperazin-1-yl)propyl)-4-(trifluoromethyl)pyridine-2,5-diamine;

- (28) 8-chloro-N-methyl-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (29) 8-chloro-5-(3-(piperidin-1-yl)propoxy)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (30) 8-chloro-N-(3-(piperidin-1-yl)propyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (31) 8-chloro-N-(2-morpholinoethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (32) 8-chloro-N-(2-(pyrrolidin-1-yl)ethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (33) 8-chloro-N-(4-morpholinobutyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (34) 8-chloro-N ⁴ -(3-(piperidin-1-yl)propyl)-N ² -(4-(trifluoromethoxy)phenyl)quinoline-2,4-diamine;

- (35) 4,8-dichloro-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (36) 8-chloro-5-(2-morpholinoethoxy)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (37) N ¹ -(4,8-dichloroquinolin-2-yl)-4-(trifluoromethoxy)benzene-1,2-diamine;

- (38) 4,8-dichloro-N-(2-morpholinoethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (39) 8-chloro-6-(2-morpholinoethoxy)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (40) 8-chloro-N ² -(2-morpholinoethyl)-N ⁴ -(3-(piperidin-1-yl)propyl)-N ² -(4-(trifluoromethoxy)phenyl)quinoline-2,4-diamine;

- (41) 8-chloro-N-(2-morpholinoethyl)-N-(2-nitro-4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (42) N ¹ -(8-chloroquinolin-2-yl)-N ¹ -(2-morpholinoethyl)-4-(trifluoromethoxy)benzene-1,2-diamine;

- (43) 8-chloro-5-(2-morpholinoethoxy)-N-(2-morpholinoethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (44) N ¹ -(8-chloro-5-(2-morpholinoethoxy)quinolin-2-yl)-4-(trifluoromethoxy)benzene-1,2-diamine;

- (46) 8-chloro-2-((4-(trifluoromethyl)pyridin-2-yl)oxy)quinoline;

- (47) 4-(2-((8-chloro-2-((4-(trifluoromethyl)pyridin-2-yl)oxy)quinolin-6-yl)oxy)ethyl)morpholine;

- (48) 8-chloro-2-(4-(trifluoromethoxy)phenoxy)quinoline;

- (49) 4-(2-((8-chloro-2-(4-(trifluoromethoxy)phenoxy)quinolin-6-yl)oxy)ethyl)morpholine;

- (50) 4-(2-((8-chloro-2-(4-(trifluoromethoxy)phenoxy)quinolin-5-yl)oxy)ethyl)morpholine;

- (51) mono-[8-chloro-2-(4-trifluoromethyl-pyridin-2-ylamino)-quinolin-6-yl] ester of phosphoric acid;

- (52) mono-[2-(8-chloro-quinolin-2-ylamino)-5-trifluoromethoxy-phenyl] ester of phosphoric acid;

- (53) mono-[8-chloro-2-(4-trifluoromethoxy-phenylamino)-quinolin-6-yl] ester of phosphoric acid

- and their pharmaceutically acceptable salts, and more particularly selected from compounds (8), (9), (10), (11), (30), (46), (47), (48), (49) and (50), as defined above, or one of their pharmaceutically acceptable salts.

The invention also relates to a pharmaceutical composition comprising at least one compound of formula (Id) or (Ie) or one of the compounds (8), (9), (10), (11), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (46), (47), (48), (49), (50), (51), (52) and (53), and more particularly one of the compounds (8), (9), (10), (11), (30), (46), (47), (48), (49) and (50), as defined above.

The invention further relates to a method for increasing the expression of miRNA-124 in vitro or ex vivo in a eukaryotic cell, comprising at least the step of:

a) provide the eukaryotic cell,

b) bringing this cell into contact with a quinoline derivative and, in particular, with a compound of formula (I).

The invention further relates to a method for screening in vitro or ex vivo a derivative compound, and in particular a compound of formula (I), suspected of being effective in the treatment and/or prevention of an inflammatory disease, wherein the method comprises at least the step of:

a) provide the eukaryotic cell,

b) bringing the cell into contact with a compound of formula (I),

c) determine the expression of miRNA-124 in the cell, and

d) selecting a candidate compound that is suspected of being effective in the treatment and/or prevention of an inflammatory disease when the expression level of miRNA-124 determined in step c) is increased relative to a reference value.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: Dextran sodium sulfate (DSS-)-induced colitis model 1 - percentage change in weight loss over time (days) in the mouse DSS model in the presence of a quinoline derivative compound (24), as defined herein below. Percentage weight loss (%) is indicated on the y-axis. DSS treatment is administered on days 3-10 (x-axis). The quinoline derivative in methylcellulose (MC) or MC alone is administered by gavage from days 3 to 29.

Fig. 2: Dextran sodium sulfate (DSS-)-induced colitis model 2 - percentage change in weight loss over time (days) in the mouse DSS model after the second cycle of DSS in the presence of a quinoline derivative compound (24), as defined herein below. Percentage weight loss (%) is indicated on the y-axis. DSS treatment began on day 12 and continued for a period of 5 days (x-axis). The quinoline derivative in methylcellulose (MC) or MC alone was administered by gavage from days 3 to 29.

Fig. 3: Dextran sodium sulfate (DSS-)-induced colitis model - colons were removed 2-3 days after the second DSS administration (for 5 days) and the change in colon size (cm) was measured. Statistically insignificant differences are indicated as "ns". Statistical analysis was performed using the Mann-Whitney test, an asterisk indicates a statistically significant difference (p<0.5), two asterisks indicate a highly statistically significant difference (p<0.05).

Fig. 4: Dextran sodium sulfate (DSS)-induced colitis model - colons were removed 3 days after the second DSS administration and analyzed for the number of lesions. Lesions were observed microscopically and measured.

Fig. 5: Dextran sodium sulfate (DSS-)-induced colitis model - colons were removed 3 days after the second DSS administration and the lesion area (mm ² ) was determined.

Fig. 6: Collagen-induced arthritis model - change in joint swelling (mm) over time (weeks) in a mean cohort of 10 animals. Joint swelling (mm) is shown on the y-axis. Changes between treated versus untreated groups were assessed over 12 weeks.

DETAILED DESCRIPTION OF THE INVENTION

There is a need to identify new compounds for use in the treatment and/or prevention of inflammatory diseases.

There is also a need for a new biomarker to assess the activity of drug candidates, such as quinoline derivatives, targeting inflammatory diseases.

The purpose of the present invention is to meet these needs.

Quinoline derivatives

According to a first aspect, the subject matter of the present invention relates to the use of a compound of formula (I):

(I)

Where:

Z represents C or N,

V is C or N,

means an aromatic ring where V is C or N, and when V is N, V is in the ortho-, meta- or para-position relative to Z, i.e. forms a pyridine, pyridazine, pyrimidine or pyrazine group, respectively,

R independently represents a hydrogen atom, a halogen atom or a group selected from a -CN group, a hydroxyl group, a -COOR ₁ group, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group, a (C ₃ -C ₆ )cycloalkyl group, a -NO ₂ group, a -NR ₁ R ₂ group, a (C ₁ -C ₄ )alkoxy group, a phenoxy group, a -NR ₁ -SO ₂ -NR ₁ R ₂ group, a -NR ₁ -SO ₂ -R ₁ group, a -NR ₁ -C(=O)-R ₁ group, a -NR ₁ -C(=O)-NR ₁ R ₂ group, a -SO ₂ -NR ₁ R ₂ group, a -SO ₃ H group, a -O-SO ₂ -OR ₃ group, a -OP(=O)-(OR ₃ )(OR ₄ ), the -O-CH ₂ -COOR ₃ group and a (C ₁ -C ₃ )alkyl group, wherein the alkyl is optionally monosubstituted with a hydroxyl group,

Q is N or O provided that R'' is absent when Q is O,

R ₁ and R ₂ independently represent a hydrogen atom or a (C ₁ -C ₃ )alkyl group,

R ₃ and R ₄ independently represent a hydrogen atom, Li ⁺ , Na ⁺ , K ⁺ , N ⁺ (Ra) ₄ or a benzyl group,

n is 1, 2 or 3,

n' is 1, 2 or 3,

R' independently represents a hydrogen atom or a group selected from a (C ₁ -C ₃ )alkyl group, a halogen atom, a hydroxyl group, a -COOR ₁ group, a -NO ₂ group, a -NR ₁ R ₂ group, a morpholinyl or morpholino group, an N-methylpiperazinyl group, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₄ )alkoxy group, a -OP(=O)-(OR ₃ )(OR ₄ ) group and a -CN group, and may further represent a group selected from:

A represents a covalent bond, an oxygen atom or NH,

B represents a covalent bond or NH,

m is 1, 2, 3, 4 or 5,

p is 1, 2, or 3,

Ra and Rb independently represent a hydrogen atom, a (C ₁ -C ₅ )alkyl group or a (C ₃ -C ₆ )cycloalkyl group,

Ra and Rb may further form, together with the nitrogen atom to which they are attached, a saturated 5- or 6-membered heterocycle, optionally containing an additional heteroatom selected from N, O and S, wherein the heterocycle is optionally substituted with one or more Ra, provided that when R' is a group (IIa) or (IIIa), n' may be equal to 2 or 3 only if the other groups R' are different from a group (IIa) or (IIIa),

R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group, or a group (IIa) as defined above,

or any of its pharmaceutically acceptable salts,

in the treatment and/or prevention of inflammatory diseases.

According to a preferred embodiment of the invention, Q is N.

According to another preferred embodiment of the invention, n is 1 or 2.

According to another preferred embodiment of the invention, n' is 1 or 2.

According to another preferred embodiment of the invention, R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group or a group ,

where m is 2 or 3 and X ₁ is O, CH ₂ or N-CH ₃ .

According to another preferred embodiment of the invention, R independently represents a hydrogen atom, a methyl group, a methoxy group, a trifluoromethyl group, a trifluoromethoxy group, an amino group, a halogen atom and a -OP(=O)-(OR ₃ )(OR ₄ ) group, and more particularly a fluorine or chlorine atom, a trifluoromethoxy group and an amino group.

According to another preferred embodiment of the invention, R' independently represents a hydrogen atom, a halogen atom, and more particularly a fluorine or chlorine atom, an amino group, a methyl group, a -OP(=O)-(OR ₃ )(OR ₄ ) group, or a group ,

where A is O or NH, m is 2 or 3, and X ₁ is O, CH ₂ or N-CH _3, provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group.

According to another aspect of this preferred embodiment of the invention, R' alternatively independently represents a hydrogen atom, a halogen atom, and more particularly a fluorine or chlorine atom, a methyl group or a group ,

where A is O or NH, m is 2, and X ₁ is O, CH ₂ or N-CH _3, provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group.

All preceding and following specific embodiments of the invention may, of course, be combined together and form part of the invention.

Compounds of formula (I) include compounds of formula (Ia), (Ib), (Ic), (Id) and (Ie), as defined herein below.

According to a specific embodiment of the invention, another object of the present invention is the use of a compound of formula (Ia):

(Ia)

where R, R', R'', n and n' are as defined above,

in the treatment and/or prevention of inflammatory diseases.

According to one aspect of this preferred embodiment of the invention, n is 1 or 2.

According to one aspect of this preferred embodiment of the invention, n' is 1 or 2.

According to one aspect of this preferred embodiment, R independently represents a hydrogen atom, a halogen atom, or a group selected from a hydroxyl group, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group, an -NR ₁ R ₂ group, a (C ₁ -C ₄ )alkoxy group, and a (C ₁ -C ₃ )alkyl group.

According to one aspect of this preferred embodiment of the invention, R' independently represents a hydrogen atom, a halogen atom, or a group selected from a (C ₁ -C ₃ )alkyl group, a hydroxyl group, a -OP(=O)-(OR ₃ )(OR ₄ ) group, a -NR ₁ R ₂ group, or a group ,

where A is O or NH, m is 2 or 3, and X ₁ is O, CH ₂ or N-CH _3, provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group.

According to another aspect of a preferred embodiment of the invention, R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group or a group ,

where m is 2 or 3, and X ₁ is O, CH ₂ or N-CH ₃ , and preferably R'' is a hydrogen atom or a methyl group.

According to a specific embodiment of the invention, another object of the present invention is the use of a compound of formula (Ib):

(Ib)

where R, R', R'', n and n' are as defined above,

in the treatment and/or prevention of inflammatory diseases.

According to one aspect of this preferred embodiment of the invention, n is 1 or 2.

According to one aspect of this preferred embodiment of the invention, n' is 1, 2 or 3.

According to one aspect of this preferred embodiment, R independently represents a hydrogen atom, a halogen atom, or a group selected from a hydroxyl group, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group, an -NR ₁ R ₂ group, a (C ₁ -C ₄ )alkoxy group, an -OP(=O)-(OR ₃ )(OR ₄ ) group, and a (C ₁ -C ₃ )alkyl group.

According to one aspect of this preferred embodiment of the invention, R' independently represents a hydrogen atom, a halogen atom, or a group selected from a (C ₁ -C ₃ )alkyl group, a hydroxyl group, a -OP(=O)-(OR ₃ )(OR ₄ ) group, a -NR ₁ R ₂ group, or a group ,

where A is O or NH, m is 2 or 3, and X ₁ is O, CH ₂ or N-CH _3, provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group.

According to another aspect of this preferred embodiment of the invention, R' alternatively independently represents a hydrogen atom, a halogen atom, or a group selected from a (C ₁ -C ₃ )alkyl group, a hydroxyl group, or a -NR ₁ R ₂ group.

According to another aspect of this preferred embodiment of the invention, R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group or a group ,

where m is 2 or 3, and X ₁ is O, CH ₂ or N-CH ₃ , and preferably R'' is a hydrogen atom or a methyl group.

According to a specific embodiment of the invention, another object of the present invention is the use of a compound of formula (Ic):

(Ic)

where R, R', R'', n and n' are as defined above,

in the treatment and/or prevention of inflammatory diseases.

According to one aspect of this preferred embodiment of the invention, n is 1.

According to one aspect of this preferred embodiment of the invention, n' is equal to 1.

According to one aspect of this preferred embodiment, R independently represents a hydrogen atom, a halogen atom, or a group selected from a hydroxyl group, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group, an -NR ₁ R ₂ group, a (C ₁ -C ₄ )alkoxy group, and a (C ₁ -C ₃ )alkyl group.

According to one aspect of this preferred embodiment, R alternatively independently represents a hydrogen atom or a halogen atom.

According to one aspect of this preferred embodiment of the invention, R' independently represents a hydrogen atom, a halogen atom, or a group selected from a (C ₁ -C ₃ )alkyl group, a hydroxyl group, an -NR ₁ R ₂ group, or a group ,

where A is O or NH, m is 2 or 3, and X ₁ is O, CH ₂ or N-CH _3, provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group.

According to one aspect of this preferred embodiment, R' alternatively independently represents a hydrogen atom or a halogen atom.

According to another aspect of this preferred embodiment of the invention, R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group or a group ,

where m is 2 or 3, and X ₁ is O, CH ₂ or N-CH ₃ , and preferably R'' is a hydrogen atom or a methyl group.

According to a specific embodiment of the invention, another object of the present invention is the use of a compound of formula (Id):

(Id)

where R, R', n and n' are as defined above,

in the treatment and/or prevention of inflammatory diseases.

According to one aspect of this preferred embodiment of the invention, n is 1.

According to one aspect of this preferred embodiment of the invention, n' is equal to 1.

According to one aspect of this preferred embodiment, R independently represents a hydrogen atom, a halogen atom, or a group selected from a hydroxyl group, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group, an -NR ₁ R ₂ group, a (C ₁ -C ₄ )alkoxy group, and a (C ₁ -C ₃ )alkyl group.

According to one aspect of this preferred embodiment of the invention, R alternatively independently represents a hydrogen atom, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group or a halogen atom.

According to one aspect of this preferred embodiment of the invention, R' independently represents a hydrogen atom, a halogen atom, or a group selected from a (C ₁ -C ₃ )alkyl group, a hydroxyl group, an -NR ₁ R ₂ group, or a group ,

where A is O or NH, m is 2 or 3, and X ₁ is O, CH ₂ or N-CH _3, provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group.

According to one aspect of this preferred embodiment, R' alternatively independently represents a hydrogen atom or a halogen atom.

According to another aspect of this preferred embodiment of the invention, R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group or a group ,

where m is 2 or 3, and X ₁ is O, CH ₂ or N-CH ₃ , and preferably R'' is a hydrogen atom or a methyl group.

According to a specific embodiment of the invention, another object of the present invention is the use of a compound of formula (Ie):

(Ie)

where R, R', n and n' are as defined above,

in the treatment and/or prevention of inflammatory diseases.

According to one aspect of this preferred embodiment of the invention, n is 1.

According to one aspect of this preferred embodiment of the invention, n' is equal to 1.

According to one aspect of this preferred embodiment, R independently represents a hydrogen atom, a halogen atom, or a group selected from a hydroxyl group, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group, an -NR ₁ R ₂ group, a (C ₁ -C ₄ )alkoxy group, and a (C ₁ -C ₃ )alkyl group.

According to one aspect of this preferred embodiment of the invention, R alternatively independently represents a hydrogen atom, a (C ₁ -C ₃ )fluoroalkyl group, a (C ₁ -C ₃ )fluoroalkoxy group or a halogen atom.

According to one aspect of this preferred embodiment of the invention, R' independently represents a hydrogen atom, a halogen atom, or a group selected from a (C ₁ -C ₃ )alkyl group, a hydroxyl group, an -NR ₁ R ₂ group, or a group ,

where A is O or NH, m is 2 or 3, and X ₁ is O, CH ₂ or N-CH _3, provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group.

According to one aspect of this preferred embodiment, R' alternatively independently represents a hydrogen atom or a halogen atom.

According to another aspect of this preferred embodiment of the invention, R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group or a group ,

where m is 2 or 3, and X ₁ is O, CH ₂ or N-CH ₃ , and preferably R'' is a hydrogen atom or a methyl group.

According to another particular embodiment of the invention, compounds (Id) and (Ie), as defined above, as such also form part of the present invention.

According to a preferred embodiment of the present invention, certain compounds of formula (I) are new, form part of the present invention and are selected from (number indicated in Table 1 below):

- (8) 8-chloro-5-(3-(piperidin-1-yl)propoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (9) 8-chloro-N ⁴ -(3-(piperidin-1-yl)propyl)-N ² -(4-(trifluoromethyl)pyridin-2-yl)quinoline-2,4-diamine;

- (10) 8-chloro-N-methyl-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (11) 8-chloro-N ⁴ -(2-morpholinoethyl)-N ² -(4-(trifluoromethyl)pyridin-2-yl)quinoline-2,4-diamine;

- (13) 4,8-dichloro-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (14) 8-chloro-N-(3-morpholinopropyl)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (15) 8-chloro-6-(2-morpholinoethoxy)-N-(3-morpholinopropyl)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (16) 8-chloro-5-(2-morpholinoethoxy)-N-(3-morpholinopropyl)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (17) 8-chloro-6-(2-(4-methylpiperazin-1-yl)ethoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (18) 8-chloro-6-(2-(piperidin-1-yl)ethoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (19) 8-chloro-6-(3-(piperidin-1-yl)propoxy)-N-(4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (20) 8-chloro-N-(3-fluoro-4-(trifluoromethyl)pyridin-2-yl)quinolin-2-amine;

- (21) N-(5-bromo-4-(trifluoromethyl)pyridin-2-yl)-8-chloroquinolin-2-amine;

- (22) N ² -(8-chloroquinolin-2-yl)-N ⁵ -(3-(4-methylpiperazin-1-yl)propyl)-4-(trifluoromethyl)pyridine-2,5-diamine;

- (28) 8-chloro-N-methyl-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (29) 8-chloro-5-(3-(piperidin-1-yl)propoxy)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (30) 8-chloro-N-(3-(piperidin-1-yl)propyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (31) 8-chloro-N-(2-morpholinoethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (32) 8-chloro-N-(2-(pyrrolidin-1-yl)ethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (33) 8-chloro-N-(4-morpholinobutyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (34) 8-chloro-N ⁴ -(3-(piperidin-1-yl)propyl)-N ² -(4-(trifluoromethoxy)phenyl)quinoline-2,4-diamine;

- (35) 4,8-dichloro-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (36) 8-chloro-5-(2-morpholinoethoxy)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (37) N ¹ -(4,8-dichloroquinolin-2-yl)-4-(trifluoromethoxy)benzene-1,2-diamine;

- (38) 4,8-dichloro-N-(2-morpholinoethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (39) 8-chloro-6-(2-morpholinoethoxy)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (40) 8-chloro-N ² -(2-morpholinoethyl)-N ⁴ -(3-(piperidin-1-yl)propyl)-N ² -(4-(trifluoromethoxy)phenyl)quinoline-2,4-diamine;

- (41) 8-chloro-N-(2-morpholinoethyl)-N-(2-nitro-4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (42) N ¹ -(8-chloroquinolin-2-yl)-N ¹ -(2-morpholinoethyl)-4-(trifluoromethoxy)benzene-1,2-diamine;

- (43) 8-chloro-5-(2-morpholinoethoxy)-N-(2-morpholinoethyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine;

- (44) N ¹ -(8-chloro-5-(2-morpholinoethoxy)quinolin-2-yl)-4-(trifluoromethoxy)benzene-1,2-diamine;

- (46) 8-chloro-2-((4-(trifluoromethyl)pyridin-2-yl)oxy)quinoline;

- (47) 4-(2-((8-chloro-2-((4-(trifluoromethyl)pyridin-2-yl)oxy)quinolin-6-yl)oxy)ethyl)morpholine;

- (48) 8-chloro-2-(4-(trifluoromethoxy)phenoxy)quinoline;

- (49) 4-(2-((8-chloro-2-(4-(trifluoromethoxy)phenoxy)quinolin-6-yl)oxy)ethyl)morpholine;

- (50) 4-(2-((8-chloro-2-(4-(trifluoromethoxy)phenoxy)quinolin-5-yl)oxy)ethyl)morpholine;

- (51) mono-[8-chloro-2-(4-trifluoromethyl-pyridin-2-ylamino)-quinolin-6-yl] ester of phosphoric acid;

- (52) mono-[2-(8-chloro-quinolin-2-ylamino)-5-trifluoromethoxy-phenyl] ester of phosphoric acid;

- (53) mono-[8-chloro-2-(4-trifluoromethoxy-phenylamino)-quinolin-6-yl] ester of phosphoric acid

- and their pharmaceutically acceptable salts.

For the purpose of the present invention, a compound of formula (I) includes any of the compounds of formula (Ia), (Ib), (Ic), (Id) and (Ie), as well as combinations thereof. Compounds of formula (I) include compounds (1) to (53), as defined in Table I, and combinations thereof.

The compounds of the invention may exist in the form of free bases or addition salts with pharmaceutically acceptable acids.

Suitable physiologically acceptable acid addition salts of the compounds of formula (I) include hydrobromide, tartrate, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate and fumarate.

The compounds of formula (I) and/or their salts may form solvates or hydrates, and the invention includes all such solvates and hydrates.

The terms "hydrates" and "solvates" simply mean that the compounds (I) according to the invention can exist as a hydrate or solvate, i.e., combined or associated with one or more water or solvent molecules. This is merely a chemical characteristic of such compounds, which can be applied to all organic compounds of this type.

Compounds of formula (I) may contain one or more asymmetric carbon atoms. They may thus exist as enantiomers or diastereoisomers. These enantiomers, diastereoisomers, and mixtures thereof, including racemic mixtures, are within the scope of the present invention.

In the context of the present invention, the term:

- "halogen atom" is understood to mean an atom of chlorine, fluorine, bromine or iodine and, in particular, means an atom of chlorine, fluorine or bromine,

- "(C ₁ -C ₅ )alkyl" as used herein refers to a C ₁ -C ₅ normal, secondary, or tertiary saturated hydrocarbon, respectively. Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, butyl, pentyl,

- "(C ₃ -C ₆ )cycloalkyl" as used herein, respectively, refers to a cyclic saturated hydrocarbon. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,

- "(C ₁ -C ₄ )alkoxy" as used herein, respectively, refers to an O-(C ₁ -C ₄ )alkyl moiety, wherein alkyl is as defined above. Examples include, but are not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy, butoxy,

- "fluoroalkyl group" and "fluoroalkoxy group" refer to an alkyl group and an alkoxy group, respectively, as defined above, wherein said groups are substituted by at least one fluorine atom. Examples are perfluoroalkyl groups such as trifluoromethyl or perfluoropropyl,

- "saturated 5- or 6-membered heterocycle" as used herein, respectively, refers to a saturated ring containing at least one heteroatom. Examples include, but are not limited to, morpholine, piperazine, thiomorpholine, piperidine, pyrrolidine,

- "patient" can refer to humans or mammals such as cats or dogs.

Compounds of formula (I) suitable for the invention may be prepared as described in WO 2010/143170, WO 2010/143169, WO 2012/080953 and WO 2010/143168, and/or as described herein below.

More particularly, compounds of formula (I) wherein R' is a group selected from:

as defined above, can be obtained according to the synthetic routes as described in WO 2012/080953, more particularly when A is O.

When A is N, the following synthetic route can be used. The quinoline derivative of formula (VII) can be synthesized as a building block before additional cross-coupling reactions.

In order to obtain a compound of formula (VII), the following sequence of reactions can be carried out as shown below in Scheme 1.

Scheme 1

A compound of formula (II), wherein R' is different from H and is as defined above and may in particular be a chlorine atom, can be placed in pyridine, then a compound of formula (III) can be added in a molar ratio in the range of from 1 to 2, for example 1.5, with respect to the compound of formula (II). The reaction mixture can be stirred at a temperature in the range of from 110 to 150 °C, for example at 130 °C, for a time in the range of from 8 hours to 18 hours, for example 14 hours. After cooling to room temperature, the reaction mixture can be concentrated under reduced pressure and the resulting residue can be diluted with an organic solvent, such as dichloromethane. The organic phase can then be washed with a saturated aqueous solution of an inorganic base, such as Na ₂ CO ₃ , dried over MgSO ₄ , filtered and concentrated under reduced pressure to obtain a compound of formula (IV).

The compound of formula (IV) can be placed in a THF/water mixture, then sodium hydroxide can be added in a molar ratio in the range of 1 to 1.5, for example 1.2, relative to the compound of formula (IV), and the reaction mixture can be stirred at room temperature for a time in the range of 8 hours to 18 hours, for example 14 hours. Concentrated hydrochloric acid can then be added until a pH of 2 is reached, and the resulting solution can be extracted with an organic solvent such as ethyl acetate. The organic phase can then be dried over MgSO ₄ , filtered and concentrated under reduced pressure to obtain the compound of formula (V).

The compound of formula (V) can be placed in polyphosphoric acid added in a molar ratio in the range of 5 to 15, for example 10, with respect to the compound of formula (V), and the reaction mixture can be stirred at a temperature in the range of 110 to 150°C, for example at 130°C, for a time in the range of 8 hours to 18 hours, for example 14 hours. After cooling to room temperature, an aqueous solution of sodium hydroxide having a molarity range of 1 to 5 M, for example 2 M, can be slowly added. The resulting precipitate can be filtered, washed with water and dried under reduced pressure in a desiccator to obtain a compound of formula (VI).

The compound of formula (VI) can be placed in _POCl3 , added in a molar ratio in the range of 5 to 15, for example 10, relative to the compound of formula (VI), and the reaction mixture can be stirred at a temperature in the range of 80 to 120°C, for example at 100°C, for a time in the range of 1 hour to 5 hours, for example 2 hours. After cooling to room temperature, water can be slowly added. The resulting precipitate can be filtered, washed with water and dried under reduced pressure in a desiccator to obtain the compound of formula (VII).

Said intermediate of formula (VII) can be used to form a compound of formula (I) by coupling it with an aniline derivative, an aminopyridine derivative, a pyrimidine derivative, a hydroxypyridine derivative, a phenol derivative or a hydroxypyrimidine derivative, as described in WO 2010/143170, WO 2010/143169, WO 2012/080953 and WO 2010/143168, and/or as further described herein below.

The chlorine atom at position 4 of the intermediate of formula (VII) can be subsequently replaced by an amine to form a quinoline derivative bearing a group of formula (IIa), where A = NH.

When Q = N and R'' differs from H, the following paths illustrated in diagrams 2 and 3 can be applied.

In order to obtain a compound of formula (IX), the following reaction can be carried out as shown below in Scheme 2.

Scheme 2

A compound of formula (VIII), wherein Z, V, n, n', R and R' are as defined above, can be placed in an anhydrous polar solvent, such as anhydrous N,N-dimethylformamide, in the presence of AklHal, where Alk is a (C ₁ -C ₄ )alkyl group and Hal is a halogen atom, in a molar ratio in the range of from 1 to 2, for example 1.1, and in the presence of an inorganic base, such as potassium tert-butoxide, in a molar ratio in the range of from 1 to 2, for example 1.1, with respect to the compound of formula (VIII). The reaction mixture can be stirred at room temperature for a time in the range of from 7 hours to 24 hours, for example 16 hours. The reaction mixture can be partitioned between water and an organic solvent, such as ethyl acetate. The organic phases can then be collected, washed with saturated aqueous brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure to give the compound of formula (IX).

In order to obtain compounds of formula (XII), the following reaction can be carried out as shown below in Scheme 3.

Scheme 3

A compound of formula (X), wherein Z, V, n, n', R and R' are as defined above, can be placed in an anhydrous polar solvent, such as anhydrous N,N-dimethylformamide, in the presence of NaH in a molar ratio in the range of 2 to 5, for example 3, and the reaction mixture can be stirred at room temperature for a time in the range of 10 minutes to 50 minutes, for example 30 minutes. Then, a compound of formula (XI), wherein m, B, Ra and Rb are as defined above, can be placed in a molar ratio in the range of 1 to 2, for example 1, in an anhydrous polar solvent, such as anhydrous N,N-dimethylformamide, in the presence of KI in a molar ratio in the range of 1 to 2, for example 1, and in the presence of an organic base, such as Et ₃ N, in a molar ratio in the range of 1 to 2, for example 1, with respect to the compound of formula (X), and the reaction mixture can be stirred at room temperature for a time in the range of 10 minutes to 50 minutes, for example 30 minutes, under an inert gas atmosphere, for example argon. The activated compound (X) can then be added to the compound (XI), and the resulting reaction mixture can be stirred at a temperature in the range of 70 to 110°C, for example at 90°C, for a time in the range of 2 hours to 10 hours, for example 4 hours. After cooling to room temperature, the reaction mixture can be concentrated under reduced pressure, and the resulting residue can be diluted with an organic solvent such as ethyl acetate. The organic phase can then be washed with saturated aqueous brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure to obtain a compound of formula (XII).

When the group carries a substitution representing an amino group of formula (IIa), where A is NH, the following route can be used, as in Scheme 4.

Scheme 4

A compound of formula (XIII), where Z, V, n', R and R' are as defined above and X is a halogen atom such as Br, can be placed in a polar solvent mixture such as a dioxane/N,N-dimethylformamide mixture. Then, a compound of formula (XIV), wherein B, R _a , and R _b are as defined above, is added in a molar ratio in the range of 1 to 2, for example 1.5, with respect to the compound of formula (XIII) in the presence of a non-nucleophilic organic base such as sodium tert-butoxide or potassium tert-butoxide in a molar ratio in the range of 2 to 5, for example 3, in the presence of a diphosphine such as Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethylxanthine) or X-Phos (2-dicyclohexylphosphino-2',4',6'-triisopropyldiphenyl) in an amount in the range of 5 mol% to 40 mol% with respect to the total amount of compound of formula (XIII), and in the presence of a catalyst such as Pd(OAc) ₂ or Pd ₂ (dba) ₃ in an amount in the range of 2 mol% to 15 mol% relative to the total amount of compound of formula (XIII). The reaction mixture can be heated in a microwave reactor at a temperature in the range of 90 to 150°C, for example at 120°C, for a time in the range of 30 minutes to 100 minutes, for example 70 minutes. The reaction mixture can be concentrated under reduced pressure, and the resulting residue can be diluted with an organic solvent such as ethyl acetate. The organic phase can be washed with water, decanted, dried over magnesium sulfate, filtered and concentrated under reduced pressure to obtain a compound of formula (XV).

Compounds of general formula (Id), as defined above, can be prepared according to scheme 5 below.

Scheme 5

A compound of formula (XVI), wherein n' and R' are as defined above and X is a halogen atom such as Cl, can be placed in a polar solvent such as N,N-dimethylformamide in the presence of an inorganic base such as _Cs2CO3 in a molar ratio in the range of 2 to 5, for example 3, and in the presence of CuI in a molar ratio in the range of 1 to 2, for example 1. Then _, a compound of formula (XVII), wherein R, V and n are as defined above, can be added in a molar ratio in the range of 1 to 2, for example 1, with respect to the compound of formula (XVI). The reaction mixture can be heated in a microwave reactor at a temperature in the range of 130 to 170°C, for example at 150°C, for a time in the range of 30 minutes to 100 minutes, for example 50 minutes. After cooling to room temperature, water can be added to the reaction mixture, the undissolved solids can be filtered through Celite, and the resulting filtrate can be extracted with an organic solvent such as ethyl acetate. The organic phase can then be washed with saturated aqueous brine, dried over _MgSO4 , filtered, and concentrated under reduced pressure to yield a compound of formula (Id).

Compounds of general formula (Ie), as defined above, can be prepared according to scheme 6 below.

Scheme 6

A compound of formula (XVI), wherein n' and R' are as defined above and X is a halogen atom such as Cl, can be placed in a polar solvent such as N,N-dimethylformamide in the presence of an inorganic base such as _Cs2CO3 in a molar ratio in the range of 2 to 5, for example 3, and in the presence of CuI in a molar ratio in the range of 1 to 2, for example 1. Then _, a compound of formula (XIX), wherein R, V and n are as defined above, can be added in a molar ratio in the range of 1 to 2, for example 1, with respect to the compound of formula (XVI). The reaction mixture can be heated in a microwave reactor at a temperature in the range of 130 to 170°C, for example at 150°C, for a time in the range of 30 minutes to 100 minutes, for example 50 minutes. After cooling to room temperature, water can be added to the reaction mixture, the undissolved solids can be filtered through Celite, and the resulting filtrate can be extracted with an organic solvent such as ethyl acetate. The organic phase can then be washed with water and saturated aqueous brine, dried over _MgSO4 , filtered, and concentrated under reduced pressure to yield a compound of formula (Ie).

More specifically, compounds of formula (I) wherein R' is an -OP(=O)-(OR ₃ )(OR ₄ ) group can be prepared starting from a compound of formula (XX) in which the R' group is an OH group, via the following route:

Scheme 7

A compound of formula (XX), wherein Z, V, n, n', R and R' are as defined above, can be placed in an anhydrous chloroalkane solvent, such as anhydrous dichloromethane, at 0°C under an inert gas atmosphere, such as argon, in the presence of diethyl chlorophosphate in a molar ratio of 1 and in the presence of an organic base, such as triethylamine, in a molar ratio in the range of from 1 to 2, for example 1.2, with respect to the compound of formula (XX). The reaction mixture can be stirred at room temperature for a time in the range of from 7 hours to 24 hours, for example 14 hours. The reaction mixture can then be concentrated under reduced pressure, and the resulting residue can be partitioned between an aqueous solution of hydrochloric acid of a molarity in the range of from 1 to 2 M, for example 1 M, and an organic solvent, such as ethyl acetate. The organic phases can then be collected, washed with a saturated aqueous solution of an _inorganic base such as _Na2CO3 , dried over _MgSO4 , filtered and concentrated under reduced pressure to give a compound of formula (XXI).

The compound of formula (XXI) can then be placed in a polar aprotic solvent such as acetonitrile in the presence of trimethylsilyl bromide in a molar ratio in the range of 1 to 5, for example 4, relative to the compound of formula (XXI). The reaction mixture can be stirred under microwave irradiation at a temperature in the range of 50 to 70°C, for example at 60°C, for a time in the range of 15 hours to 60 minutes, for example 30 minutes. After cooling to room temperature, a MeOH/water mixture (95/5) can be slowly added. The resulting precipitate can be filtered, washed with water and dried under reduced pressure in a desiccator to obtain a compound of formula (XXII).

The same procedure can be used to prepare compounds of formula (I) where R is an -OP(=O)-(OR ₃ )(OR ₄ ) group, starting from a compound of formula (I) in which the R group is an OH group.

Inflammatory diseases

Thus, the invention also relates to a compound of formula (Ia), (Ib), (Ic), (Id) and (Ie) for use in the treatment and/or prevention of an inflammatory disease.

The inventors were able to unexpectedly show that compounds of formula (I) resulted in dramatic improvements in the signs associated with inflammatory diseases such as inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) based on two in vivo mouse models.

The ability of compounds of formula (I) to modulate inflammation was assessed in two established mouse models of two inflammatory diseases: inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). The dextran sodium sulfate (DSS-)-induced colitis mouse model was used to study inflammatory bowel disease (see Perše and Cerar; “Dextran Sodium Sulfate Colitis Mouse Model: Traps and Tricks”; Journal of Biomedicine and Biotechnology (2012); 718617). The collagen-induced arthritis model was used to study rheumatoid arthritis, as shown by Brand et al. (“Collagen-induced arthritis”; Nature Protocols; (2007); 2(5):1269-75).

Indeed, the inventors demonstrated that administration of a compound of formula (I) resulted in an in vivo improvement in colon length and a reduction in changes in lymphoid organs, such as Peyer's patches, in mouse models of induced (DSS-) colitis. The inventors further demonstrated that administration of a compound of formula (I) resulted in a significant reduction in swelling and inflammatory signs in a mouse model of collagen-induced arthritis.

According to the invention, "inflammation" is characterized by pain, fever, redness and swelling, and may be the result of infection, irritation or injury.

Thus, "inflammatory disease" refers to a group of diseases and/or disorders caused by excessive inflammation or dysregulated inflammation.

According to the invention, "treatment and/or prevention of an inflammatory disease" may refer to the treatment and/or prevention of an inflammatory disease, or inflammation as such, which may occur in an individual in conjunction with an inflammatory disease.

Thus, treatment and/or prevention of an inflammatory disease also includes treatment and/or prevention of inflammation itself.

According to the invention, “treatment and/or prevention” of an inflammatory disease includes treatment, reducing the likelihood of development or delaying the onset of said inflammatory disease.

According to the invention, "individual" may refer to a human or a non-human mammal, and preferably to a human.

Inflammatory diseases include, but are not limited to: inflammatory disease due to autoimmune disease, inflammatory disease of the central nervous system (CNS), inflammatory joint disease, inflammatory disease of the gastrointestinal tract, inflammatory skin disease and other inflammatory diseases associated with epithelial cells such as bronchitis, inflammation due to cancer such as colon carcinoma, inflammation due to irritation, and inflammation due to injury.

Thus, the inflammatory disease may be selected from a list consisting of: inflammatory disease due to autoimmune disease, inflammatory disease of the central nervous system (CNS), inflammatory joint disease, inflammatory disease of the gastrointestinal tract, inflammatory skin disease and other inflammatory diseases associated with epithelial cells, inflammation due to cancer, inflammation due to irritation, and inflammation due to injury.

Specifically, the inflammatory disease was selected from a list consisting of: inflammatory bowel disease, rheumatoid arthritis, Crohn's disease, ulcerative colitis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, osteoarthritis, atherosclerosis, ankylosing spondylitis, psoriasis, dermatitis, Sjogren's syndrome, bronchitis, bronchial asthma, and inflammation due to colon carcinoma.

More specifically, the inflammatory disease is selected from a list consisting of: inflammatory bowel disease, rheumatoid arthritis, Crohn's disease, ulcerative colitis, multiple sclerosis, osteoarthritis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, bronchitis, and inflammation due to colon carcinoma.

More specifically, the inflammatory disease is selected from a list consisting of: inflammatory bowel disease, rheumatoid arthritis, Crohn's disease, ulcerative colitis, multiple sclerosis, osteoarthritis, ankylosing spondylitis, and psoriasis.

Preferably, the inflammatory disease according to the invention includes: inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis and multiple sclerosis.

Even more preferably, the inflammatory disease according to the invention includes: inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis.

Inflammatory disease may also include Alzheimer's disease, Parkinson's disease, asthma, atherosclerosis and dermatitis.

Eczema can be indicated as dermatitis.

In light of the above, the invention relates to a compound of formula (I) for use in the treatment and/or prevention of an inflammatory disease, including inflammation per se and inflammation associated with an inflammatory disease.

Thus, the invention also relates to the use of a compound of formula (I) for the treatment and/or prevention of an inflammatory disease, including inflammation per se and inflammation caused by an inflammatory disease.

According to another aspect, the subject of the present invention relates to a compound (Ie), (Id), (8), (9), (10), (11), (44), (46), (47), (48), (49), (50), (51), (52), (53) or pharmaceutically acceptable salts thereof, alone or in combination, for use as a medicine.

According to another of its objects, the invention relates to a pharmaceutical composition comprising compound (Ie), (Id), (8), (9), (10), (11), (44), (46), (47), (48), (49), (50), (51), (52), (53) or pharmaceutically acceptable salts thereof, alone or in combination.

The invention also relates to the use of a compound of formula (I) for the preparation of a composition, such as a medicament, for the treatment and/or prevention of inflammation, including inflammation per se and inflammation due to an inflammatory disease.

The invention also relates to a method for the treatment and/or prevention of an inflammatory disease, including inflammation per se and inflammation caused by an inflammatory disease, and which comprises the step of administering a compound of formula (I) to a patient in need thereof.

miRNA-124

MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs that can act in the cell cytoplasm, downregulating the expression of their cognate messenger RNA targets or mRNA protein translation. Mature miRNAs are typically approximately 19-23 nucleotides in length. This ability of miRNAs to inhibit the production of their target proteins ultimately leads to the regulation of many cellular activities, such as cell fate determination, apoptosis, differentiation, and oncogenesis.

miR-124 was first cloned in mouse. The human miR-124 precursor (or miRN-124 or miRNA-124 or microRNA 124) was cloned from embryonic stem cells. To date, nine miR-124 precursor haplotypes have been identified (Guo et al., PLoS ONE, 2009, 4(11):e7944), of which three are present in humans, hsa-miR-124-1, hsa-miR-124-2, and hsa-miR-124-3. (Respectively, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3).

The miR-124 microRNA precursor is a small noncoding RNA molecule. Mature, approximately 21-nucleotide miRNAs are processed from the precursor's hairpin sequences by the Dicer enzyme. The mature sequences are SEQ ID NO: 4 for miR-124-3p and SEQ ID NO: 5 for miR-124-5p.

According to the invention, determining the expression level of miR-124 includes both determining the expression level of precursor and mature miR-124.

The invention also relates to a compound of formula (I) for increasing the expression of miR-124 in a biological sample.

Thus, in accordance with another of its objects, the invention relates to a method for increasing the expression of miR-124 in vitro or ex vivo in a eukaryotic cell, comprising at least the step of:

a) provide the eukaryotic cell,

b) bringing said cell into contact with a derivative compound and in particular with a compound of formula (I).

Methods for screening candidate compounds

One of the key factors in the successful development of a drug or vaccine is the ability to effectively and rapidly assess its effectiveness. Therefore, it is especially important to have appropriate tools, such as specific biomarkers, on which to base the assessment of drug or vaccine efficacy.

Surprisingly, miR-124 was found to play a significant role in inflammation. Indeed, the inventors demonstrated that compounds of formula (I) are capable of modulating miR-124 expression. Specifically, the inventors demonstrated that the compounds of the invention can upregulate miR-124 expression (up to a 100-fold increase) in peripheral blood mononuclear cells (PBMCs) from donors isolated by Ficoll™ gradient centrifugation.

Since the inventors have now established that compounds of formula (I) are capable of modulating the expression of miR-124, the invention also relates to a method for screening in vitro or ex vivo a quinoline derivative, and in particular a compound of formula (I), suspected of being effective in the treatment and/or prevention of an inflammatory disease.

Thus, in accordance with another object thereof, the invention relates to the in vitro or ex vivo use of at least one miRNA, wherein at least one miRNA is miRNA-124, as a biomarker for screening a quinoline derivative, and in particular a compound of formula (I), suspected of being effective in the treatment and/or prevention of an inflammatory disease.

Thus, the invention also relates to a method for screening in vitro or ex vivo a quinoline derivative, and in particular a compound of formula (I), suspected of being effective in the treatment and/or prevention of an inflammatory disease, comprising at least the step of:

a) provide the eukaryotic cell,

b) bringing said cell into contact with a compound of formula (I),

c) determine the expression of miRNA-124 in the cell, and

d) selecting a candidate compound that is suspected of being effective in the treatment and/or prevention of an inflammatory disease when the expression level of miRNA-124 determined in step c) is modulated relative to a reference value.

According to the invention, "modulating" the expression level includes both up- and down-regulation of the expression level. For the purposes of the invention, "modulating" the expression level of miR-124 preferably refers to up-regulation.

In particular, the invention relates to a method for screening in vitro or ex vivo a quinoline derivative and, in particular, a compound of formula (I) suspected of being effective in the treatment and/or prevention of an inflammatory disease, comprising at least the step of:

a) provide the eukaryotic cell,

b) bringing said cell into contact with a compound of formula (I),

c) determine the expression of miRNA-124 in the cell, and

d) selecting a candidate compound that is suspected of being effective in the treatment and/or prevention of an inflammatory disease when the expression level of miRNA-124 determined in step c) is increased relative to a reference value.

According to one embodiment of the invention, the presence or expression level of miR-124 is determined in a eukaryotic cell from a biological sample and compared to a control reference value.

In particular, a "biological sample" suitable for the invention may be a sample of a biological fluid, such as blood, plasma or serum, saliva, interstitial fluid or urine; a cell sample, such as a cell culture, cell line, stem cell line or a sample containing peripheral blood mononuclear cells (PBMCs); a tissue biopsy sample, such as oral tissue, gastrointestinal tissue, skin, oral mucosa, or a plurality of samples from clinical studies. The sample may be an unpurified sample, or it may be purified to various degrees before storage, processing or determination.

In particular, a biological sample suitable for the invention is a sample of peripheral blood mononuclear cells (PBMCs) or a sample containing PBMCs. Thus, the biological sample according to the invention can be obtained by processing peripheral whole blood using conventional techniques in the art, such as density gradient centrifugation, and more specifically, a Ficoll™ gradient.

PBMC samples, particularly those processed in a Ficoll™ gradient, tend to contain lymphocytes (T cells, B cells, and natural killer (NK) cells), monocytes, and dendritic cells. In humans, the frequencies of these cell populations vary between individuals.

Thus, and without limitation, a eukaryotic cell includes any of the cell types as defined above, such as lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells.

The step of collecting biological samples for the uses and methods of the invention is performed before the implementation of the invention and does not constitute a step of the use or method in accordance with the invention.

Samples for mRNA testing can be collected at any desired interval. For example, samples can be collected hourly, twice daily, once daily, once weekly, once monthly, once every two months, once annually, or similarly. The sample can be tested immediately or stored for later testing.

Samples can be purified prior to testing. In some embodiments, miR-124 can be isolated from the remaining cell contents prior to testing. Furthermore, if desired, miR-124 molecules can be separated from other mRNAs in the sample. For example, miR-124 can be separated from mRNA based on size differences prior to testing.

The control reference value, which is used to compare a certain expression level of miR-124 in the biological sample under study, is obtained from the control sample.

Control samples can be obtained from various sources. In some embodiments, control samples are collected from the patient prior to treatment or prior to the presence of disease (e.g., an archived blood sample). In other embodiments, control samples are collected from a group of normal, disease-free members of the population. In another embodiment, the cellular assay can be performed on a control cell culture, for example, unexposed to the test compound or exposed to a control compound such as DMSO, methylcellulose (MC), or water.

According to one embodiment of the invention, for determining or monitoring an inflammatory disease in a patient, the control reference value may be obtained from an isolated biological sample obtained from an individual or group of individuals known not to suffer from such a condition.

According to another embodiment of the invention, for determining or monitoring the effectiveness of treatment for an inflammatory disease in a patient, a control reference value can be obtained from an isolated biological sample obtained from an individual or group of individuals known to not suffer from such condition(s) and/or not receiving the treatment whose effectiveness needs to be determined or monitored. Alternatively, a control reference value can be obtained from an isolated biological sample obtained from a patient suffering from an inflammatory disease and receiving the treatment whose effectiveness needs to be determined or monitored, wherein the isolated biological sample is taken from the patient before the treatment.

Numerous methods are available to those skilled in the art for determining the presence or expression level of the miR-124 biomarker.

For example, nucleic acid array assays or test systems can be used to assess the presence and/or expression level of miR-124 in a sample.

The miR-124 sequence can be used to generate a corresponding nucleotide that acts as a complementary probe or primer for use in various nucleic acid assays to detect the expression or presence of the miR-124 biomarker in a sample, such as, but not limited to, Northern blotting and polymerase chain reaction (PCR)-based methods (e.g., real-time reverse transcription-PCR or quantitative RT-PCR). Methods such as qRT-PCR can be used to accurately quantify miRNA in a sample.

The sense and antisense probes or primers of the invention can be prepared using any method known to those skilled in the art, in particular the methods described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, ^3rd ED., 2001, Cold Spring Harbor, NY).

Methods relating to the detection and quantification of RNA or DNA are well known in the art. One skilled in the art can refer, for example, to the articles by Wang et al. (1989, Proc Natl Acad Sci USA, Vol. 86: 917-921), de Wong et al. (2005, Bio Techniques, Vol. 39 (1): 75-85), de Nolan et al. (2006, Nat Protoc, Vol. 1 (3): 1559-1582) and de Klinck et al. (2008, Cancer Research, Vol. 68: 657-663), or also to the general review published by Bustin (2000, Journal of Molecular Endocrinology, Vol. 25: 169-193).

An isolated nucleic acid probe suitable for detecting the presence or expression level of miR-124 is a nucleic acid probe capable of specifically hybridizing with miR-124, such as precursor or mature miR-124.

Such a nucleic acid probe may contain from 18 to 30 nucleotides, in particular from 20 to 27, preferably from 20 to 25, preferably 20, 22 or 25, and more preferably about 25 nucleotides. As noted above, such nucleic acid probes can be obtained according to any known methods in the art.

In this field of technology, methods and formulas for predicting the optimal hybridization temperature for a given probe and a given target are known.

Thus, one skilled in the art can easily calculate the optimal hybridization temperature based on a series of probes on a given target sequence and specific hybridization conditions.

Preferably, the optimal hybridization temperature of said probes is from 40°C to 60°C, and more specifically from 45°C to 55°C, and is preferably about 48°C.

Examples of buffers suitable for hybridization of a nucleic acid probe according to the invention with a biomarker according to the invention, as a hybridization buffer, there may be mentioned a buffer containing 100 mM morpholinoethanesulfonic acid (MES), 1 M [Na+], 20 mM ethylenediaminetetraacetic acid (EDTA), 0.01% Tween-20, as a wash buffer under non-stringent conditions, a buffer containing 6X SSPE, 0.01% Tween-20, and as a wash buffer under stringent conditions, a buffer containing 100 mM MES, 0.1 M [Na+], 0.01% Tween-20.

In one embodiment of the invention, the method for detecting and quantifying nucleic acids may be a fluorescent dye-based method, wherein the nucleic acid concentration is estimated by determining the fluorescence intensity of ligands, such as dyes, that bind to said nucleic acids. Fluorescent dyes are well known in the art.

Alternatively, nucleic acid can be determined using spectrophotometry.

In another embodiment of the invention, the method for detecting and quantifying nucleic acids may be a hybridization-based method. Said hybridization-based methods may include PCR and quantitative PCR (qRT-PCR or q-PCR) methods or reverse transcriptase/polymerase methods. Advantageously, a sequencing step may be included in or combined with said method.

These methods may include (i) a step of extracting cellular mRNAs, (ii) a step of reverse transcription of the mRNA into DNA using reverse transcriptase, and (iii) a step of amplifying DNA from the DNA obtained in the preceding step. Typically, the following nucleic acids are amplified from the same starting sample: (a) DNA obtained after the step of reverse transcription of the target mRNA, and (b) DNA or a plurality of DNAs obtained after reverse transcription of mRNAs constitutively and constantly expressed by cells ("housekeeping genes"), such as RNAs encoded by the MRPL19, PUM1, and glyceraldehyde-3-phosphate dehydrogenase (GADPH) genes.

Amplified DNA can be quantified following separation by electrophoresis and measurement of DNA bands. Results for the target mRNA(s) are expressed as relative units compared to mRNAs encoded by housekeeping genes. In some embodiments, the amplified DNA separation step is performed following agarose gel electrophoresis, followed by staining of the DNA bands with ethidium bromide, and then the amount of DNA contained in these migration bands is determined by densitometry. In other embodiments, a microchannel device can be used in which the amplified DNA is separated by capillary electrophoresis, after which the emitted signal is quantified using a laser beam. Such a device can be a LabChip® device, such as the "GX" series, available from Caliper LifeSciences (Hopkinton, MA, USA).

Quantitative results obtained using qRT-PCR can sometimes be more informative than qualitative data and can simplify the standardization of assays and quality control. Thus, in some embodiments of the invention, qRT-PCR-based assays may be suitable for determining miRNA levels in cell-based assays. The qRT-PCR method may also be useful for monitoring patient treatment. Commercially available qRT-PCR-based methods (e.g., the Taqman®™ array assay system)

Any suitable assay platform can be used to determine the expression or presence of miRNA in a test sample. For example, the assay may be in the form of a rapid assay with an impregnated substrate, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a glass slide, a multi-well plate, or an optical fiber. The assay system may have a solid support to which an oligonucleotide corresponding to the miRNA is attached. The solid support may include, for example, plastic, silicone, metal, resin, glass, membrane, particles, sediment, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or glass slide. The components of the assay system can be prepared and packaged together as an miRNA detection kit.

In some embodiments of the invention, an oligonucleotide array assay system for testing the activity of a quinoline derivative or a drug candidate in a biological sample can be prepared or purchased. The array assay system typically comprises a solid support and at least one oligonucleotide contacted with the support, wherein the oligonucleotide corresponds to at least a region of the miR-124 biomarker. In some embodiments of the invention, the region of the miR-124 biomarker comprises at least 5, 10, 15, 20, or more bases.

According to one embodiment of the invention, the presence or expression of miR-124 can be analyzed in combination with other miRNAs also used as biomarkers. In this embodiment of the invention, an array assay system can be used to assess the expression or presence of multiple miRNAs in a test sample, including miRNA-124. Typically, the method comprises the following steps: a) contacting the test sample with an array assay system containing a series of probes under conditions sufficient to allow specific binding to occur; and b) examining the array assay system for the presence of any detectable level, resulting in an assessment of the amount of the corresponding target miRNAs in the test sample. The use of an expression array assay system makes it possible to obtain an miRNA expression profile for a given test sample.

Methods for preparing assay methods or array test systems for miRNA analysis are well known in the art and need not be described in more detail herein.

Nucleic acid array assays can be used to detect the presence or differential expression of miRNAs in biological samples. Polynucleotide array assays (such as DNA or RNA array assays) typically include polynucleotide regions, usually of varying sequences ("capture agents"), arranged in a predetermined configuration on a carrier. Array assays are "addressable" because these regions (sometimes referred to as "assay tags") have different predetermined positions ("addresses") on the array carrier. A region (i.e., a "tag" or "zone" of an array assay) at a specific predetermined position (i.e., an "address") on the array assay identifies a specific miRNA target. Polynucleotide array assays are typically created on flat supports either by applying pre-prepared polynucleotides to the support using a site-specific method or by site-specific synthesis of polynucleotides in situ on the support. Array assays for detecting miRNA expression can be created by applying (e.g., by contact, inkjet, or photolithography) either precursor units (such as nucleotide or amino acid monomers) or a pre-synthesized capture agent. After applying the polynucleotide capture agents to the support, the support is typically processed (e.g., washed or blocked) and stored until use.

An array test system for detecting miRNA expression has at least two, three, four, or five different subject probes. However, in some embodiments of the invention, the subject array test system may include a series of probes having at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 or more probes that can detect the corresponding number of miRNAs. In some embodiments of the invention, subject array test systems may include probes for detecting at least a portion or all of the identified miRNA of an organism or may include orthologous probes from multiple organisms.

A nucleic acid array assay can be contacted with a test sample or a labeled sample containing miRNA analytes under conditions that promote specific binding of the miRNA in the test sample to one or more capture agents present on the array assay to reveal an observable binding pattern. This binding pattern can be determined by detailed characterization of the array assay. For example, target miRNAs in the test sample can be labeled with a suitable label (such as a fluorescent compound), and this label can then be appropriately observed (e.g., by observing a fluorescence pattern) on the array assay after exposure of the array to the sample. The observed binding pattern can be indicative of the presence and/or concentration of one or more miRNA components of the test sample.

Labeling of siRNA can be accomplished using methods well known in the art, such as using DNA ligase, end transferase, or by labeling the RNA backbone, etc. In some embodiments of the invention, siRNA can be labeled with a fluorescent label. Illustrative fluorescent dyes include, but are not limited to, xanthine dyes, fluorescein dyes, rhodamine dyes, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2',4,7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; Cyanine dyes, such as Cy3, Cy5 and Cy7 dyes; Alexa dyes, such as Alexa-fluor-555; coumarin, diethylaminocoumarin, umbelliferone; benzimide dyes, such as Hoechst 33258; phenanthridine dyes, such as Texas red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, Bodypie dyes, quinoline dyes, pyrene, fluorescein chlorotriazinyl, R1 10, eosin, JOE, R6G, tetramethylrhodamine, lissamine, ROX, naphthofluorescein and the like.

In some embodiments of the invention, an oligonucleotide array assay for assessing immunomodulatory activity can be prepared or purchased, for example, from Affymetrix. The array assay can comprise a solid support and a plurality of oligonucleotides contacted with the support. The oligonucleotides can be present at specific, addressable positions on the solid support, each corresponding to at least a portion of miRNA sequences that can be differentially expressed upon exposure of a cell or patient to a quinoline derivative or a candidate drug. The miRNA sequences comprise at least one miR-124 sequence.

When using an array assay to evaluate miRNA, in a typical case the method may include the steps of (1) preparing an array assay containing surface-bound subject probes; (2) hybridizing a population of miRNAs with the surface-bound probes under conditions sufficient to ensure specific binding; (3) washing after hybridization to remove nucleic acids that did not bind during hybridization; and (4) detecting hybridized miRNAs. The reagents used in each of these steps and the conditions of their use may vary depending on the specific application.

Hybridization can be performed under suitable hybridization conditions, the stringency of which can be varied as desired. The characteristic conditions are sufficient to ensure the formation of probe/target complexes on the surface of the array test system between complementary binding members, i.e., between surface-bound subject probes and complementary miRNAs in the test sample. In some embodiments of the invention, stringent hybridization conditions can be used. Hybridization is typically performed under stringent hybridization conditions. Standard hybridization methods well known in the art are used to hybridize the test sample to the nucleic acid array test system (e.g., under conditions sufficient to ensure specific binding of the target miRNAs in the test sample to the probes on the array test system). The selection of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, stringency of wash conditions, and the like, depend on the experimental design, including the sample source, identity of the capture agents, the expected degree of complementarity, and so on, and can be determined through routine experimentation by those of ordinary skill in the art. Typically, "stringent hybridization conditions" and "stringent post-hybridization wash conditions" in the context of nucleic acid hybridization are typically sequence-dependent and vary under different experimental conditions. Hybridization can be carried out for a period of approximately 12 to approximately 24 hours. The stringency of wash conditions can affect the extent to which miRNA sequences specifically hybridize to complementary capture agents. Those of ordinary skill in the art will readily appreciate that alternative, but comparable, hybridization conditions can be used to ensure conditions of similar stringency.

As an illustration, in one embodiment of the invention, miRNA expression profiling experiments can be performed using the Affymetrix Genechip 2.0 miRNA array assay system, following the protocols described in the user manual.

In one specific embodiment of the invention, hybridization can be performed using the GeneChip® Hybridization, Wash, and Stain Kit (Affymetrix, No. 900720). Preferably, said hybridization is performed following the manufacturer's protocols.

Following the miRNA hybridization procedure, the surface-bound polynucleotides of the array assay are typically washed to remove unbound nucleic acids. Washing can be performed using any conventional wash protocol where the wash conditions are typically stringent, as described above. For example, the wash step can be performed using wash buffers commercially available from Affymetrix (Part Nos. 900721 and 900722). Hybridization of the target miRNAs to the probes is then detected using standard array reading methods. Reading of the resulting hybridized array assay can be accomplished, for example, by illuminating the array assay and reading the location and intensity of the resulting fluorescence for each feature of the array assay to detect miRNA/probe binding complexes.

Modulation of the presence or expression level of miR-124 relative to a control sample, such as a healthy donor control, may be indicative of an inflammatory disease. Specifically, a decrease or suppression of the presence or decreased expression level of this miRNA relative to a control sample (i.e., a healthy donor control) may be indicative of an inflammatory disease.

Thus, in one embodiment, the use of the invention may include obtaining or determining the expression level of miR-124 in an isolated biological sample and comparing said determined expression level with a control reference value. Observing modulation of said determined level relative to the control reference value may be indicative of an inflammatory disease or treatment for said inflammatory disease.

An elevated or upregulated miR-124 expression level in the presence of a drug candidate relative to a control reference value (i.e., without treatment with the drug candidate, such as a quinoline derivative) is an indicator of the putative efficacy of the drug candidate in the treatment and/or prevention of an inflammatory disease.

Accordingly, when miR-124 from a test sample is "increased in amount" or "upregulated" in a biological sample compared to a control reference value, said increase may be an increase of approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1000%, 5000% or more compared to the control reference value (i.e., without treatment with the quinoline derivative).

In particular, the determined expression level of miR-124 can be increased by at least twofold, preferably at least fourfold, preferably at least sixfold, preferably at least eightfold, preferably at least tenfold relative to the said control reference value.

According to a preferred embodiment of the invention, the determined expression level of miR-124 can be increased by at least 100 times relative to the said control reference value.

According to one embodiment of the invention, the method of the invention can be practiced to optimize the dosing regimen for a patient. Patients may have different responses to a given quinoline derivative, in particular a compound of formula (I), depending on factors such as age, health condition, genetic background, the presence of other complications, disease progression, and co-administration of other drugs. The use of a miR-124 biomarker can be suitable for assessing and optimizing the dosing regimen, such as the dose amount and/or dosing regimen, of the quinoline derivative in a patient. In this regard, a miR-124-based biomarker can also be used to track and adjust the effectiveness of treatment for an individual patient over time. The biomarker can be used to collect information necessary for adjusting the patient's treatment, increasing or decreasing the agent's dose as needed. For example, a patient receiving a quinoline derivative can be tested using a miR-124-based biomarker to observe whether the dosage is becoming effective or whether a more intensive treatment regimen is necessary. Thus, the amount of drug administered, the periodicity of administration, the frequency of administration, and the duration of administration can be adjusted depending on the determination of the miR-124 biomarker.

The miR-124 biomarker can also be used to monitor patient compliance during individualized treatment regimens or clinical trials. This can be monitored at set intervals to ensure that patients enrolled in the study are taking their medications as directed. Furthermore, patients receiving a quinoline derivative can be tested using the miR-124 biomarker to determine whether they are adhering to the dosing regimen as planned. Increased biomarker expression levels compared to an unexposed control sample indicate protocol compliance.

Thus, without departing from the scope of the invention, the control reference value may be obtained from a eukaryotic cell isolated from: a biological sample from a healthy donor and/or a donor not previously exposed to treatment with a given drug candidate, such as a given quinoline derivative.

The biomarker of the invention can be used in practice to evaluate and monitor the efficacy of quinoline derivatives, in particular compounds of formula (I). Accordingly, the presence or expression level of miR-124 can be determined in an isolated biological sample obtained from a patient previously treated with a quinoline derivative, such as a compound of formula (I).

The determined presence or expression level of miR-124 in an isolated biological sample can then be compared with a control reference value.

If an increase in a certain level of miR-124 expression is observed relative to a control reference value, this determination is indicative of the activity of the quinoline derivatives and, in particular, the compounds of formula (I).

In another embodiment of the invention, if an increase in a certain level of miR-124 expression is observed relative to a control reference value, this determination may be indicative of the presence of an effect in the patient from treatment with these quinoline derivatives and, in particular, with these compounds of formula (I).

In another embodiment of the invention, if an increase in a certain level relative to a control reference value is observed, this determination may be an indicator of the effectiveness of treatment with these quinoline derivatives and, in particular, with these compounds of formula (I).

In another embodiment of the invention, if an increase in a certain level of miR-124 expression is observed relative to a control reference value, this determination is indicative of the therapeutic efficacy of these quinoline derivatives, and in particular these compounds of formula (I), as a therapeutic agent for the prevention and/or treatment of an inflammatory disease.

In another embodiment of the invention, in the case of observing an increase in a certain level of miR-124 expression relative to a control reference value, this determination is indicative of the therapeutic efficacy of a particular dosage regimen of these quinoline derivatives, and in particular these compounds of formula (I), as a therapeutic agent for the prevention and/or treatment of an inflammatory disease, in the case of determining the control reference value from a biological sample obtained from a patient receiving treatment in accordance with a different dosage regimen.

The chemical structures and spectroscopic data of some compounds of formula (I) according to the invention are illustrated in Table 1 below and in Table 2 (see examples), respectively.

Table 1

Formula (Ia) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 51 Formula (Ib) 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 52 53 Formula (Ic) 45 Formula (Id) 46 47 Formula (Ie) 48 49 50

The examples provided herein are intended to be illustrative only, and those skilled in the art will recognize or be able to determine, using no more than routine experimentation, numerous equivalents to specific compounds, materials, and procedures. All such equivalents are considered within the scope of the invention and are encompassed by the appended claims.

EXAMPLES

Example 1: 8-chloro-N ⁴ -(3-(piperidin-1-yl)propyl)-N ² -(4-(trifluoromethyl)pyridin-2-yl)quinoline-2,4-diamine; compound (9)

Ortho-Chloroaniline (5.3 mL, 50 mmol, 1 eq) was taken up in pyridine (8 mL). Diethyl malonate (11.4 mL, 75 mmol, 1.5 eq) was then added, and the reaction mixture was stirred at 130 °C for 14 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure, and the resulting residue was diluted with dichloromethane. The organic phase was then washed with saturated aqueous Na ₂ CO ₃ solution, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give ethyl 2-[(2-chlorophenyl)carbamoyl]acetate (2.7 g, 22%).

^1H NMR (300 MHz, CDCl ₃ ) δ 9.74 (br s, 1H), 8.38 (dd, J = 8.1, 1.5 Hz, 1H), 7.40 (dd, J = 8.1, 1.5 Hz, 1H), 7.28 (td, J = 8.1, 1.5 Hz, 1H), 7.07 (td, J = 8.1, 1.5 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H), 3.54 (s, 2H), 1.34 (t, J = 7.1 Hz, 3H).

Ethyl 2-[(2-chlorophenyl)carbamoyl]acetate (2.4 g, 9.93 mmol, 1 eq) was taken up in a mixture of THF (9.9 mL)/water (3.8 mL). Sodium hydroxide (477 mg, 11.92 mmol, 1.2 eq) was then added, and the reaction mixture was stirred for 14 h at room temperature. Concentrated hydrochloric acid was then added until pH 2 was reached, and the resulting solution was extracted with ethyl acetate. The organic phase was then dried over MgSO ₄ , filtered, and concentrated under reduced pressure to give 2-[(2-chlorophenyl)carbamoyl]acetic acid (2 g, 94%).

^1H NMR (300 MHz, MeOD) δ 7.98 (dd, J = 8.1, 1.5 Hz, 1H), 7.45 (dd, J = 8.1, 1.5 Hz, 1H), 7.30 (td, J = 8.1, 1.5 Hz, 1H), 7.16 (td, J = 8.1, 1.5 Hz, 1H), 3.54 (s, 2H).

A reaction mixture of 2-[(2-chlorophenyl)carbamoyl]acetic acid (3.7 g, 17.32 mmol, 1 eq.) in polyphosphoric acid (17 g, 173.2 mmol, 10 eq.) was stirred at 130°C for 14 h. The reaction mixture was cooled to room temperature, then 2 M aqueous sodium hydroxide solution was slowly added. The resulting precipitate was filtered, washed with water, and dried under reduced pressure in a desiccator to give 8-chloroquinoline-2,4-diol (3 g, 89%).

^1H NMR (300 MHz, DMSO) δ 11.66 (br s, 1H), 10.40 (br s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 5.81(s, 1H).

Mass spectrum (MS) (electrospray ionization (ESI)) [MH] ^- = 194.1

A reaction mixture of 8-chloroquinoline-2,4-diol (1.5 g, 7.67 mmol, 1 equiv) in _POCl3 (7.1 mL, 76.7 mmol, 10 equiv) was stirred at 100°C for 2 h. The reaction mixture was cooled to room temperature, then water was slowly added. The resulting precipitate was filtered, washed with water, and dried under reduced pressure in a desiccator to yield 2,4,8-trichlorochloroquinoline (1.6 g, 90%).

¹ H NMR (300 MHz, DMSO) δ 8.21 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.11 (s, 1H), 7.78 (t, J = 8.4 Hz, 1H).

A reaction mixture of 2,4,8-trichloroquinoline (1 g, 4.30 mmol, 1 eq.), 2-amino-4-trifluoromethylpyridine (768 mg, 4.73 mmol, 1.1 eq.), Pd(OAc) ₂ (19 mg, 0.09 mmol, 2 mol%), XantPhos (50 mg, 0.09 mmol, 2 mol%) and _Cs2CO3 ( _3.9 g, 12.04 mmol, 2.8 eq.) in t-BuOH (17.2 mL) was heated at 90°C for 2 days. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure and the resulting residue was diluted with ethyl acetate. The organic phase was then washed with water, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to afford (4,8-dichloroquinolin-2-yl)-(4-trifluoromethylpyridin-2-yl)-amine (13) (588 mg, 38%).

^1H NMR (300 MHz, CDCl ₃ ) δ 9.40 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 8.06 (dd, J = 8.1, 1.5 Hz, 1H), 7.86 (dd, J = 8.1, 1.5 Hz, 1H), 7.81 (s, 1H), 7.40 (t, J = 8.1 Hz, 1H), 7.25 (s, 1H), 7.22 (d, J = 5.4 Hz, 1H).

MS (IER) [M+H] ⁺ = 358.1

A reaction mixture of (4,8-dichloroquinolin-2-yl)-(4-trifluoromethylpyridin-2-yl)-amine (200 mg, 0.54 mmol, 1 eq.), 3-(piperidin-1-yl)propan-1-amine (94 μL, 0.59 mmol, 1.1 eq.), CuI (10 mg, 0.05 mmol, 0.1 eq.), L-proline (9 mg, 0.11 mmol, 0.2 eq.), potassium carbonate (148 mg, 1.01 mmol, 2 eq.) in DMSO (1.4 mL) was stirred at 90°C for 24 h under an inert argon atmosphere. The reaction mixture was then partitioned between ethyl acetate and water. After decantation, the aqueous phase was further extracted with dichloromethane. The organic phases were collected, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel to yield 8-chloro-N ⁴ -(3-piperidin-1-yl-propyl)-N ² -(4-trifluoromethylpyridin-2-yl)-quinoline-2,4-diamine (9) (48 mg, 7%).

^1H NMR (300 MHz, CDCl ₃ ) δ 9.55 (s, 1H), 8.40 (d, J = 4.8 Hz, 1H), 7.85 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.62 (s, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 4.8 Hz, 1H), 5.88 (s, 1H), 3.41 - 3.31 (m, 2H), 2.59 (t, J = 5.4 Hz, 2H), 2.57 - 2.43 (m, 4H), 2.01 - 1.92 (m, 2H), 1.79 - 1.70 (m, 4H), 1.65 - 1.54 (m, 2H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 154.9, 154.0, 152.3, 148.5, 144.2, 132.2, 129.7, 121.7, 119.7, 118.4, 112.4, 109.8, 88.0, 59.6, 55.1, 44.9, 26.1, 24.4, 23.4.

MS (IER) [M+H] ⁺ = 464.2

Example 2: 2-N-(8-chloroquinolin-2-yl)-5-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(trifluoromethyl)pyridine-2,5-diamine; compound (22)

A reaction mixture of 2,8-dichloroquinoline (198 mg, 1.0 mmol, 1 eq.), 5-bromo-4-(trifluoromethyl)pyridin-2-amine (241 mg, 1.0 mmol, 1 eq.), Pd(OAc) ₂ (4.5 mg, 0.02 mmol, 2 mol%), XantPhos (11.6 mg, 0.02 mmol, 2 mol%) and _Cs2CO3 (782 mg, 2.4 mmol, 2.4 eq.) in t-BuOH (4 mL) was heated in a microwave reactor at 120 °C for 70 min. After cooling to room temperature _, the reaction mixture was concentrated under reduced pressure and the resulting residue was diluted with ethyl acetate. The organic phase was then washed with water, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to yield N-[5-bromo-4-(trifluoromethyl)pyridin-2-yl]-8-chloroquinolin-2-amine (21) (300 mg, 75%).

^1H NMR (300 MHz, CDCl ₃ ) δ 9.71 (s, 1H), 8.51 (s, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.81 (m, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H).

MS (IER) [M+H] ⁺ = 403.7

A reaction mixture of N-[5-bromo-4-(trifluoromethyl)pyridin-2-yl]-8-chloroquinolin-2-amine (101 mg, 0.250 mmol, 1 eq.), 3-(4-methylpiperazin-1-yl)propan-1-amine (64 μL, 0.375 mmol, 1.5 eq.), _Pd2 (dba) ₃ (28 mg, 0.030 mmol, 12 mol%), XantPhos (43.4 mg, 0.075 mmol, 30 mol%) and sodium tert-butoxide (72 mg, 0.75 mmol, 3 eq.) in dioxane (1 mL)/DMF (0.1 mL) was heated in a microwave reactor at 120°C for 70 min. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure, and the resulting residue was diluted with ethyl acetate. The organic phase was then washed with water, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 2-N-(8-chloroquinolin-2-yl)-5-N-[3-(4-methylpiperazin-1-yl)propyl]-4-(trifluoromethyl)pyridine-2,5-diamine (22) (52 mg, 43%).

^1H NMR (300 MHz, CDCl ₃ ) δ 9.39 (s, 1H), 7.97 - 7.86 (m, 2H), 7.82 (br s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 3.34 - 3.22 (m, 2H), 2.80 - 2.44 (m, 10H), 2.37 (s, 3H), 1.87 (t, J = 6.0 Hz, 2H).

MS (IER) [M+H] ⁺ = 479.0

Example 3 : 8-chloro-N-methyl-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine; compound (28)

8-chloro-N-[4-(trifluoromethoxy)phenyl]quinolin-2-amine, i.e. compound (24), was synthesized as described in WO 2010/143169, in Example 5.

A reaction mixture of 8-chloro-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine (24) (340 mg, 1.0 mmol, 1 eq), potassium tert-butoxide (124 mg, 1.1 mmol, 1.1 eq), and iodomethane (69 μL, 1.1 mmol, 1.1 eq) in DMF (2 mL) was stirred at room temperature for 4 h. The reaction mixture was then partitioned between ethyl acetate and water. The organic phases were collected, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to afford 8-chloro-N-methyl-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine (28) (247 mg, 70%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.69 (d, J = 9.0 Hz, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.36 - 7.25 (m, 4H), 7.14 (t, J = 7.8 Hz, 1H), 6.75 (d, J = 9.0 Hz, 1H), 3.69 (s, 3H).

MS (IER) [M+H] ⁺ = 353.1

Example 4: 8-chloro-N-(3-(piperidin-1-yl)propyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine; compound (30)

8-chloro-N-[4-(trifluoromethoxy)phenyl]quinolin-2-amine, i.e. compound (24), was synthesized as described in WO 2010/143169, in Example 5.

A reaction mixture of 8-chloro-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine (24) (500 mg, 1.47 mmol, 1 eq.) and NaH (177 mg, 4.43 mmol, 3 eq.) in anhydrous DMF (2 mL) was stirred at room temperature for 30 min. A reaction mixture of 1-(3-chloropropyl)piperidine hydrochloride (292 mg, 1.47 mmol, 1 eq.), KI (245 mg, 1.47 mmol, 1 eq.) and _Et3N (205 μL, 1.47 mmol, 1 eq.) in anhydrous DMF (5 mL) was stirred at room temperature for 30 min under an inert argon atmosphere. Then, active quinoline was added to the piperidine chain, and the resulting reaction mixture was stirred at 90°C for 4 hours. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with ethyl acetate. The organic phase was washed with saturated aqueous brine, dried over _MgSO4 , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 8-chloro-N-(3-(piperidin-1-yl)propyl)-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine (30) (472 mg, 69%).

^1H NMR (300 MHz, CDCl ₃ ) δ 7.73 - 7.63 (m, 2H), 7.48 (d, J = 7.9 Hz, 1H), 7.39 - 7.26 (m, 4H), 7.13 (t, J = 7.9 Hz, 1H), 6.67 (d, J = 9.1 Hz, 1H), 4.26 - 4.14 (m, 2H), 2.52 - 2.33 (m, 6H), 2.11 - 1.97 (m, 2H), 1.64 - 1.52 (m, 4H), 1.45 (s, 2H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 156.5, 147.3, 144.1, 143.5, 137.1, 130.8, 129.7, 129.1, 126.4, 124.7, 122.7, 122.4, 118.9 (t, J = 222 Hz), 112.5, 56.9, 54.5, 49.6, 25.6, 24.6, 24.3.

MS (IER) [M+H] ⁺ = 464.4

Example 5: 8-chloro-2-((4-(trifluoromethyl)pyridin-2-yl)oxy)quinoline; compound (46)

A reaction mixture of 2,8-dichloroquinoline (79 mg, 0.4 mmol, 1 eq), 2-hydroxy-4-(trifluoromethyl)pyridine (65 mg, 0.4 mmol, 1 eq), CuI (76 mg, 0.4 mmol, 1 eq), and _Cs2CO3 (391 mg, 1.2 mmol, 3 eq) in DMF (6 mL) was heated in a microwave reactor at 150 °C for 50 min. After cooling to room temperature _, water was added to the reaction mixture. Insoluble solids were filtered through celite, and the resulting filtrate was extracted twice with ethyl acetate. The organic phase was washed with water and saturated aqueous brine, dried over _MgSO4 , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 8-chloro-2-{[4-(trifluoromethyl)pyridin-2-yl]oxy}quinoline (46) (68 mg, 52%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.40 (d, J = 7.2 Hz, 1H), 8.34 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.56 (t, J = 7.9 Hz, 1H), 6.98 (s, 1H), 6.53 (d, J = 7.9 Hz, 1H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 161.6, 151.2, 143.4, 142.3, 138.7, 138.2, 137.4, 133.4, 130.6, 129.1, 127.6, 126.7, 120.2, 119.7, 102.3.

MS (IER) [M+H] ⁺ = 325.1

Example 6: 8-chloro-2-((4-(trifluoromethoxy)phenoxy)quinoline; compound (48)

A reaction mixture of 2,8-dichloroquinoline (2 x 79 mg, 2 x 0.4 mmol, 1 eq.), 4-(trifluoromethoxy)phenol (2 x 52 μL, 2 x 0.4 mmol, 1 eq.), CuI (2 x 76 mg, 2 x 0.4 mmol, 1 eq.), and _Cs2CO3 ( ₂ x 391 mg, 2 x 1.2 mmol, 3 eq.) in DMF (2 x 6 mL) was heated in a microwave reactor at 150°C for 50 min. After cooling to room temperature, water was added to the reaction mixture. Undissolved solids were filtered through celite, and the resulting filtrate was extracted twice with ethyl acetate. The organic phase was washed with water and saturated aqueous brine, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to afford 8-chloro-2-[4-(trifluoromethoxy)phenoxy]quinoline 48 (212 mg, 78%).

^1H NMR (300 MHz, CDCl ₃ ) δ 8.12 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 9.1 Hz, 2H), 7.38 - 7.22 (m, 3H), 7.12 (d, J = 8.8 Hz, 1H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 161.5, 151.9, 145.9, 142.7, 140.5, 132.0, 130.3, 127.0, 126.4, 125.1, 122.8, 122.2, 119.0 (t, J = 255 Hz), 113.6.

MS (IER) [M+H] ⁺ = 340.1

The structures of other compounds of the invention were confirmed by NMR spectra.

Table 2

Pr. Characteristics 8 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.57 (s, 1H), 8.45 (d, J = 9.0 Hz, 1H), 8.43 (d, J = 5.1 Hz, 1H), 7.90 (br s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 5.1 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 4.17 (t, J = 6.3 Hz, 2H), 2.56 (t, J = 7.8 Hz, 2H), 2.50 - 2.39 (m, 4H), 2.17 - 2.04 (m, 2H), 1.66 - 1.59 (m, 4H), 1.51 - 1.43 (m, 2H).
¹³ C NMR (75 MHz, CDCl ₃ ) δ 154.1, 153.8, 152.5, 148.7, 143.8, 133.5, 129.8, 122.9, 117.5, 113.2, 112.5, 109.9, 103.9, 67.2, 56.2, 54.8, 26.9, 26.1, 24.5.
MS (IER) [M+H] ⁺ = 465.4 9 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.55 (s, 1H), 8.40 (d, J = 4.8 Hz, 1H), 7.85 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.62 (s, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 4.8 Hz, 1H), 5.88 (s, 1H), 3.41 - 3.31 (m, 2H), 2.59 (t, J = 5.4 Hz, 2H), 2.57 - 2.43 (m, 4H), 2.01 - 1.92 (m, 2H), 1.79 - 1.70 (m, 4H), 1.65 - 1.54 (m, 2H).
¹³ C NMR (75 MHz, CDCl ₃ ) δ 154.9, 154.0, 152.3, 148.5, 144.2, 132.2, 129.7, 121.7, 119.7, 118.4, 112.4, 109.8, 88.0, 59.6, 55.1, 44.9, 26.1, 24.4, 23.4.
MS (IER) [M+H] ⁺ = 464.2 10 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.53 (d, J = 4.8 Hz, 1H), 8.10 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.76 (dd, J = 7.5, 1.2 Hz, 1H), 7.64 (dd, J = 7.5, 1.2 Hz, 1H), 7.43 (d, J = 9.0 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 4.8 Hz, 1H), 3.87 (s, 3H).
¹³ C NMR (75 MHz, CDCl ₃ ) δ 157.6, 155.8, 149.0, 143.4, 138.0, 131.9, 130.0, 126.3, 126.1, 124.4, 115.0, 113.1, 113.0, 111.6, 111.5, 36.3.
MS (IER) [M+H] ⁺ = 338.1 11 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.50 (s, 1H), 8.41 (br s, 1H), 8.14 (br s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.11 (s, 1H), 5.96 (s, 1H), 5.88 (s, 1H), 3.77 (s, 4H), 3.25 (s, 2H), 2.77 (s, 2H), 2.54 (s, 4H).
¹³ C NMR (75 MHz, CDCl ₃ ) δ 154.8, 153.7, 151.1, 148.5, 144.2, 132.4, 129.9, 122.4, 118.2, 118.0, 112.5, 109.9, 89.2, 67.2, 56.0, 53.2, 38.9.
MS (IER) [M+H] ⁺ = 452.1 13 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.40 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 8.06 (dd, J = 7.8, 1.5 Hz, 1H), 7.86 (dd, J = 7.8, 1.5 Hz, 1H), 7.81 (s, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.25 (s, 1H), 7.22 (d, J = 5.4 Hz, 1H).
MS (IER) [M+H] ⁺ = 358.1 14 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.51 (d, J = 4.8 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.14 (d, J = 4.8 Hz, 1H), 4.48 (t, J = 7.2 Hz, 2H), 3.77 - 3.59 (m, 4H), 2.57 - 2.33 (m, 6H), 2.13 - 1.97 (m, 2H).
MS (IER) [M+H] ⁺ = 451.2 15 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.47 (d, J = 4.8 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.68 (s, 1H), 7.49 (d, J = 2.7 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 4.8 Hz, 1H), 6.99 (d, J = 2.7 Hz, 1H), 4.45 (t, J = 6.9 Hz, 2H), 4.21 (t, J = 5.4 Hz, 2H), 3.83 - 3.64 (m, 8H), 2.87 (t, J = 5.4 Hz, 2H), 2.70 - 2.56 (m, 4H), 2.56 - 2.36 (m, 6H), 2.12 - 1.96 (t, J = 6.9 Hz, 2H).
MS (IER) [M+H] ⁺ = 580.4 16 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.50 (d, J = 4.8 Hz, 1H), 8.37 (d, J = 9.0 Hz, 1H), 7.80 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.13 (d, J = 4.8 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 4.47 (t, J = 6.9 Hz, 2H), 4.26 (t, J = 5.4 Hz, 2H), 3.78 - 3.64 (m, 8H), 2.92 (t, J = 5.4 Hz, 2H), 2.69 - 2.56 (m, 4H), 2.55 - 2.38 (m, 6H), 2.05 (t, J = 6.9 Hz, 2H),
MS (IER) [M+H] ⁺ = 580.2 17 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.47 (s, 1H), 8.41 (d, J = 5.1 Hz, 1H), 8.24 (br s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 2.7 Hz, 1H), 7.14 (d, J = 5.1 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 2.7 Hz, 1H), 4.17 (t, J = 5.7 Hz, 2H), 2.87 (t, J = 5.7 Hz, 2H), 2.75 - 2.61 (m, 4H), 2.57 - 2.45 (m, 4H), 2.31 (s, 3H).
MS (IER) [M+H] ⁺ = 233.6 18 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.48 (s, 1H), 8.42 (d, J = 4.8 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.78 (br s, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.16 (d, J = 4.8 Hz, 1H), 7.06 - 6.98 (m, 2H), 4.20 (t, J = 6.0 Hz, 2H), 2.84 (t, J = 6.0 Hz, 2H), 2.61 - 2.50 (m, 4H), 1.69 - 1.59 (m, 4H), 1.52 - 1.42 (m, 2H).
MS (IER) [M+H] ⁺ = 450.9 19 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.48 (s, 1H), 8.41 (d, J = 4.5 Hz, 1H), 8.34 (s, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.46 (s, 1H), 7.14 (d, J = 4.5 Hz, 1H), 7.01 - 6.89 (m, 2H), 4.08 (t, J = 5.7 Hz, 2H), 2.66 - 2.41 (m, 6H), 2.13 - 1.99 (m, 2H), 1.72 - 1.59 (m, 4H), 1.55 - 1.41 (m, 2H).
MS (IER) [M+H] ⁺ = 465.0 20 ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.70 (d, J = 9.0 Hz, 1H), 8.24 (d, J = 4.8 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.11 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.08 (t, J = 4.8 Hz, 1H).
MS (IER) [M+H] ⁺ = 341.9 21 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.71 (s, 1H), 8.51 (s, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.81 (m, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H).
MS (IER) [M+H] ⁺ = 403.7 22 ^1H NMR (300 MHz, CDCl ₃ ) δ 9.39 (s, 1H), 7.97 - 7.86 (m, 2H), 7.82 (br s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 3.34 - 3.22 (m, 2H), 2.80 - 2.44 (m, 10H), 2.37 (s, 3H), 1.87 (t, J = 6.0 Hz, 2H).
MS (IER) [M+H] ⁺ = 479.0 28 ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.69 (d, J = 9.0 Hz, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.36 - 7.25 (m, 4H), 7.14 (t, J = 7.8 Hz, 1H), 6.75 (d, J = 9.0 Hz, 1H), 3.69 (s, 3H).
MS (IER) [M+H] ⁺ = 353.1 29 ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.26 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 9.0 Hz, 3H), 6.83 (d, J = 9.0 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 4.06 (t, J = 6.0 Hz, 2H), 2.56 (t, J = 7.5 Hz, 2H), 2.52 - 2.40 (m, 4H), 2.14 - 2.01 (m, 2H), 1.69 - 1.58 (m, 4H), 1.50 - 1.41 (m, 2H).
MS (IER) [M+H] ⁺ = 480.3 30 ^1H NMR (300 MHz, CDCl ₃ ) δ 7.73 - 7.63 (m, 2H), 7.48 (d, J = 7.9 Hz, 1H), 7.39 - 7.26 (m, 4H), 7.13 (t, J = 7.9 Hz, 1H), 6.67 (d, J = 9.1 Hz, 1H), 4.26 - 4.14 (m, 2H), 2.52 - 2.33 (m, 6H), 2.11 - 1.97 (m, 2H), 1.64 - 1.52 (m, 4H), 1.45 (s, 2H).
¹³ C NMR (75 MHz, CDCl ₃ ) δ 156.5, 147.3, 144.1, 143.5, 137.1, 130.8, 129.7, 129.1, 126.4, 124.7, 122.7, 122.4, 121.8, 118.9, 112.5, 56.9, 54.5, 49.6, 25.6, 24.6, 24.3.
MS (IER) [M+H] ⁺ = 464.4 31 ^1H NMR (300 MHz, CDCl ₃ ) δ 7.74 - 7.65 (m, 2H), 7.50 (d, J = 7.8 Hz, 1H), 7.42 - 7.28 (m, 4H), 7.14 (t, J = 7.8 Hz, 1H), 6.63 (d, J = 9.0 Hz, 1H), 4.34 (t, J = 6.9 Hz, 2H), 3.66 (t, J = 4.5 Hz, 4H), 2.78 (t, J = 6.9 Hz, 2H), 2.59 (t, J = 4.5 Hz, 4H).
MS (IER) [M+H] ⁺ = 452.3 32 ^1H NMR (300 MHz, CDCl ₃ ) δ 7.70 (d, J = 9.0 Hz, 1H), 7.69 (dd, J = 7.8, 1.2 Hz, 1H), 7.50 (dd, J = 7.8, 1.2 Hz, 1H), 7.38 - 7.26 (m, 4H), 7.15 (t, J = 7.8 Hz, 1H), 6.66 (d, J = 9.0 Hz, 1H), 4.35 (t, J = 7.5 Hz, 2H), 2.91 (t, J = 7.5 Hz, 2H), 2.70 - 2.60 (m, 4H), 1.83 - 1.74 (m, 4H).
MS (IER) [M+H] ⁺ = 436.1 33 ^1H NMR (300 MHz, CDCl ₃ ) δ 7.66 (d, J = 9.3 Hz, 1H), 7.64 (dd, J = 7.8, 1.5 Hz, 1H), 7.46 (dd, J = 7.8, 1.5 Hz, 1H), 7.29 - 7.22 (m, 4H), 7.10 (t, J = 7.8 Hz, 1H), 6.59 (d, J = 9.3 Hz, 1H), 4.16 (t, J = 7.5 Hz, 2H), 3.66 (t, J = 4.8 Hz, 4H), 2.46 - 2.36 (m, 6H), 1.78 (dt, J = 15.0, 7.2 Hz, 2H), 1.59 (dt, J = 15.0, 7.2 Hz, 2H).
MS (IER) [M+H] ⁺ = 480.1 34 ^1H NMR (300 MHz, CDCl ₃ ) δ 7.80 (d, J = 9.0 Hz, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.58 (br s, 1H), 7.19 (d, J = 9.0 Hz, 2H), 7.11 (t, J = 8.4 Hz, 1H), 5.83 (s, 1H), 3.24 (t, J = 5.4 Hz, 2H), 2.61 - 2.46 (m, 6H), 1.91 (t, J = 5.4 Hz, 2H), 1.78 - 1.68 (m, 4H), 1.62 - 1.52 (m, 2H).
MS (IER) [M+H] ⁺ = 479.3 35 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.00 (dd, J = 7.8, 1.5 Hz, 1H), 7.87 (d, J = 9.0 Hz, 2H), 7.80 (dd, J = 7.8, 1.5 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 9.0 Hz, 2H), 7.05 (s, 1H), 6.84 (br s, 1H). 36 ¹ H NMR (300 MHz, MeOD) δ 8.37 (d, J = 9.0 Hz, 1H), 8.26 (d, J = 9.3 Hz, 2H), 7.60 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 9.3 Hz, 2H), 7.00 (d, J = 9.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 4.29 (t, J = 5.4 Hz, 2H), 3.73 (t, J = 4.8 Hz, 4H), 2.94 (t, J = 5.4 Hz, 2H), 2.66 (t, J = 4.8 Hz, 4H).
MS (IER) [M+H] ⁺ = 468.1 37 ^1H NMR (300 MHz, MeOD) δ 7.92 (dd, J = 7.8, 1.5 Hz, 1H), 7.71 (dd, J = 7.8, 1.5 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.05 (s, 1H), 6.83 - 6.79 (m, 1H), 6.64 - 6.58 (m, 1H).
MS (IER) [M+H] ⁺ = 388.0 38 ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.87 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 9.0 Hz, 2H), 7.32 (d, J = 9.0 Hz, 2H), 7.17 (t, J = 7.8 Hz, 1H), 6.69 (s, 1H), 4.29 (t, J = 6.9 Hz, 2H), 3.63 (t, J = 4.5 Hz, 4H), 2.73 (t, J = 6.9 Hz, 2H), 2.55 (t, J = 4.5 Hz, 4H).
MS (IER) [M+H] ⁺ = 486.1 39 ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.87 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 8.7 Hz, 1H), 7.44 (d, J = 2.7 Hz, 1H), 7.21 (d, J = 9.0 Hz, 2H), 6.95 (br s, 1H), 6.91 (d, J = 2.7 Hz, 1H), 6.84 (d, J = 8.7 Hz, 1H), 4.16 (t, J = 5.4 Hz, 2H), 3.76 (t, J = 4.5 Hz, 4H), 2.84 (t, J = 5.4 Hz, 2H), 2.61 (t, J = 4.5 Hz, 4H).
MS (IER) [M+H] ⁺ = 468.1 40 ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.67 - 7.59 (m, 2H), 7.39 (d, J = 9.0 Hz, 2H), 7.26 (d, J = 9.0 Hz, 2H), 7.07 (t, J = 7.8 Hz, 1H), 6.79 (s, 1H), 4.34 (t, J = 6.9 Hz, 2H), 3.82 (t, J = 4.5 Hz, 4H), 3.69 (t, J = 4.5 Hz, 4H), 3.05 (t, J = 5.4 Hz, 2H), 2.78 (t, J = 6.9 Hz, 2H), 2.66 - 2.60 (m, 4H), 2.57 - 2.48 (m, 6H), 1.82 (t, J = 5.4 Hz, 2H).
MS (IER) [M+H] ⁺ = 594.4 41 ^1H NMR (300 MHz, CDCl ₃ ) δ 7.95 (d, J = 2.7 Hz, 1H), 7.81 (d, J = 9.0 Hz, 1H), 7.70 (dd, J = 7.8, 1.5 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.61 - 7.56 (m, 1H), 7.54 (dd, J = 7.8, 1.5 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.49 (d, J = 9.0 Hz, 1H), 4.32 - 4.21 (m, 2H), 3.60 (t, J = 4.5 Hz, 4H), 2.81 (t, J = 6.3 Hz, 2H), 2.49 (t, J = 4.5 Hz, 4H).
MS (IER) [M+H] ⁺ = 497.1 42 ^1H NMR (300 MHz, CDCl ₃ ) δ 7.91 (d, J = 9.0 Hz, 1H), 7.73 (dd, J = 7.8, 1.2 Hz, 1H), 7.58 (dd, J = 7.8, 1.2 Hz, 1H), 7.52 (br s, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.73 (d, J = 9.0 Hz, 1H), 3.54 (t, J = 4.5 Hz, 4H), 3.21 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 6.0 Hz, 2H), 2.40 (t, J = 4.5 Hz, 4H).
MS (IER) [M+H] ⁺ = 467.2 43 ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.11 (d, J = 9.3 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 9.0 Hz, 2H), 7.30 (d, J = 9.0 Hz, 2H), 6.59 (d, J = 9.3 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 4.34 (t, J = 6.9 Hz, 2H), 4.22 (t, J = 5.7 Hz, 2H), 3.72 (t, J = 4.5 Hz, 4H), 3.65 (t, J = 4.5 Hz, 4H), 2.88 (t, J = 5.7 Hz, 2H), 2.76 (t, J = 6.9 Hz, 2H), 2.63 - 2.55 (m, 8H).
MS (IER) [M+H] ⁺ = 581.2 44 ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.25 (d, J = 9.3 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 6.64 (d, J = 9.3 Hz, 2H), 6.59 (d, J = 8.7 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 4.21 (t, J = 5.4 Hz, 2H), 3.72 (t, J = 4.5 Hz, 4H), 2.88 (t, J = 5.4 Hz, 2H), 2.61 (t, J = 4.5 Hz, 4H). 46 ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.40 (d, J = 7.2 Hz, 1H), 8.34 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.56 (t, J = 7.9 Hz, 1H), 6.98 (s, 1H), 6.53 (d, J = 7.9 Hz, 1H).
¹³ C NMR (75 MHz, CDCl ₃ ) δ 161.6, 151.2, 143.4, 142.3, 138.7, 138.2, 137.4, 133.4, 130.6, 129.1, 127.6, 126.7, 120.2, 119.7, 102.3.
MS (IER) [M+H] ⁺ = 325.1 47 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.33 (d, J = 7.5 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 6.96 (s, 1H), 6.50 (dd, J = 7.5, 1.8 Hz, 1H), 4.25 (t, J = 5.5 Hz, 2H), 3.86 - 3.69 (m, 4H), 2.89 (t, J = 5.5 Hz, 2H), 2.73 - 2.54 (m, 4H).
MS (IER) [M+H] ⁺ = 454.2 48 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.12 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 9.1 Hz, 2H), 7.38 - 7.22 (m, 3H), 7.12 (d, J = 8.8 Hz, 1H).
¹³ C NMR (75 MHz, CDCl ₃ ) δ 161.5, 151.9, 145.9, 142.7, 140.5, 132.0, 130.3, 127.0, 126.4, 125.1, 122.8, 122.2, 119.0 (t, J = 255 Hz), 113.6.
MS (IER) [M+H] ⁺ = 340.1 49 ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.03 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 2.7 Hz, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 7.10 (d, J = 8.7 Hz, 1H), 7.02 (d, J = 2.7 Hz, 1H), 4.19 (t, J = 5.7 Hz, 2H), 3.75 (t, J = 4.5 Hz, 4H), 2.85 (t, J = 5.7 Hz, 2H), 2.61 (t, J = 4.5 Hz, 4H).
MS (IER) [M+H] ⁺ = 469.1 50 ^1H NMR (300 MHz, CDCl ₃ ) δ 8.52 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 9.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 7.07 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 4.29 - 4.20 (m, 2H), 3.79 - 3.70 (m, 4H), 2.98 - 2.88 (m, 2H), 2.71 - 2.56 (m, 4H).
MS (IER) [M+H] ⁺ = 469.1 51 ^1H NMR (300 MHz, DMSO) δ 10.84 (s, 1H), 9.57 (s, 1H), 8.58 (d, J = 5.1 Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H), 7.78 (s, 1H), 7.70 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 5.1 Hz, 1H).
MS (IER) [M+H] ⁺ = 420.1 52 ¹ H NMR (300 MHz, DMSO) δ 9.23 (d, J = 9.1 Hz, 1H), 8.21 (d, J = 9.1 Hz, 1H), 7.79 (t, J = 7.5 Hz, 2H), 7.46 - 7.18 (m, 5H).
MS (IER) [M+H] ⁺ = 435.0 53 ^1H NMR (300 MHz, DMSO) δ 9.92 (s, 1H), 8.27 (d, J = 8.7 Hz, 2H), 8.15 (d, J = 9.0 Hz, 1H), 7.65 (s, 1H), 7.54 (s, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.17 (d, J = 9.0 Hz, 1H).
MS (IER) [M+H] ⁺ = 435.0

Pharmacological data

The compounds of the invention were subjected to pharmacological tests, which showed their relevance as active substances in the treatment and, in particular, in the prevention of inflammatory disease.

Example 7: Modulation of miR-124 expression by quinoline derivatives in an in vivo model of inflammatory bowel disease

A. Material and methods

Ex vivo studies

Extraction of peripheral blood mononuclear cells (PBMCs) using a Ficoll™ gradient

For this purpose, PBMCs from healthy donors were isolated by Ficoll™ gradient centrifugation according to standard protocols.

Briefly, 60-70 ml of buffy coat is poured into a 175 ^cm2 flask and the volume is adjusted to 300 ml using phosphate-buffered saline (PBS) to obtain an approximately 5-fold dilution of the buffy coat. 38 ml of the diluted buffy coat is then added to 50 ml Falcon™ tubes containing 12 ml of Ficoll™ (Histopack-1077) at ambient temperature. The preparation is centrifuged for 30 minutes at a splitting factor of 515 at ambient temperature. The lymphocyte ring is isolated from the Falcon™ tube using a transfer pipette ( ^Pastette® ) and then washed with PBS by centrifugation for 10 minutes at a splitting factor of 290 at ambient temperature until the supernatant is clear.

The cells were then resuspended at 37°C to a density of 1.5x10 ⁶ cells/ml in RPMI Glutamax medium (Life Technologies, #61870-010) supplemented with 10% fetal bovine serum (FBS) (Thermo Fischer, #SV30160.03) and without activation. The cells were incubated for 48 hours at 37°C in an atmosphere of 5% CO ₂ .

Treating cells with screened molecules

Six-well plates are used for screening. Molecules to be screened are added to each well containing ^3.106 cells/4 ml of RPMI supplemented with 10% fetal bovine serum and 40 U/ml of interleukin-2 (IL-2) (Peprotech, No. 200-02). One well is supplemented with 100% DMSO (4 µl) and tested as a negative control.

Each test condition is set up as described in this document below and the final corresponding volume is adjusted according to the well:

1) quinoline derivatives in 100% DMSO - (5 μM and final volume 4 μl),

2) antiretroviral drugs: Maraviroc, Efavirenz, Darunavir, AZT (10 μM for all - final volume 4 μL).

Wells are incubated for three days at 37°C under 5% _CO2 . The medium is replaced (day 3) according to standard protocols. Briefly, plates are centrifuged at a splitting factor of 290 for 5 minutes, and 3 ml of supernatant is collected. Then, 3 ml of RPMI medium supplemented with 10% fetal calf serum and 40 U/ml IL-2 are added, along with 3 μl of a stock solution of the molecule being screened at a concentration of 5 mM in 100% DMSO or 3 μl of 100% DMSO as a negative control.

miRNA extraction (day 6)

Cells are collected in 15 ml Falcon™ tubes, centrifuged at a splitting factor of 290 for 5 minutes, then washed in 10 ml of PBS and further centrifuged at a splitting factor of 290 for 5 minutes. The cells are then resuspended in 1 ml of PBS and counted.

6 x 10 ⁶ cells were collected and centrifuged at a splitting factor of 290 for 5 minutes. The cell pellet was lysed in 300 µl of ML Lysis Buffer from the Macherey Nagel Nucleospin® miRNA Extraction Kit (Macherey Nagel #740971) and then stored at -20°C.

For each test sample, 5 µl of ^2x108 copies/µl control RNA transcript for calibration (Ce_miR-39 from QIAGEN ^© - No. 219610 SEQ ID N°6) was added. miRNA extraction was performed using the Macherey Nagel Nucleospin ^® miRNA Extraction Kit protocol, using an elution volume of 50 µl for RNA and 30 µl for miRNA, and then stored at -20°C.

Reverse transcription of miRNA (day 6)

The reverse transcription step is performed on 12 µl of miRNA using the miScript RT II (RT) Reverse Transcription Kit from QIAGEN ^© using miScript HiSpec Buffer and then stored at -20°C.

Quantitative PCR of miRNA (day 6)

The quantitative PCR step is performed using the QIAGEN ^© miScript SYBR ^® Green PCR kit and the miScript primer assay according to the manufacturer's protocol.

miScript reaction mixture composition for 384-well plates:

Mixture µl/reaction 2X ^SYBR® Green Blend 5 10X Universal Primer 1 10X primer analysis method 1 H ₂ O 2 Total volume of the mixture: 9 Template cDNA in H ₂ O (*) 1 Final volume: 10

(*) cDNA obtained using the miScript II RT kit

The reaction is repeated in triplicate in a 384-well plate according to the manufacturer's protocol using a Roche ^{LightCycler®} 380 real-time PCR system. Cycling conditions are also set according to the manufacturer's protocol:

Relative quantification by qPCR is known in the art and is described in more detail below.

Relative quantification

From diluted to 1/10 in _H2O for miR-124 qPCR (Hs_miR-124a) or to 1/100 for reference/housekeeping qPCR genes (Hs_miR-26a and Hs_miR-191) using miScript Primer Assays (Hs_miR-124a, Hs_miR-26a and Hs_miR-191 or QIAGEN ^© - comparisons ms00006622, ms00029239 and ms00003682).

The analysis was performed using relative quantification models without efficiency adjustment (2 ^-ΔΔCp ), using the mean intercept (Cp) of triplicate miR-124 and the mean of the means of triplicate miR-26a and miR-191.

B. Results

One group of donors (1 to 7 donors were tested for each compound) was evaluated in the presence of different compounds of formula (I).

Using the protocol described above, the mean fold change (compared to DMSO) of miR-124 expression with different donors (any from 1 to 7) was estimated by relative quantification, and the data are presented below in Table 3:

Table 3

Molecule Fold change compared to DMSO-treated cells
(Relative quantification) Number of donors tested Arithmetic mean Standard deviation 1 31.96 7.25 3 23 45.09 7.44 3 45 34.21 6.22 3 2 66.68 13.86 2 3 48.52 12.71 3 24 32.48 21.76 7 12 16.33 15.12 4 4 24.37 - 1 25 14.28 6.50 2 5 26.76 8.45 3 6 30.94 12.28 3 26 6.13 1.18 2 27 1.42 0.19 3 7 7.54 - 1 28 22.20 8.25 3 8 7.97 2.66 3 30 9.88 8.75 4 9 111.77 75.69 2 10 21.08 13.46 3 48 23.98 11.01 4 46 6.23 4.38 6 11 17.42 - 1 Maraviroc 0.73 0.56 4 Efavirenz 0.46 0.27 4 Darunavir 1.08 0.89 4 AZT 0.90 0.55 4

Thus, the experimental data show that the above-mentioned quinoline derivatives of formula (I) upregulate miR-124 expression levels in PBMCs compared to the reference value established in untreated PBMCs.

In contrast, none of the known antiretroviral drugs (Maraviroc, Efavirenz, Darunavir, or AZT) had a significant effect on miR-124 overexpression in PBMCs from four donors.

Example 8: Effect of quinoline derivatives in the (DSS-)-induced colitis model

A. Material and methods

Mouse models

DSS model

A commonly used mouse model of inflammatory bowel disease is dextran sodium sulfate (DSS-)-induced colitis. Characteristic histological changes of acute DSS-induced colitis include mucin depletion, epithelial degeneration, and possible destruction of the mucosal barrier, leading to inflammation and colitis.

Three groups of 6-week-old C57BL/6 mice (8 mice in each group) were administered DSS in drinking water (2.5%) for 9 days (Figs. 1-2). Weight loss and water consumption were determined daily. The latter was determined by measuring the volume loss of drinking water in appropriate devices. Mice were administered 200 μL of 0.5 MC alone (DSS + MC group) or together with 40 mg/kg compound 24 (DSS + MC + 24 group) by gavage. When control mice had lost up to 20% of their body weight (DSS), DSS administration was stopped and replaced with drinking water. The remaining administrations were continued for 21 days.

Three groups of 16-week-old C57BL/6 mice (7 mice in each group) were administered 2.5% DSS in drinking water (Fig. 3). Mice were gavaged with 200 μL of 0.5 MC alone (DSS + MC group) or together with 40 mg/kg compound 24 (DSS + MC + 24 group). Mice were administered DSS for 6 days, and at the point when control mice had lost up to 15% of their body weight (DSS), all mice were euthanized and their colons were removed for further analysis.

Colons were defined using a ruler, and whole-gut preparations were prepared for histological analysis according to the jelly roll technique (Whittem et al.; “Murine Colitis Modeling using Dextran Sulfate Sodium (DSS),” J Vis Exp 2010 (35) 1652), fixed in formalin, and embedded in paraffin. 4-μm sections were deparaffinized and stained with hematoxylin and eosin, and lesion size and other changes were analyzed (Figs. 4–6). We compared naive and exposed mice over a cycle of DSS administration with quinoline derivatives suspended in methylcellulose or methylcellulose (MC) alone. Statistical analysis was performed using the Mann-Whitney test, with an asterisk indicating a significant difference (p<0.5) and two asterisks indicating a highly significant difference (p<0.05).

B. Results

The results presented were obtained using a quinoline derivative compound (24), the structure of which is shown below for reference.

(24)

After 5 days of DSS administration, mice treated with the quinoline derivative exhibited a body weight loss of less than 1% on average, in contrast to the MC group or untreated mice, which exhibited a body weight loss of 10% to 20% (Figs. 1 and 2).

In particular, the inventors noted higher water consumption in mice administered with the drug, reflecting the suppressive effect of quinoline derivatives on the disease. Administration of the quinoline derivative also resulted in significant control of body weight loss in mice during the second DSS administration, indicating that the treatment effect in mice did not cease and the quinoline derivative is suitable for repeated administration. After one DSS cycle, the colon length in mice administered with quinoline derivatives (6.4 cm +/- 0.6 cm) was significantly greater compared to mice administered with MC alone (5.9 cm +/- 0.8 cm) and untreated mice (5.8 cm +/- 0.8 cm) (Fig. 3). This difference was even more pronounced in mice that received two cycles of DSS - the colon length of mice treated with quinoline derivatives (5.7 cm +/- 0.9 cm) was significantly longer compared to mice treated with MC only (4.3 cm +/- 0.5 cm) and untreated mice (4.4 cm +/- 0.4 cm) (Fig. 3). Mice from different cohorts developed a comparable number of lesions (Fig. 4), but the average lesion area size was dramatically reduced in mice treated with the quinoline derivative, i.e., 2.1 ^mm2 compared to 8.4 ^mm2 in mice treated with MC only (Fig. 5). This difference was maintained in mice treated with two cycles of DSS (3.8 ^mm2 compared to 12.2 ^mm2 ).

Finally, the authors observed a reduction in changes in lymphoid organs, such as Peyer's patches, after two cycles of DSS, suggesting that administration of quinoline derivatives modulates immune responses.

Example 9: Effect of quinoline derivatives in a collagen-induced arthritis model

A. Material and methods

Mouse models

Collagen-induced arthritis model

Groups of 9- to 10-week-old DBA/1 mice were immunized intradermally with bovine collagen type II emulsified at a 1:1 ratio in Freund's complete adjuvant. Mice were tested 21 days after the first immunization and assessed for phenotypic arthritis by monitoring the ankle joint of each hind paw every other day; thickness was determined using a dial caliper (0- to 10-mm) control instrument using a thickness gauge. Mice were administered candidate quinoline derivatives suspended in methylcellulose or methylcellulose (MC) alone once daily for two weeks and then monitored for disease progression. Statistical analysis was performed using the Mann-Whitney test; an asterisk indicates a significant difference (p<0.5).

B. Results

Only one in ten mice treated with the quinoline derivative test compound (24) showed signs of inflammation (in comparison, eight out of ten mice treated with MC developed the disease). Mice treated with quinoline derivatives showed significantly reduced swelling compared to mice treated with MC alone (1.9 mm versus approximately 2.2 mm) (Fig. 6).

Thus, the results of the tests carried out on the compounds disclosed in the present invention showed that these compounds may be suitable for the treatment and/or prevention of inflammatory diseases, as further described above.

For this purpose, an effective amount of the compound can be administered to an individual suffering from inflammatory diseases.

Thus, the compound according to the present invention can be used in practice in a pharmaceutical composition, which can contain an effective amount of the given compound and one or more pharmaceutical excipients.

The above mentioned excipients are selected in accordance with the dosage form and the desired route of administration.

In this context, they may be presented in any pharmaceutical form suitable for enteral or parenteral administration in association with appropriate excipients, for example in the form of flat or film-coated tablets, hard gelatin capsules, soft-shelled capsules and other capsules, suppositories or in drinkable form such as suspensions, syrups, or in the form of injection solutions or suspensions, in doses providing administration of from 0.1 to 1000 mg of active substance per day.

Any route of administration may be used. For example, the compound of formula (I) may be administered orally, parenterally, intravenously, transdermally, intramuscularly, rectally, sublingually, intramucosally, nasally, or by other routes. Furthermore, the compound of formula (I) may be administered in the form of a pharmaceutical composition and/or in unit dosage form.

In particular, the pharmaceutical compositions of the invention can be administered orally and/or parenterally.

Suitable dosage forms include, but are not limited to, capsules, tablets (including rapidly dissolving tablets and delayed-release tablets), powder, syrups, oral suspensions, and parenteral solutions.

The pharmaceutical composition may also contain another anti-inflammatory agent well known to those skilled in the art in combination with the compound of the present invention.

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SEQUENCE LIST

<110> ABIVAX

UNIVERSITY OF MONTPELLIER

CENTER NATIONAL DE LA RECHERES SCIENTIFIC (CNRS)

INSTITUTE CURIE

<120> QUINOLINE DERIVATIVES FOR THE TREATMENT OF INFLAMMATORY DISEASES

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<150> EP14306164.6

<151> 2014-07-17

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<---

Claims (44)

1. The use of miR-124 in vitro or ex vivo as a biomarker for screening a compound of formula (I) for efficacy in the treatment and/or prevention of an inflammatory disease selected from the group consisting of inflammatory joint disease, inflammatory gastrointestinal disease, inflammatory skin disease, bronchitis, bronchial asthma, wherein formula (I) is defined below (I), Where Z represents C or N, V is C or N, means an aromatic ring where V is C or N, and when V is N, V is in the ortho-, meta- or para-position relative to Z, i.e. forms, respectively, a pyridine, pyridazine, pyrimidine or pyrazine group, R independently represents a hydrogen atom, a methyl group, a methoxy group, a trifluoromethyl group, a trifluoromethoxy group, an amino group, a halogen atom and a -OP(=O)-(OR ₃ )(OR ₄ ) group, and in particular a fluorine or chlorine atom, a trifluoromethoxy group and an amino group, Q is N or O, provided that R'' is absent when Q is O, R ₃ and R ₄ independently represent a hydrogen atom, Li ⁺ , Na ⁺ , K ⁺ , N ⁺ (Ra) ₄ or a benzyl group, Ra represents a hydrogen atom, a (C ₁ -C ₅ )alkyl group or a (C ₃ -C ₆ )cycloalkyl group, n is 1, 2 or 3, n' is 1, 2 or 3, R' independently represents a hydrogen atom, a halogen atom, and more particularly a fluorine or chlorine atom, an amino group, a methyl group, a -OP(=O)-(OR ₃ )(OR ₄ ) group, or a group , where A is O or NH, m is 2 or 3, and X ₁ is O, CH ₂ or N-CH ₃ , provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from said group, or alternatively R' independently represents a hydrogen atom, a halogen atom, and more particularly a fluorine or chlorine atom, a methyl group, or a group , where A is O or NH, m is 2, and X ₁ is O, CH ₂ or N-CH ₃ , provided that when R' is such a group, n' is 1 or 2, and when n' is 2, the other group R' is different from the said group, R'' represents a hydrogen atom, a (C ₁ -C ₄ )alkyl group, or a group , where m is 2 or 3 and X ₁ is O, CH ₂ or N-CH ₃ , or any of its pharmaceutically acceptable salts. 2. The in vitro or ex vivo use according to claim 1, wherein Q is N. 3. In vitro or ex vivo use according to claim 1 or 2, wherein the compound is selected from (Ia), (Ib) and (Ic), where R, R', R'', n and n' are as defined in paragraph 1 or 2. 4. A method for screening in vitro or ex vivo a compound of formula (I) as defined in any one of claims 1 to 3 for efficacy in the treatment and/or prevention of an inflammatory disease selected from the group consisting of an inflammatory joint disease, an inflammatory gastrointestinal disease, an inflammatory skin disease, bronchitis, bronchial asthma, comprising at least a step in which: a) provide the eukaryotic cell, b) bringing said cell into contact with a compound of formula (I), c) determining the expression of miR-124 in said cell, and d) selecting a compound when the expression level of miR-124 determined in step c) is increased relative to the reference value. 5. The method according to claim 4, wherein said eukaryotic cell is a peripheral blood mononuclear cell (PBMC). 6. A method for in vitro or ex vivo evaluation and monitoring of the efficacy of a compound of formula (I), as defined in any one of claims 1 to 3, in the treatment of an inflammatory disease selected from the group consisting of inflammatory joint disease, inflammatory gastrointestinal disease, inflammatory skin disease, bronchitis, bronchial asthma, comprising determining the presence or expression level of miR-124 in an isolated biological sample of an individual, wherein the expression level of miR-124 is compared with a control reference value, wherein an increase relative to the control reference value indicates efficacy. 7. The method of claim 6, wherein the biological sample is selected from a sample of blood, plasma, blood serum, saliva, interstitial fluid, urine; a cell sample such as a cell culture, a cell line, a stem cell line, a sample containing peripheral blood mononuclear cells (PBMCs), a tissue biopsy sample such as oral tissue, gastrointestinal tissue, skin, oral mucosa, or a plurality of samples from a clinical study; wherein said sample is an unpurified sample or purified to various degrees before storage, processing or determination. 8. The method according to claim 6 or 7, wherein the control reference value is obtained from an isolated biological sample from: - an individual or group of individuals who are known not to suffer from an inflammatory disease, - an individual or group of individuals receiving treatment whose effectiveness is to be determined or monitored; and/or - an individual suffering from an inflammatory disease and receiving treatment the effectiveness of which needs to be determined or monitored, where an isolated biological sample is obtained from the individual prior to treatment. 9. The method according to any one of claims 6-8, wherein the expression level of miR-124 is assessed using nucleic acid assays or array test systems, such as Northern blotting and polymerase chain reaction (PCR)-based methods. 10. The method according to any one of claims 6 to 9, which uses a method for detecting and quantifying nucleic acids, which may be a hybridization-based method, wherein said hybridization-based methods include PCR and quantitative PCR (qRT-PCR or q-PCR) methods or methods using reverse transcriptase/polymerase. 11. The method of claim 10, wherein the method includes or is combined with a sequencing step. 12. The method according to any one of claims 6 to 11, which comprises (i) a step of extracting cellular mRNA, (ii) a step of reverse transcribing the mRNA into DNA using reverse transcriptase, and (iii) a step of amplifying DNA with the DNA obtained in the previous step. 13. The method according to any one of claims 6-12, wherein a reduced, suppressed, or decreased level of expression of miR-124 relative to a control reference value obtained from a healthy donor is indicative of the need for treatment of an inflammatory disease. 14. The method according to any one of claims 6 to 13, wherein an increased level of expression of miR-124 relative to a control reference value obtained from an individual suffering from an inflammatory disease and not receiving or prior to receiving treatment is indicative of the efficacy of the compound of formula (I) for treating the inflammatory disease. 15. The use or method according to any one of claims 1-14, wherein the inflammatory disease is selected from inflammatory joint disease, inflammatory gastrointestinal disease, inflammatory skin disease. 16. The use or method according to any one of claims 1-15, wherein the inflammatory disease is selected from the group consisting of bronchitis, bronchial asthma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, osteoarthritis, ankylosing spondylitis, psoriasis, Sjogren's syndrome and inflammation associated with colon carcinoma. 17. The use or method according to claim 15, wherein the inflammatory disease is an inflammatory disease of the digestive tract. 18. The use or method according to claim 17, wherein the inflammatory disease of the digestive tract is inflammatory bowel disease. 19. The use or method according to claim 15, wherein the inflammatory disease is inflammatory rheumatoid arthritis.