WO2023111005A1

WO2023111005A1 - Carboxamide substituted heteroaromatic compounds for treating cancer

Info

Publication number: WO2023111005A1
Application number: PCT/EP2022/085829
Authority: WO
Inventors: Christoph Steeneck; Yansong Wang; Anika TARASEWICZ; Bartosz LESZCZYNSKI; Stefano SECHI; Iryna CHARAPITSA; George Reid; Johannes FABIAN; Claus Kremoser
Original assignee: Wmt Ag
Priority date: 2021-12-14
Filing date: 2022-12-14
Publication date: 2023-06-22

Abstract

The present invention relates to a compound of formula (I) or an enantiomer, diastereomer, N-oxide, solvate, prodrug or pharmaceutically acceptable salt thereof. The present invention further relates to a compound of formula (I) for use in a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer.

Description

Carboxamide Substituted Heteroaromatic Compounds For Treating Cancer

Field of the Invention

The present invention relates to new and inventive compounds, especially compounds useful in the treatment of cancer. The present invention further relates to a pharmaceutical composition comprising such a compound. The present invention further relates to such a compound for use as a medicament. The present invention further relates to such a compound or such a pharmaceutical composition for use in a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer. The present invention further relates to a method of treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer, in which an effective amount of such a compound or of such a pharmaceutical composition is administered to a subject in need thereof. The present invention further relates to the use of such a compound or such a pharmaceutical composition in the manufacture of a medicament for treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer.

Background of the Invention

Cancer remains the second leading cause of all deaths in mankind. Deadly cancers are those that are either diagnosed too late, i.e. at a very late stage of disease progression, or cancers that have been treated but could not be cured. After repeated relapses or at late stage, i.e. typically stage 3B or higher, many solid cancers, in particular, tend to acquire a chemo- and radiotherapy-resistant phenotype. Such advanced and treatment-resistant cancers are often characterized by a high degree of metastasis where the original tumours and I or the metastases employ a high degree of so-called Warburg metabolism (Weber et al., Front Oncol. (2016) 6:257).

Otto Warburg, after whom this effect is named, discovered about 100 years ago that cancer cells utilize anaerobic glycolysis to convert glucose into lactate. This metabolic phenotype generates ATP at low efficiency compared to oxidative phosphorylation, i.e. the mitochondrial respiration. Warburg ascribed this metabolic switch from respiration towards anaerobic glycolysis as a consequence of the lack of oxygen that is prevalent in the core of advanced cancers (Warburg et al., Biochem Zeitschr. (1924) 152:309-44; Warburg et al., J Gen Physiol (1927) 8(6):519-30). Research over the past 20 years has elucidated, however, that this metabolic switch towards high anaerobic glycolysis occurs in order to meet the huge demand for carbon-containing building blocks for cell proliferation, rather than to achieve efficient ATP generation. Multiple intermediates of the glycolytic or the pentose phosphate pathways are used by the cell to synthesize nucleosides, lipids, different carbohydrates or amino acids or to regenerate NADPH, which is needed for either lipid and cholesterol biosynthesis or for regenerating defense systems that protect against reactive oxygen species (ROS) (Vander et al., Cell (2017) 168(4):657-669). ROS are dangerous to the cell and their rate of generation is drastically increased in cancer cells (Hosios et al., J Biol Chem. (2018) 293(20):7490-7498). Thus, pathways such as the glutathione or Thioredoxin systems that detoxify ROS species are absolutely required in rapidly proliferating cells, and place a demand on the provision of NADPH-reducing equivalents, generally supplied by the first oxidative steps of the pentose phosphate pathway, using glucose as a substrate (Wang et al., Theranostics. (2011) 11(10):4839-4857).

ROS stress can also lead to damaged proteins where misfolded or chemically impaired proteins induce a so-called proteotoxic stress or unfolded protein response (UPR). If this stress situation occurs in the endoplasmic reticulum (ER), it is recognized as ER stress, a subphenomenon of proteotoxic stress (Radanovic et al., Cells. (2021) 10(11):2965). As part of the proteotoxic stress response, certain transcription factors, in particular Heat Shock Factor -1 (HSF-1), are well known as master regulators of a transcriptional program that occurs to compensate and resolve proteotoxic stress. HSF-1 is one of the first proteins to be induced upon heat shock or chemically induced proteotoxic stress response, where it coordinates transcription and hence production of proteins and systems that counteract this specific stress situation e.g. certain chaperones, ROS-detoxifying systems, certain membrane transporters or pumps (Dai et al., Philos Trans R Soc Lond B Biol Sci. (2018) 373(1738):20160525).

All of the aforementioned elements, increased anaerobic glycolysis, increased flux through the pentose phosphate pathway, increased need for ROS protection and for counteracting proteotoxic stress, can be defined as hallmarks of the Warburg metabolism in a wider, more modern definition.

The final key consequence of Warburg metabolism is high-level lactate production, an inevitable consequence of not utilizing oxidative phosphorylation. Lactate can be regarded as the waste product of anaerobic glycolysis, resulting from the action of Lactate Dehydrogenase (LDH), which reduces pyruvate into lactate by the concomitant oxidation of NADH into NAD+. This NAD+ needs to be regenerated to permit an intermediate transformation in the midst of glycolysis, catalyzed by GAPDH. Thus, converting pyruvate to lactate closes the loop for NAD- regeneration in anaerobic glycolysis, and does not allow for entry of pyruvate into mitochondria for oxidative phosphorylation metabolism. Importantly, however, cancer cells, even when predominately using Warburg metabolism and deprived of oxygen, do not fully shut down mitochondrial metabolism. This is essential as mitochondrial metabolism, in particular the citric acid cycle, allows for the use of alternative Warburg fuels, i.e. amino acids such as asparagine or glutamine, to use this central metabolic turntable to yield other amino acids and pyruvate. These can partly be converted into lactate for the aforementioned reasons or can go into “reverse” glycolysis, i.e. gluconeogenesis and the pentose phosphate pathway, to meet the demand for carbon building blocks (Baltazar et al., Front Oncol. (2020) 10:231).

Thus, lactate is the common end product of various aspects of the complex Warburg metabolic state. Moreover, it has a further function by suppressing an immune response within a heavily lactate-producing tissue environment. The primary physiological consequence of the immunosuppressive role of lactate, and the local acidic pH that comes with it, is that skeletal muscle, in particular, produces large amounts of lactate when oxygen supply is insufficient under physical exercise. In untrained muscles, microfibers tend to disrupt and intracellular components are exposed to the extracellular environment. This is a typical stimulus for tissueresident antigen presenting cells and other myeloid cells to define the damage muscle as target for the immune system. In order to prevent this, lactate and the acidic pH, both in their own right, act as immunosuppressants, thereby preventing an undesirable and unnecessary immune attack on muscle.

This physiological immunosuppressive role of lactate is hijacked or abused by solid tumors which release considerable amounts of lactate in their microenvironment. In this sense, such tumor compartments resemble a “sore” muscle, with several publications that demonstrate immunosuppressive activities on basically every relevant immune cell subtype.

In summary, lactate is foremost the waste product of Warburg metabolism. Determining its levels in the context of cancer cell proliferative capacity is probably the best indicator of the Warburg effect, as it integrates carbon flux from different sources under anaerobic and aerobic conditions.

Secondly, as lactate plays a key role as a pan-immunosuppressant in the tumor microenvironment, reducing tumor lactate production is an attractive target to achieve in clinical cancer treatment (Vaupel et al., J Physiol. (2021) 599(6):1745-1757; de la Cruz-Lopez et al., Oncol. (2019) 9:1143).

However, it has to be considered that cancer cells and transiently active skeletal muscle cells are not the only cell types in the human body that employ Warburg metabolism. It is well documented that all cell types that undergo several proliferation cycles upon certain mitogenic stimuli employ this metabolic phenotype, since it is an evolutionary predetermined pattern to support cell growth and division. In particular, T-cells that have been antigen-primed and that have received a strong proliferative signal, also consume huge amounts of glucose and turn it into lactate. It needs to be considered, though, that T-cells, whose activity is very instrumental in an immune anti-tumor response, typically proliferate in compartments where they come in close contact with antigen-presenting cells, i.e. tumor-draining lymph nodes, for example. Although it can be clearly demonstrated that T-cell proliferation upon a CD3/CD28 stimulus, for example, can be completely blunted by glycolysis inhibitors such as 2-deoxyglucose or others, it might be possible to separate anti-glycolytic effects on tumor cells from those on expanding T-cells by directing the respective pharmaceutical agent towards the tumor and not towards lymph nodes or the blood compartment. Once T-cells have proliferated and have become CD8-positive anti-tumor effector effector cells, they try to enter the tumor and thereafter, to attack cancer cells. This anti-tumor effectivity is not necessarily blunted by lowering intratumoral lactate, as recent publications have illustrated (Renner et al., Cell Rep. (2019) 29(1 ):135-15O.e9).

To date, there is no approved drug available that would inhibit lactate production in the context of cancer treatment, other than as a side effect of cytotoxic activity, in general. There have been several attempts, using various different chemical compounds, to either inhibit one or more of the glycolytic enzymes directly, or to downregulate or inhibit key transcription factors or master regulators of the Warburg program. Prominent examples of the latter mode of action or members of the family of Hypoxia-induced factors (HIF), foremost Hif-1alpha or Hif-2alpha. Hif-1 , -2 or -3 alpha heterodimerize with a common partner, Hif-1beta, which is also known as ARNT (Aryl Hydrocarbon Receptor Nuclear Translocator). All three members of the Hif-family are well described to act as transcription factors inducing the key genes that mediate the Warburg effect, i.e. GLUT-1 (glucose transporter), HK2, PK-M, LDH-A, MCT-4, the latter being hypoxia and tumor-specific isoforms of glycolytic enzymes or a lactate transporter (MCT-4) (Yu et al., J Cancer. (2017) 8(17):3430-3440).

Most of these drugs failed during clinical development, either due to a lack of efficacy with respect to anticancer effects, or because they exhibited serious and intolerable side effects.

The premier untransformed cell type that suffers from glycolytic inhibition is the erythrocyte, which relies solely on glycolysis as its energy source. Consequently, direct glycolysis inhibition leads to erythrocyte lysis, clinically manifesting as hemolytic anemia. Moreover, cancer patients under chemotherapy frequently suffer from anemia, which makes this side effect even more intolerable in the context of cancer treatment (Oshima et al., Cell Rep. (2020) 30(6):1798- 1810. e4).

Another approach to limit cancer cell proliferation targets the proteotoxic stress response. This can be achieved in manifold ways, including but not limited to inhibitors of the proteasome such as bortezomib or carfilzomib. Limiting proteasomal protein degradation leads to an accumulation of misfolded protein, ultimately leading to apoptosis.

There have been attempts to inhibit HSF-1 as a key regulator of the proteotoxic stress response in order to suppress measures by the cell to mitigate the anti-proliferative effects of proteotoxic stress. One such chemical compound that was found to downregulate HSF-1 is CCT251236, a small molecule chemical compound derived from a screen for the aforementioned effect. CCT251236 is published to limit cell proliferation in multiple different cancer cell lines. This compound also inhibited tumor growth in an ovarian cancer xenograft mouse model. CCT251236, and its closely related PROTAC analogue CCT367766, were found to tightly bind to Pirin. However, doubts remain if Pirin is the functionally relevant target of these bisamide compounds, also based on published results that show the CCT251236 displays micromolar inhibitors activity on other relevant proteins such as Acetylcholinesterase or certain kinases, as well. Nevertheless, CCT251236 has potent antitumor efficacy at a dose of 30 mg/kg every other day upon oral delivery (Cheeseman et al., J Med Chem. (2017) 60(1): 180-201; Chessum et al., J Med Chem. (2018) 61 (3):918-933).

Chemical compounds intended for pharmaceutical use, such as the aforementioned bisamides, may contain certain substructures or moieties that are known to pose potential hazardous risks for intended human use. One such potential hazard is genotoxicity, i.e. the propensity of a certain chemical structure as such, or of one of its metabolites, to induce mutations in the DNA, either by directly chemical modification, e.g. alkylating DNA or by inserting into the DNA doublehelix leading to either strandbreaks upon UV activation, or to replication errors, resulting in different types of mutations.

The aforementioned bisamide contains two aromatic amino groups at the central methylphenyl ring which are masked by being incorporated into carboxamide. It is reasonable to assume that such bisamides can be metabolized into substructures that contain at least one free aromatic amino group. Such anilinic amino groups are known to be metabolized into reactive species, such as arylnitrenium ions, that can add to DNA bases resulting in severe genotoxicity. This genotoxic potential of aromatic amines can be easily assessed by the so- called Ames test (‘General discussion of common mechanisms for aromatic amines’, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 99. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon (FR): International Agency for Research on Cancer; 2010.).

WO 2015/049535 and WO 2016/156872 disclose a series of bisamides with overlaps with certain compounds named CCT245232 and CCT251236 that were described in more detail in Cheeseman et al.; J Med Chem (2017), 60(1 ) 180-201 and in Chessum et al. J Med Chem. (2018);61 (3):918-933. CCT245232 was identified in a screen for inhibitors of the HSF-1 proteotoxic stress pathway. Using medicinal chemistry efforts, CCT245232 was developed further into CCT251236 which demonstrates better solubility and oral bioavailability in mice. The compounds of this bisamide series demonstrate potent antiproliferative activities against cancer cells with SK-OV-3 as the reference cell line to determine exact growth IC50 values. CCT251236 also shows significant anti-cancer effects in a mouse xenograft model of cancer.

CCT245232 CCT251236

The anticancer effects of this bisamide type of compounds were attributed to their inhibitory effects on the HSF-1 proteotoxic stress pathway. These compounds have a built-in structural element, a bis-aniline core, that contains a potentially genotoxic hazard because of its dual aromatic amine structure. In the final compounds these structures are masked by the assymetric bis-amidation, however, it is not unlikely that at least one of the aromatic amines will be liberated upon metabolization in the body.

Chessum et al., J Med Chem. (2018) 61 (3):918-933) disclose these compounds to bind very potently to Pirin, a non-heme iron containing cytosolic protein with so far only vaguely described cellular functions. In Meyers et al. (ACS Med Chem Lett (2018) ;9(12): 1199-1204.) it is demonstrated that these bisamide compounds promiscuously bind to different kinases as protein targets. Thus, it is not entirely clear whether these bisamide compounds really exert the anti-cancer effects solely by binding to Pirin.

In order to overcome the potentially genotoxic bis-anilinic core structure several attempts were undertaken to synthesize compounds of similar inhibitory activity on different cell lines. Accordingly, there is still a need for cancer therapeutics having at least similar inhibitory activity as compounds described in the art, combined with a reduced genotoxic potential.

Summary of the Invention

Unexpectedly, it has been found that solely compounds of the present invention, but not other heteroatom-containing core methylphenyl replacements, exhibit similar anti-proliferative activities in various cancer cell lines.

The heteroaryl-core containing compounds of the present invention also showed a broad antiproliferative activity against several cancer cell lines in vitro.

In a first aspect, the present invention relates to a compound according to formula (I)

or an enantiomer, diastereomer, N-oxide, solvate, prodrug or pharmaceutically acceptable salt thereof, wherein A, B, U¹-U³, R¹ and R³ are defined as disclosed below. In a further aspect, the present invention relates to a pharmaceutical composition comprising the compound according to formula (I) and a pharmaceutically acceptable excipient.

In a further aspect, the present invention relates to a compound according to formula (I) for use as a medicament.

In a further aspect, the present relates to the compound according to formula (I) or the pharmaceutical composition comprising the compound according to formula (I) for use in a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism.

In a further aspect, the present invention relates to a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism, the method comprising administering to a subject in need thereof an effective amount of the compound according to formula (I) or the pharmaceutical composition comprising the compound according to formula (I).

In a further aspect, the present invention also relates to the use of a compound according to formula (I) in the preparation of a medicament for the prophylaxis and/or treatment of a disease or condition mediated by the lactate/ATP mechanism.

In a further aspect, the present invention relates to a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), for use in a method of preventing and/or treating a disease or condition, preferably cancer, by inducing endoplasmic reticulum stress in cells affected by said disease or condition, thereby reducing intracellular ATP and lactate production in said cells.

In a further aspect, the present invention relates to a method of preventing and/or treating a disease or condition, preferably cancer, said method comprising administering a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), to a subject, said preventing and/or treating being achieved by inducing endoplasmic reticulum stress in cells affected by said disease or condition, thereby reducing intracellular ATP and lactate production in said cells.

In a further aspect, the present invention relates to the use of a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to formula (I), in the manufacture of a medicament for preventing and/or treating a disease or condition, preferably cancer, by inducing endoplasmic reticulum stress in cells affected by said disease or condition, thereby reducing intracellular ATP and lactate production in said cells.

In a further aspect, the present invention relates to a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), for use in a method of preventing and/or treating cancer, preferably a cancer cell and/or a cancerous tumor, wherein said compound reduces Hit 1 -alpha at the protein level in hypoxic areas of said cancer, thereby downregulating ATP and lactate production in these areas and ultimately leading to reduction, preferably efficient reduction, of hypoxic areas and of growth of said cancer.

In a further aspect, the present invention relates to a method of preventing and/or treating cancer, preferably a cancer cell and/or a cancerous tumor, said method comprising administering a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), to a subject, said preventing and/or treating being achieved by reducing Hif1 -alpha at the protein level in hypoxic areas of said cancer, thereby downregulating ATP and lactate production in these areas and ultimately leading to reduction, preferably efficient reduction, of hypoxic areas and of growth of said cancer.

In a further aspect, the present invention relates to the use of a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), in the manufacture of a medicament for preventing and/or treating cancer, preferably a cancer cell and/or a cancerous tumor, by reducing Hif1 -alpha at the protein level in hypoxic areas of said cancer, thereby downregulating ATP and lactate production in these areas and ultimately leading to reduction, preferably efficient reduction, of hypoxic areas and of growth of said cancer.

In a further aspect, the present invention relates to a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), for use in a method of preventing and/or treating a disease or condition which is susceptible to the induction of endoplasmic reticulum stress, wherein administration of said compound or of said pharmaceutical composition to a subject having or suspected of having said disease or condition results in a reduction of the protein level of Hif-1 alpha.

In a further aspect, the present invention relates to a method of preventing and/or treating a disease or condition which is susceptible to the induction of endoplasmic reticulum stress, said method comprising administering a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), to a subject, wherein administration of said compound to a subject having or suspected of having said disease or condition results in a reduction of the protein level of Hif-1 alpha.

In a further aspect, the present invention relates to the use of a compound according to Formula (I), or a pharmaceutical composition comprising the compound according to Formula (I), in the manufacture of a medicament for preventing and/or treating a disease or condition which is susceptible to the induction of endoplasmic reticulum stress, by reducing the protein level of Hif-1 alpha. In a further aspect, the present invention relates to the use of a compound according to formula (I) in the preparation of a medicament for the prophylaxis or treatment of cancer.

In further aspects of the treatments and uses set out above, the compound according to Formula (I) is comprised in a pharmaceutical composition together with a pharmaceutically acceptable excipient.

Description of the Figures

Figure 1 shows ATP levels which are downregulated in HeLa, 4T1 , LLC1 , A549, MDA-MB- 231 and MiaPaCa2 cells after 48 hours’ treatment with the compounds of Example 1 and Example 3.

Figure 2 shows lactate levels which are downregulated in HeLa, 4T1 , LLC1 , A549, MDA-MB- 231 and MiaPaCa2 cells after 48 hours’ treatment with the compounds of Example 1 and Example 3.

Figure 3 shows how Caspase 3/7 activity is induced in HeLa cells after 24 hours’ treatment with the compound of Example 1 in concentrations above 1.5 pM.

Figure 4 shows ER stress genes induced in HeLa cells after 2 hours’ treatment with 1 pM of the compounds of Example 1 and Example 3.

Figure 5 shows a diagram of results as obtained with the compounds of Example 1 and Example 3 downregulating HIF1o protein levels in HeLa cells after 24 hours’ treatment.

Detailed description of the invention

The present invention relates to a compound of formula (I)

or an enantiomer, diastereomer, N-oxide, solvate, prodrug or pharmaceutically acceptable salt thereof, wherein

A is a mono- or bicyclic Ce- -aryl or a mono- or bicyclic 5-14 membered heteroaryl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, wherein the mono- or bicyclic aryl or the mono- or bicyclic heteroaryl are unsubstituted or substituted with 1 to 5 same or different substituents R^A, or wherein two substituents R^A on adjacent carbon atoms of the monocyclic aryl or monocyclic heteroaryl ring systems, together with the carbon atoms to which they are attached, form a 5, 6 or 7 membered carbocylic or heterocyclic ring, the heterocyclic ring containing 1 or 2 heteroatoms independently selected from the group consisting of O, N and S, wherein the 5, 6 or 7 membered carbocyclic or heterocyclic ring is unsubstituted or substituted with 1 to 5 same or different substituents R⁴,

R^A is Ci-e-alkyl, halogen, CN, OH, O-Ci-e-alkyl, S-Ci-e-alkyl, S(O)>rCi-6-alkyl, NR^a-Ci-6-alkyl or C(O)NR^a-Ci-6-alkyl, wherein Ci-6-alkyl is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of Ci-e-alkyl, Ci-6-haloalkyl, halogen and oxo, or wherein two Ci-6-alkyl groups form together with the carbon atom to which they are attached a Cs-e-cycloalkyl group;

B is a bicyclic aryl or a bicyclic 8-14 membered heteroaryl containing 1 , 2, 3 or 4 heteroatoms independently selected from the group consisting of O, S and N, wherein the bicyclic aryl or the bicyclic heteroaryl is unsubstituted or substituted with 1 to 4 same or different substituents R^B;

R^B is Ci-e-alkyl, halogen, CN, OH or Ci-6-haloalkyl;

II¹, II² and II³ are independently selected from the group consisting of N and CR² with the proviso that at least one of II¹, II² and II³ must be N, and that no more than 2 of II¹, II² and II³ are allowed to be N;

R¹ is hydrogen, OH, Ci-3-alkyl or halogen;

R² is hydrogen, Ci-e-alkyl, halogen, CN, Ci-6-haloalkyl, OH or O-Ci-6-alkyl;

R³ is hydrogen, Ci-6-alkyl or V-W-X-Y-Z, wherein

V is a bond or Ci-6-alkylene,

W is a bond or O, S, S(O)_X, S(O)(=NR^a), S(O)₂NR^a, NR^a, NR^aC(O) or C(O)NR^a,

X is a bond or Ci-6-alkylene,

Y is a bond or O, S, S(O)_X, S(O)(=NR^a), S(O)₂NR^a, NR^a, NR^aC(O) or C(O)NR^a, wherein Ci-6-alkylene in V and X is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of Ci-e-alkyl, Ci-e- haloalkyl, halogen and oxo, or wherein two Ci-6-alkyl groups on the alkylene, together with the carbon atom to which they are attached, form a Cs-e-cycloalkyl group,

Z is hydrogen, halogen, OH, CN, Ci-e-alkyl, Cs-s-cycloalkyl, 4- to 8-membered mono or bicyclic heterocycloalkyl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, or

5- to 6-membered heteroaryl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, wherein alkyl, cycloalkyl, heterocycloalkyl or heteroaryl are unsubstituted or substituted with 1 to 4 substituents R^z independently selected from the group consisting of CN, OH, Ci-e-alkyl, O-Ci-6-alkyl, C(O)-Ci-6-alkyl, Ci-6-haloalkyl, halogen, oxo, spirocyclicly fused Cs-e-cycloalkyl and spirocyclicly fused 3-7 membered heterocycloalkyl containing 1 heteroatom selected from the group consisting of O, S and N;

R⁴ is Ci-6-alkyl, halogen, CN, OH, Ci-6-alkyl, O-Ci-6-alkyl, S-Ci-6-alkyl, oxo or spirocyclicly fused Cs-e-cycloalkyl;

R^a is hydrogen or Ci-3-alkyl; and x is 1 or 2.

In an embodiment in combination with any of the above or below embodiments, B is a bicyclic

8-10 membered heteroaryl containing 1 , 2, 3 or 4 heteroatoms independently selected from the group consisting of O, S and N, wherein the bicyclic heteroaryl is unsubstituted or substituted with 1 to 4 same or different substituents R^B; and wherein R^B is Ci-6-alkyl, halogen or Ci-6-haloalkyl.

9-10 membered heteroaryl containing 1 , 2, 3 or 4 heteroatoms independently selected from the group consisting of O, S and N, wherein the bicyclic heteroaryl is unsubstituted or substituted with 1 to 4 same or different substituents R^B; and wherein R^B is Ci-e-alkyl, halogen or Ci-6-haloalkyl.

Compounds of the invention according to Formula (I) exhibit a surprising biological activity.

Lactate lies at the crossroads between tumor metabolism and the late-stage cancer microenvironment, which resists the body’s natural immune response. Specifically, lactate is the end product of the so-called Warburg metabolism, a metabolism prevalent in cancer cells which favors fermentative over oxidative pathways. Warburg metabolism converts glucose into lactate, diverting carbon flux into energy to support the growth of the ever-dividing cancer cells. At the same time, lactate and the associated acidic pH in the tumor environment also exert an immuno-suppressive effect which reduces the ability of immune cells to combat nascent or preexisting cancer cells. This “lactic acid immune shield” is permissive for only those immune cells that themselves use Warburg metabolism, and that are passive against the tumor. Such so-called myeloid-derived suppressor cells (MDSCs) support the cancer by signaling other active immune cells to stay out of the tumor, thus protecting the growing tumor by helping it to evade immune attack. In total, then, an increase in lactate and its associated acidity in the tumor microenvironment promotes tumor development both directly, by driving tumor growth perse, and indirectly, by inhibiting the body’s ability to counter it.

It has been found that the compounds of the present invention inhibit lactate production. This activity reverses the tumor-promoting processes described above. Specifically, and without being bound by theory, reducing lactate concentration in cancer cells which depend on lactate as a source of energy has the effect of starving cancer cells of the energy they need for growth, thereby slowing or even halting cancer growth. Simultaneously, a reduction in lactate concentration in the tumor microenvironment weakens the “lactic acid immune shield” which otherwise normally protects cancer cells, thus advantageously exposing existing cancer cells to attack by cells of the body’s own immune system. In this way, the tumor is impeded in its ability to spawn new cells and is rendered more vulnerable to immune attack and destruction.

Specifically, a thorough investigation revealed that the compounds of the present invention not only inhibit cancerous proliferation but that this inhibition is mediated by an unprecedented lowering of ATP and lactate in cancer cell culture (Fig. 1 , 2). A unique feature of this ATP- lowering mechanism is that it comes as a biphasic effect. In the first phase, ranging from singledigit nanomolar to low single-digit micromolar concentrations, depending on the actual compounds of invention, the heteroaryl bisamide compounds of the invention lower ATP and lactate and inhibit cellular proliferation but without inducing cell death, i.e. apoptosis or necrosis. Thus, the first phase can be called “cytostatic”. In the second phase, starting at single-digit micromolar concentrations and depending on the actual embodiment of the compounds of invention, these compounds induce cell death by upregulating Caspase 3/7, a key marker of apoptosis induction (Fig.3). A further analysis revealed that compounds of the present invention induced markers of endoplasmic reticulum stress such as CHAC-1 , Chop, XBP-1 or SLC7A11 in the same concentration range where they displayed cytostatic effects (Fig. 4, H. Ren et al, Front. Aging Neurosci. (2021), 13: 691881). The induction of these ER stress markers as determined by quantitative real-time PCR preceded the concentrationdependent reduction in Hif1 -alpha protein levels as determined from the same cancer cells (Fig. 5). Since Hif1 -alpha plays a major role as master regulator of the transcriptional control of Warburg effect-related genes under hypoxic conditions in the tumor, this effect is likely one mechanism for the strong anti-proliferative effects observed for the compounds of the present invention.

The compounds of the invention further exhibit reduced electron density at the core aromatic ring and its amine residues. Without being bound by theory, the inventors thus consider that the compounds of the invention are also likely to exhibit reduced genotoxic potential relative to other compounds of the prior art.

Thus, we herewith provide not only novel heteroaryl-core containing bisamide compounds with unprecedented activity against various cancer cell lines and with no predicted genotoxic potential, but we have also identified novel biological mechanisms by which these compounds exert their anti-proliferative effects in such cancer cells.

It will be generally understood by one of ordinary skill in the art that if more than one of V, W, X and Y is simultaneously a bond, then any of V, W, X and Y which are a bond together form a single bond.

In an embodiment in combination with any of the above or below embodiments, II¹ and II² are N and II³ is CR².

In an embodiment in combination with any of the above or below embodiments, II¹ and II³ are N and II² is CR².

In an embodiment in combination with any of the above or below embodiments, II² and II³ are N and U¹ is CR².

In an embodiment in combination with any of the above or below embodiments, II¹ and II² are CR² and II³ is N.

In an embodiment in combination with any of the above or below embodiments, II¹ and II³ are CR² and II² is N.

In an embodiment in combination with any of the above or below embodiments, II² and II³ are CR² and U¹ is N.

In an embodiment in combination with any of the above or below embodiments, the compound is represented by formula (II),

wherein

R² is Ci-3-alkyl, OH, O-Ci-6-alkyl or halogen;

R^B is F or Ci-3-alkyl; m is independently 0, 1 or 2;

T¹ is C or N;

T² is CH, S, N, NH, HC=N, N=CH or HC=CH;

T³ is CH, O, S, N or NH;

T⁴ is CH, S, N or NH;

T⁵ is C or N; and

T⁶ is CH or N, with the proviso that at least one of T¹, T², T³, T⁴, T⁵ and T⁶ must be a heteroatom, and that no more than 4 of T¹, T², T³, T⁴, T⁵ and T⁶ are allowed to be N.

In an embodiment in combination with any of the above or below embodiments, the compound is represented by formula (Ila),

wherein

R² is Ci-3-alkyl, OH, O-Ci-6-alkyl or halogen;

R^B is F or Ci-3-alkyl; m is independently 0, 1 or 2;

T¹ is C or N;

T² is CH, S, N, NH, HC=N, N=CH or HC=CH;

T³ is CH, O, S, N or NH; T⁴ is CH, S, N or NH;

T⁵ is C or N; and

In an embodiment in combination with any of the above or below embodiments, the compound is represented by formula (III)

wherein

R² is Ci-3-alkyl or halogen;

R^B is F or Ci-3-alkyl; and m is independently 0, 1 or 2.

In an embodiment in combination with any of the above or below embodiments, A is phenyl or a 5-6 membered heteroaryl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, wherein the phenyl or the 5-6 membered heteroaryl are unsubstituted or substituted with

1 to 4 same or different substituents R^A, or wherein two substituents R^A on adjacent carbon atoms of the monocyclic aryl or monocyclic heteroaryl ring systems, together with the carbon atoms to which they are attached, form a 5, 6 or 7 membered carbocylic or heterocyclic ring, the heterocyclic ring containing 1 or 2 heteroatoms independently selected from the group consisting of O, N and S, wherein the 5, 6 or 7 membered carbocyclic or heterocyclic ring is unsubstituted or substituted with 1 to 5 same or different substituents R⁴; wherein R^A is Ci-e-alkyl, halogen, CN, OH, O-Ci-6-alkyl or S-Ci-e-alkyl, wherein Ci-6-alkyl is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of Ci-3-alkyl, Ci-3-haloalkyl, halogen and oxo; and wherein R⁴ is Ci-e-alkyl, halogen, CN, OH, Ci-e-alkyl, O-Ci-e-alkyl, S-Ci-e-alkyl, oxo or spirocyclic fused Cs-e-cycloalkyl.

In an embodiment in combination with any of the above or below embodiments, A is

wherein

K is O, S or C(R⁴)₂;

L is O, S or C(R⁴)₂;

R⁴ is F, Ci-3-alkyl or spirocyclicly fused Cs-e-cycloalkyl; n is 0, 1 , 2, 3 or 4; and p is 0, 1 or 2.

In an embodiment in combination with any of the above or below embodiments, the compound is represented by formula (IV)

wherein

K is O, S or C(R⁴)₂;

L is O, S or C(R⁴)₂;

In an embodiment in combination with any of the above or below embodiments, the compound is represented by formula (V)

wherein

R² is Ci-3-alkyl, OH, O-Ci-6-alkyl or halogen;

R^B is F or Ci-3-alkyl; ml is 0 or 1 ;

T⁷ is O, S or NH;

T⁸ is CH or N;

T⁹ is CH, or N;and

T is CH, or N.

In an embodiment in combination with any of the above or below embodiments, the compound is represented by formula (VI)

wherein

K is O, S or C(R⁴)₂;

L is O, S or C(R⁴)₂;

wherein

K is O;

L is O;

In an embodiment in combination with any of the above or below embodiments, V is a bond or Ci-3-alkylene.

In an embodiment in combination with any of the above or below embodiments, B is a bicyclic 8-10 membered heteroaryl containing 1 , 2, 3 or 4 heteroatoms independently selected from the group consisting of O, S and N, wherein the bicyclic heteroaryl is unsubstituted or substituted with 1 to 4 same or different substituents R^B; wherein R^B is Ci-e-alkyl, halogen, CN, OH, or Ci-6-haloalkyl.

In a further embodiment in combination with any of the above or below embodiments, B is selected from the group consisting of

wherein T¹-T¹⁰, R³, R^B, m and ml are defined as above.

In a preferred embodiment in combination with any of the above or below embodiments, B is

I ment in combination with any of the above or below embodiments, B is

In an embodiment in combination with any of the above or below embodiments, V is a bond.

In an embodiment in combination with any of the above or below embodiments, W is a bond, O, S or NR^a.

In an embodiment in combination with any of the above or below embodiments, W is a bond or O.

In an embodiment in combination with any of the above or below embodiments, X is bond or Ci-3-alkylene.

In an embodiment in combination with any of the above or below embodiments, X is C1.3- alkylene.

In an embodiment in combination with any of the above or below embodiments, Y is a bond, O, S or NR^a.

In an embodiment in combination with any of the above or below embodiments, Y is a bond.

In an embodiment in combination with any of the above or below embodiments, Z is hydrogen, halogen, OH, Ci-e-alkyl, Cs-e-cycloalkyl, 5-6 membered heterocycloalkyl containing 1 , 2 or 3 heteratoms independently selected from O, S and N or 5-6 membered heteroaryl containing 1 or 2 heteroatoms independently selected from O, S and N, wherein alkyl, cycloalkyl, heterocycloalkyl or heteroaryl are unsubstituted or substituted with 1-4 R^z independently selected from the group consisting of halogen, CN, Ci-3-alkyl, fluoro Ci-3-alkyl, OH, O-Ci-3-alkyl and spirocyclicly fused Cs-e-cycloalkyl.

In an embodiment in combination with any of the above or below embodiments, V-W-X-Y-Z is

Ci-3-alkyl, fluoro Ci-3-alkyl or H;

R^Z1 is independently selected from the group consisting of halogen, Ci-6-alkyl and spirocyclicly fused Cs-e-cycloalkyl;

R^Z2 is independently selected from the group consisting of hydrogen, Ci -3-alkyl and fluoro Ci- 3-alkyl;

R^Z3 is independently selected from the group consisting of halogen, CN, Ci-3-alkyl, fluoro C1.3- alkyl, OH and O-Ci-3-alkyl;

Q is CH₂, CHR^Z1, O, NH, N-Ci-e-alkyl, NC(O)-Ci-e-alkyl or S; t is 0, 1 or 2; u is 0, 1 or 2; and v is 0, 1 or 2.

Ci-3-alkyl or H;

R^Z1 is independently selected from the group consisting of halogen, Ci-3-alkyl and spirocyclicly fused cyclopropyl;

R^Z2 is independently selected from the group consisting of hydrogen, Ci .3-alkyl and fluoro Cis-alkyl;

R^Z3 is independently selected from the group consisting of halogen, Ci-3-alkyl, fluoro Ci-3-alkyl, OH and O-Ci-3-alkyl;

Q is CH₂; t is 1 or 2; u is 1 or 2; and v is 0 or 1.

In a preferred embodiment in combination with any of the above or below embodiments, V-W- X-Y-Z is selected from the group consisting of

-CH₃, CF₃ and H.

In a further preferred embodiment in combination with any of the above or below embodiments, V-W-X-Y-Z is selected from the group consisting of

In an embodiment in combination with any of the above or below embodiments, the compound is selected from the group consisting of

or an N-oxide, solvate, prodrug or pharmaceutically acceptable salt thereof.

In a more preferred embodiment in combination with any of the above or below embodiments, the compound is selected from the group consisting of

or an N-oxide, solvate, prodrug or pharmaceutically acceptable salt thereof.

A further aspect of the invention relates to a pharmaceutical composition comprising the compound according to any one of the aforementioned embodiments and a pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition may comprise about 5 wt% to about 98 wt% of one or more compounds of the invention, preferably from 0.5 mg to 0.5 g of active compound, even more preferably from 1 mg to 100 mg. In certain embodiments, the pharmaceutical composition is formulated to allow administration to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (e.g. intravenously, intramuscularly, subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.

A further aspect of the invention relates to the compound according to any one of the aforementioned embodiments for use as a medicament.

A further aspect of the invention relates to the compound according to any one of the aforementioned embodiments or the pharmaceutical composition comprising the compound according to any one of the aforementioned embodiments for use in a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism. In certain embodiments, the method comprises administering the compound or the pharmaceutical composition to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (for example intravenously, intramuscularly and/or subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.

A further aspect of the invention relates to a method of treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer, in which an effective amount of a compound of the invention or of a pharmaceutical composition of the invention is administered to a subject in need thereof. In certain embodiments, the method comprises administering the compound or the pharmaceutical composition to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (for example intravenously, intramuscularly and/or subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.

The present invention further relates to the use of a compound of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating a disease or condition mediated by the lactate/ATP mechanism, in particular cancer. In certain embodiments, the medicament is formulated to allow administration of the compound or of the pharmaceutical composition to a subject enterally (for example orally, sublingually, and/or buccally), parenterally (for example intravenously, intramuscularly and/or subcutaneously), intranasally, topically, rectally, vaginally and/or by inhalation.

In an embodiment in combination with any of the above or below embodiments, the disease or condition mediated by the lactate/ATP mechanism is cancer.

In an embodiment in combination with any of the above or below embodiments, the cancer is selected from the group consisting of include cancers of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart, or adrenals. More particularly, cancers include solid tumor, sarcoma, carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, a blood-borne tumor, acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, or multiple myeloma (see, e.g., Encyclopedia of Cancer, 3rd Edition (2018) Editors Paolo Boffetta, Pierre Hainaut). In an embodiment in combination with any of the above and below embodiments, the cancer is a solid tumor. In accordance with an embodiment, the cancer is selected from leukemia, melanoma, liver cancer, pancreatic cancer, lung cancer, colon cancer, brain cancer, ovarian cancer, breast cancer, prostate cancer, and renal cancer. In another embodiment, the cancer is liver cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, or renal cancer.

The type of cancer is not particularly limited, but in certain aspects, the cancer is characterized as hypoxic and/or highly glycolytic relative to normal tissue of the same type. "Hypoxic" cells or tissue areas as used herein relates to one or more cells that are exposed, transiently or permanently, to an oxygen partial pressure (pCh) that is lower than the typical pC>2 in cells in tissue that is considered as normal or healthy. Hypoxic cells or tissue areas can include, for example, cells or tissue areas with reduced or no access to vasculature, such as in a solid tumor.

The compounds of the present invention exert their anticancer and antiproliferative effects through a novel mechanism which induces cytostatic effects at lower and cytotoxic or cell killing effects at concentrations typically 100 -1000-fold higher than the cytostatic concentrations. The compounds of the present invention induce endoplasmatic reticulum stress in the first cytostatic phase which coincides with the onset of ATP and lactate lowering in cancer cell culture. This unique mechanism shown by the compounds of the invention allows for a certain therapeutic window, i.e. an effective dose of such a compound that can be used to stop cancerous growth without exerting cytotoxic side effects in the same patient.

In an embodiment in combination with any of the above or below embodiments, the compound or the pharmaceutical composition is administered

• together with one or more therapeutic agents for cancer selected from the group consisting of a PD-1 agent, a PD-L1 agent, a CTLA-4 agent, an IDO1 inhibitor, and an anticancer vaccine, or

• together with a cytokine therapy or known chemo- or pharmacotherapy, or during irradiation therapy.

Preferred agents, inhibitors, vaccines and therapies according to this embodiment are as set out hereinbelow.

All technical and scientific terms used hereafter carry the commonly understood meaning of the word unless defined otherwise.

It is to be understood that section headings as used throughout the present application are merely for organizational purposes, and do not confer exclusive character to the disclosure pertaining to aspects of the invention mentioned in a section headings. Section headings thus do not exclude the combination of the disclosure within any one section heading to a corresponding or analogous context provided under another section heading. Such intrasection disclosures are explicitly within the disclosure of the present application.

As used herein, each of the terms “comprising’, “having” and “containing”, including grammatical variants thereof, are meant in a non-exhaustive sense to mean “including”, but not necessarily “composed of”, and does not exclude elements in addition to those explicitly recited as being present. As used herein, then, the terms “comprising”, “having” and “containing”, and grammatical variants thereof, indicate that components other than those explicitly recited may, but need not, be present. As such these terms include as a limiting case embodiments in which no other elements than those recited are present, e.g. in the commonly accepted sense of “consisting of”.

As used herein, the phrase “consisting of”, and grammatically related variants thereof, means that no other elements are present in those recited. In standing with the above definition of “comprising” (and grammatically and semantically related terms), the term “consisting of” therefore denotes a limiting scenario within the meaning of “comprising”.

Unless defined otherwise, any feature within any aspect or embodiment of the invention may be combined with any feature within any other aspect or embodiment of the invention, and the skilled person understands such combination as being encompassed in the original disclosure of the present application. This applies in particular to all embodiments described within the section relating to compounds per se, in respect of other aspects, e.g. methods and uses, of those compositions. This also applies in particular, but not exclusively, to endpoints of ranges disclosed herein. For instance, if a given substance is disclosed as existing in a composition in a concentration range of about X-Y% or about A-B%, the present application is to be understood as explicitly disclosing not only the ranges about X-Y% and about A-B%, but also the ranges about X-B%, about A-Y% and, in as far as numerically possible, about Y-A% and about B-X%. Each of these ranges, and range combinations, are contemplated, and are to be understood as being directly and unambiguously disclosed in the present application.

Unless stated otherwise, the designation of a range in the present application using a hyphen separating two bracketing values X and Y, or two bracketing ratios, is to be understood as meaning and disclosing the specified range in which both endpoint values X and Y are included. The same applies to a range expressed as “from about X to about Y”. Accordingly, the expressions of ranges as “X -Y”, “of X to Y”, “from X to Y”, “of X - Y” and “from X - Y” are to be understood equivalently as meaning and disclosing a range encompassing the end value X, all values between X and Y, as well as the end value Y. In the event that the range in question specifies the number of atoms in an entity where only integral values would be technically meaningful, for instance a chemical substituent such as the term “Ci-Ce alkyl” or, equivalently, “C1-6 alkyl”, the specified range is understood as meaning and disclosing each integral value within that range. For instance, in the example of “Ci-Ce alkyl” (or equivalently “C1-6 alkyl”), this term is to be understood as meaning and clearly and unambiguously disclosing each of the separate integral options “Ci alkyl”, “C2 alkyl”, “C3 alkyl”, “C4 alkyl”, “C5 alkyl” and “C₆ alkyl”.

The designation of a range in the present application using the word “between” preceding two bracketing values X and Y, or two bracketing ratios, is to be understood as meaning and disclosing the specified range in which both endpoint values X and Y are excluded, but all values between the specified endpoint values X and Y are included. As used herein the term “about” when referring to a particular value, e.g. an endpoint or endpoints of a range, encompasses and discloses, in addition to the specifically recited value itself, a certain variation around the specifically recited value. Such a variation may for example arise from normal measurement variability, e.g. in the weighing or apportioning of various substances by methods known to the skilled person. The term “about” shall be understood as encompassing and disclosing a range of variability above and below an indicated specific value, said percentage values being relative to the specific recited value itself, as follows. The term “about” may denote variability of ± 5.0%. The term “about” may denote variability of ± 4.9%. The term “about” may denote variability of ± 4.8%. The term “about” may denote variability of ± 4.7%. The term “about” may denote variability of ± 4.6%. The term “about” may denote variability of ± 4.5%. The term “about” may denote variability of ± 4.4%. The term “about” may denote variability of ± 4.3%. The term “about” may denote variability of ± 4.2%. The term “about” may denote variability of ± 4.1 %. The term “about” may denote variability of ± 4.0%. The term “about” may denote variability of ± 3.9%. The term “about” may denote variability of ± 3.8%. The term “about” may denote variability of ± 3.7%. The term “about” may denote variability of ± 3.6%. The term “about” may denote variability of ± 3.5%. The term “about” may denote variability of ± 3.4%. The term “about” may denote variability of ± 3.3%. The term “about” may denote variability of ± 3.2%. The term “about” may denote variability of ± 3.1 %. The term “about” may denote variability of ± 3.0%. The term “about” may denote variability of ± 2.9%. The term “about” may denote variability of ± 2.8%. The term “about” may denote variability of ± 2.7%. The term “about” may denote variability of ± 2.6%. The term “about” may denote variability of ± 2.5%. The term “about” may denote variability of ± 2.4%. The term “about” may denote variability of ± 2.3%. The term “about” may denote variability of ± 2.2%. The term “about” may denote variability of ± 2.1%. The term “about” may denote variability of ± 2.0%. The term “about” may denote variability of ± 1.9%. The term “about” may denote variability of ± 1.8%. The term “about” may denote variability of ± 1.7%. The term “about” may denote variability of ± 1.6%. The term “about” may denote variability of ± 1.5%. The term “about” may denote variability of ± 1.4%. The term “about” may denote variability of ± 1.3%. The term “about” may denote variability of ± 1.2%. The term “about” may denote variability of ± 1.1 %. The term “about” may denote variability of ± 1.0%. The term “about” may denote variability of ± 0.9%. The term “about” may denote variability of ± 0.8%. The term “about” may denote variability of ± 0.7%. The term “about” may denote variability of ± 0.6%. The term “about” may denote variability of ± 0.5%. The term “about” may denote variability of ± 0.4%. The term “about” may denote variability of ± 0.3%. The term “about” may denote variability of ± 0.2%. The term “about” may denote variability of 0.1%. The term “about”, in reference to the particular recited value, may denote that exact particular value itself, irrespective of any explicit mention that this exact particular value is included; even in the absence of an explicit indication that the term “about” includes the particular exact recited value, this exact particular value is still included in the range of variation created by the term “about”, and is therefore disclosed. Unless stated otherwise, if the term “about” is recited before the first endpoint of a numerical range, this term refers to both the first endpoint of the range and the second endpoint of the range. For instance, a recited range of “about X to Y” should be read as “about X to about Y”.

As used herein, “Ci-6-alkyl” means a saturated alkyl chain having 1 , 2, 3, 4, 5 or 6 carbon atoms which may be straight chained or branched. In the context of the present invention, any subgroup falling within the term “Ci-6-alkyl” is also encompassed, such as a Ci-3-alkyl, C2-5- alkyl or Cs-6-alkyl group. Examples of the Ci-6-alkyl group include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and hexyl. A “Ci-6-alkylene” means that the respective group is divalent and connects an attached residue with the remaining part of the molecule. Moreover, in the context of the present invention, Ci- alkylene means a methylene linker, C2-alkylene means an ethylene linker or a methylsubstituted methylene linker and so on. In the context of the present invention, a Ci-6-alkylene preferably represents a methylene or ethylene group.

The term "O-Ci-6-alkyl" means that the alkyl chain is connected via an oxygen atom with the remainder of the molecule. Similarly, the term “S-Ci-6-alkyl” defines an alkyl chain which is connected via the sulphur atom with the remainder of the molecule and the term “NR^a-Ci-6- alkyl” defines an alkyl chain which is connected via the amino group with the remainder of the molecule.

The term "halo-Ci-6-alkyl" means that one or more hydrogen atoms in the alkyl chain are replaced by a halogen. A preferred example thereof is CF3.

A Cs-s-cycloalkyl group means a saturated or partially unsaturated mono- or bicyclic ring system comprising 3, 4, 5, 6, 7 or 8 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl and cyclooctyl. In a similar manner, a Cs-e-cycloalkyl group means a saturated or partially unsaturated monocyclic ring comprising 3, 4, 5 or 6 carbon atoms.

A 4-8 membered mono or bicyclic heterocycloalkyl group means a saturated or partially unsaturated 4-8 membered mono or bicyclic carbon ring wherein up to 4 carbon atoms may be replaced by up to 4 heteroatoms, and wherein the heteroatoms are independently selected from N, O, S, SO and SO2. Examples thereof include epoxidyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, 1 ,4-dioxanyl, morpholinyl, 4- quinuclidinyl, 1 ,4-dihydropyridinyl and 6-azabicyclo[3.2.1]octanyl. The heterocycloalkyl group can be connected with the remaining part of the molecule via a carbon, nitrogen (e.g. in morpholine or piperidine) or sulfur atom.

A cycloalkyl or heterocycloalkyl substituent may be spirocyclicly fused to the remainder of the molecule. In the context of the present invention, “spirocyclicly fused” means that the cycloalkyl or hetercycloalkyl group is connected through a single common atom with the remainder of the molecule, i.e. the ring shares a common atom with another moiety. An example of a spiro connection is shown below:

A 5-14-membered mono or bicyclic heteroaromatic ring system (also referred to herein as heteroaryl) containing up to 4 heteroatoms means a monocyclic heteroaromatic ring such as pyrrolyl, imidazolyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, oxadiazolyl and thiadiazolyl. It further means a bicyclic ring system wherein the heteroatom(s) may be present in one or both rings including the bridgehead atoms. Examples thereof include, benzimidazolyl, benzisoxazolyl, benzodioxanyl, benzofuranyl, benzoxazolyl, imidazo[1 ,2-a]pyridinyl, imidazo[1 ,2-c]pyrimidinyl, indazolyl, indolizinyl, indolyl, isoquinolinyl, pyrazolo[1 ,5-a]pyridinyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,2-c]pyridinyl, pyrrolo[2,3-d]thiazolyl, quinolinyl, quinoxalinyl, pyrazolo[1 ,5-a]pyrimidinyl, thiazolo[4,5- b]pyridinyl and [1 ,2,4]triazolo[1 ,5-a]pyrimidinyl. In the context of the present invention, any subgroup falling within the term “5-14 membered mono or bicyclic heteroaryl” is also encompassed such as 8-14 membered heteroaryl or 5-6-membered heteroaryl. A nitrogen atom of a heteroaryl system may also be optionally oxidized to the corresponding /V-oxide. If not stated otherwise, the heteroaryl system can be connected via a carbon or nitrogen atom.

Moreover, where not explicitly defined, the term “heteroaryl” contains 1 to 4 heteroatoms independently selected from the group consisting of N, O and S.

A 6-10-membered mono- or bicyclic aromatic ring system (within the application also referred to as aryl) means an aromatic carbon cycle such as phenyl, naphthyl or azulenyl.

The term “halogen” comprises the specific halogen atoms fluorine, bromine, chlorine and iodine.

It will be appreciated by the skilled person that when lists of alternative substituents include members which, because of their valency requirements or other reasons, cannot be used to substitute a particular group, the list is intended to be read with the knowledge of the skilled person to include only those members of the list which are suitable for substituting the particular group without contravening valency rules.

Any formula or structure given herein, is intended to represent unlabelled forms as well as isotopically labelled forms of the compounds. Isotopically labelled compounds have structures depicted by the formulas given herein, except that one or more atoms is/are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as, but not limited to ²H (deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ³⁶CI and ¹²⁵l. The present invention therefore also encompasses such isotopically labelled compounds, for example those containing radioactive isotopes such as ³H, ¹³C and ¹⁴C. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or in radioactive treatment of patients. Isotopically labelled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labelled reagent for a non- isotopically labelled reagent.

The present invention also includes “deuterated analogs” of compounds of Formula (I) in which from 1 to n hydrogens attached to a carbon atom is/are replaced by deuterium, in which n is the number of hydrogens in the molecule. Such compounds may exhibit increased resistance to metabolism and thus be useful for increasing the half-life of any compound of Formula (I) when administered to a mammal, e.g. a human. See, for example, Foster in T rends Pharmacol. Sci. 1984:5;524. Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced by deuterium, or are enriched in deuterium content at the position(s) in question.

Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and/or an improvement in therapeutic index. An ¹⁸F labelled compound may be useful for PET or SPECT studies.

The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of the invention, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated specifically as “H” or “hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition.

The compounds of the present invention can be in the form of a prodrug. "Prodrug" means a derivative that is converted into a compound according to the present invention by a reaction with an enzyme, gastric acid or the like under a physiological condition in the living body, e.g. by oxidation, reduction, hydrolysis or the like, each of which is carried out enzymatically. Examples of the prodrug are compounds in which the amino group in a compound of the present invention is acylated, alkylated or phosphorylated to form, e.g., eicosanoylamino, alanylamino, pivaloyloxymethylamino or wherein the hydroxyl group is acylated, alkylated, phosphorylated or converted into the borate, e.g. acetyloxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaryloxy, alanyloxy or wherein the carboxyl group is esterified or amidated. These compounds can be produced from compounds of the present invention according to well-known methods. Other examples of the prodrug are compounds in which the carboxylate in a compound of the present invention is, for example, converted into an alkyl-, aryl-, choline- , amino, acyloxymethylester, linolenoylester.

Metabolites of compounds of the present invention are also within the scope of the present invention. Where tautomerism, like e.g. keto-enol tautomerism, of compounds of the present invention or their prodrugs may occur, the individual forms, like e.g. the keto and enol form, are each within the scope of the invention as well as their mixtures in any ratio. The same applies for stereoisomers, like e.g. enantiomers, cis/trans isomers, conformers and the like.

If desired, isomers can be separated by methods well known in the art, e.g. by liquid chromatography. The same applies for enantiomers by using e.g. chiral stationary phases. Additionally, enantiomers may be isolated by converting them into diastereomers, i.e. coupling with an enantiomerically pure auxiliary compound, subsequent separation of the resulting diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of a compound of the present invention may be obtained from stereoselective synthesis using optically pure starting materials. Another way to obtain pure enantiomers from racemic mixtures would use enantioselective crystallization with chiral counterions, by methods known in the art.

Administration of one or more compounds of the invention “together with” another substance or substances or type of therapy, as used herein, need not denote simultaneity of administration. Rather, such administration of one compound “together with” another or others or a type of therapy encompasses and discloses multiple possibilities regarding the route and timing of administration of the respective substances and/or therapies. Encompassed and disclosed in this sense is for example the administration of two substances simultaneously by the same route, simultaneous administration by different routes, chronologically staggered administration by the same route, or chronologically staggered administration by different routes. Any of the administration routes described herein may be combined in any way. Further, the order of administration is not important. For instance, applying the generic example of two substances A and B, the chronologically staggered administration of substance A and substance B “together with” one another might encompass prior administration of substance A and subsequent administration of substance B, or prior administration of substance B and subsequent administration of substance A. For example, staggered administration of substance A and substance B by the same route may take the form of initial oral administration of substance A, followed by oral administration of substance B; administration of substances A and B in this way falls within the meaning of, and is disclosed by, administration of the two substances “together with” one another. Similarly, staggered administration of substance A and substance B by different routes might take the form of initial oral administration of substance A, followed by administration of substance B intravenously; administration of substances A and B in this way also falls within the meaning of, and is disclosed by, administration of the two substances “together with” one another.

There are no particular restrictions on the duration of time between administration of two substances “together with” one another as meant herein, as long as the two substances in question are administered as part of the same overall prophylactic and/or therapeutic regimen relating to the disease or condition in question.

Similarly, administration of one substance “during” a specified type of therapy, as used herein, is not to be understood as necessitating chronological simultaneity. Rather, administration of a substance or substances “during” a therapy encompasses and includes any timing and route of administration as part of the broader prophylactic and/or therapeutic regimen. For example, administration of one or more compounds according to the present invention “during” irradiation therapy of a subject does not require that such compound(s) be administered to the subject at the same moment as radiation is being applied to that subject, although it includes that possibility. For instance, administration “during” irradiation therapy might include administration of one or more compounds of the present invention before or after such application of radiation, or before and after such application of radiation, or before, simultaneously with and after such application of radiation.

The compounds of the present invention can be in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids. In case the compounds of the present invention contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts. Thus, the compounds of the present invention which contain acidic groups can be present on these groups and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. The compounds of the present invention which contain one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art. If the compounds of the present invention simultaneously contain acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). The respective salts can be obtained by customary methods which are known to the person skilled in the art such as, for example, by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the compounds of the present invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

Further the compounds of the present invention may be present in the form of solvates, such as those which include as solvate water, or pharmaceutically acceptable solvates, such as alcohols, in particular ethanol.

Pharmaceutical compositions and corresponding formulations

Furthermore, the present invention provides pharmaceutical compositions comprising at least one compound of the present invention, or a prodrug compound thereof, or a pharmaceutically acceptable salt or solvate thereof as active ingredient together with a pharmaceutically acceptable carrier.

"Pharmaceutical composition" means one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing at least one compound of the present invention and a pharmaceutically acceptable carrier.

In practical use, the compounds of the present invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such compositions and preparations should contain at least about 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 5 percent to about 98 percent of the weight of the unit, more preferably about 10 to about 90 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained. The active compounds can also be administered intranasally as, for example, liquid drops or spray. The tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch or gelatine; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavouring such as cherry or orange flavour.

The compounds used in the present invention may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

Further specific details regarding the above and further modes of formulation are set out below.

Formulation of the inventive compound for suitability for oral administration: A compound of the invention suitable for oral administration may be prepared, packaged, or solid in the form of a discrete solid dose unit including a sachet (e.g. a powder in a sachet), a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the compound(s) of the invention. Other formulations suitable for oral administration include a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. As used herein, an “oily” liquid comprises a carbon-containing liquid molecule that exhibits a less polar character than water.

A tablet comprising a compound of the invention may, for example, be made by compressing or molding an inventive compound, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, an inventive compound in a free flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may for example be made by molding, in a suitable device, an inventive compound, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.

Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface active agents include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the inventive compound. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically attractive and palatable preparation.

Hard capsules comprising an inventive compound may be made using a physiologically degradable composition, such as gelatin or cellulose derivatives. Such hard capsules comprise the inventive compound, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the inventive compound may be made using a physiologically degradable composition, such as gelatin combined with a plasticizer (i.e. glycerol) as basic component of the soft gelatin shell. Soft gelatin capsules may contain a liquid or semisolid solution, suspension, or microemulsion preconcentrate. The soft capsule filling comprises the inventive compound, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, olive oil, soybean oil, sunflower oil, a lecithin such as for example soy lecithin or sunflower lecithin, medium chain triglycerides, polyglycerol oleate, beeswax, mono- and diglycerides of fatty acids, or combinations of any of the above.

Liquid formulations of the inventive compound that are especially suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to ingestion.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

Powdered and granular formulations of an inventive compound, e.g. a pharmaceutical composition of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form sachet or tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. The inventive compound may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (e.g. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Formulation of the inventive compound for suitability for parenteral administration: For parenteral administration, the inventive compound may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other agents such as suspending, stabilizing and/or dispersing agents such as those mentioned above, may be used.

The inventive compound may be rendered especially suitable for parenteral administration by formulation with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules, crushable or otherwise, or in multidose containers containing a preservative. Compositions especially suitable for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of the inventive composition which is especially suitable for parenteral administration, the active ingredient is provided in dry (e.g. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.

The inventive compound may be prepared in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable compositions may be prepared using a non toxic, parenterally acceptable diluent or solvent, such as water or 1 ,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other usual parentally-administrable formulations include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulation of the inventive compound for suitability for transmucosal administration: The inventive compound may be formulated to be suitable for transmucosal administration. The formulation may include any substances or dosage unit suitable for application to mucosal tissue. For example, the selected active agent may be administered to the buccal mucosa in an adhesive tablet or patch, sublingually administered by placing a solid dosage form under the tongue, lingually administered by placing a solid dosage form on the tongue, administered nasally as droplets or a nasal spray, a non-aerosol liquid formulation, or a dry powder, placed within or near the rectum (“transrectal” formulations), or administered to the urethra as a suppository, ointment, or the like.

Formulation of the inventive compound for suitability for transurethal administration: The inventive compound may also be formulated to be suitable for transurethal administration. In this case, the inventive composition may comprise a urethral dosage form containing the active agent and one or more selected carriers or excipients, such as water, silicone, waxes, petroleum jelly, polyethylene glycol (“PEG”), propylene glycol (“PG”), liposomes, sugars such as mannitol and lactose, and/or a variety of other materials. A transurethral permeation enhancer may be included in the dosage from. Examples of suitable permeation enhancers include dimethylsulfoxide (“DMSO”), dimethyl formamide (“DMF”), N,N-dimethylacetamide (“DMA”), decylmethylsulfoxide (“C10 MSO”), polyethylene glycol monolaurate (“PEGML”), glycerol monolaurate, lecithin, the 1 -substituted azacycloheptan-2-ones, particularly 1-n- dodecyl-cyclazacycloheptan-2-one, surfactants as discussed above, including, for example TWEEN-80™, and lower alkanols such as ethanol.

Formulation of the inventive compound for suitability for transrectal administration: The inventive compound may also be formulated to be suitable for transrectal administration. Transrectal dosage forms may include rectal suppositories, creams, ointments, and liquid formulations (enemas). The suppository, cream, ointment or liquid formulation for transrectal delivery comprises an inventive compound and one or more conventional nontoxic carriers suitable for transrectal drug administration. The transrectal dosage forms of the inventive composition may be manufactured using conventional processes. The transrectal dosage unit may be fabricated to disintegrate rapidly or over a period of several hours. The time period for complete disintegration may be in the range of from about 10 minutes to about 6 hours, e.g., less than about 3 hours.

Formulation of the inventive compound for suitability for vaginal or perivaginal administration: The inventive compound may also be formulated to be suitable for vaginal or perivaginal administration. Suitable dosage forms to this end may include vaginal suppositories, creams, ointments, liquid formulations, pessaries, tampons, gels, pastes, foams or sprays. The suppository, cream, ointment, liquid formulation, pessary, tampon, gel, paste, foam or spray for vaginal or perivaginal delivery comprises a therapeutically effective amount of the selected active agent and one or more conventional nontoxic carriers suitable for vaginal or perivaginal drug administration. The vaginal or perivaginal forms of the present invention may be manufactured using conventional processes as for example disclosed in Remington: The Science and Practice of Pharmacy, supra. The vaginal or perivaginal dosage unit may be fabricated to disintegrate rapidly or over a period of several hours. The time period for complete disintegration may be in the range of from about 10 minutes to about 6 hours, e.g., less than about 3 hours.

Formulation of the inventive compound for suitability for topical formulations: The inventive compound may also be formulated to be suitable for topical administration. Suitable dosage forms to this end may include any form suitable for application to the body surface, and may comprise, for example, an ointment, cream, gel, lotion, solution, paste or the like, and/or may be prepared so as to contain liposomes, micelles, and/or microspheres. In certain embodiments, topical formulations herein are ointments, creams and gels.

Formulation of the inventive compound for suitability for transdermal administration: The inventive compound may also be formulated to be especially suitable for transdermal administration. As known to one skilled in the art, transdermal administration involves the delivery of pharmaceutical compounds via percutaneous passage of the compound into the systemic circulation of the patient. This can be effected e.g. by transdermal patches or iontophoresis devices. Other components besides the inventive compound may be incorporated into the transdermal patches as well. For example, compositions and/or transdermal patches may be formulated with one or more preservatives or bacteriostatic agents including, but not limited to, methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride, and the like. Dosage forms of the inventive compound may include creams, sprays, lotions, gels, ointments, eye drops, nose drops, ear drops, and the like. In such dosage forms, the ingredients of the inventive composition may be mixed to form a white, smooth, homogeneous, opaque cream or lotion with, for example, benzyl alcohol 1 % or 2% (wt/wt) as a preservative, emulsifying wax, glycerin, isopropyl palmitate, lactic acid, purified water and sorbitol solution. In addition, the compositions may contain polyethylene glycol 400. They may be mixed to form ointments with, for example, benzyl alcohol about 2% (wt/wt) as preservative, white petrolatum, emulsifying wax, and tenox II (butylated hydroxyanisole, propyl gallate, citric acid, propylene glycol). Woven pads or rolls of bandaging material, e.g., gauze, may be impregnated with the compositions in solution, lotion, cream, ointment or other such form may also be used for topical application. The compositions may also be applied topically using a transdermal system, such as one of an acrylic-based polymer adhesive with a resinous crosslinking agent impregnated with the composition and laminated to an impermeable backing.

Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are separate and distinct layers, with the adhesive underlying the reservoir that, in this case, may be either a polymeric matrix as described above, or be a liquid or hydrogel reservoir, or take some other form. Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dose of a compound of the present invention. For example, oral, rectal, topical, parenteral (including intravenous, intramuscular and subcutaneous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), nasal and the like may be employed. As set out above, dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. Preferably, compounds of the present invention are administered orally or intraveneously.

The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.

When treating or preventing lactate/ATP-mediated diseases for which compounds of Formula (I) are indicated, generally satisfactory results are obtained when the compounds are administered at a daily dosage of from about 0.1 mg to about 100 mg per kilogram of mammal body weight, preferably given as a single daily dose or in divided doses two to six times a day, or in sustained release form. For most large mammals, the total daily dosage is from about 1 mg to about 1000 mg, preferably from about 1 mg to about 50 mg. In the case of a 70 kg adult human, the total daily dose will generally be from about 7 mg to about 350 mg. This dosage regimen may be adjusted to provide the optimal therapeutic response. The compounds of the present invention are effective for treating cancer. Without being bound by theory, this is due to the compounds’ ability to modulate lactate production pathways.

The compound of the present invention can be administered together with one or more therapeutic agents for cancer selected from the group consisting of a PD-1 agent, a PD-L1 agent, a CTLA-4 agent, an IDO1 inhibitor and an anticancer vaccine. Alternatively, the compound may be administered together with a cytokine therapy, a known chemo- or pharmacotherapy, or during irradiation therapy.

Examples of PD-1 agents include, but are not limited to, pembrolizumab and nivolumab.

Examples of PD-L1 agents include, but are not limited to, atezolizumab, avelumab and durvalumab.

Examples of CTLA-4 agents include, but are not limited to, ipilimumab.

Examples of IDO1 inhibitors include, but are not limited to, epacadostat, navoximod and BMS- 986205.

Examples of anticancer vaccines include, but are not limited to, Hepa- VAC-101 and Sipuleucel-T.

Examples of cytokine therapy include, but are not limited to therapy involving the administration of IL-2, GM-CSF, IL-12 and/or IL-10.

Examples of drugs which may be used in chemo- or pharmacotherapy herein include: platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan, procarbazine, streptozocin, temozolomide, dacarbazine, bendamustine), antitumor antibiotics (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mytomycin C, plicamycin, dactinomycin), taxanes (e.g., paclitaxel, cabazitaxel and docetaxel), antimetabolites (e.g., 5-fluorouracil, cytarabine, premetrexed, thioguanine, floxuridine, capecitabine, gemcitabine, 6-mercatopurine and methotrexate), nucleoside analogues (e.g., fludarabine, clofarabine, cladribine, pentostatin, nelarabine), topoisomerase inhibitors (e.g., topotecan and irinotecan), hypomethylating agents (e.g., azacitidine and decitabine), proteosome inhibitors (e.g., bortezomib), epipodophyllotoxins (e.g., etoposide and teniposide), DNA synthesis inhibitors (e.g., hydroxyurea), vinca alkaloids (e.g., vicristine, vindesine, vinorelbine, and vinblastine), tyrosine kinase inhibitors (e.g., imatinib, dasatinib, nilotinib, sorafenib, sunitinib, bosutinib, ponatinib, erlotinib, gefitinib, afatinib osimertinib, lapatinib, crizotinib, ceritinib, axitinib, cabozantinib and lanvatinib), monoclonal antibodies (e.g., pertuzumab, rituximab, cetuximab, panetumumab, tositumomab, trastuzumab, alemtuzumab, gemtuzumab ozogamicin, bevacizumab, nivolumab, pembrolizumab or other immune checkpoint inhibitor monoclonal antibodies), nitrosoureas (e.g., carmustine, fotemustine, and lomustine), enzymes (e.g., L-asparaginase), biological agents (e.g., interferons and interleukins), hexamethylmelamine, mitotane, angiogenesis inhibitors (e.g., thalidomide, lenalidomide), steroids (e.g., prednisone, dexamethasone, and prednisolone), hormonal agents (e.g., tamoxifen, raloxifene, leuprolide, bicaluatmide, granisetron, flutamide), aromatase inhibitors (e.g., letrozole, exemestane and anastrozole), arsenic trioxide, tretinoin, nonselective cyclooxygenase inhibitors (e.g., nonsteroidal anti-inflammatory agents, salicylates, aspirin, piroxicam, ibuprofen, indomethacin, naprosyn, diclofenac, tolmetin, ketoprofen, nabumetone, oxaprozin), selective cyclooxygenase-2 (COX-2) inhibitors, or any combination thereof.

Examples

Abbreviations

Herein and throughout the application, the following abbreviations are used.

Ac acetyl

AIBN azobisisobutyronitrile d doublet

DCC /V,/V'-dicyclohexylcarbodiimide

DCM dichloromethane

DI PEA /V,/V-diisopropylethylamine

DMAP 4-(dimethylamino)pyridine

DMF /V,/V-dimethylformamide

DMSO dimethyl sulfoxide

Et ethyl

Et20 diethyl ether

EtOAc ethyl acetate

FBS fetal bovine serum

HATLI O-(7-azabenzotriazol-1-yl)-/V,/V,/V(/V'-tetramethyluronium hexafluorophosphate

HPLC high performance liquid chromatography m multiplet

Me methyl NBS /V-bromosuccinimide

NMI 1 -methylimidazole

PE petroleum ether prep preparative rt room temperature s singlet t triplet

TCFH chloro-/V,/V,/V',/V'-tetramethylformamidinium hexafluorophosphate

TEA triethylamine

TFAA trifluoroacetic anhydride

THF tetra hydrofuran

General Schemes

The compounds of the present invention can be prepared by a combination of methods known in the art including the procedures described in schemes 1 and 2 below. The following reaction schemes are meant only to exemplify and do not limit the invention.

Scheme 1 describes one route of preparation for the compounds of the present invention. A substituted or unsubstituted dinitro heteroaryl starting material (A-1) is reduced to the corresponding diamino intermediate A-2 with zinc and ammonium chloride. Amide coupling of A-2 with appropriate carboxylic acids using activation with thionyl chloride affords the corresponding mono amide A-3. A second amide coupling affords compounds of structure A- 4.

Scheme 2 describes an alternative route of preparation for the compounds of the present invention. A substituted or unsubstituted mononitro heteroaryl starting material (B-1) is converted to B-2 through amide coupling with appropriate carboxylic acids using activation with thionyl chloride. Reduction with zinc and ammonium chloride affords the corresponding mono amine intermediate B-3. A second amide coupling affords compounds of structure B-4.

Intermediate 1 : /V-(5-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)- 2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide (Int 1)

Int 1

Step 1 : 2-(2-(Pyrrolidin-1-yl)ethoxy)quinoline-6-carboxylic acid (Int 1a)

To a mixture of 2-(pyrrolidin-1-yl)ethanol (5.55 g, 48.2 mmol) in anhydrous DMF (25 mL) 60% NaH dispersion in mineral oil (1.93 g, 48.2 mmol) was added at 0 °C and the mixture was stirred at rtfor 1 h. 2-Chloroquinoline-6-carboxylic acid (2.00 g, 9.63 mmol) was added and the mixture was heated to 60 °C for 45 min. The solvent was removed under high vacuum. The residue was dissolved in water and the pH was adjusted to pH 12. The aqueous layer was extracted with EtOAc (3 x 50 mL). The pH of the aqueous layer was adjusted to pH 5.5. The aqueous layer was extracted with EtOAc (3 x 50 mL). The aqueous layer was freeze dried to afford the title compound as light brown solid.

Step 2: /V-(2-Methyl-5-nitropyridin-3-yl)-2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide (Int 1b)

A mixture of 2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxylic acid (Int 1a) (74.8 mg, 261 pmol) in thionyl chloride (1 mL) was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and then coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in anhydrous THF (1 mL) and the solution was added to a mixture of 2-methyl-5-nitropyridin-3- amine (20 mg, 131 pmol) and DIPEA (33.8 mg, 261 pmol) in 1 mL anhydrous THF at 0 °C. The mixture was stirred at rt overnight. The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 0-10% MeOH in DCM) to afford the title compound as a white solid.

Step 3: /V-(5-Amino-2-methylpyridin-3-yl)-2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6- carboxamide (Int 1)

A mixture of /V-(2-methyl-5-nitropyridin-3-yl)-2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6- carboxamide (Int 1 b) (20 mg, 47.5 pmol) and SnCh (45 mg, 237 pmol) in 2 EtOH (2 mL) was stirred under reflux for 2 h. The mixture was filtered through celite and rinsed with EtOAc (10 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness to afford the title compound that was used in the next step without further purification. Intermediate 2: /V-(5-Amino-2-methylpyridin-3-yl)-2-(2-cyclopentylethoxy)quinoline-6- carboxamide (Int 2)

Int 2

Step 1 : 2-(2-Cyclopentylethoxy)quinoline-6-carboxylic acid (Int 2a)

NaH (60% dispersion in mineral oil, 217 mg, 5.42 mmol) was added to a solution of 2- cyclopentylethanol (619 mg, 5.42 mmol) in 2 mL anhydrous DMF at 0 °C and the mixture was stirred at 0 °C for 1 h. 2-Chloroquinoline-6-carboxylic acid (225 mg, 1.084 mmol) was added and the mixture was stirred at 60 °C for 3 h. The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 0-10% MeOH in DCM) to afford the title compound as a white solid.

Step 2: 2-(2-Cyclopentylethoxy)-/V-(2-methyl-5-nitropyridin-3-yl)quinoline-6-carboxamide (Int 2b)

A mixture of 2-(2-cyclopentylethoxy)quinoline-6-carboxylic acid (Int 2a) (120 mg, 421 pmol) in thionyl chloride (1 mL) was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 1 mL anhydrous THF and the solution was added to a mixture of 2-methyl-5-nitropyridin-3-amine (64 mg, 421 pmol) and DIPEA (109 mg, 841 pmol) in 1 mL anhydrous THF at 0 °C. The mixture was stirred at rt overnight. The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 12-50% EtOAc in cyclohexane) to afford the title compound as a yellow solid.

Step 3: /V-(5-Amino-2-methylpyridin-3-yl)-2-(2-cyclopentylethoxy)quinoline-6-carboxamide (Int 2)

A mixture of 2-(2-cyclopentylethoxy)-/V-(2-methyl-5-nitropyridin-3-yl)quinoline-6-carboxamide (Int 2b) (29 mg, 0.069 mmol) and NH4CI (25.8 mg, 0.483 mmol) in 2 mL dioxane/H2O (3:1) was stirred for 15 min at rt. Zinc (31.6 mg, 0.483 mmol) was added at 0 °C and the mixture was stirred at rt for 2 h. The mixture was filtered through celite and rinsed with EtOAc (10 mL). The combined organic layers were dried over MgSC , filtered and concentrated to dryness to afford the title compound that was used in the next step without further purification.

Intermediate 3: /V-(5-Amino-6-chloropyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6- carboxamide (Int 3)

Int 3a Int 3

Step 1 : 2-Chloropyridine-3,5-diamine (Int 3a)

A mixture of NH4CI (1.47 g, 27.5 mmol) and 2-chloro-3,5-dinitropyridine (400 mg, 1.97 mmol) in 24 mL dioxane/water (3:1) was stirred at rt for 15 min. Zinc (1.80 g, 27.5 mmol) was added at 0 °C and the mixture was stirred at rt for 1 h. The mixture was filtered through celite and rinsed with EtOAc (10 mL). The combined organic layers were dried over MgSC , filtered and concentrated to dryness to afford the title compound that was used in the next step without further purification.

Step 2: /V-(5-Amino-6-chloropyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 3)

A mixture of 2,3-dihydro-1 ,4-benzodioxine-6-carboxylic acid (316 mg, 1.76 mmol) in 5 mL thionyl chloride was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 3 mL anhydrous THF and the solution was added to a mixture of 2-chloropyridine-3,5-diamine (Int 3a) (280 mg, 1.95 mmol) and DIPEA (504 mg, 3.901 mmol) in 3 mL anhydrous THF at 0 °C. The mixture was stirred at rt overnight. 2M HCI (20 mL) was added and the mixture was extracted with EtOAc (3 x 30 mL). The aqueous layer was basified with saturated Na2COs solution and the aqueous layer was extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness to afford the title compound which was used in the next step without further purification.

Intermediate 4: /V-(5-Amino-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6- carboxamide (Int 4)

Int 4a Int 4

Step 1 : 2-Methylpyridine-3,5-diamine (Int 4a)

A mixture of NH4CI (2.445 g, 45.71 mmol) and 2-methyl-5-nitropyridin-3-amine (1.00 g, 6.53 mmol) in 40 mL dioxane/water (3:1) was stirred at rt for 15 min. Zinc (2.99 g, 45.7 mmol) was added at 0 °C and the mixture was stirred at rt for 1 h. The mixture was filtered through celite and rinsed with EtOAc (400 mL). The combined organic layers were dried over MgSC , filtered and concentrated to dryness to afford the title compound which was used in the next step without further purification.

Step 2: /V-(5-Amino-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4)

A mixture of 2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxylic acid (789 mg, 4.39 mmol) in thionyl chloride (5 mL) was stirred at rt for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 10 mL of anhydrous DMF. The solution was added dropwise to a mixture of 2-methylpyridine-3,5-diamine (Int 4a) (540 mg, 4.39 mmol) and triethylamine (0.67 mL, 4.82 mmol) in anhydrous DMF (10 mL) at 0 °C. The mixture was stirred at rt for 30 min. The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 0-10% MeOH in DCM) to afford the title compound.

Intermediate 5: Ethyl 2-((6-((5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2- methylpyridin-3-yl)carbamoyl)quinolin-2-yl)oxy)acetate (Int 5)

Int 5

Step 1 : Benzyl quinoline-6-carboxylate (Int 5a)

A mixture of quinoline-6-carboxylic acid (300 mg, 1.73 mmol) in thionyl chloride (5 mL) was stirred at 50 °C for 1 h. The mixture was concentrated and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in anhydrous THF (10 mL) and the solution was added to a mixture of benzyl alcohol (187 mg, 1.73 mmol) and pyridine (0.014 mL, 0.173 mmol) in anhydrous THF (5 mL) at 0 °C. The mixture was stirred at rt for 1 h. Water (20 mL) was added and the mixture was extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over MgSC , filtered and concentrated to dryness to afford the title compound which was used in the next step without further purification.

Step 2: Benzyl quinoline-6-carboxylate (Int 5b) m-Chloroperbenzoic acid (400 mg, 0.912 mmol) was added to a mixture of benzyl quinoline- 6-carboxylate (Int 5a) (400 mg, 0.912 mmol) in anhydrous DCM (10 mL) at 0 °C. The mixture was stirred at rt for 1 h. The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 0-5% MeOH in DCM) to afford the title compound as a white solid.

Step 3: Benzyl 2-(2-ethoxy-2-oxoethoxy)quinoline-6-carboxylate (Int 5c) p-Toluenesulfonyl chloride (573 mg, 3.01 mmol) was added to a mixture of 6- ((benzyloxy)carbonyl)quinoline-1-oxide (Int 5b) (140 mg, 0.501 mmol) and DIPEA (972 mg, 7.52 mmol) in ethyl 2-hydroxyacetate (1.9 mL, 2.09 g, 20.1 mmol). The mixture was stirred at rt for 1 h. The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 10-100% EtOAc in cyclohexane) to afford the title compound as a white solid.

Step 4: 2-(2-Ethoxy-2-oxoethoxy)quinoline-6-carboxylic acid (Int 5d)

Triethylsilane (637 mg, 5.47 mmol) was added dropwise to a mixture of benzyl 2-(2-ethoxy-2- oxoethoxy)quinoline-6-carboxylate (Int 5c) (200 mg, 0.547 mmol) and 10% Pd/C (20 mg) in anhydrous MeOH (5 mL) at 0 °C. The mixture was stirred at rt for 1 h. The mixture was filtered through celite and rinsed with EtOAc (2 x 50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness to afford the title compound which was used in the next step without further purification.

Step 5: Ethyl 2-((6-((5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)carbamoyl)quinolin-2-yl)oxy)acetate (Int 5)

A mixture of 2-(2-ethoxy-2-oxoethoxy)quinoline-6-carboxylic acid (Int 5d) (55.0 mg, 0.20 mmol) in thionyl chloride (1 mL) was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in anhydrous DMF (1 mL) and the solution was added to a mixture of /V-(5-amino-6-methylpyridin- 3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (57.0 mg, 0.20 mmol) and DI PEA (51.6 mg, 0.4 mmol) in anhydrous DMF (1 mL) at 0 °C. The mixture was stirred at rt for 1 h. The mixture was concentrated to dryness and the residue was dissolved in 20 mL water. The aqueous layer was acidified to pH~6 with 2M HCI. The aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness to afford the title compound which was used for the next step without further purification.

Intermediate 6: /V-(5-Amino-2-methylpyridin-3-yl)-2-methylquinoline-6-carboxamide (Int 6)

Int 6a Int 6

Step 1 : 2-Methyl-N-(2-methyl-5-nitropyridin-3-yl)quinoline-6-carboxamide (Int 6a)

A mixture of 2-methyl-5-nitropyridin-3-amine (1.00 g, 6.53 mmol) and 2-methylquinoline-6- carboxylic acid (2.57 g, 13.7 mmol) was dissolved in THF (32.6 mL). TEA (5.4 mL, 19.6 mmol) was added and the mixture was stirred for 5 min. Thionyl chloride (1.4 mL, 2.33 mmol) was added and the mixture was stirred for 10 min. The mixture was cooled to 0 °C and t-BuOK (0.733 g, 6.50 mmol) was added. The mixture was stirred for 48 h at reflux. The mixture was filtered and concentrated to dryness. The residue was purified by column chromatography (gradient 0-10% MeOH in DCM) to afford the title compound as a yellow solid.

Step 2: /V-(5-Amino-2-methylpyridin-3-yl)-2-methylquinoline-6-carboxamide (Int 6)

2-Methyl-/V-(2-methyl-5-nitropyridin-3-yl)quinoline-6-carboxamide (Int 6a) (150 mg, 0.465 mmol) was dissolved in 1.2 mL EtOH/FW (5:1). Fe (181.9 mg, 3.26 mmol) and NH4CI (174 mg, 3.25 mmol) were added, and the mixture was refluxed for 3 h. The mixture was filtered through celite and rinsed with EtOAc (15 mL). The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 0-10% MeOH in EtOAc + 1 % TEA) to afford the title compound as a yellow oil.

Intermediate 7: 2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 7)

Int 7

Step 1 : 2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 7)

A mixture of 2,3-dihydro-1 ,4-benzodioxine-6-carboxylic acid (200 mg, 1.11 mmol) in 2 mL thionyl chloride was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 2 mL anhydrous THF. The solution was added dropwise to 1 mL of ammonia (28% in H2O) at 0 °C and the mixture was stirred for 30 min. The mixture was concentrated to dryness. The residue was dissolved in water (10 mL) and DCM (10 mL). The organic layer was extracted with DCM (3 x 10 mL). The combined organic layers were dried over MgSC , filtered and concentrated to dryness to afford the title compound which was used in the next step without further purification.

Intermediate 8: /\/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)- 2-((4-ethylpiperazin-1-yl)methyl)quinoline-6-carboxamide (Int 8)

Int 8c Int 8 Step 1 : Methyl 2-methylquinoline-6-carboxylate (Int 8a)

To a solution of 2-methylquinoline-6-carboxylic acid (8.00 g, 42.7 mmol) in MeOH (200 mL) was added dropwise H2SO4 (20.0 mL) and the mixture was stirred at 70 °C overnight. The mixture was concentrated to dryness, the residue was added to water (100 mL), extracted with EtOAc (3 x 80 mL). The combined organic layer was washed with water (100 mL), saturated sodium bicarbonate (100 mL) and brine (30 mL), dried with Na2SO4 and concentrated to dryness. The residue was purified by column chromatography on silica gel (gradient 20% EtOAc in PE) to give the title compound as an off-white solid.

Step 2: Methyl 2-(bromomethyl)quinoline-6-carboxylate (Int 8b)

To a solution of methyl 2-methylquinoline-6-carboxylate (Int 8a) (1.60 g, 7.96 mmol) in CCI4 (50 mL) was added NBS (1.56 g, 8.76 mmol) and AIBN (0.260 g, 1.59 mmol). The mixture was stirred at 75 °C overnight. The mixture was concentrated to dryness and the residue was purified by column chromatography on silica gel (gradient 20% EtOAc in) to give the title compound as a white solid.

Step 3: Methyl 2-((4-ethylpiperazin-1-yl)methyl)quinoline-6-carboxylate (Int 8c)

To a solution of methyl 2-(bromomethyl)quinoline-6-carboxylate (Int 8b) (1.03 g, 3.68 mmol) in THF (75 mL) was added 1 -ethylpiperazine (504 mg, 4.41 mmol), DIPEA (713 mg, 5.52 mmol) and the mixture was stirred at 55 °C for 6 h. The mixture was concentrated to dryness and the residue was purified by column chromatography on silica gel (gradient 50% EtOAc in PE) to give the title compound as a yellow solid.

Step 4: 2-((4-Ethylpiperazin-1-yl)methyl)quinoline-6-carboxylic acid (Int 8)

To a solution of methyl 2-((4-ethylpiperazin-1-yl)methyl)quinoline-6-carboxylate (Int 8c) (1.08 g, 3.45 mmol) in MeOH (100 mL) was added NaOH (603 mg, 15.1 mmol) and water (50 mL) and the mixture was stirred at rt for 3 h. To the mixture was added HOI (3 M) to adjust the pH to pH = 4 - 6. The mixture was concentrated to dryness and the residue was purified by prep- HPLC to give the title compound as a yellow solid.

Intermediate 9: 6-Methyl-1 ,5-naphthyridine-2-carboxylic acid (Int 9)

Step 1 : 6-Methyl-1 ,5-naphthyridin-2-ol (Int 9a)

To 5-amino-2-methoxypyridine (1.00 g, 0.008 mol) in concentrated HCI (5.4 mL) was added acetaldehyde (1.80 mL, 0.032 mol) at 0 °C. The mixture was stirred at 0 °C for 1 h, then refluxed for 1 h. The mixture was diluted with 20 mL water, then the pH was adjusted to ~10 with 2 M NaOH. The organic layer was extracted with EtOAc (3 x 60 mL). The combined organic layers were washed with brine (50 mL), dried over MgSC , filtered and concentrated to dryness to afford the title compound which was used in the next step without further purification.

Step 2: 2-Chloro-6-methyl-1 ,5-naphthyridine (Int 9b)

A mixture of 6-methyl-1 ,5-naphthyridin-2-ol (Int 9a) (400 mg, 2.5 mmol) in POCh (2.6 mL) was heated to reflux for 30 min. The mixture was cooled to room temperature and was added dropwise to a stirred mixture of water with ice. The aqueous phase was washed with EtOAc (3 x 30 mL). The aqueous phase was adjusted to pH ~12 with 2 M NaOH. The organic phase was extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to afford the title compound which was used in the next step without further purification.

Step 3: 2-lodo-6-methyl-1 ,5-naphthyridine (Int 9c)

To 2-chloro-6-methyl-1 ,5-naphthyridine (Int 9b) (80.0 mg, 0.45 mmol) and Nal (671 mg, 4.5 mmol) in MeCN (0.9 mL) was added acetyl chloride (0.048 mL, 0.7 mmol). The mixture was heated to 80 °C and stirred for 3 h. The mixture was cooled to rt and a 5% aqueous sodium thiosulfate solution (10 mL), followed by a saturated aqueous NaHCOs solution (10 mL) were added to the mixture. The organic layer was extracted with DCM (3 x 20mL). The combined organic layers were dried over MgSC , filtered, and concentrated to dryness to afford the title compound as yellowish solid.

Step 4: 6-Methyl-1 ,5-naphthyridine-2-carbonitrile (Int 9d)

In a microwave vial, 2-iodo-6-methyl-1 ,5-naphthyridine (Int 9c) (90.0 mg, 0.33 mmol), potassium hexacyanoferrate(ll) (56.3 mg, 0.13 mmol) and Cui (12.7 mg, 0.1 mmol) were added. Mesitylene (2.0 mL) and 1 -methylimidazole (0.080 mL, 1.0 mmol) were added. The mixture was degassed with nitrogen for 15 min. The mixture was stirred at 140 °C for 24 h. The mixture was cooled down to rt, diluted with EtOAc (10 mL) and water (10 mL). The organic layer was extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated to dryness to afford the title compound which was used in the next step without any further purification.

Step 5: 6-Methyl-1 ,5-naphthyridine-2-carboxylic acid (Int 9)

To a solution of 6-methyl-1 ,5-naphthyridine-2-carbonitrile (Int 9d) (55.0 mg, 0.325 mmol) in EtOH (2.0 mL) was added 2.0 mL of 2 M NaOH. The reaction mixture was stirred at 75 °C for 1 h. 2 M HOI was added to reach a pH ~ 7. The mixture was concentrated to dryness. The residue was purified by prep-HPLC to give 6-methyl-1 ,5-naphthyridine-2-carboxylic acid as a white solid.

Intermediate 10: 6-((4-Ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int

Int 10f Int 10g Int 10

Step 1 : Ethyl thieno[2,3-b]pyridine-2-carboxylate (Int 10a)

To a solution of 2-chloronicotinaldehyde (50.0 g, 354 mmol) in DMF (400 mL) was added TEA (74.1 g, 708 mmol) and ethyl 2-mercaptoacetate (42.5 g, 354 mmol). The mixture was stirred at 100 °C for 4 h under nitrogen atmosphere. The mixture was diluted with water (1.5 L) and extracted with EtOAc (3 x 1000 mL). The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by flash chromatography on silica (PE/EtOAc= 8:1) to give the title compound.

Step 2: 2-(Ethoxycarbonyl)thieno[2,3-b]pyridine 7-oxide (Int 10b) To a solution of ethyl thieno[2,3-b]pyridine-2-carboxylate (Int 10a) (52.8 g, 255.0 mmol) in acetic acid (400 mL) was added H2O2 (100 mL, 30% in water) portion wise. The resulting mixture was stirred at 60 °C overnight. The mixture was stirred at 0 °C for 1 h. A precipitate was formed during the reaction. The solid was collected by filtration and dried to give the title compound.

Step 3: Ethyl 6-hydroxythieno[2,3-b]pyridine-2-carboxylate (Int 10c)

To a solution of 2-(ethoxycarbonyl)thieno[2,3-b]pyridine 7-oxide (Int 10c) (15.0 g, 67.2 mmol) in DMF (150 mL) was added TFAA (28.0 g, 134 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 3 h. The mixture was poured into water (300 mL) and stirred for 5 min. A precipitate was formed. The solid was collected by filtration and dried to give the title compound as a yellow solid, which was used in the next step without further purification.

Step 4: Ethyl 6-chlorothieno[2,3-b]pyridine-2-carboxylate (Int 10 d)

To a solution of ethyl 6-hydroxythieno[2,3-b]pyridine-2-carboxylate (Int 10c) (14.8 g, 66.3 mmol) in DMF (150 mL) was added POCh (50.7 g, 332 mmol), and the mixture was stirred at 100 “C overnight. The mixture was cooled to rt and the solvent was removed under reduced pressure. The residue was diluted with water (200 mL) and adjusted to pH = 8 with saturated aqueous NaHCCh. The mixture was extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated to dryness. The crude product was purified by column chromatography (PE/EtOAc = 8:1) to give the title compound.

Step 5: Ethyl 6-methylthieno[2,3-b]pyridine-2-carboxylate (Int 10e)

A solution of ethyl 6-chlorothieno [2,3-b]pyridine-2-carboxylate (Int 10d) (5.0 g, 20.7 mmol), 2,4,6-trimethyl-1 ,3,5,2,4,6-trioxatriborinane (25 g, 200 mmol), Pd(PPhs)4 (2.3 g, 2.0 mmol), K2CO3 (8.4 g, 62.1 mmol) in dioxane (100 mL) was stirred at 100 °C for 14 h under N2 atmosphere. The mixture was cooled to rt and filtered. The filtrate was concentrated to dryness. The residue was purified by column chromatography (PE/EtOAc = 8:1) to give the title compound.

Step 6: Ethyl 6-(bromomethyl)thieno[2,3-b]pyridine-2-carboxylate (Int 10f)

A solution of ethyl 6-methylthieno[2,3-b]pyridine-2-carboxylate (Int 10e) (3.8 g,17.2 mmol), AIBN (281 mg, 1 .72 mmol) and NBS (3.04 g, 17.2 mmol) in CCI4 (50 mL) was stirred at 80 °C for 14 h. The mixture was cooled to rt and concentrated to dryness. The residue was purified by column chromatography (PE/EtOAc = 6:1) to give the title compound. Step 7: Ethyl 6-((4-ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylate (Int 10g)

To a solution of ethyl 6-(bromomethyl)thieno[2,3-b]pyridine-2-carboxylate (Int 10f) (4.6 g, 15.3 mmol) in acetonitrile (100mL) was added 1 -ethylpiperazine (2.6 g, 23 mmol) and K2CO3 (6.3 g, 45.9 mmol). The mixture was stirred at 70 °C overnight. The mixture was diluted with water (500 mL) and extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated to dryness to give the title compound which was used in the next step without further purification.

Step 8: 6-((4-Ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 10)

To a solution of ethyl 6-((4-ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylate (Int 10g) (4.1 g, 12.3 mmol) in MeOH/FW (5:1 , 50 mL) was added NaOH (984 mg, 24.6 mmol) and the mixture was stirred at rt overnight. The mixture was diluted with water and adjusted to pH = 5 using 1M HCI. The mixture was extracted with EtOAc (3 x 200 mL). The aqueous phase was concentrated to dryness and the residue was purified by reverse phase column chromatography (gradient acetonitrile / H2O = 5% - 80%) to give the title compound.

Example 1 : /\/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-2- (2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide (1)

A mixture of 2,3-dihydro-1 ,4-benzodioxine-6-carboxylic acid (9.2 mg, 51 pmol) in 1 mL thionyl chloride was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 1 mL anhydrous THF. The solution was added to a mixture of /V-(5-amino-2-methylpyridin-3-yl)-2-(2-(pyrrolidin-1- yl)ethoxy)quinoline-6-carboxamide (Int 1) (20 mg, 51 pmol) and DI PEA (13.2 mg, 102 pmol) in anhydrous THF (1 mL) at 0 °C and the mixture was stirred at rt overnight. The mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, DMSO-d₆) 5 10.29 (s, 1 H), 10.26 (s, 1 H), 8.72 (s, 1 H), 8.58 (s, 1 H), 8.39 (d, J = 8.8 Hz, 1 H), 8.30 - 8.18 (m, 2H), 7.88 (d, J = 8.9 Hz, 1 H), 7.58 - 7.50 (m, 2H), 7.13 (d, J = 8.9 Hz, 1 H), 7.00 (d, J = 8.4 Hz, 1 H), 4.57 (t, J = 5.7 Hz, 2H), 4.37 - 4.27 (m, 4H), 2.89 (t, J = 5.6 Hz, 2H), 2.63 - 2.54 (m, 4H), 2.44 (s, 3H), 1 .76 - 1.65 (m, 4H). MS: m/z 554.5 [M+H]⁺. Example 1/1 : 2-(2-Cyclopentylethoxy)-/V-(5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6- carboxamido)-2-methylpyridin-3-yl)quinoline-6-carboxamide (1/1)

The title compound was prepared similarly as described for Example 1 using A/-(5-amino-2- methylpyridin-3-yl)-2-(2-cyclopentylethoxy)quinoline-6-carboxamide (Int 2) in place of A/-(5- amino-2-methylpyridin-3-yl)-2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide (Int 1). ¹H NMR (300 MHz, CDCI₃): 6 ppm 8.71 (s, 1 H), 8.62 (s, 1 H), 8.43 (s, 1 H), 8.35-8.27 (m, 2H), 8.05 (dd, J = 14.4, 8.8 Hz, 2H), 7.89 (d, J = 8.7 Hz, 1 H), 7.47 (s ,1 H), 7.43 (d, J = 8.3 Hz, 1 H), 6.96 - 6.83 (m, 2H), 4.51 (t, J = 6.8 Hz, 2H), 4.30-4.19 (m, 4H), 2.55 (s, 3H), 2.14-1.94 (m, 1 H), 1.92-1.80 (m, 4H), 1.70-1.49 (m, 4H), 1.29-1.14 (m, 2H). MS: 553.5 m/z [M+H]⁺

Example 2: A/-(2-Chloro-5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)pyridin-3-yl)-2-(2- (pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide (2)

A mixture of 2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxylic acid (Int 1a) (45 mg, 157 pmol) in thionyl chloride (4 mL) was stirred at 50 °C for 1.5 h. The mixture was concentrated and coevaporated with dry toluene (3 x 10 mL). The solution was dissolved in anhydrous THF (1 mL) and the solution was added to a mixture of A/-(5-amino-6-chloropyridin-3-yl)-2,3- dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 3) (48 mg, 157 mmol) and DI PEA (40.6 mg, 314 pmol) in anhydrous THF (5 mL) at 0 °C. The mixture was stirred at rt overnight. The mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, CDCh): 6 ppm 9.07 (s, 1 H), 8.83 (s, 1 H), 8.55 (s, 1 H), 8.28 (s, 1 H), 8.17 (s, 1 H), 8.13 - 8.03 (m, 2H), 7.92 (d, J = 8.7 Hz, 1 H), 7.46 (s, 1 H), 7.43 - 7.37 (m, 1 H), 7.04 (dd, J = 8.9, 1 .7 Hz, 1 H), 6.96 (dd, J = 8.4, 1.8 Hz, 1 H), 4.74 (t, J = 5.6 Hz, 2H), 4.35-4.25 (m, 4H), 3.15 (t, J = 5.6 Hz, 2H), 2.97-2.83 (m, 4H), 1.96-1.84 (m, 4H). MS: m/z 574.6 [M+H]⁺. Example 3: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-2- methylquinoline-6-carboxamide (3)

Int 4 Example 3

DCC (506 mg, 2.45 mmol) was added to a mixture of A/-(5-amino-6-methylpyridin-3-yl)-2,3- dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (200 mg, 0.701 mmol), 2-methylquinoline- 6-carboxylic acid (394 mg, 2.103 mmol) and DMAP (8.6 mg, 0.070 mmol) in anhydrous DMF (2.5 mL) at rt. The mixture was stirred at 130 °C for 1 h. The mixture was concentrated to dryness and the residue was purified by column chromatography (gradient 1-5% MeOH in DCM) to afford 160 mg of a crude product which was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, CD3OD): 5 ppm 8.72 (s, 1 H), 8.60 (s, 1 H), 8.43 (d, J = 9.0 Hz, 1 H) 8.37 (s, 1 H), 8.30 (d, J = 9.0 Hz, 1 H), 8.10 (d, J = 9.0 Hz, 1 H), 7.59-7.46 (m, 3H), 6.98 (d, J = 8.3 Hz, 1 H), 4.36-4.27 (m, 4H), 2.79 (s, 3H), 2.55 (s, 3H). MS: m/z 455.5 [M+H]⁺.

Example 3/1 : /V-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)pyrazolo[1 ,5-a]pyridine-5-carboxamide (3/1)

The title compound was prepared similarly as described for Example 3 using pyrazolo[1 ,5- a]pyridine-5-carboxylic acid in place of 2-methylquinoline-6-carboxylic acid. ¹H NMR (300 MHz, DMSO-d₆): 5 ppm 10.33 - 10.25 (m, 2H), 8.85 (d, J = 7.4 Hz, 1 H), 8.73 (s, 1 H), 8.44 (s, 1 H), 8.29 (s, 1 H), 8.15 (s, 1 H), 7.58-7.48 (m, 2H), 7.39 (d, J = 7.3 Hz, 1 H), 7.02 (d, J = 8.4 Hz, 1 H), 6.91 (s, 1 H), 4.38 - 4.27 (m, 4H), 2.44 (s, 3H). MS: 430.4 m/z [M+H]⁺.

Example 4: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)benzo[d]thiazole-6-carboxamide (4)

Int 4 Example 4

N-(5-Amino-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (25 mg, 87.6 pmol) and TEA (17.7 mg, 175 pmol) was added to a mixture of benzo[d]thiazole-6- carboxylic acid (15.7 mg, 87.6 pmol) and HATLI (33.3 mg, 87.6 pmol) in anhydrous DMF (1 mL) at rt and the mixture was stirred at rt for 24 h. The mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, DMSO-d₆): 5 ppm 10.27 (d, J = 10.5 Hz, 2H), 9.58 (s, 1 H), 8.83 (s, 1 H), 8.73 (s, 1 H), 8.29 (s, 1 H), 8.24 (d, J = 8.5 Hz, 1 H), 8.14 (d, = 8.5 Hz, 1 H), 7.53 (m, 2H), 7.00 (d, J = 8.0 Hz, 1 H), 4.35-4.28 (m, 4H), 2.44 (s, 1 H). MS: m/z 447.4 [M+ H]⁺.

Example 5: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)quinoline-7-carboxamide (5)

Thionyl chloride (18.3 mg, 154 pmol) was added to a mixture of /V-(5-amino-6-methylpyridin- 3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (20 mg, 70.1 pmol), quinoline-7- carboxylic acid (25.5 mg, 147 pmol) and DIPEA (54.4 mg, 421 pmol) in anhydrous THF (4 mL) at rt. The mixture was refluxed for 3 h. The mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, DMSO-d₆): 5 ppm 10.46 (s, 1 H), 10.33 (s, 1 H), 9.03 (d, J = 4.1 Hz, 1 H), 8.74 (d, J = 11.1 Hz, 2H), 8.49 (d, J = 8.2 Hz, 1 H), 8.31 (s, 1 H), 8.15 (s, 2H), 7.67 (dd, J = 8.4, 4.2 Hz, 1 H), 7.58-7.50 (m, 2H), 7.02 (d, J = 8.3 Hz, 1 H), 4.36-4.27 (m, 4H), 2.47 (s, 3H). MS: m/z 441.5 [M+H]⁺.

Example 5/1 : /\/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)- 1 H-indole-5-carboxamide (5/1)

Int 4 Example 5/1

The title compound was prepared similar as described for Example 5 using 1/7-indole-5- carboxylic acid in place of quinoline-7-carboxylic acid. ¹H NMR (300 MHz, CD3OD): 5 ppm 8.73-8.69 (m, 1 H), 8.33-8.28 (m, 2H), 7.80 (d, J = 8.4 Hz, 1 H), 7.52-7.46 (m, 3H), 7.37 (d, J = 3.3 Hz, 1 H), 6.97 (d, J = 7.8 Hz, 1 H), 6.62 (d, J = 2.1 Hz, 1 H), 4.36-4.27 (m, 4H), 2.55 (s, 3H). MS: m/z 429.5 [M+H]⁺.

Example 5/2: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)- 1 H-indole-5-carboxamide (5/2)

The title compound was prepared similar as described for Example 5 using 1-benzothiophene- 2-carboxylic acid in place of quinoline-7-carboxylic acid. ¹H NMR (300 MHz, DMSO-de): 5 ppm 10.36 (s, 1 H), 10.27 (s, 1 H), 8.75 (s, 1 H), 8.36 (s, 1 H), 8.27 (s, 1 H), 8.11 - 7.99 (m, 2H), 8.04 - 7.94 (m, 1 H), 7.58 - 7.44 (m, 3H), 7.00 (d, J = 8.3 Hz, 1 H), 4.35 - 4.28 (m, 4H), 2.44 (s, 3H). MS: m/z 446.4 [M+H]⁺.

Example 5/3: A/-(6-Methyl-5-(4-(trifluoromethyl)benzamido)pyridin-3-yl)-2,3- d i hydro be nzo[b][1 ,4]dioxine-6-carboxamide (5/3)

Int 4 Example 5/3

The title compound was prepared similar as described for Example 5 using 2- (trifluoromethyl)quinoline-6-carboxylic acid in place of quinoline-7-carboxylic acid. ¹H NMR (300 MHz, DMSO-de): 5 ppm 10.49 (s, 1 H), 10.32 (s, 1 H), 8.94 (d, J = 8.6 Hz, 1 H), 8.85 (s, 1 H), 8.74 (s, 1 H), 8.43 (d, J = 9.0 Hz, 1 H), 8.38 - 8.27 (m, 2H), 8.11 (d, J = 8.5 Hz, 1 H), 7.60 - 7.51 (m, 2H), 7.02 (d, J = 8.1 Hz, 1 H), 4.37 - 4.26 (m, 4H), 2.47 (s, 3H). MS: m/z 509.4 [M+H]⁺.

Example 5/4: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-6- methyl-1 H-indole-2-carboxamide (5/5)

Int 4 Example 5/4

The title compound was prepared similar as described for Example 5 using 6-methyl-1/7- indole-2-carboxylic acid in place of quinoline-7-carboxylic acid. ¹H NMR (300 MHz, CD3OD): 5 ppm 11.61 (s, 1 H), 10.26 (s, 1 H), 9.99 (s, 1 H), 8.73 (s, 1 H), 8.26 (s, 1 H), 7.61 - 7.49 (m, 3H), 7.34 (s, 1 H), 7.26 (s, 1 H), 7.01 (d, J = 8.4 Hz, 1 H), 6.93 (d, J = 8.3 Hz, 1 H), 4.36 - 4.26 (m, 4H), 2.43 (s, 3H), 2.41 (s, 3H). MS: m/z 443.4 [M+H]⁺.

Example 5/5: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-5- methyl-1 /7-indole-2-carboxamide (5/5)

The title compound was prepared similar as described for Example 5 using 5-methyl-1/7- indole-2-carboxylic acid in place of quinoline-7-carboxylic acid. ¹H NMR (300 MHz, CD3OD): 5 8.70 (s, 1 H), 8.34 (s, 1 H), 7.57 - 7.41 (m, 3H), 7.37 (d, J = 8.4 Hz, 1 H), 7.23 (s, 1 H), 7.12 (d, J = 8.5 Hz, 1 H), 6.96 (d, J = 8.3 Hz, 1 H), 4.36 - 4.26 (m, 4H), 2.52 (s, 3H), 2.43 (s, 3H). MS: m/z 443.4 [M+H]⁺.

Example 5/6: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)quinoxaline-6-carboxamide (5/6)

Int 4 Example 5/6

The title compound was prepared similar as described for Example 5 using quinoxaline-6- carboxylic acid in place of quinoline-7-carboxylic acid. ¹H NMR (300 MHz, CDCh): 6 ppm 8.98 (s, 2H), 8.74 (d, J = 5.3 Hz, 2H), 8.61 (s, 1 H), 8.37 - 8.25 (m, 2H), 7.96 (s, 1 H), 7.81 (s, 1 H), 7.46 (s, 1 H), 7.41 (d, J = 8.2 Hz, 1 H), 6.96 (d, J = 8.4 Hz, 1 H), 4.37 - 4.29 (m, 4H), 2.63 (s,

3H). MS: m/z 442.5 [M+H]⁺.

Example 6: /V-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-2- (2-hydroxy-2-methylpropoxy)quinoline-6-carboxamide (6)

1M methylmagnesium bromide solution in THF (0.124mL, 14.8 mg, 124.4 pM) was added dropwise to a mixture of ethyl 2-((6-((5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2- methylpyridin-3-yl)carbamoyl)quinolin-2-yl)oxy)acetate (Int 5) (15 mg, 20.7 pmol) in anhydrous THF (1 mL) at 0 °C. The mixture was stirred at rt for 30 min. Water (2 mL) was added and the mixture was concentrated. Water (10 mL) was added and the pH was adjusted to pH 2 with 1M aqueous HCI. The aqueous layer was extracted with EtOAc (2 x 15 mL). The pH of the aqueous layer was adjusted to pH 12 with 1M NaOH solution. The aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness. The residue was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, CDCh): 6 ppm 8.65-8.61 (m, 1 H) 8.57-8.53 (m, 1 H), 8.30 (s, 1 H), 8.16 - 8.00 (m, 4H), 7.88 (d, J = 8.8 Hz, 1 H), 7.45 (s, 1 H) 7.41 (d, J = 8.6 Hz, 1 H), 7.05 (d, J = 8.9 Hz, 1 H), 6.93 (d, J = 8.4 Hz, 1 H), 4.43 (s, 2H), 4.34-4.25 (m, 4H), 2.53 (s, 3H), 1.38 (s, 6H). MS: m/z 529.5 [M+H]⁺.

Example 7: /V-(5-(Chromane-6-carboxamido)-2-methylpyridin-3-yl)-2-methylquinoline-6- carboxamide (7)

A mixture of chromane-6-carboxylic acid (7.60 mg, 43.0 pmol) in 5 mL thionyl chloride was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 1 mL anhydrous THF. The solution was added to a mixture of A/-(5-amino-2-methylpyridin-3-yl)-2-methylquinoline-6-carboxamide (Int 6) (12 mg, 43 pmol) and DIPEA (15 pL, 86 pmol) in anhydrous THF (1 mL) at 0 °C and the mixture was stirred at rt overnight. The mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, DMSO-d₆): 5 ppm 10.34 (s, 1 H), 10.28 (s, 1 H), 8.72 (s, 1 H), 8.62 (s, 1 H), 8.43 (d, J = 8.5 Hz, 1 H), 8.30 (s, 1 H), 8.25 (d, J = 8.6, 1 H), 8.05 (d, J = 8.8 Hz, 1 H), 7.80 - 7.70 (m, 2H), 7.54 (d, J = 8.4 Hz, 1 H), 6.86 (d, J = 8.4 Hz, 1 H), 4.21 (t, J = 5.1 Hz, 2H), 2.82 (t, J = 6.4 Hz, 2H), 2.71 (s, 3H), 2.45 (s, 3H), 1 .96 (p, J = 6.0 Hz, 2H). 5. MS: m/z 453.5 [M+H]⁺.

Example 7/1 : /V-(6-Methyl-5-(2-methylquinoline-6-carboxamido)pyridin-3-yl)benzo[cf]thiazole- 5-carboxamide (7/1)

The title compound was prepared similar as described for Example 7 using 1 ,3-benzothiazole- 5-carboxylic acid in place of chromane-6-carboxylic acid. ¹H NMR (300 MHz, CD3OD): 5 ppm 9.37 (s, 1 H), 8.79 (s, 1 H), 8.71 (s, 1 H), 8.60 (s, 1 H), 8.49 - 8.37 (m, 2H), 8.34 - 8.20 (m, 2H), 8.09 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.6 Hz, 1 H), 2.78 (s, 3H), 2.57 (s, 3H). MS: m/z 454.4 [M+H]⁺.

Example 7/2: /V-(5-(Chromane-7-carboxamido)-2-methylpyridin-3-yl)-2-methylquinoline-6- carboxamide (7/2)

The title compound was prepared similar as described for Example 7 using chromane-7- carboxylic acid in place of chromane-6-carboxylic acid. ¹H NMR (300 MHz, CD3OD): 5 ppm 8.74 (s, 1 H), 8.61 (s, 1 H), 8.47 - 8.36 (m, 2H), 8.31 (d, J = 8.8 Hz, 1 H), 8.11 (d, J = 8.9 Hz, 1 H), 7.57 (d, J = 8.6 Hz, 1 H), 7.44 (d, J = 7.8 Hz, 1 H), 7.37 (s, 1 H), 7.21 (d, J = 7.9 Hz, 1 H), 4.24 (t, J = 5.1 Hz, 2H), 2.88 (t, J = 6.4 Hz, 2H), 2.80 (s, 3H), 2.57 (s, 3H), 2.05 (p, J = 6.1 Hz, 2H). MS: m/z 453.4 [M+H]⁺.

Example 8: /V-(4-(Benzo[b]thiophene-2-carboxamido)-5-methylpyridin-2-yl)-2,3- d i hydro be nzo[b][1 ,4]dioxine-6-carboxamide (8)

Step 1 : /V-(2-Bromo-5-methylpyridin-4-yl)benzo[b]thiophene-2-carboxamide (8a)

2-Bromo-5-methylpyridin-4-amine (50.0 mg, 0.267 mmol) was dissolved in pyridine (2 mL) along with 1-benzothiophene-2-carboxylic acid (47.6 mg, 0.267 mmol). To the mixture was added DIPEA (0.093 mL, 0.535 mmol) and DMAP (4.9 mg, 0.040 mmol). Phosphoryl chloride (0.027 mL, 0.294 mmol) was added to the mixture at 0 °C. The mixture was stirred at rt for 16 h. Water (5 mL) and EtOAc (5 mL) were added. The organic layer was extracted with EtOAc (3 x 5 mL) and washed with 1 M HCI and saturated aqueous NaHCCh. The combined organic layers were dried over MgSC , filtered, and concentrated to dryness to afford the title compound which was used in the next step without further purification.

Step 2: /V-(4-(Benzo[b]thiophene-2-carboxamido)-5-methylpyridin-2-yl)-2,3-dihydrobenzo[b] [1 ,4]dioxine-6-carboxamide (8)

/V-(2-Bromo-5-methylpyridin-4-yl)benzo[b]thiophene-2-carboxamide (8a) (15.0 mg, 0.043 mmol), 2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 7) (7.70 mg, 0.043 mmol), caesium carbonate (21.1 mg, 0.065 mmol) and methanesulfonato(2-dicyclohexylphosphino- 2',4',6'-tri-i-propyl-1 ,T-biphenyl)(2'-methylamino-1 ,1'-biphenyl-2-yl)palladium(ll) (5.60 mg, 0.006 mmol) were placed into a nitrogen flushed microwave vial and dissolved in dry dioxane. The reaction was stirred at 130 °C overnight. The mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford the title compound as a white solid. ¹H NMR (300 MHz, CD₃OD): 5 ppm 8.48 (s, 1 H), 8.22 (d, J = 9.2 Hz, 2H), 7.96 (d, J = 7.2 Hz, 2H), 7.55 - 7.42 (m, 4H), 6.97 (d, J = 8.3 Hz, 1 H), 4.35 - 4.27 (m, 4H), 2.34 (s, 3H). MS: m/z 446.4 [M+H]⁺.

Example 8/1 : A/-(2-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-5-methylpyridin-4-yl)-2- methylquinoline-6-carboxamide (8/1 )

The title compound was prepared similar as described for Example 8 using 2-methylquinoline- 6-carboxylic acid in place of benzo[b]thiophene-2-carboxylic acid. ¹H NMR (300 MHz, DMSO- d₆): 6 ppm 10.52 (s, 1 H), 10.25 (s, 1 H), 8.63 (s, 1 H), 8.49 - 8.39 (m, 2H), 8.27 - 8.17 (m, 2H), 8.06 (d, J = 8.8 Hz, 1 H), 7.64 - 7.52 (m, 2H), 7.55 (d, J = 8.4 Hz, 1 H), 6.96 (d, J = 8.5 Hz, 1 H), 4.35 - 4.26 (m, 4H), 2.72 (s, 3H), 2.29 (s, 3H). MS: m/z 455.4 [M+H]⁺.

Example 9: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)thieno[2,3-b]pyrazine-6-carboxamide (9)

Int 4 Example 9

/V-(5-Amino-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (30.0 mg, 0.105 mmol), thieno[2,3-b]pyrazine-6-carboxylic acid (18.9 mg, 0.105 mmol) and DMAP (1.9 mg, 0.016 mmol) were dissolved in pyridine (350 pL) and the mixture was stirred for 5 min at rt. The mixture was cooled to 0°C, POC (11 pL, 0.115 mol) was added and the mixture was allowed to warm to rt and stirred for 1 h. The mixture was concentrated to dryness and the residue was purified by flash column chromatography with a gradient of EtOAc (with 1%TEA) and MeOH 0-10% to yield the title compound as a white powder. ¹H NMR (300 MHz, DMSO-d₆): 5 ppm 10.66 (s, 1 H), 10.32 (s, 1 H), 8.90 (s, 1 H), 8.80-8.75 (m, 2H), 8.57 (s, 1 H), 8.31 (s, 1 H), 7.60 - 7.49 (m, 2H), 7.02 (d, J = 8.5 Hz, 1 H), 4.36 - 4.26 (m, 4H), 2.55 (s, 3H). MS: m/z 448.4 [M+H]⁺ .

Example 9/1 : /V-(5-(Benzofuran-2-carboxamido)-6-methylpyridin-3-yl)-2,3- d i hydro be nzo[b][1 ,4]dioxine-6-carboxamide (9/1)

The title compound was prepared similar as described for Example 9 using benzofuran-2- carboxylic acid in place of thieno[2,3-b]pyrazine-6-carboxylic acid. ¹H NMR (300 MHz, DMSO- d₆): 6 ppm 10.40 (s, 1 H), 10.30 (s, 1 H), 8.74 (s, 1 H), 8.27 (s, 1 H), 7.85 (d, J = 7.9 Hz, 1 H), 7.79 (s, 1 H), 7.75 (d, J = 8.3 Hz, 1 H), 7.60 - 7.48 (m, 3H), 7.39 (t, J = 7.5 Hz, 1 H), 7.01 (d, J = 8.4 Hz, 1 H), 4.37 - 4.28 (m, 4H), 2.44 (s, 3H). MS: m/z 430.4 [M+H]⁺.

Example 9/2: A/-(5-(5-Chlorobenzofuran-2-carboxamido)-6-methylpyridin-3-yl)-2,3- d i hydro be nzo[b][1 ,4]dioxine-6-carboxamide (9/2)

Int 4 Example 9/2

The title compound was prepared similar as described for Example 9 using 5- chlorobenzofuran-2-carboxylic acid in place of 5-hydroxybenzofuran-2-carboxylic acid. ¹H NMR (300 MHz, DMSO-d₆): 5 ppm 10.47 (s, 1 H), 10.30 (s, 1 H), 8.74 (s, 1 H), 8.26 (s, 1 H), 7.96 (s, 1 H), 7.83 - 7.74 (m, 2H), 7.59 - 7.50 (m, 3H), 7.01 (d, J = 8.4 Hz, 1 H), 4.40 - 4.27 (m, 4H), 2.43 (s, 3H). MS: m/z 464.4 [M+H]⁺.

Example 9/3: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)quinoline-6-carboxamide (9/3)

The title compound was prepared similar as described for Example 9 using quinoline-6- carboxylic acid in place of benzo[b]thiophene-2-carboxylic acid. ¹H NMR (300 MHz, CD3OD): 5 ppm 8.98 (d, J = 4.3 Hz, 1 H), 8.71 (s, 1 H), 8.65 (s, 1 H), 8.55 (d, J = 8.4 Hz, 1 H), 8.38 (s, 1 H), 8.33 (d, J = 9.1 Hz, 1H), 8.18 (d, J = 8.9 Hz, 1 H), 7.66 (dd, J = 8.5, 4.3 Hz, 1H), 7.54 -7.45 (m,

2H), 6.95 (d, J = 8.3 Hz, 1 H), 4.38 - 4.24 (m, 4H), 2.54 (s, 3H). MS: m/z 441.4 [M+H]⁺.

Example 9/4: A/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-2- ((4-ethylpiperazin-1-yl)methyl)quinoline-6-carboxamide (9/4)

Int 4 Example 9/4

The title compound was prepared similar as described for Example 9 using 2-((4- ethylpiperazin-1-yl)methyl)quinoline-6-carboxylic acid (Int 8) in place of benzo[b]thiophene-2- carboxylic acid. ¹H NMR (400 MHz, DMSO-d₆): 5 ppm 10.30 (d, J= 12.6 Hz, 2H), 8.74 (s, 1 H), 8.65 (s, 1 H), 8.50 (d, J = 8.4 Hz, 1 H), 8.37 - 8.22 (m, 2H), 8.10 (d, J = 8.8 Hz, 1 H), 7.73 (d, J = 8.5 Hz, 1 H), 7.63 - 7.48 (m, 2H), 7.01 (d, J = 8.4 Hz, 1 H), 4.32 (s, 4H), 3.80 (s, 2H), 2.51 -

2.36 (m, 11 H), 2.34 - 2.26 (m, 2H), 0.99 (t, J = 7.1 Hz, 3H). MS: m/z 567.5 [M+H]⁺.

Example 10: A/-(6-Methyl-5-(6-methyl-2-naphthamido)pyridin-3-yl)-2,3-dihydrobenzo[b] [1,4]dioxine-6-carboxamide (10)

Example 10

Step 1 : /V-(5-(6-Bromo-2-naphthamido)-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b]

[1 ,4]dioxine-6-carboxamide (10a)

/V-(5-Amino-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (30.0 mg, 0.105 mmol), 6-bromo-2-naphthoic acid (31.7 mg, 0.126 mmol) and NMI (25.9 mg, 0.315 mmol) was dissolved in THF (1.1 mL) and stirred for 5 min. TCFH (32.5 mg, 0.116 mmol) was added. The mixture was stirred at rt for 16 h. EtOAc (5 mL) and water (5 mL) were added to the mixture. The organic layer was extracted with EtOAc (3 x 5 mL). The combined organic layers were dried over MgSO4 and filtered. The filtrate was concentrated to dryness to afford the title compound which was used in the next step without further purification.

Step 2: /V-(6-Methyl-5-(6-methyl-2-naphthamido)pyridin-3-yl)-2,3-dihydrobenzo[b]

[1 ,4]dioxine-6-carboxamide (10)

/V-(5-(6-Bromo-2-naphthamido)-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6- carboxamide (10a) (15.5 mg, 0.030 mmol) was placed in a microwave vial and dissolved in dioxane (0.3 mL) and H2O (0.03 mL). Pd(PPhs)4 (3.5 mg, 0.003 mmol), trimethylboroxine (5.6 mg, 0.045 mmol) and K2CO3 (12.4 mg, 0.090 mmol) were then added. The mixture was stirred at 80 °C overnight. The mixture was diluted by EtOAc (5 mL) and water (5 mL). The organic layer was then extracted with EtOAc (3 x 5 mL) and washed with brine (5 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness to provide the crude product. The residue was purified by prep-HPLC to afford the title compound. ¹H NMR (300 MHz, CD3OD): 5 ppm 8.72 (s, 1 H), 8.52 (s, 1 H), 8.34 (s, 1 H), 8.00 (d, J = 9.0 Hz, 1 H), 7.93 (t, J = 7.5 Hz, 2H), 7.75 (s, 1 H), 7.70 - 7.53 (m, 1 H), 7.54 - 7.42 (m, 2H), 6.97 (d, J = 8.3 Hz, 1 H), 4.36 - 4.26 (m, 4H), 2.56 (s, 3H), 2.54 (s, 3H). MS: m/z 454.5 [M+H]⁺.

Example 11 : /\/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-2- (2-(pyridin-2-yl)ethoxy)quinoline-6-carboxamide (11 )

Example 11

Step 1 : 2-Chloro-/V-(5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3- yl)quinoline-6-carboxamide (11a)

To a solution of 2-chloroquinoline-6-carboxylic acid (2.00 g, 9.66 mmol) and NMI (2.46 g, 30.0 mmol) in DMF (50 mL) was added TCFH (6.78 g, 24.2 mmol), and the mixture was stirred at rt for 1 h. The mixture was cooled to 0 °C. /V-(5-amino-6-methylpyridin-3-yl)-2,3- dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (2.48 g, 8.70 mmol) was added and the mixture was stirred overnight. The mixture was concentrated to dryness and the residue was triturated with MeCN/MeOH (3:2, 80 mL), filtered and the solid was dried in vacuo to give the title compound as a white solid.

Step 2: /\/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-2-(2- (pyridin-2-yl)ethoxy)quinoline-6-carboxamide (11)

To a mixture of 2-chloro-/V-(5-(2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2- methylpyridin-3-yl)quinoline-6-carboxamide (11a) (2.50 g, 5.27 mmol), Pd(OAc)2 (178 mg, 0.790 mmol), Trixiephos (210 mg, 0.53 mmol) and CS2CO3 (3.50 g, 10.7 mmol) in dioxane (60 mL) was added 2-(pyridin-2-yl)ethan-1-ol (1.30 g, 10.6 mmol) under N2. The mixture was stirred at 100 °C for 1.5 h. The mixture was filtered and the filtrate was concentrated to dryness. The residue was purified by column chromatography on silica gel (gradient 2-10% MeOH in DCM) to afford the crude product. The crude product was purified by prep-HPLC to give /\/-(5- (2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-2-(2-(pyridin-2- yl)ethoxy)quinoline-6-carboxamide as a white solid. ¹H NMR (400 MHz, DMSO-de): 5 ppm: 10.27 (s, 1 H), 10.24 (s, 1 H), 8.73 (d, J = 2.0 Hz, 1 H), 8.58 (d, J = 1.6 Hz, 1 H), 8.53 (d, J = 4.4 Hz, 1 H), 8.38 (d, J = 8.8 Hz, 1 H), 8.29 (d, J = 2.0 Hz, 1 H), 8.23 (dd, J = 8.8, 2.0 Hz, 1 H), 7.90 (d, J = 8.8 Hz, 1 H), 7.74 (td, J = 7.6, 1.6 Hz, 1 H), 7.58 - 7.51 (m, 2H), 7.40 (d, J = 7.6 Hz, 1 H), 7.25 (dd, J = 7.2, 5.4 Hz, 1 H), 7.06 (d, J = 8.8 Hz, 1 H), 7.01 (d, J = 8.4 Hz, 1 H), 4.85 (t, J = 6.8 Hz, 2H), 4.32 (q, J = 4.8 Hz, 4H), 3.28 (d, J = 7.2 Hz, 2H), 2.45 (s, 3H). MS: m/z 562.4 [M+H]⁺. Example 12: /\/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-2-methylpyridin-3-yl)-6- ((4-ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxamide (12)

Int 4 Example 12

/V-(5-Amino-6-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (Int 4) (70.0 mg, 0.245 mmol), 6-((4-ethylpiperazin-1-yl)methyl)thieno[2,3-b]pyridine-2-carboxylic acid (Int 10) (74.9 mg, 0.245 mmol) and 1 -methylimidazole (60.4 mg, 59.0 pL, 0.736 mmol) were dissolved in 3 mL of dry DMF. The mixture was stirred for 5 min at rt. Then TCFH (82.6 mg, 0.294 mmol) was added portion-wise and the mixture was stirred at rt overnight. The mixture was concentrated to dryness and the crude product was purified by preparative HPLC (10-25% acetonitrile in water) to afford the title compound as a white powder. NMR (300 MHz, DMSO-d₆): 5 ppm 10.44 (s, 1 H), 10.27 (s, 1 H), 8.72 (s, 1 H), 8.45 (d, J = 8.0 Hz, 1 H), 8.32 ( d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.4 Hz, 1 H), 7.55 (s, 1 H), 7.51 (s, 1 H), 7.02 (d, J = 8.0 Hz, 1 H), 4.32 (s, 4H), 3.83 (s, 2H), 3.24-2.66 (m, 10H), 2.44 (s, 3H), 1.16 (t, J = 5.8 Hz, 3H). MS: m/z 573.68 [M+H]⁺.

Example 13: /V-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-6-methylpyridin-3-yl)-2- (2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide (13)

Step 1 : /V-(2-Methyl-5-nitropyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (13a)

Thionyl chloride (0.1 mL) was added to a mixture of 2,3-dihydro-1 ,4-benzodioxine-6-carboxylic acid (180 mg, 0.653 mmol) in 0.5 mL of DCM. The mixture was stirred at rt for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 1 mL anhydrous DMF. The solution was added to a mixture of 2-methyl-5- nitropyridin-3-amine (100 mg, 0.653 mmol) and DIPEA (0.1 mL) in anhydrous DMF (0.5 mL) at 0 °C. The mixture was stirred at rt overnight. The mixture was concentrated to dryness to afford the title compound which was used in the next step without further purification.

Step 2: /V-(5-Amino-2-methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (13b)

/V-(2-methyl-5-nitropyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (13a) (5.0 mg, 0.016 mmol) was placed in a round bottom flask with zinc powder (7.3 mg, 0.111 mmol) and NH4CI (5.9 mg, 0.111 mmol) in dioxane:water 3:1 (1 mL). After 1 h, the reaction was filtered through celite and the celite was washed with EtOAc (5 mL). The combined organic layers were dried over MgSC , filtered, and concentrated to dryness to afford the title compound, which was used in the next step without further purification.

Step 3: /\/-(5-(2,3-Dihydrobenzo[b][1 ,4]dioxine-6-carboxamido)-6-methylpyridin-3-yl)-2-(2- (pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide (13)

Thionyl chloride (0.1 mL) was added to 2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxylic acid (Int 1a) (10 mg, 0.035 mmol) and the mixture was stirred at 50 °C for 1 h. The mixture was concentrated to dryness and coevaporated with dry toluene (3 x 10 mL). The residue was dissolved in 1 mL anhydrous THF. The solution was added to a mixture of /V-(5-amino-2- methylpyridin-3-yl)-2,3-dihydrobenzo[b][1 ,4]dioxine-6-carboxamide (13b) (10 mg, 0.035 mmol) and DIPEA (0.012 mL, 0.070 mmol) in anhydrous THF (0.5 mL) at 0 °C. The mixture was stirred at rt overnight. The crude product was purified by prep-HPLC to give the title compound as a white solid. ¹H NMR (300 MHz, CDCh): 6 ppm 8.83 (s, 1 H), 8.62 (s, 1 H), 8.41 (s, 1 H), 8.29 (s, 1 H), 8.05 (t, J = 9.4 Hz, 2H), 7.82 (d, J = 8.8 Hz, 2H), 7.45 (s, 1 H), 7.40 (d, J = 8.7 Hz, 1 H), 6.97 (q, J = 4.6 Hz, 2H), 4.84 (t, J = 4.9 Hz, 2H), 4.37 - 4.25 (m, 4H), 3.32 (t, J = 4.6 Hz, 2H), 3.20 - 3.00 (m, 4H), 2.54 (s, 3H), 2.10 - 1.97 (m, 4H). MS: m/z 554.5 [M+H]⁺.

Biological Assays

Assay for inhibition of ATP production

The assay determines the number of viable cells in culture by quantifying ATP, which indicates the presence of metabolically active cells. Luminescence readout is directly proportional to the number of viable cells in culture.

Cells were plated at 4,000 per well in 96-well plate in a medium of high glucose DMEM supplemented with 10% FBS, 2 mM glutamine-alanine, 2 mM pyruvate and penicillin- streptromycin. The next day, cells were induced with test compounds, as well as with vehicle (DMSO) only, for 48 hours. After this time, the medium was removed from the cells and intracellular ATP levels were determined using the Promega CellTiter-Glo 2.0 assay protocol (Promega, G9241). To estimate the effect of compounds on the ATP production, sample values were compared to the values of DMSO only. IC50 values were generated by plotting the luciferase counts against compound concentration.

Fig. 1 encompasses several graphs which show the inhibition activity of the compounds of Examples 1 and 3 in HeLa, 4T1 , LLC1 , A549, MDA-MB-231 and MiaPaCa2 cells after 48 hours treatment. In all cases, a dose-dependent reduction of the ATP production level is observed.

Numerical results are shown in Table 1 below, in which compound activities are grouped according to the following IC50 ranges:

+ 1 pM - 10 pM,

++ 100 nM - < 1 pM

+++ < 100 nM

Lactate production inhibition assay

Recombinant Lactate Dehydrogenase A (LDHA) was used to convert lactate in tissue culture supernatants to pyruvate, with the concomitant reduction of NAD⁺ to NADH. The electron acceptor 1 -methoxyphenazine methosulfate (MPMS) was then reduced by the generated NADH, thereby regenerating NAD⁺. Reduced 1 -methoxyphenazine methosulfate is used in turn to reduce Water Soluble Tetrazolium 8 (WST8) to an orange WST8 formazan product. The assay is adapted from the publication “Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays described in Chamchoy et al., BMC Biochemistry (2019) 20(1):4.

Specifically, cell supernatants are diluted 1 :5 in assay buffer (0.2 M tris, pH 8.2) by adding 10 pl cell supernatant + 40 pl buffer in a 96-well plate. Assay substrate mix is prepared by adding NAD+ (3.75 mM), WST-8 (0.9 mM), MPMS (10 mM) and recombinant LDHA, expressed in E. coli as an N-terminal hexahistidine tagged fusion protein, is added to a final concentration of 0.8 pg/ml in assay buffer. Thereafter, 80 pl of substrate mix is added to 20 pl of diluted cell supernatant in a 96-well flat-bottomed plate and the absorbance at 460 nm determined at one minute intervals over a 30 minute period. A standard curve of known lactate concentrations of 10, 8, 6, 4, 1 , 0.5 and 0.25 mM was constructed for each assay plate.

Fig. 2 shows the downregulation of lactate levels in HeLa, 4T1 , LLC1 , A549, MDA-MB-231 and MiaPaCa2 cells after 48 hours treatment with Example 1 and Example 3. In all cases, a dose-dependent reduction of the lactate production level is observed. Numerical results are shown in Table 1 below, in which compound activities are grouped according to the following IC50 ranges:

+ 1 pM - 10 pM,

++ 100 nM - < 1 pM

< 100 nM

Table 1

As can be seen in the above table, all tested inventive compounds showed activity in inhibiting production of ATP and/or lactate. In particular, the inventive compounds of Examples 1 , 1/1 , 2, 3, 4, 6, 5/5, 7/2 and 10-12 demonstrated high activity in inhibiting production of either one or both of ATP and lactate.

Caspase 3/7 activity assay The assay determines the activity of caspases 3 and 7 in a population of cells, which indicates apoptosis. Luminescence readout is directly proportional to the number of activated caspase molecules in cultured cells.

Cells were plated at 5.000 per well in 96-well plate in medium of high glucose DMEM supplemented with 10% FBS, 2 mM glutamine-alanine, and pen-strep, and grown at 37°C in 0.5% O2, 5 % CO2 and 75% humidity. Next day, cells were treated with test compounds, as well as with vehicle (DMSO) only, for 24 hours. After this time, the medium was removed from cell and intracellular Caspase 3/7 activity was determined according to the Promega Caspase- Glo® 3/7 assay protocol (Promega, G8093). To estimate the effect of compounds on the activation of caspases and thereby apoptosis, sample values were compared to the values of DMSO only.

The result is shown in Fig. 3 which shows an increase in caspase 3 and 7 activity in HeLa cells after a 24 hour treatment with the compound of Example 1 in concentrations above 1.5 pM.

Gene expression analysis by qPCR

Cells were plated at 40,000 cells per ml in medium of high glucose DMEM supplemented with 10% FBS, 2 mM glutamine-alanine, 2 mM pyruvate and pen-strep, and grown at 37°C in 0.5% O2, 5 % CO2 and 75% humidity. Next day, cells were induced with test compounds, as well as with vehicle (DMSO) only, for 2 hours. After this time, the cells were lysed according to protocol (Promega, SV 96 Total RNA Isolation System, #Z3505). After transferring the lysate to a 96- well-RNA binding plate, RNA was isolated and put at approx. 1 pg into cDNA synthesis using LunaScript RT SuperMix Kit (NEB, #E3010). The resulting cDNA was diluted 1 :5 and used for qPCR Analysis (Taqman). Luna Universal Probe qPCR Master Mix (NEB, #M3004) is used as 2x reaction mix to which the diluted cDNA is added.

A set of primers plus probe for each gene of interest was purchased at IDT (Integrated DNA Technologies). The corresponding sequences are summarized in Table 2:

Table 2:

Fig. 4 shows that the compounds of Examples 1 and 3 induce markers of endoplasmic reticulum stress such as CHAC-1 , CHOP, XBP1 and SLC7A11 in HeLa cells after a 2 hour treatment.

Western Blot

Western Blot was used to detect amounts of specific protein in differently treated cells. Proteins are separated by size, transferred onto a nitrocellulose membrane and incubated with protein specific antibodies. The strength of the observed band is proportional to the amount of the specific protein inside the cell.

Cells were plated at 3.5x10⁶ (for hypoxia) or 3x10⁶ (for normoxia) cells per 10cm² plate in medium of high glucose DMEM supplemented with 10% FBS, 2 mM glutamine-alanine, 2mM pyruvate and pen-strep, and grown at 37°C in 0.5% O2, 5 % CO2 and 75% humidity. For hypoxia, cells were grown and treated in the Whitley H35 Hypoxystation (Don Whitley Scientific) at 37°C in 0.5% O2, 5 % CO2 and 75% humidity. Next day, the cells were treated with test compounds, as well as with vehicle (DMSO) only, for 24h. After 24 hours, the cells were kept on ice, washed with PBS twice and scraped to collect. The lysate was centrifuged at 3,000rpm for 3 min at 4°C. The cell pellet was resuspended in 200pl Lysis Buffer (20mM Tris pH 7,8, 150mM NaCI, 0.05% Tween20, 2mM MgCI2, protease inhibitors (Roche) and sonicated at 50% for 10 sec twice. The sample was centrifuged at 13,000 rpm for 1 min at4°C, the supernatant transferred to a new tube, centrifuged again at 13,000 rpm for 1 min at 4°C and the supernatant transferred to a new tube. Protein concentration of the sample was measured using the DC Protein Assay Kit (Biorad, 5000112), 30 pg of protein extract per well was loaded onto a precast SDS page gel (Biorad, 1704273). Proteins were then transferred onto a nitrocellulose membrane (5671084, Biorad) using Biorad Trans-Blot Turbo Transfer System. The membrane then was incubated in the blocking buffer (5% dry milk, 0.1 % Tween20, Tris-buffered saline buffer) for 30 min, and after with the 1 :1000 dilution of primary antibody against HIF1a (#36169, Cell Signalling) or a-tubulin (#2125, Cell Signalling) in the blocking buffer, overnight. Next day, the membrane was washed with 0.1 % Tween20 in TBS buffer three times for 10 min and incubated with the secondary antibody anti-rabbit IgG HRP (#sc-2004, Santa Cruz Biotechnology) in the blocking buffer for 45 min. The membrane was then washed three times for 10 min. The chemiluminescence was measured using chemiluminescent substrate (Thermo Scientific #34580). The result of the Western blot with the compound of Example 1 is depicted in Fig. 5.

Claims

79 Claims

1 . A compound of formula (I)

R^A is Ci-e-alkyl, halogen, CN, OH, O-Ci-e-alkyl, S-Ci-e-alkyl, S(O)>rCi.6-alkyl, NR^a-Ci-6-alkyl or C(O)NR^a-Ci-6-alkyl, wherein Ci-6-alkyl is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of Ci-e-alkyl, Ci-6-haloalkyl, halogen and oxo, or wherein two Ci-6-alkyl groups form together with the carbon atom to which they are attached a Cs-e-cycloalkyl group;

B is a bicyclic aryl or a bicyclic 8-14 membered heteroaryl containing 1 , 2, 3 or 4 heteroatoms independently selected from the group consisting of O, S and N, wherein the bicyclic aryl or the bicyclic heteroaryl is unsubstituted or substituted with 1 to 4 same or different substituents R^B; 80

R^B is Ci-e-alkyl, halogen, CN, OH, or Ci-6-haloalkyl;

R¹ is hydrogen, OH, Ci-3-alkyl or halogen;

R² is hydrogen, Ci-e-alkyl, halogen, CN, Ci-6-haloalkyl, OH or O-Ci-e-alkyl;

R³ is hydrogen, Ci-6-alkyl or V-W-X-Y-Z, wherein

V is absent, a bond or Ci-6-alkylene,

W is absent, a bond or O, S, S(O)_X, S(O)(=NR^a), S(O)₂NR^a, NR^a, NR^aC(O) or C(O)NR^a,

X is absent, a bond or Ci-6-alkylene,

Y is absent, a bond or O, S, S(O)_X, S(O)(=NR^a), S(O)₂NR^a, NR^a, NR^aC(O) or C(O)NR^a, wherein Ci-6-alkylene in V and X is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of Ci-e-alkyl, Ci-e- haloalkyl, halogen and oxo, or wherein two Ci-6-alkyl groups on the alkylene, together with the carbon atom to which they are attached, form a Cs-e-cycloalkyl group,

Z is hydrogen, halogen, OH, CN, Ci-6-alkyl, Cs-s-cycloalkyl, 4- to 8-membered mono or bicyclic heterocycloalkyl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, or

5- to 6-membered heteroaryl containing 1 , 2 or 3 heteroatoms independently selected from the group consisting of O, S and N, wherein alkyl, cycloalkyl, heterocycloalkyl or heteroaryl are unsubstituted or substituted with 1 to 4 substituents R^z independently selected from the group consisting of CN, OH, Ci-6-alkyl, O-Ci-6-alkyl, C(O)-Ci-6-alkyl, Ci-6-haloalkyl, halogen, oxo, spirocyclicly fused Cs-e-cycloalkyl and spirocyclicly fused 3-7 membered heterocycloalkyl containing 1 heteroatom selected from the group consisting of O, S and N;

R⁴ is Ci-6-alkyl, halogen, CN, OH, Ci-6-alkyl, O-Ci-6-alkyl, S-Ci-6-alkyl, oxo or spirocyclicly fused C3-6-cycloalkyl; 81

R^a is hydrogen or Ci-3-alkyl; and x is 1 or 2.

2. The compound according to claim 1 , wherein the compound is represented by formula (II),

wherein

R² is Ci-3-alkyl, OH, O-Ci-6-alkyl or halogen;

R^B is F or Ci-3-alkyl; m is independently 0, 1 or 2;

T¹ is C or N;

T² is CH, S, N, NH, HC=N, N=CH or HC=CH;

T³ is CH, O, S, N or NH;

T⁴ is CH, S, N or NH;

T⁵ is C or N; and

3. The compound according to claim 1 or 2, wherein the compound is represented by formula (HI)

wherein

R² is Ci-3-alkyl or halogen;

R^B is F or Ci-3-alkyl; and 82 m is independently 0, 1 or 2.

4. The compound according to any one of claim 1-3, wherein the compound is represented by formula (IV)

wherein

K is O, S or C(R⁴)₂;

L is O, S or C(R⁴)₂;

5. The compound according to claim 1 , wherein the compound is represented by formula (V)

wherein

R² is Ci-3-alkyl, OH, O-Ci-6-alkyl or halogen;

R^B is F or Ci-3-alkyl; ml is 0 or 1 ;

T⁷ is O, S or NH;

T⁸ is CH or N;

T⁹ is CH, or N; and

T¹⁰ is CH, or N. 83

6. The compound according to claim 1 or 5, wherein the compound is represented by formula (VI)

wherein

K is O, S or C(R⁴)₂;

L is O, S or C(R⁴)₂;

7. The compound according to any one of claims 1 to 6, wherein

Q is CH₂, CHR^Z1, O, NH, N-Ci-6-alkyl, N-C(O)-Ci-e-alkyl or S; t is 0, 1 or 2; u is 0, 1 or 2; and v is 0, 1 or 2. 84

8. The compound according to claim 1, wherein the compound is selected from the group consisting of

85

or an N-oxide, solvate, prodrug or pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. The compound according to any one of claims 1 to 8 for use as a medicament.

11. The compound according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 for use in a method of preventing and/or treating a disease or condition mediated by the lactate/ATP mechanism.

12. The compound for use or the pharmaceutical composition for use according to claim 11 , wherein the disease or condition mediated by the lactate/ATP mechanism is cancer.

13. The compound for use or the pharmaceutical composition for use according to claim 12, wherein the compound or the pharmaceutical composition is administered

• together with a cytokine therapy or a known chemo- or pharmacotherapy, or during irradiation therapy.

14. A compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9, for use in a method of preventing and/or treating a disease or condition, preferably cancer, by inducing endoplasmic reticulum stress in cells affected by said disease or condition, thereby reducing intracellular ATP and lactate production in said cells.

15. A compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9, for use in a method of preventing and/or treating cancer, preferably a cancer cell and/or a cancerous tumor, wherein said compound or said pharmaceutical composition reduces Hif1 -alpha at the protein level in hypoxic areas of said cancer, thereby downregulating ATP and lactate production in these areas and ultimately leading to reduction, preferably efficient reduction, of hypoxic areas and of growth of said cancer.

16. A compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9, for use in a method of preventing and/or treating a disease or condition which is susceptible to the induction of endoplasmic reticulum stress, wherein administration of said compound or said pharmaceutical composition to a subject having or suspected of having said disease or condition results in a reduction of the protein level of Hif-1 alpha.