LU102907B1

LU102907B1 - Citrate transporter modulators and uses thereof

Info

Publication number: LU102907B1
Application number: LU102907A
Authority: LU
Inventors: Kay Schreiter; Russell Craft; Grit Zahn; Christopher John Yarnold; Sabine Schaertl; Someina Khor; Richard Jarjes-Pike; Steven Mark Bromidge
Original assignee: Eternygen Uk Ltd C/O Epidarex Capital
Priority date: 2022-02-03
Filing date: 2022-02-03
Publication date: 2023-08-03

Abstract

Described herein are compounds that modulate the function and activity of citrate transporters, e.g. a sodium-coupled citrate transporter such as SLC13A5 (also referred to as Indy or NaCT). Also described herein are methods of making such compounds, pharmaceutical compositions comprising such compounds, alone or in combination with other compounds, and uses thereof, including their use to treat and/or prevent a condition that is citrate transporter dependent or citrate transporter mediated, including metabolic diseases and age related diseases.

Description

P 00303-032 LU102907

Eternygen UK Ltd.

CITRATE TRANSPORTER MODULATORS AND USES THEREOF

FIELD OF THE INVENTION

BRIEF DESCRIPTION OF THE RELATED ART

Energy balance and insulin action are both closely related to life span. Caloric excess leads to obesity and insulin resistance to an increased mortality. Caloric restriction reduces adiposity and increases lipid oxidation, insulin sensitivity, and mitochondrial biogenesis. In addition, caloric restriction reverses obesity, type 2 diabetes, delays aging, and prolongs life in many species, including primates (Hursting et al, 2003, Annu. Rev. Med. 54, p. 131-152; Lopez-Lluch et al, 2006,

Proc. Natl. Acad. Sci. U. S. A 103, p. 1768-1773; Hunt et al, 2006, Ageing Res. Rev. 5, p. 125-143;

Fontana and Klein 2007, JAMA 297, p 986-994; Colman et al, 2009, Science 325, p. 201-204).

Beneficial effects of caloric restriction are mediated by decreased plasma concentrations of anabolic hormones and growth factors, 1.e. insulin and insulin like growth factors (Fontana and Klein 2007,

JAMA 297, p 986-994; Colman et al, 2009, Science 325, p. 201-204). Reduced expression of the

Indy (for I am Not Dead, Yet) gene in D. melanogaster and C. elegans has been shown to promote longevity in a manner akin to caloric restriction, however the cellular mechanism by which reduced expression of Indy leads to increased survival is unknown (Rogina et al, 2000, Science 290, p. 2137- 2140; Fei et al, 2004, Biochem. J. 379, p. 191-198; Fei et al, 2003, J. Biol. Chem. 278, p. 6136- 6144; Wang et al, 2009, Proc. Natl. Acad. Sci. U. S. A 106, p. 9262-9267).

Indy encodes in D. melanogaster a non-electrogenic dicarboxylate and citrate transporter (Knauf et al, 2006, Biochem. J. 397, p. 25-29; Knauf et al, 2002, Proc. Natl. Acad. Sci. U. S. A 99, p. 14315- 14319) and it is mainly expressed in the fat body, mid gut, and oenocyte (Rogina et al, 2000, Science 290, p. 2137-2140), the major organs of intermediary metabolism in flies. In mammals, the gene product of SLC13A5, the sodium-coupled citrate transporter NaCT (mINDY), shares the highest sequence and functional similarity with INDY of D. melanogaster (Inoue et al, 2002, Biochem. J. 367,313-319, WO 2004/048925) and it is predominantly expressed in liver cells (Inoue et al, 2002,

P 00303-032 2 LU102907

Eternygen UK Ltd.

J. Biol. Chem. 277, p. 39469-39476; Knauf et al, 2006, Biochem. J. 397, p. 25-29; Knauf et al, 2002, Proc. Natl. Acad. Sci. U. S. A 99, p. 14315-14319; Gopal et al, 2007, Am. J. Physiol

Gastrointest. Liver Physiol 292, G402-G408, WO 2004/048925).

Indy and its mammalian homolog mINDY (SIc13a5, NaCT) are transporters of tricarboxylic acid (TCA) cycle intermediates. Basically, INDY handles the uptake of citrate via the plasma membrane into the cytosol where citrate is used for the synthesis of fatty acids and cholesterol (Inoue et al, 2002, J. Biol. Chem. 277, p. 39469-39476, Birkenfeld et al, 2011, Cell Metab 14, p. 184-195).

Cytosolic citrate is known as the prime carbon source for the synthesis of fatty acids, triacylglycerols, cholesterols and low-density lipoproteins (Willmes and Birkenfeld 2013 Comput

Struct Biotechnol J. 2013 6:7). Moreover citrate leads to the activation of fatty acid synthesis and affects glycolysis and B-oxidation (Spencer and Lowenstein 1962 J Biol Chem 237: 3640-48, Bloch and Vance 1977 Ann Rev Biochem 46:263-298, Ruderman et al. 1999 Am J Physiol 276: E1-18).

Main organs for fatty acid synthesis are the liver and white adipose tissue and fatty acid synthesis has been shown to directly correlate with cytosolic citrate concentrations, partially depending on the direct import across the plasma membrane by mINDY (Inoue 2002 Biochem Biophys Res

Comm 299:465—471, Gopal et al, 2007, Am. J. Physiol Gastrointest. Liver Physiol 292, G402-

G408).

Birkenfeld and colleagues described in 2011 that deletion of the mouse homologue of INDY (mIndy) reduces citrate uptake into the liver and sterol and fatty acid synthesis in hepatocytes.

Furthermore, it reduces adiposity, prevents lipid accumulation into liver and skeletal muscle and increases insulin sensitivity under high fat diet (HFD) conditions and during aging in mIndy knock- out mice. Loss of mINDY augments energy expenditure associated with increased hepatic fat oxidation and attenuates hepatic lipogenesis (Birkenfeld et al, 2011, Cell Metab 14, p. 184-195).

Furthermore, Pesta and colleagues (Pesta et al. 2015 Aging 7(12), p. 1086-93) showed that a hepatic knockdown of mINDY in rats under HFD improved metabolism by reducing fasting plasma insulin, hepatic glucose production, liver fat accumulation and improving insulin sensitivity. Additionally,

Rong et al. showed recently (Rong et al. 2015 Conference abstract Keystone Symposia: Obesity and the Metabolic Syndrome/Liver Metabolism March 2015) that hepatic knockdown of mINDY in mice improved several metabolic parameters such as fed glucose and insulin sensitivity assessed by HOMA-IR, reduced the body weight in animals under high fat diet mainly in liver and adipose tissue weights and reduced liver triglycerides.

Expression analysis of INDY in monkey and human liver samples showed that INDY expression is increased in human obesity and fatty liver as well as in monkeys after 2 years high fat diet (Loeffelholz et al. 2017 Hepatology 66(2), p. 616-630)

P 00303-032 3 LU102907

Eternygen UK Ltd.

Reducing INDY expression by knockout and knockdown has been proven beneficial in terms of metabolic regulation and/or life span in all species tested so far. Therefore, mINDY is a drug target for the treatment of metabolic disease, such as obesity, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatis (NASH) and type 2 diabetes, but also hyperlipidemia and hypercholesterolemia (Birkenfeld et al, 2011, Cell Metab 14, p. 184-195, Pesta et al. 2015 Aging 7(12), p. 1086-93, Mancusso et al, 2012, Nature 491, p. 622-626; Frankel and Rogina, 2012, Front

Genet 3, p. 13; Schindler, 2012, Ther Adv Endocrinol Metab 3, p. 51-53, WO 2004/048925,

Neuschäfer-Rube Diabetes. 2014 63(3), p. 1048-57, Willmes 2016 Aging 2, p. 208-9). It can be expected that an inhibitor of MINDY function reducing the uptake of extracellular citrate will have similar beneficial therapeutic effect as reduction of INDY expression by knockout and knockdown mINDY (WO 2004/048925).

In fact, a very recent work from Huard et al. 2015 (Huard et al. 2015 Sci Rep. 5, p. 17391) has proven this hypothesis with a small molecule citrate analogue and have shown an improvement of metabolism via reduction of hepatic citrate uptake. In this study the inhibition of mINDY recapitulates the main features previously reported for mINDY knockout mice specifically reduction in hepatic lipid production and in plasma glucose levels following oral glucose tolerance test. It has been shown that inhibition of mINDY simultaneously reduce hepatic glucose and lipid production. However, based on the chemical properties and the resulting very high effective doses this molecule can be only considered as a tool compound but not drug like molecule for therapeutic treatment of metabolic diseases

Functionally, loss of mindy also mimics many aspects of calorically restriction. Moreover, in flies and nematodes, both, reduced expression of Indy, as well as caloric restriction, prolong life span (Rogina et al, 2000, Science 290, p. 2137-2140; Fei et al, 2004, Biochem. J. 379, p. 191-198) and

AMPK has been shown to be the mediator of longevity in response to most dietary restriction regimens in C.elegans (Schulz et al, 2007, Cell Metab 6, p. 280-293; Greer et al, 2009, Aging Cell 8, p. 113-127; Mair et al, 2011, Nature 470, p. 404-408). In addition, caloric restriction does not increase life span further in flies with reduced Indy expression (Toivonen et al, 2007, PLoS. Genet. 3, e95; Wang et al, 2009, Proc. Natl. Acad. Sci. U. S. A 106, p. 9262-9267), pointing to similar underlying mechanisms in both conditions. In fact, a very recent work by Schwarz and colleagues (Schwarz et al. 2015 Aging 7(8), p. 553-67) has been shown that knockdown of Indy in c. elegans extends life span by inducing AMPK, whereas the effect was abolished in worms without functional

AMPK. This indicates that the life extending effect of reducing mINDY is at least in part mediated by AMPK. These data suggest that m/ndy may be a key mediator of the beneficial effects of dietary energy restriction. Since prolonged caloric restriction is very difficult to achieve in humans, the

P 00303-032 4 LU102907

Eternygen UK Ltd. observations raise the tantalizing possibility that modulating the levels or function of mindy could lead to some of the health promoting effects of calorie restriction, without requiring severe caloric restriction.

Very recent work by Willmes and colleagues showed that loss of mIndy also affects blood pressure (Willmes et al. 2021, JCI Insight 6(2): e13083). Deletion of mINDY reduces blood pressure in animals fed a HFD. These finding raises the possibility that m/NDY is part of the signalling pathway linking excessive caloric intake to increased blood pressure. Therefore, it seems plausible to speculate that mINDY might be an interesting target for the treatment of hypertension.

Another recent work by Li and colleagues showing the link between mINDY and drug induced hepatic steatosis (Li et al. 2015 Mol Pharmacol. 87(4):674-82). Knockdown of INDY by antisense oligonucleotides leads to significant decrease of rifampicin induced lipid accumulation in HepG2 via PXR dependent pathway. These data were confirmed by Neuschäfer-Rube et al. (Neuschäfer-

Rube et al. 2015 Toxicology 337, p. 1-9). This work showed the link between mINDY and drug induced hepatic steatosis in vitro by benzo[a]pyrene induced lipid accumulation in primary rat hepatocytes (via arylhyrocarbon receptor). Therefore, mINDY is an interesting target for the treatment of drug induced hepatic steatosis.

Importantly, all known studies to reduce INDY expression in vivo in mice as well as rats showed consistently a reduction of liver fat accumulation in animals under high fat diet (Birkenfeld et al, 2011, Cell Metab 14, p. 184-195, Pesta et al. 2015 Aging 7(12), p. 1086-93, Rong et al. 2015

Conference abstract Keystone Symposia: Obesity and the Metabolic Syndrome/Liver Metabolism

March 2015, Huard et al. 2015 Sci Rep. 5, p. 17391, Willmes 2016 Aging 2, p. 208-9) further supporting the hypothesis that mINDY is a drug target for the treatment of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

To date, no pharmaceutically effective and specific therapeutic agents with drug-like properties to modulate mINDY function or expression are known, except an antisense oligonucleotide as tool compound. Interestingly, the stimulation of human m/NDY activity by Lithium was described in concentrations that are observed during the treatment of bipolar disorders. Aluvila and colleagues disclosed compounds, which inhibit another but not mINDY related citrate transporter, the citrate transport protein (CTP) on the inner mitochondrial membrane (Aluvila et al, 2010, Mol Pharmacol 77, p. 26-34; Irwin and Shoichet, 2005, J Chem Inf Model 45, p. 177-182). Moreover, a compound with selectivity for mINDY over CTP was identified by Sun and co-workers (Sun et al. 2010, Mol

Cell Pharmacol 2, p. 101-110). This compound shows inhibitory activity in a millimolar range in a cell free assay. However, in a cellular citrate uptake assay this compound seems to activate Indy.

P 00303-032 5 LU102907

Eternygen UK Ltd.

Pajor and colleagues disclosed an inhibitor of Indy in a high micromolar range (Pajor et al. Mol

Pharmacol. 2007 November; 72(5), p. 1330-1336). But it seems that this compound activity is related to cytotoxic side effects. Furthermore, Ganapathy and colleagues disclosed a substrate analogue, hydroxycitrate, as an inhibitor of Indy in a high micromolar range (30-40% inhibition at 0.1mM) (WO 2004/048925). However, in a cellular citrate uptake assay it was not possible to reproduce this data. À recent work by Colas and Co-workers (Colas 2015 Biochemistry 54(31), p. 4900-8) used a combined modelling and virtual screening approach to find mINDY inhibitors. One active compound has been found with an activity in MM range in a vitro assay. Finally, Huard et al. (Huardetal 2015 Sci Rep. 5, p. 17391) described a tool compound which was selective for mINDY with submicromolar activities in vitro. However, based on its characteristics and chemical properties very high doses (250 mg/kg bi-daily) are necessary to show a therapeutic effect in vivo.

Therefore, this substrate analogue compound class seems to be not a suitable drug like molecule.

Its not known so far, whether such compounds can be used in therapeutic intervention. The high concentration needed to inhibit m/NDY with these molecules or potential toxic side effects make it unlikely to become clinically relevant. The discovery of a more potent and specific compound modulating mINDY function could provide a useful tool to delineate the structure and function of mINDY and to become therapeutic drug to treat metabolic diseases. Ultimately, a putative inhibitor of mINDY holds the potential to induce the beneficial effects of caloric restriction, without requiring severe caloric restriction in mammals. (Willmes and Birkenfeld, 2013, Computational and

Structural Biotechnology Journal. 6 (7))

Thus, in view of the deficits of the prior art compounds and the severe conditions associated with a pathophysiological uptake of extracellular citrate, both acute and chronic, there is a need for compounds that inhibit the activity of citrate transporters such as Indy.

SUMMARY OF THE INVENTION

The present invention was made in view of the prior art and the needs described above, and, therefore, the object of the present invention is to provide compounds according to Formula (I), including pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof, which act as inhibitors for the activity of citrate transporters, such as Indy, and therefore, are useful as agents for the treatment and/or prevention of diseases or conditions in which citrate transporters play a role, such as diseases or conditions in which citrate transporters participate, are involved in the etiology or pathology of the disease or condition, or contribute to at least one symptom of the disease or condition. Preferably, the compounds of general formula (I), or pharmaceutically acceptable

P 00303-032 6 LU102907

Eternygen UK Ltd. salts, solvates, metabolites and prodrugs thereof, described herein have one or more improved properties, e.g. an improved pharmacokinetic and/or physiochemical property, including bioavailability, solubility, metabolic stability, and a LADME (liberation, absorption, distribution, metabolism, and excretion) property.

In one aspect, described herein are compounds of Formula (I), (IT), (Ila), (IIT), (IlTa) and (IV), pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof. In some embodiments, compounds of Formula (I), (II), (Ila), (IIT), (Illa) and (IV) are citrate transporter modulators. In some embodiments, the compound of Formula (I), (IT), (Ha), (IIT), (Ia) or (IV) is a citrate transporter inhibitor and/or a citrate transporter trafficking modulator. In some embodiments, the compound of Formula (I), (ID), (Ha), (III), (Illa) or (IV) is a citrate transporter inhibitor that inhibits the activity of the citrate transporter directly or allosterically.

In one aspect, provided herein is a compound for use in the prevention and/or treatment of a citrate transporter meditated or citrate transporter dependent disease or condition, wherein the compound is a compound of Formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof:

RL! N O

XX M CHR?

R? ©

R® (1) wherein

L'is absent, -O-, -O(CH»)-, -S-, -S(FO)»-, -C(=0)NH-,- NHS(=0),-, or -S(=0),NH-;

R! is H, halogen, CN, NO», (C1-C+)alkyl, (Ci-Cs)haloalkyl, (C1-C;)alkoxy, (Ci-Cs)haloalkoxy, substituted or unsubstituted (C»-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle;

R?is H, F, CI, CN, NO», or OCH3;

R’isH, F, Cl, Br, CN, NO», CF; or OCH;; or R° and R° are taken together to form a group -O-C(R°)-O-; mis | or 2;

R* is CHF,, CF;, CH=CH,, C=CH, or C=CCH;; each R° is independently selected from H, halogen, (Ci-Cs)alkyl, (Ci-Cs)haloalkyl and (C1-

Cz)alkoxy; provided that R! is only H when L' is present or when R° and R° are taken together to form a group -O-C(R°),-0-.

P 00303-032 7 LU102907

Eternygen UK Ltd.

In one aspect, provided herein is a compound of Formula (IIa), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof:

R'—L! N O

X 7 (CH)m—R’

RA © (Ia) wherein

L' is absent, -O-, -O(CHz)-, -S-, -S(=0),-, -C(FO)NH-,- NHS(=0):-, or -S(=0),NH-;

R! is H, halogen, CN, NO», (C1-C+)alkyl, (C1-C;)haloalkyl, (C1-C;)alkoxy, (Ci-Cs)haloalkoxy, substituted or unsubstituted (Cz-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle;

R*isF, CI, CN, NO, or OCH;; or R! and R are taken together to form a group -O-C(R®),-O-; mis | or 2;

R’ is CHF; or CFs; each RP is independently selected from H, halogen, (Ci-Cs)alkyl, (Ci-Cs)haloalkyl and (C1-

Cs)alkoxy; provided that R! is only H when L' is present; and that the moiety -L'-R! is not Cl if R* is CI, m is 2 and R is CFs.

In one aspect, provided herein is a compound of Formula (IIIa), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof:

RL! N O

U M NCH2)m-R7

O

R3 (Ia) wherein

R'is H, halogen, CN, NO», (Ci-Cs)alkyl, (Ci-Cs)haloalkyl, (Ci-Cs)alkoxy, (C1-C3)haloalkoxy, substituted or unsubstituted (C»-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle;

R° is F, Cl, Br, CN, NO», CF; or OCHs; mis | or 2;

R’is CHF; or CF;; provided that R' is only H when L' is present.

P 00303-032 8 LU102907

Eternygen UK Ltd.

In a further aspect, the present invention provides a pharmaceutical composition comprising at least one inhibitor for the activity of a citrate transporter, such as /ndy, as described herein. In some embodiments, provided is a pharmaceutical composition comprising a compound of Formula (I), (ID), (Ha), (III), (Illa) or (IV), or pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound of Formula (I), (II), (Ha), (IIT), (Ia) or (IV), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof. In some embodiments, the pharmaceutical composition also contains at least one pharmaceutically acceptable inactive ingredient, such as a carrier substance, excipient and/or adjuvant. In some embodiments, the pharmaceutical composition is formulated for intravenous injection, subcutaneous injection, oral administration, or topical administration, such as formulated as an aerosol, a cream, a gel, a pill, a capsule, a syrup, a solution, a transdermal patch or a pharmaceutical delivery device. In some embodiments, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, or a lotion.

Pharmaceutical compositions described herein are administerable to a subject in a variety of ways by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), buccal, topical or transdermal administration routes.

In a further aspect; the present invention provides a pharmaceutical composition comprising at least one of the citrate transporter inhibitors described herein and at least one further active pharmaceutical ingredient. In some embodiments, the at least one further active pharmaceutical ingredient is selected from the group comprising: a. anti-obesity agents selected from the group consisting of orlistat, lorcaserin, Phentermine,

Topiramate, sibutramine, bromocriptine, ephedrine, leptin, and pseudoephedrine, 5-HT2c receptor agonists, Bupropion, Naltrexone, methionine aminopeptidase 2 inhibitors, GLP1 agonists; b. anti-diabetes agents comprising insulin, incretin mimetics, SGLT-2 inhibitors, DPPIV inhibitors, PPAR agonist, Glucokinase activator, MTP inhibitors, Glycogen phosphorylase inhibitors, DGAT-1 inhibitor, GLP1 agonists, dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor; c. anti-NASH agents comprising insulin, incretin mimetics, statins, PPAR agonists, AMPK activators, FXR agonists, DGAT-2 inhibitors, DGAT-1 inhibitors, Bile-Acid Conjugates, methionine aminopeptidase 2 inhibitors, PDE4 inhibitors, inhibitors of acetyl-CoA carboxylase, inhibitors of ketohexokinase, inhibitors of ATP citrate lyase, GLP-1 agonist, dual GLPIl/glucagon receptor agonists, triagonist for GLP1/glucose-dependent

P 00303-032 9 LU102907

Eternygen UK Ltd. insulinotropic polypeptide/glucagon receptor, inhibitors of ASK-1, CCR2/CCR5 antagonist, inhibitors of SLC10A2, inhibitors of LOXL2, inhibitors of Galectin-3, inhibitors of caspase, FGF21, FGF19, inhibitors of CGRP, AOC3: Amine Oxidase, Copper

Containing 3, inhibitors of DPP-4, THR- agonists, anti-CD3 monoclonal antibody (mAbs), A3AR agonists, inhibitors of SGLT2, inhibitors of SGLT1, inhibitors for TGFB activation, anti-cannabinoid CDI receptor antibody, antibody agonist of the P-

Klotho/FGFR 1c receptor complex, inhibitors of the inflammasome, ATP citrate lyase inhibitors, stearoyl-CoA desaturase inhibitors, fatty acid synthesis inhibitors; d. anti-dyslipidaemia agents comprising, statins, ApoB antisense oligonucleotides, PCSK9 inhibitors, Cholesterol-absorption inhibitors, Niacin, Bile-acid-sequestering resins, MTP inhibitors, Fibrates, CETP inhibitors, ATP citrate lyase inhibitors; e. anti-cancer agents comprising chemotherapeutic drugs; and f anti aging drugs comprising vitamins.

In yet a further aspect, the present invention relates to uses of compound(s) described herein, including the use as, or for the preparation of, a medicament. In one aspect, provided herein is the use of a compound of Formula (Ha) or (Illa), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, as a medicament, In some embodiments, provided herein is the use of a compound of Formula (I), (ID), (Ia), (IIT), (Illa) or (IV), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, for the preparation of a medicament for the the prevention and/or treatment of a citrate transporter meditated or citrate transporter dependent disease or condition. In some embodiments, a compound, composition or preparation described herein for use in the treatment and/or prevention of a condition associated with the activity of a citrate transporter, including metabolic diseases, such as obesity and diabetes, in particular type 2 diabetes, and age related diseases. The compound of Formula (I), (ID), (Ia), (IIT), (Ila) or (IV) binds to citrate transporters, such as as Indy, and influences their activity. The compound of Formula (I), (ID), (Ha), (IIT), (IIIa) or (IV) inhibits the activity of such a citrate transporter directly or allosterically.

Allosteric inhibition or regulation means the regulation of the activity of a protein or enzyme by binding to one or more allosteric site(s) of said protein or enzyme, which allosteric site(s) is/are different from the active site(s) of the respective protein or enzyme.

In some embodiments, provided is a method comprising administering a compound of Formula (I), (II), (Ha), (III), (IIa) or (IV), a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, or a pharmaceutical composition described herein, to a subject, preferably a human, with a diseases or condition that is citrate transporter meditated or citrate transporter dependent. The

P 00303-032 10 LU102907

Eternygen UK Ltd. compound may be a compound of Formula (I), (ID), (Ila), (ID), (Illa) or (IV), a mixture of compounds of Formula (I), (II), (Ha), (III), (IIIa) and/or (IV), or pharmaceutically acceptable salts, solvates, metabolites or prodrugs thereof. In some embodiments, the subject (e.g. human) 1s already being administered one or more additional therapeutically active agents other than a compound of

Formula (I), (I), (Ha), (IIT), (Ia) or (IV). In some embodiments, the method further comprises administering one or more additional therapeutically active agents other than a compound of

Formula (I), (II), (Ia), (III), (IIa) or (IV).

In some embodiments, the citrate transporter is the gene product of Indy or a homologue thereof.

The term “homologues” used in this disclosure refers to genes or proteins having similar or identical biological functions. The similarity or identity of the biological functions can be reflected by sequence similarity or identity (at either the amino acid or nucleotide level) of about 45%, about 70% or about 90%. Sequence similarity or identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignments using computer software programs such as BLAST, ALIGN, DNAstar and

INHERIT which employ various algorithms to measure homology. A person skilled in the art is familiar with these alignment programs. Sequence regions that are homologous may be called conserved, consensus or canonical sequences and represent the most common choice of base or amino acid at each position.

In some embodiments, the citrate transporter meditated or citrate transporter dependent disease or condition, or the condition associated with the activity of a citrate transporter, is: a. a metabolic disease selected from the group comprising insulin resistance, alcoholic and non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hereditary diseases and metabolic syndrome; b. an eating disorder; c. a chronic liver disease; d. liver cancer and/or cancer related to obesity; e an age related disease comprising atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension and neurodegenerative diseases like Alzheimer's disease; or f drug induced hepatic steatosis.

In some embodiments, described herein is a compound or a pharmaceutical composition described herein for use in a method of diagnosis of a citrate transporter meditated or citrate transporter

P 00303-032 11 LU102907

Eternygen UK Ltd. dependent disease or condition, or a condition associated with the activity of a citrate transporter, including the diseases or conditions identified above. In some embodiments, the method of diagnosis is in vivo. In some embodiments, the method of diagnosis is in vitro or ex vivo.

In any of the aspects or embodiments disclosed herein, the mammal is preferably a human. In some embodiments, compounds, compositions or preparations described herein are administered to a human.

In some cases, compounds provided herein are used to diminish, reduce, or eliminate the activity of citrate transporters. In some embodiments, described herein is a method for altering the activity of citrate transporters in vitro or in vivo, said method comprising contacting a citrate transporter with at least one compound described herein, or a salt thereof, under conditions and in an amount sufficient to detectably diminish, reduce, or eliminate the activity of the citrate transporter. In some embodiments, the citrate transporter is contained in a cell, tissue or sample (e.g., a cell sample or tissue sample). In some embodiments, compounds or compositions described herein may be used in detection assays for localizing or detecting a citrate transporter in a cell, tissue or sample, and in some embodiments, such detection assays may use a detectably labelled compound, such as a compound of general formula (I), or a salt thereof, that is linked to a detectable label or functional moiety, such as a radio nucleotide, fluorophore or enzyme.

Articles of manufacture, which include packaging material, a compound of Formula (I), (II), (Ha), (II), (IIa) or (IV), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, within the packaging material, and a label that indicates that the compound or composition, or pharmaceutically acceptable salt, tautomers, pharmaceutically active metabolite, pharmaceutically acceptable prodrug, or pharmaceutically acceptable solvate thereof, is used for reducing, diminishing or eliminating the effects of citrate transporters, or for the treatment, prevention or amelioration of one or more symptoms of a disease or condition that would benefit from a reduction or elimination of citrate transporter activity, are provided.

Still other objects, aspects, features and advantages of the compounds, methods and compositions described herein will become apparent from the following description and examples. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only.

BRIEF DESCRIPTION OF THE FIGURES

Figurel. Body weight

P 00303-032 12 LU102907

Eternygen UK Ltd.

Fig. 1 shows data for the development of body weight of the different groups of test animals (diet- induced obese (DIO) male mice) over the seven days test period. Animals were treated orally twice a day with Compound 21 at three different doses (1, 5 and 15 mg/kg) or with corresponding vehicle.

Data shows dose dependent reduction of body weight with a statistically significant reduction for highest dose compared to its respective pair-fed group.

Figure 2. Food intake

Fig. 2 shows data for the food intake of the different groups of test animals (diet-induced obese (DIO) male mice) during the test period. Food intake of animals treated with medium dose and high dose of Compound 21 was reduced during the first days. After this initial period all compound- treated groups had a slight increase in food intake, and pair-fed groups were well adjusted to compound groups.

Figure 3. Blood glucose measurement

Fig. 3 shows data for the measurement of blood glucose levels of the different groups of test animals (diet-induced obese (DIO) male mice) during the test period. Blood glucose levels were lower for all compound treated and respective pair-fed groups over the study period when compared to vehicle. Overnight fasting blood glucose determined at the end of the study were found to be significantly reduced in the high dose group compared to vehicle treated animals as well as to the respective pair-fed group.

Figure 4. Serum insulin measurement

Fig. 4 shows data for the measurement of serum insulin levels of the different groups of test animals (diet-induced obese (DIO) male mice). The high dose group showed a remarkable strong decrease in insulin levels compared to vehicle treated animals as well as to the respective pair-fed group.

Figure S. Serum beta-hydroxybutyrate (HBA) measurement

Fig. 5 shows data for the measurement of serum beta-hydroxybutyrate (HBA) levels of the different groups of test animals (diet-induced obese (DIO) male mice). The high dose group showed a significant increase compared to vehicle treated animals as well as to the respective pair-fed group.

Figure 6. Serum cholesterol measurment

Fig. 6 shows data for the measurement of serum chloestrol of the different groups of test animals (diet-induced obese (DIO) male mice). The high dose group as well as the respective pair-fed group

P 00303-032 13 LU102907

Eternygen UK Ltd. showed a significant reduction compared to vehicle treated animals indicating amelioration of high fat diet induced hypercholesterolaemia and an improved overall lipid metabolism.

Figure 7. Western blot analysis of liver tissue samples

Fig. 7 shows results of the AMPK phosphorylation status in liver tissue samples from different groups of test animals (diet-induced obese (DIO) male mice). High dose administration of

Compound 21 (Ex 21) resulted in a significant increase in the pAMP/total AMPK ratio compared to the vehicle and its respective pair-fed group. No difference could be observed between vehicle and pair-fed to high dose group. This data is indicative for the beneficial therapeutic effect of

Compound 21 in the prevention or treatment of, for example, cardiometabolic disease, including hyperglycaemia, hyperlipidaemia, non-alcoholic fatty liver disease (NAFLD), insulin resistance (type 2 diabetes), hypertension, chronic low-grade inflammation, etc., where activation of AMPK exerts positive effects.

DETAILED DESCRIPTION OF THE INVENTION

INDY and its mammalian homolog mINDY (Slc13a5, NaCT) are transporters of tricarboxylic acid (TCA) cycle intermediates. INDY is crucial for the uptake of citrate via the plasma membrane into the cytosol where citrate is used for the synthesis of fatty acids and cholesterol. Cytosolic citrate is not only known as the prime carbon source for the synthesis of fatty acids, triacylglycerols, cholesterols and low-density lipoproteins, but also leads to the activation of fatty acid synthesis and affects glycolysis and B-oxidation. Main organs for fatty acid synthesis are the liver and white adipose tissue.

Given the central role of INDY in a variety of pathways that have been found to be critical in the development or manifestation of certain conditions or diseases, compounds modulating the acitivity of INDY are useful for the prevention and/or treatment of conditions or diseases that are INDY mediated or INDY dependent. INDY mediated or INDY dependent diseases or conditions include, but are not limited to, metabolic diseases selected from the group comprising insulin resistance, alcoholic and non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hereditary diseases and metabolic syndrome; eating disorder; chronic liver disease; liver cancer and/or cancer related to obesity; age related diseases comprising atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension and neurodegenerative diseases like Alzheimer's disease; and drug induced hepatic steatosis.

P 00303-032 14 LU102907

Eternygen UK Ltd.

The compounds of Formula (I), (IT), (Ia), (III), (Ha) or (IV) described herein, or pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof, inhibit the activity of the sodium- coupled citrate transporter INDY (also known as SLC13A5 or NaCT). In experiments performed on two different cellular systems of citrate uptake, HepG2 cells endogenously expressing human

INDY (hSLC13A5) and HEK293 cells over-expressing mouse INDY, compounds described herein showed significant inhibitory activity. This effect is thus attributed to the inhibition of INDY. Also, selectivity of the inhibitory activity within the SLC family of membrane transport proteins could be demonstrated by tests with two different cellular systems of succinate uptake, namely HEK293 cells over-expressing recombinant human SLC13A3 and HEK293 cells overexpressing recombinant human SLC13A2, where no inhibitory activity of the compounds of the invention was observed.

The relevance of the inhibitory activity for utility in the treatment of diseases and/or conditions associated with or modulated by uptake of extracellular citrate was further demonstrated under physiological conditions in vivo using diet-induced obese (DIO) mice, a well established research model to study high-fat diet-induced diabetes, and fatty liver where a dose dependent reduction of critical metabolites, e.g. serum insulin and serum cholesterol, was observed. The results of these investigations are described in more detail in the section Examples below.

The compounds described herein, as defined hereinbefore or hereinafter, including pharmaceutically acceptable salts, prodrugs, and pharmaceutically acceptable solvates thereof, are useful in the treatment or prophylaxis of diseases and/or conditions that are a citrate transporter meditated or citrate transporter dependent, i.e. associated with or modulated by uptake of extracellular citrate, including, but not limited to, those indicated hereinbefore or hereinafter, e.g. metabolic and/or age related diseases.

In one aspect, provided herein is a compound of Formua (I):

RL! N O el (CHR

R? ©

R° (D) wherein

R'is H, halogen, CN, NO», (Ci-Cs)alkyl, (C1-Cs)haloalkyl, (Ci-Cs)alkoxy, (Ci-Cs)haloalkoxy, substituted or unsubstituted (C»-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle;

R?is H, F, CI, CN, NO», or OCHs;

R° is H, F, CI, Br, CN, NO», CF; or OCH;

P 00303-032 15 LU102907

Eternygen UK Ltd. or R° and R° are taken together to form a group -O-C(R°)-O-; mis | or 2;

Caz)alkoxy; provided that R! is only H when L' is present or when R° and R° are taken together to form a group -O-C(R°)-0-; or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, for use in the prevention and/or treatment of a citrate transporter meditated or citrate transporter dependent disease or condition.

Compounds are usually described herein using standard nomenclature or the definitions presented below. For compounds having asymmetric centers, it should be understood that, unless otherwise specified, all of the optical isomers and mixtures thereof are encompassed. Compounds with two or more asymmetric elements can also be present as mixtures of diastereomers. In addition, compounds with carbon-carbon double bonds may occur in Z- and E- forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. It will be apparent that the compound of the invention may, but need not, be present as a hydrate, solvate or non-covalent complex. In addition, the various crystal forms and polymorphs are within the scope of the present invention, as are prodrugs of the compound of the invention. Recited compounds are further intended to encompass compounds in which one or more atoms are replaced with an isotope, i.e., an atom having the same atomic number but a different mass number. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include !!C, PC, and *C.

Compounds according to the formulas provided herein, which have one or more stereogenic center(s), have an enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.

P 00303-032 16 LU102907

Eternygen UK Ltd.

The compounds are described herein using a general formulae that include variables such as, e.g.

RY, R°, R°, R*, R°, RÉ, R°, R* RB, and L'. Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R”, the group may be unsubstituted, or substituted with 1 or 2 group(s) R”, wherein R" at each occurrence is selected independently from the corresponding definition of R”. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds, i.e., compounds that can be isolated, characterized and tested for biological activity. For any and all of the embodiments of the compounds of Formula (I), (ID), (Ha), (III), (IIIa) or (IV) described herein, substituents are selected from among from a subset of the listed alternatives.

As used herein a wording defining the limits of a range of length such as, e. g., “from 1 to 5” means any integer from 1 to 5,1. e. 1, 2, 3, 4 and 5. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range. For example, the term "C1-C3" refers to 1 to 3, i.e. 1,2 or 3, carbon atoms; and the term "C1-C¢" refers to 1 to 6, i.e. 1, 2, 3, 4, 5 or 6, carbon atoms. Further, the prefix "(Cxy)" as used herein means that the chain, ring or combination of chain and ring structure as a whole, indicated in direct association of the prefix, may consist of a minimum of x and a maximum of y carbon atoms (i.e. x < y), wherein x and y represent integers defining the limits of the length of the chain (number of carbon atoms) and/or the size of the ring (number of carbon ring atoms). "Pharmaceutically acceptable" as used herein, refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e, the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term "salt" or "salts" as used herein refers to compounds obtained by reacting a compound of general formula (I) with an acid or a base to form a salt. A "pharmaceutically acceptable salt" of a compound disclosed herein, including all pharmaceutically acceptable solvates (including hydrates), polymorphs, and amorphous phases thereof, refers to a formulation of the compound that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication. Such pharmaceutically acceptable salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.

P 00303-032 17 LU102907

Eternygen UK Ltd.

Examples of suitable pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH,),~-COOH where n is any integer from Oto 4 (i.e., 0, 1, 2, 3, or 4) and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmacologically acceptable salts for the compounds provided herein. In general, a pharmacologically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.

A "substituent," as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a substituent on a ring may be a moiety such as a halogen atom, an alkyl, haloalkyl, hydroxy, cyano, or amino group, or any other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member.

The term "substituted," as used herein, means that any one or more hydrogen atom(s) on the designated atom or group (e.g. alkyl, alkoxy, alkoxyalkyl, cycloalkyl, heterocycloalkyl, heteroaryl) 1s replaced with a selection from the indicated substituents, provided that the designated atom's normal valence or the group's number of possible sites for substitution is not exceeded, and that the substitution results in a stable compound, i.e. a compound that can be isolated, characterized and tested for biological activity. When a substituent is oxo, i.e., =O, then 2 hydrogens on the atom are replaced. An oxo group that is a substituent of an aromatic carbon atom results in a conversion of —

CH- to -C(=O)- and may lead to a loss of aromaticity. For example, a pyridyl group substituted by oxo is a pyridone. The indication mono-, di-, tri or tetrasubstituted denotes groups having one (mono), two (di), three (tri) or four (tetra) substituents, provided that the substitution does not exceeded the number of possible sites for substitution and results in a stable compound. For example, a monosubstituted imidazolyl group may be an (imidazolidin-2-on)yl group and a disubstituted isoxazolyl group may be a ((3,5-dimethyl)isoxazolyl) group.

P 00303-032 18 LU102907

Eternygen UK Ltd.

As used herein, "comprising", "including", "containing", "characterized by", and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method steps. Yet, "Comprising", etc. is also to be interpreted as including the more restrictive terms "consisting essentially of" and "consisting of", respectively.

As used herein, "consisting of” excludes any element, step, or ingredient not specified in the claim.

When trade names are used herein, it is intended to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product.

In general, unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and are consistent with general textbooks and dictionaries. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed. In this application, the use of "or" or "and" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

The expression alkyl or alkyl group denotes a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 6 carbon atoms, or the number of carbon atoms indicated in the prefix. If an alkyl 1s substituted, the substitution may take place, independently of one another, by mono-, di-, or tri-substitution of individual carbon atoms of the molecule, e.g. 1, 2, 3, 4, 5, 6, or 7 hydrogen atom(s) may, at each occasion independently, be replaced by a selection from the indicated substituents. The foregoing also applies if the alkyl group forms a part of a group, e.g. haloalkyl, hydroxyalkyl, alkylamino, alkoxy, or alkoxyalkyl. Examples of an alkyl group include methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, 2,2- dimethylbutyl, or n-octyl, and examples of a substituted alkyl group or a group where the alkyl forms a part of a group, include haloalkyl, e.g. a trifluoromethyl or a difluoromethyl group; hydroxyalkyl, e.g. hydroxymethyl or 2-hydroxyethyl group, and a methoxymethyl group. The term "(Cis) alkyl" includes, for example, H:C-, H3C-CH»-, H;C-CH»-CH>-, H;C-CH(CH3)-, H;C-CH>-

CH»-CH»-, H3C-CH»-CH(CHs)-, H:C-CH(CH:)-CH», H3C-C(CHs)2-, H:C-CH»-CH»-CH»>-CH>-,

H3C-CH>-CH»-CH(CH:)-, H;C-CH»-CH(CH3)-CH>-, H;C-CH(CH3)-CH»-CH>-, H;C-CH->-

P 00303-032 19 LU102907

Eternygen UK Ltd.

C(CH:)>-, H:C-C(CH3)»-CH>-, H;C-CH(CH;)-CH(CH3)-, H:C-CH»-CH(CH:CH;)-, -

CH:CH:CH,CH:CH:CH;, -CH(CH+)CH,CH:CH:CH3, (H:CH,C)CH(CH:CH:CH;3)-, -

C(CHz)»(CH2CH:CHz), -CH(CH3)CH(CH:)CH:CHz, and -CH(CHz)CH2CH(CH3)>.

The expressions alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, especially from 2 to 6 (e.g. 2, 3 or 4) carbon atoms, for example an ethenyl (vinyl), propenyl (allyl), iso-propenyl, butenyl, ethinyl, propinyl, butinyl, acetylenyl, propargyl, isoprenyl or hex-2-enyl group. Preferably, alkenyl groups have one or two (especially preferably one) double bond(s), and alkynyl groups have one or two (especially preferably one) triple bond(s).

The expression alkoxy or alkoxy group refers to an alkyl group singular bonded to oxygen, i.e. —O- alkyl, where alkyl is as defined herein. The term "(C1-C6) alkoxy" includes, for example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, sec-butoxy, iso-butoxy, fert-butoxy, n-pentyloxy , fert- amyloxy- or n-hexyloxy, and accordingly (C1-C3)alkoxy includes methoxy, ethoxy, n-propoxy, or iso-propoxy.

The expression alkoxyalkyl or alkoxyalkyl group refers to an alkyl group singular bonded to one or more alkoxy group(s), e.g. —alkyl-O-alkyl or —alkyl-O-alkyl-O-alkyl. The term "(C>-Cs) alkoxyalkyl" includes, for example, methoxymethyl, methoxyethyl, methoxy-n-propyl, methoxy- iso-propyl, methoxy-n-butyl, methoxy-sec-butyl, methoxy-iso-butyl, methoxy-zerr-butyl, methoxyethoxymethyl, methoxyethoxyethyl, ethoxymethoxymethyl, ethoxymethoxyethyl, and 1- ethoxyethyl.

The expression haloalkyl or haloalkyl group refers to an alkyl group in which one, two, three or more hydrogen atoms have been replaced independently of each other by a halogen atom. The term "(C:-C3) haloalkyl" includes, for example, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, bromomethyl, dibromomethyl, iodomethyl, (1- or 2-)haloethyl (e.g. (1- or 2-)fluoroethyl or (1- or 2-)chloroethyl), (2- or 3-) halopropyl (e.g. (2- or 3-) fluoropropyl or (2- or 3-) chloropropyl).

The expression haloalkoxy refers to an alkoxy group in which one, two, three or more hydrogen atoms of the alkyl group have been replaced independently of each other by a halogen atom.

As used herein, the expression heteroalkyl or heteroalkyl group refers to an alkyl group, straight chain or branched as defined above, in which one or more, preferably 1, 2, 3 or 4, carbon atom(s) has/have been replaced, each independently of one another, by an oxygen, nitrogen, selenium,

P 00303-032 20 LU102907

Eternygen UK Ltd. silicon or sulphur atom, preferably by an oxygen, sulphur or nitrogen atom, C(O), OC(O), C(O)O,

C(O)NH, NHC(O), NH, SO, or SO», wherein said heteroalkyl group may contain a CH=CH group and/or may be substituted. Examples of heteroalkyl groups, include, for example, groups of formulae: R*-O-Y*-, R%-S-Y?, R-SO-Y*, R*SO2-Y?., R*-N(R®)-Y?, R-CO-Y*, R-O-CO-Y*,

R%CO-0-Y%, R*-CO-N(RP)-Y*-, R*-N(R”)-CO-Y?-, R*O-CO-N(R®)-Y*, R:-N(R")-CO-O-Y?-,

RLN(R®)-CO-N(R9-Y?, R&-O-CO-O-Y*-, R*N(R®)-C(=NRY)-N(R°)-Y?-, R*-CS-Y%,

R%-0-CS-Y?-, R%CS-O-Y, R*-CS-N(R®-Y?, | R&-N(RP)-CS-Y-, R*-O-CS-N(R®)-Y%,

REN(R®)-CS-0-Y%, R&-N(R®)-CS-N(R°)-Y*, R&-O-CS-O-Y*-, R*-S-CO-Y*-, R*-CO-S-Y*,

R%-S-CO-N(R)-Y%, R&-N(RE)-CO-S-Y*-, R*-S-CO-O-Y*-, R*-O-CO-S-Y*-, R-S-CO-S-Y*,

R%S-CS-Y*, R*CS-S-Y%, | R“-S-CS-N(R®)-Y*, R*N(R)-CS-S-Y?-, | R*-S-CS-O-Y*,

R*-O-CS-S-Y"-, wherein R? being a hydrogen atom, a Ci-Cs alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group; R° being a hydrogen atom, a C1-Cs alkyl, a C»-Cs alkenyl or a C2-Cs alkynyl group;

RC being a hydrogen atom, a Ci-Cs alkyl, a C2-C6 alkenyl or a Cz-Cç alkynyl group; R? being a hydrogen atom, a C1-Cs alkyl, a C».Cs alkenyl or a C2-Ce alkynyl group and Y* being a direct bond, a CiCs alkylene, a C2-Cs alkenylene or a C»-Cs alkynylene group, wherein each heteroalkyl group contains at least one carbon atom and at least one heteroatom as defined hereinbefore. The term "(C1-C4)heteroalkyl group" refers, for example, to a group containing from 1 to 4, e.g 1, 2, 3 or 4, carbon atoms and 1, 2, 3 or 4, preferably 1, 2 or 3, heteroatoms selected from oxygen, nitrogen and sulphur (especially oxygen and nitrogen). Examples of a heteroalkyl group include alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide, alkoxycarbonyloxy, alkylcarbamoyl, alkylamido, alkylcarbamoylalkyl, alkylamidoalkyl, alkylcarbamoyloxyalkyl, alkylureidoalkyl, alkoxy, alkoxyalkyl, or alkylthio group. The expression alkylthio or alkylthio group refers to an alkyl group, in which one or more non-adjacent CH» group(s) are replaced by sulphur, wherein the alkyl moiety of the alkylthio group may be substituted. Specific examples of a heteroalkyl group include acyl, methoxy, trifluoromethoxy, ethoxy, n-propyloxy, iso-propyloxy, fert-butyloxy, methoxymethyl, ethoxymethyl, methoxyethyl, methylamino, ethylamino, dimethylamino, diethylamino, iso- propylethylamino, methylaminomethyl, ethylaminomethyl, diisopropylaminoethyl, dimethylaminomethyl, dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, isobutyrylamino-methyl, N-ethyl-N-methylcarbamoyl, N- methylcarbamoyl, cyano, nitrile, isonitrile, thiocyanate, isocyanate, isothiocyanate and alkylnitnile.

The expression cycloalkyl or cycloalkyl group refers to a saturated carbocyclic ring group comprising one or more rings (preferably 1 or 2) and containing from 3 to 14 ring carbon atoms, preferably from 3 to 10 (more preferably 3, 4, 5, 6 or 7) ring carbon atoms; the cycloalkyl group may be substituted and can be bonded as a substituent via every suitable position of the ring system.

Cycloalkyls may be fused with an aromatic ring, and the point of attachment is at a carbon that is

P 00303-032 21 LU102907

Eternygen UK Ltd. not an aromatic ring carbon atom. Examples of cycloalkyl include monocyclic hydrocarbon rings, bicyclic hydrocarbon rings and spiro-hydrocarbon rings. In a bicyclic cycloalkyl group, two rings are joined together so that they have at least two carbon atoms in common. In a spiro-hydrocarbon ring, 2 or 3 rings are linked together by one common carbon atom (spiro-atom). If a cycloalkyl is substituted, the substitution may take place, independently of one another, by mono- or di- substitution of individual ring carbon atoms of the molecule, and the cycloalkyl group as a whole may carry 1, 2, 3, or 4 substituents from the indicated selection of substituents, i.e. 1, 2, 3, or 4 hydrogen atom(s) of the carbon ring atoms may, at each occasion independently, be replaced by a substituent selected from the indicated list of substituents thereby resulting in a mono-, di-, tri-, or tetrasubstituted cycloalkyl group. If a cycloalkyl is partially unsaturated, then the carbocyclic ring contains one or more double-bonded ring-carbon atoms (e.g. one or more groups -CH=CH-), which may be unsubstituted or substituted as mentioned hereinbefore. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.0]hexyl, bicyclo[3.2.0]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[4.3.0]nonyl (octahydroindenyl), bicyclo[4.4.0]decyl (decahydronaphthyl), bicyclo[2.2.1]heptyl (norbornyl), bicyclo[4.1.0]Jheptyl (norcaranyl), bicyclo[3.1.1]heptyl (pinanyl), spiro[2.5]octyl, and spiro[3.3]heptyl. If a cycloalkyl is partially unsaturated, the group contains one, two or more double bonds, such as, for example, a cycloalkenyl group, including cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclobutadienyl, cyclopentadienyl, cyclohexadienyl, bicyclo[2.2.1 Jheptadienyl, and spiro[4,5]decenyl.

The expression heterocycloalkyl or heterocycloalkyl group refers to a cycloalkyl group, saturated or partially unsaturated, as defined above, in which one or more, preferably 1, 2 or 3, ring carbon atom(s) has/have been replaced each independently of one another by an oxygen, nitrogen or sulphur atom, preferably oxygen or nitrogen, or by NO, SO or SO», with the proviso that any ring does not contain two adjacent O or S atoms, or NO, SO or SO; moieties; the heterocycloalkyl may be substituted and can be bonded as a substituent via every suitable position of the ring system; at least one carbon atom must be present between two oxygen atoms and between two sulphur atoms or between an oxygen and a sulphur atom; and the ring as a whole must have chemical stability. A heterocycloalkyl group has preferably 1 or 2 ring(s) containing from 3 to 10 (more preferably 3, 4, 5, 6 or 7, and most preferably 5, 6 or 7) ring atoms. Examples of heterocycloalkyl include aziridinyl, oxiranyl, thiiranyl, oxaziridinyl, dioxiranyl, azetidinyl, oxetanyl, thietanyl, diazetidinyl, dioxetanyl, dithietanyl, pyrrolidinyl, tetrahydrofuranyl, thiolanyl, azolyl, thiazolyl, isothiazolyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, piperazinyl, morpholinyl, thiomorpholinyl, trioxanyl, azepanyl, oxepanyl, thiepanyl, homopiperazinyl, urotropinyl, oxazolidinonyl, dihydropyrazolyl, dihydropyrrolyl, dihydropyrazinyl, dihydropyridyl, dihydropyrimidinyl, dihydrofuryl, dihydropyranyl, and

P 00303-032 22 LU102907

Eternygen UK Ltd. examples of substituted heterocycloalkyl include lactam, lactone, and cyclic carbamate, cyclic carbamide as well as cyclic imide ring systems.

The expressions aryl, Ar or aryl group refer to an aromatic group that contains one or more aromatic rings containing from 6 to 14 ring carbon atoms (C6-C14), preferably from 6 to 10 (Cs-Cio), more preferably 6 ring carbon atoms; the aryl may be substituted and can be bonded as a substituent via every suitable position of the ring system. Examples of aryl include phenyl, naphthyl, bi-phenyl, indanyl, indenyl, anthracenyl, phenanthrenyl, tetrahydronaphthyl and fluorenyl.

The expression heteroaryl or heteroaryl group refers to an aromatic group that contains one or more aromatic rings containing from 5 to 14 ring atoms, preferably from 5 to 10 (more preferably 5 or 6) ring atoms, and contains one or more (preferably 1, 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N), with the proviso that any ring does not contain two adjacent O,

P or S atoms; the heteroaryl may be substituted and can be bonded as a substituent via every suitable position of the ring system. Examples of an unsubstituted heteroaryl group include 2-pyridyl, 2- imidazolyl, 3-phenylpyrrolyl, thiazolyl, oxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, pyridazinyl, quinolinyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3"- bifuryl, 3-pyrazolyl and isoquinolinyl.

The expression heterocycle denotes ring systems, which include the above defined heterocycloalkyl and heteroaryl ring systems, e.g. a partially unsaturated heterocycle is synonymous with a partially unsaturated heterocycloalkyl and an aromatic heterocycle, e.g. a 6-membered heteroaromatic group, is synonymous with a heteroaryl. The heterocycle may be substituted and can be bonded as a substituent via every suitable position of the ring system. Examples of a partially unsaturated or aromatic heterocycle include oxetenyl, thietenyl, azetinyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl, 2,5-dihydrothiophenyl, 2,5-dihydro-1H-pyrrolyl, furanyl, thiophenyl, pyrrolyl, benzo[b]furanyl, benzo[b]thiophenyl, indolyl, benzo[c]pyrrolyl, benzo[a]pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, dihydropyridinyl, oxazinyl, pyridinyl, dihydropyranyl, azepinyl, tetrahydropyranyl, dihydrothiopyranyl, quinolinyl, 1soquinolinyl, quinazolinyl, pyridazinyl, pyrimidinyl, pyrazinyl, purinyl, and pteridinyl.

The term "optionally substituted" or "substituted" means that the referenced group may be substituted with one or more additional group(s) individually and independently selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxy, alkoxy, aryloxy, cyano, halo, nitro, haloalkyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. By way of example optional substituents are independently selected from halide, -CN, -NO,, and -LR, wherein each L is independently selected from a bond, -O-, -C(=0)-,

P 00303-032 23 LU102907

Eternygen UK Ltd. -C(=0)0-, -S-, -S(FO)-, -S(=0),-, -NH-, -NHC(=0)-, -C(FO)NH-, S(=0),NH-, -NHS(=0),, -

OC(FO)NH-, -NHC(=0)O-, or -(C,-C,alkylene)-; and each R 1s selected from H, alkyl, haloalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl. In some embodiments, preferred substituents include halogen atoms (e.g. F, Cl, Br) and groups of formula -OH, -O-C1 alkyl (e.g. -

OMe, -OEt, -O-nPr, -O-iPr, -O-nBu, -O-iBu and -O-/Bu), -NH>, -NHC 1 alkyl, -N(C1-6 alkyl), -

COOH, -COOMe, -COMe, -NHSO»Me, -SO2NMe», -SO+H, -SO,NH;, -CONH;, -CH>NH>, -CN, -

C1 alkyl (e.g. -Me, -Et, -nPr, -iPr, -nBu, -iBu, -/Bu and -CF3), -SH, —S-CO-C14 alkyl, -S-C14 alkyl, -NHAc, -NO», -C=CH, -NHCONH;, -SO2Me and cyclopropyl. In some embodiments, a substituent independently selected from halogen (especially F and CI), -Me, -CF3, -OMe, -OH, -COOH, -

CONH,, -COOMEe, -COMe and -NO; may be particularly preferred.

Examples of most preferred compounds are compounds disclosed in the examples, or pharmaceutically acceptable salts, prodrugs, hydrates or solvates thereof.

Ttis further preferred to combine embodiments described herein, especially preferred embodiments described herein, in any desired manner (e.g., any embodiment for R' may be combined with any embodiment of R?).

The term "aromatic" refers to a planar ring having a delocalized x-electron system containing 4n+27 electrons, where n is an integer. Aromatic rings can be formed from five, six, seven, eight, nine, ten, or more than ten atoms. Aromatics are optionally substituted. The term "aromatic" includes both carbocyclic aryl ("aryl", eg, phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (Le., rings which share adjacent pairs of carbon atoms) groups.

The expression "alkylene" (or alkanediyl functional group) refers to an unsubstituted, saturated, straight chain hydrocarbon group that contains the indicated number of carbon atoms (in the form of methylene (CHz) groups) and has the free valencies at the terminal methylene groups, for example a butylene —(CH»)4-, n-pentylene —(CH»)s-, n-hexylene —CH»)s-, or n-octylene (CHa )s- group.

The expression "alkenylene" refers to an at least partially unsaturated alkanediyl functional group as defined above that contains one or more double bond(s) (i.e. the methylene groups of the alkanediyl functional group are interrupted by -CH=CH- and/or terminated by -CH>—CH=).

The term "bond" or "single bond" refers to a chemical bond between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure. In one aspect,

P 00303-032 24 LU102907

Eternygen UK Ltd. when a group described herein is a bond, the referenced group is absent thereby allowing a bond to be formed between the remaining identified groups.

The term "moiety" refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.

The expression halo, halogen or halogen atom as used herein means fluorine, chlorine, bromine, or iodine.

The expression heteroatom as used herein, preferably denotes an oxygen, nitrogen or sulphur atom, more preferably a nitrogen or oxygen atom unless specified otherwise.

In preferred embodiments, the compound of Formula (I) as defined above, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, can include one or more of the following:

In some embodiments, R° is F or Cl, and Ris H.

In some embodiments, R° is H.

In some embodiments, L' is absent, -O-, -O(CH»)- or -S(=0)-.

In preferred embodiments, R* is CHF», CF; or C=CH.

In preferred embodiments, m is 2.

In preferred embodiments, the compound of Formula (I) may be a compound of Formula (IT), (III) or (IV), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof’ 1 1 H 1 1 H

R—L NO 6 R—L NO

IN I CHmR 1 NCH2)m-R®

O

R? ©

R3 (I) (II)

H

RAT

R (IV)

P 00303-032 25 LU102907

Eternygen UK Ltd. wherein m, L!, R!, R°, R°, R* and R° are defined as hereinbefore or below, and R® is CHF», CF; or

C=CH.

In preferred embodiments, the moiety -L!-R! is selected from F, CI, Br, OH, OCH;, OCH,CHs,

OCHF,, OCF;, O(CH2),OCHjs, CN, NO», CH3, CHF», CFs, -C(FO)NHz, -NHS(=0),CH3, SCHz,

NT SGA NV O- oJ Ao iy oh -S(=0):CH3, -S(=0)2NH,, and .

In preferred embodiments, the compound of Formula (I) includes:

But-3-yn-1-yl N-(3-chlorophenyl)carbamate;

But-3-yn-1-yl N-(4-chloro-3-methoxyphenyl)carbamate;

But-3-yn-1-yl N-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)carbamate;

But-3-yn-1-yl (4-chloro-3-(trifluoromethyl)phenyl carbamate;

But-3-yn-1-yl (3,4-dichlorophenyl)carbamate;

But-3-yn-1-yl (3,5-dichlorophenyl)carbamate;

But-3-yn-1-yl (4-chloro-3-fluorophenyl)carbamate

But-3-yn-1-yl N-(4-fluoro-3-methoxyphenyl)carbamate;

But-3-yn-1-yl N-(3,5-dimethoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-methylphenyl)carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3-cyanophenyl)carbamate; 3,3,3-Trifluoropropyl N-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3,5-dichlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-fluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3-methoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-methoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(difluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-cyanophenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-4-cyanophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Tnfluoropropyl N-(4-chloro-3-nitrophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3-cyano-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3-cyano-5-fluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-nitro-3-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[3-nitro-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Tnfluoropropyl N-(3-bromo-5-chlorophenyl)carbamate;

P 00303-032 26 LU102907

Eternygen UK Ltd. 3,3,3-Trifluoropropyl N-(3-chlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3-ethoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(trifluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-3-(trifluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3,5-difluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3,5-bis(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3,4-dichlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-4-fluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-5-(morpholin-4-yl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3-carbamoyl-4-chlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(cyclopropylmethoxy) phenyl]carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(2-methoxyethoxy)phenyl] carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(pyridin-4-ylmethoxy)phenyl] carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(pyridin-4-yloxy)phenyl] carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-3-(cyclopropylmethoxy) phenyl]carbamate 3,3,3-Trifluoropropyl N-(5-chloro-3-hydroxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-3-(difluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(difluoromethyl)phenyl]carbamate; 3,3,3-Trifloropropyl N-(5-chloro-3-methanesulfonylphenyl) carbamate; 3,3,3-Trifluoropropyl N-(5-chloro-3-methanesulfonamidophenyl) carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3-sulfamoylphenyl)carbamate; 3,3,3-Trifluoroprpyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate; 3,3,3-Trifluoropropyl (3-methanesufonyl-5-nitrophenyl)carbamate; 3,3,3-Trifluoropropyl (3-chloro-5-nitrophenyl)carbamate; 3,3,3-Trifluoropropyl (5-chloro-3-(methylsulfanyl)phenyl)carbamate; 2,2,2-Trifluoroethyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate; 2,2,2-Trifluoroethyl N-[4-chloro-3-(trifluoromethyl)phenyl]carbamate; 2,2-Difluoroethyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate; 2,2-Difluoroethyl (3,5-dichlorophenyl)carbamate; 2,2-Difluoroethyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate; 2,2-Difluoroethyl (3,4-dichlorophenyl)carbamate;

Prop-2-yn-1-yl (3,4-dichlorophenyl)carbamate;

Prop-2-yn-1-yl (4-chloro-3-(trifluoromethyl)phenyl)carbamate;

Prop-2-yn-1-yl (3,5-dichlorophenyl)carbamate;

But-2-yn-1-yl (3,5-dichlorophenyl)carbamate;

Allyl (3,5-dichlorophenyl)carbamate;

Allyl (3,4-dichlorophenyl)carbamate;

P 00303-032 27 LU102907

Eternygen UK Ltd.

Allyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate; 2,2,2-Trifluoroethyl (3,5-dichlorophenyl)carbamate; 2,2,2-Trifluoroethyl (3,4-dichlorophenyl)carbamate;

Propyl (3,5-dichlorophenyl)carbamate; 3,3-Difluoropropyl (3,5-dichlorophenyl)carbamate; 3,3-Difluoropropyl (3-chloro-5-(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (3-nitro-5-(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (3,5-bis(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (3-chloro-5-nitrophenyl)carbamate; 3,3-Difluoropropyl (3-bromo-5-chlorophenyl)carbamate; 3,3-Difluororopyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate; 3,3-Difluoroprpyl N-(3-methanesulfonyl-5-nitrophenyl)carbamate; 3,3-Difluoropropyl (3-(methylsulfanyl)-5-chlorophenyl)carbamate; or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof.

In a preferred embodiment, the citrate transporter is the gene product of Indy or a homologue thereof.

According to the invention, the citrate transporter meditated or citrate transporter dependent disease or condition, includes a disease or condition selected from: (a) metabolic diseases selected from the group comprising insulin resistance, alcoholic and non- alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hereditary diseases and metabolic syndrome; (b) eating disorders; (c) chronic liver diseases; (d) liver cancer and cancer related to obesity; (e) age related diseases comprising atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension and neurodegenerative diseases like

Alzheimer's disease; and (f) drug induced hepatic steatosis.

In one aspect, provided herein is compound of Formula (Ila), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof:

P 00303-032 28 LU102907

Eternygen UK Ltd.

R'—L! N O

X 7 (CH)m—R’

RA © (Ia) wherein

R! is H, halogen, CN, NO», (C1-C+)alkyl, (Ci-Cs)haloalkyl, (C1-C;)alkoxy, (Ci-Cs)haloalkoxy, substituted or unsubstituted (C»-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cz-Ce)cycloalkyl, or substituted or unsubstituted Csheterocycle;

R* is F, CI, CN, NO», or OCH; or R! and R are taken together to form a group -O-C(R®),-O-; mis | or 2;

Ris CHF; or CFs; each RP is independently selected from H, halogen, (Ci-Cs)alkyl, (Ci-Cs)haloalkyl and (C1-

In preferred embodiments of Formula (Ha), R* is F, Cl or NO»; and m is 2.

In preferred embodiments of Formula (Ia), the moiety -L'-R! is selected from F, CI, OCH;, OCHF>,

OCF3, CN, NO», CHF», and CFs.

In preferred embodiments, the compound of Formula (Ha) includes: 3,3,3-Trifluoropropyl N-(4-chloro-3-methoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-4-fluorophenyl)carbamate; or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof.

In one aspect, provided herein is compound of Formula (IHa), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof: 4

Te

O

R3 (Ia) wherein

L'is absent, -O-, -O(CH)-, -S-, -S(FO)»-, -C(FO)NH-,- NHS(=0);-, or -S(=0),NH-;

P 00303-032 29 LU102907

Eternygen UK Ltd.

R! is H, halogen, CN, NO», (C1-C+)alkyl, (Ci-Cs)haloalkyl, (C1-C;)alkoxy, (Ci-Cs)haloalkoxy, substituted or unsubstituted (Cz-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle;

R° is F, Cl, Br, CN, NO», CF; or OCHj3; mislor2;

R’ is CHF; or CFs; provided that R! is only H when L' is present.

In preferred embodiments of Formula (IIIa), R° is Cl, NO» or CF; and m is 2.

In preferred embodiments of Formula (IIIa), the moiety -L'-R! is selected from F, CI, Br, OH,

OCHs, OCH:CH;, OCHF,, OCF;, O(CH2)OCHs, CN, NO», CH;, CHF», CF;, -C(=0O)NH,, -

NHS(#0)2CHz, SCHz, (NT SGA NV O- oJ Ao iy oh -S(=0),CH3, -S(FO)aNH>, and

In preferred embodiments, the compound of Formula (Ia), includes: 3,3,3-Trifluoropropyl N-(3,5-dichlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[3-nitro-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[3,5-bis(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl (3-chloro-5-nitrophenyl)carbamate; 3,3-Difluororopyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate; or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof.

In general, all suitable combinations of preferred embodiments of the compounds according to

Formula (I), (II), (Ila), (III), (Ila) or (IV), or pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof, which result in a stable and active compound, are encompassed and disclosed herein, and may be part of the invention disclosed herein. Compounds of Formula (I), (ID), (Ha), (III), (Illa) or (IV), or pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof, described herein can be prepared using techniques and materials described herein or otherwise known, including those found in March, ADVANCED ORGANIC CHEMISTRY 4th

Ed., (Wiley 1992 ); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4th Ed, Vols.

A and B (Plenum 2000, 2001 ), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC

SYNTHESIS 3rd Ed., (Wiley 1999 ), or by using standard synthetic techniques known in the art in combination with methods described herein. In addition, solvents, temperatures and other reaction

P 00303-032 30 LU102907

Eternygen UK Ltd. conditions presented herein may vary. In general, the starting materials used for the synthesis of the compounds disclosed herein are either synthesized using standard synthetic techniques or obtained from commercial sources, such as, but not limited to, Sigma-Aldrich, Fluka, and the like. General methods for the preparation of compounds are described in the examples, which can be modified by the use of appropriate reagents and conditions for the introduction of the various moieties found in the formulae as provided herein, and groups and substituents thereof are chosen by one skilled in the art to provide stable moieties and compounds.

The compounds provided herein exhibit high inhibitory activity against uptake of extracellular citrate, e. g. an inhibition constant ICso (half-maximal inhibitory concentration) for inhibition of a citrate transporter, such as INDY, of, for example, 10 uM or less in an assay mentioned below. As such the compounds described herein can exhibit a high inhibitory activity on human citrate transporter, such as INDY, but also on homolgues and citrate transporters of species other than human, e.g. rat, mouse, gerbil, guinea pig, rabbit, dog, cat, pig, or cynomolgus monkey.

The activity and more specifically the bioactivity of the compounds according to the present invention can be assessed using appropriate assays known to those skilled in the art, e.g. in vitro or in vivo assays. For instance, the inhibitory effect (expressed as ICso value) of a compound of the invention on the citrate transporter activity may be determined via a citrate uptake assay described in more detail in the Examples section below.

The therapeutic use of a compound of Formula (I), (II), (Ha), (III), (Illa) or (IV), or pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof, and also of a formulation or a pharmaceutical composition containing the same are within the scope of the present invention. The present invention also relates to the use of a compound of Formula (I), (IT), (Ila), (III), (Ia) or (IV), or pharmaceutically acceptable salts, solvates, metabolites and prodrugs thereof, as active ingredient in the preparation or manufacture of a medicament. In some ebodiments, the compound of Formula (Ha) or (Illa) according to the invention is used as a medicament.

A pharmaceutical composition provided herein comprises at least one compound of Formula (I), (ID), (Ha), (III), (Ia) or (IV), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, and a pharmaceutically acceptable inactive ingredient. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable inactive ingredient and one or more compound(s) of Formula (IIa) or (Illa) according to the invention, or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof. The a pharmaceutically acceptable inactive ingredient includes at least one, i.e. one or more, carrier substance, excipient and/or adjuvant. In some embodiments, the pharmaceutical composition includes other medicinal or pharmaceutical

P 00303-032 31 LU102907

Eternygen UK Ltd. agents, carriers, adjuvants, preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. In other embodiments, the pharmaceutical composition includes other therapeutically valuable substances, and, optionally, at least one carrier substance, excipient and/or adjuvant.

The pharmaceutical composition may additionally comprise, for example, one or more of water, buffers (e.g, neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g, glucose, mannose, sucrose or dextrans), mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives.

In some embodiments, the compounds described herein are provided as pharmaceutical composition in which a compound of Formula (I), (II), (Ila), (IIT), (Illa) or (IV), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, is mixed with at least one other active ingredient (i.e. one or more active ingredient(s)). For instance, one or more compound(s) described herein may advantageously be contained in a pharmaceutical composition that contains at least one further active pharmaceutical ingredient. The further or supplemental active agent or active pharmaceutical ingredient is preferably an active agent or active pharmaceutical ingredient which has utility in the prevention or treatment of one or more condition(s) associated with or modulated by uptake of extracellular citrate, including those mentioned hereinbefore, hereinafter or in the claims. Examples of further active pharmaceutical ingredients, include one or more of the following from (a) to (f): (a) anti-obesity agents selected from the group consisting of orlistat, lorcaserin, Phentermine, Topiramate, sibutramine, bromocriptine, ephedrine, leptin, and pseudoephedrine, 5-HT2c receptor agonists, Bupropion,

Naltrexone, methionine aminopeptidase 2 inhibitors, GLP1 agonists; (b) anti-diabetes agents comprising insulin, incretin mimetics, SGLT-2 inhibitors, DPPIV inhibitors, PPAR agonist,

Glucokinase activator, MTP inhibitors, Glycogen phosphorylase inhibitors, DGAT-1 inhibitor,

GLP1 agonists, dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor; (c) anti-NASH agents comprising insulin, incretin mimetics, statins, PPAR agonists, AMPK activators, FXR agonists, DGAT-2 inhibitors, DGAT-1 inhibitors, Bile-Acid Conjugates, methionine aminopeptidase 2 inhibitors, PDE4 inhibitors, inhibitors of acetyl-CoA carboxylase, inhibitors of ketohexokinase, inhibitors of ATP citrate lyase,

GLP-1 agonist, dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor, inhibitors of ASK-1, CCR2/CCRS antagonist, inhibitors of SLC10A2, inhibitors of LOXL2, inhibitors of Galectin-3, inhibitors of caspase,

FGF21, FGF19, inhibitors of CGRP, AOC3: Amine Oxidase, Copper Containing 3, inhibitors of

DPP-4, THR-B agonists, anti-CD3 monoclonal antibody (mAbs), A3AR agonists, inhibitors of

P 00303-032 32 LU102907

Eternygen UK Ltd.

SGLT2, inhibitors of SGLT1, inhibitors for TGFB activation, anti-cannabinoid CDI receptor antibody, antibody agonist of the P-Klotho/FGFRIc receptor complex, inhibitors of the inflammasome, ATP citrate lyase inhibitors, stearoyl-CoA desaturase inhibitors, fatty acid synthesis inhibitors; (d) anti-dyslipidaemia agents comprising, statins, ApoB antisense oligonucleotides,

PCSKO inhibitors, Cholesterol-absorption inhibitors, Niacin, Bile-acid-sequestering resins, MTP inhibitors, Fibrates, CETP inhibitors ATP citrate lyase inhibitors; (e) anti-cancer agents comprising chemotherapeutic drugs; and (f) anti aging drugs comprising vitamins. In some embodiments, a compound Formula (Ila) or (Illa) according to the invention, or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, is combined with one or more of the further active pharmaceutical ingredients disclosed herein in a pharmaceutical composition. In some embodiments, a pharmaceutical composition comprising a compound of Formula (IIa) or (Illa), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, and one or more of the further active pharmaceutical ingredients disclosed herein, is used as a medicament.

The pharmaceutical composition provided herein can be used as a medicament, e.g., in the prophylaxis and/or treatment of a disease or condition that is citrate transporter meditated or citrate transporter dependent, or that is associated with the activity of a citrate transporter. For this use, the pharmaceutical composition provided herein may be formulated for any appropriate manner of administration, including, for example, topical (e.g, transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use are preferred. Such forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilizate. Formulation for topical administration may be preferred for certain conditions (e.g, in the treatment of skin conditions such as burns or itch). Briefly summarized, the pharmaceutical compositions described herein include compositions formulated as an aerosol, a cream, a gel, a pill, a capsule, a syrup, a solution, a transdermal patch or a pharmaceutical delivery device.

In one embodiment, the compounds of Formula (I), (II), (Ila), (III), (Illa) or (IV), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, are used in the preparation of medicaments for the treatment of diseases or conditions in a mammal that would benefit from a reduction of citrate transporter activity. Methods for treating any of the diseases or conditions described herein in a mammal in need of such treatment, involves administration of pharmaceutical

P 00303-032 33 LU102907

Eternygen UK Ltd. compositions that include at least one compound of Formula (I), (II), (Ha), (III), (Ia) or (IV), or a pharmaceutically acceptable salt, solvate, metabolite or prodrug thereof, in therapeutically effective amounts to said subject. As used herein, the term "treatment" encompasses both disease- modifying treatment and symptomatic treatment, either of which may be prophylactic (i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms) or therapeutic (i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms). A condition is "associated with or modulated by uptake of extracellular citrate" if modulation of citrate transporter (e.g. INDY) activity results in alleviation of the condition or a symptom thereof. Patients may include but are not limited to primates (especially humans), domesticated companion animals (such as dogs, cats, horses) and livestock (such as cattle, pigs, sheep), with dosages as described herein.

In certain embodiments, the compositions containing the compound(s) described herein are administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, the compositions are administered to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest at least one of the symptoms of the disease or condition. Amounts effective for this use depend on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation clinical trial.

In prophylactic applications, compositions containing the compounds described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a "prophylactically effective amount or dose." In this use, the precise amounts also depend on the patient's state of health, weight, and the like. When used in a patient, effective amounts for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. In one aspect, prophylactic treatments include administering to a mammal, who previously experienced at least one symptom of the disease being treated and is currently in remission, a pharmaceutical composition comprising a compound of general formula (D) in order to prevent a return of the symptoms of the disease or condition.

For the prophylaxis and/or treatment of a disease or condition that is citrate transporter meditated or citrate transporter dependent, or that is associated with the activity of a citrate transporter, the dose of drug being administered may vary within wide limits and may be adjusted to individual requirements. Active compounds described herein are generally administered in a therapeutically effective amount. Preferred doses range from about 0.1 mg to about 140 mg per kilogram of body

P 00303-032 34 LU102907

Eternygen UK Ltd. weight per day (about 0.5 mg to about 7 g per patient per day). The daily dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the patient) and the severity of the particular disease undergoing therapy.

The phrase "therapeutically effective amount" means an amount of a compound described herein that (1) treats the particular disease, condition, or disorder, (11) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (111) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In certain embodiments a therapeutically effective amount may achieve one or more of lowering blood glucose level, decreasing insulin resistance and increasing insulin sensitivity, lowering hepatic lipids, lowering hepatic triglycerides, lowering hepatic diacylglycerol, lowering blood cholesterol, lowering blood triglycerides, lowering blood LDL, lowering muscle diacylglycerols.

Examples of a disease or condition that is citrate transporter meditated or citrate transporter dependent, or that is associated with the activity of a citrate transporter, include: (a) metabolic diseases selected from the group comprising insulin resistance, alcoholic and non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hereditary diseases and metabolic syndrome; (b) eating disorders; (c) chronic liver diseases; (d) liver cancer and cancer related to obesity; (e) age related diseases comprising atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension and neurodegenerative diseases like Alzheimer's disease; and (f) drug induced hepatic steatosis.

Diabetes mellitus is a metabolic disease that causes high blood sugar, and it generally refers to fasting plasma glucose values of >126 mg/dL (> 7.0 mmol/l) and insulin resistance is defined here as a fasting blood insulin level greater than 20 mcU/mL. Untreated high blood sugar from diabetes can damage your nerves, eyes, kidneys, and other organs, and types of diabetes include type 1 diabetes, type 2 diabetes, prediabetes and gestational diabetes. Adiposity and obesity both refer to amedical condition in which excess body fat has accumulated to an extent where it may increases the likelihood of various diseases, particularly heart disease, type 2 diabetes, obstructive sleep apnoea, certain types of cancer, and osteoarthritis. Generally adiposity and obesity are related to a

P 00303-032 35 LU102907

Eternygen UK Ltd.

Body Mass Index (kg/m?*) above 25. NAFLD refers to a wide spectrum of liver clinicopathologic conditions, ranging from pure fatty steatosis (fatty infiltration in >5% of hepatocytes) to non- alcoholic steatohepatitis (NASH), which may progress to cirrhosis, liver failure, and hepatocellular carcinoma and 1s characterized by excessive fat accumulation in the liver parenchyma of patients who have no history of alcohol abuse. NASH refers to a medical condition with presence of specific histological abnormalities on liver biopsy such as a characteristic pattern of steatosis, inflammation and hepatocellular ballooning in the absence of significant alcohol consumption.

Age-related diseases occur with increasing frequency with increasing senescence such as atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension and neurodegenerative diseases like Alzheimer's disease. The incidence of all of these diseases increases rapidly with aging. One aspect of age related diseases concerns pre-diabetes, a condition that raises a person's risk for developing type 2 diabetes, heart disease, and stroke. Within the context of the present invention such diseases shall be understood as age related diseases.

Potential cancers to be treated with a compound of general formula (I) comprise liver, pancreas cancer, breast cancer, oesophagus cancer, pancreas cancer, colon cancer, gallbladder cancer, colorectal cancer, endometrium cancer, kidney cancer, gallbladder cancer, thyroid cancer, rectal cancer, melanoma, leukaemia, multiple myeloma, non-Hodgkin lymphoma, prostate cancer, uterine cancer, ovarian cancer, endometrial cancer and cervical cancer.

In one aspect, the methods and compositions described herein include the use of compounds of

Formula (I), (ID), (Ia), (III), (Ia) or (IV) in form of pharmaceutically acceptable salts, prodrugs, enantiomers, diastereomers, racemic mixtures, crystalline forms, non-crystalline forms, amorphous forms, unsolvated forms and solvates. In some embodiments, pharmaceutically acceptable salts of the compound of Formula (I), (II), (Ha), (III), (IIIa) or (IV) are prepared with relatively nontoxic (Le. pharmaceutically acceptable) acids or bases, depending on the particular substituents found on the compounds of the present invention. If, for example, compounds of the present invention contain acidic functionalities, base addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.

Non-limiting examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. If compounds of the present invention contain basic functionalities, acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, phosphoric, partially neutralized phosphoric acids, sulfuric, partially neutralized sulfuric,

P 00303-032 36 LU102907

Eternygen UK Ltd. hydroiodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic. malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, methanesulfonic, and the like.

Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like. Certain specific compounds of the present invention may contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Contacting the salt with a base may regenerate the neutral forms of the compounds of the present invention or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

In some embodiments, compounds described herein are provided as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug under physiological conditions. Additionally, prodrugs can also be converted to the parent drug by chemical or biochemical methods in an ex- vivo environment. For example, prodrugs can be slowly converted to the parent drug when, for example, placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.

The compound of Formula (I), (I), (Ia), (III), (Ila) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, described herein can be administered to the subject at a suitable dose. The compound of Formula (I), (IT), (Ia), (IIT), (IIIa) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, is preferably administered to mammals such as domestic and pet animals. Non-limiting examples of domestic and pet animals are pigs, cows, buffalos, sheep, goats, rabbits, horses, donkeys, chickens, ducks, cats, dogs, genuine pigs, or hamsters. Most preferred it is administered to humans. The preferred way of administration depends on the form of the compound of Formula (I), (ID), (Ila), (IIT), (Illa) or (IV). As described herein above, the compound having the Formula (I), (I), (Ha), (III), (Ila) or (IV) can be in the form of pharmaceutically acceptable salts, prodrugs, enantiomers, diastereomers, racemic mixtures, crystalline forms, non-crystalline forms, amorphous forms, unsolvated forms or solvates. The compound of the invention may be administered orally, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, intraocular, transdermally, transmucosally, subdurally, locally or topically via iontopheresis, sublingually, by inhalation spray, aerosol or rectally and the like in dosage unit formulations optionally further comprising

P 00303-032 37 LU102907

Eternygen UK Ltd. conventional pharmaceutically acceptable excipients. The compound of Formula (I), (II), (Ha), (III), (Ia) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, for use in accordance with the present invention can be formulated as a pharmaceutical composition using one or more physiological carriers or excipient, see, for example Ansel et al, "Pharmaceutical

Dosage Forms and Drug Delivery Systems", 7th edition, Lippincott Williams & Wilkins Publishers, 1999.

For oral administration of the compound of Formula (I), (ID), (Ha), (IIT), (Illa) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, the pharmaceutical composition can take the form of, for example, a tablet or capsule prepared by conventional means with pharmaceutical acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica), disintegrants (e.g., potato starch, sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulphate). The pharmaceutical composition can be administered with a physiologically acceptable carrier to a patient. In a specific embodiment, the term "pharmaceutically acceptable” means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can be in the form of ointments, solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. A preferred form is an ointment.

The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. E.W. Martin describes examples of suitable pharmaceutical carriers in “Remington’s

Pharmaceutical Sciences”. Such compositions will contain a therapeutically effective amount of the aforementioned compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. Liquid preparations for oral administration can be in the form of, for

P 00303-032 38 LU102907

Eternygen UK Ltd. example, solutions, syrups, or suspensions, or can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparation can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol, syrup, cellulose derivatives, hydrogenated edible fats), emulsifying agents (e.g., lecithin, acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, fractionated vegetable oils), preservatives (e.g., methyl or propyl-p-hydroxycarbonates, soric acids). The preparations can also contain buffer salts, flavouring, coloring and sweetening agents as deemed appropriate.

Preparations for oral administration can be suitably formulated to give controlled release of the pharmaceutical composition of the invention.

For administration by inhalation, the pharmaceutical composition comprising a compound of

Formula (I), (ID), (Ha), (III), (Ia) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, is conveniently delivered in the form of an aerosol spray presentation from a pressurised pack or a nebulizer, with the use of a suitable propellant (e.g, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurised aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatine, for use in an inhaler or insufflator can be formulated containing a powder mix of the pharmaceutical composition of the invention and a suitable powder base such as lactose or starch.

The pharmaceutical composition comprising a compound of Formula (I), (IT), (Ia), (III), (Ia) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.

Site of injections include intra-venous, intra-peritoneal or sub-cutaneous. Formulations for injection can be presented in units dosage form (e.g., in phial, in multi-dose container), and with an added preservative. The pharmaceutical composition of the invention can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, or dispersing agents. Alternatively, the agent can be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where

P 00303-032 39 LU102907

Eternygen UK Ltd. the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

It is obvious for a person ordinary skilled in the art that the pharmaceutical compositon also encompasses sustained release dosage forms, which are designed to release a drug at a predetermined rate in order to maintain a constant drug concentration for a specific time period of time with minimum side effects. This can be achieved through a variety of formulations or devices, including microspheres, nanoparticles, liposomes, and other polymer matrices such as drug- polymer conjugates like hydrogels or biodegradables like poly(lactic-co-glycolic acid) (PLGA) encapsulating the drug. It is preferred to adapt the release to the specific needs for treating particular diseases, e.g. like sustained release of injections in treating diabetes. Sustained release's definition is more akin to a "controlled release" or “depot medication” rather than "sustained".

The pharmaceutical composition comprising the compound of Formula (I), (ID), (Ia), (III), (Ia) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, can also, if desired, be presented in a pack, or dispenser, which can contain one or more unit dosage forms containing the said agent. The pack can for example comprise metal or plastic foil, such as blister pack. The pack or dispenser device can be accompanied with instruction for administration.

The pharmaceutical composition comprising the compound of Formula (I), (ID), (ITa), (IIT), (IMa) or (IV), or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, can be administered as sole active agent or can be administered in combination with other active agents. Such additional active agents should be primarily chosen from active agents being related to the treatment of the same disease. In case that obesity shall be treated an additional active agent should be chosen from the group of anti-obesity drugs. In analogy anti-diabetes and also anti-NAFLD/NASH as well as anti-dyslipidaemia drugs may be used as further active agents. Furthermore, such additional active agent should be chosen from active agents being related to side effects such as body weight gain like anti-psychotic treatments.

For obesity combinations may comprise combination therapies that are administered in conjunction with exercise, combination therapies that are administered in conjunction with sensible diet, combination therapies with anti-obesity agents are selected from the group consisting of orlistat, lorcaserin, Phentermine, Topiramate, sibutramine, bromocriptine, ephedrine, leptin, and pseudoephedrine. Further examples of combinations with a compound of general formula (I) or (II) are lipase inhibitors (e.g. Orlistat Xenical®, Roche, Alli®, GSK, Cetilistat), 5-HT2c receptor agonists (e.g. Lorcaserin, Belviq® Arena Inc., Eisai), phentermine and topiramate (e.g. Qsymia®,

Vivus Inc), noradrenergic anorectic agents (e.g. phentermine, mazindol) , appetite suppressants (for

P 00303-032 40 LU102907

Eternygen UK Ltd. example, bupropion), bupropion and Naltrexone (e.g. Contrave®, Orexigen Inc.), drugs affecting endogenous signaling of appetite-regulating hormones, neuropeptides, neurotransmitters (e.g. targeting neuro peptide Y receptor, MOR, AgRP, MCHR 1, H3 R), human agouti-related proteins (AGRP), ghrelin receptor antagonists, histamine 3 receptor antagonists or reverse agonists, neuropeptide-Y antagonists, MCR-4 agonists, melanocyte-stimulating hormone receptor analogs, melanin concentrating hormone antagonists, apolipoprotein-B secretion/microsomal triglyceride transfer protein (apo-B/MTP) inhibitors (eg. JNJ16269110, J&J), GR-II antagonist (e.g. CORT108297, Corcept Therapeutics Inc), GLP1 agonists (e.g. Exenatide (Byetta®, Ely Lilly),

Liraglutide (Victoza®), Novo Nordisk), Glucokinase activator (e.g. AZD1656, AstraZeneca),

SGLT-2 inhibitor (e.g. gliflozines such as Invokana™ (canagliflozin), J&J; remogliflozin, Kissel,

GSK, Dapagliflozin (Forxiga®, BMS, Astra Zeneca)), PPAR alpha and —gamma agonist (e.g. glitazars such as LBM642 (cevoglitazar), Novartis, Aleglitazar, Roche), MetAP inhibitor (e.g. CKD732 (beloranib), Zafgen), cholescystokinin-A (CCK-A) agonists, serotonin and norepinephrine reuptake inhibitors (e.g. sibutramine), sympathomimetic agents, 3 adrenergic receptor agonists, dopamine agonists (e.g. bromocriptine), cannabinoid 1 receptor antagonists e.g.

SR141716: N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole- 3-carboxamide], leptons (the OB protein), leptin analogues, leptin receptor agonists, galanin antagonists, lipase inhibitors (such as tetrahydrolipstatin, 1.e., Orlistat), anorectic agents (such as a bombesin agonist), thyromimetic agents, dehydroepiandrosterone or an analogue thereof, glucocorticoid receptor agonists or antagonists, orexin receptor antagonists, urocortin binding protein antagonists, glucagon-like peptide-1 receptor agonists, ciliary neutrotrophic factors (such as

Axokine™ Regeneron Pharmaceuticals) and neuromedin U receptor agonists.

For T2DM combinations may be selected from Incretin mimetics, GLP1 agonists (e.g. Exenatide (Byetta®, Fly Lilly), Liraglutide (Victoza®), Novo Nordisk), dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor,

GPR119 agonist (e.g. PSN-821, Astra Zeneca), GPR40 agonist (e.g. Fasiglifam, Takeda, ASP5034,

Astellas), SGLT-2 inhibitor (e.g. gliflozines such as Dapagliflozin (Forxiga®, BMS, Astra Zeneca),

Canagliflozin (Ivokana® J&J)), DPPIV inhibitors (e.g. gliptine such as Sitagliptin (Januvia®,

Merck)), PPAR agonist (e.g. glitazones such as Rosiglitazone (Avandia®), GSK), Dual PPAR alpha and —gamma agonists (e.g. glitazars such as Cevoglitazar, Novartis, Aleglitazar, Roche)),

Glucokinase activator (e.g. AZD1656, AstraZeneca), MTP inhibitors (e.g. INJ16269110, J&J),

Glycogen phosphorylase inhibitor, 11-beta-HSD-1(e.g. INCB13739 Incyte, AZD4017 AZ),

DGAT-1 inhibitor, aldose reductase inhibitor, a sorbitol dehydrogenase inhibitor, a protein tyrosine phosphatase 1B inhibitor, an insulin mimetic, metformin, acarbose, a sulfonylurea, glipazide, glyburide, or chlorpropamide, glucosidase inhibitor, meglitimide and an aP2 inhibitor.

P 00303-032 41 LU102907

Eternygen UK Ltd.

For NAFLD/NASH combinations may comprise incretin mimetics, GLP1 agonists (e.g. Exenatide (Byetta®, Ely Lilly), Liraglutide (Victoza®), semaglutide, Novo Nordisk), dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor, GPR119 agonist (e.g. PSN-821, Astra Zeneca), GPR40 agonist (e.g. Fasiglifam, Takeda,

ASP5034, Astellas), statins (HMG-CoA Reductase, e.g. atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor, Altocor), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor) and simvastatin (Zocor)), PPARgamma agonists (e.g. Pioglitazone), PPAR agonist (e.g. glitazones such as Rosiglitazone (Avandia®), GSK), dual PPAR alpha and —gamma agonists (e.g. glitazars such as Cevoglitazar, panPPAR alpha/gamma/delta agonists (e.g. lanifibranor), AMPK (e.g.

Metformin, PXL770), Drugs which increase cholesterol secretion into bile by synthetic Fatty-Acid / Bile-Acid Conjugates (e.g. Aramchol (Galmed)), FXR agonist (e.g. INT747, obetecholic acid (Intercept Pharm.), MET409), Px-102/104 (Phenex)), Cysteamine bitartrate (adiponectin multimerization) (e.g. RP103 (Raptor Pharm)), DGAT-1 inhibitor (e.g. LCQ908 (Novartis)), SAMe (methionine metabolism), oral (anti-CD3 antibody) (e.g. OKT3 (NasVax)), LOXL2 Mab (e.g.

Simtuzumab (Gilead)), Omega-3 fatty acid (e.g. EPA-E (Moichida Pharm)), Dual PPARa/d agonist

GFT505 (GENFIT), PDE4 inhibitor (e.g. Roflumilast (Takeda)), immunomodulation by glucospingolipid (e.g. EGS21 (Enzo)), inhibitors of acetyl-CoA carboxylase, inhibitors of ketohexokinase (e.g. PF-06835919), inhibitors of ATP citrate lyase, GLP-1 agonist, inhibitors of

ASK-1 (e.g. GS4977), CCR2/CCRS antagonist (e.g. cenicriviroc), inhibitors of SLC10A2, inhibitors of LOXL2, inhibitors of Galectin-3, inhibitors of caspase, FGF21 (e.g. BIO89-100, BMS- 986036), FGF19 (e.g. NGM282), inhibitors of CGRP, AOC3: Amine Oxidase, Copper Containing 3, inhibitors of DPP-4 (e.g. linagliptin, sitagliptin), THR-B agonists (e.g. MGL3196, VK2809), anti-CD3 monoclonal antibody (mAbs), A3AR agonists, inhibitors of SGLT2 (e.g. canagliflozin, dapagliflozin), inhibitors of SGLT1, inhibitors for TGFB activation, anti-cannabinoid CDI receptor antibody, antibody agonist of the P-Klotho/FGFRIc receptor complex, inhibitors of the inflammasome, DGAT-2 antagonist, ATP citrate lyase inhibitors (e.g. bempedoic acid), stearoyl-

CoA desaturase inhibitors (e.g. Aramchol™, CVT-12805), fatty acid synthesis inhibitors (e.g.

TVB-2640).

For hyper/dyslipidemia combinations may comprise statins (e.g. HMG-CoA reductase inhibitor such as atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor, Altocor), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor) and simvastatin (Zocor), ApoB antisense oligonucleotides (e.g. mipomersen, Kynamro), PCSK9 inhibitors (e.g. antibodies such as AMG145 (Amgen), 1D05-IgG2 (Merck & Co.), and SAR236553/REGN727 (Aventis/Regeneron) or antisense RNA such as ALN-PCS, Amgen), cholesterol-absorption inhibitors (e.g. NPCILI inhibitors such as ezetimibe), niacin, bile-acid-sequestering resins (e.g. Cholestyramine (Questran),

Colesevelam (Cholestagel, Welchol), Colestipol (Colestid) Colestipid, MTP inhibitors (e.g.

P 00303-032 42 LU102907

Eternygen UK Ltd. lomitapide), fibrates (e.g. Bezafibrate (e.g. Bezalip), Ciprofibrate (e.g. Modalim), Clofibrate,

Gemfibrozil (e.g. Lopid), Fenofibrate (e.g. TriCor)), CETP inhibitors (e.g. dalcetrapib, torcetrapib anacetrapib and evacetrapib), ATP citrate lyase inhibitors (e.g. bempedoic acid).

For anti-psychotic treatment combinations may comprise Butyrophenones (e.g. Haloperidol),

Diphenylbutylpiperidine (e.g. Fluspirilene, Penfluridol, Pimozide), Phenothiazines (e.g.

Fluphenazine Perazine Perphenazine Promethazine Trifluoperazine), Thioxanthenes (e.g.

Clopenthixol Tiotixene) or Clozapine, Olanzapine, quetiapine, zotepine).

For use in the therapeutic applications described herein, kits and articles of manufacture are also described herein. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers are formed from any acceptable material including, e.g., glass or plastic. For example, the container(s) can comprise one or more compounds described herein, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprising a compound with an identifying description or label or instructions relating to its use in the methods described herein.

A kit will typically comprise one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of a compound described herein. Nonlimiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use. À set of instructions will also typically be included.

A label can be on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein.

Compounds of Formula (I), (II), (Ha), (IIT), (Ia) or (IV) provided herein can also be used as an inhibitor of a citrate transporter, such as INDY, in a variety of applications, both in vitro and in

P 00303-032 43 LU102907

Eternygen UK Ltd. vivo. Inhibitors of citrate transporters according to the present invention may be used to inhibit the uptake of extracellular citrate by binding to the transporter (e.g., INDY) in vitro or in vivo. This use includes, for example, a method of inhibiting binding of extracellular citrate to INDY in vitro or in vivo, wherein said method comprises contacting INDY with at least one compound or salt described herein under conditions and in an amount sufficient to detectably inhibit binding or transport of extracellular citrate or any other substance to INDY. In some embodiments, the citrate transporter is contained in a cell, tissue or sample (e.g., a cell sample or tissue sample). In some embodiments, compounds or compositions described herein may be used in detection assays for localizing or detecting a citrate transporter in a cell, tissue or sample, and in some embodiments, such detection assays may use a detectably labelled compound, such as a compound of general formula (I), or a salt thereof, that is linked to a detectable label or functional moiety, such as a radio nucleotide, fluorophore or enzyme.

The present invention is now further illustrated by the following examples from which further features, embodiments and advantages of the present invention may be taken. However, the invention should not be construed to be limited to the examples, but encompasses the subject-matter defined in the claims.

EXAMPLES

Materials and methods

Chemical names are generally IUPAC names, and abbreviations have their meanings customary per se to the skilled person. In some cases generally accepted names of commercially available reagents are used in place of [IUPAC names. Abbreviations used herein as far as they are not explained within the text body include: aq. aqueous

AcOH acetic acid

Alloc-CI allyl chloroformate

BMS borane dimethylsulfide

DAST N,N-Diethylaminosuflur trifluoride

DCE 1,2-dichloroethane

DBU 1,8-Diazabicyclo[5.4.0Jundec-7-ene

DCM dichloromethane

DIPEA N,N-diisopropyl-N-ethylamine

P 00303-032 44 LU102907

Eternygen UK Ltd.

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

DPEphos (Oxydi-2,1-phenylene)bis(diphenylphosphine)

ESI electron-spray ionization

EtO diethyl ether

EtOAc ethyl acetate

FCC flash column chromatography h. hours

HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

HCl hydrochloric acid

HPLC high-performance liquid chromatography

KHMDS Potassium hexadimethyldisilazide

KOtBu potassium tert-butoxide

IPA isopropanol

LC liquid chromatography

LCMS liquid chromatography — mass spectrometry

LiAIH4 Lithium aluminium hydride

M molar

MeCN acetonitrile

Mel iodomethane

MeMgBr methylmagnesium bromide

MeOH methanol

MgSO, magnesium sulfate

MHz mega Hertz

MnO2 Manganese dioxide uL microlitre(s) min minute(s) mg milligram(s) mL milliliter(s) mmol millimole(s)

NaH sodium hydride

NaHCO:s sodium bicarbonate

P 00303-032 45 LU102907

Eternygen UK Ltd.

NaOH sodium hydroxide

Na2SO4 sodium sulfate n-BuLi n-butyllithium

NH4C1 ammonium chloride

NMP N-methylpyrrolidinone

NMR nuclear magnetic resonance

Pd(PPh3)4 Tetrakis(triphenylphosphane)palladium(0)

RPB reverse phase biotage

Rt/ wr retention time

RT /rt. room temperature sat. saturated

SFC supercritical fluid chromatography

STAB sodium triacetoxyborohydride

TBME tert-butyl methyl ether

THF tetrahydrofuran

Specific examples for the preparation of compounds of general formula (I) are provided below.

Unless otherwise specified all starting materials and reagents are of standard commercial grade, and are used without further purification, or are readily prepared from such materials by routine methods. Those skilled in the art of organic synthesis will recognize that starting materials and reaction conditions may be varied including additional steps employed to produce compounds encompassed by the present invention. Compounds disclosed herein were commonly named using a computer program capable of naming in accordance with IUPAC rules; JChem for Excel,

MarvinSketch manufactured by ChemAxon Ltd. or ChemBioDraw Ultra 12.0 developed by

CambridgeSoft Ltd. Compounds purified by automated flash chromatography were purified using a biotage 1solera 4 system.

Analytical methods and conditions used for the chemical characterization of compounds are summarized below.

NMR Spectroscopy

Unless otherwise stated, 'H NMR spectra were recorded at 500 MHz, 400 MHz or 250 MHz on either a Bruker Avance III HD 500 MHz, VARIAN VNMR 400 MHz spectrometer or Bruker

Avance III HD 250 MHz spectrometer, respectively. Chemical shifts, 6, are quoted in parts per

P 00303-032 46 LU102907

Eternygen UK Ltd. million (ppm) and are referenced to the residual solvent peak. The following abbreviations are used to denote the multiplicities and general assignments: s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet of triplets), dq (doublet of quartets), hep (heptet), m (multiplet), pent (pentet), td (triplet of doublets), qd (quartet of doublets), app. (apparent) and br. (broad).

Chromatography / Mass spectrometry

One or more of Methods (A), (B), (C), (D), (E), (F) and (G) were used for chromatographic purification and/or characterization of compounds. The respective method employed is recorded in the analytical LC-MS results, and the solvents and conditions used for the LC-MC analysis in the individual methods are indicated below.

Method A - (1600) MS10. The conditions of method A are summarized in the table below.

Solvent(eluent

LB | B-Acctonitle

Detection | 000000

Peakwidth mins oe | 5

Pump gradient oo | 5

P 00303-032 47 LU102907

Eternygen UK Ltd.

Method B - (MSQ1). The conditions of method B are summarized in the table below.

Kinetix-XB C18 100 x 2.1 mm, 1.7um _ Solvent(eluent |__

LB |. B-Fomicacid(uetonimil)01%

Detection |)

Peakwidth

Pumpgradient |; mins oo | 5

Method C - (1416). The conditions of method C are summarized in the table below. 2.1 x 100mm, 3 jum _ Solvent(eluent |__ oe | eromcwacmovors

Detection

P 00303-032 48 LU102907

Eternygen UK Ltd.

Peakwidth |) pump gradient | 1 mins 000 | 5

Method D - (1673). The conditions of method D are summarized in the table below.

Supelco Ascentis Express C18 30 x 2.1 mm, 2.7um _ Solvent(eluent |__

LB |. B-Fomicacid(uetonimil)01%

Detection |)

Peakwidth |)

Pumpgradient |)

Tm i B 0 | 0s

P 00303-032 49 LU102907

Eternygen UK Ltd.

Method E. The conditions of method E are summarized in the table below.

C18 2.6 u 30*4.6 mm. 100 A

Solvent(eluent phase B er + 5% phase A

Détection

Peakwidth mins 00 5 pump gradient

Method F - (30-95) (Triart, Basic). The conditions of method F are summarized in the table below.

P 00303-032 50 LU102907

Eternygen UK Ltd.

X 4.6 mm. 3 um

Solvent(eluent) . B | B= Acetonitrile + 5% Solvent A +0.1% Ammonia

Detecton

Peakwidm mins 000 | 30 pump gradient

Method G - (6.5 min Basic YMC Triart ). The conditions of method G are summarized in the table below. 50 X 4.6 mm. 3 um

Solvent(eluent) . B | B=Acetonitrile + 5% Solvent A +0.1% Ammonia

Detecton

P 00303-032 51 LU102907

Eternygen UK Ltd.

Peakwidth 1.40 mL/min

Gradient Time % Organic mins 000 | 30 pump gradient

Synthesis of compounds *Compounds denoted with (*) show activity >20 uM against human INDY (hINDY) and/or mouse

INDY (mINDY).

A. Ethyl alkyne carbamates

Described herein are ethyl alkyne carbamates of the following structure:

O 2

Ars À A

N O

H .

A general method for the synthesis of ethyl alkyne carbamates can be summarized by: _ I = À LA Ar ZZ

Stage 1 Stage 2 H

Stage 1 - (But-3-yn-1-yloxy)(chloro)methanone (Tetrahedron, 63, 2007, 9153-9162):

O PP

A = cl o A0

A 3 neck round bottom flask equipped with a bubbler, N; inlet and a septum was purged with N> and then triphosgene (9.28 g, 37.7 mmol) and dry ether (200 ml) were added at r.t. and the reaction

P 00303-032 52 LU102907

Eternygen UK Ltd. stirred until the triphosgene had dissolved. Activated charcoal (0.250 g) was added and the reaction was left for 1 h. The reaction was cooled to 0 °C and but-3-yn-1-ol (5.7 ml, 75.3 mmol) in dry ether (100 ml) was slowly added dropwise. The reaction was slowly warmed to r.t. and left for 19 h. The reaction was filtered and concentrated in vacuo to give 9.280 g (93%) of crude (but-3-yn-1- yloxy)(chloro)methanone as a colourless oil which was used in subsequent reactions without further purification. 'H NMR (500 MHz, DMSO-de) & 3.47 (t, 2H), 2.74 (t, 1H), 2.27 (td, 2H)

Stage 2A - General formation of carbamates (using EtsN as a base)

X=

Ar =

H

To a vial were added (but-3-yn-1-yloxy)(chloro)methanone (0.1 g, 1 eq.) and DCM (2 ml) followed by a solution/suspension of aniline (1.1 eq.) and Et3N (2 eq.) in DCM (2 ml) at r.t. and the mixture purged with N2 and stirred for 1 h. The reaction was quenched with water (2 ml), the organic layer separated, dried over MgSO4, filtered and concentrated in vacuo to give the crude product. The product was purified by silica flash column chromatography to give the required carbamate.

Example 1: Synthesis of Compounds 1-3

Compounds 1 - 3 were prepared according to the above method.

Compound 1: But-3-yn-1-yl N-(3-chlorophenyl)carbamate*

JO LA 0 = cl N ZZ

H

Yellow oil (37%); LC/MS m 3.21 min; (ES") m/z 224 [M+H]”; (B) 'H NMR (500 MHz, DMSO-de) 5 9.95 (s, 1H), 7.60 (t, 1H), 7.42 - 7.36 (m, 1H), 7.30 (t, 1H), 7.04 (ddd, 1H), 4.19 - 4.13 (m, 2H), 2.90 (t, 1H), 2.56 (td, 2H).

Compound 2: But-3-yn-1-yl N-(4-chloro-3-methoxyphenyl)carbamate*

Cl

O 2

O N O

P 00303-032 53 LU102907

Eternygen UK Ltd.

White solid (41%); LC/MS fr 3.15 min; (ES") m/z 254 [M+H]"; (B) 'H NMR (500 MHz, DMSO-ds) § 9.87 (s, 1H), 7.38 (s, 1H), 7.30 (d, 1H), 7.03 (dd,1H), 4.16 (t, 2H), 3.80 (s, 3H), 2.91 (t, 1H), 2.57 (td, 2H).

Compound 3: But-3-yn-1-yl N-(2,2-difluoro-2H-1.3-benzodioxol-5-yl)carbamate*

XI

A

F O A AZ

H

White solid (36%); LC/MS fr 3.38 min; (ES") m/z 270 [M+H]"; (B) 'H NMR (500 MHz, DMSO-ds) § 9.96 (s, 1H), 7.56 (d, 1H), 7.32 (d, 1H), 7.18 (dd, 1H), 4.16 (t, 2H), 2.90 (t, 1H), 2.56 (td, 2H).

Stage 2B - General formation of carbamates (using NaHCOs as a base)

O 2

Ars M A0

N O

H

To a vial or round bottom flask were added aniline (0.1 g, 1 eq.), sodium hydrogen carbonate (3 eq.) and DCM (2 ml) followed by dropwise addition of a solution of chloro(3,3,3- trifluoropropoxy)methanone (1.5 eq.) in DCM (2 ml) at r.t. The mixture purged with N3 and stirred for 16-24 h. TLC and LCMS analysis after this time showed product had formed. The reaction was filtered and concentrated in vacuo to give the crude product. The product was purified by either silica flash column chromatography and/or prep-HPLC ((MeCN/Water, 2 mM NH4HCOs3) or (MeCN/Water) or (MeCN/Water, 0.1% HCO,H)) to give the carbamate.

Example 2: Synthesis of Compounds 4 - 9

Compounds 4 - 9 were prepared according to the above method utilizing Stage 2B.

Compound 4: But-3-yn-1-yl (4-chloro-3-(trifluoromethyl)phenyl)carbamate

Cl

F Zz

N ° A0

F H

F

White solid (50%); LC/MS tr 2.98 min; (ES") m/z 290 [M-H]"; (F) !H NMR (400 MHz, CDCI;): 5 7.74 (d, 1H), 7.59 - 7.52 (m, 1H), 7.43 (d, 1H), 6.79 (bs, 1H), 4.30 (t, 2H), 2.66 - 2.55 (m, 2H), 2.04 (m, 1H).

P 00303-032 54 LU102907

Eternygen UK Ltd.

Compound 5: But-3-yn-1-yl (3.4-dichlorophenyl)carbamate

CI

O ins

CI N O

H

White solid (73%); LC/MS tr 2.92 min; (ES) m/z 256 [M-H]"; (F) 'HNMR (400 MHz, CDCl:): 5 7.62 (d, 1H), 7.35 (d, 1H), 7.19 (dd, 1H), 6.67 (bs, 1H), 4.29 (t, 2H), 2.59 (m, 2H), 2.07 - 1.98 (m, 1H).

Compound 6: But-3-yn-1-yl (3,5-dichlorophenyl)carbamate

Cl

O

J A

CI N O

H

White solid (66%); LC/MS tr 3.03 min; (ES”) m/z 256 [M-H]"; (F) "HNMR (400 MHz, CDCl): 5 7.38 - 7.21 (m, 2H), 7.06 (m, 1H), 6.70 (bs, 1H), 4.29 (m, 2H), 2.60 (m, 2H), 2.05 (m, 1H).

Compound 7: But-3-yn-1-yl (4-chloro-3-fluorophenyl)carbamate*

CI

O

OU

F NT SO

H

White solid (50%); LC/MS tr 2.81 min; (ES”) m/z 240 [M-H]"; (F)

IH NMR (400 MHz, CDCI;): 6 7.46 - 7.42 (m, 1H), 7.30 - 7.28 (m, 1H), 7.03 - 6.95 (m, 1H), 6.70 (bs, 1H), 4.29 (t, 2H), 2.59 (td, 2H), 2.04 (t, 1H). (Compound 8: But-3-yn-1-yl N-(4-fluoro-3-methoxyphenyl)carbamate *

MeO

Th =

F N O7

H

White solid (42%); LC/MS fr 2.84 min; (ES") m/z 238 [M+H]"; (B) 'H NMR (500 MHz, DMSO-de) § 9.73 (s, 1H), 7.35 (d, 1H), 7.10 (dd, 1H), 7.02 - 6.91 (m, 1H), 4.15 (t, 2H), 3.78 (s, 3H), 2.89 (t, 1H), 2.55 (td, 2H).

Compound 9: But-3-yn-1-yl N-(3,5-dimethoxyphenyl)carbamate *

P 00303-032 55 LU102907

Eternygen UK Ltd.

OMe ol XNA =

MeO N ZZ

H

White solid (42%); LC/MS fr 2.86 min; (ES") m/z 250 [M+H]"; (B) !H NMR (500 MHz, DMSO-de) 8 9.66 (s, 1H), 6.72 (d, 2H), 6.16 (t, 1H), 4.13 (t, 2H), 3.69 (s, 6H), 2.89 (t, 1H), 2.55 (td, 2H).

B. Ethyl trifluoromethane carbamates

Described herein are ethyl trifluoromethane carbamates of the following structure:

X A

Ar UK “NT 0 F

H .

A general synthetic route for the preparation of ethyl trifluoromethane carbamates can be summarized by: —_— —» Ar ces ao SAXE N © F

Stage 1 Stage 2A or 2B

Stage 1 - Chloro(3,3,3-trifluoropropoxy)methanone

O F

A AN

CI O F

A 3 neck round bottom flask equipped with a bubbler, N; inlet and a septum was purged with N> and triphosgene (6.504 g, 21.9 mmol) and dry ether (100 ml) added at r.t. and the reaction stirred until the triphosgene had dissolved. Activated charcoal (0.250 g) was added and the reaction was left for 1 h. The reaction was cooled to 0 °C and 3,3,3-trifluoropropan-1-ol (3.86 ml, 43.8 mmol) in dry ether (100 ml) was slowly added dropwise. The reaction was slowly warmed to r.t. and left for 18h. The reaction was filtered and concentrated in vacuo to give 5.72 g (67%) of crude chloro(3,3,3- trifluoropropoxy)methanone as a pale yellow oil which was used in subsequent reactions without further purification. 'H NMR (500 MHz, DMSO-de) 5 3.62 (t, 2H), 2.39 (ddt, 2H)

Stage 2A - General formation of carbamates (using Et:N as a base)

P 00303-032 56 LU102907

Eternygen UK Ltd.

O F

A J AA r “NT To F

H

To a vial or round bottom flask were added chloro(3,3,3-trifluoropropoxy)methanone (0.1 g, 1 eq.) and DCM (2 ml) followed by a solution/suspension of aniline (1 eq.) and Et3N (5 eq.) in DCM (2 ml) at r.t. and the mixture purged with N2 and stirred for 2-24 h. TLC and LCMS analysis after this time showed product had formed. The reaction was quenched with water (2 ml), the organic layer separated, dried over MgSQy, filtered and concentrated in vacuo to give the crude product. The product was purified by either silica flash column chromatography and/or prep-HPLC ((MeCN/Water, 2 mM NH4HCO3) or (MeCN/Water) or (MeCN/Water, 0.1% HCO»H)) to give the carbamate.

Example 3: Synthesis of Compounds 10 - 29

Compounds 10 - 29 were prepared according to the above method.

Compound 10: 3,3,3-Trifluoropropyl N-(3-chloro-5-methylphenyl)carbamate

Cl

O F

Qi tr

N O F

H

Brown oil (5%); LC/MS fr 3.74 min; (ES) m/z 280 [M-H]"; (B) !H NMR (500 MHz, methanol-ds) 5 7.38 (s, 1H), 7.13 (s, 1H), 6.86 (s, 1H), 4.36 (t, 2H), 2.66 - 2.55 (m, 2H), 2.29 (s, 3H).

Compound 11: 3,3.3-Trifluoropropyl N-(4-chloro-3-cyanophenyl)carbamate*

CI

O F

AAA

NT H

White solid (7%); LC/MS 3.36 min; (ES”) m/z 291 [M-H]"; (B) !H NMR (500 MHz, DMSO-ds) § 10.25 (s, 1H), 7.97 (d, 1H), 7.74 (dd, 1H), 7.67 (d, 1H), 4.34 (t, 2H), 2.72 (qt, 2H).

Compound 12: 3.3 3-Trifluoropropyl N-(2.2-difluoro-2H-1,3-benzodioxol-5-yl)carbamate

XI

F

H

P 00303-032 57 LU102907

Eternygen UK Ltd.

Orange solid (20%); LC/MS tr 3.66 min; (ES") m/z 314 [M+H]"; (B) !H NMR (500 MHz, DMSO-de) § 9.95 (s, 1H), 7.56 (s, 1H), 7.32 (d, 1H), 7.18 (d, 1H), 4.32 (t, 2H), 2.71 (qt, 2H). (Compound 13: 3,3,3-Trnfluoropropyl N-[4-chloro-3-(trifluoromethyl)phenyl]carbamate

CI

À Ati

F A

N O F

F H

F

Colourless oil (7%); LC/MS fr 3.66 min; (ES") m/z 314 [M+H]"; (B) 'H NMR (500 MHz, DMSO-de) § 10.22 (s, 1H), 8.03 (s, 1H), 7.73 (d, 1H), 7.64 (d, 1H), 4.34 (t, 2H), 2.72 (qt, 2H).

Compound 14: 3.3.3-Trifluoropropyl N-(3.5-dichlorophenyl)carbamate

Cl

O F

Jel Ik

Cl N O F

H

Colourless oil (9%); LC/MS fr 4.60 min; (ES”) m/z 300 [M-H]"; (B) 'H NMR (500 MHz, DMSO-ds) § 10.15 (s, 1H), 7.57 - 7.47 (m, 2H), 7.22 (t, 1H), 4.34 (t, 2H), 2.72 (at, 2H).

Compound 15: 3.3.3-Trifluoropropyl N-(3-chloro-5-fluorophenyl)carbamate

Cl oO F 0. LL

F N Oo F

H

Colourless oil (6%); LC/MS fr 4.20 min; (ES”) m/z 284 [M-H]"; (B) 'H NMR (500 MHz, DMSO-ds) 8 10.17 (s, 1H), 7.39 (s, 1H), 7.32 (d, 1H), 7.05 (dt, 1H), 4.34 (t, 2H), 2.72 (qt, 2H).

Compound 16: 3.3.3-Trifluoropropyl N-(4-chloro-3-methoxyphenyl)carbamate™

CI

O F

À AN

O N O F

H

P 00303-032 58 LU102907

Eternygen UK Ltd.

Colourless oil (31%); LC/MS fr 3.42 min; (ES) m/z 298 [M+H]"; (B) 'H NMR (500 MHz, DMSO-d) 8 9.86 (s, 1H), 7.36 (s, 1H), 7.30 (d, 1H), 7.10 - 6.98 (m, 1H), 4.32 (t, 2H), 3.80 (s, 3H), 2.71 (qt, 2H). (Compound 17: 3.3,3-Trifluoropropyl N-(3-chloro-5-methoxyphenyl)carbamate

Cl

O F

O N O F

H

Pale brown solid (20%); LC/MS fr 3.58 min; (ES”) m/z 298 [M+H]"; (B) !H NMR (500 MHz, DMSO-d) 8 9.91 (s, 1H), 7.17 (s, 1H), 7.05 (s, 1H), 6.67 (t, 1H), 4.31 (t, 2H), 3.73 (s, 3H), 2.71 (qt, 2H).

Compound 18: 3.3.3-Trifluoropropyl N-[4-chloro-3-(difluoromethoxy)phenyl]carbamate

F

SN

CI

OL,

N SE

H

Pale yellow oil (42%); LC/MS fr 3.68 min; (ES”) m/z 332 [M-H]"; (B) 'H NMR (500 MHz, DMSO-de) 5 10.08 (s, 1H), 7.62 (s, 1H), 7.50 (d, 1H), 7.39 - 7.01 (m, 2H), 4.33 (t, 2H), 2.72 (qt, 2H).

Compound 19: 3,3.3-tnfluoropropyl N-(3-chloro-5-cyanophenyl)carbamate

Cl

O

D JAN

NZ N © F

Colourless oil (7%); LC/MS tr 3.50 min; (ES) m/z 291 [M-H]"; (B) 'H NMR (500 MHz, DMSO-de) § 10.32 (s, 1H), 7.85 (s, 1H), 7.80 (s, 1H), 7.72 - 7.62 (m, 1H), 4.36 (t, 2H), 2.74 (qt, 2H).

Compound 20: 3.3.3-Trifluoropropyl N-(3-chloro-4-cyanophenyl)carbamate*

P 00303-032 59 LU102907

Eternygen UK Ltd.

N CI

S

O F

> Le

N O F

H

White solid (12%); LC/MS fr 4.49 min; (ES") m/z 291 [M-H]"; (A) !H NMR (500 MHz, DMSO-de) 5 10.48 (s, 1H), 7.92 - 7.83 (m, 2H), 7.56 (dd, 1H), 4.37 (t, 2H), 2.74 (qt, 2H).

Compound 21: 3.3.3-Trifluoropropyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate

CI

O F F

F M AA

N O F

F H

F

Yellow oil (4%); LC/MS tr 4.02 min; (ES) m/z 336 [M+H]"; (B) 'H NMR (500 MHz, methanol-ds) 5 7.79 (s, 1H), 7.74 (s, 1H), 7.31 (s, 1H), 4.40 (t, 2H), 2.69 - 2.56 (m, 2H).

Compound 22: 3,3,3-Trifluoropropyl N-(4-chloro-3-nitrophenyl)carbamate

Cl

O F

À AA

O,N N O F

H

Yellow oil (11%); LC/MS fr 3.48 min; (ES?) m/z 311 [M-HJ’; (B) 'H NMR (500 MHz, methanol-ds) 5 8.15 (d, 1H), 7.62 (dd, 1H), 7.53 (d, 1H), 4.40 (t, 2H), 2.71 - 2.55 (m, 2H).

Compound 23: 3,3,3-Trifluoropropyl N-[3-cyano-5-(trifluoromethyl)phenyl]carbamate

F

F F

O F

J AA

CE N O F

NT H

White solid (5%); LC/MS fr 3.64 min; (ES") m/z 325 [M-H]"; (B)

P 00303-032 60 LU102907

Eternygen UK Ltd. !H NMR (500 MHz, methanol-ds) § 7.28 (s, 1H), 7.27 (s, 1H), 6.89 (s, 1H), 3.61 (t, 2H), 1.89 - 1.77 (m, 2H).

Compound 24: 3,3,3-Trifluoropropyl N-(3-cyano-5-fluorophenyl)carbamate

F

O F

À AA

= N O F

NT H

Pale yellow solid (10%); LC/MS fr 3.27 min; (ES) m/z 275 [M-H]’; (B) 'H NMR (500 MHz, DMSO-ds) § 10.34 (s, 1H), 7.70 - 7.62 (m, 2H), 7.48 (dt, 1H), 4.35 (t, 2H), 2.73 (qt, 2H).

Compound 25: 3,3,3-Trifluoropropyl N-[4-nitro-3-(trifluoromethyl)phenyl]carbamate

O,N 0 Fr

NT -O F

F H

F

Pale yellow solid (17%); LC/MS fr 3.67 min; (ES") m/z 345 [M-H]"; (B) !H NMR (500 MHz, DMSO-ds) § 10.68 (s, 1H), 8.20 (d, 1H), 8.13 (d, 1H), 7.93 (dd, 1H), 4.39 (t, 2H), 2.75 (qt, 2H).

Compound 26: 3,3,3-Trifluoropropyl N-[3-nitro-5-(trifluoromethyl)phenyl]carbamate

NO, el LA

F A

NT DO F

F H

F

Yellow solid (4%); LC/MS fr 3.81 min; (ES") m/z 345 [M-H]"; (B) !H NMR (500 MHz, methanol-ds) 5 7.84 (s, 1H), 7.37 (s, 1H), 7.32 (s, 1H), 3.63 (t, 2H), 1.90 - 1.78 (m2H).

Compound 27: 3,3,3-Trifluoropropyl N-(3-bromo-5-chlorophenyl)carbamate

P 00303-032 61 LU102907

Eternygen UK Ltd.

Cl

O F

À A

Br N O F

H

Yellow oil (14%); LC/MS fr 4.06 min; (ES") m/z 346 [M-HJ’; (B) 'H NMR (500 MHz, DMSO-d) § 10.13 (s, 1H), 7.67 (s, 1H), 7.56 (s, 1H), 7.33 (t, 1H), 4.33 (t, 2H), 2.72 (qt, 2H).

Compound 28: 3,3,3-Trfluoropropyl N-(3-chlorophenyl)carbamate *

O F

IAE

CI N O F

H

Colourless oil (24%); LC/MS fr 3.48 min; (ES) m/z 268 [M+H]"; (B) 'H NMR (500 MHz, DMSO-ds) 8 9.95 (s, 1H), 7.61 (s, 1H), 7.39 (d, 1H), 7.31 (t, 1H), 7.09 - 7.02 (m, 1H), 4.32 (t, 2H), 2.71 (qt, 2H).

Compound 29: 3.3.3-Trifluoropropyl N-(4-chloro-3-ethoxyphenyl)carbamate *

Cl

AAA

0 N SNS

H

White solid (22%); LC/MS fr 4.32 min; (ES") m/z 312 [M+H]"; (B) 'H NMR (500 MHz, DMSO-ds) 8 9.83 (s, 1H), 7.34 (s, 1H), 7.29 (d, 1H), 7.03 (d, 1H), 4.31 (t, 2H), 4.04 (q, 2H), 2.70 (qt, 2H), 1.35 (t, 3H).

Stage 2B - General formation of carbamates (using NaHCOs as a base)

O F

A J AA rs

N O F

H

To a vial or round bottom flask were added aniline (0.1 g, 1 eq.), sodium hydrogen carbonate (3 eq.) and DCM (2 ml) followed by dropwise addition of a solution of 3,3,3-trifluoropropyl chloroformate (1.5 eq.) in DCM (2 ml) at r.t. The mixture purged with No and stirred for 16 h-5 days. TLC and LCMS analysis after this time showed product had formed. The reaction was filtered and concentrated in vacuo to give the crude product. The product was purified by either silica flash column chromatography and/or prep-HPLC ((MeCN/Water, 2 mM NH4sHCO3) or (MeCN/Water) or (MeCN/Water, 0.1% HCO»H)) to give the carbamate.

P 00303-032 62 LU102907

Eternygen UK Ltd.

Example 4: Synthesis of Compounds 30 - 33

Compounds 30 - 33 were prepared according to the above method utilizing Stage 2B.

Compound 30: 3,3,3-Trifluoropropyl N-[4-chloro-3-(trifluoromethoxy)phenyl]carbamate

CI 0

FL IE A AUX "

O

F N 0 F

Colourless oil (45%); LC/MS fr 4.05 min; (ES) m/z 350 [M-HJ’; (B) 'H NMR (250 MHz, DMSO-de) 8 10.19 (s, 1H), 7.80 (s, 1H), 7.60 (d, 1H), 7.45 (dd, 1H), 4.34 (t, 2H), 2.73 (qt, 2H).

Compound 31: 3,3,3-Trifluoropropyl N-[3-chloro-5-(trifluoromethoxy)phenyl]carbamate

Cl

F O

4 2 LAN

O N

F H © F

Colourless oil (8%); LC/MS fr 4.17 min; (ES”) m/z 350 [M-H]"; (B) 'H NMR (250 MHz, DMSO-de) § 10.25 (s, 1H), 7.73 - 7.38 (m, 2H), 7.17 (s, 1H), 4.34 (t, 2H), 2.72 (qt, 2H).

Compound 32: 3,3,3-Trifluoropropyl N-(4-chloro-3,5-difluorophenyl)carbamate

F

CI °

AA

F N

H © F

White solid (97%); LC/MS tr 3.82 min; (ES) m/z 303 [M-H]"; (B) 'H NMR (500 MHz, DMSO-ds) § 10.30 (s, 1H), 7.39 (s, 1H), 7.37 (s, 1H), 4.35 (t, 2H), 2.73 (qt, 2H).

Compound 33: 3.3.3-Trifluoropropyl N-[3.5-bis(trifluoromethyl)phenyl]carbamate

P 00303-032 63 LU102907

Eternygen UK Ltd.

F

FF

0 F

F DU

O

F H F

F

White solid (63%); LC/MS fr 4.15 min; (ES") m/z 369 [M+H]"; (B) 'H NMR (250 MHz, DMSO-ds) § 10.46 (s, 1H), 8.14 (s, 2H), 7.71 (s, 1H), 4.38 (t, 2H), 2.75 (qt, 2H).

An alternative general method for the synthesis ethyl trifluoromethan carbamates from isocyanate can be summarized by:

F R—N—=0 o F ; ee Jk

HO F N © F

Stage 1 H

Stage 1 - General formation of carbamates

O F

F

Ar AR

YA, i

H

To a solution of 3,3,3-trifluoropropan-1-ol (0.1 g, 1 eq.) in DCM (1 ml) was added isocyanate (1.1 eq.). The reaction was stirred at r.t. for 1.5 h. The mixture was washed with saturated NaHCO; (1 ml) and the organic layer was dried over MgSO4, filtered and solvent removed in vacuo. The crude product was purified by silica flash column chromatography and prep-HPLC (MeCN/Water, 0.1%

HCO»H)) to give the carbamate.

Example 5: Synthesis of Compounds 34 and 35

Compounds 34 and 35 were prepared according to the alternative method indicated above.

Compound 34: 3.3.3-Trifluoropropyl N-(3.4-dichlorophenyl)carbamate

Cl

O F

M A F

N O F

H

Yellow oil (56%); LC/MS fr 3.80 min; (ES") m/z 300 [M-H]; (B) !H NMR (500 MHz, DMSO-ds) § 10.08 (s, 1H), 7.79 (d, 1H), 7.55 (d, 1H), 7.43 (dd, 1H), 4.34 (t,

P 00303-032 64 LU102907

Eternygen UK Ltd. 2H), 2.72 (qt, 2H).

Compound 35: 3,3,3-Trifluoropropyl N-(3-chloro-4-fluorophenyl)carbamate*

Cl

F oO F

DC A Le

N oO F

H

Colourless oil (94%); LC/MS tr 3.53 min; (ES”) m/z 284 [M-H]"; (B) 'H NMR (500 MHz, DMSO-de) 5 9.96 (s, 1H), 7.71 (d, 1H), 7.51 - 7.21 (m, 2H), 4.32 (t, 2H), 2.71 (qt, 2H).

Example 6: Synthesis of Compound 36

Compound 36: 3.3.3-Trifluoropropyl N-[3-chloro-5-(morpholin-4-yl)phenyl carbamate

CI

Cl

O F 9 | F

Li Jk

ON NT So F

Br N O F Stage 1 H

À o

To a pressure tube were added 3,3,3-trifluoropropyl N-(3-bromo-5-chlorophenyl)carbamate (Compound 27, 0.030 g, 0.084 mmol, 97% pure), morpholine (0.02 ml, 0.168 mmol), Pd»(dba)s (0.008 g, 0.008 mmol), XPhos (0.008 g, 0.017 mmol), NaOBu (0.020 g, 0.210 mmol) and dry toluene (1 ml) at r.t. and N; bubbled through the solution for 5 mins. The pressure tube was sealed and heated to 100 °C for 4 h. LCMS analysis after this time showed starting material was consumed and product had formed. The reaction was cooled to r.t. and diluted with EtOAc (3 ml) and water (3 ml). The organic layer was separated and the aqueous layer was extracted with EtOAc (2 x 3 ml) and the combined organic layers were dried over MgSO,, filtered and concentrated in vacuo to give the crude product. The product was purified by prep-HPLC (MeCN/Water) to give 0.008 g (27%) of 3,3,3-trifluoropropyl N-[3-chloro-5-(morpholin-4-yl)phenyl]carbamate as a brown solid.

LC/MS tr 3.50 min; (ES) m/z 353 [M-H]; (B) 'H NMR (500 MHz, methanol-d4) § 7.02 (s, 1H), 6.96 (s, 1H), 6.63 (t, 1H), 4.36 (t, 2H), 3.83 - 3.77 (m, 4H), 3.15 - 3.09 (m, 4H), 2.66 - 2.55 (m, 2H).

Example 7: Synthesis of Compound 37

Compound 37: 3.3.3-Trifluoropropyl N-(3-carbamoyl-4-chlorophenyl)carbamate *

P 00303-032 65 LU102907

Eternygen UK Ltd.

CI

CI o F O F F

À AT —— HN AN NS

NC N° DO F N

H Stage 1 O

To a vial were added 3,3,3-trifluoropropyl N-(4-chloro-3-cyanophenyl)carbamate (Compound 11, 0.015 g, 0.626 mmol, 98% pure), THF (3 ml), water (0.75 ml), LIOH (0.023 g, 0.954 mmol) and

HO: (35% w/w solution) (0.6 ml, 6.85 mmol) at r.t. and the reaction stirred for 5 days. LCMS analysis after this time showed product had formed and starting material was consumed. The reaction was diluted with water (3 ml) and EtOAc (3 ml) and the organic layer separated. The aqueous layer was extracted with EtOAc (2 x 3 ml) and the combined organic layers were washed with brine (3 ml), dried over MgSO4, filtered and concentrated in vacuo to give the crude product.

The product was purified by prep-HPLC (MeCN/Water) to give 0.013 g (83%) of 3,3,3- trifluoropropyl N-(3-carbamoyl-4-chlorophenyl)carbamate as a white solid.

LC/MS fr 2.45 min; (ES") m/z 311 [M+H]"; (B) 'H NMR (500 MHz, DMSO-de) 5 9.97 (s, 1H), 7.83 (s, 1H), 7.60 - 7.53 (m, 2H), 7.50 (dd, 1H), 7.38 (d, 1H), 4.32 (t, 2H), 2.71 (qt, 2H).

Example 8: Synthesis of Compound 38

The synthesis route can be summarized by:

Ho NF, ANG NH, ANG NO F

Stage 1 I Stage 2 I T

Cl age el age cl

Stage 1 - 4-Chloro-3-(cyclopropylmethoxy)aniline

Cl

A pressure tube was charged with 5-amino-2-chlorophenol (0.3 g, 2.09 mmol), anhydrous MeCN (5 ml), dipotassium carbonate (0.578 g, 4.18 mmol) and (bromomethyl)cyclopropane (0.564 g, 4.18 mmol) respectively. Reaction was stirred at 90 °C for 16 h. The reaction mixture was filtered and residue was washed with EtOAc (10 ml). The filtrate was concentrated in vacuo and the crude was purified by silica flash column chromatography to give the 0.209 g (51%) of 4-chloro-3- (cyclopropylmethoxy)aniline as a brown oil.

LC/MS tr 1.19 min; (ES") m/z 198 [M+H]"; (D)

P 00303-032 66 LU102907

Eternygen UK Ltd. 'H NMR (500 MHz, DMSO-de) 8 6.96 (d, 1H), 6.29 (d, 1H), 6.12 (dd, 1H), 5.17 (s, 2H), 3.77 (d, 2H), 1.35 - 1.09 (m, 1H), 0.59 - 0.54 (m, 2H), 0.35 - 0.30 (m, 2H).

Stage 2 - Compound 38: 3.33-trifluoropropyl N-[4-chloro-3-(cyclopropylmethoxy) phenyl]carbamate*

Ao Kresse

Compound 38 was prepared following Stage ZA - formation of carbamates (using EtsN as a base).

White solid (28%); LC/MS fr 4.08 min; (ES") m/z 697 [2M+Na]"; (B) !H NMR (500 MHz, DMSO-de) § 9.82 (s, 1H), 7.34 (s, 1H), 7.29 (d, 1H), 7.02 (d, 1H), 4.31 (t, 2H), 3.84 (d, 2H), 2.70 (qt, 2H), 1.25 (ddd, 1H), 0.66 - 0.50 (m, 2H), 0.36 (q, 2H).

Example 9: Synthesis of Compound 39

The synthesis route can be summarized by:

HO NH, No NH, NIN N. Lo F

AD Stage 1 SD Stage 2 QU T Vf

Stage 1- 4-Chloro-3-(2-methoxyethoxy)aniline ~ UT

CI

A pressure tube was charged with 5-amino-2-chlorophenol (0.3 g, 2.09 mmol), anhydrous MeCN (5 ml), dipotassium carbonate (0.578 g, 4.18 mmol) and 1-bromo-2-methoxyethane (0.581 g, 4.18 mmol) respectively. Reaction was stirred at 80 °C for 16 h. The reaction mixture was filtered. The reaction mixture was filtered and residue was washed with EtOAc (10 ml). The filtrate was concentrated in vacuo and the crude was purified by silica flash column chromatography to give 0.282 g (66%) of 4-chloro-3-(2-methoxyethoxy)aniline as a brown oil.

LC/MS tr 1.01 min; (ES") m/z 202 [M+H]"; (D) 'H NMR (500 MHz, DMSO-d) 6 6.97 (d, 1H), 6.31 (d, 1H), 6.14 (dd, 1H), 5.20 (s, 2H), 4.02 (dd, 2H), 3.66 (dd, 2H).

Stage 2 — Compound 39: 3.3.3-Trifluoropropyl N-[4-chloro-3-(2-methoxyethoxy)phenyl] carbamate *

P 00303-032 67 LU102907

Eternygen UK Ltd.

O N O F s ANS

O

Or

O F

CI

Compound 39 was prepared following Stage 2A - formation of carbamates (using EtsN as a base).

Pale orange oil (8%); LC/MS tr 3.40 min; (ES) m/z 342 [M+H]"; (B) 'H NMR (500 MHz, Chloroform-d) § 7.29 (s, 1H), 7.26 (d, 1H), 6.76 (dd, 1H), 6.63 (s, 1H), 4.39 (t, 2H), 4.20 - 4.17 (m, 2H), 3.80 (dd, 2H), 3.48 (s, 3H), 2.52 (qt, 2H).

Example 10: Synthesis of Compound 40

The synthesis route can be summarized by: — NS 0 NH, ___, NS 0 N_ 0 F

CI Stage 1 ry Stage 2 OU 1 "D cl cl

Stage 1 - 4-Chloro-3-(pyridin-4-ylmethoxy)aniline

N A

US | Tr

Cl

Potassium 2-methylpropan-2-olate (0.344 g, 3.07 mmol) was added to a solution of 5-amino-2- chlorophenol (0.200 g, 1.39 mmol) in anhydrous DMF (5 ml) at r.t. and the reaction stirred for 15 mins. 4-(Bromomethyl)pyridine hydrobromide (1:1) (0.423 g, 1.67 mmol) was added and the mixture was stirred for 16 h. The reaction mixture was evaporated to dryness and the residue was partitioned between EtOAc and sat. NaHCOs. The organic layer was separated, dried over MgSO4, filtered and concentrated in vacuo to give the crude product which was purified by silica flash column chromatography to give 0.025 g, (7%) 4-chloro-3-(pyridin-4-ylmethoxy)aniline as a brown solid.

LC/MS tr 1.31 min; (ES") m/z 235 [M+H]"; (D) 'H NMR (500 MHz, DMSO-de) 8 8.64 - 8.55 (m, 2H), 8.49 (dd, 1H), 7.43 (d, 2H), 7.31 (d, 1H), 7.02 (d, 1H), 5.25 (s, 2H), 5.15 (s, 2H).

Stage 2 — Compound 40: 3.3.3-trifluoropropyl N-[4-chloro-3-(pyridin-4-ylmethoxy)phenyl] carbamate *

NT

H

N o NO F

X haha 0 F

Cl

P 00303-032 68 LU102907

Eternygen UK Ltd.

Compound 40 was prepared following Stage 2A - formation of carbamates (using EtsN as a base).

Off-white solid (17%); LC/MS tr 2.52 min; (ES") m/z 375 [M+H]"; (B) 1H NMR (500 MHz, DMSO-d) 5 9.88 (s, 1H), 8.63 - 8.59 (m, 2H), 7.46 (d, 2H), 7.42 (s, 1H), 7.36 (d1H), 7.08 (dd, 1H), 5.22 (s, 2H), 4.31 (t, 2H), 2.70 (qt, 2H).

Example 11: Synthesis of Compound 41

The synthesis route can be summarized by:

HO NH, © NH, © H © F

Oo OO 0x0, cl Stage 1 cl stage2 NA qi O F

Stage 1 - 4-Chloro-3-(pyridin-4-yloxy)aniline

NO

CI

A pressure tube was charged with 5-amino-2-chlorophenol (0.168 g, 1.17 mmol), 4-iodopyridine (0.200 g, 0.976 mmol), copper (I) iodide (0.009 g, 0.049 mmol), pyridine-2-carboxylic acid (0.012 g, 0.098 mmol), anhydrous DMSO (3 ml, de-gassed with N, for 30 mins prior to use) and tripotassium phosphate (0.414 g, 1.95 mmol) respectively at r.t. The reaction was heated to 95 °C and stirred for 18 h. The reaction mixture was partitioned between EtOAc and H,O and the organic layer separated. The aqueous layer was extracted with EtOAc (5 x 50 ml). The organic layers were combined and concentrated in vacuo to give the crude product which was purified by silica flash column chromatography to give 0.100 g, (46%) of 4-chloro-3-(pyridin-4-yloxy)aniline as a pale yellow solid.

LC/MS tr 0.75 min; (ES) m/z 221 [M+H]"; (D) 'H NMR (500 MHz, DMSO-ds) § 8.45 (dd, 2H), 7.20 (d, 1H), 6.86 (dd, 2H), 6.52 (dd, 1H), 6.42 (d, 1H), 5.52 (s, 2H).

Stage 2 — Compound 41: 3.3.3-Trifluoropropyl N-[4-chloro-3-(pyridin-4-yloxy)phenyl] carbamate *

H

O N Oo F -

Di IN TO

No =z cl O F

P 00303-032 69 LU102907

Eternygen UK Ltd.

Compound 41 was prepared following Stage 2A - formation of carbamates (using EtsN as a base).

Pale yellow solid (23%); LC/MS fr 2.07 min; (ES") m/z 361 [M+H]"; (B) 'H NMR (500 MHz, DMSO-de) 5 10.08 (s, 1H), 8.71 - 8.25 (m, 2H), 7.57 (d, 1H), 7.48 (s, 1H), 7.41 (dd, 1H), 6.91 (dd, 2H), 4.31 (t, 2H), 2.70 (qt, 2H).

Example 12: Synthesis of Compounds 42, 43 and 44

The synthesis route can be summarized by:

H H H

OF OO LA

Stage 1 Stage 2

CI cl CI

Stage 1 — Compound 43: 3,3,3-Trifluoropropyl N-(3-chloro-5-hydroxyphenyl)carbamate

H

HO N_ 0 F

GY al

To a solution of 3,3,3-trifluoropropyl N-(4-chloro-3-methoxyphenyl)carbamate (Compound 17, 0.153 g, 0.51 mmol) in DCM (4 ml) was added BBrz (0.24 ml, 2.57 mmol) at -78 °C. The reaction mixture was stirred at this temperature for 30 mins, then at r.t. for 1 h. HO (5 ml) was added dropwise to quench the reaction and the mixture was extracted with DCM. The organic layer was dried over MgSO4 and concentrated in vacuo to give 0.087 g (58%) of 3,3,3-trifluoropropyl N-(3- chloro-5-hydroxyphenyl)carbamate as an off-white solid.

LC/MS fr 2.98 min; (ES) m/z 589 [2M+Na]"; (B) 'H NMR (500 MHz, DMSO-ds) 8 9.87 (s, 1H), 9.81 (s, 1H), 7.01 (s, 1H), 6.94 (s, 1H), 6.44 (t, 1H), 4.31 (t 2H), 2.70 (qt, 2H).

Stage 2 — Compound 42: 3.3.3-Trifluoropropyl N-[3-chloro-5-(cyclopropylmethoxy) phenyl]carbamate ° No F

A_ “ Y A al

A pressure tube was charged with Compound 43 (3,3,3-trifluoropropyl N-(3-chloro-5- hydroxyphenyl)carbamate; 0.040 g, 0.140 mmol), anhydrous MeCN (2 ml), potassium carbonate (0.039 g, 0.280 mmol) and (bromomethyl)cyclopropane (0.038 g, 0.280 mmol) respectively. The reaction was stirred at 80 °C for 16 h. The reaction mixture was filtered and the residue was washed with EtOAc (5 ml). The filtrate was concentrated in vacuo and the crude product was purified by

P 00303-032 70 LU102907

Eternygen UK Ltd. silica flash column chromatography to give 0.031 g (64%) of 3,3,3-trifluoropropyl N-[3-chloro-5- (cyclopropylmethoxy) phenyl]carbamate as a colourless oil.

LC/MS r 2.98 min; (ES") m/z 338 [M+H]"; (B) !H NMR (500 MHz, DMSO-de) § 9.82 (s, 1H), 7.34 (s, 1H), 7.29 (d, 1H), 7.02 (d, 1H), 4.31 (t, 2H), 3.84 (d, 2H), 2.70 (qt, 2H), 1.25 (ddd, 1H), 0.66 - 0.50 (m, 2H), 0.36 (q, 2H).

Compound 44: 3.3 3-trifluoropropyl N-[3-chloro-5-(difluoromethoxy)phenyl]carbamate

HO Ro F FeO NR. _o F

IS TK Y IS TOK

Stage cl CI

To a solution of Compound 43 (3,3,3-trifluoropropyl N-(3-chloro-5-hydroxyphenyl)carbamate; 0.087 g, 0.31 mmol) and potassium hydroxide (0.344 g, 6.14 mmol) in MeCN / HO (1:1) (4 ml) was added diethyl [bromo(difluoro)methyl phosphonate (0.11 ml, 0.610 mmol) at -78 °C. The reaction mixture was allowed to warm up to r.t. and stirred for 20 mins. The mixture was extracted with TBME (2 x 5 ml) and the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by silica flash column chromatography to give 0.031 g, (31%) of the required product as a pale yellow oil.

LC/MS tr 3.78 min; (ES?) m/z 332 [M-H]; (B) !H NMR (500 MHz, DMSO-de) 5 10.14 (s, 1H), 7.43 (s, 1H), 7.40 - 7.07 (m, 2H), 6.95 (t, 1H), 4.33 (t, 2H), 2.72 (qt, 2H).

Example 13: Synthesis of Compound 45

The synthesis route can be summarized by: 0 F F F H

Nw wm 70777 cl Stage 1 ci Stage 2 ci Stage 3 ci O F

Stage 1 - 1-Chloro-2-(difluoromethyl)-4-nitrobenzene

F

“eo”

Cl

To a solution of 2-chloro-5-nitrobenzaldehyde (0.300 g, 1.62 mmol) in DCM (5 ml) was added N- ethyl-N-(trifluoro-lambda~4~-sulfanyl)ethanamine (0.43 ml, 3.23 mmol) at -78 °C. The reaction

P 00303-032 71 LU102907

Eternygen UK Ltd. was allowed to warm up to r.t. and stirred for 16 h. The reaction was quenched with H>O (3 ml) and the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by silica flash column chromatography to give 0.298 g, (87%) of 1-chloro-2-(difluoromethyl)-4- nitrobenzene as a colourless oil. 'H NMR (500 MHz, DMSO-ds) 8 8.51 - 8.33 (m, 2H), 7.94 (d, 1H), 7.34 (t, 1H).

Stage 2 - 4-Chloro-3-(difluoromethyl)aniline

F

Or

Cl

To a solution of 1-chloro-2-(difluoromethyl)-4-nitrobenzene (0.100 g, 0.480 mmol) in MeOH (2 ml) and HO (1 ml) was added iron powder (0.081 g, 1.45 mmol) and ammonium chloride (0.103 g, 1.93 mmol). The reaction was stirred at 70 °C for 1 hr. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude product was purified by silica flash column chromatography to give 0.049 g, (57%) of 4-chloro-3-(difluoromethyl)aniline as a pale yellow solid.

LC/MS tr 1.16 min; (ES) m/z 178 [M+H]"; (D) 'H NMR (500 MHz, DMSO-ds) § 7.21 - 6.89 (m, 2H), 6.85 (d, 1H), 6.69 (dd, 1H), 5.55 (s, 2H).

Stage 3 — Compound 45: 3.3.3-Trifluoropropyl N-[4-chloro-3-(difluoromethyl)phenyl]carbamate

FF a. o FF 2.

Compound 45 was prepared following Stage 2A - General formation of carbamates (using Et:N as a base).

Colourless oil (21%); LC/MS tr 3.66 min; (ES) m/z 316 [M-HT; (B) 'H NMR (500 MHz, DMSO-de) 8 10.10 (s, 1H), 7.88 (s, 1H), 7.61 (d, 1H), 7.51 (d, 1H), 7.16 (t, 1H), 4.33 (t, 2H), 2.72 (qt, 2H).

Example 14: Synthesis of Compound 46

The synthesis route can be summarized by:

P 00303-032 72 LU102907

Eternygen UK Ltd. cl NO cl NH N cl NO, 2 2 Cl N O F

F er 070" "0%

Stage 1 _ St 2 _ Stage 3 cl 071 — we 071 — 07 —

O O o

Stage 1 - 1-Chloro-3-methanesulfonyl-5-nitrobenzene

NP

28 NO,

Cl

A mixture of 1, 3-dichloro-5-nitrobenzene (1 g, 5.21 mmol), sodium methanesulfinate (0.532 g, 5.21 mmol), copper (I) chloride (0.052 g, 0.520 mmol), quinoline (0.062 ml, 0.52 mmol) and NMP (5 ml) was stirred at 150 °C for 30 mins in the microwave. The reaction mixture was poured into ice water and extracted with DCM (2 x 50 ml). The organic layer was dried over MgSO, and concentrated in vacuo. The crude product was purified by silica flash column chromatography and further purified by prep-HPLC ((MeCN/Water, 2 mM NH4HCO;) to give 0.040 g, (3%) of 1-chloro- 3-methanesulfonyl-5-nitrobenzene as a pale yellow oil. 'H NMR (500 MHz, DMSO-de) 5 8.65 (t, 1H), 8.57 (t, 1H), 8.46 (t, 1H), 3.42 (s, 3H).

Stage 2 - 3-Chloro-5-methanesulfonylaniline 7 ase

Cl

To a solution of 1-chloro-3-methanesulfoyl-5-nitrobenzene (0.030 g, 0.130 mmol) in MeOH (1 ml) and HO (0.5 ml) was added iron powder (0.021 g, 0.380 mmol) and ammonium chloride (0.027 g, 0.510 mmol). The reaction was stirred at 70 °C for 1 hr. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude product was purified by silica flash column chromatography to give 0.025 g (74%) of 3-chloro-5-methanesulfonylaniline as an off-white solid.

LC/MS fz 1.08 min; (ES") m/z 206 [M+H]"; (D) 'H NMR (500 MHz, DMSO-de) § 7.02 (t, 1H), 6.97 (t, 1H), 6.85 (t, 1H), 6.02 (s, 2H), 3.18 (s, 3H).

Stage 3 — Compound 46: 3.3.3-Trifloropropyl N-(3-chloro-5-methanesulfonylphenyl) carbamate

P 00303-032 73 LU102907

Eternygen UK Ltd. cl N. 0 F

MF

O F oF—

O

Compound 46 was prepared following Stage 2B - General formation of carbamates (using NaHCO3 as a base).

White solid (55%); LC/MS tr 3.09 min; (ES) m/z 344 [M-H}; (B) "HNMR (500 MHz, DMSO-d) § 10.37 (s, 1H), 8.04 (s, 1H), 7.85 (t, 1H), 7.63 (t, 1H), 4.37 (t, 2H), 3.26 (s, 3H), 2.74 (qt, 2H).

Example 15: Synthesis of Compound 47

The synthesis route can be summarized by: cl Cl Cl

Stage 1 OSs NH. Stage 2 N NX

H,N NH, ° À 2 ° À H F

Stage 1 - N-(3-Amino-5-chlorophenyl)methanesulfonamide

Cl + 5 N NH,

To a stirring solution of 5-chlorobenzene-1,3-diamine (0.249 g, 1.75 mmol) and DIPEA (0.338 g, 2.62 mmol) in DCM (5 ml) was slowly added methanesulfonyl chloride (0.1 g, 0.87 mmol) at r.t. and the reaction stirred for 1 h. LCMS analysis showed the formation product and bis-addition product. The reaction was concentrated under reduced pressure and the crude product was purified by prep-HPLC (MeCN/Water, 0.1% HCO;H) to give 0.060 g (31%) of N-(3-amino-5- chlorophenyl)methanesulfonamide as a white solid.

White solid (31%); LC/MS tr 0.92 min; (ES) m/z 221 [M+H] ; (D) !H NMR (500 MHz, Chloroform-d) § 6.72 (s, 1H), 6.56 (t, 1H), 6.52 - 6.46 (m, 2H), 3.52 (s, 2H), 3.06 (s, 3H).

Stage 2 — Compound 47: 3.3.3-Trifluoropropyl N-(3-chloro-5-methanesulfonamidophenyl) carbamate *

P 00303-032 74 LU102907

Eternygen UK Ltd. cl 0 F5

Oss. J EN

NN N O F

O H H

Compound 47 was prepared following Stage 2A - General formation of carbamates (using Et:N as a base).

White solid (53%); LC/MS tr 2.88 min; (ES) m/z 361 [M+H]"; (B) 'H NMR (500 MHz, DMSO-de) § 6.62 (t, 1H), 6.56 (t, 1H), 6.42 (t, 1H), 5.61 (s, 2H), 4.36 (t, 2H), 3.54 (s, 3H), 2.72 - 2.59 (m, 2H).

Example 16: Synthesis of Compound 48

The synthesis route can be summarized by: cl ST HN — HN — = HAN

OK NO, Stage 1 A NO, Stage 2 A NH, Stage 3 A N 9 F oo oO oo oO

Stage 1 - 2-Chloro-5-nitrobenzene-1-sulfonamide

CI

HN TL

2 A NO,

OO

A mixture of 2-chloro-5-nitrobenzenesulfonyl chloride (0.3 g, 1.17 mmol) and aqueous ammonia (30%) (3 ml, 76.2 mmol) was stirred at r.t. for 2 h. The reaction was quenched with citric acid powder until pH was 5. The aqueous layer was extracted with DCM (3 x 4 ml) and the combined organic layers were dried over MgSO4 and concentrated in vacuo to give 0.257 g, (93%) of 2-chloro- 5-nitrobenzene-1-sulfonamide as a white solid.

LC/MS fr 0.91 min; (ES) m/z 235 [M-H]"; (D) 'HNMR (250 MHz, DMSO-de) 8 8.67 (d, 1H), 8.42 (dd, 1H), 7.98 (s, 2H), 7.95 (s, 1H).

Stage 2 - 5-Amino-2-chlorobenzene-1-sulfonamide

CI

HN, TL > NH,

O O

A mixture of 2-chloro-5-nitrobenzene-1-sulfonamide (0.257 g, 1.09 mmol), iron powder (0.182 g, 3.26 mmol), amine hydrochloride (0.232 g, 4.34 mmol) in EtOH (3 ml) and H>O (3 ml) was stirred

P 00303-032 75 LU102907

Eternygen UK Ltd. in a sealed tube at 70 °C for 1 hr. The reaction mixture was filtered through Celite®, washed with

MeOH and the filtrate was concentrated in vacuo. The residue was triturated with DCM (3 x 25 ml) and the combined washings were concentrated in vacuo to give 0.201 g, (90%) of 5-amino-2- chlorobenzene-1-sulfonamide as a pale brown solid.

LC/MS tr 0.72 min; (ES") m/z 207 [M+H]"; (D) 'H NMR (500 MHz, DMSO-de) § 7.30 (s, 2H), 7.22 (d, 1H), 7.18 (d, 1H), 6.68 (dd, 1H), 5.65 (s, 2H).

Stage 3 — Compound 48: 3.3.3-Trifluoropropyl N-(4-chloro-3-sulfamoylphenyl)carbamate * cl 0 Foor

HN, A AX 22 H F

IN oO

Compound 48 was prepared following Stage 2B - General formation of carbamates (using NaHCO; as a base).

Off-white solid (38%); LC/MS tr 2.59 min; (ES) m/z 693 [2M+H]"; (B) 'H NMR (500 MHz, DMSO-ds) 5 10.16 (s, 1H), 8.25 (s, 1H), 7.64 (dd, 1H), 7.54 (d, 3H), 4.33 (t, 2H), 2.72 (qt, 2H).

Example 17: Synthesis of Compound 49

The synthesis route can be summarized by: y ~&, $ $

F —_— — - = O F i No, Stage 1 F NG, Stage2 F NH, Stage 3 F ANKE

F F F F H

F F F

Stage 1 - 1-Methanesulfonyl-3-nitro-5-(trifluoromethyl)benzene ,0

Ds

O

F. - NO,

F

To a sealed tube were added 1-bromo-3-nitro-5-(trifluoromethyl)benzene (0.945 g, 3.50 mmol), sodium methanesulfinate (0.429 g, 4.20 mmol), Cul (0.067 g, 0.350 mmol), L-proline (0.081 g, 0.700 mmol), NaOH (0.028 g, 0.700 mmol) and DMSO (3 ml) at r.t. The tube was purged with N» and the reaction heated to 100 °C and stirred for 18 h. The reaction was cooled to r.t. and diluted

P 00303-032 76 LU102907

Eternygen UK Ltd. with water (10 ml). The aqueous layer was extracted with EtOAc (3 x 30 ml) and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo to give the crude product.

The product was purified by silica flash column chromatography to give 0.286 g, (25%) of 1- methanesulfonyl-3-nitro-5-(trifluoromethyl)benzene as a yellow solid.

LC/MS tr 1.18 min; (ES?) m/z 267 [M-H]; (D) "H NMR (250 MHz, CDCIs): § 8.99 (s, 1H), 8.77 (s, 1H), 8.54 (s, 1H), 3.19 (s, 3H).

Stage 2 - 3-Methanesulfonyl-5-(trifluoromethyl)aniline a

F NH,

F

To a round bottom flask were addd 1-methanesulfonyl-3-nitro-5-(trifluoromethyl)benzene (0.286 g, 0.861 mmol), iron (0.144 g, 2.58 mmol), ammonium chloride (0.184 g, 3.44 mmol), EtOH (3 ml) and water (3 ml) at r.t. and the reaction stirred and heated to 70 °C for 1 h. The reaction was cooled to r.t. and filtered through glass-fibre filter paper under suction. The filtrate was concentrated in vacuo to give the crude product. The product was purified by silica flash column chromatography to give 0.216 g, (100%) of 3-methanesulfonyl-5-(trifluoromethyl)aniline as a white solid.

LC/MS tr 1.06 min; (ES") m/z 240 [M+H]"; (D) 'H NMR (500 MHz, CDCls): 8 7.51 (s, 1H), 7.35 (t, 1H), 7.10 (s, 1H), 3.06 (s, 3H).

Stage 3 — Compound 49: 3.3.3-Trifluoroprpyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate ~ 7

S=o

O F

DO J AT

;- N° 0 F

F

Compound 49 was prepared following Stage 2B - General formation of carbamates (using NaHCO3 as a base).

White solid (28%); LC/MS tr 3.27 min; (ES) m/z 378 [M-H}; (B) 'H NMR (500 MHz, DMSO-ds): § 10.51 (s, 1H), 8.34 (s, 1H), 8.15 (s, 1H), 7.88 (s, 1H), 4.38 (t, 2H), 3.31 (s, 3H), 2.75 (qt, 2H).

Example 18: Synthesis of Compound 50

P 00303-032 77 LU102907

Eternygen UK Ltd.

The synthesis route can be summarized by:

SZ xx 0 Stage 1 Jol 0 ” 0 1 Er age Stage 2 Stage 3

ON NO, ON NO, ON NH, ON N LS

Stage 1 - 1-Methanesulfonyl-3,5-dinitrobenzene

O

Ne

ON NO,

To a round bottom flask were added 1-10do-3,5-dinitrobenzene (3.000 g, 10.2 mmol), sodium methanesulfinate (1.250 g, 12.2 mmol), Cul (3.109 g, 16.3 mmol) and DMF (37.5 ml) at r.t. and the reaction stirred and heated to 100 °C for 18 h. The reaction was cooled to r.t. and water (40 ml) was added. The precipitate which formed was collected by filtration under suction and dried in a vacuum oven at 40 °C for 2 h to give 5.40 g, (100%, 65% pure) 1-methanesulfonyl-3,5- dinitrobenzene as a brown solid which was used in subsequent reactions without further purification.

LC/MS fr 1.00 min; (ES) m/z 247 [M-H]"; (D) 'H NMR (500 MHz, DMSO-de): 8 9.22 - 8.67 (m, 3H), 3.49 (s, 3H).

Stage 2 - 3-Methanesulfonyl-5-nitroaniline

O

2,

ON : “NH,

To a round bottom flask were added 1-mthanesulfonyl-3,5-dinitrobenzene (5.40 g, 14.3 mmol, 65% pure), NaHSO; (4.41 g, 42.8 mmol), toluene (50 ml) and MeOH (250 ml) at r.t. and the reaction was stirred and heated to 100 °C and left for 18 h. The reaction was cooled to r.t., filtered and the filtrate concentrated in vacuo to give the crude product. The product was purified by silica flash column chromatography to give 1.11 g (33%) of 3-methanesulfonyl-5-nitroaniline as an orange solid.

LC/MS fr 0.87 min; (ES) m/z 215 [M-H]"; (D) 'H NMR (500 MHz, DMSO-d): 8 7.70 - 7.67 (m, 1H), 7.63 (t, 1H), 7.45 - 7.43 (m, 1H), 6.40 (s, 2H), 3.25 (s, 3H).

P 00303-032 78 LU102907

Eternygen UK Ltd.

Stage 3 — Compound 50: 3.3.3-Trifluoropropyl (3-methanesufonyl-5-nitrophenyl)carbamate

SL

Our

ON N°0 F

Compound 50 was prepared following Stage 2B - General formation of carbamates (using NaHCO3 as a base).

White solid (37%); LC/MS tr 2.95 min; (ES) m/z 355 [M-H}; (B) 'H NMR (500 MHz, DMSO-de): § 10.66 (s, 1H), 8.66 (s, 1H), 8.43 (s, 1H), 8.29 (t, 1H), 4.39 (t, 2H), 3.33 (s, 3H), 2.76 (qt, 2H).

Example 19: Synthesis of Compound 51

The synthesis route can be summarized by:

NH, cl cl ol

Ô Stage 1 OL Stage 2 Jol Stage 3 OL I A

ON NO, ON NO, ON NH, ON No F

Stage 1 - 1-Chloro-3,5-dinitrobenzene

Cl pou

To a 3 neck round bottom flask with a thermometer and dropping funnel were added 3,5- dinitroaniline (3.000 g, 16.4 mmol) and TFA (60 ml) at r.t. and the mixture stirred and cooled to 0 °C in an ice-water bath. Sodium nitrite (2.294 g, 32.8 mmol) was added in one portion and the reaction was left to cool to below 5 °C. A solution of CuCl (5.920 g, 59.8 mmol) in conc. HCI (60 ml) was added to the dropping funnel and the solution slowly added dropwise over 45 mins maintaining an internal temperature below 5 °C. After this time the reaction had become a thick slurry and vigorous stirring was required to homogenise the mixture. After complete addition the reaction was stirred for a further 30 mins. The reaction was quenched by pouring onto ice (~250 g) and extracted with EtOAc (250 ml). The organic layer was separated and sat. NaHCOs3 solution (100 ml) was added followed by NaHCO; until pH >7. The organic layer was separated and washed with brine (100 ml), dried over MgSO, filtered and concentrated in vacuo to give the crude

P 00303-032 79 LU102907

Eternygen UK Ltd. product. The product was purified by silica flash column chromatography to give 2.69 g, (81%) of 1-chloro-3,5-dinitrobenzene as a dark green solid.

LC/MS fr 1.25 min; (ES") m/z does not ionise; (D) 'H NMR (500 MHz, CDCl): 5 8.96 (t, 1H), 8.56 (d, 2H).

Stage 2 - 3-Chloro-5-nitroaniline

Cl

O,N | NH,

To a round bottom flask were added 1-chlor-3,5-dinitrobenzene (2.273 g, 11.2 mmol) and EtOH (35 ml) at r.t. and the reaction was stirred. Diammonium sulfide (20% w/w in H20) (7.4 ml, 22.4 mmol) was slowly added dropwise and the reaction heated to 80 °C for 30 mins. The reaction was cooled to r.t. and the mixture diluted with EtOAc (340 ml) and brine (135 ml). The organic layer was separated and dried over MgSO,, filtered and concentrated in vacuo to give the crude product.

The product was purified by silica flash column chromatography to give 3-chloro-5-nitroaniline 1.63 g, (80%) as an orange solid.

LC/MS tr 1.20 min; (ES) m/z 173 [M+H]"; (D) 'H NMR (500 MHz, DMSO-de): 8 7.34 (t, 1H), 7.26 (t, 1H), 6.96 (t, 1H), 6.14 (s, 2H).

Stage 3 — Compound 51: 3.3.3-Trifluoropropyl (3-chloro-5-nitrophenyl)carbamate

Cl

Our

O,N NT VO F

H

Compound 51 was prepared following Stage 2B - General formation of carbamates (using NaHCO3 as a base).

Pale yellow solid (80%); LC/MS tr 3.70 min, (ES) m/z 311 [M-H]; (B) 'H NMR (500 MHz, DMSO-de): 10.46 (s, 1H), 8.36 (s, 1H), 7.92 (t, 1H), 7.90 (t, 1H), 4.37 (t, 2H), 2.74 (qt, 2H).

Example 20: Synthesis of Compound 52

Compound 52: 3,3,3-Trifluoropropyl (3-chloro-5-(methylsulfanyl)phenyl)carbamate

P 00303-032 80 LU102907

Eternygen UK Ltd.

CI

10008:

A stirring solution of Compound 27 (3,3,3-trifluoropropyl N-(3-bromo-5-chlorophenyl)carbamate; 0.615 g, 1.77 mmol), (methylsulfanyl)sodium (0.140 g, 1.95 mmol), Xantphos (0.100 g, 0.180 mmol) and DIPEA (0.59 ml, 3.55 mmol) in toluene (10 ml) was degassed by bubbling N; through the solution for 15 mins. Pd2(dba)3 (0.081 g, 0.090 mmol) was added to the solution. The reaction mixture was heated at 110 °C for 15 h. The reaction was tested by LCMS which showed the formation of a species with the desired mass of the product. The reaction was worked up; the reaction mixture was filtered through Celite, the filtrate was concentrated under reduced pressure.

The crude residue obtained was purified silica flash column chromatography to afford 0.377 g, (68%) of 3,3,3-trifluoropropyl (3-chloro-5-(methylsulfanyl)phenyl)carbamate as a green oil.

LC/MS tr 3.90 min; (ES") m/z 313 [M+H]"; (B) 'H NMR (250 MHz, DMSO-de) § 9.96 (s, 1H), 7.35 (dd, 2H), 6.94 (t, 1H), 4.33 (t, 2H), 2.73 (dp, 2H), 2.47 (s, 3H).

C. Methyl trifluoromethane carbamates

Described herein are methyl trifluoromethane carbamates of the following structure:

X

Ar F

H F

F .

A general synthetic route for the preparation of methyl trifluoromethane carbamates can be summarized by:

O ads, - \—/ \/ Jig

NH H —_— Ar F

Ar” 2 + 0K, ~N 0" K

F Stage 1 H Er

Stage 1 - General formation of carbamates via CDI method.

To a stirring solution of aniline (0.1 g, 1 eq.) and 2,2,2-trifluoroethanol (1 eq.) in DCM (5 ml) was added di-1H-imidazol-1-ylmethanone (1.1 eq.) and the reaction stirred at r.t. for 90 h. LCMS analysis after this time showed minimal starting material remained and symmetric urea and product

P 00303-032 81 LU102907

Eternygen UK Ltd. had formed. The reaction was concentrated in vacuo to give the crude product. The product was purified by prep-HPLC (MeCN/Water, 0.1% HCO»H)) to give the carbamate.

Example 21: Synthesis of Compounds 53 and 54

Compounds 53 and 54 were prepared according to the above method.

Compound 53: 2.2.2-Trifluoroethyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate

Cl

A X À

F F

Yellow oil (15%); LC/MS 4.49 min; (ES”) m/z 321 [M-HJ; (A) !H NMR (500 MHz, DMSO-de) 5 10.60 (s, 1H), 8.01 (d, 1H), 7.74 (dd, 1H), 7.68 (d, 1H), 4.84 (q, 2H).

Compound 54: 2,2,2-Trifluoroethyl N-[4-chloro-3-(trifluoromethyl)phenyl]carbamate

CI

JO À

F F

N O

F H Ke

F F

Yellow oil (31%); LC/MS tr 4.36 min; (ES?) m/z 321 [M-H]; (A) 'H NMR (500 MHz, DMSO-de) 6 10.69 (s, 1H), 7.82 (s, 2H), 7.53 (s, 1H), 4.85 (q, 2H).

An alternative general synthetic route for the preparation of methyl trifluoromethane carbamates via chloroformate can be summarized by:

LA 1

HO F Ar F

YY — Noy — ON 7

F Stage 1 F Stage 2 H F

Stage 1 - (But-3-yn-1-yloxy)(chloro)methanone

O

F

A 3 neck round bottom flask equipped with a bubbler, N, inlet and a septum was purged with Na and then triphosgene (5.43 g, 18.3 mmol) and dry ether (60 ml) were added at r.t. and the reaction stirred until the triphosgene had dissolved. Activated charcoal (0.150 g) was added and the reaction

P 00303-032 82 LU102907

Eternygen UK Ltd. was left for 1 h. The reaction was cooled to 0 °C and 2,2-difluoroethanol (2.3 ml, 36.6 mmol) in dry ether (60 ml) was slowly added dropwise. The reaction was slowly warmed to r.t. and left for 18 h. The reaction was filtered and concentrated in vacuo to give 2.386 g (41%, 90% pure by 'H

NMR) of crude chloro(2,2-difluoroethoxy)methanone as a colourless oil which was used in subsequent reactions without further purification. 'H NMR (500 MHz, DMSO-de) 8 5.95 (tt, 1H), 3.58 (td, 2H).

Stage 2 - Formation of methyl trifluoromethane carbamates of the following general formula:

X

Ar F

H

F

To a vial were added aniline (0.2 g, 1 eq.), NaHCOs (3 eq.) and DCM (2.5 ml) at r.t. and the mixture stirred. Chloro(2,2-difluoroethoxy)methanone (1.05 eq.) in DCM (2.5 ml) was slowly added dropwise and the reaction was purged with N> and stirred for 18 h-5 days. TLC and LCMS analysis after this time showed starting amine was consumed and product had formed. The reaction was filtered and concentrated in vacuo to give the crude product. The product was purified by silica flash column and/or prep-HPLC ((MeCN/Water, 2 mM NH;HCOs) or (MeCN/Water) or (MeCN/Water, 0.1% HCO:H)) to give the carbamate.

Example 22: Synthesis of Compounds 55 — 58

Compounds 55 — 58 were prepared according to the above alternative method via chloroformate.

Compound 55: 2,2-Difluoroethyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate

CI

OL

F F

Yellow oil (25%); LC/MS tr 3.82 min; (ES) m/z 302 [M-HT; (B) 'H NMR (500 MHz, DMSO-de) § 10.54 (s, 1H), 7.81 (d, 2H), 7.49 (s, 1H), 6.33 (tt, 1H), 4.44 (td, 2H).

Compound 56: 2.2-Difluoroethyl (3.5-dichlorophenyl)carbamate

P 00303-032 83 LU102907

Eternygen UK Ltd.

CI

D I

F cl N oY

F

White solid (46%); LC/MS tr 2.98 min; (ES") m/z 268 [M-H]’; (F) 'H NMR (400 MHz, CDCIs): 8 7.34 (d, 2H), 7.10 (t, 1H), 6.76 (bs, 1H), 6.13 - 5.85 (m, 1H), 4.37 (m, 2H).

Compound 57: 2,2-Difluoroethyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate*

CI

JA

F F

White solid (35%); LC/MS tr 4.03 min; (ES) m/z 302 [M-HJ; (F) 'H NMR (400 MHz, CDCIs): § 7.74 (d, 1H), 7.56 (d, 1H), 7.46 (d, 1H), 6.83 (bs, 1H), 6.13 - 5.86 (m, 1H), 4.39 (ddd, 2H), 1.26 (s, 1H).

Compound 58: 2,2-Difluoroethyl (3,4-dichlorophenyl)carbamate*

Cl

O

TL J F cl N of

H

F

Colourless solid (24%); LC/MS tr 4.00 min; (ES) m/z 268 [M-H]; (F) 'H NMR (400 MHz, CDCI-): 8 7.61 (d, 1H), 7.38 (d, 1H), 7.24 - 7.13 (m, 1H), 6.76 (bs, 1H), 6.13 - 5.85 (m, 1H), 4.37 (td, 2H), 1.36 - 1.21 (m, 1H).

D. Substituted carbamates

Further described herein are carbamates of the following general structure:

À

Ar _R “NT To

H .

A general synthetic route for the preparation of such carbamates via chloroformate can be summarized by:

O O

R

Hoo T= À RR Ar AR

Stage 1 Cl O Stage 2 H

Stage 1 — Chloroformates

P 00303-032 84 LU102907

Eternygen UK Ltd.

O

A R

CI O

A solution of triphosgene (1 eq.) in dry ether (30 ml) and catalytic amount of activated charcoal was stirred for 0.5 h. The reaction mixture was cooled to 0 °C and the respective alcohol (1 g, 2 eq.) was added drop-wise. The resultant solution was stirred at r.t. for 12 h and filtered. The filtrate was concentrated and the liquid was used as such for the next step. The products were confirmed by 'H

NMR.

Stage 2 - General formation of carbamates (using NaHCO; as a base)

À

Ar R “NT Do

H

To a solution of the amine (1 eq.) in anhydrous DCM, sodium hydrogen carbonate (1.2 eq.) was added. The suspension was cooled to 0 °C and then the respective chloroformate (1.1 eq.) was added drop-wise over a period of 15 min. After 12 h stirring at r.t., the reaction mixture was filtered. The filtrate was washed with saturated sodium bicarbonate solution, followed by brine, dried over anhydrous sodium sulphate and evaporated to afford the corresponding carbamates after purification by either silica flash column chromatography and/or prep-HPLC ((MeCN/Water, 2 mM

NH4HCO3) or (MeCN/Water) or (MeCN/Water, 0.1% HCO»H)) to give the carbamate.

Example 23: Synthesis of Compounds 59 - 68

Compounds 59 - 68 were prepared according to the above method.

Compound 59: Prop-2-yn-1-yl (3,4-dichlorophenyl)carbamate

Cl

A

Cl

N 0x

White solid (53%); LC/MS fr 2.88 min; (ES") m/z 242 [M-HJ; (F) 'H NMR (400 MHz, CDCI-): § 7.63 (d, 1H), 7.37 (d, 1H), 7.20 (dd, 1H), 6.67 (bs, 1H), 4.79 (d, 2H), 2.53 (t, 1H).

Compound 60: Prop-2-yn-1-yl (4-chloro-3-(trifluoromethyl)phenyl)carbamate

P 00303-032 85 LU102907

Eternygen UK Ltd.

CI

De x

F

Fl À °

White solid (77%); LC/MS tr 2.93 min; (ES?) m/z 276 [M-HJ; (F) 'H NMR (400 MHz, CDCl): 5 7.75 (d, 1H), 7.56 (dd, 1H), 7.44 (d, 1H), 6.80 (bs, 1H), 4.80 (d, 2H), 2.54 (t, 1H).

Compound 61: Prop-2-yn-1-yl (3,5-dichlorophenyl)carbamate

Cl 2 x

CI N os

White solid (50%); LC/MS fr 2.93 min; no ionization; (F) "HNMR (400 MHz, CDCl): 5 7.35 (d, 2H), 7.08 (q, 1H), 6.70 (bs, 1H), 4.79 (d, 2H), 2.54 (q, 2H).

Compound 62: But-2-yn-1-yl (3.5-dichlorophenyl)carbamate

Cl 7 I

CI N “=

White solid (75%); LC/MS fr 3.14 min; (ES) m/z 258 [M-H]". (F) !H NMR (400 MHz, CDCls): § 7.34 (d, 2H), 7.06 (t, 1H), 6.65 (bs, 1H), 4.75 (m, 2H), 1.88 (t, 3H).

Compound 63: Allyl (3.5-dichlorophenyl)carbamate

Cl 2. I cl = oF

White solid (40%); LC/MS tr 3.05 min; (ES) m/z 244 [M-HJ; (F) !H NMR (400 MHz, CDCls): § 7.35 (d, 2H), 7.06 (t, 1H), 6.65 (bs, 1H), 5.96 (m, 1H), 5.43 - 5.24 (m, 2H), 4.67 (m, 2H).

Compound 64: Allyl (3.4-dichlorophenyl)carbamate

P 00303-032 86 LU102907

Eternygen UK Ltd.

Cl

Lk cl NN

H

White solid (90%); LC/MS fr 1.90 min; no ionization; (E) !H NMR (400 MHz, CDCls): § 7.62 (d, 1H), 7.35 (d, 1H), 7.20 (dd, 1H), 6.62 (bs, 1H), 5.96 (m, 1H), 5.42 - 5.24 (m, 2H), 4.67 (m, 2H).

Compound 65: Allyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate

Cl

L

F

A oF

F H

F

White solid (16%); LC/MS tr 2.98 min; (ES) m/z 278 [M-HJ; (F) "HNMR (400 MHz, CDCIs): § 7.78 - 7.71 (m, 1H), 7.60 - 7.52 (m, 1H), 7.43 (d, 1H), 6.74 (bs, 1H), 6.04 - 5.89 (m, 1H), 5.43 - 5.25 (m, 2H), 4.68 (m, 2H).

Compound 66: 2,2,2-Trifluoroethyl (3,5-dichlorophenyl)carbamate

Cl

Jol À

F

Cl

White solid (5%); LC/MS fr 2.62 min; (ES”) m/z 287 [M-H]; (F)

HNMR (400 MHz, CDCl): § 7.35 (d, 2H), 7.12 (q, 1H), 6.80 (bs, 1H), 4.62 - 4.51 (m, 2H), 1.36 - 1.23 (m, 1H).

Compound 67: 2.2 2-Trifluoroethyl (3.4-dichlorophenyl)carbamate™

Cl

Qt

F

CI N

H °F

Sticky solid (9%); LC/MS t 4.19 min; (ES) m/z 286 [M-H]"; (G) !H NMR (400 MHz, CDCl): § 7.62 (s, 1H), 7.39 (d, 1H), 7.25 - 7.17 (m, 1H), 6.77 (s, 1H), 4.56 (q, 2H), 1.28 (m, 2H).

Compound 68: Propyl (3,5-dichlorophenyl)carbamate*

P 00303-032 87 LU102907

Eternygen UK Ltd.

CI

0 I ° Age

H

White solid (39%); LC/MS tr 3.31 min; (ES) m/z 246 [M-HJ; (F) 'H NMR (400 MHz, CDCIs): 5 7.34 (d, 2H), 7.04 (d, 1H), 6.59 (bs, 1H), 4.14 (t, 2H), 1.70 (m, 2H), 0.98 (t, 3H).

E. Ethyl difluoromethane carbamates

Described herein are ethyl difluoromethane carbamates of the following structure:

À A

Ar AA “NT To F

H .

A general synthetic route for the preparation of these ethyl difluoromethane carbamates can be summarized by: 0” "0H 0X

HO” "oH —— = —

Stage 1 Stage 2

F

AA F 0 F

Stage 3 Stage4 HO F Stage5 C| O F

O F

—— a AAA

Stage 6 N O F

H

Stage 1 - 3-(Benzyloxy)propan-1-ol

NaH (60% dispersion in mineral oil, 2.31 g, 57.6 mmol) was diluted with dry DMF (75 ml) and the solution stirred at 0 °C under N». Propane-1, 3-diol (3.8 ml, 52.6 mmol) was slowly added and the reaction stirred for 10 min. Benzyl bromide (6.3 ml, 52.6 mmol) was added dropwise and the reaction was left to warm to r.t. over 18 h. The reaction was quenched with water (100 ml) and extracted with EtOAc (6 x 50 ml). The combined organic layers were washed with brine (50 ml), dried over anhydrous MgSO4, filtered and concentrated in vacuo to give the crude product which was purified by flash column chromatography using 100% heptane, then 40% EtOAc: 60% heptane

P 00303-032 88 LU102907

Eternygen UK Ltd. as eluent to give 3-(benzyloxy)propan-1-ol as a pale yellow oil (5.31 g, 61%).

LC/MS tr 1.05 min; (ES) m/z 167 [M+H]"; (D) 'H NMR (500 MHz, DMSO-de) § 7.31 (ddt, 5H), 4.44 (s, 2H), 4.38 (t, 1H), 3.52 - 3.43 (m, 4H), 1.69 (qt, 2H).

Stage 2 - 3-(Benzyloxy)propanal

To a stirred solution of 3-(benzyloxy)propanal (2.5 g, 0.015 mmol) in DCM (50 ml) at r.t. was added Dess-Martin periodinane (7.02 g, 16.54 mmol). The suspension was stirred at r.t. for 6 h.

The reaction mixture was diluted with DCM (25 ml), concentrated onto silica under reduced pressure and purified by automated flash column chromatography (FCC, 100% heptane to 50: 50 heptane : EtOAc) to afford 2.08 g (84.2%) of 3-(benzyloxy)propanal as a colourless oil.

LC/MS tr 1.10 min; (ES") m/z N/A [M+H]"; (D) 'H NMR (500 MHz, DMSO-de) § 9.67 (t, 1H), 7.41 - 7.22 (m, 5H), 4.47 (s, 2H), 3.75 (t, 2H), 2.66 (td, 2H).

Stage 3 - ((3,3-Difluoropropoxy)methyl)benzene

To a stirred solution of 3-(benzyloxy)propanal (2.08 g, 12.67 mmol) in DCM (50 ml) cooled to -78 °C was added DAST (3.35 ml, 25.33 mmol) dropwise over 10 min. The reaction was stirred at -78 °C for 1 hand allowed to warm to r.t.. Saturated NaHCOs (20 ml, aq) was added, the layers were separated, the aqueous extract was washed with DCM (3 x 20 ml), the organic extracts were combined, washed with brine (20 ml), dried over anhydrous Na:SO4 and concentrated in vacuo.

The crude residue was purified by automated FCC (100% heptane to 30:70 EtOAc: heptane) to afford 1.82 g (77.2%) of ((3,3-difluoropropoxy)methyl)benzene as a colourless oil.

LC/MS tr 1.33 min; (ES") m/z N/A [M+H]"; (D) 'H NMR (500 MHz, DMSO-de) 8 7.52 - 7.14 (m, SH), 6.14 (tt, 1H), 4.49 (s, 2H), 3.57 (t, 2H), 2.13 (ddt, 2H).

Stage 4 - 3,3-Difluoropropan-1-ol

A stirred solution of ((3,3-difluoropropoxy)methyl)benzene (1.75 g, 9.4 mmol) in DCM (100 ml) was evacuated and filled with N, (x 3). Pd(OH)» (20%, 0.66 g, 0.94 mmol) was added to the solution. The reaction mixture was evacuated and filled with H> (x 3). The reaction was stirred overnight (~15 h) at r.t.. The reaction was filtered and the filtrate concentrated under reduced pressure to give 1.89 g (85.8%) of 3,3-difluoropropan-1-ol as a colourless oil. (42% pure, in toluene derived from debenzylation and DCM). The residual solvent was not removed due to the volatility of product. The crude was used in the next step without further purification. 'H NMR (250 MHz, DMSO-de) 5 6.10 (tt, 1H), 4.75 (t, 1H), 3.65 - 3.47 (m, 2H), 1.97 (ttd, 2H).

P 00303-032 89 LU102907

Eternygen UK Ltd.

Stage 5- Chloro(3,3-difluoropropoxy)methanone

To a stirred solution of triphosgene (1.39 g, 4.7 mmol) in Et;O (20 ml) cooled to 0 °C was added activated charcoal (112.79 mg, 9.4 mmol), the mixture was stirred for 30 min at 0 °C. 3, 3- difluoropropan-1-ol (0.9 g, 9.4 mmol) in Et,O (10ml) was added. The reaction was warmed to r.t. and was stirred for 63 h. The reaction was worked up; filtered and the filtrate concentrated under reduced pressure to afford 1.54 g (57.4%) of chloro(3,3-difluoropropoxy)methanone as a colourless oil. (crude 55% pure, in Et20, toluene). The residual solvent was not removed and crude product used directly in the next step. 'H NMR (250 MHz, DMSO-d) § 7.32 - 7.08 (m, 2H), 6.09 (tt, 1H), 3.54 (t, 2H), 3.38 (q, 2H), 2.30 (s, 2H), 1.96 (ttd, 2H).

Stage 6 - General formation of carbamates (using NaHCO3 as a base)

À

Ar AA “NT To F

H

The general method is identical to Stage 2B (experimental for compounds 30 — 33), except that 3,3- difluoropropyl chloroformate is used instead of 3,3,3-trifluoropropyl chloroformate.

Example 24: Synthesis of Compounds 69 - 78

Compounds 69 - 78 were prepared according to the above method.

Compound 69: 3.3-Difluoropropyl (3.5-dichlorophenyl)carbamate

Cl

OQ À A cl N Ar

Orange oil (62%); LC/MS fr 4.46 min; (ES*) m/z 286 [M+H]"; (C) 'H NMR (250 MHz, DMSO-de) § 10.11 (s, 1H), 7.52 (d, 2H), 7.21 (t, 1H), 6.19 (tt, 1H), 4.26 (t, 2H), 2.38 - 2.10 (m, 2H).

Compound 70: 3.3-Difluoropropyl (3-chloro-5-(trifluoromethyl)phenyl)carbamate

CF,

OQ À A cl N ENN

H

Orange oil (64%); LC/MS f 4.53 min; (ES") m/z 316 [M+H]"; (C) 'H NMR (250 MHz, DMSO-ds) 8 10.27 (s, 1H), 7.82 (d, 2H), 7.47 (s, 1H), 6.21 (tt, 1H), 4.29 (t,

P 00303-032 90 LU102907

Eternygen UK Ltd. 2H), 2.37 - 2.15 (m, 2H).

Compound 71: 3,3-Difluoropropyl (3-nitro-5-(trifluoromethyl)phenyl)carbamate

CF, 2 Lu

ON N Ar

H

Orange oil (51%); LC/MS fr 3.65 min; (ES") m/z 328 [M+H]"; (B) 'H NMR (250 MHz, DMSO-d) 8 10.58 (s, 1H), 8.63 (t, 1H), 8.23 (s, 1H), 8.11 (s, 1H), 6.22 (tt, 1H), 4.32 (t, 2H), 2.42 - 2.13 (m, 2H).

Compound 72: 3,3-Difluoropropyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate*

CF,

CI

OL LA

N SAS

H

Colourless oil (50%); LC/MS fr 4.42 min; (ES") m/z 316 [M+H]"; (C) 'H NMR (250 MHz, DMSO-ds) & 10.18 (s, 1H), 8.03 (d, 1H), 7.83 - 7.57 (m, 2H), 6.20 (tt, 1H), 4.27 (t, 2H), 2.37 - 2.15 (m, 2H).

Compound 73: 3,3-Difluoropropyl (3.,5-bis(trifluoromethyl)phenyl)carbamate

CF,

OL À

CF; N Ar

H

White solid (67%); LC/MS fr 4.01 min; (ES") m/z 350 [M+H]"; (B) 'H NMR (250 MHz, DMSO-d) 8 10.42 (s, 1H), 8.13 (s, 2H), 7.70 (s, 1H), 6.21 (tt, 1H), 4.31 (t, 2H), 2.27 (ttd, 2H).

Compound 74: 3,3-Difluoropropyl (3-chloro-5-nitrophenyl)carbamate

Cl 0 JP! o,N N oN

H

Orange oil (55%); LC/MS fr 3.52 min; (ES") m/z 294 [M+H]"; (B)

P 00303-032 91 LU102907

Eternygen UK Ltd. 'H NMR (500 MHz, DMSO-de) § 10.42 (s, 1H), 8.35 (t, 1H), 7.92 (t, 1H), 7.90 (t, 1H), 6.22 (tt, 1H), 4.30 (t, 2H), 2.27 (ttd, 2H).

Compound 75: 3,3-Difluoropropyl (3-bromo-5-chlorophenyl)carbamate

CI

Q À A

Br N SE

H

Colourless oil (93%); LC/MS ?r 3.89 min; (ES") m/z 327 [M+H]"; (B) 'H NMR (500 MHz, DMSO-de) § 10.10 (s, 1H), 7.66 (t, 1H), 7.56 (t, 1H), 7.33 (t, 1H), 6.20 (tt, 1H), 4.26 (t, 2H), 2.25 (ttd, 2H).

Compound 76: 3.3-Difluororopyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate

O

>

O F

F M AA

N O F

F H

F

Compound 76 was prepared from previously described 3-methanesulfonyl-5- (trifluoromethyl)aniline.

White solid (53%); LC/MS tr 3.10 min; (ES) m/z 360 [M-H}; (B) 'H NMR (500 MHz, DMSO-de) § 10.47 (s, 1H), 8.32 (s, 1H), 8.14 (s, 1H), 7.87 (s, 1H), 6.21 (tt, 1H), 4.30 (t, 2H), 3.31 (s, 3H), 2.27 (ddt, 2H).

Compound 77: 3.3-Difluoroprpyl N-(3-methanesulfonyl-5-nitrophenyl)carbamate ~ r

Sw0o

QL XJ

ON N Ar

H

(Compound 77 was prepared from previously described 3-methanesulfonyl-5-nitroaniline.

White solid (63 %); LC/MS tr 2.76 min; (ES) m/z 337 [M-H}; (B) 'H NMR (500 MHz, DMSO-de) § 10.62 (s, 1H), 8.65 (t, 1H), 8.43 (s, 1H), 8.37 - 8.25 (m, 1H), 6.22 (tt, 1H), 4.33 (t, 2H), 3.34 (s, 3H), 2.35 - 2.22 (m, 2H). (Compound 78: 3.3-Difluoropropyl (3-chloro-5-(methylsulfanyl)phenyl)carbamate

P 00303-032 92 LU102907

Eternygen UK Ltd.

Cl 0. O F

ALIA

To a stirred solution of Compound 75 (3,3-difluoropropyl (3-bromo-5-chlorophenyl)carbamate; 0.307 g, 1.14 mmol), (methylsulfanyl)sodium (0.090 g, 1.25 mmol), Xantphos (0.070 g, 0.110 mmol) and DIPEA (0.38 ml, 2.27 mmol) were degassed with a stream of N; for 15 min. Pd»(dba)s (0.052 g, 0.060 mmol) was added. The reaction mixture was heated for 15 h at 110 °C. The mixture was filtered through a pad of celite, the filtrate was concentrated under reduced pressure and the crude residue obtained was purified by prep-HPLC (MeCN/Water, 0.1% HCO»H) to afford 0.132 g, (39%) of 3,3-difluoropropyl (3-chloro-5-(methylsulfanyl)phenyl)carbamate as a light yellow oil.

LC/MS fr 3.73 min; (ES") m/z 295 [M+H]"; (B)

HNMR (500 MHz, DMSO-de) 8 9.92 (s, 1H), 7.35 (s, 1H), 7.32 (s, 1H), 6.93 (t, 1H), 6.20 (tt, 1H), 4.25 (t, 2H), 2.47 (s, 3H), 2.25 (ttd, 2H).

Procedures and assays for characterizing biological activity

Compounds were tested using a functional citrate uptake assay with HEK293 cells overexpressing recombinant human INDY. To confirm activity of selected compounds on the target INDY another cell assay was conducted. In this assay system citrate uptake was measured using HepG2 cells, which endogenously express human INDY. Selectivity of selected compounds was tested for two other transporters in different assay systems. The first transporter was the glutamate transporter

GLT-1 tested in a glutamate uptake assays using HEK293 cells overexpressing recombinant human

GLT-1. The second transporter was the glucose transporter GLUT] tested in a glucose uptake assay using Huh-7 cells which endogenously express GLUTI. Compounds were considered to be selective with an at least 5 fold higher activity for INDY than for the other two transporters. Species specificity was controlled in HEK293 cells overexpressing mouse INDY.

Cell cultivation media and reagents were obtained from standard suppliers. Cultivation of cells was done as known by a person ordinary skilled in the art (comp. Green and Sambrook, Molecular

Cloning: A laboratory handbook, 4% Edition 2012, Cold Spring Harbour Laboratory Press).

Cells were maintained in cell medium using cell culture grade flasks (CellSTACK Coming, 500cm2 dishes or T175 depending on cell number). The selection antibiotic and G418 (800ug/ml) was added during cultivation but not for seeding into assay plates. For splitting, cells were washed with PBS (w/o Ca2+, Mg2+, phenol red) and detached with Trypsin/EDT A. Throughout cultivation cells were kept sub-confluent. Detached cells (50,000 cells/well) were seeded in 96-well plates (white, clear

P 00303-032 93 LU102907

Eternygen UK Ltd. bottom from Corning #655098) and incubated over night before usage in the assay. The following media were used. For HEK293 cells expressing INDY MEM (PAA)+ 10% FCS + 1xP/S+ 2mM L-

Gln; for HEK293 cells expressing GLT-1 DMEM (Gibco) + 10% FCS + 400ug/ml G418; for Huh7 cells DMEM (Gibco) + 10% FCS + 2mM L-Gin (+1x P/S ); for HepG2 cells MEM (PAA, Cat#

E15-024) + 10% FCS + 2mM L-Gin+ 1x NEAA+1mM Na-Pyruvat

The following assay buffers for the particular uptake reaction were used. Citrate uptake buffer: 120mM NaCl, 5.4mM KCI, 0.8mM MgSO4, SmM glucose, 1.8mM CaCl2, 25mM Hepes, 25mM

MES, pH 6.5. Glutamate uptake buffer: HBSS, ImM CaCl2, I1mM MgCI2, 20mM Hepes pH 7.4.

Glucose uptake buffer: 25mM Hepes + 25mM MES pH 7.4, 120mM NaCl, 5.4mM KCI, 1.8mM

CaCl2, 0.8mM MgSO4.

INDY mediated citrate uptake in HEK293 cells

INDY mediated citrate uptake was determined in HEK293 cells. To determine the uptake of *C labelled citrate into cells, HEK293 cells over-expressing human NaCT were used. Cloning of human NaCT was done in analogy as described in Birkenfeld et al. (Birkenfeld et al., 2011, Cell

Metabolism 14, 184-195, 2011).

Human NaCT HEK293 cells were seeded into white clear-bottom 96-well plates (50,000/well) in the presence of 4ug/ml Poly-D-Lysine and incubated at 37°C for 16-24 hours. On the days of the assay the plates were washed once with assay buffer using automated washing / LS 405 Selectors,

BioTek ending with a buffer volume of 40ul. Then Sul of compound solution in assay buffer was added and incubated for 5-30 min at 37°C in cell incubator, HERA cell Heraeus. Afterwards 10ul of substrate 14C-citrate (1.5nM / 8.7.4nCi per well) were added and incubated for another 20 - 90 min at 37°C in cell incubator, HER Acell. Then plates were washed twice with 200ul ice-cold assay buffer and residual volume was discarded. For cell lysis 50ul ice-cold NaOH (100mM) was added and incubated for 15 minutes at RT on a plate shaker. Finally, 200ul scintillation fluid (scintillator

OptiPhase Supermix, Perkin Elmer) was added. After 15 min incubation at RT without shaking, plates were measured using a TopCount reader system (Perkin Elmer). As a positive control 10mM citrate was applied. It is possible to use this assay also in analogy in a 384-well plate format.

INDY mediated citrate uptake in HepG2 cells

INDY mediated citrate uptake was also determined in HepG2 cells. To determine the uptake of !*C labelled citrate into cells, HepG2 cells endogenously expressing human NaCT were used. (Gopal etal 2007 Am J Physiol Gastrointest Liver Physiol 292).

P 00303-032 94 LU102907

Eternygen UK Ltd.

HepG2 cells were seeded into white clear-bottom 96-well plates (50,000/well) coated with collagen and incubated at 37°C for 16-24 hours. After that plates were washed once with assay buffer using automated washing / LS 405 Selectors, BioTek ending with a buffer volume of 40ul. Then Sul of compound solution in assay buffer was added and incubated for 5 min at 37°C in cell incubator,

HERA cell Heraeus. Afterwards 10ul of substrate 14C-citrate (10nM) were added and incubated for another 90 min at 37°C in cell incubator, HERAcell. Then plates were washed 3x with 200ul buffer and 60ul cell lysis induced by adding 50ul ice-cold NaOH (100mM). Plates were incubated for 25 min at RT on a plate shaker and afterwards 200ul scintillation fluid was added and incubated for 15 min on a plate shaker at RT and for another 15 min without shaking at RT. Plates were measured using a TopCount system. As a positive control SmM citrate was applied. This assay can also be used in a 384-well plate format.

Glutamate uptake assay

Glutamate uptake by the GLT-1 transporter was used as a counter assay. The principle of the assay is to measure the uptake of *H labelled glutamate into HEK cells, which overexpress human GLT- 1.

The generation of a GLT-1 cell line was performed as follows: human GLT1 1 isoform svl cDNA (Origene #RC223924) was cloned into vector pFB-Neo-CMV-hGLT1 using QIAfilter Plasmid

Midi Kit (Qiagen). Virus was generated in GP293 packaging cells (transfection of plasmid with

Lipofectamine 2000 + OptiMEM). For transfection into HEK cells GP293, supernatant containing virus was passed through a 0.45 micron sterile filter and DEAE-dextran was added at 10mg/ml final concentration. The solution was used to transfect HEK293 cells at 37°C, 8.5%CO2. After 24h selection marker G418 at 400ug/ml was added and cultivation medium performed in DMEM 10%

FBS.

The assay was performed in a 96-well format using repeated washing steps, cell lysis and addition of scintillation fluid. Assay buffer was HBSS, ImM CaCl,, ImM MgCl,, 25mM Hepes pH 7.4.

Cells were seeded into white clear-bottom 96-well plates (50,000/well) in the presence of Poly-D-

Lysine and incubated at 37°C for 16-24 hours. Subsequently the plates were washed once with assay buffer using automated washing / LS 405 Selectors, BioTek ending with a buffer volume of 40ul.

Then Sul of compound solution in assay buffer was added and incubated for 20 min at 37°C in cell incubator, HERA cell Heraeus. Afterwards 10ul of substrate 3H-Glu (3nM) were added and incubated for another 20 min at 37°C in cell incubator, HERAcell. Then the plates were washed once with 200 ul cold assay buffer and 150ul scintillation fluid was added. Then plates were shaked

P 00303-032 95 LU102907

Eternygen UK Ltd. for 30 min and incubated for another 15 min without shaker, Titramax 1000, Heidolph and finally measured using Top count, Perkin Elmer. As a positive control 1mM glutamate was applied.

Glucose uptake assay

Glucose uptake in Huh7 cells was measured as a counter assay. The principle of this assay is to determine the uptake of *H labelled glucose into Huh7 cells. Hepatocellular cell lines like Huh-7 cells overexpress endogenously several glucose transporters from the GLUT family mainly GLUT 1 (Brito et al. EASL 2011, Amman et al. 2009 Am J Pathol 174(4)). As assay buffer 25mM Hepes + 25mM MES pH 7.4, 120mM NaCl, 5.4mM KCI, 1.8mM CaCl2, 0.8mM MgSO4 was used. Huh7 cells were seeded into collagen-coated 96-well plates (white, clear bottom; 50,000/well) and incubated at 37°C for 16-24 hours. The plate were washed once with assay buffer using automated washing / LS 405 Selectors, BioTek ending with 40ul buffer followed by adding Sul of compound solution in assay buffer and incubation for 5 min at 37°C in a cell incubator, HERA cell Heraeus.

Afterwards 10ul of substrate 3H-glucose (0.113nM; 100nCi/well) were added and incubated for another 30 min at 37°C in ca ell incubator, HERAcell. The plate were washed twice with 200ul buffer. Cell lysis was induced by adding 50ul ice-cold NaOH (100mM). Plates were incubated for min at RT and afterwards 200ul scintillation fluid added. After 20 min incubation at RT plates were measured using TopCount system, Perkin Elmer. 100uM phloretin was added as positive control. 20

Mouse INDY mediated citrate uptake assay

Mouse INDY mediated citrate uptake was determined in HEK293 cells to ensure species specificity.

The assay is based on the measurement of '*C labelled citrate uptake into HEK293 cells, which over-express the mouse gene for NaCT. Cloning of mouse NaCT was performed as described in

Birkenfeld et al. 2011 (Cell Metabolism 14, 184-195, 2011).

The mouse NaCT HEK293 cells were seeded into white clear-bottom 96-well plates (50,000/well) in the presence of 4ug/ml Poly-D-Lysine and incubated at 37°C for 16-24 hours. On the days of the assay the plates were washed once with assay buffer using automated washing / LS 405 Selectors,

BioTek ending with a buffer volume of 40ul. Then Sul of compound solution in assay buffer were added and incubated for 5-30 min at 37°C in a cell incubator, HERA cell Heraeus. Afterwards 10ul of substrate 14C-citrate (2nM / 11.6nCi per well) were added and incubated for another 20 - 90 min at 37°C in cell incubator, HER Acell. The plates were washed twice with 200ul ice-cold assay buffer and residual volume was discarded. To induce cell lysis, 50ul ice-cold NaOH (100mM) were added and incubated for 15 minutes at RT on a plate shaker. Finally, 200ul scintillation fluid (scintillator

OptiPhase Supermix, Perkin Elmer) were added. After 15 min incubation at RT without shaking,

P 00303-032 96 LU102907

Eternygen UK Ltd. plates were measured using a TopCount reader system (Perkin Elmer). 10mM citrate were applied as a positive control.

Cytoxocitiy — cell imaging

In order to identify compounds with potential cytotoxic properties, cell imaging was used.

Alternatively, cell protein content was determined.

For imaging, cells were stained with propidium iodide (PI), which does not cross the cellular membrane and therefore only stains the nucleus if pores are present indicating late apoptosis. In parallel, a Hoechst stain was applied which can penetrate the cell membrane and therefore stains all cell nuclei. Early apoptotic effects are identified by condensed and rounded nuclei.

The corresponding cells (depends on assay) were seeded into imaging-compatible black clear- bottom 384-well plates (Greiner # 781956, black clear, collagen coated for HepG2 or Huh7 or

Coming #3683BC for HEK cell lines in the presence of Poly-D-Lysine) (20,000/well) and incubated at 37°C for 16-24 hours. Subsequently plates were washed twice using automated washing / LS 405 Selectors, BioTek ending with 25ul of the corresponding assay buffer. Then Sul of compound solution in assay buffer was added and incubated for 2 — 24 hours at 37°C for in cell incubator, HERA cell Heraeus. Optionally cells were fixed with 4% paraformaldehyde (PFA). Then of 45ul propidium iodide (0.05mg/ml) and Hoechst (1uM) stain were added and incubated for 30 min at RT. Afterwards plates were analysed using the Opera™ High Content Screening system (Perkin Elmer). As a positive control 20uM staurosporine was applied. The PI stain was analysed by counting the detected nuclei as an indicator for late toxicity. The Hoechst stain was analysed by combining the cell roundness factor and the detected cell area (Hoechst readout =

Cell roundness/Cell area).

For determination of the cell protein content, 10ul of cell lysate was removed after lysis with 60ul

NaOH and transferred into a fresh assay plated into 15ul PBS. Then, 200ul Pierce BCA protein assay kit (Thermo Scientific) was added and incubated according to the supplier instructions.

Absorbance at 562nm was measured on a plate reader.

Fatty acid synthesis (FAS) assay

A functional cell assay measuring the effect of selected compounds on the citrate mediated fatty acid synthesis in hepatocytes was performed to test the functional activity of selected compounds and to bridge between the in vitro assay for citrate uptake to the physiological relevant lipid accumulation in liver cells. This assay.

P 00303-032 97 LU102907

Eternygen UK Ltd.

Fatty acid synthesis (FAS) assay in HepG2: The principle of the assay is to measure the generation of “C-labelled fatty acids after uptake of *C labelled citrate into HepG2 cells. HepG2 cells were seeded into white clear-bottom 96-well plates (50,000/well) coated with collagen and incubated at 37°C for 16-24 hours. After that plates were washed once with 100ul PBS (+CaCl, +MgCl,) and 40ul assay medium (RPMI 1860 containing 11mM glucose, 10mM HEPES and InM Insulin) per well was added. Test substances in assay medium were added (Sul, 0.5% DMSO) and incubated for 20 min. After that 10ul assay medium containing '*C-citrate (final concentration 50nM) and

PrestoBlue reagent (1x final concentration; Life Technologies) were added and incubated for 60 min (37°C, 5% CO»). Cell viability was measured (Safire microplate reader, ex: 560/em: 590) and plates were incubated for another 30 min. Medium was removed by inverting the plate and cells were washed once with 100ul ice-cold PBS. Cells were lysed with 50ul lysis buffer (100mM NaOH, 0.1% Triton X-100) and plate was sealed with Tape Pads and vortexed. For the saponification reaction plates were incubated for 16 — 24 hours at 80°C. Seals were removed and 200ul 100nM

HCI was added for neutralisation of the pH. 150ul from each well was transferred into the corresponding wells of a 96-well CytoStarT plate (Perkin Elmer) and sealed with TopSeal A (Perkin

Elmer). Plates were incubated for 4h at 70°C and for 1h at room temperature in the dark. Signal of

LC radioactivity was measured (TopCount NXT scintillation counter, 1min/well). As a positive control SmM citrate or 40uM C75 (Sigma, C5490) was applied.

Citrate uptake in murine primary hepatocytes

A further functional cell assay measuring the effect of selected compounds on the citrate uptake in murine primary hepatocytes was performed to test the functional activity of selected compounds and to bridge between the in vitro assay for citrate uptake in an immortalized liver cell line to the physiological more relevant primary cells. In this assay the activity of test compounds was tested at a fixed concentration (20uM) in inhibitory efficacy expressed as % inhibition.

NaCT citrate uptake assay in primary mouse hepatocytes: The principle of the assay is to measure the uptake of 14C labelled citrate into primary mouse hepatocytes which endogenously express human NaCT. Cells were obtained from Invitrogen (cryopreserved mouse (CD-1) hepatocytes, male, plateable; cat Nr MSCP10; 4 — 8 x 106 cells / vial). Primary hepatocytes were thawed, pelleted and resuspended in 2ml medium (Willia's Medium E (1x) + 25mL FCS + 18mL Thawing/Plating

Cocktail + 70uL Dexamethansone Supplement Kit from Invitrogen). Cells count was determined in a Neubauer chamber and 250,000 cells/well/500ul were seeded manually (pipette) into a 24-well plate (Collagen-coated Coming 24-well white clear bottom; BIOCOAT#354408) and incubated at 37°C with 5% CO2. Cell medium was changed on the same day (after 5 hours) and plates were

P 00303-032 98 LU102907

Eternygen UK Ltd. incubated overnight. On the day of the assay the medium was discarded manually and 500ul buffer (25mM Hepes, 25mM MES pH 7.4, 120mM NaCl, 5.4mM KCl, 1.8mM CaCl2, 0.8mM MgS04, 5mM glucose) containing test compounds or controls (final DMSO concentration of 0.2%) were added manually with a pipette and incubated for 5 minutes (37°C, 5% CO2). Then 500ul buffer containing 10nM 14C-citrate were added manually (pipette) and incubated for 90 min (37°C, 5%

C02). Supernatant was discarded and cells were washed once with 1000ul assay buffer (manually with 8-channel pipette). Cells were lysed with 50ul ice cold lysis buffer (100mM NaOH + 0.1%

Triton X-100) for 25 minutes while shaking. The lysate was transferred (manual, pipette) to 96-well plates (white) and 200ul Scintillator (OptiPhase Supermix, Perkin Elmer) was added and incubated for 15 minutes while shaking and another 25 minutes without shaking. Plates were measured using a TopCount system, Perkin Elmer. As a positive control SmM citrate was applied.

Measurement results of selected example compounds in the above-described assays for determining activity and selectivity are summarized in Table 1. The IC50 values were ranked as follows:

A: <1uM on hINDY and < 10uM on mINDY; B: >1uM <10 uM on hINDY and < 10uM mINDY,;

C: <10 uM on hINDY but >10uM on mINDY; D: >10uM on hINDY but <10uM on mINDY; and

E: >10 uM on hINDY. None of the tested compounds showed any toxic effects in the cell-based test systems.

Table 1: Illustrative activity and selectivity data for representative compounds disclosed herein

Citrate/

Trans- porter INDY GLT-1 GLUT1 | mINDY INDY mINDY protein re [vom | vec [wor || wen | I

Cell line | HepG2 HEK293 Huh7 | HEK293 | HepG2 hepato-cytes

Cpd. Inhibition at

HEE

1 || 132 | md | 560 | 5540 | na | md | Cc 2 || 180 | na | > | >60 | na | md | Cc 3 | | 808 | na | > | 8735 | na | md | Cc 4 || 030 | >60 | > | 03 | na | md | A

P 00303-032 99 LU102907

Eternygen UK Ltd. | | 037 | >50 | >60 | 144 | na | nd | A 6 | | om | >60 | >» | ote | 057 | 50% | A 7 | | 158 | >60 | 60 | 2890 | na | md | © 8 | | 663 | nd | nd | nd | nd | nd | E_ 9 | | ow | na | né | nd | na | nd | E_ | | 402 | na | »0 | 5983 | na | md | B_

Lu || 220 | na | > | 5148 | na | md | Cc 2 | | 534 | >60 | > | >50 | na | md | Cc a | | 094 | >60 | >» | 054 | 28 | 69% | A

Cw | loss | >60 | 0 | 057 | 14 | 87% | A

L 15 | | 224 | >60 | 60 | 461 | na | md | B_ 16 | | 135 | md | > | 4692 | na | md | © wr | | 167 | nd | >50 | 646 | na | nd | B 18 | | 380 | >60 | > | 065 | na | md | B_ 19 | | 194 | md | nd | 08 | na | md | B_ | | 265 | nd | nd | 6761 | na | nd | Cc (100% 21 0.47 >50 >50 028 | 78% A inhibition at 20uM 2 | | 268 | >60 | 60 | 95 | na | nd | 0B 2 || 133 | nd | nd | 053 | nd | nd | 0B 2 | | 840 | na | nd | 268 | nd | md | B | | 156 | nd | nd | 070 | na | md | B_ 2% | | 016 | na | nd | 012 | 083 | nd | A a |__| 104 | na | né | 08 | na | md | B_ 2 | | 334 | na | nd | nd | na | nd | E 2 | | 880 | nd | nd | 53 | na | nd | E zo | | 248 | na | nd | 143 | na | nd |B 81 | | 080 | na | né | 02 | na | md | A 32 | | 182 | na | nd | 53 | na | md | B_ 33 | | 058 | nd | nd | 017 | na | md | A u | | 189 | >60 | »0 | 411 | na | 25% | B 3 | | 430 | na | »0 | 2118 | na | md | Cc 3% | | 231 | na | nd | 175 | na | nd | B_

La | [ow | na | né | nd | na | md | E

P 00303-032 100 LU102907

Eternygen UK Ltd. 8 | 1238 | nd | nd | 3365 | na | nd | E 3% | | 655 | nd | nd | 532 | nd | nd | E a | | >60 | na | né | nd | na | nd | E_ m || >50 | na | né | nd | na | ne | E_ 4 |__| 144 | na | né | 020 | na | md |B 4 || 145 | na | nd | 02 | na | md |B a | | 066 | nd | nd | 03 | na | md | A 45 | | 20 | na | nd | 190 | na | md |B 46 | | 19 | na | nd | 658 | na | nd | D ar |__| >50 | na | né | nd | na | nd | E_ 48 | | >60 | na | nd | >60 | na | nd | E 40 | | 248 | na | nd | 36 | na | nd | D | | 063 | nd | nd | 061 | na | nd | A 1 51 | | o72 | na | nd | 063 | na | md | A 52 | | 20 | na | nd | 046 | na | md | B_ 58 | | 077 | na | né | 02 | na | md | A (100% 54 1.82 n.d. n.d. 0.64 n.d. inhibition at 20uM | | 130 | nd | nd | 003 | 12 | md | 0B 56 | | 087 | >60 | 60 | 014 | na | md | A 57 | | 2442 | na | né | 443 | na | nd | D 58 | | 5044 | na | nd | 241 | na | nd | D so | | 134 | >60 | 60 | 958 | na | 50% | B 60 | | 410 | >60 | > | 412 | na | md | B_ 181 | | 055 | >60 | > | 0235 | na | md | A 62 | lest | nd | nd | 1485 | na | nd | © es | | 69 | >60 | »o | 097 | na | md | B_ 64 | | 19.44 | >60 | nd | 82 | nd | md | D es | | 15000 | >60 | 560 | 24 | na | md | D <<20 1.04 >50 >50 0.12 (100% n.d. inhibition at 20uM or |__| @M | na | né | 27 | na | md | D

P 00303-032 101 LU102907

Eternygen UK Ltd. 68 | | 4888 | nd | nd | 170 | nd | nd | D eo | | 114 | na | nd | 44 | na | nd | D zo | | 33 | na | né | 07 | na | md | B_ mn | | 20 | na | né | 23 | na | nd |B 2 | | 820 | na | nd | 77 | na | ne | D 73 || 44 | na | né | 019 | na | md | B_ 47 | na | né | 18 | na | nd | B_ 75 | | 38 | na | né | 26 | nd | md | B_ 76 | | 025 | na | nd | 054 | na | md | A m | | 175 | na | né | 03 | na | md | B_ 78 | | 134 | nd | né | 25 | na | md | D

Compounds denoted with * were measured in 384 well format.

Example 25: In vivo testing using diet-induced obese (DIO) mice — an established model to study high fat diet induced fatty liver and other metabolic complications

To test the functional activity of a selected compound in vivo and to bridge between the in vitro assay for citrate uptake and fatty acid synthesis to the physiological relevant readouts in a disease model, a diet induced obesity model was performed. More specifically, an in vive pharmacology study in male DIO mice was performed to evaluate the efficacy of Compound 21 (also referred to as Ex 21 or example 21) in an animal model of type 2 diabetes (T2DM).

Since arrival, at an age of 4 weeks mice (C57BL6/N, male, Charles River Laboratories, Sulzfeld,

Germany, n=6/group) were kept on high fat diet (D12492; 60 kcal% fat; 5.24 kcal/g (total) (Research Diets, Inc.; New Brunswick, USA) for 13 weeks to induce the DIO (Diet Induced

Obesity) status. Example 21 was administered at three different doses for 7 days twice a day by oral gavage. In addition, for each compound dosage group a respective pair-fed vehicle group was run in order to detect effects which were related to a possible dosage-dependent change in food intake.

Husbandry and group assignment: Mice were housed at a temperature of 22-24 °C, a day/night cycle of 13/11 hours (06:00-19:00-06:00) and had access to the appropriate diet and tap water ad libitum. Food was only withdrawn for overnight fasting on study day 6 (pair-fed groups: study day 7) for fasted blood glucose test on study day 7 (section) (pair-fed groups: study day 8). Groups 1-7 were randomized by choosing 42 mice from a pool of 59 mice according to random fed blood glucose levels and body weight before treatment start and housed in groups of 3 animals per cage.

P 00303-032 102 LU102907

Eternygen UK Ltd.

Control group: Group 1 received vehicle (5 % DMSO/10% Cremophor® EL/85% water) given twice daily p.o. and served as control vs. compound treatment. Like group 1, the groups 5-7 received vehicle, too, but in addition these groups received only the amount of food which was consumed the day before by the allocated compound treated group.

Test compound: Compound 21 (Ex 21) was applied in three different dosages: 1 mg/kg (low dose), 5 mg/kg (medium dose) and 15 mg/kg (high dose). The dosage was applied twice daily (8 am and 5 pm), application route was per os (p.o.) by gavage, the application volume was 5.0 ml/kg.

Experimental schedule: On day -4, body weight and blood glucose were measured and animals were assigned to groups 1-7. From study day -3 (pair-fed groups: study day -2) until start of compound treatment on study day 0, animals of all groups were treated with vehicle. Starting with study day 0, mice were treated with vehicle or compound. Group 1 served as a vehicle control group and was treated with the vehicle from study day -3 to study day 6, p.o., twice daily. Groups 2-4 comprised treatment groups that were treated with Example 21, p.o., twice daily from study day 0 to study day 6. Groups 5-7 comprised vehicle groups which were treated with vehicle from study day -2 to study day 6, p.o., twice daily, and which were pair-fed to groups 2-4 according to their cage-wise daily food consumption. Therefore groups 5-7 were started one day delayed to groups 1-4.

Animal welfare: In case of obvious and unacceptable impairment of individual animal welfare, these animals will be sacrificed immediately.

Readouts: in life (daily): Body weight, blood glucose: random-fed and fasted (section), food intake, water intake. After section: serum beta-hydroxybutyrate level, Piccolo Lipid panel plus, Tissue samples from liver for Western blotting.

The methods comprised: e Body weight was recorded daily in the morning using a Sartorius Quintix® precision balance operating with an internal automatic calibration program. e Food and water intake per cage was determined daily during the treatment period in the morning together with random-fed blood glucose and body weight. Data were recorded per cage and calculated as g/mouse*day unit. e Blood glucose levels were determined by analysing tail tip blood samples with the “Lifescan

ONETOUCH Verio” (Johnson&Johnson company, range <33.3 mmol/l). Results exceeding 33.3 mmol/l were calculated as 33.3 mmol/l. Random fed blood glucose was determined at the times indicated for body weight (see 3.2). Fasting blood glucose was determined after overnight fasting on study day 7 (section group 1-4) and study day 8 (section group 5-7).

P 00303-032 103 LU102907

Eternygen UK Ltd. e Dissection and tissue sample conservation: Mice were sectioned on study day 7 (group 1-4) and study day 8 (group 5-7). Retro-orbital bleeding was performed on anesthetized animals.

Then the animals were sacrificed by cervical dislocation. One piece of liver was collected immediately (fast prep) for analysis of AMPK phosphorylation. Tissue samples from liver were collected in small pieces and frozen in liquid nitrogen. One liver lobe was frozen in TissueTec® for cryosectioning and another liver lobe in 4% PFA for optional histology. e Collection of plasma samples: For serum sample generation the mice were anaesthetized by isofluran inhalation; blood samples were taken by retro-orbital bleeding (non-coated capillaries) and collected in non-coated tubes (Sarstedt). After centrifugation at 4000 g for 10 min the serum samples were dispensed into a 100 ul plus a (rest) aliquot. The samples were stored at — 80°C. For VetScan® system analysis (Lipid panel plus) a 110 pl-aliquot of lithium- heparin blood plasma was collected. For this purpose the blood samples were taken by retro- orbital bleeding and collected in tubes coated with lithium-heparin (Sarstedt). After incubation at RT for at least 10 min, samples were centrifuged at 4000 g for 10 min, the supernatant was then transferred to a new tube and stored at -80°C. e Liver tissue sample analysis: One liver piece (fast prep) was used to determine AMPK phosphorylation by western blot. e Serum insulin was determined with a Mouse Insulin ELISA (Mercodia Cat. No. 10-1247-01) following manufacturer’s instructions. Sample was undiluted or 1:10 diluted, respectively, and volume was 10 pl. e Serum beta-hydroxybutyrate was determined with a Beta-Hydroxybutyrate Assay kit, (Sigma,

Cat.No. MAKO041) following manufacturer’s instructions. Sample was undiluted and volume was 5 ul. e Western blotting procedure was a standard procedure using a semi-dry transfer method with

Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, #170-3940) on Cellulose

Nitrate Strips (Sartorius, #11327) following to standard SDS-PAGE. Deviating from standard protocol samples were heated for 5 min at 95°C and the blotting time was 1 hour at 20 Volt.

For target protein detection AMPK alpha (D63G4) Rabbit mAb (Cell Signaling Technology,

Inc.; Cat. No. #5832/ 3; working dilution 1:1000) and Phospho-AMPK alpha (Thr172)(40H9)

Rabbit mAb (Cell Signaling Technology, Inc.; Cat.No. #2535/16; working dilution 1:1000) was used; gel loading was controlled by anti-beta-actin (Sigma-Aldrich, Cat. No. A2228). As technical control ready-to-use AMPK control cell extracts (Cell Signaling Technology, Inc ;

Cat.No.9158) were employed. Signal detection was performed with the SuperSignal West Dura

Extended Duration Substrate (Thermo Fisher Scientific, Bonn, FRG; Cat. No. 34076). e Statistical evaluation: For each treatment group the values were compared to the vehicle values by means of the student's unpaired two-tailed t-test (D. Colquhoun, Lectures on Biostatistics, 167-9 (1971), Clarendon Press, Oxford). Multiple comparisons were done by a two-way

P 00303-032 104 LU102907

Eternygen UK Ltd.

ANOVA. These and other calculations like correlation analysis were performed either using

Excel 2010 (Microsoft Office) or GraphPad Prism 6 for Windows. The following limit was used: p=0.05. In graphs and tables significant differences from the start value, relative to vehicle were indicated as * p<0.05, ** p<0.01, *** p<0.001. Error bars were displayed as mean + SD or SEM (as indicated).

The results of the in-vivo experiments can be summarized as follows:

The body weight development throughout the study is shown in Figure I. A compound dose- dependent decrease in bodyweight was observed with a significant decrease in high dose compared to vehicle. Mice pair-fed to low and medium dose lost similar amounts of bodyweight as the respective compound groups. The high dose compound group had a significantly decreased bodyweight compared to its respective pair-fed group.

During the first days of compound treatment food intake decreased in the medium dose as well as in the high dose group. After this initial period all compound-treated groups had a slight increase in food intake. Differences between the vehicle group and the medium and high dose group mostly were significant as indicated in Figure 2. Pair-fed groups were well adjusted to compound groups besides minor deviations due to spoiled food in the cages.

Random fed blood glucose levels were lower for all compound treated and respective pair-fed groups over the study period when compared to vehicle as indicated in Figure 3. At the end of the study overnight fasting blood glucose levels were determined. Fasting blood glucose in animals treated with 15 mg/kg Compound 21 (Ex 21) twice daily were significantly reduced compared to vehicle treated animals as well as to the respective pair-fed group.

The serum insulin levels as shown in Figure 4 show a remarkable strong decrease in insulin levels for high dose treatment of Compound 21 (Ex 21), which differed significantly from vehicle as well as from its pair-fed group.

Treatment with 15 mg/kg Compound 21 (Ex 21) twice a day led to a significantly increase in serum beta-hydroxybutyrate (HBA) compared to vehicle treated animals and its respective pair-fed group as shown in Figure 5. However, a considerable number of measurements was below assay detection limit.

The serum cholesterol level was significantly lowered in the high dosage compound group and its respective pair-fed group compared to vehicle as shown in Figure 6.

P 00303-032 105 LU102907

Eternygen UK Ltd.

The western blot analysis (AMPK) of phosphor-AMPK and total AMPK were performed on liver samples directly snap frozen as first step during necropsy (Figure 7). Administration of two times 15 mg/kg Compound 21 (Ex 21) resulted in a significant increase in the pAMP/total AMPK ratio compared to the vehicle and its respective pair-fed group. No difference could be observed between vehicle and pair-fed to high dose group.

In summary: e No adverse effects with respect to general health conditions were observed with Compound 21 (Ex 21). e Treatment with 2 x 15 mg/kg Compound 21 (Ex 21) resulted in beneficial effects compared to vehicle treated animals and the respective pair-fed group for fasting blood glucose, fasting insulin, beta-hydroxybutyrate and pAMPK/AMPK ratio in liver tissue. e Treatment with 2 x 15 mg/kg Compound 21 (Ex 21) resulted in decreased food intake compared to vehicle group, decreased body weight compared to vehicle and the respective pair-fed group, decreased plasma cholesterol compared to vehicle group.

Claims

P 00303-032 106 LU102907 Eternygen UK Ltd. CLAIMS

1. A compound for use in the prevention and/or treatment of a citrate transporter meditated or citrate transporter dependent disease or condition, wherein the compound is a compound of Formula (D, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof: RH! N O X J (CHR? R? © R® D wherein L' is absent, -O-, -O(CHz)-, -S-, -S(=0),-, -C(FO)NH-,- NHS(=0):-, or -S(=0),NH-; R'is H, halogen, CN, NO», (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, (C1-Cs)haloalkoxy, substituted or unsubstituted (Cz-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle; R° is H, F, CI, CN, NO, or OCH;; R° is H, F, CI, Br, CN, NO», CF; or OCHz; or R° and R° are taken together to form a group -O-C(R°)-O-; mis | or 2; R* is CHF,, CF;, CH=CH,, C=CH, or C=CCH;; each R° is independently selected from H, halogen, (Ci-Cs)alkyl, (C1-C3)haloalkyl and (C1- C3)alkoxy; provided that R' is only H when L! is present or when R° and R° are taken together to form a group -O-C(R°),-O-.

2. The compound for use as in claim 1, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein R° is F or CI, and R°is H.

3. The compound for use as in claim 1, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein R° is H.

4. The compound for use as in any one of claims 1 to 3, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein L' is absent, -O-, -O(CHz)- or -S(=0)»-.

5. The compound for use as in any one of claims 1 to 4, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein R* is CHF», CF; or C=CH.

P 00303-032 107 LU102907 Eternygen UK Ltd.

6. The compound for use as in any one of claims 1 to 5, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein m 1s 2.

7. The compound for use as in claim 1, wherein the compound is a compound of Formula (II), (IIT) or (IV), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof: R'—L! N O RL N O D M VCHom-R® U I NCH2)m-R® =? O O RS (II) (111) H N To R6 oh O R° x; © R (IV) wherein m, L', RY, R°, R*, R* and R° are defined as in claim 1, and RS is CHF», CF; or C=CH.

8. The compound for use as in any one of claims 1 to 7, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein the moiety -L!-R! is selected from F, CI, Br, OH, OCHs, OCH:CH;, OCHF,, OCF;, O(CH2)OCHs, CN, NO», CH;, CHF», CF;, -C(=0O)NH,, - NHS(#0)2CHz, SCHz, (NT SGA NV O- EN Ra 0° 0 -S(=O)2CH,, -S(=O)2NH, NA ana NF

9. The compound for use as in claim 1, wherein the compound is: But-3-yn-1-yl N-(3-chlorophenyl)carbamate; But-3-yn-1-yl N-(4-chloro-3-methoxyphenyl)carbamate; But-3-yn-1-yl N-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)carbamate; But-3-yn-1-yl (4-chloro-3-(trifluoromethyl)phenyl carbamate; But-3-yn-1-yl (3,4-dichlorophenyl)carbamate; But-3-yn-1-yl (3,5-dichlorophenyl)carbamate; But-3-yn-1-yl (4-chloro-3-fluorophenyl)carbamate But-3-yn-1-yl N-(4-fluoro-3-methoxyphenyl)carbamate; But-3-yn-1-yl N-(3,5-dimethoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-methylphenyl)carbamate;

P 00303-032 108 LU102907 Eternygen UK Ltd.

3,3,3-Trifluoropropyl N-(4-chloro-3-cyanophenyl)carbamate; 3,3,3-Trifluoropropyl N-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3,5-dichlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-fluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3-methoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-methoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(difluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-5-cyanophenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-4-cyanophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3-nitrophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3-cyano-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3-cyano-5-fluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-nitro-3-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[3-nitro-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3-bromo-5-chlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chlorophenyl)carbamate;

3,3,3-Trifluoropropyl N-(4-chloro-3-ethoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(trifluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-3-(trifluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(4-chloro-3,5-difluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3,5-bis(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3,4-dichlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-4-fluorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-5-(morpholin-4-yl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-(3-carbamoyl-4-chlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(cyclopropylmethoxy) phenyl]carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(2-methoxyethoxy)phenyl] carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(pyridin-4-ylmethoxy)phenyl] carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(pyridin-4-yloxy)phenyl] carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-3-(cyclopropylmethoxy) phenyl]carbamate 3,3,3-Trifluoropropyl N-(5-chloro-3-hydroxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-[5-chloro-3-(difluoromethoxy)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[4-chloro-3-(difluoromethyl)phenyl]carbamate; 3,3,3-Trifloropropyl N-(5-chloro-3-methanesulfonylphenyl) carbamate; 3,3,3-Trifluoropropyl N-(5-chloro-3-methanesulfonamidophenyl) carbamate;

P 00303-032 109 LU102907 Eternygen UK Ltd. 3,3,3-Trifluoropropyl N-(4-chloro-3-sulfamoylphenyl)carbamate; 3,3,3-Trifluoroprpyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate; 3,3,3-Trifluoropropyl (3-methanesufonyl-5-nitrophenyl)carbamate; 3,3,3-Trifluoropropyl (3-chloro-5-nitrophenyl)carbamate; 3,3,3-Trifluoropropyl (5-chloro-3-(methylsulfanyl)phenyl)carbamate; 2,2,2-Trifluoroethyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate; 2,2,2-Trifluoroethyl N-[4-chloro-3-(trifluoromethyl)phenyl]carbamate; 2,2-Difluoroethyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate; 2,2-Difluoroethyl (3,5-dichlorophenyl)carbamate; 2,2-Difluoroethyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate; 2,2-Difluoroethyl (3,4-dichlorophenyl)carbamate; Prop-2-yn-1-yl (3,4-dichlorophenyl)carbamate; Prop-2-yn-1-yl (4-chloro-3-(trifluoromethyl)phenyl)carbamate; Prop-2-yn-1-yl (3,5-dichlorophenyl)carbamate; But-2-yn-1-yl (3,5-dichlorophenyl)carbamate; Allyl (3,5-dichlorophenyl)carbamate; Allyl (3,4-dichlorophenyl)carbamate; Allyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate; 2,2,2-Trifluoroethyl (3,5-dichlorophenyl)carbamate; 2,2,2-Trifluoroethyl (3,4-dichlorophenyl)carbamate; Propyl (3,5-dichlorophenyl)carbamate; 3,3-Difluoropropyl (3,5-dichlorophenyl)carbamate; 3,3-Difluoropropyl (3-chloro-5-(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (3-nitro-5-(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (4-chloro-3-(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (3,5-bis(trifluoromethyl)phenyl)carbamate; 3,3-Difluoropropyl (3-chloro-5-nitrophenyl)carbamate; 3,3-Difluoropropyl (3-bromo-5-chlorophenyl)carbamate; 3,3-Difluororopyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate; 3,3-Difluoroprpyl N-(3-methanesulfonyl-5-nitrophenyl)carbamate; 3,3-Difluoropropyl (3-(methylsulfanyl)-5-chlorophenyl)carbamate; or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof.

10. The compound for use as in any one of claims 1 to 9, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein the citrate transporter is the gene product of Indy or a homologue thereof.

P 00303-032 110 LU102907 Eternygen UK Ltd.

11. The compound for use as in any one of claims 1 to 10, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein the citrate transporter meditated or citrate transporter dependent disease or condition is selected from: (g) metabolic diseases selected from the group comprising insulin resistance, alcoholic and non- alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hereditary diseases and metabolic syndrome; (h) eating disorders; (1) chronic liver diseases; (J) liver cancer and cancer related to obesity; (k) age related diseases comprising atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension and neurodegenerative diseases like Alzheimer's disease; and (I) drug induced hepatic steatosis.

12. A pharmaceutical composition comprising a pharmaceutically acceptable inactive ingredient and one or more compound(s) according to any one of claims 1 to 9, or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof.

13. The pharmaceutical composition of claim 12, further comprising at least one further active pharmaceutical ingredient selected from the group comprising: (a) anti-obesity agents selected from the group consisting of orlistat, lorcaserin, Phentermine, Topiramate, sibutramine, bromocriptine, ephedrine, leptin, and pseudoephedrine, 5-HT2c receptor agonists, Bupropion, Naltrexone, methionine aminopeptidase 2 inhibitors, GLP1 agonists; (b) anti-diabetes agents comprising insulin, incretin mimetics, SGLT-2 inhibitors, DPPIV inhibitors, PPAR agonist, Glucokinase activator, MTP inhibitors, Glycogen phosphorylase inhibitors, DGAT-1 inhibitor, GLP1 agonists, dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor; (c) anti-NASH agents comprising insulin, incretin mimetics, statins, PPAR agonists, AMPK activators, FXR agonists, DGAT-2 inhibitors, DGAT-1 inhibitors, Bile-Acid Conjugates, methionine aminopeptidase 2 inhibitors, PDE4 inhibitors, inhibitors of acetyl-CoA carboxylase, inhibitors of ketohexokinase, inhibitors of ATP citrate lyase, GLP-1 agonist, dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor, inhibitors of ASK-1, CCR2/CCR5 antagonist, inhibitors of SLC10A2, inhibitors of LOXL2, inhibitors of Galectin-3, inhibitors of caspase, FGF21, FGF19, inhibitors of CGRP, AOC3: Amine Oxidase, Copper Containing 3, inhibitors

P 00303-032 111 LU102907 Eternygen UK Ltd. of DPP-4, THR-B agonists, anti-CD3 monoclonal antibody (mAbs), A3AR agonists, inhibitors of SGLT2, inhibitors of SGLT1, inhibitors for TGFR activation, anti- cannabinoid CD1 receptor antibody, antibody agonist of the B-Klotho/FGFR1c receptor complex, inhibitors of the inflammasome ATP citrate lyase inhibitors, stearoyl-CoA desaturase inhibitor, fatty acid synthesis inhibitors; (d) anti-dyslipidaemia agents comprising, statins, ApoB antisense oligonucleotides, PCSK9 inhibitors, Cholesterol-absorption inhibitors, Niacin, Bile-acid-sequestering resins, MTP inhibitors, Fibrates, CETP inhibitors, ATP citrate lyase inhibitors; (e) anti-cancer agents comprising chemotherapeutic drugs; and (f) anti aging drugs comprising vitamins.

14. The pharmaceutical composition of claim 12 or 13 for use in the prevention and/or treatment of a citrate transporter meditated or citrate transporter dependent disease or condition.

15. A compound of Formula (Ila), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof: 4 Poe RA © (Ila) wherein L' is absent, -O-, -O(CHz)-, -S-, -S(=0),-, -C(FO)NH-,- NHS(=0):-, or -S(=0),NH-; R! is H, halogen, CN, NO», (C1-C+)alkyl, (Ci-Cs)haloalkyl, (C1-C;)alkoxy, (Ci-Cs)haloalkoxy, substituted or unsubstituted (C»-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle; RA is F, CI, CN, NO, or OCH;; or R! and R are taken together to form a group -O-C(R®),-O-; mis | or 2; R’ is CHF; or CF; each RP is independently selected from H, halogen, (Ci-Cs)alkyl, (Ci-Cs)haloalkyl and (C1- Cs)alkoxy; provided that R! is only H when L' is present; and that the moiety -L'-R! is not Cl if R* is CI, m is 2 and R is CFs.

16. The compound according to claim 15, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein R* is F, CI or NO»; and m is 2.

P 00303-032 112 LU102907 Eternygen UK Ltd.

17. The compound according to claim 15 or 16, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein the moiety -L!-R! is selected from F, Cl, OCH;, OCHF,, OCF, CN, NO», CHF, and CFs.

18. The compound according to any one of claims 15 to 17, wherein the compound is: 3,3,3-Trifluoropropyl N-(4-chloro-3-methoxyphenyl)carbamate; 3,3,3-Trifluoropropyl N-(3-chloro-4-fluorophenyl)carbamate; or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof.

19. A compound of Formula (IIIa), or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof: 4 QT CH) RT O R® (Ia) wherein L' is absent, -O-, -O(CHz)-, -S-, -S(=0),-, -C(FO)NH-,- NHS(=0):-, or -S(=0),NH-; R! is H, halogen, CN, NO», (C1-C+)alkyl, (Ci-Cs)haloalkyl, (C1-C;)alkoxy, (Ci-Cs)haloalkoxy, substituted or unsubstituted (Cz-Cs) alkoxyalkyl, (Ci-Cs)heteroalkyl, (Cs-Cs)cycloalkyl, or substituted or unsubstituted Csheterocycle; R° is F, Cl, Br, CN, NO», CF; or OCHj3; mis | or 2; R’ is CHF; or CF; provided that R! is only H when L' is present.

20. The compound according to claim 19, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein R° is Cl, NO; or CF3; and m is 2.

21. The compound according to claim 19 or 20, or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein the moiety -L'-R! is selected from F, Cl, Br, OH, OCH, OCH,CH;, OCHF,, OCF;, O(CH:):OCHs3;, CN, NO, CHs, CHF», CF;, -C(=O)NH», - NHS(#0)2CHz, SCHs, (NT SGA NV O- oJ Ao iy oh -S(=0),CH3, -S(FO)aNH>, and

22. The compound according to any one of claims 19 to 21, wherein the compound is: 3,3,3-Trifluoropropyl N-(3,5-dichlorophenyl)carbamate; 3,3,3-Trifluoropropyl N-[3-chloro-5-(trifluoromethyl)phenyl]carbamate;

P 00303-032 113 LU102907 Eternygen UK Ltd. 3,3,3-Trifluoropropyl N-[3-nitro-5-(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl N-[3,5-bis(trifluoromethyl)phenyl]carbamate; 3,3,3-Trifluoropropyl (3-chloro-5-nitrophenyl)carbamate; 3,3-Difluororopyl (3-methanesulfonyl-5-(trifluoromethyl)phenyl) carbamate; or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof.

23. A pharmaceutical composition comprising a pharmaceutically acceptable inactive ingredient and one or more compound(s) according to any one of claims 15 to 22, or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof.

24. A pharmaceutical composition comprising one or more compound(s) according to any one of claims 15 to 22, or a pharmaceutically acceptable prodrug, hydrate, solvate or salt thereof, and at least one further active pharmaceutical ingredient selected from the group comprising: (a) anti-obesity agents selected from the group consisting of orlistat, lorcaserin, Phentermine, Topiramate, sibutramine, bromocriptine, ephedrine, leptin, and pseudoephedrine, 5-HT2c receptor agonists, Bupropion, Naltrexone, methionine aminopeptidase 2 inhibitors, GLP1 agonists; (b) anti-diabetes agents comprising insulin, incretin mimetics, SGLT-2 inhibitors, DPPIV inhibitors, PPAR agonist, Glucokinase activator, MTP inhibitors, Glycogen phosphorylase inhibitors, DGAT-1 inhibitor, GLP1 agonists, dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor; (c) anti-NASH agents comprising insulin, incretin mimetics, statins, PPAR agonists, AMPK activators, FXR agonists, DGAT-2 inhibitors, DGAT-1 inhibitors, Bile-Acid Conjugates, methionine aminopeptidase 2 inhibitors, PDE4 inhibitors, inhibitors of acetyl-CoA carboxylase, inhibitors of ketohexokinase, inhibitors of ATP citrate lyase, GLP-1 agonist, dual GLP1/glucagon receptor agonists, triagonist for GLP1/glucose-dependent insulinotropic polypeptide/glucagon receptor, inhibitors of ASK-1, CCR2/CCR5 antagonist, inhibitors of SLC10A2, inhibitors of LOXL2, inhibitors of Galectin-3, inhibitors of caspase, FGF21, FGF19, inhibitors of CGRP, AOC3: Amine Oxidase, Copper Containing 3, inhibitors of DPP-4, THR-B agonists, anti-CD3 monoclonal antibody (mAbs), A3AR agonists, inhibitors of SGLT2, inhibitors of SGLT1, inhibitors for TGFR activation, anti- cannabinoid CD1 receptor antibody, antibody agonist of the B-Klotho/FGFR1c receptor complex, inhibitors of the inflammasome ATP citrate lyase inhibitors, stearoyl-CoA desaturase inhibitor, fatty acid synthesis inhibitors; (d) anti-dyslipidaemia agents comprising, statins, ApoB antisense oligonucleotides, PCSK9 inhibitors, Cholesterol-absorption inhibitors, Niacin, Bile-acid-sequestering resins, MTP inhibitors, Fibrates, CETP inhibitors, ATP citrate lyase inhibitors; (e) anti-cancer agents comprising chemotherapeutic drugs; and

P 00303-032 114 LU102907 Eternygen UK Ltd. (f) anti aging drugs comprising vitamins.

25. The compound according to any one of claims 15 to 22, or the pharmaceutical composition of claim 23 or 24, for use as a medicament.

26. The compound according to any one of claims 15 to 22, or the pharmaceutical composition of claim 23 or 24, for use in the prevention and/or treatment of a citrate transporter meditated or citrate transporter dependent disease or condition.