IL305348B1 - Brain permeable dhodh inhibitors and uses thereof - Google Patents

Brain permeable dhodh inhibitors and uses thereof

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IL305348B1
IL305348B1 IL305348A IL30534823A IL305348B1 IL 305348 B1 IL305348 B1 IL 305348B1 IL 305348 A IL305348 A IL 305348A IL 30534823 A IL30534823 A IL 30534823A IL 305348 B1 IL305348 B1 IL 305348B1
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sln
alkyl
compound
cancer
methyl
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IL305348A
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IL305348A (en
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Pessah Neta
Getter Tamar
Yelena Mostinski
Bingor Alexey
Paker-Krush Yelena
Roussay-Maggi Claudia
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Selene Therapeutics LTD
Pessah Neta
Getter Tamar
Yelena Mostinski
Bingor Alexey
Yelena Paker Krush
Claudia Roussay Maggi
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Application filed by Selene Therapeutics LTD, Pessah Neta, Getter Tamar, Yelena Mostinski, Bingor Alexey, Yelena Paker Krush, Claudia Roussay Maggi filed Critical Selene Therapeutics LTD
Priority to IL305348A priority Critical patent/IL305348B1/en
Publication of IL305348A publication Critical patent/IL305348A/en
Publication of IL305348B1 publication Critical patent/IL305348B1/en

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Description

FUTURX-004 IL 305 348/3 BRAIN PERMEABLE DHODH INHIBITORS AND USES THEREOF TECHNICAL FIELD [0001] The present invention relates to dihydroorotate dehydrogenase (DHODH) inhibitors, which are brain permeable, and to pharmaceutical compositions comprising them. These compounds and compositions are useful in preventing, delaying onset of, or treating diseases or disorders associated with neuronal hyperexcitability, e.g., diseases or disorders which can be treated by the specific inhibition of DHODH enzyme activity such as epilepsy and Alzheimer disease.
BACKGROUND ART [0002] Excitatory and inhibitory neurons in the brain are maintained within a highly preserved balance, that has roles in the formation, development, and homeostasis of brain circuits (Telias and Segal, 2022). The highly tenuous balance between these populations is preserved by local control mechanisms, regulating presynaptic efficacy as well as postsynaptic excitability. Impairment of this delicate equilibrium may lead to different pathologies and symptoms due to neuronal hyperexcitability and network hyperactivity. Such pathologies include, e.g., epilepsy, neurodegenerative diseases such as Alzheimer’s disease, neurodevelopmental impairments, and psychiatric diseases (Telias and Segal, 2022). [0003] Epilepsy, also referred to as a seizure disorder, is one of the most common neurological disorders, characterized by recurrent transient occurrence of signs and/or symptoms. This neurological disorder is rather a group of disorders with widely varying types of symptoms, including unprovoked loss of attention or sleepiness, convulsions which are sometimes severe and accompanied by loss of consciousness, called seizures, and may be focal or generalized (Löscher and Klein, 2021, Fisher et al., 2014). These symptoms and seizures are the result of abnormal excessive or synchronous neuronal activity. Epilepsy burdens neurobiological, cognitive, psychological, and social consequences. [0004] The prevalence of epilepsy is estimated at 0.5-1% of the general population (Fisher et al., 2014), meaning that there are more than 50 million patients worldwide. While there is no cure for epilepsy, in about 70% of the patients the symptoms can be treated with antiseizure medications (ASMs).
FUTURX-004 IL 305 348/3
[0005] Nevertheless, even though there are more than 30 ASMs in market, about 30% of the patients are still not seizure free, and there is therefore a substantial need for novel therapies, that may relief those patients from the burden and consequences of epileptic seizures. Furthermore, the response to ASMs is individual, and side effects vary from one patient to another. Thus, there is a need for ASMs that will work in a novel mechanism of action and with reduced side effects (Sang Kun Lee, 2014). In addition, synergistic effects and broad spectrum novel ASMs may lead to higher seizure free rates amongst epilepsy patients. [0006] As recently reported, inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme located on the outer surface of the inner membrane of the mitochondria, either by a small molecule or by a small interfering RNA, leads to the reduction of mean firing rate (MFR) in primary hippocampal neuron cultures (mice), attenuating cortico-hippocampal hyperactivity (Styr et al., 2019; WO 2018096538). Furthermore, inhibition of DHODH using its known clinically approved inhibitor teriflunomide (AUBAGIO®) prevented hyperexcitability caused by the glutamate transport inhibitor DL-threo-β-benzyloxyaspartic acid (TBOA) in primary neuronal cultures, and according to Styr et al. (2019) created a new homeostatic set-point, in the hippocampal neuronal network, creating a hyperexcitability proof network. This method was utilized also in two epilepsy mouse models: (1) The pentylenetetrazole induced seizure model (PTZ); and (2) the genetic Dravet Syndrome heat sensitivity model, in which the epileptic seizures are induced by elevation of temperature. [0007] The DHODH enzyme catalyzes the fourth enzymatic step, i.e., the ubiquinone-mediated oxidation of dihydroorotate to orotate, in the de novo pyrimidine biosynthesis. As further shown in WO 2018096538, addition of uridine (a precursor of pyrimidines) does not occlude the reduction in MFR induced by teriflunomide at a timescale of 2 days, indicating that lack of uridine is not the factor leading to long-term reduction in the MFR, and suggesting that DHODH inhibition triggers a long-term reduction in the MFR due to its direct inhibition of mitochondrial functions (without hampering ATP levels), but not de-novo pyrimidine synthesis. [0008] Teriflunomide, approved for treatment of multiple sclerosis, inhibits DHODH in a specific, non-competitive, reversible manner, thus blocks the de novo pyrimidine synthesis which interrupts the cell cycle in S phase and exerts a cytostatic effect on proliferating T and B cells, limiting their involvement in the inflammatory processes involved in the multiple sclerosis pathogenesis (Bar Or et al., 2014). Nevertheless, the mechanism of action of FUTURX-004 IL 305 348/3 teriflunomide in multiple sclerosis is by targeting peripheral DHODH, which affects the levels of T and B cells that then penetrate the brain. Teriflunomide does not cross the blood-brain barrier (BBB) in a sufficient manner and its efficacy in epilepsy, in which brain permeability is crucial, is therefore limited. [0009] BAY-2402234 ((S)-N-(2-chloro-6-fluorophenyl)-4-(4-ethyl-3-(hydroxymethyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-5-fluoro-2-((1,1,1-trifluoropropan-2-yl)oxy) benzamide; CAS: 2225819-06-5) is a potent and selective DHODH inhibitor, developed initially for myeloid malignancies such as acute myeloid leukemia. As reported, the antitumor activity of said compound was tested in a mouse model of IDH1-mutant glioma, and the compound was reported to accumulate in the brain tumor at a concentration of 1nM, suggesting sufficient brain permeability in this model (Shi et al., 2022). Nevertheless, in naïve mice used by the present inventors in epilepsy models, BAY-2402234 failed to demonstrate sufficient permeability. [0010] DHODH inhibitors other than teriflunomide and BAY-2402234 are in different development stages for different indications; however, according to our knowledge, none of them crosses the BBB in a sufficient manner. Thus, in order to utilize the inhibition of DHODH to treat epilepsy effectively, novel soluble DHODH inhibitors that are BBB permeable are required.
SUMMARY OF INVENTION [0011] In one aspect, disclosed herein is a compound of formula I or II: , I II or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, wherein R is aryl, -(C1-C6)alkyl-aryl such as benzyl, heteroaryl, -(C1-C6)alkyl-heteroaryl, heterocyclyl, or -(C1-C6)alkyl-heterocyclyl, optionally substituted; Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 R is aryl, -(C1-C6)alkyl-aryl such as benzyl, heteroaryl, -(C1-C6)alkyl-heteroaryl, heterocyclyl, or -(C1-C6)alkyl-heterocyclyl, optionally substituted; R is selected from (C1-C6)alkyl, cycloalkyl, aryl, and heteroaryl; and R, R, R and R each independently is H, halogen, (C1-C6)alkyl, or (C1-C6)haloalkyl. [0012] In certain embodiments, disclosed herein is a compound as defined above, wherein R is phenyl optionally substituted, preferably at position 2 thereof, with at least one substituent each independently selected from deuterium, halogen, -CN, (C1-C6)alkyl, (C1-C6)haloalkyl, and -O-(C1-C6)alkyl, and optionally further substituted, preferably at position or 6 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is heteroaryl optionally substituted, e.g., pyridin-2-yl, 3-methylpyridin-2-yl, 4-methylpyridin-2-yl, 5-methylpyridin-2-yl, 6-methylpyridin-2-yl, 4,5-dimethylpyridin-2-yl, 4-trifluoromethyl pyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 4-fluoropyridine-2-yl, 4-chloropyridin-2-yl, 4-bromopyridin-2-yl, 2-hydroxypyridin-3-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyridin-3-yl, 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyrimidin-6-yl, thiazol-4-yl, 2-(methylthio)thiazol-4-yl, tetrahydrobenzo[c] isoxazole-3-yl, tetrahydro-1,2-benzisoxazol-3-yl, 5-methylisoxazol-3-yl, 5-ethylisoxazol-3-yl, 5,6-dihydro-4H-cyclopenta[d]isoxazol-3-yl, methylnicotinate-6-yl, 5-methylpyrazin-2-yl, benzofuran-2-yl, 3-methylbenzofuran-2-yl, 3-methylthiophene-2-yl, 1,3-benzoxazol-2-yl, indol-2-yl, 4-methoxyquinolin-2-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, and 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; R is (C1-C6)alkyl or (C3-C7)cycloalkyl; and R, R, R and R each independently is H, fluoro, or (C1-C3)alkyl. [0013] Specific such compounds exemplified herein are those identified herein as SLN-008, SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, and SLN-0(all of the formula I), as well as SLN-023, SLN-024, and SLN-038 (all of the formula II), or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof (see Table 1 ). [0014] In another aspect, disclosed herein is a pharmaceutical composition comprising a compound of the formula I or II as defined above, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof. Such compositions may be formulated for enteral or parenteral administration, as well as for inhalation, and are useful in preventing, delaying onset of, or treating a disease or disorder associated with impaired (abnormal) Underlined postdated 12.12.20Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 neuronal excitability, more specifically elevated neuronal excitability (i.e., neuronal hyperexcitability). In other words, said compositions are useful in preventing, delaying onset of, or treating a disease or disorder that may benefit from reduction of DHODH enzymatic activity. Particular diseases or disorders associated with neuronal hyperexcitability includes, without limiting, neurological or neurodegenerative diseases or disorders, autoimmune diseases, and cancer. [0015] In yet another aspect, disclosed herein is a compound of the formula I or II as defined above, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, for use in the preparation of a pharmaceutical composition for preventing, delaying onset of, or treating a disease or disorder associated with impaired neuronal excitability, more specifically elevated neuronal excitability, e.g., a disease or disorder associated with DHODH enzyme activity. [0016] In a further aspect, disclosed herein is a method for preventing, delaying onset of, or treating a disease or disorder associated with impaired neuronal excitability (i.e., neuronal hyperexcitability), more specifically elevated neuronal excitability, e.g., a disease or disorder associated with DHODH enzyme activity, in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a compound of the formula I or II as defined above, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof.
BRIEF DESCRIPTION OF DRAWINGS [0017] Fig. 1 shows inhibitory activity of commercially available DHODH inhibitors at µM, measured in an enzymatic assay using DCIP as a final electron acceptor and 2 nM of recombinant DHODH. The DHODH inhibitors tested were teriflunomide ((2Z)-2-cyano-3-hydroxy-N-[4-(trifluoromethyl) phenyl]but-2-enamide); brequinar (6-fluoro-2-[4-(2-fluorophenyl)phenyl]-3-methylquinoline-4-carboxylic acid); NITD-982 (methyl 5-[1-(3-chlorophenyl)-5-(trifluoromethyl)-1H-pyrazol-4-yl]-3-(2,6-dichlorophenyl)isoxazole-4-carboxylate); 21q (5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridin); 1291 (1-(3’-(dimethylamino)-2,6-difluoro-[1,1’-biphenyl]-4-yl)-6-isopropyl-1,5,6,7-tetrahydro-4H-benzo[d][1,2,3] triazol-4-one); and BAY-2402234. [0018] Fig. 2 shows IC50 determination of SLN-023 and its enantiomers SLN-023A (active enantiomer) and SLN-023B, SLN-031 and its enantiomers SLN-031A and SLN- FUTURX-004 IL 305 348/3 031B (active enantiomer), SLN-038, and SLN-042. The half maximal inhibitory concentration was determined using 6 concentrations. [0019] Fig. 3 shows IC50 determination of SLN-038 and its enantiomers SLN-038A and SLN-038B (active enantiomer), SLN-042 and its enantiomers SLN-042A (active R-enantiomer) and SLN-042B (inactive S enantiomer), and SLN-043, The half maximal inhibitory concentration was determined using 6 concentrations. [0020] Figs. 4A-4B show the influence of SLN compounds and BAY-2402234 on cell proliferation. HepG2 cells were treated with different compounds or DMSO as a control, in the presence or absence of 100μM uridine. Cell confluence as a measure of cell growth was monitored over 72h using CellTiterGlo. The plates were prepared in triplicates. ( 4A ) BAY-2402234 and SLN-031; and ( 4B ) BAY-2402234, SLN-038 and SLN-042. [0021] Figs. 5A-5D show the inhibition of DHODH by known inhibitors causes prolonged reduction MFR in hippocampal neurons. [0022] Figs. 6A-6D . Pretreatment with known DHODH inhibitors suppresses induced excitation in hippocampal neurons. [0023] Figs. 7A-7C show that SLN-023, SLN-031 and SLN-042A (active R-enantiomer) molecules reduce and stabilize MFR, and suppress excitation in hippocampal neurons. [0024] Figs. 8A-8C show that short-term exposure to SLN-023, SLN-031 or SLN-042A (active R-enantiomer) molecules provides an effect similar to long-term exposure in MFR stabilization and excitation suppression. [0025] Fig. 9 shows the effects of SLN-031 on forelimb clonus. [0026] Fig. 10 shows the effects of SLN-042A (active R-enantiomer) on forelimb clonus . [0027] Fig. 11 show percentages of protected (darkest blue), partially protected (blue), and not protected larvae (light blue) for each treatment with SLN-023 and SLN-031. [0028] Figs. 12A-12C show ( 12A ) quantification of the total distance moved during minutes of light exposure; ( 12B ) max velocity (left panel) and number of turns (right panel) reached in response to light flashes; and ( 12C ) quantification of the distance moved during the dark/light cycles per minute (left panel) and during the total duration of the trial (right panel). Dark periods in the left plot are highlighted in grey. Not injected wild-type controls are shown in the darkest blue; Control larvae (scramble) are shown in dark blue; scn1lab crispants are displayed in light blue; scn1lab crispants treated with SLN-023 are represented in green (light green = 32.25 µM; dark green = 64.5 µM); scn1lab crispants treated with SLN-031 are represented in purple (light purple = 14.5 µM; dark purple = 29 µM); scn1lab Underlined postdated 12.12.2023 Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 crispants treated with topiramate are represented in orange (light orange = 50 µM; dark orange = 100 µM). Error bars show min to max values.
DETAILED DESCRIPTION [0029] Disclosed herein are novel soluble DHODH inhibitors, which are BBB penetrable. As shown herein, these DHODH inhibitors possess microsomal stability and other favorable absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters, and demonstrate sufficient pharmacokinetic properties, and positive efficacy in epilepsy animal models. Selene’s novel compounds possess a chiral center. [0030] In one aspect, the present invention thus provides a compound of formula I or II: , I II or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, wherein R is aryl, -(C1-C6)alkyl-aryl such as benzyl, heteroaryl, -(C1-C6)alkyl-heteroaryl, heterocyclyl, or -(C1-C6)alkyl-heterocyclyl, optionally substituted; R is aryl, -(C1-C6)alkyl-aryl such as benzyl, heteroaryl, -(C1-C6)alkyl-heteroaryl, heterocyclyl, or -(C1-C6)alkyl-heterocyclyl, optionally substituted; R is selected from (C1-C6)alkyl, cycloalkyl, aryl, and heteroaryl; and R, R, R and R each independently is H, halogen, (C1-C6)alkyl, or (C1-C6)haloalkyl. [0031] The term "alkyl" typically means a linear or branched hydrocarbyl having, e.g., 1-carbon atoms and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like. Preferred are (C1-C6)alkyl groups, more preferably (C1-C3)alkyl groups, most preferably methyl, ethyl, propyl, or isopropyl. The alkyl defined herein may optionally be substituted with one or more groups each independently selected from deuterium, halogen, (C1-C6)haloalkyl such Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 as -CF3, -COR, -COOR, -CON(R)2, -CN, -OR, -O-C(=O)R, -NO2, -N(R)2, -SR, -SO2R, and -S(=O)R, wherein R each independently is H, unsubstituted (C1-C6)alkyl or (C1-C6)haloalkyl, and may further optionally be interrupted by one or more identical or different heteroatoms selected from S, O and N. [0032] The term “haloalkyl” as used herein refers to an alkyl group as defined above, substituted, at any position thereof, with at least one halogen. In case the alkyl is substituted with more than one halogens, said substituents may be linked to either the same or different carbon atoms. Particular haloalkyl groups are those consisting of a linear hydrocarbyl wherein the terminal carbon atom is substituted with one or more halogens, e.g., -(CH2)n-CH2F, -(CH2)n-CHF2, -(CH2)n-CF3, -(CH2)n-CH2Cl, -(CH2)n-CHCl2, or -(CH2)n-CCl3, wherein n is an integer of 0-5. [0033] The term “carbocyclic ring” means a mono-, bi-, or polycyclic saturated ring of carbon atoms, e.g., 3-7 carbon atoms. The term "carbocyclic group”, “carbocyclyl”, or “cycloalkyl” used herein interchangeably refers to any univalent group derived from a carbocyclic ring as defined herein by removal of hydrogen from one of the carbon atoms. Examples of such groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. The carbocyclic group may optionally be substituted, at any position of the ring, with one or more groups each independently selected from deuterium, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl such as -CF3,-COR, -COOR, -CON(R)2, -CN, -OR, -O-C(=O)R, -NO2, -N(R)2, -SR, -SO2R, and -S(=O)R, wherein R each independently is H, unsubstituted (C1-C6)alkyl or (C1-C6)haloalkyl. [0034] The term "heterocyclic ring" as used herein denotes a mono- or poly-cyclic non-aromatic ring of, e.g., 3-12 atoms containing at least two carbon atoms and at least one heteroatom selected from S, O, and N, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. Non-limiting examples of heterocyclic ring include pyrrolidine, piperidine, pyridine, dihydropyridine, tetrahydropyridine, pyrazole, pyrazoline, pyrazolidine, piperazine, imidazolidine, imidazoline, tetrahydropyrimidine, dihydrotriazine, azepane, ethylene oxide, furan, tetrahydrofuran, pyran, dihydropyran, tetrahydropyran, dioxole, dioxolane, morpholine, oxazolidine, oxazole, oxadiazole, oxazoline, dihydrooxadiazole, thiomorpholine, thiazolidine, thiazole, thiadiazole, and thiazoline. Preferred are 5- or 6-membered heterocyclic rings. The heterocyclic ring may be substituted at any of the ring atoms, with one or more groups each independently selected from deuterium, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl such as -CF3,-COR, -COOR, -CON(R)2, FUTURX-004 IL 305 348/3 -CN, -OR, -O-C(=O)R, -NO2, -N(R)2, -SR, -SO2R, and -S(=O)R, wherein R each independently is H, unsubstituted (C1-C6)alkyl or (C1-C6)haloalkyl. The term “heterocyclyl” as used herein refers to any univalent group derived from a heterocyclic ring as defined herein by removal of hydrogen atom from any ring atom. [0035] The term “aromatic ring” denotes an aromatic carbocyclic ring having, e.g., 6-14, preferably 6-10, carbon atoms, and consisting of a single ring or multiple rings either condensed or linked by a covalent bond. Non-limiting examples of aromatic rings include benzene, naphthalene, anthracene, naphthacene, phenanthrene, pyrene, chrysene, tetracene, and triphenylene. The term “aromatic group” or “aryl” used herein interchangeably denotes a univalent group derived from an aromatic ring by removal of hydrogen atom from any of the ring atoms. The aryl may optionally be substituted by at least one group each independently selected from deuterium, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl such as -CF3, -O-(C1-C6)alkyl, -COR, -COOR, -CON(R)2, -CN, -OR, -O-C(=O)R, -NO2, -N(R)2, -SR, -SO2R, and -S(=O)R, wherein R each independently is H, unsubstituted (C1-C6)alkyl or (C1-C6)haloalkyl. [0036] The term “heteroaromatic ring” denotes a mono-, bi-, or poly-cyclic aromatic ring having, e.g., 4-12 atoms, and consisting of at least one carbon atom and at least one heteroatom selected from nitrogen, oxygen, and sulfur (optionally oxidized). Preferred are mono- or bi-cyclic aromatic rings having 5-10 atoms. The term “heteroaromatic group” or “heteroaryl” used herein interchangeably refers to a univalent group derived from a heteroaromatic ring as defined herein by removal of hydrogen atom from any of the ring atoms. Examples of mono-cyclic heteroaromatic ring include, without being limited to, pyridine, pyrimidine, thiazole, oxazole, isoxazole, pyrazine, thiophene, indole, imidazole, pyrrole, furan, thiazine, pyrazole, thiazole, isothiazole, 1,2,3-triazine, 1,3,4-triazine, and 1,3,5-triazine. Polycyclic heteroaromatic rings are preferably composed of two rings such as, but not limited to, imidazo[1,2-a]pyridine, imidazo[1,5-a]pyridine, triazolo[1,5-a]pyridine, imidazo[1,5-a]pyrimidine, benzoxazole, benzoisoxazole, benzo[c]isoxazole, cyclopenta[c]isoxazole, cyclopenta[d]isoxazole, benzofuran, quinoline, isoquinoline benzofurane, isobenzofurane, benzothiophene, indole, benzimidazole, benzthiazole, pyrido[1,2-a]pyrimidine and 1,3-benzodioxole. The heteroaryl may optionally be substituted, at any position of the ring, with at least one group each independently selected from deuterium, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl such as -CF3, (C1-C6)alkyl-NR2, (C3-C7)cycloalkyl, -COR, -COOR, -CON(R)2, -CN, -OR, -O-C(=O)R, -NO2, -N(R)2, -SR, -Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 SO2R, and -S(=O)R, wherein R each independently is H, unsubstituted (C1-C6)alkyl or (C1-C6)haloalkyl. It should be understood that when a polycyclic heteroaryl is substituted, the substitution may be in any of the carbocyclic and/or heterocyclic rings. [0037] The term “deuterium” as used herein refers to a stable isotope of hydrogen, which, unlike “normal” hydrogen atoms, or protium, also contains a neutron. [0038] The term “halogen” as used herein refers to a halogen and includes fluoro, chloro, bromo, and iodo, but it is preferably fluoro or chloro. [0039] In certain embodiments, the present invention discloses a compound of the formula I or II as defined above, wherein R is phenyl optionally substituted with at least one substituent each independently selected from deuterium, halogen, -CN, (C1-C6)alkyl, (C1-C6)haloalkyl, and -O-(C1-C6)alkyl. In particular such embodiments, R is phenyl substituted, preferably at position 2 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl, and optionally further substituted, preferably at position 3 or 6 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl. [0040] In certain embodiments, the present invention discloses a compound of the formula I or II as defined above, wherein R is heteroaryl optionally substituted, such as pyridin-2-yl, 3-methylpyridin-2-yl, 4-methylpyridin-2-yl, 5-methylpyridin-2-yl, 6-methylpyridin-2-yl, 4,5-dimethylpyridin-2-yl, 4-trifluoromethylpyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 4-fluoropyridine-2-yl, 4-chloropyridin-2-yl, 4-bromopyridin-2-yl, 2-hydroxypyridin-3-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyridin-3-yl, 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyrimidin-6-yl, thiazol-4-yl, 2-(methylthio)thiazol-4-yl, tetrahydrobenzo[c] isoxazole-3-yl, tetrahydro-1,2-benzisoxazol-3-yl, 5-methylisoxazol-3-yl, 5-ethylisoxazol-3-yl, 5,6-dihydro-4H-cyclopenta[d]isoxazol-3-yl, methylnicotinate-6-yl, 5-methylpyrazin-2-yl, benzofuran-2-yl, 3-methylbenzofuran-2-yl, 3-methylthiophene-2-yl, 1,3-benzoxazol-2-yl, indol-2-yl, 4-methoxyquinolin-2-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, and 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl. Particular such embodiments are those wherein R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, tetrahydrobenzo[c]isoxazole-3-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, or 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl.
Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3
[0041] In certain embodiments, the present invention discloses a compound of the formula I or II as defined above, wherein R is (C1-C6)alkyl, preferably methyl, ethyl, propyl, or isopropyl, or (C3-C7)cycloalkyl. [0042] In certain embodiments, the present invention discloses a compound of the formula I or II as defined above, wherein R, R, R and R each independently is H, fluoro, or (C1-C3)alkyl. In particular such embodiments, R, R, R and R each is H or fluoro; or two of R, R, R and R linked to the same carbon atom (e.g., R and R, or R and R) each is H or fluoro, and the other two of R, R, R and R each is methyl or ethyl. [0043] In certain embodiments, the present invention discloses a compound of the formula I or II as defined above, wherein R is phenyl optionally substituted with at least one substituent each independently selected from deuterium, halogen, -CN, (C1-C6)alkyl, (C1-C6)haloalkyl, and -O-(C1-C6)alkyl; R is heteroaryl optionally substituted; R is (C1-C6)alkyl or (C3-C7)cycloalkyl; and R, R, R and R each independently is H, fluoro, or (C1-C3)alkyl. In particular such embodiments, R is phenyl substituted, preferably at position 2 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl, and optionally further substituted, preferably at position 3 or 6 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is selected from pyridin-2-yl, 3-methylpyridin-2-yl, 4-methylpyridin-2-yl, 5-methylpyridin-2-yl, 6-methylpyridin-2-yl, 4,5-dimethylpyridin-2-yl, 4-trifluoromethyl pyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 4-fluoropyridine-2-yl, 4-chloropyridin-2-yl, 4-bromopyridin-2-yl, 2-hydroxypyridin-3-yl, 5,6,7,8-tetrahydroimidazo [1,5-a]pyridin-1-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyridin-3-yl, 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyrimidin-6-yl, thiazol-4-yl, 2-(methylthio)thiazol-4-yl, tetrahydrobenzo[c]isoxazole-3-yl, tetrahydro-1,2-benzisoxazol-3-yl, 5-methylisoxazol-3-yl, 5-ethylisoxazol-3-yl, 5,6-dihydro-4H-cyclopenta[d]isoxazol-3-yl, methylnicotinate-6-yl, 5-methylpyrazin-2-yl, benzofuran-2-yl, 3-methylbenzofuran-2-yl, 3-methylthiophene-2-yl, 1,3-benzoxazol-2-yl, indol-2-yl, 4-methoxyquinolin-2-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, and 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; R is selected from methyl, ethyl, propyl, isopropyl, and cyclopropyl; and R, R, R and R each is H or fluoro; or two of R, R, R and R linked to the same carbon atom each is H or fluoro, and the other two of R, R, R and R each is methyl or ethyl. More particular such embodiments are those wherein R is phenyl substituted, preferably at position thereof, with fluoro or chloro, and optionally further substituted, preferably at position 3 or Underlined postdated 12.12.20Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 6 thereof, with fluoro, chloro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, tetrahydrobenzo[c] isoxazole-3-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, or 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; R is methyl; and R, R, R and R each is H; or R and R each is H, and R and R each is methyl. [0044] In certain specific embodiments exemplified herein, the compound disclosed herein is a compound of the formula I, wherein (i) R is 2-fluorophenyl; R is tetrahydrobenzo[c]isoxazole-3-yl; and R, R, R and R each is H (herein identified SLN-008); (ii) R is 2-fluorophenyl; R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl; and R, R, R and R each is H (herein identified SLN-031); (iii) R is 2-fluorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-042); (iv) R is 2-fluorophenyl; R is 6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl; and R, R, R and R each is H (herein identified SLN-043); (v) R is 2-fluorophenyl; R is 5-cyclopropyl-1-methyl-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-044); (vi) R is 2-chlorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-049); (vii) R is 2-fluoro-3-methylphenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-050); (viii) R is 2,6-difluorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-051); or (ix) R is 2-fluorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; R and R each is H; and R and R each is methyl (herein identified SLN-052). In other specific embodiments exemplified herein, the compound disclosed herein is a compound of the formula II, wherein (i) R is 2-fluorophenyl; R is tetrahydrobenzo[c]isoxazole-3-yl; and R is methyl (herein identified SLN-023); (ii) R is 2-fluorophenyl; R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl; and R is methyl (herein identified SLN-038); or (iii) R is 2-fluorophenyl; R is tetrahydrobenzo[c]isoxazole-3-yl; and R is cyclopropyl (herein identified SLN-024) (see Table 1 ). Preferred are the compounds of formula I identified herein as SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, and SLN-052, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof; and the compounds of formula II identified herein as SLN-023 and SLN-038, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof.
Underlined postdated 12.12.2023 Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 Table 1 . Specific SLN compounds exemplified herein SLN-008 SLN-023 SLN-024 SLN-031 SLN-038 SLN-042 SLN-043 SLN-044 SLN-049 Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 SLN-050 SLN-051 SLN-052
[0045] The compounds of the present invention, also referred to herein as “ the SLN molecules/compounds ”, may be synthesized according to any suitable technology or procedure known in the art, e.g., as described in the Experimental section hereinafter (procedures for the synthesis of the compounds exemplified are depicted in Schemes 1-7 in the Appendix). [0046] The compounds of the formula I or II may have one or more asymmetric centers, e.g., at the ring carbon atom linked to the -NH-CO- group, and may accordingly exist both as enantiomers, i.e., optical isomers (R, S, or racemate, wherein a certain enantiomer may have an optical purity of 90%, 95%, 99% or more) and as diastereoisomers. The present invention encompasses all such enantiomers, isomers and mixtures thereof, as well as pharmaceutically acceptable salts thereof. [0047] Optically active forms of the compounds of the invention may be prepared using any method known in the art, e.g., by resolution of the racemic form by recrystallization techniques; by chiral synthesis; by extraction with chiral solvents; or by chromatographic separation using a chiral stationary phase. A non-limiting example of a method for obtaining optically active materials is transport across chiral membranes, i.e., a technique whereby a racemate is placed in contact with a thin membrane barrier, the concentration or pressure differential causes preferential transport across the membrane barrier, and separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through. Chiral chromatography, including simulated moving bed chromatography, can also be used. A wide variety of chiral stationary phases are commercially available.
Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3
[0048] Pharmaceutically acceptable salts of the compounds disclosed herein may be either acidic or basic salts. Suitable acidic pharmaceutically acceptable salts include, without being limited to, the mesylate salt, the maleate salt, the fumarate salt, the tartrate salt, the hydrochloride salt, the hydrobromide salt, the esylate salt, the p-toluenesulfonate salt, the benzenesulfonate salt, the benzoate salt, the acetate salt, the phosphate salt, the sulfate salt, the citrate salt, the carbonate salt, and the succinate salt. Basic pharmaceutically acceptable salts include, without limiting, metal salts such as alkali metal salts, e.g., lithium, sodium and potassium salts, and alkaline earth metal salts, e.g., calcium and magnesium salts; as well as salts of ammonium (NH4+) or an organic cation derived from an amine of the formula R4N+, wherein each one of the Rs independently is selected from H, C 1-C22, preferably C1-C6 alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine. [0049] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the formula I or II according to any one of the embodiments above, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutical acceptable carrier and/or excipient. [0050] In certain embodiments, the pharmaceutical composition of the present invention comprises a compound of the formula I or II, wherein R is phenyl optionally substituted with at least one substituent each independently selected from deuterium, halogen, -CN, (C1-C6)alkyl, (C1-C6)haloalkyl, and -O-(C1-C6)alkyl; R is heteroaryl optionally substituted; R is (C1-C6)alkyl or (C3-C7)cycloalkyl; and R, R, R and R each independently is H, fluoro, or (C1-C3)alkyl. In particular such embodiments, R is phenyl substituted, preferably at position 2 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl, and optionally further substituted, preferably at position 3 or thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is selected from pyridin-2-yl, 3-methylpyridin-2-yl, 4-methylpyridin-2-yl, 5-methylpyridin-2-yl, 6-methylpyridin-2-yl, 4,5-dimethylpyridin-2-yl, 4-trifluoromethyl pyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 4-fluoropyridine-2-yl, 4-chloropyridin-2-yl, 4-bromopyridin-2-yl, 2-hydroxypyridin-3-yl, 5,6,7,8-tetrahydroimidazo [1,5-a]pyridin-1-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyridin-3- Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 yl, 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyrimidin-6-yl, thiazol-4-yl, 2-(methylthio)thiazol-4-yl, tetrahydrobenzo[c] isoxazole-3-yl, tetrahydro-1,2-benzisoxazol-3-yl, 5-methylisoxazol-3-yl, 5-ethylisoxazol-3-yl, 5,6-dihydro-4H-cyclopenta[d]isoxazol-3-yl, methylnicotinate-6-yl, 5-methylpyrazin-2-yl, benzofuran-2-yl, 3-methylbenzofuran-2-yl, 3-methylthiophene-2-yl, 1,3-benzoxazol-2-yl, indol-2-yl, 4-methoxyquinolin-2-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, and 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; R is selected from methyl, ethyl, propyl, isopropyl, and cyclopropyl; and R, R, R and R each is H or fluoro; or two of R, R, R and R linked to the same carbon atom each is H or fluoro, and the other two of R, R, R and R each is methyl or ethyl. More particular such embodiments are those wherein R is phenyl substituted, preferably at position 2 thereof, with fluoro or chloro, and optionally further substituted, preferably at position 3 or 6 thereof, with fluoro, chloro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, tetrahydrobenzo[c] isoxazole-3-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, or 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; R is methyl; and R, R, R and R each is H; or R and R each is H, and R and R each is methyl. [0051] In specific embodiments, the compound comprised within the pharmaceutical composition is a compound selected from the compounds herein identified SLN-008, SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, SLN-052, SLN-023, SLN-024, and SLN-038, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof. [0052] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein interchangeably refers to any non-active ingredient such as a solvent, dispersion medium, preservative, antioxidant, coating, isotonic and absorption delaying agent, and the like, that is compatible with pharmaceutical administration, and does not produce an adverse, allergic, or other untoward reaction when administered to a mammal or human as appropriate. For human administration, compositions should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory authorities such as the U.S. Food and Drug Administration (FDA), and the European Medicines Agency (EMA). [0053] The compositions provided by the present invention may be prepared by conventional techniques known in the art, e.g., as described in Remington: The Science and Practice of Pharmacy, 19th Ed., 1995. In particular, the compositions may be prepared, e.g., Underlined postdated 12.12.20Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 by uniformly and intimately bringing the active agent, i.e., the compound of the formula I or II, into association with a liquid or semi-liquid, e.g., gel, carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation. [0054] The pharmaceutical compositions of the present invention may be formulated for either enteral administration, e.g., oral, sublingual, buccal, or rectal administration; or parenteral administration, e.g., intravenous, intraarterial, intrathecal, intrapleural, intratracheal, intraperitoneal, intramuscular, intraosseous, intracerebroventricular, intranasal, transdermal, subcutaneous, or topical administration. The pharmaceutical composition may also be formulated for inhalation. [0055] Pharmaceutical compositions formulated for oral administration may be in the form of a liquid, e.g., a solution in an edible solvent such as ethanol, tincture, syrup, or elixir; a semi-solid such as a gel; or a solid such as tablets, caplets, pills, troches, lozenges, dispersible powder or granules, hard or soft capsules, and sachets. In some particular embodiments, the pharmaceutical composition is in the form of a bi- or multilayer tablet, in which each one of the layers comprise the active agent, and the layers are optionally separated by an intermediate, inactive layer, e.g., a layer comprising one or more disintegrants. [0056] Useful dosage forms of the pharmaceutical compositions include orally disintegrating systems including, but not limited to, solid, semi-solid and liquid systems including disintegrating or dissolving tablets, soft or hard capsules, gels, fast dispersing dosage forms, controlled dispersing dosage forms, caplets, films, wafers, ovules, granules, buccal/mucoadhesive patches, powders, freeze dried (lyophilized) wafers, chewable tablets which disintegrate with saliva in the buccal/mouth cavity and combinations thereof. Useful films include, but are not limited to, single layer stand-alone films and dry multiple layer stand-alone films. [0057] In certain embodiments, the pharmaceutical composition is formulated for oral administration, and is in the form of a matrix tablet wherein the release of the active agent is controlled by having said active agent diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity. In FUTURX-004 IL 305 348/3 other embodiments, the tablet is formulated as a bi- or multi-layer tablet, made up of two or more distinct layers of granulation compressed together with the individual layers lying one on top of another, with each separate layer containing a different active agent. Bilayer tablets have the appearance of a sandwich since the edge of each layer or zone is exposed. [0058] Pharmaceutical compositions for oral administration may be formulated for immediate release, as well as for controlled release, i.e., extended-, sustained-, or delayed-release, of the active agent. Compositions for extended or sustained release are aimed at releasing the active agent over an extended period of time following ingestion, wherein there is no rapid release (dumping) of the active agent at any point of time, and may be in the form of a controlled-release matrix, e.g., controlled-release matrix tablets, in which the release of a soluble active agent is controlled by having the active diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo). In contrast, compositions for delayed release are aimed at inhibiting the release of the active agent in the stomach, i.e., delaying the release of said active agent until at least a portion of the dosage form has traversed the stomach, in order to avoid the acidity of the gastric contents from hydrolyzing the active agent. Particular compositions for controlled release are those wherein the active agent is coated by a pH-dependent enteric-coating polymer. Examples of pH-dependent enteric-coating polymer include, without being limited to, Eudragit® S (poly(methacrylicacid, methylmethacrylate), 1:2), Eudragit® L 55 (poly (methacrylicacid, ethylacrylate), 1:1), Kollicoat® (poly(methacrylicacid, ethylacrylate), 1:1), hydroxypropyl methylcellulose phthalate (HPMCP), alginates, carboxymethylcellulose, and combinations thereof. The pH-dependent enteric-coating polymer may be present in the composition in an amount from about 10% to about 95% by weight of the entire composition. [0059] Another contemplated formulation is depot systems, based on biodegradable polymers. As the polymer degrades, the active agent is slowly released. The most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules. Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLG). [0060] Pharmaceutical compositions for oral administration may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
FUTURX-004 IL 305 348/3 In addition, said compositions may comprise one or more pharmaceutically acceptable excipients. For example, a tablet may comprise at least one filler such as lactose, ethylcellulose, microcrystalline cellulose, and silicified microcrystalline cellulose; at least one disintegrant such as cross-linked polyvinylpyrrolidinone; at least one binder such as polyvinylpyridone, and hydroxypropylmethyl cellulose; at least one surfactant such as sodium laurylsulfate; at least one glidant such as colloidal silicon dioxide; and at least one lubricant such as magnesium stearate. [0061] Pharmaceutical compositions formulated for rectal administration may be in the form of suppositories; rectal capsules; semisolids such as gels, creams and ointments; and liquid rectal preparations such as solutions, emulsions and suspensions, wherein rectal suppositories are preferred. [0062] Pharmaceutical compositions formulated for parenteral administration, i.e., for administration elsewhere in the body than the mouth and alimentary canal, may be in the form of a sterile, optionally injectable, aqueous or oleaginous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents. The sterile injectable preparation may also be an injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include, without limiting, water, Ringer's solution, polyethylene glycol (PEG), 2-hydroxypropyl- -cyclodextrin (HPCD), a surfactant such as Tween-80, and isotonic sodium chloride solution. [0063] Pharmaceutical compositions formulated for inhalation may be administered utilizing any suitable device known in the art, such as metered dose inhalers, liquid nebulizers, dry powder inhalers, sprayers, thermal vaporizers, electrohydrodynamic aerosolizers, and the like. [0064] As shown in the Experimental section herein, the compounds of the formula I or II disclosed herein have demonstrated anticonvulsant efficacy in a mouse 6Hz 32mA psychomotor seizure test (SLN-031 at 150 mg/kg, po; and SLN-042A (the active R-enantiomer) at 100 mg/kg, po) and in the Dravet Syndrome model for genetic generalized epilepsy in zebrafish larvae (SLN-023 at 32.25 and 62.5 µM; SLN-031 at 29 µM). [0065] The compounds and pharmaceutical compositions of the present invention, according to any one of the embodiments above, are thus useful for use in preventing, delaying onset of, or treating a disease or disorder associated with impaired, i.e., abnormal, Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 neuronal excitability, more specifically elevated neuronal excitability also commonly referred to as neuronal hyperexcitability. In certain embodiments, said disease or disorder associated with elevated neuronal excitability, i.e., neuronal hyperexcitability, is a disease or disorder associated with DHODH enzyme activity, e.g., a disease or disorder associated with DHODH enzyme activity in the central nervous system (CNS). In other words, the compounds and pharmaceutical compositions of the present invention, according to any one of the embodiments above, are useful in preventing, delaying onset of, or treating any disease or disorder that may benefit from reduction of DHODH enzymatic activity. [0066] The term “impaired neuronal excitability” or “abnormal neuronal excitability” as used herein interchangeably refers to unnormal activity of the brain due to imbalance between excitatory and inhibitory neurons which may lead to various pathologies and symptoms. [0067] The term “elevated neuronal excitability” or “neuronal hyperexcitability” as used herein interchangeably refers to increased probability that a neuron or neurons will be activated by a certain stimulus, leading to an increased brain activity (Targa Dias Anastacio et al., 2022). [0068] In yet another aspect, the present invention refers to a compound of the formula I or II according to any one of the embodiments above, e.g., SLN-008, SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, SLN-052, SLN-023, SLN-024, or SLN-038, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, for use in the preparation of a pharmaceutical composition for preventing, delaying onset of, or treating a disease or disorder associated with impaired neuronal excitability, more specifically elevated neuronal excitability, e.g., a disease or disorder associated with DHODH enzyme activity. [0069] In a further aspect, the present invention relates to a method for preventing, delaying onset of, or treating a disease or disorder associated with impaired neuronal excitability (i.e., neuronal hyperexcitability), more specifically elevated neuronal excitability, e.g., a disease or disorder associated with DHODH enzyme activity, e.g., in the CNS, in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a compound of the formula I or II according to any one of the embodiments above, e.g., SLN-008, SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, SLN-052, SLN-023, SLN-024, or SLN-038, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof.
Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3
[0070] In certain embodiments, the disease or disorder associated with neuronal hyperexcitability referred to herein is selected from a neurological or neurodegenerative disease or disorder, an autoimmune disease, and cancer. [0071] Non-limiting examples of neurological or neurodegenerative diseases or disorders as referred to herein include epilepsy such as temporal lobe epilepsy (TLE), Dravet syndrome (also known as Severe Myoclonic Epilepsy of Infancy, SMEI) and Lennox-Gastaut syndrome, Alzheimer's disease, both sporadic and genetic, Parkinson’s disease, mild-cognitive impairments (MCI), and multiple sclerosis. In certain particular embodiments, the compound or pharmaceutical composition of the invention, according to any one of the embodiments above, is useful in treating epilepsy such as such as TLE, Dravet syndrome and Lennox-Gastaut syndrome, or in preventing sudden unexplained death in epilepsy (SUDEP). In other particular embodiments, the compound or pharmaceutical composition of the invention, according to any one of the embodiments above, is thus useful in treating Alzheimer's disease, or preventing or delaying onset of Alzheimer's disease in a subject being at genetic risk for Alzheimer's disease, e.g., a subject carrying apolipoprotein E (APOE)-ε4. [0072] Non-limiting examples of an autoimmune disease as referred to herein include, e.g., rheumatoid arthritis and multiple sclerosis. In certain particular embodiments, the compound or pharmaceutical composition of the invention, according to any one of the embodiments above, is thus useful in treating rheumatoid arthritis and multiple sclerosis. [0073] Non-limiting examples of cancer as referred to herein include lung cancer such as bronchogenic carcinoma, alveolar or bronchiolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, and mesothelioma; gastrointestinal cancer such as esophageal cancer, stomach cancer, pancreatic cancer, small intestine cancer, colon cancer, and rectal cancer; liver cancer such as hepatoma, cholangiocarcinoma (bile duct cancer), hepatoblastoma, angiosarcoma, and hepatocellular adenoma; breast cancer; adrenal cancer; pancreatic cancer; bone cancer such as osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, multiple myeloma, malignant giant cell tumor chordoma, and osteochronfroma; genitourinary cancer such as kidney cancer, bladder cancer, urethral cancer, prostate cancer, and testicular cancer; gynecologic cancer such as cancer of the uterus, cervix, ovaries, vulva, vagina, or fallopian tubes; haematologic cancer such as cancers of the blood and bone marrow (e.g., leukemia such as acute myeloid leukemia and chronic myeloid leukemia), Hodgkin lymphoma, non-Hodgkin lymphoma, FUTURX-004 IL 305 348/3 and Burkitt lymphoma; head and neck and/or nervous system cancer such as neuroblastoma, glioblastoma, and cancers of the skull, meninges, brain, or spinal cord; and skin cancer such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, and Kaposi's sarcoma. In certain particular embodiments, the compound or pharmaceutical composition of the invention, according to any one of the embodiments above, is thus useful in treating cancer such as any one of the specific cancers listed above. [0074] The term "subject" as used herein refers to a mammal that is either a human (herein also referred to as "individual") or a non-human animal. [0075] The term "treating" as used herein with respect to a disease or disorder associated with impaired neuronal excitability, more specifically neuronal hyperexcitability, means administering an active agent, i.e., a compound of the formula I or II according to any one of the embodiments above, e.g., SLN-008, SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, SLN-052, SLN-023, SLN-024, or SLN-038, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, after the onset of at least one pathological phenotype manifested by said disease or disorder in order to treat, reduce or attenuate said pathological phenotype, and/or slow down the progression of said disease or disorder, i.e., prevent the appearance, or delay the onset, of other pathological phenotypes associated with said disease or disorder. The terms "preventing" and "delaying the onset" as used herein with respect to said disease or disorder mean administering said active agent to a subject either diagnosed as suffering from impaired, more specifically elevated, neuronal excitability or being at genetic risk for developing a disease or disorder associated with impaired, more specifically elevated, neuronal excitability, prior to the onset of at least one pathological phenotype manifested by said disease or disorder, in order to prevent the appearance, or delay the onset, of said disease or disorder. [0076] The term "therapeutically effective amount" as used herein refers to the amount or dose of an active agent as defined above, i.e., a compound of the formula I or II, e.g., SLN-008, SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, SLN-052, SLN-023, SLN-024, or SLN-038, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, that is useful to treat, attenuate, prevent, or delay the onset of at least one pathological phenotype manifested by a disease or disorder associated with impaired neuronal excitability, more specifically neuronal hyperexcitability. [0077] Considering the disclosure of WO 2018096538 discussed above, it should be noted that the active agent provided according to the method disclosed herein, i.e., the compound Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 of the formula I or II according to any one of the embodiments above, e.g., SLN-008, SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051, SLN-052, SLN-023, SLN-024, or SLN-038, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, may be administered in combination with a pyrimidine nucleobase or an intermediate in the de novo synthesis thereof. [0078] The term "pyrimidine nucleobase" as used herein refers to any of the three types of nucleobases that are pyrimidine derivatives, i.e., uracil (pyrimidine-2,4(1H,3H)-dione; 2-oxy-4-oxy pyrimidine), cytosine (4-aminopyrimidin-2(1H)-one; 4-amino-1H-pyrimidine-2-one), and thymine (5-methylpyrimidine-2,4(1H,3H)-dione; 5-methyluracil). [0079] The terms "de-novo pyrimidine synthesis" and "de novo pyrimidine nucleotide biosynthesis", used herein interchangeably, refer to the "orotate pathway", which is defined as the formation of uridine monophosphate (UMP) from carbamoyl phosphate (CP). The initial reaction in the orotate pathway catalyzed by CP synthetase is the formation of CP by combination of carbonate, adenosine triphosphate (ATP) and an amino group from glutamine. Three additional reactions are necessary to form the pyrimidine ring from CP. The phosphoribosyl group of phosphoribosyl pyrophosphate is added to the pyrimidine base, orotate, forming orotidine 5'-monophosphate that is then decarboxylated to make UMP, the first pyrimidine nucleotide. UMP is subsequently phosphorylated to uridine diphosphate (UDP) and uridine triphosphate (UTP). The transfer of an amino group from glutamine to UTP by cytidine triphosphate (CTP) synthetase leads to the synthesis of CTP. UMP is also a precursor for the synthesis of cytidine monophosphate (CMP) and deoxythymidine monophosphate (dTMP). In certain embodiments, the intermediate in the de novo synthesis of pyrimidine nucleobases is uridine, UMP, cytidine, CMP, deoxythymidine, or dTMP. In particular such embodiments, said intermediate is uridine or UMP. [0080] In certain embodiments, the method of the present invention, according to any one of the embodiments above, further comprises administering to said subject an amount of a pyrimidine nucleobase or an intermediate in the de novo synthesis thereof, e.g., uridine, UMP, cytidine, CMP, deoxythymidine, or dTMP, but preferably UMP. According to the method of the invention, the active agent, i.e., the compound of the formula I or II, and said pyrimidine nucleobase or an intermediate in the de novo synthesis thereof, may be administered via the same or different administration routes, as well as concomitantly or subsequently at any order. In particular embodiments, the two agents referred to above are administered from a sole pharmaceutical composition.
Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3
[0081] In still a further aspect, the present invention thus provides a kit comprising: (i) a first pharmaceutical composition as disclosed herein, i.e., a pharmaceutical composition comprising a compound of the formula I or II according to any one of the embodiments above, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutical acceptable carrier and/or excipient; (ii) a second pharmaceutical composition comprising a pyrimidine nucleobase or an intermediate in the de novo synthesis thereof as defined in any one of the embodiments above; and optionally (iii) instructions for co-administration of said pharmaceutical compositions for preventing, delaying onset of, or treating a disease or disorder associated with impaired neuronal excitability, more specifically elevated neuronal excitability, e.g., a disease or disorder associated with elevated DHODH enzyme activity, e.g., in the CNS. [0082] The invention will now be illustrated by the following non-limiting Examples.
EXAMPLES Abbreviations [0083] ACN , acetonitrile; DCM , dichloromethane; DIEA , N,N-diisopropylethylamine; DMA , dimethylacetamide; DMF , N,N'-dimethylformamide; DMSO , dimethylsulfoxide; EDCI , 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt , hydroxybenzotriazole; EtOAc , ethyl acetate; Rt , retention time; TFA , trifluoroacetic acid; THF , tetrahydrofuran.
Example 1. Synthesis of SLN-008 [0084] Synthesis of compound 8-2 . As depicted in Scheme 1 , to a solution of cyclopentane-1,3-dione (2.00 g, 20.3 mmol, 1.00 eq) in toluene (20.0 mL) was added DMF-DMA (2.67 g, 22.4 mmol; 2.98 mL, 1.10 eq). The mixture was stirred at 20°C for 6 hrs. The reaction mixture was distilled under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM/methanol = 1/0 to 1/2). Thin layer chromatography (TLC) (DCM/methanol = 10/1, product: Rf = 0.42). Compound 8-2 (2.g, 19.0 mmol, 93.1% yield) was obtained as a brown solid. H NMR: (400 MHz, DMSO- d 6) δ 7.43 (s, 1H), 3.61 (s, 3 H), 3.37 (s, 3 H), 2.33 (s, 4 H). [0085] Synthesis of compound 8-3 . To a solution of compound 8-2 (2.15 g, 14.0 mmol, 1.00 eq) and (2-fluorophenyl)hydrazine (1.86 g, 14.7 mmol, 1.05 eq) in 1-butanol (18.4 mL) was added HCl (18.8 g, 196 mmol, 18.4 mL, 38.0% purity, 14.0 eq). The mixture was stirred FUTURX-004 IL 305 348/3 at 110°C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO2, DCM/ methanol = 1/to 1/2). (TLC: DCM/methanol = 10/1, product: Rf = 0.50). Compound 8-3 (1.20 g, 5.mmol, 39.5% yield) was obtained as light yellow solid. LCMS: m/z =217.0 [M+H]+. H NMR: (400 MHz, DMSO- d 6) δ 7.99 (s, 1 H), 7.74 (td, J = 7.8, 1.6 Hz, 1H), 7.66-7.49 (m, 2H), 7.47-7.35 (m, 1 H), 3.03 (s, 4 H). [0086] Synthesis of compound 8-4 . To a solution of compound 8-3 (1.00 g, 4.63 mmol, 1.00 eq) in ethanol (10.0 mL) was added NH2OH•HCl (1.29 g, 18.5 mmol, 4.00 eq) and K2CO3 (3.84 g, 27.7 mmol, 6.00 eq). The mixture was stirred at 90°C for 10 hrs. The mixture was filtered, and filter liquor was dried under vacuum to give residue. The residue was purified by column chromatography (SiO2, DCM/methanol = 1/0-1/2). (TLC: DCM/methanol = 10/1, product: Rf = 0.40). Compound 8-4 (0.918 g, 3.97 mmol, 85.8% yield) was obtained as a yellow solid. LCMS: m/z = 232.0. H NMR: (400 MHz, DMSO- d 6) δ 10.42-10.32 (m, 1H), 7.86 (s, 1H), 7.72-7.66 (m, 1H), 7.55-7.48 (m, 2H), 7.38 (ddd, J = 2.8, 5.8, 8.2 Hz, 1H), 3.24-3.13 (m, 2H), 3.01-2.90 (m, 2H). [0087] Synthesis of compound 8-5 . To a solution of compound 8-4 (500 mg, 2.16 mmol, 1.00 eq) in methanol (12.5 mL) and dioxane (6.25 mL) was added NiCl 2 (140 mg, 1.mmol, 0.50 eq), then the reaction mixture was cooled to -20°C, NaBH4 (327 mg, 8.65 mmol, 4.00 eq) was added slowly. The mixture was stirred at -20°C for 2 hrs. The reaction mixture was warmed to 0°C, sat. aq. NH4Cl (20 mL) was added dropwise, the mixture was concentrated under reduced pressure to remove solvent then extracted with EtOAc (ml×3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase high-performance liquid chromatography (HPLC) (0.1% FA condition). Compound 8-5 (0.312 g, 1.44 mmol, 66.4% yield) was obtained as a yellow oil. LCMS: m/z = 218.0 [M+H]+. [0088] Synthesis of SLN-008 . To a solution of compound 8-5 (100 mg, 460 µmol, 1.eq) and 4,5,6,7-tetrahydro-2,1-benzoxazole-3-carboxylic acid (84.6 mg, 506 µmol, 1.10 eq) in DCM (2.00 mL) was added DIEA (178 mg, 1.38 mmol, 240 µL, 3.00 eq), EDCI (132 mg, 690 µmol, 1.50 eq) and HOBt (93.3 mg, 690 µmol, 1.50 eq). The mixture was stirred at 20°C for 6 hrs. The reactions were concentrated under reduced pressure to remove solvent. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150 × 40 mm × 15 um ; mobile phase : [water(FA)-ACN] ; B% : 37% - 67%, 9 min). Compound SLN-008 (86.4 mg, FUTURX-004 IL 305 348/3 235 µmol, 51.2% yield, 100% purity) was obtained as an off-white solid. LCMS: m/z = 367.1 [M+H]+. H NMR: (400 MHz, DMSO- d 6) δ 9.13 (br d, J = 7.4 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.53 (s, 1H), 7.50-7.44 (m, 2H), 7.40-7.32 (m, 1H), 5.31-5.19 (m, 1H), 3.00-2.86 (m, 2H), 2.79-2.57 (m, 6H), 1.77-1.63 (m, 4H).
Example 2. Synthesis of SLN-023 [0089] Synthesis of compound 23-b . As depicted in Scheme 2 , to a solution of compound 23-a (5.00 g, 17.7 mmol, 1.00 eq), ethyl methylglycinate (2.85 g, 18.6 mmol, 1.05 eq), Na2CO3 (7.50 g, 70.8 mmol, 4.00 eq) in ACN (50.0 mL) was degassed and purged with Nfor 3 times, the mixture was stirred at 80°C for 16 hrs. The reaction solution was concentrated to dryness to give crude product which was purified by column chromatography (SiO2, petroleum ether/EtOAc = 10/1 to 2/1 ,petroleum ether/EtOAc = 2/1, product Rf = 0.4) to give compound 23-b (5.20 g, 14.3 mmol, 80.9% yield) as brown oil. LCMS: m/z = 364.3 [M+H]+. [0090] Synthesis of compound 23-c . To a solution of compound 23-b (5.00 g, 13.8 mmol, 1.00 eq) in THF (40.0 mL) was added LiHMDS (1.00 M, 13.8 mL, 1.00 eq). The mixture was stirred at 50°C for 3 hrs. The reaction mixture was quenched by addition H 2O 120 mL at 20°C, extracted with EtOAc 200 mL (100 mL×2). The combined organic layers were washed with NaCl 120 mL (120 mL×2), dried over Na2SO4, filtered and concentrated under reduced pressure to give product which was purified by column chromatography (SiO2, petroleum ether/EtOAc = 10/1 to 2/1, petroleum ether/EtOAc = 2/1, product Rf = 0.3) to give compound 23-c (1.66 g, 5.22 mmol, 37.9% yield) as orange solid. LCMS: m/z = 318.[M+H]+. [0091] Synthesis of compound 23-d . To a solution of compound 23-c (523 mg, 1.mmol, 1.00 eq) in HCl (5.00 mL). The mixture was stirred at 80°C for 6 hrs. The reaction mixture was quenched by addition H2O 120 mL at 20°C, extracted with EtOAc 200 mL (1mL×2). The combined organic layers were washed with NaCl 120 mL (120 mL×2), dried over Na2SO4, filtered and concentrated under reduced pressure to give crude product which was purified by column chromatography (SiO2, petroleum ether/EtOAc = 10/1 to 2/1, petroleum ether/EtOAc =2/1, product Rf = 0.4) to give compound 23-d (180 mg, 734 μmol, 44.5% yield) as orange solid. LCMS: m/z = 246.1 [M+H]+. [0092] Synthesis of compound 23-e . To a solution of compound 23-d (100 mg, 407 μmol, 1.00 eq) in t-BuOH (1.00 mL) was added ammonium acetate (314 mg, 4.08 mmol, 10.0 eq) and NaBH3CN (128 mg, 2.04 mmol, 5.00 eq). The mixture was stirred at 70°C for 12 hrs.
FUTURX-004 IL 305 348/3 The reaction mixture was diluted with methanol (10.0 mL) then concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (column: Waters Xbridge 150×25mm×5µm; mobile phase: [water (ammonia hydroxide v/v)-ACN];gradient: 10%-40% B over min) and freeze-drying to give compound 23-e (22.0 mg, 89.3 μmol, 21.9% yield) as a yellow oil. LCMS : m/z = 247.2 [M+H]+. [0093] Synthesis of SLN-23 . To a solution of compound 23-e (14.9 mg, 89.3 μmol, 1.eq) and 4,5,6,7-tetrahydrobenzo[c]isoxazole-3-carboxylic acid (14.9 mg, 89.3 μmol, 1.eq) in DCM (1.00 mL) was added DIEA (52.4 mg, 406 μmol, 70.7 μL, 5.00 eq), EDCI (23.mg, 121 μmol, 1.50 eq) and HOBt (16.4 mg, 121 μmol, 1.50 eq). The mixture was stirred at 20°C for 6 hrs. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (column: Phenomenex luna C18 150×25mm×10µm; mobile phase: [water(FA)-ACN]; gradient:5%-35% B over min) and freeze drying to give compound SLN-023 (5.98 mg, 18.3% yield, 98.5% purity) as a pink solid. LCMS : m/z = 396.1 [M + H]+ . H NMR: (400 MHz, CDCl3) δ 7.74 (s, 1H), 7.50 (dt, J = 1.6, 7.8 Hz, 1H), 7.46-7.38 (m, 1H), 7.31-7.28 (m, 1H), 7.27-7.22 (m, 1H), 7.(br d, J = 7.8 Hz, 1H), 5.35-5.30 (m, 1H), 3.73-3.62 (m, 1H), 3.26 (br d, J = 14.8 Hz, 1H), 2.95-2.87 (m, 3H), 2.83-2.76 (m, 3H), 2.49 (s, 3H), 1.85-1.76 (m, 4H). [0094] The synthesis of SLN-023 was repeated in the same manner on the gram-scale and 514 mg of the racemate product were obtained. 100 mg of the racemate SLN-023 were submitted to chiral separation: 100 mg of SLN-023 was further separated by SFC (condition: column: DAICEL CHIRALCEL OJ (250mm×30mm,10µm); mobile phase: [CO2-i-PrOH(0.1%NH3H2O)]; B%:30%, isocratic elution mode) to give SLN-023A (Rt = 1.32 min, 29.4 mg, 74.35 μmol) and SLN-023B (Rt = 1.73 min, 29.7 mg, 75.11 μmol). Method details: "Column:Chiralcel OJ-3 50×4.6mm I.D., 3µm. Mobile phase:Phase A for CO2, and Phase B for IPA(0.05%DEA). Gradient elution:IPA(0.05%DEA) in CO2 from 5% to 40%. Flow rate:3mL/min;Detector:PDA. Column Temp:35C; Back Pressure:100Bar”.
Example 3. Synthesis of SLN-024 [0095] Synthesis of compound 24-b . As depicted in Scheme 3 , to a solution of compound 23-a (8.00 g, 28.3 mmol, 1.00 eq) and ethyl cyclopropylglycinate (4.25 g, 29.7 mmol, 1.eq) in ACN (80.0 mL) was added Na2CO3 (12.0 g, 113 mmol, 4.00 eq). The mixture was stirred at 80°C for 6 hrs. The reaction mixture was quenched by addition H2O (300 mL) at 20°C, extracted with EtOAc 600 mL (300 mL×2). The combined organic layers were washed FUTURX-004 IL 305 348/3 with NaCl 600 mL (300 mL×2), dried over Na2SO4, filtered and concentrated under reduced pressure to give crude product which was purified by column chromatography (SiO 2, petroleum ether/EtOAc = 10/1 to 2/1, petroleum ether/EtOAc = 2/1, product Rf = 0.4). Compound 24_b (2.50 g, 6.42 mmol, 22.7% yield) was obtained as brown oil. LCMS: m/z = 390.2 [M+H]+. [0096] Synthesis of compound 24-c . To a solution of compound 24-b (2.00 g, 5.14 mmol, 1.00 eq) in THF (20.0 mL) was added LiHMDS (1.00 M, 7.70 mL, 1.50 eq) at 0°C. The mixture was stirred at 60°C for 2 hrs. The reaction mixture was quenched with sat. NH4Cl (aq, 30.0 mL) and extracted with EtOAc (30.0 mL×2), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 24-c (2.00 g, crude) as a red oil. LCMS : m/z = 344.1 [M + H]+. [0097] Synthesis of compound 24-d . A mixture of compound 24-c (2.00 g, 5.82 mmol, 1.00 eq) in HCl (20.0 mL) was degassed and purged with N2 for 3 times, then the mixture was stirred at 80°C for 6 hrs under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (0.1% NH3•H2O condition). Compound 24-d (638 mg, 2.35 mmol, 40.3% yield) was obtained as a red solid. LCMS: m/z = 272.2 [M+H]+. H NMR: (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.70-7.60 (m, 2H), 7.59-7.52 (m, 1H), 7.47-7.41 (m, 1H), 3.92 (s, 2H), 3.41 (s, 2H), 1.98 (s, 1H), 0.49-0.42 (m, 2H), 0.40-0.32 (m, 2H). [0098] Synthesis of compound 24-e . To a solution of compound 24-d (300 mg, 1.mmol, 1.00 eq) in t-BuOH (6.00 mL) was added ammonium acetate (852 mg, 11.0 mmol, 10.0 eq) and NaBH3CN (347 mg, 5.53 mmol, 5.00 eq). The mixture was stirred at 70°C for hrs. The reaction mixture was diluted with methanol (10.0 mL) then concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (column: Waters Xbridge 150×25mm×5µm; mobile phase: [water (ammonia hydroxide v/v)-ACN]; gradient:20%-50% B over min) and freeze drying. Compound 24-e (82.0 mg, 301 μmol, 27.2% yield) was obtained as a yellow oil and confirmed by H NMR. LCMS: m/z = 256.2 [M+H - 17]+. H NMR: (400 MHz, DMSO-d6) δ 7.57 (s, 1H), 7.46-7.32 (m, 3H), 7.29-7.22 (m, 1H), 3.71 (br t, J = 5.8 Hz, 1H), 3.42 (br s, 1H), 2.95-2.84 (m, 1H), 2.43-2.31 (m, 2H), 1.78 (br dd, J = 2.8, 6.2 Hz, 1H), 0.40-0.29 (m, 2H), 0.28-0.18 (m, 2H). [0099] Synthesis of SLN-024 . To a solution of compound 24-e (80.0 mg, 293 μmol, 1.eq) and 4,5,6,7-tetrahydrobenzo[c]isoxazole-3-carboxylic acid (54.0 mg, 323 μmol, 1.10 eq) in DCM (1.00 mL) was added DIEA (189 mg, 1.47 mmol, 255 μL, 5.00 eq), EDCI (84.4 FUTURX-004 IL 305 348/3 mg, 440 μmol, 1.50 eq) and HOBt (59.5 mg, 440 μmol, 1.50 eq). The mixture was stirred at 20°C for 6 hrs. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (column: Phenomenex Luna C18 150×25mm×10µm; mobile phase: [water(TFA)-ACN]; gradient:20%-50% B over min) and freeze drying. Compound SLN-024 (5.20 mg, 3.94% yield, 93.8% purity) was obtained as a white solid. LCMS: m/z = 422.2 [M+H]+. H NMR: (400 MHz, CDCl3) δ 7.94-7.85 (m, 1H), 7.77 (s, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.47 (q, J = 7.2 Hz, 1H), 7.37-7.32 (m, 1H), 7.30 (s, 1H), 5.70-5.63 (m, 1H), 4.52 (br d, J = 14.8 Hz, 1H), 4.00 (br d, J = 15.2 Hz, 1H), 3.66-3.62 (m, 1H), 3.56-3.52 (m, 1H), 2.91-2.85 (m, 2H), 2.80 (t, J = 6.0 Hz, 2H), 2.55-2.43 (m, 1H), 1.85-1.76 (m, 4H), 1.35-1.27 (m, 1H), 1.09 (br d, J = 4.6 Hz, 1H), 0.91-0.(m, 2H).
Example 4. Synthesis of SLN-031 [00100] Synthesis of SLN-031 . As depicted in Scheme 4 , to a solution of compound h (53.mg, 322 μmol, 1.00 eq) in DCM (1.00 mL) was added HOBt (65.3 mg, 483 μmol, 1.50 eq), EDCI (92.6 mg, 483 μmol, 1.50 eq), DIEA (166 mg, 1.29 mmol, 224 μL, 4.00 eq) and compound 8-5 (70.0 mg, 322 μmol, 1.00 eq). The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150×25mm×10um; mobile phase: [water(FA)-ACN]; gradient: 22%-52% B over 9 min) to give compound SLN-031 (55.8 mg, 152 μmol, 3.78% yield, 99.8% purity) was obtained as white solid. LCMS: m/z = 366.2 [M+H]+. H NMR: (400 MHz, DMSO-d6) δ 7.75 (br d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.54 (s, 1H), 7.50 (s, 1H), 7.48-7.43 (m, 2H), 7.38-7.32 (m, 1H), 5.30-5.20 (m, 1H), 3.99 (t, J = 5.9 Hz, 2H), 3.01-2.(m, 4H), 2.76-2.65 (m, 1H), 2.46-2.38 (m, 1H), 1.90-1.82 (m, 2H), 1.80-1.72 (m, 2H). [00101] The synthesis of SLN-031 was repeated in the same manner on the gram-scale and 550 mg of the racemate product were obtained. 100 mg of the racemate SLN-031 were submitted to chiral separation: SLN-031 (100 mg, 273 µmol, 1.00 eq) was purified by SFC (column: DAICEL CHIRALPAK IG (250 mm × 30 mm, 10 um); mobile phase: [CO2-EtOH (0.1% NH3•H2O)]; B%:55%, isocratic elution mode) and concentrated under reduced pressure to give a residue. SLN-031A (45.9 mg, 125 μmol, 45.8% yield, 99.8% purity) was obtained as off-white solid, which was confirmed by LCMS, HPLC, HNMR, and SFC (Rt = 1.93 min). SLN-031B (45.2 mg, 119 μmol, 43.6% yield, 96.3% purity) was obtained as off-white solid, which was confirmed by LCMS, HPLC, HNMR, and SFC (Rt = 2.48 min).
FUTURX-004 IL 305 348/3 Method details: "Column:Chiralpak IG-3 50×4.6mm I.D., 3um. Mobile phase:Phase A for CO2,and Phase B for EtOH(0.05%DEA). Gradient elution: 40% EtOH (0.05% diethylamine (DEA) in CO2. Flow rate:3mL/min;Detector:PDA. Column Temp:35C;Back Pressure:100Bar".
Example 5. Synthesis of SLN-038 [00102] As depicted in Scheme 5 , to a solution of compound 23-e (50.0 mg, 203 μmol, 1.eq) and 5,6,7,8-tetrahydroimidazo[1,5-a]pyridine-1-carboxylic acid (40.5 mg, 244 μmol, 1.20 eq) in DCM (5.00 mL) was added EDCI (58.4 mg, 305 μmol, 1.50 eq), HOBt (41.2 mg, 305 μmol, 1.50 eq) and DIEA (78.7 mg, 609 μmol, 106 μL, 3.00 eq). The mixture was stirred at 25°C for 12 hrs. The mixture was concentrated under reduced pressure to give residue. The crude product was purified by reversed-phase HPLC (0.1% TFA condition) and lyophilization to give SLN-38 (24.66 mg, 61.46 μmol, 30.2% yield, 98.3% purity) as off-white solid. LCMS: m/z = 395.2 [M+H]+. H NMR: (400 MHz, DMSO-d6) δ 8.29 (br s, 1H), 7.61 (s, 1H), 7.58-7.51 (m, 3H), 7.51-7.45 (m, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.39-7.34 (m, 1H), 5.10-5.04 (m, 1H), 3.99 (br t, J = 5.6 Hz, 2H), 3.51 (s, 1H), 3.24-3.16 (m, 1H), 2.99 (br t, J = 5.6 Hz, 2H), 2.70 (br d, J = 4.0 Hz, 2H), 2.38 (s, 3H), 1.85 (br d, J = 4.4 Hz, 2H), 1.80-1.73 (m, 2H). [00103] The synthesis of SLN-038 was repeated in the same manner on the gram-scale and 1.10 g of the racemate product were obtained. 960 mg of the racemate SLN-038 were submitted to chiral separation: SLN-038 (960 mg, 2.43 mmol, 1.00 eq) was further separated by SFC (column: (s, s) WHELK-O1 (250mm × 30mm, 10um); mobile phase: [CO2-ACN/i-PrOH (0.1% NH3•H2O)]; B%: 45%, isocratic elution mode). SLN-038A (304 mg, 770 μmol, 31.6% yield, 100% purity) was obtained as white solid, which was confirmed by SFC (Rt = 1.99 min). SLN-038B (303 mg, 762 μmol, 31.3% yield, 99.2% purity) was obtained as off-white solid, which was confirmed by SFC (Rt = 1.83 min). Method details: "Column:Chiralcel OD-3 50*4.6mm I.D.,3um. Mobile phase:Phase A for CO2, and Phase B for IPA(0.05%DEA); Gradient elution:IPA(0.05%DEA) in CO2 from 5% to 40%, Flow rate:3mL/min; Detector:PDA; Column Temp:35C;Back Pressure:100Bar".
Example 6. Synthesis of SLN-042 [00104] As depicted in Scheme 6 , to a solution of compound 8-5 (223 mg, 1.03 mmol, 2.eq) in DCM (2.00 mL) was added compound 1-methyl-5-(trifluoromethyl)-1H-imidazole-4- FUTURX-004 IL 305 348/3 carboxylic acid (100 mg, 515 µmol, 1.00 eq), HOBt (104 mg, 772 µmol, 1.50 eq), EDCI (148 mg, 772 µmol, 1.50 eq) and DIEA (199 mg, 1.55 mmol, 269 uL, 3.00 eq). The mixture was stirred at 25°C for 2 hrs. The mixture was concentrated under reduced pressure to give a crude residue. The residue was purified by reversed-phase HPLC (column: Phenomenex luna C18 150×40 mm×15um; mobile phase: [water(FA)-ACN]; gradient:30%-60% B over min) to give SLN-042 (70.0 mg, 175 μmol, 34.0% yield, 98.6% purity) as off-white solid. LCMS: m/z = 394.1 [M+H]+. 1H NMR: (400 MHz, DMSO-d6) δ 8.49 (d, J = 7.6 Hz, 1H), 7.96 (s, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.50 (s, 1H), 7.49-7.43 (m, 2H), 7.40-7.32 (m, 1H), 5.24 (dt, J = 3.7, 7.8 Hz, 1H), 3.79 (d, J = 1.1 Hz, 3H), 3.01-2.84 (m, 2H), 2.78-2.64 (m, 1H), 2.49-2.41 (m, 1H). [00105] The synthesis of SLN-042 was repeated in the same manner on the gram-scale and 1.05 g of the racemate product were obtained. 950 mg of the racemate SLN-042 were submitted to chiral separation: SLN-042 (950 mg, 2.42 mmol, 1.00 eq) was further separated by SFC (column: DAICEL CHIRALPAK IC (250 mm×30 mm, 10 um); mobile phase: [CO2-EtOH (0.1% NH3•H2O)]; B%:30%, isocratic elution mode) and concentrated under reduced pressure to give a residue. SLN-042A (R enantiomer; 474 mg, 1.20 mmol, 49.6% yield, 99.5% purity) was obtained as white solid, which was confirmed by SFC (Rt = 1.97 min). SLN-042B (S enantiomer; 351 mg, 879 μmol, 36.4% yield, 98.6% purity) was obtained as white solid, which was confirmed by SFC (Rt =2.10 min). Method details: "Column:Chiralpak IC-3 50×4.6mm I.D., 3um. Mobile phase:Phase A for CO2, and Phase B for EtOH(0.05%DEA). Gradient elution: EtOH(0.05%DEA) in CO2 from 5% to 40% . Flow rate:3mL/min; Detector:PDA. Column Temp:35C;Back Pressure:100Bar". The identity of the specific enantiomer was validated by X-crystallography.
Example 7. Synthesis of SLN-043 [00106] Synthesis of compound 5A_2 . As depicted in Scheme 7 , to a solution of compound 5A_1 (5.00 g, 50.4 mmol, 1.00 eq) in ethanol (100 mL) was added NaHCO3 (5.08 g, 60.mmol, 2.36 mL, 1.20 eq) and compound 5a (6.85 g, 60.5 mmol, 6.62 mL, 1.20 eq). The mixture was stirred at 90°C for 12 hrs. The mixture was concentrated under reduced pressure to give a crude residue. The residue was purified by prep-HPLC (basic condition) to give compound 5A_2 (100 mg, 554 μmol, 5.50% yield, 100% purity) as brown oil. H NMR: (400 MHz, DMSO-d6) δ 7.61 (s, 1H), 4.17 (q, J = 7.1 Hz, 2H), 4.02 (t, J = 7.2 Hz, 2H), 2.(t, J = 7.4 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H).
Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3
[00107] Synthesis of compound 5A_3 . To a solution of compound 5A_2 (100 mg, 5μmol, 1.00 eq) in HCl (6 M, 4.00 mL, 43.2 eq) was stirred at 100°C for 48 hrs. The mixture was concentrated under reduced pressure to give compound 5A (87.0 mg, crude) as brown solid. [00108] Synthesis of SLN-043 . To a solution of compound 5A_3 (87.0 mg, 570 μmol, 1.eq) in DCM (1.00 mL) was added HOBt (115 mg, 856 μmol, 1.50 eq), EDCI (164 mg, 8μmol, 1.50 eq), DIEA (221 mg, 1.71 mmol, 298 μL, 3.00 eq) and compound 8-5 (124 mg, 570 μmol, 1.00 eq), the mixture was stirred at 20°C for 12 hrs. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150 × 40 mm × 15 um; mobile phase: [water (TFA)-ACN]; gradient: 10%-40% B over 15 min) to give compound SLN-043 (15.0 mg, 42.6 μmol, 7.46% yield, 99.8% purity) as off-white solid. LCMS: m/z = 352.2 [M+H]+. H NMR: (4MHz, DMSO-d6) δ 7.80 (d, J = 7.9 Hz, 1H), 7.66-7.60 (m, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 7.48-7.43 (m, 2H), 7.38-7.32 (m, 1H), 5.28-5.19 (m, 1H), 3.99 (t, J = 7.2 Hz, 2H), 2.98-2.(m, 4H), 2.76-2.64 (m, 1H), 2.56 (t, J = 7.3 Hz, 2H), 2.48-2.40 (m, 1H).
Example 8. Synthesis of SLN-044, SLN-049, SLN-050, SLN-051 and SLN-052 [00109] The synthesis of each one of the compounds SLN-044, SLN-049, SLN-050, SLN-051 and SLN-052 has been carried out using procedures similar to those used in the preparation of the compounds described in Examples 1-7 above.
Example 9. In vitro studies Cell free biochemical assay to screen and determine IC50 values of potential DHODH inhibitors [00110] The inhibitory activity of known DHODH inhibitors (teriflunomide, brequinar, NITD-982, 21q, 1291, and BAY-2402234) and newly synthesized SLN molecules against recombinant human DHODH was tested in vitro by an optimized enzymatic assay that uses decylubiquinone as a substitute for the highly insoluble coenzyme Q10. The colorimetric kinetic reaction, which detects changes of the oxidation state of 2,6-dichlorophenol indophenol (DCIP) as a final electron acceptor, was monitored for 60 min ( Fig. 1 ). ICvalues were then determined using dose response curves ( Figs. 2-3 ). The IC50 was calculated using Prism-GraphPad Software by linear regression. IC50 results were compared to the Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 known well established DHODH inhibitor BAY-2402234, which was set as a positive control in the evaluation process. [00111] DHODH inhibitor dilutions for the kinetic enzymatic assay were prepared in assay buffer (50 mM Tris, pH 8.0; 0.1% Triton X-100, 150 mM KCl, 10% glycerol) from 100% dimethylsulfoxide (DMSO) stocks. Five μL of these dilutions were mixed with 40 μL of the reaction mix (50 mM Tris, pH 8.0; 0.1% Triton X-100; 150 mM KCl; 10% glycerol - assay buffer; 1000 µM L-dihydroorotic acid (L-DHO); 100 µM decylubiquinone (Q10); 60 µM DCIP). The reaction was initiated by the addition of 5 μL of 20 nM full length human DHODH enzyme (10062-DD-020, R&D Systems), excluding blank and enzyme depletion controls. Enzymatic activity was measured by loss of absorbance of DCIP at 600 nm in a time course kinetic reaction with reads every 3 min, at 32°C.
[00112] Materials . Triton-X 100 (327371000, Acros); Tris (pH 8.0) (T2694, Sigma); KCl (P5405, Sigma); glycerol (BP229, Fisher); DHODH enzyme (10062-DD-020, R&D Systems); L-DHO (D7128-500MG, Sigma); decylubiquinone (Q10) (D7911-10MG, Sigma); DCIP (D1878-5G, Sigma); DMSO (D5879-100ML, Sigma). Microplate 96w, UVStar, Half Area (675801, Greiner).
Table 2 . IC50 of the various SLN compounds synthesized SLN compound IC 50 (nM)SLN-008 23SLN-023 3SLN-023A 1SLN-023B 261SLN-024 1SLN-031 1SLN-031A 71SLN-031B SLN-038 1SLN-038A 179SLN-038B SLN-042 3SLN-042A (R enantiomer) 1SLN-042B (S enantiomer) 211SLN-043 4SLN-044 2SLN-049 Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 SLN-050 SLN-051 1SLN-052 1
[00113] Statistical analysis . All data are reported as the mean ± standard error of the mean (SEM) with different experiments. The statistical analysis - t test was performed.
[00114] Results . The IC50 values of the various SLN compounds synthesized are shown in Table 2 . All SLN compounds listed in Table 2 have a chiral center. SLN-023, SLN-031, SLN-038, and SLN-042 were separated to their pure enantiomers. For each compound the racemic mixture was given a number, and the pure enantiomers (separated based on their retention time) are identified by the same number and either the letter A or B. As clearly shown from Table 2 and from Figs. 2-3 , one of the enantiomers is highly potent with IC value in the nanomolar range, while the opposite lacks affinity to the target DHODH enzyme and has IC50 value within the micromolar range. The racemic mixture potency of each SLN compound is an average of the two enantiomers.
Cell penetration assay [00115] DHODH catalyzes the fourth step of the “de novo pyrimidine synthesis”, i.e., the conversion of dihydroorotate to orotate. This conversion is essential for the cell’s ability to produce uridine monophosphate (UMP). Inhibition of DHODH suppresses the proliferation of cells relying on this pathway, e.g., cancer cells that are highly dependent on UMP synthesis. [00116] In order to assess the capability of the SLN compounds to penetrate into cells, and to reassure the DHODH specific mechanism of action, an in-vitro cell-based proliferation assay was used. Reduction in cell proliferation was translated to cell penetration capability of SLN compounds. [00117] Additionally, in order to assess the specificity of the mechanism of action, the experiments were based on the hypothesis that bypassing the de novo pyrimidine biosynthesis, and mimicking the pyrimidine salvage pathway, excess addition of uridine to the inhibition reaction medium may reverse DHODH proliferation-inhibition’s effect (which is relevant to cancer cells). This hypothesis was applied to the cell-based assay.
[00118] Materials . Minimum essential medium (MEM) (Sartorius), supplied with 10% fetal bovine serum (FBS) (Gibco, ThermoFisherScientific Inc., USA), 1% L-glutamine/L-alanine and 1% penicillin-streptomycin-amphotericin B solution (PSA) (Biological Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 industries both). The tested DHODH inhibitors (SLN molecules) were produced by Wuxi, China. CellTiter-Glo cell luminescent viability from Promega, Madison, WI, USA. Uridine from Sigma Aldrich and BAY-2402234 from Tocris.
[00119] Cell line . HepG2 cells (Cat# HB-8065 ATCC) were cultured in MEM supplemented with 10% FBS, 1% L-glutamine/L-alanine, and 1% PSA. Cells were maintained at 37°C and 5% CO2 in a humidified incubator. When received, cell lines were assayed for mycoplasma contamination and were confirmed to be mycoplasma-free.
[00120] Uridine rescue assay . 4500 cells per well, for HepG2, were seeded in a clear 96-well plate and incubated as described above overnight prior to treatment. The next day, the medium was replaced by fresh medium (for w/o treatment experiment) or DMSO containing medium supplemented. The cells were treated with BAY-2402234 (20µM) or different SLN molecules (concentration range: 1 µM-100µM), in the presence or absence of uridine (100µM). Cell growth was monitored 72h after treatment started. The cell confluence was quantified as a measure of cell growth using the CellTiter Glo kit where the amount of ATP was assessed.
[00121] Statistical analysis . All data are reported as the mean ± SEM with different experiments in cell lines. Experiments involving cells treated with DMSO (controls) or different SLN compounds are based on paired samples, i.e., the same sample was analyzed under different conditions; accordingly, the statistical analyses utilized were a paired t test.
[00122] Results . The effect of SLN compounds on the proliferation of HepG2 cells is shown in Fig. 4 . SLN compounds reduce the proliferation of HepG2 cells in a dose response manner at ranges of 1 to 100 µM. When uridine is added to the cell culture, the inhibition of the proliferation is diminished in all SLN compounds, with the exception of SLN-031 at the concentration of 100 µM.
[00123] Conclusions . DHODH is located on the inner membrane of the mitochondria, therefore inhibition in proliferation as a result of its inhibition, requires the presence of the different compounds within cells’ mitochondria. The capability of SLN compounds to penetrate cells and inhibit the proliferation was demonstrated in HepG2 cells in a dose dependent manner. Cancer cells are highly dependent on pyrimidine building blocks (e.g., uridine), therefore DHODH inhibitors were initially developed as anti-proliferation agents for cancer treatment, as DHODH inhibition reduces uridine de-novo synthesis. Nevertheless, FUTURX-004 IL 305 348/3 uridine’s physiological supply is highly controlled, and is not dependent solely on the de-novo pathway, but also on the salvage pathway and the catabolic pathway. This leads to the assumption that in normal tissues the levels of uridine are within normal range, and the effect of DHODH inhibition on proliferation may be reversible. We applied this hypothesis on HepG2 cells: uridine rescue, demonstrating a reversible effect on proliferation, was demonstrated for the different inhibitors (with exception of SLN-031, 100 µM). When uridine was added to the cell medium, the cells proliferation was continued, possibly indicating that cytotoxic effect due to inhibition of DHODH in normal tissues may be reduced to the minimum ( Fig. 4 ).
Evaluation of neuronal activity using electrophysiology assays [00124] The effect of the different DHODH inhibitors on the electrical activity of hippocampal neurons was tested using a Multiple Electrode Array (MEA) system, capable of recording from primary cultured neuronal network from 120 channels (electrodes) simultaneously. The electrical activity was recorded and analyzed in different conditions: non treated, DHODH/pharmacological agent treatment (short and long termed), and post pharmacological excitation using TBOA, in order to simulate and analyze the effect and response to an epileptic seizure.
Materials and Methods [00125] Primary cultures . Hippocampi were dissected from BALB/c pups (both sexes) at P0-1 in ice cold Leibovitz L-15 medium. Tissues were washed 3 times with cold HBSS and incubated in digestion solution (137mM NaCl, 5mM KCl, 7mM Na2HPO4, 25mM HEPES, mg/ml trypsin, 0.5 mg/ml DNase) for 20 min at 37°C. Next, double volume of HBSS supplemented with 20% FBS added to inactivate the protease, and once again with HBSS alone. Cells were then dissociated in HBSS supplemented with 13 mM MgSO4 and 0.mg/ml DNase by trituration with fire-polished glass pipettes. Suspension of dissociated cells was centrifugated at 1000 relative centrifugal force (RCF) for 6 min at 4°C, supernatant was removed, cells were re-suspended with plating medium (MEM supplemented with 10% FBS, 32.7 mM glucose, 25 mg/ml insulin, 2 mM glutamax, 0.1 mg/ml transferrin, 0.1% Bsupplement) and then plated on matrigel-coated multi-electrode array (MEA) plates. One day later, 50% of the serum medium was replaced with feeding medium (MEM supplemented with 32.7 mM glucose, 2 mM glutamax, 0.1 mg/ml transferrin, 2% Bsupplement). Two days later 50% of the serum medium was replaced with feeding medium FUTURX-004 IL 305 348/3 containing additional 3 mM cytosine arabinoside (ARA-C), in order to inhibit microglia cells division in the culture. Half of the medium was then replaced twice a week with a fresh feeding medium. The experiments were performed in cultures after 14-21 days in-vitro (DIV).
[00126] Electrophysiology recording and analysis . Obtained cultures were allowed to grow for 14-21 days before experiments were carried out. Electrical activity of the primary neural culture was recorded using MEA2100-System and 120-electordes MEA plates (Multi Channel Systems (MCS), Germany). Data acquisition was performed using a hardware filter cut-off of 3.3 kHz and sampling rate of 10 kHz per electrode. Recordings were carried out under constant 37⁰C and 5% CO2 levels in suitable cell culture incubator. Spontaneous spiking activity, changes in firing rates and signs of homeostatic compensation were monitored and analyzed offline using MCS software. The offline analysis of raw data included filtration with Butterworth high pass of 2nd order with 200 Hz cut-off. Spikes were detected based on a ±5 standard deviation threshold from the baseline noise level. Detected spikes arranged in 20 minutes bins for further analysis. All channels recorded during the specific experiment were normalized to their own baseline and then all recorded channels analyzed together for changes in the mean firing rate (MFR), compared to the normalized baseline. Statistical analysis performed with GraphPad Prism v.9 software (GraphPad Software, USA).
Results [00127] Inhibition of DHODH causes prolonged reduction MFR in hippocampal neurons . The effect of DHODH inhibition on the electrical activity of hippocampal neurons was tested with MEA system, which allows long-term monitoring of entire neural network activity. Cultured primary hippocampal neurons grown on MEA chip with 120 electrodes. As found, application of the known DHODH inhibitors teriflunomide (TERI)) 50 µM, brequinar (BRQ) 1 µM, and BAY-2402234 (BAY) 20 µM causes prolonged and stable decrease in the MFR of neural network ( Fig. 5A , 5B , and 5C , respectively). Vehicle-treated (DMSO 0.1% v/v) cells did not show any significant change in the MFR over the experiment timeline ( Fig. 5D ).
[00128] Pretreatment with DHODH inhibitors suppresses induced excitation in hippocampal neurons . Application of TBOA 10 µM induces a potent prolonged excitation in neural electrical activity ( Fig. 6A ). Pretreatment with the known DHODH inhibitors FUTURX-004 IL 305 348/3 suppresses this TBOA-induced excitation. As shown, application of teriflunomide 50 µM, brequinar 2 µM and BAY-2402234 20 µM ( Fig. 6B , 6C , and 6D , respectively) leads to two-factor effect in hippocampal network: first, the induced excitation amplitude is significantly smaller compared to the untreated cells; second, the induced excitation is transient and after the initial peak MFR decreased back to the baseline level and even below the baseline level in pretreated cells.
Conclusions [00129] SLN molecules reduce and stabilize MFR and suppress excitation in hippocampal neurons . As demonstrated herein, the compounds identified as SLN-023, SLN-031 and SLN-042A (R enantiomer) ( Fig. 7A , 7B , and 7C , respectively), when applied to the primary hippocampal culture, cause a significant decrease in MFR (p<0.01 for SLN-023, p<0.05 for SLN-031 and SLN-042A), similar to the previously known and described DHODH inhibitors. Additionally, SLN-023, SLN-031 and SLN-042A (R enantiomer) suppress TBOA-induced excitation in neural network and stabilize neural MFR at baseline level in a long-term perspective after the induced excitation occurs.
[00130] Short-term exposure to SLN molecules provides an effect similar to long-term exposure in MFR stabilization and excitation suppression . As previously demonstrated, long-term (>20 hours) exposure to DHODH inhibitors leads to decrease in MFR of primary hippocampal neurons’ network and suppresses pharmacologically induced neural excitation. Compounds SLN-023, SLN-031 and SLN-042A (R enantiomer) show the ability to provide an effect similar to long-term exposure in short-term (3 hours) exposure ( Figs. 8A-8C ). Short-term exposure to these DHODH inhibitors does not affect the baseline MFR of neural network, and suppresses TBOA-induced excitation in electrical activity of the hippocampal culture.
Example 10. In vivo studies [00131] The anticonvulsant activity of SLN compounds was demonstrated in two animal models for epilepsy: the 6 Hz psychomotor test in the mouse, and the genetic Dravet Syndrome model in zebrafish larvae. 6 Hz psychomotor test (32mA) in the mouse [00132] The method, which detects anticonvulsant activity of small organic molecules, follows that described by Brown et al. (1953).
Underlined postdated 12.12.20Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 Materials and methods [00133] Animals . Male Swiss mice (Janvier Labs, France), 26-35 g body weight range at the beginning of the experiment, n=12-15 per group in the 6 Hz test. Animals were acclimatized for at least 3 days after delivery, in makrolon cages (4 per cage) on wood litter with nesting and gnawing material with free access to food and water.
[00134] Seizure induction method . Before transcorneal stimulation, a drop of tetracaine solution (1%) was applied on each eye of the mouse for local anesthesia. Between 1-minutes later, the mice were administered a rectangular current (32 mA, rectangular pulse: 0.2 ms pulse width, 3 s duration, 6 Hz) via corneal electrodes connected to a constant current shock generator (Ugo Basile: type 7801). The results for the number of seizures as reflected by forelimb clonus were recorded immediately after current administration. Forelimb clonus was scored as absent (0), mild (1), or strong (2).
Results [00135] SLN-031 . SLN-031 was evaluated at one dose, administered p.o. 2 hours and hour before the test, and was compared with a vehicle control group (administered under the same experimental conditions). The vehicle group was administered p.o. with 0.5% HPMC hour before the test. Carbamazepine (50 mg/kg) was administered p.o. 1 hour before the test and was used as a reference substance. The experiment thus included 4 groups as shown in Table 3 . Quantitative data (scores) with the test substance will be analyzed by comparing treated groups with vehicle control using Kruskal-Wallis test followed by Mann-Whitney U test. Quantitative data with the reference substance will be analyzed using Mann-Whitney U test. 15 mice were studied per group, with the exception of one group: SLN-031, administered 2 hours before the test (12 mice only). The test was performed blindly.
Table 3 Group Number of animals Treatment Dose-level (mg/kg) Concentration (mg/mL) Administration volume (mL/kg)15 males Control Substance (-60 min) 0 0 5 15 males SLN-031 (-60 min) 150 15 6 12 males SLN-031 (-120 min) 150 15 7 15 males Carbamazepine (-60 min) 50 5
[00136] In vehicle controls administered with 0.5% hydroxypropylmethycellulose (HPMC) in distilled water, p.o. 1 h before the test, the forelimb seizure score was 0.9 ± 0.1. SLN-031 FUTURX-004 IL 305 348/3 (150 mg/kg), administered p.o. 1 hour before the test, tended to decrease the forelimb seizure score, as compared with vehicle controls (-44%, NS), and when administered p.o. 2 hours before the test, significantly affected the forelimb seizure score, as compared with vehicle controls (-67%, p<0.05). Carbamazepine (50 mg/kg), administered p.o. 1 hour before the test, significantly decreased the forelimb seizure score, as compared with vehicle controls (-89%, p<0.001). See Fig. 9 , and Tables 4-5 .
Table 4 . Effects of SLN-031 on forelimb clonus Treatment Forelimb clonus (mean±s.e.m) Compared with vehicle (p.o -1h) (1) (2) %vehicle (p.o -1h) 0.9 ± 0.1 SLN-031 (150 mg/kg p.o -1h) SLN-031 (150 mg/kg p.o -2h) 0.5 ± 0.0.3 ± 0.* NS * -44% -67% Carbamazepine (50 mg/kg p.o -1h) 0.1 ± 0.*** -89% Inter-group comparison: NS = not significant; * p<0.05; ** p<0.01; *** p<0.001. (1) For SLN compound-treated groups: Kruskal-Wallis test; For reference-treated group: Mann-Whitney U test (2) Dunn’s multiple comparison test when Kruskal-Wallis test is significant.
Table 5 . Individual data, mice seizure score, SLN-031 Vehicle (p.o. -1h) SLN-031 (150 mg/kg p.o. -1h) SLN-031 (150 mg/kg p.o. -2h) Carbamazepine (50 mg/kg p.o -1h)B1 1 A1 2 D1 0 C1 B2 0 A2 1 D2 0 C2 B3 1 A3 0 D3 0 C3 B4 1 A4 0 D4 1 C4 B5 0 A5 1 D5 0 C5 B6 1 A6 0 D6 0 C6 B7 1 A7 1 D7 0 C7 B8 2 A8 0 D8 1 C8 B9 1 A9 0 D9 0 C9 B10 1 A10 1 D10 1 C10 B11 0 A11 1 D11 0 C11 B12 1 A12 1 D12 1 C12 B13 1 A13 0 C13 B14 1 A14 0 C14 B15 1 A15 0 C15 0 - absence; 1 - mild; 2 - strong.
[00137] SLN-042A . SLN-042A (R enantiomer) was evaluated at one dose, administered p.o. 2 hours and 1 hour before the test, and was compared with a vehicle control group Underlined postdated 12.12.2023 FUTURX-004 IL 305 348/3 (administered under the same experimental conditions). The vehicle group was administered p.o. with 0.5% HPMC 1 hour before the test. Carbamazepine (50 mg/kg) was administered p.o. 1 hour before the test and was used as a reference substance. The experiment thus included 4 groups as shown in Table 6 . Quantitative data (scores) with the test substance will be analyzed by comparing treated groups with vehicle control using Kruskal-Wallis test followed by Mann-Whitney U test. Quantitative data with the reference substance will be analyzed using Mann-Whitney U test. 15 mice were studied per group). The test was performed blindly.
Table 6 Group Number of animals Treatment Dose-level (mg/kg) Concentration (mg/mL) Administration volume (mL/kg)15 males Control Substance (-60 min) 0 0 5 15 males SLN-042A (-60 min) 100 10 6 12 males SLN-042A (-120 min) 100 10 7 15 males Carbamazepine (-60 min) 50 5
[00138] In vehicle controls administered with 0.5% hydroxypropylmethylcellulose (HPMC) in distilled water, p.o. 1 hour before the test, 3 mice had a forelimb seizure score of 0, 9 mice had a forelimb seizure score of 1 and 3 mice had a forelimb score of 2. The mean forelimb seizure score was 1.0 ± 0.2. SLN-042A at 100 mg/kg, administered p.o. hour before the test, significantly decreased the mean forelimb seizure score as compared with vehicle controls (-90%, p<0.001); and when administered p.o. 2 hours before the test, significantly decreased the mean forelimb seizure score as compared with vehicle controls (-70%, p<0.01). Carbamazepine at 50 mg/kg, administered p.o. 1 hour before the test, significantly decreased the mean forelimb seizure score as compared with vehicle controls (-100%, p<0.001). See Fig. 10 and Tables 7 - 8 .
FUTURX-004 IL 305 348/3 Table 7 . Effects of SLN-042A on forelimb clonus Treatment Forelimb clonus (mean±s.e.m) Compared with vehicle (p.o -1h) (1) (2) %vehicle (p.o -1h) 1.0 ± 0.2 SLN-042A (100 mg/kg p.o -1h) SLN-042A (100 mg/kg p.o -2h) 0.1 ± 0.0.3 ± 0.*** *** ** -90% -70% Carbamazepine (50 mg/kg p.o -1h) 0.0 ± 0.*** -100% Inter-group comparison: NS = not significant; * p<0.05; ** p<0.01; *** p<0.001. (1) For SLN compound-treated group: Grouped analysis (non-parametric test) - Kruskal-Wallis test; For reference-treated group: Two groups analysis (non-parametric test) - Mann-Whitney U test (2) Dunn’s multiple comparison test (post-hoc) when Kruskal-Wallis test is significant.
Table 8 . Individual data, mice seizure score, SLN-042A Vehicle (p.o. -1h) SLN-042A (100 mg/kg p.o. -1h) SLN-042A (100 mg/kg p.o. -2h) Carbamazepine (50 mg/kg p.o -1h)D1 2 B1 0 A1 0 C1 D2 1 B2 0 A2 0 C2 D3 1 B3 0 A3 1 C3 D4 2 B4 0 A4 0 C4 D5 1 B5 1 A5 0 C5 D6 0 B6 0 A6 0 C6 D7 1 B7 0 A7 0 C7 D8 0 B8 1 A8 0 C8 D9 1 B9 0 A9 1 C9 D10 1 B10 0 A10 0 C10 D11 1 B11 0 A11 1 C11 D12 1 B12 0 A12 0 C12 D13 0 B13 0 A13 1 C13 D14 1 B14 0 A14 0 C14 D15 2 B15 0 A15 0 C15 0 - absence; 1 - mild; 2 - strong.
Conclusions [00139] For SLN-031, the results suggest the presence of anticonvulsant effects at the dose of 150 mg/kg p.o. 2 hours before the test. The same trend was observed at 150 mg/kg with a shorter pre-treatment time. For SLN-042A, the results suggest the presence of anticonvulsant effects for SLN-042A at the dose of 100 mg/kg p.o., when administered 1 or 2 hours before the test.
Dravet syndrome model in zebrafish larvae FUTURX-004 IL 305 348/3
[00140] Genetic model of knockout (KO) to scn1lab gene: a sodium channel 1, in which a mutation leads to developmental defects and recurrent seizures. [00141] It has been demonstrated that zebrafish is a powerful tool to model the human Dravet Syndrome, a form of genetic epilepsy caused by mutations in the SCN1A gene. Indeed, mutations in the zebrafish snc1lab gene induce epileptic-like phenotypes that could be rescued pharmacologically (Baraban et al., 2013). scn1lab crispants display an impaired locomotion but display a strong movement response if light flashes are employed to trigger epileptic seizures (scn1lab crispants show an increase of maximum velocity and abnormal changes in their trajectory (number of turns) that reflects the erratic movement typical of seizures). [00142] For each larva, the following criteria were used to define the degree of protection induced by the incubation with the test compound: • Full protection (no seizures) . The values of max velocity AND number of turns are both lower than the sum of the means of the values observed in the control group (scramble) and their standard deviation max velocity (treated larva) < mean + SD (scrambles); and number of turns (treated larva) < mean + SD (scrambles). • Partial protection (mild seizures/mild alteration of movement) . Only one value between max velocity or number of turns is lower than the sum of the means of the values observed in the control group (scramble) and their standard deviation: max velocity (treated larva) < mean + SD (scrambles) AND number of turns (treated larva) > mean + SD (scrambles); OR max velocity (treated larva) > mean + SD (scrambles) AND number of turns (treated larva) < mean + SD (scrambles). • No protection (strong seizures/strong alteration of movement) . The values of max velocity AND number of turns are both higher than the sum of the means of the values observed in the control group (scramble) and their standard deviation max velocity (treated larva) > mean + SD (scrambles); AND number of turns (treated larva) > mean + SD (scrambles). [00143] Results and conclusions:the treatment of larvae with the SLN-023 and SLN-0had an impact on the seizure induction phase, when flashes of light were employed to trigger epileptic seizures. SLN-031 demonstrated a significant anti-epileptic activity by reducing FUTURX-004 IL 305 348/3 max velocity and number of turns in a dose response manner ( Table 9 ). Treatment with SLN-023 showed a trend toward an anti-epileptic effect. [00144] Zebrafish larvae were also exposed to alternating dark/light environments, in order to test possible deviations from the zebrafish stereotyped moving pattern (larvae tend to be more active in the dark and to cover shorter distances in the light). No alteration in the response to the changes of light was detected: all experimental groups swam more actively during the dark phases and tended to rest during the intervals in which they were exposed to light, leading to the conclusion that no behavioral changes were caused. See Fig. 11 , Fig. 12A-12C and Table 9 .
Table 9 . Percentages of protected, partially protected, and not protected larvae for each treatment Full protection (%) Partial protection (%) No protection (%) scn1lab DMSO 13.5 48.6 37. scn1lab SLN-023 32.25 µM 47.6 ( p<0.05) 23.8 28. scn1lab SLN-023 64.5 µM 57.1 ( p<0.01) 31.4 11. scn1lab SLN-0031 14.5 µM 36.1 41.7 22. scn1lab SLN-0031 29 µM 61.5 ( p<0.0001) 23.1 15.
Example 11. Pharmacokinetic studies with SLN-031 [00145] The pharmacokinetic (PK) properties of SLN-031 were evaluated in CD-1 mice, in both intravenous (IV) administration (single dose, 5 mg/kg) and oral administration (q.d, for three consecutive days, 50 mg/kg).
[00146] Animals . 6-10 weeks male CD-1 mice (WTLH-BJ, China). Animals were acclimatized for at least 3 days after delivery, in polysulfon cages (4 per cage) on wood litter, with nesting and gnawing material, and with free access to food and water. The mice were divided into four groups as shown in Table 10 . Samples were collected from blood (plasma) and brain, as detailed in Table 11 , and bioanalysis was performed using LC/MS/MS.
Results [00147] The main PK parameters for SLN-031 were determined and are depicted in Table 12 . As shown, the main PK parameters of SLN-031 are: (i) half-life: 0.9h (IV), 1.4h (PO), tmax (plasma) 0.5h, tmax (brain) 1h, oral bioavailability 58%, brain to plasma ratio at tmax brain 0.11, CL 5.89 ml/min/kg. These parameters indicate that SLN-031 is well absorbed orally from the tested formulation and penetrated the BBB, yielding sufficient exposure in the brain, to result in anti-seizure activity.
FUTURX-004 IL 305 348/3 Table 10 . Mice groups for PK study with SLN-0 Group Animal No. Dose route Dose (mg/kg) Conc. (mg/mL) Dose volume (mL/kg) Vehicle 1 9 IV bolus 5 1 5% ethanol, 10% Kolliphor HSand 85% physiological saline 12 PO 50 5 10 0.5% HPMC E4M in distilled water 3 PO 50 5 10 0.5% HPMC E4M in distilled water 15 PO 50 5 10 0.5% HPMC E4M in distilled water Table 11 . Sample collection of samples from different mice. Collection was performed from blood (plasma, P) and brain (B), in order to determine brain permeability and concentration Group Phase Dosage (mg/kg) A1 A2 A3 Day Time Point (hr) B P 1 NA M01 M02 M03 0.083(5min) 0.25 0.5 1 2 4 8 1 M04 M05 M06 1 0.25 1 M07 M08 M09 1 1 1 2 NA M10 M11 M12 0.083 1 1 M13 M14 M15 0.5 2 1 M16 M17 M18 1 4 1 M19 M20 M21 8 12 1 3 NA 50 M22 M23 M24 0.083 2 1 4 NA M25 M26 M27 0 (pre-dose) 1 1 M28 M29 M30 0.083 2 1 M31 M32 M33 0.5 4 1 M34 M35 M36 3 12 1 M37 M38 M2 0 (pre-dose) 3 24 1 1 FUTURX-004 IL 305 348/3 Table 12 . Mean PK values at days 1, 2 and 3 of SLN-031 following IV and oral administration PK Parameters IV bolus1-Day 1 PO2-Day 1 PO4-Day 3 Mean brain Mean plasma Mean brain Mean plasma Mean brain Mean plasma Rsq_adj ND 0.990 1.00 0.973 0.998 1. No. points used for T 1/2ND 3.00 3.00 5.00 3.00 4. C 0 (ng/mL) -- 15311 -- -- -- -- C max (ng/mL or ng/g)ND -- 3240 37633 2265 325 T max (h) ND -- 1.00 0.500 1.00 0.5 T 1/2 (h) ND 0.932 1.37 1.34 1.56 1. Vd ss (L/kg) -- 0.426 -- -- -- -- Cl(mL/min/kg) -- 5.89 -- -- -- -- T last (h) ND 8.00 12.0 12.0 12.0 12. AUC 0-last (ng.h/mL or ng.h/g)ND 14112 6602 88168 4462 677 AUC 0-inf (ng.h/mL or ng.h/g)ND 14151 6620 88313 4484 680 MRT 0-last (h) ND 1.18 2.38 2.32 2.41 2. MRT 0-inf (h) ND 1.21 2.41 2.34 2.47 2. AUC Extra (%) ND 0.272 0.271 0.164 0.506 0.3 AUMC Extra (%) ND 2.11 1.57 0.979 2.92 2. Bioavailability (%) b -- -- -- 58.3 -- -- a AUC Ratio ND -- 0.0749 -- 0.0658 -- Results and conclusions [00148] The PK parameters obtained for SLN-031 suggest brain permeability of the compound, enabling sufficient pre-clinically, model enabling, relevant brain concentration, as deduced from electrophysiology assays. These concentrations were calculated as enough to lead to reduced seizure susceptibility in epilepsy animal models in mice. This conclusion was similar in the case of SLN-042A, and was confirmed in the high efficacy of both test compounds in the mouse 6 Hz (32mA) animal model for refractory epilepsy.
FUTURX-004 IL 305 348/3 APPENDIX Scheme 1 . Synthesis of SLN-0 Scheme 2 . Synthesis of SLN-0 FUTURX-004 IL 305 348/3 Scheme 3 . Synthesis of SLN-0 Scheme 4 . Synthesis of SLN-0 Scheme 5 . Synthesis of SLN-0 FUTURX-004 IL 305 348/3 Scheme 6 . Synthesis of SLN-0 Scheme 7 . Synthesis of SLN-0 FUTURX-004 IL 305 348/3 REFERENCESBaraban, S.C. et al., Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment. Nature Communications, 2013 , 4, Article No. 2410 Bar-Or, A. et al., Teriflunomide and its mechanism of action in multiple sclerosis, Drugs, 2014 , 74(6), 659-674 Brown, W.C. et al., Comparative assay of antiepileptic drugs by “psychomotor” seizure test and minimal electroshock threshold test. J. Pharmacol. Exp. Ther., 1953 , 107, 273-2Fisher, R.S. et al., A practical clinical definition of epilepsy, Epilepsia, 2014 , 55(4), 475-482 Löscher, W.; Klein, P., The pharmacology and clinical efficacy of antiseizure medications: from bromide salts to cenobamate and beyond, CNS Drugs, 2021 , 35, 935-963 Sang Kun Lee, Old versus new: why do we need new antiepileptic drugs?, J. Epilepsy Res., 2014 , 4(2) Shi, D.D. et al., De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma. Cancer Cell, 2022 , 40(9), P939-9 Styr, B. et al., Mitochondrial regulation of the hippocampal firing rate set point and seizure susceptibility, Neuron, 2019 , 102(5), 1009-1024 Sven, C. et al., The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 triggers differentiation and is effective in the treatment of myeloid malignancies. Leukemia, 2019 , 33(10), 2403-24 Targa Dias Anastacio, H.; Matosin, N.; Ooi, L., Neuronal hyperexcitability in Alzheimer’s disease: what are the drivers behind this aberrant phenotype? Transl Psychiatry, 2022 , 12, 257 Telias, M.; Segal, M., Editorial: Pathological hyperactivity and hyperexcitability in the central nervous system. Front. Mol. Neurosci., 2022 ,

Claims (25)

  1. FUTURX-004 IL 30 5348/3 CLAIMS1. A compound of formula I: , I or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, wherein R is aryl, -(C1-C6)alkyl-aryl such as benzyl, heteroaryl, -(C1-C6)alkyl-heteroaryl, heterocyclyl, or -(C1-C6)alkyl-heterocyclyl, optionally substituted; R is aryl, -(C1-C6)alkyl-aryl such as benzyl, heteroaryl, -(C1-C6)alkyl-heteroaryl, heterocyclyl, or -(C1-C6)alkyl-heterocyclyl, optionally substituted; and R, R, R and R each independently is H, halogen, (C1-C6)alkyl, or (C1-C6)haloalkyl.
  2. 2. The compound of claim 1, wherein R is phenyl optionally substituted with at least one substituent each independently selected from deuterium, halogen, -CN, (C1-C6)alkyl, (C1-C6)haloalkyl, and -O-(C1-C6)alkyl.
  3. 3. The compound of claim 1, wherein R is phenyl substituted, preferably at position thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl, and optionally further substituted, preferably at position 3 or 6 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl.
  4. 4. The compound of claim 1, wherein R is heteroaryl optionally substituted.
  5. 5. The compound of claim 4, wherein R is selected from pyridin-2-yl, 3-methylpyridin-2-yl, 4-methylpyridin-2-yl, 5-methylpyridin-2-yl, 6-methylpyridin-2-yl, 4,5-dimethylpyridin-2-yl, 4-trifluoromethylpyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 4-fluoro Underlined postdated 12. 12.20Underlined postdated 12. 12.2023 FUTURX-004 IL 30 5348/3 pyridine-2-yl, 4-chloropyridin-2-yl, 4-bromopyridin-2-yl, 2-hydroxypyridin-3-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyridin-3-yl, 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyrimidin-6-yl, thiazol-4-yl, 2-(methylthio)thiazol-4-yl, tetrahydrobenzo[c]isoxazole-3-yl, tetrahydro-1,2-benzisoxazol-3-yl, 5-methylisoxazol-3-yl, 5-ethylisoxazol-3-yl, 5,6-dihydro-4H-cyclopenta[d]isoxazol-3-yl, methylnicotinate-6-yl, 5-methylpyrazin-2-yl, benzofuran-2-yl, 3-methylbenzofuran-2-yl, 3-methylthiophene-2-yl, 1,3-benzoxazol-2-yl, indol-2-yl, 4-methoxyquinolin-2-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, and 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl.
  6. 6. The compound of claim 5, wherein R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, tetrahydrobenzo[c]isoxazole-3-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, or 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl.
  7. 7. The compound of claim 1, wherein R, R, R and R each independently is H, fluoro, or (C1-C3)alkyl.
  8. 8. The compound of claim 7, wherein R, R, R and R each is H or fluoro; or two of R, R, R and R linked to the same carbon atom each is H or fluoro, and the other two of R, R, R and R each is methyl or ethyl.
  9. 9. The compound of claim 1, wherein: R is phenyl optionally substituted with at least one substituent each independently selected from deuterium, halogen, -CN, (C1-C6)alkyl, (C1-C6)haloalkyl, and -O-(C1-C6)alkyl; R is heteroaryl optionally substituted; and R, R, R and R each independently is H, fluoro, or (C1-C3)alkyl.
  10. 10. The compound of claim 9, wherein: R is phenyl substituted, preferably at position 2 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl, and optionally further substituted, preferably at position 3 or 6 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is selected from pyridin-2-yl, 3-methylpyridin-2-yl, 4-methylpyridin-2-yl, 5-methylpyridin-2-yl, 6-methylpyridin-2-yl, 4,5-dimethylpyridin-2-yl, 4-trifluoromethyl pyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 4-fluoropyridine-2-yl, 4-chloropyridin-2-yl, 4- Underlined postdated 12. 12.2023 Underlined postdated 12. 12.2023 Underlined postdated 12. 12.2023 FUTURX-004 IL 30 5348/3 bromopyridin-2-yl, 2-hydroxypyridin-3-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyridin-3-yl, 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyrimidin-6-yl, thiazol-4-yl, 2-(methylthio)thiazol-4-yl, tetrahydrobenzo[c]isoxazole-3-yl, tetrahydro-1,2-benzisoxazol-3-yl, 5-methylisoxazol-3-yl, 5-ethylisoxazol-3-yl, 5,6-dihydro-4H-cyclopenta[d]isoxazol-3-yl, methylnicotinate-6-yl, 5-methylpyrazin-2-yl, benzofuran-2-yl, 3-methylbenzofuran-2-yl, 3-methylthiophene-2-yl, 1,3-benzoxazol-2-yl, indol-2-yl, 4-methoxyquinolin-2-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, and 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H or fluoro; or two of R, R, R and R linked to the same carbon atom each is H or fluoro, and the other two of R, R, R and R each is methyl or ethyl.
  11. 11. The compound of claim 10, wherein: R is phenyl substituted, preferably at position 2 thereof, with fluoro or chloro, and optionally further substituted, preferably at position 3 or 6 thereof, with fluoro, chloro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, tetrahydrobenzo[c]isoxazole-3-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, or 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H; or R and R each is H, and R and R each is methyl.
  12. 12. The compound of claim 11, wherein: (i) R is 2-fluorophenyl; R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl; and R, R, R and R each is H (herein identified SLN-031); (ii) R is 2-fluorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-042); (iii) R is 2-fluorophenyl; R is 6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl; and R, R, R and R each is H (herein identified SLN-043); (iv) R is 2-fluorophenyl; R is 5-cyclopropyl-1-methyl-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-044); (v) R is 2-chlorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-049); Underlined postdated 12. 12.2023 Underlined postdated 12. 12.20Underlined postdated 12. 12.2023 FUTURX-004 IL 30 5348/3 (vi) R is 2-fluoro-3-methylphenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-050); (vii) R is 2,6-difluorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H (herein identified SLN-051); or (viii) R is 2-fluorophenyl; R is 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; R and R each is H; and R and R each is methyl (herein identified SLN-052). SLN-031 SLN-042 SLN-043 SLN-044 SLN-049 SLN-050 Underlined postdated 12. 12.20Underlined postdated 12. 12.2023 FUTURX-004 IL 30 5348/3 SLN-051 SLN-052
  13. 13. A pharmaceutical composition comprising a compound of any one of claims 1-12, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof.
  14. 14. The pharmaceutical composition of claim 13, wherein: R is phenyl optionally substituted with at least one substituent each independently selected from deuterium, halogen, -CN, (C1-C6)alkyl, (C1-C6)haloalkyl, and -O-(C1-C6)alkyl; and R is heteroaryl optionally substituted; and R, R, R and R each independently is H, fluoro, or (C1-C3)alkyl.
  15. 15. The pharmaceutical composition of claim 14, wherein: R is phenyl substituted, preferably at position 2 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl, and optionally further substituted, preferably at position 3 or 6 thereof, with halogen, preferably fluoro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is selected from pyridin-2-yl, 3-methylpyridin-2-yl, 4-methylpyridin-2-yl, 5-methylpyridin-2-yl, 6-methylpyridin-2-yl, 4,5-dimethylpyridin-2-yl, 4-trifluoromethyl pyridin-2-yl, 5-trifluoromethylpyridin-2-yl, 4-fluoropyridine-2-yl, 4-chloropyridin-2-yl, 4-bromopyridin-2-yl, 2-hydroxypyridin-3-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyridin-3-yl, 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridin-3-yl, imidazo[1,5-a]pyrimidin-6-yl, thiazol-4-yl, 2-(methylthio)thiazol-4-yl, tetrahydrobenzo[c]isoxazole-3-yl, tetrahydro-1,2-benzisoxazol-3-yl, 5-methylisoxazol-3-yl, 5-ethylisoxazol-3-yl, 5,6-dihydro-4H-cyclopenta[d]isoxazol-3-yl, methylnicotinate-6-yl, 5-methylpyrazin-2-yl, benzofuran-2-yl, 3-methylbenzofuran-2-yl, 3- Underlined postdated 12. 12.20Underlined postdated 12. 12.2023 FUTURX-004 IL 30 5348/3 methylthiophene-2-yl, 1,3-benzoxazol-2-yl, indol-2-yl, 4-methoxyquinolin-2-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, and 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H or fluoro; or two of R, R, R and R linked to the same carbon atom each is H or fluoro, and the other two of R, R, R and R each is methyl or ethyl.
  16. 16. The pharmaceutical composition of claim 15, wherein: R is phenyl substituted, preferably at position 2 thereof, with fluoro or chloro, and optionally further substituted, preferably at position 3 or 6 thereof, with fluoro, chloro, -CN, (C1-C3)alkyl, (C1-C3)haloalkyl such as -CF3, or -O-(C1-C3)alkyl; R is 5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-1-yl, tetrahydrobenzo[c]isoxazole-3-yl, 5-cyclopropyl-1-methyl-1H-imidazol-4-yl, or 1-methyl-5-(trifluoromethyl)-1H-imidazol-4-yl; and R, R, R and R each is H; or R and R each is H, and R and R each is methyl.
  17. 17. The pharmaceutical composition of claim 16, wherein said compound is the compound herein identified SLN-031, SLN-042, SLN-043, SLN-044, SLN-049, SLN-050, SLN-051 or SLN-052, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof.
  18. 18. The pharmaceutical composition of any one of claims 13-17, formulated for enteral (e.g., oral, sublingual, buccal or rectal) or parenteral (e.g., intravenous, intraarterial, intrathecal, intrapleural, intratracheal, intraperitoneal, intramuscular, intraosseous, intracerebroventricular, intranasal, transdermal, subcutaneous, or topical) administration, or for inhalation.
  19. 19. The compound of any one of claims 1-12, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 13-18, for use in preventing, delaying onset of, or treating a disease or disorder associated with impaired neuronal excitability and selected from a neurological or neurodegenerative disease or disorder, autoimmune disease, and cancer.
  20. 20. The compound or pharmaceutical composition for use according to claim 19, wherein said neurological or neurodegenerative disease or disorder is selected from epilepsy such as Underlined postdated 12. 12.2023 Underlined postdated 12. 12.20Underlined postdated 12. 12.2023 FUTURX-004 IL 30 5348/3 temporal lobe epilepsy (TLE) and Dravet syndrome, Alzheimer's disease, Parkinson’s disease, mild-cognitive impairments (MCI), and multiple sclerosis.
  21. 21. The compound or pharmaceutical composition for use according to claim 20, for use in treating Alzheimer's disease, or preventing or delaying onset of Alzheimer's disease in a subject being at genetic risk for Alzheimer's disease.
  22. 22. The compound or pharmaceutical composition for use according to claim 20, for use in treating epilepsy, or in preventing sudden unexplained death in epilepsy (SUDEP).
  23. 23. The compound or pharmaceutical composition for use according to claim 19, wherein said autoimmune disease is rheumatoid arthritis or multiple sclerosis.
  24. 24. The compound or pharmaceutical composition for use according to claim 19, wherein said cancer is selected from lung cancer such as bronchogenic carcinoma, alveolar or bronchiolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, and mesothelioma; gastrointestinal cancer such as esophageal cancer, stomach cancer, pancreatic cancer, small intestine cancer, colon cancer, and rectal cancer; liver cancer such as hepatoma, cholangiocarcinoma (bile duct cancer), hepatoblastoma, angiosarcoma, and hepatocellular adenoma; breast cancer; adrenal cancer; pancreatic cancer; bone cancer such as osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, multiple myeloma, malignant giant cell tumor chordoma, and osteochronfroma; genitourinary cancer such as kidney cancer, bladder cancer, urethral cancer, prostate cancer, and testicular cancer; gynecologic cancer such as cancer of the uterus, cervix, ovaries, vulva, vagina, or fallopian tubes; haematologic cancer such as cancers of the blood and bone marrow (e.g., leukemia such as acute myeloid leukemia and chronic myeloid leukemia), Hodgkin lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma; head and neck and/or nervous system cancer such as neuroblastoma, glioblastoma, and cancers of the skull, meninges, brain, or spinal cord; and skin cancer such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, and Kaposi's sarcoma.
  25. 25. A compound according to any one of claims 1-12, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, for use in the preparation of a pharmaceutical composition for preventing, delaying onset of, or treating a disease or FUTURX-004 IL 30 5348/3 disorder associated with impaired neuronal excitability and selected from a neurological or neurodegenerative disease or disorder, autoimmune disease, and cancer. For the Applicant Paulina Ben-Ami Patent Attorneys
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