How to use it to treat epilepsy

Abstract

The present embodiments relate in part to compounds or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof for modulating S1P1 receptor activity, and methods of using the same for treating convulsions, epilepsy-related disorders, epilepsy-related syndromes, and the like as described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 896,116, filed on September 5, 2019, which is hereby incorporated by reference in its entirety. Technical Field

[0003] Embodiments disclosed herein relate, in part, to compounds or pharmaceutically acceptable salts thereof for modulating S1P1 receptor activity and / or methods for treating and / or preventing epilepsy, epilepsy-related syndromes, and the like as described herein. Background Art

[0004] Epilepsy is a chronic neurological disorder that presents a wide spectrum of disease, affecting 2.2 million people in the United States and >65 million people worldwide (Hirtz et al. 2007). Currently available anti-epileptic drugs suffer from a range of side effects, and there is a significant patient population, including approximately 20-30% of cases, that are resistant to currently available therapeutic agents. Therefore, new compounds and compositions for the treatment and / or prevention of epilepsy are needed. The compounds and compositions described herein meet these needs and others. Summary of the invention

[0005] Provided are methods for treating or preventing convulsions, epilepsy, or epilepsy-related syndromes, etc., as described herein, in a subject as described herein. In some embodiments, the method comprises administering to the subject a compound as described herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof. In some embodiments, the compound as described herein partially modulates S1P1 receptor activity. In some embodiments, the method comprises administering to the subject one or more compounds as described herein.

[0006] In some embodiments, a method of treating or preventing convulsions, epilepsy, or epilepsy-related syndromes, etc., as described herein, in a subject is provided, wherein the method comprises administering to the subject a compound having the formula of Formula I or Formula II:

[0007] or a pharmaceutically acceptable salt thereof, wherein AA, B1, B2, B3, B4, D1, V, R 30 and R 31 is as provided herein and can be selected, for example, from the respective groups of chemical moieties described herein. Also provided are methods for preparing these compounds.

[0008] In some embodiments, a method for treating or preventing convulsions, epilepsy, or epilepsy-related syndromes, etc., as described herein, in a subject is also provided, wherein the method comprises administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising one or more compounds as described herein, and the pharmaceutical composition may also include a pharmaceutically acceptable carrier. In some embodiments, the compounds described herein may be provided in any form, such as a solid or a solution (e.g., an aqueous solution), such as described herein. For example, the compounds described herein may be obtained and used in a lyophilized form alone or with a suitable additive. DETAILED DESCRIPTION

[0009] Unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed embodiments belong.

[0010] As used herein, the terms "a" or "an" mean "at least one" or "one or more" unless the context clearly indicates otherwise.

[0011] As used herein, the term "about" means that the numerical values ​​are approximate and small variations will not significantly affect the practice of the disclosed embodiments. Where numerical limitations are used, unless the context indicates otherwise, "about" means that the numerical values ​​may vary by ±10% and still be within the scope of the disclosed embodiments.

[0012] As used herein, the term "acylamino" refers to an amino group substituted with an acyl group (e.g., -OC(=O)-H or -OC(=O)-alkyl). Examples of acylamino groups are -NHC(=O)H or -NHC(=O)CH3. The term "lower acylamino" refers to an amino group substituted with a lower acyl group (e.g., -OC(=O)-H or -OC(=O)-C 1-6 Examples of lower acylamino groups are -NHC(=O)H or -NHC(=O)CH3.

[0013] As used herein, the term "alkenyl" means a straight or branched chain alkyl group having one or more carbon-carbon double bonds and 2-20 carbon atoms, including but not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. In some embodiments, the alkenyl chain is 2 to 10 carbon atoms in length, 2 to 8 carbon atoms in length, 2 to 6 carbon atoms in length, or 2 to 4 carbon atoms in length.

[0014] As used herein, the term "one or more anti-epileptic drugs" (also commonly called anticonvulsants or anti-seizure drugs) or "one or more AEDs" generally encompass agents that reduce the frequency or likelihood of seizures. There are many drug classes that include a group of anti-epileptic drugs (AEDs) and represent many different mechanisms of action. For example, some drugs are thought to increase the seizure threshold, making the brain less likely to initiate a seizure. Other drugs slow the spread of bursting activity and tend to prevent the spread or spread of epileptic activity. Some AEDs, such as benzodiazepines, are used to treat seizures. Class, play a role via GABA receptors and comprehensively inhibit neural activity. However, other AEDs can play a role by regulating neuronal calcium channels, neuronal potassium channels, neuronal NMDA channels, neuronal AMPA channels, neuronal metabotropic channels, neuronal sodium channels and / or neuronal kainite channels. The phrases "antiepileptic drugs that block sodium channels" and "AEDs that block sodium channels" used herein refer to antiepileptic drugs that block sodium channels. The AED that blocks sodium channels can be selected from the group consisting of carbamazepine, clonazepam, eslicarbazepine, ethosuximide, felbamate, gabapentin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabalin, primidone, rufinamide, tiagabine, topiramate, vigabatrin, valproate, and zonisamide, and other existing or new AEDs that may be identified in the future for blocking sodium channels.

[0015] The terms "alkoxy", "phenoxy", "benzoyloxy" and "pyrimidinyloxy" refer to an alkyl group, a phenyl group, a benzyl group or a pyrimidinyl group, respectively, bonded through an oxygen atom, each of which is optionally substituted. For example, the term "alkoxy" means a straight or branched-O-alkyl group of 1 to 20 carbon atoms, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like. In some embodiments, the alkoxy chain is 1 to 10 carbon atoms in length, 1 to 8 carbon atoms in length, 1 to 6 carbon atoms in length, 1 to 4 carbon atoms in length, 2 to 10 carbon atoms in length, 2 to 8 carbon atoms in length, 2 to 6 carbon atoms in length, or 2 to 4 carbon atoms in length.

[0016] As used herein, the term "alkyl" means a straight or branched saturated hydrocarbon group. The alkyl group may contain 1 to 20, 2 to 20, 1 to 10, 2 to 10, 1 to 8, 2 to 8, 1 to 6, 2 to 6, 1 to 4, 2 to 4, 1 to 3, or 2 or 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, tert-butyl, isobutyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, 2-methyl-1-propyl, 2-methyl- 2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2-methyl-1-pentyl, 2,2-dimethyl-1-propyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, etc.

[0017] As used herein, the term "allylamino" means an amino group substituted by an alkyl group having 1 to 6 carbon atoms. An example of an alkylamino group is -NHCH2CH3.

[0018] As used herein, the term "alkylene" or "alkylenyl" means a divalent alkyl linking group. An example of an alkylene (or alkylenyl) is methylene or methylenyl (-CH2-).

[0019] As used herein, the term "alkylthio" refers to an -S-alkyl group having 1 to 6 carbon atoms. An example of an alkylthio group is -SCH2CH3.

[0020] As used herein, the term "alkynyl" means a straight or branched chain alkyl group having one or more carbon-carbon triple bonds and 2-20 carbon atoms, including but not limited to acetylene, 1-propylene, 2-propylene, etc. In some embodiments, the alkynyl chain is 2 to 10 carbon atoms in length, 2 to 8 carbon atoms in length, 2 to 6 carbon atoms in length, or 2 to 4 carbon atoms in length.

[0021] As used herein, the term "amidino" refers to -C(=NH)NH2.

[0022] As used herein, the term "amino" refers to -NH2.

[0023] As used herein, the term "aminoalkoxy" refers to an alkoxy group substituted with an amino group. An example of an aminoalkoxy group is -OCH2CH2NH2.

[0024] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group. An example of an aminoalkyl group is -CH2CH2NH2.

[0025] As used herein, the term "aminosulfonyl" refers to -S(=O)2NH2.

[0026] As used herein, the term "aminoalkylthio" refers to an alkylthio group substituted with an amino group. An example of an aminoalkylthio group is -SCH2CH2NH2.

[0027] As used herein, the term "amphiphilic" means a three-dimensional structure having discrete hydrophobic and hydrophilic regions. An amphiphilic compound suitably has both hydrophobic and hydrophilic elements present.

[0028] As used herein, the term "animal" includes, but is not limited to, humans and non-human vertebrates, such as wild animals, domestic animals, and farm animals.

[0029] As used herein, the term "antagonize" or "antagonizing" means to reduce or completely eliminate an effect, such as the activity of an S1P1 receptor.

[0030] As used herein, the phrase "anti-receptor effective amount" of a compound can be measured by the anti-receptor effectiveness of the compound. In some embodiments, the anti-receptor effective amount inhibits receptor activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, an "anti-receptor effective amount" is also a "therapeutically effective amount" whereby the compound reduces or eliminates or modulates at least one effect of the S1P1 receptor. In some embodiments, the effect is a β-arrestin effect. In some embodiments, the effect is a G protein-mediated effect.

[0031] As used herein, the term "aryl" means a monocyclic, bicyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbon. In some embodiments, the aryl group has 6 to 20 carbon atoms or 6 to 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthyl, etc. Examples of aryl groups include, but are not limited to:

[0032]

[0033]

[0034] As used herein, the term "arylalkyl" refers to a C 1-6 alkyl.

[0035] As used herein, the term "arylamino" refers to an amino group substituted with an aryl group. An example of an arylamino group is -NH(phenyl).

[0036] As used herein, the term "arylene" means an aryl linking group, ie, an aryl group that links one group in a molecule to another group.

[0037] As used herein, the term "carbamyl" refers to -C(=O)-NH2.

[0038] As used herein, the term "carbocycle" means a 5- or 6-membered saturated or unsaturated cyclic ring, optionally containing O, S or N atoms as part of the ring. Examples of carbocycles include, but are not limited to, cyclopentyl, cyclohexyl, cyclopenta-13-diene, phenyl, and any heterocycle listed above.

[0039] As used herein, the term "carrier" means a diluent, adjuvant or excipient with which the compound is administered. Pharmaceutical carriers can be liquids such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical carriers can also be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used.

[0040] As used herein, the term "compound" refers to all stereoisomers, tautomers, and isotopes of the compounds described herein.

[0041] As used herein, the term "complex partial seizure" means one of the symptoms associated with intractable epilepsy, refers to a partial seizure with disturbance of consciousness, and is similar to seizures conventionally referred to as psychomotor seizures or seizures associated with temporal lobe epilepsy. In the draft international classification (1981), a complex partial seizure is defined as a seizure with disturbance of consciousness exhibited by the electroencephalogram during the seizure, wherein unilateral or bilateral discharges are attributed to diffuse or temporal or anterior temporal lesions.

[0042] As used herein, the terms "comprising" (and any form of comprising, such as, for example, "comprise," "comprises," and "comprised"), "having" (and any form of having, such as, for example, "have" and "has"), "including" (and any form of including, such as, for example, "includes" and "include"), or "containing" (and any form of containing, such as, for example, "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps.

[0043] As used herein, the term "contacting" means bringing two elements of an in vitro system or an in vivo system together. For example, "contacting" an S1P1 receptor compound with an S1P1 receptor in an individual or patient or cell includes administering the compound to an individual or patient such as a human, and, for example, introducing the compound into a sample containing cells or purified preparations containing the S1P1 receptor.

[0044] As used herein, the term "cortical epilepsy (cortex epilepsy)" means a kind of intractable epilepsy type, is the epilepsy with the focus in the cerebral cortex, and is classified as belonging to the symptomatic epilepsy of partial (localization-related) (focal) epilepsy and syndrome in the International Classification of Epilepsy. In the international classification, the convulsions related to cortical epilepsy are classified as simple partial convulsions, i.e., partial convulsions without reduced consciousness. Therefore, the electroencephalogram (EEG) taken during the convulsions related to cortical epilepsy (not always recorded on the scalp) shows the local contralateral discharge of the corresponding cortical field (cortical field). Cortical epilepsy is classified as temporal lobe epilepsy, parietal lobe epilepsy or occipital lobe epilepsy.

[0045] As used herein, the term "cyano" refers to -CN.

[0046] As used herein, the term "cycloalkyl" means a non-aromatic cyclic hydrocarbon, including cyclized alkyl, alkenyl and alkynyl groups containing up to 20 ring-forming carbon atoms. The cycloalkyl group can include a monocyclic or polycyclic system, such as a condensed ring system, a bridged ring system and a spirocyclic system. In some embodiments, the polycyclic system includes 2, 3 or 4 condensed rings. The cycloalkyl group can include 3 to 15, 3 to 10, 3 to 8, 3 to 6, 4 to 6, 3 to 5 or 5 or 6 ring-forming carbon atoms. The ring-forming carbon atoms of the cycloalkyl group can be optionally substituted by an oxysubyl or a sulfido. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinenyl, norcarnyl, adamantyl, etc. Also included within the definition of cycloalkyl are moieties having one or more aromatic rings fused (having a common bond) to a cycloalkyl ring, such as benzo or thienyl derivatives of pentane, pentene, hexane, etc. (e.g., 2,3-dihydro-1H-inden-1-yl or 1H-inden-2(3H)-on-1-yl).

[0047] As used herein, the term "cycloalkylalkyl" refers to a C 1-6 alkyl.

[0048] As used herein, the term "dialkylamino" means an amino group substituted with two alkyl groups each having 1 to 6 carbon atoms.

[0049] As used herein, the term "diazaamino" refers to -N(NH2)2.

[0050] As used herein, the terms "epilepsy," "epileptic convulsions (seizures)" and "epileptic syndromes" are meant to include all known types of epileptic convulsions and syndromes, including: partial convulsions, including simple, complex and partial convulsions, evolving into generalized tonic-clonic seizures and generalized convulsions, both convulsive and non-convulsive, and unclassified epileptic convulsions.

[0051] As used herein, the term "facially amphiphilic" or "facial amphiphilicity" refers to compounds having polar (hydrophilic) and nonpolar (hydrophobic) side chains that adopt one or more conformations that result in the separation of the polar and nonpolar side chains to opposite faces or separate regions of a structure or molecule.

[0052] As used herein, the term "guanidino" refers to -NH(=NH)NH2.

[0053] As used herein, the term "halo" means a halogen radical including, but not limited to, fluorine, chlorine, bromine, and iodine.

[0054] As used herein, the term "haloalkoxy" refers to an -O-haloalkyl group. An example of a haloalkoxy group is OCF3.

[0055] As used herein, the term "haloalkyl" refers to a C 1-6 Alkyl groups. Examples of haloalkyl groups include, but are not limited to, CF3, C2F5, CH2F, CHF2, CCl3, CHCl2, CH2CF3, and the like.

[0056] As used herein, the term "heteroaryl" means an aromatic heterocycle having up to 20 ring atoms (e.g., C) and having at least one heteroatom ring member (ring atoms) such as sulfur, oxygen, or nitrogen. In some embodiments, the heteroaryl group has at least one or more heteroatom ring atoms, each of which is independently sulfur, oxygen, or nitrogen. In some embodiments, the heteroaryl group has 3 to 20 ring atoms, 3 to 10 ring atoms, 3 to 6 ring atoms, or 3 to 5 ring atoms. In some embodiments, the heteroaryl group includes 2 to 14 carbon atoms, 2 to 7 carbon atoms, or 5 or 6 carbon atoms. In some embodiments, the heteroaryl group has 1 to 4 heteroatoms, 1 to 3 heteroatoms, or 1 or 2 heteroatoms. The heteroaryl group includes monocyclic and polycyclic (e.g., having 2, 3, or 4 condensed rings) systems. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl (such as indol-3-yl), pyrrolyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothiophenyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, pyrrolyl, 1H-pyranyl, 1H-pyrrolyl, 1H-pyrrolyl, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinazolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furanyl, phenoxazinyl, etc. Suitable heteroaryl groups include 1,2,3-triazole, 1,2,4-triazole, 5-amino-1,2,4-triazole, imidazole, oxazole, isoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 3-amino-1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine and 2-aminopyridine.

[0057] As used herein, the term "heteroarylalkyl" refers to a C 1-6 Alkyl group.

[0058] As used herein, the term "heteroarylamino" refers to an amino group substituted with a heteroaryl group. An example of a heteroarylamino group is -NH-(2-pyridyl).

[0059] As used herein, the term "heteroarylene" means a heteroaryl linking group, ie, a heteroaryl group that links one group in the molecule to another group.

[0060] As used herein, the term "heterocycle" or "heterocyclic ring" means a 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic ring system, any of which may be saturated or unsaturated, and is composed of carbon atoms and one to three heteroatoms selected from N, O and S, and wherein the N and S heteroatoms may be optionally oxidized, and the N heteroatom may be optionally quaternized, and includes any bicyclic group in which any of the above-defined heterocycles is fused to a benzene ring. Particularly useful are rings comprising one oxygen or sulfur, one to three nitrogen atoms, or one oxygen or sulfur in combination with one or two nitrogen atoms. The heterocycle may be attached to any heteroatom or carbon atom, which results in a stable structure. Examples of heterocyclic groups include, but are not limited to, piperidinyl, piperazinyl, 2-oxyylidenepiperazinyl, 2-oxyylidenepiperidinyl, 2-oxyylidenepyrrolidinyl, 2-oxyylideneazepine Base, Aza azepinyl, pyrrolyl, 4-piperidinone, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzoxazolyl, furanyl, tetrahydrofuranyl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone and oxadiazolyl. Morpholino is the same as morpholinyl.

[0061] As used herein, the term "heterocycloalkyl" means a non-aromatic heterocycle with up to 20 ring atoms, including cyclized alkyl, alkenyl and alkynyl groups, wherein one or more ring carbon atoms are replaced by heteroatoms such as O, N or S atoms. The heterocycloalkyl group can be a monocyclic or polycyclic (e.g., fused, bridged or spirocyclic system). In some embodiments, the heterocycloalkyl group has 1 to 20 carbon atoms, or 3 to 20 carbon atoms. In some embodiments, the heterocycloalkyl group includes 3 to 14 ring atoms, 3 to 7 ring atoms or 5 or 6 ring atoms. In some embodiments, the heterocycloalkyl group has 1 to 4 heteroatoms, 1 to 3 heteroatoms or 1 or 2 heteroatoms. In some embodiments, the heterocycloalkyl alkyl group includes 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group includes 0 to 2 triple bonds. Examples of heterocycloalkyl groups include, but are not limited to, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, pyrazolidinyl, thiazolidinyl, imidazolidinyl, pyrrolidin-2-one-3-yl, etc. In addition, the ring-forming carbon atoms and heteroatoms of the heterocycloalkyl group can be optionally substituted by oxysubyl or sulfenyl. For example, the ring-forming S atom can be substituted by 1 or 2 oxysubyls (forming S(O) or S(O)2). For other examples, the ring-forming C atom can be substituted by oxysubyl (forming carbonyl). The definition of heterocycloalkyl also includes moieties having one or more aromatic rings fused to a non-aromatic heterocycle (having a common bond), including, but not limited to, heterocyclic pyridyl, thienyl, phthalimidyl, naphthylimide, and benzo derivatives such as indolene, isoindolene, 4,5,6,7-tetrahydrothieno[2,3-c]pyridin-5-yl, 5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one-5-yl, isoindolin-1-one-3-yl, and 3,4-dihydroisoquinolin-1(2H)-one-3-yl groups. The ring-forming carbon atoms and heteroatoms of the heterocycloalkyl group may be optionally substituted with oxy or thio groups.

[0062] As used herein, the term "heterocycloalkylalkyl" refers to a C 1-6 alkyl.

[0063] As used herein, the term "hydroxy" or "hydroxyl" refers to an -OH group.

[0064] As used herein, the term "hydroxyalkyl" or "hydroxylalkyl" refers to an alkyl group substituted with a hydroxyl group. Examples of hydroxyalkyl groups include, but are not limited to,

[0065] -CH2OH and -CH2CH2OH.

[0066] As used herein, the terms "subject" or "patient," which are used interchangeably, mean any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates such as humans.

[0067] As used herein, the phrase "inhibiting an activity", such as enzyme activity or receptor activity, means reducing the activity of an enzyme or receptor, such as the S1P1 receptor, by any measurable amount.

[0068] As used herein, the phrase "activating an activity", such as enzyme activity or receptor activity, means increasing the activity of an enzyme or receptor, such as the S1P1 receptor, by any measurable amount.

[0069] As used herein, the phrase "in need of" means that an animal or mammal has been determined to be in need of a specific method or treatment. In some embodiments, it can be determined by any diagnostic means. In any method and treatment described herein, an animal or mammal may be in need of this. In some embodiments, the animal or mammal is in or will be in the following environment, in which a specific disease, illness or condition is common. In some embodiments, a "subject or patient in need of" is a subject who has been diagnosed with or suspected of having epilepsy or an epileptic syndrome. In some embodiments, a "subject or patient in need of" has suffered from convulsions. In some embodiments, when a compound or composition provided herein is administered or used in the currently described method, a "subject or patient in need of" currently suffers from convulsions.

[0070] As used herein, the phrase "in situ gellable" is meant to include not only low viscosity liquids that form gels upon contact with the eye or with tear fluid external to the eye, but also more viscous liquids, such as semifluids and thixotropic gels, that exhibit a substantially increased viscosity or gel stiffness upon administration to the eye.

[0071] As used herein, the phrase "integer from X to Y" means any integer including the endpoints. For example, the phrase "integer from X to Y" means 1, 2, 3, 4 or 5.

[0072] As used herein, the phrase "refractory epilepsy" refers to epilepsy or convulsions associated therewith, corresponding to the following four types of epilepsy or convulsions associated therewith:

[0073] (1) refractory epilepsy in which the suppression of convulsions associated therewith cannot be controlled by conventional drug treatment (Masako WATANABE, et al., Igaku-no Ayumi, 183(1):103-108, 1997); (2) epilepsy corresponding to the following (a) to (c): (a) partial epilepsy, such as temporal lobe epilepsy and cortical epilepsy; (b) generalized epilepsy and myoclonic epilepsy; and (c) unspecified epilepsy and syndrome, whether focal or generalized, such as severe myoclonic epilepsy; (3) convulsions associated with the above-mentioned refractory epilepsy, including tonic convulsions, tonic-clonic convulsions, atypical absence convulsions, atonic convulsions, myoclonic convulsions, clonic convulsions, simple partial convulsions, complex partial convulsions and secondary generalized convulsions; and (4) epilepsy, such as epilepsy after brain surgery, traumatic epilepsy and recurrent epilepsy after epilepsy surgery. Characteristics of refractory epilepsy include a high incidence of partial convulsions followed by generalized convulsions (particularly temporal lobe epilepsy), a high incidence of symptomatic epilepsy caused by organic lesions in the brain, and long-term untreated seizures from the onset of convulsions to specialist visits and high incidence; and a high incidence of status epilepticus in the past. The temporal lobe is likely to be the part of the brain responsible for refractory epilepsy. It is suggested that epilepsy becomes more refractory by changing its nature and evolving with the repetition of acquired convulsions. Refractory epilepsy is classified into three clinical types: (a) partial epilepsy and syndromes, including temporal lobe epilepsy, frontal lobe epilepsy, and multilobe epilepsy, of which temporal lobe epilepsy and frontal lobe epilepsy are typical examples of refractory epilepsy, and multilobe epilepsy is considered to be caused by two or more lobes; (b) generalized epilepsy and syndromes, including myoclonic epilepsy; and (c) undetermined epilepsy and syndromes, whether focal or generalized, including severe myoclonic epilepsy, which exhibits a variety of convulsion types, including tonic-clonic convulsions that occur frequently and often lead to status. There is a strong need for specific treatments for epilepsy by specialists (Masako WATANABE, et al., Igakuno Ayumi, 183(1):103-108, 1997). As used herein, the term "isolated" means that the compounds described herein are separated from other components of (a) natural sources, such as plants or cells, or (b) synthetic organic chemical reaction mixtures, such as by conventional techniques.

[0074] As used herein, the term "mammal" means a rodent (ie, a mouse, rat, or guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.

[0075] As used herein, the term "N-alkyl" refers to an alkyl chain substituted with an amine group. Non-limiting examples include, but are not limited to The alkyl chain can be linear, branched, cyclic, or any combination thereof. In some embodiments, the alkyl group includes 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 carbons.

[0076] As used herein, the term "nitro" means -NO2.

[0077] As used herein, the term "n-membered," where n is an integer, generally describes the number of ring-forming atoms in a moiety, where the number of ring-forming atoms is n. For example, pyridine is an example of a 6-membered heteroaryl ring, and thiophene is an example of a 5-membered heteroaryl ring.

[0078] As used herein, the phrase "ophthalmologically acceptable" means having no persistent detrimental effect on the treated eye or its function, or on the general health of the treated subject. However, it will be recognized that transient effects such as mild irritation or a "stinging" sensation are common when topical ophthalmologically administered drugs, and the presence of such transient effects is inconsistent with the composition, formulation, or ingredient (such as, excipient) being discussed as "ophthalmologically acceptable" as defined herein.

[0079] As used herein, the phrase "optionally substituted" means that substitution is optional, and therefore includes both unsubstituted and substituted atoms and moieties. A "substituted" atom or moiety means that any hydrogen on the designated atom or moiety can be replaced by a selection from the indicated substituent group, provided that the normal valence of the designated atom or moiety is not exceeded and that the substitution results in a stable compound. For example, if a methyl group is optionally substituted, then 3 hydrogen atoms on the carbon atom can be replaced by substituent groups.

[0080] As used herein, the phrase "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment. In some embodiments, "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly for use in humans.

[0081] In some embodiments, the salts of the compounds described herein are pharmaceutically acceptable salts thereof. As used herein, the phrase "one or more pharmaceutically acceptable salts" includes, but is not limited to, salts of acidic or basic groups. Compounds that are basic in nature are able to form a variety of salts with various inorganic and organic acids. The acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, including but not limited to sulfuric acid, thiosulfuric acid, citric acid, maleic acid, acetic acid, oxalic acid, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, bisulfite, phosphate, acid phosphate, isonicotinate, borate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, succinate ... The compounds of the present invention include benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, bicarbonate, malonate, methanesulfonate, ethanesulfonate, napsydisylate, toluenesulfonate, benzenesulfonate, orthophoshate, trifluoroacetate and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. In addition to the above acids, compounds including amino moieties can form pharmaceutically acceptable salts with various amino acids. Compounds of acidic nature can form basic salts with various pharmaceutically acceptable cations. Examples of such salts include, but are not limited to, alkali metal or alkaline earth metal salts, and particularly calcium, magnesium, ammonium, sodium, lithium, zinc, potassium and iron salts. The present embodiment also includes quaternary ammonium salts of the compounds described herein, wherein the compounds have one or more tertiary amine moieties.

[0082] As used herein, the term "phenyl" refers to -C6H5. A phenyl group may be unsubstituted or substituted with one, two or three suitable substituents.

[0083] As used herein, the term "prodrug" means a derivative of a known direct-acting drug that has enhanced delivery characteristics and therapeutic value compared to the drug and is converted to an active drug by enzymatic or chemical processes.

[0084] As used herein, the term "purified" means that when isolated, the isolate contains at least 90%, at least 95%, at least 98%, or at least 99% by weight of the isolate of a compound described herein.

[0085] As used herein, the phrase "quaternary ammonium salt" means a derivative of the disclosed compounds having one or more tertiary amine moieties, wherein at least one tertiary amine moiety in the parent compound is modified by alkylation (e.g., methylation or ethylation) to convert the tertiary amine moiety to a quaternary ammonium cation (and the cation is modified by an anion such as Cl - 、CH3COO - and CF3COO - balance).

[0086] As used herein, the term "semicarbazone" means =NNHC(=O)NH2.

[0087] As used herein, the phrase "solubilizer" means an agent that causes the formation of a micellar solution or a true solution of the drug.

[0088] As used herein, the term "secondary generalized convulsion" means one of the symptoms associated with intractable epilepsy, is a partial convulsion type, which shows clinical syndrome and electroencephalogram characteristics, observed as neuronal excitation, which shows that convulsions begin in a limited part of one cerebral hemisphere. Secondary generalized convulsions start with simple partial convulsions (no consciousness disorder) or complex partial convulsions (with consciousness disorder), and develop into whole-body convulsions by secondary comprehensive induction. Its cardinal symptom is convulsion, such as tonic-clonic convulsions, tonic convulsions or clonic convulsions.

[0089] As used herein, the term "solution / suspension" means a liquid composition wherein a first portion of the active agent is present in solution and a second portion of the active agent is present in suspension in the form of microparticles in a liquid matrix.

[0090] As used herein, the term "temporal lobe epilepsy" means refractory epilepsy type, is epilepsy with convulsive lesions in the temporal lobe, and is classified as symptomatic and partial epilepsy based on the international classification of epilepsy, which also includes frontal lobe epilepsy, parietal lobe epilepsy and occipital lobe epilepsy.Temporal lobe epilepsy syndrome is different according to the focus location and convulsion propagation type, because the temporal lobe has a complex structure anatomically, including neocortex, heteromorphic cortex and old cortex.Temporal lobe epilepsy, as previously defined as psychomotor convulsions, mainly causes complex partial convulsions, such as convulsions observed clinically, and also causes simple partial convulsions, secondary generalized convulsions and combinations thereof.Simple partial convulsions include autonomic and psychiatric symptoms and sensory symptoms such as smell, hearing or vision, sometimes accompanied by symptoms of experience such as memory illusion and recognition of the old as new.Complex partial convulsions often show as motor arrest, followed by feeding function autonomic disease, and are divided into amygdala-hippocampal convulsions and lateral temporal lobe convulsions according to positioning. In cases of temporal lobe epilepsy, 70-80% of convulsions are hippocampal convulsions, in which aura, motor arrest, lip automatism and clouding of consciousness develop in sequence to lead to amnesia. When the lesion is in the amygdala, autonomic nervous system symptoms such as upper abdominal irritability, phobia, and olfactory hallucinations are caused. Lateral temporal lobe convulsions include auditory illusions, hallucinations and dreamlike states, as well as speech disorders when the lesion is in the dominant hemisphere. In addition to other symptoms and recognition and memory disorders, temporal lobe epilepsy more frequently presents a long-term psychotic state than other epilepsy (Medical Dictionary, Nanzando). The treatment of temporal lobe epilepsy is carried out by drug therapy using a maximum dose of a combination of drugs or by surgical treatment.

[0091] As used herein, the phrase "substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it was formed or detected.

[0092] As used herein, the phrase "suitable substituent" or "substituent" means a group that does not invalidate the synthetic or pharmaceutical utility of the compounds described herein or the intermediates used to prepare them. Examples of suitable substituents include, but are not limited to, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C5-C6 aryl, C1-C6 alkoxy, C3-C5 heteroaryl, C3-C6 cycloalkyl, C5-C6 aryloxy, -CN, -OH, oxydiyl, halogen, haloalkyl, -NO2, -CO2H, -NH2, -NH(C1-C8 alkyl), -N(C1-C8 alkyl)2, -NH(C6 aryl), -N(C5-C6 aryl)2, -CHO, -CO(C1-C6 alkyl), -CO((C5-C6) aryl), -CO2((C1-C6) alkyl) and -CO2((C5-C6) aryl). One skilled in the art can readily select appropriate substituents based on the stability and pharmacological and synthetic activity of the compounds described herein.

[0093] As used herein, the phrase "therapeutically effective amount" means the amount of an active compound or agent that causes the biological or medical response sought by a researcher, veterinarian, physician or other clinician in a tissue, system, animal, individual or human. The therapeutic effect depends on the disorder being treated or the desired biological effect. Therefore, the therapeutic effect can be to reduce the severity of symptoms associated with the disorder and / or inhibit (partially or completely) the progression of the disorder, or improve the treatment, cure, prevention or elimination of the disorder or side effects. The amount required to cause a therapeutic response can be determined based on the age, health, size and sex of the subject. The optimal amount can also be determined based on monitoring the subject's response to treatment.

[0094] As used herein, the term "traumatic epilepsy", which is a type of intractable epilepsy, is broadly divided into two types of epilepsy, i.e., "early epilepsy" and "late epilepsy". "Early epilepsy" is caused by stimulation of the brain due to convulsions induced within a week after the trauma, and is not a true epilepsy. In contrast, "late epilepsy" is a true epilepsy, which is caused one or more weeks after the trauma. Most traumatic epilepsy is caused by the formation of lesions at the traumatic damaged part of the cortex, and they are considered to be typical examples of partial epilepsy.

[0095] As used herein, the terms "treat," "treated," or "treating" mean both therapeutic treatment and preventive measures, wherein the goal is to slow down (mitigate) an undesirable pathophysiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to: relief of symptoms; reduction in the extent of a condition, disorder, or disease; a stable (i.e., non-worsening) state of a condition, disorder, or disease; a delay in the onset of or a slowing of the progression of a condition, disorder, or disease; an improvement or alleviation (whether partial or total) of a condition, disorder, or disease state (whether detectable or undetectable); an improvement in at least one measurable physical parameter that is not necessarily discernible by the patient; or an enhancement or improvement of a condition, disorder, or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. "Treatment" also includes prolonging survival compared to the expected survival if not receiving treatment. Thus, "treatment of epilepsy" or "treating epilepsy" means alleviating or ameliorating the activity of any primary phenomenon or secondary symptoms associated with epilepsy or other conditions described herein.

[0096] As used herein, the term "ureido" refers to -NHC(=O)-NH2.

[0097] At various places in this specification, substituents of compounds may be disclosed in groups or in ranges. It is expressly intended that embodiments include each member of such groups and ranges and every individual subcombination thereof. For example, the term "C 1-6"Alkyl" individually discloses methyl, ethyl, propyl, C4 alkyl, C5 alkyl and C6 alkyl.

[0098] For compounds where a variable appears more than once, each variable can be a different moiety selected from the Markush group defining that variable. For example, where a structure is described as having two R groups present simultaneously on the same compound, the two R groups can represent different moieties selected from the Markush groups defined for R. In other examples, when optionally multiple substituents are present, such as When the form of is specified, it is understood that the substituent R can appear s times on the ring, and R can be a different moiety at each occurrence. In the above example, where the variable T 1 is defined to include hydrogen, such as when T 1 When it is CH2, NH, etc., any H can be replaced by a substituent.

[0099] It is further understood that certain features described herein that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, for brevity, the various features described in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

[0100] It should be understood that the present embodiment encompasses the use of stereoisomers, diastereomers and optical stereoisomers of compounds and mixtures thereof (where appropriate). In addition, it should be understood that stereoisomers, diastereomers and optical stereoisomers of compounds and mixtures thereof are within the scope of the embodiments. As non-limiting examples, the mixture may be a racemate, or the mixture may include unequal proportions of one specific stereoisomer relative to another. In addition, the compound may be provided as substantially pure stereoisomers, diastereomers and optical stereoisomers (such as epimers).

[0101] Compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended to be included within the scope of the embodiments. Compounds containing asymmetrically substituted carbon atoms may be separated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by splitting a racemic mixture or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc. may also be present in compounds described herein, and all such stable isomers are provided herein. The cis and trans geometric isomers of the compound are also included in the present embodiment and may be separated as a mixture of isomers or as separated isomeric forms. When specifying a compound capable of stereoisomerism or geometric isomerism in its structure or name without mentioning a specific R / S or cis / trans configuration, all such isomers are intended to be considered.

[0102] In some embodiments, the composition includes at least 90%, at least 95%, at least 98%, or at least 99%, or 100% enantiomerically pure compound or a pharmaceutically acceptable salt thereof, which means that the ratio of one enantiomer to the other enantiomer in the composition is at least 90:1, at least 95:1, at least 98:1, or at least 99:1, or is entirely in the form of one enantiomer relative to the other enantiomer.

[0103] The resolution of the racemic mixture of the compound can be carried out by any of many methods known in the art, including, for example, chiral HPLC, fractional recrystallization using a chiral resolution acid, which is an optically active salified organic acid. Suitable resolving agents for the fractional recrystallization method include, but are not limited to, optically active acids such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, and various optically active camphor sulfonic acids (such as β-camphor sulfonic acid) in D- and L-types. Other resolving agents suitable for the fractional crystallization method include, but are not limited to, α-methylbenzylamine in stereoisomerically pure form (e.g., S and R forms, or diastereoisomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, etc. The resolution of the racemic mixture can also be carried out by eluting on a column filled with an optically active resolution agent (e.g., dinitrobenzoylphenylglycine). One skilled in the art can determine the appropriate elution solvent composition.

[0104] Compounds may also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond along with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge. Examples of prototropic tautomers include, but are not limited to, keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms, wherein a proton can occupy two or more positions of a heterocyclic ring system, including but not limited to 1H- and 3H-imidazoles, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles, and 1H- and 2H-pyrazoles. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

[0105] The compounds also include hydrates and solvates, as well as anhydrous and non-solvated forms.

[0106] Compounds can also include all isotopes of atoms that occur in intermediates or final compounds. Isotopes include those atoms with the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0107] In some embodiments, the compound or its salt is substantially isolated. Partial separation can include, for example, a composition rich in the compound. Substantially separating can include a composition containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the compound or its salt by weight. Methods for separating compounds and their salts are conventional in the art.

[0108] Although the disclosed compounds are suitable, other functional groups can be incorporated into the compounds to expect similar results. In particular, it is expected that thioamides and thioesters have very similar characteristics. The distance between the aromatic rings can affect the geometric pattern of the compound, and the distance can be changed by incorporating fatty chains of different lengths, which can be optionally substituted or can include amino acids, dicarboxylic acids or diamines. The distance and relative orientation between monomers in the compound can also be changed by replacing the amide bond with a substitute with an extra atom. Therefore, replacing the carbonyl group with a dicarbonyl group changes the distance between the monomers and the tendency of the dicarbonyl unit to adopt the anti-arrangement of two carbonyl moieties and change the periodicity of the compound. Pyromellitic anhydride represents another alternative to simple amide bonds, which can change the conformation and physical properties of the compound. Modern methods of solid phase organic chemistry (E.Atherton and RCSheppard, Solid Phase Peptide Synthesis A Practical Approach IRL Press Oxford 1989) now allow the synthesis of homogeneous dispersed compounds with molecular weights close to 5,000 daltons. Other substitution patterns are also available.

[0109] The compounds also include derivatives known as prodrugs.

[0110] Compounds containing amine functional groups can also form N-oxides. Compounds containing amine functional groups mentioned herein also include N-oxides. When the compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form N-oxides. Examples of N-oxides include tertiary amines containing nitrogen heterocycles or N-oxides of nitrogen atoms. N-oxides can be formed by treating the corresponding amine with an oxidant such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid) (see, Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience).

[0111] Various embodiments of compounds and salts thereof are provided for use in methods of treating or preventing convulsions, epilepsy or epilepsy-related syndromes in a subject as described herein. Where a variable is not explicitly listed, the variable may be any of the options described herein unless otherwise stated or dictated by context.

[0112] In some embodiments, the compound is as described in the accompanying exemplary, non-limiting claims, or a pharmaceutically acceptable salt thereof.

[0113] In some embodiments, provided are compounds having Formula I or Formula II or pharmaceutically acceptable salts thereof:

[0114]

[0115] in:

[0116] AA is

[0117] W is O, S or NR1;

[0118] X is O, S or NR4;

[0119] V is O, S or NR 32 ;

[0120] Z is CHR 42 or NR 43 ;

[0121] n is 0, 1, 2, 3, or 4;

[0122] Y1 and Y2 are independently O, S, NR5, C=O, C=S or C=NR6;

[0123] Y3 is O, S, CH2 or NR 34 ;

[0124] m is 0, 1, 2, or 3;

[0125] A1 is O, S, NR7, C=O or C=S;

[0126] A2 and A3 are independently CR 29 or N;

[0127] B1 is an optionally substituted aryl or heteroaryl group, a carbocyclic ring or

[0128] B2, B3 and B4 are independently CR 38 or N;

[0129] D1 is H, OH, NH2, NO2, a ring, an optionally substituted aryl group, a branched or unbranched alkyl alcohol, a halogen, a branched or unbranched alkyl group, an amide, a cyano group, an alkoxy group, a haloalkyl group, an alkylsulfonyl group, a nitrite group, or an alkylsulfanyl group;

[0130] R2 and R3 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl; or R2 and R3 together are optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl;

[0131] R1, R4, R5, R6, R7, R 29 , R 31, R 32 , R 33 , R 34 , R 38 and R 43 and R is independently H, OH, NH2, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl.

[0132] R 30 is independently H, CN, CF3, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl; or optionally substituted haloalkyl;

[0133] R 42 is independently Br, Cl, F, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl;

[0134] In some embodiments of the compound of Formula I or Formula II, D1 and B1 are:

[0135] in:

[0136] Z1 and Z2 are independently N or CR 39 ;

[0137] Z3 is O, S or NR 27 ;

[0138] R 27 and R 39 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl, and

[0139] D1 is H, OH, NH2, NO2, a ring, an optionally substituted aryl group, a branched or unbranched alkyl alcohol, a halogen, a branched or unbranched alkyl group, an amide, a cyano group, an alkoxy group, a haloalkyl group, an alkylsulfonyl group, a nitrite group or an alkylsulfanyl group.

[0140] In some embodiments, one of Z1 and Z2 is N. In some embodiments, both Z1 and Z2 are N. In some embodiments, Z3 is O.

[0141] In some embodiments of the compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof, D1 and B1 have the formula

[0142] in:

[0143] Z4 is O, S or NR 28 ;

[0144] Z5 is N or CH;

[0145] R 19 and R 20 Each is independently H, OH, NH2, NO2, a ring, an aryl group, a branched or unbranched alkyl alcohol, a halogen, a branched or unbranched alkyl group, an amide, a cyano group, an alkoxy group, an alkylthio group, a haloalkyl group, an alkylsulfonyl group, a nitrite group or an alkylsulfanyl group; or R 19 and R 20 Two of together form an aromatic group or a ring connected to one or more atoms of B1;

[0146] R 28 is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl, and

[0147] D1 is H, OH, NH2, NO2, a ring, an optionally substituted aryl group, a branched or unbranched alkyl alcohol, a halogen, a branched or unbranched alkyl group, an amide, a cyano group, an alkoxy group, a haloalkyl group, an alkylsulfonyl group, a nitrite group or an alkylsulfanyl group.

[0148] In some embodiments, Z5 is N. In some embodiments, Z4 is O. In some embodiments, Z5 is N and Z4 is O.

[0149] In some embodiments of the compounds of Formula I or Formula II, D1 is

[0150] Where R 21 , R 22 and R 23 Each is independently H, OH, NH2, NO2, a ring, an aryl, a branched or unbranched alkyl alcohol, a halogen, a branched or unbranched alkyl, an amide, a cyano group, an alkoxy group, a haloalkyl group, an alkylsulfonyl group, a nitrite group or an alkylsulfanyl group; or R 21 , R 22 and R 23 Two of the together form an aromatic group or a ring connected to one or more atoms of D1.

[0151] In some embodiments, R 21 , R 22 and R 23 One is H. In some embodiments, R 21 , R 22 and R 23In some embodiments, two of R are H. 23 is Me, OH, NH2, Cl, NHSO2Me, SO2NH2, NH(CO)Me or (CO)NH2. 21 and R 22 is H and R 23 It is Me, OH, NH2, Cl, NHSO2Me, SO2NH2, NH(CO)Me or (CO)NH2.

[0152] In some embodiments of the compound of Formula I or Formula II, D1 is optionally substituted aryl or optionally substituted heteroaryl.

[0153] In some embodiments of the compounds of Formula I or Formula II, D1 is

[0154] Where R 24 , R 25 and R 26 R is independently H, OH, NH2, NO2, a ring (e.g., a carbocyclic or heterocyclic ring), an aryl group, a branched or unbranched alkyl alcohol, a halogen group, a branched or unbranched alkyl group, an amide group, a cyano group, an alkoxy group, a haloalkyl group, an alkylsulfonyl group, a nitrite group, or an alkylsulfanyl group; or 24 , R 25 and R 26 Two of the together form an aromatic group or a ring connected to one or more atoms of D1.

[0155] In some embodiments, R 24 , R 25 and R 26 One is H. In some embodiments, R 24 , R 25 and R 26 In some embodiments, two of R are H. 26 is H, Me, OH, CF3 or OMe. In some embodiments, R 24 and R 25 is H and R 26 It is H, Me, OH, CF3 or OMe.

[0156] In some embodiments of the compounds of Formula I or Formula II, AA is Wherein, the variables are as defined in the aforementioned embodiments.

[0157] In some embodiments, W is O. In some embodiments, X is O. In some embodiments, R2 and R3 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl or optionally substituted cycloheteroalkyl. In some embodiments, R2 and R3 are the same. In some embodiments, R2 and R3 are ethyl.

[0158] In some embodiments, D1 is In some embodiments, R 24 , R 25 and R 26 One is H. In some embodiments, R 24 , R 25 and R 26 Two of are H and the other members are as defined herein. In some embodiments, D1 is In some embodiments, D1 is

[0159] In some embodiments, R 24 is H. In some embodiments, R 24 In some embodiments, D1 is In some embodiments, R 24 It's OMe.

[0160] In some embodiments, D1 is In some embodiments, R 24 , R 25 and R 26 One is H. In some embodiments, R 24 , R 25 and R 26 Two of are H and the other members are as defined herein.

[0161] In some embodiments, D1 is In some embodiments, D1 is In some embodiments, D1 is In some embodiments, R 24 is halogen. In some embodiments, R 24 It's F.

[0162] In some embodiments, R 24 is Me. In some embodiments, R 24 is OMe. In some embodiments, R 24 It's OH.

[0163] In some embodiments of the compounds of Formula I or Formula II, R2 and R3 together are In some embodiments, n is 1.

[0164] In some embodiments of the compounds of Formula I or Formula II, AA is The variables are as defined in the above embodiments.

[0165] In some embodiments, Y1 is NR5. In some embodiments, R5 is H.

[0166] In some embodiments, Y2 is C═NR6. In some embodiments, R6 is H.

[0167] In some embodiments, Y2 is C=O. In some embodiments, Y3 is O. In some embodiments, Y3 is CH2. In some embodiments, m is 0. In some embodiments, m is 1.

[0168] In some embodiments of the compounds of Formula I or Formula II, AA is The variables are as defined in the above embodiments.

[0169] In some embodiments, A1 is O. In some embodiments, A1 is S. In some embodiments, A2 is N. In some embodiments, A3 is N. In some embodiments, A3 is CR 29 In some embodiments, R 29 It's H.

[0170] In some embodiments, A2 is CR 29 In some embodiments, R 29 It's H.

[0171] In some embodiments, A1 is NR7. In some embodiments, R7 is

[0172]

[0173] In some embodiments, D1 is And R 21 , R 22 and R 23 One of them is H.

[0174] In some embodiments, D1 is And R 21 , R 22 and R 23 Two of are H. In some embodiments, D1 is In some embodiments, R 21 is an optionally substituted C1-C6 alkyl. 21 is ethyl or methyl. In some embodiments, D1 is

[0175] In some embodiments of the compounds of Formula I or Formula II, D1 is

[0176]

[0177] in:

[0178] Z6 is O, S, NR 40 or CHR 37 ;

[0179] Z7, Z8, Z9 and Z 10 Independently N or CR 41 ;

[0180] R 35 , R 36 , R 37 , R 40 and R 41 Each is independently H, OH, NH2, cyclo, aryl, branched or unbranched alkyl alcohol, halogen, branched or unbranched alkyl, amide, cyano, alkoxy, haloalkyl, alkylsulfonyl, nitrite or alkylsulfanyl; or R 35 and R 36 Together they form an aromatic group or a ring connected to one or more atoms of D1.

[0181] In some embodiments, R 35 and R 36 One of them is H.

[0182] In some embodiments, R 35 and R 36 Both are H. In some embodiments, Z6 is NH. In some embodiments, one of Z7, Z8 and Z9 is N.

[0183] In some embodiments, Z7 is N. In some embodiments, Z8 is CH. In some embodiments, Z9 is CH. In some embodiments, both Z8 and Z9 are CH.

[0184] In some embodiments, AA is The variables are as defined in the above embodiments.

[0185] In some embodiments, W is O. In some embodiments, X is O. In some embodiments, R2 and R3 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hydroxyalkyl, optionally substituted C1-C6 alkoxy, optionally substituted cycloalkyl, or optionally substituted cycloheteroalkyl. In some embodiments, both R2 and R3 are the same. In some embodiments, both R2 and R3 are methyl or ethyl. In some embodiments, n is 1. In some embodiments, D1 is pyrazolyl. In some embodiments, D1 is

[0186] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is of the formula A compound of formula I or a pharmaceutically acceptable salt thereof, wherein Z1, Z2 and Z3 are as defined herein and above.

[0187] In some embodiments, Z2 is N. In some embodiments, Z1 is N. In some embodiments, Z3 is O. In some embodiments, Z2 and Z1 are N and Z3 is as defined herein. In some embodiments, Z2 and Z1 are N and Z3 is O. In some embodiments, the compound is of formula A compound of formula I or a pharmaceutically acceptable salt thereof.

[0188] In some embodiments of the compound of Formula II, D1 and B1 are

[0189] And the variables are as defined in the aforementioned embodiments.

[0190] In some embodiments, Z3 is O and Z1 and Z2 are independently N or CR 39 .

[0191] In some embodiments, Z1 is N, Z2 is N or CR 39 and Z3 is O, S or NR 27 In some embodiments, Z1 and Z2 are N and Z3 is O.

[0192] In some embodiments, the compound is of formula A compound of Formula II or a pharmaceutically acceptable salt thereof, wherein the variables are as defined in the above embodiments. In some embodiments, R 30 is CN. In some embodiments, V is NH. In some embodiments, R 31 It is a C1-C5 alkyl group.

[0193] In some embodiments, R 31 yes In some embodiments, R 31 It is a C1-C5 haloalkyl group.

[0194] In some embodiments, R 31 yes

[0195] In some embodiments of the compound of Formula II, D1, B1 and AA together are

[0196] The variables are as defined in the above embodiments.

[0197] In some embodiments, R 30 In some embodiments, V is O or NH.

[0198] In some embodiments, R 30 It's CF3.

[0199] In some embodiments, B1-D1 is wherein D1 is as defined herein and above. In some embodiments, D1 is In some embodiments, R 31 yes

[0200] In the foregoing embodiments, or as set forth below, or as set forth in the appended claims, if a variable (substituent) is not expressly defined, then the variable is as defined above, as would be apparent based on the embodiments.

[0201] In some embodiments, the compound has the formula:

[0202] or a pharmaceutically acceptable salt thereof.

[0203] In some embodiments, the present embodiment provides methods for treating or preventing convulsions, epilepsy, or epilepsy-related syndromes, etc., as described herein, in a subject, comprising administering to the subject a pharmaceutical composition comprising one or more compounds as provided or described herein, such as any compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof.

[0204] In some embodiments, the present embodiment provides methods for treating or preventing convulsions, epilepsy, or epilepsy-related syndromes, etc., as described herein, in a subject, which methods include administering to the subject a pharmaceutical composition comprising one or more compounds as provided or described herein, and a pharmaceutically acceptable carrier.

[0205] In some embodiments, the present embodiment provides a method for treating or preventing convulsions, epilepsy, or epilepsy-related syndromes, etc., as described herein, in a subject, comprising administering to the subject one or more compounds described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds described herein. In some embodiments, the present embodiment provides a method for treating convulsions, epilepsy, or epilepsy-related syndromes, etc., as described herein, in a subject, comprising administering to the subject one or more compounds described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds described herein. In some embodiments, the present embodiment provides a method for preventing convulsions or epilepsy-related symptoms or epilepsy-related syndromes, etc., as described herein, in a subject, comprising administering to the subject one or more compounds described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds described herein.

[0206] It is intractable epilepsy.

[0207] In some embodiments, the epilepsy being treated is refractory epilepsy. In some embodiments, refractory epilepsy is partial epilepsy, generalized epilepsy, or a syndrome thereof. In some embodiments, partial epilepsy is cortical epilepsy or temporal lobe epilepsy. In some embodiments, cortical epilepsy is frontal lobe epilepsy, parietal lobe epilepsy, or occipital lobe epilepsy. In some embodiments, these methods are used to treat or prevent epileptic convulsions. In some embodiments, epileptic convulsions are refractory partial epileptic convulsions, refractory secondary generalized convulsions, refractory complex partial convulsions, or refractory status epilepticus.

[0208] In some embodiments, epilepsy is refractory epilepsy. In some embodiments, wherein, refractory epilepsy is partial epilepsy, generalized epilepsy or its syndrome. In some embodiments, partial epilepsy is cortical epilepsy or temporal lobe epilepsy. In some embodiments, cortical epilepsy is frontal lobe epilepsy, parietal lobe epilepsy or occipital lobe epilepsy. In some embodiments, wherein epilepsy-related syndrome is epileptic convulsion. In some embodiments, epileptic convulsion is refractory partial epilepsy, refractory secondary generalized convulsion, refractory complex partial convulsion or refractory status epilepticus.

[0209] In some embodiments, the present embodiment provides a method for treating or preventing epilepsy or epilepsy-related syndromes in a subject, the method further comprising at least one (i.e., additional) anti-epileptic drug that is not a compound of Formula I or Formula II. In some embodiments, at least one anti-epileptic drug is selected from the group consisting of carbamazepine, clonazepam, eslicarbazepine, felbamate, gabapentin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabalin, primidone, rufinamide, tiagabine, topiramate, vigabatrin, valproic acid, and zonisamide.

[0210] In some embodiments, wherein the subject is a subject in need thereof. In some embodiments, wherein the epilepsy therapeutic agent is selected from those described herein.

[0211] In some embodiments, the disorder is prevented.

[0212] In some embodiments, the compound or its pharmaceutically acceptable salt for treating or preventing the method for convulsion, epilepsy or epilepsy-related syndrome etc. as described herein is selected from the compound as shown in the following table and / or as described herein (including in the Examples section of the present disclosure). Any compound provided herein can be prepared as a part of a pharmaceutically acceptable salt and / or pharmaceutical composition as provided herein. Examples of such salts are provided herein. As described herein, compounds can be prepared according to the schemes and methods described herein.

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[0324] Although the compounds described herein can be shown as having specific stereochemistry around certain atoms, such as cis or trans, the compounds can also be made in the opposite orientation or as racemic mixtures. Such isomers or racemic mixtures are encompassed in the present disclosure. In addition, although the compounds are collectively shown in the table, any compound or a pharmaceutically acceptable salt thereof can be selected from the table and used in the embodiments provided herein.

[0325] In some embodiments, provided are pharmaceutical compositions comprising a compound or a pharmaceutical salt thereof of any of the compounds described herein for use in a method of treating or preventing convulsions, epilepsy, or an epilepsy-related syndrome in a subject as described herein.

[0326] The compounds described herein can be manufactured according to the methods described herein and in the Examples. The methods described herein can be modified based on the compounds desired and described herein. In some embodiments, the method is performed according to the following scheme, wherein Q and L are substituents as shown and described herein, and will be apparent to those skilled in the art based on the present disclosure. In some embodiments, the method can be used to manufacture one or more compounds as described herein, and it will be apparent to those skilled in the art which compounds can be manufactured according to the methods described herein.

[0327] Conditions and temperatures can be varied, such as shown in the embodiments described herein. These schemes are non-limiting synthetic schemes, and synthetic routes can be modified, as will be apparent to those skilled in the art reading this specification. These compounds can also be prepared according to the schemes described in the embodiments.

[0328] These compounds can be used to modulate the S1P1 receptor. Therefore, in some embodiments, these compounds can be referred to as S1P1 receptor modulating compounds.

[0329] The compounds described herein can be administered in any conventional manner by any route in which they are active. Administration can be systemic, topical or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, sublingual or ocular, or intravaginal, by inhalation, by depot injection or by implantation. The mode of administration can depend on the condition or disease to be targeted or treated. The choice of a specific route of administration can be selected or adjusted by a clinician according to methods known to clinicians to obtain a desired clinical response.

[0330] In some embodiments, it may be desirable to administer one or more compounds or pharmaceutically acceptable salts thereof locally to the area in need of treatment. This may be accomplished, for example and without limitation, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), injection, by catheter, by suppository, or by implant, wherein the implant is a porous, non-porous, or gel-like material, including membranes, such as sialastic membranes, or fibers.

[0331] The compounds described herein can be administered alone or in combination (simultaneously or continuously) with other drugs. For example, the compounds can be administered in combination with other anti-epileptic drugs, etc. Examples of other drugs or drugs are known to those skilled in the art and include, but are not limited to, those described herein.

[0332] Means and methods for administration are known in the art, and the skilled artisan can refer to various pharmacology references for guidance (see, e.g., Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980)).

[0333] The amount of the compound to be administered is a therapeutically effective amount. The dosage to be administered will depend on the characteristics of the subject being treated, for example, the type and treatment frequency of the specific animal, age, body weight, health, concurrent treatment (if any) being treated, and can be easily determined by those skilled in the art (for example, clinicians). The standard dose of protamine can be used and regulated (i.e., increase or decrease) according to the above factors. The selection of a specific dosage regimen can be selected or regulated or titrated by a clinician according to methods known to clinicians to obtain the desired clinical response.

[0334] The amount of the compound described herein that will effectively treat and / or prevent a specific disease, illness or condition will depend on the nature and degree of the disease, condition or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo determinations can be optionally used to help identify the optimal dose range. The exact dose used in the composition will also depend on the severity of the route of administration and the condition, and should be determined according to the judgment of the practitioner and the situation of each patient. However, the suitable dosage range for oral administration is generally about 0.001 milligram to about 200 milligrams / kg body weight, about 0.01 milligram to about 100 milligrams / kg body weight, about 0.01 milligram to about 70 milligrams / kg body weight, about 0.1 milligram to about 50 milligrams / kg body weight, 0.5 milligram to about 20 milligrams / kg body weight or about 1 milligram to about 10 milligrams / kg body weight. In some embodiments, the oral dose is about 5 milligrams / kg body weight.

[0335] In some embodiments, the suitable dosage range for intravenous (iv) administration is about 0.01mg to about 500mg / kg body weight, about 0.1mg to about 100mg / kg body weight, about 1mg to about 50mg / kg body weight or about 10mg to about 35mg / kg body weight. The suitable dosage range of other modes of administration can be calculated based on the aforementioned dosage as known to those skilled in the art. For example, the recommended dosage for intranasal, transmucosal, intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal administration or administration by inhalation is about 0.001mg to about 200mg / kg body weight, about 0.01mg to about 100mg / kg body weight, about 0.1mg to about 50mg / kg body weight or about 1mg to about 20mg / kg body weight. The effective dose can be extrapolated from the dose-response curve derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.

[0336] The compounds described herein can be formulated for parenteral administration by injection (such as by bolus injection or continuous infusion). The compound can be administered by subcutaneous continuous infusion within a period of about 15 minutes to about 24 hours. The injection preparation can be presented in a unit dosage form, such as in an ampoule or a multi-dose container to which a preservative is optionally added. These compositions can take the form of a suspension, solution or emulsion such as in an oily or aqueous carrier, and can include a formulation (such as a suspending agent, a stabilizer and / or a dispersant). In some embodiments, the injectable is in the form of a short-acting, reservoir or implant and a spherical form for subcutaneous or intramuscular injection. In some embodiments, the parenteral dosage form is in the form of a solution, a suspension, an emulsion or a powder.

[0337] For oral administration, the compounds described herein can be formulated by combining the compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds to be formulated into tablets, pills, dragees, capsules, emulsions, liquids, gels, syrups, caches, pellets, powders, granules, slurries, lozenges, aqueous or oily suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral administration can be obtained by, for example, adding solid excipients, optionally grinding the resulting mixture after adding suitable adjuvants (if desired) and processing the mixture of particles to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0338] Orally administered compositions may include one or more optional agents, such as sweeteners such as fructose, aspartame or saccharin; flavoring agents such as peppermint, wintergreen oil or cherry; coloring agents; and preservatives to provide pharmaceutically palatable preparations. In addition, in the case of tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over an extended period of time. Selectively permeable membranes surrounding osmotic active driving compounds are also suitable for orally administered compounds. Oral compositions may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such carriers are suitably pharmaceutical grade.

[0339] Suitable coatings may be provided for dragee cores. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablets or dragee coatings to identify or characterize different combinations of active compound dosages.

[0340] Pharmaceutical preparations that can be used orally include, but are not limited to, push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. The push-fit capsules can contain active ingredients, which are mixed with fillers (such as lactose), binders (such as starch) and / or lubricants (such as talc or magnesium stearate) and optionally stabilizers. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin or liquid polyethylene glycol. In addition, stabilizers can be added.

[0341] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0342] For administration by inhalation, the compounds described herein can be delivered in the form of an aerosol spray from a pressurized bag or nebulizer using a suitable propellant (such as, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges such as gelatin used in an inhalation device or insufflator can be formulated into a powder mixture comprising the compound and a suitable powder base (such as lactose or starch).

[0343] The compounds described herein may also be formulated into rectal compositions such as suppositories or retention enemas, such as containing conventional suppository bases such as cocoa butter or other glycerides. The compounds described herein may also be formulated into vaginal compositions such as vaginal creams, suppositories, pessaries, vaginal rings, and intrauterine devices.

[0344] In transdermal administration, the compound can be applied to a plaster, or can be applied through a transdermal therapeutic system, which is thus supplied to the organism. In some embodiments, the compound is present in a cream, solution, powder, fluid emulsion, fluid suspension, semisolid, ointment, paste, gel, jelly and foam, or in a patch comprising any of these.

[0345] Compounds described herein can also be formulated into reservoir preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Reservoir injections can be administered at intervals of about 1 to about 6 months or longer. Therefore, for example, compounds can be formulated with suitable polymers or hydrophobic materials (e.g., formulated into an emulsion in an acceptable oil) or with ion exchange resins, or formulated into poorly soluble derivatives, such as formulated into poorly soluble salts.

[0346] In some embodiments, the compound can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 1987, 14, 201; Buchwald et al., Surgery, 1980, 88, 507; Saudek et al., N. Engl. J. Med., 1989, 321, 574). In some embodiments, polymer materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger et al., J. Macromol. Sci. Rev. Macromol. Chem., 1983, 23, 61; see also Levy et al., Science, 1985, 228, 190; During et al., Ann. Neurol., 1989, 25, 351; Howard et al., J. Neurosurg., 1989, 71, 105). In yet another embodiment, a controlled release system can be placed near a target of the compounds described herein, such as the liver, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, Science, 1990, 249, 1527-1533) can be used.

[0347] It is also known in the art that the compound can be included in such a preparation together with a pharmaceutically acceptable diluent, filler, disintegrant, binder, lubricant, surfactant, hydrophobic carrier, water-soluble carrier, emulsifier, buffer, wetting agent, humectant, solubilizer, preservative, etc. The pharmaceutical composition can also include a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers (such as polyethylene glycol). In some embodiments, the compounds described herein can be used with agents (including, but not limited to, local analgesics (e.g., lidocaine), barrier devices (e.g., GelClair), or irrigants (e.g., Caphosol)).

[0348] In some embodiments, the compounds described herein can be delivered in vesicles, particularly in liposomes (see Langer, Science, 1990, 249, 1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally supra).

[0349] Suitable compositions include, but are not limited to, oral non-absorbable compositions. Suitable compositions also include, but are not limited to, saline, water, cyclodextrin solutions, and buffer solutions of pH 3-9.

[0350] The compounds described herein or their pharmaceutically acceptable salts can be formulated with a variety of excipients, including but not limited to purified water, propylene glycol, PEG 400, glycerol, DMA, ethanol, benzyl alcohol, citric acid / sodium citrate (pH 3), citric acid / sodium citrate (pH 5), tris(hydroxymethyl)aminomethane HCl (pH 7.0), 0.9% saline and 1.2% saline, and any combination thereof. In some embodiments, the excipient is selected from propylene glycol, purified water, and glycerol.

[0351] In some embodiments, the formulation can be lyophilized to a solid and reconstituted with, for example, water prior to use.

[0352] When administered to mammals (eg, animals for veterinary use or humans for clinical use), the compound can be administered in isolated form.

[0353] When administered to people, the compound can be sterile. When the compound of formula I is administered intravenously, water is a suitable carrier. Saline solution and aqueous dextrose and glycerol solution can also be used as liquid carriers, particularly injectable solutions. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skim milk, glycerol, propylene, glycol, water, ethanol, etc. If necessary, the present composition can also include a small amount of wetting agent or emulsifier, or pH buffer.

[0354] The compositions described herein can take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained release formulations, suppositories, aerosols, sprays, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A.R. Gennaro (Editor) Mack Publishing Co.

[0355] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for administration to humans according to conventional procedures. Typically, the compound is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent. The composition for intravenous administration may optionally include a local anesthetic (such as lidocaine) to relieve pain at the injection site. Typically, the components are supplied separately or mixed together in a unit dosage form indicating the amount of the active agent, such as a dry lyophilized powder or anhydrous concentrate in an airtight sealed container (such as an ampoule or a sachet). If the compound is administered by infusion, for example, an infusion bottle containing sterile pharmaceutical grade water or saline is used for dispensing. If the compound is administered by injection, an ampoule of sterile water for injection or saline may be provided so that each component may be mixed before administration.

[0356] The pharmaceutical composition can be in unit dosage form. In such form, the composition can be divided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged preparation containing discrete quantities of the preparation, such as packaged tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be any of these dosage forms in appropriate number of packaged forms.

[0357] In some embodiments, the composition is in liquid form, wherein the active agent (i.e., one of the amphiphilic polymers or oligomers disclosed herein) is present in solution, suspension, as an emulsion or as a solution / suspension. In some embodiments, the liquid composition is in the form of a gel. In other embodiments, the liquid composition is aqueous. In other embodiments, the composition is in the form of an ointment.

[0358] In some embodiments, the composition is in the form of a solid article. For example, in some embodiments, the ophthalmic composition is a solid article that can be inserted into a suitable position in the eye, such as between the eye and the eyelid or in the conjunctival sac, where it releases the active agent, such as, for example, U.S. Patent No. 3,863,633; U.S. Patent No. 3,867,519; U.S. Patent No. 3,868,445; U.S. Patent No. 3,960,150; U.S. Patent No. 3,963,025; U.S. Patent No. 4,186,184; U.S. Patent No. 4,303,637; U.S. Patent No. 5,443,505 and U.S. Patent No. 5,869,079 described. Usually released from such an article to the cornea via tears soaking the corneal surface, or directly released to the cornea itself, the solid article is usually in close contact with the cornea. Solid articles suitable for implantation in the eye in this way are usually mainly composed of polymers and can be bioerodible or non-bioerodible. Bioerodible polymers that can be used to prepare ocular implants carrying one or more compounds include, but are not limited to, aliphatic polyesters, such as polymers and copolymers of poly(glycolide), poly(lactide), poly(ε-caprolactone), poly(hydroxybutyrate), and poly(hydroxyvalerate), polyamino acids, polyorthoesters, polyanhydrides, aliphatic polycarbonates, and polyether lactones. Suitable non-bioerodible polymers include silicone elastomers.

[0359] The compositions described herein may include a preservative. Suitable preservatives include, but are not limited to, mercury-containing substances such as phenylmercuric salts (e.g., phenylmercuric acetate, phenylmercuric borate, and phenylmercuric nitrate) and thimerosal; stabilized chlorine dioxide; quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride; imidazolidinyl urea; parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, and salts thereof; phenoxyethanol; chlorophenoxyethanol; phenoxypropanol; chlorobutanol; chlorocresol; phenylethyl alcohol; disodium EDTA; and sorbic acid and its salts.

[0360] Optionally, one or more stabilizers may be included in the composition to enhance chemical stability when needed. Suitable stabilizers include, but are not limited to, chelating agents or complexing agents, such as, for example, calcium complexing agent ethylenediaminetetraacetic acid (EDTA). For example, an appropriate amount of EDTA or its salt, such as the disodium salt, may be included in the composition to complex excessive calcium ions and prevent gel formation during storage. EDTA or its salt may be included in an amount of about 0.01% to about 0.5%. In those embodiments comprising a preservative other than EDTA, EDTA or its salt, more particularly disodium EDTA, may be present in an amount of about 0.025% to about 0.1% by weight.

[0361] One or more antioxidants may also be included in the composition. Suitable antioxidants include, but are not limited to, ascorbic acid, sodium pyrosulfite, sodium bisulfite, acetylcysteine, polyquaternium-1, benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, or other agents known to those skilled in the art. Such preservatives are typically used at a level of about 0.001% to about 1.0% by weight.

[0362] In some embodiments, the compound is at least partially solubilized by an acceptable solubilizing agent. Certain acceptable nonionic surfactants, such as polysorbate 80, can be used as solubilizing agents, as can ophthalmically acceptable glycols, polyglycols, such as polyethylene glycol 400 (PEG-400), and glycol ethers.

[0363] Suitable solubilizers for solutions and solution / suspension compositions are cyclodextrins. Suitable cyclodextrins may be selected from α-cyclodextrin, β-cyclodextrin,

[0364] γ-cyclodextrin, alkyl cyclodextrin (e.g., methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, diethyl-β-cyclodextrin), hydroxyalkyl cyclodextrin (e.g., hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin), carboxyl-alkyl cyclodextrin (e.g., carboxymethyl-β-cyclodextrin), sulfoalkyl ether cyclodextrin (e.g., sulfobutyl ether-β-cyclodextrin), etc. The ophthalmic applications of cyclodextrins are reviewed in Rajewski et al., Journal of Pharmaceutical Sciences, 1996, 85, 1155-1159.

[0365] In some embodiments, the composition optionally comprises a suspending agent. For example, in those embodiments where the composition is an aqueous suspension or solution / suspension, the composition may comprise one or more polymers as suspending agents. Useful polymers include, but are not limited to, water-soluble polymers such as cellulose polymers (e.g., hydroxypropyl methylcellulose), and water-insoluble polymers such as cross-linked carboxyl-containing polymers.

[0366] One or more acceptable pH adjusters and / or buffers may be included in the composition, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethylaminomethane); and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts required to maintain the pH of the composition within an acceptable range.

[0367] One or more acceptable salts can be included in the composition in an amount required to bring the osmotic pressure of the composition within an acceptable range. Such salts include, but are not limited to, those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions. In some embodiments, the salt includes sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate. In some embodiments, the salt is sodium chloride.

[0368] Optionally, one or more acceptable surfactants, preferably nonionic surfactants, or cosolvents may be included in the composition to enhance the solubility of the composition components or to impart physical stability, or for other purposes. Suitable nonionic surfactants include, but are not limited to, polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol 10, octoxynol 40; polysorbates 20, 60 and 80; polyoxyethylene / polyoxypropylene surfactants (e.g., F-68, F84 and P-103); cyclodextrin; or other agents known to those skilled in the art. Typically, such co-solvents or surfactants are used at a level of about 0.01% to about 2% by weight in the composition.

[0369] In some embodiments, a pharmaceutical pack or kit is provided that includes one or more containers filled with one or more compounds described herein. Optionally associated with such one or more containers may be a notice in a form prescribed by a governmental agency that regulates the manufacture, use, or sale of drugs or biological products, which reflects the agency's permission to manufacture, use, or sell for human administration for the treatment of conditions, diseases, or disorders described herein. In some embodiments, the kit contains more than one compound described herein. In some embodiments, the kit includes a compound described herein in a single injectable dosage form, such as a single dose in an injectable device such as a syringe with a needle.

[0370] Modulation of the S1P1 receptor has been found to be a target for the treatment of certain disorders.

[0371] In some embodiments, a compound provided herein or a pharmaceutically acceptable salt thereof is administered to a subject of any condition or indication without causing significant lymphopenia or immunosuppression. In some embodiments, these methods are performed without causing lymphopenia or immunosuppression.

[0372] In some embodiments, the methods described herein include administering to a subject one or more compounds described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof. In some embodiments, the subject is a subject in need of such treatment. As described herein, in some embodiments, the subject is a mammal, such as, but not limited to, a human.

[0373] In some embodiments, one or more compounds described above or their pharmaceutically acceptable salts or pharmaceutical compositions comprising one or more compounds described above are also provided for use in the manufacture of a drug for treating and / or preventing convulsions, epilepsy and / or epilepsy-related syndromes (including but not limited to the conditions described herein, such as those described herein) in a subject. In some embodiments, the subject is a subject in need thereof.

[0374] In some embodiments, one or more compounds described above or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising one or more compounds described above is also provided for use in the manufacture of a drug for treating convulsions, epilepsy and / or epilepsy-related syndromes (including but not limited to the conditions described herein, such as those described herein) in a subject. In some embodiments, the subject is a subject in need thereof.

[0375] In some embodiments, one or more compounds described above or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising one or more compounds described above is also provided for use in the manufacture of a drug for preventing convulsions, epilepsy and / or epilepsy-related syndromes (including but not limited to the conditions described herein, such as those described herein) in a subject. In some embodiments, the subject is a subject in need thereof.

[0376] In some embodiments, one or more compounds described above or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising one or more compounds described above are also provided for use in treating and / or preventing convulsions, epilepsy and / or epilepsy-related syndromes (including but not limited to the conditions described herein, such as those described herein) in a subject. In some embodiments, the subject is a subject in need thereof.

[0377] In some embodiments, one or more compounds described above or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising one or more compounds described above are also provided for use in treating convulsions, epilepsy and / or epilepsy-related syndromes (including but not limited to the conditions described herein, such as those described herein) in a subject. In some embodiments, the subject is a subject in need thereof.

[0378] In some embodiments, one or more compounds described above or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising one or more compounds described above are also provided for use in preventing convulsions, epilepsy and / or epilepsy-related syndromes (including but not limited to the conditions described herein, such as those described herein) in a subject. In some embodiments, the subject is a subject in need thereof.

[0379] In some embodiments, one or more compounds described above or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising one or more compounds described above are also provided for use in treating and / or preventing convulsions, epilepsy and / or epilepsy-related syndromes (including but not limited to the conditions described herein, such as those described herein) in a subject. In some embodiments, the subject is a subject in need thereof.

[0380] This embodiment also provides the use of one or more compounds described above or their pharmaceutically acceptable salts or pharmaceutical compositions comprising one or more compounds described above in regulating S1P1 receptor activity such as presence on a cell surface. In some embodiments, the compound, its pharmaceutically acceptable salt or its pharmaceutical composition regulates the internalization, transport and / or degradation of the S1P1 receptor. In some embodiments, the compound, its pharmaceutically acceptable salt or its pharmaceutical composition regulates the G protein-regulated pathway of the S1P1 receptor.

[0381] This embodiment also provides the use of one or more compounds described above or their pharmaceutically acceptable salts or pharmaceutical compositions comprising one or more compounds described above in regulating S1P1 receptor activity such as presence on a cell surface. In some embodiments, the compound, its pharmaceutically acceptable salt or its pharmaceutical composition regulates the internalization, transport and / or degradation of the S1P1 receptor. In some embodiments, the compound, its pharmaceutically acceptable salt or its pharmaceutical composition regulates the G protein-regulated pathway of the S1P1 receptor.

[0382] As used herein, "modulation" may refer to inhibiting or enhancing specific activity. For example, modulation of the S1P1 receptor may refer to inhibiting and / or activating the G protein-mediated pathway of the S1P1 receptor. In some embodiments, modulation refers to inhibiting or activating the β-arrestin-mediated pathway of the S1P1 receptor. In some embodiments, modulation refers to inhibiting or activating the internalization of the S1P1 receptor. In some embodiments, modulation refers to inhibiting or activating any cell signaling pathway, or an intracellular and / or extracellular entity that is directly or indirectly regulated by the S1P1 receptor. The activity of the S1P1 receptor can be measured by any method, including but not limited to the methods described herein.

[0383] The compounds described herein may be agonists or agonist-like or antagonists or antagonist-like of the S1P1 receptor. The ability of a compound to stimulate or inhibit S1P1 receptor signaling can be measured using any assay known in the art for detecting S1P1 receptor-mediated signaling or S1P1 receptor activity or the absence of such signaling / activity. "S1P1 receptor activity" refers to the ability of an S1P1 receptor to transduce a signal. Such activity can be measured, for example, in heterologous cells by binding an S1P1 receptor (or a chimeric S1P1 receptor) to a downstream effector such as adenylate cyclase.

[0384] As used herein, "natural ligand-induced activity" refers to activation of the S1P1 receptor by an endogenous ligand of the S1P1 receptor. Activity can be assessed using any number of endpoints to measure S1P1 receptor activity.

[0385] Generally, assays for testing compounds that modulate S1P 1 receptor-mediated signaling include determining any parameter that is affected indirectly or directly by the S1P 1 receptor, such as a functional, physical or chemical effect.

[0386] A sample or assay comprising an S1P1 receptor treated with a potential activator, inhibitor or modulator is compared to a control sample without the inhibitor, activator or modulator to examine the degree of inhibition. The control sample (not treated with an inhibitor) is assigned a relative S1P1 receptor activity value of 100%. When the S1P1 receptor activity value is about 80%, 50% or 25% relative to the control, inhibition of the S1P1 receptor is achieved. Activation of the S1P1 receptor is achieved when the S1P1 receptor activity value is 110%, 150% or 200-500% (i.e., two to five times higher than the control) or 1000% 3000% or higher relative to the control (not treated with an activator). For example, in some embodiments, an assay comprising an S1P1 receptor treated with a potential activator, inhibitor or modulator is used to measure the functional ability of a test compound to inhibit the activity of a known S1P1 receptor agonist measured in an assay or to activate a cell signaling pathway. When the measured activity value caused by a known S1P1 receptor agonist such as S1P (endogenous ligand) or fingolimod is blocked by the test compound, inhibition of the S1P1 receptor is achieved. Activation of the S1P1 receptor by the test compound is achieved when the total efficacy of the test compound on S1P1 receptor activation relative to the known agonist (S1P, fingolimod) is 50% or more.

[0387] The effect of a compound on S1P1 receptor function can be measured by examining any of the parameters described above. Any suitable physiological changes that affect S1P1 receptor activity can be used to assess the effect of a compound on S1P1 receptor and natural ligand-mediated S1P1 receptor activity. When determining functional consequences using intact cells or animals, various effects such as changes in intracellular second messengers (such as cAMP) can also be measured.

[0388] Modulators of S1P1 receptor activity can be tested using recombinant or naturally occurring S1P1 receptor polypeptides as described herein. The protein can be isolated, expressed in cells, expressed in membranes derived from cells, expressed in tissues, or expressed in animals. For example, neuronal cells, immune system cells, transformed cells, or membranes can be used to test the S1P1 receptor polypeptides described herein. Modulation can be tested using one of the in vitro or in vivo assays described herein. Signal transduction and cellular trafficking can also be examined in vitro using chimeric molecules, such as the extracellular domain of a receptor covalently linked to a heterologous signaling domain or a heterologous extracellular domain covalently linked to a transmembrane or cytoplasmic domain of a receptor. In addition, the ligand binding domain of the protein of interest can be used in vitro in a soluble or solid phase reaction to determine ligand binding.

[0389] Ligands that bind to the S1P1 receptor, domain, or chimeric protein can be tested in a variety of formats. Binding can be performed in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in a vesicle. For example, in an assay, the binding of a natural ligand to its receptor is measured in the presence of a candidate modulator (such as a compound described herein). Alternatively, the binding of a candidate modulator can be measured in the presence of a natural ligand. Typically, competitive assays are used that measure the ability of a compound to compete with a natural ligand for binding to a receptor. Binding can be tested by measuring, for example, changes in spectral characteristics (e.g., fluorescence, absorbance, refractive index), changes in fluid dynamics (e.g., shape), or changes in chromatographic or solubility properties.

[0390] Another technique that can be used to evaluate S1P1 receptor-protein interactions in living cells involves bioluminescence resonance energy transfer (BRET). A detailed discussion of BRET can be found in Kroeger et al., J. Biol. Chem., 276(16):12736 43 (2001).

[0391] After the receptor is expressed in the cell, the cell can be grown in a suitable culture medium in a suitable cell plate. The cells can be plated, for example, in a 384-well plate with 5000-10000 cells per well. In some embodiments, the cells are plated with about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 cells / well. The plate can have any number of holes, and the number of cells can be modified accordingly.

[0392] Any drug having utility in the uses described herein can be used for co-treatment, co-administration or co-formulation with the compositions described above. Thus, the compounds described herein can be administered before, simultaneously with or after such therapeutic agents are administered to a subject.

[0393] Additional drugs may be administered in co-therapy (including co-formulation) with one or more of the compounds described herein.

[0394] In some embodiments, the response of the disease or disorder to treatment is monitored, and the treatment regimen is adjusted if necessary based on such monitoring.

[0395] The dosing frequency is generally such that the dosing interval, e.g., the time period between one dosing and the next dosing during waking hours, is about 2 to about 12 hours, about 3 to about 8 hours, or about 4 to about 6 hours. One skilled in the art will appreciate that the appropriate dosing interval depends, to some extent, on the length of time the selected composition is able to maintain a concentration of one or more compounds in the subject and / or target tissue (e.g., above the EC 50 (The minimum concentration of compound that modulates receptor activity by 90%). Ideally, the concentration should remain above the EC for at least 100% of the dosing interval. 50 If this is not achievable, it is desirable that concentrations remain above the EC for at least about 60% of the dosing interval. 50 , or should remain above the EC for at least about 40% of the dosing interval 50 .

[0396] The present disclosure also provides the following non-limiting embodiments:

[0397] In order to more effectively understand the embodiments disclosed herein, examples are provided below. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the embodiments in any way. In these examples, molecular cloning reactions and other standard recombinant DNA techniques described may be present, and these are performed using commercially available reagents according to the methods described in Maniatis et al., Molecular Cloning-A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), except where otherwise noted.

[0398] The following examples are illustrative of the methods and compositions described herein and are not limiting. Other suitable modifications and adaptations of the various conditions and parameters normally encountered in the therapeutic, synthetic and other embodiments disclosed herein are within the spirit and scope of the embodiments.

[0399] Example

[0400] Example 1: Synthesis of compounds

[0401] Certain synthetic schemes are provided herein, both general and specific. Compounds disclosed herein can be made according to the methods described herein, or intermediates producing compounds disclosed herein can be made according to the methods described herein. Substitution can vary according to other modifications known to those skilled in the art or compounds made based on the following examples.

[0402] The following compounds were prepared according to the following examples, or the compounds were prepared according to modifications of the examples by those skilled in the art.

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

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[0420]

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[0427]

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[0442]

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[0456]

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[0476]

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[0508]

[0509]

[0510]

[0511]

[0512]

[0513] General Procedure A:

[0514] Solution 1

[0515]

[0516]

[0517] Example 2

[0518] Solution 2

[0519] Synthetic intermediate 2-2: N,2-dihydroxypyridine-3-carboxamidine

[0520]

[0521] At 20°C, to a mixture of 2-1 (2-hydroxypyridine-3-carbonitrile) (200 mg, 1.67 mmol) in ethanol (10 mL) was added the hydrochloride salt of hydroxylamine (174 mg, 2.50 mmol), diisopropylethylamine (430 mg, 3.33 mmol). The mixture was then heated to 90°C and stirred for 16 hours. The mixture was concentrated in vacuo to remove part of the ethanol, the resulting mixture was filtered, and the solid was dried in vacuo and used as the product in the next step without further purification (185 mg, 69% yield).

[0522] 1 H NMR (400MHz, DMSO-d6) δ=12.06(br,s,1H),9.50(br,s,1H),7.95(dd,J=7.2,2.4Hz,1H),7.51(dd,J=6.0,2.0Hz,1H),6.3–6.30(m,3H).

[0523] General Procedure B

[0524] Synthetic intermediate 3-2: 2,2-diethyl-4-oxydeoxy-3,4-dihydro-2H-1-benzopyran-6-carboxylic acid

[0525] Solution 3

[0526]

[0527]

[0528] Alternative synthetic intermediate 4-4: 2,2-diethyl-4-oxydeoxy-3,4-dihydro-2H-1-benzopyran-6-carboxylic acid

[0529] Solution 4

[0530]

[0531] Synthesis 4-2: 6-Bromo-2,2-diethyl-3,4-dihydro-2H-1-benzopyran-4-one

[0532] Solution 5

[0533]

[0534] Pyrrolidine (7.94 g, 112 mmol, 1.2 equivalents) and pentane-3-one (9.61 g, 112 mmol, 1.2 equivalents) were added to a solution of 1-(5-bromo-2-hydroxy-phenyl)ethanone (20 g, 93.0 mmol, 1 equivalent) in methanol (400 mL). The mixture was stirred at 80 ° C for 16 hours. The reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (200 mL X2), the combined organic layers were washed with brine (500 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue, which was purified by column chromatography to obtain the desired product 4-2 (12 g, 46% yield) as a yellow oil.

[0535] 1 H NMR (400MHz, CDCl3) δ = 7.93 (d, J = 2.5Hz, 1H), 7.52 (dd, J = 8.8, 2.6Hz, 1H), 6.84 (d, J = 8.8Hz, 1H), 2.70 (s, 2H), 1.86-1.62 (m, 4H), 0.92 (t, J = 7.5Hz, 6H).

[0536] Synthetic intermediate 4-3: 2,2-diethyl-4-oxydeoxy-3,4-dihydro-2H-1-benzopyran-6-carbonitrile

[0537] Solution 6

[0538]

[0539] To a solution of 6-bromo-2,2-diethyl-chroman-4-one (10g, 35.3mmol, 1 equivalent) in DMF (100mL), zinc cyanide (6.22g, 53.0mmol, 1.5 equivalents) and tetrakis triphenylphosphine palladium (4.08g, 3.53mmol, 0.1 equivalent) were added. The mixture was stirred at 130 ° C for 2 hours. The reaction mixture was diluted with water (100mL) and extracted with ethyl acetate (100mL X3). The combined organic layer was washed with brine (200mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue, which was purified by flash silica gel chromatography (petroleum ether / ethyl acetate=100 / 1 to 1 / 1) to obtain the desired product 4-3 (8g, 99% yield).

[0540] 1H NMR (400MHz, DMSO-d6) δ = 8.08 (d, J = 1.9 Hz, 1H), 7.96 (dd, J = 8.7, 2.0 Hz, 1H), 7. 20(d,J=8.7Hz,1H),2.88(s,2H),1.86-1.59(m,4H),0.86(br,t,J=7.4Hz,6H).

[0541] Solution 7

[0542] Synthetic intermediate 4-4: 2,2-diethyl-4-oxydeoxy-3,4-dihydro-2H-1-benzopyran-6-carboxylic acid

[0543]

[0544] A suspension of 2,2-diethyl-4-oxyylidene-chroman-6-carbonitrile (6.05 g, 26.4 mmol, 1 eq) in acetic acid (60 mL) and concentrated hydrochloride solution (60 mL) was stirred at 120 °C for 16 h, and the residue was triturated with water (500 mL), filtered and dried under vacuum to give the title product 4-4 (5.8 g, 89% yield).

[0545] 1 H NMR (400MHz, DMSO-d6) δ = 8.27 (d, J = 1.9 Hz, 1H), 8.06 (dd, J = 8.7, 2.0 Hz, 1H), 7.11(d,J=8.7Hz,1H),2.85(s,2H),1.77-1.68(m,4H),0.87(t,J=7.4Hz,6H).

[0546] General Procedure C:

[0547] Solution 8

[0548]

[0549] Synthesis of compound 9a-1: 2,2-diethyl-6-(3-(2-hydroxypyridin-3-yl)-1,2,4-oxadiazol-5-yl)chroman-4-one

[0550] Scheme 9a

[0551]

[0552] At 20 ° C under nitrogen atmosphere, HOBt (159 mg, 1.18 mmol, 1.2 equivalents), EDCI (225 mg, 1.18 mmol, 1.2 equivalents) were added to a mixture of 4-4 (268 mg, 1.08 mmol) in N, N-dimethylformamide (6 mL). The mixture was stirred for 30 min, then 2-2 (150 mg, 980 umol, 1 equivalent) was added, and then the resulting mixture was heated to 120 ° C and stirred for 2 hours. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL X3). The combined organic phase was washed with brine (50 mL), dried over sodium sulfate, and concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Gemini 150x25mmx10um; mobile phase: [water (0.05% ammonium hydroxide v / v)-ACN]; B%: 35%-65%, 12 min) to give the product 9-1 as a white solid (20 mg, 6% yield).

[0553] 1 H NMR (400MHz, DMSO-d6) δ=12.23(br,s,1H),8.44(d,J=2.0Hz,1H),8.33(dd,J=7.2,2.0Hz,1H),8.28(dd,J=8.8,2.4Hz,1H),7. 68(dd,J=6.0,2.0Hz,1H),7.29(d,J=8.8Hz,1H),6.42(t,J=6.8Hz,1H),2.93(s,2H),1.81–1.70(m,4H),0.90(t,J=7.2Hz,6H).

[0554] Synthesis of compound 2,2-diethyl-6-[3-(1H-pyrazol-4-yl)-1,2,4-oxadiazol-5-yl]-3H-1-benzopyran-4-one

[0555]

[0556] At 20 ° C, EDCI (15.20 g, 79.29 mmol, 1.2 equivalents) and HOBt (8.93 g, 66.08 mmol, 1.0 equivalents) were added to a solution of compound 4-4 (16.41 g, 66.08 mmol, 1 equivalent) in DMF (50 mL) and stirred for 0.5 hours. Then compound 9b-1 (10 g, 79.29 mmol, 1.2 equivalents) was added. The mixture was stirred at 20 ° C for 0.5 hours, then heated to 120 ° C and stirred for 2 hours. The mixture was diluted with water (100 mL), extracted with EtOAc (150 mL * 3), dried with sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (PE: EA = 3: 1) to obtain 9b-2 (7.2 g, yield: 30%) as a white solid.

[0557] 1 H NMR (400MHz, DMSO-d6) δ = 13.49 (br.s, 1H), 8.47 (s, 1H), 8.42 (d, J = 2.3Hz, 1H), 8.26 (dd, J = 2.3, 8.8 Hz, 1H), 8.06 (s, 1H), 7.26 (d, J = 8.7Hz, 1H), 2.91 (s, 2H), 1.79-1.69 (m, 4H), 0.89 (t, J = 7.4Hz, 6H).

[0558] General Procedure D:

[0559] Solution 10

[0560]

[0561]

[0562] Synthesis 11-1: 3-Bromo-5-(3-methoxyphenyl)-1,2,4-thiadiazole

[0563] Solution 11

[0564]

[0565] Pd(dppf)Cl2(119.06mg, 162.71umol, 0.1 equivalent), K3PO4(1.04g, 4.88mmol, 3 equivalents) and 10-1,3-bromo-5-chloro-1,2,4-thiadiazole (649.07mg, 3.25mmol, 2 equivalents) were added to a solution of (3-methoxyphenyl)boronic acid (247.25mg, 1.63mmol, 1 equivalent) in DME (5mL). The mixture was stirred at 80°C for 0.5h. The reaction mixture was diluted with water (50mL) and extracted with EA (50mL x3). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (PE / EA=10 / 1) to give 11-1(3-bromo-5-(3-methoxyphenyl)-1,2,4-thiadiazole) (200 mg, 737.64 umol, 45% yield) as a white solid.

[0566] 1 H NMR (400MHz, DMSO-d6) δ = 8.23 ​​(dd, J = 1.6, 7.8Hz, 1H), 7.78-7.57 (m, 1H), 7.38 (d, J = 8.4Hz, 1H), 7.21 (t, J = 7.6Hz, 1H), 4.13 (s, 3H).

[0567] Synthesis of 12-1 2,2-diethyl-6-[5-(2-methoxyphenyl)-1,2,4-thiadiazol-3-yl]-3,4-dihydro-2H-1-benzopyran-4-one

[0568] Solution 12

[0569]

[0570] To a solution of 3-bromo-5-(2-methoxyphenyl)-1,2,4-thiadiazole (100 mg, 368.82 umol, 1 eq.) and 2,2-diethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)chroman-4-one (146.15 mg, 442.59 umol, 1.2 eq.) in DMF (1 mL) and H2O (0.5 mL) was added K3PO4 (234.87 mg, 1.11 mmol, 3 eq.) and Pd(PPh3)4 (42.62 mg, 36.88 umol, 0.1 eq.) and stirred at 120°C for 0.25 h. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150x25x10um; mobile phase: [water (0.225% FA)-ACN]; B%: 70%-100%, 10 min) to give 2,2-diethyl-6-(5-(2-methoxyphenyl)-1,2,4-thiadiazol-3-yl)chroman-4-one (34.5 mg, 87.46 umol, 23.71% yield, 100% purity) as a yellow solid.

[0571] 1 H NMR (400MHz, DMSO-d6) δ = 8.66 (d, J = 2.1Hz, 1H), 8.52-8.41 (m, 2H), 7.71-7.62 (m, 1H), 7.39 (d, J = 8.3Hz, 1H), 7.26 (t, J =7.5Hz, 1H), 7.19 (d, J = 8.7Hz, 1H), 4.14 (s, 3H), 2.88 (s, 2H), 1.76 (quint, J = 7.2, 14.4Hz, 4H), 0.90 (t, J = 7.4Hz, 6H).

[0572] Synthesis of 10-8 2,2-diethyl-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-1-benzopyran-4-one

[0573] Solution 13

[0574]

[0575] To a solution of 6-bromo-2,2-diethyl-chroman-4-one (1 g, 3.53 mmol, 1 eq.) in dioxane (10 mL) were added 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolan (986.48 mg, 3.88 mmol, 1.1 eq.), DPPF (195.78 mg, 353.16 umol, 0.1 eq.), Pd(dppf)Cl2 (258.41 mg, 353.16 umol, 0.1 eq.) and KOAc (415.92 mg, 4.24 mmol, 1.2 eq.) and the mixture was stirred at 100 °C for 4 h. The reaction mixture was diluted with water (100 mL) and extracted with (EA 100 mL x 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (PE / EA=1 / 1) to give 2,2-diethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)chroman-4-one (1 g, 3.03 mmol, 85.75% yield) as a white solid.

[0576] 1 H NMR(400MHz,CHLOROFORM-d)δ=8.26(d,J=1.6Hz,1H),7.86-7.75(m,1H),6.85(d,J=8 .3Hz,1H),2.64(s,2H),1.82-1.58(m,4H),1.32-1.21(m,12H),0.85(t,J=7.5Hz,6H).

[0577] Synthesis of 14-5: 1-(Propan-2-yl)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-1,2,3-benzotriazole

[0578] Solution 14

[0579]

[0580] Synthesis of 14-2 4-bromo-2-nitro-N-(propan-2-yl)aniline

[0581] Solution 15

[0582]

[0583] At 10 ° C, DIEA (7.34g, 56.82mmol, 9.90mL, 2.50 equivalents) was added to a solution of 4-bromo-1-fluoro-2-nitro-benzene (5g, 22.73mmol, 2.79mL, 1.00eq) and propane-2-amine (2.02g, 34.09mmol, 2.92mL, 1.50 equivalents) in THF (250.00mL) and stirred for 1h, the reaction mixture was diluted with water (500mL) and extracted with EA (500mLx2). The combined organic layer was washed with NaHCO (500mL), dried over NaSO, filtered and concentrated under reduced pressure to obtain residue. The residue was purified by flash silica gel chromatography to obtain 4-bromo-N-isopropyl-2-nitro-aniline (3.2g, 12.35mmol, 54.34% yield), as a yellow oil.

[0584] 1 H NMR (400MHz, DMSO-d6) δ = 8.14 (d, J = 2.4Hz, 1H), 7.88 (br d, J=7.5Hz, 1H), 7.63 (dd, J=2.3, 9.3Hz, 1H), 7.07 (d, J=9.4Hz, 1H), 3.92 (sxtd, J=6.5, 13.2Hz, 1H), 1.25 (d, J=6.4Hz, 6H).

[0585] Synthesis of 14-3 4-bromo-1-N-(propane-2-yl)benzene-1,2-diamine

[0586] Scheme 16

[0587]

[0588] SnCl is added to the solution of 4- bromo-N- isopropyl -2- nitro - aniline (2g, 7.72mmol, 1 equivalent) in EtOH (20mL). 2H2O (5.23g, 23.16mmol, 1.93mL, 3 equivalents) and stirred at 80 ℃ for 16h. The reaction mixture is quenched with NaOH aqueous solution (4M, 50mL), and then diluted with water (50mL) and EA (100mL x2) is used. The organic layer combined is dried over Na2SO4, filtered and concentrated under reduced pressure to obtain residue. Residue is purified by flash silica gel chromatography (PE / EA=100 / 1 to 1 / 1) to obtain 4- bromo-N- isopropyl - benzene -12- diamine (850mg 3.71mmol 48.06% yield), as a dark brown solid.

[0589] 1H NMR (400MHz, CHLOROFORM-d) δ = 6.82 (dd, J = 2.1, 8.4Hz, 1H), 6.76 (d, J = 2.2Hz, 1H), 6.44 (d, J = 8.3Hz, 1H), 3.56-3.40 (m, 1H), 1.14 (d, J = 6.4Hz, 6H).

[0590] Synthesis of 14-4 5-bromo-1-(propan-2-yl)-1H-1,2,3-benzotriazole

[0591] Solution 17

[0592]

[0593] At 5 ° C, 4-bromo-N--isopropyl-benzene-1,2-diamine (750mg, 3.27mmol, 1.00 equivalent) was added dropwise in a solution of HCl (5mL, 6M) in H2O (2mL) NaNO2 (271.04mg, 3.93mmol, 213.42uL, 1.20 equivalent) and stirred for 0.5h. The reaction mixture was diluted with water (200mL) and extracted with EA (200mL x2). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (1 / 1) to obtain 5-bromo-1-isopropyl-benzotriazole (700mg, 2.92mmol, 89.06% yield), a dark brown solid.

[0594] 1 H NMR (400MHz, DMSO-d6) δ = 8.32 (d, J = 1.7Hz, 1H), 7.94 (d, J = 8.8Hz, 1H), 7.60-7.6 0(m,1H),7.67(dd,J=1.8,8.9Hz,1H),5.32-5.16(m,1H),1.62(d,J=6.7Hz,6H).

[0595] Synthesis of 14-5 1-(Propan-2-yl)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-1,2,3-benzotriazole

[0596]

[0597] To a solution of 5-bromo-1-isopropyl-benzotriazole (700 mg, 2.92 mmol, 1 eq) in dioxane (10 mL) was added 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolan (814.38 mg, 3.21 mmol, 1.1 eq), DPPF (161.63 mg, 291.55 umol, 0.1 eq), Pd(dppf)Cl2 (213.33 mg, 291.55 umol, 0.1 eq) and KOAc (343.35 mg, 3.50 mmol, 1.2 eq) and the mixture was stirred at 100 °C for 1 h. The mixture was diluted with water (100 mL) and extracted with (EA 100 mL x 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (PE / EA=100 / 1 to 1 / 1) to give 1-isopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzotriazole (300 mg, 1.04 mmol, 35.83% yield) as a yellow solid.

[0598] 1 H NMR(400MHz, CHLOROFORM-d)δ=8.57(s,1H),7.89(dd,J=0.7,8.3Hz,1H),7.55(dd,J= 0.8,8.4Hz,1H),5.11(spt,J=6.8Hz,1H),1.75(d,J=6.8Hz,6H),1.47-1.34(m,12H).

[0599] General Procedure E:

[0600] Scheme 18

[0601]

[0602]

[0603] Synthesis of 19-6 and 19-7 2,2-diethyl-6-{5-[1-(propan-2-yl)-1H-1,2,3-benzotriazol-5-yl]-1,3,4-thiadiazol-2-yl}-3,4-dihydro-2H-1-benzopyran-4-one; 2,2-diethyl-6-{5-[1-(propan-2-yl)-1H-1,2,3-benzotriazol-5-yl]-1,3,4-oxadiazol-2-yl}-3,4-dihydro-2H-1-benzopyran-4-one

[0604] Solution 19

[0605]

[0606] Synthesis 19-2: 1-(Propan-2-yl)-1H-1,2,3-benzotriazole-5-carboxylic acid methyl ester

[0607] Solution 20

[0608]

[0609] A mixture of 1-isopropylbenzotriazole-5-carbonitrile (1 g, 5.37 mmol, 1 eq.) in HCl / MeOH (20 mL, 4 M) was stirred at 80 ° C for 2 h. The mixture was concentrated, diluted with water (20 mL), extracted with EA (20 mL×2), dried over Na SO and concentrated to dryness. The crude product 1-isopropylbenzotriazole-5-carboxylic acid methyl ester (0.9 g, 4.11 mmol, 76.44% yield) was used in the next step without further purification.

[0610] Synthesis of 19-3 1-(Propan-2-yl)-1H-1,2,3-benzotriazole-5-carbohydrazide

[0611] Solution 21

[0612]

[0613] A mixture of 1-isopropylbenzotriazole-5-methylformate (0.5 g, 2.28 mmol, 1 equivalent) and NH2NH2·H2O (1.14 g, 22.81 mmol, 1.11 mL, 10 equivalents) in EtOH (10 mL) was stirred at 80 ° C for 2 h. The mixture was concentrated to dryness. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1: 1) to obtain 1-isopropylbenzotriazole-5-carbohydrazide (0.38 g, 1.73 mmol, 76.00% yield) as a white solid.

[0614] Synthesis of 19-5 2,2-diethyl-4-oxyylidene-N'-[1-(propan-2-yl)-1H-1,2,3-benzotriazole-5-carbonyl]-3,4-dihydro-2H-1-benzopyran-6-carbohydrazide

[0615] Solution 22

[0616]

[0617] To a mixture of 2,2-diethyl-4-oxyylidene-chroman-6-carboxylic acid (274.04 mg, 1.10 mmol, 1.1 eq) and 1-isopropylbenzotriazole-5-carbohydrazide (220 mg, 1.00 mmol, 1 eq) in THF (10 mL) was added HATU (419.70 mg, 1.10 mmol, 1.1 eq) and DIEA (142.66 mg, 1.10 mmol, 192.26 uL, 1.1 eq) and the mixture was stirred for 2 hours at 15° C. The mixture was diluted with water (50 mL), extracted with EA (50 mL×2), dried over Na 2 SO 4 and concentrated in vacuo. N'-(2,2-Diethyl-4-oxyylidene-chroman-6-carbonyl)-1-isopropyl-benzotriazole-5-carbohydrazide (420 mg, 934.37 umol, 93.12% yield) was obtained as a yellow solid without further purification.

[0618] Synthesis 19-7: 2,2-Diethyl-6-{5-[1-(propane-2-yl)-1H-1,2,3-benzotriazol-5-yl]-1,3,4-thiadiazol-2-yl}-3,4-dihydro-2H-1-benzopyran-4-one

[0619] Solution 23

[0620]

[0621] A mixture of N'-(2,2-diethyl-4-oxyylidene-chroman-6-carbonyl)-1-isopropyl-benzotriazole-5-carbohydrazide (200 mg, 444.94 umol, 1 eq.) and Lawesson's reagent (359.93 mg, 889.88 umol, 2 eq.) in THF (2 mL) was stirred at 80° C. for 2 h. The mixture was diluted with water (20 mL), extracted with EA (20 mL×2), dried over Na2SO4 and concentrated to dryness. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150x25x10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 62%-92%, 13min) to give 2,2-diethyl-6-[5-(1-isopropylbenzotriazol-5-yl)-1,3,4-thiadiazol-2-yl]chroman-4-one (61 mg, 29.99umol, 6.74% yield, 22% purity) as a white solid.

[0622] 1H NMR (400MHz, CHLOROFORM-d) δ = 8.62 (d, J = 0.6 Hz, 1H), 8.38-8.36 (m, 1H), 8.36-8.33 (m, 1H), 8.33-8.30 (m, 1H), 7.72 (d, J = 8. 8Hz,1H),7.13(d,J=8.7Hz,1H),5.22-5.11(m,1H),2.82(s,2H),1.95-1.83(m,4H),1.82-1.80(m,6H),0.99(t,J=7.5Hz,6H).

[0623] Synthesis 19-7: 2,2-Diethyl-6-{5-[1-(propane-2-yl)-1H-1,2,3-benzotriazol-5-yl]-1,3,4-oxadiazol-2-yl}-3,4-dihydro-2H-1-benzopyran-4-one

[0624] Solution 24

[0625]

[0626] A mixture of N'-(2,2-diethyl-4-oxyylidene-chroman-6-carbonyl)-1-isopropyl-benzotriazole-5-carbohydrazide (200 mg, 444.94 umol, 1 eq) and Burgess reagent (530.17 mg, 2.22 mmol, 5 eq) in DCM (2 mL) was stirred for 2 h at 15° C. The mixture was diluted with water (20 mL), extracted with EA (20 mL×2), dried over Na2SO4 and concentrated to dryness. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150x25x10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 55%-85%, 12min) to give 2,2-diethyl-6-[5-(1-isopropylbenzotriazol-5-yl)-1,3,4-oxadiazol-2-yl]chroman-4-one (56 mg, 129.78umol, 29.17% yield, 100% purity) as a white solid.

[0627] 1H NMR (400MHz, CHLOROFORM-d) δ = 8.85 (s, 1H), 8.61 (d, J = 2.3Hz, 1H), 8.35 (t, J = 2.1Hz, 1H), 8.33 (t, J = 2.1Hz, 1H), 7.75 (d, J = 8.7Hz, 1H), 7 .16(d,J=8.8Hz,1H),5.24-5.11(m,1H),2.84(s,2H),1.94-1.85(m,2H),1.82(d,J=6.8Hz,5H),1.80-1.75(m,2H),1.00(t,J=7.5Hz,6H).

[0628] Synthesis 25-2: 5-[5-(1H-1,3-benzodiazole-5-yl)-1,3,4-oxadiazole-2-yl]-2-fluorobenzonitrile

[0629] Solution 25

[0630]

[0631] A suspension of 1 (N'-(1H-1,3-benzodiazole-5-carbonyl)-3-cyano-4-fluorobenzohydrazide, 541 mg, 1.67 mmol) in phosphorus oxychloride (10 mL, excess) was heated at 105 °C for 3 hours. The mixture was concentrated and the residue was suspended in water under sonication / stirring. The resulting solid was collected, washed with saturated NaHCO3 and water, and then dried in a filter funnel under N2 / vacuum. The tan solid was suspended in MeCN and concentrated twice to remove residual water and dried under high vacuum to give 25-2 (0.55 g 107%). MH+=306.1.

[0632] Synthesis of 26-2 6-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]-3-methyl-2,3-dihydro-1,3-benzoxazol-2-one

[0633] Scheme 26

[0634]

[0635] To a suspension of 3 (6-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]-2,3-dihydro-1,3-benzoxazol-2-one trifluoroacetate, 13 mg, 0.031 mmol) in DMF (1 mL) was added potassium carbonate (8.6 mg, 0.062 mmol), followed by iodomethane (5.2 mg, 0.037 mmol), and the resulting white suspension was heated to 100 ° C for 45 min. The reaction was cooled to room temperature, filtered, and purified by reverse phase chromatography (25%-75% MeCN / water / 0.1% TFA). The product fractions were lyophilized to give 26-2 (1.6 mg 12%). MH+=324.1. 1 H NMR (400MHz, DMSO)7.93-7.91(3H,m),7.59-7.54(1H,m),7.44-7.41(1H,m),7.25-7.22(1H,m),7.11-7.07(1H,m),3.88(3H,s); 3.45(3H,s)

[0636] Synthesis of 27-3N-({6-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]-2-oxyde-2,3-dihydro-1,3-benzoxazol-3-yl}sulfonyl)carbamic acid methyl ester

[0637] Scheme 27

[0638]

[0639] To a dry mixture of 27-1 (2-methoxyphenylhydrazide, 139 mg, 0.837 mmol), 27-2 (benzoxazol-2-one-6-carboxylic acid, 150 mg, 0.837 mmol) and HATU (318 mg, 0.837 mmol) was added THF (10 mL) to produce a turbid reddish solution. DIPEA (0.29 mL, 1.67 mmol) was added and the reaction was stirred at room temperature for 2 hours. A portion of Burgess reagent (499 mg, 2.09 mmol) was added and the reaction was heated to 60 °C overnight. An additional 499 mg of Burgess reagent was added and heating was continued. After 4 hours, 2N KHSO 4 (10 mL) was added and the resulting oily mixture was extracted with 3X EtOAc. The combined organics were washed once with water, once with brine, filtered through cotton and concentrated to an orange solid which was purified by reverse phase chromatography (20%-60% MeCN / water / 0.1% TFA) to give 67 mg of 27-3 (18%) MH+=447.0. 1H NMR (400MHz, DMSO) 8.02-7.97 (3H, m), 7.74 (1H, d, J = 8.4Hz), 7.64 (1H, t, J = 8. 2Hz), 7.30 (1H, d, J = 8.4Hz), 7.18 (1H, t, J = 7.4Hz), 3.95 (3H, s), 3.39 (3H, s).

[0640] Synthesis 28-2: 2-[(2-fluoropropyl)amino]-5-[5-(2-oxyylidene-1,2,3,4-tetrahydroquinolin-6-yl)-1,3,4-oxadiazol-2-yl]benzonitrile

[0641] Scheme 28

[0642]

[0643] 10% Pd / C (14 mg) was added to a small Parr hydrogenation bottle purged with nitrogen and moistened with a small amount of EtOH. EtOH (10 mL) and EtOAc (90 mL) were added to 28-1 (2-[(2-fluoroprop-2-ene-1-yl)amino]-5-[5-(2-oxyylidene-1,2,3,4-tetrahydroquinoline-6-yl)-1,3,4-oxadiazol-2-yl]benzonitrile trifluoroacetate, 70 mg, 0.139 mmol) to produce a milky mixture, which was added to the hydrogenation bottle. The mixture was hydrogenated at 50 psi H2 for 24 hours. MeCN was added until the milky mixture was clear, then filtered through celite and concentrated. The residue was heated in a small volume of DMF, cooled, filtered, and purified by reverse phase chromatography (30%-75% MeCN / water / 0.1% TFA). The product fractions were lyophilized to give 28-2 as a fluffy white solid (12.8 mg, 18%). MH+ = 392.1. 1 H NMR(400MHz,DMSO)10.40(1H,s),8.25(1H,d,J=2.1Hz),8.11(1H,dd,J=2.0 ,9.2Hz),7.92-7.81(2H,m),7.22(1H,t,J=6.3Hz),7.11(1H,d,J=9.2Hz),7. 11(1H,d,J=9.4Hz),7.02(1H,d,J=8.7Hz),7.04-7.00(1H,m),5.00-4.80(1H ,m),3.60-3.51(2H,m),3.01(2H,t,J=7.4Hz),1.35(3H,dd,J=6.2,24.0Hz).

[0644] Synthesis 29-3: 5-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]-1,3-dihydro-2,1-benzoxazol-3-one

[0645] Scheme 29

[0646]

[0647] 10% Pd / C (12 mg) was added to a nitrogen purged hydrogenation bottle, which was wetted with EtOH. A suspension of 29-1 (methyl 5-[5-(2-methoxyphenyl)-1,3,4-oxadiazole-2-yl]-2-nitrobenzoate, prepared according to Wuxi 1,3,4-oxadiazole experiment, 112 mg, 0.281) in EtOH (25 mL) and EtOAc (20 mL) was hydrogenated under 48 psi H2. After 30 minutes, the reaction was filtered and concentrated to a solid 29-2 (113 mg, 105%), which was used without further purification. MH+ = 342.1.

[0648] To a solution of 29-2 (47 mg, 0.132 mmol) in DMF (2 mL) was added triethylamine (0.1 mL) followed by water (0.2 mL) and the resulting solution was stirred at room temperature for 60 hours. The reaction mixture was directly purified by reverse phase chromatography (20%-65% MeCN / water / 0.1% TFA) to give 8 mg of 29-3 (14%). MH+=310.1. 1 H NMR(400MHz, DMSO)12.52(1H,s),8.42-8.38(2H,m),8.03(1H,d,J=6.9Hz),7.65(1H,t,J= 8.3Hz), 7.57(1H,d,J=7.6Hz), 7.32(1H,d,J=8.3Hz), 7.17(1H,t,J=6.9Hz), 3.96(3H,s).

[0649] Synthesis 30-1: 5-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]-1-methyl-1,3-dihydro-2,1-benzoxazol-3-one

[0650] Scheme 30

[0651]

[0652] To a solution of 29-2 (8 mg, 0.019 mmol) in DMF (0.5 mL) was added potassium carbonate (2.6 mg, 0.019 mmol) followed by iodomethane (4.0 mg, 0.028 mmol) and the resulting solution was heated to 100 ° C. for 15 minutes. The reaction was cooled to room temperature, filtered, and directly purified by reverse phase chromatography (25%-75% MeCN / water / 0.1% TFA) to give 5.3 mg of 30-1 (64%) as a white solid.

[0653] MH+=324.1. 1 H NMR (400MHz, DMSO)8.40(1H,d,J=8.7Hz),8.32(1H,s),7.97(1H,d,J=8.0Hz),7.73(1H,d,J=8.7H z), 7.58 (1H, t, J = 8.3Hz), 7.25 (1H, d, J = 8.0Hz), 7.10 (1H, t, J = 7.7Hz), 3.89 (3H, s), 3.49 (3H, s).

[0654] General Procedure F:

[0655] Scheme 31

[0656]

[0657]

[0658] Synthesis of 32-2 N-hydroxy-2-oxydeoxy-2,3-dihydro-1H-indole-5-carboxamidine

[0659] Scheme 32

[0660]

[0661] To a mixture of 2-oxyylidene indoline-5-carbonitrile (200 mg, 1.26 mmol, 1 equivalent) in ethanol (5 mL) at 20 ° C under a nitrogen atmosphere, hydroxylamine (176 mg, 2.53 mmol, 2.0 equivalents), diisopropylethylamine (327 mg, 2.53 mmol, 2.0 equivalents) hydrochloride was added. The mixture was then heated to 90 ° C and stirred for 16 hours. The mixture was concentrated in a vacuum and a white solid was precipitated. The suspension was filtered and the white solid was dried in a vacuum to obtain product 32-2 (210 mg, 83% yield).

[0662] 1H NMR (400MHz, DMSO-d6) δ=10.46(br,s,1H),9.45(br,s,1H),7.51(s,1H),7.50(d,J=10.8Hz,1H),6.79(d,J=8.0Hz,1H),5.70(br,s,2H),3.49(s,2H).

[0663] Synthesis 33-1: 2-[(Cyclopropylmethyl)amino]-5-[3-(2-oxydeoxy-2,3-dihydro-1H-indol-5-yl)-1,2,4-oxadiazol-5-yl]benzonitrile

[0664] Scheme 33

[0665]

[0666] To a mixture of 3-cyano-4-(cyclopropylmethylamino)benzoic acid (107 mg, 496 umol, 1.2 equiv) in N,N-dimethylformamide (1 mL) was added HOBt (67.0 mg, 496 umol, 1.2 equiv), EDCI (95.1 mg, 496 umol, 1.2 equiv) at 20 ° C. The mixture was stirred for 30 min, then N-hydroxy-2-oxyylidene-dihydroindole-5-carboxamidine (79 mg, 413 umol, 1 equiv) was added, and then the resulting mixture was heated to 150 ° C and stirred for 1 hour. The cooled reaction mixture was directly purified by preparative HPLC (column: Boston Green ODS 150x30 5u; mobile phase: [water (0.225% FA)-ACN]; B%: 47%-77%, 10 min) to obtain product 33-1 (20 mg, 12% yield).

[0667] 1 H NMR (400MHz, DMSO-d6) δ=10.73(br,s,1H),8.23(d,J=2.4Hz,1H),8.11(dd,J=9.2,2.0Hz,1H),7.93(d,J=8.0Hz,1H),7.89(s,1H),7.17(t,J=6.4H z,1H),7.07(d,J=9.2Hz,1H),7.00(d,J=8.0Hz,1H),3.61(s,2H),3.20(t ,J=6.4Hz,2H),1.16–1.13(m,1H),0.52–0.47(m,2H),0.33–0.29(m,2H).

[0668] General Procedure G:

[0669] Scheme 34

[0670]

[0671]

[0672] Synthesis of 35-2 3-cyano-4-[(cyclopropylmethyl)amino]benzoic acid

[0673] Scheme 35

[0674]

[0675] To a mixture of 3-cyano-4-fluoro-benzoic acid (5 g, 30.3 mmol, 1 eq.) and cyclopropylmethaneamine (5.38 g, 75.7 mmol, 2.5 eq.) in dimethyl sulfoxide (30 mL) was added potassium carbonate (12.6 g, 90.8 mmol, 3 eq.) at 20 °C. The mixture was then heated to 100 °C and stirred for 16 hours. The mixture was filtered, and the filtrate was diluted with water (50 mL) and acidified to pH = 4-5 with hydrochloride solution (2N). A yellow solid was precipitated, the suspension was filtered, and the solid was washed with water (50 mL X3). The solid was dried in vacuo to obtain the desired product 35-2 (5 g, 73% yield).

[0676] 1H NMR (400MHz, DMSO-d6) δ = 7.95 (d, J = 1.2Hz, 1H), 7.90 (d, J = 9.2Hz, 1H), 6.88 (d, J = 9.2Hz, 1H), 6 .85–6.82(m,1H),3.13(t,J=6.0,2H),1.16–1.06(m,1H),0.49–0.44(m,2H),0.29–0.25(m,2H).

[0677] Synthesis 36-2 5-[3-(1H-indol-5-yl)-1,2,4-oxadiazol-5-yl]-2-[(prop-2-en-1-yl)amino]benzonitrile

[0678] Scheme 36

[0679]

[0680] To a mixture of tert-butyl 5-[5-[4-(allylamino)-3-cyano-phenyl]-1,2,4-oxadiazol-3-yl]indole-1-carboxylate (60.0 mg, 136 umol, 1.00 equiv) in dichloromethane (10.0 mL) was added trifluoroacetic acid (770 mg, 6.75 mmol, 50 equiv) at 20 ° C under nitrogen atmosphere. The mixture was stirred at 20 ° C for 16 h. The mixture was concentrated in vacuo and the residue was purified by preparative HPLC (column: Gemini 150x25 5u; mobile phase: [water (0.05% ammonium hydroxide v / v)-ACN]; B%: 47%-77%, 12 min) to give the product 36-2 (15 mg, 30% yield) as a white solid.

[0681] 1 H NMR (400MHz, DMSO-d6) δ = 8.33 (s, 1H), 8.27 (d, J = 2.0Hz, 1H), 8.14 (dd, J = 8.8, 2.0Hz, 1H), 7.80 (dd, J = 8.4, 1.2Hz, 1H), 7.56 (d, J = 8.8Hz, 1H),7.48(t,J=2.4Hz,1H),7.39(t,J=5.6Hz,1H),6.91(d,J=8.8Hz,1H),6.61(s,1H),5.94–5.85(m,1H),5.24–5.16(m,2H),3.97(s,1H).

[0682] General Procedure H:

[0683] Scheme 37

[0684]

[0685]

[0686] Synthesis of 38-4 1-cyclopentyl-N-hydroxy-1H-1,2,3-benzotriazole-5-carboximidamide

[0687] Scheme 38

[0688]

[0689] Synthesis 38-1 4-(Cyclopentylamino)-3-nitrobenzonitrile

[0690] Scheme 39

[0691]

[0692] Preparation 1: To a solution of 4-fluoro-3-nitro-benzonitrile (1.00 g, 6.02 mmol, 1.00 equiv) and cyclopentylamine (767 mg, 9.03 mmol, 1.50 equiv) in THF (20.00 mL) was added DIEA (1.95 g, 15.05 mmol, 2.63 mL, 2.50 equiv) and the mixture was stirred at 10 ° C for 16 hours. The mixture was evaporated to dryness and diluted with H2O (50 mL), extracted with (50 mLx2), dried over Na2SO4, filtered and concentrated to dryness. Compound 4-(cyclopentylamino)-3-nitrobenzonitrile (1.36 g, 5.91 mmol, 98.10% yield) was obtained as a yellow solid, which was used directly in the next step.

[0693] Synthesis of 38-2 3-amino-4-(cyclopentylamino)benzonitrile

[0694] Scheme 40

[0695]

[0696] Preparation 2: To a solution of 4-(cyclopentylamino)-3-nitro-benzonitrile (5.00 g, 21.62 mmol, 1.00 equiv) in MeOH (150.00 mL) was added Pd / C (1.00 g, 4.32 mmol, 10% purity, 0.20 equiv). The mixture was then stirred at 20° C. under H2 (50 psi) for 12 h. The mixture was filtered and the filtrate was concentrated to dryness to give 3-amino-4-(cyclopentylamino)benzonitrile (4.20 g, 20.87 mmol, 96.52% yield) as a black solid which was used directly in the next step. 1 H NMR (400MHz, DMSO-d6) δ = 8.77 (s, 1H), 8.12 (d, J = 8.7Hz, 1H), 7.91 (d, J = 8.7Hz, 1H), 5. 43(m,1H),2.37-2.21(m,2H),2.18-2.06(m,2H),1.99-1.85(m,2H),1.81-1.67(m,2H).

[0697] Synthesis of 38-4: 1-cyclopentyl-N-hydroxy-1H-1,2,3-benzotriazole-5-carboximidamide

[0698] Scheme 41

[0699]

[0700] Preparation 3: To a solution of 1-cyclopentylbenzotriazole-5-carbonitrile (1.50 g, 7.07 mmol) in ethanol (15.00 mL) was added hydroxylamine hydrochloride (736.64 mg, 10.60 mmol) and DIPEA (2.01 g, 15.55 mmol, 2.72 mL). The mixture was then stirred at 70 ° C for 4 hours. The mixture was filtered to give 1-cyclopentyl-N-hydroxy-benzotriazole-5-carboximidamide (1.20 g, 4.89 mmol, 69.20% yield) as a white solid.

[0701] 1 H NMR (400MHz, DMSO-d6)δ=9.77(s,1H),8.31(s,1H),7.97-7.89(m,1H),7.87-7.79(m,1H),5.98(s,2H ), 5.34 (q, J = 7.0Hz, 1H), 2.36-2.21 (m, 2H), 2.19-2.07 (m, 2H), 1.99-1.84 (m, 2H), 1.81-1.67 (m, 2H).

[0702] General Procedure I:

[0703] Scheme 42

[0704]

[0705]

[0706] Synthesis of 42-2 5-[5-(2-bromophenyl)-1,2,4-oxadiazol-3-yl]-1-cyclopentyl-1H-1,2,3-benzotriazole

[0707] Scheme 43

[0708]

[0709] To the solution of 3-methylbenzoic acid (66.61mg, 489.24umol) in DMF (4.00mL), add EDCI (93.79mg, 489.24umol) and HOBt (66.11mg, 489.24umol). The mixture was stirred at 20 ℃ for 1 hour and 1-cyclopentyl-N-hydroxy-benzotriazole-5-formamidine (100.00mg, 407.70umol) was added to the mixture. Then the mixture was stirred at 120 ℃ under N2 for 12 hours. The mixture was quenched with water (30mL) and extracted with ethyl acetate (30mLx2). Organic layer was washed with salt water (40mL), through Na2SO4, and concentrated to dryness. The residue was purified by preparative HPLC (TFA) to give 3-(1-cyclopentylbenzotriazol-5-yl)-5-(m-tolyl)-1,2,4-oxadiazole (96.00 mg, 277.94 umol, 68.17% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ = 8.93 (s, 1H), 8.30 (dd, J = 1.3, 8.7Hz, 1H), 8.08 (s, 1H), 8.06 (br d,J=6.8Hz,1H),7.68(d,J=8.8Hz,1H),7.51-7.42(m,2H),5.22(quin,J=7.0H z,1H),2.50(s,3H),2.43-2.32(m,4H),2.15-2.01(m,2H),1.92-1.81(m,2H).

[0710] Synthesis of 44-2 2-{5-[5-(2-bromophenyl)-1,2,4-oxadiazol-3-yl]-1H-1,2,3-benzotriazol-1-yl}acetic acid

[0711] Scheme 44

[0712]

[0713] To a solution of methyl 2-[5-[5-(2-bromophenyl)-1,2,4-oxadiazole-3-yl]benzotriazole-1-yl]acetate (40.00 mg, 96.57 umol, 1.00 equiv) in dioxane (2.00 mL) and H2O (2.00 mL) was added NaOH (15.45 mg, 386.28 umol, 4.00 equiv). The mixture was then stirred at 20 °C for 12 hours. The mixture was adjusted to pH = 2-3 with HCl (1N) and extracted with ethyl acetate (15 mL x 2). The organic layer was dried over Na2SO4 and concentrated to give 2-[5-[5-(2-bromophenyl)-1,2,4-oxadiazole-3-yl]benzotriazole-1-yl]acetic acid (17.30 mg, 43.23 umol, 44.76% yield) as a white solid.

[0714] 1 H NMR (400MHz, DMSO-d6) δ = 8.70 (s, 1H), 8.26 (m, 3H), 7.93 (m, 2H), 7.66 (m, 2H), 5.24 (br s, 2H)

[0715] Synthesis of 45-1 2-{5-[5-(2-bromophenyl)-1,2,4-oxadiazol-3-yl]-1H-1,2,3-benzotriazol-1-yl}ethan-1-ol

[0716] Scheme 45

[0717]

[0718] To 2-[5-[5-(2-bromophenyl)-1,2,4-oxadiazole-3-yl]benzotriazole-1-yl]acetic acid methyl ester (50.00mg, 120.71umol, 1.00 equivalent) in THF (1.00mL) solution, add LiBH4(5.26mg, 241.42umol, 2.00 equivalent) and stir at 10 DEG C for 16h.The mixture is diluted with H2O(20mL) and extracted with EA(30mLx2), the combined organic layer is dried over Na2SO4, filtered and concentrated to dryness.The residue is purified by preparative HPLC (TFA conditions) to obtain 2-[5-[5-(2-bromophenyl)-1,2,4-oxadiazole-3-yl]benzotriazole-1-yl]ethanol (10.00mg, 25.89umol, 21.45% yield) as a white solid.

[0719] 1H NMR (400MHz, DMSO-d6) δ = 8.74 (s, 1H), 8.43 (br s,1H),8.25(dd,J=1.1,8.8Hz,1H),8.17(dd,J=1.9,7.4Hz,1H),8.12(d,J=8.7Hz,1H ),8.01-7.92(m,1H),7.74-7.60(m,2H),4.84(t,J=5.1Hz,2H),3.93(t,J=5.1Hz,2H).

[0720] General Procedure J:

[0721] Scheme 46

[0722]

[0723]

[0724] Synthesis of 46-1 3-(2-isopropoxyphenyl)-5-(1-isopropylindol-5-yl)-1,2,4-oxadiazole

[0725] Scheme 47

[0726]

[0727] To a solution of N-hydroxy-2-isopropoxy-benzamidine (90.00 mg, 393.86 umol, 1.00 equiv) in DMF (2.00 mL) was added 1-isopropylindole-5-carboxylic acid (80.05 mg, 393.86 umol, 1.00 equiv), HOBt (63.86 mg, 472.63 umol, 1.20 equiv) and EDCI (90.60 mg, 472.63 umol, 1.20 equiv) and the reaction was stirred for 12 h at 120° C. The mixture was filtered and concentrated. The residue was purified by preparative HPLC to give 3-(2-isopropoxyphenyl)-5-(1-isopropylindol-5-yl)-1,2,4-oxadiazole (26.00 mg, 71.93 umol, 18.26% yield, 98.9% purity) as a yellow oil.

[0728] 1H NMR (400MHz, CHLOROFORM-d) δ = 8.56 (d, J = 1.1Hz, 1H), 8.10 (dt, J = 1.7, 8.6Hz, 2H), 7.53-7.43 (m, 2H), 7.35 (d, J = 3. 3Hz, 1H), 7.14-7.07 (m, 2H), 6.69 (d, J = 3.3Hz, 1H), 4.81-4.65 (m, 2H), 1.60 (d, J = 6.7Hz, 7H), 1.46 (d, J = 6.1Hz, 6H).

[0729] Synthesis of 48-2 5-[3-(2-methoxyphenyl)-1,2,4-oxadiazol-5-yl]-1-(propan-2-yl)-1H-1,2,3-benzotriazole

[0730] Scheme 48

[0731]

[0732] To a solution of 1-isopropylbenzotriazole-5-carboxylic acid (100.00 mg, 487.31 umol, 1.00 equiv) in DMF (1.00 mL) was added HOBt (79.01 mg, 584.77 umol, 1.20 equiv) and EDCI (112.10 mg, 584.77 umol, 1.20 equiv). The mixture was stirred at 10 ° C for 0.5 h, then N-hydroxy-2-methoxy-benzamidine (80.98 mg, 487.31 umol, 1.00 equiv) was added and stirred at 120 ° C for 12 h. The mixture was diluted with H2O (20 mL) and extracted with EA (30 mL x 2), and the combined organic layers were dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by preparative-HPLC (column: Welch Ultimate AQ-C18 150x30mmx5um; mobile phase: [water (0.1% TFA)-ACN]; B%: 50%-80% <13min) to give 5-(1-isopropylbenzotriazol-5-yl)-3-(2-methoxyphenyl)-1,2,4-oxadiazole (110.00 mg, 328.01 umol, 67.31% yield) as a white solid.

[0733] 1H NMR (400MHz, CHLOROFORM-d) δ = 8.99 (s, 1H), 8.36 (dd, J = 1.2, 8.7Hz, 1H), 8.17 (dd, J = 1.6, 7.7Hz, 1H), 7.74 (d, J =8.7Hz,1H),7.59-7.48(m,1H),7.20-7.05(m,2H),5.16(spt,J=6.8Hz,1H),4.03(s,3H),1.81(d,J=6.8Hz,6H).

[0734] General Procedure K:

[0735] Scheme 49

[0736]

[0737]

[0738] Synthesis 49-1: 1-(Propan-2-yl)-1H-1,2,3-benzotriazole-5-carbothioamide

[0739] Scheme 50

[0740]

[0741] To a solution of 1-isopropylbenzotriazole-5-carbonitrile (2 g, 10.74 mmol, 1 eq.) in HCl / dioxane (100 mL, 4 M) was added thioacetamide (1.61 g, 21.48 mmol, 2 eq.) and stirred for 2 h at 110° C. The reaction mixture was concentrated under reduced pressure and purified by flash silica gel chromatography (PE / EA=2 / 1 to 1 / 1) to give 1-isopropylbenzotriazole-5-thiocarboamide (2.1 g, 9.53 mmol, 88.76% yield) as a yellow solid.

[0742] Synthesis of 51-25-{5-[2-(methyl-λ 3 -oxy)phenyl]-1,3-thiazol-2-yl}-1-(propan-2-yl)-1H-1,2,3-benzotriazole

[0743] Scheme 51

[0744]

[0745] To a solution of 1-isopropylbenzotriazole-5-carbothioamide (300 mg, 1.36 mmol, 1 eq.) in DMF (3 mL) was added CsCO (443.71 mg, 1.36 mmol, 1.00 eq.) and 2-bromo-1-(2-methoxyphenyl)ethanone (311.95 mg, 1.36 mmol, 1 eq.) The mixture was stirred at 100 °C for 12 h. The residue was purified by preparative-HPLC (column: Waters Xbridge 150x25 5u; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 60%-90%, 10 min) to give 2-(1-isopropylbenzotriazol-5-yl)-5-(2-methoxyphenyl)thiazole (190 mg, 509.65 umol, 37.42% yield, 94% purity) as a yellow solid.

[0746] 1 H NMR(400MHz, CHLOROFORM-d)δ=8.60(s,1H),8.37(dd,J=1.7,7.8Hz,1H),8.22(dd,J=1.1,8.8Hz,1H),7.91(s,1H),7.55(d,J=8.8Hz ,1H),7.31-7.23(m,1H),7.05(t,J=7.5Hz,1H),6.96(d,J=8.3Hz,1H),5.04(spt,J=6.8Hz,1H),3.92(s,3H),1.70(d,J=6.7Hz,6H).

[0747] General Procedure L:

[0748] Scheme 52

[0749]

[0750]

[0751] Synthesis of 52-1: 1-(Propan-2-yl)-1H-1,2,3-benzotriazole-5-carboxamide:

[0752] To a solution of 46-1 (1.0 g, 5.4 mmol) in HOAc (10 mL) was added H2SO4 (0.5 mL) and the reaction was heated at 120 ° C with MW for 90 min. Allow to cool overnight. The reaction mixture was poured onto ice, neutralized and extracted with EtOAc. The solvent was evaporated to obtain a dark solid. Silica gel chromatography (10-50% acetone in hexane) obtained a residue, which was ground with a small amount of acetone, and the off-white solid was collected by filtration to obtain the title compound (52-1) (0.5 g, 45%).

[0753] Synthesis of 52-3: 5-(4-phenyl-1,3-oxazol-2-yl)-1-(propane-2-yl)-1H-1,2,3-benzotriazole: To a mixture of 52-1 (50 mg, 0.25 mmol), 52-2 (51 mg, 0.25 mmol) was added AgSbF6 (86 0.25 mmol), and the mixture was heated to 90°C for about 3 hours and then cooled to room temperature. The reaction was carried out with NaHCO3 and CH2Cl2. The organic layer was separated and evaporated to give a dark oil. The residue was chromatographed (0 to 5% MeOH in CH2Cl2) to give a residue, which was further purified by reverse phase HPLC. The appropriate fractions were combined and lyophilized to give the title compound as an off-white solid. (7 mg, 10%).

[0754] General Program M

[0755] Scheme 53

[0756]

[0757] Synthesis of 5-(2-methoxyphenyl)-1,3,4-thiadiazole-2-amine 53--2: DMF (0.2 mL) was added to a solution of 53-1 (1.9 g, 10.0 mmol) in CH2Cl2 (50 mL), followed by addition of oxalyl chloride (1.7 mL, 20.0 mmol) in batches, and the solution was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo. Semicarbazide (1.1 g, 15 mmol) was added to the residue, followed by addition of POCl3 (2.8 mL, 30 mmol) and the reaction mixture was heated to 90 ° C. After about 45 min to 1 hour, the heating was turned off and allowed to cool overnight. It was quenched with ice and treated with K2CO3 and EtOAc. The organic layer was washed with saturated NaHCO3 and dried over Na2SO4. It was filtered and evaporated to give a yellow residue, which was ground with CH2Cl2 and the title compound was collected as a beige solid (0.9 g, 43%). The material was used directly in the next step.

[0758] Synthesis of 2-bromo-5-(2-methoxyphenyl)-1,3,4-thiadiazole 53-3: A mixture of tert-butyl nitrite (0.9 mL, 9.6 mmol) and CuBr2 (2.2 g, 9.6 mmol) in MeCN (30 mL) was stirred for 10 min, and then 53-2 (0.9 g, 1.76 mmol) was added in 2 portions. Stir for ~1 hour and then remove the solvent in vacuo. The residue was suspended in EtOAc, washed twice with 1N HCl, then washed with brine and dried over Na2SO4. Filtered and evaporated to give the title compound as a yellow-orange solid (1 g, 86%).

[0759] Synthesis of 5-[5-(2-methoxyphenyl)-1,3,4-thiadiazol-2-yl]-1-(propane-2-yl)-1H-1,2,3-benzotriazole 53--4: 52-3 (95 mg, 0.25 mmol), 14-5 (45 mg, 0.3 mmol), K3PO4 (132 mg, 0.625 mmol) and Pd(Ph3P)4 (58 mg, 0.05 mmol) were combined in a mixture of DMF (4 mL) and water (1 mL). Heat to 120 °C in MW for 30 min. The sol was evaporated to give a residue, which was chromatographed on silica (0 to 30% EtOAc in hexanes). The appropriate fractions were combined and the solvent was evaporated. The residue was further purified by reverse phase HPLC to give the title compound (30 mg, 20%) as an off-white solid.

[0760] Synthesis of 2-{3-[1-(propane-2-yl)-1H-1,2,3-benzotriazol-5-yl]-1,2,4-thiadiazol-5-yl}phenol 53-5: 53--4 (40 mg, 0.11 mmol) was dissolved in HR and heated to 120°C. After ~48 hours, the starting material disappeared. Neutralized with NaHCO3. The residue was purified by reverse phase HPLC to give the title compound (10 mg, 22%). [M+H] + :338.0

[0761] General Program N

[0762] Scheme 54

[0763]

[0764] Synthesis 54-3: 4-amino-3-(3-ethyl-3-hydroxypent-1-yn-1-yl)benzonitrile

[0765] Compound 54-2 (683.15 mg, 6.09 mmol, 783.43 uL, 1.2 equiv.), CuI (48.33 mg, 253.77 umol, 0.05 equiv.) and Pd(PPh3)2Cl2 (178.12 mg, 253.77 umol, 0.05 equiv.) were added to a solution of compound 54-1 (1 g, 5.08 mmol, 1 equiv.) in DMF (7 mL) and TEA (2.18 g, 21.55 mmol, 3 mL, 4.25 equiv.). The mixture was stirred at 90 ° C for 3 hours under a nitrogen atmosphere. TLC showed that a new spot was formed. The reaction mixture was diluted with water (20 mL), extracted with EtOAc (20 mLx2), washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by flash silica gel column chromatography (PE:EA=5:1) to give compound 3 (1 g, yield: 86%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ = 7.52 (d, J = 1.6Hz, 1H), 7.34 (dd, J = 8.6, 1.8Hz, 1H), 6.68 (d, J = 8.4Hz, 1H) ,4.76(s,2H),2.95(s,1H),2.88(s,1H),2.32(s,1H),1.85-1.70(m,4H),1.10(t,J=7.4Hz,6H).

[0766] Synthesis of 54-4: 2,2-Diethyl-4-oxydeoxy-1,3-dihydroquinoline-6-carboxylic acid

[0767] A mixture of compound 54-3 (300 mg, 1.31 mmol) in concentrated hydrochloric acid solution (1 mL) and acetic acid (1 mL) was stirred at 115° C. for 3 hours. The mixture was basified to pH=10 with 1N sodium hydroxide solution, washed with EtOAc (20 mL×3), the aqueous phase was acidified to pH=3 with 1N hydrochloric acid solution, filtered, the filter cake was washed with water (10 mL), and dried under vacuum to obtain compound 54-4 (35 mg, yield: 11%) as a white solid.

[0768] 1 H NMR (400MHz, DMSO-d6) δ=12.08(br.s,1H),7.90(d,J=2.0Hz,1H),7.52(dd,J=2.0,8.7Hz,1H ), 7.14 (s, 1H), 6.58 (d, J = 8.7Hz, 1H), 1.73 (s, 1H), 1.36-1.19 (m, 4H), 0.58 (t, J = 7.4Hz, 6H).

[0769] Synthesis of 55-1: 2,2-diethyl-6-[3-(thiophen-3-yl)-1,2,4-oxadiazol-5-yl]-1,3-dihydroquinolin-4-one

[0770] Scheme 55

[0771]

[0772] To a solution of compound 54-4 (100 mg, 404.39 umol, 1 eq.) in DMF (1 mL) was added HOBt (65.57 mg, 485.26 umol, 1.2 eq.) and EDCI (93.02 mg, 485.26 umol, 1.2 eq.). After stirring at 20 °C for 30 min, compound 55-2 (63.24 mg, 444.82 umol, 1.1 eq.) was added and stirred for another 30 min. The mixture was then heated to 120 °C and stirred for 2 hours. The mixture was triturated with EA (20 mL), filtered, washed with EA (10 mL), and the residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150x25x10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 50%-80%, 13 min) to give 55-1 (45 mg, yield: 29%) as an orange solid. 1 HNMR (400MHz, DMSO-d6) δ=8.36-8.27(m,2H),7.96(dd,J=2.0,8.8Hz,1H),7.79(dd,J=3.0,5.0Hz,1H),7.71 (s, 1H), 7.62 (d, J = 5.0Hz, 1H), 6.99 (d, J = 8.9Hz, 1H), 2.59 (s, 2H), 1.67-1.48 (m, 4H), 0.86 (t, J = 7.3Hz, 6H).

[0773] General ProgramO

[0774] Scheme 56

[0775]

[0776]

[0777] Synthesis of 57-3: 5-(4-methoxy-1,3-benzooxazol-2-yl)-1-(2-methylpropyl)-1,2,3-benzotriazole

[0778] Scheme 57

[0779]

[0780] Synthesis 57-2: N-(2-hydroxy-6-methoxyphenyl)-1-(2-methylpropyl)-1,2,3-benzotriazole-5-carboxamide

[0781] To a solution of compound 57-1 (100 mg, 487.30 umol, 1 eq.) in DMF (3 mL), HOBt (65.85 mg, 487.30 umol, 1.2 eq.) and EDCI (112.10 mg, 584.76 umol, 1.2 eq.) were added. After addition, the mixture was stirred at 20 ° C for 0.5 hours, and then compound 57-4 (81.37 mg, 584.76 umol, 1.2 eq.) was added, and the mixture was stirred for another 12 hours at 20 ° C. LCMS showed that compound 57-1 had been consumed, and a main peak with the desired Ms was detected. The mixture was diluted with water (20 mL), extracted with EtOAc (15 mL * 2), washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated to give compound 57-2 (159 mg, crude product), which was a brown oil that was directly used in the next step without further purification. LCMS: 327.2[M+1]

[0782] Synthesis of 57-3: 5-(4-methoxy-1,3-benzooxazol-2-yl)-1-(2-methylpropyl)-1,2,3-benzotriazole

[0783] To a solution of compound 57-2 (159 mg, crude) in xylene (10 mL) was added TsOH.HO (370.70 mg, 1.95 mmol, 4 equiv). After addition, the mixture was stirred at 120 ° C for 2 hours. LCMS showed that compound 57-2 had been consumed and a main peak with the desired Ms was detected. The mixture was concentrated, diluted with saturated sodium bicarbonate solution (20 mL), extracted with EtOAc (20 mL * 2), dried over sodium sulfate, filtered and concentrated. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150 * 25 * 10 um; mobile phase: [water (0.1% TFA) -ACN]; B%: 42% -72%, 10 min) to obtain 57-3 (53 mg, yield: 35%) as a gray solid.

[0784] 1H NMR (400MHz, CDCl3) δ = 8.95 (s, 1H), 8.51 (dd, J = 1.1, 8.8Hz, 1H), 7.69 (d, J = 8.8Hz, 1H), 7.37-7.31 (m, 1 H),7.28-7.27(m,1H),6.85(d,J=7.9Hz,1H),5.18-5.08(m,1H),4.10(s,3H),1.80(s,3H),1.78(s,3H)

[0785] Synthesis of 58-3: 1-isopropyl-5-(7-methoxy-1,3-benzoxazol-2-yl)-1,2,3-benzotriazole

[0786] Scheme 58

[0787]

[0788] Synthesis 58-2: N-(2-bromo-3-methoxyphenyl)-1-isopropyl-1,2,3-benzotriazole-5-carboxamide

[0789] A mixture of compound 58-1 (200 mg, 989.86 umol, 1 equivalent), compound 58-1 (243.76 mg, 1.19 mmol, 1.2 equivalents) and EDCI (284.64 mg, 1.48 mmol, 1.5 equivalents) in pyridine (3 mL) was stirred at 20 ° C for 12 hours. The mixture was diluted with EtOAc (30 mL), washed with 1N hydrochloric acid solution (20 mL * 3), dried with sodium sulfate, filtered and concentrated. The residue was purified by preparative TLC (PE: EA = 3: 1) to give compound 58-2 (150 mg, yield: 38%) as a brown oil. LCMS: 391.1 [M + 1]

[0790] Synthesis of 58-3: 1-isopropyl-5-(7-methoxy-1,3-benzoxazol-2-yl)-1,2,3-benzotriazole

[0791] A mixture of compound 58-2 (50 mg, 128.45 umol, 1 eq.), 1,10-phenanthroline (2.31 mg, 12.85 umol, 0.1 eq.), Cs2CO3 (62.78 mg, 192.68 umol, 1.5 eq.) and CuI (1.22 mg, 6.42 umol, 0.05 eq.) in DME (2 mL) was heated to 85 ° C. and stirred for 12 hours under a nitrogen atmosphere. The mixture was diluted with EtOAc (20 mL), washed with water (10 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 45%-75%, 12 min) to give 58-3 (8 mg, yield: 19%) as a gray solid.

[0792] 1 H NMR (400MHz, DMSO-d6) δ=8.76(s,1H),8.32(dd,J=1.3,8.8Hz,1H),8.13(d,J=8.7Hz,1H),7.44- 7.28 (m, 2H), 7.07 (d, J = 7.8Hz, 1H), 5.29 (spt, J = 6.7Hz, 1H), 4.02 (s, 3H), 1.66 (d, J = 6.6Hz, 6H).

[0793] Synthesis of 59-3: 1-isopropyl-5-(6-methoxy-4-methyl-1,3-benzoxazol-2-yl)-1,2,3-benzotriazole

[0794] Scheme 59

[0795]

[0796] Synthesis of 59-3: 1-isopropyl-5-(6-methoxy-4-methyl-1,3-benzoxazol-2-yl)-1,2,3-benzotriazole

[0797] To a solution of compound 59-2 (1-isopropyl-N-(4-methoxy-2-methylphenyl)-1,2,3-benzotriazole-5-carboxamide) (50 mg, 154.14 umol, 1 equiv.) prepared according to the procedure for preparing 58-2 in o-xylene (2 mL) was added Cu(OTf)2 (11.12 mg, 30.83 umol, 0.2 equiv.). The reaction was stirred at 130 ° C. for 16 hours under an oxygen atmosphere. The mixture was diluted with water (20 mL), extracted with EtOAc (30 mL * 3), dried with sodium sulfate, filtered and concentrated. The residue was purified by preparative TLC (PE: EA = 4: 1) to give 59-3 (1.1 mg, yield: 2%) as a yellow solid.

[0798] 1 H NMR (400MHz, CDCl3) δ = 8.80 (s, 1H), 8.33 (dd, J = 1.4, 8.7Hz, 1H), 7.59 (d, J = 8.8Hz, 1H), 6.91 (d, J = 2 .2Hz,1H),6.72(d,J=1.5Hz,1H),5.16-4.93(m,1H),3.81(s,3H),2.58(s,3H),1.72(d,J=6.8Hz,6H)

[0799] Example 2: In vitro activity of compounds

[0800] Stable Cloning of Chinese Hamster Ovary K1 (CHO-K1) Cells Co-expressing EA-β-Arrestin 2 and Human Sphingosine-1-Phosphate Receptor 1 (NM_001400, S1P1) with C-terminal Prolink TM The tags were purchased from DiscoverX (Cat#: 93-0207C2).

[0801] Cell culture and assay plating

[0802] Cell lines were placed in AssayComplete TM Culture medium 6 (DiscoverX, Cat#: 920018GF2) was cultured at 37°C and 5% CO2 in a humidified CO2 and temperature-controlled incubator. To start the assay plating, the cells were washed with Dulbecco's phosphate buffered saline (CellGro, Cat#: 21-031-CV) and lifted from the culture flask by incubation with CellStripper (Cellgro, Cat#: 25-056-CI) (37°C, 5 min). The lifted cells were placed in AssayComplete TMResuspended to 250,000 cells / ml in Cell Plating11 reagent (DiscoverX, Cat#:93-0563R11B) and plated at 5,000 cells / well in white-opaque 384-well plates (Greiner Bio-One Item#:20--784080). The plated cells were incubated overnight at 37°C and 5% CO2 in a humidified CO2 and temperature-controlled incubator.

[0803] Detection of inhibition of cAMP production

[0804] By using The cAMP HiRange Kit (CisBio, Cat#: 62AM6PEJ) measures changes in intracellular cAMP based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology to determine agonist-promoted G protein responses. TM Cell Plating 11 reagent was removed and replaced with Ham's F-12 (CellGro, Cat#: 10-080-Cm) containing the desired concentrations of isobutyl-methyl-xanthine (IBMX; 500 μM; Tocris Bioscience, Cat#: 2845) and NKH-477 (1.5 μM; Tocris Bioscience, Cat#: 1603) together with the test or reference compound. After incubation for 30 minutes at 37°C and 5% CO2 in a humidified CO2 and temperature-controlled incubator, the components of the cAMP HiRange kit were added according to the manufacturer's instructions. After incubation for one hour at room temperature, the plate was analyzed using a BMG PheraStar microplate reader. The reaction was measured as the ratio of the fluorescence emission at 665 nm to the fluorescence emission at 620 nm.

[0805] β-Arrestin 2 recruitment assay

[0806] Agonist-promoted recruitment of β-arrestin 2 to sphingosine-1-phosphate 1 receptor is activated using β-arrestin Pathway In this system, β-arrestin 2 is fused to an N-terminal deletion mutant of β-galactosidase (called enzyme acceptor or EA), while the C-terminus of the target GPCR is fused to a smaller (42 amino acids) weakly complementary fragment 15 (called ProLink TM In cells stably expressing these fusion proteins, stimulation with cognate agonists resulted in the expression of β-arrestin2 and Prolink TM-labeled GPCR. This allows the two β-galactosidase fragments to complement and result in the formation of a functional enzyme with β-galactosidase activity. TM Cell Plating 11 reagent was removed and replaced with Ham's F-12 containing the desired concentrations of IBMX (500 μM) and NKH-477 (1.5 μM) along with the test or reference compound. After incubation for 60 min at 37°C and 5% CO2 in a humidified CO2 and temperature-controlled incubator, β-arrestin Path After one hour incubation at room temperature, the plates were analyzed by a BMG PheraStar microplate reader.

[0807] Active Table

[0808] Compounds as described herein are capable of modulating the activity of sphingosine-1-phosphate 1 receptors as described herein (inhibiting cAMP production and beta-inhibitory protein 2 recruitment). The following table includes the efficacy of compounds normalized to the maximum efficacy of the reference compound (referred to as "SPAN"). These values ​​are normalized to fingolimod, a known agonist of sphingosine-1-phosphate 1 receptors. The table also includes efficacy values ​​(pEC50) for modulating discrete receptor-mediated activity (inhibiting cAMP production and beta-inhibitory protein 2 recruitment). This value represents an estimated concentration to promote half of the maximum efficacy (or SPAN) observed for each compound.

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827] Example 3: In the 6 Hz 44 mA psychomotor seizure model mouse, the expected compound effectively protects the animal from induced seizures

[0828] The efficacy of the compound was tested in a 6Hz 44mA psychomotor convulsion model mouse. In the 6Hz model, the 6Hz44mA model evaluates the ability of the compound to prevent convulsions induced by 6Hz corneal stimulation at a current intensity of 44mA. These convulsions are considered to be partial convulsion models observed in humans. The 6Hz test uses the same method described in the MES test. A 44mA current (2 times that of CC97) is delivered through corneal electrodes to attack mice for 3sec to induce psychomotor convulsions (1). Typically, convulsions are characterized by an initial short-term vertigo, followed by immediate jaw clonus, forelimb clonus, vibrissa twitching, and Straub tail lifting for at least 1 second. Animals that do not exhibit this behavior are considered to be "protected".

[0829] The initial qualitative screening of anticonvulsant activity in the 6Hz convulsion model was conducted using N=4 male CF-1 mice / dose / time point. The default dose and time point were 0.1, 1, 10 mg / kg sc at 0.5 and 2 hours after administration. If other test data support, the dose and / or time point are adjusted. The quantification of ED50 is carried out at the time of peak effect (TPE). In order to determine TPE, mice are treated with research compounds at 0.25, 0.5, 1.0, 2.0 and 4.0 hours or based on the time point of previous studies. Groups of N=8 mice are tested with different doses of research compounds until at least two points (i.e., at least 4 test doses) can be clearly established between the limits of 100% protection or toxicity and 0% protection. The data for each condition are presented as N / F, wherein N=the number of protected animals, and F=the number of tested animals. ED50, 95% confidence interval, the slope of the regression line and the SEM of the slope are calculated by probability analysis.

[0830] Example 4: In both the subcutaneous Metrazol seizure model and the intravenous Metrazol seizure threshold model in mice, the expected compounds are effective in protecting animals from induced seizures.

[0831] The compounds were tested for efficacy in the subcutaneous cardiazol seizure model and the intravenous cardiazol seizure threshold model in mice.

[0832] Subcutaneous Cardiac Seizure Threshold Test (scMET)

[0833] The scMET test detects the ability of a test compound to increase the chemical convulsion-induced seizure threshold of an animal and thereby protect it from exhibiting clonic forebrain seizures. Cardiazol is injected at a dose of 85 mg / kg for mice and 56.4 mg / kg for rats into a relaxed skinfold on the midline of the neck. The animals are placed in an isolation cage to minimize stress and observed for the presence or absence of seizures over the next 30 min. The onset of clonic spasms of the forelimbs and / or hindlimbs, jaws or tentacles for approximately 3 to 5 seconds is taken as the endpoint. Animals that do not exhibit forelimb and / or hindlimb clonus, jaw chewing or tentacles twitching are considered protected.

[0834] The initial qualitative screening of anticonvulsant activity in the scMET test was performed using N = 4 male CF-1 mice / dose / time point. The default dose and time point were 1, 2.5, 5, 7.5, 10 mg / kg sc at 0.5 and 2 hr after administration. If other test data support, the dose and / or time point are adjusted. Quantification of ED50 is performed at the time of peak effect (TPE). To determine TPE, mice are treated with the study compound at 0.25, 0.5, 1.0, 2.0 and 4.0 hours or based on the time point of previous studies. Groups of N = 8 mice were tested with different doses of the study compound until at least two points (i.e., at least 4 test doses) can be clearly established between the limits of 100% protection or toxicity and 0% protection. The data for each condition are presented as N / F, where N = the number of protected animals and F = the number of tested animals. ED50, 95% confidence interval, slope of the regression line and SEM of the slope were calculated by probability analysis.

[0835] Intravenous cardiazol seizure threshold test (ivMET)

[0836] This test is not routinely performed, but it is used to evaluate the effect of research compounds on MET. That is, whether the compound increases or decreases the threshold of convulsions that can be induced by MET. Timed intravenous infusion of MET into mice is used as a chemical convulsion test to distinguish those compounds that lower the convulsion threshold and may be proconvulsant in themselves from those compounds that increase the convulsion threshold and are therefore anticonvulsants. Male CF1 mice (n=10, per dose level) were injected every 2 minutes with vehicle or one of the two doses (ED50 and TD50; ip) of the test compound previously determined to be effective in screening convulsion tests and motor impairment assessments. During dosing, animals maintained the same dosing order until all mice were injected according to the method. Under the previously determined TPE, a No.20Pe tube was then inserted into the mouse lateral tail vein to infuse a 0.5% MET solution at a constant rate of 0.34 ml / min. At the start of the MET infusion, the hemostat clamped on the catheter was removed to prevent backflow, the infusion began, and two stopwatches started. The time from the start of infusion to the onset of the "first twitch" and sustained clonus was recorded in seconds, or if this time passed without convulsion, it was recorded as 90 seconds. The mean and SEM were calculated for each of the 3 groups (vehicle, ED50 and TD50), and significant differences between the test group and the control were calculated. An increase in the first twitch or clonus (in mg / kg) indicates that the test substance increases the convulsion threshold; while a decrease indicates that the test substance lowers the convulsion threshold, or may be proconvulsive.

[0837] Example 5: In the corneal kindling seizure model mouse, the expected compound effectively protects the animal from induced seizures

[0838] The efficacy of the compound was tested in the corneal kindling convulsion model (CKM) mice. The pharmacological profile of the corneal kindling mice is consistent with partial epilepsy in humans, and effectively identifies the anticonvulsant potential of compounds useful for this condition (such as levetiracetam). Male C57BL / 6 mice were electrically ignited with 3-second stimulation, 3mA, 60Hz and corneal electrodes to the criteria of 5 consecutive stage 5 convulsions (facial clonus and head nodding progressed to forelimb clonus, and finally rearing and falling with hind legs, accompanied by generalized clonic convulsions). After receiving corneal stimulation twice a day, mice usually reached the first stage 5 convulsion between about 10-14 days. Each mouse continued to stimulate twice a day until the mouse had reached the criteria of 5 consecutive stage 5 convulsions, so that it was considered "fully kindled". The fully kindled mice were then stimulated every other day until all other mice in the group reached the criteria of 5 consecutive stage 5 convulsions. Any mice that did not achieve a fully kindled state were not included in any evaluation of the study compound.

[0839] Testing of study compounds begins at least 5-7 days after the last stimulation. Mice are stimulated one day prior to any drug evaluation to ensure that all mice used for drug studies will present stage 5 seizures. For qualification studies, test compounds are dosed at 3 and 5 mg / kg sc to groups of 8 fully kindled mice per group and tested at time points of 0.5 and 1 hour post-dose. Based on the experimental results, compounds are then retested to determine ED50 values. Following testing, corneal kindled animals are returned to their home cages. Unlike acute seizure testing performed by ETSP, each corneal kindled mouse is allowed at least 3-4 days between testing to "wash out" any study compound following testing.

[0840] Example 6: Compound 469 can inhibit convulsions

[0841] Compound 469 was tested in the method described in Example 5. The data for Compound 469 are shown in Tables 1-4. Compound 469 was found to have an ED of 6.2 mg / kg at the 2 hour time point in the corneal kindled seizure model. 50 .

[0842] Table 1. Experiment 1: Seizure scores at 0.5 hour and 1.0 hour time points for 3 and 5 mg / kg of Compound 469 in the corneal kindled seizure model.

[0843]

[0844] N: Number of animals that are protected or poisonous.

[0845] F: Total number of animals tested.

[0846] Table 2. Experiment 2: Seizure scores at 2 hours at different doses of Compound 469 in the corneal kindled seizure model.

[0847]

[0848] N: Number of animals that are protected or poisonous.

[0849] F: Total number of animals tested.

[0850] Table 3. ED50 of Compound 469 at 2 hour time point in the corneal kindled seizure model.

[0851]

[0852] Table 4. Seizure scores at 1 hour and 2.0 hour time points for 10 mg / kg of Compound 469 in the corneal kindled seizure model.

[0853]

[0854] These data demonstrate that compounds provided herein, such as Compound 469, can reduce convulsions and, therefore, can be used to treat convulsive disorders, such as those provided herein.

[0855] Example 7: In the lamotrigine (LTG)-resistant amygdala kindling seizure model rats, the expected compound effectively protects the animals from induced seizures

[0856] The efficacy of the compounds was tested in the lamotrigine (LTG)-resistant amygdala kindled seizure model rat. The LTG-resistant amygdala kindled rat model is not only used to identify compounds that are effective against secondary generalized partial seizures, but also allows for the differentiation of compounds that may be effective for therapy-resistant patients. Daily administration of lamotrigine (LTG; 5 mg / kg) during the kindling acquisition phase did not prevent the test animals from developing kindling, but resulted in a state of LTG resistance in fully kindled rats. Other sodium channel blockers, such as phenytoin and carbamazepine, also did not block kindling acquisition, although they were highly effective in fully kindled seizures in drug-naive rats. In contrast, valproate can both prevent kindling development and block the expression of fully kindled seizures. During the development of kindled seizures in this model, the addition of traditional ASD, carbamazepine or lamotrigine will ultimately impair the effectiveness of lamotrigine against fully expressed kindled seizures. These findings suggest that the presence of lamotrigine during the epileptogenic process leads to subsequent resistance to other Na+ channel blockers, making it a useful model of drug resistance. This model can serve as a means of identifying compounds that may be effective for therapy-resistant convulsions. Anesthetized male Sprague-Dawley rats (250-300g) were surgically implanted with electrodes in the left amygdala. The animals were then allowed to recover for one week before starting to kindle. The kindling procedure consisted of daily delivery of 200 μAmp stimulation (superthreshold) until all animals in the two treatment groups showed consistent 4th or 5th stage convulsions. One week after all animals were kindled, the animals received a challenge dose of LTG (30 mg / kg, ip) and then stimulated to confirm the LTG sensitivity of the control animals treated with the vehicle and the LTG resistance of the group treated with LTG. The animals were then allowed to clear for 3 days. On the 3rd day of clearing, the animals were pre-stimulated to ensure complete recovery of ignited convulsions. On the 4th day, the kindled rats were attacked with a dose of the study agent (a dose that produces minimal motor impairment), and then the rats were attacked with kindling stimulation under the predetermined TPE of the study drug. When it was observed that drug treatment significantly reduced convulsion scores and reduced afterdischarge, a dose-response study could be performed. For this study, the ability of candidate substances to reduce after-discharge duration (ADD) and behavioral seizure scores (BSS) was quantified by varying the dose between 0 and 100% effect. Unlike other acute seizure tests performed by the ETSP, each kindled rat was allowed at least 3-4 days between tests to "wash out" any study compounds after testing. The mean seizure score ± SEM and after-discharge duration (ADD) were recorded, as were the number of animals protected from seizures (defined as Racine score <3) compared to the number of animals tested.

[0857] The following procedure was used for surgical implantation of electrodes. Male Spraque-Dawey rats were anesthetized using either 1-3% isoflurane or a ketamine / xylazine mixture. Ketamine was provided as a 100 mg / ml solution and a dose of 50 mg / kg was given ip. Xylazine was provided as a 100 mg / ml solution and a dose of 20 mg / kg was given ip. The two solutions were mixed together in one syringe and given ip as a single injection. The rats were monitored surgically. If the rats reacted to tail clamping, half the dose of ketamine / xylazine or the isoflurane percentage was increased. Buprenorphine at 0.02 mg / kg was administered sc for pain management. An electric razor was used to shave the head surface. The rats were placed on an external heat source on a stereotaxic apparatus so that the height of the incisor bar and the positioning of the ear bars ensured consistency with the reference atlas. The entire scalp was rubbed with betadine (3x) and wiped with alcohol. Ophthalmic ointment was applied to each eye. Before starting surgery, all instruments are sterilized via autoclave. During surgery, the instruments are placed on sterile sheets. For subsequent surgery on the same day, the instruments can be placed in a hot bead sterilizer between surgeries. Stainless steel screws and electrodes are sterilized in 70% ethanol and placed on sterile sheets. Sterile scalp blades are used to start from a point flush with the eyes and extend back to the imaginary line connecting the ears to make a midline scalp incision. The fascia should be gently separated and pulled away from the scalp. Retractors / forceps can be used to keep the incision open. Drill screw holes without penetrating the dura mater using a Dermal drill, and four anchor screws are connected to the skull. Bipolar stimulating electrodes are implanted through the fourth hole drilled in the left amygdala (front-back, AP, +5.7mm, middle-side, ML, +4.5mm, dorsal-ventral, DV, +2.0mm from zero point in the ear). The electrode assembly is anchored to the skull via stainless steel screws with dental acrylic cementum. After the incision was closed with sutures, antibiotic ointment was applied around the incision site. Rats were given a 60,000 unit dose of penicillin and injectable 0.03 mg / kg of rimidyl sc. Rats were left on a heat source until ambulatory.

[0858] Example 8: In a preliminary study of spontaneous recurrent seizures induced by status epilepticus measured by video-EEG monitoring, in rats, the expected compounds were effective in protecting the animals from induced seizures.

[0859] The efficacy of the compounds was tested in a preliminary study in rats on spontaneous recurrent seizures induced by status epilepticus measured with video-EEG monitoring.

[0860] Chronic epilepsy triggers:

[0861] 48 Sprague-Dawley rats were induced with status epilepticus using a repeated low-dose kainic acid (KA) paradigm. 7.5 mg / kg of KA was injected intraperitoneally (ip) to the rats at 0 h, 1 h, and every subsequent half hour (up to 4 h) or until the animal had two Racine stage 5 convulsions. The dose can be reduced to 1 / 2 or 1 / 3 if necessary because the animal exhibited lower stage convulsions. At the end of the injection period, the rats were given 3 ml of Ringers solution to prevent dehydration. It is expected that approximately 36 rats will survive this treatment.

[0862] Telemeter Implantation 10 Weeks Later Rats are implanted with a Millar wireless telemeter. The telemeter is implanted in the peritoneal space. The EEG cable is routed from the stomach to the head under the skin. Three holes are drilled in the skull and three fixation screws are placed. Two additional holes are drilled and the EEG wire is placed in each. The wire is secured via super glue. The skull is sutured and the excess wire is then coiled in the peritoneal space and the stomach is sutured. The rats are placed in the EEG suite and an initial seizure rate is obtained within one week. The 24 rats with the highest seizure rate will be selected. Rats with a daily seizure burden score (the seizure burden is calculated as the sum of all seizure scores divided by the number of days tested) below 10 are removed from future studies.

[0863] Phase 1 Chronic Monitoring: In this paradigm, rats are injected (ip), subcutaneously (sc), or orally (po) with vehicle or drug twice or three times daily based on the known pharmacokinetic profile of the drug being tested (if available) or the time to peak effect (TPE) of the drug in a previously evaluated seizure model. (It is expected that the amygdala kindling model of lamotrigine resistance will be used as the primary guide for determining treatment strategies in Phase 1 chronic monitoring studies). In the first week (week 1), baseline seizure rates are established. During the following week (week 2), injections are performed over 5 days, Monday through Friday. Rats are divided into groups of 8-12 (vehicle and drug-treated groups). After treatment is completed in week 2, rats will be monitored during week 3 (washout period). This process (weeks 1-3) will be repeated for up to 5 separate test runs.

[0864] Possible design variations include (1) a crossover paradigm: one group receives drug for the first 5 days (during Week 2) and vehicle for the next 5 days (Week 3). Similarly, the second group receives vehicle first, followed by drug. The rats will then enter a one-week washout period (Week 4). (2) Group sizes can vary and will be updated once the initial power analysis is completed to verify the numbers required to achieve statistical significance. For example, the initial group of 24 rats participating in Phase 1 chronic monitoring can be divided into two (N=12 / group) or three (N=8 / group) treatment groups.

[0865] Data review and analysis

[0866] EEG data were reviewed daily in a blinded fashion. Data channel orders were randomly scrambled and unlabeled. Lists of potential detected events were automatically generated overnight by an automated seizure detection algorithm. Reviewers went through these detected lists in sequential order and scored any detected positive events. Data were accumulated at the end of the paradigm and analyzed via the MATLAB GUI. Factors analyzed included seizure load, frequency, and distribution of Racine scores.

[0867] Example 9: In an oral dosing study in rats of spontaneous recurrent seizures induced by status epilepticus as measured by video-EEG monitoring, the expected compounds were effective in protecting the animals from induced seizures.

[0868] In the oral administration study of spontaneous recurrent convulsions induced by status epilepticus measured by video-EEG monitoring, the efficacy of the compound was tested in rats. This paradigm uses an automatic feeding system to deliver drugs in food on a fixed schedule. The induction of chronic epilepsy in rats and the implantation of telemeters are carried out in the same manner as described in the preliminary study. The animals are divided into two groups and given 45g / kg of drug or control food every day for two weeks. The amount of drug in each gram of food is fixed to make an appropriate dose. The number of "meals", i.e., automatic pellet distribution, will be based on the known pharmacokinetic characteristics of the tested drug (if available) or the time and peak effect (TPE) of the drug in the previously evaluated convulsion model. In the first week (week 1), the baseline convulsion rate is determined. During the following two weeks (weeks 2-3), feeding is given based on the rat group. After the treatment is completed in week 3, the rats will be monitored during week 4 (clearance period). This process (weeks 1-4) will be repeated up to 4 separate test runs.

[0869] Data review and analysis

[0870] EEG data were reviewed daily in a blinded fashion. Data channel orders were randomly scrambled and unlabeled. Lists of potential detected events were automatically generated overnight by an automated seizure detection algorithm. Reviewers went through these detected lists in sequential order and scored any detected positive events. Data were accumulated at the end of the paradigm and analyzed via the MATLAB GUI. Factors analyzed included seizure load, frequency, and distribution of Racine scores.

[0871] Example 10: The expected compounds are effective in protecting animals from induced convulsions in the PTZ (Pentylenetetrazol) test

[0872] The efficacy of the compounds was tested in the PTZ (pentylenetetrazole) test. In the experiment, the test animals (mice; ICR and rats; SD) were administered intraperitoneally (ip) or orally; the experimental animals, male SD rats, were purchased from OrientBio or Narabiotech in South Korea, and 4-5 mice were kept in each cage for 4-5 days. The weight range of the mice used was between 19 and 26 grams, and the weight range of the rats used was between 100 and 130 grams. After the peak time of administration (0.5, 1, 2 and 4 hours), PTZ (pentylenetetrazole) was administered subcutaneously from the beginning of administration, at a concentration that could induce 97% of intermittent convulsions (mice and rats: 90-110 mg / kg·bw, 2μl / g). If no clonic convulsions were observed for at least 3 seconds in the PTZ-administered animals, the test compound was considered to have non-convulsive convulsive activity. The median effective dose (ED50) was determined using 6 animals per concentration (three different concentrations in total) and was calculated by the Litchfield and Wicoxon log-probability method, which is a dose-response relationship.

[0873] Example 11: Compounds are expected to be effective in protecting animals from induced convulsions in the minimal clonic convulsion (6 Hz) test.

[0874] The efficacy of the compound is tested in the minimal clonic seizure (6 Hz) test. Some clinically useful AEDs are ineffective in the standard MES and scPTZ tests, but still have anticonvulsant activity in vivo. In order to identify potential AEDs with this feature, compounds can be tested in the minimal clonic seizure (6 Hz or 'psychomotor') test (Barton et al., 2001). Like the maximum electroshock (EMS) test, the minimal clonic seizure (6 Hz) test is used to evaluate the efficacy of compounds on electrically induced seizures, but a lower frequency (6 Hz) and longer duration of stimulation (3 s) are used.

[0875] The test compound was pre-administered to mice via ip injection. At different times, individual mice (four at each time point) were attacked with enough current delivered by corneal electrodes to cause psychomotor convulsions in 97% of the animals (32mA or 44mA, 3s) (Toman et al., 1952). Untreated mice will show convulsions with the following characteristics: a minimal clonic period, followed by a stereotyped automatic behavior, initially described as a precursor to human patients with partial convulsions. Animals that do not show this behavior are considered to be protected. The test can be quantitatively evaluated by measuring the response of different doses at a determined peak effect time (TPE). (Reference; Barton ME, Klein BD, Wolf HH and White HS (2001) Pharmacological characterization of the 6Hz psychomotor seizure model of partial epilepsy, Epilepsy Res. 47:217-227. / Toman JE, Everett G.M. and Richards RK (1952), The search for new drugs againstepilepsy.Tex.Rep.Biol.Med.10:96-104.).

[0876] Example 12: In the lithium-pilocarpine induced status epilepticus model, the expected compounds are effective in protecting animals from induced convulsions.

[0877] Testing the efficacy of compounds in the lithium-pilocarpine-induced status epilepticus model

[0878] Prevention research

[0879] These studies used male Sprague-Dawley rats weighing 200-230 g (purchased from Orient BioInc., Korea), and 4-5 rats were housed per cage for 4-5 days. The day before status epilepticus (SE), rats received 127 mg / kg lithium chloride (Sigma St. Louis MO., USA) intraperitoneally (ip). Approximately 18-20 hours after this treatment, rats were given 43 mg / kg pilocarpine (Sigma) intraperitoneally. 2 mg / kg methyl-scopolamine (Sigma) was injected ip 30 min before pilocarpine to block the effects of muscarinic agonists on peripheral cholinergic receptors. The test drugs were administered intraperitoneally (ip) in a volume of 2 ul / g body weight. The pharmacological effects of all test materials were evaluated to compare the test group (n=6) with the control group (n=6). The control group was only administered with the vehicle. The peak time was determined by random doses of 0.5, 1, 2, and 4 hours of the test material. The most protected time is defined as the peak time, and the ED50 is determined by other doses of the peak time. The animals are then transferred to an observation cage and observed for 90 minutes. Approximately 95% of the control group elicits convulsive activity. Protection is defined as the complete absence of convulsions of grade 4 to 5 (based on the Racine grade (Racine, 1972)) during the 90-min observation period. The effective dose (i.e., ED50) of the compound required for 50% control protection from convulsions is determined by log probability analysis using the SPSS software program (SPSS Inc.).

[0880] Intervention studies

[0881] These studies used male Sprague-Dawley rats weighing 200-230 g (purchased from Orient BioInc., Korea), and 4-5 rats were housed per cage for 4-5 days. One day before SE, rats received 127 mg / kg lithium chloride (Sigma St.Louis MO.USA) intraperitoneally (ip). About 18-20 hours after this treatment, rats were given 43 mg / kg pilocarpine (Sigma) intraperitoneally. 2 mg / kg methyl-scopolamine (Sigma) was injected ip 30 min before pilocarpine to block the effects of muscarinic agonists on peripheral cholinergic receptors. The effects of test compounds dissolved in 30% polyethylene glycol 400 (Acros Organics, Haier, Belgium) or 20% Tween80 were studied at different times or 30 min after the first motor convulsion or SE attack. The drug was administered intraperitoneally in a volume of 2 ul / g body weight. The pharmacological effects were evaluated to compare the test group with the control group (n=8). The control group was only administered with the vehicle. (Reference: Racine RJ (1972). Modification of seizure activity by electrical stimulation: II Motor seizure. Electroenceph. Clin. Neurophysiol. 32: 281-294.)

[0882] Example 13: Anticipated Compounds for Benzodiazepines Potential pharmacological therapies for resistant status epilepticus.

[0883] For benzodiazepines The efficacy of the compounds was tested in studies of potential pharmacological therapies for resistance to status epilepticus.

[0884] The lithium-pilocarpine model was used to study the effects of test compounds on benzodiazepines Effect of electrographic properties of resistance to SE. Adult rats were implanted for electroencephalographic (EEG) recording and then pretreated with lithium chloride (127 mg / kg, 24 h) and scopolamine bromide (1 mg / kg; 30 min) before administration of pilocarpine (50 mg / kg). Thirty or sixty minutes after the development of the first motor convulsion, the animals received diazepam (10 mg / kg). Ten minutes after diazepam, the experimental group was given the test compound and the control group was given the vehicle. Typically, 8 animals constituted a "trial", of which 2 animals were controls (i.e., vehicle only) and 6 animals received the test compound, although this varied depending on the number of animals that actually experienced SE. In order to have a sufficient number of replications, two (or even more) trials were performed. When necessary, additional control animals were obtained from other temporarily adjacent trials using the same protocol.

[0885] Example 14: Compounds Selective for S1P1 Receptors

[0886] In vitro selectivity assay

[0887] Stable cloned Chinese hamster ovary K1 (CHO-K1) cells co-expressing EA-β-arrestin 2 and human sphingosine-1-phosphate receptor 2 (NM_004230.3, S1P2), human sphingosine-1-phosphate receptor 3 (NM_005226, S1P3), and sphingosine-1-phosphate receptor 5 (NM_001166215.1, S1P5) with C-terminal Prolink TM The tags were purchased from DiscoverX (S1P2: Cat#93-0256C2, S1P3: Cat#93-0217C2, S1P5: Cat#93-0583C2).

[0888] Cell culture and assay plating

[0889] Cell lines were placed in AssayComplete TM Culture medium 6 (DiscoverX, Cat#: 920018GF2) at 37°C and 5% CO2 in a humidified CO2 and temperature-controlled incubator. To start the assay plating, the cells were washed with Dulbecco's phosphate-buffered saline (CellGro, Cat#: 21-031-CV) and lifted from the culture flask by incubation with CellStripper (Cellgro, Cat#: 25-056-CI) (37°C, 5 min). The lifted cells were placed in AssayComplete TMCellPlating 11 reagent (S1P5 cell line) (DiscoverX, Cat#: 93-0563R11B) or AssayComplete TM Resuspended to 250,000 cells / ml in Cell Plating 2 reagent (S1P2 and S1P3 cell lines) (DiscoverX, Cat#: 93-0563R2B) and plated at 5000 cells / well (S1P3 cell line) or 7500 cells / well (S1P2 and S1P5 cell lines) in white-opaque 384-well plates (Greiner Bio-One Item#: 20-784080). The plated cells were incubated overnight at 37° C. and 5% CO2 in a humidified CO2 and temperature-controlled incubator.

[0890] Detection of inhibition of cAMP production

[0891] By using The cAMP HiRange Kit (CisBio, Cat#: 62AM6PEJ) measures changes in intracellular cAMP based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology to determine G protein responses promoted by S1P3 and S1P5 agonists. TM Cell Plating11 reagent was removed and replaced with Ham's F-12 (CellGro, Cat#: 10-080-Cm), which contained the desired concentrations of isobutyl-methyl-xanthine (IBMX; 500 μM; Tocris Bioscience, Cat#: 2845) and NKH-477 (1.5 μM; Tocris Bioscience, Cat#: 1603) together with the test or control compound. After incubation at room temperature for 30 minutes, the components of the cAMP HiRange kit were added according to the manufacturer's instructions. After incubation at room temperature for one hour, the plate was analyzed by a BMG PheraStar microplate reader. The reaction was measured as the ratio of the signal of the fluorescence emission at 665 nm relative to the background to the fluorescence emission at 620 nm.

[0892] Detection of inositol monophosphate production

[0893] The S1P2 agonist-promoted G protein response was determined by measuring changes in intracellular inositol-monophosphate using the IP-one TB kit (CisBio, Cat#: 62IPAPEJ) based on the time-resolved fluorescence resonance energy transfer (TR-FRET) technique. TMCell Plating 2 reagent is removed and replaced with 1x Ip-one stimulation buffer (according to the manufacturer's instructions) at the desired concentration along with the test or control compound. After 60 minutes of incubation in a humidified CO2 and temperature-controlled incubator at 37°C and 5% CO2, the components of the IP-one TB kit are added according to the manufacturer's instructions. After one hour of incubation at room temperature, the plate is analyzed by a BMG PheraStar microplate reader. The reaction is measured as the ratio of the signal of the fluorescence emission at 665nm relative to the background to the fluorescence emission at 620nm.

[0894] Active Table

[0895] As shown herein, these compounds are able to modulate the activity of sphingosine-1-phosphate 2, sphingosine-1-phosphate 3, sphingosine-1-phosphate 5 receptors (inhibit cAMP production or inositol-monophosphate aggregation). The following table includes the efficacy of the compound normalized to the maximum efficacy of the reference compound (called "SPAN"). These values ​​are normalized to fingolimod, an agonist of known sphingosine-1-phosphate 3 and 5 receptors or CYM5520, an agonist of known sphingosine-1-phosphate 2 receptors. The table also includes efficacy values ​​(pEC50) for modulating discrete receptor-mediated activity (inhibit cAMP production or inositol-monophosphate aggregation). This value represents an estimated concentration to promote half of the maximum efficacy (or SPAN) observed for each compound. Exemplary compounds found to be selective are shown below.

[0896]

[0897] Therefore, these compounds were found to be sufficiently selective for S1P1.

[0898] Example 15: Compounds do not inhibit hERG channel activity

[0899] A standard automated Qpatch patch clamp assay has been used and the selective hERG inhibitor E4031 served as a positive control.

[0900] Compound No. <![CDATA[IC 50 (μM)]]> E4031 0.013 26 >10 304 >10 469 >10 497 >10 520 >10 730 >10 738 >10 742 >10 743 >10 744 >10

[0901] Example 16: Compounds do not cause lymphopenia

[0902] The compounds were tested for changes in peripheral blood lymphocytes in C57bl / 6 mice. In acute studies, animals (n=5 / group) were subcutaneously administered a dose of 3 mg / kg of the test compound. Animals were sacrificed at specific time points and 500 μl of whole blood was collected in EDTA (K2) Eppendorf tubes. The blood was stored on ice and immediately shipped overnight to Charles River Laboratories for analysis. CRL ran samples through their WBC / differential panels on an Advia 120 instrument. We received peripheral lymphocyte counts (10 3 Cells / μl). The treatment group means were compared to the vehicle treated group to obtain statistical significance. In the chronic study, animals (n=6-8 / group) were subcutaneously administered the test compound for three to seven days at a dose of at least 5 times the effective dose in the pharmacology / behavior assay. On the last day, the animals were sacrificed 45 minutes to 6 hours after the last dose. Whole blood was collected and analyzed as described in the acute study. No compound showed a statistically significant reduction in peripheral blood lymphocytes in either the acute or chronic studies. Non-limiting exemplary data are provided below.

[0903] Lymphocytopenia – C57BL / 6 mice

[0904]

Claims

1. Use of a compound of the following formula or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing convulsions in a subject, 2. The use according to claim 1, wherein the convulsion is an epileptic convulsion.

3. The use according to claim 2, wherein the epileptic convulsion is a refractory partial epileptic convulsion, a refractory secondary generalized convulsion, a refractory complex partial convulsion or a refractory status epilepticus.

4. Use of a pharmaceutical composition comprising a compound of the formula below or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing convulsions in a subject, 5. The use according to claim 4, wherein the convulsion is an epileptic convulsion.

6. The use according to claim 5, wherein the epileptic convulsion is a refractory partial epileptic convulsion, a refractory secondary generalized convulsion, a refractory complex partial convulsion or a refractory status epilepticus.

7. Use of the compound of the following formula or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating epilepsy in a subject 8. The use according to claim 7, wherein The epilepsy is refractory epilepsy.

9. The use according to claim 8, wherein The intractable epilepsy is partial epilepsy, generalized epilepsy or a syndrome thereof.

10. The use according to claim 9, wherein The partial epilepsy is cortical epilepsy or temporal lobe epilepsy.

11. The use according to claim 10, wherein The cortical epilepsy is frontal lobe epilepsy, parietal lobe epilepsy or occipital lobe epilepsy.

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