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WO2020074403A1 - Appetite-modulating molecules - Google Patents

Appetite-modulating molecules Download PDF

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Publication number
WO2020074403A1
WO2020074403A1 PCT/EP2019/076992 EP2019076992W WO2020074403A1 WO 2020074403 A1 WO2020074403 A1 WO 2020074403A1 EP 2019076992 W EP2019076992 W EP 2019076992W WO 2020074403 A1 WO2020074403 A1 WO 2020074403A1
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WO
WIPO (PCT)
Prior art keywords
methyl
alkyl
compound according
item
compound
Prior art date
Application number
PCT/EP2019/076992
Other languages
French (fr)
Inventor
Florian Engert
Josua JORDI
Original Assignee
Universität Zürich
President And Fellows Of Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universität Zürich, President And Fellows Of Harvard College filed Critical Universität Zürich
Publication of WO2020074403A1 publication Critical patent/WO2020074403A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
    • C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C311/08—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the present invention relates to small molecule compounds capable of modulating appetite in a vertebrate animal, particularly in a mammal.
  • the invention further relates to the use of the compounds of the invention in methods of treatment of eating disorders, and as a food additive for human consumption or animal husbandry.
  • Mainstream drug discovery efforts focus on protein targets using simple in vitro or cellular screens to identify small molecules or biologies as modulators.
  • the quality of the target correlates with success and, consequently, an in-depth molecular characterization underlying a phenotype is a prerequisite.
  • Behaviors such as food intake are not understood at the molecular level, which strongly limits the reductionist approach of target-driven drug discovery.
  • Up to the present target-based approaches are far less successful at identifying first-in-class drugs compared to holistic and unbiased phenotypic strategies, particularly in CNS drug discovery.
  • Furthermore in vivo drug efficacy is a major obstacle, because traditional drug screening methods do not mimic whole organism dynamics e.g. the blood-brain barrier.
  • the objective of the present invention is to provide means and methods to screen for appetite regulators. This objective is attained by the subject-matter of the independent claims of the present specification.
  • the present invention relates to compounds which regulate appetite and their uses.
  • the present invention relates to a compound characterized by a general formula
  • R 4 is an unsubstituted or methyl-substituted 5- or 6-membered aryl or heteroaryl
  • R 5 is an unsubstituted or substituted aryl with the substituent being F, Cl, C1-C3 alkyl, C1-C3 alkyl ether, vinyl or allyl ether;
  • R 6 is COR c or CONHR c with R c being an unsubstituted or C1-C3 alkyl substituted heteroaryl.
  • R 4 is selected from
  • R 5 is selected from
  • the compound is characterized by a formula (201 )
  • the present invention relates to a compound characterized by a general formula (100)
  • R 1 is selected from unsubstituted C1-C4 alkyl or amino-/hydroxy- and/or fluoro-substituted C1-C4 alkyl, unsubstituted phenyl or phenyl substituted by C 1 -C 3 unsubstituted alkyl, NR n 2 , wherein each R N independently from the other is H or C 1 -C 3 unsubstituted alkyl;
  • R 21 , R 22 , R 23 , R 22‘ , and R 21 are independently selected from H, halogen, unsubstituted C 1 -C 3 alkyl or O-alkyl, fluoro-substituted C 1 -C 3 alkyl or O-alkyl, particularly R 21 , R 22 , R 23 , R 22‘ , and R 21 are independently selected from H, F, Cl, methyl, CF 3 , ethyl, O-methyl, and O-CF 3 ; more particularly R 21 and R 22 ’ are independently selected from halogen, unsubstituted C 1 -C 3 alkyl or O-alkyl, fluoro-substituted C 1 -C 3 alkyl or O-alkyl and the other ones are H, or R 23 is selected from halogen, unsubstituted C 1 -C 3 alkyl or
  • R 31 , R 32 , R 33 , R 32 ‘, and R 31 ’ are independently selected from H, F, Cl, CF 3 , methyl, ethyl, O-methyl, and O-CF 3 ;
  • D is NH, N-methyl or O
  • R 3 is NHR D orOR D , wherein R D is a 5- or 6-membered unsubstituted or amino-/hydroxy- or F-substituted cyclic alkyl.
  • R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4- methylphenyl, and N-dimethyl; and/or
  • R 2 is selected from
  • R 3 is selected from
  • the compound is characterized by a formula (101 )
  • the present invention relates to a compound as diclosed above in the first or second aspect for use for the treatment of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
  • the present invention relates to a foodstuff or food additive comprising a compound as diclosed above in the first or second aspect, particularly for use in animal farming.
  • the present invention relates to a compound characterized by a general formula (300)
  • X is S, O or N-methyl
  • R 7 is H, F, Cl or C 1 -C 3 alkyl
  • R 8 is H, F, Cl or C 1 -C 3 alkyl
  • R 9 is NH 2 , NH-methyl, OH or methyl;
  • R 10 is
  • R 101 , R 102 , R 103 , R 102 ‘, and R 101 ’ are independently selected from H, F, Cl, CF 3 , methyl, and NO2.
  • X is selected from S, O, and N-methyl
  • R 7 is selected from H and methyl; and/or R 8 is selected from H, Cl, and methyl; and/or R 9 is selected from NH 2 ; NH-methyl, OH, methyl; and/or R 10 is selected from
  • X is S; R7 is methyl; R8 is methyl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3- nitrophenyl, preferably 4-nitrophenyl.
  • the compound is characterized by a formula (302)
  • the present invention relates to a compound as disclosed in the third aspect for use for the treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • the present invention also relates to a pharmaceutical composition comprising any compound as disclosed herein and to their use as a drug.
  • the present invention further relates to a non-therapeutic method for modulating the appetite in a subject comprising administering a compound as disclosed herein.
  • the appetite can be increased and the compound is a compound according to the first or second aspect.
  • the appetite can be decreased and the compound is a compound according to the third aspect.
  • dpf is an abbreviation for days-post-fertilization.
  • the zebrafish eggs are fertilized and the growing zebrafish larvae are analysed at certain time period after their fertilization measured in dpf.
  • ANOVA is an abbreviation for analysis of variance. ANOVA comprises certain statistical methods used to analyse the difference of group means in a sample for statistical significance.
  • C 1 -C 4 alkyl in the context of the present specification signifies a saturated linear or branched hydrocarbon having 1 , 2, 3 or 4 carbon atoms, wherein in certain embodiments one carbon-carbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge).
  • Non- limiting examples for a C 1 -C 4 alkyl are methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert- butyl, but-3-enyl, prop-2-inyl and but-3-inyl.
  • a C 1 -C 4 alkyl is a methyl, ethyl, propyl or butyl moiety.
  • a C1-C6 alkyl in the context of the present specification signifies a saturated linear or branched hydrocarbon having 1 , 2, 3, 4, 5 or 6 carbon atoms, wherein one carbon-carbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge).
  • Non-limiting examples for a C1-C6 alkyl include the examples given for C 1 -C 4 alkyl above, and additionally 3-methylbut-2-enyl, 2- methylbut-3-enyl, 3-methylbut-3-enyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 1 ,1-dimethylpropyl, 1 ,2-dimethylpropyl, 1 ,2-dimethylpropyl, pent-4-inyl, 3-methyl-2-pentyl, and 4-methyl-2-pentyl.
  • a C5 alkyl is a pentyl or cyclopentyl moiety and a O Q alkyl is a hexyl or cyclohexyl moiety.
  • unsubstituted C n alkyl when used herein in the narrowest sense relates to the moiety - C n H2 n - if used as a bridge between moieties of the molecule, or -C n H2 n+i if used in the context of a terminal moiety. It may still contain fewer H atoms if a cyclical structure or one or more (non- aromatic) double bonds are present.
  • unsubstituted C n alkyl and substituted C n alkyl include a linear alkyl comprising or being linked to a cyclical structure, for example a cyclopropane, cyclobutane, cyclopentane or cyclohexane moiety, unsubstituted or substituted depending on the annotation or the context of mention, having linear alkyl substitutions.
  • N, O or other hetero atom in the linear chain or cyclical structure adds up to n.
  • Me is methyl CH3
  • Et is ethyl -CH2CH3
  • Prop is propyl -(Chh ⁇ CHs (n-propyl, n-pr) or -CH(CH3)2 (iso- propyl, i-pr), but is butyl -C 4 H 9 , -(CH 2 ) 3 CH3, -CHCH3CH2CH3, -CH 2 CH(CH 3 ) 2 or -C(CH 3 ) 3 .
  • substituted alkyl in its broadest sense refers to an alkyl as defined above in the broadest sense that is covalently linked to an atom that is not carbon or hydrogen, particularly to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which itself may be -if applicable- linked to one or several other atoms of this group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in their broadest sense).
  • substituted alkyl refers to an alkyl as defined above in the broadest sense that is substituted in one or several carbon atoms by groups selected from amine NH 2 , alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl) NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl) OCOR, carbonyl O and its ketal or acetal (OR) 2 , nitril CN, isonitril NC, cyanate CNO, isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluoride F, choride Cl, bromide Br, iodide I, phosphonate PO3H2, PO3R2, phosphate OPO3H2 and OPO3R2, sulfhydryl SH, suflalkyl SR, sulfoxide SOR, sulf
  • amino substituted alkyl or hydroxyl substituted alkyl refers to an alkyl according to the above definition that is modified by one or several amine or hydroxyl groups NH 2 , NHR, NR 2 or OH, wherein the R substituent as used in the current paragraph, different from other uses assigned to R in the body of the specification, is itself an unsubstituted or substituted C 1 to C 12 alkyl in its broadest sense, and in a narrower sense, R is methyl, ethyl or propyl unless otherwise specified.
  • An alkyl having more than one carbon may comprise more than one amine or hydroxyl.
  • the term“substituted alkyl” refers to alkyl in which each C is only substituted by at most one amine or hydroxyl group, in addition to bonds to the alkyl chain, terminal methyl, or hydrogen.
  • carboxyl substituted alkyl refers to an alkyl according to the above definition that is modified by one or several carboxyl groups COOH, or derivatives thereof, particularly carboxylamides CONH 2 , CONHR and CONR 2 , or carboxylic esters COOR, with R having the meaning as laid out in the preceding paragraph and different from other meanings assigned to R in the body of this specification.
  • Non-limiting examples of amino-substituted alkyl include -CH 2 NH 2 , -CH 2 NHMe, -CH 2 NHEt, -CH 2 CH 2 NH 2 , -CH 2 CH 2 NHMe, -CH 2 CH 2 NHEt, -(CH 2 ) 3 NH 2 , -(CH 2 ) 3 NHMe, -(CH 2 ) 3 NHEt, -CH 2 CH(NH 2 )CH 3 , -CH 2 CH(NHMe)CH 3 , -CH 2 CH(NHEt)CH 3 , -(CH 2 )3CH 2 NH 2 ,
  • -CH 2 CH(CH 2 NHEt) 2 for terminal moieties and -CH 2 CHNH 2 -, -CH 2 CHNHMe-, -CH 2 CHNHEt- for an amino substituted alkyl moiety bridging two other moieties.
  • Non-limiting examples of hydroxy-substituted alkyl include -CH 2 OH, -(CH 2 ) 2 OH, -(CH 2 ) 3 OH, -CH 2 CH(OH)CH 3 , -(CH 2 ) 4 OH, -CH(CH 2 OH)CH 2 CH 3 , -CH 2 CH(CH 2 OH)CH 3 ,
  • halogen-substituted alkyl refers to an alkyl according to the above definition that is modified by one or several halogen atoms selected (independently) from F, Cl, Br, I.
  • fluoro substituted alkyl refers to an alkyl according to the above definition that is modified by one or several fluoride groups F.
  • fluoro-substituted alkyl include -CH 2 F, -CHF 2 , -CF 3 , -(CH 2 ) 2 F, -(CHF) 2 H, -(CHF) 2 F, -C 2 F 5 , -(CH 2 ) 3 F, -(CHF) 3 H, -(CHF) 3 F, -C 3 F 7 , -(CH 2 ) 4 F, -(CHF) 4 H, -(CHF) 4 F and -C 4 F 9 .
  • Non-limiting examples of hydroxyl- and fluoro-substituted alkyl include -CHFCH 2 OH, - CF 2 CH 2 OH, -(CHF) 2 CH 2 OH, -(CF 2 ) 2 CH 2 OH, -(CHF) 3 CH 2 OH, -(CF 2 ) 3 CH 2 OH, -(CH 2 ) 3 OH, -CF 2 CH(OH)CH 3 , -CF 2 CH(OH)CF 3 , -CF(CH 2 OH)CHFCH 3 , and -CF(CH 2 OH)CHFCF 3 .
  • O-alkyl refers to an alkyloxy moiety (e.g. 0-CH 3 ) wherein the alkyl part is defined as above.
  • aryl in the context of the present specification signifies a cyclic aromatic C5-C10 hydrocarbon that may comprise a heteroatom (e.g. N, O, S).
  • heteroatom e.g. N, O, S.
  • aryl include, without being restricted to, phenyl and naphthyl, and any heteroaryl.
  • a heteroaryl is an aryl that comprises one or several nitrogen, oxygen and/or sulphur atoms.
  • heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole.
  • An aryl or a heteroaryl in the context of the specification additionally may be substituted by one or more alkyl groups.
  • a phenyl in the context of the present specification is a 6-membered aromatic carbon ring (C 6 H 6 ). Phenyl may be substituted be any substituent specified.
  • An aryl methylene in the context of the present specification signifies a CFh (-methylene) group substituted by an aryl moiety.
  • One non-limiting example of aryl methylene is a benzyl (Bn) group.
  • Bn benzyl
  • a heteroaryl methylene in the context of the present specification signifies a CFh (-methylene) group substituted by a heteroaryl moiety.
  • heteroaryl examples include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole.
  • An aryl or a heteroaryl in the context of this specification additionally may be substituted by one or more alkyl groups.
  • a carboxylic ester is a group -CO2R, with R being defined further in the description.
  • a carboxylic amide is a group -CONHR, with R being defined further in the description.
  • treating or treatment of any disease or disorder refers to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • treating or treatment refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • treating refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.
  • a first aspect of the invention relates to a compound characterized by a general formula (200)
  • R 4 is an unsubstituted or methyl-substituted 5- or 6-membered aryl or heteroaryl
  • R 5 is an unsubstituted or substituted aryl with the substituent being F, Cl, C1-C3 alkyl, C1-C3 alkyl ether, vinyl or allyl ether;
  • R 6 is COR c or CONHR c with R c being an unsubstituted or C1-C3 alkyl substituted heteroaryl.
  • the compound may be synthesised by a general route shown in scheme 1.
  • R 4 is selected from
  • R 5 is selected from
  • R 6 is selected from
  • the compound is characterized by a formula (201 )
  • the present invention also relates to a pharmaceutical composition comprising a compound according to this first aspect. It further relates to a compound according to this first aspect for its use as a drug.
  • a second aspect of the invention relates to a compound characterized by a general formula
  • R 1 is selected from
  • R 21 , R 22 , R 23 , R 22‘ , and R 21 are independently selected from H, halogen, unsubstituted C 1 -C 3 alkyl or O-alkyl, fluoro-substituted C 1 -C 3 alkyl or O-alkyl;
  • R 31 , R 32 , R 33 , R 32 ‘, and R 31 ’ are independently selected from H, F, Cl, CF 3 , methyl, ethyl, O-methyl, and O-CF 3 ;
  • R D is NH, N-methyl or O; or R 3 is NHR D orOR D , wherein R D is a 5- or 6-membered unsubstituted or amino-/hydroxy- or F-substituted cyclic alkyl.
  • the compound may be synthesised by a general route shown in scheme 2.
  • R 21 , R 22 , R 23 , R 22‘ , and R 21 are independently selected from H, F, Cl, methyl, CF 3 , ethyl, O-methyl, and O-CF 3 .
  • R 21 and R 22 ’ are independently selected from halogen, unsubstituted C 1 -C 3 alkyl or O-alkyl, fluoro-substituted C 1 -C 3 alkyl or O-alkyl and the other ones are H, or R 23 is selected from halogen, unsubstituted C 1 -C 3 alkyl or O-alkyl, fluoro-substituted C 1 -C 3 alkyl or O-alkyl and the other ones are H.
  • R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl.
  • R 2 is selected from
  • R 3 is selected from
  • the compound is characterized by a formula (101 )
  • the present invention also relates to a pharmaceutical composition comprising a compound according to this second aspect. It further relates to a compound according to this second aspect for its use as a drug.
  • a third aspect of the invention relates to the compound of to the first or second aspect for use for the treatment of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
  • the compounds according to the first and second aspect increases the appetite.
  • the invention relates to the use of a compound according to this first or second aspect for the manufacture of a medicament for treating of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
  • the invention relates to a method for increasing the appetite of a subject comprising administering a compound according to this first or second aspect to the subject. The method could be a non- therapeutic method.
  • the method could be a therapeutic method for treating an eating disorder, more particularly cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
  • the compound of the first or second aspect of the invention is comprised in a foodstuff or food additive, particularly for use in animal farming.
  • a fourth aspect of the invention relates to a compound characterized by a general formula (300)
  • X is S, O or N-methyl
  • R 7 is H, F, Cl or C 1 -C 3 alkyl
  • R 8 is H, F, Cl or C 1 -C 3 alkyl
  • R 9 is NH2, NH-methyl, OH or methyl
  • R 101 , R 102 , R 103 , R 102 ‘, and R 101 ’ are independently selected from H, F, Cl, CF 3 , methyl, and NO2.
  • the compound may be synthesised by a general route shown in scheme 3.
  • X is selected from S, O, and N-methyl.
  • R 7 is selected from H and methyl.
  • R 8 is selected from H, Cl, and methyl.
  • R 9 is selected from NH2, NH-methyl, OH, methyl.
  • R 10 is selected from
  • the compound is characterized by a formula (302)
  • the present invention also relates to a pharmaceutical composition comprising a compound according to this fourth aspect. It further relates to a compound according to this fourth aspect for its use as a drug.
  • a fifth aspect of the invention relates to the compound of the fourth aspectfor use for the treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • the invention also relates to the use of a compound of the fourth aspect for the manufacture of a medicament for the treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease,
  • the invention relates to a method for decreasing the appetite in a subject comprising administering a compound of the fourth aspect.
  • the method is a non-therapeutic method.
  • the goal could be improved the physical appearance by reducing the weight.
  • the subject has a normal body mass index.
  • the method can have a therapeutic object, for instance for treating a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • Item A1 A compound characterized by a general formula (200)
  • R 4 is an unsubstituted or methyl-substituted 5- or 6-membered aryl or heteroaryl
  • R 5 is an unsubstituted or substituted aryl with the substituent being F, Cl, C1-C3 alkyl, C1-C3 alkyl ether, vinyl or allyl ether;
  • R 6 is COR c or CONHR c with R c being an unsubstituted or C1-C3 alkyl substituted heteroaryl.
  • Item A2 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A3 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A4 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A5 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A6 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A7 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A8 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A9 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A10 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A11 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxy phenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A12 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A13 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A14 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A15 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A16 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A17 The compound according to item B1 , wherein
  • R 5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
  • Item A18 The compound according to item B1 or B2, characterized by a formula (201 )
  • Item B1 A compound characterized by a general formula (100)
  • R 1 is selected from
  • R 21 , R 22 , R 23 , R 22‘ , and R 21 are independently selected from H, halogen, unsubstituted C 1 -C 3 alkyl or O-alkyl, fluoro-substituted C 1 -C 3 alkyl or O-alkyl, particularly R 21 , R 22 , R 23 , R 22‘ , and R 21’ are independently selected from H, F, Cl, methyl, CF 3 , ethyl, O-methyl, and O-CF 3 ; more particularly R 21 and R 22 ’ are independently selected from halogen, unsubstituted C 1 -C 3 alkyl or O- alkyl, fluoro-substituted C 1 -C 3 alkyl or O-alkyl and the other ones are H, or R 23 is selected from halogen, unsubstituted C 1 -C 3 alkyl or O-alkyl, fluoro- substituted C 1 -C 3 alkyl or O
  • R 31 , R 32 , R 33 , R 32 ‘, and R 31 ’ are independently selected from H, F, Cl, CF 3 , methyl, ethyl, O-methyl, and O-CF 3 ;
  • D is NH, N-methyl or O
  • R 3 is NHR D orOR D , wherein R D is a 5- or 6-membered unsubstituted or amino-/hydroxy- or F-substituted cyclic alkyl.
  • Item B2 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-methoxy-5-fluorophenyl and R 3 is phenylamidyl.
  • Item B3 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-methoxy-5-fluorophenyl and R 3 is
  • Item B4 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-methoxy-5-fluorophenyl and R 3 is 3-chlorophenylamidyl.
  • Item B5 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-methoxy-5-fluorophenyl and R 3 is 2- methoxy-5-methylphenylamidyl.
  • Item B6 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-methoxy-5-fluorophenyl and R 3 is
  • Item B7 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methylphenyl and R 3 is phenylamidyl.
  • Item B8 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methylphenyl and R 3 is 2,5- dimethylphenylamidyl.
  • Item B9 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methylphenyl and R 3 is 3-chlorophenylamidyl.
  • Item B10 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methylphenyl and R 3 is 2-methoxy-5-methylphenylamidyl.
  • Item B1 1 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methylphenyl and R 3 is 2,5-dimethylphenyloxyl.
  • Item B12 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methoxyphenyl and R 3 is phenylamidyl.
  • Item B13 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methoxyphenyl and R 3 is 2,5-dimethylphenylamidyl.
  • Item B14 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methoxyphenyl and R 3 is 3-chlorophenylamidyl.
  • Item B15 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methoxyphenyl and R 3 is 2-methoxy-5-methylphenylamidyl.
  • Item B16 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 3-chloro-6-methoxyphenyl and R 3 is 2,5-dimethylphenyloxyl.
  • Item B17 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 4-methoxyphenyl and R 3 is phenylamidyl.
  • Item B18 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 4-methoxyphenyl and R 3 is 2,5- dimethylphenylamidyl.
  • Item B19 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 4-methoxyphenyl and R 3 is 3- chlorophenylamidyl.
  • Item B20 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 4-methoxyphenyl and R 3 is 2- methoxy-5-methylphenylamidyl.
  • Item B21 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 4-methoxyphenyl and R 3 is 2,5- dimethylphenyloxyl.
  • Item B22 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-trifluoromethoxyphenyl and R 3 is phenylamidyl.
  • Item B23 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-trifluoromethoxyphenyl and R 3 is
  • Item B24 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-trifluoromethoxyphenyl and R 3 is 3-chlorophenylamidyl.
  • Item B25 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-trifluoromethoxyphenyl and R 3 is 2-methoxy-5-methylphenylamidyl.
  • Item B26 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-trifluoromethoxyphenyl and R 3 is
  • Item B27 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-chloro-5-methoxyphenyl and R 3 is phenylamidyl.
  • Item B28 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-chloro-5-methoxyphenyl and R 3 is 2,5-dimethylphenylamidyl.
  • Item B29 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-chloro-5-methoxyphenyl and R 3 is 3-chlorophenylamidyl.
  • Item B30 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-chloro-5-methoxyphenyl and R 3 is 2-methoxy-5-methylphenylamidyl.
  • Item B31 The compound according to item A1 , wherein R 1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R 2 is 2-chloro-5-methoxyphenyl and R 3 is 2,5-dimethylphenyloxyl.
  • Item B32 The compound according to item A1 or A28, characterized by a formula (101 )
  • Item AB1 The compound according to any one of items A1 through B18 for treatment of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
  • an eating disorder cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection
  • cachexia associated with a psychological disorder particularly cachexia associated with stress, depression, or anxiety
  • cachexia associated with particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis
  • medication-induced anorexia associated with chemotherapy administration of a laxative or amphetamine drug.
  • Item AB2 A foodstuff or food additive comprising a compound according to any one of items A1 through B18, particularly for use in animal farming.
  • Item C1 A compound characterized by a general formula (300)
  • - X is S, O or N-methyl
  • R 7 is H, F, Cl or C1-C3 alkyl
  • R 8 is H, F, Cl or C1-C3 alkyl
  • R 9 is NH2, NH-methyl, OH or methyl
  • R 101 , R 102 , R 103 , R 102 ‘, and R 101 ’ are independently selected from H, F, Cl, CF3, methyl, and NO2.
  • Item C2 The compound according to item C1 , wherein
  • - X is selected from S, O, and N-methyl
  • R 7 is selected from H and methyl
  • R 8 is selected from H, Cl, and methyl
  • R 9 is selected from NH2, NH-methyl, OH, methyl;
  • R 10 is selected from
  • Item C3 The compound according to item C1, wherein X is S; R 7 is H; R 8 is H; R 9 is NH ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C4 The compound according to item C1, wherein X is O; R 7 is H; R 8 is H; R 9 is NH ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C5 The compound according to item C1, wherein X is N-methyl; R 7 is H; R 8 is H; R 9 is NH ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C6 The compound according to item C1, wherein X is S; R 7 is methyl; R 8 is H; R 9 is NH ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C7 The compound according to item C1, wherein X is O; R 7 is methyl; R 8 is H; R 9 is NH ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C8 The compound according to item C1, wherein X is N-methyl; R 7 is methyl; R 8 is H; R 9 is NH ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C9 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is Cl; R 9 is NH 2 ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C10 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is Cl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C1 1 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is Cl; R 9 is NH 2 ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C12 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is Cl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C13 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is Cl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C14 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is Cl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C15 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is methyl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C16 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is methyl; R 9 is NH 2 ;and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C17 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is methyl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C18 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is methyl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C19 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is methyl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C20 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is methyl; R 9 is NH 2 ; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C21 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is H; R 9 is NH- methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C22 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is H; R 9 is NH- methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C23 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is H; R 9 is NH-methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C24 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is H; R 9 is NH- methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C25 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is H; R 9 is NH- methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C26 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is H; R 9 is NH-methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C27 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is methyl; R 9 is NH- methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C28 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is methyl; R 9 is NH- methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C29 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is methyl; R 9 is NH-methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C30 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is methyl; R 9 is NH-methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C31 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is methyl; R 9 is NH-methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C32 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is methyl; R 9 is NH-methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C33 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is H; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C34 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is H; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C35 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is H; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C36 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is H; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C37 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is H; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C38 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is H; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C39 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is methyl; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C40 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is methyl; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C41 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is methyl; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C42 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is methyl; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C43 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is methyl; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C44 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is methyl; R 9 is OH; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C45 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is H; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C46 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is H; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C47 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is H; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C48 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is H; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C49 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is H; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C50 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is H; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C51 The compound according to item C1 , wherein X is S; R 7 is H; R 8 is methyl; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C52 The compound according to item C1 , wherein X is O; R 7 is H; R 8 is methyl; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C53 The compound according to item C1 , wherein X is N-methyl; R 7 is H; R 8 is methyl; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C54 The compound according to item C1 , wherein X is S; R 7 is methyl; R 8 is methyl; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C55 The compound according to item C1 , wherein X is O; R 7 is methyl; R 8 is methyl; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
  • Item C56 The compound according to item C1 , wherein X is N-methyl; R 7 is methyl; R 8 is methyl; R 9 is methyl; and R 10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
  • Item C57 The compound according to item C1 or C2, characterized by a formula (302)
  • Item C58 The compound according to any one of the items C1 through C57 for treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabeles, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
  • a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabeles, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression
  • any specifically mentioned drug may be present as a pharmaceutically acceptable salt of said drug.
  • Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion.
  • Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate.
  • Fig. 1 Homeostatic state modulates feeding behavior, but not spontaneous activity, arousal to visual or acoustic signals nor habituation in zebrafish larvae.
  • A Zebrafish larvae were raised under controlled conditions until 7 dpf, loaded into a well of a 96-well plate and fasted for different time-periods prior assessment of distinct behaviors.
  • B A custom-built imaging platform can quantify larval feeding behavior by measuring intestinal food content if combined with feeding fluorescently labeled live prey; larval locomotion by tracking swimming behavior; larval arousal by presenting visual and acoustic signals; and larval habituation (non- associative learning) by repeatedly presenting inconsequential stimuli.
  • the two exemplary images show data quality used for larval activity tracking and larval intestinal food content quantification using two distinct invisible light sources. All measurements were acquired under daylight conditions, scale bar - 1 mm.
  • C After different fasting periods, larvae were given access to fluorescently labeled paramecia and their intestinal food content quantified for 2 hours.
  • D total food intake
  • E initial intake rate
  • F digestion rate
  • (G) Simultaneously, larval locomotor activity was tracked for 2 hours, n 48, lc - line crosses.
  • (H) Larval zebrafish react to dark flashes with increased motor output. After different fasting periods, larvae were exposed to eight dark flashes (750ms duration, inter-stimuli interval 30s) and their locomotion quantified. The triggered average is shown, n 48.
  • (I) An acoustic stimulus triggers larval locomotion. Identical experiment as in H, but here a single tap was presented instead of a dark flash, n 48.
  • Fig. 2 Available appetite modulators trigger a pleiotropy of behaviors.
  • A Schematic of multi-behavior protocol used to test drug impact. Briefly, 2h fasted larvae were pre-exposed to a drug for 30 min prior multi-behavioral profiling. Here, the inventors tested many appetite modulating drugs used in humans. Each drug’s behavioral impact was condensed into a single, quantitative behavioral barcode, which is illustrated here for two classic anorectic drugs - nicotine and rimonabant - in detail, n>12.
  • B After giving access to live prey, the inventors quantified larvae’s intestinal food content for 2 h and condensed the fluorescent traces into two feeding periods (F1 , F2).
  • Each square represents an SSMD value (red, higher; blue, lower than control) for a single behavioral metric and jointly they form a compounds behavioral barcode.
  • the black box indicates the barcodes for nicotine and rimonabant, shown in detail in panel B-J, orange box is feeding behavior, green box - habituation; * barcodes shown for drugs tested at 100 mM.
  • pharmacology represent chemical structures with unknown biological activity.
  • Behavioral metrics F, feeding; S, spontaneous activity; V, visual response; A, acoustic response; H, habituation; L, lethargy; 1 or 2, different time periods.
  • Fig. 3 Diversity of drug-induced behavioral changes.
  • A Hierarchical clustering groups hit compounds with similar behavioral barcodes based on their correlation.
  • Dendrogram color code localizes selective clusters shown at higher magnification.
  • B tSNE groups hit compounds with similar barcodes using non-linear probability distributions and preserves local distance metrics. All 4,801 hits are shown in a two-dimensional space. The tSNE map is false-colored with the primary behavior modulated for a given compound. The magnification of the rectangle is shown in panel (C).
  • C tSNE map location of selected hierarchical clusters magnified in panel A.
  • E Histogram of the pairwise Pearson correlation ( R ) for compound barcodes sharing one or more targets, or compound barcodes with unknown target (two-tailed, two-sample Kolmogorov- Smirnov test, line depicts median).
  • Tanimoto coefficient >0.85 reflects very similar structures.
  • Fig. 4 Identification of novel and selective appetite modulators.
  • A Barcodes of all hits modulating feeding behavior selectively based on the SSMD threshold sorted by their effect size on feeding period 1 (F1 ).
  • B 27 structurally novel compounds were selected based on the displayed behavioral barcodes and unique chemical structures. All compounds were validated and the complete validation data is shown in Fig. 5.
  • C Heatmap illustration of in vitro target binding profiles for all 27 compounds across 43 different human targets. All binding assays were validated with a positive control.
  • adrenergic receptors a-1A, a-1 B, a-1 D, a-2A, a-2B, b- 1 , b-2; dopamine receptors, D1-D5; gamma- amino butyric acid (GABA)-A receptor, peripheral benzodiazepine receptor (PBR), allosteric benzodiazepine binding site on rat brain slices (BZP); histamine receptors, H1 , H3, H4; muscarinic receptors, M1-M5; serotonin receptors (5- hydroxytryptamine, 5-HT), 1A, 1 B, 1 D, 1 E, 2A, 2B, 2C, 3, 5A, 6, 7; sigma receptors 1 , 2; opioid receptors, d-opioid receptor (DOR), k-opioid receptor (KOR), m-opioid receptors (MOR);
  • DOR d-opioid receptor
  • KOR k-opio
  • biogenic amine transporters dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT).
  • DAT dopamine transporter
  • NET norepinephrine transporter
  • SERT serotonin transporter
  • Fig. 7 Orexigenic and anorectic candidate compound impact on mice behaviors.
  • Fig. 8 Spectra of candidate compounds.
  • A spectrum oC compound A.
  • B spectrum oC compound B.
  • C spectrum aC compound C.
  • Larvae from bucket crosses were collected and pooled in blue water (density of >5 ml / larvae, pH 7.2 - sodium bicarbonate buffer, 1 g/l methylene blue, 0.2 g/l instant ocean salt). Live embryos were selected ⁇ 24 hpf and transferred to the automated growth system in 2 L tanks.
  • the automated system was maintained at 28 °C and at a 14:10 h lighhdark cycle.
  • Protozoan pellets (Carolina Biological Supply, Burlington, USA) were dissolved in boiling larval growth water. After cooling, live paramecia and RotiGrow Plus (Reed Mariculture, Campbell, USA) were added to the protozoan water and the culture incubated for a minimum of 2 days at 28 °C prior to use. All paramecia strains were cultured using this protocol and were originally obtained from Ward’s science (Rochester, USA). Visual inspection of paramecia size and structure enabled to distinguish between paramecium caudatum, paramecium bursaria or bdelloidea. If not stated differently, paramecium caudatum was used for all experiments. All paramecia cultures were visually inspected for purity prior to use with light microscopy.
  • Fluorescent excitation light was generated using a single, cooled 740 nm led (LZ4, LED engine), a lens (12° uniform spot, LED engine) and a squared light diffuser (Thorlabs, Newton, USA).
  • the new generation of IDS camera was used with significantly improved sensitivity in the inventors’ hands in the 780-820 nm range [emission range of fluorophore] (IDS UI-3370CP-NIR, IDS, Obersulm, Germany).
  • a push-tap solenoid (Guardian Electric 28P-I-12D, Allied Electronis, Texas, USA) enables to deliver acoustic stimuli. Four instruments were constructed to enable higher throughput.
  • Emission light calibration was conducted using DiR dye (Molecular Probes, Eugene, USA) dissolved in DMSO at different concentrations, and recording fluorescent emission from the same probes for 100 ms on all the different behavioral imaging platforms. All image platforms showed very similar sensitivity for fluorescence detection.
  • larval zebrafish were transferred to a single well of a flat-bottom 96 well plate (Falcon, VWR, USA) in 150 mI larval growth water. Within 1 min prior to starting behavioral imaging, 50 mI of labeled paramecia culture (OD 4 90nm > 0.5, >500 paramecia/well; see Jordi et al. (Jordi et al.
  • larvae were pre-exposed to 30 mI of compounds (final concentration 10 mM in 0.15 % dimethyl sulfoxide, if not stated differently) for 30 min.
  • 50 mI of labeled paramecia culture (OD 4 90nm > 0.5, >500 paramecia/well) were added, the plate transferred to the behavioral imaging platform and the experiment initialized.
  • the following compound libraries were tested: DIVERSetE library (ChemBridge, San Diego, USA, > 95% purity); Spectrum library (Microsource Discovery Systems, Gaylordsvill, USA, >95 % purity); Prestwick library (Prestwick Chemical, lllkirch, France, >95 % purity);
  • Biomol Neurotransmitter Library (BML-2810, ENZO Life Sciences, Farmingdale, USA, >95% purity). Individual compounds were re-ordered from suppliers. For the screen, each compound was tested in 6 animals. A subset of 530 compounds was tested in 12 or 18 animals because identical compounds were contained in multiple libraries. Lorcaserin (AdooQ Bioscience LLC, CA, USA), rimonabant-HCI (Sigma-aldrich, MO, USA) and anamorelin (AdooQ) were acquired at >98% purity.
  • the experimental flow is graphically depicted in Fig. 2A.
  • the inventors collected a fluorescent image, required to measure intestinal paramecia content, with 100 ms exposure time every min for 2 h; and simultaneously use transmitted IR images to track in real time spontaneous swimming activity of all zebrafish larvae at a camera frame rate of 10 Hz.
  • 8 dark flashes 750 ms, inter-stimulus interval [ISI] 30 s
  • 8 taps ISI 30 s
  • Larval intestines were identified using intensity and size thresholds. Such thresholding led to a number of particles per well, which were sorted by signal strength (area * mean intensity) and the top two stored as intestinal traces.
  • Kinematic traces were quantified as described in Kokel et al., where the number of times an animal crosses any of three parallel imaginary lines are counted (Kokel et al. Nature Chemical Biology 6, 231-237, (2010).). The inventors counted larval line crosses for each well at a frame rate of 10 Hz.
  • the data was pre-processed as follows on a well by well basis (Fig. 2): The fluorescence measurements were binned down to two-time periods reflecting the accumulated fluorescence in the first 40 min (F1 , feeding period 1 ) and the last 80 min of the experiment (F2, feeding period 2), respectively. Concurrent larval locomotion activity was condensed into one bin for the entire 2 h (S, spontaneous locomotion).
  • the inventors aligned the repeats of the initial eight dark flashes, the eight taps, and the four post-dark flashes to construct triggered- averages, each 28 s in length, using an interpolation-based alignment method to generate a standard time base.
  • SSMD Strictly Standardized Median Difference
  • Fertilized embroys were collected following natural spawning, visually inspected and three healthy randomly-selected embryo’s placed into a well of standard flat-bottom 6-well plate (VWR) filled with larvae’s growth water. Each well was treated either with DMSO, candidate compound A to D (10 mM) or EtOH (350 mM) one-hour post-fertilization. 6-well plates were maintained on a 14:10 h lighhdark cycle at 28°C. Every 12 hours each embryo was inspected with a stereomicroscope and scored for survival till 6 dpf. At 5 dpf, images were acquired with a stereomicroscope and body length measured using ImageJ.
  • the phototaxis experiment was executed identically to the thigmotaxis assay. The main difference being that one half of the well was covered with an infrared light proof shelter (no visible light passes, McMaster, New Jersey, USA). For data analysis, each well was split into 2 equal sized areas - shelter and open space. The time periods each fish spent in an area based on its coordinates were summed and the phototaxis index calculated as follows: [Time(open space)-Time(shelter)]/Time(total).
  • One agar pad (2 % agar in larvae’s growth water) contained mustard oil (noxious stimuli, 20 % (v/v) Allyl isothiocyanate, Sigma) and the other contained no additives.
  • mice 4-month-old male C57BL/6J mice (Charles River, Sulzfeld, Germany) were individually housed in wire-mesh hanging cages or in standard mice cages at a room temperature of 21 ⁇ 1 °C and an artificial 12/12 h light/dark cycle. Animals were able to see, hear and smell their conspecifics in neighboring cages and were not socially isolated. Water, food (mice chow-3436, Kliba Nafag, Kaiseraugst, Switzerland) and bedding was provided ad libitum, if not indicated differently. All procedures for mice handling and experimental interventions were according to Swiss Animal Welfare laws, approved by the“Kantonales Veterinaramt Zurich” and conform to the principles of UK regulations. Animals were adapted to novel housing situation and feeding schedule at least for 1 week. All experiments were conducted three times with saline injections prior to drug administration to habituate the animals to the experimental procedure.
  • mice received a tail cut for immediate blood glucose measurement (Breeze 2, Bayer AG, Leverkusen, Germany). Subsequently, candidate compounds were injected
  • mice were returned to their home cage for 30 min before a second blood glucose measurement was performed as described before.
  • mice displacement was tracked online at 16 Hz by a camera (Logitech C930e HD) positioned above the chamber which was illuminated with an infrared light source from below. The camera had an infrared filter mounted to avoid interference from visible light.
  • the tracking algorithm subtracted a 10- frames running-average background image, applied a size threshold to identify the mouse (min. motion detected was 5 mm) and extracted its coordinates. Extracted coordinates with linked timestamps were used to calculate displacement. Mice behavior was recorded for 15 min in complete darkness and the final 10 min were analyzed.
  • mice 3 h fasted mice were injected intraperitoneally with a candidate compound and were tracked in complete darkness 30 min post-injection as outlined above. After recording undisturbed behavior for 15 min, eight taps (75-85 Db) were applied to the mouse cage with an inter- stimulus interval of 30 s. The inventors aligned the repeats of the eight taps to a triggered- average using an interpolation-based alignment method to generate a standard time base. These time traces were normalized to the period preceding a given stimulus (2 s) to account for differences in spontaneous locomotion and the tap response index calculated as follows:
  • mice to white light flashes were tested similarly to the tap-response. Instead of taps, eight periods of 5 s white light flashes (300 lux, UV, and IR light filtered) were repeated every 30 s. The flash response index was calculated as follows: median displacement (time period of light) / median displacement (time period of darkness).
  • mice 3 h fasted mice were injected intraperitoneally with a candidate compound and were tracked in complete darkness 30 min post-injection with the apparatus outlined above.
  • the cage was split into 2 equal sized areas - border and center square. The time periods that each mouse spent in an area based on its coordinates were summed and the thigmotaxis index calculated as follows: [Time(wall) - Time(center)] / Time(total).
  • Powdered KOH (2.9 g, 51.3 mmol) was added to a suspension of 4,6-dimethyl-2-thioxo-1 ,2- dihydropyridine-3-carbonitrile (2.8 g, 17.1 mmol) and 2-bromo-4’-nitroacetophenone (4.2 g, 17.1 mmol) in anhydrous EtOH (100 ml).
  • the resulting thick suspension was stirred at room temperature for 6 h.
  • the suspension was concentrated to half volume and diluted with ethyl acetate. The mixture was filtered and the filtrate washed with concentrated citric acid (3x100ml), water (100 ml) and brine (100 ml).
  • Fig. 1A Prior to behavioral phenotyping at 7 days-post-fertilization (dpf), larvae were fasted for different time periods and placed into a 96-well plate. Subsequently, a custom-built imaging platform detected several distinct behavioral phenotypes in all 96 animals simultaneously (Fig.
  • FIG. 1 B first, larval zebrafish hunt and ingest live paramecia, a unicellular protozoan roughly 100 pm size, and thereby enable the quantitative characterization of feeding behavior.
  • the inventors fluorescently-labeled the prey and the imaging platform quantified larval food intake by detecting intestinal fluorescence content for 2 hours (Fig. 1 C).
  • Biologically grounded curve-fitting algorithms enabled the inventors to extract key characteristics of feeding behavior, namely total food intake, initial intake rate and digestion rate. Different fasting periods modulated these parameters analogous to observation in adult rodents and humans (Fig. 1 D-F).
  • the inventors simultaneously tracked larval locomotion to quantify their swimming activity for 2 hours (Fig. 1 B, G).
  • Example 2 Current appetite modulators are not selective for feeding behavior
  • Fig. 2G responsiveness to visual
  • Fig. 2H acoustic stimuli
  • Fig. 2I habituation
  • Fig. 2J lethargy
  • Nicotine and rimonabant both significantly reduced food intake (Fig. 2B-E). Additional nutritional and endocrinal interventions were previously demonstrated to have an analogous impact on feeding behavior in fish, rodents, and humans.
  • nicotine increased spontaneous activity, yet had no significant impact on any other tested behaviors (Fig. 2F-J).
  • Rimonabant decreased spontaneous activity, reduced the habituation response and modulated lethargy, but had no impact on the response to visual stimuli (Fig. 2F-J).
  • Example 3 Multi-behavioral profiles link compounds to their molecular mechanism of action independent of the underlying chemical structure
  • the inventors ought to screen for ideal psychoactive small molecules, that is, compounds which induce only their intended, single behavioral change. To that end, the inventors performed a high-throughput chemical screen to identify such putative selective bioactive compounds.
  • the inventors acquired quantitative multi-behavioral barcodes for a total of 10,421 compounds, each individually tested in at least 6 animals (Fig. 2L-M).
  • the inventors found two compounds with an unknown mechanism of action that had a strong correlation with known drugs targeting either histamine receptor 1 (H1 ), or muscarinic acetylcholine receptor 3 (M3), respectively (Fig. 3F-H). This behavioral phenotype correlation suggested a shared mechanism.
  • H1 histamine receptor 1
  • M3 muscarinic acetylcholine receptor 3
  • Fig. 3I-J In vitro competitive binding assays confirmed an interaction of these two unknown compounds with the H1 or M3 receptor, respectively.
  • the inventors identified two novel molecules binding human H1 and M3 receptor with nanomolar affinity in vitro as well as an in vivo behavioral phenotype.
  • the measured binding affinities are in the nanomolar range, which is at least one order of magnitudes stronger compared to compounds normally identified by in vitro targeted high-throughput screens (mM - mM range, see also Fig. 4C).
  • a compound’s molecular target is a predictor for its multi- behavioral impact in vivo.
  • Example 4 ⁇ The zebrafish central nervous system controls different behaviors by an
  • Example 5 Novel appetite modulators are potent and selective for feeding behavior and the majority is mechanistically independent of the main neurotransmitter systems
  • the hit rate for selective orexigenic compounds was 1.8 % and for selective anorectic compounds 0.7 %. Consequently, the selectivity filter reduced the hit rate by roughly 50% illustrating one advantage.
  • the inventors selected, after inspection of the individual barcodes and the chemistry, eleven orexigenic and eleven anorectic candidate compounds with unique chemical structures for validation (Fig. 4B).
  • Fig. 4C in vitro competitive binding assays
  • Fig. 4C a few candidate compounds bound with nanomolar affinity to targets that are established regulators of feeding behavior in mammals (histamine receptor 1 , serotonin receptor 2B or C [5-HT2B, 5-HT2C] and peripheral benzodiazepine receptor [PBR]), thereby independently validating our discovery strategy.
  • Example 6 Orexigenic and anorectic candidate compounds enable the selective control of appetite in zebrafish and mice
  • Fig. 5A Orexigenic candidate (oC) compound A and B doubled food intake, whereas the two anorectic candidate (aC) compounds C and D reduced food intake by more than 50 % in zebrafish (Fig. 5B-E, routes of synthesis shown in scheme 1-3, spectra shown in Fig. 8). None of the candidate compounds modulated the responsiveness to visual (Fig. 5F) or acoustic stimuli (Fig. 5G), spontaneous activity (Fig. 5H), habituation (Fig. 5I) and lethargy (Fig. 5J).
  • aC compound D bound to PBR, 5-HT2B and 5-HT2C receptor with nanomolar affinity (Fig. 5K, L), whereas the other candidate small molecules did not bind any of the 43 human neuronal targets with a millimolar affinity (Fig. 4C).
  • Norfenfluramine the active metabolite of the former anti- obesity drug fenfluramine, binds 5-HT2B and 5-HT2C receptor with similar affinities as aC D.
  • 5- HT2C receptor activity is considered the driver for fenfluramine’s anorectic effect and is the primary target of the recently approved anti-obesity agent lorcaserin.
  • the inventors monitored zebrafish development under the exposure of candidate drugs in vivo using light microscopy from the single fertilized cell- stage to the 6-dpf behaving larvae. None of the candidates had a significant impact on embryological mortality, developmental defects or temporal delays nor on the body length at 5 dpf, whereas EtOH exposure significantly reduced survival after hatching and lead to lordosis as reported previously (Fig. 5M, N). As the inventors were unable to detect an impact of the candidate compounds on normal vertebrate development, they tested for a drug impact on more subtle behavioral characteristics. These behaviors required the analysis of single fish at high temporal and spatial resolution and therefore are non-suitable for high-throughput screening.
  • thigmotaxis (or“wall-loving”), which is a validated behavioral index for anxiety in animals.
  • Anxiolytic drugs reduce thigmotaxis, whereas anxiogenic compounds enhance it in zebrafish larvae.
  • Candidate compound A-D did not modulate the thigmotaxic response in daylight compared to vehicle control nor did they interfere with the characteristic reduction of thigmotaxis observed with the onset of darkness (Fig. 50).
  • the inventors assessed the impact of the candidate compounds on two sensitive visual behaviors - phototaxis and the optomotor response. Larval zebrafish are attracted by light and averse to darkness, hence, perform positive phototaxis.
  • the optomotor response is an orienting behavior evoked by visual motion.
  • a closed loop setup presented a grating moving perpendicular to the body axes of an individual zebrafish.
  • the stimulus elicits a turning behavior within the direction of the stimulus.
  • the inventors did not detect a significant impact of candidate compounds on the correct execution nor the turn-angle used during the optomotor response (Fig. 5Q).
  • Larvae avoided the presence of noxious stimuli (here, mustard oil), but had no preference if presented a non-noxious stimulus (agar). This preference was not visually mediated as it was detectable during daylight and darkness.
  • Larva treated with the candidate compounds avoided the noxious stimuli analogously to vehicle control (Fig. 5R).
  • larval zebrafish swam spontaneously in an open arena and their locomotion exhibited a characteristic segmentation into an individual burst of locomotion, also called a swim bout.
  • the inventors traced larval swim locomotion in the presence of the candidate compounds and, subsequently, performed detailed kinematic analysis. Neither larval average swim speed, bout length, bout- nor interbout duration were altered by the candidate compounds (Fig. 5S).
  • all the candidate compounds selectively modulated feeding behavior in zebrafish larvae while they showed no detectable impact on development and did not interfere with the execution of an array of behavioral tasks.
  • oC compound A and B increased food intake with distinct temporal dynamics in ad libitum feed mice (Fig. 6B, C) but had no significant impact in 16 h fasted mice, probably due to an appetite ceiling effect (Fig. 6D).
  • Anamorelin had a similar impact on food intake as the oC compounds.
  • aC compound C and D reduced food intake in ad libitum fed and 16 h fasted mice with similar kinetics (Fig. 6B-D) based on a smaller meal size (Fig. 7C).
  • Rimonabant induced a more potent anorectic effect in the dark-phase compared to aC compounds, and also only rimonabant triggered compensatory food intake in the following light-phase (Fig. 7D).
  • the inventors tracked mice locomotion to quantify their spontaneous activity. None of the candidate compounds had an impact on undisturbed mice locomotion, whereas hypoglycemia induced by insulin injection reduced locomotion (Fig. 6E, F).
  • the inventors presented short light-flashes (5s) to which mice did not show a detectable locomotion response independent of treatment (Fig. 6G).
  • mice behavioral chamber causing mice to freeze
  • Fig. 6H Only rimonabant treatment modulated the response to the acoustic tap in mice as observed in zebrafish
  • Fig. 2K zebrafish
  • the inventors wanted to further benchmark the behavioral selectivity of our compounds by selecting additional behaviors based on the previously observed problems with appetite modulators: specifically, the inventors considered glucose homeostasis, psychiatric effects, and nausea.
  • blood glucose levels were not altered 30-min post candidate compound injection in mice, whereas insulin injection induced hypoglycemia (Fig. 6E). Blood glucose levels were similar across experimental groups’ prior to injection (Fig. 7E).
  • thigmotaxis in mice which is a behavioral index for anxiety. None of the candidate compounds modulated the thigmotaxis response, whereas rimonabant increased murine anxiety as reported previously (Fig. 6I).
  • the inventors demonstrated the existence of ideal, behavior-selective small molecules in two vertebrate species.
  • appetite modulators the inventors identified 268 compounds that are novel and to date have no described in vivo bioactivity. Twenty-two of these candidates were extensively validated and their behavioral selectivity confirmed in multiple follow-up studies. Furthermore, the majority of these did not bind to known receptors from main neurotransmitter systems.
  • the two orexigenic and two anorectic candidate compounds were benchmarked in a variety of sophisticated behavioral assays and were shown to exclusively modulate appetite in zebrafish and in mice.
  • the inventors performed a similar analysis for compounds selective for habituation (Fig. 7). In sum, the inventors identified ideal, behavior-selective small molecules, which exclusively promote or inhibit distinct vertebrate behaviors in two vertebrate species.
  • phenotype screens are their superior ability to identify“first-in-class” drugs compared to target-based strategies.
  • such screens only employ effect size in their hit selection criteria.
  • the inventors considered compound selectivity at the initial stage, which reduced the hit rate by ⁇ 50 % compared to the standard effect size criterion, and led to the selection of more specific in vivo phenotypes.
  • This selectivity can arise either from the interaction of compounds with unknown molecular targets that are unique to a behavior or from systemic drug action balanced optimally across different mechanisms. Both are impossible to identify without a whole-organism approach. Taking the above into account, future strategies should combine a series of complex behaviors - e.g.
  • the inventors unbiased and large-scale behavioral findings jointly establish the advantages of multi-behavioral screening for psychoactive compounds, propose a largely independent and modular organization of neural circuits that generate behavior in the larval zebrafish, and demonstrate the feasibility to control vertebrate behavior with novel, behavior- selective compounds.

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Abstract

The present invention relates to small molecule compounds capable of modulating appetite in a vertebrate animal, particularly in a mammal. The invention further relates to the use of the compounds of the invention in methods of treatment of eating disorders, and as a food additive for human consumption or animal husbandry.

Description

Appetite-modulating molecules
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under U19NS104653, NIH grants R24 NS086601 and R43OD024879 awarded by National Institutes of Health. The government has certain rights in the invention.
Field of the Invention
The present invention relates to small molecule compounds capable of modulating appetite in a vertebrate animal, particularly in a mammal. The invention further relates to the use of the compounds of the invention in methods of treatment of eating disorders, and as a food additive for human consumption or animal husbandry.
Background of the Invention
From an evolutionary, as well as a clinical, perspective, feeding represents a fundamental behavior as nutritional supply is critical for survival. Eating disorders are a rapidly growing prevalent health problem. Extreme lack of appetite i.e. anorexia nervosa causes irreversible physiological damage, while augmented food intake primarily promotes obesity, with a sedentary lifestyle and polygenetic mutations favoring disease onset. Managing nutritional intake is a key intervention strategy, however, to date the pharmacological toolkit to selectively modulate food intake does not exist. Historically, the majority of appetite effectors provoked unintentional side-effects, illustrating one of the challenges in psychoactive drug discovery. One of such cautionary examples is the effective appetite suppressant rimonabant, a cannabinoid receptor 1 antagonist, which also causes anxiety and depression. This lack in specificity limits the usefulness of such drugs with respect to therapeutic intervention and constrains their use in the targeted perturbation of neural circuits when the goal is the discovery of general principles regulating feeding behavior. Therefore, a priority in drug discovery for therapeutic as well as basic research goals needs to include a description of a general behavioral impact as well as drug selectivity. Both should become a standard requirement in pre-clinical development.
Mainstream drug discovery efforts focus on protein targets using simple in vitro or cellular screens to identify small molecules or biologies as modulators. The quality of the target correlates with success and, consequently, an in-depth molecular characterization underlying a phenotype is a prerequisite. Behaviors such as food intake are not understood at the molecular level, which strongly limits the reductionist approach of target-driven drug discovery. Up to the present target-based approaches are far less successful at identifying first-in-class drugs compared to holistic and unbiased phenotypic strategies, particularly in CNS drug discovery. Furthermore in vivo drug efficacy is a major obstacle, because traditional drug screening methods do not mimic whole organism dynamics e.g. the blood-brain barrier.
Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to screen for appetite regulators. This objective is attained by the subject-matter of the independent claims of the present specification.
Summary of the Invention
The present invention relates to compounds which regulate appetite and their uses.
In a first aspect, the present invention relates to a compound characterized by a general formula
(200)
Figure imgf000003_0001
wherein
R4 is an unsubstituted or methyl-substituted 5- or 6-membered aryl or heteroaryl;
R5 is an unsubstituted or substituted aryl with the substituent being F, Cl, C1-C3 alkyl, C1-C3 alkyl ether, vinyl or allyl ether; R6 is CORc or CONHRc with Rc being an unsubstituted or C1-C3 alkyl substituted heteroaryl.
In a particular aspect, R4 is selected from
Figure imgf000003_0002
R5 is selected from
Figure imgf000004_0001
In a specific aspect, the compound is characterized by a formula (201 )
Figure imgf000004_0002
In a second aspect, the present invention relates to a compound characterized by a general formula (100)
Figure imgf000005_0001
wherein
R1 is selected from unsubstituted C1-C4 alkyl or amino-/hydroxy- and/or fluoro-substituted C1-C4 alkyl, unsubstituted phenyl or phenyl substituted by C1-C3 unsubstituted alkyl, NRn 2, wherein each RN independently from the other is H or C1-C3 unsubstituted alkyl;
R2 is
Figure imgf000005_0002
wherein R21, R22, R23, R22‘, and R21 are independently selected from H, halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl, particularly R21, R22, R23, R22‘, and R21 are independently selected from H, F, Cl, methyl, CF3, ethyl, O-methyl, and O-CF3; more particularly R21 and R22’ are independently selected from halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl and the other ones are H, or R23 is selected from halogen, unsubstituted C1-C3 alkyl or
O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl and the other ones are H;
R3 is
Figure imgf000006_0001
wherein, R31, R32, R33, R32‘, and R31’ are independently selected from H, F, Cl, CF3, methyl, ethyl, O-methyl, and O-CF3; and
D is NH, N-methyl or O; or
R3 is NHRD orORD, wherein RD is a 5- or 6-membered unsubstituted or amino-/hydroxy- or F-substituted cyclic alkyl.
In a particular aspect, R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4- methylphenyl, and N-dimethyl; and/or
R2 is selected from
Figure imgf000006_0002
R3 is selected from
Figure imgf000007_0001
In a specific aspect, the compound is characterized by a formula (101 )
Figure imgf000007_0002
The present invention relates to a compound as diclosed above in the first or second aspect for use for the treatment of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
The present invention relates to a foodstuff or food additive comprising a compound as diclosed above in the first or second aspect, particularly for use in animal farming.
In a third aspect, the present invention relates to a compound characterized by a general formula (300)
Figure imgf000008_0002
wherein
X is S, O or N-methyl;
R7 is H, F, Cl or C1-C3 alkyl;
R8 is H, F, Cl or C1-C3 alkyl;
R9 is NH2, NH-methyl, OH or methyl; R10 is
Figure imgf000008_0001
wherein, R101, R102, R103, R102‘, and R101’ are independently selected from H, F, Cl, CF3, methyl, and NO2.
In a particular aspect, X is selected from S, O, and N-methyl; and/or
R7 is selected from H and methyl; and/or R8 is selected from H, Cl, and methyl; and/or R9 is selected from NH2; NH-methyl, OH, methyl; and/or R10 is selected from
Figure imgf000009_0001
In a further particular aspect, X is S; R7 is methyl; R8 is methyl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3- nitrophenyl, preferably 4-nitrophenyl.
In a specific aspect, the compound is characterized by a formula (302)
Figure imgf000009_0002
The present invention relates to a compound as disclosed in the third aspect for use for the treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
The present invention also relates to a pharmaceutical composition comprising any compound as disclosed herein and to their use as a drug.
The present invention further relates to a non-therapeutic method for modulating the appetite in a subject comprising administering a compound as disclosed herein. In particular, the appetite can be increased and the compound is a compound according to the first or second aspect. Alternatively, the appetite can be decreased and the compound is a compound according to the third aspect.
Detailed description of the Invention Terms and definitions
The term dpf is an abbreviation for days-post-fertilization. The zebrafish eggs are fertilized and the growing zebrafish larvae are analysed at certain time period after their fertilization measured in dpf.
The term ANOVA is an abbreviation for analysis of variance. ANOVA comprises certain statistical methods used to analyse the difference of group means in a sample for statistical significance.
The term C1-C4 alkyl in the context of the present specification signifies a saturated linear or branched hydrocarbon having 1 , 2, 3 or 4 carbon atoms, wherein in certain embodiments one carbon-carbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge). Non- limiting examples for a C1-C4 alkyl are methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert- butyl, but-3-enyl, prop-2-inyl and but-3-inyl. In certain embodiments, a C1-C4 alkyl is a methyl, ethyl, propyl or butyl moiety.
A C1-C6 alkyl in the context of the present specification signifies a saturated linear or branched hydrocarbon having 1 , 2, 3, 4, 5 or 6 carbon atoms, wherein one carbon-carbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge). Non-limiting examples for a C1-C6 alkyl include the examples given for C1-C4 alkyl above, and additionally 3-methylbut-2-enyl, 2- methylbut-3-enyl, 3-methylbut-3-enyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 1 ,1-dimethylpropyl, 1 ,2-dimethylpropyl, 1 ,2-dimethylpropyl, pent-4-inyl, 3-methyl-2-pentyl, and 4-methyl-2-pentyl. In certain embodiments, a C5 alkyl is a pentyl or cyclopentyl moiety and a OQ alkyl is a hexyl or cyclohexyl moiety.
The term unsubstituted Cn alkyl when used herein in the narrowest sense relates to the moiety - CnH2n- if used as a bridge between moieties of the molecule, or -CnH2n+i if used in the context of a terminal moiety. It may still contain fewer H atoms if a cyclical structure or one or more (non- aromatic) double bonds are present.
The terms unsubstituted Cn alkyl and substituted Cn alkyl include a linear alkyl comprising or being linked to a cyclical structure, for example a cyclopropane, cyclobutane, cyclopentane or cyclohexane moiety, unsubstituted or substituted depending on the annotation or the context of mention, having linear alkyl substitutions. The total number of carbon and -where appropriate-
N, O or other hetero atom in the linear chain or cyclical structure adds up to n. Where used in the context of chemical formulae, the following abbreviations may be used: Me is methyl CH3, Et is ethyl -CH2CH3, Prop is propyl -(Chh^CHs (n-propyl, n-pr) or -CH(CH3)2 (iso- propyl, i-pr), but is butyl -C4H9, -(CH2)3CH3, -CHCH3CH2CH3, -CH2CH(CH3)2 or -C(CH3)3.
The term substituted alkyl in its broadest sense refers to an alkyl as defined above in the broadest sense that is covalently linked to an atom that is not carbon or hydrogen, particularly to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which itself may be -if applicable- linked to one or several other atoms of this group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in their broadest sense). In a narrower sense, substituted alkyl refers to an alkyl as defined above in the broadest sense that is substituted in one or several carbon atoms by groups selected from amine NH2, alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl) NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl) OCOR, carbonyl O and its ketal or acetal (OR)2, nitril CN, isonitril NC, cyanate CNO, isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluoride F, choride Cl, bromide Br, iodide I, phosphonate PO3H2, PO3R2, phosphate OPO3H2 and OPO3R2, sulfhydryl SH, suflalkyl SR, sulfoxide SOR, sulfonyl SO2R, sulfanylamide SO2NHR, sulfate SO3H and sulfate ester SO3R, wherein the R substituent as used in the current paragraph, different from other uses assigned to R in the body of the specification, is itself an unsubstituted or substituted C1 to C12 alkyl in its broadest sense, and in a narrower sense, R is methyl, ethyl or propyl unless otherwise specified.
The term amino substituted alkyl or hydroxyl substituted alkyl refers to an alkyl according to the above definition that is modified by one or several amine or hydroxyl groups NH2, NHR, NR2 or OH, wherein the R substituent as used in the current paragraph, different from other uses assigned to R in the body of the specification, is itself an unsubstituted or substituted C1 to C12 alkyl in its broadest sense, and in a narrower sense, R is methyl, ethyl or propyl unless otherwise specified. An alkyl having more than one carbon may comprise more than one amine or hydroxyl. Unless otherwise specified, the term“substituted alkyl” refers to alkyl in which each C is only substituted by at most one amine or hydroxyl group, in addition to bonds to the alkyl chain, terminal methyl, or hydrogen.
The term carboxyl substituted alkyl refers to an alkyl according to the above definition that is modified by one or several carboxyl groups COOH, or derivatives thereof, particularly carboxylamides CONH2, CONHR and CONR2, or carboxylic esters COOR, with R having the meaning as laid out in the preceding paragraph and different from other meanings assigned to R in the body of this specification.
Non-limiting examples of amino-substituted alkyl include -CH2NH2, -CH2NHMe, -CH2NHEt, -CH2CH2NH2, -CH2CH2NHMe, -CH2CH2NHEt, -(CH2)3NH2, -(CH2)3NHMe, -(CH2)3NHEt, -CH2CH(NH2)CH3, -CH2CH(NHMe)CH3, -CH2CH(NHEt)CH3, -(CH2)3CH2NH2,
-(CH2)3CH2NHMe, -(CH2)3CH2NHEt, -CH(CH2NH2)CH2CH3, -CH(CH2NHMe)CH2CH3, -CH(CH2NHEt)CH2CH3, -CH2CH(CH2NH2)CH3, -CH2CH(CH2NHMe)CH3,
-CH2CH(CH2NHEt)CH3, -CH(NH2)(CH2)2NH2, -CH(NHMe)(CH2)2NHMe,
-CH(NHEt)(CH2)2NHEt, -CH2CH(NH2)CH2NH2, -CH2CH(NHMe)CH2NHMe,
-CH2CH(NHEt)CH2NHEt, -CH2CH(NH2)(CH2)2NH2, -CH2CH(NHMe)(CH2)2NHMe,
-CH2CH(NHEt)(CH2)2NHEt, -CH2CH(CH2NH2)2, -CH2CH(CH2NHMe)2 and
-CH2CH(CH2NHEt)2for terminal moieties and -CH2CHNH2-, -CH2CHNHMe-, -CH2CHNHEt- for an amino substituted alkyl moiety bridging two other moieties.
Non-limiting examples of hydroxy-substituted alkyl include -CH2OH, -(CH2)2OH, -(CH2)3OH, -CH2CH(OH)CH3, -(CH2)4OH, -CH(CH2OH)CH2CH3, -CH2CH(CH2OH)CH3,
-CH(OH)(CH2)2OH, -CH2CH(OH)CH2OH, -CH2CH(OH)(CH2)2OH and -CH2CH(CH2OH)2 for terminal moieties and -CHOH-, -CH2CHOH-, -CH2CH(OH)CH2-, -(CH2)2CHOHCH2-, - CH(CH2OH)CH2CH2-, -CH2CH(CH2OH)CH2-, -CH(OH)(CH2CHOH-, -CH2CH(OH)CH2OH, - CH2CH(OH)(CH2)2OH and -CH2CHCH2OHCHOH- for a hydroxyl substituted alkyl moiety bridging two other moieties.
The term halogen-substituted alkyl refers to an alkyl according to the above definition that is modified by one or several halogen atoms selected (independently) from F, Cl, Br, I.
The term fluoro substituted alkyl refers to an alkyl according to the above definition that is modified by one or several fluoride groups F. Non-limiting examples of fluoro-substituted alkyl include -CH2F, -CHF2, -CF3, -(CH2)2F, -(CHF)2H, -(CHF)2F, -C2F5, -(CH2)3F, -(CHF)3H, -(CHF)3F, -C3F7, -(CH2)4F, -(CHF)4H, -(CHF)4F and -C4F9.
Non-limiting examples of hydroxyl- and fluoro-substituted alkyl include -CHFCH2OH, - CF2CH2OH, -(CHF)2CH2OH, -(CF2)2CH2OH, -(CHF)3CH2OH, -(CF2)3CH2OH, -(CH2)3OH, -CF2CH(OH)CH3, -CF2CH(OH)CF3, -CF(CH2OH)CHFCH3, and -CF(CH2OH)CHFCF3.
The term O-alkyl refers to an alkyloxy moiety (e.g. 0-CH3) wherein the alkyl part is defined as above.
The term aryl in the context of the present specification signifies a cyclic aromatic C5-C10 hydrocarbon that may comprise a heteroatom (e.g. N, O, S). Examples of aryl include, without being restricted to, phenyl and naphthyl, and any heteroaryl.
A heteroaryl is an aryl that comprises one or several nitrogen, oxygen and/or sulphur atoms. Examples for heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole. An aryl or a heteroaryl in the context of the specification additionally may be substituted by one or more alkyl groups.
A phenyl in the context of the present specification is a 6-membered aromatic carbon ring (C6H6). Phenyl may be substituted be any substituent specified. An aryl methylene in the context of the present specification signifies a CFh (-methylene) group substituted by an aryl moiety. One non-limiting example of aryl methylene is a benzyl (Bn) group. If used in particular, a heteroaryl methylene in the context of the present specification signifies a CFh (-methylene) group substituted by a heteroaryl moiety. Examples for heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole. An aryl or a heteroaryl in the context of this specification additionally may be substituted by one or more alkyl groups.
A carboxylic ester is a group -CO2R, with R being defined further in the description. A carboxylic amide is a group -CONHR, with R being defined further in the description. As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). Also, "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In addition, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.
A first aspect of the invention relates to a compound characterized by a general formula (200)
Figure imgf000013_0001
wherein
R4 is an unsubstituted or methyl-substituted 5- or 6-membered aryl or heteroaryl;
R5 is an unsubstituted or substituted aryl with the substituent being F, Cl, C1-C3 alkyl, C1-C3 alkyl ether, vinyl or allyl ether;
R6 is CORc or CONHRc with Rc being an unsubstituted or C1-C3 alkyl substituted heteroaryl.
The compound may be synthesised by a general route shown in scheme 1.
In certain embodiments, R4 is selected from
Figure imgf000014_0001
In certain embodiments, R5is selected from
Figure imgf000014_0002
4-prop-2-eneoxyphenyl
Figure imgf000014_0003
4-methoxyphenyl
Figure imgf000014_0004
4-methylphenyl
Figure imgf000014_0005
Figure imgf000014_0006
4-chlorophenyl
In certain embodiments, R6is selected from
Figure imgf000015_0001
In certain embodiments, the compound is characterized by a formula (201 )
Figure imgf000015_0002
(5-(4-Allyloxy-phenyl)-3-hydroxy-1-(5-methyl-[1 ,3,4]thiadiazol-2-yl)-4-(thiophene-2- carbonyl)-1 ,5-dihydro-pyrrol-2-one).
The present invention also relates to a pharmaceutical composition comprising a compound according to this first aspect. It further relates to a compound according to this first aspect for its use as a drug. A second aspect of the invention relates to a compound characterized by a general formula
(100)
Figure imgf000016_0001
wherein
R1 is selected from
o unsubstituted C1-C4 alkyl or amino-/hydroxy- and/or fluoro-substituted C1-C4 alkyl,
o unsubstituted phenyl or phenyl substituted by C1-C3 unsubstituted alkyl, o NRn 2, wherein each RN independently from the other is H or C1-C3
unsubstituted alkyl;
R2 is
Figure imgf000016_0002
wherein R21, R22, R23, R22‘, and R21 are independently selected from H, halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl;
R3 is
Figure imgf000016_0003
wherein, R31, R32, R33, R32‘, and R31’ are independently selected from H, F, Cl, CF3, methyl, ethyl, O-methyl, and O-CF3; and
D is NH, N-methyl or O; or R3 is NHRD orORD, wherein RD is a 5- or 6-membered unsubstituted or amino-/hydroxy- or F-substituted cyclic alkyl.
The compound may be synthesised by a general route shown in scheme 2.
In certain embodiments, R21, R22, R23, R22‘, and R21 are independently selected from H, F, Cl, methyl, CF3, ethyl, O-methyl, and O-CF3.
In certain embodiments, R21 and R22’ are independently selected from halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl and the other ones are H, or R23 is selected from halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl and the other ones are H.
In certain embodiments, R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl.
In certain embodiments, R2 is selected from
2-methoxy-5-fluorophenyl:
Figure imgf000017_0002
3-chloro-6-methylphenyl:
3-chloro-6-methoxyphenyl:
Figure imgf000017_0001
4-methoxyphenyl
Figure imgf000017_0003
2-trifluoromethoxyphenyl and
2-chloro-5-methoxyphenyl
Figure imgf000018_0006
In certain embodiments, R3 is selected from
Figure imgf000018_0001
2,5-dimethylphenylamidyl:
Figure imgf000018_0002
phenylamidyl:
Figure imgf000018_0003
3-chlorophenylamidyl:
Figure imgf000018_0004
2-methoxy-5-methylphenylamidyl: , and.
2,5-dimethylphenyloxyl:
Figure imgf000018_0005
In certain embodiments, the compound is characterized by a formula (101 )
Figure imgf000019_0001
(N2-(5-Chloro-2-methoxyphenyl)-N-(2,5-dimethylphenyl)-N2-(methylsulfonyl)glycinamide).
The present invention also relates to a pharmaceutical composition comprising a compound according to this second aspect. It further relates to a compound according to this second aspect for its use as a drug.
A third aspect of the invention relates to the compound of to the first or second aspect for use for the treatment of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug. Indeed, the compounds according to the first and second aspect increases the appetite. The invention relates to the use of a compound according to this first or second aspect for the manufacture of a medicament for treating of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug. The invention relates to a method for increasing the appetite of a subject comprising administering a compound according to this first or second aspect to the subject. The method could be a non- therapeutic method. Alternatively the method could be a therapeutic method for treating an eating disorder, more particularly cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug. In certain embodiments, the compound of the first or second aspect of the invention is comprised in a foodstuff or food additive, particularly for use in animal farming.
A fourth aspect of the invention relates to a compound characterized by a general formula (300)
Figure imgf000020_0001
wherein
X is S, O or N-methyl;
R7 is H, F, Cl or C1-C3 alkyl;
- R8 is H, F, Cl or C1-C3 alkyl;
R9 is NH2, NH-methyl, OH or methyl;
- R10 is
Figure imgf000020_0002
wherein, R101, R102, R103, R102‘, and R101’ are independently selected from H, F, Cl, CF3, methyl, and NO2.
The compound may be synthesised by a general route shown in scheme 3.
In certain embodiments, X is selected from S, O, and N-methyl.
In certain embodiments, R7 is selected from H and methyl.
In certain embodiments, R8 is selected from H, Cl, and methyl.
In certain embodiments, R9 is selected from NH2, NH-methyl, OH, methyl.
In certain embodiments, R10 is selected from
Figure imgf000021_0001
In certain embodiments, the compound is characterized by a formula (302)
Figure imgf000021_0002
The present invention also relates to a pharmaceutical composition comprising a compound according to this fourth aspect. It further relates to a compound according to this fourth aspect for its use as a drug.
A fifth aspect of the invention relates to the compound of the fourth aspectfor use for the treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
The invention also relates to the use of a compound of the fourth aspect for the manufacture of a medicament for the treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
The invention relates to a method for decreasing the appetite in a subject comprising administering a compound of the fourth aspect. In an aspect, the method is a non-therapeutic method. For instance, the goal could be improved the physical appearance by reducing the weight. In this context, the subject has a normal body mass index. Alternatively, the method can have a therapeutic object, for instance for treating a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
All of the above indications are directly associated with obesity; reduction of body mass is an integral part of therapy for these disease states. Numerous expert opinions have been published, which express the expectation that an improved pharmacological intervention aimed at weight loss will significantly improve morbidity associated with the above-mentioned diseases. For example, see the AACE/ACE Obesity Clinical Practice Guidelines (American Association of Clinical Endocrinologists/ American College of Endocrinology Clinical Practice Guidelines for Comprehensive Medical Care of Patients with Obesity; PMID: 27219496).
Certain aspects and embodiments of the invention are characterized by the following items:
Item A1 : A compound characterized by a general formula (200)
Figure imgf000022_0001
wherein
R4 is an unsubstituted or methyl-substituted 5- or 6-membered aryl or heteroaryl;
R5 is an unsubstituted or substituted aryl with the substituent being F, Cl, C1-C3 alkyl, C1-C3 alkyl ether, vinyl or allyl ether;
R6 is CORc or CONHRc with Rc being an unsubstituted or C1-C3 alkyl substituted heteroaryl.
Item A2: The compound according to item B1 , wherein
Figure imgf000023_0001
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A3: The compound according to item B1 , wherein
Figure imgf000023_0004
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A4: The compound according to item B1 , wherein
Figure imgf000023_0003
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A5: The compound according to item B1 , wherein
Figure imgf000023_0002
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A6: The compound according to item B1 , wherein
Figure imgf000024_0004
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A7: The compound according to item B1 , wherein
Figure imgf000024_0001
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A8: The compound according to item B1 , wherein
Figure imgf000024_0002
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A9: The compound according to item B1 , wherein
Figure imgf000024_0003
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A10: The compound according to item B1 , wherein
Figure imgf000025_0001
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A11 : The compound according to item B1 , wherein
Figure imgf000025_0002
R5 is selected from 4-prop-2-eneoxy phenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A12: The compound according to item B1 , wherein
Figure imgf000025_0003
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A13: The compound according to item B1 , wherein
Figure imgf000025_0004
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A14: The compound according to item B1 , wherein
Figure imgf000026_0002
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A15: The compound according to item B1 , wherein
Figure imgf000026_0001
and
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A16: The compound according to item B1 , wherein
Figure imgf000026_0003
and
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A17: The compound according to item B1 , wherein
Figure imgf000026_0004
R5 is selected from 4-prop-2-eneoxyphenyl, 4-methoxyphenyl, 4-methylphenyl and 4- chlorophenyl.
Item A18: The compound according to item B1 or B2, characterized by a formula (201 )
Figure imgf000027_0001
Item B1 : A compound characterized by a general formula (100)
Figure imgf000027_0002
wherein
R1 is selected from
o unsubstituted C1-C4 alkyl or amino-/hydroxy- and/or fluoro-substituted C1-C4 alkyl,
o unsubstituted phenyl or phenyl substituted by C1-C3 unsubstituted alkyl, o NRn 2, wherein each RN independently from the other is H or C1-C3 unsubstituted alkyl;
R2 is
Figure imgf000027_0003
wherein R21, R22, R23, R22‘, and R21 are independently selected from H, halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl, particularly R21, R22, R23, R22‘, and R21’ are independently selected from H, F, Cl, methyl, CF3, ethyl, O-methyl, and O-CF3; more particularly R21 and R22’ are independently selected from halogen, unsubstituted C1-C3 alkyl or O- alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl and the other ones are H, or R23 is selected from halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro- substituted C1-C3 alkyl or O-alkyl and the other ones are H;
R3 is
Figure imgf000028_0001
wherein, R31, R32, R33, R32‘, and R31’ are independently selected from H, F, Cl, CF3, methyl, ethyl, O-methyl, and O-CF3; and
D is NH, N-methyl or O; or
R3 is NHRD orORD, wherein RD is a 5- or 6-membered unsubstituted or amino-/hydroxy- or F-substituted cyclic alkyl.
Item B2: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-methoxy-5-fluorophenyl and R3 is phenylamidyl.
Item B3: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-methoxy-5-fluorophenyl and R3 is
2.5-dimethylphenylamidyl.
Item B4: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-methoxy-5-fluorophenyl and R3 is 3-chlorophenylamidyl.
Item B5: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-methoxy-5-fluorophenyl and R3 is 2- methoxy-5-methylphenylamidyl.
Item B6: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-methoxy-5-fluorophenyl and R3 is
2.5-dimethylphenyloxyl. Item B7: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methylphenyl and R3 is phenylamidyl.
Item B8: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso- butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methylphenyl and R3 is 2,5- dimethylphenylamidyl.
Item B9: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methylphenyl and R3 is 3-chlorophenylamidyl.
Item B10: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methylphenyl and R3 is 2-methoxy-5-methylphenylamidyl.
Item B1 1 : The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methylphenyl and R3 is 2,5-dimethylphenyloxyl.
Item B12: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methoxyphenyl and R3 is phenylamidyl.
Item B13: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methoxyphenyl and R3 is 2,5-dimethylphenylamidyl.
Item B14: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methoxyphenyl and R3 is 3-chlorophenylamidyl.
Item B15: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methoxyphenyl and R3 is 2-methoxy-5-methylphenylamidyl.
Item B16: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 3-chloro-6-methoxyphenyl and R3 is 2,5-dimethylphenyloxyl.
Item B17: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 4-methoxyphenyl and R3 is phenylamidyl. Item B18: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 4-methoxyphenyl and R3 is 2,5- dimethylphenylamidyl.
Item B19: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 4-methoxyphenyl and R3 is 3- chlorophenylamidyl.
Item B20: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 4-methoxyphenyl and R3 is 2- methoxy-5-methylphenylamidyl.
Item B21 : The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 4-methoxyphenyl and R3 is 2,5- dimethylphenyloxyl.
Item B22: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-trifluoromethoxyphenyl and R3 is phenylamidyl.
Item B23: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-trifluoromethoxyphenyl and R3 is
2.5-dimethylphenylamidyl.
Item B24: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-trifluoromethoxyphenyl and R3 is 3-chlorophenylamidyl.
Item B25: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-trifluoromethoxyphenyl and R3 is 2-methoxy-5-methylphenylamidyl.
Item B26: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-trifluoromethoxyphenyl and R3 is
2.5-dimethylphenyloxyl.
Item B27: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-chloro-5-methoxyphenyl and R3 is phenylamidyl.
Item B28: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-chloro-5-methoxyphenyl and R3 is 2,5-dimethylphenylamidyl. Item B29: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-chloro-5-methoxyphenyl and R3 is 3-chlorophenylamidyl.
Item B30: The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-chloro-5-methoxyphenyl and R3 is 2-methoxy-5-methylphenylamidyl.
Item B31 : The compound according to item A1 , wherein R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N-dimethyl; and R2 is 2-chloro-5-methoxyphenyl and R3 is 2,5-dimethylphenyloxyl. Item B32: The compound according to item A1 or A28, characterized by a formula (101 )
Figure imgf000031_0001
Item AB1 : The compound according to any one of items A1 through B18 for treatment of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
Item AB2: A foodstuff or food additive comprising a compound according to any one of items A1 through B18, particularly for use in animal farming.
Item C1 : A compound characterized by a general formula (300)
Figure imgf000032_0001
wherein
- X is S, O or N-methyl;
- R7 is H, F, Cl or C1-C3 alkyl;
- R8 is H, F, Cl or C1-C3 alkyl;
R9 is NH2, NH-methyl, OH or methyl;
- R10 is
Figure imgf000032_0002
wherein, R101, R102, R103, R102‘, and R101’ are independently selected from H, F, Cl, CF3, methyl, and NO2.
Item C2: The compound according to item C1 , wherein
- X is selected from S, O, and N-methyl; and/or
R7 is selected from H and methyl; and/or
R8 is selected from H, Cl, and methyl; and/or
- R9 is selected from NH2, NH-methyl, OH, methyl; and/or
R10 is selected from
Figure imgf000033_0001
Item C3: The compound according to item C1, wherein X is S; R7is H; R8is H; R9is NH ; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C4: The compound according to item C1, wherein X is O; R7is H; R8is H; R9is NH ;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C5: The compound according to item C1, wherein X is N-methyl; R7is H; R8is H; R9is NH ;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C6: The compound according to item C1, wherein X is S; R7is methyl; R8is H; R9is NH ;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C7: The compound according to item C1, wherein X is O; R7is methyl; R8is H; R9is NH ;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C8: The compound according to item C1, wherein X is N-methyl; R7is methyl; R8is H; R9is NH ;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl. Item C9: The compound according to item C1 , wherein X is S; R7 is H; R8 is Cl; R9 is NH2;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C10: The compound according to item C1 , wherein X is O; R7 is H; R8 is Cl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C1 1 : The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is Cl; R9 is NH2;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C12: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is Cl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C13: The compound according to item C1 , wherein X is O; R7 is methyl; R8 is Cl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C14: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is Cl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C15: The compound according to item C1 , wherein X is S; R7 is H; R8 is methyl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C16: The compound according to item C1 , wherein X is O; R7 is H; R8 is methyl; R9 is NH2;and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C17: The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is methyl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C18: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is methyl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C19: The compound according to item C1 , wherein X is O; R7 is methyl; R8 is methyl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl. Item C20: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is methyl; R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C21 : The compound according to item C1 , wherein X is S; R7 is H; R8 is H; R9 is NH- methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C22: The compound according to item C1 , wherein X is O; R7 is H; R8 is H; R9 is NH- methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C23: The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is H; R9 is NH-methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C24: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is H; R9 is NH- methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C25: The compound according to item C1 , wherein X is O; R7 is methyl; R8 is H; R9 is NH- methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C26: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is H; R9 is NH-methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C27: The compound according to item C1 , wherein X is S; R7 is H; R8 is methyl; R9 is NH- methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C28: The compound according to item C1 , wherein X is O; R7 is H; R8 is methyl; R9 is NH- methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C29: The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is methyl; R9 is NH-methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C30: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is methyl; R9 is NH-methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl. Item C31 : The compound according to item C1 , wherein X is O; R7 is methyl; R8 is methyl; R9 is NH-methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C32: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is methyl; R9 is NH-methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C33: The compound according to item C1 , wherein X is S; R7 is H; R8 is H; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C34: The compound according to item C1 , wherein X is O; R7 is H; R8 is H; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C35: The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is H; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C36: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is H; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C37: The compound according to item C1 , wherein X is O; R7 is methyl; R8 is H; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C38: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is H; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C39: The compound according to item C1 , wherein X is S; R7 is H; R8 is methyl; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C40: The compound according to item C1 , wherein X is O; R7 is H; R8 is methyl; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C41 : The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is methyl; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl. Item C42: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is methyl; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C43: The compound according to item C1 , wherein X is O; R7 is methyl; R8 is methyl; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C44: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is methyl; R9 is OH; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C45: The compound according to item C1 , wherein X is S; R7 is H; R8 is H; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C46: The compound according to item C1 , wherein X is O; R7 is H; R8 is H; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C47: The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is H; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C48: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is H; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C49: The compound according to item C1 , wherein X is O; R7 is methyl; R8 is H; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C50: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is H; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C51 : The compound according to item C1 , wherein X is S; R7 is H; R8 is methyl; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C52: The compound according to item C1 , wherein X is O; R7 is H; R8 is methyl; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl. Item C53: The compound according to item C1 , wherein X is N-methyl; R7 is H; R8 is methyl; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C54: The compound according to item C1 , wherein X is S; R7 is methyl; R8 is methyl; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C55: The compound according to item C1 , wherein X is O; R7 is methyl; R8 is methyl; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4- fluorophenyl, and 3-nitrophenyl.
Item C56: The compound according to item C1 , wherein X is N-methyl; R7 is methyl; R8 is methyl; R9 is methyl; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2-chloro,4-fluorophenyl, and 3-nitrophenyl.
Item C57: The compound according to item C1 or C2, characterized by a formula (302)
Figure imgf000038_0001
Item C58: The compound according to any one of the items C1 through C57 for treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabeles, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
The skilled person is aware that any specifically mentioned drug may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.
Wherever alternatives for single separable features such as, for example, a chemical moiety or medical indication are laid out herein as“embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a chemical moiety may be combined with any of the alternative embodiments of medical indication mentioned herein.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Brief description of the figures
Fig. 1 : Homeostatic state modulates feeding behavior, but not spontaneous activity, arousal to visual or acoustic signals nor habituation in zebrafish larvae. (A) Zebrafish larvae were raised under controlled conditions until 7 dpf, loaded into a well of a 96-well plate and fasted for different time-periods prior assessment of distinct behaviors. (B) A custom-built imaging platform can quantify larval feeding behavior by measuring intestinal food content if combined with feeding fluorescently labeled live prey; larval locomotion by tracking swimming behavior; larval arousal by presenting visual and acoustic signals; and larval habituation (non- associative learning) by repeatedly presenting inconsequential stimuli. The two exemplary images show data quality used for larval activity tracking and larval intestinal food content quantification using two distinct invisible light sources. All measurements were acquired under daylight conditions, scale bar - 1 mm. (C) After different fasting periods, larvae were given access to fluorescently labeled paramecia and their intestinal food content quantified for 2 hours. Using a biological inspired curve-fitting algorithm (Jordi, J. et al. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 309, R345-R357.) the inventors extracted total food intake (D), initial intake rate (E), and digestion rate (F), n=48, one- way ANOVA, Dunnett post-test, ***p<0.001. (G) Simultaneously, larval locomotor activity was tracked for 2 hours, n=48, lc - line crosses. (H) Larval zebrafish react to dark flashes with increased motor output. After different fasting periods, larvae were exposed to eight dark flashes (750ms duration, inter-stimuli interval 30s) and their locomotion quantified. The triggered average is shown, n=48. (I) An acoustic stimulus triggers larval locomotion. Identical experiment as in H, but here a single tap was presented instead of a dark flash, n=48. (J) Zebrafish larvae habituate to repetitive, inconsequential stimuli. Here, the inventors presented 30 sequential taps with a short interstimulus interval (2 s) to differently fasted larvae and quantified larvae’s locomotor response. (K) The habituation metric is the activity ratio of the initial and last three taps indicated by the black bars, n=48, one-way ANOVA, Dunnett post-test, p>0.05.
Fig. 2: Available appetite modulators trigger a pleiotropy of behaviors. (A) Schematic of multi-behavior protocol used to test drug impact. Briefly, 2h fasted larvae were pre-exposed to a drug for 30 min prior multi-behavioral profiling. Here, the inventors tested many appetite modulating drugs used in humans. Each drug’s behavioral impact was condensed into a single, quantitative behavioral barcode, which is illustrated here for two classic anorectic drugs - nicotine and rimonabant - in detail, n>12. (B) After giving access to live prey, the inventors quantified larvae’s intestinal food content for 2 h and condensed the fluorescent traces into two feeding periods (F1 , F2). (C-E) Other feeding metrics can be extracted post-hoc, one-way ANOVA, Dunnett post-test, **p<0.01 , ***p<0.001. (F) Simultaneously, larval locomotor activity was tracked and summarized in a single metric (S, spontaneous activity). (G) Following these 2 hours, the inventors presented eight consecutive dark flashes to larvae and measured their activity. The triggered average response was condensed into two time points - visual periods 1 (V1 , first 2 s) and 2 (V2, the following 26 s). (H) Next, the inventors presented eight consecutive taps and measured larval kinematic response. Acoustic period 1 (A1 ) reflects the first 2 s of the triggered average, A2 the following 26 s. (I) Subsequently, a 3 min rest period preceded a sequence of 30 non-sequential taps with short interstimulus interval (2s) used to quantify habituation (H). (J) Finally, 4 dark flashes were presented identically as before in G to determine lethargy (L1 , L2), which is the activity ratio of the initial and post dark flash triggered average. (K) All ten behavioral metrics (F1 , F2, S, V1 , V2, A1 , A2, H, L1 , L2) were acquired on a single well basis and normalized to on-plate vehicle controls (n > 24). Each compound was tested in multiple animals (n > 12), and their behavioral effect size and reproducibility condensed using strictly standardized median difference (SSMD). Each square represents an SSMD value (red, higher; blue, lower than control) for a single behavioral metric and jointly they form a compounds behavioral barcode. The black box indicates the barcodes for nicotine and rimonabant, shown in detail in panel B-J, orange box is feeding behavior, green box - habituation; *barcodes shown for drugs tested at 100 mM. (L) Overview of all compounds tested in the high-throughput screen using the above protocol. Compounds with unknown
pharmacology represent chemical structures with unknown biological activity. (M) Overview of all animals used in the high-throughput screen. Each compound was tested in multiple animals (n >6) and effects quantified relative to on-plate vehicle controls (n > 24). 62 compounds were excluded due to no detectable activity in the last two minutes of the experiment (L1 +L2 = 0), potentially based on strong anesthetic or lethal impact of the tested compound. Behavioral metrics: F, feeding; S, spontaneous activity; V, visual response; A, acoustic response; H, habituation; L, lethargy; 1 or 2, different time periods. Fig. 3: Diversity of drug-induced behavioral changes. (A) Hierarchical clustering groups hit compounds with similar behavioral barcodes based on their correlation. Dendrogram color code localizes selective clusters shown at higher magnification. (B) tSNE groups hit compounds with similar barcodes using non-linear probability distributions and preserves local distance metrics. All 4,801 hits are shown in a two-dimensional space. The tSNE map is false-colored with the primary behavior modulated for a given compound. The magnification of the rectangle is shown in panel (C). (D) tSNE map location of selected hierarchical clusters magnified in panel A. (E) Histogram of the pairwise Pearson correlation ( R ) for compound barcodes sharing one or more targets, or compound barcodes with unknown target (two-tailed, two-sample Kolmogorov- Smirnov test, line depicts median). (F) The chemical structure of two novel compounds with unknown target. PubChem Compound No., Compound Identifier (CID): unknown 1 , 1206526; unknown 2, 2836278. (G, H) Unknown compound 1 behavioral barcode showed high pairwise Pearson correlation with drugs targeting histamine receptor 1 (H1 , R = 0.70±0.13, meaniSD); unknown compound 2 with drugs targeting muscarinic acetylcholine receptor 3 (M3, R = 0.72±0.15). All the relevant barcodes are shown for illustration and the box shows the mean barcode for all H1 or M3 drug relevant for correlation, *depicts trans-tripolidine, L-hyoscyamine, and ipratropium-bromide. (I, J) Competitive in vitro human receptor binding assays for compound unknown 1 and H1 receptor; respectively for compound unknown 2 and M3 receptor with positive control; n=4, meaniSD. (K) Physiochemical properties of all tested compounds. Lines indicate the boundaries of Lipinski’s rule of five. (L) Cumulative histogram of pairwise structural similarities (2D Tanimoto coefficient, T) for compounds within behavioral clusters or shuffle control (two-tailed, two-sample Kolmogorov-Smirnov test). (M) tSNE map is falsely labeled with the pairwise structural similarity coefficient (T) for the illustrated example compound. Each dot represents a different compound (red, similar; blue, dissimilar structures).
A Tanimoto coefficient >0.85 reflects very similar structures. Behavioral metrics: F, feeding; S, spontaneous activity; V, visual response; A, acoustic response; H, habituation; L, lethargy; 1 or 2, different time periods.
Fig. 4: Identification of novel and selective appetite modulators. (A) Barcodes of all hits modulating feeding behavior selectively based on the SSMD threshold sorted by their effect size on feeding period 1 (F1 ). (B) 27 structurally novel compounds were selected based on the displayed behavioral barcodes and unique chemical structures. All compounds were validated and the complete validation data is shown in Fig. 5. (C) Heatmap illustration of in vitro target binding profiles for all 27 compounds across 43 different human targets. All binding assays were validated with a positive control. Targets: adrenergic receptors, a-1A, a-1 B, a-1 D, a-2A, a-2B, b- 1 , b-2; dopamine receptors, D1-D5; gamma- amino butyric acid (GABA)-A receptor, peripheral benzodiazepine receptor (PBR), allosteric benzodiazepine binding site on rat brain slices (BZP); histamine receptors, H1 , H3, H4; muscarinic receptors, M1-M5; serotonin receptors (5- hydroxytryptamine, 5-HT), 1A, 1 B, 1 D, 1 E, 2A, 2B, 2C, 3, 5A, 6, 7; sigma receptors 1 , 2; opioid receptors, d-opioid receptor (DOR), k-opioid receptor (KOR), m-opioid receptors (MOR);
biogenic amine transporters dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT). Behavioral metrics: F, feeding; S, spontaneous activity; V, visual response; A, acoustic response; H, habituation; L, lethargy; 1 or 2, different time periods.
Fig. 5:“Ideal” candidate compounds ideal appetite modulators identified in zebrafish. (A)
Chemical structure of four compounds, two orexigenic candidates (oC) and two anorectic candidates (aC), with to date unknown biological activity. All structure fulfill Lipinski’s rule of five. Two hours fasted individual larval zebrafish (7 dpf) were pre-exposed for 30 min to the candidate compounds prior giving access to labeled food. Subsequently, the inventors quantified larval feeding behavior (B-E), arousal to visual (F) and acoustic (G) stimuli, spontaneous activity (H), habituation (I) and lethargy (J), n=48, one-way ANOVA, Dunnett post- test, ***p<0.001. (K, L) Competitive in vitro human receptor binding assays for aC D and 5- HT2B or 5-HT2C receptor with positive control; n=4, meaniSD. (M, N) Developmental assay - Embryos were continuously exposed to drugs starting 1-hour post-fertilization and survival rates recorded till 6 dpf. Body length was recorded on day 5. EtOH (350 mM) served as positive control, n=27, one-way ANOVA, Dunnett post-test, ***p<0.001. (O) Example locomotion trace of an individual larval zebrafish performing thigmotaxis (wall-loving) in daylight and to a lesser extent in darkness. The time in the outer circle versus the inner circle was quantified as a thigmotaxic index in the presence of candidate compounds, n=21 , one-way ANOVA, Dunnett post-test, ***p<0.001 , scale bar - 10 mm. (P) The swimming path of individual larvae in the phototaxis assay during daylight and darkness, in which one half of the well is covered by a visible light proof shelter. Phototaxis was quantified as the time in the open space versus under the shelter with the treatment of the candidate compounds, n=21 , one-way ANOVA, Dunnett post-test, ***p<0.001 , scale bar - 10 mm. (Q) Turn angles triggered by perpendicular to the larvae’s body axes moving black stripes - the optomotor response - under the influence of the candidate compounds. If larvae turned in the direction of the stimulus, this was quantified as a correct turn, n = 16, two-tailed, two-sample Kolmogorov-Smirnov test, p>0.05. (R) Example swim traces of an individual larval zebrafish in an aversion experimental paradigm, where a noxious stimulus is located at one end of the swimming chamber. The time in either half of the tank was quantified as a preference index with treatment of the candidate compounds in daylight or darkness, n=16, one-way ANOVA, Dunnett post-test, ***p<0.001 , scale bar - 20 mm. (S) Larval zebrafish swim in bouts, short bursts of motor activity, and these characteristic swim kinetics were quantified under the influence of the candidate compounds in a large arena, n = 20, one-way ANOVA, Dunnett post-test, p>0.05, scale bar - 20 mm.
Fig. 6: Orexigenic and anorectic candidate compounds selectively modulated food intake in mice (A, B) Ad libitum fed or 16 h fasted mice received an intraperitoneal injection of candidate compounds (30 mg/kg) and their subsequent food intake was monitored continuously. Their cumulative food intake is shown relative to vehicle control in panel B, n = 10. Summary characteristics of food intake and food seeking behavior during the dark phase in ad libitum fed (C) or 16 h fasted (D) mice, n = 10, one-way ANOVA, Dunnet post-test, *p<0.05, ***p<0.001. Anamorelin (Ana) and rimonabant (Rim) served as positive control. (E) Blood glucose levels measured 30-min post compound application, n = 10, one-way ANOVA, Dunnet post-test, ***p<0.001. Insulin (Ins) served as positive control. (F) Spontaneous locomotion of mice quantified 30-min post compound application, n = 10, one-way ANOVA, Dunnet post-test, *p<0.05. (G) Triggered average displacement response of mice to eight light flashes of 5 s duration and the corresponding light flash index after treatment with candidate compounds, n = 10. (H) Triggered average displacement response of mice to eight acoustic taps and the corresponding tap response index after treatment with candidate compounds, n = 10, one-way ANOVA, Dunnet post-test, *p<0.05. (I) Example locomotion trace of individual mice performing thigmotaxis (anxiolytic behavior) treated with vehicle (V) or rimonabant (rim). The time in the outer versus the inner square was quantified as a thigmotaxic index after treatment with candidate compounds, n = 10, one-way ANOVA, Dunnet post-test, **p<0.01. (J) Fluid intake and saccharine preference of mice measured on the test day of a conditioned taste aversion test. LiCI served as positive control, n = 8, one-way ANOVA, Dunnet post-test, ***p<0.001.
Fig. 7: Orexigenic and anorectic candidate compound impact on mice behaviors. The microstructure of feeding behavior measured after intraperitoneal injection of candidate compounds (30 mg/kg) prior dark onset in ad libitum (A, B) or 16 h fasted mice (C, D) in the dark phase (A, C) and the following light phase (B, D). Anamorelin (Ana) and rimonabant (Rim) served as positive control, n = 10, unpaired Welch’s t-test, *p<0.05, **p<0.01 , ***p<0.001. (E) Blood glucose levels measured prior compound application, n = 10. Insulin (Ins) served as positive control. (F) Saccharine intake of mice measured on the conditioning day prior compound application. LiCI served as positive control, n = 8.
Fig. 8: Spectra of candidate compounds. (A) spectrum oC compound A. (B) spectrum oC compound B. (C) spectrum aC compound C.
Examples
Materials and methods Zebrafish
Adult zebrafish ( Danio rerio) from the mitfa~h (nacre), TL and EK strain were maintained on a 14:10 h lighhdark cycle at 28°C and fed three times daily with live artemia and/or powder food (O. range, INVE aquaculture, Netherlands). All protocols and procedures involving zebrafish were approved by the Harvard University/Faculty of Arts & Sciences Standing Committee on the Use of Animals in Research and Teaching (IACUC). If not stated differently, larvae from the mitfa~h (nacre) strain were used in the experiments.
Zebrafish larvae production and standardized growth-conditions
Larvae from bucket crosses were collected and pooled in blue water (density of >5 ml / larvae, pH 7.2 - sodium bicarbonate buffer, 1 g/l methylene blue, 0.2 g/l instant ocean salt). Live embryos were selected ~24 hpf and transferred to the automated growth system in 2 L tanks. The automated growth system exchanged 50% of the larvae’s growth water (>5 ml / larvae, pH 7.2 - sodium bicarbonate buffer, 0.2 g/l instant ocean salt, continuously oxygenated) four times daily, and fed the larvae starting at 4 dpf with a bolus of >30 ml fresh paramecia culture (optical density, OD490nm = >0.2) 10 min post each water exchange. The automated system was maintained at 28 °C and at a 14:10 h lighhdark cycle.
Paramecia production and labeling
Protozoan pellets (Carolina Biological Supply, Burlington, USA) were dissolved in boiling larval growth water. After cooling, live paramecia and RotiGrow Plus (Reed Mariculture, Campbell, USA) were added to the protozoan water and the culture incubated for a minimum of 2 days at 28 °C prior to use. All paramecia strains were cultured using this protocol and were originally obtained from Ward’s science (Rochester, USA). Visual inspection of paramecia size and structure enabled to distinguish between paramecium caudatum, paramecium bursaria or bdelloidea. If not stated differently, paramecium caudatum was used for all experiments. All paramecia cultures were visually inspected for purity prior to use with light microscopy.
Paramecia were fluorescently labeled using previously described protocols (Jordi et al.
American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 309, R345-R357, (2015).).
Zebrafish imaging platform
The basic concept comes from the infrared macroscope described in Jordi et al (Jordi et al. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 309, R345-R357, (2015).). All components were identical, except for the following changes. These were made to reduce cost and simplify construction, to improve the signal to noise, and to enable diverse behaviors: (1 ) White light (LZ4, LED engine, San Jose, USA) was filtered using a UV-IR bandpass filter (Hoya, Tokyo, Japan). (2) Fluorescent excitation light was generated using a single, cooled 740 nm led (LZ4, LED engine), a lens (12° uniform spot, LED engine) and a squared light diffuser (Thorlabs, Newton, USA). (3) The new generation of IDS camera was used with significantly improved sensitivity in the inventors’ hands in the 780-820 nm range [emission range of fluorophore] (IDS UI-3370CP-NIR, IDS, Obersulm, Germany). (4) A push-tap solenoid (Guardian Electric 28P-I-12D, Allied Electronis, Texas, USA) enables to deliver acoustic stimuli. Four instruments were constructed to enable higher throughput. Emission light calibration was conducted using DiR dye (Molecular Probes, Eugene, USA) dissolved in DMSO at different concentrations, and recording fluorescent emission from the same probes for 100 ms on all the different behavioral imaging platforms. All image platforms showed very similar sensitivity for fluorescence detection.
Zebrafish homeostatic state experiment
7 dpf larvae were collected from the automated system, washed to remove remaining paramecia and transferred into a Petri dishes. Dead and malformed larvae were discarded upon visual identification. Larvae were randomly split into a fed, 2h-fasted and 4h-fasted group. All groups were timed to enable simultaneous behavioral measurements. For the feeding assay, larval zebrafish were transferred to a single well of a flat-bottom 96 well plate (Falcon, VWR, USA) in 150 mI larval growth water. Within 1 min prior to starting behavioral imaging, 50 mI of labeled paramecia culture (OD490nm > 0.5, >500 paramecia/well; see Jordi et al. (Jordi et al. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 309, R345-R357, (2015).) for calibration) were added, the plate transferred to the behavioral imaging platform and the experiment initialized. For all other behaviors, larval zebrafish were transferred to a single well of a flat-bottom 96 well plate in 200 mI larval growth water, subsequently transferred to the behavioral imaging platform and each experiment initialized.
Zebrafish high-throughput chemical screen
7 dpf larvae were collected from the automated system 30 min after the last automatic paramecia supply, washed to remove remaining paramecia and transferred into Petri dishes. Dead and malformed larvae were discarded upon visual identification. Petri dishes were cooled at 4 °C for 5-10 min to immobilize larvae. Two immobilized, randomly selected larval zebrafish were transferred to a single randomly selected well of a flat-bottom 96 well plate in 120 mI larval growth water. Larvae fasted for 2 h at room temperature (23-24 °C) within the multi-well plate. Subsequently, larvae were pre-exposed to 30 mI of compounds (final concentration 10 mM in 0.15 % dimethyl sulfoxide, if not stated differently) for 30 min. Within 1 min prior to starting behavioral imaging, 50 mI of labeled paramecia culture (OD490nm > 0.5, >500 paramecia/well) were added, the plate transferred to the behavioral imaging platform and the experiment initialized. The following compound libraries were tested: DIVERSetE library (ChemBridge, San Diego, USA, > 95% purity); Spectrum library (Microsource Discovery Systems, Gaylordsvill, USA, >95 % purity); Prestwick library (Prestwick Chemical, lllkirch, France, >95 % purity);
Biomol Neurotransmitter Library (BML-2810, ENZO Life Sciences, Farmingdale, USA, >95% purity). Individual compounds were re-ordered from suppliers. For the screen, each compound was tested in 6 animals. A subset of 530 compounds was tested in 12 or 18 animals because identical compounds were contained in multiple libraries. Lorcaserin (AdooQ Bioscience LLC, CA, USA), rimonabant-HCI (Sigma-aldrich, MO, USA) and anamorelin (AdooQ) were acquired at >98% purity.
Validation experiments of zebrafish high-throughput screen and other multi-behavioral experiments.
Validation (Fig. 5), preliminary dose response and appetite-modulating drug (Fig. 2)
experiments were conducted similarly to above. Compounds and different doses were randomly distributed across validation plates and experiments repeated on at least three different days to acquire representative compounds effects. One larva was tested individually for each compound and dose. Larvae were not cooled to simplify platting.
Zebrafish multi-behavioral experiment
The experimental flow is graphically depicted in Fig. 2A. The inventors collected a fluorescent image, required to measure intestinal paramecia content, with 100 ms exposure time every min for 2 h; and simultaneously use transmitted IR images to track in real time spontaneous swimming activity of all zebrafish larvae at a camera frame rate of 10 Hz. After this initial 2 h feeding experiment, 8 dark flashes (750 ms, inter-stimulus interval [ISI] 30 s) followed by 8 taps (ISI 30 s) were presented to larvae. This period was followed by a 3 min rest period, followed by the presentation of 30 taps with 2 s ISI (habituation). After habituation, 4 dark flashes (750 ms, ISI 30 s) were presented to the larvae prior to completion of the experiment.
Zebrafish screen data acquisition and behavioral barcoding
The principles for data acquisition represent the code presented in Jordi et al (Jordi et al.
American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 309, R345-R357, (2015).), but was further optimized. The following adaptations were made enabling measurement of 2 larvae per well: Larval intestines were identified using intensity and size thresholds. Such thresholding led to a number of particles per well, which were sorted by signal strength (area*mean intensity) and the top two stored as intestinal traces. Kinematic traces were quantified as described in Kokel et al., where the number of times an animal crosses any of three parallel imaginary lines are counted (Kokel et al. Nature Chemical Biology 6, 231-237, (2010).). The inventors counted larval line crosses for each well at a frame rate of 10 Hz. Next, the data was pre-processed as follows on a well by well basis (Fig. 2): The fluorescence measurements were binned down to two-time periods reflecting the accumulated fluorescence in the first 40 min (F1 , feeding period 1 ) and the last 80 min of the experiment (F2, feeding period 2), respectively. Concurrent larval locomotion activity was condensed into one bin for the entire 2 h (S, spontaneous locomotion). Next, the inventors aligned the repeats of the initial eight dark flashes, the eight taps, and the four post-dark flashes to construct triggered- averages, each 28 s in length, using an interpolation-based alignment method to generate a standard time base. These time traces were normalized to the period preceding a given stimulus (2 s) to account for differences in spontaneous locomotion. These traces were then condensed into two metrics for the visual response (V, visual response) and the acoustic response (A, acoustic), the first comprising the initial 2 s of the response (period 1 ) and the second comprising the remaining 26 s (period 2). The ratio between the values for the initial and final dark flashes quantified animal’s lethargy (L1 and L2, lethargy period 1 and 2). The 30-tap habituation stimulus was condensed into a single number by taking the ratio of the maximum response values of the last 15 taps to the first 15 taps (H, habituation). Next, the entire plate was normalized to the median of on-plate vehicle controls to yield a fold change with respect to the vehicle. After this final calculation on a per-plate basis, the inventors calculated the Strictly Standardized Median Difference (SSMD) for the values from each compound by matching its replicate wells across plates and their corresponding vehicle control wells. The statistical advantages and limitations of SSMD are extensively discussed elsewhere (Zhang, Journal of Biomolecular Screening 12, 645-655, (2007).). This calculation was performed on the individual metrics coming from the feeding behavior (F1 , F2), spontaneous activity (S), visual response (V1 , V2), acoustic response (A1 , A2), habituation (H) and lethargy (L1 , L2). These ten SSMD values per compound were termed a given compound’s barcode and were used for the clustering detailed below (Kokel et al. Trends in Biotechnology 30, 421-425, (2012).). For the calculation of confidence boundaries, the inventors performed the same SSMD calculations for all vehicle-treated animals split into measurements of 6 animals analogously to the per- compound measurement outlined before. This entire process was performed in a blinded fashion, i.e. there was no a priori knowledge of compounds prior to hit determination. For the analysis of the candidates’ validation (Fig. 5, 9), preliminary dose response and anti-obesity drug (Fig. 2) experiments the inventors slightly modified the aforementioned process to enable measurement of single larvae per well: Fluorescent traces were not binned, the activity traces were condensed to 1 min bin using the fluorescent reads as a common time base. For dark flash, tap and post dark flask the inventors performed trigger-average alignment as above. Habituation reflects the activity ratio of the mean last and initial three taps, if not indicated differently. Validation barcodes were constructed accordingly.
Compound structural similarity, physiochemical properties, and target annotation
Compounds from the Biomol, Spectrum and Prestwick libraries were matched to their PubChem Compound Identification (CID) based on the Chemical Abstracts Service number (CAS), which was provided by the supplier. The Chembridge library was annotated to CIDs by the vendor. PubChem was used to calculate pairwise structural similarity (2D tanimoto coefficient) between all compounds, and to transform CIDs into chemical structure line notations, i.e. simplified molecular input line entry system (SMILE) (Kim et al. Nucleic Acids Research 44, (2016).). Compound physiochemical properties were computed based on compounds SMILE using Open Babel implemented in ChemMine (Backman et al. Nucleic Acids Research 39, W486-W491 , (201 1 ).)· Targets were annotated to CIDs curated from publications and patents using the BindingDB (Liu et al. Nucleic Acids Research 35, D198-D201 , (2007).). If no target was found, the ChemblDB was searched, followed by PDSPKi and finally PubChem (Bento et al. Nucleic Acids Research 42, D1083-D1090, (2014), Besnard et al. Nature 492, 215-220, (2012).). 2007 of the 3334 known molecules were assigned a single target. Cautionary notes: (1 ) many molecules may have a known or unknown poly-pharmacological mechanism of action. For simplicity, here the first identified target was used for annotation. (2) compound target annotation in many cases is based on in vitro or cellular screens, hence, a direct translation to in vivo may reflect an oversimplification. These and other obvious limitations must be considered when assessing the relevance of target annotation.
Clustering and Correlation
Hierarchical clustering (distance metric - correlation; linkage - average), tSNE (perplexity = 25) and Pearson correlation analysis were performed in Matlab (R2015a, The Mathworks,
Massachusetts, USA) (van der Maaten et al. Journal of Machine Learning Research 9, 2579- 2605, (2008).).
Receptor binding assay
In vitro binding assays were performed by the US National Institute of Mental Health
Psychoactive Drug Screening Program (Besnard et al. Nature 492, 215-220, (2012).). Detailed descriptions of the method and data analysis can be found at
https://pdspdb.unc.edu/pdspWeb/content/PDSP Protocols II 2013-03-28.pdf.
Zebrafish developmental assay
Fertilized embroys were collected following natural spawning, visually inspected and three healthy randomly-selected embryo’s placed into a well of standard flat-bottom 6-well plate (VWR) filled with larvae’s growth water. Each well was treated either with DMSO, candidate compound A to D (10 mM) or EtOH (350 mM) one-hour post-fertilization. 6-well plates were maintained on a 14:10 h lighhdark cycle at 28°C. Every 12 hours each embryo was inspected with a stereomicroscope and scored for survival till 6 dpf. At 5 dpf, images were acquired with a stereomicroscope and body length measured using ImageJ.
Zebrafish thigmotaxis assay
7 dpf larvae were collected from the automated system, washed to remove remaining paramecia and transferred into Petri dishes. Larvae were 2h-fasted in a standard flat-bottom 6- well plate prior exposure to the candidate compound’s (10 mM) for 30 min. Subsequently, larval swimming behavior was tracked at 10 HZ by a camera (IDS UI-3370CP-NIR) positioned above the plate, which was illuminated with an infrared light source from below. The camera had an infrared filter mounted to avoid interference from visible light. Briefly, the tracking algorithm subtracted a 10-frames running-average background image, applied a size threshold to identify the fish and extracted its coordinates, heading angle, etc. After recording 10-min of swimming behavior in daylight, the ambient light was shut-off and 10-min swimming was recorded in darkness. For data analysis, each well was split into 2 equal sized areas - border and center. The time periods each fish spent in an area based on its coordinates was summed and the thigmotaxis index calculated as follows: [Time(wall)-Time(center)]/Time(total).
Zebrafish phototaxis assay
The phototaxis experiment was executed identically to the thigmotaxis assay. The main difference being that one half of the well was covered with an infrared light proof shelter (no visible light passes, McMaster, New Jersey, USA). For data analysis, each well was split into 2 equal sized areas - shelter and open space. The time periods each fish spent in an area based on its coordinates were summed and the phototaxis index calculated as follows: [Time(open space)-Time(shelter)]/Time(total).
Zebrafish optomotor-response
7 dpf larvae were collected from the automated system, washed to remove remaining paramecia and transferred into a petri dish. Larvae were 2h-fasted in the petri-dish prior exposure to candidate compounds (10 mM) for 30 min. Subsequently, their optomotor-response was quantified using a closed-loop imaging system and code previously described (Huang et al. Current Biology 23, 1566-1573, (2010).).
Zebrafish aversion assay
Zebrafish larvae (7 dpf from the automated system) fasted for 2h in a petri-dish prior exposure to candidate compounds (10 pM) for 30 min. Next, they incl. the drug were transferred into a custom built rectangular well with the following dimensions: 1.8x9.2x0.4cm (wxlxh). A chamber at the width side allowed for the placement of a firm agar pad. One agar pad (2 % agar in larvae’s growth water) contained mustard oil (noxious stimuli, 20 % (v/v) Allyl isothiocyanate, Sigma) and the other contained no additives. After recording 10-min of swimming behavior in daylight, the ambient light was shut-off and 10-min swimming was recorded in darkness using the same setup and algorithm as for thigmo- and phototaxis. Aversion was quantified by splitting the rectangle into two equal sized half’s - noxious and agar side. The time periods each fish spent in an area based on its coordinates was summed and the aversion index calculated as follows: [Time(noxious)-Time(agar)]/Time(total). Zebrafish free-swimming
7 dpi larvae were collected from the automated system, washed to remove remaining paramecia and transferred into a petri dish. Larvae were 2h-fasted in the petri-dish prior exposure to the candidate compounds (10 mM) for 30 min. Subsequently, larval free-swimming behavior was recorded for 10-min in in daylight within a standard flat bottom petri-dish
(diameter, 100mm) using the algorithm outlined above for thigmo- and phototaxis. Extracted coordinates with associated timestamps were used to calculate average swim speed, bout length, and bout frequency.
Mice
4-month-old male C57BL/6J mice (Charles River, Sulzfeld, Germany) were individually housed in wire-mesh hanging cages or in standard mice cages at a room temperature of 21 ±1 °C and an artificial 12/12 h light/dark cycle. Animals were able to see, hear and smell their conspecifics in neighboring cages and were not socially isolated. Water, food (mice chow-3436, Kliba Nafag, Kaiseraugst, Switzerland) and bedding was provided ad libitum, if not indicated differently. All procedures for mice handling and experimental interventions were according to Swiss Animal Welfare laws, approved by the“Kantonales Veterinaramt Zurich” and conform to the principles of UK regulations. Animals were adapted to novel housing situation and feeding schedule at least for 1 week. All experiments were conducted three times with saline injections prior to drug administration to habituate the animals to the experimental procedure.
Mice compound application
Individual compounds (candidate compounds, rimonabant, anamorelin) were obtained from suppliers at a purity of >95% and dissolved in dimethylsulfoxide (Sigma). Compounds were injected intraperitoneally diluted in 0.9% NaCI (max. 8% DMSO, max. 250 pi/ 10 g BW) at a concentration of 30 mg/kg, if not stated differently. LiCI (0.15M, Sigma) and insulin (1 unit/kg, Humalog, Lilly, IN, USA) were dissolved in 0.9% NaCI directly (no DMSO, max. 250 mI/ 10 g BW).
Mice feeding-behavior
Ad libitum fed or 16 h fasted mice received an intraperitoneal injection of candidate compounds within 15 min before dark-onset. Food was made available at dark-onset to the 16 h fasted mice. Food intake was measured continuously in undisturbed mice using an automated system (BioDAQ, Research Diets, NJ, USA). This system measures food hopper weight (± 0.01 g) at 1 Hz resolution. The microstructure of feeding was analyzed using proprietary software (BioDAQ Monitoring Software) as follows: Absolute food hopper weight changes smaller than 0.02 g within a 5 s time interval were counted as food-seeking bouts. Absolute food hopper weight changes of 0.02 g or larger represent a meal and were summed into a single meal based on a 10 min inter-meal interval.
Mice blood glucose
3 h fasted mice received a tail cut for immediate blood glucose measurement (Breeze 2, Bayer AG, Leverkusen, Germany). Subsequently, candidate compounds were injected
intraperitoneally, mice were returned to their home cage for 30 min before a second blood glucose measurement was performed as described before.
Mice kinematic measurement
3 h fasted mice were injected intraperitoneally with a candidate compound and were placed into a clear plastic chamber (58X38X20 cm) 30 min post-injection. Subsequently, mice displacement was tracked online at 16 Hz by a camera (Logitech C930e HD) positioned above the chamber which was illuminated with an infrared light source from below. The camera had an infrared filter mounted to avoid interference from visible light. Briefly, the tracking algorithm subtracted a 10- frames running-average background image, applied a size threshold to identify the mouse (min. motion detected was 5 mm) and extracted its coordinates. Extracted coordinates with linked timestamps were used to calculate displacement. Mice behavior was recorded for 15 min in complete darkness and the final 10 min were analyzed.
Mice tap and white light response
3 h fasted mice were injected intraperitoneally with a candidate compound and were tracked in complete darkness 30 min post-injection as outlined above. After recording undisturbed behavior for 15 min, eight taps (75-85 Db) were applied to the mouse cage with an inter- stimulus interval of 30 s. The inventors aligned the repeats of the eight taps to a triggered- average using an interpolation-based alignment method to generate a standard time base. These time traces were normalized to the period preceding a given stimulus (2 s) to account for differences in spontaneous locomotion and the tap response index calculated as follows:
median displacement (time period 2-28 s after the tap) / median displacement (time period 0-2 s immediately after the tap). The response of mice to white light flashes was tested similarly to the tap-response. Instead of taps, eight periods of 5 s white light flashes (300 lux, UV, and IR light filtered) were repeated every 30 s. The flash response index was calculated as follows: median displacement (time period of light) / median displacement (time period of darkness).
Mice thigmotaxis assay
3 h fasted mice were injected intraperitoneally with a candidate compound and were tracked in complete darkness 30 min post-injection with the apparatus outlined above. For data analysis, the cage was split into 2 equal sized areas - border and center square. The time periods that each mouse spent in an area based on its coordinates were summed and the thigmotaxis index calculated as follows: [Time(wall) - Time(center)] / Time(total).
Mice conditioned taste aversion test
14 h water deprived mice received access to 0.15% saccharin (Sigma) solution for 2 h on the conditioning day. Next, candidate compounds were injected intraperitoneally, saccharine was removed and water access provided for 8 h. On the subsequent test day, 14 h water deprived animals received simultaneously access to water and saccharine; and their fluid intake was measured for 6 h.
Statistics
All experiments were randomized and data analysis was performed blindly. Statistical tests were used as appropriate and performed using GraphPad Prism 7.02 (GraphPad Software, California, USA) or Matlab. Data are presented as mean±SEM, if not indicated differently.
Compounds Synthesis
Figure imgf000052_0001
To a solution of diethyl oxalate (2.0 ml, 15 mmol) and 2-acetylthiophene (1.1 ml, 10 mmol) in anhydrous ethanol (50 ml) was added NaOEt (2.0 g, 30 mmol). The suspension was stirred at room temperature until TLC indicated the absence of starting material after which the reaction was quenched by addition of 1 M HCI. The solvent was removed in vacuo and the residue extracted with ethyl acetate and the organic layer dried over anhydrous Na2S04. The residue was purified by flash column chromatography (S1O2, ethyl acetate/hexanes) to afford the title compound as a brownish solid (1.8 g, 80%).
The above obtained diketone (1.2 g, 5.3 mmol), 2-amino-5-methyl-1 ,3,4-thiadiazole (0.61 mg, 5.3 mmol) and 4-allyloxybenzaldehyde (0.81 ml, 5.3 mmol) were suspended in 1 ,4-dioxane (40 ml) and heated to 70 °C for 20 h. The reaction mixture was concentrated and purified by flash column chromatography (S1O2, ethyl acetate/hexanes) to afford compound 201 as an off-white solid (1.2 g, 51 %). 1H-NMR (DMSO-d6): 7.32 (d, J = 7.4 Hz, 1 H), 7.89 (d, J = 8.2 Hz, 1 H), 7.30 (d, J = 8.0, Hz, 2H), 6.77 (d, 8.0 Hz, 2H), 6.14 (s, 1 H), 6.02 - 5.91 (m, 1 H), 5.35 (ddd, J = 10.3, 1.5, 1.4 Hz, 1 H), 5.11 (ddd, J = 17.2, 1.5, 1.4 Hz, 1 H), 4.96 (d, J = 6.6 Hz, 2H), 2.62 (s, 3H).
Figure imgf000053_0002
A solution of 4-chloro-2-methoxyaniline (2.5 g, 15.8 mmol) and triethylamine (3.4 ml_, 23.9 mmol) in anhydrous CH2CI2 (50 ml.) was added methanesulfonyl chloride (1.26 ml_, 15.8 mmol) dropwise at room temperature. The resulting suspension was stirred at room temperature until TLC indicated complete consumption of starting material. The solvent was removed under reduced pressure and the residue was diluted with ethyl acetate and filtered. The filtrate was washed with water and brine and then dried over anhydrous Na2S04. Concentration in vacuo afforded crude product which was used in the next step without further purification.
To the crude from above in acetone (100 ml.) was added K2CO3 (6.18 g, 45 mmol), Kl (248 mg, 1.5 mmol) and 2-chloro-N-2,5-dimethylphenylacetamide (2.9 g, 15 mmol) and the suspension was stirred at 55 °C for 12 h. The solvent was removed by concentration in vacuo and the residue was diluted with ethyl acetate and filtered. The filtrate was washed with water and brine (50 ml. each). The organic layer was dried over anhydrous MgS04, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (S1O2, ethyl acetate/hexanes) to afford compound 101 as a colorless solid (2.1 g, 35% yield). 1H-NMR (DMSO-d6): 9.14 (s, 1 H), 7.57 (d, J = 1.2 Hz, 1 H), 7.40 (dd, J = 7.2, 1.8 Hz, 1 H), 7.22 - 7.13 (m, 2H), 7.06 (d, J = 8.5 Hz, 1 H), 6.89 (d, J = 8.3 Hz, 1 H), 4.38 (s, 2H), 3,90 (s, 3H), 3.1 1 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H).
Figure imgf000053_0001
Powdered KOH (2.9 g, 51.3 mmol) was added to a suspension of 4,6-dimethyl-2-thioxo-1 ,2- dihydropyridine-3-carbonitrile (2.8 g, 17.1 mmol) and 2-bromo-4’-nitroacetophenone (4.2 g, 17.1 mmol) in anhydrous EtOH (100 ml). The resulting thick suspension was stirred at room temperature for 6 h. The suspension was concentrated to half volume and diluted with ethyl acetate. The mixture was filtered and the filtrate washed with concentrated citric acid (3x100ml), water (100 ml) and brine (100 ml). The organic layer was dried over Na2S04, filtered and concentrated to afford compound 301 as a yellow crystalline solid (2.2 g, 39%). 1H-NMR (DMSO- d6): 8.37 (d, J = 8.3 Hz, 2H), 8.18 (br, 2H), 7.99 (d, J = 8.2 Hz, 2H), 7.14 (s, 1 H), 2.78 (s, 3H), 2.52 (s, 3H). Example 1: The homeostatic state modulates feeding behavior in zebrafish
Towards identifying feeding selective compounds, the inventors designed a whole organism high-throughput screening system, that enabled them to quantify - in addition to feeding - a series of other vital behaviors in larval zebrafish. Larvae growth and development was standardized to guarantee similar baseline conditions in all experimental animals (Fig. 1A). Prior to behavioral phenotyping at 7 days-post-fertilization (dpf), larvae were fasted for different time periods and placed into a 96-well plate. Subsequently, a custom-built imaging platform detected several distinct behavioral phenotypes in all 96 animals simultaneously (Fig. 1 B): first, larval zebrafish hunt and ingest live paramecia, a unicellular protozoan roughly 100 pm size, and thereby enable the quantitative characterization of feeding behavior. Here, the inventors fluorescently-labeled the prey and the imaging platform quantified larval food intake by detecting intestinal fluorescence content for 2 hours (Fig. 1 C). Biologically grounded curve-fitting algorithms enabled the inventors to extract key characteristics of feeding behavior, namely total food intake, initial intake rate and digestion rate. Different fasting periods modulated these parameters analogous to observation in adult rodents and humans (Fig. 1 D-F). Second, in parallel to the fluorescent feeding assay, the inventors simultaneously tracked larval locomotion to quantify their swimming activity for 2 hours (Fig. 1 B, G). Third, the inventors presented short periods of darkness to which larval zebrafish react with an increased motor output (Fig. 1 H). Fourth, the inventors applied a single mechanical tap to the plate, causing larvae to respond with a stereotypic startle response (Fig. 11). Fifth, larvae habituated to a series of high-frequency taps (Fig. 1 J, K). Interestingly, neither spontaneous activity (Fig. 1 G), responsiveness to visual (Fig. 1 H) or acoustic stimuli (Fig. 11) nor habituation (Fig. 1 J, K) was affected by different fasting periods and their underlying homeostatic state, whereas feeding behavior (Fig. 1 C-F) was strongly modulated indicating a lack of inter-dependence of these behaviors and, possibly, the underlying neuronal circuitry. All these behaviors were quantifiable in a multi-well plate and therefore amenable to large-scale chemical screening.
Example 2: Current appetite modulators are not selective for feeding behavior
To establish the value of the here developed multi-behavioral strategy, the inventors first tested many appetite-modulating drugs previously used in humans. Briefly, we pre-exposed 2h-fasted larval zebrafish to a single compound, as shown here for nicotine and rimonabant; prior to giving them access to live, fluorescently-labeled prey (Fig. 2A). Subsequently, the custom-built imaging platform quantified larval food intake by detecting intestinal fluorescence content (Fig. 2B-E); and tracked larval locomotion to quantify their spontaneous activity (Fig. 2F),
responsiveness to visual (Fig. 2G) or acoustic stimuli (Fig. 2H), habituation (Fig. 2I) and lethargy (Fig. 2J). Nicotine and rimonabant both significantly reduced food intake (Fig. 2B-E). Additional nutritional and endocrinal interventions were previously demonstrated to have an analogous impact on feeding behavior in fish, rodents, and humans. Furthermore, nicotine increased spontaneous activity, yet had no significant impact on any other tested behaviors (Fig. 2F-J). Rimonabant decreased spontaneous activity, reduced the habituation response and modulated lethargy, but had no impact on the response to visual stimuli (Fig. 2F-J). In an effort to simplify behavioral quantification, the inventors normalized each compound’s behavioral effect size to on-plate vehicle controls (n > 24 per plate) and condensed their quantitative behavioral impact into behavioral barcodes accounting for reproducibility and effect size by an observer- independent approach (Fig. 2K, black box). Next, the inventors tested additional appetite modulating drugs previously used in humans and showed, that all of these drugs pheno-copied their behavioral effect in zebrafish larvae (Fig. 2K, first two columns, F1 and F2, orange box). Critically, all of these drugs altered a pleiotropy of larval behaviors (Fig. 2K), which is representative for the limited selectivity of current appetite-modulating drugs. Distinct side effects had led to the market retraction for the majority of these anorectic drugs. Noteworthy, the recently approved Lorcaserin, a serotonin receptor 2C agonist, reduced food intake and the habituation response in zebrafish (Fig. 2K) and also alters a pleiotropy of rodent behaviors. This lack of behavioral selectivity resembles the unspecific neuronal impact observed after rimonabant treatment in rodents. Anamorelin, a ghrelin receptor agonist, is the exception of the here tested compounds being an appetite enhancer currently undergoing clinical development. Hence, the inventors demonstrated a conserved vertebrate neuropharmacology for many mammalian appetite modulating agents and showed their lack of behavioral selectivity, which is a potential warning sign for neuronal side effects.
Example 3: Multi-behavioral profiles link compounds to their molecular mechanism of action independent of the underlying chemical structure
Motivated by these findings, the inventors ought to screen for ideal psychoactive small molecules, that is, compounds which induce only their intended, single behavioral change. To that end, the inventors performed a high-throughput chemical screen to identify such putative selective bioactive compounds. The inventors acquired quantitative multi-behavioral barcodes for a total of 10,421 compounds, each individually tested in at least 6 animals (Fig. 2L-M).
Internal and external controls established screen quality and reproducibility using a standard statistical approach. This first large-scale multi-behavioral dataset revealed a remarkable diversity in the impact that various compounds have on different behaviors (Fig. 3A) and enabled the inventors to address four general questions in drug discovery and neuroscience.
First, the inventors tested whether compounds with a similar molecular target share similar behavioral phenotypes. A high similarity between multi behavioral barcodes from a drug with known mode of action and a compound with unknown target should predict the molecular target of the unknown compound. To address this hypothesis, the inventors took advantage of the quantitative nature of the behavioral barcodes to form clusters with similar functional phenotypes. Two complementary algorithms (hierarchical clustering (Fig. 3A left) and t- distributed stochastic neighbor embedding [tSNE], (Fig. 3B - D)) segregated behavioral barcodes into compounds that selectively alter a single behavior. Molecules with multi- behavioral phenotypes populated the transition space between major clusters (Fig. 3A right,
3B). Within those, compounds triggered all combinatorial forms of behaviors, such as general loss or gain of function effects. A subset of molecules caused more complex, multi-dimensional phenotypes, such as enhanced acoustic response and reduced habituation or reduced food intake and increased spontaneous activity (Fig. 3A right, 3B). Importantly, barcodes of drugs that share one or multiple targets correlated significantly better compared to compounds with unknown mechanisms (p=6x10 24, Fig. 3E), thereby confirming the assumption that drugs sharing molecular targets trigger similar behavioral consequences. Following this logic, the inventors found two compounds with an unknown mechanism of action that had a strong correlation with known drugs targeting either histamine receptor 1 (H1 ), or muscarinic acetylcholine receptor 3 (M3), respectively (Fig. 3F-H). This behavioral phenotype correlation suggested a shared mechanism. In vitro competitive binding assays confirmed an interaction of these two unknown compounds with the H1 or M3 receptor, respectively (Fig. 3I-J). Thus, the inventors identified two novel molecules binding human H1 and M3 receptor with nanomolar affinity in vitro as well as an in vivo behavioral phenotype. Importantly, the measured binding affinities are in the nanomolar range, which is at least one order of magnitudes stronger compared to compounds normally identified by in vitro targeted high-throughput screens (mM - mM range, see also Fig. 4C). In sum, a compound’s molecular target is a predictor for its multi- behavioral impact in vivo.
Second, the inventors questioned how a compound’s chemical composition and structure influences its in vivo bioactivity and behavioral selectivity. The inventors did not detect major differences in physiochemical properties between inactive and active compounds (Fig. 3K), though it should be mentioned that we intentionally biased our screening library toward drug-like molecules. To explore whether similar chemical structures caused related behavioral phenotypes, the inventors computed structural similarity scores (2D Tanimoto similarity) for all molecules within a behavioral cluster and compared them to a shuffled control. The inventors found that functional clusters were not enriched with similar chemical structures (p=0.99; Fig. 3L-M). Hence, a compound's chemical structure is, at least in the inventors’ hands, not a good predictor for its in vivo impact on vertebrate behavior.
Example 4\ The zebrafish central nervous system controls different behaviors by an
autonomous neuronal circuitry Third, the inventors hypothesized that if two different behaviors are orchestrated by the same or overlapping neuronal circuitry, they should correlate with respect to their behavioral barcodes. For instance, a larva responding to visual stimuli quickly relative to other larvae may also respond more quickly to acoustic stimuli if both behaviors are controlled by the same neuronal circuitry. If a drug affects this shared circuit, the inventors would expect both behaviors to be altered similarly. To quantitatively test this hypothesis, the inventors investigated the
correlations among all tested behaviors across all animals. The analysis from the behavioral barcodes of more than ten-thousand control animals revealed strong linear correlation within a specific behavioral group, e.g. feeding periods F1 and F2 (Pearson correlation R>0.65; two- tailed p<1x10 250), however, different behaviors, e.g. visual and acoustic response, did not correlate linearly at a meaningful level (Pearson correlation R<0.25). Importantly, the intervention with different small molecules did not reshuffle this inter-behavioral interaction. In line with this observation, 78 % of all hits modulated a single behavior based on a standard statistical threshold, of which the majority upregulated a behavioral response relative to vehicle control. Overall, the different behaviors’ orthogonality in term of drug response suggests that each neuronal circuit has unique features targetable by small molecules. Hence, the toolbox that each separate circuit is built from doesn’t appear to be the same in the zebrafish central nervous system.
Example 5: Novel appetite modulators are potent and selective for feeding behavior and the majority is mechanistically independent of the main neurotransmitter systems
Finally, the existence of ideal, behavior-selective small molecules has been questioned, in particular for appetite modulators. Current appetite modulating drugs, which were or are used by humans, demonstrated a remarkable lack of behavioral selectivity in zebrafish (Fig. 2K). A significant advantage of the multi-behavior strategy developed here is that it allows for the selection of hits not only based on the strength of their effect but also on their selectivity. The inventors’ screen identified 373 orexigenic and 145 anorectic molecules based on a standard statistical threshold, however, only 194 and 74 were selective for feeding behavior (Fig. 4A).
The hit rate for selective orexigenic compounds was 1.8 % and for selective anorectic compounds 0.7 %. Consequently, the selectivity filter reduced the hit rate by roughly 50% illustrating one advantage. Of these 268 selective appetite modulators, the inventors selected, after inspection of the individual barcodes and the chemistry, eleven orexigenic and eleven anorectic candidate compounds with unique chemical structures for validation (Fig. 4B).
Additionally, the inventors included 5 compounds with no significant phenotype as a negative control. To date, these candidate small molecules have no known biological activity and therefore represent novel intervention options for basic research with potential for clinical development. Dose-response studies confirmed their selective impact on feeding behavior over a dose range from 0.6 to 20 mM. Generally, the drug-induced orexigenic effect was comparable to doubling the fasting period and the magnitude of the anorectic effect to a reduction of fasting times by 50-75 %. Further validation studies in two distinct zebrafish wildtype strains, TL and EK, confirmed the compounds efficacy and selectivity in two distinct genetic backgrounds. To identify underlying molecular mechanisms, the inventors tested these twenty-two candidate compounds for their binding affinity to 43 human neuronal targets using in vitro competitive binding assays (Fig. 4C). The inventors found three noteworthy features: First, a few candidate compounds bound with nanomolar affinity to targets that are established regulators of feeding behavior in mammals (histamine receptor 1 , serotonin receptor 2B or C [5-HT2B, 5-HT2C] and peripheral benzodiazepine receptor [PBR]), thereby independently validating our discovery strategy. Second, some compounds interacted with many targets indicating the presence of polypharmacology. Third, the majority of candidate molecules did not bind to any of the tested receptors with high affinity, suggesting that they act by non-tested or unknown molecular mechanisms. Consequently, these twenty-two orexigenic and anorectic compounds represent unique pharmacological tools to selectively modulate feeding behavior in zebrafish.
Example 6: Orexigenic and anorectic candidate compounds enable the selective control of appetite in zebrafish and mice
To further challenge the selectivity of these novel compounds, the inventors chose four of these for more detailed characterization. These four molecules fulfill concepts of drug-likeness (Fig. 5A). Orexigenic candidate (oC) compound A and B doubled food intake, whereas the two anorectic candidate (aC) compounds C and D reduced food intake by more than 50 % in zebrafish (Fig. 5B-E, routes of synthesis shown in scheme 1-3, spectra shown in Fig. 8). None of the candidate compounds modulated the responsiveness to visual (Fig. 5F) or acoustic stimuli (Fig. 5G), spontaneous activity (Fig. 5H), habituation (Fig. 5I) and lethargy (Fig. 5J). aC compound D bound to PBR, 5-HT2B and 5-HT2C receptor with nanomolar affinity (Fig. 5K, L), whereas the other candidate small molecules did not bind any of the 43 human neuronal targets with a millimolar affinity (Fig. 4C). Norfenfluramine, the active metabolite of the former anti- obesity drug fenfluramine, binds 5-HT2B and 5-HT2C receptor with similar affinities as aC D. 5- HT2C receptor activity is considered the driver for fenfluramine’s anorectic effect and is the primary target of the recently approved anti-obesity agent lorcaserin. To detect potential toxic and mutagenic small molecule effects, the inventors monitored zebrafish development under the exposure of candidate drugs in vivo using light microscopy from the single fertilized cell- stage to the 6-dpf behaving larvae. None of the candidates had a significant impact on embryological mortality, developmental defects or temporal delays nor on the body length at 5 dpf, whereas EtOH exposure significantly reduced survival after hatching and lead to lordosis as reported previously (Fig. 5M, N). As the inventors were unable to detect an impact of the candidate compounds on normal vertebrate development, they tested for a drug impact on more subtle behavioral characteristics. These behaviors required the analysis of single fish at high temporal and spatial resolution and therefore are non-suitable for high-throughput screening. First, the inventors assessed thigmotaxis (or“wall-loving”), which is a validated behavioral index for anxiety in animals. Anxiolytic drugs reduce thigmotaxis, whereas anxiogenic compounds enhance it in zebrafish larvae. Candidate compound A-D did not modulate the thigmotaxic response in daylight compared to vehicle control nor did they interfere with the characteristic reduction of thigmotaxis observed with the onset of darkness (Fig. 50). Second, the inventors assessed the impact of the candidate compounds on two sensitive visual behaviors - phototaxis and the optomotor response. Larval zebrafish are attracted by light and averse to darkness, hence, perform positive phototaxis. Candidate compound A-D treated larvae executed phototaxis with similar precision as vehicle-treated animals and reduced their place preference during darkness accordingly (Fig. 5P). The optomotor response is an orienting behavior evoked by visual motion. A closed loop setup presented a grating moving perpendicular to the body axes of an individual zebrafish. The stimulus elicits a turning behavior within the direction of the stimulus. The inventors did not detect a significant impact of candidate compounds on the correct execution nor the turn-angle used during the optomotor response (Fig. 5Q). Third, the inventors quantified the responsiveness of larvae within a novel aversive behavioral paradigm. Larvae avoided the presence of noxious stimuli (here, mustard oil), but had no preference if presented a non-noxious stimulus (agar). This preference was not visually mediated as it was detectable during daylight and darkness. Larva treated with the candidate compounds avoided the noxious stimuli analogously to vehicle control (Fig. 5R). Fourth, larval zebrafish swam spontaneously in an open arena and their locomotion exhibited a characteristic segmentation into an individual burst of locomotion, also called a swim bout. The inventors traced larval swim locomotion in the presence of the candidate compounds and, subsequently, performed detailed kinematic analysis. Neither larval average swim speed, bout length, bout- nor interbout duration were altered by the candidate compounds (Fig. 5S). Hence, all the candidate compounds selectively modulated feeding behavior in zebrafish larvae while they showed no detectable impact on development and did not interfere with the execution of an array of behavioral tasks.
Next, the inventors evaluated the translational potential of candidate compounds, as well as their generalized applicability to diverse vertebrate animals, by testing their efficacy in an adult mouse model. To that end the inventors followed the above logic to evaluate potency and selectivity on multiple mice behaviors. Acute systemic small molecule administration into the peritoneum (IP, 100 mg/kg, n=3) had no qualitatively detectable impact on alopecia, whisker- loss, dermatitis, tremor, kyphosis, distended abdomen, tail stiffening, nesting or diarrhea in mature mice. Initially, the inventors focused the quantitative behavioral assessment in analogy to the zebrafish behavior screen. First, the inventors measured candidate compounds’ impact (30 mg/kg IP) on the microstructure of food intake in undisturbed mice continuously (Fig. 6A). Mice had access to food ad libitum or were fasted for 16 hours prior to the experiment. These two complementary paradigms reflect distinct physiological states of appetite. oC compound A and B increased food intake with distinct temporal dynamics in ad libitum feed mice (Fig. 6B, C) but had no significant impact in 16 h fasted mice, probably due to an appetite ceiling effect (Fig. 6D). Anamorelin had a similar impact on food intake as the oC compounds. These effects originated from an increase in meal numbers and a reduction of the meal time (Fig. 7A). oC compound A also increased food-seeking behavior (Fig. 6B). aC compound C and D, on the other hand, reduced food intake in ad libitum fed and 16 h fasted mice with similar kinetics (Fig. 6B-D) based on a smaller meal size (Fig. 7C). Rimonabant induced a more potent anorectic effect in the dark-phase compared to aC compounds, and also only rimonabant triggered compensatory food intake in the following light-phase (Fig. 7D). Second, the inventors tracked mice locomotion to quantify their spontaneous activity. None of the candidate compounds had an impact on undisturbed mice locomotion, whereas hypoglycemia induced by insulin injection reduced locomotion (Fig. 6E, F). Third, the inventors presented short light-flashes (5s) to which mice did not show a detectable locomotion response independent of treatment (Fig. 6G).
Fourth, the inventors applied a single mechanical tap to the mice behavioral chamber, causing mice to freeze (Fig. 6H). Only rimonabant treatment modulated the response to the acoustic tap in mice as observed in zebrafish (Fig. 2K). Finally, the inventors wanted to further benchmark the behavioral selectivity of our compounds by selecting additional behaviors based on the previously observed problems with appetite modulators: specifically, the inventors considered glucose homeostasis, psychiatric effects, and nausea. First, blood glucose levels were not altered 30-min post candidate compound injection in mice, whereas insulin injection induced hypoglycemia (Fig. 6E). Blood glucose levels were similar across experimental groups’ prior to injection (Fig. 7E). Second, the inventors assessed thigmotaxis in mice, which is a behavioral index for anxiety. None of the candidate compounds modulated the thigmotaxis response, whereas rimonabant increased murine anxiety as reported previously (Fig. 6I). Third, the inventors evaluated if candidate compounds trigger nausea by measuring conditional taste aversion. Candidate compound treated animals maintained a strong preference for conditioned saccharine, whereas LiCI-treated animals reduced the intake of this sweet tasting fluid (Fig. 6J). Fluid intake was similar across experimental groups on the test day (Fig. 6J) as was saccharine intake on the conditioning day (Fig. 7F). Hence, candidate compounds did not induce a conditioned taste aversion. In sum, all the candidate compounds selectively modulated feeding behavior in adult mice while they showed no detectable impact on a series of complex mice behaviors.
Overall, the inventors demonstrated the existence of ideal, behavior-selective small molecules in two vertebrate species. In the context of appetite modulators, the inventors identified 268 compounds that are novel and to date have no described in vivo bioactivity. Twenty-two of these candidates were extensively validated and their behavioral selectivity confirmed in multiple follow-up studies. Furthermore, the majority of these did not bind to known receptors from main neurotransmitter systems. Four of these, the two orexigenic and two anorectic candidate compounds, were benchmarked in a variety of sophisticated behavioral assays and were shown to exclusively modulate appetite in zebrafish and in mice. To further illustrate the generalizability of this unbiased psychoactive drug discovery strategy, the inventors performed a similar analysis for compounds selective for habituation (Fig. 7). In sum, the inventors identified ideal, behavior-selective small molecules, which exclusively promote or inhibit distinct vertebrate behaviors in two vertebrate species.
Discussion
The large-scale chemical and multi-behavior screen developed here proved to be a powerful and unbiased tool for [1] the analysis of links between a compound’s function, target and structure; [2] the identification of novel and ideal neuroactive compounds for different vertebrate behaviors; and [3] the investigation of the interaction and interdependency of different vertebrate behaviors. The inventors consider these findings to have major implications for drug discovery and neuroscience.
First, modern medicine relies on drugs to control disease progression and the study of drug action has revealed many biological principles. Here, the inventors developed an unbiased in vivo drug screening strategy to identify compounds modulating a clinically relevant behavior, namely feeding. Such chemical interventions are perturbances to the molecular mechanisms underlying behavior, and therefore invaluable tools for future in-depth mechanistic
understanding. Identifying each individual compound’s in vivo mechanism is a heroic future challenge for neuropharmacology. These novel molecules, in addition to being selective pharmacological tools to the community, are defined chemical backbones for drug development targeting human disease. The conserved neurobiology and physiology between zebrafish, mice, and humans will simplify such translational ambitions. Species differences need to be considered, but they exist between all animal model systems. Overall, these novel compounds establish the existence of ideal psychoactive small molecules, namely compounds triggering only their intended, single behavioral change in vivo.
Second, the strength of phenotype screens is their superior ability to identify“first-in-class” drugs compared to target-based strategies. Traditionally, such screens only employ effect size in their hit selection criteria. Here, the inventors considered compound selectivity at the initial stage, which reduced the hit rate by ~50 % compared to the standard effect size criterion, and led to the selection of more specific in vivo phenotypes. This selectivity can arise either from the interaction of compounds with unknown molecular targets that are unique to a behavior or from systemic drug action balanced optimally across different mechanisms. Both are impossible to identify without a whole-organism approach. Taking the above into account, future strategies should combine a series of complex behaviors - e.g. sexual behavior, feeding, and social interactions - and systematically quantify the impact of a chemical or genetic intervention with unsupervised methods. If throughput is maintained at the scale established here, such a pipeline is applicable for combinatorial drug discovery, for the chemical rescue of disease- relevant monogenetic mutations, and for fragment-based chemical screens. In addition, such assays can serve as a standard drug safety control early in pre-clinical development and may prevent unexpected behavioral side-effects. Ultimately, this multi-behavioral strategy enables selection of more specific phenotypes, which may result in a more comprehensive
understanding of in vivo drug mechanisms.
Third, diverse schools of thought aim to decode the organizational principles underlying brain function and its capability to control behavior. Here, the inventors find that all tested zebrafish behaviors were decoupled from each other across thousands of control animals and a behavioral selectivity for numerous pharmacological interventions. This independence in behavior-generating output architecture favors a functional brain structure along orthogonal modules in zebrafish, which genetic studies supported even for goal-driven behaviors like feeding in distinct animal model systems. For instance, among the best-understood genetically- defined brain circuits are the hypothalamic agouti-related peptide secreting neurons, which have been studied extensively in the last twenty years and considered crucial for feeding behavior.
On the other hand, their exclusivity to feeding behavior was recently demonstrated to be an oversimplification and such behavioral findings are more compatible with the concept of a distributed neuronal processing brain architecture, which may better mirror the complexity observed in the dense anatomical reconstruction of brain volumes, the diversity of secreted neuropeptides, and the dynamics of individual neurons gene expression. Future work can contribute to the resolution of this ongoing discussion by exploiting the interaction of selective molecules with the in vivo complexity of an intact brain generating selective behavioral output.
In conclusion, the inventors’ unbiased and large-scale behavioral findings jointly establish the advantages of multi-behavioral screening for psychoactive compounds, propose a largely independent and modular organization of neural circuits that generate behavior in the larval zebrafish, and demonstrate the feasibility to control vertebrate behavior with novel, behavior- selective compounds.
Compounds synthesis
Scheme 1 : Synthetic routes oC compound 200
Figure imgf000063_0001

Claims

Claims
1 . A compound characterized by a general formula (200)
Figure imgf000064_0001
wherein
R4 is an unsubstituted or methyl-substituted 5- or 6-membered aryl or heteroaryl;
R5 is an unsubstituted or substituted aryl with the substituent being F, Cl, C1-C3 alkyl, C1-C3 alkyl ether, vinyl or allyl ether;
R6 is CORc or CONHRc with Rc being an unsubstituted or C1-C3 alkyl substituted heteroaryl.
2. The compound according to claim 1 , wherein
R4 is selected from
Figure imgf000064_0002
R6 is selected from
Figure imgf000065_0003
3. The compound according to claim 1 or 2, characterized by a formula (201 )
Figure imgf000065_0001
).
4. A compound characterized by a general formula (100)
Figure imgf000065_0002
wherein
R1 is selected from
o unsubstituted C1-C4 alkyl or amino-/hydroxy- and/or fluoro-substituted Ci-C4 alkyl,
o unsubstituted phenyl or phenyl substituted by C1-C3 unsubstituted alkyl, o NRn 2, wherein each RN independently from the other is H or C1-C3
unsubstituted alkyl; R2 is
Figure imgf000066_0002
wherein R21, R22, R23, R22‘, and R21’ are independently selected from H, halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl,
particularly R21, R22, R23, R22‘, and R21 are independently selected from H, F, Cl, methyl, CF3, ethyl, O-methyl, and O-CF3; more particularly R21 and R22’ are independently selected from halogen, unsubstituted C1-C3 alkyl or O- alkyl, fluoro-substituted C1-C3 alkyl or O-alkyl and the other ones are H, or R23 is selected from halogen, unsubstituted C1-C3 alkyl or O-alkyl, fluoro- substituted C1-C3 alkyl or O-alkyl and the other ones are H;
R3 is
Figure imgf000066_0001
wherein, R31, R32, R33, R32‘, and R31’ are independently selected from H, F, Cl, CF3, methyl, ethyl, O-methyl, and O-CF3; and
D is NH, N-methyl or O; or
R3 is NHRD orORD, wherein RD is a 5- or 6-membered unsubstituted or amino-/hydroxy- or F-substituted cyclic alkyl.
5. The compound according to claim 4, wherein
- R1 is selected from methyl, n-propyl, iso-butyl, phenyl, 4-methylphenyl, and N- dimethyl; and/or
R2 is selected from
Figure imgf000067_0001
6. The compound according to claim 4 or 5, characterized by a formula (101 )
Figure imgf000067_0002
7. The compound according to any one of claim 1 to 6 for use for the treatment of an eating disorder, cachexia or anorexia, particularly anorexia nervosa, bulimia, cachexia associated with tumour disease, viral or bacterial infection; cachexia associated with a psychological disorder, particularly cachexia associated with stress, depression, or anxiety; cachexia associated with, particularly a gastrointestinal disorder selected from peptic ulcer, GERD, and ulcerative colitis; medication-induced anorexia associated with chemotherapy, administration of a laxative or amphetamine drug.
8. A foodstuff or food additive comprising a compound according to any one of claim 1 to 6, particularly for use in animal farming.
9. A compound characterized by a general formula (300)
Figure imgf000068_0001
wherein
X is S, O or N-methyl;
R7 is H, F, Cl or C1-C3 alkyl;
- R8 is H, F, Cl or C1-C3 alkyl;
R9 is NH2, NH-methyl, OH or methyl;
- R10 is
Figure imgf000068_0002
wherein, R101, R102, R103, R102‘, and R101’ are independently selected from H, F, Cl, CF3, methyl, and NO2.
10. The compound according to claim 9, wherein
X is selected from S, O, and N-methyl; and/or
R7 is selected from H and methyl; and/or
R8 is selected from H, Cl, and methyl; and/or
- R9 is selected from NH2, NH-methyl, OH, methyl; and/or
R10 is selected from
Figure imgf000069_0001
1 1. The compound according to claim 9 or 10, wherein X is S; R7 is methyl; R8 is methyl;
R9 is NH2; and R10 is selected from 4-nitrophenyl, 4-trifluoromethyl, 4- chlorophenyl, 2- chloro,4-fluorophenyl, and 3-nitrophenyl, preferably 4-nitrophenyl.
12. The compound according to claim 9 or 10, characterized by a formula (302)
Figure imgf000069_0002
13. The compound according to any one of claims 9 to 12 for use for the treatment of a disease selected from obesity, binge eating disorder, metabolic syndrome, prediabetes, type 2 diabetes, dyslipidaemia, hypertension, cardiovascular disease, non-alcoholic fatty liver disease, polycystic ovary syndrome, female infertility, male hypogonadism, obstructive sleep apnoea, asthma/reactive airway disease, osteoarthritis, urinary stress incontinence, gastroesophageal reflux disease, and depression.
14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and 9 to 12.
15. The compound according to any one of claims 1 to 6 and 9 to 12 for use as a drug.
16. A non-therapeutic method for modulating the appetite in a subject comprising
administering a compound according to any one of claims 1 to 6 and 9 to 12 to the subject.
17. The method of claims 16, wherein the appetite is increased and the compound is a compound according to any one of claims 1 to 6.
18. The method of claims 16, wherein the appetite is decreased and the compound is a compound according to any one of claims 9 to 12.
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