WO2015069110A1 - Multiple d2 a(nta)gonists/h3 antagonists for treatment of cns-related disorders - Google Patents

Abstract

The present invention relates to compounds compound according to Formula (III); and pharmaceutically acceptable salts, hydrates and solvates thereof. These compounds have D₂receptor antagonist/(partial) agonist effects and H₃antagonistic effects, pharmaceutical compositions thereof, and methods of using them for application in the prophylaxis or treatment of CNS disorders.

Description

Multiple D₂ a(nta)gonists/H₃ antagonists for treatment of CNS-related disorders Field of the invention The present invention relates to phenyl amine compounds having D₂ receptor antagonist/(partial) agonist activity and ¾ antagonistic activity and to a method of preparing these phenyl amine compounds. The present invention also relates to pharmaceutical compositions comprising these phenyl amine compounds. The present invention further relates to methods for the prophylaxis or treatment of CNS-related disorders in a mammal in need thereof.

Background of the invention

Schizophrenia and Parkinson's disease are disorders of the central nervous system (CNS) in which the dopaminergic system is dysfunctional. They are complex disorders with numerous symptoms, some of which are treated with dopamine D₂ antagonists and agonists for schizophrenia and Parkinson's disease, respectively. In both cases patients can also present with impairments with cognitive functions, these cognitive symptoms are not successfully treated with existing medications. Quinpirole ((4aR,8aR)-5-propyl-4,4a,5,6,7,8,8a,9-octahydro-lH-pyrazolo[3,4- g]quinoline) is a selective D2 and D3 antagonist. It is disclosed in US 4.198.415, incorporated by reference.

Bromocriptine ((5 'a)-2-bromo- 12'-hydroxy-5 '-(2-methylpropyl)-3 ',6', 18-trioxo- 2'-(propan-2-yl)ergotaman) is a D2 agonist. It is disclosed in US 3.752.814, incorporated by reference.

Haloperidol (4-[4-(4-chlorophenyl)-4-hydroxy-l-piperidyl]-l-(4-fluorophenyl)- butan-l-one) is a D2 antagonist. It is disclosed in US 3.438.991, incorporated by reference.

Clobenpropit, also known as VUF-9153, (N'-[(4-chlorophenyl)methyl]-l-[3-(3H- imidazol-4-yl)propyl-sulfanyl]formamidine) is a H3 antagonist. It is disclosed in H van der Goot, MJ.P. Schepers, G.J. Sterk and H. Timmerman, Eur. J. Med. Chem. 27, 51 1, 1992, incorporated by reference. l,4-Benzodioxine-5-yl-piperazine compounds having anti-psychotic properties are for example disclosed in US 4.782.061, incorporated by reference. US 5.462.942, incorporated by reference, discloses 5-(piperazin-l-yl)-2,3- dihydrobenzo[b][l,4]dioxine derivatives having 5-HT_IA antagonistic activity. These compounds are useful for the treatment of CNS disorders.

US 6.255.312 and US 2003/0186838, both incorporated by reference, disclose 7- (piperazin-l-yl)benzo[d]oxazol-2(3H)-one derivatives having combined D₂ agonist activity and 5-HT_IA agonistic activity. These compounds can be used for the treatment of CNS-related disorders such as Parkinson's disease.

Considerable research is underway to study the role of histamine H₃ receptors in cognitive performance. These receptors are localized in the frontal cortex, hippocampus, caudate and cerebellum - areas of the brain that play a role in cognition and that are impacted by Parkinson's disease pathology [Clapham and Kilpatrick (1992) British Journal Pharmacol 107(4) :919-23.] and schizophrenia. The predominant function of H₃ receptors is to act as pre-synaptic heteroreceptors modulating the release of pro-cognitive neurotransmitters such as acetylcholine (ACh) [Fink et al (1990) Naunyn Schmiedebergs Arch Pharmacol 342(5):513-9.; Schlicker et al (1989) Naunyn Schmiedebergs Arch Pharmacol 340(6) .633-8]. Thus, activation of these receptors results in the inhibition of neurotransmitter release, and conversely, blockade of these receptors by antagonists relieves this inhibition leading to an increase in neurotransmitter release. Furthermore, in preclinical models, H₃ receptor antagonists have been shown to improve performance in a variety of animal models of cognitive function, including avoidance tasks and spatial, social and novel object recognition (NOR) memory models [Fox et al (2003) Journal Pharmacol Experimental Therapeutics 305: 897-908 ;Komater et al (2003) Psychopharmacol (Berl) 167:363-72; Chen (2000) Acta Pharmacol Sin 21:905-10; Fox et al (2005) Journal Pharmacol Experimental Therapeutics 313: 176-90; Ligneau et al (2007) Biochem Pharmacol 73:1215-24.]. These data, coupled to the autoreceptor function and broad receptor distribution within the brain, represent the primary driver for targeting ¾ receptors to treat deficits in cognitive performance [Within and Nelson (2004) Pharmacology and Therapeutics 103: 1-20; Esbenshade et al (2006) Molecular Interventions 6: 77-88].

There is a need in the art for pharmaceutically active compounds having D₂ receptor antagonist/(partial) agonist activity and H₃ antagonistic activity. Such pharmaceutically active compounds would, without being bound by theory, be suitable for the treatment of CNS-related disorders such as schizophrenia and Parkinson's disease.

Summary of the invention

The present invention relates to phenyl amine compounds having D₂ receptor antagonist/(partial) agonist activity and H₃ antagonistic activity. The present invention also relates to methods of preparing these phenyl amine compounds. The present invention further relates to pharmaceutical compositions comprising these phenyl amine compounds. The present invention further relates to methods for the prophylaxis or treatment of CNS-related disorders in a mammal in need thereof.

In particular, the present invention relates to compounds according to Formula (I) or Formula (II):

(I) (Π) wherein:

P and S are independently O or NR⁴;

Q and R are independently C(R⁴)₂ or C(O), provided that not both Q and R are C(O), or Q is absent and R is C(R⁴)₂ or C(O);

R¹, R², R³, R⁶, R⁷, R⁸ and R⁹ are independently selected from the group consisting of H, Ci - Ci2 alkyl, C2 - C12 alkenyl, C2 - C12 alkynyl, Ci - C12 alkoxy, C2 - C12 alkenoxy, C2

- C12 alkynoxy, Ci - C12 alkylthio, C2 - C₁₂ alkenylthio, C2 - C₁₂ alkynylthio, OH, halogen, N(R⁴)₂, N0₂, CN, S(0)pR⁴, S(0)₂N(R⁴)₂, C(0)R⁴, C(X)OR⁴, C(X)N(R⁴)₂, N(R⁴)-C(0)R⁴, P(0)(R⁴)OH and P(0)(R⁴)N(R⁴)₂;

R⁴ is independently selected from the group consisting of H, Ci - C₁₂ alkyl, Ce - C₂4 aryl and C3 - C24 heteroaryl;

p is 0, 1 or 2;

X is =0, =S, =N-CN, =C=C-N0₂ or =N-S0₂NH₂;

T is N or CR^4a, wherein R^4a is H or Ci - C₁₂ alkyl;

R⁵ is independently selected from the group consisting of H, Ci - C₁₂ alkyl, OH, halogen, Ci - C12 alkoxy, Ci - C₁₂ alkylthio, NH₂, NH(Ci - C₄ alkyl) and N(Ci - C₄ alkyl)₂, or two groups R⁵ represent =0, =S, =NH, =N(Ci - C₄ alkyl), =NOH or =NO(Ci

- C₄ alkyl);

n is 0, 1, 2, 3, 4, 5 or 6;

U is selected from the group consisting of [C(R⁵)₂]„, O, N(R⁵) and C(O), or U and R⁹ and the carbon atoms to which they are attached form a five-membered or six- membered ring, and when U is [C(R⁵)2]_n, [C(R⁵)2]_n may contain a carbon-carbon double bond or a carbon-carbon triple bond;

V is CHR¹⁰R^U or NR¹⁰R^U, wherein R¹⁰ and R^{1 1} are independently selected from the group consisting of H, Ci - C₁₂ alkyl, C₆ - C₂₄ aryl and C3 - C₂₄ heteroaryl, or wherein R¹⁰ and R¹¹ represent together the group [C(R⁵)₂]_m;

m is 3, 4, 5 or 6;

the groups [C(R⁵)2]_n and [C(R⁵)2]_m may be interrupted by one to three heteroatoms selected from O, NR^4a and S, or may independently be substituted by one or two =0, =S or =NR^4a; and

pharmaceutically acceptable salts, hydrates and solvates thereof Detailed description of the invention

The verb "to comprise" as is used in this description and in the claims and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The compounds disclosed in this document may comprise one or more asymmetric centres, and different diastereomers and/or enantiomers may exist of the compounds. The compounds disclosed in this document are meant to include all diastereomers, and mixtures thereof, unless stated otherwise. In addition, the compounds disclosed in this document are meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer.

The compounds disclosed in this document may occur in different tautomeric forms. The compounds disclosed in this document are meant to include all tautomeric forms, unless stated otherwise.

The compounds disclosed in this document may occur as cis and trans isomers and/or as Z- and i somers. Unless stated otherwise, the compounds disclosed in this document are meant to include both the individual cis and the individual trans isomer and/or the individual Z-isomer and i -isomer of a compound, as well as any mixture thereof. Accordingly, when the structure of a compound is depicted as a cis isomer, it is to be understood that the corresponding trans isomer or any mixture of the cis and trans isomer are not excluded from the invention of the present application. Likewise, when the structure of a compound is depicted as a £-isomer, it is to be understood that the corresponding Z-isomer or any mixture of the E- and Z- isomer are not excluded from the invention of the present application. The compounds disclosed in this document may exist in an amorphous form or in a crystalline form. Accordingly, the compounds disclosed in this document may exist in different polymorphic forms.

The compounds disclosed in this document may be in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salts of the compounds according to Formula (I) and Formula (II) may be acid addition salts or base addition salts.

Definitions of substituents

The Ci - Ci₂ alkyl group may be linear or branched. The alkyl group may further comprise one or more heteroatoms, preferably one, two or three heteroatoms, selected from the group consisting of O, N and S. The alkyl group may further be substituted by one or more substituents, preferably one, two or three substituents. Preferred substituents include halogen, hydroxy, Ci - C alkoxy, Ci - Ce alkylthio, NH₂, NH(Ci - C₄ alkyl) and N(Ci - C₄ alkyl)2, or two substituents represent =0, =S, =NH, =N(C₁ - C₄ alkyl), =NOH or =NO(Ci - C₄ alkyl). The alkyl group is preferably a Ci - C₆ alkyl group. When the alkyl group is a C₃ - C₁₂ alkyl group, preferably a C₃ - C₆ alkyl group, the alkyl group may be cyclic.

The C₂ - Co alkenyl group may be linear or branched. The alkenyl group may further comprise one or more heteroatoms, preferably one, two or three heteroatoms, selected from the group consisting of O, N and S. The alkenyl group may further be substituted by one or more substituents preferably one, two or three substituents. Preferred substituents include halogen, hydroxy, Ci - C₆ alkoxy, Ci - Ce alkylthio, H₂, NH(Ci - C₄ alkyl) and N(Ci - C₄ alkyl)₂, or two substituents represent =0, =S, =NH, =N(Ci - C₄ alkyl), =NOH or =NO(Ci - C₄ alkyl). The alkenyl group is preferably a Ci - C-6 alkenyl group. When the alkenyl group is a C5 - Ci₂ alkenyl group, preferably a C5 - C₆ alkenyl group, the alkenyl group may be cyclic.

The C₂ - C12 alkynyl group may be linear or branched. The alkynyl group may further comprise one or more heteroatoms, preferably one, two or three heteroatoms, selected from the group consisting of O, N and S. The alkynyl group may further be substituted by one or more substituents, preferably one, two or three substituents. Preferred substituents include halogen, hydroxy, Ci - C₆ alkoxy, Ci - Ce alkylthio, H₂, NH(Ci - C₄ alkyl) and N(Ci - C₄ alkyl)₂, or two substituents represent =0, =S, = H, =N(Ci - C₄ alkyl), =NOH or =NO(Ci - C₄ alkyl). The alkynyl group is preferably a Ci - C₆ alkynyl group. When the alkynyl group is a C₈ - C₁₂ alkenyl group, the alkynyl group may be cyclic.

These definitions also apply to groups which include an alkyl group, an alkenyl group or an alkynyl group, for example an alkoxy group.

The Ce - C₂4 aryl group includes monocyclic and bicyclic structures, wherein in the bicyclic structure one ring moiety may be (partly) saturated. The aryl group may be substituted by one or more substituents, preferably one, two, three, four or five substituents. The C₆ - C24 aryl group is preferably a C₆ - C₁₂ aryl group. It is preferred that the aryl group is selected from the group consisting of phenyl, indenyl (1-, 2-, 3-, 4-, 5-, 6- or 7-), indanyl (1-, 2-, 3-, 4-, 5-, 6- or 7-), naphtyl (1-, 2-, 3- or 4-), 1,2- dihydronaphtyl (1 -, 2-, 3-, 4-, 5-, 6-, 7- or 8-) and 1 ,2,3,4-tetrahydronaphtyl (1 -, 2-, 3-, 4-, 5-, 6-, 7- or 8-).

The C3 - C24 heteroaryl group comprises at least one heteroatom, preferably one, two, three to four heteroatoms, selected from the group consisting of O, N and S and includes monocyclic and bicyclic structures, wherein one or two ring moieties may be (partly) saturated. The heteroaryl group may be substituted by one or more substituents, preferably one, two, three, four or five substituents. It is preferred that the heteroaryl group is selected from the group consisting of 2-pyrrolyl, 3-pyrrolyl, 2-furanyl, 3- furanyl, 2-thienyl, 3-thienyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 1- imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-oxazolyl, 4-oxazolyl, 5- oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, triazolyl (this term includes the radical derived from 1,2,3-triazole and of 1,2,4-triazole, i.e. that "triazolyl" may be 1- triazolyl, 2-triazolyl, 3-triazolyl, 4-triazolyl or 5-triazolyl), 2-pyridinyl, 3-pyridinyl, 4- pyridinyl, 2-pyrazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 4- pyridazinyl, triazinyl, indolyl (1-, 2-, 3-, 4-, 5-, 6- or 7-), 3-H-indolyl (2-, 3-, 4-, 5-, 6-, 7-), isoindolyl (1-, 2-, 4- or 5-), indolizinyl (1-, 2-, 3-, 5-, 6-, 7- or 8-), indazolyl (1-, 3-, 4-, 5-, 6- or 7-), purinyl (2-, 6-, 8-or 9-), benzo[b]furanyl (2-, 3-, 4-, 5-, 6- or 7-), benzo[c]furanyl (1-, 4-, 5- or 6-), benzo[b]-2,3-dihydro-furanyl (2-, 3-, 4-, 5-, 6- or 7-), benzo[c]-l,3-dihydro-furanyl (1-, 3-, 4-, 5-, 6- or 7-), benzo[b]thienyl (2-, 3-, 4-, 5-, 6- or 7-), benzo[c]thienyl (1-, 4-, 5- or 6-), benzo[b]-2,3-dihydro-thienyl (2-, 3-, 4-, 5-, 6- or 7-), benzo[c]-l,3-dihydro-thienyl (1-, 3-, 4-, 5-, 6- or 7-), quinolinyl (2-, 3-, 4-, 5-, 6- , 7- or 8-), isoquinolinyl (1-, 3-, 4-, 5-, 6-, 7- or 8-), phthalazinyl (1-, 5- or 6-), naphthypyridinyl (2-, 3- or 4-), quinoxalinyl (2-, 5- or 6-), quinazolinyl (2-, 5-, 6-, 7- or 8-), cinnolinyl (3-, 4-, 5-, 6-, 7- or 8-) and pteridinyl (2-, 4-, 6- or 7-). The C₃ - C₂₄ heteroaryl group is preferably a C₃ - C₁₂ heteroaryl group.

Preferred substituents for the Ce - C₂₄ aryl group and the C3 - C24 heteroaryl group include independently halogen, hydroxy, Ci - Ce alkyl, Ci - Ce alkoxy, Ci - C alkylthio, NH₂, NH(Ci - C₄ alkyl) and N(Ci - C₄ alkyl)₂.

The group of halogens include fluorine, chlorine, bromine and iodine.

When U is [C(R⁵)₂]_n, [C(R⁵)₂]_n may contain a carbon-carbon double bond or a carbon-carbon triple bond. In other words, two groups R⁵ on two adjoining carbon atoms may form with said adjoining carbon atoms a carbon-carbon double bond and four groups R⁵ on two adjoining carbon atoms may form with said adjoining carbon atoms a carbon-carbon triple bond.

The groups [C(R⁵)₂]_n and [C(R⁵)₂]_m may independently be substituted by one or two =0, =S or =NR^4a. in other words, two groups R⁵ on the same carbon atom may form a =0, =S or =NR^4a group, e.g. an oxo group. Alternatively, two groups R on adjacent carbon atoms may also form a =0, =S or =NR^4a group, e.g. an epoxy group. Preferred embodiments

In a preferred group of compounds, the compounds are according to Formula (I).

In another preferred group of compounds, P is NR⁴, Q is absent, R is C(O) and S is O. More preferably, this group of compounds is according to Formula (I).

In another preferred group of compounds, P and S are O and Q and R are C(R⁴)₂.

More preferably, this group of compounds is according to Formula (I).

In another preferred group of compounds, P is NR⁴, S is O and Q and R are C(R⁴)₂. More preferably, this group of compounds is according to Formula (I).

1 2 3 6 7 8 9

Preferably, R , R , R , R , R , R and R are independently selected from the group consisting of H, Ci - C₆ alkyl, Ci - C₆ alkoxy, Ci - C₆ alkylthio, OH, halogen, N(R⁴)₂, N0₂, CN, S(0)_pR⁴, S(0)₂N(R⁴)₂, C(0)R⁴, C(0)OR⁴, C(0)N(R⁴)₂ and N(R⁴)- C(0)R⁴. More preferably, R¹, R², R³, R⁶, R⁷, R⁸ and R⁹ are independently selected from the group consisting of H, Ci - C₆ alkyl, Ci - C₆ alkoxy, OH, halogen, N(R⁴)₂, C(0)R⁴, C(0)OR⁴ and C(0)N(R⁴)₂.

Preferably, R⁴ is independently selected from the group consisting of H and Ci - Ci2 alkyl.

Preferably, R^4a is H.

Preferably, R⁵ is independently selected from the group consisting of H, Ci - C₁₂ alkyl, OH, halogen, Ci - C_n alkoxy, H₂, NH(Ci - C₄ alkyl) and N(Ci - C₄ alkyl)₂, or two groups R⁵ represent =0, =NH or =N(C₁ - C₄ alkyl).

Preferably, n is 0, 1, 2, 3 or 4.

U and R⁹ and the carbon atoms to which they are attached may form a five- membered or a six-membered ring, and said five-membered or six-membered ring may form a spiro compound with another five-membered or six-membered ring formed by V, when R¹⁰ and R¹¹ represent a group [C(R³)2]_m This is further illustrated below by the compounds according to Formula (VI).

A first preferred group of compounds according to the present invention is according to Formula (III):

(IH) wherein n, m, p, R¹, R², R³, R⁴, R^4a, R⁶, R⁷, R⁸, R⁹ _, T and V are as defined above, W is O or NR^4a, U is [C(R⁵)₂)]_n and R¹² is selected from the group consisting of H, Ci - C₁₂ alkyl and halogen, or wherein two groups R¹² represent =0.

A second preferred group of compounds according to the present invention is according to Formula (IV):

(IV) wherein n, m, p, R¹, R², R³, R⁴, R^4a, R⁵, R⁶, R⁷, R⁸, R⁹, T and V are as defined above and wherein U is O, NR⁵ or C(O).

A third preferred group of compounds according to the present invention is according to Formula (V):

wherein n, m, p, R¹, R², R³, R⁴, R^4a, R⁵, R⁶, R⁷, R⁸, R^y, T and V are as defined above and wherein U is O, NR⁵ or C(O).

A fourth preferred group of compounds according to the present invention is according to Formula (VI):

wherein n, m, p, R¹, R², R³, R⁴, R^4a, R⁵, R⁶, R⁷, R⁸, R^y, T and W are as defined above and wherein R¹³ and R¹⁴ are independently selected from the group consisting of H, Ci - Ci2 alkyl, C - C24 aryl and C3 - C24 heteroaryl, or wherein R¹⁰ and R¹¹ represent together the group [C(R⁵)2]₀, o is 3, 4 or 5, and the group [C(R )2]₀ may be interrupted by one or two heteroatoms selected from O, NR^4a and S, or may independently be substituted by one or two =0, =S or =NR^4a. In this fourth preferred group of compounds according to the present invention is according to Formula (VI), it is preferred that W is O. A fifth preferred group of compounds according to the present invention is according to Formula (VII):

(VII) wherein n, m, p, R¹, R², R³, R⁴, R^4a, R⁵, R⁶, R⁷, R⁸, R⁹, T, U and V are as defined above.

Pharmaceutical compositions

The present invention also relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a compound according to Formula (I) or Formula (II) and a pharmaceutically acceptable carrier. To prepare the pharmaceutical composition according to the invention, a pharmaceutically effective amount of a compound according to Formula (I) or Formula (II) is combined with a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may take a wide variety of forms depending on the form of preparation desired for administration. The pharmaceutical composition is desirably in unitary dosage form and may be suitable for administration by the oral, nasal, rectal, sublingual, transdermal, percutaneous or parenteral route. Accordingly, the pharmaceutical composition in oral dosage form may a solid dosage form or a liquid dosage form. Suitable solid oral dosage forms include tablets and capsules and these are very advantageous oral dosage forms because of their ease in administration. Suitable liquid oral dosage forms include suspensions, syrups, elixirs and solutions.

The pharmaceutical composition according to the present invention are suitable for preventing and treating CNS-disorders, preferably selected from the group consisting of depression, major depressive disorder, generalised anxiety disorder, major anxiety disorder and panic disorder.

The present invention also relates to a method for preventing or treating a CNS- disorder in a subject in need thereof, wherein a therapeutically effective amount of a compound according to the present invention or a pharmaceutical composition comprising said compound according to the present invention is administered to said subject. Prefererably, the subject is a mammal and more preferably, the subject

Examples

Exemplary compounds useful in the invention are described by reference to the illustrative synthetic schemes for their general preparation and specific examples. One skilled in the art will recognize that it may be advantageous to perform the reactions shown in the Schemes in an order different from that depicted. The substituents R¹, R², R³ etc, have the meaning as defined above.

Abbreviations

RT: ambient temperature Example 1

Synthesis of 7-(4-alkylpiperazin-l-yl)benzo[d]oxazol-2(3H)-one D₂-H₃ a(nta)gonists.

Example lal : Synthesis of 7-(piperazin-l-yl)benzo[d]oxazol-2(3H)-one

SCHEME A

A Curtius reaction of 3-nitrosalicylic acid and intramoleculair cyclization of the isocyanate formed yielded the cyclic carbamate. Reduction of the nitro-moiety by catalytic hydrogenation afforded the corresponding amine. The final target was obtained by subsequent reaction with the in situ formed bis-mesylate of N-benzyl- bis(2-hydroxyethyl)amine and finally deprotection of the benzyl group.

7-Nitrobenzo [d] oxazol-2(3H)-one

Et₃N (19 mL, 13.7 g, 0.135 mol) was added to a suspension of 3-nitrosalicylic acid (23.6 g, 0.13 mol) in toluene (0.4 L), followed by drop-wise addition of DPPA (29 mL, 37.1 g, 0.14 mol). After 1 h the mixture was heated to ~110°C and stirred overnight. The reaction mixture was allowed to cool to RT. NaHC0₃ (0.5 L) was added and the layers were separated. The organic layer was dried (Na₂S0₄) and concentrated in vacuo. The crude product was triturated with toluene to afford 7-nitrobenzo[d]oxazol- 2(3H)-one as a light brown solid (12.7 g, 70.5 mmol, 56%).

7-Aminobenzo [d] oxazol-2(3H)-one

To a solution of 7-nitrobenzo[d]oxazol-2(3H)-one (14.4 g, 80 mmol) in EtOH (125 mL) in a Berghof reactor was added under NITROGEN 10% Pd on C (0.5 g) and the mixture was placed under 8 bars of H₂. The mixture was heated to 65°C and the reactor was refilled with H₂ regularly until the uptake of gas stopped. After complete conversion the mixture was filtered over Celite and concentrated to give the product aimed for as a light brown solid (11.1 g, 73.7 mmol, 92%).

7-(4-Benzylpiperazin-l-yl)benzo[d]oxazol-2(3H)-one

A solution of Ms₂0 (32 1 g, 184.8 mmol) in MeCN (200 mL) was cooled to < -5 °C (ice/H₂0 salt) and sequentially were added a solution of N-benzyl-bis(2- hydroxyethyl)amine (16.4 g, 84.1 mmol) in MeCN (50 mL), Et₃N (36 mL, 255 mmol, 3.8 eq.), and MsOH (13.1 mL, 202 mmol, 3 eq.) while maintaining the temperature below 10°C. 7-Aminobenzo[d]oxazol-2(3H)-one (10.0 g, 67.2 mmol) was added as a solid and the mixture was heated to reflux. Et₃N (28.5 mL, 202 mmol) was added drop- wise over a period of 1.5 hour. Reflux was maintained for 18 additional hours. The resulting clear solution was allowed to cool to RT and concentrated in vacuo at 70°C. The residue was separated between NaHCC (0.8 L) and EtOAc (0.4 L). The aqueous layer was extracted with EtOAc (3 x 0.2 L). The combined organic layers were washed with brine, dried (NaiSC^) and concentrated to afford 7-(4-benzylpiperazin-l- yl)benzo[d]oxazol-2(3H)-one as a white solid (20.2 g, 65.3 mmol, 97%).

7-(Piperazin-l-yl)benzo[d]oxazol-2(3H)-one

To a solution of 7-(4-benzylpiperazin-l-yl)benzo[d]oxazol-2(3H)-one (20.0 g, 64.6 mmol) in methanol (0.9 L) was added 10% Pd/C (2.0 g) and cone. HC1 (6 mL, 71.0 mmol) and the mixture was stirred under a 1 atm H₂ pressure for 18 h. No conversion was observed, therefore it was filtered and concentrated. The residue was redissolved in MeOH (0.9 L) and 10% Pd/C (3 g) was added. The mixture was placed under H₂ and heated to 80°C external temperature. The mixture was filtered over Celite and the filtrate was concentrated in vacuo. The residue was triturated in hot EtOH and filtered to afford 7-(piperazin-l-yl)benzo[d]oxazol-2(3H)-one (12.5 g, 48.9 mmol, 76%) as a light brown solid.

Example la2: Synthesis of 4-bromoalkoxybenzylamines

SCHEME B

Reductive amination of 4-hydroxybenzaldehyde with the appropriate amine and subsequent alkylation with a dibromoalkane afforded the bromine intermediates.

General Example: Synthesis of 4-(4-(3-bromopropoxy)benzyl)morpholine.

_Br, _v ^ _^ , ,0

4-(Morpholinomethyl)phenol

To a solution of 4-hydroxybenzaldehyde (5.0 g, 41 mmol) in MeOH (10 mL) was added a solution of morpholine (7.1 mL, 82 mmol) in MeOH (10 mL). KBH₄ (1.13 g, 21 mmol) was added portion-wise (within 2 h). After stirring for 1 h the reaction mixture was acidified with 3 N HCl (aq) and concentrated. The residue was separated between 1 N HCl (aq) and EtOAc. The water layer was washed with EtOAc (2 x). The water layer was neutralized using cone. NH₄OH (aq) and extracted with EtOAc (3 x). The combined organic layers were dried (Na₂S04) and concentrated to afford the product as an off-white solid (7.2 g, 37.3 mmol, 91%). The product was used in the next step without any further purification.

4-(4-(3-Bromopropoxy)benzyl)morpholine

A mixture of 4-(moipholinomethyl)-phenol (5.0 g, 25.9 mmol), DCM (50 mL), 1 N NaOH (52 mL, 52 mmol), Bu₄NBr (838 mg, 2.6 mmol) and 1,3-dibromopropane (13.1 ml, 129.5 mmol, 5 eq.) was stirred for 3 h. The layers were separated. The water layer was extracted with DCM (2 x). The combined organic layers were dried (Na₂S04) and concentrated. The residue obtained was filtered through silica with EtOAc/heptanes, followed by EtOAc to afford the product aimed for (3.2 g, 10.2 mmol, 40%). NOTE: For longer storage all the amines prepared were converted to the corresponding HC1 salts.

Example lb: Synthesis of 7-(4-alkylpiperazin-l-yl)benzo[d]oxazol-2(3H)-ones

The compounds aimed for were prepared by alkylation of 7-(piperazin-l- yl)benzo[d]oxazol-2(3H)-one with the appropriate bromine.

General example: Synthesis of 7-(4-(3(4(morpholinomethyl)phenoxy)propyl)piperazin- l-yl)benzo[d]oxazol-2(3H)-one (AG-0029).

7-(4-(3-(4-(Morpholinomethyl)phenoxy)propyl)piperazin-l-yl)benzo[< |oxazol- 2(3H)-one (AG-0029)

7-(Piperazin-l-yl)benzo[d]oxazol-2(3H)-one (2.0 g, 7.8 mmol), 4-(4-(3- bromopropoxy)benzyl)morpholine (3.6 g, 12.0 mmol), K₂CO₃ (2.0 g, 21.0 mol) and Kl (0.2 g, 12.0 mol) were suspended in MeCN (50 mL) and heated to 80 °C overnight. After cooling to RT the mixture was filtered over Celite, washed with DCM and concentrated in vacuo. Automated column chromatography (ISCO) resulted in material with a purity <90%. A second purification by automated column chromatography and recrystallization from MeOH afforded crystals. The material obtained was dissolved in DCM (10 ml), and HC1 in dioxane (4M, 4 eq., 6 mmol, 1.5 ml) was added. The reaction mixture was stirred at RT for about 30 minutes and concentrated to afford 7-(4-(3-(4- (mo holinomethyl)phenoxy)propyl)piperazin- l-yl)benzo[ίί]oxazol-2(3H)-one (650 mg, 1.23 mmol, 16%).

^XH- MR (CD₃OD, in ppm): 2.38 (m, 2H), 3.05-4.10 (m, 18H), 4.20 (t, 2H), 4.36 (s, 2H), 6.80 (t, 2H), 7.16 (m, 3H), 7.52 (d, 2H).

(M-H)⁺ = 453.1 The above-described procedure was utilized for synthesis of AG-0087 to AG-0091 and AG-0099-0100.

Results

Table 1

R = D₂ (IC₅₀) D₂ (EC₅₀)

antagonist Agonist (nM) (nM) (nM)

AG-0029 0.08 1.1 513

(I = 1 15%)

AG-0099 >100 >1000 330

AG-0100 >100 > 1000 23 AG-0089 0.6 8 5

(E^ 107%)

AG-0091 53 >100 27

AG-0090 >100 > 1000 13

AG-0087 0.04 0.8 5

(E_max= 94%)

AG-0088 99 > 1000 >10

Quinpirole 0.8 1.0

Bromocriptine 47

Haloperidol 6.0

Clobenpropit 2.1

Example lc: Synthesis of (benzylpiperazin-l-yl)benzo[d]oxazol-2(3H)-ones

A Mitsunobu reaction of 4-hydroxybenzaldehyde with tert-butyl 4-hydroxypiperidine- 1-carboxylate afforded t-butyl 4-(4-formylphenoxy)piperidine-l-carboxylate. Subsequent reductive amination and hydrolysis of the Boc-protecting group afforded the intermediate 7-(4-(4-(piperidin-4-yloxy)benzyl)piperazin-l-yl)benzo[d]oxazol- 2(3H)-one. Reductive amination with the appropriate amine furnished the targets aimed for

General example: Synthesis of 7-(4-(4-((l-cyclobutylpiperidin-4- yl)oxy)benzyl)piperazin-l-yl)benzo[d]oxazol-2(3H)-one (AG-0056).

t-Butyl 4-(4-formylphenoxy)piperidine-l-carboxylate

A solution of tert-butyl 4-hydroxypiperidine-l-carboxylate (5.66 g, 28.1 mmol), 4- hydroxybenzaldehyde (6.87 g, 56.2 mmol) and PPh₃ (8.1 g, 30.9 mmol) in THF (100 mL) was cooled on an ice/H₂0 bath. DIAD (5.6 mL, 28.1 mmol) was added drop-wise and the mixture was stirred overnight at RT. It was concentrated and the residue was separated between 1 N NaOH (0.2 L) and EtOAc (0.2 L). The water layer was extracted with EtOAc (2 x 0.2 L). The combined organic layers were dried (Na₂S0₄) and concentrated. The residue was purified by chromatography (silica, EtOAc 15% in heptane) to afford the product as a yellow oil that solidified upon standing (5.4 g, 17.7 mmol, 62%) t-Butyl 4-(4-((4-(2-oxo-2,3-dihy drobenzo [d] oxazol-7-yl)piperazin- 1- yl)methyl)phenoxy)piperidine-l-carboxylate

NaBH(OAc)3 (3.3 g, 15.6 mmol) was added to a solution of 7-(piperazin-l- yl)benzo[d]oxazol-2(3H)-one (1.0 g, 3.9 mmol), t-butyl 4-(4- formylphenoxy)piperidine-l-carboxylate (1.77 g, 4.69 mmol) and AcOH (1 mL) in DMF (50 mL). After stirring overnight H₂0 (0.2 L) was added and the mixture was made alkaline using 2 N NaOH. It was extracted with EtOAc (3 x 0.1 L). The combined organic layers were dried (Na₂S04) and concentrated. The residue was triturated with Et₂0, filtered and washed with Et₂0 to furnish the product as a white solid (1.63 g, 3.20 mmol, 82%).

7-(4-(4-(Piperidin-4-yloxy)benzyl)piperazin-l-yl)benzo[d]oxazol-2(3H)-one

To a solution of t-butyl 4-(4-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperazin-l- yl)methyl)phenoxy)piperidine-l-carboxylate (1.63 g, 3.20 mmol) in DCM (30 mL) was added TFA (5 mL). After 2 h it was concentrated in vacuo to furnish the product as a yellowish oil (3.31 g, max 3.2 mmol, quant.) that was used as such in the following step(s). 7-(4-(4-((l-(Cyclobutyl)piperidin-4-yl)oxy)benzyl)piperazin-l-yl)benzo[d]oxazol- 2(3H)-one

To a solution of 7-(4-(4-(piperidin-4-yloxy)benzyl)piperazin-l-yl)benzo[d]oxazol- 2(3H)-one. TFA (2.0 g, max 1.93 mmol) in DMF (20 mL) were added cyclobutanone (0.73 niL, 9.67 mmol) and AcOH (1 mL). NaBH(OAc)₃ (3.3 g, 15.4 mmol) was added in 5 portions over 1 h. After stirring overnight the reaction mixture was poured into H₂0 (0.2 L) and NaOH (2 N) was added to adjust the pH to >14. The aqueous phase was extracted with EtOAc (3 x 0.1 L). The combined organic layers were dried (Na₂S0₄) and concentrated to furnish the crude product as a light brown oil. Purification by column chromatography chromatography (ISCO, silica; gradient of 7 N NH₃ in MeOH DCM) afforded the pure product with some residual DMF. The product was dissolved in MeOH (5 mL) and drop-wise added to H₂0 (100 mL) while stirring. The resulting solid was filtered off, washed with ¾0 and Et₂0 and dried using the freeze dryer overnight to give the product as a white solid (408 mg, 0.88 mmol, 46%). The residue was dissolved in dioxane (10 mL) and 1 N HC1 (1.7 mL, 1.73 mmol, 2.1 eq.) in dioxane was added. The resulting suspension was concentrated in vacuo. To remove traces of dioxane the solid was dissolved in H₂0 (5 mL) and it was lyophilized to give AG-0056 as a white solid (465 mg, 0.865 mmol, quant.).

1H-NMR (CDC1₃, in ppm): 1.6-2.25 (m, 10H), 2.60 (bs, 8H), 2.80 (m, 1H), 3.34 (bs, 4H), 3.52 (s, 2H), 4.37 (m, 1H), 6.60 (d, 2H), 6.89 (d, 2H), 7.02 (t, 1H), 7.24 (d , 2H). (M-H)⁺ = 463.2

The above-described procedure was utilized for synthesis of AG-0110, AG-0127-0132 and AG-0208.

Example Id: 7-(4-((l-alkyl-3H-spiro[benzofuran-2,4'-piperidin]-5-yl)methylpiperazin- l-yl)benzo[d]oxazol-2(3H)-ones

The Grignard reagent prepared from 2-fluorobenzyl chloride was added to benzyl- piperidone. The crude adduct formed was cyclized using NaH and the benzyl moiety was removed with Pd. Bromination of the HCl-salt of compound 4 with NBS, reductive amination with the appropriate ketone and lithium-halogen exchange followed by quenching with DMF afforded the l'-alkyl-3H-spiro[benzofuran-2,4'-piperidine]-5- carbaldehydes. Reductive amination with 7-(piperazin-l-yl)benzo[d]oxazol-2(3H)-one using NaBH(OAc)3 as a reducing agent resulted in the final targets. General example: Synthesis of 7-(4-((l'-cyclobutyl-3H-spiro[benzofuran-2,4'- piperidin]-5-yl)methyl)piperazin-l-yl)benzo[d]oxazol-2(3H)-one (AG-01 12).

l'-Benzyl-3H-spiro[benzofuran-2,4'-piperidine]

A three neck flask was dried using a hot gun under N₂ atmosphere. Mg (3.5 g, 144 mmol) and a crystal of I₂ were added and it was heated with a hot gun until the I₂ sublimed. After 1 h of vigorous stirring ether (75 mL) was added. Part (-10%) of a solution of 2-fluorobenzyl chloride (15.6 mL, 19.0 g, 131 mmol) in ether (75 mL) was added to start up the Grignard reaction. Then, the remaining solution was added at such a rate that reflux was maintained. After complete addition the mixture was heated to reflux for 2 h. The mixture was cooled to RT and poured into saturated NH₄C1 (aq.) (200 mL) and brine (200 mL). Extraction with EtOAc (3 x 0.4 L) followed by drying the combined organic fractions and concentration gave the crude product (33 g, max. 110 mmol). The material was dissolved in DMF (200 mL) and added drop-wise to a suspension of NaH (3.4 g, 71 mmol, 50% in mineral oil) in DMF (200 mL). After heating to 120°C for 5 h it was poured into H₂0 (2 L) and extracted with EtOAc (3 x 0.5 L). The combined organic fractions were dried (Na₂SC>4) and concentrated. The residue was dissolved in DCM (0.1 L) and filtered through a pad of silica (10 cm). The product was eluted with EtO Ac/heptanes (1/1) to furnish a pure fraction (1 g, 2.7%o over two steps) and a less pure fraction (12 g, 43 mmol, 32%). Part of this fraction crystallized in itself. These were isolated and washed with pentane. The mother-liquor was mixed with EtOAc/heptanes and over time crystals were formed, which were isolated, washed and dried to afford (combined) l '-Benzyl-3H-spiro[benzofuran-2,4'- piperidine] (4.2 g, 15.1 mol, 1 1%).

3H-Spiro[benzofuran-2,4'-piperidine]

l'-Benzyl-3H-spiro[benzofuran-2,4'-piperidine] (1 gram, 3.6 mmol) was dissolved in isopropanol (25 mL, heating was needed). Pd/C (catalytic, slurry in IP A) was added and the reaction flask was evacuated and loaded with H₂ via a balloon. The procedure was repeated three times. After stirring at RT, partial conversion was achieved. The mixture was transferred to a Parr vessel, H₂ pressure was applied (5 bar) and the vessel was vented. The procedure was repeated four times. The mixture was stirred for 2 h, no change in conversion was observed. Extra Pd/C was added as a slurry in IPA and the mixture was stirred overnight to reach full conversion. The reaction mixture was filtered over Celite and the filtrate was concentrated to afford of a yellow solid (847 mg (>100%)). 5-Bromo-3H-spiro[benzofuran-2,4'-piperidine] hydrochloride

To a solution of 3H-spiro[benzofuran-2,4'-piperidine] hydrochloride (150 mg, 0.66 mmol) in methanol (2.6 mL) at 0°C was added A^Lbromosuccinimide (1 14 mg, 0.64 mmol) at once. The mixture was allowed to reach RT and stirred overnight. It was concentrated and the residue was partitioned between 1 N NaOH (4-5 mL) and DCM (10 mL). The water layer was extracted with DCM (5 mL) and the combined organics were washed with brine, dried (Na₂S0₄) and concentrated to an orange oil. It was redissolved in DCM. 4 N HC1 in dioxane (0.3 mL) was added and TBME to affect precipitation. The solid formed was filtered off and dried under high vacuum to afford a white solid (139 mg, 0.47 mmol, 71%).

5-Bromo-l'-cyclobiityl-3H-spiro[benzofuran-2,4'-piperidine]

To a solution of 5-bromo-3H-spiro[benzofuran-2,4'-piperidine] (partly HCl-salt, 2.78 mmol) in methanol (14 mL) was added Et₃N (enough to liberate the HC1 salt). AcOH

(0.6 mL, 10 mmol) and cyclobutanone (1.0 mL, 14 mmol) were added and after 1-2 min stirring NaBH₃CN (1.15 g, 18 mmol) was added portion-wise with cooling. The mixture was stirred for 30 min and poured in DCM (25 mL) and sat. NaHC0₃ (15 mL).

The water layer was extracted with DCM (1 x). The combined organics were washed with brine, dried (Na₂SC>4) and concentrated. Automated column chromatography

(Alumina, 0-20% EtOAc/heptanes) resulted in a yellow oil, slowly solidifying to a yellow solid (620 mg, 1.92 mmol, 69%). l'-Cyclobutyl-3H-spiro[benzofuran-2,4'-piperidine]-5-carbaldehyde

To a solution of 5-bromo-r-cyclobutyl-3H-spiro[benzofuran-2,4'-piperidine] (200 mg, 0.62 mmol) in THF (1.0 mL) at -78°C was added drop-wise w-BuLi (2.5 M in hexanes, 0.32 mL, 0.81 mmol) drop-wise and the mixture was stirred at -78°C for 30 min. DMF (0.13 mL, 1.6 mmol) was added and the mixture was allowed to reach RT and stirred for 1 h. Subsequently, H₂0 was added and the mixture was extracted with EtOAc (3 x 15 mL). The organic extracts were dried (Na₂S0₄) and concentrated to furnish a yellow oil, slowly crystallizing (160 mg, 0.59 mmol, 95%). 7-(4-((l-Cyclobutyl-3H-spiro[benzofuran-2,4'-piperidin]-5-yl)methylpiperazin-l- yl)benzo[d]oxazol-2(3H)-one (AG-0112)

-Cyclobutyl-3H-spiro[benzofuran-2,4'-piperidine]-5-carbaldehyde (160 mg, 0.59 mmol) was mixed with DCE (5 mL). 7-(Piperazin- l-yl)benzo[d]oxazol-2(3H)-one (137 mg, 0.54 mmol) was added, followed by Et₃N (0.08 mL, 0.57 mmol) and AcOH (0.8 mL). Heating was applied until almost all solid had dissolved. The mixture was allowed to cool to RT and NaBH(OAc)₃ (229 mg, 1.08 mmol) was added and the reaction mixture was stirred at RT overnight. A gel-like system was obtained. It was diluted with DCM (15 mL) and sat. NaHC0₃ (15 mL) and stirred until gas-evolution ceased. The layers were separated and the water layer was extracted with DCM (2 x 15 mL). The combined organics were washed with H₂0, dried (Na₂SC>4) and concentrated to yield an orange oil. Automated column chromatography (Alox, 0-5% methanol in DCM) provided a light-yellow foam (47 mg, 0,106 mmol, 18%).

¹H-NMR (CDC1₃, in ppm): 1.5-2.2 (m, 10H), 2.42 (bs, 4H), 2.62 (bs, 4H), 3.81 (m, 1H), 2.99 (s, 2H), 3.36 (bs, 4H), 3.52 (s, 2H), 6.60 (d, 2H), 6.71 (d, 1H), 7.05 (t, 2H), 7.18 (s, 1H).

(M-H)⁺ = 475.0

Example le: Synthesis of 7-(4-(4-(3-(alkyamino)propoxy)benzyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-ones

SCHEME F

Alkylation of 4-hydroxybenzaldehyde with the appropriate l-(3-chloropropyl)amine and subsequent reductive amination with 7-(piperazin-l-yl)benzo[d]oxazol-2(3H)-one afforded the targets aimed for. General example: Synthesis of 7-(4-(4-(3 -(piped din- l-yl)propoxy)benzyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-one (AG-

4-(3-(Piperidin-l-yl)propoxy)benzaldehyde

4-Hydroxybenzaldehyde (1 g, 8.19 mmol), Nal (0.61 g, 4.09 mmol), K₂C0₃ (3.51 g, 25.4 mmol) and N-3'-chloropropylpiperidine.HCl (2.43 g, 12.3 mmol) were suspended in acetone (30 mL). The mixture was heated at reflux temperature overnight. The mixture was filtered over Celite and the filter cake was rinsed with acetone. The filtrate was concentrated in vacuo. The residue was taken up in EtOAc and the solids formed were removed by filtration. The filtrate was concentrated in vacuo yielding a yellow oil (2.4 g, 9.6 mmol, 100%). The product was used as such in the next step.

7-(4-(4-(3-(Piperidin-l-yl)propoxy)benzyl)piperazin-l-yl)benzo[d]oxazol-2(3H)- one (AG-0109)

4-(3-(Piperidin-l-yl)propoxy)benzaldehyde (97 mg, 0.39 mmol) and 7-(piperazin-l- yl)benzo[d]oxazol-2(3H)-one (100 mg, 0.39 mmol) were suspended in DCE (25 mL), under a nitrogen atmosphere. Et₃N (0.054 mL, 0.39 mmol), NaBH(OAc)₃ (211 mg, 0.55 mol) and AcOH (0.022 mL, 0.39 mmol) were added respectively. The reaction mixture was stirred at RT for 2 nights. The reaction mixture was poured in NaOH (I N, 50 mL) and DCM (50 mL). The layers were separated. The water layer was extracted with DCM (2 x 50 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo (166 mg) Purification by prep. HPLC afforded a brown foam (38 mg, 0.84 mmol, 22%). 1H-NMR (CDCI₃, in ppm): 1.50 (m, 2H), 1.62 (m, 4H), 2.00 (m, 2H), 2.66 (m, 6H), 2.61 (bs, 4H), 3.35 (bs, 4H), 3.57 (2H), 4.01 (t, 2H), 6.60 (d, 2H), 6.87 (d, 2H), 7.02 (t, 1H), 7.21 (d, 2H).

(M-H)⁺ = 451.2

Example If: Synthesis of 7-(4-(4-((l-alkylpiperidin-4-yl)oxy)phenyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-ones

A Mitsunobu coupling of 4-iodophenol with tert-butyl 4-hydroxypiperidine-l- carboxylate provided the corresponding aryl ether. Boc-deprotection and reductive amination with the appropriate amine afforded the l-alkyl-4-(4- iodophenoxy)piperidines. Subsequent coupling with 7-(piperazin-l-yl)benzo[d]oxazol- 2(3H)-one (Buchwald conditions) yielded the product(s) aimed for. General example: synthesis of 7-(4-(4-((l-cyclobutylpiperidin-4- yl)oxy)phenyl)piperazin-l-yl)benzo[d]oxazol-2(3H)-one (AG-0116).

t-Butyl 4-(4-iodophenoxy)piperidine- 1-car boxylate

4-Iodophenol (1.50 g, 6.8 mmol), fert-butyl-4-hydroxypiperidine-l-carboxylate (1.37 g, 6.8 mmol) and ΡΡ1¾ (2.32 g, 8.8 mmol) in THF (15 mL) were cooled in ice, while DIAL⁾ (1.73 mL, 8.8 mmol, 1.3 eq.) was added drop-wise. The resulting clear mixture was stirred overnight at RT. It was diluted with EtOAc (70 mL), washed with brine (1x30 mL, 1x20 mL), dried (Na₂S0₄) and concentrated to furnish a yellow oil. Automated column chromatography (Silica, 0-30% EtO Ac/heptanes) afforded a mixture of the aryl ether and starting phenol. The oil was dissolved in DCM (100 mL) and washed with 1 M NaOH (35 mL). The organic layer was dried

and concentrated to furnish a colourless oil (1.31 g, 3.44 mmol, 51%).

4-(4-Iodophenoxy)piperidine hydrochloride

tert-Butyl 4-(4-iodophenoxy)piperidine-l-carboxylate (1.39 g, 3.44 mmol) was mixed with 4 M HC1 in dioxane (10 mL) and stirred for 3h. The mixture was concentrated to yield a white solid (580 mg, 1.71 mmol, 50%). l-Cyclobutyl-4-(4-iodophenoxy)piperidine

4-(4-Iodophenoxy)piperidine hydrochloride (580 mg, 1.71 mmol) was mixed with methanol (9 mL). Et₃N (0.24 mL, 1.7 mmol) was added and almost all solid dissolved. Subsequently, AcOH (0.29 mL, 5.1 mmol) was added and the mixture was cooled in ice. Cyclobutanone (0.64 mL, 8.6 mmol) was added, followed by portion-wise addition of NaBH₃CN. The mixture was stirred at 0°C for 5 min, allowed to reach RT afterwards and stirred overnight. The mixture was poured in sat. NaHC0₃ (15 mL) and DCM (15 mL). The water layer was extracted with DCM (2 x 15 mL). The combined organics were washed with brine, dried (Na₂SC>4) and concentrated. The solid was treated with sat. NaHC0 , filtered, air-dried and stripped with toluene (3x) to afford white needles (600 mg, 1.68 mmol, 98%).

7-(4-(4-((l-Cyclobutylpiperidin-4-yl)oxy)phenyl)piperazin-l-yl)benzo[i/|oxazol- 2(3H)-one (AG-0116)

A mixture of l-cyclobutyl-4-(4-iodophenoxy)piperidine. (200 mg, 0.56 mmol), 7- (piperazin-l-yl)benzo[d]oxazol-2(3H)-one (143 mg, 0.56 mmol), Pd₂dba₃.CHCl₃ (58 mg, 0.056 mmol), tBu₃P (1 M in toluene, 0.34 mL, 0.34 mmol) in toluene (1 .2 mL) was degassed with nitrogen for 20 min. NaOtBu (113 mg, 1.18 mmol) was added and the vial was closed and heated at 130°C overnight. The mixture was allowed to cool, filtered and the solid filtered off was washed with EtOAc. The filtrate was extracted with 1 N HC1 (2x10 mL) and H₂0 (10 mL) and the combined aqueous extracts were washed with TBME (10 mL). The water layer was made alkaline with solid NaHC0₃ and 1 N HC1 (2 mL), extracted with EtOAc (3 x 10 mL). The organic extracts were washed with brine (10 mL), dried ( a₂S04) and concentrated to an orange oil. Automated column chromatography (Alox, 0-5% DCM in methanol) and preparative HPLC afforded a light-brown solid (15.7 mg, 0,034 mmol, 6%).

1H- MR (CDC1 , in ppm): 1.4-2.25 (m, 12H), 2.70 (m, 3H), 3.28 (bs, 4H), 3.47 (bs, 4H), 4.22 (m, 1H), 6.63 (t, 2H), 6.95 (m, 4H), 7.08 (t, 1H).

(M-H)⁺ = 449.2

Example lg: Synthesis of 7-(4-(4-(4-alkylpiperazine-l-carbonyl)benzyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-ones HEME H

Reductive amination of Boc-piperazine with the appropriate ketone and subsequent removal of the Boc-moiety (acidic conditions) afforded 1-alkylpiperazines. A peptide coupling with 4-formylbenzoic acid and reductive amination with 7-(piperazin- 1 - yl)benzo[d]oxazol-2(3H)-one furnished the final target(s).

General example: synthesis of 7-(4-(4-(4-cyclobutylpiperazine-l- carbonyl)benzyl)piperazin- 1 -yl)benzo[d]oxazol-2 3H -one (AG-0117).

t-Butyl 4-cyclobutylpiperazine-l-carboxylate

Boc-piperazine (1.86 g; 10.0 mmol) was dissolved in dichloroethane (35 mL). Acetic acid (0.57 mL; 0.60 g; 10.0 mmol) and NaBH(OAc)₃ (3.00 g; 14.2 mmol) were added. Cyclobutanone (0.71 g; 10.0 mmol) was added to the reaction mixture. After stirring overnight NaOH solution (IN, 60 mL) was added, followed by additional H₂0 (50 mL). The aqueous phase was extracted with DCM (2 x 50 mL). The combined organic phases were dried over Na₂SC>4, filtered and evaporated to dryness to yield a slightly yellow oil (2.22 g, 9.24 mmol, 92% ).

1-Cyclobutylpiperazine

t-Butyl 4-cyclobutylpiperazine-l-carboxylate (2.22 g, 9.24 mmol) was dissolved in methanol (50 mL) and cooled in an ice/H₂0 bath. HC1 in 1,4-dioxane (4N, 10 mL) was added drop-wise. The solution was stirred at 0 - 5 °C for 1 h, and then at RT for 3 days. The solution was evaporated to dryness and the white powder was used as such in the next step.

4-(4-Cyclobutylpiperazine-l-carbonyl)benzaldehyde

1-Cyclobutylpiperazine (426 mg. 2.00 mmol, 1.0 eq.) and 4-formylbenzoic acid (300 mg, 2.00 mmol) were suspended in DMF (20 mL), under a nitrogen atmosphere. Et₃N (0.9 mL, 6.39 mmol) and EDCI (460 mg, 2.40 mmol) were added and the mixture was stirred at RT overnight and concentrated in vacuo. DCM (100 mL) was added and the organic layer was washed with H₂0 (1 x 10 mL), HC1 (1 N, 2 x 10 mL), H₂0 (1 x 10 mL), sat. aq. NaHCO sol. (1 x 10 mL) and brine (1 x 10 mL), dried over Na₂SC>4 and concentrated in vacuo to provide a yellow solid. The product was suspended in DCM and the solids were isolated by filtration (16.6 mg, 0.06 mmol, 3%, not optimized).

7-(4-(4-(4-Cyclobutylpiperazine-l-carbonyl)beiizyl)piperazin-l-yl)benzo[d]oxazol- 2(3H)-one (AG-0117)

4-(4-Cyclobutylpiperazine-l-carbonyl)benzaldehyde (16.6 mg, 0.061 mmol) and 7- (piperazin-l-yl)benzo[d]oxazol-2(3H)-one (15.6 mg, 0.061 mmol) were suspended in DCE (2 mL). Et₃N (0.01 mL, 0.061 mmol) was added followed by the addition of AcOH (1 mL). NaBH(OAc)₃ (18.1 mg, 0.085 mmol) was added and the reaction mixture was stirred at RT overnight. NaOH (IN, 10 mL) was added and the water layer was extracted with DCM (3 x 5 mL). The combined organic layers were concentrated in vacuo. The crude product was purified by automated column chromatography (eluent 0 to 20% MeOH in DCM) and prep. HPLC (3.4 mg, 0.0071 mmol, 12%). 1H-NMR (CDCI₃, in ppm): 1.62 (m, 2H), 1.82 (m, 2H), 2.03 (m, 2H), 2.38 (m, 4H), 2.62 (bs, 4H), 2.88 (m, 1H), 3.35 (bs, 4H), 3.48 (m, 2H), 3.60 (s, 2H), 3.81 (m, 2H), 6.60 (d, 2H), 7.02 (t, 1H), 7.40 (bs, 4H).

(M-H)⁺ = 476.2

Example lh: Synthesis of 7-(4-((5-(4-alkyllpiperazine-l-carbonyl)pyridin-2- yl)methyl)piperazin-l-yl)benzo[d]oxazol-2(3H)-ones

A Kornblum oxidation of methyl 6-methylnicotinate with I₂ and DMSO afforded the corresponding aldehyde. Reductive amination with 7-(piperazin-l-yl)benzo[d]oxazol- 2(3H)-one furnished methyl 6-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperazin-l- yl)methyl)nicotinate. Hydrolysis was achieved with LiOH and the lithium salt was subsequently used in a HATU coupling with the appropriate amine to afford the final compound(s). General example: synthesis of 7-(4-((5-(4-isopropylpiperazine-l-carbonyl)pyridin-2- yl)methyl)piperazin-l-yl)benzo[d]oxazol-2(3H)-one (AG-0126).

Methyl 6-formylnicotinate

A mixture of methyl 6-methylnicotinate (5.0 g, 33.0 mmol), I₂ (8.4 g, 33.2 mmol) and TFA (7.5 mL, 11.5 g, 0.1 mol) in DMSO (100 mL) was heated to ~160°C for 2 h. The mixture was allowed to cool to RT and poured into a 1 N solution of Na₂S₂03 (aq.) (0.5 L) solution, made alkaline using NaOH (2 N) and extracted with EtOAc (3 x 0.2 L). The combined organic layers were dried (Na₂SC>4) and concentrated in vacuo. The residue was dissolved in DCM and applied on a short pad of silica. The product was eluted with EtOAc to afford the product as a yellowish solid (3.8 g, 23 mmol, 70%). Methyl 6-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperazin-l- yl)methyl)nicotinate

To a solution of 7-(piperazin-l-yl)benzo[d]oxazol-2(3H)-one (2.0 g, 7.8 mmol) and methyl 6-formylnicotinate (1.55 g, 9.4 mmol) in DMF (50 mL) was added NaBH(OAc)₃ (6.6 g, 31 mmol), followed by the addition of AcOH (1 mL). After stirring overnight it was poured into H₂0 (0.4 L) and made alkaline to pH 14 and stirred for 1 h. The mixture was extracted with EtOAc (3 x 0.1 L). The combined organic layers were dried (Na2SC>4) and concentrated in vacuo. Purification by automated chromatography (ISCO; silica; gradient MeOH in DCM) afforded the pure product as a yellowish solid (2.0 g, 5.42 mmol, 69%). Lithium 6-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperazin-l- yl)methyl)nicotinate

To a solution of methyl 6-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperazin-l- yl)methyl)nicotinate (300 mg, 0.81 mmol) in 10% aqueous THF (10 mL) was added LiOH H₂0 (68.4 mg, 1.62 mmol). After stirring overnight it was concentrated to yield a light brown waxy solid (546 mg, max 0.81 mmol).

7-(4-((5-(4-Isopropylpiperazine-l-carbonyl)pyridin-2-yl)methyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-one (AG-0126)

HATU (124 mg, 0.325 mmol) was added to a solution of lithium 6-((4-(2-oxo-2,3- dihydrobenzo[d]oxazol-7-yl)piperazin-l-yl)methyl)nicotinate (182 mg, max 0.27 mmol), isopropylpiperidine (52 mg, 0.41 mmol) and DIPEA (0.2 mL, 1 mmol) in DMF (5 mL) at RT. After stirring overnight the mixture was concentrated and subjected to automated column chromatography (ISCO; silica; gradient of MeOH in DCM) and, finally, preparative HPLC to furnish a a brown oil (14.4 mg, 0.031 mmol, 11%).

¹H-NMR (CDC1₃, in ppm): 1.03 (d, 6H), 2.4-2.8 (m, 9H), 3.37 (bs, 4H), 3.50 (m, 2H), 3.79 (s, 2H), 3.81 (m, 2H), 6.60 (m, 2H), 7.02 (t, IH), 7.55 (d, IH), 7.79 (d, IH), 8.64 (s, IH).

(M-H)⁺ = 465.3

Results

Table 2

Example 2

Synthesis of D2-H3 7-(l-alkylpiperidin-4-yl)benzo[d]oxazol-2(3H)-ones a(nta)gonists.

Example 2a: Synthesis of 7-(piperidin-4-yl)benzo[d]oxazol-2(3H)-

SCHEME J

Boc-protection of 3-fluoroaniline afforded tert-butyl (3-fluorophenyl)carbamate. Ortho- lithation with fert-butyllithium and reaction with benzyl piperidone furnished the alcohol 7-(l-benzyl-4-hydroxypiperidin-4-yl)benzo[d]oxazol-2(3H)-one in moderate yield. Acidic aqueous reflux resulted in elimination of H₂0 and subsequent treatment with H₂ in the presence of Pd(OH)₂ catalyst yielded 7-(piperidin-4-yl)benzo[d]oxazol- 2(3H)-one.

(3-Fluorophenyl)carbamate

3-Fluoroaniline (29.4 g, 0.26 mol) and Boc₂0 (18.5 g, 0.29 mol) were stirred in refluxing THF (200 mL) for 2 h. The mixture was allowed to cool down to RT, and stirred overnight. The reaction mixture was concentrated and the residue partitioned between EtOAc (300 mL) and 10% citric acid. The organic layer was washed with brine, dried (Na₂SC)4) and concentrated (orange oil). The oil was dissolved in boiling heptane (50 mL) and the clear hot solution was allowed to cool to RT. The crystallized material was filtered. The white solid obtained was washed with heptane and air-dried to furnish (3-fluorophenyl)carbamate (15.6 g, 28%). The mother liquor was concentrated and dissolved in THF. Boc₂0 (27.7 g and a catalytic amount of DMAP were added and the mixture was heated to reflux overnight. The mixture was worked- up as above to afford a white solid (combined yield 34.8 g, 0.16 mmol, 62%).

7-(l-Benzyl-4-hydroxypiperidin-4-yl)benzo [d] oxazol-2(3H)-one

A solution of (3-fluorophenyl)carbamate (14.80 g, 70.1 mmol) in THF (200 mL) in a flame-dried three-neck under nitrogen atmosphere was cooled to T = -70°C. tert-Butyl lithium (1.6 M in pentane, 87.6 mL, 140 mmol) was added drop-wise. After the addition was complete the mixture was stirred at -25°C for 2 h. The mixture was cooled to -70°C and a solution of Λ-benzylpiperidone (12.5 mL, 70.1 mmol) in THF (25 mL) was added drop-wise. On completion of the addition, the dry-ice bath was removed and the mixture allowed to reach RT and stirred overnight. The mixture was cooled in ice and 1 N HCl (100 mL) was added drop-wise, maintaining an internal temperature below 25°C. The mixture was diluted with 1 N HCl (100 mL). The organic layer was separated and washed with 1 N HCl (50 mL). The combined water layers were carefully made alkaline with solid NaHC0₃. The resulting solid was filtered off, washed with H₂0 and Et₂0 and air-dried, yielding the HCl salt of 7-(l-benzyl-4- hydroxypiperidin-4-yl)benzo[<i]oxazol-2(3H)-one as a white solid (9.6 g, 26.6 mmol, 38%). 7-(l-Benzyl-l,2,3,6-tetrahydropyridin-4-yl)benzo[d]oxazol-2(3H)-one

7-(l-Benzyl-4-hydroxypiperidin-4-yl)benzo[<i]oxazol-2(3H)-one (7.2 g, 22.2 mmol) was dissolved in TFA (40 mL). The mixture was refluxed for 15 h and concentrated in vacuo to give the TFA-salt of 7-(l-benzyl-l,2,3,6-tetrahydropyridin-4- yl)benzo[d]oxazol-2(3H)-one as a brown oil (12.1 g, max 22.2 mmol)). The material was used as such in the next step. 7-(Piperidin-4-yl)benzo [d] oxazol-2(3H)-one

A mixture of the TFA-salt of 7-(l-benzyl-l,2,3,6-tetrahydropyridin-4- yl)benzo[d]oxazol-2(3H)-one (12.1 g, max 22.2 mmol) and Pd(OH)₂/carbon (20%) (1.5 g) in MeOH (150 mL) was stirred overnight under a H₂ atmosphere (balloon). The mixture was filtered over Celite and the pad was washed MeOH. The filtrate was concentrated and stirred in Et₂0 (40 mL). The precipitate was filtered and dried to afford an off-white solid (6.7 g, 21.4 mmol, 96% over two steps).

Example 2b: Synthesis of 7-(l-(3-(4-((alkyllamino)methyl)phenoxy)alkyl)piperidin-4- yl)benzo[d]oxazol-2(3H)-ones

SCHEME K

The compounds aimed for were prepared by alkylation of 7-(piperidin yl)benzo[d]oxazol-2(3H)-one with the appropriate bromine (see example la2).

General example 7-(l-(3 -(4-(morpholinomethyl)phenoxy)propyl)piperidin yl)benzo[d]oxazol-2(3H)-one (AG-

7-(l-(3-(4-(Morpholinomethyl)phenoxy)propyl)piperidin-4-yl)benzo[d]oxazol- 2(3H)-one (AG-104)

A mixture of 7-(piperidin-4-yl)benzo[d]oxazol-2(3H)-one. TFA (2 g, 6.3 mmol), 4-(4- (3-bromopropoxy)benzyl)morpholine (1.6 g, 5.1 mol), K₂C0₃ (700 mg, 5.1 mol) and Nal (0.085 g, 0.6 mmol) in MeCN (50 mL) was heated at 80°C overnight. The mixture was concentrated, diluted with H₂0 and extracted with DCM (3 x 50 mL). The organics were washed with H₂0, dried (Na₂S0₄) and concentrated. Automated column chromatography (ISCO (Alox, 0-5% MeOH in DCM)) provided a light-yellow foam that was dissolved in DCM (20 - 50 ml). 4M HC1 in dioxane (2.2 ml, 8.7 mmol, 3 eq.) was added and the resulting mixture was stirred for 2 - 3 hours at RT. The solids were filtered, dried and dissolved in H₂0. Freeze-drying afforded an off-white, fluffy solid (1.20 g, 2.29 mmol, 45%).

1H-NMR (CDC1₃, in ppm): 2.2-2.4 (m, 6H), 3.2-3.45 (m, 11H), 3.6-4.1 (m, 6H), 4.20 (t, 2H), 7.0-7.2 (m, 5H), 7.50 (d, 2H).

(M-H)⁺ = 452.2

The above-described procedure was utilized for synthesis of AG-0200.

Example 2c: Synthesis of 7-(l-(4-((l-alkylpiperidin-4-yl)oxy)benzyl)piperidin-4- yl)benzo[d]oxazol-2(3H)-ones

SCHEME L

Reductive amination of 7-(piperidin-4-yl)benzo[d]oxazol-2(3H)-one with t-butyl 4-(4- formylphenoxy)piperidine-l-carboxylate (see example lc) and subsequent hydrolysis of the Boc-protecting group afforded the intermediate 7-(l-(4-(piperidin-4- yloxy)benzyl)piperidin-4-yl)benzo[d]oxazol-2(3H)-one. Reductive amination with the appropriate amine furnished the targets aimed for.

General example 7-( 1 -(4-(( 1 -cyclobutylpiperidin-4-yl)oxy)benzyl)piperidin-4- yl)benzo[d]oxazol-2(3H)-one (AG-

i-Butyl 4-(4-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperidin-l- yl)methyl)phenoxy)piperidine-l-carboxylate

NaBH(OAc)3 (6.65 g, 31 mmol) was added portion-wise to a mixture of t-butyl 4-(4- formylphenoxy)piperidine-l-carboxylate (1.68 g, 5.49 mmol), 7-(piperidin-4- yl)benzo[d]oxazol-2(3H)-one (1.14 g, 5.23 mmol) and AcOH (0.5 mL) in DMF (25 mL). The mixture was stirred at RT for 16 hr and poured into H₂0. After stirring for 1 hr the mixture was extracted with EtOAc (3x), dried over Na₂SC>4 and concentrated in vacuo. The crude product was stirred in Et₂0. The precipitate formed was filtered off. Additional drying in vacuo yielded the title compound as a pinkish solid (1.42 g, 2.80 mmol, 54%).

7-(l-(4-(Piperidin-4-yloxy)benzyl)piperidin-4-yl)benzo[d]oxazol-2(3H)-one

t-Butyl 4-(4-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperidin-l- yl)methyl)phenoxy)piperidine-l-carboxylate (1.42 g, 2.80 mmol) was suspended in DCM (50 mL) and TFA (1.04 mL, 1.59 g, 14 mmol) was added drop-wise. Upon addition of TFA everything dissolved. The mixture was stirred for 1 h and concentrated in vacuo (red oil). The compound was used as such in the next step.

7-(l-(4-((l-Cyclobutylpiperidin-4-yl)oxy)benzyl)piperidin-4-yl)benzo[d]oxazol- 2(3H)-one (AG-0114)

7-(l-(4-(Piperidin-4-yloxy)benzyl)piperidin-4-yl)benzo[d]oxazol-2(3H)-one (max. 2.80 mmol) was dissolved in DMF (25 mL) and AcOH (0.5 mL) was added. Cyclobutanone (1.26 mL, 1.18 g, 16.8 mmol) and NaBH(OAc)₃ (2.37 g, 11.2 mmol) were added portion-wise. The mixture was stirred at RT for 16 hr and poured into H₂0. The pH was adjusted to > 14 with 2 N NaOH and the mixture was extracted with EtOAc (3x). The combined organic phases were dried over Na₂S0₄ and concentrated in vacuo. The crude product was purified by automated column chromatography (ISCO/(MeOH (3.5N ¾) in DCM). The product aimed for was collected as an off-white solid (391 mg, 0.85 mmol, 30%).

¹H-NMR (CDCI₃, in ppm): 1.4-2.25 (m, 12H), 2.70 (m, 3H), 3.28 (bs, 4H), 3.47 (bs, 4H), 4.22 (m, 1H), 6.63 (t, 2H), 6.95 (m, 4H), 7.08 (t, 1H).

(M-H)⁺ = 449.2

The above-described procedure was utilized for synthesis of AG-0200.

Example 2d: Synthesis of 7-(4-((6-((l-alkylpiperidin-4-yl)oxy)pyridin-3- yl)methyl)piperazin-l-yl)benzo[d]oxazol-2(3H)-ones

Alkylation of 6-hydroxynicotinaldehyde with t-butyl 4-iodopiperidine-l-carboxylate afforded t-butyl 4-((5-formylpyridin-2-yl)oxy)piperidine-l-carboxylate. Subsequent reductive amination with 7-(piperidin-4-yl)benzo[d]oxazol-2(3H)-one and hydrolysis of the Boc-protecting group yielded the intermediate 7-(l-((6-(piperidin-4- yloxy)pyridin-3-yl)methyl)piperidin-4-yl)benzo[d]oxazol-2(3H)-one. Reductive amination with the appropriate amine furnished the targets aimed for. General example; Synthesis of 7-(l-((6-((l-cyclobutylpiperidin-4-yl)oxy)pyridin-3- yl)methyl)piperidin-4-yl)benzo[d] 0325).

f-Butyl 4-((5-formylpyridin-2-yl)oxy)piperidine- 1-carboxylate

2-Hydroxy-5-pyridinecarbaldehyde (123 mg, 1 mmol) and N-boc-4-iodopiperidine (622 mg, 2 mmol) were mixed in heptanes in a microwave vial and AgC0₃ (331 mg, 1.2 mmol) was added. The vial was capped and heated to 150°C for 70 minutes. The batch was filtered and concentrated. Purification over silica (EtOAc/Heptanes 2:3) afforded the product aimed for (92 mg, 0.3 mmol, 30%). f-Butyl 4-((5-((4-(2-oxo-2,3-dihydrobenzo[i/|oxazol-7-yl)piperidin-l- yl)methyl)pyridin-2-yl)oxy)piperidine-l-carboxylate

Z-Butyl 4-((5-formylpyridin-2-yl)oxy)piperi dine- 1-carboxylate (212 mg, 0.69 mmol), 7- (piperidin-4-yl)benzo[i/]oxazol-2(3H)-one (125 mg, 0.58 mmol), AcOH (0.14 mL) and NaBH(OAc)₃ (489 mg, 2.30 mmol) were added to DMF (30 ml). The mixture was stirred over weekend under N2 atmosphere and H₂0 was added. The mixture was made alkaline with 1 N aq. NaOH solution and extracted with EtOAc (3 x). The organic layers were dried over Na2SC>4, filtered and concentrated to afford a yellow oil, which solidified upon standing. Purification by automated column chromatography (ISCO (0 - 100% EtO Ac/heptanes) afforded a white foam (168 mg, 0.39 mmol, 57%). 7-(l-((6-(Piperidin-4-yloxy)pyridin-3-yl)methyl)piperidin-4-yl)benzo[d]oxazol- 2(3H)-one

z-Butyl 4-((5-((4-(2-oxo-2,3-dihydrobenzo[<i]oxazol-7-yl)piperidin-l- yl)methyl)pyridin-2-yl)oxy)piperidine- l-carboxylate (50 mg, 0.098 mmol) was dissolved in MeOH (4 mL) and 4 N HC1 in dioxane (0.16 niL, 0.64 mmol) was added to the solution. The mixture was stirred for 30 minutes under N₂ atmosphere and concentrated to afford a white solid (59 mg (>100%)). The product was used as such in the next step. 7-(l-((6-((l-Cyclobutylpiperidin-4-yl)oxy)pyridin-3-yl)methyl)piperidin-4- yl)benzo[</|oxazol-2(3H)-one (AG-0325)

7-(l-((6-(Piperidin-4-yloxy)pyridin-3-yl)methyl)piperidin-4-yl)benzo[d]oxazol-2(3H)- one (crude 59 mg, max. 0.10 mmol) was dissolved in DMF (5 mL) and AcOH (catalytic amount), cyclobutanone (8.4 mg, 0.12 mmol) and NaBH(OAc)3 (83 mg, 0.392 mmol) were added. The reaction mixture was stirred overnight under N₂ atmosphere, poured in H₂O and made alkaline with 1 N aq. NaOH solution. The mixture was extracted with EtOAc (3 x), dried over Na₂S0₄, filtered and concentrated. The mixture was redissolved in a DCM 1 N aq. NaOH solution and worked up as above to afford a clear oil (22 mg, 0.047 mmol, 48%).

1H-NMR (CDCI₃, in ppm): 1 .2-2.25 (m, 22H), 2.60-3.05 (m, 2H), 3.43 (s, 2H), 3.61 (m, IH), 6.63 (d, IH), 6.85 (d, IH), 6.98 (d, IH), 7.06 (t, IH), 7.60 (dd, IH), 8.01 (s, IH).

(M-H)⁺ = 463.2

Example 2e: 7-(l-(4-(3-(alkylamino)propoxy)benzyl)piperidin-4-yl)benzo[d]oxazol- 2(3H)-ones

SCHEME N

Reductive amination of 7-(piperazin-l-yl)benzo[d]oxazol-2(3H)-one with the appropriate 4-(3-(alkylamino)propoxy)benzaldehydes (see example le) afforded the targets aimed for.

General example: Synthesis of 7-(l-(4-(3-(piperidin-l-yl)propoxy)benzyl)piperidin-4- yl)benzo[d]oxazol-2(3H)-one ( -0327).

7-(l-(4-(3-(Piperidin-l-yl)propoxy)benzyl)piperidin-4-yl)benzo[d]oxazol-2(3H)- one (AG-0327)

NaBH(OAc)3 (443 mg; 2.1 mmol) was added to a solution of 4-(3-(piperidin-l- yl)propoxy)benzaldehyde (200 mg; 0.57 mmol), 7-(piperidin-4-yl)benzo[d]oxazol- 2(3H)-one (114 mg; 0.52 mmol) and AcOH (1 mL) in DMF (10 mL). This mixture was stirred overnight. H₂0 (20 mL) was added and the reaction mixture was made alkaline with 1 N NaOH. The mixture was extracted with EtOAc (2x), dried over Na₂S0₄ and concentrated in vacuo. Purification of the crude material (-300 mg) by automated column chromatograpy (ISCO; gradient DCM/MeOH) and prep. HPLC afforded a slightly brown/green solid (100 mg, 0.22 mmol, 43%).

(M-H)⁺ = 450.3 Example 2f: Synthesis of 7-(4-(4-(4-alkylpiperazine-l-carbonyl)benzyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-ones

SCHEME O

Amidation of 4-formylbenzoic acid with mono-Boc piperazine afforded tert-butyl 4-(4- formylbenzoyl)piperazine-l-carboxylate. Reductive amination of 7-(piperidin-4- yl)benzo[d]oxazol-2(3H)-one with tert-butyl 4-(4-formylbenzoyl)piperazine-l- carboxylate (see example lg) and subsequent removal of the Boc-moiety (acidic conditions) furnished 7-(l-(4-(piperazine-l-carbonyl)benzyl)piperidin-4- yl)benzo[d]oxazol-2(3H)-one. Reductive amination with the appropriate ketone/aldehyde yielded the final target(s).

General example: synthesis of 7-(4-(4-(4-cyclobutylpiperazine-l- carbonyl)benzyl)piperazin-l-yl)ben (AG-0323).

f-Butyl 4-(4-formylbenzoyl)piperazine-l-carboxylate

To a suspension of 4-carboxyaldehyde (4.95 g, 33.0 mmol) in DCM (100 mL) was added piperazine-l-carboxylic tert-butyl ester (6.2 g, 33.3 mmol), EDCI (6.4 g, 33.3 mmol), HOBt (4.5 g, 33.3 mmol) and DMAP (32 mg, 0.27 mmol). The mixture was cooled in an ice-bath for lh and stirred overnight at RT. The reaction mixture was washed with 1 N NaOH and 1 N HCl, dried over Na₂S0₄ and concentrated in vacuo yielding a white solid (10.4 g, 32.7 mmol, 98%). f-Butyl 4-(4-((4-(2-oxo-2,3-dihy drobenzo [d] oxazol-7-yl)piperidin- 1- yl)methyl)benzoyl)piperazine-l-carboxylate

NaBH(OAc) (3.1 g, 14.7 mmol) was added to a solution of t-butyl 4-(4- formylbenzoyl)piperazine-l-carboxylate (1.4 g, 4.40 mmol) and 7-(piperidin-4- yl)benzo[d]oxazol-2(3H)-one (800 mg, 3.66 mmol) in DMF (50 mL and AcOH (1 mL). The mixture was stirred overnight at RT. H₂0 (100 mL) was added and the mixture was stirred for 10 min. The mixture was made alkaline with 1 N NaOH, extracted with EtOAc (2 x) dried over Na₂S0₄ and concentrated in vacuo. Purification by automated column chromatography (Silica; DCM/MeOH (0-10%>) furnished an off-white solid (1.0 g, 1.9 mmol, 52%).

7-(l-(4-(Piperazine-l-carbonyl)benzyl)piperidin-4-yl)benzo[d]oxazol-2(3H)-one t-butyl 4-(4-((4-(2-oxo-2,3-dihydrobenzo[d]oxazol-7-yl)piperidin-l- yl)methyl)benzoyl)piperazine-l-carboxylate (800 mg, 1.5 mmol) was dissolved in DCM (10 niL). A few mL of TFA was added and the mixture was concentrated in vacuo. 7-(l-(4-(Piperazine-l-carbonyl)benzyl)piperidin-4-yl)benzo[d]oxazol-2(3H)- one (bis-TFA-salt) was obtained as an off-white foam (1.1 g). The product was used as such in the next step.

7-(l-(4-(4-Cyclobutylpiperazine-l-carbonyl)benzyl)piperidin-4-yl)benzo[d]oxazol- 2(3H)-one (AG-0323)

7-(l-(4-(Piperazine-l-carbonyl)benzyl)piperidin-4-yl)benzo[d]oxazol-2(3H)-one (200 mg, 0.41 mmol) was dissolved in DMF (10 mL) and AcOH (1 mL). Cyclobutanone (142 mg, 2.0 mmol) was added followed by a portion-wise addition of NaBH(OAc)3 (687 mg, 3.2 mmol). The reaction mixture was stirred overnight and poured on ice and made alkaline with 1 N NaOH. The reaction mixture was extracted with EtOAc (2x) dried over Na₂S04 and concentrated in vacuo. Purification by automated column chromatography (Silica; 0-10% DCM/MeOH) furnished an off-white solid (63 mg, 0.13 mmol, 33%).

1H-NMR (CD₃OD, in ppm): 1.7-2.5 (m, 18H), 2.80 (m, 1H), 3.03 (m, 1H), 3.40-3.80 (m, 6H), 6.91 (d, 1H), 6.99 (d, 1H), 7.10 (t, 1H), 7.40 (d, 2H), 7.49 (d, 2H).

(M-H)⁺ = 475.2 Results

Table 3

R = D₂ (IC₅₀) D₂ (EC₅₀) H₃ antagonist Agonist (nM)

(nM) (nM)

Example 3

Synthesis of D₂-H₃ l-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)-4-alkyllpiperazine a(nta)gonists.

Example 3a: Synthesis of l-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine

SCHEME P

A Curtius reaction of 2,3-dihydrobenzo[b][l,4]dioxine-5-carboxylic acid and removal of the Boc-moiety under acidic conditions furnished 2,3-dihydrobenzo[b][l,4]dioxin-5- amine. Subsequent reaction with bis(2-chloroethyl)amine afforded l-(2,3- dihydrobenzo[b] [ 1 ,4]dioxin-5-yl)piperazine. f-Butyl (2,3-dihydrobenzo[b][l,4]dioxin-5-yl)carbamate

2,3-Dihydro-l,4-benzodioxine-5-carboxylic acid (45 g, 0.25 mol) was dissolved in t- BuOH (250 mL). Et₃N (53.0 g, 72.9 mL, 0.52 mol) was added and the mixture was heated to reflux. DPPA (68.7 g, 53.8 mL, 0.25 mol) was added drop-wise (gas evolution) and the reaction mixture was heated to reflux overnight. The reaction mixture was concentrated and the residue obtained was dissolved in toluene and washed with 2N NaOH. The layers were separated and the organic layer was filtered over Celite and concentrated. The concentrate was dissolved in CHC1₃, filtered over Celite and concentrated to yield a yellow oil (56.5 g, 0.22 mol, 90%).

2,3-Dihydrobenzo[b][l,4]dioxin-5-amine

t-Butyl (2,3-dihydrobenzo[b][l,4]dioxin-5-yl)carbamate (56.5 g, 0.22 mol) was dissolved in DCM (150 mL). TFA (-30 mL) was added and the reaction mixture was stirred over the weekend. The mixture was concentrated, dissolved in EtOAc (350 mL) and washed with 1 N NaOH and brine. After drying over Na₂S04 concentration afforded a brown oil (34.8 g, 0.22 mol, quant.). l-(2,3-Dihydrobenzo[b] [l,4]dioxin-5-yl)piperazine

2,3-Dihydrobenzo[b][l,4]dioxin-5-amine (1.2 g, 0.008 mol) and bis(2-chloroethyl) amine.HCl (1.4 g, 8.0 mmol) where dissolved in chlorobenzene (20 ml) and heated to reflux over the weekend. The reaction mixture was concentrated in vacuo, diluted with H₂0 (50 mL), made alkaline with NaOH (5 M) and extracted with EtOAc (5 x 50 ml). The combined organic layers were dried over Na₂S0₄, filtered and concentrated to afford a purple solid (1.8 g, 8.0 mmol, 100%).

Example 3b: Synthesis of l-(4-(3-(4-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l- yl)alkoxy)phenyl)-N-alkylmethanamines

SCHEME Q

The compounds aimed for were prepared by alkylation of l-(2,3- dihydrobenzo[b][l,4]dioxin-5-yl)piperazine with the appropriate bromine (see example la2). General example 4-(4-(3-(4-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l- yl)propoxy)benzyl)morpholine (AG-

4-(4-(3-(4-(2,3-Dihydrobenzo[b] [l,4]dioxin-5-yl)piperazin-l- yl)propoxy)benzyl)morpholine (AG-0098)

l-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (300 mg, 1.18 mmol), 4-(4-(3- bromopropoxy)benzyl)morpholine (552 mg, 1.76 mmol), K2CO₃ (437 mg, 3.16 mmol) and KI (29 mg, 0.18 mmol) were suspended in MeCN (25 mL). The reaction mixture was heated at 80°C overnight, and concentrated in vacuo. The crude product obtained was purified by column chromatography (ISCO, eluent 0 to 10% MeOH in DCM) affording a greyish solid (0.28 g, 0.62 mmol, 52%).

¹H-NMR (CDC1₃, in ppm): 1.4-2.25 (m, 12H), 2.70 (m, 3H), 3.28 (bs 4H), 3.47 (bs, 4H), 4.22 (m, 1H), 6.63 (t, 2H), 6.95 (m, 4H), 7.08 (t, 1H).

(M-H)⁺ = 449.2

The above-described procedure was also utilized for synthesis of AG-0199. Example 3c: Synthesis of l-(4-((l-alkylpiperidin-4-yl)oxy)benzyl)-4-(2,3- dihydrobenzo[b][l,4]dioxin-5-yl)piperazines

SCHEME R

Alkylation of 4-fluorobenzonitril with 4-Boc-piperidinol (KHMDS) and deprotection of the Boc-group afforded the TFA salt of 4-(piperidin-4-yloxy)benzonitrile. Reductive amination with the appropriate amine and hydrolysis of the nitrile resulted in the corresponding carboxylic acid. Conversion to the ester, subsequent reduction and finally oxidation towards the aldehyde with Mn0₂ yielded (4-((l-alkyllpiperidin-4- yl)oxy)phenyl)methanols. The final target(s) was/were obtained by reductive amination with l-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine.

Most likely, the synthesis can be simplified using the synthetic strategy as depicted in e.g., Scheme CI .

General example l-(4-((l-cyclobutylpiperidin-4-yl)oxy)benzyl)-4-(2,3- dihydrobenzo[b][l,4]dioxin-5-yl)pi

t-Butyl 4-(4-cyanophenoxy)piperidine-l-carboxylate

To a solution of 4-fluorobenzonitril (6.0 gram, 49.5 mmol) and 4-Boc-piperidinol (10.0 g, 49.7 mmol) in THF at 0°C was added drop-wise a 0.5 M solution of KHMDS (100 mL, 50 mmol) in toluene. The mixture was stirred for 2h at RT. The THF was removed in vacuo and EtOAc (200 mL) was added. The organic layer was washed with 1 M HC1 (2 x 50 mL), aqueous saturated sodium bicarbonate (2 x 50 mL) and once with brine. The organic layer was dried over Na₂SC>4 and concentrated. Further purification with flash chromatography (50% EtO Ac/heptanes) afforded a clear oil (12.7 gram, 42.0 mmol, 84%). 4-((l-Cyclobutylpiperidin-4-yl)oxy)benzonitrile

A solution of t-butyl 4-(4-cyanophenoxy)piperidine-l-carboxylate (12.7 g, 42.0 mmol) in DCM (50 mL) was treated with TFA (50 mL) and stirred for 1 h at RT. The solution was concentrated in vacuo and co-evaporated with toluene. The resulting TFA-salt was suspended in DCM (350 mL) and ΕΪ3Ν (18 mL, 128 mmol) and cyclobutanone (4.2 mL, 60 mmol) were added. After 30 min sodium triacetoxyborohydride (12.4 gram, 58.9 mmol) was added and the resulting solution was stirred overnight at RT. The mixture was washed with 1 M potassium carbonate (3 x 50 mL) and once with brine. The organic layer was dried over Na₂S0₄ and concentrated. Purification by flash chromatography (gradient 0 to 5% MeOH in DCM), resulted in an off-white solid (8.66 g, 33.8 mmol, 80%, 2 steps).

4-((l-Cyclobutylpiperidin-4-yl)oxy)benzoic acid

A suspension of 4-((l-cyclobutylpiperidin-4-yl)oxy)benzonitrile (8.7 gram, 33.8 mmol) in concentrated HCl (150 mL) was refluxed for 2 h. After cooling to RT the solution was stored at -18 °C for 1 h. The mixture was filtered and washed with some cold H₂0. The residue was dried in vacuo to afford the compound aimed for as a white solid (5.4 gram, 17.3 mmol, 52%). (4-((l-Cyclobutylpiperidin-4-yl)oxy)phenyl)methanol

To a suspension of 4-((l-cyclobutylpiperidin-4-yl)oxy)benzoic acid hydrochloride (1.0 g, 3.2 mmol) in MeOH (10 mL) was added AcCl (2 mL) dropwise. The resulting mixture was refluxed for 4 h. After concentration the residue was separated between EtOAc and 0.1 N NaOH. The water layer was extracted with EtOAc (2 x). The combined organic layers were dried (Na₂S0₄) and concentrated to yield methyl 4-((l - cyclobutylpiperidin-4-yl)oxy)benzoate (709 mg, 2.6 mmol, 80%). The material was dissolved in THF (20 mL) and LiAlH₄ (200 mg, 5.2 mmol) was added at RT. The mixture was refluxed for 2 h EtOAc (100 mL) was carefully added, followed by acetone (2 mL) and H₂0 (10 mL). The organic layer was separated, dried (Na₂S0₄) and concentrated to afford (4-((l-cyclobutylpiperidin-4-yl)oxy)phenyl)methanol as a brown oil (670 mg, 2.56 mmol, 98%).

4-((l-Cyclobutylpiperidin-4-yl)oxy)benzaldehyde

(4-((l-Cyclobutylpiperidin-4-yl)oxy)phenyl)methanol (530 mg, 2.03 mmol) was dissolved in DCM (10 mL), under a N₂ atmosphere. Mn0₂ (1.4 g, 16.2 mmol) was added and the mixture was stirred at RT overnight. The mixture was filtered over Celite and the filter cake was rinsed with DCM (100 mL). The filtrate was concentrated in vacuo yielding a brown solid (0.46 g, 1.8 mmol, 88%). l-(4-((l-Cyclobutylpiperidin-4-yl)oxy)benzyl)-4-(2,3-dihydrobenzo [b] [1,4] dioxin- 5-yl)piperazine (AG-0118)

l-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (53 mg, 0.21 mmol) and 4-((l- cyclobutylpiperidin-4-yl)oxy)benzaldehyde (75 mg, 0.29 mmol) were suspended in DCE (2 mL). Et₃N (0.03 mL, 0.21 mmol) and acetic acid (~1 mL) were added and heating afforded a clear solution. After cooling to RT, NaB(OAc)₃ (88 mg, 0.41 mmol) was added and the mixture was stirred overnight. NaOH (IN, 10 mL) was added and the water layer was extracted with DCM (3 x 5 mL). The combined organic layers were washed with brine (1 x 5 mL), dried over Na₂S04 and concentrated in vacuo. The crude product obtained was purified by column chromatography (ISCO (eluent 0 to 20% MeOH in DCM) affording a white foam (23.3 mg, 0.050 mmol, 24%).

XH-NMR (CDC1₃, in ppm): 1.55-2.25 (m, 14H), 2.60-2.90 (m, 5H), 3.12 (bs, 4H), 3.58 (s, 2H), 4.24 (t, 2H), 4.32 (t, 2H), 4.50 (m, 1H), 6.57 (d, 1H), 6.60 (d, 1H), 6.79 (t, 1H), 6.87 (d, 2H), 6.91 (d, 2H).

(M-H)⁺ = 464.2

Example 3d: Synthesis of 3-(4-((4-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l- yl)methyl)phenoxy)-N-alkylpropan- 1 -amines HEME S

Reductive amination of 7-(piperazin-l-yl)benzo[d]oxazol-2(3H)-one with the appropriate 4-(3-(alkylamino)propoxy)benzaldehydes (see example le) afforded the targets aimed for. General example: Synthesis of 4-(3-(4-((4-(2,3-dihydrobenzo[b][l,4]dioxin-5- yl)piperazin-l-yl)methyl)phenoxy)propyl)moipholine (AG-0448).

4-(3-(4-((4-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l- yl)methyl)phenoxy)propyl)morpholine (AG-0448)

4-(3-Morpholinopropoxy)benzaldehyde (143 mg, 0.57 mmol), l-(2,3- dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (126 mg, 0.57 mmol), NaBH(OAc)₃ (170 mg, 0.80 mmol) and AcOH (33 μΐ_^, 0.57 mmol) were suspended in DMF (10 mL) and stirred at RT overnight. H₂0 (50 mL) and NaOH (35%) were added and the reaction mixture was extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. The product was purified by column chromatography (ISCO, eluent 0 to 20% MeOH in DCM) affording a a orange oil (138 mg, 0.30 mmol, 53%).

¹HNMR (CDC1₃): 1.99 (q, 2H) 2.45-2.55 (m, 6H), 2.65 (bs, 4H), 3.09 (bs, 4H), 3.53 (s, 2H), 3.73 (m, 4H), 4.03 (t, 2H), 4.24 (m, 2H), 4.32 (m, 2H), 6.54 (dd, 1H), 6.59 (dd, 1H), 6.77 (t, 1H), 6.85 (d, 2H), 7.25 (d, 2H).

(M-H)⁺ = 454.2

The above-described procedure was used for preparation of AG-0339. Example 3e: Synthesis of 7-(4-(4-(4-alkylpiperazine-l-carbonyl)benzyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-ones

SCHEME T

Reductive amination of l-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine with t-butyl 4-(4-fonnylbenzoyl)piperazine-l-carboxylate (see example lg) and subsequent removal of the Boc-moiety (acidic conditions) furnished (4-((4-(2,3- dihydrobenzo[b] [ 1 ,4]dioxin-5-yl)piperazin- 1 -yl)methyl)phenyl)(piperazin- 1 - yl)methanone. Reductive amination with the appropriate ketone/aldehyde yielded the final target(s).

General example: synthesis of (4-cyclobutylpiperazin-l-yl)(4-((4-(2,3- dihydrobenzo[b] [ 1 ,4]dioxin-5-yl)piperazin- 1 -yl)methyl)phenyl)methanone (AG-0338).

t-Butyl 4-(4-((4-(2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazin-l- yl)methyl)benzoyl)piperazine-l-carboxylate

l-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (1.0 g, 4.5 mmol) and tert-butyl 4- (4-formylbenzoyl)piperazine-l-carboxylate (1.7 g, 5.5 mmol) where dissolved with DMF/AcOH (50 niL/1 mL). NaBH(OAc)₃ (3.8 g, 0.0182 mol) was added in portions. The reaction mixture was stirred at RT over the weekend. H₂0 was added, followed by addition of NaOH (1 M). After extraction with EtOAc (3 x 100 mL) the combined organic layers were dried over Na₂SC>4, filtered and concentrated. The residue obtained was purified by column chromatography affording the compound aimed for (2.4 g, 4.5 mmol, 100%). (4-((4-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l- yl)methyl)phenyl)(piperazin-l-yl)methanone

t-Butyl 4-(4-((4-(2,3 -dihydrobenzo[b] [ 1 ,4]dioxin-5-yl)piperazin- 1 - yl)methyl)benzoyl)piperazine-l-carboxylate (2.3 g, 3.5 mmol) was dissolved in DCM (20 mL). To the solution was added TFA (0.8 g, 0.5 mL, 7.0 mol). The reaction mixture was stirred for 1-2 hours, then concentrated in vacuo affording a yellow foam (1.5 g, quant.).

(4-Cyclobutylpiperazin-l-yl)(4-((4-(2,3-dihydrobenzo[bJ [l,4Jdioxin-5-yl)piperazin- l-yl)methyl)phenyl)methanone (AG-0338)

(4-((4-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l-yl)methyl)phenyl)(piperazin- l-yl)methanone (1.5 g, 4.0 mmol) was dissolved in DMF/AcOH (50 mL/5 mL). Cyclobutanone (1.26 g, 1.4 mL, 18.0 mmol) was added followed by a portion-wise addition of NaBH(OAc)₃ (5.9 g, 28.0 mmol). The reaction mixture was stirred at RT overnight, poured on ice, made alkaline with 2 - 3M NaOH and extracted with EtOAc (3 x). The combined organic layers were dried, filtered and concentrated. Purification by column chromatography furnished a yellow foam (493 mg, 1.03 mmol, 26% yield). 1H MR (CDC1₃): 1.6 (m, 4H), 1.8 (m, 2H), 2.2-2.4 (m, 4H), 2.61 (s, 4H), 2.70 (t, 1H), 3.08 (s, 4H), 3.45 (m, 2H), 3.61 (s, 2H), 3.79 (m, 2H), 4.2-4.4 (m, 4H), 6.48 (q, 2H); 6.76 (t, 1H), 7.34 (m, 4H).

(M-H)⁺ = 477.2

Example 3f: Synthesis of 7-(4-(4-(4-alkylpiperazine-l-carbonyl)benzyl)piperazin-l- yl)benzo[d]oxazol-2(3H)-ones

SCHEME U

Reductive amination of 4-(4-hydroxybut-l-yn-l-yl)benzaldehyde with l-(2,3- dihydrobenzo[b][l,4]dioxin-5-yl)piperazine yielded the alcohol 4-(4-((4-(2,3- dihydrobenzo[b] [ 1 ,4]dioxin-5-yl)piperazin- 1 -yl)methyl)phenyl)but-3 -yn- 1 -ol.

Tosylation and reaction with the appropriate amine afforded the compound(s) aimed for. General example: Synthesis of l-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)-4-(4-(4- (piperidin- 1 -yl)but- 1 -yn- 1 -yl)benzyl)piperazine (AG-0374) .

4-(4-((4-(2,3-Dihydrobenzo[b] [l,4]dioxin-5-yl)piperazin-l-yl)methyl)phenyl)but-3- yn-l-ol

l-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (500 mg, 2.27 mmol) and 4-(4- hydroxybut-l-yn-l-yl)benzaldehyde (475 mg, 2.27 mmol) were suspended in DMF/AcOH (50 mL/1 mL). The mixture was stirred for 30 minutes, then NaBH(OAc)₃ (193 mg, 9.09 mmol) was added and the resulting reaction mixture was stirred at RT overnight. The reaction mixture was concentrated, diluted with H₂0 (200 ml), made alkaline with NaOH (2 M) and extracted with DCM (3 x 150 ml). The combined organic layers were dried, filtered and concentrated affording 4-(4-((4-(2,3- dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l-yl)methyl)phenyl)but-3-yn-l-ol (0.6 g, 1.59 mmol, 70%). The material obtained was used in the next step without any further purification.

4-(4-((4-(2,3-Dihydrobenzo [b] [1 ,4] dioxin-5-yl)piperazin- l-yl)methyl)phenyl)but-3- yn-l-yl 4-methylbenzenesulfonate

4-(4-((4-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)piperazin-l-yl)methyl)phenyl)but-3-yn- l-ol (60 mg, 0.16 mmol) and triethylamine (0.9 mL, 0.634 mmol) were dissolved in DCM and cooled to T = 0°C. p-TosCl (60 mg, 0.317 mmol) was added and the reaction mixture was stirred at RT overnight. The reaction mixture was washed with sat. NaHC0₃ and H₂0, dried, filtered and concentrated affording crude 4-(4-((4-(2,3- dihydrobenzofb] [ 1 ,4]dioxin-5-yl)piperazin- 1 -yl)methyl)phenyl)but-3 -yn- 1 -yl 4- methylbenzenesulfonate (90 mg, 0.17 mmol, quant.). The product was utilized next step without any further purification. l-(2,3-Dihydrobenzo[b][l,4]dioxin-5-yl)-4-(4-(4-(piperidin-l-yl)but-l-yn-l- yl)benzyl)piperazine (AG-0374)

A pressure tube was charged with 4-(4-((4-(2,3-dihydrobenzo[b][l,4]dioxin-5- yl)piperazin-l-yl)methyl)phenyl)but-3-yn-l-yl-4-methylbenzenesulfonate (90 mg, 0.17 mmol), piperidine (0.04 mL, 0.34 mmol), Na₂C0₃ (56 mg, 0.37 mmol) and DMF (10 - 20 mL). The reaction mixture was heated to 80°C for 2 h in a sand bath. The reaction mixture was cooled to RT, concentrated, diluted with H₂0 (50 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried, filtered and concentrated. Purification by column chromatography (ISCO) furnished a colourless oil (16 mg, 0.035 mmol, 22%).

1H- MR (CDC1₃): 1.28 (m, 2H) 1.42 (m, 2H), 1.60 (m, 4H), 2.45-2.80 (m, 10H), 3.05 (bs, 4H), 3.59 (s, 2H), 4.27 (bs, 2H), 4.34 (bs, 2H), 6.58 (d, 1H), 6.60 (d, 1H), 6.79 (t, 1H), 7.30 (d, 2H), 7.38 (d, 2H).

(M-H)⁺ = 446.2

A slightly modified procedure was utilized for synthesis of AG-0447. In this specific case activation of the alcohol was achieved via the mesylate.

Results

Table 5

Example 4

Synthesis of miscellaneous D₂-H₃ a(nta)gonists.

Example 4a: Synthesis of 4-(4-(3-(4-(6-chloro-2,3-dihydrobenzo[b][l,4]d yl)piperazin- 1 -yl)propoxy)benzyl)morpholine (AG-0441 ) .

SCHEME V

Chlorination of 2,3-dihydrobenzo[b][l,4]dioxin-5-amine (see example 3a) with NCS yielded 6-chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-amine. Subsequent piperazine formation with bis(2-chloroethyl)amine hydrochloride and Boc-protection (for purification) afforded tert-butyl 4-(6-chloro-2,3-dihydrobenzo[b][l,4]dioxin-5- yl)piperazine-l-carboxylate. The final target was obtained by deprotection of the Boc- group under acidic conditions and alkylation with 4-(4-(3- bromopropoxy)benzyl)morpholine. 6-Chloro-2,3-dihydrobenzo [b] [1,4] dioxin-5-amine

2,3-Dihydrobenzo[b][l,4]dioxin-5-amine (3.8 g, 25.1 mmol; see example 3a) was dissolved in CHCI₃ (75 mL) and the mixture was cooled in an ice-bath. N- Chlorosuccinimide (3.4 g, 25.1 mmol) was added portion-wise and the mixture was stirred overnight at RT. The purple suspension was filtered over Celite and the filtrate was concentrated. The concentrate was dissolved in DCM and purified over a short plug of Silica. The filtrate was concentrated and the residue was purified by column column chromatography (ISCO, silicagel). ^lJ and ³J-CH correlation HSQC NMR were taken to identify the product formed (1.8 g, 9.7 mmol, 39%). t-Butyl 4-(6-chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine-l-carboxylate 6-Chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-amine (1.35 g, 7.3 mmol) and bis(2- chloroethyl)amine hydrochloride (1.6 g, 8.7 mmol) were heated to reflux in 1.2- dichlorobenzene (180°C) for 5 days. The reaction mixture was concentrated in vacuo and the residue was suspended in THF (50 mL) and IN NaOH (50 mL). Boc₂0 (3.1 g) was added and the mixture was stirred overnight at RT. Subsequently, EtOAc was added and the mixture was washed with H₂0, brine, dried over Na₂S0₄ and concentrated in vacuo. The crude brown oil was purified by automated column chromatography (ISCO, silicagel; 1.9 g, 5.4 mmol, 74%) l-(6-Chloro-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazine hydrochloride

t-Butyl 4-(6-chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine-l-carboxylate (1.9 g, 5.4 mmol) was dissolved in dioxane (20 mL) and 4 N HC1 in dioxane (8 mL) was added. The reaction mixture was stirred overnight at RT. Concentration in vacuo afforded a white solid (1.6 g, 5.4 mmol, quant.). 4-(4-(3-(4-(6-Chloro-2,3-dihydrobenzo [b] [1,4] dioxin-5-yl)piperazin-l- yl)propoxy)benzyl)morpholine (AG-0441)

l-(6-Chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine hydrochloride (330 mg, 1.1 mmol), 4-(4-(3-bromopropoxy)benzyl)morpholine hydrochloride (596 mg, 1.7 mmol), K₂CO₃ (658 mg, 4.8 mmol) and KI (28 mg, 0.17 mmol) were suspended in MeCN (50 mL). The mixture was heated to 80°C and stirred at this temperature overnight. The mixture was concentrated in vacuo. The residue was dissolved in EtOAc, washed with H₂0 (2 x), brine, dried over Na₂S0₄ and concentrated in vacuo. The crude material was purified by column chromatography (ISCO, silicagel) affording a yellow oil (251 mg, 0.51 mmol, 45%).

1H-NMR (CDCI₃): 2.06 (m, 2H), 2.44 (m, 4H), 2.30-2.90 (m, 6H), 2.95-3.50 (m, 4H), 3.45 (s, 2H), 3.72 (m, 4H), 4.05 (t, 2H), 4.24 (m, 4H), 6.60 (d, 1H), 6.87 (m, 3H), 7.28 (d, 2H).

(M-H)⁺ = 488.1 Example 4b: Synthesis of 4-(4-(3-(4-(7-chloro-2,3-dihydrobenzo[b][l,4]dioxin-5- yl)piperazin- 1 -yl)propoxy)benzyl)morpholine (AG-0442)

SCHEME W

A Baeyer Villiger oxidation of l-(5-chloro-2-hydroxy-3-nitrophenyl)ethanone afforded 5-chl oro-3 -nitrobenzene- 1,2-diol. Subsequent reaction with dibromoethane and reduction of the nitro-moiety towards the corresponding amine furnished 7-chloro-2,3- dihydrobenzo[b][l,4]dioxin-5-amine. The compound aimed for was obtained by reaction of the amine with bis(2-chloroethyl)amine.HCl and finally alkylation with 4- (4-(3-bromopropoxy)benzyl)morpholine. 5-Chloro-3-nitrobenzene- 1,2-diol

Concentrated H₂SO₄ (4 mL) was added to a solution of 35% wt. H₂O₂ (16.3 mL, 190 mmol) in dioxane (69 mL) and the mixture was heated at 40°C for 1 h. l-(5-Chloro-2- hydroxy-3-nitrophenyl)ethanone (5.00 g, 23.2 mmol) was added in one portion (endothermic) and the mixture was heated at 40°C for 15 min. H₃BO₃ (11.76 g, 190 mmol) was added (endothermic) and the mixture was heated at 80°C for 8 h. After cooling to RT the mixture was concentrated in vacuo. H₂O (100 mL) was added and the solid was filtered off, washed with H₂0 and dried. The residue was suspended in TBME (100 mL), filtered and the filtrate was dried (Na₂S0₄) and concentrated to yield an orange solid (3.63 g). The material contained residual dioxane and was used in the next step without any further purification.

7-Chloro-5-nitro-2,3-dihydrobenzo [b] [1,4] dioxine A mixture of 5-chloro-3-nitrobenzene-l,2-diol (3.63 g, max. 19 mmol), 1,2- dibromethane (4.11 mL, 48 mmol), K₂C0₃ (10.66 g, 77 mmol) in DMF (33 mL) was heated at 80°C for 5 h. After cooling H₂0 (100 mL) was added. The mixture was extracted with EtOAc (3 x). The combined organic layers were washed with brine, dried (Na₂S0₄) and concentrated to an orange solid. After stripping with toluene and stirring up in TBME the title compound was obtained as an orange-brown solid (2.63 g, 12 mmol, 53% yield over two steps).

7-Chloro-2,3-dihydrobenzo[b] [l,4]dioxin-5-amine

To a mixture of 7-chloro-5-nitro-2,3-dihydrobenzo[b][l,4]dioxine (2.23 g, 10.3 mmol) in EtOAc (25 mL) and AcOH (25 mL) was added Fe powder (3.45 g, 61.8 mmol) at RT. The mixture was stirred overnight. The mixture was filtered over Celite and the pad was washed with EtOAc. The filtrate was made alkaline with aHC03 and then extracted with EtOAc (3 x). The combined organic layers were washed with brine, dried (Na₂S0₄) and concentrated to a black oil. The oil was treated with 10% HC1 in H₂0 and washed with TBME (2 x). The water layer was made alkaline with 1 M NaOH (35 mL) and extracted with EtOAc (3 x). The combined organic layers were washed with brine, dried (Na₂S0₄), filtered over Celite and the filtrate was concentrated to a brown oil (1.00 g, 5.4 mmol, 52%). l-(7-Chloro-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazine

7-Chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-amine (1.00 g, 5.4 mmol) and bis(2- chloroethyl)amine.HCl (1.15 g, 6.5 mmol) were heated in chlorobenzene (10 mL) at reflux for 76 h. After the cooling to RT, the residue was mixed with EtOH and Na₂C0₃ (300 mg) was added. The mixture was heated until almost all had dissolved and re- cooled to RT. After filtration the filtrate was concentrated and dissolved in THF (40 mL). Boc₂0 (1.5 g, 6.87 mmol) was added, followed by 1 M NaOH (30 mL). After 30 min. stirring the two layers were separated and the water layer was extracted with EtOAc. The organic layers were dried (Na₂S0₄) and concentrated to afford a brown solid. Automated column chromatography (ISCO, Silica, 0-20% EtOAc in heptane) provided the Boc-protected amine. It was dissolved in 4 N HC1 in dioxane (10 mL) and stirred at RT overnight. The mixture was concentrated in vacuo to provide a white solid (289 mg, 1.1 mmol, 21%). 4-(4-(3-(4-(7-Chloro-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazin-l- yl)propoxy)benzyl)morpholine (AG-0442)

l-(7-Chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (100 mg. 0.34 mmol), 4- (4-(3-bromopropoxy)benzyl)mo holine (181 mg, 0.52 mmol), K₂CO₃ (214 mg, 1.5 mmol) and KI (8.6 mg, 0.052 mmol) were suspended in MeCN (20 mL), under a N2 atmosphere. The suspension was heated at reflux temperature overnight. After concentration in vacuo, H₂0 (10 mL) was added. The water layer was extracted with EtOAc (1 x 10 mL and 2 x 5 mL). The combined organic layers were dried over NaiSC^ and concentrated in vacuo to provide a yellow oil. The crude product was purified by automated column chromatography (ISCO, eluent 0 to 5% MeOH in DCM) affording a a white solid (70.4 mg, 0.14 mmol, 42%).

¹HNMR (CDCI₃): 2.02 (m, 2H), 2.42 (m, 4H), 2.58 (t, 2H), 2.65 (bs, 4H), 3.08 (bs, 4H), 3.43 (s, 2H), 3.69 (m, 4H), 4.02 (t, 2H), 4.22 (m, 2H), 4.30 (m, 2H), 6.48 (d, 1H) 6.59 (d, 1H), 6.85 (d, 2H), 7.23 (d, 2H).

(M-H)⁺ = 488.1

Example 4c: Synthesis of 8-(4-(3-(4-(morpholinomethyl)phenoxy)propyl)piperazin-l- yl)-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile (AG-0445)

SCHEME X

Demethylation of 5-nitrovanilline with BBrg and alkylation with 1,2-dibromoethane afforded 8-nitro-2,3-dihydrobenzo[b][l,4]dioxine-6-carbaldehyde. The aldehyde functionality was converted into a nitrile group, using formic acid and hydroxylamine and, subsequently, the nitro group was reduced with Fe to the corresponding amine. The piperazine ring was prepared by refluxing 8-amino-2,3- dihydrobenzo[b][l,4]dioxine-6-carbonitrile in 1,2-dichlorobenzene in the presence of bis(2-chloroethyl)amine hydrochloride. For purification purposes the piperazine was protected with a Boc-group that was cleaved afterwards with HC1 in dioxane affording 8-(piperazin-l-yl)-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile.HCl. Final coupling with 4-(4-(3-bromopropoxy)benzyl)morpholine hydrochloride furnished the target aimed for. 3,4-Dihydroxy-5-nitrobenzaldehyde

To a suspension of 5-nitrovanilline (5.00 g, 25 mmol) in DCM (50 mL) at 0°C was added drop-wise pure BBr₃ (25.0 g, 100 mmol). The mixture was allowed to warm up to RT overnight. It was re-cooled in ice and H₂0 (60 mL) was added, initially drop- wise. NH₄CI sat. (60 mL) was added and the whole mixture was filtered over Celite. The pad was washed with EtOAc (100 mL). The filtrate separated into two layers and the pad was washed with EtOAc (2 x 100 mL). The filtrates were used to extract the water layer. The combined organic layers were washed with H₂0 (2 x 20 mL), 15% Na₂S₂0₃ (50 mL), brine, dried (Na₂S0₄) and concentrated to a brown oil/solid (6.2 g, > 100%). The product was used in the next step without any further purification.

8-Nitro-2,3-dihydrobenzo[b][l,4]dioxine-6-carbaldehyde

3,4-Dihydroxy-5-nitrobenzaldehyde (3.2 g, max. 16 mmol), 1 ,2-dibromoethane (2.77 mL, 32 mmol) and Cs₂C0₃ (15.6 g, 48 mmol) were heated in DMF (32 mL) at 80°C overnight. The mixture was concentrated and the residue stirred in CHCI₃. The suspension was filtered over Celite, the pad washed with acetone and the filtrate was concentrated. The residue obtained was stirred up in TBME and the remaining solid was filtered off (0.43 g, 2.1 mmol, 13% yield over two steps). 8-Nitro-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile

A solution of 8-nitro-2,3-dihydrobenzo[b][l,4]dioxine-6-carbaldehyde (530 mg, 2.5 mmol) and hydroxylamine hydrochloride (264 mg, 3.8 mmol) in formic acid (3 mL) was heated to reflux for 2 h. The mixture was concentrated and the brown solid obtained was combined with another batch (max. 0.48 mmol) and purified by automated column chromatography (ISCO) affording a yellow solid (300 mg, 1.46 mmol, 50%).

8-Amino-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile

8-Nitro-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile (300 mg, 1.46 mmol) was suspended in EtOH (6 mL) and AcOH (3 mL). Fe (325 mg, 5.8 mmol) was added and the mixture was stirred overnight at RT. The mixture was concentrated, dissolved in EtOAc and neutralized with sat. NaHC0₃. The mixture was filtered over Celite and the layers were separated. The organic layer was washed with brine, dried over Na₂S0₄ and concentrated in vacuo. 8-Amino-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile (250 mg, 1.42 mmol, 90%) was obtained as a brown solid and used as such in the next reaction. t-Butyl 4-(7-cyano-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine-l-carboxylate

8-Amino-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile (250 mg, 1.42 mmol) and bis(2-chloroethyl)amine hydrochloride (304 mg, 1.70 mmol) were heated to reflux in 1.2-dichlorobenzene (180°C) overnight. The reaction mixture was concentrated in vacuo and the residue obtained was suspended in THF (10 mL) and 1 N NaOH (10 mL). Boc₂0 (600 mg) was added and the mixture was stirred at RT for 20 min. Subsequently, EtOAc was added and the mixture was washed with H₂0, brine, dried over Na₂S0₄ and concentrated in vacuo. The crude brown oil was purified by automated column chromatography (TSCO, silicagel) affording t-butyl 4-(7-cyano-2,3- dihydrobenzo[b][l,4]dioxin-5-yl)piperazine-l-carboxylate (150 mg) that was used in the next step without any further purification.

8-(Piperazin-l-yl)-2,3-dihydrobenzo[b][l,4]dioxine-6-carbonitrile hydrochloride t-Butyl 4-(7-cyano-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine-l-carboxylate (150 mg) was stirred in 4 N HCl in dioxane for 1.5 h. The mixture was concentrated in vacuo (150 mg). The material was used as such in the next step.

8-(4-(3-(4-(Morpholinomethyl)phenoxy)propyl)piperazin-l-yl)-2,3- dihydrobenzo[b] [l,4]dioxine-6-carboiiitrile (AG-0445)

8-(Piperazin- 1 -yl)-2,3 -dihydrobenzo[b] [1 ,4]dioxine-6-carbonitrile hydrochloride (150 mg, 0.53 mmol), 4-(4-(3-bromopropoxy)benzyl)morpholine hydrochloride (280 mg, 0.80 mmol), K2CO3 (309 mg, 2.2 mmol) and KI (13 mg, 0.08 mmol) were suspended in MeCN (25 mL). The mixture was heated to 80°C and stirred overnight. The mixture was concentrated in vacuo and the concentrate was dissolved in EtOAc, washed with H₂0 (2 x), brine, dried over Na₂S0₄ and concentrated in vacuo. The crude material was purified by automated column chromatography (ISCO slicagel) and the compound aimed for was obtained as a yellow oil (130 mg, 0.27 mmol, 19% from 8-amino-2,3- dihydrobenzo[b][l,4]dioxine-6-carbonitrile). 1H-NMR (CDCI₃): 2.03 (m, 2H), 2.41 (bs, 4H), 2.63 (t, 2H), 2.65 (bs, 4H), 3.11 (bs, 4H), 3.46 (s, 2H), 3.72 (m, 4H), 4.05 (t, 2H), 4.28 (m, 2H), 4.40 (m, 2H), 6.78 (s, 1H), 6.88 (m, 3H), 7.26 (d, 2H).

(M-H)⁺ = 479.2

Example 4d: Synthesis of 4-(4-(3-(4-(7-fluoro-2,3-dihydrobenzo[b][l,4]dioxin-5- yl)piperazin- 1 -yl)propoxy)benzyl)morpholine (AG-0446)

SCHEME Y

Nitration of 5-fluoro-2-hydroxyacetophenone afforded l-(5-fluoro-2-hydroxy-3- nitrophenyl)ethanone. A Baeyer Villiger oxidation and alkylation of both hydroxylmoieties with dibromoethane yielded 7-fluoro-5-nitro-2,3- dihydrobenzo[b][l,4]dioxine. Catalytic reduction of the nitro-group and building up the piperazine ring with 2,2'-(benzylazanediyl)diethanol furnished l-benzyl-4-(7- fluoro-2,3-dihydrobenzo[b][l ,4]dioxin-5-yl)piperazine. The final target was obtained by deprotection of the benzylmoiety and alkylation with 4-(4-(3- bromopropoxy)benzyl)morpholine. l-(5-Fluoro-2-hydroxy-3-nitrophenyl)ethanone

5-Fluoro-2-hydroxyacetophenone (10 g, 65 mmol) was dissolved in CC1₄ (40 mL). Nitric acid (65%, 6.8 mL, 97 mmol) was added and the solution was heated at reflux temperature for 50 min and allowed to cool to RT overnight. A suspension was formed and the solids were isolated by filtration. A yellow solid was obtained (12.5 g, 62.8 mmol, 97%).

5-Fluoro-3-nitrobenzene- 1 ,2-diol

H₂0₂ (35%, 45 mL, 521 mmol) was diluted with dioxane (190 mL). H₂S0₄ (10.8 mL) was added carefully, the temperature rose to 40°C and kept 1 h at this temperature. 1- (5-Fluoro-2-hydroxy-3-nitrophenyl)ethanone (12.5 g, 62.8 mmol) was added in one portion. After 15 min boric acid (32 g, 521 mmol) was added. The reaction mixture was heated at 80°C for 8 h and cooled to RT overnight. After concentration in vacuo, FLO (250 mL) was added and the formed solids were removed by filtration. The filter cake was rinsed with TBME. Additional TBME (250 mL) was added to the filtrate and the layers were separated. The water layer was extracted with TBME (2 x 150 mL). The combined organic layers were dried with Na₂S0₄ and concentrated in vacuo to provide an orange solid (8.04 g, 46.5 mmol, 74%, still dioxane present). The product was used as such in the next step.

7-Fluoro-5-nitro-2,3-dihydrobenzo[b] [l,4]dioxine

5-Fluoro-3-nitrobenzene-l,2-diol (8.0 g, 46 mmol) was dissolved in DMF (80 mL). K₂CO₃ (26.2 g, 189 mmol) and 1,2-dibromopropane (10.0 mL, 116 mmol) were added respectively. The mixture was heated at 80°C for 5 h, the color changed from red to brown during heating, and cooled to RT overnight. H₂0 (300 mL) was added and the water layer was extracted with EtOAc (1 x 300 mL and 2 x 200 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. The crude product was purified by automated column chromatography (ISCO, eluent 0 to 100% EtO Ac/heptanes) affording a yellow solid (0.93 g, 4.7 mmol, 10%).

7-Fluoro-2,3-dihydrobenzo[b] [l,4]dioxin-5-amine

7-Fluoro-5-nitro-2,3-dihydrobenzo[b][l,4]dioxine (400 mg, 2.0 mmol) was dissolved in EtOH (50 mL). Pd/C (10%, 40 mg) was added and the mixture was put under 3 bar H₂ pressure overnight. The mixture was filtered over Celite and the filter cake was rinsed with MeOH (100 mL). The filtrate was concentrated in vacuo, yielding a dark green oil (334 mg, 2.0 mmol, 98%). l-Benzyl-4-(7-fluoro-2,3-dihydrobenzo [b] [1,4] dioxin-5-yl)piperazine

Methanesulfonic anhydride (946 mg, 5.4 mmol) was dissolved in MeCN (6 mL), under a N₂ atmosphere. The solution was cooled to -5°C with an ice/water/salt bath. A solution of N-benzyl-bis(2-hydroxyethyl)amine (482 mg, 2.5 mmol) in MeCN (2 mL) was added followed by the addition of Et₃N (1.0 mL, 7.5 mmol), methanesulfonic acid (385 iL, 5.9 mmol) and a solution of 7-fluoro-2,3-dihydrobenzo[b][l,4]dioxin-5-amine (334 mg, 2.0 mmol) in MeCN (4 mL) respectively. The temperature was kept below 2 °C during addition and heated to reflux afterwards. Et₃N (0.82 mL, 5.9 mmol) was added drop-wise. The reaction mixture was heated at reflux temperature overnight and concentrated in vacuo. Sat. aq. NaHC0₃ sol. (50 mL) was added and the water layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. The crude product was purified by column chromatography (ISCO, eluent 0 to 50% EtOAc in heptanes) affording a light yellow oil (400 mg, 1.2 mmol, 61%). l-(7-Fluoro-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazine

l-Benzyl-4-(7-fluoro-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (400 mg, 1.2 mmol) was dissolved in MeOH (10 mL). Pd/C (40 mg) was added and the mixture was put under 1 bar H₂ pressure. The mixture was stirred at RT overnight. The mixture was filtered over Celite, the filter cake was rinsed with MeOH (50 mL). The filtrate was concentrated in vacuo to provide a hazy oil (0.24 g, 1.0 mmol, 83%).

4-(4-(3-(4-(7-Fluoro-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazin-l- yl)propoxy)benzyl)morpholine (AG-0446)

l-(7-Fluoro-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (100 mg, 0.42 mmol), 4- (4-(3-bromopropoxy)benzyl)morpholine.HCl (222 mg, 0.63 mmol), K₂C0₃ (186 mg, 1.3 mmol) and KI (10.5 mg, 0.063 mmol) were suspended in MeCN (20 mL). The mixture was heated at reflux temperature overnight and cooled to RT. The mixture was concentrated and H₂0 (10 mL) was added. The water layer was extracted with EtOAc (4 x 5 mL). The combined organic layers were washed with H₂0 (2 x 2 mL), dried over Na₂S0₄ and concentrated in vacuo. The crude product was purified by automated column chromatography (ISCO, eluent 0 to 10% MeOH in DCM) furnishing a white solid (99 mg, 0.21 mmol, 50%).

XH-NMR (CDC1₃): 2.04 (m, 2H), 2.45 (bs, 4H), 2.68 (m, 6H), 3.12 (bs, 4H), 3.46 (s, 2H), 3.72 (m, 4H), 4.05 (t, 2H), 4.28 (m, 4H), 6.31 (dt, 2H), 6.88 (d, 2H), 7.26 (d, 2H). (M-H)⁺ = 472.2

Example 4e: Synthesis of 4-(4-(3-(4-(8-chloro-2,3-dihydrobenzo[b][l,4]dioxin-5- yl)piperazin- 1 -yl)propoxy)benzyl)morpholine (AG-0443 ) .

SCHEME Z

Chlorination of l-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine with AICI₃, isopropylsulfide and sulfurylchloride afforded l-(8-chloro-2,3- dihydrobenzo[b][l,4]dioxin-5-yl)piperazine. Subsequent alkylation with 4-(4-(3- bromopropoxy)benzyl)morpholine.HCl resulted in the compound aimed for. l-(8-Chloro-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazine

To a stirred solution of l-(2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (1.8 g, 8.2 mmol) in DCM (40 mL) was added a catalytic amount of AICI₃ and isopropylsulfide. The milky solution was cooled in an ice-bath and sulfuryl chloride (1.1 g, 0.66 mL, 8.2 mmol) was added drop-wise. At the end of the addition the solution became clear. The mixture was stirred overnight at RT. The reaction mixture was concentrated in vacuo. To the residue was added DCM and 1 N NaOH. The organic layer was washed with H₂0, dried over Na₂S0₄ and concentrated in vacuo. The crude material was purified by automated column chromatography (ISCO, silicagel) and part of the material (500 mg of 1.6 g) was additionally purified by prep. HPLC affording a white solid (66 mg, 0.26 mmol, 10%).

4-(4-(3-(4-(8-Chloro-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazin-l- yl)propoxy)benzyl)morpholine (AG-0443)

l-(8-Chloro-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (66 mg, 0.26 mmol, 4-(4- (3-bromopropoxy)benzyl)morpholine hydrochloride (136 mg, 0.39 mmol), K₂CO₃ (115 mg, 0.83 mmol) and KI (6 mg, 0.04 mmol) were suspended in MeCN (10 mL). The mixture was heated to 80°C and stirred overnight. The mixture was concentrated in vacuo. The concentrate was dissolved in EtOAc, washed with H₂0 (2 x), brine, dried over Na₂SC>4 and concentrated in vacuo. The crude material was purified by automated column chromatography (ISCO, silicagel). The material obtained (some TBAB residue present) was dissolved in EtOAc, washed with H₂0 (2 x), brine, dried over Na₂S0₄ and concentrated in vacuo affording a yellow oil (78.5 mg, 0.16 mmol, 62%).

1H- MR (CDCI₃): 2.07 (m, 2H), 2.45 (m, 4H), 2.64 (m, 6H), 3.09 (m, 4H), 3.43 (s, 2H), 3.71 (m, 4H), 4.05 (t, 2H), 4.38 (s, 4H), 6.50 (d, 1H), 6.87 (m, 3H), 7.28 (d, 2H), (M-H)⁺ = 488.1

Example 4f: Synthesis of 4-(4-(3-(4-(8-chloro-2,3-dihydrobenzo[b][l,4]dioxin-5- yl)piperazin- 1 -yl)propoxy)benzyl)morpholine (AG-0444)

Protection and nitration of 2,3-dihydrobenzo[b][l,4]dioxin-6-amine afforded N-(7,8- dinitro-2,3-dihydrobenzo[b][l,4]dioxin-6-yl)acetamide. Subsequent hydrolysis of the acetamide group and diazotation with NaN0₂ in H₂0 yielded 5,6-dinitro-2,3- dihydrobenzo[b][l,4]dioxine. Reaction with NaOMe and reduction of the remaining nitro-group resulted in 6-methoxy-2,3-dihydrobenzo[b][l,4]dioxin-5-amine. Under conditions as described previously the piperazine ring was built up and deprotection and alkylation with 4-(4-(3-bromopropoxy)benzyl)morpholine hydrochloride afforded the final target.

N-(2,3-Dihydrobenzo[b][l,4]dioxin-6-yl)acetamide

l,4-Benzodioxan-6-amine (20 g, 0.13 mmol) was dissolved in AcOH (100 mL) and heated at reflux temperature overnight. The solution was concentrated. Heptane (100 mL) was added to the residue and the solids obtained were isolated by filtration, stirred in Et₂0 (100 niL) and filtered again. Subsequent stirring in DCM (-100 niL) afforded a grey solid (11.2 g, 58 mmol, 44%). The mother liquor was concentrated and purified by automated column chromatography (ISCO, eluent 50 to 90% EtOAc in heptanes) yielding a purple solid (6.3 g, 0.033 mmol, 25%; total yield 69%).

N-(7,8-Dinitro-2,3-dihydrobenzo[b] [l,4]dioxin-6-yl)acetamide

HN0₃ (65%, 43 mL) and HN0₃ (90%, 11 mL) were cooled to 10°C with an ice/H₂0 bath. N-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)acetamide (5 g, 25.9 mmol) was added portion-wise. The temperature was kept below 10°C during addition. A red paste was formed. H₂0 (54 mL) was added and the mixture was cooled to RT. The solids were isolated by filtration. The filter cake was rinsed with H₂0. The solids were stirred in acetone (80 mL), isolated by filtration and stripped with toluene (3.1 g, 10.8 mmol, 42%). 7,8-Dinitro-2,3-dihydrobenzo[b] [l,4]dioxin-6-amine

N-(7,8-Dinitro-2,3-dihydrobenzo[b][l,4]dioxin-6-yl)acetamide (3.06 g, 10.8 mmol) was suspended in EtOH (20 mL). HC1 (37%, 1.1 mL) was added and the solution was heated at reflux temperature for 4 h. The mixture was cooled to RT and the solids were isolated by filtration. The filter cake was rinsed with EtOH. According to MR only -25% product was present. The mother liquor and the isolated solids were added together and HC1 (37%, 3 mL) was added. The mixture was heated at reflux temperature for 7 h. The mixture was cooled to RT and the solids were isolated by filtration. The solids were rinsed with EtOH affording an orange solid (1.5 g, 6.4 mmol, 59%).

5,6-Dinitro-2,3-dihydrobenzo[b][l,4]dioxine

7,8-Dinitro-2,3-dihydrobenzo[b][l,4]dioxin-6-amine (1.5 g, 6.2 mmol) was suspended in AcOH (19 mL), under a N₂ atmosphere. The mixture was cooled to ~15°C. A solution ofNaN0₂ (1.3 g, 19 mmol) in sulphuric acid (7.5 mL, exothermic) was added. During addition a small amount of H₂0 was added. (~4 mL). The reaction mixture was poured in EtOH (15 mL). Cu (82 mg, 1.2 mmol) was added and gas formation was visible. The mixture was stirred at RT overnight. The mixture was poured in H₂0 (100 mL). The water layer was extracted with EtOAc (4 x 50 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. The crude product was purified by column chromatography (ISCO, eluent 0 to 60% EtOAc/heptanes) yielding a brown solid (0.82 g, 3.6 mmol, 58%). 6-Methoxy-5-nitro-2,3-dihydrobenzo [b] [1,4] dioxine

5,6-Dinitro-2,3-dihydrobenzo[b][l,4]dioxine (0.81 g, 3.6 mmol) was dissolved in DMF (10 mL), under a N₂ atmosphere. NaOMe (213 mg, 3.9 mmol) was added and the colour changed from red to black. The mixture was heated at ~110°C overnight and cooled to RT. H₂0 (10 mL) was added and the water layer was acidified with 2 N HC1 to pH~5 and extracted with DCM (3 x 20 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. Analytical data showed that the methoxygroup was mainly cleaved to the corresponding phenol and therefore part of the crude material (300 mg, 1.5 mmol), Cs₂C0₃ (744 mg, 2.3 mmol) and MeT (142 pL, 2.3 mmol) were dissolved in DMF (15 mL), under a N₂ atmosphere. The solution was heated at 100°C (external temperature) overnight. The mixture was cooled to RT and concentrated in vacuo. Brine (40 mL) was added and the water layer was extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. The crude product was purified by column chromatography (ISCO, eluent 0 to 40% EtOAc in heptanes) affording a yellow powder (201 mg, 0.95 mmol, 63%).

6-Methoxy-2,3-dihydrobenzo[b] [l,4]dioxin-5-amine

6-Methoxy-5-nitro-2,3-dihydrobenzo[b][l,4]dioxine (201 mg, 0.95 mmol) was dissolved in MeOH (-20 mL). Pd/C (spatula point) was added and the mixture was put under 1 bar H₂-pressure and stirred at RT overnight. The mixture was filtered over Celite and the filter cake was rinsed with MeOH (100 mL). The filtrate was concentrated in vacuo to provide a red solid (157 mg, 0.87 mmol, 91%) l-Benzyl-4-(6-methoxy-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazine

Methane sulfonic anhydride (416 mg, 2.4 mmol) was dissolved in MeCN (6 mL), under a N₂ atmosphere. The solution was cooled to -5°C with an ice/water/salt bath. A solution of N-benzyl-£ s-(2-hydoxyethoxy)amine (212 mg, 1.1 mmol) in MeCN (2 mL) was added followed by the addition of Et₃N (0.46 mL, 3.3 mmol), methanesulfonic acid (169 μί, 2.6 mmol) and a solution of 6-methoxy-2,3-dihydrobenzo[b][l,4]dioxin-5- amine (157 mg, 0.87 mmol) in MeCN (~5 mL). The temperature was kept below 0°C during addition. The mixture was heated to reflux, Et₃N (0.36 mL, 2.6 mmol, 3.0 eq.) was added and the mixture was heated at reflux temperature overnight. The mixture was concentrated and sat. aq. NaHC0₃ sol. (10 mL) was added and the water layer was extracted with EtOAc (1 x 10 mL and 2 x 5 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo to provide a red oil. The crude product was purified by column chromatography (ISCO, eluent 0 to 50% EtOAc in heptanes) affording a colourless oil (185 mg, 0.54 mmol, 62%). l-(6-Methoxy-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazine

l-Benzyl-4-(6-methoxy-2,3-dihydrobenzo[b][l,4]dioxin-5-yl)piperazine (185 mg, 0.54 mmol) was dissolved in MeOH (-10 mL). Pd/C (spatula point) was added and the mixture was put under 1 bar H₂-pressure overnight. The mixture was filtered over Celite and the filter cake was rinsed with MeOH. The filtrate was concentrated furnishing an oil (80 mg, 0.32 mmol, 59%).

4-(4-(3-(4-(6-Methoxy-2,3-dihydrobenzo[b] [l,4]dioxin-5-yl)piperazin-l- yl)propoxy)benzyl)morpholine (AG-0444)

l -(6-Methoxy-2,3-dihydrobenzo[b][l ,4]dioxin-5-yl)piperazine (80 mg, 0.32 mmol), 4- (4-(3-bromopropoxy)benzyl)morpholine hydrochloride (168 mg, 0.48 mmol), K₂C0₃ (141 mg, 1.0 mmol) and a catalytic amount of KI were suspended in MeCN (35 mL). The reaction mixture was heated to 80°C and stirred at this temperature overnight. The reaction mixture was concentrated in vacuo. The residue was extracted with EtOAc and washed with H₂0 (2 x), brine, dried over Na₂S0₄ and concentrated in vacuo. The crude material was purified by column chromatography (silicagel). Subsequent purification by prep-HPLC yielded the compound aimed for (60 mg, 1.12 mmol, 39%).

1H-NMR (CDC1₃): 2.02 (m, 2H), 2.41 (m, 4H), 2.61 (m, 6H), 3.04 (m, 4H), 3.41 (s, 2H), 3.71 (m, 4H), 3.92 (s, 3H), 4.04 (t, 2H), 4.22 (t, 2H), 4.30 (t, 2H), 6.44 (d, 1H), 6.60 (d, 1H), 6.87 (m, 3H), 7.24 (d, 2H).

(M-H)⁺ = 484.2 Example 4g: Synthesis of 2,4-dimethyl-8-(4-(3 -(4-(piperidin- 1 - ylmethyl)phenoxy)propyl)piperazin- 1 -yl)-2H-benzo[b] [ 1 ,4] oxazin-3 (4H)-one (AG- 0198).

The target was prepared by alkylation of 2,4-dimethyl-8-(piperazin-l-yl)-2H- benzo[b][l,4]oxazin-3(4H)-one with l-(4-(3-bromopropoxy)benzyl)piperidine. 2,4-Dimethyl-8-(4-(3-(piperidin-l-ylmethyl)phenoxy)propyl)piperazin-l-yl)-2H- benzo[b][l,4]oxazin -3(4H)-one (AG-0198)

A mixture of 2,4-dimethyl-8-(piperazin-l-yl)-2H-benzo[b][l,4]oxazin-3(4H)-one (66 mg, 0.25 mmol), l-(4-(3-bromopropoxy)benzyl)piperidine (100 mg, 0.33 mmol), K₂C0₃ (66 mg, 0.48 mmol), Nal (9 mg, 0.06 mmol) in MeCN (5 mL) was heated at 80°C for 24 h under N₂ atmosphere. The mixture was concentrated. To the residue was added DCM and the suspension was filtered. The filtrate was concentrated to afford an orange oil. Purification by automated column chromatography (ISCO Alox, 0 - 5% MeOH in DCM and Silica, 0 - 10% MeOH in DCM) afforded a light-yellow sticky oil (61 mg, 0.12 mmol, 50%).

1H-NMR (CDCI₃): 1.40 (m, 2H), 1.60 (m, 7H), 2.0 (t, 2H), 2.39 (bs, 4H), 2.61 (t, 2H), 2.63 (bs, 4H), 3.15 (m, 4H), 3.38 (s, 3H), 3.41 (s, 2H), 4.01 (t, 2H), 4.6, (m, 1H), 6.62 (t, 2H), 6.81 (d, 2H), 6.98 (t, 1H), 7.22 (d, 2H).

(M-H)⁺ = 493.3 Example 4h: Synthesis of 8-(4-(4-((l-cyclobutylpiperidin-4-yl)oxy)benzyl)piperazin-l- yl)-2,4-dimethyl-2H-benzo[b][l,4]oxazin-3(4H)-one (AG-0113).

SCHEME AC

Reductive amination of 2,4-dimethyl-8-(piperazin-l-yl)-2H-benzo[b][l,4]oxazin- 3(4H)-one with 4-((l-cyclobutylpiperidin-4-yl)oxy)benzaldehyde afforded of 8-(4-(4- ((l-cyclobutylpiperidin-4-yl)oxy)benzyl)piperazin-l-yl)-2,4-dimethyl-2H- benzo[b][l,4]oxazin-3(4H)-one.

8-(4-(4-((l-Cyclobutylpiperidin-4-yl)oxy)benzyl)piperazin-l-yl)-2,4-dimethyl-2H- benzo[b][l,4]oxazin-3(4H)-one (AG-0113)

2,4-Dimethyl-8-(piperazin-l-yl)-2H-benzo[b][l,4]oxazin-3(4H)-one (54 mg, 0.21 mmol) and 4-((l-cyclobutylpiperidin-4-yl)oxy)benzaldehyde (75 mg, 0.29 mmol) were suspended in DCE (2 mL). AcOH (1 mL) was added and the mixture was heated until almost all was dissolved. NaBH(OAc)₃ (88 mg, 0.41 mmol) was added and the solution was stirred at RT overnight. NaOH (1 N, 10 mL) was added and the water layer was extracted with DCM (3 x 5 mL). The combined organic layers were dried over NaiSCu and concentrated to provide a brown solid. The product was purified by column chromatography (ISCO, eluent 0 to 20% MeOH in DCM) providing a brown foam (6.3 mg, 0.13 mmol, 6%).

1H- MR (CDC1₃): 1.60 (d, 3H), 1.63-2.40 (m, 12H), 2.6-3.0 (m, 8H), 3.15 (m, 4H), 3.59 (s, 3H), 3.56 (s, 2H), 4.60 (m, 1H), 6.63 (t, 2H), 6.83 (d, 2H), 6.98 (t, 1H), 7.25 (d, 2H).

(M-H)⁺ = 505.30 Example 4i: Synthesis of (S)-6-((4-((l-cyclobutylpiperidin-4- yl)oxy)benzyl)(propyl)amino)-5,6,7,8-tetrahydronaphthalen-l-ol (AG-0115).

SCHEME AD

Reductive amination of (S)-6-(propylamino)-5,6,7,8-tetrahydronaphthalen-l-ol with the previously described aldehyde furnished the target aimed for.

(S)-6-((4-((l-Cyclobutylpiperidin-4-yl)oxy)benzyl)(propyl)amino)-5,6,7,8- tetrahydronaphthalen-l-ol (AG-0113)

(S)-6-(propylamino)-5,6,7,8-tetrahydronaphthalen-l-ol (22 mg, 0 077 mmol), 4-((l- cyclobutylpiperidin-4-yl)oxy)benzaldehyde (20 mg, 0.077 mmol) and Et₃N (0.01 mL, 0.077 mmol) were dissolved in DCE (4 mL). The solution was cooled to 0°C with an ice/water bath. NaBH(OAc)3 (33 mg, 0.154 mmol, 2.0 eq.) was added and the mixture was warmed to RT overnight. NaOH (1 N, 10 mL) was added and the water layer was extracted with DCM (3 x 5 mL). The combined organic layers were concentrated in vacuo. The crude product was purified by prep. HPLC affording a yellow powder (10.5 mg, 0,023 mmol, 30%).

1H-NMR (CDC1₃): 0.88 (d, 3H), 1.40-2.20 (m, 14H), 2.4-3.0 (m, 12H), 3.60 (d, 2H), 4.29 (m, 1H), 6.60 (d, 1H), 6.68 (d, 1H), 6.81 (d, 2H), 6.99 (t, 1H), 7.22 (d, 2H).

(M-H)⁺ = 449.3 Results

Table 6

Table 7

R = D₂ (ICso) D₂ (ECso) H₃ antagonist Agonist (nM) (nM) (nM)

AG-0198 8.3_>E_mK= 107% 15

0 Table 8

Biological methods

In vitro assays

A functional assay (measurement of intracellular calcium by the aequorin calcium assay) was performed for the dopamine receptor D2. For this assay commercially available frozen irradiated cells expressing the human recombinant long isoform of the receptor (i.e. the D2L receptor) were used.

A functional assay (measurement of intracellular cAMP by the LANCE^® Ultra cAMP assay) was performed for the histamine receptor H3. For this assay commercially available frozen irradiated cells expressing the human recombinant receptor were used.

D2L functional assay

The development of an AequoScreen^® calcium assay for the dopamine D2L receptor was guided by an article of Brini et al (J. Biol. Chem. 1995; 270: 9896-9903). The AequoScreen^® assay is a cellular aequorin-based assay in which cells are loaded with the apoaequorin cofactor coelenterazine. Aequorin is a photoprotein originating from the jellyfish Aequorea Victoria. The apo-enzyme (apoaequorin) is a 21 kD protein that needs a hydrophobic prosthetic group, coelenterazine, to be converted to aequorin, the active form of the enzyme. This enzyme possesses three calcium binding sites which control its activity. Upon calcium binding, aequorin oxidizes coelenterazine into coelenteramide with production of C0₂ and emission of light. The consumption of aequorin is proportional to the calcium concentration and the measurement of light (luminescence) emitted upon oxidation of coelenterazine is therefore a reliable tool for measurement of intracellular calcium flux resulting from the activation of the D2L receptor by a compound.

D2L aequorin calcium assay Commercially available frozen irradiated CHO-K1 cells expressing the human recombinant D2L receptor were used. Agonist and antagonist concentration response experiments were performed.

In the agonist assay, the compounds were analyzed in duplicate at 6 concentrations (ranging from 0.01 - 1000 nM). The agonistic response of a compound was expressed as % of the control (i.e. the maximal response of the agonist bromocriptine was defined as 100%). The E_niax values were determined and EC5₀ values were fitted.

In the antagonist assay, the compounds were screened in duplicate at 4 concentrations (ranging from 0.1 - 100 nM) allowing an estimation of their IC50 values. The compounds were incubated for 2 or 15 minutes and bromocriptine was used as reference agonist. The results were expressed as % of the control response (i.e. the response of bromocriptine at its ECgo value was set as 100%) and the IC5₀ values were calculated. Eticlopride was used as reference inhibitor in each experiment (full concentration response curve 0.01 - 10 nM) for the determination of its IC5₀ value. H3 functional assay

The development of an LANCE^® Ultra cAMP assay was guided by the LANCE^® Ultra cAMP Kit enclosure of Perkin Elmer. The LANCE^® Ultra cAMP assay is a homogenous TR-FRET immunoassay designed to measure cAMP produced upon modulation of adenylyl cyclase by GPCRs. The assay is based on the competition between an europium (Eu)-chelate labeled cAMP tracer and sample cAMP for binding sites on cAMP-specific monoclonal antibodies labeled with the XJLighf™ dye. When antibodies are bound to the Eu-labeled cAMP tracer, light pulse at 340 nm excites the Eu-chelate molecule of the tracer. The energy emitted by the Eu-chelate is transferred by FRET to XJLighf™ molecules on the antibodies, which in turn emit light at 665 nm. Residual energy from the Eu-chelate will produce light at 615 nm. In the absence of free cAMP, maximal TR-FRET signal is achieved. Free cAMP produced by stimulated cells competes with the Eu-cAMP tracer for binding to the XJLighf™ antibodies, causing a decrease in TR-FRET signal.

H3 LANCE^® Ultra cAVlP assay

Commercially available frozen irradiated CHO-K1 cells expressing the human recombinant H3 receptor were used Forskolin was used as a reference antagonist (full concentration response curve ranging from 1 - 100000 nM). Histamine was used as a reference agonist (full concentration response curve ranging from 0.01 - 10000 nM).

Compounds were screened in duplicate for their antagonistic properties using 4 concentrations (ranging from 0.01 - 10 nM) allowing an estimation of their IC50 values. The results were expressed as % of the control response (i.e. the response of histamine at its EC90 value was set as 100%) and the IC50 values were calculated.

Claims

Claims

1. A compound according to according to Formula (III):

(in) wherein:

1 2 3 6 7 8 9

R , R , R\ R , R , R° and R' are independently selected from the group consisting of H, Ci - C₁₂ alkyl, C₂ - C₁₂ alkenyl, C₂ - C₁₂ alkynyl, Ci - C₁₂ alkoxy, C₂ - C₁₂ alkenoxy, C₂ - C₁₂ alkynoxy, Ci - C₁₂ alkylthio, C₂ - C₁₂ alkenylthio, C₂ - Ci2 alkynylthio, OH, halogen, N(R⁴)₂, N0₂, CN, S(0)pR⁴, S(0)₂N(R⁴)₂, C(0)R⁴, C(X)OR⁴, C(X)N(R⁴)₂, N(R⁴)-C(0)R⁴, P(0)(R⁴)OH and P(0)(R⁴)N(R⁴)₂;

R⁴ is independently selected from the group consisting of H, Ci - C₁₂ alkyl, Ce - C₂4 aryl and C3 - C24 heteroaryl;

R^4a is H or Ci - C₁₂ alkyl;

T is N or CR^4a: V is CHR¹⁰R^U or NR¹⁰Rⁿ, wherein R¹⁰ and R¹¹ are independently selected from the group consisting of H, Ci - C₁₂ alkyl, Ce - C24 aryl and C3 - C24 heteroaryl, or wherein R¹⁰ and R^{1 1} represent together the group [C(R )₂]_m;

n is 1, 2, 3, 4, 5 or 6;

m is 3, 4, 5 or 6;

p is 0, 1 or 2;

W is O or R^4a;

U is C(R⁵)„; and

R¹² is selected from the group consisting of H, Ci - C₁₂ alkyl and halogen, or wherein two groups R¹² represent =0.

The compound according to Claim 1, wherein R¹, R², R³, R⁶, R⁷, R⁸ and R⁹ are independently selected from the group consisting of H, Ci - Ce alkyl, Ci - Ce alkoxy, Ci - C₆ alkylthio, OH, halogen, N(R⁴)₂, N0₂, CN, S(0)_pR⁴, S(0)₂N(R⁴)₂, C(0)R⁴, C(0)OR⁴, C(0)N(R⁴)₂ and N(R⁴)-C(0)R⁴.

1 2 3 6 7 8 9

The compound according to Claim 2, wherein R , R , R , R , R , R and R are independently selected from the group consisting of H, Ci - Ce alkyl, Ci - Ce alkoxy, OH, halogen, N(R⁴)₂, C(0)R⁴, C(0)OR⁴ and C(0)N(R⁴)₂.

The compound according to any one of Claims 1 - 3, wherein n is 1, 2, 3 or 4. The compound according to any one of Claims 1 - 4, wherein U and R⁹ and the carbon atoms to which they are attached may form a five-membered or a six- membered ring, and said five-membered or six-membered ring may form a spiro compound with another five-membered or six-membered ring formed by V, when R¹⁰ and R¹¹ represent a group [C(R⁵)₂]_m.

The compound according to any one of Claims 1 - 5, wherein R⁴ is independently selected from the group consisting of H and Ci - C₁₂ alkyl.

The compound according to any one of Claims 1 - 5, wherein R^4a is H.

A compound according to any one of Claims 1 - 7 for use as a medicament.

A compound according to any one of Claims 1 - 7 for use in the prophylaxis or treatment of a CNS-related disorder.

A compound according to Claim 9, wherein the CNS-disorder is selected from the group consisting of depression, major depressive disorder, generalised anxiety disorder, major anxiety disorder and panic disorder.

11. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound according to any one of Claims 1 - 7 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to Claim 1 1 for preventing and treating CNS-disorders.

13. The pharmaceutical composition according to Claim 12, wherein the CNS- disorder is selected from the group consisting of depression, major depressive disorder, generalised anxiety disorder, major anxiety disorder and panic disorder.