N,N'-disubstituted benzimidazolone derivatives with affinity at the serotonin and dopamine receptors

[0001] The present invention relates to novel pharmacologically active N,N-disubstituted benzimidazolone derivatives and their addition salts which bind the serotonin or dopamine receptors, to their preparation and their use for therapeutic purposes. These compounds are able to discriminate the different serotonin and dopamine receptor subtypes like 5-HT1A, 5-HT2A, and D4 at which they can act as agonists or antagonists. Owing to this pharmacological activity, the present compounds are useful in the treatment of anxiety disorders, affective disorders such as depression, psychosis and schizophrenia, eating disorders, sexual disorders, Parkinson, stroke and traumatic brain injury.

BACKGROUND OF THE INVENTION

[0002] Serotonin (5-HT) and dopamine (DA) recognize several well defined cell surface receptor subtypes. Among these, 5-HT1A and 5-HT2A having a high and a low affinity for 5-HT, respectively, and D4 at which DA has high affinity, have been implicated in many Central Nervous System (CNS) disorders.

[0003] In the previous art, several classes of compounds able to interfere with the neurotransmission at 5-HT or DA receptor subtypes are known. Particularly, derivatives based on the core structure of the aryl piperazine and benzimidazolone have been described (e.g., GB 2023594, U.S. Pat. No. 3,472,854, U.S. Pat. No. 4,954,503, WO-9616949, WO-9501965, and WO-9833784), and targeted both to generic 5-HT or DA receptors and to a specific receptor subtype. In another patent (U.S. Pat. No. 5,576,318) are described compounds based both on the benzimidazolone and phenylpiperazine structures: in this latter case the described affinities are limited to 5-HT1A and 5-HT2A receptor subtypes.

DETAILED DESCRIPTION OF THE INVENTION

[0004] Now we describe, and this is the object of the present invention, new derivatives of a benzimidazolone core structure. The N-substituents are alkyl chains bearing additional hydrophilic functional groups whereas the N′-substituents are alkyl or alkenyl spacers connecting the benzimidazolone scaffold to a large set of secondary amines bearing other diversity points. The compounds included in this invention possess an interesting affinity profile at the said serotonin and dopamine receptor subtypes: indeed some of them have a high and preferential affinity at a given site (e.g., 5-HT1A, 5-HT2A, or D4) whereas some others have a mixed affinity at the said receptors. Moreover, a selected pool of compounds possesses an agonistic activity at the 5-HT1A receptor coupled with an antagonistic activity at the 5-HT2A receptor. Owing to their peculiar profile, the present compounds may play a role in the regulation of neurotransmission at the serotonin and/or the dopamine sites and thus may be of value in the treatment of those diseases where an altered functioning of neurosignal transmission is present. Examples of these disorders include anxiety, depression, schizophrenia, Parkinson, sleep, sexual and eating disorders, stroke and brain injury. Particularly the compounds included in the present invention can be of value in the treatment of depression according to the mounting evidence that 5-HT1A full agonists or high efficiency partial agonists are required for a robust antidepressant effect. In fact, electrophysiology studies suggest that repeated administration of a variety of antidepressant treatments facilitate 5-HT1A neurotransmission in the hippocampus, possibly through either an increased sensitivity of post-synaptic 5-HT1A receptors or a decreased sensitivity of 5-HT1A autoreceptors. Furthermore, there is some evidence from controlled clinical trials to support this suggestion. In addition the compound's ability to block the 5-HT2A receptor is also of value: indeed, the stimulation of 5-HT1A and 5-HT2A receptors lead to opposite electrical events, inhibitory and excitatory, respectively. Thus only a concurrent activation of 5-HT1A coupled with antagonism at 5-HT2A receptors may completely and rapidly inhibit 5-HT post-synaptic cells, an important physiological event for antidepressant effects.

[0005] The present invention pertains to compounds of general formula (I)

[0006] wherein:

[0007] R1 denotes C1-C6-alkyl, preferably C1-C4-alkyl, being substituted by a group selected from OH, C1-C6-alkoxy, —OCONHC1-C6-alkyl, —OCONHC1-C6-alkyl, —NHSO2C1-C6-alkyl, and —NHCOC1-C6-alkyl, or

[0008] R1 denotes C1-C6-alkyl, preferably C1-C4-alkyl, being substituted by a saturated or unsaturated 5- or 6-membered heterocycle containing one or two heteroatoms selected from the group consisting of nitrogen and oxygen, said heterocycle being optionally substituted by a group selected from C1-C4-alkyl, halogen, and benzyl;

[0009] R2 and R3 together with the nitrogen form a saturated or unsaturated 5- or 6-membered heterocyclic ring which may contain nitrogen or oxygen as an additional heteroatom, whilst the heterocyclic ring is substituted by a group selected from phenyl, benzyl, and diphenylmethyl, said group being optionally mono- or di-substituted by one or two groups selected from CF3, C1-C4-alkyl, C1-C4-alkoxy, phenyl, benzyl, halogen, and OH, or

[0010] R2 and R3 together with the nitrogen form a saturated or unsaturated 5- or 6-membered heterocyclic ring which may contain nitrogen or oxygen as an additional heteroatom, said heterocyclic ring being linked via a single bond, a methylene-bridge or spiro-connected to another saturated or unsaturated heterocyclic group containing one or two heteroatoms selected from oxygen and nitrogen, said heterocyclic group being optionally mono- or di-substituted by a group selected from CF3, C1-C4-alkyl, C1-C4-alkoxy, phenyl, benzyl, halogen, ═O, and OH, or

[0011] R2 and R3 together with the nitrogen form a saturated or unsaturated bi- or tricyclic heterocyclic ring-system which may contain nitrogen or oxygen as an additional heteroatom, said heterocyclic ring-system being optionally substituted by a group selected from CF3, C1-C4-alkyl, C1-C4-alkoxy, phenyl, benzyl, halogen, ═O, and OH;

[0012] A denotes C1-C6-alkylene, preferably C1-C4-alkylene, C2-C6-alkenylene, preferably C2-C4-alkenylene, or C2-C6-alkynylene, preferably C2-C4-alkynylene,

[0013] or a pharmaceutically acceptable salt thereof.

[0014] Preferred compounds are those of formula (I), wherein:

[0015] R1 denotes C1-C4-alkyl, preferably C2-C3-alkyl, being substituted by a group selected from OH, C1-C4-alkoxy, —OCONHC1-C4-alkyl, —OCONHC1-C4-alkyl, —NHSO2C1-C4-alkyl, and —NHCOC1-C4-alkyl, or

[0016] R1 denotes C1-C4-alkyl, preferably C2-C3-alkyl, being substituted by a saturated or unsaturated 6-membered heterocycle containing one or two heteroatoms selected from the group consisting of nitrogen and oxygen; p R2 and R3 together with the nitrogen form a saturated or unsaturated 5- or 6-membered heterocyclic ring which may contain nitrogen as an additional heteroatom, whilst the heterocyclic ring is substituted by a group selected from phenyl, benzyl, diphenylmethyl, pyridinyl, pyrimidinyl, benzimidazolonyl, and 3,4-methylenedioxibenzyl, said group being optionally mono- or di-substituted by a group selected from CF3, C1-C4-alkyl, C1-C4-alkoxy, halogen, and OH;

[0017] A denotes C1-C4-alkylene or C2-C4-alkenylene,

[0018] or a pharmaceutically acceptable salt thereof.

[0019] Also preferred compounds are those of formula (I), wherein:

[0020] R1 denotes ethyl, being substituted by a group selected from OH, OCH3, OCH2CH3, —OCONHCH3, —OCONHCH2CH3, —NHSO2CH3, —NHSO2CH2CH3, —NHCOCH3, —NHCOCH2CH3, morpholinyl, piperazinyl, and piperidinyl

[0021] R2 and R3 together with the nitrogen form a 6-membered saturated or unsaturated heterocyclic ring which may contain nitrogen as an additional heteroatom, whilst the heterocyclic ring is substituted by a group selected from phenyl, pyridinyl, pyrimidinyl, benzimidizalonyl, and substituted phenyl being mono- or di-substituted by a group selected from CF3, CH3, OCH3, F, and Cl;

[0022] A denotes C1-C4-alkylene or C2-C4-alkenylene,

[0023] or a pharmaceutically acceptable salt thereof.

[0024] Also of interest are compounds of formula (I), wherein:

[0025] R1 denotes ethyl, being substituted by a group selected from OH, OCH3, —OCONHCH2CH3, —NHSO2CH3, —NHCOCH3, morpholinyl, and piperidinyl;

[0026] R2 and R3 together with the nitrogen form a 6-membered saturated or unsaturated heterocyclic ring which may contain nitrogen as an additional heteroatom, whilst the heterocyclic ring is substituted by a group selected from pyridinyl, phenyl, and substituted phenyl being mono- or di-substituted by a group selected from CF3, CH3, OCH3, F, and Cl;

[0027] A denotes ethylene, propylene, butylene, or butenylene,

[0028] or a pharmaceutically acceptable salt thereof.

[0029] Of particular interest are compounds of formula (I), wherein:

[0030] R1 denotes ethyl, being substituted by a group selected from OH, OCH3, —OCONHCH2CH3, —NHSO2CH3, —NHCOCH3, morpholinyl, and piperidinyl;

[0031] R2 and R3 together with the nitrogen form a ring selected from the group consisting of piperazine, piperidine, and tetrahydropyridine, which is substituted by a group selected from pyridinyl, phenyl, and substituted phenyl being mono- or di-substituted by a group selected from CF3, CH3, and Cl;

[0032] A denotes ethylene, butylene, or butenylene,

[0033] or a pharmaceutically acceptable salt thereof.

[0034] Furthermore preferred are compounds of formula (I), wherein:

[0035] R1 denotes ethyl, being substituted by a group selected from OH, OCH3, —OCONHCH2CH3, and —NHSO2CH3;

[0036] R2 and R3 together with the nitrogen form a piperazine ring, being substituted by a group selected from trifluoromethylphenyl, methylphenyl, dimethylphenyl, and chlorophenyl; and

[0037] A denotes ethylene, butylene, or butenylene,

[0038] or a pharmaceutically acceptable salt thereof.

[0039] The most preferred compounds according to the invention are:

[0040] (a) 1-(2-methoxyethyl)-3-(4-{4-[3-(trifluoromethyl)phenyl]-1-piperazinyl}butyl)-1,3-dihydro-2H-benzimidazol-2-one;

[0041] (b) 1-{4-[4-(2,3-dimethylphenyl)-1-piperazinyl]butyl}-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one;

[0042] (c) 2-[2-oxo-3-(4-{4-[3-(trifluoromethyl)phenyl]-1-piperazinyl}butyl)-2,3-dihydro-1H-benzimidazol-1-yl]ethyl-ethylcarbamate;

[0043] (d) 1-(2-methoxyethyl)-3-(2-{4-[3-(trifluoromethyl)phenyl]-1-piperazinyl}ethyl)-1,3-dihydro-2H-benzimidazol-2-one;

[0044] (e) 1-{2-[4-(2,3-dimethylphenyl)-1-piperazinyl]ethyl}-3-(2-methoxyethyl)-1,3-dihydro-2H-benzimidazol-2-one;

[0045] (f) 1-{2-[4-(3-chlorophenyl)-1-piperazinyl]ethyl}-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one;

[0046] (g) 2-[2-oxo-3-(2-{4-[3-(trifluoromethyl)phenyl]-1-piperazinyl}ethyl)-2,3-dihydro-1H-benzimidazol-1-yl]ethyl-ethylcarbamate;

[0047] (h) 2-(3-{2-[4-(2,3-dimethylphenyl)-1-piperazinyl]ethyl}-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)ethyl-ethylcarbamate;

[0048] (i) N-[2-(3-{2-[4-(2,3-dimethylphenyl)-1-piperazinyl]ethyl}-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)ethyl]methanesulfonamide;

[0049] (j) N-[2-(3-{(2Z)-4-[4-(3-methylphenyl)-1-piperazinyl]-2-butenyl}-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)ethyl]methanesulfonamide; and

[0050] (k) N-[2-(3-{(2E)-4-[4-(3-chlorophenyl)-1-piperazinyl]-2-butenyl}-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)ethyl]methanesulfonamide.

[0051] If required, the compounds of general formula (I) may be converted into the salts thereof, particularly, for pharmaceutical use, into the pharmaceutically acceptable salts thereof with an inorganic or organic acid. Suitable acids for this purpose include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, or maleic acid. Moreover, mixtures of these acids may be used.

[0052] The alkyl groups meant here (including those which are components of other groups) are branched and unbranched alkyl groups having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, such as: methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl.

[0053] The alkylene groups meant here are branched and unbranched alkyl-bridges having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, such as: methylene, ethylene, n-propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, pentylene, isopentylene, and hexylene.

[0054] Alkenyl groups (including those which are components of other groups) are the branched and unbranched alkenyl groups with 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, provided that they have at least one double bond, e.g., the alkyl groups mentioned above provided that they have at least one double bond, such as for example vinyl (provided that no unstable enamines or enolethers are formed), propenyl, isopropenyl, butenyl, pentenyl, and hexenyl.

[0055] Alkenylene groups are the branched and unbranched alkenyl-bridges with 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, provided that they have at least one double bond, e.g., the alkylene groups mentioned above provided that they have at least one double bond, such as for example vinylene (provided that no unstable enamines or enolethers are formed), propenylene, isopropenylene, butenylene, pentenylene, and hexenylene.

[0056] If not otherwise specified the alkenyl- and alkenylene-groups mentioned above are to be understood as embracing optionally existing stereoisomers. Accordingly, for instance the definition 2-butenyl is to be understood as embracing 2-(Z)-butenyl and 2-(E)-butenyl, etc.

[0057] The term alkynyl groups (including those which are components of other groups) refers to alkynyl groups having 2 to 6, preferably 2 to 4 carbon atoms, provided that they have at least one triple bond, e.g., ethynyl, propargyl, butynyl, pentynyl, and hexynyl.

[0058] Examples of N-linked 5- or 6-membered heterocyclic rings of general formula NR2R3 are as follows: pyrrole, pyrroline, pyrrolidine, piperidine, piperazine, morpholine, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, and pyrazolidine, preferably morpholine, piperazine, and piperidine.

[0059] Examples of saturated or unsaturated bi- or tricyclic heterocyclic ring-system of formula NR2R3 which may contain nitrogen or oxygen as an additional heteroatom, are as follows: indole, tetrahydroindole, benzimidazole, benzoxazole, 1,2-dihydrochinoline, 1,2-dihydroisochinoline, β-carboline, 9H-1,2,3,4-tetrahydropyridoindole, and 9,10-dihydroacridine.

[0060] Halogen means fluorine, chlorine, bromine, or iodine, preferably chlorine or bromine.

[0061] “═O” means an oxygen atom linked by a double bond.

[0062] The compounds of general formula (I) may be conveniently prepared by a variety of synthetic processes analogous to those known in the art using conventional methods. For example, these compounds may be prepared by alkylating the suitable secondary amine (III) with the proper benzimidazolone (II) bearing in the alkyl or alkenyl side chain suitable leaving group X such as halogen, methanesulfonate, or 4-methylbenzenesulfonate (Scheme 1).

[0063] Scheme 1

[0064] The reaction conditions for the conventional synthesis of compounds of formula (I) according to Scheme 1 are disclosed in EP 526 434 A1. Said reference additionally describes the possible synthetic pathways for the preparation of starting compounds (II).

[0065] According to a second option, the reaction sequence according to Scheme 1 can not only be conducted via the conventional synthetic methods outlined in EP 526 434 A1 but, in the alternative, via combinatorial chemistry. For this approach a set of N-alkyl-N′-halo alkyl/alkenyl benzimidazolones of formula (II) (hereinafter identified as Building Blocks or BB; see hereto Table 1) was prepared via the traditional methods described in EP 526 434 A1 and then combinatorially reacted with the suitable secondary amines of formula (III) (Table 2). The process was carried out in a special apparatus consisting of a lower vial (reacting chamber) and an upper vial (condenser). Each compound was reacted with each amine in DMF under stirring at a temperature between 40° C. and 100° C., preferably at 60° C., for 6 to 8 hours in the presence of Na2CO3. The excess amine was then scavenged at room temperature by introducing a polystyrene isocyanatemethyl resin of formula (IV) able to catch the excess amine as an urea of formula (V) immobilized on the solid support (Scheme 2).

[0066] Scheme 2

[0067] The upper part of the reaction apparatus is substituted with another vial containing a frit inside and a connection to the vacuum. Filtration after turning over the apparatus and evaporation to dryness afforded the desired compounds of formula (I) in excellent yield and good purity. The parallel application of the aforementioned process to all of the compounds of formula (II) as shown in Table 1 and all of the selected amines (III) as shown in Table 2 allows the efficient synthesis of all of the compounds (I) according to the present invention.

1TABLE 1Building Blocks (BB) of Formula (II) Subjected to the Process of Scheme 2(II)

BuildingBuildingBlock No.StructureBlock No.StructureBB01

BB02

BB03

BB04

BB05

BB06

BB07

BB08

BB09

BB10

BB11

BB12

BB13

BB14

BB15

BB16

BB17

BB18

BB19

BB20

BB21

BB22

BB23

BB24

BB25

BB26

BB27

BB28

[0068]

2

TABLE 2

Amines (AM) of Formula (III) Subjected to the Process of Scheme 2

(III)

Amine No.
Structure
Amine No.
Structure

AM01

AM02

AM03

AM04

AM05

AM06

AM07

AM08

AM09

AM10

AM11

AM12

AM13

AM14

AM15

AM16

AM17

AM18

AM19

AM20

AM21

AM22

[0069] For pharmaceutical use, the compounds of general formula (I) may be used as such or in the form of physiologically acceptable acid addition salts. The term “physiologically acceptable acid addition salts” includes the salts resulting from both organic and inorganic acids such as maleic, citric tartaric, methanesulfonic, acetic, benzoic, succinic, gluconic, isethionic, glycinic, lactic, malic, mucoic, glutammic, sulfamic, and ascorbic acids; inorganic acids include hydrochloric, hydrobromic, nitric, sulfuric, or phosphoric acid.

[0070] According to a further feature of the present invention, there are provided pharmaceutical compositions comprising as an active ingredient at least one compound of formula (I), as before defined, or a physiologically acceptable addition salt thereof in addition to one or more pharmaceutical carrier, diluents or excipients. For pharmaceutical administration, the compounds of general formula (I) and their physiologically acceptable acid addition salts may be incorporated into the conventional pharmaceutical preparation in solid, liquid, or spray form. The composition may, for example, be presented in a form suitable for oral, rectal, or parenteral administration or for nasal inhalation: preferred forms include, for example, capsules, tablets, coated tables, ampoules, suppositories, and nasal spray. The active ingredient may be incorporated in excipients or carriers conventionally used in pharmaceutical compositions such as, for example, talc, arabic gum, lactose, gelatine, magnesium stearate, corn starch, aqueous or nonaqueous vehicles, polyvinyl pyrrolidone, semisynthetic glycerides of fatty acids, benzalcon chloride, sodium phosphate, EDTA, or polysorbate 80.

[0071] In case it is desired to further increase the solubility of the compounds of general formula (I) or of their physiologically acceptable salts, surfactants or nonionic surfactants such as PEG 400, cyclodextrin, metastable polymorphs, or inert adsorbents such as bentonite, may be incorporated. Furthermore, some techniques may be employed by preparing, for example, eutectic mixtures and/or solid dispersion by using mannitol, sorbitol, saccharose, or succinic acid or physically-modified forms by using hydrosoluble polymers, PVP, or PEG 4000-20,000. The compositions are advantageously formulated in dosage units, each dosage unit being adapted to supply a single dose of the active ingredient. Each dosage unit may conveniently contain from 0.01 mg to 100 mg, preferably from 0.1 to 50 mg.

[0072] However, it could be necessary to depart from the cited amounts, depending on the body weight or on the administration route, on the individual response to the medicament, on the type of formulation and on the time, or time range, in which the administration is carried out. Therefore, it can be sufficient, in some cases, to use a lower amount then the cited minimum amount, whereas in other cases the higher range could be exceeded. When administering higher amounts, it would be advisable to subdivide them in repeated administrations during the day. Moreover, the compounds of general formula (I) or the acid addition salts thereof can also be combined with other, different active substances.

[0073] The following examples illustrate the present invention, without limiting the scope thereof.

[0074] Examples of Pharmaceutical Formulations

3A. Tablets Containing 100 mg of Active SubstanceComponentAmount per tablet (mg)active substance100lactose140maize starch240polyvinylpyrrolidone 15magnesium stearate 5TOTAL500

[0075] The finely ground active substance, lactose, and part of maize starch are mixed. The mixture is sieved, wetted with a solution of polyvinylpyrrolidone in water, kneaded, finely granulated, and dried. The granulate, the remaining maize starch, and magnesium stearate are sieved and mixed together. The mixture is compressed to tablets of suitable form and size.

4B. Tablets Containing 80 mg of Active SubstanceComponentAmount per tablet (mg)active substance80lactose55maize starch190 polyvinylpyrrolidone15sodium carboxymethyl starch23magnesium stearate 2TOTAL400

[0076] The finely ground active substance, part of the maize starch, lactose, microcrystalline cellulose and polyvinylpyrrolidone are mixed. The mixture is sieved and worked up with the remaining maize starch and water, to obtain a granulate, which is dried and sieved. This is added to sodium carboxymethyl starch and magnesium stearate and mixed, and the mixture is then compressed to tablets of suitable size.

5C. Solutions for VialsComponentAmountactive substance50 mgsodium chloride50 mgwater for injection 5 ml

[0077] The active substance is dissolved in water, optionally at pH of 5.5 to 6.5, and treated with sodium chloride as an osmolality agent. The resulting solution is filtered apyrogenically, and the filtrate is placed in vials under aseptic conditions, then the vials are sterilized and flame sealed. The vials contain 5 mg, 25 mg, and 50 mg of active substance.

[0078] Experimental

[0079] The following examples illustrate the preparation of all the new compounds included in the present invention. It should be understood that the invention is not limited to the given examples of chemical methods and processes for the preparation of the substances, as other conventional methods well known to those skilled in the art, are suitable too. In the following descriptions, each of the 28 Building Blocks prepared is identified by its relevant Tag.

[0080] A. Preparation of the Building Blocks (BB) of Formula (II)

[0081] Description 1

[0082] 1-[2-(1-piperidinyl)ethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0083] A solution of 1-isopropenyl-1,3-dihydro-2H-benzimidazol-2-one (35 g, 0.2 moles) in DMF (250 ml) was added dropwise to a suspension of 80% sodium hydride (6 g, 0.2 moles) in DMF (50 ml). The reaction mixture was first heated at 45° C. for 30 minutes, allowed to cool at room temperature, and an additional amount of 80% sodium hydride (7.2 g, 0.24 moles) was added. Then 1-(2-chloroethyl)piperidine hydrochloride (44.16 g, 0.24 moles) was added portionwise and the reaction mixture was heated at 80° C.-90° C. for 3 hours. The mixture was then cooled at room temperature, adjusted to pH 3 with 37% aqueous HCl, and heated to 80° C. for additional 2 hours. The reaction mixture was poured into water and washed with ethyl acetate. The aqueous phase was adjusted to pH 8-9 with a saturated sodium carbonate solution and extracted into ethyl acetate. The organic layer was taken to dryness to give an ivory solid which after crystallization from isopropyl ether afforded 22.9 g of the title compound. M.p. 123° C.-125° C.

[0084] According to the above described procedure, the following compound was prepared from the suitable intermediates:

[0085] 1-[2-(4-morpholinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0086] 11.7 g; m.p. 122° C.-126° C.

[0087] Description 2

[0088] 1-(2-methoxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0089] Phenyl-2-oxo-2,3-dihydro-1H-benzimidazole-1-carboxylate (80 g, 0.315 moles) was added to a suspension of 80% sodium hydride (11.3 g, 0.378 moles) in DMF (500 ml) and heated at 35° C. for 1 hour. To the cooled solution, 2-chloroethylmethylether (43 ml, 0.472 moles) was added and the reaction mixture was heated at 100° C. for 4 hours. The reaction mixture was poured into water and extracted with ethyl acetate. The combined extracts were taken to dryness to give 52 g of the protected intermediate. This was suspended in methanol (500 ml), a solution of K2CO3 (44 g) in water (230 ml) was added, and the mixture and stirred for 2 hours at room temperature. After evaporation, the reaction mixture was acidified and extracted into ethyl acetate. The organic layer was taken to dryness and from the crude oily residue, after crystallization with isopropyl ether, 21 g of the title compound was obtained as a white solid. M.p. 88° C.

[0090] Description 3

[0091] 1-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0092] (a) Phenyl-2-oxo-2,3-dihydro-1H-benzimidazole-1-carboxylate (50 g, 0.197 moles) was added to a suspension of 80% sodium hydride (7 g, 0.236 moles) in DMF (400 ml) and stirred for 1 hour at room temperature, then 2-(2-chloroethoxy)tetrahydro-2H-pyran (34.8 ml, 0.236 moles) was added and the reaction mixture was heated at 100° C. for 7 hours. The reaction mixture was then poured into water and extracted into ethyl acetate. The organic layer was taken to dryness to give an oily residue.

[0093] (b) This residue (83 g) was dissolved in methanol, a solution of KOH (26 g in 260 ml water) was added and stirred for 2 hours at room temperature. The methanol was evaporated and the residue was extracted into ethyl acetate. The organic layer was washed with an aqueous 5% HCl solution, dried, and taken to dryness. The oily residue was crystallized from diisopropyl ether to give 26 g of the title compound. M.p. 115° C.

[0094] Description 4

[0095] 1-(2-aminoethyl)-1,3-dihydro-2H-benzimidazol-2-one hydrochloride

[0096] (a) A suspension of phenyl-2-oxo-2,3-dihydro-1H-benzimidazole-1-carboxylate (10 g, 39 mmoles) and 80% sodium hydride (1.3 g, 43 mmoles) in DMF (100 ml) was stirred at room temperature for 30 minutes. N-(2-bromoethyl)phthalimide (10 g, 39 mmoles) was added and the mixture heated at 100° C. for 12 hours. The reaction mixture was then poured into 600 ml of water and stirred for 4 hours at room temperature. The precipitated phthaloyl derivative was filtered off (white solid; 6.5 g).

[0097] (b) This intermediate was suspended in methanol (70 ml), a 10% aqueous K2CO3 solution was added and the reaction mixture stirred overnight at room temperature. The methanol was evaporated, the residue was extracted with dichloromethane, and the aqueous solution was acidified with 10% aqueous HCl to give the corresponding 2-carboxybenzamido derivative which was filtered off (5 g).

[0098] (c) To the crude intermediate was added 32 ml of a 15% aqueous HCl solution and the resulting suspension was heated at 90° C. for 3 hours. After cooling, the solid was filtered off and the acidic solution was taken to dryness to give 1.5 g of the hydrochloride of the title compound as a pinkish solid. M.p. >280° C.

[0099] Description 5

[0100] N-[2-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)ethyl]-acetamide

[0101] To a cooled solution of NaOH (0.41 g, 10 mmoles) in water (5 ml) were simultaneously added 1-(2-aminoethyl)-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (0.7 g, 3.3 mmoles) and a solution of acetic anhydride (0.37 ml, 3.9 mmoles) in dioxane (10 ml). The reaction mixture was stirred for 2 hours at room temperature and then taken to dryness. The residue was dissolved in water, adjusted to pH 4 with 10% aqueous HCl, and extracted with CHCl3. The organic layer was taken to dryness and from the crude residue 0.35 g of the title compound was obtained after crystallization from diethyl ether. M.p. 153° C.

[0102] Description 6

[0103] N-[2-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)ethyl]methanesulfonamide

[0104] To a solution of 1-(2-aminoethyl)-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (2.8 g, 13 mmoles) in THF (30 ml) and triethyl amine (5.5 ml, 39 mmoles) was added methanesulfonyl chloride (1.12 ml, 14 mmoles) and the reaction mixture was stirred for 2 hours at room temperature. The organic solvent was evaporated and the residue was partitioned into water and ethyl acetate. The organic layer was washed with saturated aqueous Na2CO3 solution and taken to dryness. The crude residue was purified by flash chromatography (CH2Cl2-methanol 96-4) to give 0.5 g of the title compound as a white solid. M.p. 162-170° C.

[0105] Description 7

[0106] 1-(2-chloroethyl)-3-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0107] Into a stirred solution of 1-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one (6.5 g, 25 mmoles) in DMF (40 ml) 80% sodium hydride (0.9 g, 30 mmoles) was added. After 30 minutes of stirring and heating to 35° C., 1-bromo-2-chloroethane (6.2 g, 48 mmoles) was added, the reaction temperature was increased to 90° C. and kept for 6 hours then cooled at room temperature. The reaction mixture was poured into water, extracted with diethyl ether, and the organic layer was taken to dryness. The residue was purified by flash chromatography (cyclohexane-ethyl acetate 50-50) to give 2.8 g of the title compound as a thick oil which was used without any further purification.

[0108] According to the above described procedure, the following compounds were prepared from the suitable intermediates:

[0109] 1-(4-chlorobutyl)-3-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0110] The compound was purified by flash chromatography (cyclohexane-ethyl acetate 50-50). Thick oil.

[0111] [BB01]: 1-(2-chloroethyl)-3-(2-methoxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0112] White solid. M.p. 55° C., from diisopropyl ether.

[0113] [BB01]: 1-(4-chlorobutyl)-3-(2-methoxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0114] The compound was purified by flash chromatography (cyclohexane-ethyl acetate 50-50). Thick oil.

[0115] [BB15]: 1-[(2Z)-4-chloro-2butenyl)]-3-(2-methoxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0116] The compound was purified by flash chromatography (cyclohexane-ethyl acetate 70-30). Thick oil.

[0117] [BB24]: N-{2-[3-(2-chloroethyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl}-acetamide

[0118] White solid. M.p. 140° C.-142° C., from acetone.

[0119] [BB06]: N-{2-[3-(4-chlorobutyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl}-acetamide

[0120] Ivory solid. M.p. 100° C., from diethyl ether.

[0121] Description 8

[0122] 1-[(2Z)-4-chloro-2-butenyl]-3-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0123] To a solution of 1-[2-(tetrahydro-2H-pyran-2yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one (12 g, 46 mmoles) in DMF (120 ml) was added 80% sodium hydride (1.7 g, 55 mmoles) and the mixture was stirred at room temperature for 1 hour. Cis-1,4-dichloro-2-butene (5.8 ml, 55 mmoles) was added dropwise and the reaction mixture was stirred for 3 hours at room temperature. The reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was taken to dryness and the residue was purified by flash chromatography (cyclohexane-ethyl acetate 70-30) to give 2.8 g of the title compound as a thick oil which was used without any further purification.

[0124] According to the above described procedure, the following compounds were prepared from the suitable intermediates:

[0125] 1-[(2E)-4-chloro-2-butenyl)]-3-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0126] The compound was purified by flash chromatography (cyclohexane-ethyl acetate 70-30). Waxy solid.

[0127] [BB16]: 1-[(2E)-4-chloro-2-butenyl)]-3-(2-methoxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0128] The compound was purified by flash chromatography (cyclohexane-ethyl acetate 70-30). Thick oil.

[0129] [BB25]: N-(2-{3-[(2Z)-4-chloro-2-butenyl]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]}ethyl)-acetamide

[0130] The compound was purified by flash chromatography (CH2Cl2-methanol 97-3). Waxy solid from diisopropyl ether, m.p. 118° C.

[0131] [BB26]: N-(2-{3-[(2E)-4-chloro-2-butenyl]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]}ethyl)-acetamide

[0132] White solid from diethyl ether, m.p. 108° C.

[0133] [BB13]: N-{2-[3-(2-chloroethyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl}methanesulfonamide

[0134] The compound was purified by flash chromatography (CH2Cl2-methanol 98-2). White low melting solid.

[0135] [BB07]: N-{2-[3-(4-chlorobutyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl}methanesulfonamide

[0136] White solid, m.p. 104° C. from diethyl ether.

[0137] [BB27]: N-(2-{3-[(2Z)-4-chloro-2-butenyl)]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl}methanesulfonamide

[0138] The compound was purified by flash chromatography (CH2Cl2-methanol 97-3). White solid, m.p. 83° C. from diethyl ether.

[0139] [BB28]: N-2-{3-[(2E)-4-chloro-2butenyl)]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl}methanesulfonamide

[0140] The compound was purified by flash chromatography (CH2Cl2-methanol 98-2). White solid, m.p. 98° C. from diethyl ether.

[0141] Description 9

[0142] [BB08]: 1-(2-chloroethyl)-3-[(2-piperidinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0143] A solution of 1-[2-(1-piperidinyl)ethyl)-1,3-dihydro-2H-benzimidazol-2-one (4 g, 16.3 mmoles) in DMF (50 ml) was added to a suspension of 80% sodium hydride (0.49 g, 16.3 mmoles) in DMF (25 ml) and the reaction mixture was heated under stirring for 30 minutes at 40° C. The solution was slowly transferred (3 hours) into a solution of 1-bromo-2-chloroethane (2.7 ml, 32.6 mmoles) in DMF (30 ml), the temperature was increased to 60° C. and stirred for 5 hours. The reaction mixture was then taken to dryness under vacuum, and the residue partitioned between 5% aqueous HCl and diethyl ether. The aqueous layer was adjusted to pH 9 to 10 with sodium carbonate and extracted with ethyl acetate. After evaporation and flash chromatography purification (CH2Cl2-methanol-NH4OH 95-5-0.5), 2.2 g of the pure title compound was obtained as a clear oil.

[0144] According to the above described procedure, the following compounds were prepared from the suitable intermediates:

[0145] [BB02]: 1-(4-chlorobutyl)-3-[(2-(1-piperidinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0146] The compound was purified by flash chromatography (CH2Cl2-methanol-NH4OH 95-5-0.5). Ivory solid, m.p. 82° C.-87° C. from diethyl ether.

[0147] [BB12]: 1-(2-chloroethyl)-3-[2-(4-morpholinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0148] The compound was purified by flash chromatography (CH2Cl2-methanol-NH4OH 95-5-0.5). Thick oil.

[0149] [BB03]: 1-(4-chlorobutyl)-3-[2-(4-morpholinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0150] The compound was purified by flash chromatography (CH2Cl2-methanol-NH4OH 95-5-0.5). Clear oil.

[0151] Description 10

[0152] [BB14]: 1-[(2Z)-4-chloro-2-butenyl)]-3-[2-(4-morpholinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0153] A solution of 1-[2-(4-morpholinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one (4 g, 16.2 mmoles) in DMF (50 ml) was added dropwise to a suspension of 80% sodium hydride (0.49 g, 16.2 mmoles) in DMF (50 ml) and the mixture was heated under stirring at 45° C. for 30 minutes. This solution was slowly transferred (4 hours) to a solution of cis-1,4-dichloro-2-butene (3.43 ml, 32.4 mmoles) in DMF (20 ml). The reaction mixture was stirred overnight at room temperature, taken to dryness under vacuum, and partitioned between ethyl acetate and water. From the organic solution after evaporation and flash chromatography purification (CH2Cl2-methanol-NH4OH 95-5-0.5), 2.4 g of the title compound were obtained as an oil.

[0154] According to the above described procedure, the following compounds were prepared from the suitable intermediates:

[0155] [BB19]: 1-[(2E)-4-chloro-2-butenyl]-3-[2-(4-morpholinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0156] The compound was purified by flash chromatography (CH2Cl2-methanol-NH4OH 95-5-0.5). Thick oil.

[0157] [BB17]: 1-[(2Z)-4-chloro-2-butenyl]-3-[2-(1-piperidinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0158] The compound was purified by flash chromatography (CH2Cl2-methanol-NH4OH 95-5-0.5). Thick oil.

[0159] [BB18]: 1-[(2E)-4-chloro-2-butenyl]-3-[2-(1-piperidinyl)ethyl]-1,3-dihydro-2H-benzimidazol-2-one

[0160] The compound was purified by flash chromatography (CH2Cl2-methanol-NH4OH 95-5-0.5). Thick oil.

[0161] Description 11

[0162] [BB10]: 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0163] A solution of 1-(2-chloroethyl)-3-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,3-dihydro-2H-benzimidazol-2-one (2.2 g) and a catalytic amount of p-toluenesulfonic acid (0.1 g) in methanol (30 ml) was stirred at room temperature for 2 hours. The reaction mixture was evaporated to dryness, the residue was dissolved in CH2Cl2 and washed with a saturated aqueous solution of K2CO3. The organic layer was taken to dryness to give 1.5 g of the title compound as white solid. M.p. 135° C.

[0164] According to the above described procedure, the following compounds were prepared from the suitable intermediates:

[0165] [BB04]: 1-(4-chlorobutyl)-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0166] Thick oil.

[0167] [BB20]: 1-[(2Z)-4-chloro-2-butenyl]-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0168] White solid, m.p. 80° C. from diethyl ether.

[0169] [BB21]: 1-[(2E)-4-chloro-2-butenyl]-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one

[0170] Ivory solid, m.p. 73° C. from diethyl ether.

[0171] Description 12

[0172] [BB11]: 2-[3-(2-chloroethyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl-ethylcarbamate

[0173] 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one (2.6 g, 11 mmoles) and ethyl isocyanate (20 ml) were refluxed under stirring for 6 hours then left overnight at room temperature. The reaction mixture was taken to dryness and the residue was crystallized from diisopropyl ether to give 3 g of the title compound. M.p. 125° C.

[0174] According to the above described procedure, the following compound was prepared from the suitable intermediate:

[0175] [BB05]: 2-[3-(4-chlorobutyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl-ethylcarbamate

[0176] White solid, m.p. 75° C. from diethyl ether.

[0177] Description 13

[0178] [BB22]: 2-{3-[(2Z)-4-chloro-2-butenyl]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl]ethyl-ethylcarbamate

[0179] 1-(4-chlorobutyl)-3-(2-hydroxyethyl)-1,3-dihydro-2H-benzimidazol-2-one (1.8 g, 6.8 mmoles) and ethyl isocyanate (6 ml) were stirred at room temperature for 48 hours. The reaction mixture was then taken to dryness and the residue was crystallized from diethyl ether to give 1.8 g the title compound. M.p. 107° C.

[0180] According to the above described procedure, the following compound was prepared from the suitable intermediate:

[0181] [BB23]: 2-{3-[(2E)-4-chloro-2-butenyl]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl}ethyl-ethylcarbamate

[0182] The compound was purified by flash chromatography (cyclohexane-ethyl acetate 50-50). White solid, m.p. 70° C. from diethyl ether.

[0183] B. General Method for the Preparation of the Compounds of Formula (I)

[0184] A solution of each building block of formula (II) (0.1 mM) was reacted under stirring with each amine (0.2 mM) in anhydrous DMF (100 μl) in the presence of Na2CO3 (0.3 mM) at a temperature ranging from room temperature to 100° C., preferably between 60° C. and 80° C., for about 6 to 8 hours. Isocyanatemethyl Polystyrene Resin (loading 0.23 meq/g) (0.2 mM) was introduced and the mixture was gently stirred at room temperature for 8 hours. The resin was then filtered off under vacuum, washed with DMF, and filtered again. The collected solutions were evaporated to dryness in a speed-vac centrifuge. The compounds which were prepared according to the above described procedure are listed in Table 3.

[0185] Table 3 collects the structural formula of the synthesized compounds along with the corresponding characterizing mass data (i.e., [M+H]+) obtained for each of the compounds according to the invention. The identification of the compounds and their purity was carried out by using positive APCI-LC/MS technique.

6TABLE 3Compounds of Formula (I)(I)

Compound No.—R1—A—

[M + H]+1

4092

5333

4234

4105

4436

4117

4198

4789

47710

40611

47712

46413

52614

100

43915

101

102

103

45716

104

105

106

47817

107

108

109

46718

110

111

112

43719

113

114

115

43920

116

117

118

46221

119

120

121

58622

122

123

124

47623

125

126

127

46324

128

129

130

49625

131

132

133

46426

134

135

136

47227

137

138

139

53128

140

141

142

53029

143

144

145

45930

146

147

148

53031

149

150

151

51732

152

153

154

57933

155

156

157

49234

158

159

160

51035

161

162

163

53136

164

165

166

52037

167

168

169

49038

170

171

172

49239

173

174

175

46440

176

177

178

58841

179

180

181

47842

182

183

184

46543

185

186

187

49844

188

189

190

46645

191

192

193

47446

194

195

196

53347

197

198

199

53248

200

201

202

46149

203

204

205

53250

206

207

208

51951

209

210

211

58152

212

213

214

49453

215

216

217

51254

218

219

220

53355

221

222

223

52256

224

225

226

39557

227

228

229

41958

230

231

232

40959

233

234

235

39660

236

237

238

42961

239

240

241

39762

242

243

244

40563

245

246

247

46464

248

249

250

46365

251

252

253

39266

254

255

256

46367

257

258

259

45068

260

261

262

51269

263

264

265

42570

266

267

268

44371

269

270

271

46472

272

273

274

45373

275

276

277

42374

278

279

280

42575

281

282

283

46676

284

285

286

59077

287

288

289

48078

290

291

292

46779

293

294

295

50080

296

297

298

46881

299

300

301

47682

302

303

304

53583

305

306

307

53484

308

309

310

46385

311

312

313

53486

314

315

316

52187

317

318

319

58388

320

321

322

49689

323

324

325

51490

326

327

328

53591

329

330

331

52492

332

333

334

49493

335

336

337

49694

338

339

340

48095

341

342

343

48096

344

345

346

50097

347

348

349

43698

350

351

352

56099

353

354

355

450100

356

357

358

437101

359

360

361

470102

362

363

364

438103

365

366

367

446104

368

369

370

505105

371

372

373

504106

374

375

376

433107

377

378

379

504108

380

381

382

491109

383

384

385

553110

386

387

388

466111

389

390

391

484112

392

393

394

505113

395

396

397

494114

398

399

400

464115

401

402

403

466116

404

405

406

450117

407

408

409

450118

410

411

412

470119

413

414

415

472120

416

417

418

596121

419

420

421

486122

422

423

424

473123

425

426

427

506124

428

429

430

474125

431

432

433

482126

434

435

436

541127

437

438

439

540128

440

441

442

469129

443

444

445

540130

446

447

448

527131

449

450

451

589132

452

453

454

502133

455

456

457

520134

458

459

460

541135

461

462

463

530136

464

465

466

500137

467

468

469

502138

470

471

472

486139

473

474

475

486140

476

477

478

506141

479

480

481

434142

482

483

484

558143

485

486

487

448144

488

489

490

435145

491

492

493

468146

494

495

496

436147

497

498

499

444148

500

501

502

503149

503

504

505

502150

506

507

508

431151

509

510

511

502152

512

513

514

489153

515

516

517

551154

518

519

520

464155

521

522

523

482156

524

525

526

503157

527

528

529

492158

530

531

532

462159

533

534

535

464160

536

537

538

381161

539

540

541

505162

542

543

544

395163

545

546

547

382164

548

549

550

415165

551

552

553

383166

554

555

556

391167

557

558

559

450168

560

561

562

449169

563

564

565

378170

566

567

568

449171

569

570

571

436172

572

573

574

526173

575

576

577

411174

578

579

580

429175

581

582

583

450176

584

585

586

450177

587

588

589

409178

590

591

592

411179

593

594

595

367180

596

597

598

491181

599

600

601

381182

602

603

604

368183

605

606

607

401184

608

609

610

369185

611

612

613

377186

614

615

616

436187

617

618

619

435188

620

621

622

364189

623

624

625

435190

626

627

628

422191

629

630

631

484192

632

633

634

397193

635

636

637

415194

638

639

640

436195

641

642

643

436196

644

645

646

395197

647

648

649

397198

650

651

652

438199

653

654

655

562200

656

657

658

452201

659

660

661

439202

662

663

664

472203

665

666

667

440204

668

669

670

448205

671

672

673

507206

674

675

676

506207

677

678

679

435208

680

681

682

506209

683

684

685

493210

686

687

688

555211

689

690

691

468212

692

693

694

486213

695

696

697

507214

698

699

700

496215

701

702

703

466216

704

705

706

468217

707

708

709

452218

710

711

712

452219

713

714

715

472220

716

717

718

436221

719

720

721

560222

722

723

724

450223

725

726

727

437224

728

729

730

470225

731

732

733

438226

734

735

736

446227

737

738

739

533228

740

741

742

504229

743

744

745

433230

746

747

748

504231

749

750

751

491232

752

753

754

553233

755

756

757

466234

758

759

760

484235

761

762

763

505236

764

765

766

494237

767

768

769

444238

770

771

772

568239

773

774

775

458240

776

777

778

445241

779

780

781

478242

782

783

784

446243

785

786

787

454244

788

789

790

513245

791

792

793

512246

794

795

796

441247

797

798

799

512248

800

801

802

499249

803

804

805

561250

806

807

808

474251

809

810

811

492252

812

813

814

513253

815

816

817

502254

818

819

820

472255

821

822

823

474256

824

825

826

458257

827

828

829

458258

830

831

832

478259

833

834

835

462260

836

837

838

586261

839

840

841

476262

842

843

844

463263

845

846

847

496264

848

849

850

464265

851

852

853

472266

854

855

856

531267

857

858

859

530268

860

861

862

459269

863

864

865

530270

866

867

868

517271

869

870

871

579272

872

873

874

492273

875

876

877

510274

878

879

880

531275

881

882

883

520276

884

885

886

407277

887

888

889

531278

890

891

892

421279

893

894

895

408280

896

897

898

441281

899

900

901

409282

902

903

904

417283

905

906

907

476284

908

909

910

475285

911

912

913

404286

914

915

916

475287

917

918

919

462288

920

921

922

524289

923

924

925

437290

926

927

928

455291

929

930

931

476292

932

933

934

465293

935

936

937

407294

938

939

940

531295

941

942

943

421296

944

945

946

408297

947

948

949

441298

950

951

952

409299

953

954

955

417300

956

957

958

432301

959

960

961

475302

962

963

964

404303

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462305

971

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524306

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437307

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455308

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476309

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465310

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694315

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1003

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1009

465318

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528319

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457320

1016

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467352

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530355

1121

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1123

517356

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1126

579357

1127

1128

1129

492358

1130

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510359

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531360

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520361

1139

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393362

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390371

1169

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1177

510374

1178

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407381

1199

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466401

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474402

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469403

1265

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532404

1268

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461405

1271

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532406

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519407

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581408

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494409

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533411

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522412

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464413

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588414

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504

[0186] The biological profile of the compounds of the invention, was assessed by evaluating their activity at the 5-HT1A, 5-HT2A, and D4 receptors, according to the methods described below.

[0187] Receptor Binding Studies

[0188] Receptor binding studies were carried out to determine the affinity of the compounds for 5-HT1A, 5-HT2A, and D4 receptors

[0189] 5-HT1A Radioligand Receptor Binding Assay

[0190] Membranes from CHO cells, expressing 5-HT1A human receptors were suspended in incubation buffer.

[0191] Binding Assay:

[0192] Binding assays were performed in MultiProbe 204 pipetting system (Packard), according to a predetermined mapping, consistent with the software Screen. The compounds were tested in singlicate at one concentration (10−1 M) in a total volume of 1000 μl. 980 μl of diluted membranes, 10 μl DMSO or unlabelled ligand and 10 μl of [3H]-8-OH-DPAT (0.6-0.7 nM) were incubated for 60 minutes at 27° C. The reaction was stopped by rapid filtration through Tomtec Cell Harvester (48 wells) using Filtermat B (presoaked in 0.1% PEI) filters. Filters were washed with ice-cold 50 mM Tris-HCl (pH 7.4) buffer (9×700 μl), dried, covered with MeltiLex B/HS scintillator sheets (Wallac) and heated at 80° C. to 90° C. for about 10 minutes, transferred into plastic sample bags (Wallac), sealed, and put into 1024 Beta Plate scintillation counter (Wallac). Non-specific binding was determined in the presence of 5-HT (10−5 M).

[0193] Data Analysis:

[0194] The specific radioligand binding to the receptor was defined by the difference between total binding and non-specific binding, determined in the presence of an excess of unlabelled ligand. Results were expressed as percentage of control specific binding obtained in the presence of the compounds. The affinity values (IC50) for the compounds were obtained by a nonlinear least squares regression analysis on the basis of a one binding site model.

[0195] 5-HT1A Functional Assay (cAMP)

[0196] CHO/5-HT1A cells were random seeded at a density of about 200,000/well in 24 well plates the day prior to the experiment. On the day of the experiment, cells were pretreated for 15 minutes at 37° C. with 500 μM isobutylmethylxantine (IBMX) dissolved in culture medium without serum. Wells were then divided in different groups in duplicate as follows: control, 10 μM FSK, 10 μM FSK+1 μM 5-HT as positive standard and 10 μM FSK+10 μM of the different compound under evaluation. Sample solutions were added and incubated for additional 15 minutes at 37° C. After incubation, the medium was aspirated and the reaction stopped by adding 200 μl of lysis buffer. Plates were shaken for 5 minutes, then the lysate was removed and samples were stored at 4° C. until the day of the assay. For the cAMP evaluation, samples were properly diluted and the cAMP content was measured by an enzyme immunoassay system.

[0197] Data Analysis:

[0198] Results are expressed as % inhibition of the cAMP accumulation induced by 10 μM FSK.

[0199] D4 Radioligand Receptor Binding Assay

[0200] Membranes from CHO cells expressing D4 human receptors, were suspended in incubation buffer.

[0201] Binding Assay:

[0202] Binding assays were performed in MultiProbe 204 pipetting system (Packard), according to a predetermined mapping, consistent with the software Screen. The compounds were tested in singlicate at one concentration (10−7 M) in a total volume of 1000 μl (980 μl of diluted membranes, 10 μl DMSO or unlabelled ligand and 10 μl of [3H] YM-09151-2 (0.15-0.25 nM). After incubation for 120 minutes at 27° C., the reaction was stopped by rapid filtration through Tomtec Cell Harvester (48 wells) using Filtermat B (presoaked in 0.1% PEI) filters. Filters were washed with ice-cold 50 mM Tris-HCl (pH 7.4) buffer (9×700 μl), dried, covered with MeltiLex B/HS (Wallac) scintillator sheets and heated in oven at 80° C. to 90° C. for about 10 minutes, transferred into plastic sample bags (Wallac), sealed, and put into 1024 Beta Plate scintillation counter (Wallac). Non-specific binding was determined in the presence of clozapine dissolved in DMSO to a final concentration of 10−5 M.

[0203] Data Analysis:

[0204] The specific radioligand binding to the receptor was defined by the difference between total binding and non-specific binding, determined in the presence of an excess of unlabelled ligand. Results were expressed as percentage of control specific binding obtained in the presence of the compounds.

[0205] 5-HT2A Radioligand Receptor Binding Assay

[0206] Tissue Preparation:

[0207] Rats (male Sprague-Dawley, 200-250 g) were used. Cerebral frontal cortex was homogenized in 10 volumes of ice cold 0.32 M sucrose in 5 mM Tris-HCl (pH 74) buffer. After centrifugation of the homogenate (1,000×g for 10 minutes) the supernatant was then recentrifuged at 48,000×g for 15 minutes. The resulting pellet was gently homogenized in an equal volume of 50 mM Tris-HCl buffer (pH 7.4) and incubated at 37° C. for 10 minutes. Membranes were then collected by centrifugation as above described and finally resuspended in 10 volumes of 50 mM Tris-HCl buffer (pH 7.4).

[0208] Binding Assay:

[0209] For displacement experiments membranes (980 μl) were diluted in 50 mM Tris-HCl buffer (pH 7.4) to a final concentration of 1:100 (w/v); the tissue suspension was then incubated at 37° C. for 10 minutes in a final volume of 1 ml in the presence of 0.5 nM [3H]-Ketanserin. Non-specific binding was determined by incubating similar samples with unlabelled methysergide (100 μM). After incubation, samples prepared in a 24 wells cell culture cluster (Costar) were rapidly filtered by Inotech Cell Harvester (IH 201 filters). The filters were washed three times with 2 ml ice-cold Tris-HCl buffer and placed in polyethylene vials, then 4 ml of Filter Count scintillation cocktail (Packard) were added. The radioactivity present was counted by liquid scintillation spectrometry.

[0210] Data Analysis:

[0211] The affinity values (IC50) for the compounds were obtained by a nonlinear least squares regression analysis on the basis of a one binding site model.

[0212] 5-HT2A Functional Assay (PI turnover)

[0213] Tissue Preparation:

[0214] Cross-chopped miniprisms (350×350 μm) were prepared from mouse whole cerebral cortices and incubated for 60 minutes at 37° C. in Krebs-Henseleit buffer containing 2 g/l glucose.

[0215] Functional Assay:

[0216] Cerebral cortex miniprisms were distributed in vials and incubated for 30 minutes with approximately 170 nM [3H]-myoinositol (10-20 Ci/mmol) and 10 mM lithium chloride. Samples were divided in different groups in triplicate: control, 100 μM 5-HT, 10 and 30 μM flibanserin+100 μM 5-HT, as standards, and 10 μM of the different compound under investigation+100 μM 5-HT. When 5-HT was added the incubation continued for 45 minutes. Compounds under investigation and flibanserin were added 10 minutes before dispensing 5-HT. Incubation was terminated by the addition of 940 μl chloroform-methanol (1:2 v/v). Further aliquots of chloroform (310 μl) and water (310 μl) were added and labeled inositol phosphates (1 Ps) were extracted from the aqueous phase by ion exchange chromatography using Dowex resin in the formate form. After addition of 10 ml of PicoFluor 40 scintillation cocktail (Packard), the radioactivity present in an aliquot (400 μl) of the aqueous extract was counted by liquid scintillation spectrometry.

[0217] Data Analysis:

[0218] Results are expressed as % inhibition of the PI turnover accumulation induced by 100 μM 5-HT.

[0219] The following Tables 4 to 6 collect the biological data at the receptors of the new compounds.

7TABLE 4% Inhibition at 5-HT1A and D4 Receptors5-HT1A ReceptorD4 Receptor5-HT1A ReceptorD4 ReceptorBinding AssayBinding AssayBinding AssayBinding AssayComp.% inhibition% inhibitionComp.% inhibition% inhibitionNo.(10−7 M)(10−7 M)No.(10−7 M)(10−7 M)156387869445925479865877791815578993328389391060478452421148908577791969329294552050609394622373489488722490679585642548449692722670941075558288935118803629578314585423044901498835379054150575238927815895723936421598550431045516496414510082169855856637117798395975511826245609382183966262739618794366492431887878655274189549966659919777437391582159848749262216924475675421989372249851332788522671323339980228953333595552296734336908124169323489951254853434973312555833361864625659513647736263924236589632655578367619028093383699659

[0220]

8

TABLE 5

5-HT1A Agonist Activity

5-HT1A

Receptor Binding
cAMP

Compound No.
IC50 (nM)
% inhibition

5
13
63

9
9.9
48

37
16
44

60
6.2
65

64
12
52

73
13
45

83
15
64

158
13
48

164
4.2
73

168
5.0
71

177
3.3
76

183
7.2
66

187
8.2
44

202
1.7
72

206
2.1
80

215
0.85
83

217
5.2
68

219
15
61

228
6.0
82

254
7.4
66

263
8.8
63

284
4.9
82

285
5.2
47

296
8.2
82

297
7.9
74

301
2.6
83

310
15
63

314
7.1
44

318
3.1
61

330
7.9
62

333
16
67

335
3.5
69

347
13
65

348
3.5
57

352
4.1
79

365
15
82

369
3.5
84

370
4.4
52

381
44
79

382
11
64

386
6.7
82

403
5.5
84

404
2.1
60

415
14
82

416
7.9
77

420
1.8
87

421
0.66
56

446
7.3
81

459
8.8
86

468
3.1
66

472
3.1
67

481
11
82

499
5.0
74

503
2.8
87

504
3.6
50

512
0.59
68

514
7.9
67

520
8.1
70

521
0.61
67

525
1.5
87

536
9.5
55

[0221]

9

TABLE 6

5-HT2A Antagonist Activity

5-HT2A

Receptor

Binding
PI Turnover

Compound No.
IC50 (nM)
% inhibition

9
16
45

73
0.90
42

83
43
86

168
46
22.00

177
7.7
39

183
27
12.00

206
17
90

215
3.2
83

254
65
64

514
74
41

521
30
48

N,N'-disubstituted benzimidazolone derivatives with affinity at the serotonin and dopamine receptors

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Priority Claims (1)

Provisional Applications (1)