EP2935254A1

EP2935254A1 - Compounds of 2,3-dihydro-4h-1,3-benzoxazine-4-one, method for preparing them and pharmaceutical form comprising them

Info

Publication number: EP2935254A1
Application number: EP13826965.9A
Authority: EP
Inventors: Alessandra Topai; Teresa Fabiola MISCIOSCIA; Fabio BARILE; Tatiana GUZZO; Franco MINISSI; Manolo SABLONE; Mauro Maccarrone
Original assignee: C4T SCARL
Current assignee: C4T SCARL
Priority date: 2012-12-21
Filing date: 2013-12-18
Publication date: 2015-10-28
Also published as: WO2014097188A1; ITMI20122221A1

Abstract

The present invention concerns new compounds of 2,3-dihydro-4H-1,3- benzoxazine-4-one, a method for preparing the said compounds, a pharmaceutical form that contains the said compounds, and their use. In particular, the present invention concerns new compounds of 2,3-dihydro-4H-1,3-benzoxazine-4-one having a selective agonist action for the type 2 cannabinoid receptor (CB2), and their use in the treatment of pathologies associated with non-activation of this receptor, particularly chronic pain.

Description

COMPOUNDS OF 2,3-DIHYDRO-4H-1 ,3-BENZOXAZINE-4-ONE, METHOD FOR PREPARING THEM AND PHARMACEUTICAL FORM COMPRISING THEM

DESCRIPTION

FIELD OF THE INVENTION The present invention concerns new derivatives of 2,3-dihydro-4H-1 ,3- benzoxazine-4-one, a method for preparing them, a pharmaceutical comprising them, and their use in a treatment method.

In particular, the present invention concerns new derivatives of 2,3-dihydro-4H-1 ,3-benzoxazine-4-one that have a selective agonist action for the type 2 cannabinoid receptor.

BACKGROUND OF THE INVENTION

The endocannabinoid system contains a group of lipid neuromodulators, which are synthesised from phospholipid precursors, in the neuronal cells as a result of physiological or pathological stimulation. The currently known lipid neuromodulators, or endocannabinoids, are anandamide (arachidonoylethanolamine), arachidonoylglycerol (2-arachidonoylglycerol), noladin (2-arachidonoyl glyceryl ether), virhodamine (arachidonic acid-(2-aminoethyl)-ester) and N-arachidonoyldopamine (NADA).

Once produced, these neuromodulators behave as autocrine mediators, when they bind to receptors that are present on the cell that produced them, or as paracrine mediators, when they bind to receptors that are present on neighbouring cells.

The cannabinoid receptors are membrane receptors, situated in both the central and peripheral nervous systems. They belong to the family of G protein-coupled receptors and consist of 7 transmembrane helices connected by 3 extracellular loops and 3 intracellular loops, with the amine termination on the extracellular side and the carboxyl termination on the intracellular side.

There are currently two known receptor sub-types, CB1 and CB2, which have an overall sequence homology of around 44%, which is higher at the seven transmembrane helices (around 68%). It is known that activation of the receptors CB1 and CB2 entails inhibition of the adenylate cyclase enzyme and stimulation of the MAP-kinase enzymes, with a consequent reduction in the secretion of various pro-inflammatory prostanoids and cytokines and a consequent reduction in the pain signal.

The CB1 receptors are present mainly in the central nervous system. The bonding of cannabinoids to these receptors induces both a pre-synaptic inhibition of the release of neurotransmitters (particularly NMDA and glutamate) and a stimulation of various regions of grey matter involved in the transmission of pain, particularly in the rostral ventromedial medulla.

The CB2 receptors are expressed mainly in the peripheral nervous system and on the T-cells of the immune system. It has been ascertained that these receptors play a role in controlling peripheral pain and inflammatory phenomena, by modulating the release of mediators such as cytokines and endorphins and the migration of immune system cells.

It has been hypothesised that activation of the CB2 receptor in immune system cells causes a reduction in the production of pro-inflammatory cytokines (Quartilho et al., Anesthesiology, 2003, 99, 955). It has also been suggested that activation of the CB2 receptor induces analgesia by reducing basal levels of the pro-algesic NGF (Nerve Growth Factor) molecule.

More recent studies have also identified the presence of the CB2 receptor in dorsal root ganglia (Beltramo et al., Eur. J. Neurosc. 2006, 23, 1530; Hsieh et al., Br. J. Pharm. 201 1 , 162, 42). This has led to suggestions that the CB2 receptors situated on the central and peripheral terminals of the nociceptive pathway may reduce both the excitability of the nociceptors and the release of pro-nociceptive mediators.

Finally, the presence of CB2 receptors on the microglia might reduce their activation, with a consequent reduction in the release of pro-algesic substances (Beltramo et al., 2006),

The development of selective agonists for the CB2 receptors could have promising effects in the treatment of neuropathic pain, without causing the central psychotropic effects mediated by the CB1 receptor (Quartilho et al., 2003; Brownjohn et al., Neurosc. 2012, 203, 180). Numerous selective agonists for the CB2 receptor are described in the literature, including AM1241 , which is capable of reducing the sensory hypersensitivity associated with neuropathic pain, without any collateral effects associated with the activity at central level (Quartilho et al., 2003). Clinical trials are also under way for CB2 agonists, such as Pharmos Corporation's Cannabinor and Glaxo Smith Kline's GW-842166X, a potent and selective CB2 agonist with an analgesic, anti-inflammatory and anti-hyperalgesic action. Of the exogenous cannabinoids, the best known is delta-9-tetrahydrocannabinol (A9-THC), the main active compound of Cannabis sativa. This molecule acts as a partial agonist for both CB1 and CB2 receptors, through which it acts as an analgesic, anti-emetic and appetite-stimulating compound. However, A9-THC has a psychoactive action on the central nervous system, wherein it causes tolerance, habituation and dependence.

Sativex™ is a sublingual spray with a standardised content of A9-THC and cannabidiol, which has been approved in a number of countries for the treatment of neuropathic pain caused by multiple sclerosis and cancer. Phase II and III clinical trials are currently under way on various models of peripheral neuropathic pain, for example where characterised by allodynia.

By modifying the structure of A9-THC, various synthetic compounds have been prepared in order to avoid undesired effects on the central nervous system while maintaining the therapeutic efficacy.

One of these is nabilone, a synthetic derivative of A9-THC, marketed as Cesamet™ for the treatment of nausea and vomiting in patients undergoing chemotherapy for tumours.

Neuropathic pain is universally recognised as one of the most difficult painful conditions to treat. It is linked to a functional anomaly of the nervous system and it is a chronic pain, characterised by constant burning sensations or perceptions of pain associated with stimuli that are either very slight (hyperalgesia) or not such as to normally provoke pain, such as contact between the skin and fabric (allodynia). It is estimated that at least 1 % of the world's population suffers from neuropathic pain, with over 600,000 of these patients being found in the United Kingdom.

Neuropathic pain is associated with numerous pathologies such as multiple sclerosis, stroke, cancer, lesions of the spinal cord, physical trauma, peripheral neuropathy caused by diabetes, and postherpetic neuralgia. The number of pharmacological therapies for the treatment of neuropathic pain is rather limited, and for this reason it is often necessary to administer combinations of drugs to alleviate its symptoms.

The traditional pharmacological therapies include the use of analgesics (generally more effective in preventing the onset of pain than in alleviating existing pain); opioids such as methadone, tramadol and oxycodone; tricyclic antidepressants such as amitriptyline, nortriptyline and desipramine; muscle relaxants and/or anticonvulsants such as gabapentin and carbamazepine; and cannabinoids such as Sativex™.

There are experimental proofs of the efficacy of various agonists for the CB2 receptors in preclinical models of neuropathic pain (Quartilho et al., 2003; Beltramo et al., 2006; Romero-Sandoval et al., Anesthesiology, 2007, 106, 787-794; Brownjohn et al., 2012).

Numerous classes of non-psychotropic cannabinoids have been described in patent applications as selective agonists for the CB2 receptor, useful in the treatment of pain, including neuropathic pain. For example, WO 2006/097808, US 2009/137584 and WO 2008/032164 describe derivatives of imidazole that are said to be effective in the treatment of pain characterised by allodynia.

In addition, WO 2010/063721 describes derivatives of 2,4-imidazolidinedione that have been demonstrated to have an anti-allodynia action in a model of neuropathic pain and an anti-algesic action in a model of inflammatory pain.

WO 201 1/025541 describes pyrazolic derivatives with demonstrated efficacy in models of post-operative pain, inflammatory pain, pain caused by cancer, chronic pain characterised by allodynia or diabetic neuropathy.

WO 01/32169 and WO 03/064359 describe derivatives of 4-phenyl pinene that have been shown to be effective in a model of cutaneous peripheral pain.

Derivatives of benzoxazin-4-one have been described both in patent applications and in literature documents.

In particular, derivatives of 4H-benzoxazin-4-one are divided between derivatives of 1 ,2-dihydro-4H-3, 1 -benzoxazin-4-one, having the general formula:

and derivatives of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one, having the general formula:

Derivatives of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one have been described, for example, in US 4,036,960 as pesticides with an ovicidal action, in US 5,071 ,850 for the treatment of cardiovascular diseases, in US 5,446, 152 for the treatment of gastrointestinal disturbances, particularly as anti-emetics with no effect on the central nervous system, and in WO 1995/004048 as therapeutic agents in the cardiovascular field, particularly in the treatment of angina and as anti-ischemics.

In addition, processes for the synthesis of 4H-1 ,3-benzoxazin-4-one have been described by Horrom BW et al., J. Am Chem Soc, 1950, 72 (2), 721 -724, by Gammil RB, J Org Chem, 1981 , 48, 3340, and more recently in EP 0 899 266 B1 . There are no known derivatives of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one with a selective agonist action for the type 2 cannabinoid receptor (CB2).

SUMMARY OF THE INVENTION

The Applicant observed that there is a strong need to find new compounds having a selective agonist action for the type 2 cannabinoid receptor (CB2). The Applicant has found new compounds derived from 2,3-dihydro-4H-1 ,3- benzoxazin-4-one.

Surprisingly, the Applicant has found that these new compounds have an agonist action and they are selective for the CB2 receptor. Consequently, the new compounds of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one according to the present invention can be used in medicine.

More in particular, said new compounds can be used in the treatment of pathological conditions associated with non-activation of the CB2 receptors, without causing any psychotropic effect in the central nervous system.

The structure of these new compounds differs both from that of the derivatives of benzoxazin-4-one described in the art and from that of the known compounds with a selective agonist action for the CB2 receptor.

In a first aspect, the present invention concerns new 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds of formula (I):

wherein:

X is -0- or -NH-,

R is H, -R₅-Ar, -S0₂-R5-Ar, -R₆ or S0₂-R6, where R₅ is σ bond, (Ci_-6)alkyl, (Ci_-6)alkyl-0-, -C(=0)-, -(Ci_-6)alkyl-C(=0)-, -C(=0)-0-, -(Ci_-6)alkyl-C(=0)-0-, and R₆ is (Ci_-6)alkyl,

Ar is an aromatic or heteroaromatic ring comprising 5 to 14 members, optionally substituted with one or more substituents selected from: halogen, -OH, -NH₂, (Ci-6)alkyl, (Ci-6)alkoxy, -CN, nitro, trialo(Ci-3)alkyl, trialo(Ci-3)alkoxy, aromatic or heteroaromatic ring comprising 5 or 6 members, benzyl;

R₂ is H, (Ci-6)alkyl, (Ci-6)alkoxy and benzyl, the said (Ci-6)alkyl, (Ci-6)alkoxy and benzyl being optionally substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-6)alkoxy, nitro and -CN;

R₃ and R₄, being the same or different from one another, are H, (Ci-6)alkyl, (Ci-6)alkoxy, or R₃ and R₄ together form a carbocycle or a heterocycle of 5 or 6 members, optionally substituted with a group (Ci-6)alkyl, (Ci-6)alkoxy, -C(=O)-(Ci_-3)alkyl -COOH, (Ci_-3)alkyl-COOH; and their addition salts with pharmaceutically acceptable organic or inorganic acids or bases.

In a second aspect, the present invention concerns a process for preparing the above-mentioned 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds of formula (I)

wherein X, R-i, R₂, R3 and R₄ are as defined earlier.

In a first embodiment, said process comprises the following steps:

(i) causing a compound of formula (a) to react in a protic polar solvent to obtain the intermediate (b):

wherein X is as defined earlier;

(ii) causing said intermediate (b) to react with an aqueous ammonia solution to obtain the intermediate (c):

(C)

wherein X is as defined earlier;

(iii) causing the said intermediate (c) to react with a reagent of formula

(III) wherein R₃ and R₄ are as defined earlier and R₇ and R₈, being identical, are each a group -0-(Ci-3)alkyl or R₇ and R₈ together with the carbon atom to which they are bonded form a group -C(=0),

to obtain a product of formula (d):

(d)

wherein X, R₃ and R₄ are as defined earlier;

(iv) causing said product (d) to react with a reagent of formula (IV)

Y-Ri

(IV) wherein Y is a halogen atom and Ri is as defined earlier, to form a product of formula (e):

(e)

wherein X, Ri, R₃ and R₄ are as defined earlier; (v) optionally, cause the said product (e) to react with a reagent of formula (V):

Y'-R₂ (V)

wherein Y' is a halogen atom and R₂ is as defined earlier, to form said product of formula (I):

(I) wherein X, R-ι, R₂, R3 and R₄ are as defined earlier.

In an alternative embodiment, the above process for preparing said compounds of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one of formula (I) comprises the following steps:

(vi) causing the product of formula (d) to react with a reagent of formula (VI):

Z-P

(VI) wherein Z is a halogen atom and P is a protective group, to obtain the intermediate (g- 1 ):

(g.1 ) wherein X, R₃ and R₄ are as defined earlier or alternatively, when X is -NH-, cause the product of formula (d) to react with a suitable oxidising agent, to obtain the intermediate (g.2)

(g.2) wherein R₃ and R₄ are as defined earlier

(vii) causing said intermediates (g.1 ) and (g.2) to react with a reagent of formula (VII)

Y"-R₂

(VII) wherein Y" is a halogen atom and R₂ is as defined earlier, to obtain, respectively, the intermediates (h.1 ) and (h.2):

wherein X, R₂, R3 and R₄ are as defined earlier;

(viii) causing the said intermediates (h.1 ) and (h.2) to react under suitable conditions to obtain a product of formula (i):

(i)

wherein X, R₂, R3 and R₄ as defined earlier;

(ix) optionally, cause the said product (i) to react with a reagent of formula (IX):

Y'"-Ri (IX) wherein Y'" is a halogen atom and Ri is as defined earlier, to obtain the said product of formula (I):

(I)

wherein X, Ri, R₂, R3 and R₄ are as defined earlier.

The skilled in the art will readily understand that the products as defined by the said formulae (d), (e) and (i) are within the scope of the present invention. In particular, the compound of formula (d) represents the compound of formula (I) when both Ri and R₂ are hydrogen; the compound of formula (e) represents the compound of formula (I) when R₂ is hydrogen; and the compound of formula (i) represents the compound of formula (I) when Ri is hydrogen. In a third aspect, the present invention concerns a pharmaceutical form comprising at least one compound of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one of formula (I)

(I)

wherein X is -O- or -NH-,

R₂ is H, (Ci-6)alkyl, (Ci-6)alkoxy and benzyl, said (Ci-6)alkyl, (Ci-6)alkoxy and benzyl being optionally substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-6)alkoxy, nitro and -CN;

R₃ and R₄, being the same or different from one another, are H, (Ci-6)alkyl, (Ci-6)alkoxy, or R₃ and R₄ together form a carbocycle or a heterocycle of 5 or 6 members, optionally substituted with a group (Ci-6)alkyl, (Ci-6)alkoxy, -C(=O)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH; or their addition salts with pharmaceutically acceptable organic or inorganic acids or bases, and at least one pharmaceutically acceptable excipient. In a fourth aspect, the present invention concerns the compounds of 2,3- dihydro-4H-1 ,3-benzoxazin-4-one of formula I):

(I)

wherein

X is -0- or -NH-,

R is H, -R₅-Ar, -S0₂-R5-Ar, -R₆ or S0₂-R6, where R₅ is σ bond, (Ci_-6)alkyl, (Ci_-6)alkyl-0-, -C(=0)-, -(Ci_-6)alkyl-C(=0)-, -C(=0)-0-, -(Ci_-6)alkyl-C(=0)-0-, and R₆ is (Ci_-6)alkyl, Ar is an aromatic or heteroaromatic ring comprising 5 to 14 members, optionally substituted with one or more substituents selected from: halogen, -OH, -NH₂, (Ci-6)alkyl, (Ci-6)alkoxy, -CN, nitro, trialo(Ci-3)alkyl, trialo(Ci-3)alkoxy, aromatic or heteroaromatic ring comprising 5 or 6 members, benzyl;

R₃ and R₄, being the same or different from one another, are H, (Ci-6)alkyl, (Ci-6)alkoxy, or R₃ and R₄ together form a carbocycle or a heterocycle of 5 or 6 members, optionally substituted with a group (Ci-6)alkyl, (Ci-6)alkoxy, -C(=O)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH; and their addition salts with pharmaceutically acceptable organic or inorganic acids or bases, for use in the treatment of pathologies related to the non-activation of the CB2 receptors, particularly inflammation and chronic pain. Advantageously, said chronic pain is neuropathic pain, post-operative pain or inflammatory pain. Finally, in a fifth aspect, the present invention concerns a method of treatment of pathologies related to the non-activation of the CB2 receptors, particularly inflammation and chronic pain, by administering to a human subject in need thereof a pharmaceutically effective amount of a compound of 2,3-dihydro-4H-1 ,3 benzoxazin-4-one of formula (I):

(I)

wherein

X is -0- or -NH-, R is H, -R₅-Ar, -S0₂-R5-Ar, -R₆ or S0₂-R6, where R₅ is σ bond, (Ci_-6)alkyl, (Ci_-6)alkyl-0-, -C(=0)-, -(Ci_-6)alkyl-C(=0)-, -C(=0)-0-, -(Ci_-6)alkyl-C(=0)-0-, and R₆ is (Ci_-6)alkyl,

Ar is an aromatic or heteroaromatic ring comprising 5 to 14 members, optionally substituted with one or more substituents selected from: halogen, -OH, -NH₂, (Ci-6)alkyl, (Ci-6)alkoxy, -CN, nitro, trialo(Ci-3)alkyl, trialo(Ci-3)alkoxy, benzyl, aromatic or heteroaromatic ring comprising 5 or 6 members;

R₂ is H, (Ci-6)alkyl, (Ci-6)alkoxy and benzyl, said (Ci-6)alkyl, (Ci-6)alkoxy and benzyl being optionally substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-6)alkoxy, nitro and -CN; R₃ and R₄, being the same or different from one another, are H, (Ci-6)alkyl, (Ci-6)alkoxy, or R₃ and R₄ together form a carbocycle or a heterocycle of 5 or 6 members, optionally replaced with a group (Ci-6)alkyl, (Ci-6)alkoxy, -C(=O)-(Ci-3)alkyl, -COOH, (Ci_-3)alkyl-COOH; or their addition salts with pharmaceutically acceptable organic or inorganic acids or bases.

Advantageously, said chronic pain is neuropathic pain, post-operative pain or inflammatory pain. The present invention also comprises esters, prodrugs, stereoisomers and enantiomers of the compounds of formula (I) described above.

DETAILED DESCRIPTION

In the present description and the claims that follow, the term "(Ci-6)alkyl" represents linear or branched alkyl groups having from 1 to 6 carbon atoms, for example the methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 3-pentyl, isopentyl, neo-pentyl, n-hexyl, sec-hexyl and neo-hexyl groups.

In the present description and the claims that follow, the term "(Ci-₄)alkyl" represents linear or branched alkyl groups having from 1 to 4 carbon atoms, for example the methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl groups.

In the present description and the claims that follow, the term "(Ci-3)alkyl" represents linear or branched alkyl groups having from 1 to 3 carbon atoms, for example the methyl, ethyl, n-propyl and isopropyl groups.

In the present description and the claims that follow, the term "(Ci-6)alkoxy" represents linear or branched alkoxy groups having from 1 to 6 carbon atoms, for example the methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, sec-pentoxy, isopentoxy and n-hexyloxy groups.

In the present description and the claims that follow, the term "(Ci_-4)alkoxy" represents linear or branched alkoxy groups having from 1 to 4 carbon atoms, for example the methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and tert-butoxy groups.

In the present description and the claims that follow, the term "(Ci-3)alkoxy" represents linear or branched alkoxy groups having from 1 to 3 carbon atoms, for example the methoxy, ethoxy, n-propoxy and isopropoxy groups.

In the present description and the claims that follow, "Ar" represents aromatic or heteroaromatic rings comprising at least one atom of nitrogen, oxygen or sulphur, said aromatic or heteroaromatic rings comprising from 5 to 14 members, more preferably from 5 to 10 members. Preferably, "Ar" represents, for example, benzene, naphthene, thiophene, furan, pyrrole, imidazol, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, benzothiophene, benzofuran, quinoline, isoquinoline, indole, indazole, purine and naphthyridine. Optionally, the said aromatic and heteroaromatic rings represented by "Ar" are substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-6)alkyl, (Ci_-6)alkoxy, -CN, nitro, trialo(Ci_-3)alkyl, trialo(Ci-3)alkoxy, benzyl, aromatic or heteroaromatic ring comprising 5 or 6 members.

In a preferred embodiment of the present invention, the meanings of X, R-i , R₂, R3 and R₄ of the above-mentioned formula (I) are defined as follows. Preferably, X is -O-.

Preferably, -X-R₁ is -OH, -O-R₆, -O-SO₂-R₆, -O-R₅-Ar or -O-SO₂-R₅-Ar.

Preferably, R₅ is σ bond, (Ci-₃)alkyl, (Ci-₃)alkyl-O-, -C(=O)-, (Ci-₃)alkyl-C(=O)-, -C(=O)-O-, (Ci_-3)alkyl-C(=O)-O-.

More preferably, R₅ is selected from σ bond, (Ci-₃)alkyl, (Ci_-3)alkyl-C(=O)-. Even more preferably, R₅ is σ bond, methyl or -CH₂-C(=O)-. Preferably, R₆ is (Ci_-3)alkyl.

Preferably, Ar is an aromatic or heteroaromatic ring comprising from 5 to 10 members, optionally substituted with one or more substituents selected from halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-3)alkoxy, -CN, nitro, trifluoromethyl, trifluoromethoxy, benzyl, aromatic or heteroaromatic ring comprising 5 or 6 members.

More preferably, Ar is an aromatic or heteroaromatic ring comprising from 5 to 10 members, optionally replaced with at least one substituent selected from halogen, (Ci_-3)alkyl, (Ci_-3)alkoxy, -CN, nitro, trifluoromethyl, trifluoromethoxy, phenyl, benzyl.

In a preferred aspect, Ar is an aromatic ring comprising from 6 to 10 members or a heteroaromatic ring comprising from 5 to 10 members and at least one heteroatom selected from N, O or S.

In a further preferred embodiment, said aromatic ring comprising from 6 to 10 members is selected from benzene and naphthene, and said heteroaromatic ring comprising from 5 to 10 members and at least one heteroatom selected from N, O or S is selected from thiophene, pyrazole, furan, pyridine, benzothiophene, quinoline, isoquinoline, benzofuran and indole.

Preferably, R₂ is H, (Ci_-4)alkyl, (Ci_-4)alkoxy or benzyl, said (Ci_-4)alkyl, (Ci_-4)alkoxy and benzyl being optionally substituted with at least one substituent selected from halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-3)alkoxy, nitro and -CN. More preferably, R₂ is H, (Ci_-4)alkyl or benzyl, said (Ci_-4)alkyl and benzyl being optionally substituted with at least one substituent selected from halogen, (Ci-3)alkyl and -CN.

Still more preferably, R₂ is H, an alkyl group selected from methyl, ethyl, n-propyl, isopropyl and n-butyl, benzyl, said alkyl groups or said benzyl being optionally substituted with F, CI, (Ci_-3)alkyl and -CN.

Preferably, R₃ and R₄, being the same or different from one another, are H, (Ci-3)alkyl, (Ci-3)alkoxy, or R₃ and R₄ together form a carbocycle or a heterocycle of 5 or 6 members, optionally substituted with a group (Ci-3)alkyl, (Ci-3)alkoxy, -C(=0)-(Ci_-3alkyl).

More preferably, R₃ and R₄, being the same or different from one another, are (Ci-3)alkyl, or R₃ and R₄ together form a carbocycle or a heterocycle of 6 members, optionally substituted with a group (Ci-3)alkyl, (Ci-3)alkoxy, -C(=0)-(Ci-3alkyl).

In a preferred aspect, said carbocycle with 6 members is cyclohexyl and said heterocycle with 6 members is tetrahydropyranyl, piperidyl or (N-acetyl)-piperidyl.

Preferably, at least one of R-i, R₂, R3 and R₄ is other than H.

Preferably, Ri is other than H. Preferably, R₂ is other than H. Preferably, R₃ is other than H. Preferably, R₄ is other than H.

The present invention also comprises addition salts of the said compounds of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one of formula (I) with pharmaceutically acceptable organic or inorganic acids or bases.

Examples of pharmaceutically acceptable organic acids are oxalic acid, maleic acid, methanesulphonic acid, paratoluenesulphonic acid, trifluoracetic acid, succinic acid, citric acid, malic acid, lactic acid. Preferably, trifluoracetic acid. Examples of pharmaceutically acceptable inorganic acids are hydrochloric acid, hydrobromic acid, phosphoric acid and sulphuric acid.

Examples of pharmaceutically acceptable organic bases are tromethamine, lysine, arginine, glycine, alanine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, dipropylamine, tripropylamine, ethylenediamine, monoethanolamine, diethanolamine, triethanolamine, guanidine, morpholine, piperidine, pyrrolidine, piperazine, 1 -butylpiperidine, 1 -ethyl-2- methylpiperidine, N-methylpiperazine, 1 ,4-dimethylpiperazine,

N-benzylphenethylamine, N-methyl glucosamine and tris(hydroxymethyl)aminomethane.

Examples of pharmaceutically acceptable inorganic bases are hydroxides or carbonates of alkaline or alkaline-earth metals, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate and calcium carbonate.

The present invention also concerns a process for preparing the said compounds of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one of formula (I). A first embodiment of said process comprises steps (i) to (v).

Preferably, said step (i) is carried out in a protic polar solvent selected from water, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, acids such as formic acid and acetic acid. More preferably, said solvent is selected from water, methanol and ethanol. Still more preferably, said solvent is methanol. Preferably, said step (i) is carried out in the presence of thionyl chloride. More preferably, the molar ratio between the said compound of formula (a) and thionyl chloride is between 1 :0.5 and 1 :3.5. Advantageously, the said molar ratio is between 1 : 1 and 1 :3.

Preferably, said step (ii) is carried out by irradiating the aqueous ammonia solution comprising said intermediate (b) with microwaves in a range of 200 to 300 W.

Preferably, the said step (iii) is carried out in a nitrogen atmosphere.

Preferably, said reagent of formula (III) is selected from 1 , 1 -dimethoxycyclohexane, cyclohexanone, 2,2-dimethoxy-propane, 3-pentanone, N-acetyl-4-piperidone, 3H-2,5-dihydropiran-4-one. Preferably, said step (iv) is carried out in an aprotic polar solvent. More preferably, the said aprotic polar solvent is selected from acetone, acetonitrile, dimethyl sulphoxide, dichloromethane and dimethylformamide. Still more preferably, the said solvent is dimethylformamide.

Preferably, in said reagent of formula (IV) Y-R-i, Y is chlorine or bromine and Ri is -S0₂- 5-Ar or -Rs-Ar, wherein R_s and Ar are as defined earlier. Preferably, said step (v) is conducted in an aprotic polar solvent. More preferably, the said aprotic polar solvent is selected from acetone, acetonitrile, dimethyl sulphoxide, dichloromethane and dimethylformamide. Still more preferably, the said solvent is dimethylformamide. Preferably, in said reagent of formula (V) Y-R2, Y' is bromine and R₂ is as defined earlier.

In a second embodiment, said process comprises steps (i) to (iii) as described earlier, and also steps (vi) to (ix).

Preferably, said step (vi) is conducted in an aprotic polar solvent. More preferably, the said aprotic polar solvent is selected from acetone, acetonitrile, dimethyl sulphoxide, dichloromethane and dimethylformamide. Even more preferably, said solvent is selected from dimethylformamide and dichloromethane.

Preferably, in the said reagent of formula (VI) Z-P, Z is chlorine and P is a protective group selected from trityl, tosyl and methoxym ethyl. Preferably, the oxidising agent used in said step (vi) is selected from hydrogen peroxide, meta-chloroperoxybenzoic acid and sodium perborate tetrahydrate. More preferably, the said oxidising agent is sodium perborate tetrahydrate.

Preferably, said step (vii) is carried out in an aprotic polar solvent. More preferably, said aprotic polar solvent is selected from acetone, acetonitrile, dimethyl sulphoxide, dichloromethane and dimethylformamide. Still more preferably, the said solvent is dimethylformamide.

Preferably, in the said reagent of formula (VII) Y"-R₂, Y" is bromine and R₂ is as defined earlier.

Preferably, the said step (viii) is carried out in a polar solvent. More preferably, the said polar solvent is selected from water, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, acids such as formic acid, acetic acid, acetone, acetronile, dimethyl sulphoxide, dichloromethane, dimethylformamide or aqueous hydrochloric acid. Still more preferably, the said solvent is selected from methanol, acetone and aqueous hydrochloric acid. Preferably, the said step (viii) is conducted in the presence of a reducing agent such as iron filings. Preferably, in the said reagent of formula (IX) Y"'-Ri, Y'" is chlorine or bromine and Ri is -S0₂-R5-Ar or -R₅-Ar.

Advantageously, said compounds of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one of formula (I) are comprised in a pharmaceutical form. The pharmaceutical form according to the present invention comprises the above-mentioned compounds of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one of formula (I) and at least one pharmaceutically acceptable excipient.

In the present description and the claims that follow, the term "excipient" means any agent known in the art that is suitable for the preparation of a pharmaceutical form. The excipients suitable for use in the present invention are known in the art, such as, for example, anti-adherents, binders, disintegrants, fillers, diluents, colorants, fluidifiers, glidants, lubrificants, preservatives, stabilisers, humectants, absorbents, solvents, surfactants, buffers, salts for regulating osmotic pressure, emulsifiers, flavourings and sweeteners. The said pharmaceutical form may be prepared in the form of a unit of dosage according to methods known in the art.

Advantageously, the said pharmaceutical form is in a form suitable for oral administration, for example tablet, capsule, coated tablet, pellet, pill, syrup, solution for oral use; for topical or transdermal administration, for example medicated plaster, solution, paste, cream, ointment; suppository for rectal administration; sterile solution for aerosol administration; for injectable administration, for example solution, suspension or aqueous emulsion, in the form of a powder to be reconstituted for the preparation of a solution, a suspension or an aqueous emulsion for endovenous, intramuscular, subcutaneous, transdermal or intraperitoneal administration. The said pharmaceutical form comprises a pharmaceutically effective amount of at least one compound of formula (I) according to the present invention.

The amount of said compounds of formula (I) may be determined by those who are experts in the field on the basis of the characteristics of the patient to be treated, for example age, weight, seriousness of illness, and so on. The following examples are intended to illustrate the present invention but without limiting it in any way. EXPERIMENTAL PART

Synthesis of compounds of formula (I) General synthesis

The synthesis of the compounds of formula (I) according to the present invention was preferably carried out as described in the following general synthesis.

(a) (b)

The hydroxybenzoic derivative (a), wherein X was -0- or -NH-, was dissolved in a suitable solvent, for example methanol, in a flask held over a flame and placed under a nitrogen atmosphere. The mixture was subjected to agitation and cooled to 0°C with an ice bath. Thionyl chloride was added drop by drop. After 30 minutes, the mixture was placed under reflux and left under agitation. The mixture was dried at reduced pressure, recovered with ethyl acetate and washed 3 times with aqueous NaHC0₃, 3 times with water and further 3 times with aqueous NaCI. The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure, obtaining the intermediate (b).

(b) (c)

The intermediate (b) was placed in a microwave reactor test-tube together with a 33% aqueous ammonia solution. The mixture was irradiated with microwaves (250 W) at 120°C for a certain period of time, and if necessary the pH was corrected with an appropriate acid or base. The mixture was then extracted 3 times with a suitable solvent and the collected organic phases were washed 2 times with aqueous NaCI, dried with Na₂S0₄ and evaporated at reduced pressure, obtaining the intermediate (c).

(C) (d)

The intermediate (c) was dissolved in a suitable chetonic solvent in a flask held over a flame and placed under a nitrogen atmosphere. A suitable reagent of formula (II) was then added

(II)

wherein R₃ and R₄ are as defined earlier and R₇ and R₈, being identical, are each a group -OCH3 or together with the carbon atom form a group -C(=0).

The mixture was left under agitation at room temperature for 10 hours. A suitable aqueous solvent was added and the organic phase was extracted with a suitable solvent. The product (d) obtained as a solid was appropriately purified.

Product (d) was then caused to react under suitable reaction conditions with one of the following reagents: a compound of general formula Br- R₅-Ar or Br-R₆ - a compound of formula Cl-S0₂-Rs-Ar or CI-SO₂-R6 a suitable protective group for the substituent R-i.

Products (e.1 ) and (e.2) thus obtained were subsequently caused to react with a suitable reagent of formula Br-R₂, obtaining the compounds of 2,3-dihydro-4H- 1 ,3-benzoxazin-4-one of formula (I) according to the present invention. The intermediate (g) was caused to react with a suitable reagent of formula Br-R₂, obtaining respectively the intermediate (h).

The intermediate (h) was then suitably treated to remove the protective group P, obtaining the product (i), which was then in due course made to react under suitable reaction conditions with a reagent selected from Br-R₅-Ar and Cl-S0₂-Rs-Ar to obtain the derivatives of 2,3-dihydro-4H-1 ,3-benzoxazin-4-one of formula (I) according to the present invention. The compounds listed in the following Table 1 were prepared as described in detail below.

4-one In the examples that follow, the analytical characterisations of the compounds were carried out with an HPLC-MS system (Gilson-ThermoFinnigan) using the Synergy Polar RP (150 x 4.6; Phenomenex) or ODS3 (150 x 4.6; GL Science) column.

Product 1

(X-R1 = -OH; R? = -H; Ra + R₄ = cyclohexyl)

(i) (ii)

The 2,4-dihydroxybenzoic acid (i) (20.0 g, 130 mmol) was dissolved in methanol (100 mL) in a 250 mL flask, held over a flame and placed under a nitrogen atmosphere. The mixture was subjected to agitation and cooled to 0°C with an ice bath. Thionyl chloride (10.0 mL, 137 mmol) was added drop by drop. After 30 minutes, the mixture was placed under reflux (65°C) and left under agitation for 4 hours. The mixture was dried at reduced pressure, recovered with ethyl acetate (100 mL) and washed with aqueous NaHCO₃ (3 x 100 mL), water (3 x 100 mL) and aqueous NaCI (3 x 100 mL). The organic phase was dried with Na₂SO₄ and evaporated under reduced pressure, obtaining methyl 2,4-dihydroxybenzoate (ii) (19.3 g, yield 88%).

LC-UV purity: 98.01 % (λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(dmso-d6) δ: 10.73 (s, 1 H), 10.49 (s, 1 H), 7,64 (dd, J=3.2,

J=8.6, 1 H), 6.39 (d, J=8.6, 1 H), 6.32 (d, J=3.2, 1 H), 3.85 (s, 3H).

microwave 250W

120°C

30 min.

(ii) (iii)

The compound (ii) (19.3 g, 1 15 mmol) was placed in a microwave reactor test-tube together with a 33% aqueous ammonia solution (20 mL). The mixture was irradiated with microwaves (250 W) at 120°C for 30 minutes, then acidified with aqueous 1 N HCI and extracted with ethyl acetate (3 x 100 mL). The collected organic phases were washed with aqueous NaCI (2 x 100 mL), dried with Na₂S0₄ and evaporated under reduced pressure, obtaining 2,4-dihydroxybenzamide (iii) (10.6 g, yield 60%).

LC-UV purity: 90.44%(λ= 220 nm), 89% (λ= 254 nm)

(iii) (1 )

The 2,4-dihydroxybenzamide (iii) (2.00 g, 13.1 mmol) was dissolved in cyclohexanone (15 mL) in a 50 mL flask held over a flame and placed under a nitrogen atmosphere. 1 , 1 -dimethoxycyclohexane (3.00 mL, 19.6 mmol) and HCI 1 .25M in MeOH (0.4 mL, 0.5 mmol) were then added. The mixture was left under agitation at room temperature for 10 hours. Aqueous NH₄CI (30 mL) was then added, and the mixture was extracted with ethyl acetate (3 times with 30 mL). The collected organic phases were washed with aqueous NaCI (3 times with 20 mL), dried with Na₂S0₄ and evaporated under reduced pressure, obtaining product (1 ) as a white solid (2.28 g, yield: 75%).

LC-MS: 234.7(M+1 ); 468,22 (2M+1 )

LC-UV purity: >99% (λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(dmso-d6) δ: 10,23 (s, 1 H), 8.28 (s, 1 H), 7.54 (d, J=7.5 Hz, 1 H),

6.46 (d, J=7.5 Hz, 1 H), 6.29 (s, 1 H), 1.94 (br s, 2H), 1 .54 (br s, 7H), 1 .22 (br s, 1 H).

Product 2

(X-R1 = -OH: R? = -H: R_a = methyl: R₄ = methyl)

(iii) (2)

The 2,4-dihydroxybenzamide (iii) (5.00 g, 32.7 mmol) obtained as described earlier for the preparation of product (1 ) was dissolved in 40 mL of acetone together with 2,2-dimethoxypropane (40.0 mL, 325 mmol) and paratoluenesulphonic acid (catalytic), in a 250 mL flask held over a flame and placed under a nitrogen atmosphere. The mixture was left under agitation at room temperature for 10 hours, after which it was filtered in a Buchner funnel. The solid obtained was washed liberally with water and dried in a stove at 40°C under reduced pressure, obtaining product (2) as a white solid (3.80 g, yield: 61 %). LC-MS: 194.6 (M+1 )

LC-UV purity: >99%(λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(dmso d6) δ; 8.29 (s, 1 H), 7.56 (d, J= 8.6, 1 H), 6.46 (dd, J=2.2, J=8.6, 1 H), 6.26 (d, J=2.2, 1 H), 1 .49 (s, 6H).

Product 3 (X-R1 = -OH: R? = -H: R¾ = ethyl: R₄ = ethyl

(iii) (3)

The 2,4-dihydroxybenzamide (iii) (3.50 g, 22.8 mmol) obtained as described earlier for the preparation of product (1 ) was dissolved in 3-pentanone (30 mL) in a 100 mL flask held over a flame and placed under a nitrogen atmosphere. 1 .25M HCI in methanol (2.0 mL, 2.5 mmol) was added. The mixture was left under agitation at room temperature for 10 hours. Aqueous NH₄CI (30 mL) was then added, and the mixture was extracted with ethyl acetate (3 times with 30 mL). The collected organic phases were washed with aqueous NaCI (3 times with 20 mL), dried with Na₂SO₄ and evaporated under reduced pressure. The solid obtained was triturated in acetone and filtered, obtaining product (3) as a white solid (1 .4 g, yield: 75%).

LC-UV purity: >99%(λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(CDCI₃) δ 7.82 (d, J= 8.2, 1 H), 6.52 (d, J=8.2, 1 H), 6.4 (s, 1 H), 1 .89 (m, 4H), 0.99 (t, J=7.6, 6H). Product 4

(X-R1 = -O-SO?-(thiophene-2-yl); R? = -H: R¾ + R₄ = cyclohexyl)

(1 ) (4)

100 mg of product (1 ) (0.429 mmol) was dissolved in anhydrous DMF (2 mL) in a 5 mL Schlenk tube, held over a flame and placed under a nitrogen atmosphere. Potassium carbonate (1 18 mg, 0.643 mmol) was added, and the mixture was left under agitation for 15 minutes. 2-thiophene-sulphonyl chloride (1 17 mg, 0.643 mmol) was then added, and the mixture was left under agitation for 10 hours. Aqueous Na₂C0₃ (3 mL) was added, and the mixture was extracted with ethyl acetate (3 times with 5 mL). The organic phases were collected and washed with water (3 times with 10 mL) and aqueous NaCI (3 times with 10 mL). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure. The residue was triturated with a mixture of CH₃CN/DMF in a 1 : 1 ratio, obtaining product (4) as a white solid (55 mg, yield 34%).

Product 5

(X-R1 = -0-SO?-(thiophene-2-yl); R? = benzyl; R¾ + R₄ = cvclohexyl)

(4) (5)

46.0 mg (0.121 mmol) of product (4) were dissolved in anhydrous DMF (2 mL) in a Schlenk tube, held over a flame and placed under a nitrogen atmosphere. The mixture, placed under agitation, was cooled to 0°C with an ice bath, and then sodium hydride (3.20 mg, 0.133 mmol) and benzyl bromide (17.3 μί, 0.145 mmol) were added. The mixture was left under agitation at room temperature for 10 hours, acidified with aqueous HCI 1 N and extracted with ethyl acetate (3 times with 5 mL). The collected organic phases were washed with aqueous NaCI (3 times with 5 mL), dried with Na₂S0₄ and evaporated under reduced pressure. The obtained residue was purified with preparative HPLC using as eluent CH₃CN in water, in a gradient from 10% to 80%, obtaining product (5) as a waxy solid (45 mg, yield: 79%). Product 6

(X-R1 = -0-SO?-i(1 -methyl)-pyrazol-3-vH; R? = -H; R¾ + R₄ = cyclohexyl)

(1 ) (6)

Product (1 ) (100 mg, 0.430 mmol) was dissolved in anhydrous DMF (2 mL) in a Schlenk tube of 5 mL, held over a flame and placed under a nitrogen atmosphere. Potassium carbonate was added (89 mg, 0.64 mmol) and the mixture was left under agitation for 15 minutes. 1 -methyl-1 H-pyrazol-3-sulphonylchloride (1 16 mg, 0.860 mmol) was added and the mixture was left under agitation for 10 hours.

Aqueous Na₂C0₃ was added (3 mL) and the mixture was extracted with ethyl acetate (3 times with 5 mL). The collected organic phases were washed with water (3 times with 10 mL) and aqueous NaCI (3 times with 10 mL). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure to obtain the raw product which was purified with preparative HPLC (ACN/H₂0 20/95, 1 % trifluoroacetic acid -TFA). The fractions were then collected and lyophilized to obtain product (6) as a trifluoroacetate salt.

LC-MS:379.0 (M+1 ) 433.1 (M+23+32); Purity LC-UV: >99%(λ= 220 nm), 92% (λ= 254 nm) Product 7

(X-R1 = -0-SO?-r(1 -methyl)-pyrazol-3-yl1; R? = (4-cyano)-benzyl; R¾ + R₄ = cyclohexyl)

(6) (7) Product (6) (59.0 mg, 0.121 mmol) was dissolved in anhydrous DMF (2 ml_) in a Schlenk tube held over a flame and placed under a nitrogen atmosphere. The mixture, placed under agitation, was cooled to 0°C with an ice bath, and then sodium hydride (6.1 mg, 0.25 mmol) and 4-(bromomethyl)benzonitrile (28.0 mg, 0.145 mmol) were added. The mixture was left under agitation at room temperature for 10 hours. A saturated aqueous solution of NH₄CI was added, and the mixture was extracted with ethyl acetate (3 times with 5 ml_). The collected organic phases were washed with aqueous NaCI (3 times with 5 ml_), dried with Na₂S0₄ and evaporated under reduced pressure. The residue obtained was purified using preparative HPLC (eluent CH₃CN from 10% to 80% in H₂0, TFA 1 %), obtaining product (7), in the form of a TFA salt, as a waxy solid (20 mg, yield: 33%).

LC-UV purity: >99%(λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(CDCI₃) δ: 7.92 (d, J=8.6 Hz, 1 H), 7.63 (d, J=7.6 Hz, 2H), 7.53 (s, 1 H), 7.41 (d, J=8.2 Hz, 2H), 6.86-6.68 (m, 3H), 4.87 (s, 2H), 4.04 (s, 3H), 2.10 (d, J=1 1 .2, 2H), 1 .80-1 .47 (m, 7H), 1 .25-1 .10 (m, 1 H).

Product 8

(X-R1 = -0-SO?-i(1 -methyl)-pyrazole-3-vH; R? = -CH?CN; R* + R₄ = cvclohexyl)

(6) (8)

Product (6) (37.3 mg, 0.0988 mmol) was dissolved in anhydrous DMF (2 ml_) in a Schlenk tube held over a flame and placed under a nitrogen atmosphere. The mixture, placed under agitation, was cooled to 0°C with an ice bath, and sodium hydride (6.6 mg, 0.27 mmol) and 2-bromoacetonitrile (14 mg, 0.12 mmol) were added. The mixture was left under agitation at room temperature for 10 hours. A saturated aqueous solution of NH₄CI was added, and the mixture was extracted with ethyl acetate (3 times with 5 ml_). The collected organic phases were washed with a saturated aqueous solution of NaCI (3 times with 5 ml_), dried with Na₂S0₄ and evaporated under reduced pressure. Product (8) was thus obtained as a white solid (20 mg; yield: 49%). Product 9

(X-R1 = -0-SO?-r4-bromo-phenyl1; R? = -H; R¾ + R₄ = cyclohexyl)

(1 ) (9)

Product (9) was obtained by means of a procedure similar to that described for product (6), using 4-bromo-phenyl-sulphonyl chloride instead of 1 -methyl-1 H-pyrazole-3-sulphonyl chloride.

LC-MS: 453.0 (M+1 )

LC-UV purity: >99% (λ= 220 nm), >99% (λ= 254 nm) ¹ H-NMR-(CDI3) 6: 7.92 (d, J=8.6, 1 H), 7.72 (s, 4H), 7.53 (s, 1 H), 6.75 (m, 2H), 4.56 (s, 2H), 2.26 (d, J=12.9, 2H), 1 .92-1 ,58 (m, 7H), 1 .40-1 ,20 (m, 1 H).

Product 10

(X-R1 = -O-CHg-naphthyl: R? = -H; R_a + R₄ = cyclohexyl)

(1 ) (10)

Compound (1 ) (100 mg, 0.429 mmol) was dissolved in anhydrous DMF (2 mL) in a 5 mL Schlenk tube held over a flame and placed under a nitrogen atmosphere. Potassium carbonate (1 18 mg, 0.643 mmol) was added, and the mixture was left under agitation for 15 minutes. 2-(bromomethyl)naphthalene (1 14 mg, 0,515 mmol) was then added, and the mixture was left under agitation for 10 hours. Aqueous NH₄CI (3 mL) was added, and the mixture was extracted with ethyl acetate (3 times with 5 mL). The collected organic phases were washed with water (3 times with 10 mL) and aqueous NaCI (3 times with 10 mL). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure. Thus, product (10) was obtained as a white solid (150 mg, yield 93%).

LC-UV purity: >99%(λ= 220 nm), >99% (λ= 254 nm) ¹H-NMR-(CDCI3) δ: 7.96-7.78 (m, 5H), 7.58-7.46 (m, 3H), 6.74 (d, J=7.6, 1 H), 6.59 (s, 1 H), 6.14 (s, 1 H), 5.26 (s 1 H), 2.1 1 (d, J= 1 1 .4, 2H), 1 .81 -1 .33 (m, 8H).

Product 1 1

(X-R1 = -OH; R? = benzyl; R¾ + R₄ = cyclohexyl

(1 ) (iv)

3.00 g of compound (1 ) (12.9 mmol) were dissolved in anhydrous DMF (10 mL) in a 50 mL flask held over a flame and placed under a nitrogen atmosphere. Potassium carbonate (5.33 g, 38.6 mmol) was then added, and after cooling the mixture to 0°C with an ice bath, chloromethoxymethane (MOMCI - 1 .95 mL, 25.7 mmol) was added drop by drop. The mixture was then left under agitation for 10 hours at room temperature, after which it was poured into a saturated aqueous solution of sodium carbonate (20 mL) and ice. The mixture was extracted with ethyl acetate (3 times with 30 mL). The organic phases were collected and washed with water (3 times with 20 mL) and aqueous NaCI (3 times with 20 mL), dried with Na₂SO₄ and evaporated under reduced pressure, obtaining the intermediate (iv) as a pink solid (3.01 g, yield: 84%) in which the hydroxyl group in position 1 is protected with the methoxymethoxy group (MOMO).

LC-UV purity: >99%(λ= 220 nm), >99% (λ= 254 nm) ¹H-NMR-(dmso-d6) δ: 8.45 (s, 1 H), 7.65 (d, J=7.6 Hz, 1 H), 6.70 (d, J=7.6 Hz, 1 H), 6.60 (s, 1 H), 5.24 (s, 2H), 3.39 (s, 3H), 1 .97 (s, 2H), 1 .57 (s, 7H), 1.24 (s, 1 H).

^{DMF "} " ^'* ο '^'" ··^:· ^'" ¾" ^' Ί

(iv) (v)

3.01 g (10.8 mmol) of intermediate (iv) were dissolved in anhydrous DMF (100 mL) in a 250 mL flask held over a flame and placed under a nitrogen atmosphere. The mixture, placed under agitation, was cooled to 0°C with an ice bath, and then sodium hydride (521 mg, 21 .7 mmol) and benzyl bromide (1 .93 mL, 16.3 mmol) were added. The mixture was left under agitation at room temperature for 10 hours, then acidified with aqueous 1 N HCI and extracted with ethyl acetate (3 times with 20 mL). The organic phases were collected and washed with aqueous NaCI (5 times with 20 mL), dried with Na₂S0₄ and evaporated under reduced pressure. The residue was purified with a silica column, using as an eluent an ether mixture of petroleum/ethyl acetate at a ratio of 9: 1 , obtaining the intermediate (v) as a white solid (3.90 g, yield: 98%).

LC-UV purity: >99%(λ= 220 nm), >99% (λ= 254 nm) ¹H-NMR-(dmso-d6) δ: 7.78 (d, J=7.6 Hz, 1 H), 7.40-7.20 (m, 5H), 6.79 (d, J=7.6 Hz, 1 H), 6.70 (s, 1 H), 5.27 (s, 2H), 4.80 (s, 2H), 3.41 (s, 3H), 2.06- 1 .88 (m, 2H), 180-1 .5 (m, 7H), 1 .28-1 .10 (m, 1 H).

(v) (1 1 )

4.00 g (10.89 mmol) of intermediate (v) were dissolved in methanol (50 mL) and aqueous 6M HCI (4 mL). The mixture was left under agitation at room temperature for 10 hours. The mixture was then concentrated under reduced pressure, recovered with ethyl acetate (100 mL) and washed with aqueous NaCI (3 times with 100 mL). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure, obtaining product (1 1 ) as a white solid (3.0 g, yield 85%).

LC-UV purity: >99%(λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(dmso-d6) δ: 7.78 (d, J=7.6 Hz, 1 H), 7.40-7.20 (m, 5H), 6.79 (d, J=7.6 Hz, 1 H), 6.70 (s, 1 H), 5.27 (s, 2H), 4.80 (s, 2H), 3.41 (s, 3H), 2.06-1 .88 (m, 2H), 1 .80-1.5 (m, 7H), 1 .28-1 .10 (m, 1 H).

Product 12

(X-R1 = -0-SO?-3-cvano)-phenyl; R? = benzyl: R₃ + R₄ = cvclohexyl)

(1 1 ) (12)

Product (1 1 ) (70 mg, 0.22 mmol) was dissolved in anhydrous DMF (2 mL) in a 5 mL Schlenk tube held over a flame and placed under a nitrogen atmosphere. Potassium carbonate (90 mg, 0.65 mmol) was added, and the mixture was left under agitation for 15 minutes. 3-cyanobenzene-1 -sulphonyl chloride (87 mg, 0.43 mmol) was then added, and the mixture was left under agitation for 10 hours. After adding aqueous Na₂C0₃ (3 mL), the mixture was extracted with ethyl acetate (3 times with 5 mL) and the organic phases were collected and washed with water (3 times with 10 mL) and aqueous NaCI (3 times with 10 mL). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure. The residue obtained was purified by means of semi-preparative HPLC using as an eluent CH₃CN in water, in a gradient from 10% to 80%, obtaining product (12) as a yellow oil (5.6 mg, yield: 5%).

Product 13 (X-R1 = -0-SO?-(4=methyl-phenyl); R? = (4-fluoro)-benzyl; R¾ = methyl: R₄ = methyl)

(2) (vi)

1 .50 g of product (2) (7.76 mmol) were dissolved in anhydrous DMF (30 mL) in a 100 mL flask held over a flame and placed under a nitrogen atmosphere. Potassium carbonate (1 .18 g, 8.54 mmol) and tosyl chloride (TsCI - 1 .63 g, 8.54 mmol) were then added, and the mixture was left under agitation for 10 hours. The mixture was then concentrated under reduced pressure, recovered with DCM (100 mL) and washed with aqueous NaHC0₃ (3 times with 100 mL), water (3 times with 100 mL) and aqueous NaCI (3 times with 100 mL). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure, obtaining the intermediate (vi) as a white solid (2.18 g, yield 81 %).

(vi) (13)

50.0 mg (0.144 mmol) of intermediate (vi) were dissolved in anhydrous DMF (2.5 mL) in a Schlenk tube held over a flame and placed under a nitrogen atmosphere. The mixture, placed under agitation, was cooled to 0°C with an ice bath, and sodium hydride (3.80 mg, 0.158 mmol) and p-fluoro-benzyl bromide (32.6 mg, 0.173 mmol) were added. The mixture was left under agitation at room temperature for 10 hours, and then acidified with aqueous 1 N HCI (5 mL) and extracted with ethyl acetate (3 times with 10 mL). The collected organic phases were washed with aqueous NaCI (5 times with 10 mL), dried with Na₂S0₄ and evaporated under reduced pressure, obtaining product (13) as a colourless oil (22.6 mg, yield: 35%).

Product 14

(X-R1 = -0-SO?-r(4-methoxy)-phenyl1; R? = benzyl; R¾ + R₄ = cvclohexyl)

(1 1 ) (14)

Product (1 1 ) (70 mg, 0.22 mmol) was dissolved in anhydrous DMF (2 mL) in a 5 mL Schlenk tube held over a flame and placed under a nitrogen atmosphere. Potassium carbonate (61 mg, 0.44 mmol) was added and the mixture was left under agitation for 15 minutes. P-methoxybenzene-sulphonyl chloride (91 mg, 0.44 mmol) was then added and the mixture was left under agitation for 10 hours. A saturated solution of aqueous NH₄CI (3 mL) was added and the mixture was extracted with ethyl acetate (3 times with 5 mL). The organic phases were collected and washed with water (3 times with 10 mL) and aqueous NaCI (3 times with 10 mL). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure. The residue obtained was purified by means of semi-preparative HPLC (eluent CH₃CN 10 to 80% in H₂0), obtaining product (14) as a yellow solid (10.7 mg). LC-MS: 516 (M+23).

LC-UV purity: >99%(λ= 220 nm), 93% (λ= 254 nm)

¹H-NMR-(dmso-d6) δ: 7,84(d, J=8.6 Hz, 1 H), 7.80 (d, J=9.1 Hz, 2H), 7.36-7.20 (m, 5H), 7.17 (d, J=9, 1 Hz, 2H) 6.87 (dd, J=8.6 Hz, J=2.2 Hz, 1 H), 6.72 (d, J=2.2Hz, IH), 4.79(s, 2H), 3.86 (s, 3H), 1 .84 (d, J=12.9 Hz, 2H), 1 .68 (t, J₁=J₂=12.9 Hz, 2H), 1 .60-1 .32 (m, 5H), 1 .20-1 .05 (m, 1 H).

Product 15

(X-R1 = -0-SO?-r(3-methoxy)-phenyl1; R? = benzyl; R_a + R₄ = cyclohexyl)

Product (15) was obtained from product (1 1 ) by following a procedure similar to that described for product (14), using (3-methoxy)-benzene-sulphonyl chloride instead of p-methoxybenzene-sulphonyl chloride.

Product 16

(X-R1 = -Q-SO?-f(3-methoxy)-phenyll: R? = (4-cvano)butyl; R¾ + R₄ = cyclohexyl)

Product (16) was obtained by causing product (1 1 ) to react by following a procedure similar to that described for product (6), using (3-methoxy)-benzene-sulphonyl chloride instead of 1 -methyl-1 H-pyrazolyl-3-sulphonyl chloride and then following a procedure similar to that described for product (8), using 1 -bromo-4-cyano-n-butyl instead of 2-bromoacetonitrile.

Product 17 (X-R1 = -0-SO?-r(2-nitro)-phenyll; R? = -H; R¾ + R₄ = cyclohexyl)

Product 17 was obtained from product (1 ) by following a procedure similar to that described for product (4), using 2-nitro-phenyl-sulphonyl chloride instead of 2-thiophene-sulphonyl chloride.

Product 18 (X-R1 = -0-SO?-r(4-cvano)-phenyl1; R? = benzyl; R¾ + R₄ = cyclohexyl)

Product (18) was obtained from product (12), using a procedure similar to that described for product (14), using (4-cyano)-benzenesulphonyl chloride instead of p-methoxybenzene-sulphonyl chloride.

Product 19 (X-R1 = -OH; R? = H; R¾ + R₄ = tetrahydropyran-4-yl)

Product (19) was obtained by following a procedure similar to that described for product (1 ), using 4,4-dimethoxy-tetrahydropyran instead of 1 , 1 -dimethoxycyclohexane.

Product 20

(X-R1 = -OH; R? = benzyl; R¾ + R₄ = tetrahydropyran-4-yl)

Product (20) was obtained from product (19) by following a procedure similar to that described for product (1 1 ), using the same reagent benzyl bromide.

Product 21

(X-R1 = -O-SO?-r(3-nitro)-phenyll; R? = benzyl; R¾ + R₄ = tetrahvdropyran-4-yl)

Product (21 ) was obtained from product (20) by following a procedure similar to that described for product (12), using (3-nitro)-phenyl-sulphonyl chloride instead of (3-cyano)-benzene-1 -sulphonyl chloride.

Product 22

(X-R1 = -O-SO?-[(3-methoxy)-phenyl1; R? = benzyl; R¾ + R₄ = tetrahydropyran-4-yl)

Product (22) was obtained from product (20) by following a procedure similar to that described for product (12), using 3-methoxy-phenyl-sulphonyl chloride instead of (3-cyano)-benzene-1 -sulphony chloride.

Product 23

(X-R1 = -O-SO?-[(4-methoxy)-phenyl1; R? = benzyl; R¾ + R₄ = tetrahydropyran-4-yl)

Product (23) was obtained from product (20) by following a procedure similar to that described for product (12), using 4-nitro-phenyl-sulphonyl chloride instead of (3-cyano)-benzene-1 -sulphonyl chloride.

Product 24

(X-R1 = -OH; R? = H; R¾ + R₄ = (N-acetyl)-piperidine-4-yl)

Product (24) was obtained by following a procedure similar to that described for product (1 ), using 4,4-dimethoxy-N-acetyl-piperidine instead of 1 , 1 -dimethoxycyclohexane.

Product 25 (X-R1 = -OH; R? = benzyl; R¾ + R₄ = (N-acetyl)-piperidine-4-yl)

Product (25) was obtained from product (24) by following a procedure similar to that described for product (1 1 ), using benzyl bromide.

Product 26 (X-R1 = -O-SO?-benzyl; R? = benzyl; R¾ + R₄ = (N-acetyl)-piperidine-4-yl)

Product (26) was obtained from product (25) by following a procedure similar to that described for product (12), using benzyl-sulphonyl chloride instead of (3-cyano)-benzene-1 -sulphonyl chloride.

Product 27 (X-R1 = -O-SQg-naphthyl: R? = methyl; R¾ = methyl; R₄ = methyl)

Product (27) was obtained from product (2) by following a procedure similar to that described for product (13), using naphthyl-sulphonyl chloride instead of 4-methyl-phenyl-sulphonyl chloride and methyl bromide instead of 4-fluoro-benzyl bromide. Product 28

(X-R1 = -O-CH?-[(4-trifluoromethoxy)-phenyl1; R? = n-propyl; R¾ = methyl; R₄ = methyl)

Product (28) was obtained from product (2) by following a procedure similar to that described for product (10), using (4-trifluoromethoxy)-(bromomethyl)-benzene instead of 2-bromomethyl-naphthene and then following a procedure similar to that described for product (8), using n-propyl-bromide instead of bromoacetonitrile.

Product 29

(X-R1 = -O-SO?-r(4-methyl)-phenyl1; R? = benzyl; R¾ = methyl; R₄ = methyl)

Product (29) was obtained from product (2) by following a procedure similar to that described for product (1 1 ), using benzyl bromide instead of p-fluorobenzyl bromide and (4-methyl)-phenyl-sulphonyl chloride instead of 4-methylphenyl-sulphonyl chloride.

Product 30

(X-R1 = -O-CH_?-C(=O)-r(4-methoxy)-phenyll; R? = (4-fluoro)-benzyl; R¾ = methyl; R₄ =

methyl Product (30) was obtained from product (2) by following a procedure similar to that described for product (10), using 2-bromo-1 -(4-methoxy-phenyl)-ethanone instead of 2-bromomethyl-naphthene and then a procedure similar to that described for product (8), using (4-fluoro)-benzyl bromide instead of bromoacetonitrile. Product 31

(X-R1 = -0-CH?-C(=Q)-benzothiophene-2-yl; R? = (4-isopropyl)-benzyl; R¾ = methyl;

R₄ = methyl)

Product (31 ) was obtained from product (2) by using a procedure similar to that described for product (10), using 2-bromo-1 -(benzothiophene-2-yl)-ethanone instead of 2-bromomethyl-naphthene and then a procedure similar to that described for product (8), using (4-isopropyl)-benzyl bromide instead of bromoacetonitrile.

Product 32

(X-R1 = -0-CH?-r(4-phenyl)-phenyll; R? = (4-fluoro)-benzyl; R¾ = methyl; R₄ = methyl)

Product (32) was obtained from product (2) by using a procedure similar to that described for product (10), using 4-phenyl-benzyl bromide instead of 2-bromomethyl-naphthene, and then a procedure similar to that described for product (8), using (4-fluoro)-benzyl bromide instead of bromoacetonitrile.

Product 33

(X-R1 = -NH?; R? = -H; R* + R₄ = cvclohexyl)

(X) (Xi)

4-amino-2-hydroxybenzoic acid (x) (35.0 g, 229 mmol) was dissolved in methanol (250 ml_) in a 250 ml_ flask held over a flame and placed under a nitrogen atmosphere. The mixture was placed under agitation and cooled to 0°C with an ice bath. Thionyl chloride (SOCI2 - 50.0 ml_, 687 mmol) was added drop by drop. After 30 minutes, the mixture was placed under reflux (65°C) and left under agitation for 10 hours. The mixture was then dried under reduced pressure, recovered with ethyl acetate (100 ml_) and washed with aqueous NaHC0₃ (3 x 100 ml_), water (3 x 100 ml_) and aqueous NaCI (3 x 100 ml_). The organic phase was dried with Na₂S0₄ and evaporated under reduced pressure, obtaining the intermediate 4-amino-2-methyl hydroxybenzoate (xi) (10.0 g, yield 26%).

LC-UV purity: >99% (λ= 220 nm), 93% (λ= 254 nm)

¹H-NMR-(dmso-d6) 6: 10.79 (s, 1 H), 7.45 (d, J=8.6 Hz, 1 H), 6.16-6.10 (m, 3H) 6.01 (s, 1 H), 3.79 (s, 3H).

(xi) (xii)

Intermediate (xi) (10.0 g, 59.8 mmol) was placed in a microwave reactor test-tube together with a 33% aqueous ammonia solution (50 ml_). The mixture was irradiated with microwaves (250 W) at 120°C for 2 hours. The mixture was then poured on to ice and brought to a pH comprised between 7 and 8 with aqueous HCl 37%, maintaining the temperature between 0 and 5°C. The mixture was extracted with ethyl acetate (3 x 100 ml_). The collected organic phases were washed with aqueous NaCI (2 x 100 ml_), dried with Na₂S0₄ and evaporated under reduced pressure, obtaining the intermediate 4-amino-2-hydroxybenzamide (xii) as a red solid (6.82 g, yield 75%).

LC-UV purity: >99% (λ= 220 nm), 93% (λ= 254 nm)

¹H-NMR-(dmso-d6) 6: 13.26 (s, 1 H), 7.47 (d, J=8.6 Hz, 1 H), 6.03 (dd, J=8.6 Hz, J=2.2 Hz, 1 H), 5.93 (d, J=2.2Hz, 1 H).

(Xii) (33)

Intermediate (xii) (1.00 g, 6.57 mmol) was dissolved in cyclohexanone (10 ml_) in a 50 ml_ flask held over a flame and placed under a nitrogen atmosphere. 1 , 1 -dimethoxycyclohexane (1 .5 ml_, 9.8 mmol) and 1 .25M HCl in methanol (0.7 ml_) were then added. The mixture was irradiated with microwaves (250W) for 60 minutes (T=80°C, P=22bar). The mixture was subjected to evaporation under reduced pressure, and the crude product was triturated in di-isopropyl ether, filtered and triturated in acetone. The filtered crude product was purified by chromatography on S1O2 (eluent: ethyl acetate/petroleum ether 9: 1 ), obtaining product (33) as a beige solid (1 .0 g, yield: 65.5%).

LC-MS: 233.34(M+1 ) LC-UV purity: >99% (λ= 220 nm), 90% (λ= 254 nm)

¹H-NMR-(dmso-d6) 6: 8.01 (s, 1 H), 7.35 (d, J=8.1 Hz, 1 H), 6.20 (d, J=8.1 Hz, 1 H), 6.02 (s, 1 H), 5.86 (s, 1 H), 2.00-1.85 (s, 2H), 1 .53 (bs, 7H), 1 .23 (s, IH).

Product 34

(X-R1 = -NH?: R? = benzyl: R_a + R₄ = cyclohexyl)

(33) (xiii)

Product (33) (200 mg, 0.861 mmol), triphenylchloromethane (240 mg, 0.861 mmol) and triethylamine (0.12 ml_, 0.086 mmol) were dissolved in 10 mL of anhydrous DCM in a 50 mL flask held over a flame and placed under a nitrogen atmosphere. The mixture was left under agitation for 4 hours, then washed with water. The organic phase was dried with anhydrous Na₂S0₄ and evaporated under reduced pressure, obtaining the crude product, which was purified by RP-HPLC (H₂0/AcCN, HCOOH 0.1 %, Phenomenex GEMINI-NX AXIA column - 150 x 21 .2mm, 5 Dm 1 10 A). The collected fractions were evaporated under reduced pressure to give the intermediate (xiii) as a whitish solid (1 15 mg, yield: 28.1 %).

LC-MS: 476.2 (M+1 )

LC-UV purity: >99% (λ= 220 nm), 90% (λ= 254 nm)

¹H-NMR-(Acetone-d6) δ: 8.1 (s, 1 H), 7.62-7.2 (m, 17H), 6.98 (s, 1 H), 6.34 (d, J= 8.6Hz, 1 H), 6.2 (s, 1 H), 1 .60 (bs, 2H), 1 .48-1 .01 (bs, 8H).

(xiii) (xiv)

In a 100 mL flask, intermediate (xiii) (1 10 mg, 0.232 mmol) was dissolved in DMF (20 mL). The mixture was cooled to 0°C and NaH (13.9 mg, 0.348 mmol) was added. After 30 minutes, benzyl bromide was added slowly. The mixture was left under agitation for 3 hours, then diluted with a saturated aqueous solution of NH₄CI and extracted with ethyl acetate. The organic phases were collected, dried with Na₂S0₄ and evaporated under reduced pressure, obtaining the crude product, which was purified by RP-HPLC (H₂0/ACN, HCOOH 0.1 %, Phenomenex GEMINI-NX AXIA column - 150 x 21 .2mm, 5pm 1 10 A). The collected fractions were evaporated at reduced pressure to give the desired intermediate (xiv) as a white solid (120 mg, yield: 45.8%).

LC-UV purity: >99% (λ= 220 nm), 90% (λ= 254 nm)

¹H-NMR-(CDCI3): 7.58 (d, J=8.6 Hz, 1 H), 7.40-7.2 (m, 20H), 6.20 (dd, Ji=8.6, J₂=2.2Hz, 1 H), 5.71 (d, J=2.2 Hz, 1 H), 5.46 (bs, 1 H), 4.73 (s, 2H), 2.1 -1 .9 (m, 2H), 1 .65-1 .22 (m, 7H), 1 .1 -0.90 (m, 1 H).

(xiv) (34)

In a 50 mL flask, intermediate (xiv) (120 mg, 0.212 mmol) was dissolved in acetone (15 mL) and then cone. HCI (20μΙ) was added. The mixture was left to react under agitation for 3 hours at room temperature. The mixture was basified with aqueous NaHC0₃ and extracted with ethyl acetate . The organic phase was dried with Na₂S04 and evaporated under reduced pressure to give the product (34) as a solid (75 mg).

LC-UV purity: 98% (λ= 220 nm), 85% (λ= 254 nm)

Product 35 (X-R1 = -NH-SO?-(thiophene-3-yl); R? = benzyl; R¾ + R₄ = cyclohexyl)

Product (35) was obtained from product (33) by following a procedure similar to that described for product (13), using 2-thiophenyl-sulphonyl chloride instead of 3-cyanobenzene-1 -sulphonyl chloride.

Product 36

(X-R1 = -NH?; R? = H; R¾ + R₄ = (N-acetyl)-piperidine-4-yl)

(xii) (36)

4-amino-2-hydroxybenzamide (xii) (1 .00 g, 6.57 mmol), obtained as described earlier for product (31 ), was dissolved in toluene (20 mL), together with N-acetyl-4-piperidone (640 μΙ_, 5.20 mmol) and pyrrolidine (50 μΙ_, 0.60 mmol), in a flask fitted with a Dean-Stark apparatus. The mixture was warmed under reflux and agitation for 10 hours. The solvent was then removed under reduced pressure and the residue was triturated with acetone, obtaining 1 .60 g of product (36), which was contaminated by 4-amino-2-methyl hydroxybenzoate at a percentage of approximately 12%.

LC-UV purity: >99% (λ= 220 nm), 93% (λ= 254 nm)

¹H-NMR-(dmso-d6) δ: 8.13 (s, 1 H), 7.38 (d, J=8.1 Hz, 1 H), 6.24 (dd, J=2.2Hz, J=8.1 Hz, 1 H), 6.1 (d, J=2.2, 1 H), 5.93 (bs, 2H), 4.07 (d, J=12.9 Hz. IH), 3.68 (d, J= 12.8 Hz, 1 H), 3.30 (t, J=12.4 Hz, 1 H), 2.98 (t, J=12.4 Hz, 1 H), 2.12-1.91 (m, 5H), 1 .82-1 .52 (m, 2H), 1 .49 (s, 3H).

Product 37

(X-R1 = -NH?; R? = benzyl; R¾ + R₄ = (N-acetyl)-piperidine-4-yl)

(36) (xv) Sodium perborate tetrahydrate (3.39 g, 22.0 mmol) was dissolved in acetic acid (100 mL) in a flask fitted with a cooler and a drip funnel, in which 1 .21 g (4.40 mmol) of product (36) was placed, having previously been dissolved in acetic acid (20 mL). The mixture was heated to 55°C under agitation, and the solution of the product was added drop by drop. After 1 hour, the solvent was removed under reduced pressure and the residue recovered with aqueous 2M HCI (100 mL). The mixture was extracted with ethyl acetate (3 x 100 mL) and the organic phases were collected, washed with aqueous NaCI (2 x 100 mL), dried with Na₂S0₄ and evaporated under reduced pressure, obtaining the desired intermediate (xv) (1 .27 g, yield 95%).

(XV) (xvi)

Intermediate (xv) (1 .27 g, 4.18 mmol) was dissolved in anhydrous DMF (40 mL) in a flask held over a flame and placed under a nitrogen atmosphere. The mixture, placed under agitation, was cooled to 0°C with an ice bath and sodium hydride (200 mg, 8.36 mmol) was added. After 15 minutes, benzyl bromide (1 .09 mL, 9.19 mmol) was slowly added, again at 0°C. The mixture was left under agitation at room temperature for 10 hours and then poured over ice and brought to a pH of between 2 and 3 with 1 M HCI, maintaining the temperature between 0 and 5°C. The mixture was extracted with ethyl acetate (3 x 100 mL). The collected organic phases were washed with aqueous NaCI (2 x 100 mL), dried with Na₂S0₄ and evaporated under reduced pressure. The residue was dried in an oven at 40°C under reduced pressure, and then purified in a silica column, using as an eluent a mixture of DCM in MeOH at a ratio of 99: 1 , obtaining the desired intermediate (xvi) (1.65 g, yield 34%).

LC-UV purity: 98% (λ= 220 nm), 87% (λ= 254 nm) ¹H-NMR-(dmso-d6) 6: 8.18-8.08 (m, 2H), 8.02 (dd, Ji=8.6, J₂=2.2 Hz, 1 H), 7.48-7.18 (m, 5H), 4.85 (d, J=4.9Hz, 2H), 4.30 (d, J=12.9Hz, 1 H), 3.74 (d, J=12.9 Hz, 1 H), 2.9-2.72 (m, 2H), 2.18-1 .80 (m, 7H).

(xvi) (37)

Iron filings (791 mg, 14.2 mmol) and aqueous 37% HCI (0.25 ml_) were dissolved in methanol (30 ml_) in a 100 ml_ flask. The mixture was warmed to 60°C under agitation for 30 minutes, after which the intermediate (xvi) (560 mg, 1 .42 mmol) was added, and the mixture was left under agitation at the same temperature until conversion was complete (approximately 2 hours) and evaluated with TLC. The mixture was filtered through celite to remove the iron, after which the solvent was removed under reduced pressure. The residue was purified by semi-preparative HPLC-UV (eluent CH₃CN from 10% to 80% in water), obtaining product (37) as a white solid (45 mg, yield: 8.7%).

LC-UV purity: >99% (λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(CDCI3) δ: 7.76 (d, J=8.1 Hz, 1 H), 7.27 (s, 5H), 6.37 (d, J=8.6 Hz, 1 H), 6.16 (s, 1 H), 4.76 (dd, J-16.2 Hz, J=31 , 3Hz, 2H), 4.50 (d, J=12.4 Hz, 1 H), 4.29 (s, 1 H), 3.60 (d, J=12.8Hz, 1 H), 3.43-3.20 (m, 1 H), 2.82 (t, Ji=J₂=12.8Hz, 1 H), 2.12 (s, 3H), 1 .82-1 .64 (m, 4H).

Product 38

(X-R1 = -NH-SO?-(benzyl): R? = benzyl: R¾ + R₄ = (N-acetyl)-piperidine-4-yl)

Product (38) was obtained from product (37) by following a procedure similar to that described for product (14), using benzyl-sulphonyl chloride instead of p-methoxybenzene-sulphonyl chloride.

Product 39

(X-R1 = -NH?: R? = H; R¾ + R₄ = tetrahvdropyran-4-yl)

(xii) (39) 4-amino-2-hydroxybenzamide (xii) (2.00 g, 13.16 mmol), obtained as described earlier, dihydro-2-pyran-4(3H)-one (972 μΙ_, 10.53 mmol) and pyrrolidine (87 μΙ_, 1 .05 mmol) were dissolved in toluene (30 ml_) in a flask fitted with a Dean-Stark apparatus. The mixture was warmed under reflux and agitation for 10 hours. The solvent was then removed under reduced pressure and the residue triturated with ethyl ether, obtaining product (39) (1 .70 g; yield 57%).

LC-MS: 235.34 (M+1 )

LC-UV purity: >99% (λ= 220 nm), >99% (λ= 254 nm)

¹H-NMR-(dmso-d6) δ: 13.2 (s, 1 H), 8.29 (s, 1 H), 7.56 (d, 2.2 Hz, 1 H), 6.46 (dd, J=2.2 Hz, J=8.6 Hz, 1 H), 6.26 (d, J=2.2 Hz, 1 H), 1 .49 (s, 3H).

Product 40

(X-R1 = -NH-SO?-r(3-methoxy)phenyll; R? = H; R¾ + R₄ = tetrahvdropyran-4-yl)

Product (40) was obtained from product (39) by following a procedure similar to that described for product (4), using (3-methoxy)phenyl-sulphonyl chloride instead of 2-thiophene-sulphonyl chloride.

Product 41

(X-R1 = -NH-SO?-r(3-methoxy)phenyll; R? = benzyl; R¾ + R₄ = tetrahvdropyran-4-yl)

Product (41 ) was obtained from product (40) by following a procedure similar to that described for product (5), using benzyl bromide.

2 - Pharmacology

A) Determination of capacity for binding with the CB2 receptor

Determination of the capacity for binding with the CB2 receptor was carried out using the "MultiScreenHTS 96-well Plates for binding assays" system on membranes that over-express the CB2 receptor (Millipore™ membranes).

The receptorial affinity was determined by evaluating the capacity of the products of formula (I) to disrupt the specific bond between [³H]CP-55940 (0.8 nM) and the receptor. In all the experiments, the non-specific bond was determined in the presence of unmarked CP-55940 in a concentration of 1 μΜ. CP-55940 is a cannabinoid that mimics the effects of THC, but is 45 times more potent than the natural molecule. It is considered to be a complete agonist for both the CB1 and CB2 receptors, with a Ki of 0.58 nM and 0.68 nM respectively.

The receptorial affinity was expressed as a percentage of residual radioactivity (average values over 3 measurements) of [³H]CP-55940 bound to the CB2 receptor in the presence of analysed compound at 100 nM.

Table 2 below shows some of the products of formula (I) according to the present invention that showed a residual radioactivity (res. rad.) of 70% or less and were therefore subjected to the functional assays described below. Table 2

B) Determination of agonist/antagonist activity of compounds

The agonist or antagonist activity of the compounds of formula (I) was evaluated by studying the G-protein coupling of the CB2 receptors, using the radioligand [ S]GTPyS (at a concentration of 0.6 nM), in the presence and absence of the reference standard CP-55940.

The membranes were made permeable with saponin (1 :1 ratio by weight) and incubated with [³⁵S]-GTPyS in a buffer containing 20 mM HEPES, pH 7.4, 100 nM NaCI, 10 mM MgCI₂ and 10 μΜ GDP (final volume 100 μΙ_), on 96-well plates for 30 minutes at 37°C. The samples were then transferred on to filter plates previously conditioned with water. The wells were washed 3 times with 10 mM of sodium phosphate with a pH of 7.4. The plate was dried and the radioactivity recorded with a β-counter. The specific bond of [³⁵S]-GTPyS to the membranes was determined by pre-incubating the membranes for 20 minutes with cold GTPyS in excess (1 μΜ).

Firstly, the agonist or antagonist profile of the studied compounds at concentrations of 10 μΜ and 100 μΜ (in triplicate for each point) was evaluated, in the presence and absence of the reference standard CP-55940 (60 nM). The compounds itemised in Table 3 were shown to have an agonist activity.

For these compounds, the dose-response curve was determined at 8 different concentrations, from 10^"4 to 10^"12 M, with respect to CB2 and CB1 .

The results are summarised in Table 3 below.

Table 3

The results of Table 3 showed that the products of formula (I) according to the present invention have a selective activity on the CB2 receptor.

Claims

1 . 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds of formula (I):

in which X is -O- or -NH-,

Ar is an aromatic or heteroaromatic ring comprising from 5 to 14 members, optionally substituted with one or more substituents selected from: halogen, -OH, -NH₂, (Ci-6)alkyl, (Ci-6)alkoxy, -CN, nitro, trihalo(Ci-3)alkyl, trihalo(Ci-3)alkoxy, aromatic or heteroaromatic ring comprising 5 or 6 members, benzyl;

R₃ and R₄, identical or different each other, are H, (Ci-6)alkyl, (Ci-6)alkoxy; or R₃ and R₄ together form a carbocycle or a heterocycle having 5 or 6 members, optionally substituted with a (Ci_-6)alkyl, (Ci_-6)alkoxy, -C(=O)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH group; and their salts of addition with pharmaceutically acceptable organic or inorganic acids or bases.

2. The 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds according to claim 1 , in which X is -O-.

3. The 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds according to claim 1 , in which -X-R₁ is -OH, -O-R₆, -O-SO₂-R₆, -O-R₅-Ar or -O-SO₂-R₅-Ar.

4. The 2,3-dihydrc-4H-1 ,3-benzoxazin-4-one compounds according to any one of the preceding claims, in which R₅ is σ bond, (Ci-3)alkyl, (Ci-3)alkyl-0-, -C(=O)-, (Ci_-3)alkyl-C(=0)-, -C(=0)-0-, (Ci_-3)alkyl-C(=0)-0-.

5. The 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds according to any one of the preceding claims, in which R₆ is (Ci-3)alkyl.

6. The 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds according to any one of the preceding claims, in which Ar is an aromatic or heteroaromatic ring comprising from 5 to 10 members, optionally substituted with one or more substituents selected from halogen, -OH, -NH₂, (Ci-3)alkyl, (Ci-3)alkoxy, -CN, nitro, trifluoromethyl, trifluorometoxy, benzyl, aromatic or heteroaromatic ring comprising 5 or 6 members.

7. The 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds according to any one of the preceding claims, in which R₂ is H, (Ci_-4)alkyl, (Ci_-4)alkoxy or benzyl, said (Ci_-4)alkyl, (Ci_-4)alkoxy and benzyl being optionally substituted with at least one substituent selected from halogen, -OH, -NH₂, (Ci-3)alkyl, (Ci-3)alkoxy, nitro and -CN.

8. The 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds according to any one of the preceding claims, in which R₃ and R₄, identical or different each other, are H, (Ci-3)alkyl, (Ci-3)alkoxy; or R₃ and R₄ together form a carbocycle or a heterocycle having 5 or 6 members, optionally substituted with a (Ci-3)alkyl, (Ci-3)alkoxy, -C(=O)-(Ci_-3alkyl) group.

9. A process to prepare the 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds of formula (I)

in which

X is -O- or -NH-, R is H, -R₅-Ar, -SO₂-R5-Ar, -R₆ or SO₂-R₆, where R₅ is σ bond, (Ci_-6)alkyl, (Ci_-6)alkyl-O-, -C(=O)-, -(Ci_-6)alkyl-C(=O)-, -C(=O)-O-, -(Ci_-6)alkyl-C(=O)-O- and R₆ is (Ci_-6)alkyl, Ar is an aromatic or heteroaromatic ring comprising from 5 to 14 members, optionally substituted with one or more substituents selected from: halogen, -OH, -NH₂, (Ci-6)alkyl, (Ci-6)alkoxy, -CN, nitro, trihalo(Ci-3)alkyl, trihalo(Ci-3)alkoxy, aromatic or heteroaromatic ring comprising 5 or 6 members, benzyl; R₂ is H, (Ci-6)alkyl, (Ci-6)alkoxy and benzyl, said (Ci-6)alkyl, (Ci-6)alkoxy and benzyl being optionally substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-6)alkoxy, nitro and -CN;

R₃ and R₄, identical or different each other, are H, (Ci-6)alkyl, (Ci-6)alkoxy; or R₃ and R₄ together form a carbocycle or a heterocycle having 5 or 6 members, optionally substituted with a (Ci_-6)alkyl, (Ci_-6)alkoxy, -C(=O)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH group; comprising the steps of:

(i) reacting a compound of formula (a) in a protic polar solvent to obtain the intermediate (b):

(a) (b)

in which X is as defined above;

(ii) reacting said intermediate (b) with an aqueous ammonia solution to obtain the intermediate (c):

(c)

in which X is as defined above;

(iii) reacting said intermediate (c) with a reagent of formula (III)

in which R₃ and R₄ are as defined above and each of R₇ and R₈, identical to each other, is a -0-(Ci-3alkyl) group or R₇ and R₈ together with the carbon atom to which they are bonded form a -C(=0) group, to obtain a compound of formula (d):

in which X, R₃ and R₄ are as defined above; (iv) reacting said product (d) with a reagent of formula (IV)

Y-Ri (IV) in which Y is a halogen atom and Ri is as defined above, to form a product of formula (e):

in which X, R-i, R₃ and R₄ are as defined above; (v) optionally, reacting said product (e) with a reagent of formula (V)

Y'-Rz (V)

in which Y' is a halogen atom and R₂ is as defined above, to form said compound of formula (I):

in which X, R-i, R₂, R3 and R₄ are as defined above.

10. A process to prepare the 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds of formula (I)

in which X is -O- or -NH-,

R is H, -R₅-Ar, -S0₂-R5-Ar, -R₆ or S0₂-R6, where R₅ is σ bond, (Ci_-6)alkyl, (Ci_-6)alkyl-0-, -C(=0)-, -(Ci_-6)alkyl-C(=0)-, -C(=0)-0-, -(Ci_-6)alkyl-C(=0)-0- and R₆ is (Ci_-6)alkyl,

R₃ and R₄, identical or different from each other, are H, (Ci-6)alkyl, (Ci-6)alkoxy; or R₃ and R₄ together form a carbocycle or a heterocycle having 5 or 6 members, optionally substituted with a (Ci_-6)alkyl, (Ci_-6)alkoxy, -C(=O)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH group; comprising the steps of:

(vi) reacting a product (d)

with a reagent of formula (VI)

Z-P (VI) in which Z is a halogen atom and P is a protective group, to obtain the intermediate (g- 1 ):

in which X, R₃ and R₄ are as defined above or alternatively, when X is -NH-, reacting said product (d) with a suitable oxidizing agent, to obtain the intermediate (g.2)

in which R₃ and R₄ are as defined above; (vii) reacting said intermediates (g.1 ) and (g.2) with a reagent of formula (VII)

Y"-R₂ (VII)

in which Y" is a halogen atom and R₂ is as defined above, to obtain the intermediates (h.1 ) and (h.2), respectively:

in which X, R₂, R3 and R₄ are as defined above;

(viii) reacting said intermediates (h.1 ) and (h.2) in suitable conditions to obtain a product of formula (i):

in which X, R₂, R3 and R₄ are as defined above; (ix) optionally, reacting said product (i) with a reagent of formula (IX)

Y"'-Ri (IX)

in which Y'" is a halogen atom and Ri is as defined above to obtain said compound of formula (I):

in which X, R-i , R₂, R3 e R₄ are as defined above.

1 1 . A pharmaceutical form comprising at

2,3-dihydro-4H-1 ,3-benzoxazin-4-one compound of formula (I)

in which

X is -0- or -NH-,

R is H, -R₅-Ar, -S0₂-R5-Ar, -R₆ or SO2-R6, where R₅ is σ bond, (Ci_-6)alkyl, (Ci_-6)alkyl-0-, -C(=0)-, -(Ci_-6)alkyl-C(=0)-, -C(=0)-0-, -(Ci_-6)alkyl-C(=0)-0- and R₆ is (Ci_-6)alkyl,

Ar is an aromatic or heteroaromatic ring comprising from 5 to 14 members, optionally substituted with one or more substituents selected from: halogen, -OH, -NH₂, (Ci-6)alkyl, (Ci-6)alkoxy, -CN, nitro, trihalo(Ci-3)alkyl, trihalo(Ci-3)alkoxy, aromatic or heteroaromatic ring comprising 5 or 6 members, benzyl; R2 is H, (Ci-6)alkyl, (Ci-6)alkoxy and benzyl, said (Ci-6)alkyl, (Ci-6)alkoxy and benzyl being optionally substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-6)alkoxy, nitro and -CN;

R₃ and R₄, identical or different from each other, are H, (Ci-6)alkyl, (Ci-6)alkoxy, or R₃ and R₄ together form a carbocycle or a heterocycle having 5 or 6 members, optionally substituted with a (Ci_-6)alkyl, (Ci_-6)alkoxy, -C(=O)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH group; or its salts of addition with pharmaceutically acceptable organic or inorganic acids or bases, and at least one pharmaceutically acceptable excipient.

12. 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds of formula (I)

in which

X is -O- or -NH-, R is H, -R₅-Ar, -SO₂-R5-Ar, -R₆ or SO2-R6, where R₅ is σ bond, (Ci_-6)alkyl, (Ci_-6)alkyl-O-, -C(=O)-, -(Ci_-6)alkyl-C(=O)-, -C(=O)-O-, -(Ci_-6)alkyl-C(=O)-O- and R₆ is (Ci_-6)alkyl,

Ar is an aromatic or heteroaromatic ring comprising from 5 to 14 members, optionally substituted with one or more substituents selected from: halogen, -OH, -NH₂, (Ci-6)alkyl, (Ci-6)alkoxy, -CN, nitro, trihalo(Ci_-3)alkyl, trihalo(Ci_-3)alkoxy, aromatic or heteroaromatic ring comprising 5 or 6 members, benzyl;

R2 is H, (Ci-6)alkyl, (Ci-6)alkoxy and benzyl, said (Ci-6)alkyl, (Ci-6)alkoxy and benzyl being optionally substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-6)alkoxy, nitro and -CN; R₃ and R₄, identical or different from each other, are H, (Ci-6)alkyl, (Ci-6)alkoxy; or R₃ and R₄ together form a carbocycle or a heterocycle having 5 or 6 members, optionally substituted with a (Ci_-6)alkyl, (Ci_-6)alkoxy, -C(=0)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH group; or their salts of addition with pharmaceutically acceptable organic or inorganic acids or bases, for use in medicine.

13. 2,3-dihydro-4H-1 ,3-benzoxazin-4-one compounds of formula (I)

in which

R₂ is H, (Ci-6)alkyl, (Ci-6)alkoxy and benzyl, said (Ci-6)alkyl, (Ci-6)alkoxy and benzyl being optionally substituted with at least one substituent selected from: halogen, -OH, -NH₂, (Ci_-3)alkyl, (Ci_-6)alkoxy, nitro and -CN; R₃ and R₄, identical or different from each other, are H, (Ci-6)alkyl, (Ci-6)alkoxy; or R₃ and R₄ together form a carbocycle or a heterocycle having 5 or 6 members, optionally substituted with a (Ci_-6)alkyl, (Ci_-6)alkoxy, -C(=O)-(Ci_-3)alkyl, -COOH, (Ci_-3)alkyl-COOH group; and their salts of addition with pharmaceutically acceptable organic or inorganic acids or bases, for use in the treatment of diseases related to the non-activation of CB2 receptors, in particular inflammation and chronic pain.