ORGANIC COMPOUNDS USEFUL FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES, USES AND METHODS FOR THE PREPARATION THEREOF

TECHNICAL FIELD

The present invention relates to organic compounds, organic compounds for use as medicaments, uses of such organic compounds for the manufacture of pharmaceutical preparations. The present invention moreover relates to methods for the synthesis of the above mentioned organic compounds.

BACKGROUND ART

Neurodegenerative diseases are a group of disorders characterized by a progressive loss of the anatomical and physiological structure of neurons. Despite their etiology has not definitively been clarified, it has been shown that among other fundamental causes, oxidative stress and protein precipitation followed by the formation of fibrillar aggregates play an important role. A higher sensibility to a state of oxidative excess (cause and consequence of mitochondrial dysfunctions) can be found above all where, such as in the brain, high level of oxygen consumption is present. At a same extent, the formation of protein fibrils is a well-known histopathological feature of Alzheimer's disease (AD) Parkinson's disease (PD), and Huntington' s disease (HD) .

In this respect, recent studies have pointed to the oxidative stress as an important factor related to cognitive impairment

and aging [Lu, T. et al . Gene Regulation and DNA damage in the ageing Human Brain. Nature 429:883-891(2004)] .

On the basis of both these experimental evidences and the fairly disappointing clinical results obtained so far, it has been shown that the use of a single antioxidant compound could not be enough for the treatment of such diseases. Indeed, since several processes are involved in the pathogenesis of these diseases, the treatment with a single compound fails. Furthermore, besides AD, PD, and HD, there are other diseases characterized by the presence of Aβ deposits. These pathologies include: Down's syndrome, hereditary cerebral haemorrhage associated with amyloidosis of the "Dutch type", amyloidosis associated with chronic inflammations, amyloidosis associated with multiple myelomas and other dyscrasias of the B lymphoid haematic cells, amyloidosis associated with diabetes type II, amyloidosis associated with diseases deriving from pryons such as Creutzfeldt-Jakob' s disease, the Gertsmann-Straussler syndrome, Kuru and scrapie in sheeps (WO 02/00603) . In the field of pharmaceutical products for the treatment of AD (PCT Int. Appl. PCT/IT03/00227) has reported on new 2,5- bis-diamine- [1 , 4]benzoquinonic derivatives, which showed, among others, high activity for the treatment of AD in mammals . Nevertheless, despite some symptomatic drugs have recently been brought to the market, therapeutic treatments able to slow down the progression of the disease, have so far not been reported. These drugs - Tacrine (Cognex®) , Donepezil

(Aricept®) and Rivastigmine (Exelon®) - share the same action mechanism, which consists of the inhibition of acetylcholinesterase (AChE) .

From the above it is clear that there is still a considerable need to make new medicaments available for the treatment of neurodegenerative diseases, of pathologies characterized by- deposits of Aβ, and of pathologies characterized by mitochondrial dysfunctions; in particular for AD.

DISCLOSURE OF INVENTION

The aim of the present invention is therefore to provide compounds that can be advantageously used in the treatment of neurodegenerative diseases, of pathologies characterized by deposits of Aβ, and of pathologies characterized by mitochondrial dysfunctions; in particular for AD.

According to the present invention, organic compounds, organic compounds as medicaments, uses of organic compounds for the manufacture of pharmaceutical preparations, synthetic methods for the preparation of such compounds are provided according to the following independent claims and, preferably, in anyone of the claims directly or indirectly dependent on the independent claims .

If not differently explicitly specified, the following terms have the following meaning. In the present text, "pharmaceutically acceptable salt" indicates a salt that maintains the biological properties of the starting compound. Non limiting examples of methodologies for the preparation of such salts include the following:

addition of inorganic acids (e.g., chloridric acid, bromidric acid, sulfuric acid, phosphoric acid, and similar) or organic (e.g., acetic acid, ossalic acid, maleic acid, methansulfonic acid, salicylic acid, succinic acid, citric acid, and similar) , and the freebase of the starting compound; substitution of the acid proton of the starting compound with a metal cation (e.g., a cation of an alkali metal, aluminium, or similar) ; acid proton transfer from the starting compound to an organic base (e.g., dimethylamine, triethylamine, and similar) and coordination with such an organic base. The compounds of the present invention, have to be considered, if not differently specified, as comprising their pharmaceutically acceptable salts. In this text, "prodrug" indicates an agent that in vivo is converted in a pharmacologically active substance. A prodrug may have some advantages over the pharmacologically active substance. For instance, it can be more easily administered to patients and/or it can have a better solubility and/or more able to cross the cell membranes. The compounds object of the present invention have to be considered as comprising their prodrugs. The compounds object of the present invention can act as prodrug of other pharmacologically active compounds. Some compounds of the present text can have one or more asymmetric centers; such compounds can, therefore, be made as stereoisomers (R)- or (S)- or as mixture. The compounds identified in the present text have to be considered, if not differently specified, as comprising the individual isomer or mixtures thereof, which may be racemic or of other kind.

Methods for the stereochemistry determination and stereoisomer separation are known in the background art (see for instance, Chapter 4 in "Advanced Organic Chemistry" IV Edition L. March, John Wiley and Sons, New York, 1992) . The compounds reported in the present text may show tautomerism; such compounds should be considered, if not differently specified, as comprising the tautomeric forms both alone or in mixtures . In the present text, "C _x -C _y " indicates a group having from x to y carbon atoms.

In the present text, "aliphatic" indicates a non-aromatic and non-substituted hydrocarbon, saturated or unsaturated, linear, ramified and/or cyclic. Non limiting examples of aliphatic groups are: t-butyl, ethenyl, 1- or 2- propenyl, cyclohexyl . In the present text, "alkyl" indicates a saturated aliphatic group (i.e. an aliphatic group devoid of double and triple carbon-carbon bonds) . Non limiting examples of alkyl groups are: methyl, n-propyl, t-butyl, cyclohexyl. In the present text, "alkoxy" indicates an aliphatic (preferably an aliphatic Ci-C ₅ , advantageously an alkyl Ci-C ₄ group) linked to the remaining part of the molecule through an oxygen atom. Non limiting example of alkoxy groups are; methoxy, ethoxy. In the present text, "Bn" indicates a benzyl. In accordance with a first aspect of the present invention, it is provided a compound having the general formula (I) :

or its geometrical isomers, its optical active forms, diastereoisomers, its racemic forms, or its pharmaceutically acceptable salts, or its prodrug, wherein R ² , R ³ , R ⁶ , R ⁷ are chosen, independently of each other, from the group consisting of: hydrogen and alkyl Ci-C ₅ group; R ⁴ and R ⁵ represent, each independently of the other, a substituent chosen from the group consisting of: hydrogen, alkyl Ci-C ₄ group, OMe, SMe, Cl, F, Br, and Ph (preferably, hydrogen, alkyl C _x -C ₄ group, OMe, and SMe) ;

Z ¹ and Z ² represent, each independently of the other, a respective aliphatic C ₂ -Cg, in particular an alkyl group; at least one of R ¹ and R ⁸ represent, each independently of the other, an anti-oxidant group and/or according to some embodiments, anti-amyloid group.

Advantageously, at least one of R ¹ and R ⁸ (both, according to some embodiments) represent, each independently of the other, an anti-oxidant able to reduce the quinone to hydroquinone . According to some embodiments, R ¹ and R ⁸ are chosen, each independently of the other, from the group consisting of:

(ID (III) (IV)

(V) (VI) wherein R ⁹ , R ¹⁰ , R ¹⁸ , R ²⁰ , R ¹³ , represent, independently of each other, a respective alkyl group, in particular Ci-C ₃ ; R ¹¹ , R ¹² , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁹ represent, independently of each other, a respective alkyl group, in particular Ci-C ₃ ; with the proviso that if one of R ¹ and R ⁸ is

the other of R ¹ and R ⁸ is chosen from the group consisting of :

Advantageoulsy, in the compound having general formula (I) , where one of R ¹ and R ⁸ is

V λ ⁰ Ct

the other of R ¹ and R ⁸ is :

According to some embodiments, R ⁴ and R ⁵ represent, each independently of the other, a substituent chosen from the group consisting of: hydrogen, alkyl Ci-C ₃ group. Advantageously, R ⁴ and R ⁵ represent, each, a respective

hydrogen. Advantageously, Z ¹ and Z ² represent, each independently of the other, an aliphatic (in particular an alky group), advantageously C3-C ₆ .

According to some embodiments, R ² , R ³ , R ⁶ , R ⁷ are chosen, each independently of the other, in a group consisting of: hydrogen and alkyl, advantageously Ci-C ₂ . According to some embodiments, R ⁸ represents:

Advantageously, R ⁹ represents an alkyl C ₃ -C ₅ group. According to particular embodiments embodiments, R ¹ represents :

Advantageously, R ⁹ represents C ₄ H ₈ , in particular linear (n- butyl) . Advantageously, R ²⁰ represents an alkyl Ci-C ₄ group. According to specific embodiments, the compound having the general formula (I) has one of the following structures:

According to some embodiments, R represents:

Advantageously, R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ represent, independently of each other, an alkyl C ₁ -C ₃ group; R ¹³ represents an alkyl C ₁ -C ₄ group .

Advantageously, R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ represent, each, a respective methyl group; R ¹³ represents CH ₂ . Advantageously, R ¹ and R ⁸ represent identical groups .

According to a specific embodiment, the compound having general formula (I) has the following structure:

According to some embodiments, R ⁸ represents:

Advantageously, R ¹⁰ represents an alkyl Ci-C ₄ group; >ii

represents an alkyl Ci-C ₃ group.

Advantageously, R ¹¹ represents a methyl group; R ¹⁰ represents

CH ₂ . Advantageously, R ¹ and R ⁸ represent identical groups.

Advantageously, Z ¹ and Z ² represent, each independently of the other, C ₆ H ₁₂ , in particular linear (n-hexyl) .

According to a specific embodiment, the compound having general formula (I) has the following structure:

According to further embodiments, R ⁸ represents:

Advantageously, R ,18 represents an alkyl C1-C4 group; R 12 represents an alkyl C ₁ -C ₃ group.

Advantageously, R ¹² represents a methyl group; R ¹⁸ represents CH ₂ ; R ¹ and R ⁸ represent identical groups.

According to a specific embodiment, the compound having genera formula (I) has the following structure:

1 ft

According to some embodiments, at least one of R and R represents, independently of the other, a respective group able to confront the amyloid aggregation. According to some embodiments, at least one of R ¹ and R ⁸ has

the general formula (XVII) :

(XVII) wherein Alo is an halogen, R ²² is an aliphatic Ci-C ₄ group and R ²¹ is chosen among: C=O and

(XVIII) wherein R ²³ is an aliphatic C ₂ -C ₇ group and the carbonyl is directly linked to the benzylic carbon. Advantageously, Alo is a fluorine. Advantageously, R ²² represents an alkyl Ci-C ₂ group and R ²³ is an alkyl C ₂ -C ₄ group.

Advantageously, R ¹ and R ⁸ represent identical groups.

According to some embodiments, R ²¹ is C=O.

According to some embodiments , the compound having general formula (I) has the following structure:

According to some embodiments, R ²¹ is:

; R ¹ and R ⁸ represent identical groups .

According to some embodiments, the compound having general formula (I) has the following structure:

The compounds having a general formula (I) as above defined are multifunctional compounds able to inhibit the oxidative stress at a mitochondrial level and simultaneously the amyloidogenic process, that characterizes neurodegenerative diseases such as AD, PD, HD, and prion diseases. When compared to the molecules (Memoquin) reported in the PCT Int. Appl. PCT/IT03/00227, the compounds having a general formula (I) as above defined show improved anti-oxidant and anti-amyloid activities. In particular, the compounds falling within the general formula (I) , wherein at least one of R ¹ and R ⁸ has the general formula (XVII) , show an improved anti- aggregating amyloid activity; compounds having a general formula (I), wherein at least one of R ¹ and R ⁸ is represented by the general formula (II), (III), (IV), (V) or (VI) show an improved anti-oxidant activity.

In particular, the compounds having general formula (I) , as above defined, combine in a single molecule two mitochondrial anti-oxidant: the coenzyme QlO and α-lipoic acid and/or curcumin and/or melatonin and/or Vitamin E. Alternatively, the compounds combine in a single molecule an anti-oxidant moiety (coenzyme QlO) with an anti-amyloid moiety such as the compound flurbiprofen. In brief, it has been used the 2 , 5-bis-diamine- [1, 4]benzoquinonic moiety as anti-oxidant scaffold, on which molecular residues both having anti-amyloidogenic activity and

able to improve the anti-oxidant profile have been mounted. In particular, since the lipoic acid is able to reduce the ubiquinone to ubiguinol, by means of the transfer of a couple of electrons, such hybrid compounds have high anti-oxidant properties. Indeed, the lipoic acid scaffold should be able to maintain the quinone moiety in the hydroquinone form, which is a potent radical scavenger. Furthermore, both these scaffolds are natural substrates of the mitochondrial enzymes that maintain both the lipoic acid and the QlO in the reduced form. All the above molecular and biochemical observations make the compounds above reported, wherein at least one of R ¹ and R ⁸ has the general formula (II), (III), (IV), (V), or (VI), good anti-oxidant compounds. At the best of our knowledge and according to the bibliographic search carried out, these compounds are the only ones that include in a single chemical structure two anti-oxidant scaffolds able to confront the oxidative process at the basis of neurodegenerative diseases in two different points. The new derivatives also show anti-aggregating activity. In the light of what has been above reported and in accordance with further aspects of the present invention, it is provided a compound having a general formula (I) for use as a medicament. In particular, for the treatment of diseases characterized by deposits of β-amyloid (Aβ) in mammals and/or in diseases characterized by mitochondrial dysfunction and/or neurodegenerative diseases; in particular, Alzheimer's disease and/or Parkinson's disease and/or Huntington's disease. In accordance with further aspects of the present invention,

it is provided a compound having the general formula (I) as above defined for the manufacture of pharmaceutical preparations for the treatment of diseases characterized by deposits of β-amyloid (Aβ) in mammals and/or of diseases characterized by mitochondrial dysfunction and/or of neurodegenerative diseases; in particular, Alzheimer's disease and/or Parkinson's disease and/or Huntington's disease. In accordance with further aspects of the present invention, it is provided a method for the treatment of diseases characterized by deposits of β-amyloid (Aβ) in mammals and/or in diseases characterized by mitochondrial dysfunction and/or neurodegenerative diseases; in particular, Alzheimer's disease and/or Parkinson's disease and/or Huntington's disease. According to the method a effective amount of a compound having general formula (I) , as above defined, is administered to said mammal .

In accordance with a further aspect of the present invention, it is provided a pharmaceutical preparation comprising a compound having general formula (I) , as above defined, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient and/or diluent. The compounds having general formula (I) can be formulated, in a known way, for parenteral administration by injection or continuous administration. Formulae for injection may be in the form of single doses, for example in ampoules or multidose containers containing preservatives. The composition may be in the form of a suspension, in aqueous or oily liquids, and it may contain

formulation elements such as dispersing and stabilizing agents. Alternatively, the active compound may be in powder form to be dissolved immediately before use in a suitable liquid, for example sterilized water. The compounds having general formula (I) can be formulated for rectal administration as suppositories or enteroclysis, for example containing excipients for suppositories of a known type such as cocoa butter or other fats . The compounds having general formula (I) can also be formulated, in a known way, as compositions with prolonged release. These compositions with prolonged release can be administered by means of an implant (for example subcutaneous, or intramuscular) or by means of an intramuscular injection. Thus, for example, the compounds having the general formula (I) can be formulated with suitable polymeric or hydrophobic materials (for example an emulsion or an oil) or resins with ionic exchange, or relatively poorly soluble derivatives, such as relatively poorly soluble salts. For intranasal administration, the compounds having general formula (I) can be formulated for administration by means of a (known) device, for example in powder form with a suitable carrier.

The dosages of the compounds having general formula (I) will depend on the age and conditions of the patient, so the precise dosage must be decided from time to time by the doctor. The dosage will also depend on the means of administration and on the particular compound selected. Usable doses may be for example between 0.1 mg/Kg and 400 mg/Kg with

respect to body weight per day.

The compounds having general formula (I) , can be administered in combination with one or more suitable therapeutic agents and formulated in every known usable way. According to a further aspect of the present invention, a method is provided for the synthesis of a compound having a general formula (I) , as above described; the method comprises a nucleophilic substitution step, in which on a p-benzoquinone having the general formula (VII) :

(VII) where LG represents a leaving group having inductive electro- withdrawing effect, a substitution is carried out with a first intermediate compound having general formula (VIII) :

(VIII) in such a way to obtain a compound having general formula (I) ; R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , Z ¹ , Z ² are defined as above described. Advantageously, during the nucleophilic substitution on a p- benzoquinone having general formula (IX) :

(IX) in which substitutions are carried out with intermediate

compounds having general formulae (VIII) and (X) :

(VIII) (X)

Advantageously, the intermediate compounds having the general formulas (VIII) and (X) are identical.

According to some embodiments, LG represents an Ci-C ₄ alkoxy group, advantageously a methoxy.

According to some embodiments, the nucleophilic substitution step is carried out in the presence of an alcoholic solvent, in particular ethanol . The nucleophilic substitution step is carried out at a temperature of from 50 ⁰ C to 65°C. According to some embodiments, the method comprises a hydrolysis step, wherein a further intermediate compound having general formula (XI) :

BnOOCN 2N _{v 8}

(XI) is hydrolyzed to obtain the above described first intermediate compound having general formula (VIII) .

According to a further aspect of the present invention, a method is provided for the synthesis of a compound having general formula (I) , as above defined; the method comprises a step in which the p-benzoquinone having general formula (XII) :

(XII) reacts with an intermediate having general formula (XIII) :

(XIII ) in such a way to obtain a compound having general formula ( I ) ; R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , Z ¹ , Z ² are defined as above reported.

According to some embodiments, the p-benzoquinone having general formula (XII) is obtained by means of a hydrolysis reaction of the p-benzoquinone having general formula (XIV) :

(XIV)

Advantageously, the p-benzoquinone having general formula (XIV) is obtained by reacting the p-benzoquinone having general formula (XV) :

(XV) in which LG is defined as above reported, with a further intermediate having general formula (XVI) :

(XVI)

Advantageously, the p-benzoquinone having general formula (XV) is obtained by reacting the diamine having general formula

(X) :

(X) with an excess of a p-benzoquinone having general formula (VII) :

(VII)

According to a further aspect of the present invention, it is provided the intermediate compound having general formula (VIII) :

(VIII) wherein R ⁶ , R ⁷ , R ⁸ , Z ² are defined according to claims 1 to 32. According to a further aspect of the present invention, it is provided the compound having general formula (VII) :

(VII) wherein LG, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , Z ¹ are defined as above. Further characteristics of the present invention will appear more clearly from the following description which refers to embodiments taken purely as examples and limitative. Examples Compound characterization was performed using:

• Nuclear magnetic resonance: ¹ H NMR spectra were recorded on Varian VXR 200 and 300 instruments. Chemical shifts are reported in parts per millions (ppm) relative to the peak for tetramethylsilane (TMS) , and spin multiplicities are given as s (singlet) , br s (broad singlet), d (doublet), t (triplet), q (quartet), sept (septet) , m (multiplet) , m complex (multiplet complex) , exch (exchangeable with D ₂ O) .

• Mass spectroscopy: direct infusion ESI-MS or EI-MS spectra were recorded on Waters Micromas ZQ 4000 and

Finnigan MAT 95 XP apparatuses, respectively.

Reactions were followed by thin-layer chromatography (TLC) on Merck (0.25 mm) glass-packed precoated silica gel plates (60 F254) and then visualized in an iodine chamber or with a UV lamp.

Chromatographic separations were performed on silica gel columns by flash (Kieselgel 40, 0.040-0.063 mm) or gravity (Kieselgel 60, 0.063-0.200 mm) column chromatography. Reagents were purchased by SIGMA-Aldrich. Compounds were named following IUPAC rules as applied by the CHEM-DRAW ULTRA software. Examples 1-22

For the synthesis of compounds 1-4 the synthetic scheme depicted below was followed:

11 : n=6 12 : n=3

Example 1

N- (benzyloxycarbonil ) -hexanediamine (5)

In a three-necked flask equipped, hexandiamine (20 g 0.172 mol) was dissolved in 30 ml of water containing bromo cresol green as an indicator. At 5 ⁰ C methanesulphonic acid was

added dropwise (23 ml, 0.33 mol) keeping ph <5, and then the resulting solution was diluted with ethanol (50 mL) and vigorously stirred.

A solution of benzylchloroformate (8 ml, 0.054 mol) in 25 mL of dimethoxyethane and a solution of 50% potassium acetate

(20 ml) were added dropwise simultaneously. The reaction mixture was heated to 40 ⁰ C, was stirred for another hour. The volatile solvents were evaporated under vacuum so as to obtain a yellow suspension, which was filtered. The yellow suspension was washed with toluene (25 x 100 mL) , made basic with KOH

(pH= 13) and extracted with toluene (2 x 150 mL) . The combined organic extracts were washed with water (100 mL) , dried and evaporated under vacuum to give 6.67 g of compound 5 as an oil. Yield: 49%. 1H-NMR (CDCl ₃ , 200 MHz) δ 1.33 (s, 2H), δ 1.40-1.60 (m, 8H), δ

2.70 (t, 2H), δ 3.25 (q, 2H), δ 4.90 (br m, IH), δ 5.05 (s,

2H) , δ 7.35 (s, 5H) .

Example 2

N- (benzyloxycarbonil) -propanediamine (6) Compound 6 was obtained by the reaction of 1, 3-diaminopropane

(10.9 ml, 0.13 mol) and methanesulphonic acid (15.5 ml, 0.24 mol), following the procedure described for 5, to give 6.94 g of 6. Yield: 30%.

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.33 (s, 2H), δ 1.58-1.67 (m, 2H), δ 2.78 (t, 2H), δ 3.30 (q, 2H), δ 5.11 (s, 2H), δ 5.38 (br m,

IH) , δ 7.35 (s, 5H) .

Example 3

[6-(2-Methoxy-benzylamino)-hexyyl]-carbamic acid benzyl ester

(7 )

A solution of 5 (3.92 g 15.6 mmol) and 2-methoxybenzaldheyde

(2.13 g 15.7 mmol) in toluene was stirred at the refluxing temperature (T 140 ⁰ C) in a Dean-Stark apparatus for 3.5 h. The solvent was evaporated, and the reddish oil residue was dissolved in EtOH (50 ml), the solution cooled and treated with NaBH ₄ (0.6 g 15.8 mmol) . The reaction mixture was stirred overnight at room temperature and then made acidic with 37%

HCl. After evaporation of the solvents under vacuum, the formed yellow-white solid was washed with diethyl ether (2 x 60 ml) filtered and dried under vacuum, affording 5 g of 7 as hydrochloride salt. Yield: 70%.

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.30-1.80 (m, 8H), δ 1.80 (br m, IH) , δ 2.78 (t, 2H), δ 3.17 (t, 2H), δ 3.60 (s, 2H), δ 3.85 (s, 3H), δ 4.90 (br m, IH), δ 5.10 (s, 2H), 6.89 (m, 2H), δ 7.23 (d, 2H) , δ 7.28 (s, 5H) . Example 4

[3- (2-Methoxy-benzylamino) -propyl] -carbamic acid benzyl ester (8) Compound 8 was synthesized from 6 (3 g 14.4 mmol) and methoxybenzaldehyde (1.9 g 14.5 mmol) . It was purified by flash chromatography. Eluting with CH ₂ Cl ₂ /CH ₃ OH/NH ₃

(9.5:0.5:0.05), afforded 1 g of 8. Yield: 21%.

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.73 (s, 2H), δ 2.7 (t, 2H), δ 3.32 (q, 2H), δ 3.78 (s, 2H), δ 3.83 (s, 3H), δ 5.11 (s, 2H), δ 5.73 (m, IH), δ 6.89 (m, 2H), δ 7.23 (d, 2H), δ 7.28 (s, 5H) . Example 5 {6- [Ethyl- (2-methoxy-benzyl) -amino] -hexyl} -carbamic acid

benzyl ester (9)

KOH was added to a solution of [6- (2-methoxy-benzylamine) - hexyl] -carbamic acid hydrochloric benzyl ester (10.2 g 0.025 mol) in methanol. Afterward acetaldehyde (2.8 ml 0.05 mol) was added. The resulting suspension was agitated at room temperature for 30 minutes and then NaBH ₃ CN (0.6 g 9.6 mmol ) was added. When addition was completed, the resulting mixture was stirred overnight. Then KOH was added and the suspension obtained was suction filtered on celite® and concentrated. A yellow oil was obtained, which was then partitioned between water and CH ₂ Cl ₂ (2 x 200 mL) . The combined organic extracts were dried and concentrated to give a reddish oil which was purified through extraction with stirred and slightly hot petroleum ether for 10 minutes. The operation was carried out three times. The combined and dried extracts provided 4.28g (43% yield) of 9.

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.07 (t, 3H), δ 1.20-1.60 (m, 8H), δ 2.30 (t, 2H), δ 2.57 (t, 2H), δ 3.17 (q, 2H), δ 3.60 (s, 2H), δ 3.90 (s, 3H), δ 4.70-4.90 (m, IH), δ 5.15 (s, 2H), δ 6.89 (m, 2H), δ 7.23 (d, 2H), δ 7.28 (s, 5H) . Example 6

{3- [Ethyl- (2-methoxy-benzy1 ) -amino] -propyl} -carbamic acid benzyl ester (10) Compound 10 was obtained from a solution of 8 (1 g 3 mmol) and acetaldehyde (1 ml 6 mmol), affording 0.8 g of 10. Yield: 78%. δ 1.07 (t, 3H), δ 1.20-1.60 (m, 2H), δ 2.30 (t, 2H), δ 2.57 (t, 2H), δ 3.17 (q, 2H), δ 3.60 (s, 2H), δ 3.90 (s, 3H), δ 4.70-4.90 (m, IH), δ 5.15 (s, 2H), δ 6.89 (m, 2H), δ 7.23 (d,

2H) , δ 7.28 (s, 5H) .

Example 7

I^-Ethyl-N ¹ - (2-methoxy-benzyl) -hexane-1, 6-diamine (11)

A solution of 9 (3.7 g 9.3 iranol) in CH ₃ COOH (50 ml) was treated with HBr in CH ₃ COOH (10 ml) . It was stirred at room temperature for 6h and then, after addition of ethyl ether (70 ml) overnight. A solution and an oil separated. The oil was taken up with water (70 ml) then washed with CH ₂ Cl ₂ (3 x 70 ml) . The aqueous solution was made basic with KOH (pH=10) and the product extracted with CH ₂ Cl ₂ (3 x 100ml) . The combined and dried organic extracts were evaporated under vacuum to provide a raw material which was purified by flash chromatography Eluting with CH ₂ Cl ₂ /Toluene/CH ₃ OH/NH ₃

(8:0.5:1.5:0.15) 1 g of 11 as a yellow-brown oil was obtained. Yield: 41%.

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.07 (t, 3H), δ 1.32 (s, 2H), δ 1.47-1.52 (m, 8H), δ 2.50 (m, 4H), δ 2.68 (t, 2H), δ 3.61 (s, 2H), δ 3.83 (s, 3H), δ 6.84-6.94 (m, 2H), δ 7.24 (t, IH), δ 7.41 (d, IH) . Example 8

N^-Ethyl-N ¹ - (2-methoxy-benzyl ) -propane-1, 3-diamine ( 12 ) Compound 12 was synthesized from 10 (0.8 g 2 mmol) and HBr/CH ₃ COOH (2 ml), affording 0.5 g of 12. Yield: 83%. 1H-NMR (CDCl ₃ , 200 MHz) δ 1.07 (t, 3H), δ 1.50 (s, 2H) δ 1.57- 1.71 (m, 5H), δ 2.49-2.60 (m, 4H), δ 2.73 (t, 2H), δ 3.59 (s, 2H), δ 3.83 (s, 3H), δ 6.85-6.98 (m, 2H), δ 7.23 (t, IH), δ 7.41 (d, IH). Example 9

2- {6- [Ethyl- (2-methoxy-benzyl) -amino] -hexylamino} -5-methoxy- [1, 4]benzoquinone (13)

A solution of 11 (0.180 g 0.168 iranol) , in CHCl ₃ (10 ml) was added dropwise to a vigorously pre-stirred preheated (for about an hour) (80 ⁰ C) mixture of 2 , 5-dimethoxy-l, 4- benzoquinone (0.80 g 4.7 mmol) ethanol (150 ml) and chloroform (50 ml). The yellow solution turned orange-red upon stirring overnight. The unreacted quinone excess was filtered off and the solvents evaporated under reduced pressure, to afford a residue which was purified by gravity column. Eluting with CH ₂ Cl ₂ /CH3OH/NH3 (9.5:0.5:0.05), 70 mg of 13 were obtained. Yield: 26%.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.07 (t, 3H), δ 1.29-1.45 (m, 8H), δ 2.40-2.60 (m, 4H), δ 3.11 (q, 2H), δ 3.61 (s, 2H), δ 3.83 (s, 3H), δ 3.85 (s, 3H), δ 5.62 (s, IH), δ 5.79 (s, IH), 5.90-6.00 (m, IH), δ 6.84-6.94 (m, 2H), δ 7.24 (t, IH), δ 7.45 (d, IH) . Example 10

2-{3- [Ethyl- (2-methoxy-benzyl) -amino] -propylamino}-5-methoxy- [1,4] benzoquinone ( 14 ) Compound 14 was synthesized from 2 , 5-dimethoxy-l, 4- benzoquinone (2.12 g 12.6 mmol) and 12 (0.4 g 1.8 mmol) . Purification by flash chromatography (eluting system: CH2CI2/CH3OH/NH3, 9.75:0.25:0.025) afforded 0.25g of 14. Yield: 40%. ¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.12 (t, 3H), δ 1.82 (m, 2H), δ 2.58 (m, 4H), δ 3.15 (q, 2H), δ 3.63 (s, 2H), δ 3.82 (s, 3H), δ 3.87 (s, 3H), δ 5.32 (s, IH), δ 5.36 (s, IH), δ 5.75 (s, IH), δ 6.84-6.94 (m, 2H), δ 7.24 (t, IH), δ 7.42 (d, IH) . Example 11

[3- (4- {6- [Ethyl- (2-methoxy-benzyl) -amino] -hexyylamino} - 3 , 6-dioxo-cyclohexa-l, 4-dienylamino) -propyl] -carbamic acid tβrt-butyl ester (15)

A solution of 13 (0.08 g 2 mmol) in CH ₂ Cl ₂ (5 ml), was added to a solution of N- (ter-butoxycarbonyl) -1, 3-propanediamine

(Fluka) (0.035 g 2 mmol), in CH ₂ Cl ₂ . The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure to afford an oil which was purified by flash chromatography. Eluting with CH ₂ CI ₂ /CH ₃ OH/NH ₃ (9.5:0.5:0.05), 0.08g of 15 were obtained. Yield: 73%.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.11 (t, 3H), δ 1.48 (s, 9H), δ 1.64 (m, 8H), δ 1.83-1.88 (m, 2H), δ 2.52 (m, 4H), δ 3.17 (q, 6H), δ 3.63 (s, 2H), δ 3.85 (s, 3H), δ 4.69 (m, IH), δ 5.32 (s, IH), δ 5.33 (s, IH), δ 5.34 (s, IH), δ 6.59 (m, IH), δ 6.84- 6.94 (m, 2H), δ 7.24 (t, IH), δ 7.43 (d, IH). Example 12

[6- (4-{3- [Ethyl- (2-methoxy-benzyl) -amino] -propylamino}- 3, 6-dioxo-cyclohexa-l, 4-dienylamino) -hexyl] -carbamic acid tert-butyl ester (16) Compound 16 was synthesized from 14 (0.120 g 3.3 mmol) and N-

( ter-butoxycarbonyl ) -1, 6-hexanediamine (Fluka) (0.070 g, 3.3 mmol), following the procedure described for 15. Yield: 84%.

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.12 (t, 3H), δ 1.48 (s, 9H), δ 1.67

(m, 8H), δ 1.81-1.88 (m, 2H), δ 2.58 (m, 4H), δ 3.14 (q, 6H), δ 3.62 (s, 2H), δ 3.82 (s, 3H), δ 4.52 (m, IH), δ 5.27 (s, IH), δ 5.29 (s, IH), δ 5.32 (s, IH), δ 6.57 (m, IH), δ 6.86- 6.99 (m, 2H), δ 7.24 (t, IH), δ 7.41 (d, IH) . Example 13

[3- (4- {3- [Ethyl- (2-methoxy-benzyl) -amino] -propylamino}- 3 , 6-dioxo-cyclohexa-l, 4-dienylamino) -propyl] -carbamic acid tert-butyl ester (17)

Compound 17 was synthesized fin 91% yield from 14 (0.125 g 3.5 mmol) and N- (ter-butoxycarbonyl) -1, 3-propanediamine (Fluka)

(0.060 g 3.5 mmol), following the procedure described for 15.

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.12 (t, 3H), δ 1.47 (s, 9H), δ

1.81-1.91 (m, 4H), δ 2.58 (m, 4H), δ 3.20 (q, 6H), δ 3.63 (s,

2H), δ 3.82 (s, 3H), δ 4.62 (m, IH), δ 5.27 (s, IH), δ 5.30 (s, IH), δ 5.32 (s, IH), δ 6.71 (m, IH), δ 6.86-6.99 (m, 2H), δ 7.24 (t, IH) , δ 7.41 (d, IH) .

Example 14

[6- (4- {6- [Ethyl- (2-methoxy-benzyl) -amino] -hexylamino} -

3 , 6-dioxo-cyclohexa-l, 4-dienylamino) -hexyl] -carbamic acid tert-butyl ester (18)

Compound 18 was synthesized in 42% yield from 13 (70 mg 1.7 mmol) and N- (ter-butoxycarbonyl) -1, 6-hexanediamine (Fluka) (37 mg 1.7 mmol), following the procedure described for 15.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.16 (t, 3H), δ 1.28-1.51 (m, 16H), δ 1.67 (s, 9H), δ 2.59 (m, 4H), δ 3.18 (q, 6H), δ 3.72 (s,

2H), δ 3.86 (s, 3H), δ 4.54 (m, IH), δ 5.32 (s, 2H), δ 6.61-

6.63 (m, 2H), δ 6.89-6.99 (m, 2H), δ 7.24 (t, IH), δ 7.52 (d,

IH) .

Example 15 2- (3-Amino-propylamino) -5- {6- [ethyl- (2-methoxy-benzyl) -amino] - hexylamino} - [1, 4] benzoquinone (19 )

A solution of 15 (0.08 g 1.5 mmol) in CH ₂ Cl ₂ (5 ml) and TFA (1 ml 13 mmol) was stirred for 1 h at room temperature. The

reaction mixture was then snaked with a saturated solution of

K ₂ CO ₃ (10 ml) . The organic layer was separated and dried over

Na ₂ SO ₄ . The solvent was removed in vacuum to give 0.06 g (90% yield) of 19 as a red wax. ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.07 (t, 3H), δ 1.27 (s, 2H), δ 1.54-1.64 (m, 8H), δ 1.74-1.87 (m, 2H), δ 2.42-2.60 (m, 4H), δ 2.87 (t, 2H), δ 3.13 (q, 2H), δ 3.27

(q, 2H), δ 3.59 (s, 2H), δ 3.83 (s, 3H), δ 5.30 (s, IH), δ

5.32 (s, IH), δ 6.59 (m, IH), δ 6.89-6.99 (m, 2H), δ 7.24 (t,

IH) , δ 7.43 (d, IH) . Example 16

2- (6-Amino-hexylamino) -5-{3- [ethyl- (2-methoxy-benzyl ) -amino] - propylamine} - [1, 4]benzoquinone (20)

Compound 20 was synthesized in 96% yield from 16 (0.150 g 2.7 mmol) and CF ₃ COOH (2 ml 26 mmol) , following the procedure described for 19: ¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.11 (t, 3H), δ 1.28 (s, 2H), δ 1.69-1.80 (m, 10H), δ 2.54-2.61 (m, 4H), δ 2.75 (t, 2H), δ 3.18 (q, 2H), δ 3.27 (q, 2H), δ 3.63 (s, 2H), δ 3.83 (s, 3H), δ 5.29 (s, IH), δ 5.31 (s, IH), δ 6.63 (m, 2H), δ 6.87-6.90 (m, 2H), δ 7.24 (t, IH), δ 7.43 (d, IH) . Example 17

2- (3-Amino-propylamino) -5- {3- [ethyl- (2-methoxy-benzyl) -amino] - propylamine} -[1,4] -benzoquinone (21)

Compound 21 was synthesized in 87% yield from 17 (0.1 g 2 mmol) and CF ₃ COOH (1 ml 13 mmol), following the procedure described for 19: ¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.11 (t, 3H), δ 1.30 (s, 2H), δ 1.69-1.80 (m, 4H), δ 2.54-2.61 (m, 4H), δ 2.75 (t, 2H), δ 3.18 (q, 2H), δ 3.28 (q, 2H), δ 3.63 (s, 2H), δ 3.83 (s, 3H), δ 5.25 (s, IH), δ 5.31 (s, IH), δ 6.63 (m, 2H),

δ 6. 87- 6 . 90 (m, 2H) , δ 7 . 24 (t , IH) , δ 7 . 43 (d, IH) . Example 18

2- (6-Amino-hexylamino) -5-{6- [ethyl- (2-methoxy-benzyl) -amino] - hexylamino} - [1, 4]benzoguinone (22) Compound 21 was synthesized in 97% yield from 18 (0.04 g 0.74 mmol) and CF ₃ COOH (0.7 ml 9 mmol) , following the procedure described for 19: ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.12 (t, 3H), δ 1.27 (s, 2H), δ 1.43-1.47 (m, 16H), δ 2.42-2.61 (m, 4H), δ 2.87 (t, 2H), δ 3.18 (q, 2H), δ 3.27 (q, 2H), δ 3.63 (s, 2H), δ 3.90 (s, 3H), δ 5.30 (s, IH), δ 5.32 (s, IH), δ 6.60 (m, 2H), δ 6.94-6.98 (m, 2H), δ 7.25 (t, IH), δ 7.43 (d, IH) . Example 19

5-[l,2]Dithiolan-3-yl-pentanoic acid [3-(4-{6- [ethyl-(2- methoxy-benzyl) -amino] -hexylamino} -3, 6-dioxo-cyclohexa-l, 4- dienylamino) -propyl] -amide (1)

A solution of 19 (0.06 g 0.135 mmol) in CH ₂ Cl ₂ (5 ml), triethylamine (60 μl) , hydroxybenzotriazole (0.035 g 0.26 mmol) , l-ethyl-3- [3- (dimethylamino) propyl] -carbodiimide hydrochloride (EDCI) (0.088 g 0.46 mol) and additional triethylamine (60 μl) were added successively to a solution of lipoic acid (LA) (0.035 g 0.17 mmol) in CH ₂ Cl ₂ (5 ml) . The reaction mixture was stirred overnight at room temperature in the darkness. It was carried out an extraction with water and organic solvents, which were dried over Na ₂ SO ₄ . The solvent was evaporated under vacuum and purified by flash

chromatography. Eluting with CH ₂ CI ₂ /CH ₃ OH/NH ₃ (9.5:0.5:0.05), 0.065 g (75%) of 1 as a red waxy solid were obtained. ESI-MS: m/z: 631 [M+H ⁺ ]

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.09 (t, 3H), δ 1.27-1.33 (m, 10H), δ 1.83-2.01 (m, 4H), δ 2.22 (t, 2H), δ 2.33 (t, 2H), δ 2.43- 2.53 (m, 4H), δ 3.12-3.25 (m, 6H), δ 3.34 (q, 2H), δ 3.59 (t, 2H), δ 3.66 (m, .1H), δ 3.69 (s, 2H), δ 3.84 (s, 3H), δ 5.31 (s, 2H), δ 5.58 (t, IH), δ 6.57 (t, IH), δ 6.85-6.89 (m, 2H), δ 6.97 (t, IH), δ 7.43 (d, IH) . Example 20

5-[l,2]Dithiolan-3-yl-pentanoic acid [6- (4- {3- [ethyl- (2- methoxy-benzyl) -amino] -propylamines} -3, 6-dioxo-cyclohexa-l, 4- dienylamino) -hexyl] -amide (2 )

Compound 2 was synthesized in 37% yield from LA (0.066 g 0.32 mmol) and 20 (0.130 g 0.3 mmol) , following the procedure described for 1: ESI-MS: m/z: 631 [M+H ⁺ ] ;

¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.14 (t, 3H), δ 1.41-1.62 (m, 10H) , δ 1.88-2.01 (m, 4H), δ 2.19 (t, 2H), δ 2.43 (t, 2H), δ 2.49- 2.60 (m, 4H), δ 3.12-3.21 (m, 6H), δ 3.29 (q, 2H), δ 3.61 (t, 2H), δ 3.66 (m, IH), δ 3.69 (s, 2H), δ 3.83 (s, 3H), δ 5.27 (s, IH), δ 5.29 (s, IH), δ 5.48 (t, IH), δ 6.58 (t, IH), δ 6.85-6.89 (m, 2H), δ 6.97 (t, IH), δ 7.40 (d, IH) . Example 21 5-[l,2]Dithiolan-3-yl-pentanoic acid [3- (4- {3- [ethyl- (2- methoxy-benzyl) -amino] -propylamino}-3, 6-dioxo-cyclohexa-l,4-

dienylamino) -propyl] -amide (3 )

Compound 3 was synthesized in 70% yield from LA (0.038 g 0.186 mmol) and 21 (0.07 g 0.17 mmol) , adding triethylamine (60μl) , Hydroxybenzotriazole (0.038 g 0.28 mmol), EDCl (0.096 g 0.5 mmol) and again triethylamine (60μl) following the procedure described for 1: ESI-MS: m/z: 589 [M+H ⁺ ] 1H-NMR (CDCl ₃ , 300 MHz) 1.14 (t, 3H), δ 1.37-1.72 (m, 8H), δ 1.81-1.98 (m, 4H), δ 2.22 (t, 2H), δ 2.47 (t, 2H), δ 2.47-2.55 (m, 4H), δ 3.12-3.30 (m, 5H), δ 3.61 (q, 2H), δ 3.66 (s, 2H), δ 3.86 (s, 3H), δ 5.25 (s, IH), δ 5.29 (s, IH), δ 5.50 (t, IH), δ 6.64 (t, 2H), δ 6.85-6.89 (m, 2H), δ 6.97 (t, IH), δ 7.40 (d, IH) . Example 22 5-[l,2]Dithiolan-3-yl-pentanoic acid [6- (4- {6- [ethyl- (2- methoxy-benzyl) -amino] -hexylamino} -3 , 6-dioxo

-cyclohexa-l,4-dienylamino) -hexyl] -amide (4)

Compound 4 was synthesized in 83% yield from LA (0.016 g 0.077 mmol) and 22 (0.035 g 0.072 mmol), adding triethylamine

(30μl), Hydroxybenzotriazole (0.015 g 0.28 mmol), EDCl (0.04 g

0.21 mmol) and again triethylamine (30μl) following the procedure described for 1: ESI-MS: m/z: 673 [M+H ⁺ ]

¹ H-NMR (CDCl ₃ , 300 MHz) 1.10 (t, 3H), δ 1.37-1.72 (m, 22H), δ 1.88-2.01 (m, 4H), δ 2.23 (t, 2H), δ 2.47-2.55 (m, 4H), δ 3.18-3.62 (m, 6H), δ 3.61 (m, 3H), δ 3.86 (s, 3H), δ 5.33 (s, 2H), δ 5.48 (t, IH), δ 6.65 (m, 2H), δ 6.85-6.98 (m, 2H), δ 7.29 (t, IH) , δ 7.40 (d, IH) . Example 23

[6- (4-Hydroxy-3-methoxy-benzylamino) -hβxyl] -carbamic acid benzyl ester (23) To a solution of N-Z-1, 6-diaminohexane (0.50 g, 1.99 mmol) in trimethylorthoformate (20 ml), vanillin (0.30 g, 1.99 mmol) was added. The reaction mixture was stirred for Ih at room temperature, then acetic acid (0.6 ml) and NaCNBH ₃ (0.13 g, 1.99 mmol) were added. After stirring overnight at room temperature, the solvents were evaporated under reduced pressure and the resulting raw material was purified by chromatography. Eluting with CH ₂ Cl ₂ /CH ₃ OH/NH ₃ (9.5:0.5:0.05) .

0.27 g of 23 as colorless oil were obtained. Yield: 35%. ¹ H-NMR

(CDCl ₃ , 200 MHz) δ 1.20-1.59 (m, 8H), δ 2.62 (t, 2H), δ 3.13 (q, 2H), δ 3.69 (s, 2H), δ 3.75 (s, 3H), δ 4.30 (br s exch, 2H), δ 5.09 (s, 2H + IH exch), δ 6.73-6.87 (m complex, 3H) . Example 24 4- [ (6-Amino-hexylamino) -methyl] -2-methoxy-phenol (24) A solution of 23 (0.27 g, 0.70 mmol) in methanol (70 ml), was hydrogenated using a 10% Pd/C catalyst (0.027 g) . The reaction mixture was stirred until the theoretical hydrogen consumption

(8 h) . Following catalyst removal (by filtering over Celite) , the solvent was evaporated affording 0.14 g of 24 as a yellow oil. Yield: 79% . ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.28-1.63 (m, 8H), δ 2.30 (br s, 3H), δ 2.60-2.74 (m, 4H), δ 3.72 (s, 2H), δ 3.89 (s, 3H), δ 6.81-6.88 (m, 3H) . Example 25

2, 5-Bis- [6- (4-hydroxy-3-methoxy-benzylamino) -hexylamino] - [ 1, 4] benzoquinone (25)

To a suspension of 2 , 5-dimethoxyquinone (0.09 g, 0.55 mmol) in boiling EtOH (15 ml ) 24 (0.23 g, 1.1 mmol) was added. The solid progressively precipitating from the reaction mixture after stirring overnight was collected by filtration and further purified by flash chromatography. Eluting with CH ₂ CI ₂ /CH ₃ OH/NH ₃ (9:1:0.1), 0.051 g (18% yield) of 25 as pink crystals were obtained: mp = 172 ⁰ C. EI-MS: 609 (M+H ⁺ ) . ¹ H-

NMR (DMSO, 300 MHz) δ 1.27-1.67 (m, 16H), δ 2.43 (t, 4H), δ

3.20-3.31 (m, 4H), δ 3.55 (s, 4H), δ 3.74 (s, 6H), δ 5.21

(s, 2H), δ 6.70-6.72 (m, 4H), δ 6.88 (s, 2H), δ 7.72 (t exch, 2H), δ 8.79 (br s exch, 2H) . Example 26

3- [ (3-Benzyloxycarbonylamino-propylamino) -methyl] -5- methoxy-indole-1-carboxylic acid tert-butyl ester

A solution of N-Z-I, 3-diaminopropane (0.52 g, 2.5 mmol) and 5- methoxy-1-tert-butoxycarbonyl-indole-3-carboxaldehyde [Chem. Pharm. Bull. 48 1872-1876 (2000)] (0.69 g, 2.5 mmol) in trimethylorthoformate (15 ml) was stirred at room temperature for Ih. Acetic acid (0.6 ml) and NaCNBH ₃ (0.16 g, 2.5 mmol) were added and the resulting mixture was stirred for further 10 min. It was then made basic with NaOH 2N and extracted with CH ₂ Cl ₂ (2 x 25 ml) . The combined and dried organic extracts were evaporated under vacuum affording 0.96 g (82% yield) of 26 as a yellow oil: ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.68 (s, 9H), δ 2.09-2.13 (m, 2H), δ 2.80 (q, 2H), δ 3.35 (q, 2H), δ 3.65 (s, 2H), δ 3.87 (s, 3H). δ 5.09 (s, 2H), δ 5.41 (br s exch, IH), δ 6.95 (dd, IH), δ 7.11 (s, IH), δ 7.30-7.40 (m, 5H), δ 7.53 (s, IH) , δ 8.03 (d, IH) . Example 27

3-{ [ (3-Benzyloxycarbonylamino-propyl) -tert- butoxycarbonyl-amino] -methyl} -5-methoxy-indole-l-carboxylic acid tert-butyl ester (27)

A solution of 26 (0.96 g, 2.05 mmol) in acetonitrile (100 ml) was treated with diterbutyldicarbonate (0.54 g, 2.46 mmol) and 4-dimethlaminopyridine (0.035 g, 0.29 mmol) . The reaction mixture was stirred at 40 ⁰ C for 12h. After cooling, the solvents were evaporated and the resulting pink solid was taken up with CH ₂ Cl ₂ (50 ml) and the solution washed with KHSO ₄ IM (1 x 50 ml) and purified by flash chromatography. Eluting

with petroleum ether/ethyl acetate (7:3) provided 0.9 g (78% yield) of 27 as a yellow gummy solid: ¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.54 (s, 9H), δ 1.59-1.68 (m, HH) , δ 3.06-3.29 (m, 4H), δ 3.86 (s, 3H), δ 4.53 (s, 2H), δ 5.11 (s, 2H), δ 6.95 (dd, IH), δ 7.28-7.36 (m, 6H), δ 7.47 (s,lH), δ 8.01 (d,lH). Example 28

3- { [ (3-amino-propyl) -tert-butoxycarbonyl-amino] -methyl} - 5-methoxy-indole-l-carboxylic acid tert-butyl ester (28)

A solution of 27 (0.9 g, 1.58 mmol) in methanol (70 ml), was hydrogenated using a 10% Pd/C catalyst (0.09 g) . The reaction mixture was stirred until the theoretical hydrogen consumption. Following catalyst removal by filtration through a pad of Celite, the solvent was evaporated affording 0.46 g (68%) of 28 as a colorless oil: ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.54(s, 9H), δ 1.65-1.78 (m, HH) , δ 2.71 (t, 2H), δ 3.24 (br s, 2H), δ 3.86 (s, 3H), δ 4.55 (s, 2H), δ 6.94 (dd, IH), δ 7.48 (s, IH), δ 7.20-7.28 (m,lH) . δ 8.00 (d, IH) . Example 29 2,5-Bis-{3- [ (5-methoxy-N-BOC-indol-3-ylmethyl) -tert- butoxycarbonyl-amino] -propylamine} - [1, 4]benzoguinone (29)

To a solution of 28 (0.059 g, 0.13 mmol) in ethanol (7 ml)

2, 5-dimetoxy-l, 4-benzoquinone (O.Ollg, 0.065 mmol) was added. The reaction mixture was stirred at 50 ⁰ C and then at room temperature overnight . The red solution was evaporated under vacuum and the obtained solid was purified by flash chromatography. Eluting with petroleum ether/ethyl acetate/CH ₃ OH/NH ₃ (7.5:2:0.5:0.05) 0.030 g (48 % yield) of 29 as a reddish oil were obtained: ESI-MS: 994 (M+Na ⁺ ) . ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.35-196 (m complex, 40H), δ 3.08-3.11 (m, 4H), δ 3.26 (m, 4H), δ 3.84 (s, 6H), δ 4.54 (s, 4H), δ 5.21 (s, 2H), δ 6.93 (dd,2H), δ 7.23 (s, 2H), δ 7.46 (s, 2H), δ 7.98 (d, 2H) . Example 30 2 , 5-Bis- {3- [ (5-methoxy-lH-indol-3-ylmethyl ) -amino] - propylamine)} - [1, 4] benzoquinone (30)

A solution of 29 (0.03 g, 0.031 mmol) in CH ₂ Cl ₂ (3 ml) was treated with trifluoroacetic acid (0.5 ml) and then stirred at room temperature for Ih. By addition of ether, a solid was formed which was collected by filtration to give 0.02 g (95% yield) of 30 as a brown solid: mp = 181°C. ES-MS:595 (M+Na ⁺ ) . ¹ H-NMR (CD ₃ OD, 200 MHz) δ 2.01-2.04 (m, 4H), δ 3.14 (t, 4H), δ 3.83 (s, 6H), δ 4.42 (s, 4H), δ 5.35 (s, 2H), δ 6.89 (d, 2H), δ 7.21 (s, 2H), δ 7.32 (d, 2H), δ 7.44 (s, 2H) . Example 31 {6- [ (6-Hydroxy-5, 7, 8-trimethyl-chroman-2-ylmethyl) -amino] - hexyl}-carbamic acid benzyl ester (31)

Compound 31 was obtained in 35% yield from Trolox aldehyde

[Bioorg. Med. Chem. Lett. 15 3012-3015 (2005)] and N-Z-I, 6- diaminohexane, following the procedure described for 23.

Example 32

2- [ (6-Amino-hexylamino) -methyl] -5, 7, 8-trimethyl-chroman-6-ol

(32)

Compound 32 was obtained from 31 by catalytic hydrogenation following the procedure described for 24.

Example 33

2,5-Bis-{6- [ (6-hydroxy-2,5,7 _# 8-tetramethyl-chroman-2- ylmethyl) -amino] -hexylamino}- [ 1, 4 ] benzoquinone (33)

Compound 33 was synthesized from 32 and 2, 5-dimethoxy-l, 4- benzoquinone following the procedure described for 25. ESI- MS: 773 (M+H ⁺ ) . '

Example 34

(R) - (3-tert-Butoxycarbonylamino-propyl) - {3- [2- (2-fluoro- biphenyl-4-yl)-propionylamino] -propyl}-carbamic acid tert- butyl ester (34)

A solution of (3 -amino-propyl) - (3-tert-butoxycarbonylamino- propyl) -carbamic acid tert-butyl ester [European Journal of Medicinal Chemistry, 38 (2003), 117-122] (0.41 g, 1.23 ramol) in dry THF (15 ml), was treated with triethyl amine (0.124 g, 1.23 mmol . ) Stirring at O ⁰ C under inert atmosphere (R) -(- ) -2-fluoro-α-mehyl-4- biphenyl-acetic acid [(R)- flurbiprofen] (0.3 g,1.23 mmol) and EDCI (0.24 g, 1.23 mmol) were added. The reaction mixture was stirred for 20' at 0 ⁰ C and then at room temperature for one day. After removal of the solvent, the residue was purified by flash chromatography. Eluting with CH ₂ C1 ₂ /CH ₃ OH (9:1) afforded 0.36 g (52% yield) of 34 as a colorless oil: ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.45(s, 18H), δ 1.56 (d, 3H), δ 1.63-1.66 (m, 4H), δ 3.08-3.19 (m, 8H), δ 3.61 (q, IH), δ 7.16-7.22 (m, 2H), δ 7.37-7.56 (m complex, 6H) . Example 35

(R) N- [3- (3-Amino-propylamino) -propyl] -2- (2-fluoro-biphenyl- 4-yl ) -propionamide (35)

A solution of 34 (0.36 g, 0.64 mmol) in methylene chloride

(15 ml) kept at 0 ⁰ C was treated with trifluoroacetic acid (0.36 g, 3.2 mmol), which was slowly added. After stirring at room temperature overnight, the solvents were eliminated

and the obtained residue was purified by flash chromatography. Eluting with a step gradient from CH ₂ Cl ₂ /CH ₃ OH/NH ₃ (7:3:0.3) to CH ₂ Cl ₂ /CH ₃ OH/NH ₃ (5:5:0.5) afforded 0.13 g of 35 (56% yield) as a colorless oil: ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.50(d, 3H), δ 1.57-1.66 (m, 4H), δ 2.58- 2.78 (m, 8H), δ 3.29 (q, IH), δ 7.12-7.52 (m complex, 8H) . Example 36

(R) -2- (2-Fluoro-biphenyl-4-yl) -N- (3-{3- [4- (3- {3- [2- (2- fluoro-biphenyl-4-yl) -propionylamino] -propylamin 0} -propylamino) -3, 6-dioxo-cyclohexa-l,4-dienylamino] - propylamino} -propyl ) -propionamide (36)

A solution of 35 (0.13 g, 0.36 mmol) in ethanol (10 ml) was treated with 2 , 5-dimethoxy-l, 4-benzoquinone (0.030 g, 0.18 mmol) . The reaction mixture was stirred at 50 ⁰ C for 3h and then at room temperature overnight. An orange solid precipitated which was collected by filtration affording 0.055 g (37% yield) of 36: mp: 136 ⁰ C; ES-MS: 819; ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.55 (d, 6H), δ 1.66-1.81 (m, 8H), δ 2.58-2.74 (m, 8H), δ 3.20 (q, 4H), δ 3.34-3.47 (m, 4H), δ 3.58 (q,2H), δ 5.29 (s, 2H), δ 6.98 (br t exch, IH), δ 7.13-7.56 (m complex, 16H), δ 8.46 (br exch, 2H) . Example 37 {3- [2- (2-Fluoro-biphenyl-4-yl) -propionylamino] -propyl}- carbamic acid benzyl ester (37)

A solution of (±) -flurbiprofen (0.87 g, 3.56 mmol) in chloroform (15 ml) was treated with thionyl chloride (1.27 g, 10.7 mmol) and stirred at 60 ⁰ C for 2h. Elimination of the solvents afforded a residue which was dissolved in chloroform (20 ml) and treated with triethyl amine (0.5 ml, 3.56 mmol) and 6 (0.74 g, 3.56 mmol). After stirring at room temperature overnight, the reaction mixture was washed with water (30 ml), K ₂ CO ₃ 20% (30 ml) and HCl 1 N (30 ml). Evaporation of the dried organic phase afforded a residue which was purified by flash chromatography. Eluting with ethyl acetate/petroleum ether (7:3) afforded 0.21 g of 37

(14% yield) as a white solid: ¹ H-NMR (CDCl ₃ , 200 MHz) δ

1.57-1.65 (m, 5H), δ 3.20 (q , 2H), δ 3.31 (q, 2H), δ 3.60 (q, IH), δ 5.11 (s, 2H), δ 5.23 (br s exch, IH), δ 6.12 (br s exch, IH), δ 7.13-7.59 (m,13H). Example 38

N- (3-aimnino-propil) -2- (2-fluoro-bifenil-4-il) -propionammide (38)

A solution of 37 (0.21 g, 0.48 mmol) in methanol (60 ml) was hydrogenated using a 10% Pd/C catalyst (0.021 g) . The reaction mixture was stirred until the theoretical hydrogen consumption. Following catalyst removal through a Celite pad,

the solvent was eliminated under reduced pressure to give 0.13 g of 38 (93% yield) as a yellow oil: ¹ H-NMR (CDCl ₃ , 200 MHz) δ 1.58-1.63 (m, 5H + 2H exch) , δ 2.78 (q, 2H), δ 3.38 (t, 2H), δ 3.49-3.63 (m, IH), δ 6.54 (br s exch, IH), δ 7.15-7.61 (m complex, 9H) . Example 39

2-(2-Fluoro-biphenyl-4-yl)-N-[3-(4-{3-[2-(2-fluoro-biphβnyl - 4-yl) -propionylamino] -propylamino}-3, 6-dioxo-cyclohexa-l _# 4- dienylamino) -propyl] -propionamide (39 )

A solution of 38 (0.13 g, 0.43 mmol) in ethanol (7 ml), was treated with 2 , 5-dimethoxy-l, 4-benzoquinone (0.037 g, 0.22 mmol) . The reaction mixture was stirred at 50 ⁰ C for 3h and then at room temperature overnight. An orange solid precipitated which was collected by filtration and recrystalized from ethanol/chloroform affording 0.043 g (29% yield) of 39 as a red solid: mp: 178 ⁰ C; ES-MS: 727 (M+Na ⁺ ) . ¹ H-NMR (CDCl ₃ , 300 MHz) δ 1.58-1.60 (s, 10H), δ 1.81-1.85 (m, 4H), δ 3.16 (q, 4H), δ 3.23-3.38 (m, 4H), δ 3.62 (q, 2H), δ 5.28 (s _f 2H), δ 5.57 (br t exch, 2H), δ 6.76 (br t exch, 2H), δ 7.19 (t, 4H), δ 7.29-7.60 (m complex, 12H) .

Determination of the inhibitory potency towards HuAChE and BChE The biological activity of compounds under examination, expressed as IC ₅₀ values, was tested following the spectrofluorometric Ellman's method [Ellman G. L., Courtney

K. D., Andrei V., Featherstone R.M. Biochem. Pharmacol. 1961, 7, 88-95] towards human recombinant acetylcholinesterase (E. C.

3.1.1.7) (AChE o HuAChE) and butyrilcholinesterase (E. C.

3.1.1.8) (BChE) from human serum. IC ₅₀ values represent the concentration of inhibitor required to decrease the enzyme activity by 50% and are the mean of two independent experiments, each one in duplicate.

The synthesized compounds (1, 2, 3, 4, 25, 30, 33, 36 e 39, in particular 36 and 39) show interesting IC ₅₀ AChE and IC ₅₀ BChE, values, at least comparable with that of anti-AD drug tacrine.

Example 33

Inhibitory effect on β-amyloid(l-40) aggregation induced by human recombinant AChE

The ability of compounds to inhibit the proaggregating action of AChE toward β-amyloid(l-40) was assessed through a thioflavin T-based fluorometric assay. (Bartolini, M. ;

Bertucci, C; Cavrini, V.; Andrisano, V. β-Amyloid aggregation induced by human acetylcholinesterase: inhibition studies.

Biochem. Pharmacol. 2003, 65, 407-416). Compounds were tested at the fixed concentration of 100 μM.

The synthesized compounds (1, 2, 3, 4, 25, 30, 33, 36 e 39, in particular 36 e 39) show interesting % of inhibition towards amyloid (1-40) aggregation induced by human recombinant AChE.

Example 34 The intracellular antioxidant activity was evaluated against formation of ROS in human neuronal-like cells (SH-SY5Y) after treatment with t-BuOOH, a compound used to induce oxidative

(the cell cultures were treated as described in example 26)

ROS intracellular formation was determined by using a fluorescent probe, DCFH-DA, as described by Wang H. et al . (H. Wang, J. A. Joseph, Free Radic. Biol. Med. 1999, 27, 612) . The synthesized compounds (1, 2, 3, 4, 25, 30, 33, 36 e 39) , in particular 1, 2, 3, 4, 25, 30 e 33, show an interesting antioxidant profile. Example 35

Toxicity effects of the synthesized compounds (in particular 1, 2, 3, 4, 25, 30, 33, 36 e 39) and LA were preliminarily determined by the MTT colorimetric method in SH-SY5Y cell lines, resembling the human cells, as described by Mosmann et al . (Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55-63) . The measurement were performed by a spectrophotometer (TECAN ^® , Spectra model Classic, Salzburg, Austria) at a wavelength of 405 nm. Cell viability was expressed as percentage over control and calculated by the formula Ft/Fnt x 100, where Ft = absorbance of treated neurones and Fnt = absorbance of untreated neurones.

Human neuronal-like cells, SH-SY5Y, were routinely grown at

37°C in a humidified incubator with 5% C02 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum

(FCS) , 2 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin.

SH-SY5Y cell treatment by the synthesized compounds (0.1-50 μM) does not affect cellular viability. Vice versa, compounds

are able to protect SH-SY5Y cells by Aβ (1-42) -induced toxicity.

Example 36 The anti-aggregating activity of the synthesized compounds (in particular 1, 2, 3, 4, 25, 30, 33, 36 e 39) towards Aβ(l-42) peptide was determined through a thioflavin T-based fluorometric assay. [Bartolini et al . , ChemBioChem, 2007, in press] . The synthesized compounds show an interesting anti- amyloid profile. Example 37

NQOl (Sigma) specificity was tested. The synthesized compounds (in particular 1, 2, 3, 4, 25, 30, 33, 36 e 39) were tested following absorbance variation of cytochrome c, as secondary acceptor. Each reaction consisted of NADH, NQOl, and the tested compounds in a final volume of Tris-HCl buffer containing bovine serum albumin. Reactions were started by the addition of NADH. The time course of the reaction was followed by monitoring the decrease of cytochrome c at 550-540 nm. Menadione was used as a reference compound.

The synthesized compounds showed interesting behaviors. Example 38

The synthesized compounds (in particular 1, 2, 3, 4, 25, 30, 33, 36 e 39) were in vivo tested, following the method described within PCT/IT03/00227 (in particular see example 55) , which is here integrally recalled for the sake of completeness in description. The synthesized compounds showed interesting in vivo profiles.