Nitric oxide releasing GABA analogs for the treatment of neuropathic pain.

The present invention relates to nitrooxyderivatives of Gamma-aminobutyric acid analogs (GABA analogs) for treating neuropathic pain and in particular diabetic neuropathy. The present invention also relates to pharmaceutical formulation comprising such derivatives, to a process for their preparation .

Neuropathic pain is a form of chronic pain that can be classified as peripheral or central. Peripheral neuropathic pain is caused by injury or infection of peripheral sensory nerves, whereas central neuropathic pain is caused by damage to the CNS or/and the spinal cord. Both peripheral and central neuropathic pain can occur without obvious initial nerve damage

Neuropathic pain is common in cancer as a direct result of cancer on peripheral nerves (e.g., compression by a tumor), or as a side effect of chemotherapy, radiation injury or surgery.

The clinical causes of neuropathic pain are widespread and include both trauma and disease. Various metabolic diseases may be the cause of neuropathic changes and may subsequently be implicated in neuropathic pain. An example of such a neuropathy is diabetic neuropathy, which occurs in a large number of patients suffering from diabetes and may be associated with a large number of clinical symptoms such as a feeling of numbness, tingling sensation, or pain. The most common form of diabetic neuropathy is distal symmetrical sensomotor polyneuropathy

There are a wide range of treatments available to physicians for neuropathic pain, some of which have specific indications, and some of which are used off-label.

Common analgesics such as opioids and NSAIDs, insufficiently address chronic abnormal pain syndromes such as peripheral and central neuropathic pain due to insufficient efficacy or limiting side effects, although a subset of patients with neuropathic pain responds to opioids. In the search for alternative treatment regimes to produce satisfactory and sustained pain relief, corticosteroids, conduction blockade, glycerol, antidepressants, local anesthetics, gangliosides and electrostimulation have been tried. Anticonvulsants have been found useful against various types of peripheral neuropathic pain conditions.

Regarding the treatment of neuropathic pain in diabetic patients, amitriptyline, gabapentin and pregabalin are among the primary treatments for painful diabetic neuropathy (PDN) , and it is clear that they produce beneficial effects, but there are questions about these treatments that have not been adequately addressed. For example, although there is a growing consensus that the therapeutic effects of amitriptyline in pain patients are independent of its effects on mood, it is not clear that amitriptyline has specific and direct effects on pain. There is also a fairly broad consensus that gabapentin is safe and well tolerated, but the side-effect profile of gabapentin has not been adequately assessed in pain populations

Pregabalin induces weight gain in Type I or II diabetes patients by edema formation. Increased weight is an established risk factor for cardiovascular disease, particularly in Type II diabetic patients.

WO 01/90052 discloses gabapentin analogues wherein the amine and/or the acid group is substituted with a labile organic group that becomes removed in the human or animal body. Said compounds, when administered to humans or animals, provide an increased duration of active compound in the plasma.

WO 03/000642 discloses Nitric oxide (NO) releasing derivatives of drugs used for the treatment of neuropathic pain, including GABA analogs. The nitric-oxide releasing part of the molecule is attached to the carboxyl group of the precursor drugs.

In particular, WO 03/000642 discloses nitrooxy derivatives of gabapentin that showed good efficacy in models of pain but they are unstable and they are rapidly converted upon contact to aqueous medium, such as plasma, to the gabapentin gamma- lactam that is toxic.

Consequently, formation of gamma-lactam during formulation and/or storage and/or administration of the drug must be minimized for safety reasons.

WO 02/100347 discloses prodrugs of the GABA analogs such as gabapentin and pregabalin; said compounds show a better bioavailability than their precursors gabapentin and pregabalin respectively.

WO 2004/054965 discloses nitrooxyderivatives of drugs for chronic pain including gabapentin. These compounds show a good efficacy in animal models of pain, however these compounds exhibit poor oral absorption consequently they require high doses to achieve a therapeutic concentration in the systemic circulation. These high doses prevent the successful clinical use of these compounds.

Despite the broad spectrum of drugs available with different modes of action, at present there is no effective treatment for neuropathic pain and in particular for painful diabetic neuropathy that addresses all the clinical unmet needs such as drugs having a reduced time to onset of action, an improved efficacy or an improved side effects profile.

It was thus object of the present invention to provide drugs having an improved therapeutic profile and/or lower side effects in the treatment of chronic pain, in particular neuropathic pain. It has been now surprisingly and unexpectedly found that this problem can be solved with the class of GABA analogs nitrooxyderivatives described below.

The present invention relates to GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or

or Rc is pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is selected from the group consisting of:

1) - (R ^u ) -CH (ONO 2,)- R ^~ /

2) - (R ^u ) -CH (ONO,) -CH (ONO,) R

3) - (R ^u ) _n - (T) _m -X- (Q)p- (CH ₂ ) _q -CH(ON0 ₂ )R

4) - (R°) - (T) -X- (Q)p- (CH,) -CH (ONOJ CH (ONOJ R

5)

ONO

7) - (R°) _n -CH(ON0 ₂ ) - (CH ₂ ) _ql -X- (CH ₂ ) _q2 CH (ON0 ₂ ) R ¹

8) - (R°) _n - (CR ⁶ R ⁷ ) - (CH ₂ ) _q CH (ON0 ₂ ) R ¹

wherein

n is 0 or 1,

m is 0 or 1,

p is 0 or 1,

nl is 0 or 1;

q is an integer from 0 to 10, preferably q is from 0 to 6;

ql is an integer from 1 to 10, preferably q is from 1 to 6; q2 is an integer from 1 to 10, preferably q is from 1 to 6; t is an is an integer from 1 to 4, preferably t is 2 or 3;

R° is a straight or branched Ci-Cio alkylene; preferably R° is a straight or branched C1-C6 alkylene, more preferably R° is

-C(CH ₃ ) ₂ - or -C(CH ₃ ) ₂ -CH ₂ -;

R ¹ is selected from hydrogen, a straight or branched C1-C4 alkyl, -(CH ₂ )OCH ₃ , preferably R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ , -CH(CH ₃ ) ₂ ;

R ^la is hydrogen or a straight or branched C1-C4 alkyl, preferably R ^la is hydrogen or -CH ₃ ;

X is selected from an oxygen atom, -NH-, -N(CH ₃ )- or a covalent bond, preferably X is oxygen or a covalent bond;

2 3 2 3

T and Q are -CR R -, wherein R and R at each occurrence are independently selected from hydrogen, C ₁ -C ₄ linear or branched alkyl, OR ⁴ , NHR ⁵ , preferably R ² and R ³ are hydrogen or R ² is hydrogen and R ³ is NHR ⁵ ;

R ⁴ is hydrogen or C ₁ -C ₄ linear or branched alkyl, preferably R ⁴ is hydrogen or -CH ₃ ;

R ⁵ is hydrogen, C ₁ -C ₄ linear or branched alkyl, -C(0)CH ₃ or -C (0) OC (CH ₃ ) 3 (Boc) , more preferably R ⁵ is -C(0)CH ₃ or

-C(0)OC(CH ₃ ) ₃ ;

R ⁶ is hydrogen or C ₁ -C ₄ linear or branched alkyl, preferably R ⁶ is -CH ₃ ;

R ⁷ is (CH ₂ ) _q CH (ONO ₂ ) R ¹ wherein q and R ¹ are as above defined.

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

1) - (R°) _n -CH (ON0 ₂ ) R ¹

wherein

n is 0 or 1

R° is a straight or branched C ₁ -C6 alkylene, preferably R° is -C(CH ₃ ) ₂ - or -C(CH ₃ ) ₂ -CH ₂ -;

R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ , or -CH(CH ₃ ) ₂ .

In one embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

3) - (R°) _n - (T) _m -X- (Q)p- (CH^ _g -CHiO O^R ¹

wherein

n is 0 or 1

m is 1

p is 1

q is from 0 to 6;

R° is a straight or branched C ₁ -C6 alkylene, more preferably R° is -C(CH ₃ ) ₂ - or -C(CH ₃ ) ₂ -CH ₂ -;

R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ , or -CH(CH ₃ ) ₂ ;

X is oxygen;

2 3 2 3

T and Q are -CR R -, wherein R and R are hydrogen.

In one embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

3) - (R°) _n - (T) _m -X- (Q)p- (CH^ _g -CHiO O^R ¹

wherein

n is 0 or 1; m is 0 ;

p is 1;

q is from 0 to 6;

R° is a straight or branched C ₁ -C6 alkylene;

R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ , or -CH(CH ₃ ) ₂ ;

X is a covalent bond;

Q is -CR ² R ³ -, wherein R ² is hydrogen and R ³ is NHR ⁵ wherein R ⁵ is -C(0)CH ₃ or -C (0) OC (CH ₃ ) ₃ .

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) 2CHCH2-, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

2) - (R°) _n -CH (ON0 ₂ ) -CH (ON0 ₂ ) R ^la

wherein

n is 1 ;

R° is a straight or branched C ₁ -C ₁ 0 alkylene;

R ^la is hydrogen or -CH ₃ ;

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) : (I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

la

4) - (R ^u ) - (T) -X- (Q) _p - (CH _? ) -CH(ONO _? )CH(ONO _? )R

wherein

n is O or l;

m is 1 ;

p is 1;

q is from 0 to 6;

R° is a straight or branched C ₁ -C6 alkylene, more preferably R' is -C(CH ₃ ) ₂ - or -C (CH ₃ ) ₂ -CH ₂ - ;

X is oxygen;

2 3 2 3

T and Q are -CR R -, wherein R and R are hydrogen;

R ^la is hydrogen or -CH ₃ .

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

6)

wherein

n is 0 ;

m is 0 ;

p is 0;

nl is 0 or 1;

q is an integer from 0 to 10;

t is 2 or 3;

R° is a straight or branched Ci-Cio alkylene;

X is a covalent bond.

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH3) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

5)

wherein

n is 0 ;

m is 0 ; p is 0 ;

nl is 0 or 1;

q is an integer from 0 to 10,

t is 2 or 3;

R° is a straight or branched Ci-Cio alkylene;

X is a covalent bond.

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

7) - (R°) _n -CH(ON0 ₂ ) - (CH ₂ ) _ql -X- (CH ₂ ) _q2 CH (ON0 ₂ ) R ¹

wherein

n is 0;

ql is an integer from 1 to 10;

q2 is an integer from 1 to 10;

X is oxygen or a covalent bond;

R ¹ is selected from hydrogen, a straight or branched C ₁ -C ₄ alkyl, -(CH ₂ )OCH ₃ , preferably R ¹ is H.

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) 2 CHCH2 - , or Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is

8) - (R°) _n - (CR ⁶ R ⁷ ) - (CH ₂ ) _q CH (ON0 ₂ ) R ¹

wherein

n is 0 ;

q is an integer from 0 to 10, preferably q is from 0 to 6;

R ⁶ is hydrogen or C ₁ -C ₄ linear or branched alkyl, preferably R ⁶ is -CH ₃ ;

R ⁷ is ( C¾ ) _q CH ( ONO2 ) R ¹ wherein q is as above defined

R ¹ is hydrogen.

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or

or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH ₃ ) ₂ CHCH ₂ -, or Ra and Rb together with the carbon atom to which they are attached a cyclohexyl;

R is selected from the group consisting of:

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra is hydrogen and Rb is (CH3) ₂ CHCH ₂ - ;

R is selected from the group consisting of:

CH ₃

In another embodiment of the invention, there is provided GABA analogs nitrooxyderivatives or salts or stereoisomers thereof having the general formula (I) :

(I)

wherein

Rc is a hydrogen, or a pharmaceutically acceptable cation;

Ra and Rb taken together with the carbon atom to which they are attached form a cyclohexyl;

R is selected from the group consisting of:

In another embodiment, the compound of formula (I) is selected from the roup of compounds shown below:

(3) (4)





(3i: (32:

(33: (34:

(35;

(36;

(37:

39)

(40) .

The compounds of the present invention have a better efficacy than the correspondent parent drugs in reducing neuropathic pain and, in particular, in reducing neuropathic pain associated with diabetes.

The compounds of the inventions have a better efficacy than gabapentin and pregabalin in counteracting the development of pain sensitization in painful diabetic neuropathy.

The compounds of the present invention induce less side effects such as less sedation than the correspondent parent drugs .

Moreover the compounds of the present inventions have a good bioavailability and better plasma stability, namely they do not form gabapentin lactam, than other known gabapentin derivatives. Pharmaceutically acceptable cations includes ammonium cation, alkali metal salts such sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, cations of organic bases such as dicyclohexylamine , N-methyl-D- glucamine, and cations of amino acids such as arginine, lysine .

In another embodiment, of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) and/or a salt or stereoisomer thereof and at least a pharmaceutically acceptable excipient. The pharmaceutical composition can be used for oral or systemic administration of the compound of formula ( I ) .

The term "excipient" is used herein to describe any ingredient other than the compound (s) of the invention. The choice of the excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on the stability, and the nature of the dosage form.

In another embodiment of the invention, there is provided a pharmaceutical composition wherein the compound of the invention is administered as a solution, suspension or emulsion in an acceptable vehicle.

In one embodiment, the invention relates to the use of the GABA analogs nitrooxyderivatives of formula (I) for treating or preventing a neuropathic pain condition, such as restless legs syndrome (RLS) , in particular to treating or preventing a diabetes-associated neuropathic pain.

In one embodiment, the invention relates to the use of the GABA analogs nitrooxyderivatives of formula (I) for treating or preventing a diabetes-associated neuropathic pain.

In one embodiment, the invention relates to a method for treating or preventing a neuropathic condition in a subject, the method consisting essentially of administering to the subject at least one GABA analogs nitrooxyderivatives of formula ( I ) .

In one embodiment, the invention relates to a method for treating or preventing diabetes-associated neuropathic pain in a subject, the method consisting essentially of administering to the subject at least one GABA analogs nitrooxyderivatives of formula ( I ) .

In one embodiment, relates to GABA analogs nitrooxyderivatives of formula (I) in neutral form, the form of acid, in the form of a salt, preferably a physiologically acceptable salt, in the form of a solvate or of a polymorph and/or in the form of its racemate, pure stereoisomers, especially enantiomers or diastereomers or in the form of mixtures of stereoisomers, especially enantiomers or diastereomers, and/or in any mixing ratio.

In yet another embodiment of the invention, there is provided a process for the preparation of compounds of formula (I) or stereoisomers thereof

(I)

wherein

Rc is a hydrogen, or

or Rc is the pharmaceutically acceptable cation as described above ; Ra is hydrogen and Rb is (C¾) 2CHCH2-, or Ra and Rb together with the carbon atom to which they are attached a cyclohexyl ring;

R is selected from the group consisting of:

1) - (R -CH(ON0 )R ,

la

2) (R -CH (ONO,) -CH (ONO,) R

3) (R (T) _m -X- (Q)p- (CH ₂ ) _q -CH(ON0 ₂ )R ,

la

4) (R (T) -X- (Q)p- (CH,) -CH (ONO,) CH (ONO,) R

5)

ONO

6)

(R°) - (T) -X- (Q) - (CH ₂ 2) ^/

7) - (R ^u ) -CH (ONO,) - (CH,) _η -X- (CH 2 I q2 CH (ONO,) R

1

8) - (R°) ( CR ^D R ' ) - (CH,)„CH (ONO,) R

wherein

n is 0 or 1,

m is 0 or 1,

p is 0 or 1,

nl is 0 or 1;

q is an integer from 0 to 10, preferably q is from 0 to 6; ql is an integer from 1 to 10, preferably q is from 1 to 6 q2 is an integer from 1 to 10, preferably q is from 1 to 6 t is an is an integer from 1 to 4, preferably t is 2 or 3; R° is a straight or branched C1-C10 alkylene; preferably R° straight or branched C1-C6 alkylene, more preferably R° is -C (CH ₃ ) ₂ - or -C (CH ₃ ) ₂ -CH ₂ -; R is selected from hydrogen, a straight or branched C1-C4 alkyl, -(CH ₂ )OCH ₃ , preferably R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ , -CH(CH ₃ ) ₂ ;

Rla is hydrogen or a straight or branched C1-C4 alkyl, preferably R ^a is hydrogen or -CH ₃ ;

X is selected from an oxygen atom, -NH-, -N(CH ₃ )- or a covalent bond, preferably X is oxygen or a covalent bond;

2 3 2 3

T and Q are -CR R -, wherein R and R at each occurrence are independently selected from hydrogen, C ₁ -C ₄ linear or branched alkyl, OR ⁴ , NHR ⁵ , preferably R ² and R ³ are hydrogen or R ² is hydrogen and R ³ is NHR ⁵ ;

R ⁴ is hydrogen or C ₁ -C ₄ linear or branched alkyl, preferably R ⁴ is hydrogen or -CH ₃ ;

R ⁵ is hydrogen, C ₁ -C ₄ linear or branched alkyl, -C(0)CH ₃ or -C (0) OC (CH ₃ ) 3 (Boc) , more preferably R ⁵ is -C(0)CH ₃ or

-C(0)OC(CH ₃ ) ₃ ;

R ⁶ is C ₁ -C ₄ linear or branched alkyl or hydrogen, preferably R ⁶ is -CH ₃ ;

R ⁷ is (C¾) qCH (ONO2) R ¹ wherein q and R ¹ are as above defined, said process, as depicted in Scheme 1

M 0 CH ₃ 0 CH ₃ 0

R ο0-X x ₁ + AX0Xz AΑ0λ0λR

iii)

(VI) _(v) (II)

(VII) (vi)

Scheme 1 comprises :

i) reacting a compound of formula (II)

(ID

wherein A is an activating group selected from the formulas (2a) - (2h)

(2a) (2b) (2c)

(2d) (2e; (2f)

(2g) (2h)

with a compound of formula (III) or (IV)

(III) (IV)

wherein P is a carboxylic protecting group such as a t-butyl group, or a silyl protecting group, in an aprotic polar/non polar solvent in the presence of an organic or inorganic base; ii) removing the protective group P by acidic work-up or with known methods depending on the group P to obtain a compound of formula (I) wherein Rc is H and all the other variable are as previously defined

(I)

iia) optionally reacting the compound of formula (I) with a compound (XIII)

(XIII)

wherein Xo is CI, Br or I, in the presence of an inorganic or organic base, in an aprotic polar/non polar, to obtain a compound of formula (I) wherein Rc is

iib) optionally reacting the compounds of formula (I) wherein Rc is H with the appropriate base to obtain a compound of formula (I) wherein Rc is the pharmaceutically acceptable cation;

iii) reacting a compound of formula (VI)

RCOOXi (VI)

wherein R is as above defined and Xi is Ag ⁺ or Hg ⁺ ,

with a compound of formula (V) (V)

wherein A is as above defined, Xo is CI, Br or I, in a molar ratio of compounds of formulas (VI) : (V) from 2 to 0.5, in an organic solvent to obtain a compound of formula (II)

(ID

wherein A and R are as above defined;

iv) reacting a nitrate carboxylic acid of formula (VII)

R-COOH (VII)

wherein R is as above defined, with Ag ₂ 0 or Hg ₂ 0 in a molar ratio compound of formula (VII) : (Ag ₂ 0 ) or Hg ₂ 0 of from 1 to 0.5 in an organic solvent, to obtain the carboxylic acid salt of formula (VI)

R-COOXi (VI)

wherein Xi is Ag ⁺ or Hg ⁺ .

In step i) the silyl protecting group is trimethylsilyl or tert-butyldimethylsilyl, the organic or inorganic base is selected from pyridine or triethylamine or C S 2CO ₃ , the aprotic polar/non polar solvent is for example THF, DMF or CH2C I 2 . The reaction is carried out at temperature ranging from -80°C to 60°C.

In step iia) the inorganic or organic base is selected from C S 2CO ₃ or pyridine or triethylamine. The aprotic polar/non polar solvent can be THF, DMF or CH2C I 2 . The reaction is carried at temperature ranging from -80°C to 60°C,

Compounds (XIII) of step iia) are commercially available or synthesized according to methods well known in the art.

In step iii) the reaction is typically carried out in a temperature range from about -20°C to about 120°C, preferably from 10°C to 30°C. Generally, the reaction is carried out in an organic solvent, generally an aprotic solvent, such as toluene, methylene chloride, tetrahydrofurane or chloroform or dipolar solvents such as, dimethylformamide (DMF) , N- methylpyrrolidone, acetonitrile or in a mixture thereof, depending on the solubility of the compounds involved in the reaction; the preferred solvent are toluene, methylene chloride, THF, a mixture of methylene chloride and THF.

Compounds of formula (V) of step iii) are prepared by known compounds following procedures well known in the art (Jose Alexander,* Robyn Cargill, Stuart R. Michelson, and Harvey Schwamt, (Acyloxy) alkyl Carbamates as Novel Bioreversible Prodrugs for Amines: Increased Permeation through Biological Membranes, J. Med. Chem., 1988, 31, 318-322)

In step iv) the organic solvent is an aprotic solvent, such as toluene, methylene chloride, or chloroform or polar/dipolar solvents such as acetonitrile, water, tetrahydrofurane, dimethylformamide (DMF) , N-methylpyrrolidone, or in a mixture thereof, depending on the solubility of the compounds involved in the reaction; the preferred solvent are acetonitrile, water a mixture of acetonitrile and water.

The reaction is generally carried out in a temperature range from about -20°C to about 80°C, preferably from 10°C to 30°C in the dark.

The compound of general formula (VII) of step iv) are known in the literature or can be obtained from the corresponding compounds of formula (VIII)

R'COOH (VIII)

wherein R' is selected from the group consisting of:

2) - (R°) _n -CH (OH) -CH (OH) R ^la

3) - (R°) _n - (T) _m -X- (Q)p-

4) - (R°) _n - (T) _m -X- (Q) _p - (CH ₂ ) _q -CH(OH)CH(OH)R ^la ,

5)

6)

(R°) _n -(T) -X- ( Q ) -(CH,)

7) (R°) _n -CH (OH) - (CH ₂ ) _ql -X- (CH ₂ ) _q2 CH (OH) R ¹

8 (R°) - (CR ⁶ R ⁷ ) - (CHJ CH (OH) R ¹

wherein

n is 0 or 1 ;

m is 0 or 1 ;

p is 0 or 1 ;

nl is 0 or 1 ;

q is an integer from 0 to 10, preferably q is from 0 to 6;

ql is an integer from 1 to 10, preferably q is from 1 to 6; q2 is an integer from 1 to 10, preferably q is from 1 to 6; t is an is an integer from 1 to 4, preferably t is 2 or 3;

R° is a straight or branched Ci-Cio alkylene; preferably R° is a straight or branched C ₁ -C6 alkylene, more preferably R° is

-C(CH ₃ ) ₂ - or -C(CH ₃ ) ₂ -CH ₂ -;

R ¹ is selected from hydrogen, a straight or branched C ₁ -C4 alkyl, -(CH ₂ )OCH ₃ , preferably R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ ,

-CH(CH ₃ ) ₂ ;

R la is hydrogen or a straight or branched C ₁ -C4 alkyl, preferably R ^a is hydrogen or -CH ₃ ;

X is selected from an oxygen atom, -NH-, -N(CH ₃ )- or a covalent bond, preferably X is oxygen or a covalent bond;

2 3 2 3

T and Q are -CR R -, wherein R and R at each occurrence are independently selected from hydrogen, C ₁ -C ₄ linear or branched alkyl, OR ⁴ , NHR ⁵ , preferably R ² and R ³ are hydrogen or R ² is hydrogen and R ³ is NHR ⁵ ; R ⁴ is hydrogen or C ₁ -C ₄ linear or branched alkyl, preferably R ⁴ is hydrogen or -CH ₃ ;

R ⁵ is hydrogen, C ₁ -C ₄ linear or branched alkyl, -C(0)CH ₃ or -C (0) OC (CH ₃ ) 3 (Boc) , more preferably R ⁵ is -C(0)CH ₃ or

-C(0)OC(CH ₃ ) ₃ ;

R ⁶ is C ₁ -C ₄ linear or branched alkyl or hydrogen, preferably R ⁶ is -CH ₃ ;

R ⁷ is (C¾) _q CH (OH) R ¹ wherein q and R ¹ are as above defined, by reacting with nitric acid and acetic anhydride in a temperature range from -50°C to 0°C, or by reacting with triflic anhydride/tetraalkylammonium nitrate salt in an aprotic polar/non polar solvent such as DMF, THF or CH2CI2 at temperature ranging from -80°C to 65°C

Compounds (VIII) are commercially available or are known in the literature or can be prepared by known compounds by methods well known in the art.

Alternatively, compounds (VII) can be prepared by reacting compound (IX)

R"COOH (IX)

wherein R" is selected from the group consisting of:

2) -(R°)i _n -CH(Xo)-CH(X ₀ Ra ¹

3) -(R°)i _n -(T) _m -X-(Q) _p -

4) -(R°)i _n -(T) _m -X-(Q) _p - (CH ₂ )i _q -CH(Xo)CH(X ₀ )R ^la ,

5)

- (R°) - (T) -X- (Q) - (CH 2 I

6)

7) - (R ^u ) _n -CH(X ₀ ) - (CH ₂ ) _ql -X- (CH ₂ ) _q2 CH(X ₀ )R

8) - (R°) _n - (CR ⁶ R ⁷ ) - (C^J _g CHiXoJR ¹

wherein

n is 0 or 1 ;

m is O or l;

p is 0 or 1 ;

nl is 0 or 1;

q is an integer from 0 to 10, preferably q is from 0 to 6;

ql is an integer from 1 to 10, preferably q is from 1 to 6; q2 is an integer from 1 to 10, preferably q is from 1 to 6; t is an is an integer from 1 to 4, preferably t is 2 or 3;

R° is a straight or branched Ci-Cio alkylene; preferably R° is a straight or branched C ₁ -C6 alkylene, more preferably R° is

-C(CH ₃ ) ₂ - or -C(CH ₃ ) ₂ -CH ₂ -;

R ¹ is selected from hydrogen, a straight or branched C ₁ -C4 alkyl, -(CH ₂ )OCH ₃ , preferably R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ , -CH(CH ₃ ) ₂ ;

R ^la is hydrogen or a straight or branched C ₁ -C4 alkyl, preferably R ^la is hydrogen or -CH ₃ ;

X is selected from an oxygen atom, -NH-, -N(CH ₃ )- or a covalent bond, preferably X is oxygen or a covalent bond;

2 3 2 3

T and Q are -CR R -, wherein R and R at each occurrence are independently selected from hydrogen, C ₁ -C ₄ linear or branched alkyl, OR ⁴ , NHR ⁵ , preferably R ² and R ³ are hydrogen or R ² is hydrogen and R ³ is NHR ⁵ ;

R ⁴ is hydrogen or C ₁ -C ₄ linear or branched alkyl, preferably R ⁴ is hydrogen or -CH ₃ ;

R ⁵ is hydrogen, C ₁ -C ₄ linear or branched alkyl, -C(0)CH ₃ or -C (0) OC (CH ₃ ) 3 (Boc) , more preferably R ⁵ is -C(0)CH ₃ or

-C(0)OC(CH ₃ ) ₃ ;

R ⁶ is C ₁ -C ₄ linear or branched alkyl or hydrogen, preferably R ⁶ is -CH ₃ ; R is (C¾) _q CH (Xo) R wherein q and R are as above defined, Xo is as above defined

with Ag 03 as described in the international application WO 2006/008196.

Alternatively (VII) can be obtained by oxidation of compound (X)

RCH ₂ OH (X)

wherein R is as above defined, by oxidation with TCCA or NaOCl or NaIC>4 in the presence of a catalyst such as TEMPO or ruthenium (IV) oxide hydrate in an aprotic polar/protic polar/non polar solvent such as acetone, CH ₃ CN, water, CH ₃ CI or

CH ₂ CI ₂ or mixtures, at temperatures ranging from -80°C to 65°C.

Compounds (X) can be obtained by nitration of compounds (XI) or (XII)

R'CH ₂ OP' (XI)

R''CH ₂ OH (XII)

wherein R' and R' ' are as above defined and P' is a suitable hydroxyl protecting group

using the procedures above described, followed by the removal of the protecting group by methods well known in the art.

Another embodiment of the invention provides a compound of formula (II):

(ID

wherein A is an activating group selected from the formulas (2a) - (2h)

(2d) (2e) (2f)

(2g) (2h)

preferably A is 2d or 2a or 2g;

R is selected from the group consisting of:

1) -(R°) _n -CH (ON0 ₂ JR ¹ ,

2) -(R°) _n -CH (ON0 ₂ ) -CH (ON0 ₂ )R ^la

3) -(R°) n- (T)' m -x- (Q)p- (CH ₂ ) _q -CH(ON0 ₂ )R ¹ ,

4) -(R°) n- (T)' m -x- (Q)p- (CH ₂ ) _q -CH (ON0 ₂ ) CH (ON0 ₂ ) R

5)

6)

7) - (R ^u ) -CH (ON0 ) - (CH ₉ )^-X- (CH 2 I q2 CH (ON0 ) R

1

- (R ^u ) _n - (CR ^D R' ) - (CH,)„CH (ON0 ) R

wherein

n is 0 or 1,

m is 0 or 1,

p is 0 or 1,

nl is 0 or 1;

q is an integer from 0 to 10, preferably q is from 0 to 6; ql is an integer from 1 to 10, preferably q is from 1 to 6; q2 is an integer from 1 to 10, preferably q is from 1 to 6; t is an is an integer from 1 to 4, preferably t is 2 or 3;

R° is a straight or branched Ci-Cio alkylene; preferably R° is a straight or branched C ₁ -C6 alkylene, more preferably R° is

-C(CH ₃ ) ₂ - or -C(CH ₃ ) ₂ -CH ₂ -;

R ¹ is selected from hydrogen, a straight or branched C ₁ -C4 alkyl, -(CH ₂ )OCH ₃ , preferably R ¹ is hydrogen, -CH ₃ , -(CH ₂ )OCH ₃ , -CH(CH ₃ ) ₂ ;

R ^la is hydrogen or a straight or branched C ₁ -C4 alkyl, preferably R ^la is hydrogen or -CH ₃ ;

X is selected from an oxygen atom, -NH-, -N(CH ₃ )- or a covalent bond, preferably X is oxygen or a covalent bond;

2 3 2 3

T and Q are -CR R -, wherein R and R at each occurrence are independently selected from hydrogen, C ₁ -C ₄ linear or branched alkyl, OR ⁴ , NHR ⁵ , preferably R ² and R ³ are hydrogen or R ² is hydrogen and R ³ is NHR ⁵ ;

R ⁴ is hydrogen or C ₁ -C ₄ linear or branched alkyl, preferably R ⁴ is hydrogen or -CH ₃ ;

R ⁵ is hydrogen, C ₁ -C ₄ linear or branched alkyl, -C(0)CH ₃ or -C (0) OC (CH ₃ ) 3 (Boc) , more preferably R ⁵ is -C(0)CH ₃ or

-C(0)OC(CH ₃ ) ₃ ;

R ⁶ is C ₁ -C ₄ linear or branched alkyl or hydrogen, preferably R ⁶ is -CH ₃ ;

R ⁷ is (CH ₂ ) _q CH (ON0 ₂ ) R ¹ wherein q and R ¹ are as above defined.

The nitrate derivatives of formula (II) are stable compounds obtainable in high yields and purity, suitable for long term storage. Said compounds can react with the compound of formula (III) or (IV

(III) (IV)

under mild reaction conditions, allowing to obtain nitrooxyderivatives of formula (I) in high yield and purity.

The compounds of formula (II)

(ID

wherein the variables A and R are as above defined are synthesized by the process, as depicted in Scheme 2

M 0 CH , 0 CH ₃ 0

Λο

R 0-Xx ₁ AA0 z AA0A0A,R

iii)

(VI) _(v) (II)

(VII) (VI)

Scheme 2 comprising :

iii ) reacting a compound of formula (VI)

RCOOXi (VI)

wherein R is as above defined and Xi is Ag ⁺ or Hg ⁺ ,

with a compound of formula (V)

(V)

wherein A is as above defined, Xo is CI, Br or I, in a molar ratio of compounds of formulas (VI) : (V) from 2 to 0.5, in an organic solvent to obtain a compound of formula (II)

(ID wherein A and R are as above defined;

iv) reacting a nitrate carboxylic acid of formula (VII)

R-COOH (VII)

wherein R is as above defined, with Ag ₂ <3 or Hg ₂ 0 in a molar ratio compound of formula (VII) : (Ag ₂ <3 ) or Hg ₂ 0 of from 1 to 0.5 in an organic solvent, to obtain the carboxylic acid salt of formula (VI)

R-COOXi (VI)

wherein Xi is Ag ⁺ or Hg ⁺ .

In step iii) the reaction is typically carried out in a temperature range from about -20°C to about 120°C, preferably from 10°C to 30°C. Generally, the reaction is carried out in an organic solvent, generally an aprotic solvent, such as toluene, methylene chloride, tetrahydrofurane or chloroform or dipolar solvents such as, dimethylformamide (DMF) , N- methylpyrrolidone, acetonitrile or in a mixture thereof, depending on the solubility of the compounds involved in the reaction; the preferred solvent are toluene, methylene chloride, THF, a mixture of methylene chloride and THF.

Compounds of formula (V) of step iii) , wherein A and Xo are as above defined, are prepared by known compounds following procedures well known in the art (Jose Alexander, * Robyn Cargill, Stuart R. Michelson, and Harvey Schwamt, (Acyloxy) alkyl Carbamates as Novel Bioreversible Prodrugs for Amines: Increased Permeation through Biological Membranes, J. Med. Chem., 1988, 31, 318-322) .

In step iv) the organic solvent is an aprotic solvent, such as toluene, methylene chloride, or chloroform or polar/dipolar solvents such as acetonitrile, water, tetrahydrofurane, dimethylformamide (DMF) , N-methylpyrrolidone, or in a mixture thereof, depending on the solubility of the compounds involved in the reaction; the preferred solvent are acetonitrile, water a mixture of acetonitrile and water. The reaction is generally carried out in a temperature range from about -20°C to about 80°C, preferably from 10°C to 30°C in the dark.

The compound of general formula (VII) of step iv) are known in the literature or can be prepared as reported above.

Example 1

Synthesis of 1-iodoethyl 4-nitrophenyl carbonate

STEP A: 1-chloroethyl 4-nitrophenyl carbonate

To a solution of 4-nitrophenol (3.0 g, 21.6 mmol) and pyridine (1.9 mL, 24 mmol) cooled to 0°C, 1-chloroethyl chloroformate (2.3 mL, 21.6 mmol) was added. The reaction was stirred at room temperature for 5-6 hours, then washed with Na¾P0 ₄ (5%) and brine, dried over Na2 S C>4 and concentrated affording the title compound (4.88 g, 92%) as a pale yellow solid.

¹ H-NMR (CDC1 ₃ ) : 8.32 (2H,m); 7.44 (2H,m); 6.52 ( 1H, q, J=5.8 Hz); 1.94 (3H, d, J=5.5Hz) .

STEP B: 1-iodoethyl 4-nitrophenyl carbonate

1-chloroethyl 4-nitrophenyl carbonate (4.88 g, 20 mmol) and Nal (20.8 g, 139 mmol) were dissolved in CH ₃ CN (40 mL) and the mixture was refluxed overnight. CH ₃ CN was removed and the residue was diluted with EtOAc, washed with brine, Na2 S 20s (10%), K ₂ CO ₃ (10%) and brine, dried over Na2 S C>4 and concentrated affording the title compound as a pale oil (3.56 g, 53%)

1H-NMR (CDCI ₃ ) : 8.34 (2H,m); 7.47 (2H,m); 6.86 ( 1H, q, J=6.2 Hz); 2.35 (3H,d, J=6.2Hz) .

Example 2

S nthesis of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid HNO ₃ (2.4 mL, 54 mmol) was dissolved in AC2O (7.5 mL, 80 mmol) , cooled to 0°C, and methyl 3-hydroxy-2 , 2-dimethylpropanoate (4 mL, 31 mmol) was added. The mixture was stirred for 1 hour, then poured into iced aHC03 (5%) and diluted with EtOAc. The organic phase was washed with brine, dried over Na2 S C>4 and concentrated affording methyl 2 , 2-dimethyl-3- (nitrooxy) propanoate as a clear oil

The residue was dissolved in NaOH (2.5 M, 20 mL) and MeOH (20 mL) and the solution was stirred at room temperature for 6 hours, and then acidified with HC1 (5%) to pH 3-4. The aqueous phase was extracted with EtOAc and the combined organic phases were washed with brine, dried over Na2 S C>4 and concentrated affording the title compound as a pale yellow solid (5.00 g, 98%)

¹ H-NMR (CDCI3) : 4.53 (2H,s); 1.34 (6H,s).

Example 3

Synthesis of 6- (nitrooxy) hexanoic acid

6-Bromohexanoic acid (1.0 g, 5.1 mmol) was dissolved in CH3CN (20 mL) and Ag 03 (1.74 g, 19.2 mmol) was added. The reaction was heated by microwave at 90 °C for 14 minutes. The precipitate was filtered off and the reaction was concentrated under reduced pressure; EtOAc (20 mL) was added and the organic phase was washed with water and brine, dried over a ₂ S0 ₄ and concentrated affording the title compound as a yellow oil (840 mg, 93%) .

¹ H-NMR (CDCI3) : 4.47 (2H, t, J=6.6Hz) ; 2.40 ( 2H, t , J=7.3Hz ) ; 1.84- 1.63 (4H,m); 1.50 (2H,m).

Example 4 S nthesis of (S) -3-methyl-2- (nitrooxy) butanoic acid

HNO ₃ (3 mL, 76 mmol) was dissolved in AC ₂ O (10 mL) , cooled to 0°C, and (S) - ( + ) -2-Hydroxy-3-methylbutyric acid (1.5 g, 12.7 mmol) was added. The mixture was stirred for 1 hour, then poured into iced NaHC0 ₃ (10%) and diluted with CH ₂ C1 ₂ . The organic phase was washed with brine, dried over Na ₂ SC> ₄ and concentrated affording the title compound as a clear oil (1.75 g, 85%)

¹ H-NMR (CDCI3) : 5.17 ( 2H, d, J=4.3Hz ) ; 2.25 (lH,m); 0.98 (6H,dd, J=6.9Hz) .

Example 5

Synthesis of 2- (2- (nitrooxy) ethoxy) acetic acid

STEP A: 2- ( 2 -hydroxyethoxy) ethyl nitrate

Dietilenglycol monochloride (10 mL, 94.3 mmol) was dissolved in CH ₃ CN (250 mL) and Nal (141 g, 943 mmol) was added. The reaction was refluxed for 16 hours. The solvent was removed and CH ₂ CI ₂ (100 mL) was added, the mixture was filtered and the organic phase was washed with a solution of Na ₂ S ₂ 0s (10%), dried and evaporated. The crude material was dissolved in CH3CN (250 mL) , Ag 0 ₃ (55 g, 330 mmol) was added and the reaction refluxed for 2 hours. The mixture was concentrated and diluted with EtOAc, then filtered. The organic phase was washed with water and brine, dried over a ₂ S0 ₄ and concentrated affording the title compound as a clear oil. (8.5 g, 60%)

1H-NMR (CDCI3) : 4.66 (2H,m); 3.80 (4H,m); 3.63 (2H,m) . STEP B: 2- (2- (nitrooxy) ethoxy) acetic acid

2- (2-hydroxyethoxy) ethyl nitrate (17 g, 112 mmol) was dissolved in acetone and cooled to 0°C; aHC03 (15%, 150 mL) , NaBr (2 g, 22 mmol) and TEMPO (170 mg, 1.12 mmol) were added. Trichloroisocyanuric acid (50 g, 224 mmol) was added in portions in 15 minutes. The reaction was stirred at room temperature for 3 hours, then cooled to 0°C and 2-propanol (60 mL) was slowly added in 15 minutes. The reaction was stirred at 0°C for 30 minutes then the precipitate was filtered. The solvent was concentrated and NaOH (10%) was added to pH=9. The aqueous phase was extracted with EtOAC, then HC1 was added to pH=3 and the aqueous phase was extracted with CH ₂ CI ₂ . The organic phase was dried over a ₂ S0 ₄ and concentrated affording the title compound as an oil. (8.7 g, 47%)

1H-NMR (DMSO) : 4.67 (2H,m); 4.06 (2H,s); 3.78 (2H,m) .

Example 6

Synthesis of (R) -5 , 6-bis (nitrooxy) hexanoic acid STEP A: tert-butyl ( 5R) -5 , 6-dihydroxyhexanoate

Tert-butyl hex-5-enoate (5 g, 29.4 mmol) was added to a suspension of AD-mix- β (41 g) in H ₂ 0/t-BuOH (125 mL/125 mL) at 0°C and the reaction was stirred overnight at 5°C. EtOAc (100 mL) was added followed by a ₂ S ₂ 0s (7.0 g) in portions and the reaction was stirred for 30 minutes at 5°C and 1 hour at room temperature. The organic phase was separated and the aqueous layer was extracted with EtOAc. The combined organic phases were dried over a ₂ S0 ₄ and concentrated affording the title compound as an oil (5.8 g, 98%)

H-NMR (CDCI3) : 3.67 (2H,m); 3.47 (lH,m); 2.27 (2H,

(4H,m) ; 1.46 (9H, s) . STEP B: tert-butyl ( 5R) -5 , 6-bis (nitrooxy) hexanoate HNO ₃ (5.23 mL, 126.2 mmol) was dissolved in AC ₂ O (20 mL) , cooled to 0°C, and tert-butyl ( 5R) -5 , 6-dihydroxyhexanoate (5.15 g, 25.2 mmol) was added. The mixture was stirred for 1 hour, then poured into iced NaOH (10%) and diluted with CH ₂ CI ₂ . The organic phase was washed with brine, dried over Na ₂ SC>4 and concentrated affording the title compound as a brown oil (7.0 g, 95%)

¹ H-NMR (CDCI3) : 5.29 (lH,m); 4.77 (lH,m); 4.50 (lH,m); 2.30 (2H,m); 1.77 (4H,m); 1.46 (9H,s) .

STEP C: (R) -5 , 6-bis (nitrooxy) hexanoic acid

Tert-butyl ( 5R) -5 , 6-bis (nitrooxy) hexanoate (7.0 g, 29.4 mmol) was dissolved in CH ₂ CI ₂ and cooled to 0°C. BF ₃ -Et ₂ <0 (3 mL, 32.3 mmol) was added and the reaction stirred 30 minutes at 0°C and 4 hours at room temperature. The organic phase was washed with brine, dried over Na ₂ SC>4 and concentrated. The crude material was purified by silica gel chromatography (CH ₂ Cl ₂ /MeOH 95:5) affording the title compound as a brown oil. (4.0 g, 60%) ¹ H-NMR (CDCI ₃ ) : 5.30 (lH,m); 4.77 (lH,m); 4.51 (lH,m); 2.46 (2H,m) ; 1.83 (4H,m) .

Example 7

Synthesis of ( S) -5 , 6-bis (nitrooxy) hexanoic acid Title compound was synthesized following the procedure of Example 6 except that in STEP A AD-mix-a was used instead of AD-mix-/3.

¹ H-NMR (CDCI3) : 5.30 (lH,m); 4.77 (lH,m); 4.51 (lH,m); 2.46 (2H,m) ; 1.83 (4H,m) .

Example 8

Synthesis of silver (I) 2 , 2-dimethyl-3- (nitrooxy) propanoate 2 , 2-dimethyl-3- (nitrooxy) propanoic acid (obtained in Example 2) (5.12 g, 31 mmol) was dissolved in CH ₃ CN (134 mL) . To the solution Ag ₂ 0 (4.86 g, 21 mmol) and ¾0 (67 mL) were added and the mixture was stirred at room temperature for 5 hours, and then filtered over celite. The filtrate was concentrated affording the title compound as a pale yellow solid (6.60 g, 78%)

¹ H-NMR (CDC1 ₃ ) : 4.55 (2H,s); 1.30 (6H,s)

Example 9

Synthesis of 1- ( (4-nitrophenoxy) carbonyloxy) ethyl-2, 2- dimethyl-3- (nitrooxy) propanoate

silver (I) 2 , 2-dimethyl-3- (nitrooxy) propanoate (obtained in Example 8) (3.13 g, 12 mmol) and 1-iodoethyl 4-nitrophenyl carbonate (obtained in Example 1) (3.56 g, 11 mmol) were dissolved in dry toluene (50 mL) and the suspension was stirred overnight, filtered and purified by BIOTAGE (eluting with EtOAc/Hexane 5 to 20%) affording the title compound (1.5 g, 38%) as a pale oil.

¹ H-NMR (CDCI3) : 8.31 (2H,m); 7.42 (2H,m); 6.87 (lH,q,J=5.4 Hz); 4.54 (2H,m); 1.65 (3H,d); 1.34 (6H,s).

Example 10

Synthesis of Silver (I) 6- (nitrooxy) hexanoate

Title compound was synthesized following the procedure of Example 8 except that 6- (nitrooxy) hexanoic acid (prepared in Example 3) was used instead of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid.

¹ H-NMR (DMSO) : 4.50 (2H, t , J=6.6Hz) ; 1.65 (2H,m); 1.53 (2H,m); 1.35 (2H,m) .

Example 11 1- ( (4-nitrophenoxy) carbonyloxy) ethyl 6- (nitrooxy) hexanoate

Starting from Silver (I) 6- (nitrooxy) hexanoate (prepared in Example 10) and 1-iodoethyl 4-nitrophenyl carbonate (obtained in Example 1), the title compound was synthesized following the procedure of Example 9.

¹ H-NMR (CDC1 ₃ ) : 8.31 (2H,m); 7.42 (2H,m); 6.87 (lH,q, J=5.4 Hz); 4.47 (2H,t, J=6.5Hz) ; 2.43 (2H,m); 1.88-1.66 (4H,m); 1.64 (3H, d, J=5.5Hz) ; 1.50 (2H,m).

Example 12

Synthesis of Silver (I) (S) -3-methyl-2- (nitrooxy) butanoate Title compound was synthesized following the procedure of Example 8 except that (S) -3-methyl-2- (nitrooxy) butanoic acid (prepared in Example 4) was used instead of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid.

¹ H-NMR (DMSO) : 5.17 ( 1H, d, J=4.3Hz ) ; 2.26 (lH,m); 0.98 (6H,dd, J=6.9Hz)

Example 13

Synthesis of (2S) -1- ( (4-nitrophenoxy) carbonyloxy) ethyl-3- meth l-2- (nitrooxy) butanoate

Starting from Silver (I) (S) -3-methyl-2- (nitrooxy) butanoate (prepared in Example 12) title compound was synthesized following the procedure of Example 9.

¹ H-NMR (CDCI3) : 8.31 (2H,m); 7.41 (2H,m); 6.93 (lH,m); 5.02 (1H, d, J=4.7Hz) ; 2.36(lH,m); 1.69 (3H,m); 1.11 (6H,m).

Example 14

Synthesis of Silver (I) 2- (2- (nitrooxy) ethoxy) acetate Title compound was synthesized following the procedure of Example 8 except that 2- (2- (nitrooxy) ethoxy) acetic acid

(prepared in Example 5) was used instead of 2 , 2-dimethyl-3-

(nitrooxy) propanoic acid.

1H-NMR (DMSO) : 4.66 (2H,m); 3.92 (2H,s); 3.77 (2H,m)

Example 15

Synthesis of 1- ( (4-nitrophenoxy) carbonyloxy) ethyl 2- (2- nitrooxy) ethoxy) acetate

1-iodoethyl 4-nitrophenyl carbonate (10.55 g, 31.3 mmol) obtained in Example 1 was dissolved in dry toluene (15 mL) under nitrogen atmosphere at r.t. To this solution Silver (I)

2- (2- (nitrooxy) ethoxy) acetate (11 g, 40.7 mmol) obtained in Example 14 was added and the suspension was stirred in the dark at room temperature for 5 hours. The precipitate was filtered and the filtrate was concentrated under reduced pressure. The crude was purified by silica gel chromatography (eluting with EtOAc/Hexane from 5 to 40%) affording the title compound (7.5 g, 68%) as a pale oil.

¹ H-NMR (CDC1 ₃ ) : 8.30 (2H,m); 7.4 (2H,m); 6.92 ( 1H, q, J=5.4Hz ) ; 4.67 (2H,m); 4.20 (2H,s); 3.90 (2H,m); 1.67 ( 3H, d, J=5.4Hz ) . Example 16

Synthesis of Silver (I) ( S ) -5 , 6-bis (nitrooxy) hexanoate

Title compound was synthesized following the procedure of Example 8 except that ( S ) -5 , 6-bis (nitrooxy) hexanoic acid (prepared in Example 7) was used instead of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid.

¹ H-NMR (DMSO): 5.42 (lH,m); 4.92 (lH,m); 4.68 (lH,m); 2.5 (2H,m); 2.16 (2H,m); 1.81-1.51 (4H,m).

Example 17 Synthesis of (5S) -1- ( (4-nitrophenoxy) carbonyloxy) ethyl-5, 6- bis (nitrooxy) hexanoate

Starting from Silver (I) (S) -5, 6-bis (nitrooxy) hexanoate (prepared in Example 16) title compound was synthesized following the procedure of Example 9.

¹ H-NMR (CDC1 ₃ ) : 8.30 (2H,m); 7.42 (2H,m); 6.85 (lH,m); 5.28 (lH,m); 4.74 (lH,m); 4.47 (lH,m); 2.47 (2H,m); 1.83 (4H,m); 1.64 (3H,m) .

Example 18

Synthesis of Silver (I) (R) -5 , 6-bis (nitrooxy) hexanoate

Title compound was synthesized following the procedure of Example 8 except that (R) -5 , 6-bis (nitrooxy) hexanoic acid (prepared in Example 6) was used instead of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid.

¹ H-NMR (DMSO) : 5.42 (lH,m); 4.92 (lH,m); 4.68 (lH,m); 2.5 (2H,m); 2.16 (2H,m); 1.81-1.51 (4H,m).

Example 19

Synthesis of (5R) -1- ( (4-nitrophenoxy) carbonyloxy) ethyl-5, 6- bis (nitrooxy) hexanoate

Starting from Silver (I) (R) -5 , 6-bis (nitrooxy) hexanoate

(prepared in Example 18), title compound was synthesized following the procedure of Example 9.

1H-NMR (CDCI3) : 8.30 (2H,m); 7.42 (2H,m); 6.85 (lH,m); 5.28 (lH,m); 4.74 (lH,m); 4.47 (lH,m); 2.47 (2H,m); 1.83 (4H,m); 1.64 (3H,m) .

Example 20 Synthesis of 2- ( 1- ((( 1- (2 , 2-dimethyl-3- (nitrooxy) propanoyloxy) ethoxy) carbonylamino) methyl) cyclohexyl) acetic acid (compound 1)

(1)

To a solution of Gabapentin (670 mg, 3.9 mmol) in CH ₂ CI ₂ (20 mL) , cooled to 0°C, triethylamine (1.4 mL, 11 mmol) and trimethylsilylchloride (1 mL, 7.8 mmol) were added. The reaction was stirred at 0°C for 2 hours, then 1- ( (4- nitrophenoxy) carbonyloxy) ethyl-2, 2-dimethyl-3- (nitrooxy) propanoate (Example 9) (1.5 g, 4 mmol) was added. The reaction was stirred at room temperature overnight, then washed with Na¾P0 ₄ (5%) and brine, dried over Na ₂ SC>4 and concentrated.

The crude material was purified by BIOTAGE (eluting with EtOAc/Hexane from 20 to 80%) affording the title compound (1.29 g, 80%) as a clear oil.

¹ H-NMR (CDCI3) : 6.81 ( 1H, q, J=5.5Hz ) ; 5.28 (1H, bt) ; 4.52 (2H,m); 3.26 (2H,m); 2.36 (2H,s); 1.51-1.29 (13H, d+m) ; 1.29 (6H, s) .

Example 21

Synthesis 2- (1- ( ( (1- (6- (nitrooxy) hexanoyloxy) ethoxy) carbonyl amino) methyl) cyclohexyl) acetic acid (compound 3)

(3) Title compound was synthesized following the procedure of Example 20, starting from compound obtained in Example 11.

1H-NMR (CDC1 ₃ ) : 6.81 (lH,m); 5.27 (1H, bt) ; 4.45 (2H,m); 3.25 (2H,m); 2.34 (4H,m); 1.82-1.63 (4H,m); 1.66-1.34 (15H,m).

Example 22

Synthesis of 2- (1- ( ( (1- ( (S) -3-methyl-2- (nitrooxy) butanoyloxy) ethoxy) carbonylamino) methyl) cyclohexyl) acetic acid (compound 7)

Title compound was synthesized following the procedure of Example 20 Starting from compound obtained in Example 13.

1H-NMR (CDCI3) : 6.92 (lH,m); 5.33 (1H, bt) ; 4.93 (lH,m); 3.27 (2H,m); 2.33 (3H,m); 1.66-1.34 (13H,m); 1.07 (6H,m).

Example 23

Synthesis of 2- (1- (5-methyl-ll- (nitrooxy) -3, 7-dioxo-4, 6, 9- trioxa-2-azaundecyl) cyclohexyl) acetic acid (corresponding to compound 12)

(12)

To a solution of Gabapentin (3.45 g, 20 mmol) in CH ₂ CI ₂ (200 mL) , cooled to 0°C, triethylamine (5.2 mL, 46 mmol) and trimethylsilylchloride (5.2 mL, 41 mmol) were added. The reaction was stirred at 0°C for 2 hours, then 1- ( (4- nitrophenoxy) carbonyloxy) ethyl 2- (2- (nitrooxy) ethoxy) acetate (Example 15) (7.5 g, 20 mmol) was added. The reaction was stirred at room temperature overnight, then washed with NaH ₂ PC>4 (5%) and brine, dried over Na ₂ SC>4 and concentrated.

The crude material was purified by BIOTAGE (eluting with EtOAc/Hexane from 20 to 80%) affording the title compound (6.0 g, 74 %) as a clear oil.

¹ H-NMR (CDC1 ₃ ) : 6.86 ( 1H, q, J=5.4Hz ) ; 5.31 (lH,bt); 4.66 (2H,m); 4.14 (2H,m); 3.88 (2H,m); 3.24 (2H,m); 2.35 (2H,s); 1.63-1.33 (13H,m) .

Example 24

Synthesis of (12S) -12-isobutyl-7-methyl-l- (nitrooxy) -5, 9- dioxo-3, 6, 8-trioxa-10-azatetradecan-14-oic acid (corresponding to compound 14)

(14)

To a solution of pregabalin (510 mg, 3.2 mmol) in CH ₂ CI ₂ (25 mL) , cooled to 0°C, triethylamine (1.2 mL, 10 mmol) and trimethylsilylchloride (800 lL, 6.3 mmol) were added. The reaction was stirred at 0°C for 2 hours, then 1- ( (4- nitrophenoxy) carbonyloxy) ethyl 2- (2- (nitrooxy) ethoxy) acetate (Example 15) (1.2 g, 3.2 mmol) was added. The reaction was stirred at room temperature overnight, then washed with Na¾P0 ₄ (5%) and brine, dried over Na ₂ SC>4 and concentrated.

The crude material was purified by BIOTAGE (eluting with EtOAc/Hexane from 15 to 60 %) affording the title compound (950 mg, 75 %) as a clear oil.

¹ H-NMR (CDCI3) : 6.86 (lH,m) ; 5.06 ( 1H, m) ; 4.66(2H,m); 4.14 (2H,m); 3.88(2H,m); 3.22(2H,m); 2.34(3H,m); 1.67 ( 1H, m) ; 1.52 (3H, m) ; 1.19 (2H,m) ; 0.92 (6H,m) .

Example 25 Synthesis of 2- (1- ( ( (1- ( (S) -5, 6-bis (nitrooxy) hexanoyloxy ethoxy) carbonylamino) methyl) cyclohexyl) acetic acid (compound 9)

(9)

Title compound was synthesized following the procedure of Example 20 starting from compound obtained in Example 17.

1H-NMR (CDC1 ₃ ) : 6.80 (lH,m); 5.89 (lH,bt); 4.77 (lH,m); 4.49 (lH,m); 3.25 (2H,m); 2.38 (4H,m); 1.81 (4H,m); 1.62-1.30 (13H,m) .

Example 26

Synthesis of (3S) -3- (( (1- ( (R) -5, 6-bis (nitrooxy) hexanoyloxy) ethoxy) carbonylamino) methyl) -5-methylhexanoic acid (compound 10)

(10)

Title compound was synthesized following the procedure of Example 24 starting from compound obtained in Example 19.

1H-NMR (CDCI3) : 6.79(lH,m); 5.303 ( 1H, m) ; 5.02 (1H, m) ; 4.78 (lH,m) ; 4.49(lH,m); 3.26(2H,m); 2.41 -2.30 ( 5H, m) ; 1.81- 1.65(5H,m); 1.49(3H,m); 1.25(2H,m); 0.94 ( 6H, m) .

Example 27

Synthesis of 5-methyl-2-oxo-l , 3-dioxol-4-yl { 2- ( 1- (5-methyl-ll- (nitrooxy) -3, 7-dioxo-4, 6, 9-trioxa-2-azaundecyl) cyclohexyl } acetate (compound 27)

(27)

To a stirred solution of 2- (1- (5-methyl-ll- (nitrooxy) -3, 7- dioxo-4, 6, 9-trioxa-2-azaundecyl) cyclohexyl) acetic acid (Example 23) (600 mg, 1.47 mmol) in dry DMF (20 mL) was added 4-chloromethyl-5-methyl-l , 3-dioxol-2-one (241 mg, 1.62 mmol) and CS ₂ CO ₃ carbonate (327 mg, 1.62 mmol) . The mixture was stirred at room temperature for 2 hours. The reaction was diluted with Na¾P0 ₄ 5% and extracted with EtOAc. The organic phase was washed with brine and dried over a ₂ S0 ₄ . The crude material was purified by silica gel chromatography (eluting with EtOAC/Hexane 20 to 80 %) affording the product (450 mg, 60 %) as a clear oil.

1H-NMR (CDCI3) : 6.86 (1H, m) ; 5.23 (1H, bt) ; 4.85 (2H, s); 4.65 (2H, t) ; 4.14 (2h, m) ; 3.86 (2H, m) ; 3.19 (2H, m) ; 2.36 (2H, s); 2.20 (3H, s) ; 1.6-1.3 (13H, m) .

Example 28

Synthesis of (R) -2- (3-methoxy-2- (nitrooxy) propoxy) acetic acid O ON0 ₂ STEP A: methyl { [ (4S) -2, 2-dimethyl-l, 3-dioxolan-4-yl] methoxy} acetate

(S) - ( + ) -1, 2-isopropylideneglycerol (14 ml, 112.8 mmol) was slowly added to a suspension of NaH (60, 9.0 g , 225 mmol) in dry THF (350 mL) at 0°C under nitrogen atmosphere. 15-crown-5 (3 mL, 0.15 mmol) was then added. The reaction was stirred for 20 minutes at 0°C and methyl bromoacetate (31 mL, 338 mmol) in THF (30 ml) was slowly added. The reaction was stirred at room temperature for 2 hours. The reaction was quenched by adding a satured solution of NH ₄ C1 (300 mL) and the aqueous phase was extracted with Et ₂ 0. The combined organic phases were dried over Na ₂ SC>4 and concentrated. The crude material was purified by silica gel chromatography (Hex/AcOEt 8:2) affording the title compound as a oil. (7.15 g, 31%) ¹ H-NMR (CDC1 ₃ ) : 4.33 (lH,m); 4.17 (2H,s); 4.08 (lH,m); 3.79 (lH,m); 3.76 (3H,s); 3.62 (2H,d); 1.43 (3H,s); 1.36 (3H,s) .

STEP B: Methyl { [ (2R) -2 , 3-dihydroxypropyl ] oxy} acetate

Methyl { [ ( 4S ) -2 , 2-dimethyl-l , 3-dioxolan-4-yl ] methoxy} acetate (8.2 g; 40 mmol) was dissolved in MeOH (100 mL) and HCl _gas was bubbled in the solution. The reaction was stirred at room temperature for 3 hours . The mixture was concentrated affording the title compound as an oil (6.4 g, 98%)

1H-NMR (CDC1 ₃ ) : 4.32 (lH,m) 4.14 (2H,s); 3.90 (2H,m); 3.76 (3H, s) ; 3.74-3.58 (2H,m) .

STEP C: methyl { [ (2S) -2-hydroxy-3- ( tert-Butyldiphenylsilyl ) propyl] oxyjacetate

To a solution of methyl { [ (2R) -2 , 3-dihydroxypropyl ] oxy } acetate (1.7 g, 10.4 mmol) in dry DMF (18 mL) , tert-butyldiphenyl silylchloride (3 mL, 11.5 mmol) and imidazole (1.42 g , 20.9 mmol) were added. The solution was stirred 6 hours at room temperature. The organic phase was diluted with Na¾P0 ₄ (5%, 60 mL) and extracted with EtOAc, dried over Na ₂ SC>4 and concentrated. The crude material was purified by silica gel chromatography (Hex/AcOEt 8:2) affording the title compound as an oil. (1.0 g, 24%)

¹ H-NMR (CDCI3) : 7.66 (4H,m); 7.41 (6H,m); 4.13 (3H,m); 3.94 (lH,m); 3.71 (5H,m); 3.61 (lH,m) 1.06 (9H,m) .

STEP D: methyl { [ (2S) -2- (nitrooxy) -3- ( tert-butyl diphenyl silyl)propyl] oxyjacetate HN0 ₃ (0.247 mL, 5.95 mmol) was dissolved in Ac ₂ 0 (0.655 mL, 6.9 mmol) and CH ₂ C1 ₂ (30 mL) , cooled to 0°C, and methyl {[(2S)-2- hydroxy-3- ( tert-Butyldiphenylsilyl ) propyl ] oxy} acetate (2.0 g, 4.96 mmol) was added. The mixture was stirred for 6 hours, then poured into iced NaOH (10%) . The organic phase was washed with brine, dried over Na ₂ SC>4 and concentrated affording the title compound as an oil (2.0 g, 90%)

¹ H-NMR (CDCI3) : 7.65 (4H,m); 7.42 (6H,m); 5.32 (lH,m); 4.10 (2H,m); 3.86 (2H,m); 3.74 (3H,m); 1.05 (9H,m).

STEP E: methyl {[ (2R) -3-hydroxy-2- (nitrooxy) propyl ] oxy } acetate To a solution of methyl {[ (2S) -2- (nitrooxy) -3- (tert- butyldiphenylsilyl ) propyl ] oxy } acetate (2.0 g, 4.47 mmol) in dry THF (30 mL) at 0°C, acetic acid (0.9 mL, 15.7 mmol) was added and a tetrabutylammonium fluoride solution (1M in THF, 13.4 mL , 14.4 mmol) was added dropwise. The mixture was stirred 4 hours at room temperature. The reaction was diluted with EtOAc (30 mL) and the organic phase was washed with NaH ₂ PC>4 (5%) , dried over Na ₂ SC>4 and concentrated. The crude material was purified by silica gel chromatography (Hex/AcOEt 1:1) affording the title compound as an oil. (600 mg, 65%) ¹ H-NMR (CDCI3) : 5.22 (lH,m); 4.15 (2H,m); 3.95 (2H,m); 3.86 (2H,m) ; 3.78 (3H, s) . STEP F: methyl {[ (2R) -3-methoxy-2- (nitrooxy) propyl ] oxy } acetate To a solution of methyl {[ (2R) -3-hydroxy-2- (nitrooxy) propyl ] oxyjacetate (0.60 g, 2.87 mmol) in dry CH ₂ C1 ₂ (18 mL) at 0°C, 2 , 6-di-tert-butyl-4-methylpyridine (2.36 g, 11.5 mmol) and MeOS0 ₂ CF ₃ (1.3 mL , 11.5 mmol) were added. The solution was stirred 30 minutes at 0°C and then irradiated by microwave for 1 hour at 80°C. The reaction was washed with NaH ₂ PC>4 (5%), dried over a ₂ S0 ₄ and concentrated. The crude material was purified by silica gel chromatography (Hex/AcOEt 8:2) affording the title compound as a oil. (520 mg, 82%) .

1H-NMR (CDC1 ₃ ) : 5.35 (lH,m); 4.15 (2H,s); 3.82 (2H,m); 3.75 (3H,s); 3.66 (2H,m); 3.39 (3H,s).

STEP G: {[ (2R) -3-methoxy-2- (nitrooxy) propyl ] oxy} acetic acid To a solution of methyl {[ (2R) -3-methoxy-2- (nitrooxy) propyl ] oxyjacetate (0.45 g, 2.0 mmol) in acetone (13 mL) , phosphate buffer (PH=8, 50 mL) , water (50 mL) and pig liver esterase in ammonium sulfate suspension (0.533 mL) were added. The reaction was stirred overnight at room temperature then acidified with HC1 (IN) to pH=3. It was extracted with EtOAc, dried over a2 S 0 ₄ and concentrated affording the title compound as an oil. (350 mg, 85%)

1H-NMR (CDCI3) : 5.33 (lH,m); 4.18 (2H,s); 3.85 (2H,m); 3.68 (2H, d) ; 3.40 (3H, s) .

Example 29

Synthesis of silver (I) ( R ) -2- (3-methoxy-2- (nitrooxy) propoxy) acetate

Title compound was synthesized following the procedure described in Example 8 except that { [ (2R) -3-methoxy-2- (nitrooxy) propyl ] oxy } acetic acid (prepared in Example 28) was used instead of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid. ¹ H-NMR (DMSO) : 5.40 (lH,m); 4.92 (2H,s); 3.73 (2H,m); 3.98 (2H,m) ; 3.26 (3H, s) .

Example 30

Synthesis of 1- ( (4-nitrophenoxy) carbonyloxy) ethyl 2- ( (R) -2- hydroxy-3-methox ropoxy) acetate

Starting from silver (I) (R) -2- (3-methoxy-2- (nitrooxy) propoxy) acetate (prepared in Example 29) and 1-iodoethyl 4-nitrophenyl carbonate (obtained in Example 1), the title compound was synthesized following the procedure of Example 9.

1H-NMR (CDC1 ₃ ) : 8.30 (2H,d); 7.42 (2H,d); 6.90 (lH,m); 5.35 (lH,m); 4.29 (2H,s); 3.85 (2H,m); 3.65 (2H,m); 3.37 (3H,s); 1.65 (3H,d) .

Example 31

Synthesis of 2- (1- ( (11R) -ll-hydroxy-5-methyl-3, 7-dioxo-4, 6, 9, 13-tetraoxa-2-azatetradecyl) cyclohexyl) acetic acid (compound 23

(23)

Title compound was synthesized following the procedure of Example 20, starting from compound obtained in Example 30.

1H-NMR (CDCI3) : 6.85 (lH,m); 5.32 (2H,m); 4.12 (2H,s); 3.83 (2H,m); 3.66 (2H,m); 3.38 (3H,s); 3.25 (2H,d); 2.34 (2H,s); 1.62-1.32 (13H,m) .

Example 32

Synthesis of 4- (nitrooxy) butanoic acid

STEP A: methyl 4-bromobutanoate

To a solution of 4-bromobutanoic acid (6.5 g , 38.9 mmol) in MeOH (120 ml) at 0°C under nitrogen atmosphere, thionyl chloride (2.8 ml, 38.9 mmol) was slowly added and the reaction was stirred overnight at r.t. Solvent was evaporated and the crude material was used directly in next step. (6.0 g, 85%)

¹ H-NMR (CDCI3) : 3.71 (3H,s); 3.49 (2H,t J=6.44); 2.53 (2H,t J=7.16) ; 2.20 (2H,m) . STEP B: methyl 4- (nitrooxy) butanoate

Methyl 4-bromobutanoate (6.0 g; 33 mmol) was dissolved in C¾CN (120 mL) and silver nitrate (14.0 g, 82.6 mmol) was added. The reaction was heated at 80°C in the dark for 4 hours. Solvent was evaporated and the crude was dissolved in EtOAc (80 ml) .Precipitate was filtered off and organic phase was washed with water (2 x 100 ml) and brine (1 x 100 ml), dried over Na ₂ SC>4 and concentrated affording the title compound as an oil. (5.0 g, 93%)

1H-NMR (CDC1 ₃ ) : 4.54 (2H,t J=6.31); 3.73 (3H,s); 2.48 (2H,t J=7.15) ; 2.08 (2H,m) .

STEP C: 4- (nitrooxy) butanoic acid

Methyl 4- (nitrooxy) butanoate (10.0 g; 61.0 mmol) was dissolved in MeOH (240 mL) at 5°C and LiOH 2N (60 ml) was added. The reaction was stirred overnight at 5°C. Solution was acidified to pH 3 with HC1 IN and MeOH was evaporated. Aqueous phase was exctracted with CH ₂ CI ₂ , dried over a ₂ S0 ₄ and concentrated affording the title compound as an oil. (7.2 g, 79%)

1H-NMR (CDCI3) : 4.55 (2H,t J=6.30); 2.54 (2H,t); 2.10 (2H,m). Example 33

Synthesis of silver (I) 4- (nitrooxy) butanoate

Title compound was synthesized following the procedure described in Example 8 except that 4- (nitrooxy) butanoic acid (prepared in Example 32)

was used instead of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid. ¹ H-NMR (DMSO) : 4.56 (2H,t J=6.71); 2.42 (2H,t J=6.54); 2.15 (2H,m)

Example 34

Synthesis of 1- ( (4-nitrophenoxy) carbonyloxy) ethyl 4- (nitrooxy) butanoate

Starting from silver (I) 4- (nitrooxy) butanoate (Example 33) and 1-iodoethyl 4-nitrophenyl carbonate (obtained in Example 1), the title compound was synthesized following the procedure of Example 9.

¹ H-NMR (CDC1 ₃ ) : 8.30 (2H, d) ; 7.42 (2H, d) ; 6.91 (1H, m) ; 4.55 (2H, t) ; 2.56 (2H,t); 2.11 (2H, m) ; 1.65 (3H, s).

Example 35

Synthesis of (3S) -5-methyl-3- (( (1- (4- (nitrooxy) butanoyloxy) ethoxy) carbonylamino) methyl) hexanoic acid (compound 30)

(30)

Title compound was synthesized following the procedure of Example 24 starting from compound obtained in Example 34.

1H-NMR (CDCI3) : 6.82 (1H, m) ; 5.05 (1H, t) ; 4.53 (2H, t) ; 3.30 (1H, m) ; 3.13 (1H, m) ; 2.50 -2.39 ( 7H, m) ; 1.67 (1H, m) ; 1.52 (3H, d) ; 1.20 (2H, t) ; 0.91 (6H, m) .

Example 36

Synthesis of 3- (2- (nitrooxy) ethoxy) propanoic acid

STEP A: 2- (but-3-en-l-yloxy) ethyl nitrate

To a solution of HNO ₃ fuming (3.2 ml, 78.3 mmol) and Acetic anhydride (6.5 ml, 68.5 mmol) in dry CH ₂ CI ₂ (30 ml) cooled at - 50 °C, a solution of 2-allyloxyethanol (5.0 g, 48.9 mmol) in CH ₂ CI ₂ (30 ml) was added dropwise. The solution was stirred at -50 °C 1.5 hours then it was allowed to rich r. t. and stirred for 4h. The solution was then poured into ice, the two phases were separated and the organic phase was washed with brine, dried on Na ₂ S0 ₄ and concentrated. The residue was purified by flash chromatography with SP1 Biotage instrument (SNAP 100 g column, n-Hex/EtOAc 95:5, 10 CV) affording the desired product as a colourless oil (6.6 g, yield: 92%) .

¹ H-NMR (CDC1 ₃ ) : 6.05 - 5.77 (1 H, m) , 5.39-5.14 (2 H, m) , 4.69 - 4.57 (2 H, m) , 4.08 - 4.00 (2 H, m) , 3.78 - 3.68 (2 H, m) .

STEP B: 2- (3-hydroxypropoxy) ethyl nitrate

To a solution of 2- (but-3-en-l-yloxy) ethyl nitrate (6.6 g,

44.8 mmol) in dry THF (100 ml) cooled at 0 °C, 9-BBN 0.5M in THF (269 ml, 134.5 mmol) was added dropwise. The solution was stirred at r.t. overnight. Then It was cooled at 0 °C and NaOH 2M (112 ml, 224.2 mmol) and H ₂ 0 ₂ 30% (77 ml) were added dropwise at the same time. The solution was stirred at 0°C for 1 hour. Then It was diluted with ¾0 (200 ml) and extracted with Et ₂ 0 (5x 200 ml) . The combined organic phases were dried on Na ₂ SC>4 and concentrated affording 20 g of a clear oil. The residue was purified by flash chromatography (SP1 Biotage instrument, SNAP 340 g column, n-Hex/EtOAc from 10:90 to 20:80 in 10 CV) affording the desired product as a clear oil (1.8 g, yield: 24%) .

^X H NMR (CDCI3) : 4.63 (t, 2H) , 3.86 - 3.71 (m, 4H) , 3.67 (t, 2H) , 1.93 - 1.81 (m, 2H) .

STEP C: 3- (2- (nitrooxy) ethoxy) propanoic acid

To a solution of 2- (3-hydroxypropoxy) ethyl nitrate (1.8 g,

10.9 mmol) in acetone (50 ml) cooled at 0°C , NaHCC>3 saturated solution (15 ml), NaBr (0.23 mg, 2.2 mmol), TEMPO (0.17 mg, 1.1 mmol) and tricholoisocyanuric acid (5.0 g, 21.8 mmol) were added. The mixture was stirred at r.t. for 3 hours. Then the solution was cooled at 0°C and Isopropanol (8 ml) was added dropwise. The solution was stirred at 0°C for 30 minutes. The precipitate was filterd off and the solution concentrated. The residue was diluted with NaOH 2N (100 ml) and washed with EtOAc (2X50 ml) . The aqueous phase was acidified with HC1 cone, until pH=2 and extracted with EtOAc (5X 30 ml) . The combined organic phases were dried on a ₂ S0 ₄ and concentrated affording the desired product as a clear oil (1.7 g, yield: 89%) .

^X H NMR (CDC1 ₃ ) : 4.70 - 4.56 (m, 2H) , 3.86 - 3.73 (m, 4H) , 2.66 (t, 2H) .

Example 37

Synthesis of silver (I) 3- (2- (nitrooxy) ethoxy) propanoate

Title compound was synthesized following the procedure described in Example 8 except that 3- (2- (nitrooxy) ethoxy) propanoic acid (prepared in Example 36)

was used instead of 2 , 2-dimethyl-3- (nitrooxy) propanoic acid. ^X H NMR ( DMSO) : 4.71 - 4.55 (m, 2H) , 3.76 - 3.53 (m, 4H) , 2.36 (t, 2H) .

Example 38

Synthesis of 1- ( (4-nitrophenoxy) carbonyloxy) ethyl 3- (2- (nitrooxy) ethoxy) propanoate

Starting from silver (I) 3- (2- (nitrooxy) ethoxy) propanoate (Example 37) and 1-iodoethyl 4-nitrophenyl carbonate (obtained in Example 1), the title compound was synthesized following the procedure of Example 9.

^X H NMR (CDCI3) : 8.40 - 8.22 (m, 2H) , 7.53 - 7.34 (m, 2H) , 6.98 - 6.80 (m, 1H) , 4.60 (t, 2H) , 3.90 - 3.70 (m, 4H) , 2.68 (t, 2H) , 1.74 - 1.59 (m, 3H) . Example 39

Synthesis of (13S) -13-isobutyl-8-methyl-l- (nitrooxy) -6, 10- dioxo-3 7, 9-trioxa-ll-azapentadecan-15-oic acid (compound 16)

(16)

Title compound was synthesized following the procedure of Example 24 starting from compound obtained in Example 38.

XH NMR (CDC1 ₃ ) : 6.93 - 6.76 (m, 1H) , 5.17 - 4.97 (m, 1H) , 4.62 (t, 2H) , 3.85-3.68 (m, 4H) , 3.41-3.21 (m, 1H) , 3.21 - 3.01 (m, 1H) , 2.59 (t, 2H) , 2.46 - 2.05 (m, 3H) , 1.79 - 1.57 (m, 1H) , 1.50 (d, 3H) , 1.19 (t, 2H) , 1.0-0.8 (t, 6H) .

Example 40

Synthesis of (3S) -3- (( (1- (2, 2-dimethyl-3- (nitrooxy) propanoyl oxy) ethoxy) carbonylamino) methyl) -5-methylhexanoic acid (compound 2)

(2)

Title compound was synthesized following the procedure Example 24 starting from compound obtained in Example 9.

1 NMR (CDCI3) : 6.88- 6.67 (m, 1H) , 5.14- 4.88 (m, 1H) , 4 4.37 (m, 2H) , 3.45 - 2.87 (m, 2H) , 2.46 - 2.03 (m, 3H) , 1 1.56 (m, 1H) , 1.48 (d, 3H) , 1.24 - 1.08 (m, 2H) , 1.01-0.76 6H) .

Example 41 Synthesis of ( 3S ) -5-methyl-3- ( ( ( 1- ( 6- (nitrooxy) hexanoyloxy) ethoxy) carbonylamino) methyl) hexanoic acid (compound 4)

(4)

Title compound was synthesized following the procedure of Example 24 starting from compound obtained in Example 11.

¾ NMR (CDC1 ₃ ) : 6.80 (lH,m) ; 5.03 ( 1H, m) ; 4.46(2H,t); 3.29 ( 1H, m) ; 3.17 (lH,m) ; 2.37-2.11 (5H, m) ; 1.70(5H,m); 1. 7 (5H, m) ;

1.19 (2H,m) ; 0.92 (6H,m) .

Example 42

Evaluation of the efficacy of the compounds in a Mouse Chronic Constriction Injury Model (CCI model)

Test compounds:

Compound 12 (prepared according to example 23) : 71 mg Kg ^_1 = 0.175 mmoli Kg ^_1

Gabapentin (reference compound) : 30 mg Kg ^_1 = 0.175 mmoli Kg ^_1 General Procedure and surgery

24 Male C57/black mice weighting 25-28 g caged with free access to food and water under 12h light/dark cycles were used. Experiments were carried out according to the Declaration of Helsinki and internally approved animal care protocols. Efforts were made to minimize animal suffering.

The method described by Bennet and Xie was generally followed (1988) . Mice were anesthetized by tribromoethanol at the dose of 400 mg kg ^-1 . The right common sciatic nerve was exposed at the level of the middle of the tight by blunt dissection through the biceps femoris. Proximal to the sciatic nerve trifurcation, about 6 mm of the nerve was freed of adhering tissue and 3 ligatures (7/0 silk suture) were tied loosely around it with about 1 mm spacing. Ligatures were tied such that the diameter of the nerve was only barely constricted. The incision was closed in layers (3/0 silk suture) .

The experiment was carried out 10 days after surgery.

Reference gabapentin (Sigma Chemicals St Louis, MO, USA) and compound 12 were administered to 8 mice at the indicated doses dissolved in a vehicle containing PEG400: water (70:30) by oral gavage, and a group of 8 mice was administered with the vehicle (PEG400: water (70:30), 5 ml/kg). 1 hour after the treatment the thermal hyperalgesia test was performed on the three groups of mice.

Thermal Hyperalgesia Assessment.

Thermal hyperlagesia to radiant heat was assessed using the Plantar analgesia meter with the heated glass provided by 2B- Biological system (Besozzo, Varese, Italy) . Mice are individually placed in a closed transparent plastic box (5 x 5 x 4 cm) on a glass heated floor which is taken 20 cm over the experimental table and with the help of a small mirror, an infrared heat source is positioned under the plantar surface of the animal hind paw.

The animal paws are tested alternatively with at least 5 minutes interval between consecutive measurements.

The withdrawal latency that is the time taken by the mice to remove or shake its hind paw from the heat source was detected.

Results

The results reported in Table I as delta withdrawal latency (sec) versus basal value (taken before treatment) showed that compound 12 is more efficacious than gabapentin in reversing established pain behavior. Table I: Thermal Hyperalgesia Assessment in a mouse CCI model

Dose lh post-treat

Treatment (delta vs basal)

(mg)

(sec)

Vehicle -0.50 ± 0.40

Gabapentin 30 1.04 ± 0.49

Compound 12 71 2.97 ± 0.48

Example 43

Evaluation of the efficacy of the compounds in a mouse STZ- induced neuropathic pain model (chronic treatment)

The streptozotocin ( STZ ) -induced diabetic mice is the most commonly employed animal model used to study mechanisms of painful diabetic neuropathy and to evaluate potential therapies .

Mice develop tactile allodynia to stimulation of the plantar surface of the hindpaw with von Frey filaments within 20 days of streptozotocin-induced diabetes.

Tactile allodynia is a common type of pain in painful diabetic neuropathy patients.

Test compounds:

Compound 12 (disclosed in example 23) : 71 mg Kg ^_1 = 0.175 mmoli Kg ^_1

Gabapentin (reference compound) : 30 mg Kg ^_1 = 0.175 mmoli Kg ^_1 General Procedure

56 Male C57/black mice weighting 25-28 g caged with free access to food and water under 12h light/dark cycles were used. Experiments were carried out according to the Declaration of Helsinki and internally approved animal care protocols. Efforts were made to minimize animal suffering. Diabetes was induced by the intraperitoneal injection of streptozotocin (STZ) at 120 mg kg ^-1 . Two days later the animals were fasted for 12h and diabetes confirmed by measuring glucose concentration in blood samples obtained by tail prick using a glucose oxidase-impregnated test strips (OneTouch ^® Ultra2 ^® ' LifeScan, CA, USA) .

Only STZ-injected animals with glucose concentration above 300 mg dl ^-1 were considered diabetic and included in further compound testing. Hyperglycaemia was also confirmed at the time of behavioral testing.

After 1 week of the single i.p. injection of STZ mice were divided in three groups and Gabapentin and compound 12 were administered daily, for 21 days consecutively, to 2 groups of mice at 0.175 mmoli kg ^-1 dissolved in a vehicle made with phosphate buffer at pH 7 by oral gavage, and a group of mice was administered with the vehicle (phosphate buffer at pH 7) . 24 hours after the last treatment the tactile allodynia in the three groups of mice was evaluated as below reported.

Tactile Allodynia Assessment.

A standardized testing procedure modified for mice was performed to address tactile allodynia (Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL, J Neurosci Methods. (1994) , 53: 55-63.) . Mice were transferred to the testing cage with a wire mesh bottom and allowed to acclimatize for 60 min.

Tactile allodynia was assessed by recording the pressure at which the affected paw was withdrawn from graded stimuli (Von Frey monofilaments bending force from 0.02 to 3.6 g) applied perpendicularly to the plantar surface of the paw.

These filaments were used to determine the 50% mechanical threshold using the up and down method previously described by Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL, J Neurosci Methods. (1994), 53: 55-63. With an algorithm, using the response of the animal to the different filaments, the threshold at which the animal withdraws the paw the 50% of the times is calculated.

All behavioral testing were performed by investigators blind to the treatments.

Results are expressed as median of the 50% mechanical threshold (g) .

The results reported in Table II, show that diabetes induced a profound allodynic effects (see vehicle value) . This study showed that after chronic administration only Compound 12 is efficacious in counteracting the development of tactile allodynia in a model of neuropathic pain.

Example 44

Evaluation of the efficacy of the compounds in a Rat Spinal Nerve Ligation Model (SNL model)

Test compounds:

Compound 12 (prepared according to example 23) : 71 mg Kg ^_1 = 0.175 mmoli Kg ^_1

Gabapentin (reference compound) : 30 mg Kg ^_1 = 0.175 mmoli Kg ^_1 General Procedure and surgery

34 Male Sprague-Dawley rats weighting 150-200g caged with free access to food and water under 12h light/dark cycles were used. Experiments were carried out according to the Declaration of Helsinki and internally approved animal care protocols. Efforts were made to minimize animal suffering.

The method described by Kim and Chung (1992, Pain. 50(3) : 355- 363) was generally followed. Rats were anesthetized by chloral hydrate at the dose of 400 mg kg ^-1 . Briefly, neuropathic injury was produced by tightly ligating the right L5 and L6 spinal nerves .

The experiment was than carried out 14 days after surgery.

Reference gabapentin (Sigma Chemicals St Louis, MO, USA) and compound 12 were administered to 10-12 rats at the indicated doses dissolved in a vehicle containing PEG400: water (70:30) by oral gavage, and a group of 12 rats was administered with the vehicle (PEG400: water (70:30), 5 ml/kg). 2 hour after the treatment the mechanical sensitivity was measured to the three groups of rats.

Mechanical allodynia Assessment.

A standardized testing procedure was performed to address tactile allodynia (Chaplan et al . , J Neurosci Methods. 53: 55- 63, 1994) . Briefly, rats were transferred to the testing cage with a wire mesh bottom and allowed to acclimatize for 60 min. Von Frey monofilaments (Stoelting, Wood Dale IL, bending force from 0.15 to 15 g applied using a single, steady > Is application) were used to determine the 50% mechanical threshold for foot withdrawal using the up and down method previously described by Chaplan et al . , 1994. All behavioral testing were performed by investigators blind to the treatments . Results

The results reported in Table III as 50% mechanical threshold (g) taken 2h post-treatment showed that compound 12 is more efficacious than gabapentin in reversing established pain behavior.

Example 45

Evaluation of the efficacy of the compounds in the Mouse Formalin test

Test compounds:

Compound 12 (prepared according to example 23) : 71 mg Kg ^_1 = 0.175 mmoli Kg ^_1

Gabapentin (reference compound) : 30 mg Kg ^_1 = 0.175 mmoli Kg ^_1 Compound 30 (prepared according to example 35) : 23.76 mg Kg ^_1 = 0.0628 mmoli Kg ^"1

Pregabalin (reference compound) : 10 mg Kg ^_1 = 0.0628 mmoli Kg ^-1 General Procedure

39 Male C57/black mice weighting 25-28 g caged with free access to food and water under 12h light/dark cycles were used. Experiments were carried out according to the Declaration of Helsinki and internally approved animal care protocols. Efforts were made to minimize animal suffering.

Mice were habituated in the test environment for 30 minutes and the behavioral testing was performed in a blinded manner. Reference gabapentin and pregabalin (Sigma Chemicals St Louis, MO, USA) and compound 12/30 were administered to 6-9 mice at the indicated doses dissolved in a vehicle containing PEG400: water (70:30) by oral gavage, and a group of 8 mice was administered with the vehicle (PEG400: water (70:30), 5 ml/kg) . 30 minutes after the treatment a solution of formalin (20 μΐ at 1.5%) was injected subcutaneously into the dorsal surface of the right hindpaw using an Hamilton syringe with a 26-gauge needle. The efficacy of the compounds in formalin- evoked spontaneous inflammatory pain was assessed by measuring the time (in seconds) spent licking and flinching the affected paw every 5 minutes for 30 minutes, starting from 15 minutes till to 45 minutes after formalin injection (second phase of the test) . The efficacy of the compounds assessed in the second phase of the test is an evaluation of their efficacy in chronic pain.

Results

The results reported in Table IV show that compound 12 and compound 30 were more efficacious than gabapentin and pregabalin respectively.

Example 46

Evaluation of the efficacy of the compounds on allodynic response in streptozotocin (STZ) -treated diabetic mice. Test compounds:

Compound 30 (prepared according to example 35) : 23.76 mg Kg ^_1 = 0.0628 mmoli Kg ^"1

Compound 16 (prepared according to Example 39) 25.65 mg Kg ^_1 = 0.0628 mmoli Kg ^-1 (single dose)

Compound Pregabalin (reference compound) : 10 mg Kg ^_1 = 0.0628 mmoli Kg ^-1 (single dose)

General procedure

Male CD1 mice, weighting 25-35 g were used. Animals were kept under standard conditions of temperature (21-22°C) and illumination (12:12-h light/darkness ) and allowed to adjust to this environment in cages with mesh bottoms with free access to tap water and food for at least 7 days before the experiment began. All experiments were performed to minimize animal suffering and in accordance with approved protocols. Diabetes was induced by injecting 200 mg/kg i.p. of streptozotocin (STZ) . Blood glucose levels were assessed from the 2 ^nd day until the end of the experiment using an Accu-Chek blood glucose monitoring system. Only animals reaching glucose plasma levels above 200mg/dl within 24-48 h post-STZ-inj ection were included in the following behavioral experiments which started 14 days after the STZ treatment.

Paw withdrawal threshold (PWT) was determined using the Dynamic Plantar Aesthesiometer (Ugo Basile, Italy) , an apparatus that generates a mechanical force linearly increasing with time. More specifically, mice were placed individually in plastic cages with a wire mesh bottom and allowed to acclimatize for at least 2 h. Increasing mechanical stimulation (0.25 g/s, cut-off force: 10 g) was applied to the plantar surface of a hind paw so to address the respective nociceptive threshold which is defined as the force, in grams, at which the mouse withdraw its paw. When a withdrawal response occurred, the stimulus was terminated and the response threshold electronically determined.

Results

The effects of test compounds were assayed following an acute challenge at the indicated dose by addressing the allodynic threshold at 30, 60, 90, 120 min time points after the oral treatment.

Compound 30 and compound 16 showed a better efficacy than reference compound pregabalin in this test as indicated by the greater mechanical threshold reached by both compounds at all time-points. (Table V)

Example 47

Evaluation of the efficacy of the compounds after repeated daily treatment on allodynic response in streptozotocin (STZ) - treated diabetic mice.

Test compounds

Compound 12 (prepared according to example 23) : 71 mg Kg ^_1 = 0.175 mmoli Kg ^_1 (repeated daily oral treatment)

Gabapentin (reference compound) : 30 mg Kg ^_1 = 0.175 mmoli Kg ^_1 (repeated daily oral treatment)

General Procedure

Male CD1 mice, weighting 25-35 g were used. Animals were kept under standard conditions of temperature (21-22°C) and illumination (12:12-h light/darkness ) and allowed to adjust to this environment in cages with mesh bottoms with free access to tap water and food for at least 7 days before the experiment began. All experiments were performed to minimize animal suffering and in accordance with approved protocols. Diabetes was induced by injecting 200 mg/kg i.p. of streptozotocin (STZ) . Blood glucose levels were assessed from the 2 ^nd day until the end of the experiment using an Accu-Chek blood glucose monitoring system. Only animals reaching glucose plasma levels above 200mg/dl within 24-48 h post-STZ-inj ection were included in the following behavioral experiments which started 14 days after the STZ treatment.

Paw withdrawal threshold (PWT) was determined using the Dynamic Plantar Aesthesiometer (Ugo Basile, Italy) , an apparatus that generates a mechanical force linearly increasing with time. More specifically, mice were placed individually in plastic cages with a wire mesh bottom and allowed to acclimatize for at least 2 h. Increasing mechanical stimulation (0.25 g/s, cut-off force: 10 g) was applied to the plantar surface of a hind paw so to address the respective nociceptive threshold which is defined as the force, in grams, at which the mouse withdraw its paw. When a withdrawal response occurred, the stimulus was terminated and the response threshold electronically determined.

Results

The effects of the test compounds were assayed by addressing the allodynic threshold 18-24 h after the last repeated (1- 2- or 3-week) daily oral treatment.

The results reported in Table VI showed that the animals treated daily for 1, 2, or 3 weeks with compound 12 developed significantly less mechanical allodynia than animals received vehicle or gabapentin with similar treatment schedule.

These data suggest that compound 12 is more efficacious in hampering the development of pain sensitization in hyperglycemic conditions.

Table VI:

Mechanical allodynia 18 h following 1, 2, or 3-week daily treatment in mice with STZ-induced diabetes

Paw withdrawal threshold (g)

Treatment

1-week 2-week 3-week

Vehicle 1.7 ± 0.1 1.5 ± 0.1 1.3 ± 0.1

Gabapentin 2.0 ± 0.1 1.9 ± 0.3 1.9 ± 0.2

Compound 12 3.2 ± 0.2* 3.8 ± 0.2* 4.1 ± 0.2*

Example 48

Measure of Pharmacokinetic parameters

This study investigated the bioavailability following oral administration of a compound of the present invention and of a compound disclosed by WO 2004/054965 (compound XVA) .

Test compounds:

- Compound 12 (disclosed in example 23) - 1 [4- (nitrooxymethyl) benzoylaminomethyl] cyclohexanacetic acid (compound (XVA) of WO 2004/054965 disclosed in example 1 of WO 2004/054965) (comparative compound)

The integral of the plasma concentration of the test compounds versus an interval of definite time were calculated (AUC)

Significant difference was obtained in the calculated areas under the curve (AUC) of the two compounds, showing that compound (XVA) exhibits poor oral absorption (AUC is 0.358 yg/yL/min) whereas compound 12 is rapidly absorbed from the gastrointestinal tract (AUC is 1.853 yg/yL*min) and it has a good bioavailability.

Pharmacokinetic assessments were performed in blood samples withdrawn intracardially from anesthetized mice (chloral hydrate 380 mg kg ^-1 , ip from Sigma Chemicals, St Louis, MO, USA) at the 5, 15, 30, 60, 120, 240 and 360 time points after oral administration of 71 mg Kg ^_1 (0.175 mmoli Kg ^_1 ) of compound 12 and of 61 mg Kg ^_1 (0.174 mmoli Kg ^_1 ) of compound (XVA). Animals were sacrificed thereafter.

Blood samples were added with eparine to avoid coagulation then blood samples were quenched with methanol to prevent further conversion and analyzed immediately by a sensitive and specific LC/MS/MS method for simultaneous determination of gabapentin, gabapentin lactam and the above reported test compounds .

The plasma levels of compound 12 was evaluated on the basis of the plasma levels of gabapentin because compound 12 forms rapidly gabapentin.

Method for simultaneous determination of gabapentin, gabapentin lactam and the test compounds

The system was an Acquity UPLC (Waters, Milford, MA) . The detector was a Quattro micro™ API LC/MS/MS (Waters, Milford, MA) .

The analytical conditions were: Column: ACQUITY BEH Phenyl 50 x 2.1 mm (5ym) @ 40 °C

Mobile Phase: A. water + 0.1% formic acid; B. methanol + 0.1% formic acid

Flow Rate: 0.5mL/min

Gradient profile

Time (min) A(%)/B(%)

0.0 95/5

1.0 95/5

2.0 0/100

3.0 0/100

3.2 95/5

Injection Volume: 2yL

Detector Wavelength: 210nm

MRM transitions (Multiple Reaction Monitoring) were m/ z 172.10 → 137.00 for gabapentin, m/ z 154.30 → 94.00 for gabapentin lactam and m/ z 350.90 → 180.15 for compound (XVA) .

The raw data (average peak area at each time point) were interpolated using Quanlynx v4.1 software to obtain the concentration of compound (XVA) , gabapentin and gabapentin lactam at each time point.

The results showed that compound (XVA) does not form gabapentin and only a little amount of gabapentin lactam (under quantification limit of ΙμΜ ) was observed at all time points .

Compound 12 forms rapidly gabapentin (within 5 minute after administration) and formation of gabapentin lactam was not observed .

The calculated total AUC of compound (XVA) is 0.358 yg/yL/min. The calculated total AUC of compound 12 is 1.853 yg/yL/min. Example 49

Evaluations of Rat and human plasma stabilities

In this study the plasma stability of some compounds of the invention and of a compound of the prior art were evaluated. Test compounds: - Compound 12 (disclosed in example 23)

- Compound 7 (disclosed in example 22)

1- (aminomethy) cyclohexan acetic acid 3- (nitrooxymethyl) phenyl (compound XVI of WO03/000642, disclosed in example 3 of WO 03/000642) (comparative compound)

The stability of drug candidates is an important parameter, which strongly can influence the in vivo efficacy. In particular it is known that gabapentin lactam, that is a degradation product of gabapentin, is toxic and the specification for the maximum allowable limit for lactam is 0.5% in any dosage form.

The data reported in Table III show that compound XVI undergoes a fast degradation via intramolecular cyclization to 3, 3-pentamethylene-4-butyrolactam (gabapentin lactam), the half -life (ti _/2 ) of compound (XVI) is less than 5 minutes in both matrix (rat and human plasma) and the only metabolite is gabapentin lactam.

Whereas the half -life of compounds 12 and 7 after incubation in human plasma is about 30 minutes and no gabapentin lactam formation was observed.

Example 50

Rat and human liver stabilities methods

A calibration curve (CC) for test compounds 12, 7 and (XVI), for gabapentin and for gabapentin-lactam in both human and rat plasma was prepared in the range 1 - 100 μΜ.

Working solutions (WS) of test compounds in DMSO and of gabapentin and gabapentin-lactam in water were prepared by serial dilution of 10 mM stock solutions to the following final concentrations:

WS (μΜ) 500 / 250 / 100 / 50 / 25 / 10 / 5

1 mL of human or rat plasma were protein precipitated using 10 mL of methanol; 550 yL of each deproteinized matrix were spiked with 10 yL of each working solution to create a calibration curve for each analyte in each matrix to the following final nominal concentrations:

CC (μΜ) 100 / 50 / 20 / 10 / 5 / 2 / 1 990yL of plasma at 37°C were spiked with 10yL of a 1 mM solution of test compound in DMSO. At each time point (5, 10, 15, 30 and 60 min) , 50yL of the test solution were protein precipitated in duplicate using 500yL of acetonitrile, vortex mixed and centrifuged 10 minutes at 4°C at 4000 rpm. The supernatant was transferred to a clean plate together with all the calibration curves and injected onto a UPLC-UV system. Analytical conditions were as follows:

Column: ACQUITY BEH Phenyl 50 x 2.1 mm (5ym) at 40 °C

Mobile Phase: A. water + 0.1% formic acid; B. methanol + 0.1% formic acid

Flow Rate: 0.5mL/min

Gradient profile

Time (min) A(%)/B(%)

0.0 95/5

1.0 95/5

2.0 0/100

3.0 0/100

3.2 95/5

Injection Volume: 2yL

Detector Wavelength: 210nm

MRM transitions (Multiple Reaction Monitoring) were:

m/ z 172.10 → 137.00 for gabapentin,

m/ z 154.30 → 94.00 for gabapentin lactam

m/ z 322.95 → 276.25 for compound (XVI)

m/ z 429.1 → 323.0 for compound 12

m/ z 427.0 → 337.9 for compound 7

The raw data (average peak area at each time point) were interpolated using Quanlynx v4.1 software to obtain the concentration of test compounds, gabapentin and gabapentin lactam at each time points. The extrapolated concentrations were used to calculate the half-life (ti _/2 ) of compound in each matrix and they are reported in Table VII.

Example 51

Measure of Pharmacokinetic parameters in Rat

In this study the bioavailability following oral administration in rats of a compound of the present invention and of gabapentin as reference compound were investigated.

Test compounds:

Test compounds

Compound 12 (prepared according to example 23) : 71 mg Kg ^_1 = 0.175 mmoli Kg ^_1

Gabapentin (reference compound) : 30 mg Kg ^_1 = 0.175 mmoli Kg ^_1 General procedure

Male Wistar rats (Harlan, Concorezzo, Italy) underwent permanent cannulation of the jugular vein to sample blood. The second day after surgery, animals were fasted overnight and in the morning they were treated with gabapentin (30 mg Kg ^_1 , equal to 0.175 mmoli Kg ^_1 ) or compound 12 (71 mg Kg ^_1 equal to 0.175 mmoli Kg ^_1 ) .

A dosing regimen of 5 ml/kg (by gavage) was adopted.

At each time point (0.08, 0.25, 0.5, 1, 2, 4, and 6 hrs) , 100 μΐ of blood were taken from the cannula and treated as described below. Animals were then sacrificed by a lethal injection (Pentothal) . Blood samples were added with eparine to avoid coagulation then blood samples were quenched with methanol to prevent further conversion and analyzed immediately by a sensitive and specific LC/MS/MS method for simultaneous determination of gabapentin, gabapentin lactam and the above reported test compounds .

The plasma levels of compound 12 was evaluated on the basis of the plasma levels of gabapentin because compound 12 forms rapidly gabapentin.

Results

The integral of the plasma concentration of the test compounds versus an interval of definite time were calculated (AUC) Significant differences were obtained in the calculated areas under the curve (AUC) of the two compounds, showing that compound 12 is rapidly absorbed from the gastrointestinal tract (AUC is 3689 yg/yL*min) and it has a good bioavailability .

Method for simultaneous determination of gabapentin, gabapentin lactam and the test compounds

The system was an Acquity UPLC (Waters, Milford, MA) . The detector was a Quattro micro™ API LC/MS/MS (Waters, Milford, MA) .

The analytical conditions were:

Column: ACQUITY BEH Phenyl 50 x 2.1 mm (5ym) @ 40 °C

Mobile Phase: A. water + 0.1% formic acid; B. methanol + 0.1% formic acid

Flow Rate: 0.5mL/min

Gradient profile

Time (min) A(%)/B(%)

0.0 95/5

1.0 95/5

2.0 0/100

3.0 0/100

3.2 95/5 Injection Volume: 2yL

MRM transitions (Multiple Reaction Monitoring) in ESI positive mode were m/z 172.10 → 137.00 for gabapentin, m/z 154.04 → 94.78 for gabapentin lactam and m/z 429.10 → 323.00 for compound 12

The raw data (average peak area at each time point) were interpolated using Quanlynx v4.1 software to obtain the concentration of compound 12, gabapentin and gabapentin lactam at each time point.

Compound 12 forms gabapentin (T _max 240 minute) and the calculated total AUC of compound 12 is 3689 yg/yL*min.