EP2344500A1

EP2344500A1 - Novel combinations of antibacterial nitrogenous heterocyclic compounds with other antibacterial compounds, and use thereof as drugs

Info

Publication number: EP2344500A1
Application number: EP09786281A
Authority: EP
Inventors: Premavathy Levasseur; John Lee Pace; Kenneth Coleman; John Lowther
Original assignee: Novexel SA
Current assignee: Novexel SA
Priority date: 2008-10-10
Filing date: 2009-09-29
Publication date: 2011-07-20
Also published as: ECSP11010973A; PE20110392A1; BRPI0919812A8; PA8845401A1; TW201026697A; WO2010041112A1; IL212180A0; FR2936951B1; FR2936951A1; AR073771A1; UY32168A; CA2740035A1; BRPI0919812A2; EA201170532A1; KR20110067148A; AU2009302153A1; ZA201102498B; CN102216301B; MX2011003812A; CN102216301A

Abstract

The invention relates to the combination of antibacterial nitrogenous heterocyclic compounds of the formula (I) with other antibacterial compounds, and to the use thereof as drugs. The nitrogenous heterocyclic compounds have the general formula (I) where R₁ is a {CH₂}_n-NH₂ or {CH₂}_n-NHR radical, R being a (C₁-C₆) alkyl and n being equal to 1 or 2, R₂ is a hydrogen atom, R₃ and R₄ form together a nitrogenous heterocycle of the aromatic type with 5 vertexes including 1, 2 or 3 nitrogen atoms and optionally substituted by one or more R’ groups, R’ being selected from the group comprising a hydrogen atom and alkyl radicals containing 1 to 6 carbon atoms, in the free form thereof, in zwitterion form, or in the form of salts with pharmaceutically acceptable mineral or organic bases and acids. The other antibacterial agents are selected from the group comprising beta-lactams, monobactams, penicillin, optionally combined with a beta-lactamase inhibitor, aminoglycosides, glycylcyclines, tetracyclines, quinolones, glycopeptides, lipopeptides, macrolides, ketolides, lincosamides, streptogramins, oxazolidinones, polymyxins, and other known compounds having a therapeutic activity on Pseudomonas aeruginosa and Enterobacteriaceae.

Description

NOVEL COMBINATIONS OF ANTIBACTERIAL NITROGENIC HETEROCYCLIC COMPOUNDS WITH OTHER ANTIBACTERIAL COMPOUNDS AND THEIR USE

USE AS MEDICINES

The invention relates to the combination of antibacterial nitrogen heterocyclic compounds with other antibacterial compounds and their use as medicaments.

The Applicant has discovered that new combinations of the compounds of formula (I) described and claimed in the French application 07 02663, with other antibacterial compounds, have antibacterial properties of a very interesting level, thereby manifest an effect. synergy as remarkable as unexpected. The uniqueness of the synergistic combinations of the invention resides in particular in the fact that they exhibit excellent activity on Pseudomonas aeruginosa and Enterobacteriaceae, which are bacterial strains frequently encountered in nocosomal infections as well as in patients suffering from cystic fibrosis.

This particularly interesting and unexpected activity is not present in the compounds of the prior art and in particular those of the application WO 02/100860 which describes compounds comprising other groups R1 than those of the nitrogenous heterocyclic compounds corresponding to the formula ( I) defined below.

These compounds of formula (I) have been active on models of animal infection, including strains usually resistant to commonly used antibiotics. They are able to counter the main mechanisms of bacterial resistance that are β-lactamases, efflux pumps and mutations of porins. These compounds have the following formula: wherein R 1 is (Ct 1) _n -NH ₂ or (CH ₂ ) _n -NHR where R is (C 1 -C 6) alkyl and n is 1 or 2; R ₂ represents a hydrogen atom;

R ₃ and R ₄ together form a nitrogenous heterocycle with a 5-membered aromatic character containing 1, 2 or 3 nitrogen atoms optionally substituted with one or more R 'groups, R' being chosen from the group consisting of a hydrogen atom; and alkyl radicals containing from 1 to 6 carbon atoms, in free form, of zwitterions, and in the form of salts with bases and pharmaceutically acceptable inorganic or organic acids.

The Applicant has discovered that the compounds of general formula (I) potentiate the activity of existing antibacterial compounds, in particular on Pseudomonas aeruginosa and Enterobacteriaceae.

The subject of the invention is thus the combination of a compound of general formula (I) as defined above, in free form, with zwitterions, or in the form of salts with bases and pharmaceutically acceptable inorganic or organic acids, with another antibacterial compound.

By another antibacterial compound is meant in particular a beta lactam, a monobactam or a penicillin, where appropriate combined with a beta lactamase inhibitor, an aminoglycoside, a glycylcycline, a tetracycline, a quinolone, a glycopeptide, a lipopeptide, a macrolide, a ketolide, a lincosamide, a streptogramin, an oxazolidinone, a polymyxin and other known compounds endowed with a therapeutic activity on Pseudomonas aeruginosa and Enterobacteriaceae. As examples of aminoglycosides which may be mentioned, mention may be made of amikacin, gentamycin and tobramycin.

As examples of beta lactams, mention may be made of carbapenems such as: Imipenem, Meropenem, Ertapenem and the compound known under the name PZ-601;

Cephalosporins such as cefazolin, cefepime, cefotaxime,

Cefoxitin, Ceftaroline, Ceftazidime, Ceftibiprole,

Ceftriaxone, Cefuroxime and Cephalexin, Monobactams such as Aztreonam, Penicillins and combinations with beta-lactamase inhibitors such as Amoxicillin,

Amoxicillin / Clavulanate, Ampicillin, Ampicillin / Sulbactam,

Oxacillin, Piperacillin, Piperacillin / Tazobactam,

Ticarcillin, Ticarcillin / Clavulanate and Penicillin. Examples of glycylcycline and tetracycline include Doxycycline, Minocycline, Tetracycline and Tigecycline.

As examples of Quinolones, ciprofloxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Moxifloxacin and Ofloxacin may be mentioned. Examples of Macrolide and Ketolide include Azithromycin, Clarithromycin, Roxythromycin and Telithromycin.

As examples of polymyxin, mention may be made of Colistin and Polymyxin B. Other antibacterial compounds such as Fosfomycin and the combination may also be mentioned.

Trimethoprim / Sulfamethoxazole.

In the compounds of general formula (I), by alkyl radical containing from 1 to 6 carbon atoms, is meant in particular the methyl, ethyl, propyl, isopropyl, and butyl, pentyl or hexyl linear or branched.

By alkenyl radical containing from 2 to 6 carbon atoms is meant in particular the allyl radical and the linear or branched butenyl, pentenyl and hexenyl radicals. Heterocycle with aromatic character, especially means those selected from the following list, the two bonds symbolizing the junction with the nitrogenous ring (R ₃ R 4):

Among the acid salts of the products of formula (I), mention may be made, inter alia, of those formed with mineral acids, such as hydrochloric, hydrobromic, hydroiodic, sulfuric or phosphoric acids, or with organic acids such as formic acid. , acetic, trifluoroacetic, propionic, benzoic, maleic, fumaric, succinic, tartaric, citric, oxalic, glyoxylic, aspartic, alkanesulfonic, such as methane and ethanesulphonic acids, arylsulfonic acids such as benzene and paratoluenesulphonic acids.

Among the base salts of the products of formula (I), mention may be made, inter alia, of those formed with mineral bases such as, for example, sodium, potassium, lithium, calcium, magnesium or ammonium or with organic bases such as, for example, methylamine, propylamine, trimethylamine, diethylamine, triethylamine, N, N-dimethylethanolamine, tris (hydroxymethyl) amino methane, ethanolamine, pyridine , picoline, dicyclohexylamine, morpholine, benzylamine, procaline, lysine, arginine, histidine, N-methylglucamine, or phosphonium salts, such as alkylphosphonium, arylphosphonium, alkylarylphosphonium, alkenylarylphosphonium or quaternary ammonium salts such as tetra-n-butylammonium salt.

Among the synergistic combinations as defined above, the subject of the invention is in particular those containing compounds of formula (I) in which R ₃ and R ₄ together form an optionally substituted pyrazolyl or triazolyl heterocycle. Among these combinations, the subject of the invention is in particular those containing compounds in which R1 is chosen from the group consisting of (CH ₂ ) _H -NH _{2 groups} and

(CH ₂ ) _n -NHCH ₃ , n being as defined above, the heterocycle formed by R ₃ and R ₄ is substituted with a (C ₁ -C ₆ ) alkyl radical.

Among these combinations, the subject of the invention is more particularly those containing compounds in which R ₁ represents a radical (CHa) _n -NH ₂ or (CH ₂ ) _n -NHCH ₃ , n being as defined previously and R ₃ and R4 together form a pyrazolyl ring substituted by a (C ₁ -C ₆ ) alkyl radical.

Among these combinations, the subject of the invention is particularly those containing a compound of formula (I) chosen from: trans-8- (aminomethyl) -4,8-dihydro-1-methyl-5- (sulfooxy) -4 7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-β (5H) -one, trans-8- (aminomethyl) -4,8-dihydro-5- (sulfooxy) -4- , 7-methano-7H-pyrazolo [3, 4-e] [I ₁ 3] diazepin-β (5H) -one, - trans 8- (methylaminomethyl) -4, 8-dihydro-5- (sulphooxy) -

4, 7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-β (5H) -one, in free form, from zwittterion and salts with bases and pharmaceutically acceptable inorganic or organic acids . Among the combinations as defined above, the subject of the invention is in particular those containing antibacterial compounds chosen from beta-lactams or penicillins if necessary combined with beta lactamase inhibitors, and polymyxins.

Among these combinations, the invention particularly relates to those containing antibacterial compounds selected from Tobramycin, Meropenem, Aztreonam, Cefepime,

Ceftazidime, Piperacillin if appropriate combined with

Tazobactam, Colistin and Polymyxin B.

The compounds of formula (I) may be prepared by a process comprising: a) a step in which a compound of formula (II) is reacted with a carbonylation agent, if appropriate in the presence of a base :

in which: R ' _! represents a radical CN, COOH protected, COOR or (CH ₂ J _n R ' ₅ ,

R ' ₅ is a protected OH, CN NH ₂ or protected NHR, protected CO ₂ H, CO ₂ R n, R, R ₃ and R ₄ are as defined above, the aminoalkyl substituent optionally present on the heterocycle formed by R ₃ and R ₄ being then optionally protected,

ZH represents a protected group -NHOH, in order to obtain an intermediate compound of formula (III):

in which : R'i, R ₃ and R ₄ have the same meanings as above and either X _x is a hydrogen atom or a protecting group and X2 represents a group -Z-CO-X ₃ , X ₃ representing the remainder of the carbonylation agent, ie X ₂ is a -ZH group and X1 represents a CO-X _{3 group} , X ₃ being defined as above; b) a step during which the intermediate obtained previously is cyclized in the presence of a base; c) where appropriate, step a) is preceded and / or step b) is followed by one or more of the following reactions, in an appropriate order:

- protection of reactive functions,

deprotection of the reactive functions,

- esterification

- saponification, - sulfation,

- reduction of esters,

- alkylation,

carbamoylation,

formation of an azido group, reduction of an azido to an amine,

- excepted,

- ion exchange,

- Duplication or separation of diastereoisomers. As the carbonylation agent, it is possible to use a reagent such as phosgene, diphosgene, triphosgene, an aryl chloroformate such as phenyl or p-nitrophenyl chloroformate, an aralkyl chloroformate such as benzyl chloroformate. an alkyl or alkenyl chloroformate such as methyl or allyl chloroformate, an alkyl dicarbonate such as tert-butyl dicarbonate, carbonyl diimidazole and mixtures thereof, disphosgene being preferred.

The reaction is preferably carried out in the presence of a base or a mixture of bases which neutralises the acid formed. It can especially be an amine such as triethylamine, diisopropylethylamine, pyridine, dimethylaminopyridine. However, it is also possible to operate using the starting material of formula II as a base. An excess is then used.

If desired, the product of formula II is used in the form of an acid salt, for example a hydrochloride or a trifluoroacetate.

As a base in step b), it is also possible to use the amines, or the hydrides, alkoxides, amides or carbonates of alkali or alkaline-earth metals.

The amines can be chosen for example from the list above.

As hydride can be used in particular sodium hydride or potassium.

As the alkali metal alcoholate, potassium t-butoxide is preferably used. As alkali metal amide can be used in particular lithium bis (trimethylsilyl) amide.

As carbonate, it is possible to use carbonate or sodium bicarbonate or potassium.

If appropriate, the intermediate of formula III can be obtained in the form of an acid salt generated during the carbonylation reaction and in particular a hydrochloride. It is then implemented in the cyclization reaction in this form.

Preferably, the cyclization is carried out without isolation of the intermediate of formula III. The reactions mentioned in step c) are generally conventional reactions, well known to those skilled in the art. Examples of conditions used are described in application WO 02/100860 or in application 04/052891.

The reactive functions that should be protected, if necessary, are the carboxylic acid, amine, amide, hydroxyl and hydroxylamine functions.

The protection of the acid function is especially carried out in the form of alkyl esters, allyl esters, benzyl, benzhydryl or p-nitrobenzyl. Deprotection is carried out by saponification, acid hydrolysis, hydrogenolysis, or cleavage using soluble complexes of Palladium O. Examples of these protections and deprotections are provided in application WO 02/100860.

The protection of amines, heterocyclic nitrogens and amides is in particular carried out, as the case may be, in the form of benzylated or tritylated derivatives, in the form of carbamates, especially of allyl, benzyl, phenyl or tertbutyl, or else in the form of silylated derivatives. such as tertbutyl dimethyl, trimethyl, triphenyl or diphenyl tert-butylsilyl derivatives, or phenylsulfonylalkyl or cyanoalkyl derivatives. Deprotection is carried out, according to the nature of the protective group, by sodium or lithium in liquid ammonia, by hydrogenolysis or with the aid of soluble complexes of Palladium O, by action of an acid, or by the action of fluoride. tetrabutylammonium or strong bases such as sodium hydride or potassium t-butoxide.

The hydroxylamine protection is carried out in particular in the form of benzyl or allyl ethers.

The cleavage of the ethers is carried out by hydrogenolysis or with the aid of soluble complexes of Palladium O. The protection of alcohols and phenols is carried out conventionally in the form of ethers, esters or carbonates. The ethers may be alkyl or alkoxyalkyl ethers, preferably methyl or methoxyethoxymethyl ethers, aryl or preferably aralkyl ethers, for example benzyl, or silylated ethers, for example derivatives thereof. silylated above. The esters may be any cleavable ester known to those skilled in the art and preferably acetate, propionate or benzoate or p-nitrobenzoate. The carbonates may be, for example, methyl, tert-butyl, allyl, benzyl or p-nitrobenzyl carbonates.

The deprotection is carried out by means known to those skilled in the art, in particular saponification, hydrogenolysis, cleavage with soluble complexes of palladium-0, hydrolysis in an acidic medium or, for the silylated derivatives, the treatment with tetrabutylammonium fluoride.

Examples are provided in the experimental part. The sulphation reaction is carried out by action of S0 ₃ complex amines such as SO ₃ -pyridine or SO ₃ - dimethylformamide, operating in pyridine, the salt formed, for example the pyridine salt, can then be exchanged for example by a salt of another amine, a quaternary ammonium or an alkali metal. An example is provided in the experimental part.

The alkylation reaction is carried out by action on the hydroxylated derivatives, the enolates of esters or ketones, the amines or the heterocyclic nitrogens, as the case may be, of an alkyl sulphate or of an alkyl halide or substituted alkyl, in particular with a free or esterified carboxy radical. Alkylation reactions can also be carried out by reductive amination. Salification with acids is optionally carried out by adding an acid in the soluble phase to the compound. Salification with the bases of the sulphooxy function can be carried out using the pyridinium salt obtained during the action of the Sθ ₃ -pyridine complex and the other salts are obtained from this pyridinium salt. One can still operate by ion exchange resin.

The carbamoylation reaction may be carried out by the use of a chloroformate or a Boc-ON type reagent and then an amine or, where appropriate, ammonia. The introduction of an azido group may be carried out for example by the action of sodium azide on a mesylate intermediate or by Mitsunobu type reactions.

The reduction of an azide group can be carried out by the action of trialkyl or triarylphosphine. The separation of enantiomers and diastereoisomers can be carried out according to the techniques known to those skilled in the art, in particular chromatography.

In addition to the processes described above, compounds of formula (I) can be obtained by methods which initially use a compound of formula (II) in which R ' ₁ , R ₃ , R ₄ and HZ have the values which directly lead to (without transformation) to those of the compounds that we wish to prepare. If appropriate, those of these values which contain reactive functions as mentioned above are then protected, the deprotection occurring at the end of the cyclization step b or at any other convenient time in the synthesis. The protections and deprotections are then carried out as described above.

The compound of formula (II) can be obtained by a process according to which a compound of formula (IV) is treated:

in which R'i, R ₃ and R ₄ are defined as above, and A represents a hydrogen atom or a nitrogen-protecting group, by a reducing agent, to obtain a compound of formula (V):

in which A, R'i, R ₃ and R ₄ retain their abovementioned meaning, in which, where appropriate, the OH group is replaced by a leaving group, to obtain a compound of formula (VI):

(VI) in which A, R '1, R ₃ and R ₄ retain their abovementioned meaning and R ₉ represents a leaving group, which is treated with a compound of formula Z ₁ H ₂ in which Z _x represents a group -HN-OH protected then, if necessary, by an agent for deprotecting the appropriate nitrogen atom.

The compound of formula (II) may also be obtained by a process according to which a compound of formula (IV) as defined above is treated with hydroxylamine protected at the level of hydroxy to obtain a compound of formula (VII ):

in which A, R ', R' ₂ , R ₃ , R ' ₄ , n and R' ₈ are defined as above, which is reacted with a reducing agent to obtain a compound of formula (VIII):

in which A, R ', R ₃ , R ₄ , n "and ZH are defined as above, which is treated, if appropriate, with a deprotecting agent of the appropriate nitrogen atom. Nitrogen is one of those mentioned above.

The reducing agent is in particular an alkaline borohydride.

The leaving group is in particular a sulphonate, for example a mesylate or a tosylate, obtained by the action of corresponding sulphonyl chloride in the presence of a base, or a halogen, more particularly chlorine, bromine or iodine, obtained for example by the action of thionyl chloride or P (C ₆ H ₅ ) ₃ CBr ₄ or PBr ₃ or, in the case of an iodine atom, by the action of an alkaline iodide on a sulfonate. The deprotection agent is in particular one of those mentioned above.

The reducing agent that is acted on the compound of formula (VII) is in particular a cyano or a sodium acetoxyborohydride. As indicated above, the compounds of general formula

(I) potentiate the activity of existing antibacterial compounds, in particular on Pseudomonas aeruginosa and

Enterobacteriaceae as well as models of animal infection with strains resistant to commonly used antibacterial agents. Such remarkable and unexpected antibiotic activity had not been observed for the compounds of the prior art.

These properties make the synergistic combinations according to the invention suitable for use as medicaments, in particular in the treatment of severe Pseudomonas and Enterobacteriaceae infections, in particular nosocomial infections and, in general, major infections in subjects at risk. . These may include respiratory tract infections, eg acute pneumonia or chronic lower respiratory tract infections, blood infections, eg septicemia, acute or chronic urinary tract infections, systemic infections. auditory, eg, malignant otitis externa, or chronic suppurative otitis, those of the skin and soft tissues, eg dermatitis, infected wounds, folliculitis, pyoderma, relapsing forms of acne, infections eyes, eg corneal ulcer, nervous system ulcers, including meningitis and brain abscesses, heart infections such as endocarditis, bone and joint infections such as stenoarticular pyoarthrosis, vertebral osteomyelitis, pubic symphysitis, infections of the gastrointestinal tract such that I ¹ necrotizing enterocolitis and perirectal infections.

The present invention also relates to, as ^• medicines including antibiotics drugs, synergistic combinations as defined above.

Among these combinations, the subject of the invention is, as medicaments, those containing compounds of formula (I) in which R ₃ and R ₄ together form an optionally substituted pyrazolyl or triazolyl heterocycle, and of these, those in which R 1 is selected from the group consisting of (CH ₂ ) _n -NH ₂ and (CH ₂ ) _n "NHCH _{3 groups} , n being as defined above, the heterocycle formed by R ₃ and R ₄ is substituted by an alkyl radical (C ₁ -C ₆ ). Among these combinations, the invention more particularly relates, as medicaments, those containing compounds in which R 1 represents a radical (CH ₂ ) n -NH ₂ or (CH 2 ) _n ~ NHCH ₃ , n being as defined above and R ₃ and R ₄ together form a pyrazolyl ring substituted with a (C ₁ -C ₆ ) alkyl radical.

Among these combinations, the subject of the invention is, as a medicament, those containing at least one of the compounds whose names follow: trans-8- (aminomethyl) -4,8-dihydro-1-methyl- 5- (Sulfooxy) -7,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepine

6 (5H) -one, trans-8- (aminomethyl) -4,8-dihydro-5- (sulfooxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepine 6 (5H) -one, or - trans-8- (methylaminomethyl) -4,8-dihydro-5- (sulfooxy) -

4, 7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-6 (5H) -one, in free form, from zwittterion and salts with pharmaceutically acceptable inorganic or organic bases and acids . Among these combinations, the subject of the invention is, in particular, as medicaments, those containing antibacterial compounds chosen from aminoglycosides, beta- lactams, penicillins where appropriate combined with beta lactamase inhibitors, and polymyxins.

Among these combinations, the invention particularly relates, as medicaments, those containing antibacterial compounds selected from Tobramycin, Meropenem, Cefepime, Ceftazidime, Aztreonam, Levofloxacin, Piperacillin if combined Tazobactam, Colistin and Polymyxin B.

The invention also relates to pharmaceutical compositions containing as active ingredients, a synergistic combination as defined above.

These compositions may be administered orally, rectally, parenterally, in particular intramuscular, or locally by topical application to the skin and mucous membranes.

The compositions according to the invention can be solid or liquid and be in the pharmaceutical forms commonly used in human medicine, for example, single or coated tablets, capsules, granules, suppositories, injectable preparations, ointments, creams, gels; they are prepared according to the usual methods. The active ingredient (s) can be incorporated into excipients usually employed in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non aqueous vehicles. fatty substances of animal or vegetable origin, paraffinic derivatives, glycols, various wetting agents, dispersants or emulsifiers, preservatives. These compositions may in particular be in the form of a lyophilizate intended to be dissolved extemporaneously in a suitable vehicle, for example sterile, pyrogen-free water.

The compositions according to the invention thus comprise at least two active principles, and these can be administered simultaneously, separately or in a staggered manner over time. For example, they can present in the form of a "kit", allowing the administration of a compound of general formula (I) and that of another antibacterial compound separately.

The dose of compound of formula (I) administered varies according to the level and nature of the condition treated, the subject, the route of administration and the other antibacterial product considered. It may be, for example, between 0.25 g and 10 g per day, orally in humans, with the product described in Example 1 or between 0.25 g and 10 g per day per day. intramuscular or intravenous route.

The dose in the other antibacterial compound is also variable depending on the condition being treated, the subject, the route of administration and the product under consideration, but usually follows the usual doses prescribed by the pratitians, for example as described in Vidal reference publication. This dose can be up to 10 g per day or more. Nevertheless, as a result of the potentiation provided by the compounds of general formula (I) to the other antibacterial compounds, the doses thereof in the combination can be reduced compared with standard doses.

The combinations according to the invention can also be used as disinfectants for surgical instruments. The following examples illustrate the preparation of compounds of formula (I). The other antibacterial compounds are known and commercial.

EXAMPLES Example 1: sodium salt and trifluoroacetate of the trans

8- (aminomethyl) -4,8-dihydro-1-methyl-5- (sulfooxy) -4,7-methanol

7H-pyrazolo [3,4-e] [1,3] diazepin-6 (5H) -one

Stage A:

4, 7-Dihydro-1-methyl-4- ((phenylmethoxy) amino) -1H-pyrazolo [3,4-c] pyridine-β [5H), 6- (1,1-dimethylethyl) -7-dicarboxylate and 7-methyl (B) The 6- (1,1-dimethylethyl) 6- (1,1-dimethylethyl) 6- (1,1-dimethylethyl) 6- (1H-dihydro-4-hydroxy-1-methyl-1H-pyrazolo [3,4-c] pyridine-6-dicarboxylate derivative) 7-methyl, described in application WO 02100860 (10 g, 32.12 mmol) is suspended in dichloromethane (100 ml) at room temperature under nitrogen and with stirring.The suspension dissolves after the addition of triethylamine (14.30 ml) 10.28 mmol, 3.2eq) To the reaction medium cooled to -78 ^° C., a solution of methanesulfonyl chloride (11.4 ml, 96.36 mmol, 3eq) in dichloromethane (12 ml, 1 volume) is added dropwise. 30 minutes of contact, alcohol A is completely converted to mesylate.

A solution of O-benzylhydroxylamine in dichloromethane is freshly prepared from O-benzylhydroxylamine hydrochloride (25.4 g, 160.6 mmol, 5eq). O-Benzylhydroxylamine hydrochloride is dissolved in a mixture of dichloromethane (100 ml) and water (50 ml). A 2N sodium hydroxide solution (85 ml, 176.66 mmol) is added at 0 ^° C. After 10 min of contact and decantation, the organic phase is dried over magnesium sulfate for 45 min and then concentrated to half volume. The addition of this solution to the mesylate prepared above is at -78 ° C dropwise over 1 hour. The reaction mixture is stirred while allowing the temperature to rise gradually to ambient temperature. It is treated by addition of water (200 ml) and the medium is diluted with dichloromethane (100 ml), stirred, decanted and then the aqueous phase is re-extracted with dichloromethane. The organic phase is washed with saturated NaCl solution (200 ml), dried and then concentrated to dryness. A white amorphous powder is recovered, which after chromatography delivers the expected derivative B (8.25 g, 66%). MS (ES (+)): m / z [M ⁺ ] = 417.2

¹ H NMR (400MHz, Cdc13) diastereomer (2 rotamers) δ (ppm)

= 1.43 (s, 9H, tBu), 3.15 (dd, 1H, N-CH 2 -CH-N), 3.68 / 3.70 (s,

3H, CH 3), 3.84 (s, 3H, CH 3), 3.98 (m, 2H, N-CH 2 -CH-N), 4.6-4.8

(solid, 3H, NH-O-CH 2 -Ph and N-CH 2 -CH-N), 5.40 / 5.8 (s, 1H, CH-CO2Me), 7.22-7.31 (solid, 5H, Ph), 7.40 ( s, IH, H pyrazole) Stage B: 4,5-trans-methyl-β-oxo-5- (phenylmethoxy) -5,6,6,8-tetrahydro-4,7-methano-1H-pyrazolo [3,4-e] [1,3] diazepine-8 ( 7H) methyl carboxylate (C)

A 4N solution of HCl / dioxane (400 ml, 15 eq) is poured onto a solution of B (21 g, 50.42 mmol) dissolved in dioxane (50 ml) at room temperature. The reaction mixture is stirred for 30 min and then the dioxane is evaporated. The residue is taken up with stirring in a mixture of water (100 ml) and ethyl acetate (500 ml). A solution of 20% concentrated ammonia (42 ml) is added at 0 ^° C. Stirring is continued for 30 min. After decantation ^, the aqueous phase is re-extracted with ethyl acetate (2 * 300 ml), the last extraction being carried out after saturation of the aqueous phase with NaCl. The organic phase is dried and concentrated. The deprotected piperidine intermediate is obtained in the form of a yellow oil (m = 15.7 g, 98%) which is taken up in acetonitrile (400 ml). To this mixture cooled to 0 ⁰ C, was added triethylamine (21 ml, 151.2 mmol, 3 eq) and then diphosgene (3.04 ml _f 25.2 mmol, 0.5eq) run in dropwise over 30 min. After a night of contact at ambient temperature, the medium is concentrated and then taken up in ethyl acetate (500 ml) and treated with a 10% tartaric acid solution (200 ml). The mixture is stirred, decanted. The organic phase is washed with a solution of 10% tartaric acid (2 * 200 ml), with a saturated NaCl solution, then dried and concentrated under reduced pressure. The white product obtained (m = 15.3 g, 89%) is taken up in dichloromethane (150 ml). 1-8-Diazabicyclo [5.4.0] undec-7-ene (7.53 mL, 50.04 mmol) is added dropwise. The mixture is stirred for 2 h, treated with water (200 ml), stirred, decanted. The organic phase is washed with water (2 × 200 ml) and then with saturated NaCl solution (1 × 200 ml), dried over MgSO ₄ and then concentrated to dryness.

The expected C derivative (m = 14.72 g, 85%) is recovered in the form of a white solid

MS (ES (+)): m / z [M ⁺ ] = 343 ¹ H NMR (400MHz, Cdc13): δ (ppm) = 3.25 (d, IH, N-CH2-CH-N), 3.45 (d, IH, N-CH2-CH-N), 3.80 (s, 3H, CH 3), 3.88 (s, 3H, CH 3), 3.9 (s, 1H, N-CH 2 -CH-N), 4.7 (d, 1H, NO-CH 2 -Ph), 5.02 (d, 1H, NO-CH 2) -Ph), 5.22 (sH, CH-CO2Me), 7.39-7.43 (solid, 6H, H pyrazole + Ph) Stage C:

4,8-dihydro-8- (hydroxymethyl) -1-methyl-5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-β (5H) - one (D)

A solution of C (5 g, 14.60 mmol) in a mixture of anhydrous tetrahydrofuran (150 ml) / methanol (50 ml), under nitrogen and with stirring, is cooled to -10 ^° C. Lithium borohydride (668 mg, 30.67 mmol, 1.2eq) is added to the reaction medium. After stirring for 2 h at -10 ^° C., an additional 1.2 eq of LiBH 4 are added. The reaction is cold-treated 2 h later with a 10% solution of NaH 2 PO 4. Tetrahydrofuran and methanol are evaporated under reduced pressure (200 mbar, 40 ⁰ C). The residual mixture is taken up in ethyl acetate (200 ml), stirred and decanted. The aqueous phase is re-extracted with ethyl acetate (100 ml). The organic phase is dried over magnesium sulfate and then concentrated to dryness. The light yellow powder obtained (6.6 g) is chromatographed on silica (eluent-ethyl acetate) to give the derivative D (3.2 g, 10.18 mmol, 64%). MS (ES (+)): m / z [M ⁺ ] = 315 1H NMR (400 MHz, DMSC-d6): δ (ppm) = 3.16 (dd, 1H, N-CH2-CH-N),

3.48 (d, 1H, N-CH 2 -CH-N), 3.71 (s, 3H, CH 3), 3.81-3.91

(solid, 2H, CH 2 OH), 4.44 (m, 1H, N-CH 2 -CH-N), 4.48 (m, 1H,

CH 2 OH), 4.88 (m, 2H, N-O-CH 2 -Ph), 5.20 (m, 1H, OH), 7.35.

7.40 (solid, 6H, H pyrazole + Ph). Stage D:

Trans-4,8-dihydro-1-methyl-8 - [(methylsulfonyl) oxymethyl)] - 5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepine 6 (5H) -one (E)

The derivative D (2.76 g, 8.78 mmol) is dissolved in dichloromethane (100 ml) at room temperature under nitrogen and with stirring. After cooling to 0 ^° C., triethylamine (1.83 ml, 13.17 mmol, 1.5eq) is added and then drop a solution of mesyl chloride (1.61 g, 14.05 mmol) in dichloromethane (100 ml). The ice bath is removed at the end of the addition. After one hour of contact at room temperature, the reaction is treated with stirring with a 10% solution of NaH2PO4 (80 ml). After stirring and decantation, the aqueous phase is re-extracted with dichloromethane (50 ml). The organic phase is dried and then concentrated under reduced pressure to give the expected derivative (3.44 g, quantitative yield). MS (ES (+)): m / z [M ⁺ ] = 393

¹ H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) = 3.23 (dd, 1H, N-CH 2 -CH-N), 3.26 (s, 3H, CH 3), 3.45 (d, 1H, N- CH2-CH-N), 3.76 (s, 3H, CH3), 4.52 (m, 1H, N-CH2-CH-N), 4.58 (dd, 1H, CH-CH2-OMs), 4.66 (dd, 1H, CH-CH2-OM5), 4.88 (m, 3H, CHCH2OMS and NO-CH2-Ph), 7.35-7.45 (solid, 6H, H pyrazole + Ph) Step E: trans 8- (azidomethyl) -4,8-dihydro 1-methyl-5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-6 (5H) -one (F)

Sodium azide is added all at once (1.71 g, 26.3 mmol) to a solution of E (3.44 g, 8.78 mmol) in dimethylformamide (70 ml) at room temperature under nitrogen and with stirring. The reaction medium is heated at 65 ° C. overnight, and then treated with a 10% aqueous solution of NaH ₂ PO ₄ (50 ml). After stirring and decantation, the aqueous phase is re-extracted with dichloromethane (2 * 50 ml). The organic phase is dried and then concentrated under reduced pressure to give 3.96 g of expected F derivative (3 g, 8.78 mmol). MS (ES (+)): m / z [M ⁺ ] = 340 1H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) = 3.20 (dd, 1H, N-CH 2 -CH-N), 3.48 (d, 1H, N-CH2-CH-N), 3.66 (dd, 1H, CH-CH2-N3), 3.72 (s, 3H, CH3), 3.92 (dd, 1H, CH-CH2-N3), 4.50 (d, 1H, N-CH2-CH-N), 4.76 (dd, 1H, CHCH2ON3), 4.89 (m, 2H, NO-CH2-Ph), 7.35-7.45 (solid, 6H, H pyrazole + Ph) Stage F: trans [[4,5,6,8-tetrahydro-1-methyl-6-oxo-5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] 1,1-Dimethylethyl diazepam-n-8-yl] methyl] carbamate (G) A molar solution of trimethylphosphine (3.4 ml, 3.4 mmol) is added dropwise to a solution of F (1.15 g, 3.39 g). mmol) in toluene (5 ml) and tetrahydrofuran (5 ml) at room temperature under nitrogen and stirring. After 3 hours of contact, a solution of BOC-ON (0.92 g, 3.6 mmol) in tetrahydrofuran (10 ml) is added dropwise to the reaction medium cooled to 0 ° C. Stirring is continued for 3 h at room temperature. The reaction medium is treated with a 10% aqueous solution of NaHCO ₃ (50 ml). After stirring and decantation, the aqueous phase is reextracted with ethyl acetate (50 ml). The organic phase is dried and then concentrated under reduced pressure to give an oil (2.2 g). The crude product is chromatographed on a silica column (eluent cyclohexane / ethyl acetate 5/5). The expected product is obtained (0.62 g, 1.49 mmol, 70%).

MS (ES (+)): m / z [M ⁺ ] = 414

¹ H NMR (400MHz, Cdc13): δ (ppm) = 1.39 (s, 9H, tBu), 3.05 (dd, IH, N-CH2_-CH-N), 3.19 (dd, IH, CH-CH2-NHBoc) , 3.27 (dd, 1H, N-CH2-CH-N), 3.72 (s, 3H, CH3), 3.78 (m, 1H, CH-CH2-NHBOC), 3.88 (d, 1H, N-CH2-CH- N), 4.48 (dd, 1H, CHCH2NHBOC), 4.79 (d, 1H, NO-CH2-Ph), 4.92 (d, 1H, NO-CH2-Ph), 5.18 (m, 1H, mobile H), 7.35 ( s, 1H, H pyrazole), 7.37-7.48 (solid, 5H, Ph) Step G: Trans [[4,5,6,8-tetrahydro-1-methyl-6-oxo-5- (sulphooxy) pyridinium salt 1,1-Dimethylethyl-4, 7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-8-yl] methyl] carbamate (H).

10% palladium on charcoal (140 mg) is added to a solution of G (0.6 g, 1.45 mmol) in methanol (10 ml). The reaction medium is hydrogenated for 3 hours. The methanol is then evaporated under reduced pressure to give the debenzylated intermediate. MS (ES (+)): m / z [M ⁺ ] = 324 The debenzylated intermediate is taken up in pyridine (3 ml) in the presence of the pyridine / trioxide sulfide complex (462 mg, 2.9 mmol). The reaction is stirred at room temperature overnight. The medium is then concentrated under reduced pressure. The crude reaction product is chromatographed on a silica column (eluent dichloromethane 100% and then gradient with methanol from 5% to 20%) to give the derivative H (0.49 g, 1.25 mmol, 84%). MS (ES (+)): m / z ^[M-] = 402 1H NMR (400 MHz, DMSO-d _6): δ (ppm) = 1.41 (s, 9H, tBu), 3.30- 3.80 (unresolved peak, 4H , 2. CH2), 3.72 (s, 3H, CH3), 4.42 (dd, 1H, CHCH2ONHBOC), 4.64 (d, 1H, N-CH2-CH-N), 7.21 (m, 1H, mobile), 7.35 ( s, 1H, H pyrazole), 8.02 (dd, 2H, pyridine), 8.54 (m, 1H, pyridine), 8.91 (m, 2H, pyridine). Step H:

Sodium salt of trans [[4,5,5,6,8-tetrahydro-1-methyl-6-oxo-5- (sulphooxy) -4,7-methano-7-pyrazolo [3,4-e] [1 , 3] diazepin-8-yl] methyl] -carbamate 1,1-dimethylethyl (I)

A suspension of 60 g of DOWEX 50WX8 resin in 2N sodium hydroxide solution (300 ml) is stirred for one hour and then poured onto a chromatographed column. The product is eluted with demineralized water to a neutral pH, and then the column is conditioned with a 90/10 water / THF mixture. The derivative H (0.49 g, 1.01 mmol) is dissolved in a minimum of water, deposited on the column, then eluted with a water / THF 90/10 mixture. The fractions containing the substrate are combined and frozen. The frozen solution is lyophilized to yield the expected product (0.44 g, 1.03 mmol, 100%). MS (ES (-)): m / z [M ^"] = 402 1H NMR (400 MHz, DMSO-d _6): δ (ppm) = 1.39 (s, 9H, tBu), 3.30-3.72 (m, 7H , 2 CH2, CH3), 4.42 (m, 1H, CHCH2ONHBOC), 4.64 (s, 1H, N-CH2-CH-N), 7.16 (m, 1H, mobile), 7.35 (s, 1H, H pyrazole) Step I: Trans 8- (Aminomethyl) -4,8-dihydro-1-methyl-5- (sulfooxy) -4,7-methano-7H-pyrazolo [3,4-e] sodium salt and trifluoroacetate salt ] [1,3] diazepin-6 (5H) -one (J) A solution of trifluoroacetic acid (10 ml) in dichloromethane (10 ml) is poured dropwise onto a solution of JC (0.15 g, 0.35 mmol) in dichloromethane (5 ml) under nitrogen and cooled to 0 ^° C. The reaction is stirred for 1 h at room temperature. The mixture is evaporated to dryness and taken up in a minimum of water. The solution is frozen and then lyophilized to give the expected derivative J (193 mg, 0.35 mmol, 100%). MS (ES (-)): m / z [M ⁺ ] = 301 1H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) = 3.32 (dd, 1H, N-CH 2 -CH-N), 3.33 -3.37 (m, 2H, 2CH), 3.43 (d, 1H, N-CH2-CH-N), 3.74 (s, 3H, CH3), 4.73 (m, 2H, CH-CH2-NH3 +), 7.41 (s). , 1H, H pyrazole), 8.10 (m, 3H, NH 3 ⁺ ). EXAMPLE 2 The sodium salt and trifluoroacetate salt of trans-8- (amino-methyl) -4,8-dihydro-5- (sulfooxy) -4 7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-6 (5H) -one Step A:

Trans-4,8-dihydro-8- (hydroxymethyl) -5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-6 (5H) -one trans-4,5,6,8-tetrahydro-6-oxo-5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepine-8-carboxylate ester Methyl described in WO 2004/052891 (Example 1, Step K) (5 g, 15.2 mmol) is dissolved in a 1: 1 methanol / anhydrous tetrahydrofuran (100 mL) under nitrogen. NaBH ₄ (2.3 g, 60.9 mmol) is then added portionwise.

After stirring overnight at room temperature, the reaction mixture is treated with an aqueous solution of 10% NaH ₂ PO _4.

(100 mL). After evaporation to dryness, the reaction mixture is taken up in water. The precipitate formed is stirred overnight in ice, then filtered and dried for at least 24 hours under vacuum in the presence of P ₂ O ₅ , to give the expected compound (3.30 g, 11.0 mmol, 72%) in the form of a white powder. MS (ES (+)): m / z [M + H] ⁺ = 301 1H NMR (400MHz, DMSO-d ₆ ): δ (ppm) = 3.18-3.50 (ABX, 2H, N-CH ₂ -CH- N), 3.65-3.76 (ABX, 2H, N-CH-CH ₂ -OH), 4.34 (t, 1H, N-CH-CH ₂ -OH), 4.46 (d, 1H, N-CH ₂ -CH- N), 4.88 (s, 2H, CH ₂ -Ph), 7.29-7.43 (m, 5H, Ph), 7.66 (s, 1H, H pyrazole), 12.72 (broad, 1H, OH). Step B: trans [[4,5,6,8-tetrahydro-6-oxo-5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-8 -yl] methyl] - carbamate ^• 1, 1-dimethyl the alcohol obtained in step A of example 2 (1.73 g, 5.76 mmol) is dissolved in anhydrous pyridine (35 mL) under nitrogen at 0 ⁰ C. Then methanesulfonyl chloride (1.78 mL, 23 mmol) was added dropwise. After stirring for 2 hours at room temperature, the reaction mixture is treated with a saturated aqueous solution of ammonium chloride (100 ml) and then extracted with ethyl acetate. The combined organic phases are then washed 5 times with a saturated aqueous solution of ammonium chloride, dried over sodium sulfate, filtered and then concentrated in vacuo to give the expected dimesylated derivative in the form of a yellow oil.

The dimesylated intermediate is dissolved in anhydrous dimethylformamide (45 ml), under nitrogen, in the presence of sodium azide (1.12 g, 17.3 mmol). The reaction mixture is heated at 70 ^° C. for 24 hours. If necessary 1 eq. Azide is added so that the conversion is complete. When the reaction is complete, the mixture is treated with a 10% aqueous solution of NaH ₂ PO ₄ (100 mL) and then extracted with dichloromethane. The combined organic phases are dried over sodium sulphate, filtered and then concentrated in vacuo to give the expected azide as a yellow oil.

The intermediate is reacted, under nitrogen, in absolute ethanol (17.5 mL). Di-tert-butyl dicarbonate (1.38 g, 6.34 mmol), triethylsilane (1.38 mL, 8.64 mmol) and 10% Degussa palladium hydroxide on charcoal (52 mg) are then successively added. After one night at room temperature, the reaction mixture is filtered and concentrated to give a crude yellow oil. This crude is purified by chromatography on a silica column (gradient eluent CH ₂ Cl ₂ ZMeOH 100/0 to 95/5 by 1%) to yield the expected compound (1.36 g, 3.40 mmol, 34%) in the form of a white solid. MS (ES (+)): m / z [M + H] ⁺ = 401 ¹ H NMR (400MHz, MeOH-d _4): δ (ppm) = 1.51 (s, 9H, C (CH ₃ J _3), 3.21- 3.59 (m, 4H, N-CH ₂ -CH-N and N- CH-CH ₂ -NHBOC), 4.36 (m, IH, N-CH-CH ₂ -OH), 4.46 (m, IH, N-CH ₂ -CH-N), 4.99 (AB, 2H, CH ₂ -Ph 7.41-7.52 (m, 5H, Ph), 7.63 (s, 1H, H pyrazole) ^• Stage C: trans [[4,5,6,8-tetrahydro-1-tert-butoxycarbamate-6-oxo] 1- (Phenyl-methoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-8-yl] methyl] -carbamate 1,1-diethylcellulose

The compound obtained in Step B of Example 2 (104 mg, 0.26 mmol) is dissolved in anhydrous dichloromethane

(2.5 mL) then di-tert-butyl dicarbonate (114 mg, 0.52 mmol) and dimethylaminopyridine (32 mg, 0.26 mmol) are added to the mixture. After stirring for 1 night at room temperature, the reaction mixture is treated with water. The phases are separated then the organic phase is washed with a saturated aqueous solution of sodium chloride, dried over sodium sulfate, filtered and then concentrated in vacuo. The crude product thus obtained is purified by chromatography on silica (eluent: CH ₂ Cl ₂ ZAcOEt 90/10) to give the expected product (76 mg, 0.15 mmol, 59%). MS (ES (+)): m / z [M + H] ⁺ = 500

Stage D: pyridinium salt of trans [[1-tert-butoxycarbamate-4,5,6,8-tetrahydro-6-oxo-5- (sulfooxy) -4,7-methano-7H-pyrazolo [3,4-de] e] [1,3] diazepin-8-yl] methyl] -carbamate 1,1-dimethyl

The compound obtained in Step C of Example 2 (76 mg, 0.15 mmol) is dissolved under nitrogen in a mixture of dimethylformamide / CH ₂ Cl ₂ 1/3 anhydrous (0.87 mL). 10% palladium on carbon at 50% water (49 mg) is added. After three vacuum / nitrogen purges, the reaction mixture is placed under a hydrogen atmosphere until the starting material is separated by HPLC. The mixture is then concentrated under vacuum and then co-evaporated three times with anhydrous dichloromethane, finally dried under a vacuum blank in the presence of P ₂ O ₅ for 2 h. The debenzylated derivative is taken up in anhydrous pyridine

(0.43 mL), under nitrogen, in the presence of the pyridine / trioxide sulfide complex (48 mg, 0.30 mmol). The mixture The reaction mixture is stirred at room temperature until complete conversion to HPLC and then evaporated to dryness after treatment with the addition of water. The crude thus obtained is purified by chromatography on silica (eluent CH ₂ Cl ₂ / MeOH 90/10) to give the expected compound (47 mg, 0.083 mmol, 55%). MS (ES (-)): m / z [M-2 * BOC-H] ^~ = 388

¹ H NMR (400MHz, MeOH-d _4): δ (ppm) = 1.52 (s, 18H, 2x C (CH ₃₎ _3), 3.50 (m, 4H, N-CHZ-CH-N and CH ₂ -NHBOC 4.62 (m, 1H, CH-CH ₂ -NHBoc), 4.85 (d, 1H, N-CH ₂ -CH-N), 7.72 (s, 1H, H pyrazole). Stage E: Trans [[8- (Amino-methyl) -4,8-dihydro-5- (sulfoxy) -4,7-methano-7H-pyrazolo [3,4-e] sodium and trifluoroacetate salt [1,3] diazepin-6 (5E) -one

A suspension of 6 g of DOWEX 50WX8 resin in a 2N sodium hydroxide solution (30 mL) is stirred at room temperature for 1 h, then poured onto a column to be chromatographed. After rinsing with H ₂ O until neutral pH, the column is conditioned with a THF / H ₂ O 10/90 mixture. The derivative obtained in Step D of Example 2 (47 mg, 0.08 mmol) is dissolved in a minimum of methanol and then deposited on the column. After elution with a THF / H ₂ O 10/90 mixture, the fractions containing the expected product are combined, frozen and then lyophilized to yield the expected sodium salt.

The sodium salt is taken up in anhydrous dichloromethane (1.04 mL) under nitrogen and then cooled to 0 ^° C. A solution of trifluoroacetic acid / anhydrous dichloromethane 1/1 (2.04 mL) is added dropwise. The reaction mixture is then stirred at room temperature for 45 minutes. After evaporation to dryness and then co-evaporation with anhydrous dichloromethane, the compound is taken up in water (~ 2 mL) and then frozen and lyophilized to give the expected salt (16 mg, 0.030 mmol, 36%) as a yellow powder. blade. MS (ES (-)): m / z [MH] ^"= 288 1H NMR (400MHz, MeOH-d _4): δ (ppm) = 3.37-3.69 (m, 4H, N-CH ₂ -CH-N and CH-CH ₂ -NH ₂ ), 4.81 (dd, 1H, CH-CH ₂ -NH ₂ ), 4.98 (d, 1H, N-CH ₂ -CH-N), 7.79 (s, 1H, H pyrazole). Example 3: Sodium salt and trifluoroacetate salt of trans [[8- (methylamnomethyl) -4,8-dihydro-1-methyl-5- (sulfooxy) -4,7-methano-7H-pyrazolo [3,4-de] e] [1,3] diazepin-6 (5H) -one Step A: Iodide of trans [[[4,5,6,8-tetrahydro-1-methyl-6-oxo-5-

(phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepin-8-yl] methyl] methylamino] trimethylphosphonium

A molar solution of trimethylphosphine (1.5 μL, 1.5 mmol) is added dropwise to a solution of the derivative obtained in Step E of Example 1 (0.5 g, 1.25 mmol) in solution in tetrahydrofuran (15 mL) at room temperature. under nitrogen and with stirring. After stirring for 2 h, the methane iodide (0.21 g, 3.75 mmol) is added to the reaction medium. A light yellow precipitate is quickly formed. After stirring overnight at ambient temperature, the reaction medium is concentrated under reduced pressure. The crude product is triturated in dichloromethane. The precipitate is filtered to give the product (0.42 g, 1.04 mmol, 84%) expected as yellowish iodine salt. MS (ES (+)): m / z [M + H] ⁺ = 402

¹ H NMR (400MHz, CDCl ₃₎ as two conformers: δ (ppm) =

2.04 (s, 3H, CH ₃ P), 2.32 (s, 3H, CH ₃ P), 2.35 (s, 3H, CH ₃ P),

3.03 (s, 3H, β-NCH ₃ (A) -CH ₂ ), 3.05 (s, 3H, β-NCH ₃ (B) -CH ₂ ), 3.37 (m, 1H, N-CH ₂ -CH-N). or CH-CH ₂ -N (CH ₃ ) P), 3.44 (m, 1H, N-CH ₂ -CH-N or CH-CH ₂ -N (CH ₃ ) P), 3.69 (m, 1H, N- CH ₂ -CH-N or CH-CH ₂ -N (CH ₃ ) P), 3.82 (s, 3H, CH ₃ ), 3.88 (m, 1H, N-CH ₂ -CH-N or CH-CH ₂ - N (CH ₃ ) P),

4.05 (d, 1H, N-CH ₂ -CH-N), 4.59 (d, 1H, CH-CH ₂ -N (CH ₃ ) P), 4.88 (d, 1H, NO-CR 2 -Ph), 5.00 ( d, 1H, NO-CH ₂ -Ph), 7.35 (s, 1H, H pyrazole), 7.37-7.45 (solid, 5H, Ph). Step B: trans 8- (methylaminomethyl) -4,8-dihydro-1- methyl-5-

(phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] diazepine

6 (5H) -one

To an aqueous solution of sodium carbonate (2.5N, 9 mL) is added the derivative obtained in Step A of Example 3 (0.42 g,

1.04 mmol). The reaction medium is stirred at 55 ° C. for 3 h 30 min. After cooling to room temperature, the medium The reaction is saturated with sodium chloride in the presence of ethyl acetate (25 mL). The aqueous phase is extracted with ethyl acetate (3x25 mL). The organic phase is dried over magnesium sulfate and then concentrated under reduced pressure to give a yellow oil (0.26 g). The crude reaction product is purified by chromatography on a silica column (100% dichloromethane eluent and then gradient with 2% to 10% methanol) to give the expected derivative (0.084 g, 0.256 mmol, 26%). MS (ES (+)): m / z [M + H] ⁺ = 328 1H NMR (400MHz, CDCl ₃ ): δ (ppm) = 2.97-3.00 (dd, 1H, N-CH ₂ -CH-N) , 3.00 (CH-CH ₂ -NCH ₃ ), 3.15 (dd, 1H, CH-CH ₂ -NCH ₃ ), 3.9 (dd, 1H, N-CH ₂ -CH-N), 3.75 (s, 3H, CH). ₃ ), 3.98 (d, 1H, CH-CH ₂ -N (CH ₃ ) Boc), 4.72 (dd, 1H, N-CH ₂ -CH-N), 4.90 (d, 1H, NO-CH ₂ -Ph). ), 5.03 (d, 1H, NO-CH ₂ -Ph), 7.30 (s, 1H, H pyrazole), 7.34-7.44 (solid, 5H, Ph)

Stage C: trans [[4,5,6,8-tetrahydro-1-methyl-6-oxo-5- (phenylmethoxy) -4,7-methano-7H-pyrazolo [3,4-e] [1,3] ] The derivative obtained in Step B of Example 3 (80 mg, 0.244 mmol) is dissolved in dichloromethane (1 mL) and then dissolved in methylene chloride. room temperature is successively added triethylamine (60 μl, 0.488 mmol) and di-tert-butyldicarbonate (106 mg, 0.488 mmol). After stirring for 4 hours at room temperature, a saturated solution of sodium chloride (5 ml) is added to the reaction medium. The aqueous phase is extracted with dichloromethane (3 × 20 mL). The organic phase is dried over magnesium sulfate and then concentrated under reduced pressure to give an amorphous white powder (157 mg). The crude reaction product is chromatographed on a silica column (100% dichloromethane eluent and then gradient with 20% to 30% ethyl acetate) to give the expected derivative (0.068 g, 0.159 mmol, 60%). MS (ES (+)): m / z [M + H] ⁺ = 428 1H NMR (400MHz, CDCl ₃ ): δ (ppm) = 1.59 (s, 9H, C (CH ₃ J ₃ ), 3.05 (s) , 3H, CH ₃ NBoC-CH ₂ ), 3.10 (m, 3H, N-CH ₂ -CH-N, CH-CH ₂ -NBoc), 3.75 (m, 1H, N-CH ₂ -CH-N), 3.85 (s, 3H, CH ₃ ), 3.99 (s, 1H, N- CH ₂ -CH-N), 4.75 (m, 1H, CH-CH ₂ -N (CH ₃ ) BoC), 4.90 (d, 1H, NO-CH ₂ -Ph), 5.02 (d, 1H, NO-CH 2 -Ph), 7.37 (s, 1H, H pyrazole), 7.40-7.46 (solid, 5H, Ph) Step D: Pyridinium salt of trans [[4,5,6,8-tetrahydro-1-methyl-6-] 1,1-dimethylethyl-5-oxo-5- (sulphooxy) -4,7-methano-7-pyrazolo [3,4-e] [1,3] diazepin-8-yl] methyl] methyl-carbamate

Proceeding as indicated in Step G of Example 1, the compound obtained in Step C of Example 3 (0.068 g, 0.159 mmol) in methanol (5 mL), in the presence of 10% palladium on carbon (25 mg ) lead to the debenzylated product. MS (ES (+)): m / z [M + H] ⁺ = 337

The debenzylated intermediate, pyridine (1 mL), the pyridine / trioxide sulfide complex (50 mg, 0.318 mmol) give the expected salt (0.045 g, 0.090 mmol, 100%). MS (ES (-)): m / z [MH] ^"= 416

¹ H NMR (400 MHz, MeOH-d ₄₎ in the form of two conformers: δ (ppm) = 1.53 (s, 9H, C (CH ₃₎ _3, 3.09 (s, 3H, CH ₃ (A) NHBoc) 3.10 (s, 3H, CH ₃ (B) NHBOC), 3.37 (m, 1H, BocN (CH ₃ ) -CH ₂ -CH or N-CH ₂ -CH-N), 3.58 (m, 1H, BoCN (CH ₃ ) -CH ₂ -CH or N-CH ₂ -CH-N), 3.75 (s, 3H, CH ₃ ), 3.84

(m, 1H, BocN (CH ₃ ) -CH ₂ -CH or N-CH ₂ -CH-N), 3.90 (m, 1H, BoCN (CH ₃ ) -

CH ₂ -CH or N-CH ₂ -CH-N), 4.90 (m, 2H, N-CH-CH ₂ -N, N-CH ₂ -CH-N + H ₂ O signal), 7.54 (s, 1H) , H pyrazole), 8.16 (dd, 2H, pyridine),

8.70 (dd, 2H, pyridine), 8.94 (d, 1H, pyridine) Step E:

Sodium salt of trans [[4,5,5,6,8-tetrahydro-1-methyl-6-oxo-5- (sulphooxy) -4,7-methano-7H-pyrazolo [3,4-e] [1 , 3] 1,1-dimethylethyl diazepin-8-yl] methyl] methyl-carbamate

By proceeding as indicated in Step H of Example 1, the salt obtained in Step D of Example 3 (0.045 g, 0.090 mmol), DOWEX 50WX8 resin (30 g) and 2N sodium hydroxide (150 mL) lead to expected sodium salt (0.039 g, 0.090 mmol, 100%). MS (ES (-)): m / z [MH] ^"= 416 1H NMR (400 MHz, MeOH-d ₄₎ in the form of two conformers: δ (ppm) = 1.56 (s, 9H, C (CH ₃₎ ₃ ), 3.09 (s, 3H, CH ₃ (A) NHBoC), 3.10 (s, 3H, CH ₃ (B) NHBoC), 3.37 (m, 1H, BocN (CH ₃ ) -CH ₂ -CH or N- CH ₂ -CH-N), 3.64 (m, 1H, BocN (CH ₃ ) -CH ₂ -CH or N-CH ₂ -CH-N), 3.75 (s, 3H, CH ₃ ), 3.84 (m, 1H, BocN (CH ₃ ) -CH ₂ -CH or N-CH ₂ -CH-N), 3.93 (m, 1H, BOcN (CH ₃ ) -CH ₂ -CH or N-CH ₂ -CH -N), 4.90 (m, 2H, N-CH-CH ₂ -N, N-CH ₂ -CH-N + H ₂ O signal), 7.55 (s, 1H, H pyrazole). Stage F: Trans B sodium and trifluoroacetate salt

(Methylaminomethyl) -4,8-dihydro-1-methyl-5- (sulfooxy) -4,7-methano-7Η-pyrazolo [3,4-e] [1,3] diazepin-6 (5H) -one

Proceeding as indicated in Step I of Example 1, the sodium salt obtained in Step E of Example 3 (0.039 g, 0.088 mmol), dichloromethane (5 mL) and a mixture of trifluoroacetic acid / anhydrous dichloromethane 1/1 (4 mL) yield the expected product (39 mg, 0.08 mmol, 100%). MS (ES (-)): m / z [MH] ^- = 315

¹ H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) = 2.76 (s, 3H, CH ₃ -NH ⁺ ₂ -CH ₂ ), 3.30-3.50 (m, 4H, N-CH ₂ -CH-N, NH ⁺ ₂ -CH ₂ -CH), 3.75 (s, 3H, CH ₃ ), 4.74 (m, 1H, N-CH ₂ -CH-N), 4.82 (d, 1H, CH-CH ₂ -NH ⁺ ₂ CH) ₃ ), 7.43 (s, 1H, H pyrazole), 8.67 (m, 2H, NH ₃ ⁺ ) • Example 4: Pharmaceutical compositions

A composition for injection containing: - compound of Example 1: 300 mg

Tobramycin: 500 mg

Sterile aqueous excipient: qs 5 cm ³ A composition for injection containing:

Compound of Example 1: 200 mg - Ceftazidime: 500mg

Sterile aqueous excipient: qs 5 cm ³

Determination of bactericidal activity

Aim: The in vitro bactericidal activity of the antibiotic is measured by demonstrating the lowest concentration that allows the survival of 0.001% of bacteria after a given single time and over time.

products

The products to be tested are weighed and solubilized, and the mother solution obtained is then diluted in medium according to concentrations to be tested, knowing that each dilution will be introduced under 0.5ml in a total volume of 20ml is a final dilution to 1/40.

Method

The Minimum Inhibitory Concentrations (MICs) of the test products (products alone and combinations) are determined beforehand.

For each concentration of product to be tested and the control strain, an Erlenmeyer flask containing 18.5 ml of Muller-Hinton medium (Ca2 ++) is prepared.

• From a night culture in broth or a bacterial suspension of OD (optical density) = 1, a 1/100 dilution is made.

• Culture is shaken for 2h at 37 ⁰ C.

• The OD is measured, if OD> 0.5, dilute to 1/10.

• Each flask is inoculated with 1 ml of the shaking culture or dilution, the initial inoculum to be 1x10 ⁶ cfu / ml. • The different antibiotic solutions are added in a volume of 0.5 ml and 0.5 ml of medium in the control flask.

• On a volume of 0.1ml, we count the control erlen = TO.

Incubated with stirring at 37 ^° C.

• At each sampling time (2, 4, 6, 24, 48 hours), a volume of 0.1 ml of each Erlenmeyer is taken and a count is made.

• All counting boxes (24h - 48h) are incubated at 37 ° C.

Measured parameters

• Colonies are counted.

• The CFU / ml curves are plotted as a function of time.

• Bactericidal effect = 3 log reduction compared to the initial inoculum.

Bibliography

PETERSON LR, SHANHOLTZER CJ. Tests for bactericidal effects of antimicrobials agents: technical performance and clinical relevance. Clin. Microb. Rev. , 1992, 5, 420-432

COURVALIN P., DRUGEON H., FLANDROIS J. P., GOLDSTEIN F. Bactericide. Theoretical and therapeutic aspects. Ed. Maloine, Paris, 1991.

Bactericidal activity was evaluated on a sensitive strain of Pseudomonas aeruginosa (391HT2)

MICs are determined on a microplate:

- Ceftazidime / CAZ: 2 μg / mL

- Ciprofloxacin / CIPRO: 1 μg / mL

Tobramycin / TOBRA: 1 μg / mL - Product of Example 1 (NXL105): 0.25 μg / mL

For bactericidal tests, the MICs are determined in a volume of 10 mL - Bactericidal conditions (exponential bacterial growth): - CAZ: 8 μg / mL

- CIPRO: 2 μg / mL

- TOBRA: 1 μg / mL

- Product of Example 1: 0.25 μg / mL

The bactericidal activities shown on plates 1 to 3 in the appendix are evaluated after 48 hours, either for the product of example 1 alone, or for a combination. They show a total absence of bacterial regrowth after 48 hours for combinations.

Highlighting the synergistic activity - Determination of MICs:

In vitro activity, dilutions method in a liquid medium: A series of 96-well microplates are prepared in which the same quantity of sterile nutrient medium is distributed. distributes in each well increasing quantities of the compound to be studied, namely the antibacterial compound alone and the combination according to the invention with the compound of formula

(I) of Example 1, in the respective proportions 2: 1 and 4: 1, then each plate is seeded with a bacterial strain: Pseudomonas aeruginosa and enterobacteriaceae.

After incubation for 24 hours in an oven at 37 ⁰ C, inhibition of growth is assessed by transillumination, which allows to determine the minimum inhibitory concentrations (MIC) expressed in μg / ml.

In all the tests below (CMI and FIC):

- Ceftazidime = CAZ

- Meropenem = MRP

- Aztreoname = AZT - Levofloxacin = LVX

- Compound of Example 1 = Compound A

C. freundii ftmpC> 32> 32 2 8 16 4 8 0.125 0.06 <= 0.03

C. freundii ftmpC + TEM-I 16> 32 1 2 16 O. 5 0. 5 0 .06 <= 0.03 <= 0.03

C. freundii AmpC 32 16 <= 0.03 <= 0.03 4 <= 0 .03 <= 0 .03 0 .06 <= 0.03 <= 0.03

E. aerogenes EAR2 Case-R + FEP-R>32>32>32> 32 16 4 4 0. 125 0.125 0.125

Demonstration of synecological activity -

Determination of Inhibitory Concentrations

Fractional. (FIC = Fractional Inhibitory Concentrations)

Chessboard technique (checkerboard) for the determination of the synergy of antibiotics:

Objective: The purpose of the study is to determine the concentration of a compound A, necessary to reduce the MIC of a compound B by one-half, one-quarter, one-eighth, one-sixteenth and 'a thirty second against strains of enterobacteriaceae and non-enterobacteriaceae species resistant to compound B.

The above objective is achieved by the so-called chessboard technique ("checkerboard"). This technique is used to evaluate antimicrobial combinations.

This technique consists in titrating the compound A, an inhibitor, in a series of dilutions (2 in 2) arranged horizontally on a microplate, while at the same time assaying the compound B in a series of dilutions arranged vertically. The plate is then inoculated with the bacterial strain and the bacteria are allowed to grow overnight. Each well on the chessboard of the microplate contains a different combination of concentrations of the inhibitor and the antibacterial compound, which allows a total determination of any synergy between the two components.

Reading the plates:

Growth is assessed for each well. The endpoints (CMIs) for which there is no Growth is determined for each row and the concentrations of compounds A and B for each well where there is no growth are then used to determine the levels of synergy.

The synergy is represented by "FIC indices", FIC being the Fractional Inhibitory Concentration ("Fractional Inhibitory Concentration") of the combination.

Calculations of Fractional Inhibitory Concentration (FIC) Combination index of two antimicrobial agents:

(A) / (CMI _A ) + (B) Z (CMIB) = FIC _A + FIC _B = FIC index

(A) is the concentration of compound A in a well corresponding to the lowest concentration of this growth inhibiting compound in the row. when the well also contains compound B (MIC _A ) is the lowest concentration of compound A alone that inhibits growth. FIC _A is the "Fractional Inhibitory Concentration" of compound A.

(B), (MIC _B ), and FIC _B are defined in the same manner as above for compound B.

If the value of the FIC index is <= 0.5, we consider that we are in the presence of a synergy.

Enterobacteriaceae

Pseudomonas

FIC <0.5 = synergy

Claims

1. A combination with a synergistic effect of an antibacterial compound of general formula (I):

in which Ri represents a radical (CHa) _n -NH ₂ or (CH ₂ ) _n -NHR, R being a (Ci-C ₆ ) alkyl and n being equal to 1 or 2; R ₂ represents a hydrogen atom;

R ₃ and R ₄ together form a nitrogen heterocycle of aromatic character with 5 vertices containing 1, 2 or 3 nitrogen atoms optionally substituted by one or more R' groups, R' being chosen from the group consisting of a hydrogen atom , alkyl radicals containing 1 to 6 carbon atoms; in free form, zwitterions and in the form of salts with pharmaceutically acceptable mineral or organic bases and acids, with another antibacterial compound.

2. A combination according to claim 1, characterized in that the other antibacterial compound is chosen from the group consisting of aminoglycosides, beta-lactams, monobactams, penicillins where appropriate combined with a beta lactamase inhibitor, glycylcyclines, tetracyclines, quinolones, glycopeptides, lipopeptides, macrolides, ketolides, lincosamides, streptogramins, oxazolidinones, polymyxins and other compounds known to have therapeutic activity against Pseudomonas aeruginosa and Enterobacteriaceae.

3. A combination according to claim 1 or 2, 5 characterized in that in the compound of general formula

(I), R ₃ and R ₄ together form an optionally substituted pyrazolyl or triazolyl radical.

4. A combination according to any one of claims 1 to 3, characterized in that in the compound of general formula (I), Ri is chosen from the group consisting of the groups (CH ₂ ) _n -NH ₂ and

(CH ₂ ) _n -NHCH ₃ , n being as defined in claim 1, the heterocycle formed by R ₃ and R ₄ is substituted by an alkyl radical (Ci-C ₆ ).

5. A combination according to any one of claims 1 to 4, characterized in that in the compound of general formula (I), Ri represents a radical 0 (CH ₂ ) _n -NH ₂ or (CH ₂ ) _n -NHCH ₃ , n being as defined in claim 1 and R ₃ and R ₄ together form a pyrazolyl ring substituted by an alkyl radical (Ci-C ₆ ).

6. A combination according to any one of claims 1 to 3, characterized in that the compound of general formula (I) is any of those whose names follow: trans 8- (aminomethyl) -4, 8 -dihydro-l-methyl-5- (sulfooxy) - 4, 7-methano-7H-pyrazolo [3, 4-e]0 [l,3]diazepin-6(5H)-one, trans 8- (aminomethyl ) -4, 8-dihydro-5- (sulfooxy) - 4, 7-methano-7H-ρyrazolo [3, 4-e] [1,3] diazepin-6 (5H) - one, trans 8- (methylaminomethyl ) -4, 8-dihydro-5-5 (sulfooxy)- 4, 7-methano-7H-pyrazolo[3, 4-e]

[l,3]diazepin-6 (5H) -one, in free form, zwittterion and salts with pharmaceutically acceptable mineral or organic bases and acids.

7. A combination according to any one of claims 1 to 6, characterized in that the other antibacterial compound is chosen from the group consisting of beta-lactams, penicillins, where appropriate combined with beta lactamase inhibitors, aminoglycosides and polymyxins.

8. A combination according to any one of claims 1 to 7, characterized in that the antibacterial compound is chosen from the group consisting of Tobramycin, Meropenem, Aztreonam, Cefepime, Ceftazidime, Piperacillin where appropriate combined with Tazobactam, Colistin and Polymyxin B.

9. A combination according to claim 1, characterized in that the compound of general formula (I) is any of those whose names follow: trans 8- (aminomethyl) -4, 8-dihydro-l-methyl -5- (sulfooxy)-4,7-methano-7H-pyrazolo[3,4-e]

[1, 3]diazepin-6(5H)-one, trans 8-(aminomethyl)-4, 8-dihydro-5-(sulfooxy)-4,7-methano-7H-ρyrazolo[3,4-e] [1, 3]diazepin-6 (5H) - one, - trans 8- (methylaminomethyl) -4, 8-dihydro-5- (sulfooxy) - 4, 7-methano-7H-pyrazolo [3, 4-e ]

[1, 3]diazepin-6 (5H) -one, in free form, of zwittterion and salts with bases and pharmaceutically acceptable mineral or organic acids, and the antibacterial compound is chosen from the group consisting of Tobramycin, Meropenem, Cefepime, Ceftazidime, Aztreonam, Levofloxacin, Piperacillin if necessary combined with Tazobactam, Colistin and Polymyxin B.

10. As medications, combinations as defined in any one of claims 1 to 8.

11. As medications, combinations as defined in claim 9.

12. Pharmaceutical compositions containing, as active principle, at least one drug according to claim 11.

13. Pharmaceutical compositions containing, as active principle, at least one drug according to claim 12.