EA043636B1 - ANTIBACTERIAL 3,4-DIHYDRO-1H-[1,8]NAPHTHYRIDINONES, SUBSTITUTED WITH CYCLOPENTA[c]pyrrole - Google Patents

Abstract

The invention relates to novel compounds of formula (I) that inhibit the activity of the Fabl enzyme and are therefore useful for the treatment of bacterial infections. It also relates to pharmaceutical compositions containing these compounds and chemical processes for their preparation.

Description

The invention relates to novel compounds of formula (I) that inhibit the activity of the Fabl enzyme and are therefore useful for the treatment of bacterial infections. It also relates to pharmaceutical compositions containing these compounds and chemical processes for their preparation.

The compounds of the present invention are antibacterial compounds that inhibit the Fabl protein, an NADH-dependent enzyme, enoyl-acyl transfer protein (ACP) reductase, in the fatty acid biosynthesis pathway. Fatty acid synthase (FAS) is involved in the common pathway for saturated fatty acid biosynthesis in all organisms, but the structural organization of FAS varies significantly among them. Distinctive characteristics of vertebrate and yeast FAS include the fact that all enzymatic activities are recorded on one or two polypeptide chains, and that the acyl transfer protein (ACP) is present as a complex. In contrast, in bacterial FAS, each stage of synthesis is catalyzed by a specific monofunctional enzyme, while ACP is a separate protein. Therefore, it is possible to selectively inhibit bacterial FAS by blocking one of the synthesis stages using an inhibitory agent. NADH-dependent enoyl-ACP reductase (Fabl) is involved in the final step of a four-step reaction that occurs in each cycle of bacterial fatty acid biosynthesis. Thus, Fabl is a biosynthetic enzyme in the general pathway of bacterial fatty acid biosynthesis.

The Fabl enzyme has been shown to be a critical target in major pathogens such as E. coli (Heath et al., J. Biol. Chern. 1995, 270, 26538; Bergler et al., Eur. J. Biochem. 2000, 275, 4654). Therefore, compounds that inhibit Fabl may be useful as antibacterial agents.

Compounds having inhibitory activity against the Fabl enzyme are disclosed in publications WO-01/26652, WO-01/26654 and WO-01/27103. Substituted naphthyridinone compounds having inhibitory activity against Fabl are disclosed in publications WO-03/088897, WO2007/043835 and WO-2008/098374. International patent application WO 2007/053131 discloses various compounds for putative use as Fabl inhibitors. International patent application WO 2011/061214 also discloses various compounds for putative use as Fabl inhibitors. However, none of these documents disclose a fused bicyclic moiety that is attached directly to the carbonyl moiety located at the a-position of the alkene.

The present invention relates to a compound of formula (I)

where A is -OC- or the bond ⁷⁷⁷⁷⁷⁷ is a single or double bond, X is carbon or nitrogen, and if X is nitrogen, the bond is a single bond;

Zi represents CH or N;

R ¹ is hydrogen, C^alkyl or halogen;

R ² is hydrogen, C alkyl or halogen;

R ³ is hydrogen, C1. ₆ alkyl, hydroxyl, or halogen;

R ⁴ _is hydrogen; halogen; _C1.6 alkyl; C ₂ balkenyl; C ₂ alkyloxy; C1.4 alkyloxycarbonyl; aminocarbonyl; mono- or di(C1.4 alkyl)aminocarbonyl; aryl; aryloxy; arylcarbonyl; arylsulfonyl; heteroaryl; _C1.6 alkyl substituted with cyano; _C1.6 alkyl substituted with aryl or aryloxy; or _C1.6 alkyl substituted with heteroaryl; aryl is phenyl; phenyl substituted with one, two or three substituents, each of which is individually selected from halogen, hydroxyl, C1.4 alkyl, C1.4 alkoxy, polyhaloC1.4 alkyl, polyhaloC1.4 alkoxy, cyano, nitro and amino groups; heteroaryl is furanyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzo[1,3]dioxolyl, benzofuranyl, benzothiazolyl, indolyl, 2,3-dihydro-1H-indolyl, tetrahydrothiophenyl, or quinolinyl, wherein each heteroaryl may be optionally substituted with one or two substituents, each individually selected from halogen, cyano, _C1.4 alkyl, _C1.4 alkoxy, _C1.4 alkylcarbonyl, or phenyl;

or a pharmaceutically acceptable acid addition salt thereof.

As used in the preceding definitions, the term halogen is a generic term for the atoms of fluorine, chlorine, bromine, and iodine;

_C1.4 alkyl denotes straight and branched chain hydrocarbon radicals containing from 1 to 4 carbon atoms, such as, for example, methyl, ethyl, propyl, butyl, 1-methylethyl, 2-methylpropyl, etc.;

_C1.6 alkyl includes C^alkyl and its higher homologues containing 5 or 6 carbon atoms, such as, for example, 2-methylbutyl, pentyl, hexyl, etc.;

- 1 ₀₄₃₆₃₆ polyhaloC1-4 alkyl is defined as polyhalo-substituted C _1-4 alkyl (as defined herein and above) substituted with 2-6 halogen atoms, such as difluoromethyl, trifluoromethyl, trifluoroethyl, etc.

As used in the description, whenever the term compound of formula (I) is used, it is intended to also include the pharmaceutical addition salts that the compounds of formula (I) are able to form and the solvates that the compounds of formula (I) are able to form or the pharmaceutically acceptable acid addition salts of the compounds of formula (I).

The definition of a compound of formula (I) essentially includes all stereoisomers of the compound of formula (I), either as a pure stereoisomer or as a mixture of two or more stereoisomers. Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or a racemic mixture. Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, that is, they do not relate as mirror images. If the compound contains a disubstituted cycloalkyl group, the substituents may be in the cis or trans configuration. Therefore, this invention includes enantiomers, diastereomers, racemates, cis isomers, trans isomers, and mixtures thereof.

The absolute configuration is determined according to the Cahn-Ingold-Prelog system. The configuration of an asymmetric atom is indicated by either R or S. Isolated compounds whose absolute configuration is not known may be designated by (+) or (-), depending on the direction in which they rotate plane-polarized light. When a particular stereoisomer is indicated, this means that the said stereoisomer is substantially free of other isomers, i.e., associated with less than 50%, preferably less than 20%, more preferably less than 10%, still more preferably less than 5%, in particular less than 2% and most preferably less than 1% of other isomers. Thus, if a compound of formula (I) is indicated as (R), this means that the compound is substantially free of the (S) isomer; if a compound of formula (I) is indicated, for example, as E, this means that the compound is substantially free of the Z isomer; if a compound of formula (I) is indicated, for example, as cis, this means that the compound contains substantially no trans isomer.

The terms stereoisomers or stereochemically isomeric forms are used interchangeably hereinafter.

Those skilled in the art can readily determine the absolute stereochemical configuration of the compounds of formula (I) and the intermediates used in their preparation using well-known methods such as, for example, X-ray diffraction.

Certain compounds of formula (I) may also exist in their tautomeric form. Such forms, although not explicitly indicated in the above formula, are intended to be included within the scope of the present invention.

Furthermore, some compounds of formula (I) and intermediates used in their preparation may exhibit polymorphism. It is understood that the present invention encompasses any polymorphic forms possessing properties useful in the therapeutic treatment of the conditions described above.

The pharmaceutically acceptable acid addition salts mentioned herein and previously include the non-toxic acid addition salt forms having therapeutic activity which can be formed by a compound of formula (I). The pharmaceutically acceptable acid addition salts can be readily prepared by treating the base form with such appropriate acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids, for example hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propionic, glycolic, lactic, pyruvic, oxalic (i.e., ethanedioic), malonic, succinic (i.e., butanedioic), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.

Conversely, the said salt forms can be converted by treatment with an appropriate base into the free base form.

Compounds of formula (I) can exist in both unsolvated and solvated forms. The term "solvate" is used herein to denote a molecular association comprising the compounds of this invention and one or more molecules of a pharmaceutically acceptable solvent, such as water or ethanol. The term "hydrate" is used when the solvent is water.

The term FabI is known in the art and refers to bacterial enzymes that presumably act as enoyl-acyl transfer protein (ACP) reductase in the final step of four reactions involved in each cycle of bacterial fatty acid biosynthesis. This enzyme is believed to be widespread in bacteria.

Compounds of formula (I) which may be mentioned include those in which:

(i) Z1 is CH and therefore the compound of formula I is as follows:

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where: (ii) if R ¹ or R ² are halogen, they are preferably F or Cl;

(iii) R ¹ is hydrogen or C1_ ₄ alkyl and/or (iv) R ² is hydrogen or C1_ ₄ alkyl.

Preferred compounds of formula (I) include those in which A is a double bond (rather than a triple bond), i.e., preferably A is V

Of interest are compounds of formula (I), which are those compounds of formula (I) where one or more of the following restrictions apply:

a) R ¹ and R ² are hydrogen; or

b) R ³ is hydrogen; or

c) R ³ is hydrogen, halogen or hydroxyl; or

d) R ⁴ is hydrogen or halogen; or

e) R ⁴ is aryl; or

f) R ⁴ is C1_ ₆ alkyl; or

g) R ⁴ is aryloxy or arylsulfonyl; or

h) R ⁴ is C1_ ₆ alkyl substituted with aryl; or

i) R ⁴ is heteroaryl; or

j) R ⁴ is C1_ ₆ alkyl substituted with heteroaryl; or

k) heteroaryl is furanyl, thiophenyl, pyrazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridinyl or pyrimidinyl; or

1) X is carbon; or

m) X is nitrogen and the ⁷⁷⁷⁷⁷¹ bond is a single bond.

The first group of compounds are compounds of formula (I)

R ¹ where A is -C=C- or ^r2 = bond is a single bond or a double bond, X is carbon or nitrogen, and if X is nitrogen then the bond ^ ⁷⁷⁷⁷ ^ is a single bond;

R ¹ is hydrogen;

R is hydrogen;

R ³ is hydrogen, hydroxyl or halogen;

R ⁴ is hydrogen, halogen; C1_ ₆ alkyl; C1_ ₆ alkyloxy; C1_ ₄ alkyloxycarbonyl; aminocarbonyl; mono- or di(C1_ ₄ alkyl)aminocarbonyl; aryl; aryloxy; arylsulfonyl; heteroaryl; C1_ ₆ alkyl substituted with cyano; C1_ ₆ alkyl substituted with aryl; or C1_ ₆ alkyl substituted with heteroaryl; aryl is phenyl; phenyl substituted with one substituent selected from halogen, C1_ ₄ alkyl, C1_ ₄ alkyloxy and cyano; heteroaryl is furanyl, thiophenyl, pyrazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridinyl or pyrimidinyl; wherein each heteroaryl may be substituted with one substituent selected from halogen, cyano, _{C1_4} alkyl, _{C1_4} alkyloxy or _{C1_4} alkylcarbonyl;

or a pharmaceutically acceptable acid addition salt thereof.

The second group of compounds of formula (I) are those compounds of formula (I) where A is -C^C-.

The third group of compounds of formula (I) are those compounds of formula (I) where A is R ¹ ϊ

Preferred compounds of formula (I) include those in which the X-containing ring is one of the following:

_c j _s single enantiomer (cis) single enantiomer (cis) i.e., double rings containing the cis configuration at the ring junction (trans configuration would cause ring strain), which may be racemic or single enantiomers. As explained here and below, if the absolute stereochemistry of a single enantiomer is/was not known, chiral carbons at the ring junction may be indicated by bold or dashed lines (rather than as a wedge).

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More preferred compounds of formula (I) include those in which the fused bicyclic X-containing ring is one of the following:

where in the above mentioned fused double rings the compounds may be racemic or one enantiomer (if the corresponding symmetry is absent and the existence of enantiomers is possible), as described herein and above. Preferably in compounds of formula (I):

(i) at least one substituent R ³ or R ⁴ is present that is not hydrogen;

(ii) one of R ³ and R ⁴ (e.g., R ³ ) is hydrogen, hydroxyl, or halogen (e.g., fluorine), and the other of R ³ and R ⁴ (e.g., R ⁴ ) is a substituent other than hydrogen;

(iii) R ³ is hydrogen, hydroxyl or halogen (e.g. fluorine) and more preferably is hydrogen (i.e. R ³ is substantially not present);

(iv) R ⁴ is a substituent other than hydrogen (i.e., there is a substituent R ⁴ that is not hydrogen);

(v) R ⁴ represents a substituent other than hydrogen that is attached to X, wherein any of the above may be taken together or in combination.

For example, (iii), (iv) and/or (v) may be taken in combination to provide particularly preferred compounds of formula (I) below

in which R ⁴ is a substituent other than hydrogen. Particularly preferred substituents that may be represented by R ⁴ (here and elsewhere in the document) include:

(i) optionally substituted aryl;

(ii) optionally substituted heteroaryl (iii) C _1-6 alkyl substituted by aryl or heteroaryl (the latter of the two aryl and heteroaryl groups are themselves optionally substituted as defined herein);

(iv) aryloxy (in which the aryl moiety is optionally substituted as defined herein);

(v) arylsulfonyl (in which the aryl moiety is optionally substituted as defined herein);

(vi) C _1-6 alkyl which is unsubstituted (e.g. ethyl, methyl, isopropyl);

(vii) di(C _1-4 alkyl)aminocarbonyl (eg -C(O)N(CH ₃ ) ₂ );

(viii) aminocarbonyl (-C(O)NH2);

(ix) C _1-4 alkyloxycarbonyl (e.g., -C(O)O-CH ₂ CH ₃ );

(x) halogen (e.g. fluorine);

(xi) C _2-6 alkynyl (e.g., -C=C);

(xii) C _1-6 alkoxy (eg -OCH ₃ ).

It is particularly preferred that the R ⁴ group contain an aromatic moiety, and therefore the above (i), (ii), (iii), (iv) and (v) are particularly preferred.

In case R ⁴ is as given in (i) above, the aryl group is preferably phenyl, which group may be unsubstituted or substituted with one or two (eg one) substituents selected from C _1-4 alkyloxy, halogen, C _1-4 alkyl or cyano (eg OCH3, chloro, fluoro, methyl or cyano).

In the case where R ⁴ is as in (ii) above, the heteroaryl group is a monocyclic 5- or 6-membered ring containing one to four heteroatoms, for example, thienyl (e.g. 2- or 3-thienyl), pyridyl (e.g. 4-pyridyl or 3-pyridyl), pyrazolyl (e.g. 5-pyrazolyl, 4-pyrazolyl or 1-pyrazolyl), furanyl (e.g. 2- or 3-furanyl), thiazolyl (e.g. 2-thiazolyl), isoxazolyl (e.g. 4-isoxazolyl), pyrrolyl (e.g. 1-pyrrolyl), triazolyl (e.g. 1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl or 1,2,4-triazol-2-yl), thiadiazolyl (e.g. 1,3,4-thiadiazol-2-yl), pyrimidinyl (e.g. 5-pyrimidinyl), tetrazolyl (e.g. 1,2,3,4-tetrazol2-yl, 1,2,3,4-tetrazol-1-yl), imidazolyl (e.g. 2-imidazolyl). Such heteroaryl groups may be unsubstituted or substituted with one or two (e.g. two or, preferably, one) substituent(s) selected from halogen, cyano, _C1-4 alkyl (e.g. _C1-2 alkyl), _C1-4 alkyloxy (e.g. _C1-2 alkyloxy) and _C1-4 alkylcarbonyl (e.g. _C1-2 alkylcarbonyl), for example, _-OCH3 , methyl, halogen (e.g. chlorine), cyano and -C(O) _-CH3 .

In the case where R ⁴ is (iii) above, the C _1-6 alkyl group is methyl, i.e., -CH3, substituted aryl (e.g., phenyl, such as unsubstituted phenyl) or heteroaryl (e.g., a 5- or 6-membered monocyclic heteroaryl group containing one or

- 4 043636 two (e.g. one) heteroatom(s), thus forming, for example, a thienyl group, such as a 2-thienyl group; and such heteroaryl group is preferably unsubstituted).

In the case where R ⁴ is (iv) or (v) above, aryl is preferably unsubstituted phenyl and therefore the R ⁴ group is -O-phenyl or -S(O) ₂ -phenyl.

More preferably, the R ⁴ group is (i) or (ii) above, i.e., aryl or heteroaryl. Even more preferably, the R ⁴ group is (i) above, especially unsubstituted phenyl.

Particularly preferred compounds of formula (I) include those in which the X-containing fused bicyclic moiety is:

r ⁴ — ^R4— wherein R ⁴ is as defined herein. Compounds that contain either an N(R ⁴ ) moiety or a C(R ⁴ ) moiety immediately adjacent to the double bond may be useful. This is because the shape of the nitrogen atom (e.g., being inherently more planar than a CR ⁴ moiety that is not immediately adjacent to the double bond) or the presence of a double bond in an X-containing ring may help orient the R ⁴ group (if present) such that the compound as a whole (e.g., in terms of the orientation of the R ⁴ substituent) exhibits better/improved binding properties to the bacterial enzyme FabI. Therefore, these compounds of the present invention may be useful in the sense that the presence of a double bond may result in improved binding to or inhibition of the FabI enzyme. Therefore, the compounds of the present invention may be useful compounds (e.g., compared to known compounds) due to these properties, which may consequently lead to better efficacy, effectiveness, etc.

Compounds of formula (I) can generally be prepared by reacting an intermediate of formula (II) with an intermediate of formula (III) in at least one inert solvent and optionally in the presence of at least one suitable coupling reagent and/or a suitable base, said process optionally further comprising converting the compound of formula (I) into its addition salt and/or obtaining its stereochemically isomeric forms.

It may be convenient to activate the carboxylic acid of formula (III) by adding an effective amount of a reaction accelerator. Non-limiting examples of such reaction accelerators include carbonyldiimidazole, N,N'-dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydroxybenzotriazole, benzotriazolyl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate, tetrapyrrolidinephosphonium hexafluorophosphate, bromotripyrrolidinephosphonium hexafluorophosphate, or a functional derivative thereof.

Compounds of formula (I) can also be prepared by reacting an intermediate of formula (II) with an intermediate of formula (IV), wherein Y is hydroxyl or halogen. The reaction can be carried out in an inert solvent, such as, for example, dichloromethane or dimethylformamide, and optionally in the presence of a suitable base, such as, for example, diisopropylethylamine (DIPEA).

? /i=\ /=°

V\Xy ^{NH + NH}

R ^3N (II) (IV)

Compounds of formula (I) in which A is -C(R ² )=C(R ¹ )- can also be prepared by reacting an intermediate of formula (V) with an intermediate of formula (VI),

where X _a1 is a suitable leaving group such as a suitable halide group (e.g. chloro-, iodine-, and especially bromo-), and other designations as defined herein and above, under suitable reaction conditions, for example under coupling reaction conditions in the presence of a metal catalyst (e.g. a coupling reaction in the presence of a precious metal, where the precious metal is, for example, palladium-based), especially under Heck reaction conditions using preferably a palladium-based catalyst such as palladium acetate, tetrakis(triphenylphosphine)palladium(0), bis-(triphenylphosphine)palladium(II) dichloride, [1,1'-bis-(diphenylphosphino)ferrocene]palladium(H) dichloride or the like (preferably the catalyst is

- 5 043636 palladium acetate), for example, optionally in the presence of a suitable solvent (e.g. acetonitrile or the like), a base (e.g. an amine base such as N,N-diisopropylamine or the like) and a ligand (e.g. triphenylphosphine, tri-O-tolylphosphine or the like). The reaction can be carried out in a sealed tube and/or in a microwave oven.

The starting materials and some intermediates are known compounds and can be purchased or prepared according to conventional reaction procedures generally known in the art.

For compounds in which Z ₁ is CH, intermediates (IV) and (VI) can be prepared as described herein or according to conventional reaction procedures generally known in the art. This may also apply in the case of the corresponding intermediates in which Z ₁ is N. However, such compounds can also be prepared according to the following scheme:

CAS 16298-03-6

Conditions:

a) NBS, ACN, reflux, 3 h, 70%;

b) LiAlH4 1M in THF, THF, 5°C to room temperature, overnight, 20%;

c) PBr ₃ , DCM, room temperature, overnight, 90%;

f) dimethyl malonate, NaOMe in MeOH, MeOH, room temperature, overnight, 25%;

g) NaOH, MeOH, reflux, 4 h, HCl, reflux, overnight;

h) DIEA, Pd(OAc) ₂ , tri-O-tolylphosphine, ACN, DMF, microwave, 180°C, 25 min.

The compounds of formula (I), obtained in the processes described herein and above, can be synthesized in the form of racemic mixtures of enantiomers, which can be separated from each other by art-known resolution procedures. These compounds of formula (I), which are obtained in racemic form, can be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. Said diastereomeric salt forms are then separated, for example, by selective or fractional crystallization, and the enantiomers are isolated therefrom by means of alkali. Alternatively, liquid chromatography using a chiral stationary phase is used to separate the enantiomeric forms of the compounds of formula (I). Said pure stereochemically isomeric forms can also be obtained from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically. If a specific stereoisomer is desired, it is preferred that said compound be synthesized by stereospecific methods of preparation. These methods predominantly use enantiomerically pure starting materials.

The compounds described herein are inhibitors of the FabI enzyme, as illustrated by the examples below (including Pharmacological Example 1). Given these FabI-inhibiting properties, the compounds described herein are useful in the treatment of bacterial infections.

For example, these compounds are useful in the treatment of bacterial infections such as, for example, upper respiratory tract infections (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory tract (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhea, splenic abscess, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, dacryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g., impetigo, folliculitis, skin abscesses, cellulitis, wound infections, bacterial myositis), as well as bones and joints (e.g., septic arthritis, osteomyelitis). In addition, the compounds may be useful in combination with known antibiotics.

Thus, the present invention also relates to compounds of formula (I) for use as a medicinal product, particularly for use in the treatment of bacterial infections, particularly bacterial infections caused by bacteria expressing the FabI enzyme. The present compounds can further be used in the manufacture of a medicinal product for the treatment of bacterial infections, particularly bacterial infections caused by bacteria expressing the FabI enzyme.

Furthermore, the present invention provides a method of treating bacterial infections which comprises administering to a subject in need thereof a compound of formula (I) which inhibits the FabI enzyme.

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A subject in need of treatment has a bacterial infection or has been in contact with an infectious bacterium, the symptoms of which can be alleviated by administering a therapeutically effective amount of the compounds of the present invention. For example, a subject in need of treatment may have an infection for which the compounds of Formula (I) can be administered as a treatment. In another example, a subject in need of treatment may have an open wound or burn for which the compounds of Formula (I) can be administered as a treatment. Typically, the subject is treated for an existing bacterial infection.

The subject may have a bacterial infection caused by

Bacillus anthracis, Citrobacter sp., Escherichia coll,

Francisella tularensis, Haemophilus influenza, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium tuberculosis, Neisseria meningitidis, Proteus mirabilis, Proteus vulgaris, Salmonella sp., Serratia sp., Shigella sp., Stenotrophomonas maltophilia, Staphylococcus aureus or Staphylococcus epidermidi s.

Preferably, the subject is treated (prophylactically or therapeutically) against a bacterial infection caused by a bacterium that expresses the FabI enzyme.

The term treat and cure as used herein refers to curative, palliative and prophylactic treatment, including reversing, alleviating, slowing the progress of the disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition.

A therapeutically effective amount of a compound of this invention is an amount that, when administered to a subject in need of treatment, improves the subject's prognosis, such as delaying the onset and/or reducing the severity of one or more symptoms associated with a bacterial infection. The amount of the disclosed compound required to be administered to a subject will depend on the specific disease, the route of administration, and the subject's characteristics, such as general health, the presence of other diseases, age, gender, genotype, body weight, and drug tolerance. One skilled in the art will be able to determine the necessary dosages based on these and other factors.

Compounds may be tested in one of several biological assays to determine the concentration of the compound required for a given pharmacological effect.

Furthermore, the present invention provides pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula (I).

To prepare pharmaceutical compositions according to the present invention, an effective amount of a particular compound, in base form or in acid addition salt form, as an active ingredient is combined in a homogeneous mixture with at least one pharmaceutically acceptable carrier, wherein the carrier can take a wide variety of forms depending on the form of the preparation desired for administration. These pharmaceutical compositions are preferably in a unit dosage form suitable, preferably, for oral administration, rectal administration, transdermal administration, or parenteral injection.

For example, in preparing compositions in oral dosage form, any of the usual liquid pharmaceutical carriers can be used, such as, for example, water, glycols, oils, alcohols, and the like, in the case of oral liquid preparations such as suspensions, syrups, tinctures, and solutions; or solid pharmaceutical carriers, such as starches, sugars, kaolin, lubricants, binders, disintegrating agents, and the like, in the case of powders, pills, capsules, and tablets. Because of their ease of administration, tablets and capsules represent the most convenient oral dosage unit form, and in such a case, solid pharmaceutical carriers are obviously used. In the case of compositions for parenteral injection, the pharmaceutical carrier will mainly comprise sterile water, although other components may be included to improve the solubility of the active ingredient. Injectable solutions can be prepared by using a pharmaceutical carrier containing saline, glucose, or a mixture of both. Injectable suspensions can be prepared using suitable liquid carriers, suspending agents, and the like. In compositions suitable for transdermal administration, the pharmaceutical carrier may optionally contain a penetration enhancer and/or an acceptable wetting agent, optionally combined with acceptable additives of any nature in minor amounts that do not have a significant adverse effect on the skin. These additives can be selected to facilitate the introduction of the active ingredient into the skin and/or to facilitate the preparation of the desired compositions. These compositions for external use can be administered in various ways, for example, in the form of a transdermal patch, spot-on application, or ointment. Due to their increased solubility in water compared to the corresponding base form, addition salts of the compounds of formula (I) are more suitable for the preparation of aqueous compositions.

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It is particularly advantageous to formulate the pharmaceutical compositions of the present invention in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scoreable or film-coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, heaping teaspoons, heaping tablespoons, and the like, and segregated multiples thereof.

For oral administration, the pharmaceutical compositions of the present invention may take the form of a solid dosage form such as tablets (both chewable and in a form to be swallowed whole), capsules or gel capsules prepared by conventional methods using pharmaceutically acceptable excipients and carriers such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.), fillers (e.g., lactose, microcrystalline cellulose, calcium phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silicon dioxide, etc.), disintegrants (e.g., potato starch, sodium starch glycolate, etc.), wetting agents (e.g., sodium lauryl sulfate), etc. Such tablets may also have a coating obtained by methods known in the art.

Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be formulated as a dry product for constitution with water and/or another suitable liquid carrier before use. Such liquid preparations may be prepared by conventional methods, optionally with other pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, hydroxypropyl methylcellulose, or hydrogenated edible fats), emulsifiers (e.g., lecithin or gum arabic), non-aqueous vehicles (e.g., almond oil, fatty esters, or ethyl alcohol), sweeteners, flavorings, masking agents, and preservatives (e.g., methyl or propyl phydroxybenzoates, or sorbic acid).

Pharmaceutically acceptable sweeteners suitable for the pharmaceutical compositions of the present invention include at least one intense sweetener such as aspartame, acesulfame potassium, sodium cyclamate, alitame, dihydrochalcone sweetener, monellin, sucralose stevioside (4,1',6'-trichloro4,1',6'-trideoxygalactosucrose) or, preferably, saccharin, sodium or calcium saccharin, and optionally at least one bulk sweetener such as sorbitol, mannitol, fructose, sucrose, maltose, isomalt, glucose, hydrogenated glucose syrup, xylitol, caramel or honey. Intense sweeteners are typically used in small amounts. For example, in the case of sodium saccharin, the concentration may vary from about 0.04% to 0.1% (w/v) of the final formulation. The bulk sweetener can be effectively used in higher concentrations ranging from about 10% to about 35%, preferably from about 10% to 15% (w/v).

Pharmaceutically acceptable flavorings that can mask bitter-tasting ingredients in low-dose formulations are preferably fruit flavorings such as cherry, raspberry, blackcurrant, or strawberry. A combination of two flavorings can produce very good results. High-dose formulations may require stronger pharmaceutically acceptable flavorings such as caramel-chocolate, mint cool, fantasy, and the like. Each flavoring can be present in the final composition at a concentration ranging from approximately 0.05% to 1% (w/v). Combinations of these strong flavorings are advantageously used. It is preferable to use a flavoring that does not undergo any change or reduction in flavor and/or color under the formulation conditions.

The compositions of formula (I) can be formulated for parenteral administration by injection, preferably by intravenous, intramuscular, or subcutaneous injection, for example, by bolus injection or continuous intravenous infusion. Formulations for injection can be presented as unit doses, for example, in ampoules or multi-dose containers, including an added preservative. They can be formulated as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as isotonizing, suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient can be presented in powder form for constitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

The compounds of formula (I) may be formulated into rectal compositions such as suppositories or retention enemas, for example containing conventional suppository bases such as cocoa butter and/or other glycerides.

Those skilled in the treatment of bacterial diseases associated with inhibition of the FabI enzyme can readily determine a therapeutically effective amount of the compound of formula (I) from the results

- 8,043,636 trials presented hereinafter. In general, it is expected that a therapeutically effective dose will be from about 0.001 mg/kg to about 50 mg/kg of body weight, more preferably from about 0.01 mg/kg to about 10 mg/kg of body weight of the patient undergoing treatment. It may be appropriate to administer the therapeutically effective dose as two or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated in unit dose forms, each containing from about 0.1 mg to about 1000 mg, in particular from about 1 to about 500 mg of the active ingredient per unit dose form.

The precise dosage and frequency of administration depend on the specific compound of formula (I) used, the specific condition being treated, the severity of the condition being treated, the age, weight, and general physical condition of the individual patient, as well as other medications the patient may be receiving, as is well known to those skilled in the art. Furthermore, the therapeutically effective amount mentioned may be reduced or increased depending on the response of the patient undergoing treatment and/or depending on the assessment of the physician prescribing the compounds of this invention. The ranges of effective daily amounts given above and below are merely recommendations.

The compounds of formula (I) may have the advantage of being more potent, less toxic, longer acting, more potent, having fewer side effects, being more readily absorbed and/or having an improved pharmacokinetic curve (e.g. having higher bioavailability and/or lower clearance), and/or having beneficial pharmacological, physical or chemical properties compared to the compounds known in the art, both when used in the above-mentioned cases and in other cases. The compounds may also exhibit such advantages due to the presence of an NR ⁴ moiety or a CR ⁴ moiety, which is located immediately adjacent to the double bond in the X-containing ring.

For example, the compounds of formula (I) may have the advantage of having good or improved thermodynamic stability (e.g., compared to compounds known in the art; and, for example, as determined using a known method and/or the method described herein). The compounds of formula (I) may also have the advantage of having a broad spectrum of activity compared to antibacterial agents (e.g., a broader spectrum of antibacterial activity compared to compounds known in the art; and, for example, as determined by known tests and/or the tests described herein). The compounds of formula (I) may also have the advantage of having good or improved in vivo pharmacokinetics and oral bioavailability. They may also have the advantage of having good or improved in vivo efficacy. For example, the compounds of the present invention may be adapted for intravenous formulation/dosage and may therefore exhibit improved in vivo efficacy when administered intravenously. Compounds can also exhibit such advantages due to the presence of an NR ⁴ moiety or a CR ⁴ moiety that is located directly adjacent to the double bond in the X-containing ring.

Experimental part.

Abbreviations.

DMF is defined as N,N-dimethylformamide, DCM or CH2Cl2 is defined as dichloromethane, MeOH is defined as methanol, EtOH is defined as ethanol, MgSO ₄ is defined as magnesium sulfate and THF is defined as tetrahydrofuran, AcOEt or EtOAc is defined as ethyl acetate, DIPEA is defined as diisopropylethylamine, EDCI is defined as N'(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine monohydrochloride, HOBT is defined as 1-hydroxy-1H-benzotriazole, DIPA is defined as diisopropylamine, K2CO3 is defined as potassium carbonate, TFA is defined as trifluoroacetic acid, NH4OH is defined as ammonium hydroxide, NaHCO ₃ is defined as sodium hydrogen carbonate, Et2O is defined as diethyl ether, Na2SO4 is defined as sodium sulfate, CH3CN is defined as acetonitrile, NaOH is defined as sodium hydroxide, n-BuLi is defined as n-butyllithium, i-PrOH is defined as isopropanol, Pd(OAc)2 is defined as palladium acetate, DMA is defined as dimethylacetamide, Et ₃ N is defined as triethylamine.

Stereochemical representation.

Compounds of formula (I) have at least two asymmetric carbon atoms as shown below, where the asymmetric carbon atoms are identified by the designation*:

n

Due to the ring strain in a system of two ring-shaped five-membered rings, only cis forms can be obtained, but not trans forms.

- 9 043636

Compounds of formula (I) wherein the system of two ring-shaped five-membered rings has a cis-configuration

N

Each of the cis compounds described above consists of a racemic mixture of two enantiomers, and the bold or dashed bonds have been used to denote these respective stereochemical configurations.

When such a cis compound resolves into two separate enantiomers, the stereochemical configuration of one of these enantiomers is designated as R* or S*, indicating the relative stereochemistry. Accordingly, one enantiomer designated as (R*,S*) can have either the absolute (R,S) configuration or the (c,R) configuration. If the absolute stereochemistry of a specific chiral carbon atom in one enantiomer is known, bonds designated in bold and dashed have been replaced with wedge bonds to indicate that the compound is a single enantiomer with a known absolute stereochemistry.

A. Synthesis of intermediate compounds.

Example A. 1.

intermediate compound (1)

A solution of 6-bromo-3,4-dihydro-1H-[1,8]naphthyridin-2-one (1.0 g, 4.4 mmol), tert-butyl acrylate (2.56 mL, 17.62 mmol), and N,N-diisopropylethylamine (1.46 mL, 8.81 mmol) in acetonitrile (20 mL) and DMF (7 mL) was stirred and degassed with nitrogen gas for 10 min. Tri-o-tolylphosphine (0.27 g, 0.88 mmol) and palladium(II) acetate (47% as Pd) (0.099 g, 0.44 mol) were added, and the resulting mixture was microwaved (1600 W, 180°C, 35 min). The reaction mixture was evaporated to dryness, dissolved in DCM/methanol (8/2) (50 ml), filtered through a thin pad of celite and washed with dichloromethane. The organic layer was washed with water, dried (MgSO ₄ ), filtered and evaporated to dryness. The residue was dissolved in cold ethanol (10 ml) and stirred at 5°C for 5 min, the precipitate was filtered off, washed with cold ethanol (3 ml) and dried under vacuum, yielding 950 mg of intermediate (1).

intermediate compound (2)

Intermediate (1) (4.1 g, 14.95 mmol) was dissolved in a mixture of trifluoroacetic acid (23.2 mL) and DCM (41 mL). The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure. The resulting solid was triturated with diethyl ether, filtered, and dried under vacuum to yield 3.97 g of intermediate (2).

intermediate compound (3)

Intermediate (2) was triturated overnight in a mixture of HCl in dioxane (4 M, 48 ml), the solid was filtered, washed with diethyl ether and dried under vacuum to yield 3.9 g of intermediate (3).

Example A.2.

a) Receipt

intermediate compound (4)

A solution of allyl prop-2-ynyl carbamic acid tert-butyl ester (CAS 147528-20-9, 45 g, 0.23 mol), cobalt carbonyl (17.5 g, 46.1 mmol) and 1,1,3,3-tetramethyl-2-thiourea (36.6 g, 0.277 mmol) in toluene (1.8 L) was stirred and heated at 70 °C for 5 h in an autoclave under CO pressure (2-3 bar). The resulting mixture was filtered through a fine pad of celite and evaporated to dryness. The residue was dissolved in DCM and filtered through a fine pad of celite to give a clear solution. It was evaporated to dryness to give 85.7 g of crude residue. It was purified by preparative liquid chromatography on (silica gel 20-45 μm, 1000 g, mobile phase (DCM/AcOEt gradient from 95/5 to 80/20). The pure fractions were collected and the solvent was evaporated, yielding 36.5 g of intermediate (4).

intermediate compound (5)

- 10 043636

A mixture of intermediate (4) (37.6 g, 0.168 mol) and 10% palladium on carbon (7.5 g) in ethyl acetate (750 mL) was hydrogenated at room temperature for 30 min under a pressure of 3 bar in a closed reactor. The resulting mixture was filtered through a fine pad of celite and evaporated to dryness, yielding 38.2 g of intermediate (5).

c) Receipt o ° / f ₃ cso—/2% a ^-0-0- \ ( ^cis > ⁰ n intermediate connection (6)

H-BuLi 1.6 M in hexane (64 mL, 0.102 mol) was added dropwise at -20 °C, under N ₂ , to a solution of diisopropylamine (14.3 mL, 0.102 mol) in dry THF (140 mL), and the mixture was stirred at -20 °C for 20 min. A solution of intermediate (5) (19.1 g, 84.8 mmol) in dry THF (190 mL) was then added at -78 °C, and the resulting mixture was stirred for 1 h at -78 °C. A solution of N-phenyltrifluoromethane sulfonimide (36.4 g, 0.102 mol) in dry THF (110 mL) was added at -78 °C, and the mixture was allowed to warm to room temperature and stirred overnight. The mixture was evaporated to dryness. The residue was dissolved in DCM, washed with aqueous NaHCO ₃ , dried (MgSO ₄ ) and evaporated to dryness, yielding 27.7 g of intermediate (6).

d) Receipt V o \ /—\ CT A ^-0-0 χ ^- ( ^cis ) n intermediate connection (7)

A solution of intermediate (6) (9.3 g, 26.0 mmol) and phenylboronic acid (3.81 g, 31.2 mmol) in a solution of 2 M potassium carbonate (26 mL) and ethylene glycol dimethyl ether (93 mL) was purged with _N2 for 10 min, then tetrakis(triphenylphosphine)palladium (3.0 g, 2.6 mmol) was added. The closed reactor was heated at 80 °C using a CEM Mars single chamber microwave oven with the power range from 0 to 400 W for 30 min. The resulting solution was cooled to room temperature, water and EtOAc were added, the organic layer was separated, washed with water and then with brine, dried (MgSO4), and evaporated to dryness. The residue was purified by flash chromatography on silica gel (330 g, 15-40 μm, heptane/EtOAc from 100/0 to 80/20). The purified fractions were collected and evaporated to dryness, yielding 4.3 g of intermediate (7).

e) Receipt \ /—xJL' ^NH ( ^cis ) Η intermediate connection (8)

Trifluoroacetic acid (44 mL) was added dropwise to a solution of intermediate (7) (14.5 g, 50.8 mmol) in CH ₂ Cl ₂ (44 mL). The resulting solution was stirred at room temperature for 30 min, then cooled to 5 °C. 3 N NaOH was added slowly until basic, and the mixture was extracted twice with CH ₂ Cl ₂ . The combined organic layer was washed with 3 N NaOH, then water, dried over MgSO ₄ , and evaporated to yield 8.8 g of racemic intermediate (8). _________________________________________________

f) Receipt n n intermediate connection (9) And and y intermediate connection (10)

Intermediate (8) was purified and separated by chiral SFC (CHIRALPAK AD-H 5 μm 250x20 mm). Mobile phase (0.3% isopropylamine, 73% CO ₂ , 27% iPrOH). The pure fractions were collected and the solvent was removed, yielding 3.9 g of intermediate (10) (R*,S*) ([a] _D ²⁰ = -53.19° (589 nm, conc. 0.3365 w/v%, DMF, 20°C)) and 4 g of intermediate (9) (S*,R*) ([a]d ²⁰ = +38.6° (589 nm, conc. 0.285 w/v%, DMF, 20°C)).

Intermediate connection (9).

¹ H NMR (400 MHz, DMSO-a ₆ ) δ (ppm) 7.43 (d, J=7.6 Hz, 2H), 7.32 (t, J=7.6 Hz, 2H), 7.20-7.26 (m, 1H), 6.07 (d, J=2.0 Hz, 1H), 3.30-3.39 (m, 1H), 2.77-2.94 (m, 4H), 2.66 (dd, J=3.0, 11.1 Hz, 1H), 2.58 (dd, J=3.0, 11.1 Hz, 1H), 2.46 (d, J=15.7 Hz, 1H).

Intermediate connection (10).

1H NMR (400 MHz, DMSO-6 ₆ ) δ (ppm) 7.43 (d, J=7.6 Hz, 2H), 7.32 (t, J=7.6 Hz, 2H), 7.20-7.26 (m, 1H), 6.07 (d, J=2.0 Hz, 1H), 3.30-3.39 (m, 1H), 2.77-2.94 (m, 4H), 2.66 (dd, J=3.0, 11.1 Hz, 1H), 2.58 (dd, J=3.0, 11.1 Hz, 1H), 2.46 (d, J=15.7 Hz, 1H).

Example A.3.

a) Receipt /Α-Ά /° Г у—/ Д' N-Д, (cis) ^{s /} о—Z_ Л \ intermediate connection (11)

A solution of intermediate (6) (44.4 g, 111.82 mmol) and 3-thiophenylboronic acid (17.17 g, 134.19 mmol) in a solution of 2 M potassium carbonate (112 ml) and ethylene glycol dimethyl ether

- 11 043636 (444 mL) was purged with N2 in an open container for 10 min, then tetrakis(triphenylphosphine)palladium (12.92 g, 223.65 mmol) was added. The solution was heated at 78 °C using a microwave oven with one multimode chamber of the CEM Mars system with the power range from 0 to 400 W for 1 h. The mixture was cooled to room temperature, water and EtOAc were added. The resulting mixture was filtered through a pad of celite. The organic layer was separated, washed with water, then brine, dried over _MgSO4 and evaporated to dryness. The residue was purified by preparative liquid chromatography (silica gel 20-45 μm, 1000 g, mobile phase (80% heptane, 20% AcOEt)). The purified fractions were collected and concentrated to yield 16 g of intermediate compound (11).

b) Receipt m S «--/ ^NH ( ^cis ) Η intermediate connection (12)

Trifluoroacetic acid (14.37 mL, 186.47 mmol) was added to a solution of intermediate (11) (5.72 g, 18.65 mmol) in _{CH2Cl2 (} 57 mL). The reaction mixture was stirred at room temperature for 3 _h . _K2CO3 (10% _aqueous solution, 50 mL) and then solid _K2CO3 were added at 0 °C to make the solution basic. The organic layer was separated, washed with water, dried ( _MgSO4 ), and evaporated to dryness. The residue was purified by preparative liquid chromatography on (silica gel 20-45 μm, 1000 g, mobile phase (1% NH4OH, 93% DCM, 7% MeOH)). The purified fractions were collected and concentrated to yield 12 g of intermediate compound (12).

c) Receipt n intermediate connection (13) And n intermediate connection (14)

Intermediate (12) was purified and resolved by chiral SFC on (CHIRALPAK

AD-H 5 μm 250x20 mm). Mobile phase (0.3% isopropylamine, 80% CO ₂ , 20% methanol). The pure fractions were collected and the solvent was removed, yielding 5.8 g of intermediate (14) (R*,S*) ([a] _D ²⁰ = -12.4° (589 nm, conc. 0.5 w/v%, DCM, 20°C)) and 5.6 g of intermediate (13) (S*,R*) ([a]D ²⁰ = +9.43° (589 nm, conc. 0.35 w/v%, DCM, 20°C)).

Intermediate connection (13).

1H NMR (500 MHz, DMSO-d ₆ ) δ (ppm) 7.49 (da, J = 2.5, 5.0 Hz, 1H), 7.31 (d, J = 5.0 Hz, 1H), 7.29 (d, J = 2.5 Hz, 1H), 5.88 (d, J = 1.9 Hz, 1H), 3.28-3.33 (br s, 1H), 2.75-2.87 (m, 4H), 2.61 (da, J = 2.8, 10.7 Hz, 1H), 2.54 (dd, J = 3.3, 10.9 Hz, 1H), 2.40-2.15 (m, 2H).

Intermediate connection (14).

1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.49 (dd, J=2.5, 5.0 Hz, 1H), 7.31 (d, J=5.0 Hz, 1H), 7.29 (d, J=2.5 Hz, 1H), 5.88 (d, J=1.9 Hz, 1H), 3.28-3.33 (br s, 1H), 2.75-2.87 (m, 4H), 2.61 (dd, J=2.8, 10.7 Hz, 1H), 2.54 (dd, J=3.3, 10.9 Hz, 1H), 2.40-2.15 (m, 2H).

Example A.4.

intermediate compound (15)

A solution of intermediate (6) (108 g, 0.302 mol) and pyridine-4-boric acid (49.5 g, 0.363 mol) in 2 M aqueous potassium carbonate (302 mL, 0.604 mol) and ethylene glycol dimethyl ether (1.1 L) was purged with _N2 for 5 min, then tetrakis(triphenylphosphine)palladium (34.9 g, 0.030 mol) was added, the mixture was heated at 78 °C using a multi-mode microwave oven (CEM Mars 5) with a power range from 0 to 800 W for 1 h, cooled to room temperature, EtOAc was added, the organic layer was separated, washed with water, then brine, dried over _MgSO4 and evaporated to dryness. The residue was purified by preparative liquid chromatography on silica gel 15-40 μm, 300 g, mobile phase (0.1% NH4OH, 97% DCM, 3% iPrOH). The pure fractions were collected and the solvent was removed, yielding 47.6 g of intermediate 15.

Intermediate (16) was purified and isolated by chromatography on Chiralpak AD (20 μm, 2000 g, 110 mm) at a flow rate of 750 ml/min. The mobile phase was 100% methanol. The pure fractions were collected and evaporated to dryness, yielding 18.7 g of intermediate (18) (R*,S*) ([a] _D ²⁰ = +55.75° (589 nm, conc. 0.339 w/v%, DMF, 20°C)) and 20.7 g of intermediate (17) (S*,R*) ([a] _D ²⁰ = -68.38° (589 nm, conc. 0.253 w/v%, DMF, 20°C)).

- 12 043636

Intermediate connection (17).

¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm) 8.52 (d, J=6.0 Hz, 2H), 7.41 (d, J=6.0 Hz, 2H), 6.50 (s, 1H), 3.36-3.61 (m, 4H), 2.81-3.02 (m, 3H), 2.61-2.53 (m, 1H), 1.36 (s, 9H).

Intermediate connection (18).

¹ H NMR (500 MHz, DMSO-d6) δ (ppm) 8.52 (d, J=6.0 Hz, 2H), 7.41 (d, J=6.0 Hz, 2H), 6.50 (s, 1H), 3.363.61 (m, 4H), 2.81-3.02 (m, 3H), 2.61-2.53 (m, 1H), 1.36 (s, 9H).

Example A.5

Receiving - ^ns| R intermediate connection (19)

Intermediate (18) (24.8 g, 86.6 mmol) was added to HCl dioxane (4 M, 108 ml) at 5°C, and the mixture was stirred at room temperature for 90 min. The resulting precipitate was filtered off, washed with diethyl ether, and dried under vacuum at 70°C, yielding 21.1 g of intermediate (19).

Receiving n —Sfu ¹⁴¹⁴ ' ^НС1 n intermediate connection (20)

Intermediate (20) was prepared similarly, starting from intermediate (17).

Example A-6.

a) Receipt ABOUT \ intermediate connection (21)

The reaction was performed on four batches of 0.5 g each of 6-bromo-3,4-dihydro-1H-[1,8]naphthyridin-2-one. A solution of 6-bromo-3,4-dihydro-1H-[1,8]naphthyridin-2-one (0.5 g, 2.20 mmol), bis(pinacolato)diboron (0.67 g, 2.64 mmol), and potassium acetate (0.648 g, 6.61 mmol) in DMF (5 mL) and CH3CN (10 mL) was stirred and degassed with nitrogen for 10 min. 1,1'-Bis-(diphenylphosphine)ferrocenedichloropalladium(II) (0.161 g, 0.22 mmol) was added and heated at 120°C using a microwave oven (Biotage initiator 60) with the power range from 0 to 400 W for 40 min. The mixture was evaporated to dryness, the residue was dissolved in DCM and water, filtered through a thin pad of celite. The organic layer of the filtrate was separated, washed with water, dried (MgSO ₄ ), and evaporated to dryness. The residue was dissolved in EtOH, filtered, and dried, yielding 0.36 g of intermediate (21).

b) Receipt 1 ° N N Y N intermediate connection (22)

Intermediate (21) (1.0 g, 3.65 mmol), tert-butyl propionate (0.426 mL, 3.04 mmol), silver(I) oxide (1.06 g, 4.56 mmol) and K2CO3 (0.84 g, 6.08 mmol) in CH3CN (10 mL) and DMF (5 mL) were purged with _N2 gas, then palladium(II) acetate (47% Pd) (0.034 g, 0.152 mmol) was added and the mixture was heated at 100 °C using a single-mode microwave oven (Biotage initiator 60) with a power range of 0 to 400 W for 20 min. Water and EtOAc were added, the mixture was filtered through a thin pad of Celite, the organic layer was separated, washed with water, then brine dried (MgSO ₄ ) and evaporated to dryness. The resulting residue was purified by flash chromatography on silica gel (15-40 μm, 30 g cartridge, CH ₂ Cl ₂ to CH ₂ Cl ₂ /CH ₃ OH/NH ₄ OH: 98.5/1.5/0.1). The pure fractions were collected and evaporated to dryness, yielding 0.037 g of intermediate (22). ________________________________________________________________

c) Receipt lYI N N O N intermediate connection (23)

Intermediate (22) (0.053 g, 0.195 mmol) was dissolved in TFA/DCM solution (0.37 mL/0.5 mL). The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure. The resulting solid was triturated with diethyl ether, filtered, and dried under vacuum (80°C) to yield 0.032 g of intermediate (23).

Example A.7 ___________________________________________

a) Receipt intermediate connection (24)

Microwave conditions: Biotage, 90°C, 25 min, slow after 30 sec preheat

- 13 043636 stirring. A solution of bromobenzene (0.228 mL, 2.64 mmol), cis-2-tert-butyloxycarbonylhexahydropyrrolo[3.4]pyrrole (0.6 g, 2.82 mmol), and sodium tert-butoxide (0.624 g, 6.5 mmol) in toluene (well dried over molecular sieves) (15 mL) was stirred and degassed with nitrogen for 10 min. Tris-(dibenzylideneacetone)dipalladium(0) (0.198 g, 0.216 mmol) and 2-di-tert-butylphosphino)biphenyl (0.065 g, 0.216 mmol) were added, and the resulting mixture was re-irradiated following the microwave treatment conditions described above. Water and ethyl acetate were added and the organic layer was separated, dried (MgSO ₄ ), filtered and concentrated. The resulting residue was purified by flash chromatography on silica gel (15-40 μm, 40 g, heptane/EtOAc 80/20). The pure fractions were collected and concentrated to give intermediate compound (24). _______________________________________________________________

b) Receipt n n intermediate connection (25)

TFA (4.54 mL, 58.95 mmol) was added to a solution of intermediate (24) (1.7 g, 5.9 mmol) in DCM (15 mL). The reaction mixture was stirred at room temperature for 2 h, water and DCM were added _{, K2CO3} (10% aqueous solution) was added for alkalinization, and the organic layer was separated, washed with water, dried (MgSO4), and evaporated to dryness to yield intermediate (25) as an oil.

The following compounds were prepared using the same procedure as in Example A.7, where bromobenzene was replaced by 2-bromothiophene, 2-bromoanisole, 2-bromo-1-methylbenzene, 2-bromo-1-chlorobenzene, 3-bromopyridine, 2-bromothiazole, 4-bromo-1-chlorobenzene, or 3-bromo-1-chlorobenzene, respectively.

n \ Р ^н f V- N Rp NH Н 0-<ph> ⁿ n intermediate connection (26) intermediate connection (27) intermediate connection (28) n n n rV ^N GK ^NH Η intermediate connection (29) intermediate connection (30) intermediate connection (31) n a— n n intermediate connection (32) intermediate connection (33)

Example A.

a) Receipt °° / F3C-S-0—С NCO-+- (cis) ⁰ n intermediate connection (34)

The reaction was carried out under N2 atmosphere. n-BuLi (1.6 M in hexane) (3.33 mL, 5.33 mmol) was added dropwise at -20 °C to a solution of DIPA (0.749 mL, 5.33 mol) in THF (8 mL), and the mixture was stirred at 20 °C for 20 min. Then, a solution of intermediate (4) (1.0 g, 4.44 mmol) in THF (10 mL) was added at -78 °C, and the resulting mixture was stirred for 30 min at -78 °C. A solution of N-phenyltrifluoromethanesulfonimide (1.4 g, 4.88 mol) in THF (6 mL) was added at -78 °C, and the mixture was allowed to warm to room temperature and stirred overnight. The mixture was concentrated and the residue was purified by flash chromatography on silica gel (40 g, 15-40 μm, heptane/EtOAc 70/30). The pure fractions were collected and evaporated to dryness, yielding intermediate compound (34).

b) Receipt Q-^T^M-C-Q—(Cis) Η intermediate connection (35)

Reaction under nitrogen atmosphere. Microwave conditions: Biotage initiator 60, 80°C, 20 min. A solution of intermediate (34) (0.42 g, 0.881 mmol) and thiophene-2-boric acid (0.135 g, 1.06 mmol) in K2CO3 (2 M, 0.88 mL) and ethylene glycol dimethyl ether (4 mL) was purged with N2 for 10 min, then tetrakis(triphenylphosphine)palladium(0) (0.102 g, 0.088 mmol) was added. The mixture was irradiated under the above microwave conditions, cooled to room temperature, water and EtOAc were added, the organic layer was separated, washed with water, then brine dried ( _MgSO4 ) and evaporated to dryness. The resulting residue was purified by flash chromatography on silica gel (10 g, 15-40 μm, heptane 100 to heptane/EtOAc 80/20). The purified fractions were collected and evaporated to dryness to yield intermediate compound (35).

- 14 043636

c) Receipt

intermediate compound (36)

A mixture of intermediate (35) (0.226 g, 0.776 mmol) in TFA (0.7 mL) and DCM (4 mL) was stirred at room temperature for 1 h, then the reaction mixture was poured into K ₂ CO ₃ (10% aqueous solution) and extracted with DCM. The organic layer was separated, washed with water, dried (MgSO ₄ ), and evaporated to dryness, yielding intermediate (36).

The following compounds were prepared using the same procedure as in Example A.8b/A.8c, where thiophene-2-boronic acid was replaced by 2-methoxyphenylboronic acid or formic acid, respectively.

intermediate compound (37)

Example A.9.

Microwave conditions: Biotage 60, 120°C, 30 min. A mixture of cis-2-tert-butyloxycarbonyl-hexahydropyrrolo[3,4]pyrrole (0.027 g, 0.13 mmol), 2-bromopropane (0.018 mL, 0.19 mmol), and triethylamine (0.088 mL, 0.64 mol) in DMF (0.2 mL) was irradiated under the microwave conditions described above. Water and EtOAc were added, the organic layer was separated, the aqueous layer was extracted twice with EtOAc, the combined organic phase was washed with water and brine, dried (MgSO ₄ ), and evaporated to dryness to give intermediate (37).

intermediate compound (38)

TFA (0.62 g, 8.02 mmol) was added to a solution of intermediate (37) (0.204 g, 0.8 mmol) in DCM (2 mL). The reaction mixture was stirred at room temperature for 3 h, water and DCM were added, 10% K ₂ CO ₃ was added to make it basic, solid NaCl was added to saturation, and the organic layer was separated, washed with water, dried (MgSO ₄ ), and evaporated to dryness to give intermediate (38) as an oil.

The following compounds were prepared using the same procedure as in Example A.9, where 2-bromopropane was replaced by propargyl bromide, benzylsulfonyl chloride, or methanesulfonate, respectively.

pF n ⁿ intermediate intermediate intermediate connection (39) connection (40) connection (41)

Example A.10.

a) Receipt _HO ^Y ? / 7\ 1 \ ( ^cis ) intermediate A- ^{n n} connections (44)

Reaction under N ₂ atmosphere. BuLi (1.6 M in hexane) (4.8 mL, 7.70 mmol) was added dropwise at -78 °C to a solution of thiazole (0.5 mL, 7.05 mol) in Et ₂ O (5 mL), then the mixture was stirred for 30 min. A solution of intermediate (5) (1.44 g, 6.41 mmol) in Et ₂ O (7 mL) was added, then the mixture was stirred and allowed to warm to room temperature over 2 h. Water and EtOAc were added, the organic layer was separated, washed with water, then with brine, dried over MgSO ₄ and evaporated to dryness. Purification of the resulting residue was performed by flash chromatography on silica gel (50 g, 15-40 μm, heptane/EtOAc 80/20 to heptane/EtOAc 50/50). The purified fractions were collected and evaporated to dryness to yield intermediate compound (44).

intermediate compound (45)

A mixture of intermediate (44) (1.05 g, 3.38 mmol) in HCl (37% in _H2O ) (7 ml) in a sealed tube was heated at 140°C using a microwave oven (Biotage Initiator EXP 60) with the power range from 0 to 400 W for 1 h. The reaction mixture was poured into _K2CO3 (10% aqueous solution), the _organic layer was separated, dried ( _MgSO4 ) and evaporated to dryness, yielding 0.23 g of residue (1). The aqueous layer was evaporated to dryness, the solid was suspended in DCM and stirred for 10 min. The suspension was filtered and the filtrate was evaporated to dryness, yielding 0.29 g of residue (2). Residues (1) and (2) were combined for purification, which was carried out with

- 15 043636 by flash chromatography on silica gel (15-40 μm, 30 g, _CH2Cl2 to _CH2Cl2 / _CH3OH / _NH4OH : 90/10/1). The pure fractions were collected and evaporated to dryness _, yielding 0.41 g of intermediate (45).

Example A.11.

a) Receipt 'ufn-s-'D- (CIS) intermediate connection (46)

Diethylaminosulfur trifluoride (1.24 mL, 10.12 mmol) was added dropwise to a solution of intermediate (5) (0.570 g, 2.53 mmol) in DCM (6 mL), cooled in an ice bath at 5 °C, and the mixture was stirred for 1 h at 5 °C and then overnight at room temperature. The mixture was cooled at 0 °C and saturated NaHCO ₃ was added. The organic layer was extracted with CH ₂ Cl ₂ , dried (MgSO ₄ ), filtered, and concentrated to give intermediate (46).

b) Receipt ы /\ 1 / ^н (as) Н intermediate connection (47)

TFA (0.39 g, 5.12 mmol) was added to a solution of intermediate (46) (0.146 g, 0.51 mmol) in DCM (1.2 mL). The reaction mixture was stirred at room temperature for 3 h, water and DCM were added, K ₂ CO ₃ (10% aqueous solution) was added for alkalinization, and the organic layer was separated, washed with water, dried (MgSO ₄ ), and evaporated to dryness to yield intermediate (47) as an oil.

Example A.12.

a) Receipt ¥ o \ /—\ ΖΙΓ /Ν ^- s—o—4— (cis) n intermediate connection (48)

A mixture of intermediate (7) (0.3 g, 1.05 mmol) and 10% dry Pd/C (0.06 g) in MeOH (15 mL) was hydrogenated at room temperature and atmospheric pressure for 2 h. The resulting mixture was filtered through a thin pad of celite, washed with DCM, and the filtrate was evaporated to dryness to yield intermediate (48).

b) Receipt \ /—\ T / ^nh ( ^cis ) Η intermediate connection (49)

A mixture of intermediate (48) (0.286 g, 0.995 mmol) and TFA (0.9 mL) in DCM (6 mL) was stirred at room temperature for 30 min, then the reaction mixture was poured into K ₂ CO ₃ (10% aqueous solution) and extracted with DCM. The organic layer was separated, washed with water, dried (MgSO ₄ ), and evaporated to dryness, yielding intermediate (49).

Example A.13.

a) Receipt C| _H __/ θ / \ /—(хУс· ⁰ \ Η intermediate connection (50)

Reaction under N2 atmosphere. Microwave treatment conditions: Biotage initiator 60, 80°C, 20 min. A solution of intermediate (38) (0.45 g, 1.26 mmol) and 2-chlorophenylboronic acid (0.236 g, 1.51 mmol) in K2CO3 (2 M, 1.2 6 mL) and ethylene glycol dimethyl ether (5 mL) was purged with N2 for 10 min, then tetrakis(triphenylphosphine)palladium(0) (0.146 g, 0.126 mmol) was added. The mixture was irradiated under the above treatment conditions, cooled to room temperature, water and DCM were added, the organic layer was separated, washed with water, dried ( _MgSO4 ) and evaporated to dryness. The residue was purified by preparative liquid chromatography (silica gel 15 μm, 150x30.0 mm). Mobile phase (100% DCM). The desired fractions were collected and the solvent was evaporated, yielding intermediate compound (50).

b) Receipt \ /—Cl X ( ^cis ) H intermediate connection (51)

A mixture of intermediate (50) (0.3 g, 0.938 mmol) and TFA (0.9 mL) in DCM (6 mL) was stirred at room temperature for 30 min, then the reaction mixture was poured into K ₂ CO ₃ (10% aqueous solution) and extracted with DCM. The organic layer was separated, washed with water, dried (MgSO ₄ ), and evaporated to dryness, yielding intermediate (51).

The following compounds were prepared using the same procedure as in Example A.13, wherein 2-chlorophenylboronic acid was replaced by 2-methylphenylboronic acid, 1-methyl-1Hpyrazole-5-boronic pinacol ester, furan-2-boric acid, 2-fluorophenylboronic acid, furan-3boric acid, 2-cyanophenylboronic acid, 5-dimethylisoxazole-4-boric acid, pyridine-3boric acid, 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)-1H-pyrazole, benzylzinc bromide, 2-chloropyridine-3-boric acid, pyrimidyl-5-boronic acid pinacolate, pinacol ester 1-BOC- 16 043636 pyrazole-4-boric acid, 5-methylfuran-2-boric acid or 4-methoxy-3-pyridinylboronic acid, respectively.

\ /—у ₇ ^nh (cis) ы /—\Т/ ^nh ( ^cis > ^Ν γ Хф/ G 2--u T NH (cis) N \ A n intermediate intermediate intermediate connection (52) connection (53) connection (54) at 2—(cis) 1 \--у ф NH (cis) Ρ ^Ν н \ /—\J(/ ^nh (cis) N N N intermediate intermediate intermediate connection (55) connection (56) connection (57) / ΐ №®\ / —W ^NH ( ^cis ) \ % (cis) ^/-у 1 _Z ^NH ( ^cis ) \ Η N N intermediate intermediate intermediate connection (58) connection (59) connection (60) ы у--у ф NH (CIS) { \—(Th (cis) ы С /—\I/ ^NH ^s) / \ n Ύι N intermediate intermediate intermediate connection (61) connection (62) connection (63) ы /—у ^(cis) £ ₂ \ J2 /NH ( ^cis ) N n O- intermediate intermediate intermediate connection (64) connection (65) connection (66)

Example A.14.

a) Receipt _s O , |G (I \-co—T (cis) H intermediate connection (67)

Intermediate (34) (2.798 mmol), palladium(II)acetate (47% Pd) (0.14 mmol), _K2CO3 (4.198 _mmol ), trimethylacetic acid (0.84 mmol), and tricyclohexylphosphonium tetrafluoroborate (0.196 mmol) were purged with _N2 in a sealed tube. Thiazole (4.198 mmol) and DMA (10 mL) were added, and the reaction mixture was heated at 100°C overnight. Water and EtOAc were added, the organic layer was separated, washed with water and brine, dried over _MgSO4 , and evaporated to dryness. The resulting residue was purified by flash chromatography on silica gel (30 g cartridge, 15-40 μm, heptane/EtOAc 80/20 to heptane/EtOAc 60/40). The pure fractions were collected and evaporated to dryness, yielding intermediate compound (67).

b) Receipt <04 intermediate connection (68)

A solution of intermediate (67) (0.24 g, 0.821 mmol) in TFA (0.8 mL) and DCM (5 mL) was stirred at room temperature for 30 min, then the reaction mixture was poured into K ₂ CO ₃ (10% aqueous solution) and extracted with DCM. The organic layer was separated, washed with water, dried (MgSO ₄ ), and evaporated to dryness, yielding 0.1 g of intermediate (68).

Example A.15.

a) Receipt n \_ /—( ^cis ) Η intermediate connection (69)

Pd(OAc) ₂ (1/3 mg, 0.0056 mmol) was added to a solution of intermediate (34) (0.1 g, 0.28 mmol), 1,3-bis-(diphenylphosphine)propane (4.6 mg, 0.011 mmol), and potassium acetate (0.041 g, 0.42 mmol) in EtOH (0.25 mL) and THF (2 mL) under nitrogen atmosphere. The mixture was stirred under carbon monoxide at 5 bar for 18 h in a stainless steel autoclave to yield intermediate (69).

b) Receipt n y --y T NH (CIS) 'Ί ^/ N intermediate connection (70)

A solution of intermediate (69) (0.2 g, 0.711 mmol) in HCl (4 M in dioxane) (2 ml) was stirred at room temperature for 30 min, then evaporated to dryness, yielding 0.13 g of intermediate (70).

Example A.16.

- 17 043636

a) Receipt o ft / /—\ JL , ^nco— C (cis) but \ n intermediate connection (71)

Pd(OAc)2 (25 mg, 0.112 mmol) was added to a solution of intermediate (34) (2.0 g, 5.6 mmol), 1,3-bis(diphenylphosphine)propane (92 mg, 0.22 mmol) and potassium acetate (0.82 g, 8.4 mmol) in EtOH (5 ml) and THF (40 ml) under nitrogen atmosphere, then the mixture was stirred under carbon monoxide at 5 bar at 100°C for 18 h in a stainless steel autoclave. The reaction mixture was poured into water and EtOAc, the organic layer was separated, washed with water and then with brine, dried ( _MgSO4 ), filtered and evaporated to dryness. Purification of the resulting residue was performed by flash chromatography on silica gel (15-40 μm, 40 g, heptane/EtOAc 90/10 to heptane/EtOAc 70/30). The pure fractions were collected and evaporated to dryness, yielding 0.61 g of intermediate (71).

b) Receipt /гфтг· ^(cis> intermediate connection (72)

A mixture of intermediate (71) (0.3 g, 1.18 mmol), dimethylamine in THF (2 M, 1.18 ml, 2.37 mmol), EDCI (0.27 g, 1.42 mmol), HOBt (0.19 g, 6.21 mmol), and triethylamine (0.25 ml, 1.78 mmol) in DCM (3 ml) and THF (3 ml) was stirred overnight at room temperature. Water and DCM were added, the organic layer was separated, dried (MgSO ₄ ), and evaporated to dryness, yielding 0.37 g of intermediate (72).

c) Receipt n / /G NH (cis) -d Ag intermediate connection (73)

A solution of intermediate (72) (0.37 g, 1.32 mmol) in HCl (4 M in dioxane) (4 mL) was stirred at room temperature for 30 min, then the reaction mixture was poured into K ₂ CO ₃ (10% aqueous solution) and extracted with DCM. The organic layer was separated, washed with water, dried (MgSO 4 ), and evaporated to dryness, yielding intermediate (73).

Example A.17.

a) Receipt ° /-^Р\ θ / -\Т/С-О-Л (cis) h ₂ n '-Д '' Η intermediate connection (74)

A mixture of intermediate (71) (0.3 g, 1.18 mmol), 1,1,1,3,3,3-hexamethyldisilazane (0.23 g, 1.42 mmol), EDCI (0.27 g, 1.42 mmol), HOBt (0.19 g, 6.21 mmol) and triethylamine (0.25 ml, 1.78 mmol) in DCM (3 ml) and THF (3 ml) was stirred overnight at room temperature. Water and DCM were added, the organic layer was separated, dried (MgSO ₄ ) and evaporated to dryness. The resulting residue was purified by flash chromatography on silica gel (15-40 μm, 10 g, CH ₂ Cl ₂ to CH ₂ Cl ₂ /CH ₃ OH/NH ₄ OH: 94/6/0.1). The purified fractions were collected and evaporated to dryness, yielding 0.16 g of intermediate (74).

b) Receipt /Н (cis) h ₂ n Н intermediate connection (75)

A solution of intermediate (74) (0.16 g, 0.634 mmol) in HCl (4 M in dioxane) (2 ml) was stirred at room temperature for 30 min, then evaporated to dryness, yielding 0.1 g of intermediate (75).

Example A.18.

a) Receipt ^N-C-O-^- (cis) intermediate connection (76)

A solution of intermediate (34) (0.2 g, 0.56 mmol), bis-(pinacolato)diboron (0.171 g, 0.67 mmol), and potassium acetate (0.165 g, 1.68 mmol) in dioxane (2 mL) was stirred and degassed with N2 for 10 min. 1,1'-Bis-(diphenylphosphine)ferrocenedichloropalladium(II) (0.041 g, 0.056 mmol) was added, and the reaction mixture was heated at 100 °C using a microwave oven (Biotage Initiator EXP 60) with a power range of 0 to 400 W for 20 min. Water and EtOAc were added, the organic layer was separated, washed with water, then with brine, dried over MgSO ₄ and evaporated to dryness. The resulting residue was purified by flash chromatography on silica gel (10 g, 15-40 μm, heptane/EtOAc 85/15 to heptane/EtOAc 70/30). The pure fractions were collected and evaporated to dryness, yielding intermediate compound (76).

b) Receipt _s /DA a / J mTTo-g ( ^cis ) ⁿ 'n mn \ n intermediate connection (77)

- 18 043636

A solution of intermediate (76) (0.45 g, 1.34 mmol) and 2-bromo-5-methyl-1,3,4-thiadiazole (0.288 g, 1.61 mmol) in _K2CO3 (2 M, 1.34 _mL , 2.69 mmol) and ethylene glycol dimethyl ether (5 mL) was stirred and degassed with _N2 for 10 min. Tetrakis(triphenylphosphine)palladium(0) (0.155 g, 0.134 mmol) was added and the reaction mixture was heated at 150 °C using a single-mode microwave oven (Biotage Initiator EXP 60) with a power range of 0 to 400 W for 5 min. Water and EtOAc were added, the organic layer was separated, washed with brine, dried (MgSO ₄ ), and evaporated to dryness. The resulting residue was purified by flash chromatography on silica gel (30 g cartridge, 15-40 μm, DCM to DCM/MeOH/NH 4 OH: 98/2/0, 1). The pure fractions were collected and evaporated to dryness, yielding intermediate compound (77).

c) Receipt ы УГ 6--/ NH (cis) ⁿ 'n / Η intermediate connection (78)

A solution of intermediate (77) (0.14 g, 0.455 mmol) in HCl (4 M in dioxane) (2 ml) was stirred at room temperature for 30 min, then the reaction mixture was poured into 10% aqueous K2CO3 and extracted with DCM. The organic layer was separated, washed with water, dried ( _MgSO4 ), and evaporated to dryness, yielding 81 mg of intermediate (78).

Example A.19.

a) Receipt ы _YI^NCO—(CIS) ^н intermediate connection (79)

BuLi (1.6 M in hexane) (4.2 mL, 6.66 mmol) was added dropwise to a solution of 1-methylimidazole (0.53 mL, 6.66 mol) in THF (5 mL) under nitrogen atmosphere at -78 °C, then the resulting mixture was stirred at 0 °C for 1 h. The reaction mixture was cooled to -78 °C, a solution of intermediate (5) (91.0 g, 4.44 mmol) in THF (10 mL) was added. The reaction mixture was stirred for 2 h at -78 °C, then allowed to cool to room temperature and stirred overnight. Water and EtOAc were added, the organic layer was separated, washed with water and brine, dried over MgSO ₄ and evaporated to dryness. The resulting residue was purified by flash chromatography on silica gel (15-40 μm, 30 g, from _CH2Cl2 to _CH2Cl2 / _CH3OH / _NH4OH : 95/5/0.1). _The pure fractions were collected and evaporated to dryness _, yielding 0.54 g of intermediate (79).

b) Receipt intermediate connection (80)

A mixture of intermediate (79) (0.54 g, 1.76 mmol) in HCl (37% in _H2O ) (5 mL) in a sealed tube was heated at 140°C using a single-mode microwave oven (Biotage Initiator EXP 60) with a power range of 0 to 400 W for 1 h. The reaction mixture was evaporated to dryness, yielding 0.47 g of intermediate (80).

Example A.20.

a) Receipt F ₃ CSQ~^^ / ( ^cis ) n \ intermediate connection (81)

The reaction was performed under anhydrous conditions under argon and its progress was monitored by thin layer chromatography (silica gel, petroleum ether/ethyl acetate 1/1, UV/phosphomolybdic acid). n-Butyllithium, 2.5 M in hexane (4.28 ml, 10.7 mmol), was added dropwise (5 min) to a solution of diisopropylamine (1.51 ml, 10.7 mmol) in THF (16 ml) at -20 °C. The mixture was stirred for 15 min at -20 °C and then cooled to -78 °C. A solution of intermediate (95) (2.00 g, 8.88 mmol) in THF (20 ml) was added (5 min) at -78 °C. The mixture was stirred at -78°C for 2 h. A solution of 2-[N,N-bis-(trifluoromethylsulfonyl)amino]pyridine (3.50 g, 9.77 mmol) in THF (12.5 mL) was added (5 min) at -78°C. The mixture was then allowed to warm back to room temperature and stirred for 17 h. The mixture was heated at 50°C for 4 h. The reaction mixture was quenched by the addition of saturated aqueous ammonium chloride (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried (sodium sulfate), filtered, and concentrated. Dichloromethane (50 ml) was added to the obtained residue (6.07 g), then the mixture was filtered to yield 1.30 g of a white solid, the filtrate was concentrated and then purified by flash column chromatography on silica gel (eluent: petroleum ether/EtOAc 100/0 to 60/40). The product fractions were collected and the solvent was evaporated, yielding 1.02 g of intermediate (81).

b) Receipt o .o X/*—SD-U' / ^(cis) n \ intermediate connection (82)

The reaction was carried out in an argon atmosphere, monitoring its progress using a thin-film

- 19 043636 chromatography (petroleum ether/ethyl acetate 8/2, UV/phosphomolybdic acid). 5-Acetyl-2-thienylboronic acid (0.057 g, 0.336 mmol) and 2 M aqueous potassium carbonate (0.280 ml, 0.560 mmol) were added to a solution of intermediate (81) (0.100 g, 0.280 mmol) in 1,2-dimethoxyethane (5 ml). The mixture was purged with argon and tetrakis(triphenylphosphine)palladium(0) (0.032 g, 0.028 mmol) was added. Then the mixture was heated at 80 °C overnight. The mixture was cooled to room temperature, water (10 ml) and EtOAc (10 ml) were added. The organic layer was separated, washed with water (10 ml), then with brine (10 ml), dried (sodium sulfate), filtered, and evaporated under vacuum to dryness. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 8/2). The desired fractions were collected and the solvent was evaporated, yielding 0.076 g of intermediate (82).

c) Receipt II n ( ^cis ) H intermediate connection (83)

The reaction was performed under anhydrous conditions under argon, monitoring its progress using thin-layer chromatography (petroleum ether/ethyl acetate 9/1, UV). HCl, 4 M in dioxane (3.33 ml, 13.3 mmol), was added to a solution of intermediate (82) (0.444 g, 1.33 mmol) in dioxane (9 ml). The reaction mixture was stirred at room temperature for 70 h and then concentrated to dryness, yielding 0.370 g of intermediate (83).

The following compounds were prepared using the same procedure as in Example A.20b/A.20c, wherein 5-acetyl-2-thienylboronic acid was replaced by 4-methylthiophene-2-boric acid, 2-chlorothiophene-3-boric acid, 4-methyl-3-thiopheneboric acid, 2-acetyl-3-thiopheneboric acid, 5-cyanothiophene-2-boric acid, 5-chlorothiophene-2-boric acid, 5-methylthiophene-2-boric acid pinacol ester, 3-methylthiophene-2-boric acid pinacol ester, or 3-methoxythiophene-2-boric acid pinacol ester, respectively.

N (cis) f \\ JC NH H (cis) L /—\ Ji ^Xnh \ H (cis) intermediate connection (84) intermediate connection (85) intermediate connection (86) (cis) N<±,s /-J% Lz—\ JL / ^NH H (cis) Cl _s ΐ T U-f TC ,NH H (cis) intermediate connection (87) intermediate connection (88) intermediate connection (89) N (cis) ы [ί><τ> \ H (cis) G f U NH o— n (cis) intermediate intermediate intermediate connection (90) connection (91) connection (92)

Example A.21.

a) Receipt —/ ( ^cis ) intermediate connection (93)

The reaction was performed under anhydrous conditions under argon and monitored by thin layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 9/1, phosphomolybdic acid). Methyl lithium 1.6 M in diethyl ether (3.29 ml, 5.26 mmol) was added to a suspension of copper(I) iodide (0.794 g, 4.17 mmol) in THF (5.0 ml) at 0 °C. After 1 h, a solution of intermediate (81) (0.355 g, 0.993 mmol) in THF (2.1 ml) was added via tube at 0 °C, washing with THF (2.1 ml). The reaction mixture was stirred at room temperature overnight. The mixture was quenched with saturated NH ₄ Cl solution (14 ml) and dried to dryness. The residue was purified by column chromatography on silica gel (eluent: pentane/ethyl acetate 95/5). The product fractions were collected and the solvent was evaporated, yielding 0.180 g of intermediate compound (93).

b) Receipt —f NH (cis) H intermediate compound (94)

The reaction was carried out under anhydrous conditions under argon atmosphere and its progress was monitored by thin layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 9/1, phosphomolybdic acid). HCl (4 M in dioxane) (2.02 g, 8.06 mmol) was added to a solution of intermediate (93) (0.180 g, 0.806 mmol) in 1,4-dioxane (4.3 ml), the solution was stirred at room temperature for 65 h, then concentrated to dryness, yielding 0.141 g of intermediate (94) (110%).

- 20 043636

Example A.22.

a) Receipt /^-C\ ° / /t/X ^- ( ^cis ) n intermediate compound (95)

The reaction was carried out under anhydrous conditions and its progress was monitored by thin layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 50/50, developer: UV/phosphomolybdic acid). A solution of intermediate (4) (6.93 g, 31.0 mmol) in THF (180 ml) was hydrogenated at room temperature (atmospheric pressure) with palladium on carbon, 10% (w/w) loading (1.65 g) as a catalyst for 15 h. The catalyst was filtered off on clarcel, the filter cake was washed with dichloromethane (50 ml) and the combined filtrates were concentrated under reduced pressure to dryness. Purification of the resulting residue (7.26 g) was performed by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 80/20 to 50/50). The product fractions were collected and the solvent was evaporated, yielding 6.70 g of intermediate (95).

b) Receipt /\ 1 n-c-o—e— (as) n intermediate compound (96)

The reaction was carried out under anhydrous conditions under argon, monitoring its progress using thin-layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 6/4, DCIP). Lanthanum trichloride lithium complex 0.6 M in THF (3.70 ml, 2.22 mmol) was added to a solution of intermediate (95) (0.500 g, 2.22 mmol) in THF (15 ml). The mixture was stirred at room temperature for 1 h, then cooled to 0°C. A solution of ethyl magnesium bromide, 0.1 M in THF (2.66 mL, 2.66 mmol) was added dropwise and the reaction mixture was allowed to warm to room temperature, stirred for 18 h. The reaction mixture was quenched by addition of saturated aqueous ammonium chloride (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried (Na ₂ SO ₄ ), filtered and concentrated. Purification of the obtained residue (0.635 g) was done by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 9/1 to 7/3). The product fractions were collected and the solvent was evaporated. Purification of the obtained residue (0.410 g) was done by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 8/2). The product fractions were collected and the solvent was evaporated, yielding 0.235 g of intermediate (96).

c) Receipt n /— ^(cis) H intermediate compound (97)

The reaction was carried out under anhydrous conditions under argon, monitoring its progress using 111 NMR. HCl in dioxane (4 M, 2.30 mL, 9.20 mmol) was added to a solution of intermediate (96) (0.235 g, 0.920 mmol) in dioxane (2 mL).

The reaction mixture was stirred at 60°C for 18 h.

After cooling to room temperature, the precipitate was filtered on a glass frit and washed with diethyl ether (20 ml) to yield 0.126 g of a solid. The filtrate was evaporated to dryness to yield 0.077 g of a residue.

The solid and residue were combined and dissolved in dioxane (2 mL). 4 M HCl in dioxane (2.30 mL, 9.20 mmol) was added, and the mixture was stirred at 60°C for 24 h, then at 100°C for 72 h. The reaction mixture was concentrated to dryness, yielding 0.158 g of intermediate (97).

Example A.23.

a) Receipt but—С I n-c-o—(cis) н intermediate compound (98)

The reaction was performed under anhydrous conditions under argon and monitored by thin layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 1/1, phosphomolybdic acid). Sodium borohydride (0.893 g, 23.6 mmol) was added portionwise over 30 min to a solution of intermediate (95) (2.66 g, 11.8 mmol) in MeOH (60 ml) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and then concentrated to dryness. The residue was diluted with ethyl acetate (200 ml) and washed with water (100 ml), 1 M aqueous hydrochloric acid (100 ml) and brine (100 ml). The organic layer was dried (Na2SO4), filtered and concentrated to yield 2.27 g of intermediate (98).

b) Receipt ° ° / —s—о -/jf/Vc-o “С ( ^cis ) ⁰ UL intermediate compound (99)

The reaction was carried out under anhydrous conditions under argon atmosphere, monitoring its progress using thin-layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 1/1, phosphomo

- 21 043636 libdic acid). Methanesulfonyl chloride (0.930 ml, 11.9 mmol) was added dropwise to a solution of intermediate (98) (2.27 g, 9.98 mmol) and triethylamine (4.17 ml, 29.9 mmol) in DCM (50 ml) at 0 °C. The reaction mixture was stirred for 1 h at room temperature and then concentrated to dryness. The residue was diluted with ethyl acetate (200 ml) and washed with water (100 ml), brine (100 ml), 1 M aqueous hydrochloric acid (100 ml) and brine again (100 ml). The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated. Purification of the obtained residue (2.52 g) was performed by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 8/2 to 5/5). The product fractions were collected and the solvent was evaporated, yielding 2.39 g of intermediate compound (99).

c) Receipt intermediate connection (100)

The reaction was performed under anhydrous conditions under argon atmosphere and its progress was monitored by thin layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 8/2, ninhydrin/phosphomolybdic acid). Intermediate (99) (0.300 g, 0.982 mmol) was dissolved in DMF (3 ml) and the mixture was cooled to 0°C. Pyrrole (0.102 ml, 1.47 mmol) and sodium hydride, 60% dispersion in mineral oil (0.0589 g, 1.47 mmol) were added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with ethyl acetate (50 ml) and washed with water (2 x 50 ml) and then with brine (3 x 50 ml). The organic layer was dried ( _Na2SO4 ), _filtered and concentrated. Purification of the obtained residue (0.290 g) was performed by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 98/2 to 95/5 and then to 90/10). The product fractions were collected and the solvent was evaporated, yielding 0.175 g of intermediate (100).

d) Receipt oA|o« I* intermediate connection (101)

The reaction was performed under anhydrous conditions under argon atmosphere and its progress was monitored by thin-layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 6/4, phosphomolybdic acid). 4 M HCl in dioxane (1.58 ml, 6.33 mmol) was added to a solution of intermediate (100) (0.175 g, 0.633 mmol) in dioxane (3 ml). The reaction mixture was stirred at 50°C for 2 h and concentrated to dryness, yielding 0.135 g of intermediate (101).

The following compounds were prepared using the same procedure as in Example A.23c/A.23b, where the pyrrole was replaced by tetrazole, pyrazole, 1,2,4-triazole, 1,2,3-triazole, or phenol, respectively.

Example A.24.

a) Receipt AA o. —o—6 JT N-c-o—e— (cis) n intermediate compound (109)

The reaction was performed under anhydrous conditions under argon and monitored by thin layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 8/2, ninhydrin/phosphomolybdic acid). A 25% (w/w) solution of sodium methoxide in methanol (0.449 ml, 1.96 mmol) was added to a solution of intermediate (99) (0.300 g, 0.982 mmol) in MeOH (4 ml). The reaction mixture was stirred at reflux for 20 h. The reaction mixture was concentrated to dryness. The residue was diluted with ethyl acetate (50 ml) and washed with water (50 ml) and then with brine (50 ml). The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated. Purification of the obtained residue was performed by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 100/0 to 97/3 and then to 1/1). The product fractions were collected and the solvent was evaporated, yielding 0.182 g of intermediate compound (109).

- 22 043636

intermediate compound (110)

The reaction was performed under anhydrous conditions under argon atmosphere and its progress was monitored by thin layer chromatography (silica gel, eluent: petroleum ether/ethyl acetate 8/2, ninhydrin/phosphomolybdic acid). 4 M HCl in dioxane (1.88 ml, 7.54 mmol) was added to a solution of intermediate (109) (0.182 g, 0.754 mmol) in dioxane (4 ml). The reaction mixture was stirred at room temperature for 18 h and then at 50 °C for 2 h. The reaction mixture was concentrated to dryness, yielding 0.139 g of intermediate (110).

Some of the intermediates used in the preparation of the final compounds are commercially available, for example.

B. Obtaining final compounds.

Example B.1.

intermediate compound (14)

A mixture of intermediate (3) (9.4 g, 44.3 mmol), intermediate (9) (8.5 g, 44.3 mmol), hydroxybenzotriazole (6.0 g, 44.3 mmol), triethylamine (15.4 mL, 0.111 mmol) in CH2Cl2 (160 mL) and THF (160 mL) was stirred overnight at room temperature. Water (175 mL) was added, the precipitate was filtered off, washed with water/EtOH (50 mL). The solid was suspended in EtOH (50 mL) and stirred for 15 min. The resulting suspension was filtered and dried under vacuum at 70°C to yield 7.3 g of compound (14) as a white powder (mp = 266°C), ([a] _D ²⁰ = -105.1° (589 nm, conc. 0.1275 w/v%, CH2Cl 20°C).

1H NMR (500 MHz, DMSO-4) δ (ppm) 10.64 (d, J=7.6 Hz, 1H), 8.33 (dd, J=1.7, 9.6 Hz, 1H), 8.04 (d, J=17.3 Hz, 1H), 7.48 (d, J=7.6 Hz, 2H), 7.31-7.42 (m, 3 H), 7.23-7.28 (m, 1H), 6.99 (t, J=15.0 Hz, 1H), 6.20 (d, J=6.0 Hz, 1H), 3.38-4.04 (m, 5H), 3.09-3.21 (m, 1H), 2.85-3.04 (m, 3H), 2.55-2.67 (m, 3H).

Example B.2.

A mixture of intermediate (14) (5.8 g, 30.32 mmol), intermediate (3) (7.72 g, 30.32 mmol), 1-hydroxybenzotriazole (4.92 g, 30.38 mmol), EDCI (6.97 g, 30.38 mmol), triethylamine (14.71 mL, 106.12 mmol) in _{CH2Cl2 (} 100 mL) and THF (100 mL) was stirred overnight at room temperature. The mixture was poured into water. The precipitate was filtered off, washed twice with EtOH and dried under vacuum at 65°C. This precipitate was recrystallized from EtOH, filtered and dried under vacuum at 62°C to give 9.02 g of compound (44) as a white powder (mp=264°C), ([a]D ²⁰ =+170.12° (589 nm, conc. 0.2075 w/v%, C^Clz, 20°C).

1H NMR (400 MHz, DMSO-a ₆ ) δ (ppm) 10.63 (d, J=4.5 Hz, 1H), 8.32 (d, J=5.1 Hz, 1H), 8.03 (d, J=10.6 Hz, 1H), 7.52 (dd, J=2.8, 4.8 Hz, 1H), 7.41 (br s, 1H), 7.36 (dd, J=4.8, 9.3 Hz, 2H), 6.98 (dd, J=9.1, 15.7 Hz, 1H), 6.01 (br s, 1H), 3.35-4.03 (m, 5H), 2.94-3.21 (m, 2H), 2.90 (q, J=7.9 Hz, 3H), 2.52-2.62 (m, 2H).

Example B.3. _____________

intermediate compound (40)

A mixture of intermediate (19) (21.1 g, 81.4 mmol), intermediate (3) (17.3 g, 61.8 mmol), 1-hydroxybenzotriazole (11.0 g, 81.4 mmol), EDCI (15.6 g, 81.4 mmol), and triethylamine (47 mL, 0.339 mmol) in _CH2Cl2 ( ₃₅₀ mL) and THF (350 mL) was stirred overnight at room temperature. Water was added to the mixture. The precipitate was filtered, washed with water/EtOH and then EtOH, and dried under vacuum at 70°C to give 12.7 g of compound (40) as a white powder (mp=271°C), ([a]D ²⁰ =+116.08° (589 nm, conc. 0.2145 w/v%, C^Ch, 20°C).

¹ H NMR (400 MHz, DMSO-d6) δ (ppm) 10.63 (d, J=5.1 Hz, 1H), 8.52 (d, J=5.6 Hz, 2H), 8.33 (d, J=6.1 Hz, 1H), 8.03 (d, J=13.6 Hz, 1H), 7.41-7.46 (d, J=15.7 Hz, 2H), 7.38 (d, J=4.0 Hz, 1H), 6.98 (dd, J=11.6, 15.7 Hz, 1H), 6.53 (d, J=8.1 Hz, 1H), 3.37-4.04 (m, 5H), 2.86-3.22 (m, 5H), 2.58-2.70 (m, 2H).

intermediate compound (41)

Compound (41) was obtained similarly by reacting intermediate (20) with intermediate (3) following the same procedure.

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Ή NMR (500 MHz, DMSO-06) δ (ppm) 10.63 (d, J=5.1 Hz, 1H), 8.52 (d, J=5.6 Hz, 2H), 8.33 (d, J=6.1 Hz, 1H), 8.03 (d, J=13.6 Hz, 1H), 7.41-7.46 (d, J=15.7 Hz, 2H), 7.38 (d, J=4.0 Hz, 1H), 6.98 (dd, J=11.6, 15.7 Hz, 1H), 6.53 (d, J=8.1 Hz, 1H), 3.37-4.04 (m, 5H), 2.86-3.22 (m, 5H), 2.58-2.70 (m, 2H).

(Md ²⁰ = -115.85° (589 nm, conc. 0.183 wt/vol%, C^Ch, 20°C)).

Example B.4. ______________________________________________

a) Receipt > ^,JU sa n n intermediate connection (111)

The reaction was carried out under argon and monitored by thin _layer chromatography (silica gel, _CH2Cl2 /methanol/triethylamine 95/5/0.1, UV/phosphomolybdic acid). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (HCl) (1.70 g, 8.87 mmol) was added to a mixture of intermediate (3) (2.02 g, 7.39 mmol), crude cis-hexahydro-cyclopenta[c]pyrrol-5(III)one (1.85 g, max 8.89 mmol), 1-hydroxybenzotriazole monohydrate (1.36 g, 8.87 mmol), and N-ethyldiisopropylamine (6.32 mL, 36.9 mmol) in DMF (75 mL). The mixture was stirred at room temperature for 18 h. The mixture was concentrated under reduced pressure, diluted with dichloromethane (150 ml) and washed with saturated aqueous NaHCO ₃ (100 ml). The aqueous layer was back-extracted with dichloromethane (2 x 150 ml). The combined organic layers were washed with brine (400 ml), dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure to dryness. Purification of the obtained residue was performed by column chromatography on silica gel (eluent: dichloromethane/methanol 100/0 to 94/6). The product fractions were collected and the solvent was evaporated. The basic aqueous layers were back-extracted with dichloromethane (3 x 300 ml). The combined organic layers were washed with brine (900 mL), dried (Na ₂ SO ₄ ), filtered, and concentrated under reduced pressure to dryness. The resulting residue was purified by column chromatography on silica gel (eluent: dichloromethane/methanol 100/0 to 94/6). The product fractions were collected, and the solvent was evaporated. The desired residues were combined to yield 1.58 g of intermediate (111).

b) Receipt '-- But intermediate connection (71)

The reaction was carried out under anhydrous conditions under argon atmosphere and its progress was monitored using thin layer chromatography (petroleum ether/ethyl acetate 95/5, UV/phosphomolybdic acid). Lanthanum trichloride lithium chloride complex 0.6 M in THF (2.38 ml, 1.43 mmol) was added to a suspension of intermediate (111) (0.464 g, 1.43 mmol) in THF (18 ml). The mixture was stirred at room temperature for 1 h, then cooled to 0°C. A solution of phenylmagnesium bromide, 0.1 M in THF (3.57 ml, 3.57 mmol) was added dropwise. The reaction mixture was stirred and allowed to warm back to room temperature over 3 days. Additionally, 1.0 M phenylmagnesium bromide solution in THF (2.85 mL, 2.85 mmol, 2 equivalents) was added dropwise. The mixture was stirred at room temperature for 2 h. The reaction mixture was quenched by addition of saturated aqueous ammonium chloride (30 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were dried (Na ₂ SO ₄ ), filtered, and concentrated under reduced pressure to dryness. The resulting residue was purified by flash column chromatography on silica gel (eluent: CH ₂ Cl ₂ /MeOH 100/0 to 95/5). The solvent from the collected product fractions was evaporated. The residue was triturated with diethyl ether (2 x 3 mL) and then dried under vacuum to yield 0.077 g of compound (71).

Example B.5

intermediate compound (73)

A mixture of intermediate (23) (0.032 g, 0.097 mmol), intermediate (8) (0.032 g, 0.145 mmol), EDCI (0.022 g, 0.0116 mmol), HOBt (0.016 g, 0.116 mmol) and triethylamine (0.049 mL, 0.349 mmol) in DCM (1 mL) and THF (1 mL) was stirred overnight at room temperature. Water was added, the mixture was extracted with DCM, the organic layer was separated, washed with water, dried (MgSO ₄ ) and evaporated to dryness. The residue was recrystallized from EtOH, the solid was filtered, washed with EtOH and dried (vacuum, 70 °C) to yield 0.015 g of compound (73).

Table F-1 lists the compounds that were obtained according to one of the examples given above.

- 24 043636

Table F-1

(cis) Compound No. 1; Note B.l Connection No. 2; Note B.1 (cis) Compound No. 3; Note B.l V-s Connection No. 4; Note B.1 X JL/\ N N o ρ η h (cis) Compound No. 5; Note B.l о S Η Η ό Compound No. 6; Note B.l NN O [| ^H H (cis) Compound No. 7; Note Bl a ||V ^H h Conn. No. 8; Note Bl c Compound No. 9; Note B.l 5¼ (cis) Compound No. 10; Note B.l Compound No. 11; Note B.l ^N ·^ (cis) Compound No. 12; Note Bl Compound No. 13; Note Bl ( [a] _D ²⁰ =+l04, 17 ° (589 nm, conc. 0.096 wt./vol.%, CH ₂ Cl ₂ , 20°C) Compound No. 14; Note B.l N-\ (Cis) Compound No. 15; Note B.1 Compound No. 16; Note B.l

- 25 043636

Compound No. 17; Note B.l Compound No. 18; Note B.l cD-^XX ^Vo (cis) ^S^xx Compound No. 19; Note B.l Compound No. 20; Note B.l o >' ^’W· O-^ (cis) Compound No. 21; Note B.l Compound No. 22; Note B.l H. JL ^x /X a ^{n n} Compound No. 23; Note B.l Compound No. 24; Note B.l H Α^^ΐχ^/ΐχ/χ^\n? ΤΧλ Jx AL NN N ^HH ° \ (cis) "YES." (cis) Compound No. 25; Note B.l Compound No. 26; Note B.l N=^ (cis) (cis) Compound No. 27; Note B.l Compound No. 28; Note B.l W Wo (Cis) Compound No. 29; Note B.l Compound No. 30; Note B.l

- 26 043636

J/Oca ^H (cis) ⁿ Compound No. 31; Note B.1 ^Vs _(cis) Compound No. 32; Note B.1 vAXV·· qp N N'^O _F ^z Η Η Connection No. 33; Note B.1 (cis) Compound No. 34; Note B.1 Connection No. 35; Note B.1 Cl Compound No. 36; Note B.1 o n X ϋO' ΓΧΐ O ^{n n} Connection No. 37; Note B.1 Connection No. 38; Note B.1 X (cis) rHP". Connection No. 39; Note B.1 Conn. No. 40; Note V.Z Hi", n n Conn. No. 41; Note V.Z Connection No. 42; Note B.1 1 JL Η Η (cis) RAKH." Connection No. 43; Note B.1 Connection No. 44; Note B.2 ° (cis)

- 27 043636

- 28 043636

C. Connection identification.

C1. LCMS.

The following methods were used to characterize the compounds of the present invention by LCMS.

General methodology A.

LC measurements were performed using an Acquity ultra-high-performance liquid chromatography (UPLC) system (Waters) equipped with a binary pump with a degasser, an autosampler, a diode array detector (DAD), and a column as described in the respective methods below, maintained at 40°C. Column flow was directed to a MS detector. The MS detector was equipped with an electrospray ionization source. The capillary needle voltage was 3 kV, and the temperature of the Quattro (a triple quadrupole mass spectrometer from Waters) was maintained at 130°C. Nitrogen was used as the nebulizer gas. Data acquisition and processing were performed using a Waters-Micromass MassLynx-Openlynx data processing system.

General methodology of V.

HPLC measurements were performed using an Alliance HT 2795 system (Waters), including a quaternary pump with a degasser, an autosampler, a diode array detector (DAD), and a column as described in the corresponding methods below. The column was maintained at 30°C. The column flow was split for the MS spectrometer. The MS detector was implemented with an electrospray ionization source. The capillary needle voltage was 3 kV, and the source temperature in the LCT (Time of Flight Zspray™ mass spectrometer from Waters) was maintained at 100°C. Nitrogen was used as the nebulizer gas. Data acquisition and processing were performed using a WatersMicromass MassLynx-Openlynx data processing system.

Method 1.

In addition to General Procedure A, reversed-phase UPLC was performed on a Waters Acquity C18 BEH (bridged ethylsiloxane/silica hybrid) column (1.7 μm, 2.1 x 100 mm) at a flow rate of 0.35 mL/min. Two mobile phases (mobile phase A: 95% 7 mM ammonium acetate/5% acetonitrile; mobile phase B: 100% acetonitrile) were used to perform a gradient condition from 90% A and 10% B (hold for 0.5 min) to 8% A and 92% B, hold for 2 min, and returning to the initial conditions after 0.5 min, hold for 1.5 min. The

- 29 043636 sample injection volume was 2 µl. Cone voltage was 20 V for both positive and negative ionization modes. Mass spectra were acquired by scanning from 100 to 1000 in 0.2 s with a delay time of 0.1 s between scans.

Method 2.

In addition to General Procedure A, reversed-phase UPLC was performed on a Waters Acquity C18 BEH (bridged ethylsiloxane/silica hybrid) column (1.7 μm, 2.1 x 100 mm) at a flow rate of 0.343 mL/min. Two mobile phases (mobile phase A: 95% 7 mM ammonium acetate/5% acetonitrile; mobile phase B: 100% acetonitrile) were used to run a gradient from 84.2% A and 15.8% B (0.49 min hold) to 10.5% A and 89.5% B, 2.18 min hold, and returning to baseline at 0.73 min, 0.73 min hold. An injection volume of 2 μL was used. The cone voltage was 20 V for both positive and negative ionization modes. Mass spectra were acquired by scanning from 100 to 1000 in 0.2 s with a 0.1 s delay time between scans.

Method 3.

In addition to General Procedure B, reversed-phase HPLC was performed on a Waters Xbridge C18 column (3.5 μm, 4.6 x 100 mm) at a flow rate of 0.8 mL/min. Two mobile phases (mobile phase A: 100% 7 mM ammonium acetate; mobile phase B: 100% acetonitrile) were used to maintain a gradient condition from 80% A to 20% B (hold for 0.5 min) to 90% B over 4.5 min, 90% B for 4 min, and re-equilibration at initial conditions for 3 min. An injection volume of 5 μL was used. The cone voltage was 20 V for both positive and negative ionization mode. Mass spectra were acquired by scanning from 100 to 1000 in 0.4 s using a delay time of 0.3 s between scans.

Method 4.

In addition to General Procedure B, reversed-phase HPLC was performed on a Waters Atlantis C18 column (5 μm, 3.9 x 100 mm) at a flow rate of 0.8 mL/min. Three mobile phases (mobile phase A: 100% 7 mM ammonium acetate; mobile phase B: 100% acetonitrile; mobile phase C: 0.2% formic acid + 99.8% ultrapure water) were used to maintain a gradient condition from 50% A and 50% C (hold for 1.5 min) to 10% A, 80% B, and 10% C in 4.5 min, hold for 4 min, and re-equilibrate at initial conditions for 3 min. An injection volume of 5 μL was used. The cone voltage was 20 V for both positive and negative ionization modes. Mass spectra were acquired by scanning from 100 to 1000 in 0.4 s with a 0.3 s delay time between scans.

Method 5.

HPLC measurements were performed using an Alliance 1100/1200 HPLC system, including a quaternary pump with a degasser, an autosampler, a diode array detector (DAD), and a column as described in the corresponding methods below. The column was maintained at room temperature. The MS detector (Agilent single quadrupole) was equipped with an APCI electrospray ionization source. Nitrogen was used as the nebulizer gas. Data acquisition and processing were performed using the Chemstation data processing system.

Reversed-phase HPLC was performed on a Nucleosil C18 column (3 μm, 3x150 mm) at a flow rate of 0.42 mL/min. Two mobile phases (mobile phase A: water/TFA (0.1%); mobile phase B: 100% acetonitrile) were used to maintain a gradient condition from 98% A over 3 min, to 100% B over 12 min, 100% B for 5 min, then returning to 98% A over 2 min, and re-equilibrating at the initial conditions with 98% A for 6 min. An injection volume of 2 μL was used. The capillary needle voltage was 3 kV, the corona discharge was held at 1 μA, and the source temperature was maintained at 250°C. A sweep voltage was used for the fragmentor. Mass spectra were obtained using electrospray ionization and APCI in positive mode, scanning from 100 to 1100 amu.

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Table C.1

LC/MS data

Connection No. Exp. MN ⁺ Method Connection No. Exp. MN ⁺ Method 2 5, 12 419 3 41 1.98 387 2 3 2.53 392 2 42 2.12 310 2 6 2.84 412 1 43 2.76 400 2 7 2.43 411 2 44 2, 63 392 2 9 2.74 420 2 45 2.58 392 2 10 2.53 392 2 46 2.16 382 2 11 2.74 400 2 47 2.24 421 2 12 1.96 387 2 48 5, 4 353 4 13 3.23 392 1 49 1.85 408 2 14 2, 63 386 2 50 1.78 388 2 15 2.92 426 2 51 1.84 376 2 17 2, 64 403 2 52 13.46 324 5 18 2, 65 423 2 53 14.13 338 5 19 2.44 376 2 54 14.38 404 5 21 2, 66 404 2 57 2.59 390 2 22 2.4 376 2 58 11.35 380 5 23 2.22 405 2 59 15.16 426 5 26 1.5 353 2 60 14.79 406 5 30 1.78 351 2 61 2.35 417 2 31 8.79 342 5 62 13.58 434 5 32 2.07 393 2 63 15, 03 406 5 33 2.12 453 2 64 1, 6 381 2 34 2.1 348 2 67 14.01 417 5 35 2.72 423 2 68 15, 62 426 5 36 2.73 423 2 69 15, 09 406 5 37 11.2 379 5 71 13, 1 404 5 38 11.4 379 5 72 14.51 422 5 39 12.26 379 5 73 2.78 384 2 40 1.99 387 2

C2. Melting points.

For some compounds, melting points were obtained using a Kofler stage, which consists of a heated plate with a linear temperature gradient, a sliding pointer, and a temperature scale in degrees Celsius.

For a number of compounds, melting points were determined using differential scanning calorimetry (DSC). Melting points were measured with a temperature gradient of 10°C/min, starting from 25°C. The maximum temperature was 350°C.

Melting points for a number of compounds were determined using a Buchi B-560 melting point apparatus. A metal block served as the heating medium. Sample melting was observed visually using a magnifying lens and strong light contrast. Melting points were measured with a temperature gradient of either 3 or 10°C/min. The maximum temperature was 300°C.

The remaining melting points were determined using open capillary tubes.

Table C.2

Melting point data Compound No. Melting point

274.95°C

Method

DSC No. Compound Melting Point Method

249.0- Buchi

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D. Pharmacological examples.

D.1. FabI enzyme inhibition: Staphylococcus aureus FabI enzyme inhibition assay.

FabI enzyme inhibition assays were performed in a half-area, 384-well microtiter plate. Compounds were assessed in 40-μl samples of mixtures containing 100 mM NaADA, pH 6.5 (ADA = N-[2-acetamido]-2-iminodiacetic acid), 250 μM crotonyl-CoA, 625 μM NADH, and 50 μg/ml S. aureus ATCC 29213 FabI. Inhibitors typically ranged from 50 to 0.39 μM. Reaction mixtures were incubated for 30 min at room temperature and stopped by adding 200 mM Tris buffer (pH 9.0) to create a pH shift. NADH composition was monitored by measuring changes in absorbance at 340°C. The percentage inhibition of enzymatic activity by the compounds was determined by comparing the measured values with those of negative (no compound) and positive (no enzyme) control samples. The best-fit curve was determined using the least-squares method. From this curve, the IC ₅₀ value (expressed in μg/mL), resulting in 50% inhibition of enzymatic activity, was obtained.

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Table D.1

S values. aureus FabI IC ₅₀ Connection No. Fabl 1C ₅₀ cg/ml Connection No. Fabl 1C ₅₀ cg/ml 1 0.32 35 1.25 2 0.78 36 0.93 3 0.29 37 3.37 4 0.70 38 2.08 5 ~0.6 39 0.56 6 3.73 40 0.39 8 0.50 41 0.44 9 0.75 42 0.83 10 0.53 43 0.60 11 0.48 44 0.46 12 0.44 45 0.45 13 0.39 46 0.54 14 0.40 47 0.43 15 0.48 48 2.93 17 0.38 49 0.44 18 0.44 51 0.54 19 ~0.62 52 0.50 20 1.07 53 0.36 21 0.65 54 1.84 22 0.58 57 0.62 23 0.41 59 0.76 24 0.58 60 0.59 25 0.51 61 0.54 26 0.41 62 0.44 27 0.6 63 0.63 29 1.04 64 1, 62 30 2, 66 67 0.63 31 1.42 68 0.99 32 0.46 71 2.55 33 3, 06 72 0.43 34 1, 67 73 0.80

D.2. In vitro method for testing the antibacterial activity of compounds against various bacterial strains.

Preparation of bacterial suspensions for susceptibility testing.

The following bacteria were used: Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus (MRSA) ATCC 700788, and Escherichia coli ATCC 35218. Bacteria used in this study were grown overnight in flasks containing 100 mL of Mueller-Hinton broth (Difco cat. #0757-17) in sterile deionized water with shaking at 37°C. The prepared solution was stored at -70°C until use.

Bacteria were incubated on a tryptic soy agar plate containing 5% sheep blood (Becton Dickinson cat. no. 254053) for 18-24 hours at 35°C under aerobic conditions (first pass). For the second pass, fresh Mueller-Hinton broth was seeded using 5-10 colonies and grown overnight at 35°C until turbidity appeared (reaching the inoculation phase) under aerobic conditions. The bacterial suspension was then adjusted to a specific gravity of 0.5 according to McFarland and further diluted 1:100 in Mueller-Hinton broth medium. This was used as the inoculum.

The results (for STA ATCC 29213) are presented in Table D2 below.

Antibacterial susceptibility testing.

Definition.

MIC (minimum inhibitory concentration) studies were performed by broth microdilution in a 96-well format (flat-bottomed microtiter plate) with a final volume of 0.1 ml of Mueller Hinton broth containing twofold serial dilutions of the compounds with a bacterial inoculum of 5x105 CFU/ml (standard inoculum size according to CLSI guidelines). Inhibitors typically range from 63 to 0.49 μM. The final DMSO concentration in the study was 1.25% (maximum allowable DMSO concentration = 6%). In studies testing the effect of human serum on compound activity against S. aureus, human serum was added at a final concentration of 10%. The plates were incubated at 35°C for 16-20 h. At the end of the incubation, bacterial growth was quantified fluorometrically. To do this, resazurin was added to all wells and the plates were re-incubated. Incubation time

- 33 043636 depends on the type of bacteria. A color change from blue to pink indicates bacterial growth. Fluorescence was measured in a computer-controlled fluorometer (Fluoroskan Ascent FL, Labsystems) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. The percentage of growth inhibition achieved by the compounds was calculated according to standard methods. IC ₅₀ (expressed in μg/mL) was defined as the 90% inhibition concentration of bacterial growth. A set of reference compounds was tested simultaneously to confirm quality control.

The results are presented in Table D2 (STA+10% HS) below.

Cytotoxicity study.

The cytotoxicity of the compounds was assessed using the MTT assay. Human HelaM cells grown in 96-well plates were subjected to serial dilutions of the test compounds (final volume of 0.2 mL) and incubated for 72 h at 37°C and 5% CO ₂ . Inhibitors typically ranged from 25 to 0.8 μM. The final DMSO concentration in the assay was 0.5%. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tetrazole) was added and reduced to purple formazan only in living cells. Solubilization of formazan crystals was achieved by the addition of 100 μL of 2-propanol. Cell viability was determined by measuring the absorbance of reduced formazan, which produces a violet color, at 540 nm and 690 nm. The absorbance measured at 690 nm was automatically subtracted from the absorbance at 540 nm to eliminate the effect of nonspecific absorption. The percentage of cytotoxicity achieved by the compounds was calculated according to standard methods. Cytotoxicity was expressed as CC ₅₀ – the concentration that causes a 50% reduction in cell viability.

The results are presented in Table D2 (TOX HELAM) below.

Table D2

Data from representative examples

Connection No. STA (361.159) IC90 dg/ml STA + 10% HS (361.169) IC90 dg/ml TOX HELAM (222.125) CC50 dg/ml 1 0.09 0.17 >3,8547 2 1, 02 1.09 >19,4696 3 0.03 0.06 >3.25636 5 0.64 1.14 7.92 8 1, 15 1.52 >10,5122 9 0.33 0.69 4.77 10 0.08 0.13 >3,915 11 0.37 1.76 6, 19 12 0.33 0.53 >9.70744 13 0.31 0.43 >9.68257 14 0.19 0.19 >9.68257 15 0.74 0.72 >9.78279 17 0.37 0.38 >10,1103 18 0.21 0.37 >10,6233 19 0.18 0.12 >9.43038 21 0.13 0.29 >4,0346 22 0.23 0.25 >9.43038 23 0.67 0.79 >10,3108 24 4, 05 2.44 >3,9549 26 1, 11 1.11 >3,8646

Example E.

E.1. Thermodynamic solubility/solubility in aqueous solution.

A pH-dependent solubility study was performed at ambient temperature over a 4-day period. A saturation solubility study was conducted to determine the maximum solubility in a given buffer solution. The compound was added to the appropriate buffer solution until saturation was reached. The flask was then shaken for 4 days at ambient temperature. After 4 days, the solutions were filtered and injected into an HPLC, and the concentration was determined using a conventional HPLC method.

Results. ____________________________________________________

Connection No. 14 Connection No. 1 Connection No. 41 Connection No. 2 pH 2 buffer <0.01 <0.002 1.18 <0.01 10% ΗΡ-β-CD 0.076 BUT BUT BUT

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pH 2 buffer 20% ΗΡ-β-CD buffer pH 2 0.20 BUT BUT BUT pH 4 buffer <0.01 <0.002 <0.01 <0.01 10% ΗΡ-β-CD buffer pH 4 0.069 0, 177 1.1 0.11 20% ΗΡ-β-CD buffer pH 4 0.18 0, 308 >1.15 0.28 PH 7.4 buffer <0.01 <0.002 0.13 <0.01 10% ΗΡ-β-CD buffer pH 7.4 0.089 0, 100 0.49 0.14 20% ΗΡ-β-CD buffer pH 7.4 0.20 0.417 0.56 0.33

BUT means not determined.

E.2. Antimicrobial spectrum of action.

Minimum inhibitory concentrations (MICs) were determined according to the Clinical and Laboratory Standards Institute (CLSI) methodology against aerobic bacteria (CLSI M07-A8) (see Clinical and Laboratory Standards Institute. 2009. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. CLSI document M07-A8, Vol. 29, No. 2.) using the cation-modified Mueller-Hinton broth microdilution method (CA-MHB) for most organisms, except for Haemophilus influenza, where Haemophilis test medium (HTM) broth was used. Descriptions of individual organisms can be found in the table. Where available, ATCC standard strains were tested.

The inoculum density for susceptibility testing was standardized to yield a final inoculum with approximately 5 x 10 ⁵ CFU/mL. The broth MIC was defined as the lowest drug concentration that prevented visible growth after 16–24 h (depending on the species) of incubation at 35–37°C.

Description of individual organisms studied

Organism Specifications MIC of the test environment Staphylococcus aureus ATCC 29213; reference strain MS SA MNV Staphylococcus aureus ATCC 43300; reference strain MRSA MNV Staphylococcus aureus NRS119; LZD-R; SCCmec IV; origin: USA MNV Staphylococcus aureus NRS120; LZD-R; SCCmec IV; Origin: USA MNV Staphylococcus aureus NRS121; LZD-R; SCCmec IV; Origin: USA MNV Escherichia coll ATCC 25922; reference strain MNV Escherichia coli Tol C mutant MNV Haemophilus influenzae ATCC 49247; reference strain NTM broth Moraxella catarrhalis ATCC 8176; β-lactamase negative MNV

Stock solutions of the compounds were prepared in DMSO at a concentration of 1 mg/mL. Linezolid was prepared in DMSO at a concentration of 2 mg/mL. Stock solutions of all compounds were diluted in CA-MNV to obtain a range of twofold dilutions, depending on the sensitivity of the test organism.

Results (if any). __________________________________________

Organism Compound No. and MIC90 (µg/ml) 14 1 44 2 41 10 22 12 S. aureus ATCC 29213 0.03 0.016 0.03 0.25 0.03 0.015 0.06 0.125 S. aureus ATCC 43300 0.03 0.016 0.03 0.5 0.03 0.03 0.125 0.125

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S. aureus NRS119 0.03 0.03 0.03 0.06 S. aureus NRS120 0.03 0.016 0.03 0.06 S. aureus NRS121 0.03 0.016 0.06 0.06 Mutant E. coll tolC 0.25 <0.03 >8 0.25 1 0.125 1 0.25 E. coll ATCC 25922 4 >32 >8 >8 8 >8 >8 >8 H. influenza ATCC 49247 0.25 >8 >8 0.5 >8 4 1 M. catarrhalis ATCC 8176 0.015 0.25 0.12

E.3. In vivo pharmacokinetics and bioavailability after oral administration.

The in vivo pharmacokinetics and oral bioavailability of the example compounds were/are being studied in male Swiss mice (fed) after single intravenous (i.v.) bolus and oral (r.o.) administration. For the i.v. and r.o. formulations, the compound was dissolved in a 20% ΗΡ-β-CD solution. The pH of the formulation was/is approximately pH 4. All i.v. formulations were isotonic.

Results.

Connection No. 14 Connection No. 1 Connection No. 10 Connection No. 44 Connection No. 12 i.v. Dose (mg/kg) 2.5 2.5 2.5 2.5 2.5 η 3 3 3 3 3 Co (ng/ml) 2929 2921 4154 4524 2333 Plasma clearance Cl (L/h/kg) 0.33 0.35 0.64 0.49 2.2 Vd _z (l/kg) 1.3 1.5 1.2 0.9 3.7 AUCo-inf (ng/h/ml) 7464 7074 3992 5037 1124 Half-life (tl/ ₂ ) (H) 2.7 2.9 1.3 1.3 1.1 r.o. Dose (mg/kg) 10 5 10 10 10 η 3 3 3 3 3 Stakh (NG/ML) 2950 1720 3537 2670 275 Tshchakh (And) 2.0 2.0 1, 0 1.0 1.0 AUCo-inf (ng/h/ml) 21394 12158 12376 14527 914 AUCo-last Half-life (ti/ ₂ ) (h) 3.2 3.1 2.2 2.8 n.d. Oral bioavailability (%) 72 86 81 59 21

E.4. In vivo efficacy.

The concept of studying the in vivo effect of an antibacterial compound by treating intraperitoneally infected mice was introduced in 1911 during a study of the effect of optochin against pneumococci (Morgenroth and Levy, 1911). The popularity of this model is explained by its ease of use, short experimental duration, reproducible infections, and simple endpoints.

Method.

Staphylococcus aureus strain ATCC 29213 was used to infect female Swiss white mice. Bacterial culture of brain heart infusion (BHI) broth was cultured the day before infection, incubated at 37°C overnight, and diluted with fresh BHI broth to the desired concentration. E.p. (intraperitoneal) administration of ~5x10 ⁸ -5x10 ⁹ colony-forming units (CFU) was performed in either of the lateral lower quadrants of the abdomen. After infection, mice were kept in cages and monitored daily for signs of infection or death. Both the p.o. and i.v. routes were used to treat mice, and each mouse was individually treated by

Claims (18)

administration through a gastric tube or by i.v. introduction. In this model, both solutions (p.o. and i.v.) and suspensions (p.o.) were tested. The parameter used to monitor the progression of infection and the effect of treatment was death or survival of animals within 3 days after infection. Because death may occur as a result of toxicity, a control group of 3 mice was included and treated with the highest dose of test compound (in suspension studies). Results. In vivo antibacterial activity in peritonitis models of S. aureus infection (ATCC 29213) following oral intravenous administration using solutions. Compound Path of infection Sowing material (magazine- nary entry 10) Compound Processing path Treatment dose (mg per kg) % survivability 44 IP 8, 9 Solution 20%CD+lHCl RO, QD 1; 5 57; : 100 14 IP 8.7 20%CD+2H2T IV, QD 2.5; 5 75; : 100 Control mice experienced mortality rates of 80 and 100% in each of the respective trials. CLAIM 1. Compound of formula (I)? / ^|= SU° ^(|) R ³ where A represents -C=C- or ^r2 bond represents a single bond or a double bond; X is carbon or nitrogen, and if X is nitrogen, then the bond is a single bond; Z1 represents CH or N; R ¹ represents hydrogen, C _1-4 alkyl or halogen; R ² represents hydrogen, C _1-4 alkyl or halogen; R ³ represents hydrogen, C _1-6 alkyl, hydroxyl or halogen; R ⁴ represents hydrogen; halogen; C _1-6 alkyl; C _2-6 alkenyl; C _2-6 alkynyl; C _1-6 alkyloxy; C _1-4 alkyloxycarbonyl; aminocarbonyl; mono- or di(C _1-4 alkyl)aminocarbonyl; aryl; aryloxy; arylcarbonyl; arylsulfonyl; heteroaryl; C _1-6 alkyl substituted with cyano group; C _1-6 alkyl substituted with aryl or aryloxy; or C _1-6 alkyl substituted with heteroaryl; aryl is phenyl; phenyl substituted with one, two or three substituents, each of them individually selected from halogen, hydroxyl, C _1-4 alkyl, C _1-4 alkoxy, polyhaloC _1-4 alkyl, polyhaloC _1-4 alkoxy, cyano-, nitro- and amino groups; heteroaryl is furanyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzo[1,3]dioxolyl, benzofuranyl, benzothiazolyl, indolyl, 2 ,3-dihydro-1Nindolyl, tetrahydrothiophenyl or quinolinyl, wherein each heteroaryl may be substituted with one or two substituents, each individually selected from halogen, cyano, C _1-4 alkyl, C _1-4 alkoxy, C _1-4 alkylcarbonyl or phenyl; or a pharmaceutically acceptable acid addition salt thereof. 2. The compound according to claim 1, where Z ₁ represents CH; R ¹ represents hydrogen or C _1-4 alkyl; R ² represents hydrogen or C _1-4 alkyl. 3. The compound according to claim 1 or 2, where A is -C=C- or the bond is a single bond or a double bond; X represents carbon or nitrogen, and if X represents nitrogen, then the ^ ⁷⁷⁷⁷ ^ bond represents a single bond; R ¹ is hydrogen; R ² is hydrogen; - 37 043636 R ³ is hydrogen, hydroxyl or halogen; R ⁴ is hydrogen; halogen; With _1-6 alkyl; C1 _-6 alkyloxy; C1 _-4 alkyloxycarbonyl; aminocarbonyl; mono- or di(C _1-4 alkyl)aminocarbonyl; aryl; aryloxy; arylsulfonyl; heteroaryl; C _1-6 alkyl substituted with a cyano group; C _1-6 alkyl substituted with aryl or aryloxy; or With _1-6 alkyl substituted with heteroaryl; aryl is phenyl; phenyl substituted with one substituent selected from halogen, C _1-4 alkyl, C _1-4 alkyloxy and cyano groups; heteroaryl is furanyl, thiophenyl, pyrazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridinyl or pyrimidinyl, wherein each heteroaryl may be substituted with one substituent selected from halogen, cyano, C _1-4 alkyl, C _1-4 alkoxy or C _1-4 alkylcarbonyl; or a pharmaceutically acceptable acid addition salt thereof. 4. A compound according to any one of claims 1 to 3, where R ¹ is hydrogen and R ² is hydrogen. 5. A compound according to any one of claims 1 to 4, where R ³ represents hydrogen. 6. A compound according to any one of claims 1 to 5, where R ⁴ is aryl. 7. A compound according to any one of claims 1 to 5, where R ⁴ is heteroaryl. 8. A compound according to any one of claims 1-5, where R ⁴ is C _1-6 alkyl substituted with aryl. 9. A compound according to any one of claims 1 to 8, wherein X represents nitrogen and the bond is a single bond. 10. Connection according to any one of claims 1-9, where A represents 11. Connection selected from the following: - 38 043636 - 39 043636 - 40 043636 - 41 043636 or a pharmaceutically acceptable salt thereof. 12. A pharmaceutical composition for the treatment of bacterial infections, containing a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound according to any one of paragraphs. 1 11. 13. A method for preparing a pharmaceutical composition according to claim 12, wherein a therapeutically effective amount of a compound according to any one of claims 1 to 11 is thoroughly mixed with a pharmaceutically acceptable carrier. 14. Use of a compound of formula (I) according to any one of claims 1 to 11 as a medicinal agent for the treatment of bacterial infections. 15. Use of a compound of formula (I) according to any one of claims 1 to 11 for the treatment of bacterial infections. - 42 043636 16. Use according to claim 15, wherein the bacterial infection is caused by a bacterium that expresses the enzyme Fabl. 17. A method for preparing a compound of formula (I) according to claim 1 or a pharmaceutically acceptable acid addition salt thereof, comprising reacting an intermediate of formula (II) with an intermediate of formula (III) (II) (III) where A, X, R ³ , R ⁴ and Ζι are as defined in claim 1, and subsequently converting the compound of formula (I) into a pharmaceutically acceptable acid addition salt by reacting the compound of formula (I) with acid. 18. A method for preparing a compound of formula (I) according to claim 1 or a pharmaceutically acceptable acid addition salt thereof, wherein in the compound of formula (I) A is -C(R ² )=C(R ¹ )-, comprising reacting an intermediate of the formula (V) with an intermediate of formula (VI) (I) where X _a i represents a leaving group representing a halide group, and X, Z _b R ¹ , R ² , R ³ and R ⁴ are as defined in claim 1; and then converting the compound of formula (I) into a pharmaceutically acceptable acid addition salt by reacting the compound of formula (I) with an acid. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2