Novel compounds for treatment of bacterial infections

Abstract

The invention relates to a compound of formula (I) wherein R1 to R7, n, X, L, A1 and A2 are as defined in the description and in the claims. The compounds of formula (I) are useful as medicaments.

Description

[0001] This invention relates to organic compounds that can be used to treat and / or prevent bacterial infections in mammals. Specifically, these molecules can inhibit the LPS synthesis pathway, particularly LpxH, and can be used to treat bacterial infections.

[0002] This invention particularly relates to a compound of formula (I).

[0003] (I)

[0004] in

[0005] L is absent or is an alkyl group;

[0006] X is -O- or -NH-;

[0007] n is 0 or 1;

[0008] R1 is an amino group, an aminoalkyl group, an alkylamino group, an aminoalkylamino group, a haloalkyl group, a diaminoalkyl group, a guanidinyl group, a (aminoalkyl)(aminoalkyl)(hydroxycarbonylalkyl)ammoniumalkyl group, an aminoalkoxyalkyl group, a (aminoalkyl)(aminoalkyl)(aminocarbonylalkyl)ammoniumalkyl group, an (amino)(hydroxy)alkyl group, an (aminoalkyl)(aminoalkyl)amino group, an (aminoalkyl)(aminoalkyl)aminoalkyl group, an (aminoalkyl)(aminoalkyl)aminoalkoxy group, an (aminoalkyl)(aminoalkyl)aminoalkylamino group, an (aminoalkyl)(aminoalkyl)(hydroxycarbonyl)ammoniumalkylamino group, an (hydroxy)(halo)alkylamino group, an (aminoalkyl)(aminoalkyl)(hydroxycarbonylalkyl)ammoniumalkoxy group, a cycloalkyl group, or a heterocycloalkyl group, wherein the heterocycloalkyl group and the cycloalkyl group are optionally substituted by one or two independent substituents selected from R6;

[0009] The prerequisite is that if R1 is amino, then L does not exist;

[0010] R2 and R3 are independently selected from halogens; or R2 and R3 together with the carbon atoms to which they are attached form cycloalkyl groups;

[0011] R4 is a halogen or alkyl group;

[0012] R5 is an amino, hydroxyalkyl, aminoalkyl, alkylamino, aminoalkylamino, or piperazine group;

[0013] R6 is independently selected from amino, alkyl, hydroxy, aminoalkyl, aminocarbonylalkyl, and aminoalkylcarbonylamino in each case;

[0014] R7 is hydrogen or hydroxyl;

[0015] A1 is -N- or -CH-; and

[0016] A2 is either -O- or -CH2-;

[0017] Or its medicinal salt.

[0018] The intensive use of antibiotics has exerted selective evolutionary pressure on microorganisms to develop gene-based resistance mechanisms. Modern medicine and socioeconomic practices exacerbate the problem of resistance development by creating slow-growing environments for pathogenic microorganisms (e.g., in artificial joints) and by supporting long-term host storage (e.g., in immunocompromised patients). In hospital settings, an increasing number of strains of Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus spp., Enterobacteriaceae (such as Klebsiella pneumoniae), Acinetobacter baumannii, and Pseudomonas aeruginosa (the main sources of infection) are becoming multidrug resistant and therefore difficult to treat. This is particularly concerning for Gram-negative organisms, as no new agents with differentiated mechanisms of action have been approved for decades. Therefore, there is a significant medical need for novel antimicrobial compounds to address Gram-negative resistant bacteria, particularly third-generation cephalosporin- and carbapenem-resistant Enterobacteriaceae, as well as multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. One approach to addressing cross-resistance to established classes of antibiotics is to inhibit essential proteins or functions that current antibiotics do not target.

[0019] Gram-negative bacteria are unique in that their outer membrane contains lipopolysaccharide (LPS), which is crucial for maintaining membrane integrity and is essential for bacterial viability (reviewed in Ann. Rev. Biochem 76: 295-329, 2007). The major lipid component of LPS is lipid A, and inhibition of lipid A biosynthesis is lethal to bacteria. Lipid A is synthesized on the cytoplasmic surface of the bacterial inner membrane via a pathway consisting of nine different enzymes. These enzymes are highly conserved in most Gram-negative bacteria. LpxH is a calcineurin-like phosphatase (CLP) that catalyzes the hydrolysis of UDP-2,3-diacyl-glucosamine (UDP-DAGn) to produce lipid X and UMP. LpxH has no mammalian homologues, making it a promising target for developing novel antibiotics targeting Gram-negative bacteria.

[0020] The term "alkyl" refers, alone or in combination, to a monovalent straight-chain or branched saturated hydrocarbon group with 1 to 12 carbon atoms, particularly 1 to 7 carbon atoms, and even more particularly 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Methyl, ethyl, and propyl are specific examples of "alkyl".

[0021] The term "cycloalkyl" refers, alone or in combination, to a cycloalkyl ring having 3 to 8 carbon atoms, particularly a cycloalkyl ring having 3 to 6 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Specific examples of "cycloalkyl" are cyclopropyl and cyclobutyl.

[0022] The term "heterocyclic alkyl" refers, alone or in combination, to a monocyclic, bicyclic, or tricyclic system consisting of 4 to 12 ring atoms, particularly 5 to 7 ring atoms, that is monovalently saturated or partially unsaturated, comprising 1, 2, or 3 heterocyclic atoms selected from N, O, and S, with the remaining ring atoms being carbon. Bicyclic means consisting of two rings having one or two common ring atoms. "Heterocyclic alkyl" may contain a carbonyl group, where the carbon of the carbonyl group is part of the ring system. The ring system can be attached to the remaining compound by atoms selected from C, N, S, and O, particularly by an N atom ("N-heterocyclic alkyl"). Examples of “heterocyclic alkyl” include, but are not limited to, morpholino, morpholino-4-yl, pyrrolyl, pyrrolidine-1-yl, pyrrolidine-3-yl, piperidinyl, 1-piperidinyl, 4-piperidinyl, 2-oxopyrrolidine-1-yl, piperazinyl, piperazin-1-yl, azirrobutyl, azirrobutyl-1-yl, azirrobutyl-3-yl, azirrobutyl-1-yl, or 5-azinonspiro[2.4]heptane. Specific examples are azinonspiro-3-yl, azinonspiro-1-yl, morpholino-4-yl, pyrrolidine-1-yl, and 5-azinonspiro[2.4]heptane.

[0023] The term "alkoxy" or "alkyloxy" alone or in combination represents a group of the formula "alkyl-O-", wherein the term "alkyl" has the meaning previously given, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. Specific examples of "alkoxy" are methoxy and ethoxy.

[0024] The term "oxygen group" refers to the -O- group, either alone or in combination.

[0025] The term "halogen" or "halogenated" alone or in combination means fluorine, chlorine, bromine or iodine, and particularly fluorine, chlorine or bromine, and more particularly fluorine. The term "halogenated" in combination with another group means that the group is substituted with at least one halogen, particularly with one to five halogens, especially one to four halogens, i.e., one, two, three or four halogens.

[0026] The term "haloalkyl" alone or in combination refers to an alkyl group substituted with at least one halogen, particularly with one to five halogens, particularly with one to three halogens, and more particularly with two to three halogens. Specific "haloalkyl" groups are fluoromethyl, fluoroethyl, fluoropropyl, fluorobutyl, difluoromethyl, difluoroethyl, trifluoromethyl, and trifluoroethyl.

[0027] The term "haloalkoxy" alone or in combination refers to an alkoxy group substituted with at least one halogen, particularly with one to five halogens, especially one to three halogens. Specific "haloalkoxy" groups are fluoromethoxy, fluoroethoxy, and fluoropropoxy.

[0028] The term "hydroxyl" (hydroxyl / hydroxy) refers to the -OH group, either alone or in combination.

[0029] The term "carbonyl" refers to the -C(O)- group, either alone or in combination.

[0030] The term "amino" alone or in combination represents a primary amino group (-NH2), a secondary amino group (-NH-), or a tertiary amino group (-N-).

[0031] The term "alkylamino" refers, alone or in combination, to an alkyl group attached to an -NH- group. The term "dialkylamino" refers to two alkyl groups attached to an -N- atom.

[0032] The term "aminoalkyl" refers alone or in combination to an amino group attached to an alkyl group, wherein the alkyl group is further attached to the compound shown. The term "diaminoalkyl" means two amino groups attached to an alkyl group, wherein the alkyl group is further attached to the compound shown.

[0033] The term "aminoalkoxy" alone or in combination refers to an amino group attached to an alkoxy group, wherein the alkoxy group is further attached to the compound shown via an -O- atom.

[0034] The term "aminoalkoxyalkyl" refers, alone or in combination, to an amino group attached to an alkyl group via an alkoxy group, wherein the alkyl group is further attached to the compound shown.

[0035] The term "ammonium alkyl" refers, alone or in combination, to an ammonium group attached to an alkyl group, wherein the alkyl group is further attached to the compound shown.

[0036] The term "alkyl carbonyl" refers to an alkyl group attached to a -C(O)- group, wherein the carbonyl group is further attached to the compound shown. Specifically, "alkyl carbonyl" refers to methyl carbonyl (also known as acetyl) and ethyl carbonyl.

[0037] The term "carbonylalkyl" refers to a carbonyl group attached to an alkyl group, wherein the alkyl group is further attached to the compound shown. Specific "alkylcarbonyl" compounds are carbonylmethyl and carbonylethyl.

[0038] The term "carbonylamino" refers to a carbonyl group attached to an amino group, wherein the amino group is further attached to the compound shown.

[0039] The term "sulfonyl" alone or in combination represents the -SO2- group.

[0040] The term "medicinal salt" refers to a salt that is not biologically or otherwise undesirable. "Medicinal salt" includes both acid addition salts and base addition salts. "Medicinal acid addition salt" refers to those medicinal salts that form with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, etc., and organic acids that can be selected from aliphatic, alicyclic, aromatic, arylaliphatic, heterocyclic, carboxylic acid, and sulfonic acid organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, dihydroxynaphthyl acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid, etc. The term "medicinal base addition salt" refers to those medicinal salts that form with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutical organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and salts of basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins). The term "pharmaceutical salt" includes, but is not limited to, the salts exemplified herein. The term "pharmaceutical salt" includes those salts in which the counterion has been exchanged by another suitable counterion, particularly by a suitable anion.

[0041] The term “one or more compounds of the present invention” refers to compounds of formula (I) and their stereoisomers, tautomers, solvates and salts (e.g., pharmaceutical salts).

[0042] Tautomers, i.e., structural isomers that are tautomers of compounds of formula (I), especially in solution, may exist in some cases and should be understood to be included in this invention.

[0043] If one of the starting materials or compounds of formula (I) contains one or more functional groups that are unstable or reactive under the reaction conditions of one or more reaction steps, then a suitable protecting group may be introduced prior to the key steps of the application of methods known in the art (e.g., in TW Greene and PGM Wuts, step 3). (As described in "Protective Groups in Organic Chemistry" by Wiley, New York, 1999). Such protecting groups can be removed using standard methods described in the literature at a later stage of synthesis. Examples of protecting groups are tert-butoxycarbonyl (Boc), 9-fluorenylmethylcarbamate (Fmoc), 2-trimethylsilylethylcarbamate (Teoc), benzyloxycarbonyl (Cbz), and p-methoxybenzyloxycarbonyl (Moz).

[0044] Compounds of formula (I) may contain several asymmetric centers and may exist in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, mixtures of diastereomers, diastereomeric racemates, or mixtures of diastereomeric racemates.

[0045] The term "asymmetric carbon atom" refers to a carbon atom with four different substituents. According to the Cahn-Ingold-Prelog sequence, an asymmetric carbon atom can be either "R" or "S" configuration.

[0046] Furthermore, the present invention includes all optical isomers of the compounds of formula (I) (where applicable), namely, diastereomers, diastereomer mixtures, racemic mixtures, all their respective enantiomers and / or tautomers, and their solvates.

[0047] If necessary, the racemic mixture of the compounds of the present invention can be separated to isolate the individual enantiomers. Separation can be carried out by methods well known in the art, such as coupling the racemic mixture of the compounds to an enantiomerically pure compound to form a diastereomer mixture, and then separating the individual diastereomers by standard methods such as fractional crystallization or chromatography.

[0048] In some embodiments where optically pure enantiomers are provided, an optically pure enantiomer means that the compound contains >90% by weight of the desired isomer, particularly >95% by weight, or more particularly >99% by weight of the desired isomer, said weight percentage based on the total weight of the compound's isomers. Chiral pure compounds or chiral-enriched compounds can be prepared by chiral selective synthesis or by enantiomer separation. Enantiomer separation can be performed on the final product, or alternatively on suitable intermediates.

[0049] The structures described herein are also intended to include compounds that differ only in the presence of one or more isotope-rich atoms. Specific examples of radioactive isotopes are... 2 H, 3 H, 13 C 14 C and 18 F. For example, one or more hydrogen atoms are replaced by deuterium or tritium, or one or more carbon atoms are enriched with... 13 C or 14 Structures with carbon substitution of C are within the scope of this invention.

[0050] The present invention therefore relates to the following numbered aspects:

[0051] 1. A compound of formula (I)

[0052] (I)

[0053] in

[0054] L is absent or is an alkyl group;

[0055] X is -O- or -NH-;

[0056] n is 0 or 1;

[0057] R1 is an amino group, an aminoalkyl group, an alkylamino group, an aminoalkylamino group, a haloalkyl group, a diaminoalkyl group, a guanidinyl group, a (aminoalkyl)(aminoalkyl)(hydroxycarbonylalkyl)ammoniumalkyl group, an aminoalkoxyalkyl group, a (aminoalkyl)(aminoalkyl)(aminocarbonylalkyl)ammoniumalkyl group, an (amino)(hydroxy)alkyl group, an (aminoalkyl)(aminoalkyl)amino group, an (aminoalkyl)(aminoalkyl)aminoalkyl group, an (aminoalkyl)(aminoalkyl)aminoalkoxy group, an (aminoalkyl)(aminoalkyl)aminoalkylamino group, an (aminoalkyl)(aminoalkyl)(hydroxycarbonyl)ammoniumalkylamino group, an (hydroxy)(halo)alkylamino group, an (aminoalkyl)(aminoalkyl)(hydroxycarbonylalkyl)ammoniumalkoxy group, a cycloalkyl group, or a heterocycloalkyl group, wherein the heterocycloalkyl group and the cycloalkyl group are optionally substituted by one or two independent substituents selected from R6;

[0058] The prerequisite is that if R1 is amino, then L does not exist;

[0059] R2 and R3 are independently selected from halogens; or R2 and R3 together with the carbon atoms to which they are attached form cycloalkyl groups;

[0060] R4 is a halogen or alkyl group;

[0061] R5 is an amino, hydroxyalkyl, aminoalkyl, alkylamino, aminoalkylamino, or piperazine group;

[0062] R6 is independently selected from amino, alkyl, hydroxy, aminoalkyl, aminocarbonylalkyl, and aminoalkylcarbonylamino in each case;

[0063] R7 is hydrogen or hydroxyl;

[0064] A1 is -N- or -CH-; and

[0065] A2 is either -O- or -CH2-;

[0066] Or its medicinal salt.

[0067] 2. The compound according to aspect 1, wherein L is absent.

[0068] 3. The compound according to aspect 1 or 2, wherein L is an alkyl group, particularly a methyl group.

[0069] 4. The compound according to any one of aspects 1 to 3, wherein n is 1.

[0070] 5. The compound according to any one of aspects 1 to 4, wherein X is -O-.

[0071] 6. The compound according to any one of aspects 1 to 5, wherein X is -NH-.

[0072] 7. The compound according to any one of aspects 1 to 3, wherein n is 0.

[0073] 8. The compound according to any one of aspects 1 to 7, wherein R1 is amino, aminomethyl, aminoethyl, methylamino, aminoethylamino, aminopropylamino, trifluoromethyl, diaminoethyl, guanidinyl, aminoethoxymethyl, (aminopropyl)(aminopropyl)(hydroxycarbonylmethyl)ammoniumpropyl, (aminopropyl)(aminopropyl)(aminocarbonylmethyl)ammoniumpropyl, (amino)(hydroxy)ethyl, (aminopropyl)(aminopropyl)amino, (aminoethyl)(aminoethyl)aminopropyl, (aminopropyl)(aminopropyl)aminopropyl, (aminopropyl)(aminopropyl)aminoethoxy, (aminoethyl)(aminoethyl)aminoethoxy, (aminoethyl)(aminoethyl)aminoethoxy, (aminoethyl)(aminopropyl)aminopropyl, (aminoethyl)(aminopropyl)aminoethoxy, (aminoethyl)(aminopropyl)aminopropyl, (aminoethyl)(aminopropyl)aminopropyl, (aminopropyl)(aminopropyl)aminoethoxy, (aminoethyl)(aminopropyl)aminopropyl)aminopropyl, (aminoethyl)(aminopropyl)aminopropyl)aminoethoxy, (aminoethyl)(aminopropyl)aminopropyl)aminopropyl)aminopropyl, (aminopropyl)(aminopropyl) ... Aminoethyl)aminoethylamino, (aminopropyl)(aminopropyl)(hydroxycarbonylmethyl)ammoniumpropylamino, (hydroxy)difluoropropylamino, (hydroxy)trifluoropropylamino, (aminopropyl)(aminopropyl)(hydroxycarbonylmethyl)ammoniumethoxy, cyclobutyl, azirmonobutane-3-yl, azirmonobutane-1-yl, morpholino-4-yl, pyrrolidine-1-yl or 5-aza-spiro[2.4]heptane, wherein cyclobutyl, azirmonobutane-3-yl, azirmonobutane-1-yl, morpholino-4-yl, pyrrolidine-1-yl or 5-aza-spiro[2.4]heptane is optionally substituted with one or two independent substituents selected from R6;

[0074] The prerequisite is that if R1 is amino, then L does not exist.

[0075] 9. The compound according to any one of aspects 1 to 7, wherein R1 is a heterocyclic alkyl group optionally substituted with one or two substituents independently selected from R6.

[0076] 10. The compound according to any one of aspects 1 to 9, wherein R2 and R3 are both fluorine; or R2 and R3 together with the carbon to which they are attached form a cyclopropyl group.

[0077] 11. The compound according to any one of aspects 1 to 10, wherein R4 is chlorine or methyl, particularly chlorine.

[0078] 12. The compound according to any one of aspects 1 to 10, wherein R4 is methyl.

[0079] 13. The compound according to any one of aspects 1 to 12, wherein R5 is an amino or hydroxyl group.

[0080] 14. The compound according to any one of aspects 1 to 12, wherein R5 is an amino group.

[0081] 15. The compound according to any one of aspects 1 to 14, wherein R6 is independently selected from amino, methyl, hydroxy, aminomethyl, aminocarbonylmethyl and aminoethylcarbonylamino in each case.

[0082] 16. The compound according to any one of aspects 1 to 15, wherein R7 is hydrogen.

[0083] 17. The compound according to any one of aspects 1 to 16, wherein A1 is –CH-.

[0084] 18. The compound according to any one of aspects 1 to 17, wherein A2 is -CH2-.

[0085] 19. A compound selected from...

[0086] cis-(4R)-4-amino-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutan-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one;

[0087] trans-(4R)-4-amino-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one;

[0088] 2-Amino-N-[1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]azacyclobutane-3-yl]propionamide;

[0089] (4R)-4-amino-1-[4-[4-[4-[[4-(3-aminopropylamino)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one;

[0090] trans-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide;

[0091] cis-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide;

[0092] cis-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide;

[0093] trans-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide;

[0094] trans-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]cyclobutanecarboxamide;

[0095] cis-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]cyclobutanecarboxamide;

[0096] cis-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidone-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-hydroxy-propionamide;

[0097] cis-1-(2-aminoethyl)-3-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidone-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]urea;

[0098] trans-(6S)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5,5-dimethyl-5-azapyronspiro[2,4]heptane-6-carboxamide;

[0099] trans-(5R,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide;

[0100] trans-(5S,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide;

[0101] Racemic-(4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one;

[0102] Racemic-(4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one;

[0103] 2-Amino-N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]acetamide;

[0104] 4-(((1r,4r)-4-((2-(4-((4-((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)amino)-N,N-bis(3-aminopropyl)-N-(carboxymethyl)-4-oxobut-1-ammonium;

[0105] N-((1r,4r)-4-((2-(4-((4-((R)-4-amino-2-oxopyrrolid-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-2-(2-aminoethoxy)acetamide;

[0106] 3-Amino-N-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-2-hydroxypropionamide;

[0107] 2,3-Diamino-N-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)propionamide;

[0108] 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(diaminomethylene)urea;

[0109] (2-Amino-2-oxo-ethyl)-[4-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]amino]-4-oxo-butyl]-bis(3-aminopropyl)ammonium;

[0110] 3-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamoylamino]propyl-bis(3-aminopropyl)-(carboxymethyl)ammonium;

[0111] 3-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-1,1-bis(3-aminopropyl)urea;

[0112] 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(3-aminopropyl)guanidine;

[0113] N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]-2,2,2-trifluoro-acetamide;

[0114] N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(2-aminoethyl)amino]butyramide;

[0115] N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(3-aminopropyl)amino]ethyl ester;

[0116] 2-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamoyloxy]ethyl-bis(3-aminopropyl)-(carboxymethyl)ammonium;

[0117] (2S,6S)-2,6-bis(aminomethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]morpholine-4-carboxamide;

[0118] N-[4-[1-[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]cyclopropyl]cyclohexyl]-4-[bis(2-aminoethyl)amino]butyramide;

[0119] N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]-N-hydroxy-butyramide;

[0120] N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]butyramide;

[0121] (4R)-4-amino-1-[4-[4-[6-chloro-4-[[4-[(2,2-difluoro-3-hydroxy-propyl)amino]cyclohexyl]-difluoro-methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one;

[0122] (3R,4S)-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-hydroxy-pyrrolidine-1-carboxamide;

[0123] 1-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-3-(2-(bis(2-aminoethyl)amino)ethyl)urea;

[0124] N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester;

[0125] (4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-methyl-pyrimidin-2-yl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one;

[0126] N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-methyl-pyrimidin-4-yl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]butyramide;

[0127] (4R)-4-amino-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)pyrrolid-2-one;

[0128] N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester;

[0129] (4R)-4-amino-1-[4-[4-[4-[[4-[[4-(azacyclobutan-1-yl)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one;

[0130] ((3R)-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidone-3-yl)tert-butyl carbamate;

[0131] Or its medicinal salt.

[0132] 20. The compound according to any one of aspects 1 to 19, which is used as a therapeutically active substance.

[0133] 21. A pharmaceutical composition comprising: a compound according to any one of aspects 1 to 19, and a therapeutically inert carrier.

[0134] 22. Use of the compound according to any one of aspects 1 to 19 for the treatment or prevention of bacterial infection, particularly wherein the bacteria are Gram-negative bacteria.

[0135] 23. Use of the compound according to any one of aspects 1 to 19 for the preparation of a medicament for the treatment or prevention of bacterial infection, particularly wherein the bacteria are Gram-negative bacteria.

[0136] 24. The compound according to any one of aspects 1 to 19, used for the treatment or prevention of bacterial infection, particularly wherein the bacteria are Gram-negative bacteria.

[0137] 25. A method for treating or preventing bacterial infections, particularly Gram-negative bacterial infections, the method comprising administering an effective amount of a compound as defined in any one of aspects 1 to 19.

[0138] 26. The use, method, or compound for use according to any one of aspects 22 to 25, wherein the Gram-negative bacteria are selected from Enterobacteriaceae, Neisseria gonorrhoeae, Haemophilus influenzae, Helicobacter pylori, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0139] 27. The use, method, or compound for use according to aspect 26, wherein the Gram-negative bacteria is Enterobacteriaceae, wherein Enterobacteriaceae is Klebsiella pneumoniae or Escherichia coli.

[0140] One embodiment of the present invention relates to a compound according to the invention, wherein the compound is a compound of formula (II).

[0141]

[0142] Or its medicinal salt.

[0143] The synthesis of compounds of formula (I) can be carried out, for example, according to the non-exhaustive procedures of the general schemes 1 to 12 described below, or according to methods known to those skilled in the art. In some cases, the order of reaction steps can be modified, and the steps of different schemes can be combined in different ways as disclosed herein. Unless otherwise indicated, all substituents are as defined in the specification and claims. Furthermore, and unless expressly specified otherwise, all reactions, reaction conditions, abbreviations, and symbols have the meanings commonly known to those skilled in the art.

[0144] Option 1

[0145]

[0146] In the above scheme, PG 1 and PG 2 The protecting group is an independently chosen group, such as benzyloxycarbonyl and tert-butoxycarbonyl groups.

[0147] Compound (IV) can be obtained from the sulfonation reaction between nitro-phenylsulfonyl chloride (II) and compound (III). The reduction of the nitro group in compound (IV) to aniline (V) can be carried out using platinum carbon in the presence of H2. Compound (V) is amidated with lactone (VI) in the presence of a base (such as LHMDS) to provide an amide (VII). The primary alcohol (VII) is cyclized via a Mitsunobu reaction to give a cyclic lactam (VIII). Compound (IX) can be obtained by deprotecting compound (VIII) with a suitable reducing agent (such as palladium on carbon) in the presence of H2.

[0148] Option 2

[0149]

[0150] In the above scheme, B 1 Halogen or -SMe; B 2 It is a halogen or H; R4 is a halogen; Al is -N- or -CH-; PG 3 and PG 4 The protecting group is an independently chosen group, such as, for example, tert-butoxycarbonyl and 2-(trimethylsilyl)ethoxycarbonyl.

[0151] Compound (X) is subjected to magnesium-halogen exchange or deprotection with a suitable Grignard reagent (such as a magnesium chloride-lithium chloride complex or lithium chloride; chloro-(2,2,6,6-tetramethyl-1-piperidinyl)magnesium), followed by the addition of compound (XI), to give compound (XII). Compound (XIII) can be obtained by oxidizing compound (XII) in the presence of a suitable oxidant (such as a Dys-Martin reagent). Compound (XIII) is fluorinated in the presence of a suitable reagent (such as DAST) to give compound (XIV). Compound (XV) can be obtained by deprotecting compound (XIV) with a suitable acid (such as trifluoroacetic acid). Compound (XVI) can be obtained by protecting amine (XV) with 1-[2-(trimethylsilyl)ethoxycarbonyloxy]pyrrolidine-2,5-dione (Teoc-OSu) using PG4.

[0152] Option 3

[0153]

[0154] In the above scheme, B 1 Halogen or -SMe; B 2 R4 is a halogen; A1 is -N- or -CH-; R is an alkyl group; PG 3 Protecting groups, such as tert-butoxycarbonyl groups.

[0155] Compounds of formula (XVII) can be obtained by alkylating compounds of formula (XIII) with alkyl halides (such as MeI) and using bases (such as NaH). Compounds of formula (XVIII) can be obtained by deprotecting compounds of formula (XVII) with a suitable acid (such as trifluoroacetic acid).

[0156] Option 4

[0157]

[0158] In the above scheme, B 1 Halogen or -SMe; B 2 R4 is a halogen or H; A1 is -N- or -CH-.

[0159] Compound (X) is subjected to magnesium-halogen exchange or deprotection with a suitable Grignard reagent (such as a magnesium chloride-lithium chloride complex or lithium chloride; chloro-(2,2,6,6-tetramethyl-1-piperidinyl)magnesium), followed by the addition of compound (XIX) to obtain compound (XX). Compound (XXI) can be obtained by oxidizing compound (XX) in the presence of a suitable oxidizing agent (such as a DAST reagent). Fluorination of compound (XXI) in the presence of a suitable reagent (such as DAST) yields compound (XXII). Compound (XXIII) can be obtained by reducing compound (XXII) with a suitable reducing agent (such as lithium aluminum hydride).

[0160] Option 5

[0161]

[0162] In the above scheme, B 1 Halogen or -SMe; B 3 R is -SO2Me; R4 is a halogen; R 4a Alkyl group; A1 is -N- or -CH-; PG 5 Protecting groups, such as benzyloxycarbonyl groups.

[0163] Compound (XXIV) can be obtained by Suzuki coupling between compound (XXII) and alkyl-boronic acid or ester using a Pd catalyst (such as Pd-XPhos-G4) and a base (such as K3PO4). Hydrolysis of compound (XXIV) under suitable conditions (such as LiBr and Et3N) yields compound (XXV). Curtius rearrangement of compound (XXV) in the presence of DPPA and Et3N, followed by isocyanate capture by benzyl alcohol, yields compound (XXVI). Compound (XXVII) can be obtained by oxidation of compound (XXVI) in the presence of a suitable oxidant (such as mCPBA).

[0164] Option 6

[0165]

[0166] In the above scheme, B 1 R4 is a halogen; A1 is -N- or -CH-; X is -O- or –NH-; Y is OH or a leaving group, such as halogens and -O-4-NO2Ph; PG 3and PG 6 Independently protected groups, such as benzyloxycarbonyl and tert-butoxycarbonyl groups.

[0167] Compound (XXIX) can be obtained by reacting compound (XIII) with a suitable reagent (such as methyltriphenylphosphine bromide) and a base (such as n-BuLi) via a Wittig reaction. Compound (XXX) can be obtained by cyclopropanating compound (XXIX) with a base (such as NaH) under suitable conditions in the presence of trimethylsulfoxonium iodide. Compound (XXXI) can be obtained by deprotecting compound (XXX) with a suitable acid (such as trifluoroacetic acid). Compound (XXXIII) can be obtained by amidating compound (XXXI) with compound (XXXII) in the presence of a base (such as DIEA) under suitable coupling conditions (such as HATU).

[0168] Option 7

[0169]

[0170] In the above scheme, B 1 It is a halogen; B 3 R4 is –SO2Me; R5 is a halogen or alkyl group; A1 is -N- or -CH-; R7 is H or –OH; R is an alkyl or hydroxyalkyl group, a haloalkyl group, a haloalkyl alcohol, an amine with a cycloalkyl group, or an amine with a cycloalkyl alcohol; PG 2 PG 3 PG 4 and PG 5 Independently protected groups, such as tert-butoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, and benzyloxycarbonyl groups.

[0171] In the presence of a suitable base (such as DIEA), nucleophilic substitution of compounds of formula (XIII, XVI, XXVII) with compounds of formula (IX) yields compounds of formula (XXXIV). Compounds of formula (XXXV) can be obtained by selectively deprotecting compounds of formula (XVI, XXVII) under suitable F anion conditions (such as tetraethylammonium fluoride (TEAF)) or reducing conditions (such as PdCl2, Et3SiH, TEA).

[0172] Compounds of formula (XXXVI) can be obtained by several methods different from those of compound (XXXV); (1) conversion to –NHOH (R7:OH) using the following three steps: cyanomethylation with cyanomethyl bromide, oxidation with mCPBA, followed by the addition of NH2OH; (2) dialkylation of -NH2 by XCH2CH(Z)CH2X to form a 4-membered ring; (3) reductive amination with various aldehydes in the presence of AcOH using a suitable reducing agent (such as NaBH(OAc)3); (4) ring-opening of epoxides in the presence of a base (such as DIEA). Deprotection of compounds of formula (XXXV) and (XXXVI) using a suitable acid (such as trifluoroacetic acid) yields compounds of formula (XXXVII) and (XXXVIII), respectively.

[0173] Option 8

[0174]

[0175] Among them B 1 It is a halide; R4 is a halogen or alkyl group; A1 is -N- or -CH-; R is an alkyl group; PG 2 and PG 3 Independently protected groups, such as tert-butoxycarbonyl groups.

[0176] Global deprotection of compound (XXXIV) can also be performed using a suitable acid (such as trifluoroacetic acid) to obtain compound (XXXVII). Alternatively, compound (XXXVI) can be obtained by substituting compound (XVIII) with compound (IX) in the presence of a suitable base (such as DIEA).

[0177] Option 9

[0178]

[0179] In the above scheme, R4 is a halogen or alkyl group; A1 is -N- or -CH-; X is -O-; R1 is a C or O-linked substituent (amide or carbamate); Y is OH or a leaving group, such as halogen and -O-4-NO2Ph; PG 2 and PG 6 Independently protected groups, such as tert-butoxycarbonyl, 2- and benzyloxycarbonyl groups. In some cases, PG is not present. 6 Protection.

[0180] Compound (XXXIX) can be obtained by two methods via coupling of compound (XXXV) and compound (XXXII): (1) amidation of compound (XXXV) and compound (XXXII) using suitable coupling conditions (such as HATU) in the presence of a base (such as DIEA). (2) Y = -O-4-NO2Ph: to make alcohol (HO-R1-PG) 6 It reacts with 4-nitrobenzene chloroformate to generate activated YCOO-R1-PG. 6 (XXXII) is reacted with an amine (XXXV) to give a carbamate (XXXIX). Deprotection / global deprotection of the compound of formula (XXXIX) can be carried out using a suitable acid (such as trifluoroacetic acid) to give a compound of formula (XXXX).

[0181] Option 10

[0182]

[0183] In the above scheme, R4 is a halogen or alkyl group; A1 is -N- or -CH-; X is -O- or -NH; Z is N-imidazole; PG 2 and PG 7 Independently protected groups, such as tert-butoxycarbonyl, 2- and benzyloxycarbonyl groups. In some cases, PG is not present. 7 Protection.

[0184] Compounds of formula (XXXXII) can be obtained by coupling compounds of formula (XXXV) and formula (XXXXI), respectively. N,N'-carbonyldiimidazole (CDI) or imidazol-1-yl(1,2,4-triazol-4-yl)methylimine is used as compound (XXXXI). Substitution of the amine (XXXXIII) with compound (XXXXII) in the presence of a base (such as K₂CO₃) or in the absence of a base yields compounds of formula (XXXXIV), respectively. Deprotection / global deprotection of compound (XXXXIV) can be performed using a suitable acid (such as trifluoroacetic acid) to obtain compound (XXXXV).

[0185] Option 11

[0186]

[0187] In the above scheme, B 1R4 is a halide or -SO2Me; A1 is -N- or -CH-; X is -O- or –NH; Y is OH or a leaving group, such as halogen and -O-4-NO2Ph; PG 2 and PG 6 The protecting group is independent, such as a tert-butoxycarbonyl group. In some cases, there is no protecting group PG. 6 .

[0188] Compound (XXXXVI) can be obtained by substituting compound (XXIII) with compound (IX) in the presence of a suitable base (such as DIEA).

[0189] The primary alcohol (XXXXVI) is converted to a leaving group such as –OTf by Tf₂O in the presence of a base (such as DIEA), and then substituted with NH₃ to obtain compound (XXXXVII). Compound (XXXXVIII) can be obtained by amidation of compound (XXXXVII) with compound (XXXII) in the presence of a base (such as DIEA) using suitable coupling conditions (such as HATU). Deprotection / global deprotection of compound (XXXXVIII) can be carried out using a suitable acid (such as trifluoroacetic acid) to obtain compound (XXXXIX).

[0190] Option 12

[0191]

[0192] In the above scheme, B 1 R4 is a halide or -SO2Me; A1 is -N- or -CH-; X is -O- or –NH; PG 2 and PG 6 The protecting group is independent, such as a tert-butoxycarbonyl group. In some cases, there is no protecting group PG. 6 .

[0193] Compound (XXXXX) can be obtained by substituting compound (XXXIII) with compound (IX) in the presence of a suitable base (such as DIEA).

[0194] Deprotection / global deprotection of the compound of formula (XXXXX) can be performed using a suitable acid (such as trifluoroacetic acid) to obtain the compound of formula (XXXXXI).

[0195] Therefore, the present invention also relates to a method for preparing the compound according to the present invention according to the above process.

[0196] Pharmaceutical Composition

[0197] Another embodiment of the invention provides a pharmaceutical composition or medicament comprising the compound of the invention and a therapeutically inert carrier, diluent, or excipient, and a method for preparing such compositions and medicaments using the compound of the invention. In one example, the compound of formula (I) can be formulated into a galenical form by mixing it with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the recipient at the dose and concentration used) at ambient temperature, at an appropriate pH, and with the desired purity. The pH of the formulation depends primarily on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, the compound of formula (I) is formulated in an acetate buffer at pH 5. In another embodiment, the compound of formula (I) is sterile. The compound can be stored, for example, as a solid or amorphous composition, as a lyophilized formulation, or as an aqueous solution.

[0198] The composition shall be prepared, measured, and administered in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery, the method of administration, the timing of administration, and other factors known to the practicing physician.

[0199] The compounds of the present invention may be administered by any suitable manner, including oral, topical (including sublingual and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, epidural, and intranasal administration, and intralesional administration if local treatment is required. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0200] The compounds of this invention can be administered in any convenient form, such as tablets, powders, capsules, solutions, dispersants, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional to pharmaceutical formulations, such as diluents, carriers, pH adjusters, sweeteners, fillers, and other active agents.

[0201] Conventional formulations are prepared by mixing the compounds of the present invention with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Ansel, Howard C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams and Wilkins, 2004; Gennaro, Alfonso R. et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams and Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, flow aids, processing aids, colorants, sweeteners, flavorings, diluents, and other known additives to provide an aesthetically pleasing presentation of the drug (i.e., the compound of the present invention or a pharmaceutical composition thereof) or to facilitate the preparation of the pharmaceutical product (i.e., the drug).

[0202] The invention will be more fully understood by referring to the following examples. However, they should not be construed as limiting the scope of the invention.

[0203] Example

[0204] abbreviation

[0205] ACN: Acetonitrile; BH3•Me2S: Borane dimethyl sulfide complex; Boc: tert-butoxycarbonyl; CaCl2: Calcium chloride; Cbz: benzyloxycarbonyl; CDI: carbonyl diimidazole; DCE: 1,2-dichloroethane; DCE: dichloroethylene; DCM: dichloromethane; DIEA: N,N-diisopropylethylamine; DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; DPPA: diphenyl azidophosphate (diphenylphosphine); EA: ethyl acetate; ESI: electrospray ionization; EtOAc: ethyl acetate; Et3SiH: triethylsilane; FA: formic acid; H2O: water; HATU: tetramethyluranium hexafluorophosphate; HCl: hydrochloric acid; HOsu: N-hydroxysuccinimide; HPLC: high performance liquid chromatography; K2CO3: carbonic acid Potassium; LCMS: Liquid Chromatography-Mass Spectrometry; LiHMDS: Lithium Bis(Trimethylsilyl)amino; MeOH: Methanol; m-CPBA: m-chloroperbenzoic acid; MS: Mass Spectrometry; MTBE: Methyl tert-butyl ether; NH4Cl: Ammonium chloride; n-Bu3P: Tributylphosphine; NaHCO3: Sodium bicarbonate; Na2SO4: Sodium sulfate; N2: Nitrogen; Na2SO3: Sodium sulfite; NH2OH•HCl: Hydroxyammonium chloride; NaH: Sodium hydride; NMP: N-Methyl-2-pyrrolidone; NMR: Nuclear Magnetic Resonance; NH3•H2O: Ammonium hydroxide; PdCl2: Palladium chloride; Pd / C: Palladium on carbon; Pd(OH)2 / C: Carbon-supported palladium hydroxide; PE: Petroleum ether; Rf: Retention factor; SFC: Supercritical Fluid Chromatography; THF: Tetrahydrofuran; T3P: 2,4,6-Tripropyl-1,3,5,2λ 5 ,4λ 5 ,6λ 5 Trioxane 2,4,6-trioxide; Teoc: 2-(trimethylsilyl)ethoxycarbonyl; TEA: triethylamine; TFA: trifluoroacetic acid; TLC: thin-layer chromatography.

[0206] Purify intermediates and final compounds using rapid chromatography using one of the following instruments: i) Biotage SP1 system and Quad 12 / 25 Cartridge module; ii) ISCO combi-flash chromatograph. Silica gel brand and pore size: i) KP-SIL 60 Å, particle size: 40-60 µm; ii) CAS Registry Number: 63231-67-4, particle size: 47-60 µm silica gel; iii) ZCX from Qingdao Ocean Chemical Co., Ltd., pore size: 200-300 or 300-400.

[0207] Intermediates and the final compound were purified by preparative HPLC on a reversed-phase column using an XBridge column. TM Prep-C18 (5 µm, OBDTM 30 × 100 mm) column, SunFire TM Prep-C18 (5 µm, OBD) TM 30 × 100 mm column, Phenomenex Synergi-C18 (10 µm, 25 × 150 mm) or Phenomenex Gemini-C18 (10 µm, 25 × 150 mm). Waters AutoP purification system (sample manager 2767, pump 2525, detectors: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium hydroxide in water; acetonitrile and 0.1% FA in water, or acetonitrile and 0.1% TFA in water). Alternatively, use the Gilson-281 purification system (pump 322, detector: UV 156, solvent system: acetonitrile and 0.05% ammonium hydroxide in water; acetonitrile and 0.225% FA in water; acetonitrile and 0.05% HCl in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water).

[0208] For SFC chiral separation, intermediates were separated using chiral columns (Daicel chiralpak IC, 5 µm, 30 × 250 mm), AS (10 µm, 30 × 250 mm), or AD (10 µm, 30 × 250 mm), employing a Mettler Toledo Multigram III SFC system, a Waters 80Q preparative SFC, or a Thar 80 preparative SFC. The solvent system was CO2 and IPA (0.5% TEA in IPA) or CO2 and MeOH (0.1% NH3∙H2O in MeOH), with a back pressure of 100 bar and a detection UV of 254 nm or 220 nm.

[0209] Using LC / MS (Waters TMThe LC / MS spectra of the compounds were obtained using Alliance 2795-Micromass ZQ, Shimadzu Alliance2020-Micromass ZQ, or Agilent Alliance 6110-Micromass ZQ. The LC / MS conditions were as follows (run time 3 or 1.5 mins):

[0210] Acidic conditions I: A: 0.1% TFA solution in H2O; B: 0.1% TFA solution in acetonitrile;

[0211] Acidic conditions II: A: 0.0375% TFA solution in H2O; B: 0.01875% TFA solution in acetonitrile;

[0212] Alkaline conditions I: A: 0.1% NH3·H2O solution in H2O; B: Acetonitrile;

[0213] Alkaline conditions II: A: 0.025% NH3·H2O solution in H2O; B: Acetonitrile;

[0214] Neutral conditions: A: H2O; B: Acetonitrile.

[0215] Mass spectrometry (MS): Typically only ions representing the parent mass are reported, and unless otherwise specified, the mass ions cited are positive mass ions (MH). + .

[0216] NMR spectra were obtained using a Bruker Avance 400 MHz.

[0217] Microwave-assisted reactions were performed in a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were carried out under an argon or nitrogen atmosphere. Unless otherwise specified, reagents were purchased as is from commercial suppliers without further purification.

[0218] Int-1-1: 2,6-Dichloro-4-[1,4-dioxaspiro[4,5]decane-8-yl(difluoro)methyl]pyridine

[0219]

[0220] Int-1-1

[0221] Step 1: Ethyl 1,4-dioxaspiro[4.5]decane-8-carboxylate

[0222]

[0223] Int-1-1a

[0224] TsOH (354 mg, 2.06 mmol) was added to a solution of ethyl 4-oxocyclohexanecarboxylate (7.0 g, 41.13 mmol) and ethylene glycol (3.4 mL, 61.7 mmol) in toluene (100 mL) under N2 atmosphere. The mixture was then... Refluxed at 120 °C for 18 hours. The reaction mixture was then cooled to room temperature and washed with 100 mL × 3 NaHCO3 aqueous solution. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to provide Int-1-1a as a pale yellow oil (8.8 g, 41.07 mmol, 99.9% yield). MS observations (ESI) + ): 215.2 [(M+H) + ].

[0225] Step 2: 1,4-Dioxaspiro[4.5]heptane-8-carboxylic acid

[0226]

[0227] Int-1-1b

[0228] Lithium hydroxide monohydrate (3.45 g, 82.14 mmol) was added to a solution of Int-1-1a (8.8 g, 41.1 mmol) in THF (70 mL) and water (10 mL). The mixture was stirred at 25 °C for 16 hours. After stirring, the mixture was concentrated under vacuum to provide Int-1-1b (7.65 g, 41.08 mmol, 100% yield) as a white solid, which was used directly in the next step. MS observations (ESI) + ): 187.1 [(M+H) + ].

[0229] Step 3: N-methoxy-N-methyl-1,4-dioxaspiro[4.5]decane-8-carboxamide

[0230]

[0231] Int-1-1c

[0232] HATU (23.0 g, 60.42 mmol) was added to a solution of Int-1-1b (7.5 g, 40.3 mmol) in DMF (100 mL). Then, O,N-dimethylhydroxylamine·HCl (5.89 g, 60.42 mmol) and TEA (16.8 mL, 120.83 mmol) were added to the reaction mixture. The mixture was stirred for 1 hour at room temperature. After completion, the resulting mixture was diluted with EA (150 mL) and washed with water (100 mL × 3). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give a crude product, which was purified by silica gel column chromatography (EA in PE = 0% to 10%) to provide Int-1-1c (8 g, 34.89 mmol, 86.63% yield) as a colorless oil. MS observations (ESI) + ): 230.1 [(M+H) + ].

[0233] Step 4: (2,6-Dichloro-4-pyridyl)-(1,4-dioxaspiro[4.5]decane-8-yl)methyl ketone

[0234]

[0235] Int-1-1d

[0236] A solution of 2,6-dichloropyridine (5.89 g, 39.78 mmol) in THF (20 mL) was added to a solution of n-BuLi (16.57 mL, 39.78 mmol) in THF (50 mL) at -78 °C under N2. After the addition, the mixture was stirred for 30 min, and a solution of Int-1-1c (7.6 g, 33.15 mmol) in THF (20 mL) was added. The mixture was then stirred at -78 °C for 5 h. After completion, the reaction mixture was quenched with an aqueous solution of NH4Cl (100 mL) and extracted with EA (150 mL × 2). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by silica gel column chromatography (EA in PE = 0% to 10%) to provide Int-1-1d as a pale yellow solid (6 g, 18.98 mmol, 57.25% yield). MS observations (ESI) + ): 316.0 [(M+H) + ].

[0237] Step 5: 2,6-Dichloro-4-[1,4-dioxaspiro[4,5]decane-8-yl(difluoro)methyl]pyridine

[0238]

[0239] Int-1-1

[0240] Add 20 mL of BAST to a solution of Int-1-1d (4.0 g, 12.65 mmol) in DCM (20 mL). Stir the mixture at 25 °C for 3 hours. After stirring, quench the mixture by slowly pouring it into cooled water (50 mL). Extract the mixture with DCM (50 mL × 3). Concentrate the combined organic layers under vacuum and purify the crude product by silica gel column chromatography (EA in PE = 0% to 10%) to provide Int-1-1 as a white solid (3.2 g, 9.46 mmol, 74.8% yield). MS observations (ESI) + ): 338.0 [(M+H) + ].

[0241] Int-2-1: 4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexanone

[0242]

[0243] Int-2-1

[0244] Compound Int-1-1 (500 mg, 1.48 mmol) was dissolved in MeOH (12 mL). 6 M HCl (250 µL, 1.48 mmol) was added to the suspension. The reaction was carried out at 90 °C. Stir overnight. After completion, cool the reaction to room temperature and dilute with EA (30 mL) and water (30 mL). Extract the aqueous layer with EA (20 mL × 2). Wash the combined organic layers with water (30 mL) and brine (30 mL), dry over anhydrous sodium sulfate, filter, and concentrate under vacuum to obtain a crude residue. Purify the crude residue by silica gel column chromatography (EA in PE = 0% to 50%) to provide Int-2-1 (300 mg, 68.99%) as a pale yellow solid. MS observations (ESI) + ) [(M+H) + ]:294.232.

[0245] Int-3-1: 4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexylamine

[0246]

[0247] Int-3-1

[0248] Ammonium acetate (4.56 g, 59.16 mmol) and sodium triacetoxyborohydride (2.51 g, 11.83 mmol) were added to a solution of Int-2 (1.74 g, 5.92 mmol) in MeOH (30 mL). The reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was poured into water (50 mL) at 0 °C. The mixture was extracted with EA (50 mL × 3). The combined organic layers were washed with brine (50 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give a crude residue, which was purified by silica gel column chromatography (MeOH in DCM = 0% to 30%) to provide Int-3-1 (1.6 g, 5.42 mmol, 91.63% yield) as a colorless oil. MS observations (ESI) + ): 295.0 [(M+H) + ].

[0249] Int-4-1: cis-4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexylamine

[0250]

[0251] Int-4-1

[0252] Similar to the preparation of compound Int-3-1, Int-4-1 was prepared by replacing Int-2-1 with cis-4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexanone. Int-4-1 (200 mg) was obtained as a white solid. MS observations (ESI) + ) [(M+H) + ]:296.6.

[0253] Int-1: N-[(3R)-5-oxo-1-(4-piperazin-1-ylsulfonylphenyl)pyrrolidine-3-yl]tert-butyl carbamate

[0254]

[0255] Int-1

[0256] Step 1: Benzyl 4-(4-nitrophenyl)sulfonylpiperazine-1-carboxylate

[0257]

[0258] Int-1a

[0259] 4-Nitrobenzenesulfonyl chloride (50.0 g, 225.6 mmol) was added to a solution of 1-Cbz-piperazine (49.7 g, 225.6 mmol) and TEA (94.3 mL, 676.8 mmol) in DCM (500 mL) at 0 °C under an ice-water bath. After stirring at 0 °C for 2 hours, H2O (500 mL) was added to the mixture, and it was extracted with DCM (500 mL × 3). The combined organic layers were washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was ground together with MTBE to give Int-1a (87.0 g, 214.6 mmol) as a white solid. MS observation (ESI) + ) [(M+H) + ]:428.1.

[0260] Step 2: Benzyl 4-(4-aminophenyl)sulfonylpiperazine-1-carboxylate

[0261]

[0262] Int-1b

[0263] Isopropanol (400 mL) and THF (400 mL) were added to a solid of compound Int-1a (87.0 g, 214.6 mmol) and Pt / C (4.0 g) at 25 °C under N2. The reaction mixture was purged three times with H2 and stirred at 45 °C under H2 (45 Psi) for 72 h. After cooling to room temperature, the mixture was degassed under vacuum and purged several times with N2. The reaction mixture was diluted with THF (1.0 L) and filtered through a diatomaceous earth mat, and washed with THF (1.0 L). The filtrate was concentrated under vacuum to give Int-1b (81.0 g, 215.8 mmol) as a white solid. MS observation (ESI+) [(M+H)+]: 376.1.

[0264] Step 3: 4-[4-[[(3R)-3-(tert-butoxycarbonylamino)-4-hydroxy-butyryl]amino]phenyl]sulfonylpiperazine-1-carboxylic acid benzyl ester

[0265]

[0266] Int-1c

[0267] A solution of LiHMDS in THF (1.0 M, 333 mL, 333 mmol) was added dropwise to a solution of compound Int-1b (50.0 g, 133.2 mmol) in THF (666 mL), and the mixture was stirred at 0 °C for 0.5 h. Next, N-[(3R)-5-oxotetrahydrofuran-3-yl]tert-butyl carbamate (40.2 g, 199.77 mmol) was added at 0 °C, and the mixture was stirred at 0 °C for 1 h. The reaction mixture was quenched by adding water (500 mL) and extracted with EtOAc (500 mL × 3). The combined organic layers were washed with saturated brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to remove the solvent. Residue was passed through a silica gel column (PE / EtOAc = 5 / 1 to 1 / 3; TLC: PE / EtOAc = 1 / 1, R...) f = 0.3) purification to give Int-1c as a white solid (55.0 g, 95.4 mmol, 71.6% yield). MS observation (ESI) + ) [(M+H) + ]:577.3.

[0268] Step 4: 4-[4-[(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazine-1-carboxylic acid benzyl ester

[0269]

[0270] Int-1d

[0271] N,N,N,N-Tetramethylazodicarbonamide (CAS: 10465-78-8, 22.4 g, 130.1 mmol) was added fractionally to a solution of Bu3P (32.5 mL, 130.1 mmol) and compound Int-1c (50.0 g, 86.7 mmol) in THF (500 mL) at 0 °C under N2 atmosphere. The mixture was then heated to 25 °C and stirred for 2 h. After adding an aqueous solution of HCl (1 M, 200 mL), the mixture was extracted with EtOAc (500 mL × 3). The combined organic layers were washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The residue was ground together with MTBE (500 mL) to give Int-1d as a white solid (45.0 g, 80.6 mmol, 92.9% yield). MS observations (ESI) + ) [(M+H) + ]:559.2.

[0272] Step 5: N-[(3R)-5-oxo-1-(4-piperazin-1-ylsulfonylphenyl)pyrrolidine-3-yl] tert-butyl carbamate

[0273]

[0274] Int-1

[0275] Wet Pd / C (5.0 g, containing 50% water) was added to a solution of compound Int-1d (45.0 g, 80.6 mmol) in isopropanol (250 mL) and THF (250 mL) at 25 °C under N2. The reaction mixture was degassed under vacuum and purged three times with H2. The suspension was then stirred at 45 °C for 12 h under H2 (45 Psi). After cooling the reaction mixture to room temperature and purging with N2, the mixture was diluted with THF (1.0 L), filtered through a diatomaceous earth mat, and washed with a solution of THF (2.0 L) and MeOH (2.0 L). The filtrate was concentrated under vacuum to give Int-1 (38.0 g, 89.5 mmol, 94.5% yield) as a brown solid. The crude product was used directly in the next step without further purification. MS observation (ESI+) ([M+H) + ): 425.2.

[0276] Int-2: N-[racemic-(3R)-1-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0277]

[0278] Int-2

[0279] Step 1: N-(4-formylcyclohexyl)carbamate tert-butyl ester

[0280]

[0281] Int-2a

[0282] A solution of DMSO (37.5 g, 479.7 mmol) in DCM (100 mL) was added dropwise to a solution of oxaloyl chloride (41.5 g, 327.1 mmol) in DCM (200 mL) under N2 at -78 °C, and the mixture was stirred under N2 at -78 °C for 1 h. Then, a solution of N-[4-(hydroxymethyl)cyclohexyl]carbamate tert-butyl ester (50.0 g, 218.0 mmol) in DCM (100 mL) was added at -78 °C and stirred for 1 h. After adding DIEA (112.9 mL, 872.1 mmol) to the mixture at -78 °C, the reaction was warmed to 20 °C over 1 h and stirred at 20 °C for another 1 h. The reaction mixture was quenched with water (300 mL) at room temperature and extracted with DCM (300 mL × 3). The combined organic layers were washed with aqueous HCl solution (1.0 M, 100 mL × 3) and saturated brine (200 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain crude Int-2a as a pale yellow solid (48.0 g, 211.2 mmol, 96.9% yield). The crude product was used directly in the next step without further purification.

[0283] Step 2: N-[4-[(2,6-dichloro-4-pyridyl)-hydroxy-methyl]cyclohexyl]tert-butyl carbamate

[0284]

[0285] Int-2b

[0286] A solution of 2,6-dichloro-4-iodopyridine (86.8 g, 316.8 mmol) in THF (200 mL) was added dropwise at -70°C to -40°C under N2. The mixture was stirred at 20°C for 2 hours. A solution of Int-2a (48.0 g, 211.2 mmol) in THF (200 mL) was added to the mixture at -70°C to -40°C, and the reaction mixture was warmed to 0°C and stirred at 0°C for 0.5 hours. The reaction mixture was poured into a saturated aqueous solution of NH4Cl (300 mL) and extracted with EtOAc (300 mL × 4). The combined organic layers were washed with saturated brine (300 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was ground twice with EtOAc (150 mL) to give compound Int-2b as a white solid (22.0 g, 58.6 mmol, 27.8% yield). MS observation (ESI+) ([{ 35 Cl}M-C4H8+H] + ): 319.0.

[0287] Step 3: N-[4-(2,6-dichloropyridine-4-carbonyl)cyclohexyl]tert-butyl carbamate

[0288]

[0289] Int-2c

[0290] DMP (49.7 g, 117.2 mmol) was added fractionally to a solution of compound Int-2b (22.0 g, 58.6 mmol) in THF (400 mL) at 0 °C. After the addition of DMP, the mixture was warmed to 20 °C and stirred for 4 hours. The reaction mixture was poured into a saturated aqueous solution of K₂CO₃ (600 mL) and stirred at 20 °C for 12 hours. The mixture was extracted with EtOAc (400 mL × 3). The combined organic layers were washed with saturated brine (150 mL × 2), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give Int-2c (17.0 g, 45.5 mmol, 77.7% yield) as a pale yellow solid. MS observation (ESI+) ([{ 35 Cl}M-C4H8+H]+ ): 316.9.

[0291] Step 4: N-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]tert-butyl carbamate

[0292]

[0293] Int-2d

[0294] DAST (34.0 mL) was added to a solution of compound Int-2c (17.0 g, 45.5 mmol) in DCM (60 mL) at 0 °C. The reaction mixture was stirred at 20 °C for 16 hours, then slowly poured into ice water (400 mL), and the mixture was extracted with DCM (200 mL × 3). The combined organic layers were washed with saturated aqueous NaHCO3 solution (200 mL), saturated brine (100 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was passed through a silica gel column (PE / EtOAc = 20 / 1 to 5 / 1; TLC: PE / EtOAc = 2 / 1, R... f = 0.5) purification to obtain Int-2d as a pale yellow solid (11.6 g, 29.4 mmol, 64.4% yield). MS observation (ESI+) ([{ 35 Cl}M-C4H9+H] + ): 339.1.

[0295] Step 5: 4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexylamine

[0296]

[0297] Int-2e

[0298] TFA (10.0 mL) was added to a solution of compound Int-2d (5.0 g, 12.7 mmol) in DCM (10 mL) at 25 °C. After stirring at 25 °C for 1 hour, the reaction mixture was concentrated under vacuum to give crude Int-2e (3.5 g, 11.9 mmol, 93.7% yield) as a dark green oil. The crude product was used directly in the next step without purification. MS observation (ESI+) ([{ 35 Cl}M+H] + ): 294.8.

[0299] Step 6: N-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]carbamate 2-(trimethyl-λ4-thio)ethyl ester

[0300]

[0301] Int-2f

[0302] Teoc-OSu (3.7 g, 14.2 mmol) was added to a solution of compounds Int-2e (3.5 g, 11.9 mmol) and DIEA (6.2 mL, 35.6 mmol) in DCM (50 mL) at 0 °C. After stirring at 25 °C for 2 hours, the reaction mixture was diluted with H₂O (100 mL), extracted with DCM (100 mL × 3) and washed with saturated brine (100 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was passed through a silica gel column (PE / EtOAc = 1 / 0 to 5 / 1; TLC: PE / EtOAc = 2 / 1, R f = 0.45) purification to give Int-2f (3.6 g, 8.1 mmol, 85.9% yield) as a yellow oil. MS observation (ESI+) ([{ 35 Cl}M-C2H4+H] + ): 411.2.

[0303] Step 7: N-[4-[[2-chloro-6-[4-[4-[racemic-(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-4-pyridyl]difluoro-methyl]cyclohexyl]carbamate-2-trimethylsilylethyl ester

[0304]

[0305] Int-2g

[0306] DIEA (4.7 mL, 26.8 mmol) was added in a single addition to a solution of compounds Int-2f (700.0 mg, 1.6 mmol) and Int-1 (743.9 mg, 1.8 mmol) in DMSO (4 mL). After stirring at 100 °C for 12 hours, the reaction mixture was cooled to 25 °C and poured into H2O (50 mL). The mixture was extracted with EtOAc (150 mL × 2) and washed with saturated brine (50 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give Int-2 g (1.5 g, 1.8 mmol, 97.9% yield) as a yellow oil. The crude product was used directly in the next step without further purification. MS observation (ESI+) ([{ 35 Cl}M+H] + ): 827.5, ([{ 35 Cl}M-C2H4+H] + ): 799.5, ([{ 35 Cl}M-Boc-C2H4+H] + ): 699.5.

[0307] Step 8: N-[racemic-(3R)-1-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0308]

[0309] Int-2

[0310] Tetraethylammonium fluoride (6.7 g, 36.3 mmol) was added in a single addition to a solution of compound Int-2 g (1.5 g, 1.8 mmol) in ACN (20 mL). After stirring at 50 °C for 2 hours, the mixture was cooled to room temperature and adjusted to pH 8 to 9 with aqueous NaHCO3 solution. The solution was extracted with EtOAc (100 mL × 2) and washed with saturated brine (50 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give Int-2 as a yellow solid (550.0 mg, 0.8 mmol, 44.4% yield). The crude product was used directly in the next step without further purification. MS observation (ESI+) ([{ 35 Cl}M+H] + ): 683.1

[0311] Int-3: N-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]tert-butyl carbamate

[0312]

[0313] Int-3

[0314] Similar to the preparation of compound Int-2d, Int-3 was prepared by replacing trans-N-[4-(hydroxymethyl)cyclohexyl]carbamate with cis-N-[4-(hydroxymethyl)cyclohexyl]carbamate in step 1. Int-3 (200 mg) was obtained as a white solid. MS observation (ESI+) ([{ 35 Cl}M-C4H9+H] + ): 339.1, ([{ 35 Cl}M-Boc+H] + ): 294.8.

[0315] Int-4: N-[racemic-(3R)-1-[4-[4-[4-[[4-(aminomethyl)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0316]

[0317] Int-4

[0318] Step 1: Methyl 4-(hydroxymethyl)cyclohexanecarboxylate

[0319]

[0320] Int-4a

[0321] BH3•Me2S (10 M, 54 mL, 540 mmol) was added dropwise to a solution of 4-(methoxycarbonyl)cyclohexane-1-carboxylic acid (50.0 g, 268.5 mmol) in THF (150 mL) at 0 to 10 °C under N2 for 1 hour. The mixture was stirred at 25 °C for 12 hours. After cooling the reaction mixture to 0 °C, MeOH (100 mL) was added dropwise to the reaction mixture at 0 °C to quench the reaction. The mixture was concentrated to dryness under vacuum at 40 °C. The residue was passed through a silica gel column (PE / EtOAc = 100 / 1 to 1 / 1; TLC: PE / EtOAc = 2 / 1, R f= 0.4) purification to obtain Int-4a as a colorless oil (28.0 g, 162.6 mmol, 60.6% yield). Int-4a was used directly in the next step without any analysis. The results were obtained by comparing with commercially available standards on TLC using R1. f We determine it by comparison. 1 ¹H NMR (400MHz, DMSO-d⁶) δ ppm 3.58 (s, 3H), 3.19 - 3.22 (d, J = 4 Hz, 2H), 2.22 - 2.23 (m, 1H), 1.77 - 1.92 (m, 2H), 1.73 - 1.75 (m, 2H), 1.27 -1.32 (m, 3H), 0.91- 0.96 (td, J = 9.6, 3.6 Hz, 2H). MS observation (ESI+) [(M+H)+]: 173.2.

[0322] Step 2: Methyl 4-formylcyclohexanecarboxylate

[0323]

[0324] Int-4b

[0325] DMSO (25.4 g, 325.2 mmol) was added to a solution of oxaloyl chloride (31.0 g, 243.9 mmol) in DCM (100 mL) at -60 °C, and the mixture was stirred at -60 °C for 0.5 h under N2. After adding a solution of compound Int-4a (28.0 g, 162.6 mmol) in DCM (100 mL) at -60 °C, the reaction mixture was stirred at -60 °C for 1 h. Then, DIEA (84.1 g, 650.3 mmol) was added at -60 °C, and the mixture was stirred from -60 °C to -20 °C for another 1 h. The mixture was then warmed to 0 °C and stirred again under N2 for 1 h. The resulting mixture was diluted with EtOAc (600 mL) and washed with CaCl2 (100 mL × 3) and saturated brine (100 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was passed through a silica gel column (PE / EtOAc = 30 / 1 to 1 / 1; TLC: PE / EtOAc = 3 / 1, R... f= 0.5) purification to obtain a pale yellow oil of Int-4b (24.0 g, 141.0 mmol, 86.7% yield). Int-4b was used directly in the next step without characterization analysis. The results were obtained by comparing with commercially available standards on TLC using Ri. f We determine it by comparison. 1 H NMR (400 MHz, DMSO-d6) δ ppm9.56 - 9.58 (s, 1 H), 2.24 - 2.29 (m, 2 H), 1.93 - 2.00 (m, 4 H), 1.26 - 1.34(m, 4 H)

[0326] Step 3: 4-[(2,6-dichloro-4-pyridyl)-hydroxy-methyl]cyclohexane carbamate

[0327]

[0328] Int-4c

[0329] The isopropyl magnesium chloride-lithium chloride complex (130.2 mL, 169.2 mmol) in THF was added dropwise to a solution of 2,6-dichloro-4-iodopyridine (42.5 g, 155.1 mmol) in THF (50 mL) at -40 °C under N2. The mixture was stirred at 25 °C for 1 hour. Then, the mixture was added dropwise to a solution of compound Int-4b (24.0 g, 141.0 mmol) in THF (100 mL) at -40 °C. The reaction mixture was stirred at 25 °C for 3 hours. The mixture was poured into an aqueous solution of NH4Cl (200 mL) and extracted with EtOAc (300 mL × 3). The combined organic layers were washed with saturated brine (100 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue passed through a silica gel column (PE / EtOAc = 100 / 1 to 1 / 2; TLC: PE / EtOAc = 1 / 1, R f = 0.4) purification to give compound Int-4c (33.0 g, 103.7 mmol, 73.6% yield) as a white solid. MS observation (ESI) + ) [({ 35 Cl}M+H) + ]:317.8.

[0330] Step 4: Methyl 4-(2,6-dichloropyridine-4-carbonyl)cyclohexanecarboxylate

[0331]

[0332] Int-4d

[0333] Under N2 at 0 °C, a solution of compound Int-4c (33.0 g, 103.7 mmol) in THF (150 mL) was slowly added to a solution of Des Martin periodane (48.4 g, 114.1 mmol). The mixture was then warmed to 25 °C and stirred at this temperature under N2 for 3 hours. The mixture was diluted with EtOAc (500 mL) and poured into an aqueous solution of K2CO3 (200 mL). The mixture was stirred at room temperature for 0.5 hours and filtered. The organic layer was separated and washed with an aqueous solution of K2CO3 (100 mL × 2) and saturated brine (100 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give crude compound Int-4d (30.0 g, 94.9 mmol, 91.5% yield) as a white solid. The crude product was used directly in the next step without further purification. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:315.8.

[0334] Step 5: Methyl 4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexanecarboxylate

[0335]

[0336] Int-4e

[0337] Diethylaminosulfur trichloride (135.4 g, 839.7 mmol) was added to a solution of compound Int-4d (29.5 g, 93.3 mmol) in DCM (20 mL), and the mixture was stirred at 45 °C for 12 h. After cooling to 25 °C, the mixture was poured into ice water at 0 °C and extracted with EtOAc (200 mL × 3). The combined organic layers were washed with aqueous K₂CO₃ solution (100 mL × 2) and saturated brine (100 mL × 2), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under vacuum. The residue was recrystallized from MeOH (150 mL) to give compound Int-4e (25.6 g, 75.7 mmol, 81.1% yield) as a white solid. MS observations (ESI) + ) [({35 Cl}M+H) + ]:337.8.

[0338] Step 6: [4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]methanol

[0339]

[0340] Int-4f

[0341] LiAlH4 (42.1 mg, 1.1 mmol) was added to a solution of compound Int-4e (250.0 mg, 0.7 mmol) in THF (5 mL) at -10 °C, and the mixture was stirred at -10 °C for 2 hours. After warming the mixture to 0 °C, the reaction mixture was diluted with THF (100 mL) and quenched with H2O (0.1 mL), followed by quenching with 15% NaOH aqueous solution (0.2 mL) and H2O (0.1 mL) sequentially. MgSO4 (10 g) was added to the suspension, and the mixture was stirred at 20 °C for 0.5 hours. The mixture was filtered through a diatomaceous earth mat, and the filtrate was concentrated under vacuum. The residue was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give Int-4f as a white solid (210.0 mg, 0.7 mmol, 91.6% yield). MS observation (ESI) + ) [({ 35 Cl}M+H) + ]:310.1

[0342] Step 7: N-[racemic-(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(hydroxymethyl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0343]

[0344] Int-4g

[0345] Int-1 (288.1 mg, 0.7 mmol) was added to a solution of compound Int-4f (210.0 mg, 0.7 mmol) in DMSO (1 mL) at 20 °C, and the mixture was stirred at 120 °C for 16 hours. After cooling to room temperature, the resulting mixture was transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to provide a crude product. This crude product was purified by preparative HPLC (column: C18 spherical, 20–35 µm, 100 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0%–100%, 15 min) and lyophilized to give Int-4 g (400.0 mg, 0.6 mmol, 77.4% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:698.2.

[0346] Step 7: N-[racemic-(3R)-1-[4-[4-[4-[[4-(aminomethyl)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0347]

[0348] Int-4

[0349] Trifluoromethanesulfonic anhydride (0.2 mL, 1.1 mmol) was added to a solution of compound Int-4 g (380.0 mg, 0.5 mmol) in DCM (5 mL) at -20 °C, and the mixture was stirred at -20 °C for 0.5 h. The mixture was then poured into a solution of ammonia in THF (20 mL) and stirred at 45 °C for 1 h. The mixture was concentrated to 10 mL and diluted by slowly adding saturated ammonium chloride aqueous solution (20 mL). The resulting mixture was transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to provide a crude product, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give Int-4 as a white solid (120.0 mg, 0.2 mmol, 31.6% yield). MS observation (ESI) + ) [({ 35 Cl}M+H) + ]:697.2.

[0350] Int-5: ((R)-1-(4-((4-(4-((((1r,4r)-4-aminocyclohexyl)difluoromethyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidine-3-yl)tert-butyl carbamate

[0351]

[0352] Int-5

[0353] Step 1: Lithium chloride; Chloro-(2,2,6,6-tetramethyl-1-piperidinyl)magnesium

[0354]

[0355] Int-5a

[0356] 2,2,6,6-Tetramethylpiperidine (12.1 mL, 71.5 mmol) was added dropwise to a solution of isopropyl magnesium chloride-lithium chloride complex (50.0 mL, 65.0 mmol) at -40 °C. The mixture was stirred at 25 °C under N2 for 24 hours to obtain a solution of Int-5a in THF (60 mL, approximately 1.1 M). This solution was used directly in the next step without further processing.

[0357] Step 2: Methyl 4-[(6-chloro-2-methylthio-pyrimidin-4-yl)-hydroxy-methyl]cyclohexanecarboxylate

[0358]

[0359] Int-5b

[0360] Int-5a (18.7 mL, 20.6 mmol) was added to a solution of 4-chloro-2-(methylthio)pyrimidine (3.0 g, 18.7 mmol) in THF (8 mL) at -40 °C. The mixture was stirred at 0 °C for 40 min. After adding the mixture to a solution of methyl 4-formylcyclohexanecarboxylate (3.5 g, 20.6 mmol) in THF (12 mL) at -40 °C, the mixture was warmed to 25 °C and stirred for 1 h. The mixture was poured into water (50 mL) and extracted with EtOAc (80 mL × 3). The organic layer was washed with saturated brine (50 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was passed through a silica gel column (PE / EtOAc = 100 / 1 to 1 / 1; TLC: PE / EtOAc = 1 / 1, R f = 0.4) purification to give Int-5b (1.8 g, 5.4 mmol, 29.1% yield) as a pale yellow oil. MS observations (ESI) + )[({ 35 Cl}M+H) + ]:331.3

[0361] Step 3: Methyl 4-(6-chloro-2-methylthio-pyrimidin-4-carbonyl)cyclohexanecarboxylate

[0362]

[0363] Int-5c

[0364] At 0 °C, a solution of compound Int-5b (1.7 g, 5.1 mmol) in THF (30 mL) was added to a solution of Des Martin periodane (2.6 g, 6.2 mmol), and the mixture was stirred at 25 °C for 2 h. The mixture was quenched with ice water (30 mL) and adjusted to pH = 9 with aqueous NaHCO3 solution. The mixture was extracted with EtOAc (100 mL × 2) and washed with saturated brine (50 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was passed through a silica gel column (PE / EtOAc = 1 / 0 to 1 / 1; TLC: PE / EtOAc = 1 / 1, R f =0.5) purification to give Int-5c (1.6 g, 4.9 mmol, 94.7% yield) as a yellow solid. (ESI) + )[({ 35 Cl}M+H) + ]:329.3.

[0365] Step 4: Methyl 4-[(6-chloro-2-methylthio-pyrimidin-4-yl)-difluoro-methyl]cyclohexanecarboxylate

[0366]

[0367] Int-5d

[0368] Diethylaminosulfur trifluoride (10.0 mL, 4.9 mmol) was added to a solution of Int-5c (1.6 g, 4.9 mmol) in DCM (10 mL) at 0 °C, and the mixture was heated to 25 °C and stirred for 2 hours. The mixture was carefully poured into ice water and the pH was adjusted to 9 with an aqueous NaHCO3 solution. The mixture was extracted with EtOAc (200 mL × 2) and washed with saturated brine (100 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was passed through a silica gel column (PE / EtOAc = 1 / 0 to 5 / 1; TLC: PE / EtOAc = 5 / 1, R f Purified with a concentration of 0.55 μL to obtain Int-5d as a yellow solid (1.5 g, 4.3 mmol, 87.9% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:351.3.

[0369] Step 5: Methyl 4-[difluoro-(6-methyl-2-methylthio-pyrimidin-4-yl)methyl]cyclohexanecarboxylate

[0370]

[0371] Int-5e

[0372] Trimethylboroxane (9.7 mL, 30.8 mmol) was added to a mixture of compound Int-5d (900.0 mg, 2.6 mmol), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (201.9 mg, 0.3 mmol), and potassium phosphate (1.6 g, 7.7 mmol) in 1,4-dioxane (5 mL) and water (2 mL). The reaction mixture was degassed under vacuum and purged several times with N2, and stirred at 80 °C for 12 hours under N2. After cooling to room temperature, the mixture was poured into H2O (20 mL), extracted with EtOAc (50 mL × 2), and washed with brine (50 mL × 2). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was passed through a silica gel column (PE / EtOAc = 30 / 1 to 10 / 1; TLC: PE / EtOAc = 5 / 1, R₂O₃). f = 0.65) purification to give Int-5e as a yellow oil (420.0 mg, 1.3 mmol, 34.7% yield). MS observation (ESI) + ) [(M+H) + ]:331.3.

[0373] Step 6: (1r,4r)-4-(difluoro(6-methyl-2-(methylthio)pyrimidin-4-yl)methyl)cyclohexane-1-carboxylic acid:

[0374]

[0375] Int-5f

[0376] LiBr (6.0 g, 70.0 mmol) and TEA (2.1 g, 21.0 mmol) were added to a mixture of Int-5e (2.3 g, 7.0 mmol) in MeCN (10 mL) and water (2 mL) at 25 °C. The reaction was stirred at 50 °C for 2 hours, and the mixture was quenched by slow addition of HCl solution (1.0 M, 20 mL). The resulting mixture was transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (100 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to provide crude Int-5f (2.0 g, 6.3 mmol, 90.3% yield) as a white solid, which was used in the next step without further purification. MS observations (ESI) + ) [(M+H) + ]:317.1.

[0377] Step 7: 4-(difluoro((1r,4r)-4-isocyanate-cyclohexyl)methyl)-6-methyl-2-(methylthio)pyrimidine

[0378]

[0379] Int-5g

[0380] DPPA (3.7 g, 13.3 mmol) and TEA (2.2 g, 22.1 mmol) were added to a solution of Int-5f (2.0 g, 6.3 mmol) in THF (10 mL) at 25 °C. The reaction was stirred at 25 °C for 2 hours, and the mixture was quenched by slow addition of a saturated aqueous solution of ammonium chloride (20 mL). The resulting mixture was transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (100 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide a crude product, which was passed through a silica gel column (PE / EtOAc = 1 / 0 to 2 / 1; TLC: PE / EtOAc = 2 / 1, R...). f = 0.5) purification to give Int-5 g (1.0 g, 3.2 mmol, 50.5% yield) as a white solid. MS observation (ESI) + ) [(M+H) + ]:314.0

[0381] Step 8: ((1r,4r)-4-(difluoro(6-methyl-2-(methylthio)pyrimidin-4-yl)methyl)cyclohexyl)benzyl carbamate

[0382]

[0383] Int-5h

[0384] Benzyl alcohol (20.0 mL) was added to Int-5 g (1.0 g, 3.2 mmol) at 25 °C, and the mixture was heated to 50 °C and stirred for 2 hours. After cooling to 25 °C, the resulting mixture was poured into water (50 mL) and transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (80 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to provide a crude product, which was then passed through a silica gel column (PE / EtOAc = 1 / 0 to 2 / 1; TLC: PE / EtOAc = 1 / 1, R f = 0.3) purification to give Int-5h as a white solid (1.1 g, 2.6 mmol, 81.8% yield). MS observation (ESI) + ) [(M+H) + ]:422.1.

[0385] Step 9: ((1r,4r)-4-(difluoro(6-methyl-2-(methanesulfonyl)pyrimidin-4-yl)methyl)cyclohexyl)tert-butyl carbamate

[0386]

[0387] Int-5i

[0388] Sodium tungstate dihydrate (172.2 mg, 0.5 mmol) and hydrogen peroxide (4.0 mL, 30% wt.) were added in a single addition to a solution of Int-5h (1.1 g, 2.6 mmol) in acetic acid (20 mL) at 0 °C under N2. The reaction was stirred at 25 °C for 16 hours, quenched with aqueous NaHCO3 solution (100 mL), aqueous Na2SO3 solution (100 mL), and then extracted with EtOAc (100 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous Na2SO4, and then filtered. The filtrate was concentrated under vacuum to provide a crude product, which was then passed through a silica gel column (PE / EtOAc = 1 / 0 to 1 / 1; TLC: PE / EtOAc = 1 / 1, R...). f = 0.5) purification to give Int-5i as a white solid (1.0 g, 2.2 mmol, 84.7% yield). MS observation (ESI) + ) [(M+H) + ]:454.1.

[0389] Step 10: ((R)-1-(4-((4-(4-(((1r,4r)-4-(((benzyloxy)carbonyl)amino)cyclohexyl)difluoromethyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidine-3-yl)tert-butyl carbamate

[0390]

[0391] Int-5j

[0392] Add DIEA (0.9 g, 6.6 mmol) and Int-1 (1.0 g, 2.4 mmol) to a solution of Int-5i (1.0 g, 2.2 mmol) in DMSO (4 mL). Stir the reaction at 100 °C for 16 hours, then cool to room temperature. Pour the resulting mixture into water (50 mL) and transfer to a separatory funnel. Extract the aqueous layer with EtOAc (80 mL × 3). Wash the combined organic layers with brine (50 mL × 3), dry to anhydrous Na₂SO₄, filter, and concentrate under reduced pressure. Pass the crude product through a silica gel column (PE / EtOAc = 1 / 0 to 0 / 1; TLC: PE / EtOAc = 1 / 1, R f= 0.4) purification to give Int-5j as a white solid (1.2 g, 1.5 mmol, 67.9% yield). MS observation (ESI) + ) [(M+H) + ]:798.6.

[0393] Step 11: ((R)-1-(4-((4-(4-((((1r,4r)-4-aminocyclohexyl)difluoromethyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidine-3-yl)tert-butyl carbamate

[0394]

[0395] Int-5

[0396] Add Et3SiH (1.9 mL, 12.0 mmol), PdCl2 (132.4 mg, 0.8 mmol), and TEA (0.6 mL, 4.5 mmol) to a solution of Int-5j (1.2 g, 1.5 mmol) in DCM (20 mL). Degas the mixture under vacuum and purge it three times with N2. Stir the reaction at 25 °C under N2 for 2 hours, then pour the resulting mixture into water (50 mL) and transfer it to a separatory funnel. Extract the aqueous layer with EtOAc (100 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to provide a crude product. This crude product was then passed through a silica gel column (EtOAc / MeOH = 10:0 to 5:1; TLC: EtOAc / MeOH = 5 / 1, R₂O₃). f = 0.5) purification to give Int-5 as a white solid (870.0 mg, 1.3 mmol, 87.2% yield). MS observation (ESI) + ) [(M+H) + ]:664.3.

[0397] Int-6: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(hydroxyamino)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0398]

[0399] Int-6

[0400] Step 1: N-[3-[[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]amino]propyl]benzyl carbamate

[0401]

[0402] Int-6a

[0403] Bromoacetonitrile (105.3 mg, 0.9 mmol) and K₂CO₃ (181.8 mg, 1.3 mmol) were added to a solution of Int-2 (0.3 g, 0.4 mmol) in DMF (2 mL). The reaction was stirred at 20 °C for 16 hours, then diluted with EtOAc (50 mL) and filtered. The filtrate was concentrated under vacuum. The residue was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give Int-6a (150.0 mg, 0.2 mmol, 47.3% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:722.3.

[0404] Step 2: N-[4-[[2-[4-[4-[(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-1-cyanomethylamine oxide

[0405]

[0406] Int-6b

[0407] At 0 °C, m-CPBA (105.1 mg, 0.6 mmol) was added to a solution of compound Int-6a (200.0 mg, 0.3 mmol) in DCM (3 mL). After stirring at 20 °C for 2 hours, the reaction mixture was quenched with saturated aqueous Na₂SO₃ solution (10 mL) and extracted with DCM (70 mL × 3). The combined organic layers were successively washed with NaHCO₃ (50 mL), saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The residue was purified by preparative HPLC (column: C18 spherical, 20 to 35 µm, 100 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give Int-6b as a white solid (100.0 mg, 0.1 mmol, 49.1% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:736.3.

[0408] Step 3: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(hydroxyamino)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0409]

[0410] Int-6

[0411] NH₂OH•HCl (34.0 mg, 0.5 mmol) was added to a solution of compound Int-6b (60.0 mg, 0.1 mmol) in methanol (1.0 mL) and DMF (1 mL) at 20 °C, and the mixture was stirred at 60 °C for 2 h. The solvent was concentrated under vacuum and purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give Int-6 as a white solid (50.0 mg, 0.1 mmol, 87.8% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:699.3.

[0412] Int-7: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(methylamino)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0413]

[0414] Int-7

[0415] Step 1: N-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]-N-methyl-carbamate tert-butyl ester

[0416]

[0417] Int-7a

[0418] NaH (89.1 mg, 2.2 mmol) was added to a solution of Int-2d (0.8 g, 2.0 mmol) and iodomethane (316.0 mg, 2.2 mmol) in DMF (2 mL) at 0 °C, and the mixture was stirred at 0 °C for 1 h. After quenching the reaction by adding ice water (10 mL), the mixture was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na2SO4, and then filtered. The filtrate was concentrated under vacuum to provide a crude product, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give Int-7a as a yellow oil (541.0 mg, 1.3 mmol, 65.3% yield). MS observation (ESI) + ) [({ 35 Cl}M+H) + ]:353.2.

[0419] Step 2: 4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]-N-methyl-cyclohexylamine

[0420]

[0421] Int-7b

[0422] TFA (1.0 mL, 13.0 mmol) was added to a solution of Int-7a (500.0 mg, 1.2 mmol) in DCM (1 mL) at 25 °C, and the mixture was stirred at this temperature for 1 hour. The mixture was concentrated under vacuum, and the residue was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give int.7b (320.0 mg, 1.0 mmol, 84.7% yield) as a yellow oil. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:309.0.

[0423] Step 3: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(methylamino)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0424]

[0425] Int-7

[0426] DIEA (190.0 mg, 1.5 mmol) was added to a solution of Int-7b (150.0 mg, 0.49 mmol) and Int-1 (206.0 mg, 0.5 mmol) in DMSO (1 mL), and the mixture was stirred at 100 °C for 16 h. After cooling to 25 °C, the mixture was concentrated under vacuum to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give Int-7 (240.0 mg, 0.3 mmol, 71.0% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:697.4.

[0427] Example 001: cis-(4R)-4-amino-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one (001A) and trans-(4R)-4-amino-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one (001B)

[0428]

[0429] 001A

[0430]

[0431] 001B

[0432] Step 1: N-[(3R)-1-[4-[4-[6-chloro-4-[1,4-dioxaspiro[4.5]decane-8-yl(difluoro)methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0433]

[0434] 001a

[0435] Compound Int-1-1 (200 mg, 591 µmol) and (R)-(5-oxo-1-(4-(piperazin-1-ylsulfonyl)phenyl)pyrrolidine-3-yl)carbamate tertiary ester (213 mg, 502 µmol) were dissolved in DMF (6 mL), followed by the addition of DIEA (263 µl, 1.51 mmol). The reaction mixture was stirred overnight at 120 °C. After cooling to room temperature, the mixture was diluted with EA (30 mL) and quenched with water (30 mL). The aqueous layer was extracted with EA (20 mL × 2). The combined organic layers were washed with water (30 mL) and brine (30 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to obtain a crude residue. This crude residue was purified by silica gel column chromatography (EA in PE = 0% to 70%) to provide 001b (0.29 g, 399 µmol, 79.6% yield). MS observations (ESI) + ): 726.201 [(M+H) + ].

[0436] Step 7: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-(4-oxocyclohexyl]methyl)-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0437]

[0438] 001b

[0439] Compound 001a (227 mg, 313 µmol) was dissolved in 36% HCl (5 mL, 25 mmol) and MeOH (5 mL). The mixture was stirred at 50 °C for 2 hours. LC-MS showed the major product formation, with de-Boc products also observed. The mixture was then adjusted to pH 8 with aqueous NaHCO3 solution and extracted with DCM (30 mL × 3). The combined organic layers were concentrated under vacuum to give a crude product (180 mg, 201 µmol, 64.3% yield) as a pale yellow solid. MS observations (ESI) + ): 582.375 [(M+H) + The resulting compound was dissolved in DCM (2.5 mL). Boc₂O (93.7 mg, 430 µmol) and Et₃N (71.8 µl, 515 µmol) were added to the stirred solution. The reaction was stirred overnight at room temperature, and LC-MS showed complete disappearance of the starting material. After completion, the reaction was extracted with DCM (30 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give crude 001b (100 mg, 147 µmol, 85.3% yield) as a yellow oil. MS observations (ESI) + ): 682.152 [(M+H) + ].

[0440] Step 8: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0441]

[0442] 001c

[0443] Compound 001b (100 mg, 172 µmol) and aziridine-3-ol hydrochloride (94.1 mg, 859 µmol) were dissolved in THF (15 mL), followed by the addition of one drop of titanium isopropoxide (IV). The reaction mixture was stirred at room temperature for 1 hour, and then sodium triacetoxyborohydride (182 mg, 859 µmol) and 5 mL of MeOH were added. The mixture was then heated at 70 °C. o Stir for 2 hours. After completion, pour the mixture into water (50 mL) and extract with EA (50 mL × 3). Wash the combined organic layers with brine (50 mL × 2), dry with anhydrous Na2SO4, filter, and concentrate under vacuum to give crude 001c (127 mg, 172 µmol, 100% yield), which was used directly in the next step. MS observation (ESI) + ): 739.3 [(M+H) + ].

[0444] Step 9: cis-(4R)-4-amino-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one (001A) and trans-(4R)-4-amino-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one (001B)

[0445]

[0446] 001A

[0447]

[0448] 001B

[0449] Compound 001c (127 mg, 172 µmol) and TFA (23.1 mg, 203 µmol) were mixed in DCM (5 mL). The reaction was stirred at room temperature for 1 hour. After completion, the reaction was concentrated under vacuum to obtain a crude compound, which was purified by preparative HPLC to give a mixture of 001A and 001B. The two epimers were separated by SFC to give Example 001A (first eluent, 93 mg, 64% yield) and Example 001B (second eluent, 14 mg, 20% yield). The cis and trans conformations were confirmed by 2D NMR.

[0450] Example 001A: MS observations (ESI) + ) [(M+H) + ]:639.3. 1 H NMR (400 MHz, DMSO-d6) δppm 7.88 - 7.98 (m, 2 H), 7.76 - 7.85 (m, 2 H), 6.71 - 6.81 (m, 1 H), 6.64 -6.69 (m, 1 H), 4.36 - 4.51 (m, 1 H), 4.18 - 4.36 (m, 3 H), 4.04 - 4.15 (m, 1H), 3.74 - 3.95 (m, 3 H), 3.59 - 3.73 (m, 4 H), 3.02 - 3.15 (m, 1 H), 2.88 -3.02 (m, 4 H), 2.54 - 2.66 (m, 1 H), 2.17 - 2.34 (m, 1H), 1.67 - 1.82 (m, 2H), 1.52 - 1.67 (m, 2H), 1.32 - 1.52 (m, 4H). 19 F NMR (376 MHz, DMSO-d6) δppm -105.95 - -101.82 (m, 2 F).

[0451] Example 001B: MS observations (ESI) + ) [(M+H) + ]:683.2. 1H NMR (400 MHz, DMSO-d6) δppm 7.87 - 8.00 (m, 2 H), 7.73 - 7.83 (m, 2 H), 6.76 - 6.83 (m, 1 H), 6.69 -6.75 (m, 1 H), 6.14 - 6.32 (m, 1 H), 4.37 - 4.50 (m, 1 H), 4.18 - 4.34 (m, 3H), 4.00 - 4.18 (m, 1 H), 3.74 - 3.89 (m, 3 H), 3.61 - 3.72 (m, 4 H), 3.02 -3.13 (m, 2 H), 2.90 - 3.02 (m, 4 H), 2.53 - 2.65 (m, 1 H), 2.09 - 2.30 (m, 1H), 1.87 - 2.04 (m, 2 H), 1.59 - 1.81 (m, 2 H), 0.98 - 1.20 (m, 4 H). 19 F NMR (376 MHz, DMSO-d6) δ ppm -108.32 - -100.42 (m, 2 F).

[0452] Example 002: 2-Amino-N-[1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]azacyclobutane-3-yl]propionamide

[0453] 002

[0455] Step 1: N-[1-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]azacyclobutane-3-yl]tert-butyl carbamate

[0456]

[0457] 002a

[0458] Compound Int-2-1 (300 mg, 1.02 mmol), N-(azacyclobutane-3-yl)carbamate tert-butyl ester (263.49 mg, 1.53 mmol), and sodium triacetoxyborohydride (648.53 mg, 3.06 mmol) were added to a mixture of DCM (15 mL) and MeOH (5 mL). The reaction was stirred at room temperature for 1 day. LC-MS showed complete consumption of the starting material. The mixture was then poured into water (50 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with brine (50 mL × 2), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give crude 002a (400 mg, 87.08%) as a yellow solid. MS observations (ESI) + ) [(M+H) + ]:450.22.

[0459] Step 3: 1-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]azacyclobutane-3-amine

[0460]

[0461] 002b

[0462] Compound 002a (300 mg, 0.666 mmol) and TFA (1.5 mL, 20.22 mmol) were dissolved in DCM (15 mL). The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction was concentrated under vacuum. The resulting residue 002b was azeotropically reacted with toluene and used directly in the next step. MS observations (ESI) + ) [(M+H) + ]:350.8.

[0463] Step 4: N-[2-[[1-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]azacyclobutane-3-yl]amino]-1-methyl-2-oxo-ethyl]tert-butyl carbamate

[0464]

[0465] 002c

[0466] Compound 002b (130 mg, 0.280 mmol) and 2-(tert-butoxycarbonylamino)propionic acid (66.23 mg, 0.350 mmol) were dissolved in DCM (15 mL). HATU (159 mg, 0.420 mmol) and DIEA (146.71 μL, 0.840 mmol) were added to the stirred solution. The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction was diluted with DCM (15 mL) and water (15 mL). The aqueous layer was extracted with DCM (50 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give crude 002c (120 mg, 82.19% yield) as a yellow solid, which was used directly in the next step.

[0467] Step 5: N-[2-[[1-[4-[[2-[4-[4-[(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]azacyclobutane-3-yl]amino]-1-methyl-2-oxo-ethyl]tert-butyl carbamate

[0468]

[0469] 002d

[0470] Compound 002c (120 mg, 0.230 mmol) was dissolved in DMF (10 mL), followed by the addition of N-[(3R)-5-one-1-(4-piperazinylsulfonylphenyl)pyrrolidine-3-yl]carbamate tert-butyl ester (97.7 mg, 0.230 mmol) and DIEA (120 µL, 0.690 mmol). The mixture was heated at 125 °C. o C. Stir overnight. After complete conversion of the starting material, dilute the reaction mixture with water (50 mL) and extract with EA (50 mL × 2). Wash the combined organic layers with water (50 mL) and brine (50 mL), dry to dryness with anhydrous Na2SO4, and concentrate to dryness to give crude 002d (150 mg, 71.67% yield) as a yellow oil, which was used directly in the next step. MS observation (ESI) + ) [(M+H) + ]:909.6.

[0471] Step 6: 2-Amino-N-[1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]difluoro-methyl]cyclohexyl]azacyclobutane-3-yl]propionamide (002)

[0472] 002

[0474] Compound 002d (100 mg, 0.110 mmol) was dissolved in DCM (5 mL), followed by the addition of TFA (2 mL, 25.96 mmol). The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction was concentrated under vacuum, and the resulting residue was purified by preparative HPLC to give Example 002 (7.3 mg, 7.26% yield) as a pale yellow solid. MS observations (ESI) + ) [(M+H) + ]:709.3. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.79- 7.89 (m, 2 H), 7.68 - 7.78 (m, 2 H), 7.39 - 7.49 (m, 1 H), 6.50 - 6.64 (m,1 H), 4.35 - 4.46 (m, 1 H), 4.20 - 4.32 (m, 1 H), 3.99 - 4.11 (m, 1 H), 3.86 - 3.97 (m, 1 H), 3.72 - 3.86 (m, 2 H), 3.56 - 3.63 (m, 4 H), 3.32 - 3.42 (m, 1 H), 3.01 - 3.11 (m, 1 H), 2.88 - 3.01 (m, 4 H), 2.47 - 2.62 (m, 1 H), 2.00- 2.13 (m, 1 H), 1.86 - 1.96 (m, 1 H), 1.72 - 1.82 (m, 1 H), 1.59 - 1.72 (m, 2 H), 1.33 - 1.50 (m, 8H), 1.13 - 1.33 (m, 3H). 19 F NMR (376 MHz, METHANOL-d4) δ ppm -113.96 - -100.71 (m, 2 F).

[0475] Example 003: (4R)-4-amino-1-[4-[4-[4-[[4-(3-aminopropylamino)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0476] 003

[0478] Step 1: N-[3-[[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]amino]propyl]benzyl carbamate

[0479]

[0480] 003a

[0481] Add (3-oxopropyl)carbamate (330 mg, 1.59 mmol) and sodium triacetoxyborate (674 mg, 3.18 mmol) to a solution of Int-3-1 (313 mg, 1.06 mmol) in DCM (5 mL). Stir the reaction mixture overnight at 25 °C. After completion, pour the reaction mixture into water and extract with DCM (50 mL × 3). Concentrate the combined organic layers under vacuum to give crude 003a (500 mg, 96% yield) as a yellow oil, which was used directly in the next step. MS observations (ESI) + ) [(M+H) + ]:486.1.

[0482] Step 2: N-[3-(benzyloxycarbonylamino)propyl]-N-[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]tert-butyl carbamate

[0483]

[0484] 003b

[0485] Compound 003a (86 mg, 177 µmol) was dissolved in DCM (10 mL). Boc₂O (117 mg, 124 µl, 535 µmol) and triethylamine (112 µl, 802 µmol) were added to the stirred solution. The mixture was stirred overnight at room temperature. After extraction, the reaction mixture was poured into water and extracted with DCM (50 mL × 3). The combined organic layers were concentrated under vacuum to give crude 003b, which was used directly in the next step. MS observations (ESI) + ) [(M+H)+ ]:586.1.

[0486] Step 3: N-[(3R)-1-[4-[4-[4-[[4-[3-(benzyloxycarbonylamino)propylamino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0487]

[0488] 003c

[0489] Compound N-[(3R)-5-one-1-(4-piperazinylsulfonylphenyl)pyrrolidine-3-yl]carbamate tert-butyl ester (100 mg, 0.236 mmol) and 003b (150 mg, 0.256 mmol) were dissolved in DMF (5 mL). DIEA (134 μL, 0.767 mmol) was added to the solution. The reaction was carried out at 120 °C. Stir overnight. After completion, cool the reaction to room temperature and dilute with EA (100 mL). Extract the aqueous layer with EA (150 mL × 3). Wash the combined organic layers with water (100 mL) and brine (200 mL), dry with anhydrous sodium sulfate and concentrate under vacuum to give crude 003c (200 mg, 80.24%) as a light brown oil, which is used directly in the next step.

[0490] Step 4: (4R)-4-amino-1-[4-[4-[4-[[4-(3-aminopropylamino)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0491] 003

[0493] Compound 003c (100 mg, 0.103 mmol) was mixed with TFA (2 mL). The mixture was heated at 90°C. o C. Heat for 2 hours. After completion, concentrate the reaction mixture under reduced pressure to obtain a crude residue, which is purified by preparative HPLC to give Example 003 (2.5 mg, 3.43% yield) as a white solid. MS observation (ESI) + ) [(M+H) + ]:640.4. 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.89 - 7.99 (m, 2H), 7.79 - 7.89 (m, 2 H), 7.48 - 7.62 (m, 1 H), 6.60 - 6.75 (m, 1 H), 5.29 -5.39 (m, 1 H), 4.34 - 4.45 (m, 1 H), 4.12 - 4.25 (m, 1 H), 3.88 - 3.99 (m, 1H), 3.62 - 3.75 (m, 4 H), 3.18 - 3.24 (m, 1 H), 2.99 - 3.18 (m, 8 H), 2.14 -2.30 (m, 3 H), 1.99 - 2.14 (m, 3 H), 1.76 - 1.99 (m, 3 H), 1.54 - 1.76 (m, 2H), 1.37 - 1.52 (m, 2 H). 19 F NMR (376 MHz, METHANOL-d4) δ ppm -112.27 - -105.22 (m, 2 F).

[0494] Example 004: trans-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide (004A) and cis-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide (004B)

[0495]

[0496] 004A

[0497]

[0498] 004B

[0499] Step 1: N-[3-[[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]amino]-3-oxo-propyl]tert-butyl carbamate

[0500]

[0501] 004a

[0502] Add 3-((tert-butoxycarbonyl)amino)propionic acid (0.128 g, 678 µmol) to a solution of Int-3-1 (0.1 g, 339 µmol) in DCM (1.5 mL), followed by the addition of HATU (0.193 g, 509 µmol) and DIEA (177 µL, 1.02 mmol). Heat the reaction mixture at 100 °C overnight. After completion, dilute the reaction mixture with EA and wash with water (50 mL). Dry the organic layer with anhydrous sodium sulfate and concentrate under vacuum to give crude 004a (150 mg, 94.94% yield), which was used directly in the next step. MS observations (ESI) + ) [(M+H) + ]:366.8.

[0503] Step 2: N-[3-[[4-[[2-[4-[4-[(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]amino]-3-oxo-propyl]tert-butyl carbamate

[0504]

[0505] 004b

[0506] Compound 004a (150 mg, 322 µmol) and (R)-(5-oxo-1-(4-(piperazin-1-ylsulfonyl)phenyl)pyrrolidine-3-yl)carbamate tert-butyl ester (100 mg, 236 µmol) were mixed in DMSO (5 mL). DIEA (123 µl, 707 µmol) was added to the stirred suspension. The reaction mixture was stirred overnight at 120 °C. After the reaction was complete, it was cooled to room temperature and diluted with EA (50 mL). The organic layer was collected, washed with water (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give crude 004b (150 mg, 176 µmol, 74.5% yield) as a light brown oil. MS observations (ESI) + ) [(M+H-BOC) + ]:854.3.

[0507] Step 3: trans-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide (004A) and cis-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide (004B)

[0508]

[0509] 004A

[0510]

[0511] 004B

[0512] Compound 004b (100 mg, 117 µmol) was dissolved in DCM (5 mL), followed by the addition of TFA (2 mL, 58.5 µmol). The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction was concentrated under vacuum to obtain a crude residue, which was purified by preparative HPLC and then chirally separated by SFC to give a single isomer: trans-example 004A as a white solid (first eluent, 28.5 mg, 74.45% yield) and cis-example 004B as a white solid (second eluent, 28 mg, 73.14% yield). Configuration was determined by 2D NMR.

[0513] Example 004A: MS Observations (ESI) + ) [(M+H) + ]:654.118. 11H NMR (400 MHz, DMSO-d6) δ ppm 7.97 - 8.08 (m, 1 H), 7.88 - 7.96 (m, 2 H), 7.77 - 7.86 (m, 2 H), 6.76 - 6.82 (m, 1 H), 6.66 - 6.74 (m, 1 H), 4.21 - 4.36 (m, 1 H), 4.00 - 4.16 (m, 1 H), 3.77 - 3.90 (m, 1 H), 3.58 - 3.69 (m, 4 H), 3.03 - 3.14 (m, 1 H), 2.89 - 3.03 (m, 6 H), 2.34 - 2.44 (m, 2 H), 2.08 - 2.24 (m, 1 H), 1.75 - 1.87 (m, 2 H), 1.58 - 1.68 (m, 2 H), 1.02 - 1.34 (m, 6 H). 19 19F NMR (376 MHz, DMSO-d6) δ ppm -107.75 - -100.42 (m, 2 F).

[0514] Example 004B: MS Observed (ESI + ) [(M + H) + : 654.118. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 7.99 - 8.07 (m, 1 H), 7.87 - 7.98 (m, 2 H), 7.75 - 7.84 (m, 2 H), 6.72 - 6.89 (m, 1 H), 6.60 - 6.75 (m, 1 H), 4.20 - 4.32 (m, 1 H), 4.03 - 4.16 (m, 1 H), 3.74 - 3.91 (m, 2 H), 3.60 - 3.74 (m, 4 H), 3.02 - 3.13 (m, 1 H), 2.85 - 3.02 (m, 4 H), 2.55 - 2.71 (m, 3 H), 2.08 - 2.28 (m, 1 H), 1.61 - 1.80 (m, 2 H), 1.35 - 1.56 (m, 5 H), 0.96 - 1.35 (m, 3 H). 19 19F NMR (376 MHz, DMSO-d6) δ ppm -111.70 - -95.35 (m, 2 F).

[0515] Example 005: cis-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide (005A) and trans-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]propionamide (005B)

[0516]

[0517] 005A

[0518]

[0519] 005B

[0520] Similar to the preparation of compounds 004A and 004B, compounds 005A and 005B were prepared by replacing 3-((tert-butoxycarbonyl)amino)propionic acid with (tert-butoxycarbonyl)alanine in step 1. Examples 005A (4.3 mg) and 005B (5.7 mg) were obtained as white solids. The configuration was determined by 2D NMR.

[0521] Example 005A: MS Observations (ESI) + ) [(M+H) + ]:654.3. 11H NMR (400 MHz, METHANOL-d4) δ ppm 7.77 - 7.91 (m, 2 H), 7.65 - 7.75 (m, 2 H), 6.51 - 6.63 (m, 2 H), 4.40 - 4.59 (m, 2 H), 3.91 - 4.11 (m, 2 H), 3.77 - 3.90 (m, 1 H), 3.64 - 3.72 (m, 1 H), 3.55 - 3.64 (m, 4 H), 3.46 - 3.55 (m, 1 H), 3.27 - 3.41 (m, 1 H), 2.91 - 3.03 (m, 4 H), 2.74 - 2.87 (m, 1 H), 2.04 - 2.36 (m, 2 H), 1.85 - 1.99 (m, 3 H), 1.64 - 1.79 (m, 2 H), 1.30 - 1.58 (m, 4 H). 19 19F NMR (376 MHz, METHANOL-d4) δ ppm -109.16 - -99.58 (m, 2 F).

[0522] Example 005B: MS Observed (ESI + ) [(M+H) + : 654.3. 1 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.76 - 7.89 (m, 2 H), 7.64 - 7.75 (m, 2 H), 6.46 - 6.66 (m, 2 H), 4.40 - 4.61 (m, 1 H), 3.99 - 4.09 (m, 1 H), 3.62 - 3.70 (m, 1 H), 3.54 - 3.61 (m, 4 H), 3.34 - 3.56 (m, 3 H), 2.92 - 3.02 (m, 4 H), 2.72 - 2.86 (m, 1 H), 2.24 - 2.38 (m,​F NMR(METHANOL-d4, 376 MHz) δ -106.71 (br d, 2 F, J=13.6 Hz)

[0523] Example 006: trans-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]cyclobutane formamide

[0524] 006

[0526] Similar to the preparation of compound 004A, compound 006 was prepared in step 1 by replacing 3-((tert-butoxycarbonyl)amino)propionic acid with trans-(tert-butoxycarbonyl)amino)propionic acid, without chiral separation. Example 006 (76 mg) was obtained as a white solid. MS observations (ESI) + ) [(M+H) + ]:680.2. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.80 - 7.88 (m, 2 H), 7.68 - 7.80 (m, 2 H), 6.52 - 6.64(m, 2 H), 4.23 - 4.36 (m, 1 H), 4.02 - 4.15 (m, 1 H), 3.78 - 3.96 (m, 2 H), 3.51 - 3.67 (m, 4 H), 3.36 - 3.51 (m, 1 H), 3.01 - 3.14 (m, 2 H), 2.89 - 3.01 (m, 4 H), 2.50 - 2.66 (m, 1 H), 2.34 - 2.50 (m, 2 H), 2.16 - 2.34 (m, 2 H), 1.60 - 2.05 (m, 4 H), 1.31 - 1.54 (m, 3 H), 1.00 - 1.31 (m, 4 H). 19 F NMR (376MHz, METHANOL-d4) δ ppm -113.39 - -101.55 (m, 2 F).

[0527] Example 007: cis-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]cyclobutane formamide

[0528] 007

[0530] Similar to the preparation of compound 004A, compound 007 was prepared in step 1 by replacing 3-((tert-butoxycarbonyl)amino)propionic acid with cis-(tert-butoxycarbonyl)amino)carboxylic acid, without chiral separation. Example 007 (47.5 mg) was obtained as a white solid. MS observations (ESI) + ) [(M+H) + ]:680.2. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.89 - 8.00 (m, 2 H), 7.78 - 7.89 (m, 2 H), 6.66 - 6.73(m, 2 H), 4.36 - 4.47 (m, 1 H), 4.14 - 4.29 (m, 1 H), 3.89 - 4.03 (m, 1 H), 3.65 - 3.79 (m, 5 H), 3.47 - 3.65 (m, 1 H), 3.15 - 3.27 (m, 1 H), 3.00 - 3.15 (m, 5 H), 2.85 - 3.00 (m, 1 H), 2.65 - 2.77 (m, 1 H), 2.50 - 2.65 (m, 2 H), 2.22 - 2.36 (m, 2 H), 1.71 - 2.22 (m, 4 H), 1.43 - 1.63 (m, 3 H), 1.07 - 1.43 (m, 4 H). 19 F NMR (376 MHz, METHANOL-d4) δ ppm -115.37 - -97.60 (m, 2 F).

[0531] Example 008: cis-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-hydroxy-propionamide

[0532] 008

[0534] Similar to the preparation of compound 004A, compound 008 was prepared by replacing Int-3-1 with Int-4-1 in step 1 and by replacing 3-((tert-butoxycarbonyl)amino)propionic acid with 3-[tert-butyl(dimethyl)silyl]oxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propionic acid. Example 008 (3.15 mg) was obtained as a white solid. MS observations (ESI) + ) [(M+H) + ]:670.3. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.79 - 7.89 (m,2 H), 7.69 - 7.79 (m, 2 H), 6.54 - 6.63 (m, 2 H), 5.22 - 5.28 (m, 1 H), 4.25- 4.36 (m, 1 H), 4.01 - 4.14 (m, 1 H), 3.73 - 3.94 (m, 4 H), 3.64 - 3.73 (m,1 H), 3.52 - 3.64 (m, 4 H), 3.42 - 3.52 (m, 1 H), 3.04 - 3.14 (m, 2 H), 2.92- 3.04 (m, 4 H), 2.49 - 2.64 (m, 1 H), 2.03 - 2.18 (m, 2 H), 1.86 - 2.03 (m, 2 H), 1.69 - 1.86 (m, 2 H), 1.33 - 1.62 (m, 2 H). 19 F NMR (376 MHz, METHANOL-d4) δ ppm -109.45 - -104.93 (m, 1 F).

[0535] Example 009: cis-1-(2-aminoethyl)-3-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]urea

[0536] 009

[0538] Step 1: cis-N-[2-[[4-[(2,6-dichloro-4-pyridyl)-difluoro-methyl]cyclohexyl]carbamoylamino]ethyl]tert-butyl carbamate

[0539]

[0540] 009a

[0541] A solution of N-(2-aminoethyl)carbamate tert-butyl ester (96.54 µL, 0.610 mmol) and Et3N (51.42 mg, 70.83 µL, 0.508 mmol) in acetonitrile (5 mL) was added dropwise to a solution of triphosgene (60.32 mg, 0.203 mmol) in acetonitrile (5 mL) cooled in an ice / water bath. Then, a solution of Int-4-1 (60 mg, 0.203 mmol) in acetonitrile (5 mL) was added to the stirred mixture. The reaction mixture was slowly warmed to room temperature and stirred overnight. Afterward, the reaction mixture was concentrated under vacuum to give crude product 009a (80 mg, 81.76% yield) as a yellow solid, which was used directly in the next step. MS observations (ESI) + ) [(M+H) + ]:481.227.

[0542] Step 2: cis-N-[(3R)-1-[4-[4-[4-[[4-[2-(tert-butoxycarbonylamino)ethylcarbamoylamino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0543]

[0544] 009b

[0545] Compound 009a (83.72 mg, 0.174 mmol) was dissolved in DMF (10 mL), and N-[(3R)-5-oxo-1-(4-piperazin-1-ylsulfonylphenyl)pyrrolidine-3-yl]carbamate tert-butyl ester (73.83 mg, 0.174 mmol) and DIEA (30 µL, 0.174 mmol) were added at room temperature. The mixture was then heated at 125 °C. o C. Stir overnight. After complete conversion of the starting material, dilute the reaction mixture with water (50 mL) and extract with EA (50 mL × 2). Wash the combined organic layers with water (50 mL) and brine (50 mL), dry with anhydrous Na2SO4, and concentrate under vacuum to give crude 009b (100 mg, 66.13%) as a pale yellow solid. MS observation (ESI)+ ) [(M+H) + ]:869.3.

[0546] Step 3: cis-1-(2-aminoethyl)-3-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]urea

[0547] 009

[0549] Compound 009b (100 mg, 0.115 mmol) was dissolved in DCM (10 mL). TFA (1 mL) was added to the stirred solution. The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction was concentrated under vacuum to obtain a crude residue, which was purified by preparative HPLC to obtain Example 009 (2.5 mg, 2.64%) as a pale yellow solid. MS observations (ESI) + ) [(M+H) + ]:669.245. 1 H NMR (400 MHz, METHANOL-d4)δ ppm 7.80 - 7.87 (m, 2 H), 7.67 - 7.78 (m, 2 H), 6.48 - 6.63 (m, 2 H), 4.23- 4.33 (m, 1 H), 4.00 - 4.14 (m, 1 H), 3.77 - 3.89 (m, 1 H), 3.69 - 3.77 (m,1 H), 3.52 - 3.66 (m, 4 H), 3.25 - 3.29 (m, 1 H), 3.02 - 3.13 (m, 2 H), 2.94- 3.02 (m, 4 H), 2.82 - 2.94 (m, 2 H), 2.45 - 2.68 (m, 1 H), 2.07 - 2.20 (m, 1 H), 1.86 - 2.07 (m, 3 H), 1.63 - 1.80 (m, 2 H), 1.40 - 1.55 (m, 5 H), 1.27- 1.40 (m, 2 H). 19 F NMR (376 MHz, METHANOL-d4) δ ppm -115.65 - -100.71 (m, 2F).

[0550] Example 010: trans-(6S)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5,5-dimethyl-5-azapyronspiro[2,4]heptane-6-carboxamide

[0551] 010

[0553] Step 1: (6S)-5,5-dimethyl-5-azapyronspiro[2,4]heptane-6-carboxylic acid

[0554]

[0555] 010a

[0556] (6S)-5-azaspiro[2.4]heptane-6-carboxylic acid (290 mg, 2.05 mmol) was dissolved in DMF (10 mL), and iodomethane (1.28 mL, 20.54 mmol) and DIEA (3.6 mL, 20.54 mmol) were added. The reaction mixture was stirred overnight at room temperature. After completion, the reaction mixture was concentrated to dryness to give crude 010a (300 mg, 85.79%) as a pale yellow oil, which was used directly in the next step.

[0557] Step 2: N-[(3R)-1-[4-[4-[6-chloro-4-[[4-[[(6S)-5,5-dimethyl-5-azapyronspiro[2,4]heptane-6-carbonyl]amino]cyclohexyl]-difluoro-methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0558]

[0559] 010b

[0560] Compound Int-5-1 (150 mg, 0.220 mmol) was dissolved in DMF (10 mL), and then compound 010a (100 mg, 0.587 mmol), HATU (208.7 mg, 0.549 mmol), and DIEA (85.12 mg, 115.03 µL, 0.659 mmol) were added at room temperature. The mixture was stirred overnight at room temperature. After completion, the reaction mixture was diluted with saturated NH4Cl aqueous solution (50 mL) and extracted with EA (50 mL × 2). The combined organic layers were washed with water (50 mL × 3) and brine (50 mL), dried over anhydrous Na2SO4, and concentrated to dryness to provide crude 010b (180 mg, 98.14% yield) as a pale yellow oil, which was used directly in the next step. MS observations (ESI) + )[(M+H) + ]:834.6.

[0561] Step 3: trans-(6S)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5,5-dimethyl-5-azapyronspiro[2,4]heptane-6-carboxamide

[0562] 010

[0564] Compound 010b (150 mg, 0.180 mmol) was dissolved in DCM (5 mL) at room temperature, followed by the addition of TFA (1.5 mL, 19.47 mmol). The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction mixture was concentrated to dryness to provide a crude product, which was purified by preparative HPLC to yield Example 010 (26 mg, 18.71% yield) as a white solid. MS observations (ESI) + ) [(M+H) + ]:734.4. 1H NMR (500 MHz, DMSO-d6) δ ppm 8.58 - 8.71 (m, 1 H), 8.27 - 8.41 (m, 2 H), 7.87 - 7.98 (m, 2H), 7.73 - 7.84 (m, 2 H), 6.76 - 6.85 (m, 1 H), 6.66 - 6.76 (m, 1 H), 4.35 -4.43 (m, 1 H), 4.21 - 4.33 (m, 1 H), 4.04 - 4.13 (m, 1 H), 3.78 - 3.90 (m, 2H), 3.63 - 3.70 (m, 4 H), 3.20 - 3.29 (m, 3 H), 3.10 - 3.20 (m, 4 H), 3.02 -3.10 (m, 1 H), 2.92 - 3.02 (m, 4 H), 2.55 - 2.64 (m, 2 H), 2.25 - 2.45 (m, 2H), 2.07 - 2.25 (m, 1 H), 1.76 - 1.98 (m, 2 H), 1.57 - 1.76 (m, 2 H), 1.04 -1.34 (m, 4 H), 0.58 - 0.89 (m, 4 H). 19 F NMR (471 MHz, DMSO-d6) δ ppm -126.70 --92.41 (m, 2 F).

[0565] Example 011: trans-(5R,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide (011A) And trans-(5S,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide (011B)

[0566]

[0567] 011A

[0568]

[0569] 011B

[0570] Step 1: (6S)-5-(2-amino-2-oxo-ethyl)-5-azaspiro[2,4]heptane-6-carboxylic acid

[0571]

[0572] 011a

[0573] Add 3-bromopropionamide (161.5 mg, 1.06 mmol) and K₂CO₃ (293.71 mg, 2.13 mmol) to a solution of (6S)-5-azaspiro[2.4]heptane-6-carboxylic acid (100 mg, 0.708 mmol) in acetonitrile (5 mL). Stir the reaction mixture at room temperature for 3 hours. After this, dilute the reaction mixture with water (50 mL). Extract the aqueous layer with EA (50 mL × 2). Wash the combined organic layers with water (50 mL) and brine (50 mL), dry to dryness with anhydrous Na₂SO₄, and concentrate to dryness to provide crude O11a (120 mg, 85.46% yield) as a pale yellow solid, which is used directly in the next step.

[0574] Step 2: N-[(3R)-1-[4-[4-[4-[[4-[[(6S)-5-(2-amino-2-oxo-ethyl)-5-azaspiro[2.4]heptane-6-carbonyl]amino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0575]

[0576] 011b

[0577] Add 011a (116.44 mg, 0.587 mmol), HATU (208.7 mg, 0.549 mmol), and DIEA (85.12 mg, 115.03 µL, 0.659 mmol) to a solution of compound Int-5 (150 mg, 0.220 mmol) in DMF (10 mL). Stir the reaction mixture overnight at room temperature. After completion, dilute the reaction mixture with saturated NH4Cl solution (100 mL). Extract the aqueous layer with EA (100 mL × 2). Wash the organic layer with water (50 mL) and brine (50 mL), dry to dryness with anhydrous Na2SO4, and concentrate to dryness. Purify the crude substance by silica gel chromatography (0–10% MeOH / DCM) to provide 011b (150 mg, 79.13% yield) as a pale yellow solid. MS observations (ESI) + ) [(M+H) + ]:863.6.

[0578] Step 3: N-[(3R)-1-[4-[4-[4-[[4-[[(6S)-5-(2-amino-2-oxo-ethyl)-5-methyl-5-azapyron[2.4]heptane-6-carbonyl]amino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0579]

[0580] 011c

[0581] Iodimethane (21.73 µL, 0.347 mmol) and DIEA (151.71 µL, 0.869 mmol) were added to a solution of compound 011b (150 mg, 0.174 mmol) in DMF (5 mL). The reaction mixture was stirred overnight at room temperature. After completion, the reaction mixture was concentrated to dryness to give crude 011c (150 mg, 98.29% yield) as a dark brown oil, which was used directly in the next step. MS observations (ESI) + ) [(M+H) + ]:877.664.

[0582] Step 4: trans-(5R,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide (011A) And trans-(5S,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide (011B)

[0583]

[0584] 011A

[0585]

[0586] 011B

[0587] TFA (1.5 mL, 19.47 mmol) was added to a solution of 011c (150 mg, 0.171 mmol) in DCM (5 mL). The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction mixture was concentrated to dryness. The crude material was purified by preparative HPLC to give 011A (first eluent, 8.1 mg, 5.49%) and 011B (second eluent, 10.1 mg, 6.84%) as white solids.

[0588] Example 011A: MS observations (ESI) + ) [(M+H) + ]:777.4. 11H NMR (500 MHz, DMSO-d6) δ ppm 7.92 (d, J = 8.70 Hz, 2 H), 7.80 (d, J = 8.70 Hz, 2 H), 6.79 (s, 1 H), 6.73 (s, 1 H), 4.57 - 4.75 (m, 1 H), 4.18 - 4.37 (m, 3 H), 4.07 (br d, J = 11.14 Hz, 2 H), 3.75 - 3.89 (m, 2 H), 3.66 (br s, 4 H), 3.17 (s, 2 H), 3.08 (br dd, J = 17.85, 8.24 Hz, 2 H), 2.98 (br s, 4 H), 2.54 - 2.62 (m, 2 H), 2.40 - 2.49 (m, 1 H), 2.15 (br dd, J = 13.73, 4.43 Hz, 2 H), 1.87 (br d, J = 10.68 Hz, 1 H), 1.74 - 1.83 (m, 1 H), 1.60 - 1.73 (m, 2 H), 1.10 - 1.26 (m, 4 H), 0.72 - 0.91 (m, 4 H). 19 19F NMR (471 MHz, DMSO-d6) δ ppm -114.06 - -97.82 (m, 2 F).

[0589] Example 011B: MS Observed (ESI + ) [(M + H) + : 777.5. 1H NMR (500 MHz, DMSO-d6) δppm 7.86 - 7.96 (m, 2 H), 7.77 - 7.84 (m, 2 H), 6.77 - 6.86 (m, 1 H), 6.65 -6.75 (m, 1 H), 4.57 - 4.66 (m, 1 H), 4.34 - 4.43 (m, 1 H), 4.22 - 4.32 (m, 1H), 4.11 - 4.22 (m, 1 H), 3.99 - 4.12 (m, 1 H), 3.71 - 3.87 (m, 2 H), 3.61 -3.71 (m, 4 H), 3.14 - 3.21 (m, 2 H), 3.02 - 3.14 (m, 2 H), 2.86 - 3.02 (m, 4H), 2.56 - 2.66 (m, 3 H), 2.10 - 2.33 (m, 1 H), 1.77 - 1.91 (m, 3 H), 1.58 -1.73 (m, 3 H), 1.11 - 1.33 (m, 4 H), 0.81 - 0.92 (m, 2 H), 0.60 - 0.80 (m, 4H). 19 F NMR (471 MHz, DMSO-d6) δ ppm -114.06 - -91.20 (m, 2 F).

[0590] Example 12: (R)-4-amino-1-(4-((4-(6-chloro-4-(difluoro((1r,4r)-4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl)methyl)pyridin-2-yl)piperazin-1-yl)sulfonyl)phenyl)pyrrolidone-2-one

[0591] 12

[0593] Step 1: ((R)-1-(4-((4-(6-chloro-4-(difluoro((1r,4r)-4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl)methyl)pyridin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidine-3-yl)tert-butyl carbamate

[0594]

[0595] 12a

[0596] At 25 °C, 1,3-dibromo-2-propanol (32.0 mg, 0.1 mmol) and DIEA (54.0 mg, 0.4 mmol) were added to a solution of compound Int-2 (100.0 mg, 0.1 mmol) in NMP (3 mL). The reaction was stirred at 50 °C for 2 hours. After cooling to room temperature, the resulting mixture was poured into water (50 mL) and transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to provide a crude product. This crude product was purified by preparative HPLC (column: C18 spherical, 20–35 µm, 100 g; mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0%–100%, 15 min) and lyophilized to give 12a as a white solid (74.0 mg, 0.1 mmol, 70.0% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:740.1

[0597] Step 2: (R)-4-amino-1-(4-((4-(6-chloro-4-(difluoro((1r,4r)-4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl)methyl)pyridin-2-yl)piperazin-1-yl)sulfonyl)phenyl)pyrrolidone-2-one

[0598] 12

[0600] TFA (1.0 mL, 12.98 mmol) was added to a solution of compound 12a (40.0 mg, 0.1 mmol) in DCM (1 mL) at 25 °C, and the mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 19% to 49%, 10 min) and lyophilized to give Example 12 (21.2 mg, 0.1 mmol, 54.5% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:640.3.

[0601] Example 41: (4R)-4-amino-1-[4-[4-[4-[[4-(azacyclobutan-1-yl)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one

[0602] 41

[0604] Similar to the preparation of compound Example 12, Example 41 was prepared by replacing 1,3-dibromo-2-propanol with 1,3-dibromo-2-propanol in step 1. Example 41 was obtained as a white solid in the form of a TFA salt (43.3 mg, 0.1 mmol, 49.0% yield). MS observation (ESI+) [({ 35 Cl}M+H) + ]:623.1.

[0605] Example 13: cis-racemic-(4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0606] 13

[0608] Step 1: N-[4-[[2-chloro-6-[4-[4-[racemic-(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-4-pyridyl]difluoro-methyl]cyclohexyl]tert-butyl carbamate

[0609]

[0610] 13a

[0611] DIEA (0.5 mL, 0.51 mmol) was added to a solution of compounds Int-3 (214.8 mg, 0.5 mmol) and Int-1 (200.0 mg, 0.5 mmol) in DMSO (2 mL) at 25 °C, and the mixture was heated to 110 °C with stirring for 12 hours. After cooling to room temperature, EtOAc (100 mL) and HCl (30 mL) were added to the mixture. The resulting mixture was transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (50 mL × 2). The combined organic layers were washed with saturated brine (80 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was analyzed by preparative TLC (PE / EtOAc = 1 / 1; TLC: PE / EtOAc = 1 / 1, R f = 0.4) purification to give compound 13a as a yellow solid (280.0 mg, 0.4 mmol, 70.6% yield). MS observation (ESI) + ) [{ 35 Cl}(M+H) + ]:783.4.

[0612] Step 2: cis-racemic-(4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0613] 13

[0615] TFA (3 mL) was added to a solution of compound 13a (250.0 mg, 0.3 mmol) in DCM (3 mL) at 25 °C, and the mixture was stirred at this temperature for 2 hours. The mixture was concentrated under reduced pressure to obtain a residue, which was redissolved in water (10 mL) and 5% (w%) NH4+ was added. 3• A 10 mL aqueous solution of H₂O was added to the mixture. A solid was formed, and the mixture was stirred at 20 °C for 16 hours. The resulting precipitate was filtered. The filter cake was collected and dissolved in 20 mL of water, and the suspension was lyophilized to give 13 (153.4 mg, 0.3 mmol, 81.5% yield) as a white solid. MS observations (ESI) + ) [{ 35 Cl}(M+H) + ]:583.0.

[0616] Example 14: trans-racemic-(4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0617] 14

[0619] Step 1: N-[4-[[2-chloro-6-[4-[4-[racemic-(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-4-pyridyl]difluoro-methyl]cyclohexyl]tert-butyl carbamate

[0620] 14

[0622] TFA (2.0 mL, 26.0 mmol) was added to a solution of compound Int-2 (200.0 mg, 0.3 mmol) in DCM (5 mL) at 20 °C, and the mixture was stirred at this temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 17% to 47%, 10 min) and lyophilized to give a product (100.0 mg) in the form of an HCl salt. The product was dissolved in water (10 mL) and 5% (w%) NH4+. 3• The solid was dissolved in 10 mL of H₂O aqueous solution. After stirring at 20 °C for 16 hours, the precipitate formed and was filtered. The filter cake was collected and redissolved in 20 mL of water. The suspension was lyophilized to give 14 (55.3 mg, 0.1 mmol, 35.4% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:583.1.

[0623] Example 15: 2-Amino-N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]acetamide

[0624] 15

[0626] Step 1: N-[2-[[4-[[2-chloro-6-[4-[4-[racemic-(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-4-pyridyl]-difluoro-methyl]cyclohexyl]methylamino]-2-oxo-ethyl]tert-butyl carbamate

[0627]

[0628] 15a

[0629] HATU (129.2 mg, 0.3 mmol) and DIEA (70.0 mg, 0.5 mmol) were added to a solution of compound Int-4 (120.0 mg, 0.2 mmol) and (tert-butoxycarbonyl)glycine (20.7 mg, 0.1 mmol) in DMF (1 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 2 hours, and the resulting mixture was diluted with H₂O (30 mL). The mixture was then transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 40 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 15a as a white solid (40.0 mg, 0.1 mmol, 59.4% yield). MS observations (ESI) + ) [{ 35 Cl}(M+H) + ]:854.3.

[0630] Step 3: 2-Amino-N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]acetamide

[0631] 15

[0633] TFA (1.0 mL, 13.0 mmol) was added to a solution of compound 15a (40.0 mg, 0.1 mmol) in DCM (1 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 h. The mixture was concentrated under reduced pressure to provide a crude product, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 18% to 48%, 10 min) and lyophilized to give Example 15 (18.0 mg, 0.1 mmol, 57.5% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:654.3.

[0634] Example 16: 4-(((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)amino)-N,N-bis(3-aminopropyl)-N-(carboxymethyl)-4-oxobut-1-ammonium

[0635] 16

[0637] Step 1: 4-(bis(3-((tert-butoxycarbonyl)amino)propyl)amino)benzyl butyrate

[0638]

[0639] 16a

[0640] 4-Bromobenzyl butyrate (2.0 g, 7.7 mmol) and NaHCO3 (1.0 g, 11.5 mmol) were added to a solution of N-[3-[3-(tert-butoxycarbonylamino)propylamino]propyl]carbamate tert-butyl (2.5 g, 7.7 mmol) in ACN (15 mL) at 25 °C, and the mixture was stirred at 25 °C for 16 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure to provide a crude product. This crude product was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 16a (1.8 g, 3.6 mmol, 46.2% yield) as a colorless oil. MS observations (ESI) + ) [(M+H) + ]:508.1.

[0641] Step 2: 4-(benzyloxy)-N-(2-(tert-butoxy)-2-oxoethyl)-N,N-bis(3-((tert-butoxycarbonyl)amino)propyl)-4-oxobut-1-ammonium bromide

[0642]

[0643] 16b

[0644] At 25 °C, tert-butyl bromoacetate (461.1 mg, 2.4 mmol) and NaHCO3 (413.7 mg, 4.9 mmol) were added to a solution of compound 16a (1.0 g, 2.0 mmol) in MeCN (3 mL) with stirring for 16 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. This crude product was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 16b (640.0 mg, 1.0 mmol, 52.2% yield) as a colorless oil. MS observations (ESI) + ) [(M+H) + ]:622.3.

[0645] Step 3: N-(2-(tert-butoxy)-2-oxoethyl)-3-((tert-butoxycarbonyl)amino)-N-(3-((tert-butoxycarbonyl)amino)propyl)-N-(3-carboxypropyl)prop-1-ammonium bromide

[0646]

[0647] 16c

[0648] Wet Pd / C (106.4 mg, 5% wt, containing 50% water) was added to a solution of compound 16b (650.0 mg, 1.0 mmol) in isopropanol (10 mL) under a nitrogen atmosphere. The suspension was degassed under vacuum, purged three times with H2, and stirred at 25 °C under a H2 atmosphere for 3 h. After dilution with MeOH (100 mL), the mixture was filtered and the filtrate was concentrated under reduced pressure to give crude 16c (550.0 mg, 1.0 mmol, 98.9% yield) as a white solid. The crude product was used directly in the next step without further purification. MS observation (ESI) + ) [(M+H) + ]:532.4.

[0649] Step 4: 4-(((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)amino)-N,N-bis(3-aminopropyl)-N-(carboxymethyl)-4-oxobut-1-ammonium (Example 16)

[0650] 16

[0652] Similar to the preparation of Example 15, Example 16 was prepared by replacing Int-4 and (tert-butoxycarbonyl)glycine with Int-2 and 16c, respectively, in step 1. Example 16 was obtained as a pale yellow solid in the form of hydrochloride (102.0 mg, 0.1 mmol, 76.3% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:840.1.

[0653] Example 17: N-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-2-(2-aminoethoxy)acetamide

[0654] 17

[0656] Similar to the preparation of compound 15, Example 17 was prepared by replacing Int-4 and (tert-butoxycarbonyl)glycine with Int-2 and 2-[2-[(2-methylprop-2-yl)oxycarbonylamino]ethoxy]acetic acid in step 1, respectively, and by replacing HATU with T3P. Example 17 was obtained as a white solid hydrochloride salt (12.2 mg, 0.1 mmol, 35.7% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:684.2.

[0657] Example 18: 3-Amino-N-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-2-hydroxypropionamide

[0658] 18

[0660] Similar to the preparation of compound Example 15, Example 18 was prepared by replacing Int-4 and (tert-butoxycarbonyl)glycine with Int-2 and 3-(((benzyloxy)carbonyl)amino)-2-hydroxypropionic acid, respectively, in step 1. Example 18 (6.0 mg, 0.01 mmol, 22.4% yield) was obtained as a white solid, in the form of hydrochloride. MS observation (ESI+) [({ 35 Cl}M+H) + ]:670.2.

[0661] Example 19: 2,3-Diamino-N-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)propionamide

[0662] 19

[0664] Similar to the preparation of compound Example 15, Example 19 was prepared by replacing Int-4 and (tert-butoxycarbonyl)glycine with Int-2 and 2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propionic acid, respectively, in step 1. Example 19 was obtained as a white solid hydrochloride salt (14.0 mg, 0.02 mmol, 41.8% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:669.2.

[0665] Example 26: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(2-aminoethyl)amino]butyramide]

[0666] 26

[0668] Step 1: 4-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)benzyl butyrate

[0669]

[0670] 26a

[0671] 4-Bromobenzyl butyrate (2.8 g, 10.9 mmol) and potassium carbonate (6.8 g, 49.4 mmol) were added to a solution of N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate tert-butyl ester (3.0 g, 9.9 mmol) in ACN (10 mL) at 20 °C, and the mixture was heated to 45 °C with stirring for 12 hours. After cooling to room temperature, the mixture was diluted with water (100 mL), extracted with EtOAc (100 mL × 3), washed with saturated brine (100 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (PE: EtOAc = 1:0 to 0:1; TLC: PE / EtOAc = 3 / 1, Rf = 0.5) to give 26a as a colorless oil (1.6 g, 3.3 mmol, 99.0% yield).

[0672] Step 2: 4-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]butyric acid

[0673]

[0674] 26b

[0675] Wet Pd / C (170.0 mg, 5% wt, containing 50% water) was added to a solution of 26a (800.0 mg, 1.7 mmol) in methanol (8 mL) at 25 °C under N2. The mixture was degassed under vacuum and then... H2 purge 3 The mixture was stirred at 25°C under a H2 atmosphere (15 psi) for 3 hours. The mixture was diluted with MeOH (100 mL), filtered, and the filtrate was concentrated under vacuum to give crude 26b as a colorless oil (600.0 mg, 1.5 mmol, 92.4% yield). The crude product was used directly in the next step without purification. MS observation (ESI) + ) [(M+H) + ]:390.4.

[0676] Step 3: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(2-aminoethyl)amino]butyramide (Example 26)

[0677] 26

[0679] Similar to the preparation of compound Example 15, Example 26 was prepared by replacing Int-4 and (tert-butoxycarbonyl)glycine with Int-2 and 26b, respectively, in step 1. Example 26 (30.4 mg, 0.03 mmol, 51.0% yield) was a yellow solid in the form of a TFA salt. MS observations (ESI) + ) ([{ 35 Cl}M+H] + ): 754.1.

[0680] Example 32: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]butyramide]

[0681] 32

[0683] Step 1: 4-[bis[3-(tert-butoxycarbonylamino)propyl]amino]benzyl butyrate

[0684]

[0685] 32a

[0686] K₂CO₃ (615.5 mg, 4.5 mmol) was added to a solution of N-[3-[3-(tert-butoxycarbonylamino)propylamino]propyl]carbamate tert-butyl ester (500.0 mg, 1.5 mmol) and 4-bromobutyrate benzyl ester (387.9 mg, 1.5 mmol) in ACN (5 mL), and the mixture was heated to 40 °C with stirring for 12 hours. The mixture was poured into water (30 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: C18 spherical, 20 to 35 µm, 100 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give compound 32a as a colorless oil (400.0 mg, 0.8 mmol, 52.2% yield). MS observation (ESI) + ) [(M+H) + ]:508.4.

[0687] Step 2: 4-[bis[3-(tert-butoxycarbonylamino)propyl]amino]butyric acid

[0688]

[0689] 32b

[0690] At 20°C, a solution of compound 32a (300.0 mg, 0.6 mmol) in methanol (5 mL) was moistened. Pd / C (100.0 mg, 5% wt, containing 50% water). The mixture was degassed under vacuum and purged with H2 for 3 minutes. The mixture was stirred at 20°C under a H2 atmosphere (15 psi) for 3 hours. After dilution with MeOH (100 mL), the mixture was filtered and the filtrate was concentrated under vacuum to give crude 32b (200.0 mg, 0.5 mmol, 81.1% yield) as a colorless oil. The crude product was used directly in the next step without further purification. MS observation (ESI) + ) [(M+H) + ]:418.0.

[0691] Step 3: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]butyramide]

[0692] 32

[0694] Similar to the preparation of compound Example 15, Example 32 was prepared by replacing Int-4 and (tert-butoxycarbonyl)glycine with Int-2 and 32b, respectively, in step 1. Example 32 was obtained as a white solid hydrochloride salt (14.7 mg, 0.02 mmol, 38.7% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:782.3.

[0695] Example 31: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]-N-hydroxy-butyramide]

[0696] 31

[0698] Similar to the preparation of compound Example 15, Example 31 was prepared by replacing Int-4 and (tert-butoxycarbonyl)glycine with Int-6 and 32b, respectively, in step 1. Example 31 was obtained as a white solid hydrochloride salt (4.1 mg, 0.01 mmol, 26.6% yield). MS observations (ESI) [({ 35 Cl}M+H) + ]:798.3.

[0699] Example 21: (2-amino-2-oxo-ethyl)-[4-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]amino]-4-oxo-butyl]-bis(3-aminopropyl)ammonium

[0700] twenty one

[0702] Step 1: 4-[bis[3-(tert-butoxycarbonylamino)propyl]amino]benzyl butyrate

[0703]

[0704] 21a

[0705] NaHCO3 (966.5 mg, 11.5 mmol) and benzyl 4-bromobutyrate (2.0 g, 7.7 mmol) were added to a solution of N-[3-[3-(tert-butoxycarbonylamino)propylamino]propyl]carbamate tert-butyl (2.5 g, 7.7 mmol) in ACN (15 mL) at 25 °C, and the mixture was stirred at 25 °C for 16 h. The mixture was filtered and the filtrate was concentrated to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 21a (3.6 g, 7.1 mmol, 92.5% yield) as a colorless oil. MS observations (ESI) + ) [(M+H) + ]:508.3.

[0706] Step 2: 4-[bis[3-(tert-butoxycarbonylamino)propyl]amino]butyric acid

[0707]

[0708] 21b

[0709] Compound 21a (400.0 mg, 0.8 mmol) was added to a suspension of wet Pd / C (20.0 mg, 5% wt, containing 50% water) in methanol (5 mL) at 25 °C under N2. The mixture was degassed, purged three times with H2, and stirred at 25 °C under an H2 balloon for 16 hours. The mixture was diluted with MeOH (100 mL) and filtered. The filtrate was concentrated to give crude 21b (330.0 mg, 0.8 mmol) as a colorless gel. MS observations (ESI) + ) [(M+H) + ]:418.2.

[0710] Step 3: N-[3-[3-(tert-butoxycarbonylamino)propyl-[4-[[4-[[2-chloro-6-[4-[4-[racemic-(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-4-pyridyl]-difluoro-methyl]cyclohexyl]amino]-4-oxo-butyl]amino]propyl]tert-butyl carbamate

[0711]

[0712] 21c

[0713] DIEA (0.4 mL, 2.2 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (409.8 mg, 1.1 mmol) were added to a solution of 21b (300.0 mg, 0.7 mmol) in DMF (2 mL) at 0 °C under N2. After stirring at 0 °C for 0.5 hours, Int-2 (490.9 mg, 0.7 mmol) was added, and the mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under vacuum to obtain a crude residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 21c (500.0 mg, 0.5 mmol, 61.1% yield) as a white solid. MS observation (ESI) + ) [({ 35 Cl}M+H) + ]:1082.6.

[0714] Step 4: (2-Amino-2-oxo-ethyl)-[4-[[4-[[2-[4-[4-[(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]amino]-4-oxo-butyl]-bis[3-(tert-butoxycarbonylamino)propyl]ammonium; iodide

[0715]

[0716] 21d

[0717] Iodoacetamide (153.7 mg, 0.8 mmol) and DIEA (0.1 mL, 0.8 mmol) were added to a solution of 21c (150.0 mg, 0.1 mmol) in ACN (2 mL) at 20 °C, and the mixture was heated to 40 °C with stirring for 16 hours. The mixture was poured into water (30 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 21d (80.0 mg, 0.1 mmol, 50.6% yield) as a white solid. MS observation (ESI) + ) [({ 35 Cl}M+H) + ]:1139.6.

[0718] Step 5: 2-Amino-N-[1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]azacyclobutane-3-yl]propionamide

[0719] twenty one

[0721] Trifluoroacetic acid (1 mL) was added to a solution of 21d (80.0 mg, 0.1 mmol) in DCM (2 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under vacuum to provide a crude residue, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 15% to 45%, 10 min) and lyophilized to give Example 21 (46.3 mg, 0.1 mmol, 82.4% yield) as a white solid in the form of hydrochloride. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:839.3.

[0722] Example 20: 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(diaminomethylene)urea

[0723] 20

[0725] Step 1: ((R)-1-(4-((4-(4-(((1r,4r)-4-(1H-imidazol-1-formamido)cyclohexyl)difluoromethyl)-6-chloropyridin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidine-3-yl)tert-butyl carbamate

[0726]

[0727] 20a

[0728] DIEA (100.0 mg, 0.8 mmol) was added to a mixture of compound Int-2 (100.0 mg, 0.2 mmol) and N,N'-carbonyldiimidazole (120.0 mg, 0.8 mmol) in anhydrous DCM (3 mL) at 20 °C, and the mixture was stirred for 12 hours. The mixture was poured into water (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to give crude product 20a (110.0 mg, 0.1 mmol, 96.7% yield) as a white solid, which was used directly in the next step without further purification. MS observation (ESI) + ) [({ 35 Cl}M+H) + ]:777.3.

[0729] Step 2: N-[(3R)-1-[4-[4-[4-[[4-[[(E)-[amino-(tert-butoxycarbonylamino)methylene]carbamoyl]amino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0730]

[0731] 20b

[0732] At 20 °C, tert-butyl N-aminoiminocarbamate (80.0 mg, 0.5 mmol) was added to a mixture of compound 20a (80.0 mg, 0.1 mmol) and potassium carbonate (240.0 mg, 1.7 mmol) in anhydrous DMF (1 mL), and the mixture was stirred for 12 hours. The mixture was poured into water (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 20b as a yellow solid (15.0 mg, 0.02 mmol, 16.8% yield). MS observation (ESI) + ) [(M+H)+ ]:868.3.

[0733] Step 3: 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(diaminomethylene)urea

[0734] 20

[0736] Trifluoroacetic acid (0.5 mL, 6.5 mmol) was added to a mixture of compound 20b (15.0 mg, 0.02 mmol) in anhydrous DCM (1 mL) at 20 °C, and the mixture was stirred for 1 hour. The mixture was concentrated under vacuum to give a crude product, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 17% to 47%, 10 min) and lyophilized to give 20b as a yellow solid in the form of hydrochloride (1.5 mg, 0.01 mmol, 12.1% yield). MS observations (ESI) + ) [(M+H) + ]:668.2.

[0737] Example 22: 3-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamoylamino]propyl-bis(3-aminopropyl)-(carboxymethyl)ammonium

[0738] twenty two

[0740] Step 1: N-[3-[3-(benzyloxycarbonylamino)propyl-[3-(tert-butoxycarbonylamino)propyl]amino]propyl]tert-butyl carbamate

[0741]

[0742] 22a

[0743] NaHCO3 (1.5 g, 18.1 mmol) was added to a solution of N-[3-[3-(tert-butoxycarbonylamino)propylamino]propyl]carbamate tert-butyl ester (2.0 g, 6.0 mmol) and N-(3-bromopropyl)carbamate benzyl ester (1.8 g, 6.6 mmol) in ACN (30 mL) at 25 °C, and the mixture was stirred at 25 °C for 12 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 330 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 25 min) and lyophilized to give 22a (1.0 g, 1.9 mmol, 31.7% yield) as a yellow oil. MS observations (ESI) + )[(M+H) + ]:523.8.

[0744] Step 2: 3-(benzyloxycarbonylamino)propyl-bis[3-(tert-butoxycarbonylamino)propyl]-(2-tert-butoxy-2-oxo-ethyl)ammonium; bromide

[0745]

[0746] 22b

[0747] To a solution of 22a (1.0 g, 1.9 mmol) in DMSO (5 mL), NaHCO3 (482.1 mg, 5.7 mmol) and tert-butyl bromoacetate (1.5 g, 7.7 mmol) were added, and the mixture was stirred at 50 °C for 15 hours. The mixture was diluted with EtOAc (100 mL) and filtered. The filtrate was concentrated to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 22b (500.0 mg, 0.7 mmol, 36.4% yield) as a yellow oil. MS observations (ESI) + ) [(M+H) + ]:637.2.

[0748] Step 3: 3-Aminopropyl-bis[3-(tert-butoxycarbonylamino)propyl]-(2-tert-butoxy-2-oxo-ethyl)ammonium; bromide

[0749]

[0750] 22c

[0751] 22b (1.0 g, 1.6 mmol) was added to a suspension of wet Pd(OH)₂ / C (0.5 g, 5% wt, containing 50% water) in THF (20 mL) at 25 °C under a N₂ atmosphere. The suspension was degassed under vacuum and purged three times with H₂. The mixture was then stirred at 50 °C for 2 hours under H₂ (15 psi). The mixture was diluted with MeOH (100 mL) and filtered. The filtrate was concentrated under reduced pressure to provide a crude product 22c (500.0 mg, 1.0 mmol, 63.3% yield) as a yellow oil. 22c was used directly in the next step without further purification. MS observation (ESI) + ) [(M+H) + ]:503.5.

[0752] twenty two

[0754] Similar to the preparation of compound Example 20, Example 22 was prepared by replacing N-aminoiminoylcarbamate with 22c in step 2 and using TEA instead of K2CO3. Example 22 was obtained as a yellow solid in the form of hydrochloride (14.0 mg, 0.02 mmol, 22.5% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:855.5.

[0755] Example 23: 3-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-1,1-bis(3-aminopropyl)urea

[0756] twenty three

[0758] Similar to the preparation of compound Example 20, Example 23 was prepared by replacing N-aminoiminocarbamate with N-[3-[3-(tert-butoxycarbonylamino)propylamino]propyl]carbamate in step 2 and using DIEA instead of K2CO3. Example 23 was obtained as a white powder in hydrochloric acid form (16.0 mg, 0.02 mmol, 40.0% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:740.4.

[0759] Example 34: (3R,4S)-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-hydroxy-pyrrolidine-1-carboxamide

[0760] 34

[0762] Similar to the preparation of compound Example 20, Example 34 was prepared by replacing N-aminoiminoylcarbamate with N-(3R,4S)-4-hydroxypyrrolidine-3-yl]carbamate in step 2 and using DIEA instead of K2CO3. Example 34 was obtained as a white solid hydrochloride salt (41.6 mg, 0.1 mmol, 53.3% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:711.3.

[0763] Example 35: 1-((1r,4r)-4-((2-(4-((4-((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-3-(2-(bis(2-aminoethyl)amino)ethyl)urea

[0764] 35

[0766] Step 1: N-[2-[2-(benzyloxycarbonylamino)ethyl-[2-(tert-butoxycarbonylamino)ethyl]amino]ethyl]tert-butyl carbamate

[0767]

[0768] 35a

[0769] At 20 °C, N-(2-bromoethyl)carbamate benzyl ester (1.0 g, 4.0 mmol) and NaHCO3 (830.6 mg, 9.9 mmol) were added to a solution of N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate tert-butyl ester (1.0 g, 3.3 mmol) in ACN (10 mL). The mixture was stirred at 20 °C for 15 hours, then diluted with EtOAc (100 mL) and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (PE / EtOAc = 10 / 1 to 1 / 1; TLC: PE / EtOAc = 3 / 1, Rf = 0.4) to give 35a (1.0 g, 2.1 mmol, 63.1% yield) as a colorless oil. MS observations (ESI) + ) [(M+H) + ]:481.1.

[0770] Step 2: N-[2-[2-aminoethyl-[2-(tert-butoxycarbonylamino)ethyl]amino]ethyl]tert-butyl carbamate

[0771]

[0772] 35b

[0773] Dry Pd / C (100.0 mg, 10% wt) was added to a solution of 35a (300.0 mg, 0.6 mmol) in THF (2 mL) and isopropanol (2 mL) at 20 °C under a N2 atmosphere. The suspension was degassed under vacuum and purged three times with H2. The mixture was then stirred at 20 °C for 1 hour under H2 (45 psi). The mixture was diluted with MeOH (200 mL) and filtered. The filtrate was concentrated under reduced pressure to give crude 35b (200.0 mg, 0.6 mmol, 92.5% yield) as a yellow oil, which was used directly in the next step without further purification. MS observation (ESI) + )[(M+H) + ]:347.1.

[0774] Step 3: 1-((1r,4r)-4-((2-(4-((4-((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-3-(2-(bis(2-aminoethyl)amino)ethyl)urea

[0775] 35

[0777] Similar to the preparation of compound Example 20, Example 35 was prepared by replacing N-aminoiminoylcarbamate tert-butyl with 35b in step 2 and using DIEA instead of K2CO3. Example 35 was obtained as a white solid hydrochloride salt (34.0 mg, 0.1 mmol, 50.0% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:755.2.

[0778] Example 29: (2S,6S)-2,6-bis(aminomethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidone-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]morpholine-4-carboxamide

[0779] 29

[0781] Step 1: N-(oxepane-2-ylmethyl)carbamate tert-butyl ester

[0782]

[0783] 29a

[0784] m-CPBA (32.8 g, 152.7 mmol) was added to a solution of N-allylcarbamate tert-butyl (20.0 g, 127.2 mmol) in DCM (100 mL) at 20 °C, and the mixture was stirred at 20 °C for 1 h. The reaction was quenched with an aqueous solution of Na₂SO₃ (100 mL), and the mixture was stirred at 20 °C for 1 h. After extraction with EtOAc (200 mL × 3), the combined organic layers were washed with saturated aqueous solution of NaHCO₃ (50 mL × 3) and saturated brine (50 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to give crude 29a (15.0 g, 86.6 mmol, 68.1% yield) as a yellow oil. The crude product was used directly in the next step without further purification.

[0785] Step 2: N-[3-(benzylamino)-2-hydroxypropyl] tert-butyl carbamate

[0786]

[0787] 29b

[0788] A solution of compound 29a (18.0 g, 103.9 mmol) and benzylamine (11.4 mL, 103.9 mmol) in isopropanol (100 mL) was stirred at 20 °C for 12 h. The mixture was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 330 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 70%, 20 min) and lyophilized to give compound 29b (12.0 g, 42.8 mmol, 41.2% yield) as a white solid. MS observations (ESI) + ) [(M+H) + ]:281.1.

[0789] Step 3: N-[3-[benzyl-(2-chloro-2-cyano-ethyl)amino]-2-hydroxy-propyl] tert-butyl carbamate

[0790]

[0791] 29c

[0792] 2-Chloroacrylonitrile (4.9 g, 56.2 mmol) was added to a solution of compound 29b (15.0 g, 53.5 mmol) in THF (200 mL) at 0 °C, and the mixture was heated to 20 °C with stirring for 16 hours. The mixture was poured into water (300 mL) and extracted with EtOAc (300 mL × 3). The combined organic layers were washed with saturated brine (300 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to give crude 29c (12.0 g, 32.6 mmol, 61.0% yield) as a pale yellow oil, which was used directly in the next step without further purification. MS observations (ESI) + ) [(M+H) + ]:368.1.

[0793] Step 4: N-[[racemic-(2S,6R)-4-benzyl-6-cyano-morpholin-2-yl]methyl]tert-butyl carbamate

[0794]

[0795] A solution of potassium tert-butoxide in THF (1.0 M, 41.0 mL, 41.0 mmol) was added to a solution of compound 29c (15.0 g, 40.8 mmol) in THF (100 mL) at 0 °C, and the mixture was heated to 20 °C with stirring for 2 hours. The mixture was poured into water (100 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: C18 spherical, 20 to 35 µm, 330 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 30 min) and lyophilized to give 29d-1 (5.2 g, 15.7 mmol, 27.5% yield) and 29d-2 (4.1 g, 12.4 mmol, 21.7% yield) as pale yellow oils. MS observations (ESI) + ) [(M+H) + ]:332.1.

[0796] Step 5: N-[[6-(aminomethyl)-4-benzyl-morpholin-2-yl]methyl]tert-butyl carbamate

[0797]

[0798] 29e

[0799] A solution of BH3•Me2S (10.0 M, 0.5 mL, 5.0 mmol) was added to a solution of compound 29d-1 (200.0 mg, 0.6 mmol) in THF (3 mL) at 0 °C, and the mixture was stirred at 20 °C for 2 h. The mixture was carefully quenched with MeOH (50 mL) at 0 °C and concentrated under reduced pressure to give crude 29e (150.0 mg, 0.5 mmol, 74.1% yield) as a colorless oil, which was used directly in the next step without further purification. MS observations (ESI) + ) [(M+H) + ]:336.1.

[0800] Step 6: N-[[4-benzyl-6-[(tert-butoxycarbonylamino)methyl]morpholin-2-yl]methyl]tert-butyl carbamate

[0801]

[0802] 29f

[0803] Compound 29e (150.0 mg, 0.5 mmol) was added to a solution of DIEA (0.3 mL) and di-tert-butyl dicarbonate (100.0 mg, 0.5 mmol) in THF (1 mL) at 20 °C, and stirred at this temperature for 12 hours. The reaction mixture was poured into EtOAc (50 mL) and water (10 mL). The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was passed through a silica gel column (PE / EtOAc = 50 / 1 to 2 / 1; TLC: PE / EtOAc = 3 / 1, R f Purification was performed at 0.4 g / mL to give compound 29f (150.0 mg, 0.3 mmol, 77.0% yield) as a colorless oil. MS observations (ESI) + ) [(M+H) + ]:436.1.

[0804] Step 7: (((2R,6R)-morpholino-2,6-diyl)bis(methylene))tert-butyl dicarboxylate

[0805]

[0806] 29 g

[0807] Wet Pd / C (50.0 mg, 5% wt, containing 50% water) was added to a solution of compound 29f (150.0 mg, 0.2 mmol) in isopropanol (5 mL) at 20 °C under a N2 atmosphere. The suspension was degassed under vacuum and purged three times with H2, and stirred at 20 °C for 12 h under H2 (15 psi). The mixture was diluted with methanol (200 mL) and filtered. The filtrate was concentrated under reduced pressure to provide crude compound 29 g (50.0 mg, 0.2 mmol, 36.3% yield) as a colorless oil, which was used directly in the next step without further purification. MS observations (ESI) + ) [(M+H) + ]:346.4.

[0808] Step 8: (2S,6S)-2,6-bis(aminomethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidone-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]morpholine-4-carboxamide (Example 29)

[0809] 29

[0811] Similar to the preparation of compound Example 20, Example 29 was prepared by replacing 29 g of N-aminoiminocarbamate tert-butyl ester and using DIEA instead of K2CO3 in step 2. Example 29 was obtained as a white solid hydrochloride salt (26.1 mg, 0.03 mmol, 56.3% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:754.5.

[0812] Example 24: 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(3-aminopropyl)guanidine

[0813] twenty four

[0815] Step 1: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(imidazol-1-iminoamino)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0816]

[0817] 24a

[0818] Imidazol-1-yl(1,2,4-triazol-4-yl)methylimine (85.4 mg, 0.5 mmol) was added to a solution of Int-2 (60.0 mg, 0.1 mmol) in DMF (0.4 mL) at 25 °C under N2, and the mixture was stirred at 45 °C for 12 h. After cooling to room temperature, the mixture was purified directly by preparative HPLC (column: C18 spherical 20 to 35 µm, 40 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 12 min) and lyophilized to give 24a (50.0 mg, 0.1 mmol, 34.5% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:776.3.

[0819] Step 2: N-[3-[[N-[4-[[2-[4-[4-[(4R)-4-(tert-butoxycarbonylamino)-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]aminoimino]amino]propyl]tert-butyl carbamate

[0820]

[0821] 24b

[0822] N-Boc-1,3-diaminopropane (157.1 mg, 0.9 mmol) was added to a solution of 24a (50.0 mg, 0.1 mmol) in DMF (0.2 mL) at 25 °C under N2, and the mixture was heated to 80 °C with stirring for 3 hours. After cooling to room temperature, the mixture was purified directly by preparative HPLC (column: C18 spherical 20 to 35 µm, 40 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 24b (30.0 mg, 0.03 mmol, 37.7% yield) as a yellow solid. MS observations (ESI) + ) [{ 35 Cl}M+H] + ]:882.5.

[0823] Step 3: 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(3-aminopropyl)guanidine

[0824] twenty four

[0826] TFA (1 mL) was added to a solution of 24b (25.0 mg, 0.03 mmol) in DCM (1 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 hour. The mixture was concentrated, and the residue was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 14% to 44%, 10 min) and lyophilized to give 24b as a yellow solid in the form of hydrochloride (5.6 mg, 0.01 mmol, 25.7% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:682.3.

[0827] Example 25: N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]-2,2,2-trifluoro-acetamide

[0828] 25

[0830] Step 1: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-[[(2,2,2-trifluoroacetyl)amino]methyl]cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0831]

[0832] 25a

[0833] Trifluoroacetic anhydride (63.0 mg, 0.3 mmol) and DIEA (129.5 mg, 1.0 mmol) were added to a solution of Int-4 (70.0 mg, 0.1 mmol) in DCM (0.7 mL) at 25 °C. The mixture was stirred at 25 °C for 2 hours, then quenched with water (30 mL) and extracted with EtOAc (60 mL × 3). The combined organic layers were washed with saturated brine (60 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under vacuum to provide a crude residue, which was subjected to preparative TLC (PE / EtOAc = 1 / 2; TLC: PE / EtOAc = 1 / 2, R f = 0.5) purification to give 25a (50.0 mg, 0.1 mmol, 62.8% yield) as a pale yellow solid. MS observation (ESI+) ([{ 35 Cl}M+H] + ): 793.3.

[0834] Step 2: N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]-2,2,2-trifluoro-acetamide

[0835] 25

[0837] TFA (2.0 mL) was added to a solution of 25a (35.0 mg, 0.04 mmol) in DCM (1 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under vacuum, and the residue was purified by preparative HPLC (column: Phenomenex Synergi Polar-RP 100 × 25 mm × 4 μm; mobile phase: [A: water (0.1% TFA) - ACN]; B%: 20% to 60%, 10 min) and lyophilized to give 25a (9.2 mg, 0.01 mmol, 24.0% yield) as a white solid in the form of TFA salt. MS observation (ESI+) ([{ 35 Cl}M+H] + ): 693.2.

[0838] Example 36: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester

[0839] 36

[0841] Step 1: (((2-((tert-butyldimethylsilyl)oxy)ethyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate ditert-butyl

[0842]

[0843] 36a

[0844] At 25 °C, tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate (7.2 g, 23.7 mmol) in acetonitrile (30 mL) was added with (2-bromoethoxy)tert-butyldimethylsilane (5.7 g, 23.7 mmol) and NaHCO3 (6.0 g, 71.2 mmol). The mixture was stirred at 45 °C for 72 hours, then diluted with EtOAc (100 mL) and filtered. The filtrate was concentrated under vacuum to give a residue, which was purified by silica gel (EtOAc / MeOH = 1:0 to 10 / 1; TLC: EtOAc / MeOH = 10 / 1, Rf = 0.3) to give 36a (2.7 g, 5.9 mmol, 24.6% yield) as a colorless oil. (ESI+) [(M+H)+]: 462.1.

[0845] Step 2: (((2-hydroxyethyl)azonidinediyl)bis(ethane-2,1-diyl))dicarbamate ditert-butyl

[0846]

[0847] 36b

[0848] AcOH (13 mL) was added to a solution of 36a (6.6 g, 14.3 mmol) in water (7.0 mL) and THF (30 mL) at 20 °C, and the mixture was stirred for 12 hours. The mixture was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 330 g, mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 25 min) and lyophilized to obtain 36b (2.7 g, 7.8 mmol, 54.4% yield) as a colorless oil. MS observation (ESI+) [(M+H)+]: 348.3.

[0849] Step 3: 2-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]ethyl carbonate (4-nitrophenyl) ester

[0850]

[0851] 36c

[0852] N,N-diisopropylethylamine (558.0 mg, 4.3 mmol) was added to a solution of 4-nitrobenzene chloroformate (319.1 mg, 1.6 mmol) and 36b (500.0 mg, 1.4 mmol) in THF (15 mL) at 25 °C, and the mixture was heated to 40 °C and stirred for 48 hours. The mixture was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to obtain 36c (270.0 mg, 0.5 mmol, 36.6% yield) as a colorless oil. MS observation (ESI+) [(M+H)] + ]:513.3.

[0853] Step 4: N-[(3R)-1-[4-[4-[4-[[4-[2-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]ethoxycarbonylamino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0854]

[0855] 36d

[0856] N,N-diisopropylethylamine (0.5 mL, 2.9 mmol) was added to a solution of Int.2 (200.0 mg, 0.3 mmol) and 36c (300.1 mg, 0.6 mmol) in THF (15 mL) at 25 °C, and the mixture was heated to 60 °C with stirring for 48 hours. The mixture was concentrated under vacuum, and the residue was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g; mobile phase: [A: water (0.1% TFA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 36d (80.0 mg, 0.1 mmol, 25.9% yield) as a white solid. (ESI) + ) [({ 35 Cl}M+H) + ]:1056.7.

[0857] Step 5: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester

[0858] 36

[0860] TFA (1 mL) was added to a solution of 36d (60.0 mg, 0.1 mmol) in DCM (2 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under vacuum to obtain a crude residue, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 14% to 44%, 10 min) and lyophilized to give 36d (17.8 mg, 0.02 mmol, 40.2% yield) as a white solid in the form of hydrochloride. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:756.1.

[0861] Example 27: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(3-aminopropyl)amino]ethyl ester

[0862] 27

[0864] Similar to the preparation of compound 36, in step 1, di-tert-butyl dicarboxylate (azidediylbis(propane-3,1-diyl))dicarboxylate was used instead of di-tert-butyl dicarboxylate (azidediylbis(ethane-2,1-diyl))dicarboxylate to prepare Example 27. Example 27 was obtained as a white powder in the form of TFA (6.3 mg, 0.01 mmol, 43.0% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:784.4.

[0865] Example 28: 2-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamoyloxy]ethyl-bis(3-aminopropyl)-(carboxymethyl)ammonium

[0866] 28

[0868] Step 1: N-[3-[3-(tert-butoxycarbonylamino)propyl-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]carbamate tert-butyl

[0869]

[0870] 28a

[0871] To a solution of N-[3-[3-(tert-butoxycarbonylamino)propylamino]propyl]carbamate tert-butyl ester (1.0 g, 3.0 mmol) in acetonitrile (10 mL), (2-bromoethoxy)tert-butyldimethylsilane (721.8 mg, 3.0 mmol) and NaHCO3 (760.4 mg, 9.1 mmol) were added. The mixture was stirred at 45 °C for 72 hours, filtered, and the filtrate was concentrated under vacuum to obtain a residue. This residue was purified by silica gel column chromatography (EtOAc / MeOH = 1 / 0 to 10 / 1; TLC: EtOAc / MeOH = 10 / 1, Rf = 0.3) to give 28a (950.0 mg, 1.9 mmol, 64.3% yield) as a colorless oil. MS observations (ESI) + ) [(M+H) + ]:490.4.

[0872] Step 2: 2-[bis(3-aminopropyl)amino]ethanol

[0873]

[0874] 28b

[0875] The mixture of 28a (500.0 mg, 1.0 mmol) in HCl / MeOH (4.0 M, 10.0 mL) was stirred at 25 °C for 1 hour. The mixture was concentrated under vacuum to give crude 28b (216.0 mg, 1.0 mmol, 99.9% yield) as a colorless oil, which was used directly in the next step without further purification. MS observations (ESI) + ) [(M+H) + ]:176.0

[0876] Step 3: N-[3-[3-(tert-butoxycarbonylamino)propyl-(2-hydroxyethyl)amino]propyl] tert-butyl carbamate

[0877]

[0878] 28c

[0879] Sodium bicarbonate (342.8 mg, 4.1 mmol) and di-tert-butyl dicarbonate (445.3 mg, 2.0 mmol) were added to a solution of 28b (216.0 mg, 1.0 mmol) in methanol (15 mL) at 25 °C. The mixture was stirred at 25 °C for 1 hour, then diluted with EtOAc (50 mL) and filtered. The filtrate was concentrated under vacuum to obtain a residue, which was then passed through a silica gel column (EtOAc / MeOH = 1 / 0 to 10 / 1; TLC: EtOAc / MeOH = 10 / 1, R...). f Purified at 0.3 g / mL to give 28c (180.0 mg, 0.5 mmol, 47.0% yield) as a yellow oil. MS observations (ESI) + )[(M+H) + ]:376.1.

[0880] Step 4: N-[(3R)-1-[4-[4-[4-[[4-[2-[bis[3-(tert-butoxycarbonylamino)propyl]amino]ethoxycarbonylamino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0881]

[0882] 28d

[0883] N,N-diisopropylethylamine (0.1 mL, 0.6 mmol) and 4-nitrobenzene chloroformate (30.0 mg, 0.2 mmol) were added to a solution of 28°C (50.0 mg, 0.1 mmol) in THF (2 mL) at 0°C under N2. After stirring the mixture at 40°C for 48 hours, Int-2 (100.1 mg, 0.2 mmol) and 4-dimethylaminopyridine (5.0 mg, 0.04 mmol) were added, and the mixture was stirred at 40°C for another 48 hours. The mixture was then concentrated under vacuum to obtain a residue. The residue was dissolved in DMF (2 mL), purified by preparative HPLC (column: C18 spherical, 20 to 35 µm, 40 g; mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 10 min) and lyophilized to give 28d (50.0 mg, 0.1 mmol, 34.6% yield) as a white solid. MS observation (ESI) + ) [({35 Cl}M+H) + ]:1084.7.

[0884] Step 5: N-(2-(tert-butoxy)-2-oxoethyl)-3-((tert-butoxycarbonyl)amino)-N-(2-((((1r,4r)-4-((2-(4-((4-((R)-4-((tert-butoxycarbonyl)amino)-2-oxopyrrolidine-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)carbamoyl)oxy)ethyl)-N-(3-((tert-butoxycarbonyl)amino)propyl)propyl-1-ammonium)

[0885]

[0886] 28e

[0887] A mixture of 28d (45.0 mg, 0.04 mmol), tert-butyl bromoacetate (24.3 mg, 0.1 mmol), and DIEA (0.2 mL, 1.2 mmol) in DMF (0.2 mL) was stirred at 70 °C for 3 h. The mixture was acidified with FA to pH = 5, then purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 40 g; mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 10 min) and lyophilized to give 28e (50.0 mg, 0.04 mmol, 87.8% yield) as a yellow solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:1198.5

[0888] Step 6: 2-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamoyloxy]ethyl-bis(3-aminopropyl)-(carboxymethyl)ammonium (Example 28)

[0889] 28

[0891] TFA (2 mL) was added to a solution of 28e (40.0 mg, 0.03 mmol) in DCM (2 mL) at 25 °C under N2. The mixture was stirred at 25 °C for 1 hour, then concentrated under vacuum to obtain a residue. This residue was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 15% to 45%, 10 min) and lyophilized to give 28e as a brown solid in the form of hydrochloride (15.0 mg, 0.02 mmol, 47.9% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:842.3.

[0892] Example 30: N-[4-[1-[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]cyclopropyl]cyclohexyl]-4-[bis(2-aminoethyl)amino]butyramide

[0893] 30

[0895] Step 1: N-[4-[1-(2,6-dichloro-4-pyridyl)vinyl]cyclohexyl]tert-butyl carbamate

[0896]

[0897] 30a

[0898] A solution of n-butyllithium (2.5 M, 7.5 mL, 18.8 mmol) was added dropwise to a mixture of methyltriphenylphosphonium bromide (7.5 g, 20.9 mmol) in THF (70 mL) at 0 °C. After adding a solution of Int-2c (3.9 g, 10.5 mmol) in THF (70 mL), the mixture was stirred at 0 °C for 2 hours. The reaction mixture was poured into 100 mL of saturated NH4Cl aqueous solution and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum to provide a residue, which was then passed through a silica gel column (PE / EtOAc = 50 / 1 to 2 / 1; TLC: PE / EtOAc = 2 / 1, R...). f= 0.4) purification to give 30a (2.9 g, 7.8 mmol, 74.8% yield) as a yellow solid. MS observation (ESI) + ) [({ 35 Cl}M-C4H8+H) + ]:315.0.

[0899] Step 2: N-[4-[1-(2,6-dichloro-4-pyridyl)cyclopropyl]cyclohexyl]tert-butyl carbamate

[0900]

[0901] 30b

[0902] Trimethyl sulfoxide iodide (200.0 mg, 0.9 mmol) was dissolved in DMSO (2 mL) at 0 °C, and sodium hydride (38.0 mg, 60% wt., 1.1 mmol) was added in a single addition. Then, 30a (200.0 mg, 0.5 mmol) was added to the solution at 0 °C, and the mixture was stirred at 25 °C for 2 hours. The mixture was poured into 20 mL of saturated NH4Cl aqueous solution and extracted with EtOAc (50 mL × 3). The organic layers were combined, washed with saturated brine (50 mL × 3), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under vacuum to obtain a crude residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to obtain 30b (160.0 mg, 0.4 mmol, 77.1% yield) as a yellow solid. MS observation (ESI+) [({ 35 Cl}M-C4H8+H) + ]:329.0.

[0903] Step 3: N-[4-[1-(2,6-dichloro-4-pyridyl)cyclopropyl]cyclohexyl]tert-butyl carbamate

[0904]

[0905] 30c

[0906] TFA (2 mL) was added in a single addition to a solution of 30b (200.0 mg, 0.5 mmol) in DCM (2 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under vacuum to obtain crude 30c (160.0 mg, 0.6 mmol) as a white solid, which was used directly in the next step without further purification. MS observation (ESI+): 295.2 [({ 35 Cl}M+H)+].

[0907] Step 4: N-(4-formylcyclohexyl)carbamate tert-butyl ester

[0908]

[0909] 30d

[0910] Sodium bicarbonate (1.4 g, 16.5 mmol) was added in a single batch to a solution of N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate tert-butyl (500.0 mg, 1.7 mmol) and 4-bromobutyrate benzyl ester (423.7 mg, 1.7 mmol) in ACN (10 mL). The mixture was stirred at 25 °C for 15 hours, then diluted with EtOAc (200 mL) and filtered. The filtrate was concentrated under vacuum to obtain a crude residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to obtain a yellow oily 30d (380.0 mg, 0.8 mmol, 48.1% yield). MS observation (ESI+) [(M+H)] + ]:480.2.

[0911] Step 5: 4-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]butyric acid

[0912]

[0913] 30e

[0914] Wet Pd / C (50 mg, 5% wt, containing 50% water) was added to a solution of 30d (180.0 mg, 0.4 mmol) in methanol (20 mL) at 20 °C under a N2 atmosphere. The suspension was degassed under vacuum and purged three times with H2. The reaction was then stirred at 40 °C for 16 h under H2 (15 psi). The mixture was diluted with methanol (200 mL) and filtered. The filtrate was concentrated under reduced pressure to provide a crude product, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 40 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 10 min) and lyophilized to give 30e (120.0 mg, 0.3 mmol, 82.1% yield) as a colorless oil. MS observations (ESI+) [(M+H)] + ]:390.2.

[0915] Step 6: N-[(3R)-1-[4-[4-[6-chloro-4-[1,4-dioxaspiro[4.5]decane-8-yl(difluoro)methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0916]

[0917] 30f

[0918] At 0 °C, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (96.0 mg, 0.3 mmol) and N,N-diisopropylethylamine (742.0 mg, 5.7 mmol) were added in a single dose to a solution of 30e (98.3 mg, 0.3 mmol) and 30b (60.0 mg, 0.2 mmol) in DMF (2 mL). The mixture was stirred at 25°C for 2 hours, then concentrated under vacuum to obtain a residue. This residue was purified by preparative HPLC (column: C18 spherical, 20 to 35 µm, 40 g; mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 10 min) and lyophilized to give 30f (60.0 mg, 0.1 mmol, 43.4% yield) as a yellow solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:656.3.

[0919] Step 7: N-[(3R)-1-[4-[4-[4-[1-[4-[4-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]butyrylamino]cyclohexyl]cyclopropyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0920]

[0921] 30 g

[0922] Int-1 (129.3 mg, 0.3 mmol) and N,N-diisopropylethylamine (116.0 mg, 0.9 mmol) were added to a solution of 30 f (200.0 mg, 0.3 mmol) in DMSO (1 mL) at 25 °C, and the mixture was heated to 100 °C with stirring for 16 hours. After cooling to 25 °C, the mixture was concentrated under vacuum to obtain a residue, which was purified by preparative HPLC (column: C18 spherical 20 to 35 µm, 100 g, mobile phase: [A: water (0.1% FA) - B: ACN]; B%: 0% to 100%, 15 min) and lyophilized to give 30 g (80.0 mg, 0.1 mmol, 25.1% yield) as a yellow solid. MS observations (ESI) + ) [({ 35 Cl}M-Boc+H) + ]:944.4.

[0923] Step 8: N-[(3R)-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazacyclobutane-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0924] 30

[0926] TFA (3 mL) was added in a single dose to a solution of 30 g (60.0 mg, 0.1 mmol) in DCM (2 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under vacuum to provide a crude residue, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 15% to 45%, 10 min) and lyophilized to give 30 as a white solid in the form of hydrochloride (8.0 mg, 0.01 mmol, 17.4% yield). MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:744.3.

[0927] Example 33: (4R)-4-amino-1-[4-[4-[6-chloro-4-[[4-[(2,2-difluoro-3-hydroxy-propyl)amino]cyclohexyl]-difluoro-methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0928] 33

[0930] Step 1: 3-[tert-butyl(dimethyl)silyl]oxy-2,2-difluoro-prop-1-ol

[0931]

[0932] 33a

[0933] Imidazole (1.2 g, 17.8 mmol) and tert-butyldimethylchlorosilane (1.6 g, 10.8 mmol) were added to a solution of 2,2-difluoropropane-1,3-diol (1.0 g, 8.9 mmol) in DMF (20 mL) at 0 °C. The mixture was stirred at 0 °C for 4 hours, then diluted with H₂O (30 mL) and transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (80 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to provide a crude product, which was passed through a silica gel column (PE / EtOAc = 1 / 0 to 5 / 1; TLC: PE / EtOAc = 5 / 1, R f= 0.4) purification to obtain 33a (2.0 g, 98.8% yield) as a colorless oil. 33a was used directly in the next step without characterization analysis.

[0934] Step 2: 3-[tert-butyl(dimethyl)silyl]oxy-2,2-difluoropropionaldehyde

[0935]

[0936] 33b

[0937] DMP (618.4 mg, 1.5 mmol) was added to a solution of 33a (300.0 mg, 1.3 mmol) in DCM (5 mL) at 0 °C, and the mixture was stirred at 0 °C for 2 hours. The mixture was quenched by the slow addition of saturated K₂CO₃ solution (20 mL), transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (40 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 2), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to provide a crude product, which was passed through a silica gel column (PE / EtOAc = 1 / 0 to 5 / 1; TLC: PE / EtOAc = 5 / 1, R f = 0.5) purification to obtain 33b as a colorless oil (250.0 mg, 1.1 mmol, 84.1% yield). 33b was used directly in the next step without characterization analysis.

[0938] Step 3: N-[(3R)-1-[4-[4-[4-[[4-[[3-[tert-butyl(dimethyl)silyl]oxy-2,2-difluoro-propyl]amino]cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]-5-oxo-pyrrolidine-3-yl]tert-butyl carbamate

[0939]

[0940] 33c

[0941] Acetic acid (32.1 mg, 0.5 mmol) and sodium triacetoxyborohydride (283.4 mg, 1.3 mmol) were added in a single addition to a solution of 33b (60.0 mg, 0.2 mmol) and Int-2 (201.0 mg, 0.3 mmol) in DCE (1 mL) at 25 °C under N2. The mixture was stirred at 25 °C for 16 hours and then diluted with EtOAc (100 mL) and water (20 mL). The organic layer was separated and subsequently washed with saturated brine (30 mL × 2), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give crude 33c (50.0 mg, 0.01 mmol, 48.4% yield) as a white solid, which was used directly in the next step without further purification. MS observations (ESI) + )[({ 35 Cl}M+H) + ]:891.2.

[0942] Step 4: (4R)-4-amino-1-[4-[4-[6-chloro-4-[[4-[(2,2-difluoro-3-hydroxy-propyl)amino]cyclohexyl]-difluoro-methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0943] 33

[0945] TFA (1 mL) was added to a solution of 33c (30.0 mg, 0.03 mmol) in DCM (1 mL) at 25 °C, and the mixture was stirred at 25 °C for 2 hours. The mixture was concentrated under reduced pressure to provide a crude product, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 20% to 50%, 10 min) and lyophilized to give 33c (7.5 mg, 0.01 mmol, 28.4% yield) as a white solid. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:677.2.

[0946] Example 37: (4R)-4-amino-1-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-methyl-pyrimidin-2-yl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0947] 37

[0949] Step 1: (4R)-4-amino-1-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-methyl-pyrimidin-2-yl]piperazin-1-yl]sulfonylphenyl]pyrrolidine-2-one

[0950] 37

[0952] Int-5j (35.0 mg, 0.04 mmol) was added to TFA (2.0 mL, 26.0 mmol) at 25 °C, and the mixture was heated to 60 °C and stirred for 16 hours. The mixture was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 12% to 42%, 10 min) and lyophilized to give 37 as a white solid in the form of hydrochloride (19.4 mg, 0.03 mmol, 75.6% yield). MS observations (ESI) + ) [(M+H) + ]:564.4.

[0953] Example 38: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-methyl-pyrimidin-4-yl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]butyramide]

[0954] 38

[0956] Similar to the preparation of compound Example 15, Example 38 was prepared by replacing 2-[2-[(2-methylprop-2-yl)oxycarbonylamino]ethoxy]acetic acid with 32b in step 1 and replacing Int-4 with Int-5. Example 38 was obtained as a white solid hydrochloride salt (3.5 mg, 0.01 mmol, 7.3% yield). MS observations (ESI) +)[(M+H) + ]:763.6.

[0957] Example 40: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester

[0958] 40

[0960] Step 1: N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidone-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester; trihydrochloride

[0961] 40

[0963] Trifluoroacetic acid (1.00 mL, 12.98 mmol) was added to a solution of Int-7 (50.0 mg, 0.1 mmol) in DCM (2 mL) at 25 °C, and the mixture was stirred for 1 hour. The mixture was concentrated under vacuum to obtain a residue, which was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 15% to 45%, 10 min) and lyophilized to give Example 40 (32.4 mg, 0.1 mmol, 70.7% yield) as a white solid in the form of hydrochloride. MS observations (ESI) + ) [({ 35 Cl}M+H) + ]:597.3.

[0964] Example 39: (4R)-4-amino-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)pyrrolid-2-one

[0965] 39

[0967] Step 1: ((3R)-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidine-3-yl)tert-butyl carbamate

[0968]

[0969] 39a

[0970] 1,1,1-trifluoro-2,3-epoxypropane (84.4 mg, 0.8 mmol) and DIEA (294.1 mg, 2.3 mmol) were added to a solution of Int-5 (500.0 mg, 0.8 mmol) in isopropanol (2 mL) at 25 °C, and the mixture was heated to 70 °C and stirred for 2 hours. The mixture was quenched by adding saturated ammonium chloride aqueous solution (20 mL), transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (60 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to provide a crude product, which was passed through a silica gel column (EtOAc / MeOH = 1 / 0 to 10 / 1, 1‰ NH₃•H₂O as additive; TLC: EtOAc / MeOH = 10 / 1, R f = 0.4) purification to give 39a as a white solid (400.0 mg, 0.5 mmol, 67.8% yield). MS observation (ESI) + ) [(M+H) + ]:776.2

[0971] Step 2: (4R)-4-amino-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)pyrrolid-2-one

[0972] 39

[0974] TFA (3 mL) was added to a solution of 39a (400.0 mg, 0.5 mmol) in DCM (5 mL) at 25 °C, and the mixture was stirred at 25 °C for 2 hours. The mixture was concentrated under reduced pressure to give crude Example 39 (304.0 mg, 0.5 mmol, 88.0% yield) as a white solid. MS observation (ESI) + ) [(M+H) + ]:676.2.

[0975] Example 42: ((3R)-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidine-3-yl)tert-butyl carbamate

[0976] 42

[0978] Step 1: ((S)-3,3,3-trifluoro-2-hydroxypropyl)carbamate ((1r,4S)-4-((2-(4-((4-(((R)-4-((tert-butoxycarbonyl)amino)-2-oxopyrrolidine-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-methylpyrimidin-4-yl)difluoromethyl)cyclohexyl) ester (42a) and ((R)-3,3,3-trifluoro-2-hydroxypropyl)carbamate ((1r,4R)-4-((2-(4-((4-(((R)-4-((tert-butoxycarbonyl)amino)-2-oxopyrrolidine-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-methylpyrimidin-4-yl)difluoromethyl)cyclohexyl) ester (42b)

[0979]

[0980] 42a

[0981]

[0982] 42b

[0983] Di-tert-butyl dicarbonate (210.0 mg, 1.0 mmol) and DIEA (148.9 mg, 1.2 mmol) were added to a solution of 39 (300.0 mg, 0.4 mmol) in DCM (5 mL) at 25 °C, and the mixture was stirred at 25 °C for 2 hours. The resulting mixture was poured into water (50 mL) and transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (80 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to provide a crude product, which was passed through a silica gel column (PE / EtOAc = 10 / 1 to 0 / 1; TLC: PE / EtOAc = 1 / 1, R f = 0.4) Purified and subsequently separated by preparative SFC (column: Chiralpak IG 25 × 30 mm I.D., 10 μm, mobile phase: 40% IPA (0.1% NH3•H2O) in supercritical CO2) to give 42a as a white solid (first eluent, 140.0 mg, 0.16 mmol, 41.54% yield), MS observation (ESI) + ) [(M+H) + ]: 876.2; and 42b (second eluent, 160.0 mg, 0.18 mmol, 47.47% yield) as a white solid. MS observations (ESI) + ) [(M+H) + ]:876.2.

[0984] Step 2: (R)-4-amino-1-(4-((4-(4-(difluoro((1R,4r)-4-(((R)-3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)pyrrolid-2-one

[0985] 42

[0987] TFA (2 mL) was added to a solution of 42b (120.0 mg, 0.1 mmol) in DCM (2 mL) at 25 °C, and the mixture was stirred at 25 °C for 2 h. The reaction was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: YMC Triart C18 150 × 25 mm × 5 μm, mobile phase: [A: water (0.1% HCl) - B: ACN]; B%: 18% to 48%, 10 min) and lyophilized to give compound 42 (71.2 mg, 0.1 mmol, 73.6% yield) as a white solid hydrochloride salt. MS observations (ESI) + ) [(M+H) + ]:676.3.

[0988] Example 43: Minimum Inhibition Concentration (MIC) Determination:

[0989] The antibacterial activity of the compounds of this invention was evaluated against commonly used quality control strains *Escherichia coli* ATCC 25922 and rifampicin-resistant mutant strain *Klebsiella pneumoniae* ATCC 43816. Both were originally derived from human clinical samples and were available from ATCC (American Center for Type Culture Collection). The in vitro efficacy of the compounds in inhibiting the growth of *Escherichia coli* (ATCC 25922) and *Klebsiella pneumoniae* (ATCC 43816) was assessed using the MIC (minimum inhibitory concentration) broth dilution method. The specific compound dilution was prepared from a 10 mM DMSO stock solution as follows: i) prepare a series of 2-fold dilutions in 20 µL of DMSO in a master plate (Greiner, catalog number: 651201); ii) add 180 µL of sterile distilled water to each aliquot; and iii) transfer 10 µL of the diluted compound to a new assay plate (Costar, 3599).

[0990] Each vial of the tested microorganism was kept frozen in the gas phase of a liquid nitrogen freezer. Disposable frozen vials of two strains of *Escherichia coli* ATCC 25922 (KWIKSTIK, 0335K) and *Klebsiella pneumoniae* ATCC 43816, with predetermined CFU / mL, were removed from the freezer, thawed at room temperature, and diluted in cationic-regulated Mueller Hinton broth (CAMHB) to achieve 5 × 10⁻⁶ CFU / mL. 5Final inoculum of CFU / mL. 90 µL of broth containing bacteria was dispensed into an assay plate containing pre-split compound dilutions and mixed 5 times by pipetting.

[0991] The assay plates were then incubated at 35°C in ambient air for 20 hours. After incubation, the MIC (μg / mL), the lowest drug concentration required to inhibit visible microbial growth, was read and recorded using a magnifying glass.

[0992] Compound F-1, disclosed in the literature (Bioorganic Chemistry 102 (2020) 104055), was selected as the reference compound of this invention. This compound is reported to have LPS synthesis pathway inhibitory activity and exhibits antibiotic activity against efflux-deficient Escherichia coli strains.

[0993]

[0994] Compound F-1

[0995] Table 1: MIC values ​​of the compounds of the present invention against Escherichia coli and Klebsiella pneumoniae

[0996]

[0997] Example A

[0998] Film-coated tablets containing the following ingredients can be manufactured using conventional methods:

[0999]

[1000] The active ingredient was sieved and mixed with microcrystalline cellulose, and the mixture was granulated together with a solution of polyvinylpyrrolidone in water. The granules were then mixed with sodium starch glycolate and magnesium stearate and pressed to obtain cores of 120 or 350 mg, respectively. The cores were coated with an aqueous solution / suspension of the above-described membrane coating.

[1001] Example B

[1002] Capsules containing the following ingredients can be manufactured using conventional methods:

[1003]

[1004] The components were sieved and mixed, and then filled into capsules of size 2.

[1005] Example C

[1006] Injection solutions may have the following compositions:

[1007]

[1008] Dissolve the active ingredient in a mixture of polyethylene glycol 400 and water for injection (partially). Adjust the pH to 5.0 by adding acetic acid. Adjust the volume to 1.0 ml by adding the remaining water. Filter the solution, fill vials with the appropriate excess, and sterilize.

Claims

1. A compound of formula (I) wherein (I) L is absent or alkyl; X is -O- or -NH-; n is 0 or 1 ; R1 is amino, aminoalkyl, alkylamino, aminoalkylamino, haloalkyl, diaminoalkyl, guanidinyl, (aminoalkyl)(aminoalkyl)(hydroxycarbonylalkyl)ammoniumalkyl, aminoalkyloxyalkyl, (aminoalkyl)(aminoalkyl)(aminocarbonylalkyl)ammoniumalkyl, (amino)(hydroxy)alkyl, (aminoalkyl)(aminoalkyl)amino, (aminoalkyl)(aminoalkyl)aminoalkyl, (aminoalkyl)(aminoalkyl)aminoalkoxy, (aminoalkyl)(aminoalkyl)aminoalkylamino, (aminoalkyl)(aminoalkyl)(hydroxycarbonyl)ammoniumalkylamino, (hydroxy)(halo)alkylamino, (aminoalkyl)(aminoalkyl)(hydroxycarbonylalkyl)ammoniumalkoxy, cycloalkyl, or heterocycloalkyl, wherein heterocycloalkyl and cycloalkyl are optionally substituted with one or two substituents independently selected from R6; with the proviso that if R1 is amino, then L is absent; R2 and R3 are independently selected from halogen; or R2 and R3 together with the carbon to which they are attached form cycloalkyl; R4 is halogen or alkyl; R5 is amino, hydroxyalkyl, aminoalkyl, alkylamino, aminoalkylamino, or piperazinyl; R6 is in each case independently selected from amino, alkyl, hydroxy, aminoalkyl, aminocarbonylalkyl, and aminoalkylcarbonylamino; R7 is hydrogen or hydroxy; A1 is -N- or -CH-; and A2 is -O- or -CH2-; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein L is absent.

3. The compound according to claim 1 or 2, wherein L is alkyl, in particular methyl.

4. The compound according to any one of claims 1 to 3, wherein n is 1.

5. The compound according to any one of claims 1 to 4, wherein X is -O-.

6. The compound according to any one of claims 1 to 3, wherein n is 0. ​ 7. The compound according to any one of claims 1 to 6, wherein R1 is amino, aminomethyl, aminoethyl, methylamino, aminoethylamino, aminopropylamino, trifluoromethyl, diaminomethyl, guanidinyl, aminoethoxymethyl, (aminopropyl)(aminopropyl)(hydroxycarbonylmethyl)ammoniopropyl, (aminopropyl)(aminopropyl)(aminocarbonylmethyl)ammoniopropyl, (amino)(hydroxy)ethyl, (aminopropyl)(aminopropyl)amino, (aminoethyl)(aminoethyl)aminopropyl, (aminopropyl)(aminopropyl)aminopropyl, (aminopropyl)(aminopropyl)aminoethoxy, (aminoethyl)(aminoethyl)aminoethoxy, (aminoethyl)(aminoethyl)aminoethylamino, (aminopropyl)(aminopropyl)(hydroxycarbonylmethyl)ammoniopropylamino, (hydroxy)difluoropropylamino, (hydroxy)trifluoropropylamino, (aminopropyl)(aminopropyl)(hydroxycarbonylmethyl)ammonioethoxy, cyclobutyl, azetidin-3-yl, azetidin-1-yl, morpholin-4-yl, pyrrolidin-1-yl, or 5-azoniaspiro[2.4]heptane, wherein cyclobutyl, azetidin-3-yl, azetidin-1-yl, morpholin-4-yl, pyrrolidin-1-yl, or 5-azoniaspiro[2.4]heptane is optionally substituted with one or two substituents independently selected from R6; with the proviso that if R1 is amino, then L is absent.

8. The compound according to any one of claims 1 to 6, wherein R1 is heterocycloalkyl optionally substituted with one or two substituents independently selected from R6.

9. The compound according to any one of claims 1 to 8, wherein R2 and R3 are both fluoro; or R2 and R3 together with the carbon to which they are attached form cyclopropyl.

10. The compound according to any one of claims 1 to 9, wherein R4 is chloro or methyl.

11. The compound according to any one of claims 1 to 10, wherein R5 is amino or hydroxyalkyl, in particular amino.

12. The compound according to any one of claims 1 to 11, wherein R6 is in each case independently selected from amino, methyl, hydroxy, aminomethyl, aminocarbonylmethyl, and aminoethylcarbonylamino.

13. The compound according to any one of claims 1 to 12, wherein R7 is hydrogen.

14. The compound according to any one of claims 1 to 13, wherein A1 is -CH-.

15. The compound according to any one of claims 1 to 14, wherein A2 is -CH2-.

16. A compound selected from cis-(4R)-4-amino-1-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazetidin-1-yl)cyclohexyl]methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolidin-2-one; Trans-(4R)-4-amino-l-[4-[4-[6-chloro-4-[difluoro-[4-(3-hydroxyazetidin-l- yl)cyclohexyl]methyl]-2-pyridinyl]piperazin-l-yl]sulfonylphenyl]pyrrolidin-2-one; 2-amino-N-[l-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] azetidin-3-yl]propanamide; (4R)-4-amino-l-[4-[4-[4-[[4-(3-aminopropylamino)cyclohexyl]-difluoro-methyl]- 6-chloro-2-pyridinyl]piperazin-l-yl]sulfonylphenyl]pyrrolidin-2-one; Trans-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] propanamide; Cis-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] propanamide; Cis-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] propanamide; Trans-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] propanamide; Trans-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] cyclobutanecarboxamide; Cis-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] cyclobutanecarboxamide; Cis-2-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl]-3- hydroxy-propanamide; Cis-l-(2-aminoethyl)-3-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidin-l- yl]phenyl]sulfonylpiperazin-l-yl]-6-chloro-4-pyridinyl]difluoro-methyl]cyclohexyl] urea; trans-(6S)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5,5-dimethyl-5-azapyronspiro[2,4]heptane-6-carboxamide; trans-(5R,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide; trans-(5S,6S)-5-(2-amino-2-oxo-ethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-5-methyl-5-azapyronspiro[2,4]heptane-6-carboxamide; Racemic-(4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one; Racemic-(4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one; 2-Amino-N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]acetamide; 4-(((1r,4r)-4-((2-(4-((4-((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)amino)-N,N-bis(3-aminopropyl)-N-(carboxymethyl)-4-oxobut-1-ammonium; N-((1r,4r)-4-((2-(4-((4-((R)-4-amino-2-oxopyrrolid-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-2-(2-aminoethoxy)acetamide; 3-Amino-N-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-2-hydroxypropionamide; 2,3-Diamino-N-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)propionamide; 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(diaminomethylene)urea; (2-Amino-2-oxo-ethyl)-[4-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]amino]-4-oxo-butyl]-bis(3-aminopropyl)ammonium; 3-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamoylamino]propyl-bis(3-aminopropyl)-(carboxymethyl)ammonium; 3-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-1,1-bis(3-aminopropyl)urea; 1-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-3-(3-aminopropyl)guanidine; N-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]methyl]-2,2,2-trifluoro-acetamide; N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(2-aminoethyl)amino]butyramide; N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(3-aminopropyl)amino]ethyl ester; 2-[[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamoyloxy]ethyl-bis(3-aminopropyl)-(carboxymethyl)ammonium; (2S,6S)-2,6-bis(aminomethyl)-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]morpholine-4-carboxamide; N-[4-[1-[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]cyclopropyl]cyclohexyl]-4-[bis(2-aminoethyl)amino]butyramide; N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]-N-hydroxy-butyramide; N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]butyramide; (4R)-4-amino-1-[4-[4-[6-chloro-4-[[4-[(2,2-difluoro-3-hydroxy-propyl)amino]cyclohexyl]-difluoro-methyl]-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one; (3R,4S)-3-amino-N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolidine-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]-4-hydroxy-pyrrolidine-1-carboxamide; 1-((1r,4r)-4-((2-(4-((4-(((R)-4-amino-2-oxopyrrolidone-1-yl)phenyl)sulfonyl)piperazin-1-yl)-6-chloropyridin-4-yl)difluoromethyl)cyclohexyl)-3-(2-(bis(2-aminoethyl)amino)ethyl)urea; N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester; (4R)-4-amino-1-[4-[4-[4-[4-[(4-aminocyclohexyl)-difluoro-methyl]-6-methyl-pyrimidin-2-yl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one; N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-methyl-pyrimidin-4-yl]-difluoro-methyl]cyclohexyl]-4-[bis(3-aminopropyl)amino]butyramide; (4R)-4-amino-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)pyrrolid-2-one; N-[4-[[2-[4-[4-[(4R)-4-amino-2-oxo-pyrrolid-1-yl]phenyl]sulfonylpiperazin-1-yl]-6-chloro-4-pyridyl]-difluoro-methyl]cyclohexyl]carbamate 2-[bis(2-aminoethyl)amino]ethyl ester; (4R)-4-amino-1-[4-[4-[4-[[4-[[4-(azacyclobutan-1-yl)cyclohexyl]-difluoro-methyl]-6-chloro-2-pyridyl]piperazin-1-yl]sulfonylphenyl]pyrrolid-2-one; ((3R)-1-(4-((4-(4-(difluoro((1r,4r)-4-((3,3,3-trifluoro-2-hydroxypropyl)amino)cyclohexyl)methyl)-6-methylpyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-5-oxopyrrolidone-3-yl)tert-butyl carbamate; Or its medicinal salt.

17. The compound according to any one of claims 1 to 16, which is used as a therapeutically active substance.

18. A pharmaceutical composition comprising: a compound according to any one of claims 1 to 16, and a therapeutically inert carrier.

19. Use of the compound according to any one of claims 1 to 16 for the treatment or prevention of bacterial infections, particularly wherein the bacteria are Gram-negative bacteria.

20. Use of the compound according to any one of claims 1 to 16 for the preparation of a medicament for the treatment or prevention of bacterial infection, particularly wherein the bacteria are Gram-negative bacteria.

21. The compound according to any one of claims 1 to 16, used for the treatment or prevention of bacterial infection, particularly wherein the bacteria are Gram-negative bacteria.

22. A method for treating or preventing bacterial infections, particularly Gram-negative bacterial infections, said method comprising administering an effective amount of a compound as defined in any one of claims 1 to 16.

23. The use, method, or compound for use according to any one of claims 19 to 22, wherein the Gram-negative bacteria are selected from Enterobacteriaceae, Neisseria gonorrhoeae, Haemophilus influenzae, Helicobacter pylori, Acinetobacter baumannii, and Pseudomonas aeruginosa.

24. The use, method, or compound for use according to claim 23, wherein the Gram-negative bacteria is Enterobacteriaceae, wherein Enterobacteriaceae is Klebsiella pneumoniae or Escherichia coli.

25. The present invention as described above.

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