ES2366432T3 - HEPATITIS VIRUS INHIBITORS C. - Google Patents

Abstract

A compound of formula I, including pharmaceutically acceptable salts thereof, wherein: (a) R1 is unsubstituted C3-7 cycloalkyl; (b) R2 is H, halo, C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkoxy, C6 or 10 aryl or heterocycle; wherein the heterocycle is a saturated, unsaturated, five, six or seven membered heterocycle, containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur; said aryl or heterocycle being substituted with R24; wherein R24 is H, halo or C1-6 alkyl; (c) R3 is H, halo, C1-6 alkoxy or C3-6 cycloalkoxy; (d) R4 is NH2 or -NH-R31; wherein R31 is -C (O) -R32, C (O) -NHR32 or C (O) -OR32, wherein R32 is C1-6 alkyl or C3-7 cycloalkyl; (e) R3 is H; and (f) Q is a saturated or unsaturated alkylene chain, with three to nine atoms, which optionally contains one to three heteroatoms independently selected from O or S.

Description

Reference to related requests

This application claims the priority benefit of U.S. Provisional Application Serial Number 60 / 382,103 filed on May 20, 2002 and 60 / 344,080 filed on December 20, 2001.

Background of the invention

Hepatitis C virus (HCV) is the main etiologic agent of 90% of all cases of non-non-B hepatitis (Choo et al., 1989, Kuo et al., 1989). The incidence of HCV infection is becoming a public health concern of increasing severity with 2-15% of infected individuals worldwide. Although primary infection with HCV is often asymptomatic, most HCV infections progress to a chronic state that can persist for decades. Of those with chronic HCV infections, it is believed that approximately 20-50% will eventually develop chronic liver disease (eg cirrhosis) and 2030% of these cases will lead to liver failure or liver cancer. As the current population infected with HCV ages, the morbidity and mortality associated with HCV is expected to triple.

An approved treatment for HCV infection uses interferon (IFN) that indirectly effects HCV infection by stimulating the host's antiviral response. IFN treatment is suboptimal, however, since a prolonged antiviral response occurs in less than 30% of treated patients. In addition, treatment with IFN induces a series of side effects of varying severity in more than 90% of patients (for example: acute pancreatitis, depression, retinopathy, thyroiditis). Therapy with a combination of IFN and ribavirin has provided a slightly longer prolonged response rate, but has not relieved the side effects induced by IFN.

The use of protease inhibitors, particularly those that selectively target HCV serine protease, has great potential to be useful in the treatment of HCV in patients by inhibition of HCV replication.

Among the compounds that have demonstrated efficacy in inhibiting HCV replication, such as selective HCV serine protease inhibitors, are the macrocyclic peptide compounds disclosed in International Application PCT / CAOO / 00353 (Publication No. WO00 / 59929), documents US 6,323 .180, WO 02/060926, WO 2004/037855, WO 03/064455 and WO 2004/039833.

The present invention describes selective inhibitors of the HCV serine protease NS3 / NS4A complex of improved chemical design in relation to the previously described invention, which have the potential to demonstrate complete cell permeability suitable for the treatment of HCV infected patients.

Summary of the invention

The present invention relates to compounds of Formula I, including pharmaceutically acceptable salts thereof,

image 1

35 in which:

image2

(to)

 R1 is unsubstituted C3-7 cycloalkyl;

(b)

 R2 is H, halo, C1-6 alkyl, C1-6 alkoxy, C1-6 cycloalkoxy, C6 or 10 aryl or heterocycle; wherein the heterocycle is a saturated, unsaturated, five, six or seven membered heterocycle, containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur; said aryl or heterocycle being substituted with R24; wherein R24 is H, halo, or C1-6 alkyl;

(C)

 R3 is H, halo, C1-6 alkoxy or C3-6 cycloalkoxy;

(d)

 R4 is NH2 or -NH-R31; where R31 is -C (O) -R32, C (O) -NHR32 or C (O) -OR3 ,; wherein R32 is C1-C6 alkyl,

or C3-7 cycloalkyl;

(and)

 R5 is H; Y

(F)

 Q is a saturated or unsaturated alkylene chain, with three to nine atoms, containing one to three heteroatoms independently selected from O or S.

The present invention also relates to a pharmaceutical composition useful for inhibiting HCV NS3 protease or for treating patients infected with hepatitis C virus, which comprises a therapeutically effective amount of a compound of the present invention and a pharmaceutically acceptable carrier. .

The present invention also relates to the use of the compounds of the invention in a method of treating mammals infected with the hepatitis C virus.

In addition, the present invention relates to the use of the compounds of the invention in a method of inhibiting HCV NS3 protease.

Detailed description of the invention

As used herein, the following definitions apply unless otherwise indicated. With respect to the cases in which (R) or (S) is used to designate the configuration of a substituent, it refers to the entire compound and not only to the substituent.

The term "halo", as used herein, refers to a halogen substituent selected from bromine, chlorine, fluorine or iodine.

The term "C1-6 alkyl", as used herein, refers to acyclic straight or branched chain alkyl substituents, containing from 1 to six carbon atoms and include, for example, methyl, ethyl, propyl. , butyl, tert-butyl, hexyl, 1-methyl ethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.

The term "C1-6 alkoxy", as used herein, refers to the radical -O (C1-6 alkyl) in which the alkyl is as defined above, containing up to six carbon atoms. Alkoxy includes, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. The last radical is commonly known as tert-butoxy.

The term "C3-7 cycloalkyl", as used herein, refers to a cycloalkyl substituent containing from three to seven carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The term "C3-7 cycloalkoxy", as used herein, refers to a C3-7 cycloalkyl group attached to an oxygen atom, such as, for example, butyloxy or cyclopropyloxy.

The term "C6 or 10 aryl," as used herein, refers to an aromatic monocyclic group containing 6 carbon atoms or an aromatic bicyclic group containing 10 carbon atoms, for example, aryl includes phenyl, 1 -naphthyl or 2-naphthyl.

The term "carboxy (C1-6 alkyl)", as used herein, refers to a carboxyl group (COOH) linked through a C1-6 alkyl group as defined above and includes, for example, butyric acid

The term "heterocycle", as used herein, refers to a monovalent radical obtained by removing a hydrogen from a saturated or unsaturated (including aromatic) heterocycle, of five, six or seven members, containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. In addition, the term "heterocycle" includes heterocycles, as defined above, that are fused to one or more other ring structures. Examples of suitable heterocycles include, but are not limited to, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, diazepine, 1H-imidazole, isoxazole, thiazole, tetrazol, piperidine, 1,4-dioxane, 4-morpholine, pyridine, pyrimidine, thiazolo [4,5-b] -pyridine, quinoline or indole, or the following heterocycles:

image3

image 1

The term "C1-6 alkyl-heterocycle", as used herein, refers to a heterocyclic radical as defined above linked through a branched alkyl chain or group, in which the alkyl as defined. It previously contains 1 to 6 carbon atoms. Examples of C1-6-Het alkyl include:

image4

In a preferred embodiment, the compounds of the present invention have the structure of Formula II

image 1

wherein R1, R2, R3, R4, R5 and Q are as defined for Formula I.

For compounds of Formula II, it is preferred that R1 is cyclopropyl. In addition, it is preferred that R2 is phenyl. It is further preferred that R3 be methoxy. In addition, it is further preferred that R 4 is NH 2 or tert-butoxycarbonyl NH-. It is also preferred that Q is - (CH2) nCH = C- in which n is 1-5. It is more preferred that n is 3 or

Four.

In a more preferred embodiment, the compounds of the present invention have the structure of Formula III

image5

image 1

where R4 is NH2 or tert-butoxycarbonylNH-, Q is (CH2) nCH = C- and n is 1-5. For compounds of Formula III, it prefer more than n be 3 or 4. In another preferred embodiment for compounds of Formula II, in which: R2 is

image6

The compounds of the present invention, by virtue of their basic moiety, can form salts by the addition of a pharmaceutically acceptable acid. Acid addition salts are formed from a compound of Formula I and a pharmaceutically acceptable inorganic acid, including, but not limited to, hydrochloric, hydrobromic, iohydric, sulfuric, phosphoric, or an organic acid, such as p-toluenesulfonic acid, methanesulfonic, acetic, benzoic, citric, malonic, fumaric, maleic, oxalic, succinic, sulfamic or tartaric. Therefore, the

Examples of such pharmaceutically acceptable salts include chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, citrate, acetate, malonate, fumarate, sulfamate and tartrate.

The salts of an amine group may also comprise quaternary ammonium salts in which the amino nitrogen has a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety.

The compounds of the present invention, which are substituted with an acidic group, may exist in the form of

20 salts formed through the addition of bases. Said base addition salts include those obtained from inorganic bases that include, for example, alkali metal salts (e.g., sodium and potassium), alkaline earth metal salts (e.g., calcium and magnesium), aluminum salts and ammonium salts In addition, suitable base addition salts include physiologically acceptable organic base salts such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N, N’-dibenzylethylenediamine, 2

Hydroxyethylamine, bis- (2-hydroxyethyl) amine, tri- (2-hydroxyethyl) amine, procaine, dibenzylpiperidine, N-benzyl-p-phenethylamine, dehydroabyethylamine, N, N'-bishydroabyethylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, ethylenediamine, ornithine, choline, N, N'-benzylphenethylamine, chloroprocaine, diethanolamine, diethylamine, piperazine, tris (hydroxymethyl) aminomethane and tetramethylammonium hydroxide and basic amino acids such as lysine, arginine and Nmethylglutamine. These salts can be prepared by procedures known to those skilled in the art.

image7

In addition, the compounds of the present invention, or a salt thereof, may show polymorphism. The present invention also includes any of said polymorphic forms.

The compounds of the present invention (Formula I, II or III) also contain two or more chiral centers and exist in different optically active forms. For example, compounds of Formula I may include a cyclopropyl of formula

image 1

in which each of C1 and C2 represents an asymmetric carbon atom in positions 1 and 2 of the ring of

10 cyclopropyl. Without taking into account other possible asymmetric centers in other segments of the compounds of formula I, the presence of these two asymmetric centers means that the compounds of formula I may exist as mixtures of diastereomers, such as the diastereomers of compounds of Formula II in the that Q is configured syn with respect to the amide or syn with respect to carbonyl, as shown below.

image 1

15 Alternatively, the structures can be seen as:

image 1

The present invention includes enantiomers and mixtures of enantiomers such as racemic mixtures.

As illustrated in the examples, racemic mixtures can be prepared and then separated into individual optical isomers, or these optical isomers can be prepared by chiral synthesis.

image8

The enantiomers can be resolved by procedures known to those skilled in the art, for example, by formation of diastereoisomeric salts that can be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of an enantiomer with a specific enantiomer reagent. It will be appreciated that when the desired enantiomer is converted to another chiral entity by a separation technique, then an additional step is required to form the desired enantiomeric form. Alternatively, specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.

Certain compounds of the present invention may also exist in different stable conformational forms that can be separable. Torsional asymmetry due to restricted rotation around an asymmetric single bond, for example, due to steric hindrance or ring tension, may allow the separation of different conformers. The present invention includes each conformational isomer of these compounds and mixtures thereof.

Certain compounds of the present invention may exist in zwitterionic form and the present invention includes each zwitterionic form of these compounds and mixtures thereof.

The compounds of the present invention are useful in the inhibition of HCV NS3 protease, as well as the prevention or treatment of hepatitis C virus infection and the treatment of subsequent pathological conditions. The treatment involves administering to a patient, in need of such treatment, a compound of the present invention, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

Those skilled in the art will recognize that the reference to treatment herein extends to prophylaxis as well as to the treatment of established infections or symptoms. This includes starting treatment before and after exposure to the virus. In addition, the present invention can be administered together with immunomodulators, such as interferons ,  or ; other antiviral agents such as ribavirin, amantadine; other HCV NS3 protease inhibitors; inhibitors of other targets in the HCV life cycle, including but not limited to, helicase, polymerase, metalloprotease or internal ribosome entry site (IRES); or combinations thereof. Additional agents may be combined with the compounds of the present invention to create a simple pharmaceutical form. Alternatively, these additional agents can be administered separately to a mammal as part of a multiple pharmaceutical form.

These procedures are useful in reducing the activity of HCV NS3 protease in a mammal. These procedures are useful for inhibiting viral replication in a mammal. If the pharmaceutical composition comprises only a compound of the present invention as the active component, such methods may further comprise the step of administering to said mammal an agent selected from an immunomodulatory agent, an antiviral agent, an HCV protease inhibitor or an inhibitor of other targets in the HCV life cycle such as helicase, polymerase or metalloprotease or IRES. Said additional agent may be administered to the mammal before, simultaneously with, or after administration of the compositions of the present invention.

The compounds of the present invention are also useful in the preparation and execution of screening or replication assays for antiviral compounds. In addition, the compounds of the present invention are useful in establishing or determining the binding site of other antiviral compounds with HCV NS3 protease, for example, by competitive inhibition.

The compounds of the present invention can be administered orally, parenterally, including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray or rectally, in unit pharmaceutical formulations containing vehicles, adjuvants and Conventional non-toxic pharmaceutically acceptable carriers.

The present invention also provides a pharmaceutical composition for use in the therapeutic procedure described above. A pharmaceutical composition of the present invention comprises an effective amount of a compound of Formula I in association with a pharmaceutically acceptable carrier, excipient or diluent.

The active ingredient in such formulations comprises from 0.1 percent to 99.9 percent by weight of the formulation. By "pharmaceutically acceptable" is meant that the vehicle, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The present pharmaceutical compositions are prepared by known procedures using well known and readily available ingredients. The compositions of the present invention can be formulated to provide rapid, prolonged or delayed release of the active ingredient after administration to the patient using procedures well known in the art. In making the compositions of the present invention, the active ingredient will usually be mixed with a vehicle, diluted in a vehicle or included in a vehicle that may be in the form of a capsule, sachet, paper or other container. When the vehicle serves as a diluent, it can be a solid, semi-solid or liquid material that acts as a vehicle, excipient or medium for the active ingredient. Therefore, the compositions may be in the form of tablets, pills, powders, beads, tablets, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), hard gelatin capsules and soft, suppositories, sterile injectable solutions, sterile packaged powders and the like.

image9

The compounds can be administered by a variety of routes including oral, intranasally, rectally, transdermally, subcutaneously, intravenously, intramuscularly and intranasally.

When administered orally, these compositions are prepared according to techniques well known in the field of pharmaceutical formulation. For oral administration, the compound is normally formulated with excipients such as binders, fillers, lubricants, expanders, diluents, disintegrating agents and the like as are known in the art.

For parenteral administration, the compound is formulated in parenterally acceptable, non-toxic and pharmaceutically acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, 5 percent dextrose, Ringer's solution or isotonic sodium chloride solution or agents suitable suspension, humectants or dispersion, such as sterile, tasteless, fixed oils, including synthetic mono or diglycerides and fatty acids, including oleic acid.

A compound of the present invention, or a salt or solvate thereof, can be formulated in unit pharmaceutical formulations comprising a dose between about 0.1 mg and about 1000 mg, or more, according to the particular treatment involved. An example of a unit pharmaceutical formulation comprises 5 mg of a compound of the present invention in a 10 ml sterile glass vial. Another example of a unit pharmaceutical formulation comprises about 10 mg of a compound of the present invention as a pharmaceutically acceptable salt in 20 ml of isotonic saline solution contained in a sterile vial.

The compounds of the present invention can also be administered to humans in a dosage range of 1 to 100 mg / kg of body weight in divided doses. A preferred dosage range is 1 to 20 mg / kg body weight orally in divided doses. It will be understood that, however, the specific dose level and dosage frequency for any particular patient may be varied and will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and the duration of action of the compound, the route of administration, age, body weight, general health, sex, diet, mode and timing of administration, excretion rate, combination of drugs, the severity of the particular condition and the host that He undergoes therapy.

General procedures for the synthesis of compounds included in the present invention are shown below. The preparations shown below are disclosed for purposes of illustration and should not be construed as limitations of the procedures for manufacturing the compounds by any other method.

It will be appreciated by those skilled in the art that numerous processes are available for the preparation of the compounds of the present invention. These compounds may be prepared by procedures that include methods known in the chemical art for the production of structurally analogous compounds or by a novel process described herein. A process for the preparation of these compounds (or a pharmaceutically acceptable salt thereof) and novel intermediates for the manufacture of these compounds provides additional features of the invention and is illustrated by the following procedures in which the meanings of the generic radicals are as defined above, unless otherwise indicated. It will be appreciated that it may be preferred or necessary to prepare said compound in which a functional group is protected using a conventional protective group and then remove the protective group to provide a compound of the present invention.

For example, the compounds of the present invention, which have the structure of Formula I, II or III, such as the compounds of Formula IIIA and IIIB, shown below, were synthesized, as shown in the following scheme, indirectly from compounds of formula IV, and directly from compounds of formula VIIA or VIIB.

image10

image 1

image 1

The -carboxylic acid of a compound of Formula VIIA or VIIB is coupled with R1SO2NH2, which was prepared by treatment of R1SO2Cl in a tetrahydrofuran solution saturated with ammonia, in the presence of a peptide coupling agent, such as CDI or EDAC, and in the presence of a base, such as 4-dimethylaminopyridine (4

5 DMAP) and / or 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), to form a compound of Formula I, II or III.

Compounds having the structure of Formula IV, VI, VIIA or VIIB can be prepared as described herein, and in the non-provisional application U.S.S.N. 10 / 001,850 filed on November 20, 2001; International Application Number PCT / CA00 / 00353, Publication No. WO 00/59929, published on October 12, 2000 entitled "Macrocyclic Peptides Active Against the Hepatitis C Virus"; and U.S. Patent 6,323,180

10 granted on November 27, 2001 (corresponding to International Application Number PCT / CA99 / 00736, Publication No. WO 00/09543), entitled "Hepatitis C Inhibitor TriPeptides". The disclosure of US Patent 6,323,180 is incorporated herein, in its entirety, by reference.

Compounds having the structure of Formula IV can also be prepared by coupling the chemical precursor A with the chemical precursor B as shown below.

image11

image 1

The chemical precursor A, which has been shown above, can be synthesized as described in the following.

image 1

In this scheme, the ethyl ester (A1) is condensed with the appropriate aniline (A2), in the presence of acid, to form an imine. Then, the imine is cycled by heating at approximately 260-280 ° C to give the 4

image12

5

10

fifteen

twenty

25

30

35

40

corresponding hydroxyquinoline (A3). Subsequently, 4-hydroxyquinoline (A3) is heated to reflux with phosphorus oxychloride to form a 4-chloroquinoline (A4).

Then, commercially available Boc-4 (R) -hydroxyproline (A5), in a suitable solvent such as DMSO, is treated with a base, for example, sodium hydride or potassium tert-butoxide, to form an A5 alkoxide which is then reacted with 4-chloroquinoline (A4) to give the chemical precursor A.

An alternative means for forming the chemical precursor A, and for forming fragments or precursors of the chemical precursor (A), is described in International Application Number PCT / CA00 / 00353, Publication No. WO 00/59929, entitled "Macrocyclic Peptides Active Against the Hepatitis C Virus ", and US Patent 6,323,180 issued on November 27, 2001 (corresponding to International Application Number PCT / CA99 / 00736, Publication No. WO 00/09543), entitled" Hepatitis C Inhibitor Tri-Peptides " . The disclosure of US Patent 6,323,180 is incorporated herein, in its entirety, by reference.

Chemical precursor B, which has also been shown above, can be synthesized as described in the following scheme with slight modifications represented in the experimental section of this document.

image 1

Treatment of the commercially available imine (B1), or which can be easily obtained with 1,4-dihalobutene (B2) in the presence of a base, provides the resulting imine (B3). The acid hydrolysis of B3 then provides B, which has an allyl syn substituent with respect to the carboxyl group. It is preferred, for compounds B3 and B, that the vinyl group be syn with respect to the ester.

Exemplification

The specific examples shown below illustrate the synthesis of the compounds of the present invention, and should not be considered as limitations of the invention in its scope or scope. The processes can be adapted to variations in order to produce compounds included by this invention but not specifically disclosed. In addition, variations in the processes for producing the same compounds in a different way will also be apparent to one skilled in the art.

Chemical abbreviations commonly used to identify chemical compounds in the literature include Bn: benzyl; Boc: tert-butyloxycarbonyl {Me3COC (O)}; BSA: bovine serum albumin; CDI: carbonyldiimidazole; DBU: 1,8diazabicyclo [5.4.0] -undec-7-eno; CH2Cl2 = DCM: methylene chloride; DEAD: diethyl azodicarboxylate; DIAD: diisopropyl azodicarboxylate; DIEA: diisopropylethylamine; DIPEA: diisopropylethylamine; 4-DMAP: 4-dimethylaminopyridine; DCC: 1,3-dicyclohexyl carbodiimide; DMF: dimethylformamide; DMSO: dimethylsulfoxide; DPPA: diphenylphosphorylazide; EDAC: ethyldimethylaminopropylcarbodiimide hydrochloride; EDTA: ethylenediaminetetraacetic acid; Et: ethyl; EtOH: ethanol; EtOAc: ethyl acetate; Et2O: diethyl ether; Grubb catalyst: bis (tricyclohexylphosphine) benzylidene ruthenium dichloride (IV); HATU: [O-7-azabenzotriazol-1-yl) -1, HBTU: [O (1H-benzotriazol-1-yl) -N, N, N ’, N’-tetramethyluronium hexafluorophosphate; PYBROP: bromo-tris-pyrrolidine-phosphonium hexafluorophosphate; HOAT, 1-hydroxy-7-azabenzotriazole; HPLC: high performance liquid chromatography; MS: mass spectrometry; Me: methyl; MeOH: methanol; NMM: N-methylmorpholine; NMP: N-methylpyrrolidine; Pr: propyl; Succ: 3-carboxypropanoyl; PPA: polyphosphoric acid; TBAF: tetra-n-butylammonium fluoride; 1,2-DCE or DCE: 1,2-dichloroethane; TBTU: 2- (1 H -benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate; TFA: trifluoroacetic acid; THF: tetrahydrofuran

Solution percentages express a weight-to-volume ratio, and solution ratios express a volume-to-volume ratio, unless otherwise indicated. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300, 400 or 500 MHz spectrometer; chemical shifts () are indicated in parts per million. Flash chromatography was performed on silica gel (SiO2) evident to a person skilled in the art. All Liquid Chromatography (CL) data was recorded on a Shimadzu LC-10AS liquid chromatograph using a SPD-10AV UV-Vis detector and Mass Spectrometry (EM) data was determined with a Micromass Platform for CL in mode electrospray (EN +).

image13

Unless otherwise indicated, each compound was analyzed, by LC / MS, using one of five methodologies, which has the following conditions.

Columns:

(Procedure A) - YMC ODS S7 C18 3.0 x 50 mm (Procedure B) - YMC ODS-A S7 C18 3.0 x 50 mm (Procedure C) - YMC S7 C18 3.0 x 50 mm

10 (Procedure D) - YMC Xterra ODS S7 3.0 x 50 mm (Procedure E) - YMC Xterra ODS S7 3.0 x 50 mm (Procedure F) - YMC ODS-A S7 C18 3.0 x 50 mm (Procedure G) - YMC C18 S5 4.6 x 50 mm] (Procedure H) - Xterra S7 3.0 x 50 mm

15 (Procedure I) - Xterra S7 C18 3.0 x 50 mm

Gradient:

100% Solvent A / 0% Solvent B a

0% Solvent A / 100% Solvent B

Gradient time: 2 min. (A, B, D, F, G, H, I); 8 min. (C, E)

20 Maintenance time: 1 min. (A, B, D, F, G, H, I); 2 min. (C, E) Flow rate: 5 ml / min Detector wavelength: 220 nm Solvent A: 10% MeOH / 90% H2O / 0.1% TFA Solvent B: 10% H2O / 90% MeOH / 0.1% TFA.

25 (Procedure J) - YMC Xterra ODS S7 3.0 x 50 mm

Gradient:

100% Solvent A / 0% Solvent B a

0% Solvent A / 100% Solvent B

Gradient time: 4 min (A)

30 Maintenance time: 1 min (A); 2 min Flow rate: 4 ml / min Detector wavelength: 220 nm Solvent A: 10% MeOH / 90% H2O / 0.1% TFA Solvent B: 10% H2O / 90% MeOH / 0.1 TFA %.

The chemical compounds and intermediates of the present invention, described in the following examples, were prepared according to the following procedures. For the description of the examples, in a synthetic stage such as 1a, the number refers to the example number and the letter refers to the relative sequential stage in the sequence.

Example 1

Step 1a: Preparation of 4-hydroxy-2-phenyl-7-methoxyquinoline

image 1

To a solution of m-anisidine (300 g, 2.44 mol) and ethyl benzoylacetate (234.2 g, 1.22 mol) in toluene (2.0 L) was added HCl (4.0 N in dioxane , 12.2 ml, 48.8 mmol). The resulting solution was heated at reflux for 6.5 h using a Dean-Stark apparatus (approximately 56 ml of aqueous solution was collected). The mixture was cooled to rt, partitioned multiple times with aqueous HCl (10%, 3 x 500 ml), aqueous NaOH (1.0 N, 2 x 200 ml) and water (3 x 200

image14

ml) and the organic phase was dried (MgSO4) and concentrated in vacuo to provide an oily residue (329.5 g). The crude product was heated in an oil bath (280 ° C) for 80 min using a Dean-Stark apparatus (approximately 85 ml of liquid was collected). The reaction mixture was cooled to rt, the solid residue was triturated with CH2Cl2 (400 ml), the resulting suspension was filtered and the filter cake was washed with more CH2Cl2 (2 x 150 ml). Solid

The resulting was dried under vacuum (50 ° C; 0.13 KPa (1 torr); 1 day), providing analytically pure 4-hydroxy-7-methoxy-2-phenylquinoline as a light brown solid (60.7 g, 20% in total): 1H NMR D (DMSO) 3.86 (s, 3H), 6.26 (s, 1H), 6.94 (dd, J = 9.0, 2.4 Hz, 1H ), 7.21, (d, J = 2.4 Hz, 1H), 7.56 7.61 (m, 3H), 7.80-7.84 (m, 2H), 8.00 (d, J = 9.0 Hz, 1H), 11.54 (s, 1H); 13C NMR (DMSO-d6)  55.38, 99.69, 107.07, 113.18, 119.22, 126.52, 127.17, 128.97, 130.34, 134.17, 142, 27, 149.53, 161.92, 176.48.

10 Stage 1b: Preparation of 4-chloro-7-methoxy-2-phenylquinoline

image 1

The 4-hydroxy-2-phenyl-7-methoxyquinoline (21.7 g, 86.4 mmol) was suspended in POCl3 (240 ml). The suspension was heated at reflux for 2 hours. After removing the POCl3 in vacuo, the residue was partitioned between EtOAc (1 L) and aqueous cooled NaOH (generated from 200 ml of 1.0 N NaOH and 20 ml of 10.0 N NaOH) and stirred for 15 min. The

The organic phase was washed with water (2 x 200 ml) and brine (200 ml), dried (MgSO4) and concentrated in vacuo to give 4-chloro-2-phenyl-7-methoxyquinoline (21.0 g, 90%) in form of a light brown solid. 1H NMR (DMSO-d6) ,9 3.97 (s, 3H), 7.36 (dd, J = 9.2, 2.6 Hz, 1H), 7.49-7.59 (m, 4H), 8.08 (d, J = 9.2 Hz, 1H), 8.19 (s, 1H), 8.26-8.30 (m, 2H): 13C NMR (DMSO-d6)  55.72, 108.00, 116.51, 119.52, 120.48, 124.74, 127.26, 128.81, 130.00, 137.58, 141.98, 150.20, 156.65, 161, 30 LC-MS (Procedure D: retention time: 1,547), MS m / z 270 (M ++ 1).

Step 1c: Preparation of N-Boc- (2S, 4R) - (2-phenyl-7-methoxy quinolin-4-oxo) proline

image 1

To a suspension of N-Boc- (2S, 4R) -hydroxyproline (16.44 g, 71.1 mmol) (Fluka) in DMSO (250 ml) was added t-BuOK (19.93 g, 177.6 mmol) at 0 ° C. The generated mixture was stirred for 1.5 hour and then 4-chloro-7-methoxy-2-phenylquinoline (21.02 g, 77.9 mmol) was added in three portions for 1 h. The reaction was stirred for one day and the reaction mixture was poured into ice-cold water (1.5 L) and washed with Et2O (4 x 200 ml). The aqueous solution was acidified to pH 4.6, filtered to obtain a white solid and dried under vacuum, yielding the product N-Boc- (2S, 4R) - (2-phenyl-7-methoxyquinolin-4-oxo) proline ( 32.5 g, 98%): 1H NMR (DMSO)  1.30, 1.32 (2s, 9H), 2.27-2.31 (m, 1H), 2,582.69 (m, 1H), 3.73 (s, 2H), 3.88 (s, 3H), 4.30-4.35 (m, 1H), 5.72 (s, 1H), 7.11-7.17 (m, 1H), 7.35-7.57 (m, 5H), 7,927.97 (m, 1H), 8.24 (d, J = 9.0 Hz, 2H), 12.76 (sa, 1H); LC-MS (Procedure D: retention time: 2.01; MS m / z

30 465 (M ++ H).

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Stage 1d: Preparation of racemic ethyl ester hydrochloride (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid (Procedure A and Procedure B) and chiral resolution of this racemate for the preparation of ethyl ester hydrochloride of the N- (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid (Method C)

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The named compound was made (stage d) racemic by each of the following Procedures A and B. This chiral racemate can also be resolved by providing the chiral Boc- (1R, 2S) -1-amino-2-vinylcyclopropylcarboxylic acid ester that was deprotected under acidic conditions, providing (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid ester hydrochloride (Method C).

10 Procedure A

A.1 Preparation of glycine ethyl ester N-benzyl imine

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Glycine ethyl ester hydrochloride (303.8 g, 2.16 mol) was suspended in tert-butylmethyl ether (1.6 L). They were added

Benzaldehyde (231 g, 2.16 mol) and anhydrous sodium sulfate (154.6 g, 1.09 mol) and the mixture was cooled to 0 ° C using an ice-cold water bath. Triethylamine (455 ml, 3.26 mol) was added dropwise over 30 min and the mixture was stirred for 48 h at rt. Then, the reaction was stopped by the addition of ice-cold water (1 L) and the organic phase was separated. The aqueous phase was extracted with tert-butylmethyl ether (0.5 L) and the combined organic phases were washed with a mixture of saturated aqueous NaHCO3 (1 L) and brine (1 L). The solution was dried over MgSO4 and

20 concentrated in vacuo to provide 392.4 g of the N-benzyl imine product as a thick yellow oil that was used directly in the next step. 1H NMR (CDCl3, 300 MHz)  1.32 (t, J = 7.1 Hz, 3H), 4.24 (c, J = 7.1 Hz, 2H), 4.41 (d, J = 1 , 1 Hz, 2H), 7.39-7.47 (m, 3H), 7.78-7.81 (m, 2H), 8.31 (s, 1H).

A.2 Preparation of racemic N-Boc- (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester

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To a suspension of lithium tert-butoxide (84.06 g, 1.05 mol) in dry toluene (1.2 L) was added dropwise a mixture of N-benzyl imine from the glycine ethyl ester (100 , 4 g, 0.526 mol) and trans-1,4-dibromo-2-butene (107.0 g, 0.500 mol) in dry toluene (0.6 l) for 60 min. After the addition was completed, the dark red mixture was quenched by the addition of water (1 L) and tert-butyl methyl ether (TBME, 1 L). The aqueous phase was separated and extracted a second time with TBME (1 L). The organic phases were combined, 1N HCl (1 L) was added and the mixture was

30 stirred at room temperature for 2 h. The organic phase was separated and extracted with water (0.8 L). Then, the aqueous phases were combined, saturated with salt (700 g), TBME (1 L) was added and the mixture was cooled to 0 ° C. The stirred mixture was then acidified to pH 14 by the dropwise addition of 10 N NaOH, the organic phase was

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separated and the aqueous phase was extracted with TBME (2 x 500 ml). The combined organic extracts were dried (MgSO4) and concentrated to a volume of 1 L. To this free amine solution was added BOC2O or di-tert-butyl dicarbonate (131.0 g, 0.6 mol) and the mixture was stirred for 4 days at rt. To the reaction was added more di-tert-butyl dicarbonate (50 g, 0.23 mol), the mixture was heated at reflux for 3 h and then allowed to cool to room temperature overnight. The reaction mixture was dried over MgSO4 and concentrated in vacuo to provide 80 g of crude material. This residue was purified by flash chromatography (2.5 kg of SiO2, eluting with 1% to 2% MeOH / CH2Cl2), providing 57 g (53%) of N-Boc acid ethyl ester (1R, 2S) / ( Racemic 1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid in the form of a yellow oil that solidified while resting in the refrigerator: 1H NMR (CDCl3, 300 MHz)  1.26 (t, J = 7.1 Hz , 3H), 1.46 (s, 9H), 1,431.49 (m, 1H), 1.76-1.82 (ma, 1H), 2.14 (c, J = 8.6 Hz, 1H) , 4.18 (c, J = 7.2 Hz, 2H), 5.12 (dd J = 10.3, 1.7 Hz, 1H), 5.25 (sa, 1H), 5.29 (dd , J = 17.6, 1.7 Hz, 1H), 5.77 (ddd, J = 17.6, 10.3, 8.9 Hz, 1H); MS m / z 254.16 (M-1)

A.3 Preparation of racemic ethyl ester hydrochloride (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid

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N-Boc- (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester (9.39g, 36.8mmol) was dissolved in 4N HCl / dioxane (90ml, 360 mmol) and stirred for 2 h at rt. The reaction mixture was concentrated to provide (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester hydrochloride in quantitative yield (7 g, 100%). 1H NMR (methanol-d4)  1.32 (t, J = 7.1, 3H), 1.72 (dd, J = 10.2, 6.6 Hz, 1H), 1.81 (dd, J = 8.3, 6.6 Hz, 1H), 2.38 (c, J = 8.3 Hz, 1H), 4.26-4.34 (m, 2H), 5.24 (dd, 10, 3, 1.3 Hz, 1H) 5.40 (d, J = 17.2, 1H), 5.69-5.81 (m, 1H).

Procedure B

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To a solution of potassium tert-butoxide (11.55 g, 102.9 mmol) in THF (450 ml) at -78 ° C was added the N, Ndibenzylimine of the commercially available ethyl glycine ester (25.0 g , 93.53 mmol) in THF (112 ml). The reaction mixture was heated to 0 ° C, stirred for 40 min and then cooled again to -78 ° C. To this solution was added trans-1,4-dibromo-2-butene (20.0 g, 93.50 mmol) and the mixture was stirred for 1 h at 0 ° C and cooled again to -78 ° C. Potassium tert-butoxide (11.55 g, 102.9 mmol) was added, the mixture was immediately heated to 0 ° C and stirred for another hour before being concentrated in vacuo. The crude product was taken up in Et2O (530 ml), a 1N aqueous solution of HCl (106 ml, 106 mmol) was added and the resulting biphasic mixture was stirred for 3.5 h at rt. The phases were separated and the aqueous phase was washed with Et2O (2 x) and made alkaline with a saturated aqueous solution of NaHCO3. The desired amine was extracted with Et2O (3 x) and the combined organic extract was washed with brine, dried (MgSO4) and concentrated in vacuo to obtain the free amine. This material was treated with a 4 N solution of HCl in dioxane (100 ml, 400 mmol) and concentrated to provide ethyl ester hydrochloride (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid in the form of a semi-solid brown color (5.3 g, yield 34%) identical to the material obtained from procedure A, except for the presence of a small unidentified aromatic impurity (8%).

Procedure C

C.1 Resolution of N-Boc- (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester hydrochloride

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A phosphate buffer solution (pH 8.0, 6.12 g, KH2PO4, 121.41 g, Na2PO4, in 9 l of water) was heated to 40 ° C in a reactor with a heating jacket with a mechanical stirrer. If necessary, the pH is adjusted to 8.0 with the addition of phosphoric acid. A solution (1.8 kg) of Bacillus globigii protease enzyme (Sigma) was added and the pH of the resulting mixture was adjusted to 8.0 by the addition of 50% NaOH. To this mixture was then added the racemic ester (180 g, 0.75 mmol) in the form of a solution in DMSO maintaining the pH at 8.0. Aliquots (1 ml) of the reaction mixture were taken several times and analyzed by the following procedure: 1) Acidification at pH 4 by the addition of sulfuric acid 6 (N). 2) The aliquot (1 ml) was extracted with MTBE (2 ml) and the MTBE extract was divided into two portions and evaporated. 3) A portion was dissolved again in acetonitrile and analyzed by HPLC to determine the conversion: HPLC column: YMC Propack C-18, 150 X 4.6 mm, S-3 µm. 40 ºC Solvent: Gradient from 70:30 to 10:90 of 0.2% H3PO4 -CH3CN in 15 minutes, Re-equilibrated back to original in 16 minutes and continuing until 20 min before the next injection. Flow rate: 1 ml / min, Detection: UV 210 nm. Elution Time: Acid 4.7 min, Ester 8 min. 4) The second portion was dissolved again in the mobile phase and analyzed by HPLC to determine the enantiomeric excess: Column: Chiralpak AD, Ambient Temperature, Solvent: Heptane: Ethanol (Absolute, graduation 200): Trifluoroacetic acid (97.4 : 2.5: 0.1) Flow rate: 1 ml / min, Detection: UV 210 nm Elution: Enantiomer (1R, 2S) desired 9.2 min, enantiomer (1S, 2R) unwanted 9.7 min

Note: The reaction was terminated when the analysis showed that the enantiomeric excess of the ester had reached the desired level (usually ee> 95% in 16-24 h). After 22 hours, the reaction was cooled to room temperature. The pH was 7.75. Analyzes of an aliquot (see above) showed an acid to ester ratio of

55:45 Sodium hydroxide (10%) was added to the reaction mixture to bring the pH to 8.5. The aqueous phase was extracted with MTBE (2 x 6 liters) until no ester remained in the aqueous phase. The MTBE extracts were washed with 5% NaHCO3 (4 liters) to remove any remaining acid and then with water (2 x 4 L). The solvent was evaporated, giving the ester 87.9 g (97%, based on a theoretical maximum yield of 90 g) as a yellow oil.

C.2 Preparation of chiral ethyl ester hydrochloride (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid

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N-BOC- (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester (8.5 g, 33.3 mmol) was stirred under an N2 atmosphere with 200 ml of 4 N HCl / dioxane (Aldrich ) at rt for 3 h. The solvent was removed under reduced pressure keeping the temperature below 40 ° C. This gave 6.57 g (~ 100%) of (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl hydrochloride as a light brown solid. 1H NMR (300 MHz, CD3OD)  1.31 (t, J = 7.0 Hz, 3 H), 1.69-1.82 (m, 2 H), 2.38 (c, J = 8, 8 Hz, 1 H), 4.29 (c, J = 7.0 Hz, 2 H), 5.22 (d, J = 10.3 Hz, 1 H), 5.40 (d, J = 17 , 2 Hz, 1 H), 5.69-5.81 (m, 1 H). LC-MS (Procedure J, retention time: 0.58 min), MS m / 2 156 (M ++ 1).

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Step 1e: Preparation of 2 (S) - (1 (R) -ethoxycarbonyl-2 (S) -vinylcyclopropylcarbamoyl) -4 (R) - (7-methoxy-2-phenyl-quinolin-4-yloxy) tert-butyl ester pyrrolidin-1-carboxylic acid (Diastereomer with high Fr) and tert-butyl ester of 2 (S) (1 (S) -ethoxycarbonyl-2 (R) -vinylcyclopropylcarbamoyl) -4 (R) - (7-methoxy-2- phenylquinolin-4-yloxy) -pyrrolidin-1-carboxylic acid (Diastereomer with low Fr)

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Procedure A: To a solution of Boc- (2S, 4R) - (2-phenyl-7-methoxyquinolin-4-oxo) proline (11.0 g, 23.7 mmol), ethyl acid ester hydrochloride (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid in which the carboxy group is syn to the vinyl residue (5.40 g, 28.2 mmol), NMM (20.8 ml; 18.9 mmol) in 500 ml of 50% CH2Cl2 / THF was added in three portions PyBrop coupling reagent or bromotrispirrolidinphosphonium hexafluorophosphate 10 (16.0 g, 34.3 mmol) for 10 min at 0 ° C. The solution was stirred at rt for one day and then washed with pH 4.0 buffer (4 x 50 ml). The organic phase was washed with saturated aqueous NaHCO3 (100 ml), the aqueous wash was extracted with ethyl acetate (150 ml) and the organic phase was washed again with buffer pH 4.0 (50 ml) and saturated aqueous NaHCO3 ( 50 ml) The organic solution was dried (MgSO4), concentrated and purified using a 65M Biotage column (eluting with 50% EtOAc / Hexanes) to provide 7.5 g of a 1: 1 mixture of diastereomers (1R, 2S) and

15 (1S, 2R) of cyclopropane (50% in total) or, alternatively, elution using a slow gradient of 15% to 60% EtOAc in hexanes, providing 3.54 g, 25% in total of the diastereomer (1R, 2S) of cyclopropane eluted with high Fr and 3.54 g, 25% in total of the diastereomer (1S, 2R) of cyclopropane eluted with low Fr.

Procedure B: A solution of (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester hydrochloride (7.5 g, 39.1 mmol) was combined with diisopropylethylamine (32.5 ml, 186 mmol) in dichloromethane (150 ml). To the resulting mixture was added HOBT hydrate (6.85 g, 44.7 mmol) and N-Boc (2S, 4R) (2-phenyl-7-methoxy-quinolin-4-oxo) proline (17.3 g , 37.3 mmol) followed by the addition of HBTU (16.96 g, 44.7 mmol). A slight exothermic reaction occurred immediately and the mixture was stirred at room temperature overnight. Then, the mixture was concentrated in vacuo and dissolved again in ethyl acetate (600 ml). The solution was washed with water (2 x 200 ml), then with 10% aqueous sodium bicarbonate (2 x 200 ml), then with water (150 ml) and finally with brine (150 ml). The organic phase was dried over anhydrous magnesium sulfate and filtered and the filtrate was concentrated in vacuo to give a beige crystalline solid. Purification was performed in several batches (7 g each) by flash chromatography on a Biotage Flash 75M cartridge (66% hexanes / ethyl acetate), providing the diastereomer (1R, 2S) of cyclopropane eluted with high Fr (9, 86 g in total, 44.0 yield

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%), followed by the cyclopropane diastereomer eluted with low Fr (10.43 g in total, yield 46.5%). A total of 1.97 g of the mixture fractions were recovered, giving a total conversion of 99.3% in the two diastereomers.

Data for 2 (S) - (1 (R) -ethoxycarbonyl-2 (S) -vinylcyclopropylcarbamoyl) -4 (R) - (7-methoxy-2-phenylquinolin-4-yloxy) pyrrolidine-1-carboxylic acid tert-butyl ester (Cyclopropane Diastereomer with High Fr): 1H NMR (CDCl3)  1.22 (t, J = 7.1 Hz, 3H), 1.44 (s, 9H), 1.48-1.58 (m, 1H), 1.89 (sa, 1H), 2.05-2.20 (m, 1H), 2.4 (sa, 1H), 2.90 (sa, 1H), 3.80, 3.87 (2bs, 2H), 3.94 (s, 3H), 4.07-4.22 (m, 2H), 4.56 (sa, 1H), 5.13 (d, J = 10.2 Hz, 1H), 5.30 (d, J = 17.1 Hz, 1H), 5.70-5.82 (m, 1H), 7.03 (sa, 1H), 7.09 (dd, J = 9 , 1, 2 Hz, 1H), 7.42-7.53 (m, 4H), 7.61 (sa, 1H), 7.96 (d, J = 9.1 Hz, 1H), 8.05 (d, J = 7.2 Hz, 2H); 1H NMR (methanol-d4)  1.23 (t, J = 7.2 Hz, 3H), 1.4 (s, 4H), 1.45 (s, 6H), 1.73 (dd, J = 7.9, 1.5 Hz, 0.4H), 1.79 (dd, J = 7.8, 2.4 Hz, 0.6H), 2.21 (c, J = 8.2 Hz, 1H ), 2.44-2.49 (m, 1H), 2.66-2.72 (m, 0.4H), 2.73-2.78 (m, 0.6H), 3.93-3 , 95 (m, 2H), 3.96 (s, 3H), 4.10-4.17 (m, 2H), 4.44 (c, J = 7.8 Hz, 1H), 5.13 ( d, J = 10.7 Hz, 1H), 5.31 (d, J = 17.7 Hz, 0.4H), 5.32 (d, J = 17.4 Hz, 0.6H), 5, 49 (sa, 1H), 5.66-5.82 (m, 1H), 7.16 (dd, J = 9.2, 2.5 Hz, 1H), 7.26 (s, 1H), 7 , 42 (d, J = 2.4 Hz, 1H), 7.48-7.55 (m, 3H), 8.02-8.05 (m, 3H); 13C NMR (CDCl3)  14.22; 22.83, 28.25, 33.14, 33.58, 39.92, 51.84, 55.47, 58.32, 61.30, 75.86, 81.27, 98.14, 107, 42, 115.00, 117.84, 118.27, 122.63, 123.03, 127.50, 128.72, 129.26, 133.39, 140.06, 151.23, 159.16, 160.34, 161.35, 169.78, 171.68. LC-MS (Procedure D: retention time: 1.62), MS m / z 602 (M ++ 1).

Data for 2 (S) - (1 (S) -ethoxycarbonyl-2 (R) -vinylcyclopropylcarbamoyl) -4 (R) - (7-methoxy-2-phenylquinolin-4-yloxy) pyrrolidin-1 acid tert-butyl ester -carboxylic (Cyclopropane Diastereomer with Low Fr): 1 H NMR (CDCl 3)  1.25 (t, J = 7.2 Hz, 3 H), 1.45 (s, 9 H), 1.35-1, 52 (m, 1 H), 1.85 (sa, 1 H), 2.13-2.22 (m, 1H), 2.4 (sa, 1 H), 2.95 (sa, 1 H) , 3.83 (sa, 2 H), 3.99 (s, 3 H), 4.13 (c, J = 7.2 Hz, 2 H), 4.59 (s, 1 H), 5, 16 (d, J = 10.8 Hz, 1 H), 5.33 (d, J = 17.2 Hz, 1 H), 5.40 (sa, 1 H), 5.74-5.86 ( m, 1 H), 7.06 (s, 1 H), 7.14 (dd, J = 9.2, 2.3 Hz, 1 H), 7.46-7.6 (m, 3 H) , 8.00 (d, J = 9.2, 1 H), 8.08, 8.10 (2s, 2H). LC-MS (Procedure D: retention time: 1.66), MS m / z 602 (M ++ 1).

Step 1f: Preparation of 1 - {[4- (7-methoxy-2-phenylquinolin-4 (R) -yloxy) pyrrolidin-2 (S) -carbonyl] -1 (R) amino} -2 ( S) -vinylcyclopropanecarboxylic dihydrochloride

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5.88 g (9.77 mmol) of 2 (S) - (1 (R) -ethoxycarbonyl-2 (S) vinylcyclopropylcarbamoyl) -4 (R) - (7-methoxy-2 acid tert-butyl ester were dissolved -phenyl-quinolin-4-yloxy) pyrrolidin-1-carboxylic acid (Cyclopropane Diastereomer with High Fr) in HCl / dioxane (4.0 M; 200 ml) and stirred for 2.5 h at rt. The reaction mixture was concentrated to provide the title compound that was used directly in the next step. 1H NMR (methanold4)  1.25 (t, J = 7.1 Hz, 3H), 1.51 (dd, J = 9.6, 5.4 Hz, 1H), 1.78 (dd, J = 8.1, 5.5 Hz, 1H), 2.30 (dd, J = 17.7.8.7 Hz, 1H), 2.27-2.67 (m, 1H), 3.06 (dd , J = 14.6, 7.4 Hz, 1H), 3.56-3.76 (m, 3H), 3.99 (s, 2H), 4.07 (s, 3H), 4.16 ( c, 2H), 4.77 (dd, J = 10.4, 7.4 Hz, 1H), 5.13 (dd, J = 10.3, 1.7 Hz), 5.33 (dd, J = 17.1, 1.7 Hz), 5.71-5.81 (m, 1H), 6.06 (sa, 1H), 7.49 (dd, J = 9.4, 2.3 Hz, 1H), 7.66-7.77 (m, 4H), 8.12-816 (m, 2H), 8.55 (d, J = 9.4 Hz, 1H). 13C NMR (methanold4) ,7 14.77, 23.23, 34.86, 37.25, 41.19, 43.90, 52.66, 60.35, 62.32, 62.83, 68.27, 72.58, 73.70, 81.21, 100.70, 102.44, 116.13, 118.67, 122.25, 126.93, 130.27, 130.94, 133.19, 134, 14, 134.89, 143.79, 158.39, 166.84, 167.44, 169.57, 171.33. LC-MS (Procedure D: retention time: 1.55), MS m / z 502 (M ++ 1)

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Examples 1.2 Preparation of Compounds 1 and 2

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Step 1 g: Preparation of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-hex-5-enoyl) -4- (7-methoxy-2-phenylquinolin-4 (R) -yloxy acid ethyl ester ) pyrrolidin-2 (S) -carbonyl] -1 (R) -amino] -2 (S) -vinylcyclopropanecarboxylic

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To a suspension of (0.600 g; 1.04 mmol) of 1 - {[4- (7-methoxy-2-phenylquinolin-4 (R) yloxy) pyrrolidin-2 (S) -carbonyl] -1-ethyl ester (R) -amino] -2 (S) -vinylcyclopropanecarboxylic dihydrochloride, 2 (S) -terbutoxycarbonyl-amino-5-hexenoic acid purchased from RSP Amino Acids (0.34 g, 1.46 mmol) and NMM (0.57 ml, 5.22 mmol)

10 in DMF (5 ml) HATU (0.56 g, 1.46 mmol) was added at 0 ° C. After stirring for 2 days, the reaction mixture was diluted with EtOAc (200 ml), washed with buffer pH 4.0 (2 x 30 ml), saturated aqueous NaHCO3 (30 ml) and brine (30 ml), dried (MgSO4) and purified by a 40M Biotage column (eluting with 30% to 60% EtOAc in Hexanes) to provide the title compound, 1 - {[1- (2 (S) -terc acid ethyl ester) -butoxycarbonylaminohex-5-enoyl) -4- (7-methoxy-2-phenyl-quinolin-4 (R) -yloxy) -pyrrolidin-2 (S) -carbonyl] -1 (R) -amino] -2 (S )

15 vinyl cyclopropanecarboxylic acid, in the form of a yellow oil (0.56 g, 76%): 1 H NMR (300 MHz, CDCl 3)  1.20 (t, J = 7 Hz, 3H), 1.39 (s, 9H), 1.43-1.48 (m, 1H), 1.64-1.92 (m, 4H), 2.10-2.20 (m, 2H), 2.36-2.44 ( m, 1H), 2,912.99 (m, 1H), 3.94 (s, 3H), 4.04-4.22 (m, 2H), 4.29 (d, J = 11 Hz, 1H), 4.46-4.51 (m, 1H), 4.81 (dd, J = 8.1, 5.5 Hz, 1H), 4.98-5.15 (m, 4H), 5.29 ( dd, J = 17.2, 1.5 Hz, 1H), 5.41 (sa, 1H), 5.67-5.86 (m, 2H), 7.04 (s, 1H), 7.08 (dd, J = 9.2, 2.6 Hz, 1H), 7.42-7.64 (m, 4H), 7.97-8.06 (m, 3H). LC-MS (E-retention time procedure: 1.59), MS m / z

20 713 (M ++ 1).

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Stage 1h: Preparation of ethyl ester of acid (1S, 4R, 6S, 11S) -7-trans-11-tert-butoxycarbonylamino-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo-3 , 13-diazatricyclo [11.3.0.04.6] hexadec-7-en-4-carboxylic

1,2-DCE degassed with Argon was added 70 mg of Grubbs catalyst, the mixture was degassed with Ar and refluxed for 3 h. The reaction mixture was cooled to rt, the mixture was degassed again, a final 70 mg portion of Grubbs catalyst was added and the mixture was heated at reflux for 12 h. The dark brown solution was concentrated in vacuo, purified by a 40M Biotage column (eluting with 30% to 65% EtOAc in Hexanes) to provide the acid ethyl ester intermediate (1S, 4R, 6S, 11S, 15R ) -7-trans-11-tert-butoxycarbonylamino-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo-3,13-diazatricyclo- [11.3.0.04.6] hexadec-7-en -4carboxylic (165 mg, 46%) in the form of a pale yellow oil: 1H NMR (500 MHz, CD3OD)  1, 12 (s, 9H), 1.23 (t, J = 7 Hz, 3H) , 1.41 (dd, J = 7.5, 5.8 Hz, 1H), 1.64-1.71 (m, 2H), 2.04-2.18 (m, 2H), 2.32 (m, 2H), 2.602.70 (m, 2H), 3.95 (s, 3H), 4.11-4.23 (m, 4H), 4.34 (m, 1H), 4.64 ( m, 1H), 5.25 (m, 1H), 5.54 (d, J = 15.3 Hz, 1H), 5.65 (s, 1H), 7.18 (dd, J = 9.2 , 2.4 Hz, 1H), 7.29 (s, 1H), 7.40 (d, J = 2.4 Hz, 1H), 7.56-7.59 (m, 3H), 8.02 -8.04 (m, 2H), 8.09 (d, J = 9.2 Hz, 1H). LC-MS (E-retention time procedure: 1.66), MS m / z 685 (M ++ 1).

Step 1i: Preparation of the acid (1S, 4R, 6S, 11S, 15R) -7-trans-11-tert-butoxycarbonylamino-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo- 3,13-diazatricyclo [11.3.0.04.6] hexadec-7-en-4-carboxylic

To a solution of ethyl ester of the acid (1S, 4R, 6S, 11S, 15R) -7-trans-11-tert-butoxycarbonylamino-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo- 3.13-diazatricyclo [11.3.0.09.6] hexadec-7-en-4-carboxylic (80 mg, 0.12 mmol) in 7 ml of 4: 2: 1 THF / H2O / MeOH was added 28 mg of LiOH (0.70 mmol), the mixture was stirred for 24 h, the solution was adjusted to pH 7, and the mixture was concentrated until only the water phase was maintained. The solution was adjusted to pH 4, the acidic aqueous solution was extracted with EtOAc (3 x 100 mL portions) and the organic extracts were combined, dried (MgSO4) and concentrated to provide the acid (1S, 4R, 6S, 11S, 15R) -7-trans-11-tert-butoxycarbonylamino-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo-3,13-diazatricyclo [11.3.0.04'6] hexa-dec -7-en-4-carboxylic (62.2 mg, 80%): 1H NMR (300 MHz, CD3OD)  1.12 (s, 9H), 1.18-2.48 (m, 8H), 2 , 65-2.69 (m, 1H), 3.94 (s, 3H), 4.18-4.34 (m, 3H), 4.83 (t, J = 8 Hz, 1H), 5, 30-5.38 (m, 2H), 5.53-5.61 (m, 2H), 7.14 (dd, J = 9.2, 2.4 Hz, 1H), 7.24 (s, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.48-7.64 (m, 3H), 8.02-8.07 (m, 3H). LC-MS (E-retention time procedure: 1.45), MS m / z 657 (M ++ 1). HRMS m / z (M + H) + calc. for C36H41N4O8: 657.2924, found 657.2924,

image24

Preparation of cyclopropylsulfonamide from cyclopropylsulfonyl chloride.

A solution of 100 ml of THF cooled to 0 ° C was bubbled in gaseous ammonium until saturation was reached. To this solution was added a solution of 5 g (28.45 mmol) of cyclopropylsulfonyl chloride (purchased from Array Biopharma) in 50 ml of THF, the solution was heated to rt overnight and stirred for another day. The mixture was concentrated until 1-2 ml of solvent remained, on a bed of 30 g of SiO2 (eluting with 30% to 60% EtOAc / Hexanes), yielding 3.45 g (100%) of cyclopropyl sulfonamide in Shape of a white solid. 1H NMR (Methanol-d4) ,9 0.94-1.07 (m, 4H), 2.52-2.60 (m, 1H); 13 C NMR (methanol-d4) ,9 5.92, 33.01.

image25

image 1

Preparation of cyclobutyl sulfonamide from cyclobutyl bromide

To a solution of 5.0 g (37.0 mmol) of cyclobutyl bromide in 30 ml of anhydrous diethyl ether (Et2O), cooled to 78 ° C, 44 ml (74.8 mmol) of tert-butyl- were added 1.7 M lithium in pentanes and the solution was slowly heated at 5 ° C for 1.5 h. This mixture was slowly cannulated in a solution of 5.0 g (37.0 mmol) of freshly distilled sulfuryl chloride in 100 ml of hexanes, cooled to -40 ° C, heated to 0 ° C for 1 h and carefully concentrated to empty. This mixture was dissolved again in Et2O, washed once with a little ice water, dried (MgSO4) and carefully concentrated. This mixture was dissolved again in 20 ml of THF, added dropwise to 500 ml of NH3 saturated in THF and allowed to stir overnight. The mixture is

10 concentrated in vacuo to give a yellow crude solid and recrystallized from the minimum amount of CH2Cl2 in hexanes with 1-2 drops of MeOH, providing 1.90 g (38%) of cyclobutyl sulfonamide as a colored solid. White. 1H NMR (CDCl3)  1.95-2.06 (m, 2H), 2.30-2.54 (m, 4H), 3.86 (p, J = 8 Hz, 1H), 4.75 ( sa, 2H); 13C NMR (CDCl3)  16.43, 23.93, 56.29, HRMS m / z (M-H) - calc. for C4H8NSO2: 134.0276, found 134.0282.

Step 1j: Preparation of compound 1, Example 1 tert-butyl acid ester (1S, 4R, 6S, 11S, 15R) -7-trans- [4

Cyclopropanesulfonyl-aminocarbonyl-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo-3,13-diazatricyclo [11.3.0.09.6] hexadec-7-en-11-yl] carbamic

image 1

To a solution of acid (1S, 4R, 6S, 11S, 15R) -7-trans-11-tert-butoxycarbonylamino-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo-3.13 -diazatricyclo [11.3.0.04.6] hexadec-7-en-4-carboxylic acid (47.7 mg, 0.0726 mmol) in THF (2 ml) is given

20 added in one portion CDI (16.5 mg, 0.102 mmol) under an argon atmosphere. The resulting solution was stirred for 30 min, refluxed for 30 min and allowed to cool to rt. Cyclopropylsulfonamide (12.3 mg, 0.102 mmol) was added in one portion before the addition of a pure DBU solution (0.016 ml, 0.102 mmol). The reaction was stirred for 24 h, diluted with EtOAc (100 ml) and washed with buffer pH 4.0 (3 x 20 ml) and brine (20 ml), dried (MgSO4) and purified using a plate Preparatory FTA (Analtech 20 x 40 cm,

25 thickness 1000 µM), providing tert-butyl acid ester (1S, 4R, 6S, 11S, 15R) -7-trans- [4-cyclopropanesulfonylaminocarbonyl-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo-3,13-diazatricyclo [11.3.0.04.6] hexa-dec-7-en-11il] carbamic. Compound 1, Example 1, in the form of a foam (31.8 mg, 58%): 1H NMR (300 MHz, CD3OD)  0.86-1.43 (m, 5H), 1.15 (s, 9H ), 1.63-1.73 (m, 2H), 2.03-2.20 (m, 2H), 2.27-2.41 (m, 2H), 2.56-2.80 (m , 3H), 3.92 (s, 3H), 4.16-4.37 (m, 3H), 4.83 (t, J = 8 Hz, 1H), 5.28-5.36 (m, 1H), 5.52-5.56 (m, 2H), 7.09 (dd, J = 9.2, 2.2 Hz, 1H),

30 7.22 (s, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.44-7.54 (m, 3H), 8.00-8.04 (m, 3H ). LC-MS (Procedure D-retention time: 1.43), MS m / z 760 (M ++ 1). HRMS m / z (M + H) + calc. for C39H46SN5O9: 760,3016, found 760,3002.

image26

Step 2k: Preparation of Compound 2, Example 2, [11-amino-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo-3,13 diazatricyclo- [11.3. 0.04.6] hexadec-7-en-4-carbonyl] acid amide (1S, 4R, 6S, 11S, 15R) -7-trans-cyclopropanesulfonic acid dihydrochloride

image 1

A solution of 2 ml (8 mmol) of 4 N HCl / dioxane was added to 11 mg (0.014 mmol) of tert-butyl acid ester (1S, 4R, 6S, 11S, 15R) -7-trans- [4 -Cyclopropanesulfonylaminocarbonyl-15- (7-methoxy-2-phenylquinolin-4-yloxy) -2,12-dioxo3,13-diazatriciclo- [11.3.0.04,6] hexadec-7-en-11-yl] carbamic, the mixture stirred for 2 h, concentrated and dried (50 ° C; 2.67 KPa (20 torr); 12 h), yielding 10.5 mg (~ 100%) of [11-amino-15- (7-methoxy -2-phenylquinolin-4-yloxy) -2,12-dioxo-3,13-diazatricyclo- [11.3.0.04.6] hexadec-7-en-4-carbonyl] acid amide (1S, 4R, 6S, 11S, 15R ) -7-trans

10 cyclopropanesulfonic dihydrochloride in the form of a white solid: 1H NMR (300 MHz, CD3OD)  0.87-1.41 (m, 8H), 1.70 (m, 1H), 2.16 (s, 1H ), 2.30 (m, 1H), 2.45 (m, 1H), 2.65 (m, 1H), 2.96 (m, 1H), 4.05 (s, 3H), 4.33 (s, 3H), 4.48 (m, 1H), 5.46-5.67 (m, 2H), 5.99 (s, 1H), 7.45 (m, 1H), 7.58- 7.85 (m, 5H), 8.12 (sa, 2H), 8.35 (m, 1H). LC-MS (E-retention time procedure: 1.09), MS m / z 660 (M ++ 1). HRMS m / z (M + H) + calc. for C34H38SN5O7: 660.2492, found 660.2492.

15 Examples 3.4

Preparation of Compounds 3 and 4 Step 3g: Preparation of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-hept-6-enoyl) -4- (7-methoxy-2-phenylquinolin-9 (R) ethyl ester ) -yloxy) pyrrolidin-2 (S) -carbonyl] -1 (R) -amino] -2 (S) -vinylcyclopropanecarboxylic

image 1

image27

image 1

Following the procedure of Step 1 g, with the exception that they were coupled (0.356 g, 1.46 mmol) of the acid

5 2 (S) -terc-butoxycarbonylamino-6-heptenoic purchased from RSP Amino Acids, instead of 2 (S) -terbutoxycarbonylamino-5-hexenoic acid, to give the title compound, 1 - {[1 - (2 (S) -terbutoxycarbonylamino-hept-6-enoyl) -4- (7-methoxy-2-phenylquinolin-4 (R) -yloxy) pyrrolidin-2 (S) -carbonyl] -1 (R) -amino ] -2 (S) vinylcyclopropanecarboxylic acid in the form of a yellow oil (0.600 g, 79%): 1 H NMR (CDCl3)  (300 MHz, CDCl3); 1.21 (t, J = 7 Hz, 3H), 1.40 (s, 9H), 1.42-2.18 (m, 9H), 2.36-2.45 (m, 1H), 2 , 91-2.99 (m, 1H), 3.94 (s, 3H),

10 4.05-4.22 (m, 2H), 4.26-4.30 (m, 1H), 4.39-4.49 (m, 1H), 4.83 (dd, J = 8, 2, 5.6 Hz, 1H), 4.92-5.14 (m, 4H), 5.29 (dd, J = 17.2, 1.4 Hz, 1H), 5.41 (sa, 1H ), 5.63-5.80 (m, 2H), 7.04 (s, 1H), 7.09 (dd, J = 9, 2 Hz, 1H), 7.42-7.54 (m, 4H), 8.00-8.05 (m, 3H). LC-MS (E-retention time procedure: 1.53), MS m / z 727 (M ++ 1).

image 1

Step 3h: Preparation of ethyl ester of acid (1S, 4R, 6S, 12S, 16R) -7-trans-12-tert-butoxycarbonylamino-16- (715 methoxy-2-phenylquinolin-4-yloxy) -2,13- dioxo-3,14-diazatriciclo [12.3.0.04.6] -heptadec-7-en-4-carboxylic

Following the experimental and purification procedure of Step 1h, 1 {[1- (2 (S) -terc-butoxycarbonylamino-hept-6-enoyl) -4- (7-methoxy-2-) acid ester was reacted phenylquinolin-4 (R) -yloxy) -pyrrolidin-2 (S) -carbonyl] -1 (R) amino] -2 (S) -vinylcyclopropanecarboxylic acid (404 mg, 0.555 mmol) with two 75 mg portions of Grubbs catalyst, providing ethyl ester of acid (1S, 4R, 6S, 12S, 16R) -7-trans-12-tert-butoxycarbonylamino-16- (7-methoxy-2

Phenylquinolin-4-yloxy) -2,13-dioxo-3,14-diazatricyclo [12.3.0.04.6] heptadec-7-en-4-carboxylic (190 mg, 49%): 1H NMR (500 MHz, CD3OD ) , 1.22 (t, J = 7 Hz, 3H), 1.26 (s, 9H), 1.32-1.43 (m, 3H), 1.59-1.64 (m, 1H ), 1.77-1.90 (m, 3H), 2.11 (sa, 1H), 2.29-2.34 (m, 1H), 2.60-2.65 (m, 1H), 2.70-2.74 (m, 1H), 3.93 (s, 3H), 4.07-4.18 (m, 3H), 4.30 (d, J = 7.6 Hz, 1H) , 4.52 (d, J = 11.3 Hz, 1H), 4.67 (t, J = 8.2 Hz, 1H), 5.35 (dd, J = 15.7, 6.0 Hz, 1H), 5.50-5.56 (m, 1H), 5.60 (sa, 1H), 7.10 (dd, J = 9.2, 2 Hz, 1H), 7.26 (s, 1H ), 7.37 (d, J = 2 Hz, 1H), 7.53-7.57 (m, 3H), 8.00, 8.01 (2s,

25 2H), 8.17 (d, J = 9.2 Hz, 1H). LC-MS (E-retention time procedure: 1.58), MS m / z 699 (M ++ 1).

image28

Step 3i: Preparation of the acid (1S, 4R, 6S, 12S, 16R) -7-trans-12-tert-butoxycarbonylamino-16- (7-methoxy-2-phenylquinolin-4-yloxy) -2,13-dioxo- 3,14-diazatricyclo- [12.3.0.04.6] heptadec-7-en-4-carboxylic

image 1

Following the experimental and purification procedure of Step 1i, ethyl acid ester was reacted

5 (1S, 4R, 6S, 12S, 16R) -7-trans-12-tert-butoxycarbonyl-amino-16- (7-methoxy-2-phenylquinolin-4-yloxy) -2,13-dioxo-3,14diazatricyclo - [12.3.0.04.6] heptadec-7-en-4-carboxylic acid (185 mg, 0.265 mmol) with 64 mg (1.6 mmol) of LiOH in 15.5 ml of 9: 5: 1.5 THF / H2O / MeOH, providing (1S, 4R, 6S, 12S, 16R) -7-trans-12-tert-butoxycarbonylamino16- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,13-dioxo -3,14-diazatricyclo [12.3.0.04.6] heptadec-7-en-4-carboxylic (180 mg, 100%): 1 H NMR (300 MHz, CD3OD)  1.24 (s, 9H), 1, 33-1.43 (m, 1H), 1.61-1.97 (m, 6H), 2.10 (sa, 1H), 2.28-2.36

10 (m, 1H), 2.70 (m, 2H), 3.94 (s, 3H), 4.17 (d, J = 10.6 Hz, 1H), 4.28 (d, J = 4 , 8 Hz, 1H), 4.65-4.70 (m, 1H), 5.34 (dd, J = 15.7, 5.9 Hz, 2H), 5.53-5.62 (m, 2H), 7.14 (d, J = 9 Hz, 1H), 7.32 (s, 1H), 7.39 (s, 1H), 7.56 (m, 3H), 8.01-8, 02 (m, 2H), 8.22 (d, J = 9.2 Hz, 1H). LC-MS (E-retention time procedure: 1.49), MS m / z 671 (M ++ 1). HRMS m / z (M + H) + calc. for C37H43N4O8: 671,3081, found 671,3080.

Step 3j: Preparation of Compound 3, Example 3, tert-butyl acid ester (1S, 4R, 6S, 12S, 16R) -7-trans- [4

Cyclopropanesulfonyl-aminocarbonyl-16- (7-methoxy-2-phenylquinolin-4-yloxy) -2,13-dioxo-3,14diazatricyclo [12.3.0.04.6] heptadec-7-en-12-yl] carbamic

image 1

Applying the procedure and purification measures of Step 1j, (144.9 mg, 0.216 mmol) of acid (1S, 4R, 6S, 12S, 16R) -7-trans-12-tert-butoxycarbonyl-amino-16 were converted - (7-Methoxy-2-phenylquinolin-4-yloxy) -2,13-dioxo-3,14

20 diazatricyclo [12.3.0.04.6] heptadec-7-en-4-carboxylic acid in 92 mg (56%) of the product tert-butyl ester of the acid (1S, 4R, 6S, 12S, 16R) -7-trans- [ 4-Cyclopropane-sulfonylaminocarbonyl-16- (7-methoxy-2-phenylquinolin-4-yloxy) -2,13-dioxo3,14-diazatricyclo [12.3.0.04.6] -heptadec-7-en-12-yl] carbamic : 1H NMR (300 MHz, CD3OD)  0.81-0.88 (m, 2H), 1,001.05 (m, 2H), 1.15-1.19 (m, 1H), 1.32 (s , 9H), 1.42 (m, 1H), 1.59-1.68 (m, 1H), 1.77-1.93 (m, 4H), 2.08-2.26 (m, 2H ), 2.60-2.73 (m, 3H), 3.94 (s, 3H), 4.21-4.34 (m, 2H), 4.43 (d, J = 11 Hz, 1H) , 4.56-4.62 (m, 1H), 5.28-5.36 (m, 1H), 5.46-5.55 (m, 2H), 7.07 (dd, J = 9, 5, 2.4 Hz, 1H), 7.27 (s, 1H), 7.37 (d, J = 2.4 Hz, 1H), 7.42-7.49 (m, 3H), 8, 01, 8.03 (2s, 2H), 8.18 (d, J = 9.5 Hz, 1H). LC-MS (Procedure D-retention time: 1.48), MS mlz 774 (M ++ 1). HRMS m / z (M + H) + calc. for C40H48SN5O9: 774.3173, found 774.3173.

image29

Step 4k: Preparation of Compound 4, Example 4, [12-amino-16- (7-methoxy-2-phenylquinolin-4-yloxy) -2,13-dioxo-3,14 diazatricyclo [12.3.0.04.6] heptadec- 7-en-4-carbonyl] acid amide (1S, 4R, 6S, 12S, 16R) -7-trans-cyclopropanesulfonic acid dihydrochloride

image 1

A solution of 5 ml (20 mmol) of 4 N HCl / dioxane was added to 10.8 mg (0.014 mmol) of tert-butyl ester of

10 (1S, 4R, 6S, 12S, 16R) -7-trans- [4-cyclopropanesulfonyl-aminocarbonyl-16- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,13 dioxo-3.14 acid -diazatricyclo [12.3.0.04.6] hexa-dec-7-en-12-yl] carbamic, the mixture was stirred for 2 h, concentrated and dried (50 ° C; 2.66 KPa (20 torr); 12 h), providing 10.6 mg (100%) of [12-amino-16- (7-methoxy-2-phenylquinolin4-yloxy) -2,13-dioxo-3,14-diazatricyclo- [12.3.0.04, 6] heptadec-7-en-4-carbonyl] acid amide (1S, 4R, 6S, 12S, 16R) -7trans-cyclopropanesulfonic acid dihydrochloride in the form of a white solid: 1H NMR (300 MHz, CD3OD)  0.89 (m, 1H),

15 1.05-2.12 (m, 11H), 2.18-2.26 (m, 1H), 2.41-2.49 (m, 1H), 2.62-2.99 (m, 2H), 4.05 (s, 3H), 4.30 (m, 2H), 4.44-4.46 (m, 2H), 5.15-5.23 (m, 1H), 5.68 -5.75 (m, 1H), 5.96 s, 1H), 7.45 (d, J = 9 Hz, 1H), 7.57 (s, 1H), 7.63-7.73 (m , 4H), 8,088.13 (m, 2H), 8.38 (d, J = 9 Hz, 1H). LC-MS (E-retention time procedure: 1.10), MS m / z 674 (M ++ 1). HRMS m / z (M + H) + calc. for C35H40SN5O7: 674.2649, found 674.2655.

image30

Examples 5, 6 Preparation of Compounds 5 and 6

image 1

Step 5 g: Preparation of 1 - {[1- (2 (S) -terc-butoxycarbonylaminoct-7-enoyl) -4- (7-methoxy-2-phenylquinolin-4 (R) -yloxy) pyrrolidine acid ester -2 (S) -carbonyl] -1 (R) -amino] -2 (S) -vinylcyclopropanecarboxylic

image 1

Following the procedure of Step 1 g, with the exception that (0.35 g, 1.4 mmol) of 2 (S) -terbutoxycarbonylamin-7-octenic acid purchased from RSP Amino Acids was coupled, instead of ethyl ester of 2 (S) -terbutoxycarbonylamino-5-hexenoic acid, giving 1 - {[1- (2 (S) -terc-butoxycarbonylamin-oct-7-enoyl) -4- (710 methoxy-2-phenyl) ethyl ester -quinolin-4 (R) -yloxy) pyrrolidin-2 (S) -carbonyl] -1 (R) -amino] -2 (S) -vinylcyclopropanecarboxylic in the form of a yellow oil (0.58 g, 75%): 1H NMR (300 MHz, CDCl3)  1.20 (t, J = 7 Hz, 3H), 1.32-1.47 (m, 5H), 1.39 (s, 9H), 1.54-1 , 68 (m, 2H), 1.68-2.05 (m, 3H), 2.09-2.18 (m, 1H), 2.35-2.48 (m, 1H), 2.89 -2.99 (m, 1H), 3.93 (s, 3H), 4.02-4.22 (m, 2H), 4.27 (d, J = 11.3 Hz, 1H), 4, 38-4.47 (m, 1H), 4.80-4.98 (m, 3H), 5.06-5.14 (m, 2H), 5.28 (dd, J = 17.2, 1 , 5 Hz, 1H), 5.41 (sa, 1H), 5.63-5.81 (m, 2H), 7.03 (s, 1H), 7.07 (dd, J = 9.2, 2.6 Hz, 1H), 7.41-7.57 (m, 4H), 7.98-8.04

15 (m, 3H). LC-MS (A-retention time procedure: 1.81), MS m / z 741 (M ++ 1).

image31

image 1

Step 5h: Preparation of ethyl ester of acid (1S, 4R, 6S, 13S, 17R) -7-trans-13-tert-butoxycarbonylamin-17- (7methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo -3,15-diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic acid and ethyl ester (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert- butoxycarbonylamino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15

5 diazatricyclo [13.3.0.04.6] octadec-7-en-4-carboxylic

Following the experimental and purification procedure of step 1 h, ethyl ester of 1 {[1- (2 (S) -ter-butoxycarbonylamin-oct-7-enoyl) -4- (7-methoxy-2) acid was reacted -phenylquinolin-4 (R) -yloxy) -pyrrolidin-2 (S) -carbonyl] -1 (R) amino] -2 (S) -vinylcyclopropanecarboxylic acid (1.16 g, 1,565 mmol) with portions of 275 mg and 225 mg of Grubbs catalyst, providing ethyl ester of acid (1S, 4R, 6S, 13S, 17R) -7-trans-13-tert-butoxycarbonyl-amino-17- (7

10-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15-diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic acid (90 mg, 8%) and ethyl ester of the acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamin-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14 dioxo-3,15-diazatricyclo [ 13.3.0.04.6] octadec-7-en-4-carboxylic (222 mg, 20%). Data for trans- or E-olefin isomer: 1H NMR (500 MHz, CD3OD)  1.14-1.48 (m, 6H), 1.21 (t, J = 7.0 Hz, 3H), 1, 34 (s, 9H), 1.51-1.63 (m, 2H), 1.76 (s, 1H), 1.81-1.91 (m, 2H), 2.00-2.08 ( m, 1H), 2.50 (c, J = 8 Hz, 1H), 2.61-2.66 (m, 1H), 2.75-2.80 (m, 1H), 3.95 (s , 3H),

15 4.04-4.18 (m, 2H), 4.40-4.44 (m, 1H), 4.54 (d, J = 11.6 Hz, 1H), 4.79 (t, J = 7.3 Hz, 1H), 5.04 (dd, J = 15.6, 7.6 Hz, 1H), 5.16 (d, J = 8.2 Hz, 1H), 5.46 (sa , 1H), 5.53-5.59 (m, 1H), 7.04 (s, 1H), 7.07 (dd, J = 9.2, 2.4 Hz, 1H), 7.42- 7.52 (m, 4H), 8.03-8.10 (m, 3H). LC-MS (E-retention time procedure: 1.89), MS m / z 713 (M ++ 1). Data for cis-or Z-olefin isomer: 1 H NMR (300 MHz, CDCl 3)  1.15-1.62 (m, 5H), 1.21 (t, J = 7 Hz, 3H), 1.38 ( s, 9H), 1.91 (dd, J = 8.1, 5.9 Hz, 1H), 1.97-2.02 (m, 5H), 2.33-2.41 (m, 1H) , 3.03-3.11 (m, 1H), 3.95 (s, 3H), 4.06-4.18 (m, 3H), 4.27 (d,

20 J = 11 Hz, 1H), 4.54 (m, 1H), 4.93 (dd, J = 8.2, 4.2 Hz, 1H), 5.27-5.40 (m, 2H) , 5.71-5.80 (m, 1H), 7.05 (s, 1H), 7.08 (dd, J = 9.2, 2.6 Hz, 1H), 7.43-7.53 (m, 4H), 8.02 (d, J = 9.2 Hz, 1H), 8.05-8.07 (2s, 2H). LC-MS (E-retention time procedure: 1.80), MS m / z 713 (M ++ 1).

image32

image 1

Step 5i: Preparation of acid (1S, 4R, 6S, 13S, 17R) -7-trans-13-tert-butoxycarbonylamin-17- (7-methoxy-2-phenylquinolin4-yloxy) -2,14-dioxo-3, 15-diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic

Following the experimental and purification procedure of Step 2i, ethyl acid ester was reacted

5 (1S, 4R, 6S, 13S, 17R) -7-trans-13-tert-butoxycarbonyl-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15 diazatricyclo - [13.3.0.04.6] octadec-7-en-4-carboxylic acid (185 mg, 0.265 mmol) with 64 mg (1.6 mmol) of LiOH in 15.5 ml of 9: 5: 1.5 THF / H2O / MeOH, providing (1S, 4R, 6S, 13S, 17R) -7-trans-13-tert-butoxycarbonylamin-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3 , 15-diazatricyclo [13.3.0.04.6] octadec-7-en-4-carboxylic (180 mg, 100%): 1H NMR (300 MHz, CD3OD)  1.14 (s, 9H), 1.20- 1.51 (m, 5H), 1.53-1.58 (m, 1H), 1.70 (dd, J = 9.9, 5.1 Hz, 1H), 1.86

10 (sa, 1H), 2.06 (sa, 1H), 2.28-2.59 (m, 3H), 2.65-2.73 (m, 1H), 3.94 (s, 3H) , 4.07 (dd, J = 11.3, 2.6 Hz, 1H), 4.23 (d, J = 8.4 Hz, 1H), 4.64-4.70 (m, 2H), 5.54-5.68 (m, 3H), 7.10 (dd, J = 9, 2 Hz, 1H), 7.26 (s, 1H), 7.39 (d, J = 2.2 Hz , 1H), 8.02, 8.04 (2s, 2H), 8.12 (d, J = 9.1 Hz, 1H). LC-MS (E-retention time procedure: 1.57), MS m / z 685 (M ++ 1). EMAR m / z (M + H) + calc. for C38H45N4O8: 685.3238, found 685.3241.

Step 5j: Preparation of Compound 5, Example 5, tert-butyl acid ester (1S, 4R, 6S, 13S, 17R) -7-trans- [4

Cyclopropanesulfonylamino-carbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15diazatricyclo [13.3.0.04.6] octadec-7-en-13-yl] carbamic

image 1

Using the process and purification conditions of Step 2j, 35.6 mg (0.052 mmol) of acid (1S, 4R, 6S, 13S, 17R) -7-trans-13-tert-butoxycarbonyl-amino-17 were converted - (7-Methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15

image33

diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic acid in 30 mg (73%) of tert-butyl acid ester (1S, 4R, 6S, 13S, 17R) -7-trans- [4 -Cyclo-propanesulfonylaminocarbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo3,15-diazatricyclo- [13.3.0.04.6] octadec-7-en-13-yl] carbamic: 1H NMR (300 MHz, CD3OD)  0.85-1.51 (m, 8H), 1.16 (s, 9H), 1.64-1.76 (m, 3H), 1.87 (m, 1H), 2.10 (m, 1H), 2.32 (m, 1H), 2.44 (m, 1H), 2.52-2.72 (m, 2H), 2.83 (sa, 1H ), 3.94

5 (s, 3H), 4.10-4.15 (m, 1H), 4.23-4.32 (m, 1H), 4.61-4.69 (m, 2H), 5.49- 5.64 (m, 3H), 7.08 (dd, J = 9.2, 2.2 Hz, 1H), 7.23 (s, 1H), 7.37 (d, J = 2.2 Hz , 1H), 7.46-7.56 (m, 3H), 8.03-8.09 (m, 3H). LC-MS (E-retention time procedure: 1.57), MS m / z 788 (M ++ 1). EMAR m / z (M + H) + calc. for C41H50SN5O9: 788.3329, found 788.3337.

Step 6k: Preparation of Compound 6, Example 6, [13-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15 diazatricyclo [13.3.0.04.6] octadec- 7-en-4-carbonyl] acid amide (1S, 4R, 6S, 13S, 17R) -7-trans-cyclopropanesulfonic acid

10 dihydrochloride

image 1

A solution of 2 ml (8 mmol) of 4 N HCl / dioxane was added to 10.5 mg (0.014 mmol) of tert-butyl acid ester (1S, 4R, 6S, 13S, 17R) -7-trans- [ 4-cyclopropanesulfonylaminocarbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo3,15-diazatricyclo- [13.3.0.04.6] octadec-7-en-13-yl] carbamic, mixture was stirred for 2 h, concentrated and dried

15 (50 ° C; 2.66 KPa (20 torr); 12 h), providing 10.0 mg (~ 100%) of [13-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2, 14-dioxo-3,15-diazatricyclo [13.3.0.04.6] octadec-7-en-4-carbonyl] amide (1S, 4R, 6S, 13S, 17R) -7-transcyclopropanesulfonic acid dihydrochloride as a solid white: 1H NMR (300 MHz, CD3OD)  0.86-2.03 (m, 14H), 2.17 (sa, 1H), 2.52-3.00 (m, 3H), 4, 06 (s, 3H), 4.16-4.53 (m, 4H), 5.36 (t, J = 10.6 Hz, 1H), 5.65-5.72 (m, 1H), 5 , 97 (sa, 1H), 7.44-7.47 (m, 1H), 7.59-7.74 (m, 5H), 8.12 (m, 2H), 8.38-8.41 (m, 1H). LC-MS (Procedure E

20 retention time: 1.16), MS m / z 688 (M ++ 1). EMAR m / z (M + H) + calc. for C36H42SN5O7: 688.2805, found 688.2793.

image34

Examples 7.8 Preparation of Compounds 7 and 8

image 1

Step 7h: Preparation of ethyl ester of acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamino-17- (7-methoxy2-phenylquinolin-4-yloxy) -2,14-dioxo -3.15-diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic

image 1

This stage of this particular sequence used the product of step 5g as a starting point. To a solution of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-oct-7-enoyl) -4- (7-methoxy-2-phenylquinolin-4 (R) -yloxy) pyrrolidin2 acid ester (S) -carbonyl] -1 (R) -amino] -2 (S) -vinylcyclopropanecarboxylic acid (1.44 g, 1.93 mmol) in 2.5 l of degassed 1,2-DCE 10 with Argon, They added 150 mg of Grubbs catalyst, the mixture was degassed under an Ar atmosphere and heated at reflux for 3 h. The reaction mixture was cooled to rt, the mixture was degassed again, a 140 mg portion of Grubbs catalyst was added and the mixture was heated at reflux for 14 h. The reaction mixture was cooled again to rt, the mixture degassed again, a 140 mg portion of Grubbs catalyst was added and the mixture was heated at reflux for a final 4 h. The dark brown solution was concentrated in vacuo, purified by a 40M Biotage column (eluting with 15% to 65% EtOAc in hexanes) to provide ethyl ester of acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamino-17- (7-methoxy-2-phenylquinolin-4-yloxy) 2,14-dioxo-3,15-diazatricyclo [13.3.0.04.6] octadec-7-en- 4-carboxylic (720 mg, 52%). Data for cis- or Zolefin isomer, ethyl ester of acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamino-17- (7-methoxy-2-phenylquinolin-4-yloxy) - 2.14-dioxo-3.15-diazatricyclo [13.3.0.04.6] octadec-7-en-4-carboxylic: 1H NMR (300 MHz, CDCl3)  1.15-1.62 (m, 20 5H) , 1.21 (t, J = 7 Hz, 3H), 1.38 (s, 9H), 1.91 (dd, J = 8.1, 5.9 Hz, 1H), 1.97-2, 02 (m, 5H), 2.33-2.41 (m, 1H), 3,033.11 (m, 1H), 3.95 (s, 3H), 4.06-4.18 (m, 3H) , 4.27 (d, J = 11 Hz, 1H), 4.54 (m, 1H), 4.93 (dd, J = 8.2, 4.2 Hz, 1H),

image35

5.27-5.40 (m, 2H), 5.71-5.80 (m, 1H), 7.05 (s, 1H), 7.08 (dd, J = 9.2, 2.6 Hz, 1H), 7.43-7.53 (m, 4H), 8.02 (d, J = 9.2 Hz, 1H), 8.05-8.07 (2s, 2H). LC-MS (E-retention time procedure: 1.80), MS m / z 713 (M ++ 1).

Step 7i: Preparation of acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3, 15-diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic

image 1

5 Following the experimental and purification procedure of Step 1i, ethyl acid ester (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamino-17- (7-methoxy) was reacted -2-phenyl-quinolin-4-yloxy) -2,14-dioxo-3,15 diazatricyclo [13.3.0.04.6] octadec-7-en-4-carboxylic (290 mg, 0.406 mmol) with 100 mg (2, 5 mmol) of LiOH in 23.9 ml of 14.4: 7.7: 1.8 of THF / H2O / MeOH, providing acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert -butoxycarbonylamino

10 17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15-diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic (279 mg, 100 %): 1H NMR (300 MHz, CD3OD)  0.97-1.05 (m, 2H), 1.14 (s, 9H), 1.20-1.55 (m, 5H), 1.60 -1.73 (m, 2H), 1.85 (sa, 1H), 2.06-2.22 (m, 1H), 2.29 (sa, 1H), 2.52-2.73 (m , 2H), 3.93 (s, 3H), 4.14-4.19 (m, 1H), 4.26 (d, J = 9.5 Hz, 1H), 4.56-4.67 ( m, 2H), 5.47-5.53 (m, 2H), 5.71-5.78 (m, 1H), 7.08 (dd, J = 9.1, 2.2 Hz, 1H) , 7.22 (s, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.45-7.56 (m, 3H), 8.02, 8.04 (2s, 2H ), 8.09 (d, J = 9.1 Hz, 1H). CL-MS (E-time Procedure of

15 retention: 1.60), MS m / z 685 (M ++ 1).

Step 7j: Preparation of Compound 7, Example 7 of tert-butyl acid ester (1S, 4R, 6S, 13S, 17R) -7-cis- [4-cyclopropanesulfonylamino-carbonyl-17- (7-methoxy-2-phenylquinolin-4 -yloxy) -2,14-dioxo-3,15 diazatricyclo [13.3.0.04.6] octadec-7-en-13-il] carbamic

image 1

20 Following the process and purification conditions of Step 1j, 219 mg (0.31 mmol) of acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamino-17- were converted (7-Methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15 diazatricyclo- [13.3.0.04.6] octadec-7-en-4-carboxylic acid in 94 mg (40%) of tert ester -butyl (1S, 4R, 6S, 13S, 17R) -7-cis- [4-cyclopropanesulfonylamino-carbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3 , 15 diazatricyclo [13.3.0.04.6] octadec-7-en-13-yl] carbamic: 1H NMR (300 MHz, CD3OD)  0.90-1.50 (m, 9H), 1.16 (s, 9H ), 1.64-1.74 (m, 2H), 1.85 (sa, 1H), 2.08 (sa, 1H), 2.30 (sa, 1H), 2.44 (sa, 1H) , 2.53-2.71 (m, 2H), 2.84 (sa, 1H), 3.92 (s, 3H), 4.05-4.12 (m, 1H), 4.24 (d , J = 9.5 Hz, 1H), 4.60-4.69 (m, 2H), 5.44-5.63 (m, 3H), 7.06 (dd, J = 9.2, 2 , 2 Hz, 1H), 7.20 (s, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.45-7.56 (m, 3H), 8.02-8 , 08 (m, 3H). LC-MS (Procedure D-retention time: 1.55), MS m / z 788 (M ++ 1).

image36

Step 8k: Preparation of Compound 8, Example 8, [13-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15 diazatricyclo [13.3.0.04.6] octadec- 7-en-4-carbonyl] acid amide (1S, 4R, 6S, 13S, 17R) -7-cis-cyclopropanesulfonic acid dihydrochloride

image 1

A solution of 5 ml (20 mmol) of 4 N HCl / dioxane was added to 21 mg (0.027 mmol) of tert-butyl acid ester

10 (1S, 4R, 6S, 13S, 17R) -7-cis- [4-cyclopropanesulfonylaminocarbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15 diazatricyclo- [13.3 .0.04.6] octadec-7-en-13-yl] carbamic, the mixture was stirred for 2 h, concentrated and dried (50 ° C; 2.66 KPa (20 torr); 12 h), yielding 20 mg (~ 100%) of [13-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14 dioxo-3,15-diazatricyclo [13.3.0.04.6] octadec-7-en-4 -carbonyl] acid amide (1S, 4R, 6S, 13S, 17R) -7-ciscyclopropanesulfonic dihydrochloride in the form of a white solid: 1H NMR (500 MHz, CD3OD)  1.03-1.16

15 (m, 3H), 1.25-1.33 (m, 2H), 1.35-1.57 (m, 3H), 1.65-1.71 (m, 2H), 1.76- 1.84 (m, 1H), 1.94-2.01 (m, 1H), 2.10-2.21 (m, 2H), 2.38-2.43 (m, 1H), 2, 67-2.72 (m, 1H), 2.85 (dd, J = 14.6, 7.9 Hz, 1H), 2.93-2.98 (m, 1H), 4.06 (s, 3H), 4.24 (dd, J = 12.2, 3.7 Hz, 1H), 4.40, 4.42 (2s, 2H), 4.80-4.81 (m, 1H), 5 , 53-5.57 (m, 1H), 5.67-5.74 (m, 1H), 5.95 (s, 1H), 7.45 (dd, J = 9, 2 Hz, 1H), 7.57 (d, J = 2 Hz, 1H) 7.61 (s, 1H), 7.70-7.77 (m, 3H), 8.09-8.10 (m, 2H), 8, 37 (d, J = 9.2 Hz, 1H). LC-MS (E-retention time procedure: 1.21), MS m / z 688 (M ++ 1).

20 Examples 9, 10 Preparation of Compounds 9 and 10

image37

image 1

Step 9g: Preparation of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4- (7-methoxy-2-phenylquinolin-4 (R) -yloxy) pyrrolidine- acid ethyl ester 2 (S) -carbonyl] -1 (R) -amino] -2 (S) -vinylcyclopropanecarboxylic

image 1

Following the procedure of Step 1g, with the exception that (0.78 g, 3.0 mmol) of 2 (S) -terbutoxycarbonylamino-8-nonenoic acid purchased from RSP Amino Acids was coupled, instead of acid 2 ( S) -terc-butoxycarbonylamino-5hexenoic acid, giving 1 - {[1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl acid) ethyl ester -4- (7-methoxy-210 phenylquinolin-4 ( R) -yloxy) pyrrolidin-2 (S) -carbonyl] -1 (R) -amino} -2 (S) -vinylcyclopropanecarboxylic acid in the form of a yellow oil (1.55 g, 82%): 1 H NMR ( 300 MHz, CDCl3)  1.15-1.78 (m, 9H), 1.20 (t, J = 7 Hz, 3H), 1.39 (s, 9H), 1.84 (dd, J = 8.1, 5.5 Hz, 1H), 1.91-2.04 (m, 2H), 2.14 (c, J = 8.8 Hz, 1H), 2.35-2.48 (m , 1H), 2.89-3.00 (m, 1H), 3.93 (s, 3H), 4.02-4.20 (m, 2H), 4.28 (d, J = 11.7 Hz, 1H), 4.39-4.47 (m, 1H), 4.80-4.99 (m, 1H), 5.06-5.14 (m, 2H), 5.27 (dd, J = 17, 1.3 Hz, 1H), 5.40 (sa, 1H), 5.65-5.81 (m, 2H), 7.03 (s, 1H), 7.07 (dd, J = 9.1, 2.6 Hz, 1H), 7.42-7.57 (m, 4H),

15 7.99-8.05 (m, 3H). LC-MS (E-retention time procedure: 1.79), MS m / z 755 (M ++ 1).

image38

image 1

Step 9h: Preparation of ethyl ester of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18- (7-methoxy2-phenylquinol-in-4-yloxy) -2.15 -dioxo-3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carboxylic

To a solution of ethyl ester of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4- (7-methoxy-2-phenylquinolin

5 4 (R) -yloxy) -pyrrolidin-2 (S) -carbonyl] -1 (R) -amino] -2 (S) -vinylcyclopropanecarboxylic acid (1.50 g, 1.99 mmol) in 2.4 l of 1,2DCE degassed with Argon, 150 mg of Grubbs catalyst was added, the mixture was degassed with Ar and heated at reflux for 3 h. The reaction mixture was cooled to rt, the mixture was degassed again, a 150 mg portion of Grubbs catalyst was added and the mixture was heated at reflux for 3 h. The reaction mixture was cooled again to rt, the mixture was degassed again, a 150 mg portion of catalyst was added.

10 Grubbs and the mixture was heated at reflux for a final 9 h. The dark brown solution was concentrated in vacuo and purified by a 40M Biotage column (eluting with 15% to 65% EtOAc in hexanes) to provide ethyl acid ester (1S, 4R, 6S, 14S, 18R) - 7-cis-14-tert-butoxycarbonylamino-18- (7-methoxy-2-phenylquinolin-4-yloxy) 2,15-dioxo-3,16-diaza-tricycle [14.3.0.04.6] nonadec-7-en -4-carboxylic (1.08 g, 75%): 1H NMR (300 MHz, CDCl3)  1.10-1.42 (m, 6H), 1.17 (t, J = 7 Hz, 3H), 1.33 (s, 9H), 1.54 (dd, J = 9.5, 5.5 Hz, 1H), 1.62 (m, 1H), 1.73 (dd, J = 8.5,

15 5.5 Hz, 1H), 1.75-1.86 (m, 1H), 2.01-2.24 (m, 3H), 2.39-2.47 (m, 1H), 2, 81-2.89 (m, 1H), 3.91 (s, 3H), 3.98-4.17 (m, 3H), 4.34 (d, J = 11.0 Hz, 1H), 4 , 46 (m, 1H), 4.77 (dd, J = 8.1, 5.5 Hz, 1H), 5.21-5.27 (m, 1H), 5.39 (m, 1H), 5.48-5.57 (m, 1H), 6.99 (s, 1H), 7.04 (dd, J = 9.1, 2.2 Hz, 1H), 7.38 (d, J = 2.2 Hz, 1H), 7.42-7.50 (m, 3H), 7.95-8.01 (m, 3H). LC-MS (E-retention time procedure: 1.71), MS m / z 727 (M ++ 1).

image 1

image39

Step 9i: Preparation of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3, 16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carboxylic

Following an experimental and purification procedure analogous to Step 1i, ethyl acid ester (1S, 4R, 6S, 19S, 18R) -7-cis-14-tert-butoxycarbonylamino-18- (7-methoxy-2) was reacted -phenylquinolin-4-yloxy) -2,15-dioxo-3,165 diazatricyclo [14.3.0.04.6] nonadec-7-en-4-carboxylic (225 mg, 0.309 mmol) with 78 mg (2.0 mmol) of LiOH in 20.8 ml of 12.4: 6.6: 1.8 THF / H2O / MeOH to provide acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino18- (7 -methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carboxylic (178 mg, 82%): 1H NMR (500 MHz, CD3OD)  1.12-1.54 (m, 8H), 1.19 (s, 9H), 1.59 (dd, J = 8.4, 5.0 Hz, 1H) , 1.64 (dd, J = 9.8, 4.9 Hz, 1H), 1.75-1.82 (m, 1H), 1.87-1.94 (m, 1H), 2.31 (c, J = 9 Hz, 1H), 2.52-2.57 (m, 1H), 2.69 (dd, J = 14, 7.5 Hz,

10 1H), 3.92 (s, 3H), 4.01 (dd, J = 11.6, 3.2 Hz, 1H), 4.19 (dd, J = 10.4, 2.4 Hz, 1H), 4.65 (t, J = 8 Hz, 1H), 4.75 (d, J = 11.6 Hz, 1H), 5.33 (t, J = 9.6 Hz, 1H), 5 , 55-5.60 (m, 2H), 7.08 (dd, J = 9.2, 2.1 Hz, 1H), 7.25 (s, 1H), 7.37 (d, J = 2 Hz, 1H), 7.51-7.57 (m, 3H), 8.02-8.05 (m, 2H), 8.15 (d, J = 9.2 Hz, 1H). LC-MS (E-retention time procedure: 1.61), MS m / z 699 (M ++ 1). EMAR m / z (M + H) + calc. for C39H47N4O8: 699.3394, found 699.3395.

Step 9j: Preparation of Compound 9, Example 9, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- [4

Cyclopropanesulfonylamino-carbonyl-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16 diazatricyclo [14.3.0.04.6] nonadec-7-en-14-yl] carbamic

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Following the procedure of Step 1j, 136.5 mg (0.195 mmol) of the acid (1S, 4R, 6S, 13S, 18R) -7cis-14-tert-butoxycarbonylamino-18- (7-methoxy-2-phenylquinolin) was converted -4-yloxy) -2,15-dioxo-3,16-diazatriciclo

[14.3.0.04.6] nonadec-7-en-4-carboxylic acid in 115 mg (76%) of tert-butyl acid ester (1S, 4R, 6S, 13S, 18R) -7-cis [4-cyclopropanesulfonylaminocarbonyl -18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14-yl] carbamic: NMR 1H (500 MHz, CD3OD) 0.97-1.54 (m, 11H), 1.20 (s, 9H), 1.59 (m, 1H), 1.71 (m, 1H), 1.75 -1.89 (m, 2H), 2.35-2.43 (m, 1H), 2.47-2.75 (m, 3H), 2.87-2.91 (m, 1H), 3 , 92 (s, 3H), 3.97-4.05 (m, 1H), 4.17 (d, J = 10.4 Hz, 1H), 4.64 (t, J = 7.6 Hz, 1H), 5.07 (m, 1H), 5.54 (sa, 1H), 5.63 (sa, 1H), 7.04 (d, J =

25 8.6 Hz, 1H), 7.21 (sa, 1H), 7.36 (s, 1H), 7.48-7.55 (m, 3H), 8.02, 8.03 (2s, 2H), 8.15 (d, J = 8.5 Hz, 1H). LC-MS (E-retention time procedure: 1.64), MS m / z 802 (M ++ 1). EMAR m / z (M + H) + calc. for C42H52SN5O9: 802.3486, found 802.3481.

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Step 10k: Preparation of Compound 10, Example 10, Preparation of [14-amino-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carbonyl] acid amide (1S, 9R, 6S, 14S, 18R) -7-ciscyclopropanesulfonic acid dihydrochloride

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A solution of 5 ml (20 mmol) of 4 N HCl / dioxane was added to 26 mg (0.032 mmol) of tert-butyl acid ester (1S, 4R, 6S, 13S, 18R) -7-cis- [4 -Cyclopropanesulfonylaminocarbonyl-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16diazatricyclo- [14.3.0.04.6] nonadec-7-en-14-yl] carbamic, the mixture stirred for 2 h, concentrated and dried (50 ° C; 2.66 KPa (20 torr); 12 h), providing 24.0 mg (100%) of [14-amino-18- (7-methoxy -2-phenylquinolin-4-yloxy) -2,15 dioxo-3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-4-carbonyl] acid amide (1S, 4R, 6S, 14S, 18R) -7-cis

10 cyclopropanesulfonic dihydrochloride in the form of a white solid: 1H NMR (300 MHz, CD3OD)  0.96-2.05 (m, 16H), 2.24-2.34 (m, 1H), 2.52 (MS, 1H), 2.73 (m, 1H), 2.91 (m, 2H), 3.57-3.74 (m, 1H), 4.06 (s, 3H), 4.17 ( m, 1H), 4.37 (m, 1H), 4.48 (m 2H), 5.07-5.14 (m, 1H), 5.66-5.75 (m, 1H), 5, 95 (m, 1H), 7.47 (m, 1H), 7.60-7.75 (m, 5H), 8.12 (m, 2H), 8.39-8.41 (m, 1H) LC-MS (E-retention time procedure: 1.24), MS m / z 702 (M ++ 1). EMAR m / z (M + H) + calc. for C37H44SN5O7: 702.2962, found 702.2976.

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Compounds 11/12, Example 11

Preparation of Compound 11, Example 11, tert-butyl acid ester (1S, 4R, 6S, 13S, 18R) -7-cis [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy-2-phenylquinolin-4) was achieved -yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nona-dec-7-en-14-yl] carbamic and Compound 12, Example 11, tert-butyl acid ester ( 1S, 4R, 6S, 13R, 18R) -7-cis- [4-cyclopropanesulfonylaminocarbonyl-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-3,16 diazatricyclo- [14.3 .0.04.6] nonadec-7-en-14-yl] carbamic using steps 11A-J.

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Stages 11A-11B: Preparation of 7-methoxyquinolin-4-ol from m-anisidine.

Step 11A) To a solution of m-anisidine (58 g, 471 mmol) in 800 ml of CH3CN was added Meldrum acid (75 g, 518 mmol) and trimethyl formate (60 g, 565 mmol). The heterogeneous mixture was heated at reflux for 2 h and entered into solution. The solvent was removed in vacuo, 30 ml of MeOH was added and the resulting precipitate was filtered and washed with 10-15 ml of MeOH. The MeOH addition / filtration procedure was repeated in concentrated mother liquors. The resulting combined solid was dried (~ 2.66 KPa (~ 20 torr), 45 ° C overnight) to provide (117.6 g, 90%) of the 5 - [(3-methoxyphenyl amino) methylene] - 2,2-dimethyl- [1,3] dioxane-4,6-dione intermediate.

Step 11B) To a solution of 500 g of Ph2O heated to 250 ° C, 108.7 g (392 mmol) of 5 - [(3-Methoxy-phenyl-amino) methylene] -2,2-dimethyl- [1,3] dioxane-4,6-dione over a period of 30 min. The mixture was heated for an additional 15 min, cooled to rt, diluted with 800 ml of hexanes and the resulting suspension was stirred overnight. The hexanes were removed by decantation, the solid residue was dissolved under reflux in 600 ml of MeOH, cooled to rt and the resulting solid was filtered and washed with a minimum amount of CH2Cl2. Analog recrystallization procedures were followed to provide a total of 20.73 g (30%) of 7-methoxyquinolin-4-ol as a light brown solid. 1H NMR (methanol-d4)  3.87 (s, 3H), 6.23 d, J = 7.3 Hz, 1H), 6.68 (d, J = 2.4 Hz, 1H), 6, 96 (dd, J = 9.0, 2.4 Hz, 1H), 8.11 (d, J = 9 Hz, 1H); LC-MS (retention time: 0.77, procedure D), MS m / z 176 (M ++ 1).

Step 11C: Preparation of 2-methyl ester of 1-tert-butyl ester of BOC-N-P2 acid [(4R) - (7-methoxy-quinolin-4oxo) prolin-methyl ester] 4- (7-methoxyquinoline- 4yloxy) -pyrrolidin-1,2-dicarboxylic acid from BOC-L-CIS-HYP-OH and 7-methoxy-quinolin-4-ol.

Step 11C) To a solution of 12.24 g (49.8 mmol) of BOC-L-CIS-HYP-OH (BOC-CIS-HYP-OME, methyl ester of N--tert-butoxycarbonyl-cis-L -4-hydroxyproline) and 26.14 g (99.7 mmol) of PPh3 in 200 ml of THF cooled to 0 ° C, a solution of 17.36 g (99.7 mmol) of DEAD and 8.73 was added g (49.8 mmol) of 7-methoxyquinolin-4-ol in 700 ml of THF over a period of 45 min. The mixture was allowed to slowly warm to rt overnight, concentrated in vacuo and the residue was purified on a 65 M Biotage column (eluting with 0% to 10% MeOH-EtOAc), yielding 12.78 g (64%) of the desired product, 2-methyl ester of 1-tert-butyl ester of BOC-N-P2 acid [(4R) - (7-methoxyquinolin-4-oxo) proline-methyl ester] -4- (7-methoxyquinoline-4-yloxy) -pyrrolidin-1,2-dicarboxylic, in the form of a colorless crystal. 1H NMR (CDCl3)  1.36 (s, 9H), 2.26-2.35 (m, 1H), 2.57-2.68 (m, 1H), 3.71 (s, 3H), 3.75-3.92 (m, 2H), 3.86, 3.87 (two s (rotamers) 3H), 4.41-4.53 (m, 1H), 5.09 (m, 1H) , 6.52 (d, J = 5.5 Hz, 1H), 7.06-7.09 (m, 1H), 7.24-7.26 (m, 1H), 7.94 (d, J = 9.1 Hz, 1H), 8.50-8.56 (m, 1H); LC-MS (retention time: 1.34, procedure D), MS m / e 403 (M ++ 1).

Step 11D: Preparation of 1-tert-butyl ester of BOC-N-P2 acid [(4R) - (7-methoxyquinolin-4-oxo) proline] -OH, 4- (7

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methoxyquinolin-4-yl-oxy) -pyrrolidin-1,2-dicarboxylic acid from 2-methyl ester of 1-tert-butyl ester of BOC-NP2 acid [(4R) - (7-methoxyquinoline-4-oxo) proline -methyl ester] 4- (7-methoxyquinolin-4-yloxy) -pyrrolidin-1,2-dicarboxylic.

Step 11D) To a solution of 8.54 g (21.2 mmol) of BOC-N-P2 [(4R) - (7-methoxyquinolin-4-oxo) proline methyl ester] in 600 ml of 5: 1 of THF / MeOH, a solution of 4.0 g (167 mmol) of LiOH in 150 ml of water was added. The mixture was stirred overnight, the pH was adjusted to pH 7 using 6 N aqueous HCl and the solution was concentrated until only the water phase remained. The solution was adjusted to pH 4 using 6 N aqueous HCl, NaCl was added to saturate the mixture and was repeatedly partitioned first with EtOAc and then with THF as the product was water soluble. The combined organic phases were dried (MgSO4) and concentrated to provide 8.18 g (99%) of BOC-N-P2 acid 1-butyl ester [(4R) - (7-methoxy-quinolin-4-oxo) proline] -OH, 4- (7-methoxyquinolin-4-yloxy) -pyrrolidin-1,2-dicarboxylic acid in the form of a white solid. 1H NMR (CDCl3-Methanol-d4)  1.42 (s, 9H), 2.40-2.49 (m, 1H), 2.68-2.77 (m, 1H), 3.88 (m , 2H), 3.94 (s, 3H), 4.41-4.53 (m, 1H), 5.32 (m, 1H), 6.86-6.92 (m, 1H), 7, 21 (dd, J = 9, 2 Hz, 1H), 7.30 (d, J = 2 Hz, 1H), 8.05-8.10 (m, 1H), 8.62 (d, J = 6 Hz, 1H); LC-MS (retention time 1.20, procedure A), MS m / z 389 (M ++ 1).

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Stage 11E: Coupling of P1 Remainder of HCl Salt of (1R, 2S / 1S, 2R) -Vinyl Acca) -CO2Et with P2 residue from Boc (4R) - (7methoxyquinoline-4-oxo) -S-proline to manufacture BOCN- P2 [(4R) -7-methoxyquinolin-4-oxo) prolin] -P1 (1R, 2S / 1S, 2R) vinylAcca-COOEt and chromatographic separation of BOCN-P2 diastereomers [(4R) - (7-methoxyquinoline-4- oxo) -S-proline] -P1 (1R, 2S, VinylAcca) -CO2Et (most bioactive isomer) and BOCN-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline) -P1 (1S , 2RVinilAcca) -CO2Et.

Step 11E) To a solution of 4.50 g (11.60 mmol) of Boc-4 (R) - (7-methoxyquinolin-4-oxo) proline, 2.66 g (13.9 mmol) of HCl salt of VinylAcca (existing in the form of a 1: 1 mixture of diastereoisomers (1R, 2S / 1S, 2R), in which the cyclopropyl carboxyethyl group is syn with respect to the vinyl moiety), 10 ml (57.4 mmol) of DIPEA, and 2.13 g (13.9 mmol) of HOBT.H2O in 150 ml of CH2Cl2 was added 5.27 g (13.9 mmol) of the HBTU coupling reagent and the mixture was stirred overnight. The solution was diluted with 200 ml of CH2Cl2 and partitioned with a buffer of pH 4.0 (2 x 50 ml). The organic phase was washed with saturated aqueous NaHCO3 (2 x 50 ml), water (2 x 50 ml) and brine (2 x 50 ml). The organic solution was dried (MgSO4), concentrated and purified using a Biotage 65M column (eluting with 0% to 9% MeOH / EtOAc) to provide BOC-NH-P2 [(4R) - (7-methoxyquinoline-4 -oxo) -S-proline] P1 (1R, 2SVinylAcca residue P1) -COOEt as the first eluting isomer (2.21 g, 36% total), followed by 1.13 g (19%) of the isomer pure minor Fr, BOC-NH-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] P1 (1S, 2RVinylAcca residue P1) -CO2Et. Mixed fractions were also obtained. Data for BOCN-P2 [(4R) - (7methoxyquinolin-4-oxo) -S-proline] -P1 (1R, 2S) - (VinylAcca) -COOEt: 1H NMR (CDCl3)  1.16 (t, J = 7 Hz, 3H), 1.35 (s, 9H), 1.37-1.47 (m, 1H), 1.74-1.88 (m, 1H), 2.04-2.13 (m , 1H), 2.32-2.46 (m, 1H), 2.58-2.69 (m, 1H), 3.76 (m, 1H), 3.87 (s, 3H), 4, 02-4.13 (m, 2H), 4.30-4.44 (m, 1H), 5.05-5.19 (m, 2H), 5.24 (d, J = 17 Hz, 1H) , 5.63-5.71 (m, 1H), 6.61 (m, 1H), 7.07 (dd, J = 9, 2 Hz, 1H), 7.22 (d, J = 2 Hz, 1H), 7.76-7.83 (m, 1H), 7.92 (d, J = 9 Hz, 1H), 8.50 (d, J = 5 Hz, 1H). LC-MS (retention time: 1.38, procedure A), MS m / z 526 (M ++ 1).

Data for BOC-NH-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] -P1 (1S, 2RVinylAcca residue P1) -CO2Et: 1H NMR (methanol-d4)  1.23 ( t, J = 7 Hz, 3H), 1.38 (s, 9H), 1.45 (dd, J = 9.5, 5.5 Hz, 1H), 1.77-1.87 (m, 1H ), 2.10-2.16 (m, 1H), 2.37 (m, 1H), 2.73 (m, 1H), 3.77 (m, 1H), 3.90 (s, 3H) , 4.07-4.15 (m, 2H), 4.46 (m, 1H), 5.10 (d, J = 10 Hz, 1H), 5.21 (m, 1H), 5.27 ( d, J = 17 Hz, 1H), 5.69-5.76 (m, 1H), 6.64 (m, 1H), 7.10 (dd, J = 9.2, 2.2 Hz, 1H ), 7.27 (m, 1H), 7.95 (d, J = 9.2 Hz, 1H), 8.56 (d, J = 5 Hz, 1H). LC-MS (retention time: 1.45, procedure B), MS m / z 526 (M ++ 1).

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Step 11F: Preparation of N-BOC-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] -P1 (1R, 2SVinylAcca) -CO2H or Isomer P1 (1R, 2S) VinylAcca of the terc ester {1- [2- (1-Cyclopropanesulfonylaminocarbon-yl-2-vinylcyclopropylcarbamoyl) -4- (7-methoxyquinolin-4-yloxy) pyrrolidin-1-carbonyl] -2,2-dimethylpropyl} -carbamic acid -butyl.

Step 11F) To a solution of 794 mg (1.51 mmol) of N-BOC-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] -P1 (1R, 2SVinylAcca) -CO2Et ( from step 11E) in 68 ml of 12% MeOH / THF a solution of 218 mg (9.08 mmol) of lithium hydroxide in 30 ml of water was added and the mixture was stirred 16 h. The pH was adjusted to neutral by the addition of 6 N aqueous HCl, concentrated until only water remained, the solution was adjusted to pH 4 using 1 N aqueous HCl and then extracted with 50% THF-EtOAc (portions of 5 x 200 ml). The combined organic phases were dried (MgSO4) and concentrated to provide 808 mg of crude product which was further dried to provide 752 mg (100%) of N-BOC-P2 [(4R) - (7-methoxyquinoline-4- oxo) -S-proline] P1 (1R, 2S VinylAcca) -CO2H. 1H NMR (Methanol-d4)  1.37-1.43 (m, 1H), 1.39 (s, 9H), 1.69-1.78 (m, 1 H), 2.16-2, 24 (m, 1H), 2.44-2.54 (m, 1H), 2.64-2.74 (m, 1H), 3.89-3.94 (m, 2H), 3.96 ( s, 3H), 4.40-4.43 (m, 1H), 5.11 (d, J = 10 Hz, 1H), 5.31 (d, J = 17 Hz, 1H), 5.40 ( m, 1H), 5.79-5.87 (m, 1H), 6.91 (s, 1H), 7.04 (d, J = 6 Hz, 1H), 7.25 (dd, J = 9 , 1, 2 Hz, 1H), 7.29 (m, 1H), 8.09 (d, J = 9.1 Hz, 1H), 8.66 (d, J = 6 Hz, 1H). LC-MS (retention time: 1.05, procedure H). MS m / z 498 (M ++ 1).

Step 11G: Preparation of N-BOC-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] -P1 (1R, 2SVinylAcca) CONHSO2Cyclopropane or Diastereomer P1 (1R, 2S) VinylAcca of tert-ester 2- (1-Cyclopropanesulfonylamino-carbonyl-2-vinylcyclopropylcarbamoyl) -4- (7-methoxy-quinolin-4-yloxy) pyrrolidine-1-carboxylic acid butyl ester.

Step 11G) To a solution of 399.5 mg (0.668 mmol) of N-BOC-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-prolin] P1 (1R, 2SVinylAcca) -CO2H in THF (4 ml), CDI (434 mg, 2.68 mmol) was added and the resulting solution was refluxed for 60 min and allowed to cool to rt. Cyclopropylsulfonamide (406 mg, 3.35 mmol) was added in one portion before the addition of a pure DBU solution (0.50 ml, 3.35 mmol). The reaction was stirred for 16 h, diluted with 50% THF-EtOAc (200 ml) and washed with a pH 4.0 buffer of saturated brine (2 x 40 ml), dried (MgSO4) and subjected to Chromatography on a Biotage 25M column (eluting with 0% to 15% MeOH in CH2Cl2) to provide 217 mg (54%) of the desired product N-BOC-P2 [(4R) - (7-methoxyquinoline-4-oxo) - S-proline] P1 (1R, 2S VinylAcca) -CONHSO2Cyclopropane. 1H NMR (Methanol-d4)  1.01-1.10 (m, 2H), 1.11-1.18 (m, 1H), 1.20-1.27 (m, 1H), 1.39 -1.48 (m, 1H), 1.44 (s, 9H), 1.87 (dd, J = 8, 5 Hz, 1H), 2.01-2.38 (m, 2H), 2, 57 (dd, J = 14, 7 Hz, 1H), 2.91-2.96 (m, 1H), 3.83-3.92 (m, 2H), 3.94 (s, 3H), 4 , 36-4.39 (m, 1H), 5.11 (d, J = 10 Hz, 1H), 5.29 (d, J = 17 Hz, 1H), 5.38 (m, 1H), 5 , 74-5.81 (m, 1H), 6.91 (d, J = 5.5 Hz, 1H), 7.20 (dd, J = 9.2, 2.4 Hz, 1H), 7, 29 (m, 1H), 8.07 (d, J = 9.2 Hz, 1H), 8.60 (d, J = 5.5 Hz, 1H). LC-MS (retention time: 1.28, procedure I). MS m / z 601 (M ++ 1)

Stages 11H and 11I: Preparation of BOCNH-P3 (2 (S) -terc-butoxycarbonylamino-8-nonenoyl) -P2 [(4R) - (7-methoxyquinolin4-oxo) -S-proline] -P1 (1R, 2S- VinylAcca) -CONHSO2cyclopropane or Isomer P1 (1R, 2S) of the tert-butyl ester of {1- [2- (1-cyclopropanosul-phylaminocarbonyl-2-vinyl-cyclopropylcarbamoyl) -4- (7-methoxyquinolin-4-yloxy) -pyrrolidin-1carbonyl] -oct-7-enyl} carbamic.

Stages 11H) A total of 600 mg (1.0 mmol) of BOC-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] P1 (1R, 2SVinylAcca-CONHSO2Cyclopropane), isomer P1 (1R , 2S) 2- (1-Cyclopropanesulfonylaminocarbonyl-2-vinylcyclopropyl-carbamoyl) -4- (7-methoxyquinolin-4-yloxy) pyrrolidine-1-carboxylic acid tert-butyl ester vinyl, dissolved in 4N HCl / dioxane (4 ml, 16 mmol) and stirred for 3 h at rt. The reaction mixture was concentrated to provide HN-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] -P1 (1R, 2SVinylAcca) -CONHSO2Cyclopropane, HCl Bis crude: P1 isomer (1R , 2S) 4- (7-Methoxyquinoline-4-yloxy) -pyrrolidine-2-carboxylic acid dihydrochloride (1-cyclopropane-sulfonylaminocarbonyl-2-vinylcyclopropyl) amide in the form of a brown solid which was used immediately in Stage 11I.

Stage 11I) HN-P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] -P1 (1R, 2SVinylAcca) -CONHSO2Cyclopropane, HCl Salt

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Bis of step 11H, was suspended in 20 ml of dichloromethane. To this mixture was added 352 mg (1.30 mmol) of 2 (S) -terc-butoxycarbonylamino-8-nonenoic acid purchased from RSP Amino Acids, HOAT (82 mg, 0.60 mmol), DIPEA (0.74 ml, 5.0 mmol) and HATU (494 mg, 1.30 mmol) at rt. The reaction mixture was stirred for 16 h, and most of the CH2Cl2 was removed in vacuo. The mixture was diluted with a saturated buffer of pH 4.0 (150 ml) and extracted in EtOAc (4 x 200 ml). The combined organic phases were dried (MgSO4), concentrated and purified on a 40M Biotage column (eluting with 0% to 15% MeOH in CH2Cl2) to provide 574 mg (76%) of BOCNH-P3 (2 (R / S) -terc-butoxycarbonylamino-8-noneic acid) -P2 [(4R) - (7-methoxyquinolin-4-oxo) -S-proline] -P1 (1R, 2SVinylAcca) -CONHSO2Cyclopropane or Isomer P1 (1R, 2S) of {1- [2- (1-cyclopropanesulfonylaminocarbonyl-2-vinyl-cyclopropylcarbamoyl) -4- (7-methoxyquinolin-4-yloxy) -pyrrolidin-1-carbonyl] -oct-7-yl} carbamic acid tert-butyl ester. LC-MS m / e 754 (retention time: 1.64, procedure I).

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Preparation of Compound 11, Example 11, tert-butyl acid ester (1S, 4R, 6S, 13S, 18R) -7-cis- [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy-2-phenyl-quinolin-4- iloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14-yl] carbamic and Compound 12, Example 11, tert-butyl acid ester (1S, 4R, 6S, 13R, 18R) -7-cis- [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16diazatricyclo [14.3.0.04.6] -nonadec-7-en-14-il] carbamic.

Step 11J) To a solution of BOCNH-P3 (2 (S) -terc-butoxycarbonylamino-8-nonenoyl) -P2 [(4R) - (7-methoxyquinolin-4oxo) -S-proline] -P1 (1R, 2S- VinylAcca) -CONHSO2Cyclopropane or P1 (1R, 2S) Isomer of {1- [2- (1-cyclopropanesulfonylaminocarbonyl-2-vinylcyclopropyl-carbamoyl) -9- (7-methoxyquinoline-4-yloxy) -pyrrolidine tert-butyl ester -1carbonyl] -oct-7-enyl} carbamic from Step 11I (537 mg, 0.712 mmol) in 0.7 l of 1,2-DCE degassed with Argon, 60 mg Grubb catalyst was added, the mixture was degassed in an atmosphere of Ar and heated at reflux for 4 h. The reaction mixture was cooled to rt, the mixture was degassed once more, additional 60 mg portions of Grubb catalyst were added and the mixture was heated at reflux for 4 h. The reaction mixture was cooled to rt, the mixture was degassed once more and a final 60 mg portion of Grubb catalyst was added and the mixture was heated at reflux for 2 h. The mixture was cooled to rt, stirred for a further 9 h and the resulting dark brown solution was concentrated in vacuo and purified using three Analtech 1000 PT PTLC plates (20 x 40 cm), eluted sequentially with 0% MeOH 3% in CH2Cl2 to provide 80 mg of the crude product. This material was further purified using four 500 E E-Merck PTLC plates to provide 10 mg of Compound 11, Example 11, tert-butyl acid ester (1S, 4R, 6S, 13S, 18R) -7-cis- [ 4-Cyclopropanesulfonylaminocarbonyl-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-14il] carbamic and Epimer P3 of Compound 12, Example 11, tert-butyl acid ester (1S, 4R, 6S, 13R, 18R) -7-cis

image46

[4-Cyclopropanesulfonylaminocarbonyl-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14-il ] carbamic. Data for Compound 11: 1H NMR (CD3OD)  0.87-1.10 (m, 4H), 1,152.01 (m, 11H), 1.21 (s, 9H), 2.19-2.29 (m, 1H), 2.45-2.73 (m, 3H), 2.83-2.92 (m, 1H), 3.93 (s, 3H), 4.11-4.17 (m , 1H), 4.20-4.29 (m, 1H), 4.60-4.69 (m, 2H), 5.21-5.27 (m, 1H), 5.47 (m, 1H ), 5.54-5.63 (m, 1H), 6.96 (d, J = 5.5 Hz, 1H), 5.10 (dd, J = 9.5, 2.5 Hz, 1H ), 7.27 (d, J = 2.5 Hz, 1H), 8.17 (d, J = 9.5 Hz, 1H), 8.61 (d, J = 5.5 Hz, 1H). LC-MS m / e 726 (retention time: 1.57, procedure A). Data for Compound 12: 1H NMR (CD3OD)  0.79-0.99 (m, 2H), 1.00-1.65 (m, 9H), 1.15 (s, 9H), 1.80 -2.05 (m, 4H), 2.32-2.57 (m, 2H), 2.59-2.83 (m, 2H), 2.84-2.92 (m, 1H), 3 , 92 (s, 3H), 4.03-4.16. (M, 1H), 4.27-4.31 (m, 1H), 4.53-4.78 (m, 2H), 5, 42 (m, 3H), 6.91 (d, J = 5 Hz, 1H), 7.11 (dd, J = 9.2, 2.2 Hz, 1H), 7.26 (d, J = 2 , 2 Hz, 1H), 8.11 (d, J = 9.2 Hz, 1H), 8.60 (d, J = 5 Hz, 1H). LC-MS m / e 726 (retention time: 1.49, procedure A).

Examples 12,13

Preparation of Compounds - 13 and 14

image 1

Compound 13, Example 12

Preparation of tert-butyl acid ester (1S, 4R, 6R, 13S, 17R) - [4-cyclopropane-sulfonylaminocarbonyl-17- (7-methoxy2-phenylquinolin-4-yloxy) -2,14-dioxo-3, 15-diazatricyclo [13.3.0.04.6] -octadec-13-yl] -carbamic, Compound 13, Example 12.

Procedure A. A solution of 30 mg (0.038 mmol) of tert-butyl acid ester (1S, 4R, 6S, 13S, 17R) -7-cis- [4-cyclopropanesulfonyl-aminocarbonyl-17- (7-methoxy-2-phenylquinolin -4-yloxy) -2,14-dioxo-3,15 diazatricyclo [13.3.0.04.6] octadec-7-en-13-yl] carbamic, Compound 7, Example 7, was dissolved in 15 ml of MeOH and then 20 it was removed under vacuum and repeatedly degassed in an atmosphere of Ar. To this solution was added 15 mg of 20% Pd (OH) 2 / C, the mixture was removed three times and placed under 1 atmosphere of H2 gas and finally allowed to stir under 1 atmosphere of H2 for 45 minutes. Then, the reaction contents were removed and placed under an argon atmosphere, filtered on celite and the resulting crude residue was purified on preparative HPLC (column: Xterra C18 S5 30 x 75 mm, gradient from 35% to 80 % of Solvent B / A for 28 min, 25 retention time 3 min; in which Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent

image47

B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 40 ml / min). Compound 13, Example 12 was eluted first (8.8 mg, 29.3%), followed by 15 mg of mixed fractions containing Compound 13 and an undefined product: 1H NMR (methanol-d4)  0, 84-2.08 (m, 26H), 2.51-2.69 (m, 1H), 2.74-2.78 (m, 1H), 2.91-3.01 (m, 1H), 3.96 (s, 3H), 4.04-4.09 (m, 1H), 4.17 (d, J = 11 Hz, 1H), 4.72 (t, J = 8 Hz, 1H), 4.97 (d, J = 12 Hz, 1H), 5.64 m, 1H), 7.13 (dd, 9, 1.6 Hz, 1H), 7.32 (s, 1H), 7.40 (d, J = 1.6 Hz, 1H), 7.53-7.62 (m, 3H), 8.02-8.05 (m, 2H), 8.15 (d, J = 9 Hz, 1 HOUR). LC-MS m / e 790 (retention time: 2.55, procedure D, except for the increased gradient time from 2 to 3 min).

image 1

Step i of Procedure B) To a solution of 400 mg (0.584 mmol) of acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert

10-butoxycarbonylamino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15-diazatricyclo [13.3.0.04.6] octa-dec-7-en-4-carboxylic acid and 1.90 g (11.7 mmol) of potassium diazodicarboxylate in 20 ml of MeOH, a solution of 1.34 ml (22.4 mmol) of glacial AcOH in 19 ml of MeOH was slowly added dropwise by a syringe pump during 5 h, followed by stirring for another 6 h. To this mixture was added an additional 380 mg of potassium diazodicarboxylate (prepared as in Org. React. 1991, 40, p. 91), followed by 400 µl of glacial AcOH in 4 ml of MeOH

15 using a syringe pump for 70 min. The mixture was stirred overnight, diluted with 200 ml of Et2O, filtered and concentrated in vacuo. The residue was chromatographed on 20 g of SiO2 (eluting with 5% MeOH / CH2Cl2), providing 450 mg of crude product that was dissolved in 12 ml of MeOH and injected six times (injections 6 x 2 ml) in a preparative HPLC column: Xterra C18 30 x 100 mm S5, 30% to 85% gradient of Solvent B / A for 25 min, retention time 5 min; in which Solvent A is 10 MeOH

20% / 90% H2O with 0.1% TFA. Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 40 ml / min). The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 using a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to provide 360 mg (90%) of acid (1S, 4R, 6R, 13S, 17R) -13-tert-butoxycarbonylamino-17- (7-methoxy-2-phenyl- quinolin-4-yloxy) -2,14-di

Oxo-3,15-diazatricyclo [13.3.0.04.6] octadecane-4-carboxylic: 1 H NMR (methanol-d4)  1.10 (s, 9 H), 1.34 (m, 12 H), 1 , 68 (m, 1 H), 1.84 (m, 1 H), 2.05 (s, 1 H), 2.58 (m, 1 H), 2.71 (m, 1 H) 3, 94 (s, 3 H), 4.05 (dd, J = 11.4, 2.9 Hz, 1 H), 4.16 (dd, J = 11.8, 2.9 Hz, 1 H), 4.67 (t, J = 8.1 Hz, 1 H), 4.90 (m, 1 H), 5.61 (s, 1 H), 7.10 (dd, J = 9, 2 Hz, 1 H), 7.29 (s, 1 H), 7.38 (d, J = 2 Hz, 1 H), 7.55 (m, 3 H), 8.03 (d, J = 6.7 Hz, 2 H), 8.13 (d, J = 9 Hz, 1 H). LC-MS m / e 687 (retention time: 2.84, procedure D, except for the increased gradient time from 2 to 4 min). LC-MS m / e 687

30 (retention time: 2.84, procedure D, except for the increased gradient time from 2 to 4 min).

image48

Step ii / 12j-Procedure B, Preparation of Compound 13, Example 12, Preparation of tert-butyl acid ester (1S, 4R, 6R, 13S, 17R) - [4-cyclopropanesulfonylaminocarbonyl-17- (7-methoxy-2- phenyl-quinolin-4-yloxy) -2,14-dioxo3,15-diazatricyclo [13.3.0.04.6] octadec-13-yl] -carbamic, Compound 13, Example 12.

image 1

5 Procedure B-Stage ii / Stage 12j) Following the procedure and purification conditions of Stage 1j, with the exception that preparative HPLC / extraction treatment was used instead of normal phase chromatography, 260 mg (0, 38 mmol) (1S, 4R, 6R, 13S, 17R) -13-tert-butoxycarbonyl-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15 -diazatricyclo [13.3.0.04.6] octa-decane-4-carboxylic acid in 192 mg (64%) tert-butyl acid ester (1S, 4R, 6R, 13S, 17R) - [4-cyclopropanesulfonylaminocarbonyl-17- (7-methoxy -2-phenylquinolin-4-yloxy) -2,14

10 dioxo-3,15-diazatricyclo [13.3.0.04.6] octadec-13-yl] carbamic, Compound 13, Example 12, spectroscopically identical to that prepared in Procedure A. Representative preparative HPLC / Extraction treatment conditions: Xterra C18 30 x 100 mm S5, gradient from 35% to 100% Solvent B / A for 25 min, retention time 5 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 40 ml / min) The pH of the combined fractions is

15 adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 using a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated, providing the aforementioned amount of Compound 13.

Procedure B-Stage iii / Step 13k: Preparation of Compound 14, Example 13, [13-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15-diazatricyclo [13.3. 0.04.6] -octadecane-4-carbonyl] acid amide

20 (1S, 4R, 6R, 13S, 17R) -cyclopropanesulfonic dihydrochloride Procedure B-Stage iii / Step 13k) A solution of 2.5 ml (10 mmol) of 4 N HCl / dioxane was added to 161 mg

image 1

(0.204 mmol) of tert-butyl acid ester (1S, 4R, 6R, 13S) - [4-cyclopropanesulfonylaminocarbonyl-17- (7-methoxy-2

Phenylquinolin-4-yloxy) -2,14-di-oxo-3,15-diazatricyclo [13.3.0.04.6] octadec-13-yl] carbamic, the mixture was stirred for 3 h,

concentrated and dried (40 ° C; 2.66 KPa (20 torr); 24 h), yielding 156 mg (~ 100%) of Compound 14,

5 Example 13, [13-amino-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15-diazatricyclo [13.3.0.04.6] octa-decane-4

carbonyl] acid amide (1S, 4R, 6R, 13S, 17R) -cyclopropanesulfonic dihydrochloride in the form of a white solid:

1H NMR (500 MHz, CD3OD)  1.03-1.26 (m, 3 H), 1.26-1.75 (m, 14 H), 1.84 (s, 1 H), 2.02 (m, 1 H), 2.75 (m, 1 H),

2.85 (m, 1 H), 2.96 (m, 1 H), 4.05 (s, 3 H), 4.23 (m, 1 H), 4.31 (s, 1 H), 4.46 (d, J = 11.29 Hz, 1 H), 5.95 (s, 1 H), 7.44

(d, J = 8.85 Hz, 1 H), 7.57 (s, 1 H), 7.61 (s, 1 H), 7.75 (dd, J = 8.55, 7.32 Hz , 3 H), 8.13 (d, J = 6.71 Hz, 1 H), 8.38 (d, 10 J = 9.16 Hz, 1 H). LC-MS m / e 690 (retention time: 2.10, procedure D, except for the gradient time

increased from 2 to 4 min).

Examples 14, 15

Preparation of Compounds 15 and 16

image 1

Step io Procedure B) To a solution of 470 mg (0.67 mmol) of acid (1S, 4R, 6S, 14S, 18R) -8-cis-14-tert-butoxycarbonylamino-18- (7-methoxy-2-phenylquinolin -4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nona-dec-7-en-4-carboxylic acid and 1.90 g (11.7 mmol) of potassium diazodicarboxylate (prepared as in Org. React. 1991, 40, p. 91) in 20 ml of MeOH, a solution of 1.34 ml (22.4 mmol) of glacial AcOH in 20 ml of MeOH was slowly added dropwise by pump of syringe for 6.5 h. This procedure was repeated twice, but using a

20 time interval of 12 h instead of 6.5 h, during which time the reaction stopped at approximately 90% of its completion. The mixture was concentrated in vacuo and then chromatographed on 20 g of SiO2 (eluting with 5% MeOH / CH2Cl2). The resulting residue was dissolved in 18 ml of MeOH and injected nine times (injections of 9 x 2 ml) in a preparative HPLC column: Xterra C18 30 x 100 mm S5, 30% to 75% gradient of Solvent B / A for 30 min, retention time 5 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 40 ml / min)

image49

5 The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 a 3 N aqueous solution and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to provide 278 mg (59%) of acid (1S, 4R, 6R, 14S, 18R) -14-tert-butoxycarbonylamino-18- (7-methoxy-2-phenyl- quinolin-4-yloxy) -2,15-dioxo-3,16 diazatricyclo [14.3.0.04.6] nonadecane-4-carboxylic: 1 H NMR (500 MHz, methanol-d4).  1.23 (s, 9 H), 1.12-1.49 (m, 11

10 H), 1.50-1.67 (m, 4 H), 1.71-1.86 (m, 2 H), 2.54-2.60 (m, 1 H), 2.65- 2.72 (m, 1 H), 3.94 (s, 3 H), 4.05 (dd, J = 11.29, 3.36 Hz, 1 H), 4.28-4.33 (m , 1 H), 4.65 (d, J = 11.29 Hz, 1 H), 4.70 (t, J = 8.09 Hz, 1 H), 5.55-5.61 (m, 1 H), 7.07-7.15 (m, 1 H), 7.28 (s, 1 H), 7.38 (d, J = 1.83 Hz, 1 H), 7.50-7, 59 (m, 3 H), 8.04 (d, J = 7.02 Hz, 2 H), 8.15 (d, J = 9.16 Hz, 1 H). LC-MS m / e 701 (retention time: 3.01, procedure D, except for the increased gradient time from 2 to 4 min).

Step II / 14j-Procedure B, Preparation of Compound 15, Example 14, Preparation of tert-butyl acid ester (1S, 4R, 6R, 14S, 18R) - [4-cyclopropanesulfonylaminocarbonyl-18- (7-methoxy-2 -phenylquinolin-4-yloxy) -2,15-dioxo-3,16 diazatricyclo- [14.3.0.04.6] nonadec-14-yl] carbamic.

image 1

Procedure B-Stage ii / Stage 14j) Following the purification and procedure conditions analogous to

Stages 1j and 12j, respectively, converted 100 mg (0.143 mmol) of acid (1S, 4R, 6R, 14S, 18R) -14-tert-butoxycarbonylamino-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) ) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nonadecane-4-carboxylic acid in 108 mg (93%) of tert-butyl acid ester (1S, 4R, 6R, 14S) - [4- Cyclopropanesulfonylaminocarbonyl18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo- [14.3.0.04.6] nonadec-14-yl] carbamic, Compound 15, Example 14 : 1 H NMR (500 MHz, methanol-d4) ,20 1.20 (m, 9 H) 0.83-1.83 (m, 21 H), 2.48 (s, 1 H), 2.73 (s , 1 H), 2.95

25 (s, 1 H), 3.94 (s, 3 H), 4.04 (m, 1 H), 4.24 (m, 1 H), 4.67 (m, 2 H), 5, 60 (s, 1 H), 7.12 (m, J = 8.85 Hz, 1 H), 7.30 (s, 1 H), 7.38 (s, 1 H), 7.57 (m , J = 6.10 Hz, 3 H), 8.03 (m, J = 5.80 Hz, 2 H), 8.16 (d, J = 8.85 Hz, 1 H). LC-MS m / e 804 (retention time: 1.73, procedure D.

image50

Procedure B-Stage iii / Stage 15k: Preparation of Compound 16, Example 15, [14-amino-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3. 0.04.6] -Nonadecane-4-carbonyl] acid amide (1S, 4R, 6R, 14S, 18R) -cyclopropanesulfonic acid dihydrochloride

image 1

5 Procedure B-Stage iii / Step 15k) A solution of 2.5 ml (10 mmol) of 4 N HCl / dioxane was added to 101 mg (0.126 mmol) of tert-butyl acid ester (1S, 4R, 6R, 14S, 18R) - [4-Cyclopropanesulfonyl-amino-carbonyl-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nona-dec- 14-yl] carbamic (Compound 15 of Example 14), the mixture was stirred for 3 h, concentrated and dried (40 ° C; 2.66 KPa (20 torr); 24 h), providing 98 mg (~ 100% ) of Compound 16, Example 15, [14-amino-18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3.16

10 diazatricyclo [14.3.0.04.6] nonadecane-4-carbonyl] amide (1S, 4R, 6R, 14S, 18R) -cyclopropanesulfonic acid dihydrochloride in the form of a white solid: 1H NMR (500 MHz, CD3OD)  1.05-1.77 (m, 19 H), 1.88 (m, 2 H), 2.60 (m, 1 H), 2.90 (m, 1 H), 2.96 (m, 1 H), 4.05 (s, 3 H), 4.21 (m, 1 H), 4.41 (m, 1 H), 4.46 (s, 1 H), 5.93 (s, 1 H), 7.44 (d, J = 8.55 Hz, 1 H), 7.58 (s, 1 H), 7.62 (s, 1 H), 7.74 (m, 3 H) , 8.10 (m, J = 7.02 Hz, 2 H), 8.33 (d, J = 9.16 Hz, 1 H). LC-MS m / e 704 (retention time: 2.24, procedure D, except for the increased gradient time

15 from 2 to 4 min).

Step 16j: Preparation of Compound 17, Example 16 acid tert-butyl ester (1S, 4R, 6S, 13S, 17R) -7-cis- [4-cyclobutanesulfonylamino-carbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy ) -2,14-dioxo-3,15 diazatricyclo, [13.3.0.04,6] octadec-7-en-13-il] carbamic

image 1

20 Step 16j) Following the analogous procedural conditions of Step 1j, with the exception that cyclobutyl sulfonamide was replaced by cyclopropylsulfonamide, as well as using the following preparative HPLC / Extraction Treatment Purification: Xterra C18 30 x 100 mm S5 HPLC column , gradient from 35% to 85% of Solvent B / A for 23 min, retention time 4 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 30

image51

ml / min The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to give the resulting conversion: 52 mg (0.076 mmol) of acid (1S, 4R, 6S, 13S, 17R) -7-cis-13-tert-butoxycarbonylamino-17- ( 7-methoxy-2-phenylquinolin-4-yloxy) 5 2,14-dioxo-3,15-diaza-tricycle [13.3.0.04.6] octa-dec-7-en-4-carboxylic acid became 12 mg ( 20%) of tert-butyl acid ester (1S, 4R, 6S, 13S, 17R) -7-cis- [4-cyclobutanesulfonylamino-carbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy) 2.14- dioxo-3.15-diazatricyclo [13.3.0.04.6] octadec-7-en-13-yl] carbamic: 1H NMR (500 MHz, CD3OD)  1.18 (s, 9 H), 1.23-1 , 56 (m, 11 H), 1.76 (m, 1 H), 1.98-2.13 (m, 3 H), 2.15-2.34 (m, 2 H), 2.48 (m, 1 H), 2.62 (m, 1 H), 2.74 (m, 1 H), 3.98 (m, 3 H), 4.29 (m, 3 H), 4.66 (m, 1 H), 5.41 (m, 1 H), 5.64 (m, 2 H), 7.12 (d, J = 8.85 Hz, 1 H), 7.29 (s, 1 H), 7.42

10 (m, 1 H), 7.56 (m, 3 H), 8.10 (m, 3 H). LC-MS (D-retention time procedure: 1.61), MS m / z 802 (M ++ 1).

Step 17j-Preparation of Compound 18, Example 17, Preparation of tert-butyl acid ester (1S, 4R, 6R, 13S, 17R) - [4-cyclobutanesulfonylaminocarbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy ) -2,14-dioxo-3,15 diazatricyclo [13.3.0.04.6] octadec-13-yl] carbamic.

image 1

Step 17j) Following the procedure and purification conditions of Step 1j, with the exception that preparative HPLC / extraction treatment was used instead of normal phase chromatography, 56 mg (0.082 mmol) of acid (1S, 4R, 6R, 13S, 17R) -13-tert-butoxycarbonylamino-17- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,14-dioxo3,15-diazatricyclo [13.3.0.04.6] octadecane -4-carboxylic acid in 12 mg (20%) of tert-butyl acid ester (1S, 4R, 6R, 13S, 17R) - [4-cyclobutanesulfonylaminocarbonyl-17- (7-methoxy-2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15

20 diazatricyclo- [13.3.0.04.6] octadec-13-yl] carbamic, Compound 18, Example 17, Representative Preparative HPLC / Extraction Treatment Conditions: Xterra C18 30 x 100 mm S5, 35% to 85% gradient Solvent B / A for 25 min, retention time 5 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 40 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed at

25 empty. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated, providing the aforementioned amount of Compound 18: 1H NMR (500 MHz, CD3OD)  1.07 (s, 9 H), 1.22-1.62 (m, 15 H), 1.63-1.78 (m, 2 H), 2.04 (m, 1 H), 2.52 (m, 4 H), 2.87 (m, 1 H), 4, 04 (s, 3 H), 4.14 (m, 2 H), 4.36 (m, 1 H), 4.78 (m, 1 H), 5.07 (m, 1 H), 5, 82 (s, 1 H), 7.32 (d, J = 9.16 Hz, 1 H), 7.51 (s, 1 H), 7.56 (d, J = 8.55 Hz, 1 H ), 7.72 (m, 3 H), 8.08 (m, 2 H), 8.32

30 (m, 1 H). LC-MS (Procedure D-retention time: 1.68), MS m / z 804 (M ++ 1).

image52

Step 18j-Preparation of Compound 19, Example 18, Preparation of tert-butyl acid ester (1S, 4R, 6R, 14S, 18R) - [4-cyclobutanesulfonylaminocarbonyl-18- (7-methoxy-2-phenylquinolin-4-yloxy ) -2,15-dioxo-3,16diazatricyclo- [14.3.0.04.6] nona-dec-14-il] carbamic.

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5 Step 18j) Following the analogous purification and procedure conditions of Step 1j and 17j, respectively, 50 mg (0.071 mmol) of acid (1S, 4R, 6R, 14S, 18R) -14-tert-butoxycarbonylamino- were converted 18 (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nonadecane-4-carboxylic acid in 22 mg (38%) of tertiary ester -butyl acid (1S, 4R, 6R, 14S, 18R) - [4-cyclobutanesulfonylaminocarbonyl-18- (7-methoxy-2-phenyl-quinolin4-yloxy) -2,15-dioxo-3,16-diazatricyclo [14.3 .0.04.6] nonadec-14-yl] carbamic, Compound 19, Example 18: 1H NMR (500

10 MHz, methanol-d4 solvent)  1.21 (s, 9 H), 1.14-1.88 (m, 20 H), 2.35 (m, 2 H), 2.53 (m, 3 H), 2.83 (dd, J = 13.4, 6.4 Hz, 1 H), 4.01 (s, 3 H), 4.12 (d, J = 11.3 Hz, 1 H) , 4.27 (m, 1 H), 4.36 (m, 1 H), 4.70 (m, 1 H), 5.73 (m, 1 H), 7.21 (d, J = 9 , 2 Hz, 1 H), 7.41 (m, 1H), 7.46 (m, 1H), 7.64 (m, 3H), 8.08 (m, 2H), 8.25 (d, J = 9.2 Hz, 1 H). LC-MS m / e818 (retention time: 1.75, Procedure D.

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Step 191: Preparation of Compound 20, Example 19 Preparation of [14- (3-tert-butylureido) -18- (7-methoxy-2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16- Diazatricyclo [14.3.0.04.6] nonadec-7-en-4-carbonyl] acid amide (1S, 4R, 6S, 14S, 18R) -7-cis-cyclopropanesulfonic acid

Step 191) To a suspension of 59 mg (0.0762 mmol) of compound 10 and 53.4 ml (0.381 mmol) of Et3N in 2 ml of CH2Cl2, 24 µl (0.208 mmol) of tert-butyl isocyanate was added (Aldrich) The mixture was stirred for 18 h, concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra C18 30 x100 mm S5 column, gradient 37% to 85% Solvent B / A for 30 min, retention time 5 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 35 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to provide 30 mg (49%) of Compound 20, Example 19: 1 H NMR (500 MHz, methanol-d4) ,0 1.03 (s, 9 H) 1.11 ( m, 3 H) 1.30 (m, 5 H) 1.49 (m, 5 H) 1.65 (m, 1 H) 1.78 (m, 2 H)

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1.91 (m, 1 H) 2.41 (m, 1 H) 2.69 (dd, J = 14.04, 4.58 Hz, 2 H) 2.85 (m, 1 H) 2.94 (m, 1 H) 4.07 (s, 3 H) 4.13 (dd, J = 12.2, 3.4 Hz, 1 H) 4.25 (d, J = 10.4 Hz, 1 H ) 4.75 (m, 1 H) 4.98 (d, J = 12.2 Hz, 1 H) 5.11 (m, 1 H) 5.74 (m, 1 H) 5.91 (s, 1 H) 7.40 (dd, J = 9.3, 2.4 Hz, 1 H) 7.55 (m, 1 H) 7.67 (s, 1 H) 7.77 (m, 3 H) 8.10 (d, J = 7.6 Hz, 1 H) 8.42 (d, J = 9.3 Hz, 1 H). LC-MS m / e 801 (retention time: 2.88, procedure D, except for the increased gradient time from 2 to 4 min).

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Step 201: Preparation of Compound 21, Example 20 Preparation of 2-fluoroethyl ester of the acid (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonylaminocarbonyl-18- (7-methoxy-2-phenylquinolin- 4-yloxy) -2,15-dioxo-3,16 diazatricyclo- [14.3.0.04.6] nonadec-7-en-14-il] carbamic

Step 201) To a suspension of 59 mg (0.0762 mmol) of compound 10 and 53.4 µl (0.381 mmol) of Et3N in 2 mL of CH2Cl2, 20 µl (0.208 mmol) of 2- chloroformate was added fluoroethyl (Aldrich). The mixture was stirred for 18 h, concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra C18 30 x 100 mm S5 column, 30% to 80% gradient of Solvent B / A for 30 min, retention time 2 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 35 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to provide 38 mg (64%) of Compound 21, Example 20: 1 H NMR (500 MHz, methanol-d4)  1.05 (m, 3 H) 1.25- 1.89 (m, 8 H) 1.62 (dd, J = 9.5, 5.49 Hz, 1 H) 1.74 (dd, J = 8.1, 5.34 Hz, 1 H) 1 , 84 (m, 2 H) 2.39 (m, 1 H) 2.66 (m, 2 H) 2.83 (m, 1 H) 2.91 (m, 1 H) 3.87 ( m, 2 H) 4.06 (s, 3 H) 4.09 (m, 1 H) 4.22 (m, 1 H) 4.30 (m, 1 H) 4.40 (m, 1 H) 4.71 (m, 1 H) 5.08 (m, 1 H) 5.70 (m, 1 H) 5.91 (s, 1 H) 7.41 (m, 1 H) 7.53 (s , 1 H) 7.65 (s, 1 H) 7.73 (m, 3 H) 8.07 (d, J = 7.6 Hz, 2 H) 8.39 (d, J = 9.2 Hz , 1 HOUR). LC-MS m / e 792 (retention time: 2.66, procedure D, except for the increased gradient time from 2 to 4 min).

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Step 211: Preparation of Compound 22, Example 21 Preparation of 2,2-dimethylpropyl ester of the acid (1S, 4R, 6R, 14S, 18R) - [4-cyclopropane-sulfonylaminocarbonyl-18- (7-methoxy-2-phenylquinolin- 4-yloxy) -2,15-dioxo-3,16 diazatricyclo [14.3.0.04.6] -nonadec-14-yl] carbamic

Step 211) To a suspension of 45 mg (0.058 mmol) of compound 16 and 42 µl (0.29 mmol) of Et3N in 2 mL of CH2Cl2, 26 µl (0.174 mmol) of neopentyl chloroformate (Aldrich) was added ). The mixture was stirred for 18 h, concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra C18 30 x 100 mm S5 column, 30% to 80% gradient of

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Solvent B / A for 29 min, retention time 2 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 35 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to provide 35 mg (74%) of Compound 22, Example 21: 1 H NMR (500 MHz, methanol-d4)  0.81 (s, 9 H), 1.10 -1.25 (m, 4 H), 1.27-1.79 (m, 16 H), 1.86 (m, 1 H) 2.60 (m, 1 H) 2.90 (m, 1 H), 3.01 (m, 1 H), 3.18 (d, J = 10.1 Hz, 1 H), 3.35 (m, 1 H), 4.07 (m, 3 H), 4.13 (m, 1 H), 4.26 (m, 1 H), 4.76 (m, 1 H), 4.90 (m, 1 H), 5.93 (m, 1 H), 7.43 (m, 1 H), 7.55 (m, 1 H), 7.68 (m, 1 H), 7.79 (m, 3 H), 8.12 (m, 2 H), 8.39 (d, J = 9.2 Hz, 1 H). LC-MS m / e 818 (retention time: 3.21, procedure D, except for the increased gradient time from 2 to 4 min).

General Procedure for the Preparation of Chloroformates

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This procedure was used for the preparation of non-commercially available chloroformates. To a solution of 5.96 g (67.6 mmol) of commercially available reagents, (S) -3-hydroxytetrahydrofuran and pyridine (5.8 ml; 72 mmol) in THF (150 ml) cooled to 0 ° C is a 1.93 M solution of phosgene in toluene (48 ml, 92.6 mmol for 10 min in an argon atmosphere was added. The resulting solution was allowed to warm to rt for 2 h, the resulting solid was filtered and the mother liquor they were carefully concentrated in vacuo at room temperature until the theoretical mass was obtained.The resulting residue was dissolved in 100 ml of THF to prepare a 0.68 M stock solution of 3 (S) -oxo-tetrahydrofuran chloroformate that could be stored in frozen until used, similarly, other commercially available alcohols can be converted into 0.68 M stock solutions of the corresponding chloroformates.

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Step 221: Preparation of Compound 23, Example 22, Preparation of tetrahydro [4H] pyran-4-yl acid ester (1S, 4R, 6R, 14S, 18R) - [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy -2-phenylquinolin-4-yloxy) -2,15-dioxo-3,16 diazatricyclo [14.3.0.04.6] nonadec-14-yl] carbamic

Step 221) To a suspension of 45 mg (0.058 mmol) of compound 16 and 42 µl (0.29 mmol) of Et3N in 2 mL of CH2Cl2, 217 µl (0.174 mmol) of tetrahydrochloroformate [4H] was added ] pyran-4-yl ~ 0.8 M (prepared from tetrahydro [4H] piran-4-ol of Aldrich quality using the same procedure as in the preparation of 3 (S) -oxo-tetrahydrofuran chloroformate) in CH2Cl2 . The mixture was stirred for 18 h, concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra C18 30 x 100 mm S5 column, 30% to 80% gradient of Solvent B / A for 29 min, retention time 2 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 35 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4), concentrated and again chromatographed on a 12M Biotage column (eluting with 0 to 8% MeOH / CH2Cl2), yielding 14.2 mg (30%) of Compound 23, Example 22: 1 H NMR (500 MHz, CDCl 3)  1.24 (m, 25 H), 2.48 (m, 1 H) 2.64 (m, 1 H), 2.93 (m, 1 H), 3.44 (m, 2 H), 3.83 (m, 2 H), 3.93 (s, 3 H), 4.05 (m, 1 H), 4.39 (d, J = 11, 3 Hz, 1 H), 4.45 (t, J = 6.9 Hz, 1 H), 4.65 (m, 2 H), 5.36 (m, 2 H,) 6.95 (s, 1 H), 7.02 (dd, J =

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9.0, 2.3 Hz, 1 H), 7.49 (m, 4 H), 7.93 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 7.3 Hz, 2 H), 10.26 (s, 1 H). LC-MS m / e 832 (retention time: 2.90, procedure D, except for the increased gradient time from 2 to 4 min).

octadec-13-yl] -2-cyclopropylacetamide

Step 231) To a suspension of 50 mg (0.065 mmol) of compound 14, 14 mg (0.139 mmol) of cyclopropylacetic acid and 97 µl of DIPEA in 2 ml of CH2Cl2, 53 mg (0.139 mmol) of HATU was added. The mixture was stirred for 18 h, concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra C18 30 x 100 mm S5 column, 30% to 80% gradient of Solvent B / A for 30 min, retention time 2 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 35 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to provide 20 mg (40%) of Compound 24, Example 23: 1 H NMR (500 MHz, methanol-d4)  -0.01 (m, 2 H), 0, 30 (m, 2 H), 0.66 (m, 1 H), 0.88 (m, 1 H), 1.08 (m, 3 H), 1.24-1.68 (m, 13 H ), 1.75 (m, 1 H), 1.91 (m, 3 H), 2.63 (m, 1 H), 2.81 (s, 1 H), 2.98 (m, 1 H ), 4.02 (s, 3 H), 4.15 (m, 1 H), 4.41 (m, 1 H), 4.73 (t, J = 8.1 Hz, 1 H), 4 , 94 (d, J = 11.6 Hz, 1 H), 5.86 (s, 1 H), 7.31 (dd, J = 9.2, 2.2 Hz, 1 H), 7.50 (d, J = 2.2 Hz, 1 H), 7.58 (s, 1 H), 7.69 (m, 3 H), 8.08 (m, 2 H), 8.27 (d, J = 9.2 Hz, 1 H). LC-MS m / e 772 (retention time: 2.77, procedure D, except for the increased gradient time from 2 to 4 min).

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Step 241: Preparation of Compound 25, Example 24 Preparation of cyclopentyl acid ester (1S, 4R, 6R, 14S, 17R) - [4-cyclopropanesulfonylaminocarbonyl-17- (7-methoxy-2-henylquinolin-4-yloxy) -2 , 14-dioxo-3,15 diazatricyclo [13.3.0.04.6] octadec-13-il] carbamic

Step 241) To a suspension of 50 mg (0.065 mmol) of compound 14 and 47 µl (0.32 mmol) of Et3N in 2 mL of CH2Cl2, 300 µl (0.20 mmol) of cyclopentyl chloroformate was added ~ 0.67 M (prepared from Aldrich grade cyclopentanol using the same procedure as in the preparation of 3 (S) -oxytetrahydrofuran chloroformate) in CH2Cl2. The mixture was stirred for 18 h, concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra C18 30 x 100 mm S5 column, 30% to 80% gradient of Solvent B / A for 30 min, retention time 2 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 35 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4) and concentrated to provide 26 mg (50%) of Compound 25, Example 24: 1 H NMR (500 MHz, methanol-d4)  1,021.17 (m, 3 H), 1.20 -1.67 (m, 22 H), 1.72 (m, 1 H), 1.88 (m, 1 H), 2.63 (m, 1 H), 2.86 (m, 1 H) , 2.98 (m, 1 H), 4.03 (s, 3 H), 4.08 (m, 1 H), 4.16 (m, 1 H), 4.28 (m, 1 H) , 4.78 (m, 1 H), 5.02 (d, J = 11.0 Hz, 1 H), 5.89 (m, 1 H), 7.38 (m, 1 H), 7, 51 (d, J = 2.1 Hz, 1 H), 7.64 (s; 1 H), 7.73 (m, 3 H), 8.06 (d, J = 7.6 Hz, 2 H ), 8.34 (d, J = 9.5 Hz, 1 H). LC-MS m / e 802 (retention time: 3.00, procedure D, except for the gradient time increased from 2 to 4 min).

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Step 251: Preparation of Compound 26, Example 25, Preparation of tetrahydrofuran-3'-yl acid ester (1S, 4R, 6R, 14S, 17R, 3'S) - [4-cyclopropane-sulfonylaminocarbonyl-17- (7-methoxy- 2-phenylquinolin-4-yloxy) -2,14-dioxo-3,15 diazatricyclo [13.3.0.04.6] -octadec-13-yl] -carbamic

Step 251) To a suspension of 50 mg (0.065 mmol) of compound 14 and 47 µl (0.32 mmol) of Et3N in 2 ml of

15 CH2Cl2, 300 µl (0.20 mmol) of 3 (S) -oxo-tetrahydrofuran ~ 0.67M chloroformate (prepared as described above) in CH2Cl2 was added. The mixture was stirred for 18 h, concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra C18 30 x 100 mm S5 column, 30% to 80% gradient of Solvent B / A for 30 min, retention time 2 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is MeOH

20 to 90% / 10% H2O with 0.1% TFA and the flow rate is 35 ml / min). The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined extracts were dried (MgSO4), concentrated and chromatographed on a 12M Biotage column (eluting with 0% to 8% MeOH / CH2Cl2), to provide 10 mg (25%) of Compound 26, Example 25: 1H NMR (500 MHz, CDCl3) 

25 1.20 (m, 18 H), 1.73 (m, 2 H), 1.93 (m, 2 H), 2.59 (m, 1 H), 2.69 (m, 1 H) , 2.95 (m, 1 H), 3.74 (m, 3 H), 3.93 (s, 3 H), 4.07 (m, 1 H), 4.31 (m, 1 H) , 4.65 (t, J = 7.48 Hz, 1 H), 4.71 (d, J = 11.3 Hz, 1 H), 4.96 (s, 1 H), 5.37 (d , J = 6.7 Hz, 1 H), 5.43 (s, 1 H), 7.02 (m, 3 H), 7.50 (m, 3 H), 8.07 (d, J = 7.3 Hz, 2 H) 10.83 (s, 1 H). LC-MS m / e 804 (retention time: 2.69, procedure D, except for the increased gradient time from 2 to 4 min).

The following Stages (A) - (G) show the preparation of the intermediate, the product of step (G).

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drop a solution of 2.4 ml (46.5 mmol) of bromine for 40 min. The mixture was stirred 1.5 h, diluted with 300 ml of pentane, washed with saturated aqueous NaHCO3, dried (MgSO4) and concentrated, yielding 5.81 g of 1-bromo-3-methyl-butan-2-one. impure that it was taken directly to stage B.

Step B) A pure solution of 5.58 g (34 mmol) of 1-bromo-3-methyl-butan-2-one and 4.50 g (34 mmol) of ethyl thioxamate (Aldrich) was heated to 70 ° C for 18 h and then cooled to room temperature. The mixture was partitioned between saturated aqueous NaHCO3 and EtOAc, the EtOAc phase was dried (MgSO4), concentrated and chromatographed on SiO2 (eluting with 2% to 40% EtOAc / hexanes), providing 3.4 g ( total 48%) of 4-isopropylthiazol-2-carboxylic acid ethyl ester in the form of an oil: 1 H NMR (500 MHz, CDCl 3)  1.32 (d, J = 7 Hz, 6 H), 1.42 ( t, J = 7.2 Hz, 3 H), 3.23 (m, 1 H), 4.46 (c, J = 7.2 Hz, 2 H), 7.18 (s, 1 H).

Step C) To a solution of 3.12 g (15.7 mmol) of 4-isopropylthiazol-2-carboxylic acid ethyl ester in 32 ml of 75% THF / MeOH, 110 mg (31.3 mmol) was added ) of LiOH in 8 ml of H2O. The mixture was stirred overnight, the solution was adjusted to pH 5 using a 1 N aqueous solution of HCl and concentrated to provide 4isopropylthiazol-2-carboxylic acid as a white solid (2.97 g including salts). which was used directly in Stage E: 1H NMR (500 MHz, methanol-d4)  1.29 (d, J = 6.7 Hz, 6H), 3.20 (m, 1 H), 7.39 ( m, 1 H).

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Step D) To a solution of 21.8 g (172 mmol) of m-anisidine in 800 ml of CH2Cl2 cooled to -50 ° C, 172 ml (172 mmol) of 1 M BCl3 in heptane was added for 20 min to produce an amber mixture that was stirred for 1 h. To this mixture was added 12.2 ml (172 mmol) of AcCl and 22.9 g (172 mmol) of AlCl3. The mixture was allowed to warm to room temperature overnight, poured into ice-cold water, the solution was adjusted to pH 9 using 10% aqueous NaOH and repeatedly extracted using EtOAC. The combined organic extracts were washed with brine, dried (MgSO4), concentrated and the resulting solid was recrystallized in the minimum amount of MeOH in CH2Cl2 / hexanes (50%), yielding 13.4 g (47%) of 2- slightly impure amino-4-methoxybenzophenone in the form of a crystalline solid. The solid could be collected directly in the following reaction or purified for absolute purity on SiO2 (eluting with 0% to 14% MeOH / CH2Cl2): 1 H NMR (500 MHz, CDCl 3)  2.50 (s, 3 H), 3.79 (s, 3 H), 6.05 (d, J = 2.4 Hz, 1 H), 6.21 (dd, J = 8.8, 2.4 Hz, 1 H), 6, 38 (m, 2 H), 7.62 (d, J = 8.8 Hz, 1 H).

Step E) To a suspension of 2.59 g (15.7 mmol) of 2-amino-4-methoxybenzophenone (product of step D) and 2.68 g (15.7 mmol) of 4-isopropylthiazole-2 acid -carboxylic (product of step C) in 75 ml of pyridine cooled to -30 ° C, 1.93 ml (23.5 mmol) of POCl 3 was added dropwise over 5 min. The mixture was stirred for 3 h, heated to room temperature and stirred overnight. The reaction mixture was poured into ice-cold water and extracted several times with EtOAc. The combined EtOAc extracts were dried (MgSO4), concentrated and chromatographed on SiO2 (eluting with 0% to 15% MeOH / EtOAc) to provide 2.57 g (51%) of (2-acetyl-5 -methoxyphenyl) 4-methylthiazol-2-carboxylic acid amide in the form of a yellow solid: 1 H NMR (500 MHz, CDCl 3)  1.41 (d, J = 6.7 Hz, 6 H), 2, 64 (s, 3 H), 3.24 (m, 1 H), 3.91 (s, 3 H), 6.67 (dd, J = 9, 2.5 Hz, 1 H), 7.18 (s, 1 H), 7.86 (d, J = 9 Hz, 1 H), 8.56 (d, J = 2.5 Hz, 1 H), 13.48 (s, 1 H).

Step F) To a solution of 2.5 g (7.85 mmol) of 4-methylthiazol-2-carboxylic acid (2-acetyl-5-methoxyphenyl) amide (product of step E) in 50 ml of THF, 19 ml (19 mmol) of 1M KOtBu in THF was added. The mixture was heated at 70 ° C for 3 h, cooled to rt and stirred overnight. The mixture was concentrated and cold water was added to form a suspension. Then, the mixture was acidified to pH 4, filtered and dried. The resulting solid was chromatographed on SiO2 (eluting with 0% to 25% MeOH in CH2Cl2) to provide 1.31 g (56%) of 2- (4-Isopropylthiazol-2-yl) -7-methoxyquinoline-4 -ol in the form of a beige solid: 1H NMR (300 MHz, DMSOD6)  1.32 (d, J = 6.6 Hz, 6 H), 3.14 (m, 1H), 3.89 ( s, 3 H), 7.06 (s, 1 H), 7.38 (s, 1 H), 7.51 (s, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 11.77 (m, 1 H). LC-MS m / e 301 (retention time: 1.53, procedure D).

Stage G) A suspension of 1.3 g (4.3 mmol) of 2- (4-Isopropylthiazol-2-yl) -7-methoxyquinolin-4-ol, product of step F, in 60 ml of POCl3 was heated at reflux for 2 h. The solvent was removed in vacuo, the residue was diluted with ice-cold water and the mixture was adjusted to pH 9 while cooling to 0 ° C. Testa aqueous solution was extracted several times with EtOAc. The combined EtOAc extracts were washed once with brine, pH 4 buffer, dried (MgSO4) and concentrated to provide 0.89 g (64%) of 4-chloro-2- (4-isopropylthiazol-2-yl ) -7methoxyquinoline in the form of a yellow solid: 1H NMR (500 MHz, CDCl3)  1.38 (d, J = 7 Hz, 6 H), 3.19 (m, 1 H), 3.98 ( s, 3 H), 7.06; (s, 1 H), 7.26 (m, 1 H), 7.47 (d, J = 2 Hz, 1 H), 8.10 (d, J = 9 Hz, 1 H), 8.31 (s, 1 H). LCMS m / e 319 (retention time: 2.20, procedure D).

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Preparation of Example 26, (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2,15-dioxo3,16-diazatricyclo [14.3.0.04.6] nonadec- 7-en-4-carboxylic using Stages 26i-26iv.

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Step 26i: Preparation of 1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4 (R) -hydroxy10 pyrrolidin-2 (S) -carboxylic acid methyl ester

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A solution of 2 (S) -terc-butoxycarbonylamino-8-nonenoic acid (purchased from RSP Amino Acids) (3.5 g, 12.9 mmol) in 200 ml of DCM was treated sequentially with 4 (R acid methyl ester ) -hydroxypyrrolidin-2 (S) -carboxylic hydrochloride (2.15 g, 11.8 mmol), N-methyl morpholine (4.25 ml, 38.6 mmol) and HATU (5.37 g, 14.1 mmol ). The reaction mixture was stirred at rt under an atmosphere of N2 for 3 days and then concentrated in vacuo. The residue was partitioned between ethyl acetate and pH 4 buffer (biftalate). The organic phase was washed with. NaHCO3 ac. sat., dried (MgSO4) and concentrated in vacuo to give the crude product. Flash chromatography (50% ethyl acetate / hexane to 100% ethyl acetate) gave 4.7 g (~ 100%) of 1- (2 (S) -terc-butoxycarbonylaminonon-8enoyl acid methyl ester) - 4 (R) -hydroxy-pyrrolidin-2 (S) -carboxylic in the form of a colorless oil: 1H NMR (500 MHz, CD3OD)  1.33-1.50

20 (m, 8 H), 1.46 (s, 9 H), 1.57 (m, 1 H), 1.72 (m, 1 H) 2.08 (m, 2 H), 2.28 (m, 1 H), 3.72 (s, 3 H,) 3.75-3.87 (m, 2 H), 4.36 (m, 1 H), 4.51 (sa, 1 H) , 4.57 (t, J = 8.2 Hz, 1 H), 4.95 (d, J = 10.4 Hz, 1 H), 5.01 (m, 1 H), 5.83 (m , 1 HOUR). LC-MS (Procedure J, retention time: 3.01 min), MS m / z 399 (M ++ 1).

Step 26ii: Preparation of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4 (R) -hydroxypyrrolidin-2 (S) carbonyl acid] - (1R) - amino} -2 (S) -vinyl-cyclopropanecarboxylic

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1- (2 (S) -ter-Butoxycarbonylamino-non-8-enoyl) -4 (R) -hydroxy-pyrrolidin-2 (S) carboxylic acid methyl ester (4.7 g, 11.8 mmol) was dissolved in THF (80 ml), methanol (20 ml) and water (40 ml). Powdered lithium hydroxide (5.6 g, 233 mmol) was added. The light yellow suspension was stirred at rt under an atmosphere of N2 for 16 h and then concentrated in vacuo. The residue was partitioned between ether and water. The ether phase was discarded and the aqueous phase was treated with 1 N HCl until the pH was 4. This acid solution was extracted with EtOAc (3 x). The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to give 4.36 g (96%) of 1- (2 (S) -terbutoxycarbonylamino-8-nonenoyl) -4 (R) -hydroxy-pyrrolidine -2 (S) -carboxylic in the form of a white solid. Then, this acid was dissolved in 150 ml of DMF and (1R, 2S) -1-amino-2-vinylcyclopropane carboxylic acid hydrochloride (2.61 g, 13.6 mmol), N-methyl morpholine (2, 5 ml, 22.6 mmol), HATU (5.2 g, 13.7 mmol). The reaction mixture was stirred at rt under an atmosphere of N2 for 16 h and then concentrated in vacuo. The residue was partitioned between ethyl acetate and pH 4 buffer (biftalate). The organic phase was washed with aq NaHCO3. sat., dried (MgSO4) and concentrated in vacuo to give the crude product. Flash chromatography (60% -80% ethyl acetate / hexane) gave 6.0 g (98%) of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl acid ethyl ester ) 4 (R) -hydroxypyrrolidin-2 (S) carbonyl] - (1R) -amino} -2 (S) -vinyl-cyclopropanecarboxylic acid in the form of a white solid: 1H NMR (500 MHz, CD3OD)  1, 25 (t, J = 7.2 Hz, 3 H), 1.33-1.80 (m, 10 H), 1.46 (s, 9 H), 2.09 (m, 3 H), 2 , 25 (m, 2 H), 3.76 (m, 2 H), 4.14 (m, 2 H), 4.27 (dd, J = 8.5, 5.2 Hz, 1 H), 4.50 (m, 2 H), 4.94 (d, J = 10.1 Hz, 1 H), 5.01 (dd, J = 17.1, 1.8 Hz, 1 H), 5, 11 (dd, J = 10.4, 1.8 Hz, 1 H), 5.30 (d, J = 15.6 Hz, 1 H), 5.80 (m, 2 H), 8.57 ( s, 1 H). LC-MS (Procedure J, retention time: 3.21 min), MS m / z 522 (M ++ 1).

Step 26iii: Preparation of ethyl ester of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy2,15-dioxo-3,16-diaza-tricycle [14.3.0.04 , 6] nonadec-7-en-4-carboxylic

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A solution of 1 - {[1- (2 (S) -terc-butoxycarbonyl-amino-non-8-enoyl) -4 (R) -hydroxy-pyrrolidin2 (S) carbonyl acid] - (1R) - amino} -2 (S) -vinylcyclopropane-carboxylic acid (800 mg, 1.53 mmol) in 2 l of methylene chloride was washed thoroughly with N2 for 0.5 h. Then, tricyclohexylphosphine [1,3-bis (2,4,6-trimethylphenyl) -4,5-dihydroimidazol-2-ylidene] [benzylidene] -ruthenium (IV) (Strem) dichloride (64 mg, 0.075 mmol) was added ) and the mixture was washed thoroughly with N2 for a further 10 min. The light orange homogeneous solution was heated at reflux for 2 h to give a dark orange solution. The reaction mixture was cooled to rt and concentrated in vacuo to give an orange oil. Flash chromatography (ethyl acetate) gave 460 mg (61%) of acid ethyl ester (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2.15- dioxo-3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carboxylic in the form of a gray solid. 1 H NMR (500 MHz, CDCl 3)  1.19 (t, J = 7.2 Hz, 3 H), 1.42 (s, 9 H), 1.22-1.8 (m, 8 H), 1.87 (m, 2 H), 2.03-2.22 (m, 4 H), 2.63 (m, 1 H), 3.65 (m, 1 H), 4.09 (m, 3 H), 4.45 (m, 1 H), 4.56 (s, 1 H), 4.82 (m, 1 H), 5.23 (m, 1 H), 5.51 (s, 1 H), 7.16 (s, 1 H). LC-MS (Procedure J, retention time: 2.97 min), MS m / z 494 (M ++ 1).

Step 26iv: Preparation of Example 26, acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy2,15-di-oxo-3,16-diazatricyclo [14.3.0.04 , 6] -nonadec-7-en-4-carboxylic

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To a solution of ethyl ester of the acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2.15 dioxo-3.16-diazatri-cycle [14.3.0.04, 6] -nonadec-7-en-4-carboxylic (493 mg, 1.0 mmol) in THF (4 ml), methanol (1 ml) and 5

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water (2 ml), lithium hydroxide powder (480 mg, 20 mmol) was added and the light yellow suspension was stirred at rt under an N2 atmosphere for 16 h. Then, the mixture was concentrated in vacuo and the residue was partitioned between ether and water. The ether phase was discarded and the aqueous phase was treated with 1 N HCl until pH 4. This acid solution was extracted three times with EtOAc. The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to give 460 mg (98%) of Example 26, acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino- 18-hydroxy-2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carboxylic acid in the form of a gray solid. 1H NMR (500 MHz, CD3OD)  ppm 1.26 (t, J = 7.2 Hz, 3 H), 1.35-1.52 (m, 15 H), 1.57-1.68 (m , 3 H), 1.79 (m, 1 H), 2.04 (m, 1 H), 2.16-2.41 (m, 3 H), 3.80 (dd, J = 10.7 , 4.3 Hz, 1 H), 3.88 (m, 1 H), 4.38 (dd, J = 8.9, 3.1 Hz, 1 H), 4.55 (m, 2 H) , 5.39 (t, J = 9.8 Hz, 1 H), 5.58 (m, 1 H). LC-MS (Procedure J, retention time: 2.64 min), MS m / z 466 (M ++ 1).

H) 1M KOtBu in THF; LaCl3

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Step H) At a suspension of 58 mg (0.125 mmol) of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino18-hydroxy-2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carboxylic acid, Example 26 and 31 mg (0.125 mmol) of LaCl3 in 1.2 ml of DMF cooled to -78 ° C was added 0.62 ml (0 , 62 mmol) of 1 M KOtBU in THF, followed by 4-chloro-2- (4-isopropylthiazol-2-yl) -7-methoxyquinoline, product of step G. The mixture was stirred for one hour and heated to rt, showing a small conversion in the product. Approximately 0.75 equivalents of solid KOtBU (10 mg) were added and the mixture was stirred overnight. After 14 h, approximately 4-chloro-2- (4-isopropylthiazol-2-yl) -7-methoxyquinoline (22 mg) and LaCl 3 (16 mg) medium was added, and the mixture was stirred overnight. . The mixture was quenched with pH 4 buffer and extracted in EtOAc. The combined EtOAc extracts were dried (MgSO4), concentrated in vacuo and completely dissolved in 2 ml of MeOH. This solution was injected into a preparative HPLC using the following conditions: Xterra 30 x 100 mm S5 column, 30% to 100% solvent gradient B / A for 18 min, retention time 5 min; wherein Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 40 ml / min) The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to provide 22 mg (31%) of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18- [ 2- (2-Isopropylthiazol-4-yl) -7-methoxyquinolin-4-yloxy] -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nona-dec-7-en-4-carboxylic: NMR 1H (500 MHz, Methanol-d4)  1.08-1.67 (m, 11 H), 1.39 (s, 9 H), 1.41 (d, J = 7.0 Hz, 6 H) , 1.78 (m, 1 H), 1.93 (m, 1 H), 2.32 (c, J = 9 Hz, 1 H), 2.74 (m, 1 H), 3.23 ( m, 1 H), 3.97 (m, 3 H), 4.07 (d, J = 9 Hz, 1 H), 4.20 (d, J = 8.2 Hz, 1 H), 4, 66 (t, J = 8.2 Hz, 1 H), 4.76 (d, J = 11.3 Hz, 1 H), 5.36 (m, 1 H), 5.59 (m, 1 H ), 5.64 (s, 1 H), 6.91 (s, 1 H), 7.13 (d, J = 8.9 Hz, 1 H), 7.42 (s, 1 H), 7 , 45 (s, 1 H), 7.70 (s, 1 H), 8.19 (d, J = 8.9 Hz, 1 H), 8.68 (s, 1 H). LC-MS m / e 748 (retention time: 203, procedure D).

Step I: Preparation of Compound 27, Example 27, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- {4cyclopropanesulfonylaminocarbonyl-18- [2- (2-isopropylthiazol-4- il) -7-methoxy-quinolin-4-yloxy] -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14-yl} carbamic

Step I) To a solution of 18 mg (0.024 mmol) of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18 [2- (2-isopropylthiazol-4-yl) -7-methoxyquinolin-4-yloxy] -2,15-dioxo-3,16-diazatricyclo [14.3.0.04.6] nona-dec-7-en-4-carboxylic (product of Step H) in 2 ml of THF, CDI (29 mg, 0.036 mmol) was added and the resulting solution was refluxed for 60 min and allowed to cool to rt. Cyclopropylsulfonamide (4.4 mg, 0.036 mmol) was added in one portion before the addition of a pure DBU solution (5.1 µL, 0.034 mmol). The reaction was stirred for 16 h, diluted in 2 ml of methanol and injected into a preparative HPLC using the following conditions: Xterra 30 x 100 mm S5 column, 30% to 100% gradient of Solvent B / A for 30 min , retention time 5 min; in which the

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Solvent A is 10% MeOH / 90% H2O with 0.1% TFA, Solvent B is 90% MeOH / 10% H2O with 0.1% TFA and the flow rate is 40 ml / min). The pH of the combined fractions was adjusted to neutral using a 6 N aqueous solution of NaOH and the MeOH was removed in vacuo. The solution was adjusted to pH 4 with a 3 N aqueous solution of HCl and repeatedly extracted with EtOAC. The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to provide 6.4 mg (31%) of Compound 27, Example 27, as a foam: 1H NMR (500 MHz, Methanol-d4)  0.96-1.56 (m, 17 H), 1.18 (s, 9 H), 1.61 (dd, J = 9.5, 5.5 Hz, 1 H), 1.72 (dd , J = 8.1, 5.3 Hz, 1 H), 1.84 (m, 2 H), 2.38 (m, 1 H), 2.61 (m, 1 H), 2.70 ( m, 1 H), 2.80 (m, 1 H), 2.90 (m, 1 H), 3.24 (m, 1 H), 3.96 (s, 3 H), 4.07 ( m, 1 H), 4.15 (d, J = 10.7 Hz, 1 H), 4.66 (m, 1 H), 4.86 (m, 1 H), 5.08 (m, 1 H), 5.69 (m, 2 H), 7.13 (d, J = 9.2 Hz, 1 H), 7.43 (s, 1 H), 7.45 (s, 1 H), 7.70 (s, 1 H), 8.20 (d, J = 9.2 Hz, 1 H). LC / MS m / e 851 (time of

10 retention: 1.98, procedure D).

Preparation of Example 30, Compound 29, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonyl-aminocarbonyl-2,15-dioxo-18- (quinolin-4-yloxy) -3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14il] carbamic

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Step 30A: Preparation of 1-tert-butyl ester of 4 (R) - (quinolin-4-yloxy) -pyrrolidin-1,2 (S) -dicarboxylic acid

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Step 30A) A suspension of Boc-L-4-hydroxyproline [N-Boc (2S, 4R) -hydroxyproline] (10 g, 43.3 mmol) (CHEMIMPEX International) in DMSO (150 ml) was cooled in a bath of ice for ~ 3 minutes and then t-BuOK (12.1 g, 108.2 mmol) was added. The mixture was stirred for a few minutes until a solid mass formed. At this time the reaction mixture was allowed to warm to rt for 1.5 h to give a clear colorless solution. 1-Chloroquinoline (7.7 g, 47.2 mmol) 10 min was added in two separate portions. The reaction was stirred for 24 h at rt. The dark reaction mixture was partitioned between ether (500 ml) and water (700 ml). The aqueous phase was acidified to pH 4 using 4 N HCl. The resulting white precipitate was filtered and then washed with water (200 ml) to give the title product of Step 30A as an off-white solid (13 g, 84%): 1 H NMR (500

25 MHz, DMSO)  1.32, 1.35 (2s, 9 H), 2.36 (m, 1 H), 2.66 (m, 1 H), 3.73 (m, 2 H), 4.35 (m, 1 H), 5.34 (s, 1 H), 7.06 (d, J = 5.2 Hz, 1 H), 7.56 (t, J = 8.2 Hz, 1 H), 7.75 (t, J = 8.2 Hz, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 8.11 (t, J = 8.2 Hz , 1 H), 8.72 (d, J = 5.2 Hz, 1 H). LC-MS (Procedure J, retention time: 1.87 min), MS m / z (M ++ 1).

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Step 30B: Preparation of tert-butyl ester of (2S) - (1 (R) -ethoxycarbonyl-2 (S) -vinylcyclopropylcarbamoyl) -4 (R) (quinolin-4-yloxy) pyrrolidine-1-carboxylic acid

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Step 30B) A stirring suspension of 1-tert-butyl ester of 4 (R) - (quinolin-4-yloxy) -pyrrolidine-1,2 (S) acid

Dicarboxylic acid (5.0 g, 14 mmol) in a mixture of 100 ml of methylene chloride and 100 ml of DMF was treated sequentially with N-methyl morpholine (3 ml, 27.3 mmol), HATU (PE biosystems) ( 6.3 g, 16.5 mmol) and 1R, 2S-1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester (3.2 g, 16.7 mmol). The solution was stirred at rt under an N2 atmosphere for 18 h and then concentrated in vacuo to give 10 g of a brown oil. This oil was partitioned between ethyl acetate and aq NaHCO3. sat. The organic phase was washed with brine, dried (MgSO4)

10 and concentrated in vacuo. Flash chromatography (10% MeOH in ethyl acetate) gave 7.0 g (100%) of the title product of Step 30B as a yellow solid: 1H NMR (300 MHz, CD3OD)  1, 25 (t, J = 7.3 Hz, 3 H), 1.44, 1.39 (2 s, 10 H), 1.79 (m, 1 H), 2.20 (m, 1 H), 2.43 (m, 1 H), 2.71 (m, 1 H), 3.92 (m, 2 H), 4.15 (c, J = 7.3 Hz, 2 H), 4.42 (m, 1 H), 5.11 (m, 1 H), 5.29-5.39 (m, 2 H), 5.75 (m, 1 H), 7.05 (d, J = 5 , 5 Hz, 1 H), 7.57 (t, J = 8.1 Hz, 1 H), 7.76 (t, J = 8.4 Hz, 1 H), 7.95 (d, J = 8.4 Hz, 1 H), 8.16 (d, J = 8.4 Hz, 1 H), 8.69 (d, J = 5.5 Hz, 1 H).

15 LC-MS (Procedure J, retention time: 2.35 min), MS m / z 496 (M ++ 1).

Step 30C: Preparation of 1 - {[4 (R) - (quinolin-4-yloxy) -pyrrolidin-2 (S) -carbonyl] -1 (R) -amino} -2 (S) vinyl- acid ethyl ester cyclopropane carboxylic bis hydrochloride

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Step 30C) A stirred suspension of tert-butyl ester of (2S) - (1 (R) -ethoxycarbonyl-2 (S)

Vinylcyclopropylcarbamoyl) -4 (R) - (quinolin-4-yloxy) pyrrolidin-1-carboxylic acid (7.0 g, 14.1 mmol) was treated with 150 ml (300 mmol) of 2 N HCl / ether (Aldrich) for 24 h. The reaction mixture was concentrated in vacuo to give 7.0 g (94%) of the title product of step 30C as a yellow solid: 1H NMR (500 MHz, CD3OD)  1.28 (t, J = 7.0 Hz, 3 H), 1.52 (dd, J = 5.5 Hz, 4.0 Hz, 1 H), 1.65 (dd, J = 7.6 Hz, 6.7 Hz , 1 H), 1.83 (dd, J = 5.5 Hz, 2.7 HZ, 1 H), 2.29 (c, J = 8.8 Hz, 1 H), 2.65 (m, 1 H), 3.02 (m, 2 H), 4.00 (s, 2 H), 4.20 (c, J = 7.0 Hz, 2 H), 4.74 (dd, J = 7 , 6 HZ, 2.7

25 Hz, 1 H), 5.17 (d, J = 10.4 Hz, 1 H), 5.35 (d, J = 17.1 Hz, 1 H), 5.81 (m, 1 H) , 5.95 (m, 1 H), 7.69 (d, J = 6.7 Hz, 1 H), 7.98 (t, J = 8.2 Hz, 1 H), 8.19 (m , 2 H), 8.66 (d, J = 8.2 Hz, 1 H), 9.14 (d, J = 6.7 Hz, 1 H). LC-MS (Procedure J, retention time: 1.34 min), MS m / z 396 (M ++ 1-2HCl).

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Step 30D: Preparation of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4 (R) - (quinolin-4-yloxy) pyrrolidin-2 (S) -carbonyl acid ethyl ester ] -1 (R) -amino} -2 (S) -vinylcyclopropanecarboxylic

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Step 30D) A solution of 2 (S) -terc-butoxycarbonylamino-8-nonenoic acid (1.3 g, 4.79 mmol, purchased from RSP

5 Amino Acids) dissolved in 100 ml of dichloromethane was treated sequentially with 1 - {[4 (R) (quinolin-4-yloxy) pyrrolidin-2 (S) -carbonyl] -1 (R) -amino} ethyl ester -2 (S) -vinyl-cyclopropanecarboxylic bis hydrochloride (2.0 g, 4.28 mmol), N-methyl morpholine (1.5 ml, 13.6 mmol) and HATU (PE biosystems) (1.92 g, 5.05 mmol). The reaction mixture was stirred at rt under an atmosphere of N2 for 16 h and then concentrated in vacuo. The residue was partitioned between ethyl acetate and pH 4 buffer (biftalate). The organic phase was washed with aq NaHCO3. sat., dried (MgSO4) and

10 concentrated in vacuo to give 2.2 g of the crude product. Flash chromatography (10% methanol / ethyl acetate) gave 2.0 g (73%) of the title product of Step 30D as a yellow solid: 1H NMR (500 MHz, CD3OD)  1, 27 (s, 9 H), 1.21-1.45 (m, 10 H), 1.63 (dd, J = 11.29, 6.71 Hz, 2 H) 1.75 (m, 1 H ) 2.04 (m, 2 H) 2.28 (c, J = 8.75 Hz, 1 H) 2.44 (m, 1 H) 2.76 (m, 1 H) 4.01-4, 25 (m, 4 H) 4.55 (d, J = 12.21 Hz, 1 H) 4.67 (t, J = 8.55 Hz, 1 H) 4.93 (d, J = 10.07 Hz, 1 H) 5.00 (d, J = 15.26 Hz, 1 H) 5.12 (dd, J = 10.38, 1.83 Hz, 1 H) 5.31 (dd, J = 17 , 24, 1.68

15 Hz, 1 H) 5.51 (s, 1 H) 5.79 (m, 2 H) 7.08 (d, J = 5.49 Hz, 1 H) 7.56 (t, J = 7, 32 Hz, 1 H) 7.77 (t, J = 7.48 Hz, 1 H) 7.97 (d, J = 8.54 Hz, 1 H) 8.27 (d, J = 8.55 Hz , 1 H) 8.73 (d, J = 5.19 Hz, 1 H). LC-MS (Procedure J, retention time: 3.06 min), MS m / z 649 (M ++ 1).

Step 30E: Preparation of ethyl ester of acid (1S, 4R, 6S, 14S, 18R) -7-cis- [14-tert-Butoxycarbonylamino-2,15-dioxo18- (quinolin-4-yloxy) -3,16- diaza-tricycle [14.3.0.04.6] nonadec-7-en-4-carboxylic

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Step 30E) A solution of 1 - {[1- (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4 (R) - (quinolin-4-yloxy) pyrrolidin-2 (S) - carbonyl] -1 (R) -amino} -2 (S) -vinylcyclopropanecarboxylic acid (1.6 g, 2.46 mmol) in 2 l of methylene chloride was washed thoroughly with N2 for 0.5 h. Then, tricyclohexylphosphine dichloride [1,3bis (2,4,6-trimethylphenyl) -4,5-dihydroimidazol-2-ylidene] - [benzylidene] -ruthenium (IV) (Strem) (150 mg, 0.17 was added mmol) 25 was added and the mixture was washed thoroughly with N2 for a further 10 min. The light orange homogeneous solution was heated at reflux for 16 h. An additional 50 mg of the Ru catalyst was added and the reaction mixture was refluxed for an additional 8 h. Finally, an additional 50 mg of the Ru catalyst was added and stirring was continued for 3 days at rt. The reaction mixture was concentrated in vacuo to give 1.7 g of an orange oil. Flash chromatography (ethyl acetate to 5% methanol in ethyl acetate) gave 1.1 g (68%) of the title product of Step 30 30E as a white solid: 1 H NMR (500 MHz, CD3OD),  1.19 (s, 9 H), 1.26 (t, J = 7.0 Hz, 3 H), 1.29-1.59 (m, 7 H), 1.63 (dd , J = 8.5 Hz, 4.8 Hz, 1 H), 1.67 (dd, J = 9.5 Hz, 4.8 Hz, 1 H), 1.78-1.85 (m, 1 H), 1.92-1.99

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(m, 1 H), 2.50 (m, 2 H), 2.70 (m, 1 H), 2.85 (c, J = 8.8 Hz, 1 H), 4.06 (d, J = 11.9 Hz, 1 H), 4.10-4.18 (m, 2 H), 4.23 (d, J = 10.7 Hz, 1 H), 4.68 (m, 1 H ), 4.79 (d, J = 11.3 Hz, 1 H), 5.37 (t, J = 10.1 Hz, 1 H), 5.53 (s, 1 H), 5.64 ( m, 1 H), 7.08 (d, J = 5.5 Hz, 1 H), 7.52 (t, J = 7.6 Hz, 1 H), 7.75 (t, J = 8, 2 Hz, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 8.72 (d, J = 5.5 Hz, 1 H). (LC-MS (Procedure J, retention time: 2.71 min), MS m / z 621 (M ++ 1).

Step 30F, Preparation of acid (1S, 4R, 6S, 14S, 18R) -7-cis- [14-tert-butoxycarbonylamino-2,15-dioxo-18- (quinolin-4-yloxy) -3,16-diaza-tricycle [14.3.0.04.6] nonadec-7-en-4-carboxylic

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Step 30F) A solution of ethyl ester of acid (1S, 4R, 6S, 14S, 18R) -7-cis- [14-tert-butoxycarbonylamino-2.15 dioxo-18- (quinolin-4-yloxy) -3.16 -diazatricyclo [14.3.0.04.6] nonadec-7-en-4-carboxylic (1.10 g, 1.77 mmol) was dissolved in the mixed solvent system: THF (8 ml), methanol (2 ml) and water (4 ml). Powdered lithium hydroxide (425 mg, 17.7 mmol) was added. The light yellow suspension was stirred at rt under an atmosphere of N2 for 16 h, and then concentrated in vacuo. The residue was partitioned between ether and water. The ether phase was discarded and the aqueous phase was treated with 1 N HCl until the pH was 4. This acid solution was extracted four times with ethyl acetate. The combined ethyl acetate extracts were dried (MgSO4) and concentrated in vacuo to give the product of step 30F 15 (0.90 g, 86%) as a white solid: 1 H NMR (300 MHz, CD3OD)  1.16 (s, 9 H), 1.24 (m, 1 H), 1.33-1.55 (m, 6 H), 1.61 (m, 2 H), 1.79 (m, 1 H), 1.92 (m, 1 H), 2.32 (c, J = 8.8 Hz, 1 H), 2.58 (m, 2 H), 2.68 (m, 1 H), 4.05 (d, J = 10.6 Hz, 1 H), 4.22 (m, 1 H), 4.64 (t, J = 8.8 Hz, 1 H), 4, 74 (m, 1 H), 5.36 (t, J = 10.6 Hz, 1 H), 5.99 (s, 1 H), 5.61 (m, 1 H), 7.07 (d , J = 5.5 Hz, 1 H), 7.49 (t, J = 7.7 Hz, 1 H), 7.73 (t, J = 7.7 Hz, 1 H), 7.92 ( d, J = 8.8 Hz, 1 H), 8.30 (d, J = 8.8 Hz, 1 H), 8.70 (d, J = 5.5 Hz, 1 H). LC-MS (Procedure J, retention time: 2.54 min), MS m / z

20 593 (M ++ 1).

Step 30G: Preparation of Compound 29 of Example 30, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonylamino-carbonyl-2,15-dioxo-18- (quinolin-4 -yloxy) -3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14-il] carbamic

image 1

Step 30G) To a solution of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-Butoxycarbonylamino-2,15-dioxo-18- (quinolin4-yloxy) -3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-4-carboxylic acid (100 mg, 0.17 mmol) dissolved in 3 ml of THF was added CDI (38 mg, 0.24 mmol) and the mixture was heated at reflux for one hour. The mixture was cooled to rt and treated sequentially with cyclopropylsulfonamide (29 mg, 0.24 mmol) and DBU (36 mg, 0.24 mmol). After stirring for 24 h at rt, the THF was removed by rotary evaporation. The residue was partitioned between ethyl acetate and buffer.

30 pH 4. The organic phase was dried (MgSO4) and concentrated in vacuo to give the crude product. Flash chromatography (10% MeOH / methylene chloride) gave 50 mg (42%) of the title product, Compound 29 of Example

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30, tert-butyl ester of (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonylaminocarbonyl-2,15-dioxo-18 (quinolin-4-yloxy) -3,16-diazatricyclo [ 14.3.0.04.6] nonadec-7-en-14-yl] carbamic in the form of a white powder: 1H NMR (500 MHz, CD3OD)  1.01 (m, 1 H), 1.18 (s , 9 H), 1.07-1.60 (m, 10 H), 1.65 (dd, J = 9.5 Hz, 5.2 Hz, 1 H), 1.76 (dd, J = 8 , 2 Hz, 5.5 Hz, 1 H), 1.86 (m, 2 H), 2.40 (m, 1H), 2.58 (m, 1 H), 2.68 (m, 1 H ), 2.76 (m, 1 H), 2.93 (m, 1 H), 4.07 (d, J = 10.7 Hz, 1 H), 4.19 (m, 1 H), 4 , 68 (dd, J = 7.3 Hz, 1.81 Hz, 1 H), 4.85 (m, 1H), 5.14 (t, J = 7.6 Hz, 1 H), 5.55 (s, 1 H), 5.70 (m, 1 H), 6.68 (d, J = 5.8 Hz, 1 H), 7.09 (d, J = 5.2 Hz, 1 H) , 7.51 (t, J = 7.0 Hz, 1 H), 7.76 (t, J = 7.0 Hz, 1 H), 7.95 (d, J = 8.2 Hz, 1 H ), 8.33 (d, J = 8.2 Hz, 1 H), 8.72 (d, J = 5.5 Hz, 1 H). LC-MS (Procedure A, retention time: 2.58 min), MS m / z 696 (M ++ 1).

Example 31, Preparation of Compound 30, tert-butyl ester of acid (1S, 9R, 6S, 14S, 18R) -7-cis- (4methanesulfonylaminocarbonyl-2,15-dioxo-18- (quinolin-4-yloxy) -3 , 16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14-il) carbamic

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Acid (1S, 4R, 6S, 14S, 18R) -7-cis- [14-tert-butoxycarbonylamino-2,15-dioxo-18- (quinolin-4-yloxy) 3.16-diazatricyclo [14.3. 0.04.6] nonadec-7-en-4-carboxylic acid, the product of Step F in Example 30 (50 mg, 0.075 mmol) in an analogous manner with methanesulfonamide (12 mg, 0.12 mmol) as described before in the previous example to give 10 mg (20%) of tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- [4-methanesulfonylaminocarbonyl-2,15-dioxo-18- ( quinolin-4-yloxy) -3,16-diazatricyclo [14.3.0.04.6] -nonadec-7-en-14-yl] -carbamine in the form of a white solid: 1H NMR (300 MHz, CDCl3)  1.26 (s, 9 H), 1.22-1.94 (m, 11 H), 2.27 (m, 1 H), 2.55 (m, 1 H), 2.67 (m, 2 H), 3.17 (s, 3 H), 4.05 (m, 1 H), 4.27 (m, 1 H), 4.61 (t, J = 8.1 Hz, 1 H) , 4.73 (d, J = 11.0 Hz, 1 H), 4.96-5.02 (m, 2 H), 5.35 (s, 1 H), 5.71 (c, J = 8.8 Hz, 1 H), 6.73 (d, J = 5.1 Hz, 1 H), 6.93 (sa, 1 H), 7.43 (t, J = 7.7 Hz, 1 H), 7.69 (t, J = 7.7 Hz, 1 H), 8.06 (d, J = 8.8 Hz, 1 H), 8, 15 (d, J = 7.7 Hz, 1 H), 8.77 (d, J = 5.5 Hz, 1 H). LC-MS (Procedure A, retention time: 2.43 min), MS m / z 670 (M ++ 1).

Preparation of Example 32, 2 (S) -terc-butoxycarbonylamino-3-pentenylsulfanylpropionic acid methyl ester (Example 32) for use in Example 33.

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Step 1: To a solution of N-Boc-cysteine methyl ester (3.36 g, 0.014 mol) in methanol (166 ml) at RT was added triethylamine (10.8 ml) and 1-bromopent-4-ene (3.19 g, 21 mmol, 1.5 equivalents) and the resulting solution was stirred at room temperature overnight. Then, the mixture was concentrated in vacuo and the resulting residual mixture was purified using flash chromatography (hexane gradient, ethyl acetate) to provide 1.76 g (41%) the desired thioether. 1H NMR (500 MHz, CDCl3)  1.43 (s, 9H), 1.64 (m, 2H), 2.11 (m, 2H), 2.51 (m, 2H), 2.95 (m , 2H), 3.75 (s, 3H), 4.51 (m, 1H), 4.95-5.03 (m, 2H), 5.34 (m, 1H), 5.80 (1H, m). LC-MS (Procedure H, with the exception that the gradient time was 3 min and that the flow rate was 4 ml / min, retention time: 2.29 min), MS m / z 304 (M ++ one).

Step 2: The thioether product from step 1 (9.51 g, 31.4 mmol) was added to a mixture of 1 M LiOH in water (200 ml) and THF (200 ml), and the resulting mixture was stirred at room temperature overnight. Then, the reaction mixture was acidified using 1 N hydrochloric acid and the resulting mixture was extracted several times with ethyl acetate. The extracts were combined, dried over magnesium sulfate and concentrated in vacuo to provide the desired acid that was used as such in the next reaction.

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Preparation of Example 33, Compound 31, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy-2-phenyl-quinolin-4- iloxy) -2,15-dioxo-12-tia-3,1-6diazatricyclo [14.3.0.04.6] nonadec-7-en-14-yl] -carbamic.

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Step 33A, Preparation of 1 - {[1- (2 (S) -ter-Butoxycarbonylamino-3-pent-4-enylsulfanylpropionyl) 4 (R) - (7-methoxy-2-phenylquinolin-4-yloxy acid ethyl ester ) pyrrolidin-2 (S) -carbonyl] -1 (R) -amino} -2 (S) -vinylcyclopropanecarboxylic.

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Step 33A) Note: the product of Step 1f, ethyl ester of (1 - {[4- (7-Methoxy-2-phenylquinolin-4 (R) -yloxy) pyrrolidine

2 (S) -carbonyl] -1 (R) -amino} -2 (S) -vinylcyclopropanecarboxylic was used as the starting material for the preparation of Compound 31. The carboxylic acid (282 mg, 976 mmol) of example 32 is added to a DMF solution containing HATU (445 mg, 1.17 mmol, 1.2 equiv.), NMR (375 µL, 3.42 mmol, 3.5 equiv.) and the product TFA salt the step If (600 mg, 976mol, 1 equiv.) And the resulting solution was stirred at room temperature overnight. The resulting mixture was quenched by the addition of pH 4 buffer and the resulting mixture was extracted (3 x) with

15 ethyl acetate. The extracts were combined, washed with brine, dried over magnesium sulfate and concentrated in vacuo. The crude product mixture was purified by flash chromatography (hexane, ethyl acetate) to provide the product of Step 33A (753 mg, 84%). 1H NMR (500 MHz, CDCl3)  1.20-1.25 (m, 3H), 1.36 (s, 9H), 1.54-1.68 (m, 4H), 1.88 (m, 1H), 2.10-2.20 (m, 3H), 2.55 -2.64 (m, 2H), 2.73-2.97 (m, 3H), 4.08-4.15 ( m, 3H), 4.35 (m, 1H), 4.68 (m, 1H), 4.90-5.04 (m, 3H), 5.10 (m, 1H), 5.27-5 , 44 (m, 3H), 5.68-5.82 (m, 2H), 7.05-7.09 (m,

2H), 7.42-7.59 (m, 4H), 7.92-8.19 (m, 3H).

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Step 33B, Preparation of 14-tert-butoxycarbonylamino-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) 2,15-dioxo-12-thia-3,16-diaza-tricyclo acid ethyl ester [ 14.3.0.04.6] nonadec-7-en-4-carboxylic

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Step 33B) The tripeid ester product of step 33A (390 mg, 505 mmol) was dissolved in dichloroethane

5 degassed (purged with nitrogen) (145 ml). Tricyclohexylphosphine [1,3-bis (2,4,6-trimethylphenyl) 4,5-dihydroimidazol-2-ylidene] - [benzylidene] -ruthenium (IV) (Strem) dichloride (45 mg, 53 mol) was added , the reaction mixture was heated to 45 ° C and maintained at this temperature for 5 h. At that time, additional ruthenium catalyst (45 mg, 53 mol) was added to the reaction mixture and the heating was maintained overnight. Then, the reaction mixture was concentrated in vacuo and purified by flash chromatography (hexanes, ethyl acetate) to

10 provide 159.4 mg (42%) of the desired olefin metathesis cyclization product of Step 33B. 1H NMR (500 MHz, CDCl3)  1.24 (t, J = 7 Hz, 3H), 1.37 (m, 1H), 1.41 (s, 9H), 1.50-1.75 (m , 3H), 1.85 (m, 1H), 1.96 (m, 1H), 2.13 (m, 1H), 2.21-2.45 (m, 3H), 2.52 (m, 1H), 2.81 (m, 1H), 3.07-3.15 (m, 3H), 4.12-4.25 (m, 3H), 4.85 (m, 1H), 5.09 (m, 1H), 5.37-5.42 (m, 2H), 5.5 (m, 1H), 5.6 (m, 1H), 7.05 (s, 1H), 7.09 ( m, 1H), 7.40-7.58 (m, 4H), 8.00-8.10 (m, 3H). ). LC-MS (Procedure H, with the exception that the gradient time was 2 min, the flow rate was 5 ml / min and the

15 retention time: 1.55 min), MS m / z 745 (M ++ 1).

Step 33C, Preparation of (1S ', 4R, 6S, 14S, 18R) -7-cis-14-tert-Butoxycarbonylamino-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo- 12-tia-3,16-diaza-tricycle [14.3.0.04.6] nonadec-7-en-4-carboxylic

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Step 33C) The macrocycle tripeptide ester product of step 33B (149.8 mg, 201mol) was dissolved in a

20 mixture of THF (4.1 ml), MeOH (1.9 ml), H2O (0.45 ml) containing LiOH (97 mg, 4.04 mmol) and the resulting mixture was stirred at room temperature overnight . The reaction was stopped by the addition of aqueous HCl (0.1 N) and the resulting mixture was extracted with ethyl acetate. The organic extracts were dried with magnesium sulfate and concentrated in vacuo to provide a mixture of crude product that was purified using flash chromatography (ethyl acetate, then 10% MeOH / CH2Cl2 as eluent) to provide the

Desired macrocycle carboxylic acid (109.3 mg, 76%). 1H NMR (500 MHz, DMSO-d6)  1.1 (m, 1H), 1.16 (s, 9H), 1.36 (m, 1H), 1.41-1.55 (m, 3H) , 1.60 (m, 1H), 1.95 (m, 1H), 2.27 (m, 1H), 2.32-2.43 (m, 2H), 2.45-2.60 (m , 3H), 2.71 (m, 1H), 2.90 (m, 1H), 3.92 (s, 3H), 4.05 (m, 1H), 4.25-4.55 (m, 2H), 5.42-5.70 (m, 3H), 7.10 (m, 1H), 7.19 (m, 1H), 7.32-7.62 (m, 3H), 7.75 (m, 1), 8.04 (m, 1H), 8.24-8.30 (m, 2H), 8.50 (s, 1H). LC-MS (Procedure H, with the exception that the gradient time was 2 min and the flow rate was 5 ml / min, retention time: 1.48 min), MS m / z

30 717 (M ++ 1).

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Step 33D, Preparation of Example 33, Compound 31, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy-2-phenyl-quinolin -4-yloxy) -2,15-dioxo-12-tia-3,16diazatricyclo [14.3.0.04.6] nonadec-7-en-14-il] -carbamic

image 1

Step 33D) The macrocycle acid product of step 33C (77.9 mg, 109 µmol, 1 equiv.) Was dissolved in THF (770 µl) and CDI (21.1 mg, 130 µmol, 1.2 equiv.). Then, the mixture was heated to reflux and maintained at that temperature for 2 h. Then, the reaction mixture was cooled to room temperature, cyclopropylsulfonamide (15.5 mg, 128 mol, 1.2 equiv.) And DBU (18.7 ,l, 125 mol, 1.2 equiv.) Were added and the resulting mixture was stirred at room temperature overnight. The CL / EM analysis indicated that the product mixture consisted of

10 a mixture of starting material and desired product. The resulting mixture was partitioned between methylene chloride and 1 N aqueous HCl, followed by back extraction of the aqueous phase. The combined extracts were dried over magnesium sulfate and concentrated in vacuo. Then, the mixture of starting material and desired product was again subjected to the reaction conditions described above using identical amounts of reagents. After the duration of the reaction, the reaction mixture was treated as before and then purified

15 further by chromatography (Biotage 12M column, 75% ethyl acetate / hexane) to provide the desired macrocycle sulfonamide, Example 33, Compound 31, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) - 7cis- [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2,15-dioxo-12-thia-3,16 diazatricyclo [14.3.0.04.6] nonadec-7 -en-14-yl] -carbamic (30.6 mg, 34%). 1H NMR (500 MHz, methanol-d4)  0.90-1.50 (m, 7H), 1.24 (2, 9H), 1.55 (m, 1H), 2.60-2.67 ( m, 2H), 2.83 (m, 1H), 2.15 (m, 1H), 2.30-2.47 (m, 2H), 2.5-2.9 (m, 7H),

20 3.8 (s, 3H), 4.22 (m, 1H), 4.35-4.41 (m, 2H), 4.7 (m, 1H), 5.38 (m, 1H), 5.48-5.68 (m, 2H), 6.82 (m, 1H), 7.06 (m, 1H), 7.25 (s, 1H), 7.35 (s, 1H), 7 , 49-7.60 (m, 3H), 8.03-8.15 (m, 3H). LC-MS (Procedure H, with the exception that the gradient time was 2 min, the flow rate was 5 ml / min and the retention time: 1.48 min), MS m / z 820 (M ++ 1) .

Example 34, Preparation of Compound 32, tert-butyl ester of acid (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonyl-aminocarbonyl-18- (7-methoxy-2-phenyl-quinolin-4- iloxy) -2,12,12,15-tetraoxo-12,16-tia-3,1625 diazatricyclo [14.3.0.04.6] nonadec-7-en-14-il] carbamic

image 1

Oxone Step) To a solution of Compound 31 (9.5 mg, 11.6 mol) in methanol (0.276 ml) was added a buffer of pH 7 (0.093 ml) and the solution was cooled in an ice bath. Oxona (21 mg, 34.8 mol, 3 equiv.) Was added and the reaction was equilibrated at room temperature and stirred for two hours. Then, water was added to the reaction mixture and the resulting mixture was extracted three times with ethyl acetate. The combined organic extracts were concentrated in vacuo and the crude product was purified by preparative TLC (eluent Hexane: ethyl acetate,

image71

1: 3), providing the desired product, Example 34, Compound 32, tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) -7-cis- [4-cyclopropanesulfonylaminocarbonyl-18- (7-methoxy- 2-Phenyl-quinolin-4-yloxy) -2,12,12,15 tetraoxo-12,16-thia-3,16-diazatricyclo [14.3.0.04.6] nonadec-7-en-14-yl] carbamic (9 , 8 mg, 32%). 1H NMR (500 MHz, methanol-d4)  0.74-0.94 (m, 3H), 1.0-1.08 (m, 2H), 1.26 (s, 9H), 1.22- 1.33 (m, 2H), 1.58 (m, 1H), 1.76-1.82 (m, 2H),

5 2.04 (m, 1H), 2.17 (m, 1H), 2.32-2.40 (m, 2H), 2.60 (m, 1H), 2.82-2.89 (m , 2H), 3.65-3.78 (m, 2H), 3.95 (s, 3H), 4.384.45 (m, 2H), 4.75-4.99 (m, 3H), 5, 48-5.62 (m, 3H), 7.14 (m, 1H), 7.29 (s, 1H), 7.42 (m, 1H), 7.46-7.58 (m, 3H) , 8,068.18 (m, 3 H). LC-MS (Procedure H, with the exception that the gradient time was 2 min, the flow rate was 5 ml / min and the retention time: 1.29 min), MS m / z 852 (M ++ 1) .

Example 35

10 Preparation of Compounds Defined in Table 1 and Table 2 of Example 37

Each of the Compounds shown in Table 1 and Table 2 of Example 35 are tripeptide acyl sulfonamides. Each of these compounds can be prepared from the corresponding carboxylic acid in a sulfonamide coupling process that converts the carboxylic acid into said acyl sulfonamide. Be

15 You find Examples of this procedure in the present application, in the synthesis of Compounds 1 to 34. The carboxylic acids that are used as starting materials for the formation of the acyl sulfonamides shown in Table 1 can be prepared (for a For example, see steps 9a-9i of Example 9 for the preparation of the carboxylic acid of step 9i) by the procedures described in the present application and also of the contents of International Application PCT / CA00 / 00353 (Publication No., WO 00/59929).

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Preparation of Compound 441

A non-limiting example of the above coupling procedure is shown for the preparation of compound 441 (in which

R1 = image 1 , R2 = image 1 , R3 = OMe, R3 = H, R4 = image 1 , Q = image 1 ). The starting material, carboxylic acid, Compound 441A as described in International Application PCT / CA00 / 00353 (Publication No. WO 00/59929) or Example 9 herein.

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In the coupling step of sulfonamide, a tripeptide carboxylic acid, for example 441A (in which R image 1 image 1 image 1 1 =

Suitable solvent, such as THF or dichloroethane, in an inert atmosphere, for example argon or nitrogen. Then a suitable coupling reagent is added. A non-limiting example of a coupling reagent is carbonyldiimidazole. Then, the resulting solution was stirred at room temperature for approximately 30 min and then heated to reflux for a period of 30 min. Then, the solution was cooled to room temperature and then a sulfonamide was added. In the present example, cyclopropylsulfonamide was added in a

15 portion, followed by the addition of a base. An example of a base used in the transformation is DBU. The resulting reaction was stirred for 24 h, after which it was subjected to a treatment procedure. This treatment procedure may involve first diluting the reaction mixture with an organic solvent, such as ethyl acetate, then washing the resulting mixture with an aqueous solution such as, for example, an aqueous solution buffered to a pH of four. Then, the organic phase was separated and washed with brine.

Then, the organic extract was dried (MgSO4) and concentrated in vacuo to provide a crude product. The crude product can be purified using conventional practices, such as, but not limited to, flash chromatography, preparative thin layer chromatography (TLC) or reverse phase chromatography to provide the desired acylsulfonamide compound 441.

Example 36

25 Biological Studies

FRET peptide assay of recombinant HCV NS3 / 4A protease complex

The purpose of this in vitro assay was to measure the inhibition of HCV NS3 protease complexes, derived from the BMS, H77C or J4L6S strains, as described below, by compounds of the present invention. This assay provides an indication of how effective the compounds of the present invention would be in inhibition.

30 of HCV proteolytic activity.

Serum was obtained from an HCV infected patient from Dr. T. Wright, San Francisco Hospital. A full length complementary cDNA (deoxyribonucleic acid) cDNA obtained by genetic engineering of the HCV genome (BMS strain) was constructed from DNA fragments obtained by reverse transcription PCR (RTPCR) of serum RNA (ribonucleic acid) and using primers selected based on homology among 35 other genotype 1a strains. From the determination of the complete genomic sequence, a genotype 1a was assigned to the HCV isolate according to the classification of Simmonds et al. (See P Simmonds, KA Rose, S Graham,

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SW Chan, F McOmish, BC Dow, EAFollett, PLYap and H Marsden, J. Clin. Microbiol., 31 (6), 1493-1503 (1993)). The amino acid sequence of the non-structural region, NS2-5B, was shown to be> 97% identical to HCV genotype 1a (H77c) and 87% identical to genotype 1b (J4L6S). Infectious clones, H77C (genotype 1a) and J4L6S (genotype 1b) were obtained from R. Purcell (NIH) and the sequences are published in Genbank (AAB67036, see Yanagi, M., Purcell, RH, Emerson, SU and Bukh, J. Proc. Natl. Acad. Sci. USA 94 (16), 8738-8743 (1997); AF054247, see Yanagi, M., St Claire, M., Shapiro, M., Emerson, SU, Purcell, RH and Bukh, J, Virology 244 (1), 161-172. (1998)).

The BMS, H77C and J4L6S strains were used for production of recombinant NS3 / 4A protease complexes. The DNA encoding the recombinant HCV NS3 / 4A protease complex (amino acids 1027 to 1711) was manipulated for these strains as described in P. Gallinari et al. (see Gallinari P, Paolini C, Brennan D, Nardi C, Steinkuhler C, De Francesco R. Biochemistry. 38 (17): 5620-32, (1999)). Briefly, a solubilizing tail of three lysines was added at the 3 ′ end of the NS4A coding region. The cysteine at the P1 position of the NS4A-NS4B cleavage site (amino acid 1711) was changed to a glycine to avoid proteolytic cleavage of the lysine marker. In addition, a cysteine to serine mutation was introduced by PCR at amino acid position 1454 to avoid autolytic cleavage in the NS3 helicase domain. The variant DNA fragment was cloned into the bacterial expression vector pET21b (Novagen) and the NS3 / 4A complex was expressed in the Escherichia coli strain BL21 (DE3) (Invitrogen) following the protocol described by P. Gallinari et al. (see Gallinari P, Brennan D, Nardi C, Brunetti M, Tomei L, Steinkuhler C, De Francesco R., J Virol. 72 (8): 6758-69 (1998)) with modifications. Briefly, expression of NS3 / 4A was induced with 0.5 mM isopropyl D-D-1-thiogalactopyranoside (IPTG) for 22 hours at 20 ° C. A typical fermentation (10 L) produced approximately 80 g of wet cell paste. The cells were resuspended in lysis buffer (10 ml / g) consisting of N- (2-Hydroxyethyl) Piperazin-N '- (2 Ethanesulfonic acid) (HEPES), 25 mM, pH 7.5, 20% glycerol, 500 mM sodium chloride (NaCl), 0.5% Triton-X100, 1 µg / ml lysozyme, 5 mM Magnesium Chloride (MgCl2), 1 g / ml DnasaI, 5 mM 5-Mercaptoethanol (ME), inhibitor Protease - free Tetraacetic Ethylenediamine Acid (EDTA) (Roche), were homogenized and incubated for 20 minutes at 4 ° C. The homogenate was subjected to ultrasound and clarified by ultracentrifugation at 235000 g for 1 h at 4 ° C. Imidazole was added to the supernatant at a final concentration of 15 mM and the pH was adjusted to 8.0. The crude protein extract was loaded on a pre-equilibrated nickel-nitrilotriacetic acid (Ni-NTA) column with buffer B (25 mM HEPES, pH 8.0, 20% glycerol, 500mM NaCl, 0.5% Triton-X100, 15 mM imidazole, ME 5 mM). The sample was loaded at a flow rate of 1 ml / minute. The column was washed with 15 column volumes of buffer C (the same as buffer B except with Triton-X100 0.2%). The protein was eluted with 5 column volumes of buffer D (the same as buffer C except with 200 mM imidazole).

Fractions containing NS3 / 4A protease complex were pooled and loaded into a Superdex-S200 desalination column pre-equilibrated with buffer D (25 mM HEPES, pH 7.5, 20% glycerol, 300 mM NaCl, Triton-X100 0.2%, ME 10 mM). The sample was loaded at a flow rate of 1 ml / min. Fractions containing NS3 / 4A protease complex were pooled and concentrated at approximately 0.5 mg / ml. The purity of the NS3 / 4A protease complexes, derived from the BMS, H77C and J4L6S strains, was considered to be greater than 90% by SDS-PAGE and mass spectrometry analysis.

The enzyme was stored at -80 ° C, thawed on ice and diluted before use in assay buffer. The substrate used for the NS3 / 4A protease assay was RET S1 (Resonance Energy Transfer Depsipeptide Substrate; AnaSpec, Inc. cat. No. 22991) (FRET peptide), described by Taliani et al. in Anal. Biochem 240 (2): 60-67 (1996). The sequence of this peptide is slightly based on the natural cleavage site of NS4A / NS4B except that there is an ester linker instead of an amine bond at the cleavage site. The peptide substrate was incubated with one of three recombinant NS3 / 4A complexes, in the absence or presence of a compound of the present invention and the formation of fluorescent reaction product was followed in real time using a Cytofluor Series 4000.

The reagents were as follows: HEPES and Glycerol (Ultrapure) were obtained from GIBCO-BRL. Dimethyl Sulfoxide (DMSO) and bovine serum albumin (BSA) were obtained from Sigma. Rad-Mercaptoethanol was obtained from Bio Rad: Assay buffer: 50 mM HEPES, pH 7.5; 0.15 M NaCl; Triton 0.1%, Glycerol 15%; ME 10 mM. Substrate: final concentration 2 mM (from a 2 mM stock solution in DMSO stored at -20 ° C). NS3 / 4A HCV type 1a (1b), final concentration 2-3 nM (from a 5 madreM stock solution in 25 mM HEPES, pH 7.5, 20% glycerol, 300 mM NaCl, Triton-X100 0, 2%, ME 10 mM). For compounds with potencies that approach the limit of the assay, the assay was made more sensitive by adding 50 g / ml BSA to the assay buffer and reducing the final protease concentration to 300 pM.

The test was performed on a black 96-well polystyrene plate from Falcon. Each well contained 25 µl of NS3 / 4A protease complex in assay buffer, 50 µl of a compound of the present invention in assay buffer / 10% DMSO and 25 µl of substrate in assay buffer. A control (without compound) was also prepared on the same test plate. The enzyme complex was mixed with the control compound or solution for 1 minute before starting the enzymatic reaction by adding substrate. The test plate was read immediately using the Cytofluor Series 4000 (Perspective Biosystems). The instrument was adjusted to read an emission of 340 nm and excitation of 490 nm at 25 ° C. The reactions were generally followed for approximately 15 minutes.

The percent inhibition was calculated with the following equation:

image75

100 - [(Finh / Fcon) x 100]

in which F is the change in fluorescence along the linear range of the curve. A non-linear curve fit was applied to the inhibition-concentration data and the 50% effective concentration (IC50) was calculated using Excel X1-fit software using the equation, y = A + ((BA) / (1 + ((C / x) ^ D))).

It was found that all the compounds tested had IC50 of 9 µM or less. In addition, it was found that the compounds of the present invention, which were tested against more than one type of NS3 / 4A complex had similar inhibitory properties although the compounds uniformly demonstrated greater potency against strains 1b compared to strains 1a.

Specificity tests

Specificity tests were performed to demonstrate the selectivity of the compounds of the present invention in the inhibition of HCV NS3 / 4A protease compared to other serine or cysteine proteases.

The specificities of compounds of the present invention were determined against a variety of serine proteases: human neutrophil elastase (HNE), porcine pancreatic elastase (PPE) and human pancreatic chymotrypsin and a cysteine protease: human liver cathepsin B. In all cases a 96-well plate format protocol was used using p-nitroaniline (pNA) colorimetric substrate for each enzyme as previously described (US Patent 6,323,180) with some modifications to the serine protease assays. All enzymes were obtained from Sigma while the substrates were from Bachem.

Each assay included a pre-incubation with a 2 h AT enzyme inhibitor followed by substrate addition and hydrolysis at approximately 30% conversion as measured in a Spectramax Pro microplate reader. The concentrations of the compounds varied from 100 to 0.4  M depending on its power.

The final conditions for each trial were as follows:

50 mM Tris (hydroxymethyl) aminomethane (Tris-HCl) Chloride, pH 8, 0.5 M Sodium Sulfate (Na2SO4), 50 mM NaCl, 0.1 mM EDTA, 3% DMSO, 0.01% Tween-20 with : succ-AAA-pNA 133 M and HNE 20 nM or PPE 8 nM; succ-AAPF-pNA 100 M and Chymotrypsin 250 pM.

100 mM NaHPO4 (Sodium Hydrogen Phosphate) pH 6, 0.1 mM EDTA, 3% DMSO, 1 mM Tris (2-Carboxyethyl) Phosphine Hydrochloride), 1 mM, Tween-20 0.01%, Z-FR-pNA 30 M and Cathepsin B 5 nM (buffer activated enzyme reserve containing 20 mM TCEP before use).

The percent inhibition was calculated using the formula:

[1 - ((UVinh-UVwhite) / (UVctl-UVwhite))] x 100

A non-linear curve adjustment was applied to the concentration-inhibition data and the 50% effective concentration (IC50) was calculated using Excel X1-fit software.

HCV Replicon Cell Based Assay

A complete HCV replicon cellular system was established as described in Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R., Science 285 (5424): 110-3 (1999). This system allowed the inventors to evaluate the effects of their HCV protease compounds on HCV RNA replication. Briefly, using the HCV strain 1B sequence described in Lohmann's article (Accession number: AJ238799), an HCV cDNA was generated encoding the 5 'internal ribosome entry site (IRES), the resistance gene to neomycin, the IRES of VEMC (encephalomyocarditis virus) and the non-structural proteins of HCV, NS3-NS5B and the 3 'untranslated region (NTR). In vitro transcripts of the cDNA were transfected into the human hepatoma cell line, Huh7. Selection of cells constitutively expressing HCV replicon was achieved in the presence of the selectable marker, neomycin (G418). The resulting cell lines were characterized with respect to positive and negative strand RNA production and protein production over time.

Huh7 cells were cultured, constitutively expressing HCV replicon in Dulbecco's Modified Eagle (DMEM) medium containing 10% Fetal calf serum (FCS) and 1 mg / ml G418 (Gibco-BRL). The cells were seeded the night before (1.5 x 104 cells / well) in sterile 96-well tissue culture plates. Controls were prepared without compound and with DMEM compound containing FCS 4%, Penicillin / Streptomycin 1: 100, L-glutamine 1: 100 and DMSO 5% on the dilution plate (final DMSO concentration of 0.5% in the test). Compound / DMSO mixtures were added to the cells and incubated for 4 days at 37 ° C. After 4 days, the plates were thoroughly rinsed with Phosphate Buffered Saline (PBS) (3 times 150 µl). The cells were lysed with 25 µl of a lysis assay reagent containing the FRET peptide (RET S1, as described for the in vitro enzyme assay). Lysis test reagent was prepared from 5X cell luciferase cell culture lysis reagent (Promega No. E153A) diluted to 1X with distilled water, final 150 mM NaCl was added, FRET peptide was diluted to 10 M final from a 2 mM reserve in 100% DMSO. The plate was then placed in the Cytofluor 4000 instrument which had been set to 340 nm excitation / 490 emission, automatic mode for 21 cycles and the plate was read in a kinetic mode. The EC50 determinations were performed as described for the IC50 determinations.

image76

Two different secondary tests were used to confirm EC50 determinations from the test of

5 FRET of the replicon. These included a quantitative RNA assay and a transient luciferase cell indicator assay. For the quantitative RNA assay, compound / controls without compound were incubated with the cells as described for the FRET assay of the replicon. After 4 days, the cells were lysed using the Rneasy kit (Qiagen). Total purified RNA was normalized using RiboGreen (Jones LJ, Yue ST, Cheung CY, Singer VL, Anal. Chem., 265 (2): 368-74 (1998)) and the relative quantification of HCV RNA expression was evaluated using

10 the Taqmann procedure (Kolykhalov AA, Mihalik K, Feinstone SM, Rice CM, Journal of Virology 74, 2046-2051 (2000)) and the Platinum Quantitative RT-PCR Thermoscript single stage kit (Invitrogen cat No. 11731-015) . Briefly, RNA carried at a volume of 5 µl (n1 ng) was added to a prepared 20 µl mixture containing the following; 1.25X Thermoscript reaction mixture (containing Magnesium Sulfate and 5'-triphosphate 5′-triphosphates (dNTP)), 3mM dNTP, 200 nM direct primer (sequence: 5’-gggagagccatagtggtctgc-3 ’), reverse primer

15 600 nM (5'-cccaaatctccaggcattga-3 '), 100 nM probe (5'-6-FAM-cggaattgccaggacgaccgg-HHQ-1-3') (FAM: Fluorescein-aminohexyl amidite; BHQ: Black Hole Switch), dye reference Rox 1 M (Invitrogen cat No. 12223-012) and mixture of Thermoscript Plus Platinum Taq polymerase. All primers were designed with ABI Prism 7700 software and were obtained from Biosearch Technologies, Novato, CA. Samples containing known concentrations of HCV RNA transcript were applied as standards. Using the following protocol

20 cyclisation (50 ° C, 30 min, 95 ° C, 5 min; 40 cycles of 95 ° C, 15 seconds, 60 ° C, 1 min), HCV RNA expression was quantified as described in the Perkin Elmer manual using the ABI Prism 7700 Sequence Detector.

The luciferase indicator test was also used to confirm the potency of the compound in the replicon. The use of a replicon luciferase indicator assay was first described by Krieger et al (Krieger N, Lohmann V, and Bartenschlager R, J. Virol. 75 (10): 4614-4624 (2001)). The replicon construct described for the inventors' FRET assay was modified by replacing the neomycin resistance gene with the blasticin resistance gene fused with the N-terminus of the humanized form of Renilla luciferase (restriction sites Asc1 / Pme1 used for subcloning). The adaptive mutation at position 1179 (serine to isoleucine) was also introduced (Blight KJ, Kolykhalov, AA, Rice, CM, Science 290 (5498): 1972-1974). The luciferase indicator assay was established by sowing huh7 cells the night before at a density of 2 x 106 cells per T75 flask. The 30 cells were washed the next day with 7.5 ml of Opti-MEM. After the Invitrogen protocol, 40 µl of DMRIE-C with 5 ml of Opti-MEM was vortexed before adding 5 µg of HCV indicator replicon RNA. The mixture was added to the washed huh7 cells and left for 4 hours at 37 ° C. Meanwhile, serial compound and control dilutions without DMEM compound containing 10% FCS and 5% DMSO on the dilution plate (final DMSO concentration of 0.5% in the assay) were prepared. Mixtures of compound / DMSO were added to each well of a 24-well plate. After 4 hours, the transfection mixture was aspirated and the cells were washed with 5 ml of Opti-MEM before trypsinization. Trypsinized cells were resuspended in 10% DMEM and seeded at 2 x 104 cells / well in 24-well plates containing compound or controls without compound. The plates were incubated for 4 days. After 4 days, the medium was removed and the cells were washed with PBS. 100 µl of Renilla Luciferase Lysis Buffer was immediately added

1x (Promega) to each well and the plates were frozen at -80 ° C for further analysis or tested after 15 minutes of lysis. Lysate (40 µl) of each well was transferred to a 96-well black plate (transparent bottom) followed by 200 µl of 1x Renilla Luciferase test substrate. The plates were read immediately on a Packard TopCount NXT using a luminescence program.

The percent inhibition was calculated using the following formula:

Four. Five

medium luciferase signal in experimental wells (+ compound)

  % control =

medium luciferase signal in DMSO control wells (-composite)

The values were plotted and analyzed using XLFit to obtain the EC50 value.

Biological Examples

The following Table lists compounds of the present invention that were tested in vitro according to the procedure described above. These IC50 intervals used in this table are: A is <50 M; B is <5 M; 50 C is <0.5 M; D is <0.05 M. It was found that all the compounds of the present invention that were tested had IC50 of 9 µM or less against the HC3 BMS NS3 / 4A complex. Preferred compounds had IC50 of 0.021 µM, as discovered for Compound 6, or less. In addition, it was discovered that compounds of the present invention, which were tested against more than one type of NS3 / 4A complex, had similar inhibitory properties; somewhat better inhibitory properties were shown against strain J4L6S. 55 representative compounds of the present invention were also evaluated in the HCV replicon cell assay, the results of the

image77

which are provided in the table. These EC50 intervals used in this table are: A is <50 M; B is <5 M; C is <0.5 M; D is <0.05 M. The compounds of the present invention were found to have EC50 of about 13.3 µM or less. Preferred compounds have EC50 of about 0.124 µM, as discovered for Compound 6, or less. It was found that the most preferred compounds of the present invention have EC50 of 0.01 µM or less.

In the specificity tests, it was discovered that the same compound had the following activity: HLE = 30 M; PPE> 100 M; Chymotrypsin> 100 M; Cathepsin B> 100 M. These results indicate that this family of compounds is highly specific for the NS3 protease and many of these members inhibit the replication of HCV replicon.

Compound 1

IC50 B CE50 A Compound 2 IC50 B EC50

3

B TO 4 TO

5

D D 6 D C

7

D D 8 D C

9

D D 10 D D

eleven

D D 12 C C

13

D D 14 D D

fifteen

D D 16 D D

17

D C 18 D D

19

D D twenty D D

twenty-one

D D 22 D D

2. 3

D D 24 D D

25

D D 26 D D

27

D D 28 D D

29

D D 30 D B

31

D D 32 D D

33

D D 3. 4 D D

image78

Claims (14)

1. A compound of formula I, including pharmaceutically acceptable salts thereof, image 1 in which: 5 (a) R1 is unsubstituted C3-7 cycloalkyl; 10 (c) R3 is H, halo, C1-6 alkoxy or C3-6 cycloalkoxy;

(d)

 R4 is NH2 or -NH-R31; wherein R31 is -C (O) -R32, C (O) -NHR32 or C (O) -OR32, wherein R32 is C1-6 alkyl or C3-7 cycloalkyl;

(and)

 R3 is H; Y

(f) Q is a saturated or unsaturated alkylene chain, with three to nine atoms, which optionally contains from one to three heteroatoms independently selected from O or S.

2.

 A compound of claim 1 having the formula

3.

 A compound of claim 1 or 2 wherein R1 is cyclopropyl.

Four.

 A compound of any one of the preceding claims, wherein R2 is phenyl.

5.

 A compound of any one of the preceding claims, wherein R3 is methoxy.

6.

 A compound of any one of the preceding claims, wherein R4 is NH2 or tert-butoxycarbonyl NH-.

7.

 A compound of any one of the preceding claims, wherein Q is - (CH2) nCH = C- and n is 1-5.

8.

 A compound of claim 7, wherein n is 3 or 4.

9.

 A compound of claim 1 having the formula

image2 image3 image4 in which:

(b)

 Q is - (CH2) nCH = C-; Y

(C)

 n is 1-5.

10.

 A compound of claim 9, wherein n is 3 or 4.

eleven.

 A compound of claim 1 or 2, wherein: R2 is

R4 is image2 and R1 is cyclopropyl. 12. A pharmaceutical composition, comprising a compound of claims 1-11 and a pharmaceutically acceptable carrier.

13.

 A compound of claims 1-11 for use in the therapeutic treatment of a mammal by inhibition of HCV NS3 protease.

14.

 A compound of claims 1-11 for use in the therapeutic treatment of an HCV infection in a mammal.

image5