Macrocyclic compounds and uses thereof

Abstract

The present invention relates to selected macrocyclic compounds and their use in the treatment or prevention of diseases and disorders. In particular, but not exclusively, the present invention relates to the use of selected macrocyclic compounds in the treatment or prevention of diseases caused by (1) cell death due to a variety of reasons and (2) inflammation. Such diseases can affect all parts of the body, including the central nervous system and peripheral nervous system, all organs and tissues, muscles, vasculature and bones.

Description

Technical Field

[0001] The present invention relates to selected macrocyclic compounds and their use in treating or preventing diseases and disorders. In particular, but not exclusively, the present invention relates to the use of selected macrocyclic compounds in treating or preventing diseases caused by: (1) cell death due to various causes and (2) inflammation. Such diseases can affect all parts of the body, including the central and peripheral nervous systems, all organs and tissues, muscles, vasculature and bones. Diseases in which inflammation is caused by cell necrosis are called necrotizing inflammatory diseases (Linkermann (Cell Death & Differentiation, https: / / doi.org / 10.1038 / s41418-018-0218-0 (November 23, 2018)).

[0002] The present invention therefore relates to selected macrocyclic compounds and their use in the treatment or prevention of disorders of the central and peripheral nervous systems, including neurodegenerative and neuroinflammatory diseases, epilepsy and stroke; necrotizing inflammatory diseases; ischemia and reperfusion injury; diseases caused by reduced blood flow; inherited mitochondrial diseases; muscular dystrophies and myopathies. Background Art

[0003] The role of mitochondria in disease (1): regulation of cell death and mitochondrial permeability transition

[0004] Mitochondria are not only the main source of intracellular adenosine triphosphate (ATP), they are also the main controllers of cell death, participating in the pathogenesis of human diseases and aging. This effect directly or indirectly depends on a wide range of signal transduction pathways (Javadov et al., Cells, 9, 1177 (2020)). Mitochondrial dysfunction can have many reasons, which are generally concentrated on a common final pathway, which involves mitochondrial permeability conversion, particularly the opening of the mitochondrial permeability conversion pore "MPTP" (Izzo et al., Trends in Cell Biology, May, 26, 9 (2016)).

[0005] Cell death is a physiological need to maintain tissue homeostasis and eliminate harmful cells. There are three distinct and morphologically different types of cell death, which can overlap partially: Type 1 (apoptosis); Type 2 (autophagy); and Type 3 (necrosis) (e.g., Green et al. (Cold Spring Harb. Perspect Biol., 7, a006080 (2015)). Apoptosis and autophagy are orderly processes that recycle cell building materials. These processes require ATP. Apoptosis can be triggered by activation of death ligand receptors ("extrinsic pathway") or by mitochondrial permeability conversion ("intrinsic pathway"). Necrosis occurs due to irreparable cell damage and is characterized by cell swelling, plasma membrane rupture, and release of cellular components into the bloodstream. It is the intrinsic and necrotic pathways through which the compounds of the present invention exert their activity.

[0006] There is a large body of scientific literature on stress factors that cause MPTP opening. These stress factors include Ca 2+ concentration, reactive oxygen species (ROS), hypoxia (ischemia, hypoxia), ATP depletion, exogenous or endogenous toxins, accumulation of unfolded or misfolded proteins (endoplasmic reticulum (ER) stress), DNA damage, radiation or infection (Wong et al. (Methods Mol. Biol., 810, 235-242 (2012)); Rottenberg et al. (Aging Cell, October, 16, 5, 943-955 (2017)); Bouhamida et al. (Biology, 11, 300 (2022)); Lemasters et al. (Mol. Cell Biochem., September, 174, 1-2, 159-165 (1997)); Kornfeld et al. (Circulation Research, May 22, 1783 (2015)); and Chaudhari et al. (Frontiers in Cellular Neuroscience, July, 8, Article 213 (2014)). The aging process is also associated with a decline in mitochondrial mass and function (Sun et al., Mol. Cell., Mar 3, 61, 5, 654–666 (2016); and Vereczki et al., Mitochondrian, 34, 115-126 (2017)).

[0007] MPTP was discovered in 1979. It is a multiprotein complex whose precise molecular composition remains unknown. It forms in the inner mitochondrial membrane after cells are exposed to stress factors (such as those mentioned above). MPTP is open to cause mitochondrial swelling, membrane potential reduction (depolarization), and may eventually lead to cell death through one of three intrinsic pathways, depending on the specific cell environment (e.g., Baines, Ann. Rev. Physiol., 72, 61-80 (2010)).

[0008] The protein that makes up MPTP is rarely identified with certainty, one of which is cyclophilin D. Cyclophilin is a large family of proteins that are ubiquitous in all living organisms. The first cyclophilin (later referred to as cyclophilin A) was identified as the specific binding protein of the immunosuppressant cyclosporin A (Handschumacher et al., Science, Nov 2, 226, 4674, 544-547 (1984)), and was later identified as a protein folding catalyst (Fischer et al., Nature, 337, 476-478 (1989)). It was reported in 1989 that the immunosuppressant cyclosporin A was an effective inhibitor of mitochondrial inner membrane permeability conversion (Broekemeier et al., J.Biol.Chem., 264, 14, 7826-7830 (1989)). Subsequently, a mitochondrial cyclophilin called cyclophilin D was identified (Griffiths et al., Biochem. J., 274, 611-614 (1991)). It was reported in 2002 that N-methyl-4-isoleucine cyclosporin (NIM811) also inhibits mitochondrial inner membrane permeability conversion, and NIM811 is a close analog of cyclosporin A, effectively binds to cyclophilin, but has no immunosuppressive activity at all (Waldmeier et al., Molecular Pharmacology, July, 62, 1, 22-29 (2002)). The results show that the immunosuppressive activity of cyclosporin A can be separated from the characteristics of inhibiting mitochondrial permeability conversion. It is worth noting that the inhibition of cyclophilin D by gene knockout of cyclosporin A does not provide absolute protection against cell death. Sufficiently strong stress still causes cell death. The effect of cyclophilin D is the effect of MPTP regulator, and its inhibition makes cells more resistant to cell death. It is also understood from these observations that inhibition of cyclophilin D affects the necrotic and intrinsic mitochondrial death pathways but not the extrinsic pathway, thereby preserving a physiologically essential part of the cell death machinery.

[0009] Table 1 lists diseases in which cellular stress and mitochondrial permeability transition have been shown to play a key role in the pathophysiology and in which inhibition of cyclophilin D may lead to significant therapeutic benefit. A particular advantage of the compounds of the invention is their property to cross the blood-brain barrier, which is a prerequisite for the treatment of diseases affecting the central nervous system.

[0010] Table 1: Diseases in which MPTP has been demonstrated to be involved

[0011]

[0012]

[0013] MPTP can occur in two different modes in response to the opening of the above-mentioned stress factors: low and / or short-term stress levels lead to low conductance opening, characterized by partial depolarization of the inner mitochondrial membrane. In this state, the amount of ATP produced is reduced, but still sufficient to support certain cellular functions requiring ATP. From this state, cells can recover or undergo regulated recycling processes leading to cell death, such as apoptosis or autophagy (Wacquier et al., Nature Research Scientific Reports, 10, 3924 (2020)).

[0014] High and continuous stress levels cause mitochondrial membrane potential to completely disintegrate, ATP synthesis stops, and cells will irreversibly enter the necrotic pathway of cell death. Necrosis involves cell membrane rupture and cell components overflowing into the bloodstream, in which they act as so-called damage-related patterns (DAMPs), to activate various weapons of the innate immune system, thereby causing inflammation very similar to that caused in bacterial sepsis (Roh et al. (Immune Netw., August, 18, 4, e27 (2018)); Gong et al. (Nature Reviews Immunology, 20, 95-112 (2020))). The inflammation caused by tissue necrosis is called necrotizing inflammation. A major member of the DAMP released from necrotic cells is cyclophilin A, which is a normal cytoplasmic member of the cyclophilin family. Because it appears in the bloodstream after necrotic cell death, cyclophilin A has been proposed as a biomarker of necrotic cell death (Christofferson et al., Cell Death and Differentiation, 17, 1942-1943 (2010)). In the circulation, cyclophilin A acts as a potent proinflammatory cytokine that is involved in many inflammatory diseases, including cardiovascular disease, viral infection, nervous system degeneration, cancer, rheumatoid arthritis, sepsis, asthma, periodontitis and aging (Nigro et al., Cell Death and Disease, 4, e888 (2013)). This proinflammatory activity of cyclophilin A is mediated by interactions with membrane receptors present on many different cells throughout the body. Of these receptors, only CD147 and TREM2 (triggering receptor expressed on myeloid cells 2) have been characterized (Yurchenko et al. (Immunology, 117, 301-309 (2005)); Kon-Young et al. (The FASEB Journal., 35, e21479 (2021))) and the pro-inflammatory relevance of the interaction has been firmly established using antibodies against the corresponding interacting proteins (e.g., Geng et al. (Signal Transduction and Targeted Therapy, 6, 347 (2021))).

[0015] Importantly, the interaction of cyclophilin A with both CD147 and TREM2 occurs via the cyclosporin A binding site (Bukrinski (BioChim. Biophys. Acta Gen. Subj., October, 1850, 10, 2087-2095 (2015))). If this site is occupied by cyclosporin A or by the compounds of the present invention, interaction with CD147 or TREM2 cannot occur, resulting in the pro-inflammatory activity of cyclophilin A being blocked at the source by activating these receptors.

[0016] The compounds of the present invention thus possess two distinct but complementary and valuable properties: inhibition of cyclophilin D results in inhibition of mitochondrial permeability transition and prevention of cell death; and binding to the cyclosporin A binding site of cyclophilin A circulating in the bloodstream prevents cyclophilin A from interacting with its cognate membrane receptors CD147 and TREM2, resulting in a broad anti-inflammatory activity. Summary of the invention

[0017] According to a first aspect of the present invention, there is provided a compound of formula (I):

[0018]

[0019] or a salt or solvate thereof, or an optical isomer, enantiomer or diastereomer thereof, wherein;

[0020] A is independently C or N;

[0021] R 1 and R 2 are independently H or methyl;

[0022] R 3 and R 4 Independently H, C 1-7 branched or unbranched alkyl, or can form a 4-7 membered carbocyclic or heterocyclic ring, wherein the ring heteroatom can be O or N, optionally wherein when the heteroatom is N, N is replaced by C 1-7 Branched or unbranched alkyl or C 1-4 Branched or unbranched acyl substituted, optionally wherein R 3 and R 4 is independently substituted by H or phenyl at any C;

[0023] R 5 H, unbranched or branched C 1-7 -alkyl, 4-7 membered ring or heterocyclic ring, optionally substituted by: carboxyl-C 5-7 -Aryl, C 5-7 -Arylamide, C 5-7 -aryl, 5-7 membered heterocyclic ring in which the heteroatom can be O or N, C5-7 -Arylamide, phenyl, 5-7 membered heteroaryl, C 5-7 - arylaminosulfonyl, halogen, amide or amino;

[0024] R 6 H, unbranched or branched C 1-7 -alkyl or C 3-7 -cycloalkyl, or amide, or carbonyl, optionally substituted by amino, carbonyl, 5-7 membered heterocyclic or heteroaryl, or C 5-7 - aryl;

[0025] R 7 H, branched or unbranched C 1-6 -alkyl, or forms a 4-7 membered heterocyclic ring with R8;

[0026] R 8 H, branched or unbranched C 1-6 -alkyl, acetyl, or together with R7 to form a 4-7 membered heterocyclic ring, optionally substituted by hydroxyl, C 5-7 - aryl or carbonyl substitution;

[0027] Dashed lines are absent or represent single bonds;

[0028] L is CH2 or a bond;

[0029] Y is CH2, NH, N-CH3 or O;

[0030] C=Z is C=O or CH2.

[0031] Thus, when the dashed line is absent, the bond is a single bond, and when the dashed line represents a single bond, the bond as a whole becomes a double bond.

[0032] Furthermore, when L is CH2, the CH2 group is linked to -C(R 3 R 4 )-groups and sometimes double bonds, and when L is a bond, in -C(R 3 R 4 There are no atoms or groups between the )-group and sometimes the double bond.

[0033] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising the compound of the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0034] In a third aspect of the present invention there is provided a compound according to the first aspect of the present invention for use as a medicament.

[0035] In a fourth aspect of the present invention, there is provided a compound according to the first aspect of the present invention for use in treating or preventing: degeneration of the nervous system; Alzheimer's disease; Parkinson's disease; X-linked adrenoleukodystrophy; epilepsy; amyotrophic lateral sclerosis (ALS); multiple sclerosis; bipolar disorder; stroke; hearing loss; diseases caused by cell necrosis and inflammation; acute pancreatitis; surgery-related acute kidney injury; acute kidney injury caused by toxic drugs; hearing loss caused by ototoxic drugs; acute kidney injury caused by aminoglycoside antibiotics; bacterial sepsis with antibiotics; sepsis without antibiotics; such diseases a disease in which the subject has experienced or is being subjected to physical trauma or crush injury, exposure to electrical current, extreme physical exertion or activity, and extreme temperatures associated with or at risk for developing rhabdomyolysis; a disease in which the subject has a pre-existing condition or disease that increases the subject's risk of developing a renal condition or disease when exposed to a nephrotoxins; a disease in which the pre-existing condition or disease increases the risk of chronic kidney disease, optionally where there is a history of renal impairment or the need for dialysis; a disease associated with reduced blood flow; cognitive dysfunction associated with surgery or hemodialysis; or myocardial stunning associated with hemodialysis.

[0036] In a fifth aspect of the present invention there is provided a compound according to the first aspect of the present invention for use in protecting a transplanted organ from ischemia and inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The invention will now be described with reference to the following drawings, which show the following:

[0038] Figure 1 , propidium iodide (PI) uptake as a measure of the number of dead pancreatic acinar cells (by necrosis) in an in vitro model based on pancreatic acinar cell resistance to the toxicity of the bile acid taurolithocholic acid-3-sulfate (TLCS), as a percentage of the number treated with compound (2) and cyclosporin A and controls;

[0039] Figure 2 , concentrations of compound (2) in blood, plasma and brain 30 minutes to 7 hours after oral treatment with compound (2) in rodent models (ng / ml);

[0040] Figure 3 , the concentrations (ng / ml) of compound (2) in blood, plasma and brain 30 minutes to 7 hours after subcutaneous administration of the same dose of compound (2) as that of oral administration in a rodent model; and

[0041] Figure 4, Mean extent of spinal cord injury (%) in a cohort of experimental autoimmune encephalomyelitis (EAE) C57BL / 6J mouse models (the most commonly used animal model of human multiple sclerosis (MS)) treated with vehicle alone or compound (2) in the presence of pertussis toxin (PT) to promote EAE induction. DETAILED DESCRIPTION

[0042] According to a first aspect of the present invention, there is provided a compound of formula (I):

[0043]

[0044] or a salt or solvate thereof, or an optical isomer, enantiomer or diastereomer thereof, wherein;

[0045] A is independently C or N;

[0046] R 1 and R 2 are independently H or methyl;

[0047] R 3 and R 4 Independently H, C 1-7 branched or unbranched alkyl, or can form a 4-7 membered carbocyclic or heterocyclic ring, wherein the ring heteroatom can be O or N, optionally wherein when the heteroatom is N, N is replaced by C 1-7 Branched or unbranched alkyl or C 1-4 Branched or unbranched acyl substituted, optionally wherein R 3 and R 4 is independently substituted by H or phenyl at any C;

[0048] R 5 H, unbranched or branched C 1-7 -alkyl, 4-7 membered ring or heterocyclic ring, optionally substituted by: carboxyl-C 5-7 -Aryl, C 5-7 -Arylamide, C 5-7 -aryl, 5-7 membered heterocyclic ring in which the heteroatom can be O or N, C 5-7 -Arylamide, phenyl, 5-7 membered heteroaryl, C 5-7 - arylaminosulfonyl, halogen, amide or amino;

[0049] R 6 H, unbranched or branched C 1-7 -alkyl or C 3-7 -cycloalkyl, or amide, or carbonyl, optionally substituted by amino, carbonyl, 5-7 membered heterocyclic or heteroaryl, or C 5-7 - aryl;

[0050] R7 H, branched or unbranched C 1-6 -alkyl, or forms a 4-7 membered heterocyclic ring with R8;

[0051] R 8 H, branched or unbranched C 1-6 -alkyl, acetyl, or together with R7 to form a 4-7 membered heterocyclic ring, optionally substituted by hydroxyl, C 5-7 - aryl or carbonyl substitution;

[0052] Dashed lines are absent or represent single bonds;

[0053] L is CH2 or a bond;

[0054] Y is CH2, NH, N-CH3 or O;

[0055] C=Z is C=O or CH2.

[0056] The first aspect of the present invention includes further substitution of the above substitutions with, for example, the following: 1-6 -alkyl, C 1-6 -alkoxy, -NH2, -OH, halogen, C 1-3 -carbonyl, naphthalene, phenyl and phenylamino.

[0057] Where the macrocyclic compounds of the first aspect of the invention have a chiral centre, all isomers, enantiomers, diastereomers, chiral forms and racemates are contemplated, whether in the form of an isomeric mixture or in the form of separated isomers.

[0058] In the case of any macrocyclic compound of the first aspect of the invention having an acid or basic center (such as a carboxylic acid group or an amino group), all salt forms of the compound are included herein. In the case of pharmaceutical use, the salts should be regarded as pharmaceutically acceptable salts.

[0059] Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts can be formed in a conventional manner, for example by reacting the free acid or free base form of the compound with one or more equivalents of an appropriate acid or base, optionally in a solvent or in a medium in which the salt is insoluble, followed by removal of the solvent or the medium using standard techniques (e.g., vacuum, by freeze drying or by filtration). Salts can also be prepared by exchanging the counter ion of a compound in salt form with another counter ion, for example using a suitable ion exchange resin.

[0060] Examples of pharmaceutically acceptable salts include acid addition salts derived from inorganic and organic acids, and salts derived from metals (such as sodium, magnesium, or preferably potassium and calcium) or organic bases (such as ethanolamine, N,N-dialkylethanolamine, morpholine, etc.).

[0061] Examples of acid addition salts include acid addition salts formed with acetic acid, 2,2-dichloroacetic acid, citric acid, lactic acid, mandelic acid, glycolic acid, adipic acid, alginic acid, arylsulfonic acids (e.g., benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic acid, gentisic acid, glucoheptonic acid, gluconic acid (e.g., D-glucose acid), glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-ketoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, isethionic acid, lactic acid (e.g., (+)-L-lactic acid and (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-)-L-malic acid), (±)-DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g., (+)-L-tartaric acid), thiocyanic acid, undecylenic acid, and valeric acid.

[0062] Specific examples of salts are salts derived from mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid; salts derived from organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, arylsulfonic acids; and salts derived from metals such as sodium, magnesium, or preferably potassium and calcium.

[0063] The macrocyclic compound of the first aspect of the present invention may be in any crystalline form, in particular any solvate of the macrocyclic compound of the first aspect of the present invention and its salt. Preferred solvates are solvates formed by incorporating molecules of a non-toxic pharmaceutically acceptable solvent (hereinafter referred to as a solvating solvent) into the solid state structure (e.g., crystal structure) of the macrocyclic compound of the first aspect of the present invention. Examples of these solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethyl sulfoxide. Solvates can be prepared by recrystallizing the macrocyclic compound of the first aspect of the present invention with a solvent or solvent mixture containing the solvating solvent. Whether a solvate has been formed in any given case can be determined by analyzing the crystals of the compound using well-known standard techniques such as thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray crystallography.

[0064] The solvate may be a stoichiometric or non-stoichiometric solvate. A specific solvate may be a hydrate, and examples of hydrates include hemihydrates, monohydrates, and dihydrates.

[0065] For a more detailed discussion of solvates and methods for preparing and characterizing them, see Bryn et al., Solid-State Chemistry of Drugs, 2nd edition, published by SSCI Inc. of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0066] The macrocyclic compounds of the first aspect of the invention may contain one or more isotopic substitutions, and reference to a particular element includes within its scope all isotopes of that element. For example, reference to hydrogen includes within its scope 1 H. 2 H(D) and 3 H(T). Similarly, references to carbon and oxygen include within their scope 12 C. 13 C and 14 C and 16 O and 18 O. In a similar manner, unless the context indicates otherwise, reference to a particular functional group also includes isotopic variants within its scope. For example, reference to an alkyl group such as an ethyl group also encompasses variants wherein one or more hydrogen atoms in the group are in the form of deuterium or tritium isotopes, for example, as in the following ethyl group wherein all five hydrogen atoms are in the form of deuterium isotopes (perdeuterated ethyl group). Isotopes may be radioactive or non-radioactive.

[0067] Preferably, when a dashed line represents a single bond, the compound adopts the trans configuration about the single bond.

[0068] Preferably, R 1 and R 2 It's H.

[0069] In an embodiment of the present invention, R 3 is a methyl group, and R 4 is methyl, benzyl or methoxymethyl, or wherein R 4 is a methyl group, and R 3 It is methyl, benzyl or methoxymethyl.

[0070] In other embodiments of the present invention, R 3 and R 4 Together they form a piperidine ring, a 1,3-dioxane ring, a cyclobutyl ring, a cyclopentyl ring or a cyclohexyl ring.

[0071] Preferably, R 5It is H, ethyl, n-propyl, isopropyl, benzyl, chlorobenzyl, methoxybenzyl, 6-(2-aminopyridyl), 3-aminopropyl, chloromethyl, β-aminocarbonylethyl, 3-piperidylmethyl, or 4-piperidylmethyl, or hexahydropyranyl-4-yl.

[0072] In one embodiment, R 7 and R 8 They are linked together to form a 4- to 6-membered heterocyclic ring.

[0073] In an embodiment of the present invention, R 8 Selected from methyl, ethyl, benzyl, acetyl and 3-hydroxypropyl.

[0074] In other embodiments of the present invention, R 6 It is 4-(β-naphthyl)-aminobutyl, β-carboxyethyl, β-aminocarbonylethyl, aminocarbonylmethyl, benzyl or pyridyl.

[0075] In some embodiments, L is a bond.

[0076] Specific examples of macrocyclic compounds according to formula (I) include, but are not limited to, compounds (1) to (82) in Table 2, including any solvate or salt thereof, and any possible optical isomers, enantiomers or diastereomers thereof.

[0077] Table 2: Specific examples of macrocyclic compounds according to formula (I)

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising the compound of the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0089] Although the macrocyclic compounds of the first aspect of the present invention can be administered alone, it may be preferred to present them as pharmaceutical compositions. The pharmaceutical compositions of the second aspect of the present invention for both veterinary and human use comprise at least one macrocyclic compound of the first aspect of the present invention as an active agent, together with one or more acceptable carriers and optionally other therapeutic ingredients. The one or more carriers must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not physiologically harmful to the recipient thereof.

[0090] In the context of the present invention, the term "pharmaceutical composition" means a composition comprising the macrocyclic compound of the first aspect of the present invention as an active agent and further comprising one or more pharmaceutically acceptable carriers. Depending on the mode of administration and the nature of the dosage form, the composition may further contain ingredients selected from, for example, the following: diluents, adjuvants, excipients, vehicles, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents (including liposomes or nanoparticles), sweeteners, flavoring agents, aromatics, antibacterial agents, antifungal agents, lubricants and dispersants. The composition may take, for example, the following forms: tablets, dragees, powders, elixirs, syrups, liquid preparations (including suspensions, sprays, inhalants), tablets, lozenges, emulsions, solutions, cachets, granules, capsules and suppositories and liquid preparations for injection (including liposome preparations).

[0091] One or more macrocyclic compounds of the first aspect of the invention may be administered by any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), etc. It should be understood that the preferred route may vary depending on, for example, the condition of the recipient.

[0092] The pharmaceutical composition of the second aspect of the present invention includes, but is not limited to, those suitable for the administration route described herein. The composition can be conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical field. Technology and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference in its entirety. Such methods include the step of combining the active ingredient with a carrier constituting one or more auxiliary ingredients. Typically, the composition is prepared in the following manner: the macrocyclic compound of the first aspect of the present invention as an active agent is uniformly and tightly combined with a liquid carrier or a finely divided solid carrier or both, and then (if necessary) the composition is shaped.

[0093] Excipients include ascorbic acid and other antioxidants, chelating agents (such as EDTA), carbohydrates (such as dextrin), hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, and the like.

[0094] The pH of the formulation ranges from about 3 to about 11, but is typically about 7-10.

[0095] The pharmaceutical compositions of the second aspect of the invention suitable for oral administration, each containing a predetermined amount of active agent, may be presented as discrete units such as capsules, cachets or tablets; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water liquid emulsions or water-in-oil liquid emulsions. The active agent may also be administered as a bolus, electuary or paste.

[0096] Tablets may optionally be coated or scored, and are optionally formulated so as to provide sustained or controlled release of the active ingredient. Tablets containing the active agent mixed with nontoxic pharmaceutically acceptable excipients suitable for tablet manufacture are acceptable. These excipients may be, for example, inert diluents such as calcium or sodium carbonate, lactose, lactose monohydrate, cross-linked sodium carboxymethylcellulose, povidone, calcium or sodium phosphate; granulating agents and disintegrants such as corn starch or alginic acid; binders such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc.

[0097] Tablets may be uncoated or may be coated by known techniques (including microencapsulation) to delay disintegration and adsorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. For example, a time-delay material may be employed, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0098] The pharmaceutical composition of the second aspect of the invention for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g. calcium phosphate or kaolin); or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g. peanut oil, liquid paraffin or olive oil).

[0099] For administration to the eye or other external tissue (e.g., mouth and skin), the pharmaceutical composition of the second aspect of the invention is preferably applied as a topical ointment or cream containing one or more active ingredients in an amount of, for example, 0.075% w / w to 20% w / w (including one or more active ingredients ranging between 0.1% and 20% in increments of 0.1% w / w, such as 0.6% w / w, 0.7% w / w, etc.), preferably 0.2% w / w to 15% w / w, and most preferably 0.5% w / w to 10% w / w.

[0100] When formulated in an ointment, the active agent may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active agent may be formulated in a cream with an oil-in-water cream base.

[0101] If desired, the aqueous phase of the cream base may include, for example, at least 30% w / w of a polyol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol (including PEG400) and mixtures thereof. The (topical) pharmaceutical composition of the second aspect of the present invention may desirably include a compound that enhances the absorption or penetration of the active agent through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

[0102] The oil phase of the emulsion of the (external) pharmaceutical composition of the second aspect of the present invention can be constituted by known ingredients in a known manner. Although the phase can only contain an emulsifier (also known as an emulgent), it desirably contains at least one emulsifier and a fat or oil or a mixture of both fat and oil. Preferably, a hydrophilic emulsifier and a lipophilic emulsifier acting as a stabilizer are included. Both oil and fat are also preferably included. In short, one or more emulsifiers constitute a so-called emulsifying wax with or without one or more stabilizers, and the wax constitutes a so-called emulsified ointment base with oil and fat, and the emulsified ointment base forms the oily dispersed phase of the cream formulation.

[0103] Suitable emulsifiers and emulsion stabilizers for use in the pharmaceutical composition of the second aspect of the present invention include Cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

[0104] The selection of suitable oil or fat for pharmaceutical composition is based on realizing the cosmetic properties of expectation.Emulsifiable paste should preferably be non-greasy, non-staining and washable product, and it has suitable consistency to avoid leakage from pipe or other containers.Straight or branched monobasic or dibasic alkyl esters can be used, such as propylene glycol diester of diisoadipate, isocetyl stearate, coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or the branched ester blend called Crodamol CAP, wherein the latter three kinds are preferred esters.These esters can be used alone or in combination, and this depends on required characteristics.Alternately, high melting point lipids are used, such as white soft paraffin and / or liquid paraffin or other mineral oils.

[0105] The pharmaceutical composition of the second aspect of the present invention in the form of an aqueous suspension contains the macrocyclic compound of the first aspect of the present invention as an active agent mixed with an excipient suitable for making an aqueous suspension. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and gum arabic; and dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecanethenoxycetanol), condensation products of ethylene oxide with partial esters and hexitol anhydrides derived from fatty acids (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives (e.g., ethyl parahydroxybenzoate or n-propyl parahydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweeteners (e.g., sucrose or saccharin).

[0106] The pharmaceutical composition of the second aspect of the present invention in the form of an oil suspension can be prepared in the following manner: the macrocyclic compound of the first aspect of the present invention as an active agent is suspended in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). Oral suspensions can contain thickeners such as beeswax, hard paraffin or cetyl alcohol. Sweeteners (such as those described herein) and flavoring agents can be added to provide a palatable oral preparation. These compositions can be preserved by adding antioxidants (such as ascorbic acid).

[0107] The pharmaceutical composition of the second aspect of the invention in the form of dispersible powders and granules suitable for preparing aqueous suspensions by adding water provides an active agent mixed with a dispersant or wetting agent, a suspending agent and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients such as sweeteners, flavoring agents and coloring agents may also be present.

[0108] The pharmaceutical composition of the second aspect of the present invention can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil (such as olive oil, castor oil or peanut oil), a long-chain or medium-chain triglyceride, a mineral oil (such as liquid paraffin) or a mixture of these. Suitable emulsifiers include naturally occurring gums (such as gum arabic and tragacanth), naturally occurring phospholipids (such as soybean, phosphatidylcholine, glycerol, lecithin), esters or partial esters or salts (such as sorbitan monooleate) derived from fatty acids and hexitol anhydrides, and condensation products of these partial esters with ethylene oxide, polyoxyethylene sorbitan monooleate or polyethoxylated castor oil (such as Kolliphor EL, formerly known as Cremophor ). The emulsion may also contain sweeteners and flavoring agents. Syrups and elixirs may be formulated with sweeteners such as glycerol, sorbitol or sucrose. Such compositions may also contain a demulcent, a preservative, a flavoring agent or a coloring agent.

[0109] The pharmaceutical composition of the second aspect of the present invention can be in the form of a sterile injectable preparation (such as a sterile injectable aqueous or oily suspension). The suspension can be prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents mentioned herein. The sterile injectable preparation can also be a sterile injectable solution or suspension (such as a solution in 1,3-butane-diol) in a non-toxic parenteral acceptable diluent or solvent, or be prepared as a lyophilized powder. Acceptable vehicles and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils can be conventionally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids (such as oleic acid) can also be used to prepare injectables.

[0110] Pharmaceutical compositions of the second aspect of the invention suitable for administration to the eye include eye drops, wherein the macrocyclic compound of the first aspect of the invention as an active agent is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such a formulation at a concentration of 0.5% to 20%, advantageously 0.5% to 10%, especially about 1.5% w / w.

[0111] Pharmaceutical compositions of the second aspect of the invention suitable for topical administration in the oral cavity include lozenges comprising the active agent in a flavored base, typically sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

[0112] The pharmaceutical compositions of the second aspect of the invention for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

[0113] The pharmaceutical composition of the second aspect of the present invention suitable for intrapulmonary or nasal administration has, for example, a particle size in the range of 0.1 to 500 μm (including a particle size in the range of 0.1 to 500 μm, increments such as 0.5 μm, 1 μm, 30 μm, 35 μm, etc.), which is administered through the nasal passage by rapid inhalation, or administered orally by inhalation to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be delivered together with other therapeutic agents as described herein (such as compounds used to date for the treatment or prevention of infection).

[0114] Pharmaceutical compositions of the second aspect of the invention suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active agent such carriers as are known in the art to be appropriate.

[0115] The pharmaceutical composition of the second aspect of the present invention suitable for parenteral administration includes aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Pharmaceutical compositions can be prepared to provide an easily measured amount for administration. For example, an aqueous solution intended for intravenous infusion may contain about 3 μg to 500 μg of active agent / ml solution, so that a suitable volume can be infused at a rate of about 30 mL / h.

[0116] The pharmaceutical composition of the second aspect of the present invention is presented in a unit dose or multi-dose container (e.g., sealed ampoule and vial), and can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier (e.g., water for injection) just before use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the aforementioned types. Preferred unit dose formulations are those containing a daily dose or unit daily subdose (as described above) or an appropriate fraction thereof of the macrocyclic compound of the first aspect of the present invention as an active agent.

[0117] The pharmaceutical composition of the second aspect of the present invention can also be formulated to provide controlled release of the active agent to allow less frequent dosing or to improve the pharmacokinetic or toxicity characteristics of the active agent. Therefore, the present invention also provides a pharmaceutical composition comprising one or more macrocyclic compounds of the first aspect of the present invention, the pharmaceutical composition being formulated for sustained release or controlled release.

[0118] The dosage can vary according to the needs of the patient, the severity of the condition being treated, and the macrocyclic compound of the first aspect of the present invention being used. The determination of the appropriate dosage for a particular situation is within the technical scope of the art. Typically, treatment begins with a smaller dose that is less than the optimal dose of the macrocyclic compound. Thereafter, the dosage is increased in small increments until the optimal effect in the situation is achieved. For convenience, if desired, the total daily dose can be divided and administered in batches throughout the day.

[0119] Of course, the magnitude of the effective dose of the macrocyclic compound will vary with the nature of the severity of the condition to be treated, as well as with the specific macrocyclic compound and its route of administration. The selection of an appropriate dose is within the capabilities of one of ordinary skill in the art without undue burden. Typically, the daily dose range may be from about 0.1 to about 100 mg per kg of human and non-human animal body weight, preferably from about 1 to about 50 mg per kg of human and non-human animal body weight, and most preferably from about 3 to about 30 mg per kg of human and non-human animal body weight.

[0120] In a third aspect of the present invention there is provided a compound according to the first aspect of the present invention for use as a medicament.

[0121] The macrocyclic compounds disclosed in the first aspect of the present invention can be used in medical treatment. A particular advantage of the macrocyclic compounds of the first aspect of the present invention is that they cross the blood-brain barrier, which is an indispensable prerequisite for the treatment of central nervous system (CNS) diseases. Therefore, the compounds of the first aspect of the present invention can be used to prevent or treat diseases with degenerative and inflammatory components of pathophysiology, including stroke, Alzheimer's disease and Parkinson's disease, other synucleinopathy, epilepsy, motor neuron disease and amyotrophic lateral sclerosis (ALS), multiple sclerosis, Huntington's disease, ataxia, multiple system atrophy, spinal muscular atrophy, X-linked adrenoleukodystrophy.

[0122] Other uses of the macrocyclic compounds of the first aspect of the invention include acute pancreatitis, sterile sepsis, multiple organ dysfunction, surgery-related ischemia / reperfusion injury, acute kidney injury associated with drug toxicity or rhabdomyolysis, and organ transplant protection.

[0123] The macrocyclic compound of the first aspect of the present invention can be used to manufacture a medicament. In particular, the macrocyclic compound of the first aspect of the present invention can be used to manufacture a medicament for treating the following: degenerative diseases of peripheral organs, including acute pancreatitis; acute kidney injury caused by ischemia and reperfusion, toxins, trauma, sepsis or rhabdomyolysis; myocardial depression associated with hemodialysis; CNS, including stroke, cognitive dysfunction associated with surgery or hemodialysis; Alzheimer's disease and Parkinson's disease; epilepsy; multiple sclerosis and amyotrophic lateral sclerosis (ALS); and bipolar disorder.

[0124] Therefore, in a fourth aspect of the present invention, there is provided a compound according to the first aspect of the present invention for use in the treatment or prevention of: degeneration of the nervous system; Alzheimer's disease; Parkinson's disease; X-linked adrenoleukodystrophy; epilepsy; amyotrophic lateral sclerosis (ALS); multiple sclerosis; bipolar disorder; stroke; hearing loss; diseases caused by cell necrosis and inflammation; acute pancreatitis; surgery-related acute kidney injury; acute kidney injury caused by toxic drugs; hearing loss caused by ototoxic drugs; acute kidney injury caused by aminoglycoside antibiotics; bacterial sepsis with antibiotics; sepsis without antibiotics; such Diseases in which the subject has experienced or is being subjected to physical trauma or crush injury, exposure to electrical current, extreme physical exertion or activity, and extreme temperatures associated with or at risk for developing rhabdomyolysis; diseases in which the subject has a pre-existing condition or disease that increases the subject's risk of developing a renal condition or disease when exposed to a nephrotoxins; diseases in which the pre-existing condition or disease increases the risk of chronic kidney disease, optionally where there is a history of renal impairment or the need for dialysis; diseases associated with reduced blood flow; cognitive dysfunction associated with surgery or hemodialysis; or myocardial stunning associated with hemodialysis.

[0125] In a fifth aspect of the present invention there is provided a compound according to the first aspect of the present invention for use in protecting a transplanted organ from ischemia and inflammation.

[0126] General Methods for Preparing Macrocyclic Compounds of the Invention

[0127] The skilled person will appreciate that macrocyclic compounds of formula (I) may be prepared in a variety of ways, all of which involve first assembling the building blocks to give linear precursor intermediates, and typically cyclization as the final step.

[0128] For the compounds of the first aspect of the invention, macrocyclic ring closure is achieved by three major processes, including the formation of (1) a carbon-oxygen bond, (2) a carbon-nitrogen bond, and (3) a carbon-carbon bond.

[0129] Carbon-oxygen bond formation is applied to linear precursors having a carboxyl group at one end ("C-terminus") and a hydroxyl group at the other end ("O-terminus"). Carbon-nitrogen bond formation is applied to linear precursors having a carboxyl group at the C-terminus and an amino group at the other end ("N-terminus"). One approach to carbon-carbon bond formation can be achieved by the so-called Heck coupling, which consists in the reaction of an unsaturated halide with an olefin in the presence of a base and a palladium catalyst.

[0130] In some cases, the building blocks required for assembling the linear precursors are commercially available. In other cases, such building blocks are known in the literature and can be synthesized using the described procedures. In some cases, the linear precursors carry protecting groups that can be removed after cyclization. The assembly of the linear precursors consists in linking the building blocks together using any of a large number of CC, CO or CN bond forming reactions available to skilled synthetic organic chemists.

[0131] The choice of the bond to be closed in the final ring-closing step depends on the individual case and the nature of the substituents near the bond to be formed by the cyclization. The best yield is obtained if there is minimal steric effect near the reactive group. The cyclization can be best achieved by forming an ester bond (macrolactonization) or by forming an amide bond (macrolactamization). Alternatively, the cyclization can be achieved by carbon-carbon bond formation using a transition metal catalyst (e.g., Pd, Ni) or a boronic acid reagent (e.g., Heck, Suzuki-Miyaura, Sonogashira or Negishi reaction).

[0132] General principle of macrocycle construction by assembly of intermediates and final cyclization (via macrolactonization)

[0133] This is illustrated in Scheme 1 below for the preparation of compound (2).

[0134] Scheme 1: The macrocyclic compound of the present invention is prepared by cyclization via the "macrolactonization" method, typically taking the preparation of compound (2) as an example.

[0135] Solution 1

[0136]

[0137] The general principle of building a macrocycle by assembly of intermediates and final cyclization (by macrolactamization) is illustrated below in Scheme 2 for the preparation of compound (16).

[0138] Scheme 2: The macrocyclic compound of the present invention is prepared by cyclization via the "macrolactamization" method, typically taking the preparation of compound (16) as an example.

[0139]

[0140] The general principle of building a macrocycle by assembly of intermediates and final cyclization (by intramolecular Heck coupling) is illustrated below in Scheme 3 for the preparation of compound (23).

[0141] Scheme 3: Preparation of the macrocyclic compound of the present invention by intramolecular Heck coupling, typically taking the preparation of compound (23) as an example.

[0142]

[0143] All three methods, i.e. macrolactonization, macrolactamization and Heck coupling, can be used independently to construct the macrocyclic compounds of the first aspect of the invention. The selection of the best method depends on the individual circumstances. All compounds illustrated in Table 2 are prepared by modification of Scheme 1, Scheme 2 or Scheme 3.

[0144] Cyclization to obtain a macrocyclic final product can be achieved by treating a linear precursor with a reagent that is commonly used to form an ester or amide bond. Typically, such ring-closing reactions are usually achieved in the following manner: the carboxylic acid group is converted into an activated form, which then reacts intramolecularly with an alcohol or amine group present at the other end of the linear precursor to form a macrocycle. Such reactions are usually carried out in an inert solvent under dilute conditions, such as, but not limited to, dichloromethane, toluene, acetonitrile, tert-butyl methyl ether, etc. Another method that can avoid the use of high dilution is to use a flow reactor.

[0145] Preferred reagents for converting carboxylic acids into activated forms include: 2-methyl-6-nitrobenzoic anhydride, dicyclohexylcarbodiimide / N-hydroxysuccinimide; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxybenzotriazole; 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate, etc.

[0146] The core structure common to all macrocyclic compounds of the present invention consists of piperazic acid linked to a 2-hydrazino-N-heterocycle bearing a carbon substituent at the 7-position, as shown below.

[0147]

[0148] The addition of a carbon substituent at the 7-position of an N-heterocycle can be conveniently achieved by a palladium-catalyzed coupling reaction (Heck reaction) between a bromine-substituted heterocycle and an appropriately substituted olefin. Typically, the reaction is carried out by mixing the two reactants together with a catalytic amount of a palladium reagent such as tris(dibenzylideneacetone)dipalladium(0) (Pd2dba3) in an inert solvent and optionally heating the mixture to about 100°C. Preferably, the solvent is toluene, dichloroethane, tetrahydrofuran, etc., and a wide variety of palladium reagents can be used, including those derived from tri-o-tolylphosphine ligands.

[0149] A compound similar to compound (16) can be obtained by forming an amide bond between the terminal of a linear precursor having piperazine acid at its C-terminal end and the 2-amino group of an N-terminal heterocyclic hydrazine.

[0150] N-heterocycles bearing hydrazines are known in the chemical literature and can be readily prepared by reacting 2-chloro-N-heterocycles with substituted hydrazines, e.g., methylhydrazine, benzylhydrazine (for compound (9)) or (3-hydroxy)-propylhydrazine (e.g. in compound (7)).

[0151] The invention will now be illustrated by the following examples.

[0152] Example 1: Preparation of compound (2)

[0153]

[0154] Step 1: Preparation of 1-(7-bromo-2-quinolyl)-1-methylhydrazine (Intermediate (I)).

[0155]

[0156] Methylhydrazine (17.9 ml, 341 mmol) was added to a solution of 7-bromo-2-chloroquinoline (27.6 g, 114 mmol) in 1,4-dioxane (115 mL). The stirred solution was heated at 90 ° C for 2 hours. The reaction mixture was cooled and evaporated, then dissolved in ethyl acetate. It was washed with water, saturated sodium bicarbonate aqueous solution and saturated brine, dried over magnesium sulfate, filtered and the solvent was evaporated. This gave a yellow jelly, which was dried under vacuum to give the title product intermediate (I) (28.36 g) (LCMS M+H + =252,254,C 10 H 10 BrN3 requires 251,253; RT = 1.97 min.; 1 H NMR (300MHz, CDCl3) δ3.4(2H,s)4.25(2H,s),7.25-7.35(2H,m),7.45(1H,d),7.8(1H,d),7.9(1H,d).

[0157] Step 2: Preparation of di-tert-butyl (3S)-3-[[(7-bromo-2-quinolyl)-methylamino]carbamoyl]hexahydropyridazine-1,2-dicarboxylate (Intermediate (II)).

[0158]

[0159] A solution of 1-(7-bromo-2-quinolyl)-1-methylhydrazine (20.0 g, 79.3 mmol) and (3S)-1,2-bis(tert-butoxycarbonyl)hexahydropyridazine-3-carboxylic acid (prepared according to the method described in Coats et al. (J. Org. Chem., 69, 1734-1737 (2004)) (26.2 g, 79.3 mmol) in dichloromethane (1 L) was cooled on an ice bath, and then N,N-diisopropylethylamine (DIPEA) (69.0 mL, 396.5 mmol) and [(dimethylamino)-1H-1, 2,3-Triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexafluorophosphate N-oxide (HATU) (36.2 g, 95.2 mmol). The ice bath was removed and the reaction was stirred at room temperature for 16 hours. The reaction mixture was washed with saturated sodium bicarbonate and saturated brine. It was dried over sodium sulfate, filtered and evaporated to give a yellow gum. The gum was purified by silica gel chromatography (eluted with 30% ethyl acetate, 70% isohexane) to give an off-white foam. It was dried under vacuum to give the title product (45.2 g) as a white solid (LCMS M+H + =564,566,C 25 H 34 BrN5O5 requires 563,565; RT = 3.65 min.).

[0160] Step 3: Preparation of (2S)-N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazin-1-yl]-1-methyl-2-oxo-ethyl]-2-hydroxy-3-methyl-butyramide (Intermediate (III)).

[0161]

[0162] A solution of (3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazine-1,2-dicarboxylic acid di-tert-butyl ester (44.2 g, 78 mmol) in dichloromethane (600 mL) was cooled to 0 ° C and 4M HCl (200 mL) in dioxane was added. The reaction was allowed to warm to room temperature and stirred for 16 hours. The mixture was evaporated to dryness and then suspended in toluene and evaporated (x2). This provided an intermediate amine hydrochloride (39.93 g) as a white solid.

[0163] The material was suspended in dichloromethane (2.2 L) and (2S)-2-[[(2S)-2-hydroxy-3-methyl-butyryl]amino]propionic acid (14.75 g, 78 mmol) was added. The stirred mixture was cooled to 0°C and then N,N-diisopropylethylamine (69.4 mL, 390 mmol) and [(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexafluorophosphate N-oxide (35.6 g, 93.6 mmol) were added. The ice bath was removed and the reaction was stirred at room temperature for 16 hours.

[0164] The reaction mixture was washed with saturated sodium bicarbonate aqueous solution and saturated ammonium chloride aqueous solution. It was dried over magnesium sulfate, filtered and evaporated to give a yellow gum. The gum was purified by silica gel chromatography (eluted with 2% methanol, 1% 880 ammonia, 97% ethyl acetate, then eluted with 4% methanol, 1% 880 ammonia, 95% ethyl acetate) to give the title product (26.18 g) in the form of yellow foam (LCMS M+H + =535,537,C 23 H 31 BrN6O4 requires 534,536; RT = 2.06 min.).

[0165] Step 4: Preparation of (E)-4-[2-[[[(3S)-1-[(2S)-2-[[(2S)-2-hydroxy-3-methyl-butanoyl]amino]propanoyl]hexahydropyridazine-3-carbonyl]amino]-methyl-amino]-7-quinolinyl]-2,2-dimethyl-but-3-enoic acid (Intermediate (IV)).

[0166]

[0167] A solution of (2S)-N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino] formamyl] hexahydropyridazine-1-yl]-1-methyl-2-oxo-ethyl]-2-hydroxy-3-methyl-butyramide (11.9 g, 20.9 mmol), 2,2-dimethylbutenoic acid (2.5 g, 22 mmol), tri(o-tolyl)phosphine (P(o-Tol) 3) (127 g, 4.18 mmol) and triethylamine (8.7 mL, 122 mmol) in tetrahydrofuran was purged with nitrogen for 20 minutes, and then tri(dibenzylideneacetone)dipalladium(0) (Pd 2 dba 3) (1.91 g, 2.09 mmol) was added. The stirred mixture was heated at reflux for 2 hours. The reaction was cooled, filtered through diatomaceous earth and evaporated to obtain an orange foam. The foam was dried under vacuum to give the title product (16.16 g) as an orange solid (LCMS M+H +=569, C 29 H 40 N6O6 requires 568; RT = 1.60 min.).

[0168] Step 5: Preparation of compound (2) via cyclization via macrolactonization.

[0169]

[0170] Toluene (5L) was loaded in a 10L flask equipped with mechanical stirring and powdered 4A molecular sieves (1g) were added. The mixture was stirred for 20 minutes and then left to stand for 16 hours under a nitrogen atmosphere. 2-methyl-6-nitrobenzoic anhydride (MNBA) (10.32g, 30mmol) and 4-dimethylaminopyridine (DMAP) (7.32g, 60mmol) were added and the stirred mixture was heated to 100°C. A syringe pump was used to dropwise add (E)-4-[2-[[[(3S)-1-[(2S)-2-[[(2S)-2-hydroxy-3-methyl-butyryl] amino] propionyl] hexahydropyridazine-3-carbonyl] amino]-methyl-amino]-7-quinolyl]-2,2-dimethyl-but-3-enoic acid (11.6g, 15mmol) in anhydrous N,N-dimethylformamide (40mL) solution. The reaction mixture was stirred for another 30 minutes at 100 ℃ and then cooled to room temperature. The mixture was filtered and the filter cake was further rinsed with dichloromethane. The combined solution was evaporated to dryness. The material was then redissolved in dichloromethane, then washed with saturated sodium bicarbonate aqueous solution and saturated ammonium chloride aqueous solution. The solution was filtered through a hydrophobic sieve plate and evaporated to obtain an orange solid. The solid was purified by silica gel chromatography (eluted with 0.5% 880 ammonia, 3% methanol and 96.5% ethyl acetate) to obtain a yellow solid (2.58g).

[0171] The solid was combined with another batch of products (9.52 g) prepared by dissolving in dichloromethane and then evaporating in a similar manner to obtain a yellow solid (12.10 g). The solid was crushed and then ether (60 mL) was added. The mixture was stirred for 16 hours to obtain a fine yellow suspension. The solid was collected by filtration and washed with ether, dried under vacuum to obtain the title product (compound (2)) (10.16 g) (LCMS M+H + =551, C 29 H 38 N6O5 requires 550; RT = 1.64min.; 1H NMR(300MHz,DMSO)δ0.9(6H,d),1.1(1H,t),1.3(3H,s),1.4(3H,s),1.45(3H,d),1. 6-1.9(3H,m),1.95-2.1(1H,m),2.7-2.85(1H,m),3.3-3.45(1H,m),3.35(3H,s),3. 5(1H,t),4.25(1H,d),4.7(1H,d),5.1(1H,d),5.7-5.85(1H,m),6.2(1H,d),6.35(1 H,d),7.15(1H,d),7.35(1H,s),7.7(1H,d),8.1(1H,d),8.6(1H,d),10.25(1H,s)).

[0172] Using the same procedure, compound (1) was prepared using but-3-enoic acid instead of 2,2-dimethyl-but-3-enoic acid as an intermediate ( 1 H NMR(CDCl3): 7.9(m,2H),7.6(m,1H),7.1(m,1H),6.95(d,1H),6.9(m,1H),6.7(m,1H),5.35(m,1H),4.9(m,1H),3.95 (m,1H),3.6-3.4(m,2H),3.5(s,3H),2.4(m,1H),2.25(m,1H),1.9(m,1H),1.8-1.6(m,4H),1.4(m,3H),1.0(m,6H)).

[0173] Compound (3) is an isomer of compound (2) ( 1 H NMR(CDCl3):8.05(m,1H),7.7(m,2H),7.3(m,1H),7.1(m,1h),6.6(d,1H),6.35(d,1H),5.75(q,1H),4.95(d,1H),4.5(m,1H) ,3.6(m,1H),3.4(s,3H),2.85(m,1H),2.3(m,1H),2.0(m,2H),1.7(m,2H),1.6(d,3H),1.55(s,3H),1.4(s,3H),0.95(m,6H)).

[0174] Example 2: Preparation of compound (5)

[0175]

[0176] Compound (2) (7 mg, 0.0127 mmol) from Example 1 was dissolved in ethanol (4 ml) and 10% Pd / C (3 mg) was added. H2 was introduced and the reaction was stirred at room temperature for 1 h. The catalyst was filtered, washed with a small amount of ethanol and the solvent was evaporated to give the title compound (5) (6 mg) as a colorless solid (LCMS M+H + =553, C 29 H 40 N6O5 requires 552; RT = 1.71 min.).

[0177] Example 3: Preparation of compound (6)

[0178]

[0179] Compound (6) was prepared in a similar manner to Example 1 by replacing 2,2-dimethylbut-3-enoic acid with 2,2-dimethylpent-4-enoic acid (Nicolai et al., Org. Lett., 12, 2, 384-7 (2010)) (LCMS M+H + =565, C 30 H 40 N6O5 requires 564; RT = 1.59 min.).

[0180] Example 4: Preparation of compound (4)

[0181]

[0182] Compound (4) was synthesized in a similar manner to Example 1 above from 1-(7-bromo-2-quinoxalinyl)-1-methylhydrazine (itself synthesized in a similar manner to Step 1 of Example 1 from commercially available 7-bromo-2-chloro-quinoxaline) ( 1 H NMR(CDCl3):8.6(s,1H),7.8(d,1H),7.6(m,1H),7.5(m,1H),6.3(m,2H),5.9(q,1H),5.1(d,1H),4.4(m,1H),3.6( LCMS M+H + =552, C 28 H 37 NO5 requires 551; RT = 2.17 min.).

[0183] Example 5: Preparation of Compound (15)

[0184]

[0185] Compound (15) was prepared via a macrolactonization route as described below.

[0186]

[0187] Step 1: Preparation of 1-(2-methylpropanoyl)-4-vinyl-piperidine-4-carboxylic acid (Intermediate (V)).

[0188] Intermediate (V) is prepared via intermediates (Va), (Vb) and (Vc) in a four-step process as depicted below.

[0189]

[0190] Step 1a: Preparation of 1-tert-butyl 4-ethyl 4-vinylpiperidine-1,4-dicarboxylate (Intermediate (Va))

[0191] This step uses the method described in the following document: Bercot et al. (Organic Letters, 10, 5251-5254 (2008)). LDA is lithium diisopropylamide.

[0192]

[0193] Step 1b: Preparation of ethyl 4-vinylpiperidine-4-carboxylate (Intermediate (Vb)).

[0194]

[0195] A solution of 1-tert-butyl 4-ethyl 4-vinylpiperidine-1,4-dicarboxylate (1.3 g, 4.5 mmol) in 1,4-dioxane (20 mL) was stirred at room temperature. 4M hydrogen chloride in 1,4-dioxane (10 mL) was added dropwise over 1 min and the reaction mixture was stirred for 2 h. The solvent was evaporated and the residue was co-evaporated with toluene (x2) to obtain the title compound (4.5 mmol) (LCMS (m / z) 184 [M+H], RT = 0.25 min.).

[0196] Step 1c: Preparation of 1-(2-methylpropanoyl)-4-vinyl-piperidine-4-carboxylic acid ethyl ester (Intermediate (Vc)).

[0197]

[0198] A solution of ethyl 4-vinylpiperidine-4-carboxylate (4.5mmol) in dichloromethane (20mL) was stirred at 0°C. Triethylamine (1.42g, 2mL, 14mmol) was added, followed by a solution of 2-methylpropanoyl chloride (750mg, 0.75mL, 7mmol) in dichloromethane (10mL). The reaction mixture was stirred at room temperature for 18 hours. Water was added and the mixture was washed with 2M hydrochloric acid. The organic layer was separated and washed with water, saturated sodium bicarbonate solution, water and brine. The organic layer was separated, filtered through a hydrophobic sieve plate and the filtrate was evaporated. The residue was purified by silica gel chromatography (eluted with 0-50% ethyl acetate in isohexane) to give the title compound (944mg, 79%) as an oil (LCMS (m / z) [M+H] 254, RT = 2.21min.).

[0199] Step 1d: Preparation of 1-(2-methylpropanoyl)-4-vinyl-piperidine-4-carboxylic acid (Intermediate (V)).

[0200]

[0201] A solution of 1-(2-methylpropanoyl)-4-vinyl-piperidine-4-carboxylic acid ethyl ester (944mg, 3.7mmol) in tetrahydrofuran (30mL) was stirred at 5°C under nitrogen. A solution of lithium hydroxide hydrate (466mg, 11.1mmol) in water (10mL) and methanol (3mL) was added and the reaction mixture was stirred at room temperature for 21 hours. Most of the organic solvent was evaporated, the mixture was acidified to pH 1-2 with 2M hydrochloric acid and the resulting suspension was extracted with ethyl acetate. The organic extracts were combined and washed with brine. The organic layer was filtered through a hydrophobic sieve and the filtrate was evaporated to obtain the title compound (815mg, 97%) (LCMS (m / z) 226 [M+H], RT=1.47min.) as an oil.

[0202] Step 2: Preparation of 4-[(E)-2-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolinyl]vinyl]-1-(2-methylpropanoyl)piperidine-4-carboxylic acid (Heck reaction) (Intermediate (VI)).

[0203]

[0204] To N-[(7-bromo-2-quinolyl)-methyl-amino] tert-butyl carbamate, 4-oxo-1-vinylcyclohexanecarboxylic acid (352mg, 1mmol) and 1-(2-methylpropanoyl)-4-vinyl-piperidine-4-carboxylic acid (225mg, 1mmol) in tetrahydrofuran (30mL) stirring solution, add tri-(o-tolyl) phosphine (61mg, 0.0.2mmol) and triethylamine (303mg, 0.42mL, 3mmol). Reactant mixture is degassed with nitrogen for 10 minutes. Add bis(dibenzylideneacetone) palladium (0) (92mg, 0.1mmol), reactant mixture is refluxed under nitrogen for 1 hour. Reaction is cooled to room temperature and mixture is filtered through sieve plate and filtrate is evaporated. The residue was co-evaporated with toluene (x 3) to give the title compound (1 mmol) as an orange oil, which was used crude in the next step (LCMS (m / z) 497 [M+H], RT = 1.56 min.).

[0205] Step 3: Preparation of 2,2,2-trichloroethyl (3S)-1-[(2S)-2-[[(2S)-2-[4-[(E)-2-[2-[(tert-butoxycarbonylamino)-methylamino]-7-quinolyl]vinyl]-1-(2-methylpropanoyl)piperidine-4-carbonyl]oxy-3-methylbutanoyl]amino]propanoyl]hexahydropyridazine-3-carboxylate (Intermediate (VII)).

[0206]

[0207] The title compound was prepared using the method described in Example 1 in 69% yield (LCMS (m / z) 910 / 912 / 914 [M+H], RT = 2.51 min.) from 4-[(E)-2-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolyl]vinyl]-1-(2-methylpropanoyl)piperidine-4-carboxylic acid and 2,2,2-trichloroethyl (3S)-1-[(2S)-2-[[(2S)-2-hydroxy-3-methyl-butanoyl]amino]propanoyl]hexahydropyridazine-3-carboxylate prepared as described in WO 2013 / 185093 (Gilead Sciences Inc. and Selcia Limited).

[0208] Step 4: Preparation of compound (15) via cyclization via macrolactamation.

[0209]

[0210] The title compound (15) was prepared from 2,2,2-trichloroethyl (3S)-1-[(2S)-2-[[(2S)-2-[4-[(E)-2-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolyl]vinyl]-1-(2-methylpropanoyl)piperidine-4-carbonyl]oxy-3-methyl-butanoyl]amino]propanoyl]hexahydropyridazine-3-carboxylate using the method described in Example 1 in 15% yield (over 3 steps) (LCMS (m / z) 662 [M+H], RT = 1.57 min.; 1 H NMR(CDCl3): δ8.05(d,1H),7.6(d,1H),7.45(m,2H),7.1(d,1h),6.4(d,1H),6.35(d,1H),6.0(m,1H),5.2(m,1H),4.4(m,2H),4.0(m ,1H),3.6(m,1H),3.4(s,3H),3.0(m,1H),2.8(m,2H),2.5(m,1H),2.2(m,2H),1.9-1.6(m,7H),1.55(d,3H),1.1(m,6H),1.0(m,6H)).

[0211] Example 6: Preparation of Compound 17

[0212]

[0213] Compound (17) was prepared in a manner similar to compound (15) in Example 5 above, but using (2R)-2-benzyl-2-methyl-but-3-enoic acid in place of 1-(2-methylpropanoyl)-4-vinyl-piperidine-4-carboxylic acid ethyl ester as described below.

[0214] Preparation of (2R)-2-benzyl-2-methyl-but-3-enoic acid intermediate (VIII).

[0215]

[0216] Intermediate (VIII) was prepared in a 3-step process as follows.

[0217] Step 1: Preparation of (4S)-4-isopropyl-3-[(E)-2-methylbut-2-enoyl]oxazolidin-2-one.

[0218] At -20 ℃ under N2, pivaloyl chloride (Piv-Cl) (6.23mL, 50.7mmol) is added dropwise to a solution of tiglic acid (4.6g, 46.07mmol) and trimethylamine (14.13mL, 101.35mmol) in THF (80mL) and the reaction is stirred for 30 minutes. Disposable addition of lithium chloride (2.34g, 55.28mmol), then the oxazolidinone (5.95g, 46.07mmol) added in THF (15mL) and the reaction is stirred overnight under slowly warming to room temperature. White suspension is quenched with saturated NH4Cl aqueous solution and stirred for 5 minutes. Solvent evaporation and residue are distributed between water and ethyl acetate. Organic layer is further washed with saturated NaHCO3 aqueous solution, dry (MgSO4) and solvent evaporation to obtain thick oil. It was purified by column chromatography (120 g, 0 to 50% EtOAc in hexanes) to give the title compound (8.19 g) as a white solid ( 1 H NMR (300MHz, MeOD) δ0.85(dd,6H),1.80(d,3H),1.90(s,3H),2.35(m,1H),4.15(m,1H),4.30(t,1H),4.50(m,1H),6.20(m,1H)).

[0219] Step 2: Preparation of (4S)-3-[(2R)-2-benzyl-2-methyl-but-3-enoyl]-4-isopropyl-oxazolidin-2-one.

[0220] At -78 ° C, sodium hexamethyldisilazide (NaHMDS) (20.5mL, 12.32mmol, 0.6M in toluene (PhMe)) is added dropwise to a solution of (4S)-4-isopropyl-3-[(E)-2-methylbut-2-enoyl]oxazolidin-2-one (2g, 9.48mmol) in toluene (50mL) and the reaction is stirred for 40 minutes. Benzyl bromide (BnBr) (1.69mL, 14.22mmol) is added dropwise and the reaction is allowed to be stirred for 3 hours at a temperature of slowly warming to 0 ° C. The reaction is quenched by adding saturated NH4Cl aqueous solution and the reaction is stirred for 5 minutes. The phases are separated and the organic layer is dried (MgSO4) and the solvent is evaporated to obtain a crude oil. It is purified by column chromatography (80g, 0 to 20% EtOAc in hexane) to obtain the title compound (493mg) as a colorless oil. The minor isomer (192 mg) and recovered starting material (801 mg) were also isolated ( 1HNMR(300MHz, CDCl3)δ0.85(m,6H),1.45(s,3H),2.30(m,1H),3.35(dt,2H),4 .20(m,2H),4.45(m,1H),4.95(m,1H),5.10(m,1H),6.30(m,1H),7.25(m,5H)).

[0221] Step 3: Preparation of (2R)-2-benzyl-2-methyl-but-3-enoic acid (Intermediate (VII)).

[0222] Hydrogen peroxide (966 μL, 8.53 mmol, 30% wt in H2O) is added to a mixture of (4S)-3-[(2R)-2-benzyl-2-methyl-but-3-enoyl]-4-isopropyl-oxazolidin-2-one (493 mg, 1.71 mmol) and lithium hydroxide hydrate (143 mg, 3.41 mmol) in THF (8 mL) and water (4 mL) and the reaction is stirred for 5 hours. The reaction is quenched with sodium metabisulfite, stirred for 5 minutes, and then 2M NaOH (pH=14) is added. The mixture is extracted with ether (2x). The aqueous layer is acidified with 1M HCl (pH about 2) and then extracted with ether (2x). The organic layer is dried (MgSO4) and the solvent is evaporated to give the title compound (185 mg) as an oil ( 1 H NMR (300MHz, CDCl3) δ1.25(s,3H),2.90(d,1H),3.10(d,1H),5.15(m,2H),6.10(dd,1H),7.25(m,5H)).

[0223] Final step: preparation of compound (17).

[0224] Then, (2R)-2-benzyl-2-methyl-but-3-enoic acid (intermediate (VII)) was further purified according to the method described in Example 5 to provide compound (17) (LCMS (m / z) 627 [M+H], RT = 2.01 min.; C 35 H 42 N6O5 requires 626).

[0225] Example 7: Preparation of Compound (81)

[0226]

[0227] Compound (81) was prepared in a multi-step process starting from (3S)-di-tert-butyl 3-[[(7-bromo-2-quinolyl)-methylamino]carbamoyl]hexahydropyridazine-1,2-dicarboxylate (synthesis described as intermediate (II) in Example 1).

[0228] Step 1: Preparation of (3S)-N'-(7-bromo-2-quinolyl)-N'-methyl-hexahydropyridazine-3-carbohydrazide dihydrochloride (Intermediate (IX)).

[0229]

[0230] To a solution of (3S)-3-[[(7-bromo-2-quinolyl)-methylamino]carbamoyl]hexahydropyridazine-1,2-dicarboxylic acid di-tert-butyl ester (22.3 g, 39.7 mmol) in dichloromethane (400 mL) at 0°C was added 4M hydrochloric acid (100 mL) in dioxane. The reaction mixture was allowed to warm to room temperature and then stirred for 16 hours. The reaction was concentrated under reduced pressure, then suspended in toluene and concentrated (x 2) to give the title compound (17.21 g, about 100%) as a yellow solid ( 1 H NMR(300MHz,CD3OD)δ1.94-2.10(m,3H),2.22(m,1H),3.22(m,1H),3.34(m,1H),3.64(s,3H),4 .11(m,1H),7.49(d,J=9.6Hz,1H),7.77(dd,J=8.6,1.7Hz,1H),7.92(d,J=8.6Hz,1H),8.50(br s, 1H), 8.65 (d, J = 9.6Hz, 1H); LCMS (m / z) 252, 254 [M+H]).

[0231] Step 2: Preparation of tert-butyl N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methylamino]carbamoyl]hexahydropyridazin-1-yl]-1-methyl-2-oxo-ethyl]carbamate (Intermediate (X)).

[0232]

[0233] To a solution of (3S)-N'-(7-bromo-2-quinolyl)-N'-methyl-hexahydropyridazine-3-carbohydrazide dihydrochloride (9.0 g, 20.7 mmol) in dichloromethane (400 mL) at 0°C was added Boc-L-alanine (4.0 g, 21.1 mmol), N,N-diisopropylethylamine (18 mL, 103.5 mmol), followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.1 g, 29.2 mmol). The reaction was allowed to warm to room temperature and then stirred for an additional 18 hours. The reaction mixture was diluted with dichloromethane and washed with water, saturated aqueous sodium bicarbonate solution, then brine, dried over magnesium sulfate, filtered and concentrated. The product was purified by silica gel chromatography (eluting with 0-100% acetone in isohexane) to give the title compound (11.29 g, ca. 100%) as a pale yellow solid ( 1 H NMR (300MHz, CD3OD) δ1.27-1.35(m,12H),1.67-1.95(m,3H),2.07(m,1H),3.14(m,1H),3.45(s,3H),3.64(m,1H),4.10(m,1H),5.16(m,1H),7 .06(d,J=9.0Hz,1H),7.37(dd,J=8.6,1.8Hz,1H),7.59(d,J=8.6Hz,1H),7.89(d,J=1.7Hz),8.00(d,J=8.7Hz,1H); LCMS(m / z)535,537[M+H]).

[0234] Step 3: Preparation of (3S)-1-[(2S)-2-aminopropanoyl]-N'-(7-bromo-2-quinolyl)-N'-methyl-hexahydropyridazine-3-carbohydrazide hydrochloride (Intermediate (XI)).

[0235]

[0236] To a solution of tert-butyl N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methylamino]carbamoyl]hexahydropyridazin-1-yl]-1-methyl-2-oxo-ethyl]carbamate (11.2 g, 21.1 mol) in dichloromethane (400 mL) at 0°C was added 4M hydrogen chloride in dioxane (30 mL, 120 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 18 hours. The reaction mixture was concentrated in vacuo and the resulting solid was triturated with diethyl ether to give the title compound (slightly contaminated with tetramethylurea) (10.2 g, >100%) as a pale yellow solid ( 1H NMR (300MHz, CD3OD) δ1.53(d,J=6.7Hz,3H)1.77(m,1H),1.92(m,2H),2.21(m,1H),3.64(m,1H),3.66(s,3H),3.86(m,1H),4.01(m,1H),4.93(m 1H),7.48(d,J=9.6Hz,1H),7.77(dd,J=8.7,1.8Hz,1H),7.92(d,J=8.8Hz,1H),8.46(br s,1H),8.57(d,J=10.0Hz,1H); LCMS(m / z)435,437[M+H]).

[0237] Step 4: Preparation of (2S)-N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazin-1-yl]-1-methyl-2-oxo-ethyl]-3-(2-chlorophenyl)-2-hydroxy-propionamide. Intermediate (XII).

[0238]

[0239] To a solution of (3S)-1-[(2S)-2-aminopropanoyl]-N'-(7-bromo-2-quinolyl)-N'-methylhexahydropyridazine-3-carbohydrazide hydrochloride (479 mg, 1.02 mmol) in dichloromethane (40 mL) at 0°C was added (2S)-3-(2-chlorophenyl)-2-hydroxy-propionic acid (210 mg, 1.05 mmol), N,N-diisopropylethylamine (1 mL, 5.75 mmol) followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (580 mg, 1.52 mmol). The reaction was allowed to warm to room temperature and then stirred for an additional 18 hours. The reaction mixture was diluted with dichloromethane and washed with water, saturated aqueous sodium bicarbonate solution, then brine, dried over magnesium sulfate, filtered and concentrated. The product was purified by silica gel chromatography (eluting with 0-100% acetone in isohexane) to give the title compound (382 mg, 95%) as a pale yellow solid ( 1H NMR(300MHz,CD3OD)δ1.36(d,J=6.9Hz,3H),1.63-2.00(m,3H),2.09(m,1H),2.84(dd,J=14.0,9.0Hz,1H),3 .1(m,1H),3.44(s,3H),3.65(m,1H),4.14(br.d,J=12.7Hz,1H),4.25(dd,J=8.9,3.8Hz,1H),5.17(d,J=10.5 Hz,1H),5.41(q,J=6.9Hz,1H),7.06(d,J=9.1Hz,1H),7.15-7.22(m,2H),7.27-7.34(m,2H),7.38(dd,J=8.7 ,1.8Hz,1H),7.60(d,J=8.7Hz,1H),7.89(d,J=1.8Hz.1H),8.01(d,J=8.7Hz,1H); LCMS(m / z)619,621[M+H]).

[0240] Step 5: Preparation of (E)-4-[2-[[[(3S)-1-[(2S)-2-[[(2S)-3-(2-chlorophenyl)-2-hydroxypropanoyl]amino]propanoyl]hexahydropyridazine-3-carbonyl]amino]-methyl-amino]-7-quinolinyl]-2,2-dimethyl-but-3-enoic acid (Intermediate (XIII)).

[0241]

[0242] To a solution of (2S)-N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)methylamino]carbamoyl]hexahydropyridazine-1-yl]-1-methyl-2-oxo-ethyl]-3-(2-chlorophenyl)-2-hydroxypropionamide (380 mg, 0.62 mmol) in tetrahydrofuran (25 mL) was added 2,2-dimethyl-3-butenoic acid (73 mg, 0.64 mmol), tri(o-tolyl)phosphine (35 mg, 0.12 mmol) and triethylamine (0.26 mL, 1.87 mmol). The reaction mixture was purged with nitrogen for 5 minutes and then treated with tri(dibenzylideneacetone)dipalladium(0) (57 mg, 0.062 mmol). The reaction was heated to reflux for 1 hour and then allowed to cool to room temperature. The mixture was filtered through a pad of celite and washed with ethyl acetate. The filtrate was concentrated under reduced pressure and the resulting residue was triturated with diethyl ether. The solid was collected by filtration, washed with diethyl ether and dried in vacuo to give the crude title compound (328 mg, 81%) as a yellow solid (LCMS (m / z) 651, 653 [M+H]).

[0243] Step 6. Preparation of compound (81).

[0244]

[0245] A solution of (E)-4-[2-[[[(3S)-1-[(2S)-2-[[(2S)-3-(2-chlorophenyl)-2-hydroxypropanoyl]amino]propanoyl]hexahydropyridazine-3-carbonyl]amino]-methyl-amino]-7-quinolinyl]-2,2-dimethyl-but-3-enoic acid (280 mg, 0.43 mmol) in dichloromethane (20 mL) and N,N-dimethylformamide (1 mL) was added to a solution of 2-methyl-6-nitrobenzoic anhydride (340 mg, 0.99 mmol) and 4-dimethylaminopyridine (262 mg, 2.15 mmol) in dichloromethane (200 mL) containing powdered 4A molecular sieves via a syringe pump over 4 hours at reflux. The reaction mixture was heated at reflux for an additional 2 hours and then stirred at room temperature for 16 hours. The mixture is filtered through a diatomaceous earth pad, and the solution is then washed successively with saturated aqueous ammonium chloride solution, saturated aqueous sodium bicarbonate solution and brine. The organic solution is passed through a hydrophobic sieve plate, and the solution is then concentrated under reduced pressure. The product is purified by reverse phase preparative HPLC (eluted with acetonitrile / water containing 0.1% formic acid). The relevant fractions are combined and alkalized with solid sodium bicarbonate, and then partially concentrated to remove acetonitrile. The product is extracted into ethyl acetate (x2), dried over magnesium sulfate, filtered and concentrated to obtain compound (81) (52mg, 19%) ( 1 HNMR(300MHz,CD3OD)δ1.24(s,3H),1.33(s,3H),1.59(d,J=7.2Hz,3H),1.64-2.00(m,4H),2.82(m,1H), 3.25-3.29(m,2H),3.40(s,3H),3.65(dd,J=11.1,2.6Hz,1H),4.45(m,1H),5.77(dd,J=9.1,5.3Hz,1H),5 .92(q,J=7.2Hz,1H),6.27(ABq,ΔδΑΒ=0.09,JAB=16.4Hz,2H),7.09(d,J=9.0Hz,1H),7.19-7.26(m,2H), 7.29-7.42(m,3H),7.46(s,1H),7.64(d,J=8.4Hz,1H),8.05(d,J=9.0Hz,1H); LCMS(m / z)633,635[M+H]).

[0246] Example 8: Preparation of Compound (61)

[0247]

[0248] Compound (61) was prepared in a multi-step process starting from commercially available (2S)-6-amino-2-(benzyloxycarbonylamino)hexanoic acid methyl ester hydrochloride as follows.

[0249] Step 1: Preparation of methyl (2S)-2-(benzyloxycarbonylamino)-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]hexanoate (Intermediate (XIV)).

[0250] It was prepared in a multi-step sequence as follows.

[0251] Steps 1a and 1b

[0252]

[0253] A solution of (2S)-6-amino-2-(benzyloxycarbonylamino)hexanoic acid methyl ester hydrochloride (661 mg, 2 mmol) in chloroform (20 mL) was washed with saturated sodium bicarbonate solution (20 mL). The organic layer was separated and the aqueous layer was extracted with chloroform. The organic extracts were combined, washed with brine, filtered through a hydrophobic sieve plate and the filtrate was evaporated to obtain (2S)-6-amino-2-(benzyloxycarbonylamino)hexanoic acid methyl ester (612 mg, 2 mmol) (LCMS (m / z) 295 [M+H], RT=0.88 min.).

[0254] The solution of (2S)-6-amino-2-(benzyloxycarbonylamino)hexanoic acid methyl ester (612mg, 2mmol) obtained above in methanol was stirred at room temperature under nitrogen. The solution of naphthalene-2-carboxaldehyde (ArCHO) (312mg, 2mmol) was added and the reaction mixture was stirred for 2 hours. Sodium triacetoxyborohydride (NaBH(OAc)3) (466mg, 2.2mmol) was added and the reaction mixture was stirred for 1 hour at room temperature. Most of the solvent was evaporated. Saturated sodium bicarbonate solution was added and the mixture was extracted with chloroform. The organic extracts were merged and washed with brine. The organic solution was filtered through a hydrophobic sieve plate and the filtrate was evaporated to obtain (2S)-2-(benzyloxycarbonylamino)-6-(2-naphthylmethylamino)hexanoic acid methyl ester (2mmol) (LCMS (m / z) 435 [M+H], RT=1.55min.).

[0255] Step 1c: Preparation of methyl (2S)-2-benzyloxycarbonylamino-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]hexanoate.

[0256] A solution of (2S)-2-(benzyloxycarbonylamino)-6-(2-naphthylmethylamino)hexanoic acid methyl ester (2 mmol) in tetrahydrofuran (20 mL) was stirred at room temperature. A solution of di-tert-butyl dicarbonate (523 mg, 2.4 mmol) in tetrahydrofuran (5 mL) was added and the reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated and the residue was purified by silica gel chromatography (eluting with 0-30% ethyl acetate in isohexane) to give the title compound (540 mg, 50%) as an oil (LCMS (m / z) [M+H] 535, RT=3.69 min.).

[0257] Step 2: Preparation of methyl (2S)-2-amino-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]hexanoate (Intermediate (XV)).

[0258]

[0259] A solution of (2S)-2-(benzyloxycarbonylamino)-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]hexanoic acid methyl ester (508 mg, 0.95 mmol) in methanol (20 mL) containing 10% palladium carbon paste (100 mg) was stirred under hydrogen at room temperature and normal pressure for 22 hours. The reaction mixture was passed through Hyflo (RTM) The mixture was filtered through a pad of Super-Cel filter aid and the filtrate pad was washed with methanol. The filtrate was evaporated to give the title compound (350 mg, 92%) as an oil (LCMS (m / z) [M+H] 401, RT = 1.70 min.).

[0260] Step 3: Preparation of methyl (2S)-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]-2-[[(2S)-2-hydroxy-3-methylbutanoyl]amino]hexanoate (Intermediate (XVI)).

[0261]

[0262] A solution of (2S)-2-(benzyloxycarbonylamino)-6-(2-naphthylmethylamino)hexanoic acid methyl ester (350mg, 0.88mmol) in dichloromethane (30mL) was stirred at 0°C under nitrogen. (2S)-2-hydroxy-3-methylbutanoic acid (104mg, 0.88mmol) and N,N-diisopropylethylamine (454mg, 0.61mL, 3.52mmol) were added, followed by (dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methanammonium hexafluorophosphate (500mg, 1.32mmol). The reaction mixture was stirred at 0°C for 30 minutes and then at room temperature for 22 hours. The solution was diluted with dichloromethane and the solution was washed with water, saturated sodium bicarbonate solution and brine. The organic layer was separated and filtered through a hydrophobic sieve and the filtrate was evaporated. The residue was purified by silica gel chromatography (eluting with a gradient of 0-60% ethyl acetate in isohexane) to give the title compound (332 mg, 75%) as a gum (LCMS (m / z) 501 [M+H], RT = 3.22 min.).

[0263] Step 4: Preparation of (2S)-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]-2-[[(2S)-2-hydroxy-3-methylbutanoyl]amino]hexanoic acid (Intermediate (XVII)).

[0264]

[0265] A solution of (2S)-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]-2-[[(2S)-2-hydroxy-3-methylbutanoyl]amino]hexanoic acid methyl ester (332 mg, 0.66 mmol) in tetrahydrofuran (5 mL) was stirred at -5 to -8 °C under nitrogen. A solution of lithium hydroxide hydrate (69 mg, 1.65 mmol) in water (2 mL) was added dropwise (maintaining internal T < -1 °C). The reaction mixture was stirred at -5 to -1 °C for 1 hour. Cold 2M hydrochloric acid was added dropwise to acidify the solution to pH 2-3. Brine (10 mL) was added and the mixture was extracted with ethyl acetate. The organic extracts were combined, the organic layer was filtered through a hydrophobic sieve and the filtrate was evaporated. The residue was dried to give the title compound (291 mg, 91%) as a foam (LCMS (m / z) 509 [M+Na], RT = 2.95 min.).

[0266] Step 5: Preparation of tert-butyl N-[(5S)-6-[(3S)-3-[[(7-bromo-2-quinolyl)-methylamino]carbamoyl]hexahydropyridazin-1-yl]-5-[[(2S)-2-hydroxy-3-methyl-butanoyl]amino]-6-oxo-hexyl]-N-(2-naphthylmethyl)carbamate (Intermediate (XVIII)).

[0267]

[0268] A suspension of (3S)-N'-(7-bromo-2-quinolyl)-N'-methyl-hexahydropyridazine-3-carbohydrazide dihydrochloride (as previously described in Example 7 (Step 1; Intermediate (IX))) (291 mg, 0.6 mmol) in dichloromethane (20 mL) was stirred at 0° C. under nitrogen. (2S)-6-[tert-butoxycarbonyl(2-naphthylmethyl)amino]-2-[[(2S)-2-hydroxy-3-methylbutanoyl]amino]hexanoic acid (260 mg, 0.6 mmol) and N,N-diisopropylethylamine (0.52 mL, 3.0 mmol) were obtained in solution. (Dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methanium hexafluorophosphate (319 mg, 0.84 mmol) was added and the reaction mixture was stirred at 0°C for 30 minutes and then at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane and the solution was washed with water, saturated sodium bicarbonate solution and brine. The organic layer was separated and filtered through a hydrophobic sieve and the filtrate was evaporated. The residue was purified by silica gel chromatography (eluted with a gradient of 0-50% acetone in isohexane). The residue was triturated with ether / isohexane (1:2, 15 mL), the solvent was removed and the residue was dried to give the title compound (266 mg, 53%) as a foam (LCMS (m / z) 832,834 [M+H], RT=3.57 min.).

[0269] Step 6: Preparation of compound (61).

[0270]

[0271] N-[(5S)-6-[(3S)-3-[[(7-bromo-2-quinolyl)-methylamino]carbamoyl]hexahydropyridazin-1-yl]-5-[[(2S)-2-hydroxy-3-methyl-butanoyl]amino]-6-oxo-hexyl]-N-(2-naphthylmethyl)carbamic acid tert-butyl ester (from step 5) is further purified by: (i) coupling reaction with 2,2-dimethyl-3-butenoic acid using the method already described in Example 7 (step 5), and then (ii) ring closing esterification / macrolactonization reaction using the method already described in Example 7 (step 6), followed by removal of the Boc protecting group using the method already described in Example 7 (step 2) to provide compound (61).

[0272] Example 9: Preparation of Compound 10

[0273]

[0274] Compound (10) was prepared from commercially available (7-bromo-2-quinolyl)hydrazine as described below.

[0275] Step 1: Preparation of (7-bromo-2-quinolyl)hydrazine (Intermediate (XX)).

[0276]

[0277] Hydrazine hydrate (1.5 ml, 30 mmol) was added to a suspension of commercially available 7-bromo-2-chloroquinoline (1.22 g, 5 mmol) in dioxane (10 ml) and the reaction was stirred at 90 ° C for 6 hours. After cooling, water and ethyl acetate were added and the phases were separated. The organic layer was passed through a hydrophobic sieve and the solvent was evaporated to give the crude title compound (1.07 g) as a pale yellow solid ( 1 H NMR (300MHz, CDCl3) δ4.1(2H,s),6.05(1H,s),6.8(1H,d),7.35(1H,d),7.5(1H,d),7.8(1H,d),7.95(1H,s)).

[0278] Step 2: Preparation of di-tert-butyl (3S)-3-[[(7-bromo-2-quinolyl)amino]carbamoyl]hexahydropyridazine-1,2-dicarboxylate (Intermediate (XXI)).

[0279]

[0280] [(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (461 mg, 1.21 mmol) was added to a suspension of (3S)-1,2-bis(tert-butoxycarbonyl)hexahydropyridazine-3-carboxylic acid (308 mg, 0.93 mmol), (7-bromo-2-quinolyl)hydrazine (222 mg, 0.93 mmol) and N,N-diisopropylethylamine (0.49 ml, 2.8 mmol) in dichloromethane (15 ml) and the reaction was stirred at room temperature for 1 hour. The reaction was washed with saturated aqueous sodium bicarbonate solution, passed through a hydrophobic sieve and the solvent was evaporated. The crude material was purified by silica gel chromatography (0 to 50% ethyl acetate / isohexane) to give the title compound (502 mg) as a yellow foam (LCMS M+H + =550,552,C 24 H 32 BrN5O5 requires 549,551; RT = 3.15 min.).

[0281] Step 3: Preparation of di-tert-butyl (3S)-3-[[acetyl-(7-bromo-2-quinolyl)amino]carbamoyl]hexahydropyridazine-1,2-dicarboxylate (Intermediate (XXII)).

[0282]

[0283] (3S)-3-[[(7-bromo-2-quinolyl)amino]carbamoyl]hexahydropyridazine-1,2-dicarboxylic acid di-tert-butyl ester (502 mg, 0.91 mmol) was dissolved in dichloromethane (20 ml) and acetic anhydride (0.17 ml, 1.82 mmol) was added. The reaction was stirred for 7 hours. Saturated aqueous sodium bicarbonate solution was added to the solution and the phases were separated, the organic layer was passed through a hydrophobic sieve and the solvent was evaporated to give the title compound (551 mg) as a yellow foam (LCMS M+H + =592,594,C 26 H 34 BrN5O6 requires 591,593; RT = 3.58 min.).

[0284] Step 4: Preparation of compound (10).

[0285]

[0286] The synthesis of the final step compound (10) was carried out in a similar manner to steps 3 to 5 of Example 1. Compound (10) (LCMS M+H + =579, C 30 H 38N6O6 requires 578; RT = 1.96 min.).

[0287] Example 10: Preparation of Compound 24

[0288]

[0289] Step 1: Preparation of ethyl 2-iodo-2-cyclopentyl-acetate (Intermediate (XXIII)).

[0290]

[0291] To a solution of 2-bromo-2-cyclopentyl-ethyl acetate (13.5 g, 57.4 mmol) in acetone (180 ml) was added sodium iodide (26 g, 172.3 mmol) and the mixture was heated under reflux for 18 hours. After cooling, the solvent was evaporated and the residue was partitioned between ether and 10% sodium thiosulfate. The phases were separated, the organic layer was dried (magnesium sulfate) and the solvent was evaporated to give the title compound (9 g) ( 1 H NMR (300MHz, CDCl3) δ1.15(1H,m),1.25(3H,t),1.3(1H,m),1.5-2.05(7H,m),2.45(1H,m),4.15(2H,m)).

[0292] Step 2: Preparation of 2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoic acid ethyl ester (Intermediate (XXIV)).

[0293]

[0294] To a solution of N,N-diisopropylamine (LDA) (2.8 ml, 19.6 mmol) in tetrahydrofuran (20 ml) was added n-butyl lithium (7.5 ml, 18.8 mmol, 2.5 M in hexanes) dropwise at -15 °C under N2 over 45 minutes. The reaction was stirred for 15 minutes and then cooled to -78 °C. To this was added 3,3-dimethylpent-4-en-2-one (Nakagawa-Goto et al., Synlett, 11, 1555-1558 (2011)) (2 g, 17.9 mmol) in tetrahydrofuran (10 ml) over 15 minutes. The reaction was stirred for 30 minutes and then 2-iodo-2-cyclopentyl-ethyl acetate (5.3 g, 19.6 mmol) in tetrahydrofuran (10 ml) was added dropwise over 15 minutes. The reaction was stirred at -78 °C for 30 minutes and then warmed to room temperature. The reaction was quenched with saturated aqueous ammonium chloride solution and diluted with ether. The phases were separated and the organic layer was dried over a hydrophobic sieve. The solvent was evaporated and the residue was purified by silica gel chromatography (isohexane / ethyl acetate, 20: 1) to give the title compound (1.2 g) as a yellow oil ( 1 H NMR (300MHz, CDCl3) δ1.25(9H,m),1.4-2.1(9H,m),2.6(2H,m),2.95(1H,dd),4.15(2H,m),5.15(2H,m),5.95(1H,dd)).

[0295] Step 3: Preparation of (E)-7-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolinyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoic acid ethyl ester (Intermediate (XXV)).

[0296]

[0297] To a stirred solution of N-[(7-bromo-2-quinolyl)-methyl-amino]carbamic acid tert-butyl ester (Example 1, Step 1, intermediate (I)) (1.45 g, 4.12 mmol) and 2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoic acid ethyl ester (1 g, 3.75 mmol) in dioxane (20 mL) was added tri-(o-tolyl)phosphine (456 mg, 1.5 mmol) and dicyclohexylmethylamine (1.3 ml, 5.99 mmol). The reaction mixture was degassed with nitrogen for 10 minutes. Palladium acetate (336 mg, 1.5 mmol) was added and the reaction mixture was heated in a microwave vial at 140 ° C for 4 hours. The reaction was cooled to room temperature and the mixture was filtered through diatomaceous earth and the filtrate was evaporated. The residue was purified by silica gel chromatography (isohexane / ethyl acetate, 2: 1) to give the title compound as a yellow oil (LCMS M+H+ =538, C 31 H 43 N3O5 requires 537; RT = 2.98 min.).

[0298] Step 4: Preparation of (E)-7-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolinyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoic acid (Intermediate (XXVI)).

[0299]

[0300] Lithium hydroxide hydrate (859mg, 20.4mmol) is added to a solution of (E)-7-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoic acid ethyl ester (1.1g, 2.04mmol) in tetrahydrofuran (20ml) and water (4ml) and the reaction is stirred at 50°C for 20 hours. The solvent is evaporated and the residue is dissolved in water. It is acidified (2M HCl, pH about 2) and the water layer is extracted with ethyl acetate (3x). The combined organic matter is passed through a hydrophobic sieve and the solvent is evaporated to obtain the title compound (780mg) in yellow foam (LCMSM+H + =510, C 29 H 39 N3O5 requires 509; RT = 2.3 min.).

[0301] Step 5: Preparation of 2,2,2-trichloroethyl (3S)-1-[(2S)-2-[[(E)-7-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolinyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoyl]amino]propanoyl]hexahydropyridazine-3-carboxylate (Intermediate (XXVII)).

[0302]

[0303] 4M HCl in dioxane (3.7 ml) was added to a solution of 2,2,2-trichloroethyl (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxylate (Steadman et al., J. Med. Chem., 60, 1000-1017 (2017)) (700 mg, 1.62 mmol) in dichloromethane (10 ml) and the reaction was stirred for 1 hour. The solvent was evaporated to give a white solid. To this was added (E)-7-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoic acid (750 mg, 1.47 mmol) in dichloromethane (7 ml), followed by N,N-diisopropylethylamine (1.06 ml, 5.89 mmol) and [(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanammonium hexafluorophosphate N-oxide (784 mg, 2.06 mmol). The reaction was stirred at room temperature for 18 hours. The reaction was washed with 0.5 M hydrochloric acid, saturated aqueous sodium bicarbonate, saturated aqueous brine, passed through a hydrophobic sieve and the solvent was evaporated. The crude material was purified by silica gel chromatography (25% to 33% ethyl acetate / isohexane) to give the title compound (700 mg) as a white solid (LCMS M+H + =825, C 39 H 53 Cl3N6O7 required 824; RT = 2.79 min, 2.84 min (mixture of diastereomers).

[0304] Step 6: Preparation of (3S)-1-[(2S)-2-[[(E)-7-[2-[amino(methyl)amino]-7-quinolyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoyl]amino]propanoyl]hexahydropyridazine-3-carboxylic acid (Intermediate (XXVIII)).

[0305]

[0306] Lithium hydroxide hydrate (54mg, 1.28mmol) is added to 2,2,2-trichloroethyl (3S)-1-[(2S)-2-[[(E)-7-[2-[(tert-butoxycarbonylamino)-methyl-amino]-7-quinolyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoyl] amino] propionyl] hexahydropyridazine-3-carboxylate (700mg, 0.85mmol) in tetrahydrofuran (12ml) and water (3ml) solution and the reaction is stirred at room temperature for 15 minutes. The mixture is acidified (2M HCl, 3-4pH) and solid sodium chloride is added. The mixture is extracted with ethyl acetate (2x) and the combined organic matter is passed through a hydrophobic sieve plate and the solvent is evaporated. The crude product is dissolved in dichloromethane (2ml) and added in the 4M HCl (2ml) in dioxane. The reaction was stirred for 30 min and the solvent was evaporated to give the title compound which was used as such in the next step (LCMS M+H + =593, C 32 H 44 N6O5 requires 592; RT = 1.64 min.).

[0307] Step 7: Preparation of compound (24).

[0308]

[0309] (3S)-1-[(2S)-2-[[(E)-7-[2-[amino(methyl)amino]-7-quinolyl]-2-cyclopentyl-5,5-dimethyl-4-oxo-hept-6-enoyl]amino]propanoyl]hexahydropyridazine-3-carboxylic acid (Intermediate (XXVIII)) as its dihydrochloride salt (0.85 mmol) was dissolved in anhydrous DCM (283 ml) and DIPEA (0.76 ml, 4.75 mmol) was added. HATU (453 mg, 1.2 mmol) was added under stirring under nitrogen. The reaction mixture was stirred at room temperature overnight and then concentrated to about 50 ml under reduced pressure. Washed with 0.5 M HCl, saturated sodium bicarbonate and then dried by passing through a hydrophobic sieve. The solvent was evaporated under reduced pressure and the residue was purified by silica gel chromatography (eluting with isohexane:acetone 3:2) to give the crude product (80 mg) and further purified by silica gel chromatography (eluting with ethyl acetate) to give the crude product (60 mg) ( 1H NMR (300MHz, CD3OD)8.1(d,1H,J=9Hz),7.7(d,1H,J=9Hz),7.65(m,1H),7.4(m,1H),7.15(d,1H,9Hz),6.4(d,1H,J=16Hz),6.15(d,1H,J=16H LC m / z[M+H]575).

[0310] Example 11: Preparation of Compound (25) and Compound (82)

[0311]

[0312] Preparation of starting material 2-isopropyl-5,5-dimethyl-4-oxo-hept-6-enoic acid ethyl ester (Intermediate (XXIX)).

[0313]

[0314] 2-Isopropyl-5,5-dimethyl-4-oxo-hept-6-enoic acid ethyl ester (WO 2007 / 096395 (Nycomed GmbH)) was prepared in a similar manner to Example 10 (Step 2) above by substituting 2-iodo-2-cyclopentyl-acetic acid ethyl ester for 2-iodo-3-methyl-butyric acid ethyl ester ( 1 H NMR (300MHz, CDCl3) δ0.9(9H,m),1.25(6H,m),1.95(1H,m),2.5(1H,dd),2.7(1H,m),2.95(1H,dd),4.15(2H,m),5.15(2H,m),5.9(1H,dd)).

[0315] The remaining preparations of compound (25) and compound (82) were completed in a similar manner to Example 10 (steps 3 to 7, inclusive). The two diastereoisomeric products were separated to give:

[0316] Compound (25): LCMS M+H + =549, C 30 H 40 N6O4 requires 548; RT = 1.78min.

[0317] Compound (82): LCMS M+H + =549, C 30 H40 N6O4 requires 548; RT = 1.65min.

[0318] NMR (300 MHz; CD3OD) δ8.1 (d, 1H), 8.0 (m, 1H), 7.7 (m, 1H), 7.25 (d, 1H), 7.1 (d, 1H), 6.6 (d, 1H), 6.2 (d, 1H), 5.85 (m, 1H), 4.5 (m, 1H), 3.9 (m, 1H), 3.4 (s, 3H), 2.8 (m, 2H), 2.4 (m, 1H), 2.1 (m, 1H), 1.9-1.7 (m, 5H), 1.5 (s, 3H), 1.35 (m, 3H), 1.35 (m, 3H), 1.3 (m, 3H), 1.0 (d, 3H) (multiple conformers).

[0319] Example 12: Preparation of Compound (7) and Compound (8)

[0320]

[0321] Step 1: Preparation of tert-butyl N-[3-[(4-methoxyphenyl)methoxy]propylamino]carbamate (Intermediate XXX).

[0322]

[0323] Commercially available 3-[(4-methoxyphenyl)methoxy]propanal (1.05g, 5.44mmol) and tert-butyloxycarbonylhydrazine (BocNHNH2) (720mg, 5.44mmol) were dissolved in methanol (30ml). Acetic acid (0.62ml, 10.88mmol) and sodium cyanoborohydride (342mg, 5.44mmol) were added thereto and the reaction was stirred at room temperature for 18 hours. Saturated aqueous sodium bicarbonate solution was added and the resulting mixture was extracted with dichloromethane (2x). The organic layer was passed through a hydrophobic sieve and the solvent was evaporated. It was purified by silica gel chromatography (0 to 40% ethyl acetate / isohexane) to give the title compound (548mg) as a solid ( 1 HNMR(300MHz,CDCl3)δ1.5(9H,s),1.8(2H,m),2.95(2H,m),3.5(2H,t),3.8(3H,s),4.0(1H,br s),4.45(2H,s),6.05(1H,br s),6.9(2H,d),7.3(2H,d)).

[0324] In contrast to the above reaction scheme, virtually no PMBCl is required.

[0325] Step 2: Preparation of tert-butyl N-[(7-bromo-2-quinolyl)-[3-[(4-methoxyphenyl)methoxy]propyl]amino]carbamate (Intermediate (XXXI)).

[0326]

[0327] A mixture of 2-iodo-7-bromoquinoline (589 mg, 1.76 mmol) and tert-butyl N-[3-[(4-methoxyphenyl)methoxy]propylamino]carbamate (548 mg, 1.76 mmol) in ethanol (20 ml) was heated at reflux for 18 hours. The solvent was evaporated and the crude material was purified by silica gel chromatography (0 to 20% ethyl acetate / isohexane) to give the title compound (597 mg) as a yellow oil ( 1 H NMR(300MHz, CDCl3)δ1.55(10H,m),2.05(2H,m),3.05(2H,t),3.8(3H,s),4.05(2H,br m),4.4(2H,s),6.85(2H,d),7.05(1H,d),7.2(2H,d),7.35(1H,d),7.45(1H,d),7.85(1H,d),7.9(1H,s)).

[0328] Step 3: Preparation of compound (7) and compound (8).

[0329] The synthesis of compound (7) and compound (8) was completed in a similar manner as described in Example 5 (steps 3 to 7). In the 4M HCl deprotection step, the 4-methoxybenzyl (PMB) protecting group was removed (see Example 5 (step 6)). The final macrocyclization step (see Example 5 (step 7)) gave compound 7 and compound 8:

[0330] Compound (7): LCMS M+H + =595, C 31 H 42 N6O6 requires 594; RT = 1.89 min.

[0331] Compound (8): LCMS M+H + =577, C 31 H 40 N6O5 requires 576; RT = 1.90min.

[0332] Example 13: Preparation of Compound (23)

[0333]

[0334] This compound (20) was prepared according to the procedure described for compound (20) in Example 14 by using N-Boc-N-methylvaline instead of N-Boc-valine in step 2. 1 H NMR(CDCl3):8.1(m,1H),7.8(m,1H),7.65(m,1H),7.3(m,1H),7.15(m,1H), 6.7(m,1H),6.45(m,1H),5.8,5.1(m,1H),4.3(m,1H),3.65(m,1H),3.45,3. 4(2s,3H),3.0-2.8(m,2H),2.75,2.6(2s,3H),2.5(m,1H),2.2(m,2H),1.8( m,H),1.5,1.0(2d,3H),1.3,1.2(2s,3H),1,15(s,3H),0.95,0.8(2m,6H)).

[0335] Example 14: Preparation of Compound (20)

[0336]

[0337] Step 1: Preparation of (3S)-1-[(2S)-2-aminopropanoyl]-N'-(7-bromo-2-quinolyl)-N'-methyl-hexahydropyridazine-3-carbohydrazide (Intermediate (XXXII)).

[0338]

[0339] Intermediate (XXXII) was prepared in a similar manner as described in Example 1, Step 3.

[0340] Step 2: Preparation of tert-butyl N-[(1S)-1-[[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazin-1-yl]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (Intermediate (XXXIII)).

[0341]

[0342] At 0 ° C, to a solution of (3S)-1-[(2S)-2-aminopropanoyl]-N'-(7-bromo-2-quinolyl)-N'-methyl-hexahydropyridazine-3-carbohydrazide (intermediate (XXXII)) hydrochloride (1.0 g, 2.3 mmol) in DCM (60 ml) was added Boc-N-methyl-L-valine (0.58 g, 2.53 mmol), DIPEA (2.0 ml, 11.5 mmol) and HATU (1.2 g, 3.16 mmol). The reaction mixture was allowed to warm to room temperature and stirred at room temperature overnight. The reaction mixture was washed with saturated ammonium chloride and saturated sodium bicarbonate. The organic layer was dried via passing through a hydrophobic sieve plate and then evaporated to dryness. The product was purified by silica gel chromatography (eluted with 0 to 30% (18:80:2 methanol:ethyl acetate:880 ammonia) and ethyl acetate). The product (1.3 g) was isolated as a colorless oil.

[0343] Step 3. Preparation of (2S)-2-amino-N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazin-1-yl]-1-methyl-2-oxo-ethyl]-3-methyl-butyramide (Intermediate (XXXIV)).

[0344]

[0345] By N-[(1S)-1-[[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazine-1-yl]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamic acid tert-butyl ester (intermediate (XXXIII)) (1.03g, 1.62mmol) was dissolved in DCM (80ml) and 4MHCl (10ml, 40mmol) in dioxane was added under stirring. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure to give the hydrochloride (1.01g) of the product as a white solid.

[0346] Step 4: Preparation of (2S)-N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazin-1-yl]-1-methyl-2-oxo-ethyl]-2-(2,2-dimethylbut-3-enylamino)-3-methyl-butanamide (Intermediate (XXXV)).

[0347]

[0348] Under agitation, 2,2-dimethylbut-3-enal (100mg, 1.02mmol) is added to a solution of intermediate (XXXIV) (600mg, 1.05mmol) in THF (20ml). Sodium triacetoxyborohydride (445mg, 2.1mmol) is added and the reaction mixture is stirred at room temperature overnight. The solvent is evaporated under reduced pressure and the residue is absorbed in DCM / sodium bicarbonate aqueous solution. After separation, the aqueous phase is extracted with DCM, the DCM extracts are merged and dried via passing through a hydrophobic sieve plate. The solvent is evaporated under reduced pressure and the residue is purified by silica gel chromatography (eluted with 0 to 30% (18:80:2 methanol in ethyl acetate: ethyl acetate: 880 aqueous ammonia)) to obtain the desired product (75mg) as a colorless jelly.

[0349] Preparation of compound (20).

[0350]

[0351] (2S)-N-[(1S)-2-[(3S)-3-[[(7-bromo-2-quinolyl)-methyl-amino]carbamoyl]hexahydropyridazine-1-yl]-1-methyl-2-oxo-ethyl]-2-(2,2-dimethylbut-3-enylamino)-3-methyl-butyramide (Intermediate (XXXV)) (118 mg, 0.19 mmol) was dissolved in THF (120 ml), and P(o-Tol)3 (12 mg, 0.039 mmol) and triethylamine (80 uL, 0.57 mmol) were added. The reaction mixture was purged with nitrogen and Pd2dba3 (17 mg, 0.019 mmol) was added. The reaction mixture was heated at reflux for 3 hours. Add P(o-Tol)3(13mg, 0.19mmol), Pd2dba3(18mg, 0.019mmol) and heat the reaction mixture for 3 hours. Add P(o-Tol)3(13mg, 0.19mmol), Pd2dba3(18mg, 0.019mmol) and triethylamine (80uL) and heat the reaction mixture under reflux for 24 hours. Add P(o-Tol)3(13mg, 0.19mmol), Pd2dba3(18mg, 0.019mmol) and triethylamine (80uL, 0.57mmol) and heat the reaction mixture under reflux overnight. The reaction mixture is filtered through diatomaceous earth and the solvent is evaporated. The residue is purified by preparative HPLC (eluted with 10%-20% acetonitrile in water containing 0.1% formic acid). The fractions are combined and partially evaporated. The residue was absorbed in DCM / sodium bicarbonate aqueous solution. After separation, the aqueous phase was extracted with DCM (x3). The extracts were combined, dried over a hydrophobic sieve plate and the solvent was evaporated to give the product (18 mg) as a pale yellow solid ( 1 H NMR (300MHz, CD3OD)8.05(d,1H,J=9Hz),7.9(s,1H),7.3(dd,1H,J=1.6,9Hz),7.1( d,1H,J=9Hz),6.45(d,1H,J=16Hz),6.15(d,1H,J=16Hz),6.1(m,1H),4.4(m,1H),3. 65(m,1H),3.4(s,3H),2.8(m,2H),2.65(m,2H),2.0(m,2H),1.7(m,2H),1.7(m,1H), 1.6(d,3H,J=7Hz),1.2(s,3H),1.1(s,3H),1.0(d,3H,J=7Hz),0.8(d,3H,J=7Hz); LC m / z [M+H] 536).

[0352] The other compounds in Table 2 were prepared by similar methods as described in the above Examples.

[0353] The LCMS data of the compounds are presented in Table 3

[0354] Table 3: LCMS retention times and [M+H].

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] Selected compounds 1 H NMR data are provided in Table 4.

[0361] Table 4: 1 H NMR data.

[0362]

[0363]

[0364] Spectrophotometric determination of functional peptidylprolyl isomerase (PPIase)

[0365] Cyclophilins are enzymes that catalyze the cis-trans isomerization of peptide bonds to the amino acid proline. This event is important in many biological processes, including protein folding and signal transduction. Cyclosporin and the compounds of the present invention inhibit this catalytic activity. The method for measuring this activity and its inhibition is similar to that described in the following literature: Jankowski (Analytical Biochemistry, 252, 2, 299-307 (1997)). The assay uses a UV / Vis spectrophotometer to measure the cis-to-trans isomerization of peptide substrates catalyzed by the PPIase enzyme. The assay is a manual single cuvette-based assay. The first-order rate equation is fitted to the absorbance data to obtain the rate constant. The catalytic rate is calculated by subtracting the background rate from the enzymatic rate. The Ki (concentration required to produce half-maximal inhibition) of the inhibitor is obtained from the rate constant plotted against the inhibitor concentration. Typically, a 6-point dose-response curve is obtained from replicates and the Ki is determined. The data for cyclophilins A and D are given in Table 5 as Ki [nM] and in column 2 of Table 5 as Ki (cypA) / Ki (cypD).

[0366] Cell death assay of thapsigargin-treated Jurkat cells: protection against MPTP opening

[0367] This assay demonstrates that the test compounds are able to protect Jurkat cells (an immortalized human T lymphocyte line; Bortner et al. (J. Biol. Chem., July, 30, 274, 31, 21953-21962 (1999))) from thapsigargin-induced cell death.

[0368] Thapsigargin effectively inhibits the endoplasmic reticulum Ca-ATPase, which sequesters Ca from the cytoplasm. 2+ ) to increase intracellular free calcium (Ca 2+ ). Mitochondria transfer Ca 2+ Exchange for K + To buffer increased cytosolic Ca 2+ concentration, resulting in depolarization of the mitochondrial inner membrane and permeability transition.

[0369] The thapsigargin-treated Jurkat cell death assay is a homogeneous 96-well plate-based cell death assay that is stimulated by the opening of the mitochondrial permeability transition pore (MPTP). Addition of thapsigargin to Jurkat cells results in an influx of calcium, the increase in intracellular calcium will initially be buffered by the mitochondria, but ultimately the increased calcium concentration will lead to the opening of the MPTP, resulting in cell death.

[0370] CellTox TM Green is a dye that cannot enter cells until cell death results in loss of membrane integrity. Once this occurs, CellTox TM Green enters the cell, binds to the cell's DNA and emits a fluorescent signal. If the compound inhibits the opening of the pore in response to thapsigargin or inhibits the cell death pathway produced, the fluorescence increase usually seen when thapsigargin is added is inhibited. Unless otherwise stated, the assay is typically performed in triplicate using a series of three compound concentrations of 1 μM, 3 μM and 10 μM, and kinetic readings are performed over a 24-hour period. Compounds were added 30 minutes before thapsigargin was added. Compound controls and positive controls cyclosporin A (CsA) (3 μM and 10 μM) are also included in the assay. Results are typically calculated at 8 hour time points, and kinetic data are also reported.

[0371] This study proves that the test compound can protect SH-SY5Y cells (human neuroblastoma cell line) from cell death induced by thapsigargin. The assay was performed as described for Jurkat cells, but SH-SY5Y (human neuroblastoma) cell line was used instead of Jurkat cells. The results are shown in the fourth column of Table 5 (SY5Y [10 μM] recovery %). Positive numbers indicate prevention of cell death, and negative numbers indicate compound toxicity.

[0372] Calcium retention capacity (CRC) assay in permeabilized HepG2 cells

[0373] This assay demonstrates that the test compounds are able to directly inhibit the opening of the MPTP in permeabilized cells. The use of permeabilized cells means that inhibitory compounds that would not normally be able to enter non-permeabilized cells are more likely to be active.

[0374] The calcium retention capacity (CRC) is a measure of the ability of mitochondria to retain calcium (primarily in the form of calcium phosphate) in the mitochondrial matrix. By storing calcium in the form of an osmotically inactive precipitate, mitochondria help buffer cytosolic free calcium levels, thereby regulating calcium-dependent cellular processes, particularly the opening of the mitochondrial permeability transition pore (MPTP).

[0375] In the CRC assay, the calcium indicator Calcium Green-5N (InVitrogen) is used. Calcium Green-5N is an extramitochondrial dye that shows an increase in fluorescence in the presence of calcium. The assay is performed in HepG2 (human hepatocellular carcinoma) cells permeabilized with digitonin. Multiple sequences of calcium pulses (typically 5 μM) are added, which result in a sudden increase in fluorescence that is then reduced by mitochondrial buffering. The cells will reach a point where calcium addition is no longer buffered, indicating that the MPTP has opened (mitochondrial permeability transition).

[0376] The experiments were performed with and without inhibitors. Compounds that act to inhibit the opening of mitochondrial pores will result in more calcium being required to reach the transition point. Results were normalized relative to the positive control (cyclosporin A) in each experiment. Results are shown in the third column of Table 5 as CRC [10 μM] (ratio compared to CsA). The method of digitonin permeabilized whole cell CRC assay is similar to that used in the following literature: Chiara et al. (PLoS One, March, 3, 3, e1852 (2008)).

[0377] Table 5: Results of biochemical and cellular assays.

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397] Anti-acute pancreatitis activity

[0398] Acute pancreatitis is mainly caused by gallstones or excessive alcohol consumption. Both causes focus on the opening of the MPTP as a common mechanism of disease (Mukherjee et al., Gut Online, June 12, 2015). Gallstones block the bile duct and prevent the outflow of bile acids, which are acutely toxic to pancreatic acinar cells, leading to Ca 2+. 2+ Excessive accumulation, MPTP opening and necrosis. Alcohol is partially converted into fatty acid ethyl esters in the body, which are also acutely toxic to pancreatic acinar cells, leading to Ca 2+Surge, MPTP opening and necrosis. Inhibition of MPTP opening by cyclophilin inhibitors, including compound (2), has been shown to be protective in animal models of acute pancreatitis (Mukherjee et al., Gut Online, June 12, 2015).

[0399] Protects cells against other toxins

[0400] The above-mentioned bile acid toxicity mechanism on pancreatic acinar cells is very similar to many other toxins, such as the toxicity mechanism of aminoglycoside antibiotics on renal tubular cells or inner ear hair cells (Dehne et al., Hear Res., July, 169, 1-2, 47-55 (2002)), or the toxicity mechanism of myosin on renal tubular cells (rhabdomyolysis; Chang et al. (The Journal of Nutritional Biochemistry, December, 110, 109134 (2022))).

[0401] WO 2021 / 115299 (Zuo) discloses compounds that protect against acute kidney injury caused by many different causes. The same compounds are disclosed in US 6 583 265 (C-Chem AG) as cyclophilin inhibitors. Apart from the inhibition of cyclophilins, these compounds have no known enzyme inhibition, receptor binding or ion channel activity. It can therefore be concluded that the compounds of the present invention are potent cyclophilin inhibitors, but have no activity against other enzymes or receptors, with the same spectrum of properties. This conclusion is confirmed by Figure 1 The activity of compound (2) shown in the present invention in protecting pancreatic acinar cells against the toxicity of bile acid taurolithocholic acid-3-sulfate (TLCS) was confirmed. Figure 1 Propidium iodide (PI) levels were measured as a measure of the number of dead pancreatic acinar cells (by necrosis) compared to the number of treatments with compound (2) and cyclosporin A and controls.

[0402] Figure 1 Two sets of results are shown. Figure 1 A single asterisk in the left panel of results indicates that the result is significant relative to the control "TLCS-treated". Figure 1 The double asterisk in the middle right panel of results indicates that the result is highly significant relative to the control "TLCS-treated".

[0403] Pharmacokinetics: Blood-brain barrier penetration

[0404] The test compound is administered orally, subcutaneously or intravenously to rodents (mice or rats). In order to determine blood or plasma concentrations, blood samples are collected at different time points and the concentration of the test compound is analyzed by LC / MS. Brain concentrations are measured by sacrificing animals and performing LC / MS analysis on brain homogenates.

[0405] The results for compounds (2), (19), (6) and (27) are summarized in Table 6, showing the concentrations of the compounds in plasma and brain. All four compounds showed that the compounds passed the blood-brain barrier.

[0406] Table 6: Brain penetration results for selected compounds of the invention.

[0407]

[0408] Figure 2 Graphs showing the concentrations (ng / ml) of compound (2) in blood, plasma and brain from 30 minutes to 7 hours after oral treatment. The calculated pharmacokinetic data are presented in Table 7.

[0409] Table 7: Pharmacokinetics of compound (2) in mice (strain FVB / N) at an oral dose of 10 mg / kg.

[0410] PK parameters unit plasma Whole blood brain <![CDATA[T 1 / 2 ]]> h 3.0 5.45 - <![CDATA[C max ]]> ng / mL 582 1490 297 <![CDATA[T max ]]> h 0.5 0.5 7 <![CDATA[AUC 0-t ]]> ng / mL / h 3180 12500 - Blood / Plasma ratio 2.87 2.87 - Blood / brain ratio 0.72 - 0.72

[0411] Figure 3 A graph showing the concentrations (ng / ml) of compound (2) in blood, plasma and brain from 30 minutes to 7 hours after subcutaneous administration of the same dose. The calculated pharmacokinetic data are presented in Table 8.

[0412] Table 8: Pharmacokinetics of compound (2) in mice (strain FVB / N) at 10 mg / kg subcutaneous administration.

[0413] PK parameters unit plasma Whole blood brain <![CDATA[T 1 / 2 ]]> h 1.773.17 1.773.17 <![CDATA[C max ]]> ng / mL 1640 2640 563 <![CDATA[T max ]]> h 0.5 0.5 2.0 <![CDATA[AUC 0-t ]]> ng / mL / h 3730 8150 3370 Blood / Plasma ratio 2.2 2.2 Blood / brain ratio 0.9 0.9

[0414] Experimental allergic encephalitis: a mouse model of multiple sclerosis

[0415] Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of human MS and is particularly useful for studying neuroinflammatory pathways (Martin R, "Immunological aspects of experimental allergic encephalomyelitis and multiple sclerosis and their application for new therapeutic strategies" in Mizuno, Y., Youdim, MBH, Calne, DB, Horowski, R., Poewe, W., Riederer, P. (editor) (Advances in Research on Neurodegeneration, Springer, Vienna, https: / / doi.org / 10.1007 / 978-3-7091-6844-8 _ 6 (1997)).

[0416] C57BL / 6 mice are the most commonly used strain. For the induction of EAE, typically, the immunogenic epitope MOG of myelin oligodendrocyte glycoprotein is expressed prior to immunization. 35-55 Suspended in complete Freund's adjuvant (CFA). Afterwards, pertussis toxin (PT) was applied on the day of immunization and a few days later to promote the induction of EAE. The animals were evaluated for both inflammation and demyelination by body weight change, EAE score and histopathological examination.

[0417] Compound (2) was dissolved (in 66.6% PEG / water) at a dose level of 75 mg / kg and a final concentration of 20 mg / ml, and orally administered BID (i.e., every 12 hours) to C57BL / 6J mice at a dose volume of 3.75 ml / kg. Treatment began at the first signs of concomitant disease (approximately day 10-12) and continued for 14 days. Animals were evaluated for general clinical signs, weight changes, and neurological scores. After treatment was completed, animals were sacrificed, brains and spinal cords were collected, fixed in 4% formalin, and processed for staining with hematoxylin and eosin.

[0418] Treatment with compound (2) did show reduced clinical scores and better weight gain in treated animals compared to the vehicle group, but the changes were not statistically significant. However, the extent of spinal cord injury was significantly reduced in the treated group compared to the untreated control.

[0419] The results are shown in Figure 4, which shows the average extent of spinal cord injury (%) of mouse groups treated with vehicle alone or compound (2).

[0420] Kainic Acid Model of Epilepsy

[0421] The kainic acid model of temporal lobe epilepsy involves intrahippocampal injection of kainic acid and presents neuropathological and electroencephalographic features seen in patients with temporal lobe epilepsy, especially behavioral seizures and neuropathological lesions (Levesque et al. (J. Neuroscience Methods, 260, 45-52 (2016))). Electrodes implanted in the brain are used to monitor seizures; hippocampal neurodegeneration is assessed by immunohistochemistry.

[0422] Male NMRI mice (n=21) were given 150 mg / kg of compound (2) (60% PEG400 / water solution) for 3 consecutive days. Two hours after the last dose, the animals were given 45 mg / kg of kainic acid subcutaneously. Behavior was monitored for 3 hours. On day 7 after receiving kainic acid, the animals were sacrificed and the brains were analyzed for markers of neurodegeneration by immunohistochemistry.

[0423] As a result, 5 animals (24%) in the untreated control group showed strong neurological degeneration, while only 2 animals (9%) in the treated group exhibited visible signs that they were affected.

Claims

1. A compound of formula (I): or a salt or solvate thereof, or an optical isomer, enantiomer or diastereomer thereof, wherein; A is independently C or N; R 1 and R 2 are independently H or methyl; R 3 and R 4 Independently H, C 1-7 branched or unbranched alkyl, or can form a 4-7 membered carbocyclic or heterocyclic ring, wherein the ring heteroatom can be O or N, optionally wherein when the heteroatom is N, N is replaced by C 1-7 Branched or unbranched alkyl or C 1-4 Branched or unbranched acyl substituted, optionally wherein R 3 and R 4 is independently substituted by H or phenyl at any C; R 5 H, unbranched or branched C 1-7 -alkyl, 4-7 membered ring or heterocyclic ring, optionally substituted by: carboxyl-C 5-7 -Aryl, C 5-7 -Arylamide, C 5-7 -aryl, 5-7 membered heterocyclic ring in which the heteroatom can be O or N, C 5-7 -Arylamide, phenyl, 5-7 membered heteroaryl, C 5-7 - arylaminosulfonyl, halogen, amide or amino; R 6 H, unbranched or branched C 1-7 -alkyl or C 3-7 -cycloalkyl, or amide, or carbonyl, optionally substituted by amino, carbonyl, 5-7 membered heterocyclic or heteroaryl, or C 5-7 - aryl; R 7 H, branched or unbranched C 1-6 -alkyl, or forms a 4-7 membered heterocyclic ring with R8; R 8 H, branched or unbranched C 1-6 -alkyl, acetyl, or together with R7 to form a 4-7 membered heterocyclic ring, optionally substituted by hydroxyl, C 5-7 - aryl or carbonyl substitution; Dashed lines are absent or represent single bonds; L is CH2 or a bond; Y is CH2, NH, N-CH3 or O; C=Z is C=O or CH2.

2. The compound of claim 1, wherein when a dashed line represents a single bond, the compound adopts a trans configuration about the single bond.

3. The compound according to claim 1 or claim 2, wherein R 1 and R 2 It's H.

4. A compound according to any one of claims 1 to 3, wherein R 3 is a methyl group, and R 4 is methyl, benzyl or methoxymethyl, or wherein R 4 is a methyl group, and R 3 It is methyl, benzyl or methoxymethyl.

5. A compound according to any one of claims 1 to 3, wherein R 3 and R 4 Together they form a piperidine ring, a 1,3-dioxane ring, a cyclobutyl ring, a cyclopentyl ring or a cyclohexyl ring.

6. A compound according to any one of claims 1 to 5, wherein R 5 It is H, ethyl, n-propyl, isopropyl, benzyl, chlorobenzyl, methoxybenzyl, 6-(2-aminopyridyl), 3-aminopropyl, chloromethyl, β-aminocarbonylethyl, 3-piperidylmethyl, or 4-piperidylmethyl, or hexahydropyranyl-4-yl.

7. A compound according to any one of claims 1 to 6, wherein R 7 and R 8 Linked together to form a 4- to 6-membered heterocyclic ring.

8. A compound according to any one of claims 1 to 6, wherein R 8 Selected from methyl, ethyl, benzyl, acetyl and 3-hydroxypropyl.

9. A compound according to any one of claims 1 to 8, wherein R 6 It is 4-(β-naphthyl)-aminobutyl, β-carboxyethyl, β-aminocarbonylethyl, aminocarbonylmethyl, benzyl or pyridyl.

10. The compound according to any one of claims 1 to 9, wherein L is a bond.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A compound according to any one of claims 1 to 10 for use as a medicament.

13. A compound according to any one of claims 1 to 10 for use in the treatment or prevention of: degeneration of the nervous system; Alzheimer's disease; Parkinson's disease; X-linked adrenoleukodystrophy; epilepsy; amyotrophic lateral sclerosis (ALS); multiple sclerosis; bipolar disorder; stroke; hearing loss; diseases caused by cell necrosis and inflammation; acute pancreatitis; surgery-related acute kidney injury; acute kidney injury caused by toxic drugs; hearing loss caused by ototoxic drugs; acute kidney injury caused by aminoglycoside antibiotics; bacterial sepsis with antibiotics; sepsis without antibiotics; such diseases, wherein the subject has experienced or is experiencing physical trauma or crush injury, exposure to electrical current, extreme physical exertion or activity, and extreme temperatures associated with or at risk for developing rhabdomyolysis; such a disease wherein the subject has a pre-existing condition or disease that increases the subject's risk of developing a renal condition or disease when exposed to a nephrotoxins; such a disease wherein the pre-existing condition or disease increases the risk of chronic kidney disease, optionally wherein there is a history of renal impairment or the need for dialysis; a disease associated with reduced blood flow; cognitive dysfunction associated with surgery or hemodialysis; or myocardial stunning associated with hemodialysis.

14. A compound according to any one of claims 1 to 10 for use in protecting a transplanted organ from ischemia and inflammation.

15. The compound of claim 14, wherein the compound is administered to an organ donor prior to removal of the organ.

16. The compound according to claim 14, wherein the transplanted organ is perfused and stored using a solution containing the compound at a concentration of 0.01-100, preferably 0.01-50, more preferably 0.01-10 micromolar.

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