EA049703B1 - BICYCLIC COMPOUNDS AND THEIR APPLICATION FOR THE TREATMENT OF DISEASES - Google Patents

Abstract

The invention provides compounds and compositions thereof for modulating hepatocyte growth factors. In some embodiments, the compounds are compounds of formula (I) or a pharmaceutically acceptable salt thereof, wherein the values of the variables (e.g., L, Rla, Rlb, R2, R3, R4, R5, R6, R7, and η) are as described herein. In some embodiments, the compounds and compositions are provided for the treatment of diseases, including neurological disorders.

Description

Cross reference to related application

This application claims priority to U.S. Provisional Patent Application No. 63/108,660, filed November 2, 2020, the contents of which are incorporated herein by reference in their entirety.

Field of technology

The present invention generally relates to compounds, compositions and methods for their preparation and use for the treatment of diseases, such as neurodegenerative diseases.

State of the art

Hepatocyte growth factor (HGF) is a pleiotropic protein factor involved in numerous biological processes including embryonic and organ development, regeneration, and inflammation. HGF is a critical factor in the development and maturation of cerebral cortex, motor, sensory, sympathetic, and parasympathetic neurons. HGF is translated and secreted as inactive pro-HGF, but after cleavage, the resulting α and β subunits are disulfide bonded to form an active heterodimer. HGF is expressed primarily in mesenchymal cells such as fibroblasts, chondroblasts, adipocytes, and endothelium. Expression has also been demonstrated in the central nervous system (CNS), including neurons, astrocytes, and ependymal cells (Nakamura and Mizuno, 2010). All biological actions of HGF are mediated through MET, a transmembrane receptor tyrosine kinase, which serves as the only known receptor for HGF. MET is known to be involved in a variety of biological processes, with demonstrated roles in development, regeneration, and injury response. Following binding of HGF to the extracellular domain of MET, homodimerization of the MET protein leads to autophosphorylation of the intracellular domain. Phosphorylation of the intracellular domains of MET leads to the recruitment and phosphorylation of various effector proteins, including Gab1, GRB2, phospholipase C, and Stat3 (Gherardi et al., 2012; Organ and Tsao, 2011). Such effector proteins then interact with downstream signaling pathways, including PI3K/Akt, Ras/Raf/MAPK, RAC1/CDC42, RAP/FAK, and others, to influence a wide range of cellular components, including gene regulation, cytoskeletal rearrangements, cell cycle progression, cell adhesion, survival, and proliferation (Organ and Tsao, 2011).

Because HGF plays an important role in development (Nakamura et al., 2011), homeostasis (Funakoshi and Nakamura, 2003), cell death suppression and regeneration (Matsumoto et al., 2014), stimulation of the HGF/MET signaling system is an ideal target for therapies in a variety of diseases. Therapies involving modulation of HGF activity have been proposed for the treatment of diseases and injuries in many different tissue types, including liver, kidney, gastrointestinal tract, cardiovascular components, lung, skin, nervous system and muscle (Matsumoto et al., 2014). However, highly effective compounds suitable for modulating HGF/MET signaling activity remain to be explored and discovered.

Despite the progress made in this area, there remains a need for improved compounds and methods for treating HGF-modulated diseases. Accordingly, in one aspect, the present document provides compounds that modulate HGF for use in the treatment of neurodegenerative diseases.

Brief summary of the invention

Certain embodiments of this document describe compounds and compositions thereof for modulating hepatocyte growth factor (HGF) for the treatment of diseases. Non-limiting illustrative embodiments include:

Implementation option 1.

Compound of formula (I)

SR® rS R ^lb k ^1e (O or its pharmaceutically acceptable salt, where

L represents -C(=O)- or -(CR ^a R ^b ) _m -;

R ^a and R ^b represent H;

R ^1a and R ^1b are independently H or C ₁ -C ₆ alkyl optionally substituted with hydroxyl, halogen or -CO ₂ H;

R ² is oxo;

R ³ is C ₂ -C ₆ alkyl, C ₃ -C ₆ alkenyl, C ₃ -C ₁₂ cycloalkyl or C ₃ -C ₆ cycloalkylalkyl;

R ⁴ is C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl optionally substituted with C ₁ -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy, hydroxyl or halogen, wherein the 5-10 membered heteroaryl contains 1-3 nitrogens;

- 1 049703

R ⁶ is H;

R ⁷ is oxo;

m represents 1; and n represents 0.

Embodiment 2. The compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein L is -C(=O)-.

Embodiment 3. The compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein L is -(CR ^a R ^b ) _m -.

Embodiment 4. A compound according to any one of embodiments 1-3, or a pharmaceutically acceptable salt thereof, wherein each R ^1a and R ^1b is independently H, methyl, or -CH2CO2H.

Embodiment 5. The compound of embodiment 4 or a pharmaceutically acceptable salt thereof, wherein R ^1a is methyl and R ^1b is H.

Embodiment 6. A compound according to any one of embodiments 1-5, or a pharmaceutically acceptable salt thereof, wherein R ³ is C ₂ -C ₆ alkyl, C ₂ -C ₆ alkenyl, or C ₃ -C ₆ cycloalkylalkyl.

Embodiment 7. The compound of embodiment 6 or a pharmaceutically acceptable salt thereof, wherein R ³ is

Implementation option 8.

A compound according to any one of embodiments 1-7, or a pharmaceutically acceptable salt thereof, wherein ^R4 is _C6 - _C10aryl optionally substituted with 1-3 substituents selected from halogen, hydroxyl, C1-C6 haloalkyl and C1-C6 haloalkoxy.

Implementation option 9.

The compound of embodiment 8 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is phenyl substituted with 1-3 substituents selected from -CF ₃ , -OCHF2, -OH, fluoro and chloro.

Embodiment 10.

The compound of embodiment 9 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is:

Implementation option 11.

A compound according to any one of embodiments 1-7, or a pharmaceutically acceptable salt thereof, wherein R ⁴ is pyridyl optionally substituted with 1-3 substituents selected from halogen, hydroxyl, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy.

Implementation option 12.

The compound of embodiment 11 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is

Implementation option 13.

A compound according to any one of embodiments 1-10, or a pharmaceutically acceptable salt thereof, wherein -LR ⁴ is

- 2 049703

Implementation option 14.

A compound according to any one of embodiments 1-13, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (V) ^B \

“Yin” (V).

Embodiment 15.

The compound of embodiment 14 or a pharmaceutically acceptable salt thereof, wherein

L represents -C(=O)- or -CH2-;

R ^1a and R ^1b independently represent H or C1-C3 alkyl optionally substituted with -CO2H;

R ³ is C4-C5 alkyl, C4-C5 alkenyl, or C1-C3 alkyl substituted with C ₃ -C ₅ cycloalkyl; and

R ⁴ is phenyl or pyridyl substituted with 1-3 substituents selected from -CF ₃ , -OCHF2, -OH, fluorine and chlorine.

Implementation option 16.

The connection selected from

- 3 049703

- 4 049703

and its pharmaceutically acceptable salts.

Embodiment 17.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 18.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

- 5 049703

Embodiment 19.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 20.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 21.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 22.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 23.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Implementation option 24.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 25.

The compound according to embodiment 1 or 16 of the following formula:

- 6 049703

or a pharmaceutically acceptable salt thereof.

Embodiment 26.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 27.

The compound according to embodiment 1 or 16 of the following formula:

or a pharmaceutically acceptable salt thereof.

Embodiment 28.

A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 27 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Embodiment 29.

A method for modulating hepatocyte growth factor in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to embodiment 28.

Embodiment 30.

A method for treating a disease, condition or injury associated with modulation of hepatocyte growth factor in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to embodiment 28, wherein the disease, condition or injury is a neurodegenerative disease, spinal cord injury or sensorineural hearing loss.

Embodiment 31.

The method of embodiment 30, wherein the disease, condition, or injury is a neurodegenerative disease, and the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

Embodiment 32.

The method of embodiment 31, wherein the disease, condition, or injury is amyotrophic lateral sclerosis (ALS).

Embodiment 33.

A method for treating or slowing the progression of dementia or preventing cognitive dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to embodiment 28.

Embodiment 34.

The method of embodiment 33, wherein the dementia or cognitive dysfunction is associated with Alzheimer's disease or Parkinson's disease.

Embodiment 35.

A method for treating, restoring or preventing a disease, condition or injury associated with nervous tissue, or treating or preventing a disease or disorder of the central nervous system in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a compound according to any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to embodiment 28.

- 7 049703

Detailed description of the essence of the invention Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following description sets forth certain specific details to provide a thorough understanding of the various embodiments of the present invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any of the claimed subjects of the invention. To the extent that any material incorporated by reference herein conflicts with the express content of the present invention, the express content controls. In this application, the use of the singular forms includes the plural, unless clearly indicated otherwise. It should be noted that as used in the specification and the appended claims, the singular forms include plural references, unless the context clearly dictates otherwise. In this application, the use of or means and/or, unless otherwise indicated. Furthermore, the use of the term including, as well as other forms such as include, includes and included, is not limiting.

Unless the context otherwise requires, throughout this application and claims the word "comprise" and its variations such as "comprises" and "containing" are to be construed in an open, inclusive sense, that is, as including without limitation.

In the present specification, any concentration range, percentage range, ratio range, or integer range shall be understood to include the value of any integer within the stated range and, where appropriate, fractions of the integer (e.g., one tenth and one hundredth of an integer), unless otherwise stated. In addition, any range of numbers given herein with respect to any physical feature, such as polymer subunits, size, or thickness, shall be understood to include any integer within the stated range, unless otherwise stated. As used herein, the terms about and approximately mean ±20%, ±10%, ±5%, or ±1% of the stated range, value, or structure, unless otherwise stated.

Throughout this specification, reference to one embodiment or embodiment means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the presence of the expressions in one embodiment or embodiment in various places in this specification does not necessarily always refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Amino refers to the -NH2 radical.

Carboxy or carboxyl refers to the radical _-CO2H .

Cyano refers to the radical -CN.

Hydroxy or hydroxyl refers to the -OH radical.

Nitro refers to the radical -NO2.

Oxo refers to the =O substituent.

Thioxo refers to the =S substituent.

Thiol refers to the -SH substituent.

Alkyl refers to a radical with a straight or branched saturated hydrocarbon chain consisting only of carbon and hydrogen atoms, containing from one to twelve carbon atoms (C1-C12 alkyl), preferably from one to eight carbon atoms (C1-C8 alkyl), from one to six carbon atoms (C1- _C6 alkyl) or from one to three carbon atoms (C1-C3 alkyl) and which is attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specifically stated in the description, the alkyl group is optionally substituted.

Alkenyl refers to a radical with a straight or branched unsaturated hydrocarbon chain consisting only of carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds, containing from two to twelve carbon atoms (C2-C12 alkenyl), preferably from two to eight carbon atoms ( _C2 - _C8 alkenyl) or from two to six carbon atoms ( _C2 - _C6 alkenyl) and which is attached to the rest of the molecule by a single bond, for example, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, etc. Unless otherwise specifically stated in the description, the alkenyl group is optionally substituted.

Alkynyl refers to a radical with a straight or branched unsaturated hydrocarbon chain consisting only of carbon and hydrogen atoms, which contains one or more carbon-carbon triple bonds, containing from two to twelve carbon atoms (C2-C12 alkynyl), preferably from two to eight carbon atoms (C2-C8 alkynyl) or from two to six carbon atoms

- 8 049703 of the type (C2-C6 alkynyl) and which is attached to the remainder of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless otherwise specifically stated in the description, the alkynyl group is optionally substituted.

Alkoxy refers to a radical of the formula -ORa, where Ra is an alkyl radical as defined above containing from one to twelve carbon atoms. Preferred alkoxy groups contain from one to six carbon atoms (i.e., _C1 - _C6 alkoxy) or from one to three carbon atoms (i.e., C1-C3 alkoxy) in the alkyl radical. Unless otherwise specifically indicated in the description, the alkoxy group is optionally substituted.

An aromatic ring refers to a cyclic planar molecular moiety (i.e., a radical) with a ring of resonant bonds that exhibit increased stability compared to other bonding arrangements with the same sets of atoms. Typically, aromatic rings comprise a set of covalently bonded coplanar atoms and contain a number of π-electrons (e.g., alternating double and single bonds) that is even but not a multiple of 4 (i.e., 4n+2 π-electrons, where n=0, 1, 2, 3, etc.). Aromatic rings include, but are not limited to, phenyl, naphthenyl, imidazolyl, pyrrolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridonyl, pyridazinyl, pyrimidonyl. Unless otherwise specifically stated in the description, an aromatic ring includes all radicals that are optionally substituted.

Aryl refers to a radical of a carbocyclic ring system containing from 6 to 18 carbon atoms and at least one aromatic ring (i.e., _C6 - _C18aryl ), preferably having from 6 to 10 carbon atoms (i.e., _C6 - _C10aryl ). For the purposes of embodiments of the present invention, the aryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused ring systems or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrilene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, asi-indacene, sym-indacene, indane, indene, naphthalene, phenalene, phenanthrene, phenyl, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated in the description, an aryl group is optionally substituted.

Arylalkyl refers to a radical of the formula -Rb-Rc, wherein Rb is an alkylene chain and _Rc is one or more aryl radicals as defined above, for example, benzyl, diphenylmethyl, etc. An arylalkyl group may comprise a C1-C10 alkylene chain attached to a _C6 - _C10 aryl radical (i.e., _C6 - _C10 arylalkyl). Unless otherwise specifically stated in the specification, an arylalkyl group is optionally substituted.

Cycloalkyl refers to a stable non-aromatic monocyclic or polycyclic carbocyclic radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems containing from three to fifteen carbon atoms (i.e., C3-C15 cycloalkyl), preferably containing from three to ten carbon atoms (i.e., C3-C10 cycloalkyl) or from three to six carbon atoms (i.e., C3-C6 cycloalkyl), and which is saturated or unsaturated and is attached to the remainder of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Cycloalkyl also includes spirocycloalkyl if two substitution positions are on the same carbon atom. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, etc. Unless otherwise specifically stated in the description, the cycloalkyl group is optionally substituted.

Cycloalkylalkyl refers to a radical of the formula -R _b -R _c , wherein R _b is an alkylene chain and R c is one or more cycloalkyl radicals as defined above, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, etc. A cycloalkylalkyl group may comprise a C1-C0 alkylene chain attached to a _C3 - _C12 cycloalkyl radical (i.e., _C3 - _C12 cycloalkylalkyl), or a C1-C10 alkylene chain attached to a _C3 - _C6 cycloalkyl radical (i.e., _C3 - _C6 cycloalkylalkyl). Unless otherwise specifically indicated in the specification, a cycloalkylalkyl group is optionally substituted.

Fused refers to any ring structure described herein that is fused to an existing ring structure in the compounds of the present invention. If the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure that will become part of the fused heterocyclyl ring or fused heteroaryl ring is replaced with a nitrogen atom.

Halo or halogen refers to bromine, chlorine, fluorine or iodine.

Haloalkyl refers to an alkyl radical as defined above that is substituted by one or more halogen radicals as defined above, such as trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc. Preferably

- 9 049703 A haloalkyl group comprises an alkyl group containing from one to six carbon atoms and substituted by one or more halogen radicals (i.e. C1- _C6 haloalkyl). The halogen radicals may be the same or the halogen radicals may be different. Unless otherwise specifically stated in the description, a haloalkyl group is optionally substituted.

Haloalkoxy refers to a radical of the formula -OR,,. where Ra is a haloalkyl radical as defined above containing from one to twelve carbon atoms. A preferred haloalkoxy group includes an alkoxy group containing from one to six carbon atoms (i.e., C1- _C6 haloalkoxy) or containing from one to three carbon atoms ( _C1 - _C3 haloalkoxy) and substituted by one or more halogen radicals. All halogen radicals may be the same or all halogen radicals may be different. Unless otherwise specifically stated in the description, a haloalkoxy group is optionally substituted.

Heteroaryl refers to an aromatic group (e.g., a 5- to 14-membered ring system) comprising a single ring, multiple rings, or multiple fused rings, with one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl comprises 1 to 10 ring carbon atoms and 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur in the ring. Preferred heteroaryl groups comprise a 5- to 10-membered ring system comprising one to four heteroatoms selected from nitrogen, oxygen, and sulfur (i.e., a 5- to 10-membered heteroaryl), and a 5- to 6-membered ring system comprising one to four heteroatoms selected from nitrogen, oxygen, and sulfur (i.e., a 5- to 6-membered heteroaryl). For purposes of embodiments of the present invention, a heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused ring systems or bridged ring systems. Examples of heteroaryl groups include pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). A heteroaryl may contain one or more N-oxide (N-O-) moieties, such as pyridine-Noxide. Unless otherwise specifically stated in the specification, a heteroaryl group is optionally substituted.

Heteroarylalkyl refers to a radical of the formula -Rb-Rc, wherein Rb is an alkylene chain and _Rc is one or more heteroaryl radicals as defined above. A heteroarylalkyl group may comprise a C1-C10 alkylene chain attached to a 5-10 membered heteroaryl group (i.e., a 5-10 membered heteroarylalkyl), or a C1-C= alkylene chain attached to a 5-6 membered heteroaryl group (i.e., a 5-6 membered heteroarylalkyl). Unless otherwise specifically indicated in the specification, a heteroarylalkyl group is optionally substituted.

Heterocyclyl refers to a saturated or unsaturated cyclic alkyl group with one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. The term heterocyclyl includes heterocycloalkenyl groups (i.e., a heterocyclyl group containing at least one double bond), bridged heterocyclyl groups, fused heterocyclyl groups, and spiroheterocyclyl groups. A heterocyclyl may be a single ring or multiple rings, wherein multiple rings may be fused, bridged, or spiro, and may contain one or more oxo (C=O) or N-oxide (N-O-) moieties. Any non-aromatic ring containing at least one heteroatom is considered a heterocyclyl, regardless of the mode of attachment (i.e., may be bonded through a carbon atom or a heteroatom). In addition, the term heterocyclyl is intended to encompass any non-aromatic ring containing at least one heteroatom that may be fused to an aryl or heteroaryl ring, regardless of attachment to the remainder of the molecule. As used herein, heterocyclyl contains from 1 to 10 ring carbon atoms, from 1 to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms and from 1 to 5 ring heteroatoms, from 1 to 4 heteroatoms, from 1 to 3 heteroatoms, or from 1 to 2 heteroatoms independently selected from nitrogen, sulfur, and oxygen. Preferred heterocyclyls contain from five to 10 members in the ring system, including from one to four heteroatoms selected from nitrogen and oxygen (i.e., a 5- to 10-membered heterocyclyl) or from five to eight members in the ring system, including from one to four heteroatoms selected from nitrogen and oxygen (i.e., a 5- to 8-membered heterocyclyl). Examples of heterocyclyl groups include dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl. Unless otherwise specifically stated in the description, a heterocyclyl group is optionally substituted.

Heterocyclylalkyl refers to a radical of the formula -Rb-Rc, where Rb is an alkylene chain and R _c is one or more heterocyclyl radicals as defined above. A heterocyclylalkyl group may contain a C ₁ -C ₁₀ alkylene chain attached to a 5- to 10-membered

- 10 049703 heterocyclyl radical (i.e. 5-10-membered heterocyclylalkyl), or a Ci-Cu alkylene chain attached to a 5-8-membered heterocyclyl radical (i.e. 5-8-membered heterocyclylalkyl). Unless otherwise specifically stated in the description, the heterocyclylalkyl group is optionally substituted.

In some embodiments, the term substituted, as used herein, means any of the above groups or other substituents (e.g., C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C 3 -C 12 cycloalkyl, C 3 -C 12 cycloalkylalkyl, aryl, and heteroaryl) wherein at least one hydrogen atom (e.g., 1, 2, 3, or all of the hydrogen atoms) is replaced by a bond to a non-hydrogen atom, such as, but not limited to: a halogen atom such as F, Cl, Br, and I (i.e., halogen); an oxygen atom in groups such as hydroxyl groups or alkoxy groups (e.g., alkoxy or haloalkoxy); a nitrogen atom in groups such as amines (e.g., -NH2), amides (e.g., -(C=O)NH2), and nitro; alkyl groups including one or more halogen atoms, such as F, Cl, Br and I (e.g. haloalkyl); and cyano.

It is intended that each selection for L, ^R1a , ^R1b , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , and ^R7 is optionally substituted as described above, unless specifically stated otherwise, and provided that all valencies are satisfied by the substitution. In particular, each selection for L, ^R1a , ^R1b , R2, ^R3 , ^R4 , ^R5 , ^R6 , and ^R7 is optionally substituted, unless specifically stated otherwise, and provided that such substitution results in a stable molecule ⁽ for example, groups such as H and halogen are optionally substituted).

An effective amount or a therapeutically effective amount of a compound or composition refers to that amount of the compound or composition that results in the desired result in accordance with the disclosure herein. Effective amounts can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, by determining the ED50 (the dose therapeutically effective for 50% of the population) and LD50 (the dose lethal to 50% of the population). In some embodiments, an effective amount of the compound results in a reduction or inhibition of symptoms or prolongation of survival in a subject (i.e., a human patient). The results may require multiple doses of the compound.

Treating or treating a disease in a subject refers to: 1) preventing the occurrence of a disease in a patient who is predisposed to or does not yet exhibit symptoms of the disease; 2) inhibiting the disease or stopping its progression; or 3) alleviating or causing regression of the disease. As used herein, treating or treating is an approach to obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired results include, but are not limited to, one or more of the following: reducing one or more symptoms caused by a disease or disorder, reducing the severity of a disease or disorder, stabilizing a disease or disorder (e.g., preventing or delaying worsening of a disease or disorder), delaying the onset or recurrence of a disease or disorder, delaying or slowing the progression of a disease or disorder, ameliorating a condition of a disease or disorder, bringing about a remission (partial or complete) of a disease or disorder, reducing the dose of one or more other drugs needed to treat a disease or disorder, enhancing the effect of another drug used to treat a disease or disorder, delaying the progression of a disease or disorder, improving the quality of life and/or prolonging the survival of a subject. Also, treatment includes reducing the pathological consequences of a disease or disorder. The methods of the present invention include any one or more of these aspects of treatment.

As used herein, the terms individual(s), subject(s), and patient(s) mean any mammal. Examples include, but are not limited to, mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, sheep, cows, and humans. In some embodiments, the mammal is a human.

A therapeutic effect, as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit. A therapeutic effect includes delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping, or reversing the progression of a disease or condition; causing partial or complete regression of a disease or condition, or any combination thereof.

As used herein, the terms co-administration, administered in combination with, and their grammatical equivalents in the context of the present description encompass the administration of two or more agents to an animal, including humans, such that both agents and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in compositions in which both agents are present.

Pharmaceutically acceptable refers to compounds, salts, compositions, medicinal

- 11 049703 forms and other materials that can be used in the preparation of a pharmaceutical composition suitable for pharmaceutical use in an animal or a human.

A pharmaceutically acceptable salt includes both acid addition salts and base addition salts.

A pharmaceutically acceptable acid addition salt refers to those salts that retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, Ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinous acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

A pharmaceutically acceptable base addition salt refers to salts that retain the biological effectiveness and properties of the free acids that are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic base or an organic base to the free acid. Salts prepared from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

In some embodiments, pharmaceutically acceptable salts include quaternary ammonium salts, such as alkyl halide quaternary amine salts (e.g., methyl bromide).

As used herein, a therapeutic agent refers to a biological, pharmaceutical, or chemical compound or other moiety. Non-limiting examples include a simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, an antibody fragment, a vitamin derivative, a carbohydrate, a toxin, or a chemotherapeutic compound. Various compounds can be synthesized, such as small molecules and oligomers (e.g., oligopeptides and oligonucleotides), as well as synthetic organic compounds based on various core structures. In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, etc.

The term in vivo refers to an event that occurs within the body of a subject.

It is also intended that embodiments of the present invention encompass all pharmaceutically acceptable compounds of formula (I) that are isotopically labeled by replacing one or more atoms with an atom of different atomic mass or mass number (i.e., an isotopic form of a compound of formula (I)). Examples of isotopes that may be included in compounds of formula (I) include isotopes of hydrogen, carbon, nitrogen, oxygen, ^phosphorus , fluorine, chlorine, and iodine, such as ^2H , ^3H , ^12C , ^13C , ^14C , ^13N , ^15N , 15O, ^17O , ^18O , ^31P , ^32P , ^35S , ^18F , ^36Cl , ^123I , and ^125I , respectively. These radiolabeled compounds may be useful in determining or measuring the potency of compounds by characterizing, for example, the site or mode of action or the binding affinity for a pharmacologically important site of action. Certain isotopically labeled compounds of formula (I), for example those containing a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. In particular, due to the ease of incorporation and the presence of

- 12 049703 of the ready-made detection means, radioactive isotopes such as tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C, are applicable for these purposes.

Substitution with heavier isotopes such as deuterium, i.e. ² H, may provide certain therapeutic advantages due to greater metabolic stability, such as increased half-life in vivo or the need for lower dosages, and is therefore preferable in some circumstances.

Substitution with positron emitting isotopes such as 11C, ^18F , ^15O and ^13N may be useful in positron emission tomography (PET) studies to study substrate receptor occupancy. Isotopically labeled compounds of formula (I) may generally be prepared by conventional methods known to those skilled in the art or by methods analogous to those described in the examples section as set forth below, using suitable isotopically labeled reagents in place of the non-isotopically labeled reagents previously used.

It is also intended that certain embodiments encompass in vivo metabolic products of the disclosed compounds. Such products may be formed by, for example, oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, especially by an enzymatic process. Accordingly, embodiments include compounds prepared by a method comprising administering a compound of the present invention to a mammal for a period of time sufficient to produce a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound of the present invention in a detectable dose to an animal such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism, and isolating its metabolites from urine, blood, or other biological samples.

A stable compound or stable structure is meant to mean a compound that is sufficiently robust to withstand isolation in sufficient purity from a reaction mixture and formulation into an effective therapeutic agent.

Often, a compound of the present invention is obtained by crystallization. As used herein, the term solvate refers to an aggregate that contains one or more molecules of a compound of formula (I) with one or more molecules of a solvent. In some embodiments, the solvent is water, in which case the solvate is a hydrate. Alternatively, in other embodiments, the solvent is an organic solvent. Thus, compounds of formula (I) can exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the like, as well as the corresponding solvated forms. In some aspects, a compound of formula (I) is a true solvate, while in other cases, a compound of the present invention only retains additional water or is a mixture of water plus some additional solvent.

The term optional or optional means that the event or circumstance described thereafter may or may not occur, and that the description includes instances where the stated event or circumstance occurs and instances where it does not. For example, optionally substituted aryl means that the aryl radical may or may not be substituted, and that the description includes both substituted aryl radicals and unsubstituted aryl radicals. Polymers or similar undefined structures prepared by defining substituents and then adding substituents indefinitely (e.g., a substituted aryl having a substituted alkyl that is itself substituted with a substituted aryl group that is further substituted with a substituted heteroalkyl group, etc.) are not intended to be included herein. Likewise, the above definitions are not intended to include invalid substitution patterns (e.g., methyl substituted with 5 fluorine atoms, or heteroaryl groups having two adjacent ring oxygen atoms). Such unacceptable substitution schemes are well known to a qualified specialist.

A pharmaceutical composition or pharmaceutically acceptable composition refers to a composition of a compound of the present invention and a vehicle generally acceptable in the art for delivering the biologically active compound to mammals, such as humans. Such a vehicle includes pharmaceutically acceptable carriers, diluents, or excipients.

The term pharmaceutically acceptable carrier, diluent or excipient includes, without limitation, any adjuvant, vehicle, excipient, glidant, sweetener, extender, preservative, colorant/pigment, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solubilizer or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or domesticated animals. The compounds of formula (I) or their pharmaceutically acceptable salts or isotopic forms may contain one or more centers that result in geometric asymmetry and thus may give rise to enantiomers, diastereomers and other stereoisomers.

- 13 049 703 isomeric forms, defined in terms of absolute stereochemistry as (R)- or (S)-, or as (D)- or (L)- for amino acids. Embodiments thus include all such possible isomers as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or separated using conventional techniques, such as chromatography and fractional crystallization. Conventional methods for preparing/isolating individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemic salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Where compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise indicated, such compounds are intended to include both the E- and Z-geometric isomers. All tautomeric forms are also intended to be included.

A stereoisomer refers to a compound consisting of the same atoms linked by the same bonds but having different three-dimensional structures that are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and also includes enantiomers, which refer to two stereoisomers whose molecules are insuperable mirror images of each other.

Diastereoisomers are stereoisomers that have at least two asymmetric atoms but which are not mirror images of each other.

A tautomer refers to a proton shift from one atom of a molecule to another atom of the same molecule. Thus, embodiments include tautomers of the disclosed compounds.

The chemical naming protocol and structural diagrams used here are a modified form of the IUPAC nomenclature system using ACD/Name software version 9.07 and/or the naming program ChemDraw Ultra version 11.0.1 (CambridgeSoft). For the compound chemical names used here, the substituent group is generally named before the group to which it is attached. For example, cyclopropylethyl contains an ethyl skeleton with a cyclopropyl substituent. Except as noted below, all bonds are identified in the chemical structure diagrams shown here, except for all bonds on some carbon atoms that are assumed to be bonded to enough hydrogen atoms to complete the valence.

Although various features of the present invention may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although for clarity the present invention may be described herein in the context of separate embodiments, the present invention may also be implemented in a single embodiment.

Connections

In one aspect, this document provides a compound of formula (I) ^R 4 (R ⁵ ) _rt

(I) or a pharmaceutically acceptable salt thereof, wherein

L represents a direct bond, -C(=O)-, -(CR ^a R ^b )mC(=O)-, -C(=O)-(CR ^a R ^b )m-, or -(CR ^a R ^b ) _m- ;

each R ^a and R ^b independently represents H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl;

R ^1a and R ^1b independently represent H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, or C6-C ₁₀ arylalkyl;

R ^(I)2 is H, oxo or thioxo;

R ³ is C2-C6 alkyl, C ₃ -C6 alkenyl, C ₃ -C6 alkynyl, C3-C12 cycloalkyl, C ₃ -C6 cycloalkylalkyl, C6-C10 arylalkyl, 5-10-membered heteroarylalkyl or 5-10-membered heterocyclylalkyl, wherein the 5-10-membered heteroarylalkyl or 5-10-membered heterocyclylalkyl contains 1-3 heteroatoms selected from nitrogen and oxygen;

R ⁴ is C6-C10 aryl, 5-10-membered heteroaryl or 5-10-membered heterocyclyl, wherein the 5-10-membered heteroaryl or 5-10-membered heterocyclyl contains 1-3 heteroatoms selected from nitrogen and oxygen;

each R ⁵ is independently C1-C6 alkyl, oxo or halogen;

R ⁶ is H, C1-C6 alkyl or oxo;

R ⁷ is H or oxo;

m represents 1 or 2 and n represents an integer from 0 to 3;

- 14 049703 wherein each C ₁ -C ₆ alkyl, C2 -C ₆ alkenyl, C2 -C ₆ alkynyl, C3 -C 12 cycloalkyl, C3 -C 12 cycloalkylalkyl, C ₆ -C ₁₀ aryl, C 6 -C ₁₀ arylalkyl, 5-10-membered heteroaryl, 5-10-membered heteroarylalkyl, 5-10-membered heterocyclyl and 5-10-membered heterocyclylalkyl is optionally substituted with one to five substituents selected from hydroxyl, halogen, amino, C1 -C 6 haloalkyl, C1 -C 6 alkoxy, C1 -C 6 haloalkoxy, cyano, -(C=O)NH2, nitro, -SO2(C ₁ -C ₆ alkyl) and -CO2H.

In some embodiments, L is a direct link. In some embodiments, L is -C(=O)- or -(CR ^a R ^b )m-. In some embodiments, L is -C(=O)-. In some embodiments, L is -(CR ^a R ^b )m-. In some embodiments, L is -(CR ^a R ^b )mC(=O)- or -C(=O)-(CR ^a R ^b )m-. In some embodiments, L is -(CR ^a R ^b ) _m -C(=O)-. In some embodiments, L is -C(=O)-(CR ^a R ^b )m-.

In some embodiments, R ^a and R ^b are each independently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl. In some embodiments, R ^a and R ^b are each independently H, C1-C3 alkyl, C2-C4 alkenyl, or C2- _C4 alkynyl. In some embodiments, R ^a and R ^b are each H. In some embodiments, R ^a is H. In some embodiments, R ^a is C1-C6 alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ^a is C2-C6 alkenyl, such as vinyl or propenyl. In some embodiments, R ^a is C ₂ -C ₆ alkynyl, such as ethynyl or propynyl. In some embodiments, R ^b is H. In some embodiments, R ^b is C1-C6 alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ^b is C2-C6 alkenyl, such as vinyl or propenyl. In some embodiments, R ^b is C2-C6 alkynyl, such as ethynyl or propynyl.

In some embodiments, R ^1a and R ^1b are independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, or C6-C10 arylalkyl. In some embodiments, R ^1a is H. In some embodiments, R ^1a is _C1C6 alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ^1a is C2-C6 alkenyl, such as vinyl or propenyl. In some embodiments, R ^1a is C2- _C6 alkynyl, such as ethynyl or propynyl. In some embodiments, R ^1a is C1-C6 alkoxy, such as methoxy, ethoxy, or propoxy. In some embodiments, R ^1a is halogen, such as fluoro, chloro, or bromine. In some embodiments, R ^1a is C6- _C10 arylalkyl, such as benzyl. In some embodiments, R ^1b is H. In some embodiments, R ^1b is C1C6 alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ^1b is C2-C6 alkenyl, such as vinyl or propenyl. In some embodiments, R ^1b is C2-C6 alkynyl, such as ethynyl or propynyl. In some embodiments, R ^1b is C1-C6 alkoxy, such as methoxy, ethoxy, or propoxy. In some embodiments, R ^1b is halogen, such as fluoro, chloro, or bromine. In some embodiments, R ^1b is C6- _C10 arylalkyl, such as benzyl.

In some embodiments, each R ^1a and R ^1b is independently H; C1-C6 alkyl optionally substituted with 1-3 substituents selected from halogen, -CO2H, and -C(=O)NH2; C1-C6 alkoxy; halogen; or C6-C10 arylalkyl optionally substituted with 1-3 substituents selected from halogen and amino. In some embodiments, R ^1a is C1- _C6 alkyl substituted with 1-3 halogens, such as fluoro or chloro. In some embodiments, R ^1a is C1-C6 alkyl substituted with 1-3 -CO2H groups. In some embodiments, R ^1a is C1- _C3 alkyl substituted with 1-2 CO2H groups, such as -CH2CO2H or -CH2CH2CO2H. In some embodiments, R ^1a is C1-C6 alkyl substituted with 1-3 -C(=O)NH2 groups. In some embodiments, R ^1a is C1- _C3 alkyl substituted with 1-2 -C(=O) _NH2 groups, such as -CH ₂ C(=O)NH ₂ or -CH ₂ CH ₂ C(=O)NH ₂ . In some embodiments, R ^1a is C6-C10 arylalkyl substituted with 1-3 substituents selected from halogen and amino. In some embodiments, R ^1a is C6- _C10 arylalkyl substituted with 1-3 halogens, such as fluoro, chloro, or bromine. In some embodiments, R ^1a is C6-C10 arylalkyl substituted with 1-3 amino. In some embodiments, R ^1b is C1- _C6 alkyl substituted with 1-3 halogens, such as fluorine or chlorine. In some embodiments, R ^1b is C1-C6 alkyl substituted with 1-3 -CO2H groups. In some embodiments, R ^1b is a C1-C ₃ alkyl substituted with 1-2 CO ₂ H groups, such as -CH2CO2H or -CH ₂ CH ₂ CO ₂ H. In some embodiments, R ^1b is a C1-C6 alkyl substituted with 1-3 -C(=O)NH2 groups. In some embodiments, R ^1b is a C1-C ₃ alkyl substituted with 1-2 -C(=O)NH ₂ groups, such as -CH ₂ C(=O)NH ₂ or -CH2CH2C(=O)NH2. In some embodiments, R ^1b is a C6-C10 arylalkyl substituted with 1-3 substituents selected from halogen and amino. In some embodiments, R ^1b is a _C6 - _C10 arylalkyl substituted with 1-3 halogens such as fluoro, chloro, or

- 15 049703 bromo. In some embodiments, R ^1b is C6-C10 arylalkyl substituted with 1-3 amino. In some embodiments, each R ^1a and R ^1b is independently H, methyl, fluoro, 2-methylbutyl, -CH2F, methoxy, -CH2CO2H, -CH2C(=O)NH2, benzyl, or 4-aminobenzyl. In some embodiments, each R ^1a and R ^1b is independently H or C1-C3 alkyl. In some embodiments, R ^1a is methyl and R ^1b is H. In some embodiments, each R ^1a and R ^1b is H. In some embodiments, one of R ^1a and R ^1b is H and the other is _C1 - _C3 alkyl, such as methyl.

In some embodiments, R ² is H, oxo, or thioxo. In some embodiments, R ² is H. In some embodiments, R ² is oxo. In some embodiments, R ² is thioxo.

In some embodiments, R ³ is C3-C6 alkyl, C3-C6 alkenyl, C3-C6 alkynyl, C3-C12 cycloalkyl, C3-C6 cycloalkylalkyl, C6-C10 arylalkyl, 5-10 membered heteroarylalkyl, or 5-10 membered heterocyclylalkyl, wherein the 5-10 membered heteroarylalkyl or 5-10 membered heterocyclylalkyl contains 1-3 heteroatoms selected from nitrogen and oxygen. In some embodiments, R ³ is C3-C6 alkyl such as propyl, butyl, pentyl, or hexyl. In some embodiments, R ³ is C4-C6 alkyl. In some embodiments, R ³ is C3-C6 alkenyl. In some embodiments, R ³ is C4-C6 alkenyl. In some embodiments, R ³ is C3-C6 alkynyl. In some embodiments, R ³ is C4-C6 alkynyl. In some embodiments, R ³ is C3-C12 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R ³ is C3-C6 cycloalkyl. In some embodiments, R ³ is C3-C6 cycloalkylalkyl, such as -(CH ₂ ) _1-3 (C ₃ -C6 cycloalkyl). In some embodiments, R ³ is C6-C10 arylalkyl, such as benzyl. In some embodiments, R ³ is 5-10 membered heteroarylalkyl, such as -(CH2) ₁₃ (5-10 membered heteroaryl) or -(CH ₂ ) _1-3 (5-6 membered heteroaryl). In some embodiments, the 5-10 membered heteroarylalkyl has 1-2 nitrogen atoms. In some embodiments, R ³ is a 5-10 membered heterocyclylalkyl, such as -(CH2) _1-3 (5-10 membered heterocyclyl) or -(CH ₂ ) _1-2 (5-6 membered heterocyclyl). In some embodiments, the 5-10 membered heterocyclylalkyl contains 1-2 nitrogen atoms.

In some embodiments, R ³ is C3-C6 alkyl optionally substituted with 1-3 substituents selected from halogen and -C(=O)NH2, C2-C6 alkenyl, or C3-C6 cycloalkylalkyl. In some embodiments, R ³ is C2- _C6 alkyl optionally substituted with 1-3 substituents selected from halogen, C1- _C3 alkoxy, hydroxy, -NH2, _-SO2 (C1- _C3 alkyl), and C(=O)NH2; C2-C6 alkenyl; _C3 - _C6 cycloalkylalkyl; 5-6-membered heteroarylalkyl; 5-6-membered heterocyclylalkyl; or _C6 arylalkyl. In some embodiments, R ³ is C ₂ alkyl substituted with 1-3 substituents selected from C 1-C 3 alkoxy, hydroxy, -NH 2 , and -SO ₂ (C 1-C ₃ alkyl). In some embodiments, R ³ is

-'γ'· I \γ I 1 , xV- , ϊ , , XV XX.. T. 0 b Gt In some embodiments, R ³ is G)

- 16 049703

In some embodiments, R ³ is 2-methylbutyl.

In some embodiments, R ⁴ is C6-C10 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, wherein the 5-10 membered heteroaryl or 5-10 membered heterocyclyl contains 1-3 heteroatoms selected from nitrogen and oxygen. In some embodiments, R ⁴ is C6- _C10 aryl, such as phenyl. In some embodiments, R ⁴ is 5-10 membered heteroaryl containing 1-2 nitrogen atoms. In some embodiments, R ⁴ is 5-10 membered heterocyclyl. In some embodiments, R ⁴ is 5-9 membered heterocyclyl containing 1-2 nitrogen atoms. In some embodiments, R ⁴ is 5-9 membered heterocyclyl containing 1-2 oxygen atoms. In some embodiments, R ⁴ is a 5-9 membered heterocyclyl containing 1 nitrogen atom and 1 oxygen atom.

In some embodiments, R ⁴ is C6-C10 aryl optionally substituted with 1-3 substituents selected from halogen, hydroxyl, C1-C6 haloalkyl, and C1-C6 haloalkoxy. In some embodiments, R ⁴ is phenyl substituted with 1-3 substituents selected from -CF3, -OCHF2, -OH, fluoro, and chloro. In some embodiments, R ⁴ is

In some embodiments, R ⁴ is

In some embodiments, R ⁴ is a 5-10 membered heteroaryl optionally substituted with 1-3 substituents selected from halogen, hydroxyl, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy. In some embodiments, R ⁴ is pyridyl or indolyl optionally substituted with 1-3 substituents selected from halogen, hydroxyl, C 1-C 6 haloalkyl, and C 1-C 6 haloalkoxy. In some embodiments, R ⁴ is

In some embodiments, R ⁴ is pyridyl substituted with 1-3 substituents selected from halogen, hydroxyl, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy. In some embodiments, R ⁴ is

BUT_

In some embodiments, R ⁴ is 5-10 membered heterocyclyl optionally substituted with 1-3 substituents selected from halogen, hydroxyl, C ₁ -C ₆ haloalkyl, and C 1 C 6 haloalkoxy. In some embodiments, R ⁴ is indolinyl.

In some embodiments, -LR ⁴ is -CH2(phenyl) or -C(O)(phenyl), wherein phenyl is substituted with 1-3 substituents selected from C1-C3 haloalkyl, C1-C3 haloalkoxy, halogen, and hydroxy. In some embodiments, -LR ⁴ is -CH2(pyridyl) or -C(O)(pyridyl), wherein pyridyl is substituted with 1-3 substituents selected from C1- _C3 haloalkyl, _C1 - _C3 haloalkoxy, halogen, and hydroxy. In some embodiments, -LR ⁴ is

- 17 049703

In some embodiments, each R ⁵ is independently C1-C6 alkyl, oxo, or halogen. In some embodiments, R ⁵ is Ο ₁ -Ο ₆ alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ⁵ is oxo. In some embodiments, R ⁵ is halogen, such as fluoro, chloro, or bromine. In some embodiments, R ⁵ is oxo or halogen. In some embodiments, R ⁵ is oxo or fluoro.

In some embodiments, R ⁶ is H, C1-C6 alkyl or oxo. In some embodiments, R ⁶ is H. In some embodiments, R ⁶ is C1-C ₆ alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ⁶ is oxo.

In some embodiments, R ⁷ is H or oxo. In some embodiments, R ⁷ is H. In some embodiments, R ⁷ is oxo.

In some embodiments, m is 1. In other embodiments, m is 2.

In some embodiments, n is 0. In other embodiments, n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

In any embodiments of formula (I) or variations thereof, each C ₁ -C ₆ alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C ₃ -C ₁₂ cycloalkyl, C ₃ -C ₁₂ cycloalkylalkyl, C ₆ -C ₁₀ aryl, C ₆ -C ₁₀ arylalkyl, 5-10 membered heteroaryl, 5-10 membered heteroarylalkyl, 5-10 membered heterocyclyl, and 5-10 membered heterocyclylalkyl is optionally substituted with one to three substituents selected from hydroxyl, halogen (such as fluorine, chloro, or bromo), amino, C ₁ -C ₆ haloalkyl (such as -CF ₃ or -CHF 2), C 1 -C 6 alkoxy (such as methoxy or ethoxy), C 1 -C 6 haloalkoxy (such as -OCHF 2 or -OCF ₃ ) and -(C=O)NH ₂ .

In some embodiments, the compound of formula (I) is a compound of formula (II), (IIa), (IIb), (IIc), (IId), or (IIe)

(II) ilia) (lib) a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IIb) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IIc) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IId) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IIe) or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is a compound of formula (IIIa), (IIIb), (IIIc), or (IIId)

- 18 049703

or a pharmaceutically acceptable salt thereof, wherein R ^1a , R ^1b , R ³ , R ⁵ , R ⁶ and n are described for formula (I), and R is one or more optional substituents such as hydroxyl, halogen, amino, C1-C6 haloalkyl, C1-C6 haloalkoxy, as described for formula (I). In some embodiments, the compound is a compound of formula (IIIa) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IIIb) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IIIc) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IIId) or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is a compound of formula (IVa), (IVb), (IVc), or (IVd)

or a pharmaceutically acceptable salt thereof, wherein R ⁵ and n are as described for formula (I), and R is one or more optional substituents such as hydroxyl, halogen, amino, C ₁ -C ₆ haloalkyl, C 1 -C 6 haloalkoxy, as described for formula (I). In some embodiments, the compound is a compound of formula (IVa) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IVb) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IVc) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (IVd) or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is a compound of formula (V)

(V) or a pharmaceutically acceptable salt thereof, wherein L, R ^1a , R ^1b , R ³ and R ⁴ are as described for formula (I). In some embodiments, L is -C(=O)- or -CH2-; R ^1a and R ^1b are independently H or C1-C3 alkyl optionally substituted with -CO2H; R ³ is C4- _C5 al

- 19 049703 kyl, C4-C5 alkenyl or C1-C3 alkyl substituted with C3-C5 cycloalkyl; and R ⁴ is phenyl or pyridyl substituted with 1-3 substituents selected from -CF ₃ , -OCHF2, -OH, fluoro and chloro. In some embodiments, one of R ^1a and R ^1b is H and the other is C1-C3 alkyl, such as methyl.

In the descriptions herein, it is intended that each description, variation, embodiment, or aspect of a fragment may be combined with each description, variation, embodiment, or aspect of other fragments as if each combination of descriptions were specifically and individually recited. For example, each description, variation, embodiment, or aspect provided herein with respect to L of formula (I) may be combined with each description, variation, embodiment, or aspect of R ^1a , R ^1b , R ² , R ³ , R ⁴ , R5, R ⁶ , R ⁷ and n in the same way as if each combination were specifically and individually recited. It is also intended that all descriptions, variations, embodiments, or aspects of formula (I), where applicable, apply equally to the other formulas detailed herein and are equally described as if each and every description, variation, embodiment, or aspect were separately and individually recited for all of the formulas. For example, all descriptions, variations, embodiments, or aspects of formula (I), where applicable, apply equally to other formulas detailed herein, such as formulas (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIIa), (IIIb), (IIIc), (IIId), (IVa), (IVb), (IVc), (IVd), and (V), and are equally described as if each description, variation, embodiment, or aspect were separately and individually recited for all formulas.

In some embodiments, a compound selected from the compounds of Table 1, or a pharmaceutically acceptable salt thereof, is provided. Although certain compounds described herein, including those in Table 1, are provided as specific stereoisomers and/or in non-stereochemical form, it is intended that any and all stereochemical forms, including any enantiomeric or diastereomeric forms, and any tautomers or other forms of any of the compounds of the present invention, including those in Table 1, are described herein.

Table 1

- 20 049703

- 21 049703

- 22 049703

19 20 ΌγΟ 9?4 21 22 Su ζ ^Ν γ^ _Ν ' ^Λ χΖ\ τ'Ά 23 L^ ^{1 1} O ^4 ⁵ 24 4- J ¹ o LL^ 25 O _.: --S _:: E F Y4 0 ¹ 26 ^F G) Ύ ^Ν γ4 O ¹ 27 y\. _x O _h ,E °-уСЗ F _Γ ^Ν γ-Ν-γ% υΛ 0 ¹ 28 °^ХД ^f _g; a\, χχζο o ¹ 29 /—\ C) O=/ Z^ .: 30 OyU T Υγ4 0 ¹

- 23 049703

31 F ^F γ ^Ν γ^° о ¹ 32 O,_,-CJ ^F Aed o ¹ 33 F p < ⁿ Y^n-^^o ^x ΥγΑ 0 ¹ 34 ^p\^ ⁰ ^ ^F °^уХД ^F P ^N Y^n^ ^oh v-unit o ¹ 35 o, _i: _,CJ ^f ^Ny^ _N ^NH ₂ O ¹ 36 \ <о LL. ° < Р ед А° о ^λ — ^/ 37 ed ed 38 Z'W°v' ^F ed ed < ⁿ y^nA0 Aed o ¹ 39 □_ / s^~ / p ed 40 ed t ρ-γ-Ν-Α? Υγ^ο O ¹ 41 F ed ϊ ed 42 ^I'm going to eat ^honey O ¹

- 24 049703

- 25 049703

- 26 049703 or its pharmaceutically acceptable salt.

In some embodiments, the compound of Formula (I) is not compound 3a, 3b, 9, 10, 13, 15, 16, 18, 21, 23-29, 31-41, 43-48, 50, 52, or 54.

In some embodiments, a compound selected from the compounds of Table 1A, or a pharmaceutically acceptable salt thereof, is provided. Although certain compounds described herein, including those in Table 1A, are provided as specific stereoisomers and/or in non-stereochemical form, it is intended that any and all stereochemical forms, including any enantiomeric or diastereomeric forms, and any tautomers or other forms of any of the compounds of the present invention, including those in Table 1A, are described herein.

Table 1A

- 27 049703

or a pharmaceutically acceptable salt thereof.

It is understood that in the present description, combinations of substituents and/or variables of the depicted formulas are permissible only if such combinations result in stable compounds.

Moreover, all compounds of formula (I) existing in free base or acid form can be converted into their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of compounds of formula (I) can be converted into their free base or acid form by standard techniques.

Methods for synthesizing a compound of formula (I) or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof can be prepared using synthetic organic chemistry methods known in the art. In general, the starting materials can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, and the like, or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described herein.

- 28 049703

General reaction scheme 1.

General Reaction Scheme 1 provides an illustrative method for preparing compounds of formula (I). R ^1a , R ^1b , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , L and n in General Reaction Scheme 1 are defined herein. X is a reactive moiety selected to facilitate the desired reaction (e.g., halogen). P1 and P2 are suitable protecting groups. L' is selected such that the desired moiety L is formed by reaction between L'-R ⁴ and a secondary amine. Compounds of structure A1 were purchased or prepared according to methods known in the art. Reaction of A1 with A2 under appropriate coupling conditions (e.g., T3P and base) provides the reaction product of the coupling between A1 and A2, A3. A3 is then reacted with A4 under appropriate coupling conditions (e.g., _T3P and base) to provide compound A5. Compound A5 was then cyclized (e.g. using formic acid) and deprotected (e.g. using piperidine) to give compound A6. Compound A6 was then reacted with compound A7 to give the final compound of formula (I) as shown.

General reaction scheme 2.

An alternative method for synthesizing compounds of formula (I) is depicted in General Reaction Scheme 2. R ^1a , R ^1b , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , L and n in General Reaction Scheme 2 are defined herein. P ₂ is a suitable protecting group. Each X is a reactive moiety selected to facilitate the desired reaction (e.g., halogen). L' is selected such that the desired moiety L is formed by reaction between L'-R ⁴ and a secondary amine. Intermediate A5 was prepared with a removable protecting group P ³ (e.g., para-methoxybenzyl) as the R ³ group to provide intermediate A8. A8 was then cyclized (e.g., using formic acid) and deprotected (e.g., using piperidine) to provide compound A9. Compound A9 was then reacted with A7 to give compound A10. Compound A10 was then deprotected (e.g. with ceric ammonium nitrate) to give compound A11. Compound A11 was then reacted with A12 to give the final compound of formula (I).

- 29 049703

General reaction scheme 3.

A process related to the process shown in General Reaction Scheme 2 is shown in General Reaction Scheme 3. In this process, two amine nitrogen atoms of the bicyclic ring are deprotected to form compound A10, then reacted with A7 to form compound A11. Subsequent reaction with A12 produces the final compound of formula (I).

It should be noted that various alternative strategies for preparing compounds of formula (I) are available to those skilled in the art. For example, other compounds of formula (I) can be prepared by analogous methods using the appropriate starting material.

Those skilled in the art will also appreciate that in the processes for preparing the compounds described herein, functional groups on the intermediates may need to be protected with suitable protecting groups. Such functional groups may include hydroxy, amino, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino and amidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups are optionally added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one skilled in the art will appreciate, the protecting group may also be a polymeric resin such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

Pharmaceutical compositions and formulations

In a further aspect, provided herein are pharmaceutical compositions. The pharmaceutical composition comprises any one (or more) of the above compounds and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration. In other embodiments, the pharmaceutical composition is formulated for injection. In still other embodiments, the pharmaceutical compositions comprise a compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, and an additional therapeutic agent. Non-limiting examples of such therapeutic agents are described herein below.

Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, inhalation, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

In certain embodiments, a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, is administered locally rather than systemically, such as by injecting the compound directly into the organ, often in a depot or sustained release formulation. In particular embodiments, sustained release formulations are administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Further, in other embodiments, the drug is delivered in a targeted drug delivery system, such as a liposome coated with an organ-specific antibody. In such embodiments, the liposomes are targeted and selectively selected by the organ. In other embodiments, a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, is in the form of a quick release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. In other embodiments, a compound described herein is administered locally. the compounds of formula (I) or their pharmaceutically acceptable salts, isotopic forms or stereoisomers are effective over a wide range of dosages. For example, in the treatment of adults, dosages of 0.01 to 1000 mg, 0.5 to 100 mg, 1 to 50 mg per day and 5 to 40 mg per day are

- 30 049703 dosage measures that are used in some embodiments. An illustrative dosage is from 10 to 30 mg per day. The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject being treated, the body weight of the subject, and the preference and experience of the treating physician.

In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof is administered as a single dose. Typically, such administration is by injection, such as intravenous injection for rapid administration of the agent. However, other routes are used if necessary. A single dose of a compound of the present invention can also be used to treat an acute condition (e.g., traumatic brain injury).

In some embodiments, a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, is administered in multiple doses. In some embodiments, the dosage may be about once, twice, three times, four times, five times, six times, or more than six times per day. In other embodiments, the dosage is about once a month, once every two weeks, once a week, or once every other day. In another embodiment, a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, and another therapeutic agent are administered together from about once a day to about 6 times per day. In another embodiment, the administration of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, and the therapeutic agent is continued for at least about 7 days. In yet another embodiment, the administration is continued for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained for as long as needed.

The administration of the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof can be continued for as long as necessary. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof is administered for more than 1, 2, 3, 4, 5, 6, 7, 14 or 28 days. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof is administered for less than 28, 14, 7, 6, 5, 4, 3, 2 or 1 day. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof is administered on a long-term, chronic basis, such as for the treatment of chronic effects (e.g., dementia).

In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof is administered in doses. It is known in the art that due to inter-subject variability in the pharmacokinetics of compounds, individualization of the dosing regimen is necessary for optimal therapy. The dosage for a compound can be determined by routine experimentation in light of the present invention.

In some embodiments, the compounds of Formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, are formulated into pharmaceutical compositions. In particular embodiments, the pharmaceutical compositions are formulated by conventional methods using one or more physiologically acceptable carriers containing excipients and adjuvants that facilitate the processing of the active compounds into preparations that can be used pharmaceutically. The appropriate formulation depends on the selected route of administration. Any pharmaceutically acceptable techniques, carriers, and adjuvants are used as appropriate for formulating the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999).

Provided herein are pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof and a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). Also provided herein are methods of administering a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof and a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s).

In certain embodiments, the compounds are administered as pharmaceutical compositions in which the compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, are mixed with other therapeutic agents, as in combination therapy. All combinations of active ingredients described in the Methods section below and throughout this disclosure are encompassed herein. In specific embodiments, the pharmaceutical composition comprises one or more compounds of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof.

- 31 049703 residual salts, isotopic forms or stereoisomers.

A pharmaceutical composition as used herein refers to a mixture of a compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, with other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents, and/or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of the compound to the body. In some embodiments, in practicing the methods of treatment or use provided herein, therapeutically effective amounts of the compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof provided herein, are administered as a pharmaceutical composition to a mammal with a disease, disorder, or health condition being treated. In particular embodiments, the mammal is a human. In certain embodiments, therapeutically effective amounts vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compound being used, and other factors. The compounds described herein are used alone or in combination with one or more therapeutic agents as components of mixtures.

In one embodiment, one or more compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, are formulated in aqueous solutions. In particular embodiments, the aqueous solution is selected, by way of example only, from a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. In other embodiments, one or more compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, are formulated for transmucosal administration. In particular embodiments, transmucosal formulations include penetrants that correspond to the barrier to be penetrated (e.g., the blood-brain barrier). In other embodiments, when the compounds described herein are formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In particular embodiments, such solutions include physiologically compatible buffers and/or excipients.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof are formulated for oral administration. The compounds are formulated by combining the active compounds with, for example, pharmaceutically acceptable carriers or excipients. In various embodiments, the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof are formulated in oral dosage forms, which include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

In certain embodiments, pharmaceutical preparations for oral use are obtained by mixing one or more solid excipients with one or more compounds of formula (I) or their pharmaceutically acceptable salts, isotopic forms or stereoisomers, optionally grinding the resulting mixture and processing the mixture of granules after adding suitable auxiliaries, if necessary, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; or others, such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In specific embodiments, disintegrants are optionally added. Disintegrating agents include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or salts thereof such as sodium alginate.

In one embodiment, dosage forms such as dragees and tablets are provided with one or more suitable coatings. In particular embodiments, concentrated sugar solutions are used to coat the dosage form. The sugar solutions optionally contain additional components such as, by way of example only, acacia, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes and/or pigments are also optionally added to the coatings for identification purposes. In addition, dyes and/or pigments are optionally used to characterize different dose combinations of the active compounds.

In certain embodiments, therapeutically effective amounts of at least one of the compounds of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof are formulated into other oral dosage forms. Oral dosage forms include hard capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. In particular embodiments, the hard capsules contain the active ingredients in admixture with one or more excipients. On

- 32 049703 Excipients include, by way of example only, lactose, binders such as starch, and/or lubricants such as talc or magnesium stearate, and optionally stabilizers. In other embodiments, the soft capsules contain one or more active compounds that are dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more of fatty oils, liquid paraffin, or liquid polyethyleneglycol. In addition, stabilizers are optionally added.

In other embodiments, therapeutically effective amounts of at least one of the compounds of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, described herein, are formulated for buccal or sublingual administration. Formulations suitable for buccal or sublingual administration include, by way of example only, tablets, lozenges, or gels. In other embodiments, the compounds of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, are formulated for parenteral administration, including formulations suitable for bolus injection or continuous infusion. In particular embodiments, the injectable formulations are provided in unit dosage form (e.g., ampoules) or in multi-dose containers. Preservatives are optionally added to the injectable formulations. In other embodiments, the pharmaceutical compositions are formulated in a form suitable for parenteral administration as sterile suspensions, solutions or emulsions in oily or aqueous carrier media. Parenteral injection formulations optionally contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In particular embodiments, pharmaceutical formulations for parenteral administration comprise aqueous solutions of the active compounds in water-soluble form. In further embodiments, a suspension of the active compound or compounds (e.g., compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof) is prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carrier media for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. In certain specific embodiments, aqueous injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension contains suitable stabilizers or agents that increase the solubility of the compounds to ensure the preparation of highly concentrated solutions. Alternatively, in other embodiments, the active ingredient is in powder form for constitution with a suitable carrier medium, such as sterile pyrogen-free water, before use.

In other embodiments, the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof are administered topically. The compounds are formulated into various compositions for topical administration, such as solutions, suspensions, lotions, gels, pastes, drug sticks, balms, creams or ointments. Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity enhancers, buffers and preservatives.

In other embodiments, the compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, are formulated for transdermal administration. In particular embodiments, the transdermal formulations are administered using transdermal delivery devices and transdermal delivery patches, and they can be lipophilic emulsions or buffered aqueous solutions dissolved and/or dispersed in a polymer or adhesive. In various embodiments, such patches are designed for continuous, pulsatile, or on-demand delivery of pharmaceuticals. In further embodiments, transdermal delivery of the compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, is accomplished using iontophoretic patches and the like. In certain embodiments, transdermal patches provide controlled delivery of the compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof. In specific embodiments, the rate of absorption is slowed using a rate-controlling membrane or by retaining the compound within a polymer matrix or gel. In alternative embodiments, absorption enhancers are used to increase absorption. Absorption enhancers or carriers include absorbable pharmaceutically acceptable solvents that facilitate passage through the skin. For example, in one embodiment, transdermal devices can be made in the form of a bandage comprising a support element, a reservoir containing the compound, optionally with carriers, optionally a rate-controlling barrier for delivering the compound into the host's skin at a controlled and predetermined rate over an extended period of time, and means for attaching the device to the skin.

In other embodiments, the compounds of formula (I) or pharmaceutically acceptable

- 33 049703 salts, isotopic forms or stereoisomers are formulated for administration by inhalation. Various forms suitable for administration by inhalation include, but are not limited to, aerosols, mists or powders. Pharmaceutical compositions of any compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof are typically delivered in the form of an aerosol spray from pressurized packs or a nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In particular embodiments, the dosage unit of the pressurized aerosol is determined by the presence of a valve for delivering a metered amount. In certain embodiments, capsules and cartridges of, for example, by way of example only, gelatin for use in an inhaler or insufflator are formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

In other embodiments, the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof are formulated as rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jellies or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG and the like. In suppository compositions, a low melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter, is first melted.

In certain embodiments, the pharmaceutical compositions are formulated by any conventional method using one or more physiologically acceptable carriers containing excipients and auxiliaries that facilitate the processing of the active compounds into preparations that can be used pharmaceutically. The appropriate formulation depends on the chosen route of administration. Any pharmaceutically acceptable methods, carriers and excipients can be used as appropriate. Pharmaceutical compositions containing a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof are prepared in a conventional manner, such as, by way of example only, by conventional mixing, dissolving, granulating, coating, elutriating, emulsifying, encapsulating, entrapping or pressing techniques.

The pharmaceutical compositions include at least one pharmaceutically acceptable carrier, diluent or excipient and at least one compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof described herein as an active ingredient. The active ingredient is in a free acid or free base form or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs), as well as active metabolites of these compounds having the same type of activity. All tautomers of the compounds described herein are included within the scope of the compounds provided herein. In addition, the compounds of formula (I) or their pharmaceutically acceptable salts, isotopic forms or stereoisomers encompass unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. Solvated forms of the compounds provided herein are also considered to be disclosed herein. In addition, the pharmaceutical compositions optionally include other medicinal or pharmaceutical agents, carriers, adjuvants such as preservatives, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating osmotic pressure, buffers and/or other therapeutically valuable substances.

Methods for preparing compositions containing compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof involve formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid substance. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, sachets and suppositories. Liquid compositions include solutions in which the compound is dissolved, emulsions containing the compound, or a solution containing liposomes, micelles or nanoparticles containing a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof. Semi-solid compositions include, but are not limited to, gels, suspensions and creams. The form of the pharmaceutical compositions described herein includes liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use or in the form of emulsions. These compositions also optionally contain minor amounts of non-toxic auxiliaries such as wetting or emulsifying agents, pH buffering agents, etc.

In some embodiments, a pharmaceutical composition comprising at least one compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, illustratively takes the form of a liquid in which the agents are present in solution, in suspension, or both. Typically, if the composition is administered as a solution or

- 34 049703 suspensions, the first part of the agent is present in solution, and the second part of the agent is present in the form of particles in suspension in a liquid matrix. In some embodiments, the liquid composition includes a gel composition. In other embodiments, the liquid composition is aqueous.

In certain embodiments, suitable aqueous suspensions contain one or more polymers as suspending agents. Suitable polymers include water-soluble polymers such as cellulosic polymers, for example, hydroxypropyl methylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein contain a mucoadhesive polymer selected from, for example, carboxymethylcellulose, carbomer (an acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran.

Suitable pharmaceutical compositions also optionally include solubilizing agents that promote the solubility of the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof. The term solubilizing agent generally includes agents that result in the formation of a micellar solution or a true solution of the agent. Certain acceptable nonionic surfactants, for example polysorbate 80, are useful as solubilizing agents, as are ophthalmologically acceptable glycols, polyglycols, for example polyethyleneglycol 400, and glycol ethers.

Moreover, useful pharmaceutical compositions optionally include one or more pH adjusting or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount necessary to maintain the pH of the composition in an acceptable range.

In addition, useful compositions also optionally include one or more salts in an amount required to adjust the osmolality of the composition to an acceptable range. These salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

Other useful pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merphen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

Other suitable compositions include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene glycerides of fatty acids and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10, octoxynol 40.

Still other suitable compositions include one or more antioxidants to enhance chemical stability, if desired. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

In certain embodiments, the aqueous suspension compositions are packaged in single-dose containers that are not resealable. Alternatively, reusable, multi-dose containers are used, in which case they typically include a preservative in the compositions.

In alternative embodiments, other delivery systems for hydrophobic pharmaceutical compounds are used. Liposomes and emulsions are examples of delivery vehicles or carriers useful herein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also used. In additional embodiments, the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof are delivered using a sustained release system such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. In this case, various sustained release materials are used. In some embodiments, sustained release capsules release compounds for several weeks to more than 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional protein stabilization strategies are used.

In certain embodiments, the formulations described herein include one or more antioxidants, metal chelating agents, thiol-containing compounds, and/or other general stabilizing agents. Examples of such stabilizing agents include, but are not limited to: (a) about 0.5 to about 2% w/v glycerol, (b) about 0.1 to about 1% w/v methionine, (c) about

- 35 049703

0.1 to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01 to about 2% w/v ascorbic acid, (f) about 0.003 to about 0.02% w/v polysorbate 80, (g) 0.001 to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) a combination thereof.

In some embodiments, the concentration of the compound of Formula (I) provided in the pharmaceutical compositions of the present invention is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of the compound of Formula (I) provided in the pharmaceutical compositions of the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, present in the pharmaceutical compositions ranges from about 0.0001 to about 50%, from about 0.001 to about 40%, from about 0.01 to about 30%, from about 0.02 to about 29%, from about 0.03 to about 28%, from about 0.04 to about 27%, from about 0.05 to about 26%, from about 0.06 to about 25%, from about 0.07 to about 24%, from about 0.08 to about 23%, from about 0.09 to about 22%, from about 0.1 to about 21%, from about 0.2 to about 20%, from about 0.3 to about 19%, from about 0.4 to about 18%, about 0.5 to about 17%, about 0.6 to about 16%, about 0.7 to about 15%, about 0.8 to about 14%, about 0.9 to about 12%, or about 1 to about 10% w/w, w/v, or v/v.

In some embodiments, the concentration of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, present in the pharmaceutical compositions is in the range of from about 0.001 to about 10%, from about 0.01 to about 5%, from about 0.02 to about 4.5%, from about 0.03 to about 4%, from about 0.04 to about 3.5%, from about 0.05 to about 3%, from about 0.06 to about 2.5%, from about 0.07 to about 2%, from about 0.08 to about 1.5%, from about 0.09 to about 1%, or from about 0.1 to about 0.9% w/w, w/v, or v/v.

In some embodiments, the amount of a compound of Formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof present in the pharmaceutical compositions is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In some embodiments, the amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, provided in the pharmaceutical compositions of the present invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

In some embodiments, the amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof present in the pharmaceutical compositions is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-36 049703 g, or 1-3 g.

Kits/industrial products

Kits and articles of manufacture are also provided for use in the therapeutic applications described herein. In some embodiments, such kits provide a carrier, package or container divided into compartments for receiving one or more containers, such as vials, tubes and the like, each of the container(s) containing one of the individual elements for use in the method described herein. Suitable containers include, for example, bottles, vials, syringes and tubes. The containers are made of various materials, such as glass or plastic.

The articles of manufacture disclosed herein comprise packaging materials. Packaging materials for use in packaging pharmaceutical products include those disclosed in, for example, U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and the intended route of administration and treatment. For example, the container(s) comprise one or more compounds disclosed herein, optionally in a composition or combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (e.g., the container is a bag of intravenous solution or a vial having a stopper that can be pierced with a hypodermic needle). Such kits further comprise a compound with an identifying description or label or instructions related to its use in the methods described herein.

For example, a kit typically includes one or more additional containers, each of which contains one or more different materials (such as reagents, optionally in concentrated form, and/or devices) necessary from a commercial and user point of view for using a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes, carriers, packages, containers, vials and/or tubes that indicate the contents and/or instructions for use, and package inserts with instructions for use. Typically, a set of instructions is also included in the kit. A label is not necessarily on or associated with a container. For example, a label is on a container if the letters, numbers or other symbols that form the label are affixed, molded or engraved on the container itself, and a label is associated with a container if it is present in a reservoir or carrier that also contains the container, such as a package insert. In addition, the label is used to indicate that the contents are to be used for a specific therapeutic application. In addition, the label provides directions for using the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical compositions are contained in a package or dispenser device that contains one or more unit dosage forms containing a compound provided herein. The package, for example, comprises a metal or plastic foil, such as a blister pack. Or, the package or dispenser is accompanied by instructions for use. Or, the package or dispenser is accompanied by a notice associated with the container, in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which reflects approval by the agency of the form of the drug for administration to a human or animal. Such a notice is, for example, labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved package insert. In some embodiments, compositions comprising a compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, formulated in a compatible pharmaceutical carrier, are prepared, placed in an appropriate container, and labeled for treating a specified condition.

Methods of application/treatment

Embodiments of the present invention relate to a method of modulating hepatocyte growth factor in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound disclosed herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof). In some embodiments, a compound described herein activates hepatocyte growth factor. Modulation (e.g., inhibition or activation) of hepatocyte growth factor can be assessed and demonstrated by a wide variety of methods known in the art. Kits and commercially available assays can be used to determine whether and to what extent hepatocyte growth factor has been modulated (e.g., inhibited or activated).

In some embodiments, provided herein are compounds of formula (I), or pharmaceutically acceptable salts thereof, for use in modulating hepatocyte growth factor in a subject in need thereof. In some embodiments, provided herein are

- 37 049703 compounds of formula (I) or their pharmaceutically acceptable salts for the manufacture of a medicinal product for modulating hepatocyte growth factor in a subject in need thereof.

The applicant has found that compounds of formula (I) exhibit promising activity against certain diseases of interest. Accordingly, in one aspect, provided herein is a method of modulating hepatocyte growth factor in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof. In some embodiments, provided herein is a method of activating hepatocyte growth factor in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof.

In certain more specific embodiments, modulating includes treating a disease, condition, or injury (e.g., traumatic brain injury). As non-limiting examples, the disease, condition, or injury includes a neurodegenerative disease, traumatic brain injury, memory or function loss, spinal cord injury, sensorineural hearing loss, nerve injury, and the like. In some embodiments, the disease, condition, or injury is a neurodegenerative disease, spinal cord injury, traumatic brain injury, or sensorineural hearing loss.

In one more specific embodiment, the disease, condition, or injury is a neurodegenerative disease. For example, in some embodiments, the neurodegenerative disease is Alzheimer's disease, dementia, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS). In one more specific embodiment, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

Also provided herein is a method for treating or progressing dementia in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof. In a particular embodiment, the dementia is associated with Alzheimer's disease or Parkinson's disease.

In a further aspect, this document provides a method for preventing cognitive dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof.

In another related embodiment, a method of treating, restoring or preventing a disease, condition or injury associated with nervous tissue in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof.

In other aspects, provided herein is a method of treating a neuropsychiatric disease or disorder, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof. Non-limiting examples of neuropsychiatric diseases or disorders include, but are not limited to, depression and anxiety.

In further aspects, provided herein is a method of treating a disease or disorder of the central nervous system, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof. In some embodiments, provided herein is a method of preventing a disease or disorder of the central nervous system, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof. A non-limiting example of a disease or disorder of the central nervous system is traumatic brain injury.

In other aspects, provided herein is a method of treating a disease or disorder of the peripheral nervous system, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof. In some embodiments, provided herein is a method of preventing a disease or disorder of the peripheral nervous system, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof. A non-limiting example of a disease or disorder of the peripheral nervous system is neuropathic pain.

The embodiments of the above methods involve the administration to a mammal

- 38 049 703 a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form or stereoisomer thereof. The methods disclosed herein are generally directed to the administration of compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof for the treatment, protection against or reversal of diseases and injuries associated with nerve cells or the nervous system. That is, embodiments of the present invention are directed to the treatment, prevention or reversal of neurodegenerative diseases, including the treatment of dementia; restoration of traumatic injuries; and/or the prevention of cognitive dysfunction.

In some embodiments, the present invention provides methods of modulating the activity of a protein (e.g., hepatocyte growth factor) in subjects, including but not limited to rodents and mammals (e.g., humans), by administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof. In some embodiments, the modulation of hepatocyte growth factor is activation of hepatocyte growth factor. In some embodiments, the percent modulation ratio is greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the percent inhibition is greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In some embodiments, the present invention relates to methods of modulating the activity of a hepatocyte growth factor in a cell by contacting said cell with an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, sufficient to modulate the activity of the hepatocyte growth factor. In some embodiments, the present invention relates to methods of modulating the activity of a hepatocyte growth factor in a tissue by contacting said tissue with an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, sufficient to modulate the activity of the hepatocyte growth factor in the tissue. In some embodiments, the present invention relates to methods of modulating the activity of a hepatocyte growth factor in an organism by contacting said organism with an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, sufficient to modulate the activity of the hepatocyte growth factor in the organism. In some embodiments, the present invention relates to methods of modulating hepatocyte growth factor activity in an animal by contacting said animal with an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, sufficient to modulate the hepatocyte growth factor activity in the animal. In some embodiments, the present invention relates to methods of modulating hepatocyte growth factor activity in a mammal by contacting said mammal with an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, sufficient to modulate the hepatocyte growth factor activity in the mammal. In some embodiments, the present invention relates to methods of modulating hepatocyte growth factor activity in a human by contacting said human with an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, sufficient to modulate the hepatocyte growth factor activity in the human. In other embodiments, the present invention relates to methods of treating a disease mediated by hepatocyte growth factor activity in a subject in need of such treatment. In some embodiments, modulating hepatocyte growth factor with a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof involves activating hepatocyte growth factor.

In other embodiments, combination therapy methods are provided in which a therapeutic agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes, is used in combination with a compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof. In one aspect, such therapy includes, but is not limited to, a combination of one or more compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof with therapeutic agents, therapeutic antibodies, and other forms of treatment to provide a synergistic or additive therapeutic effect.

Many therapeutic agents currently known in the art can be used in combination with the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof. In some embodiments, the therapeutic agent is selected from memantine, cholinesterase inhibitors, antidepressants, anxiolytics and/or antipsychotics. Some embodiments include the use of treatment methods including reminiscence therapy, cognitive stimulation therapy, reality orientation training, physical activity and the like.

Examples of cholinesterase inhibitors include donepenzil, galantamine, and rivastigmine, which help slow the breakdown of a brain chemical involved in memory and thinking. Memantine may help control another brain chemical needed for learning and

- 39 049703 memory. In some aspects, memantine may also be used together with donepezil in combination with a drug for the treatment of moderate to severe dementia. Antidepressants may include, but are not limited to, selective serotonin reuptake inhibitors (SSRIs). Anxiolytics may include, but are not limited to, lorazepam (Ativan) or oxazepam (Serax). Some embodiments of the methods described herein may include the use or administration of antipsychotic drugs such as aripiprazole (Ability), haloperidol (Haldol), olanzapine (Zyprexa), and risperidone (Risperdal).

In some embodiments, the compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof are formulated or administered together with liquid or solid tissue barriers, also known as lubricants. Examples of tissue barriers include, but are not limited to, polysaccharides, polyglycans, seprafilm, interceed, and hyaluronic acid.

In some embodiments, therapeutic agents that are administered together with the compounds of Formula (I), or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof, include any suitable therapeutic agent conveniently delivered by inhalation, for example, analgesics, for example, codeine, dihydromorphine, ergotamine, fentanyl or morphine; angina medications, for example, diltiazem; antiallergic agents, for example, cromoglycate, ketotifen or nedocromil; anti-infective agents, for example, cephalosporins, penicillins, streptomycin, sulfonamides, tetracyclines or pentamidine; antihistamines, for example, methapyrilene; anti-inflammatory agents, for example, beclomethasone, flunisolide, budesonide, tipredane, triamcinolone acetonide or fluticasone; antitussives, for example, noscapine; bronchodilators such as ephedrine, adrenaline, fenoterol, formoterol, isoprenaline, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol, reproterol, rimitrol, salbutamol, salmeterol, terbutaline, isoetharine, tulobuterol, orciprenaline or (-)-4-amino-3,5-dichloro-a-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]amino]methyl]benzenemethanol; diuretics such as amiloride; anticholinergics such as ipratropium, atropine or oxitropium; hormones such as cortisone, hydrocortisone or prednisolone; xanthines such as aminophylline, choline theophyllinate, lysine theophyllinate or theophylline; therapeutic proteins and peptides, such as insulin or glucagon. It will be clear to one skilled in the art that, in appropriate cases, the therapeutic agents are used in the form of salts (e.g., as alkali metal or amine salts or as acid addition salts) or as esters (e.g., lower alkyl esters) or as solvates (e.g., hydrates) to optimize the activity and/or stability of the therapeutic agent.

Additional therapeutic agents that can be combined with a compound of Formula (I), or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof, are provided in Goodman and Gilman's The Pharmacological Basis of Therapeutics Tenth Edition, edited by Hardman, Limbird and Gilman or the Physician's Desk Reference, both of which are incorporated herein by reference in their entireties. Compounds of Formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, can be used in combination with the therapeutic agents disclosed herein, depending on the condition being treated. Thus, in some embodiments, one or more compounds of Formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, will be co-administered with other therapeutic agents as described above. When used in combination therapy, compounds of Formula (I), or pharmaceutically acceptable salts, isotopic forms, or stereoisomers thereof, are administered with a second therapeutic agent, either simultaneously or separately. This combination administration may include simultaneous administration in a single dosage form, simultaneous administration of separate dosage forms, and separate administration. That is, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof and any of the therapeutic agents described above may be formulated together in a single dosage form and administered simultaneously. Alternatively, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof and any of the therapeutic agents described above may be administered simultaneously if both are present in separate formulations. In another alternative, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof may be administered immediately following any of the therapeutic agents described above, or vice versa. In some embodiments of the separate administration protocol, the compound of formula (I) or a pharmaceutically acceptable salt, isotopic form, or stereoisomer thereof and any of the therapeutic agents described above are administered within minutes, or within hours, or within days.

The following examples and preparations further illustrate and exemplify the compounds of formula (I), or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof, and methods for preparing such compounds. It is to be understood that the scope of the present invention is in no way limited by the scope of the following examples and preparations. In the following examples, as well as throughout the specification and claims, molecules with one stereocenter, unless otherwise specified, are

- 40 049703 otherwise stated, exist as a racemic mixture. Molecules with two or more stereocenters, unless otherwise stated, exist as a racemic mixture of diastereomers. Individual enantiomers/diastereomers can be prepared by methods known to those skilled in the art.

Examples

The following examples are provided for illustrative purposes. Methods for preparing compounds of formula (I) or pharmaceutically acceptable salts, isotopic forms or stereoisomers thereof are provided herein or can be prepared by one skilled in the art.

The examples and preparations provided below further illustrate and exemplify the compounds of the present invention and methods for testing such compounds. It should be understood that the scope of the present invention is in no way limited by the scope of the following examples.

The chemical reactions described in the examples can be readily adapted to prepare a variety of other compounds disclosed herein, and alternative methods for preparing the compounds of the present invention are considered to be within the scope of the present invention. For example, the synthesis of non-illustrated compounds of the present invention can be accomplished by modifications obvious to those skilled in the art, such as by appropriately protecting interfering groups, using other suitable reagents known in the art in addition to those described, or by making routine modifications to the reaction conditions, reagents, and starting materials. Alternatively, other reactions disclosed herein or known in the art will be found to be applicable to the preparation of other compounds of the present invention.

Unless otherwise stated in the following examples, the compounds are isolated as racemic mixtures.

The following abbreviations may be applicable to this application.

Abbreviations

AcOH: acetic acid

CAN: Ceric Ammonium Nitrate

DAST: diethylaminosulfur trifluoride

DCM: dichloromethane

DIPEA: N.N-diisopropylethylamine

DMEM: Dulbecco's modified Eagle's medium

DMF: dimethylformamide

DMSO: dimethyl sulfoxide

EMEM: Eagle's Minimum Essential Medium

EtOAc: ethyl acetate

EtOH: ethanol

FBS: fetal bovine serum

Fmoc: Fluorenylmethoxycarbonyl

HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

LC/MS: liquid chromatography/mass spectrometry

Me: methyl

MeOH: methanol

PBS: phosphate buffered saline

Pic-BH ₃ : picolineborane

RMB: para-methoxybenzyl ether

Prep. HPLC: Preparative High Performance Liquid Chromatography

r.t. or R.T.: room temperature

TFA: trifluoroacetic acid

TLC: thin layer chromatography

T ₃ P: propanephosphonic acid anhydride

Examples of synthesis

Example S1. Synthesis of (6S)-6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine4,7(6H)-dione. The synthetic route to obtain this compound is from the starting material as shown in Scheme 1.

- 41 049703

Scheme 1.

FmocHN

T ₃ P, DI PEA,

DXM, CT, 8h

Piperidine

DXM, kg, 2h

Fmoc । n

N

HCOOH G T ϊ T Piperidine, DMF, SVV y ^x ιπ,16 4 k-Ν^Αθ ¹

O O

Step 1: Synthesis of (9H-fluoren-9-yl)methyl(2S)-1-((2,2-dimethoxyethyl)(2-methylbutyl)amino)-1oxopropan-2-ylcarbamate

To a stirred solution of (N)-2-(((9H-fluoren-9yl)methoxy)carbonylamino)propanoic acid (5.0 g, 16.07) in dichloromethane (100 mL) was added _T3P (15.2 mL, 24.1) and DIPEA (5.6 mL, 32.1 mmol) at room temperature. The reaction mixture was stirred at room temperature for 15 min and N-(2,2-dimethoxyethyl)-2-methylbutan-1amine (2.81 g, 32.1 mmol) was added and stirring was continued at room temperature for 8 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was quenched with ice-cold water (100 mL) and extracted with dichloromethane (2 x 100 mL). The combined organic layers _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give the crude compound. The crude compound was purified by flash column chromatography (100-200 mesh silica gel, eluted with 40% ethyl acetate in petroleum ether) to give pure (9H-fluoren-9-yl)methyl(2S)-1-((2,2-dimethoxyethyl)(2-methylbutyl)amino)-1-oxopropan-2-ylcarbamate (5.2 g, 69.1%) as a gummy compound.

Step 2: Synthesis of N)-2-amino-M-(2,2-dimethoxyethyl)-M-(2-methylbutyl)propenamide

To a stirred solution of (9H-fluoren-9-yl)methyl(2S)-1-((2,2-dimethoxyethyl)(2-methylbutyl)amino)-1-oxopropan-2-ylcarbamate (34.0 g, 72.6 mmol) in DMF (230 mL) was added 20% piperidine in DMF (70 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After completion of the reaction, excess DMF (100 mL) was added, then washed with excess n-hexane (3 x 200 mL). The DMF layer was collected and poured into ice water (1000 mL), then extracted with 10% methanol-dichloromethane (3 x 500 mL). The combined organic layers _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give (D)-2-amino-N-(2,2-dimethoxyethyl)-N-(2-methylbutyl)propanamide (20.4 g, 68.4%) as a gummy solid.

Step 3: Synthesis of (9H-fluoren-9-yl)methyl 3-(DO)-1-((2,2-dimethoxyethyl)(2-methylbutyl)amino)-1oxopropan-2-ylamino)-3-oxopropylcarbamate

To a stirred solution of 3-(((9H-fluoren-9-yl)methoxy)carbonylamino)propanoic acid (20.2 g, 81.2 mmol) stirred in dichloromethane at room temperature (500 mL) were added T3P (80 mL, 121.8 mmol) and DIPEA (28.6 mL, 160.4 mmol) and the mixture was stirred for 10 min. To it was added (N)-2-amino-N-(2,2-dimethoxyethyl)-N-(2-methylbutyl)propanamide (25.53, 81.2 mmol) and stirring was continued at room temperature for 16 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was quenched with water (500 mL) and the mixture was extracted with dichloromethane (2 x 500 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude compound was purified by flash column chromatography (100-200 mesh silica gel, eluted with 70% ethyl acetate in petroleum ether) to give pure (9H-fluoren-9-yl)methyl 3-((2S)-1-((2,2-dimethoxyethyl)(2-methylbutyl)amino)-1-oxopropan-2-ylamino)-3-oxopropylcarbamate (21.2 g, 78.6%) as a gummy compound.

Step 4: Synthesis of (6S)-(9H-fluoren-9-yl)methyl 6-methyl-8-(2-methylbutyl)-4,7-dioxooctahydro-1Hpyrazino[1,2-a]pyrimidine-1-carboxylate

To a stirred solution of (9H-fluoren-9-yl)methyl 3-(^)-1-((2,2-dimethoxyethyl)(2methylbutyl)amino)-1-oxopropan-2-ylamino)-3-oxopropylcarbamate (21.0 g, 38.9 mmol) was added formic acid (105 mL). The reaction mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated under reduced pressure to afford the crude compound. The crude compound was dissolved in

- 42 049703 was dissolved in saturated aqueous NaHCO3 solution (200 mL) then extracted with ethyl acetate (3 x 500 mL). The combined organic layers were washed with brine (500 mL), then the combined organic layers were dried over _{anhydrous Na2SO4} , filtered and concentrated under reduced pressure. The crude compound was purified by flash column chromatography (100-200 mesh silica gel, eluted with 50% ethyl acetate in petroleum ether) to afford pure compound (6S)(9H-fluoren-9-yl)methyl 6-methyl-8-(2-methylbutyl)-4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidine-1-carboxylate (25 g, 69.0%) as a gum.

Step 5: Synthesis of (6S)-6-methyl-8-(2-methylbutyl)tetrahydro-1H-pyrazino[1,2-a]pyrimidine4,7(6H,8H)-dione

To a stirred solution of (6S)-(9H-fluoren-9-yl)methyl 6-methyl-8-(2-methylbutyl)-4,7dioxooctahydro-1H-pyrazino[1,2-a]pyrimidine-1-carboxylate (14.0 g, 29.4 mmol) at 0 °C in DMF (70 mL) was added 20% piperidine in DMF (30 mL). The reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction progress was monitored by TLC. After complete consumption of the starting material, additional DMF (50 mL) was added and the mixture was washed with excess n-hexane (3 x 200 mL). The DMF layer was poured into ice-cold water (1000 mL) and extracted with 10% methanol-dichloromethane (3 x 500 mL). The combined organic layers were dried over _{anhydrous Na2SO4} , filtered and concentrated under reduced pressure to give the desired crude compound (6S)-6-methyl-8-(2-methylbutyl)tetrahydro-1H-pyrazino[1,2a]pyrimidine-4,7(6H,8H)-dione (6.25 g, 83.8%) as a solid.

Example S2. Synthesis of compound 1a

The synthetic route to obtain compound 1a is shown in Scheme 2.

Scheme 2.

o =

1a

To a solution of 4-(trifluoromethyl)benzoic acid (0.232 g, 0.91 mmol) stirred in dichloromethane (20 ml) at room temperature were added _T3P (1.2 ml, 1.37 mmol) and DIPEA (0.42 ml, 1.82 mmol) and the mixture was stirred for 15 min. To it was added (6S)-6-methyl-8-(2-methylbutyl)tetrahydro-N-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)-dione (0.310 g, 0.91 mmol) and stirring was continued for 8 h. The reaction progress was monitored by TLC. After completion of the reaction, the mixture was quenched with water (50 ml) and extracted with dichloromethane (2 x 50 ml). The combined organic layers _{were dried over Na2SO4 ,} filtered and concentrated under reduced pressure. The crude compound was purified by prep. HPLC. Pure fractions were combined and concentrated under reduced pressure, then lyophilized to afford 1a (0.340 g, 65.3%) as a solid. Prep. HPLC method: Mobile phase A: 10 mM ammonium bicarbonate in water; mobile phase B: acetonitrile; Column: X-Select phenyl hexyl (150 x 19 mm 5 μm); Flow rate: 16 mL/min. MS (ESI) m/z [M+H]+: 426.05.

Example S3. Synthesis of compound 2a.

The synthetic route to obtain compound 2a is shown in Scheme 3.

Scheme 3.

2a

To a solution of 4-(difluoromethoxy)benzoic acid (0.37 g, 1.968 mmol) in dichloromethane (15 mL) at room temperature were added DIPEA (0.8 mL, 5.904 mmol) and _T3P (2.0 mL, 3.936 mmol). The mixture was stirred for 30 min, then (6S)-6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2a]pyrimidine-4,7(6H)-dione (0.4 g, 1.578 mmol) was added and stirring was continued for 16 h. The reaction progress was monitored by TLC and LC/MS. The reaction mixture was diluted with dichloromethane (100 mL) and washed with water (50 mL) and saturated sodium chloride solution (50 mL), then dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by prep. HPLC. The pure fractions were collected and lyophilized to give 2a (380 mg, 46%) as a solid. Prep. HPLC conditions: Mobile phase A: 10 mM ammonium bicarbonate in water; Mobile phase B: acetonitrile; Column: Kromosil phenyl (150 x 25 mm 10 μm); Flow: 25 mL/min. MS (ESI) m/z [M+H] ⁺ : 424.11.

- 43 049703

Example S4. Synthesis of compound 3 a.

The synthetic route to obtain compound 3a is shown in Scheme 4.

Scheme 4.

To a solution of ((S)-6-methyl-8-(2-methylbutyl)tetrahydro-(N-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)dione (0.500 g, 1.97 mmol) stirred in methanol (20 mL) were added 4-hydroxybenzaldehyde (0.289 g, 1.97 mmol) and acetic acid (0.23 mL, 3.95 mmol) at room temperature. The reaction mixture was stirred at room temperature for 5 min. To it was added picolineborane (0.253 g, 2.37 mmol) and stirring was continued for 48 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was quenched with ice-cold water (50 mL) and the mixture was extracted with 10% methanol-dichloromethane (3 x 40 mL). The combined organic layers _were dried over _Na2SO4 , filtered and concentrated under reduced pressure. The crude compound was purified by prep. HPLC method. Pure fractions were combined and concentrated under reduced pressure then lyophilized to afford 3a (0.180 g, 46.09%) as a solid. Prep. HPLC method: Mobile phase A: 10 mM ammonium bicarbonate in water; Mobile phase B: acetonitrile; Column: Kromosil Phenyl (150 x 25 mm 10 μm); Flow: 25 mL/min. MS (ESI) m/z [M+H]+: 360.11.

Example S5. Synthesis of compound 4a.

The synthetic route to obtain compound 4a is shown in Scheme 5.

Scheme 5.

To a solution of 6-hydroxynicotinic acid (0.340 g, 2.446 mmol) in DMF (15 mL) were added DIPEA (1.30 mL, 7.338 mmol) and HATU (1.39 g, 3.669 mmol) at room temperature. The resulting reaction mixture was stirred for 30 min, then (6S)-6-methyl-8-(2methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.495 g, 1.956 mmol) was added and the mixture was stirred for 16 h. The reaction progress was monitored by TLC and LC/MS (TLC system: 10% methanol/dichloromethane, Rf: 0.15, detection: UV). The reaction mixture was quenched with cold water (100 mL) and extracted with 10% methanol/dichloromethane (3 x 100 mL). The combined organic layers were washed with cold water (50 mL) and cold brine (50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by prep HPLC. The pure fractions were collected and lyophilized to give 4a (160 mg, 21.8%) as a solid. Prep HPLC method: Mobile phase A: 0.01 mM ammonium bicarbonate in water; Mobile phase B: acetonitrile; Column: X-Select phenyl hexyl (150 x 19 mm, 5 μm); Flow: 15 mL/min. MS (ESI) m/z [M+H]+: 375.05.

Example S6. Synthesis of compound 5a.

The synthetic route to obtain compound 5a is shown in Scheme 6.

Scheme 6.

To a solution of ((3)-6-methyl-8-(2-methylbutyl)tetrahydro-(3H,8H)-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)dione (0.5 g, 1.97 mmol) and 1-(bromomethyl)-4-(trifluoromethyl)benzene (0.470 g, 1.97 mmol) stirred in DMF (20 mL) was added K ₂ CO ₃ (0.546 g, 3.95 mmol) at room temperature and the mixture was stirred for 8 h. The reaction progress was monitored by TLC. Upon completion, the mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over

- 44 049703

Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by prep. HPLC. Pure fractions were combined and concentrated under reduced pressure, then lyophilized to afford 5a (0.270 g, 63.8%) as a gum. Prep. HPLC method: Mobile phase A: 10 mM ammonium bicarbonate in water; Mobile phase B: acetonitrile; Column: Kromosil Cig (150 x 25 mm 10 µm); Flow: 25 mL/min. MS (ESI) m/z [M+H]+: 412.2.

Example S7. Synthesis of compound 6a.

The synthetic route to obtain compound 6a is shown in Scheme 7.

Scheme 7.

To a solution of ((3)-6-methyl-8-(2-methylbutyl)tetrahydro-1H-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)dione (0.500 g, 1.97 mmol) and 1-(bromomethyl)-4-(difluoromethoxy)benzene (0.466 g, 1.97 mmol) stirred in DMF (20 mL) was added K ₂ CO ₃ (0.546 g, 9.95 mmol). The reaction mixture was stirred at room temperature for 18 h. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude compound was purified by prep HPLC. Pure fractions were pooled and concentrated under reduced pressure, then lyophilized to afford 6a (0.178 g, 41.5%) as a semi-solid. Prep HPLC method: Mobile phase A: 10 mM ammonium bicarbonate in water; Mobile phase B: acetonitrile; Column: X-Select C ₁₈ (250 x 19 mm, 5 μm); Flow rate: 18 mL/min. MS (ESI) m/z [M+H]+: 410.11.

Example S8. Synthesis of compound 7a.

The synthetic route to obtain compound 7a is shown in Scheme 8.

Scheme 8.

To a solution of (6S)-6-methyl-8-(2-methylbutyl)tetrahydro-1H-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)-dione (0.500 g, 1.97 mmol) stirred in methanol (20 mL) were added 6-hydroxynicotinaldehyde (0.243 g, 1.97 mmol) and acetic acid (0.25 mL, 3.95 mmol) at room temperature and the mixture was stirred for 5 min. To it was added picolineborane (0.318 g, 2.96 mmol) and stirring was continued for 96 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was quenched with ice-cold water (50 mL) and extracted with 10% methanol-dichloromethane (3 x 40 mL). The combined organic layers _were dried over _Na2SO4 , filtered and concentrated under reduced pressure. The crude compound was purified by prep. HPLC. The pure fractions were collected and concentrated under reduced pressure then lyophilized to afford 7a (0.164 g, 42%) as a solid. Prep. HPLC method: Mobile phase A: 10 mM ammonium bicarbonate in water; mobile phase B: acetonitrile; Column: X-BRIDGE C ₁₈ (250 x 19 mm, 5 μm); Flow rate: 18 mL/min. MS (ESI) m/z [M+H]+: 3 61.11.

Example S9. Synthesis of compound 8a.

The synthetic route to obtain compound 8a is shown in Scheme 9.

Scheme 9.

- 45 049703

Step 1: Synthesis of ^)-1-(4-(benzyloxy)benzoyl)-6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione

To a solution of 4-(benzyloxy)benzoic acid (0.360 g, 1.42 mmol) stirred in dichloromethane (20 ml) were added _T3P (1.2 ml, 1.7 mmol) and DIPEA (0.55 ml, 2.84 mmol) at room temperature and the mixture was stirred for 15 min. To it was added ((S)-6-methyl-8-(2-methylbutyl)tetrahydro-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)-dione (0.400 g, 1.42 mmol) and stirring was continued at room temperature for 16 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was quenched with water (50 ml) and extracted with dichloromethane (2 x 50 ml). The combined organic layers were dried over _Na2SO4 , filtered and concentrated under reduced pressure to yield 0.9 g _of crude material. LC/MS analysis of the crude material indicated 54.59% of the desired product. The crude material was used in the next step without purification.

Step 2: Synthesis of compound 8a.

To a solution of ((3)-1-(4-(benzyloxy)benzoyl)-6-methyl-8-(2-methylbutyl)tetrahydro-N-pyrazino[1,2a]pyrimidine-4,7(6H,8H)-dione (0.900 g) stirred in methanol (20 mL) was added 10% Pd-C (0.200 g) under N2 atmosphere at room temperature. The reaction mixture was stirred at room temperature with a balloon of _H2 for 8 h. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was filtered through celite and evaporated under reduced pressure to give the crude compound. The crude compound was dissolved in dichloromethane (50 mL), washed with aqueous _NaHCO3 (20 mL) and brine (20 mL). The filtrate was dried over _Na2SO4 , filtered and concentrated under reduced pressure. The crude compound was triturated with diethyl ether to give 8a (0.330 g, 82%) as a solid. MS (ESI) m/z [M+H]+: 374.11.

Example S10. Synthesis of compound 9.

The synthetic route to obtain compound 9 is shown in Scheme 10.

Scheme 10.

Step 1: Synthesis of (9H-fluoren-9-yl)methyl 2-(sec-butyl(2,2-dimethoxyethyl)amino)-2-oxoethylcarbamate.

To a stirred solution of 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)acetic acid (10 g, 33.6 mmol) in dichloromethane (100 mL) cooled to 0 °C were added DIPEA (11.88 mL, 67.3 mmol), N(2,2-dimethoxyethyl)butan-2-amine (10.84 g, 67.3 mmol) and _T3P (53.0 mL, 84.1 mmol) and the reaction mixture was stirred for 16 h at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, ice water (100 mL) was added and extracted with ethyl acetate (2 x 150 mL). The combined organic layers _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give the desired crude product. The crude compound was purified by flash column chromatography (100-200 mesh silica gel) and eluted with 20-25% ethyl acetate in petroleum ether to give (9H-fluoren-9-yl)methyl 2-(sec-butyl(2,2-dimethoxyethyl)amino)-2-oxoethylcarbamate (10.8 g, 72.9%) as a solid.

Step 2: Synthesis of 2-amino-M-sec-butyl-M-(2,2-dimethoxyethyl)acetamide

To a solution of (9H-fluoren-9-yl)methyl 2-(sec-butyl(2,2-dimethoxyethyl)amino)-2-oxoethylcarbamate (10.8 g, 24.5 mmol) in DMF (20 mL) cooled to 0 °C was added piperidine (2.4 mL) and the reaction mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After TLC showed complete consumption of the starting material, the reaction mixture was diluted with petroleum ether (2 x 100 mL), then water was added and the mixture was separated. The aqueous layer of ext

- 46 049703 was diluted _with dichloromethane (2 x 150 mL). The combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give the desired pure product 2-amino-N-sec-butyl-N-(2,2-dimethoxyethyl)acetamide (3.6 g, 67.2%) as a solid.

Step 3: Synthesis of (9H-fluoren-9-yl)methyl 3-(2-(sec-butyl(2,2-dimethoxyethyl)amino)-2-oxoethylamino)-3-oxopropylcarbamate

To a stirred solution of 2-amino-N-sec-butyl-N-(2,2-dimethoxyethyl)acetamide (3.6 g, 16.5 mmol) in dichloromethane (40 mL) were added DIPEA (31.91 mL, 49.5 mmol), 3-(((9H-fluoren-9yl)methoxy)carbonylamino)propanoic acid (5.14 g, 16.5 mmol) and _T3P (39.13 g, 33 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The reaction progress was monitored by TLC. After completion of the reaction, reaction water (100 mL) was added and the organic phase was separated. The aqueous phase was extracted with dichloromethane (2 x 150 mL). The combined organic layers _were dried over _Na2SO4 , filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography using silica gel (230-400 mesh; 23-25% ethyl acetate/petroleum ether as eluent). The collected pure fractions were concentrated under reduced pressure to give (9H-fluoren-9-yl)methyl 3-(2-(sec-butyl(2,2-dimethoxyethyl)amino)-2-oxoethylamino)-3-oxopropylcarbamate (4.1 g, 48.6%) as a gum.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl-8-sec-butyl-4,7-dioxooctahydro-1H-pyrazino[1,2a]pyrimidin-1-carboxylate

A solution of (9H-fluoren-9-yl)methyl 3-(2-(sec-butyl(2,2-dimethoxyethyl)amino)-2-oxoethylamino)-3-oxopropylcarbamate (4.1 g, 8.01 mmol) in acetic acid (2 mL) was stirred for 16 h at room temperature. The reaction progress was monitored by TLC. After TLC showed complete consumption of the starting material, the reaction mixture was concentrated and the resulting mass was diluted with water and extracted with dichloromethane (2 x 100 mL). The combined organic layers _{were dried over Na2SO4 ,} filtered and concentrated under reduced pressure to give the product (9Hfluoren-9-yl)methyl 8-sec-butyl 4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidine-1-carboxylate (3.2 g, 89.3%) as a gum.

Step 5: Synthesis of 8-sec-butyltetrahydro-1H-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)-dione

To a solution of (9H-fluoren-9-yl)methyl 8-sec-butyl 4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidine-1-carboxylate (3.2 g, 7.1 mmol) in DMF (20 mL) cooled to 0 °C was added piperidine (0.7 mL, 1.0 equiv) and the reaction mixture was stirred at room temperature for 2 h. After TLC showed complete consumption of the starting material, the reaction mixture was washed with petroleum ether (2 x 50 mL) to remove non-polar impurities. Cold water was added and the mixture was extracted with dichloromethane (2 x 100 mL). The combined organic layers were dried over _Na2SO4 , filtered and concentrated under reduced pressure to give pure 8-sec-butyltetrahydro-1H-pyrazino[1,2-a]pyrimidine-4,7(6H,8H)-dione (900 mg, 55.9%) as _a solid.

Step 6: Synthesis of compound 9 To a stirred solution of (8-(sec-butyl)hexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.500 g, 2.2 mmol) and 4-hydroxybenzaldehyde (0.271 g, 2.2 mmol) in methanol (10 mL) were added acetic acid (0.27 mL, 2.0 equiv) and picolineborane (0.285 g, 2.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 48 h. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with ice-cold water (10 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude product. The crude compound was analyzed by LC/MS. LC/MS of the crude material indicated 8.28% of the desired mass. The crude compound was purified by column chromatography over silica gel (100-200) and 50-70% ethyl acetate in petroleum ether and the desired compound was eluted. LC/MS of the eluted fractions indicated 72.16% of the desired mass, which was further purified by prep-HPLC. After purification by prep-HPLC, the fractions were collected and concentrated under reduced pressure, then lyophilized to afford 9 (0.168 g, 22.8%) as a solid. Prep-HPLC Method: Mobile Phase A: 10 mM ammonium bicarbonate in water; Mobile Phase B: acetonitrile; Column: X-BRIDGE C ₁₈ (150 x 19 mm 5 microns); flow: 18 ml/min. MS (ESI) m/z [M+H]+: 332.2.

- 47 049703

Example S11. Synthesis of compound 10. The synthetic route for obtaining compound 10 is shown in Scheme 11.

Scheme 11.

To a solution of 6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (250 mg, 0.98 mmol) and 4-chlorobenzoic acid (170 mg, 1.09 mmol) in DMF (4 mL) at 0 °C was added HATU (413 mg, 1.08 mmol) followed by DIPEA (0.35 mL, 1.97 mmol). The reaction mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was quenched with ice-cold water (50 mL) and the aqueous layer was extracted with EtOAc (50 mL x 2). The organic layer was washed with cold H ₂ O (30 mL) and then with brine (30 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 30% EtOAc in hexanes) to give 1-(4-chlorobenzoyl)-6-methyl-8-(2methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione 10 (150 mg, 0.383 mmol, 39.2% yield) as a solid. MS (ESI) m/z [M+H]+: 392.05. 1H NMR (400 MHz, DMSO-06) δ 0.660.89 (m, 6H) 0.91-1.42 (m, 4H) 1.57-1.78 (m, 1H) 2.16-2.35 (m, 2H) 2.55-2.65 (m, 2H) 3.08-3.23 (m, 2H) 3.283.40 (m, 1H) 3.51-3.64 (m, 2H) 4.76-4.89 (m, 1H) 5.88-6.02 (m, 1H) 7.46-7.56 (m, 4H).

Example S12. Synthesis of compound 11. The synthetic route for obtaining compound 11 is shown in Scheme 12 Scheme 12.

To a solution of 6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (250 mg, 0.98 mmol) and 4-fluorobenzoic acid (153 mg, 1.09 mmol) in DMF (4 mL) at 0 °C was added HATU (413 mg, 1.08 mmol) followed by DIPEA (0.35 mL, 1.97 mmol). The reaction mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was quenched with ice-cold water (50 mL) and the aqueous layer was extracted with EtOAc (50 mL x 2). The organic layer was washed with cold H ₂ O (30 mL) and then brine (30 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 30% EtOAc in hexanes) to give 1-(4-fluorobenzoyl)-6-methyl-8-(2methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione 11 (140 mg, 0.37 mmol, 38.0% yield) as a solid. MS (ESI) m/z [M+H]+: 376.05. 1H NMR (400 MHz, DMSO-4) δ 0.69-0.81 (m, 3H) 0.86 (t, J=7.23 Hz, 3H) 0.95-1.14 (m, 2H) 1.20-1.43 (m, 4H) 1.59-1.80 (m, 2H) 2.26 (d, J=16.95 Hz, 1H) 2.55-2.72 (m, 1H) 3.20-3.31 (m, 2H) 3.35-3.39 (m, 1H) 3.52-3.70 (m, 2H) 4.73-4.89 (m, 1H) 7.33 (t, J=8.73 Hz, 2H) 7.61 (dd, J=8.23, 5.73 Hz, 2N).

Example S13. Synthesis of compound 12. The synthetic route for obtaining compound 12 is shown in Scheme 13 Scheme 13.

To a solution of 6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (250 mg, 0.98 mmol) and 3-chloro-4-(trifluoromethyl)benzoic acid (242 mg, 1.09 mmol) in DMF (4 mL) at 0 °C was added HATU (413 mg, 1.08 mmol) followed by DIPEA (0.35 mL, 1.97 mmol). The reaction mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was quenched with ice-cold water (50 mL) and the aqueous layer was extracted with EtOAc (50 mL x 2). The organic layer was washed with cold _H2O (30 mL) and then brine (30 mL), dried over _Na2SO4 , and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 30% EtOAc in hexanes) to give 1-(3-chloro _- 4(trifluoromethyl)benzoyl)-6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione 12 (250 mg, 0.55 mmol, 55.2% yield) as a solid. MS (ESI) m/z [M+H]+: 460.0. ¹ H NMR (400 MHz, DMSO-06) δ 0.74-0.93 (m, 6H) 0.98-1.19 (m, 2H) 1.28-1.46 (m, 3H) 1.64-1.81 (m, 1H) 2.22 (d, J=17.45 Hz, 1H) 2.57-2.70 (m, 1H) 3.14 (dd, J=13.21, 6.23 Hz, 1H) 3.25-3.31 (m, 2H) 3.44-3.57 (m, 1H) 3.61

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3.87 (m, 2H) 4.78-4.90 (m, 1H) 5.89-6.05 (m, 1H) 7.72 (d, J=7.98 Hz, 1H) 7.90-8.02 (m, 2H).

Example S14. Synthesis of the intermediate 8-(4-methoxybenzyl)-6-methylhexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)-dione

The synthetic route to obtain this intermediate is shown in Scheme 14.

Scheme 14.

9 ^Et i anisaldehyde ? ^E1 H 9 ^Et I DEA, DXM _Β Ά- ^ΝΗ! --- BO^-RMB

Stage 1 ^{DMF 0} Stage

Stage 2 ~

Step 1: Synthesis of 2,2-diethoxy-(4-methoxybenzyl)ethan-1-amine

A 500 mL round bottom flask was charged with anisaldehyde (12 mL, 90.22 mmol) and 2,2-diethoxyethanamine (10 g, 75.18 mmol). The reaction mixture was heated at 100 °C for 1 h. The reaction mixture was allowed to cool at room temperature and EtOH (100 mL) was added followed by NaBH4 (4.28 g, 112.7 mmol). The resulting reaction mixture was stirred at room temperature for 16 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated under reduced vacuum. The resulting crude material was dissolved in EtOAc (300 mL). The organic layer was washed with brine (100 mL), dried over _Na2SO4 and concentrated in vacuo to give _the crude product. The resulting crude product was purified by column chromatography (silica gel 100-200 mesh; 70% EtOAc in hexanes) to give 2,2-diethoxy-(4-methoxybenzyl)ethan-1-amine (15 g, 59.28 mmol, 78% yield) as a liquid. MS (ESI) m/z [M+H]+: 254.3.

Step 2: (9H-fluoren-9-yl)methyl(1-((2,2-diethoxyethyl)(4-methoxybenzyl)amino)-1-oxopropan-2yl)carbamate

To a stirred solution of (((9H-fluoren-9-yl)methoxy)carbonyl)alanine (32 g, 102.76 mmol) in dry DMF (140 mL) maintained at 0 °C were added HATU (42 g, 110.67 mmol), DIPEA (21.06 mL, 118.57 mmol), and then 2,2-diethoxy-(4-methoxybenzyl)ethan-1-amine (20 g, 79.05 mmol). The reaction mixture was stirred at room temperature for 16 h. After complete consumption of the starting material, the reaction mixture was quenched with ice-cold water (300 mL) and the aqueous layer was extracted with EtOAc (200 mL x 2). The organic layer was washed with cold _H2O (200 mL) and then brine (100 mL), dried over _Na2SO4 , and concentrated under reduced pressure to give the crude product. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 50% EtOAc in hexanes) to give (9H-fluoren-9 _- yl)methyl (1-((2,2-diethoxyethyl)(4-methoxybenzyl)amino)-1-oxopropan-2-yl)carbamate (28 g, 51.22 mmol, 64.8% yield) as a gummy liquid. MS (ESI) m/z [M+H-EtOH]^ 501.2.

Step 3: Synthesis of 2-amino-A2,2-diethoxyethyl)-A4-methoxybenzyl)propanamide

To a solution of (9H-fluoren-9-yl)methyl (1-((2,2-diethoxyethyl)(4-methoxybenzyl)amino)-1-oxopropan2-yl)carbamate (28 g, 51.22 mmol) in _CH2Cl2 ( ₃₀ mL) was added diethylamine (200 mL). The reaction mixture was stirred at room temperature for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated and the resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 2-amino-N-(2,2-diethoxyethyl)-N-(4-methoxybenzyl)propanamide (14.5 g, 44.75 mmol, 87% yield) as a viscous liquid. MS (IER) m/z [M+N-EUN] ^a 279.05.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl(3-((1-((2,2-diethoxyethyl)(4-methoxybenzyl)amino)-1oxopropan-2-yl)amino)-3-oxopropyl)carbamate

To a stirred solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (14.78 g, 47.53 mmol) in dry DMF (120 mL) maintained at 0 °C were added HATU (18.06 g, 47.53 mmol), DIPEA (9.21 mL, 51.85 mmol) and then 2-amino-N-(2,2-diethoxyethyl)-(4-methoxybenzyl)propanamide (14 g, 43.20 mmol). The reaction mixture was stirred for 16 h at room temperature. Upon completion, the reaction mixture was quenched with ice-cold water (200 mL) and the aqueous layer was extracted with EtOAc (200 mL x 2). The organic layer was washed with cold _H2O (500 mL) and then brine (200 mL), dried over _Na2SO4 , and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 30% EtOAc in hexanes) to give (9H-fluoren-9 _- yl)methyl (3-((1-((2,2-diethoxyethyl)(4-methoxybenzyl)amino)-1-oxopropan-2-yl)amino)-3-oxopropyl)carbamate (18 g, 29.14

- 49 049703 mmol, yield 67.44%) as a viscous liquid. MS (ESI) m/z [M+H-EtOH]+: 572.

Step 5: Synthesis of (9H-fluoren-9-yl)methyl-8-(4-methoxybenzyl)-6-methyl-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate

A solution of (9H-fluoren-9-yl)methyl (3-((1-((2,2-diethoxyethyl)(4-methoxybenzyl)amino)-1-oxopropan2-yl)amino)-3-oxopropyl)carbamate (18 g, 29.14 mmol) in formic acid (120 mL) was stirred at room temperature for 12 h. Upon completion, the reaction mixture was concentrated and the resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 30% EtOAc in hexanes) to give (9H-fluoren-9-yl)methyl 8-(4-methoxybenzyl)-6-methyl-4,7dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (14.5 g, 27.58 mmol, yield 94%) as a solid. MS (ESI) m/z [M+H]+: 526.

Step 6: Synthesis of 8-(4-methoxybenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione

To a solution of (9H-fluoren-9-yl)methyl 8-(4-methoxybenzyl)-6-methyl-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (14 g, 26.63 mmol) in _CH2Cl2 ( ₁₅₀ mL) was added diethylamine (100 mL) and the reaction mixture was stirred at room temperature for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated and the resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 8-(4-methoxybenzyl)-6-methylhexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)-dione (7 g, 23.07 mmol, 87% yield) as a sticky solid. MS (ESI) m/z [M+H]+: 304.

Example S15. Synthesis of the intermediate 8-(4-methoxybenzyl)-6-methylhexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)-dione

The synthetic route to obtain this intermediate is shown in Scheme 15.

Scheme 15.

Step 1: Synthesis of 8-(4-methoxybenzyl)-6-methyl-1-(4-(trifluoromethyl)benzoyl)hexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)-dione

To a solution of 4-(trifluoromethyl)benzoic acid (5.26 g, 27.69 mmol) in DMF (100 mL) maintained at 0 °C were added HATU (10.52 g, 27.69 mmol), DIPEA (12.30 mL, 69.23 mmol) and then 8-(4methoxybenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (7 g, 23.07 mmol), and the reaction mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was quenched with ice-cold water (200 mL) and the aqueous layer was extracted with EtOAc (200 mL x 2). The organic layer was washed with cold _H2O (200 mL) and then with brine (150 mL), dried over _Na2SO4 , and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 30% EtOAc in hexanes) to give 8-(4 _- methoxybenzyl)-6-methyl-1-(4-(trifluoromethyl)benzoyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (9 g, 18.92 mmol, 82.04% yield) as a solid. MS (ESI) m/z [M+H]+: 476.15 and MS (ESI) m/z [M+Na]+: 498.05.

Step 2: Synthesis of 6-methyl-1-(4-(trifluoromethyl)benzoyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine4,7(6H)-dione

To a solution of 8-(4-methoxybenzyl)-6-methyl-1-(4-(trifluoromethyl)benzoyl)hexahydro-4H-pyrazino[1,2a]pyrimidine-4,7(6H)-dione (9 g, 18.92 mmol) in _CH3CN : _H2O (2:1, 150 mL) maintained at 0 °C was added CAN (31.15 g, 56.82 mmol) and the reaction mixture was allowed to stir at room temperature for 3 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was quenched with saturated aq. _NaHCO3 (200 mL) and extracted with EtOAc (200 mL x 2). The combined organic layer was washed with _H2O (200 mL) and then brine (150 mL), dried over _Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 10% MeOH in DCM) to give 6-methyl _- 1-(4-(trifluoromethyl)benzoyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (3.5 g, 9.85 mmol, 52.8% yield) as a solid. MS (ESI) m/z [M+H+ _CH3CN ]+: 397.0. ¹ H NMR (400 MHz, DMSO-06) δ 1.25-1.46 (m, 3H) 2.15-2.30 (m, 1H) 2.56-2.69 (m, 1H) 3.16 (d, J=4.99 Hz, 1H) 3.22-3.30 (m, 1H) 3.42-3.72 (m, 2H) 4.70-4.87 (m, 1H) 5.85-5.95 (m, 1H) 7.75 (d, J=7.98 Hz, 2H) 7.86 (d, J=7.98 Hz, 2H) 8.11 (br s, 1H).

Example S16. General procedure A for the synthesis of final compounds

To a solution of 6-methyl-1-(4-(trifluoromethyl)benzoyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (200 mg, 0.56 mmol) in DMF (2 mL) was added KOtBu (1 M in THF, 1.69 mmol, 1.69 mL) followed by alkyl halide (1.12 mmol), and the reaction mixture was microwaved at

- 50 049703

120°C for 1 h. The reaction mixture was cooled to room temperature and quenched with _H2O (25 mL). The aqueous layer was extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine and concentrated. The crude product was purified by CombiFlash.

Example S17. Synthesis of compound 15

Compound 15 was synthesized by General Procedure A using (bromomethyl)cyclopentane as the alkyl halide. MS (ESI) m/z [M+H]+: 438.65. ^1H NMR (400 MHz, _DMSOd6 ) δ 1.02-1.26 (m, 3H) 1.28-1.42 (m, 2H) 1.44-1.76 (m, 6H) 1.80-2.08-2.33 (m, 2H) 2.55-2.71 (m, 1H) 3.22 (dd, J=12.96, 7.48 Hz, 1H) 3.26-3.32 (m, 1H) 3.39 (d, J=6.98 Hz, 1H) 3.49-3.57 (m, 1H) 3.59-3.74 (m, 1H) 3.76-3.91 (m, 1H) 4.80-4.90 (m, 1H) 5.95-6.05 (m, 1H) 7.72-7.79 (m, 2H) 7.84-7.91 (m, 2H).

Example S18. Synthesis of compound 16

Compound 16 was synthesized by General Procedure A using bromomethylcyclobutane as the alkyl halide. MS (ESI) m/z [M+H]+: 424.15. 1H NMR (400 MHz, DMSO-d^δ 1.291.44 (m, 2H) 1.58-1.89 (m, 4H) 1.90-2.08 (m, 2H) 2.16-2.31 (m, 1H) 2.55-2.70 (m, 2H) 3.18-3.31 (m, 1H) 3.253.26 (m, 1H) 3.34-3.42 (m, 1H) 3.36-3.57 (m, 2H) 3.60-3.69 (n, 1H) 3.71-3.83 (m, 1H) 4.75-4.89 (m, 1H) 5.906.05 (m, 1H) 7.70-7.79 (m, 2H) 7.87 (d, J=8.31 Hz, 2H).

Example S19. Synthesis of compound 19

Compound 19 was synthesized by General Procedure A using (2bromoethyl)cyclopentane as the alkyl halide. MS (ESI) m/z [M+H]+: 452.35. ^1H NMR (400 MHz, DMSO-d^δ 0.94-1.18 (m, 3H) 1.26-1.61 (m, 9H) 1.66-1.83 (m, 2H) 2.16-2.31 (m, 1H) 2.56-2.70 (m, 1H) 3.163.28 (m, 1H) 3.35-3.56 (m, 3H) 3.60-3.73 (m, 1H) 3.77-3.90 (m, 1H) 4.72-4.92 (m, 1H) 5.94-6.06 (m, 1H) 7.77 (d, J=7.98 Hz, 2H)

7.87 (d, J=7.98 Hz, 2H).

Example S20. Synthesis of Compound 20

Compound 20 was synthesized by General Procedure A using (2bromoethyl)cyclobutane as the alkyl halide. MS (ESI) m/z [M+H]+: 438.25. 1H NMR (400 MHz, DMSO^) δ 1.27-1.44 (m, 3H) 1.50-1.71 (m, 4H) 1.71-1.88 (m, 2H) 1.93-2.09 (m, 2H) 2.13-2.34 (m, 2H) 2.562.70 (m, 2H) 3.25-3.32 (m, 1H) 3.35-3.42 (m, 1H) 3.45-3.55 (m, 1H) 3.59-3.72 (m, 1H) 3.74-3.90 (m, 1H) 4.754.89 (m, 1H) 5.94-6.05 (m, 1H) 7.71-7.79 (m, 2H) 7.87 (d, J=8.31 Hz, 2H).

Example S21. Synthesis of compound 21

Compound 21 was synthesized according to General Procedure A using 1-bromobutane as the alkyl halide. MS (ESI) m/z [M+H]+: 412.20. 1H NMR (400 MHz, DMSO-d^δ 0.81-0.97 (m, 3H) 1.15-1.57 (m, 7H) 2.15-2.31 (m, 1H) 2.57-2.69 (m, 1H) 3.14-3.28 (m, 1H) 3.35-3.60 (m, 3H) 3.62-3.73 (m, 1H) 3.74-3.92 (m, 1H) 4.75-4.91 (m, 1H) 5.94-6.06 (m, 1H) 7.76 (d, J=7.34 Hz, 2H) 7.87 (d, J=7.83 Hz, 2H).

Example S22. Synthesis of Compound 22

Compound 22 was synthesized by General Procedure A using 4-bromobut-1-ene as the alkyl halide. MS (ESI) m/z [M+H]+: 410.20. 1H NMR (400 MHz, DMSO-d^δ 1.28-1.45 (m, 3H) 2.14-2.38 (m, 3H) 2.55-2.69 (m, 1H) 3.36-3.57 (m, 4H) 3.58-3.72 (m, 1H) 3.75-3.89 (m, 1H) 4.75-4.90 (m, 1H) 4.98-5.19 (m, 2H) 5.69-5.84 (m, 1H) 5.93-6.05 (m, 1H) 7.76 (d, J=7.98 Hz, 2H) 7.88 (d, J=7.98 Hz, 2H).

Example S23. Synthesis of compound 23

Compound 23 was synthesized according to General Procedure A using 1-bromo-2-methylpropane as the alkyl halide. MS (ESI) m/z [M+H]+: 412.25. 1H NMR (400 MHz, DMSO-d^δ 0.80-0.96 (m, 6H) 1.30-1.48 (m, 3H) 1.85-2.03 (m, 1H) 2.15-2.31 (m, 1H) 2.57-2.70 (m, 1H) 3.06-3.16 (m, 1H) 3.18-3.28 (m, 1H) 3.36-3.45 (m, 1H) 3.44-3.57 (m, 1H) 3.60-3.74 (m, 1H) 3.73-3.87 (m, 1H) 4.77-4.92 (m, 1H) 5.93-6.07 (m, 1H) 7.76 (d, J=7.48 Hz, 2H) 7.87 (d, J=7.48 Hz, 2H).

Example S24. Synthesis of compound 24

Compound 24 was synthesized according to General Procedure A using 2-bromopropane as the alkyl halide. MS (ESI) m/z [M+H]+: 398.55. 1H NMR (400 MHz, DMSO-d^δ 1.10 (d, J=5.49 Hz, 6H) 1.28-1.45 (m, 3H) 2.16-2.24 (m, 1H) 2.56-2.71 (m, 1H) 3.34-3.40 (m, 1H) 3.44-3.79 (m, 3H) 4.59-4.72 (m, 1H) 4.75-4.90 (m, 1H) 5.86-6.00 (m, 1H) 7.79 (d, J=7.98 Hz, 2H) 7.83-7.92 (m, 2H).

Example S25. Synthesis of the intermediate 1-(4-(difluoromethoxy)benzoyl)-6methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione

The synthetic route to obtain this intermediate is shown in Scheme 16.

Scheme 16.

CAN, CHjCN, _H2O

NATU, DI PEA, DMF Stage 1

Step 1: Synthesis of 1-(4-(difluoromethoxy)benzoyl)-8-(4-methoxybenzyl)-6-methylhexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)-dione

- 51 049703

To a solution of 4-(difluoromethoxy)benzoic acid (1.71 g, 9.08 mmol) in DMF (25 mL) maintained at 0 °C were added HATU (3.45 g, 9.08 mmol), DIPEA (4.34 mL, 24.8 mmol) and then 8-(4methoxybenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (2.5 g, 8.25 mmol), and the reaction mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was quenched with ice-cold water (50 mL) and the aqueous layer was extracted with EtOAc (100 mL x 2). The organic layer was washed with cold _H2O (100 mL) and then brine (100 mL), dried over _Na2SO4 , and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 30% EtOAc in hexanes) to give 1- ₍ 4-(difluoromethoxy)benzoyl)-8-(4-methoxybenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (3.5 g, 7.38 mmol, 89.5% yield) as a solid. MS (ESI) m/z [M+H]+: 474.12.

Step 2: Synthesis of 1-(4-(difluoromethoxy)benzoyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine4,7(6H)-dione

To a solution of 1-(4-(difluoromethoxy)benzoyl)-8-(4-methoxybenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (3.0 g, 6.34 mmol) in _CH3CN :H2O (2:1, 45 mL) maintained at 0 °C was added CAN (12.0 g, 21.90 mmol) and the reaction mixture was allowed to stir at room temperature for 3 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was quenched with saturated aq. _NaHCO3 (100 mL) and extracted with EtOAc (200 mL x 2). The combined organic layer was washed with _H2O (250 mL) and then brine (250 mL), dried over _Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 10% MeOH in DCM) to give 1- ₍ 4-(difluoromethoxy)benzoyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione (2.0 g, 5.66 mmol, 89.6% yield) as a solid. MS (ESI) m/z [M+H]+: 353.95. ¹ H NMR (400 MHz, DMSO-4) δ 1.10-1.39 (m, 3H) 2.17-2.18 (m, 1H) 2.52-2.68 (m, 1H) 3.18-3.27 (m, 2H) 3.44-3.71 (m, 2H) 4.69-4.83 (m, 1H) 5.75-5.92 (m, 1H) 7.24 (d, J=7.83 Hz, 2H) 7.32 (t, J=72.0 Hz, 1H) 7.57 (d, J=8.31 Hz, 2H) 8.04 (br s, 1H).

Example S26. General procedure B for the synthesis of final compounds

To a solution of 1-(4-(difluoromethoxy)benzoyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (200 mg, 0.56 mmol) in DMF (4 mL) maintained at 0 °C was added NaH (122 mg, 2.8 mmol, 55% dispersion in mineral oil) and the reaction mixture was stirred at the same temperature for 15 min. To this reaction mixture was added alkyl halide (1.6 mmol) and the reaction mixture was allowed to warm to room temperature and stirred for 3 h. Upon completion, the reaction mixture was quenched with ice-cold _H2O (15 mL) and the aqueous layer was extracted with EtOAc (15 mL x 3). The combined organic layer was washed with brine and concentrated. The crude product obtained was purified by CombiFlash.

Example S27. Synthesis of compound 13

Compound 13 was synthesized by General Procedure B using (bromomethyl)cyclopentane as the alkyl halide. MS (ESI) m/z [M+H]+: 436.05. 1H NMR (400 MHz, DMSOd6) δ 1.07-1.16 (m, 3H) 1.32 (d, J=6.48 Hz, 3H) 1.41-1.73 (m, 7H) 2.06-2.21 (m, 1H) 2.21-2.34 (m, 1H) 2.54-2.70 (m, 1H) 3.14-3.29 (m, 1H) 3.35-3.45 (m, 1H) 3.52-3.69 (m, 1H) 3.75-3.93 (m, 1H) 4.75-4.91 (m, 1H) 5.88-5.99 (m, 1H) 7.27 (d, J=8.48 Hz, 2H) 7.35 (t, J=72.0 Hz, 1H) 7.61 (d, J=8.98 Hz, 2H). Example S28. Synthesis of compound 14.

Compound 14 was synthesized by General Procedure B using (bromomethyl)cyclobutane as the alkyl halide. MS (ESI) m/z [M+H]+: 421.14. 1H NMR (400 MHz, DMSOd ₆ ) δ 1.16-1.25 (m, 1H) 1.27-1.43 (m, 3H) 1.54-1.73 (m, 2H) 1.73-1.86 (m, 2H) 1.89-2.03 (m, 2H) 2.24 (d, J=17.12 Hz, 1H) 2.53-2.69 (m, 2H) 3.20-3.28 (m, 1H) 3.29-3.40 (m, 1H) 3.40-3.66 (m, 2H) 3.69-3.87 (m, 1H) 4.75-4.86 (m, 1H) 5.74-6.02 (m, 1H) 7.26 (d, J=8.31 Hz, 2H)) 7.33 (t, J=72.0 Hz, 1H) 7.59 (d, J=8.31 Hz, 2H).

Example S29. Synthesis of compound 17

Compound 20 was synthesized by General Procedure B using 1-bromobutane as the alkyl halide. MS (ESI) m/z [M+H]+: 410.0. 1H NMR (400 MHz, DMSOD ₆ ) δ 0.81-0.96 (m, 3H) 1.15-1.39 (m, 4H) 1.40-1.55 (m, 2H) 2.26 (d, J=16.95 Hz, 1H) 2.53-2.70 (m, 2H) 3.12-3.30 (m, 2H) 3.38-3.46 (m, 1H) 3.56-3.74 (m, 2H) 3.75-3.92 (m, 1H) 4.84 (k, J=6.81 Hz, 1H) 5.86-6.06 (m, 1H) 7.28 (d, J=7.98 Hz, 2H) 7.36 (t, J=72.0 Hz, 1H) 7.62 (d, J=8.48 Hz, 2H).

Example S30. Synthesis of compound 18

Compound 18 was synthesized by General Procedure B using 4-bromobut-1-ene as the alkyl halide. MS (ESI) m/z [M+H]+: 408.06. 1H NMR (400 MHz, DMSO D ₆ ) δ 1.16-1.45 (m, 3H) 2.18-2.33 (m, 3H) 2.53-2.70 (m, 1H) 3.36-3.46 (m, 3H) 3.51-3.72 (m, 2H) 3.74-3.90 (m, 1H) 4.84 (q, J=6.65 Hz, 1H) 4.91-5.15 (m, 2H) 5.67-5.84 (m, 1H) 5.86-6.03 (m, 1H) 7.29 (d, J=8.48 Hz, 2H) 7.36 (t, J=72.0 Hz, 1H) 7.61 (d, J=8.48 Hz, 2N).

Example S31. Synthesis of compound 27

Compound 27 was synthesized by General Procedure B using 2-52 049703 (bromomethyl)tetrahydrofuran as the alkyl halide. MS (ESI) m/z [M+H]+: 438.1. 1H NMR (400 MHz, CDCl ₃ ) δ 7.48-7.55 (m, 2H), 7.20-7.30 (m, 2H), 6.40-6.76 (m, 1H), 5.90-6.20 (m, 1H), 5.16-5.26 (m, 1H), 4.06-4.17 (m, 2H), 3.82-3.92 (m, 4H), 3.61-3.77 (m, 2H), 2.83-2.99 (m, 1H), 2.47-2.59 (m, 2H), 2.01-2.12 (m, 4H), 1.49 (s, 3H).

Example S32. Synthesis of compound 28

Compound 28 was synthesized by General Procedure B using (2bromoethyl)benzene as the alkyl halide. MS (ESI) m/z [M+H]+: 458.10. ^1H NMR (400 MHz, CDCl ₃ ) δ 7.40-7.50 (m, 2H), 7.20-7.28 (m, 2H), 7.33-7.43 (m, 5H), 6.40-6.76 (m, 1H), 5.90-6.20 (m, 1H), 5.16-5.26 (m, 1H), 3.72-3.96 (m, 2H), 3.44-3.52 (m, 1H), 3.25-3.35 (m, 2H), 2.83-2.99 (m, 2H), 2.47-2.59 (m, 1H), 2.42-2.60 (m, 1H), 2.30-2.57 (m, 1H), 1.49 (s, 3H).

Example S33. Synthesis of compound 29

Compound 29 was synthesized by General Procedure B using 4-(2-bromoethyl)pyridine as the alkyl halide. MS (ESI) m/z [M+H]+: 459.10. 1H NMR (400 MHz, CDCl ₃ ) δ 8.50-8.58 (m, 2H), 7.24-7.46 (m, 4H), 7.18 (d, J=7.99 Hz, 2H), 6.40-6.76 (m, 1H), 5.90-6.20 (m, 1H), 5.16-5.26 (m, 1H), 3.72-3.96 (m, 2H), 3.44-3.52 (m, 1H), 3.25-3.35 (m, 2H), 2.83-2.99 (m, 2H), 2.47-2.59 (m, 1H), 2.42-2.60 (m, 1H), 2.30-2.57 (m, 1H), 1.49 (s, 3H).

Example S34. Synthesis of compound 30

Compound 30 was synthesized by General Procedure B using (3bromopropyl)cyclopropane as the alkyl halide. MS (ESI) m/z [M+H]+: 459.10. 1H NMR (400 MHz, CDCl ₃ ) δ 8.50-8.58 (m, 2H), 7.24-7.46 (m, 4H), 7.18 (d, J=7.99 Hz, 2H), 6.40-6.76 (m, 1H), 5.90-6.20 (m, 1H), 5.16-5.26 (m, 1H), 3.72-3.96 (m, 2H), 3.44-3.52 (m, 1H), 3.25-3.35 (m, 2H), 2.83-2.99 (m, 2H), 2.47-2.59 (m, 1H), 2.42-2.60 (m, 1H), 2.30-2.57 (m, 1H), 1.49 (s, 3H).

Example S35. Synthesis of compound 31

Compound 31 was synthesized by General Procedure B using (2bromoethyl)cyclopropane as the alkyl halide. MS (ESI) m/z [M+H]+: 422.2. 1H NMR (400 MHz, _CDCl3 ) δ 7.48 (d, J=8.01 Hz, 2H), 7.20-7.28 (m, 2H), 6.40-6.76 (m, 1H), 5.90-6.20 (m, 1H), 5.16-5.26 (m, 1H), 3.72-3.96 (m, 1H), 3.46-3.64 (m, 5H), 2.46-2.64 (m, 2H), 1.43-1.56 (m, 5H), 0.43-0.65 (m, 2H), 0.75-0.85 (m, 2H).

Example S36. Synthesis of compound 32

Compound 32 was synthesized by General Procedure B using 1-bromo-2methoxyethane as the alkyl halide. MS (ESI) m/z [M+H]+: 412.1. 1H NMR (400 MHz, _DMSOD6 ) δ 7.52-7.62 (m, 2H), 7.16-7.34 (m, 3H), 5.85-5.95 (m, 1H), 4.80-4.90 (m, 1H), 3.85-3.95 (m, 1H), 3.70-3.80 (m, 2H), 3.25-3.46 (m, 5H), 3.22 (s, 3H), 2.62-2.72 (m, 1H), 2.20-2.30 (m, 1H), 1.49 (s, 3H).

Example S37. Synthesis of compound 33

Compound 33 was synthesized by General Procedure B using 1-bromo-3methoxypropane as the alkyl halide. MS (ESI) m/z [M+H]+: 426.20. 1H NMR (400 MHz, DMSOd6) δ 7.52-7.62 (m, 2H), 7.16-7.34 (m, 3H), 5.85-5.95 (m, 1H), 4.80-4.90 (m, 1H), 3.85-3.95 (m, 1H), 3.70-3.80 (m, 2H), 3.58-3.68 (m, 2H), 3.45-3.55 (m, 4H), 3.22 (s, 3H), 2.62-2.72 (m, 1H), 2.20-2.30 (m, 2H), 1.49 (s, 3H).

Example S38. Synthesis of compound 36

Compound 36 was synthesized by General Procedure B using (2bromoethyl)methylsulfone as the alkyl halide. MS (ESI) m/z [M+H]+: 459.95. 1H NMR (400 MHz, Chloroform) δ 7.49 (d, J=8.01 Hz, 2H), 7.15-7.26 (m, 2H), 6.40-6.76 (m, 1H), 5.90-6.20 (m, 1H), 5.15-5.25 (m, 1H), 3.86-3.97 (m, 3H), 3.66-3.77 (m, 2H), 3.38-3.49 (m, 3H), 2.97 (s, 3H), 2.59-2.69 (m, 2H), 1.49 (s, 3H).

Example S39. Synthesis of compound 34.

Step 1. To a solution of 1-(4-(difluoromethoxy)benzoyl)-6-methylhexahydro-4H-pyrazino[1,2a-pyrimidine]-dione (0.300 g, 0.849 mmol) in DMF (6 mL) was added _{Cs2CO3 (} 0.827 g, 2.547 mmol) followed by (2-bromoethoxy)(tert-butyl)dimethylsilane (0.243 g, 1.018 mmol) at 0 °C, and the reaction mixture was heated at 120 °C in a sealed tube for 1 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was slowly quenched with ice-cold water (30 mL) and extracted with EtOAc (50 mL).

The combined organic layer was washed with ice-cold brine (3 x 30 mL), dried _{over Na2SO4} and concentrated under reduced pressure to give 8-(2-((tert-butyldimethylsilyl)oxy)ethyl)-1-(4-(difluoromethoxy)benzoyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.250 g, crude). The crude compound was used as such for the next reaction without further purification. MS (ESI) m/z [M+H]+: 512.10.

Step 2. To a solution of 8-(2-((tert-butyldimethylsilyl)oxy)ethyl)-1-(4-(difluoromethoxy)benzoyl)-6methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.250 g, 0.4886 mmol) in THF (5 mL) was added TBAF (3 mL) at 0 °C. The reaction mixture was allowed to reach room temperature and stirred for 6 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was slowly quenched with ice-cold water (5 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layer was washed with ice-cold brine (10 mL), dried over Na ₂ SO ₄

- 53 049703 and concentrated under reduced pressure to give the crude compound. The resulting crude compound was purified by column chromatography (silica gel 60-120 mesh; 10% MeOH in DCM) to give 1-(4-(difluoromethoxy)benzoyl)-8-(2-hydroxyethyl)-6-methylhexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.102 g, 52% yield) as a white solid. MS (ESI) m/z [M+H]+: 398.2. ^1H NMR (400 MHz, DMSO-de) δ 7.52-7.62 (m, 2H), 7.16-7.34 (m, 3H), 5.92-6.02 (m, 1H), 6.78-6.88 (m, 2H), 3.86-3.92 (m, 1H), 3.47-3.62 (m, 6H), 3.21-3.31 (m, 1H), 2.57-2.67 (m, 1H), 2.25-3.35 (m, 1H), 1.49 (s, 3H).

Example S40. Synthesis of compound 35

Step 1. To a solution of 1-(4-(difluoromethoxy)benzoyl)-6-methylhexαhydro-4H-pyrazino[1,2a]pyrimidine-4,7(6H)-dione (0.300 g, 0.849 mmol) in DMF (6 mL) was added NaH (0.050 g, 1.274 mmol) followed by 2-bromoacetonitrile (0.112 g, 0.933 mmol) at 0 °C and the reaction mixture was allowed to stand at room temperature for 1 h. Upon completion, the reaction mixture was slowly quenched with ice-cold water (70 mL) and extracted with EtOAc (100 mL). The combined organic layer was washed with ice-cold brine (100 mL), dried _{over Na2SO4} and concentrated under reduced pressure to give the crude compound. The resulting crude compound was purified by column chromatography (silica gel 60-120 mesh; 10% MeOH in DCM) to give 2-(1-(4-(difluoromethoxy)benzoyl)-6-methyl-4,7-dioxooctahydro-8Hpyrazino[1,2-a]pyrimidin-8-yl)acetonitrile (0.120 g, 36% yield) as a white solid. MS (ESI) m/z [M+H]+: 393.05.

Step 2: To a solution of 2-(1-(4-(difluoromethoxy)benzoyl)-6-methyl-4,7-dioxooctahydro-8Hpyrazino[1,2-a]pyrimidin-8-yl)acetonitrile (0.120 g, 0.305 mmol) in ethanol (5 mL) was added conc. HCl (0.100 mL) followed by platinum oxide (0.012 g, 0.030 mmol) at room temperature, and the reaction mixture was heated under hydrogen gas for 3 h. Upon completion, the reaction mixture was filtered through a pad of celite. The pad of celite was washed with ethanol (20 mL), and the filtrate was concentrated under reduced pressure to give the crude compound. The crude compound was triturated with n-pentane to give 8-(2-aminoethyl)-1-(4(difluoromethoxy)benzoyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.110 g, 90% yield) as a white solid. MS (ESI) m/z [M+H]+: 397.05. 1H NMR (400 MHz, DMSO _d6 ) δ 7.96 (s, 2H), 7.55-7.65 (m, 2H), 7.20-7.35 (m, 3H), 5.90-6.20 (m, 1H), 4.85-4.95 (m, 1H), 3.82-3.92 (m, 1H), 3.55. - 3.85 (m, 2H), 3.35-3.45 (m, 3H), 2.95-3.05 (m, 2H), 2.60-2.70 (m, 1H), 2.20-2.30 (m, 1H), 1.35 (s, 3H).

Example S41. General procedure C for the synthesis of final compounds

To a solution of 1-(4-(difluoromethoxy)benzoyl)-6-methylhexahydro-4H-pyrαzino[1,2-a]pyrimidine4,7(6H)-dione (0.200 g, 0.566 mmol) in DMF (5 mL) was added _Cs2CO3 ( _0.735 g, 2.264 mmol, 4 equiv) followed by alkyl halide (0.679 mmol, 1.2 equiv) at 0 °C and the reaction mixture was heated at 50 °C under microwave irradiation for 1 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was slowly quenched with ice-cold water (6 mL) and extracted with EtOAc (20 mL x 3). The combined organic layer was washed with saturated brine (10 mL), _dried over _Na2SO4 and concentrated under reduced pressure to obtain a crude compound. The resulting crude compound was purified by column chromatography to obtain the target compound.

Example S42. Synthesis of compound 25

Compound 25 was synthesized by General Procedure C using 2-(2iodoethyl)furan as the alkyl halide. MS (ESI) m/z [M+H]+: 448.10. 1H NMR: 5 7.40-7.50 (m, 2H), 7.28-7.38 (m, 1H), 7.15-7.25 (m, 2H), 6.39-6.78 (m, 1H), 6.25-6.35 (m, 1H), 5.90-6.12 (m, 2H), 5.25-5.35 (m, 1H), 5.10-5.20 (m, 1H), 3.70-3.80 (m, 1H), 3.50-3.60 (m, 1H), 3.20-3.40 (m, 2H), 2.95-3.05 (m, 3H), 2.45-2.60 (m, 2H), 1.59 (s, 3H).

Example S43. Synthesis of compound 26

Compound 26 was synthesized by General Procedure C using 2-(2-bromoethyl)thiophene as the alkyl halide. MS (ESI) m/z [M+H]+: 464.1. 1H NMR (400 MHz, _CDCl3 ) δ 7.40-7.48 (m, 2H), 7.15-7.26 (m, 3H), 6.85-6.95 (m, 2H), 6.39-6.95 (m, 2H), 5.90-6.20 (m, 1H), 5.15-5.25 (m, 1H), 3.72-3.96 (m, 2H), 3.47-3.54 (m, 1H), 3.32-3.42 (m, 3H), 3.10-3.20 (m, 2H), 2.42-2.56 (m, 2H), 1.49 (s, 3H).

Example S44. Synthesis of the intermediate 1-(4-(difluoromethoxy)benzyl)-6-methylhexahydro4H-pyrazino[1,2-a]pyrimidine-4,7(6H-dione

- 54 049703

Step 1: Synthesis of (9H-fluoren-9-yl)methyl 6-methyl-4,7-dioxohexahydro-2H-pyrazino[1,2a]pyrimidine-1(6H)-carboxylate

A solution of (9H-fluoren-9-yl)methyl 8-(4-methoxybenzyl)-6-methyl-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (1.0 g, 26.63 mmol) in TFA (10 mL) was stirred at 130 °C for 2 h in a microwave oven. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated in vacuo and the crude product was extracted with ethyl acetate (100 mL) and saturated sodium bicarbonate solution. The organic layer was dried over anhydrous _Na2SO4 , concentrated in vacuo and purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give ( _9H -fluoren-9-yl)methyl 6-methyl-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (300 mg, 42% yield) as a sticky solid. MS (ESI) m/z [M+H]+: 406.

Step 2: Synthesis of 6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione

To a solution of (9H-fluoren-9-yl)methyl 6-methyl-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (300 mg, 0.74 mmol) in _{CH2Cl2 (} 5 mL) was added diethylamine (6 mL). The reaction mixture was stirred at room temperature for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated and the crude product was purified by column chromatography (silica gel 100-200 mesh; 10% MeOH in DCM) to give 6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (120 mg, 92% yield) as a white solid. MS (ESI) m/z [M+H] ⁺ : 184.

Step 3: Synthesis of 1-(4-(difluoromethoxy)benzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine4,7(6H)-diox

To a solution of 6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.700 g, 3.820 mmol) in DMF (8.0 mL) was added K ₂ CO ₃ (1.58 g, 11.46 mmol) at room temperature and stirred for 10 min. To the resulting reaction mixture was added 1-(bromomethyl)-4-(difluoromethoxy)benzene (1.086 g, 4.584 mmol) and the reaction mixture was heated at 80 °C for 6 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was cooled to room temperature, quenched with water (50 mL) and extracted with EtOAc (50 mL x 2).

The combined organic layers were washed with brine (20 mL), dried _{over Na2SO4} , and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 1-(4(difluoromethoxy)benzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.550 g, 43.0% yield) as a white solid. MS (ESI) m/z [M+H] ⁺ : 340.34.

Example S45. General procedure D for the synthesis of final compounds

To a solution of 1-(4-(difluoromethoxy)benzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-diox (0.100 g, 0.2949 mmol) in DMF (2 mL) was added NaH (0.021 g, 0.8847 mmol) at 0 °C followed by the appropriate alkyl halide (2 equiv) and the reaction mixture was allowed to warm to room temperature and stirred for 5 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was slowly quenched with saturated aq. NaHCO3 (2 mL) and extracted with EtOAc (10 mL x 2). The combined organic layers were washed with H2O (5 mL) and then brine (5 mL), dried _{over Na2SO4} and concentrated under reduced pressure. The crude material was purified by combiflash column chromatography (5% MeOH in DCM) to afford the final product.

Example S46. Synthesis of compound 37

Compound 37 was synthesized according to General Procedure D using 4-bromo-1,1,1-trifluorobutane as the alkyl halide. MS (ESI) m/z [M+H] ⁺ : 354.2. 1H NMR (400 MHz, CDCl ₃ ): δ 1.41 (d, J=7.13 Hz, 3H), 1.71-1.86 (m, 2H), 2.01-2.15 (m, 2H), 2.26-2.35 (m, 1H), 2.60-2.67 (m, 1H), 2.89-3.01 (m, 1H), 3.07-3.15 (m, 1H), 3.21-3.34 (m, 2H), 3.46-3.65 (m, 2H), 3.81-3.95 (m, 2H), 4.35-4.41 (m, 1H), 5.205.29 (m, 1H), 6.53 (t, J=72.0 Hz, 1H), 7.08-7.16 (m, 2H), 7.30-7.36 (m, 2H).

Example S47. Synthesis of compound 38

Compound 38 was synthesized according to General Procedure D using (2bromoethyl)cyclopentane as the alkyl halide. MS (ESI) m/z [M+H] ⁺ : 436.2. 1H NMR (400 MHz, CDCl3) δ 1.01-1.15 (m, 2H), 1.41 (d, J=7.13 Hz, 3H), 1.45-1.62 (m, 8 H), 1.66-1.80 (m, 2H), 2.23-2.34 (m, 1H),

- 55 049703

2.58-2.72 (m, 1H), 2.89-2.98 (m, 1H), 3.04-3.18 (m, 2H), 3.23-3.33 (m, 1H), 3.43-3.54 (m, 1H), 3.55-3.65 (m, 1H), 3.78-3.93 (m, 1H), 4.31-4.39 (m, 1H), 5.15-5.26 (m, 1H), 6.53 (t, J=72.0 Hz, 1H), 7.13 (d, J=8.50 Hz, 2H), 7.34 (d, J=8.50 Hz, 2H).

Example S48. Synthesis of compound 39

Compound 39 was synthesized according to General Procedure D using 4-bromobut-1-ene as the alkyl halide. MS (ESI) m/z [M+H]+: 394.2. ¹ H NMR (400 MHz, CDCl ₃ ) δ 1.41 (d, J=7.13 Hz, 3H), 2.23-2.35 (m, 3H), 2.60-2.71 (m, 1H), 2.92-3.01 (m, 1H), 3.06-3.14 (m, 1H), 3.22-3.46 (m, 3H), 3.53-3.64 (m, 1H), 3.79-3.93 (m, 2H), 4.28-4.38 (m, 1H), 4.91-5.00 (m, 2H), 5.16-5.26 (m, 1H), 5.64-5.76 (m, 1H), 6.52 (t, J=72.0 Hz, 1H), 7.10-7.16 (m, 2H), 7.30-7.36 (m, 2H).

Example S49. Synthesis of compound 40

Compound 40 was synthesized by General Procedure D using (2bromoethyl)cyclobutene as the alkyl halide. MS (ESI) m/z [M+H]+: 422.25 1H NMR (400 MHz, CDCl ₃ ) δ 1.41 (d, J=7.13 Hz, 3H), 1.56-1.65 (m, 4H), 1.73-1.92 (m, 2H), 1.95-2.07 (m, 2H), 2.14-2.25 (m, 1H), 2.26-2.35 (m, 1H), 2.59-2.72 (m, 1H), 2.91-2.99 (m, 1H), 3.04-3.14 (m, 2H), 3.23-3.43 (m, 2H), 3.53-3.63 (m, 1H), 3.87 (k, J=13.38 Hz, 2H), 4.29-4.39 (m, 1H), 5.17-5.24 (m, 1H), 6.53 (t, J=72.0 Hz, 1H), 7.13 (d, J=8.63 Hz, 2H), 7.30-7.37 (m, 2H).

Example S50. Synthesis of Compound 41

Compound 41 was synthesized according to General Procedure D using 1-bromobutane as the alkyl halide. MS (ESI) m/z [M+H]+: 396.05. 1H NMR (400 MHz, DMSOL ₆ ) δ 0.86 (t, J=7.34 Hz, 3H), 1.14-1.24 (m, 2H), 1.24-1.30 (m, 2H), 1.38-1.50 (m, 2H), 1.98-2.10 (m, 1H), 2.53-2.61 (m, 2H), 2.642.77 (m, 2H), 3.07-3.25 (m, 3H), 3.32-3.41 (m, 1H), 3.62-3.73 (m, 1H), 3.87-3.93 (m, 2H), 4.49-4.58 (m, 1H), 4.84-4.94 (m, 1H), 7.15 (d, J=8.56 Hz, 2H), 7.22 (t, J=72.0 Hz, 1H), 7.43 (d, J=8.56 Hz, 1H).

Example S51. Synthesis of compound 52

Compound 52 was synthesized according to General Procedure D using 2-trifluoromethyl-1-bromoethane as the alkyl halide. MS (ESI) m/z [M+H]+: 420.16. 1H NMR (400 MHz, CDCl ₃ ) δ ppm 7.31-7.38 (m, 2H), 7.11-7.16 (m, 2H), 6.31-6.73 (m, 1H), 5.26 (k, J=7.21 Hz, 1H), 4.23-4.44 (m, 2H), 3.98-4.13 (m, 1H), 3.80-3.93 (m, 3H), 3.59 (t, J=11.07 Hz, 1H), 3.10 (dd, J=11.51, 3.75 Hz, 1H), 2.90-2.99 (m, 1H), 2.622.72 (m, 1H), 2.32 (dd, J=4.38, 2.38 Hz, 1H), 2.28 (dd, J=4.31, 2.31 Hz, 1H), 1.48 (d, J=7.25 Hz, 1H), 1.41 (d, J=7.13 Hz, 3H).

Example S52. General procedure E for the synthesis of final compounds

To a solution of 6-methyl-8-(2-methylbutyl)hexαhydro-4H-pyrαzino[1,2-a]pyrimidine-4,7-dione (0.300 g, 1.184 mmol) in DMF (6 mL) stirred in a flask immersed in an ice-water bath was added cesium carbonate (0.771 g, 2.368 mmol, 2 equiv) followed by the appropriate alkyl halide (1.1 equiv). The flask was removed from the bath and stirred until TLC showed complete consumption of the starting material. The reaction mixture was poured into ice-water (70 mL) and the aqueous layer was extracted with EtOAc (100 mL). The organic layer was washed with ice-cold brine (50 mL x 3), dried over _{anhydrous Na2SO4} and concentrated under reduced pressure. The crude product was purified by preparative HPLC to afford the title compound.

Example S53. Synthesis of compound 42

Compound 42 was synthesized by General Procedure E using 4-(bromomethyl)-2-chloro-1-(trifluoromethyl)benzene as the alkyl halide. MS (ESI) m/z [M+H]+: 362.2. 1H NMR (400 MHz, DMSOO δ 0.75-0.89 (m, 3H), 0.82-0.87 (m, 3H), 0.96-1.13 (m, 1H), 1.23-1.31 (m, 4H), 1.64-1.75 (m, 1H), 2.06-2.09 (m, 1H), 2.55-2.62 (m, 1H), 2.65-2.76 (m, 1H), 3.05-3.15 (m, 1H), 3.15-3.26 (m, 3H), 3.64-3.74 (m, 1H), 3.84-3.95 (m, 2H), 4.52-4.60 (m, 1H), 4.86-4.94 (m, 1H), 7.17 (t, J=8.76 Hz, 2H), 7.41 (dd, J=8.19, 5.82 Hz, 2H).

Example S54. Synthesis of compound 43

Compound 43 was synthesized by General Procedure E using 4-(bromomethyl)-2-chloro-1-(trifluoromethyl)benzene as the alkyl halide. MS (ESI) m/z [M+H]+: 446.2. 1H NMR (400 MHz, DMSOL ₆ ) 0.72-0.80 (m, 3H), 0.80-0.87 (m, 3H), 0.96-1.10 (m, 1H), 1.21-1.27 (m, 1H), 1.28-1.34 (m, 3H), 1.62-1.79 (m, 1H), 2.00-2.13 (m, 1H), 2.53-2.65 (m, 1H), 2.66-2.76 (m, 1H), 3.00-3.10 (m, 1H), 3.17-3.29 (m, 3H), 3.62-3.72 (m, 1H), 4.00-4.08 (m, 2H), 4.55-4.65 (m, 1H), 4.85-4.95 (m, 1H), 7.52-7.60 (m, 1H), 7.73 (s, 1H), 7.80-7.88 (m, 1H).

Example S55. Synthesis of compound 44.

To a solution of 6-methyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7-dione (0.420 g, 1.657 mmol) and 1H-indole-3-carbaldehyde (0.264 g, 1.823 mmol) in DCE (15 mL) was added acetic acid (1 mL, 1.657 mmol) and the reaction mixture was heated at 80 °C for 1 h. To the resulting reaction mixture was added NaBH ₄ (0.188 g, 4.973 mmol) portionwise and the reaction mixture was heated at 80 °C and stirred for 4 h. When TLC analysis (5% MeOH in DCM) indicated complete consumption of the starting material, the reaction mixture was diluted with water (40 mL) and the aqueous layer was extracted with DCM (100 ml). The organic layer was washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude material

- 56 049703 was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM), then washed with water (30 mL) and dried under reduced pressure to give compound 44 (0.250 g, 39% yield) as an off-white solid. MS (ESI) m/z [M+H]+: 383.4. 1H NMR (400 MHz, DMSOd6) δ 0.70 (t, J=7.09 Hz, 3H), 0.75-0.82 (m, 3H), 0.91-1.11 (m, 1H), 1.22-1.31 (m, 3H), 1.57-1.72 (m, 1H), 1.972.07 (m, 1H), 2.55-2.70 (m, 2H), 2.83 (dt, J=10.91, 2.74 Hz, 1H), 2.95-3.07 (m, 1H), 3.10-3.26 (m, 3H), 3.54-3.69 (m, 1H), 3.96-4.04 (m, 1H), 4.06-4.15 (m, 1H), 4.54-4.64 (m, 1H), 4.84-4.95 (m, 1H), 6.94-7.02 (m, 1H), 7.047.13 (m, 1H), 7.29-7.40 (m, 2H), 7.65 (d, J=7.95 Hz, 1H), 10.95 (s, 1H).

Example S55. Synthesis of the intermediate 1-(4-fluorobenzyl)-6-methylhexahydro-4Hpyrazino[1,2-a]pyrimidine-4,7(6H)dione

To a solution of 6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (250 mg, 1.40 mmol) in DMF (3 mL) was added potassium carbonate (580 mg, 4.20 mmol) followed by 4-fluorobenzyl bromide (0.320 g, 1.70 mmol), and stirred at 80 °C for 3 h. After completion, the reaction mixture was monitored by TLC (5% MeOH in DCM). The reaction mixture was poured into ice water (50 mL), and the aqueous layer was extracted with EtOAc (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 1-(4-fluorobenzyl)-6methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione (160 mg, 70% yield) as a white solid. MS (ESI) m/z [M+H]+: 292.

Example S56. Synthesis of compound 45

To a solution of 1-(4-fluorobenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione (80 mg, 0.2739 mmol) in DMF (3 mL) in an ice water bath at 0 °C was added NaH (20 mg, 0.2739 mmol) and stirred for 20 min, then (2-bromoethyl)cyclobutane (67 mg, 0.41 mmol) was added after 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was quenched with ice cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 8-(2-cyclobutylethyl)-1-(4-fluorobenzyl)-6-methylhexahydro-4H-pyrazino[1-(pyrimidine)dione]dione (13 mg, 16% yield) as a gummy liquid. MS (ESI) m/z [M+H]+: 374. ¹ H NMR (400 MHz, CD ₃ Cl ₃ ): δ 7.30-7.40 (m, 2H), 7.00-7.10 (m, 2H), 5.15-5.25 (m, 1H), 4.25-4.35 (m, 1H), 3.80-3.95 (m, 2H), 3.55-3.65 (m, 1H), 3.25-3.45 (m, 2H), 3.05-3.20 (m, 2H), 2.90-3.0 (m, 1H), 2.60-2.70 (m, 1H), 2.15-2.40 (m, 2H), 1.75-2.10 (m, 4H), 1.55-1.65 (m, 4H), 1.20-1.30 (m, 3H).

Example S57. Synthesis of compound 46

To a solution of 1-(4-fluorobenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione (80 mg, 0.2739 mmol) in DMF (3 mL) in an ice water bath at 0 °C was added NaH (20 mg, 0.2739 mmol) and stirred for 20 min, then after 3 h (2-bromoethyl)cyclopentane (72 mg, 0.41 mmol) was added, the consumption of the starting material was monitored by TLC, the reaction mixture was quenched with ice cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 8-(2-cyclopentylethyl)-1-(4-fluorobenzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione as a gummy liquid.

Example S58. Synthesis of the intermediate compound of the hydrochloride salt of 6-(fluoromethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1-pyrimidine-N-dione]

Step 1: Synthesis of N(2,2-diethoxyethyl)-2-methylbutan-1-amine

To stirred neat 2,2-diethoxyethan-1-amine (20.0 g, 0.137 mmol) was added 2-methylbutanal (11.60 g, 0.137 mmol) at room temperature and the reaction mixture was heated to 100 °C for 3 h. To the resulting reaction mixture was slowly added ethanol (200 mL) followed by NaBH ₄ (15.40 g, 0.413 mmol) at room temperature and the reaction mixture was stirred for 16 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was cooled to room temperature and slowly quenched with saturated NH ₄ Cl solution (100 mL). The aq. layer was extracted with EtOAc (200 mL x 2). The combined organic layer was washed with brine (400 mL), dried over _Na2SO4 , _filtered and concentrated under reduced pressure to give the crude compound. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 10% MeOH in DCM) to give N-(2,2-diethoxyethyl)-2-methylbutan-1-amine (25.8 g, 88% yield) as a colorless liquid. MS (ESI) m/z [M+H]+: 204.3. 1H NMR (400 MHz, DMSO<) δ 0.80-0.89 (m, 6H) 1.11 (t, J=6.98 Hz, 6H) 1.35-1.48 (m, 2H) 2.28-2.32 (m, 1H) 2.41-2.45 (m, 1H) 2.55 (d, J=5.49 Hz, 2H) 3.42-3.52 (m, 2H) 3.57-3.65 (m, 2H) 4.49 (t, J=5.49 Hz, 1H).

Step 2: (9H-Fluoren-9-yl)methyl(1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-3-hydroxy-1 oxopropan-2-yl)carbamate

To a stirred solution of (((9H-fluoren-9-yl)methoxy)carbonyl)serine (15.0 g, 45.81 mmol) in

- 57 049703 HATU (26.0 g, 68.80 mmol), DIPEA (23.92 mL, 137.61 mmol) and then H-(2,2-diethoxyethyl)-2-methylbutan-1-amine (12.10 g, 59.63 mmol) were added to dry DMF (150 mL) maintained at 0 °C. The reaction mixture was stirred at room temperature for 4 h. After complete consumption of the starting material, the reaction mixture was quenched with ice-cold water (500 mL) and the aqueous layer was extracted with EtOAc (250 mL x 2). The combined organic layer was washed with cold H ₂ O (200 mL) and then brine (200 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude product. The crude material was purified by column chromatography (silica gel 100-200 mesh; 80% EtOAc in hexanes) to give (9H-fluoren-9-yl)methyl (1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-3-hydroxy-1-oxopropan-2-yl)carbamate (21.0 g, 89.43% yield) as a yellow sticky solid. MS (ESI) m/z [M+Na]+: 535.35.

Step 3: Synthesis of 2-amino-H-(2,2-diethoxyethyl)-3-hydroxy-N-(2-methylbutyl)propenamide

To a stirred solution of (9H-fluoren-9-yl)methyl (1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-3hydroxy-1-oxopropan-2-yl)carbamate (21.0 g, 41.01 mmol) in dry DCM (110 mL) maintained at 0 °C was added diethylamine (58 mL, 2.80 vol) and the reaction mixture was stirred at room temperature for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated under reduced pressure to give the crude product. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 2-amino-N-(2,2-diethoxyethyl)-3-hydroxy-N-(2-methylbutyl)propenamide (9.50 g, 80% yield) as a yellow sticky solid. MS (ESI) m/z [M+H]+: 291.4.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl (3-((1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-3hydroxy-1-oxopropan-2-yl)amino)-3-oxopropyl)carbamate

To a stirred solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (9.50 g, 30.54 mmol) in dry DMF (95 mL) maintained at 0 °C were added HATU (17.40 g, 45.81 mmol), DIPEA (16.0 mL, 91.62 mmol) and then 2-amino-N-(2,2-diethoxyethyl)-3-hydroxy-N-(2-methylbutyl)propanamide (13.20 g, 45.81 mmol) at room temperature and the reaction mixture was stirred for 16 h. Upon completion, the reaction mixture was quenched with ice-cold water (200 mL) and the aqueous layer was extracted with EtOAc (200 mL x 2). The organic layer was washed with cold _H2O (500 mL) and then brine (200 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude compound was purified by column chromatography (silica gel 100-200 mesh; 80% EtOAc in hexanes) to give (9H-fluoren-9-yl)methyl (3-((1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-3-hydroxy-1-oxopropan-2-yl)amino)-3-oxopropyl)carbamate (8.0 g, 31.0% yield) as a viscous yellow oil. MS (ESI) m/z [MH] ^- : 582.2.

Step 5: Synthesis of (9H-fluoren-9-yl)methyl 6-(hydroxymethyl)-8-(2-methylbutyl)-4,7dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate

A solution of (9H-fluoren-9-yl)methyl (3-((1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-3-hydroxy-1-oxopropan-2-yl)amino)-3-oxopropyl)carbamate (8.0 g, 13.77 mmol) in formic acid (48.0 mL, 6.0 vol) was stirred at room temperature and the reaction mixture was stirred for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (9Hfluoren-9-yl)methyl 6-(hydroxymethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (6.0 g, crude) as a brown semi-solid. The crude compound was used as is in the next reaction without further purification. MS (ESI) m/z [M+H]+: 492.2.

Step 6: Synthesis of 6-(hydroxymethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine4,7(6H)-dione

To a solution of (9H-fluoren-9-yl)methyl 6-(hydroxymethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (6.0 g, 12.20 mmol) in _CH2Cl2 ( _36.0 mL) was added diethylamine (18.0 mL) at 0 °C, and the reaction mixture was stirred at room temperature for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated under reduced pressure to give the crude compound. The crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 6-(hydroxymethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (3.0 g, 93.75% yield) as a viscous colorless oil. MS (ESI) m/z [M+H]+: 270.20.

Step 7: Synthesis of tert-butyl 6-(hydroxymethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate

To a solution of 6-(hydroxymethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione (3.0 g, 11.15 mmol) in _{CH2Cl2 (} 60 mL) was added triethylamine (4.5 mL, 33.45 mmol) followed by Boe's anhybrid (3.78 mL, 16.72 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 16 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was slowly quenched with ice-cold water (30 mL) and extracted with DCM (40 mL). The organic layer was washed with brine (30 mL), dried over _Na2SO4 , and _concentrated at

- 58 049703 under reduced pressure. The crude compound was purified by column chromatography (silica gel 100-200 mesh; 10% MeOH in DCM) to give tert-butyl 6-(hydroxymethyl)-8-(2-methylbutyl)-4,7dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (8.0 g, 31.0% yield) as a viscous yellow oil. MS (ESI) m/z [M+H]+: 370.25.

Step 8: Synthesis of tert-butyl 6-(fluoromethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1-hydroxycarboxylate

To a solution of 6-(hydroxymethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (1.50 g, 4.065 mmol) in DCM (30 mL) was added DAST (1.97 g, 12.19 mmol) at -78 °C and stirred for 15 min. The reaction mixture was allowed to warm to room temperature and stirred for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with saturated NaHCO ₃ solution (15 mL) and the aqueous layer was extracted with EtOAc (100 mL x 2). The combined organic layer was washed with brine (50 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound. The crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give tert-butyl 6-(fluoromethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (0.800 g, 72.0% yield) as a colorless viscous oil. MS (ESI) m/z [M+H]+: 372.2.

Step 9: Synthesis of 6-(fluoromethyl)-8-(2-methylbutyl)hexahydro-4Hpyrazino [1^^pyrimidineL^^^-dione hydrochloride salt

To a stirred solution of tert-butyl 6-(fluoromethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (1.0 g, 2.695 mmol) in 1,4-dioxane (5 mL) was added 4 M HCl in dioxane (5 mL) at 0 °C and the reaction mixture was stirred at room temperature for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layer was washed with brine (10 mL), dried _{over Na2SO4} and concentrated under reduced pressure to give 6-(fluoromethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione hydrochloride salt (0.630 g, crude) as a brown sticky oil. MS (ESI) m/z [M+H]+ free base: 271.00.

Example S59. Synthesis of compound 47

To a solution of 6-(fluoromethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2a]pyrimidine-4,7(6H)-dione hydrochloride salt (0.150 g, 0.550 mmol) in DMF (1.5 mL) was added K ₂ CO ₃ (0.381 g, 2.760 mmol) followed by 1-(bromomethyl)-4-(difluoromethoxy)benzene (0.261 g, 1.100 mmol), and the reaction mixture was stirred at room temperature for 16 h. Upon completion (monitored by TLC), the reaction mixture was slowly quenched with ice-cold water (6 mL) and extracted with EtOAc (20 mL x 3). The combined organic layer was washed with brine (10 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound. The crude compound was purified by prep. HPLC to afford 1-(4(difluoromethoxy)benzyl)-6-(fluoromethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.040 g, 17.0% yield) as a white solid. MS (ESI) m/z [M+H]+: 428.10. ¹ H NMR (400 MHz, _CDCl3 ) δ 7.34 (d, J=8.01, 2H), 7.11 (d, J=8.01, 2H), 6.32-6.69 (m, 1H), 5.14-5.25 (m, 2H), 4.60-4.76 (m, 2H), 3.84-3.97 (m, 2H), 3.35-3.45 (m, 2H), 3.12-3.40 (m, 4H), 2.85-3.05 (m, 1H), 2.65-2.75 (m, 1H), 2.29-2.34 (m, 1H), 1.65-1.75 (m, 1H), 1.30-1.40 (m, 1H), 1.05 -1.18 (m, 1H), 0.80-0.90 (m, 6 N).

Example S60. Synthesis of compound 48

To a solution of 6-(fluoromethyl)-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2a]pyrimidine-4,7(6H)-dione hydrochloride salt (0.340 g, 1.253 mmol) in DMF (3.4 mL) was added _Cs2CO3 (0.814 g, 2.506 mmol) followed by _1- (bromomethyl)-4-(trifluoromethyl)benzene (0.598 g, 2.506 mmol), and the reaction mixture was stirred at room temperature for 16 h. Upon completion (monitored by TLC), the reaction mixture was slowly quenched with ice-cold water (6 mL) and extracted with EtOAc (20 mL x 3). The combined organic layer was washed with brine (10 mL), dried over _Na2SO4 and concentrated under reduced pressure to give the crude compound. The crude compound was purified by prep-HPLC to give 6-(fluoromethyl)-8-(2-methylbutyl)-1(4-(trifluoromethyl)benzyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (0.045 g, 8.0% yield) as a white solid. MS (ESI) m/z [M+H]+: 430.10. 1H NMR (400 MHz, _CDCl3 ) δ 7.34 (d, J=8.01, 2H), 7.11 (d, J=8.01, 2H), 5.14-5.25 (m, 2H), 4.60-4.76 (m, 2H), 3.84-3.97 (m, 2H), 3.35-3.45 (m, 2H), 3.12-3.40 (m, 4H), 2.85-3.05 (m, 1H), 2.65-2.75 (m, 1H), 2.29-2.34 (m, 1H), 1.65-1.75 (m, 1H), 1.30-1.40 (m, 1H), 1.05-1.18 (m, 1N), 0.80-0.90 (m, 6 N).

Example S61. Synthesis of the intermediate methyl 2-(1-(4-(difluoromethoxy)benzyl)-8-(2methylbutyl)-4,7-dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6-yl)acetate

Step 1: Synthesis of methyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((2,2-diethoxyethyl)(2methylbutyl)amino)-4-oxobutanoate

To a solution of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methoxy-4-oxobutanoic acid

- 59 049703 (1.90 g, 9.475 mmol) stirred at 0 °C in dry DMF (30 mL) was added HATU (3.60 g, 1.137 mmol) followed by DIPEA (2.70 mL, 1.895 mmol) and the reaction mixture was stirred at the same temperature for 10 min. To the resulting reaction mixture was added N-(2.2-diethoxyethyl)-2-methylbutan-1-amine (3.50 g, 9.475 mmol) and then the mixture was allowed to warm to room temperature and stirred for 6 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was quenched with ice-cold water (100 mL) and the aqueous layer was extracted with EtOAc (50 mL x 2). The combined organic layer was washed with cold _H2O (50 mL) and then brine (50 mL), dried over _Na2SO4 and concentrated under reduced pressure to give the crude product. The crude material was purified by CombiFlash column chromatography using 50% EtOAc in n _- hexanes to give methyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-oxobutanoate (4.30 g, 83.0% yield) as a white solid. MS (ESI) m/z [M+H-EtOH]+: 509.2.

Step 2: Synthesis of methyl 3-amino-4-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-oxobutanoate

To a solution of methyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-oxobutanoate (1.36 g, 2.451 mmol) in _CH2Cl2 ( _27.0 mL) was added diethylamine (1.53 mL, 14.71 mmol) at room temperature and the reaction mixture was stirred for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated under reduced pressure to give the crude compound. The crude compound was purified by CombiFlash column chromatography using 5% MeOH in DCM to give methyl 3-amino-4-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-oxobutanoate (0.700 g, 86% yield) as a yellow viscous liquid. MS (ESI) m/z [M+H-EtOH]+: 287.68.

Step 3: Synthesis of methyl 3-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-oxobutanoate

To a stirred solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (0.490 g, 1.594 mmol) in dry DMF (10 mL) maintained at 0 °C were added HATU (0.720 g, 1.913 mmol), DIPEA (0.555 mL, 3.188 mmol) followed by the addition of methyl 3-amino-4-((2,2-diethoxyethyl)(2methylbutyl)amino)-4-oxobutanoate (0.530 g, 1.594 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 6 h. Upon completion, the reaction mixture was quenched with ice-cold water (20 mL) and the aqueous layer was extracted with EtOAc (20 mL x 2). The organic layer was washed with cold H2O (10 mL) and then brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude compound was purified by Combiflash column chromatography using 5% MeOH in DCM to give methyl 3-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-oxobutanoate (0.630 g, 70% yield) as an off-white solid. MS (ESI) m/z [M+H-EtOH] ⁺ : 580.20.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl 6-(2-methoxy-2-oxoethyl)-8-(2-methylbutyl)-4,7dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate

To a stirred solution of methyl 3-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-oxobutanoate (0.300 g, 0.4794 mmol) was added formic acid (1.5 mL) at room temperature and the reaction mixture was stirred for 16 h. Upon completion, the reaction mixture was concentrated and the resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 0-5% MeOH in DCM) to give (9H-fluoren-9-yl)methyl 6-(2-methoxy-2-oxoethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (0.200 g, 80% yield) as a yellow solid. MS (ESI) m/z [M+H]+: 534.67.

Step 5: Synthesis of methyl 2-(8-(2-methylbutyl)-4,7-dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6yl)acetate

To a solution of (9H-fluoren-9-yl)methyl 6-(2-methoxy-2-oxoethyl)-8-(2-methylbutyl)-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (0.240 g, 0.4499 mmol) in _CH2Cl2 ( _0.5 mL) was added diethylamine (0.280 mL) and the reaction mixture was stirred at room temperature for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated and the crude material was purified by combiflash column chromatography using 0-5% MeOH in DCM to give methyl 2-(8-(2-methylbutyl)-4,7dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6-yl)acetate (0.130 g, yield 93%) as a white solid. MS (ESI) m/z [M-H] ⁺ : 310.4.

Step 6: Synthesis of methyl 2-(1-(4-(difluoromethoxy)benzyl)-8-(2-methylbutyl)-4,7-dioxooctahydro-2Hpyrazino[1,2-a]pyrimidin-6-yl)acetate

To a solution of methyl 2-(8-(2-methylbutyl)-4,7-dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6yl)acetate (3.08 g, 9.890 mmol) in DMF (30 mL) was added K ₂ CO ₃ (4.10 g, 29.66 mmol) at room temperature, and the reaction mixture was stirred at 80 °C for 15 min. To the resulting reaction mixture was added 1-(bromomethyl)-4-(difluoromethoxy)benzene (3.48 g, 14.36 mmol), and the stirred mixture was heated to 80 °C for 2 h. Upon completion, the reaction mixture was quenched with ice cold water

- 60 049703 (200 mL) and the aqueous layer was extracted with EtOAc (200 mL x 2). The organic layer was washed with cold _H2O (200 mL) and then with brine (150 mL), dried over _Na2SO4 and concentrated under _reduced pressure. The resulting crude compound was purified by Combiflash column chromatography (5% MeOH in DCM) to give methyl 2-(1-(4-(difluoromethoxy)benzyl)-8-(2-methylbutyl)-4,7-dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6-yl)acetate (2.20 g, 48% yield) as a yellow solid. MS (ESI) m/z [M- _CH3 ] ⁺ : 454.10.

Example S61. Synthesis of compound 49

To a solution of methyl 2-(1-(4-(difluoromethoxy)benzyl)-8-(2-methylbutyl)-4,7-dioxooctahydro-2Hpyrazino[1,2-a]pyrimidin-6-yl)acetate (2.20 g, 4.705 mmol) in THF (22.0 mL) was added NaOH (0.560 g, 14.11 mmol) followed by water (4 mL), and the reaction mixture was stirred at room temperature for 3 h. Upon completion, the reaction mixture was concentrated under reduced pressure. The crude residue was dissolved in water (10 mL), slowly acidified with 6 N HCl (10 mL), and stirred for 5 min. The resulting solid precipitate was filtered through a Buchner funnel and dried under reduced pressure to give 2-(1-(4(difluoromethoxy)benzyl)-8-(2-methylbutyl)-4,7-dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6-yl)acetic acid (0.85 g, yield 40%) as a white solid.

MS (ESI) m/z [M+H] ⁺ : 454.10. ¹ H NMR (400 MHz, CDC13) δ 7.28-7.38 (m, 2H), 7.11 (d, J=7.99 Hz, 2H), 6.33-6.71 (m, 1H), 5.36-5.40 (m, 1H), 4.70-4.80 (m, 1H), 4.65-4.75 (m, 1H), 3.80-4.00 (m, 2H), 3.55-3.65 (m, 1H), 3.35-3.45 (m, 1H), 2.85-3.30 (m, 6 N), 2.70-2.80 (m, 1H), 2.25-2.35 (m, 1H), 1.65-1.76 (m, 1H), 1.25-1.35 (m, 1H), 1.10-1.20 (m, 1H), 0.8-0.9 (m, 6H).

Example S62. Synthesis of Compound 50

To a solution of 2-(1-(4-(difluoromethoxy)benzyl)-8-(2-methylbutyl)-4,7-dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6-yl)acetic acid (0.470 g, 1.036 mmol) in THF (5 mL) was added 1,1'carbonyldiimidazole (0.500 g, 3.109 mmol) at room temperature and the reaction mixture was stirred for 15 min. To the resulting reaction mixture was added aq. NH3 (10 mL) and the reaction mixture was stirred at room temperature for 3 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was slowly quenched with ice-cold water (6 mL) and extracted with EtOAc (20 mL x 3). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to give the crude compound. The resulting crude compound was purified by Combiflash column chromatography using 5% MeOH in DCM followed by prep. HPLC to give 2-(1-(4(difluoromethoxy)benzyl)-8-(2-methylbutyl)-4,7-dioxooctahydro-2H-pyrazino[1,2-a]pyrimidin-6-yl)acetamide (0.070 g, 15% yield) as a white solid. MS (ESI) m/z [M+H] ⁺ : 453.20. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.30-7.40 (m, 2H), 7.05-7.15 (m, 2H), 6.39-6.70 (m, 1H), 5.20-5.40 (m, 2H), 4.75-4.85 (m, 1H), 3.95-4.05 (m, 1H), 3.75-3.85 (m, 1H), 3.50-3.60 (m, 1H), 3.30-3.40 (m, 1H), 3.05-3.25 (m, 2H), 2.85-2.95 (m, 2H), 2.55-2.70 (m, 1H), 2.25-2.35 (m, 1H), 1.70-1.80 (m, 2H), 1.30-1.40 (m, 2H), 1.05-1.20 (m, 2H), 0.750.90 (m, 6H).

Example S63. Synthesis of the intermediate 1-(3-chloro-4-(trifluoromethyl)benzyl)-6methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione

To a solution of 6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (500 mg, 2.732 mmol) in DMF (7 mL) was added potassium carbonate (1.13 g, 8.196 mmol) followed by 4-(bromomethyl)-2-chloro-1(trifluoromethyl)benzene (0.894 g, 3.278 mmol) and stirred at 80 °C for 12 h. After completion of the reaction, it was monitored by TLC (5% MeOH in DCM). The reaction mixture was poured into ice water (50 mL) and the aqueous layer was extracted with EtOAc (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to give 1-(3-chloro-4-(trifluoromethyl)benzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione (320 mg, 42% yield) as a white solid. MS (ESI) m/z [M+H] ⁺ : 376.34.

Example S64. General procedure F for the synthesis of final compounds

To a solution of 1-(3-chloro-4-(trifluoromethyl)benzyl)-6-methylhexahydro-4H-pyrazino[1,2-a]pyrimidine4,7(6H)-dione (150 mg, 0.400 mmol) in DMF (2 mL) at 0 °C was added Cs ₂ CO ₃ (4 equiv) and stirred for 20 min, then the appropriate alkyl halide (1.2 equiv) was added at room temperature and the reaction mixture was heated at 80 °C and stirred for 12 h. After consumption of the starting material (monitored by TLC), the reaction mixture was quenched with ice-cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude material was purified by column chromatography (silica gel 100-200 mesh; 5% MeOH in DCM) to afford the final compounds.

Example S65. Synthesis of compound 51

Compound 51 was synthesized according to General Procedure F using (2bromoethyl)cyclobutane as the alkyl halide. MS (ESI) m/z [M+H] ⁺ : 458.2. 1H NMR (400 MHz, CDC13) δ ppm 7.70 (d, J=8.07 Hz, 1H), 7.54 (s, 1H), 7.33 (d, J=8.68 Hz, 1H), 4.34 (dd, J=10.64, 3.55 Hz, 1H),

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3.87-3.99 (m, 2H), 3.61 (t, J=11.13 Hz, 1H), 3.25-3.42 (m, 2H), 3.08-3.19 (m, 2H), 2.89-2.98 (m, 1H), 2.64-2.74 (m, 1H) 2.29-2.38 (m, 1H) 2.17-2.27 (m, 1H) 1.97-2.09 (m, 2H) 1.72-1.92 (m, 3H) 1.58-1.66 (m, 4H) 1.55 (br s, 3H).

Example S66. Synthesis of compound 54

Compound 54 was synthesized by General Procedure F using (2bromoethyl)cyclopentane as the alkyl halide. MS (ESI) m/z [M+H]+: 472.15. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 7.69 (d, J=8.11 Hz, 1H) 7.52-7.56 (m, 1H) 7.33 (d, J=7.89 Hz, 1H), 5.23 (k, J=7.23 Hz, 1H), 4.36 (dd, J=10.52, 3.29 Hz, 1H), 3.87-3.98 (m, 2H), 3.63 (t, J=11.07 Hz, 1H), 3.46-3.56 (m, 1H), 3.25-3.35 (m, 1H), 3.12-3.23 (m, 2H), 2.87-2.97 (m, 1H), 2.60-2.70 (m, 1H), 2.29-2.37 (m, 1H), 1.66-1.82 (m, 2H), 1.54-1.63 (m, 1H), 1.51 (d, J=2.63 Hz, 2H), 1.42 (d, J=7.23 Hz, 3H), 1.26 (broad s, 2H) 1.04-1.16 (m, 2H) 0.80-0.92 (m, 2H).

Example S67. Synthesis of compound 53

Step 1: Synthesis of (9H-fluoren-9-yl)methyl(1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-methyl-1oxopentan-2-yl)carbamate.

To a stirred solution of (((9H-fluoren-9-yl)methoxy)carbonyl)leucine (20.0 g, 56.58 mmol) in dry DMF (200 mL) was added HATU (21.50 g, 56.58 mmol) followed by DIPEA (10.62 mL, 61.10 mmol) at 0 °C, and the reaction mixture was stirred at the same temperature for 10 min. To the resulting reaction mixture was added N-(2,2-diethoxyethyl)-2-methylbutan-1-amine (11.48 g, 56.58 mmol) at room temperature and the reaction mixture was stirred for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was quenched with ice-cold water (100 mL) and the aqueous layer was extracted with EtOAc (50 mL x 4). The combined organic layers were washed with cold _H2O (50 mL x 2) and then brine (50 mL), dried _{over Na2SO4} and concentrated under reduced pressure to give the crude product. The crude product was purified by CombiFlash column chromatography using 5% MeOH in DCM to give (9Hfluoren-9-yl)methyl (1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (14.5 g, 47.57% yield) as a white solid. MS (ESI) m/z [M+H]+: 539.04.

Step 2: Synthesis of 2-amino-H-(2,2-diethoxyethyl)-4-methyl-N-(2-methylbutyl)pentanamide

To a solution of (9H-fluoren-9-yl)methyl(1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8.50 g, 15.77 mmol) in _{CH2Cl2 (} 50 mL) was added diethylamine (16 mL, 157.7 mmol) at room temperature and the reaction mixture was stirred for 3 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated under reduced pressure to give the crude compound. The crude compound was purified by CombiFlash column chromatography using 5% MeOH in DCM to give 2-amino-N-(2,2-diethoxyethyl)-4-methyl-N-(2-methylbutyl)pentanamide (3.60 g, 72% yield) as a yellow viscous liquid. MS (ESI) m/z [M+H-EtOH]+: 272.10.

Step 3: Synthesis of (9H-fluoren-9-yl)methyl(3-((1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-methyl1-oxopentan-2-yl)amino)-3-oxopropyl)carbamate.

To a stirred solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (3.80 g, 12.28 mmol) in dry DMF (35 mL) maintained at 0 °C were added HATU (6.48 g, 17.05 mmol) and DIPEA (4.90 mL, 28.42 mmol), followed by the addition of 2-amino-N-(2,2-diethoxyethyl)-4-methyl-N-(2-methylbutyl)pentanamide (3.60 g, 11.37 mmol). The reaction mixture was allowed to reach room temperature and stirred for 3 h. Upon completion, the reaction mixture was quenched with ice-cold water (20 mL) and the aqueous layer was extracted with EtOAc (30 mL x 2). The organic layer was washed with cold H2O (10 mL) and then brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude compound was purified by Combiflash column chromatography using 5% MeOH in DCM to give (9H-fluoren-9-yl)methyl (3-((1-((2,2-diethoxyethyl)(2-methylbutyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-oxopropyl)carbamate (3.8 g, 55% yield) as an off-white solid. MS (ESI) m/z [M+H-EtOH]+: 565.30.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl 6-isobutyl-8-(2-methylbutyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1-hydroxy-2H-carboxylate

To a stirred solution of (9H-fluoren-9-yl)methyl 6-isobutyl-8-(2-methylbutyl)-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (3.80 g, 6.231 mmol) was added formic acid (20 mL) at room temperature, and the reaction mixture was stirred for 16 h. After completion, the reaction mixture was concentrated under reduced pressure. The crude compound was purified by column chromatography (silica gel 100-200 mesh; 0-5% MeOH in DCM) to give (9H-fluoren-9-yl)methyl 6-isobutyl-8-(2-methylbutyl)-4,7-dioxohexahydro-2Hpyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (3.60 g, 94% yield) as a yellow solid. MS (ESI) m/z [M+H]+: 518.23.

Step 5: Synthesis of 6-isobutyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione

To a solution of ^^fluoren^-yl^egal 6-isobutyl-8-(2-methylbutyl)-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxylate (3.60 g, 6.954 mmol) in CH ₂ Cl ₂ (36 mL) was added diethylamine

- 62 049703 (6.8 mL, 69.54 mmol) and the reaction mixture was stirred at room temperature for 16 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was concentrated under reduced pressure and the crude product was purified by combiflash column chromatography using 10-50% ethyl acetate in n-hexane to give 6-isobutyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)-dione (1.20 g, 60% yield) as a white solid. MS (ESI) m/z [M+H]+: 296.10.

Step 6: Synthesis of 1-(4-(difluoromethoxy)benzyl)-6-isobutyl-8-(2-methylbutyl)hexahydro-4Hpyrazino[1^^pyrimidineL^^^-dione

To a solution of 6-isobutyl-8-(2-methylbutyl)hexahydro-4H-pyrazino[1,2-a]pyrimidine-4,7-dione (0.170 g, 0.576 mmol) in DMF (5 mL) was added _K2CO3 (0.159 g, 1.152 _mmol ) at 0 °C and the reaction mixture was stirred for 10 min. To the resulting reaction mixture was added 1-(bromomethyl)-4(difluoromethoxy)benzene (0.150 g, 0.632 mmol) at room temperature and stirred for 3 h. Upon completion, the reaction mixture was quenched with ice-cold water (200 mL) and the aqueous layer was extracted with EtOAc (20 mL x 2). The organic layer was washed with cold _H2O (20 mL) and then brine (15 mL), dried over _Na2SO4 and concentrated under reduced pressure. The resulting crude compound was purified by prep-HPLC to give 1-( ₄ (difluoromethoxy)benzyl)-6-isobutyl-8-(2-methylbutyl)hexαhydro-4H-pyrazino[1,2-a]pyrimidine-4,7(6H)dione (0.103 g, 40% yield) as a white solid. MS (ESI) m/z [M+H]+: 452.3. ¹ H NMR (400 MHz, DMSO d6) δ 7.42 (d, J=8.8 Hz, 2H), 7.14-7.24 (m, 3H), 5.0-5.10 (m, 1H), 4.50-4.60 (m, 1H), 3.90-4.00 (m, 2H), 3.60-3.70 (m, 1H), 3.02-3.40 (m, 4H), 2.70-2.85 (m, 2H), 2.0-2.10 (m, 1H), 1.50-1.70 (m, 4H), 1.20-1.35 (m, 1H), 1.0-1.10 (m, 1H), 0.70-0.98 (m, 12H).

Biological examples

Example B1. ELISA with phospho-MET

Compounds were screened for activity against the HGF/MET system using phospho-MET (pMET) ELISA kits (Cell Signaling). pMET levels were detected in samples with low (1 ng/mL) and high (10 ng/mL) HGF concentrations.

HEK293 cells were prepared by spiking into 6-well multiplates and grown at 37°C with 5% _CO2 in DMEM+10% FBS to approximately 90% confluency. Cells were then starved for at least 8 hours in serum-free growth medium.

Illustrative compounds were prepared in DMEM+0.1% FBS, diluted, and added to treatment medium with 1 ng/ml recombinant HGF protein (R&D Systems). Cells were incubated in triplicate at 37°C and 5% _CO2 for 15 min. Samples were then treated with 180 μl ice-cold RIPA buffer and cells were lysed on ice for 15 min. Lysates were cleared by centrifugation at 16,000 g for 15 min, and the supernatant was saved. Samples were normalized by BCA lysate assay to determine protein concentrations in all samples.

Between 50 and 100 μg of total protein lysate were loaded into ELISA wells in a pMET sandwich ELISA kit (Cell Signaling, catalog #7227C), ensuring equal protein loading in each well. ELISA was performed according to the manufacturer's instructions. After color development, absorbance was read on an optical plate reader at 450 nm.

Potency indices were determined using peak efficacy by scaling test compound doses on a 1-10 scale between 1 ng/mL and 10 ng/mL HGF according to the following formula:

y=1+(x-A)*(10-1)/(B-A) where y is the normalized data point, x is the original data point, A is the mean HGF value at 1 ng/mL and B is the mean HGF value at 10 ng/mL. The results for the calculated power are shown in Table 2.

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means the compound failed to significantly enhance MET phosphorylation + means maximal activity at or above 100 nM ++ means maximal activity at or above 10 nM +++ means maximal activity at or above 1 nM ++++ means maximal activity at or above 0.1 nM

Example B2. Analysis of cell scattering behavior

MDCK cells were grown under normal conditions and observed to spontaneously form dense colonies as they proliferated. MDCK cells respond to HGF treatment by moving away from each other (scattering), which is quantified to determine the level of HGF/MET activation in the cell population. In this experiment, MDCK cells were seeded in a 96-well format, treated with HGF and example compounds, fluorescently stained, imaged in wide fields, and quantified for scattering behavior. Quantification was determined by analyzing the number of continuous groups of cells compared to the total stained area of the image (normalized particle count).

MDCK cells were seeded at low density in black-walled imaging plates and incubated overnight at 37°C and 5% CO2 in DMEM+10% FBS. Cells were then starved for up to 2 h in DMEM without FBS (starvation medium). Samples containing example compounds were prepared in DMEM without FBS and included 5 ng/ml HGF protein (treatment medium). A control curve was also generated for each plate using HGF concentrations of 0, 5, 10, and 20 ng/ml. Starvation medium was replaced with treatment medium and cells were incubated for 24 h at 37°C and 5% CO2.

Following incubation, cells were fixed by replacing the treatment medium with cold ethanol and incubating for 20 min at 4°C. Cells were then rehydrated by washing with PBS and then with staining solution (fluorescent wheat germ agglutinin; WGA488 at 20 μg/ml in PBS). Cells were incubated with staining solution for 30 min at room temperature, after which the staining solution was replaced with fresh PBS.

Cell fields were visualized using a high-resolution imaging device.

- 64 049703 iCyte retention at the green wavelength. Images were converted to binary and analyzed for particle size and number. For analysis, single cells not touching other cells or separated colonies of cells were identified as particles, and particle numbers were normalized to the total signal area to account for differences in cell numbers. An increase in particle number indicated that individual cells were moving away from each other as a result of scattering behavior. Compound activity was assessed by a statistical increase in normalized particle number compared to HGF treatment alone. The results are presented in Table 3.

Table 3. Results of cell scattering analysis using example compounds

Connection Activity Connection Activity 1a ++++ 2a + For - 4a + 5a ++++ 6a +++ 7a ++ 8a +++ 9 - 10 - 11 ++ 12 ++ 13 - 14 +++ 15 - 16 - 17 - 18 - 19 +++ 20 +++ 21 - 22 ++ 23 - 24 - 25 - 26 - 27 NT 28 - 29 NT 30 ++ 31 - 32 - 33 - 34 - 35 - 36 - 37 - 38 NT 39 - 40 NT 41 - 42 +++ 43 - 44 - 45 nt 46 - 47 nt 48 nt 49 ++++ 50 - 51 +++ 52 - 53 +++ 54 -

indicates that the compound failed to significantly improve the cell scattering behavior + means maximum activity at or above 100 nM ++ means maximum activity at or above 10 nM +++ means maximum activity at or above 1 nM ++++ means maximum activity at or above 0.1 nM

NT means the connection has not been tested

Example B3. Solubility analysis

Aqueous solubility is a critical property of a drug that helps predict bioavailability. Generally, compounds with aqueous solubility <100 μg/mL are poorly effective drugs. To assess the solubility of compounds, a turbidimetric solubility assay was performed with the example compounds in the concentration range of 3-300 μM.

To assess the solubility of the compound by turbidity, the test compounds were first dissolved in an organic solvent (DMSO) at a concentration of 10 mM. This compound solution was then diluted in an aqueous solvent (PBS) in serial dilutions from 3 to 300 μM in a 96-well assay plate. The solutions were incubated at 37°C for 2 h.

In wells with test compounds exceeding the solubility limit, the compound will precipitate, effectively blocking the passage of light and thereby increasing the UV absorption signal at 620 nm. The compounds were considered insoluble at the concentration tested,

- 65 049703 if turbidity increased the absorbance by more than 10% compared to the control value. The results are presented in Table 4.

Table 4. Solubility of illustrative compounds

Connection Solubility Connection Solubility 1a ++++ 2a ++++ For ++++ 4a ++++ 5a ++++ 6a ++++ 7a ++++ 8a ++++ 9 ++++ 10 ++++ 11 ++++ 12 ++++ 13 ++++ 14 ++++ 15 ++++ 16 ++++ 17 ++++ 18 ++++ 19 ++++ 20 ++++ 21 ++++ 22 ++++ 23 ++++ 24 ++++ 25 ++++ 26 ++++ 27 +++ 28 +++ 29 ++++ 30 ++++ 31 ++++ 32 ++++ 33 ++++ 34 ++++ 35 ++++ 36 ++++ 37 ++++ 38 +++ 39 ++++ 40 ++++ 41 ++++ 42 ++++ 43 +++ 44 +++ 45 ++++ 46 ++++ 47 ++++ 48 +++ 49 ++++ 50 ++++ 51 +++ 52 +++ 53 +++ 54 ++

+ indicates solubility at 10 µM ++ indicates solubility at 30 µM +++ indicates solubility at 100 µM ++++ indicates solubility at 300 µM

Example B4. Permeability analysis

Bioavailable drugs must penetrate the cellular membranes of the gastrointestinal mucosa. To assess the permeability of the example compounds, a parallel in vitro artificial membrane permeability assay (PAMPA) was performed.

Test compounds must have a standard curve in the final read plate to determine the split concentration of each drug. A 6-point standard curve from 0 to 200 μM in phosphate-buffered saline (PBS) was prepared for each compound.

The test compound solution (300 µl in PBS) was added to the donor (bottom) well of the PAMPA plate in 5 replicates, and PBS carrier medium (200 µl) was added to the acceptor (top) wells of the corresponding wells to match the donor plate loading. The bottom and top halves of the PAMPA plates were then joined together. The PAMPA plates were then incubated at room temperature for 5 h. After incubation, 150 µl of the donor solution was added to a UV-compatible plate containing the appropriate standard curve. 150 µl of the acceptor well solution was added next to the appropriate standard curve and donor well samples for the compound. The plate was then read using a UV plate reader.

The membrane permeability and retention were then calculated using the following formulas:

Permeability (cm/s): (Rexcm/sec^Ln [1 - CA(t) / Ceq]} / [A * (1/VD+1/VA) * t] (equation 1) where

A=filtering surface (0.3 ^cm2 );

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VD=volume of donor well (0.3 ml);

UA=volume of the acceptor well (0.2 ml);

t=incubation time (seconds);

CA(p = concentration of the compound in the acceptor well at time t;

CD(t)=concentration of compound in donor well at time t and

Ceq=[CD(t)*VD+CA(t)*VA]/(VD+VA).

Membrane retention (R)=1-[CD(t) * VD+CA(t) * VA]/(CO * VD) (equation 2) where

CD(t), VD, CA(t) and VA are defined for equation 1 and C0=initial concentration in the donor well (200 μM).

The results are presented in Table 5.

Table 5. Permeability of illustrative compounds

NT 28 NT

27 NT 28 NT 29 nt 30 +++ 31 nt 32 NT 33 nt 34 nt 35 nt 36 nt 37 nt 38 nt 39 nt 40 nt 41 nt 42 ++ 43 nt 44 nt 45 nt 46 nt 47 nt 48 nt 49 ++ 50 nt 51 +++ 52 nt 53 +++ 54 +++

+ indicates permeability above 1 x 10' ⁵ cm/sec ++ indicates permeability above 2x10' ⁶ cm/sec +++ indicates permeability below 2 x 10' ⁶ cm/sec

NT means the connection has not been tested

Example B5. Cytotoxicity Assay

This experiment was designed to obtain a preliminary assessment of cytotoxicity. Compounds were tested at high concentrations to determine if any cytotoxic effects were observed in hepatocyte (HepG2) cell cultures by measuring the release of lactate dehydrogenase (LDH) into the culture medium as a measure of lysed/killed cells.

HepG2 cells were seeded in 96-well cell culture plates and incubated overnight at 37°C, 5% CO2 in EMEM+10% FBS. Treatments were performed in complete medium (EMEM+10% FBS) and included serial dilutions of test compounds from 0.1 to 100 μM. The known cytotoxin cerivastatin was used as a positive control for the assay and was prepared at a final concentration of 0.5 μM.

The growth medium was replaced with treatment medium (EMEM+10% FBS containing the test compound dissolved in DMSO), and the cells were incubated with the test compounds for 48 h. At the end of the incubation period, the supernatant medium from each well was transferred to a new plate and

- 67 049703 LDH assay working solution was added. The LDH assay solution was subjected to a colorimetric reaction in proportion to the amount of lactate dehydrogenase (an intracellular protein that was detected in the medium only in the presence of lysed cells) in the medium. The color reaction was quantified by measuring the absorbance at 490 nm.

The signal range of the assay was determined by the absence of manipulation of the negative control treatment and complete lysis of all cells in the lysis control sample. Compounds that increased the level of cytotoxicity by more than 20% compared to the negative control samples were considered cytotoxic in this assay. The results are presented in Table 6.

Table 6. Cytotoxicity of illustrative compounds

Connection Cytotoxicity Connection Cytotoxicity 1a ++++ 2a ++++ For NT 4a ++++ 5a ++++ 6a ++++ 7a ++++ 8a ++++ 9 ++++ 10 NT 11 ++++ 12 ++++ 13 NT 14 ++++ 15 nt 16 NT 17 ++++ 18 nt 19 ++++ 20 ++++ 21 nt 22 ++++ 23 nt 24 nt 25 nt 26 nt 27 nt 28 nt 29 nt 30 ++++ 31 nt 32 nt 33 nt 34 nt 35 nt 36 nt 37 nt 38 nt 39 nt 40 nt 41 nt 42 ++++ 43 nt 44 nt 45 nt 46 nt 47 nt 48 nt 49 ++++ 50 nt 51 +++ 52 nt 53 +++ 54 +++

+ indicates non-toxicity at 0.1 µM ++ indicates non-toxicity at 1 µM +++ indicates non-toxicity at 10 µM ++++ indicates non-toxicity at 100 µM

NT means the connection has not been tested

Example B6. In vitro stability tests

Bioavailability can be assessed by the stability of a compound when exposed to in vivo conditions. As an initial assessment of stability properties under a variety of animal conditions, the example compounds were tested for stability in a battery of simulated body compartments. The compounds were tested for stability in the following solutions: simulated gastric juice (SGF: 34.2 mM NaCl, pH 1.2), simulated gastric juice with the digestive enzyme pepsin (SGF+enzyme: SGF with 3.2 mg/mL pepsin), simulated intestinal juice with _an enzyme mixture in porcine pancreatin (SIF+enzyme: 28.7 mM _NaH2PO4 , 105.7 mM NaCl, pH 6.8, 10 mg/mL pancreatin), rat plasma, and human plasma.

Test compounds were incubated at a final concentration of 5 μM in the above solutions at 37°C with samples removed at the following time points: 0, 1, 2 and 4 h. Reactions were stopped and prepared for quantification by adding excess quencher solution.

- 68 049703 solution containing an internal standard (acetonitrile, 200 ng/mL bucetin). The test compound and internal standard in each sample were quantified by LC-MS/MS, and after internal normalization to bucetin, the concentration of the test compound was expressed as a percentage of the concentration at time 0 h. Stability in the corresponding test solution was then determined by the percentage remaining at time 4 h. The results are presented in Table 7.

Table 7. In vitro stability of the example compounds

Connection SGF SGF+Farm. SIF Rat plasma Human plasma 1a ++++ ++++ ++++ ++ ++ 2a ++++ +++ ++++ ++ ++ For NT NT NT NT NT 4a nt nt nt nt nt 5a +++ + +++ ++ ++ 6a +++ + ++ +++ ++ 7a ++++ +++ ++ nt nt 8a ++++ +++ +++ ++++ ++ 9 nt nt nt nt nt 10 nt nt nt nt nt 11 +++ +++ ++++ ++++ +++ 12 +++ +++ ++ ++++ +++

13 NT nt nt nt nt 14 ++ ++ +++ +++ +++ 15 NT nt nt nt nt 16 nt nt nt nt nt 17 +++ +++ +++ ++ +++ 18 nt nt nt nt nt 19 +++ ++ + +++ +++ 20 +++ +++ +++ ++ +++ 21 nt nt nt nt nt 22 +++ +++ +++ +++ +++

+ indicates 20-39% of compound remaining after 4 hours ++ indicates 40-79% of compound remaining after 4 hours +++ indicates 80-99% of compound remaining after 4 hours ++++ indicates 100% of compound remaining after 4 hours NT means compound was not tested

Example B7. In Vivo Pharmacokinetics

Administration of the example compounds by selected routes followed by blood collection and quantification of the compounds in plasma was used to determine the pharmacokinetic (PK) profile of the compounds. Compounds were administered to mixed-sex Sprague-Dawley rats weighing at least 250 grams by dissolving the test compound in DMSO and then diluting the compound in the appropriate vehicle, either saline or saline and polyethyleneglycol. Dosing was performed either by tail vein puncture (i.v.) or orally (p.o.), and animals were dosed with compound according to their weight at 1 ml/kg. At selected time points after administration (10, 20, 40, 60, 120, and 360 min), blood was collected by tail vein bleeding. Whole blood was then processed by centrifugation to obtain plasma. The content of compounds in plasma samples was quantified by LC-MS/MS and compared with internal standard and standard curves to accurately determine the concentration.

Plasma concentrations were then averaged for each time point and plotted as a function of time. The area under the curve was calculated by integrating the curve, C _max was the highest concentration achieved in plasma, and T _max was determined from the time to reach C _max . The results are presented in Table 8.

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Table 8. Pharmacokinetic parameters of illustrative compounds

AUC Smach Tmax Connection B/B By B/B By B/B By 1a ++ ++ +++ + +++ ++ 2a +++ ++ +++ ++ +++ ++ For NT nt nt nt nt nt 4a nt nt nt nt nt nt 5a ++ + ++ + +++ ++ 6a +++ + +++ + +++ ++ 7a ++++ + ++++ + +++ ++ 8a nt nt nt nt nt nt 9 nt nt nt nt nt nt 10 nt nt nt nt nt nt 11 +++ ++ ++ ++ +++ +++ 12 ++ ++ ++ + +++ + 13 nt nt nt nt nt nt 14 +++ +++ ++ ++ +++ + 15 nt nt nt nt nt nt 16 nt nt nt nt nt nt 17 nt nt nt nt nt nt 18 nt nt nt nt nt nt 19 nt nt nt nt nt nt 20 +++ +++ +++ ++ +++ + 21 nt nt nt nt nt nt 22 +++ ++ ++ ++ +++ ++

AUC: ++++ indicates dose-adjusted AUC above 3000 ng*h/mL; +++ indicates dose-adjusted AUC 1000-2999 ng*h/mL; ++ indicates dose-adjusted AUC 100-999 ng*h/mL; + indicates dose-adjusted AUC 1-100 ng*h/mL.

Cmax: ++++ means dose-adjusted plasma Cmax above 3000 ng/ml; +++ means dose-adjusted plasma Cmax 1000-2999 ng/ml; ++ means dose-adjusted plasma _Cmax 100-999 ng/ml; + means dose-adjusted plasma _Cmax 1-100 ng/ml.

T _max: +++ means T _max less than 30 min; ++ means T _max 30-60 min; + means T _max above 60 min.

NT means the connection has not been tested.

Example B8. Calculation of oral availability

Oral bioavailability plays a critical role in the development of small molecule therapeutics for orally administered administration. Calculations of oral bioavailability (%F) were performed by comparing in vivo pharmacokinetic data (Example B7) using the intravenous dosing as the maximum possible exposure and determining the rate of exposure after p.o. In these studies, the dose-adjusted AUC for p.o. was divided by the dose-adjusted AUC for i.v. and multiplied by 100 to obtain %F. The results are presented in Table 9.

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T Table 9. Calculated first Connection 1a 2a Za 4a 5a 6a 7a 8a 9 10 11 12 13 14 15 16 17 18 19 20 21 oral availability of compounds example Oral bioavailability NT NT + nt nt nt nt nt nt nt nt nt V 22 +++

++++ means oral bioavailability above 50% +++ means oral bioavailability 25-50% ++ means oral bioavailability 1-25% + means oral bioavailability below 1%

NT means the connection has not been tested

Example B9. Non-specific protein binding

The effects of the example compounds on plasma and tissues were assessed by their non-specific protein binding affinity in target tissues or fluids to determine the fraction of the compound available for interaction with the target. Non-specific binding was determined in plasma and brain homogenate collected from male and female Sprague-Dawley rats.

Known concentrations of test compounds were mixed with plasma or brain homogenate and incubated in the donor chamber of a rapid equilibrium dialysis (RED) device with blank PBS buffer in the receiving chamber. After incubation for 4 h at 37°C in an orbital shaking incubator, the compound in each chamber was quantified by LC-MS/MS. The fraction of unbound material (f and _f ) was calculated using the following formula:

' 1 fu, tissue ⁼ ~ “ ^{1 +} (t----- ^- T ^D \J and, homogenate / where f ctissue, is the fraction of unbound substance in the tissue;

f _and , _{the homogenate} is the ratio of the concentration in the buffer chamber to the concentration in the sample chamber and

D is the dilution factor used to obtain the sample.

The results are presented in Table 10.

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Table 10. Non-specific protein binding of example compounds

Connection Unbound fraction Plasma The brain 1a +++ ++ 2a +++ ++ For NT NT 4a NT nt 5a + + 6a ++ + 7a ++++ +++ 8a nt nt 9 nt nt 10 nt nt 11 + ++ 12 ++ ++ 13 nt nt

14 ++ ++++ 15 NT nt 16 NT nt 17 nt nt 18 nt nt 19 nt nt 20 ++ +++ 21 nt nt 22 ++ ++++

++++ means unbound fraction above 0.9 +++ means unbound fraction from 0.5 to 0.9 ++ means unbound fraction from 0.1 to 0.5 + means unbound fraction below 0.1 NT means the compound was not tested

Example B10. Tissue distribution in vivo

The distribution rate in target tissues is an important characteristic of therapeutic molecules. Tissue distribution of the example compounds was performed in Sprague-dawley rats of different sexes. The test compounds were administered by tail vein injection (IV), tissues were collected at T _max (10 minutes after administration). Animals were deeply anesthetized with isoflurane, whole blood was collected from the right atrium and processed by centrifugation to obtain plasma. The animals were then completely perfused with PBS, which was injected into the left ventricle to prevent tissue contamination with blood.

Tissues were collected and homogenized, and target tissue compound content was quantified by LC-MS/MS. Tissue distribution rates were determined by dividing the tissue compound concentration by the plasma concentration and multiplying by 100. The results are presented in Table 11.

Table 11. In vivo tissue distribution of example compounds

Connection Tissue distribution (% plasma exposure at 10 min after IV dose) Muscle Sciatic nerve Brain Hippocamp Cerebellum Cortical layer 1a NT NT ++ ++ ++ ++ 2a ++++ ++++ ++ ++ + ++ For NT nt NT NT NT NT 4a nt nt nt nt nt nt 5a nt nt ++++ ++++ ++++ ++++ 6a ++++ ++++ ++ ++ ++ ++

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7a ++ ++++ + + + + 8a NT nt nt nt nt nt 9 NT nt nt nt nt nt 10 nt nt nt nt nt nt 11 nt ++ ++ + + + 12 nt +++ ++ ++ ++ ++ 13 nt nt nt nt nt nt 14 nt ++ ++ ++ ++ ++ 15 nt nt nt nt nt nt 16 nt nt nt nt nt nt 17 nt nt nt nt nt nt 18 nt nt nt nt nt nt 19 nt nt nt nt nt nt 20 nt ++++ ++++ +++ ++++ ++++ 21 nt nt nt nt nt nt 22 nt +++ ++ ++ ++ ++

++++ means distribution above 70% +++ means distribution from 40 to 69% ++ means distribution from 5 to 39% + means distribution from 0.05 to 5% NT means that the compound was not tested

Example B11. In Vivo Efficacy: Scopolamine-Induced Spatial Memory Deficit in the Morris Water Maze

Compounds 2a and 6a were evaluated for their ability to reverse chemically induced spatial memory deficits in rats in the Morris water maze. The water maze consists of a large circular tank (2.1 m diameter) filled with water at 26-28°C to a depth of ~30 cm, the water was turbidified with white paint. A circular platform (13 cm diameter) was secured so that it was 2-3 cm below the surface of the water. High-contrast visual cues were placed around the tank to facilitate spatial orientation of the test animals. The test consisted of placing the animal in the water facing the wall of the tank at one of three randomly assigned starting positions and allowing it to swim and search for a hidden platform for up to 120 s. The time required for the animal to locate the platform was recorded as the escape latency. Animals were tested 5 times daily with a 30 s rest period between trials. The test was carried out for a total of 8 consecutive days.

Animals were divided into groups (N=8 per group) depending on the treatment method. Control animals received empty vehicle only. Scopolamine groups received 3 mg/kg scopolamine dissolved in sterile saline by intraperitoneal (IP) injection 30 min before testing. Test compound groups received test compound at various concentrations by oral gavage (OG) dissolved in 48% sterile saline, 50% polyethyleneglycol (PEG-400), and 2% DMSO 40 min before testing. Escape latencies were recorded for each animal for 5 trials each day for 8 consecutive days. Changes in escape latency curves were statistically analyzed by two-way ANOVA with Bonferroni post hoc test. Results are presented in Table 12.

Example 1a was evaluated for its ability to reverse chemically induced spatial memory deficits in rats in the Morris water maze. The water maze consists of a large circular tank (1.5 m diameter) filled with water at 23-26°C to a depth of ~30 cm, turbidified with white paint. A circular platform was secured so that it was 2-3 cm below the surface of the water. High-contrast visual cues were placed around the tank to facilitate spatial orientation of the test animals. The test consisted of placing the animal in the water facing the wall of the tank at one of three randomly assigned starting positions and allowing it to swim and search for a hidden platform for up to 90 s. The time required for the animal to locate the platform was recorded as the escape latency. Animals were tested 5 times per day with a 30 s rest period between trials. The test was conducted for a total of 5 consecutive days.

Animals were divided into groups (N=12 per group) depending on the treatment method. Control animals received empty vehicle only. Scopolamine groups received 2 mg/kg scopolamine dissolved in sterile saline by intraperitoneal (IP) injection 30 min before testing. Test compound groups received the test compound in

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Claims (31)

at various concentrations via gavage (GT) dissolved in 78% sterile saline, 20% polyethyleneglycol (PEG-400) and 2% DMSO 40 min before testing. Escape latencies were recorded for each animal for 5 trials each day for 5 consecutive days. Changes in escape latency curves were statistically analyzed using a two-way ANOVA with Bonferroni post hoc test. The results are presented in Table 12. Table 12. In Vivo Efficacy of Illustrative Compounds Compound 1a 2a Dose (mg/kg) 8 2 0.5 10 Improvement of cognitive abilities + 1 +++ 0,1 - 6a 10 ++ 1 - +++ means posterior p-value below 0.01 ++ means posterior p-value between 0.05 and 0.01 + means posterior p-value between 0.06 and 0.05 - means a posterior p-value greater than 0.06 Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present invention. The contents of all patent and scientific literature cited herein are expressly and fully incorporated by reference into this specification. CLAUSE OF INVENTION 1. Compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein L represents -C(=O)- or -(CR ^a R ^b )m-; R ^a and R ^b represent H; R ^1a and R ^1b independently represent H or C1-C6 alkyl optionally substituted with hydroxyl, halogen or -CO ₂ H; R ² is oxo; R ³ is C ₂ -C ₆ alkyl, C ₃ -C ₆ alkenyl, C ₃ -C ₁₂ cycloalkyl or C ₃ -C ₆ cycloalkylalkyl; R ⁴ is C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl optionally substituted with C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl or halogen, wherein the 5-10 membered heteroaryl contains 1-3 nitrogens; R ⁶ is H; R ⁷ is oxo; m represents 1 and n represents 0. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein L is -C(=O)-. 3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L is -(CR ^a R ^b )m-. 4. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein each R ^1a and R ^1b independently represents H, methyl or -CH ₂ CO ₂ H. 5. The compound of claim 4 or a pharmaceutically acceptable salt thereof, wherein R ^1a is methyl and R ^1b is H. 6. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R ³ is C ₂ -C ₆ alkyl, C ₂ -C ₆ alkenyl or C ₃ -C ₆ cycloalkylalkyl. 7. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein R ³ is - 74 049703 8. A compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is C ₆ -C ₁₀ aryl optionally substituted with 1 to 3 substituents selected from halogen, hydroxyl, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy. 9. The compound according to claim 8 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is phenyl substituted with 1-3 substituents selected from -CF ₃ , -OCHF ₂ , -OH, fluorine and chlorine. 10. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is 11. A compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is pyridyl optionally substituted with 1 to 3 substituents selected from halogen, hydroxyl, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy. 12. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein R ⁴ is 13. A compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, wherein -LR ⁴ is 14. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (V) 15. The compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein L represents -C(=O)- or -CH2-; R ^1a and R ^1b independently represent H or C ₁ -C3 alkyl, optionally substituted with -CO ₂ H; R3 is _C4 - _C5 alkyl, _C4 - _C5 alkenyl, or _C1 -C3 alkyl substituted with C3- _C5 cycloalkyl; and R ⁴ is phenyl or pyridyl substituted with 1-3 substituents selected from -CF3, -OCHF2, -OH, fluorine and chlorine. 16. A compound selected from - 75 049703 - 76 049703 4 co LL ^F 3 ^C \X^. Tso /N. C~E o ¹ UyO/N. /x /¾. 9?· 0 ¹ ΟγΌ·^ F уЦ 0 ¹ 'E? V/-O o ¹ O =( o o > ⁼ 4 b / ° ^δ ^Ζ/ o LL? -U o c O =( - 77 049703 or its pharmaceutically acceptable salt. 21. A compound according to claim 1 or 16 of the following formula: or a pharmaceutically acceptable salt thereof. 22. A compound according to claim 1 or 16 of the following formula: or a pharmaceutically acceptable salt thereof. 23. A compound according to claim 1 or 16 of the following formula: or a pharmaceutically acceptable salt thereof. 24. A compound according to claim 1 or 16 of the following formula: or a pharmaceutically acceptable salt thereof. 25. A compound according to claim 1 or 16 of the following formula: or a pharmaceutically acceptable salt thereof. 26. A compound according to claim 1 or 16 of the following formula: or a pharmaceutically acceptable salt thereof. 27. A compound according to claim 1 or 16 of the following formula: or a pharmaceutically acceptable salt thereof. 28. A pharmaceutical composition comprising a compound according to any one of claims 1 to 27 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 29. A method for modulating hepatocyte growth factor in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a compound according to any one of claims 1-27 - 78 049703 or its pharmaceutically acceptable salt or pharmaceutical composition according to paragraph 28. 30. A method for treating a disease, condition or injury associated with modulation of hepatocyte growth factor in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of claims 1-27 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 28, wherein the disease, condition or injury is a neurodegenerative disease, spinal cord injury or sensorineural hearing loss. 31. The method of claim 30, wherein the disease, condition or injury is a neurodegenerative disease and the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis (ALS). 32. The method of claim 31, wherein the disease, condition or injury is amyotrophic lateral sclerosis (ALS). 33. A method for treating or slowing the progression of dementia or preventing cognitive dysfunction in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of claims 1-27 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 28. 34. The method according to claim 33, wherein the dementia or cognitive dysfunction is associated with Alzheimer's disease or Parkinson's disease. 35. A method for treating, restoring or preventing a disease, condition or injury associated with nervous tissue, or treating or preventing a disease or disorder of the central nervous system in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of claims 1-27 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 28. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2 -