HK40046600B - Pyrrolo[2,3-b]pyridin derivatives as inhibitors of influenza virus replication - Google Patents

Description

Pyrrolo[2,3-B]pyridine derivatives as inhibitors of influenza virus replication

Technical Field

This disclosure generally relates to inhibitors of influenza virus replication and methods of treating or preventing influenza infection by administering said inhibitors to patients in need of treatment.

Background Technology

Influenza circulates globally in seasonal epidemics, causing hundreds of thousands of deaths each year, and millions in pandemic years. For example, the 20th century saw three influenza pandemics, killing tens of millions of people, each caused by the emergence of a novel strain of the human virus. Typically, these novel strains arise from the transmission of existing influenza viruses from other animal species to humans.

Influenza is primarily spread from person to person through large, virus-laden droplets produced when an infected person coughs or sneezes. These droplets can then remain on the mucous membranes of the upper respiratory tract of susceptible individuals nearby (e.g., within about 6 feet). Transmission can also occur through direct or indirect contact with respiratory secretions (such as touching a surface contaminated with the influenza virus and then touching the eyes, nose, or mouth). Adults can transmit influenza to others from one day before symptoms appear until about five days after the onset of symptoms. Young children and people with weakened immune systems can remain infectious for 10 days or more after the onset of symptoms.

Influenza viruses are RNA viruses belonging to the Orthomyxoviridae family, which includes five genera: Influenza A virus, Influenza B virus, Influenza C virus, Isavirus, and Thogoto virus.

Influenza A viruses can cause both seasonal and pandemic influenza epidemics. There is one species, influenza A virus, and wild waterfowl are the natural hosts for many types of influenza A. Occasionally, the virus can spread to other species and subsequently cause devastating outbreaks in poultry or lead to human influenza pandemics. Influenza A viruses are the most virulent human pathogens of the three influenza types and cause the most severe illness. Influenza A viruses can be further classified into different serotypes based on antibody responses to these viruses. The confirmed human serotypes, ranked by the number of known human pandemic deaths, are: H1N1 (which caused the Spanish flu in 1918), H2N2 (which caused the Asian flu in 1957), H3N2, H5N1 (a pandemic threat during the 2007-2008 flu season), H7N7 (a potential pandemic threat), H1N2 (an endemic disease present in humans and pigs), H9N2, H7N2, H7N3, and H10N7.

Influenza B viruses are a genus that causes seasonal influenza and comprise a single species, the influenza B virus. Influenza B infects almost exclusively humans and is less prevalent than influenza A. The only other known animal susceptible to influenza B is the seal. This type of influenza mutates at a rate 2 to 3 times slower than influenza A, resulting in lower genetic diversity and only one influenza B serotype. Due to this lack of antigenic diversity, some degree of immunity to influenza B is usually acquired early on. However, the robust mutation of influenza B makes sustained immunity impossible. This reduced rate of antigenic change, combined with its limited host range (suppressing cross-species antigenic transfer), ensures that pandemics of influenza B do not occur.

The genus *Influenza C* contains one species, *Influenza C* virus, which infects humans and pigs and can cause severe illness and localized epidemics. However, *Influenza C* is less common than other types and appears to typically cause mild illness in children.

The structures of influenza viruses across serotypes and genera are remarkably similar. The influenza virus genome consists of eight single-stranded RNAs packaged into rod-shaped structures of varying sizes, called ribonucleoprotein complexes (RNPs). Each RNP contains unique viral RNA, multiple copies of the backbone nucleoprotein, and a heterotriviral polymerase composed of PA, PB1, and PB2 subunits, which catalyzes the transcription and replication of the viral genome. Recent biochemical and structural studies of the influenza polymerase complex have provided insights into the mechanisms of cap-snatching and RNA synthesis via influenza polymerase. In simple terms, the PB2 cap-binding domain first binds to its 5' cap to isolate the host precursor mRNA. The PA, an endonuclease subunit, then cleaves 10–13 nucleotides of the captured precursor mRNA downstream of the cap. The PB2 subunit then rotates approximately 70° to guide the capping primer to the PB1 polymerase active site. The PB1 subunit interacts directly with the PB2 and PA subunits. These subunits contain highly conserved domains across different influenza virus strains and are attracted as attractive targets for anti-influenza drugs. In addition to the polymerase complex, the influenza genome encodes its own neuraminidase (NA), hemagglutinin (HA), nucleoprotein (NP), matrix proteins M1 and M2, and non-structural proteins NS1 and NS2. NA is a target of the antiviral drugs oseltamivir (Tamiflu) and zanamivir (Relenza). These drugs inhibit the enzymatic activity of NA, thereby slowing the release of sialic acid analogs from infected cells by progeny viruses.

Influenza incurs direct costs due to lost productivity and related medical treatment, and indirect costs due to prevention and control measures. In the United States, influenza costs more than $10 billion annually, and future pandemics are estimated to incur hundreds of billions of dollars in direct and indirect costs. Prevention and control costs are also high. Governments worldwide have spent billions of dollars preparing for and planning for a potential H5N1 avian influenza pandemic, with costs associated with purchasing medicines and vaccines, conducting disaster drills, and developing strategies for improved border controls.

Current treatment options for influenza include vaccination and chemotherapy or chemoprevention using antiviral drugs. Influenza vaccination is generally recommended for high-risk groups, such as children and the elderly, or people with asthma, diabetes, or heart disease. However, it is possible to contract influenza after vaccination. The vaccine is reconfigured each season against several specific influenza virus strains, but may not include all strains that are effective in infecting the global population during that season. Manufacturers spend approximately six months reconfiguring and producing the millions of doses needed to combat seasonal epidemics; occasionally, novel or neglected strains become prominent during that period and infect vaccinated individuals (such as the H3N2 Fujian influenza in the 2003-2004 flu season). It is also possible to contract the specific strain the vaccine is intended to prevent, as the vaccine takes approximately two weeks to become effective.

Furthermore, the effectiveness of these flu vaccines varies. Due to the high mutation rate of the virus, a particular flu vaccine typically provides protection for no more than a few years. A vaccine formulated for one year may be ineffective the following year because the flu virus mutates rapidly over time, and different strains become dominant.

Because it lacks an RNA-correcting enzyme, the RNA-dependent RNA polymerase of influenza vRNA produces approximately one nucleotide insertion error per 10,000 nucleotides (which is the approximate length of influenza vRNA). Therefore, almost every newly produced influenza virus is a mutant antigenic drift. Separating the genome into eight unique segments of vRNA allows for mixing or recombining of vRNAs when more than one viral strain infects a single cell. The resulting rapid changes in viral genetics produce antigenic transfer and allow the virus to infect novel host species and quickly overcome protective immunity.

Antiviral drugs can also be used to treat influenza, with norepinephrine (NA) inhibitors being particularly effective; however, viruses can develop resistance to approved NA antiviral drugs. Furthermore, the emergence of multidrug-resistant pandemic influenza A viruses has been well-documented. Drug-resistant pandemic influenza A has become a major public health threat. In addition to drug-resistant influenza A viruses, NA inhibitors are approved for the treatment of early-stage influenza infection (within 48 hours of the onset of influenza symptoms).

Therefore, there is still a need for drugs to treat influenza infections, such as those that have a prolonged therapeutic window and/or reduced sensitivity to viral titers.

Summary of the Invention

This disclosure generally relates to methods for treating influenza, methods for inhibiting the replication of influenza viruses, methods for reducing the amount of influenza viruses, and compounds and compositions that can be used in such methods.

In one aspect, this disclosure provides compounds of formulas A, B, and C and their pharmaceutically acceptable salts:

in:

^Z1 can be -R*, halogen, cyano, -OR*, _-CO2R *, _-NO2 , or -CON(R*) ₂ ;

Z ² can be -R*, -OR*, -CO ₂ R*, -NR* ₂ , or -CON(R*) ₂ ;

Z ³ is -H, hydroxyl, halogen, -NH ₂ ; -NH (C ₁ -C ₆ alkyl); -N (C ₁ -C ₆ alkyl) ₂ , C ₁ -C ₆ alkoxy or C ₁ -C ₆ alkyl, optionally substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl and C ₁ -C ₆ alkoxy;

X is -C( ^R5 ) _2- , -O-, -N( ^R5 )-, ^-NR5 -SO2-, _C6 - _C10 aryl, or a 5- to ₁₀ -membered heteroaryl group comprising 1 to 3 cyclic heteroatoms selected from O, N, and S.

When X is -N( ^R5 )-, the ^R5 portion, together with the nitrogen atom it is attached to, optionally forms a 5- or 6-membered heterocycle. In this case, L is attached to a ring atom (e.g., a cyclic carbon or nitrogen atom), preferably to a carbon or nitrogen ring atom at the β-position of nitrogen, and the 5- or 6-membered heterocycle may optionally include one or two side oxygen substituents.

^R1 is -H or _C1 - _C6 alkyl;

^R2 is -H; -F; _-NH2 ; -NH ( _C1 - _C6 alkyl); -N ( _C1 - _C6 alkyl) ₂ ; -C=N-OH; cyclopropyl, optionally substituted with one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, _-OCH3 , and _-CH3 ; or _C1 - _C6 alkyl, optionally substituted with one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, and _C1 - _C6 alkoxy; and

^R3 is -H, halogen, hydroxyl, _C1 - _C6 alkoxy, _-NH2 , -NH( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl) ₂ , -cyano, or _C1 - _C6 alkyl, wherein the _C1 - _C6 alkyl is optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, syloxy, _-NH2 , -NH( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl) ₂ , -OCO- _C1 - _C6 alkyl, -CO- _{C1 - C6} alkyl, -CO2H, _{-CO2C1 -C6 alkyl} , and _C1 - _C6 alkoxy;

Each ^R4 is independently H, _C1 - _C6 alkyl, _C3 - _C8 cycloalkyl, _C2 - _C8 alkoxy, ( _C1 - _C14 alkoxy) _r- ( _C1 - _C14 alkyl) _s , _C2-8 alkoxy- _C6 - _C10 aryl, _C1 - _C6 alkyl-C6- _C10 aryl, 5- to ₁₀ -membered heteroaryl, or _C1 - _C6 alkyl-5- to 10-membered heteroaryl, wherein the heteroaryl comprises 1 to 3 cyclic heteroatoms selected from O, N, and S;

Each R ⁵ is independently H, _C1 - _C6 alkyl, _C3 - _C8 cycloalkyl, _C2 - _C8 alkoxy, _C2 - _C8 alkoxy- _C6 - _C10 aryl, _CO2H , CO2- _C1 -C6 alkyl, _CONH2 , CONH- _C1 - _C6 alkyl, CON( _C1 - _C6 alkyl) ₂ , _{C1 - C6 alkyl} - _CO2H , C1-C6 alkyl-CO2- _C1 - _C6 alkyl, _C1 - _C6 alkyl- _CONH2 , _{C1 - C6} alkyl _- CONH( _C1 - _C6 alkyl), _C1 - _C6 alkyl-CON( _C1 - _C6 alkyl) _{2 , C1} - _C6 alkyl- _C6 - _C10 aryl, 5 to 10 heteroaryl, or _C1 -C _6- alkyl-5 to 10-membered heteroaryl, wherein the heteroaryl group comprises 1 to 3 cyclic heteroatoms selected from O, N and S;

^R9 is independently -H, halogen, cyano, hydroxyl, _-NH2 , carboxyl, _C1 - _C6 alkyl, _C1 - _C6 haloalkyl, _C1 - _C6 cyanoalkyl, _C2 - _C6 alkoxy- _C1 - _C6 alkyl, _C1 - _C6 aminoalkyl, _C1 - _C6 hydroxyalkyl, CO- _C1 - _C6 alkyl, or _C1 - _C6 alkoxy;

Each R* is independently: i) -H; ii) _C1 - _C6 alkyl, optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxy, _-NH2 , carboxyl, C3- _C8 cycloalkyl, 5- to 6 _- membered heterocyclic alkyl, phenyl, 5- to 6-membered heteroaryl, _C1 - _C6 alkoxy, and -CO- _C1 - _C6 alkyl; wherein each of the alkyl groups in _C1 - _C6 alkoxy and -CO- _C1 - _C6 alkyl is optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH( _C1 - _C4 alkyl), -N( _C1 - _C4 alkyl) ₂ , -OCO- _C1 - _C4 alkyl, -CO- _C1 - _C4 alkyl, _-CO2 H, -CO ₂ -C ₁ -C ₄ alkyl and C ₁ -C ₄ alkoxy, wherein the heterocyclic alkyl or heteroaryl group comprises 1 to 3 cyclic heteroatoms selected from O, N and S, and each of the cycloalkyl, heterocyclic alkyl, phenyl and heteroaryl groups is independently and optionally substituted by one or more (e.g., 1, 2 or 3) instances of J; or iii) C ₃ -C ₈ cycloalkyl or 4 to 8-membered heterocyclic alkyl groups comprising 1 to 3 cyclic heteroatoms selected from O, N and S, each independently and optionally substituted by one or more (e.g., 1, 2 or 3) instances of J; and

Each J is independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , carboxyl, amide, _C1 - _C6 alkyl, _-C1 - _C6 alkoxy, and -CO- _C1 - _C6 alkyl, wherein each of the alkyl groups is optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO- _C1 - _C4 alkyl, _-CO2H , _{-CO2C1 -C4 alkyl} , and _C1 - _C4 alkoxy;

L represents ( _C1 - _C14 alkoxy) _q - ^Rz , _C1 - _C14 alkyl-( _C1 - _C14 alkoxy) _q - ^Rz , ( _C1 - _C14 alkoxy) _q -Y, _C1 - _C14 alkyl-C(O) ^NR5 - _C1 - _C14 alkyl- ^Rz , _C1 - _C14 alkyl- ^NR5C (O)-C1- _C14 alkyl- ^Rz , _{C1 - C14} alkyl-OC(O)NH- _C1 - _C14 alkyl- ^Rz , -( _C1 - _C14 alkyl) _q- ( _C1 - _C14 alkoxy) _q -Y-( _C1 - _C14 alkyl) _q - ^Rz , ( _C1 - _C14 alkoxy) _q -Y-( _C1 - _C14 alkoxy) _q- (C ₁ - _C14 alkyl) _q -R ^z , ( _C1 - _C14 alkyl) _q - _YC1 - _C14 alkyl-( _C1 - _C14 alkoxy) _q -R ^z ;

Each Rz is independently selected from H, halogen, cyano, hydroxyl, C<sub> 1 -C 6  alkyl, C<sub> 1- C 6  haloalkyl, -OR 5 , (C<sub> 1 -C 6  alkoxy) r -OC<sub> 1 -C 6  haloalkyl, C<sub> 1 -C6alkyl-NHR 5 , -N(R 5 ) 2 , -NH(R 5 )-CO 2 H, C<sub> 1- C 6  alkoxy-OCHO, -CO 2 R 5 , C <sub>1 -C 6 alkyl-CO 2 R 5 , -CONHR 5 , -OY, -CONH(R 5 )-Y, CO-Y, C<sub> 1 -C 14  alkyl-Y, C <sub>1 -C 14  alkoxy-Y, and Y;

Each Y is independently selected from _C6 - _C10 aryl, _C3 - _C12 cycloalkyl, 5- to 10-membered heteroaryl, or 3- to 12-membered heterocyclic, wherein the heteroaryl or heterocyclic comprises 1 to 3 cyclic heteroatoms selected from O, N, and S, wherein Y is optionally substituted by one to three substituents selected from: halogen, cyano, hydroxyl, _C1 - _C6 alkyl, _C1 - _C6 alkoxy, _-CO2H , _{-CO2C1 -C4 alkyl} , and _-OC6 - _C10 aryl;

m can be 0, 1, 2, or 3;

q is 0, 1, 2, or 3, and if X is not -C( ^R5 ) _2- or a 5- to 10-membered heteroaryl group, then q is 1, 2, or 3; and

Each of r and s is 0, 1, 2 or 3; and r+s is 1 to 6.

In some cases, the compound is a compound of formulas 1 to 13 or a pharmaceutically acceptable salt thereof:

in:

X is -C( ^R₅ ) _₂- , -N( ^R₅ )-, ^-NR₅ - _SO₂- , or -O-;

Each Q is independently N, CH, or C*, where * indicates the junction of the L group and only one Q is C*; and n is independently 1, 2, 3, or 4.

In some cases, the compound or its medicinal salt has a structure

in

Het/Ar is a _C6 - _C10 aryl group or a 5- to 10-membered heteroaryl group having 1 to 3 cyclic heteroatoms selected from N, O, and S; and

L is _C1 - _C14alkyl - ^NR5C (O) _-C1 - _C14alkyl - ^Rz or _C1 - _C14alkyl -OC(O)NH- _C1 - _C14alkyl - ^Rz ; and

R z  is H, C<sub> 1 -C 6  alkyl, or C<sub> 1 -C 6  alkyl-CO 2 R 5 , and the alkyl group is optionally substituted with one or more (e.g., 1, 2, or 3) of the following: hydroxyl, halogen, C<sub> 1 -C 6  alkoxy, C<sub> 1 -C 6  haloalkoxy, NH 2 ,

-NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO- _C1 - _C4 alkyl, -CO _2H and -CO _{2C1 -C4 alkyl} .

In some cases, the compound or its medicinal salt has the formula

Wherein R" is -H or _C1 - _C6 alkyl, optionally substituted by one or more (e.g., 1, ₂ or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, amino, carboxyl, _C1 - _C6 alkoxy, _C1 - _C6 haloalkoxy, _C1 - _C6 aminoalkoxy, C1- _C6 cyanoalkoxy, _C1 - _C6 hydroxyalkoxy and _C2 - _C6 alkoxyalkoxy.

In some cases, the compound or its medicinal salt has the formula

Wherein R" is -H or _C1 - _C6 alkyl, optionally substituted by one or more (e.g., 1, ₂ or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, amino, carboxyl, _C1 - _C6 alkoxy, _C1 - _C6 haloalkoxy, _C1 - _C6 aminoalkoxy, C1- _C6 cyanoalkoxy, _C1 - _C6 hydroxyalkoxy and _C2 - _C6 alkoxyalkoxy.

Further, methods are provided for administering safe and effective amounts of compounds as disclosed herein, such as those represented by formula A, B, or C or formulas 1 to 13, to biological samples or patients.

This document also provides a method for reducing the amount of influenza virus in a biological sample or patient by administering a safe and effective amount of such a compound as disclosed herein, such as that represented by any one of Formula A, B or C or Formulas 1 to 13, to the biological sample or patient.

Further methods of treating or preventing influenza in patients include administering to the patient a safe and effective amount of a compound as disclosed herein, such as that represented by formula A, B or C or formulas 1 to 13.

Pharmaceutical compositions are also provided that comprise, as disclosed herein, such as compounds represented by any one of formulas A, B or C, or formulas 1 to 13, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, adjuvants, or mediators thereof.

The uses of the compounds described herein are also provided for inhibiting the replication of influenza virus in biological samples or patients, for reducing the amount of influenza virus in biological samples or patients, or for treating influenza in patients.

This document further provides the use of the compounds described herein for the manufacture of agents for treating influenza in patients, for reducing the amount of influenza virus in biological samples or patients, or for inhibiting the replication of influenza virus in biological samples or patients.

Invention Details

This document discloses compounds and their use in inhibiting influenza viruses. One aspect of this disclosure generally relates to the use of the compounds described herein or pharmaceutically acceptable salts, or pharmaceutically acceptable compositions comprising such compounds or pharmaceutically acceptable salts thereof, for inhibiting the replication of influenza virus in biological samples or patients, for reducing the amount of influenza virus in biological samples or patients (reducing viral titer), and for treating influenza in patients.

The compounds disclosed herein

This disclosure provides compounds of formula A, B or C, or formulas 1 to 13, or pharmaceutically acceptable salts thereof:

Or its pharmaceutically acceptable salt, wherein:

^Z1 can be -R*, halogen, cyano, -OR*, _-CO2R *, _-NO2 , or -CON(R*) ₂ ;

Z ² can be -R*, -OR*, -CO ₂ R*, -NR* ₂ , or -CON(R*) ₂ ;

Z ³ is -H, hydroxyl, halogen, -NH ₂ ; -NH (C ₁ -C ₆ alkyl); -N (C ₁ -C ₆ alkyl) ₂ , C ₁ -C ₆ alkoxy or C ₁ -C ₆ alkyl, optionally substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl and C ₁ -C ₆ alkoxy;

X is -C( ^R5 ) _2- , -O-, -N( ^R5 )-, ^-NR5 -SO2-, _C6 - _C10 aryl, or a 5- to ₁₀ -membered heteroaryl group comprising 1 to 3 cyclic heteroatoms selected from O, N, and S.

When X is -N( ^R5 )-, the ^R5 portion, together with the nitrogen atom it is attached to, optionally forms a 5- or 6-membered heterocycle. In this case, L is attached to a ring atom (e.g., a cyclic carbon or nitrogen atom), preferably to a carbon or nitrogen ring atom at the β position of nitrogen, and the 5- or 6-membered heterocycle may optionally include one or two oxo substituents.

^R1 is -H or _C1 - _C6 alkyl;

^R2 is -H; -F; _-NH2 ; -NH ( _C1 - _C6 alkyl); -N ( _C1 - _C6 alkyl) ₂ ; -C=N-OH; cyclopropyl, optionally substituted with one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, _-OCH3 , and _-CH3 ; or _C1 - _C6 alkyl, optionally substituted with one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, and _C1 - _C6 alkoxy; and

^R3 is -H, halogen, hydroxyl, _C1 - _C6 alkoxy, _-NH2 , -NH( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl) ₂ , -cyano, or _C1 - _C6 alkyl, wherein the _C1 - _C6 alkyl is optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl) ₂ , -OCO- _C1 - _C6 alkyl, -CO- _{C1 - C6} alkyl, -CO2H, _{-CO2C1 -C6 alkyl} , and _C1 - _C6 alkoxy;

Each ^R4 is independently H, _C1 - _C6 alkyl, _C3 - _C8 cycloalkyl, _C2 - _C8 alkoxy, ( _C1 - _C14 alkoxy) _r- ( _C1 - _C14 alkyl) _s , _C2-8 alkoxy- _C6 - _C10 aryl, _C1 - _C6 alkyl-C6- _C10 aryl, 5- to ₁₀ -membered heteroaryl, or _C1 - _C6 alkyl-5- to 10-membered heteroaryl, wherein the heteroaryl comprises 1 to 3 cyclic heteroatoms selected from O, N, and S;

Each R ⁵ is independently H, _C1 - _C6 alkyl, _C3 - _C8 cycloalkyl, _C2 - _C8 alkoxy, _C2 - _C8 alkoxy- _C6 - _C10 aryl, _CO2H , CO2- _C1 -C6 alkyl, _CONH2 , CONH- _C1 - _C6 alkyl, CON( _C1 - _C6 alkyl) ₂ , _{C1 - C6 alkyl} - _CO2H , C1-C6 alkyl-CO2- _C1 - _C6 alkyl, _C1 - _C6 alkyl- _CONH2 , _{C1 - C6} alkyl _- CONH( _C1 - _C6 alkyl), _C1 - _C6 alkyl-CON( _C1 - _C6 alkyl) _{2 , C1} - _C6 alkyl- _C6 - _C10 aryl, 5 to 10 heteroaryl, or _C1 -C _6- alkyl-5 to 10-membered heteroaryl, wherein the heteroaryl group comprises 1 to 3 cyclic heteroatoms selected from O, N and S;

^R9 is independently -H, halogen, cyano, hydroxyl, _-NH2 , carboxyl, _C1 - _C6 alkyl, _C1 - _C6 haloalkyl, _C1 - _C6 cyanoalkyl, _C2 - _C6 alkoxy- _C1 - _C6 alkyl, _C1 - _C6 aminoalkyl, _C1 - _C6 hydroxyalkyl, CO- _C1 - _C6 alkyl, or _C1 - _C6 alkoxy;

Each R* is independently: i) -H; ii) _C1 - _C6 alkyl, optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , carboxyl, C3- _C8 cycloalkyl, 5- to 6 _- membered heterocyclic alkyl, phenyl, 5- to 6-membered heteroaryl, _C1 - _C6 alkoxy, and -CO- _C1 - _C6 alkyl; wherein each of the alkyl groups in _C1 - _C6 alkoxy and -CO- _C1 - _C6 alkyl is optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH( _C1 - _C4 alkyl), -N( _C1 - _C4 alkyl) ₂ , -OCO- _C1 - _C4 alkyl, -CO- _C1 - _C4 alkyl, _-CO2 H, -CO ₂ -C ₁ -C ₄ alkyl and C ₁ -C ₄ alkoxy, wherein the heterocyclic alkyl or heteroaryl group comprises 1 to 3 cyclic heteroatoms selected from O, N and S, and each of the cycloalkyl, heterocyclic alkyl, phenyl and heteroaryl groups is independently and optionally substituted by one or more (e.g., 1, 2 or 3) instances of J; or iii) C ₃ -C ₈ cycloalkyl or 4 to 8-membered heterocyclic alkyl groups comprising 1 to 3 cyclic heteroatoms selected from O, N and S, each independently and optionally substituted by one or more (e.g., 1, 2 or 3) instances of J; and

Each J is independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , carboxyl, amide, _C1 - _C6 alkyl, _-C1 - _C6 alkoxy, and -CO- _C1 - _C6 alkyl, wherein each of the alkyl groups is optionally substituted by one or more (e.g., 1, 2, or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO- _C1 - _C4 alkyl, _-CO2H , _{-CO2C1 -C4 alkyl} , and _C1 - _C4 alkoxy;

L represents ( _C1 - _C14 alkoxy) _q - ^Rz , _C1 - _C14 alkyl-( _C1 - _C14 alkoxy) _q - ^Rz , ( _C1 - _C14 alkoxy) _q -Y, _C1 - _C14 alkyl-C(O) ^NR5 - _C1 - _C14 alkyl- ^Rz , _C1 - _C14 alkyl- ^NR5C (O)-C1- _C14 alkyl- ^Rz , _{C1 - C14} alkyl-OC(O)NH- _C1 - _C14 alkyl- ^Rz , -( _C1 - _C14 alkyl) _q- ( _C1 - _C14 alkoxy) _q -Y-( _C1 - _C14 alkyl) _q - ^Rz , ( _C1 - _C14 alkoxy) _q -Y-( _C1 - _C14 alkoxy) _q- (C ₁ - _C14 alkyl) _q -R ^z , ( _C1 - _C14 alkyl) _q - _YC1 - _C14 alkyl-( _C1 - _C14 alkoxy) _q -R ^z ;

Each Rz is independently selected from H, halogen, cyano, hydroxyl, C<sub> 1 -C 6  alkyl, C<sub> 1- C 6  haloalkyl, -OR 5 , (C<sub> 1 -C 6  alkoxy) r -OC<sub> 1 -C 6  haloalkyl, C<sub> 1 -C6alkyl-NHR 5 , -N(R 5 ) 2 , -NH(R 5 )-CO 2 H, C<sub> 1- C 6  alkoxy-OCHO, -CO 2 R 5 , C <sub>1 -C 6 alkyl-CO 2 R 5 , -CONHR 5 , -OY, -CONH(R 5 )-Y, CO-Y, C<sub> 1 -C 14  alkyl-Y, C <sub>1 -C 14  alkoxy-Y, and Y;

Each Y is independently selected from _C6 - _C10 aryl, _C3 - _C12 cycloalkyl, 5- to 10-membered heteroaryl, or 3- to 12-membered heterocyclic, wherein the heteroaryl or heterocyclic comprises 1 to 3 cyclic heteroatoms selected from O, N, and S, wherein Y is optionally substituted by one to three substituents selected from: halogen, cyano, hydroxyl, _C1 - _C6 alkyl, _C1 - _C6 alkoxy, _-CO2H , _{-CO2C1 -C4 alkyl} , and _-OC6 - _C10 aryl;

m can be 0, 1, 2, or 3;

q is 0, 1, 2, or 3, and if X is not -C( ^R5 ) _2- or a 5- to 10-membered heteroaryl group, then q is 1, 2, or 3; and

Each of r and s is 0, 1, 2 or 3; and r+s is 1 to 6.

In some cases, the compound has a structure of one of the following formulas:

Or its pharmaceutically acceptable salt, wherein:

X is -C( ^R₅ ) _₂- , -N( ^R₅ )-, ^-NR₅ - _SO₂- , or -O-;

Each Q is independently N, CH, or C*, where * indicates the junction of the L group and only one Q is C*; and

n is independently 1, 2, 3 or 4, and all remaining substituents are as described for one of formulas A, B or C.

In some embodiments, the compounds described herein may have one of the following formulas:

Wherein R" is -H or _C1 - _C6 alkyl, optionally substituted by one or more (e.g., 1, 2 or 3) substituents independently selected from the group consisting of: halogen, _cyano , hydroxyl, oxo, amino, CO2H, _C1 - _C6 alkoxy, _C1 - _C6 haloalkoxy, _C1 - _C6 aminoalkoxy, _C1 - _C6 cyanoalkoxy, _C1 - _C6 hydroxyalkoxy and _C2 - _C6 alkoxyalkoxy.

In embodiments described herein, side chain portions L not shown as connected to a specific location in the structure should be understood as connected in any open position.

In some embodiments, the compounds of formulas 7 to 13 do not contain side chains, i.e., L = H.

The side chain portion L can have one of the following formulas:

-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ -T, where T is selected from the group consisting of: OH, OR ⁵ , NHR ⁵ , CO ₂ R ⁵ , CO ₂ NHR ⁵ , C _1-6 alkyl-CO ₂ R ⁵ , C _1-6 alkyl-NHR ⁵ , C ₆ -C ₁₀ aryl, 5 to 10 heteroaryl, C _1-8 alkyl and C _3-8 cycloalkyl;

-C _1-8 alkoxy-C _1-8 alkoxy-U;

-C _1-8alkoxy -C _1-8alkoxy -C _1-8alkoxy -U,

-C _1-8 alkoxy-aryl/heteroaryl-C _1-8 alkyl-C _1-8 alkoxy

-C _6-13 alkyl-U;

-C _1-8 alkyl-C _1-8 alkoxy-U,

-C _1-8 alkyl-C _1-8 alkoxy-C _1-8 alkoxy-U,

-C _1-8 alkyl-C _1-8 alkoxy-C _1-8 alkoxy-C _1-8 alkoxy-U,

-C _1-8 alkyl-aryl/heteroaryl-C _1-8 alkoxy-C _1-8 alkoxy-U,

-C _1-8 alkyl-aryl/heteroaryl-C _1-8 alkyl-C _1-8 alkoxy-C _1-8 alkoxy-U,

-C _1-8 alkyl-aryl/heteroaryl-C _1-8 alkyl-C _1-8 alkoxy-C _1-8 alkyl-U,

-C _1-8 alkyl-aryl/heteroaryl-C _1-8 alkyl-C _1-8 alkoxy-U,

-C _1-8 alkyl-C _1-8 alkoxy-aryl/heteroaryl-C _1-8 alkoxy-aryl,

-C _1-8 alkyl-C _1-8 alkoxy-aryl/heteroaryl-C _1-8 alkyl-U,

-C _1-8 alkyl-C _1-8 alkoxy-aryl/heteroaryl-C _1-8 alkyl-alkoxy-U,

-C _1-8 alkyl-(optionally substituted)phenoxy;

-C _1-8 alkyl-C _1-8 alkoxy-(optionally substituted) phenoxy;

-C _1-8 alkyl-N(R ⁵ )-C(O)-C _1-8 alkyl-C _1-8 alkoxy-U;

-C _1-8 alkyl-OC(O)N(R ⁵ )-C _1-8 alkyl-U;

-C _1-8 alkyl-aryl/heteroaryl-C _1-8 alkoxy-U;

-aryl/heteroaryl-U;

-aryl/heteroaryl- _C1-8alkoxy -U;

-aryl/heteroaryl- _C1-8alkoxy - _C1-8alkyl -U;

-aryl/heteroaryl- _C1-8alkoxy - _C1-8alkoxy -U;

-aryl/heteroaryl- _C1-8alkyl - _C1-8alkoxy - _C1-8alkoxy -U; or

-aryl/heteroaryl- _C1-8alkoxy - _C1-8alkoxy -U;

U is selected from the group consisting of: H, _C1-8 alkoxy, _C6 - _C10 aryloxy, OR ⁵ , NHR ⁵ , CO _2R ⁵ , C(O)NHR ⁵ , _C1-6 alkyl-CO _2R ⁵ , _C1-6 alkyl-NHR ⁵ , _C6 - _C10 aryl, _C6 - _C10 aryl- _C1-6 alkyl, _C1-6 alkyl- _C6 - _C10 aryl, 3 to 12-membered heterocyclic alkyl, 5 to 10-membered heteroaryl, _C1-8 alkyl, _C1-6 haloalkyl, and _C3-8 cycloalkyl, wherein the heterocyclic alkyl and heteroaryl groups have 1 to 3 cyclic heteroatoms selected from N, O, and S.

The alkyl, alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, and cycloalkyl moieties may optionally be substituted by one to three (e.g., 1, 2, or 3) substituents selected from the group consisting of: hydroxyl, _C1-6 hydroxyalkyl, _C1-6 alkoxy, _C2-8 alkoxyalkyl, _C2-8 alkylalkoxy, C1-8 alkoxycarbonyl, _C1-8 alkyl, arylalkoxycarbonyl, _C2-6 alkenyl, _C2-6 alkynyl, _{CO₂H, halogen, C1-6 haloalkyl, N₃, cyano, N(R')₂ , SR ' ,} -C(O)NHR', OCOR', OC(O)NHR', N(CO)R', N(CO)OR', N(CO)COR', SCOR', S(O _{)₂NR'₂ ,} S(O) _₂R ', wherein each R' is independently H, C _1-6 alkyl, _C1-6 haloalkyl, C1-6 alkoxy, _C2-6 alkenyl, _C2-6 alkynyl, _C3-6 cycloalkyl, heterocyclic, aryl, aryloxy, heteroaryl, heteroaryloxy, alkylaryl _, arylalkyl, heteroarylalkyl or alkylheteroaryl, wherein the heteroaryl group has 1 to 3 cyclic heteroatoms selected from N, O and S.

The representative sidechain portion L may contain sidechains S1 to S57:

In some embodiments, for formula A, B, or C, or any of formulas 1 to 13, ^Z1 and ^R3 are F or C1, and ^R1 , ^R2 , ^Z2 , and ^Z3 are H. In some aspects, one or both of ^Z1 and ^R3 are F.

In some embodiments, for formula A, B, or C, or any one of formulas 1 to 13 or a pharmaceutically acceptable salt thereof: ^R1 is -H; ^R2 is -H; ^R3 is -H, -F, -Cl, _C1-4 alkyl, or _C1-4 haloalkyl; ^Z1 is -H, -F, or -Cl; ^Z2 is -H; ^Z3 is -H; each ^R8 is independently -H, halogen, hydroxyl, _C1 - _C4 alkyl, _C1 -C4 haloalkyl, _{C1 -C4 hydroxyalkyl} , _C2 - _C4 alkoxyalkyl, or -O ( _C1 - _C4 alkyl); and ^R9 is independently -H or _C1 - _C4 alkyl.

In some embodiments, the compounds described herein may have the following formula:

Wherein Het/Ar is a _C6 - _C10 aryl or a 5- to 10-membered heteroaryl; L is a _C1 - _C14 alkyl- ^NR5C (O)-C1 _{-C14 alkyl} - ^Rz or _C1 - _C14 alkyl-OC(O)NH- _C1 - _C14 alkyl ^{- Rz} ; and ^Rz is H, _C1 - _C6 alkyl or _C1 - _C6 alkyl- _CO2R5 , and the alkyl group is optionally substituted by one or more (e.g., 1, 2 or 3) of the following: hydroxyl, halogen, _C1 - _C6 alkoxy, _C1 - _C6 haloalkoxy, _NH2 , -NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO- _C1 - _C4 alkyl, _-CO2H and _{-CO2C1 -C4 alkyl} .

In some embodiments, Het/Ar is pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, pyrroloyl, pyrazolyl, triazolyl, furanyl, pyranyl, thiopheneyl, benzimidazolyl, benzothiopheneyl, benzofuranylbenzotriazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, indoleyl, isoindoleyl, purinyl, phenyl, or naphthyl. In some embodiments, Het/Ar is pyridyl or phenyl. In some embodiments, Het/Ar is pyridyl.

In some embodiments, L is _C1 - _C14 alkyl-OC(O)NH- _C1 - _C14 alkyl- ^Rz . In some embodiments, L is _C1 - _C6 alkyl-OC(O)NH- _C1 - _C6 alkyl- ^Rz .

In some embodiments, the compounds described herein may have the following formula:

Wherein R" is -H or _C1 - _C6 alkyl, optionally substituted by one or more (e.g., 1, ₂ or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, amino, carboxyl, _C1 - _C6 alkoxy, _C1 - _C6 haloalkoxy, _C1 - _C6 aminoalkoxy, C1- _C6 cyanoalkoxy, _C1 - _C6 hydroxyalkoxy and _C2 - _C6 alkoxyalkoxy.

In some embodiments, the compounds described herein may have the following formula:

Wherein R" is -H or _C1 - _C6 alkyl, optionally substituted by one or more (e.g., 1, ₂ or 3) substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, amino, carboxyl, _C1 - _C6 alkoxy, _C1 - _C6 haloalkoxy, _C1 - _C6 aminoalkoxy, C1- _C6 cyanoalkoxy, _C1 - _C6 hydroxyalkoxy and _C2 - _C6 alkoxyalkoxy.

It should be understood that the values of each variable are chosen to result in the formation of stable or chemically viable compounds.

The specific compounds covered include those in the table below. Compounds displaying a specific stereoisomer center indicate at least relative stereoisomerism and may indicate absolute stereoisomerism. Compounds with chiral centers that do not indicate a specific stereoisomerism indicate a mixture of stereoisomer centers at the chiral center.

The compound may be one of the compounds listed in Table A or a pharmaceutically acceptable salt thereof.

Table A

How to use

The compounds described herein, or their pharmaceutically acceptable salts, may be used to reduce viral titers in biological samples (e.g., infected cell cultures) or in humans (e.g., lung virus titers in patients).

As used in this article, the terms “influenza virus-mediated symptoms,” “influenza infection,” or “influenza” are used interchangeably to refer to illness caused by an infection with the influenza virus.

Influenza is an infectious disease affecting birds and mammals caused by influenza viruses. Influenza viruses are RNA viruses belonging to the Orthomyxoviridae family, which includes five genera: Influenza A virus, Influenza B virus, Influenza C virus, Infectious Salmon Anemia Virus, and Togovirus. The Influenza A virus genus contains one species, influenza A virus, which can be further divided into different serotypes based on antibody responses to these viruses: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H7N9, and H10N7. The Influenza B virus genus contains one species, influenza B virus. Influenza B virus almost exclusively infects humans and is less prevalent than influenza A virus. The Influenza C virus genus contains one species, influenza C virus, which infects humans and pigs and can cause severe illness and localized epidemics. However, influenza C virus is less prevalent than other types and appears to typically cause mild illness in children.

In some embodiments, influenza or influenza virus is associated with influenza A or influenza B virus. In some embodiments, influenza or influenza virus is associated with influenza A virus. In some specific embodiments, influenza A virus is H1N1, H2N2, H3N2, H7N9, or H5N1.

In humans, common symptoms of influenza include chills, fever, sore throat, muscle aches, severe headache, cough, weakness, and general malaise. In more severe cases, influenza can cause pneumonia, which can be fatal, especially in young children and the elderly. Although often confused with the common cold, influenza is a much more serious illness caused by different types of viruses. Influenza can cause nausea and vomiting, especially in children, but these symptoms are more characteristic of unrelated gastroenteritis, sometimes referred to as "stomach flu" or "24-hour flu."

Flu symptoms can appear quite suddenly, one to two days after infection. The first symptom is usually chills or coldness, but fever is also common in the early stages of infection, with temperatures ranging from 38°C to 39°C (approximately 100°F to 103°F). Many people become severely ill, requiring bed rest for several days, experiencing general aches and pains, particularly severe in the back and legs. Flu symptoms may include: general aches and pains (especially in the joints and throat), extreme chills and fever, fatigue, headache, irritated and watery eyes, red eyes, red and swollen skin (especially the face), red and swollen mouth, red and swollen throat and nose, and abdominal pain (in children with influenza B). Flu symptoms are not specific and can overlap with those of many pathogens (“influenza-like illnesses”). Laboratory data are usually required to confirm a diagnosis.

The terms “disease,” “symptom,” and “symptom” are used interchangeably in this article to refer to medical or pathological symptoms mediated by the influenza virus.

As used herein, the terms “individual” and “patient” are used interchangeably. The terms “individual” and “patient” refer to animals (e.g., birds such as chickens, quails, or turkeys, or mammals), and more specifically, “mammals” include non-primates (e.g., cattle, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs, and mice) and primates (e.g., monkeys, chimpanzees, and humans), and more precisely, humans. In some embodiments, an individual is a non-human animal, such as livestock (e.g., horses, cattle, pigs, or sheep), or a pet (e.g., dogs, cats, guinea pigs, or rabbits). In a preferred embodiment, an individual is a “human.”

As used herein, the term “biological sample” includes (but is not limited to) cell cultures or extracts thereof; biopsy material obtained from mammals or extracts thereof; blood, saliva, urine, feces, semen, tears or other bodily fluids or extracts thereof.

As used herein, the term "inhibition of influenza virus replication" includes both reducing the amount of viral replication (e.g., reducing it by at least 10%) and completely suppressing viral replication (i.e., reducing the amount of viral replication by 100%). In some embodiments, influenza virus replication is inhibited by at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, or at least 95%.

Influenza virus replication can be measured by any suitable method known in the field. For example, influenza virus titers can be measured in biological samples (e.g., infected cell cultures) or in humans (e.g., lung virus titers in patients). More specifically, for cell-based analyses, in each case where cells are cultured in vitro, the virus is added to the culture with or without the test agent, and the virus-dependent endpoint is assessed after an appropriate time period. For typical analyses, Madin-Darby canine kidney cells (MDCK) suitable for influenza virus strain A/Puerto Rico/8/34 and standard tissue cultures can be used. The first type of cell analysis that can be used depends on the death of the infected target cells, a process known as cytopathic effect (CPE), in which viral infection causes depletion of cell-derived cells and eventual lysis of the cells. In the first type of cell analysis, a small fraction of cells in the wells of a microtiter plate are infected (typically 1/10 to 1/1000), allowing the virus to undergo several rounds of replication over 48 to 72 hours, and then the amount of cell death is measured by the decrease in cellular ATP content compared to an uninfected control. The second type of cell analysis that can be used depends on the proliferation of virus-specific RNA molecules in infected cells, in which branched-strand DNA hybridization (bDNA) is used to directly measure RNA content. In this second type of cell analysis, a small number of cells are initially infected in the wells of a microtiter plate, allowing the virus to replicate in the infected cells and spread to additional rounds of cells. The cells are then lysed, and the viral RNA content is measured. This analysis is typically stopped prematurely after 18 to 36 hours, when all target cells are still viable. Viral RNA is quantified by hybridizing to specific oligonucleotide probes immobilized in the wells of the analysis plate, followed by amplification of the signal through hybridization with an additional probe linked to a reporter enzyme.

As used herein, “viral titer” or “titer” is a measure of viral concentration. Titer testing can be performed using serial dilutions to obtain approximate quantitative information from analytical procedures that evaluate only as positive or negative. The titer corresponds to the highest dilution factor that still produces a positive reading; for example, positive readings from the first eight consecutive 2-fold dilutions are converted to a titer of 1:256. A specific example is the viral titer. To determine the titer, several dilutions are prepared, such as ^10⁻¹ , ^10⁻² , ^10⁻³ , ..., ^10⁻⁸ . The lowest concentration of virus that still infects cells is the viral titer.

As used herein, the term "treat/treatment/treating" refers to both therapeutic and preventative treatment. For example, therapeutic treatment includes reducing or improving the progression, severity, and/or duration of influenza virus-mediated symptoms, or improving one or more symptoms (specifically, one or more identifiable symptoms) of influenza virus-mediated symptoms by administering one or more therapies (e.g., one or more therapeutic agents, such as compounds or compositions of the present invention). In a particular embodiment, therapeutic treatment includes improving at least one measurable physical parameter of influenza virus-mediated symptoms. In other embodiments, therapeutic treatment includes physically inhibiting the progression of influenza virus-mediated symptoms by, for example, stabilizing identifiable symptoms, and physiologically by, for example, stabilizing physical parameters, or both. In other embodiments, therapeutic treatment includes reducing or stabilizing influenza virus-mediated infection. Antiviral drugs can be used in community settings to treat individuals already suffering from influenza to reduce the severity of symptoms and the number of days they are ill.

The term "chemotherapy" refers to the treatment of a condition or disease using medications such as small molecule drugs (rather than "vaccines").

As used herein, the terms “prevention,” “preventative,” “preventative use,” and “preventative treatment” refer to any medical or public health procedure intended to prevent, rather than treat or cure, a disease. As used herein, the term “prevent/prevention/preventing” means reducing the risk of developing or exhibiting a given symptom, or reducing or suppressing a recurrence of the symptom in an individual who is not ill but has become or may become a carrier. The term “chemoprevention” refers to the treatment of a condition or disease with medication, such as small molecule drugs (not “vaccines”).

As used herein, prophylactic use includes use in situations where an outbreak has been detected to prevent contact transmission or spread of infection in places where large groups of people at high risk of severe influenza complications are in close contact with each other (e.g., hospital wards, daycare centers, prisons, nursing homes, etc.). It also includes use in individuals who need protection from developing influenza but have not received protection after vaccination (e.g., due to a weakened immune system), or when a vaccine is unavailable to them or when they cannot obtain a vaccine due to side effects. It also includes use within two weeks of vaccination or during any period after vaccination but before the vaccine becomes effective. Prophylactic use may also include treating individuals who do not have influenza or are not considered at high risk of complications to reduce the chance of contracting influenza and transmitting it to high-risk individuals in close contact with them (e.g., healthcare workers, nursing home staff, etc.).

As used herein and according to the United States Centers for Disease Control and Prevention (US CDC), an influenza “outbreak” is defined as a rapid increase in acute febrile respiratory illness (AFRI) relative to the normal prior-technical rate occurring within a 48 to 72-hour period in a population in close proximity to each other (e.g., in the same area of an assisted living facility, in the same household, etc.) or when any individual in the analyzed population tests positive for influenza. Any situation of influenza confirmed by any testing method is considered an outbreak.

As used in this article, "index case," "primary case," or "patient zero" refers to the first patient in a population sample of an epidemiological study. The index case is the first patient indicating the existence of an outbreak. Earlier cases may be found and labeled as primary, secondary, tertiary, etc.

In some embodiments, the methods of the present invention are preventative or protective measures for patients (specifically humans) susceptible to complications arising from influenza virus infection. The preventative methods described herein can be used in cases where an index case or outbreak has been confirmed to prevent the spread of infection to the rest of the community or population.

In some embodiments, the method of the present invention is applied as a preventative measure to members of a community or population, specifically humans, to prevent the spread of infection.

As used herein, "effective amount" means an amount sufficient to elicit the desired biological response. In this invention, the desired biological response is the inhibition of influenza virus replication, reduction of the amount of influenza virus, or reduction or improvement of the severity, duration, progression, or onset of influenza virus infection, prevention of the acceleration of influenza virus infection, prevention of recurrence, manifestation, onset, or progression of symptoms associated with influenza virus infection, or enhancement or improvement of the prophylactic or therapeutic effect of another therapy used against influenza infection. The precise amount of compound administered to an individual will depend on the mode of administration, the type and severity of infection, and the individual's characteristics (e.g., general health status, age, sex, weight, and tolerance to the drug). Those skilled in the art will be able to determine the appropriate dosage based on these and other factors. When administered co-administered with other antiviral agents, such as when administered co-administered with anti-influenza drugs, the effective amount of the second agent will depend on the type of drug used. The safe amount is an amount with minimal side effects, as readily determined by those skilled in the art. Suitable dosages of approved agents are known and can be adjusted by those skilled in the art based on the individual's condition, the type of condition being treated, and the amount of the compound described herein used. Where the dosage is not explicitly stated, a safe and effective amount should be assumed. For example, the compounds described herein may be administered to individuals at doses ranging from about 0.01 to 100 mg/kg body weight/day for therapeutic or prophylactic treatment.

As used herein, a “safe and effective amount” of a compound or composition is an effective amount of a compound or composition that does not cause excessive or harmful side effects in patients.

Generally, dosing regimens can be selected based on a variety of factors, including: the condition being treated and its severity; the activity of the specific compound used; the specific combination used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the individual's renal and hepatic function; the specific compound used or its salts, the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field. Those skilled in the art can readily determine and prescribe safe and effective amounts of the compounds described herein for the treatment, prevention, inhibition (complete or partial) or cessation of disease progression.

The dosage of the compounds described herein may range from about 0.01 to about 100 mg/kg body weight/day, from about 0.01 to about 50 mg/kg body weight/day, from about 0.1 to about 50 mg/kg body weight/day, or from about 1 to about 25 mg/kg body weight/day. It should be understood that the total daily dose may be administered as a single dose or may be administered multiple times, such as twice a day (e.g., every 12 hours), three times a day (e.g., every 8 hours), or four times a day (e.g., every 6 hours).

For therapeutic treatment, the compounds described herein may be administered to the patient within, for example, 48 hours (or 40 hours, or less than 2 days, or less than 1.5 days, or 24 hours) of the onset of symptoms (e.g., nasal congestion, sore throat, cough, pain, fatigue, headache, and chills/night sweats). Therapeutic treatment may continue for any suitable duration, such as 5 days, 7 days, 10 days, 14 days, etc. For prophylactic treatment during community outbreaks, the compounds described herein may be administered to the patient, for example, within 2 days of the onset of symptoms in index cases, and may continue for any suitable duration, such as 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc.

Combination therapy

The compounds described herein, or pharmaceutically acceptable salts thereof, may be administered alone or in combination with additional suitable therapeutic agents, such as antiviral agents or vaccines. When using combination therapy, a safe and effective amount may be obtained using a first amount of a compound disclosed herein, such as any one of compounds of formula A, B, or C, or any one of formulas 1 to 13, or a pharmaceutically acceptable salt thereof, and a second amount of additional suitable therapeutic agent (e.g., antiviral agent or vaccine).

In some embodiments of this disclosure, the compound described herein, or a pharmaceutically acceptable salt thereof, and an additional therapeutic agent are each administered in a safe and effective amount (i.e., an amount that would be therapeutically effective if administered alone). In some embodiments, the compound and the additional therapeutic agent are each administered in an amount that does not provide a therapeutic effect (below a therapeutic dose). In some embodiments, the compound may be administered in a safe and effective amount, while the additional therapeutic agent is administered below a therapeutic dose. In some embodiments, the compound may be administered below a therapeutic dose, while the additional therapeutic agent, such as a suitable cancer treatment agent, is administered in a safe and effective amount.

As used herein, the terms “combination therapy,” “combination,” and “co-administration” or “co-administration” are used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). The use of these terms does not limit the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to an individual.

Co-administration can cover the administration of the combined first and second amounts of the compound in a substantially simultaneous manner, such as in the form of a single pharmaceutical composition, for example, capsules or tablets having a first and second amount in a fixed ratio, or in the form of multiple separate individual capsules or tablets. Additionally, such co-administration can also cover the sequential administration of the compounds in any order.

In some embodiments, this disclosure relates to combination therapies for inhibiting influenza virus replication in biological samples or patients, or for treating or preventing influenza virus infection in patients, using compounds or pharmaceutical compositions of the present invention. Therefore, the pharmaceutical compositions described herein also include those compositions comprising combinations of inhibitors of influenza virus replication as described herein with antiviral compounds exhibiting anti-influenza virus activity.

The methods of use also include chemotherapy with the compounds described herein or in combination with other antiviral agents, and vaccination with an influenza vaccine.

When co-administration involves the individual administration of a first amount of a compound as described herein and a second amount of an additional therapeutic agent, the compounds and agents are administered within sufficiently close timeframes to achieve the desired therapeutic effect. For example, the time interval between administrations that can produce the desired therapeutic effect can range from minutes to hours, and can be determined taking into account the characteristics of each compound, such as potency, solubility, bioavailability, plasma half-life, and kinetic profile. For example, the compound as described herein and the second therapeutic agent can be administered to each other in any order, within approximately 24 hours, approximately 16 hours, approximately 8 hours, approximately 4 hours, approximately 1 hour, or approximately 30 minutes.

More precisely, the first therapy (e.g., a preventative or therapeutic agent, such as the compound of the present invention) may be administered to an individual before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to) administration of an additional therapeutic agent (e.g., an antiviral agent or an influenza vaccine), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to) administration of an additional therapeutic agent (e.g., an antiviral agent or an influenza vaccine).

It should be understood that the method of co-administering a first amount of the compound as described herein and a second amount of the additional therapeutic agent can produce an enhanced or synergistic therapeutic effect, wherein the combined effect is greater than the additive effect produced by individually administering a first amount of the compound as described herein and a second amount of the additional therapeutic agent.

As used herein, the term "synergistic" refers to a combination of the compound of the invention and another therapy (e.g., a preventative or therapeutic agent) that is more effective than the sum of the effects of the therapies. The synergistic effect of a combination of therapies (e.g., a combination of preventative or therapeutic agents) allows for the use of one or more lower doses of the therapy and/or less frequent administration of the therapy to an individual. The ability to utilize lower doses of the therapy (e.g., a preventative or therapeutic agent) and/or less frequent administration of the therapy reduces the toxicity associated with administration of the therapy to an individual without diminishing the efficacy of the therapy in preventing, treating, or treating a condition. Furthermore, the synergistic effect can improve the efficacy of the agent in preventing, treating, or treating a condition. Finally, the synergistic effect of a combination of therapies (e.g., a combination of preventative or therapeutic agents) can avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

When the combination therapy using the compounds of the present invention is combined with an influenza vaccine, the two treatments can be administered to allow for longer intervals between administrations (e.g., days, weeks, or months).

The presence of synergistic effects can be determined using appropriate methods for assessing drug interactions. Appropriate methods include, for example, the Sigmoid-Emax equation (Holford, N.H.G. and Scheiner, L.B., *Clin. Pharmacokinet* 6:429-453 (1981)), the Loewe additive equation (Loewe, S. and Muischnek, H., *Arch. Exp. Pathol. Pharmacol.* 114:313-326 (1926)), and the intermediate-effect equation (Chou, T.C. and Talalay, P., *Advances in Enzyme Regulation* 22:27-55 (1984)). The equations mentioned above can be applied to experimental data to produce corresponding graphs that help assess the effects of drug combinations. The corresponding graphs associated with the equations mentioned above are the concentration-effect curve, the equivalent line graph curve, and the combination index curve.

Influenza vaccine

The compounds described herein can be prophylactically combined with anti-influenza vaccines. These vaccines can be administered, for example, subcutaneously or intranasally. Subcutaneous vaccination typically induces neutralizing IgG antibodies in the serum and is highly effective in preventing the development of more severe symptoms such as pneumonia and similar conditions. However, IgA is the predominant prophylactic component in the upper respiratory tract mucosa, which serves as the site of infection. Since IgA is not induced by subcutaneous administration, intranasal administration may also be advantageous.

antiviral inhibitors

Various other compounds may be used in combination with the compounds described herein to treat or prevent influenza infection. Approved compounds include neuraminidase (NA) inhibitors, ion channel (M2) inhibitors, polymerase (PB1) inhibitors, and other influenza antiviral agents.

There are three FDA-approved antiviral drugs for combating influenza viruses, including zanamivir, oseltamivir phosphate, and peramivir. Earlier drugs, such as amantadine and rimantadine, are approved for the treatment and prevention of influenza A.

Neuraminidase (NA) inhibitors are a class of drugs that block neuraminidase. They are commonly used as antiviral drugs because they block the function of the influenza virus's neuraminidase by preventing the virus from replicating through budding from host cells. Representative neuraminidase inhibitors include oseltamivir, zanamivir, laninavir, and peramivir.

M2 inhibitors can also be used. Matrix-2 (M2) protein is a proton-selective ion channel protein that is entirely contained within the viral envelope of influenza A virus.

Amantadine, an antiviral drug for influenza, is a specific blocker of the M2 H+ channel. Amantadine (comprising amantadine and rimantadine) has been widely abandoned due to viral resistance, but combination therapy can mitigate the development of resistance to these drugs, as viruses that become resistant to one active agent can still be treated with other agents in the combination therapy.

Inhibitors of influenza RNA-dependent RNA polymerase (RdRp) include favipiravir and the compounds described in PCT WO2013/138236. Additional compounds disclosed by Muratore et al. in Proceedings of the National Academy of Sciences (PNAS), 109(16), 6247-6252 (April 2012) include the following:

Specific examples of compounds that can be co-administered with the compounds described herein include: neuraminidase inhibitors, such as oseltamivir and zanamivir; viral ion channel (M2 protein) blockers, such as amantadine and rimantadine; and antiviral drugs described in WO 2003/015798, including T-705, which is being developed by Toyama Chemical in Japan. (See also Ruruta et al., Antiviral Res., 82:95-102 (2009)). In some embodiments, the compounds described herein can be co-administered with conventional influenza vaccines.

The compounds described herein are applicable as inhibitors of influenza virus replication in biological samples or patients. These compounds are also applicable to reducing the amount (viral titer) of influenza virus in biological samples or patients. They are also applicable for the therapeutic and prophylactic treatment of influenza virus infections in biological samples or patients.

This disclosure also provides methods for preparing the compounds described herein. In some embodiments, the methods involve preparing compounds represented by formulas 1-13 or pharmaceutically acceptable salts thereof.

Preparation of the compounds disclosed herein

The synthesis of the compounds described herein can be simplified using one or more skeletons. For example, the skeleton of one of the following can react with a suitably functionalized pyrimidine ring to form a core structure comprising an azaindole linked to a pyrimidine ring.

This disclosure also provides methods for preparing the compounds described herein. The compounds described herein, their pharmaceutical salts, and prodrugs all contain a common core comprising an azaindole ring coupled to a pyrimidine ring. In some embodiments, these two-ring systems having suitable substitution patterns to provide the compounds described herein can be coupled using the chemicals described below.

In some embodiments, the method includes the following steps: making precursor A:

Reaction with precursor B or B1:

To form a compound represented by formula I1 (intermediate, or "I" of formula 1):

Other salts besides potassium salts can be used, and any of the leaving groups (LG) can be used, including toluenesulfonate group, 4-nitrobenzenesulfonate group, bromobenzenesulfonate group, methanesulfonate group, trifluoromethanesulfonate group, etc.

Representative examples of precursors B and B1 include the following:

Ts stands for tosylate group, also known as "tosyl" or "tosylate".

The variables in these formulas ( ^Z1 , ^Z2 , ^Z3 , ^R2 , ^R3 , ^R4 , X, and L) are identical to those defined in the section defining the compounds described herein, or, where the functional group defined by the variables would be unstable under the reaction conditions described herein, may be the protected form of the functional group or a synthetic component of such a group. Examples of protecting groups are detailed in Greene, TW, and Wuts, PG, *Protective Groups in Organic Synthesis*, 3rd edition, John Wiley & Sons, New York: 1999 (and other editions of the book), the entire contents of which are incorporated herein by reference.

In the relevant art, any suitable reaction conditions known, for example, in PCT WO 2005/095400 and PCT WO 2007/084557 concerning the coupling of dioxaborane with chloropyrimidine, can be used for the reaction between precursor (A) and (B or B1). For example, the reaction between precursor (A) and (B or B1) can be carried out in the presence of Pd( _PPh3 ) ₄ . Specific exemplary conditions are described in the examples in the Example section of this invention.

The leaving group on compound I1 can be replaced to obtain compound 1:

^R1 is as described above.

In some embodiments of the synthetic formula I1 compound, the synthesis comprises the step of reacting precursor C1 or C2 with a suitably substituted cyclohexylamine (i.e., substituted with N( ^R4 )C(O)XL) to form a compound represented by structural formula I1, as shown in the following process B:

Process B

In some embodiments, the leaving group is a toluenesulfonate group, and the toluenesulfonate group is "de-toluenesulfonated" to produce an intermediate of formula (IA). Any suitable conditions known in the art for removing the protection of the Ts group can be used. Specific exemplary conditions are described in the examples. De-toluenesulfonation can produce a compound of formula (IA), wherein ^R1 is -H. If necessary, the ^R1 position can be alkylated by any suitable method known in the art to form a compound of formula (IA), wherein ^R1 is a _C1-6 alkyl group.

Precursors (A), (B), (B1), (C1), (C2) and appropriately substituted cyclohexylamine can be prepared by any suitable method known in the art. Specific exemplary synthetic methods are described in the following examples.

Compounds of formulas 2 to 11 can be prepared using similar chemical substances. A list of suitable precursors for use in process B is provided below:

Alternatively, we can use compounds with the structure of intermediate Int-11 to prepare compounds with the structure of intermediate Int-12.

From Int-12, we can prepare compounds of formula 1 by reacting them with compounds of formula L-X-C(O)-Cl under appropriate amidation conditions (usually involving the use of trialkylamines as proton sponges).

From Int-12, we can prepare compounds of formulas 2 to 3 by reacting them with appropriately substituted azircyclopentane-C(O)Cl or azircyclohexane-C(O)Cl compounds under appropriate amidation conditions.

From Int-12, we can prepare compounds of formulas 4 to 5 by reacting them with appropriately substituted tetrahydroisoquinoline-C(O)Cl under suitable amidation conditions.

From Int-12, we can prepare compounds of formulas 8 to 9 by reacting them with appropriately substituted pyridine-C(O)Cl, pyrimidine-C(O)Cl or pyrazine-C(O)Cl under appropriate amidation conditions.

From Int-12, we can prepare compounds of formulas 10 to 13 by reacting them with appropriately substituted azinaphthalene-C(O)Cl under suitable amidation conditions.

Chiral separation

The compounds described herein may have an asymmetric center and exist as racemates, mixtures of racemates, individual diastereomers, or enantiomers, all of which are included in this invention. Compounds described herein with a chiral center may exist and be separated in both optically active and racemic forms. Some compounds may exhibit polymorphism. This disclosure covers racemic, optically active, polymorphic, or stereoisomeric forms or mixtures thereof of the compounds described herein having the applicable properties described herein. The optically active form can be prepared, for example, by resolving the racemic form using recrystallization, by synthesis from an optically active starting material, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution. We can purify the corresponding compound, then derivatize the compound to form the compound described herein or purify the compound itself.

The optically active form of the compound may be prepared using any method known in the art, including (but not limited to) resolving the racemic form by recrystallization, by synthesis from an optically active starting material, by chiral synthesis, or by separation by chiral stationary phase chromatography.

Examples of methods for obtaining optically active materials include at least the following.

i) Physical separation of crystals: A technique for manually separating macroscopic crystals of individual enantiomers. This technique can be used if crystals of the individual enantiomers are present, i.e., the substance is an aggregate and the crystals are visually apparent;

ii) Simultaneous crystallization: a technique for crystallizing individual enantiomers from a racemic solution, which is only possible when the racemic mixture is a solid aggregate;

iii) Enzymatic resolution: A technique that uses the difference in reaction rates between enantiomers and enzymes to partially or completely separate racemic mixtures;

iv) Enzymatic asymmetric synthesis: a synthetic technique in which at least one step of the synthesis uses an enzymatic reaction to obtain enantiomeric purity or synthetic precursor rich in the desired enantiomer;

v) Chemical asymmetric synthesis: a synthetic technique for synthesizing desired enantiomers from achiral precursors under conditions that produce asymmetry (i.e., chirality) in the product, which can be achieved using chiral catalysts or chiral auxiliaries.

vi) Diastereomer separation: A technique involving the reaction of a racemic compound with an enantiomeric pure reagent (chiral auxiliary agent) that converts individual enantiomers into diastereomers. The resulting diastereomers are then separated by chromatography or crystallization based on their now more pronounced structural differences, and subsequently the chiral auxiliary agent is removed to obtain the desired enantiomers;

vii) First and second-order asymmetric transformations: a technique involving diastereomeric equilibrium from the racemic mixture to preferentially resolve diastereomeric isomers from the desired enantiomer, or preferential crystallization of the diastereomeric isomers from the desired enantiomers to disrupt the equilibrium so that ultimately substantially all substances are converted into crystalline diastereomeric isomers from the desired enantiomers. The desired enantiomer is then released from the diastereomeric isomers.

viii) Kinetic resolution: This technique refers to the partial or complete resolution of a racemic compound (or further resolution of a partially resolved compound) by means of the unequal reaction rates of the enantiomer and the chiral non-racemic reagent or catalyst under kinetic conditions.

ix) Enantiomer-specific synthesis from non-racemic precursors: a synthetic technique that obtains the desired enantiomer from an achiral starting material in such a way that the stereochemical integrity is not or only minimally impaired during the synthesis.

x) Chiral liquid chromatography: a technique for separating enantiomers of racemic mixtures by means of their different interactions with a stationary phase (including (but not limited to) via chiral HPLC) in a liquid mobile phase. The stationary phase may be prepared from a chiral substance or the mobile phase may contain another chiral substance to induce different interactions;

xi) Chiral gas chromatography: a technique for separating racemic mixtures by means of their different interactions with a column containing a fixed non-racemic chiral adsorbent phase in a mobile gas phase;

xii) Chiral solvent extraction: a technique for separating enantiomers by means of an enantiomer preferentially dissolving in a particular chiral solvent;

xiii) Transport across chiral membranes: Techniques that bring a racemic mixture into contact with a membrane barrier. The barrier typically separates two miscible fluids, one containing the racemic mixture, and driving forces (such as concentration or pressure difference) induce preferential transport across the membrane barrier. Separation is achieved through the non-racemic chiral properties of the membrane, which allow only one enantiomer of the racemic mixture to pass through.

One embodiment uses chiral chromatography, including (but not limited to) simulated moving bed chromatography. Various chiral stationary phases are commercially available.

Definitions and General Terms

For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999 and March's Advanced Organic Chemistry, 5th edition, eds. Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

As described herein, the compounds described herein may optionally be substituted with one or more substituents as generally shown below or exemplified by the specific classes, subclasses, and species described herein. It should be understood that the phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” Generally, the term “substituted,” whether or not it follows the term “optionally,” refers to the substitution of one or more hydrogen groups in a given structure by a specific substituent. Unless otherwise specified, the optionally substituted group may have substituents at each substituted position of the group. When more than one position in a given structure may be substituted by more than one substituent selected from a particular group, the substituents may be the same or different at each position. When the term “optionally substituted” precedes a list, the term refers to all subsequent substituted groups in the list. If a substituent or structure is not identified or defined as “optionally substituted,” then the substituent or structure is unsubstituted. For example, if X is an optionally substituted _C1 - _C3 alkyl or phenyl; then X may be an optionally substituted _C1 - _C3 alkyl or an optionally substituted phenyl. Similarly, if the term "optionally substituted" follows a list, then unless otherwise specified, the term also refers to all substituted groups in the preceding list. For example: if X is a _C1 - _C3 alkyl or phenyl group, wherein X is optionally and independently substituted with ^JX , then the _C1 - _C3 alkyl and phenyl groups may be optionally substituted with ^JX . As will be apparent to those skilled in the art, groups such as H, halogens, _NO2 , CN, _NH2 , OH, or _OCF3 are non-substituted groups.

As used herein, the phrase “at most” refers to zero or any integer equal to or less than the number following the phrase. For example, “at most 3” means any one of 0, 1, 2, and 3. As described herein, the specified range of the number of atoms includes any integer within that range. For example, a group having 1 to 4 atoms may have 1, 2, 3, or 4 atoms.

As used herein, the phrase “substituted by one or more instances” indicates that the referred portion has at least one substituent as defined herein, as permitted by the valence of the substituted portion. For example, a 6-membered aryl ring (e.g., a benzene ring) substituted by one or more J instances may be substituted by any of 1, 2, 3, 4, 5, and 6 J.

The selection and combination of substituents covered herein refer to those selections and combinations that form stable or chemically viable compounds. As used herein, the term "stable" means that a compound remains substantially unchanged when subjected to conditions that allow for its production, detection, and, more precisely, its recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable or chemically viable compound is one that remains substantially unchanged when kept at a temperature of 40°C or lower for at least one week in the absence of moisture or other chemically reactive conditions. Only those selections and combinations of substituents that produce stable structures are covered. Such selections and combinations will be obvious to those skilled in the art and can be determined without improper experimentation.

As used herein, the term "aliphatic group" or "aliphatic group" means a straight-chain (i.e., unbranched) or branched hydrocarbon chain that is fully saturated or contains one or more unsaturated but non-aromatic units. Unless otherwise specified, an aliphatic group contains 1 to 20 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1 to 10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 6 aliphatic carbon atoms, and in other embodiments, an aliphatic group contains 1 to 4 aliphatic carbon atoms. The aliphatic group may be straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, or ynyl. Specific examples include (but are not limited to) methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl, ethynyl, and tert-butyl and acetylene.

As used herein, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon. As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon comprising one or more double bonds. As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon comprising one or more triple bonds. As used herein, each of "alkyl,""alkenyl," or "alkynyl" may optionally be substituted as described below. In some embodiments, "alkyl" is a _C1 - _C14 alkyl, _{C1 - C6} alkyl, or _{C1 - C4} alkyl. In some embodiments, "alkenyl" is a C2- _C6 alkenyl or C2- _C4 alkenyl. In some embodiments, "alkynyl" is a _C2 - _C6 alynyl or _C2 - _C4 alynyl.

The term "cycloaliphatic group" (or "cycloalkyl", "carbocyclic", or "carbocyclic group" or "carbocyclic") refers to a non-aromatic ring system containing only carbon atoms, which may be saturated or contain one or more unsaturated units, having 3 to 14 ring carbon atoms. In some embodiments, the number of carbon atoms is 3 to 12 (i.e., _C3 - _C12 cycloalkyl). In other embodiments, the number of carbon atoms is 4 to 7. In other embodiments, the number of carbon atoms is 5 or 6. The term encompasses monocyclic, bicyclic, or polycyclic fused, helical, or bridged carbocyclic systems. The term also encompasses polycyclic systems in which the carbon ring may be "fused" with one or more non-aromatic carbon rings or heterocycles or one or more aromatic rings or combinations thereof, wherein a group or connecting point is located on the carbon ring. A "fused" bicyclic system comprises two rings sharing two adjacent ring atoms. A bridged bicyclic group comprises two rings sharing three or four adjacent ring atoms. A helical bicyclic system shares one ring atom. Examples of cycloaliphatic groups include (but are not limited to) cycloalkyl and cycloalkenyl groups. Specific examples include (but are not limited to) cyclohexyl, cyclopropenyl, cyclobutyl, and cyclopropyl.

As used herein, the term "heterocyclic" (or "heterocyclic group," "heterocyclic," or "non-aromatic heterocyclic") refers to a non-aromatic ring system that may be saturated or contain one or more unsaturated units, having three to fourteen ring atoms, wherein one or more ring carbons are replaced by heteroatoms such as N, S, or O. In some embodiments, the ring system may contain 3 to 12 ring atoms (i.e., a 3 to 12-membered heterocyclic group). In some embodiments, the non-aromatic heterocyclic includes at most three heteroatoms selected from N, S, and O within the ring. In other embodiments, the non-aromatic heterocyclic includes at most two heteroatoms selected from N, S, and O within the ring system. In other embodiments, the non-aromatic heterocyclic includes at most two heteroatoms selected from N and O within the ring system. The term encompasses monocyclic, bicyclic, or polycyclic fused, helicaled, or bridged heterocyclic systems. The term also encompasses polycyclic systems in which the heterocyclic may be "fused" with one or more non-aromatic carbon rings or heterocycles or one or more aromatic rings or combinations thereof, wherein a group or connecting point is located on the heterocyclic. Examples of heterocycles include (but are not limited to): piperidinyl, piperazinyl, pyrrolyl, pyrazolidyl, imidazodiphenyl, azirheptanyl, diazaheptanyl, triazaheptanyl, azirheptanyl, diazaheptanyl, triazaheptanyl, oxazolidinyl, isoxazolidinyl, thiazodiphenyl, isothiazolidinyl, oxazolidinyl, oxazolidinyl, thiazoptanyl, thiazoptanyl, benzimidazolone, tetrahydrofuranyl, tetrahydrothiophene, N-morpholinyl (including, for example, 3-N-morpholinyl, 4-N-morpholinyl, 2-N-thiomorpholinyl, 3-N-thiomorpholinyl, 4-N-thiomorpholinyl), 1-pyrrolyl, 2-pyrrolyl , 3-pyrrolidinyl, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolinyl, 3-thiazolinyl, 4-thiazolinyl, 1-imidazolinyl, 2-imidazolinyl, 4-imidazolinyl, 5-imidazolinyl, indololinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzodithiacyclopentyl, benzodithiaalkyl, 3-(1-alkyl)-benzimidazol-2-one and 1,3-dihydro-imidazolin-2-one.

As used herein, the term "aryl" refers to a monocyclic aromatic group, such as a phenyl group. Unless otherwise specified, an aryl group may have 6 to 14 carbon atoms in the ring, such as 6 to 10 carbon atoms in the ring (i.e., _C6 - _C10 aryl). Unless otherwise specified, an aryl group may be unsubstituted or substituted with one or more, and especially one to four, independently selected from, for example, halogenated, alkyl, alkenyl, _OCF3 , _NO2 , CN, NC, OH, alkoxy, amino, CO2H, _CO2alkyl , aryl, _and heteroaryl. An aryl group may be independent (e.g., phenyl) or fused with another aryl group (e.g., naphthyl, anthracene), cycloalkyl (e.g., tetrahydronaphthyl), heterocycloalkyl, and/or heteroaryl. Exemplary aryl groups include (but are not limited to) phenyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, 2,4-methoxychlorophenyl, 1-naphthyl, 2-naphthyl, 1-anthrayl, 2-anthrayl, dihydroindenyl, phthalimide, naphthylimide, phenidyl or tetrahydronaphthyl and similar groups.

The terms “heteroaryl,” “heteroaromatic,” “heteroaryl ring,” “heteroaryl group,” “aromatic heterocycle,” or “heteroaromatic group,” used alone or as a major part of “heteroarylalkyl” or “heteroarylalkoxy,” refer to a heteroaromatic ring group having five to fourteen members, including monocyclic and polycyclic aromatic rings, wherein the monocyclic aromatic ring is fused with one or more other aromatic rings. A heteroaryl has one or more cyclic heteroatoms selected from nitrogen, oxygen, and sulfur in the aromatic ring. As used herein, a heteroaryl ring may have 5 to 14 ring atoms, such as 5 to 10 ring atoms (i.e., 5 to 10 membered heteroaryls). The scope of the term “heteroaryl” as used herein also includes groups “fused” to one or more non-aromatic rings (carbocyclic or heterocyclic), wherein the group or connecting point is on the aromatic ring. As used herein, for example, a bicyclic 6.5 heteroaromatic ring is a six-membered heteroaromatic ring fused with a second five-membered ring, wherein the group or linker is located on the six-membered ring. Examples of heteroaromatic groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazoleyl, pyrroleyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, or thiadiazolyl, including, for example, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isooxazolyl, 4-isooxazolyl, 5-imidazolyl, 3-isooxazolyl, 4-isooxazolyl, 5- -Isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-pyrazolyl, 4-pyrazolyl, 1-pyrroleyl, 2-pyrroleyl, 3-pyrroleyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl 2-Triazolyl, 5-Triazolyl, Tetrazolyl, 2-Thienyl, 3-Thienyl, Carbazole, Benzimidazolyl, Benzimidenyl, Benzifuranyl, Indolyl, Benzimidazolyl, Benzimidazolyl, Benzimidazolyl, Isoquinolinyl, Indolyl, Isoyindolyl, Acridineyl, Benzisoxazolyl, Isothiazolyl, 1,2,3-Oxadiazolyl, 1,2,5-Oxadiazolyl The compounds include 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl, 1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl) and isoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl or 4-isoquinolinyl).

As used herein, “cyclic,” “cyclic,” “cycloyl” or “cyclic moiety” includes monocyclic, dicyclic and tricyclic ring systems, bridged bicyclic systems and spirocyclic systems, and includes cycloalkyl, heterocyclic, aryl or heteroaryl or combinations thereof, each of which has been previously defined.

As used herein, “bridged bicyclic system” refers to a bicyclic heterocyclic aliphatic ring system or a bicyclic aliphatic ring system in which the rings are bridged. Examples of bridged bicyclic systems include (but are not limited to) adamantyl, norbornel, bicyclic [3.2.1]octyl, bicyclic [2.2.2]octyl, bicyclic [3.3.1]nonyl, bicyclic [3.2.3]nonyl, 2-oxa-bicyclic [2.2.2]octyl, 1-aza-bicyclic [2.2.2]octyl, 3-aza-bicyclic [3.2.1]octyl, and 2,6-dioxa-tricyclic [3.3.1.03,7]nonyl. Bridged bicyclic systems may be optionally substituted.

As used in this article, the term "screwed" refers to a ring system having a single atom (usually the fourth carbon) as the only common atom between the two rings.

The term "cyclic atom" refers to an atom such as C, N, O, or S located in a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring.

A "substitutable cyclic atom" is a cyclic carbon or nitrogen atom that can bond to a hydrogen atom but is bonded to a portion other than a hydrogen atom. Therefore, the term "substitutable cyclic atom" does not include cyclic nitrogen or carbon atoms shared when two rings are fused. Furthermore, a "substitutable cyclic atom" does not include cyclic carbon or nitrogen atoms when its structure is described as being attached to a portion other than hydrogen.

Unless otherwise stated herein, the term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or the like.

As used herein, the term "aralkyl" refers to an aryl ring attached to an alkyl chain, and "aralkylalkoxy" refers to an aryl ring attached to an alkoxy chain. Optionally substituted aralkyl groups may be substituted at both the alkyl and aryl moieties. Unless otherwise specified, as used herein, optionally substituted aralkyl groups are optionally substituted at the aryl moieties.

In some embodiments, the aliphatic or heteroaliphatic group or non-aromatic heterocycle may contain one or more substituents. Suitable substituents on the saturated carbon of the aliphatic or heteroaliphatic group or non-aromatic heterocycle are selected from those listed above, such as those listed in the definition of J. Other suitable substituents include those listed as applicable to the unsaturated carbon of the carbocyclic aryl or heteroaryl group, and additionally include the following: =O, =S, =NNHR*, =NN(R*) ₂ , =NNHC(O)R*, = _NNHCO2 (alkyl), = _NNHSO2 (alkyl), or =NR*, wherein each R* is independently selected from hydrogen or optionally substituted _C1-6 aliphatic groups. The optional substituents on the aliphatic group of R* are selected from _NH2 , NH ( _C1-4 alkyl), N ( _C1-4 alkyl) ₂ , halogen, _C1-4 alkyl, OH, O ( _C1-4 alkyl), _NO2 , CN, _CO2H , _CO2 ( _C1-4 alkyl), O (halogenated _C1-4 alkyl) or halogenated ( _C1-4 alkyl), wherein each of the aforementioned _C1-4 alkyl groups of R* is unsubstituted.

In some embodiments, optional substituents on the nitrogen of the non-aromatic heterocycle include those substituents used in the definition of J above, for example. Other suitable substituents include -R ⁺ , -N(R ⁺ ) ₂ , -C(O)R ⁺ , _-CO2R ⁺ , -C(O)C(O)R ⁺ , -C(O) _CH2C (O)R ⁺ , _-SO2R ⁺ , _-SO2N (R ⁺ ) ₂ , -C(=S)N(R ⁺ ) ₂ , -C(=NH)-N(R ⁺ ) ₂ , or -NR+ _SO2R ⁺ ; wherein R ⁺ is hydrogen, optionally substituted _C1-6 alkyl, optionally substituted phenyl, optionally substituted -O(Ph), optionally substituted _-CH2 (Ph), optionally substituted -( _CH2 ) _1-2 (Ph); optionally substituted -CH=CH(Ph); or unsubstituted 5- to 6-membered heteroaryl or heterocyclic ring having one to four independently selected cyclic heteroatoms chosen from oxygen, nitrogen, and sulfur, or two independently occurring R ⁺ groups on the same or different substituents together with the atoms attached to each R ⁺ group to form a 5- to 8-membered heterocyclic group, aryl or heteroaryl ring, or a 3- to 8-membered cycloalkyl ring, wherein the heteroaryl or heterocyclic ring has one to three independently selected cyclic heteroatoms chosen from nitrogen, oxygen, and sulfur. The optional substituents on the aliphatic group or benzene ring of R ⁺ are selected from _NH2 , NH ( _C1-4 alkyl), N ( _C1-4 alkyl) ₂ , halogen, _C1-4 alkyl, OH, O ( _C1-4 alkyl), _NO2 , CN, _CO2H , _CO2 ( _C1-4 alkyl), O (halogenated _C1-4 alkyl), or halogenated ( _C1-4 alkyl), wherein each of the aforementioned _C1-4 alkyl groups of R ⁺ is unsubstituted.

In some embodiments, the aryl or heteroaryl group may include one or more (e.g., 1, 2 or 3) substituents. Suitable substituents include: halogens; ^-Ro ; ^-ORo ; ^-SRo ; 1,2-methylenedioxy; 1,2-ethylenedioxy; optionally Ro ^- substituted phenyl (Ph); optionally ^Ro -substituted -O (Ph); optionally ^Ro- substituted -( _CH₂ ) _¹⁻² (Ph); optionally ^Ro -substituted -CH=CH (Ph); _-NO₂ ; -CN; -N( ^Ro ) _₂ ; ^-NRoC (O) ^Ro ; ^-NRoC (S) ^Ro ) _₂ ; ^-NRoC (O)N( ^Ro ) _₂ ; ^{-NRoC (} S)N( ^{Ro )} _₂ ; ^{-NRoCO₂Ro ;} _-NRoNRoC (O) ^Ro ; ^{-NRoNRoC ( O} )N( ^Ro _{) ₂} ^{; -NRoNRoCO₂Ro} ;-C(O)C(O)R ^o ;-C(O)CH ₂ C(O)R ^o ;-CO ₂ R ^o ;-C(O)R ^o ;-C(S)R ^o ;-C(O)N(R ^o ) ₂ ;-C(S)N(R ^o ) ₂ ;-OC(O)N(R ^o ) ₂ ;-OC(O)R ^o ;-C(O)N(OR ^o )R ^o ;-C(NOR ^o )R ^o ;-S(O) ₂ R ^o ;-S(O) ₃ R ^o ;-SO ₂ N(R ^o ) ₂ ;-S(O)R ^o ;-NR ^o SO ₂ N(R ^o ) ₂ ;-NR ^o SO ₂ R ^o ;-N(OR ^o )R ^o ;-C(＝NH)-N(R ^o ) ₂ ; or-(CH ₂ ) _0-2 NHC(O) ^Ro ; wherein each individually occurring ^Ro is selected from hydrogen, optionally substituted _C1-6 alkyl, unsubstituted 5- to 6-membered heteroaryl or heterocyclic, phenyl, -O(Ph) or _-CH2 (Ph), or two independently occurring ^Ros on the same or different substituents together with the atoms attached to each ^Ro group to form a 5- to 8-membered heterocyclic group, aryl or heteroaryl ring or a 3- to 8-membered cycloalkyl ring, wherein the heteroaryl or heterocyclic ring has 1 to 3 independently selected cycloheteroatoms selected from nitrogen, oxygen and sulfur. The optional substituents on the aliphatic group of ^Ro are selected from _NH2 , NH ( _C1-4 alkyl), N ( _C1-4 alkyl) ₂ , halogen, _C1-4 alkyl, OH, O ( _C1-4 alkyl), _NO2 , CN, _CO2H , _CO2 ( _C1-4 alkyl), O (halogenated _C1-4 alkyl) or halogenated ( _C1-4 alkyl), CHO, N(CO)( _C1-4 alkyl), C(O)N ( _C1-4 alkyl), wherein each of the aforementioned _C1-4 alkyl groups of ^Ro is unsubstituted.

Non-aromatic nitrogen-containing heterocycles that are substituted at a nitrogen atom on the ring and attached to the rest of the molecule at a carbon atom on the ring are called N-substituted heterocycles. For example, an N-alkylpiperidinyl ring is attached to the rest of the molecule at two, three, or four positions on the piperidinyl ring and is alkyl-substituted at a nitrogen atom on the ring. Non-aromatic nitrogen-containing heterocycles such as pyrazinyl rings that are substituted at a nitrogen atom on the ring and attached to the rest of the molecule at a second nitrogen atom on the ring are called N'-substituted -N-heterocycles. For example, an N'-acyl-N-pyrazinyl ring is attached to the rest of the molecule at one nitrogen atom on the ring and is acyl-substituted at a second nitrogen atom on the ring.

As detailed above, in some embodiments, two independently occurring R ^_o (or R_⁺, or any other variable similarly defined herein) may, together with the atoms bound to each variable, form a 5- to 8-membered heterocyclic group, aryl or heteroaryl ring, or a 3- to 8-membered cycloalkyl ring. An exemplary ring formed by two independently occurring ^Ro (or R ⁺ , or any other variable similarly defined herein) together with the atoms to which each variable is bonded includes (but is not limited to) the following: a) two independently occurring ^Ro (or R ⁺ , or any other variable similarly defined herein) bonded to the same atom and forming a ring together with said atom, such as N( ^Ro ) ₂ , wherein the two occurring ^Ros together with the nitrogen atom form a piperidin-1-yl, piperazine-1-yl, or morpholin-4-yl; and b) two independently occurring ^Ro (or R ⁺ , or any other variable similarly defined herein) bonded to different atoms and forming a ring together with both of those atoms, such as where a phenyl group is substituted by two occurring ^ORs to form a fused 6-membered ^oxygen -containing ring together with the oxygen atom to which it is bonded:

It should be understood that many other rings may be formed when two independently occurring R ^_o (or R_⁺, or any other variable similarly defined herein) are combined with the atoms bound to each variable, and the examples detailed above are not intended to be limiting.

In some embodiments, the alkyl chain may optionally be interspersed with another atom or group. This means that the methylene unit of the alkyl chain is optionally substituted with said other atom or group. Examples of such atoms or groups include (but are not limited to) those atoms or groups listed in the definition of the variables following each formula. For example, the methylene in the side chain may be substituted with the following: -NR ^4- , -O-, -S-, -CO _2- , -OC(O)-, -C(O)CO-, -C(O)-, -C(O)NR ^4- , -C(＝N-CN)-, -NR ⁴ CO-, -NR ⁴ C(O)O-, -SO ₂ NR ^4- , -NR ⁴ SO _2- , -NR ⁴ C(O)NR ^4- , -OC(O)NR ^4- , -NR ⁴ SO ₂ NR ^4- , -SO-, or -SO _2- , where R ⁴ is as defined above.

As used herein, “amino” means -NR ^{X RY} , wherein each of ^RX and ^RY is independently -H, _C1 - _C6 alkyl, _C3-7 cycloalkyl, _C6-10 aryl, 5- to 6-membered heteroaryl or 4- to 7-membered heterocycle, each independently defined herein and optionally substituted. Suitable substituents for these groups include halogens, cyano groups, hydroxyl groups, oxo groups, _-NH₂ , -NH ( _C₁ - _C₆ alkyl), -N ( _C₁ - _C₆ alkyl) _₂ , _C₁ - _C₆ alkyl, -O ( _C₁ - _C₆ alkyl), -C(O)OH, -C(O)O ( _C₁ - _C₆ alkyl), -OC(O) ( _C₁ - _C₆ alkyl), -NHC(O)( _C₁ - _C₆ alkyl), -NHC(O)O ( _C₁ - _C₆ alkyl), -C(O)NH ( _C₁ - _C₆ alkyl), and -C(O)N ( _C₁ - _C₆ alkyl) _₂ , wherein each of said alkyl groups is optionally and independently substituted by one or more substituents selected from the group consisting of: halogens, cyano groups, hydroxyl groups, oxo groups, _-NH₂ , -NH ( _C₁ - _C₄ alkyl), and -N ( _C₁ - _C₄ alkyl). _2. -OCO ( _C1 - _C4 alkyl), -CO ( _C1 - _C4 alkyl), _-CO₂H , _-CO₂ ( _C1 - _C4 alkyl), and _C1 - _C4 alkoxy. In some embodiments, each of ^RX and ^RY is independently -H, optionally substituted _C1-6 alkyl, or optionally substituted _C3-8 cycloalkyl. In some embodiments, each of ^RX and ^RY is independently -H or optionally substituted _C1-6 alkyl. In some embodiments, each of ^RX and ^RY is independently a _C1-6 alkyl group substituted with -H or optionally with one or more substituents selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH₂ , -NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) _₂ , -OCO (C1- _C4 alkyl), -CO ( _C1 - _C4 alkyl), _-CO₂H , -CO₂ ( _{C1 - C4} alkyl), and _{C1 - C4} alkoxy. Examples of amino groups include _-NH₂ , alkylamino, dialkylamino, or arylamino.

As used herein, “amide group” encompasses N(R ^X R ^Y )-C(O)- or R ^Y C(O)-N(R ^X )- when used at the end and -C(O)-N(R ^X )- or -N(R ^x )-C(O)- when used internally, where R ^X and R ^Y are as defined above. Examples of amide groups include alkylamide groups (such as alkylcarbonylamino, or alkylcarbonylamino, or alkylaminocarbonyl), (heterocyclic aliphatic)amide groups, (heteroarylalkyl)amide groups, (heteroaryl)amide groups, (heterocyclic alkyl)alkylamide groups, arylamide groups, arylalkylamide groups, (cycloalkyl)alkylamide groups, or cycloalkylamide groups. In some embodiments, the amide group is -NHC(O) ( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl)C(O) ( _C1 - _C6 alkyl), -C(O)NH2, -C(O)NH ( _{C1 - C6} alkyl), or -C(O)NH ( _C1 - _C6 alkyl) ₂ , wherein each of the alkyl groups is optionally and independently substituted by one or more substituents selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH ( _C1 - _C6 alkyl), -N ( _C1 - _C6 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO ( _C1 - _C4 alkyl), _-CO2H , _-CO2 ( _C1 - _C4 alkyl), and _C1 - _C4 alkoxy. In some embodiments, the amide group is -NHC(O) ( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl)C(O) ( _C1 - _C6 alkyl), -C(O)NH2, -C(O)NH ( _{C1 - C6} alkyl), or -C(O)NH ( _C1 - _C6 alkyl) ₂ , wherein each of the alkyl groups is optionally and independently substituted by one or more substituents selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH ( _C1 - _C6 alkyl), -N ( _C1 - _C6 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO ( _C1 - _C4 alkyl), _-CO2H , _-CO2 ( _C1 - _C4 alkyl), and _C1 - _C4 alkoxy.

As used herein, “acyl” is a methyl acyl or R ^X -C(O)- (e.g., -alkyl-C(O)-, also known as “alkyl carbonyl”), where R ^X and “alkyl” have been previously defined. Acetyl and pivaloyl are examples of acyl groups.

As used in this article, "carboxyl group" refers to -COOH, -COOR ^X , -OC(O)H, or -OC(O)R ^X when used as an end group; or -OC(O)- or -C(O)O- when used as an internal group, where R ^X is as defined above.

As used herein, “alkoxycarbonyl” as defined by the term carboxyl group, used alone or in combination with another group, refers to a group such as (alkyl-O)-C(O)-.

As used in this article, "carbonyl" refers to -C(O)-.

As used in this article, "oxo" refers to =O.

As used herein, the terms “alkoxy” or “alkylthio” as used herein refer to an alkyl group that is attached to a molecule via an oxygen (“alkoxy”, e.g., -O-alkyl) or sulfur (“alkylthio”, e.g., -S-alkyl) atom as previously defined.

As used herein, the terms “halogen”, “halogen”, and “halogen” refer to F, Cl, Br, or I.

As used herein, the terms “cyano” or “nitrile” refer to -CN.

The terms “alkoxyalkyl”, “alkoxyalkenyl”, “alkoxyaliphatic” and “alkoxyalkoxy” mean alkyl, alkenyl, aliphatic or alkoxy groups that may optionally be substituted with one or more alkoxy groups.

The terms “haloalkyl,” “haloalkenyl,” “haloaliphatic,” and “haloalkoxy” refer to an alkyl, alkenyl, aliphatic, or alkoxy group that may optionally be substituted with one or more _halogen atoms. This term includes perfluoroalkyl groups such as _-CF3 and _-CF2CF3 .

The terms “cyanoalkyl,” “cyanoalkenyl,” “cyanoaliphatic,” and “cyanoalkoxy” mean an alkyl, alkenyl, aliphatic, or alkoxy group that may optionally be substituted with one or more cyano groups. In some embodiments, the cyanoalkyl group is (NC)-alkyl-.

The terms “aminoalkyl,” “aminoalkenyl,” and “aminoalkoxy” mean an alkyl, alkenyl, or alkoxy group optionally substituted with one or more amino groups, wherein the amino group is as defined above. In some embodiments, the _C1 - _C6 alkyl group is substituted with one or more _-NH2 groups. In some embodiments, the aminoalkyl group refers to the structure ( ^RXRY )N- ^alkyl- , wherein each of ^RX and ^RY is independently as defined above. In some specific embodiments, the aminoalkyl group is a _C1 - _C6 alkyl group substituted with one or more _-NH2 groups. In some specific embodiments, the aminoalkenyl group is a _C1 - _C6 alkenyl group substituted with one or more _-NH2 groups. In some embodiments, the aminoalkoxy group is -O ( _C1 - _C6 alkyl), wherein the alkyl group is substituted with one or more _-NH2 groups.

The terms “hydroxyalkyl” and “hydroxyalkoxy” mean alkyl or alkoxy groups that may optionally be substituted with one or more -OH groups.

The terms “alkoxyalkyl” and “alkoxyalkoxy” mean an alkyl or alkoxy group that may optionally be substituted with one or more alkoxy groups. For example, “alkoxyalkyl” refers to an alkyl group such as (alkyl-O)-alkyl-, wherein the alkyl group is as defined above.

The term "carboxyalkyl" means an alkyl group substituted with one or more carboxyl groups, wherein the alkyl and carboxyl groups are as defined above.

In some embodiments, each of the amino groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formulas A, B, or C, or formulas 1 to 13) is independently _-NH₂ , -NH( _C₁ - _C₆ alkyl), -NH( _C₃ - _C₆ cycloalkyl), -N( _C₁ - _C₆ alkyl) _₂ , or -N( _C₁ - _C₆ alkyl)( _C₃ - _C₆ cycloalkyl), wherein each of the alkyl and cycloalkyl groups is optionally and independently substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH₂ , -NH( _C₁ - _C₄ alkyl), -N( _C₁ - _C₄ alkyl) _₂ , -OCO( _C₁ - _C₄ alkyl), -CO( _C₁ - _C₄ alkyl), -CO₂H, _{-CO₂ ( C₁} - _C₄ alkyl), and _C₁ -C₆ alkyl. _4. Alkoxy group; each of the carboxyl groups mentioned in the description of formulas A, B, or C or the variables of formulas 1 to 13 (e.g., ^R6 , ^R7 , J, R, R', R", R*, ^Ra , ^Rb , and ^Rc ) is independently -C(O)O ( _C1 - _C6 alkyl), -OC(O) ( _C1 - _C6 alkyl), -C(O)O ( _C3 - _C6 cycloalkyl), -OC(O) ( _C3 - _C6 cycloalkyl), or _-CO2H , wherein the alkyl group is optionally substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 , -NH ( _C1 -C4 alkyl), -N (C1- _C4 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO ( _C1 - _C4 alkyl), -CO2H, -CO2 ( _C1 - _C4 alkyl), _-CO2H , _-CO2 (C1- _C4 alkyl). ₄ -alkyl) and _C1 - _C4 alkoxy.

Each of the amide groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to 13) is independently -NHC(O) ( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl)C(O) ( _C1 - _C6 alkyl), -C(O)NH ( _C1 - _C6 alkyl), -C(O)N( _C1 - _C6 alkyl) ₂ , -NHC(O) ( _C3 - _C6 cycloalkyl), -N( _C1 - _C6 alkyl)C(O) ( _C3 - _C6 cycloalkyl), -C(O)NH ( _C3 - _C6 cycloalkyl), -C(O)N( _C1 - _C6 alkyl) ( _C3 - _C6 cycloalkyl) or -C(O) _NH2 , wherein each of the alkyl and cycloalkyl groups is optionally substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH2 -NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) ₂ , -OCO (C1- _C4 alkyl), -CO ( _{C1 - C4} alkyl), -CO _2H , -CO ₂ ( _C1 - _C4 alkyl) and _C1 - _C4 alkoxy.

Each of the aminoalkyl groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to ₁₃ ) is independently a C1 _- C6 alkyl group substituted with one or more amino groups independently selected from the group consisting of _-NH2 , -NH( _C1 - _C4 alkyl), and -N( _C1 - _C4 alkyl) ₂ .

Each of the aminoalkoxy groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to 13) is independently -O ( _C1 - _C6 alkyl), wherein the alkyl group is substituted with one or more amino groups independently selected from the group consisting of _-NH2 , -NH ( _C1 - _C4 alkyl), and -N ( _C1 - _C4 alkyl) ₂ .

In some embodiments, each of the amino groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to 13) is independently _-NH₂ , -NH( _C₁ - _C₆ alkyl) or -N( _C₁ - _C₆ alkyl) _₂ , wherein each alkyl group is optionally substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH₂ , -NH( _C₁ - _C₄ alkyl), -N( _C₁ - _C₄ alkyl) _₂ , -OCO( _C₁ - _C₄ alkyl), -CO( _C₁ - _C₄ alkyl), _-CO₂H , _-CO₂ ( _C₁ - _C₄ alkyl), and _C₁ - _C₄ alkoxy.

Each of the carboxyl groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to 13) is independently -C(O)O ( _C1 - _C6 alkyl), -OC(O) ( _C1 - _C6 alkyl), or _-CO₂H , wherein each alkyl group is optionally substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, _-NH₂ , -NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) _₂ , -OCO ( _C1 - _C4 alkyl), -CO ( _C1 - _C4 alkyl), _-CO₂H , _-CO₂ ( _C1 - _C4 alkyl), and _C1 - _C4 alkoxy.

Each of the amide groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to 13) is independently -NHC(O) ( _C1 - _C6 alkyl), -N( _C1 - _C6 alkyl)C(O) ( _C1 - _C6 alkyl), -C(O)NH ( _C1 - _C6 alkyl), -C(O)N( _C1 - _C6 alkyl) ₂ , or -C(O)NH ₂ , wherein each alkyl group is optionally substituted by one or more substituents independently selected from the group consisting of: halogen, cyano, hydroxyl, oxo, -NH ₂ , -NH ( _C1 - _C4 alkyl), -N ( _C1 - _C4 alkyl) ₂ , -OCO ( _C1 - _C4 alkyl), -CO ( _C1 - _C4 alkyl), -CO _2H , -CO ₂ ( _C1 - _C4 alkyl), and _C1 - _C4 alkoxy.

Each of the aminoalkyl groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to ₁₃ ) is independently a C1 _- C6 alkyl group substituted with an amino group independently selected from the group consisting of _-NH2 , -NH( _C1 - _C4 alkyl), and -N( _C1 - _C4 alkyl) ₂ ; and each of the aminoalkoxy groups mentioned in the description of the variables of the formulas disclosed herein (e.g., formula A, B, or C, or formulas 1 to 13) is independently -O( _C1 - _C6 alkyl), wherein the alkyl group is substituted with an amino group independently selected from the group consisting of -NH2, -NH( _{C1 - C4} alkyl), and -N( _C1 - _C4 alkyl) ₂ .

As used herein, the terms "protecting group" and "protective group" are interchangeable and refer to an agent used to temporarily block one or more desired functional groups in a compound having multiple reactive sites. In some embodiments, the protecting group has one or more, or precisely all, of the following characteristics: a) selectively added to the functional group in good yield to obtain b) a protected matrix stable to reactions occurring at one or more other reactive sites; and c) selectively removed in good yield by an agent that does not attack the regenerated deprotected functional group. As those skilled in the art will appreciate, in some cases, the agent does not attack other reactive groups in the compound. In other cases, the agent may also react with other reactive groups in the compound. Examples of protecting groups are detailed in Greene, T.W., and Wuts, P.G., *Protective Groups in Organic Synthesis*, 3rd edition, John Wiley & Sons, New York: 1999 (and other editions of the book), the entire contents of which are incorporated herein by reference. As used herein, the term "nitrogen protecting group" refers to an agent used to temporarily block one or more desired nitrogen-reactive sites in a polyfunctional compound. Preferred nitrogen protecting groups also possess the characteristics exemplified by the protecting groups described above, and certain exemplary nitrogen protecting groups are also detailed in Greene, T.W., and Wuts, P.G., *Protective Groups in Organic Synthesis*, 3rd edition, Chapter 7, John Wiley & Sons, New York: 1999, the entire contents of which are incorporated herein by reference.

As used herein, the terms “displaceable moiety” or “leaving group” refer to a group that is associated with an aliphatic or aromatic group as defined herein and undergoes displacement by nucleophilic attack by a nucleophile.

Unless otherwise specified, the structures described herein are also intended to encompass all isomers of the structures (e.g., enantiomers, diastereomers, cis-trans isomers, configurational isomers, and rotational isomers). For example, unless only one isomer is specifically depicted, the invention includes R and S configurations of each asymmetry center, (Z) and (E) double bond isomers, and (Z) and (E) configurational isomers.

Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, cis/trans isomers, configurational isomers, and rotational isomers, are within the scope of the present invention.

Unless otherwise specified, all tautomer forms of the compounds described herein are within the scope of this invention.

Furthermore, unless otherwise specified, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures of the invention, except for hydrogen replaced by deuterium or tritium or carbon replaced by carbon enriched with ^13C or ^14C , are within the scope of the invention. For example, compounds of formulas 1 to 13 having -D at the position corresponding to ^R2 are also within the scope of the invention. Such compounds are suitable as analytical tools or probes, for example, in bioanalysis. These compounds, especially deuterium analogs, may also have therapeutic applications.

The terms “one bond” and “non-existent” are used interchangeably to indicate that the group is not present.

The compounds described herein are defined by their chemical structure and/or chemical name. In cases where a compound is mentioned by its chemical structure and chemical name, and there is a conflict between the two, the chemical structure determines the compound's identity.

Pharmaceutically acceptable salts

The compounds described herein may exist in their free form or, where appropriate, as salts. Pharmaceutically acceptable salts are of particular interest because they are suitable for administering the compounds described below for medical purposes. Non-pharmaceutically acceptable salts are suitable for manufacturing processes to achieve separation and purification purposes, and in some cases, for separating stereoisomers of the compounds described herein or their intermediates.

As used herein, the term “pharmaceutically acceptable salt” refers to a salt of a compound that, within the scope of reasonable medical judgment, is suitable for contact with human and lower animal tissues without undue side effects (such as toxicity, irritation, allergic reactions, and the like) and is commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the field. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in the *Journal of Pharmaceutical Sciences*, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic acids and bases, as well as organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

In cases where the compounds described herein contain a base or a sufficiently basic bioelectron isotope, acid addition salts can be prepared by 1) reacting the purified compound, in its free base form, with a suitable organic or inorganic acid and 2) separating the resulting salt. In practice, acid addition salts may be used in a more suitable form, and the use of said salt is equivalent to the use of the free base form.

Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by amino groups with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by other methods used in the field (such as ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, hydrogen sulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, formates, transbutenedioic acid salts, glucohepanoates, glycerophosphates, glycolate salts, glucuronates, glycolate salts, hemisulfates, heptarates, hexanoates, hydrochlorides, hydrobromic acid salts, hydroiodates, and 2-hydroxyethyl salts. Ethylene sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methane sulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, p-valerate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluene sulfonate, undecanoate, valerate, etc.

In the case of compounds containing carboxyl groups or sufficiently acidic bioelectron isotopes, base addition salts can be prepared by 1) reacting a purified compound in its acidic form with a suitable organic or inorganic base and 2) separating the resulting salt. In practice, the use of base addition salts may be more suitable, and the use of the salt form itself is equivalent to the use of the free acid form. Salts derived from suitable bases include alkali metals (e.g., sodium, lithium, and potassium), alkaline earth metals (e.g., magnesium and calcium), ammonium, and N ⁺ ( _C1-4 alkyl) ₄ salts. This disclosure also contemplates the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Such quaternization yields water-soluble, oil-soluble, or dispersible products.

Base addition salts comprise pharmaceutically acceptable metal salts and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum. Sodium and potassium salts are generally preferred. Where appropriate, other pharmaceutically acceptable salts comprise non-toxic ammonium, quaternary ammonium, and amine cations formed using relative ions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, low-carbon alkyl sulfonate, and aryl sulfonate ions. Suitable inorganic base addition salts are prepared from metal bases, including sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, etc. Suitable amine base addition salts are prepared from amines commonly used in medicinal chemistry due to their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methylglucosamine, lysine, arginine, ornithine, choline, N,N'-diphenylmethylethylenediamine, chloroprocaine, diethanolamine, procaine, N-phenylmethylphenylethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, tetramethylammonium hydroxide, triethylamine, diphenylmethylamine, diphenylhydroxymethylamine, dehydrorosinamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, dicyclohexylamine, etc.

Other acids and bases, when not pharmaceutically acceptable on their own, can be used to prepare salts suitable as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

It should be understood that this disclosure includes mixtures/combinations of different pharmaceutically acceptable salts, and also includes mixtures/combinations of compounds in free form and pharmaceutically acceptable salts.

Pharmaceutical Composition

The compounds described herein can be formulated into pharmaceutical compositions further comprising pharmaceutically acceptable carriers, diluents, adjuvants, or mediators. In some embodiments, the present invention relates to a pharmaceutical composition comprising the compounds described herein and pharmaceutically acceptable carriers, diluents, adjuvants, or mediators. In some embodiments, this disclosure comprises a pharmaceutical composition comprising a safe and effective amount of the compounds described herein or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers, diluents, adjuvants, or mediators. Pharmaceutically acceptable carriers comprise, for example, pharmaceutical diluents, excipients, or carriers appropriately selected with respect to the intended form of administration and conforming to conventional pharmaceutical practice.

The term "effective dose" includes both "therapeutic effective dose" and "preventive effective dose." The term "therapeutic effective dose" refers to a dose that is effective in treating and/or improving influenza virus infection in patients. The term "preventive effective dose" refers to a dose that is effective in preventing and/or substantially reducing the chance or size of an influenza virus outbreak.

Pharmaceutically acceptable carriers may contain inert components that do not excessively inhibit the biological activity of the compound. Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or without other undesirable reactions or side effects when administered to an individual. Standard pharmaceutical compounding techniques may be used.

Pharmaceutically acceptable carriers, adjuvants, or mediators as used herein comprise any solvent, diluent or other liquid mediator, dispersant or suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant, etc., in a form suitable for the desired particular dosage form. Remington's Pharmaceutical Sciences, 16th edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for formulating pharmaceutically acceptable compositions and their known preparation techniques. Unless any conventional carrier medium is incompatible with the compounds described herein by producing any undesirable biological effect or otherwise interacting in a harmful manner with any other component of the pharmaceutically acceptable composition, its use is contemplated within the scope of this disclosure. As used herein, the phrase "side effect" encompasses an undesirable and adverse effect of a therapy (e.g., a prophylactic or therapeutic agent). Side effects are always undesirable, but undesirable actions are not necessarily undesirable. Adverse effects of therapies (such as prophylactic or therapeutic agents) can be harmful, uncomfortable, or risky. Side effects include (but are not limited to) fever, chills, drowsiness, gastrointestinal toxicity (including gastric and intestinal ulcers and erosions), nausea, vomiting, neurotoxicity, nephrotoxicity, kidney toxicity (including symptoms such as papillary necrosis and chronic interstitial nephritis), hepatotoxicity (including elevated serum liver enzyme levels), myelotoxicity (including leukopenia, bone marrow suppression, thrombocytopenia, and anemia), dry mouth, metallic taste, prolonged pregnancy, weakness, somnolence, pain (including muscle pain, bone pain, and headache), hair loss, fatigue, dizziness, extrapyramidal symptoms, akathisia, cardiovascular disturbances, and sexual dysfunction.

Some examples of substances that can serve as pharmaceutically acceptable carriers include (but are not limited to) ion exchangers, alumina, aluminum stearate; lecithin; serum proteins (such as human serum albumin); buffering substances (such as Twin 80, phosphates, glycine, sorbic acid, or potassium sorbate); mixtures of glycerides of saturated vegetable fatty acids; water, salts, or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts); colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene block polymers; methylcellulose; hydroxypropyl methylcellulose; lanolin; sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose. Ethyl cellulose and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic physiological saline; Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic and compatible lubricants such as sodium lauryl sulfate and magnesium stearate; as well as colorants, release agents, coating agents, sweeteners, flavoring agents and aromatizers, preservatives and antioxidants may also be present in the composition at the formulator's discretion.

formulations for transpulmonary delivery

In some embodiments, the pharmaceutical compositions described herein are suitable for direct administration via inhalation into the lower respiratory tract (e.g., the lungs). Compositions for inhalation administration may be in the form of an inhalable powder composition or a liquid or powder spray, and may be administered using a standard powder inhaler or aerosol dispensing device. Such devices are well known. For inhalation administration, the powder formulation typically comprises the active compound and an inert solid powdered diluent, such as lactose or starch. The inhalable dry powder composition may be present in capsules and cartridges of gelatin or similar substances, or in blister packs of laminated aluminum foil for inhalers or blow-offs. Each capsule or cartridge may typically contain, for example, from about 10 mg to about 100 g of the active compound. Alternatively, the compositions described herein may be presented without excipients.

Inhalable compositions can be packaged for single-dose or multi-dose delivery. For example, compositions can be packaged for multi-dose delivery in a manner similar to that described below: GB 2242134, U.S. Patent Nos. 6,632,666, 5,860,419, 5,873,360, and 5,590,645 (all describing “Diskus” devices); or GB 2178965, GB 2129691, GB 2169265, U.S. Patent Nos. 4,778,054, 4,811,731, and 5,035,237 (which describe “Diskhaler” devices); or EP 69715 (“Turbuhaler” device) or GB 2064336 and U.S. Patent No. 4,353,656 (“Rotahaler” device).

Spray compositions intended for delivery to the lungs via an inhalation surface can be formulated as aqueous solutions or suspensions or as aerosols delivered in self-pressurized packaging, such as in a controlled-dose inhaler (MDI), using suitable liquefied propellants comprising hydrofluoroalkanes such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and especially 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane, and mixtures thereof. Aerosol compositions suitable for inhalation can be presented in suspension or solution form.

Drugs administered via inhalation typically have a control particle size. The optimal particle size for inhalation into the bronchial system is generally from about 1 μm to about 10 μm, and in some embodiments, from about 2 μm to about 5 μm. Particles with a size greater than about 20 μm are generally too large to reach the smaller respiratory tracts during inhalation. To achieve these particle sizes, the particles of the active ingredient may undergo a particle size reduction process such as micronization. The desired particle size fraction can be separated by air separation or sieving. Preferably, the particles will be crystalline.

Nasal sprays can be formulated using aqueous or non-aqueous media with added agents such as thickeners, buffer salts or pH-adjusting acids or bases, isotropic regulators or antioxidants.

Solutions intended for inhalation via nebulization can be formulated using aqueous media by adding agents such as acids or alkalis, buffer salts, isotropic modifiers, or antimicrobial agents. They can be sterilized by filtration or heating in an autoclave, or presented as non-sterile products. The nebulizer supplies the aerosol as a mist generated from the self-contained aqueous solution.

In some embodiments, the pharmaceutical compositions described herein may be formulated together with supplemental active ingredients.

In some embodiments, the pharmaceutical compositions described herein are administered via a dry powder inhaler.

In other embodiments, the pharmaceutical composition described herein may be administered via an aerosol dispensing device optionally combined with an inhalation chamber, such as an inhalation chamber.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial activity in the compositions described herein can be achieved by adding antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. The absorption of the injectable composition can be prolonged by using an absorption-retarding agent (e.g., aluminum monostearate and gelatin) in the composition.

In some embodiments, the pharmaceutical compositions described herein may be contained within a matrix that controls the release of the composition. In some embodiments, the matrix may include: lipids, polyvinyl alcohol, polyvinyl acetate, polycaprolactone, poly(glycolic acid), poly(lactic acid), polycaprolactone, polylactic acid, polyanhydride, polylactide-co-glycolic acid, polyamino acids, polyethylene oxide, acrylic acid-terminated polyethylene oxide, polyamide, polyethylene, polyacrylonitrile, polyphosphazene, poly(orthoester), sucrose isobutyrate (SAIB), and combinations thereof, such as U.S. Patent Nos. 6,667,371 and 6,667,371. Other polymers disclosed in Nos. 13,355, 6,596,296, 6,413,536, 5,968,543, 4,079,038, 4,093,709, 4,131,648, 4,138,344, 4,180,646, 4,304,767, and 4,946,931, each of which is expressly incorporated herein by reference in its entirety. In these embodiments, the matrix sustains drug release.

Pharmaceutically acceptable carriers and/or diluents may also include any solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonics, and absorption delay agents. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

The pharmaceutical compositions described herein can be formulated for administration using conventional techniques. See, for example, Remington's *The Science and Practice of Pharmacy* (20th edition, 2000). For instance, the intranasal pharmaceutical compositions of this disclosure can be formulated as aerosols (this term includes both liquid and dry powder aerosols). As known to those skilled in the art, aerosols of liquid particles can be generated by any suitable means, such as using a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See, for example, U.S. Patent No. 4,501,729. Aerosols of solid particles (e.g., lyophilized, freeze-dried, etc.) can also be generated using any solid particulate pharmaceutical aerosol generator by techniques known in the pharmaceutical field. As another example, the pharmaceutical compositions of this disclosure can be formulated as an on-demand soluble form, providing a lyophilized portion of the pharmaceutical composition and a dissolved solution portion of the pharmaceutical composition.

In some embodiments of the invention, the pharmaceutical composition is in the form of an aqueous suspension, which may be prepared from a solution or suspension. For solutions or suspensions, the dosage form may comprise lipophilic substances, liposomes (phospholipid vesicles/membranes), and/or microcells of fatty acids (e.g., palmitic acid). In specific embodiments, the pharmaceutical composition is a solution or suspension capable of dissolving in fluids secreted by the epithelial mucosa of the tissue to which the pharmaceutical composition is administered, applied, and/or delivered, which preferably enhances absorption.

The pharmaceutical composition may be an aqueous solution, a non-aqueous solution, or a combination of aqueous and non-aqueous solutions.

Suitable aqueous solutions include (but are not limited to): hydrogels, aqueous suspensions, aqueous microsphere suspensions, aqueous microsphere dispersions, aqueous liposome dispersions, aqueous microcells of liposomes, aqueous microemulsions, and any combination thereof, or any other aqueous solution soluble in fluids secreted by the nasal mucosa. Exemplary non-aqueous solutions include (but are not limited to): non-hydrogels, non-aqueous suspensions, non-aqueous microsphere suspensions, non-aqueous microsphere dispersions, non-aqueous liposome dispersions, non-aqueous emulsions, non-aqueous microemulsions, and any combination thereof, or any other non-aqueous solution soluble in or mixable with fluids secreted by the mucosa.

Examples of powder formulations include (but are not limited to): pure powder mixtures, micronized powders, freeze-dried powders, lyophilized powders, powder microspheres, coated powder microspheres, liposome dispersions, and any combination thereof. Powder microspheres may be formed from various polysaccharides and celluloses, including (but not limited to) starch, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, polyvinyl alcohol alginate, gum arabic, polyglucosamine, and any combination thereof.

In certain embodiments, the composition is at least partially or even substantially (e.g., at least 80%, 90%, 95% or more) soluble in mucosal secretions to facilitate absorption. Alternatively or additionally, the composition may be formulated with a carrier and/or other substances that promote the dissolution of the agent in the secretions, comprising (but not limited to) fatty acids (e.g., palmitic acid), gangliosides (e.g., GM-1), phospholipids (e.g., phosphatidylserine), and emulsifiers (e.g., polysorbate 80).

Those skilled in the art will understand that, for intranasal administration or delivery, nasal secretions can alter the pH of the administered dose because the volume of the administered pharmaceutical composition is generally small, as the pH range in the nasal cavity can be as wide as 5 to 8. Such alterations can affect the concentration of the unliberated drug available for absorption. Therefore, in representative embodiments, the pharmaceutical composition further includes a buffer to maintain or adjust the pH in situ. Typical buffers include (but are not limited to) ascorbate, acetate, citrate, gluten, carbonate, and phosphate buffers.

In embodiments of the invention, the pH of the pharmaceutical composition is selected such that the internal environment of the mucosal tissue is acidic to neutral after administration, which (1) provides the active compound in an unfree form for absorption, (2) inhibits the growth of pathogenic bacteria that are more likely to occur in an alkaline environment, and (3) reduces the likelihood of mucosal irritation.

For liquid and powder sprays or aerosols, pharmaceutical compositions may be formulated to have any suitable and desired particle size or droplet size. In illustrative examples, the majority and/or average particle or droplet size ranges from about 1, 2.5, 5, 10, 15, or 20 micrometers and/or from about 25, 30, 40, 45, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 425 micrometers (inclusive of all combinations thereof). Representative examples of suitable ranges for most and/or average particle size or droplet size include (but are not limited to) about 5 to 100 micrometers, about 10 to 60 micrometers, about 175 to 325 micrometers, and about 220 to 300 micrometers, which facilitate the safe and effective deposition of active compounds, for example, in the nasal cavity (e.g., in the upper third of the nasal cavity, the superior nasal meatus, the olfactory region, and/or the sinus regions leading to the olfactory nerve pathway). Generally, particles or droplets smaller than about 5 micrometers will deposit in the trachea or even the lungs, while particles or droplets of about 50 micrometers or larger generally do not reach the nasal cavity and deposit in the anterior nasal region.

International Patent Publication WO 2005/023335 (Kurve Technology, Inc.) describes particles and droplets having diameter sizes suitable for practicing representative embodiments of this disclosure. In particular embodiments, the particles or droplets have an average diameter of about 5 to 30 micrometers, about 10 to 20 micrometers, about 10 to 17 micrometers, about 10 to 15 micrometers, about 12 to 17 micrometers, about 10 to 15 micrometers, or about 10 to 12 micrometers. The particles may “substantially” have an average diameter or size as described herein, i.e., at least about 50%, 60%, 70%, 80%, 90%, or 95% or more of the particles have the indicated diameter or size range.

The pharmaceutical compositions described herein can be delivered in a sprayed or atomized liquid form with droplet sizes as described above.

According to specific embodiments of this disclosure, including intranasal delivery methods, it may be necessary to prolong the residence time of the pharmaceutical composition in the nasal cavity (e.g., in the upper third of the nasal cavity, the superior nasal meatus, the olfactory region, and/or the sinus region), for example, to enhance absorption. Therefore, the pharmaceutical composition may optionally be formulated with: bioadhesive polymers, gums (e.g., xanthan gum), polyglucosamine (e.g., highly purified cationic polysaccharides), pectin (or any carbohydrate that thickens or emulsifies like a gel when applied to the nasal mucosa), microspheres (e.g., starch, albumin, polydextrose, cyclodextrin), gelatin, liposomes, carbomer, polyvinyl alcohol, alginate, gum arabic, polyglucosamine, and/or cellulose (e.g., methyl or propyl cellulose; hydroxy or carboxy cellulose; carboxymethyl or hydroxypropyl cellulose), which are agents that increase the residence time in the nasal cavity. As another method, increasing the viscosity of the formulation may also provide a means of prolonging the contact between the agent and the nasal epithelial cells. The pharmaceutical composition may be formulated as a nasal lotion, ointment, or gel, which offers the advantage of local application due to its viscosity.

A moist and highly vascularized membrane can facilitate rapid absorption; therefore, pharmaceutical compositions may optionally include humectants, especially in the case of gel-based compositions, to ensure adequate intranasal moisture content. Examples of suitable humectants include (but are not limited to) glycerin/glycerol, mineral oil, vegetable oil, membrane conditioners, soothing agents, and/or sugar alcohols (e.g., xylitol, sorbitol; and/or mannitol). The concentration of the humectant in the pharmaceutical composition will vary depending on the selected pharmaceutical agent and formulation.

The pharmaceutical composition may optionally include absorption enhancers, such as agents that inhibit enzyme activity, reduce the viscosity or elasticity of mucus, reduce the mucociliary clearance effect, open tightly bound and/or dissolve the active compound. Chemical enhancers are known in the art and include chelating agents (e.g., EDTA), fatty acids, bile salts, surfactants and/or preservatives. Permeation enhancers are particularly suitable when formulating compounds that exhibit poor membrane permeability, lack lipophilicity, and/or are degraded by aminopeptidase. The concentration of the absorption enhancer in the pharmaceutical composition will vary depending on the selected pharmaceutical agent and formulation.

To extend shelf life, preservatives may optionally be added to the pharmaceutical composition. Suitable preservatives include (but are not limited to) benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzylamino chloride, and combinations thereof. The concentration of the preservative will vary depending on the preservative used, the compound being formulated, the formulation, etc. In representative embodiments, the preservative is present in an amount of about 2% by weight or less.

The pharmaceutical compositions described herein may optionally contain an odorant, such as that described in EP 0 504 263B1, to provide a sensation of odor in order to facilitate inhalation of the composition, thereby promoting delivery to the olfactory region and/or triggering transmission via olfactory neurons.

As an alternative, the composition may include flavoring agents, for example, to enhance the flavor and/or individual acceptability of the composition.

Porous particles administered via the lungs

In some embodiments, the particles are porous to give them an appropriate density to avoid deposition in the back of the throat when administered via an inhaler. The combination of relatively large particle size and relatively low density avoids phagocytosis in the lungs, provides appropriately targeted delivery, avoids systemic delivery of the component, and provides a high concentration of the component within the lungs.

Representative methods for preparing such particles and for delivering such particles are described in, for example, U.S. Patent No. 7,384,649 entitled "Particulate compositions for pulmonary delivery"; U.S. Patent No. 7,182,961 entitled "Particulate compositions for pulmonary delivery"; U.S. Patent No. 7,146,978 entitled "Inhalation device and method"; and others entitled "Inhalation device and method". U.S. Patent No. 7,048,908, entitled "Particles for inhalation having sustained release properties"; U.S. Patent No. 6,956,021, entitled "Stable spray-dried protein formulations"; U.S. Patent No. 6,766,799, entitled "Inhalation device"; and U.S. Patent No. 6,732,732, entitled "Inhalation device and method".

Additional patents disclosing such particles include: U.S. Patent No. 7,279,182, entitled "Formulation for spray-drying large porous particles"; U.S. Patent No. 7,252,840, entitled "Use of simple amino acids to form porous particles"; U.S. Patent No. 7,032,593, entitled "Inhalation device and method"; and U.S. Patent No. 7,032,593, entitled "Formulation for producing dried particles". U.S. Patent No. 7,008,644, entitled "Method and apparatus for producing dry particles"; U.S. Patent No. 6,848,197, entitled "Control of process humidity to produce large, porous particles"; and U.S. Patent No. 6,749,835, entitled "Formulation for spray-drying large porous particles".

U.S. Patent No. 7,678,364, entitled "Particles for inhalation having sustained release properties," discloses a method for delivering particles to the pulmonary system, comprising: administering a safe and effective amount of dry powder to the airway of a patient requiring treatment, prevention, or diagnosis, said dry powder comprising: a) a multivalent metal cation compounded with a therapeutic, preventative, or diagnostic agent; b) a pharmaceutically acceptable carrier; and c) a component containing multivalent metal cations, wherein said dry powder is spray-dried and has a total amount of multivalent metal cations of about 10% w/w or more of the total weight of the pharmaceutical agent, a knock-tightness of about 0.4 g/ ^cm³ or less, a median geometric diameter of about 5 micrometers to about 30 micrometers, and an aerodynamic diameter of about 1 to about 5 micrometers.

The amount of the compound described herein or its salt present in the particles may range from about 0.1% by weight to about 95% by weight, although in some cases it may even be as high as 100%. For example, from about 1% to about 50%, such as from about 5% to about 30%. Particles in which the drug is distributed throughout the particle are preferred.

In some embodiments, the particles comprise surfactants other than the phospholipids described above. As used herein, the term "surfactant" refers to any agent preferably absorbed at the interface between two immiscible phases (such as the interface between water and an organic polymer solution, a water/air interface, or an organic solvent/air interface). Surfactants generally have both hydrophilic and lipophilic moieties, which facilitates the formation of an external environment that does not attract coated particles upon absorption, thus reducing particle aggregation. Surfactants can also promote the absorption of therapeutic or diagnostic agents and improve their bioavailability.

Suitable surfactants that can be used to manufacture the particles described herein include (but are not limited to): hexadecyl alcohol; fatty alcohols, such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surfactant fatty acids, such as palmitic acid or oleic acid; glycocholates; surfactant peptides; poloxamer; sorbitol fatty acid esters, such as sorbitol trioleate (Span 85); 80; and tyloxapol.

The surfactant may be present in the particles in an amount ranging from about 0 to about 5% by weight. Preferably, it may be present in the particles in an amount ranging from about 0.1 to about 1.0% by weight.

Particles with a knockdown density of less than about 0.4 g/ ^cm³ , a median diameter of at least about 5 μm, and an aerodynamic diameter of about 1 μm to about 5 μm or about 1 μm to about 3 μm are better able to avoid inertial and gravitational deposition in the oropharyngeal region and target the respiratory tract or deep lungs. The use of larger, more porous particles is advantageous because they can be atomized more effectively than smaller, denser aerosol particles (such as those currently used in inhalation therapy).

Liposome delivery

The compositions described herein are preferably delivered to the lungs to provide the compound at the site of actual or potential influenza infection. This can be achieved via transpulmonary delivery through a pre-dose inhaler or other transpulmonary delivery device, and also by allowing the particles to enter the microvascular bed surrounding the alveoli in the lungs.

Nanocarriers comprising smaller monolayer vesicles (such as liposomes) exhibit several advantages over other conventional methods for drug delivery to the lungs, including prolonged drug release and cell-specific targeted drug delivery. Nanoscale drug carriers can also facilitate the delivery of poorly water-soluble drugs, and some of the compounds described herein are poorly water-soluble. Additional advantages include their ability to provide controlled release, protection against metabolism and degradation, reduced drug toxicity, and improved targeting ability.

Liposomes (preferably monolayer vesicles) are characterized by a size of less than 200 nm as measured by dynamic light scattering, and are preferably characterized by comprising a chemically pure synthetic phospholipid composition, most preferably having aliphatic side chains of at least 16 carbons in length, and containing one or more of the compounds described herein, or a pharmaceutically acceptable salt sufficient to deliver (i.e., target) a certain amount of such compounds to the microvascular bed surrounding the alveoli. The vesicle diameter can be measured, for example, by dynamic light scattering using a helium-neon 100mW NEC gas laser and a Malvern K7027 correlator, ideally generating at least two or three measurements each time for size determination.

The term "chemically pure phospholipid" means a phospholipid that is substantially free of harmful purification components and impurities that cause polymerization of the smaller monolayer vesicles (SUVs) formed therefrom, and has a purity exceeding 97%. Preferably, the liposomes have a diameter primarily of about 50 to about 160 nm, are substantially neutral in charge, and incorporate phospholipids with side chains of 16 to 18 carbon atoms in length. More preferably, the liposomes are prepared from distearate phosphatidylcholine (DSPC) and contain cholesterol as a vesicle stabilizer (most preferably, in an amount of 10% to 50% of the total lipids).

It is also advantageous for liposomes to have a melting point above body temperature (i.e., above 37°C). Therefore, the use of pure phospholipids, preferably saturated phospholipids having a carbon chain length of at least 16 carbons, preferably between 16 and 18 carbons, is advantageous. Distearylphosphatidylcholine (DSPC) is a preferred phospholipid. Cholesterol helps stabilize the liposomes and is preferably added in an amount sufficient to provide liposome stability. Most preferably, the liposomes further comprise polyethylene glycol-modified phospholipids, such as DSPEPEG. The method involves introducing a quantity of liposomes into the bloodstream of a patient, the liposomes being less than 200 nm in size (preferably monolayer vesicles) and preferably characterized by comprising chemically pure synthetic phospholipids, most preferably having aliphatic side chains of at least 16 carbons in length, and containing the compound described herein or a pharmaceutically acceptable salt or prodrug sufficient to deliver (i.e., target) a quantity of the compound preferably to the microvascular bed surrounding the alveoli in the lungs.

The compounds described herein can be combined with other anti-influenza agents, also described herein. Such additional agents may also be present in the liposomes, in different liposomes, or administered via different routes.

The liposomes comprise one or more of the compounds described herein or pharmaceutically acceptable salts thereof, and may optionally comprise other anti-influenza agents. The liposomes are prepared by dissolving phospholipids and cholesterol in a suitable organic solvent such as chloroform and evaporating the solvent to form a lipid membrane. If an ion carrier is used to load the compounds described herein into the liposomes, the ion carrier may be added to the lipid solution prior to evaporation. The dried lipid membrane is then rehydrated in a suitable aqueous phase, such as phosphate-buffered saline or other physiologically suitable solutions. Water-soluble drugs or therapeutic agents may be contained in the hydrated solution, but if long-distance loading is required, a loading agent containing a chelating agent as described above may be added to the hydrated solution to encapsulate it within the internal water space of the liposome.

Upon addition of a hydration solution, liposomes of varying sizes spontaneously form and encapsulate a portion of the aqueous phase. Subsequently, the liposomes and the aqueous suspension are subjected to shear forces such as compression, sonication, or homogenization as described in U.S. Patent No. 4,753,788 to produce vesicles of specific sizes.

The liposomes can then be processed to remove unwanted compounds, such as unencapsulated drugs, from the self-suspended solution, which can be achieved via processes such as gel chromatography or ultrafiltration.

The use of liposomes in dry powder aerosols for targeted lung delivery is described, for example, in Willis et al., Lung, June 2012, 190(3):251-262. One advantage is that the phospholipids used to prepare the liposomes are similar to endogenous lung surfactants.

Application method

Depending on the severity of the infection being treated, the compounds and pharmaceutically acceptable compositions described above may be administered to humans and other animals via oral, rectal, non-intestinal, intracisional, vaginal, intraperitoneal, topical (as a powder, ointment, or drops), buccal, oral or nasal spray, to the pulmonary system (e.g., by using an inhaler, such as a dose-dependent inhaler (MDI)) or similar routes.

Liquid dosage forms for oral administration include (but are not limited to) pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms may contain: inert diluents commonly used in the field, such as water or other solvents; cosolvents and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, fatty acid esters of oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol; and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

For example, injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, non-enteric-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable mediators and solvents, water, Ringer's solution, U.S.P., and isotonic sodium chloride solutions can be used. Additionally, sterile non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild, non-volatile oil containing synthetic monoglycerides or diglycerides can be used. Furthermore, injectable formulations can be prepared using fatty acids (such as oleic acid).

Injectable formulations can be sterilized, for example, by filtration via a bacterial trapping filter or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media just before use.

To prolong the effects of the compounds described herein, it is generally desirable to slow the absorption of compounds administered subcutaneously or intramuscularly. This can be achieved by using liquid suspensions of crystalline or amorphous substances with poor water solubility. The absorption rate of the compound depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of compounds administered non-enterovenously can be achieved by dissolving or suspending the compound in an oil-based medium. Injectable accumulation formulations are prepared by forming microcapsule matrices of the compound in a biodegradable polymer, such as poly(lactide-polyglycolic acid). The release rate of the compound can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable accumulation formulations can also be prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are specifically suppositories, which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, or suppository waxes, which are solid at ambient temperature but liquid at body temperature and thus melt in the rectal or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with: at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate; and/or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerol; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blockers, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include buffers.

Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, which use excipients such as lactose/milk sugar and high molecular weight polyethylene glycol. Solid dosage forms of tablets, sugar-coated pills, capsules, pellets, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation field. They may optionally contain emulsifiers and may also have compositions that release or optionally delay the release of the active ingredient only or preferably in a portion of the intestine. Examples of usable encapsulation compositions include polymers and waxes. Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, which use excipients such as lactose/milk sugar and high molecular weight polyethylene glycol.

The active compound may also be in a microencapsulated form with one or more excipients as described above. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules may be prepared with coatings and shells, such as enteric coatings, release-controlled coatings, and other coatings well known in the pharmaceutical formulation field. In such solid dosage forms, the active compound may be mixed with at least one inert diluent (such as sucrose, lactose, or starch). As is common practice, such dosage forms may also include substances other than inert diluents, such as tablet-making lubricants and other tablet-making aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage form may also include a buffer. It may optionally contain an emulsifier and may also have a composition that releases or optionally releases the active ingredient only or preferably in a portion of the intestine. Examples of encapsulation compositions that can be used include polymers and waxes.

Dosage forms for the topical or transdermal application of the compounds described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, any desired preservatives or buffers. Ocular formulations, ear drops, and eye drops are also covered within the scope of this disclosure. Additionally, this disclosure covers the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispensing the compound in a suitable medium. Absorption enhancers may also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

The compositions described herein can be administered orally, non-enterically, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir. As used herein, the term "non-enterically" includes (but is not limited to) subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheath, intrahepatic, intralesional, and intracranial injection or infusion techniques. More precisely, the compositions are administered orally, intraperitoneally, or intravenously.

The sterile injectable form of the compositions described herein may be an aqueous or oily suspension. These suspensions may be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations may also be sterile injectable solutions or suspensions in non-toxic, non-enteric-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable mediators and solvents, water, Ringer's solution, and isotonic sodium chloride solution may be used. Additionally, sterile non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild non-volatile oil containing synthetic monoglycerides or diglycerides may be used. Fatty acids (such as oleic acid and its glycerol derivatives) are suitable for the preparation of injectable formulations, as are pharmaceutically acceptable natural oils (such as olive oil or castor oil, especially their polyoxyethylene forms). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used to formulate pharmaceutically acceptable dosage forms (including emulsions and suspensions). Other commonly used surfactants (such as Tween, Span, and other emulsifiers) or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

The pharmaceutical compositions described herein can be administered orally in any orally acceptable dosage form, including (but not limited to) capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, common carriers include (but are not limited to) lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When an aqueous solution is required for oral use, the active ingredient is combined with an emulsifier and a suspending agent. Certain sweeteners, flavoring agents, or coloring agents may also be added if necessary.

Alternatively, the pharmaceutical compositions described herein can be used for administration in the form of suppositories for rectal administration. These suppositories can be prepared by mixing the pharmaceutical preparation with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such substances include (but are not limited to) cocoa butter, beeswax, and polyethylene glycol.

The pharmaceutical compositions described herein can also be applied topically, especially when the therapeutic target comprises an area or organ that is easily accessible by surface application, such as diseases of the eye, skin, or lower intestine. Suitable surface formulations for each of these areas or organs are readily prepared.

For surface application to the lower intestine, it can be achieved in the form of rectal suppositories (see above) or in the form of suitable enema formulations. Topical percutaneous patches may also be used.

For surface application, the pharmaceutical composition may be formulated in a suitable ointment form containing an active ingredient suspended or dissolved in one or more carriers. Carriers used for surface application of the compounds described herein include (but are not limited to) mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the pharmaceutical composition may be formulated in a suitable lotion or cream form containing an active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include (but are not limited to) mineral oil, sorbitan monostearate, polysorbate 60, cetyl wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water.

For ophthalmic use, the pharmaceutical composition may be formulated as a micronized suspension in isotonic pH-adjusted sterile physiological saline, or more precisely, as a solution in isotonic pH-adjusted sterile physiological saline, with or without a preservative (such as benzalkonium chloride). Alternatively, for ophthalmic application, the pharmaceutical composition may be formulated in an ointment such as paraffin oil.

The compounds used in the methods described herein can be formulated in unit dosage forms. The term "unit dosage form" refers to a physically discrete unit suitable for administration to a treated individual in unit dose form, wherein each unit contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, optionally combined with a suitable pharmaceutical carrier. Unit dosage forms can be used for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage forms used for each dose may be the same or different.

This disclosure is intended to provide a more complete understanding of the exemplary embodiments described herein. However, these examples should not be construed as limiting the scope of this disclosure. All references throughout this disclosure are expressly incorporated herein by reference.

Detailed Implementation

Example

Example 1: Side chain preparation

Side chain-1

2-(2-(2-ethoxyethoxy)ethoxy)acetic acid (side chain -1)

NaOH (1.2 g, 30 mmol) was added to a stirred solution of 2-(2-ethoxyethoxyethyl-1-ol 1 (2.68 g, 20 mmol) in 1,4-dioxane (60 mL) at 0 °C. The mixture was stirred at room temperature for 15 minutes. Then, the mixture was cooled to 0 °C, and 2-bromoacetic acid tert-butyl ester 2 (7.8 g, 40 mmol) and 18-crown ether (250 mg) were added to the reaction mixture and stirred at room temperature for 16 hours. After the starting material was exhausted, the mixture was rinsed with ice water. The mixture was diluted (200 mL) and extracted with diethyl ether (2 × 100 mL). The separated aqueous layer was acidified with concentrated HCl (30 mL) (pH approximately 2) and extracted with a dichloromethane (2 × 250 mL) brine solution (50 mL). After drying with sodium sulfate and concentration under reduced pressure, a pure 2-(2-(2-ethoxyethoxy)ethoxy)acetic acid side chain-1 (1.5 g, 7.8 mmol, yield 39%) was obtained as a brown liquid. TLC system: 10% methanol in dichloromethane: _Rf : 0.10

Side chain-2

2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (2)

A solution of NaOH (2.28 g, 57.22 mmol) in water (10 mL) was added to a stirred solution of 2-(2-(2-ethoxyethoxy)ethoxy)ethyl-1-ol 1 (5.0 g, 28.05 mmol) in THF (20 mL) at 0 °C, followed by the addition of p-toluenesulfonyl chloride (6.84 g, 35.90 mmol) in THF (10 mL) at 0 °C. The mixture was then allowed to reach room temperature and stirred for 4 hours. After the starting material was exhausted, the mixture was quenched with water (100 mL) and extracted with diethyl ether (2 × 100 mL), washed with ice-cold water (2 × 25 mL) and brine solution (25 mL), dried over sodium sulfate, and concentrated to give 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid 2 (9.02 g, 27.16 mmol, yield 96%) as a colorless oil. TLC system: 40% ethyl acetate in hexane, _Rf : 0.3; LCMS: m/z = 332.83 (M+H) ⁺

2-(2-(2-(2-ethoxyethoxy)ethoxy)ethyl)isoindoline-1,3-dione (3)

Potassium phthalimide (4.418 g, 23.85 mmol) was added to a stirred solution of 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid 2 (6 g, 18.07 mmol) in DMF (15 mL) at room temperature, followed by stirring at 110 °C for 16 hours. Once the reaction was complete as indicated by TLC, the reaction mixture was cooled to room temperature, ether (50 mL) was added, and the mixture was stirred at room temperature for 15 minutes. The mixture was filtered and washed with ether. The filtrate was washed with 1 M NaOH solution (50 mL), water (50 mL), and brine solution (50 mL ₎ , dried over _Na₂SO₄ , and concentrated under reduced pressure to obtain a yellow, oily liquid, 2-(2-(2-(2-ethoxyethoxy)ethoxy)ethyl)isoindoline-1,3-dione 3 (3.5 g, 11.40 mmol, 63%). TLC system: 40% ethyl acetate in hexane, _Rf : 0.50; LCMS: m/z = 308.11 (M+H) ⁺

2-(2-(2-ethoxyethoxy)ethoxy)ethyl-1-amine (side chain -2)

Hydrazine hydrate (5 mL) was added to a stirred solution of 2-(2-(2-(2-ethoxyethoxy)ethoxy)ethyl)isoindoline-1,3-dione 3 (500 mg, 1.62 mmol) in ethanol (5 mL), and the mixture was stirred at 110 °C for 16 hours. Once the reaction was complete as indicated by TLC, the reaction mixture was cooled to room temperature, extracted with toluene (2 × 50 mL), washed with a brine solution ( ₂₅ mL), dried over _Na₂SO₄ , and concentrated under reduced pressure to obtain 2-(2-(2-ethoxyethoxy)ethoxy)ethyl-1-amine 4 (150 mg, 0.84 mmol, 52%) as a yellow, oily liquid. TLC system: 10% methanol in dichloromethane, _Rf : 0.10

Side chain-3

2-(2-(2-ethoxyethoxy)ethoxy)-N-methylethyl-1-amine (side chain -3)

A stirred solution of 2-(2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (500 mg, 1.50 mmol) and 33% methylamine in ethanol (3 mL) was heated in a sealed tube at 50 °C for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, the crude material was dissolved in water (25 mL), and extracted with dichloromethane (2 × 50 mL). The combined organic layers were washed with a brine solution (25 mL), dried over _{Na₂SO₄ ,} and concentrated to give a yellow, oily liquid, 2-(2-(2-ethoxyethoxy)ethoxy)-N-methylethyl-1-amine side chain-3 (210 mg, 1.09 mmol, 73%). TLC system: 10% methanol in dichloromethane, _Rf : 0.20.

Side chain-4

2-(2-(2-ethoxyethoxy)ethoxy)ethane-1-thiol (2)

Sodium hydrogen sulfide hydrate (3.7 g, 66.26 mmol) was added to a stirred solution of 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (2.2 g, 6.626 mmol) in ethanol (10 mL) and THF (1 mL) and stirred at room temperature for 16 hours. When the reaction was complete as indicated by TLC, the organic solvent was evaporated, the crude material was diluted with water (100 mL), and extracted into ethyl acetate (2 × 100 mL). The organic layer was washed with a saline solution (100 _mL ), dried over _Na₂SO₄ , and concentrated to obtain 2-(2-(2-ethoxyethoxy)ethoxy)ethane-1-thiol 2 (1.1 g, 5.67 mmol, 85%) as a pale brown liquid. TLC system: ethyl acetate 50% in hexane; _Rf : 0.20

2-(2-(2-ethoxyethoxy)ethoxy)ethane-1-sulfonyl chloride (side chain -4)

Thionyl chloride (1.5 mL) was added to a stirred solution of 2-(2-(2-ethoxyethoxy)ethoxy)ethane-1-thiol 2 (1.4 g, 7.21 mmol) and potassium nitrate (2.41 g, 18.04 mmol) at 0 °C and stirred at room temperature for 16 hours. After the reaction was complete as indicated by TLC, water (200 mL) was added and the mixture was extracted with DCM (2 × 200 mL). The organic layer was washed with a brine solution ( ₁₀₀ mL), dried over _Na₂SO₄ , and concentrated to give a brown liquid 2-(2-(2-ethoxyethoxy)ethoxy)ethane-1-sulfonyl chloride side chain-4 (1.4 g, 5.38 mmol, 74%). TLC system: ethyl acetate 50% in hexane; _Rf : 0.40

Side chain - 5

2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethanol(2)

Dihydropyran (20 g, 233.3 mmol) and p-toluenesulfonic acid (6.3 g, 33.33 mmol) were added to a stirred solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol 1 (50 g, 333.3 mmol) in dichloromethane (1.5 L) at 0 °C, and the mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with water (1000 mL) and extracted with dichloromethane (3 × 800 mL). The combined organic layers were washed with brine (2 × 200 mL) and dried over sodium sulfate and concentrated. The residue was purified by rapid column chromatography (100-200 silica) using 3% methanol in dichloromethane to give 2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethanol 2 (3 g, 12.82 mmol, 10% yield) as a colorless, oily liquid. TLC system: 15% acetone in dichloromethane, _Rf : 0.35

2-(2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethoxy)ethyl acetate (4)

NaH (554 mg, 23.08 mmol) was added to a stirred solution of 2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethanol 2 (2.7 g, 11.54 mmol) in N,N-dimethylformamide (5 mL) at 0 °C, and the mixture was stirred for 30 min at room temperature. Then, a solution of 2-bromoethyl acetate 3 (2.3 g, 13.84 mmol) in N,N-dimethylformamide (5 mL) was added to the above reaction mixture at 0 °C, and the mixture was stirred for 16 h at room temperature under a nitrogen atmosphere. The reaction mixture was quenched with ice-cold water and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (2 × 100 _mL ), dried over _Na₂SO₄ , and evaporated under reduced pressure. The crude residue was purified by combined rapid chromatography with ethyl acetate in 40% hexane to give ethyl acetate 2-(2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethoxy)ethyl acetate 4 (550 mg, 1.7187 mmol, 15% yield), a pale yellow, oily liquid. TLC system: ethyl acetate in 70% hexane; _Rf : 0.50

2-(2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethoxy)acetic acid (side chain-5)

Lithium hydroxide monohydrate (288 mg, 6.875 mmol) was added to a stirred solution of ethyl acetate 3 (550 mg, 1.7187 mmol) in a mixture of THF: _H₂O (3:1) (12 mL) at 0 °C and stirred at room temperature for 16 h. The reaction mixture was concentrated to remove volatile organic compounds, and water (50 mL) was added to the crude material and extracted with ethyl acetate (2 × 20 mL). The aqueous layer was acidified with saturated citric acid solution and extracted with 10% methanol in dichloromethane (2 × 50 mL). The combined organic layers were washed with 15 mL of saline solution, dried over sodium sulfate, and concentrated to give 2-(2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)acetic acid (340 mg, 1.1643 mmol, 68%), a pale yellow, colloidal liquid. TLC system: 10% methanol in dichloromethane; _Rf : 0.10

Side chain - 6

4-Methylbenzenesulfonic acid 2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl ester (2)

NaOH (3.25 g, 81.196 mmol) and p-toluenesulfonyl chloride (9.3 g, 48.718 mmol) were added to a stirred solution of 2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl-1-ol 1 (9.5 g, 40.598 mmol, the synthesis of compound 1 is reported in side chain-5)) in THF (100 mL) at 0 °C, followed by stirring at room temperature for 16 hours. After the starting material was exhausted, the mixture was diluted with ethyl acetate (50 mL) and washed with ice-cold water (2 × 20 mL) and brine (20 mL), dried over sodium sulfate and concentrated under reduced pressure to give 2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl ester 2 (14 g, 36.08 mmol, crude material) as a colorless, oily liquid. TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.50; LCMS: m/z = 305.04 (M-THP) ⁺

2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)isoindoline-1,3-dione

Potassium phthalimide (8.8 g, 47.62 mmol) was added to a stirred solution of 2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl ester 2 (14 g, 36.08 mmol) in DMF (35 mL) at room temperature, followed by stirring at 110 °C for 16 hours. Once the TLC indicated the reaction was complete, the reaction mixture was concentrated, the residue was suspended in diethyl ether and stirred for 15 minutes, and then filtered. The filtrate was washed with 1M NaOH solution (2 × 100 mL), water (100 mL), and brine solution (100 mL), dried over sodium sulfate, and concentrated under reduced pressure to give a yellow, oily liquid, 2-(2-(2-(((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)isoindoline-1,3-dione 3 (9 g, 24.79 mmol, yield 69%). TLC system: 40% ethyl acetate in petroleum ether, _Rf : 0.50; LCMS: m/z = 364 (M-THP) ⁺

2-(2-(2-((tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethyl-1-amine(4)

Hydrazine hydrate (18 mL) was added to a stirred solution of 2-(2-(2-(((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)isoindoline-1,3-dione 3 (9 g, 24.79 mmol) in ethanol, and the mixture was stirred at 110 °C for 16 h. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, water (100 mL) was added to the crude material, and the mixture was extracted with toluene (3 × 200 mL). The organic layer was washed with a brine solution (100 _mL ), dried over _Na₂SO₄ , and concentrated to give 2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl-1-amine 4 (3.4 g, 14.59 mmol, 59%) as a colorless liquid. TLC system: 15% acetone in dichloromethane, _Rf : 0.10; LCMS: m/z = 234 (M+H) ⁺

(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)tert-butyl carbamate (5)

Add (Boc)₂O (3.15 mL, 13.73 mmol) and 0.1 equivalents of DMAP (167 mg, 1.37 mmol) to a stirred solution of 2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl-1-amine ( ₄ ) (3.2 g, 13.7 mmol) in THF (30 mL), and then stir at room temperature for 2 hours. If the reaction is complete as indicated by TLC, add water (100 mL) to the reaction mixture and extract with ethyl acetate (2 × 100 mL). Wash the combined organic layers with a saline solution (100 mL), dry over _{Na₂SO₄ ,} and concentrate. The crude compound was purified by 100-200 silica gel column chromatography with elution of 25% ethyl acetate in petroleum ether to give tert-butyl (2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate 5 (2.8 g, 8.408 mmol, 61%) as a colorless liquid. TLC system: 40% ethyl acetate in petroleum ether, _Rf : 0.50

(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)tert-butyl carbamate (6)

Pyridium p-toluenesulfonic acid (1.4 g, 5.885 mmol) was added to a stirred solution of (2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)carbamate 5 (2.8 g, 8.408 mmol) in methanol (30 mL) at 0 °C and stirred at room temperature for 16 h. The organic solvent was distilled off, water (100 mL) was added to the crude compound, and it was extracted with ethyl acetate (3 × 150 mL). The combined organic layers were washed with a brine solution (100 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by rapid column chromatography (100-200 silica) using 50% ethyl acetate in petroleum ether to give (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate 6 (1.8 g, 7.229 mmol, 86% yield) as a colorless, colloidal liquid. TLC system: 40% ethyl acetate in petroleum ether, _Rf : 0.10

2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-ethyl ester (8)

A stirred solution of tert-butyl 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate 6 (125 mg, 0.502 mmol) in N,N-dimethylformamide (2 mL) was treated with NaH (30 mg, 1.255 mmol) at 0 °C and stirred for 30 min at room temperature. Ethyl 2-bromoacetate 7 (125 mg, 0.7530 mmol) in N,N-dimethylformamide (1 mL) was added to the above reaction mixture at 0 °C, and the mixture was stirred for 16 h at room temperature under an argon atmosphere. The reaction mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with a brine solution (20 mL), dried over _{Na₂SO₄ ,} and evaporated under reduced pressure. The crude residue was purified by combined rapid chromatography with 70% ethyl acetate in petroleum ether to give ethyl 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid 8 (35 mg, 0.144 mmol, 20% yield) as a colorless liquid. TLC system: ethyl acetate 70% in petroleum ether, _Rf : 0.20; direct mass: m/z = 236.1 (M-Boc) ⁺

2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid (side chain -6)

Lithium hydroxide (112 mg, 2.685 mmol) was added to a stirred solution of ethyl 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid 8 (300 mg, 0.895 mmol) in a mixture of THF: _H₂O (4:1) (20 mL) for 16 hours. The reaction mixture was concentrated to remove volatile organic compounds, and the residue was added to water (50 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with a brine solution (20 mL), dried over sodium sulfate, and concentrated under reduced pressure to give a crude compound, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid side chain-6 (280 mg, 0.9120 mmol, crude product), as a colorless liquid. TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.10; direct mass: m/z = 208.07 (M-Boc) ⁺

Side chain-7

2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethanol(2)

Dihydropyran (13.8 g, 164.85 mmol) and p-toluenesulfonic acid pyridinium (4.48 g, 23.55 mmol) were added to a stirred solution of 2,2'-oxodiethanol 1 (25 g, 235.58 mmol) in dichloromethane (750 mL) at 0 °C, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with water (600 mL), extracted into dichloromethane (3 × 250 mL), and the combined organic layers were washed with brine (2 × 100 mL), dried over sodium sulfate, and concentrated. The residue was purified by column chromatography (100-200 silica) using 2% methanol/dichloromethane to give 2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethanol 2 (7.8 g, 41.0 mmol, yield 17%) as a pale yellow, oily liquid. TLC system: 10% methanol in dichloromethane, _Rf : 0.3

Ethyl 3-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)propionate (4)

A solution of 2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethanol 2 (5.5 g, 28.947 mmol) in THF 20 mL was added to a stirred suspension of NaH (1.74 g, 43.42 mmol) in THF (70 mL) at 0 °C, and the mixture was stirred at room temperature for 30 min. Ethyl 3-bromopropionate 3 (7.85 g, 43.42 mmol) in THF (10 mL) was added to the above reaction mixture at 0 °C, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 h. The reaction mixture was quenched with ice water (200 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (2 × 100 mL), dried over _Na₂SO₄ , _and evaporated under reduced pressure. The crude residue was purified by column chromatography (100-200 silica) using 20% ethyl acetate/petroleum ether to give ethyl 3-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)propionate 4 (3.4 g, 11.72 mmol, yield 40%) as a pale yellow, oily liquid. TLC system: ethyl acetate 40% in petroleum ether, _Rf : 0.50

Ethyl 3-(2-(2-hydroxyethoxy)ethoxy)propionate (5)

Ethyl 3-(2-(2-hydroxyethoxy)ethoxy)propionate 4 (4.3 g, 14.827 mmol) was added to a stirred solution of 3-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)propionate 4 (4.3 g, 14.827 mmol) in methanol (40 mL) at 0 °C, and stirred for 16 hours at room temperature. The solvent was removed by vacuum distillation, and the residue was diluted with water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic matter was washed with brine (50 mL), dried over sodium sulfate, and concentrated. The crude residue was purified by column chromatography (100-200 silica) using 70% ethyl acetate in petroleum ether to give ethyl 3-(2-(2-hydroxyethoxy)ethoxy)propionate 5 (2.6 g, 12.62 mmol, 86% yield) as a pale yellow colloidal liquid. TLC system: ethyl acetate 70% in petroleum ether, _Rf : 0.20

3-(2-(2-(2-tert-butoxy-2-oxoethoxy)ethoxy)ethoxy)ethyl propionate

A stirred solution of ethyl 3-(2-(2-hydroxyethoxy)ethoxy)propionate 5 (2.0 g, 9.708 mmol) in N,N-dimethylformamide (20 mL) was treated with NaH (0.35 g, 14.65 mmol) at 0 °C and stirred for 30 min at room temperature. Tert-butyl 2-bromoacetate (2.27 g, 11.65 mmol) in N,N-dimethylformamide (10 mL) was added to the above reaction mixture at 0 °C and stirred for 3 h at room temperature under a nitrogen atmosphere. The reaction mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine (2 × 50 mL) and dried over _{Na₂SO₄ and} evaporated under reduced pressure. The crude residue was purified by column chromatography (100-200 silica) using 20% ethyl acetate in petroleum ether to give ethyl 3-(2-(2-(2-tert-butoxy-2-oxoethoxy)ethoxy)propionate 6 (600 mg, 1.875 mmol, yield 19%) as a pale yellow, oily liquid. TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.60

12-Oxano-3,6,9,13-tetraoxapentadecan-1-acid (side chain -7)

Ethyl 3-(2-(2-(2-tert-butoxy-2-oxoethoxy)ethoxy)propionate 6 (600 mg, 1.875 mmol) was added to a stirred solution of 4N HCl in dioxane (5 mL), followed by stirring at room temperature for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, the crude compound was diluted with water (50 mL), and extracted into ethyl acetate (2 × 30 mL). The organic layer was washed with a brine solution (30 _mL ), dried over _Na₂SO₄ , and concentrated. The crude compound was purified by washing with diethyl ether (30 mL) to obtain a pale yellow, oily liquid, 12-oxo-3,6,9,13-tetraoxapentadecano-1-acid (side chain-7) (4.0 g, 10.69 mmol, 85%). TLC system: 10% methanol in dichloromethane, _Rf : 0.20

2,2-Dimethyl-4-oxo-3,6,9,12-tetraoxapentadecan-15-acid (side chain -8)

Lithium hydroxide (26 mg, 0.625 mmol) was added to a stirred solution of ethyl 3-(2-(2-(tert-butoxy)-2-oxoethoxy)ethoxy)propionate (200 mg, 0.625 mmol, the synthesis of compound 1 is reported in side chain-7) in a mixture of THF: _H₂O (4:1) (10 mL) at 0 °C, followed by stirring at room temperature for 4 h. The reaction mixture was concentrated to remove volatile organic compounds, water (50 mL) was added, and the mixture was extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with a brine solution (20 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by column chromatography by elution with 20% ethyl acetate in petroleum ether to give 2,2-dimethyl-4-oxo-3,6,9,12-tetraoxapentadecan-15-acid side chain-8 (70 mg, 0.239 mmol, 38%) as a colorless, oily liquid. TLC system: 40% ethyl acetate in petroleum ether, _Rf : 0.40

Side chain-9

2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethanol-1-ol(2)

Dihydropyran (2.8 mL, 30.6 mmol) and p-toluenesulfonic acid (979 mg, 5.154 mmol) were added to a stirred solution of 1,2'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethanol-1-ol)1 (10 g, 51.546 mmol) in 100 mL of dichloromethane at 0 °C, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with 100 mL of water, extracted with 3 × 200 mL of dichloromethane, washed with 2 × 75 mL of brine, dried over sodium sulfate, and concentrated. The residue was purified by rapid column chromatography (100-200 silica) using 2% methanol/dichloromethane to give 2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethanol-1-ol 2 (2.2 g, 7.913 mmol, yield 15.4%) as a yellow, thick liquid. TLC system: 10% methanol in dichloromethane, _Rf : 0.20

4-Methylbenzenesulfonic acid 2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl ester (3)

Et 3 N (0.8 mL, 5.55 mmol) and p-toluenesulfonyl chloride (1.05 g, 5.55 mmol) were added to a stirred solution of _2- (2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethanol-1-ol 2 (1 g, 4.27 mmol) in DCM (20 mL) at 0 °C, and the reaction mixture was stirred at room temperature for 3 hours. After the starting material was exhausted, the mixture was diluted with ethyl acetate (50 mL), washed with ice-cold water (2 × 20 mL), _NaHCO3 solution (2 × 20 mL), and brine solution (20 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by combinatorial rapid chromatography and eluted with ethyl acetate in 50% hexane to give 2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl ester 3 (1 g, 2.31 mmol, yield 54%), a light brown liquid. TLC system: 100% ethyl acetate, _Rf : 0.50; LCMS: m/z = 455.39 (M+Na) ⁺

2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)isoindoline-1,3-dione(4)

Potassium phthalimide (282 mg, 1.52 mmol) was added to a stirred solution of 2-(2-(2-(((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl ester 3 (500 mg, 1.15 mmol) in DMF (10 mL) at room temperature, followed by stirring at 110 °C for 16 h. Once the reaction was complete as indicated by TLC, ice water (2 × 200 mL) was added to the reaction mixture and the mixture was extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with a brine solution ( ₁₀₀ mL), dried over _Na₂SO₄ , and concentrated to obtain a light brown liquid, 2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)isoindoline-1,3-dione 4 (400 mg, 9.828 mmol, 84%). TLC system: 50% ethyl acetate in hexane, _Rf : 0.20; LCMS: m/z = 429.97 (M + Na) ⁺

2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl-1-amine (side chain -9)

Hydrazine hydrate (3 mL) was added to a stirred solution of 2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)isoindoline-1,3-dione 4 (400 g, 0.98 mmol) in ethanol (3 mL) and stirred at 110 °C for 12 h. If the reaction was complete as indicated by TLC, toluene (10 mL) was added to the reaction mixture, resulting in two layers, which were then separated. The toluene layer was washed with a saline solution (10 mL), dried over _{Na₂SO₄ ,} and concentrated to obtain a brown liquid 2-(2-(2-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl-1-amine side chain-9 (150 mg, 0.541 mmol, 55%). TLC system: 10% methanol in dichloromethane, _Rf : 0.20

Side chain -10

N-Methyl-2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl-1-amine (side chain -10)

MeNH₂ in ethanol was added to a stirred solution of 2-(2-(2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl ester ₁ (500 mg, 1.15 mmol, synthesis of compound-1 is reported in side chain-9) in a sealed tube at room temperature, followed by stirring at 60 °C for 12 h. If the reaction was complete as indicated by TLC, the organic solvent was evaporated, a sodium bicarbonate solution (20 mL) was added to the residue, and the residue was extracted with 10% methanol in dichloromethane (3 × 20 mL). The combined organic layers were washed with 100 mL of saline solution, dried over anhydrous _Na₂SO₄ , and concentrated to give a brown liquid N-methyl-2-(2-(2-(2-((tetrahydro _- 2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl-1-amine side chain-10 (150 mg, 0.515 mmol, 44%). TLC system: 10% methanol in dichloromethane, _Rf : 0.20

Side chain-11

2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecane-17-ethyl ester (3)

The stirred solution of (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate tert-butyl ester 1 (1 g, 4.01 mmol) in N,N-dimethylformamide (15 mL) was treated with NaH (240 mg, 10.04 mmol) at 0 °C until room temperature was reached and maintained for 30 minutes. Ethyl 3-bromopropionate 2 (1.1 g, 6.02 mmol) in N,N-dimethylformamide was added to the above reaction mixture at 0 °C, and the mixture was stirred at room temperature under an argon atmosphere for 16 hours. The reaction mixture was quenched with ice water and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over _Na₂SO₄ , _and evaporated under reduced pressure. The crude residue was purified by column chromatography by elution with 70% ethyl acetate in petroleum ether to give ethyl 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecane-17-olate 3 (310 mg, 0.925 mmol, yield 24%) as a colorless liquid. TLC system: ethyl acetate 70% in petroleum ether, _Rf : 0.50; direct mass: m/z = 372.15(M+Na) ⁺

Ethyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionate (side chain -11)

Ethyl 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecane-17-oate 3 (310 mg, 0.888 mmol) in dioxane (1 mL) was added to a stirred solution in dioxane and stirred for 16 hours under an argon atmosphere. The organic solvent was distilled off and co-distilled with dichloromethane to give ethyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionate side chain 11 (260 mg, 1.044 mmol, crude product) as a colorless liquid. TLC system: 20% methanol in dichloromethane, _Rf : 0.10; direct mass: m/z = 250.12(M+H) ⁺

Side chain-12

3-(2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethoxy)ethyl propionate (3)

A stirred solution of 2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethanol 1 (synthesis of compound 1 reported in side chain-5) (5 g, 21.367 mmol) in N,N-dimethylformamide (30 mL) was treated with NaH (1.3 g, 53.417 mmol) at 0 °C and stirred for 30 min at room temperature. Ethyl 3-bromopropionate 2 (5.8 g, 32.05 mmol) in N,N-dimethylformamide (20 mL) was added to the above reaction mixture at 0 °C and stirred for 16 h at room temperature under a nitrogen atmosphere. The reaction mixture was quenched with ice water and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (2 × 100 mL), dried over _Na₂SO₄ , _and evaporated under reduced pressure. The crude residue was purified by combined rapid chromatography with ethyl acetate in 30% hexane to give ethyl 3-(2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethoxy)propionate 3 (1 g, 2.994 mmol, yield 14%) as a pale yellow, oily liquid. TLC system: ethyl acetate in 70% hexane; _Rf : 0.60

Ethyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propionate (4)

Ethyl 3-(2-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)propionate 3 (1 g, 2.994 mmol) was added to a stirred solution of 3-(2-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)propionate 3 (1 g, 2.994 mmol) in methanol (10 mL) at 0 °C, and the mixture was stirred at room temperature for 16 h. Volatile matter was removed under reduced pressure, water (50 mL) was added, and the mixture was extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with a brine solution (30 mL), dried over sodium sulfate, and concentrated to give ethyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)propionate 4 (580 mg, 2.32 mmol, 77% yield) as a pale yellow, oily liquid. TLC system: 5% methanol in dichloromethane, _Rf : 0.20

3-(2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)ethoxy)ethyl propionate (5)

NaOH (186 mg, 4.64 mmol) was added to a stirred solution of ethyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propionate 4 (580 mg, 2.32 mmol) in THF (5 mL) at 0 °C, and the mixture was stirred at room temperature for 15 min. The mixture was then cooled to 0 °C, and p-toluenesulfonyl chloride (530 mg, 2.784 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 16 h. After depletion of the starting material according to TLC, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with ice-cold water (2 × 40 mL) and a brine solution (20 mL). The mixture was dried over sodium sulfate and concentrated to give ethyl 3-(2-(2-(2-(toluenesulfonyloxy)ethyl)ethyl)ethoxy)propionate 5 (750 mg, 1.856 mmol, yield 17%) as a pale yellow, oily liquid. TLC system: ethyl acetate 70% in hexane; _Rf : 0.70

5,8,11-Trioxa-2-azatetradecane-14-ethyl ester (side chain -12)

Ethyl 3-(2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)propionate 5 (750 mg, 1.856 mmol) was placed in a sealed tube and dissolved in ethanol (5 mL). _MeNH₂ (0.9 mL, 9.282 mmol) containing 33% in ethanol was added at room temperature, followed by stirring at 60 °C for 16 hours. Once the reaction was complete as indicated by TLC, volatiles were removed under reduced pressure, water (50 mL) was added, and the solution was acidified to pH 2 with 1 N HCl. The aqueous layer was extracted with ethyl acetate (50 mL) and a brine solution (30 mL ₎ , dried over _Na₂SO₄ , and concentrated to give a reddish-brown, colloidal liquid containing side-chain-12 (200 mg, 0.7604 mmol, 41%). TLC system: 10% methanol in dichloromethane, _Rf : 0.10

Side chain-13

2-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl ester of 4-methylbenzenesulfonic acid (2)

Silver oxide (3.5 g, 15.46 mmol), p-toluenesulfonyl chloride (2.3 g, 12.37 mmol), and potassium iodide (342 mg, 2.06 mmol) were added to a stirred solution of 2,2'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethanol)1 (2 g, 10.30 mmol) in dichloromethane (30 mL) at 0 °C. The mixture was stirred at 0 °C for 30 minutes. After the starting material was exhausted, the mixture was filtered through a diatomaceous earth liner and the liner was washed several times with DCM. The filtrate was washed with a water-salt solution (50 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by column chromatography and eluted with 3% methanol in dichloromethane to give 2-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid 2 (2.6 g, 7.471 mmol, yield 72%) as a colorless, oily liquid. TLC system: 10% methanol in dichloromethane, _Rf : 0.50; LCMS: m/z = 371.19 (M+Na) ⁺

5,8,11-Trioxa-2-azatridecane-13-ol (3)

In a sealed tube, 33% MeNH₂ in ethanol (0.8 mL) (267 mg, 8.62 mmol) was added to a stirred solution of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl ₄ -methylbenzenesulfonic acid (600 mg, 1.724 mmol) in ethanol (5 mL) at room temperature, followed by stirring at 60 °C for 16 hours. If the reaction was complete as indicated by TLC, the organic solvent was evaporated, and the crude product was co-distilled with dichloromethane (10 mL) to give 5,8,11-trioxa-2-azatridecane-13-ol 3 (650 mg, 3.14 mmol, crude product) as a colorless, colloidal liquid. TLC system: 10% methanol in dichloromethane, _Rf : 0.20; direct mass: m/z = 208.15(M+H) ⁺

(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate tert-butyl ester (4)

Triethylamine (3.8 mL, 28.01 mmol) and di-tert-butyl dicarbonate (3.2 mL, 14.09 mmol) were added to a stirred solution of 5,8,11-trioxa-2-azatridecane-13-ol 3 (2.9 g, 14.09 mmol) in DCM (30 mL) at 0 °C, followed by stirring at room temperature for 2 hours. After the starting material was exhausted, the mixture was diluted with ethyl acetate (150 mL) and washed with ice-cold water (2 × 20 mL) and brine (20 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography at 100–200 μL by elution with 5% methanol in dichloromethane to give (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate tert-butyl 4 (1.8 g, 5.863 mmol, 42% yield) as a pale brown liquid. TLC system: 100% ethyl acetate, _Rf : 0.50; LCMS: m/z = 330.04 (M + Na) ⁺

2,2,5-Trimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-yl ester of 4-methylbenzenesulfonic acid (5)

Sodium hydroxide (485 mg, 12.052 mmol) and p-toluenesulfonyl chloride (1.63 g, 7.231 mmol) were added to a stirred solution of (2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate 4 (1.85 g, 6.026 mmol) in THF (20 mL) at 0 °C, followed by stirring at room temperature for 16 hours. Once the reaction was complete as indicated by TLC, the mixture was diluted with ethyl acetate (150 mL) and washed with ice-cold water (2 × 20 mL) and a saline solution (20 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography at 100–200 μM, eluting with 40% ethyl acetate in petroleum ether, to give 2,2,5-trimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-ester 5 of 4-methylbenzenesulfonic acid (2.5 g, 5.423 mmol, 90% yield), a colorless, colloidal liquid. TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.50; LCMS: m/z = 484.4 (M + Na) ⁺

(2-(2-(2-(2-(1,3-dioxoisoindoline-2-yl)ethoxy)ethoxy)ethoxy)ethyl)(methyl)tert-butyl carbamate (6)

Potassium phthalimide (1.3 g, 7.049 mmol) was added to a stirred solution of 2,2,5-trimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-ester 5 (2.5 g, 5.423 mmol) in DMF (10 mL) at room temperature, followed by stirring at 110 °C for 16 hours. The reaction mixture was cooled to room temperature, then ether (100 mL) was added and stirred for 15 minutes, followed by filtration. The filtrate was washed with 1M NaOH solution (50 mL), water (100 mL), and brine solution (100 mL ₎ , dried over _Na₂SO₄ , and concentrated under reduced pressure to obtain a light brown, colloidal liquid (2-(2-(2-(2-(1,3-dioxoisoindoline-2-yl)ethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate tert-butyl 6 (1.65 g, 3.784 mmol, crude product). TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.50; LCMS: m/z = 459.45 (M + Na) ⁺

2-(5,8,11-trioxa-2-azatridecane-13-yl)isoindoline-1,3-dione (side chain -13)

To a stirred solution of (2-(2-(2-(2-(1,3-dioxoisoindoline-2-yl)ethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate tert-butyl 6 (1.55 g, 3.555 mmol) in dioxane (5 mL), 4N HCl in dioxane (5 mL) was added, followed by stirring at room temperature for 30 minutes. The reaction mixture was concentrated, water (50 mL) was added to the crude material, alkalized with sodium bicarbonate solution (100 mL), and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine solution (50 mL ₎ , dried over _Na₂SO₄ , and concentrated under reduced pressure to give a pale yellow, oily liquid, 2-(5,8,11-trioxa-2-azatridecane-13-yl)isoindoline-1,3-dione side chain-13 (700 mg, 2.083 mmol, crude material). TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.10; LCMS: m/z = 337.41 (M+H) ⁺

Side chain-14

Ethyl 5-(2-ethoxyethoxy)valerate (3)

NaH (267 mg, 11.11 mmol) was added to a stirred solution of 2-ethoxyethanol 1 (500 mg, 5.555 mmol) in tetrahydrofuran (10 mL) at 0 °C and stirred for 30 min at room temperature. Ethyl 5-bromopentanoate 2 (1.74 g, 8.333 mmol) in tetrahydrofuran (5 mL) was added to the above reaction mixture at 0 °C and stirred for 16 h at room temperature under a nitrogen atmosphere. The reaction mixture was quenched with ice-cold water (50 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with brine (50 mL), dried over _{Na₂SO₄ ,} and evaporated under reduced pressure. The crude residue was purified by silica gel chromatography at 100–200 °C using 6% ethyl acetate in hexane to give ethyl 5-(2-ethoxyethoxy)pentanoate 3 (220 mg, 1.009 mmol, 18% yield) as a colorless, oily liquid. TLC system: 50% ethyl acetate in hexane; _Rf : 0.45

5-(2-ethoxyethoxy)valerate (side chain -14)

Lithium hydroxide monohydrate (127 mg, 3.027 mmol) was added to a stirred solution of ethyl 5-(2-ethoxyethoxy)valerate 3 (220 mg, 1.009 mmol) in a mixture of THF and _H₂O (3:1) (4 mL) at room temperature, and the mixture was stirred for 16 hours. The reaction mixture was concentrated to remove volatile organic compounds, water (50 mL) was added to the crude material, and the mixture was extracted with ethyl acetate (2 × 20 mL). The aqueous solution was acidified with saturated citric acid and extracted with 10% methanol in dichloromethane (2 × 30 mL). The combined organic layers were washed with a brine solution (15 mL), dried over sodium sulfate, and concentrated to give a colorless, oily liquid containing 5-(2-ethoxyethoxy)valerate side chain-14 (110 mg, 0.5789 mmol, 57%). TLC system: ethyl acetate 70% in hexane; _Rf : 0.10

Side chain - 15

5-(2-ethoxyethoxy)pentane-1 alcohol (3)

A stirred solution of 1,5-pentanediol 1 (11 g, 105.76 mmol) in N,N-dimethylformamide (30 mL) was treated with 60% NaH (4.5 g, 116.34 mmol) at 0 °C and stirred for 30 min at room temperature. 1-bromo-2-ethoxyethylene 2 (12 mL, 105.76 mmol) in N,N-dimethylformamide (20 mL) was added to the above reaction mixture at 0 °C and stirred for 16 h at room temperature under an argon atmosphere. The reaction mixture was quenched with ice water (200 mL) and extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over _{Na₂SO₄ and} evaporated under reduced pressure. The crude residue was purified by column chromatography (100-200 silica gel) using ethyl acetate in 30% hexane to give 5-(2-ethoxyethoxy)pentane-1 alcohol 3 (5.1 g, 28.97 mmol, yield 27%) as an oily liquid. TLC system: ethyl acetate in 70% hexane, _Rf : 0.50; LCMS: m/z = 199.12 (M+Na) ⁺

5-(2-ethoxyethoxy)pentyl 4-methylbenzenesulfonic acid (4)

Triethylamine (11 g, 78.40 mmol) was added to a stirred solution of 5-(2-ethoxyethoxy)pentane-1-ol 3 (4.6 g, 26.136 mmol) in DCM (45 mL) at 0 °C. The mixture was stirred at 0 °C for 10 min. The mixture was then cooled to 0 °C, p-toluenesulfonyl chloride (5.95 g, 31.36 mmol) was added, and DMAP (31 mg, 0.261 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 16 h. After depletion of the starting material, the mixture was diluted with dichloromethane (100 mL) and washed with ice-cold water (2 × 50 mL) and a brine solution (50 mL). The mixture was dried over sodium sulfate and concentrated under reduced pressure. The crude compound was purified by combinatorial rapid chromatography and eluted with ethyl acetate in 35% petroleum ether to give 4-methylbenzenesulfonic acid 5-(2-ethoxyethoxy)pentane ester 4 (3.9 g, 11.81 mmol, 45% yield) as a yellow, oily liquid. TLC system: 50% ethyl acetate in hexane, _Rf : 0.60; LCMS: m/z = 353.20 (M + Na) ⁺

1-Azide-5-(2-ethoxyethoxy)pentane (5)

_NaN₃ (441 mg, 6.787 mmol) was added to a stirred solution of 4-methylbenzenesulfonic acid 5-(2-ethoxyethoxy)pentane 4 (1.4 g, 4.242 mmol) in DMF (15 mL) at room temperature, followed by stirring at 60 °C for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, water (200 mL) was added to the crude compound, and extraction was performed with diethyl ether (2 × 50 mL). The combined organic layers were washed with a brine solution (50 mL), dried over _Na₂SO₄ , and concentrated. The crude compound was purified by column chromatography (silica gel 100-200) eluting with 30% ethyl acetate in petroleum ether to give ₁ -azido-5-(2-ethoxyethoxy)pentane 5 (750 mg, 3.731 mmol, 88%) as a liquid. TLC system: ethyl acetate 30% in hexane, _Rf : 0.40

5-(2-ethoxyethoxy)pentane-1-amine (side chain -15)

1 M P(Me)3 in THF (8.3 mL, 8.358 mmol) was added to a stirred solution of 1-azido-5-(2-ethoxyethoxy)pentane ₅ (840 mg, 4.179 mmol) in THF: _H₂O (4:1) (10 mL) at 0 °C, followed by stirring at room temperature for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated to obtain 1.1 g (crude substance) of 5-(2-ethoxyethoxy)pentane-1-amine side chain-15 in liquid form. TLC system: 10% methanol in dichloromethane, _Rf : 0.10; LCMS: m/z = 176.18(M+H) ^⁺

5-(2-ethoxyethoxy)-N-methylpentane-1-amine (side chain -16)

5-(2-ethoxyethoxy)pentyl 4-methylbenzenesulfonic acid 1 (synthesis of compound 1 reported in side chain-15) (3 g, 9.090 mmol) was stirred in a solution of 1 M methylamine in ethanol (70 mL) for 6 hours at 70 °C. After the reaction was complete as indicated by TLC, the reaction mixture was evaporated and diluted with ethyl acetate (150 mL). The organic layer was basified with triethylamine, water was added, and the mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with a brine solution (50 mL), dried over _{Na₂SO₄ ,} and concentrated to obtain a brown, oily liquid containing 5-(2-ethoxyethoxy)-N-methylpentane-1-amine side chain-16 (1.6 g, 8.465 mmol, crude). TLC system: 10% dichloromethane/triethylamine, _Rf : 0.20; direct mass: m/z = 190(M+H) ⁺

Side chain-17

N-Benzyl-5-(2-ethoxyethoxy)pentanamide (2)

Diisopropylethylamine (1.4 mL, 7.9 mmol) and HATU (2 g, 5.28 mmol) were added to a stirred solution of 5-(2-ethoxyethoxy)pentanoic acid 1 (synthesis of compound 1 is reported in side chain-14) (500 mg, 2.64 mmol) in DMF (5 mL) at room temperature, and the mixture was stirred for 10 min. Then, benzylamine (420 mg, 3.96 mmol) was added, and the mixture was stirred at room temperature for 16 h. When the reaction was complete as indicated by TLC, ice-cold water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with a saline solution (50 _mL ), dried ( _Na₂SO₄ ), and concentrated. The crude compound was purified by combinatorial rapid chromatography and eluted with 25% ethyl acetate in petroleum ether to give N-benzyl-5-(2-ethoxyethoxy)pentanamide 2 (400 mg, 1.43 mmol, 54% yield) as a pale yellow liquid. TLC system: 50% ethyl acetate in hexane, _Rf : 0.40; LCMS: m/z = 280.39 (M+H) ⁺

N-Benzyl-5-(2-ethoxyethoxy)pentane-1-amine (side chain 17)

A solution of 1 M borane in THF (5.7 mL, 5.73 mmol) was added to a stirred solution of N-benzyl-5-(2-ethoxyethoxy)pentanamide 2 (400 mg, 1.43 mmol) in THF (5 mL) at 0 °C, and the mixture was stirred at room temperature for 4 hours. When the reaction was complete as indicated by TLC, excess borane was carefully quenched with 1 mL methanol and 50 mL ice water, and the mixture was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (50 mL), _dried over _Na₂SO₄ , and evaporated under reduced pressure to give a pale yellow liquid containing N-benzyl-5-(2-ethoxyethoxy)pentan-1-amine side chain 17 (300 mg, 1.13 mmol, 79% yield). TLC system: ethyl acetate 50% in hexane, _Rf : 0.50; LCMS: m/z = 288.14(M+Na) ⁺

Side chain-18

5-Ethoxypentan-1-ol (2)

NaH (115 mg, 4.807 mmol) was added to a stirred solution of pentane-1,5-diol 1 (500 mg, 4.807 mmol) in tetrahydrofuran (10 mL) at 0 °C and stirred at room temperature for 1 hour. Iodoethane (740 mg, 4.807 mmol) in tetrahydrofuran (5 mL) was added to the above reaction mixture at 0 °C and stirred at 60 °C under a nitrogen atmosphere for 48 hours. The reaction mixture was quenched with ice water and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (20 mL), dried over _{Na₂SO₄ ,} and evaporated under reduced pressure. The crude residue was purified by silica gel chromatography at 100–200 °C using ethyl acetate in 35% hexane to give 5-ethoxypentane-1-ol 2 (310 mg, 2.348 mmol, 49% yield) as a reddish-brown oily liquid. TLC system: ethyl acetate in 70% hexane; _Rf : 0.50

2-(5-ethoxypentoxy)tert-butyl acetate (4)

Potassium carbonate (1.6 g, 11.363 mmol) and tert-butyl 2-bromoacetate (1.85 g, 9.4697 mmol) were added to a stirred solution of 5-ethoxypentyl-1-ol 2 (500 mg, 3.7878 mmol) in N,N-dimethylformamide ( ₅ mL), followed by stirring at 80 °C under nitrogen atmosphere for 16 h. The reaction mixture was quenched with ice water and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (20 mL), dried over _Na₂SO₄ , and evaporated under reduced pressure. The crude residue was purified by silica gel chromatography at 100–200 °C using ethyl acetate in 8% hexane to give tert-butyl 2-(5-ethoxypentyloxy)acetate 4 (600 mg, 2.439 mmol, 64% yield) as a colorless, oily liquid. TLC system: ethyl acetate in 30% hexane, _Rf : 0.50

2-(5-ethoxypentoxy)acetic acid (side chain-18)

4N HCl (15 mL) in dioxane was added to a stirred solution of 4-(5-ethoxypentoxy)acetic acid tert-butyl 4 (2 g, 8.13 mmol) in dioxane (10 mL), and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated, water (100 mL) was added to the crude material, and the mixture was extracted with ethyl acetate (2 × 50 mL). The aqueous layer was acidified with 1N HCl solution to pH 2 and then extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (100 mL), _dried over _Na₂SO₄ , and concentrated to give a colorless, colloidal liquid containing 2-(5-ethoxypentoxy)acetic acid side chain-18 (1.3 g, 6.842 mmol, 83%). TLC system: 100% ethyl acetate, _Rf : 0.10

Side chain-19

5-Ethoxypentan-1-ol (2)

A solution of pentane-1,5-diol 1 (10 g, 96.15 mmol) in THF (100 mL) was added to a stirred suspension of NaH (3.8 g, 96.15 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 hour. Iodoethane (3.7 mL, 96.15 mmol) in THF (50 mL) was added to the above reaction mixture at 0 °C, and the mixture was stirred at room temperature under an argon atmosphere for 16 hours. The reaction mixture was quenched with ice-cold water (200 mL) and extracted with ethyl acetate (3 × 200 mL). The combined organic layers were washed with brine (100 mL), dried over _Na₂SO₄ , and evaporated under reduced pressure. The crude residue was purified by column chromatography by elution with 40% ethyl acetate in petroleum ether to give ₅ -ethoxypentane-1-ol 2 (6.5 g, 49.24 mmol, 51% yield) as an oily liquid. TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.50; direct mass: m/z = 133.36(M+H) ⁺

((2-bromoethoxy)methanetriphenylmethyl)triphenyl(4)

Triethylamine (15.5 mL, 112 mmol) and triphenylmethyl chloride were added to a stirred solution of 2-bromoethyl-1-ol 3 (7 g, 56 mmol) in DCM (175 mL) at 0 °C, and the mixture was stirred at _room temperature for 3 h. After the reaction was complete as indicated by TLC, the reaction mixture was diluted with water (300 mL) and extracted with dichloromethane (2 × 200 mL). The combined organic layers were washed with a brine solution (100 mL), dried over _Na₂SO₄ , and concentrated under reduced pressure. The crude compound was purified by 100–200 μL silica gel column chromatography by elution with 5% ethyl acetate in petroleum ether to give ((2-bromoethoxy)methanetriphenylmethyl)triphenyl 4 (4.5 g, 12.295 mmol, 22% yield) as a grayish-white solid. TLC system: 10% ethyl acetate in petroleum ether, _Rf : 0.60; LCMS: m/z = 367.24 (M+H) ⁺

((2-((5-ethoxypentyl)oxy)ethoxy)methanetriphenylmethyl)triphenyl(5)

A solution of 5-ethoxypentan-1-ol 2 (1.25 g, 9.469 mmol) in DMF (20 mL) was added to a stirred suspension of NaH (568 mg, 23.67 mmol) at 0 °C, and the mixture was stirred at room temperature for 30 min. Then, (2-bromoethoxy)methanetriphenylmethyl)triphenyl 4 (4.5 g, 12.31 mmol) in DMF (5 mL) was added to the above reaction mixture at 0 °C, and the mixture was stirred at room temperature under an argon atmosphere for 16 h. The reaction mixture was quenched with ice-cold water (200 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (100 mL), dried over _{Na₂SO₄ ,} and evaporated under reduced pressure. The crude residue was purified by silica gel column chromatography at 100-200 μm by elution with 5% ethyl acetate in petroleum ether to give ((2-((5-ethoxypentyl)oxy)ethoxy)methanetriphenylmethyltriphenyl5 (2.5 g, 5.980 mmol, yield 63%) as a colorless, oily liquid. TLC system: 5% ethyl acetate in petroleum ether, _Rf : 0.40; LCMS: m/z = 441.46 (M+Na) ⁺

2-((5-ethoxypentyl)oxy)ethanol-1-ol(6)

HCl (4N, 24 mL) in dioxane was added to a stirred solution of ((2-((5-ethoxypentyl)oxy)ethoxy)methanetriphenylmethyl)triphenyl5 (12 g, 28.70 mmol) in dioxane (60 mL) at 0 °C, followed by stirring at room temperature for 1 hour. After the reaction was complete as indicated by TLC, the reaction mixture was concentrated, water (50 mL) was added to the residue, alkalized with sodium bicarbonate solution (100 mL), and extracted with ethyl acetate (2 × 200 mL). The combined organic layers were washed with brine solution (100 mL), dried over _{Na₂SO₄ ,} and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography at 100–200 μL, eluting with 70% ethyl acetate in petroleum ether, to give 2-((5-ethoxypentyl)oxy)ethanol-1-ol 6 (4.4 g, 25.0 mmol, 88% yield) as a colorless, oily liquid. TLC system: 50% ethyl acetate in petroleum ether, _Rf : 0.30; direct mass: m/z = 177.22(M+H) ⁺

2-((5-ethoxypentyl)oxy)ethyl 4-methylbenzenesulfonic acid (7)

NaOH (1.3 g, 34.09 mmol) and p-toluenesulfonyl chloride (3.8 g, 20.45 mmol) were added to a stirred solution of 2-((5-ethoxypentyl)oxy)ethyl-1-ol 6 (3 g, 17.04 mmol) in THF (5 mL) at 0 °C, and the mixture was stirred at room temperature for 16 h. After the starting material was exhausted, the reaction mixture was quenched with water (200 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with a brine solution (50 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography at 100–200 μL by elution with 20% ethyl acetate in petroleum ether to give 2-((5-ethoxypentyl)oxy)ethyl 4-methylbenzenesulfonic acid 7 (5.2 g, 15.75 mmol, 92% yield) in liquid form. TLC system: 20% ethyl acetate in petroleum ether, _Rf : 0.50; LCMS: m/z = 331.04 (M+H) ⁺

1-(2-Azide-ethoxy)-5-ethoxypentane (8)

To a stirred solution of 2-((5-ethoxypentyl)oxy)ethyl 4-methylbenzenesulfonic acid 7 (3.5 g, 10.60 mmol) in DMF (30 mL), NaN ₃ (1.1 g, 16.96 mmol) was added, followed by stirring at 60 °C for 16 h. Once the reaction was complete as indicated by TLC, the reaction mixture was quenched with water (200 mL) and extracted with diethyl ether (2 × 100 mL). The combined organic layers were washed with a brine solution (100 mL), dried over _Na₂SO₄ , and concentrated under reduced pressure. The crude compound was purified by column chromatography by elution with 15% ethyl acetate in petroleum ether to give _1- (2-azidoethoxy)-5-ethoxypentane 8 (1.95 g, 6.414 mmol, 92%) as a colorless, oily liquid. TLC system: 20% ethyl acetate in petroleum ether, _Rf : 0.50

2-((5-ethoxypentyl)oxy)ethyl-1-amine (side chain -19)

1 M P(Me)3 in THF (8.9 mL, 8.955 mmol) was added to a stirred solution of 1-azido-5-(2-ethoxyethoxy)pentane ₈ (900 mg, 4.477 mmol) in THF: _H₂O (4:1) (12.5 mL) at 0 °C, followed by stirring at room temperature for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, water (100 mL) was added to the residue, and the mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with a brine solution (100 mL), dried over sodium sulfate, and concentrated under reduced pressure to give a pale yellow, oily liquid containing 5-(2-ethoxyethoxy)pentane-1-amine side chain-19 (240 mg, 1.371 mmol, crude). TLC system: 5% methanol in dichloromethane, _Rf : 0.10; direct mass: m/z = 176.31(M+H) ^⁺

Side chain - 20

2-((5-ethoxypentyl)oxy)-N-methylethyl-1-amine (side chain -20)

MeNH₂ (1M, 5mL) in ethanol was added to a stirred solution of 2-((5-ethoxypentyl)oxy)ethyl ₄ -methylbenzenesulfonic acid 1 (synthesis of compound 1 is reported in side chain-19) in a sealed tube at room temperature. The mixture was then stirred in the sealed tube at 60°C for 16 hours. The organic solvent was evaporated as indicated by TLC. Water (10mL) was added to the crude mixture, which was acidified with HCl solution (6N, 50mL) and washed with dichloromethane (2×100mL). The aqueous layer was alkalized with NaOH solution (100mL) and extracted with ethyl acetate (2×100mL). The organic layer was washed with a saline solution (50 mL), dried over _{Na₂SO₄ ,} and concentrated to give a colorless, oily liquid containing 2-((5-ethoxypentyl)oxy)-N-methylethyl-1-amine side chain-20 (240 mg, 1.269 mmol, 41%). TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.10; direct mass: m/z = 190.18(M+H) ⁺

Side chain-21

N-Benzyl-2-((5-ethoxypentyl)oxy)ethyl-1-amine (side chain -21)

Aniline (63 mg, 0.5908 mmol) was added to a stirred solution of 2-((5-ethoxypentyl)oxy)ethyl 4-methylbenzenesulfonic acid 1 (synthesis of compound 1 is reported in side chain-19) (65 mg, 0.196 mmol) in ethanol (4 mL) at room temperature, followed by stirring at 70 °C for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, water (50 mL) was added to the crude compound, and extraction was performed with ethyl acetate (2 × 20 mL). The combined organic layers were washed with a brine solution (30 mL), dried over _{Na₂SO₄ ,} and concentrated to give a colorless, colloidal liquid crude compound N-benzyl-2-((5-ethoxypentyl)oxy)ethyl-1-amine side chain-21 (95 mg, 0.358 mmol, crude compound). TLC system: 10% methanol in dichloromethane, _Rf : 0.40; LCMS: m/z = 266.29 (M+H) ⁺

Side chain-22

2-(pentoxy)ethane-1-ol (3)

Sodium metal (0.6 g, 0.025 mmol) was added to ethylene glycol 1 (5 g, 0.083 mmol) at room temperature and heated to 80 °C. Then, 1-bromopentane 2 (3.7 g, 0.025 mmol) was added to the reaction mixture at the same temperature, and the mixture was stirred for 4 hours. The mixture was filtered to remove inorganic substances and diluted with ice water (200 mL), then extracted with ethyl acetate (2 × 200 _mL ). The organic layer was washed with brine, dried ( _Na₂SO₄ ), and concentrated to give a pure 2-(pentoxy)ethane-1-ol 3 (2.2 g, 0.0186 mmol, 55%) as a yellow liquid. TLC system: 10% methanol in dichloromethane, _Rf : 0.5

2-(2-(pentoxy)ethoxy)acetic acid (side chain -22)

NaOH (0.84 g, 21.12 mmol) was added to a stirred solution of 2-(pentoxy)ethoxy)-1-ol 3 (500 mg, 4.23 mmol) in 1,4-dioxane (10 mL) at room temperature. The mixture was stirred for 15 minutes at room temperature. Then, tert-butyl 2-bromoacetate 4 (1.3 mL, 8.47 mmol) and 18-crown ether (25 mg) were added to the reaction mixture, and the mixture was stirred for 18 hours at room temperature. After the starting material was exhausted, the mixture was diluted with ice-cold water (50 mL) and extracted with diethyl ether (2 × 50 mL). The separated aqueous layer was acidified with concentrated HCl (pH approximately 2) and extracted with a 10% methanol mixture in dichloromethane (2 × 100 mL). The organic layer was washed with brine (50 mL), dried over sodium sulfate, and concentrated to give a thick brown liquid of pure 2-(2-(pentoxy)ethoxy)acetic acid side chain-22 (200 mg, 1.05 mmol, yield 24%). TLC system: 10% methanol in dichloromethane, _Rf : 0.10

Side chain-23

2-(2-(pentoxy)ethoxy)ethanol-1-ol(3)

1-Bromopentane 2 was added to a stirred solution of methyl 2,2'-oxybis(ethylene-1-ol) 1 (10 g, 66.25 mmol) in 50% NaOH aqueous solution (250 mL), and the mixture was stirred at 100 °C for 16 h. If the reaction was complete as indicated by TLC, water was added to the reaction mixture (200 mL), and the mixture was extracted with ethyl acetate (2 × 500 mL). The combined organic layers were washed with a brine solution (200 mL ₎ , dried over _Na₂SO₄ , and concentrated under reduced pressure to give 2-(2-(pentoxy)ethoxy)ethylene-1-ol 3 (8.4 g, 47.72 mmol, 72%) as a brown liquid. TLC system: 100% ethyl acetate, _Rf : 0.50

2-(2-(pentoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (4)

A solution of NaOH (1.8 g, 45.45 mmol) in water (10 mL) was added to a stirred solution of 2-(2-(pentoxy)ethoxy)ethanol-1-ol 3 (4 g, 22.72 mmol) in THF (30 mL) at 0 °C. The reaction mixture was stirred at room temperature for 15 min, then cooled to 0 °C, and p-toluenesulfonyl chloride (5.1 g, 27.27 mmol) was added to the reaction mixture and stirred at room temperature for 16 h. After the starting material was exhausted, the mixture was diluted with ethyl acetate (150 mL) and washed with ice-cold water (2 × 30 mL) and brine solution (30 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography (100-200 mesh) by elution with 30% ethyl acetate in petroleum ether to give 4-methylbenzenesulfonic acid 2-(2-(pentoxy)ethoxy)ethyl ester 4 (5.4 g, 16.36 mmol, 72% yield) as a brown liquid. TLC system: 70% ethyl acetate in petroleum ether, _Rf : 0.50; LCMS: m/z = 331.09 (M+H) ⁺

1-(2-(2-azidoethoxy)ethoxy)pentane (5)

_NaN₃ (630 mg, 9.696 mmol) was added to a stirred solution of 2-(2-(pentoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (2 g, 6.06 mmol) in DMF (20 mL) at room temperature, followed by stirring at 60 °C for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, and the crude material was extracted with ice water (2 × 50 mL) and diethyl ether (2 × 100 mL). The combined organic layers were washed with a brine solution (50 mL), dried over _{Na₂SO₄ ,} and concentrated under reduced pressure to give 1-(2-(2-azidoethoxy)ethoxy)pentane 5 (1.1 g, 5.97 mmol, 91%) as a brown liquid. TLC system: ethyl acetate 50% in hexane, _Rf : 0.30; LCMS: m/z = 202(M+H) ^⁺

2-(2-(pentoxy)ethoxy)ethyl-1-amine (side chain -23)

P(Me)3 (1M, 11mL, 10.94mmol) in THF was added to a stirred solution of 5 stoichioside ( _1.1 g, 5.47 mmol) in THF: _H₂O (4:1) (10 mL) at 0 °C, and the mixture was stirred at room temperature for 16 hours. Once the reaction was complete as indicated by TLC, the organic solvent was evaporated to give a brown liquid containing 2-(2-(pentoxy)ethoxy)ethyl-1-amine side chain-23 (1.1 g, 6.285 mmol, crude product). TLC system: 10% methanol in dichloromethane, _Rf : 0.1

Side chain-24

N-Methyl-2-(2-(pentoxy)ethoxy)ethyl-1-amine (side chain -24)

500 mg (1.51 mmol) of 2-(2-(pentoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (synthesis of compound 1 is reported in side chain-23) was added to methylamine (1 M, 5 mL) in ethanol at room temperature, followed by stirring at 80 °C for 12 hours. After the reaction was complete as indicated by TLC, the reaction mixture was evaporated, diluted with water (50 mL), acidified with 1 N HCl (pH approximately 2), and washed with diethyl ether (2 × 50 mL). The separated aqueous layer was then alkalized with saturated _NaHCO3 solution and extracted with 10% methanol in dichloromethane (2 × 100 mL), washed with brine (50 _mL ), dried over _Na2SO4 , and concentrated to obtain N-methyl-2-(2-(pentoxy)ethoxy)ethyl-1-amine side chain-24 (240 mg, 1.27 mmol, 84%) as a brown, oily liquid. TLC system: 10% methanol in dichloromethane (1 drop Et ₃ N), _Rf : 0.20

Side chain -25

N-Benzyl-2-(2-(pentoxy)ethoxy)ethyl-1-amine (side chain 25)

Potassium carbonate (2 g, 15.15 mmol) and benzylamine (256 mg, 2.424 mmol) were added to a stirred solution of 2-(2-(pentoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid 1 (synthesis of compound 1 is reported in side chain-23) (500 g, 1.515 mmol) in DMF (5 mL) at room temperature, followed by stirring at 80 °C for 16 h. After the reaction was complete as indicated by TLC, ice water (30 mL) was added to the reaction mixture and the mixture was extracted with ethyl acetate (2 × 30 mL). The organic layer was washed with a brine solution ( ₃₀ mL), dried over _Na₂SO₄ , and concentrated under reduced pressure. The crude compound was purified by combinatorial rapid chromatography with elution of 10% methanol in dichloromethane to give N-benzyl-2-(2-(pentoxy)ethoxy)ethyl-1-amine side chain 25 (300 mg, 1.132 mmol, yield 74.8%) as a grayish-white solid. TLC system: 10% methanol in dichloromethane, _Rf : 0.20; LCMS: m/z = 266.43 (M+H) ⁺

Side chain-26

Undecyl 4-methylbenzenesulfonate (2)

NaOH (0.93 g, 23.2 mmol) was added to a stirred solution of undecane-1-ol 1 (2.0 g, 11.6 mmol) in THF (20 mL) at 0 °C. The mixture was stirred at room temperature for 15 min. Then, the mixture was cooled to 0 °C, and p-toluenesulfonyl chloride (2.65 g, 13.92 mmol) was added to the reaction mixture and stirred at room temperature for 16 h. After the starting material was exhausted, the mixture was diluted with ethyl acetate (150 mL), washed with ice-cold water (2 × 20 mL) and a brine solution (20 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by silica gel column chromatography (100–200 mesh) eluting with 20% ethyl acetate in hexane to give undecane 4-methylbenzenesulfonate 2 (2.6 g, 7.97 mmol, 68% yield) as a grayish-white solid. TLC system: 10% methanol in dichloromethane, _Rf : 0.70; LCMS: m/z = 325.56 (MH ⁾

N-Methylundecane-1-amine (side chain -26)

1 M _MeNH₂ in methanol (2.5 mL) was added to a stirred solution of 2-undecane 4-methylbenzenesulfonate (600 mg, 1.84 mmol) in methanol (2 mL) in a sealed tube at room temperature, followed by stirring at 80 °C for 16 hours. The organic solvent was evaporated as indicated by TLC. Water (20 mL) was added to the crude product, and extraction was performed with ethyl acetate (3 × 30 mL). The organic layer was washed with 1 N HCl solution (20 mL), saturated _NaHCO₃ solution (20 mL), and brine solution (100 mL), dried over _{Na₂SO₄ ,} and concentrated to give a brown, oily liquid N-methylundecane-1-amine (side chain -26) (190 mg, 1.02 mmol, crude product). TLC system: 10% methanol in dichloromethane, _Rf : 0.20; LCMS: m/z = 186.40 (M + H) ⁺

Side chain-27

N-Benzylundecane-1-amine (side chain -27)

Aniline (0.6 mL) was added to a stirred solution of 4-methylbenzenesulfonate 1 (synthesis of compound 1 reported in side chain-26) (600 mg, 1.84 mmol) in ethanol (10 mL) in a sealed tube at room temperature, followed by stirring at 60 °C for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, water (20 mL) was added to the crude compound, and extraction was performed with ethyl acetate (3 × 30 mL). The organic layer was washed with 1N HCl solution (20 mL), saturated _NaHCO3 solution (20 mL), and brine solution ( ₅₀ mL), dried over _Na2SO4 , and concentrated to obtain the crude compound. The crude compound was purified by 100-200 mesh silica gel column chromatography with elution of 40% ethyl acetate in hexane to give N-methylundecane-1-amine side chain-27 (350 mg, 1.34 mmol, 72% yield) as a grayish-white semi-solid. TLC system: 50% ethyl acetate in petroleum ether, _Rf : 0.20; LCMS: m/z = 262.47 (M+H) ⁺

Side chain-28

(3-(ethoxymethyl)phenyl)methanol(2)

A stirred solution of 1,3-phenylenediethanol (1) (5 g, 36.496 mmol) in THF (100 mL) was treated with NaH (1.17 g, 29.197 mmol) for 30 min at 0 °C to room temperature. Iodoethane (2.3 mL, 29.197 mmol) in THF (10 mL) was added to the reaction mixture at 0 °C, and the mixture was stirred at 60 °C under _a nitrogen atmosphere for 6 h. The reaction mixture was quenched with ice water and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over _Na₂SO₄ under reduced pressure. The crude residue was purified by combined rapid chromatography using 20% ethyl acetate in petroleum ether to give (3-(ethoxymethyl)phenyl)ethanol 3 (2.1 g, 12.65 mmol, 34% yield) as a pale yellow, oily liquid. TLC system: 40% ethyl acetate in petroleum ether, _Rf : 0.50; LCMS: m/z = 120.99 (M-46) ⁺

2-(3-(ethoxymethyl)benzoxy)tert-butyl acetate (4)

NaOH (542 mg, 13.55 mmol) was added to a stirred solution of (3-(ethoxymethyl)phenyl)methanol 2 (750 mg, 4.158 mmol) in 1,4-dioxane (10 mL) at 0 °C. The mixture was stirred at room temperature for 15 min, then cooled to 0 °C, and tert-butyl 2-bromoacetate 3 (1.76 g, 9.036 mmol) and 18-crown ether (40 mg) were added, and the mixture was stirred at room temperature for 16 h. After the starting material was exhausted, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with ice-cold water (50 mL) and brine solution (20 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by combinatorial rapid chromatography and eluted with 10% ethyl acetate in petroleum ether to give tert-butyl 2-(3-(ethoxymethyl)benzoxy)acetate 4 (900 mg, 3.21 mmol, 71.1% yield) as a colorless, oily liquid. TLC system: 30% ethyl acetate in petroleum ether, _Rf : 0.50

2-(3-(ethoxymethyl)benzoxy)acetic acid (side chain -28)

At room temperature, 4M HCl (2.5 mL) in 1,4-dioxane was added to a stirred solution of tert-butyl 2-(3-(ethoxymethyl)benzoxy)acetate 4 (300 mg, 1.0714 mmol) in 1,4-dioxane (2 mL) in a sealed tube, followed by stirring under reflux for 4 hours. Once the reaction was complete as indicated by TLC, the organic solvent was evaporated, water (50 mL) was added to the crude compound, and extraction was performed with ethyl acetate (2 × 30 mL). The organic layer was washed with a brine solution (30 mL), dried over _Na₂SO₄ , and concentrated under reduced pressure. The _crude compound was purified by wet milling with diethyl ether (30 mL) to give a pale yellow, oily liquid containing 2-(3-(ethoxymethyl)benzoxy)acetic acid side chain-28 (190 mg, 0.848 mmol, 79.1% yield). TLC system: 5% methanol in dichloromethane, _Rf : 0.20; LCMS: m/z = 225.41 (M+H) ⁺

Side chain-29

2-(3-(ethoxymethyl)benzoxy)ethanol(2)

1 M LiAlH₄ in THF (5.3 mL, 5.357 mmol) was added to a stirred solution of tert-butyl 2-(3-(ethoxymethyl)benzoxy)acetate ₁ (synthesis of compound 1 reported in side chain-28) (500 mg, 1.7857 mmol) in THF (10 mL) for 3 h at 0 °C. After TLC indicated completion, the reaction mixture was quenched with saturated _NH₄Cl solution (20 mL) and brine solution (20 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was washed with brine solution (50 mL), dried over _Na₂SO₄ , and concentrated to give _2- (3-(ethoxymethyl)benzoxy)ethanol 2 (350 mg, 1.666 mmol, 93%) as a colorless, oily liquid. TLC system: 40% ethyl acetate in hexane, _Rf : 0.20; LCMS: m/z = 233.33 (M + Na) ⁺

2-(3-(ethoxymethyl)benzenesulfonic acid)ethyl ester (3)

NaOH (0.42 g, 10.47 mmol) was added to a stirred solution of 2-(3-(ethoxymethyl)benzoxy)ethanol 2 (1.1 g, 5.23 mmol) in THF (30 mL) at 0 °C. The mixture was stirred at room temperature for 15 minutes. Then, the mixture was cooled to 0 °C, and p-toluenesulfonyl chloride (1.2 g, 6.28 mmol) was added to the reaction mixture and stirred at room temperature for 16 hours. After the starting material was exhausted, the mixture was diluted with ethyl acetate (100 mL), washed with ice-cold water (2 × 50 mL) and brine (50 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by silica gel column chromatography (100–200 mesh) and eluted with ethyl acetate in 10% hexane to give 2-(3-(ethoxymethyl)benzoxy)ethyl 4-methylbenzenesulfonic acid 3 (1.6 g, 4.39 mmol, 84% yield) as a colorless, oily liquid. TLC system: ethyl acetate in 30% hexane, _Rf : 0.70; LCMS: m/z = 386.98 (M + Na) ⁺

1-((2-azidoethoxy)methyl)-3-(ethoxymethyl)benzene(4)

_NaN₃ (0.48 g, 7.47 mmol) was added to a stirred solution of 2-(3-(ethoxymethyl)benzeneoxy)ethyl 4-methylbenzenesulfonic acid 3 (1.7 g, 4.39 mmol) in DMF (30 mL) at room temperature, followed by stirring at 60 °C for 16 h. After TLC indicated completion, water (2 × 100 mL) was added to the crude compound and it was extracted with diethyl ether (2 × 50 mL). The organic layer was washed with a brine solution (50 mL), _dried over _Na₂SO₄ , and concentrated. The crude compound was purified by washing with n-pentane (100 mL) to obtain 1-((2-azidoethoxy)methyl)-3-(ethoxymethyl)benzene 4 (1.1 g, 4.68 mmol, quantitative) as a brown, gelatinous liquid. TLC system: ethyl acetate 30% in hexane; _Rf : 0.70

2-(3-(ethoxymethyl)benzoxy)ethylamine (side chain -29)

1 M P(Me) ₃ in THF (7.7 mL, 7.69 mmol) was added to a stirred solution of 1-((2-azidoethoxy)methyl)-3-(ethoxymethyl)benzene 4 (600 mg, 2.564 mmol) in THF: _H₂O (20 mL), followed by stirring at room temperature for 16 hours. Once the reaction was complete as indicated by TLC, the organic solvent was evaporated and the crude compound was co-distilled with toluene (50 mL) to obtain 2-(3-(ethoxymethyl)benzoxy)ethylamine side chain-29 (500 mg, 2.39 mmol, 93%) as a brown semi-solid. TLC system: 10% methanol in dichloromethane, _Rf : 0.20; LCMS: m/z = 210.32 (M+H) ^⁺

Side chain - 30

2-((3-(ethoxymethyl)benzyl)oxy)-N-methylethyl-1-amine (side chain -30)

At room temperature, 500 mg (1.373 mmol) of 2-(3-(ethoxymethyl)benzyloxy)ethyl 4-methylbenzenesulfonic acid (synthesis of compound 3 is reported in side chain-29) in a sealed tube containing 3 mL of methanol was added with 5 mL of _{1 M} MeNH₂ in ethanol, followed by stirring at 80 °C for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated and the crude residue was diluted with 50 mL of water and extracted with ethyl acetate (2 × 50 mL). The organic layer was washed with ₂₀ mL of 1 N HCl solution, 20 mL of saturated NaHCO₃ solution, and 50 mL of brine solution. The residue was dried over _Na₂SO₄ and concentrated to give a yellow solid, 2-(3-(ethoxymethyl)benzyloxy)-N-methylethylamine side chain- ₃₀ (300 mg, 1.345 mmol, crude substance). TLC system: 10% methanol/dichloromethane, _Rf : 0.20; LCMS: m/z = 224.38 (M+H) ⁺

Side chain-31

2-(3-(2-ethoxyethoxy)phenyl)acetic acid (side chain -31)

2-(3-hydroxyphenyl)acetic acid 1 (2.0 g, 13.157 mmol) was added to a stirred solution of 10% NaOH solution (10 mL) and DMSO (20 mL) at room temperature. The mixture was stirred for 15 minutes at room temperature. Then, 1-bromo-2-ethoxyethylene 2 (2.01 g, 13.15 mmol) was added to the reaction mixture and stirred at 80 °C for 4 hours. After the starting material was exhausted, the mixture was quenched with 1N HCl solution (20 mL) and extracted with ethyl acetate (3 × 50 mL), washed with brine solution (50 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by column chromatography (100-200 mesh silica gel) and eluted with 40% ethyl acetate in hexane to give 2-(3-(2-ethoxyethoxy)phenyl)acetic acid side chain-31 (520 mg, 2.32 mmol, 18%) as a grayish-white solid. TLC system: 100% ethyl acetate, _Rf : 0.60; LCMS: m/z = 225.08 (M+H) ⁺

Side chain-32

3-(2-ethoxyethoxy)benzaldehyde (3)

1-Bromo-2-ethoxyethylene 2 (34 mL, 307.3 mmol) was added dropwise to a stirred solution of 3-hydroxybenzaldehyde 1 (15 g, 122.9 mmol) in DMSO (100 mL) and 10% NaOH aqueous solution in DMSO (50 mL) in DMSO (300 mL). The mixture was stirred at the same temperature for 10 hours. When the reaction was complete as indicated by TLC, the reaction mixture was poured into 1 M HCl solution (200 mL) and extracted with diethyl ether (2 × 500 mL). The combined organic layers were washed with brine (100 _mL ), dried over _Na₂SO₄ , and concentrated under reduced pressure to give 3-(2-ethoxyethoxy)benzaldehyde 3 (5.4 g, 27.83 mmol, 22%) as a colorless, oily liquid. TLC system: ethyl acetate 30% in petroleum ether, _Rf : 0.50; LCMS: m/z = 195.31 (M+H) ⁺

(E)-1-(2-ethoxyethoxy)-3-(2-nitrovinyl)benzene(4)

Ammonium acetate (88 mg, 1.133 mmol) was added to a stirred solution of 3-(2-ethoxyethoxy)benzaldehyde (200 mg, 1.03 mmol) in nitromethane (0.55 mL, 10.3 mmol) at room temperature, followed by stirring at 100 °C for 12 hours. Once the reaction was complete as indicated by TLC, the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with a brine solution (20 mL), dried over _{Na₂SO₄ ,} and concentrated under reduced pressure. The crude compound was purified by Grace column chromatography by elution with acetonitrile in 0.1% formic acid aqueous solution at 60% to give (E)-1-(2-ethoxyethoxy)-3-(2-nitrovinyl)benzene 4 (120 mg, 0.506 mmol, 49%) as a colorless, gelatinous liquid. TLC system: 100% dichloromethane, _Rf : 0.50; LCMS: m/z = 238.31 (M+H) ⁺

2-(3-(2-ethoxyethoxy)phenyl)ethyl-1-amine (side chain -32)

LAH (1 M, 26.3 mL, 26.37 mmol) in THF was added to a stirred solution of (E)-1-(2-ethoxyethoxy)-3-(2-nitrovinyl)benzene 4 (1.25 g, 5.27 mmol) in THF (15 mL) at 0 °C, followed by stirring at 75 °C for 16 h. After the reaction was complete as indicated by TLC, the reaction mixture was diluted with ice water (100 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with a brine solution (50 mL), dried over _{Na₂SO₄ ,} and concentrated to give a reddish-brown, gelatinous liquid containing 2-(3-(2-ethoxyethoxy)phenyl)ethyl-1-amine side chain-32 (310 mg, 1.483 mmol, crude). TLC system: 10% methanol in dichloromethane, _Rf : 0.10; LCMS: m/z = 210.32 (M + H) ⁺

Side chain -33

2-(3-(2-ethoxyethoxy)phenyl)-N-methylacetamide (2)

At 0 °C, 400 mg (1.785 mmol) of 2-(3-(2-ethoxyethoxy)phenyl)acetic acid 1 (synthesis of compound 1 reported in side chain-31) was added to a stirred solution of 2-(3-(2-ethoxyethoxy)phenyl)acetic acid 1 (synthesis of compound 1 reported in side chain-31) in DMF (5 mL), and the mixture was stirred at room temperature for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated, and water (50 mL) was added to the crude residue, followed by extraction with ethyl acetate (2 × 50 mL). The combined organic layers were washed with a brine solution (30 mL), _dried over _Na₂SO₄ , and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography at 100-200 μm, eluting with 2% methanol in dichloromethane, to give 2-(3-(2-ethoxyethoxy)phenyl)-N-methylacetamide 2 (270 mg, 1.139 mmol, crude product) as a grayish-white solid. TLC system: 5% methanol in dichloromethane, _Rf : 0.50; LCMS: m/z = 238.28 (M+H) ⁺

2-(3-(2-ethoxyethoxy)phenyl)-N-methylethyl-1-amine (side chain -33)

1 M BH3 _- THF (2.08 mL, 2.088 mmol) was added to a stirred solution of 2-(3-(2-ethoxyethoxy)phenyl)-N-methylacetamide 2 (165 mg, 0.696 mmol) in THF (2 mL) at 0 °C, followed by stirring at room temperature for 4 hours. If the reaction was complete as indicated by TLC, the reaction mixture was quenched with methanol (20 mL) and the organic solvent was evaporated. Water (50 mL) was added to the crude residue and the mixture was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with a _brine solution (30 mL), dried over _Na2SO4 , and concentrated under reduced pressure to give a reddish-brown liquid containing 2-(3-(2-ethoxyethoxy)phenyl)-N-methylethyl-1-amine side chain-33 (125 mg, 0.560 mmol, crude product). TLC system: 5% methanol in dichloromethane, _Rf : 0.70

Side chain -34

2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (2)

NaOH (3.89 g, 97.415 mmol) in water (10 mL) was added to a stirred solution of 2-(2-(2-ethoxyethoxy)ethoxy)ethyl-1-ol 1 (8.5 g, 47.69 mmol) in THF (30 mL) at 0 °C. The mixture was stirred at room temperature for 15 minutes. Then, the mixture was cooled to 0 °C, and p-toluenesulfonyl chloride (11.63 g, 61.04 mmol) was added to the reaction mixture and stirred at room temperature for 16 hours. After the starting material was exhausted according to TLC, the mixture was diluted with ethyl acetate (150 mL) and washed with ice-cold water (2 × 20 mL) and a saline solution (20 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by 100-200 mesh silica gel column chromatography, eluting with 20% ethyl acetate in petroleum ether, to give 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid ester 2 (4 g, 12.048 mmol, yield 25%), a brown liquid. TLC system: 40% ethyl acetate in petroleum ether, _Rf : 0.50

N-Benzyl-2-(2-(2-ethoxyethoxy)ethoxy)ethyl-1-amine (side chain -34)

Potassium carbonate (2 g, 15.06 mmol) and benzylamine (273 mg, 2.409 mmol) were added to a stirred solution of 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonic acid (500 g, 1.506 mmol) in DMF (5 mL) at room temperature and stirred at 80 °C for 16 h. After the reaction was complete (indicated by TLC), ice water was added to the reaction mixture (100 mL) and the mixture was extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with a saline solution (50 mL), dried over _{Na₂SO₄ ,} and concentrated under reduced pressure. The crude compound was purified by combinatorial rapid chromatography with elution of 10% methanol in DCM to give N-benzyl-2-(2-(2-ethoxyethoxy)ethoxy)ethyl-1-amine side chain 34 (500 mg, 0.93 mmol, yield 62%) as a yellow liquid. LC system: 10% methanol in dichloromethane, _Rf : 0.20

Side chain -35

Ethyl 5-(pentoxy)valerate (3)

NaH (450 mg, 11.63 mmol) was added to a stirred solution of pentyl-1-ol 1 (1.0 g, 11.36 mmol) in N,N-dimethylformamide (10 mL) at 0 °C and stirred for 30 min at room temperature. Ethyl 5-bromopentanoate 2 (1.99 mL, 12.49 mmol) in N,N-dimethylformamide (5 mL) was added to the above reaction mixture at 0 °C and stirred for 16 h at room temperature under nitrogen atmosphere. The reaction mixture was quenched with aqueous ammonium chloride solution and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with brine (2 × 50 mL) and dried over _Na₂SO₄ under reduced pressure. The crude residue was purified by column chromatography using 2% ethyl acetate in hexane to give ethyl 5-(pentoxy)pentanoate ₃ (200 mg, 1.7187 mmol, 8% yield) as a pale yellow oily liquid. LC system: 5% ethyl acetate in hexane, _Rf : 0.30

5-(pentoxy)valerate (side chain -35)

Lithium hydroxide (88 mg, 3.073 mmol) was added to a stirred solution of ethyl 5-(pentoxy)pentanoate (200 mg, 0.92 mmol) in a mixture of THF: _H₂O (4:1, 5 mL) at 0 °C and stirred at room temperature for 16 h. The reaction mixture was evaporated under reduced pressure, and the crude residue was washed with diethyl ether, acidified with 1 N HCl aqueous solution, and extracted with ethyl acetate (3 × 10 mL). The combined organic layers were washed with brine (2 × 15 mL), dried over _Na₂SO₄ , and concentrated. The residue was washed with pentane (2 × ₃₀ mL) to give an oily liquid side-chain-35 (150 mg, 0.797 mmol, 86% yield). LC system: ethyl acetate 50% in hexane, _Rf : 0.10; LCMS: m/z = 189.21 (M+H) ⁺

Side chain - 36

5-(pentoxy)pentan-1-ol (3)

60% NaH (4.2 g, 105.768 mmol) was added to a stirred solution of pentane-1,5-diol 1 (10 g, 96.153 mmol) in N,N-dimethylformamide (70 mL) at 0 °C and stirred for 1 h at room temperature. A solution of 1-bromopentane (14.5 g, 96.153 mmol) in N,N-dimethylformamide (30 mL) was added to the above reaction mixture at 0 °C and stirred for 16 h at room temperature under a nitrogen atmosphere. The reaction mixture was quenched with ice water and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over _Na₂SO₄ and evaporated under reduced pressure. The crude residue was purified by silica gel column chromatography at 100–200 _μL using 20% ethyl acetate in hexane to give 5-(pentoxy)pentane-1-ol 3 (7 g, 40.23 mmol, 43% yield) as a pale yellow, oily liquid. LC system: 50% ethyl acetate in hexane, _Rf : 0.50

5-(pentoxy)pentyl 4-methylbenzenesulfonic acid (4)

Triethylamine (7 g, 68.94 mmol) and DMAP (0.28 g, 2.298 mmol) were added to a stirred solution of 5-(pentoxy)pentan-1-ol 3 (4 g, 22.98 mmol) in DCM (40 mL) at 0 °C. The mixture was stirred at room temperature for 15 min. The mixture was then cooled to 0 °C, and p-toluenesulfonyl chloride (6.58 g, 34.48 mmol) was added to the reaction mixture and stirred at room temperature for 1 h. After the starting material was exhausted, the mixture was diluted with ethyl acetate (200 mL), washed with ice-cold water (2 × 200 mL) and a brine solution (100 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by combinatorial rapid chromatography by elution with 10% ethyl acetate in hexane to give 4-methylbenzenesulfonic acid 5-(pentoxy)pentyl ester 4 (6 g, 18.29 mmol, 80% yield) as a colorless, oily liquid. LC system: 20% ethyl acetate in hexane, _Rf : 0.50

1-Azide-5-(pentoxy)pentane (5)

_NaN₃ (1.2 g, 18.292 mmol) was added to a stirred solution of 4-methylbenzenesulfonic acid 5-(pentoxy)pentyl ester 4 (4.0 g, 12.195 mmol) in DMF (30 mL) at room temperature, followed by stirring at 60 °C for 16 h. The organic solvent was evaporated as indicated by TLC. Water (2 × 200 mL) was added to the crude residue, and extraction was performed with ethyl acetate (2 × 100 mL). The organic layer was washed with a brine solution (100 mL), _dried over _Na₂SO₄ , and concentrated. The crude compound was purified by washing with pentane (3 × 25 mL) to obtain 1-azido-5-(pentoxy)pentane 5 (2.3 g, 11.55 mmol, 95%) as an oily liquid. TLC system: ethyl acetate 20% in hexane; _Rf : 0.80

5-(pentoxy)pentane-1-amine (side chain -36)

1 M P(Me) ₃ in THF (23 mL, 23.11 mmol) was added to a stirred solution of 1-azido-5-(pentoxy)pentane (5) (2.3 g, 11.55 mmol) in THF: _H₂O (30 mL), and the mixture was stirred at room temperature for 16 hours. After the reaction was complete as indicated by TLC, the organic solvent was evaporated to give an oily liquid of 5-(pentoxy)pentane-1-amine side chain-36 (2.8 g, 16.185 mmol, crude product). LC system: 50% ethyl acetate in hexane, _Rf : 0.10

Side chain -37

N-Methyl-5-(pentoxy)pentane-1-amine (side chain -37)

At room temperature, 1 M methylamine (91 mL, 91.45 mmol) in ethanol was added to a stirred solution of 5-(pentoxy)pentyl 4-methylbenzenesulfonic acid ester 1 (synthesis of compound 1 reported in side chain-36) (6 g, 18.29 mmol) in ethanol (20 mL), and the mixture was stirred at 80 °C for 16 h. The organic solvent was evaporated as indicated by TLC. Water (2 × 200 mL) was added to the crude residue, and the mixture was extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with 1% triethylamine aqueous solution (200 mL) and brine solution (100 _mL ), dried over _Na₂SO₄ , and concentrated to give N-methyl-5-(pentoxy)pent-1-amine (side chain-37) (2.6 g, 13.90 mmol, 76%) as a pale yellow, oily liquid. LC system: 10% methanol in dichloromethane, _Rf : 0.10

Example 2: Compound Preparation

Intermediates 1 and 2

experiment:

5-Fluoro-3-iodopyridin-2-amine (2)

Sodium exiodate (7.5 g, 35 mmol) was added to a stirred solution of 5-fluoropyridine-2-amine (1) (10 g, 89.28 mmol) in ₂ M _H₂SO₄ aqueous solution (150 mL) at 0 °C, and the mixture was heated at 100 °C. Then, sodium iodide (13.5 g, 89.28 mmol) was added dropwise to water (30 mL) at the same temperature for 2 hours. After the starting material was exhausted, the mixture was poured into ice-cold water (300 mL), alkalized with solid _NaHCO₃ (pH approximately 8), and extracted with ethyl acetate (2 × 500 mL). The combined organic layers were washed sequentially with sodium thiosulfate solution (2 × 200 mL), water (200 mL), and brine solution (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by column chromatography using silica at 100–200 g/L and eluted with 15%–20% ethyl acetate in petroleum ether to give 5-fluoro-3-iodopyridin-2-amine (2) (12 g, 50 mmol, 56%) as a brown solid. TLC system: EtOAc in 30% hexane, _Rf : 0.5 LCMS (ESI): m/z 239 [M+H] ⁺

5-Fluoro-3-((trimethylsilyl)ethynyl)pyridine-2-amine (3)

A mixture of 5-fluoro-3-iodopyridin-2-amine (12 g, 50 mmol), trimethylsilylacetylene (10 mL, 75 mmol), and triethylamine (40 mL) in 20 mL of DMF was degassed under argon for 15 minutes. Next, dichlorobis(triphenylphosphine)palladium(II) (350 mg, 0.5 mmol) and CuI (280 mg, 1.5 mmol) were added, and the reaction mixture was heated to 50 °C and stirred for 2 hours. After the starting materials were exhausted, the mixture was filtered through a diatomaceous earth liner, which was thoroughly washed with ethyl acetate. The combined filtrate was washed with water and brine, dried ( _in anhydrous _Na₂SO₄ ), and concentrated. The crude compound was purified by column chromatography using 100–200 °C silica and eluted with 5%–10% ethyl acetate in petroleum ether to obtain 5-fluoro-3-((trimethylsilyl)ethynyl)pyridine-2-amine (3) (8 g, 38 mmol, 76%) as a yellow, thick liquid. TLC system: EtOAc in 20% hexane, _Rf : 0.4 LCMS (ESI): m/z 209 [M+H] ⁺

5-Fluoro-1H-pyrrolo[2,3-b]pyridine (4)

Potassium tert-butoxide (430 mg, 3.8 mmol) was added to a stirred solution of 5-fluoro-3-((trimethylsilyl)ethynyl)pyridine-2-amine (3) (500 mg, 2.4 mmol) in NMP (5 mL) at room temperature. The reaction mixture was stirred at 130 °C for 2 hours. When the reaction was complete as indicated by TLC, the mixture was poured into a saturated aqueous sodium chloride solution (50 mL) and extracted with diethyl ether (3 × 100 mL). The combined organic layers were washed with ice-cold water (2 × 50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain 5-fluoro-1H-pyrrolo[2,3-b]pyridine (4) (200 mg, 1.47 mmol, 61%) as a pale brown solid. TLC system: EtOAc 20% in hexane, _Rf : 0.3 LCMS (ESI): m/z 137 [M+H] ⁺ .

3-Bromo-5-fluoro-1H-pyrrolo[2,3-b]pyridine (5)

NBS (6.4 g, 36 mmol) was added fractionally to a stirred solution of 5-fluoro-1H-pyrrolo[2,3-b]pyridine (4.5 g, 33 mmol) in DMF (50 mL) at 0 °C and allowed to stand for 2 hours. When the reaction was complete as indicated by TLC, the reaction mixture was poured into ice-cold water (100 mL), the precipitated solid was filtered, and dried under vacuum to give 3-bromo-5-fluoro-1H-pyrrolo[2,3-b]pyridine (5) (4.6 g, 21 mmol, 64%) as a light brown solid. TLC system: EtOAc 20% in hexane, _Rf : 0.5 LCMS (ESI): m/z 215 [M+H] ⁺

3-Bromo-5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine (6)

3-Bromo-5-fluoro-1-toluenesulfonyl-1-pyrrolo[2,3-b]pyridine 5 (4.6 g, 21 mmol) in DMF was added to a stirred suspension of NaH (1.3 g, 34 mmol) in DMF (30 mL) at 0 °C. After 1 hour, a solution of p-TsCl (5.7 g, 30 mmol) in DMF (20 mL) was slowly added at the same temperature and stirred for 2 hours. When the reaction was complete as indicated by TLC, the mixture was poured into ice-cold water (200 mL), the precipitated solid was filtered, and dried to give 3-bromo-5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine (6) (6.2 g, 16 mmol, 76%) as a grayish-white solid. TLC system: 10% EtOAc in hexane, _Rf : 0.8 LCMS (ESI): m/z 369 [M+H] ⁺

5-Fluoro-3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine intermediate 1

Bipinnatrol diboron (2 g, 8.1 mmol), potassium acetate (0.8 g, 8.1 mmol), and PdCl₂ (dppf) (0.2 g, 0.27 mmol) were added to an argon-purified solution of 3-bromo-5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine ₆ (1 g, 2.7 mmol) in dioxane (10 mL) at room temperature. The reaction mixture was heated in a sealed tube at 60 °C for 16 hours. After the starting material was exhausted, the mixture was diluted with ice-cold water (50 mL) and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, and concentrated. The crude residue was purified by combined rapid chromatography to give 5-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine (intermediate 1) as a white solid (620 mg, 1.49 mmol, yield 55%). TLC system: EtOAc in 20% hexane, _Rf : 0.3 LCMS (ESI): m/z 417 [M+H] ⁺ .

Potassium trifluoro(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)borate (intermediate 2)

At room temperature, 4.5 M KHF 2 (29 g, 0.389 mol) was added to a stirred solution of 5-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine (intermediate ₁ ) (36 g, 0.086 mol) in MeOH (100 mL), and the mixture was stirred for 6 hours. After the starting material was exhausted, the reaction mixture was concentrated under reduced pressure and co-distilled with MeOH 3 to 4 times. The crude compound was dissolved in acetone (200 mL) and filtered to remove undissolved inorganic solids. The filtrate was concentrated under reduced pressure, and the crude compound was wet-milled with diethyl ether until the less polar spots disappeared on TLC to obtain potassium trifluoro(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)borate (intermediate 2) as a brown solid. TLC system: 10% MeOH in DCM, _Rf : 0.1 LCMS(ESI): m/z 397 [M+H] ⁺ .

Intermediate 12

(1S,3R)-N1-(2-chloro-5-fluoropyrimidin-4-yl)cyclohexane-1,3-diamine (3)

DIPEA (4 mL, 24 mmol) and 2,4-dichloro-5-fluoropyrimidine (2) (3.8 g, 23 mmol) in DMF (10 mL) were added dropwise to a stirred solution of (1R,3S)-cyclohexane-1,3-diamine (1) (5 g, 43 mmol) in DMF (40 mL) at 0 °C. The reaction mixture was stirred at the same temperature for 15 min. After depletion of the starting material according to TLC (compound 2 was not present), the mixture was diluted with ethyl acetate (200 mL) and washed with ice-cold water (3 × 150 mL), brine solution (50 mL), dried over anhydrous sodium sulfate, and concentrated. The crude compound was purified by column chromatography using 100–200 silica gel and eluted with 10% MeOH in DCM and Et ₃ N to give (1S,3R)-N1-(2-chloro-5-fluoropyrimidin-4-yl)cyclohexane-1,3-diamine (3) as a white solid (4 g, 16.3 mmol, yield 71%). TLC system: 10% MeOH in DCM (2 drops Et ₃ N), Rf: 0.1 LCMS (ESI): m/z 245.2 (M+H) ⁺

(1S,3R)-N1-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)cyclohexane-1,3-diamine (intermediate 12)

The mixture of (1S,3R)-N1-(2-chloro-5-fluoropyrimidin-4-yl)cyclohexane-1,3-diamine (3) (3 g, 12.3 mmol), intermediate 2 (7.3 g, 18.4 mmol), and sodium carbonate aqueous solution (2 M, 4 mL) in 1,2-DME (30 mL) was degassed with argon for 15 minutes. Then, tetrakis(triphenylphosphine)palladium (0.7 g, 0.6 mmol) was added, and the mixture was stirred at 100 °C for 16 hours. After the starting material was exhausted, the mixture was concentrated under reduced pressure. The crude compound was purified by column chromatography using 100-200 μL silica and eluted with 10% MeOH in DCM and Et ₃ N to give a brown solid (1S,3R)-N1-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)cyclohexane-1,3-diamine (intermediate 12) (3.5 g, 7.02 mmol, 58%). TLC system: 10% MeOH in DCM (2 drops Et ₃ N), Rf: 0.2 LCMS (ESI): m/z 498.98 (M+H) ⁺

Intermediate 13

(R)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)piperidine-1-carboxylic acid tert-butyl ester

At 0 °C, tert-butyl (R)-3-aminopiperidin-1-carboxylate (1) (1.437 g, 7.18 mmol) was added dropwise to a stirred solution of DMF (10 mL) containing DIPEA (1.56 mL, 8.98 mmol) and 2,4-dichloro-5-fluoropyrimidine (2) (1.0 g, 5.98 mmol). The reaction mixture was stirred at room temperature for 16 hours. After depletion of the starting material according to TLC (compound 2 was not present), the mixture was diluted with ethyl acetate (50 mL) and washed with ice-cold water (3 × 10 mL), a saline solution (10 mL), dried over anhydrous sodium sulfate, and concentrated. The crude compound was purified by column chromatography using 100-200 silica and eluted with 20% EA in petroleum ether to give (R)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)piperidine-1-carboxylic acid tert-butyl ester (3) (1.6 g, 4.84 mmol, yield 81%) as a grayish-white solid. TLC system: 30% EA in petroleum ether, Rf: 0.5 LCMS (ESI): m/z 330.74 (M+H) ⁺

(R)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)piperidine-1-carboxylic acid tert-butyl ester

The mixture of (R)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)piperidin-1-carboxylic acid tert-butyl ester (3) (600 mg, 1.81 mmol), intermediate Int-2 (1.0 g, 2.54 mmol), and sodium carbonate aqueous solution (2 M, 3 mL) in 1,2-DME (12 mL) was degassed with argon for 15 minutes. Next, tetrakis(triphenylphosphine)palladium (104 mg, 0.09 mmol) was added, and the mixture was again degassed with argon for 10 minutes. The reaction mixture was stirred at 90 °C for 16 hours. After the starting material was exhausted, the mixture was concentrated under reduced pressure. The crude compound was purified by column chromatography using 100-200 silica and eluted with 30% ethyl acetate-petroleum ether to give (R)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)piperidin-1-carboxylic acid tert-butyl ester 4 (750 mg, 1.28 mmol, yield 71%) as a grayish-white solid. TLC system: EA 30% in petroleum ether, Rf: 0.5 LCMS (ESI): m/z 585.12 (M+H) ⁺

(R)-5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-(piperidin-3-yl)pyrimidin-4-amine hydrochloride

(R)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)piperidine-1-carboxylic acid tert-butyl ester (4) (300 mg, 0.51 mmol), acetonitrile (4 mL), and HCl (2 mL) in dioxane at 4 N were placed in a sealed tube. The mixture was then heated at 70 °C for 16 hours. After the starting material was exhausted, the mixture was wet-milled with diethyl ether to form a white precipitate, which was filtered and further wet-milled with diethyl ether to give (R)-5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-(piperidine-3-yl)pyrimidin-4-amine hydrochloride (intermediate 13) (147 mg, 0.40 mmol, 78% yield) as a grayish-white solid. TLC system: 10% MeOH-DCMRf: 0.3 LCMS (ESI): m/z 331.37 (M+1) ⁺

Intermediate 14

(S)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)piperidine-1-carboxylic acid tert-butyl ester

tert-butyl (S)-3-aminopiperidin-1-carboxylate (1) (1.437 g, 7.18 mmol) was added dropwise to a stirred solution of DMF (10 mL) containing DIPEA (1.56 mL, 8.98 mmol) and 2,4-dichloro-5-fluoropyrimidine (2) (1.0 g, 5.98 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 16 hours. After depletion of the starting material according to TLC (compound 2 was not present), the mixture was diluted with ether (75 mL) and washed with ice-cold water (3 × 50 mL) and brine (25 mL), dried over sodium sulfate and concentrated under reduced pressure to give (S)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)piperidin-1-carboxylate (3) (1.670 g, 5.06 mmol, 84% yield) as a white solid. TLC system: EA 30% in petroleum ether, Rf: 0.5 LCMS (ESI): m/z 331.44 (M+1) ⁺

(S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)piperidine-1-carboxylic acid tert-butyl ester

The mixture of (S)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)piperidine-1-carboxylate (3) (2.5 g, 7.57 mmol), intermediate 2 (4.49 g, 11.36 mmol), and sodium carbonate aqueous solution (2 M, 10 mL) in 1,2-DME (40 mL) was degassed with argon for 15 minutes. Tetra(triphenylphosphine)palladium (0.437 g, 0.37 mmol) was added and degassed again with argon for 10 minutes. The reaction mixture was heated at 90 °C for 16 hours. After the starting material was exhausted, the mixture was concentrated under reduced pressure. The crude compound was purified by column chromatography using 100-200 silica and eluted with 30% ethyl acetate-petroleum ether to give (S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)piperidin-1-carboxylic acid tert-butyl ester 4 (3.650 g, 6.25 mmol, yield 82%) as a brown solid. TLC system: EtOAc 40% in petroleum ether, Rf: 0.5 LCMS (ESI): m/z 585.48 (M+H) ⁺

(S)-5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-(piperidin-3-yl)pyrimidin-4-amine hydrochloride

(S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)piperidine-1-carboxylic acid tert-butyl ester (4) (3.5 g, 5.99 mmol) in acetonitrile (40 mL) and HCl (20 mL) in dioxane (4 N). The mixture was then heated at 70 °C for 16 hours. After the starting material was exhausted, the mixture was wet-milled with diethyl ether. The precipitated white solid was filtered and further wet-milled with diethyl ether to give (S)-5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-(piperidine-3-yl)pyrimidin-4-amine hydrochloride (intermediate 14) (2.020 g, 5.51 mmol, 92% yield) as a grayish-white solid. TLC system: 10% MeOH-DCMRf: 0.3 LCMS (ESI): m/z 331.02 (M+H) ⁺

Intermediate 18

3-Bromo-5-chloro-1H-pyrrolo[2,3-b]pyridine (2)

NBS (7.02 g, 39.46 mmol) was added fractionally to a stirred solution of 5-chloro-1H-pyrrolo[2,3-b]pyridine 1 (5.0 g, 32.89 mmol) in acetone (50 mL) at 0 °C and stirred for 2 h. The reaction mixture was concentrated under vacuum as indicated by TLC. The crude mixture was diluted with water (50 mL) and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, and concentrated to give 3-bromo-5-chloro-1H-pyrrolo[2,3-b]pyridine 2 (7.0 g, 30.43 mmol, 92%) as a pale brown solid. TLC system: EtOAc 20% in hexane, _Rf : 0.5 LCMS (ESI): m/z 230.8 [M+H] ⁺

3-Bromo-5-chloro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine (3)

3-Bromo-5-chloro-1H-pyrrolo[2,3-b]pyridine 2 (7.0 g, 30.43 mmol) in DMF was added to a stirred suspension of NaH (1.4 g, 58.33 mmol) in DMF (30 mL) at 0 °C. After 1 hour, a solution of p-TsCl (6.3 g, 33.47 mmol) in DMF (20 mL) was slowly added at the same temperature and stirred for 2 hours. After the reaction was complete (as indicated by TLC), the mixture was poured into ice-cold water (200 mL), the precipitated solid was filtered and dried to give 3-bromo-5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine 3 (8.5 g, 22.14 mmol, 73%) as a grayish-white solid. TLC system: 10% EtOAc in hexane, _Rf : 0.8 LCMS (ESI): m/z 386.4 [M+H] ⁺

5-Chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine (4)

Bipinnatrol diboron (3.71 g, 14.58 mmol), potassium acetate (2.14 g, 21.87 mmol), and PdCl₂ (dppf) (678 mg, 0.729 mmol) were added to an argon-purified solution of 3-bromo-5-chloro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine ₃ (2.8 g, 7.29 mmol) in DMF (10 mL) at room temperature. The reaction mixture was heated at 60 °C for 16 hours in a sealed tube. After the starting material was exhausted, the mixture was diluted with ice-cold water (50 mL) and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, and concentrated. The crude residue was purified by combined rapid chromatography to give 5-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine 4 (602 mg, 1.39 mmol, 19% yield) as a white solid. TLC system: EtOAc in 20% hexane, _Rf : 0.3 LCMS (ESI): m/z 432.7 [M+H] ⁺ .

(5-Chloro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)potassium trifluoroborate (intermediate 18)

4.5 MkHF 2 (7.32 g, 93.74 mmol) was added to a stirred solution of 5-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine ₄ (9.0 g, 20.83 mmol) in MeOH (50 mL) for 6 hours. After the starting material was exhausted, the reaction mixture was concentrated under reduced pressure and co-distilled with MeOH 3 to 4 times. The crude compound was dissolved in acetone (100 mL) and the undissolved inorganic solids were filtered off. The filtrate was concentrated under reduced pressure to give the crude compound, which was wet-milled with diethyl ether until the less polar spots disappeared on TLC. The solid was then filtered and dried to obtain potassium trifluoro(5-chloro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)borate intermediate 18 (3.2 g, 7.766 mmol, 37%) as a brown solid. TLC system: 10% MeOH in DCM; _Rf : 0.1 LCMS (ESI): m/z 351 [M-60] ⁺ .

Intermediate 19

(1R,4R)-Bicyclo[2.2.2]oct-5-ene-2-carboxylic acid ethyl ester (2)

Cyclohexyl-1,3-diene 1 (15.0 g, 187.19 mmol), ethyl acrylate (18.74 g, 187.19 mmol), and methylene blue (119 mg, 0.37 mmol) were placed in a sealed tube and the mixture was heated at 140 °C for 16 hours. After depletion of the starting material, fractional distillation (at 70 °C and 0.1 mmHg) yielded (20.0 g, 111.11 mmol, 59% yield) of (1R,4R)-bicyclo[2.2.2]oct-5-en-2-carboxylate 2 as a colorless oil. ¹H NMR was consistent. TLC system: EA 5% in petroleum ether, _Rf : 0.5

(1S,4S)-Bicyclo[2.2.2]octane-2-carboxylic acid ethyl ester (3)

In a Palermo-Smithsonian apparatus, 10% Pd/C (2.0 g, 10% w/w) was added to a solution of (1R,4R)-bicyclo[2.2.2]octane-5-en-2-carboxylate 2 (20.0 g, 111.11 mmol) in EtOH (100 mL). The reaction mixture was stirred at 50 Psi under _H2 gas for 16 hours. After the starting material was exhausted, the mixture was filtered through a diatomaceous earth liner, which was thoroughly washed with ethanol. The combined filtrates were concentrated under reduced pressure to give (1S,4S)-bicyclo[2.2.2]octane-2-carboxylate 3 (18.5 g, 101.64 mmol, 91% yield) as a colorless oil. ¹H NMR was consistent, TLC system: 5% EtOAc in petroleum ether, Rf: 0.5

(1S,4S)-Bicyclo[2.2.2]oct-2-en-2-carboxylic acid ethyl ester (4)

Add 2.5 M n-BuLi solution (in hexane, 19.78 mL, 49.45 mmol) to a stirred solution of freshly distilled DIPA (7.68 mL, 54.94 mmol) in 50 mL of THF at 0 °C and stir for 30 min. Add a solution of (1S,4S)-bicyclo[2.2.2]octane-2-carboxylic acid ethyl ester 3 (5.0 g, 27.47 mmol) in 10 mL of THF at -78 °C and stir for 1 h at the same temperature. Then add a solution of PhSeBr (9.724 g, 41.20 mmol) in THF at -78 °C, and heat to 0 °C and stir for 30 min. Next, _H₂O (35 mL), _H₂O₂ ( ₂₀ mL), and AcOH (7.5 mL) were added sequentially at 0 °C, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate and the two layers were separated. The aqueous layer was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water and brine, dried over _Na₂SO₄ , and concentrated. The crude compound was purified by column chromatography using 100–200 μL silica and eluted with 5%–10% ethyl acetate in petroleum ether to give (1S,4S)-bicyclo[2.2.2]oct- ₂ -en-2-carboxylate 4 (4.02 g, 22.33 mmol, 81% yield) as a yellow liquid. TLC system: 5% EtOAc in petroleum ether, Rf: 0.5 LCMS (ESI): m/z 181.1 (M+H) ⁺

Intermediate 20

2-Chloro-5-fluoro-4-(methylthio)pyrimidine

A 15% aqueous solution of sodium methanethiol (15.36 mL, 32.93 mmol) was added to a stirred solution of 2,4-dichloro-5-fluoropyrimidine 1 (5.0 g, 29.94 mmol) in THF (40 mL) at -30 °C and stirred for 2 h. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 × 150 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 2-chloro-5-fluoro-4-(methylthio)pyrimidine 2 (5.0 g, 28.08 mmol, 93%) as a grayish-white solid. TLC system: 5% EtOAc in petroleum ether, Rf: 0.6 LCMS (ESI): m/z 179.08 (M+1) ⁺

5-Fluoro-3-(5-Fluoro-4-(methylthiomethyl)pyrimidin-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine

The mixture of 2-chloro-5-fluoro-4-(methylthio)pyrimidine 2 (1 g, 5.61 mmol), intermediate 2 (2.66 mg, 6.74 mmol), and sodium carbonate aqueous solution (2 M, 5 mL) in 1,2-DME (25 mL) was degassed with argon for 15 minutes. Next, tetra(triphenylphosphine)palladium (324 mg, 0.28 mmol) was added, and the mixture was stirred at 90 °C for 16 hours. After the starting material was exhausted, the mixture was poured into water (200 mL), and the precipitated brown solid was filtered and dried to give crude 5-fluoro-3-(5-fluoro-4-(methylthio)pyrimidin-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine 3 (2 g) and trace amounts of triphenylphosphine oxide, which were used in the next step without further purification. TLC system: 20% EtOAc in petroleum ether, Rf: 0.6 LCMS (ESI): m/z 433.15 (M+1) ⁺

5-Fluoro-3-(5-Fluoro-4-(methylsulfinyl)pyrimidin-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine and 5-Fluoro-3-(5-Fluoro-4-(methylsulfinyl)pyrimidin-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine

A mixture of 300 mg (0.69 mmol) of 5-fluoro-3-(5-fluoro-4-(methylthio)pyrimidin-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine 3 in dichloromethane (15 mL) was added fractionally over 30 minutes, followed by stirring at room temperature for 4 hours. After depletion of the starting material, the mixture was quenched with saturated ammonium chloride solution (50 mL), extracted with ethyl acetate (2 × 50 mL), washed with brine solution (25 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude compound was purified by column chromatography using 100-200 silica and eluted with 20% ethyl acetate-petroleum ether to give intermediate 20A (90 mg) of 5-fluoro-3-(5-fluoro-4-(methanesulfonyl)pyrimidin-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine as a grayish-white solid, and intermediate 20 (60 mg) of 5-fluoro-3-(5-fluoro-4-(methanesulfinyl)pyrimidin-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridine as a grayish-white solid, eluted with 80% ethyl acetate-petroleum ether. Both compounds were used in the next step without further purification. TLC system for intermediate 20a: 40% EtOAc in petroleum ether, Rf: 0.4 LCMS (ESI): m/z 433.15(M+1) ⁺ ; TLC system for intermediate 20: 40% EtOAc in petroleum ether, Rf: 0.1 LCMS (ESI): m/z 433.15(M+1) ⁺

target compound

2-(2,2,2-trifluoroethoxy)ethylcarbamate tert-butyl ester (3)

To a stirred solution of 2,2,2-trifluoroethanol 1 (3.0 g, 30.00 mmol) in toluene, tert-butyl 2-hydroxyethylcarbamate 2 (0.97 g, 6.00 mmol), 1,1'-(azadicarbonyl)piperidine (3.0 g, 12.00 mmol), and tributylphosphine (3.0 mL, 12.00 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by column chromatography (silica gel 100-200), eluting with 8% ethyl acetate in petroleum ether to give compound 2-(2,2,2-trifluoroethoxy)ethylcarbamate tert-butyl 3 (2.0 g, 8.23 mmol, 27% yield). TLC system: 20% ethyl acetate/petroleum ether; _Rf : 0.6

2-(2,2,2-trifluoroethoxy)ethylamine hydrochloride (4)

4 M HCl (5 mL) in dioxane was added to a stirred solution of 2-(2,2,2-trifluoroethoxy)ethylcarbamate (tert-butyl) (3) (2.0 g, 8.23 mmol) in dioxane at 0 °C, and the mixture was stirred in a sealed tube at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by wet milling with diethyl ether (3 × 25 mL) to give compound 2-(2,2,2-trifluoroethoxy)ethylamine hydrochloride 4 (1.4 g, 6.17 mmol, 95%). TLC system: 100% EtOAc; Rf: 0.1

2-(2,2,2-trifluoroethoxy)ethylcarbamate (2-((1R,3S)-3-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamate)pyridin-4-yl)methyl ester (5)

To a stirred solution of N-((1R,3S)-3-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexyl)-4-(hydroxymethyl)pyridinecarboxamide (intermediate 23) (200 mg, 0.315 mmol) in DMF, 1,1'-carbonyldiimidazole (130 mg, 0.789 mmol) was added and the mixture was stirred at room temperature for 1 hour. To the reaction mixture, 4-(2,2,2-trifluoroethoxy)ethylamine hydrochloride (0.11 g, 0.63 mmol) was added at room temperature and the mixture was stirred at 60 °C for 6 hours. Ice water (20 mL) was added, the mixture was filtered, dried, and the crude compound was purified by column chromatography (silica gel 100-200). The compound was eluted with 50% EtOAc/petroleum ether to give (2-((1R,3S)-3-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl 2-(2,2,2-trifluoroethoxy)ethylcarbamate (5) (180 mg, 0.224 mmol, 72% yield). TLC system: 100% EtOAc; Rf: 0.5 LCMS (ESI): m/z 803.38 (M+H) ⁺

2-(2,2,2-trifluoroethoxy)ethylcarbamate (2-((1R,3S)-3-(5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl ester

Lithium hydroxide (21 mg, 0.89 mmol) was added to Cpd-5 2-(2,2,2-trifluoroethoxy)ethylcarbamate (2-((1R,3S)-3-(5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl ester (180 mg, 0.22 mmol) at 0 °C and stirred at room temperature for 48 hours. The organic solvent was distilled off, and water (5 mL) was added to the crude material. The mixture was then acidified with an aqueous sodium thiosulfate solution and dried under reduced pressure. The filtered solid was then removed. The crude residue was purified by preparative HPLC to give a grayish-white solid (2-((1R,3S)-3-(5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl-2-(2,2,2-trifluoroethoxy)ethylcarbamate (70 mg, 0.108 mmol, yield 48%). TLC system: 100% EtOAc; Rf: 0.4; LCMS (ESI): m/z 649.14 (M+H) ⁺

4-((4-methoxybenzylcarbamoyloxy)methyl)pyridinecarboxylate (3)

At room temperature, methyl 4-(hydroxymethyl)pyridinecarboxylate 1 (150 mg, 0.898 mmol)

1,1'-carbonyldiimidazole (291 mg, 1.796 mmol) was added to a stirred solution in DMF (2 mL) and stirred at room temperature for 2 hours. (4-methoxyphenyl)methylamine (246 mg, 1.796 mmol) was added to the reaction mixture and stirred at 70 °C for 16 hours. After the reaction was complete, ice water (30 mL) was added to the mixture and it was extracted with ethyl acetate (3 × 50 mL). The separated organic layer was washed with ice water (2 × 20 mL) and brine (20 mL), dried over sodium sulfate, and concentrated under reduced pressure to obtain crude methyl 4-((4-methoxybenzylcarbamoyloxy)methyl)pyridinecarboxylate 3 (150 mg, crude). The crude compound was used directly in the next step without further purification. TLC system: 70% ethyl acetate/hexane; _Rf : 0.3; LCMS: m/z = 331.12 (M + H) ⁺

4-((4-methoxybenzylcarbamoyloxy)methyl)pyridinecarboxylic acid (4)

LiOH (32 mg, 1.363 mmol) was added to a stirred solution of methyl 4-((4-methoxybenzylcarbamoyloxy)methyl)pyridinecarboxylic acid 3 (150 mg, 0.454 mmol) in 10 mL of THF: _H₂O (2:1) and stirred at room temperature for 16 hours. The organic solvent was evaporated under reduced pressure, and the residue was acidified with 2N HCl, filtered, and dried to give 4-((4-methoxybenzylcarbamoyloxy)methyl)pyridinecarboxylic acid (4) (90 mg, 0.284 mmol, yield 63%) as a grayish-white solid. TLC system: 5% methanol/dichloromethane; _Rf : 0.1; LCMS: m/z = 317.0 (M+H) ^⁺

4-Methoxybenzylcarbamate (2-((1S,3R)-3-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl ester (5)

At 0 °C, (1R,3S)-N1-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)cyclohexane-1,3-diamine (intermediate 12, enantiomer 2 (Ent-2)) (94 mg, 0.189 mmol), HATU (120 mg, 0.316 mmol), and DIPEA (0.087 mL, 0.474 mmol) were added sequentially to a stirred solution of 4-((4-methoxybenzylcarbamoyloxy)methyl)pyridinecarboxylic acid (4) (50 mg, 0.158 mmol) in DMF (2 mL) and stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was diluted with ice-cold water (20 mL) and the precipitated solid was filtered off. This substance was purified by Grace reversed-phase chromatography to give a grayish-white solid methyl 4-methoxybenzylcarbamate (2-((1S,3R)-3-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl) ester (5) (90 mg, 0.113 mmol, yield 72%). TLC system: 100% ethyl acetate; _Rf : 0.6; LCMS: m/z = 797.2 (M+H) ⁺

4-Methoxybenzylcarbamate (2-((1S,3R)-3-(5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl ester

LiOH (13 mg, 0.565 mmol) was added to a stirred solution of 4-methoxybenzylcarbamate (2-((1S, _3R )-3-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl ester (5) (90 mg, 0.113 mmol) in a mixture of THF:H₂O (3:1) in 8 mL, and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate (3 × 25 mL), water (20 mL), and brine solution ( ₁₅ mL). The solution was dried over _Na₂SO₄ and concentrated to obtain the crude product. The crude substance was purified by Grace reversed-phase chromatography to give benzylcarbamate (2-((1R,3S)-3-(5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-ylamino)cyclohexylcarbamoyl)pyridin-4-yl)methyl ester (28 mg, 0.043 mmol, yield 38%) as a grayish-white solid. TLC system: 70% EtOAc in petroleum ether; Rf: 0.30; LCMS: m/z = 643.0 (M+H) ⁺

target compound

N-((1R,3S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)-4-(hydroxymethyl)pyridinecarboxamide (intermediate 23)

Diisopropylethylamine (0.8 mL, 4.518 mmol) and HATU (1.14 g, 3.012 mmol) were added to a stirred solution of 4-(hydroxymethyl)pyridinecarboxylic acid 1 (461 mg, 3.012 mmol) in DMF (10 mL) at 0 °C. After 20 minutes, (1S,3R)-N1-(5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)cyclohexane-1,3-diamine (intermediate 12, enantiomer 2 (Ent-2)) (750 mg, 1.506 mmol) was added and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was diluted with ice-cold water (100 mL) and the precipitated solid was filtered to obtain a pure compound, N-((1R,3S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)-4-(hydroxymethyl)pyridinecarboxamide (intermediate 23) (950 mg, 75% LCMS purity), in the form of a brown solid. TLC system: 10% MeOH in dichloromethane; Rf: 0.6; LCMS (ESI): m/z 634.31 (M+H) ⁺

(((2-(((1R,3S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)carbamoyl)pyridin-4-yl)methoxy)carbonyl)-D-valine methyl ester(3)

Add 1,1'-carbonyldiimidazole (64 mg 0.394 mmol) to a stirred solution of N-((1R,3S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)-3-(hydroxymethyl)benzamide (intermediate 23) (100 mg, 0.157 mmol) in DMF and stir for 2 hours at room temperature. Add ice water (30 mL) to the reaction mixture, filter and dry. Dissolve the crude material in DMF, add D-valine methyl ester hydrochloride 2 (53 mg, 0.3159 mmol) and stir at 70 °C for 3 hours. After the reaction is complete, dilute the mixture with ice water (30 mL), filter and dry. The crude compound was purified by column chromatography, eluting with 50% ethyl acetate/petroleum ether to give compound (((2-(((1R,3S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)carbamoyl)pyridin-4-yl)methoxy)carbonyl)-D-valine methyl ester 3 (65 mg, 0.082 mmol, yield 52%). TLC system: 70% ethyl acetate/petroleum ether; Rf: 0.6; LCMS (ESI): m/z 791.38 (M+H) ⁺

(((2-(((1R,3S)-3-((5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)carbamoyl)pyridin-4-yl)methoxy)carbonyl)-D-valine)

Potassium hydroxide (39 mg, 0.708 mmol) was added to a stirred solution of (((2-(((1R,3S)-3-((5-fluoro-2-(5-fluoro-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)carbamoyl)pyridin-4-yl)methoxy)carbonyl)-D-valine methyl ester 3 (140 mg, 0.1772 mmol) in 1,4-dioxane (1.5 mL) and water (0.5 mL), followed by stirring at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure to give a crude compound, which was diluted with water (5 mL) and acidified with citric acid solution to approximately pH 6. The precipitated solid was then filtered and dried under reduced pressure. The crude residue was purified by preparative HPLC to give a grayish-white solid (((2-(((1R,3S)-3-((5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexyl)carbamoyl)pyridin-4-yl)methoxy)carbonyl)-D-valine (17 mg, 0.0273 mmol, yield 15%). TLC system: 10% methanol in dichloromethane/acetic acid; Rf: 0.1; LCMS (ESI): m/z 623.40 (M+H) ⁺

Example 3: Data about the selected compound

In vitro antiviral analysis

Influenza antiviral analysis:

The cytopathic effect (CPE) and inhibition of cell viability induced by influenza A (virus strain A/PR/8/34, ATCC VR-95) or influenza B (cell culture-adapted virus strain B/Lee/40, ATCC VR-1535) replication in MDCK cells (female Cocker Spaniel kidney epithelial cells, ATCC CCL-34) were measured by XTT dye reduction (Appleyard et al., *J Antimicrob Chemother* 1 (Supplement 4): 49-53, 1975 and Shigeta et al., *Antimicrob Agents Chemother* 41(7): 1423-1427, 1997). MDCK cells (1 × 10⁴ cells per well) were grown into a monolayer in 96-well flat-bottom tissue culture plates using Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 ^mM sodium pyruvate, and 0.1 mM NEAA. On the day of assay setup, the cell monolayers were washed three times with DPBS. Virus was obtained from ATCC and grown in MDCK cells to generate the viral stock solution pool. The test compounds were serially diluted twice to the desired starting concentration in the assay medium (DMEM, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 ng/mL TPCK-treated trypsin, 0.1 mM NEAA, and 1 mM sodium pyruvate). Test compounds were added in triplicate at 100 μL/well for efficacy determination, in duplicate for cytotoxicity determination, and at a single per-well concentration for colorimetric evaluation before the addition of diluted virus. Ribavirin and oseltamivir carboxylate were used as control compounds for simultaneous evaluation. Pre-titrated aliquots of virus were removed from the freezer (-80°C) and rapidly thawed in a biosafety cabinet. The virus was diluted with analytical medium to a volume of 100 μL added to each well to achieve an 85% to 95% cell kill rate determined at 4 days post-infection. Cell controls containing only culture medium, virus-infected controls containing both culture medium and virus, and cytotoxicity _controls containing culture medium were all incubated at 37°C and 5% CO2 for four days. After incubation at 37°C for 4 hours, inhibition of CPE (increased cell viability) was measured by reduction with dye XTT at 450 nm (reference wavelength 650 nm) using Softmax Pro 4.6 software. The percentage reduction in CPE in virus-infected wells and the percentage of cell viability in uninfected drug-controlled wells were calculated using four-parameter curve fitting analysis with Microsoft Excel XLfit4.

(2) PB2 Binding Assay was designed as a homogeneous, high-throughput binding/displacement assay for determining the IC50 of PB2 inhibitors. The principle of the assay reverts to the competitive displacement of PB2 by a fluorescent probe (m7GTP analog) derived from its complex via the PB2 inhibitor. In simple terms, purified PB2 is incubated with a fluorescent m7GTP analog, and the test inhibitor is added to the reaction mixture. The displacement of the fluorescent m7GTP is read using a Perkin Elmer Envision. Performance is reported in Table B below: +++++: IC50, <5 μM; +++: IC50, 5–25 μM; +++: IC50, 25–50 μM; +: IC50, 50–100 μM; and +: IC50, >100 μM.

Table B

PB2 binding analyses of several influenza A virus strains were also compared. IC50 values are reported in Table C below: +++++: EC50, <5 μM; ++++: EC50, 5-25 μM; +++: EC50, 25-50 μM; ++: EC50, 50-100 μM; and +: EC50, >100 μM; -: not tested.

Table C

Claims (14)

1. A compound having the following structure: (i) in: Het/Ar can be pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, pyrroleyl, pyrazolyl, triazolyl, furanyl, pyranyl, thiopheneyl, benzimidazolyl, benzothiopheneyl, benzofuranylbenzotriazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, purinyl, phenyl, or naphthyl. L is C1 - C14 alkyl-OC(O)NH- C1 - C14 alkyl- Rz ; R z  is H, C<sub> 1 -C 6  alkyl, or C <sub>1- C 6  alkyl-CO 2 R5 , and the alkyl group is optionally substituted with one, two, or three substituents selected from: hydroxyl, halogen, C<sub> 1 -C 6  alkoxy, C<sub> 1 -C 6  haloalkoxy, -NH 2 , -NH (C<sub> 1 -C 4  alkyl), -N (C<sub> 1 -C 4 alkyl) 2 , -OCO (C<sub> 1 -C 4  alkyl), -CO-C <sub>1 -C 4  alkyl, -CO2 H, and -CO<sub>2C 1 -C 4 alkyl; and Each R 5 is independently H, C1 - C6 alkyl, C3 - C8 cycloalkyl, C2 - C8 alkoxy, C2 - C8 alkoxy- C6 - C10 aryl, CO2H , CO2- C1 -C6 alkyl, CONH2 , CONH- C1 - C6 alkyl, CON( C1 - C6 alkyl) 2 , C1 - C6 alkyl - CO2H , C1-C6 alkyl-CO2- C1 - C6 alkyl, C1 - C6 alkyl- CONH2 , C1 - C6 alkyl - CONH( C1 - C6 alkyl), C1 - C6 alkyl-CON( C1 - C6 alkyl) 2 , C1 - C6 alkyl- C6 - C10 aryl, 5 to 10 heteroaryl, or C1 -C 6- alkyl-5 to 10-membered heteroaryl, wherein the heteroaryl group comprises 1 to 3 cyclic heteroatoms selected from O, N, and S; or (ii) in R" is -H or C1 - C6 alkyl, optionally substituted by one, two, or three substituents independently selected from the group consisting of: halogen, cyano, hydroxy, oxo, amino, carboxyl, C1 - C6 alkoxy, C1 - C6 haloalkoxy, C1 - C6 aminoalkoxy, C1 - C6 cyanoalkoxy, C1 - C6 hydroxyalkoxy, and C2 - C6 alkoxyalkoxy. Or its pharmaceutically acceptable salt. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Het/Ar is pyridyl or phenyl. 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Het/Ar is pyridyl. 4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein L is C1 - C14 alkyl-OC(O)NH- C1 - C14 alkyl- Rz . 5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein L is C1 - C6 alkyl-OC(O)NH- C1 - C6 alkyl- Rz . 6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R" is a C1 - C6 alkyl group, optionally substituted by one, two or three substituents independently selected from the group consisting of: halogen, hydroxyl, amino, carboxyl, C1 - C6 alkoxy, C1 - C6 haloalkoxy, C1 - C6 aminoalkoxy, C1 - C6 hydroxyalkoxy and C2 - C6 alkoxyalkoxy. 7. A compound or a pharmaceutically acceptable salt thereof listed in Table A: Table A 8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant or mediator. 9. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 for the preparation of an agent for the treatment or prevention of influenza infection in humans. 10. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 for the preparation of an agent for reducing the amount of influenza virus in humans. 11. The use according to claim 9 or 10, wherein the pharmaceutical agent further comprises one or more additional active agents selected from the group consisting of neuraminidase inhibitors, ion channel inhibitors, and polymerase inhibitors. 12. The use according to any one of claims 9 to 11, wherein the pharmaceutical agent is in the form of a liposome formulation. 13. The use according to any one of claims 9 to 12, wherein the agent is prepared for (i) intravenous administration or (ii) intrapulmonary or nasal administration. 14. A composition comprising the compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of one or more additional active agents selected from the group consisting of neuraminidase inhibitors, ion channel inhibitors, and polymerase inhibitors, wherein the combined amount is therapeutically effective against influenza infection in the individual being treated.