RU2805211C2 - C40-, c28- and c-32-linked rapamycin analogues as mtor inhibitors - Google Patents

Abstract

FIELD: chemistry; pharmaceuticals.

SUBSTANCE: compounds - analogs of rapamycin, which have the properties of mTOR inhibitors. The compounds selected from the group specified in cl. 1-7 of claim, their pharmaceutically acceptable salts, tautomers, stereoisomers, oxepane isomers and a pharmaceutical composition based on them, inhibiting mTOR. Methods for treating and preventing diseases mediated by mTOR, including cancer, age-related conditions, immune-mediated diseases, as well as the use of such compounds in the manufacture of a drug for the treatment, prevention or reduction of the risk of a disease or condition mediated by mTOR, as well as for the treatment of cancer, age-related condition and immune-mediated disease.

EFFECT: providing mTOR inhibitory compounds that are effective in the treatment and prevention of mTOR-mediated diseases, including cancer, age-related conditions and immune-mediated diseases.

28 cl, 30 tbl, 152 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/665,435, filed May 1, 2018, and U.S. Provisional Patent Application No. 62/752,874, filed Oct. 30, 2018, and U.S. Provisional Patent Application No. 62/836,036, filed April 18. 2019, the contents of which are incorporated herein by reference in their entirety.

LINK TO LIST OF SEQUENCES

The sequence listing associated with this application is presented in text format instead of a paper copy, and is therefore included in the specification by reference. The name of the text file containing the list of sequences: REME_008_01WO_SeqList_ST25.txt. The text file is approximately 40 kilobytes, created on April 26, 2019, and is sent electronically via EFS-Web.

TECHNICAL FIELD

This disclosure relates to mTOR inhibitors. Specifically, embodiments relate to compounds and compositions that inhibit mTOR, methods of treating diseases mediated by mTOR, and methods of synthesizing these compounds.

BACKGROUND OF THE ART

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase related to lipid kinases of the phosphoinositide 3-kinase (PI3K) family. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially regulated, have different substrate specificities, and are differentially sensitive to rapamycin. mTORC1 integrates signals from growth factor receptors with cellular nutritional status and controls the level of cap-dependent mRNA translation by modulating the activity of key translation components such as cap-binding protein and the eIF4E oncogene.

mTOR signaling is being deciphered in more and more detail. The different pharmacology of mTOR inhibitors has been particularly informative. The first mTOR inhibitor described, rapamycin, is now considered an incomplete inhibitor of mTORC1. Rapamycin is a selective inhibitor of mTORC1 through binding to the FK506 rapamycin binding domain (FRB) of mTOR kinase via FK506 binding protein 12 (FKBP12). The FRB domain of mTOR is accessible in the mTORC1 complex but is less accessible in the mTORC2 complex. Interestingly, the potency of inhibitory activity against downstream mTORC1 substrates by rapamycin treatment is known to vary among mTORC1 substrates. For example, rapamycin potently inhibits phosphorylation of the mTORC1 substrate S6K and, indirectly, phosphorylation of the downstream ribosomal protein S6, which controls ribosomal biogenesis. On the other hand, rapamycin exhibits only partial inhibitory activity on phosphorylation of 4E-BP1, a master regulator of eIF4E that controls the initiation of cap-dependent translation. As a result, more comprehensive inhibitors of mTORC1 signaling are of interest.

A second class of mTOR kinase “ATP site” inhibitors has been described. This class of mTOR inhibitors will be called TORi (TOR ATP site inhibitor). The molecules compete with ATP, the substrate of the kinase reaction, at the active site of mTOR kinase (and are therefore also inhibitors of the mTOR active site). As a result, these molecules inhibit subsequent phosphorylation of a wider range of substrates.

Although inhibition of mTOR may have the effect of blocking 4E-BP1 phosphorylation, these agents may also inhibit mTORC2, resulting in blocking Akt activation due to inhibition of Akt S473 phosphorylation.

Disclosed herein are, among other things , mTOR inhibitors. In some embodiments, the compounds disclosed herein are more selective inhibitors of mTORC1 compared to mTORC2. In some embodiments, the compounds disclosed herein are more selective inhibitors of mTORC2 compared to mTORC1. In some embodiments, the compounds disclosed herein do not exhibit a difference in selectivity between mTORC1 and mTORC2.

ESSENCE OF DISCLOSURE

This disclosure relates to compounds capable of inhibiting mTOR activity. This disclosure further provides a method for preparing the compounds of this disclosure, pharmaceutical preparations containing such compounds, and methods of using such compounds and compositions in the management of mTOR-mediated diseases or disorders.

This disclosure provides a compound of formula Ic:

(Ic)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O, -OR ³ , -N ₃ or -OC(=Z ¹ )-R ^32a ;

R ²⁸ is -H, (C ₁ -C ₆ )alkyl or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where if R ²⁸ and R ⁴⁰ are H, then R ³² is not =O;

each Z ¹ is independently O or S;

R ^28a , R ^32a , and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -L ² -A ¹ -L ¹ -A ² -L ³ -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )- or L ² ; and where the bond on the right side of the A ² group, as depicted, is bonded to B or L ³ ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

each L ¹ is independently selected from

L ² and L ³ are independently absent or independently selected from

each B is independently selected from

each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-(C(R ³ ) ₂ ) _n -, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , , heteroarylene-, heterocyclylene arylene-, , NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-(C(R ³ ) ₂ ) _n -, where the connection on the left side of B ¹ , as shown, is connected to A ² , L ³ or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are each independently optional substituted -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O )NR ³ -heteroaryl or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the alkyl of (C ₁ -C ₆ )alkyl is each independently optionally substituted with -N( R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the (C ₁ -C ₆ )alkyl alkyl is each independently optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the (C ₁ -C ₆ )alkyl alkyl is each independently optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the (C ₁ -C ₆ )alkyl alkyl is each independently optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each Y is independently -C(R ³ ) ₂ or a bond;

each n is independently an integer from one to 12;

each o is independently an integer from zero to 30;

each p is independently an integer between zero and 12;

each q is independently an integer from zero to 30; And

each r is independently an integer from one to 6.

This disclosure provides a compound of formula Ia:

(Ia)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O, -OR ³ , -N ₃ or -OC(=Z ¹ )-R ^32a ;

R ²⁸ is -H, (C ₁ -C ₆ )alkyl or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where if R ²⁸ and R ⁴⁰ are H, then R ³² is not =O;

each Z ¹ is independently O or S;

R ^28a , R ^32a and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -L ² -A ¹ -L ¹ -A ² -L ³ -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

, , , , , , , , , , , , , , , , , , And ;

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )- or L ² ; and where the bond on the right side of the A ² group, as depicted, is bonded to B or L ³ ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

each L ¹ is independently selected from

,

L ² and L ³ are independently absent or independently selected from

each B is independently selected from

each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , heteroarylene-, heterocyclylene arylene- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where the connection on the left side of B ¹ , as shown, is connected to A ² , L ³ or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are each independently optional substituted -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O )NR ³ -heteroaryl or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the alkyl of (C ₁ -C ₆ )alkyl is each independently optionally substituted with -N( R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the alkyl of (C ₁ -C ₆ )alkyl is each independently optionally substituted with -N( R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the alkyl of (C ₁ -C ₆ )alkyl is each independently optionally substituted -N( R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , wherein the alkyl of (C ₁ -C ₆ )alkyl is each independently optionally substituted with -N( R ³ ) ₂ or -OR ³ ;

each Y is independently -C(R ³ ) ₂ or a bond;

each n is independently an integer from one to 12;

each o is independently an integer from zero to 30;

each p is independently an integer between zero and 12;

each q is independently an integer from zero to 30; And

each r is independently an integer from one to 6.

This disclosure provides a compound of formula I:

(I)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O or -OR ³ ;

R ²⁸ is -H or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where at least one of R ²⁸ and R ⁴⁰ is not H;

Z ¹ is independently O or S;

R ^28a and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; --L ² -A ¹ -L ¹ -A ² -L ³ -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

, , , , , , , , , , , , , , , , , , ;

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )- or L ² ; and where the bond on the right side of the A ² group, as depicted, is bonded to B or L ³ ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

each L ¹ is independently selected from

L ² and L ³ are independently absent or independently selected from

,

each B is independently selected from

each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , heteroarylene-, heterocyclylene arylene- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where the connection on the left side of B ¹ , as shown, is connected to A ² , L ³ or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are each independently optional substituted -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O )NR ³ -heteroaryl or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each Y is independently -C(R ³ ) ₂ or a bond;

each n is independently an integer from one to 12;

each o is independently an integer from zero to 30;

each p is independently an integer between zero and 12;

each q is independently an integer from zero to 30; And

each r is independently an integer from one to 6.

This disclosure provides a compound of formula II:

(II)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O or -OR ³ ;

R ²⁸ is -H or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where at least one of R ²⁸ and R ⁴⁰ is not H;

Z ¹ is independently O or S;

R ^28a and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

, , , , , , , , , , , , , , , , , , ;

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )-; and where the bond on the right side of the group A ² , as depicted, is bonded to B;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

each L ¹ is independently selected from

each B is independently selected from

,

each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , heteroarylene-, heterocyclylene arylene- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where the bond on the left side of B ¹ , as shown, is connected to A ² or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are each independently optional substituted -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O )NR ³ -heteroaryl or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each Y is independently a C(R ³ ) ₂ or bond;

each n is independently an integer from one to 12;

each o is independently an integer from zero to 30;

each p is independently an integer between zero and 12;

each q is independently an integer from zero to 30; And

each r is independently an integer from one to 6.

In some embodiments, a compound of Formula I or II is represented by the structure of Formula I-28:

(I-28)

or a pharmaceutically acceptable salt or tautomer thereof.

In some embodiments, a compound of formula Ia, Ic, I, or II is represented by the structure of formula I-28b:

(I-28b)

or a pharmaceutically acceptable salt or tautomer thereof.

In some embodiments, the compound of Formula I or II is represented by the structure of Formula I-40:

(I-40)

or a pharmaceutically acceptable salt or tautomer thereof.

In some embodiments, a compound of formula Ia, Ic, I, or II is represented by the structure of formula I-40b:

(I-40b)

or a pharmaceutically acceptable salt or tautomer thereof.

In some embodiments, a compound of formula Ia, Ic, I, or II is represented by the structure of formula I-32b:

(I-32b)

or a pharmaceutically acceptable salt or tautomer thereof.

Provided herein is a method of treating a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or susceptible to developing a disease or disorder mediated by mTOR a therapeutically effective amount of one or more disclosed compounds. Provided herein is a method of preventing a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or susceptible to developing a disease or disorder mediated by mTOR a therapeutically effective amount of one or more of the disclosed compounds. Provided herein is a method of reducing the risk of an mTOR-mediated disease or disorder, comprising administering to a subject suffering from or susceptible to developing an mTOR-mediated disease or disorder a therapeutically effective amount of one or more disclosed compounds.

Another aspect of this disclosure relates to a pharmaceutical composition containing a compound of formula I, Ia, Ib, Ic, II or IIb, or a pharmaceutically acceptable salt or tautomer of any of the foregoing, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further include an excipient, diluent or surfactant. The pharmaceutical composition may be effective for treating, preventing, or reducing the risk of an mTOR-mediated disease or disorder in a subject in need thereof.

Another aspect of this disclosure relates to a compound of Formula I, Ia, Ib, Ic, II or IIb or a pharmaceutically acceptable salt or tautomer of any of the foregoing for use in the treatment, prevention or reduction of the risk of an mTOR-mediated disease or disorder in a subject in need thereof .

Another aspect of this disclosure relates to the use of a compound of formula I, Ia, Ib, Ic, II or IIb, or a pharmaceutically acceptable salt or tautomer of any of the foregoing, in the manufacture of a medicament for the treatment, prevention or reduction of the risk of an mTOR-mediated disease or disorder in a subject needing it.

The present disclosure also provides compounds that can be used to inhibit mTOR.

DETAILED DISCLOSURE

The present disclosure relates to mTOR inhibitors. In particular, embodiments relate to compounds and compositions that inhibit mTOR, methods of treating diseases mediated by mTOR, and methods of synthesizing these compounds.

Details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, exemplary methods and materials are described below. Other features, objects and advantages of the disclosure will be apparent from the description and claims. In the specification and appended claims, the singular number may also include the plural unless the context clearly dictates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this disclosure pertains. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

Terms

The articles "a" and "an" are used in this disclosure and refer to one or more than one (i.e., at least one) grammatical object of the description, unless otherwise indicated. By way of example, "element" may mean one element or more than one element unless otherwise noted.

The term “or” means “and/or” unless otherwise noted. The term “and/or” means either “and” or “or” or both, unless otherwise noted.

The term "optionally substituted", unless otherwise indicated, means that a group may be unsubstituted or substituted with one or more (for example, 0, 1, 2, 3, 4 or 5 or more or any range derived therefrom) substituents, listed for this group, in which the specified substituents may be the same or different. In one embodiment, the optionally substituted group has 1 substituent. In another embodiment, the optionally substituted group has 2 substituents. In another embodiment, the optionally substituted group has 3 substituents. In another embodiment, the optionally substituted group has 4 substituents. In another embodiment, the optionally substituted group has 5 substituents.

The term "alkyl" by itself or as part of another substituent means, unless otherwise specified, a straight (i.e. straight) or branched non-cyclic carbon chain (or carbon) or a combination thereof, which may be fully saturated, mono- or polyunsaturated and may include di- and multivalent radicals having the specified number of carbon atoms (ie C ₁ -C ₁₀ means from one to ten carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologues and isomers, e.g. n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is a group having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers.

The term "alkylene" by itself or as part of another substituent means, unless otherwise specified, a divalent radical derived from alkyl. Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, such as groups having 10 or fewer carbon atoms.

The term "alkenyl" means an aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched, containing from 2 to 6 carbon atoms in the chain. Some alkenyl groups contain 2 to 4 carbon atoms in the chain. Branched can mean that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkenyl chain. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl and isobutenyl. A C ₂ -C ₆ alkenyl group is an alkenyl group containing from 2 to 6 carbon atoms.

The term "alkenylene" by itself or as part of another substituent means, unless otherwise specified, a divalent radical derived from an alkene.

The term "alkynyl" means an aliphatic hydrocarbon group containing a carbon-carbon triple bond, which may be straight or branched, containing from 2 to 6 carbon atoms in the chain. Some alkynyl groups contain 2 to 4 carbon atoms in the chain. Branched can mean that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkynyl chain. Examples of alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl and n-pentynyl. A C ₂ -C ₆ alkynyl group is an alkynyl group containing from 2 to 6 carbon atoms.

The term "alkynylene" by itself or as part of another substituent means, unless otherwise specified, a divalent radical derived from an alkyne.

The term "cycloalkyl" means a monocyclic or polycyclic saturated or partially unsaturated carbon ring containing 3-18 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl. A C ₃ -C ₈ cycloalkyl is a group containing from 3 to 8 carbon atoms. The cycloalkyl group may be fused (eg, decalin) or bridged (eg, norbornane).

"Cycloalkylene" by itself or as part of another substituent means a divalent radical derived from cycloalkyl.

The terms "heterocyclyl" or "heterocycloalkyl" or "heterocycle" refer to a monocyclic or polycyclic 3-24 membered ring containing carbon and at least one heteroatom selected from oxygen, phosphorus, nitrogen and sulfur, and where there are no delocalized π -electrons (aromaticity) shared by the ring carbon or heteroatom(s). Heterocyclyl rings include, but are not limited to, oxetanyl, azetadinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thio morpholinyl S-oxide, thiomorpholinyl S-dioxide , piperazinil, azepinil, oxepinil, diazepinil, tropanil and homotropanil. The heterocyclyl or heterocycloalkyl ring may also be fused or bridged, for example a bicyclic ring.

"Heterocyclylene" or "heterocycloalkylene" by itself or as part of another substituent means a divalent radical derived from "heterocyclyl", "heterocycloalkyl" or "heterocycle".

The term "aryl" means, unless otherwise specified, a polyunsaturated, aromatic hydrocarbon substituent that may be a single ring or multiple rings (preferably 1 to 3 rings) that are fused together (ie, a fused aryl ring) or linked covalently. A fused aryl ring can refer to multiple rings fused together, where at least one of the fused rings is an aryl ring.

"Arylene" by itself or as part of another substituent means a divalent radical derived from aryl.

The term "heteroaryl" refers to an aryl group (or rings) that contains at least one heteroatom, such as N, O or S, wherein the nitrogen and sulfur atom(s) are optionally oxidized and the nitrogen atom(s) are optionally quaternized . Thus, the term “heteroaryl” includes fused ring heteroaryl groups (ie, multiple rings fused together, where at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, where one ring has 5 members and the other ring has 6 members, and where at least one ring is a heteroaryl ring. Also, a 6,6-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other ring has 6 members, and where at least one ring is a heteroaryl ring. And 6,5-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other ring has 5 members, and where at least one ring is a heteroaryl ring. The heteroaryl group can be attached to the rest of the molecule via a carbon or a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl , 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2 -thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2 -quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of suitable substituents described herein.

The term “heteroaryl” may also include multiple fused ring systems that have at least one such aromatic ring, and these multiple fused ring systems are further described below. The term may also include multiple fused ring systems (e.g., ring systems containing 2, 3, or 4 rings), wherein a heteroaryl group as defined above may be fused to one or more rings selected from heteroaryls (to form, for example, naphthyridinyl , such as 1,8-naphthyridinyl), heterocycles (to form, for example, 1,2,3,4-tetrahydronaphthyridinyl, such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocycles (to form , for example, 5,6,7,8-tetrahydroquinolyl) and aryls (to form, for example, indazolyl) to form multiple fused ring systems. The rings of multiple fused ring systems can be connected to each other via fused, spiro- and bridging bonds if permitted by valence requirements. It should be understood that the individual rings of multiple fused ring systems may be connected in any order relative to each other. It should also be understood that the point of attachment of the multiple fused ring system (as defined above for heteroaryl) can be at any position of the multiple fused ring system, including the heteroaryl, heterocyclic, aryl or carbocyclic moiety of the multiple fused ring system, and on any suitable atom of the multiple fused ring system systems, including a carbon atom and a heteroatom (for example, nitrogen).

"Heteroarylene" by itself or as part of another substituent means a divalent radical derived from heteroaryl.

Non-limiting examples of aryl and heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl, benzoisoxazolyl, imidazopyri dinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzo thiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl or quinolyl. The above examples may be substituted or unsubstituted, and the divalent radicals of each example of heteroaryl given above are non-limiting examples of heteroarylene. A heteroaryl group may include a single ring heteroatom (eg, O, N, or S). A heteroaryl group may include two optionally different ring heteroatoms (eg, O, N, or S). A heteroaryl group may include three optionally different ring heteroatoms (eg, O, N, or S). A heteroaryl group may include four optionally different ring heteroatoms (eg, O, N, or S). A heteroaryl group may include five optionally different ring heteroatoms (eg, O, N, or S). The aryl group may have one ring. The aryl group may have two, optionally different rings. The aryl group may have three, optionally different rings. The aryl group may have four optionally different rings. A heteroaryl group may have one ring. The heteroaryl group may have two, optionally different rings. The heteroaryl group may have three, optionally different rings. The heteroaryl group may have four optionally different rings. The heteroaryl group may have five optionally different rings.

The terms "halo" or "halogen" by themselves or as part of another substituent mean, unless otherwise specified, a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" may include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C ₁ -C ₄ )alkyl" may include, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, 1-fluoro-2- bromoethyl and the like.

As used herein, the term “hydroxyl” means -OH.

As used herein, the term “hydroxyalkyl” means an alkyl group, as defined herein, substituted by one or more, such as one, two or three, hydroxy groups. In some cases, the same carbon atom does not carry more than one hydroxy group. Typical examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl , 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl and 2-(hydroxymethyl)-3-hydroxypropyl.

As used herein, the term "oxo" means oxygen that is double bonded to a carbon atom.

The replacement group used herein may be a group selected from the following groups:

(A) oxo, halogen, -CF ₃ , -CN, -OH, -OCH ₃ , -NH ₂ , -COOH, -CONH ₂ , -NO ₂ , -SH, -SO ₃ H, -SO ₄ H, - SO ₂ NH ₂ , -NHNH ₂ , -ONH ₂ , -NHC=(O)NHNH ₂ , -NHC=(O)NH ₂ , -NHSO ₂ H, -NHC=(O)H, -NHC(O)- OH, -NHOH, -OCF ₃ , -OCHF ₂ , -OCH ₂ F, unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, substituted with at least one substituent selected from:

(i) oxo, halogen, -CF ₃ , -CN, -OH, -OCH ₃ , -NH ₂ , -COOH, -CONH ₂ , -NO ₂ , -SH, -SO ₃ H, -SO ₄ H, - SO ₂ NH ₂ , -NHNH ₂ , -ONH ₂ , -NHC=(O)NHNH ₂ , -NHC=(O)NH ₂ , -NHSO ₂ H, -NHC=(O)H, -NHC(O)- OH, -NHOH, -OCF ₃ , -OCHF ₂ , -OCH ₂ F, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from:

(a) oxo, halogen, -CF ₃ , -CN, -OH, -OCH ₃ , -NH ₂ , -COOH, -CONH ₂ , -NO ₂ , -SH, -SO ₃ H, -SO ₄ H, - SO ₂ NH ₂ , -NHNH ₂ , -ONH ₂ , -NHC=(O)NHNH ₂ , -NHC=(O)NH ₂ , -NHSO ₂ H, -NHC=(O)H, -NHC(O)- OH, -NHOH, -OCF ₃ , -OCHF ₂ , -OCH ₂ F, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl and

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: oxo, halogen, -CF ₃ , -CN, -OH, -OCH ₃ , -NH ₂ , - COOH, -CONH ₂ , -NO ₂ , -SH, -SO ₃ H, -SO ₄ H, -SO ₂ NH ₂ , -NHNH ₂ , -ONH ₂ , -NHC=(O)NHNH ₂ , -NHC=( O)NH ₂ , -NHSO ₂ H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF ₃ , -OCHF ₂ , -OCH ₂ F, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl , unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

An "effective amount" when used in combination with a compound is an amount effective to treat or prevent a disease in a subject as described herein.

The term "carrier" as used in this disclosure includes carriers, excipients and diluents and may mean a material, composition or carrier, such as a liquid or solid excipient, diluent, excipient, solvent or encapsulating material involved in carrying or transporting a pharmaceutical agent from one organ or body part into another organ or body part of the subject.

The term "treatment" in relation to a subject refers to the reduction of at least one of the symptoms of the subject's disorder. Treatment may include cure, improvement, or at least partial relief of the condition.

The term “prevent” or “prevention” in relation to a subject refers to preventing a subject from becoming affected by a disease or disorder. Prevention may include prophylactic treatment. For example, prophylaxis may involve administering to a subject a compound described herein before the subject is affected by a disease, and administration will prevent the subject from becoming affected by the disease.

The term “disorder” is used in this disclosure to mean and is used interchangeably with the terms disease, condition, or illness unless otherwise noted.

The term “administer”, “administered” or “administration” as used in this disclosure refers to either the direct administration of a disclosed compound or a pharmaceutically acceptable salt or tautomer of a disclosed compound or composition to a subject, or the administration of a derivative of a prodrug or analogue of a compound or a pharmaceutically acceptable salt or a tautomer of a compound or composition to a subject that can form an equivalent amount of the active compound in the subject's body.

A "patient" or "subject" is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon, or rhesus.

Connections

This disclosure provides a compound having the structure of formula Ic

(Ic)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² , R ²⁸ and R ⁴⁰ are as described above.

This disclosure provides a compound having the structure of formula Ia

(Ia)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² , R ²⁸ and R ⁴⁰ are as described above.

This disclosure provides a compound having the structure of formula I

(I)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² , R ²⁸ and R ⁴⁰ are as described above.

This disclosure provides a compound having the structure of formula Ib

(Ib)

or a pharmaceutically acceptable salt or tautomer thereof, wherein ^R32 , ^R28 and ^R40 are as described above for Formula I.

This disclosure provides a compound having the structure of formula II

(II)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² , R ²⁸ and R ⁴⁰ are as described above.

This disclosure provides a compound having the structure of formula IIb

(IIb)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² , R ²⁸ and R ⁴⁰ are as described above for formula II.

In certain embodiments, the compound has the following formula:

or a pharmaceutically acceptable salt or tautomer thereof.

In certain embodiments, R ³² is =O. In certain embodiments, R ³² is -OR ³ . In certain embodiments, R ³² is H. In certain embodiments, R ³² is -N ₃ .

As described above, each R ³ is independently H or (C ₁ -C ₆ )alkyl. In certain embodiments, R ³ is H. In certain embodiments, R ³ is (C ₁ -C ₆ )alkyl. In certain embodiments, R ³ is methyl.

In certain embodiments, R ²⁸ is H. In certain embodiments, R ²⁸ is (C ₁ -C ₆ )alkyl. In certain embodiments, R ⁴⁰ is H.

In certain embodiments, the compound is represented by the structure of formula I-40b:

(I-40b)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² and R ⁴⁰ are as described above for formula Ia, Ic, I or II.

In certain embodiments, the compound is represented by the structure of Formula I-40:

(I-40)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² and R ⁴⁰ are as described above.

In certain embodiments, R ⁴⁰ is -C(=Z ¹ )-R ^40a . In certain embodiments, Z ¹ is O. In certain embodiments, Z ¹ is S.

In certain embodiments, R ^40a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl is unsubstituted or substituted with 1-5 substituents selected from NO ₂ and halogen.

In certain embodiments, R ^40a is -A ¹ -L ¹ -A ² -B. In certain embodiments, R ^40a is -A ¹ -A ² -B. In certain embodiments, R ^40a is -L ² -A ¹ -L ¹ -A ² -L ³ -B.

In certain embodiments, R ^40a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent. In certain embodiments, R ^40a is -A ¹ -L ¹ -A ² -B, where A ² is absent. In certain embodiments, R ^40a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent. In certain embodiments, R ^40a is -A ¹ -L ¹ -A ² -B. In certain embodiments, R ^40a is -A ¹ -A ² -B. In certain embodiments, R ^40a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² and A ¹ are absent. In certain embodiments, R ^40a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² is absent. In certain embodiments, R ^40a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

In certain embodiments, the compounds are represented by the structural formula I-28b:

(I-28b)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² and R ²⁸ are as described above for formula Ia, Ic, I or II.

In certain embodiments, the compound is represented by the structure of Formula I-28:

(I-28)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² and R ²⁸ are as described above.

In certain embodiments, R ²⁸ is -C(=Z ¹ )-R ^28a . In certain embodiments, Z ¹ is O. In certain embodiments, Z ¹ is S.

In certain embodiments, R ^28a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl is unsubstituted or substituted with 1-5 substituents selected from NO ₂ and halogen.

In certain embodiments, R ^28a is -A ¹ -L ¹ -A ² -B. In certain embodiments, R ^28a is -A ¹ -A ² -B. In certain embodiments, R ^28a is -L ² -A ¹ -L ¹ -A ² -L ³ -B.

In certain embodiments, R ^28a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent. In certain embodiments, R ^28a is -A ¹ -L ¹ -A ² -B, where A ² is absent. In certain embodiments, R ^28a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent. In certain embodiments, R ^28a is -A ¹ -L ¹ -A ² -B. In certain embodiments, R ^28a is -A ¹ -A ² -B. In certain embodiments, R ^28a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² and A ¹ are absent. In certain embodiments, R ^28a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² is absent. In certain embodiments, R ^28a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

In certain embodiments, the compounds are represented by the structure of Formula I-32b:

(I-32b)

or a pharmaceutically acceptable salt or tautomer thereof, wherein R ³² is as described above for formula Ia, Ic, I or II.

In certain embodiments, R ³² is -OC(=Z ¹ )-R ^32a . In certain embodiments, Z ¹ is O. In certain embodiments, Z ¹ is S.

In certain embodiments, R ^32a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl is unsubstituted or substituted with 1-5 substituents selected from NO ₂ and halogen.

In certain embodiments, R ^32a is -A ¹ -L ¹ -A ² -B. In certain embodiments, R ^32a is -A ¹ -A ² -B. In certain embodiments, R ^32a is -L ² -A ¹ -L ¹ -A ² -L ³ -B.

In certain embodiments, R ^32a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent. In certain embodiments, R ^32a is -A ¹ -L ¹ -A ² -B, where A ² is absent. In certain embodiments, R ^32a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent. In certain embodiments, R ^32a is -A ¹ -L ¹ -A ² -B. In certain embodiments, R ^32a is -A ¹ -A ² -B. In certain embodiments, R ^32a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² and A ¹ are absent. In certain embodiments, R ^32a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² is absent. In certain embodiments, R ^32a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

As described above, each L ¹ is independently selected from

As described above for Formula Ia, each L ¹ is independently selected from

As described above for formula Ic, each L ¹ is independently selected from

In certain embodiments, L ¹ is .

In certain embodiments, L ¹ is .

In certain embodiments, L ¹ is .

In certain embodiments, is . In certain embodiments, L ¹ is . In certain embodiments, L ¹ is . In certain embodiments, L ¹ is . In certain embodiments, L ¹ is .

In certain embodiments, L ¹ is . In certain embodiments, L ¹ is .

In certain embodiments, L ¹ is . In certain embodiments, L ¹ is . In certain embodiments, L ¹ is . In certain embodiments, L ¹ is .

As described above, L ² and L ³ are independently absent, or independently selected from

As described above for formula Ia and Ic, L ² and L ³ are independently absent, or independently selected from

.

In certain embodiments, L ² is absent. In certain embodiments, L ² is . In certain embodiments, L ² is . In certain embodiments, L ² is . In certain embodiments, L ² is , . In certain embodiments, L ² is . In certain embodiments, L ² is .

In certain embodiments, L ² is . In certain embodiments, L ² is . In certain embodiments, L ² is .

In certain embodiments, L ² is .

In certain embodiments, L ² is .

In certain embodiments, L ² is .

In certain embodiments, L ³ is absent. In certain embodiments, L ³ is . In certain embodiments, L ³ is . In certain embodiments, L ³ is . In certain embodiments, L ³ is , . In certain embodiments, L ³ is . In certain embodiments, L ³ is .

In certain embodiments, L ³ is . In certain embodiments, L ³ is . In certain embodiments, L ³ is In certain embodiments, L ² is . In certain embodiments , L ² is . In certain embodiments, L ² is .

In certain embodiments, L ³ is .

In certain embodiments, L ³ is .

In certain embodiments, L ³ is .

As described above, A ¹ and A ² are independently absent, or independently selected from

, , , , , , , , , , , , , , , , , , ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring.

As described above for Formula Ia, A ¹ and A ² are independently absent, or independently selected from

, , , , , , , , , , , , , , , , , , And ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring.

As described above for Formula Ic, A ¹ and A ² are independently absent, or independently selected from

, , , , , , , , , , , , , , , , , , , And ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

For Formula I, the bond on the left side of A ¹ as shown is bonded to -C(=Z ¹ )- or L ² ; and the bond on the right side of the A ² group, as shown, is bonded to B or L ³ . For Formula II, the bond on the left side of A ¹ as shown is bonded to -C(=Z ¹ )-; and the bond on the right side of the group A ² , as depicted, is bonded to B. For formula Ia and Ic, the bond on the left side of A ¹ , as depicted, is bonded to -C(=Z ¹ )- or L ² ; and where the bond on the right side of the A ² group, as depicted, is bonded to B or L ³ .

In certain embodiments, A ¹ is absent. In certain embodiments, A ¹ is . In certain embodiments, A ¹ is . In certain embodiments, A ¹ is . In certain embodiments, A ¹ is .

In certain embodiments, A ¹ is where each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene.

In certain embodiments, A ¹ is where each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene.

In certain embodiments, A ¹ is where each X is independently absent or is 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each X ¹ is a heteroarylene or heterocyclylene ring.

In certain embodiments, A ¹ is where each W is independently absent or is 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each W ¹ is a heteroarylene or heterocyclylene ring.

In certain embodiments, A ¹ is where each W is independently absent or is 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each W ¹ is a heteroarylene or heterocyclylene ring.

In certain embodiments, A ¹ is wherein each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene.

In certain embodiments, A ¹ is wherein each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring.

In certain embodiments, A ¹ is

In certain embodiments, A ¹ is . In certain embodiments, A ¹ is . In certain embodiments, A ¹ is . In certain embodiments, A ¹ is .

In certain embodiments, A ² is absent. In certain embodiments, A ² is . In certain embodiments, A ² is . In certain embodiments, A ² is . In certain embodiments, A ² is .

In certain embodiments, A ² is where each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene.

In certain embodiments, A ² is where each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene.

In certain embodiments, A ² is where each X is independently absent or is 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each X ¹ is independently a heteroarylene or heterocyclylene ring.

In certain embodiments, A ² is where each W is independently absent or is 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each W ¹ is independently a heteroarylene or heterocyclylene ring.

In certain embodiments, A ² is where each W is independently absent or is 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each W ¹ is independently a heteroarylene or heterocyclylene ring.

In certain embodiments, A ² is where each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene.

In certain embodiments, A ² is wherein each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene; and each G ¹ and G ² are independently a heteroarylene or heterocyclylene ring.

In certain embodiments, A ² is

.

In certain embodiments, A ² is . In certain embodiments, A ² is . In certain embodiments, A ² is . In certain embodiments, A ² is .

As described above, each B is independently selected from

,

.

In certain embodiments, B is .

In certain embodiments, B is .

In certain embodiments, B is , , or . In certain embodiments, B is .

As described above, each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , , heteroarylene-, heterocyclylene arylene- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where the bond on the left side of B ¹ , as shown, is connected to A ² or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl.

As described above for formula Ic, each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-(C(R ³ ) ₂ ) _n -, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , , heteroarylene-, heterocyclylene arylene-, , NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-(C(R ³ ) ₂ ) _n -; Where the connection on the left side of B ¹ , as shown, is connected to A ² , L ³ or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl.

In certain embodiments, B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -.

In certain embodiments, B ¹ is (C ₆ -C ₁₀ )arylene. In certain embodiments, B ¹ is (C ₆ -C ₁₀ )arylene, where arylene is optionally substituted with haloalkyl.

In certain embodiments, B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene- or heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-. In certain embodiments, B ¹ is or -heteroarylene.

In certain embodiments, B ¹ is heteroarylene-. In certain embodiments, B ¹ is heterocyclylene arylene-. In certain embodiments, B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-.

In certain embodiments, B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-(C(R ³ ) ₂ ) _n -. In certain embodiments, B ¹ is . In certain embodiments, B ¹ is . In certain embodiments, B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-(C(R ³ ) ₂ ) _n -.

In certain embodiments, in B ¹ , heteroaryl, heterocyclyl, and arylene are optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen, or hydroxyl.

In certain embodiments, R ³ is H. In certain embodiments, R ³ is (C ₁ -C ₆ )alkyl.

In certain embodiments, R ⁴ is H. In certain embodiments, R ⁴ is (C ₁ -C ₆ )alkyl. In certain embodiments, R ⁴ is halogen. In certain embodiments, R ⁴ is 5-12 membered heteroaryl, 5-12 membered heterocyclyl, or (C ₆ -C ₁₀ )aryl, wherein heteroaryl, heterocyclyl, and aryl are optionally substituted with -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN or -C(O)NR ³ -heteroaryl. In certain embodiments, R ⁴ is -C(O)NR ³ -heterocyclyl. In certain embodiments, R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ or -OR ³ .

As described above, each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl from (C ₁ -C ₆ )alkyl is optionally substituted - N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁵ is H. In certain embodiments, R ⁵ is (C ₁ -C ₆ )alkyl, where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁵ is -C(O)OR ³ . In certain embodiments, R ⁵ is -N(R ³ ) _2.

As described above, each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl from (C ₁ -C ₆ )alkyl is optionally substituted - N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁶ is H. In certain embodiments, R ⁶ is (C ₁ -C ₆ )alkyl, where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁶ is -C(O)OR ³ . In certain embodiments, R ⁶ is -N(R ³ ) ₂ .

As described above, each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl from (C ₁ -C ₆ )alkyl is optionally substituted - N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁷ is H. In certain embodiments, R ⁷ is (C ₁ -C ₆ )alkyl, where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁷ is -C(O)OR ³ . In certain embodiments, R ⁷ is -N(R ³ ) ₂ .

As described above, each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl from (C ₁ -C ₆ )alkyl is optionally substituted - N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁸ is H. In certain embodiments, R ⁸ is (C ₁ -C ₆ )alkyl, where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ . In certain embodiments, R ⁸ is -C(O)OR ³ . In certain embodiments, R ⁸ is -N(R ³ ) ₂ .

As described above, each Y is independently a C(R ³ ) ₂ or bond. In certain embodiments, Y is C(R ³ ) ₂ . In certain embodiments, Y is CH ₂ . In certain embodiments, Y is a link.

In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8, or any range output here. In certain embodiments, n is 1, 2, 3, or 4. In certain embodiments, n is 5, 6, 7, or 8. In certain embodiments, n is 9, 10, 11, or 12.

In certain embodiments, o is equal to an integer from zero to 10 or any range output here. In certain embodiments, o is 0, 1, 2, 3, 4, or 5. In certain embodiments, o is 6, 7, 8, 9, or 10. In certain embodiments, o is between one and 7. In certain In embodiments, o is between one and 8. In certain embodiments, o is between one and 9. In certain embodiments, o is between 3 and 8.

In certain embodiments, o is equal to an integer from zero to 30 or any range output here. In certain embodiments, o is equal to an integer from zero to 30, 29, 28, 27, or 26. In certain embodiments, o is equal to an integer from zero to 25, 24, 23, 22, or 21. In certain embodiments, o is equal to an integer from zero to 20, 19, 18, 17, or 16. In certain embodiments, o is equal to an integer from zero to 15, 14, 13, 12, or 11.

In certain embodiments, p is equal to 0, 1, 2, 3, 4, 5, or 6, or any range output here. In certain embodiments, p is 7, 8, 9, 10, 11, or 12. In certain embodiments, p is 0, 1, 2, or 3. In certain embodiments, p is 4, 5, or 6.

In certain embodiments, q is equal to an integer from zero to 10 or any range output here. In certain embodiments, q is 0, 1, 2, 3, 4, or 5. In certain embodiments, q is 6, 7, 8, 9, or 10. In certain embodiments, q is between one and 7. In certain In embodiments, q is from one to 8. In certain embodiments, q is from one to 9. In certain embodiments, q is from 3 to 8.

In certain embodiments, q is equal to an integer from zero to 30 or any range output here. In certain embodiments, q is equal to an integer from zero to 30, 29, 28, 27, or 26. In certain embodiments, q is equal to an integer from zero to 25, 24, 23, 22, or 21. In certain embodiments, q is equal to an integer from zero to 20, 19, 18, 17, or 16. In certain embodiments, q is equal to an integer from zero to 15, 14, 13, 12, or 11.

As described above, r is equal to an integer from one to 6. In certain embodiments, r is equal to one. In certain embodiments, r is 2. In certain embodiments, r is 3. In certain embodiments, r is 4. In certain embodiments, r is 5. In certain embodiments, r is 6.

As described above, if R ²⁸ and R ⁴⁰ are H, then R ³² is not =O. In certain embodiments, the compound is not rapamycin, as shown below:

In certain embodiments, in formula Ia or Ic, R ³² is -OC(=Z ¹ )-R ^32a . In certain embodiments, R ³² is -OC(=Z ¹ )-R ^32a ; where R ^32a is -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B. In certain embodiments, in formula Ia or Ic, R ²⁸ is -C(=Z ¹ )-R ^28a . In certain embodiments, R ²⁸ is -C(=Z ¹ )-R ^28a ; where R ^28a is -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B. In certain embodiments, in formula Ia or Ic, R ⁴⁰ is -C(=Z ¹ )-R ^40a . In certain embodiments, R ⁴⁰ is -C(=Z ¹ )-R ^40a , where R ^40a is -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B.

This disclosure provides a compound of formula Ia or Ic, or a pharmaceutically acceptable salt or tautomer thereof, having one, two or three of the following characteristics:

a) R ³² is -OC(=Z ¹ )-R ^32a ;

b) R ²⁸ is -C(=Z ¹ )-R ^28a ;

c) R ⁴⁰ is -C(=Z ¹ )-R ^40a .

This disclosure provides a compound of formula Ia or Ic, or a pharmaceutically acceptable salt or tautomer thereof, having one, two or three of the following characteristics:

a) R ³² is -OC(=Z ¹ )-R ^32a ; where R ^32a is -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B;

b) R ²⁸ is -C(=Z ¹ )-R ^28a ; where R ^28a is -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B;

c) R ⁴⁰ is -C(=Z ¹ )-R ^40a , where R ^40a is -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B.

This disclosure provides a compound of formula Ia or Ic, or a pharmaceutically acceptable salt or tautomer thereof, having one, two or three of the following characteristics:

a) R ⁴⁰ is -C(=Z ¹ )-R ^40a ;

b) R ^40a is -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -L ² -A ¹ -L ¹ -A ² -L ³ -B;

c) R ³² is -OR ³ such as -OH.

This disclosure provides a compound of formula Ia or Ic, or a pharmaceutically acceptable salt or tautomer thereof, having one, two, three or four of the following characteristics:

a) one of R ^28a , R ^32a and R ^40a is -A ¹ -L ¹ -A ² -B;

b) A ¹ is missing;

c) A ² is missing;

d) L ¹ is ;

e) B is ;

f) B ¹ is NR ³ -(C(R ³ ) ₂ ) _n - or ;

g) R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ or -OR ³ .

This disclosure provides a compound of the formula:

or

or a pharmaceutically acceptable salt or tautomer thereof having one, two, three or four of the following characteristics:

a) Z ¹ is O;

b) A ¹ is missing;

c) L ¹ is ;

d) B is ;

e) B ¹ is NR ³ -(C(R ³ ) ₂ ) _n - or ;

f) R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ or -OR ³ ; And

g) R ³² is =O.

Above, R ^40a can be -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B.

This disclosure provides a compound of the formula:

or

or a pharmaceutically acceptable salt or tautomer thereof having one, two, three or four of the following characteristics:

a) Z ¹ is O;

b) A ¹ is missing;

c) L ¹ is ;

d) B is ;

e) B ¹ is NR ³ -(C(R ³ ) ₂ ) _n - or ;

f) R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ or -OR ³ ; And

g) R ³² is -OH.

Above, R ^40a can be -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B.

This disclosure provides a compound of the formula:

or

or a pharmaceutically acceptable salt or tautomer thereof having one, two, three or four of the following characteristics:

a) Z ¹ is O;

b) A ¹ is ;

c) L ¹ is ;

d) B is ;

e) B ¹ is NR ³ -(C(R ³ ) ₂ ) _n - or ;

f) R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ or -OR ³ ; And

g) R ³² is =O.

Above, R ^40a can be -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B.

This disclosure provides a compound of the formula:

or a pharmaceutically acceptable salt or tautomer thereof having one, two, three or four of the following characteristics:

a) Z ¹ is O;

b) A ¹ is missing;

c) L ¹ is ;

d) B is ;

e) B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -, or NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where arylene is optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

f) R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ or -OR ³ ; And

g) R ³² is -OH.

Above, R ^40a can be -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B.

This disclosure provides a compound of the formula:

or a pharmaceutically acceptable salt or tautomer thereof having one, two, three or four of the following characteristics:

a) Z ¹ is O;

b) A ¹ is ;

c) A ² is

d) L ¹ is ;

e) B is ;

f) B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -, or NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where arylene is optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

g) R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ or -OR ³ ; And

h) R ³² is -OH.

Above, R ^40a can be -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; or -L ² -A ¹ -L ¹ -A ² -L ³ -B.

In certain embodiments, in Formula Ia or Ic, R ^40a is any organic group that may have a molecular weight (eg, the sum of the atomic masses of the substituent atoms) of less than 15 g/mol, 50 g/mol, 100 g/mol, 150 g/mol, 200 g/mol, 250 g/mol, 300 g/mol, 350 g/mol, 400 g/mol, 450 g/mol or 500 g/mol.

In certain embodiments, this disclosure provides a compound selected from the list below, or a pharmaceutically acceptable salt or tautomer thereof

Example 1 Example 2 Example 3 Example 4
Example 5
Example 6
Example 7
Example 8
Example 9
Example 10
Example 11
Example 12
Example 13
Example 14
Example 15
Example 16
Example 17
Example 18
Example 19
Example 20
Example 21
Example 22
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Example 25
Example 26
Example 27
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Example 40
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Example 61
Example 62
Example 63
Example 64
Example 65
Example 66
Example 67
Example 68
Example 69
Example 70

In certain embodiments, this disclosure is for a compound selected from the list below or a pharmaceutically acceptable salt or tautomer thereof

Example 71
Example 72
Example 73
Example 74
Example 75
Example 76
Example 77
Example 78
Example 79
Example 80
Example 81
Example 82
Example 83
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Example 125
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Example 127
Example 128
Example 129
Example 130
Example 131
Example 132
Example 133
Example 134
Example 135

In certain embodiments, this disclosure is for a compound selected from the list below or a pharmaceutically acceptable salt or tautomer thereof

Example 136
Example 137
Example 138
Example 139
Example 140
Example 141
Example 142
Example 143
Example 144
Example 145
Example 146
Example 147
Example 148
Example 149
Example 150
Example 151
Example 152

The compounds of the present disclosure may include pharmaceutically acceptable salts of the compounds disclosed herein. Exemplary "pharmaceutically acceptable salts" may include, for example, water-soluble and non-water-soluble salts such as acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate , calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavularate, dihydrochloride, edetate, edisilate, estolate, esylate, fiunarate (fiunarate?), glucepate, gluconate, glutamate, glycollarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, sethionate (isethionate?), lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2- naphthoate, oleate, oxalate, palmitate, pamoate, 1,1-methenbis-2-hydroxy-3-naphthoate, einbonate, pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, theoclate, tosylate, triethiodide and valerate.

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

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

Unless otherwise noted, the structures depicted here may also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure, except for the replacement of a hydrogen atom with deuterium or tritium, or the replacement of a carbon atom with ¹³ C or ¹⁴ C, or the replacement of a nitrogen atom with 15 N, or the replacement of an oxygen atom with ¹⁷ O or ¹⁸ O, are included within the scope of the disclosure. Such isotopically labeled compounds are useful as research or diagnostic tools.

In some embodiments, one or more deuterium atoms may be introduced into the PEG group of any compound of the present invention. The mechanisms for such modifications are known in the art, starting with commercially available starting materials such as isotopically enriched hydroxylamine building blocks. In some embodiments, tritium or deuterium can be introduced at the C32 position of the compounds of the present invention using, for example, a commercially available isotopically pure reducing agent and methods known to those skilled in the art. In some embodiments, ¹⁴ C can be introduced into the C40 carbamate group of the compounds of the present invention using commercially available materials and methods known to those skilled in the art. In some embodiments, an isotope, such as deuterium or tritium, can be introduced into the R ^40a substituent of a compound of Formula Ia, Ic, I, or II using commercially available starting materials and methods known to those skilled in the art.

Methods for the synthesis of the described compounds

The compounds of this disclosure can be prepared by a variety of methods, including standard chemical methods. Suitable synthetic routes are depicted in the schemes below.

Compounds of any of the formulas described herein can be prepared by methods known in the art of organic synthesis, which are set forth in part by the following synthetic schemes and examples. In the schemes described below, it is well understood that protecting groups for sensitive or reactive groups are used where necessary, in accordance with general principles or chemistry. Protective groups are handled according to standard methods of organic synthesis (T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis,” Third edition, Wiley, New York 1999). These groups are removed at a convenient stage in the synthesis of the compound using methods that are obvious to those skilled in the art. The selection processes, as well as the reaction conditions and the order in which they are carried out, must be consistent with the preparation of compounds of formula I, Ia, Ib, II or IIb or a pharmaceutically acceptable salt or tautomer of any of the above.

Compounds of any of the formulas described herein can be prepared by methods that avoid the use of metal-mediated cycloaddition reactions that require the use of azide-containing compounds. Azide-containing compounds pose potential safety hazards associated with their preparation and storage (eg, explosion due to high energy decomposition). In addition, the reaction schemes presented here avoid the use of copper or ruthenium metal in the penultimate or final stages of the synthesis, which can be advantageous. Avoiding the use of copper or ruthenium metal in the penultimate or final stages of synthesis reduces the likelihood of contamination of the final compounds with undesirable metallic impurities.

Since rapamycin can be an expensive starting material, good reaction yields are preferred. The reaction schemes shown here provide higher yields than other reaction schemes. The reaction schemes presented here do not require alkylation of the C40 hydroxyl of rapamycin, which is advantageous in providing a 5-fold improvement in overall yield for the preparation of divalent compounds from rapamycin compared to other reaction schemes.

There is an additional synthetic improvement associated with increased yield. Eliminating the need for alkylation at C40 hydroxyl results in a 5-fold improved overall yield for the preparation of divalent compounds from rapamycin.

Those skilled in the art will appreciate whether a stereocenter exists in any of the compounds of this disclosure. Accordingly, the present disclosure may include both possible stereoisomers (unless specified in the synthesis), and may include not only racemic compounds, and may include not only racemic compounds, but also individual enantiomers and/or diastereomers. When a compound is desired as a single enantiomer or diastereomer, it can be prepared by stereospecific synthesis or resolution of the final product or any convenient intermediate. Separation of the final product, intermediate, or starting material can be accomplished by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel, S. H. Wilen and L. N. Mander (Wiley-lnterscience, 1994).

Receiving connections

The compounds described herein can be obtained from commercially available starting materials or synthesized using known organic, inorganic and/or enzymatic processes.

The compounds of this disclosure can be prepared by a variety of methods well known to those skilled in the art of organic synthesis. As an example, the compounds of the present disclosure can be synthesized using the methods described below, together with synthetic methods known in the field of synthetic organic chemistry, or variations thereof, as understood by those skilled in the art. These methods may include, but are not limited to, the methods described below.

The term tautomers can refer to a series of compounds that have the same number and type of atoms, but differ in connectivity and are in equilibrium with each other. The "tautomer" is the only member of this series of compounds. Typically a single tautomer is drawn, but it can be understood that this single structure can represent all possible tautomers that may exist. Examples may include enol-ketone tautomerism. When a ketone is extracted, it can be understood that both the enol and ketone forms are part of the description.

In addition to tautomers, which can exist on all amide, carbonyl and oxime groups in compounds of formula I, Ia, Ib, Ic, II or IIb, compounds of this family readily interconvert via open ring compounds between two main isomeric forms known as pyran and oxepane isomers (shown below). This interconversion may be facilitated by magnesium ions, slightly acidic conditions, or alkylamine salts, as described in the following references: i) Hughes, PF; Musser, J.; Conklin, M.; Russo, R. 1992. Tetrahedron Lett. 33(33): 4739-32. ii) Zhu, T. 2007. US Patent No. 7,241,771; Wyeth. iii) Hughes, P.F. 1994. US Patent No. 5,344,833; American Home Products Corp. The scheme below shows the interconversion between the pyran and oxepane isomers in compounds of formula I, Ia, Ib, Ic, II or IIb.

Since this interconversion occurs under mild conditions and the position of thermodynamic equilibrium may vary between different members of the compounds of formula I, Ia, Ib, Ic, II or IIb, both isomers are considered for compounds of formula I, Ia, Ib, Ic, II or IIb. For brevity, the pyran isomeric form of all intermediates and compounds of formulas I, Ia, Ib, Ic, II or IIb is shown.

General Assembly Approaches for Bifunctional Rapalogues

With reference to the diagrams below, rapamycin has the formula RAP,

(RAP)

where R ¹⁶ is -OCH ₃ ; R ²⁶ is =O; R ²⁸ is -OH; R ³² is =O; and R ⁴⁰ is -OH. “Rapalog” refers to an analogue or derivative of rapamycin. For example, with reference to the diagrams below, a rapalogue can be rapamycin that is substituted at any position, such as R ¹⁶ , R ²⁶ , R ²⁸ , R ³² or R ⁴⁰ . An active site inhibitor (AS inhibitor) is an active site mTOR inhibitor. In certain embodiments, the AC Inhibitor is represented by B in Formula I, Ia, Ib, Ic, II, or IIb.

Row 1 of bifunctional rapalogs

The general structure of Row 1 bifunctional rapalogs is shown in Scheme 1 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7 and r=1-6. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. A carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine, and may include the variants found in Table 2 in the Examples section.

Scheme 1. Row 1 of bifunctional rapalogues.

Row 2 of bifunctional rapalogues

The general structure of Series 2 bifunctional rapalogues is shown in Scheme 2 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. The pre-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 2. Row of 2 bifunctional rapalogs.

Row 3 of bifunctional rapalogues

The general structure of Series 3 bifunctional rapalogues is shown in Scheme 3 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. The post-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 3. Row of 3 bifunctional rapalogs

Row 4 bifunctional rapalogues

The general structure of Series 4 bifunctional rapalogues is shown in Scheme 4 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. Each of the pre- and post-linker amines may contain substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 4. Row 4 bifunctional rapalogues

Row 5 bifunctional rapalogues

The general structure of Series 5 bifunctional rapalogues is shown in Scheme 5 below. For these types of bifunctional rapalogs, the pre-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ = O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 5. Row 5 bifunctional rapalogues

Row 6 bifunctional rapalogues

The general structure of Series 6 bifunctional rapalogues is shown in Scheme 6 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. Linker amines may include substitutions such as R=H and C1-C6 alkyl groups. The post-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ = O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 6. Row 6 bifunctional rapalogues.

Row of 7 bifunctional rapalogues

The general structure of Series 7 bifunctional rapalogues is shown in Scheme 7 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. Each of the pre- and post-linker amines includes substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 7. Row 7 bifunctional rapalogues

Row of 8 bifunctional rapalogues

The general structure of Series 8 bifunctional rapalogues is shown in Scheme 8 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. The post-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I, Ia, Ib, Ic, II or IIb), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 8. Row 8 bifunctional rapalogues

Pharmaceutical compositions

Another aspect relates to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of the present invention or a pharmaceutically acceptable salt or tautomer thereof.

In embodiments of pharmaceutical compositions, a compound of the present invention, or a pharmaceutically acceptable salt or tautomer thereof, may be included in a therapeutically effective amount.

Administration of the disclosed compounds or compositions may be accomplished by any route of administration of therapeutic agents. These methods may include systemic or local administration, such as oral, nasal, parenteral, transdermal, subcutaneous, vaginal, buccal, rectal, topical, intrathecal, or intracranial administration modes.

In some embodiments, administration may include oral administration, suppository administration, local contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or implantation of a slow-release device, such as a mini-osmotic pump. , subject. Administration may be by any route, including parenteral and transmucosal (eg, buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. The compositions of this disclosure may be delivered transdermally, locally, formulated as applicators, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, wafers, gels, syrups, pulps, suspensions, etc., suitable for oral administration by the patient. Solid form preparations include powders, tablets, pills, capsules, wafers, suppositories and dispersible granules. Preparations in liquid form include solutions, suspensions and emulsions, such as solutions in water or water/propylene glycol. The compositions of this disclosure may further include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely divided drug carrier substrates. These components are discussed in more detail in US Patent Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of this disclosure may also be delivered in the form of microspheres for slow release in the body. For example, microspheres can be administered by intradermal injection of drug-containing microspheres that are slowly released subcutaneously. (see Rao, J. Biomater Set Polym. Ed. 7:623-645, 1995); in the form of biodegradable and injectable gel formulations (see, for example, Gao Pharm. Res. 12:857-863, 1995); or in the form of microspheres for oral administration (see, for example, Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In another embodiment, the compositions of this disclosure can be delivered using liposomes that fuse with the cell membrane or undergo endocytosis, i.e. using receptor ligands attached to liposomes that bind to cell surface membrane protein receptors, leading to endocytosis. By using liposomes, particularly where the surface of the liposomes carries receptor ligands that are specific to target cells or otherwise preferentially directed to a particular organ, delivery of the compositions of the present invention to target cells in vivo can be focused. (See, for example, Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989). The compositions of this disclosure may also be delivered in the form of nanoparticles.

Depending on the intended route of administration, the disclosed compounds or pharmaceutical compositions may be in solid, semi-solid or liquid dosage form, such as, for example, injections, tablets, suppositories, pills, sustained release capsules, elixirs, tinctures, emulsions, syrups, powders , liquids, suspensions or the like, sometimes in unit dosages and in accordance with generally accepted pharmaceutical practice. Likewise, they can also be administered in intravenous (bolus and infusion), intraperitoneal, intrathecal, subcutaneous or intramuscular forms, all useful forms being well known to those skilled in the art of pharmaceuticals.

Exemplary pharmaceutical compositions are tablets and gelatin capsules containing a compound of the disclosure and a pharmaceutically acceptable carrier such as a) a diluent such as purified water, triglyceride oils such as hydrogenated or partially hydrogenated vegetable oil or mixtures thereof, corn oil, olive oil, sunflower oil, safflower oil, fish oil such as EPA or DHA, or esters thereof, triglycerides or mixtures thereof, omega-3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and/or glycine; b) a lubricant, for example silica, talc, stearic acid, its magnesium or calcium salt, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and/or polyethylene glycol; also for tablets; c) a binding agent, for example magnesium aluminosilicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, waxes and/or polyvinylpyrrolidone, if desired; d) disintegrant, for example starches, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt or effervescent mixtures; e) absorbent, coloring, flavoring and sweetening agent; f) an emulsifier or dispersing agent such as Tween 80, Labrasol, HPMC, DOSS, Caproyl 909, Labrafac, Labrafil, Peceol, Transcutol, Capmul MCM, Capmul PG-12, Captex 355, Helicur, Vitamin E TGPS or other suitable emulsifier; and/or g) an absorption enhancing agent such as cyclodextrin, hydroxypropylcyclodextrin, PEG400, PEG200.

Liquid, in particular injectable compositions can, for example, be prepared by dissolving, dispersing, etc. For example, the disclosed compound is dissolved or mixed with a pharmaceutically acceptable solvent, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like, thereby forming an isotonic injection solution or suspension. Proteins such as albumin, chylomicron particles or serum proteins can be used to solubilize the disclosed compounds.

The disclosed compounds may also be formulated as suppositories, which may be prepared from fat emulsions or suspensions; using polyalkylene glycols such as propylene glycol as a carrier.

The disclosed compounds can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, the film of lipid components is hydrogenated with an aqueous drug solution to form a lipid layer encapsulating the drug, as described, for example, in US Pat. No. 5,262,564, the contents of which are incorporated herein by reference.

The disclosed compounds can also be delivered using monoclonal antibodies as individual carriers to which the disclosed compounds are coupled. The disclosed compounds can also be associated with soluble polymers as targeted drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl methacrylamide phenol, polyhydroxyethyl aspanamide phenol, or polyethylene oxide polylysine substituted palmitoyl moieties. In addition, the disclosed compounds may be conjugated with a class of biodegradable polymers used to achieve controlled drug release, such as polylactic acid, polypsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymer hydrogels. In one embodiment, the disclosed compounds are not covalently bonded to a polymer, such as a polycarboxylic acid polymer or polyacrylate.

Administration by parenteral injection is typically used for subcutaneous, intramuscular, or intravenous injections and infusions. Injectable preparations may be prepared in conventional forms as either liquid solutions, suspensions, or solid forms suitable for dissolution in liquid prior to injection.

Another aspect of the disclosure relates to a pharmaceutical composition containing a compound or a pharmaceutically acceptable salt or tautomer thereof of the disclosure and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further include an excipient, diluent or surfactant.

The compositions can be prepared according to conventional mixing, granulating or coating methods, as appropriate, and the present pharmaceutical compositions can contain from about 0.1% to about 99%, from about 5% to about 90%, or from about 1% to about 20% of the disclosed compound by mass or volume.

The compounds described herein can be used in combination with each other, with other active agents known to be useful in the treatment of cancer, autoimmune disease, inflammatory disease, metabolic disease, neurodegenerative disease, fungal infection or transplant rejection, or with auxiliary agents that may not be effective on their own but may enhance the effectiveness of the active agent. The compounds described herein may be used in combination with other active agents known as life-prolonging agents or anti-aging agents.

In embodiments of pharmaceutical compositions, the pharmaceutical composition may include a second agent (eg, a therapeutic agent). In embodiments of pharmaceutical compositions, the pharmaceutical composition may include a second agent (eg, a therapeutic agent) in a therapeutically effective amount. In certain embodiments, the second agent is an anticancer agent. In certain embodiments, the second agent is an immunotherapeutic agent. In certain embodiments, the second agent is an immuno-oncology agent. In certain embodiments, the second agent is an anti-autoimmune agent. In embodiments, the second agent is an anti-inflammatory agent. In certain embodiments, the second agent is an anti-neurodegenerative agent. In certain embodiments, the second agent is an anti-metabolic agent. In certain embodiments, the second agent is an anti-cardiovascular agent. In certain embodiments, the second agent is an anti-aging agent. In certain embodiments, the second agent is a life-prolonging agent. In certain embodiments, the second agent is an agent for treating or preventing transplant rejection. In certain embodiments, the second agent is an agent for treating or preventing a fungal infection. In certain embodiments, the second agent is an immune system repressor. In certain embodiments, the second agent is an mTOR modulator. In certain embodiments, the second agent is an mTOR inhibitor. In certain embodiments, the second agent is an mTOR active site inhibitor. In certain embodiments, the second agent is rapamycin. In certain embodiments, the second agent is a rapamycin analog. In certain embodiments, the second agent is an mTOR pathway inhibitor. In certain embodiments, the second agent is a CDK4/6 inhibitor; anti-PD1/PD-L1, PI3K inhibitor; or AS inhibitor.

"Anticancer agent" or "anticancer drug" is used in accordance with its simple ordinary meaning and refers to a composition (eg, compound, drug, antagonist, inhibitor, modulator) having antitumor properties or the ability to inhibit cell growth or proliferation. In some embodiments, the anticancer agent is chemotherapeutic. In some embodiments, the anticancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the United States for the treatment of cancer. Examples of anticancer agents include, but are not limited to, rapamycin, rapamycin analogue, bevacizumab, PP242, ΓΝ 128, MLN0128, antiandrogens (eg, Cachodex, Flutamide, MDV3100 or ARN-509), MEK inhibitors (eg, MEK1, MEK2 or MEK1 and MEK2) (e.g. XL518, CI-1040, PD035901, selumetinib/AZD6244, GSK1 120212/trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylating agents (e.g. cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, chloromethines (e.g. mechlorethamine, cyclophosphamide, chlorambucil, melphalan), ethyleneimine and methylmelamines (e.g. hexamethylmela min, thiotepa) , alkylsulfonates (eg, busulfan), nitrosoureas (eg, carmustine, lomusitne, semustine, streptozocin, triazenes (decarbazine)), antimetabolites (eg, 5-azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analogue ( e.g. methotrexate), pyrimidine analogues (e.g. fluorouracil, floxuridine, cytarabine), purine analogues (e.g. mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g. vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (for example, irinotecan, topotecan, amsacrine, etoposide (VP 16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (for example, doxorubicin, adriamycin, daunorubicin, epirubicin , actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g. cisplatin, oxaliplatin, carboplatin), anthracenedione (e.g. mitoxantrone), substituted urea (e.g. hydroxyurea), methylhydrazine derivative (e.g. , procarbazine), adrenocortical suppressor (eg, mitotane, aminoglutethimide), epipodophyllotoxins (eg, etoposide), antibiotics (eg, daunorubicin, doxorubicin, bleomycin), enzymes (eg, L-asparaginase), nitrogen-activated protein kinase signaling inhibitors (eg , U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin or LY294002), mTOR inhibitors, antibodies (eg, Rituxan), 5-aza-2'-deoxycytidine, doxorubicin , vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), sheldanamycin, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), bortezomib, trastuzumab, anastrozole; angiogenesis inhibitors; antiandrogens, antiestrogens; antisense oligonucleotides; modulators of apoptosis genes; apoptosis regulators; arginine deaminase; BCR/ABL antagonists; beta-lactam derivatives; bFGF inhibitor; bicalutamide; camptothecin derivatives; casein kinase inhibitors (ICOS); clomiphene analogues; cytarabine dacliximab; dexamethasone; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; finasteride; fludarabine; fluorodaunorunicine hydrochloride; gadolinium texaphyrin; gallium nitrate; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfame; immunostimulating peptides; insulin-like growth factor receptor-1 inhibitor; interferon agonists; interferons; interleukins; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; matrilysin inhibitors; matrix metalloproteinase inhibitors; MIF inhibitor; mifepristone; mismatched double-stranded RNA; monoclonal antibody; mycobacterial cell wall extract; nitric oxide modulators; oxaliplatin; panomifene; pentrozole; phosphatase inhibitors; plasminogen activator inhibitor; platinum complex; platinum compounds; prednisone; proteasome inhibitors; immunomodulator based on protein A; protein kinase C inhibitor; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; ras farnesyl protein transferase inhibitors; ras inhibitor; ras-GAP inhibitor; ribozymes; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; stem cell inhibitor; stem cell division inhibitors; stromelysin inhibitors; synthetic glycosaminoglycans; tamoxifen methiodide; telomerase inhibitors; thyroid-stimulating hormone; translation inhibitors; tyrosine kinase inhibitors; urokinase receptor antagonists; steroids (eg, dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone (GnRH) agonists such as goserelin or leuprolide, adrenocorticosteroids (eg, prednisone), progestins (eg, hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (eg, diethylstilbestrol, ethinyl estradiol), anti-estrogen (eg, tamoxifen), androgens (eg, testosterone propionate, eg, fluoxymesterone), antiandrogen (eg, flutamide), immunostimulants (eg, Bacillus Calmette-Guerin (BCG), levamisole, interleukin- 2, interferon alpha, etc.), monoclonal antibodies (eg anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR and anti-VEGF monoclonal antibodies), immunotoxins (eg anti-CD33 conjugate monoclonal antibody and calicheamicin, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (eg, anti-CD20 monoclonal antibody conjugated to ^U1 ln, ⁹⁰ Y or ¹³¹ I, etc.), triptolide , homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, targeted therapy or therapeutic agent for epidermal growth factor receptor (EGFR) or (eg, gefitinib (Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992 , CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, pyrrolobenzodiazepines (eg, tomaimicin), carboplatin, CC-1 065 and CC-1065 analogs including amino-CBI, chlormethines (such as chlorambucil and melphalan), dolastatin and dolastatin analogs (including auristatins: e.g. monomethyl auristatin E), anthracycline antibiotics (such as doxorubicin, daunorubicin, etc.), duocarmycins and duocarmycin analogs, enediines (such as neocarzinostatin and calicheamicins), leptomycin derivatives, maytansinoids and maytansinoid analogs (such as mertansine), methotrexate, mitomycin C, taxoids, vinca alkaloids (such as vinblastine and vincristine), epothilones (such as epothilone B ), camptothecin and its clinical analogs topotecan and irinotecan, ΓΝΚ128, PP242, PP121, MLN0128, AZD8055, AZD2014, VP-BEZ235, BGT226, SFl 126, Torin 1, Torin 2, WYE 687, WYE 687 salt (e.g. hydrochloride reed), PF04691502, PI-103, CC-223, OSI-027, XL388, KU-0063794, GDC-0349, PKI-587, rapamycin, deforolimus (AP23573, MK-8669, ridaforolimus), temsirolimus (CCI-779), ABT478, everolimus (RAD001) or the like.

mTOR and treatments

The term “mTOR” refers to the “mechanistic target of rapamycin (serine/threonine kinase)” or “mammalian target of rapamycin” protein. The term "mTOR" may refer to the nucleotide sequence or protein sequence of human mTOR (eg, Entrez 2475, Uniprot P42345, RefSeq NM_004958 or RefSeq NP_004949) (SEQ ID NO: 1). The term “mTOR” can include both wild-type nucleotide sequences or proteins and also mutants thereof. In some embodiments, “mTOR” is wild-type mTOR. In some embodiments, “mTOR” is one or more mutant forms. The term “mTOR” XYZ can refer to the nucleotide sequence or protein of a mutant mTOR, where the amino acid number Y of mTOR, which normally has amino acid X in the wild type, instead has amino acid Z in the mutant. In embodiments, mTOR is human mTOR. In embodiments, mTOR has a nucleotide sequence corresponding to reference number GL206725550 (SEQ ID NO: 2). In embodiments, mTOR has a nucleotide sequence corresponding to RefSeq NM_004958.3 (SEQ ID NO: 2). In embodiments, mTOR has a protein sequence corresponding to reference number GL4826730 (SEQ ID NO: 1). In embodiments, mTOR has a protein sequence corresponding to RefSeq NP_004949.1 (SEQ ID NO: 1). In embodiments, mTOR has the following amino acid sequence:

MLGTGPAAATTAATTSSNVSVLQQFASGLKSRNEETRAKAAKELQHYVTMELREMSQEESTRFYDQLNHHIFELVSSSDANERKGGILAIASLIGVEGGNATRIGRFANYLRNLLPSNDPWMEMASKAIGRLAMAGDTFTAEYVEFEVKRALEWLGADRNEGRRHAAVLVLRELAISVPTFFFQQVQPFFDNIFVAVWDPKQAIRE GAVAALRACLILTTQREPKEMQKPQWYRHTFEEAEKGFDETLAKEKGMNRDDRIHGALLILNELVRISSMEGERLREEMEEITQQQLVHDKYCKDLMGFGTKPRHITPFTSFQAVQPQQSNALVGLLGYSSHQGLMGFGTSPSPAKSTLVESRCCRDLMEEKFDQVCQWVLKCRNSKNSLIQMTILNLLPRLAAFRPSAFTDT QYLQDTMNHVLSCVKKEKERTAAFQALGLLSVAVRSEFKVYLPRVLDIIRAALPPKDFAHKRQKAMQVDATVFTCISMLARAMGPGIQQDIKELLEPMLAVGLSPALTAVLYDLSRQIPQLKKDIQDGLLKMLSLVLMHKPLRHPGMPKGLAHQLASPGLTTLPEASDVGSITLALRTLGSSFEFEGHSLTQFVRHCADH FLNSEHKEIRMEAARTCSRLLTPSIHLISGHAHVVSQTAVQVVADVLSKLLWGITDPDPDIRYCVLASLDERFDAHLAQAENLQALFVAL NDQVFEIRELAICTVGRLSSMNPAFVMPFLRKMLIQILTELEHSGIGRIKEQSARMLGHLVSNAPRLIRPYMEPILKALILKLKDPDPDPNPGVINNVLATIGELAQVSGLEMRKW VDELFIIIMDMLQDSSLLAKRQVALWTLGQLVASTGYVVEPYRKYPTLLEVLLNFLKTEQNQGTRREAIRVLGLLGALDPYKHKVNIGMIDQSRDASAVSLSESKSSQDSSDYSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFIFKSLGLKCVQFLPQVMPTFLNVIRVCDGAIREFL FQQLGMLVSFVKSHIRPYMDEIVTLMREFWVMNTSIQSTIILLIEQIVVALGGEFKLYLPQLIPHMLRVFMHDNSPGRIVSIKLLAAIQLFGANLDDYLHLLLPPIVKLFDAPEAPLPSRKAALETVDRLTESLDFTDYASRIIHPIVRTLDQSPELRSTAMDTLSSLVFQLGKKYQIFIPMVNKVLVRHRINHQRYDVLICRIVK GYTLADEEEDPLIYQHRMLRSGQGDALAASGPVETGPMKKLHVSTINLQKAWGAARRVSKDDWLEWLRRLSLELLKDSSSPSLRSCWALAQAYNPMARDLFNAAFVSCWSELNEDQQDELIRSIELALTSQDIAEVTQTLLNLAEFMEHSDKGPLPLRDDNGIVLLGERAAKCRAYAKALHYKELEFQKGPTPAILESLIS INNKLQQPEAAAGVLEYAMKHFGELEIQATWYEKLHEWEDALVAYDKKMDTNKDDPELMLGRMRCLEALGEWGQLHQQCCEKWTLVNDETQAKMARMAAAAWGLGQWDSMEEYTCMIPRDTHDGAFYRAVLALHQDLFSLAQQCIDKARDLLDAELTAMAGESYSRAYGAMVSCHMLSELEEVIQYKLVPERREIIRQ IWWERLQGCQRIVEDWQKILMVRSLVVSPHEDMRTWLKYASLCGKSGRLALAHKTLVLLLGVDPSRQLDHPLPTVHPQVTYAYMKNMWKSARKIDAFQHMQHFVQTMQQQAQHAIATEDQQHKQELHKLMARCFLKLGEWQLNLQGINESTIPKVLQYYSAATEHDRSWYKAWHAWAVMNFEAVLH YKHQNQARDEKKKLRHASGANITNATTAATTAATATTTTASTEGSNSESEAESTENSPTPSPLQKKVTEDLSKTLLMYTVPAVQGFFRSISLSRGNNLQDTLRVLTLWFDYGHWPDVNEALVEGVKAIQIDTWLQVIPQLIARIDTPRPLVGRLIHQLLTDIGRYHPQALIYPLTVASKSTTTARHNAANKILKNMCEHSNTLVQQ AMMVSEELIRVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQLPQLTSLELQYVSPKLLMCRDLELAVPGTYDPNQPIIRIQSIAPSLQVITSKQRPRKLTLMGSNGHEFVFLLKGHEDLRQDERVMQLFGLVNTLLAND PTSLRKNLSIQRYAVIPLSTNSGLIGWVPHCDTLHALIRDYREKKKILLNIEHRIMLRMAPDYDHLTLMQKVEVFEHAVNNTAGDDLAKLLWLKSPSSEVWFDRRTNYTRSLAVMSMVGYILGLGDRHPSNLMLDRLSGKILHIDFGDCFEVAMTREKFPEKIPFRLTRMLTNAMEVTGLDGNYRITCHTVMEVLREHKDSVMAVLEAFV YDPLLNWRLMDTNTKGNKRSRTRTDSYSAGQSVEILDGVELGEPAHKKTGTTVPESIHSFIGDGLVKPEALNKKAIQIINRVRDKLTGRDFSHDDTLDVPTQVELLIKQATSHENLCQCYIGWCPFW

(SEQ ID NO: 1)

In embodiments, mTOR is a mutant mTOR. In embodiments, mutant mTOR is associated with a disease that is not associated with wild-type mTOR. In embodiments, mTOR may include at least one amino acid mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mutations or any range inferred here) compared to the sequence above.

The term “mTORC1” refers to a protein complex including mTOR and Raptor (mTOR regulatory associated protein). mTORC1 may also include MLST8 (mammal lethal SEC 13 protein 8), PRAS40 and/or DEPTOR. mTORC1 may function as a nutrient/energy/redox sensor and regulator of protein synthesis. The term “mTORC1 pathway” or “mTORC1 signal transduction pathway” may refer to a cellular pathway involving mTORC1. The mTORC1 pathway includes pathway components upstream and downstream of mTORC1. The mTORC1 pathway is a signaling pathway that is modulated through modulation of mTORC1 activity. In embodiments, the mTORC1 pathway is a signaling pathway that is modulated through modulation of mTORC1 activity, but not through modulation of mTORC2 activity. In embodiments, the mTORC1 pathway is a signaling pathway that is modulated more through modulation of mTORC1 activity than through modulation of mTORC2 activity.

The term “mTORC2” refers to a protein complex including mTOR and RICTOR (rapamycin-intensive companion of mTOR). mTORC2 may also include GβL, mSIN1 (mammalian stress-activated protein kinase interacting protein 1), Protor 1/2, DEPTOR, TTI1 and/or TEL2. mTORC2 can regulate cellular metabolism and cytoskeleton. The term “mTORC2 pathway” or “mTORC2 signal transduction pathway” may refer to a cellular pathway involving mTORC2. The mTORC2 pathway includes components upstream and downstream of mTORC2. The mTORC2 pathway is a signaling pathway that is modulated through modulation of mTORC2 activity. In embodiments, the mTORC2 pathway is a signaling pathway that is modulated through modulation of mTORC2 activity, but not through modulation of mTORC1 activity. In embodiments, the mTORC2 pathway is a signaling pathway that is modulated more through modulation of mTORC2 activity than through modulation of mTORC1 activity.

The term “rapamycin” or “sirolimus” refers to a macrolide produced by the bacterium Streptomyces hygroscopicus. Rapamycin can prevent T cell and B cell activation. Rapamycin has the name IUPAC (3 S , 6 R , 7E, 9 R , 10 R , 12 R , 14 S , 15E, 17E, 19E, 21 S , 23 S , 26 R , 27 R , 34a S ) -9.10 ,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1 R )-2-[(1 S ,3 R ,4 R )-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H -pyrido[2,1-c][1,4]-oxoazacyclogentriacontin-1,5,11,28,29(4H,6H,31H)-pentone. Rapamycin has CAS number 53123-88-9. Rapamycin can be produced synthetically (eg, chemical synthesis) or through the use of a production method that does not involve the use of Streptomyces hygroscopicus .

"Analogue" is used according to its usual meaning in chemistry and biology and refers to a chemical compound that is structurally similar to another compound (i.e. the so-called "reference" compound) but differs in composition, for example by replacing one atom with an atom of another element or the presence of a particular functional group or the replacement of one functional group by another functional group or the absolute stereochemistry of one or more chiral centers of the reference compound, including its isomers.

The term “rapamycin analog” or “rapalog” refers to an analogue or derivative (eg, prodrug) of rapamycin.

The terms “mTOR active site inhibitor” and “ATP mimetic” refer to a compound that inhibits mTOR activity (e.g., kinase activity) and binds to the mTOR active site (e.g., ATP binding site, superimposing the ATP binding site, blocking ATP access to the site mTOR ATP binding). Examples of mTOR active site inhibitors include, but are not limited to, ΓΝΚ128, PP242, PP121, MLN0128, AZD8055, AZD2014, NVP-BEZ235, BGT226, SF1126, Torin 1, Torin 2, WYE 687, WYE 687 salt (e.g. hydrochloride), PF04691502, PI-103, CC-223, OSI-027, XL388, KU-0063794, GDC-0349 and PKI-587. In embodiments, the mTOR active site inhibitor is asTORi. In some embodiments, “active site inhibitor” may refer to “mTOR active site inhibitor.”

The term “FKBP” refers to the protein Peptidyl-prolyl cis-trans isomerase. Non-limiting examples of FKBP are provided in Cell Mol Life Sci. 2013 Sep; 70(18): 3243-75. In embodiments, “FKBP” may refer to “FKBP-12” or “FKBP 12” or “FKBP 1A.” In embodiments, “FKBP” may refer to a human protein. The term “FKBP” includes wild type and mutant forms of the protein. In embodiments, “FKBP” may refer to a wild-type human protein. In embodiments, “FKBP” may refer to a wild-type human nucleic acid. In embodiments, the FKBP is a mutant FKBP. In embodiments, the mutant FKBP is associated with a disease that is not associated with wild type FKBP. In embodiments, FKBP includes at least one amino acid mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mutations or any range inferred here) compared to wild type FKBP.

The term “FKBP-12” or “FKBP 12” or “FKBP 1A” may refer to the protein “Peptidyl-prolyl cis-trans isomerase FKBP 1A.” In embodiments, “FKBP-12” or “FKBP 12” or “FKBP 1A” may refer to a human protein. Included in the term, “FKBP-12” or “FKBP 12” or “FKBP 1A” are the wild type and mutant forms of the protein. In embodiments, “FKBP-12” or “FKBP 12” or “FKBP 1A” may refer to a protein associated with Entrez Gene 2280, OMIM 186945, UniProt P62942 and/or RefSeq (protein) NP_000792 (SEQ ID NO:3) . In embodiments, the reference numbers immediately above may refer to protein and associated nucleic acids known as of the filing date of this application. In embodiments, “FKBP-12” or “FKBP 12” or “FKBP 1A” may refer to wild-type human protein. In embodiments, “FKBP-12” or “FKBP 12” or “FKBP 1A” may refer to wild-type human nucleic acid. In embodiments, FKBP-12 is a mutant FKBP-12. In embodiments, mutant FKBP-12 is associated with a disease that is not associated with wild-type FKBP-12. In embodiments, FKBP-12 may include at least one amino acid mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mutations or any range inferred here) compared to wild type FKBP-12. In embodiments, FKBP-12 has a protein sequence corresponding to GI reference number: 206725550. In embodiments, FKBP-12 has a protein sequence corresponding to RefSeq NP_000792.1 (SEQ ID NO:3).

The term “4E-BP1” or “4EBP1” or “EIF4EBP1” refers to the protein “eukaryotic translation initiation factor 4E binding protein 1.” In embodiments, “4E-BP1” or “4EBP1” or “EIF4EBP1” may refer to a human protein. Included in the term, “4E-BP1” or “4EBP1” or “EIF4EBP1” are the wild type and mutant forms of the protein. In embodiments, “4E-BP1” or “4EBP1” or “EIF4EBP1” may refer to a protein associated with Entrez Gene 1978, OMIM 602223, UniProt Q13541 and/or RefSeq (protein) NP_004086 (SEQ ID NO:4). In embodiments, the reference numbers immediately above may refer to protein and associated nucleic acids known as of the filing date of this application. In embodiments, “4E-BP1” or “4EBP1” or “EIF4EBP1” may refer to a wild-type human protein. In embodiments, “4E-BP1” or “4EBP1” or “EIF4EBP1” may refer to a wild-type human nucleic acid. In embodiments, 4EBP1 is a 4EBP1 mutant. In embodiments, mutant 4EBP1 is associated with a disease that is not associated with wild type 4EBP1. In embodiments, 4EBP1 may include at least one amino acid mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mutations or any range inferred here) compared to wild type 4EBP1. In embodiments, 4EBP1 has a protein sequence corresponding to reference number GL4758258. In embodiments, 4EBP1 has a protein sequence corresponding to RefSeq NP_004086.1 (SEQ ID NO:4).

The term “Akt” refers to a serine/threonine specific protein kinase involved in cellular processes such as glucose metabolism, apoptosis, proliferation and other functions, also known as “protein kinase B” (PKB) or “Akt1.” In embodiments, “Akt” or “AM” or “PKB” may refer to a human protein. Included in the term, “Akt” or “Akt1” or “PKB” are the wild type and mutant forms of the protein. In embodiments, “Akt” or “Akt1” or “PKB” may refer to a protein associated with Entrez Gene 207, OMIM 164730, UniProt P31749 and/or RefSeq (protein) NP_005154 (SEQ ID NO:5). In embodiments, the reference numbers immediately above may refer to protein and associated nucleic acids known as of the filing date of this application. In embodiments, “Akt” or “Akt1” or “PKB” may refer to a wild-type human protein. In embodiments, “Akt” or “Akt1” or “PKB” may refer to a wild-type human nucleic acid. In embodiments, Akt is a mutant Akt. In embodiments, mutant Akt is associated with a disease that is not associated with wild-type Akt. In embodiments, Akt may include at least one amino acid mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mutations or any range inferred here) compared to wild-type Akt. In embodiments, Akt has a protein sequence corresponding to GI reference number: 62241011. In embodiments, Akt has a protein sequence corresponding to RefSeq NP_005154.2 (SEQ ID NO:5).

The present disclosure provides a method of treating a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or susceptible to developing a disease or disorder mediated by mTOR a therapeutically effective amount of one or more of the disclosed compositions or compounds. Provided herein is a method of preventing a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or susceptible to developing a disease or disorder mediated by mTOR a therapeutically effective amount of one or more of the disclosed compositions or compounds. The present disclosure provides a method of reducing the risk of an mTOR-mediated disease or disorder, comprising administering to a subject suffering from or susceptible to developing an mTOR-mediated disease or disorder a therapeutically effective amount of one or more of the disclosed compositions or compounds.

In some embodiments, the disease is cancer or an immune-mediated disease. In some embodiments, the cancer is selected from brain and neurovascular cancer, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, ovarian cancer, ovarian endometriosis, testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, proliferative blood disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, vulvar cancer, prostate cancer, penile cancer, bone cancer, muscle cancer, soft tissue cancer, colon or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors. In some embodiments, the disorder is cirrhosis of the liver. In some embodiments, the immune-mediated disease is selected from transplant resistance of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine, or pancreatic islet cells; graft-versus-host disease caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis. In certain embodiments, the disease is tuberous sclerosis complex (TSC). In certain embodiments, the disease is pancreatic neuroendocrine tumor (PNT), mantle cell lymphoma (MCL), colorectal cancer or colon cancer (COCC), uterine cancer, ovarian cancer, bladder cancer, genitourinary tract or renal cancer. cell carcinoma (CCC).

The present disclosure provides a method of treating cancer, comprising administering to a subject a therapeutically effective amount of one or more of the disclosed compositions or compounds. In some embodiments, the cancer is selected from brain and neurovascular tumors, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, ovarian cancer, ovarian endometriosis , testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, blood proliferation disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, vulvar cancer, prostate cancer , penile cancer, bone cancer, muscle cancer, soft tissue cancer, colon or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors. In some embodiments, the disease is cirrhosis of the liver. In some embodiments, the disease is tuberous sclerosis complex (TSC). In certain embodiments, the disease is pancreatic neuroendocrine tumors (PNT), mantle cell lymphoma (MCL), colorectal or colon cancer (RCC), uterine cancer, ovarian cancer, bladder cancer, genitourinary tract or renal cancer -cell carcinoma (CCC).

In certain embodiments, cancer includes human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreatic cancers , stomach, brain, head and neck, skin, uterus, testis, glioma, esophageal and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin lymphoma (for example, Burkitt lymphoma, small cell and large cell), Hodgkin lymphoma, leukemia (including AML, ALL and CML) or multiple myeloma. In certain embodiments, the disease is multiple myeloma. In certain embodiments, the disease is breast cancer. In certain embodiments, the disease is triple negative breast cancer.

In certain embodiments, cancer includes cancers, neoplasms, or malignancies found in mammals (eg, humans), including leukemia, carcinomas, and sarcomas. Exemplary cancers that may be treated with the compound or method presented herein include prostate, thyroid, endocrine, brain, breast, cervical, colon, head and neck, liver, kidney, lung, non-small cell lung cancer, melanoma, mesothelioma, ovarian, sarcoma, stomach, uterus, medulloblastoma, rectal and colon cancer, pancreatic cancer. Additional examples may include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulinoma, malignant neuroendocrine tumor, bladder cancer, precancerous lesions skin, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary pancreatic carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma or prostate cancer.

In certain embodiments, the disease is leukemia. The term “leukemia” broadly refers to progressive malignant diseases of the hematopoietic organs and is usually characterized by impaired proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is usually clinically classified based on (1) the duration and nature of the disease - acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous) or monocytic; and (3) an increase or absence of an increase in the number of aberrant cells in the blood - leukemic or aleukemic (subleukemic). Examples of leukemias that can be treated with the compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocythemic leukemia, basophilic leukemia, undifferentiated leukemia, bovine leukemia, chronic myelocytic leukemia, hemoderma, embryonal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, lymphosarcoma, basophilic leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Nägeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Reeder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia or undifferentiated cell leukemia.

In certain embodiments, the disease is sarcoma. The term “sarcoma” generally refers to a tumor that is composed of a substance similar to embryonic connective tissue and usually consists of densely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that can be treated with the compound or method presented herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemeti's sarcoma, adipose tissue sarcoma, liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, botryoid sarcoma, granulocytic sarcoma to whom , choriocarcinoma, embryonal sarcoma, Wilms tumor, endometrial sarcoma, stromal sarcoma, Ewing sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic B-cell sarcoma , lymphoma, immunoblastic T- cell sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, foliate cystosarcoma, synovial sarcoma or telangiectatic sarcoma.

In certain embodiments, the disease is melanoma. The term “melanoma” refers to a tumor consisting of the melanocytic system of the skin and other organs. Melanomas that can be treated with the compound or method presented herein include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passy melanoma, juvenile melanoma, lentigo melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma.

In certain embodiments, the disease is carcinoma. The term "carcinoma" refers to a malignant neoplasm consisting of epithelial cells that tend to invade surrounding tissues and cause metastases. Examples of carcinomas that can be treated with the compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenoid cystic carcinoma, glandular cystic carcinoma, adenomatous carcinoma, carcinoma adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basocellular carcinoma, basaloid carcinoma, basospinocellular carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, medullary carcinoma, cholangiocellular carcinoma, chor ionic carcinoma, colloid carcinoma, comedonal type carcinoma, corpus carcinoma, cribriform carcinoma, en cuirasse carcinoma, skin carcinoma, columnar carcinoma, columnar carcinoma, ductal carcinoma, solid carcinoma, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ex ulcere carcinoma, fibrosum carcinoma, carcinoma gelatiniforni, mucinous carcinoma, giant cell carcinoma, adenocarcinoma, follicular cell carcinoma, basal cell carcinoma, hematoid carcinoma, hepatocellular carcinoma, oncocytic carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma , Kulczycki cell carcinoma, large cell carcinoma, lens carcinoma, lenticulare carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, medullare carcinoma, medullary carcinoma, melanotic carcinoma, molle carcinoma, mucinous carcinoma, muciparum carcinoma, mucocellulare carcinoma, mucoepidermoid carcinoma noma, mucosum carcinoma, mucous carcinoma, myxomatodes carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossificans carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, forceps cell carcinoma, softened carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatodes carcinoma, III carcinoma neider, scirrhosa carcinoma, scroti carcinoma, signet ring cell carcinoma, undifferentiated carcinoma, small cell carcinoma, solanoid carcinoma, spherical cell carcinoma, carcinosarcoma, spongiosum carcinoma, squamous cell carcinoma, string carcinoma, telangiectaticum carcinoma, telangiectodes carcinoma, transitional cell carcinoma, tuberosum carcinoma , tuberous carcinoma, verrucous carcinoma or villosum carcinoma.

The present disclosure provides a method of treating an immune-mediated disease, comprising administering to a subject a therapeutically effective amount of one or more of the disclosed compositions or compounds. In some embodiments, the immune-mediated disease is selected from transplant resistance of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerves, duodenum, small intestine, or pancreatic islet cells; graft-versus-host diseases caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis.

In certain embodiments, the disease is an autoimmune disease. As used herein, the term “autoimmune disease” refers to a disease or condition in which the immune system of a subject has an aberrant immune response against a substance that does not normally elicit an immune response in a healthy subject. Examples of autoimmune diseases that can be treated with the compound, pharmaceutical composition or method described herein include acute disseminated encephalomyelitis (ADE), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti- TBM nephritis, antiphospholipid syndrome (APS), autoimmune antineurotic edema, autoimmune aplastic anemia, autoimmune autonomic dystonia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (ADID), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune new retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathies, Balo's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP) , chronic recurrent multiple osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucinous pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, primary cryoglobulinemia of mixed type, demyelinating neuropathies, herpetiformis dermatitis, dermatomyositis, Devic's disease (opticomyelitis), discoid lupus, Dressier's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myo carditis, glomerulonephritis , Goodpasture's syndrome, granulomatosis with polyangiitis (GPA) (formerly called Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gravidarum, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP) , IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, guttate scleroderma, woody conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic, Meniere's disease, microscopic polyangiitis, mixed connective tissue lesions (MCD), Moray ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devica), neutropenia, ocular cicatricial pemphigoid, optic neuritis, migratory arthritis, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (NPH), Parry-Romberg syndrome, Personage syndrome -Turner, parsplanitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndromes type I, II and III, polymyalgia rheumatica, polymyositis, post-infarction syndrome, postcardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, true red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever , rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff muscle syndrome, subacute bacterial endocarditis (SBE), Susac syndrome, metastatic ophthalmia, Takashi's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP) ), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicolubullous dermatosis, vitiligo or Wegener's granulomatosis (i.e. granulomatosis with polyangiitis (GPA)).

The present disclosure provides a method of treating an age-related condition, comprising administering to a subject a therapeutically effective amount of one or more of the disclosed compositions or compounds. In certain embodiments, the age-related condition is selected from sarcopenia, skin atrophy, muscle atrophy, brain atrophy, atherosclerosis, arteriosclerosis, emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia, Huntington's disease, Alzheimer's disease, cataracts, age-related macular degeneration, prostate cancer, stroke, shortened life expectancy, kidney dysfunction and age-related hearing loss, aging-related disabilities (eg frailty), cognitive decline, age-related dementia, memory impairment, tendon stiffness, cardiac dysfunction such as cardiac hypertrophy and systolic and diastolic dysfunction, immune aging, cancer, obesity and diabetes.

In certain embodiments, the disclosed compositions or compounds may be used in relation to the aging immune system. Aging of the immune system can refer to a decline in immune function, resulting in an impaired immune response, such as cancer, vaccination, infectious pathogens, among others. It involves both the host's ability to respond to infections and the development of long-term immune memory, especially through vaccination. This immunodeficiency is ubiquitous and is found in both long- and short-lived species depending on their age relative to lifespan rather than chronological time. This is considered a major contributing factor to the increased incidence of morbidity and mortality among older adults. Aging of the immune system is not a random deteriorating phenomenon; rather, it appears to follow an evolutionary pattern in reverse, and most of the parameters affected by aging of the immune system appear to be under genetic control. It is sometimes possible to foresee the aging of the immune system as a result of the constant challenge of unavoidable exposure to various antigens such as viruses and bacteria. Aging of the immune system is a multifactorial condition leading to many pathologically significant health problems, for example in the elderly population. Age-related biological changes, such as hematopoietic stem cell depletion, increased PD1+ lymphocytes, decreased total phagocytes and NK cells, and decreased humoral immunity, contribute to the onset of immune system aging. In one aspect, immune system aging can be measured in an individual by measuring telomere length in immune cells (see, for example, US Pat. No. 5,741,677). Aging of the immune system can also be determined by recording a person's lower than normal number of naïve CD4 and/or CD8 T cells, T cell repertoire, number of PD1 expressing T cells, e.g. lower than normal number of PD-1 negative T cells or response to vaccination in a subject 65 years of age or older. In certain embodiments, selective mTOR modulation of certain T cell populations may improve vaccine efficacy in the aging population and enhance the effectiveness of cancer immunotherapy. Provided herein is a method of treating an aging immune system, comprising administering to a subject a therapeutically effective amount of one or more of the disclosed compositions or compounds.

In certain embodiments, the disease that can be treated by the compound, pharmaceutical composition, or method described herein is organ or tissue transplant rejection (e.g., heart, lung, heart-lung, liver, kidney, pancreas). gland, skin or cornea; graft-versus-host disease), restenosis, hamartoma syndromes (eg, tuberous sclerosis or Cowden's disease), lymphangioleiomyomatosis, retinitis pigmentosa, encephalomyelitis, insulin-dependent diabetes mellitus, lupus, dermatomyositis, arthritis, rheumatic diseases, steroid-resistant acute lymphoblastic leukemia, fibrosis, scleroderma, pulmonary fibrosis, renal fibrosis, cystic fibrosis, pulmonary hypertension syndrome, multiple sclerosis, VHL syndrome, Carney syndrome, familial adenomatous polyposis, juvenile polyposis syndrome, Birt-Hogg-Duke syndrome, familial hypertrophic cardiomyopathy, Wolff syndrome Parkinson-White, Parkinson's disease, Huntington's disease, Alzheimer's disease, dementias caused by tau mutations, spinal cerebellar ataxia type 3, motor neuron disease caused by SOD1 mutations, neural waxy lipofuscinosis/Batten disease (childhood neurodegeneration), wet macular degeneration, dry macular degeneration , muscle wasting (wasting, cachexia), myopathies (eg, Danon disease), bacterial infection, viral infection, M. tuberculosis, group A streptococcus, HSV type I, HIV infection, neurofibromatosis (eg, neurofibromatosis type 1) or Peutz syndrome -Egers.

In certain embodiments, the disease is a neurodegenerative disease. As used herein, the term “neurodegenerative disease” refers to a disease or condition in which the function of a subject's nervous system is impaired. Examples of neurodegenerative diseases that can be treated with a compound, pharmaceutical composition or method described herein include Alexander disease, Alper disease, Alzheimer's disease, amyotrophic lateral sclerosis, ataxia-telangiectasia, Batten disease (also known as Spielmeyer-Vogt disease). Sjogren-Batten syndrome), mad cow disease (BCS), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy disease, disease Krabbe, kuru, dementia with Lewy bodies, Machado-Joseph disease (spinal cerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis, Parkinson's disease, Pelitz-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion diseases , Refsum's disease, Sandhoff's disease, Schilder's disease, subacute combined degeneration of the spinal cord secondary to pernicious anemia, schizophrenia, spinocerebellar ataxia (multiple types with varying characteristics), spinal muscular atrophy, Steele-Richardson-Olszewski disease or tabes.

In some embodiments, the disease is a metabolic disease. As used herein, the term "metabolic disease" refers to a disease or condition in which a subject's metabolism or metabolic system (eg, energy storage or utilization function) is impaired. Examples of metabolic diseases that can be treated with a compound, pharmaceutical composition, or method described herein include diabetes (eg, type I or type II), obesity, metabolic syndrome, or mitochondrial disease (eg, mitochondrial dysfunction or aberrant mitochondrial function).

In certain embodiments, the disease is a fungal disease. As used herein, the term “fungal disease” refers to a disease or condition associated with a fungal infection of a subject. Examples of fungal diseases that can be treated with the compound, pharmaceutical composition or method described herein include infections by Mucor circinelloides, zygomycetes, Cryptococcus neoformans, Candida albicans, yeast and Saccharomyces cerevisiae, among others.

In certain embodiments, the disease is an inflammatory disease. As used herein, the term “inflammatory disease” refers to a disease or condition characterized by aberrant inflammation (eg, increased levels of inflammation compared to a control, such as a healthy individual without the disease). Examples of inflammatory diseases include traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile diabetes, type 1 diabetes mellitus, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behçet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addison's disease, vitiligo, asthma, allergic asthma at , acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis and atopic dermatitis.

In certain embodiments, the disease is a cardiovascular disease. As used herein, the term “cardiovascular disease” refers to a disease or condition in which the function of a subject's cardiovascular system is impaired. Examples of cardiovascular diseases that can be treated with the compound, pharmaceutical composition or method described herein include congestive heart failure; arrhythmogenic syndromes (eg, delayed paroxysomal tachycardia, ventricular tachycardia, sudden onset tachycardia, exercise-induced arrhythmias, long QT syndromes, or torsade de pointes); thromboembolic disorders (eg, arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, or cardiac thromboembolic disorders); atherosclerosis; restenosis; peripheral artery disease; coronary artery bypass surgery; carotid artery disease; arteritis; myocarditis; cardiovascular inflammation; vascular inflammation; coronary heart disease (CHD); unstable angina (UA); unstable refractory angina; stable angina pectoris (SS); chronic stable angina; acute coronary syndrome (ACS); myocardial infarction (primary or repeated); acute myocardial infarction (AMI); myocardial infarction; myocardial infarction without Q wave; myocardial infarction without ST; atherosclerotic heart disease; coronary heart disease; cardiac ischemia; ischemia; sudden ischemic death; transient ischemic attack; stroke; peripheral artery occlusive disease; venous thrombosis; deep vein thrombosis; thrombophlebitis; arterial embolism; coronary artery thrombosis; cerebral arterial thrombosis, cerebral embolism; kidney embolism; pulmonary embolism; thrombosis (eg, associated with prosthetic valves or other implants, indwelling catheters, stents, cardiopulmonary bypass, hemodialysis); thrombosis (eg, associated with atherosclerosis, surgery, prolonged immobilization, arterial fibrillation, congenital thrombophilia, cancer, diabetes, hormones, or pregnancy); or cardiac arrhythmias (eg, supraventricular arrhythmias, atrial arrhythmias, atrial flutter, or atrial fibrillation).

In one aspect, a method is provided for treating a disease associated with an aberrant level of mTOR activity in a subject in need of such treatment. The disease may be caused by increased mTOR activity. The method may include administering to a subject one or more of the compositions or compounds described herein. The method may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In one aspect, one or more compositions or compounds described herein are provided for use as a drug. In embodiments, the drug is useful for treating a disease caused by up-regulation of mTOR. Use may include administering to a subject one or more of the compositions or compounds described herein. Use may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In one aspect, one or more compositions or compounds are provided, as described herein, for use in treating a disease caused by aberrant levels of mTORC1 activity in a subject in need of such treatment. The disease may be caused by activation of mTORC1. Use may include administering to a subject one or more of the compositions or compounds described herein. Use may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTORC1 modulator (eg, inhibitor) as described above).

Upregulation of mTOR can result in increased mTOR activity above normal levels of mTOR activity in a particular subject or group of healthy subjects. An increased amount of mTOR activity can lead, for example, to excessive cell proliferation, thereby causing a disease state.

The subject of treatment of the disease is usually a mammal. The mammal treated with a compound (eg, a compound described herein, an mTOR modulator (eg, inhibitor)) may be a human, a non-human primate, and/or a non-human mammal (eg, a rodent, a dog).

In another aspect, there is provided a method of treating a disease associated with mTOR activity in a subject in need of such treatment, wherein the method comprises administering one or more compositions or compounds as described herein, including embodiments (e.g., a claim, an embodiment , example, table, figure or claim) to the subject.

In another aspect, one or more compositions or compounds are provided, as described herein, for use as a drug. In embodiments, the drug may be useful for treating a disease associated with mTORC1 activity in a subject in need of such treatment. In embodiments, use may involve administering to a subject one or more compositions or compounds as described herein, including embodiments (eg, aspect, embodiment, example, table, figure, or claim).

In another aspect, one or more compositions or compounds are provided for use in treating a disease associated with mTOR activity in a subject in need of such treatment. In embodiments, use may include administering to a subject one or more compositions or compounds as described herein, including embodiments (eg, aspect, embodiment, example, table, figure, or claim).

In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is cancer. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is an autoimmune disease. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is an inflammatory disease. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is a neurodegenerative disease. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is a metabolic disease. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is graft rejection. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is a fungal infection. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is a cardiovascular disease.

In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is aging. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is death from an age-related disease. In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is an age-related condition. In certain embodiments, the age-related condition is selected from the group consisting of sarcopenia, skin atrophy, muscle wasting, brain atrophy, atherosclerosis, arteriosclerosis, emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia, Huntington's disease, Alzheimer's disease, cataracts, age-related macular degeneration, prostate cancer, stroke, decreased life expectancy, kidney dysfunction and age-related hearing loss, aging-related disabilities (eg, frailty), cognitive decline, age-related dementia, memory impairment, tendon stiffness, cardiac dysfunction , such as cardiac hypertrophy, systolic and diastolic dysfunction, aging immune system, cancer, obesity and diabetes. In certain embodiments, selective mTOR modulation of certain T cell populations may improve vaccine efficacy in the aging population and enhance the effectiveness of cancer immunotherapy. The present disclosure provides a method of treating an aging immune system, comprising administering to a subject a therapeutically effective amount of one or more of the disclosed compounds.

In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is cancer (e.g., carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid cancers, lymphoid cancers; kidney, breast, lung, urinary bladder, colon, gastrointestinal tract, ovary, prostate, pancreas, stomach, brain, head and neck, skin, uterus, esophagus, liver; testicular cancer, glioma, hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (eg, Burkitt's, small and large cell lymphomas), Hodgkin's lymphoma, leukemia (including AML, ALL and CML), multiple myeloma and breast cancer (eg, triple negative breast cancer)).

In embodiments, the disease associated with mTOR activity, or the disease associated with aberrant levels of mTOR activity, is acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM /anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dystonia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune internal ear disease (AIUD), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreat it, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathies, Balo's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuritis pathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressier's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis ( temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA) (formerly called Wegener's granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes of pregnancy, hypogammaglobulinemia, goo opathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis , lichen planus, guttate scleroderma, woody conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Moray ulcer, Mucha-Haberman disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devica), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH) ), Parry-Romberg syndrome, Parsonage-Turner syndrome, Parsa planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndromes types I, II and III, polymyalgia rheumatica, polymyositis , post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, true erythrocyte aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome a, relapsing polychondritis , restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff man syndrome, subacute bacterial endocarditis (SBE), Susak syndrome, sympathetic ophthalmia, Takayasu arteritis , temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, Wegener's granulomatosis (i.e. e. granulomatosis with polyangiitis (GPA), traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile diabetes, type 1 diabetes mellitus, Guillain-Barré syndrome, Hashimoto's encephalitis, type 1, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjögren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behçet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addyi's disease sona, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, atopic dermatitis, Alexander disease, Alper disease, Alzheimer's disease, Amyotrophic lateral sclerosis, ataxia-telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjögren-Batten disease), mad cow disease syndrome (MBS), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru, dementia with Lewy bodies, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion diseases, Refsum's disease, Sandhoff's disease, Schilder's disease, subacute combined degeneration of the spinal cord secondary to pernicious anemia, schizophrenia, spinocerebellar ataxia (several types with different characteristics ), spinal muscular atrophy, Steele-Richardson-Olszewski disease, tabes dorsalis, diabetes (eg, type I or type II), obesity, metabolic syndrome, mitochondrial disease (eg, mitochondrial dysfunction or aberrant mitochondrial function), fungal infection, rejection transplant or cardiovascular disease (eg, congestive heart failure; arrhythmogenic syndromes (eg, paroxisomal delayed post-depolarization tachycardia, ventricular tachycardia, sudden onset tachycardia, exercise-induced arrhythmias, long QT syndromes, or torsade de pointes); thromboembolic disorders (eg, arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, or cardiac thromboembolic disorders); atherosclerosis; restenosis; peripheral artery disease; coronary artery bypass surgery; carotid artery disease; arteritis; myocarditis; cardiovascular inflammation; vascular inflammation; coronary heart disease (CHD); unstable angina (UA); unstable refractory angina; stable angina pectoris (AS); chronic stable angina; acute coronary syndrome (ACS); myocardial infarction (first or repeated); acute myocardial infarction (AMI); myocardial infarction; myocardial infarction without Q wave; myocardial infarction without ST; atherosclerotic heart disease; cardiac ischemia; cardiac ischemia; ischemia; sudden ischemic death; transient ischemic attack; stroke; peripheral artery occlusive disease; venous thrombosis; deep vein thrombosis; thrombophlebitis; arterial embolism; coronary artery thrombosis; cerebral arterial thrombosis, cerebral embolism; kidney embolism; pulmonary embolism; thrombosis (eg, associated with prosthetic valves or other implants, indwelling catheters, stents, cardiopulmonary bypass, hemodialysis); thrombosis (eg, associated with atherosclerosis, surgery, prolonged immobilization, arterial fibrillation, congenital thrombophilia, cancer, diabetes, hormones, or pregnancy); or cardiac arrhythmias (eg, supraventricular arrhythmias, atrial arrhythmias, atrial flutter, or atrial fibrillation).

In one aspect, a method of treating a disease is provided, comprising administering an effective amount of one or more compositions or compounds as described herein. In one aspect, one or more compositions or compounds are provided, as described herein, for use as a drug (eg, to treat a disease). In one aspect, one or more compositions or compounds are provided, as described herein, for use in treating a disease (eg, including administering an effective amount of one or more compositions or compounds, as described herein). In embodiments, the disease is cancer. In embodiments, the disease is an autoimmune disease. In embodiments, the disease is an inflammatory disease. In embodiments, the disease is a neurodegenerative disease. In embodiments, the disease is a metabolic disease. In embodiments, the disease is a fungal infection. In embodiments, the disease is graft rejection. In embodiments, the disease is a cardiovascular disease.

In embodiments, the disease is cancer (e.g., carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid cancers, lymphoid cancers; kidney, breast, lung, bladder, colon, gastrointestinal, ovarian, prostate, pancreatic cancer , stomach, brain, head and neck, skin, uterus, esophagus, liver; testicular cancer, glioma, hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (eg, Burkitt, small and large cell lymphomas), Hodgkin's lymphoma, leukemia (including AML, ALL and CML), multiple myeloma and breast cancer (eg triple negative breast cancer)).

In embodiments, the disease is acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune vegetative dystonia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune internal ear disease (AIUD), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), auto immune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathies, Balo's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome , cicatricial pemphigoid/benign pemphigoid of the mucous membrane, Crohn's disease, kohans syndrome, cold agglutinins disease, congenital blockade of the heart, coksaki myocarditis, Crest, essential mixed cryoglobulinemia, demyelinizing neuropathy, herpetiform dermatitis, dermatomyositis, Devika Deviki's disease (optical. ROMILITS), Discoid lupus , Dressier's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (G PA) (formerly called Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes of pregnancy, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulator new lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, guttate scleroderma, woody conjunctivitis, linear IgA disease (LAD) ), lupus (SLE), Lyme disease, chronic, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Moray ulcer, Mucha-Haberman disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devica), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, planitis Parsa (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndromes types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, true red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff man syndrome, subacute bacterial endocarditis (SBE), Susak syndrome, sympathetic ophthalmia, Takayasu arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TPP), Tolosa syndrome- Hant's, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, Wegener's granulomatosis (i.e. granulomatosis with polyangiitis (GPA), traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile diabetes, type 1 diabetes mellitus, Guillain-Barre syndrome, Hashimoto's encephalitis, type 1, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjögren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behçet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addyi's disease sona, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, atopic dermatitis, Alexander disease, Alper disease, Alzheimer's disease, Amyotrophic lateral sclerosis, ataxia-telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjögren-Batten disease), mad cow disease syndrome (MBS), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru, dementia with Lewy bodies, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion diseases, Refsum's disease, Sandhoff's disease, Schilder's disease, subacute combined degeneration of the spinal cord secondary to pernicious anemia, schizophrenia, spinocerebellar ataxia (several types with different characteristics ), spinal muscular atrophy, Steele-Richardson-Olszewski disease, tabes dorsalis, diabetes (eg, type I or type II), obesity, metabolic syndrome, mitochondrial disease (eg, mitochondrial dysfunction or aberrant mitochondrial function), fungal infection, rejection transplant or cardiovascular disease (eg, congestive heart failure; arrhythmogenic syndromes (eg, paroxisomal delayed post-depolarization tachycardia, ventricular tachycardia, sudden onset tachycardia, exercise-induced arrhythmias, long QT syndromes, or torsade de pointes); thromboembolic disorders (eg, arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, or cardiac thromboembolic disorders); atherosclerosis; restenosis; peripheral artery disease; coronary artery bypass surgery; carotid artery disease; arteritis; myocarditis; cardiovascular inflammation; vascular inflammation; coronary heart disease (CHD); unstable angina (UA); unstable refractory angina; stable angina pectoris (AS); chronic stable angina; acute coronary syndrome (ACS); myocardial infarction (first or repeated); acute myocardial infarction (AMI); myocardial infarction; myocardial infarction without Q wave; myocardial infarction without ST; atherosclerotic heart disease; cardiac ischemia; cardiac ischemia; ischemia; sudden ischemic death; transient ischemic attack; stroke; peripheral artery occlusive disease; venous thrombosis; deep vein thrombosis; thrombophlebitis; arterial embolism; coronary artery thrombosis; cerebral arterial thrombosis, cerebral embolism; kidney embolism; pulmonary embolism; thrombosis (eg, associated with prosthetic valves or other implants, indwelling catheters, stents, cardiopulmonary bypass, hemodialysis); thrombosis (eg, associated with atherosclerosis, surgery, prolonged immobilization, arterial fibrillation, congenital thrombophilia, cancer, diabetes, hormones, or pregnancy); or cardiac arrhythmias (eg, supraventricular arrhythmias, atrial arrhythmias, atrial flutter, or atrial fibrillation). In embodiments, the disease is polycystic disease. In embodiments, the disease is polycystic kidney disease. In embodiments, the disease is stenosis. In embodiments, the disease is restenosis. In embodiments, the disease is neointimal proliferation. In embodiments, the disease is neointimal hyperplasia.

In another aspect, there is provided a method of treating aging in a subject in need of such treatment, wherein the method includes administering one or more compositions or compounds as described herein, including embodiments (e.g., a claim, an embodiment, an example, a table, a figure or claim) to the subject. Provided herein is a method of treating an aging immune system, comprising administering to a subject a therapeutically effective amount of one or more of the described compounds or compositions.

In another aspect, one or more compositions or compounds are provided, as described herein, for use as a drug. In embodiments, the drug may be useful for treating aging in a subject in need of such treatment. In embodiments, use may include administering to a subject one or more compositions or compounds as described herein, including embodiments (eg, aspect, embodiment, example, table, figure, or claim).

In another aspect, one or more compositions or compounds disclosed herein are provided for use in treating aging in a subject in need of such treatment. In embodiments, use may include administering to a subject one or more compositions or compounds as described herein, including embodiments (eg, aspect, embodiment, example, table, figure, or claim).

In another aspect, there is provided a method of increasing life expectancy or inducing longevity in a subject in need of such treatment, wherein the method includes administering one or more compositions or compounds as described herein, including embodiments (e.g., claim, embodiment, example, table, figure or claim) to the subject.

In another aspect, one or more compositions or compounds are provided, as described herein, for use as a drug. In embodiments, the drug may be used to prolong life or induce longevity in a subject in need of such treatment. In embodiments, use may include administering to a subject one or more compositions or compounds as described herein, including embodiments (eg, aspect, embodiment, example, table, figure, or claim).

In another aspect, one or more compositions or compounds are provided for use in increasing lifespan or inducing longevity in a subject in need of such treatment. In embodiments, use may include administering to a subject one or more compositions or compounds as described herein, including embodiments (eg, aspect, embodiment, example, table, figure, or claim).

In one aspect, a method of treating polycystic disease in a subject in need of such treatment is provided. Polycystic disease may be polycystic kidney disease. The method may include administering to a subject one or more of the compositions or compounds described herein. The method may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In one aspect, one or more compositions or compounds are provided, as described herein, for use as a drug. In embodiments, the drug is useful for treating polycystic disease. Polycystic disease may be polycystic kidney disease. Use may include administering to a subject one or more of the compositions or compounds described herein. Use may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In one aspect, one or more compositions or compounds are provided, as described herein, for use in treating polycystic disease in a subject in need of such treatment. Polycystic disease may be polycystic kidney disease. Use may include administering to a subject one or more of the compositions or compounds described herein. Use may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In one aspect, a method of treating stenosis in a subject in need of such treatment is provided. Stenosis may be restenosis. The method may include administering to a subject one or more of the compositions or compounds described herein. In embodiments, one or more compositions or compounds are administered in a drug-eluting stent. The method may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In one aspect, one or more compositions or compounds are provided, as described herein, for use as a drug. In embodiments, the drug is useful for treating stenosis. Stenosis may be restenosis. Use may include administering to a subject one or more of the compositions or compounds described herein. In embodiments, the compound is administered in a drug-eluting stent. Use may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In one aspect, one or more compositions or compounds are provided, as described herein, for use in treating stenosis in a subject in need of such treatment. Stenosis may be restenosis. Use may include administering to a subject one or more of the compositions or compounds described herein. In embodiments, one or more compositions or compounds are administered in a drug-eluting stent. Use may include administering to a subject a therapeutically effective amount of one or more compositions or compounds described herein (eg, an mTOR modulator (eg, inhibitor) as described above).

In embodiments, the disease is a disease described herein, and the compound is a compound described herein, and the composition is a composition described herein.

Ways to modulate mTOR

In some embodiments, compounds disclosed herein are more selective inhibitors of mTORC1 compared to mTORC2. In some embodiments, compounds disclosed herein are more selective inhibitors of mTORC2 compared to mTORC1. In some embodiments, the compounds disclosed herein do not exhibit a difference in selectivity between mTORC1 and mTORC2.

In another aspect, a method is provided for modulating mTORC1 activity in a subject in need thereof, including administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof. In embodiments, the method includes inhibiting mTORC1 activity. In embodiments, the method includes inhibiting mTORC1 activity, but not inhibiting mTORC2 activity.

In embodiments, the method includes inhibiting mTORC1 activity to a greater extent than inhibiting mTORC2 activity. In embodiments, the method comprises inhibiting mTORC1 activity by at least 1.1 times greater than inhibiting mTORC2 activity (e.g., at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000 or 1000000 times).

In another aspect, a method is provided for modulating mTORC2 activity in a subject in need thereof, including administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof. In embodiments, the method includes inhibiting mTORC2 activity. In embodiments, the method includes inhibiting mTORC2 activity, but not inhibiting mTORC1 activity.

In embodiments, the method includes inhibiting mTORC2 activity to a greater extent than inhibiting mTORC1 activity. In embodiments, the method comprises inhibiting mTORC2 activity by at least 1.1 times greater than inhibiting mTORC1 activity (e.g., at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000 or 1000000 times).

In some embodiments, mTOR is located in a cell. In some embodiments, the cell is a mammalian cell, such as a human cell. A cell may be isolated in vitro , form part of a tissue in vitro , or may form part of an organism.

Exemplary Embodiments

Some embodiments of this disclosure are Embodiment I, as follows:

Embodiment I-1. Compound of formula I:

(I)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O or -OR ³ ;

R ²⁸ is -H or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where at least one of R ²⁸ and R ⁴⁰ is not H;

Z ¹ is O or S;

R ^28a and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -L ² -A ¹ -L ¹ -A ² -L ³ -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

, , , , , , , , , , , , , , , , , , ;

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )- or L ² ; and where the bond on the right side of the A ² group, as depicted, is bonded to B or L ³ ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

L ¹ is independently selected from

L ² and L ³ are independently absent or independently selected from

;

B is independently chosen from

B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , heteroarylene-, heterocyclylene arylene- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where the connection on the left side of B ¹ , as shown, is connected to A ² , L ³ or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are each independently optional substituted -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O )NR ³ -heteroaryl or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each Y is independently -C(R ³ ) ₂ or a bond;

each n is independently a number from one to 12;

each o is independently a number from zero to 30;

each p is independently a number from zero to 12;

each q is independently a number from zero to 30; And

each r is independently a number from one to 6.

Embodiment I-2. Compound of formula II:

(II)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O or -OR ³ ;

R ²⁸ is -H or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where at least one of R ²⁸ and R ⁴⁰ is not H;

Z ¹ is O or S;

R ^28a and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

, , , , , , , , , , , , , , , , , , ;

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )-; and where the bond on the right side of the group A ² , as depicted, is bonded to B;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

L ¹ is independently selected from

B is independently chosen from

,

B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , heteroarylene-, heterocyclylene arylene- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where the bond on the left side of B ¹ , as shown, is connected to A ² or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are optionally substituted - N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O)NR ³ -heteroaryl; or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each Y is independently a C(R ³ ) ₂ or bond;

each n is independently a number from one to 12;

each o is independently a number from zero to 30;

each p is independently a number from zero to 12;

each q is independently a number from zero to 30; And

each r is independently a number from one to 6.

Embodiment I-3. The connection of embodiment I-1 or I-2, where R ³² is =O.

Embodiment I-4. The compound of embodiment I-1 or I-2, wherein R ³² is -OR ³ .

Embodiment I-5. The compound of any one of embodiments I-1 to I-4, wherein the compounds are represented by the structure of formula I-40:

(I-40)

or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment I-6. The connection of embodiment I-5, where Z ¹ is O.

Embodiment I-7. The connection of embodiment I-5, where Z ¹ is S.

Embodiment I-8. The connection of any one of embodiments I-5 to I-7, wherein R ^40a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent.

Embodiment I-9. The connection according to any one of embodiments I-5 to I-7, where R ^40a is -A ¹ -L ¹ -A ² -B, where A ² is absent.

Embodiment I-10. The connection according to any one of embodiments I-5 to I-7, where R ^40a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent.

Embodiment I-11. The connection according to any one of embodiments I-5 to I-7, where R ^40a is -A ¹ -L ¹ -A ² -B.

Embodiment I-12. The connection according to any one of embodiments I-5 to I-7, where R ^40a is -A ¹ -A ² -B.

Embodiment I-13. The connection of any one of embodiments I-5 to I-7, wherein R ^40a is --L ² -A ¹ -L ^{1 -A 2} -L ³ -B, where L ² and A ¹ are absent.

Embodiment I-14. The connection of any one of embodiments I-5 to I-7, wherein R ^40a is --L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² is absent.

Embodiment I-15. The connection of any one of embodiments I-5 to I-7, wherein R ^40a is --L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

Embodiment I-16. The compound of any one of embodiments I-5 to I-7, wherein R ^40a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen.

Embodiment I-17. The compound of any one of embodiments I-1 to I-4, wherein the compounds are represented by the structure of formula I-28:

(I-28)

or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment I-18. The connection of embodiment I-17, where Z ¹ is O.

Embodiment I-19. The connection of embodiment I-17, where Z ¹ is S.

Embodiment I-20. The connection of any one of embodiments I-17 to I-19, wherein R ^28a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent.

Embodiment I-21. The connection of any one of embodiments I-17 to I-19, wherein R ^28a is -A ¹ -L ¹ -A ² -B, where A ² is absent.

Embodiment I-22. The connection according to any one of embodiments I-17 to I-19, wherein R ^28a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent.

Embodiment I-23. The connection according to any one of embodiments I-17 to I-19, where R ^28a is -A ¹ -L ¹ -A ² -B.

Embodiment I-24. The connection of any one of embodiments I-17 to I-19, wherein R ^28a is -A ¹ -A ² -B.

Embodiment I-25. The connection of any one of embodiments I-17 to I-19, wherein R ^28a is --L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² and A ¹ are absent.

Embodiment I-26. The connection of any one of embodiments I-17 to I-19, wherein R ^28a is --L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² is absent.

Embodiment I-27. The connection of any one of embodiments I-17 to I-19, wherein R ^28a is --L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

Embodiment I-28. The compound of any one of embodiments I-17 to I-19, wherein R ^28a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen.

Embodiment I-29. The connection according to any one of embodiments I-1 - I-11, I-13 - I-15, I-17 - I-23 and I-25 - I-27, where L ¹ is .

Embodiment I-30. The connection according to any one of embodiments I-1 - I-11, I-13 - I-15, I-17 - I-23 and I-25 - I-27, where L ¹ is .

Embodiment I-31. The connection according to any one of embodiments I-1 - I-11, I-13 - I-15, I-17 - I-23 and I-25 - I-27, where L ¹ is .

Embodiment I-32. The connection according to any one of embodiments I-1 - I-11, I-13 - I-15, I-17 - I-23 and I-25 - I-27, where L ¹ is .

Embodiment I-33. The connection according to any one of embodiments I-1 - I-11, I-13 - I-15, I-17 - I-23 and I-25 - I-27, where L ¹ is .

Embodiment I-34. The connection according to any one of embodiments I-1 - I-7, I-15, I-17 - I-19 and I-27, where L ² is .

Embodiment I-35. The connection according to any one of embodiments I-1 - I-7, I-13 - I-14, I-17 - I-19 and I-25 - I-26, where L ³ is .

Embodiment I-36. The connection according to any of the embodiments I-1 - I-8, I-10, I-13, I-17 - I-19, I-20, I-22, I-25 and I-29 - I-35, where A ¹ is missing.

Embodiment I-37. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is .

Embodiment I-38. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is .

Embodiment I-39. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is .

Embodiment I-40. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is .

Embodiment I-41. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is

Embodiment I-42. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is .

Embodiment I-43. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is .

Embodiment I-44. Connection according to any of the embodiments I-1 - I-7, I-9, I-11 - I-12, I-14 - I-15, I-17 - I-19, I-21, I-23 - I-24, I-26 - I-27 and I-29 - I-35, where A ¹ is .

Embodiment I-45. The connection according to any one of embodiments I-1 - I-9, I-17 - I-21 and I-29 - I-44, where A ² is absent.

Embodiment I-46. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-47. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-48. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-49. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-50. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-51. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-52. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-53. The connection according to any one of embodiments I-1 - I-7, I-10 - I-15, I-17 - I-19, I-22 - I-27 and I-29 - I-44, where A ² is .

Embodiment I-54. The connection as in any one of embodiments I-1 to I-53, wherein B is .

Embodiment I-55. The connection as in any one of embodiments I-1 to I-53, wherein B is .

Embodiment I-56. The connection according to any one of embodiments I-1 to I-53, where B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -.

Embodiment I-57. The connection according to any one of embodiments I-1 to I-53, where B ¹ is .

Embodiment I-58. The compound of any one of embodiments I-1 through I-57, wherein R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN or -C(O)NR ³ -heteroaryl.

Embodiment I-59. The compound of any one of embodiments I-1 to I-58, or a pharmaceutically acceptable salt or tautomer thereof, wherein the compound has the following formula:

Embodiment I-60. A connection selected from the group consisting of:

Example 1 Example 2 Example 3 Example 4
Example 5
Example 6
Example 7
Example 8
Example 9
Example 10
Example 11
Example 12
Example 13
Example 14
Example 15
Example 16
Example 17
Example 18
Example 19
Example 20
Example 21
Example 22
Example 23
Example 24
Example 25
Example 26
Example 27
Example 28
Example 29
Example 30
Example 31
Example 32
Example 33
Example 34
Example 35
Example 36
Example 37
Example 38
Example 39
Example 40
Example 41
Example 42
Example 43
Example 44
Example 45
Example 46
Example 47
Example 48
Example 49
Example 50
Example 51
Example 52
Example 53
Example 54
Example 55
Example 56
Example 57
Example 58
Example 59
Example 60
Example 61
Example 62
Example 63
Example 64
Example 65
Example 66
Example 67
Example 68
Example 69
Example 70

or a pharmaceutically acceptable salt or isomer thereof.

Embodiment I-61. A pharmaceutical composition comprising a compound of any one of embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof and at least one of a pharmaceutically acceptable carrier, diluent or excipient.

Embodiment I-62. A method of treating a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or at risk of developing a disease or disorder mediated by mTOR, a therapeutically effective amount of one or more compounds of any one of embodiments I-1 to I-60, or a pharmaceutically acceptable salt thereof.

Embodiment I-63. A method of preventing an mTOR-mediated disease or disorder, comprising administering to a subject suffering from or at risk of developing an mTOR-mediated disease or disorder a therapeutically effective amount of one or more compounds of any one of Embodiments I-1 to I-60, or a pharmaceutically acceptable salt thereof .

Embodiment I-64. A method of reducing the risk of an mTOR-mediated disease or disorder, comprising administering to a subject suffering from or at risk of developing an mTOR-mediated disease or disorder a therapeutically effective amount of one or more compounds of any one of Embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof .

Embodiment I-65. The method of any one of embodiments I-62 to I-64, wherein the disease is cancer or an immune-mediated disease.

Embodiment I-66. The method of Embodiment I-65, wherein the cancer is selected from brain and neurovascular tumors, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, cancer ovarian, ovarian endometriosis, testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, blood proliferation disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, cancer vulva, prostate cancer, penile cancer, bone cancer, muscle cancer, soft tissue cancer, bowel or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors.

Embodiment I-67. The method of Embodiment I-65, wherein the immune-mediated disease is selected from transplant resistance of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine, or islet pancreatic cells; graft-versus-host diseases caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis.

Embodiment I-68. A method of treating cancer comprising administering to a subject a therapeutically effective amount of one or more compounds of any one of Embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof.

Embodiment I-69. The method of Embodiment I-68, wherein the cancer is selected from brain and neurovascular tumors, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, cancer ovarian, ovarian endometriosis, testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, blood proliferation disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, cancer vulva, prostate cancer, penile cancer, bone cancer, muscle cancer, soft tissue cancer, bowel or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors.

Embodiment I-70. A method of treating an immune-mediated disease, comprising administering to a subject a therapeutically effective amount of one or more compounds of any one of Embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof.

Embodiment I-71. The method of Embodiment I-70, wherein the immune-mediated disease is selected from transplant resistance of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine, or islet pancreatic cells; graft-versus-host diseases caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis.

Embodiment I-72. A method of treating an age-related condition, comprising administering to a subject a therapeutically effective amount of one or more compounds of any one of Embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof.

Embodiment I-73. The method of Embodiment I-72, wherein the age-related condition is selected from sarcopenia, skin atrophy, muscle atrophy, brain atrophy, atherosclerosis, arteriosclerosis, emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia, Huntington's disease, Alzheimer's disease , cataracts, age-related macular degeneration, prostate cancer, stroke, shortened life expectancy, kidney dysfunction and age-related hearing loss, aging-related disabilities (eg, frailty), cognitive decline, age-related dementia, memory impairment, tendon stiffness, heart disease dysfunctions such as cardiac hypertrophy and systolic and diastolic dysfunction, immune aging, cancer, obesity and diabetes.

Embodiment I-74. The compound of any one of embodiments I-1 to I-60, or a pharmaceutically acceptable salt thereof, for use in the treatment, prevention, or reduction of the risk of a disease or condition mediated by mTOR.

Embodiment I-75. Use of a compound of any one of embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating, preventing, or reducing the risk of a disease or condition mediated by mTOR.

Embodiment I-76. The compound of any one of embodiments I-1 to I-60, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

Embodiment I-77. Use of the compound of any one of embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.

Embodiment I-78. The compound of any one of embodiments I-1 to I-60, or a pharmaceutically acceptable salt thereof, for use in the treatment of an immune-mediated disease.

Embodiment I-79. Use of a compound of any one of embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of an immune-mediated disease.

Embodiment I-80. The compound of any one of embodiments I-1 to I-60, or a pharmaceutically acceptable salt thereof, for use in treating an age-related condition.

Embodiment I-81. Use of the compound of any one of embodiments I-1 to I-60 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating an age-related condition.

Some embodiments of this disclosure are Embodiment II presented below:

Embodiment II-1. Compound of formula Ic:

(Ic)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O, -OR ³ , -N ₃ or -OC(=Z ¹ )-R ^32a ;

R ²⁸ is -H, (C ₁ -C ₆ )alkyl or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where if R ²⁸ and R ⁴⁰ are H, then R ³² is not =O;

each Z ¹ is independently O or S;

R ^28a , R ^32a and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -L ² -A ¹ -L ¹ -A ² -L ³ -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

, , , , , , , , , , , , , , , , , , , And ;

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )- or L ² ; and where the bond on the right side of the A ² group, as depicted, is bonded to B or L ³ ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

each L ¹ is independently selected from

L ² and L ³ are independently absent or independently selected from

each B is independently selected from

each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-(C(R ³ ) ₂ ) _n -, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , , heteroarylene-, heterocyclylene arylene-, , NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-(C(R ³ ) ₂ ) _n -, where the connection on the left side of B ¹ , as shown, is connected to A ² , L ³ or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are optionally substituted - N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O)NR ³ -heteroaryl or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each Y is independently a C(R ³ ) ₂ or bond;

each n is independently an integer from one to 12;

each o is independently an integer from zero to 30;

each p is independently an integer between zero and 12;

each q is independently an integer from zero to 30; And

each r is independently an integer from one to 6.

Embodiment II-1A. Compound of formula Ia:

(Ia)

or a pharmaceutically acceptable salt or tautomer thereof, wherein:

R ³² is -H, =O, -OR ³ , -N ₃ or -OC(=Z ¹ )-R ^32a ;

R ²⁸ is -H, (C ₁ -C ₆ )alkyl or -C(=Z ¹ )-R ^28a ;

R ⁴⁰ is -H or -C(=Z ¹ )-R ^40a ;

where if R ²⁸ and R ⁴⁰ are H, then R ³² is not =O;

each Z ¹ is independently O or S;

R ^28a , R ^32a and R ^40a are independently -A ¹ -L ¹ -A ² -B; -A ¹ -A ² -B; -L ² -A ¹ -L ¹ -A ² -L ³ -B; -O-(C ₁ -C ₆ )alkyl; or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen;

A ¹ and A ² are independently absent or independently selected from

, , , , , , , , , , , , , , , , , , And ;

where the bond on the left side of A ¹ , as shown, is connected to -C(=Z ¹ )- or L ² ; and where the bond on the right side of the A ² group, as depicted, is bonded to B or L ³ ;

each Q is independently 1-3 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each X ¹ is independently a heteroarylene or heterocyclylene ring;

each W is independently absent or has 1-2 rings selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each W ¹ is independently a heteroarylene or heterocyclylene ring;

each G is independently absent or is a ring selected from arylene, cycloalkylene, heteroarylene and heterocyclylene;

each G ¹ and G ² is independently a heteroarylene or heterocyclylene ring;

each L ¹ is independently selected from

,

L ² and L ³ are independently absent or independently selected from

each B is independently selected from

each B ¹ is independently selected from NR ³ -(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -(C ₆ -C ₁₀ )arylene-(C(R ³ ) ₂ ) _n -, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylene-, (C ₆ -C ₁₀ )arylene-, NR ³ -(C(R ³ ) ₂ ) _n -NR ³ C(O)-, NR ³ -(C(R ³ ) ₂ ) _n -heteroarylenheterocyclylene-(C ₆ -C ₁₀ )arylene-, heteroaryleneheterocyclylene-(C ₆ -C ₁₀ )arylene-, , -heteroarylene, , , , , (C ₆ -C ₁₀ )arylene, , heteroarylene-, heterocyclylene arylene- and NR ³ -(C(R ³ ) ₂ ) _n -S(O) ₂ -arylene-C(O)-, where the connection on the left side of B ¹ , as shown, is connected to A ² , L ³ or L ¹ ; and wherein heteroarylene, heterocyclylene and arylene are each independently optionally substituted with alkyl, hydroxyalkyl, haloalkyl, alkoxy, halogen or hydroxyl;

each R ³ is independently H or (C ₁ -C ₆ )alkyl;

each R ⁴ is independently H, (C ₁ -C ₆ )alkyl, halogen, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, (C ₆ -C ₁₀ )aryl, where heteroaryl, heterocyclyl and aryl are optionally substituted - N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN, -C(O)NR ³ -heteroaryl or -C(O)NR ³ -heterocyclyl;

each R ⁵ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁶ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁷ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each R ⁸ is independently H, (C ₁ -C ₆ )alkyl, -C(O)OR ³ or -N(R ³ ) ₂ , where the alkyl of (C ₁ -C ₆ )alkyl is optionally substituted with -N(R ³ ) ₂ or -OR ³ ;

each Y is independently a C(R ³ ) ₂ or bond;

each n is independently an integer from one to 12;

each o is independently an integer from zero to 30;

each p is independently an integer between zero and 12;

each q is independently an integer from zero to 30; And

each r is independently an integer from one to 6.

Embodiment II-2. The compound of Embodiment II-1, wherein R ³² is =O.

Embodiment II-3. The compound of Embodiment II-1, wherein R ³² is -OR ³ .

Embodiment II-4. The compound of any one of embodiments II-1 to II-3, or a pharmaceutically acceptable salt or tautomer thereof, wherein the compound is represented by the structure of formula (I-40b):

(I-40b)

where R ⁴⁰ is -C(=Z ¹ )-R ^40a .

Embodiment II-5. The connection of embodiment II-4, where Z ¹ is O.

Embodiment II-6. The connection of embodiment II-4, where Z ¹ is S.

Embodiment II-7. The connection according to any one of embodiments II-4 to II-6, where R ^40a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent.

Embodiment II-8. The connection according to any one of embodiments II-4 to II-6, wherein R ^40a is -A ¹ -L ¹ -A ² -B, where A ² is absent.

Embodiment II-9. The connection according to any one of embodiments II-4 to II-6, where R ^40a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent.

Embodiment II-10. The connection according to any one of embodiments II-4 through II-6, where R ^40a is -A ¹ -L ¹ -A ² -B.

Embodiment II-11. The connection according to any one of embodiments II-4 through II-6, where R ^40a is -A ¹ -A ² -B.

Embodiment II-12. The connection according to any one of embodiments II-4 to II-6, where R ^40a is -L ² -A ¹ -L ^{1 -A 2} -L ³ -B, where L ² and A ¹ are absent.

Embodiment II-13. The connection according to any one of embodiments II-4 through II-6, where R ^40a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² is absent.

Embodiment II-14. The connection according to any one of embodiments II-4 through II-6, where R ^40a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

Embodiment II-15. The compound of any one of embodiments II-4 to II-6, wherein R ^40a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen.

Embodiment II-16. The compound of any one of embodiments II-1 to II-3, or a pharmaceutically acceptable salt or tautomer thereof, wherein the compounds are represented by the structure of formula (I-28b):

(I-28b)

where R ²⁸ is -C(=Z ¹ )-R ^28a .

Embodiment II-17. The connection of embodiment II-16, where Z ¹ is O.

Embodiment II-18. The connection of embodiment II-16, where Z ¹ is S.

Embodiment II-19. The connection according to any one of embodiments II-16 to II-18, where R ^28a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent.

Embodiment II-20. The connection according to any one of embodiments II-16 to II-18, wherein R ^28a is -A ¹ -L ¹ -A ² -B, where A ² is absent.

Embodiment II-21. The connection according to any one of embodiments II-16 to II-18, wherein R ^28a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent.

Embodiment II-22. The connection according to any one of embodiments II-16 to II-18, where R ^28a is -A ¹ -L ¹ -A ² -B.

Embodiment II-23. The connection according to any one of embodiments II-16 to II-18, where R ^28a is -A ¹ -A ² -B.

Embodiment II-24. The connection according to any one of embodiments II-16 to II-18, where R ^28a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² and A ¹ are absent.

Embodiment II-25. The connection according to any one of embodiments II-16 to II-18, where R ^28a is -L ² -A ¹ -L ^{1 -A 2} -L ³ -B, where L ² is absent.

Embodiment II-26. The connection according to any one of embodiments II-16 to II-18, where R ^28a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

Embodiment II-27. The compound of any one of embodiments II-16 to II-18, wherein R ^28a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen.

Embodiment II-28. The compound of Embodiment II-1, or a pharmaceutically acceptable salt or tautomer thereof, wherein the compound is represented by the structure of formula (I-32b):

(I-32b)

where R ³² is -OC(=Z ¹ )-R ^32a .

Embodiment II-29. The connection of embodiment II-28, where Z ¹ is O.

Embodiment II-30. The connection of embodiment II-28, where Z ¹ is S.

Embodiment II-31. The connection according to any one of embodiments II-28 to II-30, where R ^32a is -A ¹ -L ¹ -A ² -B, where A ¹ and A ² are absent.

Embodiment II-32. The compound of any one of embodiments II-28 to II-30, wherein R ^32a is -A ¹ -L ¹ -A ² -B, where A ² is absent.

Embodiment II-33. The connection according to any one of embodiments II-28 to II-30, where R ^32a is -A ¹ -L ¹ -A ² -B, where A ¹ is absent.

Embodiment II-34. The compound of any one of embodiments II-28 to II-30, wherein R ^32a is -A ¹ -L ¹ -A ² -B.

Embodiment II-35. The compound of any one of embodiments II-28 to II-30, wherein R ^32a is -A ¹ -A ² -B.

Embodiment II-36. The connection according to any one of embodiments II-28 to II-30, where R ^32a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² and A ¹ are absent.

Embodiment II-37. The connection according to any one of embodiments II-28 to II-30, where R ^32a is -- ² -A ¹ -L ¹ -A ² -L ³ -B, where L ² is absent.

Embodiment II-38. The compound of any one of embodiments II-28 to II-30, wherein R ^32a is -L ² -A ¹ -L ¹ -A ² -L ³ -B, where L ³ is absent.

Embodiment II-39. The compound of any one of embodiments II-28 to II-30, wherein R ^32a is -O-(C ₁ -C ₆ )alkyl or -O-(C ₆ -C ₁₀ )aryl; where aryl from -O-(C ₆ -C ₁₀ )aryl is unsubstituted or substituted with 1-5 substituents selected from -NO ₂ and halogen.

Embodiment II-40. The connection according to any one of embodiments II-1 - II-10, II-12 - II-22, II-24 - II-35 and II-36 - II-39, where L ¹ is .

Embodiment II-41. The connection according to any one of embodiments II-1 - II-10, II-12 - II-22, II-24 - II-35 and II-36 - II-39, where L ¹ is .

Embodiment II-42. The connection according to any of embodiments II-1 - II-10, II-12 - II-22, 24-35 and 36-39, where L ¹ is .

Embodiment II-43. The connection according to any one of embodiments II-1 - II-10, II-12 - II-22, II-24 - II-35 and II-36 - II-39, where L ¹ is .

Embodiment II-44. The connection according to any one of embodiments II-1 - II-10, II-12 - II-22, II-24 - II-35 and II-36 - II-39, where L ¹ is .

Embodiment II-45. The connection according to any one of embodiments II-1 - II-10, II-12 II-22, II-24 - II-35 and II-36 - II-39, where L ¹ is .

Embodiment II-46. The connection according to any one of embodiments II-1 - II-6, II-12 - II-18, II-24 - II-30 and II-36 - II-45, where L ² is .

Embodiment II-47. The connection according to any one of embodiments II-1 - II-6, II-12 - II-18, II-24 - II-30 and II-36 - II-45, where L ³ is .

Embodiment II-48. The connection according to any of the embodiments II-1 - II-7, II-9, II-12, II-16 - II-19, II-21, II-24, II-28 - II-31, II-33, II-36 and II-39 - II-45, where A ¹ is absent.

Embodiment II-49. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-50. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-51. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-52. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-53. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-54. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-55. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-56. Connection according to any of the embodiments II-1 - II-6, II-8, II-10 - II-11, II-13 - II-18, II-20, II-22 - II-23, II-25 - II-30, II-32, II-34 - II-35 and II-37 - II-45, where A ¹ is .

Embodiment II-57. The connection according to any one of embodiments II-1 - II-8, II-15 - II-20, II-27 - II-32 and II-39 - II-45, where A ² is absent.

Embodiment II-58. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - II-45, where A ² is .

Embodiment II-59. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - II-45, where A ² is .

Embodiment II-60. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - II-45, where A ² is .

Embodiment II-61. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - II-45, where A ² is .

Embodiment II-62. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - Ii-45, where A ² is .

Embodiment II-63. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - II-45, where A ² is .

Embodiment II-64. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - II-45, where A ² is .

Embodiment II-65. The connection according to any one of embodiments II-1 - II-6, II-9 - II-18, II-21 - II-30 and II-33 - II-45, where A ² is .

Embodiment II-66. The connection according to any one of embodiments II-1 to II-65, where B is .

Embodiment II-67. The connection according to any one of embodiments II-1 to II-65, where B is .

Embodiment II-68. The connection according to any one of embodiments II-1 to II-65, where B ¹ is NR ³ -(C(R ³ ) ₂ ) _n -.

Embodiment II-69. The connection according to any one of embodiments II-1 to II-65, where B ¹ is .

Embodiment II-70. The compound of any one of embodiments II-1 to II-69, wherein R ⁴ is a 5-12 membered heteroaryl, optionally substituted with -N(R ³ ) ₂ , -OR ³ , halogen, (C ₁ -C ₆ )alkyl, -(C ₁ -C ₆ )alkylene heteroaryl, -(C ₁ -C ₆ )alkylene-CN or -C(O)NR ³ -heteroaryl.

Embodiment II-71. The compound of any one of embodiments II-1 to II-70, or a pharmaceutically acceptable salt or tautomer thereof, wherein the compound has the following formula:

Embodiment II-72. A connection selected from the group consisting of:

Example 1 Example 2 Example 3 Example 4
Example 5
Example 6
Example 7
Example 8
Example 9
Example 10
Example 11
Example 12
Example 13
Example 14
Example 15
Example 16
Example 17
Example 18
Example 19
Example 20
Example 21
Example 22
Example 23
Example 24
Example 25
Example 26
Example 27
Example 28
Example 29
Example 30
Example 31
Example 32
Example 33
Example 34
Example 35
Example 36
Example 37
Example 38
Example 39
Example 40
Example 41
Example 42
Example 43
Example 44
Example 45
Example 46
Example 47
Example 48
Example 49
Example 50
Example 51
Example 52
Example 53
Example 54
Example 55
Example 56
Example 57
Example 58
Example 59
Example 60
Example 61
Example 62
Example 63
Example 64
Example 65
Example 66
Example 67
Example 68
Example 69
Example 70

or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment II-73. A connection selected from the group consisting of:

Example 71
Example 72
Example 73
Example 74
Example 75
Example 76
Example 77
Example 78
Example 79
Example 80
Example 81
Example 82
Example 83
Example 84
Example 85
Example 86
Example 87
Example 88
Example 89
Example 90
Example 91
Example 92
Example 93
Example 94
Example 95
Example 96
Example 97
Example 98
Example 99
Example 100
Example 101
Example 102
Example 103
Example 104
Example 105
Example 106
Example 107
Example 108
Example 109
Example 110
Example 111
Example 112
Example 113
Example 114
Example 115
Example 116
Example 117
Example 118
Example 119
Example 120
Example 121
Example 122
Example 123
Example 124
Example 125
Example 126
Example 127
Example 128
Example 129
Example 130
Example 131
Example 132
Example 133
Example 134
Example 135

or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment II-74. A connection selected from the group consisting of:

Example 136
Example 137
Example 138
Example 139
Example 140
Example 141
Example 142
Example 143
Example 144
Example 145
Example 146
Example 147
Example 148
Example 149
Example 150
Example 151
Example 152

or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment II-75. A pharmaceutical composition comprising a compound of any one of embodiments II-1 to II-74 or a pharmaceutically acceptable salt thereof and at least one of a pharmaceutically acceptable carrier, diluent or excipient.

Embodiment II-76. A method of treating a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or at risk of developing a disease or disorder mediated by mTOR a therapeutically effective amount of one or more compounds of any one of embodiments II-1 to II-74 or a pharmaceutically acceptable salt thereof.

Embodiment II-77. A method of preventing an mTOR-mediated disease or disorder, comprising administering to a subject suffering from or at risk of developing an mTOR-mediated disease or disorder a therapeutically effective amount of one or more compounds of any one of embodiments II-1 to II-74 or a pharmaceutically acceptable salt thereof .

Embodiment II-78. A method of reducing the risk of an mTOR-mediated disease or disorder, comprising administering to a subject suffering from or at risk of developing an mTOR-mediated disease or disorder a therapeutically effective amount of one or more compounds of any one of Embodiments II-1 to II-74 or a pharmaceutically acceptable formulation thereof salt.

Embodiment II-79. The method as in any one of embodiments II-76 to II-78, wherein the disease is cancer or an immune-mediated disease.

Embodiment II-80. The method of Embodiment II-79, wherein the cancer is selected from brain and neurovascular tumors, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, cancer ovarian, ovarian endometriosis, testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, blood proliferation disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, cancer vulva, prostate cancer, penile cancer, bone cancer, muscle cancer, soft tissue cancer, bowel or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors.

Embodiment II-81. The method of Embodiment II-79, wherein the immune-mediated disease is selected from transplant resistance of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine, or islet pancreatic cells; graft-versus-host diseases caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis.

Embodiment II-82. A method of treating cancer, comprising administering to a subject a therapeutically effective amount of one or more compounds of any one of embodiments II-1 through II-74 or a pharmaceutically acceptable salt thereof.

Embodiment II-83. The method of Embodiment II-82, wherein the cancer is selected from brain and neurovascular tumors, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, cancer ovarian, ovarian endometriosis, testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, blood proliferation disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, cancer vulva, prostate cancer, penile cancer, bone cancer, muscle cancer, soft tissue cancer, bowel or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors.

Embodiment II-84. A method of treating an immune-mediated disease, comprising administering to a subject a therapeutically effective amount of one or more compounds of any one of embodiments II-1 through II-74 or a pharmaceutically acceptable salt thereof.

Embodiment II-85. The method of Embodiment II-84, wherein the immune-mediated disease is selected from transplant resistance of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine, or islet pancreatic cells; graft-versus-host diseases caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis.

Embodiment II-86. A method of treating an age-related condition, comprising administering to a subject a therapeutically effective amount of one or more compounds of any one of embodiments II-1 through II-74 or a pharmaceutically acceptable salt thereof.

Embodiment II-87. The method of Embodiment II-86, wherein the age-related condition is selected from sarcopenia, skin atrophy, muscle atrophy, brain atrophy, atherosclerosis, arteriosclerosis, emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia, Huntington's disease, Alzheimer's disease , cataracts, age-related macular degeneration, prostate cancer, stroke, shortened life expectancy, kidney dysfunction and age-related hearing loss, aging-related disabilities (eg, frailty), cognitive decline, age-related dementia, memory impairment, tendon stiffness, heart disease dysfunctions such as cardiac hypertrophy and systolic and diastolic dysfunction, immune aging, cancer, obesity and diabetes.

Embodiment II-88. The compound of any one of embodiments II-1 through II-74, or a pharmaceutically acceptable salt thereof, for use in the treatment, prevention, or reduction of the risk of a disease or condition mediated by mTOR.

Embodiment II-89. Use of a compound of any one of embodiments II-1 to II-74 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment, prevention or reduction of the risk of a disease or disorder mediated by mTOR.

Embodiment II-90. A compound according to any one of claims 1 to 74, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

Embodiment II-91. Use of the compound of any one of embodiments II-1 to II-74 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.

Embodiment II-92. The compound of any one of embodiments II-1 to II-74, or a pharmaceutically acceptable salt thereof, for use in the treatment of an immune-mediated disease.

Embodiment II-93. Use of the compound of any one of embodiments II-1 to II-74 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of an immune-mediated disease.

Embodiment II-94. The compound of any one of embodiments II-1 to II-74, or a pharmaceutically acceptable salt thereof, for use in treating an age-related condition.

Embodiment II-95. Use of the compound of any one of embodiments II-1 to II-74 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating an age-related condition.

Examples

The disclosure is further illustrated by the following examples and examples of synthesis, which should not be construed as limiting the scope or spirit of this disclosure to the specific techniques described herein. It should be understood that the presented examples illustrate certain embodiments and that they are not intended to limit the scope of the disclosure. Moreover, it should be understood that various other embodiments, modifications, and equivalents thereof may be suggested by those skilled in the art without departing from the spirit of this disclosure and/or the scope of the appended claims.

Definitions used in the following examples and elsewhere in this document:

CH ₂ Cl ₂ , DCM Methylene chloride, dichloromethane _CH3CN , MeCN Acetonitrile DIPEA
DMA Diisopropylethylamine or Huenig's base
Dimethylacetamide DME Dimethoxyethane DMF N,N-dimethylformamide EDKI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc Ethyl acetate h hour _H2O Water HCl Hydrochloric acid GOBt Hydroxybenzotriazole HPLC High performance liquid chromatography LCMS Liquid chromatography-mass spectrometry MeOH Methanol MTBE Methyl tert -butyl ether _{Na2SO4 _} Sodium sulfate PEG Polyethylene glycol TBDMS tert -butyldimethylsilyl TFC Trifluoroacetic acid THF Tetrahydrofuran TMS Tetramethylsilane

Row 1 of bifunctional rapalogs

The general structure of Row 1 bifunctional rapalogues is shown in Scheme 1 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7 and r=1-6. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. The carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine, and may include the variants found in Table 2 in the Examples section.

Scheme 1. Row 1 of bifunctional rapalogues.

Row 2 of bifunctional rapalogues

The general structure of Series 2 bifunctional rapalogues is shown in Scheme 2 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. The pre-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. The carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may be attached to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 2. Row of 2 bifunctional rapalogs.

Row 3 of bifunctional rapalogues

The general structure of Series 3 bifunctional rapalogues is shown in Scheme 3 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. The post-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. A carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may be attached to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 3. Row of 3 bifunctional rapalogs

Row 4 of bifunctional rapalogues

The general structure of Series 4 bifunctional rapalogues is shown in Scheme 4 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. Each of the pre- and post-linker amines may contain substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. The carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 4. Row 4 bifunctional rapalogues

Row 5 bifunctional rapalogues

The general structure of Series 5 bifunctional rapalogues is shown in Scheme 5 below. For these types of bifunctional rapalogs, the pre-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. The carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 5. Row 5 bifunctional rapalogues

Row 6 bifunctional rapalogues

The general structure of Series 6 bifunctional rapalogues is shown in Scheme 6 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. Linker amines may include substitutions such as R=H and C1-C6 alkyl groups. The post-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. The carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 6. Row 6 bifunctional rapalogues.

Row of 7 bifunctional rapalogues

The general structure of Series 7 bifunctional rapalogues is shown in Scheme 7 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. Each of the pre- and post-linker amines includes substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. The carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 7. Row 7 bifunctional rapalogues

Row of 8 bifunctional rapalogues

The general structure of Series 8 bifunctional rapalogues is shown in Scheme 8 below. For these types of bifunctional rapalogues, the linker may include variants where q=0-30, for example, q=1-7. The linker amine may include substitutions such as R=H and C1-C6 alkyl groups. The post-linker amine may include substitutions such as R ² =H, C1-C6 alkyl groups and cycloalkyl, including 4-8 membered rings. The carbamate group, where Z ¹ =O or S, can be attached to rapalog at R ⁴⁰ or R ²⁸ (formula I and II), including the variants found in Table 1 in the examples section. The active site mTOR inhibitor may attach to the linker via a primary or secondary amine and may include the variants found in Table 2 in the Examples section.

Scheme 8. Row 8 bifunctional rapalogues

Table 1. Carbonate- and thiocarbonate-containing monomers of rapalog.

Carbonate-bearing rapalogue Carbonate-bearing rapalogue
Monomer 1
Monomer 2
Monomer 3
Monomer 4
Monomer 5
Monomer 6
Monomer 7
Monomer 8
Monomer 9
Monomer 10
Monomer 11
Monomer 12
Monomer 13
Monomer 14
Monomer 15
Monomer 16
Monomer 17

Table 2. Active site inhibitor monomers.

Active site inhibitor monomers Active site inhibitor monomers
Monomer A
Monomer B
Monomer C
Monomer D
Monomer E
Monomer F
Monomer G
Monomer H
Monomer I
Monomer J
Monomer K
Monomer L
Monomer M
Monomer N
Monomer O
Monomer P
Monomer Q
Monomer R
Monomer S
Monomer T
Monomer U
Monomer V
Monomer W
Monomer X
Monomer Y
Monomer Z
Monomer AA
Monomer AB
Monomer AC
Monomer AD
Monomer AE
Monomer AF
Monomer AG
Monomer AH
Monomer AI

Table 3. Active site inhibitor monomers

Active site inhibitor Active site inhibitor
Monomer AJ
Monomer AK
Monomer AL
Monomer AM
Monomer AN
Monomer AO

Table 4. Amine-containing pre- and post-linkers

Amide block Amide block
Building block A
Building block B
Building block C
Building block D
Building block E
Building block F
Building block G
Building block H
Building Block I
Building block J
Building block K
Building block L
Building block M
Building block N
Building block O
Building block P
Building block Q
Building block R
Building block S
Building block T
Building block U
Building Block V
Building block W
Building Block X
Building block Y
Building block Z
Building block AA
AB building block
AC building block
Building block AD
Building block AE
Building block AF
Building block AG
Building block AH
AI building block
AJ building block
AK building block
AL building block
AM building block
AN building block
Building block AO
AP building block
AQ building block
AR building block
AS building block
AT building block
AU building block
AV building block
Building block AW
AX building block
Building block AY
Building block AZ
Building block BA
BB building block
Building block BC
Building block BD
BE building block
BF building block

Preparation of active site inhibitor monomers

Monomer A. 5-(4-amino-1-(4-(aminomethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzylcarbamate

To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3.8 g, 14.56 mmol, 1.0 equiv. ) in DMF (20 ml) add NaH (582.27 mg, 14.56 mmol, 60 wt%, 1.0 equiv ) at 0°C and the reaction solution was stirred at this temperature for 30 min, then tert- butyl 4-(bromomethyl)benzylcarbamate (4.59 g, 15.29 mmol, 1.05 equiv. ) was added to the reaction mixture at 0° C., and the reaction solution was stirred at room temperature for 2 hours. The solution was poured into H ₂ O (80 ml) and the precipitated solid was filtered. The solid cake is washed with H ₂ O (2 x 10 ml) and then dried under reduced pressure to give tert- butyl 4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl )methyl)benzylcarbamate (5 g, 53% yield) as a yellow solid. LCMS (ESI) m/z : [M+Na] calc. for C ₁₈ H ₂₁ IN ₆ O ₂ : 503.07; found 503.2.

Step 2 : Synthesis of tert- butyl 4-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzylcarbamate

To a two-phase suspension of tert- butyl 4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzylcarbamate (5 g, 7.68 mmol, 1.0 equiv .), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (2.40 g, 9.22 mmol, 1, 2 equiv.) and Pd(PPh ₃ ) ₄ (887.66 mg, 768.16 µmol, 0.1 equiv.) in DME (100 ml) and H ₂ O (50 ml) add Na ₂ CO ₃ (1, 91 g, 23.04 mmol, 3.0 equiv.) at room temperature under N ₂ . The mixture is stirred at 110°C for 3 hours. The reaction mixture is cooled at room temperature and filtered, the filtrate is extracted with EtOAc (3×50 ml). The organic phases are combined and washed with brine (10 ml), dried over Na ₂ SO ₄ , filtered and the filtrate concentrated under reduced pressure to leave a residue. The residue is purified by chromatography on silica gel (0→20% MeOH/EtOAc) to give tert- butyl 4-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4 -d]pyrimidin-1-yl)methyl)benzylcarbamate (4.5 g, 82% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₅ H ₂₆ N ₈ O ₃ : 487.22; found 487.2.

Step 3 : Synthesis of 5-(4-amino-1-(4-(aminomethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine

To a solution of tert- butyl 4-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzylcarbamate (4 .5 g, 6.29 mmol, 1.0 equiv.) to DCM (50 ml) add TFA (30.80 g, 270.12 mmol, 20 ml, 42.95 equiv.) at 0°C. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a residue, which was dissolved in 10 ml of MeCN, then poured into MTBE (100 ml). The precipitated solid is then filtered and the solid cake is dried under reduced pressure to give 5-[4-amino-1-[[4-(aminomethyl)phenyl]methyl]pyrazolo[3,4-d]pyrimidin-3-yl ]-1,3-benzoxazol-2-amine (2.22 g, 71% yield, TFA) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₁₈ N ₈ O: 387.16; found 387.1.

Monomer B. 2-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-6-ol trifluoroacetic acid salt.

Step 1 : Synthesis of tert -butyl N-(4-{4-amino-3-[6-(benzyloxy)-1H-indol-2-yl]-1H-pyrazolo[3,4-d]pyrimidin1-yl}butyl )carbamate

To a mixture of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (300 mg, 694 µmol, 1.0 equiv.) and (6-(benzyloxy)-1-( tert- butoxycarbonyl)-1H-indol-2-yl)boronic acid (763 mg, 2.08 mmol, 3.0 equiv.) in DMF (2.6 ml), EtOH (525 µl) and H ₂ O (350 µl) add Pd(OAc) ₂ (15.5 mg, 69 µmol, 0.1 equiv.), triphenylphosphine (36.1 mg, 138 µmol, 0.2 equiv.) and sodium carbonate (440 mg, 4.16 mmol, 6.0 equiv.). The reaction mixture is heated at 80°C for 20 hours, cooled at room temperature and quenched with H ₂ O (10 ml) and EtOAc (10 ml). The mixture is transferred to a separatory funnel and the aqueous phase is extracted with EtOAc (3×20 ml). The combined organic phase is washed with sat. aq. NaCl (1×20 ml), dry over Na ₂ SO ₄ , filter and concentrate under reduced pressure. The crude product was purified by silica gel chromatography (20→85% EtOAc/heptane) to give the product (201 mg, 46% yield) as an orange solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₉ H ₃₃ N ₇ O ₃ : 528.27; found 528.2.

Step 2 : Synthesis of tert- butyl (4-(4-amino-3-(6-hydroxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate

To a solution of tert- butyl N-(4-{4-amino-3-[6-(benzyloxy)-1H-indol-2-yl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl}butyl )carbamate (1.0 equiv.) in EtOH add Pd/C (10% mol.). The reaction mixture is purged with H ₂ and the reaction mixture is stirred under an H ₂ atmosphere until consumption of the starting material is determined by LCMS. The reaction mixture is then diluted with EtOAc, filtered over Celite and concentrated under reduced pressure. The resulting residue is purified by silica gel chromatography to obtain the desired product.

Step 3 : Synthesis of 2-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-6-ol

To a solution of tert- butyl (4-(4-amino-3-(6-hydroxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (1 .0 equiv.) in anhydrous DCM, add TFA (50 equiv.) dropwise at 0°C. The reaction mixture is stirred at 0°C and warmed to room temperature. After completion of the reaction, as determined by LCMS, the reaction mixture is concentrated under reduced pressure. The residue is triturated with MeCN, then dripped into MTBE for more than 10 minutes. The supernatant is removed and the precipitate is collected by filtration under N ₂ to give 2-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-6 -ola.

Monomer C. 5-(4-amino-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d ]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 6-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5 g, 19.16 mmol, 1.0 equiv.) in DMF (50.0 ml) add NaH (766.22 mg, 19.16 mmol, 60% wt., 1.0 equiv.) at 4°C. The mixture is stirred at 4°C for 30 minutes. To the reaction mixture was added tert- butyl 6-(bromomethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (6.87 g, 21.07 mmol, 1.1 equiv.) in DMF (30 mL) at 4 °C. The mixture was stirred at room temperature for 2 hours. The mixture was then cooled to 4°C and H ₂ O (400 ml) was added and the mixture was stirred for 30 minutes. The resulting precipitate was collected by filtration to yield crude tert- butyl 6-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H )-carboxylate (9.7 g, 76% yield) as a light yellow solid. The crude product is used directly in the next step.

Step 2 : Synthesis of tert- butyl 6-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)- 3,4-dihydroisoquinoline-2(1H)-carboxylate

To a two-phase suspension of tert- butyl 6-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate ( 9.7 g, 14.63 mmol, 1.0 equiv.), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2- amine (4.57 g, 17.55 mmol, 1.2 equiv.) and Na ₂ CO ₃ (7.75 g, 73.14 mmol, 5.0 equiv.) in DME (120.0 ml) and H ₂ O (60 ml) add Pd(PPh ₃ ) ₄ (1.69 g, 1.46 mmol, 0.1 equiv.) at room temperature under N ₂ . The mixture was stirred at 110°C for 3 hours. The reaction mixture was then cooled at room temperature and partitioned between EtOAc (100 ml) and H ₂ O (100 ml). The aqueous layer is separated and extracted with EtOAc (2×60 ml). The organic layers are combined, washed with brine (80 ml) and dried over anhydrous Na ₂ SO ₄ , filtered, and the filtrate is concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1→100% EtOAc/petroleum ether, then 20→50% MeOH/EtOAc) to give tert- butyl 6-((4-amino-3-(2-aminobenzo[d]oxazole-5- yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (4.5 g, 58% yield) as a light yellow solid substances.

Step 3 : Synthesis of 5-(4-amino-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyramidin-3-yl)benzo[ d]oxazol-2-amine

To pure TFA (32.5 mL, 438.97 mmol, 50.0 equiv.) add tert- butyl 6-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H- pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (4.5 g, 8.78 mmol, 1.0 equiv.) at room temperature. The mixture is stirred for 30 minutes and then concentrated under reduced pressure. The oily residue is triturated with MeCN (8 ml), then dripped into MTBE (350 ml) for over 10 min. The supernatant was removed and then the precipitate was collected by filtration under N ₂ to obtain 5-(4-amino-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d ]pyrimidin-3-yl)benzo[d]oxazol-2-amine (5.72 g, greater than 100% yield, TPA) as a light pink solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₂₀ N ₈ O: 413.18; found 413.2.

Monomer D. 2-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-7-ol salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 2-(4-amino-1-(4-(( tert- butoxycarbonyl)amino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-7-methoxy -1H-indole-1-carboxylate

To a mixture of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (1.0 equiv.) and (1-( tert- butoxycarbonyl)-7-methoxy-1H-indol-2-yl)boronic acid (3.0 equiv.) in DME and H ₂ O add Pd(PPh ₃ ) ₄ (0.1 equiv.) and sodium carbonate (6. 0 equivalent). The reaction mixture is heated at 80°C until complete, as determined by LCMS and TLC analysis. The reaction mixture is then quenched with H ₂ O and EtOAc. The mixture is transferred to a separatory funnel and the aqueous phase is extracted with EtOAc. The organic phase is washed with sat. aq. NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The desired product is isolated after chromatography on silica gel.

Step 2 : Synthesis of tert- butyl 2-(4-amino-1-(4-(( tert- butoxycarbonyl)amino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-7-hydroxy -1H-indole-1-carboxylate

To a solution of tert- butyl 2-(4-amino-1-(4-(( tert- butoxycarbonyl)amino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-7-methoxy-1H -indole-1-carboxylate (1.0 equiv.) in DCM at -10°C add BBr ₃ (2.0 equiv.). The reaction mixture is stirred until the starting material is consumed, as determined by LCMS. The reaction mixture is quenched by slowly adding sat. aq. NaHCO ₃ is transferred to a separatory funnel and the mixture is extracted with DCM. The organic phase is washed with sat. aq. NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The desired product is isolated after chromatography on silica gel.

Step 3 : Synthesis of 2-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-7-ol

To a solution of tert- butyl 2-(4-amino-1-(4-(( tert- butoxycarbonyl)amino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-7-hydroxy-1H -indole-1-carboxylate (1.0 equiv.) in DCM at 0°C, add TFA dropwise. The reaction mixture is stirred at 0°C and warmed to room temperature. After completion of the reaction, as determined by LCMS, the reaction mixture is concentrated under reduced pressure. The residue is triturated with MeCN, then dripped into MTBE for more than 10 minutes. The supernatant was removed and the precipitate was collected by filtration under N ₂ to obtain 2-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-7- ola.

Monomer E. 5-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate

To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3 g, 11.49 mmol, 1.0 equiv.) in DMA (30 ml) add tert- butyl 4-(bromomethyl )piperidine-1-carboxylate (3.36 g, 12.07 mmol, 1.05 equiv.) and K ₂ CO ₃ (4.77 g, 34.48 mmol, 3.0 equiv.), then the reaction mixture was stirred at 80°C for 3 hours. The reaction mixture is filtered to remove K ₂ CO ₃ and the filtrate is poured into H ₂ O (200 ml). The precipitated solid is then filtered to obtain tert- butyl 4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate (3 g , 57% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₆ H ₂₃ IN ₆ O ₂ : 459.10; found 459.1.

Step 2 : Synthesis of tert- butyl 4-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine -1-carboxylate

To a two-phase suspension of tert- butyl 4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate (3 g, 6.55 mmol, 1.0 equiv.) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (2.04 g, 7.86 mmol, 1.2 equiv.) and Na ₂ CO ₃ (3.47 g, 32.73 mmol, 5.0 equiv.) in DME (60 ml) and H ₂ O (30 ml) add Pd(PPh ₃ ) ₄ (756.43 mg, 654.60 µmol, 0.1 equiv.) at room temperature under N ₂ . The mixture is stirred at 110°C for 3 hours and the two batches are combined together. The reaction mixture is cooled and partitioned between EtOAc (500 ml) and H ₂ O (500 ml). The aqueous layer is separated and extracted with EtOAc (3×300 ml). All organic layers were combined, washed with brine (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the filtrate was concentrated under reduced pressure to give tert- butyl 4-((4-amino-3-(2-aminobenzo[d ]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate (4.5 g, 74% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₃ H ₂₈ N ₈ O ₃ : 465.24; found 465.2.

Step 3 : Synthesis of 5-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine

A solution of tert- butyl 4-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1- carboxylate (2.5 g, 5.38 mmol, 1.0 equiv.) in TFA (25 ml) was stirred at room temperature for 30 minutes. The reaction solution is concentrated under reduced pressure to remove TPA. The residue was added to MTBE (400 ml) and a solid precipitated, which was then filtered to give 5-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl )benzo[d]oxazol-2-amine (2.7 g, greater than 100% yield, TFA) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for _{C18H20N8O : 365.18} ; found 365.1.

Monomer F. 2-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol trifluoroacetic acid salt.

Step 1 : Synthesis of tert- butyl (4-(4-amino-3-(5-(( tert- butyldimethylsilyl)oxy)-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin- 1-yl)butyl)carbamate

To a solution of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (1.0 g, 2.31 mmol, 1.0 equiv.) in dioxane (10.5 ml) and H ₂ O (3.5 ml) add (1-( tert- butoxycarbonyl)-5-(( tert- butyldimethylsilyl)oxy)-1H-indol-2-yl) boronic acid (1.54 g, 2.78 mmol, 1.2 equiv.), K ₃ PO ₄ (1.47 g, 6.94 mmol, 3.0 equiv.), Pd ₂ (dba) ₃ (211 .84 mg, 231.34 µmol, 0.1 equiv.) and SPhos (189.95 mg, 462.69 µmol, 0.2 equiv.) at room temperature under N ₂ . The hermetically sealed tube is heated at 150°C for 20 minutes in a microwave oven. This is repeated for 9 additional games. The 10 batches are combined and the reaction mixture is cooled and partitioned between EtOAc (60 ml) and H ₂ O (80 ml). The aqueous layer was separated and extracted with EtOAc (2×50 ml). The organic layers are combined, washed with brine (60 ml) and dried over anhydrous Na ₂ SO ₄ . The suspension is filtered and the filtrate is concentrated under reduced pressure. The crude product is purified by chromatography on silica gel (1→75% EtOAc/petroleum ether). The desired fractions are combined and evaporated under reduced pressure to give tert- butyl (4-(4-amino-3-(5-(( tert- butyldimethylsilyl)oxy)-1H-indol-2-yl)-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)butyl)carbamate (10 g, 60% yield) as a light yellow solid.

Step 2 : Synthesis of tert- butyl (4-(4-amino-3-(5-hydroxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate

To a mixture of tert- butyl (4-(4-amino-3-(5-(( tert- butyldimethylsilyl)oxy)-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1- yl)butyl)carbamate (10 g, 18.12 mmol, 1.0 equiv.) in THF (100 ml) add FTBA•3H ₂ O (1 M, 54.37 ml, 3.0 equiv.) in one portion at room temperature under N ₂ . The mixture was stirred for 1 hour and then H ₂ O (100 ml) was added to the reaction mixture. The layers are separated and the aqueous phase is extracted with EtOAc (2×80 ml). The combined organic phase is washed with brine (100 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1→67% EtOAc/petroleum ether) to give tert- butyl (4-(4-amino-3-(5-hydroxy-1H-indol-2-yl)-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)butyl)carbamate (7 g, 87% yield) as a light pink solid.

Step 3 : Synthesis of 2-[4-amino-1-(4-aminobutyl)pyrazolo[3,4-d]pyrimidin-3-yl]-1H-indol-5-ol

To TFA (50.0 mL, 675.26 mmol, 38.9 equiv.) add tert- butyl (4-(4-amino-3-(5-hydroxy-1H-indol-2-yl)-1H-pyrazolo [3,4-d]pyrimidin-1-yl)butyl)carbamate (7.6 g, 17.37 mmol, 1.0 equiv.) at room temperature. The mixture is stirred for 40 minutes and then concentrated under reduced pressure. The oily residue is triturated with MeCN (20 ml), then added dropwise to MTBE (300 ml) over 10 min. The supernatant was removed and then the precipitate was collected by filtration under N ₂ to obtain 2-[4-amino-1-(4-aminobutyl)pyrazolo[3,4-d]pyrimidin-3-yl]-1H-indol-5-ol ( 7.79 g, 91% yield, TPA) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₇ H ₁₉ N ₇ O: 338.17; found 338.2.

Monomer G. 5-(4-amino-1-(azetidin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidine-1-carboxylate

To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (4 g, 15.32 mmol, 1.0 equiv. ), tert- butyl 3-(hydroxymethyl)azetidine-1-carboxylate (3.01 g, 16.09 mmol, 1.05 equiv. ) and PPh ₃ (6.03 g, 22.99 mmol, 1.5 equiv. ) in THF (80 ml) cool to 0°C, add DIAD (4.47 ml, 22.99 mmol, 1.5 equiv. ) dropwise. After addition was complete, the reaction mixture was stirred at room temperature for 14 hours. The reaction mixture was poured into H ₂ O (200 ml) and then extracted with EtOAc (3×50 ml). The organic layers are combined and washed with brine (2×50 ml). The organic phase is dried over Na ₂ SO ₄ , filtered and the filtrate is concentrated under reduced pressure to obtain a residue. The residue is purified by chromatography on silica gel (0→100% EtOAc/petroleum ether) to give tert- butyl 3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl) azetidine-1-carboxylate (4.2 g, 64% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₄ H ₁₉ IN ₆ O ₂ : 431.07; found 431.0.

Step 2 : Synthesis of tert- butyl 3-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidine -1-carboxylate

To a two-phase suspension of tert- butyl 3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidine-1-carboxylate (4 g, 9.30 mmol, 1.0 equiv.), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (2.90 g, 11.16 mmol, 1.2 equiv.) and Na ₂ CO ₃ (4.93 g, 46.49 mmol, 5.0 equiv.) in DME (100 ml) and H ₂ O (50 ml) add Pd(PPh ₃ ) ₄ (1.07 g, 929.71 µmol, 0.1 equiv.) at room temperature under N ₂ . The mixture was stirred at 110°C for 3 hours. The reaction mixture was then cooled at room temperature and filtered and the filtrate was extracted with EtOAc (3×50 ml). The organic layers were combined and washed with brine (10 mL), dried over Na ₂ SO ₄ , filtered, and the filtrate was concentrated under reduced pressure to leave a residue. The residue is purified by chromatography on silica gel (0→20% MeOH/EtOAc) to give tert- butyl 3-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4 -d]pyrimidin-1-yl)methyl)azetidine-1-carboxylate (3.5 g, 80% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₁ H ₂₄ N ₈ O ₃ : 437.20; found 437.2.

Step 3 : Synthesis of 5-(4-amino-1-(azetidin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine

To a solution of tert- butyl 3-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidine-1 -carboxylate (3.29 g, 6.87 mmol, 1.0 equiv. ) in DCM (20 ml) add TFA (7.50 ml, 101.30 mmol, 14.7 equiv. ) at 0°C. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a residue. The residue is dissolved in MeCN (6 ml) and then poured into MTBE (80 ml). A solid precipitate is filtered and the solid cake is dried under reduced pressure to give 5-[4-amino-1-(azetidin-3-ylmethyl)pyrazolo[3,4-d]pyrimidin-3-yl]-1,3 -benzoxazol-2-amine (4.34 g, greater than 100% yield, TFA) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₆ H ₁₆ N ₈ O: 337.15; found 337.1.

Monomer H. 5-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]-oxazol-2-amine salt of trifluoroacetic acid.

This monomer is synthesized according to the procedure described in Nature 2015 , 534 , 272-276, which is incorporated herein by reference in its entirety.

Monomer I. 5-(4-amino-1-(pyrrolidin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate

Suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (4.5 g, 17.24 mmol, 1.0 equiv. ), tert- butyl 3-(bromomethyl)pyrrolidine-1- carboxylate (4.78 g, 18.10 mmol, 1.05 equiv. ) and K ₂ CO ₃ (7.15 g, 51.72 mmol, 3.0 equiv. ) in DMA (40 ml) heated to 85 ° C. The reaction mixture is stirred at 85°C for 3 hours, at which point the solution is cooled to room temperature. Then, H ₂ O (80 ml) was added to the reaction mixture and a solid precipitated. The mixture is filtered and the solid cake is washed with H ₂ O (2 x 40 ml) and then dried under reduced pressure to give tert- butyl 3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine -1-yl)methyl)pyrrolidine-1-carboxylate (6 g, 78% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₅ H ₂₁ IN ₆ O ₂ : 445.08; found 445.1.

Step 2 : Synthesis of tert- butyl3-[[4-amino-3-(2-amino-1,3-benzoxazol-5-yl)pyrazolo[3,4-d]pyrimidin-1-yl]methyl]pyrrolidin-1 -carboxylate

To a two-phase suspension of tert- butyl 3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate (4 g, 9.00 mmol, 1.0 equiv. ), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (2.81 g, 10.80 mmol, 1.2 equiv. ) and Na ₂ CO ₃ (4.77 g, 45.02 mmol, 5.0 equiv. ) in DME (120 ml) and H ₂ O (60 ml) add Pd(PPh ₃ ) ₄ (1.04 g, 900.35 µmol, 0.1 equiv. ) at room temperature under N ₂ . The mixture was stirred at 110°C for 3 hours. The reaction mixture was cooled at room temperature and filtered, and the filtrate was extracted with EtOAc (3×50 ml). The organic phases are combined and washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave a residue. The residue is purified by chromatography on silica gel (0→20% MeOH/EtOAc) to give tert- butyl 3-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4 -d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate (3 g, 64% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₂₆ N ₈ O ₃ : 451.21, found 451.2.

Step 3 : Synthesis of 5-(4-amino-1-(pyrrolidin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine

To a solution of tert- butyl 3-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1 -carboxylate (3 g, 6.66 mmol, 1.0 equiv. ) in DCM (40 ml) add TFA (20 ml) at 0°C, dropwise. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction solution was then concentrated under reduced pressure to obtain a residue. The residue is dissolved in MeCN (4 ml), then poured into MTBE (100 ml) and a solid precipitates. The solid is filtered and the cake is dried under reduced pressure to obtain 5-(4-amino-1-(pyrrolidin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazole -2-amine (4.00 g, greater than 100% yield, TPA) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₇ H ₁₈ N ₈ O: 351.17; found 351.2.

Monomer J. 1-(4-aminobutyl)-3-(7-methoxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 2-(4-amino-1-(4-(( tert- butoxycarbonyl)amino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-7-methoxy -1H-indole-1-carboxylate

To a mixture of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (1.0 equiv.) and (1-( tert- butoxycarbonyl)-7-methoxy-1H-indol-2-yl)boronic acid (3.0 equiv.) in DME and H ₂ O add Pd(PPh ₃ ) ₄ (0.1 equiv.) and sodium carbonate (6. 0 equivalent). The reaction mixture is heated at 80°C until complete, as determined by LCMS and TLC analysis. The reaction mixture is then quenched with H ₂ O and EtOAc. The mixture is transferred to a separatory funnel and the aqueous phase is extracted with EtOAc. The organic phase is washed with sat. aq. NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The desired product is isolated after chromatography on silica gel.

Step 2 : Synthesis of 1-(4-aminobutyl)-3-(7-methoxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

To a solution of tert- butyl 2-(4-amino-1-(4-(( tert- butoxycarbonyl)amino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-7-hydroxy-1H -indole-1-carboxylate (1.0 equiv.) in DCM at 0°C, add TFA dropwise. The reaction mixture is stirred at 0°C and warmed to room temperature. After completion of the reaction, as determined by LCMS, the reaction mixture is concentrated under reduced pressure. The residue is triturated with MeCN, then dripped into MTBE for more than 10 minutes. The supernatant was removed and the precipitate was collected by filtration under N ₂ to give 1-(4-aminobutyl)-3-(7-methoxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4 -amine.

Monomer K. Synthesis of 1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl (4-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate

To a mixture of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (300 mg, 694 µmol, 1.0 equiv.) in MeOH (14 ml) at 0°C add zinc powder (226 mg, 3.46 mmol, 5.0 equiv.). Saturation aq. NH ₄ Cl (14 ml) was added to the reaction mixture, and the reaction mixture was warmed to room temperature and stirred for 18 hours. The reaction mixture was quenched with EtOAc (40 ml) and H ₂ O (10 ml), and the mixture was transferred to a separatory funnel. The aqueous phase is extracted with EtOAc (3×20 ml) and the combined organic phases are washed with sat. aq. NaHCO ₃ (15 ml), dry over Na ₂ SO ₄ , filter and concentrate under reduced pressure to give the product (210 mg, 99% yield) as a light yellow solid, which is used without further purification. LCMS (ESI) m/z : [M+H] calc. _{for C14H22N6O2 : 307.19} ; found 307.1.

Step 2 : Synthesis of 1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

To a solution of tert- butyl (4-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (210 mg, 691 µmol) in DCM (3.5 ml) at 0° C add TFA (3.5 ml), drop by drop. After 3 hours, the reaction mixture was warmed to room temperature and concentrated under reduced pressure to give the product trifluoroacetate (220 mg, 99% yield) as a brown oil, which was used without further purification. LCMS (ESI) m/z : [M+H] calc. for C ₉ H ₁₄ N ₆ : 207.13; found 207.1.

Monomer L. 1-[4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl]-9-(quinolin-3-yl)-1H,2H-benzo[h]1,6-naphthyridin-2- He

The preparation of this monomer has been previously described in the literature. See the following links: i) Liu, Qingsong; Chang, Jae Won; Wang, Jinhua; Kang, Seong A.; Thoreen, Carson C.; Markhard, Andrew; Hur, Wooyoung; Zhang, Jianming; Sim, Taebo; Sabatini, David M.; et al From Journal of Medicinal Chemistry (2010), 53(19), 7146-7155. ii) Gray, Nathanael; Chang, Jae Won; Zhang, Jianming; Thoreen, Carson C.; Kang, Seong Woo Anthony; Sabatini, David M.; Liu, Qingsong from int. PCT Application (2010), WO 2010044885A2, which is incorporated herein by reference in its entirety.

Monomer M. 5-(1-(4-aminobutyl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of 3-iodo-1-trityl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

A suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10.5 g, 40.23 mmol, 1.0 equiv.) in DMF (170.0 ml) was treated with Cs ₂ CO ₃ (19.7 g, 60.34 mmol, 1.5 equiv.) and [chloro(diphenyl)methyl]benzene (13.5 g, 48.27 mmol, 1.2 equiv.) at room temperature. The reaction mixture was stirred at 70°C for 4 hours under a nitrogen atmosphere. The reaction mixture was added to H ₂ O (1200 ml). The precipitate is filtered and washed with H ₂ O. The residue is purified by silica gel chromatography (0→60% EtOAc/petroleum ether) to give 3-iodo-1-trityl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (15 .40 g, 73.5% yield) as a white solid.

Step 2 : Synthesis of 3-iodo- N,N -dimethyl-1-trityl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

To a suspension of NaH (2.98 g, 74.50 mmol, 60 wt%, 2.5 equiv.) in DMF (150 ml) add a solution of 3-iodo-1-trityl-1H-pyrazolo[3,4-d ]pyrimidin-4-amine (15.0 g, 29.80 mmol, 1.0 equiv.) in DMF (50 ml) at 0°C. The mixture is stirred at 0°C for 10 minutes. Iodomethane (16.92 g, 119.20 mmol, 7.42 mL, 4.0 equiv.) was then added to the reaction mixture at 0°C. The mixture was stirred at room temperature for 2 hours, at which point H ₂ O (1400 ml) was added at 0°C. The mixture is stirred for another 10 minutes at 0°C. The resulting precipitate was collected by filtration to give the crude product, which was purified by silica gel chromatography (1%→25% EtOAc/petroleum ether) twice to give 3-iodo- N,N -dimethyl-1-trityl-1H-pyrazolo[3,4- d]pyrimidin-4-amine (9.0 g, 89% yield) as a white solid.

Step 3 : Synthesis of 3-iodo- N,N -dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

To a cooled solution of TPA (19.1 mL, 258.1 mmol, 15.0 equiv.) in DCM (100.0 mL) add 3-iodo -N,N -dimethyl-1-trityl-1H-pyrazolo[3, 4-d]pyrimidin-4-amine (9.10 g, 17.12 mmol, 1.0 equiv.) at 4°C. The mixture was stirred at room temperature for 1 hour. The residue was poured into H ₂ O (100 ml) and the aqueous phase was extracted with DCM (2×50 ml). A saturated aqueous solution of NaHCO ₃ is then added to the aqueous phase until the solution reaches a pH of 8. The resulting precipitate is collected by filtration to give 3-iodo- N,N -dimethyl-1H-pyrazolo[3,4-d]pyrimidine-4 -amine (3.40 g, 68.7% yield) as a white solid.

Step 4 : Synthesis of tert- butyl (4-(4-(dimethylamino)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate

To a suspension of 3-iodo- N,N -dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.7 g, 5.88 mmol, 1.0 equiv.) in DMF (20 ml) add NaH (247 mg, 6.17 mmol, 60% wt., 1.05 equiv.) at 4°C. The mixture is stirred at 4°C for 30 minutes. Tert- butyl N-(4-bromobutyl)carbamate (2.22 g, 8.82 mmol, 1.81 mL, 1.5 equiv.) in DMF (10 mL) was then added to the reaction mixture at 4°C. The mixture was stirred at room temperature for 2 hours. H ₂ O (100 ml) was then added to the mixture at 4°C. The mixture is stirred for an additional 30 minutes at 4°C and the resulting precipitate is collected by filtration to obtain the crude product. The residue is purified by silica gel chromatography (0→75% EtOAc/petroleum ether) to give tert- butyl(4-(4-(dimethylamino)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl) butyl)carbamate (2.0 g, 56% yield) as a white solid.

Step 5 : Synthesis of tert- butyl (4-(3-(2-aminobenzo[d]oxazol-5-yl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl )carbamate

To a biphasic suspension of tert- butyl (4-(4-(dimethylamino)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (4.0 g, 8.69 mmol, 1.0 equiv.), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (3.4 g, 13.03 mmol, 1.5 equiv.) and Na ₂ CO ₃ (4.6 g, 43.45 mmol, 5.0 equiv.) in DME (80.0 ml) and H ₂ O (40.0 ml) add Pd (PPh ₃ ) ₄ (1.0 g, 868.98 µmol, 0.1 equiv.) at room temperature under N ₂ . The mixture is stirred at 110°C for 3 hours. The reaction mixture is then cooled and partitioned between EtOAc (300 ml) and H ₂ O (600 ml). The aqueous layer is separated and extracted with EtOAc (2×100 ml). The organic layers are combined, washed with brine (2×60 ml) and dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product is purified by silica gel column chromatography (50% EtOAc/hexane, then 20% MeOH/EtOAc). The desired fractions are combined and concentrated under reduced pressure to give tert- butyl (4-(3-(2-aminobenzo[d]oxazol-5-yl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyramidin -1-yl)butyl)carbamate (3.2 g, 78.9% yield) as a light brown solid.

Step 6 : Synthesis of 5-(1-(4-aminobutyl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine

To TFA (20.82 mL, 281.27 mmol, 36.5 equiv.) add tert- butyl (4-(3-(2-aminobenzo[d]oxazol-5-yl)-4-(dimethylamino)-1H -pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (3.6 g, 7.72 mmol, 1.0 equiv.) at room temperature. The mixture is stirred for 30 minutes, at which point the mixture is concentrated under reduced pressure. The oily residue is triturated with MeCN (8 ml) and MTBE (60 ml) for 10 minutes. The supernatant was removed and the precipitate was then collected by filtration under N ₂ to give 5-(1-(4-aminobutyl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d ]oxazol-2-amine (4.0 g, crude, TPA) as a light brown solid.

To 1M NaOH (107.2 mL, 14.7 equiv.) add 5-(1-(4-aminobutyl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo [d]oxazol-2-amine (3.5 g, crude, TPA) at room temperature. The mixture is stirred for 10 minutes and then the aqueous phase is extracted with DCM (3×50 ml). The combined organic phase is washed with brine (50 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. TFA (539.37 µl, 7.28 mmol, 1.0 equiv) was added and concentrated under reduced pressure. MeCN (10 ml) is then added, followed by MTBE (150 ml). The resulting precipitate was collected by filtration to give 5-(1-(4-aminobutyl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine (1 .3 g, 36.6% yield, TPA) as a light brown product. LCMS (ESI) m/z : [M+H] calc. for _{C18H22N8O : 367.19} ; found 367.1.

Monomer N. 6-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo-[d]isoxazol-3-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl (6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]isoxazol-3-yl)carbamate

To a solution of tert- butyl (6-bromobenzo[d]isoxazol-3-yl)carbamate (1.0 equiv.) in dioxane add Pd(PPh ₃ ) ₄ (0.1 equiv.), sodium carbonate (6.0 equiv. .) and bis(pinacolato)diborone (3.0 equiv.). The reaction mixture is stirred and heated until complete, as determined by LCMS and TLC analysis. The reaction mixture is cooled at room temperature, quenched with sat. aq. NaHCO ₃ and the mixture are transferred to a separatory funnel. The aqueous phase is extracted with EtOAc and the organic phase is washed with sat. aq. NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The desired product is isolated after purification by silica gel chromatography.

Step 2 : Synthesis of tert- butyl (4-(4-amino-3-(3-(( tert- butoxycarbonyl)amino)benzo[d]isoxazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidine -1-yl)butyl)carbamate

To a mixture of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (1.0 equiv.) and tert- butyl (6- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]isoxazol-3-yl)carbamate (3.0 equiv.) in DME and H ₂ O add Pd( PPh ₃ ) ₄ (0.1 equiv.) and sodium carbonate (6.0 equiv.). The reaction mixture is heated at 80°C until complete, as determined by LCMS and TLC analysis. The reaction mixture is then quenched with H ₂ O and EtOAc. The mixture is transferred to a separatory funnel and the aqueous phase is extracted with EtOAc. The organic phase is washed with sat. aq. NaCl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The desired product is isolated after chromatography on silica gel.

Step 3 : Synthesis of 6-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo-[d]isoxazol-3-amine

To a solution of tert- butyl (4-(4-amino-3-(3-(( tert- butoxycarbonyl)amino)benzo[d]isoxazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)butyl)carbamate (1.0 equiv.) in DCM at 0°C add TFA dropwise. The reaction mixture is stirred at 0°C and warmed to room temperature. After completion of the reaction, as determined by LCMS, the reaction mixture is concentrated under reduced pressure. The residue is triturated with MeCN, then added dropwise to MTBE over 10 min. The supernatant is removed and the precipitate is collected by filtration under N ₂ to obtain 6-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo-[d]isoxazole -3-amine.

Monomer O. 4-(5-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-1H -indol-1-yl)butan-1-amine salt of trifluoroacetic acid.

The synthesis of this monomer involves alkylation of WAY-600 (CAS# 1062159-35-6) with tert- butyl (4-bromobutyl)carbamate under basic conditions, then deprotection of Boc using TPA to produce the TPA salt.

Links to obtain WAY-600: Discovery of Potent and Selective Inhibitors of the Mammalian Target of Rapamycin (mTOR) Kinase: Nowak, P.; Cole, D.C.; Brooijmans, N.; Bursavich, M. G.; Curran, K. J.; Ellingboe, J. W.; Gibbons, J.J.; Hollander, I.; Hu, Y.; Kaplan, J.; Malwitz, D. J.; Toral-Barza, L.; Verheijen, J.C.; Zask, A.; Zhang, W.-G.; Yu, K. 2009; Journal of Medicinal Chemistry Volume 52, Issue 22, 7081-89, which is incorporated herein by reference in its entirety.

Monomer P. 2-(4-(8-(6-(aminomethyl)quinolin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]quinoline- 1-yl)phenyl)-2-methylpropanenitrile salt of trifluoroacetic acid.

The synthesis of this monomer proceeds first through the synthesis of the Suzuki reaction coupling agent (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)quinolin-6-yl) -N -boc-methanamine, starting with methyl 3 -bromoquinoline-6-carboxylate. Reduction of the methyl ester with lithium aluminum hydride followed by Mitsunobu's reaction with phthalimide and hydrazine cleavage produces the benzyl amine. Protection of the benzyl amine with di- tert- butyl dicarbonate followed by the Miyaura borylation reaction gives (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)quinolin-6-yl) -N -boc-methanamine.

S _N Ar reaction of 2-(4-aminophenyl)-2-methylpropanenitrile with 6-bromo-4-chloro-3-nitroquinoline gives a substituted aminonitropyridine. Reduction of the Ni nitro group by Raney under hydrogen followed by cyclization with trichloromethyl chloroformate gives aryl-substituted urea. Substitution of free NH urea with methyl iodide mediated by tetrabutylammonium bromide and sodium hydroxide followed by Suzuki coupling (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)quinolin-6-yl) -N -boc-methanamine and then deprotecting Boc using TPA produces the TPA salt.

Reference for the preparation of 2-[4-(8-bromo-3-methyl-2-oxo-2,3-dihydroimidazo[4,5-c]quinolin-1-yl)phenyl]-2-methylpropionitrile: Vannucchi, A.M.; Bogani, C.; Bartalucci, N. 2016. JAK PI3K/mTOR combination therapy. US9358229. Novartis Pharma AG, Incyte Corporation, which is incorporated herein by reference in its entirety.

Monomer Q. 8-(6-methoxypyridin-3-yl)-3-methyl-1-[4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl]-1H,2H,3H-imidazo[4, 5-c]quinolin-2-one

This monomer is a commercially available compound known as BGT226 (CAS# 1245537-68-1). At the time of preparation of this application, it was available for purchase from several suppliers as a free amine.

Monomer R. 3-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-(4,5-dihydrothiazol-2-yl)benzamide salt trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl (4-(4-amino-3-(3-((4,5-dihydrothiazol-2-yl)carbamoyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1 -yl)butyl)carbamate

To a solution of (3-((4,5-dihydrothiazol-2-yl)carbamoyl)phenyl)boronic acid (500 mg, 1.15 mmol, 1.0 equiv.) and tert- butyl (4-(4-amino- 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (575 mg, 2.30 mmol, 2.0 equiv.) in dioxane (19.1 ml), EtOH (3 .8 ml) and H ₂ O (2.3 ml) add Pd(PPh ₃ ) ₄ (265 mg, 230 µmol, 0.2 equiv.) and sodium carbonate (730 mg, 6.89 mmol, 6.0 equiv. .). The reaction mixture is sonicated until a clear yellow solution is formed, which is then heated at 80°C for 14 hours. The reaction mixture is then diluted with sat. aq. NaCl (30 ml) and the mixture was transferred to a separatory funnel. The aqueous phase is extracted with DCM (3×25 ml). The combined organic phases are dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The desired product is isolated as a yellow solid (324 mg, 53% yield) after chromatography on silica gel (0→15% MeOH/DCM). LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₃₀ N ₈ O ₃ S: 511.22; found 511.2.

Step 2 : Synthesis of 3-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-(4,5-dihydrothiazol-2-yl)benzamide

To a solution of tert- butyl (4-(4-amino-3-(3-((4,5-dihydrothiazol-2-yl)carbamoyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl )butyl)carbamate (324 mg, 614 µmol) in DCM (4.1 ml) at 0°C add TFA (1.5 ml), dropwise. After 1 hour, the reaction mixture was warmed to room temperature and concentrated under reduced pressure to give the trifluoroacetate product as a yellow solid (320 mg, 99% yield). Can be used without further purification. LCMS (ESI) m/z : [M+H] calc. for C ₁₉ H ₂₂ N ₈ OS: 411.16; found 411.1

Monomer S. 2-(5-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-1H -indol-3-yl)ethane-1-amine.

The synthesis of this monomer is carried out through the condensation of 2,4,6-trichloropyrimidine-5-carbaldehyde with 3-((4-hydrazineylpiperidin-1-yl)methyl)pyridine hydrochloride. Reaction of the product with morpholine followed by a Suzuki reaction with a boronic ester gives the Boc-protected amine. Final deprotection of TPA yields a monomer. This synthesis method follows almost exactly the preparation of highly related structures described in the following references: i) Nowak, Pawel; Cole, Derek C.; Brooijmans, Natasja; Curran, Kevin J.; Ellingboe, John W.; Gibbons, James J.; Hollander, Irwin; Hu, Yong Bo; Kaplan, Joshua; Malwitz, David J.; et al From Journal of Medicinal Chemistry(2009), 52(22),7081-7089. ii) Zask, Arie; Nowak, Pawel Wojciech; Verheijen, Jeroen; Curran, Kevin J.; Kaplan, Joshua; Malwitz, David; Bursavich, Matthew Gregory; Cole, Derek Cecil; Ayral-Kaloustian, Semiramis; Yu, Ker; et al From PCT Int. Appl.(2008),WO2008115974A220080925, which are incorporated herein by reference.

Monomer T. 1-(4-aminobutyl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine salt of trifluoroacetic acid.

To a mixture of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)carbamate (496 mg, 1.14 mmol, 1.0 equiv. ) in DCM (5.7 ml) at 0°C add TFA (1.5 ml) dropwise. The reaction mixture was stirred at 0°C for 1 hour, at which time the reaction mixture was concentrated under reduced pressure to give a yellow solid (505 mg, 99% yield), which was taken up without further purification. LCMS (ESI) m/z : [M+H] calc. for C ₉ H ₁₃ IN ₆ : 333.02; found 332.9.

Monomer U. 5-(4-amino-1-(4-(methylamino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl (4-hydroxybutyl)(methyl)carbamate

To a solution of 4-(methylamino)butan-1-ol (0.5 g, 4.85 mmol, 104.2 mL, 1.0 equiv.) in DCM (10 mL) at room temperature add Boc ₂ O (1. 06 g, 4.85 mmol, 1.11 ml, 1.0 equiv.). The mixture is stirred for 3 hours at room temperature, and then the mixture is concentrated under reduced pressure at 30°C. The residue was purified by chromatography on silica gel (100/1-3/1 petroleum ether/EtOAc) to give tert- butyl (4-hydroxybutyl)(methyl)carbamate (0.9 g, 91.4% yield) as a colorless oil.

Step 2 : Synthesis of tert- butyl (4-bromobutyl)(methyl)carbamate

To a solution of tert- butyl (4-hydroxybutyl)(methyl)carbamate (0.9 g, 4.43 mmol, 1.0 equiv.) in THF (20 ml) at room temperature add PPh ₃ (2.21 g, 8 .41 mmol, 1.9 equiv.) and CBr ₄ (2.79 g, 8.41 mmol, 1.9 equiv.). The mixture is stirred for 1 hour, and then the reaction mixture is filtered and concentrated. The residue was purified by silica gel chromatography (1/0-4/1 petroleum ether/EtOAc) to give tert- butyl (4-bromobutyl)(methyl)carbamate (1.1 g, 93.3% yield) as a colorless oil.

Step 3 : Synthesis of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)(methyl)carbamate

To a suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.9 g, 3.45 mmol, 1.0 equiv.) in DMF (10 ml) at 4°C add NaH (137.92 mg, 3.45 mmol, 60% wt., 1.0 equiv.). The mixture was stirred at 4°C for 30 min and then a solution of tert- butyl (4-bromobutyl)(methyl)carbamate (1.01 g, 3.79 mmol, 25.92 mL, 1.1 equiv.) in DMF was added (3 ml). The mixture was stirred at room temperature for 3 hours, at which point H ₂ O (100 ml) was added. The aqueous phase is extracted with EtOAc (3×30 ml) and the combined organic phases are washed with brine (20 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/0-0/1 petroleum ether/EtOAc) to give tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl )butyl)(methyl)carbamate (1.2 g, 78% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₅ H ₂₃ IN ₆ O ₂ : 447.10; found 447.1.

Step 4 : Synthesis of tert- butyl (4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)( methyl)carbamate

To a two-phase suspension of tert- butyl (4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)(methyl)carbamate (1.2 g, 2.69 mmol , 1.0 equiv.), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (1.19 g, 3. 23 mmol, 1.2 equiv.) and Na ₂ CO ₃ (1.42 g, 13.44 mmol, 5.0 equiv.) in DME (20 ml) and H ₂ O (10 ml) add Pd at room temperature (PPh ₃ ) ₄ (310.71 mg, 268.89 µmol, 0.1 equiv.) under N ₂ . The mixture is stirred at 110°C for 3 hours and then the reaction mixture is cooled and partitioned between EtOAc (20 ml) and H ₂ O (15 ml). The aqueous layer is separated and extracted with EtOAc (3×20 ml). The combined organic layers were washed with brine (2×20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product is purified by chromatography on silica gel (1/0-4/1 EtOAc/MeOH) to give tert- butyl (4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo [3,4-d]pyrimidin-1-yl)butyl)(methyl)carbamate (0.78 g, 62.5% yield) as an orange solid.

Step 5 : Synthesis of 5-(4-amino-1-(4-(methylamino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine

Solution of tert- butyl(4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)(methyl)carbamate (0.78 g, 1.72 mmol, 1.0 equiv.) in TFA (5 ml) at room temperature and stirred for 30 minutes. The solution is concentrated under reduced pressure and the oily residue is triturated with MeCN (1 ml) and then added to MTBE (100 ml). The supernatant was removed and then the precipitate was collected by filtration under N ₂ to obtain 5-(4-amino-1-(4-(methylamino)butyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl bis-trifluorosulfonate )benzo[d]oxazol-2-amine (0.959 g, 93% yield) as an orange solid. LCMS (ESI) m/z : [M +H] calc. _{for C17H20N8O} : _353.18 ; found 353.1.

Monomer V. 1-(4-(4-(5-(aminomethyl)pyrimidin-2-yl)piperazin-1-yl)-3-(trifluoromethyl)phenyl)-8-(6-methoxypyridin-3-yl)- 3-methyl-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one.

Step 1 : Synthesis of tert- butyl N- tert- butoxycarbonyl-N-[(2-chloropyrimidin-5-yl)methyl]carbamate

To a solution of tert- butyl N- tert- butoxycarbonylcarbamate (7.33 g, 33.74 mmol, 1.0 equiv.) in DMF (80 ml) add NaH (1.62 g, 40.49 mmol, 60% wt. , 1.2 equiv.) at 0°C. The mixture was stirred at 0°C for 30 minutes and then 5-(bromomethyl)-2-chloro-pyrimidine (7 g, 33.74 mmol, 1 equiv.) was added. The reaction mixture is stirred at room temperature for 1.5 hours, and then the mixture is poured into sat. NH ₄ Cl (300 ml) and stir for 5 minutes. The aqueous phase is extracted with EtOAc (3×80 ml) and the combined organic phases are washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (20:1-1:1 petroleum ether/EtOAc) to give tert- butyl N- tert- butoxycarbonyl-N-[(2-chloropyrimidin-5-yl)methyl]carbamate (7.0 g, 60.3% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₅ H ₂₂ ClN ₃ O ₄ : 344.14; found 344.2.

Step 2 : Synthesis of tert- butyl N- tert- butoxycarbonyl-N-[[2-[4-[4-[8-(6-methoxy-3-pyridyl)-3-methyl-2-oxo-imidazo[4, 5-c]quinolin-1-yl]-2-(trifluoromethyl)phenyl]piperazin-1-yl]pyrimidin-5-yl]methyl]carbamate

To a solution of 8-(6-methoxy-3-pyridyl)-3-methyl-1-[4-piperazin-1-yl-3-(trifluoromethyl)phenyl]imidazo[4,5-c]quinolin-2-one ( 0.4 g, 748.32 µmol, 1.0 equiv.) to MeCN (7 ml) add tert- butyl N- tert- butoxycarbonyl-N-[(2-chloropyrimidin-5-yl)methyl]carbamate (514, 55 mg, 1.50 mmol, 2.0 equiv.) and K ₂ CO ₃ (413.69 mg, 2.99 mmol, 4 equiv.) at room temperature. The reaction mixture was stirred at 80°C for 14 hours, and then the mixture was cooled at room temperature, filtered and concentrated under reduced pressure. The residue was purified by washing with MTBE (5 ml) to give tert- butyl N- tert- butoxycarbonyl-N-[[2-[4-[4-[8-(6-methoxy-3-pyridyl)-3-methyl-2 -oxo-imidazo[4,5-c]quinolin-1-yl]-2-(trifluoromethyl)phenyl]piperazin-1-yl]pyrimidin-5-yl]methyl]carbamate (0.57 g, 90.5% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₄₃ H ₄₆ F ₃ N ₉ O ₆ : 842.36; found 842.7

Step 3 : Synthesis of 1-[4-[4-[5-(aminomethyl)pyrimidin-2-yl]piperazin-1-yl]-3-(trifluoromethyl)phenyl]-8-(6-methoxy-3-pyridyl) -3-methylimidazo[4,5-c]quinolin-2-one

Solution of tert- butyl N- tert- butoxycarbonyl-N-[[2-[4-[4-[8-(6-methoxy-3-pyridyl)-3-methyl-2-oxoimidazo[4,5-c]quinoline -1-yl]-2-(trifluoromethyl)phenyl]piperazin-1-yl]pyrimidin-5-yl]methyl]carbamate (0.95 g, 1.13 mmol, 1 equiv.) in TFA (10 ml) stirred at room temperature for 1 hour, at which point the solvent is concentrated. The residue is dissolved in MeCN (10 ml) and then the solution is added to MTBE (150 ml), dropwise. The precipitate is collected to give 1-[4-[4-[5-(aminomethyl)pyrimidin-2-yl]piperazin-1-yl]-3-(trifluoromethyl)phenyl]-8-(6-methoxy-3-pyridyl) trifluoromethanesulfonate )-3-methylimidazo[4,5-c]quinolin-2-one (0.778 g, 84.8% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₃₃ H ₃₀ F ₃ N ₉ O ₂ : 642.26; found 642.4

Monomer W. 1-(4-aminobutyl)-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazolo[3,4-d]pyrimidin-4-amine.

Step 1 : Synthesis of tert- butyl N-[4-[4-amino-3-(1H-indol-5-yl)pyrazolo[3,4-d]pyrimidin-1-yl]butyl]carbamate

To a two-phase suspension of tert- butyl N-[4-(4-amino-3-iodopyrazolo[3,4-d]pyrimidin-1-yl)butyl]carbamate (8 g, 18.51 mmol, 1 equiv.), 5 -(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (5.42 g, 22.21 mmol, 1.2 equiv.) and Na ₂ CO ₃ (9.81 g, 92.54 mmol, 5 equiv.) in diglyme (160 ml) and H ₂ O (80 ml) add Pd(PPh ₃ ) ₄ (2.14 g, 1.85 mmol, 0.1 equiv.) at room temperature under N ₂ . The mixture was stirred at 110°C for 3 hours. The reaction mixture was cooled at room temperature, filtered, and the filtrate was partitioned between EtOAc (500 ml) and H ₂ O (500 ml). The aqueous layer is separated and extracted with EtOAc (3×300 ml). The organic layers are combined, washed with brine (20 ml) and dried over anhydrous Na ₂ SO ₄ then filtered, and the filtrate is concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/0-0/1 petroleum ether/EtOAc, then 4/1 EtOAc/MeOH) to give tert- butyl N-[4-[4-amino-3-(1H-indole-5- yl)pyrazolo[3,4-d]pyrimidin-1-yl]butyl]carbamate (6.6 g, 84.6% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₂₇ N ₇ O ₂ : 422.22; found 423.3.

Step 2 : Synthesis of 1-(4-aminobutyl)-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazolo[3,4-d]pyrimidin-4-amine

To tert- butyl N-[4-[4-amino-3-(1H-indol-5-yl)pyrazolo[3,4-d]pyrimidin-1-yl]butyl]carbamate (6.6 g, 15. 66 mmol, 1 equiv.) add TFA (66 ml), which is then stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure to remove TFA, and then MTBE (400 ml) was added to the residue. The suspension is stirred for 15 minutes, at which time the yellow solid is filtered and the solid cake is dried under reduced pressure to give 1-(4-aminobutyl)-3-(1H-pyrrolo[2,3-b]pyridin-5-yl )pyrazolo[3,4-d]pyrimidin-4-amine (10.2 g, 97.1% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₆ H ₁₈ N ₈ : 323.17; found 323.1.

Monomer X. 2-(4-amino-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H- indol-5-ol 2,2,2-trifluoroacetate.

Step 1 : Synthesis of tert- butyl 6-((4-amino-3-(5-(( tert- butyldimethylsilyl)oxy)-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin- 1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a solution of tert- butyl 6-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (1 g, 1.97 mmol, 1.0 equiv.) in dioxane (10.5 ml) and H ₂ O (3.5 ml) add (1-( tert- butoxycarbonyl)-5-(( tert- butyldimethylsilyl)oxy )-1H-indol-2-yl)boronic acid (1.16 g, 2.96 mmol, 1.5 equiv.), K ₃ PO ₄ (1.26 g, 5.92 mmol, 3.0 equiv. ), Pd ₂ (dba) ₃ (180.85 mg, 197.50 µmol, 0.1 equiv.) and SPhos (162.16 mg, 394.99 µmol, 0.2 equiv.) at room temperature under N ₂ . The hermetically sealed tube is heated at 150°C for 20 minutes under microwaves. The reaction mixture is then cooled and the 6 separate batches are combined together. The reaction mixture is partitioned between EtOAc (100 ml) and H ₂ O (100 ml). The aqueous layer was separated and extracted with EtOAc (3×80 ml). The organic layers are combined, washed with brine (100 ml) and dried over anhydrous Na ₂ SO ₄ . The solution is filtered and the filtrate is concentrated under reduced pressure. The crude product is purified by silica gel column chromatography (100/1-1/4 petroleum ether/EtOAc) to give tert- butyl 6-((4-amino-3-(5-(( tert- butyldimethylsilyl)oxy)-1H- indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (6.17 g, 82.9% yield) as a light yellow solid.

Step 2 : Synthesis of tert- butyl 6-((4-amino-3-(5-hydroxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)- 3,4-dihydroisoquinoline-2(1H)-carboxylate

To a mixture of tert- butyl 6-((4-amino-3-(5-(( tert- butyldimethylsilyl)oxy)-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1- yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (6.17 g, 9.86 mmol, 1.0 equiv.) in THF (100 ml) add tetrabutylammonium fluoride trihydrate (1 M, 10. 84 ml, 1.1 equiv.) in one serving at 0°C under N ₂ . The mixture was stirred at 0°C for 1 h and then added to H ₂ O (100 ml). The aqueous phase is extracted with EtOAc (3×80 ml), and the combined organic phase is washed with brine (2×80 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/1-0/1 petroleum ether/EtOAc) to give tert- butyl 6-((4-amino-3-(5-hydroxy-1H-indol-2-yl)-1H-pyrazolo [3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (4 g, 79.3% yield) as a light pink solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₈ H ₂₉ N ₇ O ₃ : 512.24; found 512.3.

Step 3 : Synthesis of 2,2,2-trifluoroacetate 2-(4-amino-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidine -3-yl)-1H-indol-5-ol

To a solution of tert- butyl 6-((4-amino-3-(5-hydroxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3, 4-dihydroisoquinoline-2(1H)-carboxylate (4.5 g, 8.80 mmol, 1.0 equiv.) in MeOH (50 ml) add HCl in MeOH (4 M, 50 ml, 22.7 equiv.) at room temperature. The mixture is stirred at room temperature overnight and then concentrated under reduced pressure. EtOAc (100 ml) was added to the crude product and the resulting precipitate was collected by filtration under N ₂ to give 2-(4-amino-1-((1,2,3,4-tetrahydroisoquinoline-6-) 2,2,2-trifluoroacetate yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol (4.1 g, 85.0% yield, 3HCl) as a light yellow solid . LCMS (ESI) m/z : [M+H] calc. for C ₂₃ H ₂₁ N ₇ O: 412.19; found 412.1.

Monomer Y. 2,2,2-trifluoroacetate 3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl) -1H-pyrazolo[3,4-d]pyrimidin-4-amine.

Step 1 : Synthesis of tert- butyl 6-(bromomethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

A solution of NBS (34.07 g, 191.39 mmol, 4 equiv.) in THF (200 ml) was added portionwise to a solution of tert- butyl 6-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (12 .6 g, 47.85 mmol, 1.0 equiv.) and triphenylphosphine (37.65 g, 143.55 mmol, 3.0 equiv.) in THF (200 ml) at 0°C. After addition is complete, the mixture is stirred for 1 hour at room temperature. EtOAc (150 ml) was added and the mixture was washed with H ₂ O (200 ml) and brine (150 ml), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (100/1-10/1 petroleum ether/EtOAc) to give tert- butyl 6-(bromomethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (8.56 g, 54.8 % yield) as a light yellow solid.

Step 2 : Synthesis of tert- butyl 6-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (9.5 g, 36.40 mmol, 1.0 equiv.) in DMF (110 ml) add NaH (1.46 g, 36.40 mmol, 60% wt., 1.0 equiv.) at 0°C. The mixture was stirred at 0°C for 30 min, at which point a solution of tert- butyl 6-(bromomethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (12.47 g, 38.22 mmol, 1.05 equiv.) in DMF (40 ml) is added at 0°C. The mixture was stirred at room temperature for 1 hour and then H ₂ O (1000 ml) was added at 0°C. The mixture was stirred at 0°C for 30 minutes, and then the resulting precipitate was collected by filtration to give tert- butyl 6-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl) methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (17.8 g, 76.3% yield) as a light yellow solid, which was used immediately in the next step. LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₂₃ IN ₆ O ₂ : 507.10; found 507.1.

Step 3 : Synthesis of tert- butyl 6-((4-amino-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl )methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a two-phase suspension of tert- butyl 6-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate ( 6.5 g, 10.14 mmol, 1.0 equiv.), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3 -b]pyridine (2.97 g, 12.16 mmol, 1.2 equiv.) and Na ₂ CO ₃ (5.37 g, 50.68 mmol, 5.0 equiv.) in diglyme (100 ml) and H ₂ O (50 ml) add Pd(PPh ₃ ) ₄ (1.17 g, 1.01 mmol, 0.1 equiv.) at room temperature under N ₂ . The mixture is stirred at 110°C for 3 hours. The reaction mixture is then cooled and partitioned between EtOAc (100 ml) and H ₂ O (100 ml). The aqueous layer is separated and extracted with EtO (2×100 ml). The combined organic phase is washed with brine (100 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (0/1-1/4 MeOH/EtOAc) to give tert- butyl 6-((4-amino-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)- 1H-pyrazolo[3,4-d]pyramidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (3.77 g, 72.1% yield) as a light yellow solid . LCMS (ESI) m/z : [M+H] calc. for C ₂₇ H ₂₈ N ₈ O ₂ : 497.24; found 497.3.

Step 4 : Synthesis of 2,2,2-trifluoroacetate 3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl )-1H-pyrazolo[3,4-d]pyrimidin-4-amine

tert- Butyl 6-((4-amino-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)- 3,4-dihydroisoquinoline-2(1H)-carboxylate (3.77 g, 7.59 mmol, 1.0 equiv.) was added to TFA (85.36 ml, 1.15 mol, 151.8 equiv.) at room temperature. The reaction mixture is stirred for 1 hour. It is then concentrated under reduced pressure and the oily residue is triturated with MeCN (3 ml), then dropped into MTBE (200 ml) for 5 minutes. The supernatant was removed and the precipitate was then collected by filtration under N ₂ to obtain the product, which was dissolved in MeCN (20 ml) and finally concentrated under reduced pressure to give 2,2,2-trifluoroacetate 3-(1H-pyrrolo[2, 3-b]pyridin-5-yl)-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (4, 84 g, 85.0% yield, 3TPA) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₂₀ N ₈ : 397.19; found 397.2.

Monomer Z. 2,2,2-trifluoroacetate (4-((2-aminoethyl)sulfonyl)-3-fluoro-2-methylphenyl)(7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[f ][1,4]oxazepin-4(5H)-yl)methanone.

Step 1 : Synthesis of methyl 3,4-difluoro-2-methylbenzoate

To a solution of 3,4-difluoro-2-methylbenzoic acid (2 g, 11.62 mmol, 1.0 equiv.) in DMF (20 ml) add K ₂ CO ₃ (4.82 g, 34.86 mmol, 3 .0 equiv.) and iodomethane (3.26 ml, 52.29 mmol, 4.5 equiv.) at room temperature. The mixture was stirred at room temperature for 3 hours. A solution of methyl 3,4-difluoro-2-methylbenzoate in DMF (20 ml) was used immediately in the next step.

Step 2 : Synthesis of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)thio)-3-fluoro-2-methylbenzoate

To a solution of methyl 3,4-difluoro-2-methylbenzoate (2.16 g, 11.28 mmol, 1.0 equiv.) in DMF (20 ml) add tert- butyl (2-mercaptoethyl) carbamate (2.0 g , 11.28 mmol, 1 equiv.) and K ₂ CO ₃ (3.12 g, 22.56 mmol, 2.0 equiv.) at room temperature. The reaction mixture was stirred at 110°C for 12 hours, at which point the mixture was added to H ₂ O (50 ml). The aqueous solution is then extracted with EtOAc (3×30 ml) and the organic phase is combined and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/0-3/1 petroleum ether/EtOAc) to give methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)thio)-3-fluoro-2-methylbenzoate (3, 0 g, 76% yield) as a light yellow solid.

Step 3 : Synthesis of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-3-fluoro-2-methylbenzoate

To a solution of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)thio)-3-fluoro-2-methylbenzoate (3.3 g, 9.61 mmol, 1.0 equiv.), NaOH (2 M, 4.80 ml, 1.0 equiv.) and NaHCO ₃ (2.42 g, 28.83 mmol, 3.0 equiv.) in acetone (30 ml) add potassium peroxymonosulfate (12.35 g, 20. 08 mmol, 2.1 equiv.). The mixture is stirred for 12 hours at room temperature, and then the mixture is acidified to pH 5 by adding 1N HCl. The aqueous layer was extracted with EtOAc (3×30 ml) and the combined organic phase was washed with brine (20 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/0-3/1 petroleum ether/EtOAc) to give methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-3-fluoro-2-methylbenzoate (2, 1 g, 58.2% yield) as a yellow solid. LCMS (ESI) m/z : [M-56 +H] calc. for C ₁₆ H ₂₂ FNO ₆ S: 320.12; found 320.1

Step 4 : Synthesis of 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-3-fluoro-2-methylbenzoic acid

To a solution of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-3-fluoro-2-methylbenzoate (2.1 g, 5.59 mmol, 1.0 equiv.) in THF (20 ml), MeOH (10 ml) and H ₂ O (10 ml) add LiOH•H ₂ O (704.16 mg, 16.78 mmol, 3.0 equiv.) at room temperature. The reaction mixture was stirred at 40°C for 4 hours. The mixture was then concentrated under reduced pressure to remove THF and MeOH. The aqueous phase is neutralized with 0.5N HCl and then extracted with EtOAc (5×20 ml). The combined organic phase is washed with brine (2×20 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-3 -fluoro-2-methylbenzoic acid (2.01 g, 97.1% yield) as a white solid. LCMS (ESI) m/z : [M-100+H] calc. for C ₁₅ H ₂₀ FNO ₆ S: 262.11; found 262.1.

Step 5 : Synthesis of (4-( tert- butoxycarbonyl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-7-yl)boronic acid

To a solution of tert- butyl 7-bromo-2,3-dihydrobenzo[f][1,4]oxazepine-4(5H)-carboxylate (4 g, 12.19 mmol, 1.0 equiv.) in THF (80 ml ) at -60°C add B(OiPr) ₃ (4.58 g, 24.38 mmol, 5.60 ml, 2.0 equiv.), then add n -BuLi (2.5 M, 12, 19 ml, 2.5 equiv.) in n -hexane. The reaction mixture was stirred at -65°C for 1 hour. The reaction mixture was quenched with 1N HCl (12.25 ml) and warmed to room temperature. The reaction mixture was extracted with EtOAc (3×30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give (4-( tert- butoxycarbonyl)-2,3,4,5-tetrahydrobenzo[f][1 ,4]oxazepin-7-yl)boronic acid (3.5 g, crude) as a light yellow oil, which is used immediately in the next step. LCMS (ESI) m/z : [M-100+H] calc. for C ₁₄ H ₂₀ BNO ₅ : 194.15; found 194.2.

Step 6 : Synthesis of tert- butyl 7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[f][1,4]oxazepine-4(5H)-carboxylate

To a solution of (4-( tert- butoxycarbonyl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-7-yl)boronic acid (4.2 g, 14.33 mmol, 1.0 equiv.) in H ₂ O (20 ml) and dioxane (60 ml) add 5-bromopyridin-2-amine (2.48 g, 14.33 mmol, 1.0 equiv.), Pd(dppf)Cl ₂ • DCM (1.17 g, 1.43 mmol, 0.1 equiv.) and Et ₃ N (4.35 g, 42.99 mmol, 5.98 mL, 3.0 equiv.) at room temperature. The mixture was stirred at 85°C for 12 hours. The mixture was then cooled at room temperature and the residue was poured into H ₂ O (15 ml). The aqueous phase is extracted with EtOAc (3×40 ml) and the combined organic phase is washed with brine (2×40 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/0-1/8 petroleum ether/EtOAc) to give tert- butyl 7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[f][1,4]oxazepin- 4(5H)-carboxylate (3.3 g, 65.0% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₉ H ₂₃ N ₃ O ₃ : 342.18; found 342.2.

Step 7 : Synthesis of 5-(2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-7-yl)pyridin-2-amine

To a solution of tert- butyl 7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[f][1,4]oxazepine-4(5H)-carboxylate (3.3 g, 9.67 mmol, 1 .0 equiv.) in THF (40 ml) add HCl in EtOAc (4 M, 100 ml, 41.38 equiv.) at room temperature. The mixture was stirred for 3 hours. The reaction mixture was filtered and the filter cake was washed with EtOAc (3×15 ml) and then dried under reduced pressure to obtain 5-(2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine -7-yl)pyridin-2-amine (3 g, 95.1% yield, 2HCl) as a light yellow solid.

Step 8 : Synthesis of tert- butyl (2-((4-(7-(6-aminopyridin-3-yl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-4-carbonyl) -2-fluoro-3-methylphenyl)sulfonyl)ethyl)carbamate

To a solution of 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-3-fluoro-2-methylbenzoic acid (690.08 mg, 1.91 mmol, 1.0 equiv.) in DMF (10 ml) add GATU (1.09 g, 2.86 mmol, 1.5 equiv.) and DIPEA (1.66 ml, 9.55 mmol, 5 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, and then 5-(2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-7-yl)pyridin-2-amine (0.6 g) was added , 1.91 mmol, 1.0 equiv., 2 HCl). The mixture is stirred for 2 hours, at which point H ₂ O (40 ml) is added. The mixture was stirred for 5 minutes and the resulting precipitate was collected by filtration to obtain the crude product. The residue is purified by chromatography on silica gel (1/0-10/1 EtOAc/MeOH) to give tert- butyl (2-((4-(7-(6-aminopyridin-3-yl)-2,3,4,5- tetrahydrobenzo[f][1,4]oxazepine-4-carbonyl)-2-fluoro-3-methylphenyl)sulfonyl)ethyl)carbamate (0.538 g, 47.4% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₉ H ₃₃ FN ₄ O ₆ S: 585.22; found 585.3.

Step 9 : Synthesis of 2,2,2-trifluoroacetate (4-((2-aminoethyl)sulfonyl)-3-fluoro-2-methylphenyl)(7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[ f][1,4]oxazepin-4(5H)-yl)methanone

A solution of tert- butyl (2-((4-(7-(6-aminopyridin-3-yl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-4-carbonyl)-2- fluoro-3-methylphenyl)sulfonyl)ethyl)carbamate (0.538 g, 920.20 µmol, 1.0 equiv.) in TFA (10.35 ml, 139.74 mmol, 151.85 equiv.) was stirred at room temperature in for 2 hours. The solution is then concentrated under reduced pressure. The oily residue is triturated with MeCN (1 ml) and then dropped into MTBE (30 ml) for 10 min. The supernatant was removed and then the precipitate was collected by filtration under N ₂ to obtain 2,2,2-trifluoroacetate (4-((2-aminoethyl)sulfonyl)-3-fluoro-2-methylphenyl)(7-(6-aminopyridine-3 -yl)-2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)methanone (0.50 g, 87.4% yield) as a light brown solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₂₅ FN ₄ O ₄ S: 485.17; found 485.1.

Monomer AA. 5-(4-amino-1-(6-(piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2- amine salt of trifluoroacetic acid.

Step 1 : Synthesis of 1-(6-chloropyrimidin-4-yl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine

To a suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5 g, 19.16 mmol, 1.0 equiv) in DMF (60 ml) add NaH (804.53 mg 20.11 mmol, 60% wt., 1.05 equiv.) at 0°C. The mixture is stirred at 0°C for 30 minutes. 4,6-Dichloropyrimidine (3.42 g, 22.99 mmol, 1.2 equiv.) was then added to the reaction mixture at 0°C. The mixture was stirred at room temperature for 2.5 hours, at which point the reaction mixture was added to H ₂ O (600 ml). The suspension was then filtered to give the product (7.1 g, 99.2% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₉ H ₅ ClIN ₇ : 373.94; found 373.9.

Step 2 : Synthesis of tert- butyl 4-(6-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrimidin-4-yl)piperazine-1-carboxylate

To a solution of 1-(6-chloropyrimidin-4-yl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5 g, 13.39 mmol, 1.0 equiv.) and tert . - butyl piperazine-1-carboxylate (2.99 g, 16.06 mmol, 1.2 equiv.) in DMF (50 ml) add K ₂ CO ₃ (3.70 g, 26.77 mmol, 2.0 equiv. .). The reaction mixture is stirred at 100°C for 4 hours, at which point it is added to H ₂ O (500 ml). The suspension was then filtered to give the product (6.2 g, 88.5% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₈ H ₂₂ IN ₉ O ₂ : 524.09; found 524.2.

Step 3 : Synthesis of tert- butyl 4-(6-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrimidine -4-yl)piperazine-1-carboxylate

To a biphasic suspension of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (3.08 g, 11.85 mmol, 1. 0 equiv.), tert- butyl 4-(6-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrimidin-4-yl)piperazin-1-carboxylate ( 6.2 g, 11.85 mmol, 1.0 equiv.) and Na ₂ CO ₃ (6.28 g, 59.24 mmol, 5.0 equiv.) in H ₂ O (100 ml) and DME (200 ml) add Pd(PPh ₃ ) ₄ (1.37 g, 1.18 mmol, 0.1 equiv.) at room temperature under N ₂ . The mixture is stirred at 110°C for 24 hours, and then the mixture is filtered to obtain a solid cake. The solid was added to dioxane (20 ml) and stirred at 110°C for 60 min, then filtered to give the product (3.5 g, 55.8% yield) as a brown solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₅ H ₂₇ N ₁₁ O ₃ : 530.24; found 530.3.

Step 4 : Synthesis of 5-(4-amino-1-(6-(piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d] Oxazol-2-amine salt of trifluoroacetic acid

A solution of tert- butyl 4-(6-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrimidin-4- yl)piperazine-1-carboxylate (3.5 g, 6.61 mmol, 1.0 equiv.) in TFA (35 ml) was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and the resulting crude product was dissolved in MeCN (20 ml) and added dropwise to MTBE (500 ml). The resulting solid was then filtered to give the product (5.5 g, 91.9% yield) as a brown solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₁₉ N ₁₁ O: 430.19; found 430.1.

Monomer AB. 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(4-(5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin-1 -yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 2-(4-(4-(8-(6-methoxypyridin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5- c]quinolin-1-yl)-2-(trifluoromethyl)phenyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a mixture of 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinoline -2(3H)-one (0.3 g, 561.24 µmol, 1.0 equiv.) and tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H) -carboxylate (151.38 mg, 561.24 µmol, 1.0 equiv.) in DMF (5 ml) add K ₂ CO ₃ (193.92 mg, 1.40 mmol, 2.5 equiv.). The mixture is stirred at 100°C for 14 hours, at which point H ₂ O (20 ml) is added. The aqueous layer was extracted with EtOAc (3×40 ml) and the combined organic layers were concentrated under reduced pressure. The crude product was purified by column chromatography (30/1-15/1 DCM/MeOH) to give the product (0.30 g, 69.6% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₄₀ H ₄₀ F ₃ N ₉ O ₄ : 768.33; found 768.5.

Step 2 : Synthesis of 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(4-(5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl )piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one

Solution of tert- butyl 2-(4-(4-(8-(6-methoxypyridin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]quinoline -1-yl)-2-(trifluoromethyl)phenyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (0.8 g, 1.04 mmol , 1.0 equiv.) in TFA (8 ml) was stirred at room temperature for 2 hours. The solvent was concentrated and the residue was dissolved in MeCN (5 ml), then the solution was added dropwise to MTBE (150 ml). The precipitate is filtered and the solid is dried under reduced pressure to give the product (600 mg, 70.6% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₃₅ H ₃₂ F ₃ N ₉ O ₂ : 668.27; found 668.3.

Monomer AC. 5-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate

To a solution of tert- butyl 4-hydroxypiperidine-1-carboxylate (4 g, 19.87 mmol, 1.0 equiv.) and Et ₃ N (3.87 ml, 27.82 mmol, 1.4 equiv.) in DCM (40 ml) add MsCl (2.15 ml, 27.82 mmol, 1.4 equiv.) at 0°C. The reaction mixture was then stirred at room temperature for 1 hour. H ₂ O (50 ml) was added and the aqueous phase was extracted with DCM (3×50 ml). The combined organic phase was washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the product (5.62 g, 101% crude yield) as a yellow solid, which was used immediately in the next step.

Step 2 : Synthesis of tert- butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-carboxylate

To a suspension of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5 g, 19.16 mmol, 1.0 equiv.) and tert- butyl 4-((methylsulfonyl)oxy)piperidine- 1-carboxylate (5.62 g, 20.11 mmol, 1.05 equiv.) in DMF (100 ml) add K ₂ CO ₃ (5.29 g, 38.31 mmol, 2.0 equiv.). The mixture was stirred at 80°C for 12 hours. The reaction mixture was then added to H ₂ O (400 ml) at 0°C. The resulting precipitate was filtered to give the product (5.0 g, 58.8% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₅ H ₂₁ IN ₆ O ₂ : 445.09; found 445.1.

Step 3 : Synthesis of tert- butyl 4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1- carboxylate

To a suspension of tert- butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-carboxylate (5 g, 11.25 mmol, 1.0 equiv .), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (3.51 g, 13.51 mmol, 1, 2 equiv.) and Na ₂ CO ₃ (5.96 g, 56.27 mmol, 5.0 equiv.) in H ₂ O (50 ml) and DME (100 ml) add Pd(PPh ₃ ) ₄ (1, 30 g, 1.13 mmol, 0.1 equiv.) at room temperature under N ₂ . The mixture is stirred at 110°C for 3 hours. The reaction mixture is then cooled at room temperature and filtered. The filtrate is partitioned between EtOAc (100 ml) and H ₂ O (100 ml), and then the aqueous layer is separated and extracted with EtOAc (3×100 ml). The combined organic layer was washed with brine (20 mL) and dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was triturated with EtOAc (30 ml) and filtered to give the product (3.6 g, 71% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₂₆ N ₈ O ₃ : 451.22; found 451.3.

Step 4 : Synthesis of 5-(4-amino-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine trifluoroacetic acid salt

A solution of tert- butyl 4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-carboxylate (1 .4 g, 3.11 mmol, 1.0 equiv.) in TFA (10 ml) was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the crude solid was dissolved in MeCN (20 ml). The solution was added dropwise to MTBE (100 mL) and the resulting solid was filtered to give the product (1.6 g, 85.8% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. _{for C17H18N8O3} : _{351.17 ;} found 351.1.

Monomer AD. 1-(piperidin-4-yl)-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 4-(4-amino-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl) piperidine-1-carboxylate

To a suspension of 5-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (857.12 mg, 3.51 mmol, 1. 2 equiv.), tert- butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-carboxylate (1.3 g, 2.93 mmol , 1.0 equiv.) and Na ₂ CO ₃ (1.55 g, 14.63 mmol, 5.0 equiv.) in DME (20 ml) and H ₂ O (10 ml) add Pd(PPh ₃ ) ₄ (338.13 mg, 292.62 µmol, 0.1 equiv.) at room temperature under N ₂ . The mixture is stirred at 110°C for 3 hours. The reaction mixture is then cooled at room temperature and filtered. The filtrate is partitioned between EtOAc (50 ml) and H ₂ O (50 ml), and the aqueous layer is separated and extracted with EtOAc (3×50 ml). The combined organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was triturated with EtOAc (10 ml), filtered, and the solid cake was dried under reduced pressure to give the product (1.0 g, 78.7% yield) as a yellow solid.

Step 2 : Synthesis of 1-(piperidin-4-yl)-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine salt trifluoroacetic acid

Solution of tert- butyl 4-(4-amino-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1 -carboxylate (1.5 g, 3.45 mmol, 1.0 equiv.) in TFA (10 ml) was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the crude residue was dissolved in MeCN (20 ml). The solution was added dropwise to MTBE (100 mL) and the resulting solid was filtered to give the product (1.19 g, 74.2% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₇ H ₁₈ N ₈ : 335.18; found 335.1.

Monomer AE. (4-((2-aminoethyl)sulfonyl)-2-methylphenyl)(7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl )methanone.

Step 1 : Synthesis of methyl 4-fluoro-2-methyl benzoate

To a solution of 4-fluoro-2-methylbenzoic acid (86 g, 557.94 mmol, 1.0 equiv.) in DMF (900 ml) add K ₂ CO ₃ (231.33 g, 1.67 mol, 3.0 equiv.) and iodomethane (79.19 g, 557.94 mmol, 34.73 ml, 1.0 equiv.). The mixture was stirred at room temperature for 1 hour. A solution of methyl 4-fluoro-2-methylbenzoate in DMF (900 ml) was used immediately in the next step.

Step 2 : Synthesis of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)thio)-2-methylbenzoate

To a solution of methyl 4-fluoro-2-methylbenzoate (93.8 g, 557.94 mmol, 1.0 equiv.) in DMF (900 ml) add tert- butyl (2-mercaptoethyl) carbamate (98.91 g, 557 .97 mmol, 1.0 equiv.) and K ₂ CO ₃ (154.23 g, 1.12 mol, 2.0 equiv.). The reaction mixture was stirred at 110°C for 12 hours, at which point the mixture was cooled to room temperature and added to H ₂ O (1000 ml). The aqueous layer was then extracted with EtOAc (3×600 ml) and the combined organic layers were washed with brine, dried and concentrated under reduced pressure. Purification by chromatography on silica gel (0→25% EtOAc/petroleum ether) gave the desired product as a colorless oil (144 g, 79% yield).

Step 3 : Synthesis of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-2-methylbenzoate

To two separate batches containing a solution of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)thio)-2-methylbenzoate (72 g, 221.25 mmol, 1.0 equiv.), NaOH (2 M , 110.6 ml, 1.0 equiv.) and NaHCO ₃ (55.76 g, 663.75 mmol, 3.0 equiv.) in acetone (750 ml) add potassium peroxymonosulfate (284.28 g, 462.41 mmol, 2.1 equiv.). The mixture is stirred for 12 hours at room temperature, at which point the two batches are combined and the mixture is then acidified to pH 5 by adding 1N HCl. The aqueous layer was extracted with EtOAc (3x1500 ml) and the combined organic phases were washed with brine (2x500 ml), dried and concentrated under reduced pressure. Purification by silica gel chromatography (0→25% EtOAc/petroleum ether) gave the desired product as a white solid (120 g, 76% yield).

Step 4 : Synthesis of 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-2-methylbenzoic acid

To a solution of methyl 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-2-methylbenzoate (35 g, 97.92 mmol, 1.0 equiv.) in THF (200 ml), MeOH (100 ml) and H ₂ O (100 ml) add LiOH•H ₂ O (12.33 g, 293.77 mmol, 3.0 equiv.) at room temperature. The reaction mixture was stirred at 40°C for 1 hour. The mixture was then concentrated under reduced pressure to remove THF and MeOH. The aqueous phase is neutralized with 0.5N HCl, and the resulting precipitate is isolated by filtration. The solid cake was washed with H ₂ O (3 x 20 ml) to obtain the desired product as a white solid (25 g, 74% yield).

Step 5 : Synthesis of tert- butyl (2-((4-(7-(6-aminopyridin-3-yl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-4-carbonyl) -3-methylphenyl)sulfonyl)ethyl)carbamate

To a solution of 4-((2-(( tert- butoxycarbonyl)amino)ethyl)sulfonyl)-2-methylbenzoic acid (9.7 g, 28.25 mmol, 1.0 equiv.) and 5-(2.3, 4,5-tetrahydrobenzo[f][1,4]oxazepin-7-yl)pyridin-2-amine (8.88 g, 28.25 mmol, 1.0 equiv, 2HCl) in DMF (120 ml) was added GATU (16.11 g, 42.37 mmol, 1.5 equiv.) and DIPEA (18.25 g, 141.24 mmol, 24.60 ml, 5.0 equiv.). The reaction mixture was stirred at room temperature for 1 hour, at which point the reaction mixture was poured into H ₂ O (1000 ml). The mixture was stirred for 5 minutes and the resulting precipitate was collected by filtration to obtain the crude product. The crude product is triturated with EtOAc (100 ml), filtered and the solid cake is dried under reduced pressure to obtain the desired product as a white solid (14 g, 87% yield).

Step 6 : Synthesis of (4-((2-aminoethyl)sulfonyl)-2-methylphenyl)(7-(6-aminopyridin-3-yl)-2,3-dihydrobenzo[f][1,4]oxazepine-4( 5H)-yl)methanone

A solution of tert- butyl (2-((4-(7-(6-aminopyridin-3-yl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-4-carbonyl)-3- Methylphenyl)sulfonyl)ethyl)carbamate (19 g, 33.53 mmol, 1.0 equiv.) in TFA (100 ml) was stirred at room temperature for 30 minutes. The solution is then concentrated under reduced pressure. The residue is triturated with MeCN (30 ml) and then dropped into MTBE (600 ml) and stirred for 20 min. The suspension is filtered and the resulting solid is dissolved in MeCN (30 ml) and concentrated under reduced pressure to give the desired product as a light yellow solid (24 g, TPA salt). LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₂₆ N ₄ O ₄ S: 467.18; found 467.1.

Monomer AF. 5-(4-amino-1-((5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3- yl)benzo[d]oxazol-2-amine.

Step 1 : Synthesis of ( Z ) -tert- butyl 3-((dimethylamino)methylene)-4-oxopiperidine-1-carboxylate

A solution of tert- butyl 4-oxopiperidine-1-carboxylate (15 g, 75.28 mmol, 1.0 equiv.) and 1,1-dimethoxy- N,N -dimethylmethanamine (11.00 ml, 82.81 mmol, 1 .1 equiv.) in DMF (105 ml) was stirred at 95°C for 12 hours. The reaction mixture was then concentrated under reduced pressure and the resulting residue was dissolved in EtOAc (30 ml) and washed with brine (3 x 30 ml) . The aqueous phase was extracted with EtOAc (50 ml) and the combined organic phases were dried and concentrated under reduced pressure to give the desired product as a yellow solid (10.1 g, 53% yield).

Step 2 : Synthesis of tert- butyl 2-(hydroxymethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of NaOEt (1.98 g, 29.10 mmol, 1.0 equiv.) in EtOH (70 ml) add ( Z ) -tert -butyl 3-((dimethylamino)methylene)-4-oxopiperidine-1-carboxylate (7.4 g, 29.10 mmol, 1.0 equiv.) and 2-hydroxyacetimidamide hydrochloride (3.54 g, 32.01 mmol, 1.1 equiv. ). The reaction mixture is heated to 90°C for 12 hours, at which point the mixture is cooled at room temperature and concentrated under reduced pressure. The residue is partitioned with EtOAc (40 ml) and washed with sat. NaHCO ₃ (40 ml). The aqueous phase is extracted with EtOAc (3×20 ml) and the combined organic phases are washed with brine (2×50 ml), dried and concentrated under reduced pressure. Purification by silica gel chromatography (25% EtOAc/petroleum ether) gave the desired product as a yellow solid (7.24 g, 94% yield).

Step 3 : Synthesis of tert- butyl 2-(bromomethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of tert- butyl 2-(hydroxymethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6.24 g, 23.52 mmol, 1.0 equiv. ) and PPh ₃ (12.34 g, 47.04 mmol, 2.0 equiv.) in DCM (140 ml) add CBr ₄ (14.82 g, 44.69 mmol, 1.9 equiv.). The mixture is stirred at room temperature for 3 hours, at which point the mixture is concentrated under reduced pressure. The residue is partitioned between EtOAc (20 ml) and H ₂ O (20 ml), the aqueous phase is extracted with EtOAc (3×20 ml). The combined organic phases are washed with brine (2×50 ml), dried and concentrated under reduced pressure. Purification by silica gel chromatography (14% EtOAc/petroleum ether) gave the desired product as a yellow solid (3.6 g, 47% yield).

Step 4 : Synthesis of tert- butyl 2-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-7,8-dihydropyrido[4,3-d] pyrimidine-6(5H)-carboxylate

To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.59 g, 6.09 mmol, 1.0 equiv.) in DMF (15 ml) add NaH (243.73 mg, 6.09 mmol, 60% wt., 1.0 equiv.) at 0°C. The suspension is stirred for 30 minutes and then tert- butyl 2-(bromomethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (2.2 g, 6.70 mmol, 1.1 equivalent). The reaction mixture was warmed to room temperature and stirred for 3 hours. The mixture was poured into H ₂ O at 0°C and the precipitate was collected by filtration to give the desired product as a brown solid (2.5 g, 66% yield).

Step 5 : Synthesis of tert- butyl 2-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)- 7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of tert- butyl 2-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-7,8-dihydropyrido[4,3-d]pyrimidin- 6(5H)-carboxylate (4.55 g, 8.95 mmol, 1.0 equiv.), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo [d]oxazol-2-amine (2.79 g, 10.74 mmol, 1.2 equiv.) and Na ₂ CO ₃ (4.74 g, 44.76 mmol, 5.0 equiv.) in dioxane ( 70 ml) and H ₂ O (35 ml) add Pd(PPh ₃ ) ₄ (1.03 g, 895.11 µmol, 0.1 equiv.). The reaction mixture is heated to 110°C for 3 hours, at which point the mixture is cooled to room temperature and poured into H ₂ O at 0°C. The precipitate is filtered and the solid cake is dried under reduced pressure. The crude product was washed with EtOAc (50 mL) to obtain the desired product as a light yellow solid (3.14 g, 68% yield).

Step 6 : Synthesis of 5-(4-amino-1-((5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)methyl)-1H-pyrazolo[3,4-d] pyrimidin-3-yl)benzo[d]oxazol-2-amine

Solution of tert- butyl 2-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-7.8 -dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (3.14 g, 6.10 mmol, 1.0 equiv.) in TFA (20 ml) was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure and the resulting residue was dissolved in MeCN (7 ml) and added to MTBE (700 ml). The precipitate was collected by filtration to obtain the desired product as a brown solid (4.25 g, 92% yield, 3 TFA). LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₁₈ N ₁₀ O: 415.18; found 415.1.

Monomer AG. 5-(4-amino-1-((2-((2-aminoethyl)sulfonyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidine -3-yl)benzo[d]oxazol-2-amine.

Step 1 : Synthesis of N -Boc taurine tetrabutylammonium salt

To a solution of 2-aminoethanesulfonic acid (10.00 ml, 79.91 mmol, 1.0 equiv) in THF (60 ml) and aqueous NaOH (2 M, 40 ml, 1.0 equiv) add Boc ₂ O 18.31 g, 83.90 mmol, 1.05 equiv.). The mixture was stirred at room temperature for 15 hours, at which point the mixture was extracted with EtOAc (10 ml). The aqueous phase is diluted with H ₂ O (450 ml), treated with LiOH•H ₂ O (3.35 g, 79.83 mmol, 1.0 equiv.) and n Bu ₄ NHSO ₄ (27.13 g, 79.90 mmol , 1.0 equiv.) and stir for 30 minutes. This mixture is extracted with DCM (3 x 80 ml) and the combined organic phases are dried and concentrated under reduced pressure to give the desired product as a colorless oil (34.26 g, 91% yield).

Step 2 : Synthesis of tert- butyl (2-(chlorosulfonyl)ethyl)carbamate

To a solution of N -Boc taurine tetrabutylammonium salt (4.7 g, 10.05 mmol, 1.0 equiv.) in DCM (42 ml) add DMF (77.32 μl, 1.00 mmol, 0.1 equiv.) , then a solution of triphosgene (0.5 M, 8.04 ml, 0.4 equiv.) in DCM at 0°C. The mixture is warmed to room temperature and stirred for 30 minutes. A solution of tert- butyl (2-(chlorosulfonyl)ethyl)carbamate (2.45 g, crude) in DCM was used immediately in the next step.

Step 3 : Synthesis of tert- butyl (2-((6-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1- yl)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)ethyl)carbamate

To a solution of 5-(4-amino-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d] oxazol-2-amine (6.04 g, 9.44 mmol, 1.0 equiv., 2TFA) in DMF (40 ml) add Et ₃ N (7.88 ml, 56.63 mmol, 6.0 equiv. ). Add a solution of tert- butyl (2-(chlorosulfonyl)ethyl)carbamate in DCM (42 ml) at 0°C. The mixture was warmed to room temperature and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting solution was purified by reverse phase chromatography (15→45% MeCN/H ₂ O) to obtain the desired product as a white solid (5 .8 g, 83% yield, TPA). LCMS (ESI) m/z : [M+H] calc. for C ₂₉ H ₃₃ N ₉ O ₅ S: 620.24; found 620.3.

Step 4 : Synthesis of 5-(4-amino-1-((2-((2-aminoethyl)sulfonyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4 -d]pyrimidin-3-yl)benzo[d]oxazol-2-amine

A solution of tert- butyl (2-((6-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl )-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)ethyl)carbamate (5.8 g, 9.36 mmol, 1.0 equiv.) in TFA (48 ml) was stirred at room temperature for 0 5 hours, at which point the reaction mixture is concentrated under reduced pressure. The crude product is dissolved in MeCN (30 ml) and added dropwise to MTBE (200 ml). The mixture was stirred for 5 minutes and filtered, and the filter cake was dried under reduced pressure to obtain the desired product as a yellow solid (3.6 g, 62% yield, 2.2 TFA). LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₂₅ N ₉ O ₃ S: 520.19; found 520.1.

Monomer AH. tert- butyl ((5-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrimidin-2-yl )methyl)carbamate.

Step 1 : Synthesis of (2-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-5-yl)methyl methanesulfonate

To a solution of tert- butyl ((5-(hydroxymethyl)pyrimidin-2-yl)methyl)carbamate (4.2 g, 17.55 mmol, 1.0 equiv.) in DCM (42 ml) at 0°C add Et ₃ N (7.33 ml, 52.66 mmol, 3.0 equiv), then MsCl (2.41 g, 21.06 mmol, 1.63 ml, 1.2 equiv). The mixture was stirred at 0°C for 10 minutes and then H ₂ O (15 ml) was added. The reaction mixture was extracted with DCM (5 x 10 mL) and the combined organic phases were washed with brine (5 mL), dried, filtered and concentrated under reduced pressure to give the desired product (5.5 g, 98.7% yield) as colorless solid.

Step 2 : Synthesis of tert- butyl ((5-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrimidin-2-yl)methyl)carbamate

To a solution of (2-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-5-yl)methylmethanesulfonate (5.47 g, 17.24 mmol, 1.2 equiv.) and 3-iodo-1H-pyrazolo[3 ,4-d]pyrimidin-4-amine (3.75 g, 14.37 mmol, 1.0 equiv.) in DMF (55 ml) at room temperature add K ₂ CO ₃ (5.96 g, 43.10 mmol, 3 equiv.). The mixture was stirred at 80°C for 5 hours, at which point H ₂ O (100 ml) and brine (20 ml) were poured into the reaction mixture. The solution is extracted with EtOAc (10×30 ml) and the combined organic phases are dried, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (0→30% EtOAc/MeOH) gave the desired product (2 g, 28.9% yield) as a yellow solid.

Step 3 : Synthesis of tert- butyl ((5-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl )pyrimidin-2-yl)methyl)carbamate

To a solution of tert- butyl ((5-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrimidin-2-yl)methyl)carbamate (2 g, 4.15 mmol, 1.0 equiv.), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-benzoxazol-2-amine (1, 13 g, 4.35 mmol, 1.05 equiv.) and Na ₂ CO ₃ (688.39 mg, 8.29 mmol, 2.0 equiv.) in dioxane (20 ml) and H ₂ O (10 ml) add Pd(PPh ₃ ) ₄ (479.21 mg, 414.70 μmol, 0.1 equiv.). The mixture is stirred at 110°C for 1 hour, during which time the mixture is cooled to room temperature, filtered and the solid cake is washed with MeOH (3×10 ml). The filtrate is concentrated under reduced pressure to remove MeOH and then added dropwise to H ₂ O (50 ml). The resulting suspension is filtered and the filter cake is washed with H ₂ O (3×10 ml). The solid cake is stirred in MeOH (20 ml) for 30 minutes. The resulting suspension is filtered and the filter cake is washed with MeOH (3×8 ml). The filter cake was dried under reduced pressure to obtain the desired product (1.03 g, 48.9% yield) as a white solid. LCMS (ESI) m/z : [M+ H] calc. for C ₂₃ H ₂₄ N ₁₀ O ₃ : 489.21; found489.2.

Step 4 : Synthesis of 5-(4-amino-1-{[2-(aminomethyl)pyrimidin-5-yl]methyl}-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1,3- benzoxazole-2-amine

K tert- butyl ((5-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrimidin- 2-yl)methyl)carbamate (100 mg, 0.205 mmol, 1.0 equiv.) was added conc. HCl (850 µl, 10.2 mmol, 50 equiv.). The reaction mixture was stirred for 1 hour and then poured into acetone (3 ml). The resulting precipitate is filtered, washed with acetone and dried under reduced pressure to obtain the desired product (80 mg, 92% yield) as a brown solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₈ H ₁₆ N ₁₀ O: 389.16; found 389.0.

Monomer AI. 5-(4-(dimethylamino)-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d] Oxazol-2-amine salt of trifluoroacetic acid.

Step 1 : Synthesis of tert- butyl 6-((4-(dimethylamino)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H) -carboxylate

To a solution of 3-iodo- N,N -dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3.6 g, 12.45 mmol, 1.0 equiv.) in DMF (36 ml) at 0°C add NaH (523.00 mg, 13.08 mmol, 60 wt%, 1.05 equiv.). The mixture is stirred at 0°C for 30 minutes. A solution of tert- butyl 6-(bromomethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (4.47 g, 13.70 mmol, 1.1 equiv) in DMF (18 ml) was then added to the reaction mixture. at 0°C. The mixture was stirred at room temperature for 2 hours. The reaction mixture was then added to cold H ₂ O (200 ml) and stirred for 30 minutes. The resulting precipitate was collected by filtration to obtain the desired product (6 g, 71.9% yield) as a white solid.

Step 2 : Synthesis of tert- butyl 6-((3-(2-aminobenzo[d]oxazol-5-yl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl )-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a solution of tert- butyl 6-((4-(dimethylamino)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (2 g, 2.96 mmol, 1.0 equiv.) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2-amine (922.81 mg, 3.55 mmol, 1.2 equiv.) in dioxane (24 ml) and H ₂ O (12 ml) add Na ₂ CO ₃ (1.57 g, 14.78 mmol, 5.0 equiv.) and Pd(PPh ₃ ) ₄ (341.66 mg, 295.66 µmol, 0.1 equiv.). The mixture was stirred at 110°C for 12 hours. The reaction mixture was then poured into cold H ₂ O (200 ml) and stirred for 30 minutes. The resulting precipitate is collected by filtration. Purification by silica gel chromatography (5→100% petroleum ether/EtOAc) gave the desired product (1.2 g, 72.3% yield) as a yellow solid.

Step 3 : Synthesis of 5-(4-(dimethylamino)-1-((1,2,3,4-tetrahydroisoquinolin-6-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl) benzo[d]oxazol-2-amine

Solution of tert- butyl 6-((3-(2-aminobenzo[d]oxazol-5-yl)-4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3 ,4-dihydroisoquinoline-2(1H)-carboxylate (1.7 g, 3.14 mmol, 1.0 equiv) in TFA (10 ml) was stirred at room temperature for 30 minutes. The reaction mixture is then concentrated under reduced pressure. The residue is added to MeCN (10 ml) and the solution is added dropwise to MTBE (200 ml). The resulting solid was dissolved in MeCN (30 ml) and the solution was concentrated under reduced pressure to obtain the desired product (1.67 g, 92.9% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₂₄ N ₈ O: 441.22; found 441.2.

Monomer AJ. 4-amino-5-(2-aminobenzo[d]oxazol-5-yl)-5H-pyrimido[5,4-b]indole-7-carboxylic acid.

This monomer can be prepared from 7-methyl-5H-pyrimido[5,4-b]indol-4-ol by benzyl oxidation to carboxylic acid, conversion to ethyl ester, then O -ethylation with triethyloxonium tetrafluoroborate. Palladium-mediated arylation followed by ester hydrolysis and final ammoniolysis produces the monomer.

Monomer AK. 4-amino-5-(2-aminobenzo[d]oxazo-5-yl)-5H-pyrimido[5,4-b]indole-8-carboxylic acid.

This monomer can be prepared in the same manner as the previous monomer, but using the isomeric starting material of 8-methyl-5H-pyrimido[5,4-b]indol-4-ol. Benzyl oxidation to carboxylic acid, conversion to ethyl ester, then O -ethylation with triethyloxonium tetrafluoroborate and palladium-mediated arylation followed by ester hydrolysis and final ammoniolysis gives the monomer.

AL monomer. 3-(2,4-bis(( S )-3-methylmorpholino)-4a,8a-dihydropyrido[2,3-d]pyrimidin-7-yl)benzoic acid.

Step 1 : Synthesis of ( 3S )-4-[7-chloro-2-[( 3S )-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-4-yl]3-methyl- morpholine

To a solution of 2,4,7-trichloropyrido[2,3-d]pyrimidine (4.0 g, 17.06 mmol, 1.0 equiv.) in DMA (10 ml) add ( 3S )-3-methylmorpholine ( 4.31 g, 42.65 mmol, 2.5 equiv.) and DIPEA (5.51 g, 42.65 mmol, 7.43 ml, 2.5 equiv.). The reaction solution is heated to 70°C for 48 hours. The reaction suspension is cooled at room temperature, poured into cold H ₂ O (50 ml) until a solid precipitates. The solid is filtered and the filter cake is washed with H ₂ O and dried under reduced pressure to give the crude product, which is purified by silica gel column chromatography (0→100% petroleum ether/EtOAc) to give (3 S )-4-[7-chloro -2-[( 3S )-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-4-yl]3-methylmorpholine (3.5 g, 56.4% yield) as a yellow solid substances. LCMS (ESI) m/z : [M +H] calc. for C ₁₇ H ₂₂ ClN ₅ O ₂ : 364.15; found 364.2

Step 2 : Synthesis of 3-[2,4-bis[( 3S )-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]benzoic acid

To a solution of (3 S )-4-[7-chloro-2-[(3 S )-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-4-yl]-3-methylmorpholine (2 g, 5.50 mmol, 1.0 equiv.) and 3-boronobenzoic acid (1.09 g, 6.60 mmol, 1.2 equiv.) in 1,4-dioxane (40 ml) add a solution of K ₂ CO ₃ (911.65 mg, 6.60 mmol, 1.2 equiv.) in H ₂ O (4 ml), then Pd(PPh ₃ ) ₄ (317.60 mg, 274.85 µmol, 0.05 equiv. ). The solution is degassed for 10 minutes and refilled with N ₂ , then the reaction mixture is heated to 100°C under N ₂ for 5 hours. The reaction mixture is cooled to room temperature and filtered. The filtrate is acidified with HCl (2N) to pH 3 and the aqueous layer is washed with EtOAc (3×20 ml). The aqueous phase is concentrated under reduced pressure to give a residue, which is purified by silica gel column chromatography (50%→100% petroleum ether/EtOAc) to give 3-[2,4-bis[( 3S )-3-methylmorpholine-4 hydrochloride -yl]pyrido[2,3-d]pyrimidin-7-yl]benzoic acid (2.5 g, 89.9% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₂₇ N ₅ O ₄ : 450.21; found 450.2.

Reference for the preparation of this monomer: Menear, K.; Smith, G. C. M.; Malagu, K.; Duggan, H. M. E.; Martin, N.M.B.; Leroux, F.G.M. 2012. Pyrido-, pyrazo- and pyrimido-pyrimidine derivatives as mTOR inhibitors. US8101602. Kudos Pharmaceuticals, Ltd, which is incorporated herein by reference. fully.

Monomer AM. (1r,4r)-4-[4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[4,3-f][1,2,4]triazin-7-yl]cyclohexane -1-carboxylic acid

This monomer, also known as OSI-027 (CAS# = 936890-98-1), is a commercially available compound. At the time of writing this application, it was available for purchase from several suppliers.

AN monomer. 2-(4-(4-(8-(6-methoxypyridin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl )-2-(trifluoromethyl)phenyl)piperazin-1-yl)pyrimidine-5-carboxylic acid.

This monomer is prepared by reacting BGT226 with methyl 2-chloropyrimidine-5-carboxylate followed by hydrolysis of the ester to give the title monomer.

Monomer AO. 4-amino-5-{1H-pyrrolo[2,3-b]pyridin-5-yl}-5H-pyrimido[5,4-b]indole-8-carboxylic acid.

This monomer can be prepared from 7-methyl-5H-pyrimido[5,4-b]indol-4-ol by benzyl oxidation to carboxylic acid, conversion to ethyl ester, then O -ethylation with triethyloxonium tetrafluoroborate. Palladium-mediated arylation, followed by ester hydrolysis and final ammoniolysis produces the monomer.

Preparation of pre- and post-linkers

Building block A. tert- butyl N-[( tert- butoxy)carbonyl]-N-{[2-(piperazin-1-yl)pyrimidin-5-yl]methyl}carbamate.

Step 1 : Synthesis of 5-(bromomethyl)-2-chloropyrimidine

To a solution of 2-chloro-5-methylpyrimidine (92 g, 715.62 mmol, 1.0 equiv.) in CCl ₄ (1000 ml) add NBS (178.31 g, 1.00 mol, 1.4 equiv.) and benzoyl peroxide (3.47 g, 14.31 mmol, 0.02 equiv.). The mixture was stirred at 76°C for 18 hours. The reaction mixture was then cooled at room temperature and concentrated under reduced pressure. The reaction mixture is filtered and the solid cake is washed with DCM (150 ml). The resulting solution is concentrated under reduced pressure to obtain the crude product. The residue was purified by chromatography on silica gel (1/0-0/1 petroleum ether/EtOAc) to give the product (70.8 g, 47.7% crude yield) as a yellow oil, which was used immediately in the next step. LCMS (ESI) m/z : [M+H] calc. for C ₅ H ₄ BrClN ₂ : 206.93; found 206.9.

Step 2 : Synthesis of tert- butyl N- tert- butoxycarbonyl-N-((2-piperazin-1-ylpyrimidin-5-yl)methyl)carbamate

To a solution of tert- butyl N- tert- butoxycarbonylcarbamate (36.89 g, 169.79 mmol, 0.74 equiv.) in DMF (750 ml) add NaH (6.88 g, 172.09 mmol, 60% wt. .0.75 equiv.) at 0°C. The mixture is stirred at 0°C for 30 minutes. Then, 5-(bromomethyl)-2-chloropyrimidine (47.6 g, 229.45 mmol, 1.0 equiv.) was added at 0°C. The reaction mixture was stirred at room temperature for 15.5 hours. The mixture was then poured into H ₂ O (1600 ml) and the aqueous phase was extracted with EtOAc (3×300 ml). The combined organic phase is washed with brine (2×200 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (1/0-0/1 petroleum ether/EtOAc) to give the product (70 g, crude) as a yellow solid, which was used immediately in the next step.

Step 3 : Synthesis of tert- butyl N- tert- butoxycarbonyl-N-[(2-piperazin-1-ylpyrimidin-5-yl)methyl]carbamate

To a solution of 1-benzylpiperazine (30.44 g, 122.16 mmol, 1.0 equiv., 2HCl) in MeCN (550 ml) add tert- butyl N- tert- butoxycarbonyl-N-((2-chloropyrimidine-5- yl)methyl)carbamate (42 g, 122.16 mmol, 1.0 equiv.) and K ₂ CO ₃ (84.42 g, 610.81 mmol, 5.0 equiv.). The mixture was stirred at 80°C for 61 hours. The reaction mixture was then diluted with EtOAc (150 ml) and the mixture was filtered. The resulting solution is concentrated under reduced pressure to obtain the crude product. The residue is purified by chromatography on silica gel (1/0-0/1 petroleum ether/EtOAc) to give the product (45 g, 74% yield) as a white solid.

Step 4 : Synthesis of tert- butyl N- tert- butoxycarbonyl-N-[(2-piperazin-1-ylpyrimidin-5-yl)methyl]carbamate

To a solution of tert- butyl N-[[2-(4-benzylpiperazin-1-yl)pyrimidin-5-yl]methyl]-N- tert- butoxycarbonylcarbamate (24 g, 49.63 mmol, 1.0 equiv.) in MeOH (600 ml) add Pd/C (24 g, 47.56 mmol, 10 wt%, 1.0 equiv) under argon. The mixture is degassed under reduced pressure and purged with H ₂ three times. The mixture was stirred under H ₂ (50 psi) at 50°C for 19 hours. The reaction mixture was cooled at room temperature, filtered, and the filter cake was washed with MeOH (500 ml). The resulting solution is concentrated under reduced pressure. The residue was purified by silica gel chromatography (1/0-0/1 EtOAc/MeOH) to give the product (25.5 g, 68% yield) as a white solid.

Building block B. 2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidine-5-carboxylic acid.

Step 1 : Synthesis of ethyl 2-(4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylate

To a solution of ethyl 2-chloropyrimidine-5-carboxylate (2.37 g, 12.71 mmol, 1.0 equiv.) and tert- butyl N- tert- butoxycarbonyl-N-((2-piperazin-1-ylpyrimidin-5 -yl)methyl)carbamate (5 g, 12.71 mmol, 1.0 equiv.) in MeCN (80 ml) add K ₂ CO ₃ (5.27 g, 38.12 mmol, 3.0 equiv.). The mixture is stirred at 80°C for 16 hours. The reaction mixture is then poured into H ₂ O (200 ml) and the suspension is filtered. The filtrate was washed with H ₂ O (80 ml) and dried under reduced pressure to give the product (6.1 g, 87% yield) as a white solid.

Step 2 : Synthesis of 2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylate (5 g, 9.20 mmol, 1 .0 equiv.) in H ₂ O (50 ml), EtOH (15 ml) and THF (50 ml) add LiOH•H ₂ O (1.54 g, 36.79 mmol, 4.0 equiv.). The reaction mixture was stirred at 55°C for 16 hours. The mixture was then concentrated to remove THF and EtOH, and then the mixture was diluted with H ₂ O (55 ml) and acidified (pH=3) with aqueous HCl (1N). The mixture was filtered and the filter cake was washed with H ₂ O (36 ml). The filter cake was dried under reduced pressure to give the product (2.7 g, 69.3%) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C19H25N7O4 : 416.21} ; found 416.1.

Building block C. tert- butyl 2-(piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate.

Step 1 : Synthesis of tert- butyl 2-(4-benzylpiperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (15 g, 55.61 mmol, 1.0 equiv.) in MeCN (150 ml) add 1-benzylpiperazine (11.76 g, 66.73 mmol, 1.2 equiv.) and K ₂ CO ₃ (46.12 g, 333.67 mmol, 6.0 equiv.). The mixture was stirred at 80°C for 27 hours. The reaction mixture was diluted with EtOAc (200 ml), filtered and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (1/0-0/1 petroleum ether/EtOAc) to give the product (20.2 g, 80% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₃ H ₃₁ N ₅ O ₂ : 410.26; found 410.1.

Step 2 : Synthesis of tert- butyl2-(piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of tert- butyl 2-(4-benzylpiperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (8 g, 19.53 mmol, 1.0 equiv .) in MeOH (200 ml) add Pd/C (8 g, 19.53 mmol, 10% wt., 1.0 equiv.) under argon. The mixture is degassed and purged with H ₂ three times. The mixture was stirred under H ₂ (50 psi) at 50°C for 19 hours. The reaction mixture was cooled at room temperature, filtered through a pad of Celite and the filter cake washed with MeOH (150 ml). The resulting solution is concentrated under reduced pressure. The crude product was washed with petroleum ether (60 ml) to obtain the product (9.25 g, 72% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C16H25N5O2 :} 320.21 _; found 320.2.

Building block D. 2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of tert- butyl 2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)- carboxylate

To a solution of ethyl 2-chloropyrimidine-5-carboxylate (4.09 g, 21.92 mmol, 1.0 equiv.) in dioxane (80 ml) add tert- butyl 2-(piperazin-1-yl)-7,8 -dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (7 g, 21.92 mmol, 1.0 equiv.) and Et ₃ N (9.15 ml, 65.75 mmol, 3.0 equiv.). The mixture is stirred at 90°C for 64 hours. The solution is poured into H ₂ O (200 ml) and then the mixture is filtered and the filter cake is washed with H ₂ O (100 ml), then with petroleum ether (60 ml). The filter cake was dried under reduced pressure to give the product (10.1 g, 92% yield) as a brown solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₃ H ₃₁ N ₇ O ₄ : 470.25; found 470.4.

Step 2 : Synthesis of 2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3- d]pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5- carboxylic acid

To a solution of tert- butyl 2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate ( 6.0 g, 12.78 mmol, 1.0 equiv.) in THF (40 ml), EtOH (20 ml) and H ₂ O (40 ml) add LiOH•H ₂ O (1.07 g, 25, 56 mmol, 2.0 equiv.). The reaction mixture was stirred at 35°C for 15 hours. The mixture was then concentrated under reduced pressure to remove THF and EtOH. The mixture is then diluted with H ₂ O (500 ml) and adjusted to pH 3 with aqueous HCl (1N). The mixture is filtered and the filter cake is washed with H ₂ O (80 ml), then with petroleum ether (80 ml). The filter cake was dried under reduced pressure to give the product (3.8 g, 65% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₁ H ₂₇ N ₇ O ₄ : 442.22; found 442.3.

Building block E. tert- butyl methyl((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate.

Step 1 : Synthesis of (2-chloropyrimidin-5-yl)methanamine

To a solution of tert- butyl N- tert- butoxycarbonyl-N-((2-chloropyrimidin-5-yl)methyl)carbamate (28 g, 81.44 mmol, 1.0 equiv.) in EtOAc (30 ml) add HCl in EtOAc (260 ml). The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was filtered and the filter cake was washed with EtOAc (100 ml). The solid cake was dried under reduced pressure to give the product (14.3 g, 96.6% yield, HCl) as a white solid.

Step 2 : Synthesis of tert- butyl((2-chloropyrimidin-5-yl)methyl)carbamate

To a solution of (2-chloropyrimidin-5-yl)methanamine (13 g, 72.21 mmol, 1.0 equiv. HCl) in DCM (130 mL) add DIPEA (20.41 mL, 144.42 mmol, 1.0 equiv. 8 equiv. ) and Boc ₂ O (16.59 ml, 72.21 mmol, 1.0 equiv. ), then the mixture was stirred at room temperature for 3 hours. The reaction mixture was added to H ₂ O (100 ml) and then the aqueous layer is separated and extracted with DCM (2×100 ml). The combined organic phase is then washed with sat. NH ₄ Cl (2×200 ml) and brine (2×200 ml), dry with anhydrous Na ₂ SO ₄ , filter and concentrate under reduced pressure. The residue was purified by silica gel chromatography (1/0-1/1 petroleum ether/EtOAc) to give the product (12 g, 68.2% yield) as a white solid.

Step 3 : Synthesis of tert- butyl((2-chloropyrimidin-5-yl)methyl)(methyl)carbamate

To a solution of tert- butyl((2-chloropyrimidin-5-yl)methyl)carbamate (11 g, 45.14 mmol, 1.0 equiv. ) and MeI (14.05 ml, 225.70 mmol, 5.0 equiv . . ) in THF (150 ml) add NaH (1.99 g, 49.65 mmol, 60 wt%, 1.1 equiv. ) at 0°C. The mixture was stirred at 0°C for 3 hours, and then the reaction mixture was quenched with H ₂ O (100 ml). The aqueous phase is extracted with EtOAc (3×150 ml), and the combined organic phase is washed with brine (50 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (1/0-3/1 petroleum ether/EtOAc) to give the product (9 g, 77.4% yield) as a white solid.

Step 4 : Synthesis of tert- butyl((2-(4-benzylpiperazin-1-yl)pyrimidin-5-yl)methyl)(methyl)carbamate

To a solution of tert- butyl((2-chloropyrimidin-5-yl)methyl)(methyl)carbamate (9 g, 34.92 mmol, 1.0 equiv. ) in MeCN (90 ml) add 1-benzylpiperazine (8.70 g, 34.92 mmol, 1.0 equiv. , 2HCl) and K ₂ CO ₃ (24.13 g, 174.61 mmol, 5.0 equiv. ). The reaction mixture is stirred at 80°C for 20 hours. The mixture is then filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (1/0-1/1 petroleum ether/EtOAc) to give the product (12 g, 86.4% yield) as a yellow oil.

Step 5 : Synthesis of tert- butylmethyl((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate

To a solution of tert- butyl((2-(4-benzylpiperazin-1-yl)pyrimidin-5-yl)methyl)(methyl)carbamate (12 g, 30.19 mmol, 1.0 equiv. ) in MeOH (120 ml ) add Pd/C (2 g, 10% wt). The suspension is degassed and purged with H ₂ and then the mixture is stirred under H ₂ (15 psi) at room temperature for 3 hours. The reaction mixture is filtered through Celite and the filtrate is concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/0-1/1 petroleum ether/EtOAc) to give the semi-pure product (9 g) as a yellow oil. Petroleum ether is added to the residue and the solution is stirred at -60°C until a solid appears. The suspension is filtered and the filtrates are concentrated under reduced pressure to give the product (4.07 g, 55.6% yield) as a yellow oil. LCMS(ESI) m/ z: [M+H] calc. _{for C15H25N5O2 : 308.21} ; found 308.1.

Building block F. 2-(4-(5-((( tert- butoxycarbonyl)(methyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidine-5-carboxylic acid.

Step 1 : Synthesis of ethyl 2-(4-(5-((( tert- butoxycarbonyl)(methyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylate

To a mixture of tert- butylmethyl ((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate (4.3 g, 13.99 mmol, 1.0 equiv.) and ethyl 2-chloropyrimidine-5- carboxylate (2.87 g, 15.39 mmol, 1.1 equiv.) in MeCN (20 ml) add K ₂ CO ₃ (3.87 g, 27.98 mmol, 2.0 equiv.). The mixture is stirred at 80°C for 12 hours. The reaction mixture is then cooled at room temperature and filtered. The filtrate was concentrated under reduced pressure and the crude product was purified by silica gel chromatography (1/0-1/1 petroleum ether/EtOAc) to give the product (4.7 g, 71.3% yield) as a white solid.

Step 2 : Synthesis of 2-(4-(5-((( tert- butoxycarbonyl)(methyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(5-((( tert- butoxycarbonyl)(methyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylate (6 g, 13.11 mmol , 1.0 equiv.) in THF (100 ml), EtOH (30 ml) and H ₂ O (30 ml) add LiOH•H ₂ O (1.10 g, 26.23 mmol, 2.0 equiv.) . The mixture was stirred at room temperature for 16 hours. The mixture was then concentrated under reduced pressure to remove THF and EtOH and then neutralized by adding 1N HCl. The resulting precipitate was collected by filtration to give the product (5.11 g, 90.1% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₂₇ N ₇ O ₄ : 430.22; found 430.2.

Building block G. tert- butyl N- tert- butoxycarbonyl-N-((2-(2-(( tert- butyl(diphenyl)silyl)oxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate.

Step 1 : Synthesis of tert- butyl N-((2-(4-benzyl-2-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)-N- tert- butoxycarbonylcarbamate

To a solution of tert- butyl N- tert- butoxycarbonyl-N-((2-chloropyrimidin-5-yl)methyl)carbamate (18.33 g, 53.32 mmol, 1.1 equiv.) and (4-benzylpiperazine-2 -yl)methanol (10 g, 48.48 mmol, 1.0 equiv.) in DMF (100 ml) add K ₂ CO ₃ (13.40 g, 96.95 mmol, 2.0 equiv.). The mixture was stirred at 100°C for 12 hours. The reaction mixture was then cooled at room temperature and H ₂ O (100 ml) was added. The aqueous layer was extracted with EtOAc (2×150 ml) and the combined organic layer was washed with brine (20 ml), dried with Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure to give the product (7.3 g, 29.3% yield) as a yellow oil. LCMS (ESI) m/z : [M+H] calc. _{for C27H39N5O5 : 514.31} ; found 514.5

Step 2 : Synthesis of tert- butyl N-((2-(4-benzyl-2-(( tert- butyl(diphenyl)silyl)oxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)-N- tert - butoxycarbonylcarbamate

To a solution of tert- butyl N-((2-(4-benzyl-2-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)-N- tert- butoxycarbonylcarbamate (2.3 g, 4.48 mmol, 1.0 equiv.) to DCM (30 ml) add imidazole (609.69 mg, 8.96 mmol, 2.0 equiv.) and TBDPSCl (1.73 ml, 6.72 mmol, 1.5 equiv. .). The reaction mixture was stirred at room temperature for 2 hours. The mixture was then washed with H ₂ O (100 ml) and the aqueous phase was extracted with EtOAc (2×60 ml). The combined organic phase is washed with brine (20 ml), dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (20/1-3/1 petroleum ether/EtOAc) to give the product (4 g, 59.4% yield) as a yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₄₃ H ₅₇ N ₅ O ₅ Si: 752.42; found 752.4.

Step 3 : Synthesis of tert- butyl N- tert- butoxycarbonyl-N-((2-(2-(( tert- butyl(diphenyl)silyl)oxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate

To a solution of tert- butyl-N-((2-(4-benzyl-2-(( tert- butyl(diphenyl)silyl)oxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)-N- tert- butoxycarbonylcarbamate (3.3 g, 4.39 mmol, 1.0 equiv.) in EtOH (10 ml) add Pd(OH) ₂ /C (1 g, 10% wt.). The mixture is heated to 50°C under H ₂ (30 psi) for 30 hours. The mixture is then cooled at room temperature, filtered through Celite and concentrated under reduced pressure. The residue was purified by silica gel chromatography (20/1-3/1 EtOAc/EtOH) to give the product (1.44 g, 45.6% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₃₆ H ₅₁ N ₅ O ₅ Si: 662.38; found 662.3.

Building block H. 2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-3-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of tert- butyl N- tert -butoxycarbonyl-N-((2-(2-(hydroxymethyl)piperazin-1-yl）pyrimidin-5-yl)methyl)carbamate

To a solution of tert- butyl N-((2-(4-benzyl-2-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)-N- tert- butoxycarbonylcarbamate (3 g, 5.84 mmol, 1.0 equiv) in EtOH (40 ml) add Pd/C (2 g, 10% wt). The suspension is degassed and purged with H ₂ then stirred under H ₂ (50 psi) at 30°C for 16 hours. The reaction mixture is cooled at room temperature and filtered through Celite and then concentrated under reduced pressure to give the product (1.6 g, crude) as a yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₃₃ N ₅ O ₅ : 424.26; found 424.3.

Step 2 : Synthesis of ethyl 2-(4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-3-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-carboxylate

To a solution of tert- butyl N- tert -butoxycarbonyl-N-((2-(2-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate (1.4 g, 3.31 mmol, 1 .0 equiv.) in MeCN (20 ml) add K ₂ CO ₃ (2.28 g, 16.53 mmol, 5.0 equiv.) and ethyl 2-chloropyrimidine-5-carboxylate (616.84 mg, 3.0 equiv.) 31 mmol, 1.0 equiv.). The solution is stirred at 80°C for 4 hours. The mixture is cooled at room temperature and poured into H ₂ O (30 ml). The aqueous layer was extracted with EtOAc (2×30 ml) and the combined organic layer was washed with brine (20 ml), dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The mixture was purified by chromatography on silica gel (20/1-3/1 petroleum ether/EtOAc) to give the product (1.6 g, 66.7% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. _{for C27H39N7O7 : 574.30} ; found 574.4.

Step 3 : Synthesis of 2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-3-(hydroxymethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-3-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-carboxylate (1,4 g, 2.44 mmol, 1.0 equiv.) in THF (6 ml) and EtOH (6 ml) at 0°C add a solution of LiOH•H ₂ O (512.07 mg, 12.20 mmol, 5.0 equiv.) in H ₂ O (3 ml). The reaction mixture is warmed to room temperature and stirred for 2 hours. The mixture is then concentrated under reduced pressure to remove THF and EtOH. The aqueous phase is adjusted to pH 3 with 0.1 M HCl and the resulting suspension is filtered. The solid cake was dried under reduced pressure to obtain the product (613.14 mg, 55.6% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₂₇ N ₇ O ₅ : 446.22; found 446.2.

Building block I. tert- butyl N-[( tert- butoxy)carbonyl]-N-({2-[(3 R )-3-(hydroxymethyl)piperazin-1-yl]pyrimidin-5-yl}methyl)carbamate.

Step 1 : Synthesis of ( R ) -tert- butyl-N- tert- butoxycarbonyl-((2-(3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin-1-yl)pyrimidin-5-yl)methyl) carbamate

To a solution of tert- butyl-N- tert- butoxycarbonyl-((2-chloropyrimidin-5-yl)methyl)carbamate (24.24 g, 70.51 mmol, 1.0 equiv.) in MeCN (300 ml) add ( R )-2-((( tert- butyldiphenylsilyl)oxy)methyl)piperazine (25 g, 70.51 mmol, 1.0 equiv.) and K ₂ CO ₃ (29.24 g, 211.53 mmol, 3. 0 equivalent). The mixture was stirred at 80°C for 16 hours. The reaction mixture was then cooled at room temperature, diluted with EtOAc (200 ml), filtered and the filtrate concentrated under reduced pressure. Purification by silica gel chromatography (0→100% EtOAc/petroleum ether) gave the desired product (46.5 g, 94% yield) as a white solid.

Step 2 : Synthesis of tert- butyl N-[( tert- butoxy)carbonyl]-N-({2-[( 3R )-3-(hydroxymethyl)piperazin-1-yl]pyrimidin-5-yl}methyl)carbamate

To a solution of ( R ) -tert- butyl-N- tert- butoxycarbonyl-((2-(3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate ( 12 g, 18.13 mmol, 1.0 equiv.) to THF (120 mL) add FTBA (1 M, 23.93 mL, 1.3 equiv.). The mixture was stirred at room temperature for 2 hours. The reaction mixture was then poured into H ₂ O (300 ml) and the aqueous phase was extracted with EtOAc (3×80 ml). The combined organic phases are combined, washed with brine (80 ml), dried, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→20% MeOH/DCM) gave the desired product (5 g, 64% yield) as a yellow solid.

Building block J. 2-{4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]piperazin-1-yl}pyrimidine-5-carboxylic acid.

Step 1 : Synthesis of ( R )-ethyl 2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-2-((( tert- butyldiphenylsilyl)oxy)methyl) piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of ( R ) -tert- butyl-N- tert -butoxycarbonyl-N-((2-(3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin-1-yl)pyrimidin-5-yl)methyl) carbamate (31.5 g, 45.21 mmol, 1.0 equiv.) in MeCN (350 ml) add ethyl 2-chloropyrimidine-5-carboxylate (8.44 g, 45.21 mmol, 1.0 equiv.) and K ₂ CO ₃ (18.75 g, 135.63 mmol, 3.0 equiv.). The mixture was stirred at 80°C for 16 hours. The reaction mixture was then cooled at room temperature, diluted with EtOAc (150 ml) and filtered to remove inorganic salts. The filtrate is then concentrated under reduced pressure. Purification by silica gel chromatography (0→100% EtOAc/petroleum ether) gave the desired product (33.5 g, 89% yield).

Step 2 : Synthesis of ( R )-ethyl 2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-2-(hydroxymethyl)piperazin-1-yl)pyrimidin- 5-carboxylate

To a solution of ( R )-ethyl 2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-2-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin- 1-yl)pyrimidin-5-carboxylate (36.5 g, 44.95 mmol, 1.0 equiv.) in THF (300 ml) was added FTBA (1 M, 59.33 ml, 1.32 equiv.). The mixture was stirred at room temperature for 6 hours, at which point the reaction mixture was poured into H ₂ O (500 ml). The aqueous phase is separated and extracted with EtOAc (3×150 ml) and the combined organic layers are washed with brine (150 ml), dried, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→100% EtOAc/petroleum ether) gave the desired product (17 g, 64% yield) as a yellow oil.

Step 3 : Synthesis of ( R )-2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-2-(hydroxymethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid acids

To a solution of ( R )-ethyl 2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-2-(hydroxymethyl)piperazin-1-yl)pyrimidin-5- carboxylate (17 g, 29.64 mmol, 1.0 equiv.) in H ₂ O (160 ml), EtOH (80 ml) and THF (160 ml) add LiOH•H ₂ O (4.97 g, 118, 54 mmol, 4.0 equiv.). The reaction mixture was stirred at 55°C for 16 hours. LiOH•H ₂ O (1.01 g, 24.00 mmol, 0.81 equiv.) was then added to the mixture and the reaction mixture was stirred at 55°C for another 19 h. The mixture is cooled at room temperature, diluted with H ₂ O (150 ml) and concentrated under reduced pressure to remove THF and EtOH. The mixture is acidified (pH=5) with 1 N HCl, filtered and the filter cake is washed with H ₂ O (2×30 ml). The filter cake was dried under reduced pressure to obtain the desired product (9.2 g, 67% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₀ H ₂₇ N ₇ O ₅ : 446.22; found 446.1.

Building block K. tert- butyl N-[( tert- butoxy)carbonyl]-N-({2-[(3 S )-3-(hydroxymethyl)piperazin-1-yl]pyrimidin-5-yl}methyl)carbamate.

This building block is prepared by a procedure similar to that of Building Block I using [( 2S )-piperazin-2-yl]methanol.

Building block L. 2-[(2 S )-4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]-2-(hydroxymethyl)piperazin-1-yl]pyrimidin-5-carboxylic acid.

This building block is obtained from Building Block K using a procedure similar to that for Building Block J.

Building block M. tert- butyl 2-[(3 R )-3-(hydroxymethyl)piperazin-1-yl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidine-6-carboxylate.

Step 1 : Synthesis of ( R ) -tert -butyl 2-(3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6( 5H)-carboxylate

To a solution of ( R )-2-((( tert- butyldiphenylsilyl)oxy)methyl)piperazine (25 g, 70.51 mmol, 1.0 equiv.) in MeCN (250 ml) add K ₂ CO ₃ (29.24 g, 211.53 mmol, 3.0 equiv.) and tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (17.12 g, 63.46 mmol, 0.9 equiv.). The mixture was stirred at 80°C for 17 hours. The reaction mixture was then cooled at room temperature, filtered, and the filtrate was concentrated under reduced pressure. Purification by silica gel chromatography (0→100% EtOAc/petroleum ether) gave the desired product (31 g, 73.5% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₃₃ H ₄₅ N ₅ O ₃ Si: 588.34; found 588.2.

Step 2 : Synthesis of ( R ) -tert -butyl 2-(3-(hydroxymethyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a mixture of ( R ) -tert- butyl 2-(3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H) -carboxylate (12 g, 20.41 mmol, 1.0 equiv.) in THF (120 ml) add FTBA (1.0 M, 24.50 ml, 1.2 equiv.). The mixture was stirred at room temperature for 5 hours. The mixture was poured into H ₂ O (100 ml) and the aqueous phase was extracted with EtOAc (2×100 ml). The combined organic phases are washed with brine (100 ml), dried, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→10% MeOH/DCM) gave the desired product (6 g, 84.1% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C17H27N5O3 : 350.22} ; found 350.2.

Building block N. 2-[(2 R )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}-2-(hydroxymethyl)piperazin-1-yl]pyrimidin-5-carboxylic acid.

This building block is obtained from Building Block M using a procedure similar to that of Building Block J.

Building block O. tert- butyl 2-[(3 S )-3-(hydroxymethyl)piperazin-1-yl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-6-carboxylate.

This building block is prepared using a procedure similar to that of Building Block Ic using tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate and [( 2S )-piperazine- 2-yl]methanol.

Building block P. 2-[(2 S )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}-2-(hydroxymethyl)piperazin-1-yl]pyrimidin-5-carboxylic acid.

This building block is obtained from Building Block O using a procedure similar to that of Building Block J.

Building block Q. tert- butyl N-[( tert- butoxy)carbonyl]-N-({2-[(3 S )-3-[(dimethylamino)methyl]piperazin-1-yl]pyrimidin-5-yl}methyl)carbamate.

Step 1 : Synthesis of ( R )-dibenzyl 2-(dimethylcarbamoyl)piperazine-1,4-dicarboxylate

To a solution of CDI (12.21 g, 75.30 mmol, 1.2 equiv.) in DCM (300 ml) at 0°C add ( R )-1,4-bis((benzyloxy)carbonyl)piperazine-2- carboxylic acid (25 g, 62.75 mmol, 1.0 equiv.). The mixture was stirred at 0°C for 0.5 h, at which time dimethylamine (8.51 mL, 92.87 mmol, 1.5 equiv., HCl) was added. The reaction mixture was warmed to room temperature and stirred for 12 hours. The reaction mixture was then added to H ₂ O (200 ml) and the aqueous layer was separated and extracted with DCM (2×200 ml). The combined organic phases are washed with brine (2×50 ml), dried, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (50→100% EtOAc/petroleum ether) gave the desired product (23.5 g, 88.0% yield) as a yellow oil.

Step 2 : Synthesis of ( S )-dibenzyl 2-((dimethylamino)methyl)piperazine-1,4-dicarboxylate

To a solution of ( R )-dibenzyl 2-(dimethylcarbamoyl)piperazine-1,4-dicarboxylate (28 g, 65.81 mmol, 1.0 equiv.) in THF (300 ml) at 0°C add BH ₃ ●Me ₂ S (10 M, 13.16 ml, 2.0 equiv.). The reaction mixture was then stirred at 80°C for 3 hours. The reaction mixture was cooled at room temperature and then MeOH (50 ml) was added. After stirring for another 1 hour, the mixture is concentrated under reduced pressure. Purification by silica gel chromatography (50→100% EtOAc/petroleum ether) gave the desired product (18 g, 66.5% yield) as a yellow oil.

Step 3 : Synthesis of ( R )-N,N-dimethyl-1-(piperazin-2-yl)methanamine

To a solution of ( S )-dibenzyl 2-((dimethylamino)methyl)piperazine-1,4-dicarboxylate (18 g, 43.74 mmol, 1.0 equiv.) in EtOAc (200 ml) add Pd/C (1, 5 g, 10% wt.). The suspension is degassed under reduced pressure and purged with H ₂ three times. The suspension was stirred under H ₂ (30 psi) at 30°C for 5 hours. The reaction mixture was then filtered through Celite and the filtrate was concentrated under reduced pressure to give the desired product (6 g, 95.8% yield ) as a yellow solid.

Step 4 : Synthesis of tert- butyl N- tert- butoxycarbonyl-N-((2-(( 3S )-3-((dimethylamino)methyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate

To a solution of ( R )-N,N-dimethyl-1-(piperazin-2-yl)methanamine (2.8 g, 19.55 mmol, 1.0 equiv.) in MeCN (40 ml) add tert- butyl N - tert- butoxycarbonyl-N-((2-chloropyrimidin-5-yl)methyl)carbamate (6.72 g, 19.55 mmol, 1.0 equiv.) and K ₂ CO ₃ (5.40 g, 39. 10 mmol, 2.0 equiv.). The mixture is stirred at 80°C for 24 hours. The mixture is then cooled at room temperature, filtered and the filter cake washed with EtOAc (3×10 ml). The filtrate is then concentrated under reduced pressure. Purification by silica gel chromatography (0→100% MeOH/EtOAc) gave the desired product (5.3 g, 57.8% yield) as a yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₃₈ N ₆ O ₄ : 451.31; found 451.2.

Building block R. 2-[(2 S )-4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]-2-[(dimethylamino)methyl]piperazin-1-yl]pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ( S )-ethyl 2-(4-(5-(((bi- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)-2-((dimethylamino)methyl)piperazin-1-yl )pyrimidine-5-carboxylate

To a solution of ( S ) -tert -butyl-N- tert- butoxycarbonyl ((2-(3-((dimethylamino)methyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate (3.26 g, 7 .24 mmol, 1.0 equiv.) in DMF (30 ml) add Et ₃ N (3.02 ml, 21.71 mmol, 3.0 equiv.) and ethyl 2-chloropyrimidine-5-carboxylate (1.47 g, 7.86 mmol, 1.1 equiv.). The mixture was stirred at 50°C for 3 hours and then concentrated under reduced pressure to obtain the desired product (4.35 g, crude) as a solution in DMF (30 ml), which was used immediately in the next step. LCMS (ESI) m/z : [M+H] calc. for C ₂₉ H ₄₄ N ₈ O ₆ : 601.35; found 601.5.

Step 2 : Synthesis of ( S )-2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)-pyrimidin-2-yl)-2-((dimethylamino)methyl)piperazin-1-yl)pyrimidine -5-carboxylic acid

To a solution of ( S )-ethyl 2-(4-(5-(((bi- tert- butoxycarbonyl)amino)methyl)-pyrimidin-2-yl)-2-((dimethylamino)methyl)piperazin-1-yl) Pyrimidine 5-carboxylate (4.35 g, 7.24 mmol, 1.0 equiv.) in DMF (30 ml) was added with DMF (50 ml), EtOH (30 ml) and H ₂ O (30 ml). LiOH•H ₂ O (3 g, 71.50 mmol, 9.9 equiv.) was then added to the solution at 50°C. The reaction mixture was stirred at 50°C for 36 hours. The mixture was then cooled at room temperature, neutralized with 0.5 N HCl and concentrated under reduced pressure. Purification by reverse phase chromatography (2→30% MeCN/H ₂ O) gave the desired product (1.15 g, 34% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₃₂ N ₈ O ₄ : 473.26; found 473.3.

Building block S. tert- butyl N-[( tert- butoxy)carbonyl]-N-({2-[(3 R )-3-[(dimethylamino)methyl]piperazin-1-yl]pyrimidin-5-yl}methyl)carbamate.

This building block is prepared by a procedure similar to that of Building Block Ic using dimethyl({[(2 S )-piperazin-2-yl]methyl})amine.

Building block T. 2-[(2 R )-4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]-2-[(dimethylamino)methyl]piperazin-1-yl]pyrimidin-5-carboxylic acid.

This building block is obtained from Building Block S using a procedure similar to that of Building Block J.

Building block U. tert- butyl 2-[(3 S )-3-[(dimethylamino)methyl]piperazin-1-yl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidine-6-carboxylate.

To a solution of tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (4.80 g, 17.80 mmol, 1.4 equiv.) in MeCN (45 ml) add K ₂ CO ₃ (10.42 g, 75.40 mmol, 3.0 equiv.) and ( R )-N,N-dimethyl-1-(piperazin-2-yl)methanamine (3.6 g , 25.13 mmol, 1.0 equiv.). The mixture was stirred at 80°C for 8 hours. The mixture was then cooled at room temperature, filtered and the filter cake washed with EtOAc (50 ml). H ₂ O (50 ml) was added to the organic phase and the aqueous phase was extracted with EtOAc (3×50 ml). The combined organic phases are washed with brine (5 ml), dried, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (8→67% EtOAc/petroleum ether) gave the desired product (6.5 g, 63.5% yield) as a yellow oil.

Building block V. 2-[(2 S )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}-2-[(dimethylamino)methyl]piperazin-1-yl]pyrimidine-5-carboxylic acid.

Step 1 : Synthesis of ( S ) -tert- butyl 2-(3-((dimethylamino)methyl)-4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[ 4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of ( S ) -tert- butyl 2-(3-((dimethylamino)methyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (3 g , 7.97 mmol, 1.0 equiv.) NaH (382.44 mg, 9.56 mmol, 60 wt%, 1.2 equiv.) was added to DMF (70 mL) at 0°C. The suspension was stirred at 0°C for 0.5 h, then ethyl 2-chloropyrimidine-5-carboxylate (1.49 g, 7.97 mmol, 1 equiv.) in DMF (50 ml) was added dropwise. The mixture is warmed to room temperature and stirred for 5 hours. The mixture is then cooled to 0°C and poured into H ₂ O (360 ml). The suspension is filtered and the filter cake is washed with H ₂ O (30 ml) and dried under reduced pressure. Purification by silica gel chromatography (6%→33% EtOAc/petroleum ether) gave the desired product (1.8 g, 39.6% yield) as a brown oil.

Step 2 : Synthesis of ( S )-2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)-2-((dimethylamino )methyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ( S ) -tert- butyl 2-(3-((dimethylamino)methyl)-4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[4, 3-d]pyrimidine-6(5H)-carboxylate (1.1 g, 2.09 mmol, 1.0 equiv.) in THF (5 ml), EtOH (2.5 ml) and H ₂ O (2. 5 ml) add LiOH•H ₂ O (175.30 mg, 4.18 mmol, 2.0 equiv.). The mixture is stirred at room temperature for 2 hours, at which point the pH is adjusted to 7 by adding 1 N HCl at 0°C. The mixture is concentrated under reduced pressure to remove THF and MeOH. The resulting suspension was filtered and the filter cake was washed with H ₂ O (5 ml) and dried under reduced pressure to obtain the desired product (680 mg, 65.3% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C24H34N8O4 : 499.28} ; found 499.2.

Building block W. tert- butyl 2-[(3 R )-3-[(dimethylamino)methyl]piperazin-1-yl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidine-6-carboxylate.

This building block is prepared using a procedure similar to that of Building Block Ic using tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate and dimethyl({[( 2S ) -piperazin-2-yl]methyl})amine.

Building block X. 2-[(2 R )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}-2-[(dimethylamino)methyl]piperazin-1-yl]pyrimidine-5-carboxylic acid.

This building block is obtained from Building Block W using a procedure similar to that of Building Block J.

Building block Y. tert- butyl (2 R )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl} piperazine - 2-carboxylate.

Step 1 : Synthesis of ( R )-1,4-bis((benzyloxy)carbonyl)piperazine-2-carboxylic acid

To a solution of ( R )-piperazine-2-carboxylic acid (70 g, 344.71 mmol, 1.0 equiv., 2HCl) in dioxane (1120 ml) and H ₂ O (700 ml) add 50% aq. NaOH until the pH of the solution becomes pH=11. Benzyl chloroformate (156.82 mL, 1.10 mol, 3.2 equiv.) was added and the reaction mixture was stirred at room temperature for 12 hours. H ₂ O (1200 mL) was then added to the solution and the aqueous layer was washed with MTBE (3 ×800 ml). The aqueous layer is adjusted to pH=2 with concentrated HCl (12N) and extracted with EtOAc (2×1000 ml). The combined organic extracts are dried, filtered and the filtrate is concentrated under reduced pressure to give the desired product (137 g, 99.8% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₁ H ₂₂ N ₂ O ₆ : 399.16; found 399.2.

Step 2 : Synthesis of ( R )-1,4-dibenzyl 2- tert- butyl piperazine-1,2,4-tricarboxylate

To a solution of ( R )-1,4-bis((benzyloxy)carbonyl)piperazine-2-carboxylic acid (50 g, 125.50 mmol, 1.0 equiv.) in toluene (500 ml) at 80°C add 1 ,1-di- tert- butoxy-N,N-dimethylmethanamine (57.17 ml, 238.45 mmol, 1.9 equiv.). The solution was stirred at 80°C for 2 hours, at which point the reaction mixture was cooled to room temperature and partitioned between EtOAc (300 ml) and H ₂ O (500 ml). The aqueous layer was extracted with EtOAc (2 x 500 ml) and combined organic the layers are dried, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→25% EtOAc/petroleum ether) gave the desired product (35 g, 61.2% yield) as a white solid. LCMS (ESI) m/z : [M+Na] calc. for C ₂₅ H ₃₀ N ₂ O ₆ : 477.20; found 477.1.

Step 3 : Synthesis of ( R ) -tert- butyl piperazine-2-carboxylate

To a solution of ( R )-1,4-dibenzyl 2- tert- butyl piperazine-1,2,4-tricarboxylate (35 g, 77.01 mmol, 1.0 equiv.) in EtOAc (350 ml) add Pd/C (10 g, 10% wt.). The suspension is degassed under reduced pressure and purged with H ₂ three times. The mixture is stirred under H ₂ (30 psi) at 30°C for 4 hours. The reaction mixture is then filtered through Celite, the residue is washed with MeOH (5×200 ml) and the filtrate is concentrated under reduced pressure to obtain the desired product (14 g, 79.6% yield) as a yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₉ H ₁₈ N ₂ O ₂ : 187.15; found 187.1.

Step 4 : Synthesis of ( R ) -tert- butyl 2-(3-( tert- butoxycarbonyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of tert- butyl ( 2R )-piperazine-2-carboxylate (12 g, 64.43 mmol, 1.0 equiv.) in MeCN (200 ml) add K ₂ CO ₃ (17.81 g, 128.86 mmol, 2.0 equiv.) and tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (17.38 g, 64.43 mmol, 1.0 equiv.). The reaction mixture is heated to 80°C and stirred for 12 hours. The reaction mixture is then cooled to room temperature and filtered, the residue is washed with EtOAc (3×150 ml) and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→100% EtOAc/petroleum ether) gave the desired product (19 g, 69.2% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₁ H ₃₃ N ₅ O ₄ : 420.26; found 420.2.

Building block Z. 4-amino-2-[(2 R )-2-[( tert- butoxy)carbonyl]-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}piperazin-1-yl]pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ( R ) -tert- butyl 2-(4-(4-amino-5-(ethoxycarbonyl)pyrimidin-2-yl)-3-( tert- butoxycarbonyl)piperazin-1-yl)-7,8 -dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a stirred solution of ( R ) -tert- butyl 2-(3-( tert- butoxycarbonyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (12 g , 28.60 mmol, 1.0 equiv.) to MeCN (150 ml) add K ₂ CO ₃ (7.91 g, 57.20 mmol, 2.0 equiv.) and ethyl 4-amino-2-chloropyrimidine- 5-carboxylate (6.92 g, 34.32 mmol, 1.2 equiv.). The reaction mixture is stirred at 80°C for 12 hours, at which point the reaction mixture is filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→17% EtOAc/petroleum ether) gave the desired product (16 g, 91.6% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₈ H ₄₀ N ₈ O ₆ : 585.32; found 585.1.

Step 2 : Synthesis of ( R )-4-amino-2-(2-( tert- butoxycarbonyl)-4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d] pyrimidin-2-yl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To two separate batches prepared in parallel, each containing a solution of ( R ) -tert -butyl 2-(4-(4-amino-5-(ethoxycarbonyl)pyrimidin-2-yl)-3-( tert- butoxycarbonyl)piperazine- 1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (7 g, 11.97 mmol, 1.0 equiv.) in THF (70 ml), EtOH (35 ml) and H ₂ O (35 ml) add LiOH•H ₂ O (2.01 g, 47.89 mmol, 4.0 equiv.). The mixtures are stirred at 60°C for 3 hours, at which point the two reaction mixtures are combined and adjusted to pH=7 with 1 N HCl. The mixture is concentrated under reduced pressure to remove THF and EtOH, filtered, and the residue is dried under reduced pressure. The residue was stirred in MTBE (100 ml) for 10 min, filtered, and the residue was dried under reduced pressure to give the desired product (8.02 g, 55.1% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₆ H ₃₆ N ₈ O ₆ : 557.29; found 557.3.

Building block AA. tert- butyl (2 S )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl} piperazine -2-carboxylate.

This building block is prepared using a procedure similar to that of Building Block Ic using tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate and tert- butyl ( 2S )- piperazine-2-carboxylate.

Building block AB. 4-amino-2-[(2 S )-2-[( tert- butoxy)carbonyl]-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}piperazin-1-yl]pyrimidin-5-carboxylic acid.

This building block is prepared from Building Block AA using a procedure similar to that of Building Block J using ethyl 4-amino-2-chloropyrimidine-5-carboxylate.

AC building block. 4-amino-2-(4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}piperazin-1-yl)pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of tert- butyl 2-(4-(4-amino-5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6 (5H)-carboxylate

To a solution of tert- butyl 2-(piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (8.3 g, 25.99 mmol, 1.0 equiv .) and ethyl 4-amino-2-chloropyrimidine-5-carboxylate (5.24 g, 25.99 mmol, 1.0 equiv.) in MeCN (100 ml) were added to K ₂ CO ₃ (7.18 g, 51.97 mmol, 2.0 equiv.). The reaction mixture was stirred at 80°C for 12 hours. The reaction mixture was then cooled at room temperature, DCM (100 ml) was added and the reaction mixture was stirred for 30 minutes. The suspension is filtered and the filter cake is washed with DCM (6×100 ml). The filtrate is concentrated under reduced pressure and the residue is triturated with EtOAc (30 ml), filtered and then the filter cake is dried under reduced pressure to obtain the desired product (8.7 g, 65.9% yield) as a light yellow solid.

Step 2 : Synthesis of 4-amino-2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin-1-yl) pyrimidine-5-carboxylic acid

To a solution of tert- butyl 2-(4-(4-amino-5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H )-carboxylate (8.7 g, 17.95 mmol, 1.0 equiv.) in THF (120 ml), EtOH (60 ml) and H ₂ O (60 ml) add LiOH•H ₂ O (1.51 g, 35.91 mmol, 2.0 equiv.). The mixture was stirred at 55°C for 12 hours. The reaction mixture was then concentrated under reduced pressure to remove EtOH and THF, and the reaction mixture was adjusted to pH=6 by adding 1N HCl. The precipitate is filtered and the filter cake is washed with H ₂ O (3×50 ml) and then dried under reduced pressure to obtain the desired product (7.3 g, 89.1% yield) as a light yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₁ H ₂₈ N ₈ O ₄ : 457.23; found 457.2.

AD building block. 4-amino-2-{4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]piperazin-1-yl}pyrimidine-5-carboxylic acid.

Step 1 : Synthesis of ethyl 4-amino-2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of tert- butyl-N- tert- butoxycarbonyl-N-((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate (8.3 g, 21.09 mmol, 1.0 equiv. ) to MeCN (100 ml) add ethyl 4-amino-2-chloropyrimidine-5-carboxylate (4.04 g, 20.04 mmol, 0.95 equiv.) and K ₂ CO ₃ (8.75 g, 63. 28 mmol, 3.0 equiv.). The mixture was stirred at 80°C for 3 hours. The reaction mixture was then cooled to room temperature, DCM (150 ml) was added and the reaction mixture was stirred for 30 minutes. The suspension is filtered, the filter cake is washed with DCM (3×100 ml) and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→100% EtOAc/petroleum ether) gave the desired product (8.35 g, 67% yield) as a white solid.

Step 2 : Synthesis of 4-amino-2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 4-amino-2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylate (8.3 g , 14.86 mmol, 1.0 equiv.) in H ₂ O (70 ml), EtOH (36 ml) and THF (80 ml) add LiOH•H ₂ O (2.49 g, 59.43 mmol, 4 ,0 equivalent). The reaction mixture was stirred at 55°C for 16 hours. The mixture was then concentrated under reduced pressure to remove THF and EtOH. The mixture is diluted with H ₂ O (55 ml) and adjusted to pH=6 by adding 1N HCl. The mixture is filtered and the filter cake is washed with H ₂ O (2×20 ml). The solid cake was dried under reduced pressure to obtain the desired product (5.5 g, 84% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C19H26N8O4 : 431.22} ; found 431.4.

AE building block. 4-amino-2-[(2 R )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}-2-(hydroxymethyl)piperazin-1-yl]pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ( R ) -tert -butyl 2-(4-(4-amino-5-(ethoxycarbonyl)pyrimidin-2-yl)-3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazine-1- yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of ( R ) -tert- butyl 2-(3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H) -carboxylate (17.2 g, 29.26 mmol, 1.0 equiv.) in MeCN (200 ml) add K ₂ CO ₃ (12.13 g, 87.78 mmol, 3.0 equiv.) and ethyl 4 -amino-2-chloropyrimidine-5-carboxylate (6.37 g, 31.60 mmol, 1.08 equiv.). The mixture is stirred at 80°C for 18 hours. The reaction mixture is then cooled at room temperature, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→33% EtOAc/petroleum ether) gave the desired product (20.3 g, 90.6% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₄₀ H ₅₂ N ₈ O ₅ Si: 753.39; found 753.4.

Step 2 : Synthesis of ( R )-4-amino-2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)-2 -(hydroxymethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ( R ) -tert- butyl 2-(4-(4-amino-5-(ethoxycarbonyl)pyrimidin-2-yl)-3-((( tert- butyldiphenylsilyl)oxy)methyl)piperazin-1-yl) -7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (20.3 g, 26.96 mmol, 1.0 equiv.) in THF (200 ml) add FTBA (1.0 M, 50.75 ml, 1.9 equiv.). The reaction mixture was stirred at room temperature for 5 hours. The mixture was then poured into H ₂ O (200 ml) and the aqueous phase was extracted with EtOAc (2×150 ml). The combined organic phases are washed with brine (2 x 100 ml), dried, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (0%→20% EtOAc/petroleum ether) gave the desired product (12 g, 85.7% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₃₄ N ₈ O ₅ : 515.28; found 515.4.

Step 3 : Synthesis of ( R )-4-amino-2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)-2 -(hydroxymethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ( R )-4-amino-2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)-2-( Hydroxymethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid (12 g, 23.32 mmol, 1.0 equiv.) in THF (100 ml), EtOH (30 ml) and H ₂ O (30 ml) was added LiOH•H ₂ O (5.87 g, 139.92 mmol, 6.0 equiv.). The mixture is stirred at 50°C for 22 hours. The mixture is then concentrated under reduced pressure to remove THF and EtOH. The aqueous phase is neutralized with 1 N HCl and the resulting precipitate is filtered. The filter cake is washed with H ₂ O (50 ml) and dried under reduced pressure. The filtrate is extracted with DCM (8x60 ml) and the combined organic phases are washed with brine (2x50 ml), dried, filtered and concentrated under reduced pressure. The resulting residue was combined with the original filter cake and the solid was dissolved in DCM (150 ml) and concentrated under reduced pressure to give the desired product (9.76 g, 85.2% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₂ H ₃₀ N ₈ O ₅ : 487.24; found 487.2.

AF building block. 4-amino-2-[(2 S )-4-{6-[( tert- butoxy)carbonyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}-2-(hydroxymethyl)piperazin-1-yl]pyrimidine-5-carboxylic acid

This building block is prepared from Building Block O using a procedure similar to that of Building Block J using ethyl 4-amino-2-chloropyrimidine-5-carboxylate.

Building block AG. 2-((2-(4-(5-((di-( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethyl)(methyl)amino)acetic acid.

To a solution of tert- butyl N- tert- butoxycarbonyl-N-((2-piperazin-1-ylpyrimidin-5-yl)methyl)carbamate (4.88 g, 12.39 mmol, 1.0 equiv.) in EtOAc ( 40 ml) add 4-methylmorpholine-2,6-dione (1.6 g, 12.39 mmol, 1.0 equiv.). The reaction mixture was stirred at room temperature for 2 hours, then the reaction mixture was concentrated under reduced pressure to obtain the crude product. The residue was triturated with EtOAc (15 ml) and filtered to give the product (5.65 g, 87.2% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C24H39N6O7 : 523.28} ; found 523.3.

Building block AH. tert- butyl N- tert- butoxycarbonyl-N-((2-(4-(3-(2-piperazin-1-ylethoxy)propanoyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate.

Step 1 : Synthesis of benzyl 4-(2-(3-( tert- butoxy)-3-oxorgoroxy)ethyl)piperazine-1-carboxylate

To a solution of tert- butyl 3-(2-bromoethoxy)propanoate (35 g, 138.27 mmol, 1.0 equiv.) and benzylpiperazine-1-carboxylate (31.14 ml, 138.27 mmol, 1.0 equiv.) , HCl) in MeCN (420 ml) add K ₂ CO ₃ (57.33 g, 414.80 mmol, 3.0 equiv.). The reaction mixture is stirred at 80°C for 20 hours. The reaction mixture is cooled at room temperature and the suspension is filtered. The filter cake is washed with EtOAc (3×50 ml) and the combined filtrates are concentrated under reduced pressure to obtain the crude product. The residue was purified by chromatography on silica gel (5/1-0/1 petroleum ether/EtOAc) to give the product (46 g, 84.8% yield) as a yellow oil.

Step 2 : Synthesis of 3-(2-(4-((benzyloxy)carbonyl)piperazin-1-yl)ethoxy)propanoic acid

A solution of benzyl 4-(2-(3-( tert- butoxy)-3-oxorgoroxy)ethyl)piperazine-1-carboxylate (21 g, 53.50 mmol, 1.0 equiv.) in TFA (160 ml) was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by silica gel chromatography (1/0-4/1 EtOAc/MeOH) to give the product (20.4 g, 84.7% yield) as a yellow oil. LCMS (ESI) m/z : [M+H] calc. _{for C17H24N2O5 : 337.18} ; found 337.1.

Step 3 : Synthesis of benzyl 4-(2-(3-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl )piperazine-1-carboxylate

To a solution of 3-(2-(4-((benzyloxy)carbonyl)piperazin-1-yl)ethoxy)propanoic acid (20.2 g, 44.85 mmol, 1.0 equiv, TPA) in DCM (500 ml ) add GATU (25.58 g, 67.27 mmol, 1.5 equiv.) and DIPEA (17.39 g, 134.55 mmol, 23.44 ml, 3.0 equiv.). The reaction mixture was stirred at room temperature for 30 minutes and then tert- butyl N- tert- butoxycarbonyl-N-((2-piperazin-1-ylpyrimidin-5-yl)methyl)carbamate (14.12 g, 35.88) was added mmol, 0.8 equiv.). The reaction mixture is stirred for 2 hours and then quenched with sat. NH ₄ Cl (500 ml). The aqueous phase is extracted with DCM (3×300 ml) and the combined organic phase is washed with brine (30 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain the crude product. The residue was purified by silica gel chromatography (0/1 petroleum ether/EtOAc to 10/1 DCM/MeOH) to give the product (29 g, 90.8% yield) as a yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₃₆ H ₅₃ N ₇ O ₈ : 712.41; found 712.4.

Step 4 : Synthesis of tert- butyl N- tert -butoxycarbonyl-N-((2-(4-(3-(2-piperazin-1-ylethoxy)propanoyl)piperazin-1-yl)pyrimidin-5-yl)methyl) carbamate

To a solution of 4-(2-(3-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin- 1-carboxylate (5 g, 7.02 mmol, 1.0 equiv.) in EtOAc (150 ml) add Pd/C (2 g, 10% wt). The suspension is degassed and purged with H ₂ and then stirred under H ₂ (30 psi) at 30°C for 3 hours. The suspension is then cooled to room temperature and filtered through Celite. The filter cake was washed with MeOH (15 x 100 mL) and the combined filtrates were concentrated under reduced pressure to give the product (12 g, 89.9% yield) as a light yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₂₈ H ₄₇ N ₇ O ₆ : 578.37; found 578.5.

AI building block. ethyl 2-(piperazin-1-yl)pyrimidine-5-carboxylate .

Step 1 : Synthesis of ethyl 2-(4-( tert- butoxycarbonyl)piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of tert- butyl piperazine-1-carboxylate (11.94 g, 53.59 mmol, 1.0 equiv., HCl) and ethyl 2-chloropyrimidine-5-carboxylate (10 g, 53.59 mmol, 1.0 equiv.) to MeCN (100 ml) add K ₂ CO ₃ (7.41 g, 53.59 mmol, 1.0 equiv.). The mixture was stirred at 80°C for 17 hours and then poured into H ₂ O (200 ml). The mixture was filtered and the filter cake was washed with H ₂ O (80 ml) and dried under reduced pressure to give the product (15.76 g, 82% yield) as a white solid.

Step 2 : Synthesis of ethyl 2-(piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of ethyl 2-(4-( tert- butoxycarbonyl)piperazin-1-yl)pyrimidine-5-carboxylate (15.7 g, 46.67 mmol, 1.0 equiv.) in EtOAc (150 mL) add HCl/ EtOAc (150 ml) at 0°C. The resulting mixture was stirred at room temperature for 9 hours. The reaction mixture was filtered and the filter cake was washed with EtOAc (100 ml). The solid was dried under reduced pressure to give the product (12.55 g, 96% yield, HCl) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₁₁ H ₁₆ N ₄ O ₂ : 237.14; found 237.3.

AJ building block. 2-(4-(2-(3-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ethyl 2-(4-(2-(3-( tert- butoxy)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of ethyl 2-piperazin-1-ylpyrimidin-5-carboxylate (17.92 g, 75.85 mmol, 1.2 equiv.) and tert- butyl 3-(2-bromoethoxy)propanoate (16 g, 63.21 mmol, 1.0 equiv.) to MeCN (200 ml) add K ₂ CO ₃ (17.47 g, 126.42 mmol, 2.0 equiv.). The reaction mixture was stirred at 80°C for 12 hours, and then the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product is suspended in petroleum ether (200 ml) and stirred for 20 minutes at 0°C and then filtered. The solid was dried under reduced pressure to give the product (19.4 g, 75.1% yield) as a yellow solid.

Step 2 : Synthesis of 3-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)propanoic acid

Solution of ethyl 2-(4-(2-(3-( tert- butoxy)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylate (19.4 g, 47.49 mmol, 1.0 equiv.) in TFA (200 ml) and stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure and the residue was purified by chromatography on silica gel (50/1-1/1 EtOAc/MeOH) to give the product (18 g, 81.3% yield) as a yellow oil.

Step 3 : Synthesis of ethyl 2-(4-(2-(3-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3- oxopropoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of 3-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)propanoic acid (13 g, 27.87 mmol, 1.0 equiv.) in DCM ( 200 ml) add GATU (15.90 g, 41.81 mmol, 1.5 equiv.) and DIPEA (19.42 ml, 111.49 mmol, 4.0 equiv.). The reaction mixture was then stirred at room temperature for 30 minutes and then tert- butyl N- tert- butoxycarbonyl-N-[(2-piperazin-1-ylpyrimidin-5-yl)methyl]carbamate (10.97 g, 27. 87 mmol, 1.0 equiv.). The mixture is stirred for 2 hours and then poured into sat. NH ₄ Cl solution (200 ml). The aqueous phase is extracted with DCM (2×200 ml), and the combined organic phase is washed with brine (2×20 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (100/1-9/1 EtOAc/MeOH) to give the product (17 g, 79.0% yield) as a yellow oil.

Step 4 : Synthesis of 2-(4-(2-(3-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy )ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(2-(3-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy) ethyl)piperazin-1-yl)pyrimidin-5-carboxylate (11 g, 15.11 mmol, 1.0 equiv.) in THF (40 ml), EtOH (10 ml) and H ₂ O (20 ml) add LiOH •H ₂ O (1.27 g, 30.23 mmol, 2.0 equiv.). The mixture is then stirred at 35°C for 1.5 hours. The reaction mixture is extracted with EtOAc (30 ml) and the aqueous phase is adjusted to pH=7 by adding HCl (1 N). The mixture is then concentrated under reduced pressure. The crude product was purified by reverse phase chromatography (20/1-3/1 H ₂ O/MeCN) to give the product (6.1 g, 67.3% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₃₃ H ₄₉ N ₉ O ₈ : 700.38; found 700.4.

AK building block. 2-(4-(2-(3-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

Solution of ethyl 2-(4-(2-(3-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl )piperazin-1-yl)pyrimidin-5-carboxylate (5.4 g, 7.42 mmol, 1.0 equiv.) in THF (40 ml), EtOH (10 ml) and H ₂ O (10 ml) was added LiOH•H ₂ O (933.92 mg, 22.26 mmol, 3.0 equiv.). The mixture is then stirred at 30°C for 12 hours. The reaction mixture is then extracted with EtOAc (2×50 ml) and the aqueous phase is adjusted to pH=7 by adding HCl (1N). The solution is then concentrated under reduced pressure. The crude product was purified by reverse phase chromatography (20/1-3/1 H ₂ O/MeCN) to give the product (1.01 g, 22.5% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₈ H ₄₁ N ₉ O ₆ : 600.33; found 600.2.

AL building block. 4-{4-[2-(3-{4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]piperazin-1-yl}-3-oxopropoxy)ethyl]piperazin-1-yl}-4-oxobutanoic acid.

To a solution of tert- butyl N- tert -butoxycarbonyl-N-((2-(4-(3-(2-piperazin-1-ylethoxy)propanoyl)piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate ( 1.0 equiv.) succinic anhydride (1.2 equiv.) and Et ₃ N (2.0 equiv.) are added to DCM. The reaction mixture is stirred at room temperature until consumption of the starting material, according to LCMS analysis. The reaction mixture is then concentrated under reduced pressure to obtain the crude product. The residue is purified by chromatography on silica gel to give the product.

AM building block. 2-(4-(4-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ethyl 2-(4-(4-( tert- butoxy)-4-oxobutyl)piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of ethyl 2-(piperazin-1-yl)pyrimidine-5-carboxylate hydrochloride (10 g, 36.67 mmol, 1.0 equiv., HCl) and tert- butyl 4-bromobutanoate (8.18 g, 36. 67 mmol, 1.0 equiv.) in DMF (100 ml) add Et ₃ N (15.31 ml, 110.00 mmol, 3.0 equiv.). The mixture is stirred at 130°C for 14 hours. The mixture is then poured into H ₂ O (400 ml) and the solution is extracted with EtOAc (3×150 ml). The combined organic layer was washed with brine (200 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by silica gel chromatography (5/1-1/1 petroleum ether/EtOAc) to give the product (9.5 g, 68.5% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. _{for C19H30N4O4} : _{379.24 ;} found 379.2, 380.2.

Step 2 : Synthesis of 4-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)butanoic acid hydrochloride

To a solution of ethyl 2-(4-(4-( tert- butoxy)-4-oxobutyl)piperazin-1-yl)pyrimidin-5-carboxylate (9.5 g, 25.10 mmol, 1.0 equiv.) in EtOAc (100 ml) add HCl/EtOAc (500 ml). The mixture was stirred at room temperature for 10 hours and then the solution was concentrated under reduced pressure to give the product (9.6 g, 96.8% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C15H22N4O4 : 323.17} ; found 323.2.

Step 3 : Synthesis of ethyl 2-(4-(4-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperazine -1-yl)pyrimidin-5-carboxylic acid

To a solution of 4-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)butanoic acid hydrochloride (5 g, 15.51 mmol, 1.0 equiv.) and tert- butyl N- tert - butoxycarbonyl-N-((2-piperazin-1-ylpyrimidin-5-yl)methyl)carbamate (6.10 g, 15.51 mmol, 1.0 equiv.) in DMF (150 ml) add DIPEA (8, 11 ml, 46.53 mmol, 3.0 equiv.) and GATU (7.08 g, 18.61 mmol, 1.2 equiv.). The mixture was stirred at room temperature for 3 hours and then the solution was poured into H ₂ O (600 ml). The aqueous layer was extracted with EtOAc (3×200 ml) and then the combined organic layer was washed with brine (100 ml), dried with Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by silica gel chromatography (50/1-15/1 DCM/MeOH) to give the product (6.3 g, 58.2% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₃₄ H ₅₁ N ₉ O ₇ : 698.40; found 698.6.

Step 4 : Synthesis of 2-(4-(4-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperazine-1- yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(4-(4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperazin-1- yl)pyrimidine-5-carboxylate (4.5 g, 6.45 mmol, 1.0 equiv.) in EtOH (7 ml) and THF (28 ml) add a solution of LiOH•H ₂ O (541.17 mg, 12 .90 mmol, 2.0 equiv.) in H ₂ O (7 ml). The mixture was stirred at 30°C for 8 hours, then more LiOH•H ₂ O (541 mg, 12.90 mmol, 2.0 equiv.) was added. After stirring for a further 8 hours at 30°C, the solution is concentrated under reduced pressure. Add H ₂ O (20 ml) and adjust the solution to pH 3 with 1N HCl. The suspension is filtered and the solid is dried under reduced pressure to give the product (3.2 g, 79.1% yield) as a white solid. LCMS(ESI) m/z : [M+H] calc. _{for C27H39N9O5 : 570.32} ; found 570.3.

AN building block. 2-(4-(2-(2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid.

Step 1 : Synthesis of benzyl 4-(2-hydroxyethyl)piperazine-1-carboxylate

To a solution of benzylpiperazine-1-carboxylate hydrochloride (41.09 g, 160.04 mmol, 1.0 equiv., HCl) in MeCN (200 ml) add K ₂ CO ₃ (66.36 g, 480.13 mmol, 3 .0 equiv.) and 2-bromoethanol (20 g, 160.04 mmol, 1.0 equiv.). The reaction mixture is stirred at 80°C for 16 hours, at which point it is cooled to room temperature and filtered. The filter cake is washed with EtOAc (100 ml) and the filtrate is then washed with H ₂ O (100 ml). The aqueous phase is extracted with EtOAc (3×50 ml) and the combined organic phases are washed with brine (50 ml), dried and concentrated under reduced pressure. Purification by silica gel chromatography (5→25% MeOH/EtOAc) gave the desired product as a yellow solid (20 g, 47% yield). LCMS (ESI) m/z : [M+H] calc. for C ₁₄ H ₂₀ N ₂ O ₃ : 265.16; found 264.9.

Step 2 : Synthesis of tert- butyl 4-(2-hydroxyethyl)piperazine-1-carboxylate

To a solution of tert- butyl piperazine-1-carboxylate (198.72 g, 1.07 mol, 1.0 equiv.) in MeCN (1500 ml) add 2-bromoethanol (240 g, 1.92 mol, 1.8 equiv.). ) and K ₂ CO ₃ (221.19 g, 1.60 mol, 1.5 equiv.). The reaction mixture is stirred at 80°C for 16 hours, at which point the mixture is cooled at room temperature, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→14% MeOH/EtOAc) gave the desired product as a white solid (146 g, 59% yield).

Step 3 : Synthesis of tert- butyl 4-(2-bromoethyl)piperazine-1-carboxylate

To a solution of tert- butyl 4-(2-hydroxyethyl)piperazine-1-carboxylate (45 g, 195.39 mmol, 1.0 equiv.) in THF (600 ml) add triphenylphosphine (97.38 g, 371.25 mmol , 1.9 equiv.) and CBr ₄ (116.64 g, 351.71 mmol, 1.8 equiv.). The mixture is stirred at room temperature for 3 hours. The two separate batches are combined and the reaction mixture is filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (1→25% EtOAc/petroleum ether) gave the desired product as a light yellow solid (31 g, 27% yield).

Step 4 : Synthesis of benzyl 4-(2-(2-(4-( tert- butoxycarbonyl)piperazin-1-yl)ethoxy)ethyl)piperazine-1-carboxylate

To a solution of benzyl 4-(2-hydroxyethyl)piperazine-1-carboxylate (18 g, 68.10 mmol, 1.0 equiv.) in toluene (200 ml) add NaNH ₂ (26.57 g, 680.99 mmol, 10.0 equivalent). tert- Butyl 4-(2-bromoethyl)piperazine-1-carboxylate (25 g, 85.27 mmol, 1.25 equiv.) was added and the mixture was heated to 90°C for 18 hours. The mixture was cooled to room temperature and pour into H ₂ O (700 ml) at 0°C. The aqueous phase is extracted with EtOAc (3x240 ml) and the combined organic phases are washed successively with H ₂ O (350 ml) and brine (2x200 ml), dried, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (0→12% MeOH/EtOAc) gave the desired product as a light yellow oil (20 g, 62% yield).

Step 5 : Synthesis of tert- butyl 4-(2-(2-(piperazin-1-yl)ethoxy)ethyl)piperazine-1-carboxylate

To a solution of benzyl 4-(2-(2-(4-( tert- butoxycarbonyl)piperazin-1-yl)ethoxy)ethyl)piperazine-1-carboxylate (20 g, 41.96 mmol, 1.0 equiv.) in EtOAc (180 ml) was added to Pd/C (8 g, 10% wt). The suspension is degassed under reduced pressure and purged with H ₂ three times. The mixture was stirred under H ₂ (30 psi) at 35°C for 12 hours. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure. Purification by silica gel chromatography (0→100% MeOH/EtOAc) gave the desired product as a colorless oil (10.8 g, 75% yield).

Step 6 : Synthesis of ethyl 2-(4-(2-(2-(4-( tert- butoxycarbonyl)piperazin-1-yl)ethoxy)-ethyl)piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of tert- butyl 4-(2-(2-(piperazin-1-yl)ethoxy)ethyl)piperazine-1-carboxylate (10.8 g, 31.54 mmol, 1.0 equiv.) in MeCN (100 ml) add K ₂ CO ₃ (13.08 g, 94.61 mmol, 3.0 equiv.) and ethyl 2-chloropyrimidine-5-carboxylate (5.88 g, 31.54 mmol, 1.0 equiv.) . The mixture is stirred at 80°C for 12 hours, at which point the reaction mixture is cooled at room temperature, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→9% MeOH/DCM) gave the desired product as a white solid (13.6 g, 85% yield).

Step 7 : Synthesis of 2-(4-(2-(2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido-[4,3-d]pyrimidin-2-yl) piperazin-1-yl)ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(2-(2-(4-( tert- butoxycarbonyl)piperazin-1-yl)ethoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylate (13.6 g, 27 .61 mmol, 1.0 equiv.) in MeOH (50 ml) add a solution of HCl in MeOH (4 M, 150 ml, 21.7 equiv.). The reaction mixture was stirred at room temperature for 4 hours, at which time the mixture was concentrated under reduced pressure to give the crude desired product as a white solid (13.8 g, 4 HCl), which was taken directly to the next step. LCMS (ESI) m/z : [M+H] calc. _{for C19H32N6O3 : 393.26} ; found 393.3.

Step 8 : Synthesis of tert- butyl 2-(4-(2-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)-piperazin-1-yl)ethoxy)ethyl)piperazin-1-yl) -7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a stirred solution of 2-(4-(2-(2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin- 1-yl)ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid (10.2 g, 18.95 mmol, 1.0 equiv., 4 HCl) and DIPEA (16.50 ml, 94, 74 mmol, 5.0 equiv.) in DMF (100 ml) add tert- butyl 2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (5.11 g, 18 .95 mmol, 1.0 equiv.). The reaction mixture was stirred at 90°C for 12 hours. The reaction mixture was then cooled at room temperature and added to EtOAc (200 ml) and H ₂ O (400 ml). The aqueous phase is extracted with EtOAc (2×100 ml) and the combined organic phases are washed with aqueous NH ₄ Cl (4×100 ml), brine (2×100 ml), dried, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (0→9% MeOH/DCM) gave the desired product as a white solid (5.4 g, 45% yield). LCMS (ESI) m/z : [M+H] calc. for C ₃₁ H ₄₇ N ₉ O ₅ : 626.38; found 626.3.

Step 9 : Synthesis of 2-(4-(2-(2-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazine -1-yl)ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of tert- butyl 2-(4-(2-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl)piperazin-1-yl)-7, 8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (5.4 g, 8.63 mmol, 1.0 equiv.) in THF (50 ml), EtOH (20 ml) and H ₂ O(20 ml) add LiOH•H ₂ O (1.09 g, 25.89 mmol, 3.0 equiv.). The reaction mixture is stirred at 35°C for 12 hours, at which point the mixture is concentrated under reduced pressure to remove THF and EtOH. The aqueous phase is neutralized to pH=7 with 0.5 N HCl and concentrated under reduced pressure. Purification by reverse phase chromatography gave the desired product as a white solid (4.72 g, 92% yield). LCMS (ESI) m/z : [M+H] calc. for C ₂₉ H ₄₃ N ₉ O ₅ : 598.35; found 598.3.

AO building block. 1'-[( tert- butoxy)carbonyl]-[1,4'-bipiperidine]-4-carboxylic acid.

At the time of preparation of this application, this building block was commercially available (CAS # 201810-59-5).

AP building block. 2-((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)isoindoline-1,3-dione hydrochloride.

Step 1 : Synthesis of 2-chloro-5-(dibromomethyl)pyrimidine

To a solution of 2-chloro-5-methylpyrimidine (100 g, 777.85 mmol, 1.0 equiv.) in CCl ₄ (1200 ml) add NBS (304.58 g, 1.71 mol, 2.2 equiv.) and AIBN (51.09 g, 311.14 mmol, 0.4 equiv.). The mixture was stirred at 80°C for 16 hours. The reaction solution was then cooled at room temperature, filtered and the filtrate was poured into H ₂ O (1500 ml). The solution is diluted with DCM (3×250 ml) and the organic layer is washed with brine (300 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain the crude product as a brown oil, which is used immediately in the next step .

Step 2 : Synthesis of 5-(bromomethyl)-2-chloropyrimidine

To a solution of 2-chloro-5-(dibromomethyl)pyrimidine (229 g, 799.72 mmol, 1.0 equiv.) in THF (600 ml) add DIPEA (111.44 ml, 639.77 mmol, 0.8 equiv. .) and 1-ethoxyphosphonoyloxyethane (82.57 ml, 639.77 mmol, 0.8 equiv.). The mixture was stirred at room temperature for 19 hours. The mixture was then poured into H ₂ O (1200 ml) and the aqueous phase was extracted with EtOAc (3×300 ml). The combined organic phase is washed with brine (300 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (1/0-0/1 petroleum ether/EtOAc) to give the product as a brown oil, which is used immediately in the next step.

Step 3 : Synthesis of 2-((2-chloropyrimidin-5-yl)methyl)isoindoline-1,3-dione

To a mixture of isoindoline-1,3-dione (15 g, 101.95 mmol, 1.0 equiv.) in DMF (126 ml) add NaH (4.89 g, 122.34 mmol, 60 wt.%, 1. 2 equiv.) at 0°C. The mixture was stirred at 0°C for 30 min, then a solution of 5-(bromomethyl)-2-chloropyrimidine (30.21 g, 101.95 mmol, 1.0 equiv.) in DMF (24 ml) was added dropwise to the above mixture at room temperature. The mixture is stirred at room temperature for 2 hours and then cooled to 0°C and quenched with sat. NH ₄ Cl (600 ml). The suspension is filtered and the solid is dried under reduced pressure to obtain the crude product (27.4 g, 98.2% yield) as a gray solid, which is used immediately in the next step. LCMS(ESI) m/z : [M+H]calc. for C ₁₃ H ₈ ClN ₃ O ₂ : 274.04; found 274.0.

Step 4 : Synthesis of tert- butyl4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-carboxylate

To a solution of 2-((2-chloropyrimidin-5-yl)methyl)isoindoline-1,3-dione (27 g, 98.66 mmol, 1.0 equiv. ) and tert- butylpiperazine-1-carboxylate (20.21 g, 108.52 mmol, 1.1 equiv. ) in DMF (270 ml) add K ₂ CO ₃ (34.09 g, 246.64 mmol, 2.5 equiv. ). The mixture was stirred at 80°C for 3 hours and then the reaction mixture was cooled at room temperature and poured into H ₂ O (1200 ml). The suspension is filtered and the solid is dried under reduced pressure to obtain the crude product (35.58 g, 85.2% yield) as a white solid, which is used immediately in the next step.

Step 5 : Synthesis of 2-((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)isoindoline-1,3-dione

A solution of tert- butyl 4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazine-1-carboxylate (15 g, 35.42 mmol, 1 equiv. ) in HCl/ EtOAc (150 ml) was stirred at room temperature for 2 hours. The mixture was filtered and then the filter cake was washed with EtOAc (20 ml) and dried under reduced pressure to obtain the product (42.53 g, 92.5% yield) as white solid matter.

AQ building block. 2-[(2-{4-[2-(3-{4-[5-({bis[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]piperazin-1-yl}-3-oxopropoxy)ethyl]piperazin-1-yl}-2-oxoethyl)(methyl)amino]acetic acid.

To a solution of tert- butyl N-[( tert- butoxy)carbonyl]-N-{[2-(4-{3-[2-(piperazin-1-yl)ethoxy]propanoyl}piperazin-1-yl)pyrimidin- 5-yl]methyl}carbamate (300 mg, 519 µmol, 1.0 equiv.) in pyridine (8 ml) at 0°C add 4-methylmorpholine-2,6-dione (80.3 mg, 622 µmol, 1 ,2 equivalent). The reaction mixture is stirred at 0°C for 1 hour and then warmed to room temperature and stirred for another 12 hours. The solvent is concentrated under reduced pressure and the solid is partitioned between DCM and H ₂ O. The organic layer is separated, dried over MgSO ₄ and the solvent was concentrated under reduced pressure to obtain the product (23.0 mg, 6.28% yield). LCMS (ESI) m/z : [M+H] calc. for C ₃₃ H ₅₄ N ₈ O ₉ : 707.41; found 707.4.

AR building block. 2-(4-(2-(3-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid .

Step 1 : Synthesis of tert- butyl 2-(4-(3-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)propanoyl)piperazin-1-yl)- 7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate

To a solution of 3-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)propanoic acid (6 g, 12.86 mmol, 1.0 equiv., TPA) in DMF (55 ml) add GATU (6.36 g, 16.72 mmol, 1.3 equiv.) and DIPEA (11.20 ml, 64.32 mmol, 5.0 equiv.). After 0.5 h, tert- butyl 2-(piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (4.11 g, 12.86 mmol, 1.0 equivalent). The mixture was stirred for 3 hours, at which point it was filtered and the solid cake was dried under reduced pressure to obtain the desired product as a white solid (7.5 g, 89% yield). LCMS(ESI) m/z : [M+ H] calc. for C ₃₂ H ₄₇ N ₉ O ₆ : 654.37; found 654.4.

Step 2 : Synthesis of 2-(4-(2-(3-(4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazine -1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of tert- butyl 2-(4-(3-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)propanoyl)piperazin-1-yl)-7, 8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (7.2 g, 11.01 mmol, 1.0 equiv.) in THF (72 ml), EtOH (36 ml) and H ₂ O (36 ml) add LiOH•H ₂ O (1.85 g, 44.05 mmol, 4.0 equiv.). The reaction mixture was stirred at room temperature for 2.5 hours, at which point the mixture was filtered and the filtrate was concentrated under reduced pressure to remove THF and EtOH. The aqueous phase is neutralized to pH=7 with 1N HCl and then concentrated under reduced pressure. Purification by reverse phase chromatography (30% MeCN/H ₂ O) gave the desired product as a white solid (3.85 g, 54% yield). LCMS (ESI) m/z : [M+H] calc. for C ₃₀ H ₄₃ N ₉ O ₆ : 626.34; found 626.3.

AS building block. 2-(4-(2-(2-(3-(4-(5-((di- tert- butoxycarbonylamino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid.

Step 1 : Synthesis of 3-(2-(2-(4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethoxy)propanoic acid

Solution of ethyl 2-(4-(2-(2-(3-( tert- butoxy)-3-oxopropoxy)ethoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylate (4 g, 8.84 mmol, 1.0 equiv.) in TFA (12.29 ml, 166.00 mmol, 18.8 equiv.) was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure. Purification by silica gel chromatography (0→20% MeOH/EtOAc) gave the desired product as a brown oil (4.35 g, 95% yield, TPA salt).

Step 2 : Synthesis of ethyl 2-(4-(2-(2-(3-(4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)- 3-oxopropoxy)ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of 3-(2-(2-(4-(5-ethoxycarbonylpyrimidin-2-yl)piperazin-1-yl)ethoxy)ethoxy)propanoic acid (3.8 g, 7.44 mmol, 1.0 equiv. , TPA) in DCM (30 ml) add GATU (4.25 g, 11.17 mmol, 1.5 equiv.) and DIPEA (6.48 ml, 37.22 mmol, 5.0 equiv.). The reaction mixture was stirred at room temperature for 30 minutes and then tert- butyl N- tert- butoxycarbonyl- N -((2-piperazin-1-ylpyrimidin-5-yl)methyl)carbamate (2.93 g, 7. 44 mmol, 1.0 equiv.). The mixture is stirred at room temperature for 3.5 hours, at which point the reaction mixture is concentrated under reduced pressure. Purification by silica gel chromatography (0→20% MeOH/EtOAc) gave the desired product as a brown oil (4.14 g, 70% yield).

Step 3 : Synthesis of 2-(4-(2-(2-(3-(4-(5-((di- tert- butoxycarbonylamino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy )ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(2-(2-(3-(4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3- Oxopropoxy)ethoxy)ethyl)piperazin-1-yl)pyrimidine-5-carboxylate (1.4 g, 1.81 mmol, 1.0 equiv.) in THF (28 ml), EtOH (14 ml) and H ₂ O (14 ml) add LiOH•H ₂ O (304.44 mg, 7.25 mmol, 4.0 equiv.). The mixture is stirred at 40°C for 30 minutes, at which point the reaction mixture is concentrated under reduced pressure. Purification by reverse phase chromatography (10→40% MeCN/H ₂ O) gave the desired product as a yellow solid (500 mg, 43% yield).

AT building block. 2-{4-[2-(2-{4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]piperazin-1-yl}ethoxy)ethyl]piperazin-1-yl}pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ethyl 2-(4-(2-(2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl )piperazin-1-yl)pyrimidine-5-carboxylate

To a solution of ethyl hydrochloride 2-(4-(2-(2-(piperazin-1-yl)ethoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylate (7.3 g, 13.56 mmol, 1. 0 equiv., 4 HCl) in DMF (75 ml) add DIPEA (14.17 ml, 81.36 mmol, 6.0 equiv.) and tert- butyl-N- tert- butoxycarbonyl-N-[(2-chloropyrimidine -5-yl)methyl]carbamate (5.59 g, 16.27 mmol, 1.2 equiv.). The mixture was stirred at 80°C for 12 hours. The mixture was then cooled at room temperature and poured into H ₂ O (300 ml). The aqueous phase is extracted with EtOAc (3×80 ml). The combined organic phases are washed with sat. NH ₄ Cl (4×80 ml) and brine (150 ml), dry, filter and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→17% MeOH/EtOAc) gave the desired product (7.7 g, 81.1% yield) as a light yellow oil. LCMS (ESI) m/z : [M+Na] calc. for C ₃₄ H ₅₃ N ₉ O ₇ : 722.40; found 722.4.

Step 2 : Synthesis of 2-(4-(2-(2-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl)piperazin- 1-yl)pyrimidine-5-carboxylic acid

To a solution of ethyl 2-(4-(2-(2-(4-(5-(((di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl)piperazine -1-yl)pyrimidin-5-carboxylate (7.7 g, 11.00 mmol, 1.0 equiv.) in THF (80 ml), EtOH (20 ml) and H ₂ O (40 ml) add LiOH• H ₂ O (2.31 g, 55.01 mmol, 5.0 equiv.). The mixture is stirred at 50°C for 26 hours. The mixture is then concentrated under reduced pressure to remove THF and EtOH. The aqueous phase is neutralized with 0.5 N HCl and concentrated under reduced pressure. Purification by reverse phase chromatography gave the desired product (4.67 g, 74.3% yield) as a white solid. LCMS (ESI) m/z : [MH] calc. _{for C27H41N9O5 : 570.31} ; found 570.3.

AU building block. ( R )- tert- butyl 4-(5-((( tert- butoxycarbonyl-N- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazine-2-carboxylate.

Step 1 : Synthesis of ( R )-1,4-bis((benzyloxy)carbonyl)piperazine-2-carboxylic acid

To two separate batches containing a solution of ( 2R )-piperazine-2-carboxylic acid (70 g, 344.71 mmol, 1 equiv., 2 HCl) in H ₂ O (700 ml) and dioxane (1120 ml) add 50 % aq. NaOH to pH=11. Benzyl chloroformate (156.82 mL, 1.10 mol, 3.2 equiv.) was added and the reaction mixture was stirred at room temperature for 12 hours. The two reaction mixtures were combined and H ₂ O (1200 mL) was added. The aqueous layer is extracted with MTBE (3×1000 ml), adjusted to pH=2 with conc. HCl and then extracted with EtOAc (2×1000 ml). The combined organic phases are dried, filtered and concentrated under reduced pressure to give the desired product (280 g, 86% yield). LCMS (ESI) m/z : [M+H] calc. for C ₂₁ H ₂₂ N ₂ O ₆ : 399.16; found 399.0.

Step 2 : Synthesis of ( R )-1,4-dibenzyl 2- tert- butylpiperazine-1,2,4-tricarboxylate

To a solution of ( R )-1,4-bis((benzyloxy)carbonyl)piperazine-2-carboxylic acid (70 g, 175.70 mmol, 1.0 equiv.) in toluene (700 ml) at 80°C add 1 ,1-di- tert- butoxy- N,N -dimethylmethanamine (80.04 ml, 333.83 mmol, 1.9 equiv.). The reaction mixture is stirred at 80°C for 2 hours, at which point it is cooled to room temperature and partitioned between EtOAc (300 ml) and H ₂ O (500 ml). The aqueous layer was extracted with EtOAc (2×500 ml) and the combined organic layers were dried, filtered and concentrated under reduced pressure. Purification by chromatography on silica gel (0→25 EtOAc/petroleum ether) gave the desired product as a white solid (50 g, 57% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₂₅ H ₃₀ N ₂ O ₆ : 477.20; found 476.9.

Step 3 : Synthesis of ( R ) -tert- butylpiperazine-2-carboxylate

To a solution of ( R )-1,4-dibenzyl 2- tert- butylpiperazine-1,2,4-tricarboxylate (50 g, 110.01 mmol, 1 equiv.) in EtOAc (20 mL) add Pd/C (15 g , 10% wt.). The suspension is degassed under reduced pressure and purged with H ₂ three times. The suspension is stirred under H ₂ (30 psi) at 30°C for 4 hours. The reaction mixture is then filtered, the residue is washed with MeOH (5 x 200 ml) and the filtrate is concentrated under reduced pressure to obtain the desired product as yellow oil (17 g, 81% yield). LCMS (ESI) m/z : [M+H] calc. for C ₉ H ₁₈ N ₂ O ₂ : 187.15; found 187.1.

Step 4 : Synthesis of ( R ) -tert -butyl 4-(5-((( tert- butoxycarbonyl- N - tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazine-2-carboxylate

To a suspension of ( R ) -tert- butylpiperazine-2-carboxylate (8 g, 23.27 mmol, 1.0 equiv.) and tert- butyl- N - tert -butoxycarbonyl ((2-chloropyrimidin-5-yl)methyl) carbamate (5.20 g, 27.92 mmol, 1.2 equiv.) in MeCN (100 ml) add K ₂ CO ₃ (6.43 g, 46.54 mmol, 2.0 equiv.). The reaction mixture is heated to 80°C for 12 hours, at which point it is cooled to room temperature, filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→100% EtOAc/petroleum ether) gave the desired product as a yellow solid (9.2 g, 73% yield). LCMS (ESI) m/z : [M+H] calc. _{for C24H39N5O6 : 494.30} ; found 494.1.

AV building block. ( S )- tert- butyl 4-(5-((( tert- butoxycarbonyl-N- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazine-2-carboxylate.

This building block is prepared by a procedure similar to that of Building Block AU using ( 2S )-piperazine-2-carboxylic acid.

Building block AW. ( R )-2-(4-(2-(3-(2-( tert- butoxycarbonyl)-4-(5-((( tert- butoxycarbonyl- N - tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ( R )-ethyl 2-(4-(2-(3-(2-( tert- butoxycarbonyl)-4-(5-((( tert- butoxycarbonyl- N - tert- butoxycarbonyl)amino)methyl )pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylate

To a solution of ( R ) -tert- butyl 4-(5-((( tert- butoxycarbonyl- N - tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-2-carboxylate (5.3 g, 11. 36 mmol, 1.0 equiv., TFA) in DCM (80 ml) add GATU (6.48 g, 17.05 mmol, 1.5 equiv.) and DIPEA (7.92 ml, 45.45 mmol, 4 ,0 equivalent). The reaction mixture is stirred at room temperature for 30 minutes and then tert- butyl ( 2R )-4-(5-((bis( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-2-carboxylate is added ( 5.61 g, 11.36 mmol, 1.0 equiv.). The mixture is stirred for 1 hour, at which point it is saturated. NH ₄ Cl (80 ml). The organic phase is washed with sat. NH ₄ Cl (5×80 ml), dry, filter and concentrate under reduced pressure. Purification by silica gel chromatography (0→9% MeOH/EtOAc) gave the desired product as a yellow solid (8.4 g, 85% yield).

Step 2 : Synthesis of ( R )-2-(4-(2-(3-(2-( tert- butoxycarbonyl)-4-(5-((( tert- butoxycarbonyl- N - tert- butoxycarbonyl)amino)methyl) pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid

To two separate batches containing a solution of ( R )-ethyl 2-(4-(2-(3-(2-( tert- butoxycarbonyl)-4-(5-((( tert- butoxycarbonyl- N - tert- butoxycarbonyl) amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylate (3.4 g, 4.11 mmol, 1.0 equiv. ) in THF (16 ml), EtOH (8 ml) and H ₂ O (8 ml) add LiOH•H ₂ O (344.61 mg, 8.21 mmol, 2.0 equiv.). The mixture is stirred at room temperature for 2 hours. The two reaction mixtures are then combined and adjusted to pH=7 with 1N HCl. The solution is concentrated under reduced pressure to remove THF and EtOH. The solution is then filtered and the resulting solid is purified by reverse phase chromatography to give the desired product as a white solid (4 g, 59% yield). LCMS (ESI) m/z : [M+H] calc. for C ₃₈ H ₅₇ N ₉ O ₁₀ : 800.43; found 800.3.

AX building block. ( S )-2-(4-(2-(3-(2-( tert- butoxycarbonyl)-4-(5-((( tert- butoxycarbonyl- N - tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

This building block is obtained from Building Block AV using a procedure similar to that of Building Block AW.

Building block AY. 1'-(2-(3-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)-[1,4'-bipiperidine]-4-carboxylic acid.

Step 1 : Synthesis of 1'- tert- butyl 4-ethyl[1,4'-bipiperidine]-1',4-dicarboxylate

To a solution of ethylpiperidine-4-carboxylate (30 g, 150.57 mmol, 1.0 equiv.) and tert- butyl 4-oxopiperidine-1-carboxylate (23.67 g, 150.57 mmol, 1.0 equiv.) in DCM (300 mL) add HOAc (6.00 mL, 104.95 mmol, 0.7 equiv.). The mixture was stirred at room temperature for 30 minutes, then NaBH(OAc) ₃ (63.82 g, 301.13 mmol, 2.0 equiv.) was added. The mixture was stirred for 16 hours, at which point H ₂ O (50 ml) was added. The aqueous phase is extracted with DCM (3×15 ml) and the combined organic phases are washed with brine (10 ml), dried, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (8→100 MeOH/EtOAc) gave the desired product as a yellow oil (30 g, 59% yield).

Step 2 : Synthesis of ethyl [1,4'-bipiperidine]-4-carboxylate

To a solution of HCl in EtOAc (200 mL) add 1'- tert -butyl 4-ethyl[1,4'-bipiperidine]-1',4-dicarboxylate (20 g, 58.74 mmol, 1.0 equiv.). The mixture was stirred at room temperature for 3 hours. The mixture was then concentrated under reduced pressure to obtain the desired crude product as a white solid (15 g, HCl salt).

Step 3 : Synthesis of ethyl 1'-(2-(3-( tert- butoxy)-3-oxopropoxy)ethyl)-[1,4'-bipiperidine]-4-carboxylate

To a solution of tert- butyl 3-(2-bromoethoxy)propanoate (6.46 g, 25.54 mmol, 1.0 equiv.) in DMF (240 ml) add K ₂ CO ₃ (10.59 g, 76.61 mmol, 3.0 equiv.) and ethyl [1,4'-bipiperidine]-4-carboxylate (8 g, 25.54 mmol, 1.0 equiv., 2 HCl). The mixture is stirred at 120°C for 12 hours, at which point the reaction mixture is cooled to room temperature, filtered, the filter cake is washed with H ₂ O (20 ml) and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→11% MeOH/EtOAc) gave the desired product as a yellow oil (6.6 g, 63% yield).

Step 4 : Synthesis of 3-(2-(4-(ethoxycarbonyl)-[1,4'-bipiperidin]-1'-yl)ethoxy)propanoic acid

To a solution of HCl in EtOAc (70 ml) add ethyl 1'-(2-(3-( tert- butoxy)-3-oxopropoxy)ethyl)-[1,4'-bipiperidine]-4-carboxylate (6.6 g , 16.00 mmol, 1.0 equiv.). The mixture was stirred at room temperature for 3 hours, at which time the reaction mixture was concentrated under reduced pressure to obtain the desired product as a white solid (6.5 g, 95% yield, 2 HCl).

Step 5 : Synthesis of ethyl 1'-(2-(3-(4-(5-((( N,N -di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)- 3-oxopropoxy)ethyl)-[1,4'-bipiperidine]-4-carboxylate

To a solution of tert- butyl- tert- butoxycarbonyl((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)carbamate (2.49 g, 6.33 mmol, 1.5 equiv.) in DMF (40 ml) add DIPEA (9.74 ml, 55.89 mmol, 6.0 equiv.) and GATU (5.31 g, 13.97 mmol, 1.5 equiv.). The mixture was stirred at room temperature for 30 minutes and then 3-(2-(4-(ethoxycarbonyl)-[1,4'-bipiperidin]-1'-yl)ethoxy)propanoic acid (4 g, 9.32 mmol) was added , 1.0 equiv., 2 HCl). The mixture is stirred for 1.5 hours, at which point H ₂ O (5 ml) and EtOAc (20 ml) are added. The aqueous phase is extracted with EtOAc (3×10 ml) and the combined organic phases are washed with brine (5 ml), dried, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography gave the desired product as a brown oil (1.6 g, 23% yield). LCMS (ESI) m/z : [M+H] calc. for C ₃₇ H ₆₁ N ₇ O ₈ : 732.47; found 732.6.

Step 6 : Synthesis of 1'-(2-(3-(4-(5-((( tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)- [1,4'-bipiperidine]-4-carboxylic acid

To a solution of ethyl 1'-(2-(3-(4-(5-((( N, N -di- tert- butoxycarbonyl)amino)methyl)pyrimidin-2-yl)piperazin-1-yl)-3- Oxopropoxy)ethyl)-[1,4'-bipiperidine]-4-carboxylate (1.4 g, 1.91 mmol, 1.0 equiv.) in THF (7.5 ml), EtOH (3.8 ml) and H ₂ O (3.8 ml) add LiOH•H ₂ O (321.07 mg, 7.65 mmol, 4.0 equiv.). The mixture is stirred at room temperature for 2 hours, at which point the mixture is concentrated under reduced pressure. Purification by reverse phase chromatography (5→38% MeCN/H ₂ O) gave the desired product as a yellow solid (325 mg, 22% yield). LCMS (ESI) m/z : [M+H] calc. for C ₃₀ H ₄₉ N ₇ O ₆ : 604.38; found 604.3.

Building block AZ. 1-(4-{2-[2-(2-{[(benzyloxy)carbonyl]amino}ethoxy)ethoxy]ethyl}piperazin-1-yl)-3,6,9,12-tetraoxapentadecane-15-oic acid .

Step 1 : Synthesis of benzyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate

PPh ₃ (13, 79 g, 52.59 mmol, 1.49 equiv.) and CBr ₄ (17.44 g, 52.59 mmol, 1.49 equiv.). The mixture is then warmed to room temperature and stirred for 12 hours. The reaction mixture is then filtered and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (1%→25% EtOAc/petroleum ether) gave the desired product (10.8 g, 88.4% yield) as a yellow oil.

Step 2 : Synthesis of tert- butyl 4-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)piperazine-1-carboxylate

To a solution of benzyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate (10.8 g, 31.19 mmol, 1.0 equiv.) and tert- butyl piperazine-1-carboxylate (5.81 g , 31.19 mmol, 1.0 equiv.) to MeCN (100 ml) add K ₂ CO ₃ (4.31 g, 31.19 mmol, 1.0 equiv.). The mixture is stirred at 80°C for 1 hour. The reaction mixture is then filtered, and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→50% MeOH/EtOAc) gave the desired product (13.1 g, 93.0% yield) as a yellow oil.

Step 3 : Synthesis of benzyl (2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethyl)carbamate

Tert- Butyl 4-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)piperazin-1-carboxylate solution (5.64 g, 12.49 mmol, 1.0 equiv.) in HCl/EtOAc (50 ml, 4 M) was stirred at room temperature for 1 hour. The reaction mixture was then concentrated under reduced pressure to give the desired product (5.23 g, crude, HCl salt) as a yellow oil.

Step 4 : Synthesis of tert- butyl 1-(4-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)piperazin-1-yl)-3,6,9, 12-tetraoxapentadecane-15-oate

A solution of benzyl (2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethyl)carbamate (13.3 g, 31.34 mmol, 1.0 equiv., 2 HCl) and tert- butyl 1 -bromo-3,6,9,12-tetraoxapentadecane-15-oate in MeCN (150 ml) add K ₂ CO ₃ (21.66 g, 156.71 mmol, 5.0 equiv.). The mixture was stirred at 80°C for 12 hours. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure. Purification by silica gel chromatography (1%→17% MeOH/DCM) gave the desired product (5.4 g, 26.3% yield) as a yellow oil.

Step 5 : Synthesis of 1-(4-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecane -15-oic acid

Solution of tert- butyl 1-(4-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecane -15-oate (2.4 g, 3.66 mmol, 1.0 equiv.) in TFA (20 ml) was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure to obtain the desired product (3.03 g, TPA salt) as a yellow oil.

Building block BA. ( R )-2-(2-( tert- butoxycarbonyl)-4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

Step 1 : Synthesis of ( R ) -tert- butyl 2-(3-( tert- butoxycarbonyl)-4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[4 ,3-d]pyrimidine-6(5H)-carboxylate

To two different batches prepared in parallel, each containing a solution of ( R ) -tert -butyl 2-(3-( tert- butoxycarbonyl)piperazin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine -6(5H)-carboxylate (6 g, 14.30 mmol, 1.0 equiv.) and K ₂ CO ₃ (3.95 g, 28.60 mmol, 2.0 equiv.) in MeCN (80 ml) add ethyl 2-chloropyrimidine-5-carboxylate (3.20 g, 17.16 mmol, 1.2 equiv.). The reaction mixtures are stirred at 80°C for 12 hours. The two reaction mixtures are combined and filtered, the residue is washed with EtOAc (3×50 ml) and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0%→17% MeOH/EtOAc) gave the desired product (15 g, 91.5% yield) as a yellow solid. LCMS (ESI) m/z : [M+H] calc. for C ₂₈ H ₃₉ N ₇ O ₆ : 570.31; found 570.1.

Step 2 : Synthesis of ( R )-2-(2-( tert- butoxycarbonyl)-4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine-2- yl)piperazin-1-yl)pyrimidine-5-carboxylic acid

To a solution of ( R ) -tert- butyl 2-(3-( tert- butoxycarbonyl)-4-(5-(ethoxycarbonyl)pyrimidin-2-yl)piperazin-1-yl)-7,8-dihydropyrido[4,3 -d]pyrimidine-6(5H)-carboxylate (15 g, 26.33 mmol, 1.0 equiv.) in THF (80 ml), EtOH (40 ml) and H ₂ O (40 ml) add LiOH●H ₂ O (2.21 g, 52.66 mmol, 2.0 equiv.). The mixture is stirred at room temperature for 6 hours. The reaction mixture is then adjusted to pH=6 with 1 N HCl. The resulting suspension was filtered and the solid cake was dried under reduced pressure to obtain the desired product (10.87 g, 75.9% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. _{for C26H35N7O6 : 542.27} ; found 542.1.

BB building block. ( S )-2-(2-( tert- butoxycarbonyl)-4-(6-( tert- butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)piperazin-1-yl)pyrimidin-5-carboxylic acid.

This building block is obtained from Building Block AA using a procedure similar to that of Building Block BA.

Building block BC. 2-[(2 R )-2-[( tert- butoxy)carbonyl]-4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]piperazin-1-yl]pyrimidine-5-carboxylic acid.

This building block is obtained from Building Block AU using a procedure similar to that of Building Block BA.

BD building block. 2-[(2 S )-2-[( tert- butoxy)carbonyl]-4-[5-({[( tert- butoxy)carbonyl]amino}methyl)pyrimidin-2-yl]piperazin-1-yl]pyrimidine-5-carboxylic acid.

This building block is obtained from Building Block AV using a procedure similar to that of Building Block BA.

BE building block. 15-(6-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4- Dihydroisoquinolin-2(1H)-yl)-1-((1 S ,4 S )-5-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2,5-diazabicyclo[2,2,1]heptan-2-yl)-3,6,9,12-tetraoxapentadecane-15 -He.

Step 1 : Synthesis of ( 1S , 4S ) -tert- butyl 5-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)-2,5-diazabicyclo[2 ,2,1]heptane-2-carboxylate

To a solution of ( 1S , 4S ) -tert- butyl 2,5-diazabicyclo[2,2,1]heptane-2-carboxylate (2.85 g, 14.37 mmol, 1.0 equiv.) in MeCN ( 50 ml) add K ₂ CO ₃ (3.97 g, 28.75 mmol, 2.0 equiv.) and benzyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate (4.98 g, 14 .37 mmol, 1.0 equiv.). The mixture is stirred at 80°C for 24 hours. The reaction mixture is then filtered, and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→10% MeOH/EtOAc) gave the desired product (6.2 g, 93.0% yield) as a colorless oil. LCMS (ESI) m/z : [M+H] calc. for C ₂₄ H ₃₇ N ₃ O ₆ : 464.27; found 464.2.

Step 2 : Synthesis of benzyl (2-(2-(2-(( 1S , 4S )-2,5-diazabicyclo[2,2,1]heptan-2-yl)ethoxy)ethoxy)ethyl)carbamate

To a solution of ( 1S , 4S ) -tert- butyl 5-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)-2,5-diazabicyclo[2,2 ,1]heptane-2-carboxylate (6.2 g, 13.37 mmol, 1.0 equiv.) in DCM (60 ml) add TFA (20.7 ml, 279.12 mmol, 20.9 equiv.) . The reaction mixture was stirred for 2 hours, at which time the mixture was concentrated under reduced pressure at 45°C to obtain the desired crude product (10.5 g, 4 TFA) as a light brown oil, which was used immediately in the next step. LCMS (ESI) m/z : [M+H] calc. _{for C19H29N3O4 : 364.22} ; found 364.2.

Step 3 : Synthesis of tert- butyl 1-(( 1S , 4S )-5-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)-2,5- diazabicyclo[2,2,1]heptan-2-yl)-3,6,9,12-tetraoxapentadecane-15-oate

To a solution of benzyl (2-(2-(2-(( 1S , 4S )-2,5-diazabicyclo[2,2,1]heptan-2-yl)ethoxy)ethoxy)ethyl)carbamate (5 g, 6.10 mmol, 1.0 equiv., 4 TFA) to MeCN (80 ml) add K ₂ CO ₃ (5.06 g, 36.61 mmol, 6.0 equiv.) and tert- butyl 1-bromo- 3,6,9,12-tetraoxapentadecane-15-oate (2.35 g, 6.10 mmol, 1.0 equiv.). The reaction mixture was stirred at 80°C for 12 hours. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure. Purification by silica gel chromatography (0→15% MeOH/EtOAc) gave the desired product (5.2 g, 92.8% yield) as a light yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₃₄ H ₅₇ N ₃ O ₁₀ : 668.4; found 668.4.

Step 4 : Synthesis of 1-(( 1S , 4S )-5-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)-2,5-diazabicyclo[2 ,2,1]heptan-2-yl)-3,6,9,12-tetraoxapentadecane-15-oic acid

A solution of tert- butyl 1-(( 1S , 4S )-5-(3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)-2,5-diazabicyclo[2 ,2,1]heptan-2-yl)-3,6,9,12-tetraoxapentadecane-15-oate (5.2 g, 5.66 mmol, 1.0 equiv.) in TFA (47.3 ml, 638.27 mmol, 112.75 equiv.) stirred at room temperature for 30 minutes. The mixture is then concentrated under reduced pressure at 45°C. Purification by reverse phase chromatography (2→35% MeCN/H ₂ O (0.05% NH ₄ OH)) gave the desired product (1.88 g, 54.3% yield) as a light brown oil. LCMS (ESI) m/z : [M+H] calc. for C ₃₀ H ₄₉ N ₃ O ₁₀ : 612.34; found 612.3.

BF building block. 21-(6-((4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3,4- Dihydroisoquinolin-2(1H)-yl)-1-(piperazin-1-yl)-3,6,9,12,15,18-hexaoxagenicosan-21-one.

Step 1 : Synthesis of benzyl 4-(23,23-dimethyl-21-oxo-3,6,9,12,15,18,22-hexaoxatetracosyl)piperazine-1-carboxylate

To a solution of tert- butyl 1-bromo-3,6,9,12,15,18-hexaoxagenicosan-21-oate (5 g, 10.56 mmol, 1.0 equiv.) and benzylpiperazine-1-carboxylate (2, 62 mL, 11.62 mmol, 1.1 equiv., HCl) to MeCN (50 mL) add K ₂ CO ₃ (4.38 g, 31.69 mmol, 3.0 equiv.). The reaction mixture is stirred at 80°C for 10 hours. The mixture is then filtered, the solid cake is washed with EtOAc (3×3 ml) and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (0→10% MeOH/EtOAc) gave the desired product (4 g, 61.8% yield) as a red liquid.

Step 2 : Synthesis of 1-(4-((benzyloxy)carbonyl)piperazin-1-yl)-3,6,9,12,15,18-hexaoxagenicosan-1-oic acid

To a solution of benzyl 4-(23,23-dimethyl-21-oxo-3,6,9,12,15,18,22-hexaoxatetracosyl)piperazine-1-carboxylate (1.8 g, 2.94 mmol, 1. 0 equiv.) in DCM (10 ml) add TFA (10 ml). The solution is stirred for 0.5 hours. The solution is then concentrated under reduced pressure. DCM (30 mL) was added to the residue, and then the solution was concentrated under reduced pressure to obtain the desired product (1.6 g, 2.87 mmol, TPA) as a red liquid.

Preparation of rapamycin monomers.

Monomer 1.40( R )- O -(4-nitrophenyl)carbonate rapamycin.

To a solution of rapamycin (30.10 g, 32.92 mmol, 1.0 equiv) in DCM (148.9 ml) add pyridine (29.6 ml, 367 mmol, 11.1 equiv). The solution was cooled to -78°C and then p -nitrophenyl chloroformate (12.48 g, 61.92 mmol, 1.9 equiv.) was added. The reaction mixture was stirred at -78°C for 2 hours. DCM was then added to the reaction mixture and the solution was then poured into H ₂ O. The aqueous layer was extracted with DCM and the combined organic layers were dried over MgSO ₄ and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (0→50% EtOAc/hexane) to give the product (23.1 g, 59.2% yield) as a white solid. LCMS (ESI) m/z : [M+Na] calc. for C ₅₈ H ₈₂ N ₂ O ₁₇ : 1101.55; found 1101.6.

Monomer 2. 32( R )-hydroxy 40( R )- O -(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of 32( R )-hydroxy rapamycin

A solution of 32( R )-hydroxy-28,40-bistriethylsilyl rapamycin (3.64 g, 3.18 mmol, 1 equiv.) in THF (41.8 ml) was treated with pyridine (20.8 ml, 258 mmol, 81 equiv. .) and the reaction mixture is cooled to 0°C. The solution is treated dropwise with 70% HF-pyridine (4.60 mL, 159 mmol, 50 equiv.) and the reaction mixture is stirred at 0°C for 20 minutes, then warmed to room temperature. After 5 hours, the reaction mixture is cooled back to 0°C and ice-saturated water is carefully added. NaHCO ₃ solution (400 ml). The mixture is extracted with EtOAc (2×100 ml) and the organic phase is washed with 75 ml portions of H ₂ O, sat. NaHCO ₃ solution and saturated salt solution. The organic solution is dried over Na ₂ SO ₄ , filtered and concentrated to give a light yellow oil which gives a stiff foam under reduced pressure. The crude product was purified by silica gel chromatography (20→40% acetone/hexane) to give the desired product as a white amorphous solid (1.66 g, 57% yield). LCMS (ESI) m/z : [M+Na] calc. _{for C51H81NO13} : _938.56 ; found 938.7; m/z : [MH] calc. for C ₅₁ H ₈₁ NO ₁₃ : 914.56; found 914.7.

Step 2 : Synthesis of 32( R )-hydroxy 40( R ) -O- (4-nitrophenyl)rapamycin carbonate

To a suspension of 4Å molecular sieve powder (6.0 g) in DCM (130 mL) was added 32( R )-hydroxyrapamycin (6.00 g, 6.55 mmol, 1.0 equiv). After stirring at room temperature for 45 minutes, pyridine (5.99 mL, 74.0 mmol, 11.3 equiv.) was added. The suspension is cooled to -15°C and then 4-nitrophenyl chloroformate (1.78 g, 8.84 mmol, 1.4 equiv.) is added. The reaction mixture was stirred at -10°C for 2 hours and then filtered and the filter layer was washed with DCM (140 ml). The filtrate is washed with sat. NaHCO ₃ (130 ml), H ₂ O (130 ml) and brine (130 ml), dry over Na ₂ SO ₄ , filter and concentrate under reduced pressure. The crude product was purified by silica gel chromatography (20→50% EtOAc/hexane) to give the product (4.44 g, 63% yield) as an off-white stiff foam. LCMS (ESI) m/z : [M+Na] calc. for C ₅₈ H ₈₄ N ₂ O ₁₇ : 1103.57; found 1103.5.

Monomer 3. 32( R )-methoxy 40( R )- O -(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of 32( R )-methoxy-28,40-bistriethylsilyl rapamycin

To a stirred solution of 32( R )-hydroxy-28,40-bistriethylsilyl rapamycin (3.83 g, 3.34 mmol, 1.0 equiv.) in chloroform (95.8 ml) add Proton Sponge® (7.17 g , 33.5 mmol, 10.0 equiv.) along with freshly dried 4 Å molecular sieves (4 g). The solution was stirred for 1 hour, then trimethioxonium tetrafluoroborate (4.95 g, 33.5 mmol, 10.0 equiv., heat dried under reduced pressure at 50° C. for 1 hour before use) was added at room temperature. The reaction mixture was stirred for 18 hours and then the reaction mixture was diluted with DCM and filtered through Celite. The filtrate is washed successively with aqueous 1 M HCl (2x), sat. aqueous NaHCO ₃ solution, then dried and concentrated under reduced pressure. Purification by chromatography on silica gel (10→20% EtOAc/hexane) gives the desired product as a yellow oil with 3% wt. Proton Sponge®. The residue is taken into MTBE and washed with aqueous 1 M HCl, sat. aqueous NaHCO ₃ solution, dried and then concentrated under reduced pressure to give a yellow foam (3.15 g, 81.2% yield). LCMS (ESI) m/z : [M-TES+H ₂ O] calc. for C ₆₄ H ₁₁₁ NO ₁₃ Si ₂ : 1061.68; found 1061.9.

Step 2 : Synthesis of 32( R )-methoxy rapamycin

To a stirred solution of 32( R )-methoxy-28,40-bistriethylsilyl rapamycin (1.11 g, 0.958 mmol, 1.0 equiv.) in THF (12.6 ml) and pyridine (6.30 ml) in a plastic vial add 70% HF-pyridine (2.22 mL, 76.6 mmol, 80.0 equiv.) dropwise at 0°C. The reaction mixture is stirred at 0°C for 20 minutes, then warmed to room temperature within 3 hours when HPLC indicates complete consumption of starting material. The reaction mixture is cooled to 0°C and slowly poured into ice water. aqueous solution of NaHCO ₃ (50 ml). The aqueous layer is extracted with EtOAc (3x) and the combined organic layers are washed with sat. aqueous NaHCO ₃ solution, brine, dried and concentrated under reduced pressure . The yellow residue is dissolved in MeOH (5 ml) and added dropwise to H ₂ O (50 ml) to obtain a white precipitate. After stirring for 15 minutes, the suspension is filtered on a medium porosity funnel and the cake is washed with H ₂ O (2x). The solids were then dissolved in MeCN (50 ml) and lyophilized overnight to give the product as a white solid (780 mg, 87% yield). LCMS (ESI) m/z : [M+Na] calc. _{for C52H83NO13} : _952.58 ; found 952.4.

Step 3 : Synthesis of 32( R )-methoxy 40( R ) -O- (4-nitrophenyl)rapamycin carbonate

To a solution of 32( R )-methoxy rapamycin (4.50 g, 4.84 mmol, 1.0 equiv) in DCM (180 mL) add 4Å molecular sieves powder (6.0 g). The mixture was stirred at room temperature for 1 hour and then pyridine (3.91 mL, 48.4 mmol, 10 equiv.) was added. The mixture is cooled to -10°C and 4-nitrophenyl chloroformate (0.990 g, 4.91 mmol, 1.0 equiv.) is added in one portion. The reaction mixture was slowly warmed to room temperature and after 3 hours the reaction mixture was cooled to 0°C and 4-nitrophenyl chloroformate (250 mg, 1.24 mmol, 0.3 equiv.) was added. The mixture is warmed to room temperature and after 1 hour the reaction mixture is filtered through a pad of Celite and the layer is washed with DCM (140 ml). The filtrate is washed with H ₂ O (120 ml) and sat. NaHCO ₃ (2x120 ml). The organic phase is dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (20→50% EtOAc/hexane) to give a white stiff foam. The product is taken into MeCN, at which time a white solid is formed. The solid is filtered, washed with additional MeCN and air dried to give the product (4.51 g, 85% yield). LCMS (ESI) m/z [M+Na] calc. for C ₅₉ H ₈₆ N ₂ O ₁₇ : 1117.58; found 1117.6.

Monomers 4. 32( R )-ethoxy 40( R )- O -(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of 32( R )-ethoxy-28,40-bistriethylsilyl rapamycin

A solution of 32( R )-hydroxy-28,40-bistriethylsilyl rapamycin (773 mg, 0.675 mmol, 1.0 equiv.) in chloroform (19 ml) was treated with N, N, N', N' -tetramethyl-1,8- naphthalenediamine (1.85 g, 8.63 mmol, 12.8 equiv.) along with freshly dried 4Å molecular sieves. The mixture was stirred for 1 hour at room temperature and treated with triethyloxonium tetrafluoroborate (1.51 g, 7.95 mmol, 11.8 equiv.) in one portion at room temperature. The reaction mixture is stirred for 3 hours, at which point the reaction mixture is diluted with DCM and filtered through Celite, washing the filter layer with additional DCM. The combined filtrates are washed twice with 1M HCl, once with saturated NaHCO ₃ solution and dried over Na ₂ SO ₄ . The solution is filtered and concentrated to a residue. The crude residue is treated with MTBE and filtered to remove the polar insoluble product. The filtrate was concentrated and purified by silica gel chromatography (5→25% EtOAc/hex) to give the product as a foam (516 mg, 65% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₆₅ H ₁₁₃ NO ₁₃ Si ₂ 1194.77; found 1194.6.

Step 2 : Synthesis of 32( R )-ethoxy rapamycin

To a solution of 32( R )-ethoxyethoxy-28,40-bistriethylsilyl rapamycin (131 mg, 0.112 mmol, 1.0 equiv.) in THF (1.3 ml) at 0°C add pyridine (271 μl, 3.35 mmol , 3.4 equiv.), then 70% HF-pyridine (51 µl, 1.8 mmol, 1.8 equiv.). The reaction flask is capped and stored in the refrigerator for 3 days, at which point the reaction mixture is poured into cold saturated NaHCO ₃ (20 ml). The aqueous layer was extracted with EtOAc (3×20 mL) and the combined organic layers were washed with 1M HCl (2×20 mL), sat. NaHCO ₃ solution (20 mL) and brine. The solution is dried over Na ₂ SO ₄ , filtered and concentrated. The residue is taken up in MeOH (1.5 ml) and added dropwise to H ₂ O (20 ml). The solids are filtered and washed with additional H ₂ O to obtain the product (53 mg, 51% yield) as a white powder. LCMS (ESI) m/z : [M+Na] calc. for C ₅₃ H ₈₅ NO ₁₃ : 966.59; found 966.5.

Step 3 : Synthesis of 32( R )-ethoxy 40( R ) -O- (4-nitrophenyl)rapamycin carbonate

To a 0.03M solution of 32( R )-ethoxy rapamycin (1.0 equiv) in DCM, add 4Å molecular sieves powder. The mixture was stirred at room temperature for 1 hour and then pyridine (10 equiv.) was added. The mixture is cooled to -10°C and 4-nitrophenyl chloroformate (1.0 equiv.) is added in one portion. The reaction mixture is warmed to room temperature and stirred until 32( R )-ethoxy rapamycin is consumed, as determined by LCMS analysis. The mixture is filtered through a pad of Celite and the layer is washed with DCM. The filtrate is washed with H ₂ O and sat. _NaHCO3 . The organic phase is then dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (20→50% EtOAc/hex) to give the product.

Monomer 5.40( R )- O -(4-nitrophenyl)thiocarbonate rapamycin.

To a solution of rapamycin (4.00 g, 4.38 mmol, 1.0 equiv.) in DCM (20 ml) at -78°C add pyridine (4.0 ml, 49 mmol, 11.2 equiv.), then a solution of O- (4-nitrophenyl)chlorothiocarbonate (1.19 g, 5.47 mmol, 1.3 equiv.) in DCM (8.0 ml). The reaction mixture is heated to -20°C and stirred for 48 hours. Hexane (40 ml) is then added and the resulting suspension is purified by chromatography on silica gel (15/25/60 EtOAc/DCM/hexane then 20/25/55 EtOAc/DCM/ hexane) to give product (3.09 g, 64.4% yield) as an off-white solid. LCMS (ESI) m/z : [M+Na] calc. for C ₅₈ H ₈₂ N ₂ O ₁₆ S: 1117.53; found 1117.5.

Monomer 6. 28- O -(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of 40- O - tert- butyldimethylsilyl rapamycin

To a solution of rapamycin (1.00 g, 1.09 mmol, 1.0 equiv.) in DMF (4 ml) at room temperature, add imidazole (0.22 g, 3.2 mmol, 2.9 equiv.), then tert- butyldimethylsilyl chloride (0.176 g, 1.17 mmol, 1.07 equiv.). The reaction mixture was stirred for 18 hours. The reaction mixture was then diluted with DCM (100 ml) and washed with 20% aq. LiCl (3x20 ml). The organic layer is dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (20→40% EtOAc/hexanes) to give the product (950 mg, 75% yield) as a dull yellow glass. LCMS(ESI) m/z : [M+H ₂ O] calc. for C ₅₇ H ₉₃ NO ₁₃ Si: 1045.65; found 1045.9.

Step 2 : Synthesis of 28- O- (4-nitrophenoxycarbonyl)-40- O- ( tert- butyldimethylsilyl)rapamycin

To a solution of 40- O - tert- butyldimethylsilylrapamycin (0.845 g, 0.822 mmol, 1.0 equiv.) in DCM (10 ml) add pyridine (0.9 ml, 10 mmol, 12.1 equiv.) at room temperature, then 4-Nitrophenylchloroformate (0.373 g, 1.85 mmol, 2.25 equiv.). The reaction mixture was stirred for 2 hours. The reaction mixture was then diluted with DCM (150 ml) and the solution was washed successively with sat. NaHCO ₃ (20 ml), 10% citric acid (2x20 ml) and brine (20 ml). The organic layer is dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by reverse phase chromatography (30→100% MeCN/H ₂ O) to give the product (930 mg, 95% yield) as a pale yellow foam. LCMS(ESI) m/z : [M+H] calc. for C ₆₄ H ₉₆ N ₂ O ₁₇ Si: 1193.66; found 1193.7.

Step 3 : Synthesis of 28- O- (4-nitrophenoxycarbonyl)rapamycin

To a solution of 28- O- (4-nitrophenoxycarbonyl)-40-O-( tert- butyldimethylsilyl)rapamycin (0.930 g, 0.779 mmol, 1.0 equiv.) in THF (10.7 ml) add pyridine (3.78 ml , 46.8 mmol, 60.1 equiv.), then add 70% HF-pyridine (0.91 ml, 31.2 mmol, 40.0 equiv.) dropwise. The reaction mixture is stirred at room temperature for 48 hours. The mixture is then slowly poured into ice-cold brine. aqueous NaHCO ₃ (20 ml). The aqueous layer was extracted with EtOAc (3×20 ml) and the combined organic layer was washed with sat. NaHCO ₃ (10 ml) and brine (10 ml), dry over Na ₂ SO ₄ , filter and concentrate under reduced pressure. The residue was purified by reverse phase chromatography (30→100% MeCN/H ₂ O) to give the product (200 mg, 24% yield) as a dull yellow powder. LCMS(ESI) m/z : [M+Na] calc. for C ₅₈ H ₈₂ N ₂ O ₁₇ : 1101.55; found 1101.3.

Monomer 7. 32( R )-hydroxy 40( R )- O -(4-nitrophenyl)thiocarbonate rapamycin.

To a solution of 32( R )-hydroxy rapamycin (2.80 g, 3.06 mmol, 1.0 equiv.) in DCM (28 ml) add pyridine (27.6 ml, 34 mmol, 11 equiv.) and dried 4Å molecular sieves (2.8 g). The suspension is stirred at room temperature for 1 hour, at which point the mixture is cooled to -25°C and a solution of O -(4-nitrophenyl)chlorothioformate (0.798 g, 3.67 mmol, 1.2 equiv.) in DCM is added ( 6 ml). The reaction mixture is warmed to room temperature and after 21 hours filtered through Celite. The filtrate is partitioned between DCM and H ₂ O, and the aqueous layer is extracted with DCM. The combined organic layers are dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as a white solid (2.15 g, 64% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₈ H ₈₄ N ₂ O ₁₆ S: 1119.54; found 1120.0.

Monomer 8. 32( R )-methoxy 40( R )- O -(4-nitrophenyl)thiocarbonate rapamycin.

To a solution of 32( R )-methoxy rapamycin (6.69 g, 7.19 mmol, 1.0 equiv.) in DCM (67 ml) add pyridine (6.6 ml, 81 mmol, 11 equiv.) and dried 4Å molecular sieves (6.7 g). The suspension is stirred at room temperature for 1 hour, at which point the mixture is cooled to -25°C and a solution of O -(4-nitrophenyl)chlorothioformate (1.88 g, 8.63 mmol, 1.20 equiv.) in DCM is added (13 ml). The reaction mixture is warmed to room temperature and after 21 hours filtered through Celite. The filtrate is partitioned between DCM and H ₂ O, and the aqueous layer is extracted with DCM. The combined organic layers are dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as a white solid (5.1 g, 64% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₉ H ₈₆ N ₂ O ₁₆ S: 1133.56; found 1134.1.

Monomer 9. 32-deoxy 40( R )- O -(4-nitrophenyl)carbonate rapamycin.

To a solution of 32-deoxy rapamycin (0.623 g, 0.692 mmol, 1.0 equiv) in DCM (24.7 mL) add 4 Å molecular sieves (600 mg). The suspension was stirred for 1 hour and then pyridine (557 μl, 6.92 mmol, 10 equiv.) was added. The reaction mixture was cooled to 0°C and then O -(4-nitrophenyl)chloroformate (175 mg, 1.03 mmol, 1.7 equiv.) was added. The reaction mixture is warmed to room temperature and stirred for 2 hours, at which point the reaction mixture is concentrated under reduced pressure. Purification by silica gel chromatography (0→10% MeOH/DCM) gave the desired product as a white solid (0.61 g, 82% yield). LCMS (ESI) m/z : [M+Na] calc. for _{C58H84N2O16 : 1087.57 ;} found 1087.6.

Monomer 10. 32-deoxy 40( R )- O -(4-nitrophenyl)thiocarbonate rapamycin.

To a 0.2M solution of 32-deoxy rapamycin (1.0 equiv.) in DCM at -78°C add pyridine (11.2 equiv.), then 0.7 M solution of O -(4-nitrophenyl)chlorothiocarbonate (1.3 equiv.) in DXM. The reaction mixture is heated to -20°C and stirred until the starting material is consumed, according to LCMS analysis. Hexane is then added and the resulting suspension is purified by silica gel chromatography to give the product.

Monomer 11. 28( R )-methoxy 32( R )-hydroxy 40( R )-(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of 28( R )-methoxy 40( R ) -O - tert- butyldimethylsilyl rapamycin

To a solution of 40( R ) -O - tert- butyldimethylsilyl rapamycin (4.00 g, 4.89 mmol, 1.0 equiv.) in chloroform (67 ml) add a proton sponge (11.2 ml, 52.3 mmol, 13 equiv.) and dried 4 Å molecular sieves (5.8 g). The suspension was stirred at room temperature for 1 hour, at which point trimethyloxonium tetrafluoroborate (7.21 g, 48.8 mmol, 12.5 equiv.) was added. After 4 hours, the suspension is filtered through Celite. The filtrate is washed successively with aqueous 2 N HCl, H ₂ O, sat. aqueous NaHCO ₃ solution, then dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as a white solid (2.1 g, 52% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₈ H ₉₅ NO ₁₃ Si: 1064.65; found 1065.26.

Step 2 : Synthesis of 28( R )-methoxy 32( R )-hydroxy 40( R ) -O - tert- butyldimethylsilyl rapamycin

To a solution of 28( R )-methoxy 40( R ) -O - tert- butyldimethylsilyl rapamycin (2.13 g, 2.04 mmol, 1.0 equiv.) in THF (31 ml) at -20°C add a solution of three - lithium tert- butoxyaluminum hydride in THF (1 M, 4.09 ml, 4.09 mmol, 2.0 equiv.) dropwise. The reaction mixture is warmed to room temperature and after 3 hours added to a solution of H ₂ O (4 ml), EtOAc (31 ml) and 2M aqueous citric acid (4 ml) at 0°C. After 5 minutes, the mixture was separated and the aqueous layer was extracted with EtOAc. The combined organic layers are poured into sat. aqueous solution of NaHCO ₃ (60 ml) at 0°C. The layers are separated, and the organic layer is dried and concentrated under reduced pressure to obtain a crude white solid (2.32 g). The crude solid was dissolved in DCM (12 ml) and then pyridine (241 µl, 2.98 mmol, 1.5 equiv), dried 4Å molecular sieves (2.1 g) and copper acetate (0.27 g, 1 .49 mmol, 0.7 equiv.). The suspension is stirred at room temperature for 1 hour. The suspension is purged with O ₂ and then stored in an O ₂ atmosphere for 30 minutes. After 2 hours, the mixture is filtered through Celite and the filtrate is concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as a white solid (307 mg, 14% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₈ H ₉₇ NO ₁₃ Si: 1066.66; found 1067.0.

Step 3 : Synthesis of 28( R )-methoxy 32( R )-hydroxy rapamycin

To a solution of 28( R )-methoxy 32( R )-hydroxy 40( R )- O - tert- butyldimethylsilyl rapamycin (0.307 g, 0.294 mmol, 1.0 equiv.) in THF (4 ml) in a polypropylene vial at 0° C add pyridine (1.42 mL, 17.6 mmol, 60.0 equiv.), then 70% HF-pyridine (0.34 mL, 11.7 mmol, 40 equiv.). The solution is warmed to room temperature and stirred for 21 hours, at which point the solution is poured into sat. aqueous solution of NaHCO ₃ at 0°C. The aqueous layer is extracted with EtOAc (2x) and the combined organic layers are washed with sat. aqueous NaHCO ₃ solution and brine, then dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as a white solid (146 mg, 53% yield). LCMS (ESI) m/z : [M+Na] calc. _{for C52H83NO13} : _952.58 ; found 952.8.

Step 4 : Synthesis of rapamycin 28( R )-methoxy 32( R )-hydroxy 40( R )-(4-nitrophenyl)carbonate

To a solution of 28( R )-methoxy 32( R )-hydroxy rapamycin (0.66 g, 0.71 mmol, 1.0 equiv.) in DCM (3 ml) add pyridine (0.64 ml, 7.9 mmol , 11 equiv.) and dried 4 Å molecular sieves (0.66 g). The suspension was stirred at room temperature for 1 hour, at which point the mixture was cooled to -35°C and O -(4-nitrophenyl)chloroformate (0.17 g, 0.85 mmol, 1.2 equiv.) was added. After 3 hours, DCM (5 ml) is added and the suspension is filtered through Celite. The filtrate is washed with H ₂ O, dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as a white solid (0.44 g, 57% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₉ H ₈₆ N ₂ O ₁₇ : 1117.58; found 1118.0.

Monomer 12.28( R )-methoxy 32( R )-methoxy 40( R )-(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of 28( R )-methoxy 32( R )-methoxy 40( R ) -O - tert- butyldimethylsilyl rapamycin

To a solution of 28( R )-methoxy 32( R )-hydroxy 40( R )- O - tert- butyldimethylsilyl rapamycin (1.15 g, 1.10 mmol, 1.0 equiv.) in chloroform (19 ml) add protic acid sponge (3.22 ml, 15.0 mmol, 14 equiv.) and dried 4Å molecular sieves (2.3 g). The suspension was stirred at room temperature for 1 hour, at which point trimethyloxonium tetrafluoroborate (2.07 g, 14.0 mmol, 12.7 equiv.) was added. After 4 hours, the suspension is filtered through Celite and the filtrate is washed with 1N HCl, H ₂ O and sat. aqueous NaHCO ₃ solution, dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gives the desired product as a white solid. LCMS (ESI) m/z : [M+Na] calc. for C ₅₉ H ₉₉ NO ₁₃ Si: 1080.68; found 1081.2.

Step 2 : Synthesis of 28( R )-methoxy 32( R )-methoxy rapamycin

To a solution of 28( R )-methoxy 32( R )-methoxy 40( R )- O - tert- butyldimethylsilyl rapamycin in THF (4 ml) in a polypropylene vial at 0°C add pyridine (1.13 ml, 14.2 mmol , 12.9 equiv.), then 70% HF-pyridine (0.27 ml, 9.42 mmol, 8.6 equiv.). The solution is warmed to room temperature and stirred for 41 hours, at which point the solution is poured into sat. aqueous solution of NaHCO ₃ at 0°C. The aqueous layer is extracted with EtOAc (2x) and the combined organic layers are washed with sat. aqueous NaHCO ₃ solution and brine, then dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as a white solid (516 mg, 49% yield 2-step). LCMS (ESI) m/z : [M+Na] calc. for C ₅₃ H ₈₅ NO ₁₃ : 966.59; found 967.0.

Step 3 : Synthesis of 28( R )-methoxy 32( R )-methoxy 40( R )-(4-nitrophenyl)rapamycin carbonate

To a solution of 28( R )-methoxy 32( R )-methoxy rapamycin (0.30 g, 0.32 mmol, 1.0 equiv.) in DCM (1.4 ml) add pyridine (0.29 ml, 3. 5 mmol, 11 equiv.) and dried 4 Å molecular sieves (0.30 g). The suspension was stirred at room temperature for 1 hour, at which point it was cooled to -35°C and O -(4-nitrophenyl)chloroformate (0.08 g, 0.38 mmol, 1.2 equiv.) was added. After 3 hours, DCM (2 ml) is added and the suspension is filtered through Celite. The filtrate is washed with H ₂ O, dried and concentrated under reduced pressure. Purification by silica gel chromatography (EtOAc/hexane) gave the desired product as an off-white solid (0.20 g, 57% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₆₀ H ₈₈ N ₂ O ₁₇ : 1131.60; found 1132.1.

Monomer 13. 32( R )-(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of rapamycin 28,40- O -bis(triethylsilyl)32( R )-(4-nitrophenyl)carbonate

To a solution of 28,40- O -bis(triethylsilyl) 32( R )-hydroxy rapamycin (0.602 g, 0.526 mmol, 1.0 equiv.) in DCM (16 ml) at -20°C add pyridine (0.82 ml , 10 mmol, 19 equiv.), then O- (4-nitrophenyl)chloroformate (0.36 g, 1.8 mmol, 3.4 equiv.). The reaction mixture is warmed to room temperature and stirred for 1 hour, at which point the solution is diluted with DCM (50 ml) and poured into H ₂ O (30 ml). The aqueous layer was extracted with DCM (50 ml) and the combined organic layers were washed with brine (20 ml), dried and concentrated under reduced pressure to obtain a dull yellow foam, which was used immediately in the next step.

Step 2 : Synthesis of rapamycin 32( R )-(4-nitrophenyl)carbonate

To a solution of rapamycin 28,40- O -bis(triethylsilyl)32( R )-(4-nitrophenyl)carbonate in THF (10 ml) in a polypropylene vial at 0°C add pyridine (1.70 ml, 21.0 mmol, 40.0 equiv.), then 70% HF-pyridine (0.46 ml, 15.8 mmol, 30.0 equiv.). The solution is warmed to room temperature and stirred overnight, at which point the solution is poured into sat. aqueous solution of NaHCO ₃ at 0°C. The aqueous layer is extracted with EtOAc (3x) and the combined organic layers are washed with sat. aqueous NaHCO ₃ solution and brine, then dried and concentrated under reduced pressure. Purification by reverse phase chromatography (20→100% MeCN/H ₂ O) gave the desired product as an off-white powder (420 mg, 74% 2-step yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₈ H ₈₄ N ₂ O ₁₇ : 1103.57; found 1104.0.

Monomer 14. 32( S )-azido 40-(4-nitrophenyl)carbonate rapamycin.

Step 1 : Synthesis of 28,40- O -bis(triethylsilyl)32( R ) -O -methanesulfonyl rapamycin

To a solution of 28,40- O -bis(triethylsilyl)32( R )-hydroxy rapamycin (2.50 g, 2.18 mmol, 1.0 equiv.) in DCM (25 ml) at 0°C add Et ₃ N (0.912 ml, 6.54 mmol, 3.0 equiv.), then methanesulfonyl chloride (0.338 ml, 4.36 mmol, 2.0 equiv.). The solution is stirred at 0°C for 3 hours, at which point EtOAc is added and the solution is washed with sat. aqueous NaHCO ₃ solution. The combined organic layers were washed with brine, dried and concentrated under reduced pressure to give a yellow oil, which was used immediately in the next step.

Step 2 : Synthesis of 28- O -triethylsilyl 32( S )-azido rapamycin

To a solution of 28,40 -O -bis(triethylsilyl)32( R ) -O -methanesulfonyl rapamycin in THF (40 ml) add DIPEA (0.761 ml, 4.37 mmol, 2.0 equiv.) and tetrabutylammonium azide (3, 72 g, 13.1 mmol, 6.0 equiv.). The reaction solution is heated to reflux for 5.5 hours and then cooled to room temperature. The solution is diluted with EtOAc and washed with sat. aqueous NaHCO ₃ solution. The combined organic layers are washed with brine, dried and concentrated under reduced pressure. Purification by reverse phase chromatography (30→100% MeCN/H ₂ O) gave the desired product as a clear glass (746 mg, 33% yield 2-step). LCMS (ESI) m/z : [M+Na] calc. for C ₅₇ H ₉₄ N ₄ O ₁₂ Si: 1077.65; found 1077.8.

Step 3 : Synthesis of rapamycin 28- O -triethylsilyl 32( S )-azido 40( R )-(4-nitrophenyl)carbonate

To a solution of 28- O -triethylsilyl 32( S )-azido rapamycin (0.505 g, 0.478 mmol, 1.0 equiv.) in DCM (15 ml) add pyridine (0.75 ml, 9.3 mmol, 19 equiv.) and 4 Å molecular sieves. The suspension is cooled to -20°C and O -(4-nitrophenyl)chloroformate (0.32 g, 1.6 mmol, 3.4 equiv.) is added. The suspension is stirred at -20°C for 2 hours, at which point it is diluted with DCM (50 ml), filtered and poured into H ₂ O (20 ml). The aqueous layer was extracted with DCM (50 ml) and the combined organic layers were washed with brine (20 ml), dried and concentrated under reduced pressure to give a pale yellow foam, which was used immediately in the next step.

Step 4 : Synthesis of rapamycin 32( S )-azido 40- O- (4-nitrophenyl)carbonate

To a solution of rapamycin 28- O -triethylsilyl 32( S )-azido 40( R )-(4-nitrophenyl)carbonate in THF (10 ml) in a polypropylene vial at 0°C add pyridine (1.55 ml, 19.1 mmol , 40.0 equiv.) then 70% HF-pyridine (0.42 ml, 14.4 mmol; 30.0 equiv.). The solution is warmed to room temperature and stirred overnight, at which point the solution is poured into sat. aqueous solution of NaHCO ₃ at 0°C. The aqueous layer is extracted with EtOAc (3x) and the combined organic layers are washed with sat. aqueous NaHCO ₃ solution and brine, then dried and concentrated under reduced pressure. Purification by reverse phase chromatography (30→100% MeCN/H ₂ O) gave the desired product as an off-white powder (410 mg, 77% 2-step yield). LCMS (ESI) m/z : [M+Na] calc. _{for C58H83N5O16 : 1128.57} ; found 1129.0.

Monomer 15. 28-methoxy-40- O -(4-nitrophenoxy)carbonyl rapamycin.

Step 1 : Synthesis of 28-methoxy rapamycin

H ₂ SO ₄ ( 1 .28 µl, 0.024 mmol, 0.05 equiv.). The reaction mixture is stirred at -20°C for 48 hours. The reaction mixture is then poured into sat. aqueous solution of NaHCO ₃ (4 ml)/H ₂ O (4 ml). The aqueous layer is extracted with MTBE (2×6 ml) and the combined organic phases are dried, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography (30→100%MeCN/H ₂ O) gave the desired product as a yellow powder (270 mg, 61% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₂ H ₈₁ NO ₁₃ : 950.5; found 950.7.

Step 2 : Synthesis of 28-methoxy-40- O- (4-nitrophenoxy)carbonyl rapamycin

To a solution of 28-methoxy rapamycin (0.210 g, 0.226 mmol, 1.0 equiv.) in DCM (7.1 ml) at -20°C add pyridine (0.35 ml, 4.4 mmol, 19 equiv.) and then p -nitrophenyl chloroformate (0.15 g, 0.76 mmol, 3.4 equiv.). The reaction mixture is stirred at -20°C for 30 minutes and then warmed to room temperature. After stirring overnight, p -nitrophenyl chloroformate (0.15 g, 0.76 mmol, 3.4 equiv.) was added and the reaction mixture was stirred for an additional 2 hours. The reaction mixture was diluted with DCM (20 ml) and poured into H ₂ O (10 ml). The aqueous layer was extracted with DCM (20 ml) and the combined organic layers were washed with brine (9 ml), dried, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography (50→100%MeCN/H ₂ O) gave the desired product as a pale yellow powder (200 mg, 81% yield). LCMS (ESI) m/z : [M+Na] calc. for C ₅₉ H ₈₄ N ₂ O ₁₇ : 1115.6; found 1115.8.

Monomer 16. 32( R ),40- O, O -bis[(4-nitrophenoxy)carbonyl]rapamycin.

To a solution of 32( R ) -O -[(4-nitrophenoxy)carbonyl]rapamycin (675 mg, 0.624 mmol, 1.0 equiv.) in DCM (13 mL) add 4Å molecular sieves powder (675 mg). The suspension was stirred for 1 hour, at which point pyridine (0.56 mL, 6.90 mmol, 11.1 equiv.) was added. The mixture is cooled to -15°C and then p -nitrophenyl chloroformate (132 mg, 0.655 mmol, 1.05 equiv.) is added in one portion. The mixture is warmed to 0°C, stirred for 4 hours and then warmed to room temperature. The reaction mixture is filtered and washed with DCM (25 ml). The filtrate is washed with sat. aqueous NaHCO ₃ (15 ml), H ₂ O (15 ml) and brine (10 ml), dry, filter and concentrate under reduced pressure. Purification by silica gel chromatography (25→45% EtOAc/hexane) gave the desired product as a dull yellow solid (566 mg, 73% yield). LC-MS (ESI) m/z : [M+Na] calc. for C ₆₅ H ₈₇ N ₃ O ₂₁ : 1268.57; found 1269.3.

Monomer 17. 28( R ),32( R ),40( R )- O, O , O -tris[(4-nitrophenoxy)carbonyl]rapamycin.

To a solution of 32( R )-hydroxy rapamycin (1.00 g, 1.09 mmol, 1.0 equiv) in DCM (22 mL) add 4Å molecular sieves powder (1.0 g). The suspension is stirred for 45 minutes, at which point pyridine (0.97 mL, 12.0 mmol, 11.0 equiv.) is added. The mixture is cooled to -15°C and then p -nitrophenyl chloroformate (550 mg, 2.73 mmol, 2.5 equiv.) is added in one portion. The mixture is warmed to room temperature over 4 hours and stirred overnight. The mixture is cooled to 0°C and additional p -nitrophenyl chloroformate (220 mg, 1.09 mmol, 1.0 equiv.) is added in one portion. The reaction mixture was stirred for 1 hour, warmed to room temperature and then stirred for 2 hours. The mixture was again cooled to 0°C and additional p -nitrophenyl chloroformate (660 mg, 3.27 mmol, 3.0 equiv.) was added. The reaction mixture was stirred for 15 minutes and then at room temperature for 1 hour. The reaction mixture was filtered and washed with DCM (25 ml). The filtrate is washed with sat. aqueous NaHCO ₃ (20 ml), H ₂ O (20 ml) and brine (15 ml), dry, filter and concentrate under reduced pressure. Purification by silica gel chromatography (5→15% EtOAc/DCM) gave the desired product as a dull yellow solid (550 mg, 36% yield). DH-MS (ESI) m/z : [M+Na] calc. for C ₇₂ H ₉₀ N ₄ O ₂₅ : 1433.58; found 1434.3.

General techniques and specific examples

General Method 1: Combination of carboxylic acid and amine, then deprotection N -Boc.

Stage 1 :

To a 0.1 M solution of carboxylic acid (1.0 equiv.) in DMA, add amine (1.2 equiv.), DIPEA (4.0 equiv.), and PyBOP (1.3 equiv.). The reaction mixture is stirred until the carboxylic acid is consumed, according to LCMS data. The reaction mixture is then purified by chromatography on silica gel to obtain the product.

Stage 2 :

To a 0.07M solution of N -Boc protected amine (1.0 equiv) in dioxane add HCl (4M in dioxane) (50 equiv). The reaction mixture is stirred until the N -Boc protected amine is consumed, as determined by LCMS. The reaction mixture is then concentrated to an oil, which is then dissolved in H ₂ O and lyophilized to give the product.

Intermediate A1-7. 1-amino-27-(6-{[4-amino-3-(2-amino-1,3-benzoxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]methyl }-1,2,3,4-tetrahydroisoquinolin-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-one

Step 1 : Synthesis of tert- butyl N-[27-(6-{[4-amino-3-(2-amino-1,3-benzoxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidine -1-yl]methyl}-1,2,3,4-tetrahydroisoquinolin-2-yl)-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl]carbamatecarbamate

To a solution of 1-{[( tert- butoxy)carbonyl]amino}-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid (102 mg, 189 µmol, 1.0 equiv. ) and 6-{[4-amino-3-(2-amino-1,3-benzoxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]methyl}-1,2 ,3,4-tetrahydroisoquinolin-2-ium (120 mg, 227 µmol, 1.2 equiv.) in DMA (1.88 ml) add DIPEA (131 µl, 756 µmol, 4.0 equiv.), then PyBOP ( 127 mg, 245 µmol, 1.3 equiv.). The reaction mixture is stirred at room temperature. After 2 hours, the reaction mixture was concentrated under reduced pressure and the crude residue was purified by silica gel chromatography (0→20% MeOH/DCM) to give the product (161.5 mg, 91% yield) as a pale yellow oil. LCMS (ESI) m/z : [M+H] calc. for C ₄₆ H ₆₅ N ₉ O ₁₂ : 936.49; found 936.3.

Step 2 : Synthesis of 1-amino-27-(6-{[4-amino-3-(2-amino-1,3-benzoxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl]methyl}-1,2,3,4-tetrahydroisoquinolin-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-one

To a solution of tert- butyl N-[27-(6-{[4-amino-3-(2-amino-1,3-benzoxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1 -yl]methyl}-1,2,3,4-tetrahydroisoquinolin-2-yl)-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl]carbamate (0 .9 g, 0.9614 mmol, 1.0 equiv) in dioxane (3.20 ml) add HCl (4M in dioxane, 2.40 ml, 9.61 mmol, 10.0 equiv). The reaction mixture is stirred for 2 hours and then concentrated under reduced pressure to an oil. The oil was azeotroped with DCM (3 x 15 mL) to give the product (881 mg, 105% yield, HCl) as a tan solid, which was used immediately in the next step. LCMS (ESI) m/z : [M+H] calc. for C ₄₁ H ₅₇ N ₉ O ₁₀ : 836.43; found 836.3.

Using the general procedure 1, but using the appropriate amine active site inhibitor in Table 2 and a PEG carboxylic acid, intermediates A1 in Table 5 are prepared:

Table 5. Additional amines obtained

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection A1-1 C ₃₅ H ₅₅ N ₉ O ₁₀ [M+H] = 762.42 [M+H] = 762.3
Intermediate connection A1-2 C ₃₁ H ₄₇ N ₉ O ₈ [M+H] = 674.36 [M+H] = 674.3
Intermediate connection A1-3 _{C27H39N9O6 _ _ _} [M+H] = 586.31 [M+H] = 586.6
Intermediate connection A1-4 _{C25H35N9O5 _ _ _} [M+H] = 542.29 [M+H] = 542.3
Intermediate connection A1-5 _{C23H31N9O4 _ _ _} [M+H] = 498.26 [M+H] = 498.2
Intermediate connection A1-6 _{C21H27N9O3 _ _ _} [M+H] = 454.23 [M+H] = 454.1
Intermediate A1-7 C ₄₁ H ₅₇ N ₉ O ₁₀ [M+H] = 836.43 [M+H] = 836.3
Intermediate connection A1-8 C ₃₇ H ₄₉ N ₉ O ₈ [M+H] = 748.38 [M+H] = 748.2
Intermediate connection A1-9 C ₃₃ H ₄₁ N ₉ O ₆ [M+H] = 660.33 [M+H] = 660.2
Intermediate connection A1-10 C ₃₁ H ₃₇ N ₉ O ₅ [M+H] = 616.30 [M+H] = 616.3
Intermediate connection A1-11 _{C29H33N9O4 _ _ _} [M+H] = 572.28 [M+H] = 572.3
Intermediate connection A1-12 _{C27H29N9O3 _ _ _} [M+H] = 528.25 [M+H] = 528.2
Intermediate connection A1-13 C ₃₅ H ₅₅ N ₉ O ₉ [M+H] = 746.42 [M+H] = 746.4
Intermediate connection A1-14 C ₃₁ H ₄₇ N ₉ O ₇ [M+H] = 658.37 [M+H] = 658.3
Intermediate connection A1-15 _{C27H39N9O5 _ _ _} [M+H] = 570.32 [M+H] = 570.2
Intermediate connection A1-16 _{C23H31N9O3 _ _ _} [M+H] = 482.26 [M+H] = 482.3
Intermediate connection A1-17 _{C21H27N9O2 _ _ _} [M+H] = 438.24 [M+H] = 438.4
Intermediate connection A1-18 C ₄₉ H ₇₃ N ₉ O ₁₃ [M+H] = 996.54 [M+H] = 996.4
Intermediate connection A1-19 C ₄₅ H ₆₅ N ₉ O ₁₁ [M+H] = 908.49 [M+H] = 908.3
Intermediate connection A1-20 C ₄₁ H ₅₇ N ₉ O ₉ [M+H] = 820.44 [M+H] = 820.3
Intermediate connection A1-21 C ₃₇ H ₄₉ N ₉ O ₇ [M+H] = 732.39 [M+H] = 732.3
Intermediate connection A1-22 C ₃₅ H ₄₅ N ₉ O ₆ [M+H] = 688.36 [M+H] = 688.3
Intermediate connection A1-23 C ₃₃ H ₄₁ N ₉ O ₅ [M+H] = 644.33 [M+H] = 644.3
Intermediate connection A1-24 C ₃₁ H ₃₇ N ₉ O ₄ [M+H] = 600.31 [M+H] = 600.4
Intermediate connection A1-25 C ₃₆ H ₅₅ N ₉ O ₁₀ [M+H] = 774.42 [M+H] = 774.7
Intermediate connection A1-26 C ₃₂ H ₄₇ N ₉ O ₈ [M+H] = 686.36 [M+H] = 686.4
Intermediate connection A1-27 C ₄₀ H ₆₃ N ₉ O ₁₁ [M+H] = 846.47 [M+H] = 846.8
Intermediate connection A1-28 C ₄₃ H ₅₄ F ₃ N ₇ O ₉ [M+H] = 870.40 [M+H] = 870.4
Intermediate connection A1-29 C ₅₂ H ₆₇ F ₃ N ₁₀ O ₁₁ [M+H] = 1065.50 [M+H] = 1065.4
Intermediate connection A1-30 C ₄₈ H ₅₉ F ₃ N ₁₀ O ₉ [M+H] = 977.45 [M+H] = 977.4
Intermediate connection A1-31 C ₃₉ H ₅₇ F ₃ N ₁₂ O ₁₂ [M+H] = 853.43 [M+H] = 853.4
Intermediate connection A1-32 C ₄₀ H ₅₅ N ₉ O ₁₀ [M+H] = 822.42 [M+H] = 822.2
Intermediate connection A1-33 C ₃₉ H ₅₅ N ₉ O ₁₀ [M+H] = 810.42 [M+H] = 810.3
Intermediate connection A1-34 C ₄₃ H ₆₃ N ₅ O ₁₃ S [M+H] = 890.42 [M+H] = 890.3
Intermediate connection A1-35 C ₃₉ H ₅₄ FN ₅ O ₁₁ S [M+H] = 820.36 [M+H] = 820.3
Intermediate connection A1-36 C ₄₃ H ₆₂ FN ₅ O ₁₃ S [M+H] = 908.41 [M+H] = 908.3
Intermediate A1-37 C ₄₇ H ₆₂ F ₃ N ₇ O ₁₁ [M+H] = 958.46 [M+H] = 958.3
Intermediate connection A1-38 C ₃₄ H ₄₃ N ₉ O ₆ [M+H] = 674.34 [M+H] = 674.3
Intermediate connection A1-39 C ₄₁ H ₆₄ N ₈ O ₁₁ [M+H] = 845.48 [M+H] = 845.3
Intermediate connection A1-40 C ₃₇ H ₅₆ N ₈ O ₉ [M+H] = 757.43 [M+H] = 757.3
Intermediate connection A1-41 _{C27H36N8O4 _ _ _} [M+H] = 537.29 [M+H] = 537.2
Intermediate connection A1-42 C ₃₇ H ₅₃ N ₁₁ O ₁₀ [M+H] = 812.41 [M+H] = 812.3
Intermediate connection A1-43 C ₃₆ H ₅₆ N ₈ O ₁₀ [M+H] = 761.42 [M+H] = 761.3
Intermediate connection A1-44 C ₄₃ H ₆₁ N ₉ O ₁₀ [M+H] =864.46 [M+H] = 864.4
Intermediate connection A1-45 C ₄₃ H ₆₂ N ₁₀ O ₁₂ S [M+H] = 943.44 [M+H] = 943.3
Intermediate connection A1-46 C ₃₉ H ₅₅ N ₁₁ O ₁₀ [M+H] = 838.42 [M+H] = 838.3

Following general procedure 1, but substituting the appropriate Intermediate A1 in Table 5 and PEG carboxylic acid, Intermediates A2 in Table 6 are obtained:

Table 6. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection A2-1 C ₃₈ H ₆₀ N ₁₀ O ₁₁ [M+H] = 833.45 [M+H] = 833.8
Intermediate connection A2-2 C ₄₄ H ₆₂ N ₁₀ O ₁₁ [M+H] = 907.47 [M+H] = 908.0
Intermediate connection A2-3 C ₃₈ H ₆₀ N ₁₀ O ₁₀ [M+H] = 817.46 [M+H] = 817.4
Intermediate connection A2-4 C ₄₈ H ₇₀ N ₁₀ O ₁₂ [M+H] = 979.53 [M+H] = 979.9
Intermediate connection A2-5 C ₄₆ H ₆₆ N ₁₀ O ₁₁ [M+H] = 935.50 [M+H] = 935.3
Intermediate connection A2-6 C ₄₀ H ₆₁ N ₉ O ₁₀ [M+H] = 828.46 [M+H] = 828.3

General Procedure 2: Combination of a 4-nitrophenyl carbonate containing rapamycin monomer and an active site inhibitor containing a primary or secondary amine intermediate.

DIPEA (4.0 equiv.) is added to a 0.02M solution of rapamycin 4-nitrophenyl carbonate monomer (1.0 equiv.) and an active site inhibitor containing intermediate (2.0 equiv.) in DMA. The resulting solution is stirred at room temperature under nitrogen. After completion of LCMS analysis, the crude reaction mixture was purified by preparative HPLC to obtain the product.

Example 2: Synthesis of row 1 divalent compound rapamycin.

To a solution of 40( R ) -O- (4-nitrophenylcarbonate)rapamycin (25 mg, 23.16 µmol , 1.0 equiv. ) and intermediate A1-7 (42.0 mg, 46.32 µmol , 2.0 equiv. ) in DMA (1.15 ml) add DIPEA (16.0 µl, 92.64 µmol , 4.0 equiv. ). The reaction mixture was stirred for 18 hours, at which point the reaction mixture was purified by reverse phase chromatography (10→40→95% MeCN+0.1% formic acid/H ₂ O+0.1% formic acid) to give product (9 .92 mg, 24% yield) as a white solid. LCMS (ESI) m/z : [M+H] calc. for C ₉₃ H ₁₃₄ N ₁₀ O ₂₄ : 1775.97; found 1775.7.

Following the general procedure 2, but using the appropriate 4-nitrophenyl carbonate-containing rapamycin monomer in Table 1 and Intermediates A1 and A2 from Tables 5 and 6, Row 1 of the divalent analogues in Table 7 is synthesized:

Table 7. Row 1 divalent compounds:

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 1 C ₈₇ H ₁₃₂ N ₁₀ O ₂₄ [M+H] = 1701.95 [M+H] = 1701.8
Example 2 C ₉₃ H ₁₃₄ N ₁₀ O ₂₄ [M+H] = 1775.97 [M+H] = 1775.7
Example 3 C ₈₉ H ₁₂₆ N ₁₀ O ₂₂ [M+H] = 1687.91 [M+H] = 1687.8
Example 4 C ₈₇ H ₁₃₂ N ₁₀ O ₂₃ [M+H] = 1684.95 [M+H] = 1685.1
Example 5 C ₁₀₁ H ₁₅₀ N ₁₀ O ₂₇ [M+H] = 1936.08 [M+H] = 1936.4
Example 6 C ₉₇ H ₁₄₂ N ₁₀ O ₂₅ [M+H] = 1848.02 [M+H] = 1848.3
Example 7 C ₉₃ H ₁₃₄ N ₁₀ O ₂₃ [M+H] = 1759.97 [M+H] = 1760.1
Example 8 C ₈₇ H ₁₃₄ N ₁₀ O ₂₄ [M+H] = 1703.97 [M+H] = 1703.7
Example 9 C ₉₃ H ₁₃₆ N ₁₀ O ₂₄ [M+H] = 1777.98 [M+H] = 1777.7
Example 10 C ₉₃ H ₁₃₆ N ₁₀ O ₂₃ [M+H] = 1761.99 [M+H] = 1761.7
Example 11 C ₈₈ H ₁₃₆ N ₁₀ O ₂₄ [M+H] = 1717.98 [M+H] = 1718.0
Example 12 C ₉₄ H ₁₃₈ N ₁₀ O ₂₄ [M+H] = 1792.00 [M+H] = 1791.9
Example 13 C ₉₁ H ₁₃₃ N ₁₃ O ₂₄ [M+H] = 1792.97 [M+H] = 1793.0
Example 14 C ₉₀ H ₁₃₇ N ₁₁ O ₂₅ [M+H] = 1772.99 [M+H] = 1772.9
Example 15 C ₉₆ H ₁₃₉ N ₁₁ O ₂₅ [M+H] = 1847.00 [M+H] = 1847.0
Example 16 C ₉₆ H ₁₄₁ N ₁₁ O ₂₅ [M+H] = 1849.02 [M+H] = 1848.9
Example 17 C ₉₇ H ₁₄₃ N ₁₁ O ₂₅ [M+H] = 1863.04 [M+H] = 1863.0
Example 18 C ₉₀ H ₁₃₇ N ₁₁ O ₂₄ [M+H] = 1756.99 [M+H] = 1756.8
Example 19 C ₈₈ H ₁₃₂ N ₁₀ O ₂₄ [M+H] = 1713.95 [M+H] = 1713.9
Example 20 C ₉₂ H ₁₄₀ N ₁₀ O ₂₅ [M+H] = 1786.01 [M+H] = 1786.0
Example 21 C ₁₀₀ H ₁₄₇ N ₁₁ O ₂₆ [M+H] = 1919.06 [M+H] = 1919.0
Example 22 C ₉₃ H ₁₃₄ N ₁₀ O ₂₃ S [M+H] = 1791.94 [M+H] = 1791.8
Example 23 C ₉₃ H ₁₃₄ N ₁₀ O ₂₄ [M+H] = 1775.97 [M+H] = 1775.9
Example 24 C ₁₀₄ H ₁₄₄ F ₃ N ₁₁ O ₂₅ [M+2H]/2=1003.03 [M+2H]/2=1003.5
Example 25 C ₁₀₄ H ₁₄₆ F ₃ N ₁₁ O ₂₅ [M+2H]/2=1004.03 [M+2H]/2=1004.5
Example 26 C ₁₀₅ H ₁₄₈ F ₃ N ₁₁ O ₂₅ [M+2H]/2=1011.04 [M+2H]/2=1011.5
Example 71 C ₉₂ H ₁₃₂ N ₁₀ O ₂₄ [M+H] = 1761.95 [M+H] = 1761.8
Example 72 C ₉₂ H ₁₃₄ N ₁₀ O ₂₄ [M+H] = 1763.97 [M+H] = 1763.8
Example 73 C ₉₃ H ₁₃₆ N ₁₀ O ₂₃ [M+H] = 1761.99 [M+H] = 1762.0
Example 74 C ₉₃ H ₁₃₅ N ₁₃ O ₂₃ [M+H] = 1802.99 [M+H] = 1802.9
Example 75 C ₉₄ H ₁₃₈ N ₁₀ O ₂₄ [M+H] = 1792.00 [M+H] = 1791.7
Example 76 C ₉₅ H ₁₄₀ N ₁₀ O ₂₄ [M+H] = 1806.01 [M+H] = 1805.8
Example 77 C ₉₃ H ₁₃₆ N ₁₀ O ₂₃ S [M+H] = 1793.96 [M+H] = 1793.9
Example 78 C ₉₄ H ₁₃₈ N ₁₀ O ₂₃ S [M+H] = 1807.97 [M+H] = 1807.9
Example 79 C ₉₁ H ₁₃₂ N ₁₀ O ₂₄ [M+H] = 1749.95 [M+H] = 1749.9
Example 80 C ₉₁ H ₁₃₄ N ₁₀ O ₂₄ [M+H] = 1751.97 [M+H] = 1751.9
Example 81 C ₉₂ H ₁₃₆ N ₁₀ O ₂₄ [M+H] = 1765.98 [M+H] = 1765.8
Example 82 C ₉₁ H ₁₃₃ N ₁₃ O ₂₃ [M+H] = 1776.97 [M+H] = 1776.9
Example 83 C ₉₁ H ₁₃₃ N ₁₀ O ₂₃ S [M+H] = 1766.93 [M+H] = 1766.8
Example 84 C ₉₅ H ₁₄₀ N ₆ O ₂₇ S [M+H] = 1829.96 [M+H] = 1830.0
Example 85 C ₉₅ H ₁₄₂ N ₆ O ₂₇ S [M+H] = 1831.97 [M+H] = 1831.9
Example 86 C ₉₆ H ₁₄₄ N ₆ O ₂₇ S [M+H] = 1845.99 [M+H] = 1846.0
Example 87 C ₉₅ H ₁₄₂ N ₆ O ₂₆ S [M+H] = 1815.98 [M+H] = 1815.8
Example 88 C ₉₅ H ₁₄₁ N ₉ O ₂₆ S [M+H] = 1856.98 [M+H] = 1856.8
Example 89 C ₉₅ H ₁₄₀ N ₆ O ₂₆ S ₂ [M+H] = 1845.93 [M+H] = 1846.0
Example 90 C ₉₅ H ₁₄₂ N ₆ O ₂₆ S ₂ [M+H] = 1847.95 [M+H] = 1847.9
Example 91 C ₉₆ H ₁₄₄ N ₆ O ₂₆ S ₂ [M+H] = 1861.96 [M+H] = 1861.7
Example 92 C ₉₅ H ₁₃₉ FN ₆ O ₂₇ S [M+H] = 1847.95 [M+H] = 1848.0
Example 93 C ₉₅ H ₁₄₁ FN ₆ O ₂₇ S [M+H] = 1849.96 [M+H] = 1850.0
Example 94 C ₉₆ H ₁₄₃ FN ₆ O ₂₇ S [M+H] = 1863.98 [M+H] = 1864.0
Example 95 C ₉₅ H ₁₄₁ FN ₆ O ₂₆ S [M+H] = 1833.97 [M+H] = 1833.9
Example 96 C ₉₅ H ₁₃₉ FN ₆ O ₂₆ S ₂ [M+H] = 1863.92 [M+H] = 1863.8
Example 97 C ₉₅ H ₁₄₁ FN ₆ O ₂₆ S ₂ [M+H] = 1865.94 [M+H] = 1865.8
Example 98 C ₉₆ H ₁₄₃ FN ₆ O ₂₆ S ₂ [M+H] = 1879.96 [M+H] = 1879.9
Example 99 C ₉₃ H ₁₃₆ N ₁₀ O ₂₄ [M+H] = 1777.98 [M+H] = 1777.8
Example 100 C ₉₇ H ₁₄₄ N ₁₀ O ₂₄ [M+H] = 1834.04 [M+H] = 1833.8
Example 101 C ₉₉ H ₁₄₁ F ₃ N ₈ O ₂₅ [M+H] = 1900.00 [M+H] = 1899.9
Example 102 C ₉₉ H ₁₄₇ N ₁₁ O ₂₅ [M+H] = 1891.06 [M+H] = 1890.9
Example 136 C ₈₉ H ₁₃₅ N ₉ O ₂₃ [M+H] = 1698.97 [M+H] = 1698.8
Example 137 C ₉₃ H ₁₄₃ N ₉ O ₂₅ [M+H] = 1787.03 [M+H] = 1786.7
Example 138 C ₉₃ H ₁₄₁ N ₉ O ₂₅ [M+H] =1785.01 [M+H] = 1784.9
Example 139 C ₈₉ H ₁₃₂ N ₁₂ O ₂₄ [M+H] = 1753.96 [M+H] = 1753.7
Example 140 C ₉₃ H ₁₃₆ N ₁₀ O ₂₄ [M+H] = 1777.98 [M+H] = 1777.7
Example 141 C ₈₈ H ₁₃₅ N ₉ O ₂₄ [M+H] = 1702.97 [M+H] = 1702.9
Example 142 C ₉₅ H ₁₄₀ N ₁₀ O ₂₄ [M+H] = 1806.01 [M+H] = 1805.8
Example 143 C ₉₇ H ₁₄₃ N ₁₃ O ₂₄ [M+H] = 1875.04 [M+H] = 1874.9
Example 144 C ₉₅ H ₁₄₁ N ₁₁ O ₂₆ S [M+H] = 1884.98 [M+H] = 1884.9
Example 145 C ₉₁ H ₁₃₄ N ₁₂ O ₂₄ [M+H] = 1779.97 [M+H] = 1779.8
Example 146 C ₁₃₅ H ₁₉₁ N ₁₉ O ₃₅ [M+2H]/2=1320.19 [M+2H]/2=1320.8
Example 147 C ₉₃ H ₁₄₃ N ₉ O ₂₄ [M+H] = 1771.03 [M+H] = 1770.8
Example 148 C ₉₂ H ₁₃₈ N ₁₀ O ₂₄ [M+H] = 1768.00 [M+H] = 1767.8

General Method 3: Combination of a halide-containing PEG ester and an amine-containing pre-linker followed by deprotection of the ester

Stage 1:

To a 0.1 M solution of the amine pre-linker (1.0 equiv.) in MeCN, add K ₂ CO ₃ (2.0 equiv.), followed by the PEG ester containing the halide (1.0 equiv.). The reaction mixture is stirred at 80°C until the amine-containing pre-linker is consumed, as determined by LCMS analysis. The reaction mixture is then purified by chromatography on silica gel to obtain the product.

Stage 2:

To a 0.1M solution of PEG tert-butyl ether (1.0 equiv.) in EtOAc, add a solution of HCl in EtOAc. The resulting suspension is stirred at room temperature until the PEG ester is consumed, as determined by LCMS analysis. The reaction mixture is then concentrated under reduced pressure to obtain the product.

Intermediate B1-1,1-(4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecane -15-oic acid

Step 1 : Synthesis of tert-butyl 1-(4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12- tetraoxapentadecane-15-oate

To a mixture of 2-((2-(piperazin-1-yl)pyrimidin-5-yl)methyl)isoindoline-1,3-dione (7.97 g, 24.66 mmol, 1.0 equiv.) in MeCN ( 200 ml) add K ₂ CO ₃ (6.82 g, 49.31 mmol, 2.0 equiv.) then tert-butyl 1-bromo-3,6,9,12-tetraoxapentadecane-15-oate (9.5 g, 24.66 mmol, 1.0 equiv.). The reaction mixture is heated to 85°C and stirred for 15 hours. The mixture is then cooled to room temperature and filtered. The filtrate is concentrated under reduced pressure and the residue is purified by silica gel chromatography (0→20% EtOAc/MeOH) to give the product (11.5 g, 74.3% yield) as a light yellow liquid.

Step 2 : Synthesis of 1-(4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecane-15 -oic acid

To a solution of tert-butyl 1-(4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecane- 15-oate (3.5 g, 5.58 mmol, 1.0 equiv.) in EtOAc (50 ml) add a solution of HCl in EtOAc (500 ml). The mixture was stirred at room temperature for 3 hours. The mixture was then concentrated under reduced pressure to give the product (5.3 g, 78.2% yield, HCl) as a white solid. LCMS(ESI) m/z: [M+H] calc. _{for C28H37N5O8 : 572.27} ; found 572.4.

Following the general procedure 3, but using the appropriate PEG-containing halide and amine-containing pre-linkers in Table 4, Intermediates B1 in Table 8 are prepared:

Table 8. Additional protected amines obtained.

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection B1-1 _{C28H37N5O8 _ _ _} [M+H] = 572.27 [M+H] = 572.4

General Method 4: Combine a PEG carboxylic acid and an amine-containing active site inhibitor, then deprotect the amine.

Stage 1:

To a 0.15M solution of PEG carboxylic acid (1.0 equiv.) in DMF add GATU (1.3 equiv.) and DIPEA (5.0 equiv.). After stirring for 30 minutes, an amine-containing active site inhibitor (1.2 equiv.) is added. The reaction mixture is stirred at room temperature until the PEG carboxylic acid is consumed, as measured by LCMS. The reaction mixture is then purified by reverse phase chromatography to obtain the product.

Stage 2:

To a 0.1M solution of phthalimide-protected amine (1.0 equiv.) in MeOH at 0°C add NH ₂ NH ₂ •H ₂ O (4.0 equiv.). The resulting mixture was stirred at 60°C until the phthalimide-protected amine was consumed, as determined by LCMS analysis. The reaction mixture is then purified by reverse phase chromatography to obtain the product.

Intermediate B2-1. N-(4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)-1-(4- (5-(aminomethyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecan-15-amide

Step 1 : Synthesis of N-(4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)-1 -(4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecan-15-amide

To a mixture of 1-(4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecane-15-oic acid (3 g, 4.93 mmol, 1.0 equiv. , HCl) in DMF (30 ml) add GATU (12.11 μl, 6.41 mmol, 1.3 equiv.) and DIPEA (4.30 ml , 24.67 mmol, 5.0 equiv.). After 30 min, add 5-(4-amino-1-(4-aminobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]oxazol-2-amine (4.03 g, 5.92 mmol, 1.2 equiv., 3 TFA). The mixture was stirred at room temperature for 3 hours. The reaction mixture was then purified by pre-HPLC (MeCN/H ₂ O) to give the product (5.4 g, 81.2% yield, 4 TFA) as a light red solid. LCMS (ESI) m/z: [M+2H]/2 calc. for C ₄₄ H ₅₃ N ₁₃ O ₈ : 446.71; found 447.0.

Step 2 : Synthesis of N-(4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)-1 -(4-(5-(aminomethyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecan-15-amide

To a mixture of N-(4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butyl)-1-( 4-(5-((1,3-dioxoisoindolin-2-yl)methyl)pyrimidin-2-yl)piperazin-1-yl)-3,6,9,12-tetraoxapentadecan-15-amide (4 g, 2 .97 mmol, 1.0 equiv., 4 TFA) in MeOH (25 ml) at 0°C add NH ₂ NH ₂ •H ₂ O (588.63 μl, 11.87 mmol, 4.0 equiv.). The mixture was stirred at 60°C for 2 hours. The mixture was then cooled at room temperature and filtered, and the filter cake was washed with MeOH (5 ml). The filtrate was concentrated under reduced pressure and the residue was purified by pre-HPLC (MeCN/H ₂ O) to give the product (700 mg, 24.5% yield, TPA) as a white solid. LCMS (ESI) m/z: [M+2H]/2 calc. for C ₃₆ H ₅₁ N ₁₃ O ₆ : 381.71; found 381.8.

Following the general procedure 4, but using the appropriate Intermediate B1 in Table 8 and the amine-containing active site inhibitors in Table 2, intermediates B2 in Table 9 are prepared:

Table 9. Additional amines obtained

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate B2-1 C ₃₆ H ₅₁ N ₁₃ O ₆ [M+2H]/2=381.71 [M+2H]/2=381.8
Intermediate B2-2 C ₄₂ H ₅₃ N ₁₃ O ₆ [M+H] = 836.43 [M+H] = 836.4
Intermediate connection B2-3 C ₃₆ H ₅₁ N ₁₃ O ₅ [M+2H]/2=373.72 [M+2H]/2=737.7

General Procedure 5: Combination of a PEG halide-containing carboxylic acid and an amine-containing active site inhibitor.

To a 0.1 M solution of an amine-containing active site inhibitor (1.0 equiv.) and a carboxylic acid containing PEG (1.2 equiv.) in DMA, add DIPEA (4.0 equiv.) followed by PyBOP (1.3 equiv.). The reaction mixture is stirred until the amine-containing active site inhibitor is consumed, as determined by LCMS. The reaction mixture is then purified by reverse phase HPLC to obtain the product.

Intermediate B3-1. 18-{6-[(4-amino-3-{1H-pyrrolo[2,3-b]pyridin-5-yl}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl]- 1,2,3,4-tetrahydroisoquinolin-2-yl}-1-bromo-3,6,9,12,15-pentaoxaoctadecan-18-one

To a solution of 1-bromo-3,6,9,12,15-pentaoxaoctadecane-18-oic acid (105 mg, 282 µmol, 1.2 equiv.) and 3-{1H-pyrrolo[2,3-b]pyridine -5-yl}-1-[(1,2,3,4-tetrahydroisoquinolin-6-yl)methyl]-1H-pyrazolo[3,4-d]pyrimidin-4-amine (120 mg, 235 µmol, 1 .0 equiv.) in DMA (2.34 ml) add DIPEA (163 µl, 940 µmol, 4.0 equiv.), then PyBOP (158 mg, 305 µmol, 1.3 equiv.). The resulting solution was stirred at room temperature for 3 hours, then purified by reverse phase HPLC (10→98% MeCN + 0.1% formic acid/H ₂ O + 0.1% formic acid) to obtain the product (82.7 mg , 47% yield). LCMS (ESI) m/z : [M+H] calc. for C ₃₅ H ₄₃ BrN ₈ O ₆ : 751.26; found 751.2.

Following the general procedure 5, but using the appropriate PEG halide-containing carboxylic acid and amine-containing active site inhibitors in Table 2, intermediates B3 in Table 10 are prepared:

Table 10. Additional PEG halides produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate B3-1 C ₃₅ H ₄₃ BrN ₈ O ₆ [M+H] = 751.26 [M+H] = 751.2
Intermediate B3-2 C ₂₇ H ₂₇ BrN ₈ O ₃ [M+H] = 591.15 [M+H] = 591.2

General Procedure 6: Replacement of the PEG halide with an amine-containing post-linker and deprotection of the amine.

Stage 1:

To a 0.1M solution of halide containing PEG (1.0 equiv.) in MeCN add K ₂ CO ₃ (3.0 equiv.), followed by an amine post-linker (1.2 equiv.). The resulting suspension is heated to 80°C and stirred until the PEG halide is consumed, according to LCMS analysis. The reaction mixture is cooled at room temperature and then purified by silica gel chromatography to obtain the product.

Stage 2:

To a 0.07 M solution of N-Boc substituted amine (1.0 equiv.) in dioxane add HCl (4 M in dioxane, 10.0 equiv.). The reaction mixture is stirred until the N-Boc protected amine is consumed, as determined by LCMS analysis. The reaction mixture is then concentrated under reduced pressure to obtain the product.

Intermediate B2-4. 18-{6-[(4-amino-3-{1H-pyrrolo[2,3-b]pyridin-5-yl}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl]- 1,2,3,4-tetrahydroisoquinolin-2-yl}-1-(4-{5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}piperazin-1-yl)- 3,6,9,12,15-pentaoxaoctadecan-18-one

Step 1 : Synthesis of tert-butyl 2-[4-(18-{6-[(4-amino-3-{1H-pyrrolo[2,3-b]pyridin-5-yl}-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-yl}-18-oxo-3,6,9,12,15-pentaoxaoctadecan-1-yl)piperazin- 1-yl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidine-6-carboxylate

To a suspension of 18-{6-[(4-amino-3-{1H-pyrrolo[2,3-b]pyridin-5-yl}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl ]-1,2,3,4-tetrahydroisoquinolin-2-yl}-1-bromo-3,6,9,12,15-pentaoxaoctadecan-18-one (82.7 mg, 110 µmol, 1.0 equiv. ) in MeCN (1.09 ml) add K ₂ CO ₃ (45.6 mg, 330 µmol, 3.0 equiv.), then tert-butyl 2-(piperazin-1-yl)-5H,6H,7H, 8H-pyrido[4,3-d]pyrimidine-6-carboxylate (42.1 mg, 132 µmol, 1.2 equiv.). The resulting suspension was heated to 80°C for 8 hours, then purified by chromatography on silica gel (0→20% MeOH/DCM) to give the product (75.1 mg, 70% yield). LCMS (ESI) m/z: [M+H] calc. for _{C51H67N13O8 : 990.53 ;} found 990.5.

Step 2 : Synthesis of 18-{6-[(4-amino-3-{1H-pyrrolo[2,3-b]pyridin-5-yl}-1H-pyrazolo[3,4-d]pyrimidin-1-yl )methyl]-1,2,3,4-tetrahydroisoquinolin-2-yl}-1-(4-{5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-2-yl}piperazin-1 -yl)-3,6,9,12,15-pentaoxaoctadecan-18-one

To a solution of tert-butyl 2-[4-(18-{6-[(4-amino-3-{1H-pyrrolo[2,3-b]pyridin-5-yl}-1H-pyrazolo[3,4- d]pyrimidin-1-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-yl}-18-oxo-3,6,9,12,15-pentaoxaoctadecan-1-yl)piperazin-1- yl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidine-6-carboxylate (75.1 mg, 75.8 µmol, 1.0 equiv.) in dioxane (1 ml) add HCl ( 4M in dioxane, 472 µl, 1.89 mmol, 10.0 equiv.). The solution is stirred at room temperature for 45 minutes, then concentrated under reduced pressure to obtain the product. LCMS (ESI) m/z: [M+Na] calc. for C ₄₆ H ₅₉ N ₁₃ O ₆ : 912.46; found 912.5.

Following the general procedure 6, but using the appropriate PEG carboxylic acid and amine-containing active site inhibitors in Table 2, intermediates B2 in Table 11 are prepared:

Table 11. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate B2-4 C ₄₆ H ₅₉ N ₁₃ O ₆ [M+H] = 890.48 [M+H] = 890.5

Following the general procedure 1, but using the appropriate PEG carboxylic acid tert- butyl ester and the amine-containing active site inhibitors in Table 2, Intermediates B4 in Table 12 are prepared:

Table 12. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate B4-1 C ₂₈ H ₃₈ N ₈ O ₈ [M+H] = 615.29 [M+H] = 615.1

Following the general procedure 1, but using the appropriate Intermediates B4 in Table 12 and the amine-containing pre-linkers in Table 4, Intermediates B2 in Table 13 are prepared:

Table 13. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection B2-5 C ₃₉ H ₅₃ N ₁₃ O ₇ [M+H] = 816.43 [M+H] = 816.4

Following the general procedure 1, but using the appropriate Intermediates A1 and amine-containing pre-linkers in Table 4, Intermediates B2 in Table 14 are prepared:

Table 14. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate B2-6 C ₄₁ H ₅₂ N ₁₆ O ₆ [M+H] = 865.44 [M+H] = 865.2
Intermediate B2-7 C ₃₉ H ₄₈ N ₁₆ O ₅ [M+H] = 821.41 [M+H] = 821.2
Intermediate connection B2-8 C ₃₇ H ₄₄ N ₁₆ O ₄ [M+H] = 777.38 [M+H] = 777.3
Intermediate connection B2-9 C ₄₉ H ₅₈ N ₁₆ O ₇ [M+H] = 983.48 [M+H] = 983.4
Intermediate connection B2-10 C ₄₃ H ₄₆ N ₁₆ O ₄ [M+H] = 851.40 [M+H] = 851.4
Intermediate connection B2-11 C ₄₃ H ₅₆ N ₁₆ O ₆ [M+H] = 893.47 [M+H] = 893.3
Intermediate connection B2-12 C ₄₉ H ₅₈ N ₁₆ O ₆ [M+Na] = 989.46 [M+Na] = 989.4
Intermediate connection B2-13 C ₄₇ H ₅₄ N ₁₆ O ₅ [M+H] = 923.46 [M+H] = 923.4
Intermediate connection B2-14 C ₃₉ H ₅₀ N ₁₆ O ₆ [M+H] = 839.42 [M+H] = 839.3
Intermediate connection B2-15 C ₄₇ H ₅₆ N ₁₆ O ₇ [M+H] = 957.46 [M+H] = 957.7
Intermediate connection B2-16 C ₄₅ H ₅₂ N ₁₆ O ₆ [M+Na] = 935.42 [M+Na] = 935.3
Intermediate connection B2-17 C ₄₃ H ₄₈ N ₁₆ O ₅ [M+Na] = 891.39 [M+Na] = 891.4
Intermediate connection B2-18 C ₄₁ H ₅₄ N ₁₆ O ₆ [M+H] = 867.45 [M+H] = 867.3
Intermediate connection B2-19 C ₄₇ H ₅₆ N ₁₆ O ₆ [M+H] = 941.47 [M+H] = 941.2
Intermediate connection B2-20 C ₄₈ H ₅₈ N ₁₆ O ₆ [M+H] = 955.48 [M+H] = 955.2
Intermediate connection B2-21 C ₄₅ H ₅₂ N ₁₆ O ₅ [M+H] = 897.44 [M+H] = 897.3
Intermediate connection B2-22 C ₄₈ H ₅₈ N ₁₆ O ₈ [M+H] = 987.47 [M+H] = 987.42
Intermediate connection B2-23 C ₄₈ H ₅₈ N ₁₆ O ₇ [M+H] = 971.48 [M+H] = 971.31
Intermediate connection B2-24 C ₄₄ H ₅₅ N ₁₃ O ₇ [M+H] = 878.44 [M+H] = 878.5
Intermediate connection B2-25 C ₃₇ H ₅₁ N ₁₅ O ₆ [M+H] = 802.42 [M+H] = 802.4
Intermediate connection B2-26 C ₄₃ H ₅₃ N ₁₅ O ₆ [M+H] = 876.44 [M+H] = 876.4
Intermediate connection B2-27 C ₃₇ H ₅₁ N ₁₅ O ₅ [M+H] = 786.43 [M+H] = 786.5
Intermediate connection B2-28 C ₅₀ H ₅₈ N ₁₆ O ₉ [M+H] = 1027.47 [M+H] = 1027.1
Intermediate connection B2-29 C ₃₅ H ₄₃ N ₁₇ O ₄ [M+H] = 766.38 [M+H] = 766.3
Intermediate connection B2-30 C ₄₁ H ₄₅ N ₁₇ O ₄ [M+H] = 840.39 [M+H] = 840.4
Intermediate connection B2-31 C ₄₇ H ₅₇ N ₁₇ O ₇ [M+H] = 972.47 [M+H] = 972.5
Intermediate connection B2-32 C ₄₈ H ₅₉ N ₁₇ O ₇ [M+H] = 986.49 [M+H] = 986.4
Intermediate connection B2-33 C ₄₇ H ₅₇ N ₁₇ O ₆ [M+H] = 956.48 [M+H] = 956.3
Intermediate connection B2-34 C ₄₉ H ₅₉ N ₁₇ O ₆ [M+H] = 982.49 [M+H] = 982.2
Intermediate connection B2-35 C ₄₄ H ₆₁ N ₁₁ O ₉ [M+H] = 888.48 [M+H] = 888.3
Intermediate connection B2-36 C ₄₄ H ₅₁ N ₁₇ O ₄ [M+H] = 882.44 [M+H] = 882.4
Intermediate B2-37 C ₄₃ H ₅₂ N ₁₄ O ₄ [M+H] = 829.44 [M+H] = 829.3
Intermediate connection B2-38 C ₄₁ H ₅₆ N ₁₀ O ₈ [M+H] =817.44 [M+H] = 817.2
Intermediate connection B2-39 C ₄₃ H ₅₉ N ₁₁ O ₉ [M+H] = 874.46 [M+H] = 874.3

Following the general procedure 2, but using the appropriate rapamycin 4-nitrophenyl carbonate-containing monomers in Table 1 and Intermediates B2 from Tables 9, 11 and 13 and 14, synthesize Series 2 divalent analogues in Table 15:

Table 15. Row 2 divalent compounds:

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 27 C ₉₉ H ₁₃₃ N ₁₇ O ₂₁ [M+H] = 1896.99 [M+H] = 1896.7
Example 28 C ₉₉ H ₁₃₅ N ₁₇ O ₂₁ [M+H] = 1899.01 [M+H] = 1899.1
Example 29 C ₁₀₀ H ₁₃₇ N ₁₇ O ₂₁ [M+H] = 1913.03 [M+H] = 1913.0
Example 30 C ₉₇ H ₁₂₉ N ₁₇ O ₂₀ [M+H] = 1852.97 [M+H] = 1852.9
Example 31 C ₉₅ H ₁₂₅ N ₁₇ O ₁₉ [M+H] = 1808.94 [M+H] = 1809.0
Example 32 C ₉₃ H ₁₃₁ N ₁₇ O ₂₀ [M+H] = 1806.98 [M+H] = 1806.7
Example 33 C ₉₉ H ₁₃₃ N ₁₇ O ₂₀ [M+H] = 1881.00 [M+H] = 1881.0
Example 34 C ₁₀₀ H ₁₃₅ N ₁₇ O ₂₀ [M+H] = 1895.01 [M+H] = 1895.1
Example 35 C ₉₅ H ₁₃₃ N ₁₇ O ₂₀ [M+H] = 1833.00 [M+H] = 1832.9
Example 36 C ₁₀₁ H ₁₃₅ N ₁₇ O ₂₀ [M+H] = 1907.01 [M+H] = 1907.0
Example 37 C ₉₆ H ₁₃₂ N ₁₄ O ₂₁ [M+H] = 1817.98 [M+H] = 1817.9
Example 38 C ₉₈ H ₁₃₆ N ₁₄ O ₂₀ [M+H] = 1830.01 [M+H] = 1830.1
Example 39 C ₁₀₁ H ₁₃₅ N ₁₇ O ₂₁ [M+H] = 1923.01 [M+H] = 1923.1
Example 40 C ₁₀₀ H ₁₃₅ N ₁₇ O ₂₂ [M+H] = 1927.01 [M+H] = 1927.0
Example 41 C ₁₀₀ H ₁₃₅ N ₁₇ O ₂₁ [M+H] = 1911.01 [M+H] = 1911.0
Example 42 C ₉₅ H ₁₃₀ N ₁₆ O ₂₀ [M+H] = 1815.97 [M+H] = 1816.0
Example 43 C ₉₄ H ₁₃₀ N ₁₄ O ₂₀ [M+H] = 1775.97 [M+H] = 1775.9
Example 44 C ₉₅ H ₁₃₄ N ₁₄ O ₂₀ [M+H] = 1791.99 [M+H] = 1791.8
Example 45 C ₈₉ H ₁₃₂ N ₁₄ O ₂₀ [M+H] = 1717.98 [M+H] = 1717.9
Example 103 C ₉₆ H ₁₃₄ N ₁₆ O ₂₀ [M+H] = 1832.00 [M+H] = 1832.0
Example 104 C ₉₀ H ₁₃₂ N ₁₆ O ₂₀ [M+H] = 1757.99 [M+H] = 1757.9
Example 105 C ₁₀₃ H ₁₃₉ N ₁₇ O ₂₃ [M+H] = 1983.03 [M+H] = 1983.0
Example 106 C ₁₀₀ H ₁₃₈ N ₁₈ O ₂₁ [M+H] = 1928.04 [M+H] = 1927.9
Example 107 C ₁₀₁ H ₁₄₀ N ₁₈ O ₂₁ [M+H] = 1942.05 [M+H] = 1942.1
Example 108 C ₉₉ H ₁₃₄ N ₁₈ O ₂₀ [M+H] = 1896.01 [M+H] = 1896.0
Example 109 C ₁₀₁ H ₁₃₆ N ₁₈ O ₂₀ [M+H] = 1922.03 [M+H] = 1922.1
Example 110 C ₉₆ H ₁₄₀ N ₁₂ O ₂₃ [M+H] = 1830.02 [M+H] = 1829.9
Example 111 C ₉₇ H ₁₄₂ N ₁₂ O ₂₃ [M+H] = 1844.04 [M+H] = 1843.9
Example 149 C ₉₃ H ₁₃₅ N ₁₁ O ₂₂ [M+H] = 1758.99 [M+H] = 1758.9
Example 150 C ₉₅ H ₁₃₈ N ₁₂ O ₂₃ [M+H] = 1816.01 [M+H] = 1815.9

Following the general procedure 1, but using suitable amine-containing active site inhibitors in Table 2 and amine-containing pre-linkers in Table 4, Intermediates C1 in Table 16 are prepared:

Table 16. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate C1-1 C ₃₆ H ₃₅ N ₁₅ O ₂ [M+H] = 710.32 [M+H] = 710.2
Intermediate C1-2 C ₃₀ H ₃₃ N ₁₅ O [M+H] = 620.31 [M+H] = 620.2
Intermediate C1-3 C ₃₈ H ₃₇ N ₁₅ O ₂ [M+H] = 736.34 [M+H] = 736.2
Intermediate C1-4 C ₂₅ H ₂₈ N ₁₂ O ₂ [M+H] = 529.26 [M+H] = 529.5
Intermediate C1-5 C ₃₃ H ₃₈ N ₁₀ O ₂ [M+H] = 607.33 [M+H] = 607.3
Intermediate C1-6 C ₃₁ H ₃₀ N ₁₂ O ₂ [M+H] = 603.27 [M+H] = 603.3
Intermediate C1-7 C ₃₁ H ₃₀ N ₁₂ O [M+H] = 587.28 [M+H] = 587.3
Intermediate C1-8 C ₂₉ H ₃₂ N ₁₀ O ₂ [M+H] = 553.28 [M+H] =

Following the general procedure 1, but using the PEG carboxylic acids and Intermediates C1 in Table 16, Intermediates C2 in Table 17 are obtained:

Table 17. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate C2-1 C ₄₇ H ₅₆ N ₁₆ O ₇ [M+H] = 957.46 [M+H] = 957.7
Intermediate C2-2 C ₄₁ H ₅₄ N ₁₆ O ₆ [M+H] = 867.45 [M+H] = 867.2
Intermediate C2-3 C ₄₃ H ₄₆ N ₁₆ O ₄ [M+H] = 851.40 [M+H] = 851.2
Intermediate C2-4 C ₃₆ H ₄₉ N ₁₃ O ₇ [M+H] = 776.40 [M+H] = 776.3
Intermediate C2-5 C ₃₀ H ₃₇ N ₁₃ O ₄ [M+H] = 644.32 [M+H] = 644.3
Intermediate C2-6 C ₄₈ H ₆₇ N ₁₁ O ₉ [M+H] = 942.52 [M+H] = 943.2
Intermediate C2-7 C ₄₄ H ₆₁ N ₁₁ O ₉ [M+H] = 888.48 [M+H] = 888.3

Following the general procedure 2, but using the appropriate rapamycin 4-nitrophenyl carbonate monomer in Table 1 and Intermediates C2 from Table 17, synthesize Row 3 of the divalent analogues in Table 18:

Table 18. Row 3 divalent compounds

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 46 C ₉₉ H ₁₃₃ N ₁₇ O ₂₁ [M+H] = 1896.99 [M+H] = 1897.3
Example 47 C ₉₃ H ₁₃₁ N ₁₇ O ₂₀ [M+H] = 1806.98 [M+H] = 1806.8
Example 48 C ₁₀₀ H ₁₄₄ N ₁₂ O ₂₃ [M+H] = 1882.06 [M+H] = 1882.1
Example 49 C ₉₉ H ₁₃₅ N ₁₇ O ₂₁ [M+H] = 1899.01 [M+H] = 1899.1
Example 50 C ₁₀₀ H ₁₃₇ N ₁₇ O ₂₁ [M+H] = 1913.03 [M+H] = 1913.1
Example 112 C ₉₆ H ₁₄₀ N ₁₂ O ₂₃ [M+H] =1830.02 [M+H] = 1829.9
Example 113 C ₁₀₀ H ₁₄₆ N ₁₂ O ₂₃ [M+H] = 1884.07 [M+H] = 1883.7

Following the general procedure 1, but using the appropriate Intermediates C2 in Table 17 and the amine-containing pre-linkers in Table 4, Intermediates D1 in Table 19 are prepared:

Table 19. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection D1-1 C ₄₄ H ₅₂ N ₂₀ O ₅ [M+H] = 941.45 [M+H] = 941.5

Following the general procedure 1, but using suitable amine-containing active site inhibitors in Table 2 and amine-containing pre-linkers in Table 4, Intermediates D1 in Table 20 are prepared:

Table 20. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection D1-2 C ₃₉ H ₄₉ N ₁₇ O ₄ [M+H] = 820.43 [M+H] = 820.4
Intermediate connection D1-3 C ₄₅ H ₅₁ N ₁₇ O ₄ [M+H] = 894.44 [M+H] = 894.4
Intermediate connection D1-4 C ₃₉ H ₄₉ N ₁₇ O ₃ [M+H] = 804.43 [M+H] = 804.4
Intermediate connection D1-5 C ₄₅ H ₅₁ N ₁₇ O ₃ [M+H] = 878.45 [M+H] = 878.4
Intermediate connection D1-6 C ₄₀ H ₄₉ N ₁₇ O ₄ [M+H] = 832.43 [M+H] = 832.4
Intermediate connection D1-7 C ₅₈ H ₆₃ F ₃ N ₁₈ O ₅ [M+H] = 1149.53 [M+H] = 1149.4
Intermediate connection D1-8 C ₄₅ H ₅₆ N ₁₆ O ₅ [M+H] = 901.47 [M+H] = 901.4
Intermediate connection D1-9 C ₃₈ H ₅₁ N ₁₅ O ₄ [M+H] = 782.43 [M+H] = 782.4
Intermediate connection D1-10 C ₄₇ H ₅₃ N ₁₇ O ₄ [M+H] = 920.46 [M+H] = 920.4
Intermediate connection D1-11 C ₄₇ H ₅₆ FN ₁₃ O ₇ S [M+H] = 966.42 [M+H] = 966.3
Intermediate connection D1-12 C ₄₇ H ₅₇ N ₁₃ O ₇ S [M+H] = 948.43 [M+H] = 948.4
Intermediate connection D1-13 C ₄₇ H ₅₅ N ₁₇ O ₄ [M+H] = 922.47 [M+H] = 922.4
Intermediate connection D1-14 C ₄₇ H ₅₃ N ₁₇ O ₃ [M+H] = 904.46 [M+H] = 904.4
Intermediate connection D1-15 C ₄₄ H ₅₁ N ₁₇ O ₃ [M+H] = 866.45 [M+H] = 866.3
Intermediate connection D1-16 C ₄₆ H ₅₃ N ₁₇ O ₃ [M+H] = 892.46 [M+H] = 892.3
Intermediate connection D1-17 C ₄₄ H ₅₁ N ₁₇ O ₂ [M+H] = 850.45 [M+H] = 850.3
Intermediate connection D1-18 C ₄₆ H ₅₃ N ₁₇ O ₂ [M+H] = 876.47 [M+H] = 876.3
Intermediate connection D1-19 C ₄₄ H ₅₃ N ₁₅ O ₅ [M+H] = 872.45 [M+H] = 872.3
Intermediate connection D1-20 C ₄₆ H ₅₁ N ₁₇ O ₆ [M+H] = 938.61 [M+H] = 938.3
Intermediate connection D1-21 C ₄₆ H ₅₁ N ₁₇ O ₅ [M+H] = 922.44 [M+H] = 922.3
Intermediate connection D1-22 C ₄₇ H ₅₉ N ₁₅ O ₄ [M+H] = 898.50 [M+H] = 898.4

Following the general procedure 2, but using the appropriate rapamycin 4-nitrophenyl carbonate monomer in Table 1 and Intermediates D1 from Tables 19 and 20, synthesize Row 4 of the divalent analogues in Table 21:

Table 21. Row 4 divalent compounds

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 51 C ₉₇ H ₁₂₈ N ₁₈ O ₁₈ [M+H] = 1833.98 [M+H] = 1834.1
Example 52 C ₉₇ H ₁₂₈ N ₁₈ O ₁₇ [M+H] = 1817.98 [M+H] = 1817.9
Example 53 C ₉₆ H ₁₂₉ N ₂₁ O ₁₉ [M+H] = 1880.99 [M+H] = 1881.0
Example 54 C ₉₁ H ₁₂₆ N ₁₈ O ₁₈ [M+H] = 1759.96 [M+H] = 1760.0
Example 55 C ₉₇ H ₁₃₀ N ₁₈ O ₁₈ [M+H] = 1835.99 [M+H] = 1835.8
Example 56 C ₉₈ H ₁₃₂ N ₁₈ O ₁₈ [M+H] = 1850.01 [M+H] = 1849.8
Example 57 C ₉₂ H ₁₃₀ N ₁₈ O ₁₈ [M+H] = 1775.99 [M+H] = 1775.9
Example 58 C ₉₀ H ₁₂₈ N ₁₆ O ₁₈ [M+H] = 1721.97 [M+H] = 1721.9
Example 114 C ₁₀₀ H ₁₃₄ N ₁₈ O ₁₈ [M+H] = 1876.02 [M+H] = 1876.1
Example 115 C ₉₇ H ₁₃₀ N ₁₈ O ₁₇ [M+H] = 1819.99 [M+H] = 1820.1
Example 116 C ₉₇ H ₁₂₈ N ₁₈ O ₁₇ S [M+H] = 1849.95 [M+H] = 1849.9
Example 117 C ₉₇ H ₁₃₀ N ₁₈ O ₁₇ S [M+H] = 1851.97 [M+H] = 1851.9
Example 118 C ₉₈ H ₁₃₂ N ₁₈ O ₁₇ S [M+H] = 1865.98 [M+H] = 1865.7
Example 119 C ₉₉ H ₁₃₅ FN ₁₄ O ₂₁ S [M+H] = 1907.97 [M+H] = 1908.0
Example 120 C ₁₀₀ H ₁₃₇ FN ₁₄ O ₂₁ S [M+H] = 1921.99 [M+H] = 1922.0
Example 121 C ₉₉ H ₁₃₄ N ₁₄ O ₂₁ S [M+H] = 1887.96 [M+H] = 1888.0
Example 122 C ₉₉ H ₁₃₆ N ₁₄ O ₂₁ S [M+H] = 1889.98 [M+H] = 1890.0
Example 123 C ₉₉ H ₁₃₂ N ₁₈ O ₁₈ [M+H] = 1862.00 [M+H] = 1861.9
Example 124 C ₉₉ H ₁₃₀ N ₁₈ O ₁₇ [M+H] = 1843.99 [M+H] = 1844.1
Example 125 C ₉₇ H ₁₃₂ N ₁₈ O ₁₇ [M+H] = 1822.01 [M+H] = 1822.0
Example 126 C ₉₉ H ₁₃₄ N ₁₈ O ₁₇ [M+H] = 1848.03 [M+H] = 1848.0
Example 127 C ₉₆ H ₁₂₈ N ₁₈ O ₁₆ [M+H] = 1789.98 [M+H] = 1789.9
Example 128 C ₉₈ H ₁₃₀ N ₁₈ O ₁₆ [M+H] = 1816.00 [M+H] = 1815.9
Example 129 C ₉₆ H ₁₃₂ N ₁₆ O ₁₉ [M+H] = 1813.99 [M+H] = 1814.0
Example 130 C ₉₇ H ₁₃₄ N ₁₆ O ₁₉ [M+H] = 1828.01 [M+H] = 1828.0
Example 131 C ₉₉ H ₁₃₂ N ₁₈ O ₂₀ [M+H] = 1893.99 [M+H] = 1894.0
Example 132 C ₉₈ H ₁₂₈ N ₁₈ O ₁₉ [M+H] = 1861.97 [M+H] = 1861.9
Example 133 C ₉₇ H ₁₂₈ N ₁₈ O ₁₈ [M+H] = 1833.97 [M+H] = 1833.9
Example 151 C ₉₇ H ₁₂₉ N ₂₁ O ₁₇ [M+H] = 1861.00 [M+H] = 1860.8

Following the general procedure 1, but using the appropriate Intermediates C1 in Table 16 and the amine-containing pre-linkers in Table 4, Intermediates E1 in Table 22 are prepared:

Table 22. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection E1-1 C ₃₉ H ₄₃ N ₁₉ O ₃ [M+H] = 826.39 [M+H] = 826.5
Intermediate connection E1-2 C ₄₅ H ₄₅ N ₁₉ O ₃ [M+H] = 900.41 [M+H] = 900.2
Intermediate connection E1-3 C ₄₅ H ₄₅ N ₁₉ O ₂ [M+H] = 884.41 [M+H] = 884.4
Intermediate connection E1-4 C ₄₇ H ₄₇ N ₁₉ O ₃ [M+H] = 926.42 [M+H] = 926.6
Intermediate connection E1-5 C ₄₇ H ₄₇ N ₁₉ O ₂ [M+H] = 910.43 [M+H] = 910.2
Intermediate connection E1-6 C ₃₈ H ₄₇ N ₁₇ O ₃ [M+H] = 790.41 [M+H] = 790.4
Intermediate connection E1-7 C ₄₄ H ₄₉ N ₁₇ O ₃ [M+H] = 864.43 [M+H] = 864.3
Intermediate connection E1-8 C ₃₉ H ₄₇ N ₁₇ O ₃ [M+H] = 802.41 [M+H] = 802.3

Following the general procedure 2, but using the appropriate rapamycin monomers containing 4-nitrophenyl carbonate in Table 1 and Intermediates E1 from Table 22, synthesize Series 5 divalent analogues in Table 23:

Table 23. Row 5 divalent compounds

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 59 C ₉₁ H ₁₂₀ N ₂₀ O ₁₇ [M+H] = 1765.92 [M+H] = 1765.9
Example 60 C ₉₇ H ₁₂₂ N ₂₀ O ₁₇ [M+H] = 1839.94 [M+H] = 1840.0
Example 61 C ₉₇ H ₁₂₂ N ₂₀ O ₁₆ [M+H] = 1823.94 [M+H] = 1823.9
Example 62 C ₉₉ H ₁₂₄ N ₂₀ O ₁₇ [M+H] = 1865.95 [M+H] = 1865.8
Example 134 C ₉₉ H ₁₂₄ N ₂₀ O ₁₆ [M+H] = 1849.96 [M+H] = 1850.0

Table 24. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection F1-1 C ₄₃ H ₆₁ N ₁₁ O ₈ [M+H] = 860.48 [M+H] = 860.4
Intermediate connection F1-2 C ₄₄ H ₆₁ N ₁₁ O ₈ [M+H] =872.48 [M+H] = 872.2

Following the general procedure 2, but using the appropriate 4-nitrophenyl carbonate-containing rapamycin monomers in Table 1 and Intermediates F1 from Table 24, synthesize Series 6 divalent analogues in Table 25:

Table 25. Row 6 divalent compounds

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 135 C ₉₅ H ₁₄₀ N ₁₂ O ₂₂ [M+H] = 1802.03 [M+H] = 1802.5
Example 152 C ₉₆ H ₁₄₀ N ₁₂ O ₂₂ [M+H] = 1814.03 [M+H] = 1813.9

Following the general procedure 1, but using the appropriate Intermediates A1 in Table 5 and the amine-containing pre-linkers in Table 4, Intermediates G1 in Table 26 are prepared:

Table 26. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection G1-1 C ₄₄ H ₅₈ N ₁₈ O ₆ [M+H] = 935.49 [M+H] = 935.5
Intermediate connection G1-2 C ₅₀ H ₆₀ N ₁₈ O ₆ [M+H] = 1009.50 [M+H] = 1009.5
Intermediate connection G1-3 C ₄₄ H ₅₈ N ₁₈ O ₅ [M+H] = 919.49 [M+H] = 919.5

Following the general procedure 6, but using appropriate Intermediates B3 in Table 10 and amine-containing pre-linkers in Table 4, Intermediates G1 in Table 27 are prepared:

Table 27. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection G1-4 C ₄₅ H ₅₇ N ₁₅ O ₅ [M+H] = 888.48 [M+H] = 888.4

Following the general procedure 2, but using the appropriate rapamycin 4-nitrophenyl carbonate monomer in Table 1 and Intermediates G1 from Tables 26 and 27, synthesize Series 7 divalent analogues in Table 28:

Table 28. Row 7 divalent compounds

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 63 C ₉₆ H ₁₃₅ N ₁₉ O ₂₀ [M+H] = 1875.02 [M+H] = 1874.9
Example 64 C ₁₀₂ H ₁₃₇ N ₁₉ O ₂₀ [M+H] = 1949.04 [M+H] = 1949.0 Example 65 C ₁₀₃ H ₁₄₁ N ₁₉ O ₂₀ [M+H] = 1965.07 [M+H] = 1965.0
Example 66 C ₉₆ H ₁₃₅ N ₁₉ O ₁₉ [M+H] = 1859.03 [M+H] =
1859.0

Following the general procedure 1, but using the appropriate Intermediates D1 in Tables 19 and 20 and PEG carboxylic acids, Intermediates H1 in Table 29 are prepared:

Table 29. Additional amines produced

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Intermediate connection H1-1 C ₄₄ H ₅₈ N ₁₈ O ₆ [M+H] = 935.49 [M+H] = 935.5
Intermediate connection H1-2 C ₅₀ H ₆₀ N ₁₈ O ₆ [M+2H]/2=505.23 [M+2H]/2=505.4
Intermediate connection H1-3 C ₄₄ H ₅₈ N ₁₈ O ₅ [M+2H]/2=460.25 [M+2H]/2=460.3

Following the general procedure 2, but using the appropriate rapamycin 4-nitrophenyl carbonate monomer in Table 1 and Intermediates H1 from Table 29, synthesize the Series 8 divalent analogues in Table 30:

Table 30. Row 8 divalent compounds:

STRUCTURE MOLECULAR FORMULA CALCULATED MM FOUND MM
Example 67 C ₁₀₂ H ₁₃₇ N ₁₉ O ₂₀ [M+H] = 1949.04 [M+H] = 1949.0
Example 68 C ₉₆ H ₁₃₅ N ₁₉ O ₁₉ [M+H] = 1859.03 [M+H] = 1859.0
Example 69 C ₁₀₂ H ₁₃₉ N ₁₉ O ₂₀ [M+H] = 1951.05 [M+H] =
1951.1
Example 70 C ₁₀₃ H ₁₄₁ N ₁₉ O ₂₀ [M+H] = 1965.07 [M+H] = 1965.1

Biological examples

Cellular AlphaLISA assays to determine IC50 for inhibition of P-Akt (S473), P-4E-BP1 (T37/46) and P-P70S6K (T389) in MDA-MB-468 cells

Cellular mTOR kinase assay

To measure the functional activity of mTORC1 and mTORC2 in cells, phosphorylation of 4EBP1 (Thr37/46) and P70S6K (Thr389) and AKT1/2/3 (Ser473) was performed using AlphaLisa SureFire Ultra Kits (Perkin Elmer). MDA-MB-468 cells (ATCC® HTB-132) were cultured in 96-well tissue culture plates and treated with opening compounds at concentrations of 0.017-1,000 nM for two hours at 37°C. The incubation is terminated by removing the assay buffer and adding the lysis buffer provided with the assay kit. Samples are processed according to the manufacturer's instructions. The alpha signal from the corresponding phosphoproteins was measured in duplicate and using a microplate reader (Envision, Perkin-Elmer or Spectramax M5, Molecular Devices). Inhibitor concentration response curves were analyzed using a normalized IC ₅₀ regression curve consistent with control-based normalization.

As an example, measured IC ₅₀ values for selected compounds are presented below:

IC ₅₀ inhibition of mTORC1 and mTORC2 substrate phosphorylation (nM) Compound p-P70S6K-(T389) p-4E-BP1-(T37/46) p-AKT1/2/3-(S473) MLN-128 1.4 16 3.7 Rapamycin 0.2 >1,000 >1,000

As an example, measured IC ₅₀ values for selected compounds are presented below:

Example IC ₅₀ inhibition of mTORC1 and mTORC2 substrate phosphorylation p-P70S6K-(T389) p-4E-BP1-(T37/46) p-AKT1/2/3-(S473) 1 +++ +++ ++ 2 +++ +++ ++ 3 +++ +++ ++ 4 +++ +++ ++ 5 +++ +++ + 6 +++ +++ ++ 7 +++ +++ ++ 8 +++ +++ ++ 9 +++ +++ ++ 10 +++ - - eleven +++ +++ ++ 12 +++ +++ + 13 +++ +++ ++ 14 ++ +++ ++ 15 +++ +++ ++ 16 +++ +++ +++ 17 +++ +++ ++ 18 +++ ++ + 19 +++ +++ +++ 20 +++ +++ +++ 21 +++ +++ + 27 +++ +++ ++ 28 +++ +++ - 29 +++ +++ - thirty +++ +++ ++ 31 +++ +++ ++ 32 +++ +++ ++ 33 +++ +++ - 34 +++ - - 35 +++ +++ ++ 36 +++ +++ - 37 +++ +++ - 38 +++ - - 39 +++ +++ +++ 40 +++ +++ ++ 41 +++ +++ - 42 +++ +++ ++ 43 +++ +++ ++ 46 +++ +++ ++ 47 +++ +++ ++ 48 +++ +++ ++ 49 +++ +++ ++ 50 +++ +++ + 51 +++ +++ +++ 52 +++ +++ - 53 +++ +++ ++ 54 +++ +++ +++ 55 +++ +++ ++ 56 +++ +++ - 59 +++ +++ +++ 60 +++ ++ + 61 +++ - - 62 ++ - - 63 +++ +++ +++ 64 +++ +++ ++ 65 +++ +++ + 66 +++ +++ ++ 67 + + + 68 + + - 69 +++ +++ ++ 70 +++ +++ + 71 +++ +++ ++ 72 +++ +++ +++ 73 +++ +++ + 74 +++ +++ + 75 +++ +++ ++ 76 +++ +++ ++ 77 +++ ++ + 78 +++ +++ +++ 79 +++ +++ + 80 +++ +++ +++ 81 +++ ++ - 82 +++ +++ - 83 +++ +++ ++ 84 +++ +++ ++ 85 +++ +++ ++ 86 +++ +++ ++ 87 +++ +++ ++ 88 +++ +++ ++ 89 +++ +++ - 90 +++ - - 91 +++ +++ ++ 92 +++ +++ ++ 93 +++ +++ ++ 94 +++ +++ +++ 95 +++ +++ ++ 96 +++ +++ + 97 +++ +++ ++ 98 +++ +++ ++ 99 +++ +++ - 100 +++ +++ ++ 101 +++ +++ ++ 102 +++ +++ ++ 103 +++ - - 104 +++ +++ - 105 +++ ++ - 106 +++ ++ - 107 +++ - - 108 ++ - - 109 + - - 110 ++ - - 111 +++ +++ - 112 +++ - - 113 +++ +++ ++ 114 +++ +++ - 115 +++ +++ +++ 116 +++ +++ - 117 +++ +++ ++ 118 +++ +++ ++ 119 +++ +++ ++ 120 +++ +++ ++ 121 +++ +++ - 122 +++ +++ ++ 123 +++ +++ + 124 +++ +++ ++ 125 +++ +++ ++ 126 +++ ++ - 127 +++ ++ - 128 +++ ++ + 129 +++ +++ - 130 +++ +++ - 131 +++ +++ ++ 132 +++ +++ ++ 133 +++ +++ ++ 134 +++ +++ - 135 +++ - - 136 +++ ++ ++ 137 +++ +++ ++ 138 +++ +++ +++ 139 +++ +++ + 140 + + - 141 +++ +++ + 142 ++ - - 143 +++ ++ - 144 +++ +++ ++ 145 +++ ++ + 146 - - - 147 +++ +++ ++ 148 +++ +++ ++ 149 +++ +++ ++ 150 +++ +++ + 151 +++ ++ - 152 +++ +++ +

Note:

pIC50 (p-P70S6K-(T389)) ≥9 +++ 9>pIC50 ≥8 ++ 8>pIC50 ≥6 + <6 - pIC50 (p-4E-BP1-(T37/46) or p-AKT1/2/3-(S473)) ≥8.5 +++ 8.5>pIC50 ≥7.5 ++ 7.5>pIC50 ≥6.0 + <6 -

Equivalents

Although the present disclosure has been described in connection with the specific embodiments set forth above, many alternatives, modifications, and other variations thereof will be apparent to those skilled in the art. All such alternatives, modifications and variations are within the spirit and scope of this disclosure.

--->

LIST OF SEQUENCES

<110> Revolution Medicines, Inc.

<120> C40-, C28- AND C-32-LINKED RAPAMYCIN ANALOGUES AS MTOR INHIBITORS

<130> REME-008/01WO 323913-2136

<150> US 62/836,036

<151> 2019-04-18

<150> US 62/752,874

<151> 2018-10-30

<150> US 62/665,435

<151> 2018-05-01

<160> 5

<170> Patent In version 3.5

<210> 1

<211> 2549

<212>PRT

<213> Homo sapiens

<400> 1

Met Leu Gly Thr Gly Pro Ala Ala Ala Thr Thr Ala Ala Thr Thr Ser

1 5 10 15

Ser Asn Val Ser Val Leu Gln Gln Phe Ala Ser Gly Leu Lys Ser Arg

20 25 30

Asn Glu Glu Thr Arg Ala Lys Ala Ala Lys Glu Leu Gln His Tyr Val

35 40 45

Thr Met Glu Leu Arg Glu Met Ser Gln Glu Glu Ser Thr Arg Phe Tyr

50 55 60

Asp Gln Leu Asn His His Ile Phe Glu Leu Val Ser Ser Ser Asp Ala

65 70 75 80

Asn Glu Arg Lys Gly Gly Ile Leu Ala Ile Ala Ser Leu Ile Gly Val

85 90 95

Glu Gly Gly Asn Ala Thr Arg Ile Gly Arg Phe Ala Asn Tyr Leu Arg

100 105 110

Asn Leu Leu Pro Ser Asn Asp Pro Val Val Met Glu Met Ala Ser Lys

115 120 125

Ala Ile Gly Arg Leu Ala Met Ala Gly Asp Thr Phe Thr Ala Glu Tyr

130 135 140

Val Glu Phe Glu Val Lys Arg Ala Leu Glu Trp Leu Gly Ala Asp Arg

145 150 155 160

Asn Glu Gly Arg Arg His Ala Ala Val Leu Val Leu Arg Glu Leu Ala

165 170 175

Ile Ser Val Pro Thr Phe Phe Phe Gln Gln Val Gln Pro Phe Phe Asp

180 185 190

Asn Ile Phe Val Ala Val Trp Asp Pro Lys Gln Ala Ile Arg Glu Gly

195 200 205

Ala Val Ala Ala Leu Arg Ala Cys Leu Ile Leu Thr Thr Gln Arg Glu

210 215 220

Pro Lys Glu Met Gln Lys Pro Gln Trp Tyr Arg His Thr Phe Glu Glu

225 230 235 240

Ala Glu Lys Gly Phe Asp Glu Thr Leu Ala Lys Glu Lys Gly Met Asn

245 250 255

Arg Asp Asp Arg Ile His Gly Ala Leu Leu Ile Leu Asn Glu Leu Val

260 265 270

Arg Ile Ser Ser Met Glu Gly Glu Arg Leu Arg Glu Glu Met Glu Glu

275 280 285

Ile Thr Gln Gln Gln Leu Val His Asp Lys Tyr Cys Lys Asp Leu Met

290 295 300

Gly Phe Gly Thr Lys Pro Arg His Ile Thr Pro Phe Thr Ser Phe Gln

305 310 315 320

Ala Val Gln Pro Gln Gln Ser Asn Ala Leu Val Gly Leu Leu Gly Tyr

325 330 335

Ser Ser His Gln Gly Leu Met Gly Phe Gly Thr Ser Pro Ser Pro Ala

340 345 350

Lys Ser Thr Leu Val Glu Ser Arg Cys Cys Arg Asp Leu Met Glu Glu

355 360 365

Lys Phe Asp Gln Val Cys Gln Trp Val Leu Lys Cys Arg Asn Ser Lys

370 375 380

Asn Ser Leu Ile Gln Met Thr Ile Leu Asn Leu Leu Pro Arg Leu Ala

385 390 395 400

Ala Phe Arg Pro Ser Ala Phe Thr Asp Thr Gln Tyr Leu Gln Asp Thr

405 410 415

Met Asn His Val Leu Ser Cys Val Lys Lys Glu Lys Glu Arg Thr Ala

420 425 430

Ala Phe Gln Ala Leu Gly Leu Leu Ser Val Ala Val Arg Ser Glu Phe

435 440 445

Lys Val Tyr Leu Pro Arg Val Leu Asp Ile Ile Arg Ala Ala Leu Pro

450 455 460

Pro Lys Asp Phe Ala His Lys Arg Gln Lys Ala Met Gln Val Asp Ala

465 470 475 480

Thr Val Phe Thr Cys Ile Ser Met Leu Ala Arg Ala Met Gly Pro Gly

485 490 495

Ile Gln Gln Asp Ile Lys Glu Leu Leu Glu Pro Met Leu Ala Val Gly

500 505 510

Leu Ser Pro Ala Leu Thr Ala Val Leu Tyr Asp Leu Ser Arg Gln Ile

515 520 525

Pro Gln Leu Lys Lys Asp Ile Gln Asp Gly Leu Leu Lys Met Leu Ser

530 535 540

Leu Val Leu Met His Lys Pro Leu Arg His Pro Gly Met Pro Lys Gly

545 550 555 560

Leu Ala His Gln Leu Ala Ser Pro Gly Leu Thr Thr Leu Pro Glu Ala

565 570 575

Ser Asp Val Gly Ser Ile Thr Leu Ala Leu Arg Thr Leu Gly Ser Phe

580 585 590

Glu Phe Glu Gly His Ser Leu Thr Gln Phe Val Arg His Cys Ala Asp

595 600 605

His Phe Leu Asn Ser Glu His Lys Glu Ile Arg Met Glu Ala Ala Arg

610 615 620

Thr Cys Ser Arg Leu Leu Thr Pro Ser Ile His Leu Ile Ser Gly His

625 630 635 640

Ala His Val Val Ser Gln Thr Ala Val Gln Val Val Ala Asp Val Leu

645 650 655

Ser Lys Leu Leu Val Val Gly Ile Thr Asp Pro Asp Pro Asp Ile Arg

660 665 670

Tyr Cys Val Leu Ala Ser Leu Asp Glu Arg Phe Asp Ala His Leu Ala

675 680 685

Gln Ala Glu Asn Leu Gln Ala Leu Phe Val Ala Leu Asn Asp Gln Val

690 695 700

Phe Glu Ile Arg Glu Leu Ala Ile Cys Thr Val Gly Arg Leu Ser Ser

705 710 715 720

Met Asn Pro Ala Phe Val Met Pro Phe Leu Arg Lys Met Leu Ile Gln

725 730 735

Ile Leu Thr Glu Leu Glu His Ser Gly Ile Gly Arg Ile Lys Glu Gln

740 745 750

Ser Ala Arg Met Leu Gly His Leu Val Ser Asn Ala Pro Arg Leu Ile

755 760 765

Arg Pro Tyr Met Glu Pro Ile Leu Lys Ala Leu Ile Leu Lys Leu Lys

770 775 780

Asp Pro Asp Pro Asp Pro Asn Pro Gly Val Ile Asn Asn Val Leu Ala

785 790 795 800

Thr Ile Gly Glu Leu Ala Gln Val Ser Gly Leu Glu Met Arg Lys Trp

805 810 815

Val Asp Glu Leu Phe Ile Ile Ile Met Asp Met Leu Gln Asp Ser Ser

820 825 830

Leu Leu Ala Lys Arg Gln Val Ala Leu Trp Thr Leu Gly Gln Leu Val

835 840 845

Ala Ser Thr Gly Tyr Val Val Glu Pro Tyr Arg Lys Tyr Pro Thr Leu

850 855 860

Leu Glu Val Leu Leu Asn Phe Leu Lys Thr Glu Gln Asn Gln Gly Thr

865 870 875 880

Arg Arg Glu Ala Ile Arg Val Leu Gly Leu Leu Gly Ala Leu Asp Pro

885 890 895

Tyr Lys His Lys Val Asn Ile Gly Met Ile Asp Gln Ser Arg Asp Ala

900 905 910

Ser Ala Val Ser Leu Ser Glu Ser Lys Ser Ser Gln Asp Ser Ser Asp

915 920 925

Tyr Ser Thr Ser Glu Met Leu Val Asn Met Gly Asn Leu Pro Leu Asp

930 935 940

Glu Phe Tyr Pro Ala Val Ser Met Val Ala Leu Met Arg Ile Phe Arg

945 950 955 960

Asp Gln Ser Leu Ser His His His Thr Met Val Val Gln Ala Ile Thr

965 970 975

Phe Ile Phe Lys Ser Leu Gly Leu Lys Cys Val Gln Phe Leu Pro Gln

980 985 990

Val Met Pro Thr Phe Leu Asn Val Ile Arg Val Cys Asp Gly Ala Ile

995 1000 1005

Arg Glu Phe Leu Phe Gln Gln Leu Gly Met Leu Val Ser Phe Val

1010 1015 1020

Lys Ser His Ile Arg Pro Tyr Met Asp Glu Ile Val Thr Leu Met

1025 1030 1035

Arg Glu Phe Trp Val Met Asn Thr Ser Ile Gln Ser Thr Ile Ile

1040 1045 1050

Leu Leu Ile Glu Gln Ile Val Val Ala Leu Gly Gly Glu Phe Lys

1055 1060 1065

Leu Tyr Leu Pro Gln Leu Ile Pro His Met Leu Arg Val Phe Met

1070 1075 1080

His Asp Asn Ser Pro Gly Arg Ile Val Ser Ile Lys Leu Leu Ala

1085 1090 1095

Ala Ile Gln Leu Phe Gly Ala Asn Leu Asp Asp Tyr Leu His Leu

1100 1105 1110

Leu Leu Pro Pro Ile Val Lys Leu Phe Asp Ala Pro Glu Ala Pro

1115 1120 1125

Leu Pro Ser Arg Lys Ala Ala Leu Glu Thr Val Asp Arg Leu Thr

1130 1135 1140

Glu Ser Leu Asp Phe Thr Asp Tyr Ala Ser Arg Ile Ile His Pro

1145 1150 1155

Ile Val Arg Thr Leu Asp Gln Ser Pro Glu Leu Arg Ser Thr Ala

1160 1165 1170

Met Asp Thr Leu Ser Ser Leu Val Phe Gln Leu Gly Lys Lys Tyr

1175 1180 1185

Gln Ile Phe Ile Pro Met Val Asn Lys Val Leu Val Arg His Arg

1190 1195 1200

Ile Asn His Gln Arg Tyr Asp Val Leu Ile Cys Arg Ile Val Lys

1205 1210 1215

Gly Tyr Thr Leu Ala Asp Glu Glu Glu Asp Pro Leu Ile Tyr Gln

1220 1225 1230

His Arg Met Leu Arg Ser Gly Gln Gly Asp Ala Leu Ala Ser Gly

1235 1240 1245

Pro Val Glu Thr Gly Pro Met Lys Lys Leu His Val Ser Thr Ile

1250 1255 1260

Asn Leu Gln Lys Ala Trp Gly Ala Ala Arg Arg Val Ser Lys Asp

1265 1270 1275

Asp Trp Leu Glu Trp Leu Arg Arg Leu Ser Leu Glu Leu Leu Lys

1280 1285 1290

Asp Ser Ser Ser Pro Ser Leu Arg Ser Cys Trp Ala Leu Ala Gln

1295 1300 1305

Ala Tyr Asn Pro Met Ala Arg Asp Leu Phe Asn Ala Ala Phe Val

1310 1315 1320

Ser Cys Trp Ser Glu Leu Asn Glu Asp Gln Gln Asp Glu Leu Ile

1325 1330 1335

Arg Ser Ile Glu Leu Ala Leu Thr Ser Gln Asp Ile Ala Glu Val

1340 1345 1350

Thr Gln Thr Leu Leu Asn Leu Ala Glu Phe Met Glu His Ser Asp

1355 1360 1365

Lys Gly Pro Leu Pro Leu Arg Asp Asp Asn Gly Ile Val Leu Leu

1370 1375 1380

Gly Glu Arg Ala Ala Lys Cys Arg Ala Tyr Ala Lys Ala Leu His

1385 1390 1395

Tyr Lys Glu Leu Glu Phe Gln Lys Gly Pro Thr Pro Ala Ile Leu

1400 1405 1410

Glu Ser Leu Ile Ser Ile Asn Asn Lys Leu Gln Gln Pro Glu Ala

1415 1420 1425

Ala Ala Gly Val Leu Glu Tyr Ala Met Lys His Phe Gly Glu Leu

1430 1435 1440

Glu Ile Gln Ala Thr Trp Tyr Glu Lys Leu His Glu Trp Glu Asp

1445 1450 1455

Ala Leu Val Ala Tyr Asp Lys Lys Met Asp Thr Asn Lys Asp Asp

1460 1465 1470

Pro Glu Leu Met Leu Gly Arg Met Arg Cys Leu Glu Ala Leu Gly

1475 1480 1485

Glu Trp Gly Gln Leu His Gln Gln Cys Cys Glu Lys Trp Thr Leu

1490 1495 1500

Val Asn Asp Glu Thr Gln Ala Lys Met Ala Arg Met Ala Ala Ala

1505 1510 1515

Ala Ala Trp Gly Leu Gly Gln Trp Asp Ser Met Glu Glu Tyr Thr

1520 1525 1530

Cys Met Ile Pro Arg Asp Thr His Asp Gly Ala Phe Tyr Arg Ala

1535 1540 1545

Val Leu Ala Leu His Gln Asp Leu Phe Ser Leu Ala Gln Gln Cys

1550 1555 1560

Ile Asp Lys Ala Arg Asp Leu Leu Asp Ala Glu Leu Thr Ala Met

1565 1570 1575

Ala Gly Glu Ser Tyr Ser Arg Ala Tyr Gly Ala Met Val Ser Cys

1580 1585 1590

His Met Leu Ser Glu Leu Glu Glu Val Ile Gln Tyr Lys Leu Val

1595 1600 1605

Pro Glu Arg Arg Glu Ile Ile Arg Gln Ile Trp Trp Glu Arg Leu

1610 1615 1620

Gln Gly Cys Gln Arg Ile Val Glu Asp Trp Gln Lys Ile Leu Met

1625 1630 1635

Val Arg Ser Leu Val Val Ser Pro His Glu Asp Met Arg Thr Trp

1640 1645 1650

Leu Lys Tyr Ala Ser Leu Cys Gly Lys Ser Gly Arg Leu Ala Leu

1655 1660 1665

Ala His Lys Thr Leu Val Leu Leu Leu Gly Val Asp Pro Ser Arg

1670 1675 1680

Gln Leu Asp His Pro Leu Pro Thr Val His Pro Gln Val Thr Tyr

1685 1690 1695

Ala Tyr Met Lys Asn Met Trp Lys Ser Ala Arg Lys Ile Asp Ala

1700 1705 1710

Phe Gln His Met Gln His Phe Val Gln Thr Met Gln Gln Gln Ala

1715 1720 1725

Gln His Ala Ile Ala Thr Glu Asp Gln Gln His Lys Gln Glu Leu

1730 1735 1740

His Lys Leu Met Ala Arg Cys Phe Leu Lys Leu Gly Glu Trp Gln

1745 1750 1755

Leu Asn Leu Gln Gly Ile Asn Glu Ser Thr Ile Pro Lys Val Leu

1760 1765 1770

Gln Tyr Tyr Ser Ala Ala Thr Glu His Asp Arg Ser Trp Tyr Lys

1775 1780 1785

Ala Trp His Ala Trp Ala Val Met Asn Phe Glu Ala Val Leu His

1790 1795 1800

Tyr Lys His Gln Asn Gln Ala Arg Asp Glu Lys Lys Lys Leu Arg

1805 1810 1815

His Ala Ser Gly Ala Asn Ile Thr Asn Ala Thr Thr Ala Ala Thr

1820 1825 1830

Thr Ala Ala Thr Ala Thr Thr Thr Ala Ser Thr Glu Gly Ser Asn

1835 1840 1845

Ser Glu Ser Glu Ala Glu Ser Thr Glu Asn Ser Pro Thr Pro Ser

1850 1855 1860

Pro Leu Gln Lys Lys Val Thr Glu Asp Leu Ser Lys Thr Leu Leu

1865 1870 1875

Met Tyr Thr Val Pro Ala Val Gln Gly Phe Phe Arg Ser Ile Ser

1880 1885 1890

Leu Ser Arg Gly Asn Asn Leu Gln Asp Thr Leu Arg Val Leu Thr

1895 1900 1905

Leu Trp Phe Asp Tyr Gly His Trp Pro Asp Val Asn Glu Ala Leu

1910 1915 1920

Val Glu Gly Val Lys Ala Ile Gln Ile Asp Thr Trp Leu Gln Val

1925 1930 1935

Ile Pro Gln Leu Ile Ala Arg Ile Asp Thr Pro Arg Pro Leu Val

1940 1945 1950

Gly Arg Leu Ile His Gln Leu Leu Thr Asp Ile Gly Arg Tyr His

1955 1960 1965

Pro Gln Ala Leu Ile Tyr Pro Leu Thr Val Ala Ser Lys Ser Thr

1970 1975 1980

Thr Thr Ala Arg His Asn Ala Ala Asn Lys Ile Leu Lys Asn Met

1985 1990 1995

Cys Glu His Ser Asn Thr Leu Val Gln Gln Ala Met Met Val Ser

2000 2005 2010

Glu Glu Leu Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His

2015 2020 2025

Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn

2030 2035 2040

Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met

2045 2050 2055

Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala

2060 2065 2070

Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr

2075 2080 2085

Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala Trp Asp Leu

2090 2095 2100

Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gln Leu Pro Gln Leu

2105 2110 2115

Thr Ser Leu Glu Leu Gln Tyr Val Ser Pro Lys Leu Leu Met Cys

2120 2125 2130

Arg Asp Leu Glu Leu Ala Val Pro Gly Thr Tyr Asp Pro Asn Gln

2135 2140 2145

Pro Ile Ile Arg Ile Gln Ser Ile Ala Pro Ser Leu Gln Val Ile

2150 2155 2160

Thr Ser Lys Gln Arg Pro Arg Lys Leu Thr Leu Met Gly Ser Asn

2165 2170 2175

Gly His Glu Phe Val Phe Leu Leu Lys Gly His Glu Asp Leu Arg

2180 2185 2190

Gln Asp Glu Arg Val Met Gln Leu Phe Gly Leu Val Asn Thr Leu

2195 2200 2205

Leu Ala Asn Asp Pro Thr Ser Leu Arg Lys Asn Leu Ser Ile Gln

2210 2215 2220

Arg Tyr Ala Val Ile Pro Leu Ser Thr Asn Ser Gly Leu Ile Gly

2225 2230 2235

Trp Val Pro His Cys Asp Thr Leu His Ala Leu Ile Arg Asp Tyr

2240 2245 2250

Arg Glu Lys Lys Lys Ile Leu Leu Asn Ile Glu His Arg Ile Met

2255 2260 2265

Leu Arg Met Ala Pro Asp Tyr Asp His Leu Thr Leu Met Gln Lys

2270 2275 2280

Val Glu Val Phe Glu His Ala Val Asn Asn Thr Ala Gly Asp Asp

2285 2290 2295

Leu Ala Lys Leu Leu Trp Leu Lys Ser Pro Ser Ser Glu Val Trp

2300 2305 2310

Phe Asp Arg Arg Thr Asn Tyr Thr Arg Ser Leu Ala Val Met Ser

2315 2320 2325

Met Val Gly Tyr Ile Leu Gly Leu Gly Asp Arg His Pro Ser Asn

2330 2335 2340

Leu Met Leu Asp Arg Leu Ser Gly Lys Ile Leu His Ile Asp Phe

2345 2350 2355

Gly Asp Cys Phe Glu Val Ala Met Thr Arg Glu Lys Phe Pro Glu

2360 2365 2370

Lys Ile Pro Phe Arg Leu Thr Arg Met Leu Thr Asn Ala Met Glu

2375 2380 2385

Val Thr Gly Leu Asp Gly Asn Tyr Arg Ile Thr Cys His Thr Val

2390 2395 2400

Met Glu Val Leu Arg Glu His Lys Asp Ser Val Met Ala Val Leu

2405 2410 2415

Glu Ala Phe Val Tyr Asp Pro Leu Leu Asn Trp Arg Leu Met Asp

2420 2425 2430

Thr Asn Thr Lys Gly Asn Lys Arg Ser Arg Thr Arg Thr Asp Ser

2435 2440 2445

Tyr Ser Ala Gly Gln Ser Val Glu Ile Leu Asp Gly Val Glu Leu

2450 2455 2460

Gly Glu Pro Ala His Lys Lys Thr Gly Thr Thr Val Pro Glu Ser

2465 2470 2475

Ile His Ser Phe Ile Gly Asp Gly Leu Val Lys Pro Glu Ala Leu

2480 2485 2490

Asn Lys Lys Ala Ile Gln Ile Ile Asn Arg Val Arg Asp Lys Leu

2495 2500 2505

Thr Gly Arg Asp Phe Ser His Asp Asp Thr Leu Asp Val Pro Thr

2510 2515 2520

Gln Val Glu Leu Leu Ile Lys Gln Ala Thr Ser His Glu Asn Leu

2525 2530 2535

Cys Gln Cys Tyr Ile Gly Trp Cys Pro Phe Trp

2540 2545

<210> 2

<211> 8733

<212> DNA

<213> Homo sapiens

<400> 2

gctcccggct tagaggacag cggggaaggc gggcggtggg gcagggggcc tgaagcggcg 60

gtaccggtgc tggcggcggc agctgaggcc ttggccgaag ccgcgcgaac ctcagggcaa 120

gatgcttgga accggacctg ccgccgccac caccgctgcc accacatcta gcaatgtgag 180

cgtcctgcag cagtttgcca gtggcctaaa gagccggaat gaggaaacca gggccaaagc 240

cgccaaggag ctccagcact atgtcaccat ggaactccga gagatgagtc aagaggagtc 300

tactcgcttc tatgaccaac tgaaccatca catttttgaa ttggtttcca gctcagatgc 360

caatgagagg aaaggtggca tcttggccat agctagcctc ataggagtgg aaggtgggaa 420

tgccacccga attggcagat ttgccaacta tcttcggaac ctcctcccct ccaatgaccc 480

agttgtcatg gaaatggcat ccaaggccat tggccgtctt gccatggcag gggacacttt 540

taccgctgag tacgtggaat ttgaggtgaa gcgagccctg gaatggctgg gtgctgaccg 600

caatgaggc cggagacatg cagctgtcct ggttctccgt gagctggcca tcagcgtccc 660

taccttcttc ttccagcaag tgcaaccctt ctttgacaac atttttgtgg ccgtgtggga 720

ccccaaacag gccatccgtg agggagctgt agccgccctt cgtgcctgtc tgattctcac 780

aacccagcgt gagccgaagg agatgcagaa gcctcagtgg tacaggcaca catttgaaga 840

agcagagaag ggatttgatg agaccttggc caaagagaag ggcatgaatc gggatgatcg 900

gatccatgga gccttgttga tccttaacga gctggtccga atcagcagca tggagggaga 960

gcgtctgaga gaagaaatgg aagaaatcac acagcagcag ctggtacacg acaagtactg 1020

caaagatctc atgggcttcg gaacaaaacc tcgtcacatt acccccttca ccagtttcca 1080

ggctgtacag ccccagcagt caaatgcctt ggtggggctg ctggggtaca gctctcacca 1140

aggcctcatg ggatttggga cctcccccag tccagctaag tccaccctgg tggagagccg 1200

gtgttgcaga gacttgatgg aggagaaatt tgatcaggtg tgccagtggg tgctgaaatg 1260

caggaatagc aagaactcgc tgatccaaat gacaatcctt aatttgttgc cccgcttggc 1320

tgcattccga ccttctgcct tcacagatac ccagtatctc caagatacca tgaaccatgt 1380

cctaagctgt gtcaagaagg agaaggaacg tacagcggcc ttccaagccc tggggctact 1440

ttctgtggct gtgaggtctg agtttaaggt ctatttgcct cgcgtgctgg acatcatccg 1500

agcggccctg cccccaaagg acttcgccca taagaggcag aaggcaatgc aggtggatgc 1560

cacagtcttc acttgcatca gcatgctggc tcgagcaatg gggccaggca tccagcagga 1620

tatcaaggag ctgctggagc ccatgctggc agtgggacta agccctgccc tcactgcagt 1680

gctctacgac ctgagccgtc agattccaca gctaaagaag gacattcaag atgggctact 1740

gaaaatgctg tccctggtcc ttatgcacaa acccttcgc cacccaggca tgcccaaggg 1800

cctggcccat cagctggcct ctcctggcct cacgaccctc cctgaggcca gcgatgtggg 1860

cagcatcact cttgccctcc gaacgcttgg cagctttgaa tttgaaggcc actctctgac 1920

ccaatttgtt cgccactgtg cggatcattt cctgaacagt gagcacaagg agatccgcat 1980

ggaggctgcc cgcacctgct cccgcctgct cacaccctcc atccacctca tcagtggcca 2040

tgctcatgtg gttagccaga ccgcagtgca agtggtggca gatgtgctta gcaaactgct 2100

cgtagttggg ataacagatc ctgaccctga cattcgctac tgtgtcttgg cgtccctgga 2160

cgagcgcttt gatgcacacc tggcccaggc ggagaacttg caggccttgt ttgtggctct 2220

gaatgaccag gtgtttgaga tccgggagct ggccatctgc actgtgggcc gactcagtag 2280

catgaaccct gcctttgtca tgcctttcct gcgcaagatg ctcatccaga ttttgacaga 2340

gttggagcac agtgggattg gaagaatcaa agagcagagt gcccgcatgc tggggcacct 2400

ggtctccaat gccccccgac tcatccgccc ctacatggag cctattctga aggcattaat 2460

tttgaaactg aaagatccag accctgatcc aaacccaggt gtgatcaata atgtcctggc 2520

aacaatagga gaattggcac aggttagtgg cctggaaatg aggaaatggg ttgatgaact 2580

ttttattatc atcatggaca tgctccagga ttcctctttg ttggccaaaa ggcaggtggc 2640

tctgtggacc ctgggacagt tggtggccag cactggctat gtagtagagc cctacaggaa 2700

gtaccctact ttgcttgagg tgctactgaa ttttctgaag actgagcaga accagggtac 2760

acgcagagag gccatccgtg tgttagggct tttaggggct ttggatcctt acaagcacaa 2820

agtgaacatt ggcatgatag accagtcccg ggatgcctct gctgtcagcc tgtcagaatc 2880

caagtcaagt caggattcct ctgactatag cactagtgaa atgctggtca acatgggaaa 2940

cttgcctctg gatgagttct acccagctgt gtccatggtg gccctgatgc ggatcttccg 3000

agaccagtca ctctctcatc atcacaccat ggttgtccag gccatcacct tcatcttcaa 3060

gtccctggga ctcaaatgtg tgcagttcct gccccaggtc atgcccacgt tccttaacgt 3120

cattcgagtc tgtgatgggg ccatccggga atttttgttc cagcagctgg gaatgttggt 3180

gtcctttgtg aagagccaca tcagacctta tatggatgaa atagtcaccc tcatgagaga 3240

attctgggtc atgaacacct caattcagag cacgatcatt cttctcattg agcaaattgt 3300

ggtagctctt gggggtgaat ttaagctcta cctgccccag ctgatcccac acatgctgcg 3360

tgtcttcatg catgacaaca gcccaggccg cattgtctct atcaagttac tggctgcaat 3420

ccagctgttt ggcgccaacc tggatgacta cctgcattta ctgctgcctc ctattgttaa 3480

gttgtttgat gcccctgaag ctccactgcc atctcgaaag gcagcgctag agactgtgga 3540

ccgcctgacg gagtccctgg atttcactga ctatgcctcc cggatcattc accctattgt 3600

tcgaacactg gaccagagcc cagaactgcg ctccacagcc atggacacgc tgtcttcact 3660

tgtttttcag ctggggaaga agtaccaaat tttcattcca atggtgaata aagttctggt 3720

gcgacaccga atcaatcatc agcgctatga tgtgctcatc tgcagaattg tcaagggata 3780

cacacttgct gatgaagagg aggatccttt gatttaccag catcggatgc ttaggagtgg 3840

ccaaggggat gcattggcta gtggaccagt ggaaacagga cccatgaaga aactgcacgt 3900

cagcaccatc aacctccaaa aggcctgggg cgctgccagg agggtctcca aagatgactg 3960

gctggaatgg ctgagacggc tgagcctgga gctgctgaag gactcatcat cgccctccct 4020

gcgctcctgc tgggccctgg cacaggccta caacccgatg gccagggatc tcttcaatgc 4080

tgcatttgtg tcctgctggt ctgaactgaa tgaagatcaa caggatgagc tcatcagaag 4140

catcgagttg gccctcacct cacaagacat cgctgaagtc acacagaccc tcttaaactt 4200

ggctgaattc atggaacaca gtgacaaggg ccccctgcca ctgagagatg acaatggcat 4260

tgttctgctg ggtgagagag ctgccaagtg ccgagcatat gccaaagcac tacactacaa 4320

agaactggag ttccagaaag gccccacccc tgccattcta gaatctctca tcagcattaa 4380

taataagcta cagcagccgg aggcagcggc cggagtgtta gaatatgcca tgaaacactt 4440

tggagagctg gagatccagg ctacctggta tgagaaactg cacgagtggg aggatgccct 4500

tgtggcctat gacaagaaaa tggacaccaa caaggacgac cccagctga tgctgggccg 4560

catgcgctgc ctcgaggcct tgggggaatg gggtcaactc caccagcagt gctgtgaaaa 4620

gtggaccctg gttaatgatg agacccaagc caagatggcc cggatggctg ctgcagctgc 4680

atggggttta ggtcagtggg acagcatgga agaatacacc tgtatgatcc ctcgggacac 4740

ccatgatggg gcattttata gagctgtgct ggcactgcat caggacctct tctccttggc 4800

acaacagtgc attgacaagg ccaggacct gctggatgct gaattaactg cgatggcagg 4860

agagagttac agtcgggcat atggggccat ggtttcttgc cacatgctgt ccgagctgga 4920

ggaggttatc cagtacaaac ttgtccccga gcgacgagag atcatccgcc agatctggtg 4980

ggagagactg cagggctgcc agcgtatcgt agaggactgg cagaaaatcc ttatggtgcg 5040

gtcccttgtg gtcagccctc atgaagacat gagaacctgg ctcaagtatg caagcctgtg 5100

cggcaagagt ggcaggctgg ctcttgctca taaaacttta gtgttgctcc tgggagttga 5160

tccgtctcgg caacttgacc atcctctgcc aacagttcac cctcaggtga cctatgccta 5220

catgaaaaac atgtggaaga gtgcccgcaa gatcgatgcc ttccagcaca tgcagcattt 5280

tgtccagacc atgcagcaac aggcccagca tgccatcgct actgaggacc agcagcataa 5340

gcaggaactg cacaagctca tggcccgatg cttcctgaaa cttggagagt ggcagctgaa 5400

tctacaggc atcaatgaga gcacaatccc caaagtgctg cagtactaca gcgccgccac 5460

agagcacgac cgcagctggt acaaggcctg gcatgcgtgg gcagtgatga acttcgaagc 5520

tgtgctacac tacaaacatc agaaccaagc ccgcgatgag aagaagaaac tgcgtcatgc 5580

cagcggggcc aacatcacca acgccaccac tgccgccacc acggccgcca ctgccaccac 5640

cactgccagc accgagggca gcaacagtga gagcgaggcc gagagcaccg agaacagccc 5700

caccccatcg ccgctgcaga agaaggtcac tgaggatctg tccaaaaccc tcctgatgta 5760

cacggtgcct gccgtccagg gcttcttccg ttccatctcc ttgtcacgag gcaacaacct 5820

ccaggataca ctcagagttc tcaccttatg gtttgattat ggtcactggc cagatgtcaa 5880

tgaggcctta gtggaggggg tgaaagccat ccagattgat acctggctac aggttatacc 5940

tcagctcatt gcaagaattg atacgcccag acccttggtg ggacgtctca ttcaccagct 6000

tctcacagac attggtcggt accaccccca ggccctcatc tacccactga cagtggcttc 6060

taagtctacc acgacagccc ggcacaatgc agccaacaag attctgaaga acatgtgtga 6120

gcacagcaac accctggtcc agcaggccat gatggtgagc gaggagctga tccgagtggc 6180

catcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt tgtactttgg 6240

ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta tgatggaacg 6300

gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag atttaatgga 6360

ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc tcacccaagc 6420

ctgggacctc tattatcatg tgttccgacg aatctcaaag cagctgcctc agctcacatc 6480

cttagagctg caatatgttt ccccaaaact tctgatgtgc cgggaccttg aattggctgt 6540

gccaggaaca tatgacccca accagccaat cattcgcatt cagtccatag caccgtcttt 6600

gcaagtcatc acatccaagc agaggccccg gaaattgaca cttatgggca gcaacggaca 6660

tgagtttgtt ttccttctaa aaggccatga agatctgcgc caggatgagc gtgtgatgca 6720

gctcttcggc ctggttaaca cccttctggc caatgaccca acatctcttc ggaaaaacct 6780

cagcatccag agatacgctg tcatcccttt atcgaccaac tcgggcctca ttggctgggt 6840

tccccactgt gacacactgc acgccctcat ccgggactac agggagaaga agaagatcct 6900

tctcaacatc gagcatcgca tcatgttgcg gatggctccg gactatgacc acttgactct 6960

gatgcagaag gtggaggtgt ttgagcatgc cgtcaataat acagctgggg acgacctggc 7020

caagctgctg tggctgaaaa gccccagctc cgaggtgtgg tttgaccgaa gaaccaatta 7080

tacccgttct ttagcggtca tgtcaatggt tgggtatatt ttaggcctgg gagatagaca 7140

cccatccaac ctgatgctgg accgtctgag tgggaagatc ctgcacattg actttgggga 7200

ctgctttgag gttgctatga cccgagagaa gtttccagag aagattccat ttagactaac 7260

aagaatgttg accaatgcta tggaggttac aggcctggat ggcaactaca gaatcacatg 7320

ccacacagtg atggaggtgc tgcgagagca caaggacagt gtcatggccg tgctggaagc 7380

ctttgtctat gaccccttgc tgaactggag gctgatggac acaaatacca aaggcaacaa 7440

gcgatcccga acgaggacgg attcctactc tgctggccag tcagtcgaaa ttttggacgg 7500

tgtggaactt ggagagccag cccataagaa aacggggacc acagtgccag aatctattca 7560

ttctttcatt ggagacggtt tggtgaaacc agaggcccta aataagaaag ctatccagat 7620

tattaacagg gttcgagata agctcactgg tcgggacttc tctcatgatg acactttgga 7680

tgttccaacg caagttgagc tgctcatcaa acaagcgaca tcccatgaaa acctctgcca 7740

gtgctatatt ggctggtgcc ctttctggta actggaggcc cagatgtgcc catcacgttt 7800

tttctgaggc ttttgtactt tagtaaatgc ttccactaaa ctgaaaccat ggtgagaaag 7860

tttgactttg ttaaatattt tgaaatgtaa atgaaaagaa ctactgtata ttaaaagttg 7920

gtttgaacca actttctagc tgctgttgaa gaatatattg tcagaaacac aaggcttgat 7980

ttggttccca ggacagtgaa acatagtaat accacgtaaa tcaagccatt cattttgggg 8040

aacagaagat ccataacttt agaaatacgg gttttgactt aactcacaag agaactcatc 8100

ataagtactt gctgatggaa gaatgaccta gttgctcctc tcaacatggg tacagcaaac 8160

tcagcacagc caagaagcct caggtcgtgg agaacatgga ttaggatcct agactgtaaa 8220

gacacagaag atgctgacct cacccctgcc acctatccca agacctcact ggtctgtgga 8280

cagcagcaga aatgtttgca agataggcca aaatgagtac aaaaggtctg tcttccatca 8340

gacccagtga tgctgcgact cacacgcttc aattcaagac ctgaccgcta gtagggaggt 8400

ttattcagat cgctggcagc ctcggctgag cagatgcaca gaggggatca ctgtgcagtg 8460

ggaccaccct cactggcctt ctgcagcagg gttctgggat gttttcagtg gtcaaaatac 8520

tctgtttaga gcaagggctc agaaaacaga aatactgtca tggaggtgct gaacacaggg 8580

aaggtctggt acatattgga aattatgagc agaacaaata ctcaactaaa tgcacaaagt 8640

ataaagtgta gccatgtcta gacaccatgt tgtatcagaa taatttttgt gccaataaat 8700

gacatcagaa ttttaaacat atgtaaaaaa aaa 8733

<210> 3

<211> 108

<212>PRT

<213> Homo sapiens

<400> 3

Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu

20 25 30

Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

35 40 45

Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

50 55 60

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala

85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu

100 105

<210> 4

<211> 118

<212>PRT

<213> Homo sapiens

<400> 4

Met Ser Gly Gly Ser Ser Cys Ser Gln Thr Pro Ser Arg Ala Ile Pro

1 5 10 15

Ala Thr Arg Arg Val Val Leu Gly Asp Gly Val Gln Leu Pro Pro Gly

20 25 30

Asp Tyr Ser Thr Thr Pro Gly Gly Thr Leu Phe Ser Thr Thr Pro Gly

35 40 45

Gly Thr Arg Ile Ile Tyr Asp Arg Lys Phe Leu Met Glu Cys Arg Asn

50 55 60

Ser Pro Val Thr Lys Thr Pro Pro Arg Asp Leu Pro Thr Ile Pro Gly

65 70 75 80

Val Thr Ser Pro Ser Ser Asp Glu Pro Pro Met Glu Ala Ser Gln Ser

85 90 95

His Leu Arg Asn Ser Pro Glu Asp Lys Arg Ala Gly Gly Glu Glu Ser

100 105 110

Gln Phe Glu Met Asp Ile

115

<210> 5

<211> 480

<212>PRT

<213> Homo sapiens

<400> 5

Met Ser Asp Val Ala Ile Val Lys Glu Gly Trp Leu His Lys Arg Gly

1 5 10 15

Glu Tyr Ile Lys Thr Trp Arg Pro Arg Tyr Phe Leu Leu Lys Asn Asp

20 25 30

Gly Thr Phe Ile Gly Tyr Lys Glu Arg Pro Gln Asp Val Asp Gln Arg

35 40 45

Glu Ala Pro Leu Asn Asn Phe Ser Val Ala Gln Cys Gln Leu Met Lys

50 55 60

Thr Glu Arg Pro Arg Pro Asn Thr Phe Ile Ile Arg Cys Leu Gln Trp

65 70 75 80

Thr Thr Val Ile Glu Arg Thr Phe His Val Glu Thr Pro Glu Glu Arg

85 90 95

Glu Glu Trp Thr Thr Ala Ile Gln Thr Val Ala Asp Gly Leu Lys Lys

100 105 110

Gln Glu Glu Glu Glu Met Asp Phe Arg Ser Gly Ser Pro Ser Asp Asn

115 120 125

Ser Gly Ala Glu Glu Met Glu Val Ser Leu Ala Lys Pro Lys His Arg

130 135 140

Val Thr Met Asn Glu Phe Glu Tyr Leu Lys Leu Leu Gly Lys Gly Thr

145 150 155 160

Phe Gly Lys Val Ile Leu Val Lys Glu Lys Ala Thr Gly Arg Tyr Tyr

165 170 175

Ala Met Lys Ile Leu Lys Lys Glu Val Ile Val Ala Lys Asp Glu Val

180 185 190

Ala His Thr Leu Thr Glu Asn Arg Val Leu Gln Asn Ser Arg His Pro

195 200 205

Phe Leu Thr Ala Leu Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys

210 215 220

Phe Val Met Glu Tyr Ala Asn Gly Gly Glu Leu Phe Phe His Leu Ser

225 230 235 240

Arg Glu Arg Val Phe Ser Glu Asp Arg Ala Arg Phe Tyr Gly Ala Glu

245 250 255

Ile Val Ser Ala Leu Asp Tyr Leu His Ser Glu Lys Asn Val Val Tyr

260 265 270

Arg Asp Leu Lys Leu Glu Asn Leu Met Leu Asp Lys Asp Gly His Ile

275 280 285

Lys Ile Thr Asp Phe Gly Leu Cys Lys Glu Gly Ile Lys Asp Gly Ala

290 295 300

Thr Met Lys Thr Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val

305 310 315 320

Leu Glu Asp Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly

325 330 335

Val Val Met Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln

340 345 350

Asp His Glu Lys Leu Phe Glu Leu Ile Leu Met Glu Glu Ile Arg Phe

355 360 365

Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser Leu Leu Ser Gly Leu Leu

370 375 380

Lys Lys Asp Pro Lys Gln Arg Leu Gly Gly Gly Ser Glu Asp Ala Lys

385 390 395 400

Glu Ile Met Gln His Arg Phe Phe Ala Gly Ile Val Trp Gln His Val

405 410 415

Tyr Glu Lys Lys Leu Ser Pro Pro Phe Lys Pro Gln Val Thr Ser Glu

420 425 430

Thr Asp Thr Arg Tyr Phe Asp Glu Glu Phe Thr Ala Gln Met Ile Thr

435 440 445

Ile Thr Pro Pro Asp Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu

450 455 460

Arg Arg Pro His Phe Pro Gln Phe Ser Tyr Ser Ala Ser Gly Thr Ala

465 470 475 480

<---

Claims (41)

1. A connection selected from the group consisting of: Example 1 Example 2 Example 3 Example 4
Example 5
Example 6
Example 7
Example 8
Example 9
Example 10
Example 11
Example 12
Example 13
Example 14
Example 15
Example 16
Example 17
Example 18
Example 19
Example 20
Example 21
Example 22
Example 23
Example 24
Example 25
Example 26
Example 27
Example 28
Example 29
Example 30
Example 31
Example 32
Example 33
Example 34
Example 35
Example 36
Example 37
Example 38
Example 39
Example 40
Example 41
Example 42
Example 43
Example 44
Example 45
Example 46
Example 47
Example 48
Example 49
Example 50
Example 51
Example 52
Example 53
Example 54
Example 55
Example 56
Example 57
Example 58
Example 59
Example 60
Example 61
Example 62
Example 63
Example 64
Example 65
Example 66
Example 67
Example 68
Example 69
Example 70 or a pharmaceutically acceptable salt, isomer, stereoisomer or oxepane isomer thereof. 2. A connection selected from the group consisting of:
Example 71
Example 72
Example 73
Example 74
Example 75
Example 76
Example 77
Example 78
Example 79
Example 80
Example 81
Example 82
Example 83
Example 84
Example 85
Example 86
Example 87
Example 88
Example 89
Example 90
Example 91
Example 92
Example 93
Example 94
Example 95
Example 96
Example 97
Example 98
Example 99
Example 100
Example 101
Example 102
Example 103
Example 104
Example 105
Example 106
Example 107
Example 108
Example 109
Example 110
Example 111
Example 112
Example 113
Example 114
Example 115
Example 116
Example 117
Example 118
Example 119
Example 120
Example 121
Example 122
Example 123
Example 124
Example 125
Example 126
Example 127
Example 128
Example 129
Example 130
Example 131
Example 132
Example 133
Example 134
Example 135 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 3. A connection selected from the group consisting of:
Example 136
Example 137
Example 138
Example 139
Example 140
Example 141
Example 142
Example 143
Example 144
Example 145
Example 146
Example 147
Example 148
Example 149
Example 150
Example 151
Example 152 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 4. A compound having the formula or its stereoisomer. 5. A compound having the formula or its tautomer. 6. A compound having the formula or its oxepane isomer. 7. A compound having the formula . 8. A pharmaceutical composition for inhibiting the mammalian target of rapamycin (mTOR), containing an effective amount of a compound according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof, and at least one of a pharmaceutically acceptable carrier, diluent or excipient. 9. A method of treating a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or at risk of developing a disease or disorder mediated by mTOR, a therapeutically effective amount of one or more compounds according to any one of claims. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 10. A method of preventing a disease or disorder mediated by mTOR, comprising administering to a subject suffering from or at risk of developing a disease or disorder mediated by mTOR, a therapeutically effective amount of one or more compounds according to any one of claims. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 11. A method of reducing the risk of an mTOR-mediated disease or disorder, comprising administering to a subject suffering from or at risk of developing an mTOR-mediated disease or disorder a therapeutically effective amount of one or more compounds according to any one of claims. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 12. Method according to any one of paragraphs. 9-11, where the disease is cancer or an immune-mediated disease. 13. The method of claim 12, wherein the cancer is selected from brain and neurovascular tumors, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, cancer ovarian, ovarian endometriosis, testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, blood proliferation disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, cancer vulva, prostate cancer, penile cancer, bone cancer, muscle cancer, soft tissue cancer, bowel or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors. 14. The method of claim 12, wherein the immune-mediated disease is selected from resistance in transplantation of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine or islet pancreatic cells; graft-versus-host diseases caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis. 15. A method of treating cancer, comprising administering to a subject a therapeutically effective amount of one or more compounds according to any one of claims. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 16. The method of claim 15, wherein the cancer is selected from brain and neurovascular tumors, head and neck cancer, breast cancer, lung cancer, mesothelioma, lymphoid cancer, gastric cancer, kidney cancer, renal carcinoma, liver cancer, cancer ovarian, ovarian endometriosis, testicular cancer, gastrointestinal cancer, prostate cancer, glioblastoma, skin cancer, melanoma, nerve cancer, spleen cancer, pancreatic cancer, blood proliferation disorders, lymphoma, leukemia, endometrial cancer, cervical cancer, cancer vulva, prostate cancer, penile cancer, bone cancer, muscle cancer, soft tissue cancer, bowel or rectal cancer, anal cancer, bladder cancer, bile duct cancer, eye cancer, gastrointestinal stromal tumors and neuroendocrine tumors. 17. A method of treating an immune-mediated disease, comprising administering to a subject a therapeutically effective amount of one or more compounds according to any one of claims. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 18. The method of claim 17, wherein the immune-mediated disease is selected from resistance in transplantation of heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine or islet pancreatic cells; graft-versus-host diseases caused by bone marrow transplantation; rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis and glomerulonephritis. 19. A method of treating an age-related condition, comprising administering to a subject a therapeutically effective amount of one or more compounds according to any one of claims. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof. 20. The method of claim 19, wherein the age-related condition is selected from sarcopenia, skin atrophy, muscle atrophy, brain atrophy, atherosclerosis, arteriosclerosis, emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia, Huntington's disease, Alzheimer's disease , cataracts, age-related macular degeneration, prostate cancer, stroke, shortened life expectancy, kidney dysfunction and age-related hearing loss, aging-related disabilities (eg, frailty), cognitive decline, age-related dementia, memory impairment, tendon stiffness, heart disease dysfunctions such as cardiac hypertrophy and systolic and diastolic dysfunction, immune aging, cancer, obesity and diabetes. 21. Connection according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof for use in the treatment, prevention or reduction of the risk of a disease or condition mediated by mTOR. 22. Use of a compound according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof in the manufacture of a medicament for the treatment, prevention or reduction of the risk of a disease or condition mediated by mTOR. 23. The connection according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof for use in the treatment of cancer. 24. Use of a compound according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof in the manufacture of a drug for the treatment of cancer. 25. Connection according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof for use in the treatment of an immune-mediated disease. 26. Use of a compound according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof in the manufacture of a medicament for the treatment of an immune-mediated disease. 27. The connection according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomerb thereof for use in the treatment of an age-related condition. 28. Use of a compound according to any one of paragraphs. 1-7 or a pharmaceutically acceptable salt, tautomer, stereoisomer or oxepane isomer thereof in the manufacture of a medicament for treating an age-related condition.