EA039109B1 - Bridged bicyclic inhibitors of menin-mll interaction and methods of use - Google Patents

Abstract

The invention provides compounds of formula (I-A) that are useful for inhibiting the interaction of menin with MLL1, MLL2 oncoproteins and MLL fusion oncoproteins. Also provided are compositions and methods. The methods are useful for the treatment of hematologic malignancy or solid tumor cancer, in particular leukemia, prostate cancer, breast cancer, liver cancer, brain tumor and other diseases dependent on activity of MLL1, MLL2 proteins, MLL fusion proteins and/or menin.

Description

cross reference

This application claims priority to U.S. Provisional Application No. 62/309362, filed March 16, 2016, and U.S. Provisional Application No. 62/431387, filed December 7, 2016, which are incorporated herein by reference.

Prerequisites for the creation of the invention

The mixed lineage leukemia (MLL) protein is a histone methyltransferase key to the epigenetic regulation of gene transcription. Many acute leukemias, including acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and mixed line leukemia (MLL), are characterized by the presence of chimeric MLL fusion proteins that result from chromosomal translocations of the MLL gene located on chromosome 11, band q23 (11q23 ). Chimeric MLL fusion proteins contain approximately 1400 amino acids of the MLL N-terminus, but are fused to one of approximately 80 partner proteins (eg, AF4, AF9, ENL, AF10, ELL, AF6, AF1p, GAS7). MLL fusion proteins lack the original MLL C-terminal histone methyltransferase activity and acquire the ability to regulate the transcription of numerous oncogenes, including HOX and MEIS1, leading to increased cell proliferation, decreased cell differentiation, and ultimately leads to leukogenesis.

The protein menin, which is encoded by the Multiple Endocrine Neoplasia (MEN) gene, is a ubiquitously expressed nuclear protein that is involved in interactions with DNA processing and repair proteins, chromatin-modifying proteins, and numerous transcription factors (Agarwal, et al.; Horm Metab Res, 2005, 37(6): 369-374). Association of menin to the N-terminus of MLL fusion proteins is required for the observed oncogenic activity of MLL fusion proteins. This association has been shown to constitutively increase the expression of the HOX and MEIS 1 oncogenes and impair the proliferation and differentiation of hematopoietic cells, leading to the development of leukemia. Since menin acts as a common oncogenic cofactor in MLL-associated leukemias, the interaction between menin and MLL and MLL fusion proteins has been shown to represent a potential chemotherapeutic target.

Patients, especially neonates, with leukemias carrying chromosomal translocations of the MLL gene have a poor prognosis with less than 40% five-year survival (Slany; Haematologica, 2009, 94(7): 984-993). A new therapeutic strategy is urgently needed to treat these leukemias. Thus, small molecule inhibitors that block meninMLL interaction are valuable targets for the treatment of diseases associated with MLL fusion proteins.

Brief description of the invention

The present disclosure relates to a need that exists in the art to provide compositions and methods for inhibiting the protein-protein interaction of menin with the MLL1, MLL2 oncoproteins and the MLL oncoprotein fusion. The compositions and methods of the present invention may be useful in the treatment of diseases dependent on the activity of MLL1, MLL2, MLL fusion protein and/or menin proteins such as leukemia, solid cancers and diabetes. In some embodiments, a compound of the present invention interacts non-covalently with menin and inhibits the interaction of menin with MLL. In some embodiments, a compound of the present invention covalently binds to menin and inhibits the interaction of menin with MLL.

In one aspect, a compound of formula (I-A):

or a pharmaceutically acceptable salt thereof, wherein

R ¹ is C _1-3 haloalkyl;

R ² is hydrogen, -CH ₃ , -NH ₂ or -NHCH3;

R ³ is hydrogen;

each of R ^a and R ^B is independently selected from -OH, -CN and -CH3;

^RC selected from

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L ² and L ³ are each independently selected from C _1-4 alkylene;

each of m and n is independently an integer from 0 to 3;

W ¹ , W ² and W ³ are each independently selected from C _1-4 alkylene;

R ⁵¹ is hydrogen.

In one embodiment, the compound has the structure of formula (I-C):

In yet another embodiment, the compound has the structure of formula (I-E):

and W is C ₁ -alkylene. In still

In one embodiment, each of W ¹ , W' in one embodiment of the invention RC is selected from

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In yet another embodiment, R ¹ is -CH2CF3. In yet another embodiment, m is 0. In yet another embodiment, n is an integer from 1 to 3. In yet another embodiment, L2 is C1-2 alkylene. In yet another embodiment, L ² is -CH2. In yet another embodiment, L ³ is C _1-2 alkylene. In yet another embodiment, L ³ is -CH2-. In yet another embodiment, the compound of formula (I) is selected from:

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In yet another aspect of the invention, a pharmaceutical composition is provided comprising a compound as described herein and a pharmaceutically acceptable carrier.

In yet another aspect of the invention, a method is provided for inhibiting the interaction of menin with one or more of MLL1, MLL2, MLL fusion protein, and partial tandem duplication MLL, comprising contacting menin with an effective amount of a compound described herein.

In yet another aspect of the invention, there is provided a method of treating a hematologic or solid cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound described herein, or a pharmaceutical composition thereof. In one embodiment, the hematologic or solid cancer is selected from leukemia, prostate cancer, breast cancer, liver cancer, and brain tumor.

Inclusion by reference

All publications, patents, and patent applications referred to in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually identified as being incorporated by reference.

Brief description of the drawings

The novel features of the invention are detailed in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which presents illustrative embodiments using the principles of the invention, and the accompanying drawings, of which: FIG. 1 shows the amino acid sequence of human menin isoform 1 (SEQ ID NO: 1);

in fig. 2 shows the amino acid sequence of human menin isoform 2 (SEQ ID NO: 2);

in fig. 3 shows the amino acid sequence of human menin isoform 3 (SEQ ID NO: 3);

in fig. 4 shows the change in volume of MV4 tumors; 11 in mice treated with vehicle and Compound A.

in fig. 5 shows the change in volume of MV4 tumors; 11 in mice treated with vehicle and Compound B.

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Detailed description of the invention

In some embodiments of a compound provided herein, the compound non-covalently or covalently binds to any one or more isoforms of menin, e.g. isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2 (SEQ ID NO: 2 ) or menin isoform 3 (SEQ ID NO: 3) In certain embodiments, the menin protein has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity with isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2) or isoform 3 (SEQ ID NO: 3).In one aspect, the present invention provides compound of formula (I)

or a pharmaceutically acceptable salt, isotopic form or prodrug thereof, wherein

H is selected from C ₅ -l ₂ carbocycle and 5-12 membered heterocycle, each of which is optionally substituted with one or more R ⁵⁰ ;

A is selected from a bond, a C _3-12 carbocycle, and a 3-12 membered heterocycle;

B is selected from C _3-12 carbocycle and 3-12 membered heterocycle;

each of L ¹ , L ² and L ³ is independently selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH ₂ -, -C(O)-. -C(O)O-, -OC(O)-. -OC(O)O-, -C(O)N(R ⁵¹ )-, -C(O)N(R ⁵¹ )C(O)-. C(O)N(R ⁵¹ )C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-,

-N(R ⁵¹ )C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)O-, -OC(O)N(R ⁵¹ )-, -C(NR ⁵¹ )-, -N(R ⁵¹ )C(NR ⁵¹ )-, -C(NR ⁵¹ )N(R ⁵¹ )-, -N(R ⁵¹ )C(NR ⁵¹ )N(R ⁵¹ )-, -S(O)2 -, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R ⁵¹ )S(O) 2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)-, -S(O)N(R ⁵¹ )-, -N(R ⁵¹ )S(O) ₂ N(R ⁵¹ )-, -N(R ⁵¹ )S(O)N(R ⁵¹ )-; alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene and heteroalkynylene, each of which is optionally substituted with one or more R ⁵⁰ ;

each of RA, RB and RC is independently selected at each occurrence from R ⁵⁰ , or two RA groups or two R ^B groups attached to the same or different atoms together may optionally form a bridge or ring;

each of m and n is independently an integer from 0 to 6;

W ¹ is C _1-4 alkylene optionally substituted with one or more R ⁵⁰ ;

W ² is selected from the link; and C _1-4 alkylene optionally substituted with one or more R ⁵⁰ ;

W ³ is selected from absence; and C _1-4 alkylene optionally substituted with one or more R ⁵⁰ ;

R ⁵⁰ is independently selected at each occurrence from halogen, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2 , -NR53R ⁵⁴ , -S(=O)R ⁵² , -S(=O )2R ⁵² , -S(=OEN(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² ) 2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² ,

-C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C (O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =o, =s, =N(R ⁵² );

C _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halogen. -NO ₂ , -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S( =O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2, -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² c(o)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -nr ⁵² c(o)nr ⁵³ r ⁵⁴ , -c(o)n(r ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, ═O, ═S, ═N(R ⁵² ), C3-12 carbocycle and 3-12 membered heterocycle; and

C _3-12 carbocycle and 3-12 membered heterocycle;

wherein each C _3-12 carbocycle and 3-12 membered heterocycle in R ⁵⁰ is independently optionally substituted with one or more substituents selected from halogen. -NO2, -CN, -OR ⁵² . -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, - S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , - C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -nr ⁵² c(O)r ⁵² , -nr ⁵² c(O)or ⁵² , -nr ⁵² c(O)N(r ⁵² )2, -nr ⁵² c(O)nr ⁵³ r ⁵⁴ . -c(O)N(r ⁵² )2, -c(o)nr ⁵³ r ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C _1-6 -alkyl. C _1-6 haloalkyl. C _2-6 alkenyl and C _2-6 alkynyl;

R ⁵¹ is independently selected at each occurrence from hydrogen. -C(O)R ⁵² , -C(O)OR ⁵² , -C(O)N(R ⁵² ) ₂ , -C(O)NR ⁵³ R ⁵⁴ ;

C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halogen, -NO ₂ , -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, - S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , - C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2 , -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , - C(O)N(R ⁵² )2,

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-C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C _3- l ₂ -carbocycle and 3-12-membered heterocycle;

and a C ₃ -l ₂ carbocycle and a 3-12 membered heterocycle, wherein each C ₃ -l ₂ carbocycle and 3-12 membered heterocycle in R ⁵¹ is independently optionally substituted with one or more substituents selected from halo, -NO2 , -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O) 2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(= O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C( O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2 , ═O, ═S, ═N(R ⁵² ), Ci-6-alkyl, Ci-6-haloalkyl, C _2-6 alkenyl, and C _2-6 alkynyl;

R ⁵² is independently selected at each occurrence from hydrogen; and C _1-20 -αlkyl, C _2-20 -αlkenyl, C _2-20 alkynyl, 2-6 membered heteroalkyl, 03-12-carbocycle and 3-12 membered heterocycle, each of which is optionally substituted with halogen, -CN , -NO2, -NH ₂ , -NHCH ₃ , -NHCH2CH3, =O, -OH, -OCH ₃ , -OCH ₂ CH ₃ , C _3-12 carbocycle or 3-6-membered heterocycle; and

R ⁵³ and R ⁵⁴ taken together with the nitrogen atom to which they are attached form a heterocycle optionally substituted with one or more R ⁵⁰ , while for a compound or salt of formula (I), when W ³ is absent,

W ¹ is C ₁ -αkylene, W ² is a bond, and L ³ is not a bond;

W ¹ is C _2-4 alkylene and W ² is a bond; or each of W ¹ and W ² is C1-alkylene and L ³ is not a bond, each C1-alkylene is optionally substituted with one or more R ⁵⁰ , for a compound of formula (I) each of W ¹ , W ² and W ³ may be independently selected from C1-4-alkylene, with each C1-4-alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ¹ , W ² , and W ³ are each independently C1-alkylene. In some embodiments, W ¹ and W ² are each C1-alkylene and W ³ is absent. ^In some embodiments, RC is selected from -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -C(O)R ⁵² , -C(O)OR ⁵² , - NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O) N(R ⁵² )2 and -C(O)NR ⁵³ R ⁵⁴ . In some embodiments, for a compound of formula (I), H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; and B is a 3-12 membered heterocycle. In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ . In some embodiments, H is thienopyrimidinyl optionally substituted with one or more R ⁵⁰ .

In some embodiments, for a compound of formula (I), H is

each of X ¹ and X ² is independently selected from CR ² and N; each of X ³ and X ⁴ is independently selected from C and N; each of Y ¹ and Y ² is independently selected from CR ³ , N, NR ⁴ , O and S; each of R ¹ , R ² and R ³ is independently selected at each occurrence from hydrogen and R ⁵⁰ ; and R ⁴ is selected from R ⁵¹ . In some embodiments, X ³ and X ⁴ are each C. In some embodiments, X ¹ is CR ² and R ² is selected from hydrogen, halo, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C _1-3 -alkyl, C _1-3 -alkyl-N(R ⁵² )2, C1-3 haloalkyl, C2-3 alkenyl and C2-3 alkynyl. In some embodiments, X ² is N. In some embodiments, X ¹ is CR ² and R ² is selected from hydrogen, halo, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN , C1-3-αalkyl, -CH2OH, -CH2OR ⁵² , -CH2NH2, -CH2N(R ⁵² )2, C1-3-αalkyl-N(R ⁵² )2, C1-3-haloalkyl, C2-3-alkenyl and C2-3 alkynyl. In some embodiments, Y ² is CR ³ and R ³ is selected from hydrogen, halogen, -OH, -N(R ⁵² )2, -CN, -C(O)OR ⁵² , C1-3-αkyl, and C _{1 -3} -haloalkyl. In some embodiments, R ¹ is C 1 _-3 haloalkyl.

For a compound of formula (I), A may be a 5- to 8-membered heterocycle, such as a 6-membered monocyclic heterocycle. In some embodiments, the implementation of the heterocycle contains at least one nitrogen atom. In some embodiments, A is selected from piperidinylene and piperazinylene, such as

For a compound of formula (I), B may be a 6-12 membered bicyclic heterocycle. In some embodiments, the implementation of the heterocycle contains at least one nitrogen atom. In some embodiments, B is an indolylene such as

- 9 039109 optionally substituted with one or more R ^B .

In some embodiments of a compound of formula (I), H is a thienopyrimidinyl substituted with one or more R ⁵⁰ ; A is selected from piperidinylene and piperazinylene; and B is indolylene.

In some embodiments, for a compound of formula (I), H is substituted with -CH ₂ CF ₃ . In some embodiments, m is 0. In some embodiments, n is an integer from 1 to 3. In some embodiments, L ¹ contains less than 10 atoms. In some embodiments, L ¹ is -N(R ⁵¹ )-. In some embodiments, L2 contains less than 10 atoms. In some embodiments, L2 is C1_4 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L2 is selected from -CH2-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -N(R ⁵¹ )C(O)- and -N(R ⁵¹ )S(O) 2-. In some embodiments, L ³ contains less than 20 atoms. In some embodiments, L ³ is C1_4 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ³ is -CH2-. In some embodiments, the compound of formula (I) is selected from the compounds shown in Table 1. one.

In certain aspects, the present invention provides a pharmaceutical composition containing a compound or salt of formula (I) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for injection.

In some aspects, the present invention provides a method for inhibiting the interaction of menin with one or more of an MLL1, MLL2 protein, MLL fusion protein, and partial tandem duplication MLL protein, comprising contacting menin with an effective amount of a compound or salt of formula (I). In some aspects, the present invention provides a method for inhibiting a menin-MLL interaction, comprising contacting menin with an effective amount of a compound or salt of formula (I), inhibition of the interaction being evidenced by a decrease in expression of the MLL fusion protein target gene. In some aspects, the present invention provides a method for stabilizing menin, comprising contacting menin with a compound or salt of formula (I).

In the practice of any of the contemplated methods, the MLL fusion protein target gene may be HOXA9, DLX2, or MEIS1. Bringing into contact may be contact with a cell that expresses menin. In some embodiments, the method includes administering a second therapeutic agent. In some embodiments, the implementation of bringing into contact occurs in vivo. In some embodiments, the implementation of bringing into contact occurs in vitro.

In some aspects, the present invention provides a method of treating a disease or condition associated with MLL fusion proteins, comprising administering to a subject in need thereof an effective amount of a compound or salt of formula (I). In some aspects, the present invention provides a method of treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of a compound or salt of formula (I). In some embodiments, the disease or condition includes leukemia, hematologic cancer, solid cancer, prostate cancer, breast cancer, liver cancer, brain tumor, or diabetes. In some embodiments, the leukemia includes AML, ALL, mixed line leukemia, or MLL partial tandem duplication leukemia.

In some aspects, the present invention provides a method of treating a disorder mediated by a chromosomal rearrangement on chromosome 11q23 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound or salt of formula (I). In some aspects, the present invention provides a method of treating a disorder mediated by an interaction between menin and another protein, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or salt of formula (I). In some embodiments, the subject is a human.

In some aspects, the present invention provides a kit containing a pharmaceutical composition described herein and instructions for using the composition to treat a subject suffering from a disease or condition mediated by an interaction between menin and another protein.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as generally understood by a person skilled in the art to which this invention pertains.

An MLL fusion protein refers to a protein with an N-terminal MLL fragment fused to a partner protein. Non-limiting examples of a partner protein include 11q23, 11q23.3, 11q24, 1p13.1, 1p32 (EPS15), 21q22, 9p13.3, 9p22 (MLLT3/AF9), ABI1, ABI2, ACACA, ACTN4, AFF1/AF4, AFF3/ LAF4, AFF4/AF5, AKAP13, AP2A2, ARHGEF12, ARHGEF17, BCL9L, BTBD18, BUD13, C2CD3, CASC5,

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CASP8AP2 CBL CEP164 CEP170B CREBBP DCP1A DCPS EEFSEC/SELB ELL EPS15 FLNA FNBP1 FOXO3 GAS7 GMPS KIAA1524 LAMC3 LOC100131626 MAML2 ME2 MLLT1/ENL MLLT10/AF10 MLLT11/AF1Q, MLLT3/AF9, MLLT4/AF6, MLLT6/AF17, MYH11, MY01F, NA, NEBL, NRIP3, PDS5A, PICALM, PRPF19, PTD, RUNDC3B, SEPT11, SEPT2, SEPT5, SEPT6, SEPT9, SMAP1, TET1, TNRC18, TOP3A, VAV1 and Xq26.3 (CT45A2). MLL fusion proteins can be created by joining a gene encoding an MLL protein with a gene encoding a partner protein to form a fusion gene. Translation of this fusion gene can result in one or more polypeptides with functional properties derived from each of the original proteins.

The term C _xy or C _x -C _y when used in conjunction with a chemical moiety such as alkyl, alkenyl, or alkynyl includes groups that contain x to y carbon atoms in the chain. For example, the term Cx-y-alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight and branched chain alkyl groups, which contain from x to y carbon atoms in the chain. The terms Cx-y-alkenyl and Cx-y-alkyiyl refer to straight or branched substituted or unsubstituted unsaturated hydrocarbon groups that contain at least one double or triple bond, respectively. Unless otherwise indicated in the description, C _x _ _y alkyl, Cx.y-alkenyl, or Cx.y-alkynyl are optionally substituted with one or more substituents, such as those described herein.

Carbocycle refers to a saturated, unsaturated, or aromatic ring in which each ring atom is a carbon atom. The carbocycle may include 3-10 membered monocyclic rings, 6-12 membered bicyclic rings and 6-12 membered bridged rings. Each bicyclic carbocycle ring can be selected from saturated, unsaturated and aromatic rings. In some embodiments, the carbocycle is aryl. In some embodiments, the carbocycle is cycloalkyl. In some embodiments, the carbocycle is a cycloalkenyl. In an exemplary embodiment, an aromatic ring, such as phenyl, may be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings, if valence permits, is included in the definition of carbocyclic. Examples of carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl and naphthyl. Unless otherwise specifically stated in the description, the carbocycle is optionally substituted with one or more substituents, such as the substituents described herein.

A heterocycle refers to a saturated, unsaturated, or aromatic ring containing one or more heteroatoms. Examples of heteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3-10 membered monocyclic rings, 6-12 membered bicyclic rings, and 6-12 membered bridged rings. Each bicyclic heterocycle ring can be selected from saturated, unsaturated and aromatic rings. The heterocycle can be attached to the rest of the molecule through any atom of the heterocycle, if the valence permits, such as the carbon or nitrogen atom of the heterocycle. In some embodiments, the heterocycle is heteroaryl. In some embodiments, the heterocycle is heterocycloalkyl. In an exemplary embodiment, a heterocycle, such as pyridyl, may be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene.

Heteroaryl refers to a 3-12 membered aromatic ring that contains at least one heteroatom, where each heteroatom can be independently selected from N, O, and S. As used herein, the heteroaryl ring can be selected from monocyclic or bicyclic and fused or bridged ring systems, wherein at least one of the rings in the ring system is aromatic, i. it contains a cyclic, delocalized (4n+2) π-electron system in accordance with Hückel's theory. The heteroatom(s) in heteroaryl may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl may be attached to the rest of the molecule through any heteroaryl atom, if valency permits, such as the carbon or nitrogen of the heteroaryl. Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzdolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]-dioxepinyl, benzo[b] [1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxynyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]-pyrimidinyl, benzotriazolyl, benzo[4 ,6]imidazo[1,2a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5Hcyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[I ]quinazolinyl, 5,6dihydrobenzo[I]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2u]pyridinyl , 5,6,7,8,9,10hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10hexahydrocycloocta[d] pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, in dolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8

- 11 039109 tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H -pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl , pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl , 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido-[4,5c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno-[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl and thiophenyl (ie thienyl). Unless otherwise specifically stated in the specification, the term heteroaryl means heteroaryls, as defined above, which are optionally substituted with one or more substituents, such as those substituents described herein.

The compounds of the present invention also include crystalline and amorphous forms of these compounds, pharmaceutically acceptable salts and active metabolites of these compounds having the same type of activity, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, non-solvated polymorphs (including anhydrates), conformational polymorphs and amorphous forms of compounds, as well as their mixtures.

The compounds described herein may exhibit their natural isotopic abundance, or one or more atoms may be artificially enriched in a particular isotope having a similar atomic number but an atomic mass or mass number different from the atomic mass or mass number of the atom predominantly found in nature. All isotopic variants of the compounds of the present invention, whether radioactive or not, are within the scope of the present invention. For example, hydrogen has three naturally occurring isotopes, designated 1H (protium), 2H ⁽ deuterium), and 3H (tritium). Protium is the predominant isotope of hydrogen found in nature. Deuterium enrichment may confer certain therapeutic benefits, such as increased in vivo half-life and/or dosage reduction, or may provide a compound useful in in vivo studies of drug elimination and metabolism pathways. Compounds enriched in isotopes can be prepared by conventional methods well known to those skilled in the art.

Isomers are different compounds that have the same molecular formula. Stereoisomers are isomers that differ only in the arrangement of atoms in space. Enantiomers are a pair of stereoisomers that are non-overlapping mirror images of each other. A 1:1 mixture of enantiomer pairs is a racemic mixture. The term (±) is used to refer to a racemic mixture when appropriate. Diastereoisomers or diastereomers are stereoisomers that have at least two asymmetric atoms, but which are not mirror images of each other. Absolute stereochemistry was established according to the Cahn-Ingold-Prelog R-S system of rules. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon atom may be indicated by either R or S. Separated compounds whose absolute configuration is unknown are denoted (+) or (-) depending on the direction (right- or left-handed) rotation of the polarized plane. light at a wavelength of the sodium D line. Some of the compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers and other stereoisomeric forms whose asymmetric centers can be defined in terms of absolute stereochemistry as (R)- or (S)-. Chemical compounds, pharmaceutical compositions, and methods are intended to include all such possible stereoisomers, including racemic mixtures, optically pure forms, mixtures of diastereomers, and mixtures of intermediates. Optically active (R)- and (S)-isomers can be obtained using chiral synthons or chiral reagents or separated using conventional techniques. The optical activity of a compound can be analyzed by any suitable method, including, but not limited to, chiral chromatography and polarimetry, and the degree of predominance of one stereoisomer over the other isomer can be determined.

Chemical compounds having carbon-carbon double bonds or carbon-nitrogen double bonds can exist in Z- or E-form (or cis- or trans-form). In addition, some chemical compounds may exist in various tautomeric forms. Unless otherwise indicated, the chemical compounds described herein also include all Z-, E- and tautomeric forms.

The term substituted refers to moieties having substituents replacing hydrogen on one or more carbon atoms or heteroatoms of the structure. It should be understood that substitution or substituted by something includes the specific condition that such substitution occurs in accordance with the allowable valence of the atom being replaced and the substituent, and that the substitution results in a stable compound, for example one that does not undergo spontaneous transformation, such as rearrangement, cyclization, elimination, etc. As used herein, the term substituted is intended to include all permissible substituents on organic compounds. Broadly acceptable substituents include acyclic and cyclic, branched and

- 12 039109 linear, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Allowed substituents may be the same or different for suitable organic compounds. For the purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any valid substituents of the organic compounds described herein that satisfy the valences of the heteroatoms. Substituents may include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate , amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, carbocycle, heterocycle, cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic moiety. In some embodiments, the substituents may include any of the substituents described herein, such as halogen, hydroxy, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazino (=N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC(O)-R ^a , -R ^b -OC(O)-OR ^a , -R ^b -OC(O)-N(R ^a )2, -R ^b -N(R ^a )2, -R ^b -C(O)R ^a , -R ^b -C(O)OR ^a , -R ^b - C(O)N(R ^a )2, -R ^b -OR ^c -C(O)N(R ^a )2, -R ^b -N(R ^a )C(O)OR ^a , -R ^b -N (R ^a )C(O)R ^a , -R ^b -N(R ^a )S(O)tR ^a (where t is 1 or 2), -R ^b -S(O)tR ^a (where t is 1 or 2), -R ^b -S(O)tOR ^a (where t is 1 or 2), and -R ^b -S(O)tN(R ^a )2 (where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl and heteroarylalkyl, any of which may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R ^b - OR ^a , -R ^b -OC(O)-R ^a , -R ^b -OC(O)-OR ^a , -R ^b -OC(O)-N(R ^a )2, -R ^b -N(R ^a )2, -R ^b -C(O)R ^a , -R ^b -C(O)OR ^a , -R ^b -C(O)N(R ^a )2, -R ^b -OR ^c -C( O)N(R ^a )2, -R ^b -N(R ^a )C(O)OR ^a , -R ^b -N(R ^a )C(O)R ^a , -R ^b -N(R ^a ) S(O)tR ^a (where t is 1 or 2), -R ^b -S(O)tR ^a (where t is 1 or 2), -R ^b -S(O)tOR ^a (where t is 1 or 2) and -R ^b -S(O)tN(R ^a )2 (where t is 1 or 2); where each R ^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, where each R ^a , if valency allows, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), - R ^b -OR ^a , -R ^b -OC(O)-R ^a , -R ^b -OC(O)-OR ^a , -R ^b -OC(O)-N(R ^a )2, -R ^b - N(R ^a )2, -R ^b -C(O)R ^a , -R ^b -C(O)OR ^a , -R ^b -C(O)N(R ^a )2, -R ^b -OR ^c -C(O)N(R ^a )2, -R ^b -N(R ^a )C(O)OR ^a , -R ^b -N(R ^a )C(O)R ^a , -R ^b -N( R ^a )S(O)tR ^a (where t is 1 or 2), -R ^b -S(O)tR ^a (where t is 1 or 2), -R ^b -S(O)tOR ^a (where t is 1 or 2) and -R ^b -S(O)tN(R ^a ) ₂ (where t is 1 or 2); and wherein each R ^b is independently selected from a direct bond or a straight or branched alkylene, alkenylene or alkynylene chain, and each R ^c is a straight or branched alkylene, alkenylene or alkynylene chain.

Those skilled in the art will appreciate that the substituents themselves may be substituted if desired. Unless specifically indicated as unsubstituted, references to chemical moieties herein are to be understood as including substituted variants. For example, reference to a heteroaryl group or moiety is implicitly inclusive of both substituted and unsubstituted variants.

Where substituent groups are designated by their conventional left-to-right chemical formulas, they equally cover chemically identical substituents that result from right-to-left writing of the structure, for example -CH ₂ O- implies also reading -OCH2-.

The term salt or pharmaceutically acceptable salt refers to salts derived from various organic and inorganic counterions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be formed include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be formed include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be obtained by adding inorganic and organic bases. Salts derived from inorganic bases include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, for example, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as, for example, isopropylamine, trimethylamine, diethylamine , triethylamine, tripropylamine and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.

The term effective amount or therapeutically effective amount refers to

- 13 039109 the amount of the compound described herein, which is sufficient to affect the intended use, including, but not limited to, the treatment of diseases, as defined below. The therapeutically effective amount may vary depending on the intended use in the treatment (in vivo) or on the subject and condition of the disease being treated, such as the weight and age of the subject, the severity of the disease, the route of administration, etc., which can be easily determined by a person skilled in the art. in this area. The term also refers to a dose that will elicit a specific response in target cells, such as reduced platelet adhesion and/or cell migration. The specific dosage will vary depending on the specific compounds chosen, the dosage regimen to be followed, whether administered in combination with other compounds, the time of administration, the tissue into which it is administered, and the physical delivery system that carries it.

As used herein, the term treatment or cure refers to an approach to obtain beneficial or desired results with respect to a disease, disorder, or medical condition, including, but not limited to, therapeutic benefit and/or prophylactic benefit. Therapeutic benefit refers to the eradication or reduction of the underlying disease being treated. In addition, the therapeutic effect is achieved by eradicating or alleviating one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the patient despite the fact that the patient may still be affected by the underlying disorder. In some embodiments, for prophylactic benefit, the compositions are administered to a subject at risk for developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, although a diagnosis of the disease may not have been made.

The term therapeutic effect as used herein encompasses a therapeutic effect and/or a prophylactic effect as described above. A prophylactic effect includes delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, arresting, or reversing the progression of a disease or condition, or any combination thereof.

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

The terms antagonist and inhibitor are used interchangeably and refer to a compound having the ability to inhibit the biological function (e.g., activity, expression, binding, protein-protein interaction) of a target protein (e.g., menin, MLL1, MLL2 and/or MLL fusion protein). Accordingly, the terms antagonist and inhibitor are defined in the context of the biological role of the target protein. Although the preferred antagonists herein specifically interact (eg, bind) to the target, compounds that inhibit the biological activity of the target protein by interacting with other members of the signal transduction pathway in which the target protein is a participant are also specifically included in this definition. The preferred biological activity inhibited by the antagonist is associated with tumor development, growth or spread.

The term agonist as used herein refers to a compound having the ability to initiate or enhance the biological function of a target protein, whether by inhibiting the activity or expression of the target protein. Accordingly, the term agonist is defined in the context of the biological role of the target polypeptide. Although the preferred agonists herein specifically interact (eg, bind) to the target, compounds that initiate or enhance the biological activity of the target polypeptide by interacting with other members of the signal transduction pathway in which the target polypeptide is a participant are also specifically included in this definition.

Signal transduction is the process by which stimulatory or inhibitory signals are transmitted into and within the cell to elicit an intracellular response. A signaling pathway modulator refers to a compound that modulates the activity of one or more cellular proteins that map to the same specific signaling pathway. The modulator can enhance (agonist) or suppress (antagonist) the activity of the signaling molecule.

An anticancer agent, antineoplastic agent, or a chemotherapeutic agent refers to any agent useful in the treatment of a neoplastic condition. One class of anticancer agents includes chemotherapeutic agents. Chemotherapy means the administration of one or more chemotherapeutic drugs and/or other agents to a cancer patient by various routes, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal, or inhaled, or in the form of a suppository.

The subject refers to an animal, such as a mammal, such as a human. ways, opi

- 14 039109 sledge in this document, can be useful in both therapeutic and veterinary applications. In some embodiments, the subject is a mammal, and in some embodiments, the subject is a human. Mammal includes humans and tame animals such as laboratory animals and pets (e.g. cats, dogs, pigs, cattle, sheep, goats, horses, rabbits) and non-domesticated animals such as those from the wild, etc. .P.

Prodrug means a compound that can be converted under physiological conditions or by solvolysis into a biologically active compound described in this application (for example, a compound of formula (I)). Thus, the term prodrug refers to a precursor of a biologically active compound that is pharmaceutically acceptable. In some aspects, the prodrug is an inactive compound when administered to a subject, but is converted to the active compound in vivo, such as by hydrolysis. The prodrug often provides advantages in solubility, tissue compatibility, or sustained release in mammals (see, for example, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam); Higuchi , T., et al., Pro-drugs as Novel Delivery Systems, (1987) A.C.S. Symposium Series, Vol. 14; and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press), each of which are incorporated herein by reference in their entirety. The term prodrug is also intended to include any covalently linked carriers that release the active compound in vivo when such a prodrug is administered to a mammalian subject. Active substance prodrugs as described herein are typically prepared by modifying the functional groups present on the active compound such that the modifications are cleaved either by routine manipulation or in vivo into the parent active compound. Prodrugs include compounds in which a hydroxy, amino, or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a mammal, is cleaved to form the free hydroxy, free amino, or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of the hydroxy functional group or acetamide, formamide, and benzamide derivatives of the amino functional group in the active compound, and the like.

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

The term in vitro refers to an event that occurs outside the subject's body. For example, an in vitro assay encompasses any assay performed outside of a subject. In vitro assays include cellular assays that use live or dead cells. In vitro assays also cover cell-free assays, which do not use intact cells.

Optional or optional means that the occurrence of the circumstance described subsequently may or may not occur and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, optionally substituted aryl means that the aryl group may or may not be substituted and that the description includes both substituted aryl groups and unsubstituted aryl groups.

A pharmaceutically acceptable carrier, diluent, or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, coloring agent, coloring agent, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent , stabilizer, isotonic agent, diluent or emulsifier that has been approved by the US Food and Drug Administration as acceptable for use in humans or pets.

The present invention provides compounds for modulating the interaction of menin with proteins such as MLL1, MLL2 oncoproteins and MLL oncoproteins. In some embodiments, the present invention provides compounds and methods for inhibiting the interaction of menin with its upstream or downstream signaling molecules, including, but not limited to, MLL1, MLL2 oncoproteins, and MLL fusion oncoproteins. The compounds of the present invention may be used in methods of treating a wide variety of cancers and other diseases associated with one or more of MLL1, MLL2, MLL fusion proteins, and menin. In some embodiments, a compound of the invention covalently binds to menin and inhibits the interaction of menin with MLL. In some embodiments, a compound of the invention interacts non-covalently with menin and inhibits the interaction of menin with MLL.

The compounds of the invention are useful in methods of treating a wide range of diseases associated with MLL1, MLL2, MLL fusion proteins, and menin. In some embodiments, a compound of the invention interacts non-covalently with menin and inhibits the interaction of menin with MLL. In some embodiments, a compound of the invention interacts covalently with menin and inhibits the interaction of menin with MLL.

In some aspects, the present invention provides a compound or salt that selectively binds to the menin protein and/or modulates the interaction of menin with the MLL protein (e.g.

- 15 039109 measures, MLL1 protein, MLL2, MLL fusion protein). In some embodiments, the compound modulates the menin protein by binding or interacting with one or more amino acids and/or one or more metal ions. Some compounds may occupy the F9 and/or P13 pocket on the menin. Binding of these compounds can interfere with subsequent menin or MLL signaling (eg, MLL1 protein, MLL2, MLL fusion protein).

In one aspect, the present invention provides a compound of formula (I)

or a pharmaceutically acceptable salt, isotopic form or prodrug thereof, wherein

H is selected from C ₅ . ₁₂ -kαrbocycle and 5-12-membered heterocycle, each of which is optionally substituted with one or more R ⁵⁰ ;

A is selected from the bond, C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle;

B is selected from C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle;

each of L ¹ , L ² and L ³ is independently selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH ₂ , -C(O)-, -C( O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R ⁵¹ )-, -C(O)N(R ⁵¹ )C(O)-, C( O)N(R ⁵¹ )C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-,

-N(R ⁵¹ )C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)O-, -OC(O)N(R ⁵¹ )-, -C(NR ⁵¹ )-, -N(R ⁵¹ )C(NR ⁵¹ )-, -C(NR ⁵¹ )N(R ⁵¹ )-, -N(R ⁵¹ )C(NR ⁵¹ )N(R ⁵¹ )-, -S(O)2 -, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R ⁵¹ )S(O) 2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)-, -S(O)N(R ⁵¹ )-, -N(R ⁵¹ )S(O)2N (R ⁵¹ )-, -N(R ⁵¹ )S(O)N(R ⁵¹ )-; alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene and heteroalkynylene, each of which is optionally substituted with one or more R ⁵⁰ ;

each of RA, R ^B and ^RC is independently selected at each occurrence from R ⁵⁰ , or two RA groups or two R ^B groups attached to the same or different atoms together may optionally form a bridge or ring;

each of m and n is independently an integer from 0 to 6; W ¹ is C1.4 alkylene optionally substituted with one or more R ⁵⁰ ;

W ² is selected from the link; and _C1 . ₄ -αkylene optionally substituted with one or more R ⁵⁰ ;

W ³ is selected from absence; and _C1 . ₄ -alkylene optionally substituted with one or more R ⁵⁰ ;

R ⁵⁰ is independently selected at each occurrence from halogen, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2 , -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S( =O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , NR ⁵² S(=O)2N( R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2, -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N (R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² ) ₂ , -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2 , -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² );

_C1 . ₁₀ -αkyl, C ₂ . ₁₀ -alkenyl and C ₂ . ₁₀ -alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halogen, -NO ₂ , -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2 , -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O) ₂ NR ⁵³ R ⁵⁴ , -NR ⁵² S(= O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , - OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2 , -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² ) ₂ , -C(O) NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle;

C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle, each C ₃ . ₁₂ -karbocycle and 3-12-membered heterocycle in R ⁵⁰ are independently optionally substituted with one or more substituents selected from halo, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S( =O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O )NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C ₁ . ₆ -alkyl, C ₁ . ₆ -haloalkylα, C ₂ - ₆ -alkenyl and C ₂ . ₆ -alkynyl;

R ⁵¹ is independently selected at each occurrence from hydrogen, -C(O)R ⁵² , -C(O)OR ⁵² , -C(O)N(R ⁵² )2 , -C(O)NR ⁵³ R ⁵⁴ ; C^-alkyl, C2.6-alkenylα and C2.6-alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halo, -NO2, -CN, -OR ⁵² , -SR ⁵² , - N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O) 2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2 , -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N (R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ) , C3. ₁₂ -kα-rbocycle and 3-12-membered heterocycle; and

C ₃ - ₁₂ -carbocycle and 3-12-membered heterocycle, each C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle in R ⁵¹ are independently optionally substituted with one or more substituents selected from halo, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S( =O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2,

- 16 039109

-NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O) N(R ⁵² )2, -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2 , -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O )(R ⁵² )2, ═O, ═S, ═N(R ⁵² ), C _1-6 alkyl, C _1-6 haloalkyl, C _2-6 alkenyl, and C _2-6 alkynyl;

R ⁵² is independently selected at each occurrence from hydrogen; and C _1-20 alkyl, C _2-20 alkenyl, C _2-20 alkynyl, 2-6 membered heteroalkyl, C _3-12 carbocycle, and 3-12 membered heterocycle, each of which is optionally substituted with halogen , -CN, -NO ₂ , -NH ₂ , -NHCH ₃ , -NHCH ₂ CH ₃ , =O, -OH, -OCH ₃ , -OCH ₂ CH ₃ , C ₃ . ₁₂ -carbocycle or 3-6-membered heterocycle;

R ⁵³ and R ⁵⁴ are taken together with the nitrogen atom to which they are attached to form a heterocycle optionally substituted with one or more R ⁵⁰ , while for a compound or salt of formula (I), when W ³ is absent,

W ¹ is C ₁ -alkylene, W ² is a bond, and L ³ is not a bond;

W ¹ is C ₂ . ₄ -alkylene and W ² is a bond; or each of W ¹ and W ² is C1 alkylene and L ³ is not a bond, with each C1 alkylene optionally substituted with one or more R ⁵⁰ .

In some embodiments, H is a 5-12 membered heterocycle, such as a 6-12 membered bicyclic heterocycle, optionally substituted with one or more R ⁵⁰ . In some embodiments, the implementation of H contains one or more heteroatoms, such as 1, 2, 3, 4, 5 or 6 heteroatoms in the ring. In some embodiments, H contains at least 1, 2, 3, 4, or 5 ring nitrogen atoms. In some embodiments, H is thienopyrimidinyl optionally substituted with one or more R ⁵⁰ . In some embodiments, H is substituted with C ₁ . ₄ -haloalkyl such as -CH ₂ CF ₃ . In some embodiments, H is substituted with one or more R ⁵⁰ (eg, by replacing the hydrogen atom attached to the ring atom with a bond to R ⁵⁰ ). H may be substituted with 0, 1, 2, 3, 4, 5, 6 or more R ⁵⁰ groups. H may be substituted with 1, 2, 3, 4, 5 or 6 R ⁵⁰ groups, for example H is substituted with 1 or 2 R ⁵⁰ groups. In some embodiments, H is substituted with at least 1, 2, 3, 4, 5, or 6 R ⁵⁰ groups. In some embodiments, H is substituted with 6, 5, 4, 3, 2, or 1 R ⁵⁰ groups.

In some embodiments, H is

where each of X ¹ and X ² is independently selected from CR ² and N; each of X ³ and X ⁴ is independently selected from C and N; each of Y ¹ and Y ² is independently selected from CR ³ , N, NR ⁴ , O and S; each of R ¹ , R ² and R ³ is independently selected at each occurrence from hydrogen and R ⁵⁰ ; and R ⁴ is selected from R ⁵¹ . In some embodiments, X ³ and X ⁴ are each C. In some embodiments, X ¹ is CR ² and R ² is selected from hydrogen, halo, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C ₁ . ₃ -alkyl, -Ch ₂ OH, -CH2OR ⁵² , -CH2NH2, -CH2N(R ⁵² )2, C ₁ . ₃ -alkyl-N(R ⁵² )2, C1.3-haloalkyl, C2.3-alkenyl and C2_3-alkynyl. In some embodiments, X ² is N. In some embodiments, Y ² is CR ³ and R ³ is selected from hydrogen, halo, -OH, -N(R ⁵² )2, -CN, -C(O)OR ⁵² , C1.3-alkyl and C1.3-haloalkyl. In some embodiments, Y ¹ is S. In some embodiments, at least one of Y ¹ and Y ² is selected from N, NR ⁴ , O, and S. In some embodiments, R ¹ is C1. ₃ -haloalkyl, such as -CH ₂ CF ₃ . In some embodiments, X ¹ is CR ² , X ² is N, X ³ and X ⁴ are each C, Y ¹ is S, Y ² is CR ³ , and R ¹ is selected from R ⁵⁰ . In some embodiments, X ¹ is CR ² ; X ² is N; each of X ³ and X ⁴ is C; Y ¹ is S; Y ² is CH; R ¹ is C1.3 haloalkyl; and R ² is selected from hydrogen, halogen, -OH, -NH2, -CN, C ₁ . ₃ -alkyl, C ₁ . ₃ -haloalkyl, C ₂ . ₃ -alkenyl and C ₂ . ₃ -alkynyl. In some embodiments, H is such as

In some embodiments, H is

- 17 039109

and R ² is selected from hydrogen, halo, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C ₁ . ₃ -alkyl, -CH ₂ OH, -CH2OR ⁵² , -CH2NH2, -CH2N(R ⁵² )2, C ₁ . ₃ -alkyl-N(R ⁵² ) ₂ , C ₁ . ₃ -haloalkyl, C ₂ . ₃ -alkenyl and C ₂ . ₃ -alkynyl.

In some embodiments, H is

where

R ¹ is selected from H, halogen, hydroxyl, amino, cyano, dialkylphosphine oxide, oxo, carboxyl, amido, acyl, alkyl, cycloalkyl, heteroalkyl and haloalkyl such as alkyl and haloalkyl; R ² is selected from H, halogen, hydroxyl, amino, cyano, dialkylphosphine oxide, oxo, carboxyl, amido, acyl, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aminoalkyl, hydroxyalkyl, alkoxy and alkylamino, e.g. from H, halogen, hydroxyl and amino ; and each of Y ¹ and Y ² is independently selected from S, CR ³ , N, NR ⁴ and O. In some embodiments, one of Y ¹ and Y ² is O or S.

In some embodiments, L ¹ contains less than 20 atoms, such as less than 10 atoms. In some embodiments, L ¹ contains less than 20, 15, 10, 9, 8, 7, 6, 5, 4, or less than 3 atoms. In some embodiments, L ¹ contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or at least 20 atoms. In some embodiments, L ¹ contains at least one heteroatom, for example L ¹ contains at least one nitrogen atom. In some embodiments, L ¹ is substituted with one or more R ⁵⁰ . In some embodiments, L ¹ is unsubstituted. In some embodiments, L ¹ is selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O)N(R ⁵¹ )-, -S (O)2-, -S(O)-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)2N( R ⁵¹ )-, alkylene, alkenylene, heteroalkylene and heteroalkenylene. In some embodiments, L ¹ is selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O)N(R ⁵¹ )-, -S (O)2-, -S(O)-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)2N( R ⁵¹ )-, C1. ₆ -alkylene and C ₂ . ₆ -alkenylene, with each of C ₁ . ₆ -alkylene and C ₂ . ₆ -alkenylene is optionally substituted with one or more R ⁵⁰ . In some embodiments, L ¹ is N(R ⁵¹ )-, such as -NH-. In some embodiments, L ¹ is selected from -O-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)-, -C(O)N(R ⁵¹ )-, -N (R ⁵¹ )C(O)-, -N(R ⁵¹ )S(O)2-, -S(OhN(R ⁵¹ )-, ^-alkylene, C2.4-alkenylene and C1. ₄ -heteroalkylene. B in some embodiments, L ¹ is -N(R ⁵¹ )-, where R ⁵¹ is selected from hydrogen and alkyl.

In some embodiments, A is a 3-12 membered heterocycle, such as a 5-8 membered heterocycle. In some embodiments, A is a 6-membered monocyclic heterocycle. In some embodiments, the implementation of the heterocycle contains at least one nitrogen atom. In some embodiments, A contains at least one ring nitrogen atom. In some embodiments, A is selected from piperidinylene and piperazinylene, such as (R ^A ) _m

In some embodiments, A is

In some embodiments, A is an aromatic, non-aromatic, saturated, or unsaturated ring. In some embodiments, A is selected from arylene, cycloalkylene, heterocycloalkylene, N-heterocycloalkylene, heteroarylene, and N-heteroarylene. In some embodiments, A is a 5-8 membered heterocycle, wherein the heterocycle contains at least 1, 2, 3, or 4 ring heteroatoms selected from N, O, and S.

In some embodiments, A is selected from

- 18 039109

In some embodiments, A is substituted with one or more RAs (eg, by replacing a hydrogen bonded to a ring atom with a bond to an RA). A may be substituted with 0, 1, 2, 3,4, 5, 6 or more RA groups A may be substituted with 1, 2, 3, 4, 5 or 6 RA groups, eg A may be substituted with 1 or 2 R ^a groups. In some embodiments, A is substituted with at least 1, 2, 3, 4, 5, or 6 RA groups. In some embodiments, A is unsubstituted. In some embodiments, A is substituted with m RA groups, where m is an integer from 0 to 6. In some embodiments, m is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, m is at least 1, 2, 3, 4, 5, or 6. In some embodiments, m is up to 6, 5, 4, 3, 2, or 1. In some embodiments, m is 0.

In some embodiments, RA is independently selected at each occurrence from halo, hydroxyl, amino, cyano, dialkylphosphine oxide, oxo, carboxyl, amido, acyl, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aminoalkyl, hydroxyalkyl, alkoxy, alkylamino, cycloalkylalkyl, cycloalkyloxy, cycloalkylalkyloxy . In some embodiments, two RA groups attached to the same atom or different atoms can jointly form a ring.

In some embodiments, L ² contains less than 20 atoms, such as less than 10 atoms. In some embodiments, L ² contains less than 20, 15, 10, 9, 8, 7, 6, 5, 4, or less than 3 atoms. In some embodiments, L ² contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or at least 20 atoms. In some embodiments, L ² contains at least one heteroatom, for example L ² contains at least one nitrogen atom. In some embodiments, L ² is C1-1o alkylene, such as C1-4 alkylene, optionally substituted with one or more R ⁵⁰ . In some embodiments, L ² is substituted with one or more R ⁵⁰ . In some embodiments, L ² is unsubstituted. In some embodiments, L ² is selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O)N(R ⁵¹ )-, -S (O)2-, -S(O)-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)2N( R ⁵¹ )-, alkylene, alkenylene, heteroalkylene and heteroalkenylene. In some embodiments, L ² is selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O)N(R ⁵¹ )-, -S (O)2., -S(O)-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)2N( R ⁵¹ )-, C _1-6 alkylene and C _2-6 alkenylene, wherein each of C _1-6 alkylene and C _2-6 alkenylene is optionally substituted with one or more R ⁵⁰ . In some

- 19 039109 In other embodiments, L ² is selected from -O-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C (O)-, -N(R ⁵¹ )S(O)2-, -S(O) ₂ N(R ⁵¹ )-, Ω-alkylene and C1-4-heteroalkylene. In some embodiments, L ² is selected from -CH2-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -N(R ⁵¹ )C(O)-, and -N(R ⁵¹ )S(O )2-. In some embodiments, L ² is -CH ₂ -.

In some embodiments, B is a 3-12 membered heterocycle, such as a 6-12 membered bicyclic heterocycle. In some embodiments, the implementation of the heterocycle contains at least one nitrogen atom. In some embodiments, B is a 6-12 membered heterocycle, wherein the heterocycle contains at least 1, 2, 3, or 4 heteroatoms selected from N, O, and S. In some embodiments, B is 6.5- or 6, 6 bicyclic heterocycle. In some embodiments, B contains at least one ring nitrogen atom. In some embodiments, B is an indolylene such as

R ^B optionally substituted with one or more is

In some embodiments, B is such as

In some embodiments, B is selected from ^zt s _o X _o

Z®

Z ¹¹ where each of Z ¹ , Z ² , Z ³ and Z ⁴ is independently selected from CR ⁷ , N and NR ⁹ ;

Z ⁵ is selected from C and N; each of Z ⁶ , Z ⁷ and Z ⁸ is independently selected from CR ⁸ , N, NR ⁹ , O and S;

each of Z ⁹ , Z ¹⁰ and Z ¹¹ is independently selected from CR ¹⁰ , CR ^U R ¹² , NR ¹³ , O and S; each of R ⁷ , R ⁸ , R ¹⁰ , R ¹¹ and R ¹² is independently selected from hydrogen and R ⁵⁰ ; and each of R ⁹ and R ¹³ is independently selected from R ⁵¹ , whereby B can be connected at any ring atom to L ² or L ³ (for example, by replacing the hydrogen attached to the ring atom with a bond to L ² or L ³ ).

In some embodiments, B is selected from

- 20 039109

- 21 039109

In some embodiments, B is substituted with one or more R ^B (eg, by replacing the hydrogen atom attached to the ring atom with a bond to R ^B ). B may be substituted with 0, 1, 2, 3, 4, 5, 6 or more R ^B groups. B may be substituted with 1, 2, 3, 4, 5 or 6 R ^B groups, for example B is substituted with 1 or 2 R ^B groups. In some embodiments, B is substituted with at least 1, 2, 3, 4, 5, or 6 R ^B groups. In some embodiments, B is substituted with n R ^B groups, where n is an integer from 0 to 6. In some embodiments, n is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, n is at least least 1, 2, 3, 4, 5, or 6. In some embodiments, n is up to 6, 5, 4, 3, 2, or 1. In some embodiments, n is an integer from 1 to 3.

In some embodiments, R ^B is independently selected at each occurrence from halo, hydroxyl, amino, cyano, dialkylphosphine oxide, oxo, carboxyl, amido, acyl, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aminoalkyl, hydroxyalkyl, alkoxy, alkylamino, cycloalkylalkyl, cycloalkyloxy, cycloalkylalkyloxy, cycloalkylamino, cycloalkylalkylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkyloxy, heterocyclylalkylamino, heterocyclylalkylamino, aryl, aralkyl, aryloxy, aralkyloxy, arylamino, aralkylamino, heteroaryl, heteroarylalkyl, heteroaryloxy, heteroarylalkyloxy, heteroarylamino and heteroarylalkylamino. In some embodiments, R ^B is independently selected at each occurrence from halogen, hydroxyl, amino, cyano, dialkylphosphine oxide, oxo, carboxyl, amido, acyl, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aminoalkyl, hydroxyalkyl, alkoxy, alkylamino, heterocyclylalkyl, and heteroarylalkyl. In some embodiments, two R ^B groups attached to the same atom or different atoms can jointly form a ring.

In some embodiments, L ³ contains less than 30 atoms, such as less than 20 atoms. In some embodiments, L ³ contains less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, or less than 3 atoms. In some embodiments, L ³ contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or at least 20 atoms. In some embodiments, L ³ contains at least one heteroatom, for example L ³ contains at least one nitrogen atom. In some embodiments, L ³ is C ₁ . ₁₀ -αkylene, such as C ₁ . ₄ -alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ³ is substituted with one or more R ⁵⁰ . In some embodiments, L ³ is unsubstituted. In some embodiments, L ³ is selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O)N(R ⁵¹ )-, -S (O)2-, -S(O)-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)2N( R ⁵¹ )-, alkylene, alkenylene, heteroalkylene and heteroalkenylene. In some embodiments, L ³ is selected from -CH2-, -CH2CH2-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -N(R ⁵¹ )C(O)-, and -N(R ⁵¹ )S(O)2-. In some embodiments, L ³ is -CH2- or -CH2CH2-. In some embodiments, L ³ is -CH ₂ CH(CH ₃ )-. In some embodiments, L ³ is substituted with C _1-4 alkyl or C _1-4 haloalkyl, eg -CH ₃ .

In some embodiments, each of W ¹ , W ² and W ³ is independently selected from C _1-4

- 22 039109 alkylene, with each C _1-4 -alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ¹ , W ² , and W ³ are each C1 alkylene. In some embodiments, W ¹ and W ² are each C1 alkylene and W ³ is absent. In some embodiments, W ¹ , W ² and W ³ form a ring selected from

each of which is optionally substituted with one or more R ⁵⁰ . In some embodiments, W ¹ , W ² and W ³ in combination form

^In some embodiments, RC is selected from halo, -OR ⁵² , -N(R ⁵² ) ₂ , -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(= O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S (=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2, -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2,

-P(O)(R ⁵² )2;

C _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halogen, -NO ₂ , -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, - S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , - C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2, -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , - C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² ) ₂ , -P(O)(R ⁵² )2, =O, =S, = N(R ⁵² ), C3-12-carbocycle and 3-12-membered heterocycle;

wherein each ^C _3-12 carbocycle and 3-12 membered heterocycle in RC is independently optionally substituted with one or more substituents selected from halo, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C (O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O )R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² ) 2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C _{1- 6} alkyl, C _1-6 haloalkyl, C _2-6 alkenyl and C _2-6 alkynyl.

In some embodiments, R ^C is selected from

H H H

A ^N y^ 4 ^N y 4 ^N γ\

About OO ,5 5

In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; and B is a 3-12 membered heterocycle. In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; and B is a 3-12 membered heterocycle. In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; and B is a 6-12 membered bicyclic heterocycle. In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; and B is a 6-12 membered bicyclic heterocycle. In some embodiments, H is thienopyrimidinyl optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; and B is a 3-12 membered heterocycle. In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is selected from piperidinylene and piperazinylene; and B is a 3-12 membered heterocycle. In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A before

- 23 039109 is a 3-12-membered heterocycle; and B is indolylene, the membered heterocycle H is thienopyrimidinyl substituted with one or more R ⁵⁰ ; A is selected from piperidinylene and piperazinylene; and B is indolylene.

In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 3-12 membered heterocycle; W ¹ is C1.4-alkylene optionally substituted with one or more R ⁵⁰ ; W ² is C1. ₄ -alkylene optionally substituted with one or more R ⁵⁰ ; W ³ is selected from absence; and _C1 . ₄ -alkylene optionally substituted with one or more R ⁵⁰ ; m is an integer from 0 to 3; and n is an integer from 1 to 3.

In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 6-12 membered bicyclic heterocycle; W ¹ is C1.4-alkylene optionally substituted with one or more R ⁵⁰ ; W ² is C1. ₄ -alkylene optionally substituted with one or more R ⁵⁰ ; W ³ is selected from absence; and _C1 . ₄ -alkylene optionally substituted with one or more R ⁵⁰ ; m is an integer from 0 to 3; and n is an integer from 1 to 3.

In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 6-12 membered bicyclic heterocycle; W ¹ is C1.2-alkylene; W ² is C1. ₂ -alkylene; W ³ is selected from absence and C ₁ . ₂ -alkyleneα; m is an integer from 0 to 3; and n is an integer from 1 to 3.

In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 3-12 membered heterocycle; W ¹ is C1.2-alkylene optionally substituted with one or more R ⁵⁰ ; W ² is C1. ₂ -alkylene optionally substituted with one or more R ⁵⁰ ; and W ³ is C ₁ . ₂ -alkylene optionally substituted with one or more R ⁵⁰ .

In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 3-12 membered heterocycle; W ¹ is O-alkylene; W ² is C1 alkylene; and W ³ is C ₁ -alkylene.

In some embodiments, H is a 5-12 membered heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 3-12 membered heterocycle; W ¹ is C ₁ -alkylene; W ² is C1 alkylene; and W ³ is missing.

In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 6-12 membered bicyclic heterocycle; W ¹ is C1.2-alkylene optionally substituted with one or more R ⁵⁰ ; W ² is C1. ₂ -alkylene optionally substituted with one or more R ⁵⁰ ; W ³ selected C ₁ . ₂ -αkylene optionally substituted with one or more R ⁵⁰ .

In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 6-12 membered bicyclic heterocycle; W ¹ is C1-alkylene; W ² is C1-alkylene; and W ³ is C ₁ -alkylene.

In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 6-12 membered bicyclic heterocycle; W ¹ is C1-alkylene; W ² is C1-alkylene; and W ³ is missing.

In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is selected from piperidinylene and piperazinylene; B is a 6-12 membered bicyclic heterocycle; W ¹ is C1.2-alkylene; W ² is C1. ₂ -alkylene; and W ³ is selected from the absence of and C1.2-alkylene. In some embodiments, H is a 6-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is selected from piperidinylene and piperazinylene; B is a 6-12 membered heterocycle; W ¹ is C1. ₂ -alkylene; W ² is C1.2-alkylene; W ³ is selected from absence and C1. ₂ -alkylene; m is an integer from 0 to 3; and n is an integer from 1 to 3.

In some embodiments, H is thienopyrimidinyl optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 6-12 membered heterocycle; W ¹ is C1.2-alkylene; W ² is C1. ₂ -alkylene; and W ³ is selected from absence and C ₁ . ₂ -alkylene. In some embodiments, H is thienopyrimidinyl optionally substituted with one or more R ⁵⁰ ; A is a 3-12 membered heterocycle; B is a 6-12 membered bicyclic heterocycle; W ¹ represents

- 24 039109 a C _1-2 -alkylene; W ² is C1-2 alkylene; and W ³ is selected from the absence of and C1-2-alkylene; m is an integer from 0 to 3; and n is an integer from 1 to 3.

In some embodiments, H is a 9-10 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 5-7 membered heterocycle; B is a 9-membered bicyclic heterocycle; W ¹ is C1.2-alkylene; W ² is C1_ ₂ -alkylene; and W ³ is selected from the absence of and C1_2-alkylene, with each of these heterocycles containing at least one nitrogen atom. In some embodiments, H is a 9-10 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 5-7 membered heterocycle; B is a 9-membered bicyclic heterocycle; W ¹ is C1. ₂ -alkylene; W ² is C1.2-alkylene; W ³ is selected from absence and C1. ₂ -αkylene; m is an integer from 0 to 3; and n is an integer from 1 to 3, wherein each of said heterocycles contains at least one nitrogen atom.

In some embodiments, H is a 9-10 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 5-7 membered heterocycle; B is a 9-membered bicyclic heterocycle; W ¹ is C1_2-alkylene optionally substituted with one or more R ⁵⁰ ; W ² is C1_ ₂ alkylene optionally substituted with one or more R ⁵⁰ ; and W ³ is C ₁ _ ₂ alkylene optionally substituted with one or more R ⁵⁰ ; wherein each of said heterocycles contains at least one nitrogen atom.

In some embodiments, H is a 9-10 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 5-7 membered heterocycle; B is a 9-membered bicyclic heterocycle; W ¹ is C1-alkylene; W ² is C1-alkylene; W ³ is C ₁ . ₂ -alkylene, wherein each of these heterocycles contains at least one nitrogen atom.

In some embodiments, H is a 9-10 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ; A is a 5-7 membered heterocycle; B is a 9-membered bicyclic heterocycle; W ¹ is C1-alkylene; W ² is C ₁ -alkylene; W ³ is absent, while each of these heterocycles contains at least one nitrogen atom.

In some embodiments, L ¹ contains less than 10 atoms, L ² contains less than 10 atoms, and L ³ contains less than 20 atoms. In some embodiments, each of L ¹ , L ² and L ³ contains less than 10 atoms. In some embodiments, each of L ¹ , L ² and L ³ is independently selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)- , -C(O)O-, -OC(O)-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O) N(R ⁵¹ )-, -S(O)2-, -S(O)-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-, -N( R ⁵¹ )S(O)2N(R ⁵¹ )-, alkylene, alkenylene, heteroalkylene and heteroalkenylene. In some embodiments, each of L ¹ , L ² and L ³ is independently selected from -CH2-, -CH2CH2-, -N(R ⁵¹ )-, -N(R ⁵¹ )Ch2-, -N(R ⁵¹ )C( O)- and -N(R ⁵¹ )S(O)2-. In some embodiments, L ¹ is selected from -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )cH2-, -C(O)-, -C(O)O-, -OC (O)-, -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O)N(R ⁵¹ )-, -S(O )2-, -S(O)-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-,

-N(R ⁵¹ )S(O)2N(R ⁵¹ )-, alkylene, alkenylene, heteroalkylene and heteroalkenylene; and L ² and L ³ are independently selected from C1-4 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, each of L ¹ , L ² and L ³ is independently selected from -O-, -S-, -N(R ⁵¹ )-; C1-4-alkylene and 1-4-membered heteroalkylene, each of which is optionally substituted with one or more R ⁵⁰ . In some embodiments, L ¹ is -NH-, L ² is -CH2-, and L ³ is _C1-4 alkylene optionally substituted with one or more R ⁵⁰ .

In some aspects, for a compound of formula (I)

H is a 5-12 membered bicyclic heterocycle optionally substituted with one or more R ⁵⁰ ;

A is a 3-12 membered heterocycle;

B is a 3- to 12-membered heterocycle;

each of L ¹ , L ² and L ³ is independently selected from a bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH ₂ -, -C(O)-, -C (O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R ⁵¹ )-, -C(O)N(R ⁵¹ )C(O)-, C (O)N(R ⁵¹ )C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-,

-N(R ⁵¹ )C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)O-, -OC(O)N(R ⁵¹ )-, -C(NR ⁵¹ )-, -N(R ⁵¹ )C(NR ⁵¹ )-, -C(NR ⁵¹ )N(R ⁵¹ )-, -N(R ⁵¹ )C(NR ⁵¹ )N(R ⁵¹ )-, -S(O)2 -, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R ⁵¹ )S(O) 2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)-, -S(O)N(R ⁵¹ )-, -N(R ⁵¹ )S(O) ₂ N(R ⁵¹ )-, -N(R ⁵¹ )S(O)N(R ⁵¹ )-; alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene and heteroalkynylene, each of which is optionally substituted with one or more R ⁵⁰ ;

each of R ^a , R ^b and RC is independently selected at each occurrence from R ⁵⁰ , or two R ^A groups or two R ^{B groups} attached to the same atom or different atoms may together optionally form a bridge or ring;

m is an integer from 0 to 3;

n is an integer from 1 to 3;

W ¹ is _C1-4 alkylene optionally substituted with one or more R ⁵⁰ ;

W ² is _C1-4 alkylene optionally substituted with one or more R ⁵⁰ ;

- 25 039109

W ³ is selected from absence; and Ci-4-alkylene optionally substituted with one or more R ⁵⁰ ;

R ⁵⁰ is independently selected at each occurrence from halogen, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2 , -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S( =ObR ⁵² , - S(=ObN(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² ) 2, NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O )N(R ⁵² )2, -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² ) 2, NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O )(R ⁵² )2, =O, =S, =N(R ⁵² );

_C1 . ₁₀ alkyl, C ₂ _ ₁₀ alkenyl, and C ₂ _ ₁₀ alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halogen, -NO ₂ , -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(= O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O )R ⁵² , -C(O)OR ⁵² , -OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2 , NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O) N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle; and

C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle, each C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle in R ⁵⁰ are independently optionally substituted with one or more substituents selected from halo, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S( =O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , - OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C ₁ _ ₆ -alkyl, C ₁ . ₆ -haloalkulα, C ₂ . ₆ -alkenyl and C ₂ _ ₆ alkynyl; R ⁵¹ is independently selected at each occurrence from hydrogen, -C(O)R ⁵² , -C(O)OR ⁵² , -C(O)N(R ⁵² )2 , -C(O)NR ⁵³ R ⁵⁴ ;

_C1 . ₆ -alkyl, C ₂ .6-alkenyl and C ₂ . ₆ -alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halo, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2 , -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O) 2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, -NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC( O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2, -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C( O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C ₃ _ ₁₂ -carbocycle and 3-12-membered heterocycle ; and

C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle, each C ₃ . The ₁₂ -carbocycle and 3-12-membered heterocycle in R ⁵¹ is independently optionally substituted with one or more substituents selected from halo, -NO2, -CN, -OR ⁵² , -SR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -S(=O)R ⁵² , -S(=O)2R ⁵² , -S(=O)2N(R ⁵² )2, -S(=O)2NR ⁵³ R ⁵⁴ , -NR ⁵² S( =O)2R ⁵² , -NR ⁵² S(=O)2N(R ⁵² )2, NR ⁵² S(=O)2NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , - OC(O)R ⁵² , -OC(O)OR ⁵² , -OC(O)N(R ⁵² )2 , -OC(O)NR ⁵³ R ⁵⁴ , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , -P(O)(OR ⁵² )2, -P(O)(R ⁵² )2, =O, =S, =N(R ⁵² ), C ₁ . ₆ -αlkulα, C ₁ . ₆ -haloalkyl, C ₂ .6-alkenyl and C ₂ . ₆ -alkynyl;

R ⁵² is independently selected at each occurrence from hydrogen; and _C1 . ₂₀ -alkyl, C ₂ . ₂₀ -alkenyl, C ₂ . ₂₀ alkynyl, 2-6 membered heteroalkyl, C ₃ . ₁₂ -carbocycle and 3-12-membered heterocycle, each of which is optionally substituted with halogen, -CN, -NO ₂ , -NH ₂ , -NHCH ₃ , -NHCH2CH3, =O, -OH, -OCH ₃ , -OCH ₂ CH ₃ , C ₃ . ₁₂ -carbocycle or 3-6-membered heterocycle; and

R ⁵³ and R ⁵⁴ taken together with the nitrogen atom to which they are attached to form a heterocycle are optionally substituted with one or more R ⁵⁰ , while for a compound or salt of formula (I), when W ³ is absent, each of W ¹ and W ² is C1 alkylene and L ³ is not a bond, with each C1 alkylene optionally substituted with one or more R ⁵⁰ .

In some aspects, for a compound of formula (I):

H is thienopyrimidinyl optionally substituted with one or more R ⁵⁰ ;

A is selected from piperidinylene and piperazinylene;

B is indolylene;

each of L ¹ and L ² is independently selected from -O-, -S-, -NH- and -CH2-;

L ³ is selected from the bond, -O-, -S-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -C(O)-, -C(O)O-, -OC(O )-, -OC(O)O-, -C(O)N(R ⁵¹ )-, -C(O)N(R ⁵¹ )C(O)-, -C(O)N(R ⁵¹ )C (O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)O -, -OC(O)N(R ⁵¹ )-, -C(NR ⁵¹ )-, -N(R ⁵¹ )C(NR ⁵¹ )-, -C(NR ⁵¹ )N(R ⁵¹ )-, -N (R ⁵¹ )C(NR ⁵¹ )N(R ⁵¹ )-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O )2-, -S(O)2O-, -N(R ⁵¹ )S(O)2-, -S(O)2N(R ⁵¹ )-, -N(R ⁵¹ )S(O)-, - S(O)N(R ⁵¹ )-, -N(R ⁵¹ )S(O) ₂ N(R ⁵¹ )-, -N(R ⁵¹ )S(O)N(R ⁵¹ )-; alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene and heteroalkynylene, each of which is optionally substituted with one or more R ⁵⁰ ;

each of R ^a , R ^b and RC is independently selected at each occurrence from R ⁵⁰ , or two R ^A groups or two R ^{B groups} attached to the same atom or different atoms may together optionally form a bridge or ring; m is an integer from 0 to 3;

n is an integer from 1 to 3;

W ¹ is C ₁ . ₄ -alkylene optionally substituted with one or more R ⁵⁰ ;

W ² is C ₁ . ₄ -alkylene optionally substituted with one or more R ⁵⁰ ; and

W ³ is C ₁ _ ₄ alkylene optionally substituted with one or more R ⁵⁰ .

In some aspects, a compound of formula (I) may be represented

- 26 039109 e.g.

In some embodiments, R ¹ is selected from R ⁵⁰ . In some embodiments, R ¹ is C1.3-haloαalkyl, such as -CH2CF3. In some embodiments, R2 is selected from R ⁵⁰ . In some embodiments, R2 is selected from hydrogen, halogen, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C1-3-alkyl, C1-3-αkyl-OR ⁵² , C1-3 -alkyl-N(R ⁵² )2, C1-3-haloalkyl, C2-3-alkenyl and C2-3-αkynyl. In some embodiments, R ² is selected from halo, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C ₁ . ₃ -alkyl, -CH2OH, -CH2OR ⁵² , -CH2NH2, -CH2N(R ⁵² )2, C ₁ . ₃ -αkyl-N(R ⁵² )2, C1.3-haloalkyl, C2.3-αalkenyl and C2.3-αkynyl, e.g. R ² is selected from -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN and C ₁ . ₂ -alkyl. Optionally, R ² is selected from -NH2, -CH3, -OCH3, -CH2OH and -NHCH3. In some embodiments, R ³ is selected from hydrogen, halogen, -OH, -N(R ⁵² )2, -CN, -C(O)OR ⁵² , C1. ₃ -alkyl and C ₁ . ₃ -haloalkyl. In some embodiments, R ⁵¹ is selected from hydrogen and alkyl, for example R ⁵¹ is hydrogen. In some embodiments, R ^A is selected from halo, -CN, -OR ⁵² , -N(R ⁵² )2 , -NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC( O)R ⁵² , -NR ⁵² C(O)R ⁵² , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , =O, C ₁ . ₁₀ -alkyl, C ₂ . ₁₀ -αalkenyl, C ₂ . ₁₀ -αkynyl optionally substituted with C ₁ . ₁₀ -alkyl optionally substituted with C ₂ . ₁₀ -alkenyl and optionally substituted C ₂ . ₁₀ -αkynyla. In some embodiments, m is an integer from 0 to 3. In some embodiments, m is 0. In some embodiments, L ² is selected from -O-, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2- , -C(O)N(R ⁵¹ )-, -N(R ⁵¹ )C(O)-, -N(R ⁵¹ )S(O)2-, -S(O) ₂ N(R ⁵¹ )- , C ₁ . ₄ -αkylene and C ₁ . ₄ -heteroalkylene. In some embodiments, L ² is C1.4 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ² is C1. ₂ -alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ² is selected from -CH ₂ -, -N(R ⁵¹ )-, -N(R ⁵¹ )CH2-, -N(R ⁵¹ )C(O)- and -N(R ⁵¹ )S( O)2-. In some embodiments, L ² is -CH ₂ -. In some embodiments, R ^B is present at one or more positions of the indole, such as the 2, 3, 4, or 6 position of the indole. In some embodiments, R ^B is selected from halo, -CN, -OR ⁵² , -N(R ⁵² )2 , -NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC( O)R ⁵² , -NR ⁵² C(O)R ⁵² , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , =O, C1.10-alkyl, C2. ₁₀ -αalkenyl, C ₂ . ₁₀ -αkynyl optionally substituted with C ₁ . ₁₀ -alkyl optionally substituted with C ₂ . ₁₀ -alkenyl and optionally substituted C ₂ . ₁₀ -αkynyla. In some embodiments, R ^B is selected from halo, -CN, -OR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , C1. ₃ -alkyl and optionally substituted C ₁ . ₃ -alkyl, for example R ^B is selected from halogen, -CN, -OR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ and C1. ₂ -alkyl. In some embodiments, n is an integer from 1 to 4, such as an integer from 2 to 3. In some embodiments, n is 2. In some embodiments, L ³ is C1.4-αkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ³ is C1. ₂ -alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ³ is -CH2-. In some embodiments, W ¹ is C1.4 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ¹ is C1. ₂ -alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ¹ is C1.2-αkylene, such as C1-alkylene or -CH2-. In some embodiments, W ² is C1. ₄ -alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ² is C1.2-alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ² is C1. ₂ -alkylene, such as C ₁ .αkylene or -CH2-. In some embodiments, W ³ is absent. In some embodiments, W ³ is C1.4-alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ³ is C1. ₂ -alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ³ is C ₁ . ₂ -alkylene, such as C ₁ -alkylene or -CH2-. ^In some embodiments, RC is selected from -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -C(O)R ⁵² , -C(O)OR ⁵² , - NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² ,

- 27 039109

-NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N(R ⁵² )2 and -C(O)NR ⁵³ R ⁵⁴ . In some embodiments, R ^C is selected from

In some aspects, a compound of formula (I) may be represented

for example

In some embodiments, R ² is selected from R ⁵⁰ . In some embodiments, R ² is selected from hydrogen, halogen, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C _1-3 -alkyl, C _1-3 -alkyl-OR ⁵² , C1-3-αlkylN(R ⁵² )2, C _1-3 haloalkyl, C _2-3 alkenyl and C _2-3 -alkynylα. In some embodiments, R ² is selected from halo, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C^-alkyl, -CH2OH, -CH2OR ⁵² , -CH2NH2, -CH2N(R ⁵² )2, C1-3-alkyl-N(R ⁵² )2, C _1-3 -haloalkyl, C _2-3 -alkenyl and C _2-3 -αkynyl, for example, R ² is selected from -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN and C _1-2 -alkyl. Optionally R ² is selected from -NH2, -CH3, -OCH3, -CH2OH and -NHCH3. In some embodiments, R ^B is selected from halo, -CN, -OR ⁵² , -N(R ⁵² )2 , -NR ⁵³ R ⁵⁴ , -C(O)R ⁵² , -C(O)OR ⁵² , -OC( O)R ⁵² , -NR ⁵² C(O)R ⁵² , -C(O)N(R ⁵² )2, -C(O)NR ⁵³ R ⁵⁴ , =O, C1-10-alkyl, C _2-10 alkenyl, C _2-10 alkynyl, optionally substituted C _1-10 alkyl, optionally substituted C _2-10 alkenyl, and optionally substituted C _2-10 alkynyl. In some embodiments, R ^B is selected from halo, -CN, -OR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , C1-3 alkyl, and optionally substituted C _1-3 alkyl, e.g., R ^B selected from halogen, -CN, -OR ⁵² , -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ and C1-2-alkyl. In some embodiments, L ³ is C1-4 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ³ is C1-2 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, L ³ is -CH2-. In some embodiments, W ¹ is C1-4-alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ¹ is C1-2 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ¹ is C1-2-alkylene, such as C1 alkylene or -CH ₂ -. In some embodiments, W ² is C1-4-alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ² is C1-2 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ² is C1-2 alkylene, such as C1 alkylene or -CH2-. In some embodiments, W ³ is absent. In some embodiments, W ³ is C1-4-αkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ³ is C1-2 alkylene optionally substituted with one or more R ⁵⁰ . In some embodiments, W ³ is C1-2 alkylene, such as C ₁ alkylene or -CH ₂ -. ^In some embodiments, RC is selected from -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -C(O)R ⁵² , -C(O)OR ⁵² , - NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2, -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O) N(R ⁵² )2 and -C(O)NR ⁵³ R ⁵⁴ . In some embodiments, R ^C is selected from

- 28 039109

In some aspects, a compound of formula (I) may be represented

In some embodiments, R ² is selected from R ⁵⁰ . In some embodiments, R ² is selected from hydrogen, halogen, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C _1-3 -alkyl, C _1-3 -alkyl-OR ⁵² , C1-3-alkylN(R ⁵² )2, C _1-3 haloalkyl, C _2-3 alkenyl and C _2-3 alkynyl. In some embodiments, R ² is selected from halo, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C _1-3 alkyl, -CH ₂ OH, -CH2OR ⁵² , -CH2NH2, -CH2N(R ⁵² )2, C _1-3 -alkyl-N(R ⁵² )2, C1-3 haloalkyl, C2-3 alkenyl and C2-3 alkynyl, e.g. R ² is selected from -OH, - OR ⁵² , -NH2, -N(R ⁵² )2, -CN and Ci-2-alkyl. Optionally, R ² is selected from -NH2, -CH ₃ , -OCH ₃ , -CH ₂ OH and -NHCH ₃ . In some embodiments, RC is selected from -N(R ⁵² )2, -NR ⁵³ R ⁵⁴ , -NR ⁵² S(=O)2R ⁵² , -C(O)R ⁵² , -C(O)OR ⁵² , -NR ⁵² C(O)R ⁵² , -NR ⁵² C(O)OR ⁵² , -NR ⁵² C(O)N(R ⁵² )2 , -NR ⁵² C(O)NR ⁵³ R ⁵⁴ , -C(O)N (R ⁵² )2 and -C(O)NR ⁵³ R ⁵⁴ . In some embodiments, the RC is selected from

In some aspects, a compound of formula (I) may be represented

In some embodiments, R ² is selected from hydrogen, halogen, -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN, C _1-3 -alkyl, -CH ₂ OH, -CH2OR ⁵² , - CH2NH2, -CH2N(R ⁵² )2, C _1-3 alkyl-N(R ⁵² ) ₂ , C _1-3 haloalkyl, C _2-3 alkenyl and C _2-3 alkynyl, e.g. R ² is selected from -OH, -OR ⁵² , -NH2, -N(R ⁵² )2, -CN and C _1-2 -alkyl. In some embodiments, R ² is methyl or -NHCH3. In some embodiments, R ² is H.

In some aspects, the compound of the present invention covalently binds to menin and inhibits the interaction of menin with MLL. This association can lead to an increase

- 29 039109 affinity of the compound for menin, which is an advantageous property for many applications, including therapeutic and diagnostic applications. In some embodiments, the compounds of the present invention include electrophilic groups capable of reacting with a nucleophilic group present in the menin protein. Suitable electrophilic groups are described herein, while suitable nucleophilic groups include, for example, cysteine residues present in the binding domain of the menin protein. Without wishing to be bound by theory, a cysteine residue in the menin-binding domain may react with the electrophilic group of the compound of the present invention, resulting in the formation of a conjugate. In some embodiments, the compounds of the present invention are capable of covalently binding to the cysteine residue at position 329 of menin isoform 2 (SEQ ID NO: 2) or to cysteine 334 of menin isoform 1 (SEQ ID NO: 1). In some embodiments, the present invention provides a conjugate of a compound of the present invention with the menin protein. For example, the invention relates to a menin conjugate of a compound of the invention linked to cysteine 329 of menin isoform 2 (SEQ ID NO: 2) or cysteine 334 of menin isoform 1 (SEQ ID NO: 1).

In some embodiments, one or more of R ^A , R ^B , and R ^C , when present, contains a functional group covalently interacting with one or more residues on the menin. In some embodiments, the functional group covalently interacts with one or more cysteine residues on the menin. In some embodiments, the functional group covalently interacts with a cysteine on the menin at position 329 relative to SEQ ID NO: 2 when optimally aligned, or position 334 relative to SEQ ID NO: 1 when optimally aligned. In some embodiments, the functional group covalently interacts with one or more residues on the menin selected from cysteine 329, cysteine 241 and/or cysteine 230 on the menin relative to SEQ ID NO: 2 in optimal alignment. In some embodiments, the functional group covalently interacts with cysteine 329 relative to SEQ ID NO: 2 in optimal alignment.

In some embodiments, one or more of R ^A , R ^B , and R ^C , when present, contains a residue that covalently interacts with one or more residues on the menin. In some embodiments, one or more of R ^A , R ^B and R ^C , when present, contain a residue that covalently interacts with any one or more isoforms of menin, such as isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2) or isoform 3 (SEQ ID NO: 3) of menin. In some embodiments, one or more of R ^A , R ^B and R ^C , when present, contain a fragment covalently interacts with menin, wherein the menin protein contains 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity with isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2), or isoform 3 (SEQ ID NO: 3).

In some embodiments, one or more of R ^A , R ^B , and R ^C , when present, contains an electrophilic group, is suitable for nucleophilic attack from the residue on the menin. All electrophilic moieties known to the person skilled in the art that bind to nucleophilic residues are included herein, for example, any electrophilic moiety known to bind to cysteine residues. In some embodiments, one or more of R ^A , R ^B , and R ^C , when present, contain a moiety other than an electrophile, said moiety being able to bind or covalently interact with a residue on the menin. In some embodiments, the compound or salt of formula (I) is capable of (a) covalently binding to menin and (b) inhibiting the interaction of menin with MLL.

^In some embodiments, RC contains a functional group that covalently interacts with one or more residues on the menin. In some embodiments, the functional group covalently interacts with one or more cysteine residues on the menin. In some embodiments, the functional group covalently interacts with a cysteine on the menin at position 329 relative to SEQ ID NO: 2 when optimally aligned, or position 334 relative to SEQ ID NO: 1 when optimally aligned.

^In some embodiments, RC is a moiety containing an alpha, beta-unsaturated carbonyl; alpha, beta unsaturated sulfonyl; epoxide; aldehyde; sulfonyl fluoride; halomethylcarbonyl; dihalomethylcarbonyl; or trihalomethylcarbonyl.

In some embodiments, R ^C is selected from

- 30 039109

where

L ⁵ is selected from the link; and C _1-6 alkylene, C _1-6 heteroalkylene, C _2-6 alkenylene and C _2-6 alkynylene, each of which is optionally substituted with one or more R ³² ;

each of R ²² and R ²³ is independently selected from hydrogen, halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O)R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, - N(R ²⁰ )S(O)2R ²⁰ , -no ₂ , =o, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ )2, -P(O)(R ²⁰ )2 , -OP(O)(OR ²⁰ )2 and -CN;

C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N( R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O )R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ )2, -P(O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C3-10-carbocycle, and 3-10- membered heterocycle; and

C _3-10 carbocycle and 3-10 membered heterocycle, wherein each C _3-10 carbocycle and 3-10 membered heterocycle R ²² and R ²³ are independently optionally substituted with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O)R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ )2, -P(O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C _1-6 -αkyl, C _2-6 -αlkenyl and C _2-6 -αkynyl; or R ²² and R ²³ , together with the carbon atoms to which they are attached, form a carbocyclic ring;

R ²⁴ is selected from hydrogen, -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2 , -OC(O)R ²⁰ , -S(O)2R ²⁰ , and -S(O)2N( ^R20 )2;

C _1-6 -αlkyl, C _2-6 -αlkenyl, and C _2-6 -αkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N( R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O )R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ ) ₂ , -P(O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C _3-10 -carbocycle and 3-10- membered heterocycle; and

C _3-10 carbocycle and 3-10 membered heterocycle, wherein each C _3-10 carbocycle and 3-10 membered heterocycle R ²⁴ is independently optionally substituted with one or more substituents selected from halogen, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2 , -OC(O)R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S , =N(R ²⁰ ), -P(O)(OR ²⁰ )2, -P(O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2j -CN, C _1-6 -αkyl, C _2-6 -αalkenyl and C _2-6 -αkynyl; R ²⁰ is independently selected at each occurrence from R ⁵² ; and R ³² is independently selected at each occurrence of R ⁵⁰ .

In some embodiments, L ⁵ is a bond. In some embodiments, L ⁵ is an optionally substituted C1-6-αkylene. In some embodiments, L ⁵ is selected from methylene, ethylene, or propylene. In some embodiments, L ⁵ is substituted with one or more substituents selected from halo, -NO2, ═O, ═S, -OR ²⁰ , -SR ²⁰ , and -N(R ²⁰ )2. In some embodiments, R ²³ is selected from hydrogen;

C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N( R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O )R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ ) ₂ , -P(O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C _3-10 -carbocycle and 3-10- membered heterocycle; and

C _3-10 carbocycle and 3-10 membered heterocycle, wherein each C _3-10 carbocycle and 3-10 membered heterocycle is independently optionally substituted with one or more substituents selected from halogen, -OR ²⁰ , -SR ²⁰ , - N(R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC (O)R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N (R ²⁰ ), -P(O)(OR ²⁰ )2, -P(O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C _1-6 -alkyl, C _{2 -6} -alkenyl; and C _2-6 -alkynyl.

- 31 039109

In some embodiments, R ²³ is selected from hydrogen;

_C1 . ₆ -αalkyl optionally substituted with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2, ═O, ═S, ═N(R ²⁰ ) and -CN; and

3-10 membered heterocycle optionally substituted with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C( O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O)R ²⁰ , -S(O)2R ²⁰ , -S(O)2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ )2, -P(O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C ₁ . ₆ -αkyl, C ₂ . ₆ -alkenyl and C ₂ . ₆ -αkinila.

In some embodiments, R ²³ is selected from hydrogen and C1.6-αkyl optionally substituted with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2, =O, =S, = N(R ²⁰ ) and -CN.

In some embodiments, R ²² is selected from: halo and -CN;

C ₁ .6-αlkulα, C ₂ . ₆ -alkenyl and C ₂ . ₆ -alkynyl, each independently optionally substituted at each occurrence with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ ) ₂ , -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O)R ²⁰ , -S(O)2R ²⁰ , -S(O) 2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ )2, -P( O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C3.1o-carbocycle and 3-10-membered heterocycle; and

_C3 . ₁₀ -carbocycle and 3-10-membered heterocycle, where each O ₃ . ₁₀ -carbocycle and 3-10-membered heterocycle are independently optionally substituted with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2, -N(R ²⁰ )C(O)R ²⁰ , -C(O)R ²⁰ , -C(O)OR ²⁰ , -C(O)N(R ²⁰ )2, -OC(O)R ²⁰ , -S(O)2R ²⁰ , -S(O) 2N(R ²⁰ )2, -N(R ²⁰ )S(O)2R ²⁰ , -NO2, =O, =S, =N(R ²⁰ ), -P(O)(OR ²⁰ )2, -P( O)(R ²⁰ )2, -OP(O)(OR ²⁰ )2, -CN, C ₁ . ₆ -αkyl, C ₂ . ₆ -alkenyl and C ₂ . ₆ -αkinila.

In some embodiments, R ²² is selected from hydrogen, -CN; and C1.6-αkyl optionally substituted with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ and -N(R ²⁰ )2.

In some embodiments, R ²² and R ²³ , together with the carbon atoms to which they are attached, form a 5-, 6-, or 7-membered carbocyclic ring.

In some embodiments, R ²⁴ is selected from hydrogen and C1.6 alkyl optionally substituted with one or more substituents selected from halo, -OR ²⁰ , -SR ²⁰ , -N(R ²⁰ )2 , -NO ₂ , =O and -CN.

In some embodiments, R ²¹ is selected from

Any combination of the above groups for various variables is considered in this document. In the description of the group and substituents can be chosen to obtain stable

- 32 039109 fragments and compounds.

The chemical compounds described herein may be synthesized according to one or more of the illustrative schemes provided herein and/or methods known in the art. The materials used here are either commercially available or obtained by synthetic methods known to those skilled in the art. These schemes are not limited to the compounds listed in the examples, or any specific substituents that are used for illustrative purposes. Although the various steps are described and depicted in Scheme 1 and Examples 1-6, the steps may in some cases be performed in a different order than that shown in Scheme 1 and Examples 1-6. Various modifications to these synthesis reaction schemes can be made and will be offered to those skilled in the art having read the description contained in this application. The R numbers or groups in each scheme do not necessarily correspond to those of the claims or other schemes or tables herein.

Unless otherwise indicated, the reactions described herein proceed at atmospheric pressure, typically at temperatures ranging from -10 to 200°C. In addition, unless otherwise noted, the reaction times and conditions are assumed to be approximate, for example, proceeding at atmospheric pressure at a temperature in the range from about -10 to about 110 ° C for a period of time from about 1 to 24 h; the reaction is allowed to run overnight for about 16 hours.

In general, the compounds of the invention can be prepared using the following reaction scheme.

Scheme 1.

1-6

In some embodiments, a compound of Formula 1-6 may be prepared according to Scheme 1. For example, methanesulfonyl chloride may be added to a solution of alcohol 1-1 and triethylamine to form mesylate 1-2. Adding mesylate 1-2 to a solution of Cs ₂ CO ₃ and amine 1-3 can provide a compound of formula 1-4. The coupling of aldehyde 1-4 with amine 1-5 can proceed in the presence of a suitable reducing agent such as NaBH(OAc) ₃ to give a compound of formula 1-6.

In some embodiments, the implementation of the connection according to the present invention, for example, the compound of the formula presented in table. 1 is synthesized according to one of the general methods shown in Scheme 1 in Examples 1-6, or by methods known in the art. In some embodiments, exemplary compounds may include, but are not limited to, a compound or salt thereof selected from Table. one.

- 33 039109

Table 1

no. Structure MW (calculated) m/z (found) one hCG-^L ³ HN-C ° 633.73 634.45 [M+H] ⁺ 2 < ^N Y ^S \/ ^{CF3 HN} ^ ° 621.72 622.40 [M+H] ⁺ 3 < ^Ν ν ^δ \ / ^CFs hn-4 ⁰ 635.75 636.45 [M+H] ⁺ four 671.8 672.45 [M+H] ⁺ 5 /W^ ¹¹ //-^ 607.74 608.55 [M+H] ⁺ 6 CF, (rWj ^CF hn4 w ⁰ 675.69 676.40 [M+H] ⁺ 7 HN^ ^{ΗΝ Ό} ^>^ 636.73 637.30 [M+H] ⁺

- 34 039109

eight BUT 649.77 650.30 [M+H] ⁺ 9 rTN ^<? r' ^s x__/ ^CFs NyXX ° HN./\ /%/< T 1 H | p-CN 647.76 648.30 [M+H] ⁺ ten k'Vvx HN ^- Ν^χ-χ ΗΝ^χ\ ΟχΛ/™ 593.71 594.30 [M+H] ⁺ And nh ₂ ζΧΗ ^Ρ ' f HNy-h Vn/LX/ ^cn 593.71 594.35 [M+H] ⁺ 12 ^ην ^Γ ^XP ^CF> f HN^ZX >κ^ _Ν ' Un/LA/“ 649.77 650.35 [M+H] ⁺ 13 f ^N y\x ^CF3 nh ₂ Un^XXX ^cn 579.68 580.25 [M+H] ⁺ fourteen <i \__ ~Ul ° 4< τι τ' ^\L i 1 M ^z o ^ \ 2 /7^~> 647.76 648.35 [M+H] ⁺

- 35 039109

fifteen ζX>+ X 607.69 608.30 [M+H] ⁺ 16 HN<° ^H ° ^ΗΝ η+φ>™ 637.72 638.30 [M+H] ⁺ 17 N<=rc^ ^NH2 636.73 637.30 [M+H] ⁺ eighteen 621.72 622.35 [M+H] ⁺ 19 twenty yp ^CF 608.68 609.20 [M+H] ⁺ 21 iVY^ ³ H _N y ^N yU/ ^m OJ^

- 36 039109

22 iVVY HN-O 23 ^- ^N Y ^S _W ^CF 3 hn^Q° ^Νχ ]^ 24 LI' Y. ^Ν χυ ^{Η ΗΝ} η^φ>- 25 Η ,Ν___ _{_} Ν ο nc / ^- ΝΗ ί YW ^{3 ΗΝ} γΧ ΑΥ ' 26 s CF3 _ΗΝ ^ ° 27 |_| ΥνΑ / ^СРз ΐι /)~^ \/^\ Ν /С/ ° 28 |_| ^Y ^CF dg

- 37 039109

29 V PF ₃ 11 — L / ^HN n^9>- 621.72 622.40 [M+H] ⁺ thirty V X1L^ 621.72 622.40 [M+H] ⁺ 31 o CF ₃ > ^NH Il a—' 1 in^A</ —1 ^HN n^9>" 609.71 610.40 [M+H] ⁺ 32 02^ ί^ν ^ρ n ^{NH ΗΝ} η^9>» 609.71 610.40 [M+H] ⁺ 33 Vh |f ^N ^ ^S K_/ ^CF 3 HN^~ ^NH ° 664.74 665.55 [M+H] ⁺ 34 672.77 673.45 [M+H] ⁺

- 38 039109

35 O n o \\ ^{N HN} n^9>- 700.78 699.45 [M-H]' 36 iY^ ³ n1mGy ° 714.82 715.40 [M+H] ⁺ 37 nh ₂ QJ^b 650.76 651.45 [M+H] ⁺ 38 °nb <V ^s v^ ^CF3 n/ 673.75 674.35 [M+H] ⁺ 39 nh ₂ rf' ^N 'V ^S \ / ^CF 3 HN ^- Z Ν^Λ/ ° 650.76 651.25 [M+H] ⁺ 40 fYXJ^ hn^ ⁿ \ ° 664.29 665.35 [M+H] ⁺

- 39 039109

41 CL ^hn dj9>- 676.29 677.45 [M+H] ⁺ 42 from 2° 676.29 677.40 [M+H] ⁺ 43 / 651.26 652.30 [M+H] ⁺ 44 Γγ\^ ³ A^Syn Λΐ 45 ^VAY ^{1 3} hn-^nh ^Nx r^ 3t/ 46 N-, _X N^ _S CF, _UM Y ' [| \__/ ³ H, ^N X^NH ⁿ ^A<Z 3(/ 47 ζΧΗ ^{Ρ ΗΝ} η^φ>-

- 40 039109

48 hnX™ 650.28 651.40 [M+H] ⁺ 49 iYV ^F 5-< 659.30 660.30 [M+H] ⁺ fifty ^%^ ³ ηνΛ ^{0 ΗΝ} η^φ>- 51 rO < ^N V ^S \_/ ^CF3 HN% ⁰ 52 if ^N >r ^s \ / ^CF3 HN- ° ^HN n^9>" 53 ^X-^L ³ nTz zT ^{0 HN} n^9>™

- 41 039109

54 Η ₂ Ν _γ Ν _s CF _{3 HN} ^ ° 650.28 651.25 [M+H] ⁺ 55 V- ^K ”V'OJA 649.28 650.45 [M+H] ⁺ 56 hl 2 ?T ⁵ + H'H ^HN xW /V ^N ujy> ^cN 706.30 707.45 [M+H] ⁺ 57 ^VV^ ³ HN^C^ ° 690.31 691.50 [M+H] ⁺ 58 p 4xH ^{F> HN} >W Wv-N UAp> ^CN 690.31 691.50 [M+H] ⁺ 59 s ^' p ” 664.29 665.45 [M+H] ⁺ 60 ₄ of /^ _Ν η ₂ i rW ^F3 pW Λι 662.28 663.40 [M+H] ⁺

- 42 039109

61 _Ν , _CF Y™, A/ 636.26 637.40 [Μ+Η] ⁺ 62 Ν, ς CF / ^ΝΗ2 ιί YV "Η ^ΗΝ η^φ>- 678.31 679.45 [Μ+Η] ⁺ 63 iw „„^° ^η 665.28 666.40 [Μ+Η] ⁺ 64 Ν ₄ CF / ^ΝΗ2 ιί TW ^{3 Ν} ' ⁴ Τ^ A/ ° 650.28 651.40 [Μ+Η] ⁺ 65 ο Ν with ρρ / ^ΝΗ 2 ί W νΧΛ ^Ν γ^ ΛΙ ° 712.29 713.45 [Μ+Η] ⁺ 66 [^Y'S-V^ ^{3 Η} , ^Ν ^~ ^ΟΗ 651.26 652.35 [Μ+Η] ⁺

- 43 039109

67 C if ^N Y ^s \Y ^F3 ° 667.26 668.35 [Μ+Η] ⁺ 68 ΥγΑ_Υ ³ ΗΝ^~ ^ΟΗ ΝγΥ/ ° ^{ΗΝ Ό} ^9>- 663.26 664.45 [Μ+Η] ⁺ 69 F ₃ C Ν c CF / ^ΝΗ 2 if VA / ^F ' ην4 ^{ΗΝ Ό} ^φ>- 704.25 705.40 [Μ+Η] ⁺ 70 y- ΝΗ __/ ^CF3 ° ^{ΝΗ 11} // ^- ' ^/ ^Γ/ 678.31 679.3 [Μ+Η] ⁺ 71 Vnh ₂ < ^N V ^S V^ ^CF3 ° ΝΗ 664.29 665.2 [Μ+Η] ⁺ 72 Υνη ₂ ί/χν Y QJ2>·· 692.32 693.3302

- 44 039109

73 I r ^N \ 706.34 707.3461 74 c O 718.26 719.40 [M+H] ⁺ 75 ΑγΥΥ hnX n ^N γ ^χ Υ ^Η ° YV ^N T 1 11 1 Y- cn 637.24 638.35 [M+H] ⁺ 76 iV^ ^F> Ar ° ^HN n^9>" 680.29 681.3 [M+H] ⁺ 77 aO N o nc o+x ii YW ^F3 U1 ^h A, ^HN n^9>- 697.28 698.2879 78 nh ₂ HD ^ ^N V ^S V ^ ^{CF3 0} NH 712.29 713.2988

- 45 039109

79 nh ₂ H — 'YYV* NH 741.32 742.3 [M+H] ⁺ 80 ⁿ 2*k /L N n=Y ΎυΜ* ³ ι™ 741.32 742.3 [M+H] ⁺ 81 Η γΌ Ν _S Cf, _? ( lg° 762.27 82 ^YWJ^ ^{3 ΗΝ} ^ ° ^ΗΝ η^>- 650.28 651.45 [Μ+Η] ⁺ 83 ρ 'ν ^Ν ν _% ^{F1 ην} Ί ^Η 621.25 622.26 [M+H] ⁺ 84 p ΥΑ' γ 635.27 636.27 [M+H] ⁺

- 46 039109

Pharmaceutical compositions

Compositions and methods according to the present invention can be used to treat an individual in need thereof. In some embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition containing, for example, a compound or salt of formula (I) and a pharmaceutical

- 47 039109 ski acceptable carrier.

In some embodiments, the pharmaceutical composition is formulated for oral administration. In other embodiments, the pharmaceutical composition is formulated for injection. In even more preferred embodiments, the pharmaceutical compositions comprise a compound disclosed herein and an additional therapeutic agent (eg, an anti-cancer agent). Non-limiting examples of such therapeutic agents are described below.

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

In some embodiments, the compound or salt composition of formula (I) is administered by a local rather than systemic route, for example, by injecting the compound directly into an organ, often in a depot or sustained release formulation. In specific embodiments, the long acting formulations are administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. In addition, in other embodiments, the compound or salt of formula (I) is delivered in a targeted drug delivery system, for example, in a liposome coated with an organ-specific antibody. In such embodiments, liposomes are targeted to and selectively selected by an organ. In other embodiments, the composition is in the form of an immediate release composition, in the form of an extended release composition, or in the form of an intermediate release composition. In other embodiments, the composition is administered topically.

The compound of formula (I), or a pharmaceutically acceptable salt thereof, may be effective over a wide dosage range. For example, in the treatment of adults, dosages of 0.01 to 1000 mg per day, 0.5 to 100 mg per day, 1 to 50 mg per day, and 5 to 40 mg per day are examples of dosages that can be used in some embodiments. The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

In some embodiments, the compound or salt of formula (I) is administered in a single dose. Typically, such administration is by injection, eg intravenous injection, for rapid administration of the agent. However, other methods are used as needed. In some embodiments, a single dose of a compound or salt of formula (I) is used to treat an acute condition.

In some embodiments, the compound or salt of formula (I) is administered in multiple doses. In some embodiments, the dosage is one, two, three, four, five, six, or more than six times a day. In other embodiments, dosing is once a month, once every two weeks, once a week, or once every two days. In another embodiment, the compound or salt of formula (I) and another agent are administered together from about once a day to about 6 times a day. In another embodiment, the administration of the compound or salt of formula (I) and the agent is continued for less than about 7 days. In yet another embodiment, administration continues for more than about 6 days, more than about 10 days, more than about 14 days, more than about 28 days, more than about two months, more than about six months, or one year or more. In some cases, continuous dosing is achieved and maintained for as long as necessary.

Administration of the compound or salt of the formula may be continued for as long as necessary. In some embodiments, a compound of the present invention is administered for more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 14, or more than 28 days. In some embodiments, a compound of the present invention is administered for 28 days or less, 14 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day, or part of it. In some embodiments, a compound or salt of formula (I) is administered on a long-term, ongoing basis, such as for the treatment of chronic effects.

In some embodiments, a compound or salt of formula (I) is administered in doses. It is known in the art that, due to intersubject variability in the pharmacokinetics of compounds, individualization of the dosing regimen is necessary for optimal therapy. The dosage of the compound or salt of formula (I) can be determined by routine experimentation in light of the present disclosure.

In some embodiments, the compound or salt of formula (I) is formulated into pharmaceutical compositions. In specific embodiments, pharmaceutical compositions are formulated in the usual manner using one or more physiologically acceptable carriers containing excipients and excipients that facilitate processing of the active compounds into formulations that can be used pharmaceutically. The correct composition depends on

- 48 039109 the chosen route of administration. Any pharmaceutically acceptable techniques, carriers and excipients are used as appropriate for formulating the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

The pharmaceutical compositions provided herein comprise a compound or salt of formula (I) and a pharmaceutically acceptable diluent(s), excipient(s) or carrier(s). In some embodiments, the described compounds or salts are administered as pharmaceutical compositions in which the compound or salt of formula (I) is mixed with other active ingredients, as in combination therapy. What is included herein encompasses all combinations of active agents provided in the combination therapy section below and throughout. In specific embodiments, the implementation of the pharmaceutical compositions include one or more compounds of formula (I) or their pharmaceutically acceptable salts.

A pharmaceutical composition as used herein refers to a mixture of a compound or salt of formula (I) with other chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickeners and/or excipients. In some embodiments, the implementation of the pharmaceutical composition facilitates the introduction of the compounds into the body. In some embodiments of the invention relating to the practice of the methods of treatment or use provided herein, a therapeutically effective amount of a compound or salt of formula (I) is administered in a pharmaceutical composition to a mammal having a disease, disorder or medical condition to be treated. In specific embodiments, the mammal is a human. In some embodiments, the therapeutically effective amounts will vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compound being used, and other factors. The compounds or salt of the formula may be used alone or in combination with one or more therapeutic agents as components of mixtures.

In one embodiment, the compound or salt of formula (I) is formulated in an aqueous solution. In specific embodiments, the aqueous solution is selected, by way of example only, from a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. In other embodiments, the compound or salt of formula (I) is formulated for transmucosal administration. In specific embodiments, transmucosal formulations include penetrants that are suitable for barrier penetration. In other embodiments where the compound or salt of formula (I) is formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In specific embodiments, such solutions include physiologically compatible buffers and/or excipients.

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

In some embodiments, oral pharmaceutical preparations are prepared by mixing one or more solid excipients with a compound or salt of formula (I), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable excipients, if required, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, microcrystalline cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose; or others such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In particular embodiments of the invention, disintegrating agents are optionally added. Disintegrating agents include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

In one embodiment, dosage forms such as dragee cores and tablets have one or more suitable coatings. In specific embodiments, concentrated sugar solutions are used to coat the dosage form. The sugar solutions optionally contain additional components such as, for example, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes and/or pigments are also optionally added to the coatings for identification purposes. In addition, dyes and/or pigments are optionally used

- 49 039109 to characterize various combinations of doses of the active compound.

In some embodiments, a therapeutically effective amount of a compound or salt of formula (I) is formulated into other oral dosage forms. Oral dosage forms include hard capsules (push-fit) made from gelatin, as well as soft, sealed capsules made from gelatin and a plasticizer such as glycerol or sorbitol. In specific embodiments, hard capsules (push-fit) contain the active ingredients in admixture with one or more excipients. Fillers include, by way of example only, lactose, binders such as starches and/or lubricants such as talc or magnesium stearate, and optional stabilizers. In other embodiments, soft capsules contain one or more active compounds that are dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers are optionally added.

In other embodiments, a therapeutically effective amount of a compound or salt of formula (I) is for buccal or sublingual administration. Formulations suitable for buccal or sublingual administration include, by way of example only, tablets, lozenges, or gels. In yet other embodiments, the compound or salt of formula (I) is formulated for parenteral injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, the compositions for injection are presented in unit dosage form (eg, in ampoules) or in multi-dose containers. Preservatives are optionally added to injectable preparations. In yet other embodiments, the pharmaceutical compositions are formulated in a form suitable for parenteral injection as sterile suspensions, solutions or emulsions in oily or aqueous vehicles. Compositions for parenteral administration optionally contain adjuvants such as suspending, stabilizing and/or dispersing agents. In specific embodiments, the implementation of pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In additional embodiments, a suspension of the compound or salt of formula (I) is provided as suitable injectable oily suspensions. Suitable lipophilic diluents or carriers for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. In some specific embodiments, the implementation of injectable aqueous suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension contains suitable stabilizers or agents that increase the solubility of the compounds in order to provide highly concentrated solutions. In some embodiments, the active agent is in the form of a powder for solution with a suitable vehicle, such as sterile, pyrogen-free water, prior to use.

In yet other embodiments, a compound or salt of formula (I) is administered topically. The compound or salt of formula (I) can be formulated into a variety of topical compositions such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity agents, buffers and preservatives.

In still other embodiments, the compound or salt of formula (I) is formulated for transdermal administration. Transdermal formulations may use transdermal delivery devices and transdermal delivery patches and may be lipophilic emulsions or buffered aqueous solutions dissolved and/or dispersed in a polymer or adhesive. In various embodiments, such patches are designed for continuous, pulsatile, or on-demand delivery of pharmaceutical agents. In further embodiments, transdermal delivery of a compound or salt of formula (I) is via iontophoretic patches and the like. In some embodiments, transdermal patches provide controlled delivery of a compound or salt of formula (I). In specific embodiments, the rate of absorption is retarded by the use of rate-regulating membranes or by retention of the compound in a polymer matrix or gel. In alternative embodiments, absorption enhancers are used to enhance absorption. Absorption enhancers or carriers include absorbable pharmaceutically acceptable solvents that aid in skin passage. For example, in one embodiment, the transdermal devices are in the form of a dressing comprising a support member, a reservoir containing a compound or salt of formula (I), optionally with carriers, optionally a rate control barrier to deliver the compound to a subject's skin at a controlled and predetermined rate over a period of time. a long period of time and a means for fixing the device on the skin.

In other embodiments, the compound or salt of formula (I) is formulated for administration by inhalation. Various forms suitable for administration by inhalation include, but are not limited to

- 50 039109 sprays, mists or powders. Pharmaceutical compositions of the compound or salt of formula (I) are usually delivered as an aerosol spray from sealed packages or a nebulizer using a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In specific embodiments, the pressurized aerosol dosage unit is determined by providing a valve to deliver a metered amount. In some embodiments, capsules and cartridges, such as, by way of example only, gelatin for use in an inhaler or insufflator, contain a powder mixture of a compound or salt of formula (I) and a suitable powder base such as lactose or starch.

In still other embodiments, the compound or salt of formula (I) is formulated into rectal compositions, such as enemas, rectal gels, rectal foams, rectal sprays, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or others. glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository form compositions, a low melting wax is first melted, such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter.

In some embodiments, the pharmaceutical compositions are formulated by any conventional method using one or more physiologically acceptable carriers containing excipients and excipients that facilitate processing of the active compounds into formulations that can be used pharmaceutically. The correct composition depends on the chosen route of administration. Any pharmaceutically acceptable techniques, carriers and excipients may optionally be used as appropriate. Pharmaceutical compositions containing a compound or salt of formula (I) are prepared in a conventional manner, such as, by way of example only, by conventional mixing, dissolving, granulating, drageeing, levigating, emulsifying, encapsulating, encapsulating or compressing processes.

Pharmaceutical compositions contain at least one pharmaceutically acceptable carrier, diluent or excipient and a compound or salt of formula (I), sometimes referred to herein as an active agent or ingredient. The active ingredient may be in free acid or free base form, or in the form of a pharmaceutically acceptable salt. In addition, the compound or salt of formula (I) may be in unsolvated or solvated forms with pharmaceutically acceptable solvents such as water and ethanol. In addition, the pharmaceutical compositions optionally contain other drug or pharmaceutical agents, carriers, adjuvants such as preservative, stabilizing, wetting or emulsifying agents, dissolution promoters, salts for adjusting osmotic pressure, buffers and/or other therapeutically useful substances.

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

In some embodiments, a pharmaceutical composition containing a compound or salt of formula (I) is in the form of a liquid in which the agents are present in solution, suspension, or both. Typically, when the composition is administered as a solution or suspension, the first part of the agent is present in solution and the second part of the agent is present as particles in suspension in a liquid matrix. In some embodiments, the implementation of the liquid composition includes a gel composition. In other embodiments, the liquid composition is aqueous.

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

Pharmaceutical compositions also optionally include solubilizing agents to facilitate the solubility of the compound described herein. The term solubilizing

- 51 039109 agent usually includes agents that lead to the formation of a micellar solution or a true solution of the agent. Some suitable non-ionic surfactants, such as polysorbate 80, are useful as solubilizing agents, and ophthalmologically acceptable glycols, polyglycols, such as polyethylene glycol 400, and glycol ethers can also be used.

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

In addition, suitable compositions also optionally contain one or more salts in an amount necessary to bring the osmolality of the composition within an acceptable range. Such salts include salts containing sodium, potassium or ammonium cations and anions such as chloride, citrate, ascorbate, borate, phosphate, bicarbonate, thiosulfate or bisulfite; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

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

Pharmaceutical compositions may contain one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid and vegetable oil glycerides, for example, polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, for example octoxynol 10, octoxynol 40.

Pharmaceutical compositions may contain one or more antioxidants to improve chemical stability where needed. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulphite.

In some embodiments, aqueous suspension compositions are packaged in disposable containers without reclosing. Alternatively, multi-dose resealable containers are used, in which case a preservative is usually included in the composition.

In some embodiments, delivery systems for hydrophobic pharmaceutical compounds are used. Liposomes and emulsions are examples of delivery vehicles or delivery vehicles useful herein. In some embodiments, organic solvents such as N-methylpyrrolidone are also used. In additional embodiments, a compound or salt of formula (I) is delivered using a sustained release system, such as semi-permeable matrices of solid hydrophobic polymers containing a therapeutic agent. Various sustained release materials may be used herein. In some embodiments, the slow release capsules release the compounds for several weeks up to more than 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional protein stabilization strategies are used.

In some embodiments, the compositions described herein contain one or more antioxidants, metal chelating agents, thiol-containing compounds, and/or other general stabilizing agents. Examples of such stabilizing agents include, but are not limited to: (a) about 0.5 to about 2% w/v glycerol, (b) about 0.1 to about 1% w/v methionine, (c) from about 0.1 to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01 to about 2% w/v ascorbic acid, (f) from 0.003 to about 0.02% w/v polysorbate 80, (g) 0.001 to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulphate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

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

In some embodiments, the concentration of the compound or salt of formula (I) in the pharmaceutical composition is greater than about 90, 80, 70, 60, 50, 40, 30, 20, 19.75, 19.50, 19.25, 19, 18 .75, 18.50, 18.25, 18, 17.75, 17.50, 17.25, 17, 16.75, 16.50, 16.25, 16, 15.75, 15.50, 15 .25, 15, 14.75, 14.50, 14.25, 14, 13.75, 13.50, 13.25, 13, 12.75, 12.50, 12.25, 12, 11.75 , 11.50, 11.25, 11, 10.75, 10.50, 10.25, 10, 9.75, 9.50, 9.25, 9, 8.75, 8.50, 8.25 , 8, 7.75, 7.50, 7.25, 7, 6.75, 6.50, 6.25, 6, 5.75, 5.50, 5.25, 5, 4.75, 4 .50, 4.25, 4, 3.75, 3.50, 3.25, 3, 2.75, 2.50, 2.25, 2, 1.75, 1.50, 1.25, 1 , 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04,

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0.03 0.02 0.01 0.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.0009 0.0008 0.0007 0.0006 0.0005 0.0004, 0.0003, 0.0002 or 0.0001% w/w, w/v or rev./rev.

In some embodiments, the concentration of a compound or salt of formula (I) is in the range of from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, about 0.03 to about 28%, about 0.04 to about 27%, about 0.05 to about 26%, about 0.06 to about 25%, about 0.07 to about 24%, about 0.08 to about 23%, about 0.09 to about 22%, about 0.1 to about 21%, about 0.2 to about 20%, about 0.3 to about 19%, about 0.4 to about 18%, about 0.5 to about 17%, about 0.6 to about 16%, about 0.7 to about 15%, about 0.8 to about 14%, from about 0.9 to at about 12%, from about 1 to about 10% wt./wt., wt./about. or rev./rev.

In some embodiments, the concentration of the compound or salt of formula (I) is in the range of from about 0.001 to about 10%, from about 0.01 to about 5%, from about 0.02 to about 4.5%, from about 0.03 to about 4%, from about 0.04 to about 3.5%, from about 0.05 to about 3%, from about 0.06 to about 2.5%, from about 0.07 to about 2%, from about 0.08 to about 1.5%, from about 0.09 to about 1%, from about 0.1 to about 0.9% w/w, w/v. or rev./rev.

In some embodiments, the amount of a compound or salt of formula (I) is equal to or less than about 10, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0 , 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.95, 0.9, 0 .85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25 , 0.2, 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009 , 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001, 0.0009, 0.0008, 0.0007, 0.0006, 0.0005, 0.0004, 0.0003, 0.0002 or 0 .0001

In some embodiments, the amount of a compound or salt of formula (I) is greater than about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0, 0009 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045 0.005 0.0055 0.006 0.0065 0.007 0.0075 0.008 0.0085 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0 .08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.45 , 0.5, 0.55, 0.6, 0.65, 0.7, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.95, 1 , 1.5, 2, 2.5, 3, 3.5, 4.5, 5, 5.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 , 9.5 or 10 g.

In some embodiments, the amount of one or more compounds of the present invention is in the range 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05- 5 g, 0.1-4 g, 0.5-4 g or 1-3 g.

Kits and articles of manufacture are also provided for use in the therapeutic applications described herein. In some embodiments, such kits comprise a carrier, package, or container that is divided into parts to receive one or more containers, such as vials, test tubes, and the like, with each container(s) containing one of the individual items to be used in the method described in this document. Suitable containers include, for example, bottles, vials, syringes and test tubes. Containers are made from various materials such as glass or plastic.

Products made in accordance with the present invention contain packaging materials. Packaging materials for use in pharmaceutical packaging products include those described in, for example, US Pat. , containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended route of administration and treatment. For example, the container(s) contains a compound or salt of formula (I), optionally in a composition or in combination with another agent, as described herein. The container(s) optionally has a sterile access point (eg, the container is an intravenous solution bag or a vial with a hypodermic stopper). Such kits optionally contain a compound with an identifying description or label, or instructions relating to its use in the methods described herein.

For example, a kit typically contains one or more additional containers, each containing one or more different materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user point of view for the use of a compound described herein. document. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; labels on media, packages, containers, vials and/or tubes listing contents and/or instructions for use, and inserts with instructions for use. Typically, an instruction set is also included. The label is not necessarily on or associated with the container. For example, the label is

- 53 039109 is attached to a container when the letters, numbers or other symbols forming the label are affixed, shaped or engraved on the container itself, the label is associated with the container when it is present in a slot or on a carrier which also contains the container, for example in the form of an insert . In addition, the label is used to indicate that the contents are to be used for a particular therapeutic application. In addition, the label indicates directions for the application of the content, such as the methods described in this document. In some embodiments of the present invention, the pharmaceutical composition is presented in a packaging or dispenser device that contains one or more unit dosage forms containing the compound. The package, for example, contains a metal or plastic foil, such as a blister pack. In addition, the packaging or dispensing device is accompanied by instructions for administration. In addition, the package or dispenser is accompanied by a notice associated with the container in the form prescribed by the government agency regulating the manufacture, use or sale of pharmaceuticals, the notice reflecting the agency's approval of the form of the medicinal product for human or veterinary administration. For example, such a notice is a US Food and Drug Administration-approved label for prescription drugs or an approved product insert. In some embodiments, compositions containing a compound as provided herein are formulated in a compatible pharmaceutical carrier, placed in a suitable container, and labeled for the treatment of the indicated condition.

Ways

The present invention provides a method for inhibiting the interaction of menin and one or more proteins (e.g., MLL1, MLL2 protein, MLL fusion protein, or partial tandem duplication MLL protein) comprising contacting the cell with an effective amount of a compound or salt of formula (I). Inhibition of the interaction of menin and one or more proteins (eg, MLL1 protein, MLL2 protein, MLL fusion protein, or partial tandem duplication MLL protein) can be assessed and demonstrated using a wide range of methods known in the art. Non-limiting examples include reference to a) reduced binding of menin to one or more proteins or protein fragments (eg, MLL1, MLL2 protein, MLL or MLL fusion protein with partial tandem duplication, or a peptide fragment thereof); (b) reducing cell proliferation and/or cell viability; (c) increasing cell differentiation; (d) reducing levels of downstream targets of MLL1 protein, MLL2, MLL fusion protein and/or partial tandem duplication MLL) (eg Hoxa9, DLX2 and Meis1); and/or (e) reducing tumor volume and/or tumor growth rate. Kits and commercially available assays can be used to determine one or more of the above.

The invention also relates to methods of using the compounds or pharmaceutical compositions of the present invention for the treatment of diseases, including, but not limited to, conditions associated with menin, MLL, MLL1, MLL2 and/or MLL fusion proteins (eg, cancer).

In some embodiments, a method of treating cancer is provided, comprising administering an effective amount of any of the above pharmaceutical compositions containing a compound or salt of formula (I) to a subject in need thereof. In some embodiments, the cancer is mediated by an MLL fusion protein. In other embodiments, the cancer is leukemia, breast cancer, prostate cancer, pancreatic cancer, lung cancer, liver cancer, skin cancer, or brain tumor. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is a solid tumor.

In some embodiments, the invention relates to a method of treating a disorder in a subject in need thereof, the method comprising determining if the subject has an MLL fusion protein and, if the subject is determined to have an MLL fusion protein, comprising administering to the subject a therapeutically effective dose of a compound or salts of formula (I).

MLL fusion proteins have also been identified in hematological malignancies (eg, types of cancer that affect the blood, bone marrow, and/or lymph nodes). Accordingly, some embodiments are directed to administering a compound or salt of formula (I) to a patient in need of treatment for hematologic cancer. Such cancers include, but are not limited to, leukemias and lymphomas. For example, the compounds disclosed herein may be used to treat diseases such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), hairy cell leukemia and/or other leukemias. In other embodiments, the compounds may be used to treat lymphomas such as all subtypes of Hodgkin's lymphoma or non-Hodgkin's lymphoma.

Determining whether a tumor or cancer may contain an MLL fusion protein can be done by assessing the nucleotide sequence encoding the MLL fusion protein, by assessing the amino acid sequence of the MLL fusion protein, or by assessing the characteristics of the putative MLL fusion protein.

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Methods for detecting the nucleotide sequence of an MLL fusion protein are known to those skilled in the art. These methods include, but are not limited to, polymerase chain reaction - DNA restriction fragment length polymorphism (PCR-RFLP), polymerase chain reaction - single strand conformational polymorphism (PCR-SSCP) assays, real-time PCR analysis, PCR sequencing, mutant allele assays. -specific PCR amplification (MASA), direct sequencing, primer extension reactions, electrophoresis, oligonucleotide ligation assays, hybridization assays, TaqMan assays, SNP genotyping assays, high resolution melt assays, and microarray analysis. In some embodiments, the MLL fusion protein is identified, for example, using a direct sequencing method for specific regions (eg, exon 2 and/or exon 3) in the MLL or partner fusion gene. This technique identifies all possible mutations in the sequencing region.

Methods for detecting an MLL fusion protein are known to those skilled in the art. These methods include, but are not limited to, detection of an MLL fusion protein using a binding agent (eg, antibody) specific for the fusion protein, electrophoresis and Western blotting of the protein, and direct peptide sequencing.

Various samples can be used in methods for determining whether a tumor or cancer contains an MLL fusion protein. In some embodiments, the sample is taken from a subject having a tumor or cancer. In some embodiments, the sample is taken from a subject having a cancer or tumor. In some embodiments, the sample is a fresh tumor/cancer sample. In some embodiments, the sample is a frozen tumor/cancer sample. In some embodiments, the sample is a formalin-fixed sample embedded in paraffin. In some embodiments, the sample is processed into a cell lysate. In some embodiments, the sample is processed into DNA or RNA.

The invention also relates to a method for treating a hyperproliferative disorder in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound or salt of formula (I). In some embodiments, the method relates to the treatment of cancer, such as acute myeloid leukemia, adolescent cancer, childhood adrenocortical carcinoma, AIDS-associated cancer (e.g., lymphoma and Kaposi's sarcoma), anal cancer, appendix cancer, astrocytoma, atypical teratoid, carcinoma basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain glioma, brain tumor, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumor, atypical teratoid, embryonic tumors, germ cell tumor, primary lymphoma, cervical cancer uterus, childhood cancers, chordoma, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS) , embryonic tumors, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, esthesionuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extraganal germ cell tumor, eye cancer, fibrous bone histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendogenous tumors, kidney cancer, laryngeal cancer, lip and mouth cancer, liver cancer, lobar carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous cell carcinoma of the neck with an unidentified primary tumor with detected metastases, midline carcinoma, oral cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, fungoid mycosis, myelodysplastic syndromes, myelodysplastic/ myeloproliferative neoplasms neoplasms, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous bone histiocytoma and osteosarcoma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), oral cavity cancer, lip and oral cancer cavities, orthopharyngeal cancer, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach (gastrointestinal) cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, T-cell lymphoma, testicular cancer, throat cancer , thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the kidney and ureter, trophoblastic tumour, unusual types of childhood cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or virus-induced cancer. In some embodiments, the method relates to the treatment of a benign hyperproliferative disorder, such as uncomplicated skin hyperplasia (eg, psoriasis), restenosis, or prostate (eg, benign prostatic hypertrophy (BPH)).

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In some cases, the method relates to the treatment of leukemia, hematological malignancy, solid tumors, prostate cancer (e.g., castration-resistant prostate cancer), breast cancer, Ewing's sarcoma, bone sarcoma, primary bone sarcoma, T-cell prolymphocytic leukemia, glioma, glioblastoma, liver cancer (eg, hepatocellular carcinoma), or diabetes. In some cases, leukemia includes AML, ALL, leukemia of mixed origin, or leukemia with MLL Partial Tandem Duplications.

In some specific embodiments, the invention relates to methods for treating lung cancer, the methods comprising administering an effective amount of any of the above compounds (or a pharmaceutical composition containing such a compound) to a subject in need thereof. In some embodiments, the lung cancer is a non-small cell lung carcinoma (NSCLC), such as adenocarcinoma, squamous cell lung carcinoma, or large cell lung carcinoma. In other embodiments, the lung cancer is small cell lung carcinoma. Other lung cancers treatable with the disclosed compounds include, but are not limited to, glandular tumors, carcinoid tumors, and undifferentiated carcinomas.

Subjects that may be treated with a compound of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, solvate, tautomer, stereoisomer, isotopologue, hydrate, or derivative of a compound thereof, in accordance with the methods of this invention, include, for example, subjects who have been diagnosed with acute myeloid leukemia, cancer in adolescents, adrenocortical carcinoma in children, AIDS-related cancers (eg, lymphomas and Kaposi's sarcomas), anal cancer, cancer of the appendix, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain glioma, brain tumor, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumor, atypical teratoid, embryonic tumors, germ cell tumor, primary lymphoma, cervical cancer, childhood cancers, chordoma , cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia oz (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonic tumors, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, esthesionuroblastoma, sarcoma Ewing, extracranial germ cell tumor, extraganal germ cell tumor, eye cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, leukemia hairy cell, head and neck cancer, heart cancer, liver cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendogenous tumors, kidney cancer, laryngeal cancer, lip and mouth cancer, liver cancer, lobar carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous cell carcinoma of the neck with unidentified metastatic primary tumor, midline carcinoma, oral cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, Merkel cell carcinoma, malignant mesothelioma, malignant fibrous bone histiocytoma and osteosarcoma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), oral cavity cancer, lip and mouth cancer, orthopharyngeal cancer, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma , paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, cancer salivary gland cancer, skin cancer, gastric (gastrointestinal) cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cancer kidney and ureteral cells, trophoblastic tumor, uncommon childhood cancers, urethral cancer, uterine sarcoma, vaginal cancer, vulvar or virus-induced cancer, leukemia, hematologic malignancy, solid tumor, prostate cancer, castration-resistant cancer, cancer breast, Ewing's sarcoma, bone sarcoma, primary bone sarcoma and T-cell prolymphocytic leukemia, glioma, glioblastoma, hepatocellular carcinoma, liver cancer or diabetes. In some embodiments, subjects being treated with compounds of the present invention include subjects who have been diagnosed as having a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e.g., psoriasis), restenosis, or prostate (e.g., benign prostatic hypertrophy (BPH) ).

In addition, the invention relates to methods for modulating the interaction of menin and one or more proteins (e.g., MLL1 protein, MLL2 protein, MLL fusion protein, or partial tandem duplication MLL) by contacting menin with an effective amount of a compound or salt

- 56 039109 formula (I). The modulation may inhibit or activate the protein activity of menin, one or more of its binding partners and/or one or more downstream targets of menin or one or more of its binding partners. In some embodiments, the invention relates to methods of inhibiting the interaction of menin and one or more proteins (e.g., MLL1 protein, MLL2 protein, MLL fusion protein, or partial tandem duplication MLL) by contacting menin with an effective amount of a compound or salt of formula (I) . In some embodiments, the invention relates to methods for inhibiting the interaction of menin and one or more proteins (e.g., MLL1 protein, MLL2 protein, MLL fusion protein, or partial tandem duplication MLL protein) by bringing into contact a cell, tissue, or organ that expresses menin, the MLL1 protein , MLL2, MLL and/or MLL fusion protein with partial tandem duplication. In some embodiments, the present invention relates to methods for inhibiting the activity of a protein in a subject, including, but not limited to, rodents and mammals (eg, humans) by administering to the subject an effective amount of a compound or salt of formula (I). In some embodiments, the modulation percentage is greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the percentage of inhibition is greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In some embodiments, the invention provides methods for inhibiting the interaction of menin and one or more proteins (e.g., MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL protein) in a cell by contacting the cell with an amount of a compound of the invention that is sufficient to inhibit the interaction of menin and one or more proteins (eg, MLL1, MLL2, MLL fusion protein, or MLL with partial tandem duplication) in a cell. In some embodiments, the invention provides methods for inhibiting the interaction of menin and one or more proteins (e.g., MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL protein) in tissue by contacting the tissue with an amount of a compound or salt of formula (I) , which is sufficient to inhibit the interaction of menin and one or more proteins (eg, MLL1, MLL2, MLL fusion protein or partial tandem duplication MLL) in tissue. In some embodiments, the invention provides methods for inhibiting the interaction of menin and one or more proteins (e.g., MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL protein) in the body by contacting the body with an amount of a compound or salt of formula (I) , which is sufficient to inhibit the interaction of menin and one or more proteins (eg, MLL1, MLL2, MLL fusion protein or partial tandem duplication MLL) in the body. In some embodiments, the invention provides methods for inhibiting the interaction of menin and one or more proteins (e.g., MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL protein) in an animal by contacting the animal with an amount of a compound of the present invention, which is sufficient to inhibit the interaction of menin and one or more proteins (eg, MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL) in an animal. In some embodiments, the invention provides methods for inhibiting the interaction of menin and one or more proteins (e.g., MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL protein) in a mammal by contacting the mammal with an amount of a compound of the invention sufficient to inhibit interactions of menin and one or more proteins (eg, MLL1, MLL2, MLL fusion protein or partial tandem duplication MLL) in a mammal. In some embodiments, the invention provides methods for inhibiting the interaction of menin and one or more proteins (e.g., MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL protein) in a human by contacting the human with an amount of a compound of the invention that is sufficient to inhibit the interaction of menin and one or more proteins (eg, MLL1, MLL2, MLL fusion protein, or MLL with partial tandem duplication) in humans. The present invention provides methods for treating a disease mediated by the interaction of menin and one or more proteins (eg, MLL1, MLL2, MLL fusion protein, or partial tandem duplication MLL) in a subject in need of such treatment.

The invention also provides methods of treating a disorder mediated by the interaction of menin with one or more proteins (e.g., MLL1, MLL2, MLL fusion protein, or MLL partial tandem duplication) by administering to a subject in need thereof a therapeutically effective amount of a compound or salt of formula (I ).

In addition, the present invention provides methods for treating a disorder mediated by a chromosomal rearrangement on chromosome 11q23 in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound or salt of formula (I).

The invention also provides methods for treating a disease or condition by administering an effective amount of a compound or salt of formula (I) to a subject suffering from the disease or condition.

The invention further provides methods for treating a disease or condition by administering

- 57 039109 administering a compound or salt of formula (I) to a subject suffering from a disease or condition, wherein the compound binds to menin and inhibits the interaction of menin with one or more proteins (for example, MLL1, MLL2, MLL fusion protein or MLL with partial tandem duplication) .

The invention further provides methods for stabilizing menin comprising contacting the menin with a compound or salt of formula (I). In some embodiments, the contacting step comprises contacting the menin with an amount of the compound sufficient to stabilize the menin. In some embodiments, the contacting step occurs in vivo. In some embodiments, the contacting step occurs in vitro. In some embodiments, the implementation step of bringing into contact occurs in the cell.

The present invention also provides methods of combination therapies in which an agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes, is used in combination with a compound or salt of formula (I). In one aspect, such therapy includes, but is not limited to, the combination of one or more compounds of the present invention with chemotherapeutic agents, therapeutic antibodies, and radiation therapy to provide a synergistic or additive therapeutic effect.

If desired, a compound or pharmaceutical composition of the present invention may be used in combination with Notch inhibitors and/or c-Myb inhibitors. If desired, a compound or pharmaceutical composition of the present invention may be used in combination with MLL-WDR5 and/or Dot11 inhibitors.

Many chemotherapeutic agents are currently known in the art and can be used in combination with a compound of the present invention. In some embodiments, the chemotherapeutic agent is selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antihormones, angiogenesis inhibitors, and antiandrogens.

Non-limiting examples are chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (Imatinib Mesilate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib) and Adriamycin, as well as a carrier of chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamin; nitrogen mustard derivatives such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, novembicine, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carsinophilin, CasodexTM, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxoA-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, cinostatin; zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; a folic acid supplement such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfomitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitrocrine; pentostatin; phenamet; pyrarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxanes, eg paclitaxel (TAXOLTM, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERETM, RhonePoulenc Rorer, Antony, France); retinoic acid; esperamycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In addition, suitable chemotherapeutic cell conditioners included are anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogen including, for example, tamoxifen (NolvadexTM), raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onpriston and toremifene (Far

- 58 039109 Estonia); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbin; novantron; teniposide; daunomycin; aminopterin; Yeseloda; ibandronate; camptothecin-11 (CPT-11); inhibitor of RFS 2000 topoisomerase; difluoromethylornithine (DMFO). If desired, the compounds or pharmaceutical composition of the present invention may be used in combination with commonly prescribed anticancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridinocarboxamide, Adecatumumab, 17-N-allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, anti-CD22 immunotoxins, antineoplastic agents, antineoplastic herbs, Apaziquone, Atiprimol, Azathioprine, Bendamustine, Belotecan , BIBW 2992, Birikodar, Brostallicin, Briostatin, Butionine sulfoximine, CBV (chemotherapy), Calculin, non-cell cycle specific antineoplastic agents, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocytabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesin, Phosfestrol, ICE chemotherapy regimen, IT-101, Imexon, Imichimod, Indolocarbazole, Irofulve n, Lanihidar, Larotaxel, Lenalidomide, Lucanton, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, Proteasome Inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN38, Salinosporamide A, Sapacitabine, Stanford V , Schwainsonin, Talaporfin, Tarikhidar, Tegafur-uracil, Temodar, Tesetaxel, Triplatin tetranitrate, tris(2-chloroethyl)amine, Troxcitabine, Uramustine, Vadimezan, Vilflunin, ZD6126, or Zosuhidar.

The present invention also relates to a method of using a compound or salt of formula (I) or a pharmaceutical composition provided herein in combination with radiation therapy to inhibit abnormal cell growth or treat a hyperproliferative disorder in a mammal. Radiation therapy techniques are known in the art and these techniques can be used in the combination therapy described herein. Administration of a compound of the present invention in this combination therapy may be determined as described herein.

Radiation therapy can be applied in one of several ways or a combination of ways, including, without limitation, external beam therapy, internal beam therapy, implantation radiation, stereotactic radiosurgery, systemic beam therapy, radiotherapy, and permanent or temporary interstitial brachytherapy. As used herein, the term brachytherapy refers to radiation therapy delivered by spatially confined radioactive material placed in the body in or near a tumor or other proliferating tissue in a diseased area. The term is intended to include, without limitation, exposure to radioactive isotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and radioactive isotopes of Lu ). Suitable radiation sources for use as a cell conditioner according to the present invention include both solids and liquids. As a non-limiting example, the radiation source can be a radionuclide such as I-125, I-131, Yb-169, Ir-192 as a solid source, I-125 as a solid source, or other radionuclides that emit photons, beta particles , gamma radiation or other therapeutic beams. The radioactive material may also be a liquid prepared from any solution of the radionuclide(s), such as a solution of I-125 or I-131, or the radioactive liquid may be prepared using a suspension of a suitable liquid containing fine particles of radionuclide solids such as Au-198 , Y-90. Moreover, the radionuclide(s) may be in the form of a gel or radioactive microspheres.

The compounds or pharmaceutical compositions of the present invention may be used in combination with an amount of one or more of antiangiogenesis agents, signaling inhibitors, antiproliferative agents, glycolysis inhibitors, or autophagy inhibitors.

Angiogenesis inhibitors such as MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors and COX-11 (cyclooxygenase 11) inhibitors can be used in combination with the compound of the present invention and the pharmaceutical compositions described in this document. Angiogenesis inhibitors include, for example, rapamycin, temsirolimus (CCI-779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab. Examples of suitable COX-II inhibitors include CELEBREXTM (alecoxib), valdecoxib and rofecoxib. Examples of suitable matrix metalloproteinase inhibitors are described in WO 96/33172 (published October 24, 1996), WO 96/27583 (published March 7, 1996), European patent application No. 97304971.1 (filed July 8, 1997), European patent application No. 99308617.2 (filed October 29, 1999), WO 98/07697 (published February 26, 1998), WO 98/03516 (published January 29, 1998), WO 98/34918 (published August 13, 1998), WO 98/34915 ( published August 13, 1998), WO 98/33768 (published August 6, 1998), WO 98/30566 (published

- 59 039109 forged 16 July 1998), EP 606046 (published 13 July 1994), EP 931788 (published 28 July 1999), WO 90/05719 (published 31 May 1990), WO 99/52910 (published October 21, 1999), wO 99/52889 (published October 21, 1999), WO 99/29667 (published June 17, 1999), PCT International Application No. PCT/IB 98/01113 (filed July 21, 1998), EP 99302232.1 (filed 25 March 1999), UK 9912961.1 (filed 3 June 1999), U.S. Provisional Application 60/148464 (filed 12 August 1999), US Patent 5863949 (issued 26 January 1999) 1999), US Pat. Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity in inhibiting MMP-1. More preferred are those that selectively inhibit MMP-2 and/or AMP-9 over other matrix metalloproteinases (e.g. MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12 and MMP-13). Some specific examples of MMP inhibitors suitable for the invention are AG-3340, RO 32-3555 and RS 13-0830.

Autophagy inhibitors include, but are not limited to, chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil™), bafilmicin A1, 5-amino-4-imidazole carboxamide riboside (AICAR), okadaic acid, autophage-inhibiting algal toxins that inhibit protein phosphatases type 2A or type 1, cAMP analogs, and drugs that increase cAMP levels such as adenosine, LY204002, N6-mercaptopurine riboside, and vinblastine. In addition, an antisense or siRNA that inhibits the expression of proteins, including but not limited to ATG5 (which are involved in autophagy), can also be used.

In some embodiments, the compounds described herein are formulated or administered in combination with liquid or solid tissue barriers, also known as lubricants. Examples of tissue barriers include, but are not limited to, polysaccharides, polyglycans, seprafilm, interside, and hyaluronic acid.

In some embodiments, drugs that are administered in combination with the compounds described herein include any suitable drugs effectively administered by inhalation, such as analgesics, such as codeine, dihydromorphine, ergotamine, fentanyl, or morphine; anginal drugs, such as diltiazem; antiallergic agents such as cromoglycate, ketotifen or nedocromil; anti-infectives, eg cephalosporins, penicillins, streptomycin, sulfonamides, tetracyclines or pentamidine; antihistamines, eg metapyrilene; anti-inflammatory agents, eg beclomethasone, flunisolide, budesonide, tipredan, triamcinolone acetonide or fluticasone; antitussives, such as noscapine; bronchodilators, eg ephedrine, epinephrine, fenoterol, formoterol, isoprenaline, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol, reproterol, rimiterol, salbutamol, salmeterol, terbutaline, isoetharine, tulobuterol, orciprenaline, or (-)-4-amino-3,5- dichloro-alpha-[6-[2(2-pyridinyl))-ethoxy]hexyl]amino]methyl]benzenemethanol; diuretics, eg amiloride; anticholinergics, eg ipratropium, atropine or oxitropium; hormones such as cortisone, hydrocortisone or prednisone; xanthines, for example aminophylline, choline theophyllinate, lysine theophyllinate or theophylline; and therapeutic proteins and peptides such as insulin or glucagon. The person skilled in the art will appreciate that, where appropriate, drugs are used in the form of salts (for example, as alkali metal or amine salts, or as acid addition salts), or esters (for example, lower alkyl esters), or solvates ( e.g. hydrates) to optimize drug activity and/or stability.

Other exemplary therapeutic agents suitable for combination therapy include, but are not limited to, radiation therapy, hormone antagonists and their releasing factors, thyroid and antithyroid drugs, estrogens and progestins, androgens, adrenocorticotropic hormone; adrenocortical steroids and their synthetic analogues; inhibitors of the synthesis and action of adrenocortical hormones, insulin, oral hypoglycemic agents and endocrine pancreatic pharmacology, agents affecting bone calcification and remodeling: calcium, phosphate, parathyroid hormone, vitamin D, calcitonin, vitamins such as water-soluble vitamins, vitamin B complex , ascorbic acid, fat-soluble vitamins, vitamins A, K and E, growth factors, cytokines, chemokines, agonists and antagonists of muscarinic receptors; anticholinesterase agents; agents acting in the neuromuscular junction and/or autonomic ganglia; catecholamines, sympathomimetic drugs and agonists or antagonists of adrenergic receptors; and 5-hydroxytryptamine (5-HT, serotonin) receptor agonists and antagonists.

Therapeutic agents may also include agents for pain and inflammation such as histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances that are formed by biotransformation of products of selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes , aspirin, carry

- 60 039109 roid anti-inflammatory agents, analgesic - antipyretic agents, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of inducible cyclooxygenase, selective inhibitors of inducible cyclooxygenase-2, autocoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions associated with humoral and cellular immune responses, lipid-induced autacoids, eicosanoids, beta-adrenergic agonists, ipratropium, glucocorticoids, methylxanthines, sodium channel blockers, opioid receptor agonists, calcium channel blockers, membrane stabilizers, and leukotriene inhibitors.

Additional therapeutic agents contemplated herein include diuretics, vasopressin, agents affecting renal water conservation, renin, angiotensin, agents useful in the treatment of myocardial ischemia, antihypertensive agents, angiotensin-converting enzyme inhibitors, beta-adrenergic receptor antagonists, agents for treating hypercholesterolemia; and agents for the treatment of dyslipidemia.

Other contemplated therapeutic agents include drugs used to control gastric acidity, agents for the treatment of gastric ulcers, agents for the treatment of gastroesophageal reflux disease, prokinetic agents, antiemetic agents, agents for irritable bowel syndrome, agents for diarrhea, agents for for the treatment of constipation, agents used in inflammatory bowel disease, agents used in the treatment of diseases of the biliary tract, agents used in the treatment of diseases of the pancreas. Therapeutic agents used to treat protozoal infections, drugs used to treat malaria, ambiosis, giardiasis, trichomoniasis, trypanosomiasis and/or leishmaniasis, and/or drugs used in the chemotherapy of helminthiasis. Other therapeutic agents include antimicrobial agents, sulfonamides, trimethoprimsulfamethoxazole quinolones and agents for the treatment of urinary tract infections, penicillins, cephalosporins and other beta-lactam antibiotics, an agent containing an aminoglycoside, protein synthesis inhibitors, drugs used in chemotherapy of tuberculosis, mycobacterium avium complex disease and leprosy, antifungal agents, antiviral agents including nonretroviral agents and antiretroviral agents.

Examples of therapeutic antibodies that can be combined with a compound of the present invention include, but are not limited to, anti-receptor tyrosine kinase antibodies (cetuximab, panitumumab, trastuzumab), anti-CD20 antibodies (rituximab, tositumomab) and other antibodies such as alemtuzumab, bevacizumab and gemtuzumab.

Moreover, the methods described herein contemplate therapeutic agents used for immunomodulation, such as immunomodulators, immunosuppressants, tolerogens, and immunostimulants. In addition, therapeutic agents acting on the blood and hematopoietic organs, hematopoietic agents, growth factors, minerals and vitamins, anticoagulant, thrombolytic and antiplatelet drugs.

For the treatment of renal carcinoma, a compound of the present invention may be combined with sorfenib and/or avastin. For the treatment of an endometrial disorder, a compound of the present invention may be combined with doxorubicin, taxotere (Taxol) and/or cisplatin (carboplatin). For the treatment of ovarian cancer, a compound of the present invention can be combined with cisplatin (carboplatin), taxotere, doxorubicin, topotecan and/or tamoxifen. For the treatment of breast cancer, a compound of the present invention can be combined with taxotere (taxol), gemcitabine (capecitabine), tamoxifen, letrozole, tarceva, lapatinib, PD0325901, avastin, herceptin, OSI-906, and/or OSI-930. For the treatment of lung cancer, a compound of the present invention can be combined with Taxotere (Taxol), gemcitabine, cisplatin, pemetrexed, tarceva, PD0325901 and/or Avastin.

Other therapeutic agents that can be combined with a compound of the present invention can be found in Goodman and Gilman's The Pharmacological Basis of Therapeutics Tenth Edition edited by Hardman, Limbird and Gilman or Physician's Desk Reference, both of which are incorporated herein by reference in their entirety. .

The compounds described herein may be used in combination with the agents described herein or other suitable agents, depending on the condition being treated. Therefore, in some embodiments, one or more compounds of the present invention will be co-administered with other agents as described above. When used in combination therapy, the compounds described herein are administered with a second agent simultaneously or separately. This administration in combination may include simultaneous administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the compound described herein and any of the agents described above may be formulated together in the same dosage form and administered simultaneously. Alternatively, a compound of the present invention and any of the agents described above may be administered simultaneously, with both agents present in

- 61 039109 separate preparations. In another alternative, a compound of the present invention may be administered immediately after any of the agents described above, or vice versa. In some embodiments of a separate administration protocol, the compound of the present invention and any of the agents described above are administered within minutes of each other, or within hours of each other, or within days of each other.

The following examples are presented for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any way. The present examples, as well as the methods and compositions described herein, represent preferred embodiments, are illustrative and are not intended to limit the scope of the invention. Changes to this document and other uses that fall within the scope of the invention as defined by the scope of the claims will occur to those skilled in the art.

Examples

Example 1. Synthesis of compounds 13 and 3 from table. one.

³

Step A. Preparation of Compound 3-2.

To a solution of compound 3-1 (6 g, 25 mmol) in THF (100 ml) was added LiAlH4 (1.5 g, 37 mol) in small portions at 0°C. The reaction mixture was stirred until TLC indicated completion of the reaction (about 2 hours). The reaction mixture was quenched with EtOAc and partitioned between EtOAc and H2O. The organic layer was washed with brine and dried over Na2SO4. The solvent was removed in vacuo to give compound 3-2 as a yellow solid (5.2 g, yield: 97%).

Stage B. Preparation of compound 3-4.

_MsCl ( ₄₂₈ mg, 4.4 mmol) was added at ₀ °C The reaction mixture was stirred at room temperature for 30 min, then quenched by adding NaHCO ₃ , washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo to give compound 3-3, which was used in the next step without further purification.

_{The compound} 3-3 in DMF. The reaction mixture was stirred at 100°C for 10 h. The reaction mixture was then partitioned between EtOAc and H2O. The organic layer was washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (petroleum ether/EtOAc=5:1-3:1) to give compound 3-4 as a yellow solid (600 mg, 42% yield according to alcohol).

Step C. Preparation of Compound 3-5.

A mixture of compound 3-4 (2.2 g, 5.8 mmol), 6-(2,2,2-trifluoroethyl)-N-(piperidin-4-yl)thieno-[2,3d]pyrimidine-4-amine (2.3 g, 6.9 mmol) and Et ₃ N (3.5 g, 34 mmol) in CH ₂ Cl ₂ (50 ml) were stirred at room temperature for 1 h before adding NaBH(OAc) ₃ (7 .3 g, 34 mmol) to the reaction mixture. The reaction mixture was stirred at room temperature overnight. Then the reaction mixture was divided between CH ₂ Cl ₂ and NaHCO ₃ . The organic layer was washed with brine and

- 62 039109 was dried over Na2SO4. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (CH2Cl2:MeOH=50:1~20:1) to give compound 3-5 as a yellow solid (3.9 g, 98% yield).

Step D Preparation of Compound 13

To a solution of compound 3-5 (3.9 g, 5.7 mmol) in CH2C12 (30 ml) was added TFA (20 ml). The reaction mixture was stirred for 4 hours at room temperature. The solvent was removed in vacuo to give a residue which was diluted with CH2C12 and washed with NaHCO ₃ . The organic layer was washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (CH ₂ Cl ₂ :MeOH=10:1) to give compound 13 as a white foam (2.6 g, 79% yield).

Stage E. Preparation of compound 3.

Compounds 13 ( ₂ .7 g, 4.6 mmol). The reaction mixture was stirred at room temperature for 30 min before being quenched with NaHCO ₃ , washed with brine and dried over Na2SO4. The solvent was removed and the residue was purified by silica gel column chromatography (CH2Cl2:MeOH=10:1) to give compound 3 (1.8 g, yield: 61%). 1H NMR (400 MHz, CDCI3): 8.49 (s, 1H), 7.34 (d, 1H), 7.21 (s, 1H), 7.11 (d, 1H), 7.08 (s, 1H), 5.78 (s, 1H) , 5.07 (d, 1H), 4.45 (s, 2H), 4.25 (m, 1H), 3.61-3.70 (m, 4H), 2.93 (m, 2H), 2.57 (s, 3H), 2.33-2.20 (m , 2H), 2.00-2.13 (m, 2H), 2.02 (s, 6H), 1.90 (s, 3H), 1.50-1.70 (m, 2H).

Example 2. Synthesis of compound 29 from the table. one.

29-4 29-5 29

Step A: Preparation of Compound 29-2.

To a solution of compound 29-1 (200 mg, 1.0 mmol) and Et3N (202 mg, 2.0 mmol) in CH2Cl2 (10 ml) was added MsCl (172 mg, 1.5 mmol) at 0°C. The reaction mixture was stirred at room temperature overnight before water was added to the reaction mixture. The reaction mixture was extracted 3 times with CH2Cl2. The organic layer was washed with brine and dried over Na2SO4. The solution was filtered and concentrated to give compound 29-2 as a white solid (250 mg, 90% yield).

Step B: Preparation of Compound 29-3.

Mixture of compound 29-2 (250 mg, 0.9 mmol), 5-formyl-4-methyl-1H-indole-2-carbonitrile (82 mg, 0.45 mmol) and Cs2CO3 (438 mg, 1.35 mmol) in DMF (6 ml) was stirred at 60°C for 6 h before adding water (15 ml). The reaction mixture was extracted with ethyl acetate (20 ml x3). The combined organic solution was washed with brine and dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (33% EtOAc in petroleum ether to 50% EtOAc in petroleum ether) to give compound 29-3 as a yellow solid (110 mg, 33% yield).

Step C: Preparation of Compound 29-4.

A mixture of compound 29-3 (110 mg, 0.3 mmol), 6-(2,2,2-trifluoroethyl)-N-(piperidin-4-yl)thieno[2,3d]pyrimidin-4-amine hydrochloride (116 mg, 0.3 mmol) and Et ₃ N (185 mg, 1.8 mmol) in CH ₂ Cl ₂ (20 ml) were stirred at room temperature for 1 h before adding NaBH(OAc) ₃ (381 mg, 1, 8 mmol) to the reaction mixture in an ice bath. The reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (2.5% MeOH in CH2Cl2) to give compound 29-4 as a solid (180 mg, 90% yield).

Step D: Preparation of Compound 29-5.

A solution of tert-butyl carbamate 29-4 (180 mg, 0.27 mmol) in HCl/MeOH (10 ml) was stirred at room temperature for 2 h. The solvent was removed and a solution of NH ₃ (7N) in MeOH (10 ml) was added . The reaction mixture was stirred for 10 min before removing the solvent and the residue was purified

- 63 039109 silica gel column chromatography (10% MeOH in CH2C12) to give compound 29-5 as an oil (100 mg, 65% yield).

Step E: Preparation of Compound 29.

_{Acryloyl chloride} (19 _mg , 0.21 mmol) at -78°C under N2 atmosphere. The mixture was stirred at room temperature for 15 minutes, then NH ₃ -MeOH was added. The solvent was removed and the residue was purified by silica gel column chromatography (10% MeOH in CH ₂ Cl ₂ ) to give the final product 29 as a solid (78 mg, yield: 71%). 1H NMR (400 MHz, DMSO) δ: 8.32 (s, 1H), 7.81-7.80 (d, 1H), 7.64 (s, 1H), 7.55 (s, 1H), 7.39 (s, 1H), 7.34-7.32 (m, 2H), 6.16-6.01 (m, 2H), 5.57-6.54 (m, 1H), 4.33-4.31 (d, 2H), 4.09-4.00 (m, 4H), 3.68 (s, 3H), 2.86 -2.85 (m, 2H), 2.45-2.41 (m, 1H), 2.26-2.24 (m, 2H), 2.10 (brs, 2H), 1.99 (s, 1H), 1.89 (brs, 2H), 1.75-1.67 (m, 2H), 1.57 (brs, 2H); ESI-MS m/z: 622.40 (M+H).

Example 3. Synthesis of compound 10 from the table. one.

n

10.4 10-5 10

Step A: Preparation of Compound 10-1.

To a solution of compound 3-1 (300 mg, 1.24 mmol) in DMF (15 ml) was added NaH (210 mg, 2.5 mmol) at 0°C. The reaction mixture was stirred at the same temperature for 20 minutes before the addition of methane (50 mg, 2.5 mmol). The resulting mixture was stirred at room temperature for 3 hours before water was added. The reaction mixture was extracted with ethyl acetate. The combined organic layer was concentrated to dryness. The residue was purified on a silica gel column (petroleum ether/EIAc=5:1) to obtain compound 10-1 (310 mg, yield: 97%) as a colorless oil.

Step B: Preparation of Compound 10-2.

To a mixture of methyl ester 10-1 (310 mg, 1.21 mmol) in THF (10 ml) was slowly added LiAlH ₄ at 0°C. The reaction mixture was stirred at room temperature for 1 h before adding water (0.2 ml) followed by EtOAc. The reaction mixture was filtered and concentrated to dryness. The residue was purified on a silica gel column (petroleum ether/EIAc=3:1) to give compound 10-2 (237 mg, 86% yield).

Step C: Preparation of Compound 10-3.

To a solution of compound 10-2 (230 mg, 1.01 mmol) in CH2Cl2 was added Et3N (0.42 ml, 3.03 mmol) at 0°C followed by methanesulfonyl chloride (231 mg, 2.02 mmol). The resulting mixture was stirred at room temperature for 1 h. CH ₂ Cl ₂ was added, the mixture was washed with NaHCO ₃ and the organic layer was washed with brine and dried over Na2SO4. The solvent was removed to give compound 10-3 (330 mg) as a brown oil.

Step D: Preparation of Compound 10-4.

A mixture of crude compound 10-3 (330 mg), 5-formyl-4-methyl-1H-indole-2-carbonitrile (200 mg, 1.08 mmol) and Cs ₂ CO ₃ (1 g, 3.24 mmol) in DMF (10 ml) was stirred at 100°C overnight. Water was added and the reaction mixture was extracted with ethyl acetate. The organic layer was dried over Na ₂ SO ₄ and concentrated to dryness. The residue was purified on a silica gel column (petroleum ether/EIAc=4:1) to give compound 10-4 (177 mg, yield: 41%). ESI-MS m/z. 394 (M+H).

Step E Preparation of Compound 10-5.

A mixture of compound 10-4 (177 mg, 0.45 mmol), N-(piperidin-4-yl)-6-(2,2,2-trifluoroethyl)thieno[2,3d]pyrimidin-4-amine hydrochloride (238 mg, 0.68 mmol), Et ₃ N (0.2 ml, 1.3 mmol) and NaBH(OAc) ₃ in CH2Cl2 were stirred at room temperature overnight. The reaction mixture was diluted with CH ₂ Cl ₂ , washed with brine and concentrated. The residue was purified on a silica gel column (CH ₂ Cl ₂ /MeOH=30:1) to give compound 10-5 (210 mg, yield: 67%). ESI-MS m/z. 694 (M+H).

Step F Preparation of Compound 10

A solution of compound 10-5 (100 mg, 0.14 mmol), TFA (1 ml) in CH2Cl2 (5 ml) was stirred at room temperature for 3 h. The mixture was concentrated and the residue was dissolved in CH ₂ Cl ₂ , washed with NaHCO ₃ , dried over Na ₂ SO ₄ and concentrated. The residue was purified on a silica gel column.

- 64 039109 (CH2Cl ₂ /MeOH=30:1) to give compound 10 (50 mg, yield: 58%). ESI-MS m/z: 594 (M+H). ¹ H NMR (400 MHz, CDCl3) δ: 8.48 (s, 1H), 7.38 (d,1H), 7.22 (s, 1H), 71.5 (s, 1H), 7.13 (s, 1H), 5.23 (brs, 1H), 4.46 (s, 2H), 4.26-4.28 (m, 1H), 3.62-3.69 (m, 4H), 2.97 (d, 2H), 2.63 (s, 3H), 2.31-2.37 (m, 5H) , 2.08-2.14 (m, 2H), 1.65-1.73 (m, 8H).

Example 4. Synthesis of compounds 11 and 12 from the table. one.

Step A: Preparation of Compound 12-1.

A mixture of compound 3-3 and Bu4CN (3.5 g, 13 mmol) in CH3CN (30 ml) was stirred at reflux for 10 h until TLC indicated completion of the reaction. The solvent was removed and the residue was purified by silica gel chromatography (petroleum ether/EtOAc=3:1) to give compound 12-1 as a white solid (1.0 g, 86% yield according to alcohol).

Step B: Preparation of Compound 12-2.

DIBAL-H (6 mmol) was added dropwise to a solution of compound 12-1 (460 mg, 2 mmol) in CH ₂ Cl ₂ at -78°C and the reaction mixture was stirred at the same temperature for 2 h. The reaction mixture was quenched with NH4Cl and dried over Na ₂ SO ₄ . The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (petroleum ether/EULs=5:1-3:1) to give compound 12-2 as a white solid (200 mg, 44% yield).

Step C: Preparation of Compound 12-3.

To a solution of compound 12-2 (200 mg, 1 mmol) in THF was added BH3/THF (4 mmol) dropwise at 78°C. The reaction mixture was stirred for 10 hours before being quenched with MeOH. The solvent was removed in vacuo to give compound 12-3 as a white solid (200 mg, 99% yield) which was used in the next step without further purification.

Stage D. Preparation of compound 12-5.

_MsCl (73 mg, _0.63 mmol) was added at ₀ °C The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched with NaHCO ₃ , washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo to give crude product 12-4 which was used in the next step without further purification.

_{12-4 _} in DMF. The reaction mixture was stirred at 100° C. for 10 hours. The reaction mixture was partitioned between EtOAc and H ₂ O. The organic layer was washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo to give a residue which was purified by silica gel column chromatography (petroleum ether/EtOAc=5:1-3:1) to give compound 12-5 as a white solid (100 mg, 52% yield 2 steps).

Step E Preparation of Compound 12-6.

Mixture 12-5 (30 mg, 0.1 mmol), 6-(2,2,2-trifluoroethyl)-N-(piperidin-4-yl)thieno-[2,3-d]pyrimidine-4amine (50 mg , 0.12 mmol) and Et ₃ N (60 mg, 0.6 mmol) in CH ₂ Cl ₂ (10 ml) were stirred at room temperature for 1 h before adding NaBH(OAc) ₃ (130 mg, 0.6 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between CH ₂ Cl ₂ and NaHCO ₃ and the organic layer was washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo to give a residue, which was purified by silica gel column chromatography (CH ₂ Cl ₂ :MeOH=50:1~20:1) to give compound 12-6 as a yellow solid (40 mg, 60% yield).

Step F. Preparation of Compound 11.

To a solution of compound 12-6 (130 mg, 0.19 mmol) in CH ₂ Cl ₂ (3 ml) was added TFA (2 ml). The reaction mixture was stirred for 4 hours at room temperature. The solvent was removed in vacuo with

- 65 039109 obtaining a residue, which was diluted with CH2C12 and washed with NaHCO ₃ . The organic layer was washed with brine and dried over Na2SO4. The solvent was removed in vacuo to give compound 11 as a yellow foam (100 mg, wet weight).

Step G. Preparation of Compound 12.

_{Compound 11 (} 35 mg, 0.06 mmol), then the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched by adding NaHCO3, washed with brine and dried over Na2SO4. The solvent was removed to give a residue which was purified by preparative TLC (CH ₂ Cl ₂ :MeOH=10:1) to give compound 12 (10 mg, 30% yield). ¹ H NMR (400 MHz, CDCl ₃ ) 8.46 (s, 1H), 7.51 (d, 1H), 7.17~7.22 (m, 3H), 5.85 (s, 1H), 5.79 (br, 1H), 4.23~4.32 (m, 3H), 3.86 (s, 2H), 3.66 (q, 2H), 3.12 (m, 2H), 2.58 (s, 3H), 2.53~2.40 (m, 2H), 2.20~2.14 (m, 6H ), 1.99 (s, 6H), 1.86~1.90 (m, 2H), 1.12 (t, 3H). ESI-MS m/z: 650.25 (M+H).

Example 5. Synthesis of compounds 20 and 18 from the table. one.

Step A: Preparation of Compound 18-2.

A mixture of compound 18-1 and Et ₃ N (600 mg, 6 mmol) in CH ₂ Cl ₂ was stirred at 0° C. and then MsCl (460 mg, 4 mmol) was added slowly. The reaction mixture was stirred at 0° C. under N2 for 2 hours. TLC indicated completion of the reaction. The reaction mixture was partitioned between CH ₂ Cl ₂ and H ₂ O and the organic layer was washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo and the resulting compound (18-2) was used without further purification as a light yellow oil (460 mg, 99% yield).

Step B: Preparation of Compound 18-3.

A mixture of crude compound 18-2 (460 mg, 2 mmol), 5-formyl-4-methyl-1H-indole-2-carbonitrile (440 mg, 2.4 mmol) and Cs ₂ CO ₃ (1.3 g, 4 mmol) in DMF (10 ml) was stirred at 60°C for 4 h. The reaction mixture was cooled and the solid was removed by filtration. The reaction mixture was partitioned between EtOAc and H2O and the organic layer was washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo to give a residue, which was purified by silica gel column chromatography (petroleum ether: EtOAc=10:1~4:1) to give compound 18-3 as a light yellow solid (280 mg, 43% yield). ESI-MS m/z: 323 (M+H).

Step C: Preparation of Compound 18-4.

A mixture of compound 18-3 (280 mg, 0.87 mmol), N-(piperidin-4-yl)-6-(2,2,2-trifluoroethyl)thieno[2,3d]pyrimidin-4-amine hydrochloride (435 mg, 1.35 mmol) and Et ₃ N (400 mg, 4 mmol) in CH ₂ Cl ₂ (30 ml) were stirred at room temperature for 2 h before adding NaBH(OAc) ₃ (570 mg, 2.7 mmol ) upon cooling in an ice bath. The reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between CH ₂ Cl ₂ and NaHCO ₃ and the organic layer was washed with brine and dried over Na2SO4. The solvent was removed in vacuo to give a residue, which was purified by silica gel column chromatography (petroleum ether: EtOAc=10:1~1:1) to give compound 18-4 as a yellow solid (300 mg, 56% yield). ESI-MS m/z: 623 (M+H).

Step D Preparation of Compound 20

To a solution of compound 18-4 (180 mg, 0.3 mmol) in water (4 ml) and THF (10 ml) was added LiOH (24 mg, 0.6 mmol). The reaction mixture was stirred at room temperature for 16 hours. TLC indicated completion of the reaction. The pH of the mixture was adjusted to pH 4 with HCl (aq. 1N). The reaction mixture was diluted with EtOAc and the organic layer was dried over Na ₂ SO ₄ . The solvent was removed in vacuo to give Compound 20 which was used without further purification as a yellow solid (130 mg, 75% yield).

Step E. Preparation of Compound 18.

A mixture of crude compound 20 (40 mg, 0.07 mmol), methylamine hydrochloride (30 mg, 0.44 mmol), EDCI (40 mg, 0.28 mmol), HOBT (15 mg, 0.11 mmol) and Et3N ( 50 mg, 0.5 mmol) in CH2Cl2 (10 ml) was stirred at room temperature for 40 h. The reaction mixture was partitioned between CH ₂ Cl ₂ and Na- 66 039109

HCO ₃ and the organic layer were washed with brine and dried over Na ₂ SO ₄ . The solvent was removed in vacuo to give a residue which was purified by preparative TLC (CH2Cl2:MeOH=10:1) to give compound 18 as a white solid (15 mg, 35% yield). 1H NMR (400 MHz, MeOD) 8.31 (s, 1H), 7.54 (s, 1H), 7.41~7.32 (m, 3H), 4.45 (s, 2H), 4.24~4.17 (m, 1H), 3.89~3.81 (m, 2H), 3.74 (s, 2H), 3.08~3.05 (m, 2H), 2.66 (s, 3H), 2.60 (s, 3H), 2.40~2.34 (m, 2H), 2.07~2.03 (m , 2H), 1.88 (s, 6H), 1.76~1.70 (m, 2H). ESI-MS m/z: 622 (M+H).

Example 6. Synthesis of compounds 17 and 33 from the table. one.

Step A: Preparation of Compound 33-1.

Mixture of compound 13 (190 mg, 0.33 mmol), 2-(tert-butoxycarbonyl)acetic acid (79 mg, 0.43 mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (229 mg, 0.5 mmol ) and iPr ₂ NEt (0.3 ml, 1.65 mmol) in CH ₂ Cl ₂ (10 ml) were stirred at room temperature for 30 min. Water was added and the resulting mixture was extracted with CH ₂ Cl ₂ . The organic layer was concentrated and the residue was purified on a silica gel column (CH ₂ Cl ₂ /MeOH=20:1) to give compound 33-1 (210 mg, 87% yield) as a solid. ESI-MS m/z: 737 (M+H).

Step B. Preparation of Compound 17.

A mixture of Compound 33-1 (230 mg, 0.34 mmol) in CH ₂ Cl ₂ (5 ml) and trifluoroacetic acid (5 ml) was stirred at room temperature for 2 h. The reaction mixture was concentrated to dryness and the residue was dissolved in NH3 /MeOH(7N). The mixture was concentrated to dryness. The residue was purified on a silica gel column to give compound 17 as a yellow solid (210 mg, 83% yield). ESI-MS m/z: 637 (M+H).

Step C. Preparation of Compound 33.

A mixture of compound 17 (50 mg, 0.08 mmol), formic acid (8 mg, 0.16 mmol), benzotriazol-1yloxytris(dimethylamino)-phosphonium hexafluorophosphate (52 mg, 0.12 mmol) and iPr ₂ NEt (0.07 ml , 0.4 mmol) in CH2Cl2 (5 ml) was stirred at room temperature for 30 min. Water was added and the resulting reaction mixture was extracted with CH ₂ Cl ₂ . The organic layer was concentrated and the residue was purified on a silica gel column (CH ₂ Cl ₂ /MeOH=15:1) to give compound 33 as a solid (40 mg, yield: 77%). 1H NMR (400 MHz, CD3OD) δ: 8.30 (s, 1H), 8.09 (s,1H), 7.52 (s, 1H), 7.30-7.34 (m, 3H), 4.51 (s,2H), 4.20 (m , 1H), 3.67-3.85 (m, 6H), 3.07-3.10 (m, 2H), 2.59 (s, 3H), 2.34-2.44 (m, 2H), 2.05-2.08 (m, 2H), 1.96 (s , 6H), 1.62–1.76 (m, 2H). ESI-MS m/z: 664 (M+H).

- 67 039109

Example 7. Synthesis of compound 82 from the table. one.

3-4 82-1 82-2

82-3 82

Step A: Preparation of Compound 82-2.

To a mixture of compound 3-4 (1.0 g, 2.6 mmol) and compound 82-1 (1.0 g, 2.9 mmol) in DCM (30 ml) mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 h and then NaBH(OAc) ₃ (3.35 g, 15.8 mmol) was added. The reaction mixture was stirred at room temperature overnight. TLC indicated completion of the reaction. The reaction mixture was washed with water and the aqueous layer was extracted with 50 ml DCM. The combined organic solution was washed with 50 ml of brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by silica gel column chromatography (eluted with 2.5-5% MeOH in DCM) to give compound 82-2 as a white solid (1.4 g, 75% yield). ESI-MS m/z: 709.50 (M+H).

Step B: Preparation of Compound 82-3.

A solution of compound 82-2 (1.4 g, 1.98 mmol) in HCl/MeOH (20 ml) was stirred at room temperature for 1 h, then the solution was concentrated in vacuo. A solution of NH3 in MeOH was added to bring the pH to neutral. The solvent was removed and the residue was purified by silica gel column chromatography (eluted with 2.5% MeOH in DCM) to give compound 82-3 as a white solid (1.0 g, 83% yield). ESI-MS m/z: 609.40 (M+H).

Step C: Preparation of Compound 82.

BOP (1.25 g, 2 .96 mmol) and the mixture was stirred at room temperature for 2 hours. TLC indicated the reaction was complete. The reaction mixture was washed with water and the aqueous layer was extracted with 50 ml DCM. The combined organic solution was washed with 50 ml of brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by silica gel column chromatography (eluted with 35% MeOH in DCM) to give compound 82 as a solid (475 mg, 49% yield). ESI-MS m/z: 651.45 (M+H).

Example 8 Fluorescent Polarization Analysis.

This example illustrates an assay effective for monitoring MLL binding to menin. Competitive fluorescent polarization (FP) experiments are performed to determine the effectiveness with which a compound inhibits the menin-MLL interaction, reported as the IC ₅₀ value. A fluorescein-labeled peptide containing a high affinity binding motif for menin found in MLL was prepared according to Yokoyama et al. (Cell, 2005, 123(2): 207-218), incorporated herein by reference in its entirety. Binding of a labeled peptide (1.7 kDa) to a much larger menin (~67 kDa) is accompanied by a significant change in the rotational correlation time of the fluorophore, which leads to a significant increase in fluorescent polarization and fluorescent anisotropy (excitation at 500 nm, emission at 525 nm). The potency with which a compound inhibits the menin-MLL interaction is measured in a competitive FP experiment where the reduction in fluorescence anisotropy correlates with inhibition of the interaction and is used as a readout to determine the IC ₅₀ .

In table. 2 shows the biological activities of selected compounds in a fluorescent polarization assay. The numbers of compounds correspond to the numbers and structures presented in table. 1 and examples 1-7.

- 68 039109

table 2

Less than 50 nM (++++) 50 nM to less than 250 nM (+++) 250 nM to 1000 nM (++) More than 1000 nM (+) Menin MLL 4-43 IC ₅₀ (nM) 7, 15, 17,35,37, 39, 40,41,48, 54, 56, 57, 58, 59, 62, 64, 65, 70, 71, 76, 79, 80, 82, 83, 84, 85, 86 1,2,3,8, 9, 10,13, 16, 18, 29,30, 32, 34, 36, 38, 42, 43, 55, 60, 63, 66, 67, 68, 72, 73, 75 4.5, 6, 11,14,31, 33, 61, 69, 74, 77, 78 12, 20, 49

Example 9 Homogeneous Time Resolved Fluorescence (HTRF) Analysis.

Time resolved homogeneous fluorescence (HTRF) analysis is used as a secondary analysis to confirm the results of the FP analysis. In some embodiments, the HTRF assay is the primary assay and the FP assay is used as a secondary assay to confirm the results. HTRF is based on the non-radiative energy transfer of long-lived emission from the Europium cryptate donor (Eu ³⁺ -cryptate) to the allophycocyanin acceptor (XL665) combined with time-resolved detection. The Eu ³⁺ cryptate is conjugated to a mouse monoclonal antibody against 6His (which binds to His-menin) and the XL665 acceptor is conjugated to streptavidin (which binds to the biotinylated MLL peptide). When these two fluorophores are brought together by the interaction of menin with the MLL peptide, the transfer of energy to the acceptor results in an increase in fluorescent emission at 665 nm and an increase in the HTRF ratio (emission intensity at 665 nm/emission intensity at 620 nm). Inhibition of the menin-MLL interaction separates the donor from the acceptor, resulting in a decrease in emission at 665 nm and a decrease in the HTRF ratio.

Example 10 Menin interaction analysis.

Sample preparation: 2.5 µl of 100 µM compound was added to 47.5 µl of 526 nM menin in PBS (5 µM of 500 nM menin compound in 5% DMSO final concentration). The reaction mixture was incubated at room temperature for various periods of time and quenched with 2.5 μl of 4% formic acid (FA, 0.2% final concentration). Method: Thermo Finnigan Surveyor Autosampler, PDA Plus UV detector and MS Pump were used to collect these samples along with an LTQ linear mass spectrometric ion trap controlled by XCalibur software. 5 µl of sample in no waste mode was injected into Phenomenex Jupiter 5u 300A C5 (guard column) 2x4.00 mm at 45°C. Mobile phase composition: buffer A (95:5 water:acetonitrile, 0.1% FA) and buffer B (acetonitrile, 0.1% FA). Gradient elution was used with an initial mobile phase of 85:15 (buffer A:B) and a flow rate of 250 µl/min. After injection, 85:15 A:B was held for 1.3 min, buffer B was increased to 90% over 3.2 min, held for 1 min, and then returned to initial conditions after 0.1 min and held for 2.4 min. The total running time was 8 minutes. During the first 2 min of sampling, a post-column transfer valve was used to direct salts from the headspace in the waste. A blank of buffer A was used between each sample injection. A needle wash of 1:1 acetonitrile:water with 0.1% FA was used. An electrospray ionization (ESI) source used a capillary temperature of 300°C, 40 units of shield gas, 20 units of auxiliary gas, 3 units of purge gas, 3.5 kV nebulizer voltage, 120 V tubular lens. Data Acquisition: Data Acquisition were performed in the mode of full scanning of positive ions 550-1500 Da, 10 microscans, maximum ionization time 200 ms. Data analysis: Protein mass spectra were obtained as XCalibur data. The best scans were summarized using the XCalibur Qual Browser. The spectra were displayed using the View/Spectrum List with the Display option to display all peaks. The Edit/Copy menu was used to copy the mass spectrum to the PC clipboard. The spectrum on the PC clipboard was pasted into Excel. The first two columns (m/z and Intensity) have been kept, while the third column (Relative) has been removed. The remaining two columns were then saved from Excel as a tab delimited file (m/z and intensity) as filename.txt. The Masslynx Databridge program was then used to convert the tab-delimited file filename.txt to the Masslynx format. In some cases, Masslynx used an external calibration using a (similarly transformed) myoglobin spectrum to correct the m/z data values for the menin protein. The MaxEntl software from the MassLynx software package was used to deconvolve the mass spectrum to obtain the average MW of the protein(s). The percentage of covalent adduct formation was determined from the deconvolution spectrum and used to calculate the reaction rate (k) of the covalent reaction.

Example 11 Cell Proliferation Assay.

The ability of a compound of the present invention to inhibit the growth of cells such as human leukemia, human leukemia, VCaP, LNCaP, 22RV1, DU145, LNCaP-AR, MV4;11, KOPN8, ML-2, MOLM-13, RS4;11, SEM, bone marrow cells (BMCs), MLL-AF9, MLL-AF4, MLL-ENL, MLL-CBP, MLL-GAS7, MLL-AF1p, MLL-AF6, HM-2, E2A-HLF, REH, U937, K562, KG -1, HL-60 and NB4 tested using a viability assay such as Promega CellTiter-Glo® Luminescent Cell Viabil

- 69 039109 ity Assay (Promega Technical Bulletin, 2015, CellTiter-Glo® Luminescent Cell Viability Assay: 1-15, incorporated herein by reference in its entirety). Cells were seeded at appropriate concentrations, eg about 1x105-2x105 cells per well in a 96-well plate. The compound of the present invention was added at concentrations up to about 2 μM using eight 2-fold serial dilutions for each compound. Cells were incubated at 37°C for a certain period of time, for example 72 h, then counted the cells in the control wells. The medium was changed to restore the number of viable cells to the original concentration, and the compounds were resubmitted. Proliferation was measured after 72 hours using Promega CellTiter-Glo® reagents according to kit instructions.

Example 12 RT-PCR analysis of downstream MLL fusion protein targets.

The effect of a compound of the present invention on the expression of one or more downstream MLL fusion protein targets was assessed by RT-PCR. Cells such as VCaP, LNCaP, 22RV1, DU145, LNCaP-AR, MV4;11, KOPN-8, ML-2, MOLM-13, RS4;11, SEM, bone marrow cells (BMC), MLL-AF9, MLL -AF4, MLL-ENL, MLL-CBP, MLL-GAS7, MLL-AF1p, MLL-AF6, HM-2, E2A-HLF, REH, U937, K562, KG-1, HL-60 and NB4, treated with effective concentration compounds described in this application for about 7 days or less and then the total RNA was extracted from the cells using any available kit, such as RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. Total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and determination of the relative abundance of relevant gene transcripts (eg Hoxa9, DLX2 and Meis1) was performed using real time PCR. Effective inhibition of the menin-MLL interaction is expected to result in down-regulation of downstream MLL targets including Hoxa9, DLX2 and Meis1.

Example 13 Pharmacokinetic studies in mice.

The pharmacokinetics of the menin-MLL inhibitors were determined in female C57BL/6 mice after an intravenous (iv) dosing of 15 mg/kg and an oral dosing (po) of 30 mg/kg. Compounds were dissolved in a vehicle containing 25% (v/v) DMSO, 25% (v/v) PEG-400 and 50% (v/v) PBS. Serial blood samples (50 µl) were collected over 24 h, centrifuged at 15,000 rpm for 10 min, and stored for analysis. Plasma concentrations of compounds were determined by the LC-MS/MS method developed and validated for this study. The LC-MS/MS method consists of an Agilent 1200 HPLC system and chromatographic separation of the test compound is achieved using an Agilent Zorbax Extend-C18 column (5 cm x 2.1 mm, 3.5 µm; Waters). For detection, an AB Sciex QTrap 3200 mass spectrometer equipped with an electrospray ionization source (ABI-Sciex, Toronto, Canada) in positive ion multiple reaction monitoring (MRM) mode is used. All pharmacokinetic parameters were calculated by compartmentless methods using WinNonlin® version 3.2 (Pharsight Corporation, Mountain View, CA, USA).

Example 14 Efficacy Study in a Mouse Xenograft Tumor Model.

Immunodeficient mice such as 8-10 week old female athymic (nu/nu) mice were used for in vivo efficacy studies according to IACUC approved guidelines. Leukemia cells, such as human VM4-11 leukemic cells available from ATCC, were implanted subcutaneously via needle into female nude mice (5x106 cells/mouse). When the mouse tumor reached a size of approximately 150 to 250 mm ³ , tumor-bearing mice were randomly assigned to a vehicle control group or a treatment group (8 animals per group). Animals were treated with a compound of the present invention by oral gavage or intraperitoneal injection at appropriate amounts and frequencies as may be determined by one skilled in the art without undue experimentation. Subcutaneous tumor volume in nude mice and body weight of mice were measured twice a week. Tumor volumes were calculated by measuring two perpendicular diameters with calipers (V=(length×width ² )/2). The percentage of tumor growth inhibition (% TGI=1-[treatment group tumor volume change/control group tumor volume change]*100) was used to evaluate antitumor efficacy. Statistical significance was assessed using a one-tailed two-sample t-test. P<0.05 is considered statistically significant.

Example 15 Efficacy Study in a Xenograft Prostate Tumor Model.

Immunodeficient mice such as 4-6 week old male severe combined immunodeficiency (SCID) CB17 mice were used for in vivo efficacy studies according to IACUC approved guidelines. Parental prostate cancer cells such as VCaP or LNCaP-AR cells were implanted subcutaneously in male CB.17.SCID mice (3-4x106 cells in 50% Matrigel). When the tumor reached a measurable size of approximately 80 mm ³ , tumor-bearing mice were randomly assigned to a vehicle control group or a treatment group (6 or more animals per group). Animals were treated with a compound of the present invention by

- 70 039109 intraperitoneal injection with the appropriate amount and frequency, which can be determined by a person skilled in the art without undue experimentation. In one example, mice were treated daily with 40 mg/kg of a compound of the present invention by intraperitoneal injection for two weeks, then 5 days a week. Subcutaneous tumor volume and body weight of the mice were measured twice a week. Tumor volumes were calculated by measuring two perpendicular diameters with calipers (V=(length×width ² )/2).

Example 16 Efficacy Study in a Xenograft Model of Castration-Resistant Prostate Tumor (VCaP).

For in vivo efficacy studies according to IACUC approved guidelines, immunodeficient mice such as 4-6 week old male severe combined immunodeficiency (SCID) CB17 mice were used. Parental prostate cancer cells, such as VCaP cells, were implanted subcutaneously in male CB.17.SCID mice (3-4x106 cells in 50% Matrigel). When the tumor reached a size of approximately 200-300 mm ³ , the tumor-bearing mice were physically castrated and the tumor was observed to regress and recover to approximately 150 mm ³ . Tumor-bearing mice were randomly assigned to a vehicle control group or a compound treatment group (6 or more animals per group). Animals were treated with a compound of the present invention by intraperitoneal injection at appropriate amounts and frequencies as may be determined by one of skill in the art without undue experimentation. In one example, mice were treated daily with 40 mg/kg of a compound of the present invention by ip injection. Subcutaneous tumor volume and body weight of the mice were measured twice a week. Tumor volumes were calculated by measuring two perpendicular diameters with calipers (V=(length×width ² )/2).

Example 17 Efficacy study in a xenograft model of castration-resistant prostate tumor (LNCaP-AR).

Immunodeficient mice such as 4-6 week old male severe combined immunodeficiency (SCID) CB17 mice were used for in vivo efficacy studies according to IACUC approved guidelines. CB.17 SCID mice were surgically castrated and allowed to recover for 2-3 weeks prior to implantation of parental prostate cancer cells, such as LNCaP-AR cells, subcutaneously in (3-4x106 cells with 50% Matrigel). When the tumor reached a size of approximately 80-100 mm ³ , tumor-bearing mice were randomly assigned to a vehicle control group or a compound treatment group (6 or more animals per group). Animals were treated with a compound of the present invention by intraperitoneal injection at an appropriate amount and frequency as may be determined by one of skill in the art without undue experimentation. In one example, mice were treated daily with 60 mg/kg of a compound of the present invention by intraperitoneal injection for 27 days. Subcutaneous tumor volume and body weight of the mice were measured twice a week. Tumor volumes were calculated by measuring two perpendicular diameters with a caliper (V=(length×width ² )/2).

Example 18 Cellular Thermal Shift Analysis (CETSA). For CETSA cell lysate experiments, cultured cells from cell lines (eg, HEK293, bone marrow samples) were harvested and washed with PBS. Cells were diluted in kinase buffer (KB) (25 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM sodium vanadium oxide, 10 mM magnesium chloride) or in phosphate buffered saline (PBS) (10 mM phosphate buffer (pH 7.4), 2.7 mM potassium chloride and 137 mM sodium chloride). All buffers were supplemented with a complete mixture of protease inhibitors. Cell suspensions were frozen-thawed three times using liquid nitrogen. The soluble fraction (lysate) was separated from the cell debris by centrifugation at 20000xg for 20 min at 4°C. Cell lysates were diluted with the appropriate buffer and divided into two aliquots, with one aliquot treated with drug and the other aliquot treated with inhibitor diluent (control). After a 10-30 minute incubation at room temperature, the respective lysates were divided into smaller (50 μl) aliquots and heated individually at different temperatures for 3 minutes followed by cooling for 3 minutes at room temperature. Appropriate temperatures were determined in preliminary CETSA experiments. The heated lysates were centrifuged at 20,000xg for 20 min at 4°C to separate the soluble fractions from the precipitates. The supernatants were transferred to new microtubes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western Blot analysis.

For intact cell experiments, drug-treated cells from the above in vitro experiments were heated as previously described followed by the addition of KB (30 μl) and lysed using 2 freeze-thaw cycles with liquid nitrogen. Soluble fractions were isolated and analyzed by Western Blotting.

Frozen tissue lysates were used for in vivo experiments in mice. frozen

- 71 039109 organs (eg liver or kidney) were thawed on ice and washed briefly with PBS. The organs were homogenized in cold PBS using tissue grinders and then 3 freeze-thaw cycles were applied using liquid nitrogen. Cell lysates were separated from cell debris and lipids. Cell lysates were diluted with PBS containing protease inhibitors, divided into 50 μl aliquots, and heated at various temperatures. Soluble fractions were isolated and analyzed by Western Blotting.

Aliquots treated with one or more of the compounds disclosed herein are expected to exhibit increased menin thermal stability compared to control aliquots.

Example 19 CETSA-like dot blot experiments on purified proteins. Purified protein (0.5 μg) was added to the wells of the PCR plate and the volume was adjusted to 50 μl by adding buffer or cell lysates and ligands, depending on the experimental model. The samples were heated for the specified time and temperature in a thermal cycler. After heating, the samples were immediately centrifuged for 15 min at 3000xg and filtered using a 0.65 µm 96-well Multiscreen HTS filter plate. 3 μl of each filtrate was blotted onto a nitrocellulose membrane. Primary antibody and secondary conjugate were used for immunoblotting. All membranes were blocked with blocking buffer; standard transfer and western blotting protocols recommended by the manufacturers. All antibodies were diluted with blotting buffer. Developed dot blotting. Chemiluminescence intensities were detected and visualized. Images with clear dot blots were processed. The background pattern was subtracted and the intensities were quantified. Graphs were built and adjusted using a sigmoidal dose-response curve (variable slope).

Example 20 Cell Proliferation Assays

The ability of a compound of the present invention to inhibit cell growth was tested in MLL leukemia cell lines (eg MV4;11) using an MTT cell proliferation assay (ATCC® 30-1010K). One or more of the compounds described herein, such as the compound shown in table. 2 having an IC ₅₀ value of less than 1 μM, preferably 50 nM or less (a measure of the ability of a compound to disrupt the menin-MLL interaction, measured according to Example 8), inhibited the proliferation of MV4 cells;11. Cells were seeded at about 1x105 cells per well in a 96-well plate. The compound of the present invention was added at concentrations up to about 2 μM using seven 2-fold serial dilutions for each compound. Cells were incubated at 37°C for 72 h, then the cells in the control wells were counted. The medium was changed to restore the number of viable cells to the original concentration, and the compounds were reapplied. Proliferation was measured after 96 hours using MTT reagents according to the instructions supplied with the kit. The GI ₅₀ value of a compound of the present invention, such as compound A in FIG. 4 was 40 nM in MV4;11 cells as measured in the MTT cell proliferation assay. Used in the examples and figures in this document, compound A refers to compound 3 from table. 1. As used in the examples, the GI ₅₀ value of a compound is the concentration of the compound required for 50% maximum inhibition of cell proliferation.

Example 21 Efficacy study in xenograft tumor model.

One or more compounds disclosed in this document, such as the compound shown in table. 2 having an IC ₅₀ value of less than 1 μM, preferably 50 nM or less (a measure indicating the ability of a compound to disrupt the menin-MLL interaction, measured according to Example 8), provides suppression of MV4;11 (human leukemia) tumor growth in xenograft mouse model . Immunocompromised athymic (nu/nu) female mice aged 8-10 weeks were used for in vivo efficacy studies according to IACUC guidelines. Human leukemia cells MV4;11 available from ATCC were implanted subcutaneously in athymic female mice (5x106 cells/mouse). After reaching a tumor size of approximately 150-250 mm ³ , tumor-bearing mice were assigned randomly to a vehicle control or compound treatment group (8 mice per group). Mice in the treatment group were administered a compound of the present invention by gavage (150 mg/kg, twice a day). Subcutaneous tumor volume and body weight of the mice were measured twice a week. Tumor volumes were calculated by measuring two perpendicular diameters with calipers (V=(length×width ² )/2). As shown in FIG. 4, the connection presented in table. 2 has an IC ₅₀ value of 50 nM or less (a measure indicating the ability of a compound to disrupt the menin-MLL interaction, measured according to Example 8), marked Compound A in the figure inhibited tumor growth and induced tumor regression compared to the control group treated with the carrier in a dose-dependent manner. As shown in FIG. 5, the connection presented in table. 2 having an IC ₅₀ value of 50 nM or less (a measure indicating the ability of a compound to disrupt the meninMLL interaction, measured according to Example 8), marked Compound B in the figure inhibited growth

- 72 039109 tumors and induced tumor regression relative to the control group treated with the carrier, dose-dependent manner. Used in the examples and figures, compound B refers to compound 82 in table. one.

Example 22 Efficacy study in xenograft mouse model of MLL leukemia.

One or more compounds disclosed in this document, such as the compound shown in table. 2 having an IC50 value of less than 1 μM, preferably 50 nM or less (an indicator of the compound's ability to disrupt the menin-MLL interaction, measured according to Example 8), provides suppression of MV4;11 tumor growth in the mouse xenograft model of MLL leukemia. Immunocompromised female NSG mice aged 8-10 weeks were used for in vivo efficacy studies in accordance with IACUC guidelines. Luciferase-expressing human leukemic cells MV4;11 (MV4;11-luc) were inoculated intravenously by tail vein injection (1 x 10 ⁷ cells/animal). When the average cell luminescence reached approximately 1.5 x ^{10 6} , tumor-bearing mice were randomly assigned to either the vehicle control group or the compound treatment group (5 animals per group). Animals in the treatment group were treated with another compound of the present invention by oral gavage at selected dose levels. Body weight was measured daily and mean luminescence was measured 6 days after the start of compound or vehicle treatment.

Animals were sacrificed on day 7 of treatment and bone marrow samples were collected and prepared for gene expression analysis. Expression levels of MLL fusion protein target genes HOXA9, DLX2, and MEIS1 were measured by qRT-PCR and presented as fold changes normalized to GAPDH expression.

Example 23 Survival study in a xenograft mouse model of MLL leukemia.

For survival studies in MV4 xenograft models; 11 female NSG mice aged 6-8 weeks were intravenously injected with 1x10 ⁷ MV4;11 cells expressing luciferase containing the MLL-AF4 translocation. On the 12th day after transplantation, treatment was started with the compound disclosed herein, for example, the compound shown in Table. 2 having an IC50 value of less than 1 μM, preferably 50 nM or less (a measure of the compound's ability to disrupt the menin-MLL interaction measured according to Example 8), 120 mg/kg, twice a day, po, or vehicle (20% 2-hydroxypropyl-L-cyclodextrin with 5% Cremophor) and continued for 22 consecutive days.

For survival studies in the MOLM13 xenograft xenograft model, 6-8 week old female NSG mice were intravenously injected with 0.5 x 10 ⁶ MOLM13 cells carrying the MLL-AF9 translocation. On the 4th day after transplantation, treatment was started with the compound disclosed in this document, for example, the compound presented in table. 2 having an IC ₅₀ value of less than 1 μM, preferably 50 nM or less (a measure of the ability of a compound to disrupt the menin-MLL interaction, measured according to Example 8), 75 mg/kg, twice a day, po, or vehicle (20% 2-hydroxypropyl-L-cyclodextrin with 5% Cremophor) and continued for 16 consecutive days in compound-treated mice or until end-stage leukemia develops in vehicle-treated mice.

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are presented by way of example only. Numerous variations, changes and substitutions can be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be used in the practice of the invention. It is intended that the following claims define the scope of the invention and that methods and structures falling within the scope of these claims and their equivalents are also covered.

Claims (17)

1. Compound of formula (I-A) or a pharmaceutically acceptable salt thereof, wherein R ¹ is C _1-3 haloalkyl; R ² is hydrogen, -CH ₃ , -NH ₂ or -NHCH3; R ³ is hydrogen; - 73 039109 each of R ^a and R ^B is independently selected from -OH, -CN and -CH3; RC selected from L ² and L ³ are each independently selected from C ₁ _ ₄ -αkylene; each of m and n is independently an integer from 0 to 3; W ¹ , W ² and W ³ are each independently selected from C _1-4 alkylene; and R ⁵¹ is hydrogen. 2. The compound according to claim 1, having the structure of formula (I-C) R ^B (IC) 3. The compound according to claim 1, having the structure of formula (I-E) - 74 039109 4. The compound of claim 1, wherein W ¹ , W ² and W ³ are each C ₁ alkylene. 5. The compound according to claim 1, characterized in that R ^C is selected from 6. Connection by 7. Connection by 8. Connection by 9. Connection by 1, characterized in that R ¹ is -CH2CF3. 1, characterized in that m is equal to 0. 1, characterized in that n is an integer from 1 to 3. 1, characterized in that L2 is C ₁ . ₂ -alkylene. 10. A compound according to claim 1, wherein L ² is -CH2. 11. The compound according to claim 1, characterized in that L ³ is C ₁ . ₂ -alkylene. 12. A compound according to claim 1, wherein L ³ is -CH2-. 13. The compound according to claim 1, characterized in that the compound of formula (I) is selected from - 75 039109 - 76 039109 - 77 039109 - 78 039109 14. A pharmaceutical composition containing a compound according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier. 15. A method of inhibiting the interaction of menin with one or more of MLL1, MLL2, MLL fusion protein, and partial tandem duplication MLL, comprising contacting menin with an effective amount of a compound according to any one of claims 1-13. 16. A method of treating a hematologic or solid cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 14. 17. The method of claim 16, wherein the hematologic or solid malignant tumor is selected from leukemia, prostate cancer, breast cancer, liver cancer, and brain tumor. Amino acid sequence of human menin isoform 1 (SEQ ID NO: 1): bTFQFSiaroWGGLTI ·· ·: 'T.-:; : .z-dz·: mz: .g: :>.· -· t · z zz : %v · vv zl,-. / : :: zz: : one..... . ·. yut.. 4 yut :· 3-. . 3'. : . g.' : H:. 3 ·”.·.... ... „ · ... . V , , ; .... ..... . _{7 L} ; ; ; _{, :} .....: yut/: .. ; · yut . : : yut: . : < .1. . A .. < ( ' g. .... .ut : . Yut. ¹ '' , . : 'ut', Yut.- , z- Ch zyut . .. 33 .-, , .3 Ch yut .·.-·..·· yut ; yut . <з· зз: ЗЗ: ;3 343 “·“·' : - l: :з - '3-3 . '/'·h::;:··: · z ·' 33 · . 3..; ..3 _: 3, .33 . ut, 3 33 ut.. . yut.·.....,·\ . . . I,.- A : 3 3. ... :-..4.:- . . Fig. one - 79 039109 Amino acid sequence of human menin, isoform 2 (SEQ ID NO: 2): .With. a al aaa. _: : : -λ % al BUT/ ·. uu:. d·· U ... . ; L y - - % y · < AL : . . N.A / TV / :: ATAST A- A « S El AAT ASA AA A·-:/% /...11. //- / A. X LS № '/L KX aasta a%aa:aal.......1l.l\,mx1/:lg^ . ϊ . , .. ..-..g.·..,·:.'.: = AAC ' _{? L} ..... ,., „L , , . _{t (} -. A<T AAA-A'A TA. ν t .· : . - γ-d yy y . U” / A - :... u. at vu TATT - L. ^ Aa .: A' Ά,.: Ά; A .g/Cx. С·..,.<</7/ al with r. a ·: : a ^τ ”: · % % r.--ά-α: :: ” : τάα g/· ; With·. l. ts, a-a ,αΑα laaaaaa .. l a·....·. _ . .. ,. < γ. : us ./ aa .slattts/l. Fig. 2 Amino acid sequence of human menin isoform 3 (SEQ ID NO: 3): :s.. TA / s .......'% ,,· ...-u „A u. y .. \ . .V ., . ST : — . u A ’ C’ / „C : ALA AVAATAA-AAAATA -. .-. LL / A : _ C ’,, : T : ...... / . .· ,,,. %. .. ...v. /l,- ... ... _r . _v ... . ........... ...... ....... , . . ... . _v . ..... , _? : C, % A . : A. /S ........ A. . BUT: .. . ; . .: .....^ AAAA :< ”,А _-:/.yy _V y _: . _V: yy -.y // _: - · y _Г y ,y _; ; y - - - _L ~ _: - _H y, - ; - ₌ A, A '-C ... ^. AA. ......../G .. . A. / tA A , . ' A%“^ C A. ’ > . ' S AAA GAAAAAAAAAAGSAAS ^AAAAAAASGUU Ά .. '.A X. -U C-.u A.t. A:.:/ “'A / .· _¥ - A? /A.':... A A . A 'A BUT . AC '. ^: AA AA CV A A “A AA : Fig. 3 Efficiency in model MV4;11 sc. Antitumor efficacy in the MV4;11 model Treatment period (days) Fig. 5 carrier Control -^-Connection B 50 mg/kg/1 time per day “^“ Compound B 100 mg/kg/1 time per day Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2