METHODS OF PREDICTING CNS CANCER RESPONSE TO TREATMENT WITH EGFR INHIBITORS

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 63/352,004, filed June 14, 2022, the contents of which are hereby incorporated by reference its entirety.

BACKGROUND

Central Nervous System malignancies, including brain tumors, often have mutations in the epidermal growth factor receptor (EGFR) gene. For example, EGFR is mutated and/or amplified -60% in glioblastoma (GBM), the most lethal form of brain cancer. Although in EGFR is a bona fide driver of GBM tumorigenesis, conventional EGFR tyrosine kinase inhibitors (TKIs) (e.g. Erlotinib, Gefitinib, Lapatinib) have failed in GBM patients. This is likely due to the fact that nearly all EGFR TKIs cannot adequately penetrate the blood-brain- barrier (BBB) to achieve sufficient levels for an anti-tumor response.

In view of the foregoing, there exists a need for methods using potent, brain-penetrant EGFR TKIs for the treatment of malignant glioma or other CNS malignancies with EGFR as a tumor driver.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides method of treating a cancer in a subject, comprising: obtaining one or more biological sample(s) from the subject; analyzing the biological sample(s) for the presence of a first genetic marker in the biological sample(s); and administering an EGFR inhibitor to the subject if the presence of the first genetic marker is detected in the biological sample(s); wherein the first genetic marker is selected from NF1 WT, PTEN WT, a NF1 mutant, or a PTEN mutant. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flowchart showing stratification of patients based on their EGFR, NF1, and PTEN status. For example, 100% of patients with EGFR Polysomy, NF1 WT, and PTEN WT showed a response to treatment with Compound 1.

Figure 2 displays the gene status of responders (top) and nonresponders (bottom) for all models. For example, out of a total of 18 responders, 2 responders (11.11%) had EGFR amplification only.

Figure 3A displays the PTEN and NF1 statuses of EGFR amplified responders (top) and nonresponders (bottom).

Figure 3B displays the PTEN and NF1 statuses of EGFR polysomy responders (top) and nonresponders (bottom).

Figure 4 is a chart displaying treatment outcome contingency on EGFR status.

Figure 5 is a chart displaying treatment outcome contingency on NF1 status.

Figure 6 is a chart displaying treatment outcome contingency on NF1 status in subjects with EGFR polysomy.

Figure 7 is a chart displaying treatment outcome contingency on NF1 or PTEN status in subjects with EGFR polysomy.

Figure 8 is a chart displaying treatment outcome contingency on PTEN status.

Figure 9 is a chart displaying treatment outcome contingency on NF1 or PTEN status.

Figure 10 is a chart displaying treatment outcome contingency on PTEN status in subjects with EGFR polysomy.

Figure 11 is a chart displaying exemplary mutations and the subjects’ responses to administration of compound 1.

DETAILED DESCRIPTION OF THE INVENTION

Gliomas are the most commonly occurring form of brain tumor, with glioblastoma multiforme (GBM) being the most malignant form, causing 3-4% of all cancer-related deaths (Louis et al. (2007) Acta. Neuropathol. 114: 97-109.). The World Health Organization defines GBM as a grade IV cancer characterized as malignant, mitotically active, and predisposed to necrosis. GBM has a very poor prognosis with a 5-year survival rate of 4-5% with the median survival rate of GBM being 12.6 months (McLendon et al. (2003) Cancer. 98 : 1745-1748.). This can attributed to unique treatment limitations such as a high average age of onset, tumor location, and poor current understandings of the tumor pathophysiology (Louis et al. (2007) Acta. Neuropathol. 114: 97-109). The standard current standard of care for GBM includes tumor resection with concurrent radiotherapy and chemotherapy and in recent years there have been few marked improvements that increase survival rates (Stewart, et al. (2002) Lancet. 359:1011-1018.).

The standard for GBM chemotherapy is temozolomide (TMZ), which is a brainpenetrant alkylating agent that methylates purines (A or G) in DNA and induces apoptosis (Stupp, et al. (2005) N. Engl. J. Med. 352:987-996). However, TMZ use has drawbacks in that significant risk arises from DNA damage in healthy cells and that GBM cells can rapidly develop resistance towards the drug (Carlsson, et al. (2014) EMBO. Mol. Med. 6: 1359-1370). As such, additional chemotherapy options are urgently required.

EGFR is a member of the HER superfamily of receptor tyrosine kinases together with ERBB2, ERBB3, and ERBB4. A common driver of GBM progression is EGFR amplification, which is found in nearly 40% of all GBM cases (Hynes et al. (2005) Nat. Rev. Cancer. 5: 341-354; Hatanpaa et al. (2010) Neoplasia. 12 :675-684). Additionally, EGFR amplification is associated with the presence of EGFR protein variants: in 68% of EGFR mutants; there is a deletion in the N-terminal ligand-binding region between amino acids 6 and 273. These deletions in the ligand-binding domains of EGFR can lead to ligandindependent activation of EGFR (Yamazaki et al. (1990) Jpn. J. Cancer Res. 81: 773-779.).

Small molecule tyrosine kinase inhibitors (TKIs) are the most clinically advanced of the EGFR-targeted therapies, and both reversible and irreversible inhibitors are in clinical trials. Examples of the reversible inhibitors and irreversible inhibitors include erlotinib, gefitinib, lapatinib, PKI166, canertinib and pelitinib (Mischel et al. (2003) Brain Pathol. 13: 52-61). Mechanistically, these TKIs compete with ATP for binding to the tyrosine kinase domain of EGFR, however, these EGFR-specific tyrosine kinase inhibitors have been relatively ineffective against gliomas, with response rates only reaching as high as 25% in the case of erlotinib (Mischel et al. (2003) Brain Pathol. 13: 52-61; Gan et al. (2009) J. Clin. Neurosci. 16: 748-54). Although TKIs are well tolerated and display some antitumor activity in GBM patients, the recurrent problem of resistance to receptor inhibition limits their efficacy (Leam et al. (2004) Clin. Cancer. Res. 10: 3216-3224; Rich et al. (2004) Nat. Rev. Drug Discov. 3: 430-446). Additionally, recent studies have shown that brain plasma concentrations of gefitinib and erlotinib following therapy were only 6-11% of the starting dose, suggesting that these compounds may be failing to cross the blood-brain barrier as illustrated in table 1 (Karpel-Massler et al. (2009) Mol. Cancer Res. 7 : 1000-1012). Thus, insufficient delivery to the target may be another cause of the disappointing clinical results.

Table 1 : Brain Penetration Rates of the Current Standard of Care Drugs

In light of this evidence, there remains an unmet clinical need for potent tyrosine kinase inhibitors that have the ability to cross the blood brain barrier and inhibit EGFR and its isoforms.

Methods of Treatment

In certain aspects, the present disclosure provides methods of treating a cancer in a subject, comprising: obtaining one or more biological sample(s) from the subject; analyzing the biological sample(s) for the presence of a genetic marker in the biological sample(s); and administering an EGFR inhibitor to the subject if the presence of the genetic marker is detected in the biological sample(s); wherein the genetic marker is selected from NF1 WT, PTEN WT, an NF1 mutant, or a PTEN mutant.

In certain embodiments, the methods of the disclosure comprise analyzing the biological sample(s) for the presence of a second genetic marker in the biological sample(s); and administering an EGFR inhibitor to the subject only if the presence of the first genetic marker and the second genetic marker is detected in the biological sample(s); wherein the second genetic marker is selected from NF1 WT, PTEN WT, an NF1 mutant, or a PTEN mutant.

In some embodiments, the first genetic marker is NF1 WT. In other embodiments, the first genetic marker is a NF1 mutant. In yet other embodiments, the first genetic marker is PTEN WT. In still other embodiments, the first genetic marker is a PTEN mutant. In some embodiments, the second genetic marker is NF1 WT. In other embodiments, the second genetic marker is an NF1 mutant. In yet other embodiments, the second genetic marker is PTEN WT. In other embodiments, the second genetic marker is a PTEN mutant. In certain preferred embodiments, the cancer has EGFR amplification. In other preferred embodiments, the cancer has EGFR polysomy.

In certain embodiments, the PTEN mutant is a PTEN genetic marker with Glycine 129 altered to a glutamic acid residue. In certain embodiments, the PTEN mutant is a. PTEN genetic marker with Tyrosine 139 altered to a histidine residue. In certain embodiments, the PTEN mutant is a PTEN genetic marker with Methionine 134 altered to an isoleucine residue. In certain embodiments, the PTEN mutant is a PTEN genetic marker with Proline 38 altered to a leucine residue. In certain embodiments, the PTEN mutant is a PTEN genetic marker with Arginine 15 altered to a lysine residue. In certain embodiments, the PTEN mutant is a PTEN genetic marker with Proline 96 altered to a serine residue. In certain embodiments, the PTEN mutant is a PTEN genetic marker with Cysteine 105 altered to an arginine residue. In certain preferred embodiments, the PTEN mutant is a PTEN genetic marker with a mutation that causes loss of function. In certain embodiments, the NF1 mutant is an NF1 genetic marker with X464 splice, Aspartic Acid 1644 altered to an asparagine, and Valine 1909 altered to isoleucine. In certain preferred embodiments, the NF 1 mutant is an NF1 genetic marker with a mutation that causes loss of function.

EGFR inhibitors

In certain embodiments, the EGFR inhibitor is compound 1:

In certain embodiments, the EGFR inhibitors of the disclosure are compounds of formula I-a or I-b: or a pharmaceutically acceptable salt thereof, wherein:

Z is aryl or heteroaryl, and is optionally substituted with one or more R ⁶ ;

R ¹ is hydrogen, alkyl, halo, CN, NO2, OR ⁷ , cycloalkyl, heterocyclyl, aryl or heteroaryl;

R ² is hydrogen, alkyl, halo, CN, NO2, OR ⁸ , cycloalkyl, heterocyclyl, aryl or heteroaryl; or R ¹ and R ² complete a carbocyclic or heterocyclic ring;

R ³ is hydrogen, alkyl, or acyl;

R ⁴ is alkoxy;

R ⁵ is alkyl; each instance of R ⁶ is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl; and

R ⁷ and R ⁸ are each independently selected from hydrogen, alkyl, such as alkoxy alkyl, aralkyl, or arylacyl. In certain embodiments of Formula I-a or Formula I-b, if R ⁷ and R ⁸ are alkoxyalkyl and R ³ is hydrogen, then Z is not 3-ethynylphenyl.

In certain embodiments of Formula I-a or Formula I-b, Z is optionally substituted with R ⁶ selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

In certain embodiments of Formula I-a or Formula I-b, R ⁷ and R ⁸ are each independently selected from hydrogen, aralkyl, or arylacyl; each instance of R ⁶ is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl; or R ¹ and R ² taken together complete a carbocyclic or heterocyclic ring.

In certain embodiments of Formula I-a or Formula I-b, if R ⁷ and R ⁸ are combined to form a heterocylic ring and R ³ is hydrogen, then Z is not 2-fluoro,4-bromophenyl, 3- bromophenyl, 3 -methylphenyl, 3-trifluoromethylphenyl, or 3-chloro,4-fluorophenyl.

In certain embodiments of Formula I-a or Formula I-b, the compound is a compound of Formula (Il-a) or formula (Il-b):

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, or Formula Il-b, R ¹ is hydrogen. In other embodiments, R ¹ is OR ⁷ .

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, or Formula Il-b, R ⁷ is hydrogen. In certain embodiments, R ⁷ is alkyl. In certain embodiments, R ⁷ is alkoxyalkyl. In certain embodiments, R ⁷ is arylacyl.

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, or Formula Il-b, R ² is heteroaryl, such as furanyl. In certain embodiments, the heteroaryl of R ² is substituted with alkyl, alkoxy, OH, CN, NO2, halo, other embodiments, R ² is OR ⁸ .

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, or Formula Il-b, R ⁸ is hydrogen. In certain embodiments, R ⁸ is alkoxyalkyl. In certain embodiments, R ⁸ is alkyl substituted In certain embodiments, R ⁸ is acyl. In certain embodiments, R ⁸ is arylacyl.

In certain preferred embodiments of Formula I-a, Formula I-b, Formula Il-a, or Formula Il-b, R ¹ and R ² combine to form a carbocylic or heterocyclic ring, such as a 5- member, 6-member, or 7-member carbocyclic or heterocyclic ring. In certain embodiments, the carbocyclic or heterocyclic ring is substituted with hydroxyl, alkyl (e.g., methyl), or alkenyl (e.g., vinyl). In certain embodiments, the compound is the compound is certain embodiments, the carbocyclic or heterocyclic ring is substituted with alkyl (e.g., methyl) and the alkyl moieties are trans relative to each other.

In certain embodiments, the compound is the compound i certain embodiments, the carbocyclic or heterocyclic ring is substituted with alkyl (e.g., methyl) and the alkyl moieties are cis relative to each other. In certain embodiments, the compound is the compound

In even more preferred embodiments of Formula I-a, Formula I-b, Formula Il-a, or Formula Il-b, the compound is a compound of Formula (Ill-a), (Ill-b), (III-c), (Ill-d), (Ill-e), or (Ill-f):

Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, or Formula Ill-f, R ³ is hydrogen. In certain embodiments, R ³ is acyl. In certain embodiments, R ³ is alkylacyl. In certain embodiments, R ³ is alkyloxyacyl. In certain embodiments, R ³ is acyloxyalkyl. In certain embodiments, alkyl.

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, Formula Il-b,

Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, or Formula Ill-f, Z is aryl or heteroaryl optionally substituted with one or more R ⁶ ; and each instance of R ⁶ is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In certain preferred embodiments, Z is phenyl substituted with 1, 2, 3, 4, or 5 R ⁶ . In certain embodiments, each R ⁶ is independently selected from halo, alkyl, alkynyl, or arylalkoxy. In even more preferred embodiments, Z is 2-fluoro- 3-chlorophenyl, 2-fluorophenyl, 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl, 2,4,6-trifluorophenyl, pentafluorophenyl, 2-fluoro-3 -bromophenyl, 2- fluoro-3-ethynylphenyl, and 2-fluoro-3-(trifluoromethyl)phenyl. In other even more preferred embodiments, Z is 3-ethynylphenyl. In yet other even more preferred embodiments, Z is 3-chloro-4-((3-fluorobenzyl)oxy)benzene. In yet other even more preferred embodiments, Z is 3-chloro-2-(trifluoromethyl)phenyl. In yet other even more preferred embodiments, Z is 3-bromophenyl. In yet other even more preferred embodiments, Z is 2- fluoro,5-bromophenyl. In yet other even more preferred embodiments, Z is 2,6-difluoro,5- bromophenyl. In certain embodiments, Z is substituted with one R ⁶ selected from are independently selected from alkyl.

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, Formula Il-b,

Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, or Formula Ill-f, the compound is a compound of Formula (IV-a):

each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, Formula Il-b, Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, or Formula Ill-f, the compound is a compound of Formula (IV-b): and each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, Formula Il-b, Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, or Formula Ill-f, the compound is a compound of Formula (IV-c): and each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, Formula Il-b, Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, Formula Ill-f, Formula IV-a, Formula IV-b, or Formula IV-c, the compound is a compound of Formula (V- a):

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, Formula Il-b, Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, Formula Ill-f, Formula IV-a, Formula IV-b, or Formula IV-c, the compound is a compound of Formula (V- b): and each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments of Formula I-a, Formula I-b, Formula Il-a, Formula Il-b,

Formula Ill-a, Formula Ill-b, Formula III-c, Formula Ill-d, Formula Ill-e, Formula Ill-f, Formula IV-a, Formula IV-b, or Formula IV-c, the compound is a compound of Formula (V- b): and each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments, the compound of Formula I-a or I-b is selected from a compound in Table 2. Table 2: Exemplary Compounds of the Present Disclosure

or a pharmaceutically acceptable salt or stereoisomer thereof.

In certain embodiments, the EGFR inhibitors of the disclosure are compounds of

Formula I or Formula I*:

(VI) (VI*) or a pharmaceutically acceptable salt thereof, wherein:

Z is aryl or heteroaryl;

R ^2a and R ^2b are each independently selected from hydrogen, alkyl, halo, CN, and NO2;

R ³ is hydrogen, alkyl, or acyl;

R ⁴ is alkoxy;

R ⁵ is alkyl; R ⁷ and R ⁸ are, each independently, selected from hydrogen, alkyl, such as alkoxyalkyl, aralkyl, or arylacyl;

R ¹¹ is hydrogen, alkyl, halo, CN, NO2, OR ⁷ , cycloalkyl, heterocyclyl, aryl or heteroaryl; and R ¹² is hydrogen, alkyl, halo, CN, NO2, OR ⁸ , cycloalkyl, heterocyclyl, aryl or heteroaryl; or R ¹¹ and R ¹² taken together complete a carbocyclic or heterocyclic ring.

In certain preferred embodiments of Formula VI or Formula VI*, at least one of is R ^2a and R ^2b not H. In certain such embodiments of Formula VI or Formula VI*, if R ^2a is hydrogen, then R ^2b is selected from alkyl, halo, CN, and NO2. In other such embodiments of Formula I or Formula VI*, if R ^2b is hydrogen, then R ^2a is selected from alkyl, halo, CN, and NO2.

In certain embodiments of Formula VI or Formula VI*, the compound is a compound of Formula (Vila) or Formula (Vllb):

(Vila) (Vllb) or a pharmaceutically acceptable salt thereof, wherein each instance of R ⁶ is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

In certain embodiments, of Formula VI, VI*, Vila, and Vllb, R ¹¹ is hydrogen. In other preferred embodiments, R ¹¹ is OR ⁷ .

In certain embodiments, of Formula VI, VI*, Vila, and Vllb, R ⁷ is hydrogen. In other embodiments, R ⁷ is alkyl. In yet other embodiments, R ⁷ is alkoxyalkyl. In yet other embodiments, R ⁷ is arylacyl.

In certain embodiments, of Formula VI, VI*, Vila, and Vllb, R ¹² is heteroaryl, such as furanyl. In certain embodiments, the heteroaryl is substituted with alkyl, alkoxy, OH, CN,

In certain embodiments of Formula VI, VI*, Vila, and Vllb, R ⁸ is hydrogen. In other embodiments, R ⁸ is alkyl. In yet other embodiments, R ⁸ is alkoxyalkyl. In certain

In certain preferred embodiments, of Formula VI, VI*, Vila, and Vllb, R ¹¹ and R ¹² combine to form a carbocylic or heterocyclic ring, such as a 5-member, 6-member, or 7- member carbocyclic or heterocyclic ring. In certain embodiments, the carbocyclic or heterocyclic ring is substituted with hydroxyl, alkyl (e.g., methyl), or alkenyl (e.g., vinyl).

In certain embodiments, of Formula VI, VI*, Vila, and Vllb, the compound is a compound of Formula Via, VIb, Vic, or Vid:

(Vic) (Vid) or a pharmaceutically acceptable salt thereof, wherein:

X is O, S, or NH;

Z is aryl or heteroaryl;

R ¹ is hydrogen or alkyl; R ^2a and R ^2b are each independently selected from hydrogen, alkyl, halo, CN, and NO2;

R ³ is hydrogen, alkyl, or acyl;

R ⁴ is alkoxy;

R ⁵ is alkyl; and n is 0-3.

In certain embodiments of Formula Via, VIb, Vic, or Vid, either R ^2a or R ^2b is selected from alkyl, halo, CN, and NCh. In certain preferred embodiments of Formula Via, VIb, Vic, or Vid, Z is phenyl. In certain preferred embodiments of Formula Via, VIb, Vic, or Id, X is O. In certain preferred embodiments of Formula Via, VIb, Vic, or Vid, n is 1.

In certain embodiments of Formula Via, VIb, Vic, or Vid, the compound is a compound of Formula (Villa) or Formula (Vlllb):

(Villa) (Vlllb) or a pharmaceutically acceptable salt, wherein each instance of R ⁶ is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

In certain embodiments, wherein R ¹ is represented by Formula IX:

(IX) wherein,

R ^7a and R ⁷¹¹ are each independently selected from alkyl, alkenyl, alkynl, cycloalkyl, heterocyclyl, aryl or heteroaryl; or R ^7a and R ^7b combine to form a heterocyclyl; and y is 0-3.

In certain embodiments of Formula Villa or Vlllb, R ¹ is alkyl (e.g., methyl or ethyl). In certain embodiments, R ¹ is substituted with heterocyclyl (e.g., morpholinyl, piperidinyl, pyrrolodinyl, or piperazinyl, such as N-methyl piperazinyl). In other embodiments, R ¹ is substituted with amino (e.g., dimethyl amino). In other embodiments, R ¹ is alkyl substituted with hydroxyl. In certain preferred embodiments, R ¹ is in the S configuration. In other embodiments, R ¹ is in the R configuration.

In certain preferred embodiments of Formula Villa or Vlllb, R ³ is hydrogen. In other embodiments, R ³ is acyl. In certain embodiments, R ³ is alkylacyl. In certain embodiments, R ³ is alkyloxyacyl. In certain embodiments, R ³ is acyloxyalkyl. In certain embodiments, R ³

In certain embodiments of Formula Villa or Vlllb, Z is aryl or heteroaryl optionally substituted with one or more R ⁶ ; and each instance of R ⁶ is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In certain preferred embodiments, Z is phenyl substituted with 1, 2, 3, 4, or 5 R ⁶ . In certain embodiments, each R ⁶ is independently selected from halo, alkyl, alkynyl, or arylalkoxy. In even more preferred embodiments, Z is 2-fluoro-3 -chlorophenyl, 2-fluorophenyl, 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 2,6- difluorophenyl, 2,4,6-trifluorophenyl, pentafluorophenyl, 2-fluoro-3 -bromophenyl, 2- fluoro-3-ethynylphenyl, and 2-fluoro-3-(trifluoromethyl)phenyl. In other even more preferred embodiments, Z is 3-ethynylphenyl. In yet other even more preferred embodiments, Z is 3-chloro-4-((3-fluorobenzyl)oxy)benzene. In yet other even more preferred embodiments, Z is 3-chloro-2-(trifluoromethyl)phenyl. In yet other even more preferred embodiments, Z is 3-bromophenyl. In yet other even more preferred embodiments, Z is 2-fluoro,5-bromophenyl. In yet other even more preferred embodiments, Z is 2,6-difluoro,5-bromophenyl. In certain embodiments, Z is substituted with one R ⁶ selected from are independently selected from alkyl. In certain embodiments of Formula Via or VIb, the compound is a compound of Formula (Illa): and each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments of Formula Xa or Xb, the compound is a compound of

Formula (Xb): and each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments of Formula Xa or Xb, the compound is a compound of

Formula (Xc): and each R ⁶ is independently selected from fluoro, chloro, or bromo.

In certain embodiments of Formula Via, VIb, Vic, Vid, Villa, Vlllb, Xa, Xb, or Xc, R ^2a is halo (e.g., fluoro). In other preferred embodiments, , R ^2a is hydrogen.

In certain embodiments of Formula Via, VIb, Vic, Vid, Villa, Vlllb, Xa, Xb, or Xc, R ^2b is halo (e.g., fluoro). In other preferred embodiments, R ^2b is hydrogen. In certain embodiments of Formula Via, VIb, Vic, Vid, Villa, Vlllb, Xa, Xb, or Xc, pharmaceutically acceptable salt thereof.

In certain embodiments of Formula Via, VIb, Vic, Vid, Villa, Vlllb, Xa, Xb, or Xc, In certain embodiments of Formula VI, the compound pharmaceutically acceptable salt thereof.

In certain embodiments of Formula Via, VIb, Vic, Vid, Villa, Vlllb, Xa, Xb, or Xc, pharmaceutically acceptable salt thereof.

In certain embodiments, the EGFR inhibitors of the disclosure are compounds having a structure represented by Formula (XI): wherein:

R ¹ is selected from the group consisting of

R ² is selected from Ci-Ce alkyl and Cs-Ce cycloalkyl, each of which is optionally substituted with one or more halogen, or a pharmaceutically acceptable salt thereof. In certain embodiments of Formula pharmaceutically acceptable salt thereof. In other embodiment pharmaceutically acceptable salt thereof. In yet other embodiments, pharmaceutically acceptable salt thereof. In yet other embodiments, pharmaceutically acceptable salt thereof. In still other embodiments, pharmaceutically acceptable salt thereof. In other embodiments, pharmaceutically acceptable salt thereof.

In certain embodiments of Formula (XI), R ² is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, trifluoromethyl, fluoroethyl, and difluoroethyl, or a pharmaceutically acceptable salt thereof. In certain preferred embodiments, R ² is selected from methyl, ethyl, n-propyl, isopropyl, tert-butyl, fluoroethyl, and difluoroethyl, or a pharmaceutically acceptable salt thereof. In the most preferred embodiment, R ² is methyl, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the EGFR inhibitors of the disclosure are compounds having a structure represented by Formula (Xia):

wherein:

R ¹ is selected from

R ² is selected from Ci-Ce alkyl and Cs-Ce cycloalkyl, each of which is optionally substituted with one or more halogen, or a pharmaceutically acceptable salt thereof. salt the thereof. In yet other embodiments, pharmaceutically acceptable salt thereof.

In still other embodiments, pharmaceutically acceptable salt thereof. In other embodiments, pharmaceutically acceptable salt thereof. In yet other embodiments, i pharmaceutically acceptable salt thereof.

In certain embodiments of Formula (Xia), R ² is selected from methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, trifluoromethyl, fluoroethyl, and difluoroethyl, or a pharmaceutically acceptable salt thereof. In certain preferred embodiments, R ² is selected from methyl, ethyl, n-propyl, isopropyl, tert-butyl, fluoroethyl, and difluoroethyl, or a pharmaceutically acceptable salt thereof. In the most preferred embodiments, R ² is methyl, or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present disclosure provides compounds having a structure represented by Formula (Xlb): wherein:

R ¹ is selected from

R ² is selected from Ci-Ce alkyl and Cs-Ce cycloalkyl, each of which is optionally substituted with one or more halogen, or a pharmaceutically acceptable salt thereof. In certain embodiments of Formula pharmaceutically acceptable salt thereof. In other embodiment , pharmaceutically acceptable salt thereof. In yet other embodiments, pharmaceutically acceptable salt thereof. In yet other embodiments, pharmaceutically acceptable salt thereof. In yet other embodiments, pharmaceutically acceptable salt thereof. In yet other embodiments, pharmaceutically acceptable salt thereof.

In certain embodiments of Formula (Xlb), R ² is selected from methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, trifluoromethyl, fluoroethyl, and difluoroethyl, or a pharmaceutically acceptable salt thereof. In certain preferred embodiments, R ² is selected from methyl, ethyl, n-propyl, isopropyl, tert-butyl, fluoroethyl, and difluoroethyl, or a pharmaceutically acceptable salt thereof. In the most preferred embodiments, R ² is methyl, or a pharmaceutically acceptable salt thereof.

In cetain embodiments, the compound of Formula (XI), (Xia), or (Xlb) is enantiomerically enriched. In cetain embodiments, the compound of Formula (XI), (Xia), or (Xlb) is diastereomerically enriched.

In cetain embodiments, the compound of Formula (XI), (Xia), or (Xlb) is in the form of a pharmaceutically acceptable salt. In other embodiments, the compound is in the form of a free base.

In certain embodiments, the compound of the disclosure is selected from or a pharmaceutically acceptable salt thereof.

Compounds, and synthetic procedures for the preparation thereof, related to those disclosed herein are disclosed in, e.g., published US Application Nos.: 16/651,256 & 18/027,313 and US Patent No.: 11,377,451, the contents of each of which are fully incorporated by reference herein.

Cytoplasmic p53 Stabilizers

Pharmacological p53 stabilization, such as with a CNS-penetrant small molecule, for example, is synergistically lethal with the inhibition of EGFR-driven glucose uptake in patient-derived, primary GBM models. The non-transcriptional functions of p53 can have a critical role in stimulating intrinsic apoptosis in metabolic responders. Accordingly, certain methods of treatment described herein comprise the administration of cytoplasmic p53 stabilizer(s) in combination with glucose metabolism inhibitors. Cytoplasmic p53 stabilizer(s) and glucose metabolism inhibitors can be administered in the same or in different compositions, cocomitantly or sequentially. It is contemplated that in some embodiments a single p53 stabilizer is used and in other embodiments more than on p53 stabilizer is used. For example, the combination of nutlin with ABT 737 (which binds BCL-2 and BCL-XL) is reported to synergistically target the balance of pro-apoptotic and anti-apotptoic proteins at the mitochondrial level, thereby promoting cell death. (Hoe etal. 2014. Nature Reviews. Vol. 13. pp. 217) As intended herein, a cytoplasmic p53 stabilizer is any small molecule, antibody, peptide, protein, nucleic acid or derivatives thereof that can pharmacologically stabilize or activate p53 directly or indirectly. The stabilization of cytoplasmic p53 leads to priming cells, such as cancer cells, for apoptosis.

MDM2 antagonists

Protein levels of p53 within cells are tightly controlled and kept low by its negative regulator, the E3 ubiquitin protein ligase MDM2. In embodiments of the methods or composition of the current disclosure, the cytoplasmic p53 stabilizer is an MDM2 antagonist/inhibitor. In some embodiments, the MDM2 antagonist is a nutlin. In further embodiments, the nutlin is nutlin-3 or idasanutlin. In other embodiments, the MDM2 antagonist is RO5045337 (also known as RG7112), RO5503781, RO6839921, SAR405838 (also known as MI-773), DS-3032, DS-3032b, or AMG-232 or any other MDM2 inhibitor.

Other compounds within the scope of the current methods known to bind MDM-2 include Ro-2443, MI-219, MI-713, MI-888, DS-3032b, benzodiazepinediones (for example, TDP521252), sulphonamides (for example, NSC279287), chromenotriazolopyrimidine, morpholinone and piperidinones (AM-8553), terphenyls, chaicones, pyrazoles, imidazoles, imidazole-indoles, isoindolinone, pyrrolidinone (for example, PXN822), priaxon, piperidines, naturally derived prenylated xanthones, SAH-8 (stapled peptides) sMTide-02, sMTide-02a (stapled peptides), ATSP-7041 (stapled peptide), spiroligomer (a-helix mimic). Other compounds that are known to cause protein folding of MDM2 include PRIMA-1MET (also known as APR-246) Aprea 102-105, PK083, PK5174, PK5196, PK7088, benzothiazoles, stictic acid and NSC319726.

BCL-2 Inhibitors

In further embodiments of the current methods or compositions, the cytoplasmic p53 stabilizer is a BCL-2 inhibitor. In some embodiments, the BCL-2 inhibitor is, for example, antisense oligodeoxynucleotide G3139, mRNA antagonist SPC2996, venetoclax (ABT-199), GDC-0199, obatoclax, paclitaxel, navitoclax (ABT-263), ABT-737, NU-0129, S 055746, APG-1252 or any other BCL-2 inhibitor.

Bcl-xL Inhibitors

In yet further embodiments of the current methods or compositions, the cytoplasmic p53 stabilizer is a Bcl-xL inhibitor. In some embodiments, the Bcl-xL inhibitor is, for example, WEHI 539, ABT-263, ABT-199, ABT-737, sabutoclax, AT101, TW-37, APG- 1252, gambogic acid or any other Bcl-xL inhibitor.

EGFR Inhibitors

In certain embodiments, the one or more additional therapeutic agents is selected from one or more EGFR inhibitors. In certain such embodiments, the one or more EGFR inhibitors is selected from osimertinib, afatinib, erlotinib, gefitinib, lazertinib, nazartinib, dacomitinib, BLU-945, icotinib, cetuximab, paninitumab, amivantamab, lapatinib, neratinib, zorifertinib, and mobicertinib. In certain embodiments, the one or more EGFR inhibitors is osimertinib. In other embodiments, the one or more EGFR inhibitors is afatinib. In yet other embodiments, the one or more EGFR inhibitors is erlotinib. In still other embodiments, the one or more EGFR inhibitors is gefitinib. In other embodiments, the one or more EGFR inhibitors is lazertinib. In yet other embodiments, the one or more EGFR inhibitors is nazartinib. In still other embodiments, the one or more EGFR inhibitors is dacomitinib. In other embodiments, the one or more EGFR inhibitors is BLU-945. In yet other embodiments, the one or more EGFR inhibitors is icotinib. In still other embodiments, the one or more EGFR inhibitors is cetuximab. In other embodiments, the one or more EGFR inhibitors is paninitumab. In yet other embodiments, the one or more EGFR inhibitors is amivantamab. In still other embodiments, wherein the one or more EGFR inhibitors is lapatinib. In other embodiments, the one or more EGFR inhibitors is neratinib. In yet other embodiments, the one or more EGFR inhibitors is zorifertinib. In still other embodiments, wherein the one or more EGFR inhibitors is mobicertinib

SHP2 Inhibitors

In certain embodiments, the one or more additional therapeutic agents is selected from one or more SHP2 inhibitors. In some such embodiments, the one or more SHP2 inhibitors is selected from ERAS-601, TNO155, RMC-4630, JAB-3068, JAB-3312, and RLY-1971. In certain embodiments, the one or more SHP2 inhibitors is ERAS-601. In other embodiments, the one or more SHP2 inhibitors is TNO155. In yet other embodiments, the one or more SHP2 inhibitors is RMC-4630. In still other embodiments, the one or more SHP2 inhibitors is JAB-3068. In other embodiments, the one or more SHP2 inhibitors is JAB-3312. In yet other embodiments, the one or more SHP2 inhibitors is RLY-1971.

CDK4/6 Inhibitors

In certain embodiments, the one or more additional therapeutic agents is selected from one or more CDK4/6 inhibitors. In some such embodiments, the one or more CDK4/6 inhibitors is selected from palbociclib, abemaciclib, and ribociclib. In certain embodiments, the one or more CDK4/6 inhibitors is palbociclib. In other embodiments, the one or more CDK4/6 inhibitors is abemaciclib. In yet other embodiments, the one or more CDK4/6 inhibitors is ribociclib.

ERK Inhibitors

In certain embodiments, the one or more additional therapeutic agents is selected from one or more ERK inhibitors. In some such embodiments, the one or more ERK inhibitors is selected from ulixertinib, ASN007, LY3214996, and LTT462. In certain embodiments, the one or more ERK inhibitors is ulixertinib. In other embodiments, the one or more ERK inhibitors is ASN007. In yet other embodiments, the one or more ERK inhibitors is LY3214996. In still other embodiments, the one or more ERK inhibitors is LTT462.

MEK Inhibitors

In certain embodiments, the one or more additional therapeutic agents is selected from one or more MEK inhibitors. In certain such embodiments, the one or more MEK inhibitors is selected from trametinib, binimetinib, cobimetinib, and selumetinib. In some embodiments, the one or more MEK inhibitors is trametinib. In other embodiments, the one or more MEK inhibitors is binimetinib. In yet other embodiments, the one or more MEK inhibitors is cobimetinib. In still other embodiments, the one or more MEK inhibitors is selumetinib.

MET Inhibitors

In certain embodiments, the one or more additional therapeutic agents is selected from one or more MET inhibitors. In some such embodiments, the one or more MET inhibitors is selected from capmatinib, crizotinib, and savolitinib. In certain embodiments, the one or more MET inhibitors is capmatinib. In other embodiments, the one or more MET inhibitors is crizotinib. In yet other embodiments, the one or more MET inhibitors is savolitinib. Diseases Treated by Methods of the Disclosure

Types and stages of Gliomas

Primary malignant brain tumors are tumors that start in the brain or spine are known collectively as gliomas. Gliomas are not a specific type of cancer but are a term used to describe tumors that originate in glial cells. Examples of primary malignant brain tumors include astrocytomas, pilocytic astrocytomas, pleomorphic xanthoastrocytomas, diffuse astrocytomas, anaplastic astrocytomas, GBMs, gangliogliomas, oligodendrogliomas, ependymomas. According to the WHO classification of brain tumors, astrocytomas have been categorized into four grades, determined by the underlying pathology. The characteristics that are used to classify gliomas include mitoses, cellular or nuclear atypia, and vascular proliferation and necrosis with pseudopalisading features. Malignant (or highgrade) gliomas include anaplastic glioma (WHO grade III) as well as glioblastoma multiforme (GBM; WHO grade IV). These are the most aggressive brain tumors with the worst prognosis.

GBMs is the most common, complex, treatment resistant, and deadliest type of brain cancer, accounting for 45% of all brain cancers, with nearly 11,000 men, women, and children diagnosed each year. GBM (also known as grade-4 astrocytoma and glioblastoma multiforme) are the most common types of malignant (cancerous) primary brain tumors. They are extremely aggressive for a number of reasons. First, glioblastoma cells multiply quickly, as they secrete substances that stimulate a rich blood supply. They also have an ability to invade and infiltrate long distances into the normal brain by sending microscopic tendrils of tumor alongside normal cells. Two types of glioblastomas are known. Primary GBM are the most common form; they grow quickly and often cause symptoms early. Secondary glioblastomas are less common, accounting for about 10 percent of all GBMs. They progress from low-grade diffuse astrocytoma or anaplastic astrocytoma, and are more often found in younger patients. Secondary GBM are preferentially located in the frontal lobe and carry a better prognosis.

GBM is usually treated by combined multi-modal treatment plan including surgical removal of the tumor, radiation and chemotherapy. First, as much tumor as possible is removed during surgery. The tumor’s location in the brain often determines how much of it can be safely removed. After surgery, radiation and chemotherapy slow the growth of remaining tumor cells. The oral chemotherapy drug, temozolomide, is most often used for six weeks, and then monthly thereafter. Another drug, bevacizumab (known as Avastin®), is also used during treatment. This drug attacks the tumor’s ability to recruit blood supply, often slowing or even stopping tumor growth.

Novel investigational treatments are also used and these may involve adding treatments to the standard therapy or replacing one part of the standard therapy with a different treatment that may work better. Some of these treatments include immunotherapy such as vaccine immunotherapies, or low-dose pulses of electricity to the area of the brain where the tumor exists and nano therapies involving spherical nucleic acids (SNAs) such as NU-0129. In some embodiments, the methods of the current disclosure are used in combination with one or more of the aforementioned therapies.

Emodiments of the methods and compositions discussed herein are also contemplated to be applicable to other types of cancers, including but not limited to lung cancer, non-CNS cancers, CNS cancers, and CNS metastases such as brain metastases, leptomeningeal metastases, choroidal metastases, spinal cord metastases, and others.

In certain embodiments, the present disclosure provides methods of treating cancer comprising of administering an amount of an EGFR inhibitor of the disclosure to a subject in need thereof. In some embodiments, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, cardiac cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spine and neck cancer, Kaposi’s sarcoma, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, multiple myeloma, pancreatic cancer, penile cancer, testicular germ cell cancer, thymoma carcinoma, thymic carcinoma, lung cancer, ovarian cancer, prostate cancer, glioma, astrocytoma, glioblastoma, CNS cancer, non-CNS cancer, or CNS metastases.

In certain embodiments, the cancer is glioma, astrocytoma or glioblastoma. In some such embodiments, the cancer is glioma. In certain such embodiments, the cancer is glioblastoma. In other such embodiments, the cancer is glioblastoma multiforme. In certain embodiments, the cancer is astrocytoma. In certain such embodiments, the astrocytoma is low-grade astrocytoma, mixed oligoastrocytoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, or anaplastic astrocytoma.

In some embodiments, the cancer is lung cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, and pancreatic cancer. In certain such embodiments, the cancer is lung cancer. In other such embodiments, the cancer is colon cancer. In yet other such embodiments, the cancer is rectal cancer. In still other such embodiments, the cancer is colorectal cancer. In certain such embodiments, the cancer is esophageal cancer. In other such embodiments, the cancer is pancreatic cancer.

In certain embodiments, the method reduces cancer cell proliferation. In certain embodiments, the cancer is relapsed or refractory. In other embodiments, the cancer is treatment naive.

Pharmaceutical Compositions

The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In certain 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 the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

The phrase “pharmaceutically acceptable” Is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety -nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as poly lactide-poly glycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

For use in the methods of this disclosure, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patien”s condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the disclosure. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in the compositions and methods of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present disclosure, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

In certain embodiments, compounds of the disclosure may be used alone or conjointly administered with another type of therapeutic agent.

The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the disclosure in the compositions and methods of the present disclosure. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N- methylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2- hydroxyethyl)morpholine, piperazine, potassium, l-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, 1- hydroxy-2 -naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2- oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1- ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)- camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthal ene-l,5-disulfonic acid, naphthal ene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1- pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid acid salts.

The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Definitions

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4 ^th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7 ^th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6 ^th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

“Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted. It is understood that substituents and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2- O-alkyl, -OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to Ci-Ce straight-chain alkyl groups or Ci-Ce branched- chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1- octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted.

The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

The term “C _x-y ” or “C _x -C _y ”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A Ci-ealkyl group, for example, contains from one to six carbon atoms in the chain.

The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

The term “amide”, as used herein, refers to a group

0 JI o9 '

R ¹⁰ 5 wherein R ⁹ and R ¹⁰ each independently represent a hydrogen or hydrocarbyl group, or R ⁹ and R ¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R ⁹ , R ¹⁰ , and R ¹⁰ ’ each independently represent a hydrogen or a hydrocarbyl group, or R ⁹ and R ¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7- membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

The term “carbamate” is art-recognized and refers to a group wherein R ⁹ and R ¹⁰ independently represent hydrogen or a hydrocarbyl group.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0] octane, 4, 5,6,7- tetrahydro-lH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group -OCO2-.

The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R ¹⁰⁰ ) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like.

The term “ester”, as used herein, refers to a group -C(O)OR ⁹ wherein R ⁹ represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O- heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6- membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the poly cycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group -OSOsH, or a pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae 5 wherein R ⁹ and R ¹⁰ independently represents hydrogen or hydrocarbyl.

The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

The term “sulfonate” is art-recognized and refers to the group SOsH. or a pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group -S(O)2-.

The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

The term “thioester”, as used herein, refers to a group -C(O)SR ⁹ or -SC(O)R ⁹ wherein R ⁹ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the general formula

O A ,R N N ¹⁰ R ⁹ R ⁹ 5 wherein R ⁹ and R ¹⁰ independently represent hydrogen or a hydrocarbyl.

The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds represented by Formula I. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds represented by Formula I or any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01/062726. Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

“Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of formula I). Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphorami date as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound of Formula I. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

The term “Log of solubility”, “LogS” or “logS” as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption. LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol/liter.

The term “amplification” or “amplified” used herein is an art-recognized term that refers to an increase in the number of a given species. For example, in certain embodiments, as used herein the term “amplification” or “amplified” refers to the increase in the number of copies of a gene in the subject(s) (e.g., a cancer patient’s genome). The term “EGFR amplification” is used herein to describe the presence of an increased number of copies of the EGFR gene in a tumor, cancer cell, biological sample or subject, without a corresponding increase in the number of copies of chromosome 7. In preferred embodiments, “EGFR amplification” may be assayed by the presence of a number of EGFR copies that is at least 8, as determined by exome sequencing.

The term “expression” as used herein refers to the process by which the information encoded in a gene is used in a cell to produce RNA molecules. For example, the expression of an NF1 gene leads to the production of RNA molecules coding for the production of NF1 proteins.

The terms “mutation”, “mutated”, or “mutant” as used herein are an art-recognized terms that refer to a gene with a sequence difference from the wild-type DNA sequence of an organism, including the deletion of a certain gene. As a non-limiting example, in certain embodiments, the term “NF 1 mutant” as used herein refers to an NF1 gene that has an altered sequence compared to the wild-type NF1 gene, or to the absence of the NF1 gene. As a result, an NF1 mutant gene would not produce NF1 proteins, or would produce abnormal NF1 proteins, e.g. NF1 proteins that have a lower enzymatic activity level as compared to the enzymatic activity of wild-type NF1. As a second non-limiting example, in certain embodiments, the term “PTEN mutant” as used herein refers to a PTEN gene that has an altered sequence compared to the wild-type PTEN gene, or to the absence of the PTEN gene. As a result, a PTEN mutant genotype would not produce PTEN proteins, or would produce abnormal PTEN proteins, e.g. PTEN proteins that have a lower activity enzymatic activity level than as compared to the enzymatic activity of wild-type PTEN.

The term “polysomy” as used herein is an art-recognized term that refers to the presence of at least one more copy of a chromosome than is normal for said chromosome. The term “EGFR polysomy” is used herein to describe the presence of an increased number of copies of chromosome 7. In certain preferred embodiments, “EGFR Polysomy” may be assayed by the presence of any number of EGFR copies less than 8, as determined by exome sequencing.

EXAMPLES

The various aspects of the disclosure now being generally described, they will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.

Example 1: Evaluation of the Impact of Specific Tumor Genotypes on Response to Exemplary Compounds

These experiments aimed to investigate the hypothesis that PTEN and/or NF1 alterations may impact the response of glioblastomas (GBMs) harboring EGFR polysomy alterations, using GBM Patient-Derived Orthotopic Xenograft (PDOX) models with/without alterations in PTEN and NF1. Moreover, this study aimed to further validate the potential for fluorodeoxyglucose-positron emission tomography (FDG-PET) to serve as an early predictive biomarker for response to Compound 1.

Protocol: Patient GBM models were orthotopically injected into NSG mice. Mice were randomized into 25 mg/kg Compound 1 treatment arms or vehicle treatment arms and treated until endpoints were reached. At endpoints, tumors from vehicle treated mice were extracted and whole exome sequencing was performed to identify copy number and mutations of EGFR, PTEN, and NF 1.

Models: 6 Glioma PDOX models that harbor chromosome 7 polysomy with or without mutations or deletions in PTEN/NF1. For each model, 15 mice were injected for efficacy studies (6 mice vehicle, 6 mice Compound 1 (25mg/kg), and 3 mice for variance in growth error). An additional 5 mice per model were injected for FDG studies (3 mice FDG, 2 mice for error).

Genomic Characterization: Tumors from untreated Glioma PDOX models were sequenced via exome and RNA sequencing to determine EGFR mutation status, EGFR copy number status, MGMT promoter methylation status and chromosome 7 copy number status were characterized for all tested models. Sequencing data also confirmed the presence of a PTEN and/or NF1 alteration (e.g., mutation, copy number loss, or RNA expression levels).

Efficacy Metrics: Efficacy of Compound 1 was measured by two metrics: survival and secreted gaussia luciferase. Levels of secreted gaussia luciferase, which is exclusively secreted by implanted glioma cells, acted as a surrogate for tumor size.

Tolerability Data: Body weight and health observations were recorded during the treatment period.

FDG-PET Implanted mice were scanned prior and post first dose to measure changes in the implanted glioma cell glucose uptake activity. Changes in FDG-PET will be correlated with efficacy metrics to determination the relationship between changes in glucose uptake, as measured by FDG-PET, and Compound 1 efficacy.

Example 2: Efficacy and pharmacodynamic evaluation of exemplary compounds on an Intracranial NSCLC PDX

These experiments evaluated and compared the efficacy of Osimertinib against Compound 1 in an intracranial patient-derived xenograft (PDX) model of EGFR exon 19 mutant non-small cell lung cancer (NSCLC) (i.e., PC9) to evaluate whether fluorodeoxyglucose-positron emission tomography (FDG-PET) can serve as a rapid, predictive biomarker of response to inhibition of EGFR signaling.

Protocol: Patient GBM models were orthotopically injected into NSG mice. Mice were randomized into 25 mg/kg Compound 1 treatment arms or vehicle treatment arms and treated until endpoints were reached. At endpoints, tumors from vehicle treated mice were extracted and whole exome sequencing was performed to identify copy number and mutations of EGFR, PTEN, and NF 1.

Models: PC9 (lu746-Ala750 deletion mutation in exon 19 of EGFR) inoculated in the brain of NSG mice. For this study, 43 mice were injected for efficacy studies (8 mice vehicle, 8 mice lOmg/kg Compound 1, 8 mice 25 mg/kg Compound 1, 8 mice lOmg/kg Osimertinib, 8 mice 25 mg/kg Osimertinib + 3 mice for error. An additional 17 mice were injected for FDG studies, (15 mice FDG, 2 mice for error).

Efficacy Metrics: Efficacy of Compound lor Osimertinib were measured by two metrics: survival and secreted gaussia luciferase. Levels of secreted gaussia luciferase, which is exclusively secreted by implanted by tumor cells, acted as a surrogate for tumor size.

Tolerability Data: Body weight and health observations were recorded during the treatment period.

FDG-PET: Implanted mice were scanned prior and 24 hours post first dose of Compound 1 or Osimertinib to measure changes in the implanted glioma cell glucose uptake activity. Changes in FDG-PET were correlated with inhibition of EGFR signaling and efficacy metrics to determination the relationship between changes in glucose uptake, as measured by FDG-PET, and target inhibition of Compound 1/ Osimertinib and/or efficacy.

Pharmacodynamic evaluation: Following post-treatment FDG evaluation, mice were sacrificed and tumors dissected via GFP-guided microdissection. Tumors were immediately snapfrozen and prepped for immunoblot analysis. Evaluation of EGFR activity (i.e., pEGFR) and downstream signaling components (e.g., pAKT, pERK, and ps6) were tested for vehicle, Compound 1 (both doses) and Osimertinib (both doses).

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the present disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.