WO2018165569A1 - Therapeutic compounds and methods - Google Patents

Abstract

Disclosed herein are compounds of formula II: II or a salt thereof. Also disclosed are pharmaceutical compositions and therapeutic methods for treating certain diseases including cancer such as lung cancer.

Description

THERAPEUTIC COMPOUNDS AND METHODS

PRIORITY OF INVENTION

This application claims priority from United States Provisional Patent Application

Number 62/470,045 filed March 10, 2017, which is hereby incorporated by reference in its entirety.

BACKGROUND

An estimated 170,000 people this year in the U.S. will die from lung cancer. More people will die from lung cancer then prostate, colon, breast, and kidney cancer combined.

Despite advances in targeted therapy 85% of patients diagnosed with lung cancer will succumb to their disease. It is clear better treatment options are needed for the treatment of lung carcinomas.

Bone Morphogenetic proteins (BMP) are members of the Transforming Growth Factor superfamily (TGFP) that are phytogenetically conserved morphogens required for embryonic development across species from insects to humans (Weaver M, et al., Development.

1999; 126(18):4005-15, Sountoulidis A, et al., 2012;7(8):e41460. Epub 2012 Aug 20). BMP2 and BMP4 regulate a plethora of activities during embryogenesis, including the development of the lung. Following the development of the lungs there is little expression of BMP signaling in normal adult lung tissue (Sountoulidis A, 2012). Many studies have reported that the bone morphogenetic signaling cascade has a significant role in promoting tumorigenesis in lung and other cancers. The BMP signaling cascade is reactivated in lung cancer and inflammation (Sountoulidis A, et al., 2012, Langenfeld EM, et al., Carcinogenesis. 2003; 24(9): 1445-54. Epub 2003 Jun 19). The BMP2 ligand is over-expressed in 98% of non-small cell lung cancers but not in benign lung tumors. BMP signaling is also reported to enhance tumorigenesis in many other cancers including prostate (Lai TH, et al., Prostate. 2008; 68(12): 1341-53), breast (Clement JH, et al., International journal of oncology. 2005; 27(2):401-7, Owens P, et al., Oncogene. 2015; 34(19):2437-49. Epub 2014/07/08. doi: 10.1038/onc.2014.189. PubMed PMID: 24998846), pancreas (Kleeff J, et al., Gastroenterology. 1999; 116(5): 1202-16 ), melanoma (Rothhammer T, et al., Cancer research. 2005;65(2):448-56 ), and sarcomas (Nguyen A, et al., International orthopaedics. 2014;38(11):2313-22. Epub 2014/09/12. doi: 10.1007/s00264-014-2512-x. PubMed PMID: 25209345). Aberrant BMP signaling has been reported to enhance cancer migration, invasion, metastasis, proliferation, tumor angiogenesis, and is associated with a worse prognosis (Langenfeld EM, 2003, Langenfeld EM, et al., Mol Cancer Res. 2004;2(3): 141-9, Langenfeld EM, et al., . Oncogene. 2006;25(5):685-92, Ye L, et al., Front.16:865-97, Le Page et al., J Ovarian Res. 2009;2).

Several BMP ligands were identified and categorized into several subclasses. BMPs signal through transmembrane serine/threonine kinases composed of type I and type II receptors. The type I receptors are ALK2 (ActR-1), ALK3 (BMPR-IA), and ALK6 (BMPR-IB) (Nickel J, et al., Cytokine Growth Factor Rev. 2009;20(5-6):367-77). The type Π receptors are BMPR-Π and activin type Π receptors ActR-Π and AcR-IIB ((Nickel J, et al., Cytokine Growth Factor Rev. 2009;20(5-6):367-77). Each BMP receptor can be activated by several different BMP ligands (Nickel J, et al., Cytokine Growth Factor Rev. 2009;20(5-6):367-77). There are different affinities of the BMP ligands to each type Receptors ( Hahm E, et al., Nat Med 2017; 23 : 100- 106 ). Binding of the BMP ligand to the type I receptor leads to phosphorylation by the constitutively active type Π receptor. This receptor complex then phosphorylates Smad-1/5 (Attisano L, Wrana JL. Signal transduction by the TGF-beta superfamily. Science (New York, NY). 2002;296(5573): 1646-7 ) and activates the transcription of downstream target genes including inhibitor of differentiation proteins (Idl, Id2, and Id3) through BMP response elements on their promoter (Katagiri T, et al., Genes Cells. 2002;7(9):949-60. Epub 2002/09/26. doi: 573 [pii]. PubMed PMID: 12296825, Korchynskyi O, et al., The Journal of biological chemistry. 2002;277(7):4883-91, Kurooka H, et al., Biochem Biophys Res Commun. 2012;420(2):281-7. doi: 10.1016/j .bbrc.2012.02.150. Epub Mar 6, Lyden D, et al., Nature. 1999;401(6754):670-7, Hollnagel A, et al., J Biol Chem. 1999;274(28): 19838-45).

Recently, it has been reported that the BMP signaling cascade regulates several anti- apoptotic proteins in lung cancer cells through evolutionarily conserved signaling pathways, which include x-linked inhibitor of apoptosis protein (XIAP), TGFP activated kinase (TAK1), and inhibitor of differentiation proteins (Idl-Id3) (Augeri D J, et al., Molecular cancer.

2016;15:27. Epub 2016/04/07. doi: 10.1186/sl2943-016-0511-9. PubMed PMID:

27048361; PMCID: Pmc4822253). During development, XIAP binds to the BMP type I and type II receptors preventing their ubiquitination and subsequent degradation via proteasomes, thus increasing their expression (Liu Z, et al., Biochimica et biophysica acta. 2009;1793(12):1819-27. Epub 2009/09/29. doi: 10.1016/j.bbamcr.2009.09.012. PubMed PMID: 19782107). XIAP binds to TAB1, leading to the activation of TAK1 (Yamaguchi K, et al., Embo J. 1999; 18(1): 179-87 ). XIAP is the most potent inhibitor of apoptosis and is the only anti-apoptotic protein that inactivates caspases (Obexer P, et al., Frontiers in oncology.

2014;4:197. Epub 2014/08/15. doi: 10.3389/fonc.2014.00197. PubMed PMID: 25120954; PMCID: Pmc4112792 ). XIAP binds and inactivates effector caspase-3 and caspase-7 and initiator caspase-9 (Kaufmann T, Strasser ACell death and differentiation. 2012; 19(l):42-50. Epub 2011/10/01. doi: 10.1038/cdd.2011.121. PubMed PMID: 21959933; PMCID:

Pmc3252833 ). XIAP has been shown to block apoptosis induced by many pro-apoptotic agents. TAK1 also potently inhibits apoptotic cell death through the activation of NF-kappa B (NF-KB) (Mihaly SR, et al., Cell death and differentiation. 2014;21(11): 1667-76. Epub 2014/08/26. doi: 10.1038/cdd.2014.123. PubMed PMID: 25146924; PMCID: Pmc4211365) and by preventing reactive oxygen species (ROS) production (Vanlangenakker N, ret al., Cell death and

differentiation. 2011; 18(4):656-65. Epub 2010/11/06. doi: 10.1038/cdd.2010.138. PubMed PMID: 21052097; PMCID: Pmc3131911). NF-κΒ inhibition of apoptotic cell death involves the induction of cellular FLICE-like protein (c-FLIP), XIAP, c-IAP-1, and c-IAP-2 ((Mihaly SR, et al.,).

Dorsomorphin was identified in a zebrafish library screen to be a small molecule inhibitor of the BMP receptors (Zon LI, et al., Nat Rev Drug Discov. 2005;4(l):35-44. ).

Several generations of BMP inhibitors have been synthesized based on substitutions to this pyrazolo[l,5-a]pyrimidine core with varying affinities to the kinase domain of the BMP type I and type Π receptors (Hao J, et al., ACS Chem Biol.5(2):245-53. Epub 2009/12/22. doi:

10.1021/cb9002865 [doi]. PubMed PMID: 20020776; PMCID: 2825290, Yu PB, et al., Nat Med. 2008;14(12): 1363-9. Epub 2008 Nov 30).

The BMP analogs, LDN-193189 (LDN) and DMH1 have shown in tumors in mice to decrease Idl expression and suppress metastatic growth without demonstration of toxicity (Balboni AL, et al., Cancer Res. 2013;73(2): 1020-30. doi: 10.158/0008-5472.CAN-12-2862. Epub 012 Dec 14, Owens P, et al., Oncogene. 2015;34(19):2437-49. Epub 2014/07/08. doi: 10.1038/onc.2014.189. PubMed PMID: 24998846). In vitro studies showed that the BMP analog DMH2 is significantly more potent in downregulating XIAP, pTAKl, and Idl expression and inducing death of lung cancer cells than LDN and DMH1 (Augeri DJ, et al., Molecular cancer. 2016;15:27. Epub 2016/04/07. doi: 10.1186/sl2943-016-0511-9. PubMed PMID:

27048361; PMCID: Pmc4822253 ). However, DMH2 does not downregulate XIAP, ID1, or pTAKl in tumor xenografts likely because of its poor pharmacokinetic profile in mice (Augeri DJ, et al., Molecular cancer. 2016). The development of stable BMP inhibitors that have potent inhibition of BMP regulated anti-apoptotic proteins Idl, pTAKl, and/or XIAP in tumor xenografts is needed to better evaluate the role of BMP inhibitors as a cancer therapeutic. The BMP signaling pathway is also known to regulate the activation and development of dendritic cells (Martinez VG, et al., Immunology and cell biology. 2011;89(5):610-8. Epub 2010/11/26. doi: 10.1038/icb.2010.135. PubMed PMID: 21102536 ), T cells (Martinez VG, et al., PloS one. 2015; 10(6):e0131453. Epub 2015/06/26. doi: 10.1371/journal.pone.0131453. PubMed PMID: 26110906; PMCID: Pmc4481406), and natural killer cells (Robson NC, et al.,. Cancer research. 2014;74(18):5019-31. Epub 2014/07/20. doi: 10.1158/0008-5472.can-13-2845. PubMed PMID: 25038228; PMCID: Pmc4167038 ). The effects of BMP inhibitors on immune cells within the tumor microenvironment are not known. Since BMP signaling regulates immune cells it essential to understand how BMP inhibitors affect the immune cells within the tumor microenvironment and determine whether this enhances or attenuates its effect on tumor growth.

Accordingly, there is a need for new agents to treat cancer (e.g., lung cancer). There is also a need for chemotherapeutic agents that have improved pharmacokinetic profiles.

SUMMARY OF INVENTION

Compounds of formulas I and II are disclosed herein. One representative compound, the compound of formula I (also discussed herein as Compound 1 and JL5) (4-(3-(4-(3-(quinolin-4- yl)pyrazolo[l,5-a]pyrimidin-6-yl)phenyl)propyl)morpholine) inhibits BMP type I and type II receptors and in vitro induces cell death and downregulates Idl, XIAP, and pTAKl . Compound 1 induces tumor regression and downregulate Idl and pTAKl in lung tumor xenografts.

Compound 1 has also been shown to have improved pharmacokinetic characteristics (e.g., relative to DMH2).

One embodiment provides a compound of formula II:

wherein:

R¹ is H, halogen, or (Ci-C₄)alkyl;

R² is H, halogen, or (Ci-C₄)alkyl;

R³ is a 5-10 membered monocyclic or bicyclic heteroaryl, wherein the heteroaryl is optionally substituted with one or more halogen, (Ci-C4)alkyl or -0(Ci-C4)alkyl;

R⁴ is H, halogen or (Ci-Ce)alkyl;

L is (Ci-Ce)alkyl optionally substituted with one or more halogen; and

W is a 4-7 membered heterocyclyl optionally substituted with one or more halogen or C4>alkyl; or

a salt thereof.

One embodiment p

I

or a salt thereof.

One embodiment provides a pharmaceutical composition comprising a compound of formula I or Π or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

One embodiment provides a method for treating cancer in a mammal (e.g., a human) in need thereof , comprising administering to the mammal a compound of formula I or Π or a pharmaceutically acceptable salt thereof.

One embodiment provides a method for downregulating one or more anti-apoptotic proteins in a mammal in need thereof comprising administering to the mammal a compound of formula I or Π or a pharmaceutically acceptable salt thereof. One embodiment provides a method for inducing an influx of immune cells into a tumor cell in vitro or in vivo, comprising contacting the cell with a compound of formula I or Π or a salt thereof.

One embodiment provides method for inducing an influx of immune cells into a mammal, comprising administering to the mammal a compound of formula I or II or a pharmaceutically acceptable salt thereof.

One embodiment provides a compound of formula I or II or a pharmaceutically acceptable salt thereof for use in medical treatment.

One embodiment provides a compound of formula I or II or pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of cancer.

One embodiment provides a compound of formula I or II or pharmaceutically acceptable salt thereof for the preparation of a medicament for treating cancer.

One embodiment provides processes and intermediates disclosed herein that are useful for preparing a compound of formula I or II or a salt thereof.

BRIEF DESCRIPTION OF FIGURES

Figures 1 A-1B show DNA amplifications, deep deletions, truncating mutations, missense mutations, and mRNA expression for: Figure 1 A BMP ligands in lung adenocarcinomas (n=l 17) and Figure IB BMP receptors, BMP transcription factors (Smad-1/5/9), and BMP downstream targets (XIAP, MAP3K7/TAK1, ID1, ID2, ID3) in lung adenocarcinomas (n=135).. Gray boxes indicate expression without alteration.

Figure 2A shows structures of BMP inhibitors (DMH2, DMH1, LDN) based on the substitution at the R position of the pyrazolo[l,5-a]pyrimidine core.

Figure 2B shows that DMH2 was found to be chemically and metabolically unstable. LCMS of a sample of stored DMH2 for 4 months revealed the phenolic byproduct due to morpholine side-chain hydrolysis. Figure 2C shows the structure of DMH2 analogs Compound 1 (JL5) and the analog Compound 2 (JL12). The side chain of DMH2 was modified by replacing a carbon atom with an oxygen atom to create Compound 1. Compound 1 was modified by replacing the quinolone substituent with a pyrazole substituent to create compound Figures 3A-3G show that Compound 1 but not Compound 2 regulate BMP signaling and induce cell death. Figures 3A and 3B show western blot analysis of H1299 cells treated with Compound 1 or Compound 2 for 72 hours. Figure 3C shows an Idl-luciferase assay in H1299 cells treated with Compound 1 and Compound 2 for 48 hours. Figure 3D shows percent dead cells and number of live cells of treated H1299 cells after 72 hours. Figure 3E shows percent dead cells and number of live cells of treated H1299 cells after 7 days. Figure 3F shows a western blot analysis of H1299 cells treated with Compound 1 for 3 days demonstrated apoptotic cell death. Figure G shows a TUNEL assay of H1299 cells treated with Compound 1 for 24 hour demonstrating an increase in DNA double stranded breaks. All experiments were performed at least 3 times except (C), which was performed twice.

Figures 4A-C show that Compound 1 suppresses BMP Signaling and decreases tumor Growth. Figure 4A shows a Western blot analysis of established H1299 tumor xenografts in NSG mice without immune cells treated with Compound 1 for 4 days. Figures 4B and 4C show growth curves and tumor weights of established H1299 xenografts in NSG mice without immune cells

Figures 5A-5C show that Compound 1 suppresses growth of tumor xenografts in NSG mice with immune cells and induces infiltration of immune cells. Figure 5 A shows a Western blot analysis of established H1299 xenografts in NSG mice receiving transferred human immune cells and treated for 21 days. Figures 5B and 5C show growth curves and tumor weights of established H1299 xenografts in NSG mice with immune cells

Figures 6A-6D show that Compound 1 induces death of cancer cells on treatment day 13. NSG mice received adoptively transferred human immune cells and then H1299 cells were injected intradermally into the flanks. Five days later after tumors had reached approximately 5 mm2 mice were treated with DMSO or 10 mg/kg JL5 (twice daily) (n=7 mice for each group) for 13 days. Figures 6A and 6B show Compound 1 causes tumor regression after 13 days.

Figures 6C and 6D demonstrate Compound 1 induces death of cancer cells (cells shown by arrows) in tumor xenografts.

Figure 7 shows Compound 1 induces the infiltration of immune cells within the tumor microenvironment. DETAILED DESCRIPTION

The following definitions are used, unless otherwise described.

The term halo or halogen is fluoro, chloro, bromo, or iodo.

Alkyl and alkoxy, etc. denote both straight and branched groups but reference to an individual radical such as propyl embraces only the straight chain radical (a branched chain isomer such as isopropyl being specifically referred to).

As used herein, the term "(C_a-Cb)alkyl" wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms. Thus when a is 1 and b is 6, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t- butyl, n-pentyl and n-hexyl.

The term "heteroaryl" as used herein refers to a single aromatic ring or a multiple condensed ring system. The term includes single aromatic rings of from about 1 to 6 carbon atoms and about 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the rings. The sulfur and nitrogen atoms may also be present in an oxidized form provided the ring is aromatic. Such rings include but are not limited to pyridyl, pyrimidinyl, oxazolyl or furyl. The term also includes multiple condensed (e.g., fused) aromatic ring systems (e.g. ring systems comprising 2 rings) comprising about 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the rings. In one embodiment a monocyclic or bicyclic heteroaryl has 5 to 10 ring atoms comprising 1 to 9 carbon atoms and 1 to 4

heteroatoms. It is to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heteroaryl) can be at any position of the multiple condensed ring system including a carbon atom and heteroatom (e.g., a nitrogen). Exemplary heteroaryls include but are not limited to pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, quinazolyl, benzofuranyl, and benzimidazolyl.

The term "heterocyclyl" or "heterocycle" as used herein refers to a single saturated or partially unsaturated ring. The term includes single saturated or partially unsaturated rings (e.g., 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. The ring may be substituted with one or more (e.g., 1, 2 or 3) oxo groups and the sulfur and nitrogen atoms may also be present in their oxidized forms. Such rings include but are not limited to azetidinyl, tetrahydrofuranyl or pipendinyl. It is to be understood that the point of attachment for a heterocycle can be at any suitable atom of the heterocycle. Exemplary heterocycles include, but are not limited to azindinyl, azetidinyl, pyrrolidinyl, pipendinyl, homopipendinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl and tetr ahy dr othi opyr anyl .

Specific values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.

It is to understood that the embodiments provided below are for compounds of formula II and that two or more embodiments may be combined.

In one embodiment R¹ is H.

In one embodiment R² is H.

In one embodiment R⁴ is H.

In one embodiment R³ is 5-membered monocyclic heteroaryl or 9-10-membered bicyclic heteroaryl, wherein the 5-membered monocyclic heteroaryl or 9-10-membered bicyclic heteroaryl is optionally substituted with one or more halogen, (Ci-C4)alkyl or -0(Ci-C4)alkyl.

In one embodiment R³ is 5-membered monocyclic heteroaryl including 1 or 2 nitrogen atoms or 9-10-membered bicyclic heteroaryl including 1 or 2 nitrogen atoms, wherein the 5- membered monocyclic heteroaryl or 9-10-membered bicyclic heteroaryl is optionally substituted with one or more halogen, (Ci-C4)alkyl or -0(Ci-C4)alkyl.

In one embodiment R³ is quinolinyl, indazolyl, or pyrazolyl, wherein the quinolinyl, indazolyl, or pyrazolyl is optionally substituted with one or more halogen, (Ci-C4)alkyl or -0(Ci- C₄)alkyl.

In ne embodiment R³ is:

In one embodiment L is (Ci-Ce)alkyl.

In one embodiment L is ethyl or propyl.

In one embodiment L is ethyl or propyl. In one embodiment W is a 6 membered heterocyclyl optionally substituted with one or more halogen or (Ci-C4)alkyl.

In one embodiment W is a morpholinyl optionally substituted with one or more halogen or (Ci-C₄)alkyl.

In one embodiment W is a morpholin-4-yl.

One embodiment provides the compound:

or a salt thereof.

One embodiment p

or a salt thereof.

In one embodiment the salt is a pharmaceutically acceptable salt.

The terms "treat", "treatment", or "treating" to the extent it relates to a disease or condition includes inhibiting the disease or condition, eliminating the disease or condition, and/or relieving one or more symptoms of the disease or condition. The terms "treat", "treatment", or "treating" also refer to both therapeutic treatment and/or prophylactic treatment or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as, for example, the development or spread of cancer. For example, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease or disorder, stabilized (i.e., not worsening) state of disease or disorder, delay or slowing of disease progression, amelioration or palliation of the disease state or disorder, and remission (whether partial or total), whether detectable or undetectable. "Treat", "treatment", or "treating," can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in which the disease or disorder is to be prevented. In one embodiment "treat",

"treatment", or "treating" does not include preventing or prevention,

The phrase "therapeutically effective amount" or "effective amount" means an amount of a compound of the present invention that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

The term "patient" as used herein refers to any animal including mammals such as humans, higher non-human primates, rodents, domestic and farm animals such as cows, horses, pigs, sheep, dogs and cats. In one embodiment, the patient is a human patient. In one embodiment, the mammal is a human. In one embodiment, the patient is a human patient.

It is understood by one skilled in the art that this invention also includes any compound claimed that may be enriched at any or all atoms above naturally occurring isotopic ratios with one or more isotopes such as, but not limited to, deuterium (²H or D). As a non-limiting example, a -CH3 group may be substituted with -CD₃.

Additionally, administration of a compound of formula I or formula II as a

pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts include organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate. Suitable inorganic acid addition salts may also be formed, which include a physiological acceptable anion, for example, chloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The compounds of formula I and II can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

The pharmaceutical compositions of the invention can comprise one or more excipients. When used in combination with the pharmaceutical compositions of the invention the term "excipients" refers generally to an additional ingredient that is combined with the compound of formula I or formula II or the pharmaceutically acceptable salt thereof to provide a

corresponding composition. For example, when used in combination with the pharmaceutical compositions of the invention the term "excipients" includes, but is not limited to: carriers, binders, disintegrating agents, lubricants, sweetening agents, flavoring agents, coatings, preservatives, and dyes. Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable excipient such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of

microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the compounds of formula I or formula II to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat.

No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I or formula II can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

In some embodiments, one or more of the compounds disclosed herein are coadministered with one or more other active therapeutic agents (e.g., antibacterial agents). Coadministration of a compound disclosed herein with one or more other active therapeutic agents generally refers to simultaneous or sequential administration of a compound disclosed herein and one or more other active therapeutic agents, such that therapeutically effective amounts of the compounds disclosed herein and one or more other active therapeutic agents are both present in the body of the patient.

In some embodiments, one or more of the compounds disclosed herein are coadministered with one or more active therapeutic agents (e.g., antibacterial agents) by combining the compounds disclosed herein with the other therapeutic agents in a unitary dosage form for simultaneous or sequential administration to a patient. Thus, this combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

The invention will now be illustrated by the following non-limiting Examples.

Example 1

Methods

A. Cell Culture and Reagents.

Lung cancer cell lines A549 and H1229 were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma Aldrich, St Louis) supplemented with 5% fetal bovine serum (FBS) (Langenfeld EM, et al., Mol Cancer Res 2005; 3 : 679-68430). DMH2 was synthesized at Rutgers- New Jersey Medical School. ZVAD-FMK and necrostatin-1 were obtained from Sigma-Aldrich, and utilized as per manufacture instructions B. Western Blot Analysis.

Total cellular protein was prepared as previously described ( Langenfeld EM, et al., . Carcinogenesis 2003; 24: 1445-1454. Epub 2003 Jun el419.). Briefly, the protein concentration was calculated using the BCA assay. Protein was separated by SDS-PAGE and then transferred to nitrocellulose. The blots were blocked for at least 2 hr and then incubated overnight at 4°C with the selected primary antibody. Next, secondary antibodies were added for 1 hr at room temperature (RT). A chemiluminescence system (Amersham, Arlington Heights, IL) was used to detect selected proteins. Primary antibodies purchased from Cell Signaling Technology (Danvers MA) used, including anti-pTAKl (rabbit), XIAP (rabbit), anti -activated caspase-3 (rabbit), anti- PARP.

C. Chemical Synthesis of Compound 1 (JL5).

Scheme 1

To synthesize 4-(6-bromopyrazolo[l,5-a]pyrimidin-3-yl)quinolone (2) a solution of 4- (quinolin-4-yl)-lH-pyrazol-3 -amine (535 mg, 2.54 mmol, 1 eq) in acetic acid (20 ml) was added 2-bromomalonaldehyde (383 mg, 2.54 mmol, 1 eq). After stirring for 16 hours at room temperature, the reaction was diluted in water up to 150 ml total solvent. The solution was adjusted to a pH of 5-6 with careful addition of sodium hydroxide when a solid began to precipitate. After 30 minutes, the suspended solid was subjected to sonication, filtered and washed with water. The solid was recrystallized in MeOH to yield the title compound (752 mg, 91% yield) as a white solid. ¾ NMR (500 MHz, DMSO-^e) δ 9.77 (d, J= 2.2 Hz, 1H), 8.94 (d, J= 4.5 Hz, 1H), 8.78 - 8.71 (m, 1H), 8.69 (s, 1H), 8.09 (td, J= 8.3, 1.4 Hz, 2H), 7.78 (ddd, J = 8.2, 6.7, 1.4 Hz, 1H), 7.71 (d, J= 4.4 Hz, 1H), 7.58 (ddd, J= 8.3, 6.8, 1.3 Hz, 1H). MS:

324.75, 326.80 [M + H]⁺.

To synthesize 4

yl)phenyl)propyl)morpholine (3) a solution of 4-(3-(4-bromophenyl)propyl)morpholine (110 mg, 0.387 mmol, 1 eq), bis(pinacolato)diboron (147 mg, 0.581 mmol, 1.5 eq), PdCl2(dppf)CH₂Cl2 (16 mg, 0.0194 mmol, 0.05 eq), and KOAc (104 mg, 1.16 mmol, 3 eq) in dioxane (4 ml) was heated in a microwave reactor for 15 minutes at 130°C. The crude reaction was diluted in hexanes and filtered over a pad of celite to remove inorganics. The filtrate was purified by silica gel chromatography (50% -> 100% EtO Ac/Hex) and concentrated to afford the title compound (115 mg, 90%) as an oil that was used in the subsequent coupling without further purification. MS: 332.05 [M + H]⁺.

To synthesize Compound 1(JL5) (Scheme 1) a mixture of 4-(6-bromopyrazolo[l,5- a]pyrimidin-3-yl)quinolone (300 mg, 0.810 mmol, 1 eq), 4-(3-(4-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)phenyl)propyl)morpholine (402 mg, 1.22 mmol, 1.5 eq), PdCl₂(PPh₃)2 (28 mg, 0.405 mmol, 0.05 eq) in dioxane (9 ml) and 2M aqueous Na₂C0₃ (6 ml) was heated in a microwave reactor for 10 minutes at 130°C. The reaction was partitioned in EtOAc and water followed by filtration over celite to remove insoluble impurities. The filtered organic was separated and the filtered aqueous was extracted 2 x EtO Ac. The combined organic was dried over Na₂S04, filtered and concentrated. The residue was dissolved in dilute aqueous HC1 and washed 2 x DCM. The acidic aqueous was made basic with 1M NaOH and extracted 4 x DCM. The combined organic was dried over Na₂S04, filtered and concentrated. The residue was purified by silica gel chromatography (2% -> 5% MeOH/DCM) and product containing fractions were combined and concentrated. Recrystallization from EtOAc afforded the title compound (175 mg, 48%) as a white solid. ¾ NMR (500 MHz, OMSO-d₆) δ 9.61 (d, J= 2.3 Hz, 1H), 9.05 (d, J= 2.3 Hz, 1H), 8.95 (d, J= 4.5 Hz, 1H), 8.71 (s, 1H), 8.18 (ddd, J= 8.4, 1.4, 0.6 Hz, 1H), 8.09 (ddd, J= 8.5, 1.3, 0.6 Hz, 1H), 7.84 - 7.75 (m, 4H), 7.61 (ddd, J= 8.3, 6.8, 1.3 Hz, 1H), 7.40 - 7.35 (m, 2H), 3.56 (t, J= 4.7 Hz, 4H), 2.66 (dd, J= 8.3, 7.0 Hz, 2H), 2.33 (d, J= 5.5 Hz, 4H), 2.30 - 2.26 (m, 2H), 1.76 (dt, J= 14.6, 7.5 Hz, 2H). MS: 449.90 [M + H]+.

D. Evaluation of Genetic abnormalities of BMP signaling in NSCLC.

The Cancer Genome Atlas (TCGA) database available through cBioportal

(http://www.cbioportal.org/public-portal/; accessed on 3/18/2016), a publicly available data portal was used to investigate the frequency of alterations and expression of the BMP family ligands, receptors, transcription factors, and BMP downstream mediated targets regulating cell survival [PMID: 23550210; 22588877]. Data sets were queried for lung adenocarcinoma and lung squamous cell cancers (provisional for both). Only cases with complete data on mutations, copy-number alterations, and mRNA expression were included. Alterations in BMP signaling in non-small cell lung cancer cell lines (CCLE, Cancer Cell Line Encyclopedia) were queried through cBioportal [PMID: 22460905]. Dysregulated gene expression was defined by Z > 2 (over-expression) or Z <2 (reduced expression). Software tools embedded within cBioPortal were used to determine the proportion of samples within each dataset with alterations or up/down regulation.

E. Quantifying Live and Dead Cells.

Lung cancer cells were plated into 6 well plates and the following day treated for the designated amount of time with a BMP inhibitor. The number of live and dead cells was determined using Vi-CELL cell analyzer (Beckman Coulter), which analyzed 500 cells/sample. The Vi-CELL utilizes trypan blue dye exclusion to determine dead cells.

F. In vitro Kinase IC50.

The IC50 of various compounds including DMH2 for alk2, alk3, alk6, alk5, BMPRII, and

TGFP were performed at Reaction Biology Corporation (Malvern, PA). This was a 10-point assay starting from 100 μΜ to 100 nM performed in duplicate. The ATP concentration was 10 Micromolar.

G. Plasma protein binding.

Human and mouse protein plasma binding of compound 1 and DMH2 was performed using equilibrium dialysis (Sai Life Sciences Limited, Pune India).

H. Metabolic Stability.

Mouse liver microsomes were treated with DMSO or Compound 1 for 0,5, 15, 30, and 60 minutes. The plates were centrifuged and 100 μL aliquots analyzed by liquid

chromatography-mass spectrometry (LC-M/MS (Sai Life Sciences Limited, Pune India).

I. Pharmacokinetics.

The pharmacokinetics of Compound 1 was examined in BALB/c mice following intravenous and intraperitoneal (i.p.) administration (Sai Life Sciences Limited, Pune India). Three mice at each time point were dosed with a 2 mg/kg tail vein injection and 10 mg/kg i.p. Blood samples were taken 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours and analyzed with

LC/MS/MS. Plasma half-life, clearance, AUC, and volume of distribution were then determined (Sai Life Sciences Limited, Pune India).

J. Humanized and non-humanized tumor xenograft studies.

In a first experiment tumor xenografts from H1299 cells were established in the flanks of ΝΟΌ-scid IL2Rgamma^nu11 (NSG) mice, which have no functional mouse immune system, thus they readily accept human immune cells and tissues. When tumors reached 4 mm², mice were treated with DMSO, 3 mg/kg, and 10 mg/kg of Compound 1 twice daily for 4 days (n=8 tumors in each group). In a second experiment xenografts from HI 299 cells (approximately 4 mm² ) were done in NSG mice and mice transplanted with donor HLA-compatible human immune cells and treated with DMSO or 10 mg/kg of Compound 1 twice daily for 21 days (n=8 tumors in each group). In a third experiment established xenografts from H1299 in NSG mice transplanted with donor human immune cells were treated with DMSO or 10 mg/kg of

Compound 1 twice daily for 12 days (n= 8 tumors in each group). K. Quantifying tumor necrosis.

To quantify necrosis H & E slides from each tumor were analyzed using CellSens imaging software (Olympus Life Science). The area of tumor necrosis and the area of the entire tumor were measured after manual delineation. The ratios of the area of tumor necrosis to the area of the entire tumor were then calculated.

L. Toxicity studies.

Mice treated with DMSO, 3 mg/kg, and 10 mg/kg of Compound 1 twice daily for 4 days were euthanized and postmortem examinations were performed at the scheduled necropsy. At the time of necropsy, lungs, kidneys, and livers were collected and fixed in 10% neutral -buffered formalin. Microscopic examination of hematoxylin and eosin-stained paraffin sections were performed by a board certified veterinary pathologist with light microscopy. Mice undergoing 21 -day experiments treated with DMSO and 10 mg/kg of Compound 1 were examined 4 times weekly for lethargy, weight loss, loss of appetite. At the end of the experiment mice were euthanized and spleen and livers were weighed.

M. Statistical Analysis.

The mean of the control group was compared to the mean of each treated group using paired student t-test assuming unequal variances. Differences with p values <0 .05 were considered statistically significant.

N. List of Acronyms.

BMP: Bone Morphogenetic Protein

TGFP: Transforming Growth Factor Beta:

TAK1 : TGFP activated kinase

TAB: TAK1 binding protein

XIAP: X-link inhibitor of apoptosis protein

Idl : Inhibitor of differentiation

NSCLC: non-small cell lung

MEK-1/2: mitogen-activated protein kinases

Egr-1 : early growth response protein

TRAF4: necrosis factor receptor-associated factor 4

TRAF6: necrosis factor receptor-associated factor 6

LDN: LDN-193189 5Z-7-oxozeaenol (5Z)

SB: SB-505124

LY: LY2109761

VEGF Π: vascular endothelial growth factor

AMP -kinase: adenosine monophosphate-activated protein kinase

siRNA: short interfering RNA

O. Luciferase assay.

H1299 cells were stably transfected with the Id-1 promoter, which drives the expression of the luciferase reporter. Cells were treated with BMP inhibitors for 48 hours then cells lysed and luminescence measured by the TD-20/20 Luminometer (Turner Designs/Turner BioSystems, Sunnyvale, CA) (Augeri DJ, et al., Molecular cancer 2016; 15: 27). Results

Genetic alterations of the BMP signaling cascade in non-small cell lung carcinoma (NSLC).

Genetic events that could cause resistance to a target specific therapeutic include alterations that inactivate the signaling pathway, mutations of the receptor that effect its interaction with the small molecule, and amplification of an essential downstream signaling event. The Cancer Genome Atlas (TCGA) was queried to examine the genetic alterations effecting the BMP ligands, receptors, transcription factors, and downstream targets in NSCLC. In a prior study, it was shown that the BMP2 protein is highly overexpressed in 98% of lung carcinomas. Only 2 of the 228 adenocarcinomas examined had either a deep deletion or missense mutation of BMP2 (Figure 1A). In addition, the expression of the family of BMP ligands is redundant in NSCLC with a high rate of upregulation of the mRNA (86%) and amplification (26%) (Figure 1A)

Deep deletions, truncating mutations, or missense mutations were present in 12 (5%) of the BMP type I and 9 (4%) of the BMP type Π receptors (Figure IB). A missense mutation of any one BMP receptor was no more than 1.3%. None of the tumors had mutations in all 3 of the type I or type II receptors (Figure IB). Mutations of Smad 1/5/8-9 is also infrequent and never occurred in all 3 transcription factors (Figure IB).

Amplification of the downstream BMP targets XIAP, TAK1, and Idl has the potential to cause resistance to a BMP inhibitor if it upregulates expression. Amplification of XIAP occurred in 1 (0.4%), 0 for TAK1/MAP3K7, 8 (3.5%) for Idl, 0 for H2, and 3 (1.3%) for H3 (Figure IB). However, upregulation of the mRNA was not seen in any of the amplified downstream BMP targets (Figure IB).

Similar genetic alterations were identified in squamous carcinomas of the lung and lung cancer cell lines. The low rate of mutations and the redundancy of the BMP signaling cascades suggest that resistance to BMP receptor inhibitors in lung cancer because of genetic alterations is likely to be a rare event.

Metabolic Instability of DMH2.

The pyrazolo [1,5-a] pyrimidine core of the BMP dorsomorphin (Figure 2A) has been utilized as a heterocyclic core to synthesize BMP inhibitors (Yu PB, et al., Nat Chem Biol.

2008;4(1):33-41. Epub 2007 Nov 18 ). Analogs of Dorsomorphin, DMH1, DMH2 (Hao J, et al., . ACS Chem Biol.5(2):245-53. Epub 2009/12/22. doi: 10.1021/cb9002865 [doi].

PubMed PMID: 20020776; PMCID: 2825290 ), and LDN (Cuny GD, et al., Bioorganic & medicinal chemistry letters. 2008;18(15):4388-92. PubMed PMID: 18621530) differ in the substitutions made at the R-position of the pyrazolo [1,5-a] pyrimidine core (Figure 2A). It was found that after approximately 4 months aliquoted samples of DMH2 had decreased potency to downregulate Idl expression and induce death of lung cancer cells. DMH2 was found to be chemically and metabolically unstable. Analysis using liquid chromatography-mass

spectrometry (LCMS) of a sample of DMH2 stored as solid in a desiccator over 4 months revealed the phenolic byproduct due to morpholine side-chain hydrolysis (Figure 2B).

Design and Synthesis of Compound 1 (JL5).

The instability discussed above led to the design of a compound devoid of this oxygen by replacement with a carbon atom to generate Compound 1 (JL5) as shown in Figure 2. The carbon analog 1 was synthesized in a convergent fashion by palladium-catalyzed coupling using microwave reactor to couple the brominated pyrazolo[l,5-a]pyrimidine 2 to the borate ester of the morpholine side chain 3 to generate Compound 1 in 48% yield as a white crystalline solid (Scheme 1). An additional substitution of Compound 1 was made at the R2 position of the core with an imidapyrazole creating Compound 2 (JL12) (Figure 2C). Inhibitory Concentration.

Compound 1 has single digit nanomolar (nM) half maximal inhibitory concentration (IC50) for the BMP type I receptors alk2, alk3, and alk6, which is lower than previously reported for DMH2 (Table 1). Although Compound 1 only had an approximately 8 μΜ IC50 for the BMP type II receptor BMPR2, it was similar to that of DMH2 (Table 1). Prior studies suggested that DMH2 inhibition of BMPR2 increased its potency by enhancing the downregulation of XIAP (Augeri DJ,et al., Molecular cancer. 2016; 15:27. Epub 2016/04/07. doi: 10.1186/sl2943- 016-0511-9. PubMed PMID: 27048361; PMCID: Pmc4822253 ). DMHl and LDN are reported to not inhibit BMPR2 (Engers DW, et al., Bioorg Med Chem Lett. 2013;23(11):3248-52. doi: 10.1016/j .bmcl.2013.03.113. Epub Apr 11). Compound 2 demonstrated very little inhibition of the BMP type I and type II receptors and therefore was used as a negative control in subsequent studies (Table 1). These studies show that the inhibition of the BMP type I and type II receptors of Compound 1 is very similar to that of DMH2.

Pharmacokinetic Profile of Compound 1

Mouse liver microsomes demonstrated the intrinsic clearance of Compound 1 was 49% lower than that of DMH2 (Table 2). Both Compound 1 and DMH2 demonstrated high binding to mouse plasma proteins (Table 2). Following a single intravenous administration of

Compound 1 to male BALB/c mice at 2 mg/kg dose, Compound 1 showed very high plasma clearance (194 mL/min/kg) exceeding normal hepatic clearance, likely due to a high volume of distribution (Vss) of 8.75 L/kg, an elimination half-life of 0.57 hr, indicative of high tissue penetration (Table 3). The area under the curve (AUC) (i.p. injection, 10 mg/kg) was determined to be 1.6 μΜ hr with a Cmax of 1.2 μΜ (Table 3). The Vss of DMH2 was that of plasma so its distribution into the tissue is significantly lower than that of Compound 1 (Table 3). Since the pharmacokinetic properties of Compound 1 are improved over DMH2, further in vitro and in vivo xenograft studies were conducted.

Compound 1 Does Not Induce Toxicity In Mice

BALB-c mice were injected intraperitoneally (IP) with 0, 3 mg/kg, 10 mg/kg of

Compound 1 twice daily for 4 days. Mice showed no evidence of systemic toxicity such as loss of appetite, anorexia, and lethargy. Histological examination of the livers, lungs, and kidneys by a veterinarian pathologist did not reveal any evidence of toxicity (data not shown). In addition, 21 -day experiments injecting 10 mg/kg of Compound 1 twice daily also did not demonstrate evidence of toxicity as demonstrated by lack of anorexia, lethargy, or loss of weight of spleen and liver. Compound 1 Inhibits BMP Signaling and Induces Death of Lung Cancer Cells

Compound 1 caused a dose-related decrease in the expression of Idl, XIAP, and pTAKl in H1299 lung cancer cells (Figure 3 A) in same manner as previously reported for DMH2 (Augeri DJet al., Molecular cancer. 2016; 15:27). Like DMH2, Compound 1 at lower concentrations caused an increase in the expression of pTAKl, which became undetectable at higher concentrations (Figure 3A) (Augeri DJet al., Molecular cancer. 2016; 15:27). Compound 2 had no effect on the expression of Idl, XIAP, or pTAKl (Figure 3B). Since BMP signaling is a direct transcriptional regulator of the Idl promoter, it was examined whether Compound 1 regulated the Idl promoter. H1299 cells stably expressing the Idl promoter regulating the luciferase reporter were treated with Compound 1. Compound 1 caused a dose-responsive decrease in the expression of the Idl -luciferase reporter, while Compound 2 had no effect (Figure 3C). Compound 1 induced a significant dose-responsive increase in cell death (Figure 3D) and a decrease in the number of live cells (Figure 3D) of H1299 cells treated for 3 days. Compound 2 had no effect on either cell death or cell growth of the H1299 cells (Figure 3D). Compound 1 and DMH2 caused the same amount of cell death at 2.5 μΜ after 3 days (Figure 3D). After 7 days, the majority of the H1299 cells treated with Compound 1 were dead with few remaining live cells in comparison to DMSO control (Figure 3E). Compound 1 induced the activation of caspase-3 and cleavage of PARP, suggesting like DMH2, it induces apoptotic cell death (Figure 3F) (Augeri DJ,et al., . Molecular cancer. 2016; 15:27. Epub 2016/04/07. doi: 10.1186/sl2943-016-0511-9. PubMed PMID: 27048361; PMCID: Pmc4822253).

Compound 1 Inhibits Tumor Growth in NSG Mice Without Immune Cells

Compound 1 was examined to determine if it downregulated BMP downstream targets in established tumor H1299 xenografts in ΝΟΌ-scid IL2Rgamma^nuU (NSG) mice that do not have immune cells. After 4 days, Compound 1 (10 mg/kg) treated tumors had a decreased protein expression of Idl and TAKl but not XIAP (Figure 4 A). The decrease in TAKl has never been achieved with other BMP inhibitors. Similar to what was reported for DMH2, Compound 1 at lower concentration (3 mg/ml) caused a feedback increase in the expression of Idl after 4 days (Figure 4 A). However, DMH2 was not able to downregulate Idl or TAKl in tumor xenografts. The effects of Compound 1 (10 mg/ml) on H1299 tumor xenografts in NSG mice were examined in mice treated for 21 days (Figure 4B). Tumors treated with Compound 1 were significantly smaller than controlled treated tumors (Figure 4B and 4C).

Compound 1 Inhibits Tumor Growth in NSG Mice With Immune Cells

The effects of Compound 1 (10 mg/ml) on H1299 tumor xenografts in NSG that received passively transferred ULA compatible human immune cells (humanized) were also studied. Tumors in the humanized mice also had a decrease in expression of Idl and pTAKl but not XIAP (Figure 5 A). Compound 1 caused a reduction in tumor size in mice with transferred immune cells treated for 21 days (Figure 5B-C).

Compound 1 Induces Tumor Regression

Since cell death after 21 days of treatment was not observed, despite xenografts demonstrating tumor regression after approximately 13 days of treatment, the humanized xenograft study was repeated but the tumors were analyzed after 13 of treatment with Compound 1. Again a significant difference in the size and weight of the tumors of mice treated with

Compound 1 compared to vehicle control (Figure 6A-B) was observed. There was significant tumor regression in the mice treated with Compound 1 (Figure 6A-B). Examination of the H & E stains by light microscopy revealed more dead cells in tumors treated with Compound 1 compared to controls (Figure 6C). Dead cells are depicted by cells that stained pink (shown by arrows) (Figure 6C). Live cells demonstrated in controls stained purple (Figure 6C). Computer based image analysis of the H & E slides was used to quantitate the percentage of dead cells within the tumor. There was a 100% increase in the amount of dead cells in the Compound 1 treated tumors vs controls (Figure 6D). This is the first BMP inhibitor demonstrating significant death of cancer cells in a xenograft model.

Compound 1 Induces Infiltration of Immune Cells

Immunohistochemisry demonstrated that Compoundl significantly increased the number of immune cells within the tumor microenvironment (Figure 7). Quantitative image analysis demonstrated that Compound 1 induced a 67% increase in CD3 cells, 80% increase in CD4, and a 70%) increase in CD8 cells in comparison to DMSO control (Figure 7). This is the first time a BMP inhibitor has been shown to increase the infiltration of immune cells into a tumor, suggesting a possible use with immunotherapy.

Discussion

Drugs targeting specific receptors are frequently only effective if that receptor has an activating mutation, which typically occur in less than 5% of cancers. Targeted therapy is also limited by the development of mutations that are not recognized by the drug or the receptor itself is deleted. Mutations of downstream effector genes can also render a drug inactive. Analysis supports that BMP signaling cascade is active in the majority of NSCLC and genetic alterations are not likely to induce resistance to small molecules targeting the BMP receptors. The BMP ligands and receptors expression are highly redundant in NCSLC. Over 10 different BMP ligands were expressed in NSCLC. BMP type I and type Π receptors were expressed in all of the NSCLC examined. It has been shown that all 3 of the type I BMP receptors can effectively induce downstream signaling in lung cancer cell lines. The mutation rate of the BMP receptors was low (<5%) and no tumor had mutations in all of the type I or type II BMP receptors. The very low incidence of amplification of XIAP, TAK1, or Idl suggests that these downstream effectors would not be a mechanism inducing resistance to BMP targeted therapy. Overall, these data suggest that targeting the BMP signaling cascade will not be limited by genetic alterations found in either squamous or adenocarcinomas of the lung.

There are only a few studies examining the effects of BMP receptor inhibitors in tumor xenografts. The BMP inhibitors most frequently used have been DMH1, DMH2, and LDN. Studies described herein have shown that DMH2 in vitro is significantly more potent than DMH1 or LDN in decreasing the downstream targets Idl, TAK1, and XIAP and inducing cell death of cancer cells. DMH1 and LDN in tumor xenograft studies decrease tumor growth and reduce metastasis but have not demonstrated tumor regression or significant death of cancer cells. DMH2 has a half-life of only 60 minutes with a low volume of distribution. DMH2 caused an increase in Idl expression in tumor xenografts likely from low level of suppression of BMP signaling allowing for activation of TAK1, which can cause a feed-forward activation of BMP signaling. Substituting a carbon for the oxygen on the morphine side-chain improved the stability of DMH2. The potency of Compound 1 to inhibit BMP receptors and regulate BMP signaling of cancer cells is very similar to that of DMH2. Although the volume of distribution was significantly better than that DMH2, which likely contributed to the improved anti-tumor effects in vivo.

Thus, it has been shown that a BMP inhibitor induces tumor regression and causes significant cell death in tumor xenografts in mice. This was associated with a downregulation of Idl and TAK1 but not XIAP. The binding of XIAP to the BMP receptors stabilizes XIAP leading to increased expression. XIAP can be stabilized by other pathways including its binding to survivin and phosphorylation by PI3 kinase. XIAP is an upstream activator of TAK1, which can phosphorylate Smad-1/5 leading to the activation of BMP signaling. The ability to downregulate XIAP is likely to further inhibit BMP signaling leading to greater cell death. Smac mimetics have been designed to bind and inactivate inhibitor of apoptosis proteins IAP-1, IAP-2, and XIAP. During apoptotic cell death smac is released from the mitochondria, which binds and inactivates inhibitor of apoptosis proteins. Combinational therapies utilizing inhibitors of survivin, PI-3 kinase, or smac mimetics may be a potential strategy to further enhance the downregulation of BMP signaling in cancer cells.

The immune system can induce or inhibit the growth of tumors. Immune cells within the tumor micronenvironment become "exhausted" by checkpoint blockade. Inhibitors of the immune blockade have demonstrated sustained tumor regression in lung and other tumors.

Therefore, the effects of cancer therapeutics on immune cells need to be carefully examined. Despite Compound 1 causing significant cell death of cancer cells it did not cause any death of immune cells. Depending on the type of immune cell, BMP signaling can induce differentiation and activation. Tumors treated with Compound 1 had a significant increase in the number of immune cells within the tumor microenvironment. The mechanism(s) by which this occurs is not known but may be secondary to an increase secretion of BMP2, which is a chemotactic factor. The presence of immune cells within the tumor microenvironment is reported to be the best predictor of PDL-1 blockade inducing a response. Further studies are needed to examine whether BMP inhibition can enhance the anti-tumor effects of check-point inhibitors.

BMP signaling is active in the majority of lung cancers and genetic mutations in NSCLC are unlikely to mitigate the effects of BMP receptor inhibitors. In addition, Compound 1 provides a useful tool to examine the mechanisms in vivo by which the BMP signaling regulates the survival of cancer cells and develop therapeutic strategies. Since Compound 1 induces the influx of immune cells into the tumor microenvironment, raises the possibility it can be used in conjunction with check-point inhibitors. These studies demonstrate that BMP signaling is growth promoting in cancer, which is targetable supporting the need for further drug

development and design of therapeutic strategies.

Thus, the site of chemical instability of DMH2 was identified and a compound

(Compound 1) was designed to circumvent the chemical hydrolysis of the morpholine side- chain. Compound 1 has similar inhibitory concentrations to BMP type I and type II receptors and in vitro to induces cell death and downregulates Idl, XIAP, and pTAKl with similar potency as DMH2. Compound 1 is more metabolically stable than DMH2, induced tumor regression and downregulates Idl and pTAKl in lung tumor xenografts, which demonstrates it is an uniquely improved compound.

Example 2. Compounds 2, 3, 4, 5, and 6 were prepared by similar procedures discussed in Example 1 for the preparation of Compound 1.

The compounds were analyzed by mass spectrometry (Table 4) and tested for inhibition of certain kinases (Table 5). Table 4

Example 3. The following illustrate representative pharmaceutical dosage forms, containing a compound of formula I or formula Π ('Compound X'), for therapeutic or prophylactic use in humans (i) Tablet 1 mg/tablet

Compound X= 100.0

Lactose 77.5

Povidone 15.0

Croscarmellose sodium 12.0

Microcrystalline cellulose 92.5

Magnesium stearate 3.0

300.0

The above formulations may be obtained by conventional procedures well known in the pharmaceutical art. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

1. A compound of formula Π:

wherein:

R¹ is H, halogen, or (Ci-C₄)alkyl;

R² is H, halogen, or (Ci-C₄)alkyl;

R³ is a 5-10 membered monocyclic or bicyclic heteroaryl, wherein the heteroaryl is optionally substituted with one or more halogen, (Ci-C₄)alkyl or -0(Ci-C₄)alkyl;

R⁴ is H, halogen or (Ci-Ce)alkyl;

L is (Ci-Ce)alkyl optionally substituted with one or more halogen; and

W is a 4-7 membered heterocyclyl optionally substituted with one or more halogen or (Ci- C₄)alkyl; or

a salt thereof.

2. The compound of claim 1, wherein R¹ is H.

3. The compound of claim 1 or claim 2, wherein R² is H.

4. The compound of any one of claims 1-3, wherein R⁴ is H.

5. The compound of any one of claims 1-4, wherein R³ is 5-membered monocyclic heteroaryl or 9-10-membered bicyclic heteroaryl, wherein the 5-membered monocyclic heteroaryl or 9-10-membered bicyclic heteroaryl is optionally substituted with one or more halogen, (Ci-C₄)alkyl or -0(Ci-C₄)alkyl.

6. The compound of any one of claims 1-4, wherein R³ is 5-membered monocyclic heteroaryl including 1 or 2 nitrogen atoms or 9-10-membered bicyclic heteroaryl including 1 or 2 nitrogen atoms, wherein the 5-membered monocyclic heteroaryl or 9-10-membered bicyclic heteroaryl is optionally substituted with one or more halogen, (Ci-C4)alkyl or -0(Ci-C4)alkyl.

7. The compound of any one of claims 1-4, wherein R³ is quinolinyl, indazolyl, or pyrazolyl, wherein the quinolinyl, indazolyl, or pyrazolyl is optionally substituted with one or more halogen, (Ci-C4)alkyl or -0(Ci-C4)alkyl.

8. The compound of any one of claims 1-4, wherein R³ is:

9. The compound of any one of claims 1-8, wherein L is (Ci-Ce)alkyl.

10. The compound of any one of claims 1-8, wherein L is ethyl or propyl.

11. The compound of any one of claims 1-8, wherein L is ethyl or propyl.

12. The compound of any one of claims 1-11, wherein W is a 6 membered heterocyclyl optionally substituted with one or more halogen or (Ci-C4)alkyl.

13. The compound of any one of claims 1-11, wherein W is a morpholinyl optionally substituted with one or more halogen or (Ci-C4)alkyl.

14. The compound of any one of claims 1-11, wherein W is a morpholin-4-yl.

15. The compound of claim 1 that is:

or a salt thereof.

16. The compound of claim 1 that is

or a salt thereof.

17. The compound of any one of claims 1-16, wherein the salt is a pharmaceutically acceptable salt.

18. A pharmaceutical composition comprising the compound of any one of claims 1-16 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

19. A method for treating cancer in a mammal in need thereof, comprising administering to the mammal the compound of any one of claims 1-16 or a pharmaceutically acceptable salt thereof.

20. The method of claim 19, wherein the cancer is lung cancer, breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, melanoma, or sarcoma.

21. The method of claim 19, wherein the cancer is lung cancer.

22. The method of claim 19, wherein the cancer is non-small cell lung cancer.

23. The method of any one of claims 19-22, further comprising administering to the patient one or more additional chemotherapeutic agents.

24. The method of any one of claims 19-22, further comprising administering to the patient one or more additional agents selected from inhibitors of survivin, inhibitors of PI-3 kinase, and smac mimetics.

25. A method for downregulating one or more anti-apoptotic proteins in a mammal in need thereof comprising administering to the mammal the compound of any one of claims 1-16 or a pharmaceutically acceptable salt thereof.

26. The method of claim 25, wherein the anti-apoptotic proteins are selected from XIAP, TAK1 and ldl/ld3.

27. A method for inducing an influx of immune cells into a tumor cell in vitro or in vivo, comprising contacting the cell with the compound of any one of claims 1-16 or a salt thereof.

28. A method for inducing an influx of immune cells into a mammal, comprising

administering to the mammal the compound of any one of claims 1-16 or a pharmaceutically acceptable salt thereof.

29. A compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-16 for use in medical treatment.

30. A compound or a pharmaceutically acceptable salt thereof as described in of any one of claims 1-16 for the prophylactic or therapeutic treatment of cancer.

31. The use of a compound or a pharmaceutically acceptable salt thereof as described in any one of any one of claims 1-16 for the preparation of a medicament for treating cancer.