COMPOUNDS AND METHODS FOR THE TREATMENT OF CANCER STEM

CELLS

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.

61/527,930, filed on August 26, 201 1. The entire teachings of the above application is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under U54 HG005032 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer stem cells (CSCs), which drive tumor growth, are known to be resistant to standard chemotherapy and radiation treatment. (Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006). However, since CSCs generally comprise only small minorities within cancer cell populations, standard high-throughput cell viability assays applied to bulk populations of cancer cells cannot identify agents with CSC-specific toxicity. (Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008 May 16; 133(4):704-15: Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009 Aug 21; 138(4):645-59). CSCs are resistant to many current cancer treatments, including chemo- and radiation therapy. This suggests that many cancer therapies, while killing the bulk of tumor cells, may ultimately fail because they do not eliminate CSCs, which survive to regenerate new tumors. As such, there is a very significant unmet need to find therapies that can target CSCs within tumors because these cells are responsible for recurrence, the primary cause of patient mortality. SUMMARY OF THE INVENTION

The invention relates to compounds of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof:

"SUBSTITUTE SHEET"

Formula I

Wherein n is 0, 1, 2, 3, 4 or 5;

Gi is absent or a linker;

G ₂ is -C(R ₁₀ )(Rii)-, - Rio-, -S-, -0-;

Wherein each R ₁₀ and Rn is independently absent, hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl;

G ₃ is -CRio-, -NRio-, -S0 ₂ -, -C=C-, -S- or -O-;

Each Ri is absent, hydrogen, halogen, -OR ₂₀ , -SR ₂₀ , -NR ₂₀ R ₂ i, -CF ₃ , -CN, -N0 ₂ , -N ₃ , - C(O)OR ₂₀ , -C(O)R ₂₀ , -C(0)N R ₂₀ R ₂ i, -S(O)R ₂₀ , -S(O)NR ₂₀ , -S(O) ₂ R ₂₀ , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of Ri groups together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring;

Wherein each R ₂₀ and R ₂ i is independently hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl; and,

R ₂ is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic or substituted heterocyclic.

The invention further relates to the treatment of cancer comprising the step of administering a compound of Formula IA to a patient in need thereof:

R ₂ -Gi-Cyi (Formula IA)

Wherein R ₂ and Gi are as defined above; and,

Cyi is a an aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic or substituted heterocyclic.

The invention further relates to the treatment of cancer comprising the step of administering a compound of Formula I or IA to a patient in need thereof. In one embodiment, the cancer is an epithelial cancer. In one embodiment, the cancer is a lung, breast, prostate, gastric, colon, pancreatic, sarcoma, brain, esophagus, kidney, gastrointestinal, urogenital, medulloblastoma, T-cell acute lymphoblastic leukemia,

retinoblastoma, glioma or melanoma cancer. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

Figure 1A-C: Heat map comparisons of differential gene expression in cells treated with Compound-27: Genes that displayed greater than a 2-fold difference in expression were identified and plotted in a heat map, and the gene expression in all six conditions was compared.

Figure 2A and B: Heat Map Comparisons of Differential Gene Expression. Genes that displayed greater than 2-fold difference in expression were identified and plotted in a heat map, and the gene expression in all six conditions was compared.

HMLE_shECad_DMSO vs. HMLE_shGFP(A), HMLE_shECad(shECad)_DMSO versus HMLE_shECAD_CMPD(B). The triplicate samples (A, B, and C) are indicated. In panel B, "— " indicates where DMSO was added and "++" indicates where 2 μΜ of Compound- 62 was added to the cells.

Figure 3 A and B: Heat Map Comparisons of Differential Gene Expression. Genes that displayed greater than 2-fold difference in expression were identified and plotted in a heat map, and the gene expression in all six conditions was compared.

HMLE_shECad_DMSO vs. HMLE_shGFP(A), HMLE_shECad(shECad)_DMSO versus HMLE_shECAD_CMPD(B). The triplicate samples (A, B, and C) are indicated. In panel B, "— " indicates where DMSO was added and "++" indicates where 2 μΜ of Compound- 124 was added to the cells.

Figures 4A-C: Dose curve for Compound-27 (A) or Salinomycin (B and C) inhibition of HMLE_sh_eGFP, HMLE_sh_Ecad , and HMLE_sh_Twist.

Figure 5: Dose Inhibition Assay of HMLE_sh_eGFP, HMLE_sh_Ecad, and HMLE_sh_Twist by Compound-62. Figure 6: Dose Inhibition Assay of HMLE_sh_eGFP, HMLE_sh_Ecad, and HMLE_sh_Twist by Compound- 124.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compounds of Formula I or a pharmaceutically acceptable salt, ester or prodrug thereof:

Formula I

wherein n is 0, 1, 2, 3, 4 or 5;

Gi is absent or a linker;

G ₂ is -C(R ₁₀ )(Rii)-, - Rio-, -S-, -0-;

Wherein each R ₁₀ and Rn is independently absent, hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl;

G ₃ is -CRio-, -NRio-, -S0 ₂ -, -C=C-, -S- or -0-;

Each Ri is absent, hydrogen, halogen, -OR ₂₀ , -SR ₂₀ , -NR ₂₀ R ₂ i, -CF ₃ , -CN, -N0 ₂ , -N ₃ , - C(O)OR ₂₀ , -C(O)R ₂₀ , -C(0)N R ₂₀ R ₂ i, -S(O)R ₂₀ , -S(O)NR ₂₀ , -S(O) ₂ R ₂₀ , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of Ri groups together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring;

Wherein each R ₂₀ and R ₂ i is independently hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl; and,

R ₂ is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic or substituted heterocyclic.

In a preferred embodiment, the invention relates to a compound of Formula II or III or a pharmaceutically acceptable salt, ester or prodrug thereof:

Formula II

wherein m is 0, 1, 2, 3 or 4.

In a more preferred embodiment, the invention relates to a compound of Formula IV or a pharmaceutically acceptable salt, ester or prodrug thereof:

Formula IV Formula V

X is -S- or -0-;

Wherein s is 0, 1, 2, 3, 4 or 5;

do is -C(R ₁₀ )(Rii)-, -NR ₁₀ -, -S-, or -0-;

Gn is -0-, -S-, -[C(R ₁₀ )(Rii)]r -N(R ₁₀ )-, -N(R ₁₀ )[C(R ₁₀ )(Rii)]t, -O[C(R ₁₀ )(Rii)]r,

Each R ₃ is independently absent, hydrogen, halogen, -OR20, -SR20, -NR20R21, -CF ₃ , -CN, -NO2, -N _3> -C(O)OR ₂₀ , -C(O)R ₂₀ , -C(0)N R20R21, -S(O)R ₂₀ , -S(O)NR ₂₀ , - S(0)2R2o, acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of R3 groups together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring; and,

G ₅ is -0-, -S-, -[C( io)( n)]t- -N(R ₁₀ )-, -N(R ₁₀ )[C(R ₁₀ )(Rii)]t, -O[C(R ₁₀ )(Rii)]r, or - S[C(Rio)(Rn)]t-; Alternatively two Rio or two Rn groups together with the atoms to which they are attached may for an optionally substituted ring.

In a preferred embodiment, R2 is selected from:

wherein each p and g is independently 0, 1, 2, 3, 4, or 5;

Each R12 and R13 is independently hydrogen, halogen, -OR20, -SR20, -NR20R21, -CF ₃ , -CN, -NO2, -N _3> -C(O)OR ₂₀ , -C(O)R ₂₀ , -C(0)N R _{20 2} i, -S(O)R ₂₀ , -S(O)NR ₂₀ , -S(0) ₂ R ₂ o, acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of R12 groups together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring.

In one embodiment, Gi is a direct bond or straight- or branched-, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,

alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,

alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl,

alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl,

alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, or alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO ₂ , N(R«), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, wherein Rs is hydrogen, aliphatic, substituted aliphatic, aryl or substituted aryl.

In one embodiment, Gi is selected from alkyl, alkenyl, alkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkoxyaryl, alkylaminoaryl, alkoxyalkyl, alkylaminoalkyl, alkylheterocycloalkyl, alkylheteroarylalkyl, alkylamino, N(Rs)alkenyl, N(Rs)alkynyl, N(R ₈ )alkoxyalkyl, N(R ₈ )alkylaminoalkyl, N(R ₈ )alkylaminocarbonyl, N(R ₈ )alkylaryl, N(Rs)alkenylaryl, N(R ₈ )alkynylaryl, N(Rs)alkoxyaryl, N(R ₈ )alkylaminoaryl,

N(R ₈ )cycloalkyl, N(R ₈ )aryl, N(R ₈ )heteroaryl, N(R ₈ )heterocycloalkyl,

N(R ₈ )alkylheterocycloalkyl, alkoxy, O-alkenyl, O-alkynyl, O-alkoxyalkyl, O- alkylaminoalkyl, O-alkylaminocarbonyl, O-alkylaryl, O-alkenylaryl, O-alkynylaryl, O- alkoxyaryl, O-alkylaminoaryl, O-cycloalkyl, O-aryl, O-heteroaryl, O-heterocycloalkyl, O- alkylheterocycloalkyl, C(0)alkyl, C(0)-alkenyl, C(0)alkynyl, C(0)alkylaryl,

C(0)alkenylaryl, C(0)alkynylaryl, C(0)alkoxyalkyl, C(0)alkylaminoalkyl,

C(0)alkylaminocarbonyl, C(0)cycloalkyl, C(0)aryl, C(0)heteroaryl,

C(0)heterocycloalkyl, CON(R ₈ ), CON(R ₈ )alkyl, CON(R ₈ )alkenyl, CON(R ₈ )alkynyl, CON(R ₈ )alkylaryl, CON(R ₈ )alkenylaryl, CON(R ₈ )alkynylaryl, CON(R ₈ )alkoxyalkyl, CON(R ₈ )alkylaminoalkyl, CON(R ₈ )alkylaminocarbonyl, CON(R ₈ )alkoxyaryl,

CON(R ₈ )alkylaminoaryl, CON(R ₈ )cycloalkyl, CON(R ₈ )aryl, CON(R ₈ )heteroaryl, CON(R ₈ )heterocycloalkyl, CON(R ₈ )alkylheterocycloalkyl, N(R ₈ )C(0)alkyl,

N(R ₈ )C(0)alkenyl, N(R ₈ )C(0)- alkynyl, N(R ₈ )C(0)alkylaryl, N(R ₈ )C(0)alkenylaryl, N(R ₈ )C(0)alkynylaryl, N(R ₈ )C(0)alkoxyalkyl, N(R ₈ )C(0)alkylaminoalkyl,

N(R ₈ )C(0)alkylaminocarbonyl, N(R ₈ )C(0)alkoxyaryl, N(R ₈ )C(0)alkylaminoaryl, N(R ₈ )C(0)cycloalkyl, N(R ₈ )C(0)aryl, N(R ₈ )C(0)heteroaryl, N(R ₈ )C(0)heterocycloalkyl, N(R ₈ )C(0)alkylheterocycloalkyl, NHC(0)NH, NHC(0)NH-alkyl, NHC(0)NH-alkenyl, NHC(0)NH-alkynyl, NHC(0)NH-alkylaryl, NHC(0)NH-alkenylaryl, NHC(0)NH- alkynylaryl, NHC(0)NH-alkoxyaryl, NHC(0)NH-alkylaminoaryl, NHC(0)NH- cycloalkyl, NHC(0)NH-aryl, NHC(0)NH-heteroaryl, NHC(0)NH-heterocycloalkyl, NHC(0)NH-alkylheterocycloalkyl, S-alkyl, S-alkenyl, S-alkynyl, S-alkoxyalkyl, S- alkylaminoalkyl, S-alkylaryl, S-alkylaminocarbonyl, S-alkylaryl, S-alkynylaryl, S- alkoxyaryl, S-alkylaminoaryl, S-cycloalkyl, S-aryl, S-heteroaryl, S-heterocycloalkyl, S- alkylheterocycloalkyl, S(0)alkyl, S(0)alkenyl, S(0)alkynyl, S(0)alkoxyalkyl,

S(0)alkylaminoalkyl, S(0)alkylaminocarbonyl, S(0)alkylaryl, S(0)alkenylaryl,

S(0)alkynylaryl, S(0)alkoxyaryl, S(0)alkylaminoaryl, S(0)cycloalkyl, S(0)aryl, S(0)heteroaryl, S(0)heterocycloalkyl, S(0)alkylheterocycloalkyl, S(0) ₂ alkyl,

S(0) ₂ alkenyl, S(0) ₂ alkynyl, S(0) ₂ alkoxyalkyl, S(0) ₂ alkylaminoalkyl,

S(0) ₂ alkylaminocarbonyl, S(0) ₂ alkylaryl, S(0) ₂ alkenylaryl, S(0) ₂ alkynylaryl,

S(0) ₂ alkoxyaryl, S(0) ₂ alkylaminoaryl, S(0) ₂ cycloalkyl, S(0) ₂ aryl, S(0) ₂ heteroaryl, S(0) ₂ heterocycloalkyl, S(0) ₂ alkylheterocycloalkyl, S0 ₂ NH, S0 ₂ NH-alkyl, S0 ₂ NH- alkenyl, S0 ₂ NH-alkynyl, S0 ₂ NH-alkylaryl, S0 ₂ NH-alkenylaryl, S0 ₂ NH-alkynylaryl, S0 ₂ NH-cycloalkyl, S0 ₂ NH-aryl, S0 ₂ NH-heteroaryl, S0 ₂ NH-heterocycloalkyl, S0 ₂ NH- alkylheterocycloalkyl, alkylaryloxyalkoxy, alkylaryloxyalkylamino,

alkylarylaminoalkoxy, alkylarylaminoalkylamino, alkylarylalkylaminoalkoxy, alkylarylalkylaminoalkoxy, alkenylaryloxyalkoxy, alkenylaryloxyalkylamino, alkenylarylaminoalkoxy, alkenylarylaminoalkylamino, alkenylarylalkylaminoalkoxy, alkenylarylalkylaminoalkylamino.

In a more preferred embodiment, Gi is selected from -0-, -S-, -N(Rio)C(0)-, - C(R ₁₀ )=C(R _n )-, -C(R ₁₀ )=C(R ₁₁ )C(O)N(R ₁₀ )- and -C=N-N(R ₁₀ )C(O)C(R ₁₀ )(Rn)O-.

The invention further relates a compound of Formula IA or a pharmaceutically acceptable salt, ester or prodrug thereof, and the treatment of cancer comprising the step of administering a compound of Formula IA to a patient in need thereof:

R ₂ -Gi-Cyi (Formula IA)

Wherein R ₂ and Gi are as defined above; and,

Cyi is a an aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic or substituted heterocyclic.

In a preferred embodiment, Cyi is selected from:

Wherein G ₇ is -C(Ri ₀ )( n)-, -CRi ₀ =CRn-, - R ₁₀ -, -S-, or -0-; and, Each Gs and Gg is independently selected from -N- and -C-.

In a preferred embodiment, the invention relates to a compound of Table A-C, or a pharmaceutically acceptable salt, ester or prodrug thereof:

NA

NA

0.52

0.19

NA

7.83

0.58

>50 >50

NA

>50

2.92

3.79

>50

>50

2.71 >50

>50

0.95

>50

NA

NA

NA

23.8 

NA

1.22

2.73

5.96

4.49

>50

NA

2.38

98. I I 6.74

99. I II 6.96

100. I II 6.93

TABLE C 

NA

NA

NA

NA

NA

NA

NA NA

NA

NA

NA

NA

The pharmacological activity of compounds is presented for compounds of Tables A-C above. Their activity is characterized as I (Ο.ΟΟΟΙμΜ <10μΜ), II (ΙΟμΜ <50μΜ), III (50μΜ <100μΜ) and IV (>100μΜ). A represents no measured activity.

In one embodiment, the invention provides for the use of one or more compounds of the invention for halting or decreasing diseases involving aberrant proliferation, differentiation, or survival of cells. In one embodiment, the invention relates to a method of treating cancer in a subject in need of treatment comprising administering to said subject a therapeutically effective amount of a compound of the invention. The invention further relates to the treatment of cancer comprising the step of administering a compound of Formula I or IA to a patient in need thereof. In one embodiment, the cancer is an epithelial cancer. In one embodiment, the cancer is a lung (non-small cell lung cancer ( SCLC) or small-cell lung cancer (SCLC)), breast, prostate, gastric, colon, colorectal, pancreatic, sarcoma, brain, esophagus, stomach, kidney, gastrointestinal, urogenital, ovarian, medulloblastoma, T-cell acute lymphoblastic leukemia, hepatocellular, retinoblastoma, glioma, malignant pleural or peritoneal mesothelioma, or melanoma cancer. In one embodiment, the cancer is a blood borne metastases.

In one embodiment, the invention provides for the use of one or more compounds of the invention for the treatment of cancer where overexpression of one or more genes from Table 1 is present. Overexpression of genes in Table 1 (or any suitable subset thereof) indicates an increased likelihood that the epithelial cancer will be resistant to standard-of-care therapies such as paclitaxel but sensitive to a cancer stem-cell selective agent ("CSS agent"). As such, in one embodiment, the invention provides a method of cancer therapy where overexpression of one or more of the genes from Table 1 is present, by administering a compound of Formula I, IA, II, III, IV or V to a subject in need thereof. In a preferred embodiment, a compound according to Formula I, IA, II, III, IV or V, preferably a compound according to Table A-C is administered to a patient. Moreover, in various embodiments, the subset of genes may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the genes in Table 1.

TABLE 1. Genes identified that are over-expressed in cancer populations having undergone an EMT, relative to cancer populations that have not undergone an EMT.

Mean Fold OverExpression

Symbol Description GenBank Upon EMT

DCN Decorin AF138300 137.6156 collagen, type III, alpha 1 (Ehlers-Danlos

COL3A1 syndrome type IV, autosomal dominant) AU144167 132.1195

COL1A2 collagen, type I, alpha 2 AA788711 88.05054

FBN1 fibrillin 1 (Marfan syndrome) NM_000138 76.51337 gremlin 1, cysteine knot superfamily, homolog

GREM1 (Xenopus laevis) NM_013372 75.35859

POSTN periostin, osteoblast specific factor D13665 73.18114

NIDI nidogen 1 BF940043 51.91502

FBLN5 fibulin 5 NM_006329 34.4268 syndecan 2 (heparan sulfate proteoglycan 1, cell

SDC2 surface-associated, fibroglycan) AL577322 32.48001

COL5A2 collagen, type V, alpha 2 NM_000393 26.66545

PRG1 proteoglycan 1, secretory granule J03223 23.46014 transcription factor 8 (represses interleukin 2

TCF8 expression) AI806174 22.83413 ectonucleotide

pyrophosphatase/phosphodiesterase 2

ENPP2 (autotaxin) L35594 22.72739 nuclear receptor subfamily 2, group F, member

NR2F1 1 AI951185 20.64471

COL6A1 collagen, type VI, alpha 1 AA292373 17.36271

RGS4 regulator of G-protein signalling 4 AL514445 16.63788 CDH11 cadherin 11 , type 2, OB-cadherin (osteoblast) D21254 16.61483

PRRX1 paired related homeobox 1 NM_006902 14.73362

OLFML3 olfactomedin-like 3 NM_020190 14.0984 sparc/osteonectin, cwcv and kazal-like domains

SPOCK proteoglycan (testican) AF231124 13.99112 wingless-type MMTV integration site family,

WNT5A member 5A NM_003392 13.33384

MAP IB microtubule-associated protein IB AL523076 13.0877

BG109855 12.44401 pentraxin-related gene, rapidly induced by IL-1

PTX3 beta NM_002852 12.01196

C5orfl3 chromosome 5 open reading frame 13 U36189 11.95863

IGFBP4 insulin-like growth factor binding protein 4 NM_001552 11.09963

PCOLCE procollagen C-endopeptidase enhancer NM_002593 11.04575

TNFAIP6 tumor necrosis factor, alpha-induced protein 6 NM_007115 11.02984

LOC51334 NM_016644 10.91454 cytochrome P450, family 1, subfamily B,

CYP1B1 polypeptide 1 NM_000104 10.47429 tissue factor pathway inhibitor (lipoprotein-

TFPI associated coagulation inhibitor) BF511231 10.42648

PVRL3 poliovirus receptor-related 3 AA129716 10.30262

ROR1 receptor tyrosine kinase-like orphan receptor 1 NM_005012 10.10474

FBLN1 fibulin 1 NM_006486 10.09844

BIN1 bridging integrator 1 AF043899 9.928529 LUM Lumican NM_002345 9.727574 ral guanine nucleotide dissociation stimulator¬

RGL1 like 1 AF 186779 9.643922

PTGFR prostaglandin F receptor (FP) NM_000959 8.939536 transforming growth factor, beta receptor III

TGFBR3 (betaglycan, 300kDa) NM_003243 8.838

COL1A1 collagen, type I, alpha 1 Y15916 8.667645

DLC1 deleted in liver cancer 1 AF026219 8.610518

PMP22 peripheral myelin protein 22 L03203 8.560648

PRKCA protein kinase C, alpha AI471375 8.338108 matrix metallopeptidase 2 (gelatinase A, 72kDa

MMP2 gelatinase, 72kDa type IV collagenase) NM_004530 8.268926

CTGF connective tissue growth factor M92934 8.168776

CDH2 cadherin 2, type 1, N-cadherin (neuronal) M34064 7.987921 guanine nucleotide binding protein (G protein),

GNG11 gamma 11 NM_004126 7.953115

PPAP2B phosphatidic acid phosphatase type 2B AA628586 7.907272

NEBL Nebulette AL157398 7.817894

MYL9 myosin, light polypeptide 9, regulatory NM_006097 7.780485 potassium large conductance calcium-activated

KCNMA1 channel, subfamily M, alpha member 1 AI129381 7.747227

IGFBP3 insulin-like growth factor binding protein 3 BF340228 7.57812

CSPG2 chondroitin sulfate proteoglycan 2 (versican) NM_004385 7.318764

SEMA5A NM_003966 7.298702 sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain

(TM) and short cytoplasmic domain,

(semaphorin) 5A

Cbp/p300-interacting transactivator, with

CITED2 Glu/Asp-rich carboxy-terminal domain, 2 AF109161 7.220907 membrane metallo-endopeptidase (neutral

MME endopeptidase, enkephalinase, CALLA, CD 10) AI433463 7.05859

DOCK10 dedicator of cytokinesis 10 NM_017718 6.972809

DNAJB4 DnaJ (Hsp40) homolog, subfamily B, member 4 BG252490 6.782043

PCDH9 protocadherin 9 AI524125 6.711987

NID2 nidogen 2 (osteonidogen) NM_007361 6.54739

HAS2 hyaluronan synthase 2 NM_005328 6.520398

PTGER4 prostaglandin E receptor 4 (subtype EP4) AA897516 6.396133

TRAM2 translocation associated membrane protein 2 AI986461 6.275542

SYT11 synaptotagmin XI BC004291 6.149546

BGN Biglycan AA845258 5.838023

CYBRD1 cytochrome b reductase 1 NM_024843 5.710828

CHN1 chimerin (chimaerin) 1 BF339445 5.687127

DPT Dermatopontin AI146848 5.573023 integrin, beta-like 1 (with EGF-like repeat

ITGBL1 domains) AL359052 5.511939

FLJ22471 NM_025140 5.364784

LOC22136

2 AL577024 5.35364 MLPH Melanophilin NM_024101 5.296062

ANXA6 annexin A6 NM_001155 5.18628 ecmnoderm microtubule associated protein like

EML1 1 NM_004434 5.138332 cAMP responsive element binding protein 3-

CREB3L1 like 1 AF055009 5.073214

FLJ 10094 NM_017993 4.998863 leucine-rich repeats and immunoglobulin-like

LRIG1 domains 1 AB050468 4.9963

SNED1 sushi, nidogen and EGF-like domains 1 N73970 4.993945 serpin peptidase inhibitor, clade F (alpha-2

antiplasmin, pigment epithelium derived factor),

SERPINF1 member 1 NM_002615 4.969153 disabled homolog 2, mitogen-responsive

DAB2 phosphoprotein (Drosophila) NM_001343 4.913939

Wiskott-Aldrich syndrome protein interacting

WASPIP protein AW058622 4.882974

FN1 fibronectin 1 AJ276395 4.869319

C10orf56 chromosome 10 open reading frame 56 AA131324 4.795629

DAPK1 death-associated protein kinase 1 NM_004938 4.726984

LOXL1 lysyl oxidase-like 1 NM_005576 4.720305 inhibitor of DNA binding 2, dominant negative

ID2 helix-loop-helix protein NM_002166 4.672064 prostaglandin E receptor 2 (subtype EP2),

PTGER2 53kDa NM_000956 4.427892 COL8A1 collagen, type VIII, alpha 1 BE877796 4.38653

DDR2 discoidin domain receptor family, member 2 NM_006182 4.338932

SEPT6 septin 6 D50918 4.30699

HRASLS3 HRAS-like suppressor 3 BC001387 4.281926 pleckstrin homology domain containing, family

PLEKHC1 C (with FERM domain) member 1 AW469573 4.272913

THY1 Thy-1 cell surface antigen AA218868 4.253587 ribosomal protein S6 kinase, 90kDa,

RPS6KA2 polypeptide 2 AI992251 4.225143

GALC galactosylceramidase (Krabbe disease) NM_000153 4.222742 fibrillin 2 (congenital contractural

FBN2 arachnodactyly) NM_001999 4.205916

FSTL1 follistatin-like 1 BC000055 4.175243

NRP1 neuropilin 1 BE620457 4.162874

TNS1 tensin 1 AL046979 4.131713

TAGLN Transgelin NM_003186 4.131083 cyclin-dependent kinase inhibitor 2C (pi 8,

CDKN2C inhibits CDK4) NM_001262 4.124788

MAGEH1 melanoma antigen family H, 1 NM_014061 4.094423 latent transforming growth factor beta binding

LTBP2 protein 2 NM_000428 4.000998

PBX1 pre-B-cell leukemia transcription factor 1 AL049381 3.997339

TBX3 T-box 3 (ulnar mammary syndrome) NM_016569 3.992244 Previous work has shown that agents that selectively target cells induced into EMT also selectively kill cancer stem cells. (Gupta et. al, Cell, 2009) Since cancer cells induced into EMT are also highly invasive, the hypothesis is that anti-cancer therapies that target invasive and/or metastatic cancer cells are likely to also target cancer cells induced into EMT.

According to one embodiment, the invention provides a method for treating patient subpopulations that harbor tumors responsive to three classes of essentially overlapping anti-cancer therapies or treatments— i.e., (a) therapies that target invasive/metastatic cells, (b) therapies that target cancer stem cells and (c) therapies that target cells post-EMT. Specifically, the invention provides methods for treating cancer patient subpopulations that express genetic biomarkers that are upregulated in cancer cells post-EMT (Table 1).

In still one embodiment, the invention provides compounds of Formula I, IA, II, III, rv or V that target cancer stem cells or epithelial cancers that have undergone an epithelial to mesenchymal transition wherein administration of a compound of I, IA, II, III, IV or V decreases the levels of expression of at least one of the genes in Table 1.

In another embodiments, the invention provides methods of treating a patient following surgical removal of a primary tumor comprising the step of administering a compound of I, IA, II, III, IV or V. In one embodiment, the expression level in cancer of at least one gene in Table 1 is increased before, during or after the removal of the primary tumor. Preferably, the subset of the genes whose expression is evaluated is one for which a statistical test demonstrates that the genes in the subset are differentially expressed in populations treated with a cancer therapy less than 0.1, relative to an appropriate control population (e.g., DMSO treatment). Those skilled in the art will recognize that the subset of genes can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the genes in Table 1.

The invention further relates to a method of reducing the rate of tumor seeding in a subject comprising the step of administering a compound according to the invention a subject in need thereof. The invention further relates to a method of reducing or treating metastatic nodule-forming in a subject comprising the step of administering a compound according to the invention to a subject in need thereof.

In one embodiment, the invention relates to the administration of a compound of Formula I, IA, II, III, IV or V in combination with a second pharmacological agent for the treatment of a cell proliferative disease. In one embodiment, the second pharmacological agent is selected from vatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-1 1248, SU-14813, AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 or GW- 786034, gefitinib, erlotinib, HKI-272, CI-1033 or GW-2016, iressa (ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, herceptin, BAY-43-9006, BAY-57-9006, atrasentan, rituximab, cetuximab, bevacizumab, bivatuzumab mertansine, IMC-1C11, erbitux (C- 225), DC-101, EMD-72000, vitaxin, imatinib or dasatinib, VEGFtrap, melphalan, cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin, oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin, streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide, streptozocin, thiotepa, chlorambucil, mechlorethamine, an ethyleneimine compound, an alkylsulphonate, daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil), epirubicin, idarubicin, mitoxantrone, amsacrine, dactinomycin, distamycin or a derivative thereof, netropsin, pibenzimol, mitomycin, CC- 1065, a duocarmycin, mithramycin, chromomycin, olivomycin, propamidine or stilbamidine, an anthramycin, an aziridine, a nitrosourea or a derivative thereof, cytarabine, 5-fluorouracile (5-FU), pemetrexed, tegafur/uracil, uracil mustard, fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin, hydroxyurea, or folic acid, a phleomycin, a bleomycin or a derivative or salt thereof, CHPP, BZPP, MTPP, BAPP, liblomycin, an acridine or a derivative thereof, a rifamycin, an actinomycin, adramycin, a camptothecin such as irinotecan (camptosar) or topotecan, an amsacrine or analogue thereof, a tricyclic carboxamide, an

histonedeacetylase inhibitor such as SAHA, MD-275, trichostatin A, CBHA, LAQ824, or valproic acid, an anti-cancer drug from plants such as paclitaxel (taxol), docetaxel or taxotere, navelbine, vinblastin, vincristin, vindesine, vinorelbine, colchicine or a derivative thereof, maytansine, an ansamitocin or rhizoxin, phomopsin, dolastatin, an

epipodophyllotoxin or a derivative of podophyllotoxin, etoposide, teniposide, a steganacin, combretastatin, amphetinile, procarbazine, bortezomib, asparaginase, pegylated asparaginase (pegaspargase), a thymidine-phosphorylase inhibitor, a gestagen, an estrogen, estramustine (T-66), megestrol, an anti-androgen, flutamide, casodex, anandron or cyproterone acetate, aminogluthetimide, anastrozole, formestan, exemestane, letrozole, leuprorelin, buserelin, goserelin, triptorelin, an anti-estrogen, tamoxifen or its citrate salt, droloxifene, trioxifene, raloxifene, zindoxifene, an estrogen receptor antagonist such as fulvestrant, a derivative of 17. beta. -estradiol, ICI 164,384, ICI 182,780,

aminoglutethimide, formestane, fadrozole, finasteride, ketoconazole, a LH-RH antagonist, leuprolide, a steroid, prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, fluocortolone, triamcinolone, interferon .beta., IL-10, IL-12, an anti- TNF. alpha, antibody, etanercept, TNF-. alpha, (tasonermin), thalidomide and its R- and S- enantiomers and its derivatives, revimid (CC-5013), a leukotrien antagonist, mitomycin C, BMY-42355, AZQ or EO-9, a 2-nitroimidazole misonidazole, NLP-1 or NLA-1, a nitroacridine, a nitroquinoline, a nitropyrazoloacridine, RSU-1069, RB-6145, CB-1954, nitromin, an anti-CD3 or anti-CD25 antibody, a tolerance induction agent, minodronic acid and its derivatives (YM-529, Ono-5920, YH-529), zoledronic acid monohydrate, ibandronate sodium hydrate, clodronate disodium, metronidazole, misonidazole, benznidazole, nimorazole, RSU-1069, SR-4233, bromodeoxyuridine, iododeoxyuridine, WR-2721, porfimer, photofrin, a benzoporphyrin derivative, a pheophorbide derivative, merocyanin 540 (MC-540), tin etioporpurin, an ant-template, an anti-sense RNA or DNA, oblimersen, a non-steroidal inflammatory drug, acetylsalicyclic acid, mesalazin, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, nifluminic acid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam, lornoxicam, nimesulide, meloxicam, celecoxib, rofecoxib, a pharmaceutically acceptable salt of a non-steroidal inflammatory drug, a cytotoxic antibiotic, an antibody targeting the surface molecules of cancer cells, apolizumab, 1D09C3, TIMP-1, TIMP-2, Zinc, an inhibitor of oncogenes, P53, heterocyclic complexes of lanthanides, PUVA, an inhibitor of the transcription factor complex ESX/DRIP 130/Sur-2, an inhibitor of HER-2 expression, the heat shock protein HSP90 modulator geldanamycin and its derivative 17- allylaminogeldanamycin or 17-AAG, therapeutic agent selected from IM-842,

tetrathiomolybdate, squalamine, combrestatin A4, TNP-470, marimastat, neovastat, bicalutamide, abarelix, oregovomab, mitumomab, TLK-286, alemtuzumab, ibritumomab, temozolomide, denileukin diftitox, aldesleukin, dacarbazine, floxuridine, plicamycin, mitotane, pipobroman, plicamycin, tamoxifen and testolactone. ABBREVIATIONS

Abbreviations which may appear in the synthetic schemes and examples are:

Ac for acetyl;

Alloc for allyloxycarbonyl;

Boc for tert-butoxycarbonyl; DCM for dichloromethane;

DMF for dimethyl formamide;

DMSO for dimethyl sulfoxide;

EtOAc for ethyl acetate;

iPr for isopropyl;

IPA for isopropyl alcohol;

MeOH for methanol;

TEA for triethylamine; and

TFA for trifluoroacetic acid.

Definitions

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

The term "aliphatic group" or "aliphatic" refers to a non-aromatic moiety that may be saturated (e.g. single bond) or contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched or cyclic, contain carbon, hydrogen or, optionally, one or more heteroatoms and may be substituted or unsubstituted. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted. It is understood that aliphatic groups may include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and substituted or unsubstituted cycloalkyl groups as described herein.

The term "acyl" refers to a carbonyl substituted with hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl, or heteroaryl. For example, acyl includes groups such as (Ci-Ce) alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C ₃ - C6)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl,

cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g.,

pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl (e.g., thiophenyl-2- carbonyl, thiophenyl-3 -carbonyl, furanyl-2-carbonyl, furany 1-3 -carbonyl, lH-pyrroyl-2- carbonyl, lH-pyrroyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be any one of the groups described in the respective definitions. When indicated as being "optionally substituted", the acyl group may be unsubstituted or optionally substituted with one or more substituents (typically, one to three substituents) independently selected from the group of substituents listed below in the definition for "substituted" or the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be substituted as described above in the preferred and more preferred list of substituents, respectively.

The term "alkyl" is intended to include both branched and straight chain, substituted or unsubstituted saturated aliphatic hydrocarbon radicals/groups having the specified number of carbons. Preferred alkyl groups comprise about 1 to about 24 carbon atoms ("C1-C24"). Other preferred alkyl groups comprise at about 1 to about 8 carbon atoms ("Ci-Cs") such as about 1 to about 6 carbon atoms ("C1-C6"), or such as about 1 to about 3 carbon atoms ("C1-C3"). Examples of C1-C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, w-butyl, tert-butyl, n-pentyl, neopentyl and n- hexyl radicals.

The term "alkenyl" refers to linear or branched radicals having at least one carbon- carbon double bond. Such radicals preferably contain from about two to about twenty- four carbon atoms ("C2-C24"). Other preferred alkenyl radicals are "lower alkenyl" radicals having two to about ten carbon atoms ("C2-C1 ₀ ") such as ethenyl, allyl, propenyl, butenyl and 4-methylbutenyl. Preferred lower alkenyl radicals include 2 to about 6 carbon atoms ("C2-C6"). The terms "alkenyl", and "lower alkenyl", embrace radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations.

The term "alkynyl" refers to linear or branched radicals having at least one carbon- carbon triple bond. Such radicals preferably contain from about two to about twenty-four carbon atoms ("C2-C24"). Other preferred alkynyl radicals are "lower alkynyl" radicals having two to about ten carbon atoms such as propargyl, 1-propynyl, 2-propynyl, 1- butyne, 2-butynyl and 1-pentynyl. Preferred lower alkynyl radicals include 2 to about 6 carbon atoms ("C2-C6").

The term "cycloalkyl" refers to saturated carbocyclic radicals having three to about twelve carbon atoms ("C3-C12"). The term "cycloalkyl" embraces saturated carbocyclic radicals having three to about twelve carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The term "cycloalkenyl" refers to partially unsaturated carbocyclic radicals having three to twelve carbon atoms. Cycloalkenyl radicals that are partially unsaturated carbocyclic radicals that contain two double bonds (that may or may not be conjugated) can be called "cycloalkyldienyl". More preferred cycloalkenyl radicals are "lower cycloalkenyl" radicals having four to about eight carbon atoms. Examples of such radicals include cyclobutenyl, cyclopentenyl and cyclohexenyl.

The term "alkylene," as used herein, refers to a divalent group derived from a straight chain or branched saturated hydrocarbon chain having the specified number of carbons atoms. Examples of alkylene groups include, but are not limited to, ethylene, propylene, butylene, 3-methyl-pentylene, and 5-ethyl-hexylene.

The term "alkenylene," as used herein, denotes a divalent group derived from a straight chain or branched hydrocarbon moiety containing the specified number of carbon atoms having at least one carbon-carbon double bond. Alkenylene groups include, but are not limited to, for example, ethenylene, 2-propenylene, 2-butenylene, l-methyl-2-buten-l- ylene, and the like.

The term "alkynylene," as used herein, denotes a divalent group derived from a straight chain or branched hydrocarbon moiety containing the specified number of carbon atoms having at least one carbon-carbon triple bond. Representative alkynylene groups include, but are not limited to, for example, propynylene, 1-butynylene, 2-methyl-3- hexynylene, and the like.

The term "alkoxy" refers to linear or branched oxy-containing radicals each having alkyl portions of one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkoxy radicals are "lower alkoxy" radicals having one to about ten carbon atoms and more preferably having one to about eight carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert- butoxy.

The term "alkoxyalkyl" refers to alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals.

The term "aryl", alone or in combination, means an aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused. The term "aryl" embraces aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indane furanyl, quinazolinyl, pyridyl and biphenyl.

The terms "heterocyclyl", "heterocycle" "heterocyclic" or "heterocyclo" refer to saturated, partially unsaturated and unsaturated heteroatom-containing ring-shaped radicals, which can also be called "heterocyclyl", "heterocycloalkenyl" and "heteroaryl" correspondingly, where the heteroatoms may be selected from nitrogen, sulfur and oxygen. Examples of saturated heterocyclyl radicals include saturated 3 to 6-membered heteromonocyclic group containing 1 to 4 nitrogen atoms (e.g. pyrrolidinyl,

imidazolidinyl, piperidino, piperazinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g. morpholinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl, etc.). Examples of partially unsaturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole. Heterocyclyl radicals may include a pentavalent nitrogen, such as in tetrazolium and pyridinium radicals. The term "heterocycle" also embraces radicals where heterocyclyl radicals are fused with aryl or cycloalkyl radicals. Examples of such fused bicyclic radicals include benzofuran, benzothiophene, and the like.

The term "heteroaryl" refers to unsaturated aromatic heterocyclyl radicals.

Examples of heteroaryl radicals include unsaturated 3 to 6 membered heteromonocyclic group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-l,2,4-triazolyl, lH-l,2,3-triazolyl, 2H-l,2,3-triazolyl, etc.) tetrazolyl (e.g. lH-tetrazolyl, 2H-tetrazolyl, etc.), etc.; unsaturated condensed heterocyclyl group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo[l,5-b]pyridazinyl, etc.), etc.;

unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, furyl, etc.; unsaturated 3 to 6-membered heteromonocyclic group containing a sulfur atom, for example, thienyl, etc.; unsaturated 3- to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.) etc.; unsaturated condensed heterocyclyl group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g. benzoxazolyl, benzoxadiazolyl, etc.);

unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl (e.g., 1,2,4- thiadiazolyl, 1,3,4- thiadiazolyl, 1,2, 5 -thiadiazolyl, etc.) etc.; unsaturated condensed heterocyclyl group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., benzothiazolyl,

benzothiadiazolyl, etc.) and the like. The term "heterocycloalkyl" refers to heterocyclo-substituted alkyl radicals. More preferred heterocycloalkyl radicals are "lower heterocycloalkyl" radicals having one to six carbon atoms in the heterocyclo radical.

The term "alkylthio" refers to radicals containing a linear or branched alkyl radical, of one to about ten carbon atoms attached to a divalent sulfur atom. Preferred alkylthio radicals have alkyl radicals of one to about twenty- four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkylthio radicals have alkyl radicals which are "lower alkylthio" radicals having one to about ten carbon atoms. Most preferred are alkylthio radicals having lower alkyl radicals of one to about eight carbon atoms.

Examples of such lower alkylthio radicals include methylthio, ethylthio, propylthio, butylthio and hexylthio.

The terms "aralkyl" or "arylalkyl" refer to aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl.

The term "aryloxy" refers to aryl radicals attached through an oxygen atom to other radicals.

The terms "aralkoxy" or "arylalkoxy" refer to aralkyl radicals attached through an oxygen atom to other radicals.

The term "aminoalkyl" refers to alkyl radicals substituted with amino radicals. Preferred aminoalkyl radicals have alkyl radicals having about one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred aminoalkyl radicals are "lower aminoalkyl" that have alkyl radicals having one to about ten carbon atoms. Most preferred are aminoalkyl radicals having lower alkyl radicals having one to eight carbon atoms. Examples of such radicals include aminomethyl, aminoethyl, and the like.

The term "alkylamino" denotes amino groups which are substituted with one or two alkyl radicals. Preferred alkylamino radicals have alkyl radicals having about one to about twenty carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkylamino radicals are "lower alkylamino" that have alkyl radicals having one to about ten carbon atoms. Most preferred are alkylamino radicals having lower alkyl radicals having one to about eight carbon atoms. Suitable lower alkylamino may be monosubstituted N-alkylamino or disubstituted Ν,Ν-alkylamino, such as N-methylamino, N-ethylamino, Ν,Ν-dimethylamino, N,N-diethylamino or the like.

The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent may be further substituted.

For simplicity, chemical moieties that are defined and referred to throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, an "alkyl" moiety can be referred to a monovalent radical (e.g. CH ₃ -CH ₂ -), or in other instances, a bivalent linking moiety can be "alkyl," in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH ₂ -CH ₂ -), which is equivalent to the term "alkylene." Similarly, in circumstances in which divalent moieties are required and are stated as being "alkoxy", "alkylamino", "aryloxy", "alkylthio", "aryl",

"heteroaryl", "heterocyclic", "alkyl" "alkenyl", "alkynyl", "aliphatic", or "cycloalkyl", those skilled in the art will understand that the terms alkoxy", "alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic", "alkyl", "alkenyl", "alkynyl",

"aliphatic", or "cycloalkyl" refer to the corresponding divalent moiety.

The terms "halogen" or "halo" as used herein, refers to an atom selected from fluorine, chlorine, bromine and iodine.

The terms "compound" "drug", and "prodrug" as used herein all include pharmaceutically acceptable salts, co-crystals, solvates, hydrates, polymorphs, enantiomers, diastereoisomers, racemates and the like of the compounds, drugs and prodrugs having the formulas as set forth herein.

Substituents indicated as attached through variable points of attachments can be attached to any available position on the ring structure.

As used herein, the term "effective amount of the subject compounds," with respect to the subject method of treatment, refers to an amount of the subject compound which, when delivered as part of desired dose regimen, brings about management of the disease or disorder to clinically acceptable standards.

"Treatment" or "treating" refers to an approach for obtaining beneficial or desired clinical results in a patient. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of symptoms, diminishment of extent of a disease, stabilization (i.e., not worsening) of a state of disease, preventing spread (i.e., metastasis) of disease, preventing occurrence or recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total).

The term "cancer" refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas and the like. For example, cancers include, but are not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, and multiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodisplastic syndrome, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal), genito urinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular), lung cancer (e.g., small-cell and non-small cell), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors related to Gorlin's syndrome (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Additional exemplary forms of cancer which may be treated by the subject compounds include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

Additional cancers that the compounds described herein may be useful in preventing, treating and studying are, for example, colon carcinoma, familiary

adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, or melanoma. Further, cancers include, but are not limited to, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma. In one aspect of the invention, the present invention provides for the use of one or more compounds of the invention in the manufacture of a medicament for the treatment of cancer.

In one embodiment, the present invention includes the use of one or more compounds of the invention in the manufacture of a medicament that prevents further aberrant proliferation, differentiation, or survival of cells. For example, compounds of the invention may be useful in preventing tumors from increasing in size or from reaching a metastatic state. The subject compounds may be administered to halt the progression or advancement of cancer. In addition, the instant invention includes use of the subject compounds to prevent a recurrence of cancer.

This invention further embraces the treatment or prevention of cell proliferative disorders such as hyperplasias, dysplasias and pre-cancerous lesions. Dysplasia is the earliest form of pre-cancerous lesion recognizable in a biopsy by a pathologist. The subject compounds may be administered for the purpose of preventing said hyperplasias, dysplasias or pre-cancerous lesions from continuing to expand or from becoming cancerous. Examples of pre-cancerous lesions may occur in skin, esophageal tissue, breast and cervical intra-epithelial tissue.

"Combination therapy" includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention. The compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy. A "biomarker" in the context of the present invention is a molecular indicator of a specific biological property; a biochemical feature or facet that can be used to detect and/or categorize an epithelial cancer. "Biomarker" encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein-ligand complexes, elements, related metabolites, and other analytes or sample-derived measures. Biomarkers can also include mutated proteins or mutated nucleic acids. In the instant invention, measurement of mRNA is preferred.

A "biological sample" or "sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, whole blood, blood fraction, serum, plasma, blood cells, tissue biopsies, a cellular extract, a muscle or tissue sample, a muscle or tissue biopsy, or any other secretion, excretion, or other bodily fluids.

The phrase "differentially expressed" refers to differences in the quantity and/or the frequency of a biomarker present in a sample taken from patients having for example, epithelial cancer as compared to a control subject. For example without limitation, a biomarker can be an mRNA or a polypeptide which is present at an elevated level (i.e., overexpressed) or at a decreased level (i.e., underexpressed) in samples of patients with cancer as compared to samples of control subjects. Alternatively, a biomarker can be a polypeptide which is detected at a higher frequency (i.e., overexpressed) or at a lower frequency (i.e., underexpressed) in samples of patients compared to samples of control subjects. A biomarker can be differentially present in terms of quantity, frequency or both.

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention comprise a

therapeutically effective amount of a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers or excipients.

As used herein, the term "pharmaceutically acceptable carrier or excipient" means a non-toxic, inert solid, semi-solid, gel or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; cyclodextrins such as alpha- (a), beta- (β) and gamma- (γ) cyclodextrins; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. In a preferred embodiment, administration is parenteral

administration by injection.

The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous,

intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl 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, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable suspension or emulsion, such as INTRALIPID®, LIPOSY ® or OMEGA VEN®, or solution, in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. INTRALIPID® is an intravenous fat emulsion containing 10-30% soybean oil, 1-10% egg yolk phospholipids, 1-10% glycerin and water. LIPOSYN® is also an intravenous fat emulsion containing 2-15% safflower oil, 2-15% soybean oil, 0.5-5% egg phosphatides 1-10% glycerin and water. OMEGA VEN® is an emulsion for infusion containing about 5-25% fish oil, 0.5-10% egg phosphatides, 1-10% glycerin and water. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form 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 sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or

preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.

Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, 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 the compounds of this invention, 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.

Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

For pulmonary delivery, a therapeutic composition of the invention is formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system. Solid or liquid particulate forms of the active compound prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics is known in the art (see, for example U.S. Pat. No. 5,767,068 to VanDevanter et al, U.S. Pat. No. 5,508,269 to Smith et al, and WO 98/43650 by Montgomery).

EXAMPLES

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference.

All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

General methodology for evaluating the effectiveness of compounds against cancer cell lines can be found in the following publicaitons: Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 2009 Aug 21; 138(4):645-59; Gupta et. al, U.S. Patent Application No. 201 10191868.

General synthetic methodology:

All oxygen and/or moisture-sensitive reactions were carried out under N2 atmosphere in glassware that had been flame-dried under vacuum (approximately 0.5 mm Hg) and purged with nitrogen (N ₂ ) prior to use. All reagents and solvents were purchased from commercial vendors and used as received, or synthesized according to methods already reported. NMR spectra were recorded on a Bruker 300 (300 MHz ^X H, 75 MHz ¹³ C) or Varian UNITY INOVA 500 (500 MHz 125 MHz ¹³ C) spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) referenced to the NMR solvent. Data are reported as follows: chemical shifts, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet; coupling constant(s) in Hz). Unless otherwise indicated, NMR data were collected at 25 °C. Flash

chromatography was performed using 40-60 μιη Silica Gel (60 A mesh) on a Teledyne Isco Combiflash Rf. Tandem Liquid Chromatography/Mass Spectrometry (LC/MS) was performed on a Waters 2795 module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with ultraviolet (UV) light and aqueous potassium permanganate (KMn0 ₄ ) stain followed by heating.

High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility (Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer).

Example 1 : Synthesis of Compound-27

2-(2,4,6-Trichlorophenoxy)acetohydrazide (50 mg, 0.19 mmol), lH-pyrrole-2- carbaldehyde (35 mg, 0.37 mmol) and magnesium sulfate (45 mg, 0.37 mmol) were dissolved in ethanol (1.5 mL). The reaction mixture was heated to 40°C and stirred for 8 hours. The reaction was taken up in ethyl acetate (50 mL) and washed with water (2 x 25 mL) followed by brine (1 x 25 mL). The combined organic layers were dried over sodium sulfate ( a ₂ S0 ₄ ), filtered, and concentrated. Purification by column chromatography provided (E)-N'-((lH-pyrrol-2-yl)methylene)-2-(2,4,6-trichlorophenoxy )acetohydrazide as a light yellow solid (41 mg, 65% yield).

H NMR (300 MHz, CDC1 ₃ ) δ 9.68 (overlap, 2H), 8.03 (s, 1H), 7.38 (overlap, 2H),

6.97 (s, 1H), 6.54 (s, 1H), 6.28 (s, 1H), 4.67 (s, 2H); ¹³ C NMR (75 MHz, CDC13) δ 179.3, 163.1, 148.8, 140.8, 131.0, 129.6, 129.13, 126.4, 122.8, 121.3, 1 15.5, 11 1.3, 1 10.1, 71.1; M+ 345.9925

Compounds 1-26 and 28-43 were synthesized using similar procedures. Example 2: Synthesis of Compound-62

5-(hydroxymethyl)furan-2-carboxylic acid (1.15 g, 8.09 mmol) was diluted with Benzene (81 ml). Thionyl chloride (3.54 ml, 48.6 mmol) was added and then the reaction is heated to reflux. The reaction was stirred overnight. The reaction was concentrated and carried on directly to the next step. The dichloride was dissolved in dichloromethane (1 12 mL) and 4- methylthiazol-2-amine (1.302 g, 1 1.17 mmol) was added followed by DMAP (0.138 g, 1.117 mmol) and triethylamine (3.89 ml, 27.9 mmol). The reaction was stirred at room temperature until complete by LC-MS analysis. The reaction was concentrated and purified by silica gel chromatography to yield the furanyl chloride (1.61 g) in 56% yield over two steps. The furanyl chloride (63.1 mg, 0.246 mmol) was dissolved in

dimethylformamide (2.5 mL). Sodium iodide (3.68 mg, 0.025 mmol), potassium carbonate (51.0 mg, 0.369 mmol) and 3-chlorophenol (31.6 mg, 0.246 mmol) were added and the reaction was stirred at room temperature until complete by LC-MS analysis. The reaction was concentrated under vacuum and purified by silica gel chromatography to provide 5- ((3-chlorophenoxy)methyl)-N-(4-methylthiazol-2-yl)furan-2-ca rboxamide (56 mg) (Compound-62) in 65% yield.

H NMR (300 MHz, CDC1 ₃ ) δ 7.76 (d, 1H, J=15 Hz), 7.43 (d, 2H, J=6 Hz), 7.19 (d, 2H, J=6 Hz), 6.56 (d, 1H, J=15 Hz), 3.96 (t, 2H, J=9 Hz), 3.31 (s, 2H, J=9 Hz), 2.66 (q, 2H, J=9 Hz), 1.23 (t, 3H, J=9 Hz); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 179.3, 163.1, 148.8, 140.8, 131.0, 129.6, 129.13, 126.4, 122.8, 121.3, 1 15.5, 11 1.3, 1 10.1, 71.1 ; calculated [M ⁺ H] 261.1056 experimental [M ⁺ H] 261.1067.

Compounds 42-60 and 62-1 18 were synthesized using similar procedures. Example 3 : Synthesis of Compound- 124

(E)-3-(4-ethylphenyl)acrylic acid (100 mg, 0.567 mmol), 3- ((ethylimino)methyleneamino)-N,N-dimethylpropan- 1 -aminium chloride (163 mg, 0. 851 mmol), and N,N-dimethylpyridin-4-amine (13 mg, 0.19 mmol) were dissolved in dichloromethane (10 mL). N-ethyl-N-isopropylpropan-2-amine (161 mg, 1.248 mmol) was added followed by 4,5-dihydrothiazol-2-amine (64 mg, 0.624 mmol). The reaction mixture was stirred for 12 hours at room temperature. The reaction was concentrated under vacuum. Purification by column chromatography provided (E)-N-(4,5- dihydrothiazol-2-yl)-3-(4-ethylphenyl)acrylamide as a white powder (58 mg, 40% yield). H NMR (300 MHz, CDQ ₃ ) δ 7.76 (d, 1H, J=15 Hz), 7.43 (d, 2H, J=6 Hz), 7.19 (d, 2H, J=6 Hz), 6.56 (d, 1H, J=15 Hz), 3.96 (t, 2H, J=9 Hz), 3.31 (s, 2H, J=9 Hz), 2.66 (q, 2H, J=9 Hz), 1.23 (t, 3H, J=9 Hz); ¹³ C NMR (75 MHz, CDC13) δ 179.3, 163.1, 148.8, 140.8, 131.0, 129.6, 129.13, 126.4, 122.8, 121.3, 1 15.5, 11 1.3, 1 10.1, 71.1 ; calculated [M+H] 261.1056 experimental [M+H] 261.1067.

Compounds 1 19-123 and 125-144 were synthesized using similar procedures as described above.

Example 4: Primary Cell Viability Protocol with HMLE sh Ecad Cells Using CellTiter-

Glo

HMLE_sh_Ecad cells were prepared as previously described (Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 2009 Aug 21; 138(4):645-59). Procedures: Briefly, HMLE cells expressing either shRNA targeting E-cadherin

(sh_Ecad), control shRNA targeting eGFP (sh_eGFP), or shRNA targeting Twist

(sh_Twist) were propagated in 1 : 1 mixture of 10% fetal bovine serum (FBS; HyClone), 1% Penicillin/Streptomycin (Pen/Strep; Cellgro), 1% Glutamax-1 (Invitrogen), 70 nM Hydrocortisone (Sigma), 12 μg/mL Insulin (Sigma), 50 μg/mL Gentamicin (Sigma), 12.5 μg/mL Plasmocin (InVivogen), 10 ng/mL EGF in DMEM (Cellgro) with Mammary Epithelial Cell Growth Medium (MEGM complete medium; Lonza, Basel, Switzerland) at 37 °C, 5% C02. For screening, the cells were counted and resuspended in complete media without serum. Next, 2,000 cells in 50 μΐ _^ were plated per well in white, tissue culture- treated, 384-well plates (Corning). The cells were incubated at 37 °C, 5% CO ₂ for at least 4 hours and pinned with 100 nl of compounds. The cells were incubated approximately 72 hours, then 30 μΐ, of CellTiter-Glo (Promega) diluted 1 :3 with PBS was added to the well. The plates were read using the EnVision (PerkinElmer; Luminescence 0.1 sec/well) after 12 minutes.

Example 5: Secondary Orthogonal Assay for In Vitro Inhibition of Tumorspheres with Suml59 Cells

Tumorsphere assays were performed as previously described (Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003) with minor changes. Suml59 cells were propagated in 5% FBS (HyClone), 1 % Pen/Strep, 1% Glutamax-1, 12 μg/mL Insulin, 50 μg/mL Gentamicin in F12/DMEM (Cellgro). Next, 96- well, ultra-low adhesion plates (Costar) were plated with 100 μΐ _^ media respective to cell type. Then, 400 nL of compounds were pinned into the media. The cells were harvested, counted, and resuspended in their propagation media with 1% methylcellulose (ES- CultM3120, Stem Cell Technologies). Then, 100 μΐ _^ of resuspended cells were added to the plates containing media with compound for a final count of 2000 cells/well in 200 μϊ _^ with 0.5% methylcellulose. Tumorspheres were allowed to form for 9 days incubated at 37 °C, 5% C02. Tumorspheres were imaged using a 2X objective on the ImageXpress Micro (Molecular Devices, Sunnyvale, CA). Cell clusters greater than 100 μΜ in diameter were identified using MetaXpress software (version 3.1 ; Molecular Devices). Example 6: Gene Expression Assays with HMLE sh Ecad and HMLE sh eGFP Cells Triplicate samples of HMLE_sh_Ecad and HMLE_sh_eGFP cells were treated with vehicle (DMSO) or compound at an IC50 dose for 24 hours prior to isolation of RNA. Total RNA was isolated using the RNeasy Protect Mini Kit (Qiagen). Quality control (QC) processing of the RNA samples and the gene expression analysis were performed by the Genome Analysis Platform (GAP) at the Broad Institute.

Procedures: Briefly, RNA samples were analyzed for quality using Aglient Bioanalyzer Chips. cRNA synthesis from the total RNA samples passing QC was prepared for analysis on a HumanHT-12 Expression BeadChip (Illumina, San Diego, CA) according to the manufacturer's instructions. Normalization of the raw gene expression data, QC checks, and analyses were done with GenomeStudio (Illumina, San Diego, CA). All of the samples were run in triplicate and the replicate samples were normalized using cubic spline normalizations. After the samples were normalized, genes were selected as having greater than 2-fold change in expression levels.

Example 7: Plasma protein binding

Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology, Rockford, IL) for both human and mouse plasma. Each compound was prepared in duplicate at 5 μΜ in plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 μΐ) with PBS pH 7.4 added to the other side (350 μΚ). Compounds were incubated at 37 °C for 5 hours in a 250-rpm orbital shaker. Following the incubation, the samples were analyzed by UPLC- MS.

(Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Compound-27 was found to be 98.8% bound in human plasma. The percentage bound in mouse plasma was unable to be determined due to poor plasma stability.

Plasma Stability. Plasma stability was determined at 37 °C for 5 hours in both human and mouse plasma. Each compound was prepared in duplicate at 5 μΜ in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). The compounds were incubated at 37 °C for 5 hours with a 250-rpm orbital shaker with time points taken at 0 hours and 5 hours.

The samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer. Compound-27 was found to be modestly stable in human plasma with 49.2% remaining after 5 hours. The compound was not stable in mouse plasma as no compound was detected after 5 hours.

Results

Compounds 1, 19, 27, 30 and 39 were tested in a tumorsphere assay. In this assay, Suml59 cells (a human breast cancer cell line: Gupta PB, Onder TT, Jiang G, Tao K,

Kuperwasser C, Weinberg RA, Lander ES. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 2009 Aug 21 ; 138(4):645-59) were grown in the presence or absence of a compound of interest for 9 days in a low adhesion environment. Under these conditions, Suml59 cells, which contain a mixture of breast CSCs and differentiated breast cancer cells, form tumorspheres. The number of tumorspheres was counted in each of the conditions. The signal (number of tumorspheres) was normalized to neutral (DMSO) and positive (Puromycin) controls, and a 30% inhibition cut-off at an average screening concentration of 20 μΜ was used to define a hit. Compounds 1, 27 and 30 did not significantly inhibited tumorsphere formation. However, two compounds (19 and 39) did inhibit SUM 159 tumorsphere formation.

Compounds 1, 27 and 30 preferentially inhibit HMLE_sh_Ecad over

HMLE_sh_eGFP, (Table A), while the compounds active in the tumorsphere assay (19 and 39) potently inhibited both cell lines. Therefore, it is possible that only compounds that suppress both populations of cells (i.e., differentiated breast cancer cells and CSCs) can actively suppress SUM159 tumorsphere formation in this assay model. Using breast cancer cell lines with higher percentages of CSCs may yield a different result. Although Compound-27 did not inhibit SUM159 Tumorsphere formation, Salinomycin also did not inhibit tumorsphere formation in vitro (Gupta et. al). However, Salinomycin-treated mice did inhibit SUM159 tumor formation and growth in mice. SUM159 tumorsphere experiments may not be indicative of tumor suppressing compounds and other in vivo studies would be required to determine if Compound-27 inhibits tumor formation. In order to confirm results obtained by the probe Screening Center, several additional experiments were conducted. Compounds 1, 5, 14, 27, 30, 39 and 41 were investigated for their effects on viability in HMLE_sh_Ecad and HMLE_sh_eGFP cell lines. Although different absolute IC50 values obtained varied, Compound-27 had the best potency and greatest selectivity of all the compounds tested. Two additional cell lines,

HMLE_sh_TWIST and MDA231, were used to corroborate the findings.

HMLE_sh_TWIST, a second model of EMT-induced, breast CSC-like cells, was also significantly inhibited by Compound-27. Furthermore, Compound-27 activity was tested against a breast cancer cell line, MDA231. Compound-27 potently (IC ₅₀ approximately 2.8 μΜ) inhibited MDA231 viability (Table 2), suggesting that the compound can be active against tumors. TABLE 2

Gene expression studies: Compound-27 (1 μΜ) was added to HMLE_sh_Ecad or HMLE_sh_eGFP cells for 6 hours or 24 hours. The concentration of 1 μΜ was chosen 5 because it was the concentration at which 80% of the cells were killed after 3 days in culture (ICso). At this concentration, it was likely that gene expression would be significantly altered, but it would not be too toxic to recover viable mRNA.

All results were compared with the two cell types treated with DMSO only for 24 hours. Furthermore, 54 genes had differential expression with treatment of the probe 0 (ML239) in the HMLE_sh_Ecad cell line. All but one gene of these genes were specific to the HMLE sh Ecad cell line and and did not change in the HMLE sh eGFP cell line with treatment. Genes involved in protein processing within the ER, MAPK signaling, and inflammatory cytokines pathways were significantly altered.

While this invention has been particularly shown and described with references to 5 preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.