WO2013131010A2 - Function of chemokine receptor ccr8 in melanoma metastasis - Google Patents

Function of chemokine receptor ccr8 in melanoma metastasis Download PDF

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WO2013131010A2
WO2013131010A2 PCT/US2013/028683 US2013028683W WO2013131010A2 WO 2013131010 A2 WO2013131010 A2 WO 2013131010A2 US 2013028683 W US2013028683 W US 2013028683W WO 2013131010 A2 WO2013131010 A2 WO 2013131010A2
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Mihaela Skobe
Suvendu Das
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Icahn School Of Medicine At Mount Sinai
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Abstract

This invention provides a method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCL1 to the CCR8 receptors so as to thereby treat the subject. This invention also provides a method of reducing, or reducing the likelihood of, metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCL1 to the CCR8 receptors so as to thereby reduce, or reduce the likelihood of, metastases in the subject.

Description

FUNCTION OF CHEMOKINE RECEPTOR CCR8 IN MELANOMA METASTASIS

This application claims priority of U.S. Provisional Application No. 61/606,169, filed March 2, 2012, entire contents of which are hereby incorporated herein by reference.

This invention was made with government support under grant number BC044819 awarded by the Department of Defense, Breast Cancer Research Program, grant number 5R24 CA095823-04 awarded by the National Institutes of Health and the National Cancer Institute, grant number DBI-9724504 awarded by the National Science Foundation, and grant number 1 S10 RR0 9145-01 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention .

This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named "130301_0028_83810_A_PCT_Sequence_Listing_BI.txt," which is 8.15 kilobytes in size, and which was created March 1, 2013 in the IBM-PC machine format, having an operating system compatibility with MS- Windows, which is contained in the text file filed March 1, 2013 as part of this application.

Throughout this application, various publications are referenced within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Background of Invention

Metastasis of tumor cells to the regional lymph nodes is one of the key indicators of tumor aggressiveness. Lymph node status is a powerful predictor of patient survival and it is one of the key parameters used for determining the stage of disease progression and treatment options (Green, 2006; Morton et al . , 2006) . Despite the paramount importance of lymph node status for the patient outcome, the mechanisms by which tumor cells are recruited to the lymph nodes are poorly understood.

According to the current paradigm, once tumor cells gain access to the lymphatic vessels, they are carried with the flow of lymph into the sentinel lymph nodes, where they subsequently reside. Entry of tumor cells into the lymphatics has been thought to occur at random, as a consequence of tumor cell invasion through tissue. However, recent findings indicate that tumor cells are guided into the lymphatic vessels by chemokines produced by lymphatic endothelium (Ben-Baruch, 2008; Das and Skobe, 2008) . The CCL21-CCR7 ligand- receptor pair is thought to play a central role in directing tumor cells to the lymph nodes. CCL21 is constitutively expressed by the lymphatic vessels (Gunn et al . , 1998; Kerjaschki et al . , 2004; Podgrabinska et al . , 2002; Shields et al . , 2007a) and its receptor CCR7 , is expressed by melanoma and breast cancer cells (Houshmand and Zlotnik, 2003; Muller et al . , 2001) . Overexpression of CCR7 in melanoma has been shown to facilitate tumor metastasis to the lymph nodes in a mouse model (Wiley et al . , 2001) and clinical studies have confirmed the association between CCR7 expression in tumors and lymph node metastasis (Cabioglu et al . , 2005; Ishigami et al . , 2007; Mashino et al . , 2002) . Another chemokine receptor important for metastasis is CXCR4. It is the most widely expressed chemokine receptor in cancer and it has been shown to direct tumor cells to the lung and other distant organs, as well as to the lymph nodes (Muller et al . , 2001) . CCR8 is a G-protein-coupled receptor which in humans, is selectively activated by the CC chemokine CCLl/I-309 (Goya et al . , 1998; Roos et al . , 1997; Tiffany et al . , 1997) . In mice, novel chemokine CCL8 has recently been identified as a second agonist for CCR8, but no human ortholog has been found yet (Islam et al . , 2011) . CCR8 plays a rather unique role in the regulation of the immune response. It is preferentially expressed by activated TH2 cells (T helper type 2) (D'Ambrosio et al . , 1998; Islam et al . , 2011; Zingoni et al . , 1998) and it mediates TH2 cell recruitment to the sites of inflammation (Chensue et al . , 2001; Gombert et al . , 2005; Islam et al . , 2011) . Since TH2 cells are primary drivers of allergy and asthma, CCR8 activation has been implicated in allergic inflammation and pulmonary hypersensitivity (Chensue et al . , 2001; Gombert et al . , 2005; Islam et al . , 2011) . Other functions of CCR8 include T-cell homing to the skin in the steady-state (Ebert et al . , 2006; Schaerli et al . , 2004), the role in DC migration to the lymph nodes (Miller and Krangel, 1992; Qu et al . , 2004) and the role in thymic development (Louahed et al., 2003) . Consistent with its role in recruitment of T-cells to tissues, CCL1 is constitutively expressed by the dermal blood vasculature (Gombert et al., 2005; Schaerli et al . , 2004) . In the skin, CCL1 is also expressed by melanocytes and by Langerhans cells, but not by keratinocytes (Gombert et al . , 2005; Schaerli et al . , 2004) . Inflammatory cytokines and microbial products dramatically induce CCL1 expression (Gombert et al . , 2005) .

Summary of the Invention

The present invention provides a method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCL1 to the CCR8 receptors so as to thereby treat the subject.

The present invention provides a method of reducing, or reducing the likelihood of, metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCL1 to the CCR8 receptors so as to thereby reduce, or reduce the likelihood of, metastases in the subject.

The present invention provides a method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCR8 receptor produced by tumor cells present in the solid tumor, so as to thereby treat the subject.

The present invention provides a method of reducing, or reducing the likelihood of, metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCR8 receptor produced by tumor cells present in the solid tumor, so as to thereby reduce, or reduce the likelihood of, metastases in the subject.

The present invention provides a method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCL1 in the subject, so as to thereby treat the subj ect .

The present invention provides a method of reducing, or reducing the likelihood of, metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of CCL1 in the subject, so as to thereby reduce, or reduce the likelihood of, metastases in the subject.

Brief Description of the Drawings

Figure 1. CCLl secreted by LECs induces tumor cells chemotaxis .

(A) Migration of breast cancer and melanoma cells to LEC-CM in a Boyden chamber assay. MCF-10F and 184-B5, non-tumorigenic human breast epithelial cell lines; MCF-7, MDA-MB-231 and MDA-MB-435, breast cancer cell lines; SK-MEL-28, SK-MEL-25 and MEL-501, melanomas. (B) Migration of MDA-MB-435 cells to LEC-CM in presence of pertussis toxin (PTx) or cholera toxin (CTx) . (C) RT-PCR for CCLl mRNA expression in cultured LECs and BECs . cDNA from PMA-stimulated Jurkat cells was used as a positive control. (D) Western blot analysis of CCLl protein in conditioned media from LECs and BECs. 10 ng of rhCCLl was used as a positive control. (E) Inhibition of MDA- MB-435 and SK-MEL-25 chemotaxis to LECCM by a monoclonal anti-CCLl neutralizing antibody. (F) Tumor cell migration to LEC-CM depleted of CCLl with a polyclonal anti-CCLl antibody. (G) Migration of tumor cells to rhCCLl (50 ng/ml) , in comparison to LEC-CM. Migration data are representative of three experiments . Error bars indicate SEM, n=3. *, P < 0.05; **, P <0.01; P<0.001.

Figure 2. Inflammatory cytokines increase CCLl production by LECs and tumor cell migration to LEC-CM.

(A) Real-time Q-PCR analysis for CCLl mRNA upon treatment of LECs with IL-Ιβ (50 ng/ml), TNF- (50 ng/ml) or LPS (500 ng/ml) . Bars indicate mean ± STD, n=3. (B) Western blot analysis of CCLl protein in conditioned medium from LECs treated with TNF-a, LPS or IL-Ιβ. (C, D) Effect of CCLl-depletion on tumor cell (MDA-MB-435) migration to LEC-CM generated in presence of (C) TNF-a or (D) LPS. Note increase in tumor cell migration to TNF-a/LEC-CM or LPS/LEC-CM, as compared to basal LEC-CM; CCLl depletion abolishes this effect. Bars indicate mean ± SEM, n=3. Data are representative of three experiments. *, P< 0.05

Figure 3. CCR8 expression and function in tumor cell lines.

(A) Expression of CCR8 mRNA (RT-PCR, in vitro) and protein (Western blot, in vitro and in vivo) by MDA-MB-435 and SK-MEL-25 cells. FACS analysis was done on cultured tumor cells and primary melanocytes.

(B) CCR8 antagonist MC148, inhibits tumor cell chemotaxis to LECCM. Bars indicate mean ± SEM, n=3. *, P< 0.05. (C) Effects of CCR8 antagonist MC148 and pertussis toxin (PTx) on cytoskeletal rearrangements induced by rhCCLl in MDA-MB-435 cells. Cells are stained with phalloidin (red) and Hoechst (nuclei, blue) . Scale bar: 10 μιη. Data are representative of three experiments.

Figure 4. CCR8 expression in human malignant melanoma.

Immunohistochemistry for CCR8 on (A-H) human melanoma tissue array, (I) another sample of primary melanoma in the skin, and (J-L) melanoma metastases in the human lymph node. (A-H) Various staining patterns for CCR8 in melanoma. (A, B) Tumor cells showing strong membrane staining for CCR8. (B) , magnified area of (A) . (C, D) Focally strong pattern of membrane stain. (D) Magnified area of C. (E, F) Moderate intensity cytoplasmatic staining. (F) , magnified area of E. (G) Weak cytoplasmatic stain. (H) No CCR8 expression. (I) Melanoma in the dermis staining strongly positive for CCR8 (ep, epidermis) . (J, K) High expression of CCR8 in the metastatic melanoma localized in the subcapsular zone of the lymph node (c, capsule) . (K) magnified area of J. (L) CCR8 expression by metastatic melanoma localized in the follicular zone of the lymph node (f, follicle) . Scale bar: 100 μΜ.

Figure 5. Expression of CCLl in the mouse skin, tumor and the lymph node lymphatic vessels .

(A-F) Double-immunostaining for LYVE-1 (lymphatic vessels, red), and mouse CCLl (green) in frozen sections of (A-C) mouse skin overlying a tumor and (D-E) MDAcl.13 tumor xenograft. Note that both, dermal lymphatics and tumor associated lymphatics are devoid of CCLl expression. There is no significant expression of CCLl in other cell types. (G-I) Doubleimmunostaining for podoplanin (lymphatic vessels, red) and mouse CCLl (green) of tumor draining lymph nodes. (I) Merged image of G and H. (G-L) Capsule and the lymph node cortex of the naive (G-I) and the tumor-draining lymph node (J-L) . Note strong immunoreactivity for CCLl by the lymphatic vessels of the subcapsular sinus. (M-O) Lymph node medullary sinuses are negative for CCLl, some immune cells are positive. Images are representative at least n=5 samples per group, ep, epidermis. Scale bar: 100 μΜ. Figure 6. CCLl expression in human tissues.

Immunohistochemical staining for podoplanin (lymphatic vessels) and for human CCLl in consecutive sections of normal human skin (A-D) , in melanoma (E, F) and in lymph nodes with metastatic melanoma (G- J) . (A, B) CCLl is not expressed by lymphatic vessels (arrows) . Occasional blood vessels (arrowheads) stain positively for CCLl. (C, D) Higher magnifications of A and B, respectively. (E, F) Lymphatic vessels associated with malignant melanoma in the skin are not stained for CCLl. Note the absence of CCLl staining in the inflammatory infiltrate associated with the tumor. (G-J) The lymphatic endothelium (arrows) of the lymph node subcapsular sinuses is strongly positive for CCLl. (I, J) Higher magnification of G and H, respectively. T, tumor; ep: epidermis. Scale bar: 100 μΜ.

Figure 7. Blocking CC 8 inhibits tumor cell entry into the lymph node from collecting lymphatics .

(A-D) Metastases from control MDAcl.13 cells (GFP, green) in the sentinel lymph nodes. (A) Stereomicroscope captured epi-fluorescent image of the whole lymph node, showing metastases in the afferent lymphatic vessel and inside the lymph node parenchyma (boxed area) . (B-D) Multiphoton imaging of GFP-labeled metastases (green) combined with second harmonic generation (SHG, red) , showing metastases scattered throughout the lymph node cortex. (B, C) View from the top of the lymph node (from the outside) . (D) View from the lymph node parenchyma (from the inside) . Second harmonic generation allows visualization of collagen fibers in the lymph node capsule (red) . (E-H) Metastases from MDAcl .13/MC148 tumors. (E) Stereomicroscope captured epi-fluorescent image showing metastasis in the afferent lymphatic vessel (in-transit metastasis), but not in the lymph node parenchyma. Note the sharply demarcated junction of the lymphatic vessel and the lymph node, indicating tumor cell arrest at that location (boxed area) . (F-H) Multiphoton imaging of metastases (GFP, green) in the afferent lymphatic vessel combined with SHG (red) , which visualizes collagen fibers of the lymphatic vessel wall. (F, G) Lateral view of the vessel. GFP only (F) , GFP and SHG (G) . (H) View into the vessel lumen, from the lymph node. Note densely packed tumor cells completely obstructing the vessel lumen. (I, J) Immunostaining for podoplanin (red) shows metastasis in the SCS and immediately below in the LN cortex. Arrow points to the "floor" of the SCS, i.e. lymphatic endothelium at the border to the lymph node cortex. (K, L) Metastasis of the control cells which invaded into the LN parenchyma. Arrow points to the SCS stained with LYVE-1 (red) . (M, N) Cross-section of an afferent lymphatic vessel stained with podoplanin (red) , showing large intransit metastasis of MDAcl .13/MC148 cells. Note that the entire lesion is adherent to the lymphatic vessel wall, c, capsule; t, tumor; LV, Lymphatic vessels; LN, lymph node. H, Hoechst (blue) . Scale bars: 100 μΜ. (0, P) Bar graphs depict the incidence of intra-nodal metastases (0) and in- transit metastases (P) by MDAcl.13 control vs. MC148 tumors. Incidence is calculated as a percent of all samples positive for either intra-nodal or in-transit metastases (control, n=15; MC148, n=15) .

Figure 8. Expression of hCCLl, hCCR8 and MC148 by different clones of MDA-MB-435 cells and in vivo tumor growth. (A) RT-PCR of cultured cells for hCCLl, MC148 and hCCR8 mRNA. cDNA from LECs was used as a positive control for CCL1. β-actin served as a loading control. (B) Western blot of cultured cell lines for hCCR8 protein expression (panel 1), Western analysis for MC148 protein in tumor cell CM (panel 3) and in vivo tumor lysates (panel 4) . β-actin served as a loading control. (C, D) Tumor growth of control and MC148-expressing MDAcl.6 (C) or MDAcl.13 cell lines (D) . Each data point represents mean tumor volume +SEM, n = 10 mice per group.

Figure 9. Expression of hCCLl, hCCR8 and MC148 by different clones of SK-MEL-25 cells. (A) RT-PCR on cultured cells for hCCLl (panel 1) , MC148 (panel 3) and hCCR8 (panel 5) mRNA. cDNA from LECs was used as a positive control for CCL1. β-actin served as a loading control. (B) Western blot analysis of cultured cell lines for the expression of hCCR8 (panel 1) and tumor cell CM for the expression of MC148 (panel 2) . β-actin served as a loading control. Figure 10. Ectopic expression of MC148 does not alter histopathological features of MDA cl.13 tumors. (A, B) H & E staining of sections of paraffin-embedded tumor specimens. (C-H) Immunofluorescent staining of frozen tumor sections for LYVE1 (green) and CD31 (red), to visualize lymphatics and total vessels, respectively. (C, D) Appearance of dermal lymphatics (arrows) at the periphery of control (C) and MC148 tumors (D) was similar. Intratumoral lymphatics (E, F) and blood vessels (G, H) were similar in number and appearance when MC148 was expressed. H, Hoechst nuclear stain (blue) . Scale bars: 100 μιτι. (I) Quantification of lymphatic and blood vessel densities in tumors. Bars represent the mean vessel area ± SEM, n=4 (LYVE-1+, lymphatics; CD31+ blood and lymphatic vessels) . Figure 11. Ectopic expression of MC148 did not alter angiogenesis or lymphangiogenesis in SK-MEL-25 tumors: (A, B) LYVE-1- stained dermal lymphatic vessels (green) at the tumor periphery appear similar in control (A) and in MC148 (B) tumors. SK-MEL-25 control and MC148 tumors do not show intratumoral lymphangiogenesis, hence tumor images are not shown. (C-F) CD31+ vessels in the tumor-associated skin (C, D) and in tumors (E, F) . H, Hoechst nuclear stain (blue) . Scale bars: 100 μιτι. (G) Quantification of lymphatic and blood vessel densities. Bars represent the mean vessel area ± SEM, n=3 (LYVE-1+ , lymphatics; CD31+ blood and lymphatic vessels) .

Figure 12. Effects of recombinant CCLl and MC148 on calcium mobilization and F-actin rearrangement in tumor cells. (A, F)

Changes of cytosolic Ca2+ concentration measured by FACS in Fluo-4- labeled cells upon addition of human (A-C) or mouse (D-F) CCLl. Black arrow indicates the time of CCLl addition. MC148 was added at 25 nM. Data are expressed as percentile fluorescence intensity of Fluo-4 dye bound to Ca+2. Data are representative of at least two experiments. (G, H) Cell shape change of MDA-MB-435 cells stimulated with human CCLl (G) or mouse CCLl, added at 50 ng/ml (H) . Cells are stained with rhodamin-labeled phalloidin (red) . Scale bar: 10 μιτι Figure 13. Evidence of in-transit and intra-nodal metastases in the MDAcl .13 mouse model .

(A, B) Stereomicroscopy images showing in-transit metastases in the proximity of a MDAcl.13 control tumor three weeks after injection. Epi-fluorescent (A) and bright-field image (B) of metastases in the tumor-draining lymphatics (arrows) at about 1cm away from the primary tumor (T) . (C-F) In-transit metastases of MDAcl .13/MC148 cells at 12 weeks. Epi-fluorescent (C, E) and bright-field (D, F) images of metastases in the afferent lymphatic vessels in the proximity of the LN. Note that LNs are free of metastases. (G, H) Epi-fluorescent (G) and bright-field image (H) of intra-nodal metastases (arrows) by MDAcl.13 cells at 12 weeks. Note multiple metastatic foci scattered throughout the LN cortex. Tumor cells are GFP-labeled, green (arrows) . Scale bars: 1 cm (A, B) ; 500 μιτι (C-H) .

Figure 14. Effects of CCR8 blockade on intra-nodal and in-transit metastases of MDAcl.6 cells. (A) Decrease of intra-nodal metastases in MC148-treated mice. (B) Increase of in-transit metastases in MC- 148-treated mice. Metastasis incidence is calculated as a percentage of all samples positive for either intra-nodal or in-transit metastases (control, n=13; MC148, n=9) .

Figure 15. CCR8 downregulation with shRNA inhibits lung metastasis in the mouse model. Downregulation of CCR8 by shRNA inhibits lung metastases. (A-C) MDA-MB-435/VEGF-C cells tagged with GFP were injected into the mammary fatpads of immunodeficient mice and metastases evaluated when tumors reached 10mm in diameter. Two shRNA constructs were evaluated along with the scrambled shRNA control . (A) Stereomicroscopy evaluation of the intact mouse lung for the presence of metastases (GFP, green) . (B) Metastases in the lung were quantified by measuring the total lung area positive for GFP in Image J, n=10 mice. (C) Size of the individual metastatic foci in the lung was determined by measuring the foci area ( m2) , and the foci were classified as small (<104 μιτι2) or large (>104 μιτι2) . At least 100 foci were analyzed in each group. Note that both, total lung area with metastases and the size of the individual metastatic foci are significantly decreased with CCR8 knockdown. Figure 16. Inflammatory cytokines increase CCLl production by LECs and tumor cell migration to LEC-CM. (F-K) Immunostaining of mouse skin for either LYVE-1 or podoplanin (both green) and for CCLl (red); boxed areas in I and K depict the overlay. Note that CCLl is not detected on lymphatic capillaries in control skin (F, G) , but it is strongly upregulated in inflammation, upon treatment with TNF- (H, I) or with FITC (J, K) . Images are representative of at least 6 skin samples per group. Arrows, lymphatic vessels, ep, epidermis. Scale bars, 50 μιτι.

Figure 17. CCR8 expression in human malignant melanoma.

Immunohistochemistry for CCR8 on human melanoma tissue array (A-D) , samples of primary melanomas in the skin (E-I), and melanoma metastases in the human lymph node (J-M) . Tumors showed various staining patterns for CCR8 : strong membrane staining throughout (A) , or focally (B) , moderate intensity cytoplasmatic staining (C) , or no CCR8 (D) . (E-H) Serial sections of human melanoma in the skin immunostained for CCR8 (E, G) , or secondary antibody control (F, H) . Note that the pattern of CCR8 stain (red) does not overlap with the distribution of melanin (brown) . (G, H) Higher magnification of the boxed area in E and F. (I) Highly pigmented melanoma negative for CCR8. Note the distinct color of endogenous melanin (brown) and lack of CCR8 staining (red) . (J-L) Metastatic melanoma localized in the subcapsular zone of the lymph node stained for CCR8 (J,L) or secondary antibody control (K) on the serial sections. (L) Higher magnification of the boxed area in J. (M) CCR8 expression in metastatic melanoma localized in the follicular zone of the lymph node. Images are representative of 104 primary tumors and 8 lymph node samples, c, capsule; t, tumor; f, follicle. Scale bars, 200 μιτι (J, K) , 100 μιτι (A-F, I, L, M) and 25 μιτι (G, H) .

Figure 18. CCLl expression in human tissues. Immunohistochemical staining for podoplanin (lymphatic vessels) and for human CCLl on serial sections of normal human skin (A, B) , melanoma in the skin (C-F) and lymph nodes with melanoma metastases (G-J) . CCLl is not expressed by the lymphatic vessels in the skin (A, B) or by the lymphatic vessels in primary malignant melanoma (C-F) . (E, F) Higher magnification of the boxed area in C and D. (G-J) Serial sections of human lymph node with melanoma metastases stained with H&E (G) or immunostained for podoplanin (H) or CCL1 (I, J) . (H) Podoplanin identifies lymphatic vessels of the SCS (arrows) and lymphatics associated with melanoma metastases in the cortex. (I) Lymphatics of the SCS are stained for CCL1 (arrows), but not the tumor-associated lymphatics in the cortex. (J) Higher magnification of the boxed area in I, showing lymphatic vessel in the SCS positive for CCL1. Images are representative of at least 10 skin samples, 14 primary tumors and 8 lymph node samples. Arrows, lymphatic vessels; T, tumor; ep, epidermis; c, capsule. Scale bars, 100 μιτι (A-F, H, I), 250 μιτι (G) , 25 ]i (J) .

Figure 19. Blocking CC 8 inhibits tumor cell entry into the lymph node from collecting lymphatics. (A) Bar graph depicts the incidence of intra-nodal metastases and in-transit metastases by MDAcl.13 tumors upon inhibition of CCR8 with MC148 or upon downregulation with shRNA. Incidence is calculated as percent of all samples positive for either intra-nodal or in-transit metastases (control, n=15; MC148, n=15; sh control, n=14; shCCR8(l), n=12; shCCR8(2), n=8) . (B) Scanning electron microscopy (SEM) micrograph of a lymph node crosssection showing MDAcl .13/ shCCR8 ( 1 ) metastasis in the lymphatic vessel at the junction with the SCS (boxed area) ; same site as depicted in E. (C, D) Lymphatic endothelium at the floor of the SCS bordering the tumor (t) visualized by SEM at high magnification (C) or by immunofluorescent staining for LYVE-1. Images are representative of at least n=9 mice per group. Arrows, lymphatic endothelium; t, tumor. Scale bars: 100 μιτι (B) , 50 μιτι (D) and 5 μιη (C) .

Figure 20. Steps of lymph node metastasis. (A-C) SCS of the sentinel lymph node stained with Abs against VEGFR-3 or CD31 as indicated. (A) Dilation of lymphatic sinuses prior to the entry of tumor cells. Note VEGFR-3+ columns connecting the floor and the roof of the dilated sinus. (B) Single tumor cells present in the lumen of the SCS or attached to the lymphatic endothelium. (C) Early stage of metastatic growth in the SCS . Note that tumor cells adhere to the lymphatic endothelium. (D-F) SEM micrographs depicting architecture of the SCS at high resolution. (D) Lymphatic endothelia at the roof and the floor of the SCS are connected by numerous columnlike structures. (E) Subcapsular space is further divided by horizontal bridges between the columns. (F) Metastatic lesion confined to the SCS. (G) Metastasis in the afferent lymphatic vessel (dotted line) at the junction with the lymph node (boxed area) . (H) Metastasis in the LN parenchyma. Lymphatic vessels are stained with podoplanin (H) . MDAcl.6, (A); MDA cl.13 (B-L) . t, tumor; c, capsule. Scale bars, 10 μιτι (D, E) , 20 μιτι (F) , 100 μιτι (A-C, H-L) , 200 μιτι (G) .

Figure 21. Intravital imaging of metastasis at the junction of the afferent lymphatic vessel and the lymph node. (A-D) Still images from the time-lapse movies obtained by multiphoton microscopy. (A) 10-25μιη deep maximum intensity projection image showing an afferent lymphatic vessel leading into the SCS (junction is indicated by *) . Collagen fibers of the lymph node capsule and the lymphatic vessel wall are visualized by SHG (blue) . SCS contains a large cluster of tumor cells (GFP, green), which show very little movement. Evan's Blue tracer (white) labels the lymphatic vessel and the SCS. Some LN cells have taken up the Evan's blue dye and appear white. Blood vessels were labeled by i.v. injection of 2 MDa rhodamine dextran (red) . (B) Higher magnification of the boxed area in A (25μιη wide) showing a tumor cell with an extended and retracted protrusion at different time-points as indicated. (C, D) Tumor cells (green) in the SCS (white), in the single plane 35μιτι beneath the LN capsule. Leukocytes in the SCS appear black on the white background. Blue line indicates movement of one leukocyte tracked over 61min (distance traveled: 210 μιη) (C) . (D) Higher magnification view of C, showing one tumor cell which moved to a different position (arrows) and another tumor cell in the proximity which remained stationary. LV, lymphatic vessel; SCS, subcapsular sinus. Figure 22. Characteristics of SK-MEL-25 tumors expressing CC 8 inhibitor MC148. Blood and lymphatic vasculature in SK-MEL-25 tumors expressing MC148. (C) Quantification of lymphatic and blood vessel densities. Bars represent the mean vessel area ± SEM per lxlO6 μιτι2 total area, n=3.

Figure 23. Efficiency of shRNA-mediated CCR8 knockdown in MDA-MB-435 and SKMEL-25 tumor cells. (A, B) RT-PCR for human CCR8 in different cell lines as indicated (A) and in tumors in vivo (B) . (C, D) Western blot analysis of CCR8 in different tumor cell lines in vitro (C) and in tumors in vivo (D) . CCR8 knockdown in MDA-MB-435 cells was performed with shCCR8 sequence 1, in SK-MEL-25 with the sequence 2, and in MDA cl . 13 with shCCR8 sequences 1 and 2.

Figure 24. Expression of lymphatic markers by the lymph node lymphatic vessels. (A-C) Double immunofluorescent staining for LYVE- 1 (green) and podoplanin (red) in lymph node sections of athymic mice. Note that the lymphatics of the SCS are podoplaninhigh /LYVE- llow, whereas the medullary sinuses are podoplaninlow /LYVE-lhigh. (D-F) Double immuno-fluorescent staining for LYVE-1 (green) and VEGFR3 (red) shows comparable expression pattern of these two markers. Scale bar: 100 μΜ. c, capsule; m, medulla.

Figure 25. Expression of CCL8 in the mouse skin, tumor and the lymph node lymphatic vessels. (A-I) Double immunostaining for either LYVE- 1 or podoplanin (both green, lymphatic vessels) and CCL8 (red) on tissue sections of mouse skin overlying a tumor (A-C), MDAcl.13 tumor xenograft (D-F), and tumor-draining lymph nodes (G-I) . Note that CCL8 is not detected in lymphatic vessels, ep, epidermis. Scale bars, 100 μιτι.

Figure 26. Intravital imaging of an in transit metastasis in the collecting, afferent lymphatic vessel. (A-C) Still images from the time-lapse movies obtained by multiphoton microscopy. (A) An afferent lymphatic vessel containing a large cluster of MDA/cl.13 tumor cells (GFP, green) . Evans blue tracer (white) labels lymphatic vessels and indicates that the lymph flow was maintained in the presence of the tumor. Images demonstrate no change in the position of a cell cluster within the 5 min observation period, indicating that the tumor cells were not moving with the flow. (B, C) Tumor cell clusters in the collecting lymphatic vessel on both sides of the valve. Lymphatic valve (dashed line) is shown closed and partially open at indicated time points. Note that the tumor cell cluster has not changed its position within the 65 min observation period (B) . Tumor cell motility is restricted to a single cell (arrow, dotted line) which migrates into a different plane within the 20 min from the onset of cell shape change (C) . Collagen fibers of the lymphatic vessel wall are visualized by SHG (blue) . Scale bars, 100 μιτι (A, B) , 30 μιτι (C) .

Detailed Description of the Invention

The present invention provides a method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCLl to the CCR8 receptors so as to thereby treat the subject.

The present invention provides a method of reducing, or reducing the likelihood of, metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCLl to the CCR8 receptors so as to thereby reduce, or reduce the likelihood of, metastases in the subject.

In some embodiments, lymph node metastases are reduced.

In some embodiments, the solid tumor is a melanoma.

In some embodiments, the antagonist of the CCR8 receptor is an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, a polypeptide, or an antibody, which antagonist binds to the extracellular domain of the CCR8 receptor at a site which prevents binding of a CCLl ligand to the CCR8 receptor.

In some embodiments, the antagonist is a polypeptide.

In some embodiments, the antagonist is an anti-CCR8 antibody. Anti- CCR8 antibodies are described in U.S. Patent No. 6, 762, 341, the entire contents of which are hereby incorporated herein by reference .

In some embodiments, the anti-CCR8 antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is 414B, 141C, 141E, 433H, 459M, 464A, 464B, 433B or 455AL. These monoclonal antibodies, as well as processes of producing these antibodies are described in PCT International Application No. WO/2007/044756, the entire contents of which are hereby incorporated herein by reference.

In some embodiments, the monoclonal antibody is a human or a humanized monoclonal antibody. In some embodiments, the human or humanized antibody comprises or is derived from the CCR8-binding region of one of monoclonal antibody 414B, 141C, 141E, 433H, 459M, 464A, 464B, 433B or 455AL.

In some embodiments, the CCR8 antagonist is MC148.

In some embodiments, the CCR8 antagonist is an organic compound having a molecular weight less than 1000 Daltons .

Organic Compound Structure I

In some embodiments, the organic compound has the structure:

Figure imgf000019_0001

wherein

B represents the group

Figure imgf000019_0002

ring D, together with the two benzene carbon atoms to which it is fused, is a 5- or 6- membered, non-aromatic ring containing one or two ring-oxygen atoms, and optionally containing a carbon-carbon double bond between two ring carbon atoms other than said benzene carbon atoms, ring D being optionally substituted with one or more substituents independently selected from Ci-C6 alkyl, C3-C6 cycloalkyl, or phenyl (said phenyl being optionally substituted with one or more substituents independently selected from halogen, hydroxyl or Ci-C4 alkoxy) ;

and additionally wherein when ring D is a 5-membered, non-aromatic ring containing two ring-oxygen atoms that are 1,3 disposed, ring D may be optionally substituted with group E, wherein group E together with a single carbon atom on ring D, represents a 4- to 8-membered cycloalkyl ring, such that group E forms a spiro structure with ring D; w, x, y and z are independently 1, 2 or 3;

each R represents a group independently selected from halogen or Ci-C4 alkyl;

n is 0, 1 or 2;

A represents a group selected from phenyl, a 5- or 6-membered heteroaromatic ring containing at least one ring heteroatom independently selected from nitrogen, oxygen or sulphur, or pyridine-N-oxide , each group being optionally substituted with one or more substituents independently selected from hydroxyl, -CN, halogen, oxo (=0) , Ci-C6 aminoalkyl, Ci-C6 alkylamino-Ci-C6 alkyl, N,N- di (Ci-C6) alkylamino-Ci-C6 alkyl, Ci-C6 alkoxy, Ci-C6 alkylcarbonyl , NRiR2, -C(0)-NR3R4, -CI-C6 alkyenyl-C (0) -NR3R4, -C1-C4 alkyl-C (0) -NR5R6, -NHSO2-R7, -NHC(0)R8, -S02NH2, carboxyl, carboxyl-Ci-C6 alkyl, Ci-C6 alkoxycarbonyl , C1-C4 alkoxycarbonyl-Ci-C4 alkyl, C3-C6 cycloalkylamino, phenyl, pyridyl (said phenyl and pyridyl being optionally further substituted with one or more groups independently selected from halogen, hydroxyl, carboxy or C1-C4 alkyl), Ci-C6 alkyl or C3-C6 cycloalkyl (said latter two Ci-C6 alkyl and C3-C6 cycloalkyl substituents being optionally further substituted with one or more substituents independently selected from halogen, hydroxyl, or -CN) ; or A represents a 9- or 10-membered bicyclic ring system containing one or more ring heteroatoms independently selected from nitrogen, oxygen or sulphur and which is optionally substituted with one or more substituents independently selected from hydroxyl, -CN, halogen, oxo, Ci-C6 alkoxy, -NR9R10, carboxyl, or Ci-C6 alkyl;

p is 0, 1 or 2; R1 and R2 each independently represent a hydrogen atom, a Ci-C6 alkyl, C3-C6 cycloalkyl or R1 and R2 together with the nitrogen atom to which they are attached form a hydantoin group or form a 4- to 7- membered saturated heterocycle, said heterocycle being optionally substituted with hydroxyl, Ci-C4 alkoxy, or Ci-C4 alkoxy-Ci-C4 alkyl;

R3 and R4 each independently represent a hydrogen atom, Ci-C6 alkyl, or C3-C6 cycloalkyl, or R3 and R4 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocycle, said heterocycle being optionally substituted with aminocarbonyl;

R5 and R6 each independently represent a hydrogen atom, Ci-C6 alkyl, or C3-C6 cycloalkyl, or R5 and R6 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocycle, said heterocycle being optionally substituted with aminocarbonyl;

R7 represents Ci-C6 alkyl, or a 6-membered saturated or unsaturated heterocyclic ring, the ring containing at least one nitrogen atom, the ring being optionally substituted with one or more substituents independently selected from halogen, oxo, Ci-C6 alkoxy, or Ci-C6 alkyl;

R8 represents pyridine-N-oxide optionally substituted with one or more substituents independently selected from halogen, or Ci-C6 alkyl, or R8 represents Ci-C6 alkyl, Ci-C6 hydroxyalkyl , or a 5- or 6- membered saturated heterocyclic ring containing at least one heteroatom independently selected from nitrogen and oxygen, which ring being optionally substituted with one or more substituents independently selected from halogen, Ci-C6 alkoxy, oxo, or Ci-C6 alkyl ;

R9 and R10 each independently represent a hydrogen atom or Ci-C6 alkyl;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2007/030061 and U.S. Patent Application No. US 2009-0156575, the entire contents of each of which are incorporated herein by reference. Organic Compound Structure II

In some embodiments, the organic compound has the structure:

Figure imgf000022_0001

wherein R represents pyridine N-oxide;

R1 represents the roup:

Figure imgf000022_0002

R is a methoxy or ethoxy

R4 is a hydrogen, methoxy or ethoxy;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2006/107252 and U.S. Patent Application Publication No. US 2009-0215807, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the organic compound has the structure:

Figure imgf000023_0001
or a pharmaceutically active salt or ether of any of the foregoing. Organic compounds having these structures are described in Connolly et al . , (2012) "Orally bioavailable allosteric CCR8 antagonists inhibit dendritic cell, T cell and eosinophil migration" Biochemical Pharmacology 83:778-787, the entire contents of which are incorporated herein by reference.

The organic compound having the structure

Figure imgf000024_0001

The organic compound having the structure

Figure imgf000024_0002
is also known as AZ491.

The organic compound having the structure

Figure imgf000024_0003
is also known as AZ442.

The organic compound having the structure

Figure imgf000024_0004

The organic compound has the structure

Figure imgf000025_0001
is also known as AZ435.

The organic compound having the structure

Figure imgf000025_0002

Organic Compound Structure III

In some embodiments, the organic compound has the structure:

Figure imgf000025_0003
wherein R represents
Figure imgf000026_0001

wherein R2 and R3 independently represent -NR8-C (0) -COOH, -0- (Ci- 4alkyl) -C00H, -Ci_4alkyl-C00H, or -COOH;

each R4 and R5 independently represent halogen, CF3 or Ci_ alkyl;

p and q are independently 0, 1 or 2;

R8 represents hydrogen or Ci_4alkyl;

R1 represents the group:

Figure imgf000026_0002

or

and R6 and R7 are independently hydrogen, methoxy or ethoxy;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2006/107253 and U.S. Patent Application Publication No. US 2009-0118318, the entire contents of which are incorporated herein by reference.

Organic Compound Structure IV

In some embodiments, the organic compound has the structure:

Figure imgf000026_0003

R ,¾1 (Formula II of Structure IV) wherein R is pyridin-2-one or iV-Ci-C alkyl pyridin-2-one;

or R is pyridin-2-one or iV-Ci-C alkyl pyridin-2-one each of which is substituted with a group selected from CF3, halogen or Ci-C4 alkyl; R1 represents the group:

Figure imgf000027_0001

and R3 and R4 are independently methoxy or ethoxy;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2006/107254, the entire contents of which are incorporated herein by reference.

According to the present invention there is also provided a process for the preparation of compounds of formula (II) above and salts thereof which comprises

(a) reacting a compound of formula (III) :

Figure imgf000027_0002

where R1 is as defined in formula (II), with a compound of formula

(N)

Figure imgf000027_0003

where R is as defined in formula (II) and LG is a suitable leaving group, or

(b) reaction of a compound of formula (V)

Figure imgf000028_0001
srein R is as defined in formula (II), with an aldehyde compound formula (VI ) :
Figure imgf000028_0002

1 (VI) wherein R1 is as defined in formula (II), or

(c) reaction of a compound of formula (V) defined above with compound of formula (VII)

Figure imgf000028_0003

R1 (VII) wherein R1 is as defined in formula (II) and LG is a leaving group.

A compound of formula (III) can be prepared by process (d) by reacting a compound of formula (VIII)

Figure imgf000028_0004

in which P is a protecting group, with a compound of formula (VI) as defined above, and subsequently removing the protecting group P.

A compound of formula (III) can also be prepared by process (e) by reacting a compound of formula (VIII) with a compound of formula (VII), and subsequently removing the protecting group P. A compound of formula (V) can be prepared by process (f) by reacting a compound of formula (IX) :

Figure imgf000029_0001

where P is a suitable protecting group with a compound of formula (IV) as defined above, and subsequently removing the protecting group P. Process (a) may be carried out using standard coupling reactions that are well know in the art. A suitable leaving group LG is, for example OH or chlorine, preferably OH. The coupling reaction may typically carried out using activating reagents such as N-[(1H- 1,2, 3benzotriazol-l-yloxy) (dimethylamino ) methylene] -N- methylmethanaminium hexafluorophosphate (HBTU) , N-

[ (dimethylamino) (3H- [l,2,3]triazolo[4,5-b] pyridin-3-loxy) methylene] - iV-methylmethanaminium hexafluorophosphate (HATU) , or (benzotriazol- 1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PYBOP) .

Typically, the reaction is carried out in the presence of a suitable base (e.g. triethylamine ) and an organic solvent (e.g. dichloromethane) at a suitable temperature (e.g. room temperature) .

Process (b) may be carried out using standard reductive amination procedures which are well known in the art. Typically, the reaction is carried out in the presence of sodium triacetoxyborhydride [NaBH (OAc) 3] . Typically, the reaction is carried out in the presence of a suitable base (e.g. triethylamine) and an organic solvent (e.g. dichloromethane) at a suitable temperature (e.g. room temperature) . Process (c) may be carried out in a suitable organic solvent (e.g. DMF) at a suitable temperature (e.g. room temperature) . The use of leaving groups are well known in the art for this type of reaction. Examples of typical leaving groups are halo, alkoxy, trifluoromethanesulfonyloxy, methanesulfonyloxy, or p- toluenesulfonyloxy . Typically, the leaving group is a halogen such as chlorine or bromine. The coupling step of process (d) may be carried out according to the conditions described for process (b) above. The coupling step of process (e) may be carried out according to the conditions described for process (c) above. The coupling step of process (f) may be carried out according to the conditions described for process (a) above. An example of a typical protecting group P used in processes (d) , (e) and (f) is tert-butyloxycarbonyl (t-boc) . However, other suitable protecting groups may be used. In this regard, the protection and deprotection of functional groups is fully described in 'Protective Groups in Organic Chemistry', edited by J. W. F. McOmie, Plenum Press (1973), and 'Protective Groups in Organic Synthesis', 2nd edition, T. W. Greene & P. G. M. Wuts, Wiley- Interscience (1991) . After the coupling the protecting group P can be removed. Compounds of formulae (IV), (VI), (VII), (VIII), and (IX) are either commercially available, are well known in the literature or may be prepared easily using known techniques, for example as shown in the accompanying Examples. U.S. Patent No. 5, 451, 578 (Claremon et al . ) describes, under example 1 of the patent, a process for synthesising tert-butyl 3 , 9-diazaspiro [ 5.5 ] undecane-3-carboxylate (corresponding to compound (IX) with P as tert-butyloxycarbonyl) .

In so far as the intermediates referred to in the processes of the present invention are capable of forming salts, the processes of the invention described above encompass the use of the intermediates in salt form or free form. The compounds of formula (II) above may be converted to a pharmaceutically acceptable salt thereof, preferably an acid addition salt such as a hydrochloride, hydrobromide, phosphate, acetate, fumarate, maleate, tartrate, citrate, oxalate, methanesulphonate orptoluenesulphonate . The compounds of formula (II) and pharmaceutically acceptable salts thereof may exist in solvated, for example hydrated, as well as unsolvated forms, and the present invention encompasses all such solvated forms.

Organic Compound Structure V

In some embodiments, the organic compound has the structure:

Figure imgf000031_0001

or a pharmaceutically acceptable salt thereof, wherein:

Xi is a covalent bond, C=0 or CRaRb;

X is a covalent bond, 0, or NR5;

C=Z is C=0, CH2, C=NH, C=S or absent, provided that when X1 is a covalent bond then C=Z is not CH2, provided that when C=Z is absent then X is a covalent bond, and provided that when X is NR5 and C=Z is C=0 then R4 is not an aliphatic or substituted aliphatic group;

Ra and Rb are independently H or a C1-C3 alkyl;

R1 is: i) a substituted or unsubstituted aromatic group; ii) a substituted or unsubstituted non-aromatic ring; or iii) when X is

NR5, then NR5- (CH2) mR1, taken together, is optionally a substituted or unsubstituted non-aromatic heterocyclic group;

R2 is -H or a C1-C3 alkyl group;

R3 is -H; and R4 is: i) a substituted or unsubstituted aliphatic group; ii) a substituted or unsubstituted aromatic group; iii) a substituted or unsubstituted non-aromatic ring or a substituted or unsubstituted non-aromatic bridged bicyclic group; or iv) R3 and R4 taken together with the nitrogen atom to which they are bonded are a substituted or unsubstituted nitrogen-containing non- aromatic heterocyclic group; R5 is -H, or a C1-C3 alkyl group;

Ring B is a phenyl group or five or six membered carbocyclic non-aromatic ring fused to Ring A;

Rings A and B are optionally substituted with alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyl, halogen, cyano or nitro; m is 0, 1, 2 or 3;

n is 1 or 2 ;

each substitutable carbon atom of the aromatic group represented by R1 is optionally substituted with a group represented by R10, wherein R10 is halogen, R°,-OH, -OR0, -0 (haloalkyl ) , -SH, - SR°, 1 , 2-methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted - CH2(Ph), -CH2CH2(Ph), substituted -CH2CH2(Ph), -N02, -CN, -N(R')2, - NR'C02R°, -NR'C(0)R°, -NR ' NR ' C (0 ) R°, -N (R ' ) C (0) N (R ' ) 2 , NR'NR'C(0)N(R' )2, -NR ' NR ' C02R°, -C(0)C(0)R°, -C (0) CH2C (0) R°, -C02R°, - C(0)R°, -C(0)N(R°)2, -0C(0)R°, -OC (0) N (R°) 2, -S (0) 2R°, -S02N(R')2, S(0)R°, -NR'S02N(R' )2, -NR'S02R°, -C (=S ) N (R ' ) 2 , - ( CH2 ) yN (R ' ) 2 , -C(=NH)- N(R')2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, -V-N02, -V-CN, -V-N(R')2, -V-NR'C02R°, -V-R'C(0)R°, -V-NR ' NR ' C (0 ) R°, -V- N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V-C (0 ) C (0 ) R°, - V-C (0) CH2C (0) R°, -V-C02R°, -V-C (0) R°, -V-C (0) N (R°) 2, -V-OC (0) R°, -V- 0C(0)N(R°)2, -V-S(0)2R°, -V-S02N(R' ) 2, -V-S (0) R°, -V-NR ' S02N (R ' ) 2 , -V- NR'S02R°, -V-C (=S) N (R' ) 2, -V- ( CH2 ) yN (R ' ) 2 , or -V-C (=NH) -N (R ' ) 2, and each group represented by R10 is independently selected;

each substitutable carbon atom of the aliphatic group represented by R4 is optionally substituted with a group represented by R11, wherein R11 is halogen, R°, -OH, -OR0, -0 (haloalkyl ) , -SH, - SR°, 1 , 2-methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted - CH2(Ph), -CH2CH2(Ph), substituted -CH2CH2(Ph), -N02, -CN, -N(R')2, - NR'C02R°, -NR'C(0)R°, -NR ' NR ' C (0 ) R°, -N (R ' ) C (0) N (R ' ) 2 ,

NR'NR'C(0)N(R' )2, -NR ' NR ' C02R°, -C (0) C (0) R°, -C (0) CH2C (0) R°, -C (0) R°, -C(0)N(R°)2, -0C(0)R°, -0C(0)N(R°)2, -S(0)2R°, -S02N(R')2, -S (0) R°, - NR' S02N (R' ) 2, -NR'S02R°, -C (=S) N (R' ) 2, - (CH2 ) yN (R ' ) 2 , ~C (=NH) -N (R' ) 2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, -V-N02, -V-CN, -V- N(R')2, -V-NR'C02R°, -V-NR' C (0) R°, -V-NR ' NR ' C (0 ) R°, -V-

N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V-C (0) C (0) R°, - V-C (0) CH2C (0) R°, -V-C(0)R°, -V-C (0) N (R°) 2, -V-OC (0) R°, -V-OC (0) N (R°) 2, -V-S(0)2R°, -V-S02N(R' ) 2, -V-S(0)R°, -V-NR ' S02N (R ' ) 2, -V-NR'S02R°, -V- C(=S)N(R')2, -V- (CH2) yN (R' ) 2, -V-C (=NH) -N (R ' ) 2 , =NNHR* , =NN(R*)2, =NNHC(0)R*, =NNHC02 (alkyl) , =NNHS02 (alkyl), or =NR* , and each group represented by R11 is independently selected;

each substitutable carbon atom of the aromatic group represented by R4 is optionally substituted with a group represented by R12, wherein R12 is halogen, R°, -OH, -OR0, -0 (haloalkyl ) , -SH, - SR°, 1 , 2-methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted - CH2(Ph), -CH2CH2(Ph), substituted -CH2CH2 (Ph) , -CN, -NR'C02R°, NR'NR' C (0) R°, -N (R')C(0)N(R')2, -NR ' NR ' C (0) N (R ' ) 2, -NR ' NR ' C02R°, C(0)C(0)R°, -C (0) CH2C (0) R°, -C02R°, -C(0)R°, -OC (0) R°, -OC (0) N (R°) 2, - S(0)2R°, -S02N(R')2, -S(0)R°, -NR ' S02N (R ' ) 2 , -NR'S02R°, -C (=S ) N (R ' ) 2 , - (CH2) yN (R' ) 2, -C (=NH) -N (R' ) 2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, -V-CN, -V-NR'C02R°, -V-NR ' NR ' C (0) R°, -V-N (R ' ) C (0) N (R ' ) 2 , -V- NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V-C (0) C (0) R°, -V-C (0) CH2C (0) R°, -V- C02R°, -V-C(0)R°, -V-0C(0)R°, -V-OC (0) N (R°) 2, -V-S(0)2R°, -V-S02N (R ' ) 2, -V-S(0)R°, -V-NR' S02N (R' ) 2, -V-NR'S02R°, -V-C (=S) N (R' ) 2, -V- (CH2)yN(R')2 or -V-C (=NH) -N (R ' ) 2 , and each group represented by R12 is independently selected;

each substitutable carbon atom of: i) the non-aromatic ring represented by R1 or R4; ii) the non-aromatic heterocyclic group formed from NR5(CH2)mR1; iii) the nitrogen-containing non-aromatic heterocyclic group formed from NR3R4; and iv) the non-aromatic bridged bicyclic group represented by R4 is optionally and independently substituted with -R°, -OH, -OR0, -0 (haloalkyl ) , -SH, - SR°, 1 , 2-methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted - CH2(Ph), -CH2CH2(Ph), substituted -CH2CH2(Ph), -CN, -N02, -N(R')2, - C(0)N(R°)2, -NR'C(0)R°, -NR'C(0)R°, -V-N(R')2, -V-NR ' C (0) R°, -V- C(0)N(R°)2, -NR'C02R°, -NR ' NR ' C (0) R°, -N (R ' ) C (0) N (R ' ) 2 ,

NR'NR'C(0)N(R' )2, -NR ' NR ' C02R°, -C(0)C(0)R°, -C (0) CH2C (0) R°, -C02R°, - C(0)R°, -0C(0)R°, -0C(0)N(R°)2, -S(0)2R°, -S02N(R')2, -S(0)R°, NR' S02N (R' ) 2, -NR'S02R°, -C (=S) N (R' ) 2, - (CH2 ) yN (R ' ) 2 , -C (=NH) -N (R' ) 2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, -NR'C(0)R°, -V-N02, - V-CN, -V-N(R')2, -V-NR'C02R°, -V-NR ' C (0) R°, -V-NR ' NR ' C (0) R°, -V- N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V-C (0 ) C (0 ) R°, - V-C (0) CH2C (0) R°, -V-CO2R0, -V-C(0)RO, -V-C (0) N (R°) 2, -V-OC(0)R°, -V- OC(0)N(R°)2, -V-S(0)2R°, -V-S02N(R' ) 2, -V-S (0) R°, -V-NR ' S02N (R ' ) 2 , -V- NR'S02R°, -V-C (=S) N(R' ) 2, -V- ( CH2 ) yN (R ' ) 2 , -V-C (=NH) -N (R ' ) 2 , =0, =S, =NNHR* , =NN(R*)2, =NNHC(0)R*, =NNHC02 (alkyl ) , =NNHS02 (alkyl) or =NR* ;

each R° is independently hydrogen or substituted or unsubstituted aliphatic group, a substituted or unsubstituted cycloaliphatic , a substituted or unsubstituted non-aromatic heterocyclic group or a substituted or unsubstituted aromatic group selected from phenyl, naphthyl, 2-furanyl, 3-furanyl, N-imidazolyl , 2-imidazolyl , 4-imidazolyl, 5-imidazolyl , 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl , 2- oxadiazolyl, 5-oxadiazolyl , 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2- pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- pyrimidyl, 3-pyridazinyl , 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2- triazolyl, 5-triazolyl, tetrazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl , benzothienyl , benzofuranyl , indolyl, quinolinyl, benzotriazolyl , benzothiazolyl , benzooxazolyl , benzimidazolyl, isoquinolinyl , indolyl, isoindolyl, acridinyl, or benzoisazolyl;

each R' is independently R°, -C02R°, -S02R° or -C (0) R°;

each R* is independently hydrogen, an unsubstituted aliphatic group or a substituted aliphatic group;

V is a C1-C6 alkylene group;

each substitutable carbon atom of: i) the aliphatic, cycloaliphatic, non-aromatic heterocyclic group and aromatic group represented by R°; and ii) the aliphatic group represented by R* is optionally and independently substituted with amino, alkylamino, dialkylamino , aminocarbonyl , halogen, alkyl, aminoalkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl , alkylcarbonyl , hydroxy, haloalkoxy, or haloalkyl; each substitutable nitrogen atom of: i) the non-aromatic ring represented by R1 or R4; ii) the non-aromatic heterocyclic group formed from NR5(CH2)mR1; iii) the nitrogen-containing non-aromatic heterocyclic group formed from NR3R4; iv) the non-aromatic heterocyclic group represented by R°; and v) the non- aromatic bridged bicyclic group represented by R4 is optionally and independently substituted with R+, -N(R+)2, -C (0) R+, -C02R+, C(0)C(0)R+, -C (0) CH2C (0) R+, -S02R+, -S02N(R+)2, -C (=S ) N (R+) 2, -C (=NH) - N(R+)2, -NR+S02R+, -C(0)-NHR+, -C (0) -N (R+) 2, -C (0) -CH [N (R+) 2] R+ or - C(0) -CH[0R+]R+;

each R+ is independently hydrogen, an unsubstituted heteroaryl or an aliphatic, cycloaliphatic , non-aromatic heterocyclic group, phenyl or benzyl group, wherein each substitutable carbon atom of the aliphatic, cycloaliphatic, nonaromatic heterocyclic ring, phenyl or benzyl group represented by R+ is optionally substituted with amino, alkylamino, dialkylamino, aminocarbonyl , halogen, alkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl , alkylcarbonyl , hydroxy, haloalkoxy, or haloalkyl or N(R+)2 is a non-aromatic heterocyclic group; and

each substitutable nitrogen atom of the non-aromatic heterocyclic group represented by R+ is optionally substituted with alkyl, alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl or dialkylaminocarbonyl ,

provided that the compound is other than N-[4-[[(4- methoxyphenyl ) amino] sulfonyl ] -1-naphthalenyl ] -benzamide, N- [4- [ ( 2- propenylamino) sulfonyl] -1-naphthalenyl] -benzamide, N- [4- (4- morpholinylsulfonyl) -1-naphthalenyl] -benzamide or N-[4-(l- piperidinylsulfonyl) -1-naphthalenyl] -benzamide . Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2004/058736, and U.S. Patent No. 7,329,755, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the organic compound has the structure:

Figure imgf000036_0001

Organic Compound Structure VI

In some embodiments, the organic compound has the structure:

Figure imgf000036_0002

or a pharmaceutically acceptable salt thereof, wherein:

Ring A is unsubstituted;

Ring B is an unsubstituted phenyl, cyclohexyl or cyclopentyl ring fused to Ring A;

R1 is cyclohexyl or phenyl, furanyl, thienyl or pyridyl optionally substituted with C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, methylenedioxy , ethylenedioxy , halogen, cyano or nitro;

Figure imgf000037_0001

NHR' -C (0) -N (R22) 2 or -C(0)CH[N(R2J)2]R'

Rz is methyl, ethyl, 2-hydroxyethyl or iso-propyl;

Rz is -H or C1-C4 alkyl or -N (R^) taken together is N- pyrollidinyl or iV-piperidinyl , provided that R is not -H when R is -COOR22;

R23 is -H, methyl or ethyl;

R24 is -H, methyl, ethyl, phenyl, benzyl, 4-hydroxyphenyl or 4- hydroxybenzyl ; and s is 0, 1, 2, 3 or 4. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2004/058736, and U.S. Patent No. 7,329,755, the entire contents of each of which are hereby incorporated herein by reference .

Organic Compound Structure VII

I some embodiments, the organic compound has the structure:

Figure imgf000037_0002
(Formula I of Structure VII) wherein

n is 0, to 6;

m is 1, to 4 ;

p is 1, to 4; Ar is unsubstituted naphtha-2-yl, benzo [ 1 , 3 ] dioxolyl , 2 , 3- dihydro-benzo [ 1 , 4 ] dioxinyl , benzothiophenyl , benzofuranyl , or quinolinyl; or naphth-2-yl, benzo [ 1 , 3 ] dioxolyl , 2,3-dihydro- benzo [ 1 , 4 ] dioxinyl , benzothiophenyl, benzofuranyl , quinolinyl substituted with one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano, hydroxy, amino, -Ci-Ce alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-C8 alkyl carbamoyl, Ci-C8 alkoxy carbonyl and trihalomethyl;

Ri and R6 are independently selected from the group consisting of hydrogen, Ci-C8 branched or unbranched alkyl, C3-C8 cycloalkyl lower alkyl, and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci~C8 alkyl, halo, cyano and trihalomethyl;

R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, Ci-C8 branched or unbranched alkyl, C3-C8 cycloalkyl lower alkyl, Ci-C8 alkoxy, halo, cyano, trihalomethyl, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-C8 alkyl carbamoyl, Ci-C8 alkoxy carbonyl and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano and trihalomethyl;

R7 is hydrogen, unsubstituted or substituted C1-C10 branched or unbranched alkyl, unsubstituted or substituted C3-C8 cycloalkyl lower alkyl, or unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci~C8 alkyl carbamoyl, Ci~C8 alkoxy carbonyl and trihalomethyl ;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2004/074438, the entire contents of which are hereby incorporated herein by reference. Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of the present invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984) .

The term "Ci-C8 alkyl" and "Ci-Cio alkyl" is used herein includes both straight or branched chain radicals of 1 to 8 or 10 carbon atoms. By example this term includes, but is not limited to methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl and the like. "Lower alkyl" has the same meaning as Ci-C8 alkyl .

In the organic compound structure above, "Ci-C8 alkoxy" includes straight and branched chain radicals of the likes of 0-CH3, -0- CH2CH3, and the n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentoxy, and hexoxy, and the like.

"C3-C8-cycloalkyl" as applied herein is meant to include substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane and cyclohexane .

"Halogen" or "halo" means F, CI, Br, and I. Synthetic Methods

Synthetic methods to prepare the compounds of this invention frequently employ protective groups to mask a reactive functionality or minimize unwanted side reactions. Such protective groups are described generally in Green, T.W, Protective Groups in Organic Synthesis, John Wiley & Sons, New York (1981) .

Scheme 1 represents a method for making compounds of Formula (I) Scheme 1

Figure imgf000040_0001

Conditions: a) DMHB resin, Na(OAc)3BH, AcOH, NMP, rt; b) R^OzCl, pyridine, DMAP, DCE, rt; c) TMSOK, THF, rt; d) cyclic diamines, PyBOP, DIEA, NMP, rt; e) R2CHO, Na(OAc)3BH, AcOH, NMP, rt; f) 20% TFA in DCM, rt.

In Scheme 1, ethyl-3-aminobenzoates (1) are loaded onto 2,6- dimethoxy-4-polystyrenebenzyloxy-benzaldehyde (DMHB resin) via reductive amination. Sulfonylation of the resulting resin-bound anilines with various sulfonyl chlorides gives sulfonamides 2 . After hydrolysis, the resulting resin-bound acids are coupled with cyclic diamines to afford intermediates 3 . Reductive amination of 3 with various aldehydes, followed by cleavage from resin, affords the targeted compounds 4.

In some embodiments the organic compound has the structure:

Figure imgf000040_0002

or a pharmaceutically active salt or ether thereof.

Organic Compound Structure VIII

In some embodiments, the organic compound has the structure:

Figure imgf000041_0001

w (Formula I of Structure

VIII)

wherein

R1 represents a saturated or unsaturated 5- to 15-membered ring system optionally comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted with at least one substituent selected from halogen, cyano, oxo, mercapto, nitro, hydroxyl, carboxyl, -S02NH2, -NR5R6, C(0)NR7R8, Ci-C6 alkoxy, Ci-C6 alkoxycarbonyl , Ci-C6 alkylcarbonyl , Cx- C6 alkyl (optionally substituted by at least one substituent selected from halogen, hydroxyl, cyano, carboxyl, Ci-C6 alkoxycarbonyl, -NR9R10 and -C (0) NRX1R12) , C2-C6 alkenyl (optionally substituted by -C (0) NR13R14) , C3-C6 cycloalkyl (optionally substituted by at least one substituent selected from halogen, hydroxyl and cyano) , -NHS02-R15, and a saturated or unsaturated 5- to 6-membered heterocyclic ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur, the heterocycylic ring itself being optionally substituted by at least one substituent selected from halogen, hydroxyl, carboxyl and Ci-C6 alkyl;

n is O or l;

R2 and R3 each independently represent hydrogen, hydroxyl, Ci-C6 alkyl, Ci-C6 alkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkoxy, C3-C6 cycloalkyl or C6-Ci0 aryl, the aryl group being optionally substituted with at least one substituent selected from halogen, hydroxyl, Ci-C6 alkyl and Ci-C6 alkoxy, or

R2 and R3 together with the carbon atom to which they are attached form a 3- to 6-membered saturated carbocyclic ring;

R4 represents a group

Figure imgf000041_0002
in which ring B, together with the two carbon atoms of ring A to which it is fused, represents a 5- to 6-membered, saturated or unsaturated, non-aromatic heterocyclic ring comprising one ring oxygen atom and optionally one or more ring heteroatoms independently selected from nitrogen, oxygen and sulphur, the group (Ι') being optionally substituted with at least one substituent selected from halogen, Ci-C6 alkyl, C3-C6 cycloalkyl and phenyl, the phenyl itself being optionally substituted with at least one substituent selected from halogen, hydroxyl and Ci-C6 alkoxy;

R5 and R6 each independently represent hydrogen, Ci-C6 alkyl or

C3-C6 cycloalkyl, or R5 and R6 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocyclic ring optionally substituted by at least one substituent selected from hydroxyl, Ci-C6 alkoxy and Ci-C6 alkoxy-Ci-C6 alkyl;

R7 and R8 each independently represent hydrogen, Ci-C6 alkyl or

C3-C6 cycloalkyl, or R7 and R8 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocyclic ring optionally substituted by at least one aminocarbonyl ;

R9 and R10 each independently represent hydrogen, Ci-C6 alkyl or

C3-C6 cycloalkyl, or R9 and R10 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocyclic ring optionally substituted by at least one substituent selected from hydroxyl, Ci-C6 alkoxy and Ci-C6 alkoxy-Ci-C6 alkyl;

R11 and R12 each independently represent hydrogen, Ci-C6 alkyl or

C3-C6 cycloalkyl, or R11 and R12 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocyclic ring optionally substituted by at least one aminocarbonyl ;

R13 and R14 each independently represent hydrogen, Ci-C6 alkyl or

C3-C6 cycloalkyl, or R13 and R14 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocyclic ring optionally substituted by at least one aminocarbonyl; and

R15 represents a Ci-C6 alkyl group or a 6-membered saturated or unsaturated heterocyclic ring comprising at least one ring nitrogen atom, the heterocyclic ring being optionally substituted with at least one substituent selected from halogen, oxo, Ci-C6 alkyl and Cx- C6 alkoxy;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2008/099165, the entire contents of which are hereby incorporated herein by reference.

The present invention further provides a process for the preparation of a compound of formula I above or a pharmaceutically acceptable salt thereof as defined above which comprises reacting a compound of formula (II)

Figure imgf000043_0001

wherein L represents a leaving group (e.g. a hydroxyl group or halogen atom) and n, R1, R2 and R3 are as defined in formula (I), with a compound of formula (III)

Figure imgf000043_0002
wherein R4 is as defined in formula (I) , in the presence of a coupling agent (such as carbonyldiimidazole and 1- hydroxybenzotriazole ) , and optionally thereafter carrying out one or more of the following procedures:

· converting a compound of formula (I) into another compound of formula ( I ) , removing any protecting groups,

forming a pharmaceutically acceptable salt.

Compounds of formula (III) may be prepared by reacting a compound of formula (IV)

Figure imgf000044_0001

wherein Pi represents a protecting group (an amine protecting group such as tert-butyl carbamate (BOC) , with a compound of formula (V) , R4-C(0)H, wherein R4 is as defined in formula (III), in the presence of a reducing agent (such as sodium borohydridetriacetate ) , and thereafter removing the protecting group Px (e. g. by contacting the compound obtained with methanolic hydrogen chloride) . The reaction between the compounds of formula (IV) and (V) is conveniently carried out in an organic solvent (e.g. dichloromethane) and at room temperature (20 °C) . The deprotection step may conveniently be carried out in an organic solvent (e.g. methanol) and at room temperature (20°C) .

Compounds of formulae (II), (IV) and (V) are either commercially available, are well known in the literature or may be prepared easily using known techniques.

It will be appreciated by those skilled in the art that in the processes of the present invention certain functional groups such as hydroxyl or amino groups m the reagents may need to be protected by protecting groups. Thus, the preparation of the compounds of formula (I) may involve, at an appropriate stage, the removal of one or more protecting groups.

The protection and deprotection of functional groups is described in ' Protective Groups in Organic Chemistry', edited by J.W.F. McOmie, Plenum Press (1973) and 'Protective Groups in Organic Synthesis', 3rd edition, T.W. Greene and P.G.M. Wuts, Wileylnterscience (1999) .

The compounds of formula (I) above may be converted to a pharmaceutically acceptable salt thereof, preferably a basic addition salt such as a sodium, potassium, calcium, aluminium, lithium, magnesium, zinc, benzathine, chloroprocaine, choline, diethanolamine , ethanolamine, ethyldiamine , meglumine, tromethamine or procaine salt, or an acid addition salt such as a hydrochloride, hydrobromide , phosphate, acetate, trifluoroacetate, benzenesulfonate, fumarate, maleate, tartrate, citrate, oxalate, methanesulphonate or p-toluenesulphonate salt.

The compounds of formula (I) and pharmaceutically acceptable salts thereof may exist in solvated, for example hydrated, as well as unsolvated forms, and the present invention encompasses all such solvated forms .

Compounds of formula (I) are capable of existing in stereoisomeric forms. It will be understood that the invention encompasses the use of all geometric and optical isomers (including atropisomers ) of the compounds of formula (I) and mixtures thereof including racemates. The use of tautomers and mixtures thereof also form an aspect ofthe present invention. Enantiomerically pure forms are particularly desired.

It will be appreciated that the compounds of formula (I) and pharmaceutically acceptable salts thereof may exist as zwitterions. In this regard, the representation of formula (I) and covers zwitterionic forms and mixtures thereof in all proportions.

Organic Compound Structure IX

In some embodiments, the organic compound has the structure:

Figure imgf000046_0001

or physiologically acceptable salt thereof; wherein

L is selected from the group consisting of a 0, S, NRa, a bond, S02, -C(=0), and (CR'R")m;

Ra is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkylaryl, and optionally substituted cycloalkyl;

a is 0 to 3;

b is 0 to 3;

m is 1 to 8;

R' and R" are independently selected from the group consisting of hydrogen, optionally substituted alkyl, cyano and optionally substituted alkenyl;

R6, R7, R8, R9 and R10 are independently selected from the group consisting of hydrogen, hydroxyl, halogen, optionally substituted Ci-Cio alkyl, optionally substituted C2-Ci0 alkenyl, optionally substituted C2-Ci0 alkynyl, optionally substituted C3-Ci0 cycloalkyl, optionally substituted C3-Ci0 cycloalkenyl , optionally substituted C3-C10 cycloalkynyl , optionally substituted C3-C10 cycloalkoxy, cyano, C1-C10 alkoxy, C2-Ci0 alkenyloxy, C2-Ci0 alkynyloxy, benzyloxy, optionally substituted amino, optionally substituted amido, -0 (CF3) , -C(=0)0(R1), -C(=0) (R1), -S02 R!R2, trifluoromethyl , aryl, aralkyl, heteroaryl and heteroaralkyl ;

R1 and R2 are independently selected from the group consisting of hydrogen and optionally substituted alkyl;

Q3 is optionally substituted alkyl;

R11, R12, R13, R14, R15, R16, R17, R18 and R19 are each independently selected from the group consisting of hydrogen, hydroxyl, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, cyano, alkoxy, alkenyloxy, alkynyloxy, benzyloxy, optionally substituted amino, optionally substituted amido, -0 (CF3) , -C (=0) 0 (R4i) , -C(=0) (R4i), -S02NR4iR , tri fluoromethyl , aryl, aralkyl, heteroaryl and heteroaralkyl;

R41 and R42 are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl , optionally substituted cycloalkynyl , optionally substituted amino, trifluoromethyl , aryl, aralkyl, heteroaryl and heteroaralkyl; or R41 and R42 may be linked via a C2-C8 optionally substituted alkyl or alkenyl bridge where one or more carbons may be replaced by 0, S or

NR

selected from the group consisting of

Figure imgf000047_0001
-CH2 and a bond; e is 1 to 3;

f is 1 to 7;

g is 0 to 3;

h is 0 to 3;

i is 0 or 1 ;

R20 and R46 are independently hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted amino, optionally substituted amido, -C(=0)0(R4i) , C(=0) (R ), -S02NR4iR , trifluoromethyl, aryl, aralkyl, heteroaryl or heteroaralkyl ; and

Q6 is selected from the group consisting of optionally substituted aromatic ring, optionally substituted non-aromatic heterocycle, and optionally substituted heteroaromatic ring; or

R18 or R19 together with Q5Q6 and the atoms to which they are bonded form an optionally substituted non-aromatic carbocyclic group, optionally substituted nonaromatic heterocyclic group, optionally substituted aryl ring or optionally substituted heteroaryl ring;

with the proviso that the compound is not

or

Figure imgf000048_0001
Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2003/037271 and U.S. Patent Application Publication No. US 2005-0143372, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the organic compound has the structure:

Figure imgf000048_0002

or a pharmaceutically active salt or ether thereof.

Organic Compound Structure X

In some embodiments, the organic compound has the structure:

Figure imgf000048_0003

or a pharmaceutically acceptable salt thereof, wherein:

Xi is a covalent bond, C=0 or CRaRb; X is a covalent bond, 0, or NR5;

C=Z is C=0, CH2, C=NH, C=S or absent, provided that when Xi is a covalent bond then C=Z is not CH2, provided that when C=Z is absent then X is a covalent bond;

Ra and Rb are independently H or a C1-C3 alkyl;

R1 is: i) a substituted or unsubstituted aromatic group; ii) a substituted or unsubstituted non-aromatic ring; or iii) when X is NR5, then NR5 (CH2 ) mR1 , taken together, is optionally a substituted or unsubstituted non-aromatic heterocyclic group;

R2 is -H or a C1-C3 alkyl group;

R3 is -H;

R4 is: i) a substituted or unsubstituted phenyl group, benzyl group or phenethyl group; or ii) a substituted or unsubstituted non- aromatic ring;

R5 is -H, or a C1-C3 alkyl group;

Ar is a bicyclic aromatic group comprising a first six membered aromatic group fused to a second six membered aromatic group or a five or six membered non-aromatic ring, wherein the group represented by Ar is optionally substituted with one or more substituents selected from alkyl, haloalkyl, halogen, cyano, nitro, hydroxy, haloalkoxy and alkoxy;

m is 0, 1, 2 or 3;

n is 1 or 2 ;

each substitutable carbon atom of the aromatic group represented by R1 is optionally substituted with a group represented by R10, wherein R10 is halogen, -R°, -OH, -OR0, -0 (haloalkyl ) , -SH, - SR°, 1 , 2-methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted - CH2(Ph), -CH2CH2(Ph), substituted -CH2CH2 (Ph) , -N02, -CN, -N(R')z, - NR'C02R°, -NR'C(0)R°, -NR ' NR ' C (0 ) R°, -N (R ' ) C (0) N (R ' ) 2

NR'NR'C(0)N(R' )2, -NR ' NR ' C02R°, -C (0) C (0) R°, -C (0) CH2C (0) R°, -C02R°, C(0)R°, -C(0)N(R°)2, -0C(0)R°, -0C (0) N (R°) 2, -S (0) 2R°, -S02N(R')2, S(0)R°, -NR' S02N (R' ) 2, -NR'S02R°, -C (=S ) N (R ' ) 2 , - ( CH2 ) yN (R ' ) 2 , -C(=NH) N(R')2, haloalkyl, V-R°, -V-0H, -V-0R0, -V-SH, -V-SR0, -V-N02, -V-CN -V-N(R')2, -V-NR'C02R°, -V-NR ' C (0) R°, -V-NR ' NR ' C (0) R°, -V

N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V-C (0 ) C (0 ) R°, V-C (0) CH2C (0) R°, -V-C02R°, -V-C (0) R°, -V-C (0) N (R°) 2, -V-0C(0)R°, -V- OC(0)N(R°)2, -V-S(0)2R°, -V-S02N(R' ) 2, -V-S (0) R°, V-NR ' S02N (R ' ) 2 , -V- NR'S02R°, -V-C (=S) N(R' ) 2, -V- (CH2 ) yN (R ' ) 2 , or -V-C (=NH) -N (R' ) 2;

each substitutable carbon atom of the phenyl, benzyl or phenethyl group represented by R4 is optionally substituted with a group represented by R12, wherein R12 is halogen, -R°, -OH, -OR0,

0 (haloalkyl ) , -SH, -SR°, 1 , 2-methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2 (Ph) , substituted -CH2(Ph), -CH2CH2 (Ph) , substituted -CH2CH2(Ph), -CN, - NR'C02RO, -NR'NR'C (0)R°, -N (R')C(0)N(R')2, -NR ' NR ' C (0) N (R ' ) 2, NR'NR'C02R°, -C(0)C(0)R°, -C (0) CH2C (0) R°, -C02R°, -C(0)R°, -OC (0) R°, - 0C(0)N(R°)2, -S(0)2R°, -S02N(R')2, -S (0) R°, -NR ' S02N (R ' ) 2 , -NR'S02R°, - C(=S)N(R')2, - (CH2) yN (R' ) 2, -C (=NH) -N (R' ) 2, haloalkyl, -V-R°, -V-OH, - V-0R°, -V-SH, -V-SR0, -V-CN, -V-NR'C02R°, -V-NR ' NR ' C (0) R°, -V- N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V-C (0) C (0) R°, - V-C (0) CH2C (0) R°, -V-C02R°, -V-C (0) R°, -V-0C(0)R°, -V-OC (0) N (R°) 2, -V- S(0)2R°, -V-S02N (R' ) 2, -V-S (0) R°, -V-NR' S02N (R' ) 2, -V-NR'S02R°, -V- C(=S)N(R')2, -V- (CH2) yN(R' ) 2 or -V-C (=NH) -N (R ' ) 2, wherein each group represented by R12 is independently selected;

each substitutable carbon atom of: i) the non-aromatic ring represented by R1 or R4, ii) the non-aromatic heterocyclic group formed from NR5(CH2)mR1 is optionally and independently substituted with -R°, -OH, -OR0, -0 (haloalkyl ) , -SH, -SR°, 1 , 2-methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted -CH2 (Ph) , -CH2CH2 (Ph) , substituted - CH2CH2(Ph), -CN, -N02, -N(R')2, -C(0)N(R°)2, -NR'C(0)R°, -NR'C(0)R°, - V-N(R')2, -V-NR' C (0) R°, -V-C (0) N (R°) 2, -NR'C02R°, -NR ' NR ' C (0) R°, - N (R' ) C (0) N (R' ) 2, -NR'NR'C (0)N (R1 ) 2, -NR ' NR ' C02R°, -C (0) C (0) R°, C (0) CH2C (0) R°, -C02R°, -C(0)R°, -OC (0) R°, -OC (0) N (R°) 2, -S(0)2R°, - S02N(R')2, -S(0)R°, -NR' S02N(R' ) 2, -NR'S02R°, -C (=S) N (R' ) 2, (CH2) yN (R' ) 2, -C (=NH) -N (R' ) 2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, -NR'C(0)R°, -V-N02, -V-CN, -V-N(R')2, -V-NR'C02R°, -V- NR'C(0)R°, -V-NR'NR' C (0) R°, -V-N (R ' ) C (0) N (R ' ) 2 , -V-NR ' NR ' C (0) N (R ' ) 2 , -V-NR'NR'C02R°, -V-C (0) C (0) R°, -V-C (0) CH2C (0) R°, -V-C02R°, -V-C (0) R°, - V-C(0)N(R°)2, -V-0C(0)R°, -V-0C(0)N(R°)2, -V-S (0) 2R°, -V-S02N (R' ) 2, -V- S (0) R°, -V-NR' S02N (R' ) 2, -V-NR'S02R°, -V-C (=S ) N (R ' ) 2, -V- (CH2 ) yN (R ' ) 2 , - V-C (=NH) -N (R' ) 2, =0, =S, =NNHR* , =NN(R*)2, =NNHC(0)R*,

=NNHC02 (alkyl) , =NNHS02 (alkyl), or =NR* ; each R° is independently hydrogen or substituted or unsubstituted aliphatic group, a substituted or unsubstituted cycloaliphatic , a substituted or unsubstituted non-aromatic heterocyclic group or a substituted or unsubstituted aromatic group selected from phenyl, naphthyl, 2-furanyl, 3-furanyl, N-imidazolyl , 2-imidazolyl , 4-imidazolyl, 5-imidazolyl , 2-oxadiazolyl , 5- oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2- pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl , 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-triazolyl, 5-triazolyl, tetrazolyl, 2-thienyl, 3- thienyl, carbazolyl, benzimidazolyl , benzothienyl , benzofuranyl , indolyl, quinolinyl, benzotriazolyl , benzothiazolyl , benzooxazolyl , benzimidazolyl, isoquinolinyl , indolyl, isoindolyl, acridinyl or benzoisazolyl;

each R' is independently R°, -C02R°, -S02R° or -C (0) R°;

each R* is independently hydrogen, an unsubstituted aliphatic group or a substituted aliphatic group;

V is a C1-C6 alkylene group; and

each substitutable carbon atom of: i) the aliphatic, cycloaliphatic, nonaromatic heterocyclic group and aromatic group represented by R°; and ii) the aliphatic group represented by R* is optionally and independently substituted with amino, alkylamino, dialkylamino , aminocarbonyl , halogen, alkyl, aminoalkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl , alkylcarbonyl , hydroxy, haloalkoxy, or haloalkyl; each substitutable nitrogen atom of: i) the non-aromatic ring represented by R1 or R4; ii) the non-aromatic heterocyclic group formed from NR5(CH2)mR1; and iii) the non-aromatic heterocyclic group represented by R° is optionally and independently substituted with R+, -N(R+)2, -C(0)R+, -C02 R+, -C(0)C(0)R+, -C (0) CH2 C(0)R+, -S02R+, - S02N(R+)2, -C (=S) N (R+) 2, -C (=NH) -N (R+) 2, -NR+S02R+, -C(0)-NHR+, -C(0)- N(R+)2, -C(0)-CH[N(R+)2]R+ or -C (0) -CH [0R+] R+;

each R+ is independently hydrogen, an unsubstituted heteroaryl or an aliphatic, cycloaliphatic, non-aromatic heterocyclic group, phenyl or benzyl group, wherein each substitutable carbon atom of the aliphatic, cycloaliphatic, non-aromatic heterocyclic ring, phenyl or benzyl group represented by R+ is optionally substituted with amino, alkylamino, dialkylamino , aminocarbonyl , halogen, alkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl , alkylcarbonyl , hydroxy, haloalkoxy, or haloalkyl or N(R+)2 is a non-aromatic heterocyclic group; and

each substitutable nitrogen atom of the non-aromatic heterocyclic group represented by R+ is optionally substituted with alkyl, alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl or dialkylaminocarbonyl;

provided that the compound is other than N-[4-[[(4- methoxyphenyl ) amino] sulfonyl ] -1-naphthalenyl ] -benzamide, N- [4- [ ( 2- propenylamino) sulfonyl] -1-naphthalenyl] -benzamide, N- [4- (4- morpholinylsulfonyl) -1-naphthalenyl] -benzamide or N-[4-(l- piperidinylsulfonyl) -1-naphthalenyl] -benzamide . Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2004/058709 and U.S. Patent No. 7,378,525, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments the organic compound has the structure:

Figure imgf000052_0001

or a pharmaceutically active salt or ether thereof.

Organic Compound Structure XI

In some embodiments, the organic compound has the structure:

Figure imgf000053_0001

or a pharmaceutically acceptable salt thereof, wherein:

R1 is cyclohexyl or phenyl, furanyl, thienyl or pyridyl optionally substituted with C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, methylenedioxy, ethylenedioxy , halogen, cyano or nitro;

Figure imgf000053_0002

-NHR , -C(0)-N(R^)2 or -C (0) CH [N (R^) 2] R ;

R21 is methyl, ethyl, 2-hydroxyethyl or iso-propyl;

R is -H or C1-C4 alkyl or -N(R )2 taken together is N- pyrollidinyl or iV-piperidinyl , provided that R is not -H when R is -COOR22;

R23 is -H, methyl or ethyl;

R24 is -H, methyl, ethyl, phenyl, benzyl, 4-hydroxyphenyl or 4- hydroxybenzyl ; and s is 0, 1, 2, 3 or 4. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2004/058709 and U.S. Patent No. 7,378,525, the entire contents of each of which are hereby incorporated herein by reference .

Organic Compound Structure XII

In some embodiments, the organic compound has the structure:

Figure imgf000054_0001

(Formula I of Structure XII) wherein

n is 0, to 6;

m is 1, to 4 ;

p is 1, to 4 ;

Ar is unsubstituted phenyl, thiophenyl, furanyl, or pyridinyl; or phenyl, thiophenyl, furanyl, or pyridinyl substituted with one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci~ C8 alkyl, halo, cyano, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di- Ci-C8 alkyl amino, carboxyl, Ci-C8 alkyl carbamoyl, Ci-C8 alkoxy carbonyl and trihalomethyl;

Rx and R6 are independently selected from the group consisting of hydrogen, Ci~C8 branched or unbranched alkyl, C3-C8 cycloalkyl lower alkyl, and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano and trihalomethyl;

R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, Ci-C8 branched or unbranched alkyl, C3-C8 cycloalkyl lower alkyl, Ci~C8 alkoxy, halo, cyano, trihalomethyl, hydroxy, amino, N-Ci~C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-C8 alkyl carbamoyl, Ci-C8 alkoxy carbonyl and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano and trihalomethyl;

R7 is hydrogen, unsubstituted or substituted Ci-Cio branched or unbranched alkyl, unsubstituted or substituted C3-C8 cycloalkyllower alkyl, or unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-C8 alkyl carbamoyl, Ci-C8 alkoxy carbonyl and trihalomethyl;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2004/073619, the entire contents of which are hereby incorporated herein by reference.

Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of the present invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984) .

The term "Ci-C8 alkyl" and "Ci-Ci0 alkyl" is used herein includes both straight or branched chain radicals of 1 to 8 or 10 carbon atoms. By example this term includes, but is not limited to methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl and the like. "Lower alkyl" has the same meaning as Ci-C8 alkyl . In the organic compound structure above, "Ci-C8 alkoxy" includes straight and branched chain radicals of the likes of 0-CH3, -0- CH2CH3, and the n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentoxy, and hexoxy, and the like. "C3-C8-cycloalkyl" as applied herein is meant to include substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane and cyclohexane . "Halogen" or "halo" means F, CI, Br, and I.

Synthetic Methods

Synthetic methods to prepare the compounds of this invention frequently employ protective groups to mask a reactive functionality or minimize unwanted side reactions. Such protective groups are described generally in Green, T.W, Protective Groups in Organic Synthesis, John Wiley & Sons, New York (1981) .

Methyl-3-aminobenzoates ( 1 ) are loaded onto 2 , 6-dimethoxy-4- polystyrenebenzyloxy-benzaldehyde (DMHB resin) via reductive amination. Sulfonylation of the resulting resin-bound anilines with various sulfonyl chlorides gives sulfonamides 2 . After hydrolysis, the resulting resin-bound acids are coupled with cyclic diamines to afford intermediates 3 . Reductive amination of 3 with various aldehydes, followed by cleavage from resin, affords the targeted compounds 4 .

Scheme 1

Figure imgf000056_0001

Conditions: a) DMHB resin, Na(OAc)3BH, AcOH, NMP, rt; b) RiS02Cl, pyridine, DMAP, DCE, rt; c) TMSOK, THF, rt; d) cyclic diamines, PyBOP, DIEA, NMP, rt; e) R2CHO, Na(OAc)3BH, AcOH, NMP, room temp.; f) 20% TFA in DCM, rt .

In some embodiments, the organic compound has the structure:

Figure imgf000057_0001

or a pharmaceutically active salt or ether thereof.

Organic Compound Structure XIII

In some embodiments, the organic compound has the structure:

Figure imgf000057_0002

in which: w, x, y and z are independently 1, 2 or 3;

A is a phenyl, benzyl, alkyl, C3_6 saturated or partially unsaturated cycloalkyl, a 6-membered-cycloheteroalkyl ring containing 1 or 2 heteroatoms selected from 0 or N, alkyl-aryl, naphthyl, a 5- to 7- membered heteroaromatic ring containing 1 to 3 heteroatoms, a 9- or 10-membered bicyclic heteroaromatic ring containing 1 to 4 heteroatoms, a phenyl-fused-5 to 6-membered cycloheteroalkyl containing at least one heteroatom selected from 0, S or N, or pyridone ; A being optionally substituted by one or more groups selected from halogen, cyano, CF3, 0CF3, Ci_6 alkoxy, hydroxy, Ci_6 alkyl, Ci_6 thioalkyl, S02Ci_6 alkyl, NR2R3, amide, Ci_6 alkoxycarbonyl , -N02, Ci_6 acylamino, -C02H, Ci_6 carboxyalkyl , morpholine;

phenoxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl;

phenyl or diphenyl, said phenyl and diphenyl indepedently being optionally substituted with one or more groups indepedently selected from halogen, Ci_6 alkoxy, Ci_6 alkyl, or COOH;

benzyloxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl; or a 5 to 7 membered heteroaromatic ring containing 1 to 4 heteroatoms selected from 0, S or N optionally substituted with one or more groups indepedently selected from halogen, Ci_6 alkoxy, Ci_6 alkyl ;

R2 and R3 are independently halogen or Ci_6 alkyl, or R2 and R3 together with the nitrogen to which they are attached form a 6- membered saturated ring optionally containing a further heteroatom; B is a group R4-R5 where

R4 is a bond, -N(R6)-, -R7-N(R8)-, -N(R9)-R10-, 0, Ci_4 alkyl optionally interrupted by N(RX1) or 0, C2_4 alkenyl or 1 , 3-butadienyl , or -S02- N(R12) -;

R5 is C=0 or S02;

R6, R8, R11, and R12 are each independently H or Ci_6 alkyl; R9 is H, Ci- 6 alkyl or Ci_6 carboxyalkyl ;

R7 and R10 are independently Ci_4 alkyl or C3_5 cycloalkyl; D is Ci- 4 alkyl;

E is phenyl, or a 5- or 6-membered aromatic ring containing one or two heteroatoms;

Each R1 independently represents Ci_6 alkoxy optionally substituted with one or more halogens, C4_6cycloalkylalkoxy, C2 - 6 alkenyloxy, halogen, 0CH2CN, COCi- g alkyl, OR11, 0CH2R11, or -S-R12;

R11 is a phenyl or 5- or 6-membered saturated or aromatic ring containing one or two heteroatoms and each optionally substituted by one or more groups selected from Ci_6 alkyl, halogen, Ci_6 alkoxy, CF3, or cyano; R12 is Ci-6 alkyl or R12 is phenyl optionally substituted with one or more halogens, and n is 0, 1, 2, 3 or 4; provided that when E is phenyl, w + x is greater than 2 and n is 1 then R1 is not a phenoxy group at the meta-position of the phenyl ring E; and provided that when A-B is acetyl, tosyl or tertiary butyloxy- carbonyl (t-boc) , then D-E-(R1)n is not benzyl, or a pharmaceutically acceptable salt, solvate, or N-oxide thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2005/040167 and U.S. Patent Application Publication No. US 2007-0249648, the entire contents of each of which are hereby incorporated herein by reference .

In some embodiments, the organic compound has the structure:

Figure imgf000059_0001

or a pharmaceutically active salt or ether thereof.

Organic Compound Structure XIV

In some embodiments, the organic compound has the structure:

Figure imgf000059_0002

in which: w, x, y and z are independently 1, 2 or 3;

A is a phenyl, benzyl, alkyl, C3_6 saturated or partially unsaturated cycloalkyl, a 6-membered-cycloheteroalkyl ring containing 1 or 2 heteroatoms selected from 0 or N, alkyl-aryl, naphthyl, a 5- to 7- membered heteroaromatic ring containing 1 to 3 heteroatoms, a 9- or 10-membered bicyclic heteroaromatic ring containing 1 to 4 heteroatoms, a phenyl-fused-5 to 6-membered cycloheteroalkyl containing at least one heteroatom selected from 0, S or N, or pyridone;

A being optionally substituted by one or more groups selected from halogen, cyano, CF3, 0CF3, Ci_6 alkoxy, hydroxy, Ci_6 alkyl, Ci_6 thioalkyl, S02Ci_6 alkyl, NR2R3, amide, Ci_6 alkoxycarbonyl , -N02, Ci_6 acylamino, -C02H, Ci_6 carboxyalkyl , morpholine;

phenoxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl;

phenyl or diphenyl, said phenyl and diphenyl indepedently being optionally substituted with one or more groups indepedently selected from halogen, Ci_6 alkoxy, Ci_6 alkyl, or COOH;

benzyloxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl;

or a 5 to 7 membered heteroaromatic ring containing 1 to 4 heteroatoms selected from 0, S or N optionally substituted with one or more groups independently selected from halogen, Ci_6 alkoxy, Ci_6 alkyl ;

R2 and R3 are independently halogen or Ci_6 alkyl, or R2 and R3 together with the nitrogen to which they are attached form a 6- membered saturated ring optionally containing a further heteroatom;

B is a group R4-R5 where

R4 is a bond, -N(R6)-, -R7-N(R8)-, -N(R9)-R10-, 0, Ci_4 alkyl optionally interrupted by N(RX1) or 0, C2_4 alkenyl or 1 , 3-butadienyl , or -S02- N(R12) -; R5 is C=0 or S02;

R6, R8, R11, and R12 are each independently H or Ci_6 alkyl; R9 is H, Ci_6 alkyl or Ci_6 carboxyalkyl ;

R7 and R10 are independently Ci_4 alkyl or C3-5 cycloalkyl; D is Ci-4 alkyl;

E is phenyl, or a 5- or 6-membered aromatic ring containing one or two heteroatoms;

Each R1 independently represents Ci_6 alkoxy optionally substituted with one or more halogens, C4-6cycloalkylalkoxy, C2_6 alkenyloxy, halogen, OCH2CN, COCi_6 alkyl, OR11, OCH2R11, or -S-R12;

R11 is a phenyl or 5- or 6-membered saturated or aromatic ring containing one or two heteroatoms and each optionally substituted by one or more groups selected from Ci_6 alkyl, halogen, Ci_6 alkoxy, CF3, or cyano;

R12 is Ci_6 alkyl or R12 is phenyl optionally substituted with one or more halogens, and n is 0, 1, 2, 3 or 4; provided that when E is phenyl and n is 1 then R1 is not a phenoxy group at the meta position of the phenyl ring E; and provided that when A-B is acetyl, tosyl or tertiary butyloxy- carbonyl (t-boc) , then D-E-(R1)n is not benzyl or a pharmaceutically acceptable salt, solvate, or N-oxide thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2005/040167 and U.S. Patent Application Publication No. US 2007-0249648, the entire contents of each of which are hereby incorporated herein by reference . Organic Compound Structure XV

In some embodiments, the organic compound has the structure:

Figure imgf000062_0001
(Formula I of structure XV) wherein

n is 0 or 1 ;

m is O or l;

p is 1,2 or 3;

Ar is unsubstituted quinolinyl, [ 1 , 5 ] naphthyridinyl or pyridinyl; or quinolinyl, [ 1 , 5 ] naphthyridinyl or pyridinyl substituted with one or more radicals selected from the group consisting of Ci-C 6 alkoxy, Ci-C 6 alkyl, halo, cyano and trihalomethyl ;

R is Ci-Ce branched or unbranched alkyl, C3-C6 cycloalkyl lower alkyl, unsubstituted or substituted phenyl lower alkyl, unsubstituted or substituted pyridyl lower alkyl, unsubstituted or substituted indolyllower alkyl, unsubstituted or substituted N- (lower alkyl) indolyl lower alkyl, unsubstituted or substituted quinolinyl lower alkyl, unsubstituted or substituted naphthyl lower alkyl, unsubstituted or substituted benzofuranyllower alkyl, unsubstituted or substituted benzothiophenyllower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C 6 alkoxy, Ci-C 6 alkyl, halo, cyano and trihalomethyl;

or a pharmaceutically acceptable salt thereof. Organic compounds of this structure, as well as processes of synthesizing organic compounds of this structure are described in PCT International Application Publication No. WO/2004/032856, the entire contents of which are hereby incorporated herein by reference. Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of the present invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984) .

The term "d-C6 alkyl" and "Ci-C8 alkyl" is used herein includes both straight or branched chain radicals of 1 to 6 or 8 carbon atoms. By example this term includes, but is not limited to methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl and the like. "Lower alkyl" has the same meaning as Ci-C6 alkyl .

In the organic compound structure above, "Ci-C6 alkoxy" includes straight and branched chain radicals of the likes of 0-CH3, -0- CH2CH3, and the n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentoxy, and hexoxy, and the like.

"C3-C6-cycloalkyl" as applied herein is meant to include substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane and cyclohexane .

"Halogen" or "halo" means F, CI, Br, and I. Synthetic Methods

Synthetic methods to prepare the compounds above frequently employ protective groups to mask a reactive functionality or minimize unwanted side reactions. Such protective groups are described generally in Green, T.W, Protective Groups in Organic Synthesis, John Wiley & Sons, New York (1981) .

Acid addition salts of the compounds of Formula I are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic . Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg+ and NH4 are specific examples of cations present in pharmaceutically acceptable salts. Halides, sulfates, phosphates, alkanoates (such as acetate and trifluoroacetate ) , benzoates, and sulfonates (such as mesylate) are examples of anions present in pharmaceutically acceptable salts.

Compounds of Formula I may be prepared by two methods detailed in Schemes I and II below. These methods are illustrative, as it is believed other preparatory methods can be used to make one or more of the compounds of Formula I. Scheme I provides a first graphical representation of one of the general synthetic methods used to make the present compounds. The definition of n, m, p, and P in this scheme is the same as given above for Formula I, unless noted otherwise .

Figure imgf000064_0001

Figure imgf000064_0002
In this scheme, the epoxide ring opening of 1 is carried out with mono-Boc protected diamines under reflux conditions to give alcohol 2 . Oxazolidinone ring formation leads to 3 , which upon removal of the Boc protecting group yielded secondary amines 4 . Reductive alkylation of 4 with various aldehydes affords targeted compound 5 .

More specifically, in Scheme I, the conditions are as follows: a) mono-Boc protected diamines, LiC104, CH3CN, reflux; b) 1,1- carbonyldiimidazole, 4-dimethylamino pyridine, dichloromethane, room temperature; c) 30% trifluoroacetic acid in dichloromethane, room temperature; d) RCHO, Na(OAc)3BH, dichloromethane, room temperature.

Other compounds of Formula I are prepared by the steps and chemistries outlined in Scheme II. "Ar" and "R" in this scheme are equivalent to or the same as the definitions given for same for Formula I above.

Scheme II

Figure imgf000065_0001

In Scheme II, the conditions are as follows: a) Me2S, Me2S04, NaOMe, CH3CN, rt; b) 4-amino-piperidine-l-carboxylic acid tert-butyl ester, LiC104, CH3CN, reflux; c) 1 , lcarbonyldiimidazole, 4-dimethylamino pyridine, dichloromethane, room temperature; d) 30% trifluoroacetic acid in dichloromethane, room temperature; e) RCHO, Na(OAc)3BH, dichloromethane, room temperature . More specifically, epoxides 7 are synthesized from the corresponding aldehydes 6 via one-pot process under mild conditions. Epoxide ring opening of 7 with 4-aminopiperidine-l-carboxylic acid tert-butyl ester, followed by oxazolidinone ring formation, gives 8 , which upon hydrolysis and reductive alkylation affords the targeted compounds 9 .

In some emb iments, the organic compound has the structure:

Figure imgf000066_0001

or a pharmaceutically active salt or ether thereof.

Additional Organic Compound Structures

In some embodiments, the organic compound has the structure:

Figure imgf000066_0002

or a pharmaceutically active salt or ether of any of the foregoing.

Processes for synthesizing the compound:

Figure imgf000066_0003

are described in Jin, J., et al . , (2007) "Oxazolidinones as novel human CCR8 antagonists." Bioor Med Chem Lett 17:1722-5, the entire contents of which are hereby incorporated herein by reference.

Processes for synthesizing the compound:

Figure imgf000066_0004
are described in Ghosh, S, et al . , (2006) "Design, synthesis, and progress toward optimization of potent small molecule antagonists of CC chemokine receptor 8 (CCR8 ) . " J Med Chem 49:2669-72, the entire contents of which are hereby incorporated herein by reference.

The present invention includes all hydrates, solvates, complexes and prodrugs of the organic compounds above. Prodrugs are any covalently bonded compounds that release the active parent drug according to the organic compounds described herein in vivo. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers , are intended to be covered herein. Organic compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well known techniques and an individual enantiomer may be used alone. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form. The meaning of any substituent at anyone occurrence in an organic compound described herein or any subformula thereof is independent of its meaning, or any other substituent ' s meaning, at any other occurrence, unless specified otherwise.

Antagonists That Bind to CCL1

In some embodiments, the antagonist of the CCR8 receptor is an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, a polypeptide, or an antibody, which antagonist binds to CCL1 at a site which reduces binding of CCL1 to the CCR8 receptor.

In some embodiments, the antagonist of the CCR8 receptor is an anti- CCL1 antibody. In some embodiments, the anti-CCLl antibody is a monoclonal antibody .

In some embodiments, the monoclonal antibody is a humanized monoclonal antibody.

In some embodiments, the antagonist of the CCR8 receptor is a polypeptide . In some embodiments, the polypeptide is a soluble form of the CCR8 receptor .

In some embodiments, the soluble form of the CCR8 receptor comprises a portion of the extracellular domain of the CCR8 receptor sufficient to bind CCLl and reduce binding of CCLl to the CCR8 receptor .

In some embodiments, the soluble form of the CCR8 receptor comprises the entire extracellular domain of the CCR8 receptor which binds to CCLl and reduces the binding of CCLl to the CCR8 receptor.

In some embodiments, the polypeptide is a fusion protein which comprises a soluble form of the CCR8 receptor bound to an immunoglobulin (Ig) or Ig fragment.

In some embodiments, the fusion protein comprises an Ig which is a human Ig.

In some embodiments, the fusion protein comprises an Ig fragment which is a human Ig fragment.

In some embodiments, the antagonist of the CCR8 receptor is an RNA aptamer which binds to CCLl .

In some embodiments, the RNA aptamer is T48. The present invention provides a method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCR8 receptor produced by tumor cells present in the solid tumor, so as to thereby treat the subject.

Agents That Decrease The Amount of CCR8 or CCL1

The present invention provides a method of reducing, or reducing the likelihood of, metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCR8 receptor produced by tumor cells present in the solid tumor, so as to thereby reduce, or reduce the likelihood of, metastases in the subject.

In some embodiments, the oligonucleotide comprises nucleotides in a sequence that is complementary to CCR8 receptor-encoding mRNA.

In some embodiments, the oligonucleotide is an antisense oligodeoxynucleotide .

In some embodiments, the oligonucleotide is an RNA interference inducing compound.

In some embodiments, the oligonucleotide is a ribozyme.

The present invention provides a method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCL1 in the subject, so as to thereby treat the subj ect .

The present invention provides a method of reducing, or reducing the likelihood of, metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of CCL1 in the subject, so as to thereby reduce, or reduce the likelihood of, metastases in the subject. In some embodiments, the oligonucleotide comprises oligonucleotides in a sequence that is complementary to CCLl receptor-encoding mRNA.

In some embodiments, the oligonucleotide is an antisense oligodeoxynucleotide .

In some embodiments, the oligonucleotide is an RNA interference inducing compound.

In some embodiments, the oligonucleotide is a ribozyme.

In some embodiments, the oligonucleotide is modified to increase its stability in vivo.

Terms

"About" in the context of a numerical value or range means ±10% o the numerical value or range recited or claimed, unless the contex requires a more limited range .

The following abbreviations are used herein:

BEC, Blood endothelial cells;

CCL, CC Chemokine ligand;

CCR, CC Chemokine receptor;

CXCR, CXC chemokine receptor;

CM, Conditioned media;

DC, Dendritic cell;

GPCR, G protein-coupled receptor;

LEC, Lymphatic endothelial cells;

LN, Lymph node;

rh, recombinant human;

RFC, Reticular fibroblastic cell;

SCS, Subcapsular sinus;

TNF- , Tumor necrosis factor alpha.

As used herein, "a CCR8 antagonist" or "an antagonist of CCR8 receptors" means either an agent that directly reduces binding of a CCLl ligand to the CCR8 receptor by binding to the extracellular domain of CCR8, or an agent that indirectly reduces binding of a CCLl ligand to the CCR8 receptor by binding to CCLl . Non-limiting examples of CCR8 antagonists which are organic compounds are described, for example, in the following publications: PCT International Application Publication No. WO/2003/037271; WO/2004/032856; WO/2004/058709; WO/2004/058736; WO/2004/073619; WO/2004/074438; WO/2005/040167; WO/2006/107252; WO/2006/107253; WO/2006/107254; WO/2007/030061; WO/2008/099165; Norman P., (2007) "CCR8 antagonists." Expert Opin Ther Patents 17 (4 ) : 465-469; and Pease, J.E., & Horuk R., (2009) "Chemokine receptor antagonists: part 2." Expert Opin Ther Patents 19 ( 2 ) : 199-221 , all of which are hereby incorporated by reference in their entireties.

Antibodies

Examples of CCR8 antagonists that can be employed in the methods described herein are anti-CCR8 antibodies (see, e.g. PCT International Application Publication No. WO/2007/044756, which is hereby incorporated by reference in its entirety) . In some embodiments the anti-CCR8 antibody is a monoclonal antibody. Examples are those disclosed in PCT International Application Publication No. WO/2007/044756. Other examples of CCR8 antagonists that can be employed in the methods described herein are polypeptides (see, e.g. Luttichau, H.R., et al . , (2000) "A highly selective CC chemokine receptor (CCR) 8 antagonist encoded by the poxvirus molluscum contagiosum. " J Exp Med 191:171-180, which is hereby incorporated by reference in its entirety) . Methods of the present invention relate to the administration of monoclonal antibodies that recognize the human chemokine receptor CCR8 , i.e., "anti-CCR8" monoclonal antibodies. Methods of the present invention relate to the administration of monoclonal antibodies that recognize the human chemokine receptor CCLl, i.e., "anti-CCLl" monoclonal antibodies. An "antibody" as used herein is defined broadly as a protein that characteristically immunoreacts with an epitope (antigenic determinant) of an antigen. As is known in the art, the basic structural unit of an antibody is composed of two identical heavy chains and two identical light chains, in which each heavy and light chain consists of amino terminal variable regions and carboxy terminal constant regions. The anti-CCR8 and anti-CCLl antibodies of the invention include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, CDR-grafted antibodies, humanized antibodies, human antibodies, catalytic antibodies, multispecific antibodies, as well as fragments, regions or derivatives thereof provided by known techniques, including, for example, enzymatic cleavage, peptide synthesis or recombinant techniques .

As used herein, "monoclonal antibody" means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495-97 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567) . The monoclonal antibodies may also be isolated from phage display libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al . , J. Mol . Biol. 222 (3) :581-97 (1991) . The term "hybridoma" or "hybridoma cell line" refers to a cell line derived by cell fusion, or somatic cell hybridization, between a normal lymphocyte and an immortalized lymphocyte tumor line. In particular, B cell hybridomas are created by fusion of normal B cells of defined antigen specificity with a myeloma cell line, to yield immortal cell lines that produce monoclonal antibodies. In general, techniques for producing human B cell hybridomas, are well known in the art [Kozbor et al . , Immunol. Today 4:72 (1983); Cole et al . , in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. 77-96 (1985) ] .

The term "epitope" refers to a portion of a molecule (the antigen) that is capable of being bound by a binding agent, e.g., an antibody, at one or more of the binding agent's antigen binding regions. Epitopes usually consist of specific three-dimensional structural characteristics, as well as specific charge characteristics.

Humanized anti-CCR8 and anti-CCLl antibodies are also encompassed by the terms "anti-CCR8 antibody" and "anti-CCLl antibody", respectively as used herein. "Humanized antibodies" means antibodies that contain minimal sequence derived from non-human immunoglobulin sequences. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hyper variable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205, each herein incorporated by reference. In some instances, framework residues of the human immunoglobulin are replaced by corresponding non-human residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762, each herein incorporated by reference) . Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain desired affinity) . In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. For further details see Jones et al . , Nature 331:522- 25 (1986); Riechmann et al . , Nature 332:323-27 (1988); and Presta, Curro Opin. Struct. Biol. 2:593-96 (1992), each of which is incorporated herein by reference.

Also encompassed by the term anti-CCR8 antibodies are xenogeneic or modified anti-CCR8 antibodies produced in a non-human mammalian host, more particularly a transgenic mouse, characterized by inactivated endogenous immunoglobulin (Ig) loci. In such transgenic animals, competent endogenous genes for the expression of light and heavy subunits of host immunoglobulins are rendered non-functional and substituted with the analogous human immunoglobulin loci. These transgenic animals produce human antibodies in the substantial absence of light or heavy host immunoglobulin subunits. See, for example, U.S. Pat. No. 5,939,598, the entire contents of which are incorporated herein by reference.

Those skilled in the art will be aware of how to produce anti-CCR8 and anti-CCLl antibody molecules of the present invention. For example, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the CCR8 or CCL1 which elicits an antibody response in the mammal. For instance, a mammal can be immunized with irradiated cells that were transfected with a nucleic acid encoding CCR8 or CCL1 such that high levels of CCR8 or CCL1 were expressed on the cell surface. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained, and, if desired IgG molecules corresponding to the polyclonal antibodies may be isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the oligopeptide, and monoclonal antibodies isolated.

Examples of CCR8 antagonists which bind CCL1 that can be employed in the methods described herein include but are not limited to DNA or RNA aptamers, such as the RNA aptamer T48 (see, e.g. Pease, J.E., & Horuk R. , 2009. Chemokine receptor antagonists: part 2. Expert Opin Ther Patents 19(2) : 199-221) . Examples of RNA aptamers are those disclosed in Marro, M.L., et al . , (2006) "In vitro selection of RNA aptamers that block CCL1 chemokine function." Biochem Biophys Res Commun 349:270-276, which is hereby incorporated by reference in its entirety.

Oligonucleotides

Non-limiting examples of oligonucleotides which decrease the amount of the CCR8 receptor produced by cells that can be employed in the methods described herein are described, for example, in PCT International Application Publication No. WO/O3/096020 which is hereby incorporated by reference in its entirety.

The amino acid sequence of CCR8 is accessible in public databases by the accession number AAH69067 and is set forth herein as SEQ ID NO: 7. The amino acid sequence of CCR8 is also accessible in public databases by the accession number P51685. The nucleotide sequence which encodes CCR8 is accessible in public databases by the accession number BC069067 and is set forth herein as SEQ ID NO: 8.

The amino acid sequence of CCL1 is accessible in public databases by the accession number EAW80204 and is set forth herein as SEQ ID NO: 9. The amino acid sequence of CCL1 is also accessible in public databases by the accession number P22362. The nucleotide sequence which encodes CCL1 is accessible in public databases by the accession number BC111914 and is set forth herein as SEQ ID NO: 10. Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of CCR8 or CCL1 gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides , ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester intemucleotide linkages such alkylphosphonates , phosphorothioates , phosphorodithioates , alkylphosphonothioates , alkylphosphonates, phosphoramidates , phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.

Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (Nicholls et al . , 1993, J Immunol Meth 165:81-91) . An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes .

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a CCR8 or CCL1 polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a CCR8 or CCL1 polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent CCR8 or CCL1 nucleotides, can provide sufficient targeting specificity for CCR8 or CCL1 mRNA, respectively. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Noncomplementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular CCR8 polynucleotide sequence. Antisense oligonucleotides can be modified without affecting their ability to hybridize to a CCR8 or CCL1 polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 ' -substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art.

Ribozymes

Ribozymes are RNA molecules with catalytic activity (Uhlmann et al . , 1987, Tetrahedron. Lett. 215, 3539-3542) . Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences. The coding sequence of a polynucleotide can be used to generate ribozymes which will specifically bind to rnRNA transcribed from the polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art. For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.

Specific ribozyme cleavage sites within an RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC . Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NOs : 8 and 10 and their complements provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease CCR8 expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or VAS element, and a transcriptional teminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673) . Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

RNA Interference

As used herein, "RNA interference inducing compound" refers to a compound capable of inducing RNA interference or "RNAi" of CCR8 or CCL1 expression, depending on the context. RNAi involves mRNA degradation, but many of the biochemical mechanisms underlying this interference are unknown. The use of RNAi has been described in Fire et al., 1998, Carthew et al., 2001, and Elbashir et al., 2001, the contents of which are incorporated herein by reference.

Isolated RNA molecules can mediate RNAi. That is, the isolated RNA molecules of the present invention mediate degradation or block expression of mRNA that is the transcriptional product of the gene, which is also referred to as a target gene. For convenience, such mRNA may also be referred to herein as mRNA to be degraded. The terms RNA, RNA molecule (s) , RNA segment(s) and RNA fragment(s) may be used interchangeably to refer to RNA that mediates RNA interference. These terms include double-stranded RNA, small interfering RNA (siRNA) , hairpin RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) , as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA) . Nucleotides in the RNA molecules of the present invention can also comprise nonstandard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides . Collectively, all such altered RNAi molecules are referred to as analogs or analogs of naturally- occurring RNA. RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi .

As used herein the phrase "mediate RNAi" refers to and indicates the ability to distinguish which mRNA molecules are to be afflicted with the RNAi machinery or process. RNA that mediates RNAi interacts with the RNAi machinery such that it directs the machinery to degrade particular mRNAs or to otherwise reduce the expression of the target protein. In one embodiment, the present invention relates to RNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or blocking expression of the target mRNA.

As noted above, the RNA molecules of the present invention in general comprise an RNA portion and some additional portion, for example a deoxyribonucleotide portion. The total number of nucleotides in the RNA molecule is suitably less than in order to be effective mediators of RNAi. In preferred RNA molecules, the number of nucleotides is 16 to 29, more preferably 18 to 23, and most preferably 21-23.

Administration

"Administering" the antagonists described herein can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be, for example, intravenous, oral, intramuscular, intravascular, intra-arterial , intracoronary, intramyocardial , intraperitoneal, and subcutaneous. Other non-limiting examples include topical administration, or coating of a device to be placed within the subject. In embodiments, administration is effected by injection or via a catheter.

Injectable drug delivery systems may be employed in the methods described herein include solutions, suspensions, gels. Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc) . Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA) .

The administration of CCR8 antagonists described herein may be by way of compositions containing one of the antagonists and a pharmaceutically acceptable carrier. As used herein, a "pharmaceutical acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering an active compound to a mammal, including humans. The carrier may be liquid, aerosol, gel or solid and is selected with the planned manner of administration in mind. In an embodiment, the pharmaceutical carrier is a sterile pharmaceutically acceptable solvent suitable for intravenous administration. In an embodiment, the pharmaceutical carrier is a pharmaceutically acceptable solid suitable for oral administration .

As used herein, the term "effective amount" refers to the quantity of a component that is sufficient to treat a subject without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention, i.e. a therapeutically effective amount. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the structure of the compounds or its derivatives. By treating the subject there are multiple possible outcomes. For instance, treating a subject may comprise substantially reducing, slowing, stopping, preventing or reversing the progression of a disease, particularly a solid tumor. Additionally, treating a subject may comprise substantially reducing, slowing, stopping, preventing or reversing a symptom of a disease. In some embodiments, an outcome of treating the subject is substantially reducing, slowing, stopping, preventing, or reversing metastasis, wherein the subject has, or has been treated for, a solid tumor. In some embodiments, treating the subject comprises reducing the likelihood of metastasis in the subject. In some embodiments the subject is treated after a solid tumor has been removed from the subject. In some embodiments, the CCR8 antagonist is used for prevention and treatment of melanoma metastasis and recurrence. In the most favorable case, reduction is equivalent to prevention.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention .

It is understood that where a parameter range is provided, all integers within that range. For example, "0.2-5 mg/kg/day" is a disclosure of 0.2 mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/day etc. up to 5.0 mg/kg/day.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter. Experimental Details

EXAMPLE 1. Materials and Methods.

Cell culture .

Human lymphatic and blood endothelial cells (LECs and BECs) were isolated and cultured in endothelial basal medium (EBM, Lonza) supplemented with 20% FBS (Invitrogen) as previously described (Podgrabinska et al . , 2002) . The breast epithelial cells MCF10F were cultured in DMEM/Ham' s F12 (Soule, 1990; Shafie, 1980) and 184B5 cells in MEBM/MEGM (Walen, 1989) (Lonza/Clonetics Corporation) . Primary human melanocytes isolated from human foreskins (gift from Dr. Aaronson) were cultured in Medium 254 (Cascade Biologicals) . The human breast cancer cell lines MDA-MB-231, MDA-MB-435 and MCF-7 were cultured in DMEM with 10% FBS. Melanoma cell lines SK-MEL-25, SK- MEL-28 and MEL-501, and Jurkat cells, were cultured in RPMI-1640 containing 5% FBS (Zakut, 1993; Podgrabinska, 2009) . Conditioned medium (CM) was generated from just confluent LECs or BECs in passage 4-6, cultured for 48 hrs in EBM/1% FBS. For some experiments LECs were treated with TNF-a (50ng/ml) or LPS (500ng/ml) for 48 hours. CM from tumor cells was generated serum-free in their respective media.

Reagents and antibodies .

Recombinant human CCL1, mouse CCL1 and viral MCV-type II chemokine- like protein MC148 were purchased from R&D systems. We also used E coli LPS (serotype 0127 :B8, Sigma), recombinant human TNF-a (Peprotec) , IL-Ιβ (R&D systems), pertussis toxin (List Biological), cholera toxin (Sigma) , Alexa Flour 594 phallodin (Life Technologies) and Hoechst bisbenzimide ( Sigma-Aldrich) . Anti-human antibodies: goat anti-CCR8 (Alexis Biochemicals, ALX-210-762-C200) , rabbit anti- CCR8 (Abeam, ab32399) , goat anti-CD31 (Clone C20; Santa Cruz Biotechnology, sc-1505) , mouse anti-podoplanin (Angiobio, 11-003; Fitzgerald, 70R-PR007X) , goat anti-CCLl (Clone CI 9; Santa Cruz Biotechnology, sc-1412), mouse anti-CCLl (Clone 35305; R&D Systems, MAB272), isotype control IgGl (R&D Systems) . Anti-mouse antibodies: rabbit anti-LYVE-1 (Abeam, abl4917), rat anti-CD31 (Clone RAM34 ; BD Pharmingen, 550274), goat anti-VEGFR-3 (R&D Systems, AF743), hamster anti-podoplanin (AngioBio, 11-033), goat anti-CCLl (R&D Systems, AF845) , goat anti-CCL8 (R&D Systems, AF790) . Rabbit anti-His antibody Clone H15 was from Santa Cruz Biotechnology (sc-803) , chicken anti-GFP antibody was from Abeam (ab6662) . Secondary antibodies were labeled with AlexaFluor-488, 555, 594, 647 (Life Technologies), or with biotin (Vector Laboratories) . T PCR and Real- time PCR.

RT-PCR was performed as described (Podgrabinska et al . , 2002) . The expression levels of hCCLl mRNA were quantified by SYBR Green-Based real-time PCR using the Opticon2 detection system (Bio-Rad Laboratories) as described previously (Podgrabinska et al . , 2009) . On the RNA isolated from LECs treated with TNF-a (50ng/ml), LPS (500ng/ml) or IL-Ιβ (50ng/ml) for 3, 6 and 18hr. Primers used for RT-PCR and qPCR:

hCCR8 Fp: 5 CCCTGTGATGCGGAACTTAT 3' (SEQ ID NO : 1 ) ;

hCCR8 Rp: 5' GATGGCCTTGGTCTTGTTGT 3' (SEQ ID NO : 2 ) ;

hCCLl Fp: 5' GGAAGATGTGGACAGCAAGAGC 3' (SEQ ID NO:3);

hCCLl Rp: 5' TGTAGGGCTGGTAGTTTCGG 3' (SEQ ID NO : 4 ) ;

MC148 primers are shown below.

Cloning and cell transfections .

MC148 was PCR amplified from a pTej-MC148 vector (gift from Dr. Luttichau) using the Expand High Fidelity plus PCR system (Roche) , with the forward primer containing a Nhe I restriction site and the reverse primer containing an Apa I restriction site, following a His tag sequence (underlined) .

MC148 Fp: 5' AAAAGCTAGCACCATGAGGGGCGGAGACGTCTTC 3' (SEQ ID NO : 5 ) ; MCI 48 Rp: 5' AAAAGGGCCCTCAATGATGATGATGATGArGCAGAGACTCGCACCCGGACCAT AT 3' (SEQ ID NO:6) . The PCR amplified product was cloned into a pcDNA 3.1-Hygro (+) vector (Life Technologies) . Tumor cells were transfected with the expression construct pcDNA3. l/MC148-His or with an empty vector using the Lipofectamin 2000 reagent (Life Technologies), selected and maintained in media with Hygromycin (100 μg/ml) . MDAcl.13 and MDAcl .6 cells were selected and used as a whole population. SK-MEL-25 cells were subcloned and two control and two MC148 clones were used in all experiments. HEK 293 cells were transfected with the hCCR8 plasmid (Tiffany et al . , 1997) . For silencing CCR8 gene expression, shRNA oligos were cloned into the pLKO.1-25 puro lentiviral vector (Addgene 19119) according to the standard protocol. For generation of the lentiviral particles, these constructs were transfected into 293T cells along with the packaging plasmids psPAX2 (Addgene 12260) and pMD2. G (Addgene 12259) . Culture supernatants containing viral particles were collected 48hrs after transfection . Stable shRNA-expressing cells were generated by infection with viral particles in presence of 8 g/ml polybrene and selection with 2 g/ml puromycin. The shRNA sequences used are as follows :

shCCR8(l) : 5 ' -GGATTATACACTTGACCTCAGTG-3 ' (SEQ ID NO: 11);

shCCR8(2) : 5 ' -CCTCCAGCGTAGACTACATTT-3 ' (SEQ ID NO:12) ;

shScrambled: 5 ' -CCTAAGGTTAAGTCGCCCTCGC-3 ' (SEQ ID NO:13) .

MDA-MB-435/GFP cells used in shRNA knockdown experiments were generated by infecting cells with the pBabe-derived retroviral vector containing CMV promoter-driven EGFP (gift from Dr. Aaronson) and selecting for the population resistant to hygromycin.

Western blot analysis .

For CCL1 detection, 5 ml CM (from ~2xl06 ECs) was incubated with heparin-Sepharose beads (Pharmacia) and eluted by boiling with 40 μΐ Laemmli sample buffer. Whole sample was loaded onto the gel. For MC148 detection, CM (5ml, from ~7xl06 tumor cells) was concentrated 10-fold using Amicon Ultra Centrifugal Filter Units (Ultracel-3K, Millipore) and 10 μΐ was loaded on the gel. Western blot analyses for detection of CCR8, CCL1 and MC148-his expression were performed as previously described (Skobe et al . , 2001b) . Antibodies used were rabbit anti-human CCR8, goat antihuman CCL1 and rabbit anti-His for detection of His-tagged MC148. hCCLl in the culture supernatants was measured with RayBio ELI SA Kit (Cat#ELH-l309-001 ) .

Chemotaxis assays .

Tumor cell migration was assessed using 24-well plate Transwell system with 8.0 μιτι pore inserts (BD Biosciences) . EBM with 1% FBS or LEC-CM media (700 μΐ) was added into the well and tumor cells (2xl05 in 200 μΐ) into the insert. In experiments with SK-MEL-25 cells, inserts were coated with collagen (50 μg /ml) and fibronectin (1 μg /ml) . For blocking studies, tumor cells were pretreated with PTx or CTx (both lOOng/ml) for 16hr. Anti-CCLl Ab (2 μg/ml) or isotype control IgGi (2 μg/ml) were added to LEC-CM for duration of the experiment. For CCL1 immunodepletion, LEC-CM was pre-incubated with either anti-CCLl Ab (2 μg /ml) or isotype control IgGi for 2hr, followed by incubation with protein G sepharose beads (Santa Cruz Biotechnnology) . Sepharose beads were removed by centrifugation and CCLl-depleted conditioned media used in the assays. For CCR8 blocking, MC148 antagonist (100 ng/ml) (Luttichau et al . , 2001)was pre-incubated with tumor cells for 30 minutes prior to the assay. In the experiments where LEC-CM-generated in presence of TNF-a or LPS was used, EBM supplemented with TNF-a or LPS was used as a control . Tumor cells were allowed to migrate for 6hr, fixed, stained with Hoechst and examined as described (Skobe et al., 2001a) using Nikon E-600 microscope. Images from four different fields in each membrane were captured with Nikon Eclipse E600 fluorescence microscope (lOx) equipped with a Nikon DS-QilMC camera and quantitative analysis performed using IPLab software. All experiments were performed in triplicate. Statistical significance was determined using two-tailed student's t-test.

Flow cytometry and Calcium Fluorimetry.

For CCR8 expression, melanocytes and tumor cell lines were detached, stained with anti-human CCR8 (1:400) in PBS/10% FBS for 45 min, washed, incubated with FITC-conj ugated corresponding secondary Ab, washed and analyzed by flow cytometry (Becton Dickinson) . Ca+2-flux assay was essentially performed as described (Luttichau et al., 2000) . Briefly, cells were mechanically detached from the culture dish, washed and loaded with Fluo-4, AM dye (Molecular Probes) in serum-free DMEM with 4% BSA. Cells were incubated for 60min, washed in Krebs ringer buffer several times and the fluorescence kinetics were measured on a Becton Dickinson FACSCalibur. Ionomycin served as a positive control. For blocking studies, MC148 (25 nM) was added to the cell suspension prior to the addition of rCCLl . Data were analyzed using the FlowJo software. Data were plotted as time vs. percentile Fluo-4 intensity, with moving average smoothing option.

Metastasis assay and evaluation.

To examine the role of CCR8 in tumor metastasis we employed three human tumor cell lines (1) SK-MEL-25 melanoma cells, (2) highly metastatic subclone of MDA-MB-435 cells which expresses VEGF-C (MDAcl .13) , and (3) parental MDA-MB-435 cells (Skobe et al . , 2001b; Roberts et al . , 2006; Das et al., 2010) . In the experiments with MC148 antagonists we used a subclone of MDA-MB-435 cells tagged with GFP (MDAcl.6) (Skobe et al . , 2001b; Roberts, 2006; Das et al . , 2010) . In the experiments with shRNA knockdown we employed parental MDA-MB-435 cells tagged with GFP as a whole population. MDA-MB-435 (parental, cl .6 or cl.13) and SK-MEL-25 cells were injected into immunodeficient mice (Athymic Ncr Nu/Nu, NCI Frederick) , into the 2nd mammary fatpads (2x10s cells) and intradermally (4xl05 cells), respectively. Metastases were evaluated at 13 weeks and at 5 weeks, respectively (when tumors reached 10mm in diameter) ; tumors and lymph nodes were collected and processed as described (Skobe et al . , 2001b; Das et al., 2010) . LNs were carefully dissected to preserve the lymphatic vessels in the surrounding tissue and analyzed in toto with a Leica MZ16F stereomicroscope equipped for epi-fluorescence . Images were captured with DFC300Fx camera using LAS V3.6 software (Leica Microsystem) . Metastases were classified in two groups: (i) intranodal metastasis refers to the samples which contain metastases inside the lymph node (with or w/out additional metastases in the collecting lymphatic vessel); (ii) in-transit refers to the metastases in the collecting, afferent lymphatic vessels attached to the LN, but without metastases in the lymph node itself. Absence or presence of LN metastases was also confirmed by examination of 100 μιτι LN sections for the presence of GFP signal in the LN (Skobe et al . , 2001b; Das et al . , 2010) . Specific sublocalization of tumor cells in the lymphatic vessels or in the LNs was further examined by immunostaining of thin sections (6 μιτι) with lymphaticspecific markers and an anti-GFP antibody. Overall incidence of LN metastases was calculated as the number of positive LN relative to the total number of lymph nodes examined. Ratio of in transit to intranodal metastasis was calculated relative to the number of samples positive for metastases in either localization, not to the total lymph nodes examined. Ten mice (40 lymph nodes) were analyzed in each experimental group. All animal experiments were performed in accordance with the protocols approved by the MSSM' s Institutional Animal Care and Use Committee.

Multiphoton microscopy.

Lymph nodes were imaged using a Bio-Rad Radiance 2100MP Multiphoton microscope equipped with a Tsunami pulsed laser (Spectraphysics) and controlled by Lasersharp 2000 software (Bio-Rad) . Formalin-fixed lymph nodes were immobilized in a Petri dish with agar, immersed in PBS and viewed with the 20x water-immersion objective (NA 0.8) . To image EGFP and collagen (by second harmonic imaging, SHG) laser excitation source was tuned to 840nm. EGFP signal was collected by using a 515/30 nm filter. Stacks of x-y sections were acquired at 5 μιτι vertical spacing. 2D image processing and subsequent 3D reconstruction was performed with Volocity software (Improvision) .

Immunofluorescence staining.

Immunofluorecent staining procedures were performed as described (Das et al . , 2010; Roberts et al., 2006), with the following modifications: Formalin-fixed (1 hr, 10 %) cryosections of lymph nodes were treated with 0.3% Triton X-100 (in PBS) for 30 minutes, washed and stained as described. GFP detection was done using formalin-fixed cryosections, with an anti-GFP Ab .

For CCL1 detection, paraffin-embedded tumor and lymph node tissue sections were used and AlexaFluor -555 and -647 conjugated secondary antibodies. Tissue was examined using a Nikon Eclipse E600 fluorescence microscope equipped with a Nikon DS-QilMC camera. NIS Elements software (Nikon) was used for capture and quantification of images (tumor vasculature) (Roberts et al., 2006) .

Actin polymerization assay.

Tumor cells (3 X 104) were incubated overnight in 8-well chamber slides (BD pharmingen) with 1% FBS and then treated with 50 ng/ml rCCLl (human or mouse) for the indicated times. Cells were fixed (4% PFA) , permeabilized (0.1% Titron X 100) and stained with rhodamin- labelled phalloidin. Images were captured by a Nikon microscope (as mentioned above) . For the blocking experiments, cells were incubated with MC148 (50 ng/ml) and PTx (50 ng/ml) for one hour prior to the addition of rCCLl.

Human tissues and Immunohistochemistry .

Two malignant melanoma tissue arrays (ME801 and ME241) containing a total of 90 cases of malignant melanoma were purchased from Biomix US. Archived samples of paraffin-embedded human melanoma tissues (n=19), lymph nodes with melanoma metastases (n=8) and normal skin were obtained from the files of the Department of Pathology at Columbia University. Paraffin-embedded specimens were analyzed as described (Das et al . , 2010) . For CCR8 detection, goat anti-human CCR8 antibody and the Vectastain ABC kit were used. For CCL1 detection, mouse anti-human CCL1 antibody and CSA amplification method (Dako) were used. Use of human tissue samples was approved by the Institutional Review Board of Columbia University.

Scanning Electron Microscopy (SEM) .

Mice were anesthetized, perfused with PBS at the controlled rate of 5ml/min through the left ventricle and subsequently with 2% glutaraldehyde/2% paraformaldehyde. Upon sacrifice, lymph nodes were excised and post-fixed in the above fixative for 24 hr at 4°C. Lymph nodes were then embedded in 4% agarose and sectioned at 250 μιτι intervals using a vibratome (Leica VT100) . Sections were examined with a Nikon Eclipse Ti microscope to identify regions of interest and selected sections were processed for SEM according to the standard protocol (Ohtani, 2003), with the following modifications: primary fixation in 3% glutaraldehyde/0.2M cacodylate buffer (pH 7.4, 3 hr) , secondary fixation in 1% OsO4/0.2M cacodylate buffer for 1 hr. After washing tissues were dehydrated in the ethanol series, critical point dried with liquid C02 and coated with gold. Specimens were examined with the Hitachi S4300 field emission scanning electron microscope (Hitachi, Tokyo) under an accelerating voltage of 10KV. Multiphoton intravital microscopy.

MDAcl.13 or MDAcl .13/MC148 cells tagged with EGFP were injected into the 5th mammary fatpads of immunodeficient mice (2x10s cells) . Imaging of metastases was performed between 7 and 20 weeks after the tumor cell injection. To visualize sentinel lymph node and the afferent lymphatics draining the tumor, mice were anesthetized with 5% isofluorane and 10 μΐ of Evan's blue (1%) (Tsopelas, 2002) was injected at the tumor site. In some experiments, Evan's blue was injected laterally at the tail base, to identify inguinal lymph node. To visualize blood vasculature, 200 μΐ of 2 MDa rhodamine dextran was administered into the tail vein. Lymph node and the tumor-draining lymphatic vessels were exposed for imaging by performing a skin flap surgery as described (Lindquist, 2004; Wyckoff, 2011), with some modifications. Briefly, mice were anesthetized with 5% isofluorane and a medial incision through the skin was performed. Skin was gently separated from the underlying fascia and the adipose tissue covering the lymph node was carefully removed to spare lymphatic and blood vessels. Mouse was then placed on the microscope stage within a heated chamber and the temperature was maintained at 37°C. Lymph node and the surrounding tissues were submerged in saline and covered with a glass coverslip. Intravital imaging was performed with a custom-built, inverted multiphoton microscope based around an Olympus 1X71 stand (Entenberg, 2011) . The microscope's four detectors allow simultaneous imaging of collagen fibers (blue, second harmonic generation), GFP (green, tumor cells), rhodamine dextran (red, blood vasculature) and Evan's blue (far red, lymphatic vessels) . The 880nm wavelengthwas used for excitation of all fluorophores . 4D images were acquired at lfps with 5μιτι z-steps spanning -100-150 μιτι and with ~1 min between time points. Images were analyzed in ImageJ.

All animals were used accordir protocols that have been reviewed and approved by Mount Sinai' Albert Einstein' s Institutional Animal Care and Use Committee. EXAMPLE 2. Identification of CCLl as the major tumor cell chemoattractant produced by lymphatic endothelial cells

The effects of conditioned media generated by lymphatic endothelium on chemotaxis of several human melanoma and breast carcinoma cell lines were examined. LEC-conditioned media (LEC-CM) was chemotactic for most metastatic cell lines tested (Fig. 1 A) . Highly metastatic cells MDA-MB-435 and SK-MEL-25 showed the highest increase in migration, followed by MCF-7 and MDA-MB-231 cells. In contrast, non- tumorigenic human breast epithelial cell lines MCF-10F and 184-B5 and a low metastatic melanoma cell line MEL-501, did not show a significant increase in chemotaxis in response to LECs. SK-MEL-28 cells did not show increased migration to LEC-CM; however, this cell line has very high basal migration levels (data not shown) so that an increase was difficult to detect. These results indicate that metastatic tumor cells acquire responsiveness to the chemotactic signals derived from the lymphatic endothelium.

The induction of tumor cell chemotaxis by LEC-CM was completely abolished by pretreatment of tumor cells with pertussis toxin, indicating that the chemotactic effect was mediated through G- protein coupled receptor (s) (GPCRs) (Fig. 1 B) . Chemokine production by lymphatic endothelial cells was therefore screened, and it was found that CCLl mediated much of the chemotactic activity found in the LEC-CM. RT-PCR showed constitutive expression of CCLl mRNA by cultured LECs (Fig. 1 C) and Western blot analysis showed CCLl protein in LEC-CM as a heparin-bound fraction (~ 1 ng/ml CM) (Fig. 1 D) . A function-blocking monoclonal antibody to CCLl reduced chemotaxis of MDA-MB-435 and SK-MEL-25 cells to LEC-CM by 73% and 60%, respectively (Fig. 1 E) . Depletion of CCLl with a different specific polyclonal antibody reduced tumor cell migration to a similar extent (Fig. 1 F) . CCLl was not expressed by MDA-MB-435 and SK-MEL-25 cells (Fig. 8 and 9), confirming its paracrine role in this model. In agreement, chemotaxis in response to LEC-CM which was depleted by heparin was decreased by 62%. (data not shown) . Conversely, addition of rhCCLl showed increase in tumor cell chemotaxis (Fig. 1 G) , but not haptotaxis (data not shown) . CCLl had no effect on tumor cell growth or survival (data not shown) . Consistent with its function in cell migration, rhCCLl induced cytoskeletal re-arrangements and altered in vitro tumor cell morphology from elongated to round, as determined by F-actin staining (Fig. 12) . These effects, which are a prerequisite for cell motility (Burger et al . , 1999), were transient in nature: first changes were observed at 20min, the effect was most pronounced at 40min, and at 60min of exposure to CCLl cell shape largely returned to normal. Taken together, these data identify CCLl as a potent tumor chemotactic factor produced by LECs.

EXAMPLE 3. CCLl mediates tumor cell migration to inflamed LECs

Treatment of LECs with pro-inflammatory cytokines or with LPS resulted in a dramatic increase of CCLl mRNA expression (Figs. 2 and 16) . Within 18 hrs, both TNF-a and IL-Ιβ increased CCLl mRNA 543- fold and 340-fold, respectively, as determined by qPCR. LPS increased CCLl mRNA expression 400-fold (Fig. 2 A) . ELI SA and Western blot analysis of the conditioned media collected from LECs treated with TNF-a, IL-Ιβ, or LPS showed increased amounts of the secreted CCLl protein (8, 6 and 3.6-fold respectively) (Fig. 2 B) . CCLl protein levels were also increased two fold by serum (Fig. 1 D) .

Conditioned media collected from LECs that were treated with TNF-a or LPS, stimulated tumor cell chemotaxis to an even greater extent than CM obtained from untreated LECs (~ 12-fold vs. 8-fold increase, respectively) (Fig. 2 C and D) . Neither TNF-a nor LPS had a direct effect on tumor cell migration (data not shown) . Increased tumor cell chemotaxis to inflamed LECs was completely abolished by depleting CCLl (Fig. 2C and D) . These results indicate that proinflammatory cytokines facilitate tumor cell migration indirectly, by upregulating CCLl expression by LECs.

In agreement with the in vitro data, CCLl protein was strongly upregulated on lymphatic vessels in mouse skin upon treatment with TNF-a or application of FITC (Fig. 16) . CCLl protein was also strongly upregulated on dermal lymphatic vessels in human foreskins treated ex vivo with TNF- a, but was not detectable on dermal lymphatics of normal mouse or human skin by immunostaining (Fig. 16 A, B, and data not shown) . Taken together, these data demonstrate that pro-inflammatory stimuli strongly upregulate CCLl expression in vitro and in vivo.

EXAMPLE 4. Cancer cells express functional CCR8 receptor

Human CCLl binds to and exclusively activates G protein-coupled receptor CCR8 (Goya et al . , 1998; Roos et al . , 1997; Tiffany et al . , 1997) . The expression of CCR8 by the tumor cells that responded to CCLl with increased migration was examined (Fig. 3 A) . SK-MEL-25 and MDA-MB-435 cells expressed CCR8 mRNA, as determined by RT-PCR. Western blot analysis showed expression of CCR8 protein by both tumor cell lines in vitro and in vivo (Fig. 3A, Fig. 8 and 9), and this was further confirmed by FACS. Primary melanocytes did not show any detectable levels of CCR8 by FACS (Fig. 3 A) .

To determine whether CCR8 mediates cell migration, the effects of MC148, a specific CCR8 antagonist, on chemotaxis of tumor cells to LEC-CM were examined. MC148 is a highly selective CCR8 antagonist derived from the human poxvirus Molluscum contagiosum which that binds to CCR8 with the same affinity as its endogenous ligand CCLl (Luttichau et al . , 2000) . Pre-incubation of the tumor cells with MC148 potently blocked tumor cell migration (Fig. 3 B) , indicating that CCR8 mediates most of the chemotactic response to LEC-CM. MC148 also prevented the cytoskeletal rearrangements and cell shape change of tumor cells (MDA-MB-435) stimulated with rhCCLl (Fig. 3 C) , and the effect was comparable to that of pertussis toxin, a broad inhibitor of GPCR signaling. These data demonstrate that CCLl mediates its effects on tumor cell migration by activating CCR8 and GPCR signaling in tumor cells.

Chemoattraction induced by chemokines is associated with a transient rise in intracellular calcium following receptor activation, and is a hallmark of GPCR-mediated signal transduction (Houshmand and Zlotnik, 2003) . To further assess the functionality of CCR8 expressed by tumor cells, intracellular calcium flux was measured upon addition of CCL1 to three cell lines: SK-MEL-25 and MDA-MB-435 that endogenously express CCR8 and HEK293 which were transfected to overexpress CCR8 (Fig. 12) . Treatment of cells with CCL1 lead to a rapid increase of the intracellular calcium level which reached a maximum within approximately 36 seconds and then decreased steadily. MC148 completely inhibited influx of [Ca2+] induced by rhCCLl . In the absence of CCL1, MC148 had no effect on [Ca2+] mobilization. These data establish mouse CCL1 as an agonist for human CCR8.

MC148 also prevented the cytoskeletal rearrangements and cell shape change in tumor cells stimulated with rhCCLl (Fig. 3 C) . The effect was comparable to that of pertussis toxin, a broad inhibitor of GPCR signaling. Together, these data demonstrate that CCL1 mediates its effects on tumor cell migration by activating CCR8 and GPCR signaling in tumor cells.

EXAMPLE 5. CCR8 is expressed in human metastatic melanoma

The expression of CCR8 by immunohistochemistry on a tissue array containing 90 cases of human malignant melanomas, in another cohort of 19 samples of human melanoma in the skin, and in eight lymph nodes with melanoma metastases was examined. Analysis of the tissue array showed CCR8 expression in 71% of the samples, at variable levels of intensity (Fig. 4) . Approximately half of the positive samples (34%) showed strong membrane stain. Some melanomas showed intense staining for CCR8 throughout the specimen, whereas others showed focal pattern of expression (Fig. 4 A-D) . Melanomas showing weak or moderate levels of CCR8 expression mostly exhibited homogenous cytoplasmic stain (Fig. 4 E-H) . Analysis of a different cohort of patient samples (n=19) also showed that CCR8 is expressed in melanoma: 18/19 samples (95%) were stained for CCR8, while 11/19 samples (57%) were strongly positive (Figure 41) . In the lymph nodes, 7 out of 8 metastases were positive for CCR8 (Fig. 4 J-L) . In accordance with in vitro data, melanocytes did not express the CCR8 receptor. Lymphatic vessels in the normal or tumor-associated skin were CCR8 negative. A subset of blood vessels (arterioles) was CCR8 positive, which is in agreement with the published data (Haque et al., 2004) . A subset of tumor-infiltrating lymphocytes in the skin and in the lymph nodes was CCR8 positive

(data not shown) .

EXAMPLE 6. Blocking CCR8 inhibits lymph node metastasis

To investigate the role of CCR8 in tumor metastasis, three tumor cell lines were tranfected to constitutively express soluble CCR8 antagonist MC148: SK-MEL-25 melanoma cells, a highly metastatic subclone of MDA-MB-435 cells which expresses VEGF-C (MDAcl .13) , and a moderately metastatic subclone of parental MDA-MB-435 cells (MDAcl.6) (Das et al . , 2010; Roberts et al . , 2006; Skobe et al . , 2001b) . Expression of MC148 mRNA was confirmed by RT-PCR, while Western blot analysis showed high levels of MC148 protein in the conditioned media of transfected cells as well as in tumor lysates. The levels of CCR8 were not altered upon transfection (Fig. 8, 9) . High levels of MC148 protein were detected in the conditioned media of transfected cells as well as in tumor lysates. MC148 expression did not change levels of endogenous CCR8 and it did not alter tumor growth of any of the three cell lines in mice or in vitro (Fig. 8, 9, and data not shown) . CCR8 knockdown with shRNA was confirmed in vitro and in vivo by qPCR and by Western blotting (Fig. 23) .

Notably, inhibition of CCR8 significantly reduced the incidence of lymph node metastases in orthotopic tumor models, as determined by fluorescence microscopy (Table 1) . In the SK-MEL-25 tumor model, metastasis to sentinel lymph nodes was decreased from 42% in the parental cells to 0% in the MC148-expressing cells. In the MDAcl.13 model, incidence of lymph node metastases was 78% in the control vs. 20% in mice bearing MC148-expressing tumors. MDAcl .6 parental tumors showed a 35% incidence of lymph node metastases in the control group and 10% in the MC148 transfectants . Metastasis to the brachial lymph nodes was, in MDAcl.13, decreased to the same extent as to the sentinel lymph nodes (control 78% vs. MC148 20%) . In the SK-MEL-25 and MDAcl.6 tumor model the trend was the same, but the baseline rate of metastases to the next set of lymph nodes after the sentinel was too low for a difference to be significant (SK-MEL-25: control 21% vs. MC148 0%; MDA cl.6: control 10% vs. MC148 5%) . Furthermore, knockdown of CCR8 in vivo using two independent shRNA constructs significantly reduced lymph node metastases in all three tumor models, and the results were very similar to those obtained with the MC148 inhibition approach (Table 1) .

Table 1. incidence of intra-rtodai metastases:

Ceil line Control C148

SK-MEL-25 6 /14 (43%) 0/18 {0%)

MDA cl.13 14/18 (78%) 4/20 (20%)

MDA cl.6 7/20 (35%) 2/20 ( 10%)

Examination of primary tumors for various histopathological parameters and angiogenesis did not reveal any major differences between the control and treated groups in all tumor models (Fig. 10, 11) . Intratumoral lymphangiogenesis, which was induced only in MDAcl.13 tumors (Skobe et al . , 2001a), was not altered with MC148 (Fig. 10) or with shRNA knockdown (data not shown) .

Since it has not been unequivocally demonstrated that mCCLl activates human CCR8, calcium flux studies upon addition of mouse CCL1 to both SK-MEL-25 and MDA-MB-435 cells were performed (Fig. 12A-F) . Addition of recombinant mouse CCL1 lead to a rapid increase of the intracellular calcium level, while the CCR8 antagonist MC148 completely inhibited CCLl-induced [Ca2+] flux. Similar to human CCL1, mouse CCL1 also induced cytoskeletal rearrangements in human tumor cells (Fig. 12 H, I) . These data establish mouse CCL1 as an agonist for human CCR8.

Together, these findings demonstrate that inhibition of CCR8 leads to a significant reduction of lymph node metastases. Since none of the tumor cell lines expressed CCL1 (Fig. 8, 9), these data suggest that CCL1 exerts its effects on tumor cells in vivo by a paracrine mechanism and that CCR7 on tumor cells is activated by host-derived CCL1. EXAMPLE 7. CCLl is expressed by the lymph node subcapsular sinuses

Because CCLl induced tumor cell chemotaxis in vitro and because it was highly upregulated by inflammatory stimuli in vitro and in vivo, it was hypothesized herein that CCLl may be expressed by tumor- associated lymphatic vessels and facilitate tumor cell entry into the lymphatics at the primary tumor site. Surprisingly, CCLl expression in the lymphatic capillaries associated with the primary tumor in the mouse could not be detected (Fig. 5) . Neither intratumoral lymphatics, nor lymphatics in the skin overlying tumors were positive for CCLl by immunostaining . Other stromal cells were also negative for CCLl. Unexpectedly, CCLl was mainly localized in the subcapsular sinuses of the lymph nodes (Fig. 5 G-I) .

Unexpectedly, CCLl was detected mainly in the subcapsular sinuses of the lymph nodes; specifically, on the lymphatic endothelium of the SCS which was VEGFR-3high/Podoplaninhigh/LYVE-llow (Fig. 5 G-L, Fig.

24) . The staining pattern of CCLl in the tumor draining lymph nodes and in the naive LN was similar, indicating constitutive expression. Furthermore, CCLl was immunostained without permeabilization, indicating that it was displayed on the cell surface, and it was clearly present on the luminal side of the vessels (Fig. 5 J-L, Fig. 16) . Medullary sinuses (VEGFR-3high/Podoplaninlow/LYVE-lhigh, Fig. 24) were negative for CCLl, and only occasionally few lymphocytes stained positive (Fig. 5 M-O) .

Chemokine CCL8 has been recently identified in mice (but not in humans) as a second agonist for CCR8 (Islam, 2011) . Mouse tissues were therefore examined for the presence of CCL8 protein by immunostaining. Tumors and lymph nodes were negative for CCL8, while CCL8 protein was detected in skin keratinocytes , consistent with the previous findings (Islam, 2011) . CCL8 was not detected in the lymphatic vessels in the skin, tumors or in the lymph nodes (Fig.

25) .

The localization of CCLl in mouse and in human tissues was very similar (Fig. 6) . In normal human skin CCLl was detected by immunostaining only in a small subset of blood vessels, and not in the dermal lymphatics. In human melanoma, CCLl was detected in a subset of blood vessels and occasionally in tumor cells, but not in the lymphatic vessels. In the lymph nodes with melanoma metastases, subcapsular sinuses were strongly positive for CCLl (Fig. 6 G-J) . In addition to the lymphatic endothelium, a small subset of lymphocytes and a small fraction of blood vessels were also CCLl positive. These results indicate that the CCL1-CCR8 interaction is not required for tumor cell entry into the lymphatic vasculature and suggest that CCR8-mediated tumor cell interaction with LECs occurs downstream from the primary tumor.

EXAMPLE 8. Blocking CCR8 arrests metastases in the afferent lymphatic vessels and inhibits tumor cell entry into the lymph node

Because the lymph node subcapsular sinus was identified as a main site of CCLl production, the characteristics of metastases in the lymph node and at the lymph node interface were examined in detail . Remarkably, lymph nodes draining tumors expressing MC148 were mostly free of metastases; however, large clusters of metastatic cells were present in the afferent lymphatic vessels leading to the lymph node (Fig. 7) . In the MDAcl .13/MC148 group metastases were arrested at the junction of the afferent lymphatic vessel and the lymph node, and this border was sharply demarcated (Fig. 7 E) . Analysis of the junction by SEM and by immunostaining for lymphatic endothelial markers showed that LECs were the cellular boundary at which the tumor was arrested (Fig. 19B-D) . The collecting lymphatic vessels were greatly distended, particularly in the proximity of the lymph node. Metastatic cells formed large clusters that were attached to the luminal wall of lymphatic vessels, and in most cases metastases occupied (resided in) the collecting lymphatics over long distances.

Presence of metastases in the regional lymphatics distant from the primary tumor but before the regional lymph node basin, is referred to as in-transit metastasis (Fig. 13) . It is a particularly common phenomenon in melanoma and it has also been described in other types of cancer, such as breast cancer (Balch et al., 2001; Pawlik et al., 2005; van Deurzen et al . , 2008) . Both, inhibition of CCR8 with MC148 and CCR8 knockdown with shRNA, increased the ratio of in-transit to intranodal metastases (Fig. 19A) . In the MDAcl.13 control group, 93% of the mice showing metastases were lymph node positive and only 7% presented with in- transit metastases. In contrast, when CCR8 antagonist was expressed this ratio was reversed: as many as 73% presented with in-transit metastases, whereas only 27% had intra-nodal metastases (Fig. 7) . Similarly, in the MDAcl.6 group in-transit metastases increased from 46% to 78%, while intra-nodal metastases decreased (Fig. 14) . In the MC148 group, metastases were arrested at the junction of the afferent lymphatic vessel and the lymph node, and this border was sharply demarcated (Fig. 7 E and Fig. 13) . The collecting lymphatic vessels were greatly distended, in particular in the proximity of the lymph node (Fig. 7) . Knockdown of CCR8 using two different shRNA constructs confirmed these results (% in-transit metastases: control 0% vs. shCCR8(l) 83% vs. CCR8(2) 75%; Fig. 19A) . In the MDAMB-435 parental and SK-MEL-25 tumor models the trend was similar; however, these tumors did not induce lymphangiogenesis and therefore did not have as much metastatic burden in the lymphatic vessels as MDAcl.13 tumors. In the MDAcl.6 tumors in-transit metastases increased from 46% (6/13) to 78% (7/9) (n=x) , and in SK-MEL-25 from 0 (0/14) to 33% (4/12) (n=x) . These percentages are of positive samples only, not total LN. Metastatic cells formed large clusters that were attached to the lymphatic vessel wall. In some cases, the vessel lumen was partially obstructed, but in most cases tumor cells filled the lymphatic lumen over long distances (Fig. 7 F-H, M, N) . These data suggest that tumor cells may not enter lymph nodes as floating emboli, but as adherent cells. Upon entry into the lymph node, metastases resided first in the subcapsular sinus, and then invaded the lymph node cortex (Fig. 7 A-D, Fig. 13 G, H) .

The incidence of lymph node metastasis was significantly decreased in all tumor models when CCR8 was inhibited (Table 1) . Taken together, these observations demonstrate that the junction of the afferent lymphatic vessel and the lymph node sinus represents a barrier for tumor cell entry into the lymph node. Furthermore, the data show that blocking CCR8 on tumor cells prevents tumor cell extravasation from the collecting lymphatics into the lymph node, but it does not affect the tumor cell entry into the lymphatics at the primary tumor site.

EXAMPLE 9. Sequential steps of lymph node metastasis

The sequence of events leading to lymph node metastasis was characterized by immunostaining, SEM and by multiphoton intravital imaging. Dilation of the subcapsular sinus preceded the arrival of MDAcl.13 tumor cells into the sentinel lymph nodes (Fig. 20 A-C, Fig. 5J) . The SCS was lined by the continuous layer of lymphatic endothelial cells which stained positive for CD31, VEGFR-3, podoplanin and LYVE-1 (Fig. 7, I-K; Fig. 20 A-C, H; Fig. 24) . Furthermore, the SCS was traversed by the columns connecting the roof and the floor of the sinus, which were continuous with the lymphatic endothelium and also stained positive for CD31, VEGFR-3, podoplanin and LYVE-1 (Fig. 20 A, B and data not shown) . High resolution analysis using SEM showed numerous columns within the sinus spaced 11.9 μιτι apart from each other on average (range: 6.2 - 20.8 μιτι; average column diameter: 2.0 μιτι + 0.61) (Fig. 20 D) . The average height of the dilated sinus was 8.6 μιτι (range: 4.5 - 13.0 μιη) . Some columns were transversally interconnected (Fig. 20 E) , dividing the space within the sinus further into smaller compartments with an average diameter of 5.25 μιη (range: 3.6 - 7.8 μιη) . These studies revealed a complex microanatomy of the subcapsular sinus, and suggested that the small dimensions of the SCS and the columns bridging the SCS may pose a barrier for the free movement of cell clusters laterally into the sinus.

Multiphoton imaging studies in live mice showed that the tumor lesion at the junction of the lymphatic vessel and the subcapsular sinus exhibited very little movement: only occasional single cells were seen extending and retracting protrusions (Fig. 21) . In the subcapsular sinus, which was visualized by Evans blue lymphatic tracer and imaged at 35 μιτι depth under the lymph node capsule, small tumor clusters consisting of as few as five cells were stationary, and did not move through the sinus with the flow of lymph within the observation period of several hours. In contrast, many leukocytes were actively crawling through the sinus with mean velocity of 3.4 m/min (Fig. 21 C, and data not shown) . Lymph flow into the sinus was not hindered by the tumor metastasis since the SCS readily filled with the Evans blue tracer and it was evident that some leukocytes were passively drifting through the sinus with the flow of lymph at much higher velocities. Several single tumor cells present in the SCS were devoid of any movement, suggesting that they were adherent to the lymphatic endothelial lining. In addition, individual tumor cells were seen slowly moving through the SCS independently of lymph flow, with a mean velocity of 0.5 m/min (Fig. 21 D and data not shown) . In agreement with these real-time in vivo observations, immunostaining of tissue sections showed single cells and small clusters of tumor cells attached to the lymphatic endothelium of the SCS, mostly to the roof of the sinus (Fig. 20 B- D) .

Intravital imaging of metastases further upstream in the collecting lymphatic vessels showed small and large tumor cell clusters that were not moving with the flow of lymph during the time examined, but were attached to the luminal surface of the lymphatic endothelium

(Fig. 26) . Within the tumor cell clusters, some cells exhibited amoeboid movement and were seen migrating into a different position

(Fig. 26 B, C) . Tumor cell clusters were only partially obstructing the lymphatic vessel lumen, suggesting that the tumor cell arrest at the lymphatic vessel wall occurred by an active mechanism, and not because the tumor cell emboli occluded the vessel lumen.

Once tumor cells reached the lymph node, in some cases they seemed to have crossed the floor of the SCS right at the junction of the afferent lymphatic vessel and the lymph node (Fig. 7 I, J,; Fig. 20, G) . In most cases, however, metastases were first seen in the SCS (Fig. 20 B, C, F) and later in the lymph node cortex (Fig. 7, Fig 20, H) . Metastases continued to grow within the subcapsular space and eventually completely occluded the SCS (Fig. 20 F) . Remarkably, the integrity of the lymphatic endothelial layer was not disrupted even when very large lesions were present within the sinus. Taken together, these data indicate that the egress of tumor cells from the afferent lymphatic vessel into the lymph node consists of at least two distinct steps: (i) tumor cells entry into the SCS and (ii) subsequent transmigration across the floor of the SCS into the lymph node cortex. Without wishing to be bound by any scientific theory, these data support the conclusion that the entry of tumor cells into the lymph node cortex requires active cell migration.

Discussion

The lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system. It is widely believed that tumor cells enter lymph nodes passively, by the flow of lymph. As demonstrated herein, lymph node lymphatic sinuses control tumor cell entry into the lymph node. In vitro, tumor migration to lymphatic endothelium (LECs) was inhibited by blocking CCR8 or CCL1. Recombinant CCL1 promoted migration of CCR8+ tumor cells. Pro-inflammatory mediators TNF-a, IL-Ιβ and LPS increased CCL1 production by LECs as well as tumor cell migration to LECs. Blocking studies showed that CCL1 is a key molecule mediating tumor cell chemotaxis to inflamed lymphatic endothelium. In mouse and human tissues CCL1 protein was detected in lymph node lymphatic sinuses, but not in the peripheral lymphatics. In addition, CCR8 was strongly expressed by human malignant melanoma. In a mouse model, blocking CCR8 function decreased lymph node metastasis. Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus. These data identify a novel function for CCL1/CCR8 in metastasis and lymph node LECs as a critical checkpoint for entry of metastases into the lymph nodes.

In cancer, the CCL1-CCR8 axis has been implicated in leukemia and in lymphoma. The CCL1-CCR8 autocrine loop has been shown to protect lymphoma and T-cell leukemia cells from apoptosis in vitro (Louahed et al., 2003; Ruckes et al . , 2001; Van Snick et al . , 1996) and to play a role in T-cell transformation (Tamguney et al . , 2004) . Whether the CCL1-CCR8 axis plays a role in solid tumors is not yet known .

A novel function for CCL1-CCR8 in controlling the egress of tumor cells from the afferent lymphatics into the lymph node is demonstrated herein. Disclosed herein is the unexpected discovery that the lymphatic endothelial cells at the junction of the collecting lymphatic vessel and the lymph node represent a barrier for entry of tumor cells into the lymph node. Activation of CCR8 on tumor cells by CCL1, produced by lymphatic endothelial cells in the lymph node, allows entry of tumor cells into the node and subsequent formation of lymph node metastases. These data identify a novel mechanism of regulation of cell entry into the lymph node. As demonstrated herein, the entry of tumor cells from the afferent lymphatics into the lymph nodes requires activation of the CCR8 receptor on tumor cells. Blocking CCR8 activation led to arrest of tumor cells in the collecting lymphatic vessels and prevented lymph node metastases. CCL1, the ligand for CCR8, was expressed by lymph node lymphatic sinus, but not by lymphatic capillaries, indicating that the signals from lymph node subcapsular sinus LECs control tumor cell entry into the node. These data identify lymph node LECs as a critical check-point for entry of metastases into the lymph nodes .

While the recent data indicate that the entry of cells from peripheral tissues into the lymphatics is regulated by chemokines (Debes et al., 2005; Shields et al . , 2007b), the exit of cells from the collecting lymphatics into the lymph node has been considered a passive process (Achen et al . , 2005; Alitalo et al . , 2005; Nathanson, 2003) . Conventional wisdom implies that tumor cells which enter lymphatics will be delivered into the draining lymph nodes by the flow of lymph. The data herein challenge the current paradigm, and indicate that the exit from the afferent lymphatics into the lymph nodes is a distinct, controlled step. The data also suggests that tumor cells do not enter the lymph nodes as floating emboli. Instead, tumor cells may adhere to the luminal side of the collecting lymphatics and the SCS, and from there undertake migration into the LN parenchyma. This adds the rate of tumor cell egress from the afferent lymphatics as another rate limiting step for metastasis.

Lymph node junction with the afferent lymphatics may also be a check-point for entry of other cells which traffic from the periphery to lymph nodes. Dendritic cells and memory T-cells express CCR8 (Gombert et al . , 2005; Soler et al . , 2006), and, in support of this concept, blocking CCR8 has been shown to decrease a number of dendritic cells in the draining lymph nodes (Qu et al . , 2004) . Hence, tumor cells might be exploiting the mechanisms of cell traffic which operate in the physiological setting. Given the complexity of the lymph node architecture and the tight control of leukocyte traffic in the lymph nodes, it is surprising that tumor cells arriving from the afferent lymph are thought to have unrestricted access to the lymph node parenchyma. Notably, lymph-borne molecules do not have free access into the lymph node (von Andrian and Mempel, 2003) . Studies using fluorescently labeled tracers have demonstrated that soluble antigens do not freely diffuse into the lymph node parenchyma, but are retained within the subcapsular or medullary sinuses (Fossum, 1980; Gretz et al . , 2000; Szakal et al., 1983; von Andrian and Mempel, 2003) . While small molecules with a MW below 70 kDa (or a diameter smaller than 5nm) can gain access to the LN cortex through fibroblastic reticular cell (RFC) conduit, molecules larger than 5.5 nm remain confined to the sinus (Gretz et al . , 2000; von Andrian and Mempel, 2003) . Another study found that small FITC-dextran (lOkDa) was retained within the RFC conduit system and never diffused into the lymphocyte compartment. Large FITC-dextran (2000kDa) was retained in the lymphatic sinuses and excluded from both the tissue parenchyma and the conduit system (Gretz et al . , 2000; Pfeiffer et al . , 2008) . Hence, small molecules, such as chemokines, can travel through the LN conduit system, whereas antigens and immune complexes remain in the SCS (Fossum, 1980; Gretz et al . , 2000; Szakal et al . , 1983) and require cellular transport (Phan et al . , 2007) . However, select macromolecules , such as antibodies, can gain access to the LN parenchyma (von Andrian and Mempel, 2003), indicating that the selectivity is not based solely on size exclusion, but that other criteria apply. In addition to preventing diffusion of soluble molecules into the lymphocyte compartment, the subcapsular sinus is an important site for preventing systemic spread of microorganisms and viruses (Junt et al . , 2007) . These reports are consistent with the conclusion disclosed herein that lymphatic vessel sinus represents a barrier for entry of tumor cells into the lymph node. The LN cortex is protected from the direct contact with the afferent lymph by subcapsular sinus-lining lymphatic endothelial cells (Dullmann et al . , 2002; Junt et al . , 2007; Pfeiffer et al . , 2008; Phan et al . , 2007; Sainte-Marie et al . , 1982), which are also referred to as the floor of the SCS (Gretz et al . , 2000; Junt et al . , 2007) . LECs lining the sinus possess tight junctions and the basal membrane, indicating a diffusion barrier (Pfeiffer et al . , 2008) . One study showed that high MW dextrane (2000 kDa) was taken up by lymphatic capillaries but did not cross SCS LEC layer (Pfeiffer et al . , 2008), suggesting that SCS LECs have different barrier properties than peripheral lymphatics. SCS LECs also differ from lymphatic capillaries by surface markers and junctional molecules expression (Martens et al., 2006; Pfeiffer et al . , 2008); however, the true functional differences between the peripheral lymphatics and SCS LECs are yet to be determined. Based on the data herein, a model in which LECs of the SCS floor provide signals that permit or inhibit tumor cell entry into the LN parenchyma is proposed. Taken together, these data point to an emerging concept; SCS LECs play a critical role in regulating the access of soluble molecules as well as cells into the LN.

As shown herein, the CCL1 chemokine is constitutively expressed by SCS LECs, but not by lymphatic capillaries in the skin. This is in agreement with the previous reports in which CCL1 was not detected in the peripheral lymphatics (Qu et al . , 2004; Schaerli et al . , 2004) . Based on these findings, it has been concluded that this chemokine does not play a role in the exit of CCR8+ T-cells and DCs from the tissue (Qu et al . , 2004; Schaerli et al . , 2004) . Similarly, the conclusion conclude disclosed herein that CCR8 does not play a role in intravasation of tumor cells into the peripheral lymphatics. Thus far, constitutive CCL1 expression has been detected by several types of immune cells (Gombert et al . , 2005; Miller et al . , 1989; Schaerli et al . , 2004), by blood endothelium of the skin, and by melanocytes (Schaerli et al . , 2004) . Expression of CCL1 by blood endothelium raises a possibility that CCL1/CCR8 interaction could also be important for tumor cell homing to distant organs, as well for the formation of secondary skin metastases, which are frequent in melanoma. In addition, finding described herein that CCL1 was expressed by a subset of malignant melanomas (data not shown) , suggesting that CCL1 may also have an autocrine function in cancer. The importance of CCL1/CCR8 axis in distant metastasis remains to be investigated .

CCR8 is a G-protein-coupled receptor which is in humans selectively activated by the CC chemokine CCLl/I-309. As disclosed herein, the CCR8 receptor is expressed by a large subset of human malignant melanomas (71%) and that CCR8 activation on melanoma cells is important in metastasis. Blocking CCR8 signaling in mouse models of melanoma inhibited lymph node metastasis. Blocking CCR8 in vitro inhibited melanoma cell chemotaxis towards lymphatic endothelium and its ligand, chemokine CCL1.

The work disclosed herein identifies CCR8 and its ligand CCL1 as novel therapeutic targets for treatment of malignant melanoma. This includes prevention and inhibition of lymph node metastases, and disease recurrence. CCR8 may represent an attractive therapeutic target in melanoma for the following reasons: (1) CCR8 is expressed in a large number of malignant melanomas examined; (2) CCR8 shows very restricted expression pattern in normal tissues. To date, CCR8 expression has been reported primarily on a subset of regulatory T- cells and on eosinophils with role in allergic inflammation, suggesting that blocking CCR8 for therapeutic purposes could have relatively little undesired effects. This is further supported by the fact that CCR8 has only one known human ligand; chemokine CCL1 (3) Because CCR8 has been implicated in allergic disorders and chronic inflammatory diseases such as asthma, several small molecule antagonists of CCR8 have already been developed for clinical use by companies such as AstraZeneca, Smithkline Beecham and Millennium Pharmaceuticals. This offers a great opportunity for rapidly adapting these inhibitors for use in melanoma.

To date, CCR8 has been known to play a rather unique role in the regulation of the immune response. It is preferentially expressed by activated TH2 cells (T helper type 2) and it mediates TH2 cell recruitment to the sites of inflammation. Since TH2 cells are primary drivers of allergy and asthma, CCR8 activation has been implicated in allergic inflammation and pulmonary hypersensitivity. Other functions of CCR8 include T-cell homing to the skin in the steady-state, the role in DC migration to the lymph nodes and the role in thymic development. Small molecule antagonists of CCR8 have been developed for their potential use in treating chronic inflammatory diseases such as asthma.

Study disclosed herein is the first to report expression and function of CCR8 in a solid tumor, specifically, in melanoma. CCR8 expression in solid tumors has not been demonstrated to date. A role for CCR8 in lymphoma and T-cell leukemia has been suggested from in vitro studies.

Key evidence supporting our claim that CCR8 has a function in melanoma metastasis is: (1) CCR8 is expressed in a large subset of malignant melanomas (71%); (2) CCL1 is chemotactic for CCR8+ melanoma cells in vitro; (3) Lymphatic endothelium promotes melanoma cell migration by secreting CCL1; (3) CCL1 is a major chemoattractant produced by lymphatic endothelium; (4) Migration of tumor cells to lymphatic endothelium or to CCL1 can be significantly reduced by inhibiting either CCL1 or CCR8 ; (8) Soluble antagonist of CCR8 significantly reduced melanoma metastases to the lymph node in three mouse xenograft models. See Experimental Details.

The data herein imply an important link between inflammation and lymphatic metastasis. The disclosure herein shows dramatic increase of CCL1 expression in the lymphatic endothelium upon inflammatory stimulus, in agreement with the previous work which found a marked induction of CCL1 expression in BECs, DCs and mast cells in inflammation (Gombert et al . , 2005) . Remarkably, data disclosed herein show that CCL1 was a key molecule mediating chemotaxis of tumor cells to inflamed lymphatic endothelium. In contrast, CCL1 was only partially responsible for tumor cell migration to unstimulated LECs, pointing to a role of additional chemokines in the process. These data suggest that CCL1 may play a particularly important role in facilitating LN metastases in the setting of inflammation.

Human CCL1 is a selective ligand for CCR8 (Goya et al . , 1998; Roos et al., 1997; Tiffany et al . , 1997) . This selectivity is unusual, as most chemokine receptors are able to bind to more than one chemokine and vice versa (Houshmand and Zlotnik, 2003) . Very restricted pattern of CCR8 expression in tissues is also quite unusual, which makes CCR8 an attractive therapeutic target (Schaerli et al . , 2004) . The data disclosed herein showing expression of CCR8 in malignant melanoma and its role in lymph node metastasis suggest that CCR8 could be explored as a potential therapeutic target in melanoma. To date, CCR8 expression has been reported primarily on a subset of T- cells with role in allergic inflammation, further supporting the concept that blocking CCR8 for therapeutic purposes could have relatively little undesired effects.

There is a discrepancy in the literature regarding the ability of mouse CCL1 to activate human CCR8. One study found that mCCLl stimulates Ca mobilization in human THP-1 cells. (Luo et al . , 1994) . In agreement, vMIP-I, viral homologue of CCL1, is an agonist for human and mouse CCR8 (Luttichau et al . , 2001) . However, another study reported that mCCLl did not induce Ca-flux in 293-EBNA cells expressing hCCR8 (Goya et al . , 1998) . Without wishing to be bound by any scientific theory, the conclusion disclosed herein that mCCLl is an agonist for human CCR8 is based on several lines of evidence: (i) mCCLl induced Ca-flux in three human cell lines; in HEK293 cells overexpressing CCR8 and in SK-MEL-25 and MDA-MB-435 cells, which endogenously express CCR8; (ii) Ca-flux induced by mCCLl was blocked with a specific CCR8 antagonist, MC148; and mCCLl triggered cytoskeletal rearrangement in human cells. A recent study found that mouse CCL8 is a second agonist for CCR8 ; however, a human orthologue has not yet been found (Islam et al . , 2011) . The relevance of this finding for human setting is not clear; nevertheless, the relative role of the two ligands has to be considered when interpreting the data from the mouse model. In conclusion, a novel function for CCL1/CCR8 and lymph node LECs in controlling tumor cell entry into the lymph node is demonstrated herein. The work disclosed herein adds to the growing body of evidence indicating that lymphatic vessels are not only a drainage and transportation system, but that lymphatic endothelial cells play an active role in a number of important biological events, including metastasis (Cohen et al., 2010; Debes et al., 2005; Jamieson et al . , 2005; Podgrabinska et al . , 2009; Skobe et al . , 2001b) .

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Claims

What is claimed
A method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCLl to the CCR8 receptors so as to thereby treat the subject.
A method of reducing metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an antagonist of CCR8 receptors on the surface of tumor cells present in the solid tumor in an amount effective to reduce binding of CCLl to the CCR8 receptors so as to thereby reduce metastases in the subject.
The method of claim 2, wherein lymph node metastases are reduced.
The method of any one of claims 1-3, wherein the solid tumor is a melanoma.
The method of any one of claims 1-4, wherein the antagonist of the CCR8 receptor is an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, a polypeptide, or an antibody, which antagonist binds to the extracellular domain of the CCR8 receptor at a site which prevents binding of a CCLl ligand to the CCR8 receptor.
The method of claim 5, wherein the antagonist is a polypeptide .
The method of claim 5, wherein the antagonist is an anti-CCR8 antibody .
The method of claim 7, wherein the anti-CCR8 antibody is a monoclonal antibody.
9. The method of claim 8, wherein the monoclonal antibody is 414B, 141C, 141E, 433H, 459M, 464A, 464B, 433B or 455AL.
10. The method of claim 8, wherein the monoclonal antibody is a human or a humanized monoclonal antibody.
11. The method of claim 10, wherein the human or humanized antibody comprises or is derived from the CCR8-binding region of one of monoclonal antibody 414B, 141C, 141E, 433H, 459M, 464A, 464B, 433B or 455AL.
12. The method of claim 6, wherein the CCR8 antagonist is MC148.
The method of claim 5, wherein the CCR8 antagonist is an organic compound having a molecular weight less than 1000 Daltons .
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000120_0001
wherein
B represents the group
Figure imgf000120_0002
ring D, together with the two benzene carbon atoms to which it is fused, is a 5- or 6- membered, non-aromatic ring containing one or two ring-oxygen atoms, and optionally containing a carbon-carbon double bond between two ring carbon atoms other than said benzene carbon atoms, ring D being optionally substituted with one or more substituents independently selected from Ci-C6 alkyl, C3-C6 cycloalkyl, or phenyl (said phenyl being optionally substituted with one or more substituents independently selected from halogen, hydroxyl or C1-C4 alkoxy) ;
and additionally wherein when ring D is a 5-membered, non- aromatic ring containing two ring-oxygen atoms that are 1,3 disposed, ring D may be optionally substituted with group E, wherein group E together with a single carbon atom on ring D, represents a 4- to 8-membered cycloalkyl ring, such that group E forms a spiro structure with ring D; w, x, y and z are independently 1, 2 or 3;
each R represents a group independently selected from halogen or Ci-C4 alkyl;
n is 0, 1 or 2;
A represents a group selected from phenyl, a 5- or 6- membered heteroaromatic ring containing at least one ring heteroatom independently selected from nitrogen, oxygen or sulphur, or pyridine-N-oxide, each group being optionally substituted with one or more substituents independently selected from hydroxyl, -CN, halogen, oxo (=0) , Ci~C6 aminoalkyl, Ci-C6 alkylamino-Ci-C6 alkyl, N,N-di (Ci~
C6) alkylamino-Ci-C6 alkyl, Ci-C6 alkoxy, Ci-C6 alkylcarbonyl , NRiR2, -C(0)-NR3R4, -CI-C6 alkyenyl-C (0) -NR3R4, -CI-C4 alkyl-C (0) - NR5R6, -NHSO2-R7, -NHC(0)R8, -S02NH2, carboxyl, carboxyl-Ci-C6 alkyl, Ci-C6 alkoxycarbonyl , C1-C4 alkoxycarbonyl-Ci-C4 alkyl, C3-C6 cycloalkylamino , phenyl, pyridyl (said phenyl and pyridyl being optionally further substituted with one or more groups independently selected from halogen, hydroxyl, carboxy or C1-C4 alkyl) , Ci-C6 alkyl or C3-C6 cycloalkyl (said latter two Ci-C6 alkyl and C3-C6 cycloalkyl substituents being optionally further substituted with one or more substituents independently selected from halogen, hydroxyl, or -CN) ;
or A represents a 9- or 10-membered bicyclic ring system containing one or more ring heteroatoms independently selected from nitrogen, oxygen or sulphur and which is optionally substituted with one or more substituents independently selected from hydroxyl, -CN, halogen, oxo, Ci-C6 alkoxy, NR9R10, carboxyl, or Ci-C6 alkyl;
p is 0, 1 or 2 ;
R1 and R2 each independently represent a hydrogen atom, a Ci-C6 alkyl, C3-C6 cycloalkyl or R1 and R2 together with the nitrogen atom to which they are attached form a hydantoin group or form a 4- to 7-membered saturated heterocycle, said heterocycle being optionally substituted with hydroxyl, C1-C4 alkoxy, or C1-C4 alkoxy-Ci-C4 alkyl;
R3 and R4 each independently represent a hydrogen atom, Ci-C6 alkyl, or C3-C6 cycloalkyl, or R3 and R4 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocycle, said heterocycle being optionally substituted with aminocarbonyl;
R5 and R6 each independently represent a hydrogen atom, Ci-C6 alkyl, or C3-C6 cycloalkyl, or R5 and R6 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocycle, said heterocycle being optionally substituted with aminocarbonyl;
R7 represents Ci-C6 alkyl, or a 6-membered saturated or unsaturated heterocyclic ring, the ring containing at least one nitrogen atom, the ring being optionally substituted with one or more substituents independently selected from halogen, oxo, Ci-C6 alkoxy, or Ci-C6 alkyl;
R8 represents pyridine-N-oxide optionally substituted with one or more substituents independently selected from halogen, or Ci-C6 alkyl, or R8 represents Ci-C6 alkyl, Ci-C6 hydroxyalkyl , or a 5- or 6-membered saturated heterocyclic ring containing at least one heteroatom independently selected from nitrogen and oxygen, which ring being optionally substituted with one or more substituents independently selected from halogen, Ci-C6 alkoxy, oxo, or Ci-C6 alkyl;
R9 and R10 each independently represent a hydrogen atom or Ci-C6 alkyl;
or a pharmaceutically acceptable salt thereof. The method of claim 13, wherein the organic compound has the structure :
Figure imgf000123_0001
wherein R represents pyridine N-oxide;
R1 represents the group:
Figure imgf000123_0002
or
R is a methoxy or ethoxy
R is a hydrogen, methoxy or ethoxy;
or a pharmaceutically acceptable salt thereof.
16. The method of claim 13, wherein the organic compound has the structure :
Figure imgf000123_0003
wherein R represents
Figure imgf000123_0004
wherein R2 and R3 independently represent -NR8-C (0) -C00H, -0- (Ci_4alkyl) -C00H, -Ci_4alkyl-C00H, or -C00H;
each R4 and R5 independently represent halogen, CF3 or Ci_4alkyl; p and q are independently 0, 1 or 2;
R represents hydrogen or Ci_4alkyl; R1 re resents the group:
Figure imgf000124_0001
or
and R6 and R7 are independently hydrogen, methoxy or ethoxy; or a pharmaceutically acceptable salt thereof.
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000124_0002
wherein R is pyridin-2-one or IV-C1-C4 alkyl pyridin-2-one;
or R is pyridin-2-one or IV-C1-C4 alkyl pyridin-2-one each of which is substituted with a group selected from CF3, halogen or C1-C4 alkyl;
R1 represents the group:
Figure imgf000124_0003
R3 and R4 are independently methoxy or ethoxy;
pharmaceutically acceptable salt thereof.
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000125_0001
or a pharmaceutically acceptable salt thereof, wherein:
Xi is a covalent bond, C=0 or CRaRb;
X is a covalent bond, 0, or NR5;
C=Z is C=0, CH2, C=NH, C=S or absent, provided that when Xi is a covalent bond then C=Z is not CH2, provided that when C=Z is absent then X is a covalent bond, and provided that when X is NR5 and C=Z is C=0 then R4 is not an aliphatic or substituted aliphatic group;
Ra and Rb are independently H or a C1-C3 alkyl;
R1 is: i) a substituted or unsubstituted aromatic group; ii) a substituted or unsubstituted non-aromatic ring; or iii) when X is NR5, then NR5- (CH2 ) mR1 , taken together, is optionally a substituted or unsubstituted non-aromatic heterocyclic group;
R2 is -H or a C1-C3 alkyl group;
R3 is -H; and R4 is: i) a substituted or unsubstituted aliphatic group; ii) a substituted or unsubstituted aromatic group; iii) a substituted or unsubstituted non-aromatic ring or a substituted or unsubstituted non-aromatic bridged bicyclic group; or iv) R3 and R4 taken together with the nitrogen atom to which they are bonded are a substituted or unsubstituted nitrogen-containing non-aromatic heterocyclic group;
R5 is -H, or a C1-C3 alkyl group; Ring B is a phenyl group or five or six membered carbocyclic non-aromatic ring fused to Ring A;
Rings A and B are optionally substituted with alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyl, halogen, cyano or nitro;
m is 0, 1, 2 or 3;
n is 1 or 2 ;
each substitutable carbon atom of the aromatic group represented by R1 is optionally substituted with a group represented by R10, wherein R10 is halogen, R°,-OH, -OR°, 0 (haloalkyl) , -SH, -SR°, 1 , 2-methylene-dioxy, 1,2- ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted -CH2(Ph), -CH2CH2 (Ph) , substituted -CH2CH2(Ph), -N02, -CN, -N(R')2, -NR'C02R°, NR'C(0)R°, -NR'NR'C (0)R°, -N (R ' ) C (0) N (R ' ) 2, -NR ' NR ' C (0) N (R ' ) 2, - NR'NR'C02R°, -C(0)C(0)R°, -C (0) CH2C (0) R°, -C02R°, -C(0)R°, C(0)N(R°)2, -0C(0)R°, -0C(0)N(R°)2, -S (0) 2R°, -S02N(R')2, S(0)R°, -NR ' S02N (R ' ) 2 , -NR'S02R°, -C (=S) N (R' ) 2, - (CH2 ) yN (R ' ) 2 , -C (=NH) - N(R')2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, -V-N02, -V-CN, -V-N(R')2, -V-NR'C02R°, -V-R'C(0)R°, -V-NR ' NR ' C (0) R°, -V- N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V- C(0)C(0)R°, -V-C (0) CH2C (0) R°, -V-C02R°, -V-C(0)R°, -V- C(0)N(R°)2,-V-0C(0)R°, -V-0C(0)N(R°)2, -V-S(0)2R°, -V-S02N (R ' ) 2, - V-S(0)R°, -V-NR' S02N (R' ) 2, -V-NR'S02R°, -V-C (=S) N (R' ) 2, -V- (CH2) yN (R' ) 2, or -V-C (=NH) -N (R' ) 2, and each group represented by R10 is independently selected;
each substitutable carbon atom of the aliphatic group represented by R4 is optionally substituted with a group represented by R11, wherein R11 is halogen, R°, -OH, -0R°, 0 (haloalkyl) , -SH, -SR°, 1 , 2-methylene-dioxy, 1,2- ethylenedioxy, phenyl (Ph) , substituted Ph, -0(Ph), substituted -0(Ph), -CH2(Ph), substituted -CH2(Ph), -CH2CH2 (Ph) , substituted -CH2CH2(Ph), -N02, -CN, -N(R')2, -NR'C02R°, NR'C(0)R°, -NR'NR'C (0)R°, -N (R ' ) C (0) N (R ' ) 2, -NR ' NR ' C (0) N (R ' ) 2, - NR'NR'C02R°, -C(0)C(0)R°, -C (0) CH2C (0) R°, -C (0) R°, -C(0)N(R°)2, - 0C(0)R°, -0C(0)N(R°)2, -S(0)2R°, -S02N(R')2, -S (0) R°, NR' S02N (R' ) 2, -NR'S02R°, -C (=S) N (R' ) 2, - (CH2 ) yN (R ' ) 2 , -C(=NH)- N(R')2, haloalkyl, -V-R°, -V-OH, -V-OR0, -V-SH, -V-SR0, -V-N02, -V-CN, -V-N(R')2, -V-NR'C02R°, -V-NR ' C (0) R°, -V-NR ' NR ' C (0) R°, - V-N (R')C(0)N(R')2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V- C(0)C(0)R°, -V-C (0) CH2C (0) R°, -V-C(0)R°, -V-C (0) N (R°) 2, -V- 0C(0)R°, -V-0C(0)N(R°)2, -V-S(0)2R°, -V-S02N (R' ) 2, -V-S (0) R°, -V- NR' S02N (R' ) 2, -V-NR'S02R°, -V-C (=S ) N (R ' ) 2 , -V- (CH2 ) yN (R ' ) 2 , -V- C (=NH) -N (R' ) 2, =NNHR* , =NN(R*)2, =NNHC(0)R*, =NNHC02 (alkyl ) , =NNHS02 (alkyl), or =NR*, and each group represented by R11 is independently selected;
each substitutable carbon atom of the aromatic group represented by R4 is optionally substituted with a group represented by R12, wherein R12 is halogen, R°, -OH, -OR0, 0 (haloalkyl) , -SH, -SR°, 1 , 2-methylene-dioxy, 1,2- ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted -CH2(Ph), -CH2CH2 (Ph) , substituted -CH2CH2(Ph), -CN, -NR'C02R°, -NR ' NR ' C (0) R°, N (R' ) C (0) N (R' ) 2, -NR'NR' C (0) N (R' ) 2, -NR ' NR ' C02R°, -C(0)C(0)R°, - C (0) CH2C (0) R°, -C02R°, -C(0)R°, -0C(0)R°, -OC (0) N (R°) 2, -S (0) 2R°, -S02N(R')2, -S(0)R°, -NR'S02N(R' )2, -NR'S02R°, -C (=S ) N (R ' ) 2 , - (CH2) yN (R' ) 2, -C (=NH) -N (R' ) 2, haloalkyl, -V-R°, -V-OH, -V-0R0, - V-SH, -V-SR0, -V-CN, -V-NR'C02R°, -V-NR ' NR ' C (0) R°, -V- N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V- C(0)C(0)R°, -V-C (0) CH2C (0) R°, -V-C02R°, -V-C(0)R°, -V-0C(0)R°, - V-OC (0)N(R°)2, -V-S(0)2R°, -V-S02N(R' ) 2, -V-S (0) R°, -V- NR' S02N (R' ) 2, -V-NR'S02R°, -V-C (=S ) N (R ' ) 2 , -V- (CH2 ) yN (R ' ) 2 or -V- C (=NH) -N (R ' ) 2 , and each group represented by R12 is independently selected;
each substitutable carbon atom of: i) the non-aromatic ring represented by R1 or R4; ii) the non-aromatic heterocyclic group formed from NR5(CH2)mR1; iii) the nitrogen-containing non- aromatic heterocyclic group formed from NR3R4; and iv) the non- aromatic bridged bicyclic group represented by R4 is optionally and independently substituted with -R°, -OH, -OR0, 0 (haloalkyl) , -SH, -SR°, 1 , 2-methylene-dioxy, 1,2- ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted -CH2(Ph), -CH2CH2 (Ph) , substituted -CH2CH2(Ph), -CN, -N02, -N(R')2, -C(0)N(R°)2, NR'C(0)R°, -NR'C(0)R°, -V-N(R')2, -V-NR ' C (0) R°, -V-C (0) N (R°) 2, - NR'C02R°, -NR'NR'C (0)R°, -N (R ' ) C (0) N (R ' ) 2 , -NR ' NR ' C (0) N (R ' ) 2, - NR'NR'C02R°, -C(0)C(0)R°, -C (0) CH2C (0) R°, -C02R°, -C(0)R°, OC(0)R°, -OC(0)N(R°)2, -S(0)2R°, -S02N(R')2, -S (0) R°, NR ' S02N (R ' -NR'S02R°, -C (=S) N (R' ) 2, - (CH2 ) yN (R ' ) 2 , -C(=NH)- N(R')2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, NR'C(0)R°, -V-NO2, -V-CN, -V-N(R')2, -V-NR'C02R°, -V-NR ' C (0) R°, - V-NR'NR' C (0) R°, -V-N (R' ) C (0) N (R' ) 2, -V-NR ' NR ' C (0) N (R ' ) 2, -V- NR'NR'C02R°, -V-C (0) C (0) R°, -V-C (0) CH2C (0) R°, -V-C02Ro, -V- C(0)R0, -V-C (0)N(R°)2, -V-0C(0)R°, -V-OC (0) N (R°) 2, -V-S (0) 2R°, - V-S02N(R')2, -V-S(0)R°, -V-NR' S02N (R' ) 2, -V-NR'S02R°, -V- C(=S)N(R')2, -V- (CH2) yN (R' ) 2, -V-C (=NH) -N (R' ) 2, =0, =S, =NNHR* , =NN(R*)2, =NNHC(0)R*, =NNHC02 (alkyl ) , =NNHS02 (alkyl) or =NR* ; each R° is independently hydrogen or substituted or unsubstituted aliphatic group, a substituted or unsubstituted cycloaliphatic , a substituted or unsubstituted non-aromatic heterocyclic group or a substituted or unsubstituted aromatic group selected from phenyl, naphthyl, 2-furanyl, 3-furanyl, N- imidazolyl, 2-imidazolyl, 4-imidazolyl , 5-imidazolyl , 3- isoxazolyl, 4-isoxazolyl , 5-isoxazolyl , 2- oxadiazolyl, 5- oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl , 2- thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-triazolyl, 5-triazolyl, tetrazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl , benzothienyl , benzofuranyl , indolyl, quinolinyl, benzotriazolyl , benzothiazolyl , benzooxazolyl , benzimidazolyl, isoquinolinyl , indolyl, isoindolyl, acridinyl, or benzoisazolyl;
each R' is independently R°, -C02R°, -S02R° or -C (0) R°;
each R* is independently hydrogen, an unsubstituted aliphatic group or a substituted aliphatic group;
V is a C1-C6 alkylene group;
each substitutable carbon atom of: i) the aliphatic, cycloaliphatic, non-aromatic heterocyclic group and aromatic group represented by R°; and ii) the aliphatic group represented by R* is optionally and independently substituted with amino, alkylamino, dialkylamino, aminocarbonyl , halogen, alkyl, aminoalkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl , alkylcarbonyl , hydroxy, haloalkoxy, or haloalkyl;
each substitutable nitrogen atom of: i) the non-aromatic ring represented by R1 or R4; ii) the non-aromatic heterocyclic group formed from NR5 (CH2) mR1; iii) the nitrogen-containing non- aromatic heterocyclic group formed from NR3R4; iv) the non- aromatic heterocyclic group represented by R°; and v) the non- aromatic bridged bicyclic group represented by R4 is optionally and independently substituted with R+, -N(R+)2, -C(0)R+, -C02R+, -C(0)C(0)R+, -C (0) CH2C (0) R+, -S02R+, -S02N(R+)2, -C (=S ) N (R+ ) 2 , - C (=NH) -N (R+) 2, -NR+S02R+, -C(0)-NHR+, -C (0) -N (R+) 2, -C(0)- CH[N(R+)2]R+ or -C (0) -CH [0R+] R+;
each R+ is independently hydrogen, an unsubstituted heteroaryl or an aliphatic, cycloaliphatic , non-aromatic heterocyclic group, phenyl or benzyl group, wherein each substitutable carbon atom of the aliphatic, cycloaliphatic, nonaromatic heterocyclic ring, phenyl or benzyl group represented by R+ is optionally substituted with amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl or N(R+)2 is a non-aromatic heterocyclic group; and
each substitutable nitrogen atom of the non-aromatic heterocyclic group represented by R+ is optionally substituted with alkyl, alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl or dialkylaminocarbonyl,
provided that the compound is other than N-[4-[[(4- methoxyphenyl ) amino] sulfonyl ] -1-naphthalenyl ] -benzamide, N- [ 4- [ (2-propenylamino) sulfonyl ] -1-naphthalenyl ] -benzamide, N- [ 4- (4-morpholinylsulfonyl) -1-naphthalenyl] -benzamide or N-[4-(l- piperidinylsulfonyl) -1-naphthalenyl] -benzamide .
19. The method of claim 13, wherein the organic compound has the structure :
Figure imgf000130_0001
or a pharmaceutically acceptable salt thereof, wherein:
Ring A is unsubstituted;
Ring B is an unsubstituted phenyl, cyclohexyl or cyclopentyl ring fused to Ring A;
R1 is cyclohexyl or phenyl, furanyl, thienyl or pyridyl optionally substituted with Ci-C4 alkyl, Ci-C4 alkoxy, Ci-C4 haloalkyl, C1-C4 haloalkoxy, methylenedioxy, ethylenedioxy, halogen, cyano or nitro;
Figure imgf000130_0002
-C (0) -NHR -C (0) -N (R22) 2 or -C(0)CH[N(R23)2]R24;
R is methyl, ethyl, 2-hydroxyethyl or iso-propyl;
R22 is -H or C1-C4 alkyl or -N(R22)2 taken together is N- pyrollidinyl or iV-piperidinyl , provided that R22 is not -H when
R20 is -COOR22; R is -H, methyl or ethyl;
R24 is -H, methyl, ethyl, phenyl, benzyl, 4-hydroxyphenyl or 4-hydroxybenzyl ; and s is 0, 1, 2, 3 or 4.
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000131_0001
wherein
n is 0, to 6;
m is 1, to 4 ;
p is 1, to 4 ;
Ar is unsubstituted naphtha-2-yl , benzo [ 1 , 3 ] dioxolyl , 2 , 3- dihydro-benzo [ 1 , 4 ] dioxinyl , benzothiophenyl , benzofuranyl , or quinolinyl; or naphth-2-yl, benzo [ 1 , 3 ] dioxolyl , 2,3-dihydro- benzo [ 1 , 4 ] dioxinyl , benzothiophenyl, benzofuranyl , quinolinyl substituted with one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-Ce alkyl carbamoyl, Ci-C8 alkoxy carbonyl and trihalomethyl;
Ri and R6 are independently selected from the group consisting of hydrogen, Ci-C8 branched or unbranched alkyl, C3- C8 cycloalkyl lower alkyl, and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci~C8 alkyl, halo, cyano and trihalomethyl ;
R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, Ci-C8 branched or unbranched alkyl, C3-C8 cycloalkyl lower alkyl, Ci-C8 alkoxy, halo, cyano, trihalomethyl, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-C8 alkyl carbamoyl, Ci-C8 alkoxy carbonyl and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano and trihalomethyl;
R7 is hydrogen, unsubstituted or substituted Ci-Ci0 branched or unbranched alkyl, unsubstituted or substituted C3- C8 cycloalkyl lower alkyl, or unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-Ce alkyl carbamoyl, Ci-C8 alkoxy carbonyl and trihalomethyl; or a pharmaceutically acceptable salt thereof.
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000132_0001
wherein
R1 represents a saturated or unsaturated 5- to 15- membered ring system optionally comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur, the ring system being optionally substituted with at least one substituent selected from halogen, cyano, oxo, mercapto, nitro, hydroxyl, carboxyl, -S02NH2, -NR5R6, -C(0)NR7R8, Ci-C6 alkoxy, Ci-C6 alkoxycarbonyl , Ci-C6 alkylcarbonyl , Ci-C6 alkyl (optionally substituted by at least one substituent selected from halogen, hydroxyl, cyano, carboxyl, Ci-C6 alkoxycarbonyl, -NR9R10 and -C (0 ) NRX1R12 ) , C2-C6 alkenyl (optionally substituted by -C (0) NR13R14) , C3-C6 cycloalkyl (optionally substituted by at least one substituent selected from halogen, hydroxyl and cyano) , -NHS02-R15, and a saturated or unsaturated 5- to 6- membered heterocyclic ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulphur, the heterocycylic ring itself being optionally substituted by at least one substituent selected from halogen, hydroxyl, carboxyl and Ci-C6 alkyl;
n is 0 or 1 ;
R2 and R3 each independently represent hydrogen, hydroxyl, Ci-C6 alkyl, Ci-C6 alkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkoxy, C3- C6 cycloalkyl or C6-Ci0 aryl, the aryl group being optionally substituted with at least one substituent selected from halogen, hydroxyl, Ci-C6 alkyl and Ci-C6 alkoxy, or
R2 and R3 together with the carbon atom to which they are attached form a 3- to 6-membered saturated carbocyclic ring;
R4 represents a group
Figure imgf000133_0001
in which ring B, together with the two carbon atoms of ring A to which it is fused, represents a 5- to 6-membered, saturated or unsaturated, non-aromatic heterocyclic ring comprising one ring oxygen atom and optionally one or more ring heteroatoms independently selected from nitrogen, oxygen and sulphur, the group (Ι') being optionally substituted with at least one substituent selected from halogen, Ci-C6 alkyl, C3-C6 cycloalkyl and phenyl, the phenyl itself being optionally substituted with at least one substituent selected from halogen, hydroxyl and Ci-C6 alkoxy;
R5 and R6 each independently represent hydrogen, Ci-C6 alkyl or C3-C6 cycloalkyl, or R5 and R6 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocyclic ring optionally substituted by at least one substituent selected from hydroxyl, Ci-C6 alkoxy and Ci-C6 alkoxy-Ci-C6 alkyl;
R7 and R8 each independently represent hydrogen, Ci-C6 alkyl or C3-C6 cycloalkyl, or R7 and R8 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocyclic ring optionally substituted by at least one aminocarbonyl;
R9 and R10 each independently represent hydrogen, Ci-C6 alkyl or C3-C6 cycloalkyl, or R9 and R10 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocyclic ring optionally substituted by at least one substituent selected from hydroxyl, Ci-C6 alkoxy and Ci-C6 alkoxy-Ci-C6 alkyl;
R11 and R12 each independently represent hydrogen, Ci-C6 alkyl or C3-C6 cycloalkyl, or R11 and R12 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocyclic ring optionally substituted by at least one aminocarbonyl;
R13 and R14 each independently represent hydrogen, Ci-C6 alkyl or C3-C6 cycloalkyl, or R13 and R14 together with the nitrogen atom to which they are attached form a 4- to 7- membered saturated heterocyclic ring optionally substituted by at least one aminocarbonyl; and
R15 represents a Ci-C6 alkyl group or a 6-membered saturated or unsaturated heterocyclic ring comprising at least one ring nitrogen atom, the heterocyclic ring being optionally substituted with at least one substituent selected from halogen, oxo, Ci-C6 alkyl and Ci-C6 alkoxy;
or a pharmaceutically acceptable salt thereof.
22. The method of claim 13, wherein the organic compound has the structure :
Figure imgf000134_0001
or physiologically acceptable salt thereof; wherein
L is selected from the group consisting of a 0, S, NRa, a bond, S02, -C(=0), and (CR'R")m; Ra is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkylaryl, and optionally substituted cycloalkyl;
a is 0 to 3;
b is 0 to 3;
m is 1 to 8;
R' and R" are independently selected from the group consisting of hydrogen, optionally substituted alkyl, cyano and optionally substituted alkenyl;
R6, R7, R8, R9 and R10 are independently selected from the group consisting of hydrogen, hydroxyl, halogen, optionally substituted Ci-Ci0 alkyl, optionally substituted C2-Ci0 alkenyl, optionally substituted C2-Ci0 alkynyl, optionally substituted C3-Cio cycloalkyl, optionally substituted C3-Ci0 cycloalkenyl , optionally substituted C3-C10 cycloalkynyl , optionally substituted C3-Ci0 cycloalkoxy, cyano, C1-C10 alkoxy, C2-Ci0 alkenyloxy, C2-Ci0 alkynyloxy, benzyloxy, optionally substituted amino, optionally substituted amido, -0(CF3), -C(=0)0(R1), C(=0) (R1), -S02NRiR2, trifluoromethyl, aryl, aralkyl, heteroaryl and heteroaralkyl;
R1 and R2 are independently selected from the group consisting of hydrogen and optionally substituted alkyl;
Q3 is optionally substituted alkyl;
R11, R12, R13, R14, R15, R16, R17, R18 and R19 are each independently selected from the group consisting of hydrogen, hydroxyl, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, cyano, alkoxy, alkenyloxy, alkynyloxy, benzyloxy, optionally substituted amino, optionally substituted amido, -0 (CF3) , C(=0)0(R41), -C(=0) (R41) , -S02NR41R42, trifluoromethyl , aryl, aralkyl, heteroaryl and heteroaralkyl;
R41 and R42 are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl , optionally substituted cycloalkynyl , optionally substituted amino, trifluoromethyl , aryl, aralkyl, heteroaryl and heteroaralkyl; or R41 and R42 may be linked via a C2-C8 optionally substituted alkyl or alkenyl bridge where one or more carbons may be replaced by 0, S or NR46;
Q5 is selected from the group consisting of
Figure imgf000136_0001
-CH. and a bond; e is 1 to 3;
f is 1 to 7;
g is 0 to 3;
h is 0 to 3;
i is 0 or 1 ;
R20 and R46 are independently hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted amino, optionally substituted amido, -C (=0) 0 (R41 ) , -C(=0) (R41), -S02NR41R42, trifluoromethyl , aryl, aralkyl, heteroaryl or heteroaralkyl; and
Q6 is selected from the group consisting of optionally substituted aromatic ring, optionally substituted non-aromatic heterocycle, and optionally substituted heteroaromatic ring; or R18 or R19 together with Q5Q6 and the atoms to which they are bonded form an optionally substituted non-aromatic carbocyclic group, optionally substituted nonaromatic heterocyclic group, optionally substituted aryl ring or optionally substituted heteroaryl ring;
with the proviso that the compound is not
Figure imgf000137_0001
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000137_0002
or a pharmaceutically acceptable salt thereof, wherein:
Xi is a covalent bond, C=0 or CRaRb;
X is a covalent bond, 0, or NR5;
C=Z is C=0, CH2, C=NH, C=S or absent, provided that when Xi is a covalent bond then C=Z is not CH2, provided that when C=Z is absent then X is a covalent bond;
Ra and Rb are independently H or a C1-C3 alkyl;
R1 is: i) a substituted or unsubstituted aromatic group; ii) a substituted or unsubstituted non-aromatic ring; or iii) when X is NR5, then NR5 (CH2) mR1, taken together, is optionally a substituted or unsubstituted non-aromatic heterocyclic group;
R2 is -H or a C1-C3 alkyl group;
R3 is -H;
R4 is: i) a substituted or unsubstituted phenyl group, benzyl group or phenethyl group; or ii) a substituted or unsubstituted non-aromatic ring;
R5 is -H, or a C1-C3 alkyl group;
Ar is a bicyclic aromatic group comprising a first six membered aromatic group fused to a second six membered aromatic group or a five or six membered non-aromatic ring, wherein the group represented by Ar is optionally substituted with one or more substituents selected from alkyl, haloalkyl, halogen, cyano, nitro, hydroxy, haloalkoxy and alkoxy;
m is 0, 1, 2 or 3;
n is 1 or 2 ;
each substitutable carbon atom of the aromatic group represented by R1 is optionally substituted with a group represented by R10, wherein R10 is halogen, -R°, -OH, -OR0, - 0 (haloalkyl) , -SH, -SR°, 1 , 2-methylene-dioxy, 1,2- ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted -CH2(Ph), -CH2CH2 (Ph) , substituted -CH2CH2 (Ph) , -N02, -CN, -N(R')z, -NR'C02R°, NR'C(0)R°, -NR'NR'C (0)R°, -N (R ' ) C (0) N (R ' ) 2, -NR ' NR ' C (0) N (R ' ) 2, - NR'NR'C02R°, -C (0) C (0) R°, -C (0) CH2C (0) R°, -C02R°, -C (0) R°, C(0)N(R°)2, -0C(0)R°, -0C(0)N(R°)2, -S(0)2R°, -S02N(R')2, -S(0)R°, -NR ' S02N (R ' ) 2 , -NR'S02R°, -C (=S) N (R' ) 2, - (CH2) yN (R' ) 2, -C (=NH) - N(R')2, haloalkyl, V-R°, -V-0H, -V-0R0, -V-SH, -V-SR0, -V-N02, - V-CN, -V-N(R')2, -V-NR'C02R°, -V-NR ' C (0) R°, -V-NR ' NR ' C (0) R°, -V- N (R' ) C (0) N (R' ) 2, -V-NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V- C(0)C(0)R°, -V-C (0) CH2C (0) R°, -V-C02R°, -V-C (0) R°, -V-C (0) N (R°) 2, -V-0C(0)R°, -V-0C(0)N(R°)2, -V-S (0) 2R°, -V-S02N (R ' ) 2 , -V- S (0) R°, V-NR' S02N (R' ) 2, -V-NR'S02R°, -V-C (=S ) N (R ' ) 2 , -V-
(CH2)yN(R' )2, or
-V-C (=NH) -N (R' ) 2;
each substitutable carbon atom of the phenyl, benzyl or phenethyl group represented by R4 is optionally substituted with a group represented by R12, wherein R12 is halogen, -R°, - OH, -OR0, -0 (haloalkyl) , -SH, -SR°, 1 , 2-methylene-dioxy, 1,2- ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph), substituted -CH2(Ph), CH2CH2 (Ph) , substituted -CH2CH2 (Ph) , -CN, -NR'C02R0, NR'NR' C (0) R°, -N (R' ) C (0) N (R' ) 2, -NR ' NR ' C (0) N (R ' ) 2, -NR ' NR ' C02R°, -C(0)C(0)R°, -C (0) CH2C (0) R°, -C02R°, -C(0)R°, -OC (0) R°, 0C(0)N(R°)2, -S(0)2R°, -S02N(R')2, -S (0) R°, -NR ' S02N (R ' ) 2 , NR'S02R°, -C (=S ) N (R ' ) 2, - (CH2) yN (R' ) 2, -C (=NH) -N (R ' ) 2, haloalkyl, -V-R°, -V-OH, -V-0R0, -V-SH, -V-SR0, -V-CN, -V-NR'C02R°, -V- NR'NR' C (0) R°, -V-N (R' ) C (0) N (R' ) 2, -V-NR ' NR ' C (0) N (R ' ) 2, -V- NR'NR'C02R0, -V-C (0) C (0) R°, -V-C (0) CH2C (0) R°, -V-C02R°, -V- C(0)R°, -V-OC(0)R°, -V-OC(0)N(R°)2, -V-S (0) 2R°, -V-S02N (R' ) 2, -V- S (0) R°, -V-NR ' S02N (R ' ) 2, -V-NR'S02R°, -V-C (=S ) N (R ' ) 2 , -V-
(CH2)yN(R')2 or -V-C (=NH) - (R ' ) 2 , wherein each group represented by R12 is independently selected;
each substitutable carbon atom of: i) the non-aromatic ring represented by R1 or R4, ii) the non-aromatic heterocyclic group formed from NR5(CH2)mR1 is optionally and independently substituted with -R°, -OH, -OR0, -0 (haloalkyl ) , -SH, -SR°, 1,2- methylene-dioxy, 1 , 2-ethylenedioxy, phenyl (Ph) , substituted Ph, -O(Ph), substituted -O(Ph), -CH2(Ph),
substituted -CH2(Ph), -CH2CH2 (Ph) , substituted -CH2CH2(Ph), -CN, -N02, -N(R')2, -C(0)N(R°)2, -NR'C(0)R°, -NR'C(0)R°, -V-N(R')2, - V-NR'C(0)R°, -V-C (0) N (R°) 2, -NR'C02R°, -NR ' NR ' C (0) R°,
-N (R')C(0)N(R')2, -NR'NR' C (0) N (R' ) 2, -NR ' NR ' C02R°, -C (0) C (0) R°, -C (0) CH2C (0) R°, -C02R°, -C(0)R°, -0C(0)R°, -OC (0) N (R°) 2, -S (0) 2R°, -S02N(R')2, -S(0)R°, -NR'S02N(R' )2, -NR'S02R°, -C (=S ) N (R ' ) 2 , - (CH2) yN (R' ) 2, -C (=NH) -N (R' ) 2, haloalkyl, -V-R°, -V-OH, -V-0R0, - V-SH, -V-SR0, -NR'C(0)R°, -V-N02, -V-CN, -V-N(R')2, -V-NR'C02R°, -V-NR' C (0) R°, -V-NR'NR'C (0)R°, -V-N (R ' ) C (0) N (R ' ) 2, -V-
NR'NR' C (0) N (R' ) 2, -V-NR ' NR ' C02R°, -V-C (0) C (0) R°, -V-
C (0) CH2C (0) R°, -V-C02R°, -V-C (0) R°, -V-C (0) N (R°) 2, -V-0C(0)R°, - V-OC (0)N(R°)2, -V-S(0)2R°, -V-S02N(R' ) 2, -V-S (0) R°, -V-
NR' S02N (R' ) 2, -V-NR'S02R°, -V-C (=S ) N (R ' ) 2 , -V- (CH2 ) yN (R ' ) 2 , -V- C (=NH) -N (R' ) 2, =0, =S, =NNHR* , =NN(R*)2, =NNHC(0)R*, =NNHC02 (alkyl) , =NNHS02 (alkyl), or =NR* ;
each R° is independently hydrogen or substituted or unsubstituted aliphatic group, a substituted or unsubstituted cycloaliphatic , a substituted or unsubstituted non-aromatic heterocyclic group or a substituted or unsubstituted aromatic group selected from phenyl, naphthyl, 2-furanyl, 3-furanyl, N- imidazolyl, 2-imidazolyl, 4-imidazolyl , 5-imidazolyl , 2- oxadiazolyl, 5-oxadiazolyl , 2-oxazolyl, 4-oxazolyl, 5- oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3- pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3- pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2- triazolyl, 5-triazolyl, tetrazolyl, 2-thienyl, 3-thienyl, carbazolyl , benzimidazolyl , benzothienyl, benzofuranyl , indolyl , quinolinyl , benzotriazolyl, benzothiazolyl, benzooxazolyl , benzimidazolyl, isoquinolinyl , indolyl , isoindolyl, acridinyl or benzoisazolyl ;
each R' is independently R°, -C02R°, -S02R° or -C (0) R°;
each R* is independently hydrogen, an unsubstituted aliphatic group or a substituted aliphatic group;
V is a C1-C6 alkylene group; and
each substitutable carbon atom of: i) the aliphatic, cycloaliphatic , nonaromatic heterocyclic group and aromatic group represented by R°; and ii) the aliphatic group represented by R* is optionally and independently substituted with amino, alkylamino, dialkylamino, aminocarbonyl , halogen, alkyl, aminoalkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl , alkylcarbonyl , hydroxy, haloalkoxy, or haloalkyl;
each substitutable nitrogen atom of: i) the non-aromatic ring represented by R1 or R4; ii) the non-aromatic heterocyclic group formed from NR5(CH2)mR1; and iii) the non-aromatic heterocyclic group represented by R° is optionally and independently substituted with R+, -N(R+)2, -C(0)R+, -C02 R+, - C(0)C(0)R+, -C(0)CH2 C(0)R+, -S02R+, -S02N(R+)2, -C (=S ) N (R+) 2, - C (=NH) -N (R+) 2, -NR+S02R+, -C(0)-NHR+, -C (0) -N (R+) 2, -C(0)- CH[N(R+)2]R+ or -C (0) -CH [0R+] R+;
each R+ is independently hydrogen, an unsubstituted heteroaryl or an aliphatic, cycloaliphatic, non-aromatic heterocyclic group, phenyl or benzyl group, wherein each substitutable carbon atom of the aliphatic, cycloaliphatic, non-aromatic heterocyclic ring, phenyl or benzyl group represented by R+ is optionally substituted with amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl , dialkylaminocarbonyl , alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl or N(R+)2 is a non-aromatic heterocyclic group; and each substitutable nitrogen atom of the non-aromatic heterocyclic group represented by R+ is optionally substituted with alkyl, alkoxycarbonyl , alkylcarbonyl , alkylaminocarbonyl or dialkylaminocarbonyl ;
provided that the compound is other than N-[4-[[(4- methoxyphenyl ) amino] sulfonyl ] -1-naphthalenyl ] -benzamide, N- [ 4- [ (2-propenylamino) sulfonyl] -1-naphthalenyl] -benzamide,
N- [4- (4-morpholinylsulfonyl) -1-naphthalenyl] -benzamide or N- [4- (1-piperidinylsulfonyl) -1-naphthalenyl] -benzamide .
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000141_0001
or a pharmaceutically acceptable salt thereof, wherein:
R1 is cyclohexyl or phenyl, furanyl, thienyl or pyridyl optionally substituted with Ci-C4 alkyl, Ci-C4 alkoxy, Ci-C4 haloalkyl, C1-C4 haloalkoxy, methylenedioxy, ethylenedioxy, halogen, cyano or nitro;
Figure imgf000142_0001
C(0)OR , -C(0)-NHR22, -C(0)-N(R )2 or -C (0) CH [N (R ) 2] R ;
R21 is methyl, ethyl, 2-hydroxyethyl or iso-propyl;
Rz is -H or Ci-C4 alkyl or -N(R )2 taken together is N- pyrollidinyl or iV-piperidinyl , provided that R is not -H when R20 is -COOR22;
R23 is -H, methyl or ethyl;
R24 is -H, methyl, ethyl, phenyl, benzyl, 4-hydroxyphenyl or 4-hydroxybenzyl; and
s is O, 1, 2, 3 or 4.
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000142_0002
wherein
n is 0, to 6;
m is 1, to 4;
p is 1, to 4;
Ar is unsubstituted phenyl, thiophenyl, furanyl, or pyridinyl; or phenyl, thiophenyl, furanyl, or pyridinyl substituted with one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-Ce alkyl carbamoyl, Ci-C8 alkoxy carbonyl and trihalomethyl;
Ri and R6 are independently selected from the group consisting of hydrogen, Ci-C8 branched or unbranched alkyl, C3- C8 cycloalkyl lower alkyl, and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano and trihalomethyl ;
R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, Ci-C8 branched or unbranched alkyl, C3-C8 cycloalkyl lower alkyl, Ci-C8 alkoxy, halo, cyano, trihalomethyl, hydroxy, amino, N-Ci~C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci~C8 alkyl carbamoyl, Ci~C8 alkoxy carbonyl and unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano and trihalomethyl;
R7 is hydrogen, unsubstituted or substituted C1-C10 branched or unbranched alkyl, unsubstituted or substituted C3- C8 cycloalkyllower alkyl, or unsubstituted or substituted phenyl lower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C8 alkoxy, Ci-C8 alkyl, halo, cyano, hydroxy, amino, N-Ci-C8 alkyl amino, N,N-di-Ci-C8 alkyl amino, carboxyl, Ci-C8 alkyl carbamoyl, Ci-C8 alkoxy carbonyl and trihalomethyl; or a pharmaceutically acceptable salt thereof.
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000143_0001
in which: , x, y and z are independently 1, 2 or 3;
A is a phenyl, benzyl, alkyl, C3_6 saturated or partially unsaturated cycloalkyl, a 6-membered-cycloheteroalkyl ring containing 1 or 2 heteroatoms selected from 0 or N, alkyl- aryl, naphthyl, a 5- to 7-membered heteroaromatic ring containing 1 to 3 heteroatoms, a 9- or 10-membered bicyclic heteroaromatic ring containing 1 to 4 heteroatoms, a phenyl- fused-5 to 6-membered cycloheteroalkyl containing at least one heteroatom selected from 0, S or N, or pyridone;
A being optionally substituted by one or more groups selected from halogen, cyano, CF3, 0CF3, Ci_6 alkoxy, hydroxy, Ci_6 alkyl, Ci_6 thioalkyl, S02Ci_6 alkyl, NR2R3, amide, Ci_6 alkoxycarbonyl , - N02, Ci-6 acylamino, -C02H, Ci_6 carboxyalkyl , morpholine;
phenoxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl;
phenyl or diphenyl, said phenyl and diphenyl indepedently being optionally substituted with one or more groups indepedently selected from halogen, Ci_6 alkoxy, Ci_6 alkyl, or COOH;
benzyloxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl;
or a 5 to 7 membered heteroaromatic ring containing 1 to 4 heteroatoms selected from 0, S or N optionally substituted with one or more groups indepedently selected from halogen, Ci-6 alkoxy, Ci_6 alkyl;
R2 and R3 are independently halogen or Ci_6 alkyl, or R2 and R3 together with the nitrogen to which they are attached form a 6-membered saturated ring optionally containing a further heteroatom;
B is a group R4-R5 where R4 is a bond, -N(R6)-, -R7-N(R8)-, -N(R9)-R10-, 0, Ci_4 alkyl optionally interrupted by N(RX1) or 0, C2_4 alkenyl or 1,3- butadienyl, or -S02-N (R12 ) - ;
R5 is C=0 or S02;
R6, R8, R11, and R12 are each independently H or Ci_6 alkyl; R9 is H, Ci-6 alkyl or Ci_6 carboxyalkyl ;
R7 and R10 are independently Ci_4 alkyl or C3-5 cycloalkyl; D is Ci_4 alkyl;
E is phenyl, or a 5- or 6-membered aromatic ring containing one or two heteroatoms;
Each R1 independently represents Ci_6 alkoxy optionally substituted with one or more halogens, C4-6cycloalkylalkoxy, C2-6 alkenyloxy, halogen, 0CH2CN, C0Ci_6 alkyl, OR11, 0CH2R11, or -S- R12
R11 is a phenyl or 5- or 6-membered saturated or aromatic ring containing one or two heteroatoms and each optionally substituted by one or more groups selected from Ci_6 alkyl, halogen, Ci_6 alkoxy, CF3, or cyano;
R12 is Ci-6 alkyl or R12 is phenyl optionally substituted with one or more halogens, and n is 0, 1, 2, 3 or 4; provided that when E is phenyl, w + x is greater than 2 and n is 1 then R1 is not a phenoxy group at the meta-position of the phenyl ring E; and provided that when A-B is acetyl, tosyl or tertiary butyloxy-carbonyl (t-boc) , then D-E- ( R1 ) n is not benzyl, or a pharmaceutically acceptable salt, solvate, or N-oxide thereof .
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000146_0001
in which: w, x, y and z are independently 1, 2 or 3;
A is a phenyl, benzyl, alkyl, C3_6 saturated or partially unsaturated cycloalkyl, a 6-membered-cycloheteroalkyl ring containing 1 or 2 heteroatoms selected from 0 or N, alkyl- aryl, naphthyl, a 5- to 7-membered heteroaromatic ring containing 1 to 3 heteroatoms, a 9- or 10-membered bicyclic heteroaromatic ring containing 1 to 4 heteroatoms, a phenyl- fused-5 to 6-membered cycloheteroalkyl containing at least one heteroatom selected from 0, S or N, or pyridone;
A being optionally substituted by one or more groups selected from halogen, cyano, CF3 , 0CF3, Ci_6 alkoxy, hydroxy, Ci_6 alkyl, Ci-6 thioalkyl, S02Ci_6 alkyl, NR2R3 , amide, Ci_6 alkoxycarbonyl , - N02, Ci_6 acylamino, -C02H, Ci_6 carboxyalkyl , morpholine;
phenoxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl;
phenyl or diphenyl, said phenyl and diphenyl indepedently being optionally substituted with one or more groups indepedently selected from halogen, Ci_6 alkoxy, Ci_6 alkyl, or COOH; benzyloxy optionally substituted with one or more groups selected from halogen, Ci_6 alkoxy, Ci_6 alkyl;
or a 5 to 7 membered heteroaromatic ring containing 1 to 4 heteroatoms selected from 0, S or N optionally substituted with one or more groups independently selected from halogen, Ci_6 alkoxy, C1-6 alkyl;
R2 and R3 are independently halogen or Ci_6 alkyl, or R2 and R3 together with the nitrogen to which they are attached form a 6-membered saturated ring optionally containing a further heteroatom;
B is a group R4-R5 where
R4 is a bond, -N(R6)-, -R7-N(R8)-, -N(R9)-R10-, 0, Ci_4 alkyl optionally interrupted by N(RX1) or 0, C2_4 alkenyl or 1,3- butadienyl, or -S02-N (R12 ) - ;
R5 is C=0 or S02;
R6, R8, R11, and R12 are each independently H or Ci_6 alkyl; R9 is H, Ci_6 alkyl or C1-6 carboxyalkyl ;
R7 and R10 are independently Ci_4 alkyl or C3-5 cycloalkyl; D is Ci-4 alkyl;
E is phenyl, or a 5- or 6-membered aromatic ring containing one or two heteroatoms;
Each R1 independently represents Ci_6 alkoxy optionally substituted with one or more halogens, C4-6cycloalkylalkoxy, C2-6 alkenyloxy, halogen, 0CH2CN, C0Ci_6 alkyl, OR11, 0CH2R11, or -S- R12;
R11 is a phenyl or 5- or 6-membered saturated or aromatic ring containing one or two heteroatoms and each optionally substituted by one or more groups selected from Ci_6 alkyl, halogen, Ci_6 alkoxy, CF3, or cyano;
R12 is Ci-6 alkyl or R12 is phenyl optionally substituted with one or more halogens, and n is 0, 1, 2, 3 or 4; provided that when E is phenyl and n is 1 then R1 is not a phenoxy group at the meta position of the phenyl ring E; and provided that when A-B is acetyl, tosyl or tertiary butyloxy-carbonyl (t-boc) , then D-E-(R1)n is not benzyl, or a pharmaceutically acceptable salt, solvate, or N-oxide thereof .
The method of claim 13, wherein the organic compound has the structure :
Figure imgf000148_0001
wherein
n is 0 or 1 ;
m is 0 or 1 ;
p is 1, 2 or 3;
Ar is unsubstituted quinolinyl, [ 1 , 5 ] naphthyridinyl or pyridinyl; or quinolinyl, [ 1 , 5 ] naphthyridinyl or pyridinyl substituted with one or more radicals selected from the group consisting of Ci-C6 alkoxy, Ci-C6 alkyl, halo, cyano and trihalomethyl ;
R is Ci-Ce branched or unbranched alkyl, C3-C6 cycloalkyllower alkyl, unsubstituted or substituted phenyl lower alkyl, unsubstituted or substituted pyridyl lower alkyl, unsubstituted or substituted indolyllower alkyl, unsubstituted or substituted N- (lower alkyl) indolyl lower alkyl, unsubstituted or substituted quinolinyl lower alkyl, unsubstituted or substituted naphthyl lower alkyl, unsubstituted or substituted benzofuranyllower alkyl, unsubstituted or substituted benzothiophenyllower alkyl; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Ci-C6 alkoxy, Ci-C6 alkyl, halo, cyano and trihalomethyl ;
or a pharmaceutically acceptable salt thereof.
The method of claim 18, wherein the organic compound has the structure :
Figure imgf000149_0001
or a pharmaceutically active salt or ether thereof.
30. The method of claim 20, wherein the organic compound has the structure :
Figure imgf000149_0002
or a pharmaceutically active salt or ether thereof.
31. The method of claim 22, wherein the organic compound has the structure :
Figure imgf000149_0003
or a pharmaceutically active salt or ether thereof.
32. The method of claim 23, wherein the organic compound has the structure :
Figure imgf000150_0001
or a pharmaceutically active salt or ether thereof.
33. The method of claim 25, wherein the organic compound has the structure :
Figure imgf000150_0002
or a pharmaceutically active salt or ether thereof.
34. The method of claim 26, wherein the organic compound has the structure :
Figure imgf000150_0003
or a pharmaceutically active salt or ether thereof.
35. The method of claim 28, wherein the organic compound has the struc re :
Figure imgf000150_0004
or a pharmaceutically active salt or ether thereof.
36. The method of claim 13, wherein the organic compound has the structure :
Figure imgf000151_0001
or a pharmaceutically active salt or ether of any of the foregoing .
37. The method of claim 13, wherein the organic compound has the structure :
Figure imgf000151_0002
or a pharmaceutically active salt or ether of any of the foregoing .
The method of any one of claims 1-4, wherein the antagonist of the CCR8 receptor is an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, a polypeptide, or an antibody, which antagonist binds to CCLl at a site which reduces binding of CCLl to the CCR8 receptor.
39. The method of claim 38, wherein the antagonist of the CCR8 receptor is an anti-CCLl antibody.
40. The method of claim 39, wherein the anti-CCLl antibody is a monoclonal antibody.
41. The method of claim 40, wherein the monoclonal antibody is a humanized monoclonal antibody.
42. The method of claim 38, wherein the antagonist of the CCR8 receptor is a polypeptide.
43. The method of claim 42, wherein the polypeptide is a soluble form of the CCR8 receptor.
44. The method of claim 43, wherein the soluble form of the CCR8 receptor comprises a portion of the extracellular domain of the CCR8 receptor sufficient to bind CCLl and reduce binding of CCLl to the CCR8 receptor.
45. The method of claim 43, wherein the soluble form of the CCR8 receptor comprises the entire extracellular domain of the CCR8 receptor which binds to CCLl and reduces the binding of CCLl to the CCR8 receptor.
46. The method of claims 42, wherein the polypeptide is a fusion protein which comprises a soluble form of the CCR8 receptor bound to an immunoglobulin (Ig) or Ig fragment.
47. The method of claim 46, wherein the fusion protein comprises an Ig which is a human Ig.
48. The method of claim 46, wherein the fusion protein comprises an Ig fragment which is a human Ig fragment.
49. The method of claim 38, wherein the antagonist of the CCR8 receptor is an RNA aptamer which binds to CCLl. The method of claim 49, wherein the RNA aptamer is T48.
A method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCR8 receptor produced by tumor cells present in the solid tumor, so as to thereby treat the subject.
A method of reducing metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCR8 receptor produced by tumor cells present in the solid tumor, so as to thereby reduce metastases in the subj ect .
The method of claim 51 or 52, wherein the oligonucleotide comprises nucleotides in a sequence that is complementary to CCR8 receptor-encoding mRNA.
The method of claim 53, wherein the oligonucleotide is an antisense oligodeoxynucleotide .
The method of claim 51 or 52, wherein the oligonucleotide is an RNA interference inducing compound.
The method of claim 51 or 52, wherein the oligonucleotide is a ribozyme .
A method of treating a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of the CCL1 in the subject, so as to thereby treat the subject.
A method of reducing metastases in a subject who has, or has been treated for, a solid tumor which comprises administering to the subject an oligonucleotide which decreases the amount of CCL1 in the subject, so as to thereby reduce metastases in the subject.
The method of claim 57 or 58, wherein the oligonucleotide comprises oligonucleotides in a sequence that is complementary to CCL1 receptor-encoding mRNA.
The method of claim 59, wherein the oligonucleotide is an antisense oligodeoxynucleotide.
The method of claim 57 or 58, wherein the oligonucleotide is an RNA interference inducing compound.
The method of claim 57 or 58, wherein the oligonucleotide is a ribozyme .
The method of any one of claims 57 to 62, wherein the oligonucleotide is modified to increase its stability in vivo.
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