阅读说明：本技术 作为抗癌药物的选择性且有效抑制Hakai介导的泛素化的化合物 (Compounds that selectively and efficiently inhibit Hakai-mediated ubiquitination as anti-cancer agents ) 是由 安吉莉卡·菲格罗亚·康德-瓦尔维斯 费德里科·加戈·巴德纳斯 奥莱亚·安蒂亚·马蒂尼斯·伊格莱西 于 2019-11-15 设计创作，主要内容包括：本发明提供了一类化合物,其包括其对映异构体及其药学上可接受的盐,它们选择性地和有效地抑制Hakai介导的泛素化,优选地不影响Hakai蛋白水平,从而代表用于治疗多种癌症的优异抗癌药物,例如癌,特别是源自胃肠道的上皮层的肿瘤,胃肠道包括口(口腔癌)、食道、胃以及小肠和大肠(例如直肠癌或结肠癌)。它还包括皮肤癌、乳腺(乳腺癌)、胰腺癌、肺癌、头颈癌、肝癌、卵巢癌、宫颈癌、子宫癌、胆囊癌、阴茎癌和尿膀胱癌(例如肾癌、前列腺癌或膀胱癌)。(The present invention provides a class of compounds, including enantiomers thereof and pharmaceutically acceptable salts thereof, which selectively and effectively inhibit Hakai-mediated ubiquitination, preferably without affecting Hakai protein levels, and thus represent excellent anti-cancer drugs for the treatment of various cancers, such as cancers, particularly tumors derived from the epithelial layer of the gastrointestinal tract, including the mouth (oral cancer), esophagus, stomach, and small and large intestine (e.g., rectal or colon cancer). It also includes skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (e.g., renal cancer, prostate cancer, or bladder cancer).)

1. A compound for use in the treatment of cancer, represented by formula (I):

wherein:

-a represents a group selected from aryl, heteroaryl and cyclic amide, said aryl, heteroaryl and cyclic amide being optionally substituted with 1 or 2 groups independently selected from:

i) halogen atom, NO₂、-CN、-N(R^a)R^b、-OR^a、-C(＝O)R^a、-C(＝O)OR^a、-C(＝O)N(R^a)R^b、-OC(＝O)-R^a、-N(R^c)C(＝O)R^b、-NR^cSO₂R^a、-SO₂N(R^a)R^b、-SR^a、-S(O)R^a、-S(O)₂R^a；

j) Straight or branched chain C optionally substituted by 1, 2 or 3 halogen atoms₁-C₆An alkyl group;

k) c optionally containing 1 or 2 heteroatoms selected from O, S and N₃-C₆Cycloalkyl, and wherein the ring is optionally substituted by C₁-C₃Alkyl substitution;

l) are each optionally substituted by halogen atoms, cyano groups, C₁-C₃Alkyl or cyclopropyl substituted phenyl or C₅-C₆A heteroaryl group;

-R^a、R^band R^cEach independently represents:

i) a hydrogen atom;

j) straight or branched chain C optionally substituted with 1, 2 or 3 substituents selected from₁-C₁₂Alkyl radical, C₃-C₆Cycloalkyl and C₄-C₆Heterocycloalkyl group: carbonyl, halogen, hydroxy, phenyl, C₃-C₆Cycloalkyl, straight or branched C₁-C₆Alkoxy, amino, alkylamino, dialkylamino, straight or branched C₁-C₆An alkylcarbonyl group;

k) phenyl or C optionally substituted with 1, 2 or 3 substituents selected from₅-C₆Heteroaryl group: halogen atom, cyano group, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Haloalkyl, hydroxy, straight or branched C₁-C₆Alkoxy, amino, alkylamino, dialkylamino;

l)R^aand R^bTogether with the nitrogen atom to which they are attached form a 3 to 8 membered ring, which 3 to 8 membered ring optionally further comprises a further heteroatom selected from O, N and S, and wherein the ring is optionally substituted with 1, 2 or 3 substituents selected from: carbonyl, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Haloalkyl, straight-chain or branched C₁-C₆An alkylcarbonyl group;

-x and y are integers independently selected from 0 and 1;

-R¹represents a radical selected from hydrogen, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups, wherein the alkyl groups are optionally substituted with 1, 2 or 3 halogen atoms;

when y is 0, then R¹May form a 5-or 6-membered ring together with the adjacent nitrogen atom and 2 adjacent carbon atoms of the aromatic ring to which the nitrogen is attached;

-R²and R³Each independently represents a cyclopropyl group or a linear or branched C₁-C₆An alkyl group;

-R²and R³May form a 3-or 4-membered spirocyclic ring together with the carbon atoms to which they are both attached;

-z is an integer selected from 0, 1, 2 or 3;

-R⁴represents a group selected from-CN, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups, said alkyl groups being optionally substituted with 1, 2 or 3 halogen atoms; wherein the group R⁴If present, a substitution is present with R⁴A hydrogen atom of a group CH in the attached benzene ring.

2. The compound for use according to claim 1, wherein the cancer is a carcinoma.

3. The compound for use according to claim 2, wherein the cancer is a cancer selected from the list consisting of: tumors derived from the epithelial layer of the gastrointestinal tract including mouth (oral cancer), esophagus, stomach, and small and large intestines (e.g., rectal or colon cancer), skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (e.g., kidney cancer, prostate cancer, or bladder cancer).

4. A compound for use according to any one of claims 1 to 3, wherein R²And R³Each independently represents a group selected from cyclopropyl or straight or branched C₁-C₆The radical of an alkyl group.

5. A compound for use according to any one of claims 1 to 3, wherein R²And R³Together with the carbon atoms to which they are both attached form a 3-or 4-membered spirocyclic ring.

6. A compound for use according to any one of claims 1 to 3, wherein z is an integer selected from 1, 2 or 3; wherein R is⁴Represents a group selected from-CN, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups, wherein the alkyl groups are optionally substituted with 1, 2 or 3 halogen atoms; and wherein the radical R⁴The substitution being present with R⁴A hydrogen atom of a group CH in the attached benzene ring.

7. A compound for use according to any one of claims 1 to 3, wherein one or both of the integers x and y are equal to 1, and a represents a group selected from aryl, heteroaryl and cyclic amide, which are optionally substituted with 1 or 2 groups independently selected from: a halogen atom, -CN, -n (Ra) Rb, -ORa, -C (═ O) Ra, -C (═ O) ORa, -C (═ O) n (Ra) Rb, -OC (═ O) -Ra, -n (rc) C (═ O) Rb, -NRcSO2Ra, -SO2N (Ra) Rb, -SRa, -s (O) Ra, -s (O)2 Ra; linear or branched C1-C6 alkyl, wherein the alkyl is optionally substituted with 1, 2, or 3 halogen atoms; C3-C6 cycloalkyl optionally containing 1 or 2 heteroatoms selected from O, S and N, and wherein the ring is optionally substituted with C1-C3 alkyl; phenyl or C5-C6 heteroaryl, each optionally substituted by a halogen atom, a C1-C3 alkyl group or a cyclopropyl group.

8. The compound for use according to any one of claims 1 to 3, wherein the compound is selected from the list consisting of any of the following compounds:

a)

b)

c)

d)

e)

f)

g)

h)

i)

j)

k)

l)

m)

n)

o)

or

p)

9. A compound for use according to any one of claims 1 to 3, wherein the compound is 4- (5- ((2- (4-nitrophenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid.

10. The compound for use according to any one of claims 1 to 3, wherein the compound is selected from the list consisting of any of the following compounds:

a)

b)

c)

d)

e)

f)

11. the compound for use according to any one of claims 1 to 3, wherein the compound is selected from the list consisting of any of the following compounds:

a)

b)

c)

d)

e)

f)

g)

h)

i)

or

j)

12. A compound for use according to any one of claims 1 to 3, wherein the compound is

Wherein R represents a group selected from hydrogen, cyclopropyl or a linear or branched C1-C6 alkyl group, wherein said alkyl group is optionally substituted with 1, 2 or 3 halogen atoms.

13. A compound for use according to any one of claims 1 to 3, wherein the compound is

Wherein R is methyl.

14. The compound for use according to any one of claims 1 to 3, wherein the compound is selected from the list consisting of any of the following compounds:

Technical Field

The present invention relates to a novel class of compounds and compositions comprising the compounds. The compounds and compositions (e.g., pharmaceutical compositions) of the invention are useful as agents for treating cancer.

Background

Cancer (Carcinoma) is the most common type of cancer, and it originates in epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin (E-cadherin), as a result of which the cell-cell contact is disrupted. E-cadherin is a tumor suppressor that is down-regulated during Epithelial-to-mesenchymal transition (EMT); in fact, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin ligase protein that mediates E-cadherin ubiquitination, endocytosis, and eventual degradation, leading to alterations in cell-cell contact. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets of Hakai may be associated with cancer cell plasticity during tumor progression. In other work, the authors of the present invention have used the iTRAQ method to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Their findings suggest that Hakai may have a major impact on cytoskeletal-associated proteins, extracellular-exosome-associated proteins, RNA-associated proteins and proteins involved in metabolism. Furthermore, it is emphasized that the expression of several proteasome subunits is significantly reduced during Hakai-driven EMT. As proteasome inhibitors are increasingly used in cancer therapy, these findings suggest that E3 ubiquitin ligase, such as Hakai, may be a better target than proteasomes for using novel specific inhibitors in tumor subtypes that undergo EMT (e.g., carcinomas, tumors of mesenchymal phenotype or tumors in which increased Hakai expression relative to normal tissues is detected). However, until now, no compounds have been disclosed that are effective in inhibiting Hakai-mediated ubiquitination, which are particularly suitable as therapeutic tools for the treatment of cancer.

The present invention provides such compounds, including enantiomers and pharmaceutically acceptable salts thereof, which selectively and effectively inhibit Hakai-mediated ubiquitination, preferably without affecting Hakai protein levels, while they represent excellent anti-cancer drugs useful in the treatment of various cancers (e.g., carcinomas).

Disclosure of Invention

The present invention provides a class of compounds, including enantiomers thereof and pharmaceutically acceptable salts thereof, which selectively and effectively inhibit Hakai-mediated ubiquitination, preferably without affecting Hakai protein levels, and thus represent excellent anti-cancer drugs useful in the treatment of various cancers, such as cancers, particularly tumors derived from the epithelial layer of the gastrointestinal tract, including the mouth (oral cancer), esophagus, stomach, and small and large intestines (e.g., rectal or colon cancer). It also includes skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (e.g., renal cancer, prostate cancer, or bladder cancer). The compounds of the present invention also exhibit lower toxicity, which makes the compounds of the present invention very attractive. This compound is represented by the following formula (I).

Accordingly, a first aspect of the present invention relates to a compound of formula (I) and pharmaceutically acceptable salts thereof:

wherein:

-a represents a group selected from aryl, heteroaryl and cyclic amide, optionally substituted with 1 or 2 groups independently selected from:

a) halogen atom, -NO₂、-CN、-N(R^a)R^b、-OR^a、-C(＝O)R^a、-C(＝O)OR^a、-C(＝O)N(R^a)R^b、-OC(＝O)-R^a、-N(R^c)C(＝O)R^b、-NR^cSO₂R^a、-SO₂N(R^a)R^b、-SR^a、-S(O)R^a、-S(O)₂R^a；

b) Straight or branched chain C optionally substituted by 1, 2 or 3 halogen atoms₁-C₆An alkyl group;

c) c optionally containing 1 or 2 heteroatoms selected from O, S and N₃-C₆Cycloalkyl, and wherein the ring is optionally substituted by C₁-C₃Alkyl substitution;

d) each optionally substituted by halogen atoms, cyano groups, C₁-C₃Alkyl or cyclopropyl substituted phenyl or C₅-C₆A heteroaryl group;

-R^a、R^band R^cEach independently represents:

a) a hydrogen atom;

b) straight or branched chain C optionally substituted with 1, 2 or 3 substituents selected from₁-C₁₂Alkyl radical, C₃-C₆Cycloalkyl and C₄-C₆Heterocycloalkyl group: carbonyl radicalHalogen atom, hydroxy group, phenyl group, C₃-C₆Cycloalkyl, straight or branched C₁-C₆Alkoxy, amino, alkylamino, dialkylamino, straight or branched C₁-C₆An alkylcarbonyl group;

c) phenyl or C optionally substituted with 1, 2 or 3 substituents selected from₅-C₆Heteroaryl group: halogen atom, cyano group, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Haloalkyl, hydroxy, straight or branched C₁-C₆Alkoxy, amino, alkylamino, dialkylamino;

d)R^aand R^bTogether with the nitrogen atom to which they are attached form a 3 to 8 membered ring, which 3 to 8 membered ring optionally further comprises a further heteroatom selected from O, N and S, and wherein the ring is optionally substituted with 1, 2 or 3 substituents selected from: carbonyl, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Haloalkyl, straight-chain or branched C₁-C₆An alkylcarbonyl group;

-x and y are integers independently selected from 0 and 1;

-R¹represents a radical selected from hydrogen, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups, wherein the alkyl groups are optionally substituted with 1, 2 or 3 halogen atoms;

when y is 0, then R¹May form a 5-or 6-membered ring together with the adjacent nitrogen atom and 2 adjacent carbon atoms of the aromatic ring to which the nitrogen is attached;

-R²and R³Each independently represents hydrogen, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups;

-R²and R³May form a 3-or 4-membered spirocyclic ring together with the carbon atoms to which they are both attached;

-z is an integer selected from 0, 1, 2 or 3;

-R⁴represents a group selected from-CN, cyclopropyl or a linear or branched C₁-C₆Radical of an alkyl radical, the alkyl radical optionally beingSubstituted by 1, 2 or 3 halogen atoms; wherein the radical R⁴If present, a substitution is present with R⁴A hydrogen atom of a group CH in the attached benzene ring;

the compounds and pharmaceutically acceptable salts thereof are useful for the treatment of cancer, particularly for the treatment of cancer, more particularly for the treatment of cancers including tumors derived from the epithelial layer of the gastrointestinal tract including the mouth (oral cancer), esophagus, stomach, and small and large intestines (e.g., rectal or colon cancer). It also includes skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (e.g., renal cancer, prostate cancer, or bladder cancer).

It is to be noted herein that in the context of the present invention, the term "cancer" is understood to be a cancer originating from epithelial tissue. They cover the outside of the body, just like the skin, and also cover and line all organs inside the body, such as the organs of the digestive system. In addition, they line body cavities, such as the interior of the thoracic and abdominal cavities. Cancer is the most common type of cancer. These tumors cause more than 80% of cancer-related deaths in the western world.

In a preferred embodiment of the first aspect of the invention, R²And R³Each independently represents a group selected from cyclopropyl or straight or branched C₁-C₆The radical of an alkyl group.

In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, R²And R³Together with the carbon atoms to which they are both attached form a 3-or 4-membered spirocyclic ring.

In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, z is an integer selected from 1, 2 or 3; wherein R is⁴Represents a group selected from-CN, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups, wherein the alkyl groups are optionally substituted with 1, 2 or 3 halogen atoms; and wherein the radical R⁴The substitution being present with R⁴A hydrogen atom of a group CH in the attached benzene ring.

In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, one or both of the integers x and y is equal to 1, and a represents a group selected from aryl, heteroaryl and cyclic amide, which aryl, heteroaryl and cyclic amide are optionally substituted with 1 or 2 groups independently selected from: a halogen atom, -CN, -n (Ra) Rb, -ORa, -C (═ O) Ra, -C (═ O) ORa, -C (═ O) n (Ra) Rb, -OC (═ O) -Ra, -n (rc) C (═ O) Rb, -NRcSO2Ra, -SO2N (Ra) Rb, -SRa, -s (O) Ra, -s (O)2 Ra; a linear or branched C1-C6 alkyl, wherein the alkyl is optionally substituted with 1, 2, or 3 halogen atoms; C3-C6 cycloalkyl optionally containing 1 or 2 heteroatoms selected from O, S and N, and wherein the ring is optionally substituted with C1-C3 alkyl; phenyl or C5-C6 heteroaryl, each optionally substituted by a halogen atom, a C1-C3 alkyl group or a cyclopropyl group.

In another preferred embodiment of the first aspect of the invention or any preferred embodiment thereof, the compound is selected from the list consisting of any of the following compounds:

the above-specified compounds are further shown below:

more preferably, the compound is

4- (5- ((2- (4-nitrophenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid.

Even more preferably, the integers x and y are both equal to 0, and a represents a benzyl group, preferably substituted in para position by 1 group selected from: a halogen atom, -ORa, -OC (═ O) -Ra, -n (rc) C (═ O) Rb, -NRcSO2Ra, -SO2N (Ra) Rb, -SRa, -s (O) Ra or-s (O)2 Ra. Preferably, the group is selected from a halogen atom or ORa. Preferably, the compound is selected from the group consisting of hit 5 to hit 9 described above. More preferably, the compound is hit 7.

In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the compound is a ketoheteroaryl group, preferably selected from the list consisting of any of the following compounds:

the above-specified compounds are further shown below:

more preferably, a more preferred embodiment of the present invention refers to any ketoheteroaryl class of compounds as described herein that can be used in the practice of the present invention. In particular, preferably, the integers x and y are both equal to 0 and a represents heteroaryl optionally substituted by a halogen atom or ORa. Preferably, the compound is selected from the group consisting of hit 23 to hit 25 described above.

In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the compound is a cyclic amide, preferably selected from the list consisting of any of the following compounds:

the above-specified compounds are further shown below:

preferably, the compound is a substituted indoline (and indole) analogue as shown below:

wherein R represents a group selected from hydrogen, cyclopropyl or a linear or branched C1-C6 alkyl group, wherein said alkyl group is optionally substituted with 1, 2 or 3 halogen atoms. More preferably, R is methyl. Preferably, the compound is selected from the group consisting of hit 10 to hit 16 as described above.

In another preferred embodiment of the first aspect of the present invention or any preferred embodiment thereof, the compound is a benzamide, preferably selected from the list consisting of any of the following compounds:

other compounds useful in the present invention are shown in the specification.

Drawings

FIG. 1 computer simulation and in vitro screening of E3 ubiquitin ligase Hakai inhibitors. (A) Chemical structures of Hakin-1 and Hakin-5. (B) Predicted binding poses of Hakin-1 (left panel, yellow) and Hakin-5 (right panel, orange) molecules docked within the Hakai dimer (in blue and green), as determined by the CRDOCK docking program. (C) In vivo Hakai-dependent ubiquitination assay in 293T cells transfected with Flag-Hakai, v-Src and HA-ubiquitin in the presence of DMSO or the compound Hakin-1. (D) In vivo Hakai-dependent ubiquitination assay in 293T cells transfected with Flag-Hakai, v-Src and HA-ubiquitin in the presence of DMSO or the compound Hakin-5. (E) In vivo ubiquitination assay in 293T cells transfected with v-Src and HA-ubiquitin in the presence of DMSO or the compound Hakin-1. (F) Effect of Hakin-1 on Hakai-dependent ubiquitination of the E-cadherin complex. pcDNA-Flag-Hakai, pcDNA-myc-E-cadherin, pSG-v-Src and pBSSR-HA-ubiquitin were transiently transfected into 293T cells. Immunoprecipitation with anti-E-cadherin antibody prior to western blotting with the indicated antibody;

FIG. 2 Hakin-1 induces cytotoxicity and epithelial phenotype of epithelial tumor cell lines. (A) HT29 and LoVo cells were treated at increasing concentrations of Hakin-1 or Hakin-5 and cell viability was measured by MTT assay. The assay was performed in 6 replicates and expressed as mean ± SD of three independent experiments. (B) The cell viability of MDCK, Hakai-MDCK cell lines (clone 4 and clone 11) was measured as shown in (A) using either Hakin-1 (top panel) or Hakin-5 (bottom panel). (C-D) phase contrast images of HT29 and LoVo cell lines (C) and MDCK, Hakai-MDCK cell lines (clone 4 and clone 11) (D) under Hakin-1 or Hakin-5 treatment. Images were acquired using a 20-fold objective lens;

FIG. 3 Hakin-1 induces mesenchymal-epithelial transformation of epithelial tumor cell lines. (A) Western blot analysis of EMT markers after treatment with Hakin-1 in HT-29 cell line (left panel) and shows quantification by densitometry (right panel). (B) Western blot analysis of EMT markers after treatment with Hakin-1 in LoVo cells (left panel) and shows quantification by densitometry (right panel). GAPDH was used as loading control. Quantification was performed as indicated in materials and methods, and western blot data are representative of three experiments. Data show the mean of three independent experiments and are expressed as mean ± SD (. + -. p < 0.05;. p < 0.01;. p < 0.001). (C) Immunofluorescence of E-cadherin in HT-29 and LoVo cell lines in the presence of DMSO or 48h after Hakin-1 treatment. The image was acquired with a 40 x objective lens. Quantification was performed using the Image J program and results were expressed as mean ± SD of three independent experiments (. p.. p < 0.01;. p < 0.001). The scale bar is 250. mu.M for HT29 cells and 175. mu.M for LoVo cells;

FIG. 4 anti-proliferative and anti-cancer effects of Hakin-1 in tumor epithelial cells. (A) HT29 cells and LoVo cells were treated with Hakin-1 for 48h and proliferation was measured by BrdU assay as indicated in materials and methods. Results are expressed as mean ± SD of eight replicates, and the experiment was repeated three times (. p < 0.05;. p < 0.01;. p < 0.001). (B) HT29 cells and LoVo cells were treated with Hakin-5 for 48h and proliferation was measured as indicated in A. (C) MDCK cells and Hakai-MDCK cells were treated with increasing concentrations of Hakin-1 for 48h and proliferation was measured as indicated in A. (C) Soft agar assay of HT29 (left panel) cell line and Hakai-MDCK (right panel) cell line. Colonies were grown for 28 days (HT29) or 21 days (Hakai-MDCK) and counted as indicated in the materials and methods. Quantification of colonies was performed in triplicate and expressed as mean ± SD of three independent experiments (. p < 0.01;. p < 0.001).

FIG. 5 Hakin-1 reduces cell invasion and cell migration of epithelial tumor cells. (A) The invasion assay in the LoVo cell line was performed as described in materials and methods. Cells were treated in the presence of DMSO or Hakin-1 for 48h and then seeded into the invasion chamber. Representative images were taken using a 20-fold objective lens (top panel) and quantification of the invasive cells taken is shown (bottom panel). (B) The invasion assay was performed as shown in A using MDCK cells and Hakai-MDCK cells. (C) Migration assays in HT29 cells were analyzed within 48h after treatment with DMS or Hakin-1. Cells are seeded in the migration chamber as described in the materials and methods. Representative images are shown (top panel) and quantification of migrating cells is shown (bottom panel). Results are expressed as mean ± SD (. p < 0.001) of triplicates from three independent experiments;

FIG. 6 Hakin-1 inhibits tumor growth in xenograft mice. (A) Effect of Hakin-1 on tumor growth in nude mice inoculated with Hakai-MDCK cells in the flank (n ═ 6 tumors). Tumor growth curves are shown in the upper panel. Error bars represent mean ± SEM (. sp. < 0.05). A schematic of the experimental design is shown in the following figure. (B) At the end point, H & E staining was performed on Hakai-MDCK tumors treated with DMSO (left panel) or Hakin-1 (right panel). The image was acquired with a 20 x objective lens. The scale bar is 300. mu.M. (C) H & E staining shows infiltration of blood vessels by tumor cells. The image was acquired with a 20 x objective lens. The scale bar is 500. mu.M. (D) Immunohistochemistry for Ki67 marker in Hakai-MDCK tumors treated with DMSO (left panel) or Hakin-1 (right panel). Representative images were obtained with a 40 x objective. The scale bar is 500. mu.M. Quantification of the percentage of positive cells is shown in the following figure. (E) Representative images of Hakai-MDCK tumors in H & E stained nude mice are shown. Pictures (upper) were taken with a 40 x objective. Quantification of mitotic numbers in high power fields is shown (lower panel). The scale bar is 500. mu.M. (F) Immunohistochemistry for the CD31 marker in Hakai-MDCK tumors in nude mice post injection treated with DMSO (left panel) or Hakin-1 (right panel). The image was acquired with a 20 x objective lens. The scale bar is 500. mu.M. Quantification of the number in the field of view is shown in the lower graph and expressed as mean ± SEM (× p < 0.001);

figure 7 Hakin-1 treatment reduced mesenchymal markers and micrometastases formation of tumor xenografts in the lungs of nude mice. (A-C) immunohistochemical staining of Hakai (A), E-cadherin (B) and N-cadherin (C). Representative images were obtained using a 20 x objective lens (upper panel). The lower panel shows quantification of significant protein expression intensity. (D) Immunohistochemical staining of the cortical actin (Cortactin) antibody and quantification of protein expression are shown (upper and lower panels, respectively). The image was acquired with a 40 x objective lens. (E) H & E staining of mouse lungs. Representative images were obtained with a 10 x objective. (F) Real-time quantitative PCR was performed using primers for the HA epitope and Hakai to detect the presence of DNA from Hakai-MDCK cells entering the mouse lung. Results are expressed as mean ± SEM (. p < 0.001). The scale bar is 500 MuM;

FIG. 8 Hakin-5 did not affect EMT marker expression. (A) Western blot of E-cadherin, cortical actin and Hakai in HT29 cells 48h after Hakin-5 treatment. (B) Immunofluorescence of E-cadherin in HT29 cells treated with Hakin-5 for 48 h. The image was taken with a 40 x objective lens. The scale bar is 250 MuM;

FIG. 9A role of Hakin-1 in human cancer cells. Breast cancer MCF7 cells, prostate cancer PC-3 cells, bladder cancer 5637 cells, kidney cancer ACHN and liver cancer HepG2 cells were treated with Hakin-1 for 48h, proliferation was measured by BrdU assay as shown in materials and methods. Results are expressed as mean ± SD of eight replicates, with experiments repeated three times (.;) p < 0.05;. p < 0.01;. p < 0.001);

FIG. 10 Hakin-1 does not affect apoptosis in vivo in a xenograft mouse model. Tunel assay was performed as indicated in materials and methods. Representative images are shown (left panel), quantification of the number of positive cells (right panel) is also expressed as mean ± SEM. The image was taken with a 20 x objective lens. The scale bar is 125 MuM;

FIG. 11 intact cell morphology and tissue structure of liver and kidney in vivo in a xenograft mouse model treated with Hakin-1. H & E staining of liver (upper panel) and kidney (lower panel) of nude mice treated with 5mg/kg DMSO or Hakin-1. The image was taken with a 10 x objective lens. The scale bar is 500 MuM;

FIG. 12. Effect of selected analogs on the cytotoxicity of HT29 epithelial tumor cell line. Cells were treated with increasing concentration ranges (50 μ M, 100 μ M, 250 μ M and 500 μ M) of: (A) ketophenyl groups: a-1, A-7, A-8 and A-9; (B) ketoheteroaryl groups: a-23 and A-25; (C) cyclic amides: a-10 and A-16; and (D) benzamides: a-6.1. Cell viability was measured by MTT assay. The assay was set at 6 replicates and expressed as mean ± SD of two independent experiments;

FIG. 13. Effect of analogue inhibitors on Hakai-dependent ubiquitination. In vivo Hakai-dependent ubiquitination assay in 293T cells transfected with Flag-Hakai, v-Src and HA-ubiquitin in the presence of DMSO or selected analogues. (A) Ketophenyl groups: a-7 and A-9; (B) ketophenyl groups: a-9, and cyclic amides: a-10 and A-16; and (C) ketoheteroaryl groups: a-23.

Detailed Description

The invention provides 4-tetrazolyl benzoic acid Hakin-1, Hakin-2 and Hakin-6 (from the following compounds #1, #2 and #6)

Identified as Hakai inhibitors that are able to compete for HYB binding sites present only in the Hakai dimer. Hakai has been reported to be associated with tumor progression. Therefore, inhibitors of the interaction between Hakai and E-cadherin may be useful in the treatment of cancer. In this sense and as shown in the examples, inhibition of tumor progression has been demonstrated herein in vitro and in vivo using compound # 1. Analogs #2 and #6 have not been tested, but they are closely related in structure to compound #1, and it is reasonable for this specification that these compounds also inhibit tumor progression.

Accordingly, the present invention solves the technical problem of providing a compound having excellent anticancer effect and low toxicity. Thus, the compounds of the present invention may be advantageously used as a medicament, in particular for the treatment of various cancers, such as cancers, in particular cancers of the gastrointestinal tract including the mouth (oral cancer), the esophagus, the stomach, and the small and large intestine (e.g. rectal or colon cancer). It also includes skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (e.g., renal cancer, prostate cancer, or bladder cancer). Indeed, our data (as shown in the examples) collectively show that Hakin-1 is a specific inhibitor of Hakai-mediated ubiquitination without affecting the Hakai protein level (example 1). Furthermore, Hakin-1 was able to inhibit proliferation in Hakai-MDCK cells, while no effect was detected in MDCK cells (FIG. 4 c). Hakin-1 also inhibits cell proliferation in other epithelial cell lines, such as breast cancer MCF7 cells, prostate cancer PC-3 cells, bladder cancer 5637 cells, liver cancer HepG2 cells, and renal cancer ACHN cells. All these findings support the antitumor effect of Hakin-1 by acting on cell proliferation, oncogenic potential, cell motility and invasion. Hakin-1 treatment significantly inhibited tumor growth in nude mice without systemic toxicity (FIG. 11). Furthermore, Hakin-1 resulted in a significant reduction in the micrometastases detected in the lungs of Hakai-MDCK xenografted mice, but not in the lungs of untransformed MDCK-injected mice, compared to control DMSO-treated mice (fig. 7 f). This result underscores that Hakin-1 inhibits lung metastasis in vivo.

Compound #1 (also known as Hakin-1, as described earlier) is a 1, 5-disubstituted Tetrazole, a chemically and metabolically stable pharmacophore fragment commonly used in drug discovery (Tetrazole Derivatives as formulating, E.A. Popova et al, Anticancer Agents Med chem.2017Mar 27.doi:10.2174/1871520617666170327143148, Epub ahead of print). The 1-position of the tetrazole ring is substituted with a 4-carboxyphenyl group. In position 5, it is attached to another benzene ring via a mercaptomethylcarbonyl linker.

Important physicochemical parameters, such as molecular weight, calculated lipophilic logP and polar surface area are all within the range required for oral absorption of the drug unlikely to cross the blood-brain barrier. From a snapshot of docking studies on compound 1 in the Hakai HYB site, the carboxyphenyl moiety generates 3 important hydrogen bond interactions with proteins and may mimic the phosphotyrosine substrate binding pattern. The other hydrogen bond is formed by two of the tetrazole nitrogen atoms, which provides additional stability and holds the tetrazole-carboxyphenyl unit at both ends in a defined binding pattern. Thus, the following subunits appear to be essential and should be considered to identify the core structure of the derivative of compound # 1:

common structural features of compounds #1, #2 and # 6.

Starting from this common structural core, many modifications can be made to optimize the activity (and other properties) of these compounds and to provide analogs of these compounds. In this sense, there are multiple opportunities to introduce modifications in different parts of the molecule, which represents an important goal for optimizing the process and providing analogues of compounds #1, #2 and # 6. In this sense, the pharmaceutical chemistry program will begin to focus on the chemical space around compound #1, and less preferentially around compounds #2 and # 6. In view of this, we provide herein a group of different analogues of compounds #1, #2 and #6 useful in the present invention. Notably, it is useful as a medicament, in particular for the treatment of various cancers, such as cancers, in particular tumours originating from the epithelial lining of the gastrointestinal tract including mouth (oral cancer), oesophageal cancer, stomach cancer and the small and large intestine (e.g. rectal or colon cancer). It also includes skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (e.g., renal cancer, prostate cancer, or bladder cancer).

To find those analogues similar to compounds #1 and #2, the first class of analogues was provided by linking the core structure to a ring structure (Cy ═ any ring).

These types of compounds are grouped as follows according to the following structural subclasses a) to c).

a) Ketophenyls (including compound # 1):

it is noted that these structures may contain substituents on the phenyl ring or in another part of the molecule, for example, on the carboxyphenyl ring or on the carbon atom between the sulfur and carbonyl groups. The latter case is exemplified as follows:

furthermore, the following examples show the introduction of modifications, such as cyclopropyl bridges, or the addition of substituents on the carboxyphenyl ring:

specific examples of ketophenyl compounds useful in the practice of the invention are shown below:

4- (5- ((2- (4-nitrophenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-acetylphenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4- (methylsulfonyl) phenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (2- ((1- (4-carboxyphenyl) -1H-tetrazol-5-yl) thio) acetyl) benzoic acid

4- (5- ((2- (4-carbamoylphenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- (4-sulfamoylphenyl) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4- (methanesulfonamido) phenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4- (cyclopropanecarboxamido) phenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-bromophenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-cyanophenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- (4-trifluoromethyl) phenyl) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-cyclopropylphenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4- (cyclopropylamino) phenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-morpholinophenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4- (4-methylpiperazin-1-yl) phenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-methoxyphenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4- (5-fluoropyridin-3-yl) phenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

b) Ketoheteroaryl groups:

other substructures provided by linking the core structure to a ring structure (Cy ═ any ring) are listed herein as ketoheteroaryl groups. In this sense, a typical search in pharmaceutical chemistry is to replace the phenyl group present in compound #1 with a heteroaryl group, which represents a similar aromatic ring with an additional heteroatom. Here we provide 6 examples of analogues belonging to this class:

6-membered heteroaryl groups structurally closer to phenyl (e.g., pyridine, pyrimidine, pyridazine) as shown below also form part of the invention:

in addition, specific examples of ketoheteroaryl compounds useful in the practice of the present invention are shown below:

4- (5- ((2-oxo-2- (pyridin-4-yl) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (6-aminopyridin-3-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (6-hydroxypyridin-3-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (6-morpholinopyridin-3-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (5-cyanopyridin-2-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- (pyrimidin-4-yl) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- (pyridazin-4-yl) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- (pyrazin-2-yl) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

c) Cyclic amides (including compound # 2):

yet further substructures provided by linking the core structure to a ring structure (Cy ═ any ring) are listed herein as cyclic amides, as shown below:

in particular, substituted indoline (and indole) analogs are shown below:

more particularly, any compound substituted on the benzene ring is shown below:

4- (5- ((2- (5-bromoindolin-1-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (5-cyanoindolin-1-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (5-cyclopropylindolin-1-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (5-morpholinoindolin-1-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- (5- (pyridin-3-yl) indolin-1-yl) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (5-fluoroindolin-1-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

1- (2- ((1- (4-carboxyphenyl) -1H-tetrazol-5-yl) thio) acetyl) indoline-5-benzoic acid

4- (5- ((2- (5-carbamoylindolin-1-yl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

With modifications in other parts of the molecule:

4- (5- ((1- (5-cyanoindoline-1-carbonyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

2-fluoro-4- (5- ((1- (5-morpholinoindoline-1-carbonyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- (5-carbamoylindoline-1-carbonyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

3-cyclopropyl-4- (5- ((2- (5-fluoroindolin-1-yl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

3-cyano-4- (5- ((2- (5-cyclopropylindolin-1-yl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

Furthermore, the different sub-structures a) to c) provided above by linking the core structure to a ring structure (Cy ═ any ring) may be further substituted as follows:

1. examples of structures having substitutions on the mercaptoacetyl linker

4- (5- ((2-methyl-1- (4-nitrophenyl) -1-oxoprop-2-yl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- (4-carbamoylphenyl) -2-methyl-1-oxopropan-2-yl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-methyl-1- (4- (methanesulfonamido) phenyl) -1-oxopropan-2-yl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- (4-cyanophenyl) -2-methyl-1-oxopropan-2-yl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- (4-nitrobenzoyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- (4-carbamoylbenzoyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- (4-methanesulfonamido) benzoyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- (4-cyanobenzoyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

2. Examples of structures having substitution on the carboxyphenyl moiety

2-cyano-4- (5- ((2- (4-nitrophenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-carbamoylphenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) -2-fluorobenzoic acid

2-cyclopropyl-4- (5- ((2- (4- (methanesulfonamido) phenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (4-cyanophenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) -3-fluorobenzoic acid

3-cyano-4- (5- ((2- (4-nitrophenyl) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

3-cyclopropyl-4- (5- ((2- (4- (methanesulfonamido) phenyl) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

On the other hand, a completely new group or class of compounds derived from the extension of the core structure with nitrogen atoms, carbon atoms and cyclic groups to provide compounds similar to compound #6 is provided herein under the subclass of benzamides:

compounds belonging to this class are shown herein below:

other compounds belonging to this class of compounds are:

4- (5- ((2- ((3-cyanobenzyl) amino) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- ((3-cyclopropylbenzyl) amino) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- ((3-morpholinobenzyl) amino) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- ((3- (pyridin-3-yl) benzyl) amino) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- ((3-carbamoylbenzyl) amino) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- ((3-fluorobenzyl) amino) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

With modifications in other parts of the molecule:

4- (5- ((1- ((3-cyanobenzyl) carbamoyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

3-cyano-4- (5- ((2- ((3-cyclopropylbenzyl) amino) -2-oxyethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((1- ((3-morpholinobenzyl) carbamoyl) cyclopropyl) thio) -1H-tetrazol-1-yl) benzoic acid

2-fluoro-4- (5- ((2-oxo-2- ((3- (pyridin-3-yl) benzyl) amino) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- ((3-carbamoylbenzyl) amino) -2-oxyethyl) thio) -1H-tetrazol-1-yl) -3-cyclopropylbenzoic acid

4- (5- ((1- ((3-fluorobenzyl) amino) -2-methyl-1-oxopropan-2-yl) thio) -1H-tetrazol-1-yl) benzoic acid

Having a heteroaryl group:

4- (5- ((2-oxo-2- ((pyridin-4-ylmethyl) amino) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- ((pyrimidin-4-ylmethyl) amino) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2-oxo-2- ((pyridazin-4-ylmethyl) amino) ethyl) thio) -1H-tetrazol-1-yl) benzoic acid

4- (5- ((2- (((5-cyanopyridin-2-yl) methyl) amino) -2-oxoethyl) thio) -1H-tetrazol-1-yl) benzoic acid

All of the above compounds #1, #2 and #6, along with those structures classified as provided by linking the core structure to a ring structure (Cy ═ any ring) to provide compounds similar to compound #1 or #2, or as a class of compounds derived from expanding the core structure with a nitrogen atom, a carbon atom and a cyclic group to provide compounds similar to compound #6, are referred to herein as "compounds of the invention".

It is noted that all compounds of the present invention, as well as any pharmaceutically acceptable salts thereof, are encompassed by the following general formula:

wherein:

-a represents a group selected from aryl, heteroaryl and cyclic amide, optionally substituted with 1 or 2 groups independently selected from:

e) halogen atom, NO₂、-CN、-N(R^a)R^b、-OR^a、-C(＝O)R^a、-C(＝O)OR^a、-C(＝O)N(R^a)R^b、-OC(＝O)-R^a、-N(R^c)C(＝O)R^b、-NR^cSO₂R^a、-SO₂N(R^a)R^b、-SR^a、-S(O)R^a、-S(O)₂R^a；

f) Straight or branched chain C optionally substituted by 1, 2 or 3 halogen atoms₁-C₆An alkyl group;

g) c optionally containing 1 or 2 heteroatoms selected from O, S and N₃-C₆Cycloalkyl, and wherein the ring is optionally substituted by C₁-C₃Alkyl substitution;

h) each optionally substituted by halogen atoms, cyano groups, C₁-C₃Alkyl or cyclopropyl substituted phenyl or C₅-C₆A heteroaryl group;

-R^a、R^band R^cEach independently represents:

e) a hydrogen atom;

f) straight or branched chain C optionally substituted with 1, 2 or 3 substituents selected from₁-C₁₂Alkyl radical, C₃-C₆Cycloalkyl and C₄-C₆Heterocycloalkyl group: carbonyl, halogen, hydroxy, phenyl, C₃-C₆Cycloalkyl, straight or branched C₁-C₆Alkoxy, amino, alkylamino, dialkylamino, straight or branched C₁-C₆An alkylcarbonyl group;

g) phenyl or C optionally substituted with 1, 2 or 3 substituents selected from₅-C₆Heteroaryl group: halogen atom, cyano group, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Haloalkyl, hydroxy, straight or branched C₁-C₆Alkoxy, amino, alkylamino, dialkylamino;

h)R^aand R^bTogether with the nitrogen atom to which they are attached form a 3 to 8 membered ring, which 3 to 8 membered ring optionally further comprises a further heteroatom selected from O, N and S, and wherein the ring is optionally substituted with 1, 2 or 3 substituents selected from: carbonyl, straight or branched C₁-C₆Alkyl, straight or branched C₁-C₆Haloalkyl, straight-chain or branched C₁-C₆An alkylcarbonyl group;

-x and y are integers independently selected from 0 and 1;

-R¹represents a radical selected from hydrogen, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups, wherein the alkyl groups are optionally substituted with 1, 2 or 3 halogen atoms;

when y is 0, then R¹May form a 5-or 6-membered ring together with the adjacent nitrogen atom and 2 adjacent carbon atoms of the aromatic ring to which the nitrogen is attached;

-R²and R³Each independently represents hydrogen, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups; optionally, R²And R³May form a 3-or 4-membered spirocyclic ring together with the carbon atoms to which they are both attached;

-z is an integer selected from 0, 1, 2 or 3;

-R⁴represents a group selected from-CN, cyclopropyl or a linear or branched C₁-C₆A group of alkyl groups, said alkyl groups being optionally substituted with 1, 2 or 3 halogen atoms; wherein the radical R⁴If present, a substitution is present with R⁴A hydrogen atom of a group CH in the attached benzene ring.

Preferably, R2 and R3 each independently represent a group selected from cyclopropyl or a linear or branched C1-C6 alkyl group.

More preferably, R2 and R3 together with the carbon atom to which they are both attached form a 3-or 4-membered spirocyclic ring.

More preferably, z is an integer selected from 1, 2 or 3; r4 represents a group selected from-CN, cyclopropyl or linear or branched C1-C6 alkyl, wherein said alkyl is optionally substituted by 1, 2 or 3 halogen atoms; and wherein the group R4 substitutes for a hydrogen atom of a group CH present in the phenyl ring to which R4 is attached.

More preferably, one or both of the integers x and y are equal to 1, and a represents a group selected from the list consisting of aryl, heteroaryl and cyclic amide substituted with 1 or 2 groups independently selected from: a halogen atom, -CN, -n (Ra) Rb, -ORa, -C (═ O) Ra, -C (═ O) ORa, -C (═ O) n (Ra) Rb, -OC (═ O) -Ra, -n (rc) C (═ O) Rb, -NRcSO2Ra, -SO2N (Ra) Rb, -SRa, -s (O) Ra, -s (O)2 Ra; linear or branched C1-C6 alkyl, wherein the alkyl is optionally substituted with 1, 2, or 3 halogen atoms; C3-C6 cycloalkyl optionally containing 1 or 2 heteroatoms selected from O, S and N, and wherein the ring is optionally substituted with C1-C3 alkyl; phenyl or C5-C6 heteroaryl, each optionally substituted by a halogen atom, a C1-C3 alkyl group or a cyclopropyl group.

Still more preferably, the compound of the present invention is any of compounds #1, #2 and #6, or any compound identified above and classified as a structure provided by linking the core structure to a ring structure (Cy ═ any ring) to provide a compound similar to compound #1 or #2, or any compound identified above and classified as a compound derived from a class of compounds in which the core structure is extended with a nitrogen atom, a carbon atom and a cyclic group to provide a compound similar to compound # 6.

In a preferred embodiment, a "compound of the invention" useful in the practice of the invention is selected from any one of the following lists:

the compounds of the invention may be in free form or in the form of a pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include: inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, or hydrobromide salts, and the like; organic acid salts such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, maleate, or the like. In addition, when the compound has a substituent such as a carboxyl group, a salt with a base (for example, an alkali metal salt such as a sodium salt, a potassium salt, or the like, or an alkaline earth metal salt such as a calcium salt, or the like) can be cited.

The compound of the present invention or an enantiomer or a pharmaceutically acceptable salt thereof may be any of an intramolecular salt or adduct thereof, or a solvate or hydrate thereof.

When the compound of the present invention or a pharmaceutically acceptable salt thereof of the present invention is used as an effective ingredient for medical use, it may be used together with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is an inert carrier suitable for each administration method, and may be formulated into conventional pharmaceutical preparations (tablets, granules, capsules, powders, solutions, suspensions, emulsions, injections, infusions, and the like). Examples of such carriers include pharmaceutically acceptable binders (e.g., gum arabic, gelatin, sorbitol, and polyvinylpyrrolidone), excipients (e.g., lactose, sugar, corn starch, and sorbitol), lubricants (e.g., magnesium stearate, talc, and polyethylene glycol), and disintegrants (e.g., potato starch). When they are used as injection solutions or infusion solutions, they can be formulated by using distilled water for injection, physiological saline, an aqueous glucose solution.

The method of administration of the compound of the present invention and/or the pharmaceutically acceptable salt thereof of the present invention is not particularly limited, and conventional oral or parenteral administration methods (intravenous, intramuscular, subcutaneous, transdermal, intranasal and others, transmucosal, enteral and the like) can be used.

The dose of the compound of the present invention or a pharmaceutically acceptable salt thereof may be optionally set within a range of an effective amount sufficient to exhibit a pharmacological effect, depending on the potency or properties of the compound to be used as an effective ingredient. The dosage may vary depending on the administration method, age, weight or condition of the patient.

The following examples are intended to illustrate the invention only and are not intended to limit the invention.

Examples

Example 1 Synthesis

The compounds listed throughout this specification can be prepared according to the following general synthetic routes:

example 2.

2.1. Materials and methods

Protein and ligand models. The X-ray crystal structure of the phosphotyrosine binding domain of Hakai (PDB 3VK6) was downloaded from a protein database and the dimer was modeled using appropriate symmetry manipulations. The amino acid protonation was performed using pdb2pqr server at pH 7.2. The 3D model of the ligand was constructed using a Virtual Screening and Data Management Integrated Platform (VSDMIP), as described elsewhere. Briefly, the initial 3D coordinates of each ligand were generated using CORINA [ Sadowski, j.; gastiger, j.; klebe, G.Compuison of Automatic Three-Dimensional Model building Using 639X-Ray structures.J.chem.Inf.Compuit.Sci.1994, 34,1000-1008(DOI:10.1021/ci00020a039) ]. Thereafter, ALFA [4] was used to generate a wide variety of conformers, each of which was assigned a MOPAC calculated atomic partial charge by using the AM1 semi-empirical model and the ESP method. All ligand models are stored in the VSDMIP database for different virtual screening activities.

And (6) virtual screening. Html in the eMolecules catalog [ https:// www.emolecules.com/info/products-screening-compositions ] ligands were downloaded and processed as described in the previous section. Only molecules presenting a carboxylic acid moiety and/or a phosphate group capable of mimicking a phosphotyrosine residue are contemplated. Next, selected molecules were embedded into the binding pocket of Hakai with CRDOCK using CRScore scoring function and BFGS energy minimizer. Ligands are then ranked according to predicted scores and the top 350 molecules are re-evaluated by using the internal implementation of the HYDE scoring function. Finally, the best 20 molecules were visually examined to select the final set of 6 molecules.

Binding pocket analysis. To better analyze the results of the virtual screening campaign, we generated an affinity map within the binding pocket of the phosphotyrosine binding domain of Hakai using our internal cGRILL software [6] based on the hypothesized van der waals, coulombic, and hydrogen bonding interactions of the atom probe. The potential positions for molecular recognition of phosphotyrosine residues were mapped using negatively charged receptor probes (═ O) to help filter docking protocols during visual inspection of the pose.

Plasmids, inhibitors and antibodies

pcDNA-Flag-Hakai, pBSSR-HA-ubiquitin, pSG-v-Src and pcDNA-myc-E-cadherin plasmids were previously described. The compounds Hakin-1[4- (5- { [2- (4-nitrophenyl) -2-oxyethyl ] thio } -1H-tetrazol-1-yl) benzoic acid ] and Hakin-5[ (2E,4E,8E) -7, 13-dihydroxy-4, 8, 12-trimethyl-2, 4, 8-tetradecatrienoic acid ] were obtained from ChemBridge Corporation and TimTec or Analyticon Discovery, respectively. The remaining analogs tested (ketophenyl A-1, A-7, A-8, A-9; ketoheteroaryl: A-23, A-25; cyclic amides: A-10, A-16; and benzamides: A-6.1) were obtained from Vitasmlab. Compounds were resuspended at 100mM in DMSO (Sigma) for in vitro assays, while Hakin-1 was assayed at 100mM in vivo. The highest concentration of DMSO was used as the vehicle control for the experiment. Note that the chemical structure of Hakin-5 is shown in FIG. 1.

Cell culture

MDCK, HEK293T, HepG2, MCF7 and ACHN cells were cultured in Dulbecco's Modified Eagles Medium (DMEM). MDCK (Hakai-MDCK) stably expressing Hakai cells has been previously reported and grown in DMEM containing G418 (800. mu.g/ml). As previously described, different clones of Hakai-MDCK cells showed similar phenotypes and characteristics. LoVo cells and PC-3 cells were cultured in F-12K Medium (Kaighn's Modification of Ham's F-12Medium) and HT-29 cells were cultured in McCoy's 5a Medium Modified. 5637 cells were cultured in RPMI medium. All media were supplemented with 1% penicillin/streptomycin and 10% heat-inactivated Fetal Bovine Serum (FBS) at 37 ℃ in a humidified incubator with 5% CO 2. Cells were tested for mycoplasma contamination once a month and were used only 1-3 months after thawing. LoVo cells and HT29 cells were verified using the StemElite ID system (Promega). For phase contrast images, cultured cells were photographed using a Nikon Eclipse-TI microscope.

Ubiquitination assay

For ubiquitination assay, 750000 HEK293T cells were seeded in 6 well cell culture plates and 24h later transfected with 0.25. mu.g Src, 0.75. mu.g Flag-Hakai and 0.5. mu.g HA-ubiquitin with Lipofectamin 2000(Invitrogen, Uk). 6h after transfection, cells were treated with the indicated concentrations of Hakin-1, Hakin-5 or the remaining analogs tested for 36 h. Whole cell extracts were obtained in lysis buffer (20mM Tris-HCl pH7.5, 150mM NaCl and 1% Triton X-100) containing 10. mu.g/ml leupeptin, 10. mu.g/ml aprotinin and 1mM phenylmethanesulfonyl fluoride (PMSF) supplemented with 10mM N-ethylmaleimide. Cells were harvested and western blotted with anti-HA antibodies to detect ubiquitination.

Immunoprecipitation

For immunoprecipitation experiments 293 cells were transfected with 3. mu.g Src, 4. mu.g Flag-Hakai, and 2. mu.g Ha-ubiquitin and 3. mu. g E-cadherin with Lipofectamin 2000(Invitrogen, UK). 24h after transfection, cells were lysed in 1ml of 1% Triton X-100 lysis buffer (20mM Tris-HCl pH7.5, 150mM NaCl and 1% Triton X-100) supplemented with 10mM N-ethylmaleimide and 2.5mM sodium orthovanadate and containing 10. mu.g/ml leupeptin, 10. mu.g/ml aprotinin and 1mM phenylmethanesulfonyl fluoride (PMSF) for 20 minutes. After centrifugation at 18000G for 10 min, the supernatant was immunoprecipitated with 2. mu.g of anti-E-cadherin antibody bound to 60. mu.l of protein G PLUS-agarose beads for 2h, followed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western immunoblotting with the antibodies specified as described previously.

Determination of viability

For cytotoxicity assays, cells were plated at 1 × 10⁴One/well was inoculated into 96-well plates. After 24h, cells were treated with the indicated inhibitors for 72h and MTT colorimetric cell viability assay was performed according to the manufacturer's instructions (Sigma Aldrich, stouis, MO). Absorbance was measured at 570nm and 630nm using a Multiskan Plus Reader (Nanoquant Infinite M200 Tecan tracing AG, Switzerland)And (4) luminosity. Dose response curves were designed using GraphPad Prism software and half maximal Inhibitory Concentration (IC) was calculated₅₀) The value is obtained. Data shown are mean ± SEM of at least three independent experiments, with six replicates per condition.

Western blotting and immunofluorescence

For western blot analysis, cells were treated with the indicated inhibitors for 48h and whole cell extracts were obtained as described previously. 20 μ g of lysate were resolved on 10% polyacrylamide SDS-PAGE and then subjected to Western blot analysis as described previously. For immunofluorescence assays, cells were grown on glass coverslips for 24h and treated with the indicated inhibitors for 48 h. Cells were fixed with 4% PFA for 15min, permeabilized with 0.5% Triton X-10 and incubated with E-cadherin antibody for 2 h. Coverslips were incubated with fluorescein-labeled secondary antibodies (Dakopatts, Sweden) for 1 h. Finally, the coverslips were fixed with ProLong Gold anti-quenching reagent (LifeTech, UK) and images were taken in an epifluorescence microscope (Olympus) using a 40-fold objective.

Proliferation assay

For the BrdU assay, the designated cells were measured at 1X 10⁴One/well was plated into 96-well plates. After 24h, cells were treated with the indicated inhibitors for 48 h. Three independent experiments were repeated six times for each condition. Cells were treated with 10mM BrdU for 2 h. BrdU incorporation in newly synthesized DNA was measured using a cell proliferation colorimetric immunoassay kit according to the manufacturer's instructions (Roche, switzerland). Results are expressed as mean ± s.d. Results are expressed as the percentage of positive cells (mean ± s.d) in three independent experiments.

Soft agar colony formation assay

On a 12-well plate at 5X 10³MDCK and MDCK-Hakai cells/well or 12X 10³The density of individual HT29 cells/well was performed in triplicate in a soft agar colony formation assay. Cells were seeded in media with 0.5% low melting agarose overlaid on a 0.75% low melting agarose layer (Lonza Rockland, ME, USA). Cells were treated with the indicated inhibitors and DMSO was used as vehicle. The treatment was renewed every 3 days, at 21 days (for MDCK and MDCK-Hakai cells) or 28 days (for HT29 cells)) Thereafter, the number of colonies was quantified. Quantification of five randomly selected fields of view under each condition was taken using a Nikon Eclipse-TI microscope (objective 4-fold). Experiments were performed in triplicate and repeated three times. Data are presented as mean ± SD.

Migration and invasion assay

For the invasion assay, cells were treated with Hakin-1 or DMSO as vehicle for 48h, with 1% FBS for the last 24 h. Will be 3X 10⁵Individual MDCK, MDCK-Hakai, or LoVo cells were seeded in a cell invasion chamber (cell invasion assay kit, Chemicon International) containing medium containing 2% FBS. After 72h (for MDCK and MDCK-Hakai invasion) and 16h (for LoVo cells), invaded cells reaching the lower chamber containing 30% FBS were fixed and stained with crystal violet (Sigma Aldrich, StLouis, MO) according to the manufacturer's instructions. For the migration assay, HT29 cells were cultured with Hakin-1 or DMSO as vehicle for 48h, and finally treated with serum-free medium for 24 h. Seeding of 3X 10 in cell migration Chambers containing serum free Medium⁵HT29 cells (cell migration kit, Millipore, Bedford, Mass.). After 16h, migrated cells in the lower chamber of serum containing 30% FBS were stained with crystal violet and counted according to the manufacturer's instructions. For invasion and migration assays, cells were counted in five fields of view taken with an Olympus microscope using a 20-fold objective, experiments were performed in triplicate for each condition, and experiments were repeated at least three times. Results are expressed as mean ± SD.

Tumor xenograft model

The xenograft experiments were carried out in the surgical laboratory Unit of INIBIC-technical Training Center (Experimental Surgery Unit-technical Training Center) according to European Community Law (86/609/EEC) and Spanish Law (R.D. 53/2013). The experiment yielded Xerencia de Xestion Integrada da(XXIAC) approval by the Animal Experimental Ethics Committee (Ethics Committee for Animal experiment). Mice were in light/dark cycles of 12h/12hWater and food are available ad libitum. Athymic nu/nu mice at six weeks of age were randomly grouped. One million MDCK cells resuspended in DMEM without serum and antibiotics were inoculated subcutaneously into both flanks of animals, which were divided into two groups of 3 animals each. The same number of Hakai-MDCK cells were injected into animals, which were in two groups of 4 cells each. Tumors in Hakai-MDCK were palpable 20 days after inoculation. Then, half of the animals were treated every 3 days with Hakin-1(5mg/kg), and the other half with the same concentration of DMSO. Tumor growth was monitored twice weekly by measuring tumor length (L) and width (W) with electronic calipers. Tumor volumes were calculated as pLW 2/6. Forty days after inoculation, the animals were sacrificed. Tumors, lung, kidney and liver were collected and fixed in 4% PFA and embedded in paraffin blocks for histological and/or Immunohistochemical (IHC) analysis.

Histology and immunohistochemistry

Tumors and tissues were deparaffinized, rehydrated and stained with hematoxylin and eosin (H & E) as previously described. Tumor sections (4 μm) were also deparaffinized and hydrated for immunohistochemistry. Antigen retrieval was performed by heating the samples (2100 Retriever; Pickcell Laboratories) in citrate buffer (DakoREAL, Denmark) or EDTA buffer. Endogenous peroxidase activity was then blocked with a peroxidase blocker (DakoCytomation, Denmark). Samples were blocked and permeabilized with 0.2% BSA and 0.1% Tx-100 for 1 hour and incubated overnight at 4 ℃ in a wet chamber with the indicated primary antibody. Slides were incubated with secondary antibodies for 1 hour at room temperature and detected using DAB (DakoReal Envision kit) according to the manufacturer's instructions. Finally, nuclei were counterstained with Gill hematoxylin and fixed with DePeX. Photographs were taken with an Olympus microscope. 5 pictures were taken of each animal and Image quantification was performed using the Image J program, and the results shown are shown as mean. + -. SEM. The number of mitoses was counted in sections stained with H & E. In this case ten pictures were taken for each tumor using an Olympus BX50 microscope (objective lens 40 x) and the number of mitoses was counted manually. Results are expressed as mean ± SEM and representative photographs for each condition are shown.

Quantification of lung metastasis from an in vivo mouse model

Real-time PCR was used to study the presence of metastases in the mouse lungs. Quantification is performed using primers (5'-TCTGGGACGTCGTATGGGTA-3'; 5'-TTCTTCATCACCTTGCGGG-3') directed against the HA epitope present in ectopic HA-tagged Hakai expressed in MDCK-Hakai cells and Hakai. Primers (5'-CGTGGGCTCCAGCATTCTA-3'; 5'-TCACCAGTCATTTCTGCCTTTG-3') of mouse apolipoprotein B (apob) were used as endogenous controls. MDCK cell line was used as negative control. Lung DNA was extracted from 10-15 sections of paraffin blocks (4 μm) using the QIAamp DNA Mini kit (Qiagen). Amplification and quantification of DNA was performed in technical triplicate (technical triplicates) by quantitative PCR using a LightCycler 480 real-time light cycler (Roche). Relative DNA level through 2^-ΔΔCtAnd (4) calculating by using the method.

Statistical analysis

Normal distributions were investigated using the Shapiro-Wilk test, and homogeneity of variance was assessed using the Levene test. Statistical significance of the data was determined using ANOVA with Bonferroni test or Kruskal-Wallis with Tukey correction test. The significance between the experimental groups shown in the figures is shown as p < 0.05, p < 0.01 and p < 0.001. As shown, the results obtained are expressed as mean ± SD or mean ± SEM. Survival plots in the xenograft assay were analyzed using GraphPad Prism software and p-values were calculated using Breslow assay. Results are expressed as fold change in the values obtained for treated cells compared to untreated cells.

List of antibodies used in the practice of the invention.

In vivo TUNEL assay

Tissue sections from tumors were deparaffinized and rehydrated using standard protocols. Slides were washed twice with PBS and treated with citrate buffer (DakoREAL, Denmark) in microwave at 350W for 5 min. The tissue sections were then analyzed using the Fluorescein in situ Cell Death Detection Kit (Cell Death Detection Kit, fluorochein) (Roche) according to the manufacturer's instructions. The slides were then incubated with Hoechst in the dark for 5 min. The reaction was observed under an epifluorescent Olympus microscope using a 20-fold objective lens. Five representative photographs were taken for each section. The percentage of positive cells was calculated and the results are expressed as mean ± SEM.

2.2. Results

Identification of putative Selective Hakai inhibitors

To find candidate molecules with the potential required to inhibit Hakai, we designed a virtual screening workflow based on the available structural information and the nature of the phosphotyrosine binding pocket, the exploration of which was conducted with the help of affinity probes. As a first step we consider only molecules in the chemical library that show negatively charged carboxylate or phosphate groups, which are complementary to the high positive molecular electrostatic potential of the binding pocket. Selected molecules were then docked into the Hakai dimer to assess all possible binding poses, and then ranked using the HYDE post-processing scoring function to estimate the interaction energy of hypothetical Hakai-inhibitor complexes. The pre-ranking 20 molecules were visually inspected and two of them were selected for subsequent experimental validation, i.e., Hakin-1 and Hakin-5 (FIG. 1 a). According to our binding model, the benzoate moiety present in Hakin-1 will be a replacement for phosphotyrosine (FIG. 1b, top panel). According to our affinity diagram, the carboxylate groups are located in a region that is highly favorable for negatively charged probes. This region is formed by the arrangement of residues Lys-126, Tyr-176, His-185 and Arg-189, while the phenyl ring will be sandwiched between the guanidino group and the side chains of Arg-174 and Arg-189. The remainder of the molecule will be able to establish hydrogen bonds with the side chains of the Arg-174 residues from both monomers, as well as with the Gln-170 backbone, while maintaining the desired shape. While Hakin-5 (fig. 1b, bottom panel) carries carboxylate groups and exhibits the same number of potential groups for hydrogen bonding interactions, it lacks a benzene ring that can mimic phosphotyrosine.

Effect of Hakin-1 inhibitors on Hakai-induced ubiquitination

We first investigated the effect of Hakin-1 inhibitor on ubiquitination induced by E3 ubiquitin ligase Hakai by using cultured tumor cells. 293T cells were transfected with Src, Hakai and ubiquitin in the presence of a Hakin-1 inhibitor or DMSO as a control. Hakin-1 strongly reduced ubiquitination mediated by Hakai in a dose-dependent manner (FIG. 1c), and no effect was seen at the Hakai protein level. However, when Hakai is not overexpressed, Hakin-1 does not affect ubiquitination, confirming that Hakin-1 reduces ubiquitination in a Hakai-dependent manner (FIG. 1 d). Furthermore, no effect on Hakai-mediated ubiquitination was detected when cells were treated in the presence of another Hakai inhibitor, Hakin-5, identified by virtual screening (fig. 1e), which further supports the specific effect of Hakin-1 on Hakai-induced ubiquitination. Finally, when cells were treated with Hakin-1, we observed a reduction in Hakai-dependent ubiquitination of the E-cadherin complex (FIG. 1 f). In summary, our data indicate that Hakin-1 inhibits Hakai-mediated ubiquitination without affecting Hakai protein levels.

Inhibition of Hakai by Hakin-1 activates epithelial differentiation of tumor cells

Next, we investigated the effect of Hakai inhibition on the cell viability of cancer cells. To this end, we generated dose response curves by using Hakin inhibitors in several epithelial cells, as we have previously reported. First, we analyzed the cytotoxic effects of Hakin-1 on HT-29 and LoVo colon tumor cell lines, showing an important inhibitory response (FIG. 2 a). We extended our studies by using a normal epithelial MDCK cell line that has been widely used as an in vitro model system to study EMT. As previously described, Hakai overexpression in MDCK cells (Hakai-MDCK) induces a fibroblast-like phenotype and the disappearance of E-cadherin-based cell-cell contacts. When these cell lines were treated with either a Hakin-1 or Hakin-5 inhibitor, the two clones tested for the Hakai-MDCK cell line were more sensitive to Hakin-1 and Hakin-5 treatment than the more resistant normal epithelial MDCK (FIG. 2 b). These results further indicate that Hakin-1 may be particularly effective on Hakai-overexpressing cancer cells, as seen in human colon cancer, where Hakai is significantly increased compared to adjacent normal tissues. Given the previously reported role of Hakai in epithelial-mesenchymal transition, we next analyzed the effect of Hakin-1 on tumor cell phenotype. By phase contrast, we observed that Hakin slightly induced the epithelial phenotype of HT-29 and LoVo cells, however, it is important to note that both cell lines already showed epithelial morphology (fig. 2 c). Furthermore, we tested the effect of Hakin-1 and Hakin-5 inhibitors on the mesenchymal phenotype of Hakai transformed MDCK cells. We observed induction of an epithelial phenotype, increasing cell-cell contact, with a concomitant decrease in protrusion formation. In contrast, no effect was observed when epithelial MDCK cells were treated with specific inhibitors (fig. 2 d). Given the reported effect of Hakai on EMT reversal by its molecular effects on E-cadherin ubiquitination, endocytosis and degradation during EMT, leading to altered cell-cell contact, we further investigated the effect of Hakin-1 inhibitors on EMT reversal. As shown in FIG. 3a, by Western blotting, we observed that Hakin-1 was able to increase the level of E-cadherin in HT-29 cells, while the mesenchymal vimentin marker decreased. We also analyzed the effect of Hakin-1 on cortical protein (another reported Hakai substrate), confirming its effect on increasing expression levels. We also confirmed these results by using another epithelial tumor cell, LoVo cells (fig. 3 b). Furthermore, since loss of E-cadherin at cell-cell contacts was considered a marker for EMT, we investigated the expression of E-cadherin by immunofluorescence, showing a significant increase in the level of E-cadherin at cell-cell contacts in HT-29 and LoVo cells (FIG. 3 c). However, when Hakin-5 was used, no effect on E-cadherin levels was detected by Western blotting or immunofluorescence (FIG. 8). Finally, to test the effect of Hakin-1, mesenchymal Hakai-MDCK cells were also used. Hakai-MDCK cells do not express E-cadherin basal protein levels, so no recovery was detected under Hakin-1 treatment. Taken together, these results indicate that Hakin-1 induces epithelial differentiation in different tumor epithelial cells, with a concomitant decrease in mesenchymal markers in vivo.

Hakin-1 inhibits proliferation, carcinogenic potential and invasion of tumor culture cells

We next characterized the role of Hakin-1 in tumor cell lines using standard proliferation and soft agar colony formation assays. Given that Hakai affects not only cell-cell contact, but also proliferation of fibroblasts and epithelial cells, we decided to determine the possible effect of Hakin-1 in cell proliferation. Although no effect was seen with the Hakin-5 inhibitor (FIG. 4b), Hakin-1 reduced cell proliferation in HT29 and LoVo cell lines (FIG. 4a), which further supports the antitumor effect of Hakin-1 through its control of cell proliferation. We extended our analysis by using Hakai-MDCK cells in comparison to normal epithelial MDCK cells. As reported previously, we confirmed that Hakai-transformed MDCK cells strongly increased cell proliferation compared to normal MDCK cells (fig. 4 c). Interestingly, Hakin-1 was able to inhibit the proliferation of Hakai-MDCK cells, while no effect was detected in untransformed epithelial MDCK cells (fig. 4 c). Hakin-1 also inhibits cell proliferation in other epithelial cell lines, such as breast cancer MCF7 cells, prostate cancer PC-3 cells, bladder cancer 5637 cells, liver cancer HepG2 cells, and renal cancer ACHN cells. (FIG. 9). These results indicate that Hakin-1 may act as an antiproliferative agent when high levels of Hakai are expressed, as occurs in human adenocarcinoma. By using a soft agar colony formation assay, we assessed the effect of Hakin-1 in HT-29 and Hakai-MDCK tumor cell lines, showing a strong inhibition of colony formation in both cell lines (fig. 4 d). As we have previously published, no colony formation was detected when MDCK untransformed cells were treated with Hakin-1. The EMT process is characterized by the ability to acquire migration and invasion. We demonstrated that Hakin-1 strongly reduced cell invasion in LoVo cells (FIG. 5 a). Furthermore, although no cell invasion was detected in MDCK cells, Hakai overexpression induced the ability to invade in normal epithelial MDCK cells, where Hakin-1 also reduced cell invasion (fig. 5 b). Finally, considering the inability of HT-29 cells to invade under these experimental conditions, we tested the effect of Hakin-1 on cell motility, showing a significant reduction in cell migration under Hakin-1 treatment (fig. 5 c). All these findings support the antitumor effect of Hakin-1 by acting on cell proliferation, oncogenic potential, cell motility and invasion.

In vivo anti-tumor effect of Hakin-1 in tumor xenografts

Acquiring the ability to migrate and invade during EMT is a key event in the formation of distant metastases, and therefore targeting these events is an ideal approach for cancer treatment. Since we have demonstrated that Hakin-1 effectively inhibits cell proliferation, oncogenic potential, and cell invasion and motility in cell culture, we decided to investigate the efficacy of Hakin-1 in inhibiting pre-existing tumors in vivo. For this purpose, MDCK and Hakai-MDCK cells were injected subcutaneously into the flanks of nude mice. As previously reported, Hakai-MDCK cells form primary tumors, whereas parental MDCK cells do not. Hakin-1 showed a potent effect in inhibiting the growth of xenograft tumors in vivo (FIG. 6 a). Morphologically, xenograft tumor cells exhibit an undifferentiated and spindle-shaped phenotype, large nuclei and reduced cytoplasmic size. This morphology was strongly altered by Hakin-1 treatment, showing induction of tumor differentiation and an increase in cytoplasmic size (fig. 6 b). Furthermore, we found that there was tumor cell infiltration in the blood vessels, whereas no infiltration was detected in the Hakin-1 treated xenograft tumors (fig. 6 c). Furthermore, by analyzing the two proliferation markers Ki67 and mitotic index, it was shown that Hakin-1 significantly reduced the number of Ki67 positive cells and the mitotic index (fig. 6d-e), while no effect on apoptosis was detected (fig. 10), further emphasizing the inhibitory effect of Hakin-1 on cell proliferation in vivo. We also visualized blood vessels in tumor sections by immunohistochemistry using CD31 angiogenic markers. A significant reduction in the number of blood vessels in Hakin-1 treated xenograft tumors was quantified compared to untreated tumors, indicating its inhibitory effect on angiogenesis (fig. 6 f). Interestingly, no lesions were observed in lung and kidney tissues of Hakin-1 treated nude mice, showing normal morphological structures, which supports that Hakin-1 treatment inhibits tumor growth in nude mice, and apparently no systemic toxicity (fig. 11).

Hakin-1 treatment reduces in vivo tumor xenograft N-cadherin mesenchymal markers and micrometastases formation in the lung

We further evaluated the in vivo effects of Hakin-1 on EMT reversal, a key process for tumor progression and cell invasion. First, we demonstrated that the Hakai protein expression level is not affected by the Hakin-1 action in xenografted tumors in nude mice (fig. 7a), which further supports previous in vitro results showing that Hakin-1 inhibits Hakai activity by acting on its ubiquitin ligase activity without altering Hakai protein expression (fig. 1). Given the complete disappearance of E-cadherin in Hakai-MDCK cells, as expected, no E-cadherin was detected in Hakai-MDCK xenograft tumors in nude mice in the presence or absence of Hakin-1 (FIG. 7 b). However, when we investigated the expression level of the mesenchymal marker of N-cadherin (another marker of EMT), we found that the expression of N-cadherin was strongly reduced in tumor xenografts treated with Hakin-1 compared to DMSO-treated mice (fig. 7 c). These data further support that Hakin-1 inhibits mesenchymal tumor cells by reducing the mesenchymal marker of N-cadherin. Given that the best described target E-cadherin of Hakai is completely absent in Hakai-MDCK mesenchymal tumors, we also extended our study by analyzing another described target cortical protein of Hakai. The cortical protein is a cytoskeletal protein and is also one of the major substrates of Src kinase. Interestingly, cortical protein expression was detected only in the cytoplasm of the Hakai-MDCK xenograft tumors and its expression was restored by Hakin-1 treatment (fig. 7d), further supporting that Hakin-1 could inhibit the induction of ubiquitination and degradation of cortical proteins by Hakai. To determine whether Hakin-1 may affect cancer metastasis, lung tissue from nude mice was analyzed by H & E staining, however, no distant metastasis was detected under experimental conditions (fig. 7E). Therefore, to detect possible micrometastases, we performed quantitative PCR by analyzing the lung for the presence of Hakai-MDCK DNA. For this purpose, HA-tagged Hakai present in Hakai-MDCK cells was measured by using two different specific primers, one designed for the HA epitope and the second designed for Hakai. Compared to control DMSO-treated mice, Hakin-1 resulted in a significant reduction in the micrometastases detected in the lungs of Hakai-MDCK xenografted mice, whereas no detection was found in the lungs of MDCK-injected mice (fig. 7 f). This result underscores that Hakin-1 inhibits metastasis to the lung in vivo.

Effect of Hakin-1 analogs on cytotoxicity and Hakai-induced ubiquitination

Next, we investigated the effect of selected analogs on HT29 colon cancer cells. First, we analyzed the cytotoxic effects of the following analogs on HT-29 colon tumor cell line: ketophenyl groups: a-1, A-7, A-8 and A-9; ketoheteroaryl groups: a-23 and A-25; cyclic amides: a-10 and A-16; and benzamides: a-6.1. Show an important inhibitory response of the ketophenyls A-7, A-8, A-9 and ketoheteroaryls A-23, A-25, but no cytotoxic effect was detected by the effects of the ketophenyls A-1, cyclic amides A-10 and A-16, and benzamides A-6.1 (FIG. 12). We then investigated the effect of specifically selected analogues on E3 ubiquitin ligase Hakai induced ubiquitination by using cultured tumor cells. 293T cells were transfected with Src, Hakai and ubiquitin in the presence of specific analogue inhibitors or DMSO as a control. By using the ketophenylated a-7 analogues, a significant decrease in ubiquitination mediated by Hakai was observed in a dose-dependent manner (fig. 13), showing an effect on the decrease in Hakai activity without affecting Hakai protein levels. On the other hand, the analog ketophenyla-9 reduced Hakai-mediated ubiquitination at the concentrations tested, but reduced Hakai protein levels. In addition, the cyclic amides A-10 and A-16 slightly reduced Hakai-mediated ubiquitination without affecting protein levels. Finally, the inhibitory effect of low concentrations of ketoheteroaryl A-23 on Hakai activity was also detected.