WO2019111265A1 - Compounds and methods for treating cancer - Google Patents

Abstract

The present invention relates to novel compounds, compositions and methods of preparation thereof, as well as uses thereof for treating cancer of various types, including: pancreatic, liver, breast, colon, brain and lung cancer.

Description

COMPOUNDS AND METHODS FOR TREATING CANCER

FIELD OF THE INVENTION

[001] The present invention relates to novel compounds, compositions and methods of preparation thereof, as well as uses thereof for treating cancer of various types, including: pancreatic, liver, breast, colon, brain and lung cancer.

BACKGROUND OF THE INVENTION

[002] Cancer is a maj or public health problem worldwide and is the second leading cause of death in the United States. Among the diversity of cancer types, pancreatic adenocarcinoma is one of the most lethal due to its rapid growth and propensity to invade adjacent organs and metastasize.

[003] The treatments for cancer are usually one or more of surgery, radiation, and gene- or drug- based therapy. Nevertheless most of the available drugs today are not really cures, they merely slow down cancer growth, providing a few more months of life and many times with worse symptoms than the cancer itself. Moreover, these drugs and treatments are expensive, in many cases too expensive for many people to afford. Additionally, most cancer treatments are useful against a particular type of cancer.

[004] Ephedra is a genus of gymnosperm shrubs which belongs to the family of Ephedracea with over 30 registered species. As the majority of the gymnosperm plants, instead of fruits, it has bisexual strobilus which serves as food for birds. The leaves are degenerated and located in a sort of reddish membranous sheath on the stem which is green all year round. The various species of Ephedra are widespread throughout many lands. However, they are found in more abundance in southern Europe, Middle West, central Asia and northern China.

[005] A diversity of Ephedra species, especially E. sinica, have traditionally been used by the Chinese for a variety of medical purpose for at least 5000 years (Dewick, P.M.; 2009, Medicinal natural products, a biosynthetic approach, 3^rd edition, Wiley). Last studies have revealed to have anti-invasive, antiangiogenic and antitumor activities of Ephedra sinica extract (Nam, N.H. et al.; Phytother. Res. 2003 ,17, 70-76). Nevertheless, most of the plants from the Ephedra genus contains alkaloids as ephedrine (E) and pseudoephedrine (PE) which have been considered the primary pharmacologically active substances in Ephedra. These alkaloids are known to induce palpitation, hypertension, insomnia, dysuria, bronchodilation, and pronounced effects on the central nervous system (CNS) by binding to adrenergic receptors (Oshima, N. et al.; J. Nat. Med. 2016, 70, 554-562)). Therefore, the administration of dmgs containing Ephedra to patients with cardiovascular-related diseases is severely contraindicated. According to the Food and Drug Administration (FDA) assessment in 2004, food supplements containing E-type alkaloids represent an unacceptable health risk, bearing in mind the conditions of use. Consequently, FDA banned all over the counter dmgs containing ephedrine (Ibragic, S. et ah; Bosn. J. Basic. Med. Sci., 15).

[006] In the last two years, a particular species of the genus Ephedra, namely Ephedra foeminea, draw attention and interest as a potential agent for cancer treatment mainly due to two reasons: a) Surprisingly, no ephedrine alkaloids were identified in its chemical profile composition (Ibragic, S. et al.; Bosn. J. Basic. Med. Sci., 15), b) reports from patients diagnosed with different types of cancer that were apparently healed from their ailments by drinking an infusion prepared with Ephedra foeminea stalks, and a report on reducing various cancerous cells’ viability (Ben-Arye, E.; et al.; J. Cancer Res. Clin. Oncol. 2016, 142(7), 1499-1508; and Mendelovich, M. et al.; J. Med. Pla. Res. 2017, 11(43), 690-702, respectively).

[007] Due to its rapid growth and prognostic to invade adjacent organs and metastasize pancreatic adenocarcinoma is considered one of the most lethal forms of cancer. The early stages of this cancer do not usually produce symptoms, so the disease is generally advanced when it is diagnosed (Lillemoe, K. D. et al.; Ca. Can. J. Clin. 2000, 50, 241-268). It is estimated that ninety-nine percent of patients with adenocarcinoma of the pancreas will die within 5 years of diagnosis (Jemal, A. et al.; Can. stat. 2003, 53, 5-26). Pancreatic cancer is the fourth leading cause of cancer-related death in the United States and is the seventh most common cause of death worldwide (Lillemoe, K. D. et al.; Ca. Can. J. Clin. 2000, 50, 241- 268). Treatment for pancreatic cancer is rarely effective because surgical excision offers the only possibility for a cure, and fewer than 15% of patients are candidates for tumor resection at the time of diagnosis. Chemotherapy provides only a slight survival advantage over surgical resection alone, and radiation therapy has not been demonstrated to be beneficial (Hazard, L.; Gast. Can. Res., 2009, 3, 20-28). More effective treatments are needed to improve the prognosis of patients with pancreatic cancer. The renewed interest in the investigation of biologically active compounds of natural origin in the last decades has led to the introduction of several essential dmgs, especially in the oncologic field. Anticancer substances such as vinblastine, vincristine, and taxol are the main examples among others (Queiroz, E. F. et al.; Contemporary Methodological Apparoaches In The Search For New Lead Compounds From Higher Plants. I.). [008] Accordingly, this invention provides compounds, compositions, their preparation and uses in the treatment of cancer, especially their use in the treatment of pancreatic cancer.

SUMMARY OF THE INVENTION

[009] In one aspect, this invention provides a compound represented by the structure of formula (I): x'-x² -x

n

0) wherein

X¹ is a cinnamic acid moiety or derivative thereof;

X² is an amino acid or an amine linker;

X³ is a hydroxy or polyhydroxy-substituted flavone, flavanone or derivative thereof; and

n is an integer between 1 and 4; or

a pharmaceutically acceptable salt thereof.

[0010] In another embodiment, n is 1-4. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment of this invention, n is 4. Each possibility represents a separate embodiment of this invention.

[0011] In one embodiment, the compound of this invention is selected from:

[0012] In one further aspect, this invention provides a pharmaceutical composition comprising a compound as described hereinabove and a pharmaceutical acceptable carrier.

[0013] In one additional aspect, this invention provides a method of treating a disease or a medical condition in a subject, comprising administering to the subject a therapeutically effective amount of a compound as described hereinabove. In one embodiment, the disease is cancer. In another embodiment, the cancer is pancreatic, liver, breast, colon, brain or lung cancer. In another embodiment, the cancer is pancreatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The subject matter regarded as the invention is particularly pointed out and distinctly claimed 25 in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0015] Figures 1A-1B depict morphology and viability evaluation of Aspcl cells at the end point of the real time cytotoxic detection assay, 32 hours after treatment with compounds 1 or 2. Figure 1A: Representative micrographs before MTT assay; and Figure IB: MTT assay.

[0016] Figures 2 depicts real time cytotoxicity detection assay during the course of 72 hours on Aspcl cells treated with 300 mM of compound 10 (grey) or 2 (black). Measurement of fluorescent signals was calculated from the percentage of the control.

[0017] Figures 3A-3B depict Western blot analysis of protein expression profile of Aspcl cells treated with 150mM of compounds of this invention for 24 hours. Control and treated groups were analyzed in triplicates. Figure 3A: compound 10; and Figure 3B: compound 2.

[0018] Figures 4A-B depict ROS formation in Aspcl cells treated with compound 10 or 2 and then with dichlorofluorescin (DCFH). Aspcl cells were treated with compound 10 or 2 and the fluorescence intensity of dichlorofluorescein (DCF) was measured after 2 hours of incubation. Figure 4A: 24 hours treatment with compound 10 or 2; or Figure 4B: 96 hours treatment with compound 10 or 2. Solid - treatment, diagonal lines - control.

[0019] Figure 5 depicts effect of compound 10 on tumor formation. Treatment with compound 10 started 24 hours after cells seeding (before tumor formation) and persisted for 72 hours. White arrows indicate examples of secondary tumor formation.

[0020] Figure 6 depicts effect of compound 10, irinotectan or carboplatin on tumor formation. Treatment with compound 10, irinotectan or carboplatin started 72 hours after cells seeding (before tumor formation) and persisted for additional 72 hours. Black dots represent necrotic areas.

[0021] Figure 7 depicts effect of compound 2, naringenin and p-coumaric acid on tumor formation. Treatment with compound 2, naringenin and p-coumaric acid started 24 hours after cells seeding (before tumor formation) and persisted for 72 hours. Black dots represent necrotic areas.

[0022] Figure 8 depicts tumor area size of subcutaneous Aspc 1 tumors daily treated with compound 10 (30mg/Kg mice) (T) or treated only with vehicle (C).

[0023] Figure 9 depicts subcutaneous Aspcl tumors removed from nude mice treated with compound 10 after 3 weeks. A necrosis area is observed in the center of treated tumors.

[0024] Figure 10 depicts average necrosis percentage of subcutaneous Aspcl tumors removed from nude mice daily treated with compound 10.

[0025] Figure 11 depicts average percentage of natural killers (NK) cells from blood of nude mice daily treated with compound 10 (Treated) or alternatively with vehicle only (Control).

[0026] Figure 12 depicts tumor area size of subcutaneous A549 tumors daily treated with compound 10 (30mg/Kg mice) (treated) or treated only with vehicle (control).

[0027] Figure 13 depicts average of tumor size area from day 15 of subcutaneous A549 tumors daily treated with compound 10 (30mg/Kg mice) (Treated) or alternatively treated with vehicle only (Control).

[0028] Figure 14 depicts tumor size area of subcutaneous T98G tumors daily treated with compound 10 (30mg/Kg mice) (Treated) or alternatively treated with vehicle only (Control).

[0029] Figure 15 depicts average of tumor size area from day 7 of subcutaneous T98G tumors daily treated with compound 10 (30mg/Kg mice) (Treated) or alternatively treated with vehicle only (Control).

[0030] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of this invention

[0031] In one aspect, this invention provides a compound represented by the structure of formula (I): x'-x² - X³

n

wherein

X¹ is a cinnamic acid moiety or derivative thereof;

X² is an amino acid or an amine linker;

X³ is a hydroxy or polyhydroxy- substituted flavone, flavanone or derivative thereof; and

n is an integer between 1 and 4; or

a pharmaceutically acceptable salt thereof.

[0032] In another embodiment, n is 1-4. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment of this invention, n is 4. Each possibility represents a separate embodiment of this invention.

[0033] In another embodiment,“X'-X²” is attached to X³ in one point of attachment (n=l). In another embodiment, “X'-X²” is attached to X³ in more than one point of attachment (n>l). In another embodiment,“C'-c²” j_s attached to X³ via ester bond ( -C(0)0- ). In another embodiment,“X'-X²” is attached to X³ via ether bond ( -CH2-O- ).

[0034] In some embodiments, X¹ within formula (I) of the compound of this invention is represented by the structure of formula (Ila):

wherein

R'-R³ are each independently H, OH, OR, F, Cl, Br or I; and

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group.

[0035] In another embodiment, X¹ is represented by the structure of formula (lib):

wherein

R ' -R³ are each independently H, OH, OR, F, Cl, Br or I; and

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group.

[0036] In another embodiment, X¹ is represented by the structure of formula (He):

[0037] In some embodiments, X¹ of the compound of this invention is any derivative of formula IIC (i.e. p-coumaric acid) .

[0038] In some embodiments, the term“amino acid” of X² within formula (I) of the compound of this invention is herein defined as any compound having at least one N-terminal (amino moiety, NH2/NH₃ ⁺ depending on pKa/pH) and one C-terminal (carboxyl moiety, CO2H/CO2 depending on pKa/pH). In one embodiment, the amino acid of X² within formula (I) of the compound of this invention is standard or non standard. In another embodiment, non-limiting examples of the amino acids consist of:

glycine, serine, leucine, tyrosine, threonine, alanine, 5-aminovaleric acid, 2-aminocaprylic acid, carnosine, arginine, histidine, lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, selenocysteine, proline, valine, isoleucine, methionine, phenylalanine, tryptophan, gama- aminobutyric acid, carnitine, levo thyroxine, hydroxyproline, selenomethionine, hypusine, 2- aminoisobutyric acid, ornithine, citrulline, and b-alanine. In another embodiment, the amino acid is selected from glycine, serine, leucine, tyrosine, threonine, alanine, 5-aminovaleric acid, 2- aminocaprylic acid, carnosine. Each possibility represents a separate embodiment of this invention.

[0039] In some embodiments, X² of the compound of this invention is any derivative of the amino acids as described hereinabove.

[0040] In some embodiments, X² of the compound of this invention is an amine linker. In another embodiment, the amine is alkyl amine or dialkylamine. In another embodiment, one of the carbon atoms of the alkyls within the alkylamine or dialkylamine is oxygen or nitrogen. In another embodiment, non limiting examples for amines are selected from methylamine, ethylamine, propylamine, butylamine, dimethylamine, diisopropylamine, diethylamine, N-ethylethylenediamine and 2-ethoxyethaneamine. Each possibility represents a separate embodiment of this invention.

[0041] In some embodiments, X² of the compound of this invention is any derivative of the amines as described hereinabove.

[0042] In some embodiments, X³ within formula (I) of the compound of this invention is represented by the structure of formula (Ilia):

wherein

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, NO₂, CF3 or an oxygen atom which is directly attached to X²;

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

at least one of R⁴-R¹³ is an oxygen atom which is directly attached to X²; and

is a single or a double bond. In another embodiment,“

is a single bond. In another

embodiment,“

is a double bond.

[0043] In another embodiment, X³ is represented by the structure of formula Illb, IIIc, Hid, Hie, Illf or Illg:

(lllf) ; or (mg)

Each possibility represents a separate embodiment of this invention.

[0044] In some embodiments, non- limiting examples of hydroxy or polyhydroxy-substituted flavone or flavanone of X³ of the compound of this invention consist of: apigenin, acacetin, genkwanin, wogonin, norwogonin, tangeretin, 3-hydroxyflavone, kaempferol, morin, quercetin, rhamnazin, butin, hesperetin, naringenin and sterubin. Each possibility represents a separate embodiment of this invention. [0045] In one embodiment, the compound of this invention is selected from:

Each possibility represents a separate embodiment of this invention.

In another embodiment, the compound of this invention is selected from:

Each possibility represents a separate embodiment of this invention.

Definitions

[0046] In some embodiments, R ¹ - R^¾ of formulae Ila and lib are each independently H, OH, OR, F, Cl, Br or I. In another embodiment, R¹, R² and/or R³ are each independently H. In another embodiment, R¹, R² and/or R³ are each independently OH. In another embodiment, R¹, R² and/or R³ are each independently OR. In another embodiment, R¹, R² and/or R³ are each independently F. In another embodiment, R¹, R² and/or R³ are each independently Cl. In another embodiment, R¹, R² and/or R³ are each independently Br. In another embodiment, R¹, R² and/or R³ are each independently I. Each possibility represents a separate embodiment of this invention.

[0047] In some embodiments, R⁴-R¹³ of formula Ilia are each independently H, OH, R, OR, F, Cl, Br, I, NO2, CF3 or an oxygen atom which is directly attached to X². In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently H. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently OH. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently R. In another embodiment R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently OR. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently F. In another embodiment R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently Cl. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently Br. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently I. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently NO2. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently CF3. In another embodiment, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ are each independently an oxygen atom which is directly attached to X² _. Each possibility represents a separate embodiment of this invention.

[0048] In some embodiments, R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a Ci-Cs substituted or unsubstituted cycloalkyl group. In another embodiment, R is Ci-Cs linear or branched, substituted or unsubstituted alkyl group. In another embodiment, R is C-Cs substituted or unsubstituted cycloalkyl group. _. Each possibility represents a separate embodiment of this invention.

[0049] As used herein, the term“alkyl” group refers, in some embodiments, to any straight- or branched-chain alkyl group containing up to about 30 carbons unless otherwise specified. In some embodiments, an alkyl includes C1-C5 carbons. In some embodiments, an alkyl includes Ci-C₆ carbons. In some embodiments, an alkyl includes Ci-Cs carbons. In some embodiments, an alkyl includes C1-C10 carbons. In some embodiments, an alkyl is a C1-C12 carbons. In some embodiments, an alkyl is a C1-C20 carbons. In some embodiments, branched alkyl is an alkyl substituted by alkyl side chains of 1 to 5 carbons. In some embodiments, the alkyl group may be unsubstituted. In some embodiments, the alkyl group may be substituted by F, Cl, Br, I, CF₃, OH, O(alkyl) (i.e. alkoxy), CHO, C(0)NH₂, C(0)NH(alkyl), C(0)N(alkyl)₂, CN, NO2, CO2H, NH₂, NH(alkyl), N(alkyl)₂, SH, and/or S(alkyl).In another embodiment, the alkyl group is methyl. In another embodiment, the alkyl group is ethyl. In another embodiment, the alkyl group is propyl. In another embodiment, the alkyl group is butyl. In another embodiment, the alkyl group is t-Butyl. In another embodiment, the alkyl group is neopentyl. In another embodiment, the alkyl group is isopropyl. In another embodiment, the alkyl group is isobutyl. _.Each possibility represents a separate embodiment of this invention.

[0050] As used herein, the term“cycloalkyl” group refers, in some embodiments, to a ring structure comprising carbon atoms as ring atoms, which may be either saturated or unsaturated, substituted or unsubstituted, single or fused. In some embodiments the cycloalkyl is a 3-8 membered ring. In some embodiments the cycloalkyl is a 3-10 membered ring. In some embodiments the cycloalkyl is a 3-12 membered ring. In some embodiments the cycloalkyl is a 6 membered ring. In some embodiments the cycloalkyl is a 5-7 membered ring. In some embodiments the cycloalkyl is a 3-8 membered ring. In some embodiments, the cycloalkyl group may be unsubstituted or substituted by F, Cl, Br, I, CFs, OH, O(alkyl) (i.e. alkoxy), CHO, C(0)NH₂, C(0)NH(alkyl), C(0)N(alkyl)₂, CN, N0₂, C0₂H, NH₂, NH(alkyl), N(alkyl)₂, SH, and/or S(alkyl).In some embodiments, the cycloalkyl ring may be fused to another saturated or unsaturated cycloalkyl. In some embodiments, the cycloalkyl ring is a saturated ring. In some embodiments, the cycloalkyl ring is an unsaturated ring. In another embodiment, the cycloalkyl ring is cyclohexyl. In another embodiment, the cycloalkyl ring is cyclohexenyl. In another embodiment, the cycloalkyl ring is cyclopropyl. In another embodiment, the cycloalkyl ring is cyclopropenyl. In another embodiment, the cycloalkyl ring is cyclopentyl. In another embodiment, the cycloalkyl ring is cyclopentenyl. In another embodiment, the cycloalkyl ring is cyclobutyl. In another embodiment, the cycloalkyl ring is cyclobutenyl. In another embodiment, the cycloalkyl ring is cycloctyl. In another embodiment, the cycloalkyl ring is cycloctadienyl (COD). In another embodiment, the cycloalkyl ring is cycloctaene (COE). Each possibility represents a separate embodiment of this invention.

[0051] In some embodiments, this invention provides compounds of formula I in the form of salts, in particular base salts. Suitable salts include those formed with both organic and inorganic bases. Such base addition salts will normally be pharmaceutically acceptable although non- pharmaceutically acceptable salts may be of utility in the preparation and purification of the compounds in question.

[0052] As used herein, the term’’derivative” refers to compound which can be provided based on a previous compound (which is derived from) following a few known synthetic steps; e.g. 1-3 steps selected from: alkylation reactions, nucleophilic, electrophilic or aromatic substitution reactions, acid/base reactions, aldol reaction and similar condensations, esterification, trans-esterification, animation, amidation, hydrolysis, Schiff base/imine/enamine formation reactions etc.

Methods of preparation

[0053] In one further aspect, this invention provides methods for preparing the compounds as described hereinabove. In one embodiment, a method for the preparation of compound of formula (I), beginning from compound of formula (IVa), is illustrated below in Scheme 1.

Scheme 1 : Preparation of compound of formula (I) from (IVa).

DCC is dicyclohexylcarbodiimide;

NHS is N-hydroxysuccinimide;

R'-R³ is H, OH, OR, F, Cl, Br or I;

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, N0₂ or CF₃;

R is a Ci-C₈ linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

n is an integer between 1 and 4;

at least one of R⁴-R¹³ is OH; and is a single or a double bond. In another embodiment,“■” is a single bond. In another

embodiment,“

” is a double bond.

[0054] In another embodiment, a method for the preparation of compound of formula (I), beginning from compound of formula (Va), is illustrated below in Scheme 2.

Scheme 2: Preparation of compound of formula (I) from (Va).

DCC is dicyclohexylcarbodiimide;

NHS is N-hydroxysuccinimide;

RfrR³ is H, OH, OR, F, Cl, Br or I;

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, N0₂ or CF₃; R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

n is an integer between 1 and 4;

at least one of R⁴-R¹³ is OH; and a ·_>·_> is a single or a double bond. In another embodiment, a

·_>·_> is a single bond. In another

embodiment,“

” is a double bond.

[0055] In another embodiment, a method for the preparation of compound of formula (I), beginning from compound (a), is illustrated below in Scheme 3.

Scheme 3: Preparation of compound of formula (I) from a.

0

HO^'

Step I Step 2

(a)

(b)

wherein

H₂N— <— CO₂H

5 is an amino acid;

DCC is dicyclohexylcarbodiimide; NHS is N-hydroxysuccinimide;

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, NO2 or CF₃;

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

n is an integer between 1 and 4;

at least one of R⁴-R¹³ is OH; and a

g . , ·_>·_> is a single bond. In another

embodiment,“

” is a double bond.

[0056] In another embodiment, a method for the preparation of compound of formula (I), beginning from compound of formula (IVa), is illustrated below in Scheme 4.

is an amine substituted with LG, wherein LG is Cl, Br, I, mesylate, tosylate or triflate; DCC is dicyclohexylcarbodiimide; NHS is N-hydroxysuccinimide;

R'-R³ is H, OH, OR, F, Cl, Br or I;

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, NO2 or CF₃;

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

n is an integer between 1 and 4;

at least one of R⁴-R¹³ is OH; and

g . , is a single bond. In another

embodiment,“

” is a double bond.

[0057] In another embodiment, a method for the preparation of compound of formula (I), beginning from compound of formula (Va), is illustrated below in Scheme 5.

Scheme 5: Preparation of compound of formula (I) from (Va).

wherein

is an amine substituted with LG, wherein LG is Cl, Br, I, mesylate, tosylate or triflate;

DCC is dicyclohexylcarbodiimide;

NHS is N-hydroxysuccinimide;

RhR³ is H, OH, OR, F, Cl, Br or I;

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, N0₂ or CF₃;

R is a Ci-C₈ linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

n is an integer between 1 and 4;

at least one of R⁴-R¹³ is OH; and

is a single or a double bond. In another embodiment,“

is a single bond. In another embodiment,“

” is a double bond.

[0058] In another embodiment, a method for the preparation of compound of formula (I), beginning from compound (a), is illustrated below in Scheme 6.

Scheme 6: Preparation of compound of formula (I) from (a).

x^l-x² X³

11

0) wherein

is an amine substituted with LG, wherein LG is Cl, Br, I, mesylate, tosylate or triflate;

DCC is dicyclohexylcarbodiimide;

NHS is N-hydroxysuccinimide;

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, N0₂ or CF₃;

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

n is an integer between 1 and 4;

at least one of R⁴-R¹³ is OH; and is a single or a double bond. In another embodiment,

is a single bond. In another

embodiment,“

” is a double bond.

[0059] In some embodiments, a solvent(s) is/are employed in the methods which are described in Schemes 1-6. In another embodiment, any solvent as known in the art can be employed. In another embodiment, non limiting examples of solvents include: water, tetrahydrofuran (THF), acetonitrile, sulfolane, diethyl ether, N,N-dimethylformamide (DMF), dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), dioxane, ethanol, methanol and any combination thereof. Each possibility represents a separate embodiment of this invention. In another embodiment, the solvent is water. In another embodiment, the solvent is tetrahydrofuran (THF).

[0060] In some embodiments, a base is employed in step 2 of the methods which are described in schemes 1-3. In another embodiment, any base as known in the art can be employed. In another embodiment, non limiting examples of bases include: Na CCh, NaHCCh, K2CO3, KHCO3, LbCCh, LiHC(¾, NaOH, LiOH, KOH and any combination thereof. Each possibility represents a separate embodiment of this invention. In another embodiment, the base is Na₂C0₃.

[0061] In some embodiments, steps 1-4 of the methods which are described in scheme 1-3 are performed without an excess of employed reagent (compared to reactant). In on embodiment, an excess of reagent is used. In another embodiment, an excess of between 5-150% is used. In another embodiment, an excess of between 5-15% is used. In another embodiment, an excess of between 15- 30% is used. In another embodiment, an excess of between 30-50% is used. In another embodiment, an excess of between 50-75% is used. In another embodiment, an excess of between 75-100% is used. In another embodiment, an excess of between 100-125% is used. In another embodiment, an excess of between 125-150% is used. In another embodiment, an excess of 30% is used. Each possibility represents a separate embodiment of this invention.

[0062] In another embodiment, a method for the preparation of compound (1) beginning from compound (a), is illustrated below in Scheme 7.

Scheme 7: Preparation of compound ( 1) from (a).

ld)

m

wherein

DCC is dicyclohexylcarbodiimide;

NHS is N-hydroxysuccinimide;

THF is tetrahydrofuran; and

DMF is N,N-dimethylformamide.

Pharmaceutical compositions

[0063] In one further aspect, this invention provides a pharmaceutical composition comprising a compound of this invention as described hereinabove and a pharmaceutical acceptable carrier.

[0064] In some embodiments, the formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend upon the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of this invention (an "active ingredient") with the carrier which constitutes one or more suitable diluents, adjuvants, excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions. Each possibility represents a separate embodiment of this invention.

[0065] In one embodiment, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation. In another embodiment, the composition may be prepared in the form of a ready-made drink, or as a powder that can be combined with hot water and then ingested like a tea by an individual. Each possibility represents a separate embodiment of this invention.

[0066] In some embodiments, formulations of the compound of this invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non- aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Each possibility represents a separate embodiment of this invention.

[0067] In some embodiments, a tablet may be made by compression or molding, optionally with one or more carriers. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein. Each possibility represents a separate embodiment of this invention.

[0068] In some embodiments, formulations for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose of multi- dose containers, for example sealed ampoules and vials, and may be stored in a freeze- dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Each possibility represents a separate embodiment of this invention.

[0069] In some embodiments, formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.

[0070] In some embodiments, formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia. Each possibility represents a separate embodiment of this invention.

[0071] In one embodiment, preferred unit dosage formulations are those containing an effective dose, as hereinbelow recited, or an appropriate fraction thereof, of the active ingredient.

Uses

[0072] In one aspect, this invention provides a method of treating a disease or a medical condition in a subject, comprising administering to the subject a therapeutically effective amount of a compound of this invention, as described hereinabove. In one embodiment, the method comprises administering more than one compound of this invention. In one embodiment, the disease is cancer. In another embodiment, the cancer is pancreatic, liver, breast, colon, brain or lung cancer. In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is liver cancer. In another embodiment, the cancer is breast cancer. In another embodiment, the cancer is colon cancer. In another embodiment, the cancer is brain cancer. In another embodiment, the cancer is lung cancer. In another embodiment, the disease is a hyper-proliferative and/or a neoplastic disease. In another embodiment, "treating" is alleviating a neoplastic or cancerous symptoms and/or disturbances. In another embodiment, "treating" is inhibiting tumor growth. In another embodiment, "treating" is reducing tumor size. In another embodiment, "treating" is destroying the tumor. In another embodiment, "treating" is inhibiting malignancy. Each possibility represents a separate embodiment of this invention.

[0073] In one additional aspect, this invention provides one or more compounds to be used in the methods as described above. In one embodiment, the compound is utilized as a medicament. In another embodiment, the compound is used as a chemotherapeutic agent, targeted at a cancerous cell, a neoplastic cell, a malignant cell or any combination thereof. In another embodiment, the chemotherapeutic agent has low toxicity to non-malignant tissues. In another embodiment, the compound has a chemotherapeutic effect. Each possibility represents a separate embodiment of this invention.

[0074] As used herein, subject or patient refers to any mammalian patient, including without limitation, humans and other primates, dogs, cats, horses, cows, sheep, pigs, rats, mice, and other rodents. In some embodiments, the subject is male. In some embodiments, the subject is female. Each possibility represents a separate embodiment of this invention. In some embodiments, while the methods as described herein may be useful for treating either males or females.

[0075] When administering the compounds of the present invention, they can be administered systemically or, alternatively, they can be administered directly to a specific site where cancer cells or precancerous cells are present. Thus, administering can be accomplished in any manner effective for delivering the compounds or the pharmaceutical compositions to the cancer cells or precancerous cells. Exemplary modes of administration include, without limitation, administering the compounds or compositions orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. Each possibility represents a separate embodiment of this invention.

[0076] In some embodiments, the compounds of this invention shows strong biological activity by significant reducing viability of many different cancer cell lines where the human pancreatic Aspcl cells present the most sensitive behavior. In one embodiment, in vitro results (see examples below, e.g. Example 5) show that the compounds are able to significantly change the expression pattern of key proteins in cancer metabolism such as GLUT-4 receptor and E-cadherin. GLUT-4 is responsible for the glucose uptake which was showed to be impaired in treated cells generating a significant stress response. E-cadherin upregulation impairs cancer cells migration and invasion of the surrounding tissues (i.e. metastasis). Moreover, in vivo tests (see e.g. Example 8 below) performed on nude mice daily treated with compounds of this invention showed a significant increase of NK (natural killer) cells production which are very functional in the process of tumor regression. By presenting these features, NK cells inhibit angiogenesis process. It is therefore contemplated, without being bound to any mechanism, that treated cells remain attached in situ lacking essential nutrients for normal growth and recede through necrosis. It is noteworthy that the involvement of NK cells and therefore the immune system to combat cancer is just one additional feature of this small molecule which behaves as a target therapy.

[0077] In some embodiments, without being bound by any mechanism or theory, the compounds of this invention could be classified as belonging to a class of chemotherapeutic compounds known as antimetabolites, which are defined as molecules able to interfere with mechanisms related to cell growth and replication and their efficacy as anticancer agents has been widely demonstrated (Shewach, D. S. et al.; Chem Rev. 2009, 109, 2859-2861; and Mihlon, F. et al.; Semin. Intervent. Radiol. 2010, 27, 384-390). Antimetabolite compounds can induce ligands for NK cell-activating receptors in mammalian tumor cells and promote NK cell-mediated tumor cytotoxicity (e.g. increased expression of the NK-stimulating ligand NKG2D on tumor cells, leading to enhanced NK lysis of tumors: Romagne, F. et al.; Blood, 2009, 114, 2667-2677). In one embodiment, compound 10 may also be inducing the expression of NK receptors ligands, which can explain the significantly increased amount of NK cells in treated mice serum (e.g. Example 8, Figure 11). Nonetheless, the mechanisms involved in the induction of ligands for NK cell- activating receptors are only partially understood.

[0078] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention.

EXAMPLES EXAMPLE 1

Preparation of compound (1)

Step 1

Chemical Formula: CgHgO , Chemical Formula: CigH^NOg

THF, rt

Exact Mass: 164.05 Exact Mass: 261.06

a b

[0079] Compound a was dissolved in tetrahydrofuran (THF) in a ratio of 70 ml for each lg followed by addition of N-hydroxysuccinimide (NHS) and N,N’-dicyclohexylcarbodiimide (DCC) both in an equivalence of 1 :3. The reaction mixture was stirred overnight at room temperature. After filtration and evaporation, THF was added, and the mixture was kept at 4°C for 5 hours. The mixture was then filtered and evaporated until dryness to afford b, in 95% yield.

Step 2

[0080] Compound b was dissolved with THF and added to a solution with glycine and sodium bicarbonate in water. The reaction mixture was stirred overnight at room temperature. Work up was performed by evaporating THF followed by acidification (HC1 1N) of the mixture (pH2) and further extraction with Ethyl acetate (4 times: 3x150 ml + lxlOOml), to afford c, in 95% yield.

Steps 3 and 4

[0081] Compound c, NHS (N-hydroxysuccinimide) and DCC (N,N’-dicyclohexylcarbodiimide) (1: 1.3: 1.3) were dissolved in THF or DMF and stirred for 4 hours at room temperature. The reaction mixture was filtered, rinsed with THF or DMF and evaporated to dryness to afford compound d, which was then redissolved with THF or DMF before the addition of an aqueous solution of bicarbonate-containing a polyphenol. The reaction mixture was stirred overnight at room temperature. Work up was performed by evaporating THF followed by acidification (HC1 1N) of the mixture (pH 2) and further extraction with Ethyl acetate (4 times: 3x150 ml + lxl00ml).The combined organic phases were evaporated to dryness. The crude mixture was then firactioned on flash column with Water:MeOH:AcOH (5:95:0.1) to afford compound 1 with 20% yield.

EXAMPLE 2

General methods and techniques Experimental protocols of cell culture and treatment

[0082] Adherent Human carcinoma cell lines were cultured according to standard mammalian tissue culture protocols and sterile technique. Caco2, HepG2, MCF-7 cell lines were cultured in high glucose Dulbecco's Modified Eagle Medium, HT29 cells were cultured in McCoy's 5 A medium, Aspcl, A549, Mia Paca, U87MG and T98MG cells line were cultured in RPMI medium 1640 and HFF was culture in BIOAMF media. All media was supplemented with 10% fetal bovine semm, streptomycin (100 mg/ml), penicillin (100 U/ml) and Nystatin (12.5 U/ml). Cells were incubated in 5% C02 at 37 °C. All tissue culture were maintained in 25cm^Nunc^^ cell culture treated EasYFlask^M (ThermoFisher scientific) and all the media and supplements were obtained from Biological Industries. Treatment were performed by plating cells in a Nunc^M 95 micro _well delta surface plates (ThermoFisher scientific) in a starting confluence of 1 x 10^ cells/well. After 24h of incubation, the compound of interest was added in different concentrations as shown in the presented results.

MTT assay

[0083] Viability of the cells following treatment was determined using a commercially available MTT assay kit (ABCAM, abl46345) and performed according to manufacturer's instmctions.

Briefly, cells were seeded in a 96-well plate at a density of I x I O^cells/well (n=4). After overnight plating, cells were exposed to varying concentrations of the tested compound (50-500 mM). Then, plates were incubated in a humidified atmosphere containing 5% C02 +in air at 37°C for 24 hours. According to the MTT standard protocol, after 24, 48 or 72 h treatment, the media was removed and all cells were incubated with semm-free media containing 0.5 mg/ml MTT for 4 hours in the incubator. The MTT purple crystals formed by the viable cells were diluted using isopropanol containing 0.04 mol/F HC1. The quantification was determined by measuring the optical density at 570 nm in an enzyme-linked immunosorbent assay (Spark, Tecan) reader. Data was presented as proportional viability (%) by comparing the group treated with the tested compound with the untreated cells, the viability of which is assumed to be 100%.

Spheroids assembly and viability [0084] The spheroid formation potential of HT29 human colon cancer cell line was evaluated in 3D nonadherent culture condition. Initially the HT29 human cells were grown as a monolayer after which they were counted, re- suspended and plated with 1,500 cells per well in a 96-well plate pre-coated with a thin layer of 1.5% agarose (w/v) in McCoy's 5A medium containing 10% FBS and incubated at 37°C in a humidified atmosphere of 5% C02. Cell culture media was replaced every 2-3 days with fresh medium to remove cellular debris and the spheroids that were not well-formed. After the beginning of the multicellular tumor spheroid formation, the tested compound was added at various concentrations (0.2, 0.4 and 0.6 mg/ml) and incubated for 24, 48 and 72 h. The number and diameter of colonies within each well were photographed and counted every day under the microscope (Olympus BX-51 ; Olympus, Hamburg, Germany), and the images of the representative fields were captured. Each sample was analyzed in triplicate, and all the experiments were performed three times. The viability of cells growing on the spheroids was measured by the APH (acid phosphatase) assay, which was performed according to manufacturer's instructions.

Protein extraction and Western blot analysis

[0085] Whole cell lysate was prepared by washing cells pellets with IX Phosphate buffer saline (Biological Industries), resuspending it in ice cold T lysis buffer [50mM Tris-Cl (pH 7.5), l50mM

NaCl, lmM EDTA, 1% Triton X and IX halt^M p_rotease and phosphatase inhibitor cocktail] and incubating for 30 minutes in ice. The lysate was centrifuged at l3,800g for 10 min at 4^C to clear the cellular debris. Total protein was quantified using the Bradford protein assay kit (Biorad,

Hercules, CA). Equal amount of protein was resolved on precast Bolt^M 4_12% Bis-Tris Plus polyacrylamide gel (Invitrogen), electro- transferred to precast nitrocellulose stacks using iBlot®2 system (Invitrogen) and western blot analysis was performed using the antibodies described above. Immuno-detection was performed by blocking the membranes for lh in TNT buffer [10 mM Tris- Cl(pH 7.5), 150 mM NaCl, 0.05% Tween-20] containing 5% powdered non-fat milk followed by addition of the primary antibody (as indicated) in TNT for 2h at room temperature. Specifically bound primary antibodies were detected with peroxidase-coupled secondary antibodies and developed by enhanced chemiluminescence (Biological Industries) according to manufacturer’s instructions and quantitated using ImageQaunt LAS 4000 mini, General Electric). All experiments were performed at least three-five times using independent biological replicates. Quantitative real-time PCR

[0086] Quantitative polymerase chain reaction (PCR) was carried out using the ABI 7700 instrument from Applied Biosystems, according to the manufacturer’s instructions. Human PPARy Taqman probe and primers (cat# HsOl l l55l3_ml), human GAPDH Taqman probe and primers (cat# Hs99999905_ml) and human GLUT-4 taqman probe and primers (cat# Hs00l68966_ml) were purchased from Applied Biosystems. Briefly, the reaction conditions consisted of 3 mΐ of cDNA and IX Taqman assay primers in a final volume of 20 mΐ of supermix. The cycle conditions for real-time PCR were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 1 min. The experiment was performed by three independent experiments with triplicate.

Immunoblot analysis

[0087] Immunoblot analysis was performed using antibodies against GAPDH (1:6000 dilution; abl289l5, Abeam), E-cadherin (1: 10000 dilution; ab40772, Abeam), GLUT-4 (1:1000 dilution; G4048, SIGMA), and then species-specific HRP-labeled secondary antibodies were added. The blots were visualized using enhanced chemiluminescence (biological industries) and quantitated using ImageQaunt LAS 4000 mini, General Electric).

Subcutaneous tumor implantation

[0088] All the experiments were performed under strict compliance and regulations. 3-4 x 10⁶ AsPC- 1, A549 and T98G cells in 1: 1: 18 mixture of Methanol, Tween ® 20 and saline were implanted in right and left flank of mice respectively. Once tumor volume was around 0.25 cm², mice were randomly divided into two groups with 4 mice in each group. Group I served as control and received the vehicle only whereas Group II received 30 mg/kg of the tested compound by peritoneal (i.p) injection. Tumor area was measured twice a week until day 24. At day 24, mice were humanely sacrificed, and tumors were removed aseptically. Solid tumors were harvested in buffered formalin for further molecular and histological analyses.

2-DG uptake assay

[0089] After treatment with the tested compound, cells were starved in low-glucose DMEM

(Dulbecco's Modified Eagle Medium) with 0.5% semm for 16 h; cells were washed twice with 37°C Krebs-Ringer phosphate (KRP) buffer (pH 7.4) (128 mM NaCl, 4.7 mM KCl,T65 mM CaCT, 2.5 mM MgS0₄, and 5 mM Na₂HP0₄). Glucose uptake was started by addition of 1 mM 2-deoxy-D- glucose (2-DG) for an additional 20 min at 37°C. Cells were gently washed three times with ice-cold DPBS and lysed. Sample was assayed using the Colorimetric Glucose Uptake Assay kit from Abeam (Cambridge, United Kingdom). Measurements were performed at least three replicates and then averaged.

Cell viability

[0090] Tested compounds were evaluated for their ability to influence on cell viability and proliferation. A broad screening was performed using ten different cell lines as described in table 1, and four different concentrations were used (37.5, 75, 150 and 300mM) to treat cells for 24 hours and determine the proliferation inhibitory profile of each novel chemical entity.

EXAMPLE 3

Assessment of cell viability following treatment with compounds of this invention

Table 1: viability of cells treated with specific, indicated molecules

^* Asterisk points to high biologic active molecules. In this case 35mM of the indicated molecules was used only instead of 150mM. Emphasized, underlined boxes represent viability values under 70%.

^■ compound 13

[0091] Biologically, compound 13 showed high specificity against Aspcl cells by reducing cell viability to 42% using only 35 mM for 24 hours. Further viability assays are ongoing to evaluate the response of other cell lines.

^■ compound 10

[0092] This molecule showed a substantial effect in decreasing cell viability on pancreatic (Aspcl) and lung (A549) cancer cell lines. A concentration of 150mM reduced cell viability by around 50% in both cell lines. Additionally, compound 10 was capable to significantly reduce cell viability of MiaPaca

(61% with 150mM), another type of pancreatic cells and T98G cells (51.5% with 150mM) which are derived from a relatively resistant brain tumor. Normal human skin/foreskin-derived fibroblasts (HFF) were barely influenced by compound 10 (around 80% with 150mM), indicating the specificity of this molecule in treating cancer cells.

^■ compound 2

[0093] Regarding the structure of this chemical entity, the polyphenol naringenin was linked to p- coumaric acid by glycine as opposed to the previous molecule, compound 2, which is made up by linkage of quercetin in the polyphenol position and aminovaleric acid as the amino acid moiety. Despite the structure differences, compound 2 showed similar effects in Aspcl cells (56%) and a moderate effect in A549 cells (71%) when treated at a 150mM concentration. No significant effect was observed in MiaPaca and T98G brain cell lines (Table 1). Nonetheless, this molecule showed significant effects in reducing cell viability of a mammary gland/breast (MCF-7), a colorectal (FIT-29) and a liver (HepG2) cancer cell lines, with 66.5%, 57.5% and 48.5% of viability respectively when treated with a concentration of 150mM. Compound 2 can exert its effect on a broad range of cancer cell lines without influencing the viability of normal cell lines as HFF (81% with 150mM).

^■ compound 1

[0094] Compound 1 is structurally very similar to compound 2, the only difference between these compounds are two hydroxyl moieties (OH) as compound 1 has quercetin in the polyphenol position instead of Naringenin (see compounds’ stmcture described hereinabove). This small modification barely alters the decrease in viability of Aspcl cells when comparing the treatment with compound 2 (56%) to compound 1 (62.5%). However, this modification grants to this novel entity a high specificity to HepG2 cells, leading to a remarkable reduction in cell viability (21%) when these cells were treated with 150mM of compound 1 for 24 hours in comparison to healthy cells (HFF) where there was almost no effect (90% of viability).

^■ compound 8

[0095] Compound 8 is composed of quercetin linked to p-coumaric acid by a long chain molecule namely aminocapryic acid (see structure defined hereinabove). This compound was effective in reducing viability in Aspcl cells (52% viability with 150mM) with similar strength as the other molecules mentioned above (1, 2 and 10). Nonetheless, this novel entity was able to significantly reduce cell viability (62%) in U87MG, another brain cancer cell line.

^■ Compound 11

[0096] Compound 11 is structurally similar to compound 10. Instead of quercetin in the polyphenol position, compound 11 has a molecule of Naringenin (see structure defined hereinabove). Despite the high similarity, it was only able of reducing cell viability in Aspcl cells in a very moderate fashion (80% viability with 150mM). Regarding the other entities shown in table 1 (compounds 3-6 and 9), no significant results in cell viability were observed.

EXAMPLE 4

A real time cytotoxicity detection assay of Aspcl treated with compounds 2 or 10 To better evaluate the anticancer effect of the novel entities, a real time cytotoxicity detection assay was performed during the course of 72 hours on Aspcl cells after treatment with compounds 2 or 10. As elucidated above, Aspcl cells treated with compounds 2 or 10 for 24 hours at various doses (18-300mM) results in a significant decrease on cell viability in a time and dose-dependent manner, nonetheless, apoptotic bodies were not identified by microscopy analysis (Figure 1A), indicating that probably treated cells are inducing cell cycle arrest and stopping proliferation instead of inducing death by apoptosis.

[0097] Aspcl cells were incubated with a non-toxic dye that binds to the DNA of died or damage cells. After 24 hours of incubation, cells were treated with compound 1 or 2. The effect observed in cells treated with compound 10 was immediately, after 30 minutes of treatment the measurement of the fluorescence of died cells achieved its maximum level and persists constant until the end of the assay (70 hours after cells seeding) (Figure 2, grey). Compound 2 exhibits a similar pattern only that its effect was more delayed and moderate (Figure 2, black).

[0098] MTT viability assay was performed with the cells at the end point of the real-time cytotoxic detection assay in order to confirm the results (Figure 1B). The level of the fluorescence signal was correlated with the viability level. EXAMPLE 5

Protein expression patterns in GLUT-4 receptor and E-cadherin

[0099] Chemical structure of the new compounds revealed strong similarity to the known liavonoids quercetin and kaempferol. It has been suggested that a transport inhibition mechanism in which flavonoids and GLUT4 (glucose transporter) interact directly, rather than by a mechanism related to protein-tyrosine kinase and insulin signaling inhibition (Strobel, P.; et ak; Biochem. J. 2005, 386, 471-478). This direct interaction of flavonoids with GLUT4 occurs at the same residues as glucose, thus inhibiting the glucose uptake into the cells. Indeed, Aspcl cells showed a significant decrease in GLUT-4 protein expression after treatment with compound 10 (Figure 3A). In contrast, GLUT-4 expression was upregulated in cells treated with compound 2 (Figure 3B). It is worth to note that these molecules are clearly influencing GLUT-4 regulation.

[00100] The small GTPases of the RHO subfamily (Rho, Rac, and Cdc42) are signaling molecules that are primarily involved in several critical cellular processes, including cell proliferation, motility, and invasion (Bishop, L. A. et al.;Biochem. J. 2000. 348 part 2, 241-255). Recent reports revealed that the activity of RHO protein could be modulated by a functional interaction with catenin pl20, which is found in cytosolic pools. Moreover, it was demonstrated that cadherins could titrate out pl20 molecules from the cytosol, reducing their ability to affect RHO signaling (Anastasiadis, P. Z. et al. J. Cell Sci. 2000, 113 pt. 8, 1319-1334). Treating Aspcl with the new compounds resulted in similar results compared to GLUT-4 expression, regarding the expression pattern of E-cadherin. In all the cases was observed a significant upregulation in comparison to control cells (Figure 3A-B).

[00101] The results presented herein show that incubation of Aspcl cells with compound 2 or 10 for 24 h generated a significant increase in E-cadherin expression (Figures 3A-B). Therefore, it is inferred that this upregulation may impair Aspc 1 cancer cell invasion and migration by preventing the interaction between pl20 and RHO proteins. Further in vivo experiments (Example 8) with the new compounds have proven their ability to inhibit cancer migration and invasion observed by the increased necrotic areas in treated groups (Figure 9). As the cells were unable to migrate and invade the surrounding tissue, they remained attached in situ, lacking essential nutrients for normal growth and therefore inducing death by necrosis. EXAMPLE 6

Oxidative stress induction

[00102] Seeking to understand the mechanism of action by which the compounds of this invention are significantly reducing cancer cell viability as demonstrated above, it was further evaluated whether reactive oxygen species (ROS) accumulation may also be a pathway involved in the anticancer effect of these compounds, as cancer cells may be more sensitive than normal cells to the accumulation of ROS.

[00103] Several characters of cancer cell behavior have been reported to be affected by oxidative stress-mediated signaling events (Sztarowski, T. P. et ak; Cancer Res. 1991, 51, 794-798; Storz, P.; Front. Biosci. 2005, 10, 1881-1896; and Gupta, A. et al.; Carcinogenesis 1999, 20, 2063- 2073). For instance, ROS in cancer are involved in cell cycle progression and proliferation, cell survival and apoptosis, energy metabolism, cell morphology, cell-cell adhesion, cell motility, angiogenesis and maintenance of tumor sternness. Despite the elevated level of antioxidant defense mechanism in cancer cells, ROS levels are still higher than those observed in normal cells. Therefore, cancer cells may be more sensitive than normal cells to the accumulation of ROS, which offers an interesting therapeutic window (Liu, J. et ak; Oxid. Med. Cell. Longev. 2015, 294303). Targeting the enhanced antioxidant mechanisms and directly increasing ROS to reach a threshold that is incompatible with cell viability can selectively kill cancer cells, without affecting normal cells (Sosa, V. et ak; Ageing Res. Rev. 2013, 12, 376-390; and Gorrini, C. et al; Nat. Rev. drug Discov. 2013, 12, 931-947).

[00104] In this study, cells were co-treated with compound 2 or 10 and dichlorofluorescin (DCFH), an organic dye that is easily oxidized to the fluorescent dichlorofluorescein (DCF) by oxidative species inside the cells. Oxidative stress was determined by direct measuring of the fluorescence intensity generated by DCF. Aspcl cells treated with compound 10 showed a robust increase in oxidative stress after 24 hours of incubation in comparison to control cells (Figure 4A), and this output persisted during 96 hours when high concentrations were applied (75 to 300mM) (Figure 4B). Lower concentrations of 37 and 18mM were effective only in short incubation times (Figure 4A). In contrast, Aspcl cells treated with compound 2 showed almost no oxidative stress even after 96 hours of incubation, except when treated with 300mM, where a significant increase in ROS formation is observed after 96 hours of incubation (Figures 4A-B).

[00105] [00106] Compound 10 significantly increased the internal levels of ROS above the already high levels found in cancer cells (Figures 4A-B), and this effect was observed very early and persisted even after long times of incubation. Concomitantly, the level of cell toxicity measured by the real-time cytotoxic detection assay (Example 4) was also immediately increased, reaching its apex after 30 minutes after treatment (Figure 2). MTT assay revealed that this cytotoxic level corresponds to 35% of viability (Figure 1B),. Thus, it seems that cells were arrested and stopped proliferating. Daily treatment would probably culminate in cell death. A cell cycle analysis would be required to evaluate in which step of cell cycle cells are being arrested. The loss of effect of compound 2 in generating oxidative stress (Figures 4A-B) corresponds with the observed cytotoxic pattern (Figure 2) which was very moderate and delayed, reaching its apex only after 4 hours after treatment. However, treated cells also showed an antiproliferative pattern.

[00107]

EXAMPLE 7

Inhibition of tumor growth in 3D spheroids

[00108] The cellular assays described above were performed in a monolayer system. Although monolayer systems partially reflect the microenvironment of cancer cells, there are currently more advanced culture methods that mimic in a better way the cellular interactions by the cells comprising the tumor (Freidrich, J. et. al.; Nat. Prot. 2009, 4, 309-324). Seeking to elucidate whether the compounds of this invention would also be able to hinder the growth of cancer cells, it was decided to proceed with assays in a 3D cell spheroid model. This assay can be applied to test whether the compounds treatment is able of impairing tumors formation or alternately to test the effect of the treatment in growing tumors. FIT-29 cell line was used for this model due to its capacity to form spheroids in vitro. Single dose (150mM) treatment of compound 10, 24 hours after cells seeding (before spheroid formation) did not significantly impair tumor formation However, compound 10 avoided secondary tumors formation (Figure 5).

[00109] Alternatively, growing tumors were affected when a single dose of compound 10 (150mM) was applied 72h after cells seeding (after spheroid formation), the spheroid basal membrane was visibly disrupted with the emergence of black dots representing necrotic areas (Figure 6). Furthermore, spheroids were severely affected when treated with a single dose of compound 2, 24 hours after seeding (Figure 7). In this regards, it should be noted that treatment using chemotherapeutic agents like irinotecan and carboplatin doesn't generate any effect in tumor development (Figure 6).

[00110] Additionally, no effect was observed when spheroids were treated with the molecule backbones namely naringenin and p-coumaric acid (Figure 7), indicating that the pharmacological activity is not caused by the moieties separately but by the compounds (e.g. 2 and 10) themselves, through interaction with specific molecular targets in the tumor.

EXAMPLE 8

In-vivo experiments

[00111] To further investigate the therapeutic potential of compound 10, the activity of this compound was studied against Aspcl, A549, and T98G cell lines subcutaneous injected in nude mice based on the most favorable in vitro conditions (150mM at 24h).

Aspcl - Pancreas; Derived From Metastatic Site: Ascites

[00112] Compound 10 at 30 mg/ kg showed significant (P=0.0l) antitumor activity against subcutaneous Aspcl tumors. Daily doses of compound 10 in treated groups showed a robust inhibition in the tumor size growth along the three weeks of treatment compared to untreated controls (Figure 8). This difference was more striking at day 24 with almost two-fold reduction of the tumor area as compared to the beginning of the experiment. The experiment was stopped at this point and mice were sacrificed due to increasing local necrosis observed mainly in the tumors of treated mice (Figure 9), otherwise, if the treatment would be continued more extended, it is plausible that based on the graphic tendencies, the difference between these groups could be even higher.

[00113] The average necrosis percentage was determined as 77.5% in the treated group (Figure 10). Additionally, a significant (p=0.0002) increase (5.4 fold) was found in the percentage of natural killers (NK) cells in nude mice treated with compound 10 in comparison to control group (Figure 11).

A549-Lung

[00114] Daily doses of compound 10 at 30 mg/ kg in subcutaneous A549 tumors was less effective in reducing tumor area compared to Aspc 1. However, the difference between the tumor area from control to treated groups still significant (p=0.02) after two weeks of treatment (Figure 12). Despite the high standard deviation values, tumor size average of treated mice was reduced almost twice in comparison with control (Figure 13). No significant differences were observed either in necrosis or NK cells percentage between the groups.

T98G-Brain

[00115] T98G subcutaneous tumors were less stable and spontaneously started to reduce after one week from cell injection, even without any treatment. Thus the evaluation of the effect of tumor reduction between control and treated groups was performed at week 1 of treatment (Figure 14). A very significant (p=0.0008) reduction in tumor area was observed in the treated group in comparison to the control (almost 2 fold reduction) (Figure 15). Also, in this case, no necrosis was observed, and the percentage of NK cells was similar in both groups.

Claims

CLAIMS What is claimed is:

1. A compound represented by the structure of formula (I):

x'-x² - X²

n wherein

X¹ is a cinnamic acid or derivative thereof;

X² is an amino acid or an amine linker;

X³ is a hydroxy or polyhydroxy- substituted flavone, flavanone or derivative thereof; and n is an integer between 1 and 4; or

a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein X¹ is represented by the structure of formula (Ila):

Ila

wherein

R^1_R³ are each independently H, OH, OR, F, Cl, Br or I; and

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group.

3. The compound of claim 2, wherein X¹ is represented by the structure of formula (lib):

4. The compound of claim 3, wherein X¹ is represented by the structure of formula (He):

5. The compound according to any one of the preceding claims, wherein said amino acid or amine are selected from the group consisting of:

glycine, serine, leucine, tyrosine, threonine, alanine, 5-aminovaleric acid, 2-aminocaprylic acid, carnosine, arginine, histidine, lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, selenocysteine, proline, valine, isoleucine, methionine, phenylalanine, tryptophan, gama- aminobutyric acid, carnitine, levothyroxine, hydroxyproline, selenomethionine, hypusine, 2- aminoisobutyric acid, ornithine, citrulline, b-alanine, and propyl amine.

6. The compound of claim 5, wherein said amino acid or amine is selected from the group consisting of:

glycine, serine, leucine, tyrosine, threonine, alanine, 5-aminovaleric acid, 2-aminocaprylic acid, carnosine and propyl amine.

7. The compound according to any one of the preceding claims, wherein X³ is represented by the structure of formula (Ilia):

wherein

R⁴-R¹³ are each independently H, OH, R, OR, F, Cl, Br, I, N0₂, CF3 or an oxygen atom which is directly attached to X²;

R is a Ci-Cs linear or branched, substituted or unsubstituted alkyl group or a C₃-Cs substituted or unsubstituted cycloalkyl group;

at least one of R⁴-R¹³ is an oxygen atom which is directly attached to X²; and

single or a double bond.

8. The compound of claim 7, wherein n is 1.

9. The compound of claim 8, wherein said X³ is represented by the structure of formula Illb, IIIc, Hid, Hie, Illf or Illg:

(lllf) ; or (mg)

10. The compound according to any one of the preceding claims, wherein said compound is selected from:

11. The compound of claim 10, wherein said compound is selected from: