Trimeric flavon-3-ol derivatives and their use in the activation of insulin signaling

Abstract

The present disclosure relates to compounds and uses thereof, for example for treating disease associated with impaired insulin signaling and / or a metabolic disorder and / or an insulin resistance disease.

Description

[0001] COMPOUNDS AND USES THEREOF

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to compounds and uses thereof.

[0004] BACKGROUND ART

[0005] [1] Ali MK, Pearson- Stuttard J, Selvin E, Gregg EW. Interpreting global trends in type 2 diabetes complications and mortality. Diabetologia. 2022;65(l):3-13.

[0006] [2] Zhu R, Zhou S, Xia L, Bao X. Incidence, Morbidity and years Lived With Disability due to Type 2 Diabetes Mellitus in 204 Countries and Territories: Trends From 1990 to 2019. Frontiers in Endocrinology. 2022; 13.

[0007] BACKGROUND

[0008] Various diseases including Type 2 diabetes (T2D), are characterized by insulin resistance and glucose intolerance, which lead to chronic hyperglycemia. When left unmanaged, T2D might lead to severe complications and stands as a major risk factor for the development of various morbidities, such as cardiovascular and fatty liver diseases [1, 2].

[0009] GENERAL DESCRIPTION

[0010] In accordance with some aspects, the present disclosure provides a compound comprising at least three flavan-3-ol ring systems, each independently optionally substituted, wherein at least one flavan-3-ol ring system is in a trans configuration and at least one flavan-3-ol ring system is in a cis configuration and wherein the compound exhibits insulin mimetic activity.

[0011] In accordance with some other aspects, the present disclosure provides a compound having the general Formula (III),

[0012] or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein each of Ri, R2, R4, Rs, R7, and Rs, is independently hydrogen, cyano, amino, amide, nitro, hydroxy, oxo, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, or OR10, each of R3, Rs, R9, and Rio is independently hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, n is 1 to 4, m is 1 to 5, wherein at least one flavan-3-ol ring system is in a trans configuration and at least one flavan-3-ol ring system is in a cis configuration and wherein the compound is an insulin mimetic compound.

[0013] In accordance with some further aspects, the present disclosure provides a compound having the general Formula (IV): or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein each of Ri, R2, R4, Rs, R7, and Rs, is independently hydrogen, cyano, amino, amide, nitro, hydroxy, oxo, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, or OR10, each of R3, Rs, R9, and Rio is independently hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2- Cualkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, n is 1 to 4, m is 1 to 5 and wherein the compound is an insulin mimetic compound.

[0014] In accordance with some other aspects, the present disclosure provides a compound represented by Formula (X) wherein each of R3, Re, R9, Ru, R12, R13, R14, Ris, Rie, R17, Ris, R19, R20, R21 and R22 is independently hydrogen, cyano, amino, amide, nitro, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy and wherein the compound is an insulin mimetic compound.

[0015] In accordance with yet some other aspects, the present disclosure provides a compound represented by Formula (XX) or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof, wherein the compound is an insulin mimetic compound.

[0016] In accordance with some other aspects, the present disclosure provides a composition comprising an effective amount of at least one compound as described herein or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with impaired insulin signaling in a subject in need thereof.

[0017] In accordance with some other aspects, the present disclosure provides a composition comprising an effective amount of at least one compound as described herein or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an insulin-resistance disease in a subject in need thereof.

[0018] In accordance with some further aspects, the present disclosure provides a method of any one of (i) inducing insulin singling in a cell, (ii) inducing insulin receptor phosphorylation in a cell, (iii) inducing AKT phosphorylation in a cell, wherein the method comprises the step of contacting the cell with an effective amount of at least one compound as described herein.

[0019] In accordance with some further aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with impaired insulin signaling in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one compound as described herein or any composition comprising the same.

[0020] In accordance with some further aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an insulin-resistance disease in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one compound as described herein or any composition comprising the same.

[0021] In accordance with some further aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a metabolic disorder in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one compound as described herein or any composition comprising the same.

[0022] In accordance with some further aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of type 2 diabetes in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one compound as described herein or any composition comprising the same.

[0023] In accordance with some further aspects, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of type 1 diabetes in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of at least one compound as described herein or any composition comprising the same.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0026] Figure 1 is a scheme of the separation of compounds 1-4 described herein from the crude Sarcopoterium spinosum extract (SSE).

[0027] Figures 2A-2C show active components of SSE found within the phenol-rich fractions, SSE was separated based on polarity on a C18 column to nine fractions, activity of the fractions was measured by three different bioassays, Figure 2A shows differentiated 3T3-L1 adipocytes treated with SSE or its fractions at 100 pg / mL, using insulin (100 nM) as a positive control, [3J7]-2-Deoxy-d-glucose uptake into cells was determined as described below, Figure 2B shows L6 myotubes treated with 100 nM insulin, SSE, or its fraction (100 pg / mL) for 10 min, western blot analysis of the whole lysate was performed using specific antibodies, Figures 2C shows phenolic content in the SSE fractions determined by the Folin-Ciocalteu method, data represent the mean ± SEM of measurement made on at least three independent experiments, *p< 0.05, ****p<0.0001 compared to untreated cells by one-way ANOVA, followed by Tukey’s post-testing.

[0028] Figure 3 shows a typical electrospray ionization (ESI)-liquid chromatography coupled with mass spectrometry (LCMS) annotated chromatogram of SSE.

[0029] Figures 4A-4B shows structures, Figure 4A are structures of fragments a-d deduced from the ^^H COSY correlations, Figure 4B shows the structure of Sarcocyanidin A (1).

[0030] Figure 5 Structure assignment of Sarcocyanidin A (1), supported by the HMBC correlations.

[0031] Figure 6 Structure assignment of Sarcocyanidin A (1), supported by the NOE’s from a ROES Y experiment

[0032] Figures 7 A and 7B show dose-response and time course of SSE-induced IR and AKT phosphorylation, Figure 7A shows CHO cells overexpressing insulin receptor (CHO-IR) cells treated with insulin (Ins) or SSE at the indicated doses for 10 min, Figure 7B shows CHO-IR cells treated with insulin (10 and 100 nM) or SSE (lOOpg / ml) for the indicated time, western-blot analysis of whole lysate was performed using specific antibodies.

[0033] Figures 8A-8F show bioactivity of monomers, dimers and trimer catechins, Figure 8A shows CHO-IR cells treated with epicatechin (-), catechin (+), and a mix of these catechins, Figure 8B-8D shows CHO-IR cells treated with dimers of catechins, Figure 8E shows CHO-IR cells treated with trimer catechin isolated from SSE for 10 min, insulin (10 nM) was used as a positive control, western-blot analysis of whole lysate was performed using specific antibodies; Figure 8F shows differentiated 3T3-L1 adipocytes treated with SSE or with monomers, dimers and trimer Procyanidin (PC) at 100 pg / ml, with insulin (100 nM) as a positive control, the uptake of [3H]2-Deoxy -D-glucose into cells was determined as described below, the data represents the Mean±SEM of measurement made on at least 3 independent experiments. *p<0.05, **p<0.005, ****p<0.0001 compared to untreated cells by one-way ANOVA, followed by Tukey's post-testing.

[0034] Figures 9A to 9C shows the effect of Tormentic and Ursolic acids insulin signaling CHO-IR cells were treated with ursolic acid (Figure 9A), tormentic acid (Figure 9B) or hydroxy -tormentic acid (Figure 9C) at the indicated doses for 10 min. Insulin (10 nM) was used as a positive control, western-blot analysis of whole lysate was performed using specific antibodies.

[0035] Figures 10A to 10F shows IR phosphorylation in SSE-induced AKT phosphorylation, Figure 10A IR is phosphorylated on tyrosine residues by SSE, L6 myotubes were treated with 100 nM insulin or SSE (100 pg / ml) for 10 min, Cell lysate was prepared and IR phosphorylation was measured by ELISA, according to the manufacturer's instruction, Figure 10B shows L6 myotubes treated with 100 nM insulin or SSE (100 pg / ml) for the indicated time, Figure 10C to Figure 10F show Tyrosine phosphatase activity in CHO-IR cells (Figure 10C and Figure 10D) and L6 myotubes (Figure 10E and Figure 10F), treated with HNMPA-(AM)3 for 60 min, followed by treatment with insulin, SSE or Sarco-A (lOOpg / ml) for additional 10 min, Western-blot analysis of whole lysate was performed using specific antibodies and optical densitometry was measured by Image J, the data represents the Mean±SEM of measurement made on at least 3 independent experiments. *p<0.05, **p<0.005, compared to untreated cells or as indicated in the graphs by one-way ANOVA, followed by Tukey's post-testing.

[0036] Figures HA to 11G show that SSE directly interacts with IR, Figures 11A-11D shows autofluorescence of IR measured in the presence of Figure HA 100 nM insulin, Figure 11B SSE, Figure 11C SSE (20 pg / ml) in the presence of guanidine (IM), Figure HD sarcocyanidin A (1), CHO-IR cells were treated with insulin (10 nM), SSE or sarcocyanidin A (1, 10 pg / ml) for 10 min. Cellular thermal-shift assay (CTSA) was conducted as described below, Figure HE is a schematic illustration of the protocol. Figure HF shows IR expression in lysates after incubation at a gradient of temperature showing a representative result of 5 independent experiments. (Sarcocyanidin A (1): Sarco A), Figure 11G shows Optical density of IR expression. *p<0.05, ***p<0.001, compared to OD of IR in untreated cells at the same temperature, by one-way ANOVA, followed by Tukey's post-testing.

[0037] Figures 12A to 121 show the effect of a single dose SSE, phenol-rich fraction and sarcocyanidin A (Sarco-A) on blood glucose in male C57bl / 6J mice, Figure 12A blood glucose and serum insulin were measured once a hour for 7 hours, and additional measures after 24 hours, Figure 12. shows the reduction in blood glucose 6 h after SSE administration, Figure 12C shows plasma insulin 6 h after SSE administration, Figure 7D shows western blot analysis and densitometry of pAKT expression in liver and subcutaneous white adipose tissue (SC WAT) of treated mice, 6 h after SSE administration, Figures 12E and 12F show administration of Sarcocyanidin A or phenol- rich fraction (0.4 pg) and blood glucose was measured, Figure 12G shows Western blot analysis from harvested liver, Figures 12H and 121 show Blood glucose monitored for 7 h from Male C57bl / 6J given high fat diet (HFD) for 6 weeks before intraperitoneal administration of SSE (1.5 mg / ml), sarco-A or phenol-rich fraction (0.4 pg each). The data represents the Mean±SEM of measurement made on at least 3 independent experiments. Figure 12A, Figure 12B, Figure 12E, Figure 12F, Figure 12H and Figure 121: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0005, compared to time 0 of the same group by paired / -test. D and G were analyzed by one-way ANOVA, followed by Tukey's post-testing.

[0038] DETAILED DESCRIPTION OF EMBODIMENTS

[0039] The present disclosure is based on the isolation and identification of novel procyanidin compounds that modulate insulin signaling and, in particular, exert potent insulin-mimetic activity through direct interaction with the insulin receptor (IR).

[0040] As shown herein below, these compounds activate downstream insulin signaling pathways, including phosphorylation of the insulin receptor and the AKT kinase, even in the absence of endogenous insulin or in the presence of impaired insulin responsiveness.

[0041] As disease associated with impaired insulin activity such as insulin resistance is a hallmark of numerous pathological conditions, the compounds of the present disclosure are proposed to bypass defective insulin signaling mechanisms and restore insulin- responsive glucose uptake and signaling in affected tissues.

[0042] Hence, in accordance with some aspects, the present disclosure provides a compound comprising at least three flavan-3-yl ring systems, each independently optionally substituted. As described herein, the compound is an insulin mimetic compound.

[0043] The compound may comprise flavan-3-yl ring systems in a uniform configuration, such as a trans configuration (e.g., catechin-type) or a cis configuration (e.g., epicatechintype). Alternatively, the compound may comprise both cis- and trans-configured flavan- 3-yl ring systems, thereby forming a stereochemically mixed compound.

[0044] In accordance with some aspects of the present disclosure, there is provided a compound comprising at least three flavan-3-yl ring systems, each independently substituted, wherein the at least three flavan-3-yl ring systems are in a cis configuration and wherein the compound is an insulin mimetic compound.

[0045] In accordance with some aspects of the present disclosure, there is provided a compound comprising at least three flavan-3-yl ring systems, each independently substituted, wherein the at least three flavan-3-yl ring systems are in a trans configuration and wherein the compound is an insulin mimetic compound.

[0046] In accordance with some aspects of the present disclosure, there is provided a compound comprising at least three flavan-3-yl ring systems, each independently substituted, wherein least one ring system is in a trans configuration and at least one other ring system is in a cis configuration. The compound is an insulin mimetic compound.

[0047] In accordance with certain aspects of the present disclosure, there is provided a compound comprising at least three flavan-3-yl ring systems, each independently substituted, wherein at least one ring system is in a trans configuration and at least one ring system is in a cis configuration and wherein the compound exhibits insulin mimetic activity.

[0048] Unless otherwise stated, the term compounds as used herein encompasses both natural, semi -synthetic and synthetic compounds.

[0049] In the following text, when referring to at least compound it is to be understood as also referring to the oligomers, compositions, methods and uses disclosed herein. Thus, whenever providing a feature with reference to at least one compound, it is to be understood as defining the same feature with respect to the oligomers, compositions, methods, and uses, mutatis mutandis.

[0050] As used herein, the term "flavan-3-yl ring system " or "flavan-3-yl ring " or ""flavan-3-yl monomer" refers to a ring system comprising a fused bicyclic ring system with a pendant ring attached at C2, forming a three-ring scaffold. Specifically, the three- ring system comprises a benzopyran structure having a saturated heterocyclic ring (chroman) (C-ring) fused to a resorcinol-type aromatic ring (A-ring) and a phenyl ring (B-ring) attached at position C2 of the chroman nucleus.

[0051] The flavan-3-yl ring system may be represented by Formula (I): any enantiomer thereof.

[0052] The flavan-3-yl ring system may be substituted at any of the positions as further described herein. The flavan-3-yl ring system may be unsubstituted or substituted.

[0053] In some examples, the flavan-3-yl ring system may be represented by Formula (I- 1) or Formula (1-2): or any enantiomer thereof, wherein of Rx, and Ry, is independently hydrogen, cyano, amino, amide, nitro, hydroxy, oxo, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, n is 1 to 4, m is 1 to 5 and the curved line represent a point of connection.

[0054] The stereochemistry at the C2 and C3 positions may be either cis or trans, as described herein, giving rise to forms such as catechin, epicatechin, analogues, functional derivatives or enantiomers thereof.

[0055] In some embodiments, the at least three flavan-3-yl ring systems are covalently connected by two C4^C8 interflavan linkages, forming a linear trimer.

[0056] As interflavan linkage refer to chemical bond that connect two or more flavan-3-ol units (such as catechin or epicatechin) to form oligomeric or polymeric proanthocyanidins, also known as condensed tannins. Each interflavan bond connects the C4 position of an upstream unit (typically part of the saturated C-ring) to the C8 position of a downstream unit (part of the A-ring). The stereochemistry at the C4 carbon may be either a or p, thereby defining the linkage as a 4a^8 or 4p^8 bond, respectively.

[0057] The stereochemistry at C2 and C3 of the flavan-3-yl ring system may be a cis configuration or a trans configuration.

[0058] The flavan-3-yl ring system is characterized by having a substitutable C3 position, typically bearing a hydroxyl group in flavan-3-ols, but which

[0059] In some examples, the flavan-3-yl ring system is or comprises a flavan-3-ol ring system.

[0060] As used herein, "flavan-3-ol ring system" or "flavan-3-ol ring" or "flavan-3-ol monomer" refers to a polyphenolic compound having a chroman-based tricyclic scaffold comprising an A-ring, a C-ring (a saturated heterocycle), and a B-ring attached at position C2. The compound includes a hydroxyl substituent at the 3-position of the C-ring.

[0061] The flavan-3-ol ring system may be represented by Formula (II): any enantiomer thereof. The flavan-3-ol ring system may be substituted at any of the positions as further described herein.

[0062] Hence, in accordance with some aspects, the present disclosure provides a compound comprising at least three flavan-3-yl ring systems, each independently optionally substituted, wherein at least one of the flavan-3-yl ring systems is a flavan-3- ol ring system. At least one flavan-3-yl ring system is in a trans configuration and at least one is in a cis configuration. The compound is an insulin mimetic compound.

[0063] In accordance with some aspects, the present disclosure provides a compound comprising at least three flavan-3-ol ring systems, each independently optionally substituted, at least one flavan-3-ol ring system is in a trans configuration and at least one flavan-3-ol ring system is in a cis configuration. The compound is an insulin mimetic compound.

[0064] In accordance with some aspects, the present disclosure provides a compound comprising at least three flavan-3-ol ring systems, each independently optionally substituted, wherein at least one flavan-3-ol ring system is in a trans configuration and at least one flavan-3-ol ring system is in a cis configuration and wherein the compound exhibits insulin mimetic activity.

[0065] As used herein, the terms "c / .s configuration" and "trans configuration" refer to the relative stereochemistry of substituents at the 2- and 3-positions of a flavan-3-ol or flavan- 3-yl ring system.

[0066] A cis configuration indicates that these substituents are on the same face of the C- ring, while a trans configuration indicates that they are on opposite faces. These terms apply both to standalone flavan-3-ol monomers and to flavan-3-yl units incorporated into oligomeric or polymeric structures, such as procyanidins.

[0067] In some examples, the compound may be considered as an oligomer comprising at least three flavan-3-ols rings. In some examples, the compound comprises three flavan- 3-ols rings.

[0068] In accordance with some aspects, the present disclosure provides a compound represented by Formula (III),

[0069] or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein each of Ri, R2, R4, Rs, R7, and Rs, is independently hydrogen, cyano, amino, amide, nitro, hydroxy, oxo, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, or OR10, each of R3, Rs, R9, and Rio is independently hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2- Cualkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, n is 1 to 4, m is 1 to 5 and the compound is an insulin mimetic compound.

[0070] In some examples, in the compound of Formula (III), at least one flavan-3-ol ring system is in a trans configuration and at least one flavan-3-ol ring system is in a cis configuration.

[0071] In accordance with some aspects, the present disclosure provides a compound represented by Formula (III) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein each ofRi, R2, R4, Rs, R7, and Rs, is independently hydrogen, cyano, amino, amide, nitro, hydroxy, oxo, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2- Cualkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, or OR10, each of R3, Re, R9, and Rio is independently hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, n is 1 to 4, m is 1 to 5, wherein at least one flavan-3-ol ring system is in a trans configuration and at least one flavan-3-ol ring system is in a cis configuration and wherein the compound is an insulin mimetic compound.

[0072] In accordance with some embodiments which may be considered as aspects of the invention, the compound of Formula (III) is represented by Formula (IV),

[0073] or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein the compound is an insulin mimetic compound.

[0074] In accordance with some embodiments which may be considered as aspects of the invention, the compound of Formula (III) is represented by Formula (V), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein the compound is an insulin mimetic compound.

[0075] In accordance with some embodiments which may be considered as aspects of the invention, the compound of Formula (III) is represented by Formula (VI),

[0076] or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein the compound is an insulin mimetic compound.

[0077] In accordance with some embodiments which may be considered as aspects of the invention, the compound of Formula (III) is represented by Formula (VII), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein the compound is an insulin mimetic compound.

[0078] In accordance with some embodiments which may be considered as aspects of the invention, the compound of Formula (III) is represented by Formula (VIII),

[0079] or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein the compound is an insulin mimetic compound.

[0080] In accordance with some embodiments which may be considered as aspects of the invention, the compound of Formula (III) is represented by Formula (IX), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional

[0081] The present disclosure is not limited to a specific compound comprising a flavan- 3-ols ring.

[0082] In some examples, the flavan-3-ol ring is one or more of catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, epigallocatechin gallate, catechin-3 -O-glucoside, epicatechin-3 -O-rutinoside, 3'-O-methylcatechin, 4'-O- methylepicatechin, any functional derivative thereof or any combination thereof.

[0083] As used herein, a "functional derivative" of a compound described herein refers to any chemical entity that is structurally modified relative to the parent compound but retains at least one key biological or functional activity of the parent compound, such as insulin receptor activation, glucose uptake stimulation, or AKT phosphorylation.

[0084] A functional derivative may include, but is not limited to substituted analogues (e.g., hydroxyl replaced by alkoxy, halogen, sulfate, glycoside, etc.), enantiomers, diastereomers, tautomers, esterified, etherified, acylated, or glycosylated forms, prodrugs, salts, solvates, and metabolites. The term encompasses derivatives that differ by substitution at one or more positions of the flavan-3-ol scaffold, including the A-ring, firing, or C-ring, provided the biological activity (e.g., insulin mimetic function) is retained or enhanced. The functional derivative may also include oxidized, hydrogenated, or ring- modified substitutes, provided that insulin-mimetic activity is retained.

[0085] In some examples, in the compounds represented by any one of Formulas (III)-(IX), each of R2, Rs, and Rs is OR10, Rio is independently selected from hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy.

[0086] In some examples, in the compound represented by any one of Formulas (III)-(IX) the phenyl ring substituted with R2, Rs, and Rs is substituted at one or more of positions 3', 4', 5' of this ring.

[0087] In some examples, in the compound represented by any one of Formulas (III)-(IX) the phenyl ring substituted with R2, Rs, and Rs is substituted at one or more of positions 3', 4' of this ring.

[0088] In some examples, in the compound represented by any one of Formulas (III)-(IX), each one of Ri, R4, and R7 is OR10, Rio is independently hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy.

[0089] In some examples, in the compound represented by any one of Formulas (III)-(IX), the phenyl ring substituted with Ri, R4, and R7 is substituted at one or more of positions 5, 7.

[0090] In some examples, in the compound represented by any one of Formulas (III)-(IX), n is 2. In some examples, in the compound represented by any one of Formulas (III)-(IX), m is 2.

[0091] In accordance with some embodiments which may be considered as aspects of the invention, the compound is represented by Formula (X), wherein the compound exhibits insulin mimetics activity.

[0092] In accordance with some embodiments which may be considered as aspects of the invention, the compound is represented by Formula (XI), wherein the compound exhibits insulin mimetics activity.

[0093] In accordance with some embodiments which may be considered as aspects of the invention, the compound is represented by Formula (XII),

[0094] wherein the compound exhibits insulin mimetics activity.

[0095] In accordance with some embodiments which may be considered as aspects of the invention, the compound is represented by Formula (XIII), wherein the compound exhibits insulin mimetics activity.

[0096] In accordance with some embodiments which may be considered as aspects of the invention, the compound is represented by Formula (XIV),

[0097] wherein the compound exhibits insulin mimetics activity.

[0098] In accordance with some embodiments which may be considered as aspects of the invention, the compound is represented by Formula (XV), wherein the compound exhibits insulin mimetics activity.

[0099] In the compounds represented by Formula (X)-(XV), each of R3, Rs, R9, Ru, R12, R13, RU, R15, Ri6, R17, Ri8, R19, R20, R21 and R22 is independently hydrogen, cyano, amino, amide, nitro, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy.

[0100] In the compounds represented by Formula (X)-(XV), each of R3, Rs, R9, Ru, R12, R13, RU, R15, Ri6, R17, Ri8, R19, R20, R21 and R22 is independently hydrogen or C1-C12 alkyl.

[0101] In the compounds represented by Formula (X)-(XV), each of R3, Rs, R9, Ru, R12, RB, Ri4, R15, Ri6, R17, Ri8, R19, R20, R21 and R22 is independently hydrogen or Ci-Csalkyl.

[0102] In the compounds represented by Formula (X)-(XV), each of R3, Rs, R9, Ru, R12, R13, RU, R15, R16, R17, Ri8, R19, R20, R21 and R22 is hydrogen.

[0103] In some examples, in the compounds represented by Formula (X)-(XV), wherein each of R3, Rs, R9, Ru, R12, R13, R14, R15, Ri6, R17, Ris, R19, R20, R21 and R22 is hydrogen, the compound is or comprises catechin (C) or epi catechin (EC).

[0104] In some examples, flavan-3-ols ring may be catechin (C) or any functional derivative thereof. As used herein, catechin ring refers to a flavan-3-ol having IUPAC name (2A,35)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-2Z7-chromene-3,5,7-triol

[0105] In some examples, flavan-3-ols ring may be epicatechin (EC) or any functional derivative thereof. As used herein, epicatechin ring refers to a flavan-3-ol having IUPAC name (2A,3A)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2Z7-chromene-3,5,7-triol

[0106] As described herein, the compound may comprise one or more ring in cis configuration (epi catechin-type) and one or more rings in trans configuration (catechintype).

[0107] In some examples, the compound is or comprises (+)-catechin-(4a^8)-(-)- epicatechin-(4p^8)-(-)-epicatechin (C-EC-EC) and exhibits insulin mimetic activity.

[0108] In some embodiments, which may be considered as aspects of the present disclosure the compound represented by Formula (XX): pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof wherein the compound is an insulin mimetic compound. The compound represented by Formula (XX) is denoted herein as Sarcocyanidin A.

[0109] Specifically, phenol-rich fractions isolated from Sarcopoterium spinosum extract (SSE) were found to stimulate glucose uptake in 3T3-L1 adipocytes and activate insulin signaling in L6 myotubes, indicating their functional mimicry of insulin. As shown in Figure 3, the active component in the SSE was characterized as a terminer comprising a catechin unit and two epicatechin units: (+)-catechin-4a-8-(-)-epicatechin-4P-8-(-)- epicatechin.

[0110] In some aspects, the present disclosure provides an insulin receptor agonist represented by Formula (XX). The compound represented in Formula (XX) has a chemical name 2,2',2"-Tris-(3,4-dihydroxy-phenyl)-3,4,3',4',3",4"-hexahydro- 2H,2'H,2"H-[4,8';4',8"]terchromene-3,5,7,3',5',7',3",5",7"-nonaol. This compound is also referred herein as (+)-catechin-4a-8-(-)-epicatechin-4P-8-(-)-epicatechin. In some aspects, the compound represented by Formula (XX) exhibits insulin mimetic activity.

[0111] In some examples, the compound represented by Formula (XX) comprises (+)- catechin-(4a^8)-(-)-epicatechin-(4p^8)-(-)-epicatechin (C-EC-EC) and exhibits insulin mimetic activity.

[0112] In some embodiments, which may be considered as aspects of the present disclosure the compound represented by Formula (XXI): or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof wherein the compound is an insulin mimetic compound. In some examples, the compound represented by Formula (XXI) comprises (-)- epicatechin-(4p— >8)-(-)-epicatechin-(4p— >8)-(+)-catechin (EC-EC-C) and exhibits insulin mimetic activity.

[0113] In some embodiments, which may be considered as aspects of the present disclosure the compound represented by Formula (XXII): or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof wherein the compound is an insulin mimetic compound.

[0114] In some examples, the compound represented by Formula (XXII comprises (-)- epicatechin-(4p^8)-(+)-catechin-(4a^8)-(+)-catechin (EC-C-C) and exhibits insulin mimetic activity.

[0115] In some embodiments, which may be considered as aspects of the present disclosure the compound represented by Formula (XXIII):

[0116] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof wherein the compound is an insulin mimetic compound.

[0117] In some examples, the compound represented by Formula (XXIII comprises (-)- epicatechin-(4p^8)-(+)-catechin-(4a^8)-(-)-epicatechin (EC-C-EC) and exhibits insulin mimetic activity.

[0118] In some embodiments, which may be considered as aspects of the present disclosure the compound represented by Formula (XXIV): or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof wherein the compound is an insulin mimetic compound. In some examples, the compound represented by Formula (XXIV comprises (+)- catechin-(4a^8)-(-)-epicatechin-(4p^8)-(+)-catechin (C-EC-C) and exhibits insulin mimetic activity.

[0119] In some embodiments, which may be considered as aspects of the present disclosure the compound represented by Formula (XXV): or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof wherein the compound is an insulin mimetic compound.

[0120] In some examples, the compound represented by Formula (XXV (+)-catechin- (4a^8)-(+)-catechin-(4a^8)-(-)-epicatechin (C-C-EC) and exhibits insulin mimetic activity.

[0121] In these examples, the stereochemical configuration at the C4 position may define the interflavan bond as a or depending on whether the linkage projects below or above the C-ring plane of the donor monomer, respectively. Each monomer unit may be further independently optionally substituted, and the B-ring substitution may vary (e.g., catechol- or gallol-type).

[0122] In accordance with some aspect, the present disclosure provides a compound comprising three flavan-3-ol units selected from catechin and epicatechin, connected via C4^C8 interflavan linkages, optionally with stereochemistry at the C4 position of each linkage (i.e., 4a^8 or 4 — >8).

[0123] In accordance with some aspect, the present disclosure provides a compound comprising three flavan-3-ol units selected from catechin and epicatechin, connected via C4^C8 interflavan linkages, optionally with stereochemistry at the C4 position of each linkage (i.e., 4a^8 or 4p^8), wherein the compound is an insulin mimetic compound.

[0124] As described herein and shown in the Examples, the compounds of the invention act as insulin mimetic compounds.

[0125] As used herein, the term "insulin mimetic compound" or "a compound exhibiting insulin mimetic activity" refers to a compound that functionally mimics the biological activity of insulin, either partially or fully (i.e. exhibits insulin mimetic activity). Such compound binds to, interacts with, or activates the insulin receptor and / or its downstream signaling pathways.

[0126] In some examples, the insulin mimetic compound exhibits one or more of the following activities: (i) binds directly to the IR and induces autophosphorylation; (ii) activates downstream signaling components in the insulin signaling cascade; (iii) induces cellular responses typically mediated by insulin, such as glucose uptake, glycogen synthesis, or inhibition of lipolysis; (iv) restores insulin-sensitive signaling in conditions of insulin resistance or insulin deficiency; (v) acts as an IR agonist, partial agonist, or allosteric activator; (vi) enhances insulin signaling in the presence or absence of insulin; and (vii) any combination thereof.

[0127] In some examples, the compound binds to the IR. In some examples, the compound binds directly to the IR. In some examples, the compound induces IR autophosphoryl ati on .

[0128] In some examples, the compound activates one or more downstream signaling components in the insulin signaling cascade.

[0129] As used herein, "downstream signaling components in the insulin signaling cascade" refer to intracellular molecules and signaling proteins that are functionally activated following engagement of the insulin receptor (IR) and that mediate the biological effects of insulin. These components facilitate the transmission of the insulin signal and regulate key metabolic and cellular processes.

[0130] In some examples, the compound activates one or more of: insulin receptor substrates (IRS), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), glucose transporter type 4 (GLUT4), and mitogen-activated protein kinases (MAPKs), or any combination thereof.

[0131] In some examples, the compound induces cellular responses typically mediated by insulin. In some examples, the compound exhibits one or more of enhanced glucose uptake into insulin-responsive tissues, promotion of glycogen synthesis, or suppression of lipolysis in adipose tissue.

[0132] In some examples, the compound bypass impaired endogenous insulin function. In some examples, the compound bypass impaired endogenous insulin function, as may occur in insulin resistance, by restoring insulin-sensitive signaling. In some examples, the compound restores insulin signaling under conditions of impaired or insufficient endogenous insulin function, such as in insulin resistance.

[0133] In some examples, the insulin mimetic compound encompasses full and partial agonist of the insulin receptor, direct activators of insulin receptor phosphorylation, and compounds capable of restoring or enhancing insulin signaling in insulin-resistant conditions.

[0134] In some examples, the compound acts as an IR agonist, partial agonist, or allosteric activator.

[0135] The term agonist as used herein refers to a compound / molecule that binds to and activates a receptor, triggering a physiological response. In the context of the present disclosure, an insulin receptor agonist refers to a molecule that binds and activates the insulin receptor and initiates downstream signaling similar to or mimicking insulin, regardless of whether insulin is present.

[0136] As used herein, a compound that “acts as an IR agonist, partial agonist, or allosteric activator” refers to a molecule that directly or indirectly stimulates insulin receptor activity by binding to the insulin receptor and initiating downstream signaling. This may occur via: (i) full agonism, mimicking insulin and producing a maximal response; (ii) partial agonism, resulting in a submaximal activation; or (iii) allosteric activation, whereby the compound binds at a non-orthosteric site and enhances the receptor's responsiveness to insulin or signaling efficacy.

[0137] An IR agonist as used herein refers to a compound that binds to the insulin receptor (IR) and activates it in a manner similar to insulin, inducing receptor autophosphorylation and triggering downstream signaling cascades (e.g., IRS, PI3K, AKT). Full agonists produce a maximal biological response comparable to insulin.

[0138] A partial agonist as used herein refers to a compound that binds to the insulin receptor and activates it, but elicits a submaximal response even at full receptor occupancy. That is, it stimulates insulin signaling, but to a lesser extent than a full agonist like insulin. Partial agonists can be beneficial in settings where a more controlled or attenuated insulin-like effect is desired.

[0139] An allosteric activator as used herein refers to a compound that binds to the insulin receptor at a site distinct from the insulin binding site (i.e., an allosteric site), and enhances receptor activity either on its own or in combination with insulin. Allosteric activators may increase receptor sensitivity to insulin or amplify downstream signaling without directly mimicking insulin at the primary binding site.

[0140] In some examples, the compound enhanced insulin signaling in the presence of insulin.

[0141] In some examples, the compound enhanced insulin signaling in the absence of insulin.

[0142] In some examples, the compound may function independently of insulin.

[0143] As shown in Figures 8A-8F, the compound represented by Formula (XI) induces Insulin receptor and AKT phosphorylation and glucose uptake dose-dependently, in the absence of insulin.

[0144] Insulin receptor (IR) as used herein refers to a transmembrane tyrosine kinase receptor for insulin, comprising a and P subunits. Upon ligand binding to the IR, the receptor undergoes autophosphorylation of tyrosine residues, initiating signaling cascades that include IRS-1 / 2, PI3K, and AKT.

[0145] Further, as shown in Figures 10A-10C, IR phosphorylation by the compound represented by Formula (XI) is reduced / inhibited by an IR inhibitor Hydroxy-2- naphthalenylmethylphosphonic acid tri-acetoxymethyl ester (HNMPA(AM)3). These results suggest that the activity of the compound is IR-dependent and moreover that the compound is active in the absence of insulin, and its effect is blocked by an IR-specific antagonist.

[0146] As shown in Figures 11A-11G, the compound represented by Formula (XI) binds directly to IR (as shown using autofluorescence and cellular thermal shift assay (CTSA)), stabilizes IR, induces the same conformational effects (indica ted by loss of autofluorescence) as insulin, thermally stabilizes IR as insulin and reduces fluorescence, hence mimicking insulin binding.

[0147] As such, the compound represented by Formula (XX) was suggested to have a direct effect on IR activation (as shown via IR phosphorylation), to act as an IR agonist (as shown by IR-dependent response) and to work independently of insulin.

[0148] As described herein, the compounds of the invention are considered as insulin receptor agonists.

[0149] As shown in Figures 12A-12F, the compound represented by Formula (XI), has a glucose-lowering effect with increased AKT phosphorylation in insulin-responsive tissues (no increased insulin).

[0150] A further aspect of the present disclosure relates to a composition comprising as an active ingredient an effective amount of at least one compound of the invention, the composition optionally further comprises at least one of pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

[0151] In accordance with some other aspects, there is provided a compound or composition as defined herein for use in one or more of: (i) inducing insulin signaling; (ii) inducing insulin receptor phosphorylation; (iii) inducing AKT phosphorylation; (iv) activating downstream signaling components in the insulin signaling cascade; (v) stimulating glucose uptake in insulin-responsive tissues; (vi) promoting glycogen synthesis; (vii) inhibiting lipolysis (for example in adipose tissue); (viii) enhancing insulin receptor activity in the presence or absence of insulin; (ix) restoring or enhancing insulin sensitivity in insulin-resistant tissues or cells; (x) improving glucose homeostasis; (xi) reducing blood glucose levels in vivo; or (xii) any combination of the above.

[0152] In accordance with some other aspects, it is provided a compound or composition as defined herein for use in inducing insulin singling. In accordance with some other aspects, it is provided a compound or composition as defined herein for use in inducing insulin receptor phosphorylation. In accordance with some other aspects, it is provided a compound or composition as defined herein for use in inducing AKT phosphorylation. In accordance with some aspects, it is provided a compound or composition as defined herein for use in stimulating glucose uptake. In accordance with some aspects, it is provided a compound or composition as defined herein for use in stimulating glucose uptake in insulin-responsive tissues. In accordance with some aspects, it is provided a compound or composition as defined herein for use in restoring insulin sensitivity in insulin-resistant cells or tissues. In accordance with some aspects, it is provided a compound or composition as defined herein for use in reducing blood glucose levels in vivo.

[0153] In accordance with some aspects, it is provided a compound represented as one or more of Formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof, or any combination thereof or a composition comprising one or more of these compounds for use in one or more of (i) inducing insulin signaling; (ii) inducing insulin receptor phosphorylation; (iii) inducing AKT phosphorylation; (iv) activating downstream signaling components in the insulin signaling cascade; (v) stimulating glucose uptake in insulin-responsive tissues; (vi) promoting glycogen synthesis; (vii) inhibiting lipolysis (for example in adipose tissue); (viii) enhancing insulin receptor activity in the presence or absence of insulin; (ix) restoring or enhancing insulin sensitivity in insulin-resistant tissues or cells; (x) improving glucose homeostasis; (xi) reducing blood glucose levels in vivo; or (xii) any combination of the above.

[0154] In accordance with some aspects, it is provided a compound represented as one or more of Formulas (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof, or any combination thereof or a composition comprising one or more of these compounds for use in one or more of (i) inducing insulin signaling; (ii) inducing insulin receptor phosphorylation; (iii) inducing AKT phosphorylation; (iv) activating downstream signaling components in the insulin signaling cascade; (v) stimulating glucose uptake in insulin-responsive tissues; (vi) promoting glycogen synthesis; (vii) inhibiting lipolysis (for example in adipose tissue); (viii) enhancing insulin receptor activity in the presence or absence of insulin; (ix) restoring or enhancing insulin sensitivity in insulin-resistant tissues or cells; (x) improving glucose homeostasis; (xi) reducing blood glucose levels in vivo; or (xii) any combination of the above.

[0155] In accordance with some aspects, it is provided a compound represented as one or more of Formulas (XX) any functional derivative thereof, or any combination thereof or a composition comprising one or more of these compounds for use in one or more of ((i) inducing insulin signaling; (ii) inducing insulin receptor phosphorylation; (iii) inducing AKT phosphorylation; (iv) activating downstream signaling components in the insulin signaling cascade; (v) stimulating glucose uptake in insulin-responsive tissues; (vi) promoting glycogen synthesis; (vii) inhibiting lipolysis (for example in adipose tissue); (viii) enhancing insulin receptor activity in the presence or absence of insulin; (ix) restoring or enhancing insulin sensitivity in insulin-resistant tissues or cells; (x) improving glucose homeostasis; (xi) reducing blood glucose levels in vivo; or (xii) any combination of the above.

[0156] In accordance with some other aspects, the present disclosure provides a method for inducing one or more of (i) insulin singling, (ii) insulin receptor phosphorylation, (iii) AKT phosphorylation.

[0157] The method comprises the step of contacting a cell comprising at least one insulin receptor with an effective amount of at least one compound as described herein.

[0158] In accordance with some examples, the methods comprise contacting a cell comprising at least one insulin receptor with an effective amount of at least one compound represented as one or more of Formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0159] In accordance with some examples, the methods comprise contacting a cell comprising at least one insulin receptor with an effective amount of at least one compound represented as one or more of Formulas (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0160] In accordance with some examples, the methods comprise contacting a cell comprising at least one insulin receptor with an effective amount of at least one compound represented as one or more of Formulas (XX) any functional derivative thereof or any combination thereof.

[0161] The present disclosure is not limited to a specific cell and may refer to any cell comprising an insulin receptor. In some examples, the cell is one or more of 3T3-L1 adipocytes, L6 myotubes, CHO-IR cells, HepG2 hepatocytes, C2C12 myotubes, primary human adipocytes or myocytes, HEK293 cells transiently or stably expressing insulin receptor, or other human or animal-derived fibroblasts or endothelial cells expressing insulin receptors.

[0162] The term “contacting” means to bring, put, incubate or mix together. As such, a first item is contacted with a second item when the two items are brought or put together, e.g., by touching them to each other or combining them. In the context of the present invention, the term "contacting" includes all measures or steps which allow interaction between at least one compound and a cell, comprising in accordance with some embodiments at least one insulin receptor.

[0163] In some embodiments, the methods of the invention comprise contacting a cell comprising the insulin receptor with an effective amount of at least one compound of the invention in at least one of in vitro, in vivo, ex vivo methods. In some embodiments, the method is an in vitro method. In some other embodiments, the method is an in in vivo method. In some embodiments, the method is an ex vivo method.

[0164] According with some aspects of the present disclosure, the present disclosure provides a method of inducing insulin receptor activity in a subject in need thereof.

[0165] As used herein "inducing insulin receptor activity" refers to initiating, enhancing, restoring, or mimicking any functional response of the insulin receptor pathway.

[0166] In some examples, inducing insulin receptor activity may be by one or more of the following (i) binding of a ligand (e.g., an insulin mimetic compound) to the insulin receptor, (ii) inducing insulin signaling; (iii) inducing insulin receptor phosphorylation; (iv) inducing AKT phosphorylation; (v) activating downstream signaling components in the insulin signaling cascade; (vi) stimulating glucose uptake in insulin-responsive tissues; (vii) promoting glycogen synthesis; (viii) inhibiting lipolysis (for example in adipose tissue); (ix) enhancing insulin receptor activity in the presence or absence of insulin; (x) restoring or enhancing insulin sensitivity in insulin-resistant tissues or cells; (xi) improving glucose homeostasis; (xii) reducing blood glucose levels in vivo; or (xii) any combination of the above.

[0167] It should be noted that inducing insulin receptor activity encompasses receptor activation by insulin itself, by insulin receptor agonists, or by insulin mimetic compounds that act in the presence or absence of endogenous insulin.

[0168] Inducing insulin receptor activity, as defined herein, is suggested to provide a therapeutic mechanism applicable to a broad range of insulin-related disorders.

[0169] Hence, the compounds of the present disclosure may be suitable for treating insulin- related disease or disorder.

[0170] Insulin-related disease or disorder as used herein relate to disease or disorder characterized by impaired insulin signaling, insulin resistance, insulin deficiency, or dysregulated glucose metabolism.

[0171] It was suggested that the compounds of the present disclosure may be suitable for treating disease or disorders associated with impaired insulin signaling.

[0172] Hence, in accordance with some aspects, there is provided at least one compound for use in treating a disease or disorder associated with impaired insulin signaling.

[0173] It was also suggested that the compounds of the present disclosure may be suitable for treating disease or disorders associated with insulin receptor resistance.

[0174] Hence, in accordance with some aspects, there is provided at least one compound for use in treating a disease or disorder associated with insulin receptor resistance.

[0175] The present disclosure relates to method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying insulin related diseases or disorders. As described herein, in such disease insulin receptor activity or downstream signaling is impaired, deficient, or dysregulated.

[0176] The methods of the invention may be applicable for treating, inhibiting, arresting or delaying insulin related diseases or disorders.

[0177] The methods of the invention may be applicable for treating, inhibiting, arresting or delaying disease or disorder associated with impaired insulin signaling.

[0178] In yet a further aspect, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with impaired insulin signaling in a subject in need thereof. The method comprises in some embodiments the step of administering to the subject a therapeutically effective amount of at least one compound as defined by the present disclosure, or composition comprising the compound.

[0179] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with impaired insulin signaling in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0180] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with impaired insulin signaling in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0181] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with impaired insulin signaling in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX) any functional derivative thereof or any combination thereof.

[0182] As used herein, the term "a disease or disorder associated with impaired insulin signaling" refers to any pathological condition in which insulin-mediated biological responses are reduced, disrupted, or insufficient. Such impairment may be due to defects in insulin availability, insulin receptor function, or downstream signaling components. Such impairment may result from insulin resistance, insulin deficiency, or molecular abnormalities affecting the insulin receptor or its signaling pathway. Such disease encompasses conditions in which insulin-mediated biological effects, such as glucose uptake, glycogen synthesis, or lipid regulation, are diminished due to receptor-level, downstream, or systemic impairments.

[0183] In some examples, the disease or disorder associated with impaired insulin signaling is one or more of (i) diabetes mellitus and pre-diabetic conditions, (ii) hormonal or reproductive disorders, (iii) hepatic or metabolic disorders, (iv) cardiovascular or renal disorders, (v) neurological or cognitive disorders, (vi) genetic or congenital disorders, (vii) acute or stress-related conditions involving transient insulin resistance or (viii) any combination thereof.

[0184] In some examples, the disease is a form of diabetes mellitus or a pre-diabetic condition. In some examples, the diabetes mellitus and pre-diabetic conditions is one or more of type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), prediabetes, impaired glucose tolerance (IGT), impaired fasting glucose (IFG), latent autoimmune diabetes in adults (LADA) or any combination thereof.

[0185] In some examples, the disease is a hormonal or reproductive disorder. In some examples, the hormonal or reproductive disorder is one or more of polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), Cushing’s syndrome, acromegaly, hypothalamic obesity, congenital or acquired lipodystrophy or any combination thereof.

[0186] In some examples, the disease is a hepatic or metabolic disorder. In some examples, the hepatic or metabolic disorder is one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, obesity with insulin resistance, dyslipidemia associated with insulin signaling defects or any combination thereof.

[0187] The methods of the invention may be applicable for treating, inhibiting, arresting or delaying disease or disorder associated with insulin receptor resistance.

[0188] In yet a further aspect, the present disclosure provides a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with insulin receptor resistance in a subject in need thereof. The method comprises in some embodiments the step of administering to the subject a therapeutically effective amount of at least one compound as defined by the present disclosure, or composition comprising the compound.

[0189] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with insulin receptor resistance in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0190] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with insulin receptor resistance in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0191] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease or disorder associated with insulin receptor resistance in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX) any functional derivative thereof or any combination thereof.

[0192] As used herein, “a disease or disorder associated with insulin receptor resistance" refers to a pathological condition in which the insulin receptor exhibits reduced responsiveness to insulin, despite the presence of normal or elevated insulin levels. Disease or disorder associated with insulin receptor resistance may result from one or more of the following (i) impaired insulin receptor binding, (ii) reduced receptor expression, (iii) defective receptor autophosphorylation, (iv) increased inhibitory signaling (e.g., via phosphatases like PTP1B) or (v) any combination thereof.

[0193] This resistance can result from impaired insulin receptor function, including reduced IR binding affinity, diminished receptor autophosphorylation, defective downstream signaling (e.g., via IRS / PI3K / AKT pathways), or increased inhibitory regulation (e.g., via PTP1B). Additionally, or alternatively, the insulin resistance may include insulin resistance due to mutation in insulin receptor, insulin resistance not caused by mutation in the insulin receptor, insulin resistance caused by a mutation in downstream signaling pathways or induced by other disease or disorder as further described herein.

[0194] The present disclosure is not limited to a specific disease or disorder and is applicable to any disease or disorder that involves, is linked to, is associated with or caused by insulin resistance.

[0195] In accordance with some aspects which may be considered as embodiments of the invention, the present invention provides methods for treatment of metabolic disorders in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof. In some examples, the present invention provides methods for treatment of metabolic disorders in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof. In some examples, the present invention provides methods for treatment of metabolic disorders in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of at least one compound represented by Formula (XX) any functional derivative thereof or any combination thereof.

[0196] A metabolic disorder is a condition in which abnormal chemical reactions interfere with the body’s ability to maintain normal metabolic processes.

[0197] One key class of metabolic disorders involves insulin resistance, wherein cells fail to respond appropriately to insulin, a hormone critical for glucose uptake and utilization. Insulin resistance may arise due to dysfunction in metabolic organs such as the liver, skeletal muscle, or adipose tissue, or due to impaired insulin production or action associated with pancreatic dysfunction.

[0198] In some examples, the metabolic disorder is one or more of type 2 diabetes mellitus (T2DM), prediabetes, metabolic syndrome, obesity, impaired glucose tolerance (IGT), impaired fasting glucose (IFG), dyslipidemia, and lipodystrophy syndromes.

[0199] In some examples, the methods of the invention may be specifically relevant for metabolic disorder, specifically, at least one of diabetes mellitus type II and any diabetic related conditions.

[0200] The methods of treatment and uses of the invention may also be utilized for the benefit of subjects suffering from diabetes-related or associated diseases or disorders, comprising hyperinsulinaemia, dyslipidaemia, hypercholesterolemia, impaired glucose tolerance, hypertension, cardiovascular disease, diabetic cardiomyopathy, diabetic cardiac dysrhytmia, atherosclerosis, diabetic nephropathy, glomerulonephritis, glomerular sclerosis, nephrotic syndrome, hypertensive nephrosclerosis, end stage renal disease, microalbuminuria and albuminuria.

[0201] In accordance with some aspects which may be considered as embodiments of the invention, the present invention provides methods for treatment of diabetes-related or associated diseases or disorders.

[0202] In accordance with some aspects which may be considered as embodiments of the invention, the present invention provides methods for treatment of one or more of a liver disorder, a cardiovascular disorder, an endocrine and reproductive disorder, a neurological and cognitive disorder, a dermatological manifestation, or any combination thereof.

[0203] In some examples, the liver disorder is one or more of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0204] In some examples, the cardiovascular disorder is one or more of insulin-resistant hypertension, atherosclerosis, and metabolic-associated cardiovascular disease.

[0205] In some examples, the endocrine and reproductive disorder is one or more of polycystic ovary syndrome (PCOS) and gestational diabetes mellitus (GDM).

[0206] In some examples, the neurological and cognitive disorder is one or more of alzheimer's disease, insulin-resistant dementia, and neurodegenerative disorders sometimes collectively referred to as “type 3 diabetes".

[0207] In some examples, the dermatological manifestation is acanthosis nigricans.

[0208] In some examples, the disease may involve chronic inflammation that contributes to insulin resistance.

[0209] In some examples, the disease or disorder is one or more of type 2 diabetes mellitus (T2DM); metabolic syndrome; obesity; non-alcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH); polycystic ovary syndrome (PCOS); cardiovascular disease (CVD) associated with metabolic dysfunction;; dyslipidemia; hypertension; impaired glucose tolerance (IGT); impaired fasting glucose (IFG); insulin-resistant states during pregnancy including gestational diabetes mellitus (GDM); chronic inflammation- related insulin resistance; lipodystrophy syndromes; prediabetes; Alzheimer's disease or other insulin-resistant brain states (sometimes termed "type 3 diabetes"); acanthosis nigricans; and other endocrine, hepatic, or neurometabolic disorders wherein insulin resistance is a contributing or defining factor.

[0210] In accordance with some aspects, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T2DM in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0211] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T2DM in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0212] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T2DM in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX) any functional derivative thereof or any combination thereof.

[0213] In accordance with some aspects, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T1DM in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0214] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T1DM in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX), (XXI), (XXII), (XXIII), (XIV), (XV), any functional derivative thereof or any combination thereof.

[0215] In accordance with some examples, the methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T1DM in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound represented by Formulas (XX) any functional derivative thereof or any combination thereof.

[0216] The subject to be treated with one or more of the compounds described herein is suffering from or diagnosed with a disease associated with impaired insulin signaling. In some examples, the subject to be treated with one or more of the compounds described herein is suffering from or diagnosed with a disease associated with insulin resistance.

[0217] As described herein, the present disclosure encompasses a pharmaceutical composition. The pharmaceutical compositions of the invention can be administered and dosed by the methods of the invention, in accordance with good medical practice, systemically, for example intravenously. It should be noted however that the invention may further encompass additional administration modes. In other examples, the pharmaceutical composition can be introduced to a site by any suitable route including oral, intranasal, or intraocular administration, intraperitoneal, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal.

[0218] In yet some further embodiments, the composition of the invention may optionally further comprise at least one of pharmaceutically acceptable carrier / s, excipient / s, additive / s diluent / s and adjuvant / s. More specifically, pharmaceutical compositions used to treat subjects in need thereof according to the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients, specifically, the compound of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. The pharmaceutical compositions of the present invention also include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question.

[0219] As noted above, the invention provides methods for treating diseases or disorders specified above.

[0220] As used herein, “disease”, “disorder”, “condition” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. It is understood that the interchangeably used terms "associated" and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder, condition or pathology causes a second disease, disorder, condition or pathology.

[0221] The term “treatment” as used herein refers to the administering of a therapeutic amount of the compound or composition of the present invention which is effective to improve one or more undesired symptoms associated with a disease or condition as described herein. Further, the terms "treat”, “treating”, “treatment" or forms thereof, as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from a pathologic disorder. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease. The term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, a condition and illness associated with resistance of insulin receptor as described herein.

[0222] The present invention relates to the treatment of subjects or patients, in need thereof. By “patient” or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the therapeutic and prophylactic methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and rodents, specifically, murine subjects. More specifically, the methods of the invention are intended for mammals. By “mammalian subject” is meant any mammal for which the proposed therapy is desired, including human, livestock, equine, canine, and feline subjects, most specifically humans.

[0223] The term "effective amount” relates to the amount of an active agent being at least one compound present in a composition, that is needed to provide a desired level of active agent in the bloodstream or at the site of action in an individual to be treated to give an anticipated physiological response when such composition is administered. The precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e., the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein. It should be noted that the composition / s of the invention and any components thereof may be applied as a single daily dose or multiple daily doses, or every other day, once a week, once in 10 days, once in 2 weeks, etc.

[0224] The term “alkyl” as used herein refers to a linear, branched saturated hydrocarbon having from 1 to 20 carbon atoms. The term “C7-C72 alkyl” or "C1-C12 alkylene" refers to a linear (straight), branched saturated hydrocarbon having from 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, in some embodiments, contain from 2 to 8 carbons, in yet some embodiments from 2 to 5 carbons, in yet some further embodiments, from 1 to 3 carbon atoms. It should be noted that alkyl refers to an alkyl end chain and alkylene refers to a middle chain alkyl. Representative C1-C12 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, sec-butyl, iso-butyl, tertbutyl, cyclobutyl, pentyl, iso-pentyl, neo-pentyl, tert-pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, sec-octyl (1 -methylheptyl), and cyclooctyl. The alkyl can be substituted or unsubstituted. When substituted, the substituent can be as described herein.

[0225] The term “C7-C72 haloalkyl” as used herein refers to a C1-C12 alkyl as defined above, with one or more hydrogens substituted by halogen atoms.

[0226] The term “alkenyl” as used herein refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon double bond. The term “C2-C12 alkenyl” or "C2-C12 alkenylene" as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms and at least one carbon-carbon double bond, in some embodiments from 3 to 8 carbons, in yet some further embodiments, from 3 to 5 carbon atoms and at least one double bond. It should be noted that alkenyl refers to an alkyl end chain and alkenylene refers to a middle chain alkyl.

[0227] The term “C2-C12 haloalkenyF as used herein refers to a C2-Ci2alkenyl as defined above, with one or more hydrogens substituted by halogen atoms.

[0228] The term “alkynyl” as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon triple bond. The term “C2-C72 alkynyF or "C2-C12 alkynylene" as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms in certain embodiments, from 3 to 8 carbons, and at least one triple bond (at least one carbon-carbon triple bond). It should be noted that alkynyl refers to an alkyl end chain and alkynylene refers to a middle chain alkyl.

[0229] The term “C2-C72 haloalkynyl” as used herein refers to a C2-C12 alkynyl as defined above, with one or more hydrogens substituted by halogen atoms.

[0230] As used herein “alkoxy” refers to an alkyl group bonded to an oxygen atom. Similarly, the term “C1-C12 alkoxyF as used herein refers to a C1-C12 alkyl group linked to an oxygen. At times, the alkyl group may include one to twelve carbon atoms, at times between one to eight carbon atoms, at times one to five carbon atoms and at times one to three carbon atoms. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like. In certain embodiments, the alkoxy is ethoxy.

[0231] The term “C1-C12 haloalkoxy” as used herein refers to a C1-C12 alkoxy as defined above, with one or more hydrogens substituted by halogen atoms.

[0232] The term “halogen” (halo or halide) refers to F, Cl, Br or I.

[0233] The term "cyano" describes a -C=N group.

[0234] The term "amino” as used herein encompass primary, secondary, tertiary or quaternary amines where the point of attachment is through the nitrogen atom which is substituted.

[0235] The term "amide" describes a -C(=O)-NR'R" group, where R' and R" are any substituent as defined herein (for example any one of “R” substitutions).

[0236] As used herein, “nitro” refers to -NO2.

[0237] The term “hydroxy”, as used herein, refers to an -OH group.

[0238] As used herein, “oxo” refers to =0. Each one of the substitutions described herein may be optionally substituted by one or more substituents. The term “optionally substituted" refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated. The term substituted as used herein means that the compounds may contain one or more substituents, including, but not limited to, optionally substituted OH, CF3, halogen, C(=O), -COOH, -NH2, CN, alkyl, alkenyl, alkynyl, alkylene, straight alkenylene, alkynylene, haloalkyl, haloalkenyl, haloalkynyl, alkoxy, carboxyl, halogen, ring system including five to twelve atoms, aromatic or heteroaromatic ring, C(=O)- alkyl.

[0239] It should be noted that the carbon number, as used herein, refers to the carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like.

[0240] As described herein, reference to a compound in accordance with the present disclosure encompass any one of a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof or any combinations thereof.

[0241] The term "pharmaceutically acceptable salt" refers to salts derived from organic and inorganic acids of a compound described herein. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, succinate, fumarate, maleate, malonate, mandelate, malate, phthalate, and pamoate. The term “pharmaceutically acceptable salt” as used herein also refers to a salt of a compound described herein having an acidic functional group, such as a carboxylic acid functional group, and a base. Exemplary bases include, but are not limited to, hydroxide of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or trialkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(Ci-C6)-alkylamine), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D- glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like. The term “pharmaceutically acceptable salt” also includes hydrates of a salt of a compound described herein. The term "small molecule" or “compound” may include pharmaceutically acceptable forms of the recited compounds, including chelates, non-covalent complexes, and mixtures thereof.

[0242] The term “solvate” refers to an aggregate of a molecule with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.

[0243] The term “hydrate” refers to a compound formed by the addition of water. The hydrates may be obtained by any known method in the art by dissolving the compounds in water and recrystallizing them to incorporate water into the crystalline structure.

[0244] The term "stereoisomer" as used herein refers to any isomer that has the same molecular formula and atomic connectivity as a corresponding isomer, but differs in the three-dimensional arrangement of atoms in space. This term includes enantiomers, diastereomers, and geometric (cis-trans) isomers. It should be noted that a compound comprising three flavan-3-ol units such as (+)-catechin-(4a— >8)-(-)-epicatechin-(4p— >8)- (-)-epicatechin (C-EC-EC) is considered a stereoisomer of the compound (-)- epicatechin-(4 — >8)-(-)-epicatechin-(4 — >8)-(+)-catechin (EC-EC-C), due to the differing stereochemistry at the interflavan linkages and at the C2 and C3 positions of each monomer. These stereoisomeric forms exhibit distinct spatial arrangements while maintaining the same molecular formula and connectivity.

[0245] In accordance with some embodiments, the compounds of this invention may include mixtures of enantiomers (possibly as a racemic mixture) as well as purified enantiomers or enantiomerically enriched mixtures. The present invention also encompasses the individual enantiomer(s) (i.e. R or S) of the compounds being represented by the formulas above as racemic mixtures. Methods of preparing substantially isomerically pure compounds are known in the art. If, for instance, a particular enantiomer of a compound of the present disclosure is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. Alternatively, enantiomerically enriched mixtures and pure enantiomeric compounds can be prepared by using synthetic intermediates that are enantiomerically pure in combination with reactions that either leave the stereochemistry at a chiral center unchanged or result in its complete inversion. Techniques for inverting or leaving unchanged a particular stereocenter, and those for resolving mixtures of stereoisomers are well known in the art, and it is well within the ability of one of skill in the art to choose an appropriate method for a particular situation.

[0246] It should be further noted that a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof in the context of the present disclosure are considered to have similar biological or physiological activity as the small molecule to which they relate or any small molecule related thereof, for example, in modulating activity of at least one insulin receptor.

[0247] It should be appreciated that the terms "enhancement", "activation", "stimulation", "increase", "promotion", or "upregulation", as used herein, refer to an elevation or amplification of a biological or biochemical activity or process, such as but not limited to insulin receptor (IR) phosphorylation, AKT activation, glucose uptake, or insulin signaling. The enhancement may be quantified relative to a suitable control (e.g., untreated cells, vehicle-treated cells, or basal activity). Specifically, enhancement may refer to an increase of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to the control. Additionally or alternatively, enhancement may refer to an increase of about 1% to 100% or more, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, about 95% to 99%, about 99% to 100%, or greater than 100% relative to a suitable control (e.g., untreated cells or basal levels). For example, a compound that increases insulin receptor (IR) phosphorylation or AKT activation by 50% compared to vehicle control is considered to activate or enhance such signaling.

[0248] It should be appreciated that the terms "inhibition", "moderation", "reduction", "decrease", "attenuation", or "suppression", as used herein, refer to a diminution, delay, or downregulation of a biological or biochemical activity or process, such as but not limited to insulin receptor (IR) phosphorylation, AKT activation, glucose uptake, inflammatory signaling, or gene expression. The inhibition may be quantified relative to a suitable control (e.g., untreated or stimulated cells). Specifically, inhibition may refer to a decrease of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the control. In some embodiments, complete inhibition (i.e., 100%) may be achieved. These values may be measured using standard quantitative assays, such as ELISA, immunoblotting, kinase assays, or functional cellular readouts. Additionally or alternatively, inhibition may refer to a decrease or reduction of about 1% to 100% or more, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, about 95% to 99%, about 99% to 100%, or greater than 100% relative to a suitable control (e.g., untreated, stimulated, or disease-state cells or tissues).

[0249] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0250] With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.

[0251] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %.

[0252] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0253] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0254] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0255] It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0256] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0257] Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

[0258] Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0259] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention. NON-LIMITING EXAMPLES

[0260] Materials

[0261] IBMX, dexamethasone, insulin, 2-deoxy-D-glucose (2-DG), cytochalasin-B, gallic acid, folin & ciocalteu’s phenol reagent and inhibitors of proteases and phosphatases were all purchased from Merck. BSA and media for cell cultures were obtained from Biological Industries (Beit Haemek, Israel). [3H]-2-deoxy-D-glucose (1 mCi) and Optiphase scintillation solution were purchased from Perkin-Elmer. Hygromycin B was purchased from Thermo Fisher scientific. A recombinant insulin receptor was ordered from R&D Biosystems. HNMPA-(AM)3 was ordered from Abeam. Tormentic acid, ursolic acid, and procyanidins (PCs) were purchased from Cayman. Anti-actin was obtained from MP Biomedicals. Other primary antibodies were obtained from Cell-Signaling Technology. Secondary antibodies were purchased from Jackson ImmunoResearch.

[0262] Methods

[0263] SSE preparation

[0264] S. spinosum root extract was prepared from the plant (Sarcopoterium spinosum (L.) Sp. Rosaceae family) roots, collected from the wild area around Ariel, Israel. The plants were identified by the botanical staff of the University (voucher specimen is deposited in the Israel National Herbarium at the Hebrew University of Jerusalem, No. HUJ 102531). The SSE was prepared and lyophilized as described before (1). The dose for in-vitro and in- vivo studies was selected according to previous publications (1, 2) and cytotoxicity measurements (3).

[0265] Chemical characterization: mass spectrometry

[0266] SSEs of samples collected from the wild area nearby Ariel, Israel, over several months were dissolved in di-deionized water (DDW), filtered through 0.22 pm PTFE Filter and injected to the LCMS. The HRESILCMS spectra were recorded on a Waters (Milford, MA, USA) Xevo G2-XS QTOP instrument equipped with Acquity Hi Class UPLC (binary solvent manager) with an FTN sample manager, column manager, and PDA UV detector, using a 2.1 * 50 mm BEH C18 (1.7 pm) column and a flow of 0.3 mL / minutes. Samples of 10 pl were injected and eluted with a gradient of solvents composed of 100% H2O + 0.1% FA (solvent A) and Acetonitrile + 0.1 % formic acid (solvent B). The elution started with 100% solvent A for five minutes then a linear increase to 100% solvent B over 20 minutes and then return to the starting conditions for additional 2 minutes.

[0267] Bioactivity guided isolation of sarcocyanidin A (1), epicatechin (2), catechin (3) and procyanidin B 1 (4).

[0268] SSE (22.6 g) was separated on a CombiFlash AZ Prep equipped with a reversed-phase C- 18 column (Teledyne ISCO, HP C18Aq, 150 g) in ten consecutive portions. The SSE (2.26 g) was dissolved in 150 mL of di-deionized water (DDW), absorbed on HP C-18 (20 g), evaporated to dryness and packed in a pre-column. The pre-column was installed on top of the column and eluted with a linear gradient from 100% water to 100% methanol and then to 100% ethyl acetate within 60 minutes at a flow of 20 mL / min. The eluant was collected by sample auto-collector to 45 tubes, which were initially combined by the UV absorption into 17 fraction and after examination of their 'H NMR spectra were further combined into 9 fractions. Fractions 4 and 5 (Figure 1), which contained catechin monomers and oligomers and presented a higher percentage of glucose uptake than the SSE (as presented in Figure 2), were thus further separated by size-exclusion chromatography on a Sephadex LH-20 equilibrized and eluted with 1 : 1 water / methanol. Fraction 4 (4.38 g) was separated in five consecutive portions on a Sephadex LH-20 (250 ml, F x h, 3.4 x 28 cm) to yield 15 fractions. Fractions 8 (65.1 mg) and 9 (230.5 mg) from this separation process contained almost pure procyanidin trimer. Fraction 8 was purified on a preparative reversed phase HPLC column (YMC Pack ODS-A, 10 mm, 250 x 20 mm) eluted under isocratic condition (9: 1 aq. formic acid / acetonitrile, 5 mL / min) to produce pure active sarcocyanidin A (1) (3.0 mg, Rt16.5 min). While collecting the NMR data of 1 overnight we noticed that 1 was converted to another product(s), thus we examined other separation conditions (without formic acid) and HPLC column for the separation of fraction 9. Finally, fraction 9 was purified on a preparative reversed phase HPLC column (Phenomenex Luna 5 mm, Phenyl-Hexyl 100A, 250 x 21.2 mm) eluted under isocratic condition (9: 1 water / acetonitrile, 5 mL / min) to produce pure active sarcocyanidin A (1) (10.1 mg, Rt 23.5 min). Fraction 5 from the initial separation (7.83 g) was separated in seven consecutive portions on the Sephadex LH-20 column to afford in fraction 8 (out of fourteen fractions) almost pure catechin derivative (16.2 mg) that was identified by ID and 2D NMR and HRESI MS to be c / vcat echin (2). Combined fractions 6 and 7 (450 mg) from the latter Sephadex LH-20 column were separated on a preparative reversed phase HPLC column (YMC Pack ODS-A, 10 mm, 250 x 20 mm) eluted under isocratic condition (85:15 water / acetonitrile, 10 mm, 5 mL / min) to produce in fraction 5 (22.8 mg, Rt 11.3 min) pure catechin monomer (3) and in fraction 7 (13.6 mg, Rt 13.7 min) pure catechin dimer (4). Compound 3 was identified by ID and 2D NMR and HRESI MS to be catechin, while compound 4 was identified by ID and 2D NMR and HRESI MS to be procyanidin B 1.

[0269] Sarcocyanidin A (1). [a]o20-38.0 (c 0.050, H2O); UV (H2O) Ama\ (loge) 279 (4.4) nm; IR (ATR Diamond) nmax 3233br, 2912, 1708 cm-1; for 'H and13C NMR data, see Table 1; HRESIMS [M-H]“, m / z 865.1970 (calc for C45H380i8, 865.1985).

[0270] Epz-catechin (2). [a]D20-12.4 (c 0.098, MeOH); UV (MeOH) Amax(logs) 280 (3.2) nm; IR (ATR Diamond) vmax 323 Ibr, 2929, 1607, cm-1; for 'H and13C NMR data, see Table 2; HRESIMS [M-H]“, m / z 289.0718 (calc for CI5HI3O6, 289.0712).

[0271] Catechin (3). [a]D2057.1 (c 0.029, MeOH); UV (MeOH) kmax(logs) 279 (3.2) nm; IR (ATR Diamond) Vmax 3256br, 2919, 1610, cm-1; for 'H and13C NMR data, see Table 3; HRESIMS [M-H]“, m / z 289.0711 (calc for CI5HI3O6, 289.0718).

[0272] Procyanidin Bl (4). [a]D2091.4 (c 0.013, H2O); UV (H2O) kmax(logs) 279 (4.1) nm; IR (ATR Diamond) Vmax 3377br, 2922, 1610, cm-1; for 'H and13C NMR data, see Table 4; HRESIMS [M-H]“, m / z 577.1351 (calc for C30H25Oi2, 577.1346).

[0273] Table 2. 2D NMR Data of (-)-epicatechin (2) in DMSO-rU' a500 MHz for 'H. 125 MHz for13C;bH (number) to C in the row;CH (number) to H in the row. a500 MHz for 'H. 125 MHz for13C;bH (number) to C in the row;CH (number) to H in the row.

[0274] Table 4. Procyanidin Bl (4) in Acetone-t4>a a500 MHz for 'H. 125 MHz for13C;bH (number) to C in the row;CH (number) to H in the row.dassigned by comparison with compounds 2 and 3.

[0275] Total polyphenol content analysis

[0276] Polyphenols levels were measured using the Folin-Ciocalteu method (4). The fraction (25 pl) was added to a same volume of Folin-Ciocalteu Reagent and 200 pl water. The mixture was allowed to equilibrate for 5 min and then mixed with 100 pl sodium carbonate solution (20%). The mixture was incubated at 37 °C for 40 min, and the absorbance was read at 750 nm. A standard curve was prepared with Gallic acid (5-40 mg / ml). The results were expressed as pg of catechin equivalents per mg dry material.

[0277] Cell culture

[0278] 3T3-L1 pre-adipocytes (ATCC) were cultured and induced to differentiate as described before (5). L6 myoblasts (ATCC) were grown in MEM-a containing 25 mM glucose, 10% FCS, 2 mM glutamine, and 1% ampicillin. Experiments were performed on differentiated myotubes. L6 differentiation was induced, as described in our previous studies (5). CHO cells overexpressing insulin receptor (CHO-IR) were ordered from ATCC (ATCC CRL-3307). Cells were maintained in growing media (F-12K medium containing 1.26 g / L D-glucose, glutamine (2 mM), Pen-strep (0.9%), inactivated fetal bovine serum (10%), and Hygromycin B (0.3 mg / ml). For experiments, cells were seeded at a concentration of 2.5xl05cell / ml, in media without Hygromycin B. Cells were starved for 18h before analysis in media lacking serum and Hygromycin B.

[0279] Glucose uptake

[0280] Differentiated adipocytes were preincubated for 2h in serum-free DMEM. Starvation media was replaced, and cells were treated with SSE or its fractions (100 pg / ml) for 30 min. Insulin (100 nM) was used as a positive control. Glucose uptake was measured as described in our previous publication (2).

[0281] Preparation of cell lysates and Western blot analysis

[0282] Protein lysates of CHO-IR cells, L6, and 3T3-L1 cells were prepared using Radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Merck) as described. Bradford method was utilized to measure protein concentration. Protein was separated (20 pg per lane) by Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as described before (2).

[0283] Measurement of pIR by ELISA

[0284] L6 myotubes were treated with insulin (100 nM) or SSE (100 pg / ml) for 10 min. Protein lysate was prepared and tyrosine phosphorylation of IR was measured by ELISA kit (Phospho-Insulin receptor P Sandwich ELISA, C70S2, Cell Signaling Technologies) according to the manufacturer's instructions.

[0285] Protein Tyrosine phosphatase activity assay L6 myotubes were treater with insulin (100 nM) or SSE (100 pg / ml) for 5- and 10-min. cells were washed twice in cold PBS, scraped in the presence of anti -proteases and Dithiothreitol (DTT) (2 mM), incubated in ice for 5 min, and vortexed. Cells were centrifuged (4 °C, 10,000 g, 15 min), and supernatant was collected. Phosphatase activity was measured with protein phosphatase assay kit (Abeam, ab241032), according to the manufacturer's instructions. Suramin and phosphatase inhibitor were used as positive control and negative controls, respectively.

[0286] Cellular thermal shift assay

[0287] The assay was done based on the protocol described by Molina DM et. al. (6). CHO-INSR cells were treated for 10 min with insulin, SSE or trimer catechin. Cells were washed with cold PBS, scraped gently and centrifuged (300 g, 3 min), cells were then washed in PBS and centrifuged again. Cells were then diluted in 1 ml PBS, containing antiproteases. The cell suspension was divided into 10 Eppendorf tubes, each was incubated in a PCR machine for 3 min at different temp (43-52 °C). Cells were immediately transferred to ice and lysed by 2 cycles of freeze-and-though. Cells were centrifuged (4 °C, 20,000 g, 20 min), sample buffer was added to the supernatant. The samples were incubated at 70 °C for 10 min, and gel electrophoresis was performed for the detection of IR expression level.

[0288] Fluorescence Quenching measurement

[0289] This measurement is based on autofluorescence of aromatic amino acids, which is affected by protein structure and its binding to small molecules. Fluorescence of IR (0.3 pM) in phosphate buffer (pH 7.4) was measured (Ex / Em=280 / 300-500nm) in the presence or absence of SSE or sarcocyanidin A (1). Fluorescence of denaturized IR was measured by the addition of Guanidine (1 M) to the mixture.

[0290] Effect of SSE, active fraction (Fr 4+5) and trimeric catechin on glucose level in mice

[0291] The study was carried out following the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and it complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The protocol was approved by the Committee on the Ethics of Animal Experiments of Ariel University (permit numbers: IL-2207-124). The effect of a single dose of SSE, its active fraction (Fr 4+5), and trimeric catechin was measured in male C57 / bl6 mice (Envigo, Israel). Experiments had been conducted on male mice, which are more prone for developing obesity-related glucose intolerance than female. The mice were housed in an animal laboratory with a controlled environment of 20-24°C, 45-65% humidity, and a 12 h light / dark cycle. SSE, Fr 4+5 and trimeric catechin were given (intraperitoneal) to non-fasting STD-fed mice (STD, 18% of total calories derived from fat, 24% from proteins, and 58% from carbohydrates. Harlan, Teklad TD.2018). SSE was also given to mice after 8 weeks of HFD feeding (HFD, 60% of total calories derived from fatty acids, 36% saturated, 41% monounsaturated and 23% polyunsaturated; 18.4% from proteins, and 21.3% from carbohydrates, Teklad TD.06414). N=6 mice in each group. Blood glucose was measured each hour for 6 hours and after 24 h. An additional experiment was performed in which mice were killed 6 h after SSE, Fr 4+5, or trimeric catechin administration. Liver and adipose tissue were removed, protein lysate was prepared and Western blot analysis for the detection of insulin signaling was conducted.

[0292] Data and Statistical analysis

[0293] The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. Values are presented as means+SEM. Statistical differences between the treatments and controls were tested by unpaired two- tailed Student's / -test or one-way analysis of variance (ANOVA), followed by Bonferroni's post-hoc testing when appropriate. Analysis was performed using the GraphPad Prism 10.0 software. A difference of p<0.05 or less in the mean values was considered statistically significant.

[0294] Results

[0295] Example 1: Isolation and identification of active compounds.

[0296] Isolation and identification of active compounds were performed using the bioguided fractionation strategy (Figure 1).

[0297] The whole extract was loaded on a Cl 8 column and fractionated by a gradient of H20 / Me0H. According to previous studies, the activity of the isolated fractions was characterized based on their potency in the induction of glucose transport and activation of insulin signaling (2, 3).

[0298] As presented in Figures 2A-2C, fractions 4 and 5 stimulated glucose uptake in 3T3-L1 adipocytes (Figure 2 A) and activated the cascade of insulin signaling in L6 myotubes (Figure 2B), while other fractions were utterly inactive. These two active fractions were found to be rich in phenolic compounds (Figure 2C). Among these phenolics, catechin, epicatechin, procyanidin (PC) dimers and trimers were identified by LCMS (Table 1, Figure 3). Specifically, the active dimer was identified as the known procyanidin Bl, which is (-)-Epicatechin-(4P-8)-(+)-catechin dimer, and the trimer structure was decoded to be a new trimer, (+)-catechin-4a-8-(-)-epicatechin-4P-8-(-)- epicatechin (Sarcocyanidin A), as detailed below.

[0299] Table 1. Retention times and molecular ions of typical components of SSE. a23-HTAEG: 23-hydroxytormentic acid ester glycoside;b3 -Gal-23 -HT AEG: 3-galoyl-23- hydroxytormentic acid ester glycoside;c23-HTAEG-24-oate: 23-hydroxytormentic acid ester glycoside- 24-oate;d23-HTA: 23-hydroxytormentic acid;e[M+formate].

[0300] Structure elucidation of sarcocyanidin A (1)

[0301] Sarcocyanidin A (1) was isolated as a reddish amorphous solid which exhibited a negative HRESIMS molecular ion, [M-H]’ at m / z 865.1970 corresponding to the molecular formula C45H38O18 and 27 degrees of unsaturation. The latter molecular formula was in agreement with a catechin trimer. The13C NMR spectrum of 1 in D2O (Table 5) presented signals of 45 carbons: eighteen SP2quaternary carbon between 159 and 132 ppm; nine SP2methine carbons between 123 and 113 ppm, five SP2quaternary carbon between 111 and 101 ppm; four SP2methine carbons between 99 and 96 ppm; eight SP3methine carbons between 85 and 38 ppm and a single methylene carbon at 23.7 ppm. The4H NMR spectrum of 1 in D2O (Table 5) presented signals of thirteen SP2methine protons between 6.70 and 5.09 ppm, six of which broad doublet and the rest singlets, eight SP3methine protons between 4.90 and 3.45 ppm, and two broad doublet protons of a single SP3methylene resonating at 2.38 and 1.95 ppm. The correlation from the 2D NMR COSY spectrum allowed to propose four fragments (Figure 4A): (a, H-I-4 to H-I-3, H-I-3 to H-I-2, H-I-2 (4. / ) to H-I-2’, H-I-2’( ) to H-I-6’, and H-I-6’ to H- 1-5’), (b, H-II-4 to H-II-3, H-II-3 to H-II-2, H-II-2 (4. / ) to H-II-2’ and H-II-6’, H-II-2’(4J) to H-II-6’, and H-II-6’ to H-II-5’), (c, H-III-4 to H-III-3, H-III-3 to H-III-2, H-III-2 (4. / ) to H-III-2’ and H-III-6’, H-III-2’(4J) to H-III-6’, and H-III-6’ to H-III-5’), and (d, H-I-6 (4. / ) to H-I-8). Two singlet SP2protons didn’t show any correlation in the COSY spectrum. Analysis of the correlations from the HSQC and HMBC 2D spectra (Table 5 and Figure 5) allowed us to construct from the latter four fragments and the two unpaired protons three catechin-type fragments. Comparison of the . / -values of the protons connected to the SP3carbons of the latter three fragments of 1 with those of e z-catechin (2), catechin (3) and procyanidin C4 (7) allowed to identify the three fragments as: da- substituted (+)-catechin, 4b,8-disubstituted (-(-e zcatechin and 8-substituted (-)- e / zzcatechin. HMBC correlation of H-I-3 and -4 with C-II-8 and of H-I-4 with C-II-8a allow to connect the (+)-catechin to the disubstituted (-(-e zcatechin and HMBC correlations of H-II-4 with C-III-7 and -8 allowed to connect the disubstituted (-)- e zcatechin with the (-(-c / vcatechin. The structure of sarcocyanidin A (1) was thus established as catechin-(4a-8)-e / ?zcatechin-(4b-8)-e / ?zcatechin (Fig 4B). The structure assignment was supported as well by the NOE’s from a ROESY experiment (Figure 6).

[0302] Table 5. NMR data of sarcocyanidin A (1) in D2Oa

[0303]

[0304] a500 MHz for 'H, 125 MHz for13C;bH (number) to C in the row;CH (number) to H in the row.dAssigned by comparison with compounds 2 and 3.

[0305] Example 2: Evaluation of the effect of the isolated components

[0306] To evaluate the contribution of each specific polyphenolic compound to the antidiabetic properties of the whole extract, the activity of these compounds was measured by a set of analyses. The capability of these compounds to activate insulin signaling was measured in Cho-IR cells, which are highly sensitive to the stimulation of insulin signaling. Initial, dose-response and time-course of insulin signaling activation induced by insulin and SSE in these cells were characterized (Figures 7A and B, supplementary data). A dose-dependent effect of insulin-induced IR phosphorylation is presented, showing that 10 nM of insulin is sufficient to achieve a maximal effect. IR phosphorylation was minimal with 1 nM insulin, although AKT phosphorylation was highly stimulated also upon this low-dose insulin treatment, suggesting that the limited extent of IR phosphorylation induced by 1 nM insulin is sufficient to induce maximal phosphorylation of AKT.

[0307] SSE treatment stimulated IR phosphorylation only at the highest dose used (100 pg / ml), while AKT was phosphorylated in a dose-dependent manner within the 2-100 pg / ml range. IR phosphorylation was detected following insulin and SSE treatment at all time points measured (2-15 min, Fig 6B). Accordingly, all additional experiments were conducted following 10 min incubation of insulin or SSE.

[0308] Monomeric, dimeric, and trimeric forms of PCs were detected in the active fraction of SSE. Among the monomers, (-)-epicatechin, isolated from the extract, was found to be effective in the stimulation of IR and AKT phosphorylation and glucose uptake, with higher potency than (+)-catechin (Figures 8A and 8F). Next, the activity of PC-B1, which was detected in the extract, was compared to other known dimers; PC-B2 and B3. The activity of PC-B1 was compared to other catechin dimers (PCs-B2 and B3); all were commercially purchased (Cayman Chemical, Ann Arbor, USA, purity >98%). PC-B 1 was found to be the most effective among the three di-catechins investigated. This was concluded based on its capability to induce IR and AKT phosphorylation at lower dose than that of PC-B2 and B3, and to stimulate glucose uptake in 3T3-L1 adipocytes (Figures 8B-D and E). Lastly, the trimeric PC isolated, sarcocyanidin A (1), was highly effective in the induction of IR and AKT phosphorylation and stimulation of glucose transport, (Figures 8E and 8F).

[0309] Previous studies demonstrated that the pentacyclic terpenoids tormentic acid and hydroxy -tormentic acid, ursolic acid, and hydroxy -ursolic acid are major components of SSE (8). Derivatives of tormentic acid were also detected by us by LCMS (Table 1, Figure 3). Although these compounds were not detected in the active fractions (fractions 4 & 5), because of their high presence in SSE, as shown here for 23-HTAE, their bioactivity was also measured (Figures 9A-9C). As shown in Figures 9A-9C, these molecules did not stimulate insulin signaling in CHO-IR cells. Therefore, their effect on glucose transport was not measured.

[0310] In the next part of the study, the mechanism of action of SSE, and its active composite, sarcocyanidin A (1) was investigated.

[0311] IR phosphorylation was measured by sandwich ELISA, in which the wells are coated with a specific anti-IR antibody, and phosphorylation of bound receptors is detected by an antibody specific for phosphorylated tyrosine. This approach is required to validate the specific phosphorylation of IR because of the cross-reactivity of the pIR antibodies with pIGF. As shown (Figure 10A), SSE induced a 2.17-fold increase in IR phosphorylation compared to a 15.89-fold increase by insulin.

[0312] The increase in IR phosphorylation might result from a direct binding and receptor activation or from inhibition of Phospho-tyrosine phosphatase IB (PTP1B), which is the specific phosphatase of IR. To elucidate whether the effect of SSE on IR is mediated through phosphatase inhibition, the activity of PTP was measured in L6 myocytes (Figure 10B). As observed, both insulin and SSE increased PTP activity, presumably representing the activation of the downregulating response required to attenuate insulin signaling following its stimulation. Therefore, it might be concluded that SSE-induced IR phosphorylation is not mediated through phosphatase inhibition and, hence, is presumably achieved through direct activation of the receptor by specific composites of SSE.

[0313] To clarify whether activation of IR mediates the stimulatory effect of SSE and trimeric sarcocyanidin A (1) on insulin signaling, phosphorylation of IR and AKT was measured in the presence of an IR inhibitor (HNMPA(AM)3) in both CHO-IR and L6 cells (Fig 10B and C). HNMPA(AM)3 at a concentration of 100 pM inhibited insulin- induced IR and AKT phosphorylation (1 nM or 10 nM insulin in CHO-IR or L6 cells, respectively). Interestingly, SSE-induced IR and AKT phosphorylation were stimulated with HNMPA(AM)3 given at a dose of 50 pM, while a higher dose (100 pM) abolished SSE effects. Similarly, the phosphorylation of Akt, induced by sarcocyanidin A (1), was abolished by IR inhibitor. These results indicate that insulin receptor activation mediates SSE-induced AKT phosphorylation.

[0314] The presence of direct binding between SSE and IR was investigated by the measurements of IR autofluorescence and by the cellular thermal shift assay (CTSA). Like the insulin effect, SSE attenuated IR autofluorescence in a dose-dependent manner, indicating that a composite of SSE directly binds IR, leading to a change in its conformation and the resulting autofluorescence (Figures 11 A and 1 IB). This effect was abolished in the presence of the denaturizing agent guanidine, indicating that the accurate protein structure of IR is required for SSE binding (Figure 11C). A similar effect of a reduction in IR autofluorescence was observed with sarcocyanidin A (Figure 11D). Cellular thermal-shift assay (CTSA) also validated the binding of IR to a certain composite of SSE.

[0315] This assay is based on the principle of heat inducing protein denaturation, forming aggregates that sediment upon centrifugation, in contrast to native proteins that are soluble and are preserved in the supernatant. The stability of proteins is expected to be enhanced upon binding to other molecules, leading to a delay in heat-dependent denaturation and sedimentation (Figure 1 IE)

[0316] In this assay, CHO-IR cells were treated with SSE or sarcocyanidin A (1), and then cell lysates were heated to different temperatures (43-52 °C). The denatured proteins were removed by centrifugation, and only the soluble proteins were analyzed by Western blot.

[0317] As presented in Figures 1 IF and 11G, heating the control lysates to temperatures over 43 °C led to a lower level of IR in the supernatant, indicating the denaturation of the receptor at this temperature. However, pretreatment with either insulin, SSE, or sarcocyanidin A (1) increased the stability of IR to heat-induced denaturation, indicating the binding of sarcocyanidin A (1) or some additional components of SSE to the receptor.

[0318] The ability of SSE to activate insulin signaling was validated in-vivo. SSE was administrated to normoglycemic c57 / bl mice at increasing doses, and blood glucose was monitored (Figures 12A and 12B). A significant reduction in blood glucose was observed in SSE-treated mice after 5-7 h at doses of 1.5 and 3 mg, while it was not observed by 6 mg SSE, a dose that even induced an elevation in blood glucose, presumably indicating a toxic effect of this dose. Glucose levels returned to baseline after 24 h.

[0319] An elevation in insulin levels did not accompany the reduction in blood glucose (Figure 12C), indicating that the glucose-lowering properties resulted from the activation of insulin signaling rather than stimulating the secretion of the hormone. This assumption is supported by the results of Western blot analysis (Figure 12D), showing a higher AKT phosphorylation in the liver and subcutaneous (SC) WAT in response to SSE administration. Similar results were obtained in response to administering the phenol-rich fraction and sarcocyanidin A (1) (Figures 12E and 12F). The glucose-lowering properties of SSE, phenol-rich fraction, and Sarcocyanidin A (1) were also demonstrated in HFD- fed mice (Fig 12F). While SSE induced an early hyperglycemic response, which precedes the glucose-lowering effect, this phenomenon was not found in response to phenol-rich fraction and sarcocyanidin A administration.

[0320] Example 3: Mouse model of insulin depletion.

[0321] T1D is induced by of streptozotocin (STZ)-treated mice in male C57BL6J mice (10-12 weeks old) according to the multiple low-dose regimen. According to this protocol, STZ (55 mg / kg) is administered intraperitoneally for 5 consecutive days. Blood glucose is e measured after 7 days, and mice with blood glucose >250 mg / dL will be included into experimental groups as follows (n=6 in each group): Untreated, Sarcocyanidin A, SSE (50 and 100 mg / day) and Sarcocyanidin A-rich fraction (SRF) (15 and 30 mg / day). Acute and chronic effects is measured. For the measurement of the acute effect, Sarcocyanidin A, SSE and SRF are given by gavage, and blood glucose is measured for the following 6 h, assuming that blood glucose will be reduced upon Compound A, SSE and SRF administration (set 1). In an additional cohort (set 2), mice are given one-dose treatment and are sacrificed at the optimal time (according to set 1). Activation of insulin signaling is measured by Western blot analysis in the liver and adipose tissues. Blood insulin is measured to confirm the absence of the hormone.

[0322] To measure chronic effects, Sarcocyanidin A, SSE and SRF are given for 7 days in drinking water (set 3). Daily measurements are performed for body weight and blood glucose (a drop taken from the tail using a glucometer). GTT is conducted after 7 days of treatment. These experiments enable to evaluate the efficacy of Sarcocyanidin A, SSE and SRF in activating insulin signaling in the absence of insulin and mitigating severe STZ-induced hyperglycemia.

[0323] Discussion

[0324] Sarcopoterium spinosum root extract is used by Bedouin traditional medicinal practitioners as an antidiabetic drug. Experiments conducted in cells and in mice models support the potential therapeutic properties of this plant extract for the management of blood glucose (1, 8, 9, 2). However, despite the progress achieved in the research of its anti-diabetic properties, the active composites responsible for the therapeutic function of SSE have not been identified before. In this study, the inventors successfully isolated the active fraction, identified a novel active compound, and demonstrated its direct stimulatory effect on the insulin receptor.

[0325] Bio-guided fractionation approach revealed that phenolic-rich fraction is responsible for the anti-diabetic properties of SSE. This fraction stimulated the transmission of insulin signaling and induced glucose uptake in cells, even with a higher potency than achieved by the whole extract, indicating that components of this fraction are sufficient, while those of other fractions are not required, to achieve the insulinmimetic properties of SSE.

[0326] Additional fractionation steps were conducted after identifying the phenol-rich fractions as the active ones. These fractions (combined fraction 4+5) were separated by size-exclusion chromatography to several subfractions, and their bioactivity was analyzed. This bio-guided fractionation strategy enables the identification of active compounds, all of which are phenolics of the flavanols family. The inventors found that monomers, dimers, and trimers of flavonols, including a novel procyanidin trimer, activated insulin signaling.

[0327] The novelty of this study stems from the identification of a novel procyanidin trimer. This PC trimer, isolated from SSE, was found to be highly effective in the induction of IR and Akt phosphorylation. This trimer was named sarcocyanidin A (1), and identified as (+)-catechin-4P-8-(-)-epicatechin-4P-8-(-)-epicatechin, which differs from the already -known natural procyanidin trimers PC-C1 (an epicatechin trimer), and PC-C2 (a catechin trimer). In addition, it also differs from the procyanidin trimers epicatechin-(4P-8)-epicatechin-(4P-8)-catechin trimer and epicatechin-(4P-8)-catechin- (4a-8)-epicatechin trimer which were synthetically attained with a condensation reaction 10). Therefore, the identification of sarcocyanidin A (1) in SSE is the first documentation of this PC trimer, either naturally or synthetically achieved.

[0328] The monomer (-)-epicatechin was more effective than its enantiomer (+)-catechin, demonstrating a higher induction of Akt phosphorylation and glucose uptake. However, both these two enantiomers did not stimulate the phosphorylation of IR over control. Among PC dimers, PC Bl, a dimer of (-)-epicatechin- and (+)-catechin, which was the PC detected in the active fraction of SSE, was the most effective PC dimer and the only one that successfully stimulated IR phosphorylation and glucose uptake.

[0329] These results, demonstrating that dimer and oligomer of flavanols are more active in the activation of insulin signaling cascade than monomers, are in line with previous studies, showing that oligomeric PC-rich extracts stimulated glucose uptake through an IR and Akt-dependent mechanism (11, 12). Oligomer-rich fraction isolated from cocoa beans (enriched for dimers and PCs with 3-6 polymerization degree) was proved to be more effective in preventing glucose intolerance and insulin resistance in mice than either monomeric or polymeric-enriched fractions (13). These results support the advantage of dimeric and oligomeric PCs over monomers and polymers in activating insulin signaling.

[0330] However, while most studies investigated the potency of PC-rich fractions rather than isolated compounds [10, 11], in this study, the inventors could isolate and identify the specific PC dimer and trimer exerting the insulin-mimetic function. The inventors found that PC-B1 is more potent than PC-B2 and PC-B3 in activating insulin signaling. To the best of our knowledge, there is no previous study in which the insulin-mimetic activity of PC dimers was compared. The identification of PC-B1 in SSE, rather than the less potent PC-B2 and PC-B3, might explain its high efficacy as an anti-diabetic plant. Regarding the trimer, a previous study demonstrated that PC-C1, an epicatechin trimer, induced Akt phosphorylation and glucose transport and facilitated glucose disposal from the blood after glucose load more efficiently than monomer and dimer of epicatechin (14, 15). However, the effect of PC-C1 on IR phosphorylation was not presented, in difference from this data, demonstrating that the SSE-isolated trimer (+)-catechin-4P-8-(-)- epicatechin-4P-8-(-)-epicatechin mimics insulin action through its binding to the IR.

[0331] Interestingly, the pentacyclic triterpenes tormentic and ursolic acids, known constituents of SSE 16), did not activate insulin signaling at all. Several biological activities had been attributed to tormentic and ursolic acid in previous studies, including hypoglycemic properties, which are achieved through stimulation of insulin secretion (17-20). The capability of SSE to induce insulin secretion in a cell-line of beta cells was demonstrated by us in a previous study (12). However, in-vivo experiments conducted on mice models of glucose intolerance and type 2 diabetes clearly demonstrated that the antidiabetic properties of SSE are mainly achieved through amelioration of insulin sensitivity rather than stimulating insulin secretion (11, 13, 14). The innvetors assumed that the positive effects of tormentic and ursolic acid on managing blood glucose in insulinresistant conditions stem from the anti-inflammatory properties of these compounds (18, 21-25).

[0332] Since T2D is accompanied by an elevated, chronic inflammation, which worsens the metabolic alterations and promotes the development of diabetic complications, molecules with anti-inflammatory properties might work synergistically with insulinmimetic molecules to improve the metabolic health of people with T2D (26). The unique composition of SSE, including insulin-mimetic compounds and anti-inflammatory characteristics, as demonstrated in the inventors previous studies (9, 2, 28), provides this extract its high anti-diabetic potency. However, SSE likely contains additional components that either do not contribute to its glucose-lowering effects or may even counteract the actions of sarcocyanidin A, PC-B1, and other antidiabetic compounds within the extract. Notably, administration of the isolated active fraction or sarcocyanidin A (1) to mice enhances the glucose-lowering effect of Sarcopoterium spinosum. Therefore, identifying the active fraction and active molecules, achieved in this study, may facilitate the development of more potent SSE-based antidiabetic formulations.

[0333] In this study, the inventors demonstrated that IR is a target of SSE and sarcocyanidin A (1). IR inhibition abrogated SSE-induced phosphorylation of Akt, demonstrating the role of IR in mediating SSE effect on insulin signaling. A direct binding of SSE and sarcocyanidin A (1) was demonstrated by CTSA, in which an elevated thermal stability of IR was achieved in the presence of SSE and the trimeric PC. In addition, autofluorescence of native IR was reduced in the presence of SSE and the trimeric PC, while this was not observed with denaturized IR, indicating that the three-dimensional structure of IR is necessary to enable binding. This result of IR being activated by sarcocyanidin A (1) is in line with a previous study, showing a similar effect of grape-seed procyanidin extract (GSPE) (11). However, while the GSPE is a mix composed of monomers, dimers, trimers, tetramers, and longer oligomers, our data demonstrated for the first time the stimulatory effect of an isolated procyanidin trimer on IR phosphorylation.

[0334] In summary, this study identified the active components of SSE. PC-B1 and the novel PC trimer sarcocyanidin A (1) are the most potent of these molecules, leading to the activation of insulin signaling. The insulin receptor is stimulated by sarcocyanidin A (1).

[0335] References:

[0336] 1. Rozenberg K, Rosenzweig T. Sarcopoterium spinosum extract improved insulin sensitivity in mice models of glucose intolerance and diabetes. PloS one. 2018;13(5):e0196736.

[0337] 2. Ben-Shachar M, Rozenberg K, Skalka N, Wollman A, Michlin M, Rosenzweig T. Activation of Insulin Signaling in Adipocytes and Myotubes by Sarcopoterium Spinosum Extract. Nutrients. 2019; 11 (6).

[0338] 3. Elyasiyan U, Nudel A, Skalka N, Rozenberg K, Drori E, Oppenheimer R, et al. Anti-diabetic activity of aerial parts of Sarcopoterium spinosum. BMC Complement Altern Med. 2017; I7(I):356.

[0339] 4. Dag A, Ben-Gal A, Yermiyahu U, Basheer L, Nir Y, Kerem Z. The effect of irrigation level and harvest mechanization on virgin olive oil quality in a traditional rain- fed ‘Souri’ olive orchard converted to irrigation. Journal of the science of food and agriculture. 2008;88(9): 1524-8. 5. Rozenberg K, Smirin P, Sampson SR, Rosenzweig T. Insulin-sensitizing and insulin-mimetic activities of Sarcopoterium spinosum extract. Journal of ethnopharmacology. 2014.

[0340] 6. Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundback T, Nordlund P, et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat Protoc. 2014;9(9):2100-22.

[0341] 7. Wang C-M, Hsu Y-M, Jhan Y-L, Tsai S-J, Lin S-X, Su C-H, et al. Structure Elucidation of Procyanidins Isolated from Rhododendron formosanum and Their AntiOxidative and Anti-Bacterial Activities. Molecules. 2015;20(7): 12787-803.

[0342] 8. Smirin P, Taler D, Abitbol G, Brutman-Barazani T, Kerem Z, Sampson SR, et al. Sarcopoterium spinosum extract as an antidiabetic agent: in vitro and in vivo study. Journal of ethnopharmacology. 2010; 129(1): 10-7.

[0343] 10. Saito A, Tanaka A, Ubukata M, Nakajima N. Efficient stereoselective synthesis of proanthocyanidin trimers with TMSOTf-catalyzed intermolecular condensation. Synlett. 2004(6): 1069-73.

[0344] 11. Montagut G, Onnockx S, Vaque M, Blade C, Blay M, Fernandez -Larrea J, et al. Oligomers of grape-seed procyanidin extract activate the insulin receptor and key targets of the insulin signaling pathway differently from insulin. The Journal of nutritional biochemistry. 2010;21(6):476-81.

[0345] 12. Pinent M, Blade MC, Salvado MJ, Arola L, Ardevol A. Metabolic Fate of Glucose on 3T3-L1 Adipocytes Treated with Grape Seed-Derived Procyanidin Extract (GSPE). Comparison with the Effects of Insulin. Journal of Agricultural and Food Chemistry. 2005;53(15):5932-5.

[0346] 13. Dorenkott MR, Griffin LE, Goodrich KM, Thompson-Witrick KA, Fundaro G, Ye L, et al. Oligomeric cocoa procyanidins possess enhanced bioactivity compared to monomeric and polymeric cocoa procyanidins for preventing the development of obesity, insulin resistance, and impaired glucose tolerance during high-fat feeding. J Agric Food Chem. 2014;62(10):2216-27.

[0347] 14. Yamashita Y, Wang L, Nanba F, Ito C, Toda T, Ashida H. Procyanidin Promotes Translocation of Glucose Transporter 4 in Muscle of Mice through Activation of Insulin and AMPK Signaling Pathways. PloS one. 2016;! l(9):e0161704. 15. Sun P, Li K, Wang T, Ji J, Wang Y, Chen K-X, et al. Procyanidin Cl, a Component of Cinnamon Extracts, Is a Potential Insulin Sensitizer That Targets Adipocytes. Journal of Agricultural and Food Chemistry. 2019;67(32):8839-46.

[0348] 16. Reher G, Reznicek G, Baumann A. Triterpenoids from Sarcopoterium spinosum and Sanguisorba minor. Planta Med. 1991;57(5):506.

[0349] 17. Ivorra MD, Paya M, Villar A. Effect of tormentic acid on insulin secretion in isolated rat islets of langerhans. Phytotherapy Research. 1989;3(4): 145-7.

[0350] 18. Olech M, Ziemichod W, Nowacka-Jechalke N. The Occurrence and Biological Activity of Tormentic Acid-A Review. Molecules. 2021;26(13).

[0351] 19. Ivorra MD, Paya M, Villar A. Hypoglycemic and insulin release effects of tormentic acid: a new hypoglycemic natural product. Planta Med. 1988;54(4):282-5.

[0352] 20. Castro AJ, Frederico MJ, Cazarolli LH, Mendes CP, Bretanha LC, Schmidt EC, et al. The mechanism of action of ursolic acid as insulin secretagogue and insulinomimetic is mediated by cross-talk between calcium and kinases to regulate glucose balance. Biochim Biophys Acta. 2015; 1850(1): 51 -61.

[0353] 21. Wang YL, Sun GY, Zhang Y, He JJ, Zheng S, Lin JN. Tormentic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NF-kappaB signaling pathway. Mol Med Rep. 2016;14(4):3559-64.

[0354] 22. Yang Y, Wang Y, Wang Y, Zhao M, Jia H, Li B, et al. Tormentic Acid Inhibits IL-lbeta-Induced Inflammatory Response in Human Osteoarthritic Chondrocytes. Inflammation. 2016;39(3): 1151 -9.

[0355] 23. Gonzalez-Garibay AS, Lopez-Vazquez A, Garcia-Banuelos J, Sanchez-Enriquez S, Sandoval-Rodriguez AS, Del Toro Arreola S, et al. Effect of Ursolic Acid on Insulin Resistance and Hyperinsulinemia in Rats with Diet-Induced Obesity: Role of Adipokines Expression. J Med Food. 2020;23(3):297-304.

[0356] 24 Jian CX, Li MZ, Zheng WY, He Y, Ren Y, Wu ZM, et al. Tormentic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts via inhibition of TLR4-mediated NF-kappaB and MAPK signalling pathway. Arch Oral Biol. 2015;60(9): 1327-32. 25. Checker R, Sandur SK, Sharma D, Patwardhan RS, Jayakumar S, Kohli V, et al. Potent anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression ofNF-KB, AP-1 and NF-AT. PloS one. 2012;7(2):e31318.

[0357] 26. Ramirez-Rodriguez AM, Gonzalez-Ortiz M, Martinez-Abundis E, Acuna Ortega N. Effect of Ursolic Acid on Metabolic Syndrome, Insulin Sensitivity, and Inflammation. J Med Food. 2017;20(9):882-6.

[0358] 27. Rozenberg K, Wollman A, Ben-Shachar M, Argaev-Frenkel L, Rosenzweig T. Anti-inflammatory effects of Sarcopoterium spinosum extract. Journal of ethnopharmacology. 2019: 112391.

Claims

CLAIMS:

1. A compound represented by Formula (III),or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof, wherein each of Ri, R2, R4, Rs, R7, and Rs, is independently hydrogen, cyano, amino, amide, nitro, hydroxy, oxo, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, or OR10, each of R3, Rs, R9, and Rio is independently hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy, n is 1 to 4, m is 1 to 5, wherein at least one flavan-3-ol ring system is in a trans configuration and at least one flavan-3-ol ring system is in a cis configuration and wherein the compound is an insulin mimetic compound.

2. The compound of claim 1 having the general Formula (IV):or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof, or physiologically functional derivative thereof.

3. The compound of claim 1 or 2, wherein each of R2, Rs, and Rs is OR10, Rio is independently selected from hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy.

4. The compound of claim 1 or 2, wherein each one of Ri, R4, and R7 is OR10, Rio is independently hydrogen, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy.

5. The compound of any one of claims 1 to 4, wherein n is 2.

6. The compound of any one of claims 1 to 4, wherein m is 2.

7. The compound of any one of claims 1 to 6, represented by Formula (X)Wherein each of R3, Re, R9, R11, R12, R13, R14, Ris, Rie, R17, Ris, R19, R20, R21 and R22 isindependently hydrogen, cyano, amino, amide, nitro, halo, C1-C12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 haloalkenyl, C2-Ci2alkynyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C12 haloalkoxy.

8. The compound of claim 7, wherein each of Rn, R12, R13, R14, R15, Ri6, R17, Ris, R19, R20, R21 and R22 is independently hydrogen or C1-C12 alkyl.

9. The compound of claim 8, wherein each of Rn, R12, R13, R14, R15, Ri6, R17, Ris, R19, R20, R21 and R22 is hydrogen.

10. The compound of any one of claims 1 to 9, wherein each of R3, Rs, R9, is hydrogen or Ci-C 12 alkyl.

11. The compound of any one of claims 1 to 10, wherein each of R3, Re, R9, is hydrogen.

12. The compound of any one of claims 1 to 11, represented by Formula (XX)or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or physiologically functional derivative thereof.

13. The compound of any one of claims 1 to 12 for use in a method of inducing insulin singling.

14. The compound of any one of claims 1 to 12 for use in a method of inducing insulin receptor phosphorylation.

15. The compound of any one of claims 1 to 12 for use in a method of inducing AKT phosphorylation.

16. The compound of any one of claims 1 to 12 for use in a method of reducing bloodglucose level.

17. The compound of any one of claims 1 to 12 for use in a method of increasing glucose uptake.

18. The compound of any one of claims 1 to 12 for use in a method of treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with impaired insulin signaling in a subject in need thereof.

19. The compound of any one of claims 1 to 12 for use in a method of treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an insulin-resistance disease in a subject in need thereof.

20. The compound of any one of claims 1 to 12 for use in a method of treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a metabolic disorder in a subject in need thereof.

21. The compound for use of any one of claims 18 to 20, wherein said disease is one or more of (i) diabetes mellitus and pre-diabetic conditions, (ii) hormonal or reproductive disorders, (iii) hepatic or metabolic disorders, (iv) cardiovascular or renal disorders, (v) neurological or cognitive disorders, (vi) genetic or congenital disorders, (vii) acute or stress-related conditions involving transient insulin resistance or (viii) any combination thereof.

22. The compound for use of claim 21, wherein said diabetes mellitus or a pre-diabetic condition is one or more of type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), prediabetes, impaired glucose tolerance (IGT), impaired fasting glucose (IFG), latent autoimmune diabetes in adults (LADA) or any combination thereof.

23. The compound for use of claim 22, wherein said diabetes mellitus or a pre-diabetic condition is T1DM.

24. The compound for use of claim 22, wherein said diabetes mellitus or a pre-diabetic condition is T2DM.

25. The compound of any one of claims 1 to 24 for use in a method of treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T2DM in a subject in need thereof.

26. The compound of any one of claims 1 to 24 for use in a method of treating,preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of T1DM in a subject in need thereof.

27. A composition comprising an effective amount of at least one compound of any one of claims 1 to 12 or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with impaired insulin signaling in a subject in need thereof.

28. A composition comprising an effective amount of at least one compound of any one of claims 1 to 12 or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an insulin-resistance disease in a subject in need thereof.

29. A method of any one of (i) inducing insulin singling in a cell, (ii) inducing insulin receptor phosphorylation in a cell, (iii) inducing AKT phosphorylation in a cell, wherein said method comprises the step of contacting said cell with an effective amount of at least one compound of any one of claims 1 to 12.

30. The method of claim 29, wherein said cell is from a subject suffering from or diagnosed with a disease associated with impaired insulin signaling and / or a metabolic disorder and / or an insulin resistance disease.

31. The method of claim 29 or 30, wherein said cell is one or more of 3T3-L1 adipocytes, L6 myotubes.

32. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a disease associated with impaired insulin signaling in a subject in need thereof, said method comprises administering to said subject a therapeutically effective amount of at least one compound of any one of claims 1 to 12 or any composition comprising the same.

33. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an insulin-resistance disease in a subject in need thereof, said method comprises administering to said subject a therapeutically effective amount of at least one compound of any one of claims 1 to 12 or any composition comprising the same.

34. The method of claim 32 or 33, wherein said subject is suffering or diagnosed with a disease associated with impaired insulin signaling and / or a metabolic disorder and / or an insulin resistance disease.

35. The method of any one of claims 32 to 34, wherein said disease is T1DM or T2DM.

36. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a metabolic disorder in a subject in need thereof, said method comprises administering to said subject a therapeutically effective amount of at least one compound of any one of claims 1 to 12 or any composition comprising the same.

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