ES2866225A1 - TRITERPENES DERIVATIVES WITH THERAPEUTIC ACTIVITY (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Triterpene derivatives with therapeutic activity. The present invention relates to compounds of formula (1) exhibiting therapeutic activity, as well as to uses thereof to treat inflammatory diseases, chronic diseases, metabolic syndrome, obesity, diabetes and/or cancer. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

TRITERPENE DERIVATIVES WITH THERAPEUTIC ACTIVITY

FIELD OF THE INVENTION

The present invention belongs to the field of biology and medicine. In particular, it refers to triterpene derivatives with therapeutic activity.

BACKGROUND OF THE INVENTION

Triterpenes are the most abundant group of natural plant products, with a wide variety of biological activities. Many triterpene derivatives, known as triterpenoids, are biologically active and have shown interesting anti-inflammatory properties. Examples of such triterpenoids include CDDO, avicins, amyrins, betulinic acid, boswellic acid, celastrol, diosgenin, madecasic acid, maslinic acid, momordine, saikosaponins, plathicodone, pristimerin, ursolic acid, and withanolide.

However, the pharmacology of triterpenoids is complex. These compounds show highly varied pharmacokinetic properties and biological profiles, some of which may hamper the exploitation of their therapeutic and industrial potential. For example, cyano-3,12-dioxolean-1,9 (11) -diene-28-oic methyl ester (CDDO-Me), a semisynthetic derivative of oleanoic acid with inhibitory activity of the NF- ^k B pathway, subsequently presented important adverse effects that led to the termination of a phase 3 clinical study. Betulinic acid, with proven anti-inflammatory and antineoplastic properties, has been shown to have limited industrial potential due to its low water solubility and pharmacokinetic properties (absorption, distribution , metabolism, elimination).

Since the pharmacology and biological activity profiles of known triterpenoid derivatives vary, and because there is an unmet medical need for a variety of diseases, it is desirable to synthesize new compounds with various structures that are useful for the treatment of diseases.

Maslinic acid is a pentacyclic terpenic acid that is widely present in edible plants, and is found abundantly in the skin of the fruit of the olive tree and hawthorn. The compound has attracted much interest due to its proven pharmacological safety and its anti-inflammatory, anti-viral, anti-oxidant, anti-diabetogenic, anti-colon cancer and astrocytoma activity. In the state of the art there are patents that describe maslinic acid as antitumor, apoptosis inducer and carcinogen, such as US2003153538A, WO03057224A1, CN102106861A and CN102008715B. However, to the best of the inventors' understanding, no derivatives of maslinic acid have been synthesized.

Oleanolic acid is another triterpenoid present in natural plants in the form of the free acid or aglycones for triterpenoid saponins. It has weak anti-inflammatory and anti-tumor properties in vivo; however, although very potent synthetic derivatives have been synthesized, to date, none have passed the clinical studies necessary to produce a real therapeutic solution. Examples of this type of oleanolic derivatives can be found in WO14040060A1.

The technical problem can be posed as obtaining new triterpenoids with improved therapeutic effect. The solution of the present invention are the compounds of Formula (I).

DESCRIPTION OF THE INVENTION

The present invention relates to a compound of Formula I:

where R1 is H or OH, and R2 is selected from the group consisting of

y

and

In another aspect, Ri is H and R2 is

the resulting compound having the common name oleanoyl tyramide. In another aspect, R1 is OH and R2 is

the resulting compound having the common name maslinyl tyramide. In another aspect, R1 is OH and R2 is

the resulting compound having the common name maslinic tyrosol ester. In another aspect, Ri is OH and R2 is

the resulting compound having the common name maslin phenethyl ester. In a further aspect, Ri is OH and R2 is

the resulting compound having the common name of vanillyl maslinic ester.

Therefore, in a preferred aspect, the compounds of the invention are selected from maslinyl

Tyramide, Maslinyl Phenethylamide, Tyrosol Maslin Ester, Vanillyl Maslin Ester, Phenethyl Ester

maslinic, oleanoyl phenethylamide and oleanoyl tyramide.

Tyrosol Maslinic Ester Vanillyl Maslinic Ester Phenethyl Maslinic Ester

The compounds of Formula (I) are derived from maslinic acid and oleanolic acid. What

it is known to a person skilled in the art, maslinic acid and oleanolic acid are terpenoids

analogs.

Thus, in one aspect, the compounds of the present invention can be obtained by

means of chemical modification of maslinic acid or oleanolic acid at the sites

suitable. Particularly preferred compounds maslinyl tyramide, maslinyl Phenethylamide, tyrosol maslinic ester, vanillyl maslinic ester and phenethyl maslinic ester are derivatives of maslinic acid that can be synthesized using maslinic acid as starting material; whereas the compounds oleanoyl phenethylamide and oleanoyl tyramide are derivatives of oleanolic acid that can be synthesized using oleanolic acid as a starting material.

The compounds of the invention can be synthesized using the methods described in the Examples. Said methods can be modified and used using principles and techniques of organic chemistry known to one of ordinary skill in the art.

The compounds of the invention can be in free form or as a pharmaceutically acceptable salt thereof. According to the present invention, a pharmaceutically acceptable salt is one that, according to reasonable medical judgment, is acceptable to be administered to the human or animal body without an undesirable complication or response with a reasonable risk-benefit ratio. Pharmaceutically acceptable salts within the scope of the present invention possess the desired therapeutic activity, and include, for example, addition salts with organic acids, with organic bases and inorganic bases.

It should be noted that the amount of terpene compounds described in the state of the art is very large, with many different families identified. Many of these families and derivatives have some therapeutic activity, although it is typically weak. In addition, the possibility of generating chemical derivatives is immense, and there is no certainty about the therapeutic improvement. Testing all or even most of these compounds by the expert would have involved an unapproachable amount of work.

The compounds of the present invention are particularly advantageous because they have improved antitumor effects compared to the base compounds (maslinic acid and oleanolic acid), including cytotoxicity and apoptotic capacity of tumor cells, being in some cases 10 times higher. Additionally, the compounds have TGR agonistic effects, inhibit cyclooxygenase 2, and promote GLP-1 release.

Thus, in one aspect, the compounds of the invention are useful for treating disease. Thus, the compounds of the invention are useful for making a medicament. In preferred aspects, the compounds of the invention are used in the treatment of inflammatory diseases, chronic diseases, metabolic syndrome, obesity, diabetes and / or cancer. In a further aspect, the compounds of the invention are used to treat lung cancer, stomach cancer, colon cancer, cervical cancer, breast cancer, and / or leukemia.

In an even more preferred aspect, each of the compounds maslinyl tyramide, maslinic phenethylamide, oleanoyl tyramide, and oleanoyl phenethylamide is used for the treatment of lung cancer, stomach cancer, colon cancer, cervical cancer, breast cancer and / or leukemia. More preferably, the compound maslinyl tyramide is used for the treatment of colon cancer, the compound maslinic phenethylamide is used for the treatment of stomach cancer, colon cancer and breast cancer, the compound oleanoyl tyramide is used for the treatment of cancer. stomach, colon cancer, breast cancer, and leukemia, and the compound oleanoyl phenethylamide is used to treat stomach cancer, colon cancer, cervical cancer, breast cancer, and leukemia.

The present invention also relates to a composition comprising a compound of Formula (I) as described. In preferred aspects, the composition comprises any of the compounds maslinyl tyramide, maslinyl phenethylamide, tyrosol maslinic ester, vanillyl maslinic ester, phenethyl maslinic ester, oleanoyl phenethylamide and oleanoyl tyramide, or combinations thereof. Said composition can be nutraceutical or pharmaceutical. If it is a pharmaceutical composition, said composition comprises a pharmaceutically acceptable carrier.

Within the scope of the present invention, a pharmaceutically carrier is a material, composition or vehicle involved in carrying or transporting an active ingredient, and which, according to reasonable medical judgment, is acceptable to be administered to the human or animal body without complication or undesirable response with a reasonable risk-benefit ratio. Examples of pharmaceutically acceptable excipients include diluents, solubilizers, emulsifiers, binders, preservatives, and / or adjuvants.

The pharmaceutical composition of the present invention can be formulated for administration by any suitable route, including oral, sublingual, buccal, topical, transdermal, or parenteral.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Cytotoxic activity of maslinyl tyramide, maslinic phenethylamide and maslinic acid, in Jurkat leukemic cells and in human peripheral blood mononuclear cells (PBMC). Panels A and B show the percentages of (necrotic) cells stained with propidium iodide (PI) after treatment with maslinyl tyramide and maslinic phenethylamide, respectively. Panel C shows a representative image of frequency cytometric histograms for 10 pM maslinyl tyramide, 10 pM maslinic phenethylamide, and 25 pM maslinic acid, compared to untreated cells (control). The percentage of necrotic cells is indicated.

Figure 2. Cellular apoptosis of maslinyl tyramide and maslinic phenethylamide in the Jurkat lymphoid cell line. Panel A shows the percentage of necrotic cells after treatment with different doses (pM) of maslinic acid, maslinyl tyramide and maslinic phenethylamide. Panel B is a representative image of frequency histograms obtained from the cytometer for cells treated with 10 pM Maslinyl Tyramide and 10 pM Maslinyl Tyramide. The percentage of subdiploid cells (sub-G0 / G1 fraction), which corresponds to apoptotic cells, is indicated.

Figure 3. Effect of maslinic acid and maslinyl tyramide on the activation of caspase-3 and PARP-1.

Figure 4. Effect of maslinic acid derivatives on the activation of the TGR5 receptor in the stable cell line CHO-TGR5-CRE-LUC. Values are presented in reference to the observed activation for 10 pM LCA, presented as 100% activation, and measured as luciferase activity. The value obtained in the absence of stimulus is shown as a control. The concentrations of the substances tested are in micromoles.

Figure 5. Effect of maslinic acid and its derivatives (pM) on the release of GLP-1 (pM / pg protein) expressed in percentage with respect to the control of untreated NCI-H716 cells.

Figure 6. Effects of maslinic acid and oleanolic acid derivatives (pM), and of maslinic extract (pg / ml) on the transcriptional activation of COX-2 in murine macrophages RAW264.7 transfected with the plasmid COX-2- LUC and treated for 16 hours with the compounds in the presence and absence of LPS (1 pg / ml).

DESCRIPTION OF IMPLEMENTATION MODES

Example 1. Cell lines and reagents used

Several cell lines were used in the present invention: Jurkat, human T lymphoblastic leukemia cells; AGS, human gastric adenocarcinoma cells (AGS); HCT116, human colorectal carcinoma cells; HeLa, human cervical adenocarcinoma epithelial cells; A549, human lung adenocarcinoma epithelial cells; AGS, human gastric adenocarcinoma cells; MDA-MB-231 human breast adenocarcinoma cells; and mouse leukemic monocyte macrophage cell line RAW 264.7. All cell lines were cultured at 37 ° C and 5% CO2 in supplemented Dulbecco's Eagle's medium (DMEM) (Cambrex Co., Barcelona, Spain), containing 10% heat-inactivated fetal bovine serum (FBS) (Cambrex Co., Barcelona, Spain), 2 mM glutamine, penicillin (50 U / ml) and streptomycin (50 mg / ml).

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy adult volunteer donors, by centrifugation of venous blood in Ficoll-HyPaque density gradients, and kept at 37 ° C and 5% CO2 in RPMI medium supplemented with glutamine and antibiotics. as stated above.

Compound preparation: the compounds of the invention and 5-fluorouracil were dissolved in DMSO and stored at -20 ° C. Cis-diaminoplatin dichloride (cDDP) was dissolved in a 0.9% NaCl saline buffer and it was prepared fresh before use.

Example 2. Cytotoxicity of maslinic acid derivatives on different tumor cell lines.

This example demonstrates the in vitro antitumor effects of maslinic acid by illustrating the cytotoxic effects of the compounds in various cell lines adherent to murine and human tumors.

The cytotoxic effect of the compounds was investigated by the 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazo-lium bromide) (MTT) assay. Briefly, cells were cultured at a density of 1.0 x 104 cells / ml in 96-well plates, 100 gl of cell suspension per well, and cultured in DMEM containing 10% fetal calf serum for 12 h. The cells were treated with or without increasing concentrations of the compounds for 24 h. After that, 50 gl of MTT (5 mg / ml) of a mixed MTT: DMEM solution (1: 2) was added to each well, and the cells were incubated for 4 h at 37 ° C in the dark. The reaction was then stopped, the supernatant was removed and 100 gl of dimethylsulfoxide (DMSO) was added to each well for 10 minutes, with gentle stirring. Finally, the absorbance was measured at 550 nm using a TriStar LB 941 (Berthold Technologies, GmbH & Co. KG). The mean concentration in each set of three wells was measured. The absorbance of the untreated controls was taken as 100% survival, and the inhibition percentage was calculated as the cell survival rate (% viability) = 100 (T - B) / (U - B), where T ( treated) is the absorbance of drug-treated cells, U (untreated) is the absorbance of untreated cells and B (blank) is the absorbance in the absence of drug and MTT.

Table 1 shows the IC50 values of the MTT cytotoxicity assay in a panel of 5 solid tumor human tumor cell lines (A549, AGS, HCT116, HELA, MDA-MB-231) and a mouse leukemic monocyte macrophage cell line. (RAW 264.7) after 24 hours of treatment with maslinic acid and oleanolic acid derivatives.

Table 1. IC50 values of the MTT cytotoxicity assay.

All derived compounds showed a greater cytotoxic effect compared to maslinic acid, and the strongest effect was observed with maslinyl tyramide, maslinic phenethylamide, oleanoyl phenethylamide and oleanoyl tyramide. From these results, the phenethylamide and tyramide groups are considered responsible for the potent antitumor activity.

Example 3. Cytotoxic effect of the combination with 5-FU and cDDP on HCT116 human colorectal cells.

This example demonstrates that maslinic phenethylamide and maslinyl tyramide potentiate the cytotoxic effect of 5-fluorouracil (5-FU) and cis-diamineplatin dichloride (cDDP) by increasing the cell death of HCT116 when using two selected doses of compounds in combination with 5 -FU and cDDP.

5-FU and cDDP are two conventionally used chemotherapeutics. 5-FU inhibits DNA and RNA synthesis during the S phase of the cell cycle, while cDDP forms DNA-platinum complexes, both of which ultimately cause cell cycle arrest and apoptosis. The cells were treated for 24 hours with increasing concentrations of the maslinic derivatives and with a single low toxic dose of 5-FU or cDDP ( ~ 20% cell death). Results are shown in percent (%) cell death compared to untreated cells (0% cell death). The MTT assay was performed as described in Example 1.

Table 2 shows the results of the cytotoxicity analysis with MTT in the HCT116 cell line after 24 hours of treatment with maslinyl tyramide and maslinic phenethylamide, both combined with 5-FU 25 or cDDP. Maslinic phenethylamide and maslinyl tyramide combined with 5-FU and 25 pM cDDP were found to enhance their cytotoxic effects. The strongest effect was observed with 2.5 and 5 pM of both maslinic acid derivatives combined with 5-FU and 25 pM cDDP, however, it was not an additive or synergistic effect.

Table 2. Cytotoxicity of maslinic acid derivatives combined with 5-FU or cDDP.

Example 4. Cytotoxicity on Jurkat human lymphoblastic leukemia cells and primary peripheral blood mononuclear cells (PBMC).

This example demonstrates that maslinyl tyramide and maslinic phenethylamide are more cytotoxic in Jurkat lymphoblastic leukemia cells than in PBMC cells, by the percentage of cells that undergo necrosis after treatment with both compounds.

To compare the cytotoxic effect caused by selected compounds on leukemic cells and primary mononuclear cells, a flow cytometric necrosis analysis was performed. Briefly, Jurkat cells and PBMC (2.5x104 cells) were treated with different doses of the maslinic derivatives for 15 hours in 96-well plates.

The treated cells were incubated with propidium iodide (PI) (10 pg / ml) and the percentage of necrotic cells was determined by flow cytometry on FACSCantolI (Becton Dickinson).

In Figure 1A and B it is shown that maslinyl tyramide and 2.5 pM maslinic phenethylamide induced higher values of necrosis in the Jurkat cell line than in the PBMC. After nonlinear regression analysis of the data, in PBMC, the cytotoxic IC50 is 10.6 pM and 7.7 pM for maslinyl tyramide and maslinic phenethylamide, respectively, while in the Jurkat leukemic cell line, the cytotoxic IC50 it is 3.2 pM and 2.4 pM. A representative image of frequency cytometric histograms for 10 pM maslinyl tyramide, 10 pM maslinic phenethylamide, and 25 pM maslinic acid is shown in Figure 1C, compared to untreated cells (control).

The comparative analysis of the cytotoxic activity of maslinyl tyramide and maslinic phenethylamide in Jurkat leukemic cells and in PBMC, showed that both compounds at 5 pM concentration reached values close to 100% cell necrosis in Jurkat cells, while against PMBC they were significantly lower (6.7% and 36.6%, respectively). The IC50 in PMBC was 10.6 pM and 7.7 pM, and in Jurkat cells 3.2 pM and 2.4 pM respectively. The percentage of necrotic cells after treatment with 25 pM maslinic acid was similar to that of untreated cells (control).

These results indicate that the mechanism of antitumor action of maslinyl tyramide and maslinic phenethylamide is mediated by necrotic pathways.

Example 5. Activation of mechanisms in Jurkat T lymphoblastic leukemia cells.

This example demonstrates that the cytotoxic effect of maslinyl tyramide and maslinic phenethylamide on Jurkat T lymphoblastic leukemia cells is due in part to the induction of apoptotic pathways, as shown in cell cycle analysis after treatment with both compounds.

In summary, 1x105 Jurkat cells were treated with different micromolar doses of the maslinic derivatives as indicated in Figure 2, for 15 hours in 96-well plates, followed by fixation with ethanol (70%, for 24 h at 4 ° C) . The cells were then washed twice with PBS and subjected to RNA digestion (RNase-A, 50U / ml) and PI staining (20 pg / ml) in PBS for 2 hours at room temperature. The percentage of cells subjected to chromatinysis (subdiploid cells) was determined by flow cytometry on a FACSCantoII flow cytometer (Becton Dickinson). With this method, the DNA of low Molecular weight escapes from ethanol-fixed apoptotic cells and subsequent staining allows determination of the percentage of subdiploid cells (sub-G0 / G1 fraction).

The results, presented in Figure 2, show that both compounds induce apoptosis in Jurkat cells.

Example 6. Activation of caspase-3 and PARP-1.

This example demonstrates that maslinyl tyramide and maslinic acid induce apoptotic pathways by illustrating the accumulation of processed forms of caspase-3 and PARP-1 in Jurkat cells treated with these compounds.

Caspase-3 and PARP-1 are molecules that are activated in the early stages of apoptosis. These proteins break down or cleave key cellular components that are required for normal cellular function, including structural proteins in the cytoskeleton and nuclear proteins, such as DNA repair enzymes.

To analyze the proapoptotic influence of maslinic acid and maslinyl tyramide in Jurkat cells, they were treated for 3 hours with two doses of these compounds and the activation of caspase-3 and PARP-1 was analyzed by Western Blot. Briefly, Jurkat cells (1x10 6 cells / ml) were incubated with two doses of maslinyl tyramide and maslinic acid for 3 hours. After treatment, cells were washed with PBS and proteins were extracted into 100 µl of lysis buffer (20 mM Hepes, pH 8.0, 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, Na3VÜ4 0 , 5 mM, 5 mM NaF, 1 mM DTT, 1 pg / ml leupeptin, 0.5 pg / ml pepstatin, 0.5 pg / ml aprotinin and 1 mM PMSF) containing 0.5% NP-40. Proteins were boiled in Laemmli buffer and electrophoresed on 10% SDS / polyacrylamide gels. The separated proteins were transferred to nitrocellulose membranes at 20 V (0.5 A) for 30 minutes. The blots were blocked in TBS solution, containing 0.1% Tween 20 and 5% nonfat dry milk for 1 hour at room temperature. Immunodetection of specific proteins was carried out with primary antibodies (anti-caspase 3 (Dako) 1: 1000; anti-PARP (cell signaling) 1: 1000). Anti-mouse (Jackson Laborat) 1: 10000 and an ECL system (GE Healthcare) were used as secondary antibodies.

Figure 3 illustrates the accumulation of forms processed by Caspase and PARP, indicating that the derivatives activate the pro-apoptotic pathway. The results further confirm that the derivatives maslinyl tyramide and oleanoyl phenethylamide induce the processing of caspase-3 and PARP at lower doses than their respective chemical precursors. It also highlights that maslinyl tyramide is at least ten times more potent than maslinic acid.

From the above examples it is concluded that the compounds of the invention have a differential cytotoxicity in Jurkat lymphoblastic leukemia cells and in PBMC, indicating their potential use as antitumor agents.

Example 7. Effect on TGR5

This example studied the ability of maslinic acid derivatives to induce TGR5. TGR5 is a G protein coupled cell surface receptor. It is expressed in macrophages and Kuppfer cells, where it acts as an immunomodulator, decreasing the release of certain pro-inflammatory cytokines (TNFa, IL-1, IL-6, etc.) and activating endothelial NO synthase (eNOS), and therefore with an anti-inflammatory effect in liver and expressed mainly in liver. It is also a key metabolic target in the control of energy homeostasis and in the management of diet-induced obesity and insulin resistance.

CHO-TGR5-CRE-LUC cells were used, which stably express TGR5 and whose activation can be quantified as luciferase activity directed by the CRE promoter, responding to cAMP. For this, the cells were treated with increasing doses of the compounds for 6 hours, after which the luciferase activity was quantified.

The result, shown in Figure 4, indicated that maslinic acid is a TGR5 agonist compound, being able to reach receptor activation values higher than those presented by lithocholic acid (LCA) at 10 pM (this being a dose similar to that of LCA). physiological). Of the synthetic derivatives, only the vanillyl maslinic ester presented values higher than those of the maslinic acid. On the other hand, maslinic phenethylamide stood out as the compound with the ability to activate TGR5 at a lower concentration, reaching 40% activation at 1 pM. Also maslinic tyrosol ester showed a significant TGR5 activation capacity.

Example 8. Effect on the release of GLP-1

The effect of maslinic acid derivatives on the release of GLP-1 protein was studied in the human enteroendocrine cell line NCI-H716, a standard model for the analysis of GLP-1 regulation. The GLP-1 protein is the product of post-transcriptional processing of proglucagon, a peptide synthesized by distal cells of the ileum and colon in response to the intake of nutrients (carbohydrates, amino acids, and lipids such as palmitic acid, oleic acid, etc.) and that it is rapidly degraded by amino dipeptidyl peptidase IV (DPP-IV). GLP-1 participates very prominently in the homeostasis of the glucose metabolism, since it induces the gene expression of insulin, its biosynthesis and its secretion, in addition to favoring the development of pancreatic p cells.

For this, the cells were treated with increasing doses of the selected compounds for 2 hours in the presence of a DPP-IV inhibitor, and subsequently the GLP-1 protein released to the extracellular medium was quantified by ELISA (Figure 5).

Thus, it was observed that the TGR5 agonists, maslinic acid, tyrosol maslinic ester, vanillyl maslinic ester and maslinic phenethylamide increase the release of GLP-1 in a dose-dependent manner. Furthermore, the semisynthetic derivatives gave rise to a greater release of GLP-1 than that observed with maslinic acid, especially in the case of maslinic phenethylamide.

Example 9. Synthesis of the derivatives of maslinic acid

This example details the synthesis of maslinic acid amides and esters.

Maslinic acid can be amidated with PEA (phenylethylamine) using the N, N'-dicylohexylcarbodiimide (DCC) protocol to provide 5a, but direct amidation of natural acid (or its dimethoxyacetal) with other biogenic amines that have phenolic hydroxyls in the presence of carbodiimide condensing agents (DCC, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), alone or in the presence of 4-dimethylaminopyridine (DMAP)) gave a complex mixture, as a result of the non-chemoselective nature of the reaction. Under these conditions, acylation of the phenolic hydroxyl (s) and the free hydroxyl at C-2 was also observed. Surprisingly, these reactions were also observed with phosphorous reagents, such as propylphosphonic acid amide (PPAA). So, a multi-step protocol was devised, based on protecting the glycol system as an acetal and activating the carboxyl group as a hydroxysuccinimide ester. Compound 3 was acylated with various unprotected biogenic amines (tyramine, vanillamine, dopamine) using the carbodiimide protocol. The reaction was successful with tyramine and vanillamine, and after deprotection, the target compounds 5b and 5c were obtained, but failed with dopamine.

c d

Maslinic Acid Acetonide (2)

6.35 mmol (3.00 g, 1 eq) of maslinic acid is suspended in 60 mL of a 1: 1 (v / v) solution of dichloromethane: 2,2-dimethoxypropane, and the solution is stirred until it is obtained a clear solution (approximately 10 minutes). Then 0.16 mmol of p-toluenesulfonic acid (PTSA) is added, and the solution is stirred at room temperature for 4 h. The course of the reaction is monitored by thin layer chromatography (TLC), using toluene / ethanol (4: 1) as eluent. Maslinic acid acetonide (Rf = 0.69) and maslinic acid (Rf = 0.35) are detected with H ² SO ⁴ and heat (deep violet stain). The product of this reaction (maslinic acid acetonide) is hereinafter referred to as "protected maslinic acid".

After 4 h, the reaction is stopped by the addition of NaHCO ³ / brine and DCM. The organic phase is dried (Na ² SO ⁴ ) and evaporated. After trituration with DCM, the crude protected maslinic acid can be used as such for the esterification and amidation reactions. Alternatively, the protected maslinic acid can be purified by gravity column chromatography. In both ways, the performance is practically quantitative.

N-Hydroxysuccinimide Masilinylester Acetonide (3):

To a solution of N-hydroxysuccinimide (NHS) (MW 115.10, 10.06 mmol, 3.5 eq) in 40 mL of ethyl acetate (EtOAc) (4 mL / mmol), protected maslinic acid (1.496 g, MW 512.76, 2.9 mmol, 1 eq) and a solution of N, N'-dicyclohexylcarbodiimide (DCC) (2.431 g, MW 206.33, 11.78 mmol, 4.1 eq) in EtOAc ( 12 mL, 1.6 mL / mmol). The course of the reaction was verified by TLC with petroleum ether (PE) and EtOAc in a ratio of 6: 4 (Rfsm 0.62, Rfproduct 0.52). After 16 h, the reaction was filtered off to remove dicyclohexylurea (DCU), and the solvent was evaporated. The residue was purified by gas column chromatography on neutral alumina (30 ml, PE / EtOAc 8: 2 to 7: 3 as eluent) to obtain 1,753 g (99%) of activated ester as a white powder.

1H NMR (300 MHz, CDCl3) 55.31 (1H, t), 3.68 (1H, m), 3.18 (1H, m), 3.03 (1H, d, J = 9.3), 2.79 (4H, m), 1.41 (3H , s), 1.39 (3H, s), 1.15 (3H, s), 1.04 (3H, s), 1.00 (3H, s), 0.92 (3H, s), 0.91 (3H, s), 0.86 (3H, s), 0.81 (3H, s).

To a solution of activated maslinyl ester (271 mg, MW 609.84, 0.44 mmol, 1 eq) in dichloromethane (DCM) (2.5 mL) was added tyramine (prepared from the hydrochloride, MW 137.18, 0.88 mmol, 2.0 eq) and DMAP (cat). The reaction was stirred at room temperature and verified by TLC (PE / EtOAc 4: 6, Rfsm 0.60, Rfprod 0.27). After 72 h, the reagent was treated by dilution with brine (7.5 ml) and DCM (25 ml). The organic phase was dried (Na2SO4), evaporated and purified by gas column chromatography on silica gel (6 ml, PE / EtOAc 5: 5 to 4: 6 as eluent), yielding 107 mg (39%) of maslinoylamide.

1H NMR (300 MHz, CDCl3) 57.02 (2H, dd, J = 8.4 / 1.8), 6.76 (2H, dd, J = 2.1 / 8.4), 5.85 (1H, bs), 5.28 (1H, bs), 3.68 (1H, m), 3.44 (2H, dt, J = 6.9 / 6.9), 3.03 (1H, d, J = 9.3), 2.86 (1H, m), 2.71 (2H, t, J = 6.9), 2.50 (4H, bm), 1.41 (3H, s), 1.40 (3H, s), 1.12 (3H, s), 1.03 (3H, s), 0.96 (3H, s), 0.91 (3H, s), 0.89 ( 3H, s), 0.86 (3H, s), 0.73 (3H, s).

Maslinyl tyramide (5b).

with p-toluenesulfonic acid (PTSA) (30 mg, MW 190.22, 0.16 mmol, 1.1 eq). The reaction was stirred at room temperature and verified by TLC (PE / EtOAc 2: 8 max, Rfsm 0.55, Rfprod 0.38). After dilution with EtOAc and brine, drying and evaporation of the organic phase, the residue was crystallized from ether to provide 72 mg (86%) of maslininoyltyramine as a white powder.

1H NMR (300 MHz, (CD3COCD3) 57.03 (2H, d, J = 8.4), 6.73 (2H, d, J = 8.4), 5.26 (1H, t), 3.58 (1H, m), 3.34 (2H, dt , J = 6.9 / 7.8), 2.67 (2H, t, J = 7.8), 2.46 (3H, m), 1.16 (3H, s), 1.00 (3H, s), 0.97 (3H, s), 0.93 (3H , s), 0.91 (3H, s), 0.79 (3H, s), 0.76 (3H, s). 13C NMR (75 MHz, (CD3COCD3), 5 176.3, 172.4, 171.7, 157.0, 144.7, 131.5, 130.9, 123.9, 116.4, 84.3, 69.2, 56.6, 48.9, 48.0, 47.7, 47.0, 43.0, 42.6, 42.3, 40.7, 40.2, 39.7, 39.3, 36.0, 34.7, 33.9, 33.7, 33.6, 31.7, 31.6, 28.8, 26.6, 24.7, 24.3, 19.5, 18.1, 17.8, 17.5.

Tyrosol Maslinic Ester

To a DCM solution of protected maslinic acid (0.585 mmol, 1 eq), tyrosol (1.19 mmol, 2.02 eq), triphenylphosphine (TPP) (1.76 mmol, 3.02 eq) and diisopropyl were added sequentially azodicarboxylate (DIAD) (1.14 mmol, 1.95 eq). The solution was stirred at room temperature, and its course was followed by TLC using PE / EtOAc (8: 2) as eluent. The detection was carried out with H ² SO ⁴ and heat, obtaining Rf of 0.39 for the protected maslinic acid, Rf of 0.38 for the protected tyrosyl ester. After 90 minutes, the reaction was worked up by evaporation of the solvent. The residue was taken up in tetrahydrofuran (THF) (5 ml) and stored overnight in the refrigerator. An abundant precipitate of Mitsunobu by-products (TPPO-H2DIAD) formed, which was removed by filtration.

To a methanolic solution of the protected tyrosyl ester (0.51 mmol, 1 eq), PTSA (0.51 mmol, 1 eq) is added, and the reaction is stirred at room temperature, following its course by TLC. The eluent was PE / EtOAc (4: 6) and detection was done with H ² SO ⁴ and heat. A protected tyrosyl ester Rf of 0.81 and a deprotected ester (maslinic tyrosol ester) Rf of 0.27 were obtained. After 90 minutes, the reaction was worked up by adding EtOAc (5 ml) and brine (5 ml). The organic phase was dried (Na ² SO ⁴ ) and evaporated.

Maslinic phenethylester

To a DCM solution of protected maslinic acid (0.19 mmol, 1 eq), phenethyl alcohol (0.29 mmol, 1.5 eq), TPP (0.39 mmol, 2 eq) and DIAD (0 , 41 mmol, 2 eq). The solution was stirred at room temperature, and its course was followed by TLC. The eluent was PE / EtOAc (8: 2) and detection was done with H2SO4 and heat. A protected maslinic acid Rf of 0.39 and a protected phenethyl ester Rf of 0.67 were obtained. After 90 minutes, the reaction was worked up by evaporation of the solvent. The residue was taken up in THF (5 ml) and stored overnight in the refrigerator. An abundant precipitate of Mitsunobu by-products (TPPO-H2DIAD) formed, which was removed by filtration.

To a methanol solution of the protected ester (0.15 mmol, 1 eq), PTSA (0.15 mmol, 1 eq) was added, and the reaction was stirred at room temperature, following its course by TLC. The eluent was PE / EtOAc (7: 3) and detection was done with H2SO4 and heat. An Rf of the protected phenethyl ester of 0.70 and an Rf of the deprotected phenethyl ester of maslinic acid of 0.15 were obtained. After 50 minutes, the reaction was worked up by dilution with EtOAc (8 ml) and brine (8 ml). The organic phase was dried (Na2SO4) and evaporated.

Vanillyl Maslinic Ester

stirred at room temperature and verified by TLC. The eluent was PE / EtOAc (8: 2) and detection was done with H ² SO ⁴ and heat. A starting compound Rf of 0.39 and a protected vanillyl ester Rf of 0.32 were obtained. After 2 h, the reaction was worked up by evaporation. Crystallization of TPPO-H2DIAD from THF gave only a relatively small amount of precipitate. After filtration and evaporation, final purification was achieved by gravity column chromatography.

To a solution of the protected vanillyl ester (0.18 mmol, 1 eq) in methanol, PTSA (0.18 mmol, 1 eq) was added, and the reaction was stirred at room temperature, following its course by TLC. The eluent was PE / EtOAc (6: 4) and detection was done with H ² SO ⁴ and heat. An Rf of the protected ester of 0.77 and an Rf of the deprotected ester (vanillyl ester of maslinic acid) of 0.26 were obtained. After 90 minutes, the reaction was worked up by dilution with EtOAc (2 ml) and brine (2 ml). The organic phase was dried (Na ² SO ⁴ ) and evaporated, and the residue was purified by gravity column chromatography.

Claims (7)

1. A compound of Formula (I)

where R1 is H or OH, and R2 is selected from the group consisting of

2. The compound of claim 1, wherein R1 is H and R2 is

3. The compound of claim 1, wherein Ri is OH and R2 is

4. A pharmaceutical composition comprising the compound of any of claims 1-3 and a pharmaceutically acceptable excipient. The compound of any of claims 1-3, or the pharmaceutical composition of claim 4, for use as a medicine. 6. Use of the compound of any of claims 1-3 or of the composition of claim 4 for the manufacture of a medicament for the treatment of inflammatory diseases, chronic diseases, metabolic syndrome, obesity, diabetes and / or cancer. 7. The use of claim 6, wherein the cancer is selected from the group consisting of lung cancer, stomach cancer, colon cancer, cervical cancer, breast cancer, and leukemia.