Hybrid of sclareol and doxorubicin as well as synthesis and application thereof

Abstract

The present invention represents a novel hybrid of the two natural products sclareol and doxorubicin, in the form of their conjugates. These compounds are in the form of conjugates wherein doxorubicin and sclareol are covalently linked by a linker in a molar ratio of 1: 1. The hybrid has been shown to have anti-cancer properties and is effective in the treatment of cancer cells resistant to a P-glycoprotein membrane transporter responsible for resistance to doxorubicin. Hybrid have been tested on different types of cell lines, including human glioblastoma, non-small cell lung cancer, and colorectal cancer. The invention further relates to a method for the production thereof and to the use thereof in pharmaceutical products or pharmaceutical preparations. The research results include the cytotoxic activity of single compounds, combined compounds, and conjugated compounds in sensitive and resistant cancer cell pairs with and without P-glycoprotein expression. The selectivity to cancer cells is determined by comparison with commercially available normal human lung fibroblasts. The study also studies the nanoparticle properties of the hybrids, their intracellular localization and in vivo toxicity.

Description

Technical Field

[0001] The present invention relates to the synthesis of novel hybrids (conjugates) of sclareol (SC) and doxorubicin (DOX), and their use in anticancer therapy.

[0002] Technical problem:

[0003] The technical problem solved by the present invention is to discover novel hybrids that exhibit effective anticancer effects against different types of cancer cells, such as human glioblastoma, non-small cell lung cancer, and colorectal cancer. In addition, the present invention provides a method for preparing these hybrids from suitable ligands, and the use of the compounds thus obtained in pharmaceutical products or drug formulations / compositions. Background Art

[0004] The study conducted by Borges GSM et al. evaluated the effect of the combined administration of SC and DOX in anticancer therapy. The study found that the combination of doxorubicin (DOX) and sclareol (SC) at a molar ratio of 1:1.9 had a better synergistic anticancer effect on cancer cell lines in the breast than a molar ratio of 1:7.5. Compared with free DOX, the use of nanolipid carriers loaded with DOX and SC further improved the cytotoxicity in cell lines and animal models. However, the combination without nanolipid carriers caused weight loss, behavioral changes, and hematological toxicity in animals. On the other hand, the combined application with nanolipid carriers did not cause any side effects. The study recommended the delivery of DOX and SC at a ratio of less than 1:2 in nanoparticles to obtain the desired anticancer effect (Life Sci. 2019 Sep 1; 232:116678. doi: 10.1016 / j.lfs.2019.116678. Epub 2019 Jul 22. PMID: 31344429).

[0005] The study conducted by Oliveira MS et al. demonstrated that the combination of DOX and SC in solid lipid nanoparticle suspensions increased their cytotoxic effect on cancer cells. The study found that DOX was released from the lipid nanoparticles at pH 7.4, with a higher release at lower pH values. However, SC packaged alone in lipid nanoparticles did not exhibit any significant anticancer effect (J Nanosci Nanotechnol. 2018 Aug 1; 18(8):5609-5616. doi: 10.1166 / jnn.2018.15418. PMID: 29458616).

[0006] In addition, studies conducted by Perce F and Torchilin VP have shown that SC does not enhance the accumulation of DOX in spheroids of doxorubicin-resistant ovarian cancer cell lines. However, it does increase the cytotoxicity of DOX by 4.5-fold (Cancer Biol Ther. 2012 Oct;13(12):1205-13. doi: 10.4161 / cbt.21353. Epub 2012 Aug 15. PMID: 22892843).

[0007] Finally, studies by Dimas K et al. have revealed that SC significantly enhances the cytotoxic effect of DOX in breast cancer cell lines. When co-administered, 50 μM SC and 1 μM DOX have been shown to be highly cytotoxic compared to when SC or DOX is administered alone. In fact, the combination of 50 μM SC and 0.1 μM DOX also significantly reduces the cell growth rate, showing 76% when DOX is administered alone, while only 4% when combined with SC (Biomed Pharmacother. 2006 Apr;60(3):127-33. doi: 10.1016 / j.biopha.2006.01.003. Epub 2006 Feb 21. PMID: 16527443).

[0008] The present invention describes the results of the effects of combinations of two different concentrations of SC and five different concentrations of DOX on eight different human cell lines, as well as the results of the specific effects on different types of cancer compared to normal non-cancerous cells.

[0009] There is no description in the existing patent literature of a conjugate of covalently linked SC and DOX for anti-cancer treatment. A search of the patent database shows some patent literature that discloses the use of SC and its derivatives in the treatment of infections caused by microorganisms and in cosmetic formulations, such as EP1083895A2.

[0010] In addition, as prior art literature, NZ581183A describes the use of DOX in combination with major cytotoxic agents such as paclitaxel, docetaxel, and gemcitabine for the treatment of cancer.

[0011] Data on the SC-derived effect from the literature:

[0012] The method of chemically modifying SC is a well-known technique as described in the literature. For example, Rehman et al. demonstrated the synthesis of anti-cancer SC analogs through chemical transformation in their article "Design and Synthesis of Heck-Coupled Sclareol Analogs: Modulation of BH3 Family Members by SS-12 in Autophagy and Apoptotic Cell Death" published in the Journal of Medicinal Chemistry in April 2015. The authors reported that the resulting derivatives were more effective than SC in terms of their biological activity (J. Med. Chem. 2015 Apr 23; 58(8): 3432-44. doi: 10.1021 / jm501942m. Epub 2015 Apr 9. PMID: 25825934). Summary of the Invention

[0013] The present invention provides novel hybrids (conjugates) of SC and DOX that have been synthesized and obtained, which have improved selectivity for cancer cells compared to DOX alone. These conjugates are expected to have less systemic and cardiac toxicity than DOX, thus addressing its severe limitations. The present invention provides a novel method for treating different types of cancer, including but not limited to non-small cell lung cancer, colorectal cancer, and glioblastoma, which has great promise in overcoming resistance to existing treatments and improving patient outcomes.

[0014] The present invention provides novel hybrids (conjugates) of SC and DOX for treating different types of cancer, including but not limited to non-small cell lung cancer, colorectal cancer, and glioblastoma. These malignancies are known to be resistant to available and experimental treatments due to intrinsic or induced resistance. The blood-brain barrier contains P-glycoprotein, which is a key obstacle in glioblastoma treatment as it prevents the penetration of xenobiotics into the brain. Fourth-generation P-glycoprotein inhibitors, represented by natural products, have shown promise in overcoming the resistance of these malignancies. Non-small cell lung cancer and colorectal cancer are common malignancies that affect almost half of cancer patients. On the other hand, glioblastoma is the most common type of brain cancer and, unfortunately, it is also the most resistant to available and experimental treatments.

[0015] Resistance can be intrinsic or induced, and it is the main obstacle to the effective treatment of these three malignancies. Currently, for non-small cell lung cancer and colorectal cancer, there are different innovative types of therapies, such as targeted therapies and immunotherapies. However, the only chemotherapeutic agent approved for glioblastoma therapy is temozolomide.

[0016] Since 2005, no new chemical therapy has been approved for the treatment of glioblastoma, which is a major challenge.

[0017] One of the key obstacles in glioblastoma treatment is the blood-brain barrier. This barrier contains P-glycoprotein, a membrane transporter protein that does not allow foreign substances to penetrate into the brain. P-glycoprotein is found on the cell membranes of resistant cancer cells, including glioblastoma, non-small cell lung cancer, and colorectal cancer, where P-glycoprotein has a protective effect against chemotherapy and radiotherapy. P-glycoprotein also protects cancer cells from innovative targeted therapies.

[0018] Natural products, such as bioactive substances isolated from plants and microorganisms, represent the fourth generation of P-glycoprotein inhibitors. Combining natural product-P-glycoprotein inhibitors with chemotherapy and innovative therapies is expected to solve the problems in the treatment of malignancies mentioned, such as resistance.

[0019] Sclareol (SC) is a natural product that inhibits the activity of P-glycoprotein.

[0020] Doxorubicin (DOX), an anticancer drug that is a P-glycoprotein substrate, is used to treat various types of cancer. However, its use has serious limitations, such as the development of resistance, inability to cross the blood-brain barrier, and cardiotoxicity.

[0021] The hybrid (conjugate) of SC and DOX synthesized and obtained according to the present invention has better selectivity for cancer cells than DOX alone. This indicates that the conjugate may have lower systemic and cardiotoxicity compared to DOX alone.

[0022] The present invention provides a method for synthesizing new hybrids CON1 and CON2 of SC and DOX for the treatment of resistant cancer. The hybrids are covalently linked in a 1:1 molar ratio through a linker to ensure the simultaneous delivery of the two chemical entities to cancer cells. It has been shown that the conjugate has higher selectivity for cancer cells and lower resistance than DOX. The present invention also describes the synthesis of new SC derivatives named ligands LIG1 and LIG2, which are used as precursors for synthesizing the hybrids.

[0023] Cancer cells expressing P-glycoprotein have lower resistance to the hybrid (conjugates CON1 and CON2) of SC and DOX than to DOX alone. This indicates that the conjugates can be used for the treatment of resistant cancers, including brain cancer, because they can cross the blood-brain barrier.

[0024] The results of the present invention show that conjugates of P-glycoprotein inhibitors such as SC and anticancer drugs such as DOX cause less damage to normal cells compared to DOX alone and exhibit lower systemic toxicity. The intracellular localization of the conjugate of SC and DOX is in the cytoplasm and nucleus, while the intracellular localization of DOX is in the nucleus, indicating that the conjugate causes less DNA damage than doxorubicin alone, which is consistent with the lower systemic toxicity of the conjugate as expected.

[0025] The present invention relates to the synthesis of new hybrids (conjugates) in which DOX and SC are covalently linked in a 1:1 molar ratio through a linker, ensuring the simultaneous delivery of both chemical entities to cancer cells.

[0026] The present invention also describes the synthesis of new SC derivatives named ligand LIG1 and LIG2, which represent precursors in the synthesis of hybrids (CON1 and CON2 respectively) of SC and DOX.

[0027] The newly synthesized hybrids (CON1 and CON2) exhibit favorable anticancer characteristics, including higher selectivity for cancer cells compared to the starting compounds SC and DOX, and a lower degree of resistance than DOX. These hybrids were found to be effective against different types of cell lines, including human glioblastoma, non-small cell carcinoma, and colorectal cancer.

[0028] Although the combination of SC and DOX as an anticancer therapy is known in the literature, the novelty of the present invention lies in the first disclosure of the synthesis of hybrid molecules of SC and DOX, in which these structures are covalently linked, and their anticancer properties.

[0029] A method for producing a hybrid, which is a conjugate of SC and DOX, is disclosed. The method comprises a convergent synthesis of three reaction steps. The hybrid is characterized by the covalent linkage of SC and DOX. The hybrids (CON1 and CON2) surprisingly avoid P-glycoprotein activity and contribute to increased accumulation of DOX in cells while increasing the expression of P-glycoprotein. In particular, the hybrids (CON1 and CON2), the conjugates of SC and DOX, are expected to produce a breakthrough in the treatment of glioblastoma. It has been shown that hybrid structures based on natural products and / or drugs have significantly better pharmacokinetic and pharmacodynamic properties compared to individual molecules.

[0030] The following invention outlines a method for producing a hybrid, which combines SC and DOX in a convergent synthesis comprising three reaction steps. Two new hybrids, CON1 and CON2, were synthesized, which are conjugates containing different ligand derivatives, namely SC derivatives LIG1 and LIG2. These conjugates were characterized and their antitumor effects were tested on different human cell lines.

[0031] The simultaneous administration of the single compounds SC and DOX as an anticancer treatment has been previously documented in the literature. This has been achieved by combining the two compounds and by delivering them in the same lipidic nanocarrier.

[0032] The present invention describes for the first time the synthesis of the hybrid molecules SC and DOX. This means that compounds have been produced in which these structures are covalently linked, and their in vitro anticancer effects have been tested on different types of cell lines, including human glioblastoma, non-small cell lung cancer, and normal human fibroblasts.

[0033] The experimental results show that the combination of sclareol and doxorubicin used in a ratio closer to 1:1 has a better synergistic antitumor effect. Thus, by covalently linking two new antitumor compounds through a linker to form a hybrid molecule (conjugate), a reagent has been obtained that is characterized by overcoming the obstacles and limitations in the application of individual compounds.

[0034] The obstacles and limitations in drug use are the development of resistance and the cytotoxic effect on normal cells. The hybridization of SC with DOX overcomes these obstacles, showing an enviable repression of P-glycoprotein activity and contributing to an increase in the accumulation of DOX in cells, thus increasing the expression of P-glycoprotein.

[0035] In addition, it is expected that these hybrids, the conjugates of SC and DOX, will produce a breakthrough in the treatment of glioblastoma. This is because the main obstacle to the application of DOX is the blood-brain barrier, including the transport pump P-glycoprotein.

[0036] Therefore, the conjugates of SC and DOX embody the good characteristics of an antitumor strategy that combines these two compounds. It is important to note that the combination of two special structures into one molecule follows the latest medical trends in chemistry. It has been shown that hybrid structures based on natural products and / or drugs have significantly better pharmacokinetic and pharmacodynamic properties compared to individual molecules.

[0037] In the design and development of the hybrid molecule, the inventors of the present invention chose succinic acid as the linker, i.e., the structural motif that covalently links sclareol and doxorubicin. The conformational flexibility of the succinate ester has been determined, which allows the free rotation of the carbon skeleton, facilitates the passage of the molecule through biological membranes, and enhances the bioavailability of the hybrid. The amide bond was chosen as the basis for linking sclareol and doxorubicin into a unified structure through the linker due to its high stability under in vivo conditions. In addition, the high thermodynamic stability of the amide bond allows the formation of the amide bond with the desired product in high yield under mild reaction conditions.

[0038] The present invention mainly provides hybrids of sclareol and doxorubicin, CON1 and CON2, with the general formula:

[0039]

[0040] wherein the ligands LIG1 and LIG2 are defined as

[0041]

[0042] described hereinafter in the specification.

[0043] More specifically, the present invention relates to hybrids CON1 and CON2 of the following formula:

[0044]

[0045] wherein doxorubicin and sclareol are covalently linked in a 1:1 molar ratio via a linker, as further described hereinafter in the specification.

[0046] In addition, the present invention provides compounds LIG1 and LIG2 of the following formula:

[0047]

[0048] as defined hereinafter, which can be particularly used as precursors in the synthesis of compounds CON1 and CON2.

[0049] Furthermore, the present invention provides hybrids of sclareol and doxorubicin, which are particularly useful in medical products or pharmaceutical compositions / formulations.

[0050] The compounds CON1 and CON2 of the present invention obtained as described herein are defined as follows:

[0051] CON1: N1-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-1-yl)oxy)tetrahydro-2H-pyran-4-yl)-N4-(3-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)propyl)succinamide and / or a pharmaceutically acceptable salt or hydrate or solvate thereof; and

[0052] CON2: N1-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-1-yl)oxy)tetrahydro-2H-pyran-4-yl)-N4-(6-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)hexyl)succinamide and its pharmaceutically acceptable salts and / or hydrates and / or solvates.

[0053] In addition, compounds LIG1 and LIG2 of the present invention, used as ligands covalently bound to doxorubicin, are defined as:

[0054] LIG1: 4-((3-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)propyl)amino)-4-oxobutanoic acid, and

[0055] LIG2: 4-((6-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)hexyl)amino)-4-oxobutanoic acid.

[0056] In addition, the present invention includes the synthesis of sclareol derivative 1, having the formula: 4-{(1E,3R)-3-hydroxy-5-[(2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl]-3-methylpent-1-en-1-yl}benzaldehyde, which is used as a starting material for the synthesis of precursors LIG1 and LIG2 and thus as a starting material for the synthesis of hybrids CON1 and CON2. Description of the Drawings

[0057] Figure 1The inhibitory (cytotoxic) effect of cell growth in different cell lines in response to increasing SC concentration (5, 10, 20, 50 and 100 μM) is described. The cell lines include U87 and U87-TxR (sensitive and resistant glioblastoma cell lines), U251 (another glioblastoma cell line), NCI-H460 and NCI-H460 / R (sensitive and resistant non-small cell lung cancer cell lines), DLD1 and DLD1-TxR (sensitive and resistant colorectal cancer cell lines) and MRC-5 (human lung fibroblasts - normal non-cancer cells). The left figure shows the non-linear function of cell growth inhibition, while the right figure shows the "IC50" values. IC50 is the concentration that causes a 50% cell growth inhibition rate relative to the control untreated cells. These values were obtained by non-linear regression using GraphPad software Prism 8.0.2. Using MTT assay, the treatment lasted for 72 h.

[0058] Figure 2 The interaction between SC and DOX at cytotoxic levels in different cells is shown. SC (10 and 20 μM) was combined with increasing concentrations of DOX (20, 50, 100, 200 and 500 nM). The co-treatment lasted for 72 hours, and MTT assay was used to determine the non-linear regression obtained using GraphPad Prism 8.0.2 software (line graph). Additionally, the combination index (CI) of the test co-treatment of SC and DOX was obtained using CalcuSyn software (dot graph). The CI value reflects the type of interaction of the two combined compounds, where additive effect is indicated by CI≈1, synergistic effect is indicated by CI<1, and antagonistic effect is indicated by CI>1.

[0059] Figure 3 It is illustrated that the cumulative increase of DOX in all glioblastoma cell lines is independent of the expression and activity of P-glycoprotein, which is only present in excess in U87-TxR. It was found that the combination treatment with 20 μM and 50 μM SC increased the accumulation of 20 μM DOX, which was analyzed on a flow cytometer 60 minutes after the combined exposure.

[0060] Figure 4 It is shown that in the resistant cell NCI-H460 / R, the accumulation of DOX increased with the accumulation of rhodamine 123, indicating the inhibition of P-glycoprotein activity in the presence of 50 μM SC. However, the P-glycoprotein-deficient NCI-H460 cells did not show any increase in the accumulation of DOX or rhodamine 123. The accumulation of 20 μM DOX was measured on a flow cytometer 60 minutes after the treatment with the combination of SC and DOX (left figure), while the accumulation of 5 μM rhodamine 123 was measured on a flow cytometer 30 minutes after the treatment with the combination of SC and rhodamine 123 (right figure).

[0061] Figure 5 Shows the resistance profiles of two conjugates, CON1 and CON2, and DOX against sensitive U87 and resistant U87-TxR. This study used CON1 and CON2 at a range of concentrations (2.5, 5, 10, 25, and 50 μM), and DOX at a range of concentrations (0.25, 0.5, 1, 2.5, and 5 μM), and performed MTT assays for 72 h. Nonlinear regression and IC50 values were obtained using GraphPad Prism 8.0.2 software.

[0062] Figure 6 Shows the localization of DOX-derived fluorescence in U87 glioblastoma cells after treatment. Cells treated with 20 μM DOX showed fluorescence in the nucleus after only 2 h of treatment, as shown in the upper panel of the image. The fluorescence appeared in the red channel. Cells treated with CON1 and CON2 (2, 5, 10 μM) showed perinuclear fluorescence, indicating that the fluorescence originated from around the nucleus. The lower panel of the image shows the fluorescence observed after 72 h of treatment. The nucleus was visualized in the blue channel in contrast to Hoechst 33342.

[0063] Figure 7 Describes the physicochemical characteristics of CON1 and CON2. As shown in the upper panel above, a Zetasizer was used to determine the nanoparticle properties of CON1 and CON2. The size and dispersity of CON1 and CON2 were measured independently three times, and the results are shown in the upper panel. Additionally, the lower panel shows the chemical models of the nanoparticles. Chemical modeling predicted that CON1 and CON2 spontaneously "protein-like" fold due to strong intermolecular forces.

[0064] Figure 8 shows the TEM characterization of CON1 and CON2.

[0065] Figure 8A Illustrates the size and shape of the nanoparticles obtained using TEM with a digital camera SIS MegaView III and iTEM software.

[0066] Figure 8B Shows representative images of the cellular uptake and subcellular distribution of CON1 or CON2 in U87 cells after 24 h of treatment. It was found that both CON1 and CON2 entered U87 cells. Large and small nanoparticles were found inside U87 cells according to the depth of the cell cross-section. These nanoparticles were detected on the cell membrane, in the cytoplasm, in the mitochondria, and mainly at the nuclear membrane level and in the nucleus. Notably, CON1 was more abundant in the nucleolus. The nucleus is abbreviated as "Nu", and the nucleolus is abbreviated as "No". The scale bar is 500 nm.

[0067] Figure 9 shows the toxicity profiles of DOX and CON1 in Balb / c mice.

[0068] Figure 9A 、 9B and 9C illustrate the biochemical analysis performed by the Critical Care Panel (900 - 330).

[0069] Figure 9D Shows a part of the whole blood analysis using the Scil vet ABC TM Hematology Analyzer. Balb / c mice were treated, with the control group receiving the vehicle (10% DMSO and 5% Tween80), and 7 mg / kg body weight of DOX and 14 mg / kg body weight of CON1 were injected intraperitoneally (i.p.). Three time points were evaluated: 24 h, 48 h, and 72 h. Abbreviations: albumin (ALB), total protein (TP), glucose (GLU), alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatine phosphokinase (CPK), lactate (LAC), blood urea nitrogen (BUN), creatinine (CREA), total bicarbonate (tCO2).

[0070] Figure 10 Structures of the hybrids (conjugates) CON1 and CON2 of sclareol and doxorubicin. Detailed Description of the Invention

[0071] The present invention describes the synthesis of two hybrids (conjugates) CON1 and CON2, which contain different sclareol derivative ligands LIG1 and LIG2.

[0072] The key synthetic step according to the present invention is the oxidative Heck cross - coupling reaction catalyzed by a palladium complex to introduce a 4 - formylphenyl group at the C15 position of sclareol. The resulting product, compound 1 (Scheme 1), is new. Subsequently, this product undergoes further derivatization. In addition, the present invention includes the synthesis of compounds LIG1 and LIG2 by reductive amination (Scheme 3), and then their conversion to the final products CON1 and CON2 (Scheme 4).

[0073] According to the present invention, compound 1, ligands LIG1, LIG2, and the hybrid conjugates CON1 and CON2 are synthesized and characterized for the first time. Additionally, the biological activities of compounds LIG1, LIG2, CON1, and CON2 are described.

[0074] The synthesis method includes the following reaction steps:

[0075] a) Natural product sclareol (SC) reacts with 4-formylphenylboronic acid in the presence of a palladium-based catalyst to give the corresponding derivative 1 of sclareol in 80% yield (Scheme 1). The configurations at the chiral carbon atoms (C-5, C-8, C-10, and C-13) of the sclareol subunit remain unchanged under the reaction conditions applied.

[0076]

[0077] Scheme 1. Synthesis of sclareol derivative 1.

[0078] b) Compounds 2 and 3 were synthesized in good yields using alkyl diamines, 1,3-diaminopropane, 1,6-diaminohexane, and succinic anhydride (Scheme 2).

[0079]

[0080] c) In the presence of titanium(IV) isopropoxide, sclareol derivative 1 and compounds 2 and 3 were converted to the corresponding ligands LIG1 and LIG2 in good yields by reductive amination using sodium borohydride (Scheme 3).

[0081]

[0082] Scheme 3. Synthesis of ligands LIG1 and LIG2.

[0083] d) Conjugates CON1 and CON2 were obtained by forming amide bonds between doxorubicin and ligands LIG1 and LIG2. To activate the carboxylic acid functional groups of ligands LIG1 and LIG2, 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were used (Scheme 4).

[0084]

[0085] Scheme 4. Synthesis of conjugates CON1 and CON2.

[0086] Examples

[0087] The following examples illustrate the synthesis of some compounds of the present invention. These examples are not limiting and are provided for illustrative purposes.

[0088] Melting points were determined using a Boetius PMHK apparatus and are uncorrected. IR spectra were recorded on a Thermo-Scientific Nicolet 6700 FT-IR “diamond crystal” spectrophotometer. Absorption band positions are given in cm -1 and are expressed. 1 1H NMR and 1313C NMR spectra were recorded on a Varian spectrometer (at 400 and 100 MHz) in the specified solvent. Chemical shifts are reported in ppm, coupling constants (J) in Hz, and signal multiplicities as s (singlet), d (doublet), t (triplet), q (quartet), sext (sextet), dd (doubledoublet), dq (doubletquartet), and m (multiplet). HRMS-HESI spectra were recorded on an LTQ Orbitrap XL (Thermo Fisher Scientific, USA) mass spectrometer. Samples were dissolved in pure HPLC-grade CH3CN and directly injected into the instrument. Ionization was carried out in positive mode on a heated electrospray ionization probe. The following HESI parameters were used: spray voltage 4.7 kV, evaporation temperature 60 °C, drying gas and auxiliary gas flow rates 24 and 10 (arbitrary units), capillary voltage 49 V, capillary temperature 275 °C, tube lens voltage 80 V, resolution (m / z 400): 30,000. For thin-layer chromatography, SiO2 and RP-18 plates (Merck) were used. SiO2 (0.018 - 0.032 mm) was used for "dry-flash" chromatography.

[0089] Complete structural characterization of the synthesized compounds was achieved by determination of melting points and by using infrared spectroscopy (IR ATR), nuclear magnetic resonance (1D NMR (1H and 13C) methods and 2D NMR (COSY (1H-1H correlation spectroscopy), NOESY (nuclear Overhauser effect spectroscopy), HSQC (1H-13C heteronuclear single quantum coherence), HMBC (1H-13C heteronuclear multiple bond correlation)) methods, as well as high-resolution mass spectrometry (HRMS).

[0090] The purity of the compounds (HPLC) was determined using an Agilent 1200 HPLC system equipped with a quaternary pump (G1311B), injector (G1329B, 1260 ALS, TCC 1260 (G1316A)), and detector 1260 DAD VL+ (G1315C). HPLC analysis was carried out in two different systems.

[0091] Method A: InfinityLab Poroshell 120 CS-C18 4.6×100 mm 2.7 μ, S.N. was used as the stationary phase. The eluent consisted of the following solvents: 0.1% HCOOH in water (A) and MeOH (B). Analysis was performed at the UV maximum (254 nm) of the compounds to obtain maximum selectivity. The compounds were dissolved in MeOH at a final concentration of approximately 1 mg / mL, and the injection volume was 5 μL. The flow rate was 0.6 mL / min. The following gradient was used to elute compounds LIG1, LIG2, CON1, and CON2: 0 - 1 min 95% A, 1 - 6 min 95% A → 5% A, 6 - 11 min 5% A, 11 - 14 min 5% A → 95% A, 14 - 15 min 95% A.

[0092] Method B: InfinityLab Poroshell 120 CS-C18, 4.6×100 mm, 2.7 μm, serial number (S.N.), was used as the stationary phase. The eluent consisted of the following solvents: aqueous solution of 0.1% HCOOH (A) and acetonitrile (ACN) (B). Analysis was performed at the UV maximum (254 nm) of the compounds to obtain maximum selectivity. The compounds were dissolved in methanol (MeOH) at a final concentration of approximately 1 mg / mL, and the injection volume was set to 5 μL. The flow rate was maintained at 0.6 mL / min. The following gradient was used to elute compounds LIG1, LIG2, CON1, and CON2: 0 - 1 min, 95% A; 1 - 6 min, 95% A → 5% A; 6 - 11 min, 5% A; 11 - 14 min, 5% A → 95% A; 14 - 15 min, 95% A.

[0093] The experimental conditions applied did not affect the stereoisomerism of the sclareol subunit and the endo-chiral carbon atoms of doxorubicin.

[0094] Example of the synthesis of sclareol derivative 1: 4-{(1E,3R)-3-hydroxy-5-[(2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl]-3-methylpent-1-en-1-yl}benzaldehyde.

[0095] In an anaerobic environment, add dimethylformamide (DMF) (1.5 mL), sclareol SC (30 mg, 0.097 mmol, 1 equivalent), 4-formylphenylboronic acid (22 mg, 0.146 mmol, 1.5 equivalents), Pd(OAc)2 (2.2 mg, 0.010 mmol, 0.1 equivalent), Cu(OAc)2 (35.3 mg, 0.194 mmol, 2.0 equivalents), and NaOAc (23.9 mg, 0.292 mmol, 3 equivalents) to a round-bottom flask. Stir the reaction mixture at 80 °C for 2 hours. Then filter the reaction mixture through a sintered funnel, wash it with 3 × 20 mL of ethyl acetate (EtOAc), and dry it over anhydrous MgSO4. Purify the crude product by "dry-column flash" chromatography (SiO2:Hex / EtOAc = 7 / 3) to obtain Compound 1 as a yellow oil (32 mg, 80%). 1 1H NMR (400 MHz, CDCl3): δ = 9.97 (s, 1H, -CHO), 7.81 (d, J = 7.9 Hz, 2H, 2×Ar-H), 7.50 (d, J = 7.9 Hz, 2H, 2×Ar-H), 6.69 (d, J = 16.0 Hz, 1H, =CH), 6.46 (d, J = 16.2 Hz, 1H, =CH), 2.45 (brs, 2H, 2×OH), 1.84 (d, J = 12.1 Hz, 1H), 1.80 - 1.72 (m, 2H), 1.68 - 1.53 (m, 4H), 1.47 - 1.39 (m, 3H), 1.38 (s, 3H), 1.37 - 1.33 (m, 1H), 1.29 - 1.25 (m, 1H), 1.24 - 1.21 (m, 1H), 1.17 (s, 3H, Me), 1.15 - 1.07 (m, 1H), 1.02 - 0.88 (m, 2H), 0.85 (s, 3H, Me), 0.78 (s, 6H, 2×Me) ppm. 13 13C NMR (100 MHz, CDCl3): δ = 191.9, 143.7, 142.0, 135.3, 130.3, 127.0, 125.7, 75.2, 73.7, 61.4, 56.2, 45.1, 44.6, 42.1, 39.9, 39.4, 33.5, 33.4, 27.6, 24.5, 21.6, 20.6, 19.2, 18.5, 15.4 ppm. HRMS (HESI / Orbitrap) m / z: C 27 H 41 O3Na + for [M + Na] +Calculated value 435.28697; obtained value 435.28704. IR (ATR): ν = 3379, 2927, 2868, 2735, 1698, 1601, 1568, 1461, 1388, 1306, 1266, 1214, 1167, 1133, 1099, 1085, 1065, 1039, 997, 972, 938, 908, 863, 812, 786, 737, 702, 661 cm -1 .[α] 25 D +36.2 (c = 0.21 g / mol, MeOH).

[0096] Example of the synthesis of compound 2: 4-[(6-aminopropyl)amino]-4-oxobutyric acid

[0097] A solution of succinic anhydride (271 mg, 2.70 mmol) in tetrahydrofuran (THF) (5 mL) was slowly added to a solution of 1,3-diaminopropane (200 mg, 2.70 mmol) in THF (10 mL) over 1 hour. The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched and the solvent was removed under reduced pressure to give the crude product. The crude product was purified by "dry-flash" chromatography (SiO2: DCM → DCM / MeOH 1 / 1 → MeOH) to give compound 2 as a colorless solid (376 mg, 80%). 1 1H NMR (400 MHz, D2O) δ = 3.15 (t, J = 6.6 Hz, 2H, -CH2NHCO-), 2.91 - 2.82 (m, 2H, -CH2NH2), 2.32 (s, 4H, -CH2COOH and -CH2CONH), 1.78 - 1.67 (m, 2H, -CH2CH2NH2). 13 13C NMR (101 MHz, D2O) δ = 180.82, 176.34, 36.84, 35.78, 32.82, 32.15, 26.61.

[0098] Example of the synthesis of compound 3: 4-[(6-aminohexyl)amino]-4-oxobutyric acid

[0099] To a solution of succinic anhydride (200 mg, 2.00 mmol) in tetrahydrofuran (THF) (5 mL), a solution of 1,3-diaminohexane (232 mg, 2.00 mmol) in THF (10 mL) was slowly added over 1 hour. The reaction mixture was stirred at room temperature for 2 hours. The reaction was terminated and the solvent was removed under reduced pressure to give the crude product. The crude product was purified by "dry - flash" chromatography (SiO2: DCM → DCM / MeOH 1 / 1 → MeOH) to give compound 3 as a colorless solid (290 mg, 78%). 1 1H NMR (400 MHz, D2O) δ = 3.19 (t, J = 6.7 Hz, 2H, -CH2NHCO-), 3.01 (d, J = 7.5 Hz, 2H, -CH2NH2), 2.46 (s, 4H, -CH2COOH and -CH2CONH), 1.73 - 1.62 (m, 2H, -CH2CH2NH2), 1.57 - 1.47 (m, 2H, -CH2CH2NHCO-), 1.46 - 1.36 (m, 4H, -CH2CH2CH2NH2 and -CH2CH2CH2CH2NH2). 13 13C NMR (101 MHz, D2O) δ = 180.80, 175.64, 39.29, 38.95, 33.05, 32.38, 27.89, 26.47, 25.21, 24.99.

[0100] Synthesis of ligand LIG1: 4 - ((3 - ((4 - ((R,E) - 3 - hydroxy - 5 - ((1R,2R,4aS,8aS) - 2 - hydroxy - 2,5,5,8a - tetramethyldecahydronaphthalen - 1 - yl) - 3 - methylpent - 1 - en - 1 - yl)benzyl)amino)propyl)amino) - 4 - oxobutanoic acid

[0101] In a flame-dried round-bottom flask, dry MeOH (4 mL) was added, and then 4-{(1E,3R)-3-hydroxy-5-[(2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl]-3-methylpent-1-en-1-yl}benzaldehyde 1 (80 mg, 0.194 mmol) and Ti(Oi-Pr)4 (235 μL, 0.776 mmol) were added. Then, a solution of amine 2 (101 mg, 0.586 mmol) in anhydrous MeOH (0.5 mL) was added dropwise. The reaction mixture was stirred overnight at room temperature, and then NaBH4 (13 mg, 0.388 mmol) was added, and the reaction mixture was stirred for another 2 h at room temperature. The reaction was quenched by adding water (3 mL), and the reaction mixture was filtered through a silica gel column and then washed thoroughly with DCM. The solvent was removed under reduced pressure to give the crude product. Compound LIG1 was obtained as a colorless solid (94 mg, 85%) by dry flash chromatography on silica gel (SiO2: DCM → DCM / MeOH(NH3) = 8 / 2), with a melting point of 108 - 110 °C. 1 1H NMR (400 MHz, CD3OD): δ = 7.43 (d, J = 8.2 Hz, 2H, 2×Ar-H), 7.37 (d, J = 8.3 Hz, 2H, 2×Ar-H), 6.60 (d, J = 16.1 Hz, 1H, =CH), 6.36 (d, J = 16.2 Hz, 1H, =CH), 3.88 (s, 2H, Ar-CH2-N), 3.26 (t, J = 6.5 Hz, 2H, -CH2NHCO-), 2.80 (t, J = 7.2 Hz, 2H, -CH2NHCH2Ar-), 2.46 - 2.35 (m, 4H, -CH2COOH and -CH2CONH), 1.88 - 1.79 (m, 2H), 1.78 - 1.73 (m, 2H), 1.72 - 1.60 (m, 4H), 1.60 - 1.50 (m, 1H), 1.49 - 1.37 (m, 4H), 1.36 (s, 3H, Me), 1.36 - 1.28 (m, 1H), 1.25 - 1.15 (m, 1H), 1.15 - 1.12 (m, 1H), 1.12 (s, 3H, Me), 1.03 - 0.93 (m, 2H), 0.89 (s, 3H, Me), 0.83 (s, 3H, Me), 0.82 (s, 3H, Me) ppm. 1313C NMR (101 MHz, CD3OD): δ = 180.67, 176.74, 164.67, 162.52, 138.83, 130.37, 127.65, 127.57, 75.17, 74.35, 62.73, 57.56, 53.45, 49.85, 49.28, 47.08, 46.56, 45.17, 43.25, 41.18, 40.56, 37.44, 34.64, 34.18, 33.90, 33.88, 29.19, 27.82, 23.97, 21.95, 21.54, 20.84, 19.50, 16.05. HRMS (HESI / Orbitrap) m / z: C 34 H 55 O5N2 [M+H] + calculated value 571.41055; obtained value 571.41120. IR (ATR): ν = 3292, 2929, 2867, 1640, 1562, 1459, 1388, 1301, 1270, 1219, 1188, 1157, 1132, 1085, 1033, 996, 970, 938, 908, 865, 640 cm -1 . HPLC purity, method E: t R = 7.608 min, area 99.42%. Method B: t R = 6.137 min, area 99.07% (λ = 254 nm). [α] 25 D +0.054 (c = 3.3 x 10 -3 g / mol, MeOH).

[0102] Synthesis of ligand LIG2: 4 - ((6 - ((4 - ((R,E) - 3 - hydroxy - 5 - ((1R,2R,4aS,8aS) - 2 - hydroxy - 2,5,5,8a - tetramethyldecahydronaphthalen - 1 - yl) - 3 - methylpent - 1 - en - 1 - yl)benzyl)amino)hexyl)amino) - 4 - oxobutyric acid

[0103] In a flame-dried round-bottom flask, dry MeOH (4 mL) was added, and then 4-{(1E,3R)-3-hydroxy-5-[(2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl]-3-methylpent-1-en-1-yl}benzaldehyde 1 (95 mg, 0.231 mmol) and Ti(Oi-Pr)4 (262 μL, 0.924 mmol) were added. Then, a solution of amine 3 (150 mg, 0.694 mmol) in anhydrous MeOH (0.5 mL) was added dropwise. The reaction mixture was stirred overnight at room temperature, and then NaBH4 (17.4 mg, 0.462 mmol) was added, and the reaction mixture was stirred for another 2 h at room temperature. The reaction was quenched by the addition of water (3 mL), and the reaction mixture was filtered through a silica gel column and then washed thoroughly with DCM. The solvent was removed under reduced pressure to give the crude product. Compound LIG2 was obtained as a colorless solid (141 mg, 83%) by dry flash chromatography (SiO2: DCM → DCM / MeOH(NH3) = 8 / 2), with a melting point of 124 - 126 °C. 1 1H NMR (400 MHz, CD3OD): δ = 7.49 (d, J = 8.1 Hz, 2H, 2×Ar-H), 7.44 (d, J = 8.2 Hz, 2H, 2×Ar-H), 6.61 (d, J = 16.1 Hz, 1H, =CH), 6.41 (d, J = 16.1 Hz, 1H, =CH), 4.14 (s, 2H, Ar-CH2-N), 3.19 (t, J = 6.3 Hz, 2H, -CH2NHCO-), 2.99 (t, J = 7.7 Hz, 2H, -CH2NHCH2Ar-), 2.50 - 2.35 (m, 4H, -CH2COOH and -CH2CONH), 1.93 - 1.77 (m, 2H), 1.75 - 1.56 (m, 6H), 1.55 - 1.46 (m, 3H), 1.44 - 1.37 (m, 6H), 1.35 (s, 3H, Me), 1.35 - 1.28 (m, 1H), 1.22 - 1.16 (m, 1H), 1.15 - 1.10 (m, 1H), 1.11 (s, 3H, Me), 1.00 - 0.91 (m, 2H), 0.88 (s, 3H, Me), 0.82 (s, 3H, Me), 0.81 (s, 3H, Me) ppm. 13¹³C NMR (101 MHz, CD₃OD): δ = 180.53, 176.00, 140.04, 139.84, 132.01, 131.16, 127.95, 127.20, 75.17, 74.35, 62.73, 57.56, 54.80, 52.08, 49.29, 48.18, 47.03, 45.17, 43.25, 41.17, 40.55, 39.75, 34.61, 34.18, 33.94, 33.90, 29.84, 27.92, 27.06, 26.83, 26.69, 23.95, 21.94, 21.53, 20.82, 19.51, 16.05. HRMS (HESI / Orbitrap) m / z: C 37 H 61 O₅N₂ of [M + H] + Calculated value 613.45750; obtained value 613.45804. IR (ATR): ν = 3289, 3089, 2927, 2862, 1635, 1561, 1437, 1387, 1300, 1268, 1216, 1178, 1132, 1084, 1032, 995, 970, 938, 908, 864, 802, 724, 642, 563 cm -1 . HPLC purity, method A: t R = 7.683 min, area 99.32%. Method B: t R = 6.191 min, area 99.11% (λ = 254 nm). [α] 25 D +0.054 (c = 1.3 x 10 -3 g / mol, MeOH).

[0104] Synthesis of CON1: N1-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-1-yl)oxy)tetrahydro-2H-pyran-4-yl)-N4-(3-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)propyl)succinamide

[0105] To a solution of doxorubicin hydrochloride (21 mg, 0.036 mmol) in anhydrous DMF (500 μL), diisopropylethylamine (6 μL, 0.050 mmol) was added. The addition of DIEA caused the color of the solution to change from light red to dark red. The reaction mixture was stirred in argon at room temperature for 30 minutes, and then compound LIG1 (21 mg, 0.036 mmol) was added. After cooling the reaction mixture in an ice bath (0 °C), solutions of HOBT (6.1 mg, 0.050 mmol) and EDCI (9.4 mg, 0.050 mmol) in anhydrous DMF (100 μL each) were added successively. The reaction mixture was stirred at 0 °C for 30 minutes and then stirred at room temperature in the dark for 18 hours. The reaction progress was monitored using an Agilent 1200 HPLC system equipped with a reversed-phase analytical InfinityLab Poroshell120 CS-C18 column (4.6 × 100 mm, 2.7 μ, S.N. USKBM01053). The mobile phase consisted of a mixture of 0.1% aqueous formic acid and ACN, with isocratic and gradient elution programs as follows: 0 - 1 min, 5% ACN; 1 - 6 min, 5% → 95% ACN; 6 - 12 min, 95% ACN; 12 - 14 min, 95% A → 5% ACN; 14 - 15 min, 5% ACN. The flow rate was 0.6 mL / min. Signal detection was performed using a DAD detector in the wavelength range of 254 / 280 nm. After 12 hours, the reaction was stopped, and the reaction mixture was concentrated under reduced pressure. The crude reaction mixture was purified on an HPLC-DAD system using a semi-preparative ZORBAX Eclipse XDB-C18 column (9.4 × 250 mm, 5 μ, S.N. USSY004729) (DAD detector: 254 nm). The mobile phase consisted of a mixture of 0.1% aqueous formic acid and MeOH. The flow rate was 3.5 mL / min, and gradient elution was carried out as follows: 0 - 1 min, 5% MeOH; 1 - 6 min, 5 → 100% MeOH; 6 - 10 min, 100% MeOH, and CON1 (Rf = 8.31 min) was isolated. The solvent was removed under reduced pressure, and the residue was dissolved in DCM. The organic layer was washed several times with aqueous NaHCO3 solution and saturated aqueous NaCl solution and dried over anhydrous Na2SO4. After filtration, the solvent was removed by rotary evaporation under reduced pressure. Compound CON1 was obtained as a dark red powder (29.7 mg, 75%), which softened at 178 - 180 °C. 11H NMR (400 MHz, CDCl3 + CD3OD): δ = 7.90 (d, J = 6.7 Hz, 1H, H-C4Ar(DOX)), 7.70 (t, J = 8.1 Hz, 1H, H-C3Ar(DOX)), 7.32 (d, J = 8.4 Hz, 1H, H-C2Ar(DOX)), 7.25 - 7.10 (m, 4H, 4x H-Ar(scl)), 6.40 (d, J = 16.4 Hz, 1H, =CH), 6.19 (d, J = 16.3 Hz, 1H, =CH), 5.40 - 5.35( 1H, H-2’), 5.17 - 5.10 (m, 1H, H-C 10 Ar(DOX)), 4.61 (s, 2H, CH2-OH), 4.02 (d, 1H, J = 6.7 Hz, H-6’), 4.00 - 3.90 (m, 1H, H-4’), 3.97 (s, 3H, -O-CH3), 3.86 (s, 2H, Ar-CH2-NH), 3.50 - 3.45 (brs, 1H, H-5’), 3.20 - 3.00 (m, 3H, CH2NHCO-, H-C7Ar(DOX)), 3.00 - 2.85 (m, 1H, H-C7Ar(DOX)), 2.80 - 2.70 (m, 2H, -CH2NHCH2Ar-), 2.40 - 2.10 (m, 5H, -NHCOCH2CH2CONH and H-C9Ar(DOX)), 2.05 - 1.90 (m, 1H, H-C9Ar(DOX)), 1.85 - 1.65 (m, 4H, H-C 3' Ar(DOX)), 1.65 - 1.35 (m, 6H, H-C 3' Ar(DOX)), 1.30 - 1.20 (m, 3H), 1.23 (s, 3H, Me), 1.05 - 0.90 (m, 2H), 1.16 (d, J = 6.7 Hz, 3H, 6’-CH3), 1.04 (s, 3H, Me), 0.90 - 0.75 (m, 2H), 0.74 (s, 3H, Me), 0.68 (s, 3H, Me), 0.67 (s, 3H, Me). 13 13C NMR (101 MHz, CDCl3 + CD3OD): δ = 213.65 (C 13 =O), 187.14 (C 12=O), 186.71 (C5=O), 174.33 (NHC=O), 172.20 (NHC=O), 161.00 (C1), 155.92 (C6), 155.19 (C11), 139.42 (=CH), 138.48, 135.88 (C3), 135.36 (C4a), 133.86 (10a), 133.60 (C6a), 129.82 (2x Ar(scl)), 126.83 (2xAr(scl)), 126.53, 125.26 (=CH), 120.71 (C12a), 119.75 (C4), 118.61 (C2), 111.46 (5a), 111.25 (11a), 100.69 (C2’), 77.36, 76.33 (C8), 74.49, 73.06, 69.37 (C10), 68.28 (C5’), 67.41 (C6’), 65.05 (C14), 61.38, 56.54 (O-CH3), 56.05, 51.31 (Ar-CH2-NH), 45.55, 45.00, 44.50 (NH-C4’), 43.68, 41.90, 39.66, 39.18, 36.03, 35.69 (C9), 33.54 (C7), 33.21 (Me), 33.11, 31.03, 29.60, 29.28 (C3’), 26.20, 23.64 (Me), 21.32 (Me), 20.32 (Me), 18.91, 18.34, 16.64 (6’-CH3), 15.30 (Me). HRMS (HESI / Orbitrap) m / z: C 61 H 82 O 15 [M+H] of N3 + Calculated value 1096.57405; Obtained value 1096.57532. IR (ATR): ν = 3360, 2926, 1724, 1618, 1577, 1444, 1411, 1387, 1284, 1236, 1209, 1170, 1116, 1083, 1018, 985, 794, 765, 611, 463 cm -1 . HPLC purity, Method A: t R = 8.309 min, area 96.13%. Method B: t R = 6.688 min, area 95.32% (λ = 254 nm). [α] 25 D +0.012 (c = 1.7 x 10 -4 g / mol, MeOH).

[0106] Synthesis of CON2: N1-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-1-yl)oxy)tetrahydro-2H-pyran-4-yl)-N4-(6-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)hexyl)succinamide

[0107] To a solution of doxorubicin hydrochloride (15 mg, 0.052 mmol) in anhydrous DMF (500 μL), diisopropylethylamine (13 μL, 0.036 mmol) was added. The addition of DIEA caused the color of the solution to change from light red to dark red. The reaction mixture was stirred in argon at room temperature for 30 minutes, and then compound LIG2 (16 mg, 0.026 mmol) was added. After cooling the reaction mixture in an ice bath (0 °C), anhydrous DMF (100 μL each) solutions of HOBT (8.9 mg, 0.036 mmol) and EDCI (14 mg, 0.036 mmol) were added successively. The reaction mixture was stirred at 0 °C for 30 minutes and then stirred at room temperature in the dark for 18 hours. The reaction progress was monitored using an Agilent 1200 HPLC system equipped with a reversed-phase analytical InfinityLab Poroshell 120 CS-C18 column (4.6 × 100 mm, 2.7 μ, S.N. USKBM01053). The mobile phase consisted of a mixture of 0.1% formic acid aqueous solution and ACN, with isocratic elution and gradient elution programs: 0 - 1 min, 5% ACN; 1 - 6 min, 5% → 95% ACN; 6 - 12 min, 95% ACN; 12 - 14 min, 95% A → 5% ACN; 14 - 15 min, 5% ACN. The flow rate was 0.6 mL / min. Signal detection was performed using a DAD detector in the wavelength range of 254 / 280 nm. After 12 hours, the reaction was stopped and the reaction mixture was concentrated under reduced pressure. The crude reaction mixture was purified on an HPLC-DAD system using a semi-preparative ZORBAX Eclipse XDB-C18 column (9.4 × 250 mm, 5 μ, S.N. USSY004729) (DAD detector: 254 nm). The mobile phase consisted of a mixture of 0.1% formic acid aqueous solution and MeOH. The flow rate was 3.5 mL / min, and gradient elution was carried out as follows: 0 - 1 min, 5% MeOH; 1 - 6 min, 5 → 100% MeOH; 6 - 10 min, 100% MeOH, and CON2 (Rf = 8.33 min) was isolated. The solvent was removed under reduced pressure, and the residue was dissolved in DCM. The organic layer was washed several times with aqueous NaHCO3 solution and saturated aqueous NaCl solution and dried over anhydrous Na2SO4. After filtration, the solvent was removed by rotary evaporation under reduced pressure. Compound CON2 was obtained as a dark red powder (21.3 mg, 75%), which softened at 170 - 172 °C. 11H NMR (400 MHz, CDCl3 + CD3OD): δ = 7.86 (d, J = 8.7 Hz, 1H, H-C4Ar(DOX)), 7.65 (t, J = 8.1 Hz, 1H, H-C3Ar(DOX)), 7.28 (d, J = 8.7 Hz, 1H, H-C2Ar(DOX)), 7.21 (d, J = 8.2 Hz, 2H, 2x H-Ar(scl)), 7.14 (d, J = 7.9 Hz, 2H, 2x H-Ar(scl)), 6.38 (d, J = 16.4 Hz, 1H, =CH), 6.14 (d, J = 16.1 Hz, 1H, =CH), 5.35 - 5.30 (brs, 1H, H-2’), 5.13 - 5.07 (m, 1H, H-C 10 Ar(DOX)), 4.59 (s, 2H, CH2-OH), 3.99 - 3.95 (m, 1H, H-6’), 3.95 - 3.85 (m, 1H, H-4’), 3.91 (s, 3H, -O-CH3), 3.74 (s, 2H, Ar-CH2-NH), 3.40 - 3.35 (brs, 1H, H-5’), 3.12 - 3.02 (m, 1H, H-C7Ar(DOX)), 2.96 (t, J = 6.6 Hz, 2H, -CH2NHCO-), 2.92 - 2.82 (m, 1H, H-C7Ar(DOX)), 2.57 (t, J = 7.3 Hz, 2H, -CH2NHCH2Ar-), 2.40 - 2.10 (m, 5H, -NHCOCH2CH2CONH and H-C9Ar(DOX)), 2.10 - 1.90 (m, 1H, H-C9Ar(DOX)), 1.85 - 1.75 (m, 1H, H-C 3' Ar(DOX)), 1.70 - 1.50 (m, 4H, H-C 3' Ar(DOX)), 1.50 - 1.35 (m, 6H), 1.35 - 1.15 (m, 6H), 1.18 (s, 3H, Me), 1.15 - 1.05 (m, 5H), 1.05 - 0.95 (m, 2H), 1.12 (d, J = 6.6 Hz, 3H, 6’-CH3), 0.85 - 0.70 (m, 2H), 0.98 (s, 3H, Me), 0.85 - 0.70 (m, 2H), 0.69 (s, 3H, Me), 0.63 (s, 3H, Me), 0.61 (s, 3H, Me). 13 13C NMR (101 MHz, CDCl3 + CD3OD): δ = 213.56 (C 13 =O), 187.14 (C 12=O), 186.67 (C5=O), 172.89 (NHC=O), 172.30 (NHC=O), 160.94 (C1), 155.88 (C11), 155.17 (C6), 138.95 (=CH), 135.83 (C3), 135.30 (C4a), 133.92 (C10a), 133.62 (C6a), 129.39 (2x Ar(scl)), 126.67 (2x Ar(scl)), 125.33 (=CH), 120.63 (C12a), 119.65 (C4), 118.58 (C2). 111.41 (C11a), 111.17 (C5a), 100.64 (C2’), 77.36, 76.24 (C8), 74.32, 72.96, 69.33 (C10), 68.22 (C5’), 67.29 (C6’), 64.94 (C14), 61.21, 56.40 (O-CH3), 55.98, 51.48 (Ar-CH2-NH), 45.47 (NH-C4’), 44.98, 43.55, 41.81, 39.58, 39.10, 38.93, 35.70 (C9), 33.39 (C7), 33.07 (Me), 33.00, 31.18, 31.02, 29.48, 29.16 (C3’), 28.52, 26.07 (Me), 25.94, 23.46 (Me), 21.18 (Me), 20.21, 18.92, 18.23, 16.50 (6’-CH3), 15.18 (Me). HRMS (HESI / Orbitrap) m / z: C 64 H 88 O 15 For [M+H] of N3 + Calculated value 1138.62100; obtained value 1138.62158. IR (ATR): ν = 3314, 2932, 2865, 1721, 1582, 1444, 1412, 1385, 1347, 1285, 1209, 1115, 1084, 1017, 986, 792, 764, 587, 478 cm -1 . HPLC purity, method A: t R = 8.336 min, area 98.18%. Method B: t R = 6.735 min, area 98.75% (λ = 254 nm). [α] 25 D +0.019 (c = 1.7 x 10 -4 g / mol, MeOH).

[0108] Biological tests

[0109] Materials and Methods

[0110] Compound

[0111] The compounds used in this study were doxorubicin (DOX, Sigma - Aldrich, Germany), sclareol (SC), and hybrids (conjugates) CON1 and CON2, and their corresponding ligands (LIG1 and LIG2). All compounds were dissolved in dimethyl sulfoxide (DMSO) at a stock solution of 20 mM and stored in aliquots at -20 °C. Before treatment, the compounds were dissolved in sterile deionized water.

[0112] Chemicals and reagents

[0113] The following chemicals and reagents were used in the experimental work: rhodamine 123 (Rho123), Hoechst 33342, DMSO, 3 - [4,5 - dimethylthiazol - 2 - yl] - 2,5 - diphenyltetrazolium bromide (MTT) (Sigma - Aldrich Chemie GmbH, Germany), Minimal Essential Medium (MEM), RPMI 1640 medium, Dulbecco's Modified Minimal Essential Medium (DMEM), fetal bovine serum (FBS), a mixture of antibiotics penicillin - streptomycin (Capricorn Scientific, Germany), trypsin / EDTA (Biological Industries, USA), L - glutamine, a mixture of antibiotics and antifungals: penicillin, streptomycin, amphotericin B (Gibco, ThermoFisher Scientific, USA), MEM non - essential amino acids (Biowest, USA).

[0114] Cell line

[0115] The U87 human glioblastoma cell line, DLD1 human colorectal cancer cell line, and NCI-H460 human non-small cell lung cancer cell line were obtained from the American Type Culture Collection (ATCC, USA). The U87-TxR resistant human glioblastoma cell line was obtained by continuously exposing U87 cells to increasing concentrations of paclitaxel (100 - 300 nM) for six to nine months and is characterized by high expression of P-glycoprotein. The DLD1-TxR resistant human colorectal cancer cell line was selected from the DLD1 cell line by exposure to gradually increasing concentrations of paclitaxel (60 - 600 nM) for ten months and is also characterized by higher expression of P-glycoprotein compared to the parental cell line. The NCI-H460 / R resistant human non-small cell lung cancer cell line was obtained by continuously exposing NCI-H460 cells to increasing concentrations of DOX for three months and is characterized by extremely high expression of P-glycoprotein. The U251 human glioblastoma cell line and MRC-5 human lung fibroblast cell line were obtained from the European Collection of Authenticated Cell Cultures (ECACC, UK). The U87, U87-TxR, and MRC-5 cell lines were grown in MEM medium supplemented with 10% FBS, 1% L-glutamine, 1% antibiotic mixture of penicillin and streptomycin, and 1% non-essential amino acids. NCI-H460, NCI-H460 / R, DLD1, and DLD1-TxR were grown in RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamine, and 1% antibiotic-antifungal mixture. The U251 cell line was grown in DMEM medium supplemented with 10% FBS, 1% L-glutamine, and 1% antibiotic mixture of penicillin and streptomycin. All cell lines were grown in an incubator at 37°C in a humid atmosphere containing 5% CO2.

[0116] In 25 cm 2 and 75 cm 2 flasks (ThermoFisher Scientific, USA), after reaching 80 - 90% confluence, cells were passaged using 0.25% trypsin / EDTA. After trypsin digestion, cells were counted using a Burker-Turk hemocytometer on an inverted microscope. To determine the cell number, 10 μL of cell suspension was injected into two chambers of the hemocytometer. Four fields of view were counted in both chambers. To calculate the total number of cells per milliliter, the following formula was used: average number of cells per square chamber × cell dilution factor × 10 4 (chamber factor). After counting, cells were seeded at an appropriate density for further experiments or to maintain in culture (for NCI-H460, NCI-H460 / R, DLD1, and DLD1-TxR, the density was 8000 cells / cm 2; For U87, U87-TxR, U251, and MRC-5, the density was 16,000 cells / cm 2 ).

[0117] Cytotoxic effects of SC, DOX, their combinations and their new hybrids (CON1 and CON2)

[0118] The MTT assay was used to evaluate the effect of the compounds on cell viability. This assay relies on the reduction of the tetrazolium salt (MTT) to formazan, which is catalyzed by the mitochondrial enzyme succinate dehydrogenase. The activity of succinate dehydrogenase, which indicates mitochondrial respiration, is considered an indirect measure of cell metabolic activity and thus an indirect measure of its viability. The resulting MTT-formazan is a purple product, and its absorbance is measured at a wavelength of 570 nm.

[0119] To determine the inhibitory effect of SC on cell growth, different cell lines were exposed to increasing concentrations of SC (5, 10, 25, 50, and 100 μM). In 100 μL of appropriate medium in 96-well microtiter plates, cells were seeded at a density of 2,000 cells per well for NCI-H460, NCI-H460 / R, DLD1, and DLD1-TxR, and at a density of 4,000 cells per well for MRC-5, U87, U87-TxR, and U251. Untreated control cells were also seeded. After 24 h, the cells were treated with various concentrations of SC to acclimatize. After 72 h of treatment, 0.2 mg / ml MTT was added to each well in the appropriate cell medium. After incubation at 37 °C with 5% CO2 for 4 h, the medium was removed, and 100 μL of DMSO was added to each well to dissolve the formazan. The absorbance of the samples was measured at 570 nm using a Multiskan Sky microplate spectrophotometer (ThermoFisher Scientific, USA). The results were analyzed using GraphPad Prism 8.0.2 software, and the IC50 values were determined by non-linear regression.

[0120] b) To test the interaction between SC and DOX in terms of cytotoxicity in different cell lines, cells were seeded at a density of 2,000 cells per well in 100 μL of appropriate medium in a 96-well microtiter plate for NCI-H460, NCI-H460 / R, DLD1, and DLD1-TxR, and at a density of 4,000 cells per well for MRC-5, U87, U87-TxR, and U251. SC (10 and 20 μM) was combined with increasing concentrations of DOX (20, 50, 100, 200, and 500 nM). Cells were treated with the simultaneous compositions for 72 h. After this period, 0.2 mg / ml MTT was added to each well and then incubated for 4 h at 37 °C and 5% CO2. Then the medium was removed and DMSO was added to each well to dissolve formazan. The absorbance of the samples was measured at 570 nm using a Multiskan Sky microplate spectrophotometer.

[0121] Analysis was performed using GraphPad Prism software version 8.0.2 to determine the nature of the interaction between the compounds, i.e., synergistic, antagonistic, or additive. This was done using the computer software CalcuSyn, which is based on the combination index method. This method takes into account the concentrations of the compounds, the individual actions of each compound, and the combined action of the two compounds. The value of the combination index (CI) describes the nature of the interaction: CI < 0.9 indicates synergistic action, CI > 1.1 indicates antagonistic action, and CI = 0.9 - 1.1 indicates additive action.

[0122] c) To compare the resistance and selectivity profiles in SC, DOX, CON1, and CON2, the human glioblastoma cell lines U87, U87-TxR, and MRC-5 were seeded at a density of 4,000 cells per well in 100 μL of appropriate medium. In contrast, NCI-H460, NCI-H460 / R, and MRC-5 were seeded at a density of 2,000 cells per well in 100 μL of appropriate medium. The effects of SC derivatives LIG1 and LIG2 were also examined. The concentration ranges of cell lines and compounds were different. For LIG1 and LIG2, the concentration range for all cell lines was 50, 100, 200, 300, and 400 μM, while for SC, CON1, and CON2, the same range was used for MRC-5. For U87 and NCI-H460, the DOX concentration range was 0.1, 0.25, 0.5, 1, and 2.5 μM; for U87-TxR, NCI-H460 / R, and MRC-5, the DOX concentration range was 0.25, 0.5, 1, 2.5, and 5 μM. The five concentrations used for SC, CON1, and CON2 treatments varied in the range of 1, 2.5, 5, 10, 25, 50, and 100 μM. The treatment lasted for 72 h. After the treatment period, 0.2 mg / ml MTT in appropriate cell medium was added to each well. After incubation at 37 °C and 5% CO2 for 4 h, the medium was removed, and DMSO was added to each well to dissolve formazan. The absorbance of the samples was measured at 570 nm on a Multiskan Sky microplate spectrophotometer. The results were analyzed using GraphPad Prism 8.0.2 software, and the IC50 values were determined by non-linear regression.

[0123] Detection of P-glycoprotein substrate accumulation (DOX and rhodamine 123)

[0124] a) First, the DOX accumulation assay was performed on three glioblastoma cell lines U87, U87-TxR, and U251. The cells were trypsinized and counted, and then 100,000 cells per sample were collected in tubes for flow cytometry and resuspended in 500 μL of medium. The cells were treated with 20 μM and 50 μM SC. After SC treatment, the cells were immediately treated with 20 μM DOX, and the samples were incubated at 37 °C in a humid atmosphere with 5% CO2 for 60 min. After the accumulation period, the samples were centrifuged, washed twice with cold phosphate-buffered saline (PBS), and finally resuspended in 1 mL of PBS. The fluorescence of the samples was read on FL2 (red channel) of a cell fluorometer (Partec, Munster, Germany), and the results were analyzed using the software package Summit 4.3.

[0125] b) Secondly, DOX and rhodamine 123 accumulation assays were performed on non-small cell lung cancer cell lines NCI-H460 and NCI-H460 / R. The cells were trypsinized and counted, and then 100,000 cells of each sample were collected in tubes for flow cytometry and resuspended in 500 μL of medium. The cells were treated with 50 μM SC. Immediately after treatment, 5 μM Rho123 was added to one set of samples and 20 μM DOX was added to the other set. They were incubated at 37 °C in a humid atmosphere of 5% CO2 for 30 min or 60 min, respectively. After the accumulation period, the samples were centrifuged, washed twice with cold PBS, and finally resuspended in 1 mL of PBS. The samples were analyzed using a flow cytometer, and rhodamine 123 on FL1 (green channel) and DOX on FL2 (red channel) were read. The results were analyzed using Summit 4.3 software.

[0126] Intracellular localization of conjugates (CON1 and CON2) and DOX

[0127] This study investigated the localization of fluorescence derived from DOX on the sensitive glioblastoma cell line U87. The cells were seeded at a density of 24,000 cells per well in 600 μL of appropriate growth medium in 24-well plates (ThermoFisher Scientific, USA). After 24 h, the cells were treated with increasing concentrations of CON1 and CON2 (2, 5, and 10 μM) for 72 h. Two wells with untreated cells were used, one as an untreated control and the other treated with 20 μM DOX for 2 h immediately before imaging. The cells were stained with Hoechst 33342 for 15 min. Then, all wells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and imaged in the blue and red channels on a ZOE Fluorescent Cell Imager (BIO-RAD, USA).

[0128] Physicochemical properties of CON1 and CON2

[0129] The Malvern Zetasizer Nano ZS (Malvern Instruments, UK) was used to analyze the physicochemical stability characteristics of the conjugates. The measurement range was from 0.6 nm to 6 mm. The average size, polydispersity index (PDI), and ζ (Zeta) potential of the conjugates were measured. All measurements were carried out at a temperature of 25 °C, and each sample was diluted 100-fold with ultrapure water. The measurements were repeated three times, and the results were expressed as the mean. The PDI is a parameter used to define the particle size range. The term "polydispersity" (or "dispersity" recommended by IUPAC) describes the degree of inhomogeneity of the particle size distribution. The ζ potential represents the surface charge of the nanoparticles, indicating their long-term stability.

[0130] Chemical modeling

[0131] Using from Epik v5.7 of Suite 2021-3 Release 2021-3: Epik, LLC, New York, NY, 2021] to predict pKa. Using from MacroModel v13.3 module of Suite 2021-3 Release 2021-3: MacroModel, LLC, New York, NY, 2021], conformational searches were carried out with the OPLS4 force field, water as the solvent, and the hybrid torsion / low / mole sampling method. The number of steps was 1000, and the energy window (cutoff) for saving structures was 21 kJ / mol.

[0132] Transmission electron microscopy for the characterization and intracellular localization of CON1 and CON2

[0133] a) The nanoparticle properties of CON1 and CON2 were analyzed using transmission electron microscopy. For preparation of the analysis, CON1 or CON2 was diluted 100-fold with ultrapure water, and a poly(vinyl formal) (Formvar) or carbon-coated glow-discharge nickel grid was placed on top of the droplet and left for 2 - 5 minutes to absorb the excess fluid. The particles were examined on a Philips CM12 transmission electron microscope (Philips / FEI, Netherlands), which was operated at 80 kEV and equipped with a digital camera SIS MegaView III (Olympus Soft Imaging Solutions, Germany). The diameters of the CON1 and CON2 particles were measured using iTEM software.

[0134] b) For transmission electron analysis of CON1 and CON2 U87 cell uptake, 2,000,000 U87 cells were treated with 5 μM CON1 or CON2 for 24 h, and untreated cells served as controls. After treatment, the cells were immediately rinsed with PBS, fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4 °C for 2 h, and rinsed again with cacodylate buffer. The cells were then post-fixed in 2% osmium tetroxide in the same buffer, dehydrated using increasing concentrations of ethanol, and embedded in resin. Semi-thin sections were processed for electron microscopy, and ultra-thin cell sections were obtained using a Leica UC6 ultramicrotome (Leica Microsystems, Germany) and fixed on copper grids. More than 20 cell sections were examined on a Philips CM12 transmission electron microscope equipped with a digital camera SIS MegaView III. The resulting electron micrographs were used for overall U87 cell morphology analysis and CON1 or CON2 cell uptake analysis.

[0135] Comparative toxicology study of DOX and CON1 in Balb / c mice

[0136] Male Balb / c mice, 7–11 weeks old, were obtained from EL-42BIO / Br-01 (Laboratory of Pharmacology, School of Medicine, University of Thessaly, Larissa, Greece). The mice were housed in an environment with a 12-h light / dark cycle, humidity of approximately 50–60%, and temperature of 20–22 °C. They had free access to food and water. The handling and experiments on the mice were conducted in accordance with Greek law (PD 56 / 2013 and Circle 2215 / 117550 / 2013) and EU guidelines (2013 / 63 / EU). The protocol was approved by the IACUC and the Greek authorities (permit number 58376 / 13.02.2023). The mice were divided into three groups of two mice each, for a total of 18 mice. They were treated as follows: Group 1 received the vehicle (10% DMSO and 5% Tween 80); Group 2 received 7 mg / kg body weight of DOX by intraperitoneal injection (i.p.); Group 3 received 14 mg / kg body weight of CON1 (to achieve a DOX concentration of 7 mg / kg body weight) i.p. The study was conducted at three time points: 24 h, 48 h, and 72 h. At the end of each period, blood samples were collected from the tails and cheeks of the mice, and the mice were sacrificed by cervical dislocation following CO2 inhalation. Fresh blood samples were analyzed using a scil Vet ABC TMAnalyzed by a hematology analyzer (Scil Animal Care Company GmbH, Germany), and the serum of all samples was extracted and analyzed using a Skyla VB1 Veterinary Clinical Chemistry Analyzer (Skyla Corporation, Taiwan, China) equipped with a Critical Care Panel (900-330) (Skyla Corporation, Taiwan, China).

[0137] This invention relates to the cytotoxicity testing of SC against various cell types, including human glioblastoma U87, U87-TxR, and U251, human non-small cell lung cancer NCI-H460 and NCI-H460 / R, human colorectal cancer DLD1 and DLD1-TxR, and human lung fibroblast MRC-5( Figure 1 ). It also includes the study of the interaction between SC and DOX on the above cell lines in a simultaneous combined treatment( Figure 2 ). Additionally, this invention includes the cumulative study of DOX in human glioblastoma U87, U87-TxR, and U251, as well as human non-small cell lung cancer NCI-H460 and NCI-H460 / R( Figure 3 and 4 ), and the comparison of the selectivity and resistance profiles of CON1 and CON2 with DOX in glioblastoma U87 and U87-TxR, as well as human non-small cell lung cancer NCI-H460 and NCIH460 / R (Table 1, Figure 5 ). This invention also includes the analysis of the different intracellular localizations of CON1 and CON2 compared to DOX( Figure 6 ), the analysis of the unforeseen nanoparticle properties of CON1 and CON2 (Table 2, Figure 7 and 8), and the analysis of the different toxicological curves of CON1 compared to DOX (Figure 9).

[0138] As Figure 1 shown, SC alone has a relatively weak anti-cancer effect, with IC50 values achieving 50% cell growth inhibition rates in the range of 20 to 70 μM depending on the cell type. In contrast, DOX acts in the nanomolar range in sensitive cancer cell lines and in the micromolar range in resistant cancer cell lines, but remains below 5 μM. The interaction between SC and DOX has been tested on different cell types to determine the nature of their combined treatment( Figure 2 ). The results show that SC is extremely synergistic with DOX in glioblastoma cells U87-TxR and U251, as well as non-small cell lung cancer NCI-H460 / R that are resistant to DOX( Figure 2)。However, the combination of SC and DOX has an antagonistic effect on MRC-5 lung fibroblasts, indicating that SC reduces the effect of DOX on normal cells( Figure 2 )。

[0139] SC has been found to increase the accumulation of DOX in glioblastoma cells, independent of the expression and activity of P-glycoprotein. This effect was observed in all glioblastoma cell lines examined, including U87, U87-TxR, and U251( Figure 3 )。In contrast, in non-small cell lung cancer cell lines, the increased accumulation was only due to the inhibition of P-glycoprotein activity by SC in the resistant NCI-H460 / R cells( Figure 4 )。

[0140] The cytotoxicity of newly synthesized SC and DOX conjugates against different cell lines was tested. The results showed that CON1 and CON2 were more effective in inhibiting the growth of glioblastoma cells than non-small cell lung cancer cells (Table 1, Figure 5 )。The ligands themselves do not show anti-cancer activity because their IC50 values are greater than 50 μM, even for the sensitive cancer cell lines U87 and NCI-H460 (Table 1). The hybrids were found to be selective for cancer cells because they had no significant effect on the growth of lung fibroblasts MRC-5 (Table 1). In addition, CON1 and CON2 showed better selectivity in both glioblastoma and non-small cell lung cancer cell models than DOX. This indicates that the selectivity index of CON1 and CON2 is greater than that of DOX (Table 1). Additionally, CON1 and CON2 showed better resistance characteristics because the relative resistance to CON1 and CON2 was lower than that of DOX in both glioblastoma and non-small cell lung cancer cell models (Table 1).

[0141] According to fluorescence localization studies, CON1 and CON2 were located near the nuclear membrane, while DOX was found inside the nucleus( Figure 6 )。

[0142] When studying the physicochemical properties of CON1 and CON2, it was found that these molecules have the ability to spontaneously form nanoparticles. This finding was confirmed using a Zetasizer, which showed uniform nanoparticle sizes for CON1 and CON2, with a low polydispersity index of less than 0.2. A positive ζ potential greater than 20 indicates that the formation of larger aggregates is unlikely (Table 2, Figure 7 )。Furthermore, chemical modeling predicted a spontaneous "protein-like" folding for CON1 and CON2( Figure 7), which is the result of strong intramolecular forces. Prediction of the pKa of CON1 and CON2 indicates that both molecules have protonated aliphatic nitrogens under the given conditions. These structures were used for conformational searches to find stable conformational structures in solution. The most stable conformers of both molecules show several intramolecular H-bonds, as well as additional hydrophobic interactions. Some functional groups (-OH, amide, -NH2 + -) and hydrophobic structures can be used for further supramolecular organization.

[0143] The formation of CON 1 and CON 2 nanoparticles was confirmed by transmission electron microscopy ( Figure 8A ). Additionally, transmission electron microscopy showed that CON 1 and CON 2 nanoparticles can penetrate cancer cells and can be found in different parts of the cell such as the cytoplasm, cell membrane, nucleolar membrane, mitochondrial membrane, nucleus, and nucleolus ( Figure 8B ). Together with the findings obtained by fluorescence microscopy analysis, the transmission electron microscopy results indicate that the intracellular distribution of CON 1 and CON 2 compounds is different from that of DOX, which is usually located in the nucleus. This also suggests that the hybrids may have a different mechanism of action compared to DOX.

[0144] Toxicity tests conducted on Balb / c mice revealed different toxic effects of CON 1 and DOX. It was found that DOX significantly decreased the activity of alkaline phosphatase (ALP) over time and increased the level of alanine aminotransferase (ALT). This indicates that DOX affects the function of the liver by reducing its activity or damaging its activity ( Figure 9A ). However, CON 1 did not change the ALT level but increased the ALP level after 48 h and had a tendency to return to the control level after 72 h ( Figure 9A ). Both DOX and CON 1 increased the level of creatine phosphokinase (CPK) after 72 h. However, only DOX had a significant effect on CPK, indicating damage to the myocardium or other muscles ( Figure 9A ). CON 1 transiently increased the total bicarbonate (tCO2) level at 48 h, but this effect disappeared at 72 h ( Figure 9B ). On the other hand, DOX continuously increased the level of K, which implies kidney damage ( Figure 9C ). CON 1 showed a temporary increase in the Ca level and a decrease in the Na level at 48 h, but these changes disappeared after 72 h ( Figure 9C ). The most significant toxic effect of DOX is its effect on the bone marrow, as it decreases the percentage of lymphocytes (%LYM, LYM, and white blood cells - WBC) over time and increases the percentage of monocytes and granulocytes (%MON and %GRA). This indicates a toxic effect on the bone marrow responsible for producing these cells ( Figure 9D)。Myelosuppression and lymphocytopenia are common side effects of DOX in cancer patients. In contrast, CON 1 had no effect on the percentages of lymphocytes, monocytes, and granulocytes. The increase in the percentage of eosinophils (%EOS) caused by CON 1 was not significantly correlated ( Figure 9D ). Neither the test compound DOX nor CON 1 had any effect on other blood parameters, such as red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), or mean platelet volume (MPV). This data is not shown.

[0145] Table 1. Anticancer effects of DOX, SC, CON 1, CON 2, LIG1, and LIG2 expressed as IC50* values

[0146]

[0147] *The IC50 value is the concentration that causes 50% cell growth inhibition.

[0148] 1 The IC50 value of DOX is in nanomoles, and those of the other compounds are in micromoles.

[0149] 2 Multidrug-resistant (MDR) cancer cell lines obtained by continuous exposure to antitumor drugs (paclitaxel in the case of U87-TxR and doxorubicin in the case of NCI-H460 / R).

[0150] 3 Relative resistance, which is obtained as the ratio between the IC50 values of the MDR cancer cell line and the corresponding sensitive cell line.

[0151] 4 In experiments with glioblastoma cells, MRC-5 cells were initially seeded at a density of 4,000 cells per well, while in experiments with non-small cell cancer cells, they were seeded at a density of 2,000 cells per well so that their density was the same as that of the cancer cells they were being compared to.

[0152] 5 Selectivity index, which is obtained as the ratio between the IC50 values of MRC-5 and the corresponding sensitive cell line.

[0153] Table 2. Nanoparticle properties of CON 1 and CON 2 studied by Zetasizer

[0154]

[0155] The results showed that the SC and DOX hybrids, CON 1 and CON 2 ( Figure 10 ) were more effective than DOX in their anticancer properties.

[0156] The results showed that the SC and DOX hybrids, CON 1 and CON 2, were more effective than DOX in their anticancer properties. They were taken up by cancer cells, had a lower level of resistance, and a higher selectivity for cancer cells. They had nanoparticle properties and different intracellular distributions, as well as better toxicological characteristics than DOX. The study showed that by conjugating SC and DOX, the anticancer properties of DOX were improved, the resistance was reduced, and the selectivity for cancer cells was increased.

[0157] The present invention relates to a new pair of hybrids formed by the hybridization of SC and DOX, named CON 1 and CON 2. These compounds showed excellent anticancer properties against various cell lines, including glioblastoma, non-small cell lung cancer, and colorectal cancer. The hybrids had a higher selectivity level for cancer cells than DOX, and they also showed a lower level of resistance in resistant cancer cells. In addition, the intracellular localization of CON 1 and CON 2 helped to prevent extensive double-stranded DNA damage caused by the binding of DOX to DNA strands. The unexpected nanoparticle properties of CON 1, CON 2, and CON 1 with a lower toxicity distribution compared to DOX in mice further enhanced their medical value.

[0158] The hybrids of SC and DOX can be used as drugs in pharmaceutical products and drug formulations for the treatment of various types of cancer. These compounds can be formulated into various dosage forms according to the specific needs of patients, such as injection solutions, liposomal formulations, and solid dosage forms. The compounds can also be used in combination with other chemotherapeutic drugs or radiotherapy to enhance their effectiveness in treating cancer.

[0159] The present invention confirmed an unexpected ability to inhibit cancer growth in various cell lines, including human glioblastoma, non-small cell lung cancer, and colorectal cancer. Therefore, it was found that it can be used as a drug in both pharmaceutical products and drug formulations.

[0160] The present invention focuses on the conjugate derivatives of sclareol and doxorubicin, their medicinal salts, synthesis, and application in cancer treatment. These compounds have been found to have antitumor and antiproliferative activities against cancer cells, especially human glioblastoma, non-small cell lung cancer, and colorectal cancer.

[0161] Formulation

[0162] The invention of the conjugate derivatives of sclareol and doxorubicin and their pharmaceutically acceptable salts has brought about a revolutionary change in the treatment of cancer. These compounds are synthesized to exhibit in vitro anti-cancer and anti-proliferative activities against cancer cells, especially human glioblastoma, non-small cell lung cancer, and colorectal cancer.

[0163] The main objective of this invention is to provide new compounds that can be used as active agents in cancer treatment. These compounds are designed for the treatment and / or prevention of proliferative and / or neoplastic diseases. They are also developed to inhibit the growth of cancer cells, especially in humans.

[0164] Drug compositions / formulations containing the compound conjugates of sclareol and doxorubicin, as well as pharmaceutically acceptable carriers and excipients, can now be used as drugs for the treatment of cancer, especially human glioblastoma, non-small cell lung cancer, and colorectal cancer. These conjugates offer new hope to cancer patients by providing effective treatment options.

[0165] Furthermore, the invention provides conjugates of sclareol and doxorubicin for use in the manufacture of a medicament for the treatment of cancer in mammals. These compounds are specifically designed to kill cancer cells and / or inhibit cancer cell replication in mammals, especially humans.

[0166] Industrial applicability

[0167] The invention describes a three-step method for preparing the conjugates by convergent synthesis. It has been found that the conjugates obtained by this method have favorable anti-cancer properties and exhibit higher selectivity for cancer cells compared to the starting compounds sclareol and doxorubicin. Additionally, they show a lower degree of resistance compared to doxorubicin. These compounds can be used to prepare pharmaceutical products, especially for the treatment of cancer, such as glioblastoma, non-small cell lung cancer, and colorectal cancer.

[0168] The invention provides pharmaceutical products containing sclareol derivatives and doxorubicin or its pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers. These pharmaceutical products are used in cancer treatment therapies.

[0169] The pharmaceutical formulations / compositions of the invention contain an effective dose of the conjugate of sclareol and doxorubicin in the form of a pharmaceutically acceptable salt, solvate, or hydrate, and at least one pharmaceutically acceptable excipient. The excipient is selected according to the desired mode of administration and drug form.

[0170] The said drug form can be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, topically, intratracheally, intranasally, transdermally, rectally, or intravitreally. The conjugate of the main active ingredients sclareol and doxorubicin can be administered as a single dosage form or as a mixture with at least one pharmaceutical excipient.

[0171] The pharmaceutical form for administration can be in the form of tablets, gel capsules, granules, powders, oral or injectable solutions or suspensions, transdermal patches, sublingual, buccal, intratracheal, intraocular, intranasal or by inhalation, topical, transdermal, subcutaneous, intramuscular, intravenous administration, rectal administration, or implants. For topical administration, creams, gels, ointments, lotions or eye drops can be considered.

[0172] These pharmaceutical forms are prepared using known conventional methods.

Claims

1. Hybrids CON1 and CON2 conjugates of sclareol and doxorubicin having the following formula and / or pharmaceutically acceptable salts or hydrates or solvates thereof: Wherein the ligands LIG1 and LIG2 are: The CON1 and CON2 compounds are:

2. The compound according to claim 1, wherein CON1 is N1-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-1-yl)oxy)tetrahydro-2H-pyran-4-yl)-N4-(3-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)propyl)succinamide; and CON2 is N1-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-1-yl)oxy)tetrahydro-2H-pyran-4-yl)-N4-(6-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)hexyl)succinamide, and pharmaceutically acceptable salts, hydrates and / or solvates thereof.

3. The hybrid CON1 and CON2 conjugates according to claims 1 to 2, wherein, Doxorubicin and sclareol are covalently linked in a 1:1 molar ratio through a linker.

4. A method for preparing hybrids (conjugates) CON1 and CON2 of sclareol and doxorubicin, CON1 and CON2 having the following formula The method comprises the following reaction steps: a) Reacting sclareol with 4-formylphenylboronic acid in the presence of a palladium-based catalyst to obtain the corresponding derivative 1, b) Reacting an alkylenediamine, 1,3-diaminopropane, 1,6-diaminohexane and succinic anhydride to synthesize compounds 2 and 3, c) Reacting the sclareol derivative 1 with compounds 2 and 3 by adding sodium borohydride in the presence of titanium(IV) isopropoxide to form the corresponding ligands LIG1 and LIG2, d) Forming an amide bond connection between doxorubicin and ligands LIG1 and LIG2, in particular by adding 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, to produce conjugates CON1 and CON2, 5. Ligand compounds LIG1 and LIG2 having the following formula Which are prepared by steps a)-c) of the method according to claim 4.

6. The compound according to claim 5, wherein LIG1 is 4-((3-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)propyl)amino)-4-oxobutanoic acid, and LIG2 is 4-((6-((4-((R,E)-3-hydroxy-5-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)-3-methylpent-1-en-1-yl)benzyl)amino)hexyl)amino)-4-oxobutanoic acid.

7. A method for preparing the compounds LIG1 and LIG2 according to claim 6, comprising steps a)-c) according to claim 4.

8. A composition comprising a hybrid according to claims 1 to 3 or a pharmaceutically acceptable salt, hydrate or solvate thereof, optionally in combination with at least one pharmaceutically acceptable excipient.

9. Use of a hybrid according to claims 1 to 3 or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a composition according to claim 8, as a medicament.

10. Use of a hybrid according to claims 1 to 3 or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a composition according to claim 8, for the treatment of cancer in humans, including but not limited to non-small cell lung cancer, glioblastoma, and colorectal cancer.

11. A method for preparing sclareol derivative 1, comprising step a) according to claim 4.

12. The sclareol derivative 1 prepared according to claim 11, characterized in that, It is 4-{(1E,3R)-3-hydroxy-5-[(2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl]-3-methylpent-1-en-1-yl}benzaldehyde.

Images