Tricin derivatives and compositions thereof for cancer treatment

Abstract

Provided are compositions and methods for treatment of cancer and for adoptive T cell therapy (ACT). The compositions comprise one or more derivatives of tricin 7-O-β-D-glucopyranoside (ANTARTINA®). The compositions comprise one or more pharmaceutically acceptable excipients convenient for delivery of the tricin 7-O-β-D-glucopyranoside derivatives. Tricin 7-O-β-D-glucopyranoside derivatives described herein include acetylated tricin 7-O-β-D-glucopyranoside, as well as glucose, maltose, cellobiose, lactose, xylose, and galactose derivatives. The methods include treatment, or reduction in the risk of suffering from, colorectal cancer and hepatocellular cancer.

Claims

1. A method of treating colorectal cancer or hepatocellular carcinoma in a subject in need thereof, which comprises: administering a pharmaceutically effective composition comprising one or more derivatives of tricin 7-O-β-D-glucopyranoside, wherein the one or more derivatives of tricin 7-O-β-D-glucopyranosidc is one or more of: hexa-acylated tricin 7-O-β-D-glucopyranoside (6a), [3,4,5-triacetoxy-6-[5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-4-oxo-chromen-7-yl]oxy-tetrahydropyran-2-yl]methyl acetate (5b), xylose tricin 7-O-β-D-glucopyranoside (1c), 7-[(3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxyrnethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]oxy-5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)chromen-4-one (1d), 7-[(3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]oxy-5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)chromen-4-one (1e), and 7-[(2S,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]oxy-5hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)chromen-4-one.

2. The method of claim 1, wherein the subject is human.

3. The method of claim 1, wherein the colorectal cancer is an adenocarcinoma, a gastrointestinal carcinoid tumor, a colorectal lymphoma, or a gastrointestinal stromal tumor.

4. The method of claim 1, wherein one of the one or more derivatives of tricin 7-O-β-D-glucopyranoside is hexa-acylated tricin 7-O-β-D-glucopyranoside.

5. The method of claim 1, wherein the cancer is a hepatocellular carcinoma.

6. The method of claim 5, wherein one of the one or more derivatives of tricin 7-O-β-D-glucopyranosidc is hexa-acylated tricin 7-O-β-D-glucopyranoside (6a).

7. The method of claim 1, wherein the method of treating is capable of reducing proliferation of colon cancer cells or hepatocellular carcinoma cells, and/or reducing size of a tumor.

8. The method of claim 1, further comprising administering to the subject one or more additional antitumor agents in a combination anticancer therapy.

9. The method of claim 8, wherein the one or more additional antitumor agents is one or more checkpoint inhibitors.

10. A method of reducing proliferation of colorectal cancer cells or hepatocellular carcinoma cells, and/or reducing size of a tumor, wherein the method comprises administering to the subject in need thereof a pharmaceutically effective amount of hexa-acylated tricin 7-O-β-D-glucopyranoside (6a).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 depicts the chemical structure of tricin and ANTARTINA® and related derivatives and analogues synthesized and tested as described herein (“Antartina 1a” corresponds to ANTARTINA®).

(2) FIGS. 2A, 2B, and 2C depict MTT assay absorbance data obtained from incubation of ANTARTINA® and related derivatives with HEPA129 cells at various time points, including: 24 hrs (FIG. 2A), 48 hrs (FIG. 2B), and 72 hrs (FIG. 2C).

(3) FIGS. 3A, 3B, and 3C depict MTT assay absorbance data obtained from incubation of ANTARTINA® and related derivatives with CT26 cells at various time points, including: 24 hrs (FIG. 3A), 48 hrs (FIG. 3B), and 72 hrs (FIG. 3C).

(4) FIG. 4 depicts cell size (mm.sup.3) in mice of CT26 tumors after CT26 cell line injection into mice. From left to right, first panel are the results of a control containing only carrier, second panel is treatment of the mouse with tricin, third panel shows the results of treatment of the mouse with ANTARTINA®, and the last panel on the right shows treatment of the mouse with acetylated ANTARTINA®. The bottom panel provides a summary of all results in one graph.

(5) FIG. 5A shows tumor volume vs. treatment for C3H/He mice that were injected with 200 mg/kg of thioacetamide. On day 28, mice were anesthetized and orthotopic tumors were established by sub-capsular inoculation of 1.25×10.sup.5 Hepa129 cells into the left liver lobe by laparotomy (day 0). Five days after tumor implantation, mice were distributed in groups (n=6-8/group) and received: i) saline (control, FIG. 5A, left panel); ii) acetylated ANTARTINA® (50 mg/kg, 3 times per week, FIG. 5A, right panel). Fifteen days after tumor implantation, mice were sacrificed and tumor volume was quantified, as depicted in FIG. 5B (control, left panel; acetylated ANTARTINA®, right panel).

(6) FIG. 6 shows effect of ANTARTINA® and IL-12 gene therapy in combination on a murine model of CRC (CT26 cells) established in BALB/c mice. Mouse CT26 cells (5×10.sup.5) were subcutaneously inoculated on day 0. On day 7, when tumors reached a diameter of 5-7 mm, animals were divided into 4 groups: saline, ANTARTINA®, AdIL12, and the combination of ANTARTINA® and AdIL-12. Tumor volume is shown in the left panel vs. days after CT26 inoculation. Excised tumors are depicted in the right panel.

DETAILED DESCRIPTION

(7) In colorectal carcinoma (CRC) patients, distant metastatic disease is present at initial diagnosis in nearly 25% of patients. The majority of patients with metastatic CRC have an incurable disease; therefore, new therapies are needed to address this cancer. Agents derived from medicinal plants have already demonstrated therapeutic activities in human cancer cells. ANTARTINA® is an antitumor agent isolated from Deschampsia antarctica Desv. In that study, the aim was to evaluate the antitumor properties of ANTARTINA® in CRC models. Human and murine CRC cell lines were employed to investigate proliferation, apoptosis, and cell cycle effects of ANTARTINA® therapy in vitro. Immune response of ANTARTINA® against cancer cells in the CRC model was also investigated using a cytotoxic T lymphocyte (CTL) assay, analyzing dendritic cell activation and intratumor T cell sub-population, and by tumor re-challenge experiments.

(8) ANTARTINA® inhibits in vitro human CRC cell proliferation. In an immunocompetent CRC mouse model, ANTARTINA® potently inhibited tumor growth and liver metastases, leading to complete tumor regressions in >30% of mice, along with an increased animal survival rate. In addition, ANTARTINA® induced a potent specific cytotoxic T cell response against CRC and a long lasting antitumor immunity. Interestingly. ANTARTINA® increased tumor immunogenicity and stimulated dendritic cell activation. No toxic effects were observed at the doses employed. These findings show that ANTARTINA® has the ability to induce antitumor immunity against CRC and is useful for the treatment of CRC.

(9) While various aspects and embodiments have been illustrated and described in detail, above, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications can be made by those of ordinary skill within the scope and spirit of the following claims. In particular, this description is intended to encompass further embodiments with any combination of features from different embodiments described above and below.

(10) The compositions and methods described herein are additionally described by way of the following illustrative non-limiting examples that provide a better understanding of these embodiments and their many advantages. The following examples are included to demonstrate certain useful embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent compositions and methods that function well, and thus can be considered to constitute preferred embodiments. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of this disclosure.

EXAMPLES

(11) Melting points were determined in open capillary tubes with a Stuart Scientific SMP3 melting point apparatus. Infrared (IR) spectra were obtained with Fourier-transform infrared (FTIR) Cary 630 (Agilent Technologies, Santa Clara, Calif., US) with diamond attenuated total reflectance (ATR) spectrophotometer. Nuclear magnetic resonance (NMR).sup.1H and .sup.13C spectra were recorded using Varian Gemini 200, Varian Unity 300/500 MHz, or Varian Mercury 300/400 systems at room temperature. (Varian Medical Systems, Palo Alto, Calif., US).

(12) Chemical shifts are given in ppm (δ) downfield from tetramethylsilane (TMS). Coupling constants (J) are in Hertz [Hz] and signals are described as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; br, broad; app, apparent. Analysis of the NMR free induction decays (FIDs) were performed using Mestrenova 6.0.2 software (Mestrelab Research, Santiago de Compostela, Spain). Low resolution mass spectra were obtained on Agilent Technologies 6120 Quadrupole Liquid Chromatography-Mass Spectrometry (LC-MS) in electrospray ionization (ESI) with an Agilent Technologies 1260 Infinity LC system using SeQuant Zic-Hilic (Merck-Millipore) 150 mm×4.6 mm, 5 am column (Agilent Technologies, Santa Clara, Calif., US). Agilent formula predictor software was used to process the data. MS was reported in units of mass to charge ratio (m/z). The analysis was performed with a flow rate of 1 mL/min maintaining the column temperature at 25° C. The detector was set at 214-254 nm±16 nm. Mass Detection: Scan 50-1000 m/z. The mobile phase consisted of phase A (200 mM ammonium acetate, pH 5.2, without ethylenediaminetetraacetic acid (EDTA)) and phase B (acetonitrile 80% and solution of ammonium acetate 20%), isocratic method. High-resolution mass spectrometry analysis (time-of-flight, TOF) was performed using an Agilent 6210 time-of-flight LC/MS. (Agilent Technologies, Santa Clara, Calif., US).

(13) All chemicals were obtained from commercial sources, and used as received without extra purification. Palladium on carbon 10 wt % loading matrix activated carbon support (Pd (C), 205699), ammonia solution 32% (105426) were purchased from Sigma-Aldrich, St. Louis, Mo., US. Thin layer chromatography (TLC) analyses were performed using silica gel (Kieselgel 60 7254, Macherey-Nagel, Duren, Germany) and the spots were visualized under ultraviolet (UV) light. The column chromatography was performed with silica gel 60 (40-63 μm, Merck & Co., Kenilworth, N.J., US) columns, using the eluent reported in each case.

(14) Synthesis of ANTARTINA® was previously described in U.S. Pat. No. 10,058,582 (the entire contents of which are incorporated herein by reference, especially those sections outlining the synthesis of ANTARTINA®).

(15) Adenovirus IL-12 constructs are as previously reported. (Mazzolini et al., Cancer Gene Therapy, 6(6):514-522, 1999).

(16) ANTARTINA® was detected and obtained by isolation using semi-preparative high performance liquid chromatography (HPLC) from aqueous extracts of Deschampsia antarctica Desv. (Poaceae). The purity and structural identification of the isolated peak 10 was performed by Nuclear Magnetic Resonance (NMR) and analyzed by mass spectrometry (MS). Peak 10 isolated from Deschampia antartica extract, is tricin 7-O-P-D-glucopyranoside, also called 5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-7-[(2S, 3S, 4R, 5S, 6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydropyran-2-yl] oxychromen-4-one.

(17) Mouse CT26 tumor cell line, an undifferentiated murine colorectal carcinoma (CRC) cell line established from N-nitroso-N-methylurethan-induced transplantable tumor in BALB/c (H-2d) mice, BNL cells (an hepatoma cell line) were used in the studies below and can be obtained from the University of Navarra, Spain. Human cell lines were from American Type Culture Collection (ATCC, Manassas, Va., US), including LoVo (ATCC-CCL229), ASG (ATCC-CCRL-79) and lung fibroblast Wi38 (ATCC-CCL75). All lines were tested for mycoplasma and cultured in complete DMEM (GIBCO, Themo Fisher Scientific, mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% heat inactivated fetal bovine serum (FBS, GIBCO, Thermo Fisher Scientific) and incubated 37° C. in 5% CO.sub.2.

(18) All animals were from Envigo (Barcelona, Spain) and were maintained at the Spanish National Cancer Research Center (CNIO) Animal Facility (AAALAC accredited) in accordance with guidelines stated in the International Guiding Principles for Biomedical Research Involving Animals (CIOMS). All animal experiments were approved by the Competent Authority of Communidad de Madrid (project PROEX 104/16).

Example 1: Synthesis of ANTARTINA® Derivative Tricin 7-O-β-D-glucopyranoside, Per Acetylated (AST-1603-59S2, 6a, FIG. 1)

(19) ##STR00001##

(20) A mixture of Tricin 7-O-β-D-glucopyranoside 1a (0.5 g, 1.02 mmol, 1.0 eq.) and pyridine (14 mL, 173.4 mmol) in a water: ice bath (4° C.) was treated with Ac.sub.2O (14 mL) dropwise. The reaction mixture is warmed up at room temperature and stirred overnight. After that, the solvents were removed under reduced pressure and the residue was treated with methyl-t-butyl ether (MTBE) (50 mL) to obtain 0.5 g (56%) of 6a as a white solid. M. p.: 239.1-241.8° C. 1H-NMR (CDCl3, 500 MHz) δ (ppm): 2.05 (m, 6H), 2.06 (s, 3H), 2.08 (s, 3H), 2.37 (s, 3H), 2.44 (s, 3H), 3.91 (s, 6H), 3.99 (br s, 1H), 4.20 (d, J=11.8 Hz, 1H), 4.28 (dd, J=12.6 Hz, J=5.6 Hz, 1H), 5.17 (t, J=9.0 Hz, 1H), 5.36-5.23 (m, 3H), 6.56 (s, 1H), 6.70 (s, 1H), 7.02 (s, 1H), 7.05 (s, 2H). 13C-RMN (CDCl3, 125 MHz) δ (ppm): 178.8, 173.9, 172.7, 172.3, 172.0, 171.9, 170.9, 164.4, 162.5, 160.8, 155.3, 153.3, 134.2, 131.9, 115.4, 111.6, 111.4, 105.7, 105.6, 100.5, 75.1, 74.8, 73.5, 70.7, 64.6, 59.0, 23.7, 23.2, 23.0. HPLC-UV-MS: tR=15.96 min, Purity (at 254 nm): 98%; MS (ESI, positive mode): m/z=745.2, [M+H]+; MS (ESI, negative mode): m/z=743.1 (M−H)−. HRMS calculated for C.sub.35H.sub.36O.sub.18, 744.1902 [M+H]+, found, 744.1902.

Example 2: Synthesis of ANTARTINA® Derivative [3,4,5-triacetoxy-6-[5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-4-oxo-chromen-7-yl]oxy-tetrahydropyran-2-yl]methyl Acetate (AST-1603-097, 5b, FIG. 1)

(21) ##STR00002##

(22) A solution of 4b (6.0 g; 7.14 mmol, 1.0 eq.) in AcOEt:EtOH:THF (1:1:1, 250 mL, 1 g/42 mL) was treated with Pd (C) (10%) (10% w/w, 0.6 g). The reaction mixture was purged with N.sub.2 (3×2 bars) and H.sub.2 (3×2 bars). Finally, the reaction was treated with H.sub.2 (2.3 bars) at room temperature for 48 hours. After that, the reaction mixture was filtered through celite and the solvents were removed under reduced pressure. The residue was treated with Et.sub.2O (50 mL) to obtain 3.50 g (75%) of 3 as a brown-red solid. M. p.: 117.2-121.8° C. 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 1.95 (s, 3H), 2.01 (s, 3H), 2.05 (s, 3H), 2.15 (s, 3H), 3.88 (s, 6H), 4.12 (br s, 2H), 4.54 (br s, 1H), 5.24 (m, 2H), 5.35 (br s, 1H), 5.68 (br s, 1H), 6.47 (s, 1H), 6.85 (s, 1H), 7.39 (s, 1H), 7.36 (s, 2H), 9.39 (br s, 1H), 13.04 (br s, 1H); 13C-RMN (DMSO-d6, 125 MHz) δ (ppm): 185.2, 173.0, 172.9, 172.6, 172.3, 167.5, 164.7, 164.5, 159.9, 154.9, 151.3 (2C), 143.3, 142.2, 131.0, 127.9, 123.2, 108.9, 107.7 (2C), 106.9, 101.9, 99.9, 98.6, 73.8, 73.3, 71.1, 70.3, 64.6, 59.6, 33.5. HPLC-UV-MS: tR=15.67 min, Purity (at 254 nm): 98%; MS (ESI, positive mode): m/z=661, [M+H]+; MS (ESI, negative mode): m/z=659.0 (M−H)−. HRMS calculated for C.sub.31H.sub.33O.sub.16, 661.1763 [M+H]+, found, 661.1731.

Example 3: Synthesis of ANTARTINA® Derivative 5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-7-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-chromen-4-one (AST-1603-102 SP, 1b, FIG. 1)

(23) ##STR00003##

(24) [3,4,5-triacetoxy-6-[5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-4-oxo-chromen-7-yl]oxy-tetrahydropyran-2-yl]methyl acetate (AST-1603-097, 5b, 2.0 g, 3.03 mmol, 1.0 eq.) was dissolved in MeOH (140 mL; 1 g/70 mL) and finally treated with 32% aqueous NH.sub.3 solution (62 mL). The reaction mixture was stirred at room temperature overnight. After that, the solvents were removed under reduced pressure. The residue was treated with DMSO (1 mL) and distilled H.sub.2O (10 mL). The solid was removed after filtration isolating 1.0 g (67%) of 1b as a brown solid. M. p.: 260.2-263.3° C. 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.50-3.62 (m, 4H), 3.69 (dt, J=14.0 Hz, J=7.9 Hz, 2H), 3.87 (s, 6H), 4.54 (d, J=4.6 Hz, 1H), 4.71 (t, J=5.5 Hz, 1H), 4.89 (d, J=5.8 Hz, 1H), 4.99 (d, J=7.7 Hz, 1H), 5.22 (d, J=5.2 Hz, 1H), 6.45 (d, J=2.2 Hz, 1H), 6.92 (d, J=2.2 Hz, 1H), 7.06 (s, 1H), 7.35 (s, 2H), 9.36 (br s, 1H), 12.9 (br s, 1H). 13C-RMN (DMSO-d6, 125 MHz) δ (ppm): 185.3, 167.3, 166.3, 163.9, 159.9, 151.3 (2C), 142.9, 123.2, 108.5, 107.4 (2C), 106.9, 104.1, 102.6, 98.1, 78.9, 76.3, 73.2, 71.3, 63.6, 59.5 (2C). HPLC-UV-MS: tR=10.09 min; Purity (at 254 nm): 98.5%; MS (ESI, positive mode): m/z=493, [M+H]+; MS (ESI, negative mode): m/z=491 (M−H)−. HRMS calculated for C.sub.23H.sub.25O.sub.12, 493.1340 [M+H]+.

Example 4: Synthesis of ANTARTINA® Xylose Derivative 1c (AST-1608-28, 1c, FIG. 1)

(25) ##STR00004##

(26) The xylose derivative precursor, (AST1608-13S3) 5c, (2.9 g, 4.9 mmol, 1.0 eq.) was dissolved with MeOH (200 mL, 1 g/70 mL) and treated with 32% aqueous NH.sub.3 solution (100 mL). The reaction mixture was stirred at room temperature overnight. After that, the solvents were removed under reduced pressure. The residue was treated with DMSO (2 mL) and distilled H.sub.2O (40 mL). After precipitation, the solid was collected after filtration yielding 1.5 g (66%) of 1c as a pale yellow-brown solid. M. p.: 245.3-249.8° C. 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.19-3.46 (m, 5H), 3.76 (d, J=5.8 Hz, 1H), 3.88 (s, 6H), 5.08 (m, 2H), 5.42 (br s, 1H), 6.42 (d, J=1.6 Hz, 1H), 6.88 (d, J=1.6 Hz, 1H), 7.05 (s, 1H), 7.36 (s, 2H), 9.26 (br s, 2H), 12.90 (br s, 1H). 13C-RMN (DMSO-d6, 125 MHz) δ (ppm): 185.2, 167.3, 165.7, 164.2, 160.0, 151.2 (2C), 143.2, 123.2, 108.5, 107.7 (2C), 106.8, 103.4, 102.5, 98.1, 79.3, 75.9, 72.3, 68.9, 59.5 (2C). HPLC-UV-MS: tR=11.9 min; Purity (at 254 nm): 98.1%; MS (ESI, positive mode): m/z=463.0 [M+H]+, 485 [M+Na]+; MS (ESI, negative mode): m/z=461.0 (M−H)−. HRMS calculated for C.sub.22H.sub.23O.sub.11, 463.1234 [M+H]+, found, 463.1208.

Example 5: Synthesis of ANTARTINA® Derivative 7-[(3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]oxy-5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)chromen-4-one (AST-1608-77s, 1d, FIG. 1)

(27) ##STR00005##

(28) [(2R,3S,4R,5R)-5-acetonyl-4-acetoxy-6-[5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-4-oxo-chromen-7-yl]oxy-3-[(3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxy methyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]methyl acetate (AST-1608-69) 5d (4.0 g, 4.22 mmol, 1.0 eq.) was dissolved with MeOH (280 mL, 1 g/70 mL) and finally treated with 32% aqueous NH.sub.3 solution (104 mL). The reaction mixture was stirred overnight at room temperature. After that, the solvents were removed under reduced pressure. The residue was treated with DMSO (2 mL) and distilled H.sub.2O (20 mL). The solid was removed after filtration isolating 1.6 g (58%) of 1d as a pale yellow-brown solid. M. p.: 275.4-279.2° C. 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.38 (dd, J=15.7 Hz, J=6.4 Hz, 2H), 3.44-3.57 (m, 5H), 3.63 (m, 4H), 3.80 (dd, J=10.3 Hz, J=5.2 Hz, 1H), 3.88 (s, 6H), 4.23 (d, J=7.2 Hz, 1H), 4.52 (d, J=4.6 Hz, 1H) 4.67 (dd, J=12.6 Hz, J=5.7 Hz, 2H), 4.78 (d, J=5.1 Hz, 1H), 4.83 (d, J=1.3 Hz, 1H), 5.08 (d, J=4.5 Hz, 1H), 5.15 (d, J=7.8 Hz, 1H), 5.55 (d, J=5.3 Hz, 1H), 6.47 (d, J=2.0 Hz, 1H), 6.93 (d, J=2.0 Hz, 1H), 7.07 (s, 1H), 7.35 (s, 1H), 9.36 (br s, 2H), 12.9 (br s, 1H). 13C-RMN (DMSO-d6, 125 MHz) δ (ppm): 185.0, 167.2, 165.9, 164.1, 159.9, 151.3 (2C), 143.1, 123.2, 108.5, 107.6 (2C), 106.7, 102.5, 98.3, 83.1, 78.7, 78.3, 77.8, 76.4, 75.9, 73.7, 71.3, 63.5, 63.0, 59.6 (2C). HPLC-UV-MS: tR=10.06 min; Purity (at 254 nm): 95.4%; MS (ESI, positive mode): m/z=655 [M+H]+; MS (ESI, negative mode): m/z=653.3 (M−H)−. HRMS calculated for C.sub.29H.sub.35O.sub.17, 665.1868 [M+H]+, found, 655.1835.

Example 6: Synthesis of ANTARTINA® Derivative 7-[(3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]oxy-5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)chromen-4-one (AST-1608-21, 1e, FIG. 1)

(29) ##STR00006##

(30) [(2R,3S,4R,5R)-5-acetonyl-4-acetoxy-6-[5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-4-oxo-chromen-7-yl]oxy-3-[(3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxy methyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]methyl acetate (AST-1608-16) 5e (3.89 g, 4.1 mmol, 1.0 eq.) was dissolved with MeOH (280 mL, 1 g/72 mL) and finally treated with 32% aqueous NH.sub.3 solution (104 mL). The reaction mixture was stirred at room temperature overnight. After that, the solvents were removed under reduced pressure. The residue was treated with DMSO (3 mL) and distilled H.sub.2O (30 mL). The solid was collected after filtration, isolating 1.7 g (64%) of 1e as a pale yellow-brown solid. M. p.: 243.2-246.5° C. 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 2.96-3.10 (m, 2H), 3.19 (m, 3H), 3.40 (dd, J=19.9 Hz, J=11.9 Hz, 2H), 3.47 (dt, J=12.0 Hz, J=9.5 Hz, 1H), 3.64 (m, 2H), 3.67-3.74 (m, 2H), 3.79 (dd, J=10.2 Hz, J=4.7 Hz, 1H), 3.87 (s, 6H), 4.28 (d, J=7.9 Hz, 1H), 4.60 (t, J=5.2 Hz, 1H), 4.69 (t, J=5.6 Hz, 1H), 4.82 (d, J=1.9 Hz, 1H), 5.06-4.94 (m, 2H), 5.14 (d, J=7.8 Hz, 1H), 5.22 (d, J=4.7 Hz, 1H), 5.54 (d, J=5.1 Hz, 1H), 6.46 (d, J=2.1 Hz, 1H), 6.92 (d, J=2.1 Hz, 1H), 6.46 (d, J=2.1 Hz, 1H), 6.92 (d, J=2.1 Hz, 1H), 7.06 (s, 1H), 7.35 (s, 1H), 9.33 (br s, 2H), 12.9 (br s, 1H). 13C-RMN (DMSO-d6, 125 MHz) δ (ppm): 185.1, 167.2, 165.9, 164.2, 159.9, 151.3 (2C), 143.1, 123.0, 108.5, 107.6 (2C), 106.9, 106.2, 102.7, 102.5, 98.3, 82.9, 79.8, 79.5, 78.4, 77.9, 76.4, 75.9, 73.1, 64.2, 63.0, 59.4 (2C). HPLC-UV-MS: tR=10.20 min; Purity (at 254 nm): 100%; MS (ESI, positive mode): m/z=655 [M+H]+, 677 [M+Na]+; MS (ESI, negative mode): m/z=653.3 (M−H)−. HRMS calculate d for C.sub.29H.sub.35O.sub.17, 665.1868 [M+H]+, found, 665.1833.

Example 7: Synthesis of ANTARTINA® Derivative 7-[(2S,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]oxy-5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)chromen-4-one (AST-1608-73s, 1f, FIG. 1)

(31) ##STR00007##

(32) [(2R,3S,4R,5R,6S)-5-acetonyl-4-acetoxy-6-[5-hydroxy-2-(4-hydroxy-3,5-dimethoxy-phenyl)-4-oxo-chromen-7-yl]oxy-3-[(3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-2-yl]methyl acetate (AST1608-67) 5f (2.89 g, 2.95 mmol, 1.0 eq.) was dissolved with MeOH (202 mL, 1 g/70 mL) and finally treated with 32% aqueous NH.sub.3 solution (75 mL). The reaction mixture was stirred at room temperature overnight. After that, the solvents were removed under reduced pressure. The residue was treated with DMSO (2 mL) and distilled H.sub.2O (20 mL). The solid was collected after filtration yielding 1.6 g (58%) of 1f as a pale yellow-brown solid. M. p.: 114.7-194.4° C. 1H-NMR (DMSO-d6, 500 MHz) δ (ppm): 3.06 (t, J=8.1 Hz, 2H), 3.20-3.52 (m, 5H), 3.54-3.65 (m, 5H), 3.74 (d, J=10.9 Hz, 1H), 3.86 (s, 6H), 4.63 (br s, 3H), 4.91 (br s, 2H), 5.05 (d, J=3.6 Hz, 1H), 5.11 (d, J=7.7 Hz, 1H), 5.55 (br s, 4H), 6.44 (d, J=1.9 Hz, 1H), 6.90 (d, J=1.9 Hz, 1H), 7.03 (s, 1H), 7.31 (s, 2H). 13C-RMN (DMSO-d6, 125 MHz) δ (ppm): 185.1, 167.2, 166.0, 164.2, 159.9, 151.3 (2C), 143.3, 123.2, 108.5, 107.6 (2C), 106.8, 103.8, 102.8, 102.6, 98.3, 82.0, 79.2, 78.6, 76.6, 76.4, 75.8, 75.5, 72.9, 63.9, 63.3, 59.4 (2C). HPLC-UV-MS: tR=10.09 min; Purity (at 254 nm): 96.1%; MS (ESI, positive mode): m/z=655 [M+H]+; MS (ESI, negative mode): m/z=653.3 (M−H)−. HRMS calculated for C.sub.29H.sub.35O.sub.17, 665.1868 [M+H]+, found, 655.1836.

Example 8: Cell Viability Assays in HEPA129 and CT26 Cells

(33) After synthesis of ANTARTINA® and derivatives, the molecules were tested for cytotoxicity in various cancer cell lines. For this, a cell viability test was performed. Briefly, gastric cancer cells, AGS, colon cancer, Lovo (human CRC cell line), and as a control the Wi38 lung fibroblast line were employed. The cells were exposed to decreasing concentrations of molecules 1 and 3 from 1000 μM for 72 hrs. The negative control was just dimethyl sulfoxide (DMSO), the solvent in which the molecules to be tested were dissolved, with no active agent. Afterwards the cells were washed and the MTS (tetrazolium dye) colorimetric compound was added for the MTT assay. It was determined that molecules 1 and 3 were able to inhibit approximately in 50% the cell viability of the AGS and Lovo cancer lines at a concentration of 1000 and 500 μM, as shown in FIG. 2.

(34) Hepa129 (FIGS. 2A, 2B, and 2C) and CT26 (FIGS. 3A, 3B, and 3C) cells were plated onto 96-well plates at a density of 5×10.sup.3 cells/well and cultured with Dulbecco's Modified Eagle Medium (DMEM), 0.1% DMSO, or ANTARTINA® analogues at different doses (0.1, 1.0, 10 μg/ml). Twenty four hours (FIGS. 2A and 3A), 48 hr (FIGS. 2B and 3B) and 72 hrs (FIGS. 2C and 3C) after treatment, viability was determined by the MTT assay (Promega Corp., Madison, Wis., US). The plates were incubated for 4 h and the absorbance of each well was read at 490 nm. All assays were performed in quadruplicate, and each assay was repeated at least twice.

Example 9: Acetylated ANTARTINA® Exerts a Potent Antitumoral Effect in Mice with CRC that is Superior to Native ANTARTINA®

(35) Subcutaneous CRC model: Six-to-eight-week-old male BALB/c mice were used. CT26 cells were injected at a dose of 5×10.sup.5 cells subcutaneously (s.c.) into the right flank of BALB/c mice (day 0). Tumors were allowed to reach .sup.˜90 mm.sup.3 in size before treatment. Animals were distributed in different groups and then treated with 0.1% DMSO in sterile water i.p. (control group, n=5), ANTARTINA® (50 mg/kg i.p., day 8, n=5), tricin (50 mg/kg n=8), or acetylated ANTARTINA® (50 mg/kg i.p., day 8, n=5), 3 times per week over the course of 3 weeks. (See, FIG. 3, top graphs, left to right: control, ANTARTINA®, tricin, and acetylated ANTARTINA®). Tumor growth was assessed by caliper measurement. Tumor volume was calculated using the following formula: tumor volume (mm.sup.3)=length×(width) 2/2. FIG. 4 shows that acetylated ANTARTINA® clearly and markedly reduced tumor size at a higher rate than observed for any of the other tested agents.

Example 10: Antitumoral Effects of Acetylated ANTARTINA® in an Orthotopic Hepatocellular Carcinoma Model in Mice

(36) Experimental model of HCC associated with fibrosis: C3H/He mice were treated intraperitoneally (i.p) with 200 mg/kg of thioacetamide (TAA) (Sigma-Aldrich, St. Louis, USA) 3 times a week for 4 weeks. On day 28, mice were anesthetized and orthotopic tumors were established by sub-capsular inoculation of 1.25×10.sup.5 Hepa129 cells into the left liver lobe by laparotomy (day 0). Five days after tumor implantation, mice were distributed in groups (n=6-8/group) and received: i) saline (control); ii) acetylated ANTARTINA® (50 mg/kg, 3 times per week). (See, FIG. 5A). Fifteen days after tumor implantation, mice were sacrificed and tumor volume was quantified. (See, FIG. 5B). Acetylated ANTARTINA® markedly reduced the tumor volume.

Example 11: ANTARTINA® in Combination with Immuno-Gene Therapy (Adenovirus-IL12) Shows a Synergistic Antitumoral Effect

(37) In preclinical models of cancer, gene therapy with interleukin 12 (IL-12) has reached unprecedented levels of success when combined with immunotherapy approaches such as gene transfer of other cytokines and/or chemokines, costimulatory molecules, or adoptive cell therapy. (Melero et al., Trends Immunol., 22(3):113-115, 2001).

(38) This is a murine model of CRC (CT26 cells) established in BALB/c mice. Mouse CT26 cells (5×10.sup.5) were subcutaneously inoculated on day 0. On day 7, when tumors reached a diameter of 5-7 mm, animals were divided into 4 groups: saline, ANTARTINA® 50 mg/kg, 3 doses per week (systemically), AdIL12 (adenovirus encoding murine IL-12, dose: 10.sup.9 TCID50, intratumorally, one dose at day 15 after tumor inoculation), and the combination of ANTARTINA® and AdIL-12. Tumor volume was measured with a caliper 3 times per week. A synergistic antitumor effect was observed for AdIL-12+ANTARTINA®. Complete tumor regressions: saline 0/0, AdIL-12 1/5, Antartina 2/5, and combined therapy: 4/5. (FIG. 6).

(39) The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.