AU2011274194B2 - Phenyl nitrone compounds containing stilbene segment and use thereof - Google Patents

Abstract

Phenyl nitrone compounds containing stilbene segment and pharmaceutically acceptable salts thereof are provided. The above compounds can be used for treating cancer and also can be used as neuroprotective agent. Preparation method of the above compounds, pharmaceutical compositions comprising the above compounds and their use in the preparation of anti-tumor medicines are also provided.

Description

PHENYL NITRONE COMPOUNDS CONTAINING STIBENE SEGMENT AND USE THEREOF FIELP OF THE INVENTION 5 The present invention relates to phenyl nitrone compounds containing stilbene segment and their pharmaceutically acceptable salts, and such compounds may be used in the treatment of hyperproliferative diseases in mammalian cells, such as cancer, and may also be used as neuroprotective agents. The present invention also relates to a method of preparing the above compounds, pharmaceutical compositions comprising the above compounds and use of the 10 compounds in the preparation of antitumor drugs. BACKGROUND OF THE INVENTION Tumor treatment has been a worldwide problem. With the increasing knowledge of tumor cell signal transduction pathways, people have paid more and more attention to anticancer drug 15 design directed against tumor-specific molecular targets. In addition, the development of crystal diffraction technique, combinatorial chemistry, molecular modeling, high-throughput screening technology as well as computer chemistry lead to a rapid progress of targeted drugs, which offers a new strategy for the treatment of tumors. Structure and mechanism-based drug design has become a major way for the development of anticancer drugs. 20 Drug resistance in cancer is one of the most intractable problems in the treatment of cancer at present. To solve this problem, we must start from understanding the mechanism of cancer cell resistance so as to develop new selectively targeting drugs. Protein tyrosine kinases is one of the tumor targets which are studied deeply in recent years. Protein tyrosine kinases are involved in some key cell activities, such as cell proliferlation, apoptosis and aiding cancer cells against 25 chemotherapeutic drugs. Thus, the activity inhibition of protease may help to solve the problem of drug resistance in cancer treatments. Tyrosine proteasome inhibitor is currently one of the development directions of anti-cancer drugs. It has shown that many compounds such as styrene derivatives have tyrosine kinase inhibitory properties, for example, natural stilbene compounds resveratrol reported in the literature (Drug Resist Updat 2006, 9, 263-2 73). 30 Due to the factors such as abnormal vascular structure and fast growth of cancer cells in the cancer tissue, local hypoxia state may easily occur. Hypoxic cells are common in various cancer tissues, and their presence help cancer cells resist chemotherapy and radiotheraphy. Therefore, drug research targeted to hypoxic cancer cells becomes one of the research topics of the current anti-cancer drugs. Listed below are some compounds targeting hypoxic cancer cells currently 35 being developed in the world. -- -- -2- - OH 0 HN 0- 0 N OH 0 NN C( OH 0 HN N N NH 2 A Q4N +N-O quinoxaline 1,4-di-N-oxide Tirapazamine In which, AQ 4 N is currently under clinical research by the cooperation of Novacea Inc. and Kudos Pharmaceuticals Inc., and is suitable for various cancers. The report of quinoxaline 1,4-di-N-oxide may refer to Bioorg Med Chem, 2001, 9, 2395-2401. Tirapazamine is now under 5 clinical research by Sanofi-Aventis Inc., and is suitable for various cancers. Its mechanism of action may be expressed as follows: Firstly being activated by various reductases in vivo and generating free radicals in hypoxic cells, and then inducing single chain and double chains of DNA to burst and making base pairs damaged, and ultimately leading to cell death. Hypoxic cells are common in solid tumors. Tirapazamine is only activated by hypoxic cells to play its role, and 10 thus the drug has better targeting selectivity. The research of phenyl nitrone compounds may refer to an anti-radical drug NXY-059, which is newly developed by AstraZeneca Inc.. It has a strong ability to capture free radical. Once it is combined with a free radical to form a new stable compound, the free radical will lose its activity and cannot destroy tissue cells or participate in a series of biochemical changes. NaO 3 S + NaO 3 S Xr 0 15 NXY-059 In recent years, according to the analysis of bioactivity of phenyl nitrone compounds, such compounds are small molecule compounds with multiple pharmacological effects, which face many problems to be solved: how to make such agents only act on the intended target of the tumor cells, but not act on the same target of normal cells;how to achieve additive or synergistic 20 efficacy in combination therapy, and the like. All these have hindered the clinical application of targeted anticancer drugs. Therefore, the study has far-reaching implications on the mechanism of action and the application scope of targeted anticancer drugs. It has been pointed out that many other compounds, such as styrene derivatives, have tyrosine kinase inhibiting properties. Some Patent Publication EP556226A1, EP602851A1, 25 EP635507A1, EP635489AI and EP520722A1 disclose that certain quinazoline derivatives have anti-cancer properties caused by tyrosine kinase inhibiting properties. PCT application W092/20642 also discloses monocyclic or bicyclic aryl and heteroaryl compounds as tyrosine kinase inhibitor. 3 The above-mentioned anti-cancer compounds had made a great contribution to the field, based on which our study try to obtain better anti-cancer drugs with improved pharmaceutical benefits. The present invention relates to a series of phenyl nitrone derivatives containing stilbene fragment, which have new structural features, mainly displayed in their different bonding ways of 5 chemical composition fragments such as substituted stilbene fragment, substituted phenyl nitrone fragment and substituted aryl ring fragment from that of the compounds in above described patent documents. The present inventors have surprisingly found that the novel compounds obtained by these differences have prominent anti-tumor properties, and thus may be used as anticancer drugs to be developed. 10 SUMMARY OF THE INVENTION The present invention relates to phenyl nitrone compounds of the following formula (I) and pharmaceutically acceptable salts and prodrugs thereof. First, in one aspect of the present invention, compounds of the following formula (I) are 15 provided: AP\ b,, A'q (I) Wherein, p and q independently represent integers from 0 to 4 provided that p < 4 and q < 4; b represents a double bond which may be present or absent; when the double bond is present, the compounds of formula (I) may be in the E or Z configuration, and when the double bond is 20 absent, the stereocenters of the compound of formula (I) may have the R- or S- configuration; R and R' independently represent H, C 1

-C

2 0 linear or branched alkyl; C 2

-C

2 0 linear or branched alkenyl; -CO 2 Z', wherein Z' is H, sodium, potassium, or other pharmaceutically acceptable counter-ion such as calcium, magnesium, ammonium, tromethamine, tetramethylammonium, and the like; -CO 2 R.'; -NH 2 ; -NHR.'; -NR 2 .'; -OH; halo; substituted 25 C 1

-C

2 0 linear or branched alkyl or substituted C 2

-C

2 0 linear or branched alkenyl; -OR"', wherein R.' represents C 1

-C

20 linear or branched alkyl, linear or branched alkenyl, or aralkyl -(CH 2 )x-Ar, wherein X is an integer from 1 to 6; -CO 2 R"", wherein R"" represents H, optionally substituted

C

1

-C

20 alkyl, optionally substituted C 1

-C

2 0 linear or branched alkoxyl, optionally substituted

C

2

-C

2 0 alkenyl, or optionally substituted C 6 -Cio aryl or represents a cyclic moiety such as 30 morpholine, piperidine, piperazine and the like; A and A' represent mono- or multi-substituted groups, and independently represent H,

C

1

-C

2 0 acylamino, C 1

-C

2 0 acyloxyl, C 1

-C

2 0 alkanoyl, C 1

-C

2 0 alkoxycarbonyl, C 1

-C

2 0 alkoxyl, 4 6846813 1 (GHMatters) P92452.AU INNAM

C

1

-C

2 o alkylamino, C 1

-C

2 o alkylcarboxylamino, aroyl, aralkanoyl, carboxyl, cyano, halo, hydroxyl; or A and A' represent optionally substituted linear or branched C 1

-C

2 0 alkyl or C 2

-C

2 0 alkenyl, or may be joined to form a methylenedioxy or ethylenedioxy group. 5 In a further aspect of the present invention, the following preferred compounds of formula (I) are provided: Compounds of the following general formula (I): AP\ R' bA'q Rt Wherein, p and q independently represent integers from 0 to 4 provided that p < 4 and q < 4; 10 b represents a double bond which may be present or absent; when present, the compounds may be in the E or Z configuration, and when the double bond is absent, the resulting stereocenters may have the R- or S- configuration; R and R' independently represent H; -CO 2 Z', wherein Z' is H, sodium, potassium, or other pharmaceutically acceptable counter-ion such as calcium, magnesium, ammonium, tromethamine, 15 tetramethylammonium, and the like; -CO 2 R"", wherein R"" represents H, optionally substituted

C

1

-C

2 0 alkyl, optionally substituted C 1

-C

2 0 linear or branched alkoxyl, preferably optionally substituted C 1

-C

6 alkoxyl (such as methoxyl, ethoxyl or propoxyl), optionally substituted C 2

-C

20 alkenyl, or optionally substituted C 6

-C

1 o aryl or represents a cyclic moiety such as morpholine, piperidine, piperazine and the like; 20 A and A' represent mono- or multi-substituted groups, and may be H, C 1

-C

2 0 acylamino,

C

1

-C

2 0 acyloxyl, C 1

-C

2 0 alkanoyl, C 1

-C

2 0 alkoxycarbonyl, C 1

-C

2 0 alkoxyl, C 1

-C

2 0 alkylamino,

C

1

-C

2 0 alkylcarboxylamino, aroyl, aralkanoyl, carboxyl, cyano, halo, hydroxyl; or A and A' may also represent optionally substituted linear or branched C 1

-C

2 0 alkyl or C2-C20 alkenyl, or may be joined to form a methylenedioxy or ethylenedioxy group. 25 More preferred compounds: The representative compounds of the present invention include: [1] (E)-N-((E)-4-((E)-3, 5-dimethoxyl)-phenyl)-N-tert-butyl-3-nitrone (6); [2] (E)-N-((E)-4-((E)-3, 5-dibenzyloxyl)-phenyl)-N-tert-butyl-3-nitrone (7); [3] (E)-N-((E)-4-((E)-3-methoxyl-4-benzyloxy)-phenyl)-N-tert-butyl-3-nitrone (8); [4] (E)-N-((E)-4-((E)-3-methoxyl-4-benzoate)-phenyl)-N-tert-butyl-3-nitrone (9); 30 [5] (E)-N-((E)-4-((E)-3-methoxyl-4-acetate)-phenyl)-N-tert-butyl-3-nitrone (10); [6] (E)-N-((E)-4-((E)-3-methoxyl-4-hydroxyl)-phenyl)-N-tert-butyl-3-nitrone (11); [7] (E)-N-((E)-4-((E)-4-fluoro)-phenyl)-N-tert-butyl-3-nitrone (12); [8] (E)-N-((E)-4-((E)-4-benzyloxyl)-phenyl)-N-tert-butyl-3-nitrone (13); [9] (E)-N-((E)-4-((E)-4-hydroxyl)-phenyl)-N-tert-butyl-3-nitrone (14); 5 6846813 1 (GHMatters) P92452.AU INNAM [10] (E)-N-((E)-4-((E)-3-chloro-4-fluoro)-phenyl)-N-tert-butyl-3-nitrone (15); [11] (E)-N-((E)-4-((E)-3-cyano)-phenyl)-N-tert-butyl-3-nitrone (16); [12] (E)-N-((E)-4-((E)-3-fluoro)-phenyl)-N-tert-butyl-3-nitrone (17). Secondly, the present invention further provides a method of preparing the compounds of 5 formula (I). Hereinafter the representative compound 6 is used as an example to describe the preparation method of the compounds of the present invention. The synthetic route 1 according to the present invention is as follows: OMe COOCH 3

CH

2 OH

COOCH

3

COOCH

3 OHC P(OEt) 3 |OMe

LAIH

4 toluene NaOMe/Et 2 O / Et 2 O or THF rO or NaH/THF Br O MeO OMe MeO OMe 1 2 3 4 CHO N NHOH MnO 2 NHOH

CH

2 C1 2 CH 3 0H, N 2 MeO OMe 5 MeO OMe 6 10 According to the method of route 1, the following representative compounds are synthesized: J < 4+< +< N N N OMe OMe Me BnO OBn OBn 8O {O ON 010 6Ph 1 ---- -- - ---- - -6 OMe OH F OBn OH 11 12 13 14 -0 NN CI C N F 15 16 17 F Further, the present invention provides pharmaceutical compositions containing compounds of formula (I). Said compositions comprise a therapeutically effective amount of the compound 5 of formula (I) together with a physiologically acceptable carrier. Said therapeutically effective amount means that the amount of the compound of formula (I) contained in the pharmaceutical composition is sufficient to produce a clinically desirable therapeutic efficacy, for example, making the tumor of the subject decrease to a clinically acceptable extent. 10 The pharmaceutical compositions of the present invention may be administered by any route including intravenous, intradermal, intramuscular, subcutaneous, oral and the like. The pharmaceutical compositions may be formulated into a dosage for gastrointestinal tract administration such as tablets, capsule, pills and the like, and may also be formulated into a dosage for parenteral administration such as injection, external preparation and the like. 15 Further, the present invention also provides a method of treating tumors and related diseases. The said method comprises administrating a therapeutically effective amount of the compound of formula (I)to a subject suffering from cancer or related conditions.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter the examples are provided to further illustrate the present invention, but should not be construed as limiting the present invention in any way. General Methods: Melting points were measured on a RT-1 melting point apparatus (Tianjin 5 Analytical Instrument Factory) and the temperatures are corrected. The 'HNMR spectra were recorded on a Bruker AV400 (400 MHz) spectrometer and are reported as parts per million (ppm) downfield from TMS. The infrared spectra were recorded on a Nicolet Magna 550 FT-IR Fourier spectrophotometer. The mass spectra were recorded on HP1 100 Esquire2000 liquid chromatography/mass spectrometer. UV spectra were recorded on a Shimadzu UV2410 UV 10 spectrophotometer. TLC was done on silica gel GF 2 54 plates for high efficient thin-layer chromatography (Yantai Zhifu Silica Gel Development Co., Ltd., China). Optical rotation measurement was performed on WZZ- lS optical rotation meter (Shanghai Precision & Scientific Instrument Co., Ltd., China). 15 Example 1 Synthesis of Compounds of Formula (I) According to Routel 1.1 Synthesis of Compound_(2} Bromomethyl methyl benzoate 1 (5 g, 0.022 mol) and triethyl phosphite (5,4 g, 0.033 mol) were added to 50 ml of toluene, heated to 105*C and refluxed for 24 hours, and then cooled to room temperature. Toluene was evaporated under reduced pressure to give 2 as pale yellow liquid 20 (5.8 g, 93%). HNMR (400 MHz, CDCl 3 ) 8 1.11-1.15 (m, 6H), 3.03-3.05 (m, 2H), 3.88 (s, 3H), 4.07-4.10 (in, 4H), 7.17 (m, 2H), 7.85 (m, 2H); MS (EI) m/z 166 [M]. 1.2 Synthesis of Compound (3) Sodium methoxide (2.5 g, 0.046 mol) was dissolved in 50 ml of diethyl ether, and compound 2 (5.8 g, 0.035 mol) and 3, 5-dimethoxyl benzaldehyde (2.5 g, 0.015 mol) were 25 dissolved in 25 ml of diethyl ether and then slowly added dropwise to the sodium methoxide solution within about 0.5 hours. After the mixture was stirred for 20 min at room temperature and the reaction was completed as indicated by TLC analysis, the reaction solution was added with 70mL of CH 2 C1 2 and 100 mL of water and neutralized with dilute hydrochloric acid. The solution was seperated and the aqueous layer was washed with 150 ml of CH 2 Cl 2 for three times. The 30 CH 2 C1 2 phase was combined, dried on anhydrous magnesium sulfate, and purified by column chromatogaphy, to obtain 3 as white solid (4.2 g, 96%), mp (melting point): 101-102 0 C. 'HNMR (400MHz, CDCl 3 ) 6 3.83 (s, 6H), 3.92 (s, 3H), 6.42 (in, 1H), 6.69-6.72 (m, 2H), 7.07-7.17 (m, 2H), 7.54-7.56 (d, J= 7.8 Hz, 211), 8.01-8.03 (d, J= 7.8 Hz, 2H); MS (EI) m/z 298 [M]*. 1.3 Synthesis of Compound (4) 35 Compound 3 (4 g, 0.013 mol) was dissolved in 100 mL of dry diethyl ester, and added with lithium aluminum hydride (0.5 g, 0.013) by batches under stirring. After the reaction was completed as indicated by TLC analysis, water was slowly added dropwise under ice bath to stop the reaction, and dilute sulfuric acid was used to adjust pH to 5-7. The solution was extracted 8 with CH 2 C1 2 , dried on anhydrous magnesium sulfate, and purified by column chromatography to obtain 4 as white solid (3.2 g, 90%). 'HNMR (400 MHz, CDCl 3 ) 8 3.84 (s, 6H), 4.79 (s, 2H), 6.40 (m: 1H), 6.68-6.73 (m, 2H), 7.06-7.15 (m, 2H), 7.50- 7.52 (d, J = 7.8 Hz, 2H), 8.02-8.04 (d, J = 7.8 Hz, 2H); MS (EI) m/z 270 [M]+. 5 1.4 Synthesis of Compound (5) Compound 4 (1.8 g, 0.007 mol) was dissolved in 50 ml of CH 2 Cl 2 , added with active dioxide manganese (7.3 g, 0.084 mol), and stored at room temperature for 7 hours. The reaction was completed as indicated by TLC analysis. The reaction solution was filtrated and dioxide manganese was washed repeatedly with CH 2 Cl 2 . The filtrate was evaporated to dryness to obtain 10 1.6 g of yellow solid. Yield: 90%. Mp (melting point): 94-95 0 C. 'HNMR (400MHz, CDCl 3 ) 8 3.84 (s, 6H), 4.79 (s, 2H), 6.44-6.45 (m, 1H), 6.69-6.70 (m, 2H), 7.09-7.26 (m, 2H), 7.64-7.66 (d, J= 7.8 Hz, 2H), 7.87-7.88 (d, J= 7.8 Hz, 2H), 9.99 (s, 1H); MS (EI) m/z 268 {M]+. 1.5 Synthesis of Compound (6) In a three-necked flask, compound 5 (1.0 g, 0.004 mol) was dissolved in 50 ml of methanol, 15 added with hydroxylamine methanol (0.435 g, 0.005 mol), and heated under reflux for 2 hours under nitrogen atmosphere, then added with hydroxylamine methanol (0.435 g, 0.05 mol) and refluxed for another 18 to 24 hours. After the reaction was completed as indicated by TLC analysis, the reaction solution was purified by column chromatography to give yellow solid (0.9 g, 75%). Mp (melting point): 130-131*C. 'HNMR (400MHz, DMSO-d 6 ) a 1.59 (s, 9H), 3.79 (s, 6H), 20 6.44 (in, 1H), 6.52-6.54 (m, 2H), 7.07-7.17 (in, 2H), 7.63-7.66 (d, J= 11.4 Hz, 2H), 7.84 (m, 1H), 8.34-8.36 (d, J= 11.4 Hz, 2H); MS (EI) m/z 339 [M]*. Example 2 Selection of Compounds by Caliper Method In this example, 12 compounds were selected using in vitro kinase EGFR, HER2, PDGFRA, 25 PDGFRB, and SRC by Caliper method. Compound staurosporine was used as standard control. Each compound was diluted to obtain 10 concentration points and subjected to duplicate detection. The reaction conditions and results are shown in Table 1 and Table 2, respectively. Table 1 Kinase Enzyme ATP Concentration Contain MnCl 2 or not Reaction Time Concentration (nM) (pM) EGFR 8 2.3 contain 10mM MnC1 2 1 hour HER2 18 15 contain 10mM MnC1 2 1 hour PDGFRa 3.5 134 5 hours PDGFRp 6 38 5 hours SRC 1 36 1 hour Table 2 Compounds IC50(tM) 9 EGFR HER2 PDGFRa PDGFRO SRC 6 >100 0.90 7 21 16 28 8 <0.005 0.20 2.4 2.9 0.83 9 17 7.4 57 10 12 11 16 18 11 20 14 40 12 22 19 13 28 >100 14 9.6 30 99 15 5.4 5.1 4.0 8.3 3.8 16 9.4 12 13 18 17 11 11 14 21 staurosporine 0.18 0.36 0.0011 0.00075 0.016 Example 3 Effect of Compounds on Cell Proliferative Activity Anti-proliferation activities of the above compounds 6-17 on four cell lines including HepG2, A549, BGC-823 and HCT1 16 were assayed with MTT method. 5 Compounds at the concentration of 10mM were prepared with DMSO, diluted at the ratio of 1:5. Each compound was detected starting from a final concentration of 50M. 0.5pil of each compound was transferred from the corresponding plate to a cell culture plate and incubated in a 37 0 C incubator for 72 hours. Then cell morphology was observed under an inverted microscope. To each well, 20pl of 5mg/ml MTT solution prepared with sterile PBS was added, and incubated 10 in a 37*C incubator for 5 hours, and then added with 100 l of triple lysis solution, and dissolved overnight in a 37 0 C biochemistry incubator. Absorbance values were detected with Flexstation3.

IC

50 values were recorded and analyzed. The cell proliferation inhibition rates of the above 12 compounds are shown in Table 3. Table 3 Proliferation Inhibition Rates of Compounds 6-17 on Four Cell Lines Compounds HepG2 A549 HCT 116 BGC-823 CPD ID %Inhibition SD % Inhibition SD % Inhibition SD % Inhibition SD CPD 6 73.39 2.15 73.94 0.88 59.45 3.26 55.32 0.65 CPD 7 47.60 4.65 30.66 0.91 47.02 7.08 23.53 1.10 CPD 8 51.68 3.28 52.43 0.16 55.21 6.64 32.73 1.93 CPD 9 44.18 0.25 40.36 0.57 35.13 3.81 28.62 0.82 CPD 10 28.39 2.93 31.21 3.55 37.26 15.85 31.63 0.63 CPD 11 33.92 1.29 39.32 0.79 55.50 9.22 32.46 1.69 CPD 12 59.46 2.30 50.82 5.33 26.89 3.22 35.32 3.05 10 CPD 13 65.75 1.54 42.30 2.79 27.29 3.74 29.09 5.40 CPD 14 45.71 0.34 34.74 2.02 53.70 1.90 42.60 4.58 CPD 15 68.44 2.66 62.59 0.82 57.52 1.33 46.29 1.81 CPD 16 31.92 0.54 36.59 1.46 46.98 3.96 44,14 2.95 CPD 17 37.72 2.51 46.60 6.09 51.58 5.29 43.48 0.49 Conclusion According to the results of the effect of the compounds on cell proliferation inhibition rate, it can be seen that compounds 6 and 15 have moderate anti- proliferative activity on HepG2 and A549 cell lines; and compounds 7, 11, 14, 16 and 17 have moderate anti- proliferative activity 5 on HCT1 16 cell line. Example 4 Anti-tumor Effect of Compound 6 and 15 on A549 tumor-bearing nude mice Experimental methods: A549 cells in logarithmic growth phase were collected and re-suspended in a serum-free medium, allowing the final concentration to be 5.0x10 7 cells/ml. 70 10 BALB/c nude mice (at 6-7 weeks of age) were inoculated subcutaneously with 0.2ml A549 cell suspension with a density of 1.0x10 7 cells/mouse. The animals were monitored daily to observe their behavior. Tumor areas (length x width) were measured three times each week with Vernier caliper, and tumor volumes were calculated according to the formula (length x width 2 ) / 2. When the average tumor volumes reached approximately 150 mm 3 , the animals were randomly grouped 15 in accordance with 60%-70% screening ratio, and the remaining animals were euthanized. 48 animals were randomly divided into 6 groups for administration: there were 3 compound samples to be tested, including one positive control drug, which were administered once daily for three continuous weeks. Compounds treatment method: the compounds were dissolved in DMSO to obtain mother liquor with a concentration of 10 times of the final concentration, and diluted with 20 physiological saline in a ratio of 1: 9 before administration. + Group I: N = 8; solvent control (saline); + Group 2: N = 8; positive drug (medium dose = 20 mg/Kg), DMSO mother liquor was 20 mg/ml; + Group3: N = 8; compound 6, (high dose = 35 mg/Kg), DMSO mother liquor was 35 25 mg/ml; + Group 4; N 8; compound 6, (low dose= 5 mg/Kg), DMSO mother liquor was 5 mg/ml; + Group: N 8; compound 15, (high dose = 35 mg/Kg), DMSO mother liquor was 35 mg/ml; + Group 6: N = 8; compound 15, (low dose = 5 mg/Kg), DMSO mother liquor was 5 mg/ml; 30 The tested drugs were administered intragastrically. The administration volumes were 0.1 ml/1 0g. The animals were weighed before administration every day, and the tumor volumes were measured twice a week. The animals' behaviors were monitored daily. Health observation 1 1] included animal mortality, appearance, spontaneous activity, posture, food and water intake. Any side effects observed on the above aspects were recorded. Once there were phenomena that seriously affect- animal vitality (food and water intake, action capacity), such as significant weight reduction (with a relative weight change rate higher than 20%), cachexia or large tumor, the 5 animals would be euthanized; if there were serious ulcers or bloody tumor, the animals would also be euthanized. After completion of the experiment, the tumors were peeled off and weighed, and samples were collected for subsequent test. The whole in vivo research course was 28 days. Tumor volume, tumor weight, inhibition rate of tumor growth and the relative change rate of body weight are shown in Table 4 below. 10 Table 4 Antitumor Effect and Toxicity Analysis of Compounds 6 and 15 on A549 Tumor-bearing Nude Mice Toxicity Tumor Tumor TGIl Average Average max Group Number volumn weight Death (%) RCBW(%) RCBW(%)/Administratio (D27) n days Negative 8 1546.45 1.39 - 25.05 0.00 (D 0) 0 control Compound 15 8 1110.33' 1.09 30.94 20.83 0.00 (D 0) 0 high dose Compound 15 8 1342.36 1.32 14.60 23.69 0.00 (D 0) 0 low dose Compound 6 8 894.55** 0.91 46.47 19.48 0.00 (D 0) 0 high dose Positive 1026.04 0.95 37.21 21.94 -0.31 (D1) 0 control Compound 6 8 1168.25* 1.16 27.01 24.30 0.00 (D 0) 0 low dose **p<0.01 vs. Vehicle; *p<0.05 vs. Vehicle; One-way ANOVA followed by Dunnet t test. The administration day was defined as day 0, i.e. Do. TGI% = (1-(T 1 --To)/(Ti-To)) x100%; Ti is the tumor volume on the day of measurement; To is the tumor volume when randomly grouped; 15 RCBW% (relative change ratio of body weight) = (BWi-BWo)/BWox100%; BWi is the body weight on the day of measurement; BWo was the body weight when randomly grouped. Conclusion After administration for 28 days, compared with the control group, high dose group of compound 6, high dose group of compound 15 and low dose group of compound 6 had statistical 20 significance in the inhibition of tumor growth of A549 tumor-bearing mice. Low dose group of compound 15 had no statistical significance, that is, did not achieve a significant inhibition of 12 tumor growth. Example 5 Co-Administration The compounds according to the present invention may be combined with a physiologically 5 acceptable carrier or vehicle to provide a pharmaceutical composition, such as lyophilized powder in the form of tablet or capsule with various fillers and binders. Similarly, the compounds may be co-administered with other agents. Co-administration shall mean the administration of at least two agents to a subject so as to provide the beneficial effects of the combination of both agents. For example, the agents may be administered simultaneously or sequentially over a period 10 of time. The effective dosage of a compound in the composition can be widely varied as selected by those of ordinary skill in the art and may be empirically determined. Moreover, the compounds of the present invention can be used alone or in combination with one or more additional agents depending on the indication and the desired therapeutic effect. The combination therapy contemplated in the present invention include, for example, administration of the 15 inventive compound and additional agent(s) in a single pharmaceutical formulation as well as administration of the inventive compound and additional agent(s) in separate pharmaceutical formulations. The compounds of the present invention may be administered alone, or with a pharmaceutically acceptable carrier or dilute agent, optionally together with known adjuvants, for 20 example, microcrystalline cellulose in a pharmaceutical composition according to standard pharmaceutical practice to mammals, preferably human beings. The compounds may be administered orally or parenterally, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical administration route. For the oral application of the chemotherapeutic compounds of the present invention, the 25 selected compounds may be administered, for example, in the form of tablets or capsules or as aqueous solution or suspension. For oral tablets, common carriers are usually added, including lactose and corn starch, and lubricating agents such as magnesium stearate. For oral application in the form of capsules, diluents including lactose and dry corn starch may be used. When aqueous suspension is required for oral application, the active ingredient is mixed with emulsifying agents 30 and suspending agents. If desired, certain sweetening agents and/or flavoring agents may be added. For intramuscular, intraperitoneal, subcutaneous or intravenous application, the total concentration of solutes should be controlled in order to render the formulation isotonic. 5.1 Pharmaceutical Composition Containing Compound 6 35 Formulation 1: Compound 6 80 mg Microcrystalline cellulose 40 mg Lactose 50 mg . .... . ... 13 Low substituted hydroxypropylcellulose 8 mg Magnesium stearate 2 mg Total 180 mg Formulation 2: 5 Compound 6 60 mg Microcrystalline cellulose 40 mg Lactose 50 mg Cross-linked carboxymethyl cellulose sodium 8 mg Magnesium stearate 2 mg 10 Total 160mg Formulation 3: Compound 6 100 mg Microcrystalline cellulose 40 mg Lactose 50 mg 15 Cross-linked polyvidone 8 mg Magnesium stearate 2 mg Total 200 mg Formulation 4: Compound 6 100 mg 20 Lactose 94 mg Cross-linked polyvidone 4 mg Magnesium stearate 2 mg Total 200 mg Formulation 5: 25 Compound 6 100 mg Microcrystalline cellulose 40 mg Lactose 58 mg Magnesium stearate 2 mg Total 200 mg 30 Formulation 6: Compound 6 160 mg Microcrystalline cellulose 41mg Lactose 50 mg Cross-linked polyvidone 6 mg 35 Magnesium stearate 3 mg Total 260 mg Formulation 7: Compound 11 120 mg

.....

Microcrystalline cellulose 40 mg Lactose 48 mg Cross-linked polyvidone 8 mg Magnesium stearate 4 mg 5 Total 220mg 5.2 Preparation Method of Formulations: The active compound of the present invention, disintegrating agents and fillers were taken in proportion, passed through 60-100 mesh sieve, and mixed uniformly. 2-20% polyvidone K30 solution in ethanol was used to prepare the soft materials, pelletized through 20-50 mesh sieve, 10 and dried at 40-90'C, with particle moisture to be controlled within 3%, granulated, then added with the right amount of lubricant, mixed uniformly, and pressed to obtain tablets. Particularly, the pharmaceutical compositions of the above examples may also be prepared by the following method: Compound 6, fillers and disintegrants were taken by 50 times of the prescribed amount, passed through 60 and 80 mesh sieve sequentially, and mixed uniformly. 15 2-20% polyvidone K30 solution in 50% ethanol was used to prepare the soft materials, pelletized through 30 mesh sieve and dried at 60'C, with particle moisture to be controlled within 3%, granulated with 20 mesh sieve, then added with the right amount of lubricant, mixed uniformly, and pressed to obtain tablets. It will be appreciated that various modifications may be made in the invention as described 20 above and as defined in the following claims. INDUSTRIAL APPLICABILITY The compounds and compositions of the present invention have anti-tumor activity, and have a therapeutic effect in subjects suffering from cancer or related conditions. The present 25 invention provides their preparation method and application. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to 30 specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. 35 15 6846813 1 (GHMatters) P92452.AU INNAM

Claims (15)

1. A compound represented by the following formula (I): AP\ b,, A'q R (I) 5 Wherein, p and q independently represent integers from 0 to 4 provided that p < 4 and q K 4; b represents a double bond which may be present or absent; when the double bond is present, the compounds of formula (I) may be in the E or Z configuration, and when the double bond is absent, the stereocenters of the compound of formula (I) may have the R- or S- configuration; R and R' independently represent H, C 1 -C 2 0 linear or branched alkyl; C

2 -C 2 0 linear or 10 branched alkenyl; -CO 2 Z', wherein Z' is H, sodium, potassium, or other pharmaceutically acceptable counter-ion selected from the group consisting of calcium, magnesium, ammonium, tromethamine, tetramethylammonium; -CO 2 R.'; -NH 2 ; -NHR.'; -NR 2 .'; -OH; halo; substituted C 1 -C 2 0 linear or branched alkyl or substituted C 2 -C 2 0 linear or branched alkenyl; -OR"', wherein R.' represents C 1 -C 2 0 linear or branched alkyl, linear or branched alkenyl, or aralkyl -(CH 2 )x-Ar, 15 wherein X is an integer from 1 to 6; -CO 2 R"", wherein R"" represents H, optionally substituted C 1 -C 2 0 alkyl, optionally substituted C 1 -C 2 0 linear or branched alkoxy, optionally substituted C 2 -C 2 0 alkenyl, or optionally substituted C 6 -Ci 0 aryl or represents a cyclic moiety of morpholine, piperidine, or piperazine; A and A' represent mono- or multi-substituted groups, and independently represent H, 20 C 1 -C 2 0 acylamino, C 1 -C 2 0 acyloxyl, C 1 -C 2 0 alkanoyl, C 1 -C 2 0 alkoxycarbonyl, C 1 -C 2 0 alkoxyl, C 1 -C 2 0 alkylamino, C 1 -C 2 0 alkylcarboxylamino, aroyl, aralkanoyl, carboxyl, cyano, halo, hydroxyl; or A and A' represent optionally substituted linear or branched C 1 -C 2 0 alkyl or C 2 -C 2 0 alkenyl, or may be joined to form a methylenedioxy or ethylenedioxy group. 25 2. The compound represented by the following formula (I) of claim 1: AA' b,, Aq RtN (I) Wherein, p and q independently represent integers from 0 to 4 provided that p < 4 and q < 4; b represents a double bond which may be present or absent; when the double bond is present, the compounds of formula (I) may be in the E or Z configuration, and when the double bond is 30 absent, the stereocenters of the compound of formula (I) may have the R- or S- configuration; 16 6846813 1 (GHMatters) P92452.AU INNAM R and R' independently represent H; -CO 2 Z', wherein Z' is H, sodium, potassium, or other pharmaceutically acceptable counter-ion selected from the group consisting of calcium, magnesium, ammonium, tromethamine, tetramethyl ammonium; -CO 2 R"", wherein R"" represents H, optionally substituted C 1 -C 20 alkyl, optionally substituted CI-C 2 0 linear or branched alkoxyl 5 optionally substituted C 2 -C 2 0 alkenyl, or optionally substituted C 6 -Cio aryl or represents a cyclic moiety such as morpholine, piperidine, piperazine; A and A' represent mono- or multi-substituted groups, and represent H, C 1 -C 2 0 acylamino, CI-C 2 0 acyloxyl, CI-C 2 0 alkanoyl, CI-C 2 0 alkoxycarbonyl, CI-C 2 0 alkoxyl, C 1 -C 2 0 alkylamino, C 1 -C 2 0 alkylcarboxylamino, aroyl, aralkanoyl, carboxyl, cyano, halo, hydroxyl; or 10 A and A' represent optionally substituted linear or branched CI-C 2 0 alkyl or C 2 -C 2 0 alkenyl, or may be joined to form a methylenedioxy or ethylenedioxy group.

3. The compound of claim 2 wherein R"" represents optionally substituted C 1 . 6 alkoxyl.

4. The compound of claim 1 selected from the group consisting of: (E)-N-((E)-4-((E)-3, 5-dimethoxyl)-phenyl)-N-tert-butyl-3-nitrone (6); 15 (E)-N-((E)-4-((E)-3, 5-dibenzyloxyl)-phenyl)-N-tert-butyl-3-nitrone (7); (E)-N-((E)-4-((E)-3-methoxyl-4-benzyloxy)-phenyl)-N-tert-butyl-3-nitrone (8); (E)-N-((E)-4-((E)-3-methoxyl-4-benzoate)-phenyl)-N-tert-butyl-3-nitrone (9); (E)-N-((E)-4-((E)-3-methoxyl-4-acetate)-phenyl)-N-tert-butyl-3-nitrone (10); (E)-N-((E)-4-((E)-3-methoxyl-4-hydroxyl)-phenyl)-N-tert-butyl-3-nitrone (11); 20 (E)-N-((E)-4-((E)-4-fluoro)-phenyl)-N-tert-butyl-3-nitrone (12); (E)-N-((E)-4-((E)-4-benzyloxyl)-phenyl)-N-tert-butyl-3-nitrone (13); (E)-N-((E)-4-((E)-4-hydroxyl)-phenyl)-N-tert-butyl-3-nitrone (14); (E)-N-((E)-4-((E)-3-chloro-4-fluoro)-phenyl)-N-tert-butyl-3-nitrone (15); (E)-N-((E)-4-((E)-3-cyano)-phenyl)-N-tert-butyl-3-nitrone (16); 25 (E)-N-((E)-4-((E)-3-fluoro)-phenyl)-N-tert-butyl-3-nitrone (17).

5. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of claims 1 to 4 together with a physiologically acceptable carrier.

6. The pharmaceutical composition of claim 5, characterized in that said pharmaceutical composition is administered by intravenous, intradermal, intramuscular, subcutaneous, oral route; 30 and the pharmaceutical compositions is formulated into a dosage for gastrointestinal tract administration selected from the group consisting of tablets, capsule and pills, or a dosage for parenteral administration selected from the group consisting of injection and external preparation.

7. A tablet containing the compound of any one of claims 1 to 4 as active ingredient, characterized in that said tablet comprises, by weight, 30-80% of active ingredient, 2-20% of 35 disintegrating agent, 0.2-2% of lubricant, and the rest of filler.

8. Use of the compound of any one of claims 1 to 4 or a pharmaceutical composition of any one of claims 5 to 7 in the preparation of medicines for treating a tumor.

9. A method of treating a tumor comprising administering to a subject a compound of any 17 6846813 1 (GHMatters) P92452.AU INNAM one of claims I to 4 or a pharmaceutical composition of any one of claims 5 to 7.

10. A method of preparing the tablet of claim 7, characterized in that the active ingredient, disintegrating agent and filler are passed through 60-100 mesh sieve and mixed uniformly and 2-20% polyvidone K30 solution in ethanol is used to prepare soft materials, pelletized through 5 20-50 mesh sieve, dried at 40-90 0 C, and then added with the lubricant mixed uniformly, and pressed to obtain tablets,

11. A method for preparing a compound of claim 1 comprising the steps of: i) treating Methyl 4 bromornethylbenzoate with triethyl phosphite in toluene to obtain a compound of formula 2, COOCH 3 0.0 ii) treating compound 2 with a substituted benzaldehyde under Wittig Horner reaction to provide an alkene compound 3: COOCH 3 RR where R' is selected from hydrogen, benzyloxy or methoxy, R2 is selected from hydrge 15 benzyloxy, phenylcarbonyloxy, acetyloxy, hydroxy or fluoro, and R is selected from hydrogen methoxy, benzyloxy, chloro and cyano, iii) treating compound 3 with Lithium aluminium hydride in ether or tetrahydrofuran to provide alcohol 4 CH 2 OH R1 R 3 R2 4 iv) treating compound 4 with manganese dioxide in dichloromethane at room temperature to give compound 5, and CHO R1 R 3 2 5 5 v) treating compound 5 with tertiary butyl hydroxylamine in methanol to give nitrone compound 6 0 R1 R 3 R2 6.

12. A compound according to claim 1, substantially as hereinbefore described and/or exemplified. 10

13. A pharmaceutical composition according to claim 5, substantially as hereinbefore described and/or exemplified.

14. A use according to claim 8 or a method according to claim 9, substantially as hereinbefore described and/or exemplified.

15. A method according to claim 11, substantially as hereinbefore described and/or 15 exemplified. 19 6846813 1 (GHMatters) P92452.AU INNAM