ITMI20111463A1 - 1,4-DIARYL-2-AZETHYDINONS FOR ANTITUMORAL ACTIVITY - Google Patents

Description

Description of the patent for industrial invention entitled:

"L, 4-DIARYL-2-AZETIDINONES WITH ANTI-TUMOR ACTIVITY"

Summary of the invention

Analogues of combretastatin A4 are disclosed having a 1,4-diaryl-2-azetidinone structure substituted in position 3 by a hydroxyl or amino group. The compounds are useful as anticancer agents.

Field of the invention

The present invention relates to the use of 1,4-diaryl-2-azetidinones substituted in position 3 by a hydroxyl or amino group in the treatment of tumor pathologies. A further object of the invention are new enantiomeric forms of the aforementioned 1,4-diaryl-2-azetidinones and pharmaceutical compositions that contain them.

State of the art

Combretastatine are a series of natural compounds extracted in the late 1980s by Pettit (Pettit et al., 1989) from an African plant called Combretum cajfrum. These compounds have a stilbenic structure, in the double bond cis geometry, and have, as a constant, the presence of three methoxy groups on an aromatic ring (ring A) and of a methoxy on the second ring (ring B), as shown in Figure 1.

Combretastatins have mainly anticancer activity and act on tubulin, where they bind to the binding site of colchicine, preventing its polymerization. Their action is anti-vascular. The active compounds produce rupture of the vascular endothelium with consequent hemorrhage in the tumor area. To date, a large number of derivatives have been prepared where changes have been made in the substituents present in the A and B rings. See for example G. Tron et al., Journal of Medicinal Chemistry., 2006, 49 (11) , 3033-3044.

Among the compounds studied, those of greatest interest for their biological activities are combretastatin A4 and its amino derivative AC7739, both in an advanced clinical trial phase in cancer patients in the form of water-soluble pro-drugs, known as A4P (combretastatin A4 phosphate; Fosbretabulin) and AVE8062 (Ombrabulin), respectively.

Among the numerous structural modifications examined, alongside those of the substituents on rings A and B, there was the preparation of analogs with reduced conformational mobility, in order to prevent the easy isomerization of the double bond from cis to trans with consequent loss of biological activity. . In particular, the cis double bond was replaced by various rigid heterocyclic structures that allowed to maintain the cis relationship between the two aromatic rings A and B.

In particular, rigid analogues of combretastatin A4 in which the cis double bond has been replaced by a 2-azetidinone ring are described by Carr et al, European Journal of Medicinal Chemistry, 2010, 45 (12), 5752-5766. The compounds have a 1,4-diaryl-2-azetidinone structure in which position 3 of the azetidinone ring is not replaced or is replaced with one or two methyl groups.

O Boyle et al, Journal of Medicinal Chemistry 2010, 53 (24), 8569-8584 describe 1,4-diaryl-2-azetidinones substituted at position 3 of the azetidinone ring with an aryl group and, more recently, with a heteroaryl group , naphthyl, vinyl, functionalized alkyl, Biorg. Med. Chem.

2011, 19, 2306-2325, WO 201 107321 1 Al.

1,4-diaryl-2-azetidinones substituted in position 3 by a hydroxyl, methoxy or acetoxyl group are described by Sun et al, Bioorganic & Medicinal Chemistry Letters, 2004,14 (9), 2041-2046. The products are obtained exclusively as cis cyclo-adducts, in which the reaction products are present as an equimolar mixture of enantiomers (R, S) and (S, R).

Effects of inhibition of tubulin polymerization and antiproliferative activity against tumor lines are reported for all the azetidinone derivatives described above.

List of figures

FIGURE 1 shows the structures of combretastatin A4, AC7739 and their prodrug combretastatin A4 phosphate (Fosbretabulin) A4-P and AVE8062 (Ombrabulin).

FIGURE 2 shows the survival rate of HuTu-80 duodenal adenocarcinoma cells and normal small intestine cells Fhs74 after 72 h of incubation with a 10 μΜ concentration of compounds representative of the invention or of combretastatin A4.

FIGURE 3 shows the survival rate of human cell lines belonging to different tumor histotypes in the presence of concentrations of 30 nM of compounds representative of the invention and of combretastatin A4.

Figure 4 shows the DNA content evaluated by flow cytometry (FACS) of HuTu-80 duodenal adenocarcinoma cells treated for 48 or 72 hours with concentrations of 30 nM of compounds representative of the invention and of combretastatin A4.

FIGURE 5 shows the sub-Gl DNA content, assessed by flow cytometry (FACS), of HuTu-80 duodenal adenocarcinoma cells treated for 48 or 72 hours at 30 nM concentrations of representative compounds of the invention and combretastatin A4 .

Figure 6 shows the proteolytic fragments of caspase 3, evaluated by western blot analysis with specific antibodies (anti-caspase 3 purchased from Celi Signaling Technologies) on cell extracts obtained from HuTu-80 duodenal adenocarcinoma cells treated for 24 hours with concentrations of 30 nM of compounds representative of the invention and of combretastatin A4.

FIGURE 7 shows the proteolytic fragments of PARP (poly-ADP-ribose polymerase) evaluated by western blot analysis with specific antibodies (anti-PARP bought from Celi Signaling Technologies) of cell extracts obtained from HuTu-80 duodenal adenocarcinoma cells treated for 24 hours with concentrations of 30 nM of compounds representative of the invention and of combretastatin A4.

Figure 8 shows the phosphorylation of AMPK on Thrl72, evaluated by western blot analysis with specific antibodies (anti-phospho-AMPKa, Thrl72, bought from Celi Signaling Technologies) on cell extracts obtained from HuTu-80 duodenal adenocarcinoma cells treated for 24 hours with concentrations of 30 nM of compounds representative of the invention and of combretastatin A4.

FIGURE 9 shows the phosphorylation of p53 on Seri 5 evaluated by western blot analysis with specific antibodies (anti-phospho-p53, Serl5, purchased from Celi Signaling Technologies) on cell extracts obtained from HuTu-80 duodenal adenocarcinoma cells treated for 24 hours with concentrations of 30 nM of compounds representative of the invention and of combretastatin A4.

Description of the invention

It has now been found that analogues of combretastatin A4 having a 1,4-diaryl-2-azetidinone structure substituted in position 3 by a hydroxyl or amino group are endowed with advantageous properties with respect to the combretastatin and the azetidinone derivatives of the latter previously described, such their use as anticancer agents is particularly useful.

An object of the present invention are compounds of formula (I)

where one of A and B is the group:

in which Ri is chosen between H and OCH3, and the other is the group:

wherein R2 is selected from H, OH, N02, NH2;

R3 is selected from OH and NH2;

their salts, enantiomers and diastereomers;

with the exclusion of the following compounds:

3.4-cw-3-hydroxy-4- (3-nitro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) -azetidine-2-one (compound N);

3.4-cw-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4,5-trimethoxyphenyl) -azetidine-2-one (compound O);

for use as anticancer agents.

Examples of the compounds of the invention are:

(±) 3,4-cij'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one (rac-7-cis; compound A);

(±) 3,4-ir <ms'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) -azetidin-2-one (rac-7 -trans; compound B);

(±) 3,4-cw-3-amino-1- (3,5-dimethoxyphenyl) -4- (4-methoxyphenyl) -azetidin-2-one (rac-15-cw; compound U);

(±) 3,4-iranj'-3-amino-1- (3,5-dimethoxyphenyl) -4- (4-methoxyphenyl) -azetidin-2-one (rac-1 5-trans; compound V);

(±) 3,4-tran s-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) - 1 - (3, 4,5 -trimethoxyphenyl) azetidin-2-one (rac-8-ir <msv compound E);

(±) 3,4-cw-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-8-cw; compound F );

(±) 3,4-ir <ms'-3-amino-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-1 6- trans, compound M);

(±) 3, A-cis-3 -amino-4- (3-hydroxy-4-methoxyphenyl) - 1 - (3, 4,5 -trimethoxy phenyl) azetidin-2-one (rac-16-czs; compound K);

(±) 3,4-irans-3-hydroxy-4- (3-nitro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-26-trans; compound Z );

3,4-trans-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-27 -trans; compound Y);

(±) 3,4-irans-4- (3,5-dimethoxyphenyl) -3-hydroxy-1- (4-methoxyphenyl) -azetidin-2-one (rac-trans; compound C);

(±) 3,4-czs-4- (3,5-dimethoxyphenyl) -3-hydroxy-1- (4-methoxyphenyl) azetidin-2-one (rac-11-czs; compound D);

(±) 3,4-czs-3-hydroxy-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-12-cis; compound G );

(±) 3,4-trans-3-hydroxy- 1 - (3-hydroxy-4-methoxyphenyl) -4- (3, 4,5 -trimethoxyphenyl) azetidine-2-one (rac-12-trans; compound T );

(±) 3,4-trans-3 -animino- 1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-20-trans; compound I );

(±) 3,4-cis-3-amino-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-20 -cis; compound R ).

Preferred compounds of the invention are:

(±) 3,4-trans-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) -azetidin-2-one (rac-7 -trans; compound B);

(±) 3,4-trans-3-amino-1- (3,5-dimethoxyphenyl) -4- (4-methoxyphenyl) -azetidin-2-one (rac-15-trans; compound V);

(±) 3,4-trans-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) - 1 - (3, 4,5 -trimethoxyphenyl) azetidine-2-one (rac-8-trans; compound E );

(±) 3,4-trans-3-amino-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5trimethoxyphenyl) azetidine-2-one (rac-1 6-trans; compound M) ;

(±) 3,4-iran.s'-3-hydroxy-4- (3-nitro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-26-trans ; compound Z);

(±) 3,4-tr <ms'-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-27 -trans ; compound Y);

(±) 3,4-ir <ms'-4- (3,5-dimethoxyphenyl) -3-hydroxy-1- (4-methoxyphenyl) -azetidin-2-one (rac-1 1-trans; compound C) ;

(±) 3,4-irara> s'-3-hydroxy-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-12-trans ; compound T);

(±) 3,4-ir <ms'-3-amino-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one (rac-20-trans ; compound I).

Particularly preferred compounds of the invention are:

(±) 3,4-ir <ms'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) -azetidin-2-one (rac-7 -trans; compound B);

(±) 3, A-trans-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) - 1 - (3, 4,5 -trimethoxyphenyl) azetidin-2-one (rac-8 -trans; compound E ).

The compounds of formula (I) can form basic or acid addition salts acceptable for pharmaceutical use with inorganic or organic bases, or with inorganic or organic acids, respectively. Said salts are also included in the purposes of the present invention. Examples of inorganic or organic bases useful for this purpose are sodium, potassium or ammonium hydroxides; aliphatic amines such as for example triethylamine; amino alcohols such as for example ethanolamine: amino acids, such as for example glyein; aminoglycosides such as glucosamine. Examples of inorganic or organic acids are hydrochloric, sulfuric or phosphoric acid; citric acid, maleic acid, fumaric acid, tartaric acid, succinic acid and methanesulfonic acid.

The compounds of the invention can be prepared according to known methods, for example through the Staudinger reaction which involves the cyclo-addition of a suitable imine of formula (II):

A-N = CH-B

(II)

wherein A and B are as defined for the compounds of formula (I), with a ketene derivative generated in situ from acetoxy acetyl chloride or N-phthaloylglycinyl chloride, to give a compound of formula (I) in which R3 is an acetoxy group or an N-phthalimide group, respectively, which is subsequently subjected to hydrazinolysis to give the compounds of formula (I) in which R3 is hydroxy or amino.

The synthesis schemes 1-5 exemplify the preparation of specific compounds of the invention.

Scheme 1 describes the synthesis of imines 1-4 belonging to the general formula (II).

Scheme 2 describes the synthesis of the cis and trans (racemic) isomers of the compounds of the invention 7, 8, 11, 12 (compounds A, B, E, F, C, D, G, T).

Scheme 3 describes the synthesis of the cis and trans isomers of the compounds of the invention 15, 16, 20 (compounds V, U, M, K, I, R).

Scheme 4 describes the synthesis of the trans isomers of the nitro-derivative 26 and of the amino-derivative 27 of the invention (compounds Z and Y).

Scheme 4

Depending on the experimental conditions adopted and detailed in the examples of the present invention, the compounds of the invention can be obtained in the form of cis or trans isomers with reference to the substituents present in positions 3 and 4 of the azetidinone ring. Using the known conditions of the Staudinger reaction, the cis and trans isomers of the compounds of the invention are obtained as racemic mixtures, which can be resolved in the single enantiomers using conventional methods, such as chromatography on chiral supports or derivatization with chiral reagents. to give diastereomeric mixtures which can be separated into the single diastereomers by crystallization or chromatographic techniques and then converted into the single enantiomers.

An example of this last method is illustrated in diagram 5, where the resolution of compounds 7 and 8 (B and E) and Γ obtaining of the enantiomers 7a, 7b, 8a, 8b (also indicated with the letters B (+) are reported , B (-), E (+), E (-)). With the appropriate variations, well known to those skilled in the art, this scheme can also be used for the preparation of the single enantiomers of the racemic mixtures of the other compounds of general formula (I).

Compounds 7a, 7b, 8a, 8b are new. A further object of the invention is therefore a compound chosen from:

(+) - 3,4-iranj'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidine-2-one (() trans 7a; compound B (+));

(-) - 3,4-iran5'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4

methoxyphenyl) azetidine-2-one ((-) trans 7b; compound B (-));

(+) - 3,4-iran.s'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one ((+) trans 8a; compound E (+));

(-) - 3,4-iran.s'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1 - (3,4,5-trimethoxyphenyl) azetidine-2-one ((-) trans 8b; compound E (-)).

Particularly preferred are the compounds: (+) - 3,4-trans - \ - (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidine-2-one {{+) trans 7a; compound B (+));

(+) - 3,4-iran> s'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one ((+) trans 8a; compound E (+)).

The compounds according to the invention were pharmacologically tested against human tumor lines. After 72 hours of cell incubation with the compound to be tested, cytotoxicity was determined using the MTT assay which allows to quantify cell viability through the assay of mitochondrial dehydrogenases (L. Fang et. Al., J Cancer Res Clin Oncol ., 2008, 134 (12): 1337-45). From the data obtained it emerged that the compounds according to the present invention are endowed with a marked activity against cell lines belonging to different human tumor histotypes, such as, for example, duodenal adenocarcinoma, colon adenocarcinoma, rectal adenocarcinoma, cervical cancer , breast cancer and neuroblastoma, while they do not show cytotoxic effects against normal cell lines. In particular, the compounds of the invention are markedly more potent than the azetidinone derivatives 3,4-cri-3-hydroxy-4- (3-nitro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine- 2-one (compound N) and 3,4-cri-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4,5-trimethoxyphenyl) azetidin 2-one (compound O) described from Sun et al, Bioorganic & Medicinal Chemistry Letters, 2004,14 (9), 2041-2046.

The compounds of the invention determine the arrest of cell proliferation and a consistent induction of cell death due to apoptosis, mediated by the activation of the protein kinase AMPK and by the oncosuppressant p53. This effect is specific in tumor cell lines.

The compounds of the invention, when administered to mammals carrying tumors, are therefore useful in the control of tumor growth and the formation of metastases, in particular in the treatment of tumors whose growth is supported by abnormal neo-vascularization processes.

Examples of tumors that can be advantageously treated with the compounds of the invention are: duodenal adenocarcinoma, colorectal adenocarcinoma, cervical cancer, breast cancer, neuroblastoma. In addition, the compounds could also be used for the treatment of lung cancer, large cell lung cancer, hepatocarcinoma, pancreatic adenocarcinoma, kidney cancer, prostate cancer, testicular cancer, ovarian cancer, bladder cancer and melanoma. .

The compounds according to the present invention can be administered in doses ranging from 0.01 mg to 1 g per kg of body weight per day. A preferred method of administration is that which uses a dosage of from about 1 mg to about 50 mg per kg of body weight per day, using unit doses such as to be administered in 24 hours from about 70 mg to about 3.5 g of the active substance. a patient with a weight of about 70 kg. Such a mode of administration can be adjusted to obtain a better therapeutic effect. For example, the doses can be adjusted in consideration of the patient's therapeutic situation. The active compounds according to the invention can be administered orally, intravenously, intramuscularly or subcutaneously.

The compounds of the invention, when administered, according to well-known therapeutic methods, in combination with other agents used to induce the regression of tumors, increase the antitumor effects of said compounds in a synergistic way. Examples of the compounds that can be used in combination with the compounds of the invention are represented by cisplatin, carboplatin, doxorubicin, topotecan, taxol, taxotere, vincristna, 5-fluorouracil, decarbazine or in combination with radiotherapy.

A further aspect of the present invention relates to a compound selected from:

(+) - 3,4-iranj'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidine-2-one (() trans 7a; compound B (+));

(-) - 3,4-iranj'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidine-2-one ((-) trans 7b; compound B (-)) ;

(+) - 3,4-iranj'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one ((+) trans 8a; compound E (+));

(-) - 3,4-iranj'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) - 1 - (3,4,5-trimethoxyphenyl) azetidine-2-one ((-) trans 8b; compound E (-));

as a drug, in particular as an anticancer agent.

Particularly preferred are the compounds:

(+) - 3,4-iranj'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidine-2-one (() trans 7a; compound B (+));

(+) - 3,4-iran.s'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one ((+) trans 8a; compound E (+));

In another aspect, the invention relates to a pharmaceutical composition containing a therapeutically effective amount of a compound selected from:

(+) - 3,4-iran> s'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one (() trans 7a; compound B (+) );

(-) - 3,4-iran.s'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one ((-) trans 7b; compound B (- ));

(+) - 3,4-iran.s'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one ((+) trans 8a; compound E (+));

(-) - 3,4-iran.s'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1 - (3,4,5-trimethoxyphenyl) azetidine-2-one ((-) trans 8b; compound E (-));

mixed with a compatible excipient for pharmaceutical use.

For therapeutic use, the compounds of the invention can be suitably formulated with physiologically acceptable excipients or vehicles. Suitable pharmaceutical forms may vary depending on the specific compound and route of administration. The dosage of the active ingredient will be determined from time to time, based on the severity of the disease to be treated and the general condition of the patient. Suitable pharmaceutical compositions can be prepared following the instructions given in Remington's Pharmaceutical Sciences, XVIII Ed. Mack Publishing Co.

The pharmaceutical compositions according to the present invention contain therapeutically effective amounts of at least one compound according to the invention mixed with excipients compatible with pharmaceutical use.

The oral compositions generally comprise an inert diluent or an edible carrier and can be enclosed in gelatin capsules or reduced into tablets. Other possible forms of oral administration are represented by capsules, pills, elixirs, suspensions or syrups.

The tablets, pills, capsules and similar compositions may contain the following ingredients, in addition to the compound of formula I: a binder, such as microcrystalline cellulose, tragacanth or gelatin; a carrier such as starch or lactose, a disintegrant such as alginic acid, primogel, corn starch and the like; a lubricant such as magnesium stearate; a fluidifier such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin or a flavoring such as mint flavor, methyl salicylate or orange flavor. When the selected composition is in capsule form, it may additionally contain a liquid carrier such as a fatty oil. Other compositions may contain various materials, for example coating agents (for tablets and pills) such as sugar or shellac. The material used in the preparation of the compositions should be pharmaceutically pure and non-toxic at the dosages employed.

For the preparation of pharmaceutical compositions for parenteral administration the active ingredient can be included in solutions or suspensions, which may additionally contain the following components: a sterile diluent such as water for injections, saline, oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol; antioxidants such as ascorbic acid or sodium bisulfite; ketant agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting the tonicity of the solution, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, single-dose syringes, glass or plastic ampoules.

The invention is further described by the following examples.

Example 1 - Preparation of the compounds of formula (I)

Summary of Immin l (Scheme 1)

3,5-dimethoxy aniline (3.0 g, 19.6 mmol) and 4-methoxy benzaldehyde (2.67 g, 19.6 mmol) are dissolved in 6 mL of EtOH under stirring and at room temperature and Na2SO4 anhydrous ( 4 g). After 48 hours the solvent is filtered and evaporated at low pressure. 1 is obtained as brown oil (5.0 g, 18.4 mmol, yield = 94%).

1H-NMR (CDC13, 200 MHz): δ = 8.41 (IH, s), 7.86 (2H, d, J = 8.77), 7.00 (2H, d, J = 8.77), 6.45-6.35 (3H, m), 3.88 (3H, s), 3.83 (6H, s). EI-MS (m / z): 271

Synthesis of imine 2 (Scheme 1)

In 6 mL of EtOH, 3,4,5-trimethoxy aniline (1.9 g, 10.1 mmol) and 3-hydroxy-4-methoxy benzaldehyde (1.5 g, 10.1 mmol) are dissolved under stirring and a room temperature and Na2SO4anhydrous (4 g) is added. After 48 hours, a yellow precipitate is obtained together with the drying agent. The solid is filtered and 10 mL of cold EtOH is washed. The precipitate is solubilized in methylene chloride and filtered to separate it from the desiccant. The solvent is evaporated under reduced pressure and 3 is obtained as a yellow amorphous solid (3.1 g, 9.9 mmol, yield = 98%).

1H-NMR (CDC13): δ 8.36 (IH, s), 7.60-6.80 (3H, m), 6.53 (2H, s), 5.77 (IH, s), 4.1 (3H, s), 3.93 (3H, s ), 3.80 (6H, s) EI-MS (m / z): 318 (M <+>).

- Synthesis of azetidine-2-oni (trans + cis) of formula (I) (Procedure a) 3,4-frans-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4 , 5-trimethoxyphenyl) azetidine-2-one, (8 -trans) (E), and 3,4-cis-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4, 5-trimethoxyphenyl) azetidine-2-one (8-cis) (compound F)

Imine 2 (1.8 g, 5.8 mmol) and TEA (4.7 g, 6.4 mL, 46.2 mmol) are dissolved in 140 mL methylene chloride in a nitrogen atmosphere at 0 ° C anhydrous. Then 2-acetoxy acetyl chloride (3.9 g, 28.9 mmol) diluted in 30 mL of anhydrous methylene chloride is added dropwise. It is left to rise to room temperature and left under stirring for 24 hours. The volume of solvent is reduced under reduced pressure and purified by flash chromatography using the mixture hexane: ethyl acetate = 1: 1 as eluent. A mixture of rac-6-cis and rac -6-trans is obtained as a white amorphous solid. (1.14 g, 3.34 mmol, yield = 60%).

rac-6 -cis. 1H-NMR (CDC13,200 MHz), δ: 7.22 (IH, dd, J = 8.41, 2.04), 7.05 (IH, d, J = 2.04), 6.96 (IH, d, J = 8.41), 6.57 (2H, s), 5.90 (1H, d, J = 4.82), 5.28 (IH, d, J = 4.82), 3.82 (3H, s), 3.78 (3H, s), 3.73 (6H, s), 2.19 (3H, s), 1.79 (3H, s). <13> C-NMR (CDC13, 50.3 MHz), δ: 170.37, 169.57, 166.00, 161.81, 153.81, 151.71, 139.27, 132.98, 127.17, 124.86, 122.54, 113.28, 95.45, 76.48, 61.20, 61.11, 60.48, 56.32, 20.62, 20.06. ESI-MS, (m / z): 417

rac-6 -trans. 1H-NMR (CDC13,200 MHz), δ: 7.23 (IH, dd, J = 8.49, 2.19), 7.00 (IH, d, 8.49), 6.53 (2H, s), 5.38 (IH, d, J = 1.74 ), 4.68 (2H, s), 4.58 (1H, d, J = 1.74), 3.85 (3H, s), 3.79 (3H, s), 3.74 (6H, s), 2.19 (6H, s). ESI-MS, (m / z): 417

In a nitrogen atmosphere, under stirring and at 0 ° C, 6 (trans cis) (1.14 g, 3.34 mmol) is dissolved in 50 mL of methanol, then hydrazine dichloride (2.4 g, 23 mmol) is added. TEA (6.6 g, 9.1 mL, 65 mmol) is added dropwise and it is left to soak at room T and then reflux for 4 hours. The solvent is evaporated and a 0.1 N HCl solution is treated with 30 mL and extracted with ethyl acetate (3 x 30 mL). The organic phases are combined and anhydrified (Na2S04). The solvent is evaporated under reduced pressure and the mixture is separated by gradient flash chromatography (eluent hexane: terbutyl-methyl ether = 6: 4 to 2: 8). Isomers 8 (trans) (rac-E) (0.125 g, 0.33 mmol, yield = 9.9%) and 8 (cis) (rac-F) (0.350 g, 0.93 mmol, yield = 28%) as white amorphous solids.

rac-8 -trans. 1H-NMR (CDC13,200 MHz) δ: 6.8-7.0 (2H), 6.49 (IH, s), 5.91 (IH, s, disappears by deuteration), 4.83 (IH, s, disappears by deuteration), 4.74 (IH , d, J = 1.56), 4.71 (1H, d, J = 1.56), 3.86 (3H, s), 3.74 (3H, s), 3.67 (6H, s). <13> C-NMR (CDC13,50.3 MHz), 5: 167.68, 154.05, 146.94, 135.39, 133.80, 129.81, 118.80, 112.91, 111.68, 96.14, 84.14, 66.35, 61.57, 55.67. ESI-MS (m / z): 398 (M + Na <+>).

rac-8 -cis. 1H-NMR (CDC13 + D20, 200 MHz): δ 6.8-7.0 (3H), 6.62 (2H, s), 5.20 (IH, d, J = 5.2), 5.13 (IH, d, J = 5.2), 3.92 (3H, s), 3.78 (3H, s), 3.76 (3H, s). <13> C-NMR (CDC13I50.3 MHz), 5: 167.25, 153.28, 145.02, 143.25, 133.51, 126.47, 119.17, 114.49 , 111.14, 95.13, 76.50, 62.69, 59.65, 54.93. ESI-MS (m / z): 398 (M + Na <+>).

3,4-irans-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin2-one (7 -trans) (compound B) and 3,4-cis-1- (3 , 5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidine-2-one (7-cis) (compound A)

In a nitrogen atmosphere, at 0 ° C and under stirring Γ imine 1 (2.97 g, 10.9 mmol) and TEA (17.6 g, 24.3 mL, 174 mmol) are dissolved in 90 mL of anhydrous methylene. Then 2-acetoxy acetyl chloride (7.44 g, 54.5 mmol) diluted in 30 mL of anhydrous methylene chloride is added dropwise. It is left to rise to room temperature and left under stirring for 24 hours. The reaction mixture is concentrated and purified by flash chromatography using the mixture hexane: ethyl acetate = 1: 1 as eluent. A mixture of the two rac-5 -cis and rac -5-trans diastereoisomers (1.67 g , 4.5 mmol, yield = 41%) as a white amorphous solid.

rac-5- trans. 1H-NMR (CDC13,200 MHZ) δ: 7.23 (2H, d, J = 7.69), 6.85 (2H, d, J = 7.69), 6.42 (2H, d, J = 2.29), 6.13 (1H, t, J = 2.29), 5.30 (1H, d, J = 2), 4.82 (IH, d, J = 2), 3.73 (3H, s), 3.63 (6H, s), 2.12 (3H, s). <13> C-NMR (CDC13,50.3 MHz) δ: 169.81, 162.20, 161.30, 160.30, 138.66, 127.80, 127.22, 114.75, 97.10, 96.43, 82.72, 63.74, 55.49, 55.45, 20.59 EI-EI-MS (m / z): 371

rac-5 -cis. 1H-NMR (CDCl3i200 MHz) δ: 7.23 (2H, d, J = 7.70), 6.87 (2H, d, J = 7.70), 6.53 (2H, d, J = 2.18), 6.22 (IH, t, J = 2.18), 5.89 (1H, d, J = 4.91), 5.29 (IH, d, J = 4.91), 3.81 (3H, s), 3.73 (6H, s), 1.74 (3H, s).

<13> C-NMR (CDCI3,50.3 MHz) δ: 169.81, 162.43, 161.36, 160.15, 138.82, 129.32, 124.05,114.14, 97.12, 96.31, 77.82, 61. 53, 55.52, 55.49, 20.02 EI-MS (m / z): 371.

In a nitrogen atmosphere, under stirring and at 0 ° C, 5 (trans cis) (1.67 g, 4.5 mmol) is dissolved in 65 mL of methanol, then hydrazine dichloride (3.3 g, 31.5 mmol) is added ). TEA (9.11 g, 12.5 mL, 90 mmol) is dropped and it is left to soak at room T and then reflux for 4 hours. The solvent is evaporated, treated with a saturated solution of KHS04 and extracted with ethyl acetate (3 x 30 mL). The organic phases are combined and anhydrified (Na2S04). The solvent is evaporated under reduced pressure and the two diastereomers separated by gradient flash column chromatography (eluent hexane: terbutyl-methyl ether = 7: 3 to 3: 7). The isomers rac-7 (trans) (0.050 g, 0.15 mmol, yield = 3.3%) and rac-7 (cis) (0.246 g, 0.75 mmol, yield = 17%) as amorphous solids whites.

rac-7 -trans. 1H-NMR (CDC13J200 MHz) δ: 7.27 (2H, d, J = 8.6), 6.94 (2H, d, J = 8.6), 6.54 (2H, d, J = 2.2), 6.20 (IH, t, J = 2.2), 5.22 (1H, d, J = 5.2), 5.16 (2H), 3.81 (3H, s), 3.72 (6H, s). <13> C-NMR (CDC13,50.3 MHz) δ: 166.67, 161.33 , 138.80, 128.90, 124.77, 114.80, 96.93, 96.41, 77.10, 65.69, 55.57. EI-MS (m / z): 329 (M <+>), 272.

rac-7 -cis. 1H-NMR (CDC13,200 MHz) δ: 7.23 (2H, d, J = 8.7), 6.88 (2H, d, J = 8.7), 6.42 (2H, d, J = 2.2), 6.15 (IH, t, J = 2.2), 4.9-4.Ó (3H), 3.80 (3H, s), 3.67 (6H, s). <13> C-NMR (CDC13> 50.3 MHz) δ: 167.77, 161.16, 160.09, 138.67, 128.13, 127.60, 114.75, 97.07, 96.52, 83.77, 65.82, 55.49. EI-MS (m / z) 329 (M <+>), 272.

- Synthesis of azetidine-2-oni (trans) of formula (I) (Procedure b) 3,4-frans-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5 -trimethoxyphenyl) azetidin-2-one (8 -trans) (compound E)

In a nitrogen atmosphere, at 0 ° C and under stirring, compound 2 (1.0 g, 3.15 mmol) and 2-acetoxy acetyl chloride (0.86 g, 0.68 mL) are dissolved in 20 mL of anhydrous toluene. , 6.3 mmol). It is left to reach room temperature and heated to 100 ° C. Anhydrous TEA (0.70 g, 0.96 mL, 6.94 mmol) is dropped. After 5 hours the reaction mixture is concentrated and purified by flash chromatography using the mixture hexane: ethyl acetate = 1: 1 as eluent. Only the rac-6 {trans) diastereomer is obtained (0.578 g, 1.11 mmol, yield = 35%) as a white amorphous solid.

In a nitrogen atmosphere, under stirring and at 0 ° C, rac-6 trans (4.3 g, 8.3 mmol) is dissolved in 100 mL of methanol, then hydrazine dichloride (7.0 g, 66.4 mmol) is added. . TEA (13.4 g, 18.5 mL, 133 mmol) is added dropwise and it is left to soak at room T and then reflux for 4 hours. The solvent is evaporated under reduced pressure and 30 mL is treated with a 0.1 N HCl solution and extracted with ethyl acetate (3 x 50 mL). The organic phases are combined and anhydrified (Na2S04) then the solvent evaporated under reduced pressure. The crude product is purified by flash chromatography (eluent hexane: ethyl acetate = 1: 1). Rac-8 - {trans) (compound E), (1.0 g, 2.7 mmol, 32%) is isolated as a white amorphous solid.

3,4-trans-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one (1-trans) (compound B)

In a nitrogen atmosphere, at 0 ° C and under stirring, amine 1 (5.0 g, 18.4 mmol) and 2-acetoxy acetyl chloride (2.5 g, 1.97) are dissolved in 50 mL of anhydrous toluene mL, 18.4 mmol). It is then left to reach room temperature and heated to 100 ° C. Anhydrous TEA (2.0 g, 2.75 mL, 20.2 mmol) is dropped. After 5 hours the reaction mixture is concentrated and purified by flash chromatography using the mixture hexane: ethyl acetate = 1: 1 as eluent. Only the 5 -trans diastereomer (3.4 g, 9.2 mmol, yield = 50%) as a white amorphous solid.

In a nitrogen atmosphere, under stirring and at 0 ° C, 5-trans (2.5 g, 6.8 mmol) is dissolved in 30 mL of methanol, then hydrazine dichloride (2.85 g, 27.2 mmol) is added. TEA (5.5 g, 7.6 mL, 54 mmol) is dropped and it is left to soak at room T and then reflux for 4 hours. The solvent is evaporated, treated with a saturated solution of KHS04 and extracted with ethyl acetate (3 x 50 mL). The organic phases are combined and anhydrified (Na2S04) then the solvent evaporated under reduced pressure and the product is purified by flash chromatography (eluent hexane: ethyl acetate = 1: 1). Rac-7- (trans) (compound B) (2.2 g, 6.8 mmol, quantitative yield) is isolated as a white amorphous solid.

Resolution of rac-7 - (trans) (Scheme 5)

In nitrogen atmosphere and under stirring, rac-7 - (trans) (2.2 g, 6.8 mmol) and (L) -Boc-proline (3.0 g, 14 mmol) are dissolved in 30 mL of anhydrous acetonitrile . HBTU (5.6 g, 15 mmol) is added and DIPEA (72.3 mL, 420 mmol) is dropped. After 24 hours the solvent is evaporated and diluted with water. It is extracted with methylene chloride (3 x 50 mL). The combined organic phases are washed with saturated solution of KHS04, subsequently with saturated solution of NaHC03 and finally with brine. After drying (Na2SO4) and evaporation of the solvent under reduced pressure, the product is purified by gradient flash chromatography (eluent hexane: ethyl acetate = 8: 2 to 6: 4). A mixture of the two diastereomers of 21a 21b is isolated as a white amorphous solid (3.1 g, 5.9 mmol, yield = 84%). The two diasteroisomers are separated by gradient flash chromatography (eluent hexane: terbutylmethylether = 8: 2 to 2: 8) as white amorphous solids.

21a. (diastereomer with Rf = 0.36; 2 runs in Hexane / Methyl t-Butylether 4: 6; Si02): [a] D = 1.25 ((c = 30 mg / 10 ml). 1H-NMR (DMSO-d6, 80 ° C, 500 MHz), δ: 7.38 (2H, d, J = 8.5), 6.97 (2H, d, J = 8.5), 6.43 (2H, d, J = 2.2), 6.27 (IH, t, J = 2.2 ), 5.48 (1H, bs), 5.12 (IH, d, J = 1.85), 4.32 (IH, dd, J = 8.6, 4.3), 3.79 (3H, s), 3.67 (6H, s), 3.41-3.37 (2H, m), 2.35-2.26 (1H, m), 2.06-1.98 (1H, m), 1.90-1.88 (2H, m), 1.41 (9H, s).

<13> C-NMR (CDC13, 25 ° C, 125 MHz) (two conformers): δ 171.68 and 171.48, 161.03 and 160.76, 160.45 and 160.38, 159.51 and 159.27, 153.81 and 152.86, 137.80 and 137.76, 127.2 and 126.9, 126.96 and 126.83, 126.41 and 126.08, 113.96 and 113.73, 96.39 and 96.28, 95.68 and 95.58, 82.23 and 82.11, 79.50 and 79.38, 62.90, 57.87, 54.66, 45.94 and 45.68, 30.30 and 29.17, 27.74, 23.81 and 23.0.

ESI-MS (m / z): 549

21b (diastereomer with Rf = 0.32; 2 runs in Hexane / Methyl t-Butylether 4: 6; Si02), [a] D = - 5.94 (c = 30 mg / 10 ml). 1H-NMR ((DMSO-d6, 80 ° C, 500 MHz) δ: 7.38 (2H, d, J = 8.41), 6.98 (2H, d, J = 8.41), 6.43 (2H, d, J = 2.2) , 6.27 (1H, t, J = 2.2), 5.48 (IH, bs), 5.12 (IH, d, J = 1.75), 4.35 (IH, dd, J = 8.5, 4.3), 3.79 (3H, s), 3.67 (6H, s), 3.43-3.36 (1H, m), 2.35-2.26 (IH, m), 2.06-1.99 (IH, m), 1.83-1.86 (2H, m), 1.39 (9H, s). <13> C-NMR (CDC13, 25 ° C, 125 MHz) (two conformers): δ 171.35 and 171.19, 161.10 and 160.80, 160.42 and 160.42, 159.51 and 159.29, 153.80 and 152.86, 137.86 and 137.73, 127.19 and 126.89, 126.35 and 126.13, 113.97 and 113.82, 96.34 and 95.52, 82.04 and 82.04, 79.63 and 79.36, 62.71 and 62.66, 58.39 and 58.07, 54.66, 45.92 and 45.65, 30.36 and 29.32, 27.74, 23.92 and 22.99.

In a nitrogen atmosphere, under stirring and at 0 ° C (-) - 21b (0.33 g, 0.63 mmol) is dissolved in 30 mL of methanol, then hydrazine dichloride (0.53 g, 5 mmol) is added. TEA (1.0 g, 1.4 mL, 10 mmol) is added dropwise and it is left to temper at room T and then refluxed for 4 hours. The solvent is evaporated under reduced pressure, treated with a saturated solution of KH2S04 and extracted with ethyl acetate (3 x 30 mL). The organic phases are combined, washed with a saturated solution of NaHC03 and subsequently with brine. After drying, the solvent is evaporated under reduced pressure and the product is purified by flash chromatography (eluent hexane: ethyl acetate = 1: 1). (-) 7b (compound B (-)) (lllm g, 0.34 mmol, yield = 54%) is isolated as a white amorphous solid. [a] D = - 29.64 (CHC13, c = 111 mg / 10 mL).

The same protocol applied to 21a provided the enantiomer 7a (+) (compound B (+)): [a] D = 29.10 (CHC13, c = 101 mg / 5 mL). (81 mg, yield = 52%)

The enantiomers (+) 7a (compound B (+)) and (-) 7b (compound B (-)) were checked in Hplc with chiral column against their raceme and showed a purity of 97%

Disclosure resolution 8 (Scheme 5)

Rac-8-trans (2.66 mmol, 1.00 g), (L) -Boc-proline (7.98 mmol, 1.7 g), DIPEA (29.9 mmol, 4.6 g) and HBTU (7.98 mmol, 3.0 g) in 75 mL anhydrous acetonitrile). After 24 hours the solvent is evaporated and diluted with water. It is extracted with methylene chloride (3 x 50 mL). The combined organic phases are washed with saturated solution of KHS04, subsequently with saturated solution of NaHC03 and finally with brine. After drying (Na2SO4) and evaporation of the solvent under reduced pressure, the product is purified by gradient flash chromatography (eluent hexane: ethyl acetate = 8: 2 to 6: 4). A mixture of the two diastereomers of 22a 22b is isolated as a white amorphous solid (1.47 g, 1.9 mmol, yield = 71.4%). The two diasteroisomers are separated by gradient flash chromatography (eluent hexane: ethyl acetate = 8: 2 to 1: 1) as white amorphous solids.

22a (diastereomer with Rf = 0.66; Hexane / Ethyl Acetate 5: 5, Si02): [a] D = -5.83 (MeOH, c = 120 mg / 10 mL). 1H-NMR (DMSO-d6, 80 ° C, 500 MHz), δ: 7.38 (IH, dd, J = 8.41, 1.5), 7.19 (IH, d, J = 8.41), 7.19 (IH, m), 6.57 (2H, s), 5.52 (IH, bs), 5.18 (IH, d, J = 1.5), 4.45 (IH, dd, J = 8.76, 3.83), 4.33 (IH, dd, J = 8.61, 4.22), 3.80 (3H, s), 3.68 (6H, s), 3.64 (3H, s), 3.44-3.37 (4H, m), 2.36-2.29 (2H, m), 2.17-2.10 (IH, m), 2.00- 1.87 (5H, m), 1.41 (18H, bs).

<13> C-NMR (CDC13, 25 ° C, 125 MHz) (two conformers): δ 172.32 and 172.29, 171.08 and 170.84, 161.53 and 161.45, 154.08 and 153.97, 153.62, 153.30 and 153.17, 151.65 and 151.57, 139.63 and 139.50, 134.91 and 134.87, 132.71, 128.27 and 128.17, 126.30 and 126.22, 121.78, 113.92 and 113.85, 95.94, 82.36 and 82.31, 79.72 and 79.68, 62.21 and 62.07, 60.55, 58.84 and 58.76, 56.51 and 56.21, 46.85 and 46.67, 38.71, 30.91 and 30.87, 29.94, and 29.82, 28.53 and 28.38, 24.54 and 24.34, 23.69 and 23.55. EI-MS (m / z): 769.

22b (diastereomer with Rf = 0.57 (Hexane / Ethyl Acetate 5: 5) in Si02:

[a] D = -56.12 (MeOH, c = 131 mg / 10 mL). 1H-NMR (DMSO-d6, 80 ° C, 500 MHz), δ: 7.38 (IH, d, J = 7.87), 7.20 7.16 (IH, m), (IH, d, J = 7.87), 6.56 (2H , s), 5.52 (IH, bs), 5.18 (IH, bs), 5.52 (IH, bs), 5.18 (IH, d, J = 1.5), 4.45 (IH, dd, J = 8.98, 3.91), 4.34 (1H, dd, J = 8.98, 4.27), 3.80 (3H, s), 3.66 (6H, s), 3.63 (3H, s), 3.44-3.37 (4H, m), 2.36-2.31 (2H, m) , 2.17-2.15 (1H, m), 2.04-2.00 (1H, m), 1.93-1.88 (4H, m), 1.37 (18H) .. <13> C-NMR (CDC13, 25 ° C, 125 MHz) (two conformers), δ: 172.38 and 171.99, 171.10 and 170.88, 161.46 and 161.37, 154.03 and 153.97, 153.71, 153.31 and 153.21, 151.65 and 151.59, 139.65 and 139.49, 134.89, 132.74 and 132.66, 128.28 and 128.17, 126.46 and 126.14 , 122.42 and 122.26, 121.73 and 121.62, 113.79, 96.03 and 95.83, 82.29 and 82.05, 79.66 and 79.37, 62.38 and 61.93, 60.54, 58.87 and 58.63, 56.46 and 56.23, 46.84 and 46.44, 38.79, 30.92 and 30.74, 29.97 and 29.77 , 28.49 and 28.17, 24.48 and 24.37, 23.64 and 23.52 EI-MS (m / z): 769.

In a nitrogen atmosphere, under stirring and at 0 ° C, 22a (0.227 g, 0.30 mmol) is dissolved in 30 mL of methanol, then hydrazine dichloride (0.478 g, 2.36 mmol) is added. TEA (0.478 g, 0.66 mL, 4.72 mmol) is added dropwise and it is left to rise to room temperature and then refluxed for 4 hours. The solvent is evaporated under reduced pressure, treated with a saturated solution of KHS04 and extracted with ethyl acetate (3 x 20 mL). The organic phases are combined, washed with a saturated solution of NaHC03 and subsequently with brine. After drying, the solvent is evaporated under reduced pressure and the product is purified by flash chromatography (eluent hexane: ethyl acetate = 1: 1). (+) 8a (compound E (+)) (94 mg, yield = 83.5%) is isolated as a white amorphous solid. [a] D = 16.20 (CHC13, c = 75 mg / 5 mL).

The same protocol applied to 22b provided the enantiomer (-) 8b (compound E (-)): [a] D = -15.43 (CHC13, c = 97 mg / 10 mL). Yield: (97 mg, 58% yield).

Example 2 - Evaluation of the cytotoxic effects of the compounds of the invention

The representative compounds of the invention rac-7-cw (compound A), T2LQ.-1 -trans (compound B), rac-8-trans (compound E), rac-8 -cis (compound F), rac-12 -cis (compound G), rac-1 2-trans (compound T), the reference compound combretastatin A4 and the azetidinone derivatives previously described 3,4-cw-3-hydroxy-4- (3-nitro-4-methoxyphenyl ) -1- (3,4,5trimethoxyphenyl) azetidine-2-one (compound N) and 3,4-cw-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4, 5-trimethoxyphenyl) azetidin-2-one (compound O), dissolved in DMSO, were tested for their ability to inhibit the growth of human cells in culture, through a spectrophotometric assay, which allows to quantify cell viability through the assay the activity of mitochondrial dehydrogenases (MTT test, Fang et al., 2008, J. Cancer Res. Clin. Oncol. 134 (12): 1337-45). The following cell lines were used: duodenal adenocarcinoma cells, HuTu-80 and healthy small intestine cells, Fhs74. The compounds were initially supplied at a concentration of 10 μΜ for 72 h and the DMSO (0.1% w / v) was used as a negative control.

As shown in Figure 2, at a concentration of 10 μΜ the compounds of the invention showed a high ability to inhibit the cell viability of HuTu-80 cells (approximately 98%), equal to that of the reference compound combretastatin A4. On the contrary, none of these compounds had an effect on the viability of Fhs74 cells, suggesting a specificity of inhibition of the aforementioned compounds towards the tumor line.

When the MTT assay was repeated by varying the concentration of the compounds in order to create dose-response curves, the values of IC50 (concentration necessary to inhibit 50% of cell viability) reported in Table 1 were obtained. IC50 values ranging from a concentration of 9 nM to a concentration of 2.5 μΜ (for compound G). Compounds B and E were found to be the most efficient compounds in inhibiting cell viability, with IC50 of about 9 nM for compound B and 13 nM for compound E. It is interesting to note that compounds N and O, previously described by Sun et al, Bioorganic & Medicinal Chemistry Letters, 2004,14 (9), 2041-2046 have an activity of about 50 and 10 times lower than the compounds B and E object of the present invention.

Table 1

The results reported in Table 1 were obtained on the racemic mixtures of the products.

Table 2 reports the results obtained in the same experimental model with the single enantiomers of the two most active compounds B and E. For both compounds the (+) enantiomer is the most active, with an IC50 value of 8.02 nM for B (+) and 3.05 nM for E (+).

Table 2

The effect of compounds B and E in comparison to combretastatin A4 was also evaluated on the following human tumor cell lines:

SW48: colon adenocarcinoma cells

HeLa: cervical cancer cells

MCF-7: breast cancer cells

SKNBE: neuroblastoma cells

The above lines were treated with compounds B and E at a concentration of 30 nM. DMSO (0.1% w / v) was used as a negative control. The results are shown in Figure 3. As already shown in Figure 2, the growth of normal Fhs74 cells was not altered. On the contrary, the cell viability of all tumor lines, to varying degrees depending on the line and compound, was drastically reduced by compounds B and E.

Example 3- Cell death induced by the compounds of the invention From the observation under the microscope of the cell lines treated with the compounds of the invention according to Example 2, it was possible to detect an evident cell death in the tumor lines treated with the compounds. This cell death effect was analyzed in detail using Hutu-80 cells as a model system.

Hutu-80 cells were treated with a 30 nM concentration of compounds B, E and combretastatin A4 for 48 h and 72 h and their DNA content was analyzed by flow cytometry (FACS).

From the FACS analysis (Figure 4) it was evident that the treated cells showed an increase in the fraction of cells with 4C DNA content (post-replicative) and a clear reduction of cells with 2C DNA content (Gl phase of the cell cycle) , in agreement with when reported in the literature for combretastatin A4 (Shen et al., 2010, Br. J. Pharmacol. 160 (8): 2008-27). The most relevant finding was the appearance of a large fraction of cells with sub-Gl DNA content (about 25% at 48 h and 35% at 72 h), due to the DNA fragmentation typical of dead cells (Figure 5).

To distinguish whether this cell death effect was due to necrosis or apoptosis, a molecular level analysis was conducted on the effector caspase 3. It is known, in fact, that caspase 3 is involved in the activation of the apoptotic process. In fact, it is responsible for the proteolytic cleavage of many key proteins including the nuclear enzyme poly (ADP) ribose polymerase (PARP), which is involved in the repair mechanisms of DNA damage in response to environmental stress. PARP is required to maintain cell viability and its proteolytic cleavage by caspase 3 is an important marker of cells that have activated an apoptotic process (Nicholson et al., 1995, Nature 376 (6535): 37-43). Activation of caspase 3 also requires the proteolytic processing of its inactive form (35 kDa) in its two active fragments pl7 and pl2 (Nicholson et al., 1995, Nature 376 (6535): 37-43; Femandes-Alnemri et al. ., 1994, J Biol Chem.

269 (49): 30761-4).

HuTu-80 tumor cells were treated for 24 h with compounds B, E and combretastatin A4 (30 nM) and a western blot analysis was then conducted to assess whether proteolytic fragments of caspase 3 and PARP were present. markers of apoptotic cells. Western analysis clearly revealed that all three compounds induced the cleavage of caspase 3 (Figure 6) and PARP (Figure 7). This data, in association with the previous one showing the appearance of cells with sub-Gl DNA content, indicates that compounds B and E, as well as combretastatin A4, induce apoptosis in HuTu-80 duodenal cancer cells.

Example 4 - Induction of the activation of the protein kinase AMPK and of the accumulation and phosphorylation of the oncosuppressant p53 p53 plays a key role in tumor suppression, mainly by inducing arrest of cell proliferation, apoptosis and senescence (Wang et al., 2010, Transl . Oncol. 3 (1): 1-12.). It is also known that in response to different types of stress, p53 accumulates and / or is extensively phosphorylated on various residues, including Serl5 (Wang et al., 2010, Transl. Oncol. 3 (1): 1-12) . The AMPK (AMP-activated protein kinase) -dependent activation of p53 plays an essential role in inducing the apoptosis process; this event is in turn closely related to the activating phosphorylation of the AMPK kinase on the Thrl72 residue (Wang et al., 2009, Acta Physiol. 196 (l): 55-63).

The compounds of the invention B and E induce the activation of the protein kinase AMPK, similarly to what is reported for combretastatin A4 (Zhang et al., 2008), as well as the accumulation and phosphorylation of the p53 oncosuppressant. Western blot analysis conducted on protein extracts of HuTu-80 tumor cells treated for 24 hours with concentrations of 30 nM of the three compounds showed a clear phosphorylation of AMPK on Thrl72 (Figure 8). It was also observed that p53 accumulates and is phosphorylated on Serl5 in cells treated with compounds B, E and combretastatin A4 (Figure 9).

Claims (1)

CLAIMS 1. Compounds of formula I: where one of A and B is the group: in which Ri is chosen between H and OCH3, and the other is the group: wherein R2 is selected from H, OH, N02, NH2; R3 is selected from OH and NH2; their salts, enantiomers and diastereomers; with the exclusion of the following compounds: 3.4-cij'-3-hydroxy-4- (3-nitro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) -azetidine-2-one; 3.4-ci5-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4,5-trimethoxyphenyl) -azetidine-2-one; for use as anticancer agents 2. A compound according to claim 1 selected from: (±) 3,4-cij'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one; (±) 3,4-ir <ms'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one; (±) 3,4-cw-3-amino-1- (3,5-dimethoxyphenyl) -4- (4-methoxyphenyl) -azetidin-2-one; (±) 3,4-iran> s-3-amino-1- (3,5-dimethoxyphenyl) -4- (4-methoxyphenyl) -azetidin-2-one; (±) 3,4-iran.s'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-cw-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) -azetidine-2-one; (±) 3,4-ir <ms'-3-amino-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-cij'-3-amino-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxy phenyl) azetidine-2-one; (±) 3,4-iran.s'-3-hydroxy-4- (3-nitro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-tran> s-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-ir <ms'-4- (3,5-dimethoxyphenyl) -3-hydroxy-1- (4-methoxyphenyl) -azetidin-2-one; (±) 3,4-cw-4- (3,5-dimethoxyphenyl) -3-hydroxy-1- (4-methoxyphenyl) azetidin-2-one; (±) 3,4-cw-3-hydroxy-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxy phenyl) azetidine-2-one; (±) 3,4-iran.s'-3-hydroxy-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-iran.s'-3-amino-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-cij'-3-amino-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one. 3. A compound according to claims 1 and 2, selected from: (±) 3,4-ir <ms'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one; (±) 3,4-ir <ms <,> - 3-amino-1- (3,5-dimethoxyphenyl) -4- (4-methoxyphenyl) azetidine-2-one; (±) 3,4-ir <ms'-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-ir <ms'-3-amino-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-ir <ms'-3-hydroxy-4- (3-nitro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-tr <ms'-4- (3-amino-4-methoxyphenyl) -3-hydroxy-1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-iran.s'-4- (3,5-dimethoxyphenyl) -3-hydroxy-1- (4-methoxyphenyl) azetidin-2-one; (±) 3,4-iram.s'-3-hydroxy-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5-trimethoxyphenyl) azetidine-2-one; (±) 3,4-iran.s'-3-amino-1- (3-hydroxy-4-methoxyphenyl) -4- (3,4,5trimetosyphenyl) azetidine- 2- one. 4. A compound according to claims 1, 2 or 3, selected from: (±) 3,4-tran5-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one; (±) 3,4-rran5-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one. 5. A compound chosen from: (+) - 3,4-mms'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) -azetidin-2-one; (-) - 3,4-tran5-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) azetidin-2-one; (+) - 3,4-irans-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one; (-) - 3,4-rran5-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one. 6. A compound according to claim 5 selected from: (+) - 3,4-mms'-1- (3,5-dimethoxyphenyl) -3-hydroxy-4- (4-methoxyphenyl) -azetidin-2-one; (+) - 3,4-irans-3-hydroxy-4- (3-hydroxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenyl) azetidine-2-one. 7. A compound according to claim 5 or 6 as a drug. A compound according to claim 5 or 6 as an antitumor agent. 9. Pharmaceutical composition containing a compound according to claim 5 or 6 and at least one excipient suitable for pharmaceutical use. Milan, 29 July 2011