HAVING ANTI-CANCEROGENIC EFFICIENCY

SPECIFICATION

The present invention relates to (3aS,4S,4aR,7aS,8S,8aS)-6-(4-acetyl-phenyl)-3-methyl- 4, 4a, 8, 8a tetrahydro-3aH-4,8-methanoisoxazolo [4,5-f]isoindol-5,7(6H,7aH)-dione compound shown by formula 1 , the preparation methods of this compound, the formulations comprising this compound and the usage of this compound in treatment of cancer.

KNOWN STATE OF THE ART

One of the most important problems, faced in treatment of neo-plastic diseases like cancer which have become very frequent in the recent years, is that the patient becomes immune against the drugs used in chemotherapy during treatment and that the cancer cells continue to increase despite of the treatment.

Moreover, since most of the anti-neo-plastic molecules are very toxic and lead to important side effects for the patient, drug development in this field becomes difficult. An important problem faced in development of such drugs is that these molecules structurally comprise toxic functional groups.

Another problem faced in usage of such molecules is that some of the molecules detected to show anti-neo-plastic effect shows these effects only when very high doses are used, and as a result of this, this leads to pluralities of side effects.

As a result of this, pluralities of different antineoplastic molecule alternatives are needed. OBJECT OF THE INVENTION

The object of the present invention is to develop new molecules to be used in cancer treatment. Another object of the present invention is to develop anti-neoplastic agents which do not have toxic effect. Another object of the present invention is to develop anti-neoplastic agents or in other words, anti-cancerogenic agents which are effective even at low doses.

DETAILED DESCRIPTION OF THE INVENTION

As a result of the studies made by the applicants, the applicants have found that the molecules having the structure shown by formula 1 have anti-cancerogenic characteristic although they do not have the functional groups known to have toxic effect. Accordingly, the present invention relates to the molecule shown by formula 1 and which is ment.

The general chemical name of the compounds shown by Formula 1 is (3aS,4S,4aR,7aS,8S,8aS)-6-(4-acetyl-phenyl)-3-methyl-4,4a,8, 8a tetrahydro-3aH-4,8- methano-isoxazolo [4,5-f]isoindol-5,7(6H,7aH)-dione. In order to generally describe these molecules within the scope of the present invention, the terms of "formula 1 " and/or (3aS,4S,4aR,7aS,8S,8aS)-6-(4-acetyl-phenyl)-3-methyl-4,4a,8, 8a tetrahydro-3aH-4,8- methano-isoxazolo [4,5-f]isoindol-5,7(6H,7aH)-dione" or "molecule shown by formula 1 " or "subject matter molecule" can be interchangeably used.

As a result of the in vitro studies whose details are given below, the applicants have found that the subject matter molecule show favorable anti-cancerogenic effect on various cancer cell lines and that the subject matter molecule shows favorable effect even at low concentrations when compared with known anti-cancerogenic agents.

From another perspective, the present invention relates to a method to be used in preparation of the compounds shown by formula 1 , said method comprises the following steps;

(i) (3aR,4S,7R,7aS)-2-(4-acetyl-phenyl)-3a,4,7,7a-tetrahydro-1 H-4,7-methano-isoindol- 1 ,3(2H)-dione compound (formula 3) is dissolved with organic solvent,

3

(ii) Acetaldehyde oxim, shown by Formula 2, is added to the obtained mixture,

OH

I

2

(iii) The formed mixture is cooled down to 0 C,

(iv) NaOCI is added to the cooled mixture,

(v) The reaction is mixed at room temperature,

(vi)The reaction mixture is extracted and the organic phase is separated.

(vii) The organic solvent is removed.

(viii) The obtained substance is crystallized.

The statement of "organic solvent" used here defines Ci-Ci ₀ hydrocarbon or substituted C _r Cio hydrocarbon structured compounds. Within the scope of the present invention, the organic solvent can be selected from the group consisting of acetic acid, acetone, aceto- nitrile, benzene, 1 -butanol, 2-butanol, 2-butanone, t-butyl-alcohol, carbon tetra-chloride, chloro-benzene, chloro-form, cyclo-hexane, 1 ,2-dichloro-ethane, dietilen-glycol, diethyl-ether, dimethyl-formamid, dimethyl-sulphoxide, 1 -4 dioxan, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexane, methanol, methylene chloride, N-methyl-2-pyrrolidon, nitro- methane, pentane, 1 -propanol, 2-propanol, pyridine, tetra-hydrofuran, toluene, trietil amine but not limited with these. In a preferred application of the present invention, the organic solvent is selected from the group consisting of benzene, toluene, hexane and methylene- chloride. In a particularly preferred application of the present invention, methylene chloride and/or hexane is/are used as the organic solvent.

In a preferred application of the present invention, in step (i), methylene chloride is used as the organic solvent.

In a preferred application of the present invention, the crystallization process mentioned in step viii) is realized by means of crystallization of the solid substance, obtained in step vii), through CH ₂ CI ₂ and Hexane mixture. From another perspective, the present invention relates to the usage of molecules, shown by formula 1 , in the treatment of various diseases which involves application of anti- cancerogenic or similar drugs. From another perspective, the present invention relates to the usage of molecules, shown by formula 1 , in preparation of a drug to be used in the treatment of various diseases which involves application of anti-cancerogenic or similar drugs.

From one perspective, the present invention relates to usage of the molecules, shown with formula 1 , as drug in the treatment of cancer.

From another perspective, the present invention relates to usage of the molecules, shown with formula 1 , in production of a drug which is suitable for use in the treatment of cancer. As used here, the "treatment" or "treating" statements describe prevention, reduction, diminishing, healing or blocking of at least one symptom characterizing a pathologic disease in a test subject having a disorder or threatened by a disorder. A non-limiting list related to different cancer types is as follows: carcinoma, carcinoma of solid tissues, squamous celled carcinoma, adenocarcinoma, sarcoma, glioma, high degreed glioma, blastoma, neuro- blastoma, plasmocytoma, histiocytoma, melanoma, adenoma, hypoxic tumors, myeloma, metastatic cancer or general cancers. Among the specific cancer examples where the compounds described in this document can be used, the following can be mentioned: B celled lymphoma, T celled lymphoma, mycosis fungoides, Hodgkin disease, bladder cancer, brain cancer, nerve system cancer, head and neck cancer, squamous celled carcinoma of head and neck, kidney cancer, lung cancers like small cell lung cancer and non-small cell lung cancer, neuro-blastoma/glio-blastoma, ovarian cancer, pancreas cancer, prostate cancer, skin cancer, liver cancer, melanoma, carcinoma of mouth, throat and esophageal with squamous cell, colon cancer, cervical cancer, cervical carcinoma, breast cancer and epithelial cancer, kidney cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, rectal cancer, hematopoietic cancer; testis cancer; colon and rectal cancer, prostate cancer or pancreas cancer.

The subject matter molecules shown by formula 1 can moreover be used for the treatment of diseases of cervical and anal dysplasia, other dysplasia, heavy dysplasia, hyperplasia, atypical hyperplasia and diseases prior to neoplasia-like cancer. As shown in the below mentioned examples, the subject matter molecules shown by formula 1 has been tested in the in-vitro medium in L929 cell lines in a comparative manner with floro-uracil (5-FU) which is an anti-cancerogenic agent. As a result of this, it has been detected that the compounds are effective at low doses when compared with the drugs used.

The "cancer" statement used within the scope of the present invention describes malign tumors or a physiological condition characterized by uncontrolled cell growth. Cancer examples comprise carcinoma, lymphoma, blastoma, sarcoma and leukemia but not limited to these.

Carcinoma herein describes a cancer type comprising epithelial cells.

Lymphoma herein describes a cancer type formed from lymphocytes. Blastoma herein describes a cancer type formed from precursor cells also known as blast cells.

Sarcoma herein describes a cancer type resulting from mesenchymal based altered cells. Leukemia herein describes a cancer type which begins in bone marrow and which leads to high number of abnormal white blood cells.

More particular examples of cancer types comprise breast cancer, prostate cancer, colorectal cancer, skin cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, gastrointestinal cancer, pancreas cancer, glio-blastoma, vulva cancer, vaginal cancer, endometrial carcinoma, ovarian cancer, liver cancer, hepatoma, bladder cancer, kidney cancer, salivary gland carcinoma, thyroid cancer and various head and neck cancers.

As mentioned here, the subject matter molecules, which are compliant to formula 1 , find usage area in the treatment of cancer and/or in protection from cancer disease. Within this context, the subject matter molecules, which are compliant to formula 1 , are going to be applied in the form of a pharmaceutical composition.

From another perspective, the present invention relates to pharmaceutical compositions comprising at least one pharmaceutically acceptable excipient together with the molecules shown by formula 1 . Said pharmaceutical compositions can have any suitable form depending on the preferred method in terms of application of this composition to the patient.

The subject matter pharmaceutical compositions may comprise at least one second active substance in addition to the molecules shown by formula 1 .

In accordance with the present invention, in pharmaceutical compositions comprising the molecules shown by formula 1 , at least one other active substance in addition to this active substance can be selected from the active substances showing anti-cancerogenic and/or anti-metastatic effect. The second active substance can be formulated together with the molecules shown by formula 1 or it can be formulated in a separate manner from the molecules shown by formula 1 , and it can be put into sale in packages which are suitable for usage together. In case at least one other active substance is used in addition to this active substance in pharmaceutical compositions comprising molecules shown by formula 1 , the second active substance can be selected from a group comprising cyclo-phosphamide, methotrexate, 5- fluorouracil, doxorubicin, cyclophosphamide, mustine, vincristine, procarbazine, prednisolone, bleomycin, dacarbazine, bleomycin, etoposide, cisplatin, epirubicin, capecitabine, folinic acid, oxaliplatin, carboplatin, mechloramine, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methyl-urea, carmustine, lomustine, semustine, fotemustine, streptozotocin, mitozolomide, temozolomide, thiotepa, mytomycin, procarbazine, cytarabine, gemcitabine, decitabine, vidaza, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, etoposide, teniposide, novobiocin, merbarone, aclarubicin. The subject matter molecules can be used together with at least one or two or more of the agents mentioned above.

According to the present invention, this active substance can be used at a dose between 1 mcm/kg and 1000 mg/kg in pharmaceutical compositions comprising molecules shown by formula 1 .

In accordance with the present invention, the pharmaceutical compositions comprising the molecules shown by formula 1 can be applied together with one or more therapeutic active substance, for instance together with active substances used in cancer treatment in a combined manner, in a simultaneous manner, in a sequenced manner or in a separate manner. In accordance with the present invention, the pharmaceutical compositions comprising the molecules shown by formula 1 can be formulated in solid or liquid dosage forms.

In accordance with the present invention, the pharmaceutical compositions comprising the molecules shown by formula 1 can be formulated in dosage forms which are suitable for oral application.

In accordance with the present invention, in case the pharmaceutical compositions comprising the molecules shown by formula 1 are formulated in dosage forms which are suitable for oral application, said dosage form can be selected from a group consisting of tablet, capsule, modified-release tablet, prolonged-release tablet, orally disintegrating tablet, sublingual tablet, effervescent tablet, liquid solution, liquid suspension.

In accordance with the present invention, the pharmaceutical compositions comprising the molecules shown by formula 1 can be formulated in dosage forms which are suitable for reception by means of inhalation.

In accordance with the present invention, in case the pharmaceutical compositions comprising the molecules shown by formula 1 are formulated in dosage forms which are suitable for reception by means of inhalation, said dosage form can be selected from a group consisting of aerosol, inhaler, nebulizer and vaporizer.

In accordance with the present invention, the pharmaceutical compositions comprising the molecules shown by formula 1 can be formulated in dosage forms which are suitable for reception by means of injection.

In accordance with the present invention, in case the pharmaceutical compositions comprising the molecules shown by formula 1 are formulated in dosage forms which are suitable for reception by means of injection, said dosage form can be formulated in one of the dosage forms which are suitable for intradermal, intramuscular, intravascular and intraabdominal application.

In accordance with the present invention, the pharmaceutical compositions comprising the molecules shown by formula 1 can be formulated in dosage forms which are suitable for intradermal reception. In case the pharmaceutical compositions comprising the molecules shown by formula 1 are formulated in dosage forms which are suitable for intradermal reception, it can be selected from a group consisting of cream, gel, lotion, ointment, ear drop, eye drop and transdermal plaster.

The applicants present the data confirming the structure of the molecules shown by formula 1 , the examples related to the production method of the molecule and the experimental data proving the anti-cancerogenic effect of the subject matter molecule. When all the presented data and the known state of the art are taken into consideration, it is shown that the molecules shown by formula 1 are new molecules which have not been disclosed before and as also shown by the tests, the subject matter molecules are more effective and have lower toxicity when compared with the anti-cancerogenic molecules which are present in the known state of the art, and within this context, the subject matter molecules are novel and they have inventive step by surpassing the known state of the art.

Now the invention will be described with reference to the below mentioned examples which are for exemplary purposes and which shall not be interpreted to delimit the scope of the invention in any manner. EXAMPLES

Example 1 :General Method for the Synthesis of (3aS,4S,4aR,7aS,8S,8aS)-6-(4-acetyl- phenyl)-3-methyl-4,4a,8,8a tetrahydro-3aH-4,8-methanoisoxazolo [4,5-f]isoindol- 5,7(6H,7aH)-dione compound

(3aR,4S,7R,7aS)-2-(4-acetyl-phenyl)-3a,4,7,7a-tetrahydro- 1 H-4,7-methanoisoindol-1 ,3(2H)- dione compound (3) was dissolved in CH ₂ CI ₂ and acetaldehyde oxim (2) was added. NaOCI was added drop by drop to the mixture cooled in ice bath. The reaction was mixed for 6 hours at room conditions. At the end of the reaction, the mixture was extracted with CH ₂ CI ₂ . Organic phase was dried with Na ₂ S0 ₄ , and the solvent was removed in the evaporator. The product was crystallized with CH ₂ CI ₂ -Hexane.

1

Schema 1 : Synthesis method of molecules shown by Formula 1 . The purity of the obtained compound was checked in a spectroscopic manner. NMR spectrum was taken in Gaziosmanpasa University at Bruker 400 MHz ¹ H and ¹³ C NMR. CDCI3, DMSO was used as the solvent. The obtained NMR spectrum data was compliant with the structure. The melting point of the molecule shown by formula 1 was determined by means of the electro-thermal melting point determination device. In the IR analyses of the subject matter molecules shown by formula 1 , the examples were prepared with KBr, and the measurements were taken from Transform Infrared Spectrometer (JASCO FT/IR^I-30) device in Gaziosmanpasa University. The characterization information of subject matter molecule characterized in the manner whose details are given above is given below.

E.N 242-246°C Efficiency: 80%

¹ H NMR (400 MHz, CDCI ₃ )5 ppm: 8.09 (brd, J = 8.63 Hz, 2H), 7.41 (brd, J = 8.66 Hz, 2H), 4.66 (d, J = 8.06 Hz, 1 H), 3.43-3.36 (m, 2H), 3.21 (d, J = 8.93 Hz, 2H), 3.02 (d, J = 4.67 Hz, 1 H), 2.65 (s, 3H), 1 .98 (s, 3H), 1 .86 (brd, J = 67.21 , 1 1 .19 Hz, 1 H), 1 .70 (brd, J = 1 1 .19 Hz, 1 H)

¹³ C-NMR (100 MHz, CDCI ₃ ) δρρην 196.8, 175.3, 174.7, 154.5, 137.0, 135.3, 129.3, 126.5, 81 .5, 56.3, 46.6, 46.1 , 44.4, 41 .3, 35.9, 26.7, 1 1 .8.

IR Spectrum (KBr, cm ^"1 ): 3471 , 2975, 2956, 2892, 1776, 1708, 1675, 1602, 1509, 1411 , 1384, 1272, 1 178.

Example 2: Anti-Cancer Procedures

a) Cancer Cell Lines and Cell Culture

L929 cancer cell lines have been used for the in vitro tests in the study. All cell preparation processes have been realized in sterile medium in laminar cabinet. The cells are incubated for 4-5 days in 5% C0 ₂ medium at 37°C in sterile cell culture flasks of T75 comprising additive DMEM (Sigma, Germany) nutrient medium including 10% FBS (Fetal Bovine Serum) and 2% PenStrep (Penicillin-Streptomycin) solution. When the cells become confluent, the nutrient medium in the flasks is changed with 10 ml Trypsin-EDTA solution and it is incubated for 5 minutes. After incubation, the flask is slightly shaken and the cells are removed from the surface, and the formed cell suspension is transferred to 50 ml sterile falcon tubes, and 10 ml fresh nutrient medium (DMEM) is added onto the cells and Trypsin is neutralized and the cells are centrifuged for 10 minutes at 1500xg. After the supernatant section is aspired, 2 ml fresh nutrient medium is added to the cell pellet and it is turned into suspension by means of sterile pastor pipette.10 μΙ cell, taken from the cell suspension for cell counting, is mixed with 10 μΙ trypan blue solution, and 10 μΙ of this mixture is pipetted to the Thoma lam and the cells are counted under microscope and the cell concentration is determined (Cell number = total number of cells counted from the 5 wells x dilution factor x 25,000). After the counting process, the cell stock suspension (20-30 000 cells/100 ul), which is to be studied, is prepared inside a new sterile 15 ml Falcon tube. 96 cells are pipetted to the culture plates from the 100 ul cell suspension. The cells provided on the plates are incubated for 24 hours at 37°C after adding test substances with control and different concentrations. b) BrdU Cell ELIZA Cell Proliferation Test Procedure

In order to determine the anti-proliferative activity (anti-cancer activity) and IC50 concentrations of the substances tested within the scope of the invention, colorimetric BrDU (5-bromo-2'-deoxy-uridine) Cell ELISA kit (Roche) was used which provides detection of the cellular DNA amount. Therefore, 100 μΙ cell suspension from the prepared cell stock is pipetted to the related wells of the 96 welled sterile cell culture plates in a triple manner. Nutrient medium for cell control, solvent substance for negative control (solvent control, generally DMSO), a suitable anti-cancer compound (5-Floro-urasil, Sigma F6627) for positive control are added onto the cells with the same concentrations (50, 75, 100, 125, 150, 175, 200, 250 ug/ml) as the test substance. The nutrient medium is added such that the final volume in the wells is 200 μΙ, and it is incubated for 24 hours in an incubator which provides a medium of 37C and 5%C0 ₂ .

After incubation, 10 ul/well of BrDU marking solution is added onto the cells, and the cells are incubated again for 2 hours at 37 degrees. Afterwards, the medium on the plates is aspired and the marking solution is removed from the medium, and it is blotted to the filter paper and the liquid residue is taken and the cells are waited and dried for 1 hour at 60 degrees. FixDenat solution is added to the dried cells with 200 μΙ_Λ/νβΙΙ, and it is incubated for 30 minutes between 15 and 25 degrees. FixDenat fixes the cells and denatures the DNA, thus, anti-BrdU-POD antibodies can easily reach BrdU which has entered into the DNA. After incubation, FixDenat solution is removed by means of aspiration and blotting. Anti-BrdU-POD solution is added onto the cells with 100 μΙ_Λ/νβΙΙ and it is incubated for approximately 90 minutes at 22 degrees. Afterwards, the plates are blotted and the antibody conjugate is removed and the wells are washed three times with the washing solution between 200 and 300 μΙ_Λ/νβΙΙ. Afterwards, the washing solution is poured and removed by means of blotting and substrate solution is added with 100 μΙ_Λ/νβΙΙ. Until the color is formed, it is incubated for 30 minutes at 22 degrees. Finally, 25 μΙ_ 1 M sulphuric acid (stopping solution) is added to each well and it is micro-plate incubated for approximately 1 minute at 300 rpm on a shaker. Afterwards, by using an ELIZA reader (Ryto, China), the proliferation amount of the cells on the plates is determined by measuring the absorbance of the cell culture medium at 450 nm. The results are reported as cell % inhibition, and the optical density of the cells subjected to solvent will be accepted as 100%. Accordingly, inhibition % will be calculated according to the formula of [1 -(A test substance/A solvent control) ^χ 100]. c) BrdU Cell ELISA Cell Proliferation Test

In the SRB test applied as the pre-test, colorimetric BrDU (5-bromo-2'-deoxy-uridine) Cell ELISA (Roche) test, which provides a more sensitive measurement and which provides "cellular DNA amount detection", has been applied to the test substances showing anti- proliferative activity of 40% or above.

Table 1 : Colorimetric BrDU Cell ELISA test steps and parameters

For each plate whose processes are completed, absorbance values are recorded with wavelength between 450-650 nm. The anti-cancer effect of the subject matter molecule, shown by formula 1 , on L929 cells is shown in Figure 1 . It has been determined that the subject matter molecule shows favorable anti-cancerogenic effect on said cell type. In Figure 1 , the comparison of the subject matter formula 1 molecule is also given. As can also be seen in the graphics, the subject matter molecules have inhibition characteristic which is higher than 5-FU even at low concentrations. This shows that said formula 1 molecule has higher anti-cancerogenic effect when compared with the known anti-cancer agent.

Brief description of the figures given in the scope of the present invention and mentioned above is given below; Figure 1: Anti-cancer graphic of (3aS,4S,4aR,7aS,8S,8aS)-6-(4-acetyl-phenyl)-3-methyl- 4,4a,8,8a tetrahydro-3aH-4,8-methanoisoxazolo [4,5-f]isoindol-5,7(6H,7aH)-dione molecule on L929 cells