Tamoxifen derivatives for treatment of neoplastic diseases, especially with high HER2 protein level

Abstract

The subject of the invention are new mitochondrially targeted E/Z isomers of aliphatic triphenylphosphonium derivatives of tamoxifen where the aliphatic chain is alkyl or alkenyl, and their corresponding tertiary amine salts and/or their mixture (MitoTAX). Alkyl triphenylphosphonium derivatives of tamoxifen have the general formula (I), where n=8 to 12 and where Z is selected from the group of organic salts or inorganic salts. Alkenyl triphenylphosphonium derivatives of tamoxifen have the general formula IA, where n=6 to 10 and where Z has the above mentioned meaning. These compounds are applicable for the treatment of neoplastic disease, especially those with high HER2 protein levels. The drug for the treatment of neoplastic diseases according to the invention contains at least one E/Z isomer of aliphatic triphenylphosphonium derivatives of tamoxifen of the general formula (I) and/or IA or their corresponding salts of tertiary amine.

Claims

1. A mitochondrially targeted E/Z isomer of an aliphatic triphenylphosphonium derivative of tamoxifen of general formula I or IA, ##STR00027## wherein n=8 to 12, wherein Z is selected from the group consisting of anions of organic salts, anions of inorganic salts, and mixtures thereof, and wherein the crossed double bond in general formula I indicates that the double bond may have E and/or Z configuration, ##STR00028## wherein n=6 to 10, wherein Z is as defined above, and wherein the crossed double bond in general formula IA indicates that the double bond may have E and/or Z configuration.

2. The mitochondrially targeted E/Z isomer of the aliphatic triphenylphosphonium derivative of tamoxifen of general formula I or IA according to claim 1, wherein Z is selected from the group consisting of citrate, acetate, lactate, tartrate, oxalate, ascorbate, mesylate, tosylate, sulphate, halogenide, phosphate, and mixtures thereof.

3. A method of preparing the mitochondrially targeted E/Z isomer of an alkyl triphenylphosphonium derivative of tamoxifen of general formula I according to claim 1, the method comprising the steps of: generating, under the treatment of an organic base in tetrahydrofuran under an argon atmosphere at a temperature of 78 C., an ylide from tert-butyldimethylsilyl-oxy-alkyl-triphenylphosphonium of general formula II: ##STR00029## wherein n=5 to 9, and wherein X is I, Br, Cl or mesyl, condensing the ylide with an aldehyde of general formula III to form a silylated derivative of general formula IV: ##STR00030## treating the silylated derivative of general formula IV with tetrabutylammonium fluoride to form an alkenol of general formula V: ##STR00031## reducing the alkenol of general formula V in a hydrogen atmosphere in the presence of a catalyst to form an alcohol of general formula VI, ##STR00032## substituting the alcohol of general formula VI to form a derivative of general formula VII, ##STR00033## and converting the derivative of general formula VII to the mitochondrially targeted E/Z isomer of an alkyl triphenylphosphonium derivative of tamoxifen of general formula I by heating with triphenylphosphine.

4. A method of preparing the mitochondrially targeted E/Z isomer of an alkyl triphenylphosphonium derivative of tamoxifen of general formula I according to claim 1, the method comprising the steps of: condensing a (hydroxyalkyl)triphenylphosphonium bromide with an aldehyde of general formula III under the treatment of a base in a mixture tetrahydrofuran and dimethylsulphoxide at room temperature to form an alkenol of general formula V: ##STR00034## wherein n=5 to 9, and wherein X is I, Br, Cl or mesyl, reducing the alkenol of general formula V in a hydrogen atmosphere in the presence of a catalyst to form an alcohol of general formula VI, ##STR00035## substituting the alcohol of general formula VI to form a derivative of general formula VII, ##STR00036## and converting the derivative of general formula VII to the mitochondrially targeted E/Z isomer of an alkyl triphenylphosphonium derivative of tamoxifen of general formula I by heating with triphenylphosphine.

5. A method of preparing the mitochondrially targeted E/Z isomer of an alkylenyl triphenylphosphonium derivative of tamoxifen of general formula IA according to claim 1, the method comprising the steps of: generating, in a mixture of tetrahydrofuran and dimethylsulphoxide in an argon atmosphere at room temperature under the treatment of organic base, an ylide from alkyl bis(triphenylphosphonium) with the general Formula XII: ##STR00037## wherein n=7 to 11, and wherein X is I, Br, Cl or mesyl or their combination, and subsequently condensing the ylide with an aldehyde of formula III to form the mitochondrially targeted E/Z isomer of an alkylenyl triphenylphosphonium derivative of tamoxifen of general formula IA: ##STR00038##

6. A method of treating neoplastic disease comprising administering the mitochondrially targeted E/Z isomer of an aliphatic triphenylphosphonium derivative of tamoxifen of general formula I or IA according to claim 1, wherein the neoplastic disease is selected from the group consisting of carcinoma, sarcoma, lymphoma and leukemia.

7. A method of treating neoplastic disease comprising administering the mitochondrially targeted E/Z isomer of an aliphatic triphenylphosphonium derivative of tamoxifen of general formula I or IA according to claim 1, wherein the neoplastic disease is selected from the group consisting of astrocytoma, neuroblastoma, glioblastoma, mesothelioma, breast cancer, prostate cancer, non-small cell lung cancer, cervical cancer, osteosarcoma, colorectal cancer, hepatocarcinoma, and leukemia.

8. A method of killing cancer cells in various regions of breast tumors, regardless of expression levels of HER2, ER, GATA3 and Ki67 proteins comprising administering the mitochondrially targeted E/Z isomer of an aliphatic triphenylphosphonium derivative of tamoxifen of general formula I or IA according to claim 1.

9. A method of inhibiting respiration via the mitochondrial complex I comprising administering the mitochondrially targeted E/Z isomer of an aliphatic triphenylphosphonium derivative of tamoxifen of general formula I or IA according to claim 1.

10. A drug for the treatment of neoplastic disease, the drug comprising at least one mitochondrially targeted E/Z isomer of an aliphatic triphenylphosphonium derivative of tamoxifen of general formula I or IA according to claim 1.

11. The drug according to claim 10, wherein the neoplastic disease is breast cancer with a high HER2 protein level.

12. The drug according to claim 10, wherein the neoplastic disease is breast cancer with a low HER2 protein level.

13. The drug according to claim 10, wherein the drug is efficient against neoplastic disease with either low or high HER2 protein levels.

Description

OVERVIEW OF FIGURES

(1) FIG. 1: illustrates classification of individual classes of mitocans, potentially anti-cancer substances acting on mitochondria.

(2) FIG. 2: illustrates preparation of sublines of human breast cancer MCF7.

(3) FIG. 3: illustrates the effects of MitoTAX and TAX on the growth of experimental tumours with high HER2 expression.

(4) FIG. 4: illustrates apoptosis induced by MitoTAX and TAX in different cell lines.

(5) FIG. 5: illustrates the concentration-dependent induction of apoptosis by MitoTAX in various breast cancer cell lines with high HER2 level.

(6) FIG. 6: illustrates how MitoTAX at different concentrations affects the respiration via the mitochondrial complex I and II in tumour cells.

(7) FIG. 7: shows the comparison of the formation of oxygen radicals in breast cancer cells exposed to MitoTAX and TAX.

(8) FIG. 8: illustrates to decrease in mitochondrial potential in response to MitoTAX and TAX.

(9) FIG. 9: shows that HER2 is localised preferentially in mitochondria of breast cancer cells with high expression of HER2.

(10) FIG. 10: illustrates the effect of the HER2 protein level on the length of mitochondria.

(11) FIG. 11: shows the influence of the HER2 protein level on formation of lactate and mitochondrial respiration.

(12) FIG. 12: shows that cells with high HER2 protein level feature increased uptake of glucose.

(13) FIG. 13: shows that MitoTAX but not TAX reduces expression of the oestrogen receptor ER.

(14) FIG. 14: shows that the HER2 protein is localised in mitochondria of cancer cells in spontaneous tumours with high HER2 level.

(15) FIG. 15: shows that individual areas of mammary gland cancer in the FVB/N c-neu transgenic mouse differ in the expression of genes important for the development and treatment of breast cancer (HER2, ER, GATA3, Ki67).

(16) FIG. 16: illustrates sections of individual areas of the breast cancer stained by using the eosin-hematoxylin method to reveal the tumour morphology of the sections.

(17) FIG. 17: shows sections of individual parts of the same tumours with a markedly diverse HER2 protein levels.

(18) FIG. 18: illustrates level of apoptosis in MCF7 (A) and MCF7 HER2.sup.+ cells (B) exposed to various MitoTAX derivatives

EXAMPLES

(19) Aldehyde of the formula III, which was prepared according to the procedure published in 2003 ((Z)-Tamoxifen and Tetrasubstituted Alkenes and Dienes via a Regio- and Stereospecific Three-Component Magnesium Carbometalation Palladium(0) Cross-Coupling Strategy; Pierre E. Tessier, Andrea J. Penwell, Fabio E. S. Souza, and Alex G. Fallis*; ORGANIC LETTERS, 2003, Vol. 5, No. 17, 2989-2992.), was used as the starting material for preparation of alkyl triphenylphosphonium derivatives of tamoxifen of the general formula I and/or IA (MitoTAX),

(20) ##STR00010##

(21) The starting aldehyde III, as the authors of the invention presented found out, can be prepared with the use of another oxidation agent than the one used in the above mentioned publication. They found out that application 2-iodobenzoic acid (IBX) instead of Dess-Martin agents forms only one double bond isomer. The yield is comparable.

(22) IBX (12.460 g, 44.498 mmol) and the starting allyl alcohol (5.54 g, 14.833 mmol) (see the above mentioned publication) was dissolved in ethyl acetate (120 ml). The suspension was refluxed for the time of 3 hours under a constant stirring. The reaction mixture was cooled down to the room temperature, diluted with diethyl ether (1 l) and washed with saturated solution of sodium carbonate (3100 ml). Combined aqueous layers were reextracted with ethyl acetate (380 ml) again. Combined ethyl acetate layers were dried over magnesium sulphate. The desiccant was filtered and the solution was concentrated under reduced pressure to yield 4.850 g (88%) of aldehyde III in the form of a brownish solid.

Example 1

(23) (9-((tert-butyldimethylsilyl)oxy)nonyl)triphenylphosphonium bromide (634 mg, 1.057 mmol) was dissolved in dry tetrahydrofuran (THF) (6 ml), covered with argon atmosphere and cooled down to 78 C. Butyl lithium (1.2 ml, 0.9 M solution in THF) was slowly added dropwise to the reaction mixture under argon atmosphere. The solution was allowed to warm up to 0 C., colour was changed to dark red, cooled to 78 C. again and aldehyde of the formula III (160 mg, 0.430 mmol) dissolved in dry THF (3 ml) was added dropwise. Then the reaction mixture was allowed to warm up to the laboratory temperature and stirred for 16 hours under argon atmosphere. Progress of the reaction was monitored with thin layer chromatography (TLC) in the mixture of chloroform-methanol (10:1). Then saturated solution of ammonium chloride and water was added to the reaction mixture and extracted with ethyl acetate. The ethyl acetate layer was washed with brine and dried over magnesium sulphate. The solution was filtered and concentrated under reduced pressure. Chromatography of the concentrate on the column of silica gel in the system of dichloromethane (DCM)/methanol (gradient 0 to 10% of methanol) yielded 147 mg of product of the formula 4 (56% yield).

(24) ##STR00011##

(25) .sup.1H NMR (500 MHz, cdcl3) 7.42-7.36 (m, 5H), 7.18-7.28 (m, 5H), 6.94 (d, J=8.7, 2H), 6.73 (d, J=8.7, 2H), 6.19 (d, J=11.5, 1H), 5.47 (dt, J=11.5, 7.4, 1H), 4.09 (t, J=5.8, 2H), 3.72 (t, J=6.6, 2H), 2.80 (t, J=5.8, 2H), 2.42 (s, 6H), 1.69-1.57 (m, 4H), 1.48-1.13 (m, 10H), 1.03 (s, 9H), 0.18 (s, 6H). Electrospray ionization mass spectrometry (ESI MS): 612.

(26) (9-((tert-butyldimethylsilyl)oxy)nonyl)triphenylphosphonium bromide was prepared according to the procedure published in the literature. (Tetrahedron Letters, 2010, 51, 49, 6426-6428.)

Example 2

(27) Silylated derivative of the formula 4 (147 mg, 2.240 mmol) was dissolved in THF (5 ml), then covered with argon atmosphere and tetrabutylammonium fluoride (TBAF) (260 l, 1M solution in THF) was added dropwise at a temperature of 0 C. under the stirring. Then the reaction mixture was allowed to warm up to laboratory temperature and stirred for another 6 hours. Progress of the reaction was monitored with TLC in the mixture of chloroform-methanol (10:1). Then water was added and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed with saturated solution of soda and brine and dried over magnesium sulphate. The desiccant was filtered and the solution was concentrated under reduced pressure. The concentrate was purified with the column chromatography on silica gel in the system chloroform/methanol (gradient 0 to 10% of methanol) to yield 115 mg (96% yield) of the required alkenol of the formula 5.

(28) ##STR00012##

(29) .sup.1H NMR (500 MHz, cdcl3) 7.43-7.14 (m, 5H), 6.94 (d, J=8.5, 2H), 6.72 (d, J=8.5, 2H), 6.20 (d, J=11.5, 1H), 5.48 (dt, J=11.5, 7.4, 1H), 4.12 (t, J=5.9, 2H), 3.72 (t, J=6.6, 2H), 2.86 (t, J=5.9, 2H), 2.46 (s, 6H), 1.71-1.58 (m, 4H), 1.51-1.10 (m, 10H). ESI MS: 498.

Example 3

(30) Alkenol derivative of the formula 5 (115 mg, 0.231 mmol) was dissolved in absolute ethanol (6 ml) and covered with argon atmosphere. 10% Pd/C (10 mg) was added to the mixture and the flask with reaction suspension was evacuated and covered with hydrogen atmosphere repeatedly for several times. Then the reaction mixture was stirred at the laboratory temperature under the hydrogen atmosphere for 24 hours. Progress of the reaction was monitored with TLC in the mixture of chloroform-methanol (10:1). The mixture was filtered through a layer of Celite and washed several times with ethanol. Ethanol was evaporated to yield 101 mg (87% yield) of the required alcohol of the formula 6, which is used without any further purification for the next step of the synthesis.

(31) ##STR00013##

(32) .sup.1H NMR (500 MHz, cd3od) 7.40-7.01 (m, 10H), 6.85 (d, J=8.1, 2H), 6.68 (d, J=8.1, 2H), 4.20 (s, 2H), 3.55 (t, J=6.4, 2H), 3.46 (s, 2H), 2.89 (s, 6H), 2.42 (t, J=7.8, 2H), 1.57-1.48 (m, 2H), 1.38-1.11 (m, 12H). ESI MS: 500.

Example 4

(33) Alcohol of the formula 6 (230 mg, 0,460 mmol) was dissolved in DCM (10 ml). CBr.sub.4 (480 mg, 1.447 mmol) was added to the mixture at the laboratory temperature under argon atmosphere. Then triphenylphosphine (400 mg, 1.525 mmol) dissolved in DCM (3 ml) was added dropwise. The mixture was stirred at the laboratory temperature for 2 hours and then concentrated under reduced pressure. Progress of the reaction was monitored with TLC in the mixture of chloroform-methanol (10:1). Chromatography of the concentrate on the column of silica gel in the DCM/methanol system (gradient 0-10%) afforded 273 mg (92% yield) of required bromide of the formula 7. Bromide was subjected to the next reaction without any long storage.

(34) ##STR00014##

(35) .sup.1H NMR (400 MHz, cdcl3) 7.46-6.96 (m, 10H), 6.78 (d, J=8.9 Hz, 2H), 6.53 (d, J=8.8 Hz, 2H), 4.29 (t, J=6.6 Hz 2H), 3.47-3.28 (m, 4H), 2.82 (s, 6H), 2.38 (t, J=7.8 Hz, 2H), 1.80 (q, J=7.8 Hz, 2H), 1.46-0.98 (m, 14H). ESI MS: 561.

Example 5

(36) Alcohol of the formula 6 (102 mg, 0.204 mmol) was dissolved in DCM (6 ml). Triphenylphosphine (83 mg, 0.316 mmol) and imidazol (27 mg, 0.397 mmol) were added to the mixture at laboratory temperature, and the reaction mixture was cooled in an ice bath to 4 C. Iodine (76 mg, 0.302) was added to the cooled reaction mixture and stirred at the laboratory temperature for the time of 4 hours. Progress of the reaction was monitored with TLC in the mixture of chloroform-methanol (10:1). The reaction mixture was diluted with dichloromethane and extracted with thiosulphate. The organic phase was further washed with saturated solution of soda and brine and dried over magnesium sulphate. Chromatography of the concentrate on the column of silica gel in the DCM/methanol system (gradient 0 to 10%) afforded 100 mg (80% yield) of the required iodide of the formula 8. Iodide was subjected to the next reaction without any long storage.

(37) ##STR00015##

(38) .sup.1H NMR (400 MHz, cdcl3) 7.40-7.32 (m, 2H), 7.31-7.22 (m, 4H), 7.22-7.08 (m, 4H), 6.78 (d, J=8.8 Hz, 2H), 6.57 (d, J=8.8 Hz, 2H), 3.95 (t, J=5.8 Hz, 2H), 3.19 (t, J=7.0 Hz, 2H), 2.68 (t, J=5.8 Hz, 2H), 2.47-2.37 (m, 2H), 2.31 (s, 6H), 1.81 (q, J=7.0 Hz, 2H), 1.52-1.00 (m, 14H). ESI MS: 610.

Example 6

(39) Triphenylphosphine (300 mg, 1.144 mmol) was added to bromide of the formula 7 (273 mg, 0.425 mmol), and the mixture was stirred at the temperature of 85 C. under argon atmosphere for the time of 12 hours. Progress of the reaction was monitored with TLC in the mixture of chloroform-methanol (10:1). The reaction mixture was cooled to the laboratory temperature, dissolved in the minimum quantity of DCM and added dropwise to the hexane solution (50 ml) under a constant stirring at the temperature of 0 C. The formed precipitate was filtered, dissolved in a minimum quantity of DCM again and added dropwise to the diethyl ether solution (50 ml), under a constant stirring at the temperature of 0 C. The precipitate was filtered and dried under vacuum to obtain 281 mg (73% yield) of the required compound of the formula 9 in the form of yellowish powder.

(40) ##STR00016##

(41) .sup.1H NMR (500 MHz, cd3od) 7.89-7.74 (m, 15H), 7.37-7.05 (m, 10H), 6.85 (d, J=8.7, 2H), 6.71 (d, J=8.7, 2H), 4.24 (t, J=5.0, 2H), 3.57 (t, J=5.0, 2H), 3.43 (m, 2H), 2.97 (s, 6H), 2.40 (t, J=7.9, 2H), 1.74-1.60 (m, 2H), 1.59-1.49 (m, 2H), 1.36-1.05 (m, 12H). ESI MS: 744.

Example 7

(42) Application of a procedure similar to that stated in example 6 enables to obtain the compound of the formula 10 from iodide of the formula 8.

(43) ##STR00017##

Example 21

(44) The compound of the formula 5 can be obtained directly from aldehyde of the formula III by reaction with (9-hydroxynonyl)triphenylphosphonium bromide instead of (9-((tert-butyldimethylsilyl)oxy)nonyl)triphenylphosphonium bromide. Such synthesis is shorter and more cost-efficient. The main change is the use of the THF and DMSO mixture to increase solubility and the reaction can be carried out directly with (9-hydroxynonyl)triphenylphosphonium bromide, which was impossible in the actual THF. The procedure is carried out at room temperature instead of 78 C. This procedure leads also to a significant reduction of the total time of preparation of the compound required.

(45) Preparation of the Compound of the Formula 5

(46) ##STR00018##

(47) (9-hydroxynonyl)triphenylphosphonium bromide (3.920 g, 8.082 mmol) was dissolved in DMSO (10 ml) and then THF (30 ml) was added. LiHMDS solution (14.800 ml, 1M in THF) was added dropwise into the reaction mixture for the time of 3 minutes. The colour of the reaction mixture changed to bright orange. Then solution of aldehyde of the formula III (1.000 g, 2.694 mmol) in THF (15 ml) was added dropwise to the reaction mixture, and the reaction was stirred for another ten minutes at laboratory temperature. Progress of the reaction was monitored with TLC in the mixture of chloroform-methanol (10:1). The reaction mixture was poured to the cold saturated solution of ammonium chloride (100 ml) and extracted with diethyl ether (5100 ml). Combined organic layers were dried over magnesium sulphate. The desiccant was filtered and the product was concentrated under vacuum. Raw material was dissolved in diethyl ether (10 ml) and saturated ether solution of HCl (5 ml) was added dropwise. Precipitated product was filtered and extracted by the solution of NaOH (5 ml, 1M) and diethyl ether (25 ml). The organic layer was dried over magnesium sulphate. The desiccant was filtered and the product was concentrated under vacuum to yield 1,102 g (82%) of the product of the formula 5 in the form of slightly yellowish oil which was thus ready for further reactions.

Example 22

(48) From the compound of the formula 6 it is possible to prepare the compound of the formula 9a (tertiary amine hydrochloride) without the necessity to isolate the compound 7. The preparation time is reduced and the yield is higher.

(49) Preparation of the Compound of the Formula 9a

(50) Saturated ether solution of HCl (6 ml) was added to the alcohol of the formula 6 (300 mg, 0.600 mmol) dissolved in diethyl ether (6 ml) The mixture was concentrated in vacuum and dissolved in DCM (6 ml). CBr.sub.4 (298 mg, 0.901 mmol) was added to the reaction mixture and after its complete dissolution triphenylphosphine (252 mg, 960 mmol) was added. The reaction was quenched after 5 minutes with addition of methanol (1 ml) and saturated ether solution of HCl (3 ml). The solution was concentrated in vacuum and triphenylphosphine (2.000 g, 7.625 mmol) was added. The reaction mixture was mixed overnight at the temperature of 100 C. The mixture was cooled down to room temperature and then it was dissolved in DCM (10 ml). The mixture was then cooled to room temperature, dissolved in DCM (10 mL) and added dropwise to a cold and vigorously stirred diethyl ether (100 mL). The precipitate was filtered and dried in vacuum to yield 334 mg of (74%) product of the formula 9a in the form of white, slightly oily solid. The product may be re-purified through recurrent dissolution in DCM (2 ml) and subsequent precipitation in diethyl ether (20 ml).

(51) ##STR00019##

Example 23

Preparation of the Compound of the Formula 11-Isomeric Alkenyl Triphenylphosphonium Derivative of Tamoxifen

(52) Nonan-1,9-diylbis(triphenylphosphonium)bromide was prepared from 1,9-dibromnonan and triphenylphosphine mixture stirred in the solution of dimethylformamide at the temperature of 100 C. for 16 hours and subsequent crystallisation from ethyl acetate.

(53) Nonan-1,9-diylbis(triphenylphosphonium)bromide (545 mg, 674 mmol) was dissolved in DMSO (1 ml) and then THF (3 ml) was added. A solution of LiHMDS (670 l, 1M in THF) was added dropwise into the reaction mixture for the time of 3 minutes. The colour of the reaction mixture changes to bright orange. Then a solution of aldehyde of the formula III (100 mg, 0.269 mmol) in THF (1 ml) was added to the reaction mixture dropwise and the reaction was stirred for another ten minutes at room temperature. Progress of the reaction was monitored with TLC in the mixture of chloroform-methanol (10:1). The reaction mixture was poured into a cold saturated solution of ammonium chloride (10 ml) and extracted with dichloromethane (520 ml). Combined organic layers were dried over magnesium sulphate. The desiccant was filtered and the product was concentrated in vacuum. Chromatography of the concentrate on the column of silica gel in the chloroform/methanol system (gradient 0-10%) yielded 56 mg (30%) of the required product of the formula 11.

(54) ##STR00020##

(55) .sup.1H NMR (400 MHz, cdcl3) 8.00-7.52 (m, 15H), 7.25-7.11 (m, 6H), 7.11-6.96 (m, 4H), 6.72 (d, J=8.3 Hz, 2H), 6.53 (d, J=8.3 Hz, 2H), 6.00 (d, J=11.5 Hz, 1H), 5.26 (dt, J=11.5, 7.4 Hz, 1H), 4.02 (t, J=4.8 Hz, 1H), 3.80-3.53 (m, 2H), 2.88 (t, J=5.3 Hz, 2H), 2.42 (s, 6H), 2.06-1.79 (m, 2H), 1.64-1.36 (m, 4H), 1.38-1.05 (m, 4H), 1.06-0.73 (m, 4H). ESI MS: 742.

(56) Biological Tests of the Mitochondrially Targeted Alkyl Triphenylphosphonium Derivative of Tamoxifen (MitoTAX), Comparison Study with Tamoxifen (TAX)

(57) The following examples 8-20 were carried out with the MitoTAX substance of the general formula I, where n=10.

Example 8

(58) MitoTAX prepared according to Example 6 was tested for its effect on breast cancer cell lines. Lines with different levels of HER2 protein expression and oestrogen receptor . (ER) were used. The cell line MCF7 features a relatively low expression of the HER2 protein. For the testing of killing of breast cancer cells with different HER2 protein levels by MitoTAX, we prepared HER2.sup. and HER.sup.+ MCF7 cells. MCF7 cells were transfected with the vector with a non-silencing sequence (NS), with a short hairpin sequence attenuating the expression of HER2 (sh) and with the vector with a gene for HER2. FIG. 2 shows the expression of the HER2 protein in the various sublines using the western blotting method. In the subsequent work, the sublines NS, Sh1 26 (clone 26) and +11 (clone 11) were used.

Example 9

(59) We evaluated the IC.sub.50 values for TAX and MitoTAX for various breast cancer cell lines. The individual values were determined from the survival curves of cells at various concentrations of both substances using the crystal violet method. We used cellular lines with various levels of the HER2 and ER protein ER.sup.+/HER2.sup.low (MCF7.sub.par), ER.sup.+/HER2.sup.+ (MCF7.sub.HER2+, BT474, NeuTLmurine line of mammary gland cancer), ER.sup.+/HER2.sup. (MCF7.sub.HER2, T47D, ZR75-1), ER.sup./HER2.sup.+ (SK-BR-3), ER.sup./HER.sup. (MDA-MB-231, MDA-MB-453, MDA-MB-436). From Table I, it is clear that the IC.sub.50 value is significantly lower for MitoTAX, approximately by one order of magnitude. The most sensitive is the MCF7.sub.HER2+ subline with the ER.sup.+/HER2.sup.+ genotype. The corresponding lines with the ER.sup.+/HER2.sup. (MCF7.sub.HER2) and ER.sup.+/HER2.sup.low genotype (MCF7.sub.par) feature IC.sub.50 values which are approx. twice higher, which points o the fact that increased HER2 protein level leads to an increase in the sensitivity to MitoTAX. On the other hand and in contrary to MitoTAX, the sensitivity of HER2-high cells to TAX decreases. This indicates a unique property of MitoTAX that (to the best of our knowledge) has not been reported for any other anti-cancer substance.

(60) TABLE-US-00001 TABLE I IC.sub.50 values (M) for breast cancer cellular lines with different expressions of the HER2 and ER protein. Cellular line Status TAX MitoTAX MCF7.sub.par ER.sup.+/HER2.sup.low 15.2 1.25 MCF7.sub.HER2 ER.sup.+/HER2.sup. 14.1 1.45 MCF7.sub.HER2+ ER.sup.+/HER2.sup.+ 21.6 0.65 T47D ER.sup.+/HER2.sup. 17.3 3.4 MDA-MB-231 ER.sup./HER.sup. 35.8 6.2 MDA-MB-453 ER.sup./HER.sup. 17.5 2.5 MDA-MB-436 ER.sup./HER.sup. 12.6 3.4 ZR75-1 ER.sup.+/HER2.sup. 16.9 2.7 SK-BR-3 ER.sup./HER2.sup.+ 28.3 3.5 BT474 ER.sup.+/HER2.sup.+ 29.8 2.4 NeuTL ER.sup.+/HER2.sup.+ 35.6 4.5

Example 10

(61) We also investigated whether MitoTAX suppresses growth of tumours. The anti-cancer efficacy of MitoTAX was tested using the transgenic mouse strain FVB/N c-neu that is born tumour-free and that in the adult age features increased HER2 protein expression due to the action of oestrogen (Guy C T et al. Expression of the neu proto-oncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci USA 1992; 89:10578-10582.). These mice develop dysplasia and then hyperplasia in the region of mammary gland at 3 to 4 months after birth and form palpable tumours after 6 months. Importantly, this occurs in the context of the functional immune system. This model of breast cancer (mammary gland) is a very good approximation of the human breast cancer with a high HER2 protein level of the ductal in situ type. Our results (FIG. 3) indicate a very good efficacy of MitoTAX on the growth of these tumours. Mice were administered a dose of 3 mol of TAX and 0.5 mol of MitoTAX twice a week for the time of two weeks. The volume of the tumours was quantified using ultrasound imaging that can visualise tumours with high precision and in a non-invasive way, including the embedded parts. It is clear that MitoTAX is approximately 20 to 30 times more efficient than TAX, and the differences between the action of both agents are highly significant. The symbol * indicates significant differences between treated and reference animals, the symbol ** indicates significant differences between animals treated with TAX and those treated with MitoTAX. No apparent toxicity was observed in the experimental animals. The photographs below the chart show representative tumours from individual groups of animals.

Example 11

(62) An important aspect of MitoTAX is its higher growth-suppressing activity towards the lines with increased expression of the HER2 oncogene. This is shown in FIG. 4, which also documents that the line with reduced expression of the HER2 oncogene (clone 26) is less responsive to MitoTAX, whilst it is exactly opposite for TAX. For these experiments we also prepared an MCF7 subline resistant to TAX by long-term exposure of the parental MCF7 cells to escalating doses of TAX. It is possible to see that these cells, resistant to TAX, were sensitive to MitoTAX (FIG. 4). The results in FIG. 4 illustrate the survival of breast cancer sublines derived from MCF7 cells with various genotypes (ER.sup.+/HER2.sup.low, MCF7.sub.par; ER.sup.+/HER2.sup.+, MCF7.sup.HER2+-clone 26; ER.sup.+/HER2.sup., MCF7.sup.HER2-clone 11; ER.sup.+/HER2.sup.low, MCF7.sup.TAX-R). The results were obtained using the crystal violet method, which makes it possible to discriminate living and dead cells, in the presence of various concentrations of MitoTAX and TAX.

Example 12

(63) An important characteristic of anti-cancer substances that cause death of cancer cells is the mode of cell death. For this reason we tested whether MitoTAX causes apoptosis, i.e. programmed cell death when a cell is dies in a controlled way and its residual apoptotic bodies are removed from the tissue by phagocytic cells without inflammatory reactions. FIG. 5 shows that the agent, indeed, caused apoptosis. Apoptosis was evaluated on the basis of assessing of percentage of cells with annexin V in the external part of the plasma membrane by means of flow cytometry. Once again, the results document increased efficacy of MitoTAX to cells with high HER2 protein, while cells with a reduced HER2 protein level are more resistant (albeit still undergoing apoptosis).

Example 13

(64) Previous publication (Moreira P I et al. Tamoxifen and estradiol interact with the flavin mononucleotide site of complex I leading to mitochondrial failure. J Biol Chem 2006; 281:10143-10152.) indicated that the target for TAX in mitochondria is, at a high level of the agent, complex I. We have found out that this holds also for MitoTAX, which is documented in FIG. 6. This documents also in inhibitory effect of TAX (on the left) and MitoTAX (on the right) on respiration via the mitochondrial complexes I and II. It is possible to see that TAX inhibits preferably complex I to complex II, at concentrations exceeding 20 M. MitoTAX also inhibits complex I preferably to complex II, but at significantly lower concentrations of about 1 to 2 M. For these assays, MCF7 cells were placed in the chamber of the Oxygraf instrument and the respiration was determined at increasing doses of TAX (on the left) and MitoTAX (on the right). Respiration (oxygen consumption linked with ATP formation) is related to 10.sup.6 cells and is shown as a relative value with the beginning level of respiration marked with the relative value of 1.

Example 14

(65) A property typically associated a number of mitocans is their ability to increase oxidative stress (formation of reactive oxygen species, ROS), selectively in cancer cells, especially associated with their action on mitochondrial complexes participating with oxidative phosphorylation. This is usually connected with a decrease in the mitochondrial potential (Neuzil J et al. Classification of mitocans, anti-cancer drugs acting on mitochondria. Mitochondrion 2013; 13:199-208. Kluckova K et al. Mitochondrial complex II, a novel intriguing target for anti-cancer agents. Biochim Biophys Acta 2013; 1827:552-564.). We tested formation of ROS also for MitoTAX. FIG. 7 shows generation of ROS for MCF7 sublines of differing in HER2 levels, after 1 h exposure to TAX or MitoTAX (both at 5 M). It is possible to see that TAX is markedly less effective at the same concentration than MitoTAX. Another important finding is that MitoTAX induces formation of more ROS in cells with high HER2 levels, whilst a lower production of ROS occurs in cells with low HER2. TAX does not follow this trend. In all cases, the uncoupler of mitochondrial respiration (CCCP), reduces the mitochondrial potential to its basal value. FIG. 8 shows that MitoTAX (but not TAX) reduces the mitochondrial potential already at the concentration of 5 M and within 1 h.

Example 15

(66) In breast cancer cells with high level of the HER2 protein, the protein is localised preferably in mitochondria. This is shown in FIG. 9, where the western blot of the original line MCF7 as well as sublines HER2.sup.+ MCF7 (clone 11), HER2.sup. MCF7 (clone 26) is represented, and where it is seen that the actual sublines are resistant to TAX (clone TAM-R). It is possible to see that clone 11 cells feature increased expression of the HER2 protein (marked with an arrow) in the mitochondrial, cytoplasmic (it contains plasmatic membrane) as well as nuclear fractions. The lower figure shows the mitochondrial fraction when the membrane was exposed for a longer period of time, so that it can be clear that in mitochondria, albeit at a significantly lower level, the HER2 protein is present also in parental MCF7 cells and in cells resistant to TAX, but not in cells with reduced HER2 (clone 26). This surprising result is in agreement with the recently published work (Ding Y et al. Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism. Nat Commun 2012; 3:1271). This publication also shows that breast cancer cells with high expression of the HER2 protein localised predominantly in mitochondria, are resistant to trastuzumab. During application of trastuzumab to cancer cells, more HER2 protein was mobilised to mitochondria (Ding Y et al. Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism. Nat Commun 2012; 3:1271.). It is possible that breast cancer cells mobilise the HER2 protein away from their surface (plasma membrane), so that the protein cannot be affected by trastuzumab. One of the HER2 inhibition results is the activation of the p27 protein, which is an inhibitor of the cell cycle, reducing the malignant nature of the cells (Yang H Y, Shao R, Hung M C, Lee M H. p27 Kip1 inhibits HER2/neu-mediated cell growth and tumorigenesis. Oncogene 2001; 20:3695-3702.). This has a negative impact on cancer cells with high level of the HER2 protein, because cancer cells are evolutionally programmed to maintain high proliferative status (Hanahan D, Weinberg R A. The hallmarks of cancer. Cell 2000; 100:57-70). Therefore we can speculate that, since the HER2 protein, the target of trastuzumab, is not present in the membrane at a major scale, the cell will acquire resistance to trastuzumab. Nevertheless, during this process it will increase its sensitivity to MitoTAX, which is able to penetrate into mitochondria, which further highlights its exceptional nature.

Example 16

(67) One of the reasons for an increased sensitivity of breast cancer cells with high HER2 protein is their changed mitochondrial bioenergetics and morphology. The high level of the HER2 protein in mitochondria changes their morphology as well as function. FIG. 10 shows that mitochondria in the HER2.sup.+ cells (clone 11) are approximately twice shorter than those in the HER2.sup. cells (clone 26). The mitochondrial length was estimated with the help of confocal microscopy of cellular lines transfected by the mitochondrially targeted GFP protein (which visualises mitochondria by means of green fluorescence). The length was determined by the analysis of mitochondria in 50 cells selected in a random manner using the Fuji Freehand Lines Measurement Tools software. This is linked to the reduced mitochondrial respiration and is associated with lower mitochondrial potential and higher production of lactate (a symptom of a shift towards aerobic glycolytic metabolism) (FIG. 11). It is shown that in this case, cells with increased HER2 protein levels produce approximately twice more lactate than parental cells and cells with reduced HER2 protein levels. In the case of respiration, it is exactly opposite. Cells with increased HER2 protein levels respire less (ATP production is associated with lower consumption of oxygen). A higher share of glycolysis in the ATP generation for cells featuring increased HER2 protein level is associated also with their increased uptake of glucose (FIG. 12).

Example 17

(68) Another possible reason for increased sensitivity of HER2.sup.+ cells with high HER2 protein levels to MitoTAX is the effect of this agent on the oestrogen receptor ER, having anti-apoptotic effects (Thomas C, Gustaffson J. The different roles of ER subtypes in cancer biology and therapy. Nat Rev Cancer 2011; 11:597-608. Deblois D, Giguere V. Oestrogen-related receptors in breast cancer: control of cellular metabolism and beyond. Nat Rev Cancer 2013; 13:27-36.). This is shown in FIG. 13, where it is possible to see that MitoTAX reduces the ER expression already at a concentration of 1 M approximately three times, while TAX is ineffective. These results were obtained using the real-time PCR methodology.

Example 18

(69) The above mentioned high efficacy of MitoTA against tumours with high expression of the HER2 protein in the murine strain FVB/N c-neu is of high importance. This tumour, which corresponds to human tumours with high expression of the HER2 protein, was analysed for the expression of the HER2 protein and several other genes. FIG. 14 shows a representative FVB/N c-neu mouse with a tumour (the upper figure on the left) and also the excised tumour (the figure in the lower left corner). The results of the tumour analysis by western blotting documents that the tumour contains a high level of the HER2 protein, which is almost undetectable in the normal tissue of the mammary gland. The figure also shows results of the analysis of the mitochondrial (Mito) and cytosolic (Cyto) fractions. Antibodies against specific proteins are used as markers for the mitochondrial fraction. It is clear that an absolute majority of the HER2 protein is localised in mitochondria. These results obtained from an experimental tumour correspond to results from breast cancer cells with high expression of HER2.

Example 19

(70) It has been shown recently in kidney tumours that the same tumour contains areas that differs in their mutation profile (Gerlinger M et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012; 366:883-892.). Tumour heterogeneity (Stingl J, Caldas C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer 2007; 7:791-799.), and this phenomenon was identified in the case of breast carcinomas as well. This is correlated, interestingly, with the finding that spontaneous tumours of the mammary gland in the FVB/N c-neu transgenic mouse contain areas with different expression of several important genes at the level of mRNA, which may considerably affect breast cancer treatment. This concerns the genes ER, HER2, Ki67, a marker proliferation which is higher in case of higher levels of HER2) and GATA3 (transcription activator which positively affects HER2 expression). This is shown in FIG. 15. In this experiment, two tumours were divided into several parts, which were analysed using real-time PCR for the expression of the above mentioned genes. The results illustrate very different expression of the genes in the individual areas of the tumour, varying up to 5 times. Another proof of the different expression of the HER2 gene in individual parts of the tumour in the experimental FVB/N c-neu mice is shown in the following Figures, where it is possible to see the tumour morphology on the basis of staining with haematoxylin and eosin (FIG. 16), as well as an immunohistochemical analysis of the HER2 protein expression (FIG. 17). These unambiguous differences correspond to a different expression of HER2 in individual parts of the tumour at the level of mRNA and are consistent with published data on intratumour heterogeneity (Gerlinger M et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012; 366:883-892. Stingl J, Caldas C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer 2007; 7:791-799.). FIG. 17 shows that there are very large differences in the HER2 protein expression between the external part of the tumour (part 1a), middle part (part 1 b) and internal part (part 1c). This means that some tumour areas will be resistant to TAX therapy (areas with high HER2 protein expression), others will be resistant to the trastuzumab therapy (areas with low HER2 protein levels). Moreover, it is possible to expect that the trastuzumab action will be accompanied by an increased transfer of the HER2 protein to mitochondria, whereby the tumour areas with high HER2 protein expression acquire resistance to this type of therapy. On the other hand, MitoTAX, which acts on mitochondria and kills cells featuring high HER2 protein expression more efficiently than cells with low expression of this protein, is able to cope with the areas of tumours resistant to trastuzumab.

Example 20

(71) MitoTAX, efficiently killing the breast cancer cells, is effective also against other types of cancer cells. This is shown in Table 2, where it is possible to see IC.sub.50 values for MitoTAX and TAX for killing various types of cancer, including carcinomas, sarcomas and leukaemias. The IC.sub.50 values were lower for MitoTAX than for TAX in all cases.

(72) TABLE-US-00002 TABLE 2 Cellular line - tumour type TAX MitoTAX 1321n1 - astrocytoma 17.97 1.54 SHSY5Y - neuroblastoma 11.16 1.76 U87 - glioblastoma 32.44 1.96 H28 - mesothelioma 39.74 2.53 LnCAP - prostate cancer 36.70 0.86 H1299 - non-small cell lung cancer 38.53 1.80 Hela - cervical cancer 30.28 2.68 MG-63 - osteosarcoma 19.94 1.47 HCT116 - colorectal cancer 28.91 1.81 HepG2 - hepatocarcinoma 17.56 1.05 MOLT-4 - leukaemia 12.9 0.37

Example 24

(73) FIG. 18 shows apoptosis induction by the effect of alkyl and alkenyl triphenylphosphonium derivatives of MitoTAX, as documented in Table 3, in breast cancer cells MCF7 (A) and the MCF7 cell subline with increased HER2 protein level (B). The percentage of apoptotic cells was determined using the specific apoptosis essay based on evaluation of the level of externalised annexin V by using flow cytometry. MCF7 and MCF7 HER2.sup.+ cells were exposed to individual MitoTAX derivatives at the concentration of 2 M for 24 h. The CTRL column indicates the percentage of apoptotic cells in the cell population without addition of the tested substances, and thus it corresponds to the basal level of apoptosis. All tested derivatives of MitoTAX induced apoptosis.

(74) TABLE-US-00003 TABLE 3 The compound of the formula 9 The compound of the formula 9a The compound of the formula 9b The compound of the formula 10 The compound of the formula 10a The compound of the formula 11

(75) In conclusion it is possible to sum up that we have prepared brand new compounds which are based on TAX, which is a frequently used drug for the treatment of breast cancer, i.e. a disease with a rising incidence (DeSantis C et al. Breast cancer statistics, 2011. CA Cancer J Clin 2011; 1:409-4018.). The above described alkyl and alkenyl triphenylphosphonium derivatives of tamoxifen (MitoTAX) according to the invention are preferably accumulated in mitochondria, where their target site, the mitochondrial complex I, is located. The MitoTAX interaction with complex I will result in an interruption of the flow of electrons that then interact with molecular oxygen. This leads to the enhanced formation of ROS that, in turn, trigger cellular death.

(76) MitoTAX is efficient to breast cancer with both low and high levels of the HER2 protein that considerably complicates the existing methods of treatment. Thus, MitoTAX can supplement or replace both TAX and trastuzumab in cancer therapies.

USE OF THE INVENTION

(77) The new tamoxifen derivatives, of the general formulas I and IA according to the invention, are applicable for the treatment of cancer in the clinical setting and in the pharmaceutical industry for the preparation of drugs for efficient treatment of cancer.