CALIX[4]ARENES WITH HIGH ANTICANCER ACTIVITY

Abstract

A calix[4]arenes of the formula (1a),

##STR00001##

wherein: Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are selected from hydrogen, halogen, NO.sub.2, N.sub.3, CN, CHO, COOR, CONR.sub.2, NR.sub.2, triazole moiety and C.sub.1-C.sub.3-alkyl group, the C.sub.1-C.sub.3-alkyl group being optionally substituted, Z is a heterocyclic moiety which is selected from the group including of imidazole, benzimidazole, benzothiazole, benzoxazole, purine, tetrazine, oxazole, pyrazole and thiazole, the heterocyclic moiety being optionally substituted, R.sub.1 and R.sub.2 are selected from hydrogen and C.sub.1-C.sub.8-alkyl, the C.sub.1-C.sub.8-alkyl group being optionally substituted, n being an integer between 1 and 4, They exhibit high cytotoxicity against cancer cells.

Claims

1. A compound of the formula (1a), ##STR00032## or a physiologically tolerable salt thereof wherein: Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are each independently selected from the group consisting of: hydrogen, halogen, NO.sub.2, N.sub.3, CN, CHO, COOR with R being alkyl group, CONR.sub.2 with R being independently H or alkyl group, NR.sub.2 with R being independently hydrogen, alkyl or acetyl group, triazole moiety which is optionally substituted by alkyl group or phenyl, C.sub.1-C.sub.3-alkyl group, the C.sub.1-C.sub.3-alkyl group being optionally substituted with one substituent selected from the group consisting of N.sub.3, halogen, OR with R being alkyl group, COOR with R being alkyl, CONR.sub.2 with R being independently H or alkyl group, Z is a heterocyclic moiety which is selected from the group consisting of imidazole, benzimidazole, benzothiazole, benzoxazole, purine, tetrazine, oxazole, pyrazole and thiazole, the heterocyclic moiety being optionally substituted with 1 to 5 substituents which are selected from the group consisting of hydrogen, halogen, alkyl alkenyl, alkynyl, phenyl, benzyl, NO.sub.2, nitrile, OR with R being alkyl group, COOR with R being alkyl group, R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen and C.sub.1-C.sub.8-alkyl group, the C.sub.1-C.sub.8-alkyl group being optionally substituted with one or more substituents each independently selected from the group consisting of alkenyl, alkynyl, phenyl, hydroxyl, N.sub.3, halogen, COOR with R being alkyl group, CONHR with R being alkyl group or CONR.sub.2 with R being independently alkyl group, n being an integer comprised between 1 and 4, with the proviso that when R.sub.1 and R.sub.2 are each hydrogen and n is equal to 1, Z is not a benzothiazole group, or wherein: Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are each hydrogen, R.sub.1 and R.sub.2 are each C.sub.3-alkyl group, n is equal to 1 and Z is a pyridine group.

2. The compound or salt as claimed in claim 1, wherein Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are each independently selected from the group consisting of hydrogen, halogen, N.sub.3, NO.sub.2, CHO, NH.sub.2, N(CH.sub.3).sub.2, NHCOCH.sub.3, NCH.sub.3COCH.sub.3, CH.sub.2OCH.sub.3, COOCH.sub.3, COOC.sub.2H.sub.5, CONHCH.sub.3, CON(CH.sub.3).sub.2, triazole, triazole substituted by a C.sub.2-alkyl and triazole substituted by a phenyl.

3. The compound or salt as claimed in claim 2, wherein Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are all hydrogen.

4. The compound or salt as claimed in claim 1, wherein R.sub.1 and R.sub.2 are selected from the group consisting of hydrogen, C.sub.1-C.sub.3-alkyl group, ethyl acetate group, C.sub.1-C.sub.3-alkyl group substituted by N.sub.3, C.sub.1-C.sub.3-alkyl group substituted by hydroxyl, C.sub.1-C.sub.3-alkyl group substituted by at least one halogen, C.sub.1-C.sub.3-alkyl group substituted by alkynyl and C.sub.1-C.sub.3-alkyl group substituted by alkenyl.

5. The compound or salt as claimed in claim 4, wherein R.sub.1 and R.sub.2 are C.sub.1-C.sub.3-alkyl group.

6. The compound or salt as claimed in claim 1, wherein Z is selected from the group consisting of imidazole, purine, benzimidazole, oxazole, thiazole, benzoxazole, benzothiazole and tetrazine group.

7. The compound or salt as claimed in claim 6, wherein Z is a 1-C.sub.1-C.sub.6-alkyl-imidazole group or a 1-benzylimidazole group.

8. The compound or salt as claimed in claim 1, wherein n is equal to 1.

9. The compound or salt as claimed in claim 1 of the formula (2) or (3) or (4): ##STR00033##

10. A 1:1 complex formed from a calix[4]arene or a physiologically tolerable salt thereof as claimed in claim 1 and a cation.

11. A pharmaceutical composition, wherein it comprises at least one calix[4]arene of the formula (1a) ##STR00034## or a physiologically tolerable salt thereof or a 1:1 complex formed from a calix[4]arene of the formula (1a) or from a physiologically tolerable salt thereof and a cation wherein: Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are each independently selected from the group consisting of: hydrogen, halogen, NO.sub.2, N.sub.3, CN, CHO, COOR with R being alkyl group, CONR.sub.2 with R being independently H or alkyl group, NR.sub.2 with R being independently hydrogen, alkyl or acetyl, triazole moiety which is optionally substituted by alkyl group or phenyl, C.sub.1-C.sub.3-alkyl group, the C.sub.1-C.sub.3-alkyl group being optionally substituted with one substituent each independently selected from the group consisting of N.sub.3, halogen, OR with R being alkyl group, COOR with R being alkyl, CONR.sub.2 with R being independently H or alkyl group, Z is a heterocyclic moiety which is selected from the group consisting of imidazole, benzimidazole, benzothiazole, benzoxazole, pyridine, purine, oxazole, pyrazole, thiazole, triazole, tetrazine, tetrazole, pyrimidine, pyrazine and pyridazine, the heterocyclic moiety being optionally substituted with 1 to 5 substituents which are selected from the group consisting of hydrogen, halogen, alkenyl, alkynyl, phenyl, benzyl, NO.sub.2, nitrile, OR with R being alkyl group, COOR with R being alkyl group, R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen and C.sub.1-C.sub.5-alkyl group, the C.sub.1-C.sub.8-alkyl group being optionally substituted with one or more substituents each independently selected from the group consisting of alkenyl, alkynyl, phenyl, hydroxyl, N.sub.3, halogen, COOR with R being alkyl group, CONHR with R being alkyl group or CONR.sub.2 with R being independently alkyl group, n being an integer comprised between 1 and 8, and together with at least one pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11, wherein: Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are each independently selected from the group consisting of hydrogen, halogen, N.sub.3, NO.sub.2, CHO, NH.sub.2, N(CH.sub.3).sub.2, NHCOCH.sub.3, NCH.sub.3COCH.sub.3, COOCH.sub.3, COOC.sub.2H.sub.5, CONHCH.sub.3, CON(CH.sub.3).sub.2, CH.sub.2OCH.sub.3, triazole, triazole substituted by a C.sub.2-alkyl and triazole substituted by a phenyl; R.sub.1 and R.sub.2 are selected from the group consisting of hydrogen, C.sub.1-C.sub.3-alkyl group, ethyl acetate group, C.sub.1-C.sub.3-alkyl group substituted by N.sub.3, C.sub.1-C.sub.3-alkyl group substituted by hydroxyl, C.sub.1-C.sub.3-alkyl group substituted by at least one halogen, C.sub.1-C.sub.3-alkyl group substituted by alkynyl and C.sub.1-C.sub.3-alkyl group substituted by alkenyl Z is selected from the group consisting of imidazole, purine, benzimidazole, oxazole, thiazole, benzoxazole, benzothiazole, tetrazine group, pyridine and triazole; n is an integer comprised between 1 and 4.

13. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition further comprises at least one compound selected from the group consisting of chemotherapy compounds, immunotherapy compounds, antiviral compounds, antiparasitic compounds, antifungal compounds and antibiotics.

14. A calix[4]arene of the formula (1a) or a physiologically tolerable salt thereof or a 1:1 complex formed from the calix[4]arene of the formula (1a) or from the physiologically tolerable salt thereof and a cation as defined in claim 1 for use in the treatment of a disease selected from the group consisting of cancer or metastasis thereof, viral, bacterial, parasitic and fungal infections.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0212] FIG. 1 is a graph showing the profile of the proliferation index N.sub.t/N.sub.0 of the A549 cells after exposure or not with the calix[4]arene of the formula (2).

[0213] FIG. 2 is a graph showing the profile of the proliferation index N.sub.t/N.sub.0 of the PC9 cells after exposure or not with the calix[4]arene of the formula (2).

[0214] FIG. 3 is a graph showing the profile of the proliferation index N.sub.t/N.sub.0 of the H322 cells after exposure or not with the calix[4]arene of the formula (2).

EXAMPLES

IDescription and Preparation of Calix[4]Arenes of the Present Invention and Two Comparative Examples

Example 1: Synthesis and Characterization of the Calix[4]Arene of the Formula (2)

##STR00019##

[0215] In a 50 mL round bottom flask, the calix[4]arene of the formula (B) (309 mg, 0.61 mmol, 1.0 equiv.) was stirred in anhydrous THF (12 mL) with NaH (60% dispersion in mineral oil, 520 mg, 13.0 mmol, 36 equivalents) for 30 minutes under inert atmosphere. 2-(chloromethyl)-1-methyl-1H-imidazole hydrochloride (407 mg, 3.11 mmol, 5.1 equiv.) dissolved in anhydrous DMF (3 mL) was then added. The mixture was stirred at reflux for 24 hours under inert atmosphere. After reaction, the mixture was brought to room temperature and the solvent was evaporated under reduced pressure. The residue was dissolved in DCM (20 mL) and then washed with water (310 mL). The organic layer was evaporated under reduced pressure. The crude product was purified by flash chromatography (DCM/MeOH 95:5 v/v). The resulting product was washed with H.sub.2O affording the calix[4]arene of the formula (2) (269 mg, 0.38 mmol) as a white solid. Yield 64%.

[0216] .sup.1H NMR (400 MHz, CDCl.sub.3): .sub.H (ppm)=7.04 (s, 2H, ImH), 6.88-6.94 (m, 6H, ArH and ImH), 6.80 (bt, 2H, ArH), 6.33 (bt, 2H, ArH), 6.24 (s, 4H, ArH), 4.92 (s, 4H, OCH.sub.2Im), 4.34 (d, J=13 Hz, 4H, ArCH.sub.ax), 3.81 (t, J=8 Hz, 4H, OCH.sub.2Et), 3.50 (s, 6H, NCH.sub.3), 3.04 (d, J=13 Hz, 4H, ArCH.sub.eq), 1.71-1.77 (m, 4H, CH.sub.2), 0.75 (t, J=8 Hz, 6H, CH.sub.3); HRMS (ESI): calculated for C.sub.44H.sub.49N.sub.4O.sub.4.sup.+ [M+H.sup.+]: 696.3676, measured: 696.3674.

Example 1a: Synthesis and Characterization of the Calix[4]Arene CuBF.SUB.4 .Complex of the Formula (2a)

##STR00020##

[0217] The Cu.sup.+ complex of the calix[4]arene of the formula (2) was formed by mixing (2) with 1.5 equivalent of Cu.sup.+(MeCN).sub.4BF.sub.4 in a mixture of CHCl.sub.3 and MeCN, and the solvents were evaporated to obtain the salt of the formula (2a).

[0218] .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.3CN 1/4, 298 K): (ppm)=7.41 (s, ImH, 2H), 7.22 (bd, ArH, 4H), 7.11 (s, ImH, 2H), 7.09 (bt, ArH, 2H), 6.20 (bt, ArH, 2H), 6.03 (bd, ArH, 4H), 5.33 (s, OCH.sub.2Im, 4H), 3.78 (bd, ArCH.sub.2, 4H), 3.70 (t, .sup.3J=7.6 Hz, OCH.sub.2Et, 4H), 3.10 (bd, J=11.6 Hz, ArCH.sub.2, 4H), 2.85 (s, NCH.sub.3, 6H), 1.75-1.86 (m, CH.sub.2, 4H), 0.96 (t, J=7.6 Hz, CH.sub.3, 6H).

Example 2: Synthesis and Characterization of the Calix[4]Arene of the Formula (5)

##STR00021##

[0219] In a 100 mL RBF was added the calix[4]arene of the formula (A) (1.0 g, 2.21 mmol, 1 equivalent) and the reagent was dried under high vacuum for 1 hour, then purged under argon and dissolved in 25 mL of freshly distilled THF. The mixture was cooled to 0 C. and then NaH (washed 3 times with petroleum ether and then dried under high vacuum, 1.11 g, 77.3 mmol, 35 equivalents) was added. The reaction mixture was stirred at this temperature for 15 minutes then 2-(chloromethyl)-1-methyl-1H-imidazole hydrochloride (dried under high vacuum, 1.84 g, 11.05 mmol, 5 equivalents) was added in one portion. The reaction was refluxed overnight and monitored by .sup.1H NMR. Afterwards, the reaction was cooled to room temperature and the volatiles were removed under reduced pressure. 50 mL of dichloromethane were added followed by 50 mL of distilled water and the product was extracted with additional 250 mL of dichloromethane. The organic layers were combined and the volatiles were removed under reduced pressure. The resulting crude was then purified by column chromatography on silica (DCM/MeOH:98/2) to afford the calix[4]arene of the formula (5) as a white solid in 35% yield (500 mg, 0.77 mmol).

[0220] HRMS (ESI): calculated for C.sub.40H.sub.41N.sub.4O.sub.4.sup.+ [M+H.sup.+]: 641.3122, measured: 641.3129 [M+H].sup.+ and 663.2951 [M+Na].sup.+. The .sup.1H NMR spectrum of the calix[4]arene of the formula (5) in CDCl.sub.3 has broad features, thus 1 eq. of Cu.sup.+(MeCN).sub.4BF.sub.4 was added to characterise the Cu.sup.+ complex of the calix[4]arene of the formula (5). .sup.1H NMR (CDCl.sub.3/CD.sub.3CN 4/1, 400 MHz) .sub.H (ppm)=7.44 (s, 2H, ImH), (bd, J=7.2 Hz, 4H, ArH), 7.13 (s, 2H, ImH), 7.09 (bt, J=7.2 Hz, 2H, ArH), 6.26 (bt, J=6.8 Hz, 2H, ArH), 6.16 (bd, .sup.2J=7.2 Hz, 4H, ArH), 5.41 (s, OCH.sub.2, 4H), 3.8-3.57 (m, 10H, ArCH.sub.2 and NCH.sub.3), 3.12 (bd, J=12.8 Hz, 4H, ArCH.sub.2), 2.88 (s, 6H, CH.sub.3).

Example 3: Synthesis and Characterization of the Calix[4]Arene of the Formula (3)

##STR00022##

[0221] In a 100 mL RBF was added the calix[4]arene of the formula (C) (0.200 g, 0.40 mmol, 1 eq.) and the compound was dried under high vacuum for 1 hour, then purged under argon and dissolved in 25 mL of freshly distilled THF. The mixture was cooled to 0 C. and then NaH (washed 3 times with petroleum ether and then dried under high vacuum, 0.1427 g, 9.9 mmol, 25 equivalents) was added. The reaction mixture was stirred at this temperature for 15 minutes then 2-(chloromethyl)-1-methyl-1H-imidazole hydrochloride (dried under high vacuum, 0.3310 g, 1.98 mmol, 5 equivalents) was added in one portion. The reaction was refluxed overnight and monitored by .sup.1H NMR. Afterwards, the reaction was cooled to room temperature and the volatiles were removed under reduced pressure. 50 mL of dichloromethane were added followed by 50 mL of distilled water. The product was extracted with additional 250 mL of dichloromethane. The organic layers were collected and the volatiles were removed under reduced pressure. The resulting brownish oil was purified by column chromatography on silica (DCM to DCM/MeOH 98/2) to afford the calix[4]arene of the formula (3) as a white solid with 70% yield (0.1922 g, 0.30 mmol).

[0222] .sup.1H NMR (CDCl.sub.3, 300 MHz) .sub.H (ppm)=7.06-7.04 (m, ArH and ImH, 6H), 6.94-6.84 (m, ArH and ImH, 4H), 6.32 to 6.14 (m, ArH and CH.sub.2CH, 8H), 5.02-4.90 (m, =CH.sub.2, 4H), 4.88 (s, OCH.sub.2, 4H), 4.49 (d, J=6.7 Hz, CH.sub.2CH, 4H), 4.33 (d, J=13.4 Hz, ArCH.sub.ax, 4H), 3.49 (s, NCH.sub.3, 6H), 3.02 (d, J=13.5 Hz, ArCH.sub.eq, 4H); HRMS (ESI): calculated for C.sub.44H.sub.45N.sub.4O.sub.4.sup.+ [M+H.sup.+]: 693.3435, measured: 693.3448 [M+H].sup.+ and 715.3276 [M+Na].sup.+.

Example 4: Synthesis and Characterization of the Calix[4]Arene of the Formula (6)

##STR00023##

[0223] In a RBF were added the calix[4]arene of the formula (3) (0.400 g, 0.58 mmol), Pd(OAc).sub.2 (0.0194 g, 0.09 mmol, 0.15 equivalents) and PPh.sub.3 (0.0454 g, 0.173 mmol, 0.3 equivalents) with 25 mL of freshly distilled THF and 5 mL of distilled water. NEt.sub.2H (3.5 mL, 1.478 g, 20.2 mmol, 35 equivalents) was added and the reaction was refluxed under argon overnight. The conversion was monitored by .sup.1H NMR and then stopped upon completion of the reaction. The mixture was cooled to room temperature, filtered through celite followed by washing with dichloromethane 330 mL. The combined organic fractions were washed with 325 mL of distilled water and the volatiles were removed under reduced pressure. Afterwards, the resulting oil was purified by column chromatography on silica (DCM to DCM/MeOH 95/5) to afford the calix[4]arene of the formula (6) as a pinkish solid with 60% yield (0.213 g, 0.35 mmol).

[0224] .sup.1H NMR (CDCl.sub.3, 300 MHz) .sub.H (ppm)=7.49 (s, OH, 2H), 7.11-7.01 (m, ArH and ImH, 6H), 6.98 (s, ImH, 2H), 6.87 (d, J=7.4 Hz, ArH, 4H), 6.79 to 6.62 (m, ArH, 4H), 5.05 (s, OCH.sub.2, 4H), 4.22 (d, J=13.1 Hz, ArCH.sub.ax, 4H), 3.77 (s, NCH.sub.3, 4H), 3.37 (d, J=13.1 Hz, ArCH.sub.eq, 4H); HRMS (ESI): calculated for C.sub.38H.sub.37N.sub.4O.sub.4.sup.+ [M+H.sup.+]:613.2809, measured: 613.2833 [M+H].sup.+ and 635.2654 [M+Na].sup.+.

Example 5: Synthesis and Characterization of the Calix[4]Arene of the Formula (7)

##STR00024##

[0225] The calix[4]arene of the formula (2) (0.050 g, 0.072 mmol, 1 equivalent) was dissolved in CH.sub.2Cl.sub.2 (2.5 mL). A mixture of glacial CH.sub.3COOH/fuming HNO.sub.3 (1:1, 0.54 mL) was added at 0 C. and the reaction mixture was stirred for 25 hours at room temperature. The reaction mixture was concentrated under reduced pressure. The crude residue was dissolved in CH.sub.2Cl.sub.2 and washed with H.sub.2O until pH6. The solvent was then evaporated under reduced pressure to yield the calix[4]arene of the formula (7) (0.042 g, 74%) as a solid.

[0226] .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.3CN 1/4, 298 K): (ppm)=7.53 (s, ImH, 2H), 7.3-7.38 (m, ArH, 4H), 7.16-7.25 (m, ArH and ImH, 4H), 6.88 (s, ArH, 4H), 5.31 (s, OCH.sub.2Im, 4H), 3.91 (bd, J=14 Hz, ArCH.sub.2, 4H), 3.67 (t, .sup.3J=7.2 Hz, OCH.sub.2Et, 4H), 3.28 (d, J=14 Hz, ArCH.sub.2, 4H), 3.16 (s, NCH.sub.3, 6H), 1.61-1.72 (m, CH.sub.2, 4H), 0.90 (t, .sup.3J=7.2 Hz, CH.sub.3, 6H).

Example 6: Synthesis and Characterization of the Calix[4]Arene of the Formula (8)

##STR00025##

[0227] In a 50 mL RBF was added the calix[4]arene of the formula (B) (0.200 g, 0.39 mmol), dried under high vacuum and then purged under argon, with 25 mL of freshly distilled THF. The solution was cooled to 0 C. and then NaH (freshly washed with 215 mL of petroleum ether, dried under high vacuum and then purged under argon, 0.236 g, 9.8 mmol, 25 eq.) was added to the solution. The reaction mixture was stirred at 0 C. for 30 minutes and then 2-(chloromethyl)-1-methyl-1H-benzimidazole (0.213 g, 1.18 mmol, 3 eq.) dried and purged under argon, was added. The reaction mixture was heated to room temperature and then refluxed overnight under argon. Afterwards, the reaction was cooled to room temperature, quenched with slow addition of 30 mL of distilled water into the solution and then the product was extracted with 350 mL of dichloromethane. The organic layers were combined then the volatiles were removed under reduced pressure. The product was purified by column chromatography on silica (DCM to DCM/MeOH 98/2) and the volatiles were removed under reduced pressure. The product was dissolved in a minimum amount of chloroform and then precipitated by slow addition of 100 mL of pentane, to afford the calix[4]arene of the formula (8) as a purple solid with 59% yield (0.184 g, 0.23 mmol).

[0228] .sup.1H NMR (CDCl.sub.3, 300 MHz) .sub.H (ppm)=7.78 (d, J=6.7 Hz, BzImH, 2H), 7.35-7.21 (m, BzImH, 6H), 6.93 (d, J=7.3 Hz, ArH, 4H), 6.80 (t, J=7.4 Hz, ArH, 2H), 6.35 (t, J=7.3 Hz, ArH, 2H), 6.25 (d, J=7.4 Hz, ArH, 4H), 5.14 (s, OCH.sub.2, 4H), 4.41 (d, J=13.4 Hz, ArCH.sub.ax, 4H), 3.76 to 3.70 (m, NCH.sub.3+OCH.sub.2, 10H), 3.09 (d, J=13.4 Hz, ArCH.sub.eq, 4H), 1.59 (sext, J=8.3 Hz, CH.sub.2CH.sub.3, 4H), 0.44 (t, J=7.4 Hz, CH.sub.3, 6H); HRMS (ESI): calculated for C.sub.52H.sub.53N.sub.6O.sub.6.sup.+ [M+H.sup.+]: 797.4061 measured: 797.4070 [M+H].sup.+ and 819.3891 [M+Na].sup.+.

Example 7: Synthesis and Characterization of the Calix[4]Arene of the Formula (4)

##STR00026##

[0229] In a 100 mL RBF was added the calix[4]arene of the formula (D) (0.500 g, 0.84 mmol, 1 eq.), 2-(chloromethyl)-1-methyl-1H-imidazole hydrochloride (dried under high vacuum, 0.700 g, 4.19 mmol) and the mixture was dried under high vacuum for 1 hour, then purged under argon and dissolved in 25 mL of freshly distilled THF. The mixture was cooled to 0 C. and then NaH (washed 3 times with petroleum ether and then dried under high vacuum, 0.503 g, 20.9 mmol, 25 equivalents) was added. The reaction was refluxed overnight and monitored by .sup.1H NMR. Afterwards, the reaction was cooled to room temperature and the volatiles were removed under reduced pressure. 50 mL of dichloromethane were added followed by 50 mL of distilled water. The product was extracted with additional 250 mL of dichloromethane. The organic layers were collected and the volatiles were removed under reduced pressure. The resulting brownish oil was purified by column chromatography on silica (DCM to DCM/MeOH 90/10) to afford the calix[4]arene of the formula (4) as a white solid (0.104 g, 0.13 mmol) with 15% yield.

[0230] .sup.1H NMR (CDCl.sub.3, 300 MHz) .sub.H (ppm)=7.03 (bs, ImH, 2H), 6.93 to 6.74 (m, ImH and ArH, 6H), 6.36 (t, J=7.2 Hz, ArH, 2H), 6.26 (bd, J=7.6 Hz, ArH, 4H), 5.01 (s, OCH.sub.2, 4H), 4.65 (s, OCH.sub.2, 4H), 4.59 (d, J=13.9 Hz, ArCH.sub.ax, 4H), 4.10 (q, J=7.1 Hz, CH.sub.2CH.sub.3, 4H), 3.44 (s, NCH.sub.3, 6H), 3.05 (d, J=13.9 Hz, ArCH.sub.eq, 4H), 1.23 (t, J=7.2 Hz, CH.sub.2CH.sub.3, 6H); HRMS (ESI): calculated for C.sub.46H.sub.49N.sub.4O.sub.8.sup.+ [M+H.sup.+]: 785.3545, measured: 785.3542 [M+H].sup.+ and 807.3366 [M+H.sup.+Na].sup.+.

Example 8: Synthesis and Characterization of the Calix[4]Arene of the Formula (9)

##STR00027##

[0231] In a 10 mL round bottom flask, the calix[4]arene of the formula (B) (85.6 mg, 0.17 mmol, 1.0 equivalent) was stirred in anhydrous DMF (3 mL) with NaH (60% dispersion in mineral oil, 135 mg, 3.37 mmol, 22 equivalents) for 30 minutes under inert atmosphere. 2-(Chloromethyl)oxazole (89.5 mg, 0.67 mmol, 5.0 equivalents) was then added. The mixture was stirred at reflux for 24 hours under inert atmosphere. After reaction, the mixture was brought to room temperature. The solvent was evaporated under reduced pressure. The residue was dissolved in DCM and then washed with water. The organic layer was evaporated under reduced pressure. The crude product was purified by flash chromatography (DCM/MeOH 95:5 v/v) affording the calix[4]arene of the formula (9) (57 mg, 0.08 mmol) as an oil in 50% yield.

[0232] .sup.1H NMR (400 MHz, CDCl.sub.3): .sub.H (ppm)=7.65 (s, 2H, OxH), 7.11 (s, 2H, OxH), 6.91 (d, 8 Hz, 4H, ArH), 6.79-6.83 (m, 2H, ArH), 6.31-6.39 (m, 6H, ArH), 5.6 (s, 4H, OCH.sub.2Ox), 4.29 (d, J=14 Hz, 4H, ArCH.sub.2), 3.67 (t, J=8 Hz, 4H, OCH.sub.2Et), 3.14 (d, J=14 Hz, 4H, ArCH.sub.2), 1.76-1.85 (m, 4H, CH.sub.2), 0.95 (t, J=8 Hz, 6H, CH.sub.3).

Example 9: Synthesis and Characterization of the Calix[4]Arene of the Formula (10)

##STR00028##

[0233] In a 10 mL round bottom flask, the calix[4]arene of the formula (B) (105 mg, 0.21 mmol, 1.0 equivalent) was stirred in anhydrous DMF (3 mL) with NaH (60% dispersion in mineral oil, 165 mg, 4.11 mmol, 23 equiv.) for 30 minutes under inert atmosphere. 2-(Chloromethyl)thiazole (123 mg, 0.82 mmol, 5.6 equivalents) was then added. The mixture was stirred at reflux for 24 hours under inert atmosphere. After reaction, the mixture was brought to room temperature. The solvent was evaporated under reduced pressure. The residue was dissolved in DCM and then washed with water. The organic layer was evaporated under reduced pressure. The crude product was purified by flash chromatography (DCM/MeOH 95:5 v/v) affording the calix[4]arene of the formula (10) (84.0 mg, 0.12 mmol) as an oil in 58% yield.

[0234] .sup.1H NMR (400 MHz, CDCl.sub.3): .sub.H (ppm)=7.82 (d, J=3.2 Hz, 2H, thioazoleH), 7.39 (d, J=3.2 Hz, 2H, thioazoleH), 6.84 (d, J=8 Hz, 4H, ArH), 6.71-6.76 (m, 2H, ArH), 6.47-6.51 (m, 2H, ArH), 6.41 (d, J=8 Hz, 4H, ArH), 5.27 (s, 4H, OCH.sub.2thioazole), 4.43 (d, J=14 Hz, 4H, ArCH.sub.2), 3.89 (t, J=8 Hz, 4H, OCH.sub.2Et), 3.18 (d, J=14 Hz, 4H, ArCH.sub.2), 1.77-1.87 (m, 4H, CH.sub.2), 0.82 (t, J=8 Hz, 6H, CH.sub.3).

Example 10: Synthesis and Characterization of the Calix[4]Arene of the Formula (38)

##STR00029##

[0235] In a 10 mL round bottom flask, the calix[4]arene of the formula (B) (101 mg, 0.199 mmol, 1.0 equiv.) was solubilized in anhydrous THF (5 mL) under inert atmosphere. Then, NaH (60% dispersion in mineral oil, 151 mg, 6.29 mmol, 32 equiv.) and chloromethylpyridine hydrochloride (328 mg, 2.00 mmol, 10 equiv.) were added with stirring. The mixture was stirred at reflux overnight under inert atmosphere. After 16 h, the mixture was brought to room temperature and the solvent was evaporated under reduced pressure. Distilled water (5 mL) was added to the residue, the mixture was sonicated for 1 min and finally filtered. The resulting solid was purified by flash chromatography (DCM/acetone 90:10 v/v), affording the calix[4]arene of the formula (38) (72.8 mg, 0.105 mmol) as a white solid. Yield 53%.

[0236] .sup.1H NMR (300 MHz, CDCl.sub.3): .sub.H (ppm)=8.60 (dt, J=4.7 Hz, 1.2 Hz, 2H, PyrH), 7.73-7.70 (m, 4H, PyrH), 7.25-7.21 (m, 2H, PyrH), 6.98 (d, J=7.4 Hz, 4H, ArH), 6.83 (dd, J=7.9, 6.9 Hz, 2H, ArH), 6.36 (dd, J=8.5, 6.5 Hz, 2H, ArH), 6.26 (d, J=7.4 Hz, 4H, ArH), 4.94 (s, 4H, OCH.sub.2Pyr), 4.48 (d, J=13.4 Hz, 4H, ArCH.sub.ax), 3.90-3.85 (m, 4H, OCH.sub.2Et), 3.16 (d, J=13.5 Hz, 4H, ArCH.sub.eq), 1.82-1.65 (m, 4H, CH.sub.2), 0.67 (t, J=7.6 Hz, 6H, CH.sub.3).

1.SUP.ST .Comparative Example: The Calix[4]Arene of the Following Formula (X) is a Comparative Example of the Present Invention

##STR00030##

[0237] In contrast of the calix[4]arenes of the invention, in the compound of the formula (X) comprises, Y.sub.1, Y.sub.2, Y.sub.3, and Y.sub.4 are tert-butyl groups.

[0238] The Ag.sup.+ complex of the compound of the formula (X) was formed by mixing 600 L of 0.5 mM solutions of X in CDCl.sub.3 with 600 L of 0.5 mM AgNO.sub.3 in H.sub.2O for 60 minutes to obtain complex (Xa).

[0239] .sup.1H NMR (400 MHz, CDCl.sub.3, 298 K): (ppm)=7.57 (s, ImH, 2H), 7.10 (s, ArH, 4H), 6.92 (s, ImH, 2H), 6.28 (s, ArH, 4H), 4.94 (s, OCH.sub.2Im, 4H), 3.78 (bd, ArCH.sub.2, 4H), 3.68 (bt, OCH.sub.2Et, 4H), 2.99 (bd, ArCH.sub.2, 4H), 2.85 (s, NCH.sub.3, 6H), 1.69-1.75 (m, CH.sub.2, 4H), 1.33 (s, .sup.tBu, 18H), 0.90 (bt, CH.sub.3, 6H), 0.76 (s, .sup.tBu, 18H).

2.SUP.nd .Comparative Example: The Calix[4]Arene of the Following Formula (XI) is a Comparative Example of the Present Invention

##STR00031##

IIExperiments on c Log P, Binding of Cu.SUP.+., Transport and Deliverability, and Cytotoxicity of Calix[4]Arenes of the Present Invention and the Comparative Examples

IIADetermination of the c Log P of the Calix[4]Arenes of the Invention and the Comparative Examples

[0240] A key requirement for the usability of synthetic transporters (such as the above mentioned ionophores) is their ability to be delivered to the targeted cell. Highly lipophilic molecules which can strongly bind an ion are usually considered good candidates for synthetic transporters. But a high lipophilicity can also prevent the delivery of a compound into a membrane. Thus, a balance has to be achieved between the ability of a molecule to remain in the membrane and carry ions across and getting to this membrane in the first place. For drugs, Log P is the chosen value to assess lipophilicity, which is the logarithm of P, the partition coefficient between water and octanol. A Log P value can be either measured in water/octanol systems or calculated, in the latter case it is referred to as c Log P. Chemical drawing tools such as ChemDraw are able to provide a c Log P value.

[0241] In order to assess the lipophilicity of the examples of calix[4]arenes of the present invention and the comparative examples, their c Log P values were determined using the chemical properties function in ChemDraw 20.0.

[0242] The here below table 1 details these c Log P values for the examples of calix[4]arenes of the invention and the comparative examples (i.e. column c Log P).

IIBDetermination of the Binding of Cu.SUP.+

[0243] Binding abilities and complexation constants of the examples of calix[4]arenes of the present invention and the comparative examples with Cu.sup.+ were evaluated by .sup.1H NMR titrations at 298 K using copper(I)tetrakis(acetronitrile)tetrafluoroborate.

[0244] For example, for each separate calix[4]arene a stock solution of the tested calix[4]arene was prepared in a CD.sub.3CN/CDCl.sub.3, 4/1 mixture. Using this stock solution, a Cu(MeCN).sub.4BF.sub.4 solution was prepared and added by steps of 0.25 equivalent of copper(I) up until 1 equivalent and then by steps of 0.5 equivalent until a total of at least 2 equivalents. .sup.1H NMR spectra were recorded at a 400 MHz spectrometer at 298 K for the stock solutions of the calix[4]arenes and after each addition of Cu(MeCN).sub.4BF.sub.4. Additions were continued until the .sup.1H NMR signals showed no significant changes anymore.

[0245] Calix[4]arenes for which a new set of signals was observed that completely replaced the initial set of signals after the addition of 1 equivalent of Cu(MeCN).sub.4BF.sub.4 were considered to have an affinity constant (K.sub.a) equal or greater than 10.sup.5 M.sup.1.

[0246] Calix[4]arenes for which a gradual change of the chemical shifts of the .sup.1H NMR signals (>0.1 ppm for at least one of the signals) was observed upon addition of up to 4-5 equivalents of Cu(MeCN).sub.4BF.sub.4 were considered to show weak binding.

[0247] The here below table 1 details the binding abilities with Cu.sup.+ for the examples of calix[4]arenes of the invention and the comparative examples (i.e. column Cu.sup.+ binding).

[0248] More precisely, in this table 1: [0249] strong binding means an affinity constant (K.sub.a) equal or greater than 10.sup.5 M.sup.1, [0250] weak binding means a change of the chemical shifts of the .sup.1H NMR signals (>0.1 ppm for at least one of the signals) is observed upon addition of up to 4-5 equivalents of Cu(MeCN).sub.4BF.sub.4, [0251] not significant means that no significant changes of chemical shifts (<0.1 ppm) were observed upon addition of 4-5 equivalents of Cu(MeCN).sub.4BF.sub.4.

IIC1Determination of the Transport of Cu.SUP.+ by the Examples of Calix[4]Arenes

[0252] The ability of the examples of calix[4]arenes of the invention and the comparative examples to be delivered into a lipidic bilayer membrane of a liposome from a solution and their capacity to then act as a transporter of Cu.sup.+ was evaluated using bathocuproine disulphonate (hereafter abbreviated BCS) encapsulated in large unilamellar vesicles (LUVs, also referred to as liposomes) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol in a 7:3 ratio.

[0253] BCS is a fluorescent water soluble phenantroline derivative that can be used as fluorescent dye for the sensing of Cu.sup.+ through the formation of a non-fluorescent 2:1 complex.

[0254] Stock solution of lipids were prepared using deacidified CHCl.sub.3 and stored in a freezer. CHCl.sub.3 was deacidified by passing through activated basic alumina. The lipids used to prepare the liposomes were 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), (purity 99%) and cholesterol (purity of 95%). 50 mM sodium phosphate buffer were prepared with Millipore water at pH 7 (0.1) by mixing the solution of the mono- and the di-protonated phosphate salts. The solution of the dye (BCS) was prepared at 0.5 mM concentration in phosphate buffer.

[0255] The lipids solutions were mixed in different ratios in a 5 mL round bottom flask. The POPC/cholesterol molar ratio was 7:3 in all experiments. The total amount of lipids per batch varied depending on the amount of liposomes solution required for the experiment. After mixing the lipid solutions in a round bottom flask, the chloroform was evaporated under a gentle air flow and the resulting lipid film was dried at high vacuum for at least an hour. 500 L of the BCS solution in phosphate buffer and a magnetic stir bar were added to the flask. The flask was then sonicated for 30 seconds and placed under magnetic agitation for at least an hour. The solution was frozen with liquid nitrogen and heated back to room temperature with warm water ten times to form large unilamellar vesicles (LUVs). The liposome solution was extruded 29 times, using an extrusion kit equipped with a membrane with 200 nm pores. Subsequently, the solution was eluted with the buffer solution used for the experiment through a size exclusion column (Sephadex G-25) to remove the external BCS and then diluted with phosphate buffer to obtain a concentration of 0.4 mM in lipids.

[0256] Solutions of the calix[4]arenes were prepared at a concentration of 0.24 mM in DMSO and 5 L of the solution was added 2 minutes before each transport measurement. This results in a ratio of calix[4]arene to lipid of 1:1000, corresponding to a 0.4 M concentration of the tested calix[4]arenes.

[0257] Alternatively, calix[4]arenes were pre-incorporated by combining the calix[4]arene (dissolved in chloroform) with the lipids prior to the evaporation of the chloroform to obtain a lipid film. The concentrations and volumes were chosen to obtain a calix[4]arene to lipid of 1:1000.

[0258] Fluorescence measurements were performed with a Horiba FluoroMax 4 spectrometer using a 4-faced quartz cuvette (10 mm10 mm). For the Cu.sup.+ transport assay, the excitation was set at 278 nm and emission was recorded at 393 nm. These values correspond to the excitation and emission maxima of BCS encapsulated inside LUVs.

[0259] The Cu.sup.+ solutions added to the liposomes were obtained by dissolving Cu(MeCN).sub.4BF.sub.4 in MeCN at 15.25 mM concentration. The copper solution was then diluted by a factor 5 in a phosphate buffer during the first 30 seconds of the experiment to reach a copper concentration of 3.05 mM. 50 L of the diluted copper solution was added to 3 mL of liposomes, resulting in a copper gradient of 50 M. The fluorescence level was monitored for 600 seconds after the addition of the copper solution.

[0260] For each run the initial plateau and the initial vertical drop (due to the binding of the exterior dye with Cu.sup.+ and the dilution, 1-4 seconds after the addition) were removed. Then the signal was normalised by dividing the fluorescence values (F) by the value at t=0 second (F.sub.0). The average of 3 normalised runs (F/F.sub.0) was then calculated and plotted over time and final F/F.sub.0 levels (after 600 s) were determined.

[0261] The here below table 1 details the abilities of Cu.sup.+ transport for the examples of calix[4]arenes of the invention and the comparative examples (i.e. column Cu.sup.+ transport).

[0262] More precisely, in this table 1: [0263] Yes means that a significant decrease of the fluorescence of encapsulated BCS was observed upon addition of Cu.sup.+ to the liposomes (F/F.sub.0<0.65 after 600 s), [0264] No means that no significant decrease of the fluorescence of encapsulated BCS was observed upon addition of Cu.sup.+ to the liposomes (F/F.sub.0 >0.65 after 600 s) when the calix[4]arene was either delivered from a solution in DMSO to liposomes or pre-incorporated into the liposomes.

IIC2Determination of the Transport of Ag.SUP.+ by the Calix[4]Arene of the Formula (2)

[0265] Liposomes with BCS encapsulated were prepared as described above in the part entitled IIC1Determination of the transport of Cu.sup.+by the examples of calix[4]arenes and measurements were performed as described in this part.

[0266] A Ag.sup.+ solution was prepared by dissolving AgNO.sub.3 in a phosphate buffer at 3.05 mM concentration. After the first 30 seconds of the experiment, 50 L of the Ag.sup.+ solution was added to 3 mL of liposomes, resulting in a silver gradient of 50 M. The fluorescence level was monitored for 600 seconds after the addition of the copper solution.

[0267] When a solution of DMSO with the calix[4]arene of the formula (2) was added to the liposomes (5 L, to obtain a 1:1000 transport to lipid ratio) the decrease in fluorescence intensity of BCS was much faster (F/F.sub.0=0.55 after 100 s) compared to when DMSO without calix[4]arene of the formula (2) (5 L) was added (F/F.sub.0=0.85 after 100 s).

IIDDetermination of the Deliverability

[0268] The ability of the examples of calix[4]arenes of the present invention and the comparative examples to be inserted into the membrane of liposomes after their formation was tested.

[0269] For that, the procedure in which calix[4]arenes were added to the liposomes from a solution in DMSO, as detailed in part IIC1Determination of the transport of Cu.sup.+ by the examples of calix[4]arenes, was followed and transport curves were recorded.

[0270] Calix[4]arenes showing poor or no activity via this procedure were tested via pre-incorporation.

[0271] More precisely, the calix[4]arene (dissolved in chloroform) was combined with the lipids prior to the evaporation of the chloroform to obtain a lipid film. The concentrations and volumes were chosen to obtain a calix[4]arene to lipid of 1:1000.

[0272] The here below table 1 details the deliverability of the tested calix[4]arenes and the comparative examples to liposomes from a solution in DMSO (i.e. column deliverability).

[0273] More precisely, in this table 1: [0274] Yes means that a significant decrease of the fluorescence of encapsulated BCS was observed upon addition of Cu.sup.+ to the liposomes when the calix[4]arene was added to the liposomes from a solution in DMSO (F/F.sub.0<0.65 after 600 s), [0275] Medium means that the final level reached was much lower upon pre-incorporation (F/F.sub.0<0.4 after 600 s) than when adding the calix[4]arene to the liposomes from a solution in DMSO (F/F.sub.0=0.4-0.65 after 600 s), [0276] No means that no significant decrease of the fluorescence of encapsulated BCS was observed (F/F.sub.0>0.65 after 600 s) when adding the calix[4]arene to the liposomes from a solution in DMSO, but that upon pre-incorporation good transport was observed (F/F.sub.0<0.4 after 600 s), [0277] means that no transport was observed either by pre-incorporation or when adding the calix[4]arene to the liposomes from a solution in DMSO, indicating that the calix[4]arene is not active as Cu.sup.+ transport in the described method and that this method can thus not be used to verify the deliverability of the calix[4]arene.

TABLE-US-00001 Table 1 detailing the cLogP values and abilities of binding or transporting of some calix[4]arenes of the invention and the comparative examples Formula of the calix[4]arene/ Cu.sup.+ Deliver- example cLogP Cu.sup.+ binding transport ability (2)/example 1 9.2 Strong binding Yes Yes (5)/example 2 7.1 Strong binding Yes Yes (3)/example 3 8.7 Strong binding Yes Yes (6)/example 4 5.9 Strong binding Yes Yes (7)/example 5 8.7 Strong binding Yes Yes (8)/example 6 12.4 Strong binding Yes Yes (4)/example 7 7.4 Strong binding Yes Yes (9)/example 8 9.4 Weak binding Yes Medium (10)/example 9 10.4 Weak binding Yes Medium (38)/example 10 11.1 Strong binding Yes Yes (X)/1.sup.st comparative 16.5 Strong binding Yes No example (XI)/2.sup.nd comparative 12.7 Not significant No example

[0278] Furthermore, as alternative to the addition of the calix[4]arene of the formula (2) and the 1.sup.st comparative example of the formula (X) from a solution in DMSO, the delivery from liposomes was tested.

[0279] For the delivery studies with liposomes, two batches of liposomes were prepared. Liposomes with BCS encapsulated were prepared as above described and liposomes without any BCS but with either the calix[4]arene of the formula (2) or the 1.sup.st comparative example of the formula (X) pre-incorporated at a 1:500 transporter to lipid ratio were prepared as described above. 1.5 mL of each batch of liposomes were added to a cuvette and left to stir for 5 minutes in the spectrometer before starting the experiments, using the settings as described above.

[0280] When liposomes with BCS were mixed with liposomes containing the calix[4]arene of the formula (2), a good transport activity was observed, indicating that the calix[4]arene of the formula (2) can be delivered to liposomal membranes from other liposomes. In contrast, when liposomes with BCS were mixed with liposomes containing the 1.sup.st comparative example of the formula (X), no transport activity was observed, indicating that this lipophilic comparative example of the formula (X) cannot be delivered from liposomes either.

[0281] In view of the table 1, the calix[4]arenes of the formulas (2), (3) and (4) exhibit good abilities for binding and transport of Cu.sup.+ and to be delivered from a DMSO solution.

[0282] Moreover, their lipophilicities are not too high (i.e. between 7.4 and 9.2). These calix[4]arenes are preferred calix[4]arenes of the invention.

[0283] In contrast, although the comparative example of formula (X) binds Cu.sup.+ strongly and is able to transport, it is not an interesting calix[4]arene for anticancer therapy because of its high lipophilicity (c Log P=16.5) and the absence of deliverability. Indeed, the comparative example of the formula (X) is neither deliverable from a solution of DMSO nor from liposomes.

IIIExperiments of Cell Proliferation

IIIAInhibition of A549 Cell Proliferation

[0284] The inhibition of a A549 human lung cancer cell line proliferation by the calix[4]arene of the formula (2) was determined by exposure of said A549 human lung cancer cell line to a concentration of 2 M of the calix[4]arene of the formula (2) until 72 hours. The absolute number of cells were counted in the treated and untreated conditions before treatment (N.sub.0) and after 72 hours (N.sub.t).

[0285] The FIG. 1 is a graph showing: [0286] the profile of the proliferation index N.sub.t/N.sub.0 of the A549 cells after exposure with the calix[4]arene of the formula (2) with respect to the time (i.e. the curve entitled calix[4]arene (2)), [0287] the profile of the proliferation index N.sub.t/N.sub.0 of the A549 cells without any exposure with the calix[4]arene of the formula (2) with respect to the time (i.e. the curve entitled untreated).

[0288] N.sub.t is number of cells at time t and No is number of cells before treatment.

[0289] In view of the curves of the graph of the FIG. 1, the calix[4]arene of the formula (2) clearly inhibits the proliferation of the A549 human lung cancer cells.

IIIBInhibition of PC9 Cell Proliferation

[0290] The inhibition of a PC9 human lung cancer cell line proliferation by the calix[4]arene of the formulation (2) was determined by exposure of said PC9 human lung cancer cell line to a concentration of 3 M of the compound of the formula (2) until 72 hours. The absolute number of cells were counted in the treated and untreated conditions before treatment (N.sub.0) and after 72 hours (N.sub.t).

[0291] The FIG. 2 is a graph showing: [0292] the profile of the proliferation index N.sub.t/N.sub.0 of the PC9 cells after exposure with the calix[4]arene of the formula (2) with respect to the time (i.e. the curve entitled calix[4]arene (2)), [0293] the profile of the proliferation index N.sub.t/N.sub.0 of the PC9 cells without any exposure with the calix[4]arene of the formula (2) with respect to the time (i.e. the curve entitled untreated).

[0294] In view of the curves of the graph of the FIG. 2, the calix[4]arene of the formula (2) clearly inhibits the proliferation of the PC9 human lung cancer cells.

IIICInhibition of H322 Cell Proliferation

[0295] The inhibition of a H322 lung carcinoma cell line proliferation by the calix[4]arene of the formula (2) was determined by exposure of said H322 lung carcinoma cell line to a concentration of 3 M of the calix[4]arene of the formula (2) until 72 hours. The absolute number of cells were counted in the treated and untreated conditions before treatment (N.sub.0) and after 72 hours (N.sub.t).

[0296] The FIG. 3 is a graph showing: [0297] the profile of the proliferation index N.sub.t/N.sub.0 of the H322 cells after exposure with the calix[4]arene of the formula (2) with respect to the time (i.e. the curve entitled calix[4]arene (2)), [0298] the profile of the proliferation index N.sub.t/N.sub.0 of the H322 cells without any exposure with the calix[4]arene of the formula (2) with respect to the time (i.e. the curve entitled untreated).

[0299] In view of the curves of the graph of the FIG. 3, the calix[4]arene of the formula (2) clearly inhibits the proliferation of the H322 lung carcinoma cells.

IVExperiments of Cytoxicity on the Calix[4]Arene of the Formula (2), Determination of IC.SUB.50

IVAExperiments on Malignant H322, A549 and PC9 Cells

[0300] H322 lung carcinoma cells were seeded in 96-well plates together with the calix[4]arene of the formula (2). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the calix[4]arene of the formula (2). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0301] A549 human lung cancer cells were seeded in 96-well plates together with the calix[4]arene of the formula (2). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the calix[4]arene of the formula (2). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0302] PC9 human lung cancer cells were seeded in 96-well plates together with the calix[4]arene of the formula (2). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the calix[4]arene of the formula (2). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0303] The here below table 2 details the mean IC.sub.50s using MTS cytotoxicity assay on H322, A549 and PC9 cells for the calix[4]arene of the formula (2) and the confidence interval (statistical significance 95%) of the IC.sub.50 obtained with GraphPad Prism software.

TABLE-US-00002 Table 2 of the IC.sub.50s using MTS assay on H322, A549 and PC9 cells for the calix[4]arene of the formula (2) Confidence Interval Mean IC.sub.50 (95%) for IC.sub.50 (M) Cell lines (M) values H322 1.84 1.69 to 2.042 A549 1.56 1.45 to 1.69 PC9 2.06 1.79 to 2.38

[0304] In view of the table 2, the calix[4]arene of the formula (2) exhibits a high antitumor activity on H322, A549 and PC9 cells in the micromolar range, which are values in the same range as for other anticancer chemotherapeutic drugs.

IVBExperiments on Non-Malignant MRC5-SVII, MSC and HaCaT Cells

[0305] Non-malignant MRC5-SVII cells were seeded in 96-well plates together with the calix[4]arene of the formula (2). Cells were grown in Minimal Essential MediumNon Essential Amino Acids (MEM NEAA) culture medium from Gibco, supplemented with sodium pyruvate (1 M), and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the calix[4]arene of the formula (2). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0306] Non-malignant MSC cells were seeded in 96-well plates together with the calix[4]arene of the formula (2). Cells were grown in Mesenchymal Stem Cell Growth Medium (C-28009 from Promega), and treated with a broad range of concentrations of the calix[4]arene of the formula (2). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0307] Non-malignant HaCaT cells were seeded in 96-well plates together with the calix[4]arene of the formula (2). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 20%) and treated with a broad range of concentrations of the calix[4]arene of the formula (2). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0308] The here below table 3 details the IC.sub.50s using MTS cytotoxicity assay on MRC5-SVII, MSC and HaCaT cells for the calix[4]arene of the formula (2) and the confidence interval (statistical significance 95%) of the IC.sub.50 obtained with GraphPad Prism software.

TABLE-US-00003 Table 3 of the IC.sub.50s using MTS assay on MRC5-SVII, MSC and HaCat cells for the calix[4]arene of the formula (2) Confidence Mean IC.sub.50 Interval (95%) for Cell lines (M) IC.sub.50 (M) values MRC5-SVII 1.03 0.99 to 1.06 MSC 1.01 0.93 to 1.09 HaCaT 1.05 0.93 to 1.18

[0309] In view of the table 3, the calix[4]arene of the formula (2) exhibits a toxicity on non-malignant MRC5-SVII, MSC and HaCat cells, in the same range as for cancer cells (see Table 2).

IVCExperiments on 60 Malignant Cell Lines

[0310] In these experiments, the IC.sub.50s values were determined for the calix[4]arene of the formula (2) and for a reference compound which was bortezomib (i.e. an anticancer compound which inhibits proteasome).

[0311] The cytotoxicity was measured by means of the IC.sub.50 value, using the PROLiFiler-Cell Panel Screening (Reaction Biology).

[0312] Cells were cultured in different media. The here below table 4 details the corresponding medium for each tested malignant cell.

[0313] For the assays, cells were seeded in white cell culture-treated flat and clear bottom 384-well multiwall plates and incubated at 37 C. overnight before the calix[4]arene of the formula (2) or the reference compound were added. After incubation for 72 hours at 37 C. at 5% or 10% CO.sub.2 depending on the medium, cell plates were equilibrated to room temperature (i.e. 20 C.) for one hour, CellTiterGlo reagent from the firm Promega was added and luminescence was measured approximately an hour later using a luminometer.

[0314] Raw data were converted into percent cell viability relative to the high and low control, which were set to 100% and 0%, respectively. IC.sub.50 calculation was performed using GraphPad Prism software with a variable slope sigmoidal response fitting model using 0% viability as bottom constraint and 100% viability as top constraint.

[0315] The here below table 4 details the medium of culture for each tested malignant cell line.

TABLE-US-00004 Table 4 detailing the medium of culture of the calix[4]arene of the formula (2) and the reference compound for each malignant cell line No. Entity Cell line medium 1 Brain A172 DMEM + 10% FCS 2 Skin A2058 DMEM + 10% FCS 3 Skin A375 DMEM + 10% FCS 4 Kidney A498 DMEM + 10% FCS 5 Lung A549 DMEM + 10% FCS 6 Pancreas AsPC-1 DMEM + 10% FCS 7 Lung BEN DMEM + 10% FCS 8 Breast BT-20 RPMI-1640 + 10% FCS 9 Kidney Caki-1 DMEM + 10% FCS 10 Kidney Caki-2 DMEM + 10% FCS 11 Colon Colo 205 DMEM + 10% FCS 12 Lung COR-L279 RPMI-1640 + 10% FCS 13 Ovary COV434 DMEM + 10% FCS 14 Colon DLD-1 DMEM + 10% FCS 15 Prostate DU-145 DMEM + 10% FCS 16 Lung DV90 RPMI-1640 + 10% FCS 17 Breast EFM-192A RPMI-1640 + 10% FCS 18 Ovary EFO-27 RPMI-1640 + 10% FCS 19 Lung EPLC-272H RPMI-1640 + 10% FCS 20 Lung H1299 DMEM + 10% FCS 21 Brain H4 DMEM + 10% FCS 22 Lung H460 DMEM + 10% FCS 23 Breast HCC 1569 RPMI-1640 + 10% FCS 24 Breast HCC38 RPMI-1640 + 10% FCS 25 Lung HCC827 RPMI-1640 + 10% FCS 26 Colon HCT116 DMEM + 10% FCS 27 Colon HCT-15 RPMI-1640 + 10% FCS 28 Ovary HeLa DMEM + 10% FCS 29 Liver Hep3B2.1-7 DMEM + 10% FCS 30 Blood HL-60 RPMI-1640 + 10% FCS 31 Fibrosarcoma HT-1080 DMEM + 10% FCS 32 Colon HT-29 DMEM + 10% FCS 33 Liver HuH7 DMEM + 10% FCS 34 Breast JIMT-1 DMEM + 10% FCS 35 Blood K562 RPMI-1640 + 10% FCS 36 Blood KARPAS 299 RPMI-1640 + 10% FCS 37 Prostate LnCap RPMI-1640 + 10% FCS 38 Lung LOU-NH91 RPMI-1640 + 10% FCS 39 Skin MDA MB 435 RPMI-1640 + 10% FCS 40 Breast MDA-MB-468 DMEM + 10% FCS 41 Stomach MKN-1 RPMI-1640 + 10% FCS 42 Blood MOLM-13 RPMI-1640 + 10% FCS 43 Blood MV4-11 RPMI-1640 + 10% FCS 44 Lung NCI-H1563 RPMI-1640 + 10% FCS 45 Lung NCI-H1573 RPMI-1640 + 10% FCS 46 Lung NCI-H1838 RPMI-1640 + 10% FCS 47 Ovary SiHa DMEM + 10% FCS 48 Ovary SK-OV3 DMEM + 10% FCS 49 Stomach SNU-1 RPMI-1640 + 10% FCS 50 Ovary SNU840 RPMI-1640 + 10% FCS 51 Brain SW-1783 DMEM + 10% FCS 52 Colon SW480 DMEM + 10% FCS 53 Colon SW620 DMEM + 10% FCS 54 Colon SW948 DMEM + 10% FCS 55 Colon T84 DMEM + 10% FCS 56 Brain T98G DMEM + 10% FCS 57 Brain U118MG DMEM + 10% FCS 58 Brain U251MG DMEM + 10% FCS 59 Bone U2OS DMEM + 10% FCS 60 Brain U87MG DMEM + 10% FCS

[0316] In this table 4: [0317] DMEM is the abbreviation for Dulbecco's Modified Eagle's Medium, [0318] FCS is the abbreviation for fetal calf serum, [0319] RPMI-1640 is the abbreviation for Roswell Park Memorial Institute.

[0320] The here below table 5 details for each tested malignant cell line the IC.sub.50 values for the calix[4]arene of the formula (2) and the reference compound bortezomib.

TABLE-US-00005 Table 5 detailing the IC50 values for the calix[4]arene of the formula (2) and the reference compound for each tested malignant cell line IC50 [M] calix[4]arene IC50 [M] No. Entity Cell line (2) bortezomib 1 Brain A172 1.21E06 4.77E09 2 Skin A2058 9.38E07 9.24E09 3 Skin A375 1.17E06 5.44E09 4 Kidney A498 1.72E06 4.53E09 5 Lung A549 1.04E06 1.37E08 6 Pancreas AsPC-1 8.34E07 1.25E08 7 Lung BEN 3.68E06 5.31E08 8 Breast BT-20 1.27E06 2.95E08 9 Kidney Caki-1 3.24E06 5.37E09 10 Kidney Caki-2 2.61E06 1.28E08 11 Colon Colo 205 1.81E06 6.79E09 12 Lung COR-L279 1.51E06 4.94E09 13 Ovary COV434 7.32E07 9.24E09 14 Colon DLD-1 1.08E06 6.83E09 15 Prostate DU-145 1.93E06 6.80E09 16 Lung DV90 2.16E06 7.37E09 17 Breast EFM-192A 5.45E06 4.75E09 18 Ovary EFO-27 1.52E06 1.32E08 19 Lung EPLC-272H 1.47E06 7.17E09 20 Lung H1299 1.30E06 8.42E09 21 Brain H4 2.90E06 8.36E09 22 Lung H460 7.27E07 2.88E08 23 Breast HCC 1569 2.96E06 5.15E09 24 Breast HCC38 2.53E06 5.41E09 25 Lung HCC827 1.72E06 8.40E09 26 Colon HCT116 9.50E07 6.78E09 27 Colon HCT-15 2.02E06 9.25E09 28 Ovary HeLa 1.70E06 1.87E08 29 Liver Hep3B2.1-7 7.81E07 7.98E09 30 Blood HL-60 1.45E06 3.73E09 31 Fibrosarcoma HT-1080 1.90E06 1.38E08 32 Colon HT-29 1.20E06 5.99E09 33 Liver HuH7 5.43E07 4.30E09 34 Breast JIMT-1 2.70E06 8.57E09 35 Blood K562 1.89E06 1.46E08 36 Blood KARPAS 299 1.48E06 3.96E09 37 Prostate LnCap 1.19E06 1.41E08 38 Lung LOU-NH91 6.52E07 7.04E09 39 Skin MDA MB 435 2.77E06 4.30E09 40 Breast MDA-MB-468 7.41E07 4.20E09 41 Stomach MKN-1 2.16E06 4.32E09 42 Blood MOLM-13 1.71E06 3.91E09 43 Blood MV4-11 6.71E07 3.13E09 44 Lung NCI-H1563 3.44E06 1.95E08 45 Lung NCI-H1573 2.21E06 1.67E08 46 Lung NCI-H1838 3.48E06 3.41E08 47 Ovary SiHa 1.25E06 7.41E09 48 Ovary SK-OV3 1.62E06 2.40E08 49 Stomach SNU-1 2.55E06 2.88E08 50 Ovary SNU840 1.15E06 5.89E09 51 Brain SW-1783 3.38E06 8.60E09 52 Colon SW480 1.52E06 5.29E09 53 Colon SW620 1.76E06 4.74E09 54 Colon SW948 2.15E06 8.88E09 55 Colon T84 2.44E06 1.21E08 56 Brain T98G 3.11E06 1.14E08 57 Brain U118MG 2.03E06 4.14E09 58 Brain U251MG 3.53E06 9.16E09 59 Bone U2OS 1.06E06 5.83E09 60 Brain U87MG 2.46E06 8.02E09

[0321] In view of the table 5, the calix[4]arene of the formula (2) exhibits for all the tested malignant cells an antitumor activity, in the same micromolar range as for the lung cancer cell lines (see table 2).

VExperiments of Cytoxicity on the 2.SUP.ND .Comparative Example of the Formula (XI), Determination OF IC.SUB.50

VAExperiments on Malignant H322, A549 and PC9 Cells

[0322] H322 lung carcinoma cells were seeded in 96-well plates together with the compound of the 2.sup.nd comparative example of the formula (XI). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the 2.sup.nd comparative example of the formula (XI). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0323] A549 human lung cancer cells were seeded in 96-well plates together with the compound of the 2.sup.nd comparative example of the formula (XI). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the 2.sup.nd comparative example of the formula (XI). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0324] PC9 human lung cancer cells were seeded in 96-well plates together with the 2.sup.nd comparative example of the formula (XI). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the compound of the 2.sup.nd comparative example of the formula (XI). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0325] The here below table 6 details the IC.sub.50s using MTS cytotoxicity assay on H322, A549 and PC9 cells for the 2.sup.nd comparative example of the formula (XI).

TABLE-US-00006 Table 6 of the IC.sub.50s using MTS assay on H322, A549 and PC9 cells for the 2.sup.nd comparative example of the formula (XI) Cell lines IC.sub.50 (M) H322 >50 M A549 >50 M PC9 >50 M

[0326] The 2.sup.nd comparative example of the formula XI shows no signs of significant cytotoxicity on the lung cancer cell lines.

VBExperiments on Non-Malignant MRC5-SVII Cells

[0327] Non-malignant MRC5-SVII cells were seeded in 96-well plates together with the 2.sup.nd comparative example of the formula (XI). Cells were grown in Minimal Essential MediumNon Essential Amino Acids (MEM NEAA) culture medium from Gibco, supplemented with sodium pyruvate (1 M), and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the 2.sup.nd comparative example of the formula (XI). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0328] The here below table 7 details the IC.sub.50 using MTS cytotoxicity assay on MRC5-SVII cells for the 2.sup.nd comparative example of the formula (XI):

TABLE-US-00007 Table 7 of the IC.sub.50s using MTS assay on MRC5-SVII cells for the 2nd comparative example of the formula (XI) Cell lines IC.sub.50 (M) MRC5-SVII >20 M

[0329] The 2.sup.nd comparative example of the formula (XI) shows no signs of significant cytotoxicity on MRC5-SVII cell lines.

VIExperiments of Cytoxicity on the 1.SUP.ST .Comparative Example of the Formula (X) Based on IC.SUB.50

Experiments on Malignant H322, A549 and PC9 Cells

[0330] H322 lung carcinoma cells were seeded in 96-well plates together with the 1.sup.st comparative example of the formula (X). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the compound of the formula (X). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0331] A549 human lung cancer cells were seeded in 96-well plates together with 1.sup.st comparative example of the formula (X). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the 1.sup.st comparative example of the formula (X). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0332] PC9 human lung cancer cells were seeded in 96-well plates together with the 1.sup.st comparative example of the formula (X). Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the 1.sup.st comparative example of the formula (X). After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0333] The here below table 8 details the IC.sub.50s using MTS cytotoxicity assay on H322, A549 and PC9 cells for the 1.sup.st comparative example of the formula (X):

TABLE-US-00008 Table 8 of the IC.sub.50s using MTS assay on H322, A549 and PC9 cells for the 1st comparative example of the formula (X) Cell lines IC.sub.50 (M) H322 >50 M A549 >50 M PC9 >50 M

[0334] The 1.sup.st comparative example of the formula (X) shows no signs of significant cytotoxicity on the lung cancer cell lines.

VIIExperiments of Cytoxicity on Other Calix[4]Arenes of the Invention Based on IC.SUB.50

Experiments on Malignant H322 Cells

[0335] H322 lung carcinoma cells were seeded in 96-well plates together with the tested calix[4]arenes. Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) and treated with a broad range of concentrations of the tested calix[4]arenes. After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0336] The here below table 9 details the IC.sub.50s using MTS cytotoxicity assay on H322 cells for the 6 below detailed calix[4]arenes of the invention:

TABLE-US-00009 Table 9 of the IC.sub.50s using MTS assay on H322 cells for 6 calix[4]arenes of the invention Compound of the formula/ Example IC.sub.50 (M) (2a)/Example 1a 5 (5)/Example 2 >10 (3)/Example 3 5 (6)/Example 4 >10 (7)/Example 5 5 (4)/Example 7 6

[0337] Compounds with IC50>10 M (Table 9) are considered inactive on H322 cancer cell line.

VIIIExperiments of Cytoxicity on Calix[4]Arenes of the Invention Based on DL.SUB.50

[0338] Cytotoxicity was evaluated using the MTT assay. 1.10.sup.4 cells per well were seeded in a flat-bottom 96-well plate and were incubated for 48 hours at 37 C. with 5% CO.sub.2 before addition of the different calix[4]arenes at concentrations up to 200 M. In all experiments, each condition was performed in triplicate in the plate. Besides, each experiment was done three times independently. After 24 hours of incubation in presence of the compounds, medium was discarded, cells were rinsed with PBS and 100 L of medium containing 0.5 mg/mL of MTT was added to each well. After 30 minutes of incubation at 37 C., MTT solution was discarded and formazan crystals formed were dissolved in 150 L of 4 mM HCl, 0.1% NP40 in isopropanol. The absorbance in the wells were read in a microplate reader (Tecan microplate reader-550) at 570 nm.

[0339] The here below table 10 details the DL.sub.50 on Hepatocellular carcinoma (HepG2/C.sub.3A) cells for: [0340] 9 calix[4]arenes of the invention, [0341] the comparative examples of the formulas (X) and (XI), [0342] disulfiram, [0343] the complex disulfiram/Cu.sup.2+ (1:1), [0344] clioquinol, [0345] the complex of clioquinol/Cu.sup.2+ (1:1).

[0346] As explained above, disulfiram and clioquinol are well-known ionophores for anticancer therapy.

TABLE-US-00010 Table 10 detailing the DL.sub.50 on Hepatocellular carcinoma (HepG2/C3A) cells for calix[4]arenes of the invention, the comparative examples and other ionophores Tested compound Formula/example DL.sub.50 (M) (2)/example 1 3.1 (2a)/example 1a 4.0 Disulfiram 98.0 Complex disulfiram/Cu (1:1) 4.5 Clioquinol 67.0 Complex clioquinol/Cu (1:1) 24.0 (6)/example 4 20.7 (5)/example 2 23.0 (3)/example 3 4.5 (7)/example 5 10.9 (8)/example 6 127.7 (4)/example 7 3.1 (9)/example 8 70.0 (X)/1.sup.st comparative example 148.7 (XI)/2.sup.nd comparative example 184.0

[0347] In view of the table 10: [0348] The DL.sub.50 values of the complexes clioquinol/Cu (1:1) and disulfiram/Cu (1:1) are lower than the DL.sub.50 values of clioquinol and disulfiram. These two compounds are more toxic when they are in the form of a complex with Cu.sup.2+. [0349] In contrast, the calix[4]arenes of the present invention have the same toxicity alone or in complex with Cu.sup.+. DL.sub.50 value of 3.1 versus 4.0 for the example 1. [0350] The calix[4]arenes of the formulas (2), (3) and (4) have the lowest DL.sub.50 values. They are the most toxic calix[4]arenes.

IXExperiments of Cytoxicity on the Calix[4]Arene of the Formula (2) in Combination with CuCl.SUB.2

IXAExperiments on Malignant H322 Cells

[0351] H322 lung carcinoma cells were seeded in 96-well plates together with the calix[4]arene of the formula (2) and CuCl.sub.2. Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) treated with a broad range of concentrations of the calix[4]arene of the formula (2) or/and CuCl.sub.2 from Merck. After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0352] The here below table 11 details the percentage of cell viability depending on the concentration of the calix[4]arene of the formula (2) and the concentration of Cu.sup.2+.

TABLE-US-00011 TABLE 11 detailing the percentage of cell viability Concentration of the calix[4]arene of the formula (2) (M) 0 0.1 0.5 1 1.5 Concentration 0 100.0 96.5 94.1 87.0 62.6 of Cu.sup.2+ (M) 1 87.2 86.5 85.0 62.2 39.4 5 83.3 78.3 45.1 12.4 0.5 10 79.5 67.6 26.2 2.4 0 25 72.9 58.5 7.7 0 0 50 60.6 42.1 0 0 0

[0353] The table 11 details the effect of combining the calix[4]arene of the formula (2) with CuCl.sub.2 using the MTS cytotoxicity assay in H322 cell line. More precisely, in view of the detailed percentages of cell viability in this table 11, it shows that the combination of the calix[4]arene of the formula (2) with Cu (in the form of CuCl.sub.2) increases the efficacy of said calix[4]arene of the formula (2) in H322 cell line.

IXBExperiments on Malignant A549 Cells

[0354] A549 human lung cancer cells were seeded in 96-well plates together with the calix[4]arene of the formula (2) and CuCl.sub.2. Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) treated with a broad range of concentrations of the calix[4]arene of the formula (2) or/and CuCl.sub.2 from Merck. After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0355] The here below table 12 details the percentage of cell viability depending on the concentration of the calix[4]arene of the formula (2) and the concentration of Cu.sup.2+.

TABLE-US-00012 TABLE 12 detailing the percentage of cell viability Concentration of the calix[4]arene of the formula (2) (M) 0 0.1 0.5 1 1.5 Concentration 0 100.0 102.5 96.4 79.9 52.2 of Cu.sup.2+ (M) 1 89.4 90.2 87.6 45.7 22.7 5 88.1 89.0 14.7 0 0 10 84.6 86.4 3.9 0 0 25 85.6 72.3 0 0 0 50 79.4 36.1 0 0 0

[0356] The table 12 details the effect of combining the calix[4]arene of the formula (2) with CuCl.sub.2 using the MTS cytotoxicity assay in A549 cells. More precisely, in view of the detailed percentages of cell viability in this table 12, it shows that the combination of the calix[4]arene of the formula (2) with Cu (in the form of CuCl.sub.2) increases the efficacy of said calix[4]arene of the formula (2) in A549 cells.

IXCExperiments on Malignant PC9 Cells

[0357] PC9 human lung cancer cells were seeded in 96-well plates together with the calix[4]arene of the formula (2) and CuCl.sub.2. Cells were grown in RPMI-1640 supplemented with GlutaMAX (Gibco) and fetal calf serum (FCS, 10%) treated with a broad range of concentrations of the calix[4]arene of the formula (2) or/and CuCl.sub.2 from Merck. After 72 hours of treatment, the cytotoxicity was measured by means of the IC.sub.50 value, using the kit CellTiter 96R AQ.sub.uous One Solution Cell Proliferation Assay (MTS) from Promega. Following 30 minutes of incubation with the MTS reagent, the endpoint measurements were done in triplicate at 485 nm, using a fluorescence plate reader.

[0358] The here below table 13 details the percentage of cell viability depending on the concentration of the calix[4]arene of the formula (2) and the concentration of Cu.sup.2+.

TABLE-US-00013 TABLE 13 detailing the percentage of cell viability Concentration of the calix[4]arene of the formula (2) (M) 0 0.1 0.5 1 1.5 Concentration 0 100.0 99.9 93.1 75.8 24.4 of Cu.sup.2+ (M) 1 90.9 85.2 80.3 18.3 11.5 5 87.1 50.2 4.7 0 0 10 81.0 22.2 0 0 0 25 77.1 4.3 0 0 0 50 68.8 0 0 0 0

[0359] The table 13 details the effect of combining the calix[4]arene of the formula (2) with CuCl.sub.2 using the MTS cytotoxicity assay in PC9 cells. More precisely, in view of the detailed percentages of cell viability in this table 13, it shows that the combination of the calix[4]arene of the formula (2) with Cu (in the form of CuCl.sub.2) increases the efficacy of said calix[4]arene of the formula (2) in PC9 cells.

[0360] In conclusion, In view of these tables 11 to 13, the combination of the calix[4]arene of the formula (2) with Cu (in the form of CuCl.sub.2) increases the efficacy of said calix[4]arene of the formula (2) in different lung adenocarcinoma cell lines. Thus we observe a synergistic effect of the calix[4]arene of the formula (2) with Cu in the form of a salt.

XExperiments on Bacteria and Yeast

[0361] The efficiency of a compound of interest on a bacterial strain is evaluated by measuring its minimal inhibitory concentration (hereafter abbreviated MIC). MICs are measured by following the bacterial growth in a 96 wells plate exposed to a range of concentrations of the compound of interest.

[0362] All the below described procedures were done in proximity to an open flame to assure sterility. All contact surfaces were cleaned with ethanol.

[0363] The MIC of the calix[4]arene of the formula (2) was evaluated on [0364] Staphylococcus aureus (hereafter abbreviated SA), [0365] methicillin resistant Staphylococcus aureus (hereafter abbreviated MRSA), [0366] Escherichia coli (hereafter abbreviated E. coli), [0367] vancomycin resistant enterococcus (hereafter abbreviated VRE) bacterial strains andCandida albicans yeast cells.

[0368] A small amount of the frozen bacteria (stored at 80 C. in glycerol) was mixed in 25 mL of Lysogeny broth (i.e. a nutritionally rich medium used for the growth of bacteria which comprises 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1 L of distilled water; with the pH adjusted to 7.0 with 1 N NaOH; and autoclaved for 25 min at 120 C.) in a falcon tube and then placed in an incubation chamber at 180 rpm and 37 C. After 16 to 24 hours, 25 mL of Lysogeny broth was added to the growth media and placed back in the incubation chamber for two hours. The absorption of the resulting solution was measured at 600 nm (hereafter abbreviated OD.sub.600) using Lysogeny broth as blank and then the solution was diluted to 0.10.01.

[0369] 96 Well plates were filled with 20 L of 1 mM CuSO.sub.4 solution, then 160 L of growth media was added. Finally, 20 L of DMSO solutions containing a range of concentrations of the tested calix[4]arene of the formula (2) was added. The concentrations of the calix[4]arene of the formula (2) were: 200 M, 100 M, 50 M, 25 M, 12.5 M, and 6.2 M, 3.1 M, 1.6 M, 0.8 M, 0.4 M, and 0.2 M. For each condition three wells were used. The top and bottom rows were used as control, the top row only contained Lysogeny broth and the bottom one contained the growth media, DMSO and CuSO.sub.4. The plates were placed in the incubator for 16 to 24 hours. The absorbance of each well was measured at 600 nm with a multiplate reader (Fluostar from Optima).

[0370] Additionally, the same protocol was repeated with Candida albican yeast cells. Instead of Lysogeny broth, Yeast Extract-Peptone-Dextrose (Bacteriological peptone, 20 g/L; Glucose, 20 g/L; Yeast extract, 10 g/L) was used as growing medium.

[0371] The ratio between the average absorption value of three wells (OD) with the same conditions minus the average of the 12 negative controls (OD(negative)) and the average of the 12 positive control (OD(positive)) minus the negative control yielded the growth value which was subtracted from one and multiplied by a hundred to give the inhibition percentage (Equation 1).

[00001]$\begin{array}{cc}\mathrm{Inhibition}\%=100\left(1-\left[\mathrm{OD}-\mathrm{OD}\left(\mathrm{negative}\right)\right]/\left[\mathrm{OD}\left(\mathrm{positive}\right)-\mathrm{OD}\left(\mathrm{negative}\right)\right]\right)& \left(\mathrm{Equation}1\right)\end{array}$

[0372] The MIC50 or MIC90 is defined by the lowest concentration for which the inhibition value is at/over 50 or 90%. For a given experimental conditions three rows are used in a plate. The whole process was also repeated three times to obtain triplicates. MIC values were the determined by the average values obtained.

[0373] The table 14 details the values of MIC50 and MIC90 of different tested strain of SA, MRSA, E. coli and VRE obtained with the calix[4]arene of the formula (2) and 100 M CuSO.sub.4.

TABLE-US-00014 TABLE 14 detailing the MIC50 and MIC90 values obtained with the calix[4]arene of the formula (2) MIC50 MIC90 E. E. Tested strain SA MRSA coli VRE SA MRSA coli VRE Calix[4]arene 0.8 0.8 0.8 0.8 1.6 0.8 1.6 1.6 of the formula (2)

[0374] Furthermore, the minimal inhibitory concentrations values for calix[4]arene of the formula (2) in presence of 100 M CuSO4 for Candida albicans yeast was determined and found to be 0.8 M.

[0375] The lowest values measured of 0.8 M correspond to 0.5 L/mL, which is considered to be low for an antibiotic. The fact that the MIC values for each bacterial strain are similar shows that the calix[4]arene of the formula (2) is an efficient antibacterial agent regardless of the resistance of the various strains and regardless of the class of the bacteria.