Implantable material grafted with a cell antiproliferative and/or antibacterial film synthetized from a bifunctional molecule

Abstract

The present invention relates to an implantable material having at least one external surface grafted with a film including carboxylate and sulfonate functions wherein the film is simultaneously synthesized and grafted directly on the external surface by radical reaction of a bifunctional adhesion primer of Formula (I) or by radical reaction of an adhesion primer and a bifunctional polymerizable monomer of Formula (II). ##STR00001## The invention also relates to a process for directly synthesizing and grafting of a film according to the invention onto at least one external surface of an implantable material. The invention further relates to the use of a grafted implantable material for the manufacture of an antiproliferative and/or antibacterial implantable medical device. The invention also relates to compounds of Formula (I) and Formula (II).

Claims

1. An implantable material having at least one external surface grafted with a film comprising carboxylate and sulfonate functions wherein the film is simultaneously synthesized and grafted directly on said external surface by radical reaction of: a bifunctional adhesion primer of Formula (I) ##STR00024## wherein R.sub.1 represents a group substituted by one carboxylate function and one sulfonate function, said group being selected from linear or branched alkyl, aryl, or heteroaryl; said groups being optionally substituted by one or more substituent selected from alkyl, aryl, halogen or carbonyl; R.sub.2 represents an aryl or heteroaryl group substituted by at least one diazonium salt group and optionally further substituted by one or more substituent selected from alkyl, aryl or halogen; R.sub.3 is a bond or represents or a linear or branched C1-C3 alkyl group, optionally substituted by one or more substituent selected from alkyl, aryl, heteroaryl, halogen or nitro; R.sub.4 represents a hydrogen atom or a group selected from alkyl, aryl, heteroaryl or alkylcarbonyl.

2. The implantable material according to claim 1, wherein the film is simultaneously synthesized and grafted directly on the external surface by radical chemisorption and polymerization of a bifunctional adhesion primer of Formula (I) ##STR00025## wherein R.sub.1 represents a group substituted by one carboxylate function and one sulfonate function, said group being selected from a C2-C4 alkyl; R.sub.2 represents a benzodiazonium group; R.sub.3 is a bond; R.sub.4 represents a hydrogen atom.

3. The implantable material according to claim 1, wherein the thickness of the film is from 1 nm to 50 nm.

4. A process for simultaneously synthesizing and grafting a film directly onto at least one external surface of an implantable material, comprising a step of contacting, under conditions enabling the formation of radical entities, said external surface with a solution comprising: a bifunctional adhesion primer of Formula (I) ##STR00026## wherein R.sub.1 represents a group substituted by one carboxylate function and one sulfonate function, said group being selected from linear or branched alkyl, aryl, or heteroaryl; said groups being optionally substituted by one or more substituent selected from alkyl, aryl, halogen or carbonyl; R.sub.2 represents an aryl or heteroaryl group substituted by at least one diazonium salt group and optionally further substituted by one or more substituent selected from alkyl, aryl or halogen; R.sub.3 is a bond or represents a linear or branched C1-C3 alkyl group, optionally substituted by one or more substituent selected from alkyl, aryl, heteroaryl, halogen or nitro; R.sub.4 represents a hydrogen atom or a group selected from alkyl, aryl, heteroaryl or alkylcarbonyl.

5. The process according to claim 4, wherein the solution comprises water, deionized water, distilled water, acidified or not, acetic acid, hydroxylated solvents, low-molecular-weight liquid glycols and mixtures thereof.

6. The process according to claim 4, wherein the conditions enabling the formation of radical entities comprise the use of a reducing agent.

7. The process according to claim 4, wherein the solution comprises a precursor of a bifunctional adhesion primer of Formula (I) selected from: ##STR00027##

8. An antiproliferative and/or antibacterial implantable medical device comprising the implantable material according to claim 1.

9. A kit comprising the implantable material according to claim 1 and an inserting and/or implantation device.

10. A compound of Formula (I) ##STR00028## wherein R.sub.1 represents a group substituted by one carboxylate function and one sulfonate function, said group being selected from linear or branched alkyl, aryl, or heteroaryl; said groups being optionally substituted by one or more substituent selected from alkyl, aryl, halogen or carbonyl; R.sub.2 represents an aryl or heteroaryl group substituted by at least one diazonium salt group or at least one primary amine group and optionally further substituted by one or more substituent selected from alkyl, aryl or halogen; R.sub.3 is a bond or represents a linear or branched C1-C3 alkyl group, optionally substituted by one or more substituent selected from alkyl, aryl, heteroaryl, halogen or nitro; R.sub.4 represents a hydrogen atom or a group selected from alkyl, aryl, heteroaryl or alkylcarbonyl.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a scheme representing the principles of radical copolymerization and of Graftfast copolymerization-like reaction.

(2) FIG. 2 is a graph representing the number of LEC cells present at the surface of materials grafted according to the present invention (Statistics: Control vs Test; **p<0.001; ***p<0.0001).

EXAMPLES

(3) The present invention is further illustrated by the following examples.

(4) Materials

(5) All standard chemicals were purchased from Sigma Aldrich.

(6) The following examples were performed in glass cell and otherwise stated they were conducted in normal conditions of pressure in ambient air.

1. Synthesis of Adhesion Primer of Formula (I)

(7) The synthesis of 1-((4-aminophenyl)amino)-3-carboxy-1-oxopropane-2-sulfonate prec-Ia and et 3-((4-aminophenyl)amino)-1-carboxy-3-oxopropane-1-sulfonate prec-Ib was performed using two different routes of synthesis.

(8) 1.1. Method A

(9) ##STR00017##

(10) Step A1. Synthesis of (Z)-4-((4-aminophenyl)amino)-4-oxobut-2-enoic Acid 1

(11) During this first step, maleic anhydride (5,4 g, 55 mmol), dissolved in 100 mL of an aprotic solvent (DCM, THF, acetonitrile), is added dropwise in a solution of p-phenylene diamine (6.2 g, 57.4 mmol) in 200 mL of an aprotic solvent, at room temperature under vigourous stirring. The resulting solution is stirred overnight. The solution is filtrated to provide a crude yellow product which is with diethylether and dried under reduced pressure, affording a 92% yield.

(12) RMN .sup.1H (400 MHz, DMSO-d6) (ppm/TMS): 3.32 (bs, 3H NH.sub.2(C.sub.6H.sub.5)NH), 6.27 (d, 1H, NH(CO)CHCHCOOH), 6.47 (d, 1H, NH(CO)CHCHCOOH), 6.52 (d, 2H, C.sub.6H.sub.4), 7.28 (d, 2H, C.sub.6H.sub.4), 10.46 (s, 1H, COOH).

(13) Step A2. Sulfonation Leading to -prec-Ia and prec-Ib

(14) (Z)-4-((4-aminophenyl)amino)-4-oxobut-2-enoic acid (5 g, 24 mmol) is dissolved in 100 mL propan-2-ol at 50 C. Sulfite sodium salt (4 g, 31 mmol) in water is added dropwise to the solution under vigourous stirring. The mixture is heated to 82 C. overnight. The final mixture is filtered to provide a white powder, which is washed abundantly with warm ethanol and finally dried under reduced pressure. The final yield is over 90%.

(15) RMN .sup.1H (400 MHz, DMSO-d6) (ppm/TMS): 2.65-2.94 (m, 2H, CH(SO.sub.3)CH.sub.2), 3.65 (dd, 1H CH(SO.sub.3)CH.sub.2), 6.44 (d, 2H, C.sub.6H.sub.4), 7.16 (d, 2H, C.sub.6H.sub.4), 9.57 (s, 1H, COOH).

(16) 1.2. Method B

(17) ##STR00018##

(18) Step B1. Synthesis of 2,5-dioxotetrahydrofuran-3-sulfonate 2

(19) Acetic anhydride (60 mL) is poured in large excess (8:1) in a reactor containing sulfosuccinic acid (15 ml, 70% w in H.sub.2O) and the mixture is stirred during 2 hours at 80 C. The solvents are removed under heating and reduced pressure to afford a dark red oil, which is used directly in step 2 without any further purification.

(20) RMN .sup.1H (400 MHz, DMSO-d6) (ppm/TMS): 2.65-2.94 (m, 2H, CH(SO.sub.3)CH.sub.2), 3.65 (dd, 1H CH(SO.sub.3)CH.sub.2).

(21) Step B2. Synthesis of prec-Ia and prec-Ib

(22) The crude sulfosuccinic anhydride (15 g, 84 mmol) diluted in an aprotic solvent (THF) is added dropwise to a solution of p-phenylene diamine (16 g, 89 mmol) also dissolved in an aprotic solvent (THF). The reaction is stirred overnight and the solution is finally filtrated to provide a white/light brown powder, which is washed with ethanol, diethylether and dried under reduced pressure. Yield is about 90%.

(23) RMN .sup.1H (400 MHz, DMSO-d6) (ppm/TMS): 2.65-2.94 (m, 2H, CH(SO.sub.3)CH.sub.2), 3.65 (dd, 1H CH(SO.sub.3)CH.sub.2), 6.44 (d, 2H, C.sub.6H.sub.4), 7.16 (d, 2H, C.sub.6H.sub.4), 9.57 (s, 1H, COOH).

2. Synthesis of Polymerizable Monomer of Formula (II)

(24) 2.1. Synthesis of 2-carboxy-2-methacrylamidoethanesulfonate (IIa)

(25) The synthesis of 2-carboxy-2-methacrylamidoethanesulfonate IIa was performed according to Method C (scheme C):

(26) ##STR00019##

(27) Methacryloyl chloride (10 mL, 89 mmol) is added dropwise to a stirred solution of cysteic acid (18.3 g, 98 mmol) in dimethylformamide (200 mL), at 4 C. during 4 h. At the end, the crude product is separated by filtration and washed with THF. The final product is dried under reduced pressure to afford a yield of 76%.

(28) RMN .sup.1H (400 MHz, DMSO-d6) (ppm/TMS): RMN .sup.1H (400 MHz, D.sub.2O) (ppm/TMS): 1.85 (s, 3H, CH.sub.2C(CH.sub.3)), 3.44-3.56 (m, 2H, .sup.O.sub.3SCH.sub.2), 4.48 (m, 1H, (CO)NHCHCOOH) 5.38 (d, 1H, CH.sub.2C(CH.sub.3)), 5.72 (d, 1H, CH.sub.2C(CH.sub.3)).

(29) The synthesis of 2-carboxy-2-methacrylamidoethanesulfonate IIa may also be performed according to Method D, using an activated ester (scheme D):

(30) ##STR00020##

(31) Step D1. Synthesis of N-methacryloyloxysuccinimide (NMAS)

(32) Methacryloyl chloride (10 mL, 89 mmol) was added dropwise to a stirred solution of N-hydroxysuccinimide (11 g, 98 mmol) and triethylamine (13 mL, 98 mmol) in chloroform at 0 C. After stirring for 4 h at 0 C., the reaction mixture was washed with ice-cold saturated sodium bicarbonate solution four times and dried on MgSO4 overnight. Then, the solution was filtered and chloroform was removed by evaporation. Yield ca. 75%.

(33) RMN .sup.1H (400 MHz, DMSO-d6) (ppm/TMS): 1.95 (s, 3H, CH.sub.2C(CH.sub.3), 2.80 (s, 4H, (CO)CH.sub.2CH.sub.2(CO)), 5.68; 6.28 (d, CH.sub.2C(CH.sub.3)).

(34) Step D2. Synthesis of 2-carboxy-2-methacrylamidoethanesulfonate

(35) A solution of cysteic acid in water is added dropwise to a stirred solution of NMAS in DMF at room temperature. The solution was allowed to stir overnight and then the pH of the solution was lowered with concentrated hydrochloric acid. The final mixture is filtered to provide a white powder which is washed with diethylether and dried under reduced pressure. Yield is about 85%.

(36) RMN .sup.1H (400 MHz, DMSO-d6) (ppm/TMS): RMN .sup.1H (400 MHz, D.sub.2O) (ppm/TMS): 1.85 (s, 3H, CH.sub.2C(CH.sub.3)), 3.44-3.56 (m, 2H, .sup.O.sub.3SCH.sub.2), 4.48 (m, 1H, (CO)NHCHCOOH) 5.38 (d, 1H, CH.sub.2C(CH.sub.3)), 5.72 (d, 1H, CH.sub.2C(CH.sub.3)).

(37) 2.2. Synthesis of 4-[(2-[(2-methyl-1-oxo-2-ethenyl)oxy]ethyl]ester of Sulfobutanoic Acid (IIc-1 and IIc-2)

(38) The synthesis of the 4-[(2-[(2-methyl-1-oxo-2-ethenyl)oxy]ethyl]ester of sulfobutanoic acid was performed according to the method E:

(39) ##STR00021##

(40) Step E1. Synthesis of the sulfosuccinic snhydride

(41) Sulfosuccinic acid (28 g, 127 mmol) was poured into acetic anhydride (35 mL, 370 mmol) and the slurry was stirred during 4 h at 80 C. The mixture is cooled down and filtrated. The crude solid is washed first with acetic acid during 30 min and then with diethyl ether. The white solid is dried under vacuum and the anhydride formation is controlled by FTIR (CO stretching modes at 1770 and 1860 cm.sup.1).

(42) Step E2. Synthesis of 4-[2-[(2-methyl-1-oxo-2-ethenyl)oxy]ethyl]ester of sulfobutanoic acid

(43) Sulfosuccinic anhydride (5 g, 24 mmol), Hydroxyethyl acrylate (10 mL, 87 mmol) and triethylamine (10 mL, 72 mmol) are stirred together at room temperature during 16 h. The mixture is filtrated and the crude product is precipitated in an acetone/diethyl ether 1:1 solution. The solution is filtrate and the white solid is dried under vacuum.

(44) RMN .sup.1H (D.sub.2O, (ppm)/TMS): 6.50 (dd, 1H, CH.sub.2CH), 6.25 (dd, 1H, CH.sub.2CH), 6.04 (dd, 1H, CH.sub.2CH), 4.55-4.35 (m, 4H, CH.sub.2CH.sub.2), 4.20-3.97 (dd2, 1H, CH(SO.sub.3.sup.)), 3.12-2.78 (m, 2H, CH(SO.sub.3.sup.)CH.sub.2).

(45) 2.3. Synthesis of the 4-[2-[(2-methyl-1-oxo-2-propenyl)oxy]ethyl]ester of 4-sulfophthalic Acid (IId)

(46) The synthesis of the 4-[2-[(2-methyl-1-oxo-2-propenyl)oxy]ethyl]ester of 4-sulfophthalic acid was performed according to the method F.

(47) ##STR00022##

(48) Step F1. Synthesis of the 4-sulfophthalic Anhydride Sodium Salt

(49) First, sodium carbonate (6.26 g, 59 mmol) is added to the 4-sulfophthalic acid (15 mL, 39 mmol, 50% wt in water). The 4-sulfophthalic acid trisodium salt forms a white solid, which is washed in 100 mL acetone, filtrated and dried under vacuum. A part of the white solid, (4.6 g, 14 mmol) is stirred with acetic anhydride (40 mL, 423 mmol) at 80 C. during 4 h. The mixture is cooled down and filtrated. The crude solid is washed first with acetic acid during 30min and then with diethyl ether. The white solid is dried under vacuum and the anhydride formation is controlled by FTIR (CO stretching modes at 1770 and 1860 cm.sup.1).

(50) Step F2. Synthesis of the 4-[2-[(2-methyl-1-oxo-2-propenyl)oxy]ethyl]ester of 4-sulfophthalic acid

(51) 4-sulfophthalic anhydride sodium salt (5 g, 20 mmol), Hydroxyethyl methacrylate (10 mL, 82 mmol) and triethylamine (10 mL, 72 mmol) are stirred together at room temperature during 16 h. The mixture is filtrated and the crude product is precipitated in an acetone/diethyl ether 1:1 solution. The solution is filtrate and the white solid is dried under vacuum. The resulting white solid is dissolved into a small amount of water, and a 0.1 mol/L NaOH solution is slowly added to reach pH 9. The solution is poured into an acetone/diethyl ether 1:1 solution to form a white solid, which is isolate and dried under vacuum.

(52) RMN .sup.1H (D.sub.2O, (ppm)/TMS): 8.20-7.50 (m, 3H, aryl group), 6.13 (s, 1H, CH.sub.2C), 5.69 (s, 1H, CH.sub.2C), 4.65-4.45 (m, 4H, CH.sub.2CH.sub.2), 1,90 (s, 3H, CH.sub.3C).

(53) 2.4. Synthesis of the 4-[2-[(2-methyl-1-oxo-2-ethenyl)oxy]ethyl]ester of 4-sulfophthalic Acid was Performed According to the Method G (IIe)

(54) ##STR00023##

(55) Step G1. Synthesis of the 4-sulfophthalic anhydride sodium salt

(56) First, sodium carbonate (6.26 g, 59 mmol) is added to the 4-sulfophthalic acid (15 mL, 39 mmol, 50% wt in water). The 4-sulfophthalic acid trisodium salt forms a white solid, which is washed in 100 mL acetone, filtrated and dried under vacuum. A part of the white solid, (4.6 g, 14 mmol) is stirred with acetic anhydride (40 mL, 423mmol) at 80 C. during 4 h. The mixture is cooled down and filtrated. The crude solid is washed first with acetic acid during 30 min and then with diethyl ether. The white solid is dried under vacuum and the anhydride formation is controlled by FTIR (CO stretching modes at 1770 and 1860 cm.sup.1).

(57) Step G2. Synthesis of the 4-[2-[(2-methyl-1-oxo-2-ethenyl)oxy]ethyl]ester of 4-sulfophthalic acid

(58) 4-sulfophthalic anhydride sodium salt (5 g, 20 mmol), Hydroxyethyl acrylate (10 mL, 87 mmol) and triethylamine (10 mL, 72 mmol) are stirred together at room temperature during 16 h. The mixture is filtrated and the crude product is precipitated in an acetone/diethyl ether 1:1 solution. The solution is filtrate and the white solid is dried under vacuum. The resulting white solid is dissolved into a small amount of water, and a 0.1 mol/L NaOH solution is slowly added to reach pH 9. The solution is poured into an acetone/diethyl ether 1:1 solution to form a white solid, which is isolate and dried under vacuum.

(59) RMN .sup.1H (D.sub.2O, (ppm)/TMS): 8.20-7.50 (m, 3H, aryl group), 6.45 (dd, 1H, CH.sub.2CH), 6.23 (dd, 1H, CH.sub.2CH), 5.98 (dd, 1H, CH.sub.2CH), 4.65-4.45 (m, 4H, CH.sub.2CH.sub.2).

3. IOL Grafting

(60) Hydrophilic plots grafting

(61) Cylinders of polyhydroxyethyl methacrylate (PHEMA) having a diameter of 13 mm and a high of 3 mm were used for grafting tests. These cylinders are precursors of hydrophilic intraocular implants.

(62) 3.1. Diazonium Salt One-Pot Synthesis

(63) The grafting of 20 PHEMA cylinders was tested in presence of Hydroxyethylmethacrylate (HEMA) 4-((4-aminophenyl)amino)-4-oxo-2-sulfo-butanoic acid and 4-((4-aminophenyl)amino)-4-oxo-3-sulfo-butanoic acid (noticed as compounds prec-Ia and prec-Ib) with the one pot synthesis of the corresponding diazonium salt.

(64) Pre-Treatment

(65) The PHEMA cylinders were first polished with colloidal alumina powder to a Ra=0.02 m roughness. The PHEMA cylinder were placed in a beaker, covered by deionized water and treated by ultrasonic for 210 minute. The cylinders were then covered with ethanol and sonicated again for 10 min. Finally they are dried under nitrogen flux.

(66) HEMA (0.25 mol/L) and compounds prec-Ia and prec-Ib (0.05 mol/L) are mixed in deionized water, acidified with HCl (pH=1). NaNO.sub.2 (0.05 mol/L) is added dropwise under mechanical agitation. PHEMA cylinders are placed in the solution and ascorbic acid (0.01 mol/L) is added in the mixture. The reaction is let for 1 hour. The PHEMA cylinders are then sonicated in a deionized water bath and an ethanol bath for 15 minutes, before being dried under a nitrogen flux and dried at 100 C. for 10 minutes.

(67) 3.2. Diazonium Salt One-Pot Synthesis Without Monomeric Dilution

(68) The grafting of two series of 20 PHEMA cylinders was tested in presence of 4-((4-aminophenyl)amino)-4-oxo-2-sulfo-butanoic acid and 4-((4-aminophenyl)amino)-4-oxo-3-sulfo-butanoic acid (noticed as compounds prec-Ia and prec-Ib) with the one pot synthesis of the corresponding diazonium salt.

(69) Pre-Treatment

(70) The PHEMA cylinders were first polished with colloidal alumina powder to a Ra=0.02 roughness. The PHEMA cylinder were placed in a beaker, covered by deionized water and treated by ultrasonic for 210 minute. The cylinders were then covered with ethanol and sonicated again for 10 min. Finally they are dried under nitrogen flux.

(71) Grafting

(72) Compounds prec-Ia and prec-Ib (0.05 mol/L) are mixed in deionized water, acidified with HCl (pH=1). NaNO.sub.2 (0.05 mol/L) is added dropwise under mechanical agitation. PHEMA cylinders are placed in the solution and ascorbic acid (0.01 mol/L) is added in the mixture. The reaction is let for 1 hour. The PHEMA cylinders are then sonicated in a deionized water bath and an ethanol bath for 15 minutes, before being dried under a nitrogen flux and dried at 100 C. for 10 minutes.

4. Cellular Proliferation Assessment

(73) 4.1. Method

(74) Study of the growth of human lens cells (LEC) onto grafted material obtained according to the methods described at paragraphs 3.1 and 3.2 above.

(75) Human eye lens epithelial cells (LEC, CRL-11421,ATCC, USA) are seeded onto grafted implants in 24-well microplates at a rate of 20 000 cells per well in RPMI medium (VWR, France). After 2, 7 and 10 days of culture, LEC cells are counted using the MTT colorimetric assay. Control corresponds to LEC culture onto non-grafted material.

(76) Batch 1 are plots grafted with compounds Ia and Ib.

(77) Batch 2 are plots grafted with compounds Ia and Ib in presence of HEMA.

(78) 4.2. Results

(79) A general observation is that human lens cells do not proliferate onto grafted materials and that grafted materials of the invention have cytostatic but no cytotoxic activities.