Compounds that are analogs of squalamine, used as antibacterial agents

Abstract

The invention relates to compounds of formula (I), to the pharmaceutical compositions comprising same, and to the use thereof in the treatment of bacterial, fungal, viral and parasitic infections or in the treatment of cancer in humans or animals. In formula (I), R1 and R2 are as defined in claim 1. ##STR00001##

Claims

1. A method of treating a disease in a human or animal comprising administering to the human or animal a compound of formula (I): ##STR00035## in which: R.sub.1 is chosen from H, SO.sub.3H, a C.sub.1-C.sub.8 alkyl group, a C.sub.6-C.sub.10 aryl group or a C(O)R.sub.13 group, R.sub.2 is (CR.sub.3R.sub.4).sub.m(X).sub.p(CR.sub.5R.sub.6).sub.n[(Y)CR.sub.7R.sub.8).sub.o].sub.qNR.sub.9R.sub.10, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are, in each case, identical or different, each independently chosen from H, C.sub.1-C.sub.8 alkyl, C.sub.6-C.sub.10 aryl and C(O)OR.sub.14; R.sub.9 and R.sub.10, which are identical or different, are each independently chosen from H, C.sub.1-C.sub.8 alkyl or a (CH.sub.2).sub.rNH.sub.2 group or together form a 5- to 7-membered heterocyclyl group optionally substituted by one to three R.sub.14 groups; X and Y, which are identical or different, are in each case each independently chosen from NR.sub.13, O or a 5- to 6-membered nitrogenous heterocyclyl group, R.sub.11 and R.sub.12 are each independently chosen from a C.sub.1-C.sub.8 alkyl group or a C.sub.6-C.sub.10 aryl group, R.sub.13 is H, a C.sub.1-C.sub.6 alkyl group or a (CH.sub.2).sub.sNH.sub.2 group; R.sub.14 is a O or S group; m is an integer between 1 and 5; n is an integer between 1 and 5, o is an integer between 1 and 5, p is 0 or 1, q is 0, 1, 2 or 3, r is an integer between 1 and 4, s is an integer between 1 and 5, or a stereoisomer, mixture of stereoisomers or pharmaceutically acceptable salt thereof, the compound of formula I excludes 3-(norspermino)-7-hydroxy-5-cholestane and compounds having the formula: ##STR00036## wherein the disease is selected from the group consisting of bacterial infections, fungal infections, parasitic infections, dental infections, mastitis, metritis, pyodermatitis, and otitis.

2. The method as claimed in claim 1, in which the compounds for which p=1, q=0, m=3, n=3 or 4, and X=NH are excluded.

3. The method as claimed in claim 1, wherein: when p=1 and q=0, then m+n6 or 7, when p=0 and q=1, then m+n+o6 and 7.

4. The method as claimed in claim 1, in which R.sub.1 is H.

5. The method as claimed in claim 1, in which X is NR.sub.13.

6. The method as claimed in claim 1, in which R.sub.9 and/or R.sub.10 are H.

7. The method as claimed in claim 1, in which m is 2, 3, 4, or 5.

8. The method as claimed in claim 1, in which the NHR.sub.2 group is chosen from: ##STR00037## ##STR00038##

9. The method as claimed in claim 1, wherein the compound is chosen from: 3-spermino-7-hydroxy-5-cholestane (SA-1) 3-spermino-7-hydroxy-5-cholestane (SA-2) 3-norspermidino-7-hydroxy-5-cholestane (SA-3) 3-(1,3-diaminopropane)-7-hydroxy-5-cholestane (SA-4) 3-(1,4-diaminobutane)-7-hydroxy-5-cholestane (SA-5) 3-(tris(2-aminoethyl)amine)-7-hydroxy-5-cholestane (SA-6) 3-(1,5-diaminopentane)-7-hydroxy-5-cholestane (SA-7) 3-(1,4-bis(3-aminopropyl)piperazine)-7-hydroxy-5-cholestane (SA-8) 3-(1,4-bis(3-aminopropoxy)butane)-7-hydroxy-5-cholestane (SA-9) 3-(1-(3-aminopropyl)imidazole)-7-hydroxy-5-cholestane (SA-11) 3-(1-(3-aminopropyl)morpholine)-7-hydroxy-5-cholestane (SA-12).

10. The method as claimed in claim 1, in which the bacterial infection is a Gram+ or Gram bacterial infection.

11. The method as claimed in claim 1, wherein the animal is chosen from dogs, cats or ruminants.

12. The method as claimed in claim 1, in which the compound of formula (I) is administered in combination with an antibiotic compound.

13. The method of claim 1, wherein the human or animal is human.

Description

EXAMPLES

I. Synthesis of the Compounds of Formula (I)

(1) All the syntheses were carried out with solvents purified according to the usual methods. The commercial reactants are used directly without prior purification.

(2) The chemical structures synthesized were all confirmed by a proton (.sup.1H) and/or carbon (.sup.13C) NMR analysis in deuterated chloroform CDCl.sub.3 or deuterated methanol CD.sub.3OD on a device of Bruker AC 300 type. The chemical shifts are expressed in ppm. The recording frequencies of the nuclei and also the references used are as follows:

(3) .sup.1H NMR: 300 MHz, Si(CH.sub.3).sub.4

(4) .sup.13C NMR: 75 MHz, Si(CH.sub.3).sub.4

(5) The abbreviations used for writing the .sup.1H NMR spectrum are as follows: s=singlet d=doublet t=triplet q=quartet m=broad unresolved peak

(6) The mass spectra were produced at the Spectropole of Aix-Marseille III. They are produced on the dry product using a Triple Quadrupole API III Plus spectrometer from Sciex. The sample is dissolved in 500 l of CH.sub.2Cl.sub.2 and then diluted to 1/10.sup.4 in a 3 mM solution of ammonium acetate in MeOH. The solution of the extract is introduced into the ionization source by infusion (syringe driver pump, Harvard Apparatus) at a flow rate of 5 l/min.

(7) The compounds of formula (I) were prepared according to the reaction scheme below:

Synthesis of 7-ketocholest-5-en-3-ol 1

(8) ##STR00017##

(9) 50 g of cholesterol (0.129 mol), 22 g of hydroxyphthalamide (0.135 mol) and 1.5 l of an ethyl acetate/acetone (1/1) mixture are placed in a reactor provided with a mechanical stirrer. The mixture is brought to 50 C. 200 mg of benzoyl peroxide are added and air is bubbled through for 72 h while adjusting the level of the solvent and while monitoring the handling by thin-layer chromatography. After 72 h, the solvent is evaporated under vacuum. The residue is dissolved in petroleum ether and washed with sodium carbonate until the orange coloration disappears. The organic phases are washed with a saturated NaCl solution and dried over MgSO.sub.4. The solvent is driven off under vacuum and the sterol is dissolved in pyridine (200 ml). Cooling is carried out at 0 C. and 1 g of CuCl.sub.2 is added. The solution is stirred for 24 h (return from 0 C. to ambient temperature). The solution obtained is poured onto a water/ice mixture. Extraction is carried out with ethyl acetate and washing is carried out with a saturated CuSO.sub.4 solution. After separation of the phases, the organic phase is washed with a 0.1N HCl solution and dried over MgSO.sub.4. After evaporation of the solvent, the residue is chromatographed on silica gel (petroleum ether/ethyl acetate 1/1). The expected ketone is obtained in the form of an off white solid with a yield of 70%.

(10) .sup.1H NMR: =5.45-5.75 (m, 1H), 3.475-3.75 (m, 1H), 2.125-2.75 (m, 4H), 1.75-2.075 (m, 6H), 0.8-1.7 (m, 37H), 0.45-0.75 (m, 4H); .sup.13C NMR: =202.81, 165.59, 126.49, 70.89, 55.17, 50.34, 45.80, 43.49, 39.87, 38.67, 36.57, 36.11, 28.40, 26.72, 24.22, 23.22, 22.96, 21.61, 19.26, 17.71, 12.37.

Synthesis of 7-ketocholest-5-en-3-yl acetate 2

(11) ##STR00018##

(12) 3 mmol of 7-ketocholest-5-en-3-ol 1 are dissolved in pyridine (25 ml) in a two-necked round-bottomed flask equipped with a reflux condenser, and 9 mmol of acetic anhydride are added. The mixture is left under magnetic stirring in an ice bath for 24 h. The pyridine is evaporated under high vacuum and then the solid obtained is resuspended in CH.sub.2Cl.sub.2 (15 ml). An extraction with copper sulfate is then carried out and the organic phase is dried over MgSO.sub.4, filtered and then dried under high vacuum. The product is purified by chromatography on silica gel (eluent: petroleum ether/ethyl acetate 9/1) (Yd 94%).

(13) .sup.1H NMR: =5.65-5.7 (d, 1H), 4.6-4.75 (m, 1H), 0.6-2.6 (m, 44H); .sup.13C NMR: =201.90, 170.24, 163.81, 126.68, 72.19, 54.75, 49.93, 49.78, 45.38, 43.08, 39.44, 38.64, 38.28, 37.72, 36.15, 35.98, 35.69, 31.90, 28.50, 27.96, 27.32, 26.28, 23.80, 22.77, 22.52, 21.24, 21.14, 18.84, 17.22, 11.93.

Synthesis of 7-ketocholestan-3-yl acetate 3

(14) ##STR00019##

(15) 3.04 g (6.87 mmol) of 7-ketocholest-5-en-3-yl acetate 2 and 1.1 g (approximately 15 mol %) of Pd/C (10%) are introduced into 50 ml of CH.sub.2Cl.sub.2 in a stainless steel reactor. The reactor is placed under 50 bar of hydrogen and under vigorous stirring for 12 h. After filtration through celite and evaporation of the solvent under vacuum, the product is obtained virtually pure and with a quantitative yield. It will be used as is in the following stage.

(16) .sup.1H NMR: =4.62-4.70 (m, 1H), 0.64-2.39 (m, 47H); .sup.13C NMR: =211.44, 170.38, 72.71, 54.95, 49.90, 48.82, 46.43, 45.34, 42.45, 39.42, 38.65, 36.09, 35.89, 35.60, 33.79, 28.35, 27.93, 26.34, 24.93, 23.72, 22.74, 22.51, 22.15, 21.27, 18.73, 12.01, 11.65.

Synthesis of the mixture of 7-hydroxycholestan-3-ol 4a and 7-hydroxycholestan-3-ol 4b

(17) ##STR00020##

(18) 530 mg of lithium aluminum hydride (13.8 mmol) are placed in 100 ml of anhydrous THF in a two-necked round-bottomed flask. A solution of 7-ketocholestan-3-yl acetate 3 (1.5 g, 3.3 mmol) dissolved in 15 ml of THF is slowly added at 0 C. After stirring at ambient temperature for 12 h, hydrolysis is carried out with KOH (30%) solution (1.2 ml). Stirring is continued for 1 h, then filtration is carried out through celite and rinsing is carried out with MeOH, and then the solvents are evaporated under vacuum. The product in the form of a mixture of isomers (/ 50/50) is purified by chromatography on silica gel (eluent: petroleum ether/ethyl acetate 8/2) (Yd 95%).

(19) .sup.1H NMR: =0.6-3.82 (m, 48H); .sup.13C NMR: =75.11, 71.08, 70.99, 67.96, 62.57, 56.45, 56.08, 55.68, 55.19, 51.85, 50.51, 45.83, 43.95, 43.35, 42.60, 42.00, 39.46, 37.62, 37.11, 36.54, 36.24, 35.73, 35.51, 34.88, 31.30, 29.82, 28.05, 27.95, 26.86, 23.79, 23.71, 22.76, 22.51, 21.56, 20.96, 18.60, 12.40, 12.11, 11.79, 11.20.

Synthesis of 3-keto-7-hydroxycholestane 5 and 3-keto-7-hydroxycholestane 5b

(20) ##STR00021##

(21) 1.28 g of mixture of 7-hydroxycholestan-3-ol 4a and 7-hydroxycholestan-3-ol 4b are placed in 150 ml of toluene and 3.6 g of silver carbonate on celite in a single-necked round-bottomed flask surmounted by a Dean & Stark apparatus. The system is brought to reflux of the toluene for 24 h. After cooling the mixture is filtered through celite. Purification is carried out by chromatography on silica gel (eluent: petroleum ether/ethyl acetate 7/3) and the two pure isomers 5b (fraction 1) and 5a (fraction 2) are thus obtained with nonoptimized yields of 31% and 25% respectively.

(22) 5a .sup.1H NMR: =3.05-3.55 (m, 1H), 2.2-2.6 (m, 3H), 0.4-1.92 (m, 42H); .sup.13C NMR: =211.55, 74.66, 55.61, 55.24, 51.83, 44.18, 43.94, 43.66, 39.90, 39.54, 38.12, 35.72, 35.16, 28.75, 28.06, 23.89, 22.87, 22.62, 21.80, 18.84, 12.22, 11.63.

(23) 5b .sup.1H NMR: =3.3-3.85 (m, 1H), 0.60-2.65 (m, 45H); .sup.13C NMR: =211.36, 71.08, 57.18, 56.53, 54.33, 47.13, 39.86, 38.32, 37.07, 36.49, 36.08, 31.10, 30.44, 28.43, 28.39, 24.38, 24.20, 23.20, 22.93, 21.92, 19.03, 13.53, 12.40.

Synthesis of the Aminosteroidal Derivatives SA-1 to SA-12

(24) The aminosteroidal derivatives were all produced according to the same procedure. Let us consider the example of the molecule SA-1.

(25) 3.3 equivalents of spermine (171 mg, 0.82 mmol) are dissolved in 5 ml of MeOH in a two-necked round-bottomed flask placed under argon and then 300 l of Ti(O(i-Pr)).sub.4 (1 mmol) are added. After stirring for 5 minutes, 102 mg of 3-keto-7-hydroxycholestane 5b (0.25 mmol) are added to the mixture. After stirring for 24 hours, the round-bottomed flask is placed at 78 C. and then 40 mg of NaBH.sub.4 (1 mmol) are added with stirring. After 2 hours and returning to ambient temperature, 1 ml of water is added in order to terminate the reaction. After stirring for an additional 1 h, the mixture is filtered through celite. The filtrate is evaporated under vacuum and the product is purified by chromatography on silica gel (eluent: CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH (7/3/1)).

3-Spermino-7-hydroxy-5-cholestane SA-1

(26) ##STR00022##

(27) Yd: 54%. .sup.1H NMR: =3.16 (m, 1H), 2.44-2.65 (m, 13H), 1.96 (m, 5H), 0.89-1.64 (m, 57H); .sup.13C NMR: =71.65, 57.46, 56.03, 51.04, 47.98, 47.64, 46.61, 45.34, 42.76, 40.80, 40.60, 40.02, 39.82, 39.74, 38.62, 37.16, 34.58, 33.12, 29.45, 28.30, 28.12, 25.11, 24.30, 24.28, 22.68, 20.91, 18.73, 13.56, 11.92.

3-Spermino-7-hydroxy-5-cholestane SA-2

(28) ##STR00023##

(29) Yd: 49%. .sup.1H NMR: =3.05 (m, 1H), 2.44-2.65 (m, 13H), 1.95 (m, 5H), 0.89-1.63 (m, 57H); .sup.13C NMR: =73.95, 57.46, 56.03, 51.04, 47.98, 47.64, 46.84, 45.84, 42.66, 41.80, 40.75, 40.39, 39.82, 39.65, 37.45, 37.26, 36.28, 26.26, 34.58, 33.97, 29.45, 28.30, 28.08, 25.35, 25.12, 24.30, 24.28, 21.98, 20.90, 18.73, 13.54, 11.89.

3-Norspermidino-7-hydroxy-5-cholestane SA-3

(30) ##STR00024##

(31) Yd: 46%. .sup.1H NMR: =3.14 (m, 1H), 2.55-2.65 (m, 8H), 2.01 (m, 4H), 1.01-1.83 (m, 50H); .sup.13C NMR: =73.46, 57.64, 56.03, 52.39, 47.47, 46.54, 45.98, 42.79, 41.20, 40.78, 40.54, 40.22, 40.10, 37.12, 37.02, 36.28, 36.26, 34.58, 33.91, 29.41, 29.12, 28.23, 25.62, 23.41, 23.12, 22.68, 20.84, 18.74, 13.68, 11.97.

3-(1,3-Diaminopropane)-7-hydroxy-5-cholestane SA-4

(32) ##STR00025##

(33) Yd: 54%. .sup.1H NMR: =3.21 (m, 1H), 2.53-2.60 (m, 4H), 0.89-1.81 (m, 51H); .sup.13C NMR: =71.22, 57.33, 55.89, 52.02, 46.01, 41.78, 40.96, 40.35, 39.92, 39.25, 39.02, 37.98, 37.11, 36.58, 36.45, 34.75, 34.25, 29.68, 28.64, 28.02, 23.56, 23.12, 22.68, 20.87, 18.98, 14.02, 11.92.

3-(1,4-Diaminobutane)-7-hydroxy-5-cholestane SA-5

(34) ##STR00026##

(35) Yd: 48%. .sup.1H NMR: =3.17 (m, 1H), 2.55-2.63 (m, 4H), 0.99-1.83 (m, 53H); .sup.13C NMR: =71.23, 56.03, 55.66, 51.02, 48.29, 42.78, 41.19, 40.98, 40.02, 39.74, 39.65, 38.54, 37.14, 36.54, 36.45, 35.02, 34.89, 29.54, 29.45, 28.30, 28.08, 25.33, 24.85, 24.12, 22.67, 20.91, 18.76, 13.56, 11.90.

3-(Tris(2-aminoethyl)amine)-7-hydroxy-5-cholestane SA-6

(36) ##STR00027##

(37) Yd: 29%. .sup.1H NMR: =3.12 (m, 1H), 2.42-2.75 (m, 10H), 1.89 (m, 6H), 1.01-1.87 (m, 47H); .sup.13C NMR: =71.35, 57.70, 56.32, 55.24, 54.44, 51.04, 43.59, 42.98, 40.80, 40.12, 40.02, 40.00, 38.88, 38.32, 36.27, 35.97, 35.85, 35.02, 34.87, 29.18, 28.30, 28.08, 24.22, 23.85, 22.67, 20.89, 18.79, 13.54, 11.94.

3-(1,5-Diaminopentane)-7-hydroxy-5-cholestane SA-7

(38) ##STR00028##

(39) Yd: 32%. .sup.1H NMR: =3.14 (m, 1H), 2.54-2.60 (m, 5H), 0.95-1.89 (m, 54H); .sup.13C NMR: =71.90, 56.87, 52.43, 51.92, 48.71, 47.38, 43.37, 42.57, 41.02, 40.51, 39.71, 36.81, 36.51, 36.46, 36.38, 34.38, 34.12, 32.10, 29.84, 29.34, 28.68, 24.89, 22.44, 18.56, 12.34.

3-(1,4-Bis(3-aminopropyl)piperazine)-7-hydroxy-5-cholestane SA-8

(40) ##STR00029##

(41) Yd: 48%. .sup.1H NMR: =3.21 (m, 1H), 2.52-2.66 (m, 6H), 0.97-1.95 (m, 63H); .sup.13C NMR: =73.12, 56.42, 52.4, 51.91, 48.70, 47.32, 43.35, 42.55, 41.0, 40.57, 39.77, 36.89, 36.59, 36.40, 36.34, 34.38, 34.14, 32.12, 29.85, 29.37, 28.62, 24.88, 22.41, 22.41, 18.13, 12.33.

3-(1,4-Bis(3-aminopropoxy)butane)-7-hydroxy-5-cholestane SA-9

(42) ##STR00030##

(43) Yd: 42%. .sup.1H NMR: =3.13 (m, 1H), 2.51-2.60 (m, 4H), 0.97-1.98 (m, 63H); .sup.13C NMR: =73.80, 68.33, 68.12, 66.44, 66.06, 57.23, 51.95, 51.72, 48.84, 43.35, 43.02, 41.92, 41.63, 39.32, 38.25, 36.45, 35.77, 35.62, 35.12, 34.80, 33.42, 33.12, 29.87, 28.75, 26.33, 26.01, 22.12, 21.84, 18.63, 11.92.

3-(Norspermino)-7-hydroxy-5-cholestane SA-10

(44) ##STR00031##

(45) Yd: 48%. .sup.1H NMR: =3.13 (m, 1H), 2.55-2.66 (m, 8H), 1.01-2.02 (m, 54H); .sup.13C NMR: =71.9, 57.64, 56.03, 52.39, 47.50, 46.54, 45.98, 42.79, 41.20, 40.78, 40.54, 40.22, 40.10, 37.12, 37.02, 36.27, 36.26, 34.58, 33.81, 29.41, 29.12, 28.23, 25.62, 23.41, 23.12, 22.67, 20.84, 18.04, 13.50, 11.87.

3-(1-(3-Aminopropyl)imidazole)-7-hydroxy-5-cholestane SA-11

(46) ##STR00032##

(47) Yd: 22%. .sup.1H NMR: =3.18 (m, 1H), 2.56-2.66 (m, 4H), 1.09-2.01 (m, 42H); .sup.13C NMR: =139.81, 126.32, 123.27, 71.68, 57.28, 52.39, 47.57, 46.78, 45.98, 42.79, 41.14, 40.88, 40.14, 40.02, 40.00, 37.12, 37.02, 36.26, 36.26, 34.53, 33.82, 29.41, 29.12, 28.23, 25.62, 23.41, 23.12, 22.66, 20.84, 18.14, 13.50, 11.87.

3-(1-(3-Aminopropyl)morpholine)-7-hydroxy-5-cholestane SA-12

(48) ##STR00033##

(49) Yd: 34%. .sup.1H NMR: =3.14 (m, 1H), 2.56-2.66 (m, 6H), 0.94-2.02 (m, 55H); .sup.13C NMR: =72.01, 71.09, 70.95, 55.44, 55.12, 55.03, 52.39, 47.42, 47.24, 45.88, 42.19, 41.00, 40.87, 40.54, 40.22, 40.11, 37.12, 37.02, 36.27, 36.26, 35.58, 34.81, 29.41, 29.72, 28.21, 25.62, 23.41, 23.03, 22.87, 21.82, 19.04, 13.26, 11.19.

II. Evaluation of the Biological Activities

A. Intrinsic Antibacterial Activities of the Compounds of Formula (I)

1) Preparation of the Preculture

(50) Two tubes were prepared:

(51) A negative control (2 ml of sterile culture medium)

(52) A positive control (1940 l of culture medium+40 l of DMSO+20 l of the bacterial suspension) from a defrosted biological strain (the preservation of the biological strains is carried out at 80 C. in glycerol). The strains used are S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, C. albicans CIP 1180-79 and E. faecalis CIP 103015.

(53) The tubes were incubated in an Infors at 37 C. for 24 hours at 100 revolutions per minute.

(54) The microorganisms were handled under a hood in the laboratory of L2 type and, before any handling operation, a UV cycle was programmed and only sterile material was used. A test of toxicity of the solvents (methanol, ethanol, DMSO) was carried out and the latter proved to be nontoxic at concentrations of less than or equal to 2%. The chemical molecules to be tested were prepared in a DMSO/methanol (50/50) mixture at a concentration of 5 mg/ml.

2) Preparation of the Microplate for the Determination of the Minimum Inhibitory Concentration (MIC)

(55) After incubating for 24 h, the optical density was measured using a spectrophotometer at 600 nm by withdrawing 100 l of the bacterial suspension diluted in 900 l of the sterile culture medium. This test required the use of a 96-well plate and the necessary volume of the microbial suspension to be inoculated was calculated for an OD corresponding to a value equal to 0.01 in each well. In this plate, the first line corresponded to the negative control (195 l of sterile culture medium in each well), the second line to the positive control (inoculated culture medium with the addition of 2% of DMSO), the third line was charged twice with bacterial suspension, 8 l of product to be tested was placed in each well. Subsequently, cascade half dilution was carried out starting from this line.

(56) The first column was used as inhibition control. A sterile filter was subsequently placed on the microplate, allowing the passage of gases but not of contaminants. The microplate was incubated at 37 C. in a humid atmosphere for 24 h.

(57) NB: The medium used is the Mueller-Hinton (MH) medium for the bacteria. All the tests were carried out in duplicate.

3) Reading of the Results

(58) After incubation, the filter was replaced with a transparent film and subsequently reading of OD was carried out in an IEMS plate spectrophotometer at 620 nm. A calculation of the minimum inhibitory concentration (MIC) was carried out.

(59) TABLE-US-00001 TABLE I Intrinsic antibacterial activities of the compounds of formula (I) MIC (g/ml) S. S. aureus E. P. aureus Methi- coli aeruginosa Com- ATCC cillin- S. C. ATCC ATCC pounds 25923 resistant faecalis albicans 25922 27853 Squala- 4 4 4 2 12 20 mine SA-1 4 2 2 2 16 32 SA-2 4 2 2 2 32 32 SA-3 5 2.5 2.5 2.5 20 40 SA-4 4 2 2 2 32 32 SA-5 4 4 4 4 32 32 SA-6 5 5 2.5 2.5 20 40

B. Comparison of the Cytotoxicity and of the Antibacterial Activities of the Compounds of Formula (I) with Those of the Compounds (IIa), (IIb), (IIc) and (IId) Disclosed in the Application WO 2011/067501

(60) The WST-1 test was used to measure the cytotoxic activity of the products. This is a colorimetric test which makes it possible to measure the viability and the degree of cell proliferation. It is based on the cleaving of colorless tetrazolium salts WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) by mitochondrial dehydrogenases to give a yellow-colored formazan derivative, which are quantifiable by spectrophotometry at 420-480 nm.

(61) The WST-1 test was carried out on Chinese hamster ovary cells. The CHO-K1 cells (ATCC, USA) are kept cultured in McCoy's 5 A medium additivated with 10% of fetal calf serum, with 2 mM of L-glutamine and with a penicillin/streptomycin mixture (100 U/ml: 10 g/ml). Incubating is carried out at 37 C. under an atmosphere enriched in CO.sub.2 (5%) and subculturing is carried out every two days.

(62) The cells are transferred into 96-well plates (25 000 cells/ml) in complete McCoy's 5 A medium and are maintained at 37 C. for 24 h under a humid atmosphere enriched in CO.sub.2 (5%). Increasing concentrations of test products are added to the wells in duplicate tests and 8 growth controls containing the cells in the medium alone are included in each series of tests. After 24 hours at 37 C. (5% of CO.sub.2), the culture medium is removed, the cells are rinsed in phosphate buffer (PBS) and 50 l of PBS containing 10% of WST-1 reactant are added to each well. After incubating at 37 C. for 20 minutes, the results are read by spectrophotometry at 450 nm.

(63) The results are expressed in the form of dose-response relationships, modeled by a nonlinear regression analysis using the TableCurve software. The 50% Inhibitory Concentration (IC.sub.50) represents the concentration of product capable of reducing cell viability by 50%.

(64) The following compounds (IIa), (IIb), (IIc) and (IId) were prepared by following the synthesis protocol disclosed in the application WO 2011/067501:

(65) ##STR00034##

(66) The minimum inhibitory concentrations (MICs) and the cytotoxicity (IC.sub.50) were evaluated according to the experimental protocols above.

(67) TABLE-US-00002 TABLE II Comparison of the intrinsic antibacterial activities of the compounds of formula (I) according to the invention with those of the compounds of the state of the art MIC (g/ml) S. aureus (Methicillin- IC.sub.50 CHO Compound resistant) E. coli (M) IIa1 or IIb1 5 10 <5 IIa2 or IIb2 3.125 3.125 <5 IIa3 or IIb3 3.125 3.125 <5 IIc1 or IId1 3.125 >50 <5 IIc2 or IId2 12.5 >50 <5 IIc3 or IId3 12.5 >50 <5 SA1 2 16 15 SA2 2 32 50 SA3 2.5 20 32 SA4 2 32 9 SA5 4 32 15 SA6 5 20 46

(68) It is found that the compounds of formula (I) are less cytotoxic than the compounds IIa, IIb, IIc and IId tested and exhibits a greater antibacterial activity than that of the compounds IIc and IId with regard to the E. coli Gram-negative bacteria.

C. Potentiating of the Activity of Conventional Antibiotics in the Presence of the Aminosteroidal Derivatives of Formula (I)

Example of Preparation of the Microplate for the Determination of the Minimum Inhibitory Concentration (MIC) of Doxycycline in the Presence of an Aminosteroidal Derivative

(69) This method requires the use of a 96-well plate; 100 l of a liquid culture medium are deposited in each well and then inoculated with the microbial suspension prepared above. The necessary volume to be inoculated is calculated for an OD of 0.01, which corresponds to approximately 510.sup.6 bacteria in each well. In this plate, the first line corresponds to a negative control (200 l of sterile culture medium in each well), the second line to a positive control (100 l of sterile culture medium+100 l of a bacterial suspension), the third line contains 192 l of culture medium; 8 l of aminosteroidal product to be tested are placed in each well. Subsequently, cascade dilution is carried out starting from this line. 8 l of a doxycycline solution (1 mg dissolved in 20 ml) are subsequently added to each well of lines 3 to 8, in order to obtain a final concentration of antibiotic of 2 g/ml. 92 l of bacterial suspension are subsequently added to lines 3 to 8. The results (the determination of the MIC (2 g/ml of doxycycline) in the presence of X g/ml of aminosteroidal derivative) are read after incubating at 37 C. for 24 h in a humid atmosphere. After incubating at 37 C. for 24 h, 40 l of nitrotetrazolium iodide are added to each well, making it possible to reveal the presence of living bacteria by coloring the medium pink.

(70) In these first tests, the aim was to demonstrate or not a synergy of the aminosteroidal derivatives in the presence of a low concentration of two conventional antibiotics: doxycycline and chloramphenicol.

(71) It should first of all be mentioned that, with regard to the Gram-negative strain of P. aeruginosa (PAO1), doxycycline has an MIC of 40 g/ml and chloramphenicol an MIC of 1024 g/ml.

(72) The use of low amounts of aminosteroidal derivatives makes it possible to restore (to reduce) the necessary concentration of antibiotic to kill the strain under consideration. The results are recorded in the table below:

(73) TABLE-US-00003 TABLE III Potentiating of the activity of the conventional antibiotics in the presence of the aminosteroidal derivatives of formula (I) Concentration of compound necessary in order to restore the activity of the antibiotic under consideration (g/ml) Doxycycline Chloramphenicol concentration used concentration used Compounds (2 g/ml) (4 g/ml) Squalamine 1.75 1.75 SA-1 4 16 SA-2 4 16 SA-3 5 10 SA-6 2.5 20

(74) A very good synergy of certain compounds with doxycycline is observed, thus restoring the activity of this antibiotic at low concentrations of use (2 g/ml). In the case of chloramphenicol, the results are also encouraging since the MIC changes from 1024 to 4 g/ml for fairly low doses of compounds to be added.

REFERENCES

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