Compounds for the treatment of influenza

Abstract

The invention relates to oseltamivir derivatives as influenza neuraminidase inhibitors for treating influenza infections and to a method for producing said compounds.

Claims

1. A compound, wherein the compound is amidoxime (3R,4R,5S)-4-Acetamido-5-[N—(N′-hydroxy)acetimidamido]-3-(1-ethylpropoxy) cyclohex-1-en-1-carboxylic acid ethyl ester having the chemical structure: ##STR00011## or (3R,4R,5S)-4-acetamido-5-(N-acetimidamido)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid having the chemical structure: ##STR00012## and/or pharmaceutically acceptable salts, solvates, R/S enantiomers and/or prodrugs thereof.

2. A composition containing a compound according to claim 1 formulated for administration to a mammal.

3. An influenza neuraminidase inhibitor, wherein the inhibitor is amidoxime (3R,4R,5S)-4-acetamido-5-[N—(N′-hydroxy) acetimidamido]-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester having the chemical structure: ##STR00013## or (3R,4R,5S)-4-acetamido-5-(N-acetimidamido)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid having the chemical structure: ##STR00014## as well as pharmaceutically acceptable salts, solvates or prodrugs thereof.

4. A method for preparing a compound according to the general structural formula ##STR00015## as well as the pharmaceutically acceptable salts, solvates, R/S enantiomers and/or prodrugs thereof, wherein R.sup.1 is H, or a branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1 to 12, R.sup.7 is H or OH, R.sup.8 is H, R.sup.9, NH.sub.2, NHR.sup.9, N(R.sup.9).sub.2 or NHCOOR.sup.1, R.sup.9 is a branched or unbranched, substituted or unsubstituted alkyl having a chain length of 1 to 4 carbon atoms and wherein substituents for R.sup.1 and R.sup.9 are selected from the group consisting of fluorine, chlorine, bromine, iodine, oxygen, sulfur, alkoxy, acyloxy, hydroxyl, mercapto, cyano, nitro, or thio alkoxy group, and a functionality which is blocked with a protecting group, comprising reacting an oseltamivir compound having the chemical formula: ##STR00016## with an acyl hydroximoyl chloride, in an organic solvent at room temperature, to form said compound.

5. A method according to claim 4, wherein the organic solvent is a dichloromethane.

6. A method for preparing a compound according to the general structural formula: ##STR00017## as well as the pharmaceutically acceptable salts, solvates, R/S enantiomers and/or prodrugs thereof, wherein R.sup.1 is H, or a branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1 to 12, R.sup.7 is H or OH, R.sup.8 is H, R.sup.9, NH.sub.2, NHR.sup.9, N(R.sup.9).sub.2 or NHCOOR.sup.1, R.sup.9 is a branched or unbranched, substituted or unsubstituted, alkyl having a chain length of 1 to 4 carbon atoms wherein substituents for R.sup.1 and R.sup.9 are selected from the group consisting of fluorine, chlorine, bromine, iodine, oxygen, sulfur, alkoxy, acyloxy, hydroxyl, mercapto, cyano, nitro, or thio alkoxy group, and a functionality which is blocked with a protecting group, comprising reacting (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester with cyanogen bromide in an organic solvent at room temperature to form a cyanamide derivative of the compound, and reacting the cyanamide derivative compound with hydroxylamine in dioxane at room temperature to form the compound.

7. A method for preparing an influenza neuraminidase inhibitor according to the general structural formula ##STR00018## as well as the pharmaceutically acceptable salts, solvates, R/S enantiomers, or prodrugs thereof, wherein R.sup.1, R.sup.4 and R.sup.5 may be identical or different and is hydrogen, or a branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1 to 12, R.sup.2 is H R.sup.3 is H, OH, or OR.sup.1 R.sup.6 is H, R.sup.1, NH.sub.2, NHR.sup.1, N(R.sup.1).sub.2, NHCOOR.sup.1, X is OR.sup.1 Z is C and wherein substituents for R.sup.1 are selected from the group consisting of fluorine, chlorine, bromine, iodine, oxygen, sulfur, alkoxy, acyloxy, hydroxyl, mercapto, cyano, nitro or thio alkoxy group, and a functionality which is blocked with a protecting group, comprising reacting an oseltamivir compound having the chemical formula: ##STR00019## with an acyl hydroximoyl chloride, in an organic solvent at room temperature, to form said compound.

8. A method according to claim 7, wherein the organic solvent is a dichloromethane.

9. A method according to claim 7, wherein the oseltamivir compound is an amidoxime with R.sup.3 is OH.

10. A method for preparing an influenza neuraminidase inhibitor according to the general structural formula: ##STR00020## as well as the pharmaceutically acceptable salts, solvates, R/S enantiomers, or prodrugs thereof, wherein R.sup.1, R.sup.4 and R.sup.5 may be identical or different and is hydrogen, a branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1 to 12, R.sup.2 is H R.sup.3 is H, OH, or OR.sup.1 R.sup.6 is H, R.sup.1, NH.sub.2, NHR.sup.1, N(R.sup.1).sub.2, NHCOOR.sup.1, X is OR.sup.1 Z is C and wherein substituents for R.sup.1 are selected from the group consisting of fluorine, chlorine, bromine, iodine, oxygen, sulfur, alkoxy, acyloxy, hydroxyl, mercapto, cyano, nitro or thio alkoxy group, and a functionality which is blocked with a protecting group comprising reacting (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester with cyanogen bromide in an organic solvent at room temperature to form a cyanamide derivative of the compound, and reacting the cyanamide compound with hydroxylamine in dioxane at room temperature to form the compound.

11. A method according to claim 10, wherein R.sup.6 is NH.sub.2, NHR.sup.1 or N(R.sup.1).sub.2, and R.sup.3 is OH.

12. The method of claim 4, wherein the compound is amidoxime (3R,4R,5S)-4-Acetamido-5-[N—(N′-hydroxy)acetimidamido]-3-(1-ethylpropoxy) cyclohex-1-en-1-carboxylic acid ethyl ester, or hydroxyguanidine (3R,4R,5S)-4-acetamido-5-[N—(N′hydroxy)-guanidino]-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester, or (3R,4R,5S)-4-acetamido-5-(N-acetimidamido)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid, or (3R,4R,5S)-4-acetamido-5-(N-guanidino)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid and/or pharmaceutically acceptable salts, solvates, R/S enantiomers and/or prodrugs thereof.

13. The method of claim 7, wherein the inhibitor is amidoxime (3R,4R,5S)-4-acetamido-5-[N—(N′-hydroxy) acetimidamido]-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester, or hydroxyguanidine (3R,4R,5S)-4-acetamido-5-[N—(N′hydroxy)guanidino]-3-(1-ethylpropoxy) cyclohex-1-en-1-carboxylic acid ethyl ester, or (3R,4R,5S)-4-acetamido-5-(N-acetimidamido)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid, or (3R,4R,5S)-4-acetamido-5-(N-guanidino)-3-(1-ethylpropoxy) cyclohex-1-en-1-carboxylic acid as well as pharmaceutically acceptable salts, solvates or prodrugs thereof.

Description

MATERIAL AND METHODS: EMBODIMENTS

Syntheses

(3R,4R,5S)-4-acetamido-5-(N-acetimidamido)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester hydrobromide (2)

(1) 1 g of oseltamivir (3.2 mmol) is dissolved in 10 ml of ethanol and the mixture is cooled to 0° C. 1.04 g of S-(naphthylmethyl)acetimidobromid (1.1 equivalents) are added to this solution and then stirred for one hour at room temperature. The mixture is concentrated subsequently in vacuo and taken up in about 80 ml water. This solution is washed with a little diethyl ether and concentrated in vacuo. The product (85%) contains at this point still small amounts of the parent compound, which could be removed by column chromatography (DCM/MeOH, 5-10%) only. Yield: 960 mg (71%) of a white solid.

(2) DC: R.sub.f=0.65 (DCM/MeOH, 9:1)

(3) .sup.1H-NMR (DMSO-d.sub.6, 300 MHz): δ/ppm=0.79 (t, .sup.3J=7.4 Hz, 3H), 0.85 (t, .sup.3J=7.4 Hz, 3H), 1.23 (t, .sup.3J=7.1 Hz, 3H), 1.44 (m.sub.c, 4H), 1.83 (s, 3H), 2.11 (s, 3H), 2.33 (m.sub.c, 1H), 2.67 (dd, .sup.2J=17.6 Hz, .sup.3J=4.7 Hz, 1H), 3.42 (quin, .sup.3J=5.6 Hz, 1H), 3.82 (m.sub.c, 1H), 4.05 (m.sub.c, 1H), 4.17 (q, .sup.3J=7.1 Hz, 2H), 4.35, (m.sub.c, 1H), 6.69 (m.sub.c, 1H), 8.04 (br d, .sup.3J=9.0 Hz, 1H), 8.63 (br s, 1H), 9.25, 9.35 (2×br s, 1H).

(4) MS (ESI): m/z=354 [M+H].sup.+

3R,4R,5S)-4-acetamido-5-(N-acetimidamido)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid (3)

(5) The amidine ethyl ester of oseltamivir (217 mg, 0.5 mmol) in 10 ml of MeOH is mixed with 1.5 ml of a 1 M methanolic KOH (3 equivalents) and stirred for 1 hour at 40° C. until no starting material is longer detectable on the DC. The solution is diluted with water and the pH value is adjusted with 1 M HCl to 7-8. The solution is concentrated to dryness then, and the residue is purified by flash chromatography on reverse phase (RP-18 column, eluent: water, detection: iodine chamber) purified. After lyophilization, the product is isolated as white powder.

(6) Yield: 88% of a fine white powder.

(7) .sup.1H-NMR (D.sub.2O, 300 MHz):

(8) δ/ppm=0.84 (t, .sup.3J=7.4 Hz, 3H), 0.89 (t, .sup.3J=7.4 Hz), 3H, 1.38-1.63 (m, 4H), 2.03 (s, 3H), 2.23 (s, 3H), 2.43 (m.sub.c, 1H), 2.82 (dd, .sup.2J=17.5 Hz, .sup.3J=4.8 Hz, 1H,), 3.53 (quin, .sup.3J=5.4 Hz, 1H), 3.93-4.09 (m, 2H), 4.36 m.sub.c, 1H), 6.71 (br s, 1H).

(9) MS (ESI): m/z=348 [M+Na].sup.+, 326 [M+H].sup.+.

(10) HRMS (ESI): m/z calcd. for C.sub.16H.sub.27N.sub.3O.sub.4 [M+H].sup.+: 326.20743. found: 326.20737.

(3R,4R,5S)-4-acetamido-5-[N—(N′-hydroxy)acetimidamido]-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester (4)

(11) 465 mg of oseltamivir (1.49 mmol), 290 mg of DIPEA (389 μl, 1.5 equiv) are dissolved in 5 ml of dichloromethane, and cooled to 0° C. Freshly prepared acethydroximoyl chloride (209 mg, 1.5 equiv) is added slowly (dropwise) to this solution. The mixture is stirred for four hours at room temperature, mixed with 15 ml of water, stirred for an additional hour and then separated in a separating funnel. In order to increase the yield of the desired amidoxime, the aqueous phase is extracted four times with dichloromethane. The combined organic phases are dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude product is purified by column chromatography on silica gel (DCM/MeOH, 9:1).

(12) Yield: 70% of colorless, crystalline solid

(13) DC: R.sub.f=0.29 (DCM/MeOH, 9:1)

(14) .sup.1H-NMR (DMSO-d.sub.6, 300 MHz):

(15) δ/ppm=0.80 (t, .sup.3J=7.4 Hz, 3H), 0.85 (t, .sup.3J=7.4 Hz, 3H), 1.23 (t, .sup.3J=7.1 Hz, 3H), 1.43 (m.sub.c, 4H), 1.80 (s, 3H), 1.95 (s, 3H), 2.38 (m.sub.c, 1H), 2.62 (dd, .sup.2J=17.4 Hz, .sup.3J=5.0 Hz, 1H), 3.40 (quin, .sup.3J=5.6 Hz, 1H), 3.65 (m.sub.c, 1H) 3.78 (dd, .sup.2J=17.4 Hz, .sup.3J=8.7 Hz, 1H), 4.15 (q, .sup.3J=7.1 Hz, 2H), 4.19 (m.sub.c, 1H), 6.67, (m.sub.c, 1H), 6.81 (br d, 1H, .sup.3J=9.1 Hz), 7.99 (d, 1H, .sup.3J=8.6 Hz), 9.73 (br s, 1H).

(16) MS (ESI): m/z=392 [M+Na].sup.+, 370 [M+H].sup.+, 354 [M−OH+H].sup.+.

(17) HRMS (ESI): m/z calcd. for C.sub.18H.sub.31N.sub.3O.sub.5 [M+H].sup.+: 370.23365. found: 370.23379.

(3R,4R,5S)-4-acetamido-[N—(N′ hydroxy)guanidino]-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester (6)

(18) 213 mg of cyanamide (0.6 mmol) are dissolved in 5 ml of dry dioxane and exactly one equivalent of free hydroxylamine (20 mg) is added. It is stirred for 30 minutes at room temperature, concentrated and, after several fold addition and removal of dichloromethane and diethyl ether, a white solid is obtained.

(19) Yield: 222 mg (100%) of a white solid

(20) DC: R.sub.f=0.20 (EtOAc/MeOH, 6:4)

(21) .sup.1H-NMR (DMSO-d.sub.6, 300 MHz):

(22) δ/ppm=0.80 (t, .sup.3J=7.3 Hz, 3H), 0.84 (t, .sup.3J=7.4 Hz, 3H), 1.24 (t, .sup.3J=7.1 Hz, 3H), 1.40 (m, 4H), 1.83 (s, 3H), 1.99-2.07 (m, 1H), 2.86 (dd, .sup.2J=16.7 Hz, .sup.3J=2.5 Hz, 1H), 3.38 (quin, .sup.3J=5.5 Hz, 1H), 3.49 (m, 1H), 3.80 (m, 1H), 4.01, (m, 1H), 4.14 (q, .sup.3J=7.1 Hz, 2H), 4.24 (m, 1H), 4.92 (s, 2H), 6.64 (m, 1H), 7.72 (br s, 1H), 7.79 (d, .sup.3J=8.8 Hz, 1H).

(23) MS (ESI):

(24) m/z=741 [2M+H].sup.+, 393 [M+Na].sup.+, 386, 371 [M+H].sup.+.

(25) HRMS (ESI): m/z calcd. for C.sub.17H.sub.30N.sub.4O.sub.5 [M+H].sup.+: 371.22890. found: 371.22911.

(3R,4R,5S)-4-acetamido-5-[N—(N′-n-hexyloxycarbonyl)thioureido]-3-(1-ethylpropoxy)-cyclohex-1-en-1-carboxylic acid ethyl ester (7)

(26) 500 mg of oseltamivir (1.6 mmol) are dissolved in 50 ml of dry dichloromethane and equimolar amounts of hexyloxycarbonyl isothiocyanate (from an approximately 0.5 M solution in dichloromethane) are slowly added dropwise. After stirring for 2 hours at room temperature it is washed with 1% HCl, water, NaCl solution. The organic phase is dried over Na.sub.2SO.sub.4 and concentrated on a rotary evaporator. The crude product can be triturated or washed with cyclohexane and is sufficiently pure for the next reaction by this. For elemental analysis, the compound was further purified by silica gel column chromatography (Cy/EtOAc, 6:4).

(27) Yield: 600 mg (75%) of a white-yellowish solid

(28) DC: R.sub.f=0.20 (Cy/EtOAc, 6:4)

(29) .sup.1H-NMR (DMSO-d6, 300 MHz):

(30) δ/ppm=0.79 (t, .sup.3J=7.4 Hz, 3H), 0.84 (t, .sup.3J=7.3 Hz, 3H), 0.87 (t, .sup.3J=6.8 Hz, 3H), 1.23 (t, .sup.3J=7.2 Hz, 3H), 1.30 (m.sub.c, 6H), 1.45 (m.sub.c, 4H), 1.57 (m.sub.c, 2H) 1.80 (s, 3H), 2.30 (dd, 1H, .sup.2J=17.8 Hz, .sup.3J=6.8 Hz), 2.90 (dd, 1H, .sup.2J=17.8 Hz, .sup.3J=5.0 Hz), 3.43 (quin, 1H, .sup.3J=5.4 Hz), 4.07 (m.sub.c, 4H), 4.16 (q, 2H, .sup.3J=7.1 Hz), 4.55 (m.sub.c, 1H), 6.74 (br s, 1H), 7.91 (br d, 1H, .sup.3J=8.0 Hz), 9.98 (d, 1H, .sup.3J=7.6 Hz), 10.90 (s, 1H).

(31) MS (ESI):

(32) m/z=500 [M+H].sup.+, 483, 412

(3R,4R,5S)-4-acetamido-5-[N—(N′-n-hexyloxycarbonyl)-(N″-(2-methoxypropane-2-yl)oxy) guanidino]-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid ethyl ester (8)

(33) 358 mg of oseltamivir hexylthiourea (0.72 mmol) are dissolved in 10 ml of dry dichloromethane and 151 mg of O-(2-Methoxypropan-2-yl)hydroxylamine (2 equivalents), 251 μl DIPEA (2 equivalents), 276 mg of EDCI (2 equivalents) are added. The mixture is stirred for 1.5 days at room temperature, concentrated and worked up by column chromatography over silica gel (DCM/MeOH, 0-2%).

(34) DC: R.sub.f=0.39 (DCM/MeOH, 98:2)

(35) Yield: 374 mg (91%) of a white solid, which is stored at −20° C.

(36) .sup.1H-NMR (DMSO-d.sub.6, 300 MHz):

(37) δ/ppm=0.83 (m.sub.c, 9H) 1.27 (m.sub.c, 12H), 1.27 (m.sub.c, 12H), 1.45 (m.sub.c, 4H), 1.57 (m.sub.c, 2H), 1.80 (s, 3H), 2.30 (dd, 1H, .sup.2J=18.1 Hz, .sup.3J=7.1 Hz), 2.90 (dd, 1H, .sup.2J=18.1 Hz, .sup.3J=5.2 Hz), 3.06 (s, 3H), 3.43 (quin, 1H, .sup.3J=5.6 Hz), 4.06 (m.sub.c, 4H), 4.16 (q, 2H, .sup.3J=7.1 Hz), 4.55 (m.sub.c, 1H), 6.74 (s, 1H), 7.91 (d, 1H, .sup.3J=8.1 Hz), 9.98 (d, 1H, .sup.3J=7.8 Hz), 10.90 (s, 1H).

(38) HRMS (ESI): m/z calcd. for C.sub.28H.sub.50N.sub.4O.sub.8 [M+H].sup.+: 571.37014. found: 571.37034.

(3R,4R,5S)-4-acetamido-5-(N-guanidino)-3-(1-ethylpropoxy)cyclohex-1-en-1-carboxylic acid (9)

(39) a) Pharmacokinetic Characterization of the Inhibitors and Prodrugs

(40) 1. Stability Studies for the Amidine Effective Form (3) and Prodrugs Thereof (2, 4) as Well as and the Guanidine Effective Form (9) and Prodrugs Thereof (6, 10) Over 6 h

(41) The stability tests were carried out in 50 mM potassium phosphate buffer at a concentration of 0.2 mM. For this purpose, a 2 mM stock solution was prepared in 10 mM potassium phosphate buffer pH 7.4 and diluted 1:10 with phosphate buffer of respective pH value. Each compound was tested at pH 2.0, 7.4 and 9.0. For this purpose, every 30 min a sample was analyzed by HPLC and the stability was tested over 6 h. The concentration at t=0 min was set as 100%.

(42) In addition, the substances were tested in human and murine plasma. Therefor, 630 μl of the plasma as mixed with 70 μl of a 2 mM stock solution of each compound in 10 mM phosphate buffer pH 7.4. The incubations were performed in a shaking water bath at 37° C. The incubation was terminated at the times 15, 30, 45, 60, 90 and 120 min by removal of 100 μl sample and addition of 100 μl acetonitrile. The samples were centrifuged (12,000 rpm/10 min) and the supernatant surveyed by HPLC.

(43) The stability studies were analyzed using the following HPLC method.

(44) HPLC Method

(45) TABLE-US-00018 HPLC-System Waters Autosampler 717plus, Waters 600 Controller, Waters 600 Pump, Waters 2487 Dual λ Absorbance Detector and EZChrom Elite Client/Server recording and analysis software (version 2.8.3) Column: LiChrospher 60 RP-select B (125 × 4 mm, 5 μm) with a RP-select B guard column (4 × 4 mm). Flow: 1 ml/min Eluant: for 3, 9 60% 10 mM KH.sub.2PO.sub.4/0.1% TFA pH 3.0 40% MeOH for 2, 4, 50% 10 mM KH.sub.2PO.sub.4/0.1% TFA pH 3.0 6, 10 50% MeOH Running time: 7.5 min Detection: 230 nm Injection 10 μl volume: Retention  4 4.2 ± 0.1 min times:  9 4.4 ± 0.2 min  3 4.5 ± 0.2 min  6 4.6 ± 0.1 min  2 4.8 ± 0.1 min 10 3.7 ± 0.1 min
Stability Studies for the Amidine Effective Form (3) and Guanidine Effective Form (9) Over 2 Weeks
Studies at pH 7.4:

(46) The storage stability in dissolved form was determined at a concentration of 0.2 mg/ml. For this purpose, the compound was dissolved in 50 mM KH.sub.2PO.sub.4 buffer pH 7.4 or in aqua bidest and stored for the investigation period at RT (pH 7.4) or in a refrigerator at 4° C. (pH 7.4 or aqua bidest). The concentration of the effective form was determined by HPLC after 12 h, 1 d, 2 d, 4 d, 7 d and 14 d.

(47) 2. Solubility Assays of the Compounds Tested

(48) Determining the Solubility at Different pH Values:

(49) The solubility of the compounds was determined in phosphate buffer at different pH values (2.0, 7.4 and 9.0). Therefor, a few mg of the compounds were weighed and mixed with the volume of 50 mM KH.sub.2PO.sub.4 buffer of respective pH value for a 50 mM solution. If the compound had not completely dissolved, the suspension was shaken for 30 min. Subsequently, the undissolved portion was removed by centrifugation at 10.000 rpm for 15 min, and the concentration in the supernatant was determined by HPLC.

(50) 3. Determination of Protein Binding of the Amidine Effective Form (3) as Well as of Prodrugs Thereof (2, 4) and of the Guanidine Effective Form (9) as Well as of Prodrugs Thereof (6, 10)

(51) The plasma protein binding was carried out in three different concentrations (10, 25, and 50 μM). A 4% albumin solution was used as protein solutions. In each case, 50 μl of a 10-fold concentrated substance solution were pipetted in 450 μl of the protein solution. The incubation was carried out for 15 minutes in the shaking water bath at 37° C. Subsequently, the samples were transferred in ultra-filtration units (Vivaspin 500, 10 kDa cut-off) and centrifuged for 15 min at 10.000 rpm. The filtrate was analyzed by HPLC. In addition, a control was run for each concentration, which was not treated with protein and not centrifuged. Another control without protein supplement, however centrifuged through the filtration unit, showed that the prodrugs are not retained by the membrane and were used to validate the methodology.

(52) In addition, the protein binding of the two effective forms of oseltamivir-amidine (3) and oseltamivir-guanidine was (9) examined in human plasma. Therefor, instead of the 4% albumin solution, human plasma was used. A protein binding of 3.7±1.4% for compound 3 and 8.6±3.0% for compound 9 was determined. As anticipated, the values are somewhat higher than the values obtained with the 4% albumin solution and are due to the presence of other plasma proteins (e.g., α.sub.1-acidic glycoprotein) besides the albumin.

(53) 4. Investigation of the Bioactivation of the Various Prodrugs (2, 4, 6, 10)

(54) Determination of the Activation of the Prodrugs with Different Subcellular Enzyme Systems:

(55) The activation of the prodrugs into their effective forms was determined in vitro by subcellular enzyme preparations. As enzyme preparations 9000G supernatants, microsomes and mitochondria from human and porcine liver tissues were used. The incubation mixtures were composed of 500 μM prodrug, 500 μM NADH, 1 U of esterase and 0.3 mg of enzyme preparation dissolved in 150 μl 100 mM phosphate buffer pH 6.3. The incubation was carried out for 30 min at 37° C. in the shaking water bath. By adding 150 μl acetonitrile, the incubation was terminated. Afterwards, the samples were shaken for 10 min and the precipitated protein removed by centrifugation at 10.000 rpm for 15 min. The supernatant was measured with the help of HPLC.

(56) HPLC Method for the Determination of the Effective Forms (3/9) in Addition to the Prodrugs (2, 4/6, 10)

(57) TABLE-US-00019 HPLC system Waters Alliance HPLC system with Waters e2695 XC separations module, Waters 2998 photodiode array detector and Empower 2 software Column: LiChrospher 60 RP-select B (125 × 4 mm, 5 μm) with C18 guard column (4 × 4 mm) Flow: 1 ml/min Mobile phase: 70% 10 mM KH.sub.2PO.sub.4/0.1% TFA pH 6.5 30% MeOH Running time: 12 min Detection: 210 nm Injection 10 μl volume: Retention  3  4.8 ± 0.2 min time:  9  4.7 ± 0.2 min 10 24.8 ± 0.4 min  6 25.2 ± 0.3 min  4 26.6 ± 0.3 min  2 26.6 ± 0.3 min
Antiviral Effectivity
Determination of the Antiviral Effectivity in the Chemiluminescence Based Neuraminidase (NA)—Inhibition Assay

(58) Following influenza viruses were used for the studies:

(59) H1N1 viruses: A/Jena/5258/2009, A/Jena/5555/09, A/HH/1580/09, A/342/2009 (oseltamivir resistant)

(60) H.sub.2N.sub.3 viruses: Hong Kong/8/68, Saxony/6/02, Berlin/10/04, Rhineland-Palatinate/3911/03.

(61) The inhibition of viral neuraminidase by the test compounds 3 and 9 as well as by the control substances was checked using the commercially available NA-Star kit (Tropix, Applied Biosystems, Darmstadt).

(62) According to the recommendations of the manufacturer, the optimal dilution of the test viruses for the subsequent inhibition assay was determined in a preliminary test first. For this, the virus suspensions were diluted in NA-Star buffer (dilution factor of 3) in the absence of neuraminidase inhibitors (NAI). The virus dilution leading to a signal and background ratio of 40:1 was subsequently used in the NA inhibition assays for determining the 50% inhibitory concentrations.

(63) In NA-inhibition assay for the 6 virus controls per plate 25 μl assay buffer or 25 μl of the test (three parallels per dilution) or the control substance (two parallels per dilution) in assay buffer were applied into the individual wells of the microtiter plate with 96 wells. Then, 25 μl of a virus dilution was added to each well. After a 20 minute incubation time at 37° C., the substrate was diluted 1:500 in assay buffer and 10 μl was added to each well respectively. The measurement of chemiluminescence was performed 30 min later in a plate reader (microtiter plate luminometer, Dynex Technology). For the evaluation of the assays, the average of the measured chemiluminescence of the 6 untreated virus controls was taken as 100% value for the NA activity and used for the calculation of the relative NA-activity of the substance treated individual wells. From the obtained average dose-response curve of two independent assays, subsequently the 50% inhibitory concentration (IC50) of the test and control substances was calculated by linear interpolation in EXCEL.

(64) Determination of the Antiviral Effectivity of the Test Substances in Virus Yield (VY)-Inhibition Assay

(65) Cells: MDCK cells

(66) Influenza viruses: a) A/Jena/5258/2009 (pandemic H1N1; oseltamivir-sensitive)

(67) b) A/342/2009 (H1N1; oseltamivir-resistant)

(68) By adding antiviral effective agents (100 μl/well; 3 parallels/concentration/test substance and two parallels/concentration/control substance, dilution factor of 10) virus replication can be selectively inhibited. This can be determined experimentally on the basis of reduced viral titer in the supernatant.

(69) In the assay, 2 days old closed cellular monolayers were inoculated with a dose of virus, which leads 48 hours post infection to an incomplete cytopathic effect in the 3 untreated virus controls. After an incubation for one hour at 37° C., the virus which was not bound to the cells was removed by 3 consecutive washings of each well and 100 μl test medium (cell and virus controls) or of the substance dilutions was added. Following a 48 hour incubation at 37° C., the supernatants of each well were removed for the subsequent determination of the virus titer.

(70) The determination of the virus titer was performed in 2 days old MDCK cell monolayers in microtiter plates. First, logarithmic dilution series (maximum dilution factor of 10; maximal dilution 10.sup.−7) were created from the supernatants from the VY inhibition assay for this purpose. These were inoculated on cells (4 wells/virus dilution respectively) and incubated for 4 days at 37° C. During this time the cytopathic effect was formed. After fixing and staining the cells with a crystal violet formalin solution, the visual evaluation was carried out on a light box.

(71) Subsequently, the virus titers were calculated according to Reed and Muench. The average of the virus titers of the three virus controls was taken as 100% for the calculation of the titer reduction.

(72) Determination of the Antiviral Effectivity of the Test Substances in cpE Inhibition Assay

(73) The replication of the viruses used in the assay leads through a strongly pronounced cytopathic effect (cpE) to a total destruction of the host cells. By adding antiviral effective substances (100 μl/well; 3 parallels/concentration, dilution factor of 2) the virus-induced cpE can be selectively inhibited. In the assay, untreated and substance-treated enclosed cell lawns were inoculated with a dose of virus that leads 48 h after infection to a complete cpE in the untreated virus controls. At this time, the remaining adherent cells were fixed and stained with a crystal violet/formalin solution. After dye elution, the inhibition of virus-induced cpE was quantified photometrically in a Dynatech plate reader.

(74) Calculating the antiviral effect was carried out by comparing the optical densities of the substance-treated and untreated, virus infected cells with the average optical density of the cell controls, which was set as 100%. Based on the mean dose-response curve of 2 experiments, the dilution was calculated by linear interpolation in EXCEL, which prevented the formation of the virus-induced cpE by 50% (IC50).

(75) Cytotoxicity Assay for Determination of the 50% Cytotoxic Dose (CC.sub.50) of the Test Substances in MDCK (Madin Darby Canine Kidney) Cell Lawn

(76) MDCK cells were seeded in microtiter plates and incubated for 48 h in an incubator at 5% CO.sub.2, 37° C. and 95% humidity to form a closed cell lawn. Thereafter, the medium was removed and the substances were applied in culture medium in various concentrations (100 μl/well, 3 parallels/concentration, dilution factor 2). For control value determination (six untreated cell controls) 100 μl medium were used respectively. 72 h after substance administration and incubation the staining of the cells is carried out with crystal violet/methanol. After the dissolution of the dye, the optical density (OD) of each well was measured in a plate photometer from Dynatech (550/630 nm) and compared with the average of the cell controls. The average of the controls was taken as 100%.

(77) Animal Study

(78) Operation/Preparation of Animals

(79) Sprague Dawley (SD) rats were supplied for habitation 10 days before the start of the experiment, weighing ˜300-350 g and kept in an air conditioned room with a constant temperature of 20° C. and a humidity of 50%. In this room there was a day-night rhythm of twelve hours. The dark phase began daily at 18 o'clock and turned over to the light phase at 6 clock. The rats were kept over the acclimatization period in standard cages of the size 3 (length: 42 cm, width 26 cm, height: 15 cm) and transferred three days before commencement of an experiment into a special experimental room, placed in the identical environmental conditions. They received a maintenance diet (maintenance diet for rats and mice; No. 1320; Altromin) and tap water ad libitum.

(80) The animal experiments described herein were conducted according to the “NIH Guideline” and the corresponding policy on handling and use of experimental animals after approval by the Ministry of Agriculture, Environment and Rural Areas of Schleswig-Holstein.

(81) A catheter was implanted in the vein as well as in the arteria femoralis of rats receiving an i.v. administration. Rats receiving only oral administration of substances received a venous catheter only.

(82) The rats were anesthetized with pentobarbital (60 mg/kg i.p.) and were additionally narcotized with diethyl ether in case of insufficient depth of anesthesia. After shaving the neck area and the right inguinal region, the rats were placed on an electrical heating stage (EBERLE, type 52102) to maintain body temperature in the supine position and the hind legs were fixed. Along the groin an about 1.5 cm long incision was set. Subsequently, the vascular strand of the arteria femoralis, vena femoralis and nervus femoralis was set free by blunt preparation for a length of about 1 cm.

(83) After separation of the vena femoralis, a cotton thread was placed proximally around it and the vessel was closed reversibly by tightening. Approx. 5 mm in distal direction the vessel has been ligated by means of a second thread, so as to create a congestion. With a vascular scissors, a small incision was carried out in the vessel in the area of the congestion (about ⅓ of the total congestion length from the distal ligature away) and polyethylene tube (length: 26 cm; ID: 0.58 mm, OD 0.96 mm) filled with a heparin solution (250 IU/ml) was introduced 3 cm towards proximal up to the vena cava by means of a container spreader. With the proximal and distal threads the catheter was fixed to the vessel.

(84) The arteria femoralis was, in contrast to the vena femoralis, at first closed by a distal ligature and then impounded proximally by tightening the thread. Here, too, a catheter was implanted as described above. Due to the small inner diameter of the artery, a specially made artery catheter was used, consisting of a polyethylene tube (length: 26 cm, ID: 0.58 mm, OD 0.96 mm) and of a 3 cm long welded polyethylene tube (ID: 0.28 mm, OD: 0.61 mm).

(85) After including the catheter, the animal was placed in the prone position and a 5 mm wide incision was placed in the neck. By means of a metal rod and a tube, the catheters, which were sealed with wire pins, were pulled from the ledge to the neck, fixed in the neck with cotton thread and cut to approximately 3 cm in length.

(86) In the supine position again, the subcutaneous fat and then the epidermis were first sewn together with three to four double buttonhole stitches and disinfected with Betaisadonna® solution. On the following days the catheters were rinsed in the morning and the evening with 300 μl heparin solution (250 IU/ml) each. The operated rats were kept from the day of catheter insertion individually in experimental cages made of plexiglas with the dimensions height: 20 cm, width: 22 cm, and length: 25 cm or kept in standard cages of size 3.

(87) The catheterized animals were kept after surgery for a day in the experimental room and individually in their experimental cages. The application of the test compounds was carried out on the second day after the operation. On the experimental day, the rats were weighed one hour prior to the test and the arterial catheter was flushed with 300 μl of heparin solution. Subsequently, the i.v. or oral administration of the compounds was carried out.

(88) Implementation of the Animal Study

(89) The oseltamivir derivative 3 was administered intravenously to 5 rats at a concentration of 10 mg/kg. Oral administration of the neuraminidase inhibitors (2, 4) has been carried out to 5 or 6 rats at a dosage of 50 mg/kg. In addition, derivative 3 was administered (50 mg/kg) to 3 rats orally. The oral administrations were performed as a suspension or solution made with gum arabic (10% w/v) by gavage.

(90) The oseltamivir derivative 9 was administered intravenously to 5 rats at a concentration of 10 mg/kg. Oral administration of the neuraminidase inhibitor (6, 10) has been carried out at 4, or 5 rats at a dose of 50 mg/kg. Additionally, derivative 9 was administered (50 mg/kg) to 3 rats orally. The oral administrations were made as suspension or solution with gum arabic (10% w/v) by gavage.

(91) After i.v. administration, plasma samples were taken at 5, 10, 20, 45, 90, 150, 240 and 360 min, respectively after oral administration after 30, 60, 90, 120, 180, 240 and 360 min. To this, in each case 300 μl of whole blood were taken using an insulin syringe and transferred into EDTA-coated Microvettes CB 300 (Sarstedt, Nümbrecht). After each withdrawal it was rinsed with 100 μl 0.9% saline or every 60 min with heparin solution (250 IU/ml). The blood sample was shaken briefly and put until centrifugation (4° C.; 14000 U/min; 10 min) on ice. Subsequently, the samples were frozen at −80° C.

(92) The killing was carried out by decapitation 6 h after drug administration with a guillotine. In the following, the organs were removed. All organs were cleaned and frozen in dry ice cooled 2-methyl butane. Liver, kidney, and lung were harvested.

(93) Analysis of Plasma Samples

(94) The plasma samples were processed and analyzed by HPLC. Therefor, the plasma samples were thawed at room temperature. In each case, 80 μl methanol (+0.2% TFA) were prepared and subsequently 80 μl of the plasma samples were pipetted into. The samples were shaken for 45 min to precipitate plasma proteins. The samples were frozen at −80° C., thawed and shaken for another 15 min. The samples were centrifuged for 15 min at 13.000 RPM and the supernatant was transferred into HPLC vials. In each case 50 μl were used for the determinations by LC/MS.

(95) The animal studies were evaluated using the following LC/MS method.

(96) LC/MS Method

(97) TABLE-US-00020 HPLC System: Agilent 1100 binary pump, Agilent 1100 diode array detector, Agilent 1100 well-plate autosampler, Degasser G1322A Column: LiChrospher 60 RP-select B (125 × 3 mm, 5 μl) with a RP-select; B guard column (4 × 4 mm) Mass spectrometer Esquire-LC Interface: ESI (electron impact ionization) Nebulizer: 40.0 psi Dry gas: 8.0 ml/min Dry temperature: 350° C. HV capillary 5000 V Mobile phase: A 0.1% TFA in aqua bidest (pH 2.5) B 0.1% TFA in MeOH Gradient profiles: time A [%] B [%]  0 55 45  8 25 75 10 25 75 11 55 45 17 55 45 Flow rate: 0.3 ml/min Running time: 17 min Detection: PDA (190-400 nm) Injektion volume: 50 μl Retention times:  3 5.1 ± 0.3 min  9 5.1 ± 0.3 min 11 5.2 ± 0.3 min 12 5.2 ± 0.3 min

BRIEF DESCRIPTION OF THE DRAWINGS

(98) FIG. 1: Stability of some compounds of the invention at different pH values in murine or human plasma

(99) FIG. 2: Stability of the active forms of A) 3 and B) 9 in different media and with different temperatures

(100) FIG. 3: Plasma levels of derivative 3 after intravenous administration of derivative 3 into 5 rats in total

(101) FIG. 4: Plasma levels of derivative 3 after oral administration of derivative 4

(102) FIG. 5: Plasma levels of derivative 3 and metabolite 11 after oral administration of derivative 4

(103) FIG. 6: Plasma levels of derivative 3 after oral administration of derivative 3

(104) FIG. 7: Plasma levels of derivative 3 after oral administration of derivative 2

(105) FIG. 8: Plasma levels of derivative 3 after administration of amidine based neuramidase inhibitors

(106) FIG. 9: Plasma levels of derivative 9 after i.v. administration of derivative 9

(107) FIG. 10: Plasma levels of derivative 9 after oral administration of derivative 9

(108) FIG. 11: Plasma levels of derivative 9 after oral administration of derivative 6

(109) FIG. 12: Plasma levels of derivative 9 and metabolite 12 after oral administration of derivative 6

(110) FIG. 13: Plasma levels of derivative 9 after application of the guanidine based neuramidase inhibitors

LITERATURE

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