Aminophosphinic derivatives that can be used in the treatment of pain

Abstract

The present invention relates to a compound of the following general formula (I): R.sub.1NHCH(R.sub.2)P(O)(OR.sub.3)CH.sub.2C(R.sub.4)(R.sub.5)CONHCH(R.sub.6)COOR.sub.7 (I) or a pharmaceutically acceptable salt of the latter, an isomer or a mixture of isomers in any proportions, especially a mixture of enantiomers, and in particular a racemic mixture, for which R.sub.1 represents a C(O)OC(R.sup.8)(R.sup.9)OC(O)R.sup.10 group; R.sub.2 represents an optionally substituted hydrocarbon-based chain, an aryl or heteroaryl group or a methylene group substituted by a heterocycle; R.sub.3 represents a hydrogen atom or a C(R.sup.12)(R.sup.13)OC(O)R.sup.14 group; R.sub.4 and R.sub.5 form, together with the carbon that bears them, a saturated hydrocarbon-based ring or an optionally substituted piperidine ring or R.sub.4 represents a hydrogen atom and R.sub.5 represents a phenyl or a benzyl that is optionally substituted, a heteroaromatic ring or a methylene group substituted by a heterocycle; R.sub.6 represents an optionally substituted hydrocarbon-based chain or a phenyl or a benzyl that is optionally substituted; and R.sub.7 represents a hydrogen atom or a benzyl, alkyl, heteroaryl, alkylheteroaryl, CHMe-COOR.sup.18, CHR.sup.19OC(O)R.sup.20 and CHR.sup.19OC(O)OR.sup.20 group. The present invention also relates to the use of these compounds as a medicinal product, and in particular for the treatment of pain, more advantageously neuropathic and neuroinflammatory pain, to their method of synthesis and also to the compositions containing them.

Claims

1. A compound having the following general formula (I):
R.sub.1NHCH(R.sub.2)P(O)(OR.sub.3)CH.sub.2C(R.sub.4)(R.sub.5)CONHCH(R.sub.6)COOR.sub.7(I) or a pharmaceutically acceptable salt thereof, where: R.sub.1 represents the group (CO)OCHMe-OC(O)CHMe.sub.2; R.sub.2 represents a linear or branched saturated hydrocarbon chain, comprising 1 to 6 carbon atoms; R.sub.3 represents a hydrogen atom; R.sub.4 represents a benzyl substituted on the phenyl nucleus by a phenyl group or a heteroaromatic 5- or 6-membered ring; R.sub.5 represents a hydrogen atom; R.sub.6 represents a linear or branched saturated hydrocarbon chain, comprising 1 to 6 carbon atoms; and R.sub.7 represents a radical selected from the group consisting of a hydrogen atom, a benzyl, and CHR.sup.18OC(O)OR.sup.19, wherein R.sup.18 and R.sup.19 represent, independently from each other, an alkyl group.

2. The compound according to claim 1, wherein R.sub.7 represents a hydrogen atom, a benzyl, or a CH(CH.sub.3)OC(O)OEt.

3. The compound according to claim 1, wherein it is selected from the group consisting of the following compounds: 2-(2-Biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid benzyl ester; 2-(2-Biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid; Ethoxycarbonyloxy 2-(2-Biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid ester.

4. A method for treating pain comprising the administration of an effective quantity of at least one compound having the following general formula (I):
R.sub.1NHCH(R.sub.2)P(O)(OR.sub.3)CH.sub.2C(R.sub.4)(R.sub.5)CONHCH(R.sub.6)COOR.sub.7(I) or a pharmaceutically acceptable salt thereof, where: R.sub.1 represents the group (CO)OCHMe-OC(O)CHMe.sub.2; R.sub.2 represents a linear or branched saturated hydrocarbon chain, comprising 1 to 6 carbon atoms; R.sub.3 represents a hydrogen atom; R.sub.4 represents a benzyl substituted on the phenyl nucleus by a phenyl group or a heteroaromatic 5- or 6-membered ring; R.sub.5 represents a hydrogen atom; R.sub.6 represents a linear or branched saturated hydrocarbon chain, comprising 1 to 6 carbon atoms; and R.sub.7 represents a radical selected from the group consisting of a hydrogen atom, a benzyl, and CHR.sup.18OC(O)OR.sup.19, wherein R.sup.18 and R.sup.19 represent, independently from each other, an alkyl group.

5. A pharmaceutical composition comprising at least one compound having formula (I) according to claim 1 and at least one pharmaceutical acceptable vehicle.

6. The pharmaceutical composition according to claim 5, wherein the vehicle is suitable for oral administration.

7. The pharmaceutical composition according to claim 5, further comprising at least one further active substance analgesic selected from the group consisting of morphine, tetrahydrocannabinol, or a P2X3 purinergic receptor antagonist.

8. A pharmaceutical composition comprising: (i) at least one compound having formula (I) according to claim 1, and (ii) at least one further active substance, an analgesic selected from the group consisting of morphine, tetrahydrocannabinol, or a P2X3 purinergic receptor antagonist, as combination products for simultaneous, separate or staggered use.

9. A method for treating neuropathic or inflammatory to the pain caused by diabetes mellitus type I or II, viral or retroviral infection, cancer chemotherapy, radiotherapy, a surgical procedure including amputee illusion and the after-effects of mastectomy, alcoholism, facial neuralgia, trauma, radiculopathy or radiculagia, cruralgia or thoracic outlet syndrome, fibromyalgia, restless leg syndrome, inflammatory joint pain caused particularly by arthritis or an acute phase of arthrosis, degenerative joint pain caused particularly by arthrosis, or lumbago, comprising the administration of an effective quantity of at least one pharmaceutical composition according to claim 5 to a patient requiring same.

10. The pharmaceutical composition according to claim 5, wherein the vehicle is suitable for administration by the oral, sublingual, parenteral, subcutaneous, pulmonary, nasal, intramuscular, intravenous, intrathecal, intra-articular or transdermal route.

11. The pharmaceutical composition according to claim 10, comprising at least one further active substance analgesic selected from the group consisting of morphine, tetrahydrocannabinol, or a P2X3 purinergic receptor antagonist.

12. A method for treating pain comprising administering an effective quantity of at least one pharmaceutical composition according to claim 5 to a patient in need thereof.

13. The compound according to claim 1, wherein R.sub.6 represents a methyl group.

14. The compound according to claim 2, wherein R.sub.7 represents a hydrogen atom.

15. The method according to claim 4, wherein the pain is selected from the group consisting of sharp pain, neuropathic and neuroinflammatory pain.

Description

FIGURES

Key of Figures

(1) In all the figures, the statistical analyses (p, Student's test) are indicated as follows:

(2) .star-solid.p<0.1 versus control

(3) .star-solid..star-solid.p<0.01 versus control

(4) .star-solid..star-solid..star-solid.p<0.001 versus control

(5) FIG. 1 represents the total paw licking time (expressed in seconds during the 5 min examination period) by a mouse receiving formalin as a function of time (minutes) after oral administration of the vehicle () or a compound according to the invention (.square-solid.), graphs (A), (B) and (C) corresponding to the compounds from examples 8, 3 and 4, respectively.

(6) FIG. 2 represents the total paw licking time (expressed in seconds during the 5 min examination period) by a mouse receiving formalin as a function of time (minutes), 90 min after oral administration: (A) of the vehicle () or of compound 8 (50 mg/kg) according to the invention (.square-solid.) or (B) of the vehicle () or of the reference molecule (100 mg/kg) (.square-solid.).

(7) FIG. 3 represents the response obtained in a neuropathic pain model induced by partial and unilateral ligation of the sciatic nerve of a mouse (Plantar test), for evaluating thermal hyperalgia. After oral administration of the vehicle () or the compound from examples 3 (50 mg/kg) (.square-solid.), paw withdrawal is measured (in seconds) as a function of time (in minutes) 14 days after surgery. Graph (A) represents the response observed for the ipsilateral paw, whereas graph (B) represents the response obtained for the contralateral paw.

(8) (mean of contralateral paws at 90 min: vehicle (7.85), compound 3=9.4 s)

(9) FIG. 4 represents the response obtained in a Von Frey test (allodynia measurement) at a pressure (in g) of a mouse paw using filaments of increasing hardness, as a function of time (min) 14 days after surgery and after oral administration of the vehicle () or the compound from example 3 (50 mg/kg) (.square-solid.). Graph (A) represents the response observed for the ipsilateral paw, whereas graph (B) represents the response obtained for the contralateral paw.

(10) FIG. 5 represents the responses obtained at 60 min in a Von Frey test, as described in the key in FIG. 4. The compound from example 4 is administered alone orally at a dose of 10 mg/kg () and gabapentin, also orally at a dose of 30 mg/kg (). These two compounds are administered orally, in association, at the above doses (). The control () corresponds to the oral administration of the vehicle.

EXAMPLES

(11) The following examples are used to illustrate the invention without limiting same. The following abbreviations have been used:

(12) TLC Thin Layer Chromatography

(13) HPLC High Performance Liquid Chromatography

(14) DMSO Dimethylsulphoxide

(15) eq. Equivalent

(16) ESI Electrospray ionisation

(17) min Minutes

(18) NMR Nuclear Magnetic Resonance

(19) TFA Trifluoroacetic acid

Example 1: (R)(1-Benzyloxycarbonylamino-ethyl)-phosphinic acid

Step 1: (1-((diphenylmethyl)amino-ethyl)phosphinic acid

(20) A mixture of 200 g (0.91 mole) of diphenylmethylamine hydrochloride in 600 ml of water and 132 ml (1.0 mole) of phosphinic acid (50% in water) is boiled under reflux with stirring. 56 ml (1.0 mole) of acetaldehyde in solution in 350 ml of water is added drop by drop in 30 min.

(21) The reaction is followed by HPLC. After 2 hours, the reaction is returned to ambient temperature. The white precipitate obtained is filtered, washed with water (2300 ml) and acetone (2300 ml) and dried. White solid, 225 g (90%).

Step 2: (1-aminoethyl)-phosphinic acid

(22) A mixture of 225 g of the compound from step 1 and 2 l of 6N HCl is boiled under reflux for 5 hours. After cooling, the reaction medium is concentrated by half and extracted with 31.5 l of ethyl ether. The solution is evaporated to dryness and the oily residue (130 g) is taken up with 1.3 l of ethanol. The solution is cooled to 0 C. and 450 ml of propylene oxide is added. A white solid precipitates. The precipitate is dewatered, washed with ethanol (2100 ml), ethyl ether (2100 ml) and dried. White solid, 65 g (73%).

(23) (1H) NMR DMSO d6 (ppm): 1.26 (3H d, d); 3.32 (1H m); 6.98 (1H d); 8.23 (3H s).

Step 3: (R)(1-benzyloxycarbonylamino-ethyl-phosphinic acid

(24) In 300 ml of water, 65 g (0.59 mole) of the compound from step 2 is solubilised and the pH is adjusted to 93.5 by adding sodium hydroxide. 85 ml of benzyl chloroformiate is added under stirring. After 1 hour at 0 C., the mixture is returned to ambient temperature and is poured onto a mixture of ice (1 l) and concentrated HCl (300 ml). The white precipitate formed is dewatered, washed with water (2100 ml) and dried. White solid, 131 g (91%).

(25) HPLC(CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 30:70, Kromasil C18 column), Rt 4.6 min.

(26) The resolution of the racemic phosphinic acid is performed by recrystallisation of the salt obtained with the (R)(+) -methylbenzylamine, as described in Baylis et al. (J. Chem. Soc. Perkin Trans 11984, 2845) in an ethyl acetate/isopropanol mixture=3.5:1. White solid, 48 g (86%).

(27) (1H) NMR DMSO d6 (ppm): 1.19 (d, 3H); 3.66 (m, 1H); 5.03 (s, 2H); 6.78 (d, 1H); 7.35 (s, 5H); 7.62 (d, 1H)

Example 2: 2-biphenyl-4-ylmethyl-acrylic acid benzyl ester

Step 1: 2-biphenyl-4-ylmethyl-malonic acid

(28) The protocol used is that described in Organic Synthesis Coll. Vol. 3 p. 337. Using 48.4 g of diethylmalonate and 50 g of diphenyl-4-carboxaldehyde, 88.2 g of diethyl 2-biphenyl-4-ylmethylene-malonic acid ester is obtained.

(29) This compound (88 g) is placed in solution in ethanol (500 ml) and the double bond is reduced by the action of sodium borohydride (10.3 g). After 1 hour, the reduction is completed and the reaction mixture is diluted in ethanol (500 ml) and 1.36 l of 1N NaOH is added at 0 C.

(30) After evaporating the ethanol and acidifying the aqueous phase, 2-biphenyl-4-ylmethyl-malonic acid is obtained in the form of a white solid, 64.8 g (88%).

(31) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 30:70, Kromasil C18 column), Rt 3.5 min.

Step 2: 2-biphenyl-4-ylmethyl-acrylic acid

(32) The diacid obtained in step 1 (60 g) is solubilised in 300 ml of THF and 23.5 ml (2 eq.) of diethylamine and 19.5 ml (4 eq.) of formaldehyde are added. The reaction medium is boiled under reflux with stirring for 20 hours. After cooling, the THF is evaporated and the residue is taken up with ethyl acetate (500 ml) and acidified with 100 ml of 6N HCl. The organic phase is retrieved, washed with 2350 ml of water, 1300 ml of sat. NaCl and dried over Na.sub.2SO.sub.4. A white powder is obtained, 51 g (97%).

(33) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 30:70, Kromasil C18 column), Rt=6.2 min.

Step 3: 2-biphenyl-4-ylmethyl-acrylic acid benzyl ester

(34) 2-biphenyl-4-ylmethyl-acrylic acid (30 g) is suspended in 300 ml of acetonitrile. 21 g of K.sub.2CO.sub.3 followed by 16.5 g of benzyl bromide are added and heated to 80 C. for 15 hours. The reaction medium is evaporated and the residue taken up with ethyl acetate. The organic phase is washed with a Na.sub.2CO.sub.3 solution, water and with a saturated NaCl solution, and dried on Na.sub.2SO.sub.4. After filtration, the organic phase is evaporated to dryness. Oily product, 40 g (96%).

(35) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 80:20, Kromasil C18 column), Rt=12.7 min.

(36) NMR DMSO d6 (ppm): 3.6 (2H s); 5.1 (2H s); 5.5 (1H d); 6.2 (1H d); 7.1-7.5 14H m).

Example 3: 2-(2-biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid benzyl ester

(37) ##STR00009##

Step 1: 3-[(2-benzyloxycarbonyl-3-biphenyl-4-yl-propyl)-hydroxy-phosphinoyl]-butyric acid benzyl ester

(38) The aminophosphinic synthon from example 1 (9.6 g) and the 2-biphenyl-4-ylmethyl-acrylic acid benzyl ester from example 2, step 3 (14.3 g) are added to 21 ml of bis-trismethylsilylacetamide (BSA) and the reaction mixture is stirred for 5 hours at 70 C. After cooling, the mixture is diluted in ethyl acetate. A few drops of water are added until a precipitate is formed. The suspended solid is stirred for 1 hour at ambient temperature and is dewatered, washed with ethyl acetate and vacuum-dried. 11.6 g (79%) of (R)(S) configuration diastereoisomer is obtained.

(39) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 55:45, Kromasil C18 column), Rt=17.8 min.

(40) 1H NMR (DMSO) d6 (ppm) 1.26 (3H dd); 1.81-2.16 (2H m); 2.78-3.12 (3H m); 3.78 (1H qt); 4.94-5.08 (22H 2 q); 7.12-7.68 (19H arom.) +NH (m).

Step 2: 1-[(3-biphenyl-4-yl-2-carboxy-propyl)hydroxy-phosphinoyl]-ethyl ammonium hydrobromide

(41) The compound from step 1 (10.6 g) is suspended in THF (100 ml) and 7.4 g of sodium hydroxide in solution in 50 ml of water is added with stirring at 0 C. The mixture is stirred at ambient temperature overnight. The THF is evaporated, the aqueous phase is acidified to pH 1 with 1N HCl, and extracted with ethyl acetate. The organic phase is dried on Na.sub.2SO.sub.4, filtered and evaporated to dryness. White solid, 7.4 g (83%).

(42) The acid obtained (6 g)) is placed in solution in 60 ml of hydrobromic acid (45% in CH.sub.3CO.sub.2H) and the solution is stirred for 1 hour at ambient temperature.

(43) The reaction mixture is then evaporated to dryness and vacuum-dried to produce an orangey gummy product, 6.9 g (99%).

Step 3: 2-biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionic acid

(44) The compound from step 2 (6.23 g) is suspended in acetonitrile (70 ml). 9.8 g (8 eq.) of NaHCO.sub.3 in solution in 70 ml of water and 4.76 g (1.1 eq.) of isobutyric acid 1-[2-(4-nitro-phenyl)-acetoxy]-ethyl ester are successively added. The mixture is stirred at ambient temperature overnight.

(45) After evaporation of the acetonitrile, the aqueous phase is extracted with ethyl acetate. The organic phase is washed with 10% citric acid, a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4, filtered and evaporated to dryness. The unprocessed product is chromatographed on silica gel using the 9:1 CH.sub.2Cl.sub.2/MeOH mixture followed by 7:3 as an eluent. White solid, 4.42 g (60%).

(46) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, Kromasil C18 column), Rt=3.7 min.

(47) NMR (DMSO d6) ppm: 0.95-1.45 (12H m); 1.63-2.15 (2H m); 2.45 (1H m); 2.81-3.03 (3H m); 3.68 (1H m); 6.63 (1H m); 7.20-7.82 (10H m).

Step 4: 2-(2-biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphonoyl}-propionylamino)-propionic acid benzyl ester

(48) The compound from step 3 (500 mg) is placed in solution in dimethylformamide (DMF) (10 ml) and 945 mg (3 eq.) of TBTU, 1 ml of DIEA (6 eq.) and 257 mg (1.2 eq.) of the alanine benzyl ester hydrochloride are successively added. The mixture is stirred at ambient temperature for 15 min and the DMF is evaporated. The residue is taken up with ethyl acetate, and successively washed with a 10% citric acid solution, water, a 10% NaHCO.sub.3 solution, a saturated aqueous NaCl solution and is dried over Na.sub.2SO.sub.4. After filtering, the solution is evaporated in a vacuum. White solid, 1 g (95%).

(49) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, Kromasil C18 column), Rt=8.2 min.

(50) 1H NMR (DMSO-d6) (ppm): 0.98-1.42 (15H m); 1.48-1.82 (2H m); 2.45 (1H m); 2.69-3.05 (3H m); 4.33 (1H m); 5.06 (2H s); 6.62 (1H m); 7.21-7.67 (15H m); 8.45 (1H t).

Example 4: 2-(2-biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethyloxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid

(51) ##STR00010##

(52) 250 mg of the compound from example 3 is solubilised in 10 ml of MeOH and 125 mg of 10% Pd/C is added. The mixture is hydrogenolysed at standard temperature and pressure in a hydrogen atmosphere. After 1 hour, 20 ml of CH.sub.2Cl.sub.2 is added and the whole is filtered on Celite. It is evaporated to dryness and vacuum-dried. White solid 184 mg (89%).

(53) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 60:40, Kromasil C18 column), Rt=4.1 min.

(54) ESI(+) [M+H].sup.+ mass=577

(55) (1H) NMR DMSO d6, (ppm): 1-1.5 (15H m); 1.5-2.0 (2H m); 2.5 (1H, m); 3.0 (3H m); 3.7 (1H m); 4.2 (1H m); 6.2 (1H m); 7.3-7.7 (9H+1H m); 8.4 (1H dd).

Example 5: 2-(2-biphenyl-4-ylmethyl-3{hydroxy-[1-(1-isobutyrloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid sodium salt

(56) 87 mg of the compound from example 4 is dissolved in a mixture of 1 ml of water and 1 ml of acetonitrile. 12.6 mg of NaHCO.sub.3 is added. The solution obtained is freeze-dried and produces 87 mg of the expected compound.

Example 6: 2-(2-biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid ethyl ester

(57) ##STR00011##

(58) The compound from example 3 (500 mg) is coupled under the conditions in step 4 of example 3 with alanine ethyl ester hydrochloride (152 mg). The crude product obtained is chromatographed on silica gel using the 7:3:0.2 CH.sub.2Cl.sub.2/MeOH/AcOH mixture. White solid, 530 mg (88%).

(59) ESI(+) [M+H].sup.+ mass=605

(60) 1H NMR (DMSO d6) (ppm): 1.0-1.5 (18H m); 1.6-1.9 (2H m); 2.5 (1H m); 2.7-3.0 (3H m); 3.7 (1H m); 4.0 (2H q); 4.2 (1H qt); 6.6 (1H m); 7.2-7.7 (9H+1H m); 8.4 (1H dd).

Example 7: Alanine 1-ethoxycarbonyloxyethyl ester trifluoroacetate

Step 1: N-terbutyloxycaronyl-L-alanine 1-ethoxycarbonyloxyethyl ester

(61) N-boc-L-alanine (boc=tert-butyloxycarbonyl) (10 g) is placed in solution in 100 ml of ethyl acetate in the presence of triethylamine (6.4 g, 1.2 eq.) in an inert atmosphere and the mixture is stirred for 15 min at ambient temperature. NaI (1.9 g, 0.2 eq.) is then added and 1-chloroethylethylcarbonate (9.38 g, 1.1 eq.). The mixture is boiled under reflux for 15 hours. When the reaction is complete, the mixture is washed with a 10% aqueous NaHCO.sub.3 solution (twice), H.sub.2O, and a saturated aqueous NaCl solution. Slightly yellow oil. 12.9 g (80%).

(62) TLC (cyclohexane/EtOAc: 6:4) Rf=0.73.

Step 2: L-alanine 1-ethoxycarbonyloxyethyl ester trifluoroacetate

(63) The compound from step 1 (12.9 g) is solubilised in 25 ml of CH.sub.9Cl.sub.2 and 25 ml of trifluoroacetic acid is added at 0 C. The mixture is stirred at ambient temperature for 3 hours. When the reaction is complete, the reaction mixture is evaporated to dryness. The residue is taken up 3 times with cyclohexane and the mixture is re-evaporated to remove the excess TFA. Slightly brown oil, 13.4 g (99%).

(64) 1H NMR (DMSO d6) (ppm): 1.2 (3H t); 1.35 (3H dd); 1.45 (3H d); 4.1 (2H q); 6.7 (1 h m); 8.3 (3H s).

Example 8: 2-(2-biphenyl-4-ylmethyl-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid 1-ethoxycarbonyloxyethyl ester

(65) ##STR00012##

(66) The compound from example 3 (500 mg) is coupled, under the conditions of step 4 of example 3, with the compound from example 7, (500 mg, 1.2 eq.)). The crude product is chromatographed on silica gel (CH.sub.2Cl.sub.2/MeOH/AcOH: 7:3:0.2). White solid, 330 mg (55%).

(67) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column), Rt=5.4 min.

(68) ESI(+) [M+H].sup.+ mass=693

(69) 1H NMR (DMSO-d6) (ppm): 1.0-1.4 21H m); 1.5-2.0 (2H m); 2.5 (1H m); 2.7-3.0 (3H m); 3.65 (1H m), 4.15 (2H q); 4.25 (2H m); 6.6 (2H m); 7.2-7.7 (9H+1H m); 8.5 (1H dd).

Example 9: 2-(2-biphenyl-4-ylmethyl-3-{(1-isobutyryloxy-ethoxy)-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid

(70) ##STR00013##

Step 1: Isobutyric acid 1-{1-[[2-(1-benzyloxycarbonyl-ethylcarbamoyl)-3-biphenyl-4-yl-propyl]-(1-isobutyryloxy-ethoxy)-phosphinoyl]-ethylcarbamoyloxy}-ethyl ester

(71) The final compound from example 3 (350 mg) is placed in solution in toluene. In an inert atmosphere, 90 mg of tetrabutylammonium sulphate, (nBu).sub.4N.sup.+SO.sub.4H.sup. (0.5 eq.), NaI (80 mg, 1 eq.), isobutyric acid 1-chloroethyl ester (160 mg, 4 eq.) and 0.9 ml of DIEA are added. The mixture is heated to 120 C. for 3 hours. The reaction mixture is returned to ambient temperature and is diluted in 50 ml of ethyl acetate, the solution obtained is washed with water, with a 10% NaHCO.sub.3 aqueous solution, with a saturated aqueous NaCl solution and dried over Na.sub.2SO.sub.4. After filtration, the solvents are evaporated and the residue (400 mg) is purified by means of silica gel chromatography with the 90:10 CH.sub.2Cl.sub.2/MeOH mixture as the eluent. Yellow oil, 200 mg.

(72) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 80:20, Kromasil C18 column), Rt=8.8 min.

(73) ESI(+) [M+H].sup.+ mass=781.

Step 2: 2-(2-biphenylk-4-ylmethyl-3-{(isobutyryloxy-ethoxy)-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid

(74) The compound from step 1 (200 mg) is solubilised in MeOH (10 ml). 40 mg of 10% is added and the mixture is hydrogenolysed at standard temperature and pressure for 1 hour in a hydrogen atmosphere. The mixture is diluted in CH.sub.2Cl.sub.2 and filtered on celite. The solvent evaporated to dryness and the residue is freeze-dried. White solid, 106 mg (60%).

(75) ESI(+) [M+H].sup.+ mass=691.

(76) 1H NMR (DMSO-d6) (ppm): 1.0-1.5 (24H m); 1.5-1.7 (2H m); 2.5 (2H m); 2.7-3.0 (3H m); 3.9 (1H m); 4.2 (1H m); 6.4 (1H m); 6.65 (1H m); 7.2-7.7 (9H+1H m); 8.3 (1H m).

Example 10: 2-(4-bromo-benzyl)-acrylic acid benzyl ester

Step 1: 2-(4-bromo-benzyl)-acrylic acid

(77) This compound is synthesised according to the protocol described for the synthesis of 2-Biphenyl-4-ylmethyl-acrylic acid (example 2), replacing biphenyl-4-yl-acetaldehyde by 4-bromo benzaldehyde. White solid.

(78) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column) Rt=4.9 min.

Step 2: 2-(4-bromo-phenyl-4-ylmethyl-acrylic) acid benzyl ester

(79) Esterification is performed according to the protocol described in step 3 of example 2. Colourless oil.

(80) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column), Rt=22.0 min.

(81) NMR (CDCl.sub.3) (ppm): 3.6 (2H s); 5.2 (2H s); 5.6 (1H d); 6.4 (1H d); 7.1 (2H d); 7.4 (7H m).

Example 11: 2-(2-(4-bromo-benzyl)-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid benzyl ester

(82) ##STR00014##

(83) This compound is synthesised according to the protocol described for the synthesis of 2-biphenyl-4-ylmethyl-acrylic acid benzyl ester with 2-(4-bromo-benzyl)-acrylic acid benzyl ester.

Step 1: 3-[(1-benzyloxycarbonylamino-ethyl)-hydroxy-phosphinoyl]-2-(4-bromo-benzyl)-propionic acid benzyl ester

(84) The R configuration compound from example 1 (5.15 g) and the compound from example 10 (7.21 g) are placed in solution in BSA (18 ml) and the mixture is heated for 30 hours at 75 C. The reaction mixture is returned to ambient temperature and is diluted in ethyl acetate. The organic phase is washed with water and evaporated to dryness. A white solid product is obtained, corresponding to the mixture of the two 1R,2S and 1R/2R diastereoisomers in relative proportions of 65:35. By recrystallisation from acetonitrile, the 1R,2S isomer is obtained with an enantiomeric excess ee>97%. White solid, 3.35 g (42%).

(85) NMR (DMSO-d6) (ppm): 1.2 (3H dd); 1.7-2.2 (2H m); 2.7-3.1 (3H m); 3.8 (1H p); 4.95 (2H dd); 5.05 (2H s); 7.0-7.4 14H m); 7.55 (1H m).

Step 2: 3-((1-aminoethyl)-hydroxy-phosphinoyl)-2-(4-bromo-benzyl)-propionic acid

(86) Using 3.3 g of the compound from step 1 and according to the protocol described in step 2 of example 3, the expected compound in white solid form, 2.5 g (98%), is obtained.

Step 3: 2-(4-bromo-benzyl)-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionic acid

(87) Using 2.5 g of the compound from step 2 and according to the protocol described in step 3 of example 3, the expected compound is obtained. White solid, 2.6 g.

(88) (M+H).sup.+ mass=508-510

(89) NMR (DMSO-d6) (ppm): 1.0-1.5 (12H m); 1.7 (2H m); 2.5 (1H m); 2.9 (3H m); 3.8 (1H m); 6.6 (1H m); 7.1 (2H d); 7.4 (2H d); 7.75 (1H m).

Step 4: 2-(2-(4-bromo-benzyl)-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid benzyl ester

(90) Using 1 g of the compound from step 3 and 388 mg of alanine benzyl ester, the expected compound is obtained according to the protocol in step 4 of example 3. White solid.

(91) (M+H).sup.+ mass=669-671

(92) NMR (DMSO-d6) (ppm): 1.0-1.3 (6H d); 1.25 (6H m); 1.45 (3H d); 1.6-1.9 (2H m); 2.5 (1H m); 2.7-3.0 (3H m); 3.7 (1H m); 4.3 (1H m); 5.0 2H dd)); 6.6 (1H m); 7.10 (2H d); 7.3 (4H s, +2H d); 7.5 (1H m), 8.4 (1H dd).

Example 12: 2-(2-(4-bromo-benzyl)-3-{hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid

(93) ##STR00015##

Step 1: 3-((1-benzoylcarbonylamino-ethyl)-hydroxy-phosphinoyl)-2-(4-bromo-benzyl)-propionic acid

(94) The compound from step 1 of example 11 (1.2 g) is solubilised in THF (10 ml) and 0.7 g of NaOH in solution in 5 ml of water is added. The mixture is stirred at ambient temperature for 3 hours. The THF is evaporated, the aqueous phase is acidified to pH=1 with a 1N HCl solution, and extracted with ethyl acetate. The organic phase is dried over Na.sub.2SO.sub.4, filtered and evaporated to dryness. White solid, 0.9 g (90%).

Step 2: 2-(3-((1-benzyloxycarbonylamino-ethyl)-hydroxy-phosphinoyl)-2-(4-bromo-benzyl)-propionylamino)-propionic acid t-butyl ester

(95) The compound from step 1 (0.9 g) is solubilised in 10 ml of DMF in nitrogen, and t-butyl alaninate hydrochloride (450 mg), 2.5 ml of DIEA (6 eq) and 1.98 g of TBTU (3 eq) are successively added. The mixture is stirred for 15 min at ambient temperature and the reaction is processed as in step 4 of example 3. 1.10 g of expected product (97%) is obtained.

Step 3: 2-(3-((1-amino-ethyl)-hydroxy-phosphinoyl)-2-(4-bromo-benzyl)-propionylamino)-propionic acid

(96) The compound obtained in step 2 is solubilised in a 50:50 CH.sub.2Cl.sub.2/TFA mixture (40 ml) and the mixture is stirred for 1 hour at ambient temperature. The residue is evaporated to dryness, taken up with water and freeze-dried. 1.0 g of the expected compound is obtained.

(97) The compound is solubilised in an HBR/AcOH mixture (10 ml) and the solution is stirred for 1 hour at ambient temperature. The mixture is then evaporated at reduced pressure. The residue is taken up with water and freeze-dried. 930 mg of the expected product is obtained.

Step 4: 2-(2-(4-bromo-benzyl)-3-{hydroxy-[1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoyl}-propionylamino)-propionic acid

(98) The above compound is solubilised in 15 ml of CH.sub.2Cl.sub.2. 0.76 g of isobutyloxyethyl succinimidyl carbonate and 1.87 ml of DIEA are added and the mixture is stirred for 1 hour at ambient temperature. The mixture is then washed with H.sub.2O and with a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The crude product is purified by HPLC.

(99) HPLC (ACE C18 column, 40:60 CH.sub.3CH (0.1% TFA)/H.sub.2O (0.1% TFA) solvent) Rt 8.66 min.

(100) ESI: (M+H)+=578-580

(101) NMR (DMSO d6) (ppm): 1.0 (6H d); 1.1 (3H m); 1.2 (3H d); 1.35 (3H d); 1.6-1.9 (2H m); 2.5 (1H m); 2.7-2.9 (2H m); 3.7 (1H m); 4.1 (1H m); 6.6 (1H m); 7.1 (2H d); 7.4 (2H, d); 7.6 (1H dd); 8.2 (1H dd).

Example 13: (R)-benzyloxycarbonylamin)(thiophen-3-yl)methyl-phosphinic acid

(102) ##STR00016##

Step 1: (Benzhydrylamino)(thiophen-3-yl)methyl-phosphinic acid

(103) A mixture of 20.0 g (80 mmole) of diphenylmethylamine orthophosphate in 60 ml of anhydrous ethanol is boiled under reflux with stirring. 160.0 mmole of 3-Thiophene carboxaldehyde in solution in 19 ml of ethanol is added drop by drop in 30 min.

(104) The reaction is followed by HPLC. After 2.5 hours, the reaction is returned to ambient temperature. An Et.sub.2O/Acetone mixture (60 ml) is added to the mixture. The white precipitate obtained is filtered, washed with water (250 ml) and acetone (250 ml) and dried. White solid, 15.4 g (56%).

Step 2: Amino(thiophen-3-yl)methylphosphinic acid

(105) A mixture of 15 g of the compound from step 1 and 122 ml of 6N HCl is boiled under reflux for 2 hours. After cooling, the reaction medium is concentrated by half and extracted with 31.5 l of ethyl ether. The solution is evaporated to dryness and the oily residue is taken up with 120 ml of ethanol. The solution is cooled to 0 C. and 30 ml of propylene oxide is added. A white solid precipitates. The precipitate is dewatered, washed with ethanol (220 ml), ethyl ether (210 ml) and dried. White solid, 7.32 g (95.1%).

(106) (1H) NMR DMSO d6, (ppm): 4.92 (1H dd); 6.77 (1H, d); 7.10-7.50 (3H, m); 8.23 (3H m).

Step 3: (Benzyloxycarbonylamino)(thiophen-3-yl)methyl-phosphinic acid

(107) The compound from step 2 is suspended in 25 ml of diaxane. The pH is adjusted to 9.5 by adding sodium hydroxide (23 ml). 6.46 ml of benzyl chloroformiate is added with stirring. After 3 hours at 0 C., the mixture is returned to ambient temperature and is poured onto a mixture of ice (1 L) and concentrated HCl (30 ml). The white precipitate formed is dewatered, washed with water (220 ml) and dried. White solid, 7.09 g (47%).

(108) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column), Rt: 12.19 min.

(109) The resolution of the racemic phosphinic acid is performed by recrystallising the salt obtained with the (R)(+) -methylbenzylamine, as described in Baylis et al. (J. Chem. Soc. Perkin Trans 11984, 2845) in an ethyl acetate/isopropanol mixture=3.5:1. White solid, 1.42 g (27%).

(110) (1H) NMR DMSO d6 (ppm): 4.92 (1H dd); 5.05 (2H, s); 6.77 (1H, d); 7.10-7.50 (8H, m); 8.20 (1H, d).

Example 14: (R)-(benzoylcarbonylamino)(phenyl)methyl-phosphinic acid

(111) ##STR00017##

Step 1: (Benzhydrylamino)(phenyl)methylphosphinic acid

(112) A mixture of 24.95 g (100 mmole) of diphenylmethylamine orthophosphate in 75 ml of anhydrous ethanol is boiled under reflux with stirring. 200 mmole of benzaldehyde in solution in 24 ml of ethanol is added drop by drop in 30 min. The reaction is followed by HPLC. After 2.5 hours, the reaction is returned to ambient temperature. An Et.sub.2O/Acetone mixture (75 ml) is added to the mixture. The white precipitate obtained is filtered, washed with water (260 ml) and acetone (260 ml) and dried. White solid, 20.2 g (60.1%).

Step 2: Amino(phenyl)methylphosphinic acid

(113) A mixture of 20 g of the compound from step 1 and 160 ml of 6N HCl is boiled under reflux for 2 hours. After cooling, the reaction medium is concentrated by half and extracted with 31.5 l of ethyl ether. The solution is evaporated to dryness and the oily residue is taken up with 160 ml of ethanol. The solution is cooled to 0 C. and 40 ml of propylene oxide is added. A white solid precipitates. The precipitate is dewatered, washed with ethanol (220 ml), ethyl ether (210 ml) and dried. White solid, 7.25 g (95.3%).

(114) (1H) NMR DMSO d6 (ppm): 4.92 (1H dd); 6.77 (1H, d); 7.10-7.50 (5H, m); 8.23 (3H, m).

Step 3: (Benzyloxycarbonylamino)(phenyl)methyl-phosphinic acid

(115) The compound from step 2 is suspended in 25 ml of diaxane. The pH is adjusted to 9.5 by adding sodium hydroxide (23 ml). 6.46 ml of benzyl chloroformiate is added with stirring. After 3 hours at 0 C., the mixture is returned to ambient temperature and is poured onto a mixture of ice (1 L) and concentrated HCl (30 ml). The white precipitate formed is dewatered, washed with water (220 ml) and dried. White solid, 11.44 g (88.5%).

(116) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 40:60, Kromasil C18 column), Rt 3.90 min.

(117) The resolution of the racemic phosphinic acid is performed by recrystallising the salt obtained with the (R)(+) -methylbenzylamine, as described in Baylis et al. (J. Chem. Soc. Perkin Trans 11984, 2845) in an ethyl acetate/isopropanol mixture=3.5:1. White solid, 3.9 g (34.0%).

(118) (1H) NMR DMSO d6 (ppm): 4.92 (1H dd); 5.0 (2H, s); 6.77 (1H, d); 7.10-7.50 (8H, m); 8.31 (1H, d).

Example 15: 2-(4-Thiophen-3-yl-benzyl)-acrylic acid methyl ester

(119) This compound is synthesised according to the protocol described for the synthesis of 2-Biphenyl-4-ylmethyl-acrylic acid (example 2) by replacing biphenyl-4-yl-acetaldehyde by 4-bromo benzaldehyde. White solid (48.0%).

(120) HPLC (CH.sub.3CN (0.1% TFA) H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column) Rf=11.28 min.

Example 16: 2-[2-{Hydroxy-[(1-isobutyryloxy-ethoxycarbonylamino)-phenyl-methyl]-phosphinoylmethyl}-3-(4-thiophen-3-yl-phenyl)-propionylamino]-propionic acid

(121) ##STR00018##

(122) This compound is synthesised according to the protocol described in example 3 by replacing 2-biphenyl-4-ylmethyl-acrylic acid benzyl ester with 2-(4-thiophen-3-yl-benzyl)-acrylic acid methyl ester (example 15).

Step 1: 3-[(1-benzyloxycarbonylamino-ethyl)-hydroxy-phosphinoyl]-2-(4-Thiophen-3-yl-benzyl)-propionic acid ethyl ester

(123) The R configuration compound from example 14 (15.42 g; 1.2 eq) and the compound from example 15 (13.82 g; 1.0 eq) are placed in solution in BSA (50 ml) and the mixture is heated for 15 hours at 75 C. The reaction mixture is returned to ambient temperature and is diluted in ethyl acetate. The organic phase is washed with water and evaporated to dryness. A white solid product is obtained, corresponding to the mixture of the two 1R,2S and 1R,2R diastereoisomers in relative proportions of 65:35. White solid, 17.26 g (68%).

(124) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column), Rt=6.51 min.

(125) NMR (DMSO-d6) (ppm): 1.50 (3H, t), 1.81-2.16 (2H, m); 2.78-3.12; (2H, m), 3.68 (3H, s); 3.78 (1H, qt); 4.50 (2H, q); 4.90 (1H, m); 5.20 (2H, s); 7.12-8.15 (17H arom. +NH, m).

Step 2: (R)-amino(phenyl)methyl-3-ethoxy-3-oxo-2-(4-thiophen-3-yl)benzyl)propyl)phosphinic acid ethyl ester hydrobromide

(126) 17.86 g of the compound from step 1 is solubilised in 180 ml of 48% HBr in AcOH. The mixture is stirred for 1 hour at ambient temperature and the mixture is evaporated at reduced pressure to produce the crude product in oil form (100%).

(127) NMR (DMSO-d6) (ppm): 1.50 (3H, t), 1.81-2.16 (2H, m); 2.78-3.12 (2H, m); 3.68 (3H, s); 3.78 (1H, qt); 4.50 (2H, q); 4.90 (1H, m); 5.20 (2H, s); 7.12-8.15 (12H, m); 8.15 (3H, m).

Step 3: (R)-(tert-butoxycarbonylamino)(phenyl)methyl((S)-3-ethoxy-3-oxo-2-(4-(thiophen-3-yl)benzyl)propyl)phosphinic acid ethyl ester

(128) The compound from step 2 (30.9 mmol) is solubilised in 300 ml of DMF. To this solution, Et.sub.3N (35 ml, 250 mmol, 8 eq) and Boc.sub.2O (6.75 g, 1 eq) in 50 ml of DMF. The mixture is stirred overnight at ambient temperature. The DMF is evaporated at reduced pressure and the mixture is taken up with EtoAc. The organic phase is washed with a 10% citric acid solution, a 10% NaHCO.sub.3 solution, a sat. NaCl solution, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure to produce 15.16 g of product (90.2%).

(129) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column), Rt=5.42 min.

(130) NMR (DMSO-d6) (ppm) 1.50 (3H, t); 1.45 (9H, s); 1.81-2.16 (2H, m); 2.78-3.12 (2H, m); 3.78 (1H, qt); 4.50 (2H, q); 4.90 (1H, m); 7.12-8.0 (12H aromatic+NH, m).

Step 4: 3-(((R)-(tert-butoxycarbonylamino)(phenyl)methyl)(hydroxy)phosphoryl)-2-(4-(thiophen-3-yl)benzyl)propanoic acid

(131) The compound from step 3 (15.16 g; 27.88 mmol) is solubilised in 280 ml of acetone and 1N NaOH (278.8 ml. 10 eq) is added drop by drop. The mixture is stirred overnight at ambient temperature and the acetone is evaporated at reduced pressure. The mixture is taken up with EtoAc. The aqueous phase is extracted and is acidified with 1N HCl. The aqueous phase is then extracted with EtoAc. The organic phase is then washed with H.sub.2O, sat. NaCl, dried over Na.sub.2SO.sub.4 and evaporated at reduced pressure to produce 13.73 g of oil (95%).

(132) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, Kromasil C18 column), Rt=11.40 min.

(133) NMR (DMSO-d6) (ppm) 1.45 (9H, s); 1.81-2.16 (2H, m); 2.78-3.12, (2H, m); 3.78 (1H, qt); 4.90 (1H, m); 7.12-8.0 (12H aromatic+NH, m).

Step 5: (R)-(tert-butoxycarbonylamino)(phenyl)(methyl((S)-3-((S)-1-methoxy-1-oxopropan-2-ylamino)-3-oxo-2-(4-(thiophen-3-yl)benzyl)propyl)phosphinic acid methyl ester

(134) Using 150 mg of the compound from step 4 and 52 mg of alanine methyl ester, the expected compound is obtained according to the protocol of step 4 of example 3.

(135) The expected diastereoisomer is obtained by semi-preparative HPLC on a Kromasil C18 column with 50:50 CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) as the elution system. White solid: 50 mg (30.0%).

(136) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 70:30, Kromasil C18 column), Rt=6.0 min.

(137) NMR (DMSO-d6) (ppm): 1.10-1.50 (12H, m); 1.81-2.16 (2H, m); 2.78-3.12, (3H, m); 3.48 (3H, s); 3.78 (1H, qt); 4.25 (1H, d); 4.90 (1H, m); 7.12-7.81 (12H+NH, m); 8.5 (1H, d).

Step 6: (R)-(tert-butoxycarbonylamino)(phenyl)methyl((S)-3-((S)-1-methoxy-1-oxypropan-2-ylamino)-3-oxo-2-(4-(thiophen-3-yl)benzyl)propyl)phosphinic acid

(138) 50 mg of the compound from step 5 is placed in solution in 2 ml of acetone. 800 l of 1N NaOH (10 eq) is added and the mixture is stirred for 2.5 hours at ambient temperature and the acetone is evaporated at reduced pressure. The mixture is taken up with EtoAc. The aqueous phase is extracted and is acidified with 1N HCl. The aqueous phase is then extracted with EtoAc. The organic phase is then washed with H.sub.2O, sat. NaCl, dried over Na.sub.2SO.sub.4 and evaporated at reduced pressure to produce 48 mg (98%).

(139) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, Kromasil C18 column), Rt=7.91 min.

(140) NMR (DMSO-d6) (ppm): 1.10-1.50 (12H, m); 1.81-2.16 (2H, m); 2.78-3.12, (3H, m); 3.78 (1H, qt); 4.25 (1H, d); 4.90 (1H, m); 7.12-7.81 (12H+NH, m); 8.5 (1H, d).

Step 7: (2S)-2-((2S)-3-(((R)-amino(phenyl)methyl)(hydroxy)phosphoryl)-2-(4-(thiophen-3-yl)benzyl)propanamido)propanoic acid trifluoroacetate

(141) 48 mg of the compound from step 6 is placed in solution in 4 ml of DCM. 60 l of trifluoroacetic acid is added and the mixture is stirred for 45 min at 0 C. The mixture is evaporated at reduced pressure to produce 49 mg (100%).

(142) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, Kromasil C18 column) Rt=2.60 min.

(143) NMR (DMSO-d6) (ppm): 1.20 (3H, d); 1.81-2.16 (2H, m); 2.78-3.12, (3H, m); 3.78 1H (qt.); 4.25 (1H, d); 4.90 (1H, m); 7.12-7.81 (12H, m); 8.4 (1H, d); 8.7 (3H, m).

Step 8: 2-[2-{Hydroxy-[(1-isobutyryloxy-ethoxycarbonyl-amino)-phenyl-methyl]-phosphinoylmethyl}-3-(4-thiophen-3-yl-phenyl)-propionylamino]-propionic acid

(144) The above compound is solubilised in 1 ml of CH.sub.3CN and 340 l of 2N NaHCO.sub.3 and 34 mg of isobutyloxyethyl succinimide carbonate (1.2 eq.) is added and the mixture is stirred for 1 hour at 60 C. The mixture is taken up with ethyl acetate and is washed with H.sub.2O and with a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The unprocessed product is purified with HPLC.

(145) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 38:62, Atlantis T3 column) Rt=10.52 min.

(146) NMR (DMSO-d6) (ppm): 1.0-1.3 (6H d); 1.20 (3H, d); 1.25 (3H, d); 1.81-2.16 (2H, m); 2.78-3.12, (3H, m); 3.78 1H (qt.); 4.25 (1H, d); 4.90 (1H, m); 6.64 (1H m); 7.12-7.81 (13H, m); 8.28 (1H, d).

Example 17: 2-[2-{Hydroxy-[(1-isobutyryloxy-ethoxycarbonylamino)-phenyl-methyl]-phosphinoylmethyl}-3-(4-thiophen-3-yl-phenyl)-propionylamino]-3-hydroxypropionic acid

(147) ##STR00019##

(148) This compound is synthesised according to the protocol described in example 16 by replacing alanine methyl ester by serine methyl ester (O-tBu).

(149) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 38:62, Atlantis T3 column) Rt=10.02 min.

(150) NMR (DMSO-d6) (ppm): 1.0-1.3 (6H, d); 1.20 (3H, d); 1.81-2.16 (2H, m); 2.78-3.12, (3H, m); 3.40-3.60 (2H, m); 3.78 (1H, qt); 4.25 (1H, d); 4.90 (1H, m); 6.64 (1H m); 7.12-7.81 (13H, m); 8.28 (1H, d).

Example 18: 2-(3-Biphenyl-4-yl-2-{hydroxy-[(1-isobutyryloxy-ethoxycarbonylamino)-thiophen-3-yl-methyl]-phosphinoylmethyl}-propionylamino)-propionic acid

(151) ##STR00020##

Step 1: 3-(((R)-(benzyloxycarbonylamino)(thiophen-3-yl)methyl)(hydroxy)phosphoryl)-2-(biphenyl-4-ylmethyl)propanoic

(152) The R configuration compound from example 13 (660 mg; 1.0 eq) and the compound from example 2 (642 mg; 1.2 eq) are placed in solution in BSA (5 ml) and the mixture is heated for 15 hours at 75 C. The reaction mixture is returned to ambient temperature and is diluted in ethyl acetate. The organic phase is washed with water and evaporated to dryness. A white solid product is obtained, corresponding to the mixture of the two 1R,2S and 1R,2R diastereoisomers in relative proportions of 65:35. White solid, 1.4 g (100%).

(153) NMR (DMSO-d6) (ppm): 1.60-2.00 (2H, m); 2.78-3.12, (2H, m); 3.78 (1H, qt); 4.90 (1H, m); 5.20 (2H, s); 7.12-8.5 (17H arom. +NH, m).

Step 2: (R)-(benzyloxycarbonylamino)(thiophen-3-yl)methyl(2-(biphenyl-4-ylmethyl)-3-((S)-1-methoxy-1-oxopropan-2-ylamino)-3-oxopropyl)phosphinic acid

(154) Using 2.12 mmol of the compound from step 1 and 2.75 mmol of alanine methyl ester, the expected compound is obtained according to the protocol of step 4 of example 3. White solid: 1.40 g (90.0%).

(155) NMR (DMSO-d6) (ppm): 1.25-1.40 (3H, m); 1.60-2.00 (2H, m); 2.78-3.12, (2H, m); 3.40 (3H; s); 3.78 (1H, qt); 4.25 (1H, m); 4.90 (1H, m); 5.20 (2H, s); 7.12-8.15 (17H arom. +NH, m).

Step 3: 2-{2-[(Benzyloxycarbonylamino-thiophen-3-yl-methyl)-hydroxy-phosphinoylmethyl]-3-biphenyl-4-yl-propionylamino}-propionic acid

(156) 100 mg of the compound from step 2 is placed in solution in 4 ml of acetone. 1.6 ml of 1N NaOH (10 eq) is added and the mixture is stirred for 2.5 hours at ambient temperature and the acetone is evaporated at reduced pressure. The mixture is taken up with EtoAc. The aqueous phase is extracted and is acidified with 1N HCl. The aqueous phase is then extracted with EtoAc. The organic phase is then washed with H.sub.2O, sat. NaCl, dried over Na.sub.2SO.sub.4 and evaporated at reduced pressure to produce 98 mg (98%).

(157) NMR (DMSO-d6) (ppm): 1.25-1.40 (3H, m); 1.60-2.00 (2H, m); 2.78-3.12, (2H, m); 3.78 (1H, qt); 4.25 (1H, m); 4.90 (1H, m); 5.20 (2H, s); 7.12-8.15 (17 h arom. +NH, m).

Step 4: (2S)-2-((2S)-3-(((R)-amino(thiophen-3-yl)methyl)(hydroxy)phosphoryl)-2-(biphenyl-4-ylmethyl)propanamido)propanoic acid trifluoroacetate

(158) The compound from step 3 is placed in solution in 4 ml of 48% HBr in solution in acetic acid. The mixture is stirred for 2 hours at ambient temperature. The mixture is evaporated at reduced pressure and the residue is purified by semi-preparative HPLC on a Kromasil C18 column with 50:50 CH.sub.3CH (0.1% TFA)/H.sub.2O (0.1% TFA) as the elution system. White solid: 28 mg (57.1%).

(159) NMR (DMSO-d6) (ppm): 1.25-1.40 (3H, m); 1.60-2.00 (2H, m); 2.78-3.12, (2H, m); 3.78 (1H, qt); 4.25 (1H, m); 4.90 (1H, m); 7.12-8.15 (12H, m); 8.50 (3H, m).

Step 5: 2-(3-Biphenyl-4-yl-2-{hydroxy-{(1-isobutyryloxy-ethoxycarbonylamino)-thiophen-3-yl-methyl]-phosphinoylmethyl}-propionylamino)-propionic acid

(160) The above compound is solubilised in 1 ml of CH.sub.3CN and 195 l of 2N NaHCO.sub.3 and 20 mg of isobutyloxyethyl succinimide carbonate (1.2 eq.) is added. The mixture is stirred for 1 hour at 60 C. The mixture is taken up with ethyl acetate and is washed with H.sub.2O and with a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The crude product is purified with HPLC.

(161) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 38:62, Atlantis T3 column) Rt=10.42 min.

(162) NMR (DMSO-d6) (ppm): 1.0-1.3 (6H d); 1.20 (3H, d); 1.25 (3H, d); 1.80-2.20 (2H, m); 2.80-3.20, (3H, m); 3.75 1H (qt.); 4.25 (1H, d); 4.90 (1H, m); 6.65 (1H m); 7.10-7.80 (13H, m); 8.28 (1H, d).

Example 19: 2-{3-Biphenyl-4-yl-2-[hydroxy-(1-isobutyryloxymethoxy carbonylamino-ethyl)-phosphinoylmethyl]-propionylamin}-propionic acid

(163) ##STR00021##

Step 1: 2-[(1-Benzyloxycarbonylamino-ethyl)-hydroxy-phosphinoylmethyl]-3-biphenyl-4-yl-propionic acid

(164) The compound from step 1 of example 3 (1.2 g) is solubilised in THF (10 ml) and 0.7 g of NaOH in solution in 5 ml of water is added. The mixture is stirred at ambient temperature for 3 hours. The THF is evaporated, the aqueous phase is acidified to pH 1 with 1N HCl, and extracted with ethyl acetate. The organic phase is dried over Na.sub.2SO.sub.4, filtered and evaporated to dryness. White solid, 0.9 g (90%).

(165) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 38:62, ACE C18 column) Rt=17.16 min.

(166) 1H NMR (DMSO-d6): (ppm) 1.26 (3H, dd); 1.81-2.16 (2H, m); 2.78-3.12 (3H, m); 3.78 (1H, qt); 5.20 (2H, q); 7.12-7.68 (14H arom. +NH (m).)

Step 2: 2-{2-[(1-Benzyloxycarbonylamino-ethyl)-hydroxy-phosphinoylmethyl]-3-biphenyl-4-yl-propionylamino}-propionic acid t-butyl ester

(167) The compound from step 1 (0.9 g) is solubilised in 10 ml of DMF in nitrogen, and t-butyl alaninate hydrochloride (450 mg), 2.5 ml of DIEA (6 eq) and 1.98 g of TBTU (3 eq) are successively added. The mixture is stirred for 15 min at ambient temperature and the reaction is processed as in step 4 of example 3. 1.10 g of expected product (97%) is obtained.

(168) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, ACE C18 column) Rt=11.93 min.

(169) NMR (DMSO d6): (ppm) 1.15-1.35 (6H, m); 1.42 (9H, s); 1.81-2.16 (2H, m); 2.78-3.12 (3H, m); 3.78 (1H, qt); 4.22 (1H, q); 5.20 (2H, q); 7.12-7.68 (14H arom. +NH (m).

Step 3: 2 (2S)-2-((2S)-3-(((R)-1-aminoethyl)(hydroxy)phosphoryl)-2-(biphenyl-4-ylmethyl)propanamido)propanoic acid

(170) The compound obtained in step 2 is solubilised in an HBr/AcOH mixture (10 ml) and the solution is stirred for 1 hour at ambient temperature. The mixture is then evaporated at reduced pressure. The residue is taken up with water and freeze-dried. 930 mg of the expected compound is obtained (100%).

(171) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, ACE C18 column) Rt=11.93 min.

(172) 1H NMR (DMSO-d6): (ppm) 1.15-1.35 (6H, m), 1.42 (9H, s), 1.81-2.16 (2H, m), 2.78-3.12 (3H, m), 3.78 (1H, qt), 4.22 (1H, q), 5.20 (2H, q), 7.12-7.68 (14H arom. +NH (m).

Step 4: 2-{3-Biphenyl-4-yl-2-[hydroxy-(1-isobutyryloxymethoxy carbonylamino-ethyl)-phosphinoylmethyl]-propionylamino}-propionic acid

(173) 300 mg of the compound from step 3 is solubilised in 2 ml of CH.sub.3CN and 2 ml of 2N NaHCO.sub.3 and 186 mg of isobutyloxyethyl succinimide carbonate (1.2 eq.) is added. The mixture is stirred for 1 hour at 60 C. The mixture is taken up with ethyl acetate and is washed with H.sub.2O and with a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The crude product is purified by HPLC to produce 170 mg of the desired product (50.5%).

(174) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 38:62, Atlantis T3 column) Rt=8.15 min.

(175) NMR (DMSO-d6) (ppm): 1.0-1.3 (6H d); 1.25 (3H, dd); 1.45 (3H, d); 1.6-1.9 (2H, m); 2.5 (1H, m); 2.7-3.0 (3H, m); 3.7 (1H, m); 4.3 (1H, m); 5.65 (2H, s); 7.10-7.80 (9H, m); 8.4 (1H dd).

Example 20: 2-Dimethyl-propionic acid 1-(1-{[3-biphenyl-4-yl-4-yl-2-(1-carboxy-ethylcarbamoyl)-propyl]-hydroxy-phosphinoyl}-ethylcarbamoyloxy)-ethyl acid

(176) ##STR00022##

(177) 394 mg of the compound from step 3, example 19 is solubilised in 2 ml of CH.sub.3CN and 2 ml of 2N NaHCO.sub.3 and 272 mg of isobutyloxyethyl succinimide carbonate (1.2 eq.) is added and the mixture is stirred for 1 hour at 60 C. The mixture is taken up with ethyl acetate and is washed with H.sub.2O and with a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The crude product is purified by HPLC to produce 330 mg of the desired product (71.6%).

(178) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 38:62, Atlantis T3 column) Rt=16.93 and 18.41 min.

(179) NMR (DMSO-d6) (ppm): 1.20 (9H, d); 1.25 (3H, dd); 1.45 (3H, d); 1.6-1.9 (2H, m); 2.5 (1H, m); 2.7-3.0 (3H, m); 3.7 (1H, m); 4.3 (1H, m); 6.6 (1H, m); 7.10-7.80 (9H, m); 8.4 (1H dd).

Example 21: 2-[(1-{Hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoylmethyl}-cyclopentanecarbonyl)-amino]-propionic acid

(180) ##STR00023##

Step 1: cyclopentanoic acid t-butyl ester

(181) Cyclopentanoic acid (15 g, 0.131 mol), tBuOH (2.1 ml) and H.sub.2SO.sub.4 (750 l) are placed in a thick-walled flask. Approximately 150 ml condensed isobutylene at 78 C. is then added. The flask is sealed, allowed to return to ambient temperature and stirred for 4 nights at ambient temperature.

(182) After evaporating the excess isobutylene, the mixture is taken up which Et.sub.2O and washed with 10% NaHCO.sub.3. The organic phase is dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure to produce 12.03 g of the expected product (yield: 80.6%).

(183) NMR (CDCl.sub.3) (ppm): 1.40 (9H, d); 1.45-1.90 (8H, m); 2.55 (1H, m).

Step 2: 1-(hydroxymethyl)cyclopentanecarboxylic acid t-butyl ester

(184) To a solution at 0 C. of iPR.sub.2NH (7.91 ml, 56.11 mmol) in THF (190 ml), nBuLi in hexane (2.5 M, 23.3 ml, 58.17 mmol, 1.04 eq) is added. After 30 min, the mixture is cooled to 78 C. and a solution of the compound from step 1 (9.54 g, 56.11 mmol) in 25 ml of THF is added in nitrogen in 30 min. The mixture is stirred for 1 hour at 78 C. and paraformaldehyde (5 eq, 8.37 g) is added and the mixture is stirred for 1 hour at 78 C. and for 12 hours at ambient temperature.

(185) 190 ml of a sat. NH.sub.4Cl solution is added and the mixture is extracted with EtoAc (2200 ml). The organic phase is washed with 1N HCl, sat. NaCl, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure to produce the desired product (yield: 60%).

(186) NMR (CDCl.sub.3) (ppm): 1.40 (9H, d); 1.45-1.90 (8H, m); 3.50 (2H, s).

(187) HPLC Atlantis T3 4.6100 mm, 3 m, gradient: CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 10 to 90% CH.sub.3CN in 30 min, Rt: 12.43 min.

Step 3: Benzyl(1R)-1-(methoxyhydrophosphoryl)ethylcarbamate

(188) Under nitrogen, the compound from step 2, example 3 (6 g, 24.67 mmol) is solubilised in a MeOH/Toluene mixture. A trimethylsilyldiazomethane solution is added drop by drop until the colour persists (corresponding to the end of the gas emission). The mixture is stirred for 1 hour at ambient temperature and the toluene is evaporated at reduced pressure. The mixture is extracted with EtoAc. The organic phase is washed with 10% NaHCO.sub.3, sat. NaCl, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure to produce 5.75 g of desired product (yield 90.7%).

(189) NMR (DMSO d6) (ppm): 1.20-1.35 (3H, m); 3.60 (2H, m); 3.70-4.10 (1H, m); 5.05 (2H, d); 6.90 (1H, m); 7.20-7.50 (5H, m); 7.75 (1H, m).

(190) HPLC Atlantis T3 4.6100 mm, 3 m, CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 30 to CH.sub.3CN in 30 min, Rt: 4.30 and 4.48 min.

Step 4: 1-((trifluoromethylsulfonyloxy)methyl)cyclopentanoic tert-butyl ester

(191) A mixture of 6.80 ml (84.2 mmol) of pyridine and of 50 ml of dichloromethane is cooled to 78 C. in nitrogen. A triflic anhydride solution, 6.80 g (24.14 mmol) in 7 ml of CH.sub.2Cl.sub.2 is added using a bromine vial. After 10 min, a solution of 3.4 g of the compound from step 2 (17 mmol) in 14 ml of CH.sub.2Cl.sub.2 is added drop by drop. The mixture is stirred for 30 min at 78 C. and is returned to ambient temperature. 300 ml of hexane is added. The organic phase is washed with 1N NaOH, H.sub.2O, sat. NaCl, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure to produce 5.67 g of desired product (yield: 100%) used as it is for the next step.

(192) NMR (CDCl.sub.3) (ppm): 1.40 (9H, d); 1.45-1.90 (8H, m); 4.65 (2H, s).

Step 5: 1((((R)-1-benzyloxycarbonylamino)ethyl)(hydroxy)phosphoryl)methyl)cyclopentane carboxylic acid

(193) A solution of iPr.sub.2NH (2.19 ml, 15.64 mmol) in THF (17 ml) is cooled to 0 C. and 6.23 ml of 2.5 M nBuLi in hexane (15.64 mmol, 1.04 eq) is added. After 15 min, the mixture is cooled to 78 C. and a solution of the compound from step 3 (3.64 g, 14.16 mmol) in 28 ml of THF is added under nitrogen to keep the temperature below 60 C. The mixture is stirred for 10 min at 78 C. and the compound from step 4 (17 mmol) is added in 15 ml of THF and the mixture is stirred for 15 min at 78 C. and for 4 hours at ambient temperature.

(194) The mixture is diluted with EtoAc. The organic phase is washed with 1N HCl, 10% NaHCO.sub.3, sat. NaCl, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure.

(195) The crude product obtained is placed in solution in 10 ml of CH.sub.2Cl.sub.2 and 3.2 ml of TFA is added. The mixture is stirred for 2 hours at 0 C. After evaporating to dryness, the expected product is purified by semi-preparative HPLC on an ACE C18 column, CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 40:60 to produce 120 mg of pure product (yield: 29.4%).

(196) NMR (DMSO d6) ) ppm): 1.20-1.35 (3H, m); 1.50-2.20 (10H, m); 3.75 (1H, q); 5.05 (2H, d); 5.05 (2H, d); 7.20-7.60 (6H, m).

(197) HPLC ACE C18 4.6250 mm, 5 m, CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 40:60, Rt: 5.57 min.

Step 6: (R)-1-(benzyloxycarbonylamino)ethyl((1-(S)-1-tert-butoxy-1-oxypropan-2-ylcarbamoyl)cyclopentyl)methyl)phosphinic acid

(198) The compound from step 5 (0.118 g, 0.319 mmol) is solubilised in 2 ml of DMF under nitrogen, and t-butyl alaninate hydrochloride (70 mg), 279 l of DIEA (5 eq) and 308 g of TBTU (3 eq) are successively added. The mixture is stirred for 15 min at ambient temperature, and the reaction is processed as in step 4 of example 3. 111 mg of the expected product is obtained (70.3%).

(199) HPLC (CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 50:50, ACE (C18) column) Rt: 7.71 min.

(200) 1H NMR (DMSO-d6) (ppm): 1.15-1.35 (6H, m); 1.42 (9H, s); 1.40-2.20 (10H, m); 3.85 (1H, q); 4.15 (1H, q); 5.05 (2H, s); 7.1-7.45 (5H, m); 7.90 (1H, d).

Step 7: (2S)-2-(1-((((R)-1-aminoethyl)(hydroxy)phosphoryl methyl)cyclopentanecarboxamido)propanoic acid trifluoroacetate

(201) The compound obtained in step 6 is solubilised in an HBr/AcOH mixture (2 ml) and the solution is stirred for 1 hour at ambient temperature. The mixture is then evaporated at reduced pressure. The product is purified by semi-preparative HPLC on an ACE 018 column with a gradient of 0 to 60% CH.sub.3CN in 30 min of a CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) mixture to produce 57.2 mg of the expected product (yield: 61.9%).

(202) HPLC ACE C18 gradient: 0 to 60% CH.sub.3CN in 30 min of a CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) mixture Rt: 9.97 min.

(203) 1H NMR (DMSO-d6) (ppm): 1.15-1.35 (6H, m); 1.50-2.20 (10H, m); 3.40 (1H, q); 4.25 (1H, q); 7.85 (1H, d); 8.10 (3H, m).

Step 8: 2-[(1-{Hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoylmethyl}-cyclopentane carbonyl)-amino]-propionic acid

(204) 55 mg of the compound from step 7 is solubilised in 1 ml of CH.sub.3CN and 366 l of 2N NaHCO.sub.3. 35 mg of isobutyloxy succinimide carbonate (1.2 eq.) is added. The mixture is stirred for 1 hour at 60 C. The mixture is taken up with ethyl acetate and is washed with H.sub.2O and with a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The unprocessed product is purified by HPLC to produce 45 mg of the desired product (71.4%).

(205) HPLC ACE C18 CH.sub.3CN (0.1%)/H.sub.2O (0.1% TFA) 70:30 Rt=4.53 min.

(206) 1H NMR (DMSO-d6) (ppm): 1.10-2.20 (19H, m); 3.40 (1H, q); 3.7 (1H, m); 4.25 (1H, q); 6.20 (m, 1H); 7.85 (1H, d); 8.4 (1H, dd)

Example 22: 2-[(1-Acetyl-4-{Hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoylmethyl}-piperidine-4-carbonyl)-amino]-propionic acid

(207) ##STR00024##

Step 1: 1-Acetyl-piperidine-4-carboxylic acid t-butyl ester

(208) 1-acetylpiperidine-4-carboxylic acid (2.97 g), suspended in 25 ml of a THF/toluene mixture and heated to 85 C. in a nitrogen stream. N,N dimethylformamide di-t-butyl acetal (25 ml, 6 eq) is added drop by drop and heating is maintained for 30 min. The mixture is evaporated to dryness and the residue is taken up with ethyl acetate. The organic phase is washed with water, with a saturated NaCl solution, dried over Na.sub.2SO.sub.4, filtered and evaporated to dryness. Yellow oil p=3.45 g (yield 88%).

(209) HPLC Atlantis T3 10-90 gradient of CH.sub.3CN (0.1% TFA/H.sub.2O (0.1% TFA) in 15 min Rt=10.3 min.

(210) ESI(+)(M+H).sup.+=228

(211) NMR (DMSO d6) (ppm): 1.20-1.80 (13H, m), 2.10 (3H, s), 2.40 (1H, m). 2.60-3.20 (4H, m), 3.60-4.30 (4H, m).

Step 2: 1-Acetyl-4-hydroxymethyl-piperidine-4-carboxylic acid t-butyl ester

(212) To a solution at 0 C. of iPR.sub.2NH (2.10 ml, 14.89 mmol) in THF (16 ml), nBuLi in hexane (2.5 M) (6.8 ml, 16.92 mmol, 2.5 eq) is added. After 30 min, the mixture is cooled to 78 C. and a solution of the compound from step 1 (1.54 g, 6.77 mmol) in 9 ml of THF is added under nitrogen in 30 min. The mixture is stirred for 1 hour at 78 C. and paraformaldehyde (5 eq, 1.01 g) is added and the reaction mixture returned to ambient temperature with stirring. After 30 min at AT, the mixture is split between a saturated NH.sub.4Cl (120 ml) and EtoAc (60 ml) solution. The aqueous phase is extracted with EtoAc (240 ml). The organic phase is washed with 1N HCl, sat. NaCl, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure to produce a yellow oil P=1.20 g.

(213) The crude product is purified by semi-preparative HPLC on a 30100 mm Atlantis column, CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 15:85 eluent. Yield: 27%.

(214) HPLC Atlantis T3 20% for 10 min followed by 20-90 gradient of CH.sub.3CN (0.01% TFA)/H.sub.2O (0.01% TFA) in 15 min Rt=8.85 min.

(215) ESI(+)(M+H).sup.+=258.2

(216) NMR (DMSO d6) (ppm): 1.20-1.80 (13H, m), 2.10 (3H, s), 2.60-3.20 (4H, m), 3.40 (2H, s), 3.60-4.30 (4H, m).

Step 3: 1-Acetyl-4-trifluoromethanesulfonyloxymethyl-piperidine-4-carboxylic acid tert-butyl ester

(217) A mixture of 2.0 ml (250 mmol) of pyridine and of 20 ml of dichloromethane is cooled to 78 C. under nitrogen. Triflic anhydride (2.10 ml, 12.5 mmol) is added drop by drop. After 10 min, a solution of 1.29 g of the compound from step 2 (5.0 mmol) in 15 ml of CH.sub.2Cl.sub.2 is added drop by drop. The mixture is stirred for 2 hours at 78 C. The organic phase is washed with 1N HCl, H.sub.2O, sat. NaCl, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure to produce 1.68 g of the expected product (yield: 86%) used as it is for the next step.

(218) HPLC Atlantis T3 (4.6*100 mm, 3 m) 20% for 10 min followed by 20-90% gradient of CH.sub.3CN (0.1% TFA/H.sub.2O (0.1% TFA) in 15 min Rt=22.7 min.

(219) ESI(+)(M+H).sup.+=390.2

(220) NMR (CDCl.sub.3) (ppm): 1.40-1.60 (11H, m), 1.90-2.30 (5H, m), 2.90 (2H, m), 3.40-3.80 (2H, m), 4.30-4.60 (2H, m).

Step 4: 1-acetyl-4-((((R)-1-(benzyloxycarbonylamino)ethyl)(hydroxy)phosphoryl)methyl)piperidine-4-carboxylic acid

(221) A solution of iPr.sub.2NH (0.61 ml, 4.31 mmol) in THF (5 ml) is cooled to 0 C. and 3.1 ml of 1.5 M nBuLi in hexane (4.67 mmol, 3.0 eq) is added. After 15 min, the mixture is cooled to 78 C. and a solution of the compound from step 3 (925 mg, 3.59 mmol) in 8 ml of THF is added under nitrogen to keep the temperature below 60 C. The mixture is stirred for 10 min at 78 C. and the compound from step 4 (4.31 mmol) is added in 10 ml of THF and the mixture is stirred for 10 min at 78 C. and for 5 hours at ambient temperature.

(222) The mixture is diluted with EtoAc. The organic phase is washed with 1N HCl, 10% NaHCO.sub.3, sat. NaCl, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure.

(223) The crude product obtained is placed in solution in 31 ml of CH.sub.2Cl.sub.2 and 12 ml of TFA is added. The mixture is stirred for 2 hours at ambient temperature. After evaporating to dryness, the expected product is purified by semi-preparative HPLC on an Atlantis T3 column, CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 15:85 to produce 367 mg of pure product (yield: 23%).

(224) NMR (DMSO d6) (ppm): 1.17 (3H, m), 1.40-1.95 (9H, m), 3.00 (1H, m), 3.23 (1H, m), 3.55-3.81 (2H, m), 5.03 (dd, 2H), 7.33 (5H, m), 7.46 (1H, d).

(225) HPLC Atlantic T3 4.6100 mm, 3 m, CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) 20:80, Rt: 7.67 min.

Step 5: (2S)-2-(1-acetyl-4-((((R)-1-aminoethyl)(hydroxy)phosphoryl)methyl)piperidine-4-carboxamide)propanoic acid trifluoroacetate

(226) The compound from step 4 (0.175 g, 0.41 mmol) is solubilised in 2 ml of DMF under nitrogen, and t-butyl alaninate hydrochloride (89 mg, 1.2 eq), 360 l of DIEA (5 eq) and 395 mg of TBTU (3 eq) are successively added. The mixture is stirred for 15 min at ambient temperature, and the reaction mixture is evaporated to dryness. The crude residue obtained is solubilised in an HBr/AcOH mixture (4 ml) and the solution is then stirred for 1 hour at ambient temperature. The mixture is then evaporated at reduced pressure. The product is purified by semi-preparative HPLC on an Atlantis T3 column with a 0 to 30% gradient of CH.sub.3CN in 30 min of a CH.sub.3CN (0.1% TFA)/H.sub.2O (0.1% TFA) mixture to produce 80.8 mg of the expected product (yield: 40.9%).

(227) HPLC Atlantis T3 with a 0 to 30% gradient of CH.sub.3CN in 30 min Rt: 8.59 min.

(228) 1H NMR (DMSO-d6) (ppm): 1.15-1.35 (9H, m), 1.50-2.20 (10H, m), 3.23 (1H, m), 3.40 (1H, q), 4.25 (1H, q), 7.85 (1H, d), 8.10 (3H, m).

Step 6: 2-[(1-Acetyl-4-{Hydroxy-[1-(1-isobutyryloxy-ethoxycarbonylamino)-ethyl]-phosphinoylmethyl}-piperidine-4-carbonyl)-amino]-propionic acid

(229) 80 mg (0.166 mmol) of the compound from step 5 is solubilised in 2 ml of CH.sub.3CN and 560 l of 2N NaHCO.sub.3. 52 mg of isobutyloxy succinimide carbonate (1.2 eq.) is added and the mixture is stirred for 1 hour at 60 C. The mixture is taken up with ethyl acetate, washed with H.sub.2O and with a saturated aqueous NaCl solution, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The crude product is purified with HPLC to produce 70 mg of the expected product (81.4%).

(230) HPLC ACE C18 CH.sub.3CN (0.01%)/H.sub.2O (0.1% TFA) 50:50 Rt: 5.37 min.

(231) 1H NMR (DMSO-d6) (ppm); 1.10-2.20 (29H, m), 3.20 (1H, m), 3.40 (1H, q), 3.7 (1H, m), 4.25 (1H, q), 6.20 (m, 1H), 7.85 (1H, d), 8.10 (3H, m).

(232) Summary of the Molecules Described

(233) This list is not exhaustive.

(234) ##STR00025##

(235) TABLE-US-00001 Example R.sub.10 R.sub.8 R.sub.3 R.sub.2 R.sub.4 R.sub.5 R.sub.6 R.sub.7 3 iPr CH.sub.3 H (R)CH.sub.3 (S)-4- H CH.sub.3 CH.sub.2Ph Phenyl- Benzyl 4 iPr CH.sub.3 H (R)CH.sub.3 (S)-4- H CH.sub.3 H Phenyl- Benzyl 6 iPr CH.sub.3 H (R)CH.sub.3 (S)-4- H CH.sub.3 C.sub.2H.sub.5 Phenyl- Benzyl 8 iPr CH.sub.3 H (R)CH.sub.3 (S)-4- H CH.sub.3 CH(CH.sub.3)OCOOEt Phenyl- Benzyl 9 iPr CH.sub.3 CH(CH.sub.3)OCOiPr (R)CH.sub.3 (S)-4- H CH.sub.3 H Phenyl- Benzyl 11 iPr CH.sub.3 H (R)CH.sub.3 (S)-4-Bromo- H CH.sub.3 CH.sub.2Ph Benzyl 12 iPr CH.sub.3 H (R)CH.sub.3 (S)-4-Bromo- H CH.sub.3 H Benzyl 16 iPr CH.sub.3 H (R)- (S)-4-(3- H CH.sub.3 H Phenyl Thiophen)- Benzyl 17 iPr CH.sub.3 H (R)- (S)-4-(3- H CH.sub.2OH H Phenyl Thiophen)- Benzyl 18 iPr CH.sub.3 H (R)-3- (S)-4- H CH.sub.3 H Thiophen Phenyl- Benzyl 19 iPr H H (R)CH.sub.3 (S)-4- H CH.sub.3 H Phenyl- Benzyl 20 tBu CH.sub.3 H (R)CH.sub.3 (S)-4- H CH.sub.3 H Phenyl- Benzyl 21 iPr CH.sub.3 H (R)CH.sub.3 Cyclopentane CH.sub.3 H 22 iPr CH.sub.3 H (R)CH.sub.3 4(1- CH.sub.3 H acetyl)piperidine

Example 23: Study of the Stability of Phosphinic Derivatives Protected with an Acyloxyalkyl Group on the Phosphinic Function and in which the Amine Function is Free

(236) The inhibition of neprilysin (NEP) and aminopeptidase N (APN) which is required to protect enkephalins completely from the catabolism thereof requires the recognition of the active sites of both zinc metallopeptidases by the same molecule in the present invention. This was obtained, in the prior art, with phosphinic derivatives particularly described in patent application FR 2 755 135. However, for parenteral route bioavailability reasons, it is necessary to protect the phosphinic function temporarily with an acylalkyl group (pro-drug) and, in some cases also the carboxyl function of the inhibitors described in the prior art.

(237) Moreover, it is essential to note that the recognition of APN and the inhibition thereof necessarily requires the presence of a free amine function.

(238) However, the study in solution of the pro-drugs described in the prior art demonstrates that protection of the phosphinic group by an acyloxyalkyl residue gives rise to the transfer of a part of this group to the amine function to produce a molecule having an amide function instead of the free amine necessary for APN affinity.

(239) For this reason, the compound formed, which is not suitable for hydrolysis, is completely inactive with respect to said enzyme. These compounds are thus not suitable for use due to the instability thereof in preparations used in human clinical practice.

(240) The study in solution of a compound according to the prior art was conducted as follows:

(241) The compound according to the prior art is placed in solution in various mixtures used for parenteral administration, the solution subsequently being followed by HPLC (Kromasil C18 4.5*250 mm column, 50% CH.sub.3CN (0.1% TFA)/50% H.sub.2O (0.1% TFA)) to determine the composition of the solution. In any case, the formation of both products, free phosphonic acid (active compound) and the transfer product, is observed over time as illustrated in the diagram below:

(242) ##STR00026##

(243) In this way, for a prior art compound solution in an ethanol/cremophor/H.sub.2O mixture (1:1:8), the initial product content in the vehicle declines over time, as demonstrated in table 1 below:

(244) TABLE-US-00002 Time after solubilisation 0 hrs 3 hrs 6 hrs 96 hrs % initial compound 90.5% 82.0% 75.7% 25.1%

Example 25: Pharmacological Results

(245) The molecules according to the present invention were studied for the analgesic action thereof on the most predictive animal models of the response in humans. The preferred tests are those targeting neuroinflammatory (NI) and neuropathic (NP) pain in rats and mice.

(246) The molecules according to the present invention were found to be active on the following tests on mice:

(247) i) pain induced by administering formalin in the paw and study of the analgesic response in the first phase considered to reflect a peripheral action on the nociceptors, and

(248) ii) hyperalgesia and allodynia induced by partial and unilateral compression of the sciatic nerve (Seltzer model) (Bennett G. J. and Xie Y. K., Pain (1998) 33, 87-107).

(249) The techniques used in these tests are described in detail and listed in reviews such as: M. J. Millan. The induction of pain: an integrative review, in Progress in Neurobiology (1999), 52, 1-164.

(250) The association and synergy between an opioid and gabapentin has been reported, particularly in the reference: Menendez et al (2008), Eur. J. Pharmacol, 596, 50-55.

(251) The following tests were thus performed.

(252) A/ Formalin Test (Phase I)

(253) The molecules were studied at one time (90 min) for comparative tests and at two times, 90 and 150 minutes, so as to observe the period of action thereof.

(254) Test Description:

(255) The animals (OF1 male mice) were obtained from the Charles River breeding centre (France) and weighed 25.35 g at the start of the experiment. The weight of each mouse is taken into account for the administration of the product.

(256) The test is based on the protocol described by S. HUNSKAAR et al., Formalin test in mice, a useful technique for evaluating mild analgesics, J. Neurosci. Methods (1995), 14, 69-75. The early part of the test (5 to 10 min after injecting formalin), is considered to reflect neuropathic pain, which is under study.

(257) The mice (n=8) are placed individually in a transparent container (5025 cm.sup.2) and are acclimatised to this environment for 20 minutes. After this period, 20 l of formalin (5% HCHO) in solution in physiological saline solution (H.sub.2O, 0.9% NaCl) is injected subcutaneously on the plantar face of the right paw of the animal. A 26 syringe connected to a micro-syringe is used. Each mouse is then immediately returned to the test container and pain (nociceptive) responses are measured for 5 minutes (early phase). Only the number of times the paw is licked is counted.

(258) The analgesic activity is tested after force-feeding the animals at different times (generally 20 min, 90 min and 150 min) after injecting formalin, with: the vehicle only (ethanol, 0.5% methylcellulose in water) the vehicle and a compound according to the invention (50 mg/kg).

(259) The analgesic action of the product is measured by the decline in the number of times the injured paw is licked, compared to the number of times the animal receiving the vehicle only licks its paw. The total (discontinuous) licking time, expressed in seconds for each mouse, is counted for 4 min. The cumulative values for n mice are then divided by the number n of mice studied.

(260) The results are given for the three compounds from examples 3, 4 and 8 in FIG. 1.

(261) The three compounds exhibit powerful analgesic effects (40 to 60%) characterised by a very significant reduction in the number of licks compared to the vehicle (control) and the effects are relatively constant for the test period. In this way, an analgesic activity of the compounds according to the invention is observed at times of up to 150 min indicating the long period of action of the compounds according to the invention.

(262) The analgesic action is blocked by pre-administering an antagonist, methyl-naloxonium, which, at the dose used (2 mg/kg), is unable to pass the blood-brain barrier (Milne R. J. et al., Neurosci. Lett. (1990), 114, 25-32), demonstrating that the activity of these molecules is performed in the peripheral region (nociceptors) where they increase the enkephalins released from the injured site. These results clearly demonstrate that the compounds according to the invention do not pass the blood-brain barrier.

(263) B/ Comparative Study of the Analgesic Effect of the Compound from Example 8 and a Reference Molecule According to the Prior Art

(264) The reference molecule according to the prior art used for this study has the following structure:

(265) ##STR00027##

(266) Test A described above was repeated with the compound from example 8 (50 mg/kg; FIG. 2A) and the reference molecule according to the prior art (100 mg/kg; FIG. 2B) in order to compare the action thereof over time, 90 min after injecting formalin.

(267) It is thus observed that the reference molecule has no activity at 90 minutes of a 100 mg/kg dose (per os) whereas, on the other hand, the compound from example 8 gives rise to a very significant analgesic activity 90 minutes at a 50 mg/kg dose (per os) which is two times lower (see FIG. 2).

(268) The present invention is thus characterised by the development of molecules having analgesic properties by the oral route with along period of action.

(269) C/ Anti-Allodynia and Anti-Hyperalgesia Effects of the Compound from Example 3 after Oral Administration in Mice.

(270) This test was described in detail by A. B. Malmberg and A. I. Basbaum, Partial Sciatic nerve injury in the mouse as a model of neuropathic pain: behavioural and neuroanatomical correlates. Pain, (1998) 76, 215-222.

(271) It was performed on OF1 male mice (Charles River, n=39), weighing 18 to 20 g, by partial ligation of the sciatic nerve on the ipsilateral side. The animals are tested over a period of 3 to 26 days after the operation.

(272) The hyperalgesia was measured according to the method described by K. Hargreaves et al., A new sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain (1988), 32, 77-88, using the Plantar test device (Bioseb, France) as the heat source. The intensity of the nociceptive stimulus is calibrated at 8-10 s with an automatic cut-off time of 20 seconds. The mean heat-induced paw withdrawal was measured on the ipsilateral (damaged nerve) and contralateral (intact nerve) paws. Each measurement is made 3 times on each paw.

(273) The mechanical allodynia is measured as described by S. R. Chaplan et al., Quantitative assessment of tactile allodynia in the rat paw, J. Neurosci. Meth. (1994), 53, 55-63. The ipsilateral (lesion) and contralateral (control) paws are tested as above, the mechanical anti-allodynia effect being measured using the Von Frey method (Malmerg A. B. and Basbaum A. I., 1998, Pain, 76, 215-222) with filaments of increasing size likewise applying an increasing pressure.

(274) Anti-Hyperalgesia Effect:

(275) The results obtained, represented in FIG. 3, demonstrate that, administered orally at 50 mg/kg, the compound from example 3 gives rise to a very significant reduction (65-100%) in thermal hyperalgesia induced by a partial ligation of the sciatic nerve in the period 45-120 min with a peak effect at 90 min.

(276) Anti-Allodynia Effect:

(277) The effect of the compound from example 3 on mechanical allodynia is measured using the Von Frey test. The results obtained, represented in FIG. 4, demonstrate a significant long-term (45-120 min) anti-allodynia effect with a peak at 60 min equivalent to 75% of the peak response (untreated control).

(278) D/ Potentiation of Anti-Allodynia Effects of the Compound from Example 4 by Associating with Gabapentin.

(279) The effect of the association of the compound from example 4 and gabapentin, with both products administered orally, is measured using the Von Frey test. The results obtained, after 60 min, represented in FIG. 5, demonstrate a very high potentiation of the association (>300%) compared to the compound from example or gabapentin used alone, which prove to be inactive at the same doses. No effect is obtained on the uninjured contralateral paw.