PHOSPHORODIAMIDATES AND OTHER PHOSPHORUS DERIVATIVES OF FINGOLIMOD AND RELATED S1 P RECEPTOR MODULATORS
20200255460 ยท 2020-08-13
Inventors
- Emyr Lloyd-Evans (Cardiff, South Glamorgan, GB)
- Fabrizio Pertusati (Cardiff, GB)
- Edward James (Derby, Derbyshire, GB)
- Emily Maguire (Cardiff, South Glamorgan, GB)
- Christopher McGuigan (Cardiff, South Wales, GB)
Cpc classification
C07F9/5728
CHEMISTRY; METALLURGY
International classification
Abstract
Compounds of general formula (I): (Formula I)) wherein R.sup.1, Q, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and Ar.sup.1 are as defined herein are inhibitors of class I histone deacetylases and are of use in the treatment of lysosomal storage disorders, especially Niemann-Pick type C disease, as well as other lysosomal storage disorders, defective autophagy, accumulation of free cholesterol and mycobacterial diseases.
Claims
1. A compound of general formula (I) including all stereoisomers thereof and all isotopic variants thereof: ##STR00055## wherein R.sup.1 is OAr.sup.2 or -QR.sup.3; wherein Ar.sup.2 is a C.sub.6-10 aryl or a 5-10 membered heteroaryl group optionally substituted with one or more substituents selected from OH, halo, nitro, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl), O(C.sub.1-6 haloalkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl).sub.2 or SF.sub.5; Q and Q are each independently O, S or NR.sup.2; R.sup.2 is H or C.sub.1-6 alkyl optionally substituted by one or more halo, OH or phenyl substituents; R.sup.3 and R.sup.3 are each independently C.sub.1-10 alkyl or C.sub.1-10 alkyl-C(O)OR.sup.11, either or which is optionally substituted by one or more substituents R.sup.12, R.sup.11 is C.sub.1-6 alkyl, C.sub.1-6 haloalkyl or benzyl; R.sup.12 is OR.sup.13, SR.sup.13, Z, ZOR.sup.13, OZR.sup.13, ZR.sup.13, C(O)R.sup.13, C(O)OR.sup.13, NR.sup.13R.sup.14, C(O)NR.sup.13R.sup.14, NHC(O)R.sup.13, NHC(O)OR.sup.13, NH(CNH)NR.sup.13R.sup.14, OC(O)R.sup.13, SC(O)R.sup.13 or SSR.sup.13; R.sup.13 and R.sup.14 are each independently H or C.sub.1-6 alkyl; Z is a C.sub.6-10 aryl or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituent selected from halo or OH; or when Q or Q is NR.sup.2, R.sup.2 and R.sup.3 or R.sup.2 and R.sup.3 together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring substituted with C(O)OR.sup.11, wherein R.sup.11 is as defined above; R.sup.4 is OH or a group: ##STR00056## where R.sup.1, Q and R.sup.3 are as defined above; each of R.sup.5 and R.sup.6 is independently selected from hydrogen or C.sub.1-4 alkyl; or R.sup.5 and R.sup.6 together with the nitrogen atom to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from N, O or S; Ar.sup.1 is a phenyl or a 5- or 6-membered heteroaryl group, either of which is optionally substituted with one or more substituents selected from halo, OH, C.sub.1-4 alkyl or C.sub.14 haloalkyl; and R.sup.7 is C.sub.1-10 alkyl optionally substituted with phenyl or a 5- or 6-membered heteroaryl group, wherein the phenyl or heteroaryl groups are optionally substituted with one or more substituents selected from halo, NO.sub.2, OH, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, O(C.sub.1-4 alkyl) or phenyl optionally substituted with halo, OH, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl or O(C.sub.1-4 alkyl) and optionally labelled with a detectable label; or a pharmaceutically or veterinarily acceptable salt or hydrate thereof.
2. A compound according to claim 1 wherein the asymmetric carbon atom (*) to which the NR.sup.5R.sup.6, CH.sub.2R.sup.4, CH.sub.2CH.sub.2Ar.sup.1R.sup.7 and the phosphate moiety: ##STR00057## are attached is in the S-orientation.
3. A compound according to claim 1 wherein R.sup.1 is OAr.sup.2.
4. A compound according to claim 3 wherein Ar.sup.2 is phenyl, naphthyl, or tetrahydronaphthyl, any of which is optionally substituted with one or more substituents as defined in claim 1.
5. A compound according to claim 1 wherein R.sup.1 is -QR.sup.3.
6. A compound according to claim 1 wherein Q and/or Q (when present) is NR.sup.2 where R.sup.2 is H or C.sub.1-4 alkyl optionally substituted with one or more halo, OH or phenyl substituents.
7. A compound according to claim 1 wherein R.sup.3 and/or R.sup.3 (when present) is a group C.sub.1-10 alkyl-C(O)OR.sup.11.
8. A compound according to claim 7 wherein R.sup.3 and/or R.sup.3 (when present) is C(R.sup.12aR.sup.12b)C(O)R.sup.11 or C(R.sup.12aR.sup.12b)CH.sub.2C(O)OR.sup.11; wherein R.sup.11 is as defined above; R.sup.12a is H or C.sub.1-6 alkyl optionally substituted by a group R.sup.12 as defined above; and R.sup.12b is H, methyl or ethyl, more suitably H or methyl and especially H.
9. A compound according to claim 8 wherein R.sup.12a is a side chain of a naturally-occurring amino acid selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine or proline or a non-natural amino acid such as D-alanine.
10. A compound according to claim 8 wherein R.sup.12b is C.sub.1-4 alkyl or H, especially H.
11. A compound according to claim 9 wherein the amino acid side chain is modified such that OH and/or SH groups are replaced with OC.sub.1-6 alkyl or SC.sub.1-6 alkyl and/or carboxylic acid groups are esterified as a C.sub.1-6 alkyl or benzyl ester.
12. A compound according to claim 1 wherein Q is NR.sup.2, and R.sup.2 and R.sup.3 together with the nitrogen atom to which they are attached, form a pyrrolidin-1-yl ring substituted at the 2-position with C(O)OR.sup.11, wherein R.sup.11 is as defined in claim 1; and/or wherein R.sup.1 is QR.sup.3, Q is NR.sup.2, and R.sup.2 and R.sup.3 together with the nitrogen atom to which they are attached, form a pyrrolidin-1-yl ring substituted at the 2-position with C(O)OR.sup.11, wherein R.sup.11 is as defined in claim 1.
13. A compound according to claim 1 wherein Q is O or S and R.sup.3 is C.sub.1-10 alkyl substituted with, OC(O)R.sup.13, SC(O)R.sup.13 or SSR.sup.13, where R.sup.13 is H or C.sub.1-6 alkyl; and/or wherein R.sup.1 is QR.sup.3, Q is O or S and R.sup.3 is C.sub.1-10 alkyl substituted with, OC(O)R.sup.13, SC(O)R.sup.13 or SSR.sup.13, where R.sup.13 is H or C.sub.1-6 alkyl.
14. A compound according to claim 1 wherein at least one of R.sup.5 and R.sup.6 is H.
15. A compound according to claim 1 wherein, independently or in any combination: R.sup.4 is OH; R.sup.5 is H; R.sup.6 is H; R.sup.1 is OAr.sup.1 and Ar.sup.1 is phenyl; the R.sup.7 moiety is positioned at the 4-position of the phenyl ring with respect to the CH.sub.2CH.sub.2 linker group; R.sup.7 is C.sub.6-10 alkyl or C.sub.3-5 alkyl substituted with (C.sub.1-2 alkyl) phenyl; the C* centre has S stereochemistry; and the moiety OCH.sub.2C(CH.sub.2OH)(NR.sup.5R.sup.6)CH.sub.2CH.sub.2Ar.sup.1R.sup.7 is ##STR00058## and especially the S enantiomer: ##STR00059##
16. A compound according to claim 1 selected from the group consisting of: (2S)-methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)propanoate; benzyl 2-(((2-amino-2(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)amino)acetate; (2S) benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-4-methylpentanoate; (2S) benzyl-1-((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)pyrrolidine-2-carboxylate; (2S) ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)amino)-propanoate; (2R)-benzyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (naphthalen-1-yloxy)phosphoryl)amino)propanoate; (2R)-neopentyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (naphthalen-1-yloxy)phosphoryl)amino) propanoate; methyl 3-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate; (2R)-neopentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) ((5,6,7,8-tetrahydronaphthalen-1-yl)oxy)phosphoryl) amino)propanoate; isopropyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-2-methylpropanoate; (2R)-benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (naphthalen-1-yloxy)phosphoryl)amino)propanoate; (2S)-ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-4-(methylthio) butanoate; (2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-3-(4-(tert-butoxy)phenyl)propanoate; (2R)-dimethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)pentanedioate; (2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-3-(1H-indol-3-yl)propanoate; (2S,3R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (naphthalen-1-yloxy)phosphoryl)amino)-3-(tert-butoxy)butanoate; (2R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-6-((tert-butoxycarbonyl) amino)hexanoate; S-(2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)oxy)ethyl) 2,2-dimethylpropanethioate; pentyl (2S)-2- [({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate; methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)amino)acetate; ethyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)amino)acetate; (3S)-methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-3-methylpentanoate; (2S) pentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-3-phenylpropanoate; and their pharmaceutically acceptable salts, esters and hydrates and all stereochemistries.
17. A process for the preparation of a compound according to claim 1 comprising: I. for compounds of general formula (I) in which R.sup.5 and R.sup.6 are both H: reacting an analogue of the compound of general formula (I) in which one of R.sup.5 and R.sup.6 is replaced with C(O)OR.sup.15, wherein le is C.sub.1-6 alkyl or C.sub.6-14 aryl optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, C.sub.1-6 haloalkyl or halo; to remove the protecting group, for example by hydrogenation using a suitable catalyst, such as palladium/carbon; ii. reacting a compound of general formula (II): ##STR00060## wherein R.sup.5, R.sup.6, Ar.sup.1 and R.sup.7 are as defined for general formula (I); with a compound of general formula (III): ##STR00061## wherein: Q, R.sup.1 and R.sup.3 are as defined for general formula (I); and X.sup.1 is halo, particularly chloro; wherein the compound of general formula (II) may firstly be reacted with a hindered base, for example a Grignard reagent, following which the product is reacted with the compound of general formula (III).
18. (canceled)
19. (canceled)
20. (canceled)
21. A method for the treatment of lysosomal storage disorders selected from the group consisting of: Niemann-Pick type C1, Niemann-Pick type C2, Niemann-Pick types A and B, neuronal ceroid lipofuscinoses (NCL), mucolipidoses, lipidoses and sphingolipidoses, Gaucher disease, Fabry disease and Tay-Sachs disease; defective autophagy, accumulation of free cholesterol and endocytic transport defects; and mycobacterial diseases, tuberculosis and BCG, the method comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1.
22. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable excipient or carrier.
23. A pharmaceutical composition according to claim 22 which is formulated for oral administration.
Description
FIGURE LEGENDS
[0186]
[0187]
[0188]
[0189]
[0190]
[0191]
MATERIALS AND METHODS
General Procedures
[0192] All experiments involving water-sensitive compounds were conducted under scrupulously dry conditions. Anhydrous tetrahydrofuran (THF) and dichloromethane were purchased from Aldrich and used directly. Column chromatography refers to flash column chromatography carried out using Merck silica gel 60 (40-60 m) as stationary phase. Proton, carbon, and phosphorus nuclear magnetic resonance (.sup.1H, .sup.13C, .sup.31P NMR) spectra were recorded on Bruker Avance spectrometers operating either at 500, 125, and 202 MHz. The solvents used are indicated for each compound. All .sup.13C and .sup.31P spectra were recorded proton decoupled. Chemical shifts for .sup.1H and .sup.13C spectra are in parts per million downfield from tetramethylsilane. Coupling constants are referred to as J values. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), broad signal (br), doublet of doublet (dd), doublet of triplet (dt), or multiplet (m). Chemical shifts for 31P spectra are in parts per million relative to an external phosphoric acid standard. Some of the proton and carbon NMR signals were splitted because of the presence of (phosphate) diastereoisomers in the samples. Electrospray mass spectra were obtained using a Bruker MicroTOF coupled to an Agilent 1100 HPLC system. The electrospray source was operated at a temperature of 130 C. with a desolvation temperature of 300 C., a capillary voltage of 3 kV, and cone voltage of 30 V. Data were collected in the continuum mode over the mass range 100-2000 amu. Analytical HPLC was performed on a Thermo Fisher Spectra system 4000 using a RP C-18 column Varian Pursuit, 150 mm4.6 mm, 5.0 m with detection wavelength was 220 nm Mobile phases: Eluent A=H.sub.2O (+0.1% HClO.sub.4), Eluent B=Acetonitrile, gradient [time (min.)/% eluent B]: (0/50, 20/85, 22/85, 24/00, 25/100, 28/50), flow rate: 0.8 mL/min.
Synthesis of 1-naphthyl Dichlorophosphate
[0193] ##STR00018##
[0194] Phosphorus oxychloride (2.59 ml, 27.74 mmol, 1 eq) and 1-naphthol (4 g, 27.74 mmol, 1 eq) were stirred in anhydrous Et.sub.2O under an argon atmosphere. Anhydrous Et.sub.3N was added (3.87 ml, 27.74 mmol, 1 eq) at 78 C. After 30 minutes the solution was allowed to warm to room temperature. After 3.5 hours after checking the disappear of starting material peak and concomitant formation of the desired products peak by .sup.31P NMR, the mixture was subjected to vacuum filtration. The solid salt mixture was discarded and Et.sub.2O solvent was removed from the solution in vacuo yielding the yellow oil product in 72% yield (5.18 g). C.sub.10H.sub.7Cl.sub.2O.sub.6P; M.W: 260.0; .sup.1H NMR (CDCl.sub.3, 500 MHz): 8.13-7.37 (7H, m, ArH); .sup.31P NMR (CDCl.sub.3, 202 MHz): 3.73.
Phosphorochloridate Synthesis Standard Procedure
[0195] To a stirred solution of the appropriate amino acid ester salt (1 equivalent) and the appropriate aryl dichlorophosphate (1 equivalent) in anhydrous CH.sub.2Cl.sub.2 was added dropwise at 78 C. anhydrous Et.sub.3N (2 equivalents). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound and disappearance of the starting material was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. Most of the aryl phosphorochloridates synthesised were purified by flash column chromatography on silica gel (eluting with hexane-ethyl acetate 70:30 v/v).
Synthesis of Phenylbenzyloxy-L-leucinyl) Phosphorochioridate
[0196] ##STR00019##
[0197] To a stirred solution of L-leucine benzyl ester p-tosylate (3.34 g. 8.5 mmol, 1 eq) and phenyl dichlorophosphate (1.27 ml, 8.5 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (20 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.37 ml, 17 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 42% yield (1.41 g). C.sub.19H.sub.23ClNO.sub.4P; M.W: 395.1; .sup.31P NMR (CDCl.sub.3, 202 MHz): 8.45, 8.15.
Synthesis of Phenyl-(benzyloxy-L-prolinyl) Phosphorochioridate
[0198] ##STR00020##
[0199] To a stirred solution of L-Proline benzyl ester hydrochloride (2.05 g. 8.5 mmol, 1 eq) and phenyl dichlorophosphate (1.27 ml, 8.5 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (20 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.37 ml, 17 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 72% yield (2.33 g). C.sub.18H.sub.19ClNO.sub.4P; M.W: 379.1; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.38-7.16 (10H, m, ArH), 5.16 (2H, m, OCH.sub.2Ph), 4.55 (1H, m, CHNP), 3.51 (2H, m, CH.sub.2NP), 2.21 (2H, m, CHCH.sub.2CH.sub.2), 1.96 (2H, m, CHCH.sub.2CH.sub.2); .sup.31P NMR (CDCl.sub.3, 202 MHz): 7.74, 7.67.
Synthesis of Phenyl-(ethoxy-L-alaninyl) Phosphorochioridate
[0200] ##STR00021##
[0201] To a stirred solution of L-Alanine ethyl ester hydrochloride (1.31 g. 8.5 mmol, 1 eq) and phenyl dichlorophosphate (1.27 ml, 8.5 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (20 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.37 ml, 17 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 84% yield (2.1 g). C.sub.11H.sub.15ClNO.sub.4P; M.W: 291.0; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.36-7.21 (5H, m, ArH), 4.38 (1H, m, CHCH.sub.3), 4.22 (2H, m, OCH.sub.2CH.sub.3), 1.52 (3H, m, CH.sub.2CH.sub.3), 1.25 (3H, m, CHCH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz): 8.02, 7.70.
Synthesis of 1-Naphthylethoxy-L-alaninyl) Phosphorochioridate
[0202] ##STR00022##
[0203] To a stirred solution of L-Alanine ethyl ester hydrochloride (1.31 g. 8.5 mmol, 1 eq) and 1-naphthyl dichlorophosphate (1.51 ml, 8.5 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (20 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.37 ml, 17 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 64% yield (1.87 g). C.sub.15H.sub.17ClNO.sub.4P MW: 341.0 .sup.1H NMR (CDCl.sub.3, 500 MHz): 8.19-7.22 (7H, m, ArH), 4.52 (1H, m, CHCH.sub.3), 4.22 (2H, m, OCH.sub.2CH.sub.3), 1.52 (3H, m, CH.sub.2CH.sub.3), 1.25 (3H, m, CHCH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz): 8.28, 8.00.
Phenyl-(methoxy-L-methioninyl) Phosphorochloridate
[0204] ##STR00023##
[0205] To a stirred solution of L-methionine methyl ester hydrochloride (3.0 g. 14.10 mmol, 1 eq) and phenyl dichlorophosphate (2.11 ml, 14.10 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (100 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (3.94 ml, 28.21 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 80% yield (4.00 g). C.sub.13H.sub.19ClNO.sub.4P; M.W: 351.79; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.42-7.35 (m, 2H, ArH), 7.33-7.23 (m, 3H, ArH), 4.64 (t, J=10.4 Hz, 2H, CHNH), 4.30-4.26 (m, 2H, CH.sub.2CH.sub.3), 2.70-2.54 (m, 2H, CHCH.sub.2), 2.242.13 (m, 2H, CH.sub.2S), 2.10 (s, 3H, SCH.sub.3), 1.32 (t, J=7.1 Hz, 2H, CH.sub.2CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz): 8.49, 8.36.
[0206] Phenyl-(methoxy-O-tert-butyl-L-tyrosinyl) Phosphorochloridate
##STR00024##
[0207] To a stirred solution of O-tert-butyl L-tyrosine methyl ester hydrochloride (3.0 g. 10.42 mmol, 1 eq) and phenyl dichlorophosphate (1.56 ml, 10.42 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (100 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.91 ml, 20.85 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 67% yield (2.98g,). C.sub.20H.sub.25ClNO.sub.5P; M.W: 425.84; .sup.1H NMR (500 MHz, CDCl.sub.3) 7.42-7.36 (m, 2H, ArH), 7.25 (m, 3H, ArH), 7.08 (d, J=8.5 Hz, 1H, ArH), 7.04 (d, J=8.5 Hz, 1H, ArH), 6.96-6.92 (m, 2H, ArH), 4.50-4.32 (m, 1H, CHNH), 4.24-4.07 (m, 1H, CHNH), 3.75 (s, 1H, OMe), 3.73 (s, 1H, OMe), 3.17-3.09 (m, 2H), 1.35 (s, 5H), 1.33 (s, 4H); .sup.31P NMR (202 MHz, CDCl.sub.3) 7.90, 7.87.
Phenyl-(dimethoxy-L-glutamyl) Phosphorochloridate
[0208] ##STR00025##
[0209] To a stirred solution of L-glutamic acid dimethyl ester hydrochloride (3.0 g. 12.52 mmol, 1 eq) and phenyl dichlorophosphate (1.87 ml, 12.52 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (100 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (3.40 ml, 25.03 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 76% yield (3.59 g). C.sub.15H.sub.21ClNO.sub.6P; M.W: 377.76; .sup.1H NMR (500 MHz, CDCl.sub.3) 7.42-7.37 (m, 2H, ArH), 7.30-7.26 (m, 3H, ArH), 4.50-4.39 (m, 1H, CHNH), 4.31-4.18 (m, 1H, CHNH), 3.83 (s, 1H, OCH.sub.3), 3.81 (s, 1.5H, OCH.sub.3), 3.70 (s, 1.5H, OCH.sub.3), 3.67 (s, 1.5H, OCH.sub.3), 2.64-2.35 (m, 2H, CH.sub.2CO.sub.2Me), 2.32-2.22 (m, 1H, CHCH.sub.2), 2.13-2.00 (m, 1H, CHCH.sub.2); .sup.31P NMR (202 MHz, CDCl.sub.3) 8.34, 8.24.
[0210] Phenyl-(methoxy-L-tryptophanyl) Phosphorochioridate
FG-33 -tryptophan
[0211] ##STR00026##
[0212] To a stirred solution of L-Tryptophane methyl ester hydrochloride (2.0 g. 7.85 mmol, 1 eq) and phenyl dichlorophosphate (1.17 ml, 7.85 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (100 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.13 ml, 15.70 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 72% yield (2.24g). C.sub.18H.sub.18ClN.sub.2O.sub.4P; M.W: 392.77; .sup.1H NMR (CDCl.sub.3, 500MHz) 8.11 (s, 1H, NH), 7.51-7.44 (m, 1H, ArH), 7.32-7.20 (m, 3H, ArH), 7.19 (s, 1H, ArH), 7.17-6.97 (m, 4H, ArH), 4.51-4.30 (m, 1H, CHNH), 4.24-4.05 (m, 1H, CHNH), 3.61 (s, 3H, OCH.sub.3), 3.60 (s, 3H, OCH.sub.3), 3.34-3.21 (m, 2H, CH.sub.2); .sup.31P NMR (202 MHz, CDCl.sub.3) 8.08, 7.99.
Phenyl-(methoxy-O-tert-butyl-L-threoninyl) Phosphorochloridate
[0213] ##STR00027##
[0214] To a stirred solution of O-tert-butyl L-Threonine methyl ester hydrochloride (2.0 g. 8.86 mmol, 1 eq) and phenyl dichlorophosphate (1.32 ml, 8.86 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (100 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.41 ml, 17.72 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 96% yield (3.10 g). C.sub.15H.sub.23ClNO.sub.5P; M.W: 363.77; .sup.1H NMR (CDCl.sub.3, 500 MHz) 7.40-7.35 (m, 2H, Ph), 7.31-7.26 (m, 2H, Ph), 7.26-7.21 (m, 1H, Ph), 4.45 (m, 1H, NH), 4.20 (m, 1H, CH.sub.3CH), 3.97 (m, 1H, CHNH), 3.76 (s, 1.5H, OCH.sub.3), 3.74 (s, 1.5H, OCH.sub.3), 1.29 (d, J=6.2 Hz, 1.5H, CH.sub.3), 1.27 (d, J=6.2 Hz, 1.5H, CH.sub.3), 1.12 (s, 9H, C(CH.sub.3)3); .sup.31P NMR (CDCl.sub.3, 202 MHz) 9.71, 8.98.
Phenyl-(methoxy-N-Boc-L-lysinyl) Phosphorochioridate
[0215] ##STR00028##
[0216] To a stirred solution of N-Boc-L-lysine methyl ester hydrochloride (2.2 g. 7.41 mmol, 1 eq) and phenyl dichlorophosphate (1.11 ml, 7.41 mmol, 1 eq) in anhydrous CH.sub.2Cl.sub.2 (100 ml) was added dropwise at 78 C. anhydrous Et.sub.3N (2.01 ml, 14.83 mmol, 2 eq). Following the addition, the reaction mixture was stirred at 78 C. for 30 min and then at room temperature for 1 h. Formation of the desired compound was monitored by .sup.31PNMR. After this period the solvent was removed under reduced pressure to give an oil. The product was then purified by flash column chromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving the desired compound in 89% yield (2.88 g). C.sub.18H.sub.28ClN.sub.2O.sub.6P; M.W: 674.81; .sup.1H NMR (500 MHz, CDCl.sub.3) 7.43-7.37 (m, 2H, ArH), 7.31-7.25 (m, 3H, ArH), 4.59 (d, J=27.6 Hz, 1H, CHNH), 4.41-4.27 (m, 1H, CH.sub.2aNHBOC), 4.23-4.05 (m, 1H, CH.sub.2bNHBOC), 3.82 (s, 3H, OCH.sub.3), 3.78 (s, 3H, OCH.sub.3), 3.12 (d, J=6.6 Hz, 2H, CH.sub.2CHNHP), 1.90 (m, 1H, CH.sub.2), 1.85-1.73 (m, 1 H, CH.sub.2), 1.57-1.48 (m, 1H, CH.sub.2), 1.48 (s, 9H, C(CH.sub.3).sub.3); .sup.31P NMR (202 MHz, CDCl.sub.3) 8.40, 8.28.
Phosphoramidate Synthesis Using tBuMgCI Standard Procedure
[0217] tBuMgCl (1 equivalent) was added dropwise to a solution of primary alcohol (e.g. fingolimod hydrochloride/1-(4-{[(2S)-2-Amino-3-hydroxy-2-methyl-propoxy]methyl}phenyl)-4-(4-methylphenyl)butan-1-one) (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time the appropriate phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was dry-loaded to a column and isolated using flash chromatography (eluting with methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v).
EXAMPLE 1
Synthesis of (2S)-methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)propanoate[Phenyl-(methyloxy-L-alaninyl) Phosphoramidate Fingolimod]
[0218] ##STR00029##
[0219] tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Phenyl-(methyloxy-L-alaninyl) phosphorochloridate (403 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (2.6 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hour and then the solvent was removed in vacuo and the desired product was isolated using flash chromatography (methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v) giving the desired compound as a mixture of four diastereoisomers in 44% yield (0.35g). C.sub.25H.sub.45N.sub.2O.sub.6P; M.W: 548.3; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.29-7.02 (9H, m, ArH), 5.19 (1H, m, CHNH), 4.39 (3H, m, OCH.sub.3), 4.18 (1H, m, CHCH.sub.3), 3.81 (2H, m, POCH.sub.2), 3.61 (2H, m, CH.sub.2OH), 2.64 (2H, m, CH.sub.2CH.sub.2Ph), 2.54 (2H, m, CH.sub.2C.sub.7H.sub.15), 2.05 (2H, m, CH.sub.2CH.sub.2Ph), 1.61 (2H, m, CH.sub.2C.sub.6H.sub.13), 1.42 (1.5H, m, CHCH.sub.3), 1.41 (1.5H, m, CHCH.sub.3), 1.33-1.27 (10H, m, 5 CH.sub.2 C.sub.5H.sub.10CH.sub.3), 0.81 (3H, m, CH.sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz): 3.60, 3.47, 3.14, 3.06. MS [ES+] m/z 549.3 [M+H].sup.+.
EXAMPLE 2
Synthesis of Benzyl 2-(((2-amino-2(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate [Phenyl-(benzyloxy-glycinyl) Phosphoramidate Fingolimod]
[0220] ##STR00030##
[0221] tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Phenyl-(benzyloxy-glycinyl) phosphorochloridate (493 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (2.75 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated using flash chromatography (methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v) giving the desired compound as a mixture of two diastereoisomers in 17% yield (0.15 g). C.sub.34H.sub.47N.sub.2O.sub.6P; M.W: 610.3; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.32-6.98 (14H, m, ArH), 5.26 (2H, m, OCH.sub.2Ph), 5.19 (1H, m, CHNH), 4.22 (2H, m, CH.sub.2NH), 3.81 (2H, m, POCH.sub.2), 3.72 (2H, m, CH.sub.2OH), 2.64 (2H, m, CH.sub.2CH.sub.2Ph), 2.54 (2H, m, CH.sub.2C.sub.7H.sub.15), 2.05 (2H, m, CH.sub.2CH.sub.2Ph), 1.61 (2H, m, CH.sub.2C.sub.6H.sub.13), 1.42 (3H, m, CHCH.sub.3), 1.33-1.27 (10H, m, 5 CH.sub.2, C.sub.5H.sub.10CH.sub.3), 0.81 (3H, m CH.sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz): 4.27, 3.99; MS [ES+] m/z 611.3 [M+H].sup.+.
EXAMPLE 3
Synthesis of (2S) benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate [Phenyl-(benzyloxy-L-leucinyl) Phosphoramidate Fingolimod]
[0222] ##STR00031##
[0223] tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (5 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After one hour the Phenyl-(benzyloxy-L-leucinyl) phosphorochloridate (574 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (3.2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours. After 24 hours the solvent was removed in vacuo and the desired product was isolated using flash chromatography (methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v) giving the desired compound as a mixture of four diastereoisomers in 23%, yield (0.22 g). C.sub.38H.sub.55N.sub.2O.sub.6P; M.W: 666.4; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.28-6.29 (14H, m, ArH), 4.96 (2H, m, OCH.sub.2Ph), 4.39 (2H, m, NH.sub.2), 4.13 (2H, m, POCH.sub.2), 3.91 (1H, m, CHCH.sub.3), 4.02 (1H, m, CHCH.sub.2), 3.42 (2H, m, CH.sub.2OH), 2.52 (2H, m, CH.sub.2CH.sub.2Ph), 2.48 (2H, m, CH.sub.2C.sub.7H.sub.15), 1.71 (2H, m, CH.sub.2CH.sub.2Ph), 1.48 (2H, m, CCH.sub.2CH), 1.40 (2H, m, CH.sub.2C.sub.6H.sub.13), 1.22-1.17 (10H, m, 5 CH.sub.2, C.sub.5H.sub.10CH.sub.3), 0.82 (3H, m, CHCH.sub.3), 0.81 (3H, m, CH.sub.3), 0.75 (3H, m, CHCH.sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz): 4.66, 4.46, 4.20, 3.95; MS [ES+] m/z 667.4 [M+H].sup.+.
EXAMPLE 4
Synthesis of (2S) benzyl-1-((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)pyrrolidine-2-carboxylate [Phenyl-(benzyloxy-L-prolinyl) Phosphoramidate Fingolimod]
[0224] ##STR00032##
[0225] tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (5 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After one hour Phenyl-(benzyloxy-L-prolinyl) phosphorochloridate (551 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (4.5 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours. After 24 hours the solvent was removed in vacuo and the desired product was isolated using flash chromatography (methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v) giving the desired compound as a mixture of four diastereoisomers in 20% yield (0.19 g). C.sub.37H.sub.51N.sub.2O.sub.6P; M.W: 650.4; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.32-6.98 (14H, m, ArH), 5.16 (2H, m, OCH.sub.2Ph), 4.55 (1H, m, CHN), 3.81 (2H, m, POCH.sub.2), 3.61 (2H, m, CH.sub.2OH), 3.51 (2H, m, CH.sub.2NP), 2.64 (2H, m, CH.sub.2CH.sub.2Ph), 2.54 (2H, m, CH.sub.2C.sub.7H.sub.13), 2.21 (2H, m, CH.sub.2CH.sub.2Ph), 2.05 (2H, m, CH.sub.2CH.sub.2Ph), 1.96 (2H, m, CHCH.sub.2CH.sub.2), 1.61 (2H, m, CH.sub.2CH.sub.2CH.sub.2), 1.42 (3H, m, CHCH.sub.3), 1.33-1.27 (10H, m, 5 CH.sub.2 C.sub.5H.sub.10CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz): 2.99, 2.94. MS [ES+] m/z 651.4 [M+H].sup.+.
EXAMPLE 5
Synthesis of (2S) ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-propanoate [Phenyl-(ethoxy-L-alaninyl) Phosphoramidate Fingolimod]
[0226] ##STR00033##
[0227] tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (5 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After one hour the Phenyl-(ethoxy-L-alaninyl) phosphorochloridate (423 mg, 1.45 mmol, 1 equivalent) in anhydrous THF (2.8 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours. After 24 hours the solvent was removed in vacuo and the desired product was isolated using flash chromatography (methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v) giving the desired compound as a mixture of four diastereoisomers in 20%, yield (0.16 g). C.sub.30H.sub.47N.sub.2O.sub.6P; M.W: 562.3; .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.36-7.05 (9H, m, ArH), 4.22 (3H, m, OCH.sub.2CH.sub.3), 4.05 (2H, m, OCH.sub.2C), 3.65 (1H, m, CHCH.sub.3), 3.45 (2H, m, HOCH.sub.2C), 2.61 (2H, m, CH.sub.2CH.sub.2Ph), 2.58 (2H, m, CH.sub.2CH.sub.2Ph), 1.71 (2H, m, CCH.sub.2CH.sub.2), 1.65 (2H, m, CH.sub.2CH.sub.2CH.sub.2), 1.45 (3H, m, CHCH.sub.3), 1.38 (2H, m, CH.sub.2CH.sub.2CH.sub.2), 1.37 (2H, m, CH.sub.2CH.sub.2CH.sub.2), 1.36 (2H, m, CH.sub.2CH.sub.2CH.sub.2), 1.29 (2H, m, CH.sub.2CH.sub.2CH.sub.2), 1.27 (2H, m, CH.sub.2CH.sub.2CH.sub.2), 1.22 (3H, m, CHCH.sub.3), 0.98 (3H, m, CH.sub.2CH.sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz): 4.05, 3.84, 3.73, 3.59. MS [ES+] m/z 563.3 [M+H].sup.+.
EXAMPLE 6
Synthesis of (2R)-benzyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino) Propanoate
[0228] ##STR00034##
[0229] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Naphthyl-(benzyloxy-L-Alaninyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 26% yield (0.1 g) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.28, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.39H.sub.51N.sub.2O.sub.6P; M.W: 674.81; .sup.1H NMR (CDCl.sub.3, 500 MHz) 8.07-7.98 (m, 1H, Naph), 7.80-7.68 (m, 1H, Naph), 7.55 (d, J=8.2 Hz, 1H, Naph) 7.41-7.39 (m, 3H, ArH), 7.26-7.17 (m, 6H, ArH), 7.09-6.87 (m, 2H, ArH), 6.83 (m, 2H, ArH), 5.30-4.83 (m, 2H, CH.sub.2OBn), 4.23-4.16 (1H, m, CHCH.sub.3) 4.02-3.85 (m, 2H, POCH.sub.2), 3.58-3.19 (m, 2H, CH.sub.2OH,), 2.53-2.31 (m, 4H, CH.sub.2C.sub.7H.sub.15, CH.sub.2CH.sub.2Ph), 1.59-1.41 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.6H.sub.13), 1.39-1.29 (m, 3H, CHCH.sub.3), 1.28-1.13 (m, 10H, 5 CH.sub.2, C.sub.5H.sub.10CH.sub.3,), 0.80 (m, 3H, CH.sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz) 4.59, 4.36, 4.29, 4.12; .sup.13C NMR (CDCl.sub.3, 125 MHz) (several signals overlaps) 173.61, 173.56, 173.45, 173.40, 146.46, 140.51, 140.49, 138.64, 138.58, 135.17, 135.15, 134.74, 128.66, 128.59, 128.51, 128.44, 128.39, 128.30, 128.18, 128.11, 127.86, 127.81, 125.66, 125.52, 125.52, 125.15, 125.11, 125.10, 121.60, 121.46, 115.79, 115.65, 77.31, 77.05, 76.80, 68.68, 68.63, 68.48, 67.37, 67.34, 67.32, 65.52, 64.74, 64.67, 50.50, 36.86, 35.56, 35.45, 31.95, 31.93, 31.91, 31.61, 31.59, 31.58, 29.72, 29.52, 29.50, 29.41, 29.39, 29.37, 29.31, 29.29, 29.06, 28.61, 28.56, 28.54, 22.70, 20.82, 14.14; MS [ES+] m/z 675 [M+H].sup.+, HPLC r.t. 14.1, 14.5, 15.24, 15.80 min.
EXAMPLE 7
Synthesis of (2R)-neopentyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino) Propanoate (Prodrug D)
[0230] ##STR00035##
[0231] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Naphthyl-(neopentyloxy-L-Alaninyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 29% yield (110 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.28, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v). Many signals overlap in .sup.1H, .sup.13C spectra. C.sub.37H.sub.55N.sub.2O.sub.6P; M.W: 654.81; .sup.1H NMR (CDCl.sub.3, 500 MHz,) 8.05-8.03 (m, 1H, Naph), 7.77-7.73 (m, 1H, Naph), 7.57 (d, J=8.2 Hz, 1H, Naph), 7.43-4.41 (m, 3H, Naph), 7.31-7.29 (m, 1H, Naph), 6.99-6.33 (m, 4H, ArH), 4.12-4.04 (m, 1H, CHCH.sub.3), 4.00-3.87 (m, 2H, POCH.sub.2,), 3.83-3.72 (m, 1H,CH.sub.2aC(CH.sub.3).sub.3), 3.62-3.67 (m, 1H, CH.sub.2bC(CH.sub.3).sub.3), 3.26-3.17 (2H, m, CH.sub.2OH), 2.49-2.45 (m, 2H, CH.sub.2C.sub.7H.sub.15), 2.43-2.36 (m, 2H, CH.sub.2CH.sub.2Ph,), 1.57-1.48 (m, 2H, CH.sub.2C.sub.6H.sub.13), 1.46-1.30 (m, 5H, CH.sub.2CH.sub.2Ph CHCH.sub.3,), 1.30-1.14 (m, 10H, C.sub.5H.sub.10CH.sub.3), 0.85-0.76 (m, 12H, CH.sub.3, C(CH.sub.3).sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz,) 4.83, 4.62, 4.58, 4.39; .sup.13C NMR (CDCl.sub.3, 125 MHz) 173.64, 173.59, 146.49, 146.43, 140.46, 138.72, 138.67, 134.76, 128.42, 128.40, 128.39, 128.12, 128.08, 127.89, 127.86, 127.82, 125.64, 125.54, 125.48, 125.52, 125.14, 125.08, 121.61, 121.51, 121.48, 115.84, 115.71, 115.70, 115.55, 74.83, 68.82, 64.98, 50.48, 35.56, 31.92, 31.61, 31.43, 29.71, 29.51, 29.39, 29.29, 29.28, 28.51, 26.33, 26.28, 22.69, 22.69, 21.21, 21.19, 14.12; MS [ES+] m/z 655.80 [M+H].sup.+, HPLC r.t. 22.30, 22.80 min.
EXAMPLE 8
Synthesis of methyl 3-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate (Prodrug A)
[0232] ##STR00036##
[0233] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Naphthyl-(methoxy-L--Alaninyl) phosphorochloridate 1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 33% yield (115 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.26, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); Many signals overlap in .sup.1H, .sup.13C spectra; C.sub.33H.sub.47N.sub.2O.sub.6P, MW: 598.71; .sup.1H NMR (CDCl.sub.3, 500 MHz) 8.06-8.02 (m, 1H, Naph), 7.75-7.74 (m, 1H, Naph), 7.57 (d, J=8.2 Hz, 1H, Naph), 7.45-7.38 (m, 3H, Naph), 7.31-7.29 (m, 1H, Naph), 7.00-6.91 (m, 4H, ArH), 3.98-3.79 (m, 2H, POCH.sub.2), 3.54 (s, 3H, CH.sub.3), 3.28-3.14 (m, 4H, CH.sub.2OH, CH.sub.2NH), 2.45-2.35 (m, 6H, CH.sub.2CO, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.13), 1.57-1.45(m, 2H, CH.sub.2C.sub.6H.sub.13), 1.44-1.36 (m, 2H, CH.sub.2CH.sub.2Ph), 1.29-1.14 (m, 10H, 5 CH.sub.2, C.sub.5H.sub.10CH.sub.3), 0.80-0.78 (m, 3H, CH.sub.3). .sup.31P NMR (202 MHz, Chloroform-d) 6.45, 6.20; .sup.13C NMR (CDCl.sub.3, 125 MHz) 172.43, 146.55 (d, J.sub.PH=7.5 Hz), 146.53 (d, J.sub.PH=7.5 Hz), 140.49, 140.45, 138.95, 138.90, 134.76, 128.66, 128.42, 128.38, 128.13, 128.10, 127.90, 127.88, 125.65, 125.59, 125.54, 125.45, 125.59, 125.54, 125.07, 125.00, 121.52, 121.49, 115.67 (d, J.sub.PC=2.5 Hz), 115.51, (d, J.sub.PC=2.5 Hz), 69.43, 68.94, 65.35, 65.27, 55.88, 51.82, 37.44, 37.41, 36.08, 35.96, 35.73, 35.56, 31.92, 31.61, 29.51, 29.40, 29.29, 28.59, 28.56, 22.69, 14.13; MS [ES+] m/z 599.71 [M+H].sup.+, 621.7 [M+Na].sup.+.
EXAMPLE 9
Synthesis of (2R)-neopentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)((5,6,7,8-tetrahydronaphthalen-1-yl)oxy)phosphoryl)amino)propanoate
[0234] ##STR00037##
[0235] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time 5,6,7,8-tetrahydro-1-naphthyl-(neopenthyloxy-L-Alaninyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 21% yield (100 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.42, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); Many signals overlap in .sup.1H, .sup.13C spectra; C.sub.37H.sub.59N.sub.2O.sub.6P; MW: 658.85; .sup.1H NMR (CDCl.sub.3, 500 MHz,) 7.78-7.05 (m, 7H, ArH), 4.33-3.94 (m, 1H, CHCH.sub.3), 3.94-3.62(m, 4H, POCH.sub.2, CH.sub.2C(CH.sub.3).sub.3), 3.45-3.08 (m, 2H, CH.sub.2OH), 2.64-2.61 (4H, m, 2 CH.sub.2 tetrahydronaph), 2.47-2.43 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 1.73-1.43 (m, 6H, 2 CH.sub.2 tetrahydronaph, CH.sub.2C.sub.6H.sub.13), 1.38-1.31 (m, 2H, CH.sub.2CH.sub.2Ph) 1.28-1.09 (m, 10H, 5 CH.sub.2, C.sub.5H.sub.10CH.sub.3), 0.85, 0.84, 0.83, 0.83 (4s, 9H, C(CH.sub.3).sub.3), 0.84 -0.78 (m, 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz,) 4.62, 4.46, 4.23, 4.12; .sup.13C NMR (CDCl.sub.3, 125 MHz) 173.79, 173.73, 173.68, 173.66, 173.62, 173.60, 148.76, 140.52, 140.51, 139.57, 139.54, 139.50, 138.97, 138.95, 138.87, 128.71, 128.67, 128.62, 128.43, 128.13, 128.10, 128.09, 117.13, 74.81, 65.36, 65.00, 50.48, 50.37, 36.10, 35.98, 35.56, 31.91, 31.60, 31.44, 29.50, 29.47, 29.37, 29.28, 28.61, 28.59, 26.35, 23.55, 23.49, 21.26, 21.22, 21.17, 21.12, 14.11; MS [ES+] m/z 659.4 [M+H].sup.+, HPLC r.t. 23.50, 24.70 min.
EXAMPLE 10
Synthesis of isopropyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate
[0236] ##STR00038##
[0237] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time the Phenyl-(isopropoxy-dimethylglycinyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 24% yield (100 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.31, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.32H.sub.51N.sub.2O.sub.6P; M.W: 590.73; .sup.1H NMR (CDCl.sub.3, 500MHz) 7.26-7.22 (m, 2H, Ph), 7.16-7.13 (m, 2H, Ph), 7.10-7.07 (m, 1H, Ph), 7.01-6.95 (m, 2H, ArH), 4.97-4.91 (m, 1H, CH(CH.sub.3)2), 4.24 (t, J=9.1 Hz, OH), 4.03-3.79 (m, 2H, POCH.sub.2), 3.43-3.17 (m, 2H, CH.sub.2OH), 2.60-2.44 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 1.62-1.42 (m, 10H, CH.sub.2C.sub.6H.sub.13, CH.sub.2CH.sub.2Ph, C(CH.sub.3).sub.2), 1.22-1.15 (m, 16H, 5 CH.sub.2 C.sub.5H.sub.10CH.sub.3, CH(CH.sub.3).sub.2), 0.81-0.79 (m, 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz,) 3.37, 3.15; .sup.13C NMR (CDCl.sub.3, 125 MHz) 174.95, 174.86, 174.78, 150.84, 150.78, 150.73, 140.51, 138.95, 138.91, 129.71, 129.67, 128.43, 128.16, 128.13, 125.10, 125.04, 120.54 (d, J.sub.PC=5.0 Hz), 120.43(d, J.sub.PC=5.0 Hz), 69.58, 69.57, 69.21, 69.16, 68.75, 68.71, 65.26, 65.13, 57.02, 57.01, 56.98, 56.97, 56.24, 56.19, 56.15, 56.11, 35.56, 31.90, 31.59, 29.49, 29.38, 29.27, 28.62, 27.04, 26.92, 26.87, 26.84, 26.76, 26.74, 26.71, 22.67, 21.60, 21.58; MS [ES+] m/z 591.72 [M+H].sup.+, HPLC r.t. 17.172 min.
EXAMPLE 11
Synthesis of (2R)-benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino) Propanoate
[0238] ##STR00039##
[0239] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Naphtyl-(benzyloxy-L-alanilyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 26% yield (128 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.41, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.39H.sub.51N.sub.2O.sub.6P; M.Wt: 674.81; .sup.1H NMR (CDCl.sub.3, 500 MHz) 8.05-7.99 (m, 1H, Naph), 7.75-7.72 (m, 1H, Naph), 7.56 (d, J=8.3 Hz, 1H, Naph), 7.42-7.47 (m, 3H, Naph), 7.30-7.18 (m, 6H, ArH), 6.99-6.94 (m, 2H, ArH), 6.82 (d, J=6.6 Hz, 1H, ArH), 5.08-4.92 (m, 2H, CH.sub.2Ph), 4.10-3.75 (m, 3H, CHCH.sub.3, POCH.sub.2), 3.37-3.07 (m, 2H), 2.47 (td, J=7.5, 3.5 Hz, 3H), 2.51-2.34 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 1.57-1.45 (m, 2H, CH.sub.2C.sub.6H.sub.13), 1.42-1.29(m, 5H, CH.sub.2CH.sub.2Ph, CHCH.sub.3), 1.22-1.18 (m, 10H, 5 CH.sub.2, CH.sub.5H.sub.10CH.sub.3), 0.80 (m,1H 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz) 4.71, 4.51, 4.47, 4.24; .sup.13C NMR (CDCl.sub.3, 125 MHz) 173.42, 173.37, 146.40, 140.49, 140.48, 138.71, 138.64, 138.55, 135.64, 135.18, 135.15, 134.74, 134.61, 128.65, 128.59, 128.52, 128.50, 128.43, 128.39, 128.37, 128.29, 128.26, 128.18, 128.13, 128.10, 128.04, 127.90, 127.85, 127.52, 127.41, 125.64, 125.50, 125.03, 125.92, 125.51, 125.13, 125.08, 115.81, 115.78, 115.69, 115.66, 115.55, 115.52, 108.51, 67.34, 66.56, 64.69, 50.82, 50.62, 50.49, 50.46, 35.56, 31.92, 31.61, 29.52, 29.41, 29.30, 28.53, 22.69, 20.86, 20.82, 20.76, 14.13; MS [ES+] m/z 675.8 [M+H.sup.]+, HPLC r.t. 17.172 min.
EXAMPLE 12
Synthesis of (2S)-ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-(methylthio) Butanoate (Prodrug E)
[0240] ##STR00040##
[0241] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Phenyl-(ethoxy-L-methionyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 37% yield (140 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.36, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.32H.sub.51N.sub.2O.sub.6PS; M W: 622.80; .sup.1H NMR (CDCl.sub.3, 500 MHz) 6 7.25-7.21 (m, 2H, Ph), 7.17-7.13 (m, 2H, Ph), 7.09-7.04 (m, 1H, Ph), 7.10-6.96 (m, 4H, ArH), 4.16-3.78 (m, 5H, POCH.sub.2, OCH.sub.2CH.sub.3, CHNH), 3.50-3.17 (m, 2H, CH.sub.2OH), 2.56-2.33 (m, 6H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15, CH.sub.2S), 1.98-1.91 (m, 4H, SCH.sub.3, CH.sub.2aCH.sub.2S), 1.87-1.69 (m, 1H, CH.sub.2bCH.sub.2S), 1.62-1.50 (m, 6H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.6H.sub.13,), 1.22-1.13(m, 13H, 5 CH.sub.2, C.sub.5H.sub.10CH.sub.3, OCH.sub.2CH.sub.3), 0.80 (m, 3H, CH.sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz) 4.56, 4.30, 4.24, 3.98; .sup.13C NMR (CDCl.sub.3, 125 MHz) 6 172.88, 172.84, 172.79, 172.78, 172.76, 172.74, 150.68, 150.61, 150.56, 140.51, 140.49, 138.99, 138.92, 129.77, 129.73, 128.43, 128.17, 128.15, 128.13, 125.25, 125.21, 125.16, 120.50, 120.46, 120.40, 120.38, 120.36, 120.34, 120.25, 120.21, 69.51, 69.46, 69.36, 69.31, 69.20, 69.16, 65.32, 65.17, 61.80, 61.77, 61.08, 61.03, 58.26, 56.00, 55.84, 53.79, 53.72, 53.52, 53.39, 36.11, 36.05, 35.56, 33.53, 33.48, 33.43, 31.90, 31.59, 29.75, 29.70, 29.65, 29.49, 29.38, 29.27, 28.63, 28.60, 22.67, 15.41, 15.32, 14.25, 14.18, 14.14, 14.11; MS [ES+] m/z 623.3 [M+H].sup.+
EXAMPLE 13
Synthesis of (2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(4-(tert-butoxy)phenyl)propanoate
[0242] ##STR00041##
[0243] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Phenyl-(methoxy-O-tert-butyl-L-tyrosinyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 35% yield (150 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.32, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.39H.sub.57N.sub.2O.sub.7P; MW: 696.85; .sup.1H NMR (CDCl.sub.3, 500 MHz) 7.22-7.17 (m, 2H, ArH), 7.06 (d, J=8.4 Hz, 1H, ArH), 7.01-6.86 (m, 7H, ArH), 6.82-6.79 (m, 2H, ArH), 4.14-3.98 (m, 1H, CHNH), 3.96-3.61 (m, 2H, POCH.sub.2), 3.57, 3.53, 3.51 , 3.50 (4s, 4H, OCH.sub.3), 3.47-3.24 (m, 2H, CH.sub.2OH), 2.92-2.87 (m, 2H, CHCH.sub.2), 2.64-2.45 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 1.74-1.64 (m, OH), 1.75-1.49 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.6H.sub.13), 1.32-1.11 (m, 19H, 5 CH.sub.2, CH.sub.5H.sub.10CH.sub.3, C(CH.sub.3).sub.3), 0.81 (m, 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz,) 4.11, 3.89, 3.75, 3.11; .sup.13C NMR (CDCl.sub.3, 125 MHz,) 173.01, 172.98, 172.93, 172.90, 172.56, 172.51, 154.57, 150.60, 150.54, 150.47, 140.89, 140.71, 140.64, 140.52, 138.82, 138.61, 138.28, 138.18, 130.55, 130.48, 130.40, 129.99, 129.95, 129.90, 129.72, 129.70, 129.47, 129.24, 128.64, 128.50, 128.43, 128.16, 128.13, 125.19, 125.16, 124.20, 124.16, 123.97, 120.55, 120.51, 120.45, 120.43, 120.41, 120.40, 120.35, 120.31, 115.55, 78.44, 75.20, 75.15, 74.95, 74.90, 69.04, 69.00, 68.75, 68.71, 68.58, 68.54, 65.37, 64.91, 64.82, 64.79, 64.63, 56.14, 55.83, 55.77, 55.43, 52.30, 51.66, 49.93, 49.89, 49.27, 39.83, 39.79, 39.77, 39.75, 39.72, 39.70, 36.71, 36.56, 35.72, 35.67, 35.57, 31.91, 31.59, 29.71, 29.50, 29.40, 29.28, 28.85, 28.62, 28.58, 22.68, 14.12 ; MS [ES+] m/z 697.3 [M+H].sup.+ m/z 697.80 [M+H].sup.+
EXAMPLE 14
Synthesis of (2R)-dimethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)pentanedioate
[0244] ##STR00042##
[0245] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Phenyl-(dimethoxy-L-glutamyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 39% yield (148 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.31 CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.32H.sub.49N.sub.2O.sub.8P; M Wt: 620.71; .sup.1H NMR (CDCl.sub.3, 500 MHz,) 7.24-7.19 (m, 2H, Ph), 7.13-7.12 (m, 2H, Ph), 7.01-6.95 (m, 4H, ArH), 4.13-4.03 (m, 1H, CHNH), 4.00-3.83 (m, 3H, CHNH POCH.sub.2), 3.64-3.53 (m, 6H, 2 CO2CH.sub.3), 3.48-3.20 (m, 2H, CH.sub.2OH), 2.54-2.46 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 2.41-2.23 (m, CH.sub.2CH.sub.2CH), 2.14-1.78 (m, 2H, CH.sub.2CH.sub.2CH), 1.63-1.49 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.6H.sub.13), 1.22-1.18(m, 10H, 5 CH.sub.2, CH.sub.5H.sub.10CH.sub.3), 0.81 (m, 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz,) 4.34, 4.18, 4.08, 3.95; MS [ES+] m/z 621.30 [M+H].sup.+
EXAMPLE 15
Synthesis of (2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(1H-indol-3-yl)propanoate (Prodrug B)
[0246] ##STR00043##
[0247] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Phenyl-(methoxy-L-trypthophanyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 29% yield (110 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.18, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.37H.sub.50N.sub.3O.sub.6P; M. W: 663.78; .sup.1H NMR (CDCl.sub.3, 500MHz) 8.49 (bs, 1H, NH), 7.46-7.40 (m, 1H, ArH), 7.24 (m, 2H, ArH), 7.1-6.88 (m, 1H, 10H), 4.28-4.16 (m, 1H, CHNH), 3.94-3.64 (m, 3H, CHNH, POCH.sub.2), 3.27-3.01 (m, 4H, CH.sub.2OH, CHCH.sub.2), 3.57, 3.56,3.55, 3.53 (4s, 3H, OCH.sub.3), 2.51-2.38 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 1.52-1.27 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.6H.sub.13), 1.22-1.14 (m, 10H, 2 CH.sub.2, C.sub.5H.sub.10CH.sub.3), 0.80 (t, J=6.8 Hz, 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz) 4.43, 4.37, 4.19, 4.14; MS [ES+] m/z 664.5 [M+H].sup.+.
EXAMPLE 16
Synthesis of (2S,3R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)-3-(tert-butoxy)butanoate
[0248] ##STR00044##
[0249] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time Naphthyl-(methoxy-O-tert-butyl-L-threoninyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 4% yield (160 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.30, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.34H.sub.55N.sub.2O.sub.7P; M W: 634.78; .sup.1H NMR (CDCl.sub.3, 500 MHz) 7.35-7.31 (m, 2H, Ph), 7.18-7.154 (m, 2H, Ph), 7.01-7.06 (m, 4H, ArH), 4.16-4.12 (m, 1H, CHCH.sub.3), 4.07-3.77 (m, 3H, CHNH POCH.sub.2), 3.73-3.66 (m, 3H, OCH.sub.3), 3.53-3.141 (m, 2H, CH.sub.2OH), 2.72-2.55 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 1.70-1.55 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.6H.sub.13), 1.31-1.25 (m, 10H, 5 CH.sub.2, CH.sub.5H.sub.10CH.sub.3), 1.21-1.16 (m, 3H, CHCH.sub.3), 1.11-1.10 (m, 9H, C(CH.sub.3).sub.3), 0.91-0.88 (m, 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz) 5.45, 5.18, 4.93, 4.71; MS [ES+] m/z 635.37 [M+H].sup.+
EXAMPLE 17
Synthesis of (2R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-6-((tert-butoxycarbonyl) amino)hexanoate (Prodrug C)
[0250] ##STR00045##
[0251] tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimod hydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After this time the Phenyl-(methoxy-N-Boc-L-lysinlyl) phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours and then the solvent was removed in vacuo and the desired product was isolated in 39% yield (160 mg) as a mixture of four diastereoisomers using flash chromatography on a Biotage Isolera, eluting with CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v). (R.sub.f=0.40, CH.sub.2Cl.sub.2/CH.sub.3OH 95:5 v/v); C.sub.37H.sub.60N.sub.3O.sub.8P; M.W: 705.86; .sup.1H NMR (CDCl.sub.3, 500 MHz) 7.35-7.32 (m, 2H, Ph), 7.24-7.22 (m, 2H, Ph), 7.20-7.17 (m, 1H, Ph), 7.12-7.06 (m, 4H, ArH), 4.67-4.56 (m, 1H, CHNH), 4.06-4.95 (m, 4H, CH.sub.2NH, POCH.sub.2), 3.71, 3.69, 3.68 (3s, 3H, OCH.sub.3), 3.61-3.42 (m, 2H, CH.sub.2OH), 3.07-3.06 (m, 2H, CH.sub.2CH), 2.64-2.56 (m, 4H, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.7H.sub.15), 1.84-1.59 (m, 6H, CH.sub.2CH.sub.2NH, CH.sub.2CH.sub.2Ph, CH.sub.2C.sub.6H.sub.13), 1.46 (s, 9H, C(CH.sub.3).sub.3), 1.43-1.39 (m, 2H, CH.sub.2CH.sub.2CH), 1.32-1.24 (m, 10H, C.sub.5H.sub.10CH.sub.3), 0.90 (t, J=6.8 Hz, 3H, CH.sub.3); .sup.31P NMR (CDCl.sub.3, 202 MHz) 4.56, 4.32, 4.06, 3.86; .sup.13C NMR (CDCl.sub.3, 125 MHz) 173.67, 173.62, 173.45, 156.05, 150.61, 140.56, 138.91, 129.77, 129.74, 128.67, 128.52, 128.50, 128.44, 128.16, 128.13, 128.10, 128.09, 125.24, 125.20, 125.15, 120.50, 120.46, 120.43, 120.39, 120.37, 120.34, 69.24, 69.08, 65.36, 65.30, 58.41, 54.62, 54.40, 52.49, 50.79, 40.15, 40.08, 36.05, 33.88, 33.84, 33.78, 31.90, 31.60, 29.50, 29.38, 29.28, 28.63, 28.44, 22.68, 22.24, 22.20, 18.44, 14.11; MS [ES+] m/z 706.8 [M+H].sup.+
EXAMPLE 18
Synthesis of S-(2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)oxy)ethyl) 2,2-dimethylpropanethioate (SATE Prodrug)
A. Synthesis of 2,2-Dimethyl-thiopropionic acid S-(2-hydroxy-ethyl) Ester
[0252] ##STR00046##
[0253] Pivaloyl chloride (2.5 ml, 20.3 mmol) was added to a stirred solution of 2-mercaptoethanol (1.42 ml, 20.3 mmol) and triethylamine (2.83 ml, 20.3 mmol) in DCM, cooled at 78 C. The mixture was stirred at 78 C. for 1 h. After 1 h the mixture was extracted with water and DCM (320 ml). The combined organic extracts were dried over MgSO4and concentrated in vacuo. The oily residue was purified by flash column chromatography (eluting with hexane-ethyl acetate 80:20 v/v v/v increasing to 70:30 v/v) giving the desired compound (75%, 2.481 g). .sup.1H NMR (CDCl.sub.3, 500 MHz): 3.98 (2H, m, HOCH.sub.2), 3.03 (2H, m, SCH.sub.2), 2.85 (1H, b, OH), 1.23 (9H, s, CCH.sub.3).
B. Synthesis of 2,2-Dimethyl-thiopropionic acid S-[chloro-phenoxy-phosphoryloxy)-ethyl Ester
[0254] ##STR00047##
[0255] Dichloro phenyl phosphonate (2.29 ml, 15.31 mmol) was added dropwise into a cooled solution at 78 C. of 2,2-Dimethyl-thiopropionic acid S-(2-hydroxy-ethyl) ester (2.481 g, 15.31 mmol) and triethylamine (2.13 ml, 15.31 mmol) in THF (20 ml). The reaction was left to warm to room temperature and stirred overnight. The white precipitate was filtered off and the solution was concentrated in vacuo. The crude oil was used for the next step without further purification. .sup.31P NMR (CDCl.sub.3, 202 MHz): 0.69. .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.31 (2H, m, ArH), 7.18 (3H, m, ArH), 4.24 (2H, m, OCH.sub.2), 3.13 (2H, t, SCH.sub.2), 1.17 (9H, s, CCH.sub.3).
C. S-(2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)oxy)ethyl) 2,2-dimethylpropanethioate (SATE Prodrug)
[0256] ##STR00048##
[0257] tBuMgCl (1.45 ml, 1.454 mmol) was added dropwise to a solution of fingolimod HCl (500 mg, 1.454 mmol) in anhydrous THF (20 ml) under anhydrous conditions. The mixture was stirred at room temperature for one hour. After one hour 2,2-Dimethyl-thiopropionic acid S-[chloro-phenoxy-phosphoryloxy)-ethyl ester (489 mg, 1.454 mmol) in anhydrous THF (5 ml) was added dropwise to the stirring reaction mixture. The reaction was left to stir for 24 hours. After 24 hours the solvent was removed in vacuo and the desired product was isolated using flash chromatography (CH.sub.3OH/CH.sub.2Cl.sub.2 0:100 v/v increasing to 10:90 v/v) giving the desired compound as a mixture of four different diastereoisomers in 7% yield (0.076 g). C.sub.32H.sub.50N.sub.2O.sub.6PS; M.W: 607.31; .sup.1H NMR (CDCl.sub.3, 500MHz): 7.17 (4H, m, ArH), 7.02 (4H, s, ArH), 6.98 (1H, m, ArH), 6.25 (2H, b, CNH.sub.2) 3.95 (2H, q, SCH.sub.2CH.sub.2), 3.72 (4H, m, CCH.sub.2O), 3.42 (1H, CH.sub.2OH) 2.95 (2H, t, OCH.sub.2CH.sub.2), 2.55 (4H, m, ArCH.sub.2), 1.89 (2H, m, CCH.sub.2CH.sub.2), 1.59 (2H, quin, CH.sub.2CH.sub.2CH.sub.2), 1.31 (10H, m, CH.sub.2CH.sub.2CH.sub.2), 1.16 (9H, s, CCH.sub.3), 0.91 (3H, m, CH.sub.2CH.sub.3). .sup.31P NMR (CDCl.sub.3, 202 MHz): -5.88. .sup.13C NMR (CDCl.sub.3, 500 MHz): 206.41, 152.38, 140.60, 137.89, 129.40, 128.43, 128.21, 123.52, 120.07, 64.98, 62.77, 60.81, 46.37, 35.60, 33.80, 31.94, 31.63, 29.72, 29.53, 29.48, 29.32, 28.74, 28.67, 28.61, 27.63, 27.31, 22.69, 14.13; MS [ES+] m/z 608.8 [M+H].sup.+
[0258] Other compounds synthesized by methods analogous to those described above include:
[0259] pentyl (25)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate:
##STR00049##
[0260] methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)amino)acetate:
##STR00050##
[0261] Ethyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)amino)acetate:
##STR00051##
[0262] (3S)-methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy)phosphoryl)amino)-3-methylpentanoate:
##STR00052##
[0263] (2S) pentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy) (phenoxy) phosphoryl)amino)-3-phenylpropanoate:
##STR00053##
EXAMPLE 19
Enzymatic Experiments on pentyl (2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy) phosphoryl)amino]propanoate
[0264] ##STR00054##
A. Carboxypeptidase Experiment
[0265] Blank in acetone d6 alone (No trizma): 1-2 mg of pentyl (2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenypethyl]propoxy}(4-methoxyphenoxy) phosphoryl)amino]propanoate is dissolved in 150 L of acetone d6 and the solution transferred into an NMR tube. The .sup.31P spectrum is recorded (16 scans are sufficient).
[0266] The sample in the NMR tube is diluted with 100 L of Trizma buffer (pH=7.6). 0.1 mg of Carboxypeptidase Y (Aldrich) is then dissolved in 150 L of Trizma buffer and the resulting solution is added to the NMR tube. .sup.31P NMR spectra are then recorded (512 scans, 600 sec delay, 20 experiments.)
B. Half-Life Determination
[0267] The half-life of the compound was also determined in the presence of Carboxypeptidase Y (see
C. Human Serum Stability
[0268] Blank in DMSO d6 alone (No trizma).
[0269] 1-2 mg of pentyl (2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylpheny)ethyl] propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate is dissolved in a mixture of 300 L of DMSO d6 and 100 L of Trizma buffer (pH=7.6). The solution is transferred into an NMR tube. The .sup.31P spectrum is recorded (64 scans).
[0270] To the blank sample in the NMR tube 150 L of stock solution of cell lysate are added and then .sup.31P spectra are recorded (512 scans, 600 sec delay). The results of the above experiment are shown in
D. B95a Cell Lysate Experiment
[0271] 4 mg of pentyl (2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylpheny)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate are dissolved in a mixture of 150 L of acetone d6 and 100 L of trizma buffer (pH=7.6). The solution is transferred into an NMR tube. The .sup.31P spectrum is recorded (64 scans).
[0272] To the blank sample in the NMR tube 150 L of B95a cell lysate (6.000.000 cell/m L) are added and then .sup.31P spectra are recorded at 37 Celsius (512 scans, 600 sec delay, 20 experiments).
[0273] The overlaid .sup.31P spectra are shown in
[0274] The results of the above experiment confirm that in B95a cell lysate pentyl (2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate is processed with a half-life of 664 minutes.
EXAMPLE 20
Effects of FINGOLIMOD PRODRUGS on Npc1.SUP./ .fibroblasts (FIG. 6)
Cell Culture
[0275] Glia (mouse astrocytes) were primary cells cultured by Dr E Lloyd-Evans from wild-type (Npc1+.sup.+/+) and NPC1-null (Npc1.sup./) mice. Wild-type (NPC1.sup.+/+) and NPC1-null (NPC1.sup./) human fibroblasts were obtained from the coriell cell bank. All cells were grown as monolayers in a humidified incubator at 37 C. and 5% CO.sub.2 in complete Dulbecco's Modified Eagle's medium (DMEM). Flask's were used for maintenance and chamber slides (ibidi) and 24-well plates (Greiner) were used for cell treatments.
Cholesterol Staining Using Filipin
[0276] Cholesterol was visualized using filipin (filipin complex from Streptomyces filipinenses), a naturally fluorescent antibiotic that specifically binds cholesterol [ref]. PFA-fixed glia were incubated in complete DMEM with 187.5 g/ml filipin at room temperature for 30 minutes, then washed 3 times in PBS (
Lysotracker Staining for Lysosomes
[0277] Human fibroblasts grown in ibidi chamberslides were washed once in complete Hank's Balanced Salt Solution (HBSS+1 mM HEPES pH7.2, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2) prior to incubation with Lysotracker green (Invitrogen, 200 nM in HBSS), which loads specifically into lysosomes, for 10 minutes at room temperature. Cells were then washed twice in complete HBSS and imaged live (