Patent Application
20080027062
2,4-diamino-8-methyl-6-phenyl-7,8-dihydropteridine as an example for an pteridine compound of the invention carrying an alkyl substitution at the N8 atom can be synthesized in accordance with the following protocol.
Suitable amounts of 2,4-diamino-6-chloro-5-nitro-pyrimidine, for example 6 g and ω-methylamino-acetophenone-hydrochloride, for example 11.8 g can be dissolved in an appropriate solvent, for example 200 ml boiling ethanol. Then an appropriate amount of triethylamine, for example 16 ml, is added and the solution is refluxed for an suitable time, leading to the precipitation of 2,6-diamino-4-[methyl-phenacyl-amino]-5-nitro-pyrimidine. The precipitate is filtered from the solution, washed with a solvent such as ethanol and ether, dried and purified. The purification may be carried out by dissolving the crude product in boiling dimethylformamide, filtering the solution and mixing with a precipitating solvent such as ethanol.
Then 1 g of the purified 2,6-diamino-4-[methyl-phenacyl-amino]-5-nitro-pyrimidine is dissolved in an suitable solvent such as ethanol which has been alkalised by addition of NaOH. 2,6-diamino-4-[methyl-phenacyl-amino]-5-nitro-pyrimidine can then be reduced with Raney-nickel and hydrogen gas until three equivalents of hydrogen are consumed. The catalyst is filtered off and the solution neutralized, for example, with acetic anhydride and cooled overnight at −20° C. The precipitated 4-amino-8-methyl-6-phenyl-7,8-dihydropteridin is filtered, dried and can be purified by re-crystallization, for example.
In a first step, 2-Amino-4-chloro-6-N-cyclohexylmethylamino-5-nitro-pyrimidine can be prepared starting from 2-amino-4-chloro-6-hydroxy-5-nitro-pyrimidine using the Mitsunobu reaction as described in Hanaya T et al, Pteridines (1995) Vol. 6 pp. 1-7 (see also Example 2.2b)
A suitable amount of 2-amino-4-chloro-6-hydroxy-5-nitro-pyrimidine, for example 5 mmol, is mixed with acetic anhydride (20 ml) and a solvent such as pyridine (40 ml) and this mixture is then heated to an appropriate temperature such as 100° C. till completion of the reaction. After the starting material has disappeared as judged by TLC, the mixture is evaporated to dryness and the residue chromatographed on silica gel using a suitable eluent such as dichloromethane (DCM) to DCM:MeOH (95:5) in order to obtain the product 2-N2-acetylamino-4-chloro-6-hydroxy-5-nitro-pyrimidine.
Suitable molar amounts of triphenylphosphine and 2-phenylethanol are then sequentially added to a solution of 2-N2-acetylamino-4-chloro-6-hydroxy-5-nitro-pyrimidine in a solvent such as 1,4-dioxane (cf. also Example 2.2b). Diisopropylazodicarboxylate is added dropwise to this mixture and the reaction mixture then stirred, usually at room temperature for a suitable period of time. The resulting intermediate product carrying the O6-2-phenylethyl group at the 6-position can be purified by column chromatography on silica gel, using an appropriate eluent such as dichloromethane (DCM): AcOEt (1:2) followed by AcOEL
The reaction mixture so obtained can be reacted with cyclohexylmethylamine in 1,4-dioxane (20 ml) under reflux for 2 h. This volume of the reaction is then reduced, and concentrated ammonia is added and the mixture stirred for another suitable time period such as 18 h. The so obtained 2-amino-4-chloro-6-N-cyclohexylmethylamino-5-nitro-pyrimidine can be isolated from the reaction mixture by evaporation of the solvent and chromatography of the residue on silica gel.
2-Amino-4-chloro-6-N-cyclohexylmethylamino-5-nitro-pyrimidine and 1-amino-1,5-dideoxy-L-erythro-pentulose are reacted in aqueous alcohol in the presence of sodium hydrogen carbonate as described in Andrews et al., Chemical Communications, pages 120-121 (1968) to yield the corresponding nitropyrimidinyl-aminoketose. This aminoketose is then hydrogenated in water or an ethanol/aqueous NaOH mixture, using Raney nickel as catalyst to produce 2-amino-4-N-cyclohexylmethylamino-6-(L-erythro-1,2-dihydroxypropyl)-7,8-dihydro-biopteridin. The crude product can then be precipitated, for example by addition of glacial acetic acid or any other suitable precipitating agent and further purified by chromatography.
The use of 1-N-alkylamino-1,5-dideoxy-L-erythro-pentulose or 1-N-arylamino-1,5-dideoxy-L-erythro-pentulose instead of 1-amino-1,5-dideoxy-L-erythro-pentulose leads to compounds with an alkyl or aryl substituted N8-nitrogen atom.
The compounds 4-N-cyclohexylmethylamino-5,6,7,8-tetrahydrobiopterin (compound A), 2-amino-4-piperidino-6-phenyl-(R,S)-5,6,7,8-tetrahydropteridin (compound B) and 2-amino-4-di-n-propylamino-6-(4-methoxyphenyl)-(R,S)-5,6,7,8-tetrahydropteridin (compound C) were intravenously injected into male Sprague-Dawley rats (1-10 mg/kg). Venous blood samples were taken up to eight hours after the injections and analyzed for the injected tetrahydro-compounds and for their corresponding dihydro-derivatives, which are spontaneously formed in vivo, by LC-MS/MS. While the tetrahydro-compounds were oxidized with a half-life time of less than 5 minutes, the corresponding dihydro-compounds were cleared from the bloodstream at significantly slower rates (see table 1):
a) Synthesis of 2-amino-4-piperidino-6-phenyl-(R,S)-5,6,7,8-tetrahydropteridin (Compound B) and 2-anino-4-di-n-propylamino-6-(4-methoxyphenyl)-(R,S)-5,6,7,8-tetrahydropteridin (Compound C)
2-amino-4-piperidino-6-phenyl-(R,S)-5,6,7,8-tetrahydropteridin and 2-amino-4-di-n-propylamino-6-(4-methoxyphenyl)-(R,S)-5,6,7,8-tetrahydropteridin were prepared as described in Matter et al., Journal of Medical Chemistry, 2002, 45, 14, pages 2923-2941 or WO 01/21619.
b) Synthesis of 4-N-cyclohexylmethylamino-5,6,7,8-tetrahydrobiopterin (Compound A):
1. Synthesis of tri-N2,1′,2′—O-acetyl-L-biopterin
Biopterin (1 g, 4.21 mmol) dissolved in pyridine (40 ml) and acetic anhydride (20 ml) was heated to 100° C. After 3 h, the starting material disappeared as judged by TLC, the mixture was evaporated to dryness and the residue was chromatographed on silica gel eluting with dichloromethane (DCM) to DCM:MeOH (95:5). The product tri-N2, 1, 2′—O-acetyl-L-biopterin was obtained as brown foam in quantitative yield (1.5 g). The product was characterized by NMR and mass spectrometry:
NMR (DMSO-D6): 12.32 (1H, s, NH), 12.01 (1H, s, NH), 8.95 (1H. s, H-7). 5.93 (1H, d, J=4.2 Hz, H-1′), 5.34 (1H, dq, J=6 and 4 Hz, H-2′), 2.20 (3H, s, Ac); 2.16 (3H, s, Ac), 1.96 (3H, s, Ac), 1.19 (3H, d, J=6.9Hz, CH3,-3′). MS (APCI): 364 [M+H]+
2. Synthesis of tri-N2,1,2′—O-acetyl-O4-2-phenylethyl-L-biopterin
Triphenylphosphine (1.2 g, 5.4 mmol) and 2-phenylethanol (0.65 g, 5.4 mmol) were sequentially added to a solution of tri-N2,1,2′-O-acetyl-L-biopterin (1.5 g, 4.21 mmol) in 1,4-dioxane (7 ml). To this mixture diisopropylazodicarboxylate (1.05 ml, 5.4 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 18 h. Evaporation and column chromatography on silica gel eluting with AcOEt (1:2) followed by AcOEt gave the expected product together with triphenylphosphine oxide.
3. Synthesis of 4-N-cyclohexylmethylamino-4-desoyx-L-biopterin
The reaction mixture obtained in step 2 was then heated with cyclohexylmethylamine (2.2 ml, 16.8 mmol) in 1,4-dioxane (20 ml) under reflux for 2 h. This volume of the reaction was reduced by 50% by evaporation and concentrated ammonia (32%) (30 ml) was then added. Thereafter, the mixture was stirred for 18 h. The reaction mixture was evaporated and chromatographed on silica gel eluting with dichloromethane DCM:MeOH (9:1) to DCM:MeOH:NH4OH (90:10:1) to get the expected product as a yellow solid (0.9 g, 64% yield). The product was characterized by NMR and mass spectrometry.
NMR (DMSO-D6): 8.70 (1H, s, H-7), 8.10 (1H, t, J=6.3 Hz, NH), 6.60 (2H, s, NH2), 5.42 (1H, br s, OH), 4.65 (1H, br s, OH), 4.40 (1H, d, J=3.9 Hz, H-1′), 3.81 (H, m, H-2′), 3.34 (2H, m, CH2). 1.71 (5H, m, cyclohexyl), 0.95-1.27 (6H, m, cyclohexyl), 1.13 (3H, d, J=6.3 Hz, CH3-3). MS (APC1: 333 [M+H)+
4. Synthesis of 4-N-cyclohexylmethylamino-5,6,7,8-tetrahydrobiopterin
4-N-Cyclohexylmethylamino-4-deoxy-L-biopterin (1.1 g, 3.3 mmol) dissolved in trifluoracetic acid (15 ml, TFA) was added to a suspension of PtO2 (0.18 g) in TFA (10 ml) previously hydrogenated to metallic platinum. The reaction mixture was hydrogenated for 3 h, filtered through celite and evaporated. The residue was dissolved in HCl (1.25 M in methanol) (20 ml) and stirred overnight. Evaporation to dryness and trituration with AcOEt gives the expected product by filtration as a green powder (1.3 g, 97% yield). The product was characterized by NMR, mass spectrometry and elemental analysis.
NMR (CDC13-CD30D): 3.80-3.95 (SH, m), 2.78 (1H, d, J=6.9 Hz), 0,9-1.8 (1611, m). MS (APC1): 337 [M+]+. Anal. (l16H28N6O2×2 HCl after drying); calculated: 46.95% C, 7.39% H, 20.53% N, 17.32% Cl. Found: 47.1 1% C, 7.36% H, 20.51% N, 17.08% Cl. The water content was calculated to be 8.15% before drying. The water content increased after five successive measurements as follows: 8.15, 8.70, 10.71, 11.28, and 11.53% (showing the hygroscopicity of the compound).
The inhibition of NO release by the compounds of the general formula (I) can be determined by an activity assay based on the studies of Knipp und Vasak (Analytical Biochemistry 286, 257-264 (2000)). In this assay for purified NO synthase (NOS) the coproduct L-citrulline obtained during NO formation is determined quantitatively. This is carried out by the use of the color developing reaction between the carbamide group of citrulline with the reagents diacetyl-monoxime. and thiosemicarbazide. After this reaction, the colored product can be quantified directly by measuring the absorbance at 540 nm.
In this assay, 60 μl of substrate-cofactor-mix (1 mM CaCl2, 1 mM MgCl2, 1 mM arginine, 1 mM β-nicotinamide adenine dinucleotide phosphate, 5 μM flavin adenine dinucleotide, 5 μM riboflavin 5′-monophosphate, 2 μM tetrahydrobiopterin in HEPES, 100 mM, pH 7.4), 2 μl of a compound of formula (I) in dimethylsulfoxide and 0.5 or 1 μl of the purified enzyme NOS are incubated for two hours at 37° C. Then, 100 μl of the colour developing reagent mix (20 mM diacetyl-monoxime, 0,5 mM thiosemicarbazide, 4,5 M H2SO4, 2,25 M H3PO4, 1,5 mM NH4Fe(SO4)2) are added, the samples are incubated for 15 minutes at 95° C. and subsequently centrifuged for 10 minutes at >1000 g. During the centrifugation, the samples cool down to room temperature. 130 μl of each supernatant are transferred into low-volume 96-well-plates and the absorbance at 540 nm is measured. Comparison with values from uninhibited NOS (100%-values) and samples containing no enzyme (0-values) yields the inhibitor-effect of each tested compound.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/14970 | 12/30/2003 | WO | 00 | 6/11/2007 |
