In recent years,S1P₁ agonists have been developed as novel immunosuppressive agents for the treatment of autoimmune diseases. The unique mode of action of this class of drugs has evoked the interests of several researchers ^{[1, 2]}. Compound 1 (FTY720),a synthetic amino diol prodrug,has been clinically approved for the treatment of multiple sclerosis,which is phosphorylated in vivo by sphingosine kinase 2 to generate the corresponding 1-P (S-FTY720-P) that is a potent agonist of S1P_1,3-5 ^[3]. Activation of the S1P₁ receptors leads to modulating lymphocyte trafficking from peripheral blood and tissues to lymphoid organs without affecting the function of the lymphocytes ^[4]. However,clinical trials also showed related cardiovascular adverse effects,including bradycardia,which is attributed to S1P₃ activation ^{[4, 5]}. Compound 2 (KRP203,Fig. 1),with structural similarity to 1,andalsophosphorylated in vivo to 2-P(KRP203-P),is a potent selective agonist of S1P₁. Compound 2 which embraces considerable immunosuppressive effects with lower potential to induce heart rate reduction has entered phase II clinical trials for cutaneous lupus erythematosus ^[6].

Fig. 1

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Fig. 1. Structures of FTY720 (1),S-FTY720-P (1-P),KRP203 (2),KRP203 (2-P).

Recent publications have reported on some convenient and efficient methods of the mono-phosphorylation of amino diol agents to allow in vitro testing for their S1P subtype activity. Shuzo first reported the direct mono-phosphorylation of amino diol agents using tetrabenzyl pyrophosphate (TBPP) as a phosphatic donor,and following hydrogenolysis,give the corresponding phosphorylated products ^[7]. Unfortunately,compounds which have hydrogenolysis sensitive atoms or groups,such as Cl,S,and benzyl,limited its application. Hamada reported another method involving the four steps of oxazoline protection,phosphorylation, oxidation and deprotection under acidic conditions ^[8]. However,it failed when applied to compounds with oxidation sensitive groups. To our best knowledge,there are no publications on the synthesis of 2-P (KRP203-P),since it has both hydrogenolysis and oxidation sensitive groups.

To develop a general method for the mono-phosphorylation of amino diol compounds,it is crucial to choose an appropriate reagent to cleave protective groups without affecting sensitive groups or atoms. Applying this theory,dibenzylphosphoryl chloride was selected as a phosphatic donor,and TMSI was used to remove the benzyl group in the sensitive deprotective step to yield the corresponding mono-phosphorylation products,as represented in 2-P and its analogs.

All the solvents and chemicals were obtained from commercial sources and used without further purification. NMR spectra were recorded on a Mercury-400 and Mercury-300 spectrometer; HRESI- MS data were measured on Micromas AutoSpec Ultima-TOF spectrometer. Flash column chromatography was performed on Biotage Isolera one. Melting points were determined on Yanaco MP-J3 microscope melting point apparatus.

Typical procedure for the preparation of 3: Compound 2 (128 mg, 0.266 mmol) was added to a 50-mL round-bottomed flask equipped with magnetic stirrer. EtOAc (5 mL) and saturated aqueous NaHCO₃ solution (5 mL) were added. The mixture was stirred for 20 min at room temperature. Benzyl chloroformate (114 μL,0.798 mmol) was then added,and stirred for additional 30 min at room temperature (Scheme 1). The organic layer was separated and the aqueous layer was washed with ethyl acetate. The organic layers were combined,dried and concentrated under vacuum. The colorless oil (145 mg,yield: 94.4%) was purified by flash column chromatography (PE:EA,4:1 v/v).

Scheme 1

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Scheme 1. Reagents and conditions: (a) Cbz-Cl,10% NaHCO3,ethyl acetate,r.t.,50 min,94.4%,(b) dibenzylphosphoryl chloride,DMAP,dichloromethane,r.t.,6 h,67%,(c) TMSI,dichloromethane,10 ℃,15 min,90%.

Typical procedure for the preparation of 4: Compound 3 (145 mg, 0.251 mmol),CH₂Cl₂ (5 mL) and DMAP (46 mg,0.38 mmol) were added to a 20-mL round-bottomed flask equipped with magnetic stirrer. Dibenzylphosphoryl chloride (100 mg,0.337 mmol) was then added dropwise with stirring. The mixture was stirred for 6 h at room temperature (Scheme 1). The organic layer was washed with water,dried and concentrated under vacuum. The colorless oil (110 mg,yield: 67%) was purified by flash column chromatography (CH₂Cl₂:CH₃OH,100:1,v/v).

Typical procedure for the preparation of 2-P: The reaction is carried out in a 50-mL,three-necked,round-bottomed flask equipped with a magnetic stirrer,a thermometer,and a 100-μL microsyringe under an atmosphere of argon. The flask was charged with dry CH₂Cl₂ (10 mL) and intermediate 4 (92 mg,0.110 mmol). TMSI (75μL,0.528 mmol) was added dropwise over 3 h. The temperature of the reaction was maintained at 10-12 ℃ for 15 min (Scheme 1). The solvent was removed under vacuum. MeOH (1 mL) was added to the residue,followed by addition of NaS₂O₃ aqueous solution (6 mL). The mixture was stirred for 20 min,and then concentrated. MeOH (4 mL) was added to the residue. The light yellow solid (66 mg,yield: 90%) was collected by filtration.

Compound 2-P: ¹H NMR (400 MHz,DMSO-d6): δ 7.41 (m,8H), 7.21 (d,1H,J = 8 Hz),6.99 (m,2H),6.90 (d,1H,J = 7.6 Hz),5.09 (s, 2H),3.86 (s,2H),3.62 (m,2H),2.73 (m,2H),1.79 (s,2H). 13C NMR (100 MHz,DMSO-d6): δ159.32,138.61,137.08,135.67,134.78, 134.05,131.96,131.21,131.07,130.21,128.92,128.45,128.13, 123.76,117.51,114.93,69.79,64.26,61.04,59.77,31.61,26.39. HRMS (m/z): calcd. for C24H2℃lNO6PS: 524.1058 [M+H]⁺; found: 524.1062. Mp: 191-193 ℃.

Compound 6-P: ¹H NMR (300 MHz,CD₃OD): δ 7.51 (d,2H, J = 8.4 Hz),7.44(d,2H,J = 8.1 Hz),7.19(m,4H),6.92(m,4H),3.94(m, 2H),3.66 (m,2H),2.61 (m,4H),1.95 (m,2H),1.20 (t,3H,J = 7.6 Hz). 13C NMR (125MHz,CD₃OD): δ 158.63,157.45,144.76,139.52, 137.83,131.15,129.62,129.43,128.05,120.53,120.14,62.82,61.65, 58.63,35.21,29.82,18.74,16.57. HRMS (m/z): calcd. for C₂₅H₃₁NO₆P:472.1884 [M+H]⁺; found: 472.1872. Mp: 190-192 ℃.

Compound 7-P: ¹H NMR (300 MHz,CD₃OD): δ 7.70 (m,4H),7.62 (d,2H,J = 8.7 Hz),7.24 (d,2H, J = 8.7 Hz),7.01 (d,2H,J = 8.4Hz),6.94 (d,2H,J = 8.7 Hz),3.96 (m,2H),3.67 (m,2H),2.66 (m,2H),1.95 (m, 2H). 13C NMR (125MHz,CD3OD): δ 159.91,156.83,145.95,138.31, 135.95,131.23,130.01,128.58,127.04,120.92,120.81,120.08, 119.83,66.15,62.84,61.73,35.22,29.63. HRMS (m/z): calcd. for C₂₄H₂₆F₃NO₆P: 512.1444[M+H]⁺; found: 512.1430.Mp:236-238 ℃.

Compound 8-P: ¹H NMR (400 MHz,CD₃OD): δ 7.17 (m,4H), 7.09 (m,4H),6.80 (m,4H),3.92 (m,2H),3.85 (m,2H),3.64 (m,2H), 2.60 (m,2H),1.90 (m,2H). 13C NMR (125 MHz,CD3OD): δ 157.43, 157.15,141.73,137.72,137.11,132.95,131.43,131.25,130.72, 129.50,119.92,119.71,66.11,63.11,60.75,41.24,35.43,29.42. HRMS (m/z): calcd. for C₂₄H₂₈ClNO₆P: 492.1337 [M+H]⁺; found: 492.1324. Mp: 215-217 ℃.

Compound 9-P: ¹H NMR (300 MHz,CD₃OD): δ 8.01 (s,1H) 7.61 (d,2H,J = 8.4 Hz) 7.21 (d,2H,J = 8.4 Hz) 6.91 (t,4H,J = 6.9 Hz) 3.95 (brs,2H) 3.66 (s,2H) 2.64 (q,2H,J = 7.8 Hz) 2.43 (s,3H) 1.94 (q,2H,J = 5.7 Hz); ¹³C NMR (100 MHz,CD₃OD): δ 163.94,159.00,156.65, 141.23,137.84,134.94,130.86,127.99,127.17,120.39,119.58, 65.79,65.74,62.48,61.39,61.32,34.91,29.33,13.54; HRMS (m/z): calcd. for C₂₁H₂₆N₂O₇P: 449.1472 [M+H]⁺,found: 449.1465. Mp: 218-220 ℃

Compound 10-P: ¹H NMR(300 MHz,CD₃OD): δ 8.12 (s,1H) 7.74 (d,2H,J = 7.5 Hz) 7.60 (d,2H,J = 7.5 Hz) 7.54 (d,2H,J = 8.4 Hz) 7.30 (d,2H,J = 7.8 Hz) 3.98-3.68 (m,4H) 2.82 (q,2H,J = 7.5 Hz) 2.71- 2.58 (m,2H) 1.99-1.97 (m,2H) 1.33 (t,3H,J = 7.5 Hz); ¹³C NMR (100 MHz,CD₃OD): δ 168.20,141.75,135.38,130.08,129.96, 129.45,128.90,128.11,127.96,127.66,126.93,65.81,63.45,62.50, 34.76,29.61,22.45,11.60; HRMS (m/z): calcd. for C₂₂H₂₈N₂O₆P: 447.1679 [M+H]⁺,found: 447.1676. Mp: 265-270 ℃.

In our initial investigation,benzyl chloroformate was used to give the intermediate 3. The phosphorylation method was evaluated,modified and led to the current procedure as follows: dibenzylphosphoryl chloride (1.3 eq) was added to a solution of 3 and DMAP (1.5 eq). The mixture was stirred at room temperature for 6 h. The corresponding mono-phosphate was obtained after purification. Dibenzylphosphoryl chloride was considered as a better phosphatic donor,and is more reactive than TBPP and achieved a higher yield with the same selectivity compared to the traditional procedure. The results are summarized in Table 1.

Table 1

Table 1 Optimization of phosphorylation conditions.

Table 1 Optimization of phosphorylation conditions.

After the optimized reaction conditions were established,we investigated the methods to deprotect the -PO(OBn)₂ and -N-Cbz in the presence of Bn groups and S atom. To our delight,compound 4 was successfully converted to the corresponding monophosphate 2-P in 90% yield after adding TMSI (3.8 eq) for 15 min at 10-12 ℃. Therefore,TMSI was found to be an efficient reagent to remove benzyl esters without affecting sensitive groups.

With optimized conditions in hand,this reaction was applied to the formation of a variety of amino diol derivatives (analogs). Five examples of mono-phosphorylation products are summarized in Table 2. We were pleased to find these substrates were generally effective and halogen substituted compounds were well tolerated.

Table 2

Table 2 Mono-phosphorylation of KRP203 derivatives.

Table 2 Mono-phosphorylation of KRP203 derivatives.

An efficient procedure for the synthesis of 2-P was developed. The results indicate that dibenzylphosphoryl chloride is an efficient phosphatic donor,and TMSI is an ideal deprotective reagent for the selective removal of benzyl esters to give 2-P and its derivatives in moderate yields. Furthermore,this procedure is applicable to the mono-phosphorylation of diols with diverse structures.

This work was financially supported by National Natural Science Foundation of China (No. 81102322).