抗病毒药物替诺福韦（Tenofovir）的最新合成工艺解析
本文主要介绍氧代甲基膦酸二叔丁酯用于活性药物成分替诺福韦（Tenofovir，PMPA）合成的相关研究。首先，开发了一种简单且有效的合成二叔丁基（羟甲基）膦酸酯的方法。同时，通过O-甲磺酰化以高收率生成氧代甲基膦酸二叔丁酯。随后，使用Mg(OtBu)2作为碱，通过两步法实现(R)-9-(2-羟丙基)腺嘌呤（HPA，4）和13的烷基化以及脱保护反应，从而合成PMPA，收率为68%。若进行5g实验，可将收率提高至72%。此外，该策略也可用于乙型肝炎药物阿德福韦（Adefovir）的合成，收率为64％。
正文：
1993年，Balzarini报道了核苷酸逆转录酶抑制剂替诺福韦（1，PMPA），用于人类免疫缺陷病毒（HIV）药物。为实现口服应用，并增加生物利用度，从而开发出TDF（2），于2001年被FDA批准用于HIV治疗，并于2008年用于乙肝病毒的治疗。2010年，又开发一种TAF（3），与TDF相比，具有更少的副作用和更好的耐受性（Figure 1）。此外，在2020年COVID-19大流行期间，替诺福韦作为治疗SARS-CoV2的潜在药物，并用于各项研究。
2010年，Ripin等报道了一种(R)-9-(2-羟丙基)腺嘌呤（HPA，4）和DESMP（5）的烷基化以及脱保护反应，其中使用Mg(OtBu)2作为碱时，转化率> 90％。虽然该反应使用TMCSI/NaBr代替昂贵的TMSBr（降低脱保护的成本），但后处理操作复杂，需多次过滤和萃取，且滤饼中损失高达15％等。最终，以两步59％的总收率获得PMPA·H2O。2016年，Riley等报道了通过MeMgCl和tBuOH原位生成Mg(OtBu)2，并对脱保护（HBr/乙酸）工艺进行改进，最终以两步57%收率获得PMPA·H2O。2020年，Derstine等报道了一种以NaOtBu作为碱，于DMF溶剂中实现HPA与7的烷基化反应，从而获得70％收率的PMPA。
对于PMPA的合成，主要集中于DESMP（5）或衍生的游离酸7作为HPA的烷基化试剂。同时，DESMP可由廉价的化学品合成，且具备成熟的合成工艺，但从亚磷酸二乙酯（8）开始，总收率仅为60-70％（Scheme 2）。此外，使用DESMP的最大缺点为二乙酯的脱保护。
在此，本文主要研究了氧代甲基膦酸二叔丁酯用于PMPA的合成研究（Scheme 3）。
一、亚磷酸二叔丁酯（9）的合成
亚磷酸二叔丁酯（9）虽然可商购，但价格昂贵。据文献报道，可在非极性溶剂（石蜡、石油醚和乙醚）的碱性（三乙胺、吡啶或二甲基苯胺）条件下，通过将PCl3加入到冷却的叔丁醇溶液中来合成，收率为40-77％，但也存在化学选择性的问题（如三酸酯10和11，Figure 2）。
为了避免上述副反应的发生，作者以PCl3为底物，KOtBu作为碱，于THF溶剂中进行反应条件的优化（Table 1）。反应结果表明，当KOtBu的摩尔量降低时，会导致粗收率降低，但纯度会增加。
然而，上述使用的THF，需大量的硫酸钠干燥。因此，作者研究了极性较小溶剂（CPME、2-Me-THF和MTBE溶剂），反应均取得相似的结果（Table 2）。
随后，使用MTBE和2-Me-THF作为溶剂，廉价的NaOtBu为碱，进行了放大实验（Table 3）。当在MTBE中放大时，副产物增加，而在Me-THF中，纯度保持恒定。
二、亚磷酸二叔丁酯的羟甲基化反应（12的合成）
据文献报道，在甲醛溶液、三乙胺和水存在下，9可在室温下反应获得99％收率的粗品12，但31P NMR检测存在10％的杂质（Scheme 4）。
为了寻找一种更为便捷的方法，也可通过在多聚甲醛与K2CO3的乙腈溶剂中反应，可获99％收率的12，仅涉及一次过滤和脱溶剂（Scheme 5）。
受此启发，作者设想，以PCl3为底物，直接将上述两步法相结合，从而实现一锅法反应，以合成12。因此，作者对反应条件进行了再次优化（Table 4）。反应结果表明，多聚甲醛的量对于反应至关重要，稍微过量的低聚甲醛有利于反应的进行。将反应放大至11g，可获得71%收率的12。
化合物12的具体合成路线如下：
According to a method reported by Grimmond et al., a round-bottomed flask was charged with di-tert-butyl phosphite (commercially available, 96%, 3.24 g, 16.0 mmol, 1.0 equiv), NEt3 (2.6 mL, 19 mmol, 1.2 equiv), and H2O (1 mL). Aqueous formaldehyde solution (37%, 1.20 mL, 16.0 mmol 1.0 equiv) was added afterward, and the solution was stirred for 24 h at r.t. (reaction control by GC or 31P NMR spectroscopy). MeOH (5 mL) was added, and all volatiles were removed in vacuo at 40 °C. This procedure was repeated twice and then performed again with DCM (3 × 5 mL), furnishing the crude product as a slight yellowish waxy solid (3.51 g, 14.5 mmol, 91%, yield corrected based on 31P NMR). Variant 2: A roundbottomed flask was charged with crude di-tert-butyl phosphite (95%, purity estimated by 31P NMR, 11.25 g, 55.0, mmol, 1.0 equiv), paraformaldehyde (97%, 1.70 g, 1.0 equiv), K2CO3 (anhydrous, ground, stored in a desiccator, 1.60 g, 11.59 mmol, 0.2 equiv), and MeCN (HPLC grade, 170 mL). The gas-filled compartment of the flask was purged with argon for 15 s and immediately closed with a septum equipped with an argonfilled balloon. The colorless suspension was heated at 70 °C for 20 h (reaction control by GC or 31P NMR spectroscopy). The reaction mixture was cooled to r.t., filtered, and concentrated in vacuo at 40 °C to half of the original volume. The flask was
stored in a freezer overnight at −24 °C when 12 crystallized out (if no crystallization took place, slight shaking or inoculating helped). The supernatant mother liquor was decanted, and the solid was washed twice with cold MeCN (−24 °C, 2 × 5 mL). The solid was dried in vacuo at 30 °C to afford 12 (8.90 g). Concentrating the mother liquor and storing in a freezer overnight yielded a second pure crystallisate (1.97 g). In total, 10.87 g (48.47 mmol, 71% related to PCl3) of 12 was obtained.
三、化合物12的甲苯磺酸化和甲磺酰化反应（合成13和14）
据文献报道，12中的羟基磺酰化反应，使用昂贵的三氟甲磺酸酯。因此，作者开发一种可替代且更经济的方法。在对反应条件优化之后，可从EtOH中重结晶，获得87％收率的13（Scheme 6）。同时，在酸条件下，易于快速脱烷基化。若无DMAP时，粗产物中存在的甲苯磺酰氯足以在EtOH中重结晶期间缓慢释放HCl，从而导致脱烷基化。
在相似条件下，12的甲磺酰化比甲苯磺酸化更有效。此外，不需要重结晶，因为过量的甲磺酰氯在含水后处理过程中完全水解并且没有副产物形成。最终，获得97％收率的14（Scheme 7）。
化合物13的具体合成路线如下：
In an oven-dried Schlenk flask, di-tertbutyl(hydroxymethyl)phosphonate (3.00 g, 13.4 mmol, 1.0 equiv), NEt3 (2.1 mL, 14.7 mmol, 1.1 equiv), and DMAP(0.16 g, 1.34 mmol, 0.1 equiv) were dissolved in dry DCM (100 mL) under a nitrogen atmosphere. Tosyl chloride (2.81 g, 14.7 mmol, 1.1 equiv) was added, and the solution was stirred under a nitrogen atmosphere at r.t. for 24 h (99% conversion by 31P NMR spectroscopy). The reaction mixture was washed with saturated NaHCO3 solution (70 mL), and the organic phase was separated. After drying over NaSO4, all volatiles were removed in vacuo at 30 °C [CAUTION! High temperatures can lead to decomposition (dealkylation)]. The crude product (4.93 g) was dissolved in warm EtOH (17 mL, max 50 °C), cooled to r.t., and stored in a freezer (−24 °C) overnight. The supernatant mother liquor was decanted, and the crystallized solid was washed with two portions of cold (−24 °C) EtOH (2 × 3 mL). After drying in vacuo at 30 °C,13 was obtained as a colorless solid in 87% yield (4.41 g, 11.7 mmol, 87%). For longer storage, it is recommended to store the compound in a refrigerator or freezer.
化合物14的具体合成路线如下：
In an oven-dried Schlenk flask, di-tert-butyl-(hydroxymethyl)phosphonate (12.94 g, 57.71 mmol, 1.0 equiv) was dissolved in dry DCM (200 mL) under a nitrogen atmosphere. NEt3 (9.70 mL, 69.2 mmol, 1.2 equiv) was added, and the solution was cooled in an ice bath. Mesyl chloride (4.90 mL, 63.5 mmol, 1.1 equiv) was added drop-wise to the solution within 4 min, the ice bath was removed, and the solution was stirred at r.t. for 19 h (reaction control by thinlayer chromatography). The reaction mixture was washed twice with sat. NaHCO3 solution (2 × 150 mL), and the organic phase was separated. After drying over Na2SO4, all volatiles were removed in vacuo at 30 °C [CAUTION! High temperatures can lead to decomposition (dealkylation)]. The title compound 14 was obtained as an orange-brown oil (16.92 g, 55.97 mmol, 97%), which solidified after a while. For longer storage, it is recommended to store the compound in a refrigerator or freezer.
四、替诺福韦（PMPA）的合成
作者以4和14为底物，对反应条件进行了再次优化后，进行了相关的放大实验（Table 5）。当使用5 g底物4时，可获得72%收率的PMPA。
此外，使用甲苯磺酸酯13与HPA进行烷基化反应时，可在DMA、DMSO和DMF中观察到相似的结果。当在DMF中进行1 g规模实验时，可获得68％收率的PMPA（Scheme 8）。总而言之，当使用甲磺酸酯14或甲苯磺酸酯13合成PMPA时，收率没有明显的差异。
替诺福韦的具体合成路线如下：
An oven-dried Schlenk flask was charged with HPA (4, >98%, 1.00 g, 5.18 mmol, 1.0 equiv) and magnesium tert-butoxide (93%, 2.85 g, 15.5 mmol, 3.0 equiv) under a nitrogen atmosphere. Dry DMF (12 mL) was added, and the suspension was stirred at 80 °C for 25−30 min. While purging with nitrogen, (di-tert-butoxyphosphoryl)-methyl 4-methylbenzenesulfonate (13, 2.94 g, 7.76 mmol, 1.5 equiv) was added portion-wise within 1 min. The reaction mixture was stirred at 80 °C for 23 h (95% conversion of HPA detected by HPLC at λ = 254 nm). All volatiles were removed in vacuo, and 1.5 M H2SO4 (10 mL) was added to the orangebrownish residue. During heating to 60 °C, a yellowish solution formed. After 4 h (complete dealkylation to PMPA detected by HPLC at λ = 254 nm), the solution was cooled in an ice bath and the pH was adjusted to pH = 2.8−3 using conc. NH3 solution (25%, 0.6 mL, additional water (2−3 mL) was added for better stirring). A colorless to slight yellowish suspension formed, which was stirred for one further hour while cooling and then stored in a refrigerator overnight. The suspension was vacuum-filtered, and the filter cake was washed with ice-cold water (3 × 2 mL) and ice-cold acetone (3 × 2 mL). The filtered solid was first dried on the air and then at 80°C under high vacuum for 4 h. PMPA was obtained as a colorless powder (1.01 g, 3.52 mmol, 68%).
五、阿德福韦（Adefovir）的合成
在无溶剂条件下加热几分钟，也可很容易地将二叔丁基膦酸酯13和14定量地转化为相应的游离膦酸（Scheme 9）。
此外，该策略也成功用于乙型肝炎抑制剂阿德福韦（22，Adefovir）的合成，并与替诺福韦仅有一个甲基的差异。通过9-(2-羟乙基)腺嘌呤（HEA，23）与14在90 ℃下反应22 h后，获得87％的转化率。随后，经脱保护可获得64％收率的阿德福韦（Scheme 10）。
阿德福韦的具体合成路线如下：
An oven-dried Schlenk flask was charged with HEA (23, >98%, 1.00 g, 5.58 mmol, 1.0 equiv) and magnesium tert-butoxide (93%, 3.07 g, 16.7 mmol, 3.0 equiv) under a nitrogen atmosphere. Dry DMA (12 mL) was added, and the suspension was stirred at 90 °C for 30 min.While purging with nitrogen, (di-tert-butoxyphosphoryl)-methyl methanesulfonate (14, 2.53 g, 8.37 mmol, 1.5 equiv) was added portion-wise within 1 min. The reaction mixture was stirred at 90 °C for 22 h (conversion of HEA 87% detected by HPLC at λ = 254 nm). All volatiles were removed in vacuo at 50−60 °C, and 3 N HCl (10 mL) was added to the orangebrownish residue. During heating to 60 °C, an orange solution formed. After 4 h (complete dealkylation to PMEA detected by HPLC at λ = 254 nm), the solution was cooled in an ice bath and the pH was adjusted to pH = 2.8−3.0 using NaOH solution (40 wt %, 4−5 drops). A colorless to slight yellowish thick suspension formed [additional water (3 mL) was added for better stirring], which was stirred while cooling for 1 h and then stored in a refrigerator overnight. The suspension was vacuum-filtered and washed with ice-cold water (4 × 2 mL) and ice-cold acetone (3 × 2 mL). The filtered solid was first dried on the air and then at 80 °C under high vacuum for 3 h to obtain PMEA [0.98 g, 3.59 mmol, 64%, HPLC-purity (254 nm): ≥99%].
总结：本文首先介绍烷基化试剂13和14的合成。随后，通过两步法实现(R)-9-(2-羟丙基)腺嘌呤（HPA，4）和13的烷基化以及脱保护反应，从而合成替诺福韦（Tenofovir），收率为68%。若进行5g实验，可将收率提高至72%。此外，通过9-(2-羟乙基)腺嘌呤（HEA，23）与14的烷基化以及脱保护反应，可合成乙型肝炎药物阿德福韦（Adefovir），收率为64%。
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