JAK激酶抑制剂是现今药物化学研究中的重点之一，在治疗炎症和免疫系统疾病如银屑病、肠炎、类风湿性关节炎、器官移植排斥等病症时有着非常很好的疗效。目前FDA已批准两种类型JAK抑制剂分别用于治疗血液病（ruxolitinib）和类风性关节炎（tofacitinib）。Ruxolitinib，一种激酶抑制剂,抑制Janus相关激酶(JAKs)JAK1和JAK2，介导对造血和免疫功能重要的若干细胞因子和生长因子信号。JAK信号涉及细胞因子受体对STATs(信号转导物和转炉激活的补充，激活和随后STATs定位至细胞核导致基因表达的调控。Tofacitinib（CP-690550）是辉瑞公司研发的一种新型口服JAK通路抑制剂。与当前多数其他RA治疗药物主要作用于细胞外靶点不同的是，tofacitinib以细胞内信号转导通路为靶点，作用于细胞因子网络的核心部分。Tofacitinib对JAK3的抑制强度是对JAK1及JAK2的5~100倍。Tofacitinib是开发用于类风湿性关节炎（rheumatoid arthritis）治疗的首创药物（first-in-class drug）。自从tofacitinib在辉瑞自己的试验室诞生以来，该药就被寄予了重磅药物的厚望，该药的成功也将为辉瑞公司备受诟病的研发业务迎来重大胜利。

同时还有数十种类的JAK激酶抑制剂药物已经处于审批的各个阶段。在这些潜在的药物分子当中，有一种编号为ASP3627，由阿斯利康公司开发，口服后具有非常高的生物活性，对过敏和器官移植排斥等病症有着非常好的疗效。这里我们将对文献报道的ASP3627的原始合成路线进行分析，并针对该化合物提供切实可行的公斤级制备工艺。

2017年，阿斯利康首次公布了ASP3627化合物的构型以及相关药理药代数据，证实了该化合物对器官移植免疫排斥的良好抑制作用。在这篇报道中，他们给出的合成路线如下图所示(Scheme 1)。他们用商业可得的化合物2作为起始原料，首先使用SEMCl保护化合物2，得到化合物3。随后用消旋的胺类化合物(1-benzyl-4-methylpiperidin-3-amine)4与化合物3在高温(180℃)下发生S_NAr反应，得到化合物5。随后用DIBAL还原氰基，得到醛基化合物6。再通过接下来的Wittig反应，得到含有Z/E异构体的消旋化合物7。接下来发生一个两步的环化反应，得到化合物8。随后通过两步去保护，得到化合物10。在这里需要注意，化合物手性的引入是通过HPLC使用手性柱对消旋化合物进行手性拆分所得。最后用化合物11与10发生缩合反应获得终产物——药物分子ASP3627(化合物1)，具体路线如下图所示。

在这一条合成路线当中，有许多的问题存在，制约了其再放量合成当中的应用：首先是化合物5的合成当中，需要再非常高的温度下发生S_NAr反应；其次，从DIBAL还原到去保护的过程(化合物5到9)非常的繁琐，且效率极低；同时反应中需要经过多步柱层析，以及化合物10的制备过程中需要考虑Pd的去除问题；最后，反应中需要用到的许多试剂间的副反应问题，比如氢化钠和DMF，TFA，DCM的共存问题等。这些问题对于该化合物的放量合成而言都是急需解决的问题。以下我们将针对这些问题进行具体分析与改进。

1、化合物(3R,4R)-5的工艺优化

合成优化

在之前的路线当中，使用了NaH对胺基去质子化以后用SEMCl保护。这里存在几个潜在的隐患，首先是NaH在大规模合成时会产生大量的氢气，有很大的安全隐患；同时，在一定温度下，NaH可以与DMF溶剂发生副反应，所以在大规模合成时并不是适用。这里我们改用NaHMDS替代NaH来进行这个反应。

随后的SNAr反应当中，存在两个问题，首先是反应时的温度过高，同时反应得到的是消旋产物(后续通过HPLC进行手性拆分)。为了解决上述两个问题，我们在这一步选用了市售的手性胺类化合物(3R,4R)-4作为反应底物，同时改用蒸汽加热系统来进行放量反应。通过优化溶剂、碱以及温度(Table 1),我们最终确定了适用碳酸钠、环丁砜、120℃为最佳反应条件。

提纯优化

在反应结束后，小量制备采取的提纯方式是哟个柱层析进行提纯。而放量合成后，柱层析提纯并不适用，这里我们改用重结晶的方式进行提纯。因为反应当中大部分副产物以及杂质都是比产物极性更加大的化合物，所以使用合适溶剂进行重结晶，可以去除大部分的杂质。在进行了一系列的溶剂筛选后，我们发现使用乙醇对粗产物进行重结晶可以得到高纯度的化合物5(手性纯度99.3%)。

Table 1

2、从化合物5到化合物9的工艺优化

在微量合成路线当中，从化合物5到化合物9的转化过程需要经过多步反应，同时每一步都需要使用柱层析分离纯化中间体。为了在放量合成当中简化操作，我们优化了其中的每一步反应条件，同时在反应过程中不经提纯直接完成从化合物5到9的转化过程。在这中策略当中，我们发现以下几点对于整个路线的优化至关重要：1）化合物5的纯度对于合成路线的综合产率以及可操作性至关重要，2）反应当中每一步转化的产率都要足够高，3）在整个转化完成后，如何提纯得到终产物(3R,4R)-9。

首先是对从化合物5到6的这一步还原后水解反应的优化，通过优化反应条件，我们发现Entry 2所使用的条件为最佳，其中包括了使用DIBAL在-50℃下还原化合物5，之后用甲醇中止反应，随后加入Rochelle salt，以便更方便地去除铝络合物。之后使用3 M的盐酸在室温下加入反应溶液中搅拌45min，随后使用5 M NaOH溶液中和过量的酸，分离提纯，用食盐水和水洗涤后，可以99%收率的到化合物6。接下来的wittig反应(6到7)中，对于反应后的三苯氧磷杂质去除，我们使用正己烷对反应粗产物进行重结晶，成功将杂质含量从3.9A%降低至0.2A%.

接下来是针对环化反应和去SEM保护的条件研究。考虑到放量合成过程中的安全因素，我们选用浓盐酸和甲醇替代酰氯进行环化反应，并简化了后处理方法。在随后的去保护反应中，弃用了污染环境的TFA和DCM，改为使用6M盐酸在DME溶液中进行去保护，随后加入氢氧化钠溶液处理去保护后产物14，得到化合物9。将上述两步可以使用‘一锅法’进行，效率更高。

关于产物9的提纯，我们采用了如下方法：首先用EA溶解粗产物，在50℃条件下用NH₄Cl水溶液洗一遍，去除水溶的大极性杂质；然后使用乙醇对产物进行重结晶提纯。经过我们的实际检测，可以在产率损失低10%的前提下，得到纯度为94.1%的化合物9（产率81.9%）。

终产物1的合成

在得到了化合物9的提纯方法以后，我们想进一步优化最后的两步关键反应。在由化合物9到10的过程中，需要使用到Pd(OH)₂,反应后如何去除残留的重金属杂质是这一步的一大问题。通过实验探究，我们发现使用L-半胱氨酸吸收杂质Pd十分有效，同时省略了过滤等操作，只需要通过简单的重结晶即可祛除。（具体的操作流程见下图）

由10到终产物1的过程中，使用EDC·HCl即可进行该反应。我们发现反应中加入稀盐酸可以更好的促进反应的进行，提高产率。

在这里，我们针对化合物1的粗产物提纯方法进行了系统的研究。在我们的研究中，化合物1在大部分溶液当中的溶解性都非常差，除了DMF和DMSO。但我们认为DMF和DMSO都不适用，因为通过上述两种溶液重结晶后，产物中的溶剂残留会激起的高，而且无法通过其他方法去除！因此在这里，我们选择将化合物1转化为盐酸盐的形式，在乙醇和稀盐酸的混合溶液当中进行重结晶，再过滤后用NaOH的水溶液进行处理，得到纯品化合物1(>99.0%纯度，乙醇残留量<< span=''>0.2%).

最后，通过整条优化后的路线，在我们的测算下综合产率可以达到52.1%。

文章来源：A Practical and Scalable Method for Manufacturing JAK Inhibitor ASP3627

具体实验操作

General methods Unless otherwise noted, all reactions were performed under a nitrogen atmosphere. All reagents and solvents purchased from suppliers were used as received unless otherwise noted. (3R,4R)-4 was obtained commercially from a custom synthesis supplier. NMR spectra were recorded on a Bruker ADVANCE III HD500. Chemical shifts (δ) are reported in ppm in reference to the residual solvent signal (δ 2.50 for 1 H NMR in DMSO-d6, δ 39.52 for 13C{1 H} NMR in DMSO-d6, and δ 77.16 for 13C{1 H} NMR in CDCl3). High-resolution mass spectra were obtained on a Thermo Scientific EXACTIVE Plus. Elemental analyses were performed on a Elementar Vario MICRO cube and DIONEX ICS-3000-I. IR spectra were recorded on a SHIMADZU IRAffinity-1S.

4-Chloro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile (3). A 500 L reactor-1 was charged with 2 (36.0 kg, 203 mol) and DMF (170.1 kg, 5 vol). NaHMDS (38.1% in THF, 107.3 kg, 1.1 equiv, 223 mol) was added to the resulting slurry at ≤10 °C, and the mixture was stirred for ≥30 min at 0−10 °C. Following the addition of SEMCl (37.2 kg, 1.1 equiv, 223 mol) over ≥30 min at 0−10 °C, the reaction mixture was stirred for ≥1 h at 0−10 °C (IPC: 2 ≤ 1.0 A% by HPLC Method A). The reaction mixture was transferred to a 2000 L reactor-2 and quenched with aqueous NH4Cl (prepared from 72.0 kg (2 wt) of NH4Cl and 360 L (10 vol) of water), and EtOAc (97.4 kg, 3 vol) and Radiolite (18.0 kg, 0.5 wt) were added at ≤30 °C. The mixture was stirred for ≥1 h at 20−30 °C, filtered, and the wet cake was washed with EtOAc (64.9 kg, 2 vol). The filtrate was transferred to a 1500 L reactor-3. The biphasic mixture was separated. The organic layer was washed with aqueous NH4Cl (prepared from 36.0 kg (1 wt) of NH4Cl and 360 L (10 vol) of water) (×2) at 10−30 °C, transferred to a 500 L reactor- 4 and reactor-3 was rinsed with EtOAc (20.0 kg, 0.6 vol). The mixture was concentrated under vacuum at 50 °C to ≤108 L (3 vol). EtOH (113.6 kg, 4 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to ≤108 L (3 vol). More EtOH (113.6 kg, 4 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to 97−119 L (3 vol). EtOH (113.6 kg, 4 vol) was again added, the mixture was warmed to 45−55 °C, and stirred for ≥1 h. The solution was cooled to 20−30 °C (approximately 10 °C/ h), and stirred for ≥1 h. Water (108 L, 3 vol) was added over ≥30 min at 20−30 °C. The slurry was aged for ≥2 h, cooled to −5 to 5 °C (approximately 10 °C/h), aged for ≥5 h (IPC: 3 ≤ 4 g/L by HPLC Method A), and then filtered. The wet cake was washed with cooled aqueous EtOH (prepared from 73.9 kg (2.6 vol) of EtOH and 47 L (1.3 vol) of water), dried at 50 °C (internal temperature) for ≥1 h under reduced pressure (IPC: loss on drying (LOD) ≤ 1.0%) to give 3。(54.2 kg, 176 mol, 86.9% yield) as a pale yellow solid. HPLC purity: 98.0 A% (specification: ≥95.0 A% by HPLC Method A). 1 H NMR (DMSO-d6, 500 MHz): δ –0.13 (s, 9H), 0.80 (t, 2H, J = 8.0 Hz), 3.51 (t, 2H, J = 8.0 Hz), 5.66 (s, 2H), 6.74 (d, 1H, J = 3.7 Hz), 7.97 (d, 1H, J = 3.7 Hz), 8.70 (s, 1H). 13C{1 H} NMR (DMSO-d6, 125 MHz): δ –1.5, 17.1, 65.8, 73.0, 99.7, 101.5, 115.7, 118.8, 133.0, 137.3, 146.6, 148.5. HRMS–ESI (m/z): [M + H]+ calcd for C14H19N3OClSi, 308.0980; found, 308.0981. Anal. Calcd for C14H18N3OClSi: C, 54.62; H, 5.89; N, 13.65; Cl, 11.52. Found: C, 55.00; H, 5.99; N, 13.66; Cl, 11.11. IR (ATR, cm–1): 3088, 2955, 2926, 2220, 1589, 1346, 1248, 1233, 1070, 858, 833, 737, 698. DSC (onset): 66.4 °C.

4-{[(3R,4R)-1-Benzyl-4-methylpiperidin-3-yl]amino}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1Hpyrrolo[2,3-b]pyridine-5-carbonitrile (5). A 500 L reactor-1 was charged with 3 (52.0 kg, 169 mol), (3R,4R)-4 (41.4 kg, 1.2 equiv, 203 mol), and sulfolane (524.2 kg, 8 vol). Na2CO3 (21.5 kg, 1.2 equiv, 203 mol) was added to the resulting solution, and the reaction mixture was warmed to 115−125 °C and stirred for ≥18 h (IPC: 3 ≤ 1.0 A% by HPLC Method A). The reaction mixture was cooled to 10−30 °C, transferred to a 2000 L reactor-2, diluted with EtOAc (469.0 kg, 10 vol), and quenched with aqueous NaCl (prepared from 15.6 kg (0.3 wt) of NaCl and 780 L (15 vol) of water). The biphasic mixture was separated. The organic layer was washed with aqueous NaCl (prepared from 10.4 kg (0.2 wt) of NaCl and 520 L (10 vol) of water) and aqueous NaCl (prepared from 104.0 kg (2 wt) of NaCl and 520 L (10 vol) of water) at 10−30 °C. The resulting solution was transferred to a 1500 L reactor-3 and reactor-2 was rinsed with EtOAc (20.0 kg, 0.4 vol). The solution was concentrated under vacuum at 50 °C to ≤156 L. EtOH (164.1 kg, 4 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to ≤156 L (3 vol). More EtOH (164.1 kg, 4 vol) was added the residue and the mixture was concentrated

under vacuum at 50 °C to 146−166 L (3 vol). EtOH (205.1 kg, 5 vol) was again added to the residue, the mixture was cooled to −5 to 5 °C, seeded with (3R,4R)-5 (1.0 g), and aged for ≥1 h. Water (156 L, 3 vol) was added over ≥30 min, the slurry was aged for ≥1 h at −5 to 5 °C, warmed to 40−50 °C, and aged for ≥1 h. The slurry was cooled to −5 to 5 °C (approximately 10 °C/h), aged for ≥5 h (IPC: 5 ≤ 5 g/L by HPLC Method A), and then filtered. The wet cake was washed with cooled aqueous EtOH (prepared from 114.9 kg (2.8 vol) of EtOH and 62 L (1.2 vol) of water), dried at 50 °C (internal temperature) for ≥1 h under reduced pressure (IPC: LOD ≤ 1.0%) to give (3R,4R)-5 (74.2 kg, 156 mol, 92.3% yield) as a pale yellow solid. HPLC purity: 99.5 A% (specification: ≥97.0 A% by HPLC Method A). 1 H NMR (DMSO-d6, 500 MHz): δ – 0.13 (s, 9H), 0.77 (t, 2H, J = 8.0 Hz), 0.83 (d, 3H, J = 6.7 Hz), 1.42–1.56 (m, 2H), 1.81–1.93 (m, 1H), 2.05–2.16 (m, 1H), 2.27 (d, 1H, J = 10.6 Hz), 2.73–2.87 (m, 2H), 3.43 (d, 1H, J = 13.4 Hz), 3.47 (t, 2H, J = 8.0 Hz), 3.56 (d, 1H, J = 13.4 Hz), 4.39–4.47 (m, 1H), 5.53 (s, 2H), 6.31 (d, 1H, J = 9.9 Hz), 6.84 (d, 1H, J = 3.8 Hz), 7.17 (t, 1H, J = 7.3 Hz), 7.24 (t, 2H, J = 7.5 Hz), 7.35 (d, 2H, J = 7.3 Hz), 7.43 (d, 1H, J = 3.8 Hz), 8.19 (s, 1H). 13C{1 H} NMR (DMSO-d6, 125 MHz): δ – 1.5, 17.1, 17.4, 28.8, 33.7, 51.8, 52.6, 57.4, 61.6, 65.4, 72.5, 84.2, 101.3, 104.3, 118.5, 125.9, 126.9, 128.1, 128.5, 138.3, 147.5, 149.4, 149.8. HRMS–ESI (m/z): [M + H]+ calcd for C27H38N5OSi, 476.2840; found, 476.2842. Anal. Calcd for C27H37N5OSi: C, 68.17; H, 7.84; N, 14.72. Found: C, 68.74; H, 7.98; N, 14.73. [α]D23 – 27.7 (c 1.00, EtOH). IR (ATR, cm–1): 3339, 2951, 2922, 2818, 2199, 1591, 1557, 1524, 1512, 1225, 1080, 858, 833, 712, 698. DSC (onset): 75.2 °C.

resulting solution was cooled to −65 to −45 °C, and DIBAL (17% in toluene, 300.4 kg, 2.5 equiv, 363 mol) was added at −65 to −45 °C. The reaction mixture was warmed to −5 to 5 °C, and stirred for ≥1 h (IPC: 5 ≤ 0.3 A% by HPLC Method A). The reaction was cooled to −65 to −45 °C, quenched with MeOH (27.3 kg, 0.5 vol), and transferred to a 5000 L reactor-2. Aqueous Rochelle salt (prepared from 207.0 kg (3 wt) of Rochelle salt and 690 L (10 vol) of water) was added to the solution at ≤10 °C. The biphasic mixture was stirred vigorously for ≥1 h at 15−25 °C and separated to give (3R,4R)-12 in toluene solution (the organic layer). Aqueous HCl (prepared from 162.8 kg (1599 mol) of concd aqueous HCl and 414 L (6 vol) of water) was added to the toluene solution of (3R,4R)-12, and the reaction mixture was stirred for ≥3 h at 15−25 °C. The pH of the aqueous layer was adjusted to 8−9 by adding 5 M aqueous NaOH (359.8 kg, 1497 mol) at 15−25 °C. The biphasic mixture was separated. The organic layer was washed with aqueous NaCl (prepared from 69.0 kg (1 wt) of NaCl and 345 (5 vol) L of water) (×2) at 15−25 °C, transferred to a 1500 L reactor-3, and reactor-2 was rinsed with THF (20.0 kg, 0.3 vol). The solution was concentrated under vacuum at 50 °C to ≤138 L (2 vol). Toluene (298.8 kg, 5 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to ≤138 L (2 vol). More toluene (298.8 kg, 5 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to 128−148 L (2 vol, IPC: residual water ≤ 0.10%). THF (184.0 kg, 3 vol) was added to the residue to give (3R,4R)-6 in THF solution. A 1500 L reactor-4 was charged with (methoxymethyl)triphenylphosphonium chloride (74.6 kg, 1.5 equiv, 218 mol) and THF (184.0 kg, 3 vol). The resulting slurry was stirred, cooled to −20 to −10 °C, and NaHMDS (38.7% in THF, 137.5 kg, 2.0 equiv, 290 mol) was added over ≥30 min at −20 to −10 °C. The mixture was stirred for ≥1 h, the THF solution of (3R,4R)-6 was added over ≥30 min at −20 to −10 °C, and reactor-3 was rinsed with THF (122.7 kg, 2 vol). The reaction

mixture was warmed to 15−25 °C and stirred (IPC: 6 ≤ 1.0 A% by HPLC Method B). The reaction was quenched with aqueous NH4Cl (prepared from 69.0 kg (1 wt) of NH4Cl and 345 L (5 vol) of water), transferred to a 5000 L reactor-5, and reactor-4 was rinsed with THF. The biphasic mixture was separated at 0−30 °C. The organic layer was washed with aqueous NaCl (prepared from 69.0 kg (1 wt) of NaCl and 345 L (5 vol) of water) at 0−30 °C, transferred to a 1500 L reactor-6, and reactor-5 was rinsed with n-heptane (13.7 kg, 0.3 vol). The mixture was concentrated under vacuum at 50 °C to ≤207 L (3 vol). n-Heptane (236.0 kg, 5 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to ≤207 L (3 vol). More nheptane (236.0 kg, 5 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to 197−217 L (3 vol). n-Heptane (424.8 kg, 9 vol) was again added to the residue at 45−55 °C. The slurry was cooled to −5 to 5 °C (approximately 10 °C/h), aged for ≥10 h, warmed to approximately 50 °C, stirred for 3 h, cooled to approximately 30 °C (IPC: triphenylphosphine oxide ≤ 0.7 A%, by HPLC Method B), and then filtered. The wet cake was washed with n-heptane (188.8 kg, 4 vol). The filtrate was transferred to a 2000 L reactor-7 and concentrated under vacuum at 50 °C to ≤138 L (2 vol). DME (297.7 kg, 5 vol) was added to the residue and the mixture was concentrated under vacuum at 50 °C to 128−148 L (2 vol). More DME (238.2 kg, 4 vol) was added to the residue to give an E/Z mixture of (3R,4R)-7 in DME solution. Aqueous HCl (3.0 equiv, 436 mol, prepared from 44.5 kg of concd aqueous HCl and 37 L of water) was added to the E/Z mixture of (3R,4R)-7 in DME solution, the reaction mixture was warmed to 45−55 °C, and stirred for ≥3 h (IPC: 7 ≤ 1.0 A% by HPLC Method B) to give (3R,4R)-8 in aqueous DME solution.

The wet cake was washed with cooled aqueous EtOH (prepared from 141.5 kg (2.6 vol) of EtOH and 90 L (1.3 vol) of water), dried at 50 °C (internal temperature) for ≥1 h under reduced pressure (IPC: LOD ≤ 1.0%) to give (3R,4R)-95 (40.4 kg, 117 mol, 80.8% yield) as a white solid. HPLC purity: 96.8 A% (specification: ≥94.0 A% by HPLC Method A). 1 H NMR (DMSO-d6, 500 MHz): δ 0.51 (d, 3H, J = 6.8 Hz), 1.58–1.70 (m, 2 H), 2.13–2.31 (m, 2H), 2.67 (dd, 1H, J = 11.8, 3.2 Hz), 2.84–2.94 (m, 1H), 3.04 (dd, 1H, J = 11.8, 3.8 Hz), 3.53 (d, 1H, J = 13.2 Hz), 3.59 (d, 1H, J = 13.2 Hz), 5.01 (q, 1H, J = 4.0 Hz), 6.61 (d, 1H, J = 3.3 Hz), 6.74–6.77 (m, 1H), 7.22 (tt, 1H, J = 7.2, 1.4 Hz), 7.28–7.37 (m, 5H), 7.99 (br s, 1H), 8.43 (s, 1H), 11.6 (br s, 1H). 13C{1 H} NMR (CDCl3, 125 MHz): δ 16.0, 30.2, 34.1, 52.2, 56.1, 56.8, 63.5, 97.6, 101.5, 105.1, 118.3, 121.3, 126.3, 127.3, 128.4, 129.1, 135.8, 137.7, 138.3, 145.2. HRMS–ESI (m/z): [M + H]+ calcd for C22H25N4, 345.2074; found, 345.2071. Anal. Calcd for C22H24N4: C, 76.71; H, 7.02; N, 16.27. Found: C, 76.70; H, 7.08; N, 16.19. [α]D23 – 28.5 (c 1.00, 0.1 M aq HCl). IR (ATR, cm–1): 3111, 3028, 2926, 2808, 1612, 1582, 1452, 1350, 1327, 885, 837, 820, 731, 719, 698. DSC (onset): 185.0 °C. 1-[(3R,4R)-4-Methylpiperidin-3-yl]-1,6-dihydrodipyrrolo[2,3-b:2',3'-d]pyridine (10). A 500 L reactor-1 was charged with (3R,4R)-9 (39.7 kg, 115 mol), EtOH (62.6 kg, 2 vol), and water (119 L, 3 vol). Aqueous HCl (2.0 equiv, 230 mol, prepared from 23.4 kg of concd aqueous HCl and 19 L of water) and 20% Pd(OH)2/C (wetted with water, 2.0 kg, 0.05 wt, dry basis) were added to the resulting mixture. The reaction mixture was stirred for 6 h at 30−40 °C under a H2 atmosphere (0.15−0.25 MPa) (IPC: 9 ≤ 1.0 A% by HPLC Method C). The reaction mixture was cooled to 20−30 °C, filtered, and the wet cake was washed with aqueous EtOH (prepared from 31.3 kg (1 vol) of EtOH and 79 L (2 vol) of water). The filtrate was transferred to a 500 L reactor-2 and ʟ-cysteine (4.0 kg, 0.1 wt) was added. The mixture was stirred for ≥1 h at 20−30 °C. The pH of the solution was adjusted to 9.5−10.0 by adding 5 M aqueous NaOH (63.1 kg, 263 mol) at 20−30 °C. The slurry was aged for ≥1 h at 20−30 °C (IPC: 10 ≤ 2 g/L by HPLC Method C), and then filtered. The wet cake was washed with aqueous EtOH (prepared from 37.6 kg (1.2 vol) of EtOH and 111 L (2.8 vol) of water), dried at 50 °C (internal temperature) for ≥1 h under reduced pressure (IPC: LOD ≤ 1.0%) to give (3R,4R)-105 (27.9 kg, 110 mol, 95.1% yield) as a pale yellow solid. HPLC purity: 98.0 A% (specification: ≥94 A% by HPLC Method C). 1 H NMR (DMSO-d6, 500 MHz): δ 0.59 (d, 3H, J = 7.0 Hz), 1.51–1.60 (m, 1H), 1.67–1.75 (m, 1H), 2.27–2.54 (m, 2H), 2.70 (ddd, 1H, J = 12.2, 7.4, 3.7 Hz), 2.96 (ddd, 1H, J = 12.1, 7.3, 3.3 Hz), 3.08 (dd, 1H, J = 12.2, 3.7 Hz), 3.29 (dd, 1H, J = 12.3, 6.6 Hz), 4.89 (dt, 1H, J = 6.5, 4.1 Hz), 6.58 (d, 1H, J = 3.3 Hz), 6.71 (dd, 1H, J = 3.3, 1.6 Hz), 7.32 (t, 1H, J = 2.9 Hz), 7.76 (br s, 1H), 8.44 (s, 1H), 11.58 (br s, 1H). 13C{1 H} NMR (DMSO-d6, 125 MHz): δ 14.7, 30.9, 33.2, 43.2, 47.8, 55.7, 96.9, 100.7, 104.3, 117.7, 121.7, 125.1, 134.3, 137.2, 144.5. HRMS–ESI (m/z): [M + H]+ calcd for C15H19N4, 255.1604; found, 255.1603. Anal. Calcd for C15H18N4: C, 70.84; H, 7.13; N, 22.03. Found: C, 70.77; H, 7.24; N, 21.68. [α]D23 − 159.0 (c 1.00, 0.1 M aq HCl). IR (ATR, cm–1): 3310, 3109, 2899, 1614, 1352, 1339, 822, 716. DSC (onset): 84.3, 260.1 °C.

Crude 1. A 500 L reactor-1 was charged with (3R,4R)-10 (13.63 kg, 53.6 mol) and DMF (128.8 kg, 10 vol). HOBt·H2O (0.82 kg, 0.1 equiv, 5.4 mol) and cyanoacetic acid (11) (5.01 kg, 1.1 equiv, 58.9 mol) were added to the resulting slurry, followed by EDC·HCl (16.44 kg, 1.6 equiv, 85.8 mol) over ≥0.5 h, and the reaction mixture was stirred for ≥1 h at 20−30 °C (IPC: 10 ≤ 1.0 A% by HPLC Method D). Next, 1 M aqueous HCl (54.4 kg, 1.0 equiv, 53.3 mol) was added to the reaction mixture and the mixture was stirred for ≥1 h at 45−55 °C (IPC: overreacted。compound ≤ 1.0 A% by HPLC Method D). The pH of the solution was adjusted to 8.5 by adding 5 M aqueous NaOH (23.3 kg, 98.7 mol) at 45−55 °C. The slurry was aged for ≥1 h at 45−55 °C and water (72 L, 5.3 vol) was added over ≥1 h. The slurry was aged for ≥1 h at 45−55 °C, cooled to 20−30 °C (approximately 10 °C/h), aged for ≥12 h (IPC: 1 ≤ 3 g/L by HPLC Method D), and then filtered. The wet cake was washed with aqueous DMF (prepared from 12.9 kg (1 vol) of DMF and 14 L (1 vol) of water) and aqueous EtOH (prepared from 53.8 kg (5 vol) of EtOH and 68 L (5 vol) of water), and dried at 50 °C for ≥12 h under reduced pressure (IPC: LOD ≤ 1.0%) to give crude 1 (15.67 kg, 48.8 mol, 91.0% yield) as a pale yellow solid. HPLC purity: 98.7 A% (specification: ≥95 A% by HPLC Method D).

3-{(3R,4R)-3-[Dipyrrolo[2,3-b:2',3'-d]pyridin-1(6H)-yl]-4-methylpiperidin-1-yl}-3- oxopropanenitrile (1). A 200 L reactor-1 was charged with crude 1 (31.06 kg, 96.6 mol), EtOH (98.0 kg, 4 vol), water (41.9 kg, 1.35 vol), and 6 M aqueous HCl (17.62 kg, 1.0 equiv, 96.1 mol) at 0−30 °C. The resulting solution was transferred to a 500 L reactor-2 via a cartridge filter (≤1 μm) and reactor-1 was rinsed with aqueous EtOH (prepared from 49.0 kg (2 vol) of EtOH and 31 L (1 vol) of water). The solution was warmed to 45−55 °C. Next, 1 M aqueous NaOH (20.1 kg, 0.2 equiv, 19.3 mol) was added over ≥0.5 h and the mixture was stirred for ≥1 h at 45−55 °C. The pH of the mixture was adjusted to 8.5 by adding more 1 M aqueous NaOH (81.6 kg, 0.8 equiv, 78.5 mol). The slurry was stirred for ≥1 h and cooled to 20−30 °C (approximately 10 °C/h), stirred for ≥1 h (IPC: 1 ≤ 2 g/L by HPLC Method D), and then filtered. The wet cake was washed with aqueous EtOH (prepared from 122.5 kg (5 vol) of EtOH and 155 L (5 vol) of water) and water (311 L, 10 vol), and dried at 50 °C for ≥12 h under reduced pressure (IPC: residual EtOH ≤ 1.0%, DMF ≤ 0.088%) to give 15 (30.25 kg, 94.1 mol, 97.4% yield) as a pale。

yellow solid. HPLC purity: 99.2 A% (HPLC Method D), ≥99.80% ee (HPLC Method E). 1 H NMR (DMSO-d6, 500 MHz, 6/4 mixture of rotamers): δ 0.62 (d, 1.8H, J = 7.0 Hz), 0.68 (d, 1.2H, J = 7.1 Hz), 1.61−1.76 (m, 1.0H), 1.83−1.92 (m, 1.0H), 2.38−2.48 (m, 1.0H), 3.36−3.43 (m, 0.6H), 3.51−3.58 (m, 0.4H), 3.58−3.67 (m, 1.0H), 3.70−3.78 (m, 0.4H), 3.78−3.87 (m, 1.0H), 3.97 (dd, 0.4H, J = 13.7, 8.1 Hz), 4.07 (d, 0.4H, J = 19.0 Hz), 4.09 (d, 0.6H, J = 18.8 Hz), 4.18 (dd, 0.6H, J = 13.4, 6.4 Hz), 4.30 (d, 0.6H, J = 18.9 Hz), 4.95−5.01 (m, 0.6H), 5.04−5.10 (m, 0.4H), 6.64 (d, 0.6H, J = 3.3 Hz), 6.67 (d, 0.4H, J = 3.3 Hz), 6.74−6.78 (m, 1.0H), 7.19 (d, 0.4H, J = 3.3 Hz), 7.28 (d, 0.6H, J = 3.3 Hz), 7.34 (t, 1.0H, J = 2.9 Hz), 8.46 (s, 0.6H), 8.48 (s, 0.4H), 11.64 (br s, 1H). 13C{1 H} NMR (DMSO-d6, 125 MHz, 6/4 mixture of rotamers): δ 13.3, 14.2, 24.8, 25.2, 28.9, 29.1, 31.8, 32.4, 38.7, 43.4, 43.5, 46.3, 54.3, 55.0, 96.9, 97.0, 101.8, 101.9, 104.1, 104.2, 116.0, 116.2, 117.5, 117.7, 122.0, 122.1, 123.1, 123.7, 134.2, 134.4, 137.2, 137.2, 144.6, 144.6, 161.8, 162.1. HRMS–ESI (m/z): [M + H]+ calcd for C18H20N5O, 322.1662; found, 322.1661. Anal. Calcd for C18H19N5O: C, 67.27; H, 5.96; N, 21.79. Found: C, 67.31; H, 6.08; N, 21.70. [α]D23 + 51.2 (c 0.750, 0.1 M aq HCl) (lit.5 [α]D25 + 50.1 (c 0.733, 0.1 M aq HCl)). IR (ATR, cm–1): 3296, 2934, 2263, 1645, 1611, 1462, 1335, 1252, 1211, 1182, 885, 710. DSC (onset): 310.6 °C.

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