Article pubs.acs.org/OPRD
Cite This: Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Development of a Synthesis of Kinase Inhibitor AKN028 Ulf Bremberg,† Johan Eriksson-Bajtner,‡,§ Fredrik Lehmann,† Viveca Oltner,‡ Ellen Sölver,‡ and Johan Wennerberg*,‡ ‡
R&D Department, Magle Chemoswed, P.O. Box 839, SE 201 80 Malmö, Sweden Recipharm OT Chemistry, Virdings Allé 32 B, SE 754 50 Uppsala, Sweden
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ABSTRACT: The novel tyrosine kinase inhibitor AKN028 has demonstrated promising results in preclinical trials. An expedient protocol for the synthesis of the compound at kilogram scale is described, including an SNAr reaction with high regioselectivity and a Suzuki coupling. Furthermore, an efficient method for purification and removal of residual palladium is described. KEYWORDS: tyrosine kinase inhibitor, SNAr reaction, Suzuki coupling, removal of palladium
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INTRODUCTION It has been observed that abnormally expressed tyrosine kinases may be promising targets when developing drugs against acute myeloid leukemia (AML). This life-threatening disease is characterized by several acquired genetic abnormalities, and multiple signaling pathways are involved in the pathogenesis. Present therapy is still based on cytotoxic chemotherapy, but there is an emerging interest in identifying molecular drug targets, such as tyrosine kinases, that may present opportunities for more targeted treatment. A novel tyrosine kinase inhibitor, N-3-(1H-indol-5-yl)-5-pyridin-4-ylpyrazine-2,3-diamine (AKN028, 1), has shown broad in vitro activity. Exposure to AKN028 caused cell death in tumor samples from a wide range of AML patients.1,2 In order to continue the clinical program, we have developed an expedient route for the synthesis of AKN028 in kilogram quantities that includes an SNAr reaction with high regioselectivity and a Suzuki coupling (Scheme 1).3,4 Furthermore, a purification procedure involving removal of residual Pd was developed to ensure that the material was suitable for clinical trials.
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and considered to be a potential problem because purging was troublesome. The propensity for dimer formation was tested by heating all of the reagents except 5-aminoindole at 130 °C in DMSO. After 16 h, less than 2% yield of dimer 3 had been formed, and when the faster-reacting 5-aminoindole was introduced, formation of the dimer was suppressed to even lower levels. Other solvents tested gave rise to larger amounts of dimer 3. The reaction conveniently turned out to be insensitive to air and moisture. For example, when water (2.5% v/v) was added to the reaction mixture, no significant change in purity or reaction rate was observed. After the reaction was complete, excess triethylamine was boiled off, and acetic acid was added. Product 2 was then easily precipitated by addition of water. This workup method was both convenient and robust. When reaction mixtures with only partial conversion were subjected to precipitation the ratio of product 2 to 2amino-3,5-dibromopyrazine was still 50:1. When the reaction was run at multikilogram scale, the conversion was 88%, as evidenced by HPLC analysis, and prolonged heating did not increase the conversion. Workup followed by drying returned 2 in 81% yield with 96% purity according to HPLC. The SNAr reaction between 2-amino-3,5-dibromopyrazine and 5-aminoindole turned out to be highly regioselective. Such selectivity of the reaction may appear counterintuitive, since the more sterically hindered position is preferentially attacked by the nucleophile. However, the 2-amino group has a weak electrondonating effect that decreases the reactivity of the bromine atoms, and this effect is more pronounced at the 5-position. We believe that this explains the high degree of selectivity of the reaction, and in fact, we were not able to detect any regioisomeric product. The reaction between 2-amino-3,5dibromopyrazine and 5-aminoindole has been described in a journal article5 and in a few patents.6−8 The descriptions are brief, and there are no comments on the regiochemistry of the reaction. Furthermore, the literature reaction was performed in ethanol with diisopropylethylamine as the base, and the yields
RESULTS AND DISCUSSION
SNAr Reaction. Initially, the starting material 2-amino-3,5dibromopyrazine was synthesized by bromination of the parent aminopyrazine with N-bromosuccinimide. Later, we were able to source this raw material commercially. In the first step, 2-amino-3,5-dibromopyrazine and 5aminoindole were reacted in an SNAr-reaction to give 2. The reaction was performed in dimethyl sulfoxide (DMSO) with excess triethylamine as the base at elevated temperature. Initially, a 5% solution based on the dibromo compound in DMSO was used, but it was found that a 10-fold reduction in DMSO gave the same purity and increased the reaction rate. Neat conditions increased the level of impurities, as did a switch to dimethylformamide (DMF) as the solvent. Not surprisingly, larger amounts of 5-aminoindole increased the reaction rate, but no effects on purity were seen in the investigated range (1.2−3 equiv). Considering the cost of 5aminoindole, 1.3 equiv appeared to give optimal performance. During early developments, dimer 3 (Figure 1) was detected © XXXX American Chemical Society
Received: April 3, 2018
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DOI: 10.1021/acs.oprd.8b00092 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Scheme 1. Route to AKN028
Figure 1. Dimer and dehalogenated intermediate.
were substantially lower. With one exception,8 all of the related work was published after we performed our studies. Suzuki Coupling. Selecting a palladium catalyst for a Suzuki reaction may seem straightforward, but it turned out to be somewhat challenging in our case. For practical reasons, we wanted to use a heterogeneous catalyst that could be easily separated after completion of the reaction. An obvious choice was palladium on activated charcoal,9 but to our dismay, we were not able to measure any conversion of the starting material despite many attempts with different catalyst loadings and temperatures (Table 1, entry 1). Instead, we examined a couple of palladium catalysts supported on silica with different linkers (Figure 2). With four of the five tested catalysts, no or very low conversion was observed (Table 1, entries 2−5). In one case, with the trioxaphosphaadamantane-based catalyst
Figure 2. Catalysts and ligand.
Table 1. Palladium Catalysts Applied in the Suzuki Reaction entry
catalyst
loading (mol %)
solvent
conversiona
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Pd/C SCRPd SEM2Pd SPM3Pd PAPd1r PAPd2r PAPd2r PAPd2r PdCl2 Pd(OAc)2 (PPh3)2PdCl2 Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(OAc)2 + DTB-PPSb Pd(dppf)Cl2
10% w/w 4 4 4 4 4 4 4 4 4 4 2 2 1 1.5 2 2 2 2 2 2 4
DMF DMF DMF DMF DMF DMF NMP NEP DMF DMF DMF DMF DMA DMA DMA Me-THF THF MeCN NEP DMSO NMP DMF
no conversion 0.3% (2 h), 1.0% (5 h), 0.4% (24 h) 0.2% (2 h), 0.1% (5 h), 0.4% (24 h) 0% (2 h), 0.2% (5 h), 0.4% (24 h) 1.4% (2 h), 1.2% (5 h), 4% (24 h) 19% (2 h), 22% (5 h), 33% (24 h) 7%, (2 h), 12% (4 h), 16% (20 h) 5% (2 h), 11% (4 h), 18% (20 h) no conversion no conversion no conversion 100% (2 h) 98% (2.5 h), 100% (4 h) 14% (1.5 h), 34% (4 h), 99% (20 h) 17.6% (2 h), 40% (4 h), 100% (23 h) no conversion 1% (24 h) 1.5% (24 h) 9.7% (3 h), 11% (22 h) 25% (2 h), 26% (4 h) 40% (2 h), 42% (4.5 h) 100% (2 h)
a
All of the reactions were performed at 0.3−1 mmol scale with 2 M K2CO3 (1.1 equiv, 10% conc.). Conversion was monitored by HPLC. bThe Pd(OAc)2/DTB-PPS ratio was 1:1. The temperature was 100 °C except with MeCN and THF, where reflux temperature was used. B
DOI: 10.1021/acs.oprd.8b00092 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Hydrocarbons, ethers, ketones, alcohols, esters, acetonitrile, and dichloromethane were tested, all with discouraging outcomes. Apparently useful exceptions were acetone (16 mg/mL), DMF (53 mg/mL), and DMSO (114 mg/mL). At a first glance, most of the solid samples of 1 appeared to be amorphous, but small amber-colored crystals also appeared in the material. Small crystals were formed when 1 was crystallized from acetone/n-heptane. The yield was low, and 1 turned out to become degraded in acetone, especially at elevated temperature or when stored in solution. When water was added slowly to a solution of 1 in DMF, large crystals were formed, but to our dismay, the assay and purity remained unsatisfactory. Numerous other attempts were performed before a useful procedure could be identified. Finally, it was decided to use a solution of 1 in 2 M acetic acid and treatment with Hyflo Super Cel followed by filtration. This treatment increased the purity and decreased the palladium level (vide infra). The filtrate was then mixed with a large amount 2methyltetrahydrofuran and heated to 40 °C. Concentrated potassium hydroxide was added, followed by phase separation. The phase separation was facilitated by the elevated temperature. As mentioned earlier, the solubility of pure 1 in 2methyltetrahydrofuran is low (3.3 mg/mL). On the other hand, the solubility of crude 1 is much higher (21 mg/mL at both rt and 40 °C). Addition of DMF and scavengers followed by stirring at elevated temperature resulted in an acceptable residual palladium level of 5 ppm. After filtration, almost complete removal of 2-methyltetrahydrofuran was performed via distillation at reduced pressure. To remove residual water, toluene was added, followed by azeotropic distillation. An additional amount DMF was added, and a clear solution was obtained. Toluene was added slowly at 25 °C, and after 12 h a yellow to faint-orange product was isolated. We found that it was important to ensure dry material prior to the recrystallization procedure, since residual solvents exerted a negative influence on purity. For the same reason, rinsing with toluene was important to avoid residual DMF in the product. Removal of Palladium. With the increased use of metalcatalyzed reactions, the removal of metal residues from crude reaction mixtures is now a common challenge in the pharmaceutical industry.10,11 In Suzuki couplings, where palladium is used in catalytic amounts, quantities of residual metal ranging from a few hundred to several thousand parts per million are typically left after initial workup. The present case is no exception. Crude 1 may contain around 5000 ppm Pd, and wide variation among batches was observed. We made a late attempt to address the problem by using an encapsulated palladium source in order to avoid palladium in the product. Pd(II)EnCat30NP, a microencapsulated Pd(II) catalyst in a polyurea matrix available as beads was tested. A Suzuki coupling with Pd(II)EnCat30NP was performed in DMF with DTB-PPS as the ligand at 75 °C. After 20 h, the conversion was 79%, and the reaction was terminated and the catalyst filtered off. The product was isolated, dried, and analyzed. The product contained about 7000 ppm palladium, which indicated very substantial leakage of palladium from the beads. According to the product guide, coordinating solvents such as DMF and DMA may cause swelling of the matrix followed by leaching, especially at temperatures above 80 °C. Also, it is suggested that if a ligand is used, the preferred ratio should be 98% as indicated by HPLC. Workup was convenient, with addition of water followed by isolation and drying of the precipitated material. The product purity was 96% according to HPLC, and the residual palladium level was around 5000 ppm, which suggested that purification was required. Crystallization. Even though the synthetic transformations worked well, the major challenge was to obtain a pure, crystalline material with low levels of palladium. The desired kinase inhibitor 1 has very low solubility in most solvents (below or well below 10 mg/mL in almost all solvents). C
DOI: 10.1021/acs.oprd.8b00092 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
MHz (13C). Thin-layer chromatography was performed on Merck precoated TLC plates, which were visualized with UV light or sprayed with a solution of p-methoxybenzaldehyde (26 mL), glacial acetic acid (11 mL), concentrated H2SO4 (35 mL), and ethanol (960 mL) followed by heating. Solvents and reagents were obtained from commercial sources and used as such without any further purification. All of the reactors used were standard multipurpose equipment, either glass-lined or stainless steel. All of the reactions at pilot-plant scale were for safety reasons routinely carried out under an atmosphere of nitrogen. 5-Bromo-N-3-(1H-indol-5-yl)pyrazine-2,3-diamine (2). A glass-lined 100 L reactor was charged with dimethyl sulfoxide (10 L), 2-amino-3,5-dibromopyrazine (4.50 kg, 17.8 mol), 5-aminoindole (3.06 kg, 23.2 mol, 1,3 equiv), and triethylamine (5.40 kg, 53.4 mol, 3 equiv). The mixture was stirred and heated at 130−135 °C (jacket temperature) for 12 h, after which it was left to cool overnight. After in-process control (HPLC), triethylamine was distilled off under reduced pressure. Acetic acid (18.4 kg, 50% in water) was added to the remaining mixture with stirring for 20 min, followed by the addition of water (61 L) for 60 min. The solids were isolated on a filter and washed with acetic acid (2 × 20 L, 1% in water) and water (2 × 20 L). The product was dried to constant weight at 40 °C and reduced pressure to give the title compound (4.36 kg, 81%) as dark-green crystals. The time for drying was 19 h, and the purity was 96% according to HPLC. Mp 209−211 °C; IR 3275 broad, 1660, 1606, 1542, 1466, 1314 cm−1; 1H NMR (DMSO-d6) δ 11.05 (s, 1H), 8.28 (s, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.38 (s, 1H), 7.35 (s, 1H), 7.33 (t, J = 2.7 Hz, 1H), 7.23 (dd, J1 = 8.7 Hz, J2 = 2.0 Hz, 1H), 6.49 (broad s, 2H), 6.40 (m, 1H); 13C NMR (DMSO-d6) δ 143.5, 140.3, 132.6, 131.6, 129.0, 127.6, 125.8, 120.9, 116.2, 111.5, 111.3, 101.0. N-3-(1H-Indol-5-yl)-5-pyridin-4-ylpyrazine-2,3-diamine (1). A glass-lined 630 L reactor was charged with N,Ndimethylformamide (42.5 L), 4-pyridinylboronic acid (2.36 kg, 19.2 mmol, 1.5 equiv), and 2 (3.90 kg, 12.8 mol) under stirring. The reactor was flushed with nitrogen gas prior to addition of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride (Pd(dppf)Cl2) (0.42 kg, 0.57 mol, 4.4 mol %). Then a 2 M solution of potassium carbonate in water (22.3 kg, 44.6 mol) was added over a period of 20 min. After an additional flush with nitrogen, the reaction mixture was heated to 110−115 °C and kept at this temperature for 1.5 h, after which in-process control (HPLC) showed >98% conversion of the starting material. The reaction was quenched by addition of water (160 L) under vigorous stirring. The precipitated product was isolated on a filter and washed with water (2 × 25 L). Drying to constant weight at 40 °C under reduced pressure (18 h was required) gave the title compound (3.64 kg, 85%) as a red solid. Purification of 1. A glass-lined 100 L reactor was charged with a 2 M aqueous acetic acid solution (59.9 L) followed by 1 (3.64 kg, 12.1 mol) and Hyflo Super Cel (5 kg). The resulting slurry was stirred for 15 min and then filtered through a Hastelloy filter. The reactor and filter were rinsed with 2 M acetic acid (5 L) which was added to the filtrate. The solution was added to a glass-lined 600 L reactor charged with 2methyltetrahydrofuran (100 L). After heating at 41 °C for 20 min, aqueous potassium hydroxide (30%, 25 kg) was added slowly under vigorous agitation. After 15 min of stirring, the phases were allowed to separate, and the organic phase was
20% DMA did not cause any change, and this heterogeneous approach was abandoned. Instead, a number of scavengers containing thiol groups were examined (Table 2). In all cases, Table 2. Palladium Removal with Scavengersa
a
Conditions A: 1 (50 mg) was mixed with the scavenger (50 mg) in 2 M HOAc (10 mL), stirred for 1 h, and then filtered and analyzed. Conditions B: 1 (150 mg) was dissolved in DMF, and the scavenger (75 mg) was added, followed by stirring for 3 h. After filtration, water was added, and the precipitated product was analyzed.
a substantial reduction of palladium occurred, but it was not possible to reach the desired level of
