A Scalable Route to 5-Substituted 3-Isoxazolol Fibrinolysis Inhibitor

Article pubs.acs.org/OPRD

A Scalable Route to 5‑Substituted 3‑Isoxazolol Fibrinolysis Inhibitor AZD6564 Søren M. Andersen,† Martin Bollmark,‡ Robert Berg,‡ Christofer Fredriksson,§ Staffan Karlsson,∥ Catarina Liljeholm,§ and Henrik Sörensen*,∥ †

Biopharmaceutical API Support, Novo Nordisk A/S, Hagedornsvej 1, DK-2880 Gentofte, Denmark SP Process Development, Box 36, SE-151 21 Södertälje, Sweden § Pharmaceutical Development, Global Medicines Development and ∥Medicinal Chemistry, Cardiovascular & Metabolic Diseases iMed, AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden ‡

S Supporting Information *

ABSTRACT: A practical and chromatography-free multikilogram synthesis of a 3-isoxazolol containing antifibrinolytic agent, AZD6564, has been developed in eight steps and 7% overall yield starting from methyl 2-chloroisonicotinate. Highlights in the synthesis are a Negishi coupling and an enzymatic resolution of a racemic ester.

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INTRODUCTION Fibrinolysis is an important and carefully tuned mechanism whereby blood clots are dissolved or prevented from growing.1 The serine protease plasmin is mainly responsible for fibrinolysis. Plasmin is formed from its proenzyme plasminogen by the action of a variety of enzymes. Activation is partly mediated by protein−protein interactions between fibrin and plasminogen via lysine binding sites in plasminogen. Under certain conditions inhibition of fibrinolysis may be desirable, e.g., under excessive and long menstrual periods, in trauma situations, and for some types of surgery. Tranexamic acid (TXA) and 6-aminohexanoic acid (ε-aminocaproic acid, EACA, Figure 1) are used clinically for inhibiting the activation of

chromatographic purifications and a late stage enantiomer separation by chiral chromatography makes the route unattractive for further scale-up. To support preclinical and early clinical studies, a synthetic route capable of delivering multiple kilograms of 1 was needed. Synthesis of 3-isoxazolols has been studied thoroughly by others,5 and in spite of a moderate yield for the formation of such systems we did not believe that this part of the synthesis could be radically improved. Instead we decided to invest time to develop stereoselective methods to obtain precursor 6 to avoid the later chromatographic isomer separations. We also directed our efforts to find a more efficient approach to introduce the neopentyl side chain.

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RETROSYNTHESIS Working towards our target, 1, we thus reasoned that the generation of the 3-isoxazolol group should originate from the carboxylic acid intermediate (2R,4S)-5 via the previously established routes which had also been shown viable in the medicinal chemistry route (Figure 2).4 We hoped to be able to resolve either intermediate (±)-5 or a derivative thereof. Compound (2R,4S)-5 was imagined to originate from pyridine 10 by catalytic hydrogenation followed by N-protection. Hydrogenation of 10 was expected to result in a cisconfiguration between the alkyl and ester group.6 We believed intermediate 10 should be accessible via Negishi-coupling7 or iron-mediated coupling8 of 9 and an organometallic neopentyl reagent.

Figure 1. Plasmin inhibitors EACA, TXA, and the current target 1.

plasminogen by reversibly blocking specific lysine binding sites on plasminogen. Their potency is low, and the pharmacokinetics are unfavorable leading to high and frequent dosing (typically 1.5 g of TXA, 3−4 times/day). Treatments with both TXA and EACA are associated with side effects. AZD6564 (1)2 was identified as a novel oral fibrinolysis inhibitor with potent activity in an in vitro human clot lysis assay and good DMPK properties for oral dosing.3,4 In the first generation medicinal chemistry synthesis (Scheme 1), AZD6564 (1) was originally prepared in nine synthetic transformations starting from 4-methoxypyridine. This route suffers from a low overall yield of less than 0.3%, and the Van Leusen reaction between TosMIC and (±)-3 gives a poor 1:8 ratio of the desired cis-isomer to trans. In addition, two © XXXX American Chemical Society

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RESULTS AND DISCUSSION First Scale-up Campaign, GLP Campaign. For our initial studies 300 g of 1 was required. The synthesis started with a C−C coupling between methyl 2-chloroisonicotinate (9) and neopentyl metal halide. Two different couplings were tested: Received: June 14, 2014

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dx.doi.org/10.1021/op500193s | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Scheme 1. Medicinal chemistry route to AZD6564 (1)

Figure 2. Retrosynthesis of 3-isoxazolol 1.

5% Fe(acac)3 in a THF−NMP solution, 9 was alkylated using a slight excess of neopentylmagnesium chloride at 0 °C. The workup was troublesome as substantial precipitation of iron hydroxides occurred. The difficulty was circumvented by washing the organic phase with aqueous citric acid and EDTA during workup. Compound 10 was then isolated in 73% yield as its HCl salt. Hydrogenation of 10 HCl salt using platinum(IV) oxide as a catalyst in methanol at 20 °C gave predominantly the cis-(±)-11 piperidine (11:1 ratio of cis/trans piperidine 5).11−14 Crystallization from methanol and MTBE improved the diastereomeric ratio to 340:1, and cis-(±)-11 was isolated in 82% yield (see Scheme 2). We found that cis-(±)-11 was easily resolved by salt formation with phosphoric acid (+)-12-H (Scheme 3). On a

(1) palladium-catalyzed Negishi coupling of 9 with neopentylzinc bromide and (2) iron(III) catalyzed crosscoupling8,9 between neopentylmagnesium chloride and 9. Initially we developed the Negishi coupling using 1% bis(tritert-butylphosphine)palladium10 as a catalyst and ester 9 and neopentylzinc bromide as reactants. On a 100 g scale the reaction was fast, and complete conversion was achieved in less than 1 h at 60 °C. After extractive workup, crude methyl 2neopentylisonicotinate (10) was isolated as an oil. Ester 10 was converted into the corresponding HCl salt and then purified through crystallization from MTBE to give 10 as a HCl salt in 61% yield. Iron-catalyzed cross-coupling between methyl 2chloroisonicotinate (9) and neopentylmagnesium chloride was investigated in parallel and scaled up to a 2 kg scale. By using B



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Scheme 2. Synthesis of cis-(±)-11

the need for hydrogenation.16 Using 1.1 equiv of methyl chloroformate, piperidine (2R,4S)-11 was protected in 10 volumes of 2-MeTHF at 0 °C in the presence of 1.1 equiv of DIPEA affording 13 in 99% assay yield (Scheme 4). In the subsequent step, when using NaOH in methanol or LiOH in THF, hydrolysis of 13 to (2R,4S)-5 resulted in epimerization to give 5% (2R,4R)-5. The best result was obtained using a mild Et3N/LiBr hydrolysis which reduced the epimerization to 1% (Scheme 4).17 After workup (2R,4S)-1-(methoxycarbonyl)-2neopentylpiperidine-4-carboxylic acid (5) was isolated in 99% yield over two steps on a 1 kg scale. CDI coupling between (2R,4S)-5 and magnesium ethyl malonate was based on previously published procedures18 but using 2-MeTHF as solvent. On a 760 g scale (2R,4S)-5 was activated in 2-MeTHF at 20 °C by adding CDI in portions until complete conversion into the corresponding acyl imidazole was obtained (confirmed by 1H NMR).19,20 Excess of the preformed magnesium ethyl malonate complex was then added to the acyl imidazole of (2R,4S)-5 and stirred until completion of the reaction (5 days at 20 °C). After workup, βketo ester 6 was isolated as a crude mixture. Some epimerization had taken place, and the amount of trans-isomer had now increased from 1% in (2R,4S)-5 to 6% in 6. Next, for the conversion of 6 to 8 known literature procedures were followed.5 Hence we treated 6 with one equivalent of sodium hydroxide at −40 °C and subsequently added one equivalent of hydroxylamine, to give the corresponding hydroxamate 14 Na salt (Scheme 4).21 Rapid addition of the intermediate hydroxamate 14 Na salt to 6 M hydrochloric acid at 60 °C gave the cyclized product (2R,4S)-8 along with byproduct 5isoxazolone 7. Typically a 7:3 ratio of 3-isoxazolol (2R,4S)-8 to byproduct 5-oxazolone 7 was obtained. After workup and crystallization from MeOH/water (2R,4S)-8 was isolated in 65% yield. The 3-isoxazolol (2R,4S)-8 was then deprotected on a 0.5 kg scale using 33% HBr in AcOH (3 equiv) at 50 °C.22 Removal of the HBr/acetic acid in vacuo, eventually with the aid of water followed by neutralization with 25% aqueous ammonia, resulted in precipitation of zwitterionic product 1. After a slurry wash with 50% aqueous iPrOH and finally water, 1 was obtained as the monohydrate in 73% yield Second Scale-up Campaign, GMP Campaign. To support phase I studies, multiple kilograms of 1 were needed.

Scheme 3. Resolution of cis-(±)-11

200 g scale, cis-(±)-11 was thus treated with 0.5 equiv of (+)-12-H in a mixture of methanol and ethyl acetate (2.1 L, 1:2 ratio). This afforded the corresponding phosphate salt with (2R,4S)-11 in 35% yield. An ee of 97% was measured on the free base of 11 after extraction. As an alternative to the above successful resolution, we tested the enzymatic resolution of cis(±)-11 using lipase from Candida antarctica (Novozyme 435) and found that predominantly the undesired (2S,4R)-11 (Scheme 3) was hydrolyzed. After removal of the immobilized enzyme, unreacted (2R,4S)-11 could be extracted with MTBE and isolated in 34% yield and 97% ee.15 Since acid (+)-12-H was expensive and the enzymatic resolution was practical to run on a large scale, the chiral salt resolution was abandoned. With the correct stereochemistry in place, methyl carbamate was chosen as a robust protecting group that would withstand the downstream chemistry and that could be removed without Scheme 4. First scale-up, the GLP route

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Scheme 5. GMP campaign

After the GLP campaign it was found that PEPPSI-IPr23 was an even better catalyst for the Negishi coupling between neopentylzinc bromide and 9. The yields were comparable to the Fe(acac)3 coupling used in the GLP campaign, but the reaction profile was cleaner with less byproducts being formed, and therefore the Negishi cross-coupling was selected for the GMP campaign. Reaction of 10 kg of methyl 2-chloroisonicotinate (9) and 1.05 equiv of neopentylzinc bromide in the presence of 0.5% mol of PEPPSI-IPr catalyst afforded methyl 2neopentylisonicotinate hydrochloride (10) in 66% yield after crystallization from HCl/isopropanol/MTBE (Scheme 5). The coupling reaction was exothermic (166 kJ/mol, max adiabatic temperature rise = 58 K), and the reaction mixture was kept below 40 °C during the entire operation. After workup, hydrogenation of 10 HCl salt using the GLP procedure yielded cis-(±)-11 in 93% yield and with dr >99:1. Next, enzymatic resolution using immobilized lipase from Candida antarctica (Immozyme CALB-T3-150) gave (2R,4S)-11. By using 1.57 equiv of dipotassium phosphate and an initial pH of 8, pH adjustment during the course of the reaction was unnecessary. After extractive workup the methyl ester (2R,4S)-11 was directly N-protected with methyl chloroformate in the presence of DIPEA and subsequently hydrolyzed using Et3N/LiBr in a two-step telescoped sequence. The carboxylic acid (2R,4S)-5 was crystallized from 2-MeTHF−heptane in 31% overall yield over three steps.

Like in the GLP campaign, the magnesium salt of ethyl malonate was prepared by refluxing magnesium chloride and potassium ethyl malonate in 2-MeTHF. For the CDI activation of (2R,4S)-5 the order of addition of CDI was reversed. Thus, instead of successively adding CDI to a solution of (2R,4S)-5, the latter was added as a solution to 1.2 equiv of CDI in 2MeTHF, resulting in a more practical operation.17,24 After reacting the resulting acyl imidazole with the magnesium ethyl malonate complex followed by extractions and concentration, 6 was isolated in 77% yield. The rate of reaction was slow, and 97% conversion into 6 was obtained after two and a half days at 25 °C and 2 days at 35 °C. The prolonged reaction time could be due to the poor solubility of the magnesium salt of ethyl malonate in 2-MeTHF combined with the high dilution (20 volumes of solvent). After the scale-up campaigns were completed, this hypothesis was tested by carrying out the formation of 6 in THF, in which the magnesium ethyl malonate complex has better solubility. With THF it was possible to carry out the reaction in less solvent (12 volumes) and a radical change in reaction time was observed. Completeness of reaction was seen in less than 4 h at 25 °C (on a 10 g scale), and after extractive workup a 76% yield of 6 was obtained. This yield was comparable to our large scale synthesis using 2-MeTHF in which a 77% yield was obtained. We next turned our attention to the formation of 14 (Scheme 5) which in the GLP procedure was performed by treating 6 with sodium D



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hydroxide and hydroxylamine at −30 °C in methanol. In one of our test reactions on medium scale, the Na salt of 6 precipitated, resulting in a failed reaction. We tried to circumvent precipitation by using Et3N as a base instead. To our delight this resulted in a soluble reaction mixture, and we developed a robust procedure which could be carried out at −10 °C.25 In the current campaign we also reversed the order of addition of hydroxylamine by adding the methanolic solution of (2R,4S)-6 and Et3N to a methanolic solution of hydroxylamine at −10 °C, since it was noticed that the order of addition did not affect the outcome. After complete formation of the hydroxamate 14 triethylammonium salt as a solution, the reaction mixture was rapidly added to 12 M hydrochloric acid at 70 °C to give the crude 3-isoxazolol product (2R,4S)-8. After extractive workup followed by crystallization 3-isoxazolol (2R,4S)-8 was obtained in 55% yield from 6. The methoxycarbonyl protective group was removed using HBr/ AcOH at 30 °C in the same fashion as the GLP procedure. Finally, slurrying the product in water afforded the monohydrate of 1 in 89% yield on a 3 kg scale.

methyl ether (23 L) was added, and the solution was cleared by polish filtration followed by rinse of the filter with more tertbutyl methyl ether (11.5 L). To the solution was added 5 M HCl/iPrOH (2.7 L, 13.5 mol), resulting in precipitation of the HCl salt of 10. The suspension was cooled to 10 °C, and after 1 h, the solids were filtered, washed with tert-butyl methyl ether (11.5 L) and dried under reduced pressure at 40 °C to give 2.4 kg (73%) of the product 10 as a white crystalline solid. 1H NMR (400 MHz, CD3OD) δ 8.94 (d, 1H), 8.34−8.47 (m, 2H), 4.06 (s, 3H), 3.07 (s, 2H), 1.05 (s, 9H). 13C NMR (101 MHz, MeOD) δ 164.12, 158.22, 146.62, 143.90, 129.56, 125.50, 54.35, 47.91, 34.12, 29.36. HRMS (ESI): [M + H]+ m/z Calcd for C12H18NO2 208.1338; Found 208.1328. Methyl 2-Neopentylisonicotinate (10), Hydrochloride Salt by Negishi Coupling. A reactor was charged with methyl 2-chloroisonicotinate (9) (9.8 kg, 57.1 mol) and MTBE (30 L). PEPPSI-IPr (199 g, 0.292 mol) was added, and 5 min later, 1 M neopentylzinc bromide in THF (60 L, 60 mol) was added over 2.5 h (exothermic by 166 kJ/mol, max adiabatic temperature rise = 58 K), keeping the reaction temperature below 40 °C. The reaction mixture was agitated for 12 h at 25 °C and quenched with a solution of citric acid (2.8 kg, 15 mol) in water (30 L, slightly exothermic by 30 kJ/mol, max adiabatic temperature rise = 5.8 K). The reaction mixture was polishfiltered and the filter rinsed with MTBE (15 L). The phases were separated and the organic phase washed with an aqueous solution of EDTA tetrasodium hydrate (12.75 kg in 30 L water) and subsequently with water (30 L). The organic phase was concentrated under reduced pressure to approximately 20 L. Methanol (30 L) was added, and the organic phase was concentrated under reduced pressure to a volume of 20 L. The temperature was lowered to 5 °C, and 5 M HCl in isopropanol (10.5 L, 52.5 mol) was added over 45 min. Upon addition of MTBE (70 L) the product precipitated, and after 2.5 h the solids were filtered off and washed with MTBE (5 °C, 40 L) to yield 9.5 kg (97.0 wt %, 66% yield) of the product 10 as a white crystalline solid. The analytical data were identical to the data in the previous procedure. cis-Methyl 2-Neopentylpiperidine-4-carboxylate Hydrochloride (11). Caution: No precautions are necessary with platinum(IV) oxide,26 but after exposure to H2, platinum black is formed, which is is very pyrophoric. A reactor was charged with methyl 2-neopentylisonicotinate (10) hydrochloride salt (14.8 kg, 96 wt %, 58.3 mol), platinum(IV) oxide monohydrate (107.2 g, 0.437 mol), and methanol (44.4 L). The temperature was increased to 40 °C, and the reaction mixture was stirred under an atmosphere of hydrogen (pressure 20 bar). After 10 h, when the hydrogen consumption had ceased, the mixture was filtered, and the filtrate was concentrated under reduced pressure to approximately 30 L. The temperature was adjusted to 40 °C, and upon addition of MTBE (30 L) the product started precipitating. More MTBE (89 L) was added over a period of 1.5 h. The temperature was lowered to 0 °C over 3 h and stirred for 14 h. An IPC of the supernatant showed a cis/trans ratio of 3:7. The slurry was filtered and washed with MTBE (31 L) to afford 13.86 kg of cis-(±)-11 (97.4 wt %, 92.7% yield) as a white crystalline solid. 1H NMR (400 MHz, CD3OD) δ 3.70 (s, 3H), 3.43 (ddd, J = 12.9, 4.3, 2.2 Hz, 1H), 3.23−3.35 (m, 3H), 3.12 (td, J = 13.3, 13.2, 3.2 Hz, 1H), 2.79 (tt, J = 12.4, 12.4, 3.7, 3.7 Hz, 1H), 2.29−2.39 (m, 1H), 2.13−2.23 (m, 1H), 1.75 (ddd, J = 26.7, 13.8, 4.3 Hz, 1H), 1.48−1.65 (m, 3H), 1.01 (s, 9H).

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CONCLUSION A chromatography-free scalable eight step route to fibrinolysis inhibitor 1 has been developed in 7% overall yield starting from commercially available methyl 2-chloroisonicotinate (9). Highlights include use of two different cross couplings for attachment of the neopentyl side chain to 9: An iron catalyzed coupling using neopentylmagnesium chloride and a Negishi coupling using neopentylzinc bromide, of which the latter was chosen for the GMP campaign. Also, resolution of cis-(±)-11 was demonstrated using both chemical and enzymatic methods. Ultimately, 3 kg of 1 was synthesized.

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EXPERIMENTAL SECTION

Commercially available solvents and reagents were used without purification. All reactions and operations were conducted under a nitrogen atmosphere. Lipase was purchased from Chiralvision. LC analyses were performed using a Waters 600 instrument, C8 kromasil 100 column (50 × 4.6 mm, df = 5 μm), mobile phase 25 mM H3PO4 buffer/acetonitrile, monitoring UV at 220 nm. Enantiomeric excess was determined by chiral HPLC using a Waters 2695 Alliance Separation Module equipped with a 2996 PDA detector Chiralpak IC or Chirobiotic V2 columns (250 × 4.6 mm), and mobile phase hexane/i-PrOH/TFA. HRMS analyses were performed using a Micromass LCT mass spectrometer. DSC was measured on Metter Toledo 843 at 30−450 °C (30−350 °C for the reaction mixtures) at 5 °C/min in 40 μL gold plated capsules. Benzylbenzoate, 2,3,5,6-tetrachloronitrobenzene, or maleic acid were used for NMR assays. Methyl 2-Neopentylisonicotinate (10), Hydrochloride Salt by Fe(acac)3 Catalyzed Coupling. A reactor was charged with methyl 2-chloroisonicotinate (9) (2.3 kg, 13.4 mol), Fe(acac)3 (0.25 kg, 0.67 mol), THF (35 L), and NMP (3.5 L). The mixture was cooled to 0 °C, and a 1 M solution of neopentylmagnesium chloride in diethyl ether (18 L, 18 mol) was added during 4 h (EXOTHERMIC), keeping the reaction temperature around +5 °C. 10% aqueous citric acid (11.5 L) was added (EXOTHERMIC), and the aqueous phase was discarded. The organic phase was washed with 20% EDTA tetrasodium salt (11.5 L) and water (11.5 L) and then concentrated under reduced pressure at 40 °C to 5 L. tert-Butyl E



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(90% conversion) and 35 °C for 48 h (97% conversion). The reaction mixture was cooled to 20 °C, and 2 M aqueous HCl (31 L) was added. The organic phase was washed twice with 8% aqueous sodium bicarbonate (19.5 and 16 L) and concentrated under reduced pressure to 35 L, polish filtered to remove salts, and concentrated to 10 L. MeOH (19.5 L) was added and the solution of product concentrated to dryness. The residue was taken up in MeOH (23.5 L) to give a total volume of 27.5 L. An NMR assay showed 139 mg product 6/mL indicating a yield of 3.82 kg (77%). The mixture was used directly in the next step. 1 H NMR (400 MHz, CDCl3) δ 4.26 (m, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.78−3.96 (m, 1H), 3.68 (s, 3H), 3.51 (dd, J = 15.8 Hz, 2H), 3.10 (ddd, J = 14.2, 11.3, 4.7 Hz, 1H), 2.62−2.75 (p, J = 6.2 Hz, 1H), 2.11 (m, 1H), 1.84−1.97 (m, 1H), 1.65−1.82 (m, 2H), 1.56 (dd, J = 14.2, 7.4 Hz, 1H), 1.27 (t, J = 7.1 Hz, 3H), 1.17 (m, 1H), 0.89 (s, 9H). 13 C NMR (151 MHz, MeOD) δ 207.68, 169.23, 157.71, 62.24, 53.15, 50.68, 47.03, 44.96, 37.71, 32.65, 31.42, 30.18, 30.03, 25.77, 14.45. HRMS (ESI): [M + H]+ m/z Calcd for C17H30NO5 328.2124; Found 328.2116. (2R,4S)-Methyl 2-neopentyl-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1-carboxylate ((2R,4S)-8). A reactor was charged with MeOH (15 L) and hydroxylamine (50% in water, 1.85 kg, 28.0 mol) and cooled to −10 °C. In another reactor a solution of (2R,4S)-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-neopentylpiperidine-1-carboxylate (6)) (7.65 kg, 23.4 mol) in MeOH (to a total volume of 55 L) was cooled to −5 °C and Et3N (6.5 L, 46.6 mol) added. The mixture of βketoester 6 and Et3N in MeOH was added to the solution of hydroxylamine over 1.5 h and agitated at −10 °C for 5 h. The reaction mixture was added to 36.5% aqueous HCl (39 L, 429 mol) at 70 °C with MeOH (4 L) as line wash. After 0.5 h the temperature was lowered to 25 °C and water (38 L) added. 50% Aqueous NaOH was slowly added (EXOTHERMIC) until pH 10−11 (33.2 kg, 415 mol), keeping the temperature below 35 °C. MTBE (71 L) was added, and the organic phase was extracted once with water (24 L). To the combined aqueous phases was added MTBE (64 L) and then 36.5% aqueous HCl until pH was below 3. After separation the aqueous phase was extracted once more with MTBE (61 L), and the combined organic extracts were concentrated under reduced pressure until a semisolid was formed. The crude product was dried by distilling off MTBE (2 × 62 L). MTBE (62 L) was added, and the mixture was concentrated to 30 L. The temperature was set to 40 °C and after 2 h lowered to −5 °C over 6 h. The solids were washed with cold MTBE (15 L) and dried under reduced pressure at 40 °C to give 3.99 kg of (2R,4S)-8 (95.2 wt %, 55% yield) as a white crystalline solid with an ee of 99.9%. 1 H NMR (400 MHz, CDCl3) δ 5.71 (s, 1H), 4.18−4.34 (m, 1H), 3.87−4 (m, 1H), 3.71 (s, 3H), 3.08−3.24 (m, 1H), 2.91− 3.04 (m, 1H), 1.96−2.19 (m, 2H), 1.77−1.93 (m, 2H), 1.45 (dd, J = 14.3, 7.0 Hz, 1H), 1.17 (dd, J = 14.3, 5.6 Hz, 1H), 0.87 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 176.45, 171.04, 155.96, 92.59, 52.52, 49.28, 46.48, 36.45, 33.39, 30.37, 30.14, 29.63, 27.01. HRMS (ESI): [M + H]+ m/z Calcd for C15H25N2O4 297.1814; Found 297.1817. 5-((2R,4S)-2-Neopentylpiperidin-4-yl)isoxazol-3-ol (1) Monohydrate. A reactor connected to a scrubber (containing 1 L of ethylenediamine, 1.5 kg of sodium thiosulfate, 4 L of 50% aqueous NaOH, and 20 L of water) was charged with

C NMR (151 MHz, MeOD) δ 174.91, 54.73, 52.60, 48.84, 45.05, 40.15, 34.81, 31.19, 29.99, 25.83. HRMS (ESI): [M + H]+ m/z Calcd for C12H24NO2 214.1807; Found 214.1799. (2R,4S)-1-(Methoxycarbonyl)-2-neopentylpiperidine4-carboxylic Acid ((2R,4S)-5). A reactor was charged with cismethyl 2-neopentylpiperidine-4-carboxylate hydrochloride (cis(±)-11) (13.1 kg, 95.7% wt, 50.2 mol), dipotassium phosphate (13.7 kg, 78.66 mol), and water (80 L) and agitated until a solution was obtained, pH 8. Immobilized lipase from Candida Antartica (Immozyme CALB-T3-150, 3.95 kg) was added, and the reaction mixture was stirred at 35 °C for 40 h (91% conversion after 16 h, 96% conversion after 24 h, 99% conversion after 40 h). The mixture was cooled to 5 °C and 2MeTHF (39.5 L), and aqueous potassium hydroxide (4.33 kg 85 wt % potassium hydroxide in 13.10 L water) was added. The mixture was filtered, and the filter was washed with 2-MeTHF (35 L) followed by separation of the phases.27 To the organic phase containing the (2R,4S)-methyl 2-neopentylpiperidine-4carboxylate (2R,4S-11) was added diisopropylethylamine (9.5 L, 54.5 mol) and more 2-MeTHF (43 L). At 5 °C, methyl chloroformate (2.05 L, 26.5 mol) was added over 1.5 h, keeping the temperature below 10 °C. After a reaction time of 16 h, water (52 L) was added to the reactor, and the temperature increased to 25 °C. After separation of the phases the aqueous phase was discarded. To the organic phase containing 13 was added Et3N (11 L, 78.9 mol) and lithium bromide (11.4 kg, 131 mol). The temperature was increased to 85 °C and the reaction mixture was agitated for 41 h (more than 95% conversion) and then cooled to 25 °C. Water (39 L) followed by concentrated aqueous HCl (12 L) were added to obtain pH 1. The organic phase was washed with water (26 L) and concentrated under reduced pressure to approximately 10 L. More 2-MeTHF (10 L) was added and the temperature was increased to 50 °C. Heptane (55 L) was added, and the temperature was lowered to 40 °C. Once crystallization had started, the temperature was lowered to 0 °C over 12 h. More heptane (13 L) was added, and the solids were filtered off, washed with cold heptane (26 L), and dried under reduced pressure at 40 °C to give 4.02 kg of (2R,4S)-5 (98.9 wt %, 30.8% yield) as a white crystalline solid. 1H NMR (400 MHz, CDCl3) δ 4.28−4.39 (m, 1H), 3.84−3.96 (m, 1H), 3.70 (s, 3H), 3.09−3.23 (m, 1H), 2.63−2.70 (m, 1H), 1.91−2.11 (m, 3H), 1.72 (tt, J = 12.1, 12.1, 5.8, 5.8 Hz, 1H), 1.61 (dd, J = 14.3, 7.9 Hz, 1H), 1.31 (dd, J = 14.3, 4.9 Hz, 1H), 0.90 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 181.02, 155.98, 52.69, 48.84, 45.42, 36.47, 35.66, 32.33, 30.73, 29.76, 25.55. HRMS (ESI): [M + H]+ m/z Calcd for C13H24NO4 258.1705; Found 258.1710. (2R,4S)-Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-neopentylpiperidine-1-carboxylate (6). A reactor was charged with ethyl potassium malonate (7.9 kg, 98 wt %, 45.5 mol), magnesium dichloride (4.4 kg, 97.5 wt %, 45.1 mol), and 2MeTHF (39 L). The reaction mixture was refluxed for 6 h, and then the temperature was lowered to 20 °C. Another reactor was charged with 1,1′-carbonyldiimidazole (3 kg, 98 wt %, 18.1 mol) and 2-MeTHF (13 L). A solution of (2R,4S)-1-(methoxycarbonyl)-2-neopentylpiperidine-4-carboxylic acid ((2R,4S)-5) (3.9 kg, 15.2 mol) in 2-MeTHF (32 L) was added to the CDI solution (effervescent) over 30 min, and the mixture was stirred at 25 °C for 2.5 h. The resulting mixture of CDI activated acid (2R,4S)-5 was added to the ethyl malonate mixture during 0.5 h and agitated at 25 °C for 61 h 13

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(5) (a) Sørensen, U. S.; Krogsgaard-Larsen, P. Org. Prep. Proced. Int. 2001, 33, 515−564. and references cited therein. (b) Katritzky, A. R.; Barczynski, P.; Ostercamp, D. L.; Yousaf, T. I. J. Org. Chem. 1986, 51, 4037−4042. (c) Jacobsen, N.; Kolind-Andersen, H.; Christensen, J. J. Can. J. Chem. 1984, 62, 1940−1944. (6) Whelligan, D. K.; Solanki, S.; Taylor, D.; Thomson, D. W.; Cheung, K.-M. J.; Boxall, K.; Mas-Droux, C.; Barillari, C.; Burns, S.; Grummitt, C. G.; Collins, I.; van Montfort, R. L. M.; Aherne, G. W.; Bayliss, R.; Hoelder, S. J. Med. Chem. 2010, 53, 7682−7698. (7) Elan Pharmaceuticals Inc. Method of Treatment of Amyloidosis using Aspartyl-Protease Inhibitors. WO2005070407, Aug 4, 2005, p 150. (8) Fürstner, A.; Leitner, A.; Méndez, M.; Krause, H. J. Am. Chem. Soc. 2002, 124, 13856−13863. (9) Whelligan, D. K.; Solanki, S.; Taylor, D.; Thomson, D. W.; Cheung, K.-M. J.; Boxall, K.; Mas-Droux, C.; Barillari, C.; Burns, S.; Grummit, C. G.; Collins, I.; van Montfort, R. L. M.; Aherne, G. W.; Bayliss, R.; Hoelder, S. J. Med. Chem. 2010, 53, 7682−7698. (10) Dai, C.; Fu, G. C. J. Am. Chem. Soc. 2001, 123, 2719−2724. (11) For asymmetric hydrogenations of pyridines see for example (a) Wang, D.-S.; Chen, Q.-A.; Lu, S.-M.; Zhou, Y.-G. Chem. Rev. 2012, 112, 2557−2590. (b) Glorius, F.; Spielkamp, N.; Holle, S.; Goddard, R.; Lehmann, C. W. Angew. Chem., Int. Ed. 2004, 43, 2850−2852. (c) Rueping, M.; Antonchick, A. P. Angew. Chem., Int. Ed. 2007, 46, 4562−4565. (12) The cis/trans ratio is determined by integration of the methyl signals appearing at 3.70 and 3.75 ppm in CD3OD, respectively. (13) In spite of precautions we did experience an incident with platinum black (which is formed during exposure of PtO2 to H2) in combination with the methanolic reaction media. (14) Hydrogenation in water gave a less favorable cis/trans selectivity (4:1). (15) The GLP enzymatic hydrolysis was performed on 13.8 mol scale at 20 °C using Novozyme 435 (155 g, >5000 U/g) in 0.66 M potassium dihydrogen phosphate buffer (22 L). The pH was kept between 6.7 and 7.7 by portionwise addition of 3.8 M sodium hydroxide solution until the reaction was completed. After extractive workup 4.7 mol (2R,4S)-11 was isolated. (16) 5-Substituted 3-isoxazololes are easily cleaved under hydrogenolytic conditions: Oster, T. A.; Harris, T. M. J. Org. Chem. 1983, 48, 4307−4311. (17) Mattsson, S.; Dahlström, M.; Karlsson, S. Tetrahedron Lett. 2007, 48, 2497−2499. (18) Clay, R. J.; Collom, T. A.; Karrick, G. L.; Wemple, J. Synthesis 1993, 290−292. (19) In CDCl3, the disappearance of the multiplet in 8 observable at 2.63−2.70 ppm was used as an indication for complete formation of the intermediate imidazolylcarbonyl activated 8. See also ref 22. (20) Engstrom, K. M.; Sheikh, A.; Ho, R.; Miller, R. W. Org. Process Res. Dev. 2014, 18, 488−494. (21) It was found that excess hydroxylamine, prolonged reaction time, and higher temperatures resulted in more byproduct 7 being formed in the subsequent cyclization step. Furthermore, reversing the order of addition during the acidification was also detrimental with more byproduct being formed. (22) Some N-methylation of the 3-isoxazolol had taken place. This byproduct disappeared downstream. (23) Hadei, N.; Kantchev, E. A. B.; O’Brien, C. J.; Organ, M. G. Org. Lett. 2005, 7, 3805. (24) In the GLP campaign 2−3 equivalents of CDI was used. The CDI was used as supplied. The quality of the commercial CDI has been shown to vary (ref 20), and we are uncertain of the assay of the CDI used in our GLP campaign. Whether poor quality is the cause of the high consumption of CDI in our first campaign is consequently uncertain. (25) Some epimerization to give (2R,4R)-8 was observed during the HCl quench. Lowering the temperature of the HCl quench and using less concentrated HCl did reduce the amount of (2R,4R)-8, but also increased the amount of byproduct 5-isoxazolone 7 (see Scheme 1).

(2R,4S)-methyl 2-neopentyl-4-(3-oxo-2,3-dihydroisoxazol-5yl)piperidine-1-carboxylate (2R,4S)-8) (3.98 kg, 95.2 wt %, 12.8 mol) and 33% hydrogen bromide in acetic acid (12.3 L, 16.97 kg, 69.2 mol). The reaction mixture was agitated at 30 °C for 16 h and then concentrated under reduced pressure to approximately 8 L. Water (38 L) was added followed by concentration under reduced pressure to 6 L, addition of more water (39 L), and concentration under reduced pressure to 12 L. Isopropanol (12 L) was added, and the solution was polish filtered together with 50% aqueous isopropanol (2 L) as line wash. The resulting solution was neutralized to pH 8.0 with 10% ammonium hydroxide (approximately 5 L). The temperature was lowered to 2 °C, and after 1.5 h, the solids were filtered off and washed with cold 50% aqueous isopropanol (2 × 7.5 L). The crude product was slurried in cold water (12 L) to form the monohydrate. After 20 h the solids were isolated by filtration and dried under reduced pressure to give 3.04 kg 1 monohydrate (89.8 wt % 1, 10.2% water, 89% yield) as a white crystalline solid with an ee of 99.9%. 1 H NMR (600 MHz, CD3OD) δ 5.44 (s, 1H), 3.34−3.42 (m, 1H), 3.26−3.31 (m, 1H), 3.14−3.23 (m, 1H), 3.07 (dt, J = 13.1, 3.2 Hz, 1H), 2.94 (m, 1H), 2.19−2.28 (d, J = 14.2 Hz, 1H), 2.02−2.12 (d, J = 14.2 Hz, 1H), 1.70 (qd, J = 13.6, 12.3, 4.2 Hz, 1H), 1.45−1.59 (m, 3H), 0.97 (s, 9H). 13 C NMR (101 MHz, MeOD) δ 177.39, 174.72, 95.42, 54.83, 49.32, 45.50, 37.13, 34.75, 31.19, 30.07, 28.06. HRMS (ESI): [M + H]+ m/z Calcd for C13H23N2O2 239.1760; Found 239.1764.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR, 13C NMR, HRMS, and HPLC analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank our lead optimization group for valuable discussions, our NMR specialist group for help with structure elucidation, our separation science laboratory for chiral analyses, and our Structure Analysis group for HRMS and HPLC purity analyses. We also thank Dr Alleyn Plowright for linguistic advice on this manuscript.

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REFERENCES

(1) Cesarman-Maus, G.; Hajjar, K. A. Br. J. Hamaetol. 2005, 129, 307−321. (2) (a) Structure 1 may also be drawn as its tautomeric 3isoxazolinone. The present structure is drawn in accordance with spectrometric findings and calculations. See ref 2b and 2c. (b) Boulton, A. J.; Katritsky, A. R.; Majid Hamid, A.; Øksne, S. Tetrahedron 1964, 20, 2835−2840. (c) Woodcock, S.; Green, D. V. S.; Vincent, M. A.; Hillier, I. H.; Guest, M. F.; Sherwood, P. J. Chem. Soc., Perkin Trans. 2 1992, 12, 2151−2154. (3) Boström, J.; Grant, J. A.; Fjellström, O.; Thelin, A.; Gustafsson, D. J. Med. Chem. 2013, 56, 3273−3280. (4) Cheng, L.; Pettersen, D.; Ohlsson, B.; Schell, P.; Karle, M.; Evertsson, E.; Pahlén, S.; Jonforsen, M.; Plowright, A. T.; Boström, J.; Fex; Thelin, A.; Hilgendorf, C.; Xue, Y.; Wahlund, G.; Lindberg, W.; Larsson, L.-O.; Gustafsson, D. Med. Chem. Lett. 2014, 5, 538−543. G



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(26) Paquette, L. A. Encyclopedia of Reagents for Organic Synthesis; Wiley: Chichester (England), 1995. (27) 1H-NMR, 13C-NMR, ee determination, HPLC, and HRMS analysis of the material isolated at this stage in the GLP campaign is supplied in the Supporting Information (S48−S56).

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A Scalable Route to 5-Substituted 3-Isoxazolol Fibrinolysis Inhibitor

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