Synthesis of BACE Inhibitor LY2886721. Part I. An Asymmetric Nitrone

Article pubs.acs.org/OPRD

Synthesis of BACE Inhibitor LY2886721. Part I. An Asymmetric Nitrone Cycloaddition Strategy Stanley P. Kolis,*,† Marvin M. Hansen,† Enver Arslantas,‡ Lukas Bran̈ dli,‡ Jonas Buser,† Amy C. DeBaillie,† Andrea L. Frederick,† David W. Hoard,† Adrienne Hollister,† Dominique Huber,‡ Thomas Kull,‡ Ryan J. Linder,† Thomas J. Martin,‡ Rachel N. Richey,† Alfred Stutz,‡ Michael Waibel,‡ Jeffrey A. Ward,† and Alexandru Zamfir‡

Downloaded by NEW YORK MEDICAL COLLEGE on September 4, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/op500351q

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Small Molecule Design and Development, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States ‡ Dottikon Exclusive Synthesis AG, P.O. Box 5605, Dottikon, Switzerland ABSTRACT: A scalable, asymmetric synthesis of (3aS,6aS)-6a-(5-bromo-2-fluorophenyl)-1-((R)-1-phenylpropyl)tetrahydro1H,3H-furo[3,4-c]isoxazole, a key intermediate in the synthesis of LY2886721, is reported. Highlights of the synthesis include the development of an asymmetric [3 + 2] intramolecular cycloaddition facilitated by trifluoroethanol, and the development of a new synthesis of (R)-N-(1-phenylpropyl)hydroxylamine tosylate which proceeds through a p-anisaldehyde imine and avoids the formation of toxic hydrogen cyanide gas as a byproduct. The synthesis proceeds over four steps and provides the product in 36% overall yield.

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Y2886721 (1, Scheme 1)1 is a potent and selective inhibitor of beta-amyloid cleaving enzyme (BACE) and was in Phase 2 clinical trials as a potential treatment for Alzheimer’s disease. The synthetic route used by the Lilly discovery chemistry group was developed to provide the first-generation development scaleup route shown in Scheme 1. This route was suitable for providing the active pharmaceutical ingredient (API) to support toxicology studies and early clinical trials, but an improved synthetic route was required to provide larger amounts of API for clinical and product development. Key issues with the firstgeneration route included:

these two disclosures describe an efficient asymmetric synthesis of this complex API. Asymmetric intramolecular [3 + 2] cycloaddition reactions are well-established in the literature,4 but our substrate (ketone 3, Scheme 1) was not optimally constructed for such an approach. Typically, the asymmetric cycloaddition reaction is promoted by a chiral Lewis acid which complexes to an olefin bearing a carbonyl substituent. In the synthesis of 4, attaching a suitable activating functional group to the olefin in ketone 3 would require removal in a subsequent step. This approach was unattractive from an efficiency and waste generation perspective and was not pursued. As a result, we focused our efforts on the use of a chiral auxiliary for the asymmetric synthesis of compound 5 (Figure 1). Two strategies were pursued: Use of a phenylglycinol-derived hydroxylamine chiral auxiliary (Path a) or use of a chiral hydroxylamine derived from an unsubstituted chiral amine (Path b). In addition to development of the cycloaddition chemistry, both approaches necessitated the development of an efficient process to install the aryl group via addition of an aryl nucleophile to either an early- or late-stage intermediate. The use of a phenylglycinol chiral auxiliary to promote the intramolecular [3 + 2] cycloaddition shown in Path a has been disclosed in the literature.5 The cycloadduct 5′ (shown in Figure 1) was synthesized as a single diastereomer following this precedent,6 but it was not possible to open the aminal using the desired aryl anion. Minimal conversion was observed using PhLi and BF3·OEt2 at 50 °C, but ortho-fluoro anions gave no ring opening, presumably due to decomposition of the anion via a benzyne intermediate (vide infra). With the failure of retrosynthetic strategy a, we focused on retrosynthesis b. In

· Long synthesis and low overall yield (∼1%); · Moderate yield of the ketone in Step 4, despite significant efforts at optimization; · Use of a full equivalent of metallic zinc as a reducing agent; concerns with zinc waste disposal; · A “late” resolution in Step 8. While some transformations in the discovery synthesis were unattractive from the perspective of large-scale processing, a particularly attractive aspect of the synthesis was the manner in which the required cis stereochemistry was established via a [3 + 2] intramolecular dipolar cycloaddition between a nitrone and pendant olefin (Steps 5 and 6, Scheme 1).2 Building on this successful cycloaddition approach, our initial strategy was to develop an asymmetric variant of the intramolecular [3 + 2] cycloaddition to establish the absolute and relative stereochemistry. Described herein is the development of a new synthetic route to key intermediate 19 (Figure 1) that addressed the issues above, culminating in the use of an asymmetric nitrone cycloaddition for installation of the absolute and relative stereochemistry of LY2886721. Other issues with the later steps in the early development route are addressed in the companion paper immediately following in this issue.3 Together © XXXX American Chemical Society

Received: November 7, 2014

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DOI: 10.1021/op500351q Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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Organic Process Research & Development


Scheme 1. Synthesis of LY2886721a

a

Index of abbreviations used: TBAHS = tetra-n-butyl ammonium hydrogen sulfate; NMM = N-methyl morpholine; LDA = lithium diisopropyl amide; L-DPTTA = L-di-p-toluoyltartaric acid; BSTFA = N,O-bis(trimethylsilyl)trifluoroacetamide; BzNCS = benzoylisothiocyanate; DTAD = ditert-butyl-azodicarboxylate; EDCI·HCl = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; HOBt = hydroxybenzotriazole.

Figure 1. Retrosynthetic approaches to isoxazolidines 5 and 19.

option, but do note that a process based on this reagent could likely be developed. We also examined solid potassium carbonate as a base to replace Hünig’s base and were gratified to find that 2 equiv of potassium carbonate were sufficient for promoting the alkylation reaction at 60 °C, with a reaction profile similar to that reported by Patel. This allowed us to omit filtration of the byproduct ammonium salts and remove the excess potassium carbonate via aqueous extraction. The oxidation reaction to form compound 10 was typically complete within an hour after addition of the m-chloroperoxybenzoic acid (mCPBA). Good temperature control between 0 and 5 °C was crucial for the successful operation of this reaction, and this was achieved through control of the addition of mCPBA. On plant scale, this translated to an addition time of 10−11.5 h to hold the temperature in the desired range. As in Patel’s case, we

order to understand the development needs for the asymmetric nitrone cycloaddition to synthesize 19 (Table 2), we first discuss the development and scale up of the key synthetic intermediates 2, 3, and 6b. Development of the Synthesis of Chiral Hydroxylamine 6b. For the synthesis of the chiral hydroxylamine 6b, we chose to further develop the process of Patel and co-workers (Scheme 2, First Generation Route).7 We began with an examination of whether the expensive bromoacetonitrile (8a) could be replaced with chloroacetonitrile (8b). As expected, the SN2 alkylation reaction with the less reactive alkyl chloride was much slower than the alkyl bromide in ethyl acetate.8 We did find that the reaction proceeded faster using acetonitrile or dimethylformamide (DMF) as a solvent at 60 °C. Due to time constraints, we ceased development of the chloroacetonitrile B


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Scheme 2. Synthetic Routes to 6b

Development of an Alternative Synthesis of 6b. As noted above, the process developed by Patel for the synthesis of 6b exhibits the safety hazard of HCN generation. While this hazard can be mitigated on scale, we sought to develop a process that did not possess the limitation of toxic gas generation. The chemistry for the second generation route is shown in Scheme 2.9 The expensive and toxic bromoacetonitrile was replaced with panisaldehyde (11), and the imine 12 was readily synthesized through condensation with (R)-1-phenylpropyl amine (7) in nbutanol (n-BuOH) with distillative removal of water. The unisolated imine 12 was oxidized with peracetic acid in a biphasic n-BuOH/water mixture to produce oxaziridine 13 which was decomposed to the hydroxylamine free base using aqueous hydroxylamine. After a solvent swap to n-butyl acetate (nBuOAc), compound 6b was isolated in 86% overall yield. The process was executed on pilot scale to produce 2296 kg of 6b in four batches with ≥99.4% (HPLC) purity and nondetectable levels of the enantiomer. The two processes for preparing 6b are compared in Table 1. We judged the second generation route superior based on quantitative and qualitative comparisons along a number of process chemistry and engineering metrics. Development of the Synthesis of Weinreb Amide 2. The synthesis of Weinreb amide 2 (Scheme 3) is known and proceeds from either a haloacetic acid 10 or tert-butyl

found that sodium carbonate was the most efficient base for removing the residual mCPBA and m-chlorobenzoic acid byproduct, but loss of 10 to the aqueous phase was dependent on the concentration of the aqueous base solution used. When a 1 M solution of sodium carbonate was employed, roughly 5−6 wt % product 10 was lost to the aqueous layer, whereas product losses of 11−14 wt % were realized when a 2 M solution was used. We found that (regardless of base concentration) two extractions were required for the removal of the residual mCPBA and m-chlorobenzoic acid to acceptable (≤5 HPLC area %) levels in the organic phase. To minimize extraction product losses, the extraction solvent was changed from ethyl acetate to isopropyl acetate. Typically product losses were greatest during the first sodium carbonate wash, so a single back-extraction of the first aqueous phase was implemented. The organic phase containing 10 was then forward processed into the next step directly to avoid isolation of the nitrone intermediate. The hydrolysis of 10 and isolation of 6b followed Patel’s procedure. In the Patel publication Experimental Section, the procedure for this step warned that HCN would form. In order to probe whether or not HCN was actually being formed, an alkaline scrubber was attached to the laboratory reactors, and we conf irmed that approximately 4.8 g HCN/kg 6b was released during the reaction. Since care must be taken to avoid exposure of laboratory scientists and operators to HCN gas during the reaction, we introduced an in-process control (IPC) in the headspace of the Nutsche filter during large-scale processing to ensure that high levels of HCN were not accumulating in the dryer. The IPC would typically be taken after each wash of the product filter cake, and the specification was set as not more than 2 ppm of HCN in the headspace. It was found that the cake wash was necessary to ensure acceptable purity of the isolated product, and to ensure that any residual HCN present in the filter cake was removed from the product. The process sequence above was executed in three batches to produce 1227 kg of 6b, for an overall yield of ∼80% starting from commercially available (R)-1phenylpropyl amine (7). For all batches, enantiomeric purities of >99.9% were achieved, and chemical purities ranged from 98.4 to 98.6 HPLC area %.

Table 1. Criteria for comparison of alternate routes to 6b criteria overall yield (%) purity (%, HPLC area) processing time high priced raw materials hazardous reagents and byproducts extractions solvent exchanges dilution (L/kg product) C

second generation route

first generation route 80 98.4−98.6

86 99.4−99.6

96 h (R)-phenylpropylamine, bromoacetonitirile, mCPBA mCPBA, bromoacetonitrile, HCN 5 0 10

62 h (R)phenylpropylamine CH3CO3H, NH2OH 3 1 7


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Scheme 3. Synthesis of Weinreb amide 2 and ketone 3

bromoacetate11 as starting materials. For these routes, the ether bond was constructed first and then the Weinreb amide bond in a subsequent step. These procedures possessed liabilities from a large scale-processing perspective: the use of bromoacetic acid as a starting material typically required forcing conditions and the use of sodium hydride as a base for the SN2 displacement with allyl alcohol, which was undesirable for development of a safe process. Use of tert-butyl bromoacetate required a separate deprotection step of the tert-butyl ester, which was undesirable from both a throughput and environmental (Process Mass Intensity12) perspective. Therefore, we chose to operate in the opposite direction and form the Weinreb amide bond first, followed by construction of the ether bond in a subsequent step. The synthesis of the bromo Weinreb amide 15 employing bromoacetyl bromide 14 under biphasic conditions (Et2O/H2O) using potassium carbonate as an inorganic base is known, and this route was selected for further development (Scheme 3).13 For the purposes of scale-up, we wanted to replace diethyl ether with an alternative solvent. Toluene was an acceptable replacement, and full conversion was typically achieved for the alkylation reaction at the conclusion of the addition of the bromoacetyl bromide. The reaction was observed to be temperature-dependent; controlling the internal reaction temperature to ≤10 °C was necessary to prevent the formation of unidentified byproducts. Due to the apparent instability of the bromo Weinreb amide, the toluene solution of 15 was telescoped into the next step of the sequence. While the synthesis of 2 via reaction of the bromo Weinreb amide with allyl alcohol is not known, allyl alcohol is known to react with similarly substituted compounds under phase transfer conditions (n-Bu4NBr, benzene, NaOH).14 The initial results substituting toluene for benzene were promising. Use of n-Bu4NCl (10 mol %) as a phase transfer catalyst with potassium carbonate as a base and a toluene/water mixture resulted in >80% conversion to desired product after 13 h at room temperature. Screening the variables of temperature, phase transfer catalyst (PTC) load and solvent led to the interesting observation that the presence of toluene actually retards the reaction rate, and allyl alcohol can serve as an excellent solvent for the reaction. In addition, it was found that the phase transfer catalyst was NOT required, and the reaction proceeded to excellent conversion (90%) with no catalyst in the biphasic allyl alcohol/water mixture at 25−30 °C. Isolation of 2 began with removal of the inorganic material by filtration, after heptanes were added and the mixture was cooled to 0 °C. The filtrate was diluted with toluene and washed with aqueous KHSO4 to ensure that the organic layer was neutral. The excess allyl alcohol and water were azeotropically removed with toluene to afford crude Weinreb amide 2. Use of this crude

material in the next step led to low yields (vide infra), so the product was distilled using a wiped film evaporator. Purified 2 was isolated in 66% yield from bromoacetyl bromide in approximately 90 area % purity and wt% assay. Since the next step involved aryl anion chemistry, we realized the importance of controlling the allyl alcohol and water levels in the next step, and this was accomplished through the azeotropic distillation for the product. Surprisingly, we found that the presence of >10% residual toluene caused a yield decrease of 8−16% in the isolation of 19. As a result of this observation, the levels of toluene were also minimized to 97% range, whereas using a molar equivalent or excess (1.1 equiv) of 2 resulted in yields in a range covering 60−90%. When using toluene as a delivery solvent for 2, we noted an ∼5− 10% yield increase over reactions where THF was used as a diluent for 2. Given the upside in yield noted with the substoichiometric amount of 2 and the incorporation of toluene in the process, we intended to implement both these changes for D


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Organic Process Research & Development execution of the chemistry at large scale. As mentioned above, however, we noted that toluene had an adverse effect on the crystallization for the isolation of 19, so we made the decision to employ a process that employed THF alone as the solvent for 2. To make the process more scalable, an inverse quench (i.e., the reaction solution was added to saturated NH4Cl) was developed to keep the reaction tank dry. Slow addition of 2 to a solution containing the 16·MgI resulted in a higher consistency and in situ purity for the reaction, and a 1 h addition time at 0 °C was optimal for the process. In an effort to improve throughput and selectivity, the concentration of the Grignard reagent preparation was examined. At high concentration (2.6 vols THF), lower in situ purities and yields were observed, whereas at lower concentration (5.2 vols THF), purity and yield were observed to increase. So, despite the negative impact on throughput, the decision was made to run at lower concentration to make higher quality product. The product yield and purity was also correlated with the quality of 2 used in the reaction, and this led to the development of the distillative purification procedure for 2 (vide supra). The water specification of 0.1 wt%) caused the cycloaddition reaction to stall. Recourse to the literature indicated that Ti(OiPr)4 could be activating the ketone substrate to imine formation and assisting in the dehydration of the hemiaminal intermediate.10 We examined other dehydrating agents and distillative removal of water, and under a variety of conditions, no product was observed.30 Ultimately these experiments served to strengthen the hypothesis that the titanium was critical in the dehydration process to form the nitrone; however, it is unclear whether titanium is involved in the cycloaddition step. The role of Ti(OiPr)4 in the reaction was examined using a combination of 1H and 19F NMR spectroscopy. Reactions at the typical reaction temperature (60 °C) allowed us to observe the formation of the cycloadducts and a small amount of a nitrone isomer (vide infra). In an effort to glean more information regarding reactive intermediates, we decreased the reaction temperature. Running the cycloaddition with no Ti(OiPr)4 present at temperatures 10 °C, the hemiaminals 3a were unobservable, and at temperatures >40 °C the Z-nitrones 3c and 3c′ were unobservable, indicating the rapid rate at which these compounds were transformed into the desired cycloadducts. The Z-nitrone species reacted more quickly than the Enitrone, as would be expected from geometric considerations. The literature supports the proposal that both the E- and Znitrone isomers participate in intramolecular nitrone cycloadditions, with the Z-isomer showing higher reactivity,32 but at present we cannot conclusively state that the E-isomer is participating directly in the cycloaddition reaction. In order to gain more insight into the relationship between the nitrone rotamers, we examined a model substrate which could not cyclize (Figure 2, 20). The hemiaminal intermediates and nitrones formed as for the unsaturated substrate 3, but since no cycloaddition was possible we were able to observe the relationship between the three nitrone isomers. The behavior of the saturated model compound is summarized pictorially in Figure 3. The initial ratio of E- to Z-nitrones (20c + 20c′:20d) formed at −10 °C is ∼6:1 in favor of the E-isomer (6:1) and after 72 h at −10 °C, the ratio equilibrates to ∼1:1 (Z = 13.1 + 34.3 = 47.4%, vs 51% E). As the mixture was heated, the ratio changed in favor of the Z-isomers (20c + 20c′): ∼63:37 at 55 °C for 4 h and ∼86:14 at 16 h. The ratio of Z-rotamers stayed constant at about 3:1 over this temperature range. Higher temperature appears to influence the reaction rate by enabling conversion of the rotamer mixture from 50:50 to ∼85:15. For the unsaturated substrate (3), the rate of cycloaddition is so rapid at 60 °C that the Z-nitrones (3c and 3c′) are unobservable, and the rate of reaction is determined by the time required for the E-nitrone to convert to the Z-nitrones,33 or by the time it takes for the E-nitrone to G



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EXPERIMENTAL SECTION General. HPLC Method A: Agilent 1100 HPLC with a Zorbax SB-C18 column, 50 mm × 4.6 mm I.D., 1.8 μm packing, 40 °C, 2 mL/min. Gradient: A = CH3CN, B = 10 mM NH4OAc in H2O, 30% to 95% A in 6 min, hold 95% A for 2 min. The sample diluent was 1:1 MeOH:10 mM aqueous NH4HCO3. Detection at 225 nm. HPLC Method B: Agilent 1100 HPLC with an XBridge C18 column, 75 mm × 4.6 mm I.D., 2.5 μm packing, 45 °C, 1.5 mL/min. Gradient: A = MeOH, B = 10 mM NH4HCO3 in H2O, 5% to 95% A in 10 min, hold 95% A for 2 min. The sample diluent was 1:1 ACN/H2O. Detection at 225 nm. Chiral HPLC method for 6b: Agilent 1100 or 1200 with a Kromasyl AmyCoat RP column, 150 mm × 4.6 mm I.D., 3.0 μm packing, 45 °C, 1.0 mL/min. Isocratic method, 10 mM 10 mM NH4HCO3 in H2O: CH3CN (65:35 v/v). The sample diluent was 5:95 MeOH:H2O. Detection at 210 nm. 2-(Allyloxy)-N-methoxy-N-methylacetamide (2). A mixture of H2O (760 kg) and K2CO3 (306 kg, 2.21 kmol, 1.14 equiv) was cooled to 0−15 °C, and N,O-dimethylhydroxylamine·HCl (190 kg, 1.95 kmol, 1.0 equiv) was added. After 15 min, toluene (879 kg) was added, and the mixture was cooled to 0 °C. Bromoacetyl bromide (413 kg, 2.05 kmol, 1.05 equiv) was added over 60 min (gas evolution). After 15 min, the mixture was warmed to 25 °C, and the layers were separated. The aqueous layer was washed with 331 kg of toluene. The combined organic layers were concentrated under vacuum at 20−40 °C to afford a 70−80 wt %/wt solution of bromide 7 in toluene (approximately 1090 kg of distillate was collected). Allyl alcohol (711 kg, 12.2 kmol, 6.3 equiv) and powdered K2CO3 (532 kg, 3.85 kmol, 2.0 equiv) were combined in a separate reactor, and the mixture was heated at 30 °C as the 70− 80% toluene solution of bromide 7 was added over 2−3 h. After 2 h at 30 °C, 536 kg of heptanes was added, and the mixture was cooled to 0 °C. After 2 h, the mixture was filtered, and the filter cake was washed with 463 kg of toluene. The filtrate was warmed to rt and washed with a mixture of 76 kg of H2O and 53 kg of a 10% aqueous solution of KHSO4. Excess allyl alcohol and H2O were removed from the organic layer via a continuous addition/ distillation using 1850 kg of toluene and collecting approximately 3125 kg of distillate (KF and allyl alcohol each

Synthesis of BACE Inhibitor LY2886721. Part I. An Asymmetric Nitrone

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