Discovery and Development of the Natural Product ... - ACS Publications

Chapter 11

Downloaded by COLUMBIA UNIV on December 12, 2016 | http://pubs.acs.org Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1239.ch011

Discovery and Development of the Natural Product Derivative SPI-1865 as a Gamma Secretase Modulator for Alzheimer’s Disease Ruichao Shen,*,1 Nathan O. Fuller,2 Jed L. Hubbs,3 Wesley F. Austin,4 and Brian S. Bronk5 Satori Pharmaceuticals Inc. Cambridge, Massachusetts 02139, United States 1Present address: Enanta Pharmaceuticals, 500 Arsenal Street, Watertown, Massachusetts 02472, United States 2Present address: Rodin Therapeutics, 300 Technology Square, Cambridge, Massachusetts 02139, United States 3Present address: Laboratory of Organic Chemistry, ETH Zürich, HCI G336, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland 4Present address: Celgene, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140, United States 5Present address: Sanofi, 640 Memorial Drive, Cambridge, Massachusetts 02139, United States *E-mail: [email protected].

This chapter describes the scale-up synthesis of the natural product derivative SPI-1865, employing a semi-synthetic approach. The challenge of securing a good quantity of starting material was solved by preparation of two cycloartenol glycosides in multikilogram scale via extraction/isolation from biomass and a ZrCl4-catalyzed reaction. By developing a protecting group-free ether formation reaction and a significantly improved five-step/3 pot process, we achieved kilogram-scale synthesis of SPI-1865 in good overall yield.

As SPI-1865 (1) was identified as the development candidate of our GSM program (1–5), it became our initial task to scale up the synthesis to kilogram scale for clinical studies.

© 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The medicinal chemistry (Scheme 1) synthesis to 1 commenced with a double oxidative cleavage of the sugar moiety of cycloartenol triterpenoid glycosides 2 and 3 (6). Double reductive amination of the resulting dialdehyde 4 with 1-Boc-3-aminoazetidine hydrochloride provided N-Boc-azetidinyl morpholine compound 5. This compound was reduced with NaBH4 to produce C15 alcohol 6 with high diastereoselectivity (>95:5 dr). Protection of the C15 hydroxyl with TESCl followed by hydrolysis of the C24 acetate produced the diol 7. Selective alkylation of the C24 hydroxyl using NaH/EtI in DMF provided C24 ethyl ether 8. Finally, a double deprotection of the N-Boc-carbamate and the C15 TES ether using HCl in 1:1 aq MeOH at 50 °C, followed by reductive amination with acetone, provided 1. This route, requiring eight synthetic and four chromatography steps, was employed to supply 1 on multi-gram scale for in vivo PK/PD studies.

Scheme 1. Initial Synthetic Route to 1. (Reproduced with permission from reference (16). Copyright 2014 American Chemical Society).

As shown in Scheme 1, cycloartenol triterpenoid glycosides 2 and 3 served as required starting materials for the synthesis of 1. Therefore, before any optimization of the synthetic route, the first challenge we were facing was to obtain sufficient amount of 2 and 3; a non-trivial task. 272 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 2. Preparation of Compound 2/3

Our method to prepare 2 and 3 started from extraction and isolation of 24-O-acetylhydroshengmanol 3-O-β-D-xyloside (10) (7) and 24-Oacetylhydroshengmanol 3-O-α-L-arabinoside (11) (1) from the roots and rhizomes of Actaea racemosa, commonly known as “black cohosh” (Scheme 2). Glycosides 10 and 11 share the same aglycone, having the isomeric xylose and arabinose at the C3 position, respectively. Treatment of 10 and 11 with a catalytic amount of ZrCl4 in dichloromethane stereoselectively afforded 2 and 3, respectively, in 80% yield (2–4). Compounds 2/3 possessed a stable tetrahydropyran E-ring and a C15-ketone. We propose that a pinacol-type 1,2-hydride shift pathway is likely involved in this transformation (8). To meet the goal of producing one kilogram of GMP quality 1 for development studies, 10 kg of combined cycloartenol glycosides 2 and 3 would be needed. In consideration of the structural intricacy and very limited effort in total synthesis of cycloartenol natural products, the most practical approach would be an optimized process of extraction and separation of compounds 10 and 11 followed by the ZrCl4-catalyzed reaction. We set out to do so. Our original process commenced with methanol extraction of ground powder of black cohosh (Scheme 3). The concentrated methanol solution was precipitated by slow addition of 5% aqueous KCl solution. The collected solid, containing 10 and 11 in 10-15% combined HPLC purity was purified by reverse phase (RP) C-18 chromatography. A mixture of 10 and 11 in 32% combined purity was obtained. Treatment of this mixture with catalytic ZrCl4 in dichloromethane provided 2 and 3 as major products. Precipitation from aqueous ethanol further upgraded the purity of 2 and 3 to 96 HPLC %. Despite working well at the discovery stage, two major limitations prevented this process from serving as a practical one for developmental quantities. First, solids obtained from precipitation with 5% aqueous KCl had varying particle sizes. This made it difficult to maintain consistently a satisfactory recovery from filtration. Second, C-18 chromatography is obviously not practical for large scale preparations. During an effort to optimize the extraction process, a liquid/liquid extraction of the alcoholic extract of black cohosh was upgraded from 2.5% to a combined purity of 10 / 11 of 13-15%. This was accomplished using dichloromethane and 11.6% NaCl solution. Other major components in the extract were either assigned by comparison with authentic samples on HPLC or by previous analysis in the 273

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


literature (9). These compounds include actein 12 (two C26 epimers) (10, 11), 23-epi-26-deoxyactein (13) (12), 23-O-acetylshengmanol 3-O-β-D-xyloside (14) (13), cimigenol 3-O-β-D-xyloside (15) (14), cimigenol 3-O-α-L-arabinoside (16) (15), and 25-O-acetylcimigenol 3-O-β-D-xyloside (17) (16) (Figure 1).

Scheme 3. Flow Chart of Original Process To Prepare 2/3

Figure 1. Structures of the identified compounds in DCM extract. (Reproduced with permission from reference (8). Copyright 2014 American Chemical Society).

At the ZrCl4 step of the original process, we found that in addition to 10 / 11, additional components in the black cohosh extract participated in the reaction to produce 2 / 3. Among the above identified components, compound 14 could be one of the productive components, as it may transform to 10 under acidic conditions (9, 13). On the other hand, other major components in the extract were not expected to interfere with the ZrCl4 reaction. Based on this analysis, it was reasonable to treat the DCM extract directly with ZrCl4 to generate compounds 2 / 3. In a typical batch, ZrCl4 (7% w/w) was added to a DCM solution obtained from liquid/liquid extraction containing 13.6% of 10 and 11. The water content of the DCM solution was pre-adjusted to an appropriate level (0.4% relative to solids) 274 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


as it is critical for initiating the reaction (8). The mixture was stirred at 23 °C for 70 min and quenched with triethylamine. An aqueous work up with 4% NaHCO3 solution followed by filtration of the organic layer through Celite® provided a DCM solution containing 10.7% of 2 and 6.4% of 3 (HPLC). As predicted, actein (12) and cimigenol compounds (15, 16) were not affected by the ZrCl4 treatment. Compounds 2 / 3 were present as the major components in the crude product. Although purification remained as a challenge, encouraged by reasonable resolution between 2 / 3 and other major components, we identified a fractionation/precipitation process. The DCM solution from above was charged on a silica gel column (3 times of the solid weight) and eluted with a gradient of 0-12% MeOH/DCM. Collection of the desired fractions provided a solution containing 17.8% of 2 and 10.5% of 3. This solution was loaded on silica gel column (8 times of the solid weight) and eluted with a gradient of 2-15% MeOH/DCM. Combination of desired fractions provided 2 and 3 in 64% combined purity (HPLC), with actein as the major remaining impurity. Similar to our original process, precipitation from an aqueous ethanol solution produced additional purification by removal of the actein compounds. In addition, we found it crucial to add Celite® in the precipitation process to speed up the filtration. The precipitated solid together with Celite® was redissolved in DCM and filtered. Concentration of the filtrate afforded a pale brown solid containing 2 and 3 in over 90% HPLC purity.

Scheme 4. Flow Chart for the Production of 1 and 2 Using the Optimized Process A production campaign at multikilogram scale of 2 and 3 was initiated with the optimized process, (see Scheme 4 for the flow chart for this process). This campaign originated with 1330 kg of dried solids obtained from ethanol extractoin of 7 metric tons of black cohosh biomass. Using the optimized process, 11 kg of combined compounds 2 and 3 were produced (8). Overviewing the whole process, one can promptly point out that the silica gel fractionation step is the rate-limiting step, consuming the most time and effort. Although this is an inevitable step before a synthetic route is developed, it can be improved by investigating additional precipitation methods, reusable absorbents, and more efficient eluents. Having solved the supply issue of starting material 2 / 3, we focused our attention on optimizing the synthetic route (Scheme 1). In this route, three normal phase and one reverse phase column chromatographies were 275



employed as purification methods. In addition, NaH and DMF were used for the C24-O-alkylation, which generated a safety concern for scale-up. These aspects required us to improve this route before starting the kilogram-scale synthesis of 1. First we endeavored to find a safe process for the C24 hydroxy alkylation by investigating bases/alkylating reagents/solvents combinations. Treatment of diol 7 with NaOtBu (5.0 eq) and diethyl sulfate (2.5 eq), afforded a 93% conversion to 8 after only 30 min at 0 °C (Equation 1 ).

On the other hand, we were additionally encouraged by a separate reaction to explore the relative reactivity of the alcohols in tetrol 18. When the unprotected aglycone 18 was treated with NaH (4.0 eq) and EtI (1.5 eq) in THF at room temperature for 15 h, the favored product was the C24 mono-O-alkylation product 19 (41%), (Equation 2). While there was a significant amount of unreacted starting material (42%) and C15,C24 bis-ether 20 formed (17%), this experiment suggested that C24 alcohol is the most active one for alkylation. It might be possible to selectively alkylate the C24 hydroxyl without protecting the C15 hydroxyl, saving significant time. This result inspired us to apply the above optimized alkylation conditions to 21 which doesn’t have a protecting group at C15 alcohol. With NaOtBu (5.0 eq) as base, diethylsulfate (1.1 eq) as alkylating agent, and more eco-friendly toluene/DMF (3:1) as a mixed solvent, the alkylation conducted at 0 ºC for 2 h provided a 79% conversion to 22 and only 2% of bis-ether 23 (Equation 3).

276 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


In the original route, Boc was used to protect the azetidine, which was designed to synthesize a series of analogs with different substitutions at the azetidine (4). The promising result from alkylation of 21 encouraged us to further explore whether the carbamate protecting group for the azetidine was necessary. In previous experiments we found that the N-methyl azetidine compound could form the quaternary ammonium salt 24 (Figure 2) under alkylation conditions with strong base (NaH / EtI). We hoped that a weaker base (NaOtBu) along with the added steric bulkiness by the isopropyl group could suppress the formation of quaternary salts.

Figure 2. Structure of 24.

Therefore, we investigated the alkylation conditions on the functionalized N-isopropyl azetidine 25 (Table 1). When using 2.7 eq of NaOtBu, 1.1 eq of Et2SO4, and toluene/DMF (3:1) as solvent, we were delighted to see that the reaction gave 72% of C24-O-alkylation product 1 with no quaternization of the azetidine nitrogen (entry 1). Increasing the amount of base to 5 eq with a lower substrate concentration (0.1 M) improved the conversion of 1 to 80%, but also increased the formation of diethyl ether 26, in spite of being performed at lower temperature (entry 2). Further increasing the substrate concentration to 0.2 M provided a 91% conversion to SPI-1865, indicating the reaction is sensitive to substrate concentration (entry 3). Increasing the amount of diethylsulfate (1.25 eq) with a lesser amount of DMF as solvent slightly improved the conversion to 1 (93%), while keeping di-ethyl ether 26 at about the same level (entry 4). These highly selective alkylation results are exciting because they allowed us to skip the use of both protecting groups and therefore to eliminate three steps (protection/deprotection/reductive amination) from the original sequence. Applying these improvements, the optimized synthesis of the candidate SPI1865 (1) is shown in Scheme 5. Following oxidative cleavage of the 1,2,3-triol on the sugar of 2 / 3, the crude dialdehyde 4 was used directly after work-up in the double reductive amination with amine 27. Using NaBH3CN as the reducing agent is critical for a clean reaction as we found incomplete reduction products formed when NaBH(OAc)3 was used. The fact that we were now running the reaction in EtOH allowed us to add NaBH4 directly to the reaction mixture to reduce the C15 ketone to furnish the C15 alcohol, upon completion of the reductive amination. Since C15 alcohol protection is no longer required for the alkylation step, the C24 acetate was hydrolyzed in the same reaction vessel by subsequently adding aqueous NaOH upon the completion of the ketone reduction. Therefore, three reactions were efficiently telescoped in one pot to yield triol 25. 277



Table 1. Selective Alkylation of Compound 25

Scheme 5. Optimized Route for Kilogram-Scale Synthesis of Compound 1. Reproduced with permission from reference (16). Copyright 2014 American Chemical Society. In light of the excessive amount of boron reagent used, it is critical to monitor the boron level during purification of triol 25. A surprising result came when subjecting a solid obtained from precipitation of crude 25 in EtOAc to the alkylation conditions, which afforded the C-15 ethyl ether product 28 instead of 1. We proposed a cyclic boronate ester 29 formed at the C24,25-diol could be the major component in the precipitated solid in EtOAc. The boronate ester 278 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


would act as a protecting group for the C24 alcohol and leave only the C15 alcohol available for alkylation (Equation 4). To ensure complete degradation of the proposed cyclic boronate and consistent performance on scale, we decided to add diethanolamine to the reaction mixture after the hydrolysis of the C24 acetate. The new work up procedure was followed by filtration through a plug of silica gel, eliminating both lingering boron species and other impurities. At last crystallization in acetone/H2O further upgraded the purity of triol 25. This system provided triol 25 in 34% yield over four steps from glycoside 2 / 3.

In a typical alkylation batch, triol 25 (1.842 kg) was dried by aceotropic distillation and dissolved in toluene (11.6 L) and DMF (2.31 L). This solution was cooled to -1 °C and NaOtBu (1.32 kg, 5 eq) was added before further cooling to -20°C. Diethylsulfate (0.85 kg, 2 eq) was added over 15 min. The reaction was stirred at -20 °C until HPLC indicated the reaction had progressed a sufficient amount. After 205 min, the reaction was quenched with water (5.5 L) over 15 min, and warmed to 2 °C. A solution of 3:1 toluene:THF (9.2 L) was added and the mixture was warmed to 40 °C, whereupon the layers were separated. The aqueous layer was re-extracted twice with 3:1 toluene:THF (2 × 9.2 L) at 40 °C. The combined organic layer was washed with brine at 40 °C and concentrated under reduced pressure at 44 - 60 °C to provide crude 1 (1.88 kg, 66% purity by HPLC-CAD). Initial purification of crude 1 was achieved by plug chromatography on aminosilica gel (19.4 kg), using gradient elution (heptane/toluene/EtOAc). The fractions containing clean product were combined and concentrated to provide 1.24 kg of 1 (88.5% purity, HPLC-CAD). Next, crystallization of 1 (1.24 kg) was carried out for final purification by adding TBME (12.5 L), toluene (5.6 L) and water (0.063 L), and heating the mixture to reflux (~70 °C) to produce a clear solution. Seeding with crystalline 1 was necessary to initiate the crystallization process. The formed white suspension was cooled to 10 °C over 90 min, stirred for 15 min, and filtered to collect the crystallized product. The filter cake was washed with cold TBME (2.5 L) and dried under vacuum with heating to provide 0.99 kg of SPI-1865 (1, 95.3% purity HPLC-CAD). This purity was sufficient for our purposes. In summary, through development of a highly selective alkylation using a protecting group-free substrate and substantial optimization, we condensed the original route to a five step / three pot synthesis that involved two silica gel plugs and two crystallizations. These enhancements enabled us to accomplish kilogram scale syntheses of the gamma-secretase modulator 1 (SPI-1865) (17). 279


Acknowledgments The authors sincerely thank all coworkers at former Satori Pharmaceuticals, and colleagues at Carbogen-Amcis for their contributions in this project.

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280 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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