Article pubs.acs.org/OPRD
Multikilogram-Scale Production of Cycloartenol Triterpenoid Glycosides as Synthetic Intermediates for a γ‑Secretase Modulator Ruichao Shen,*,†,§ Nathan O. Fuller,†,∥ Gerd Osswald,‡ Wesley F. Austin,†,⊥ Jed L. Hubbs,†,# Steffen P. Creaser,†,∇ Mark A. Findeis,†,○ Jeffrey L. Ives,† and Brian S. Bronk†,◆ †
Satori Pharmaceuticals Inc., 281 Albany Street, Cambridge, Massachusetts 02139, United States Carbogen Amcis AG, Schachenallee 29, CH-5001 Aarau, Switzerland
‡
ABSTRACT: The process development and production of two cycloartenol triterpenoid glycosides on a multikilogram scale are described. The two compounds were used as key intermediates for the synthesis of a γ-secretase modulator and a novel potential therapeutic agent for Alzheimer’s disease (SPI-1865). This practical and efficient process includes extraction of precursor triterpenoid glycosides from Actaea racemosa (black cohosh) and a ZrCl4-catalyzed rearrangement reaction.
■
in 50% MeOH)6 or 4 (aq. HCl in DCM)4b (Scheme 1). This transformation involves acyl transfer from C24 to the C25 alcohol followed by C16 bicyclic ketal formation. Screening of additional Lewis acids revealed that ZrCl4 is the optimal catalyst for the formation of compound 1. Although the exact mechanism of the ZrCl4-catalyzed rearrangement reaction has not been fully studied, we propose two potential reaction pathways for this reaction (Scheme 2). It is conceivable that exposing the enol ether in compound 4 or the C16 hemiketal in 3 to a catalytic amount of acid generated by ZrCl 4 with trace amount of water would afford oxocarbenium ion intermediate A. In one scenario, oxocarbenium ion A could go through a pinacol-type 1,2-hydride shift pathway8 that would stereospecifically generate intermediate B and subsequently compound 1. In an alternative scenario, E1 elimination could be followed by deprotonation at C15 to generate enol intermediate C, which would then tautomerize via protonation at the more accessible β-face to form the transfused E ring. Although it is not clear how ZrCl4 is involved in either pathway, on the basis of the observation that only a modest amount of 5 is formed, we believe that the ZrCl4 reaction conditions disfavor acyl transfer from C24 to C25 and therefore dramatically suppress the formation of bicyclic ketal 5. In addition to compound 3, we later isolated from black cohosh extract the arabinoside isomer 6,4d which can be converted to compound 2 under the same ZrCl4 conditions (Scheme 3). Our analysis showed that black cohosh contains approximately equal amounts of 6 and 3 [0.2−0.3 area %, as determined by HPLC with an evaporative light scattering detector (HPLC−ELSD)]. Because both 1 and 2 can afford bisaldehyde 7,4a,b an important intermediate to compound I, the identification of compound 6 dramatically boosted the overall output of compound I from black cohosh (Scheme 3). Our early development program required approximately 1 kg of GMP-quality compound I. Therefore, as key intermediates, approximately 10 kg of combined cycloartenol glycosides 1 and
INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder affecting an estimated 5.4 million Americans1 and 36 million people worldwide.2 There are several disease-modifying approaches targeting this devastating disease that are currently under extensive investigation, including γ-secretase modulators (GSMs). Because GSMs selectively lower brain levels of amyloid-β42 (Aβ42) without significantly affecting the overall Aβ levels and other important physiological pathways, they represent a promising approach for AD therapy.3 Our GSM program4 led to the identification of SPI-1865 (I)5 (Figure 1) as a development candidate. As key intermediates, the cycloartenol triterpenoid glycosides 1 and 2 were needed for the synthesis of compound I on a multikilogram scale under an aggressive timeline (Figure 1). Structurally, compounds 1 and 2 are nearly equivalent, with the only difference being the isomeric xylose and arabinose sugars at the C3 position, respectively.
■
RESULTS AND DISCUSSION Our initial method for preparing compound 1 is shown in Scheme 1. Extraction of the Actaea racemosa root and rhizome, commonly known as “black cohosh” in North America, was followed by chromatography to afford naturally existing 24-Oacetylhydroshengmanol 3-O-β-D-xyloside (3).4d,6 Treatment of compound 3 with trifluoroacetic acid afforded enol ether 4, which was also the initial lead compound identified in our medicinal chemistry program.4d We then discovered that treatment of compound 4 with a catalytic amount of ZrCl4 in dichloromethane (DCM) stereoselectively afforded the rearranged compound 1 in over 80% yield.4a−c This compound possesses a more stable tetrahydropyran E ring and a newly generated C15 ketone. The D and E rings are trans-fused in compound 1, and the stereochemistry at C16 and C17 was confirmed by X-ray crystallography of the corresponding C15 alcohol.4b It was also found that compound 3 can be directly transformed to 1 in 80% yield upon treatment with a catalytic amount of ZrCl4 (Scheme 1). It was also noted that under strongly acidic conditions, 25-O-acetylcimigenol 3-O-β-D-xyloside (5)7 was obtained as the major product from 3 (5% H2SO4 © XXXX American Chemical Society
Received: March 2, 2014
A
dx.doi.org/10.1021/op5000732 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Figure 1. Structures of triterpenoid glycosides 1 and 2 and compound I.
Scheme 1. Initial method to prepare compound 1
Scheme 2. Potential reaction pathways to form 1
B
dx.doi.org/10.1021/op5000732 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Scheme 3. Identification of triterpenoid glycoside 6 and downstream chemistry
2 was needed. Considering the structural complexity of compounds 1 and 2 and the fact that black cohosh biomass is readily available, we believed that the most effective approach for preparing 1 and 2 would be extraction and isolation of compounds 3 and 6 followed by the ZrCl4-catalyzed reaction. In this article, we report the development of a practical process to produce 1 and 2 on a multikilogram scale starting from black cohosh (A. racemosa) biomass. Initial Process To Produce Compounds 1 and 2. Our original process to produce compounds 1 and 2 is illustrated in Scheme 4. Black cohosh root was ground to powder and extracted with methanol. The methanol solution was concentrated and slowly added to 5% aqueous KCl solution. The resulting mixture was cooled, and the precipitated solid was collected, affording crude 3 and 6 in 10−15% combined
purity (based on area percent) by HPLC−ELSD analysis. To further upgrade the purity of 3 and 6, the crude precipitate was purified by reversed-phase C-18 chromatography. This yielded a mixture of 3 and 6 with 32% combined purity (by HPLC− ELSD area percent), which was subsequently dried and treated with a catalytic amount of ZrCl4 in dichloromethane. The reaction provided triterpenoid glycosides 1 and 2 as major products. Further purification by precipitation in aqueous ethanol provided compounds 1 and 2 in sufficient purity for further manipulations (96% purity by HPLC−ELSD area percent). Although this approach provided sufficient amounts of 1 and 2 for our discovery program, key limitations prevented it from serving as a practical process for development. For example, precipitation with 5% aq. KCl solution afforded 3 and 6 as solids with inconsistent particle sizes. In some cases, the small particle size made it impossible to achieve satisfactory recovery by filtration. In addition, C-18 chromatography was expensive and time-consuming. During our research efforts, we made an observation that we wanted to exploit in an improved process: in the ZrCl4catalyzed conversion of compounds 3 and 6 to ketones 1 and 2, the products were consistently obtained in higher than theoretical yield considering only 3 and 6 as the precursors to 1 and 2. This result suggested that in addition to 3 and 6, other components in the black cohosh extract productively participate in the ZrCl4-catalyzed reaction to produce compounds 1 and 2. Therefore, the challenge was to define the optimal purity of the mixture of 3 and 6, since overpurification of these two compounds would remove the other productive components and reduce the overall yield, while insufficient purification complicated the subsequent transformations. Process Optimization. On the basis of the above analysis, we initiated research efforts to improve the process to produce cycloartenol glycosides 1 and 2. Starting from an alcoholic extract of black cohosh containing 2.5% compounds 3 and 6 (combined purity, HPLC−ELSD area percent), we found that a liquid/liquid extraction using dichloromethane/aq. NaCl upgraded the combined purity of 3 and 6 to 13−15%. This was presumed to result from excellent partitioning of the cycloartenol glycosides into dichloromethane.9 A thorough
Scheme 4. Flowchart for initial production of compounds 1 and 2
C
dx.doi.org/10.1021/op5000732 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
analysis to identify major components in the dichloromethane extract was performed to help us better design the next manipulation. A typical HPLC chromatogram of the DCM extract obtained after liquid/liquid extraction is shown in Figure 2. Major
conditions. Although we did not identify the arabinoside analogue of 11 in our extract, it was very likely present and also able to participate in the desired chemical pathway. Independent experiments in these laboratories showed that the actein isomers 8 and 9 are inert to the ZrCl4 conditions. It has also been reported that treatment of compound 3 with 5% H2SO4 provides 5 and 12 as major products,6 which shows the stability of the cimigenol-type skeleton under acidic conditions. Therefore, along with 8 and 9, compound 5, its hydrolyzed C25 alcohol 12, and isomer 13 were expected to be inert to the ZrCl4 conditions. On the other hand, under our reaction conditions, 26-deoxyactein 10 would likely generate an epoxide-opened product, albeit as a nonproductive component, requiring subsequent diligence in being able to purge these undesired compounds from our product mixture. On the basis of the above analysis, we hypothesized that direct treatment of the DCM extract with ZrCl4 would be a plausible next step to generate compounds 1 and 2 because other major components in the extract were not expected to interfere with the desired reactions. By direct use of the DCM extract, other productive components such as 11 would not be discarded. To our delight, preliminary results showed that treatment of the DCM extract with 7−9% (w/w) ZrCl4 in DCM did indeed provide compounds 1 and 2 in good conversion. Figure 4 shows a typical HPLC chromatogram of
Figure 2. Typical HPLC−ELSD chromatogram of the DCM extract with peak assignments.
components in the extract were assigned by HPLC comparison with authentic samples (3, 5, 6, 8, 9, 12, and 13) and by previous analysis in the literature10 with the assistance of LC/ MS data (10 and 11). In addition to compounds 3 and 6, the major components identified in the extract include actein (two epimers at C26, 8 and 9),11 23-epi-26-deoxyactein (10),12 23O-acetylshengmanol 3-O-β-D-xyloside (11),13 cimigenol 3-O-β14 D-xyloside (12), cimigenol 3-O-α-L-arabinoside (13),15 and 25-O-acetylcimigenol 3-O-β-D-xyloside (5)7 (Figure 3). Actein isomer 9 and compound 11 overlapped and appeared as a single peak. The structures of components 14 and 15 were not assigned, but the MS data of these compounds showed they have the same molecular weights as 12 and 13, indicating they do not have the acetate group; therefore, they were not considered as productive components. Among the major components identified in the DCM extract, compound 11 was reported to be transformed to cimigenol 5 under acidic conditions,13 indicating that it may go through compound 3 as an intermediate.10 Therefore, it is highly likely that 11 can be converted to compound 1 under ZrCl4
Figure 4. Typical HPLC−ELSD chromatogram of the crude product of the ZrCl4 reaction.
the crude product. As predicted, the actein compounds 8 and 9 and cimigenols 12 and 13 were not affected by the ZrCl4
Figure 3. Structures of the compounds identified in the DCM extract. D
dx.doi.org/10.1021/op5000732 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
treatment. Compounds 1 and 2 were present as the major components in the crude mixture. Recognizing that purification remained as a challenge, we were additionally encouraged that the HPLC chromatogram of the reaction mixture showed reasonable resolution between our target molecules 1 and 2 and the other components. A key observation made during the reaction was that water appears to play an important role in the ZrCl4 reaction. When the water content of the starting material was kept below 0.1 wt % of the dissolved crude solids, no reaction occurred after the addition of ZrCl4. In order to initiate the reaction, the water content of the starting material solution was controlled to be in the range of 0.3−0.5 wt % of the dissolved solids (as measured by Karl Fisher analysis), which is about 55−91 mol % of the ZrCl4 added. It has been documented that water reacts with ZrCl4 to generate HCl,16 which is likely required for the formation of oxocarbenium ion intermediate A in our proposed reaction pathway (Scheme 2). In addition, water may be involved in the transformation of epoxide component 11 to compounds 3 and 1.10,13 Turning our attention to purification, we were able to identify an efficient two-step process to afford 1 and 2 of sufficient quality for downstream processing. Filtration and fractionation of the crude reaction product from silica gel provided compounds 1 and 2 in ca. 60% combined purity (HPLC−ELSD area percent), with actein as a major remaining impurity. As discovered in our initial process, precipitation in aqueous ethanol afforded a further purity upgrade by removal of the actein compounds. However, on kilogram scales, filtration of the precipitated solids became problematic because of its sluggishness. Therefore, other precipitation conditions, including different solvents and the addition of filter aids, were explored on the lab scale. It was found that addition of Celite in the precipitation process significantly improved the filtration process. Extraction with DCM to remove the Celite followed by filtration and concentration provided compounds 1 and 2 in over 90% combined purity (HPLC−ELSD). Production. To prepare large amounts of triterpenoid glycosides 1 and 2 for synthesis of compound I, a production campaign on a multikilogram scale employing the optimized process was performed. The flowchart for this process is shown in Scheme 5. This campaign commenced with 1330 kg of ethanol extract (dried solids), which was obtained from 7 metric tons of black cohosh biomass. By means of the optimized process, 11 kg of combined compounds 1 and 2 was produced. Figure 5 shows a typical HPLC−ELSD chromatogram of the obtained product.
Scheme 5. Flowchart for the production of 1 and 2 using the optimized process
Figure 5. Typical HPLC−ELSD chromatogram of purified 1 and 2.
preparation of compound I on a kilogram scale from compounds 1 and 2 will be reported in the companion paper.17
■
EXPERIMENTAL SECTION General Information. Black cohosh (A. racemosa) was purchased as whole root from Famarco Limited, Inc., and crushed and extracted with ethanol at Bernett S.r.l. The content of compounds 3, 6, 1, and 2 was measured by HPLC−ELSD area percent employing the following conditions: Phenomenex Luna C18(2) column, 3 μm, 4.6 mm × 150 mm; flow rate, 1.0 mL/min; detector, ELSD; temperature, 55 °C; gain, 11. Eluent gradient conditions: 0.0 min, 40% H2O, 35% MeCN, 25% MeOH; 10.0 min, 25% H2O, 50% MeCN, 25% MeOH; 15.0 min, 5% H2O, 70% MeCN, 25% MeOH; 18.0 min, 5% H2O, 70% MeCN, 25% MeOH; 18.1 min, 40% H2O, 35% MeCN, 25% MeOH; 23.0 min, 40% H2O, 35% MeCN, 25% MeOH. Retention times: 1, tR = 8.2 min; 2 tR = 7.6 min. Liquid/Liquid Extraction. Black cohosh ethanol extract (dried solid, 335 kg, containing 1.18% 3 and 1.35% 6) was taken up in dichloromethane (1337 kg), and aqueous 11.6% NaCl solution (1100 kg) was added. The resulting mixture was stirred at 23 °C for 1 h and allowed to settle. The organic layer
■
CONCLUSIONS Cycloartenol glycosides 1 and 2 are important intermediates for the synthesis of our development candidate compound I. To provide sufficient material for our preclinical and early clinical development efforts, we devised a practical and effective process to produce compounds 1 and 2 in good yield and purity. A critical step in this process is the ZrCl4-catalyzed reaction to form the tetrahydropyran ring. This robust reaction is carried out at a relatively early stage of the process on a minimally purified extract, which ensures that the majority of productive components are conserved and transformed into compounds 1 and 2. Flash silica gel fractionation followed by precipitation with Celite is employed to upgrade the purity of 1 and 2 in the optimized process. Further studies of the E
dx.doi.org/10.1021/op5000732 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Data for 1: Prepared from compound 3 in 80% yield. 1H NMR (400 MHz, C5H5N): δ 7.05 (br s, 2H), 5.90 (br s, 1H), 5.33 (s, 1H), 4.88 (d, J = 7.2 Hz, 1H), 4.38 (dd, J = 11.2, 4.8 Hz, 1H), 4.25 (m, 2H), 4.18 (dd, J = 8.8, 8.0 Hz, 1H), 4.06 (dd, J = 8.8, 7.2 Hz, 1H), 3.80 (d, J = 11.6 Hz, 1H), 3.76 (dd, J = 11.2, 10.0 Hz, 1H), 3.53 (dd, J = 12.0, 4.0 Hz, 1H), 2.38 (br d, J = 10.8 Hz, 1H), 2.27 (br d, J = 11.6 Hz, 1H), 2.18 (s, 3H), 1.97 (m, 2H), 1.81−1.71 (m, 3H), 1.63 (s, 6H), 1.61 (m, 3H), 1.51 (m, 3H), 1.34 (s, 3H), 1.30 (m, 1H), 1.20 (m, 1H), 1.18 (s, 3H), 1.08 (m, 2H), 1.05 (s, 3H), 1.03 (s, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.62 (m, 1H), 0.50 (d, J = 3.2 Hz, 1H), 0.27 (d, J = 3.2 Hz, 1H). 13C NMR (100 MHz, C5H5N): δ 214.2, 171.4, 107.9, 88.7, 84.6, 80.2, 79.4, 79.0, 75.9, 72.4, 71.6, 67.4, 55.4, 52.6, 47.8, 44.0, 41.7, 40.3, 39.0, 33.6, 32.8, 31.7, 31.5, 30.5, 28.7, 27.4, 27.2, 26.4, 26.2, 26.1, 21.4, 21.1, 20.6, 20.3 (2C), 17.9, 15.8. 13C NMR (100 MHz, CDCl3): δ 213.1, 171.1, 105.0, 88.8, 83.8, 79.6, 75.3, 73.1, 72.6, 72.2, 69.6, 64.7, 54.9, 51.7, 47.1, 43.3, 40.7, 39.8, 37.0, 32.8, 32.1, 31.2, 31.1, 29.6, 29.1, 27.5, 26.7, 26.6, 26.0, 25.4, 20.8, 20.5, 20.2, 19.8 (2C), 17.6, 15.0. MS m/z: [M − H2O] = 645, [M + H] = 663. Data for 2: 1H NMR (400 MHz, C5H5N): δ 5.33 (s, 1H), 4.81 (d, J = 7.2 Hz, 1H), 4.47 (dd, J = 8.4, 7.2 Hz, 1H), 4.34 (br s, 1H), 4.26 (m, 2H), 4.20 (m, 1H), 3.80 (m, 2H), 3.53 (dd, J = 12.0, 4.0 Hz, 1H), 2.38 (br d, J = 10.8 Hz, 1H), 2.27 (br d, J = 11.6 Hz, 1H), 2.18 (s, 3H), 1.97 (m, 2H), 1.81−1.71 (m, 3H), 1.63 (s, 6H), 1.61 (m, 3H), 1.51 (m, 3H), 1.34 (s, 3H), 1.30 (m, 1H), 1.20 (m, 1H), 1.18 (s, 3H), 1.08 (m, 2H), 1.05 (s, 3H), 1.03 (s, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.62 (m, 1H), 0.50 (br s, 1H), 0.27 (br s, 1H). 13C NMR (100 MHz, CDCl3): δ 213.1, 171.1, 105.0, 89.1, 83.8, 79.6, 75.3, 72.7, 72.6, 71.4, 67.8, 65.1, 54.9, 51.7, 47.1, 43.3, 40.8, 39.8, 37.0, 32.8, 32.1, 31.2, 31.1, 29.6, 29.1, 27.5, 26.7, 26.6, 26.0, 25.4, 20.8, 20.5, 20.2, 19.8 (2C), 17.6, 15.0. MS m/z: [M − H2O] = 645, [M + H] = 663.
was separated and washed with aqueous 11.6% NaCl solution (248 kg). The collected organic layer (1000 kg) was concentrated by distillation to remove DCM, giving a solution (211 kg) containing 28% solids. The solids (59 kg) contained 8.1% compound 3 and 5.5% compound 6 (HPLC−ELSD). ZrCl4 Reaction. Four batches of the above-obtained solution of crude 3 and 6 were combined (792 kg, 30.8% solids). The water content of the solution (relative to the dissolved solids) was measured as 2.66 wt % by Karl Fischer titration. To adjust the water content to 0.3−0.5 wt %, the solution was azeotroped with DCM (2085 kg of DCM added in five portions and 1623 kg of solvent removed) to afford a solution (1239 kg) with 0.39 wt % water (relative to solids) and 17.15% solids. This solution was divided into six batches for the ZrCl4 reaction. To 207 kg of the above-obtained solution was added ZrCl4 (2.7 kg). The mixture was stirred at 23 °C for 70 min and cooled to 10 °C over 25 min. Triethylamine (5.6 kg) was added over 10 min. The vessel was warmed to 23 °C and stirred for 10 min. The mixture was diluted with DCM (204 kg) and washed with 3.7% aqueous NaHCO3 solution (99 kg). The organic layer was collected (390 kg), and the aqueous layer was extracted with DCM (102 kg). The combined DCM layers were filtered through Celite and concentrated to give a solution (221 kg) containing 17% solids (10.7% 1 and 6.4% 2 as determined by HPLC−ELSD). The DCM solution was loaded on a silica gel plug (40−63 μm, Zeoprep 60; Zeochem, 100 kg, conditioned with DCM) and eluted with a gradient of 0−12% MeOH in DCM. Among 13 collected fractions (50 kg/ fraction), five desired fractions were combined and concentrated to afford 54 kg of a solution having 35% solids containing 17.8% 1 and 10.5% 2 (HPLC−ELSD). Silica Gel Fractionation. Three batches of solution obtained from the above procedure (147 kg) were divided into 10 portions (5.2 kg of solids/portion). Each portion was loaded on a preconditioned silica gel column (Biotage KP-Sil flash 400L cartridge, 40 kg SiO2, installed on Biotage Flash 400) and eluted with a gradient of 2−15% MeOH in DCM. The desired fractions were combined and concentrated in vacuo to afford a brown solution (70.6 kg) that had 11.44 kg of solids containing 55.9% 1 and 8.0% 2 (HPLC−ELSD). Precipitation with Celite and Extraction with DCM. The above solution of crude 1 and 2 (69.05 kg, 11.19 kg of solids) was diluted with EtOH (165 L) and distilled (132 L of solvent removed), and the volume was adjusted with EtOH to 154 L. The brown turbid solution was filtered through a charcoal cartridge (Zeta Carbon R55S, part no. C08PX R55SP from 3M) and a downstream inline filter (Polycap HD 75 1.0 μm, part no. 6703-7510 from Whatman), and the filter was rinsed with 11 L of EtOH. The combined filtrate was heated to 50 °C, and 44 kg of Celite was added. To the solution at 50 °C was slowly added water (198 L) over 80 min. The resulting mixture was cooled to 1 °C over 120 min and stirred for 14 h, from which precipitates of compounds 1 and 2 were formed. The product together with Celite was filtered on a Nutsche filter. The filter cake was rinsed with 5:6 EtOH/H2O (44 L) at 5 °C and purged with nitrogen to ca. 60% dry weight content. The crude product was transferred into a reactor, taken up in DCM (176 L), and stirred at 20 °C for 19 h. The mixture was filtered, and the filter cake was rinsed with DCM (110 L). The combined filtrate was concentrated in vacuo to afford 5.1 kg of pale-brown solid containing 81.4% compound 1 and 13.0% compound 2 (HPLC−ELSD).
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Addresses
§ R.S.: Enanta Pharmaceuticals, 500 Arsenal Street, Watertown, MA 02472, USA. ∥ N.O.F.: AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Waltham, MA 02451, USA. ⊥ W.F.A.: Celgene Avilomics Research, 45 Wiggins Avenue, Bedford, MA 01730, USA. # J.L.H.: Laboratory of Organic Chemistry, ETH Zürich, HCI G336, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland. ∇ S.P.C.: Genzyme Corporation, 500 Kendall Street, Cambridge, MA 02142, USA. ○ M.A.F.: Resilientx Therapeutics, 431 School Street, Belmont, MA 02478, USA. ◆ B.S.B.: Sanofi, 640 Memorial Drive, Cambridge, MA 02139, USA.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank our colleagues at Carbogen Amcis, Indena S.p.A., and Avoca Inc. for their contributions to this project.
■
REFERENCES
(1) Alzheimer’s Association. Alzheimer’s Dementia 2012, 8, 131−168.
F
dx.doi.org/10.1021/op5000732 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
(2) World Alzheimer’s Report 2010: The Global Economic Impact of Dementia; Alzheimer’s Disease International: London, 2010. (3) Oehlrich, D.; Berthelot, D. J.-C.; Gijsen, H. J. M. J. Med. Chem. 2011, 54, 669−698. (4) (a) Hubbs, J. L.; Fuller, N. O.; Austin, W. F.; Shen, R.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M. B.; Tate, B.; Xia, W.; Ives, J. L.; Bronk, B. S. J. Med. Chem. 2012, 55, 9270−9282. (b) Fuller, N. O.; Hubbs, J. L.; Austin, W. F.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M. B.; Tate, B.; Xia, W.; Ives, J. L.; Findeis, M. A.; Bronk, B. S. ACS Med. Chem. Lett. 2012, 3, 908−913. (c) Austin, W. F.; Hubbs, J. L.; Fuller, N. O.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M. B.; Findeis, M. A.; Tate, B.; Ives, J. L.; Bronk, B. S. Med. Chem. Commun. 2013, 4, 569−574. (d) Findeis, M. A.; Schroeder, F.; McKee, T. D.; Yager, D.; Fraering, P. C.; Creaser, S. P.; Austin, W. F.; Clardy, J.; Wang, R.; Selkoe, D.; Eckman, C. B. ACS Chem. Neurosci. 2012, 3, 941−951. (5) Loureiro, R. M. B.; Dumin, J. A.; McKee, T. D.; Austin, W. F.; Fuller, N. O.; Hubbs, J. L.; Shen, R.; Jonker, J.; Ives, J.; Bronk, B. S. Alzheimer’s Res. Ther. 2013, 5, 19. (6) Sakurai, N.; Kimura, O.; Inoue, T.; Nagai, M. Chem. Pharm. Bull. 1981, 29, 955−960. Data for 3: 1H NMR (400 MHz, C5H5N): δ 5.68 (d, J = 8.0 Hz, 1H), 4.86 (d, J = 7.6 Hz, 1H), 4.37 (dd, J = 11.2, 5.2 Hz, 1H), 4.35 (m, 1H), 4.23 (m, 1H), 4.17 (dd, J = 8.4, 8.4 Hz, 1H), 4.15 (s, 1H), 4.03 (dd, J = 8.4, 7.6 Hz, 1H), 3.75 (dd, J = 10.8, 10.4 Hz, 1H), 3.50 (dd, J = 11.2, 4.0 Hz, 1H), 2.36 (m, 1H), 2.20 (m, 1H), 2.14−1.90 (m, 4H), 2.01 (s, 3H), 1.82−1.50 (m, 7H), 1.47 (s, 3H), 1.45 (s, 3H), 1.32 (m, 1H), 1.30 (s, 3H), 1.25 (s, 3H), 1.24 (s, 3H), 1.22 (m, 1H), 1.08 (m, 2H), 1.02 (s, 3H), 0.97 (d, J = 6.0 Hz, 3H), 0.72 (m, 1H), 0.51 (d, J = 3.2 Hz, 1H), 0.26 (d, J = 3.2 Hz, 1H). MS m/z: [M + Na] = 703, [M − 2H2O + H] = 645. (7) Takemoto, T.; Kusano, G.; Kawahara, M. Yakugaku Zassi 1970, 90, 64−67. (8) Paquette, L. A.; Lanter, J. C.; Johnston, J. N. J. Org. Chem. 1997, 62, 1702−1712. (9) A pilot study employing DCM as the solvent for extraction of black cohosh was implemented. A. racemosa root and rhizome (232 kg) was ground (3 mm output) and moistened with water (300 L) for 2 h. A volume increase was observed, and partial water (31 L) was removed. DCM (450 L) was added to the moistened biomass, and the mixture was heated to reflux (45 °C) for 3 h. The DCM solution was filtered (10 μm) and concentrated. The extraction process was repeated eight times, and the recovered DCM was reused. All of the extracts were combined, dried in vacuo, and ground (1 mm output) to provide a powder (11.3 kg) containing 13.4% compounds 3 and 6 (HPLC−ELSD). (10) He, K.; Pauli, G. F.; Zheng, B.; Wang, H.; Bai, N.; Peng, T.; Roller, M.; Zheng, Q. J. Chromatogr., A 2006, 1112, 241−254. (11) (a) Kusano, A.; Takahira, M.; Shibano, M.; In, Y.; Ishida, T.; Miyase, T.; Kusano, G. Chem. Pharm. Bull. 1998, 46, 467−472. (b) Jamroz, M. K.; Bak, J.; Glinski, J. A.; Koczorowska, A.; Wawer, I. J. Mol. Struct. 2009, 933, 118−125. (12) Chen, S.-N.; Li, W.; Fabricant, D. S.; Santarsiero, B. D.; Mesecar, A.; Fitzloff, J. F.; Fong, H. H. S.; Farnsworth, N. R. J. Nat. Prod. 2002, 65, 601−605. (13) Sakurai, N.; Inoue, T.; Nagai, M. Chem. Pharm. Bull. 1979, 27, 158−165. (14) Sakurai, N.; Nagai, M.; Inoue, T. Yakugaku Zassi 1975, 95, 1354−1360. (15) Shao, Y.; Harris, A.; Wang, M.; Zhang, H.; Cordell, G. A.; Bowman, M.; Lemmo, E. J. Nat. Prod. 2000, 63, 905−910. (16) Beden, B.; Croissant, M. J.; Valensi, G. Bull. Soc. Chim. Fr. 1974, 3−4, 366−370. (17) Fuller, N. O.; Hubbs, J. L.; Austin, W. F.; Shen, R.; Ives, J. L.; Osswald, G.; Bronk, B. S. Org. Process Res. Dev. 2014, DOI: 10.1021/ op500072b.
G
dx.doi.org/10.1021/op5000732 | Org. Process Res. Dev. XXXX, XXX, XXX−XXX
