Article Cite This: Org. Process Res. Dev. XXXX, XXX, XXX−XXX
pubs.acs.org/OPRD
Route Optimization and Manufacture of Multihundred Grams of a Ghrelin Receptor Agonist Staffan Karlsson,*,† Cristina Gardelli,‡ Marika Lindhagen,† Grigorios Nikitidis,† and Tor Svensson‡ †
Downloaded via DURHAM UNIV on September 5, 2018 at 11:47:54 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Early Chemical Development, Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca Gothenburg, SE-431 83 Mölndal, Sweden ‡ Medicinal Chemistry, Respiratory, Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca Gothenburg, SE-431 83 Mölndal, Sweden S Supporting Information *
ABSTRACT: A linear 14-step sequence was developed for the synthesis of an oxaspirocyclic cyclopentane-based candidate drug 1 containing four chiral centers. Compared with the first-generation synthesis with an overall yield of 0.7%, which also included several chromatographic purifications, the large-scale approach furnished >800 g of API 1 in 19% overall yield, and chromatography was avoided in all but two steps. The major achievements were the development of a Curtius rearrangement where hazards were minimized, a robust and safer dose-controlled allylzinc addition to a ketone, and a selective monohydrolysis of a diester. KEYWORDS: enzymatic resolution, desymmetrization, Curtius rearrangement, allylzinc, selective hydrolysis, hydroboration
■
INTRODUCTION Ghrelin plays an important role in cachexia syndrome,1 associated with many chronic diseases such as cancer2 and chronic obstructive pulmonary disease (COPD).3 For this reason, the ghrelin receptor has been identified as a promising target to increase lean body mass and to improve functional capacity, leading to better quality of life and improved morbidity. Compound 1 is a novel small molecule based on a spirocyclopentane scaffold that acts as a potent full agonist of the ghrelin receptor in vitro (Figure 1). Furthermore, it is efficacious
phase, a few hundred milligrams had been prepared using the first-generation synthesis depicted in Scheme 1. Thus, starting from commercially available anhydride 2, a literature procedure was used to prepare rac-6. Methanolysis of 2 followed by epimerization gave the trans-configurated racemic compound rac-3 in good yield. Esterification of rac-3 gave the diester rac-4, which through oxidation with KMnO4 gave diacid rac-5, which finally was ring-closed to give racemic compound rac-6.5 Chiral chromatography was then employed to separate the enantiomers. The pure (1S,2S) enantiomer 7 obtained was selectively reacted at the ketone using a large excess of the zinc reagent obtained from 1-bromo-3-methylbut-2-ene and Zn(0). This resulted in an ∼50:50 diastereomeric mixture of compound 8, and chiral chromatography was again employed to separate these to give the pure stereoisomer 9. Next, a hydroboration−oxidation reaction of the double bond of 9 gave diol 10, which subsequently was treated with MsCl/Et3N to give ring-closed 11 in low yield. Hydrogenation of this gave carboxylic acid 12, which was converted to CBz-protected amine 13 via a Curtius rearrangement. Hydrolysis of methyl ester 13 to give 14 was followed by amide coupling with commercially available amine 15 to give 16. Hydrogenolysis of 16 gave primary amine 17, which subsequently was coupled with the commercially available ethyl ester 18 to give 19. Finally, deprotection of the BOC protecting group using HCl gave the desired compound 1 as the HCl salt (1×HCl) in good yield. The overall yield for the 16 linear step sequence is 0.7%. We were a bit concerned about the scale-up of the firstgeneration synthesis and our ability to deliver >0.8 kg of API 1 within a short time frame. Two different approaches were considered: (1) utilization of the first-generation multistep synthesis but with significant improvement of the yields since
Figure 1. Strategy for the synthesis of API 1
in promoting increase of growth hormone and IGF-1 in the preclinical pharmacokinetic/pharmacodynamic model.4 We wanted to evaluate compound 1 in further preclinical studies, and >800 g was required for this purpose. A prerequisite to obtain 1 is to apply a strategy where the two amide bonds are formed as late as possible in the sequence to prevent them from interfering with the installation of the spirocyclic THF ring (Figure 1). Furthermore, since one of the amide bonds is quite labile, the order in which they are introduced is important. Previously, in the lead optimization © XXXX American Chemical Society
Received: May 30, 2018
A
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Scheme 1. First-Generation Synthesis of 1
the capacity in our large-scale facilities (up to 100 L) was limited; (2) development of a new, shorter asymmetric route with improved overall yield and the exclusion of chiral chromatography. We believed that more time would be required to use the latter approach, and we were not willing to take the risk of delaying the project. Thus, we chose to utilize the major part of the first-generation synthesis also for our large-scale purposes. However, we strived to improve the yield significantly in every single step and, for safety reasons, to make some necessary adjustments of the first-generation synthesis.
(ii) The allylzinc addition to ketone 7 required very large excesses of zinc (6−12 equiv) and 1-bromo-3-methylbut2-ene (4−8 equiv), and therefore, a troublesome workup was foreseen on a large scale. Also, inconsistent conversions were frequently obtained. The “all in” approach used in the first-generation synthesis also constituted safety concerns. Moreover, since the addition was non-diastereoselective, an unfavorable mixture of diastereomers was obtained. We sought an approach in which such selectivity issues could be avoided. (iii) Instead of time-consuming enantiomer separation through chiral chromatography as well as frequent achiral purifications by conventional silica gel chromatography, we aimed for a chromatography-free process in which intermediates could either be used as such in the next step without further purification or be purified by crystallization. (iv) The hydroboration−oxidation reaction of compound 9 is a potential hazardous step, and safety measures had to be addressed. Also, although it had not been confirmed, we suspected the formation of undesired regioisomer in this
■
RESULTS AND DISCUSSION Apart from the fact that the first-generation synthesis was long, linear, and low-yielding (0.7% overall yield), the main issues that had to be addressed before the scale-up were the following: (i) The synthesis of the diester rac-6 included a troublesome exothermic oxidation of intermediate rac-4, and the workup was believed to be impractical and timeconsuming on a large scale. Therefore, to save time, an external supplier of rac-6 or an analogue was desired. B
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Scheme 2. Resolution of Racemate rac-20
Scheme 3. Allylzinc Addition to Dimethyl Ester 21 and Attempt To Perform a Selective Hydrolysis of Diester 27
compared with the first-generation synthesis (Scheme 2). In the subsequent steps, we hoped to find suitable conditions for desymmetrization of dimethyl ester 21 that would give us the opportunity to selectively introduce the amide bonds in the desired order. Gratifyingly, starting from a racemic mixture of rac-20 on a gram scale in the presence of Novozym 435 (20− 30% w/w), we were able to obtain the desired enantiomer 21 in 40% yield with >99% ee after extractive removal of carboxylic acid 22. We considered this resolution approach robust, and consistent results were also obtained. Gratifyingly, we found that using a lower loading (15% w/w) did not have a negative effect on the enantioselectivity, and a similar rate of reaction was also observed. Given the low price of Novozym 435, we did not invest more time in trying to reduce the loading further. In the subsequent step, we hoped that after reaction with allylzinc reagent 23, obtained from Zn(0) and 1-bromo-3-methylbut-2ene, C2-symmetric dimethyl ester 21 would spontaneously furnish lactone 24 as the single product (Scheme 3). A similar strategy was reported in the synthesis of simeprevir.7 This would lead to differentiation of the two ester groups, making further selective manipulations probably a simple task. In the first-generation synthesis, allylzinc reagent 23 was prepared from 1-bromo-3-methylbut-2-ene8 and Zn(0) and then reacted with ketone 7. Large excesses of the zinc (6−12 equiv) and the bromide (4−8 equiv) were used to reach full conversion, and consequently, a very troublesome workup was foreseen using these conditions on a multihundred gram scale.
step that might complicate the workup and have a negative effect on the yield. (v) To be able to run the Curtius rearrangement of intermediate 12 safely on a multihundred gram scale, analyses of potential hazardous intermediates were required. (vi) The amide bond formed in the reaction of 17 with 18 is labile and therefore should be introduced late in the sequence. Moreover, it was found to be crucial to oxidize alkene 9 prior to amide bond formation to avoid the formation of byproducts. Thus, these factors limit the flexibility of changing the order in which these transformations are executed. (vii) The presence of four chiral centers demands a good method for their introduction and full control over the preservation of stereoisomeric purity. Starting from a meso-C2-symmetric substituted cyclopentane with no need for resolution of enantiomers is preferred, but a resolution-based approach is also acceptable provided that the resolution is performed early in the sequence. Resolution and Development of a Method for Differentiation of Ester Groups. A literature survey showed that cyclopentane-based C2-symmetric diesters can be resolved into pure enantiomers using a kinetic lipase-mediated resolution (Scheme 2).6 By the same methodology starting from the commercially available racemic dimethyl ester rac-20, two out of the four chiral centers of 1 could potentially be introduced early in the sequence, and the number of steps would be reduced C
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Also, the accessibility of 1-bromo-3-methylbut-2-ene in multihundred gram quantities was limited, and therefore, as little as possible of this should be used. Furthermore, since many zinc reagents are pyrophoric, excess use should be avoided, and they should be carefully handled under an atmosphere of nitrogen. We considered other allylmetal reagents instead, and it was found that in the presence of indium, 1-bromo-3-methylbut-2ene was efficiently transformed into the corresponding allylindium reagent, which upon reaction with ketone 21 furnished 25 in high yield.9 The reaction could also be performed in aqueous mixtures, which resulted in a safer operation. However, considering the high price of indium and its poor accessibility on a larger scale compared with zinc, we instead wanted to re-evaluate the performance of the allylzinc reagent and develop a safe and robust process. We suspected that activation of the zinc surface was crucial to obtain an efficient transformation to the desired zinc reagent 23. In the first-generation synthesis, the activation was performed using HCl or aqueous ammonium chloride solutions. Our previous good experiences of using 1,2-dibromoethane/trimethylsilyl chloride (TMSCl) for the activation of zinc led us to try this combination in the present example also.10 Fortunately, we found that after quick activation of zinc using 1,2-dibromoethane and TMS-Cl sequentially followed by dropwise addition of the bromide, a controlled exotherm was obtained, and the amounts of zinc and bromide required were reduced to 3 and 1.1 equiv, respectively. In the reaction of this zinc reagent 23 with ketone 21, we found that the most convenient way to perform the reaction was to add ketone 21 prior to addition of the bromide. This resulted in no accumulation of zinc reagent 23, which made the reaction safer to execute on a larger scale. On a small gram scale using a slight excess of 1-bromo-3-methylbut-2ene (1.1 equiv) and 3 equiv of zinc, the reaction with ketone 21 furnished a mixture of the two compounds 24 and 25 in a typical 90:10 ratio in >90% combined yield.11 We hoped to be able to utilize desymmetrized lactone ester 24 directly in the next spiroTHF installation step, and thus, we strived to drive the 90:10 mixture of 24 and 25 to 100:0. However, a prolonged reaction time and/or heating did not change the ratio. We were disappointed that we could not obtain pure lactone ester 24, but at the same time, we found that the lactone moiety was more sensitive than anticipated and easily hydrolyzed in the hydroboration−oxidation step. Thus, we searched for other ways to differentiate the two ester groups. We hoped that selective hydrolysis of the methyl ester group on the same side as the tetrahydrofuran oxygen in intermediate 27 would be possible. We planned to apply the mild LiBr/Et3N hydrolysis method previously used in several of our projects.12 We hoped that LiBr would function as a weak Lewis acid coordinating to the THF oxygen and the carbonyl of the ester group on the same side, faciliting the hydrolysis of this ester.13 To test this approach, we prepared compound 27 via the sequence described in Scheme 3. Thus, methanolysis of lactone 24 in the presence of NaCN as a catalyst followed by a sequential hydroboration− oxidation reaction gave compound 26 which after ring closure afforded 27.14 By applying the LiBr/Et3N-mediated hydrolysis method on this diester, we preferentially obtained monoester 12, as anticipated.12 However, the selectivity (∼90:10) and isolated yield were too low to be useful on a large scale, and therefore, this approach was abandoned. We instead planned to generate isopropyl methyl diester 31 (Scheme 4). The two ester groups of this intermediate were anticipated to exhibit a significant difference in reactivity, making selective hydrolysis of
Scheme 4. Differentiation of the Two Ester Groups in 31
the methyl ester possible. Thus, standard hydrolysis of compound 24 furnished diacid 28 as a solid in high yield. Upon treatment of 28 with carbonyldiimidazole (CDI) (2.5 equiv), ring closure occurred, furnishing activated ester 29, which was not isolated but directly treated with 2-propanol to give lactone ester 30 in good yield. The lactone moiety was stable under these conditions, and no alcoholysis of this by 2propanol was detected. However, we found that NaCN was an efficient catalyst for opening of the lactone, and in combination with MeOH as the solvent, the desired diester 31 was rapidly formed and then was ready for the hydroboration−oxidation step.15 Regioselective Hydroboration−Oxidation and MonoHydrolysis of Diester. Following the same procedure as used in the first-generation synthesis, the hydroboration−oxidation of compound 31 was attempted (Scheme 5). As we initially suspected, it was found after careful analysis of the crude reaction mixture that when BH3·THF was used as the boron source, a regioisomeric mixture of compounds 32 and 33 was obtained in a typical ratio of 75:25. Although the undesired regioisomer 33 does not react in the subsequent step and therefore was easy to remove further downstream in the sequence, we reasoned that the yield obtained (∼50%) was too low to be acceptable in such a long linear sequence. Instead, we tried to improve the selectivity in the hydroboration by applying 9-borabicyclo[3.3.1]nonane (9-BBN) as the boron source.16 Gratifyingly, probably as a result of steric hindrance, the desired diol 32 was obtained as a single regioisomer in good isolated yield (80%) on a gram scale as a viscous oil after silica gel chromatography. In the workup procedure after the oxidation using H2O2, we chose to destroy residual amounts of peroxides in the extracts (as checked by the starch/iodide test) through treatment with Na2SO3 solution. Also, we applied a strong nitrogen flow during the course of the reaction to keep the concentrations of hydrogen gas evolved in the hydroboration and also potential oxygen gas evolved from H2O2 to a minimum in the headspace. With these measures, the process for oxidation was considered safe on the scale on which we planned to operate.17 Next, we wanted to study the ring closure of diol 32. Starting from chemically pure diol 32 and applying the conditions used in the first-generation synthesis, we obtained the ring-closed product 34 in a very good isolated yield (Scheme 6). On the contrary, a very low yield was reported in the first-generation synthesis. We suspected that the low yield reported was due to additional impurities present in the starting material that interfered in the ring-closing step. D
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Scheme 5. Hydroboration−Oxidation of Compound 31
Scheme 6. Ring Closure and Selective Ester Hydrolysis
deemed to be too slow to be productive on a larger scale. Instead, we focused on finding a good analytical method for detection of all reactive and hazardous intermediates. Having established analytical methods for this would give us a better understanding of the reaction kinetics for the entire process and enable us to make adjustments of reaction parameters if needed. Since most of the intermediates in the Curtius transformation are reactive ones, we believed that 1H NMR measurement was the best way to monitor the reaction. Thus, aliquots were collected from the crude reaction mixture during the course of the reaction and directly dissolved in CDCl3 without further processing. It was found that the window 3−4.1 ppm was free from interfering signals, and thus, good control over the relative amount of each component was possible (Figure 2). Having optimized the long 14-step linear sequence, we were ready for the scale-up. As shown above, in the optimization of the route to 1 on a gram scale, the yields in most of the steps were close to or better than 90%. We hoped to obtain such good and consistent results also on a multihundred gram scale to be able to deliver the required amount in one single batch rather than through repeated synthesis. Since most of the intermediates were noncrystalline, we hoped to be able to use most of the crude intermediates as such with no purifications other than extractions, but chromatography was considered as a backup plan if needed. Installation of the Diamide Functionalities. Next, standard hydrolysis of 36 gave compound 14 as a viscous oil, and no epimerization of the α-center was observed according to HPLC analyses performed. Next, in the coupling with commercially available amine 15, we chose propylphosphonic anhydride (T3P) as the coupling reagent instead of EDC as used in the first-generation synthesis. The good performance in other large-scale couplings in our laboratories in combination of the ease of workup made us choose this reagent. By using a slight excess of T3P and Et3N as the base, after an extractive workup we obtained a good yield of amide 16 as a highly viscous oil with good quality. Further purification was unnecessary, and we chose to use this amide as such in the next step. What remained to be done to obtain API 1 was to remove the CBz protecting group in 16, carry out the amide coupling with 18, and remove the BOC protecting group (Scheme 8). The hydrogenolysis of compound 16 was easily performed using Pd/C as the catalyst. After removal of the catalyst followed by concentration of the mixture, crude amine 17 was ready for the next step, and no further purification was undertaken.
In our hands the result of the ring-closure step was consistent, and we believed that the method would also be robust on a larger scale. The product 34 was obtained as a viscous oil, and no further purification was necessary. We next applied the mild hydrolysis conditions on 34 using 2.5 equiv of Et3N and 5−10 equiv of LiBr in wet CH3CN.12 As we anticipated, the hydrolysis proceeded smoothly and selectively at room temperature to give acid 35 as a viscous oil in almost quantitative yield after an extractive workup. No trace of the byproduct originating from hydrolysis of the isopropyl ester was detected.18 Curtius Rearrangement. In the subsequent step, we wanted to transform acid 35 to the corresponding CBzprotected amine 36 (Scheme 7) via a Curtius rearrangement.19 Scheme 7. Curtius Rearrangement, Hydrolysis, and T3PMediated Amide Formation
Because of the high hazard associated with this reaction, we felt that it was necessary to take some precautionary measures prior to the scale-up. It is known that Curtius reactions proceed via an often highly energetic acyl azide that also might be explosive.20 Thus, it is important to avoid an accumulation of this intermediate. Also, in the presence of water, hydrazoic acid might be formed, which is a known volatile, highly toxic, explosive compound.21 Therefore, to minimize the accumulation of hydrazoic acid in the headspace, acidic conditions must be avoided, and it is essential to have control of the amount of water present in all reagents and solvents used by means of Karl Fischer titration (KF) and to avoid the use of a condenser to allow small quantities formed to escape from the reaction mixture.22 Furthermore, nitrogen evolves in the rearrangement step and must be released in a controlled manner. One way to minimize these risks is to apply continuous processes instead.23 We considered this as an alternative, but the reaction was E
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Figure 2. 1H NMR spectrum of a crude reaction mixture of the Curtius rearrangement.
Scheme 8. Hydrogenolysis, Amide Formation, and Removal of the BOC Protecting Group
The coupling with commercially available ethyl ester 18 deserves some attention. We were informed by the early chemistry team that a standard amide coupling of amine 17 and the corresponding carboxylic acid of 18 was not possible because this acid was prone to undergo decarboxylation. Therefore, attempts were made to isolate the sodium salt of this acid obtained via NaOH hydrolysis of 18. A few attempts were made to couple this sodium salt with amine 17 using various standard coupling reagents. However, we found that because of the very low solubility of this sodium salt in most organic solvents, a practical and high-yielding coupling could not be obtained. Therefore, we instead focused on finding optimal conditions for the direct aminolysis of ethyl ester 18 (Table 1). Direct thermal aminolysis of ester 18 by amine 17 in the absence of additives resulted in incomplete conversion (entries 1−3) also after a prolonged heating, and decomposition of 18
was observed under such conditions. When methanol was used as the solvent, the rate of aminolysis increased as a result of in situ transformation to the corresponding methyl ester of 18 (entry 3). However, the rate was still too low for the reaction to be productive on a multihundred gram scale, and byproduct formation was a major problem. In the first-generation synthesis, the strong amine base 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as an additive in the aminolysis, and by applying this we did indeed observe a rate enhancement, but the quality of the product mixture was low, and we were afraid that under the strongly basic conditions obtained, epimerization would occur (entry 4). Using an inorganic base such as K2CO3 instead did not improve the yield (entry 5). By using a Lewis acid additive such as lanthanum(III) trifluoromethanesulfonate, we did obtain significant conversion also at 20 °C, but the reaction stalled at ∼50% conversion, and it was not possible to obtain full F
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Table 1. Optimization of the Conditions for Aminolysis of 18 by Amine 17
entry
additive
solvent
temperature
time (h)
conversion of 17 (%)a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
− − − TBDc (50 mol %) K2CO3 (50 mol %) La(OTf)3 (10 mol %) La(OTf)3 (10 mol %) N-hydroxysuccinimide, K2CO3 (50 mol % each) 4-chlorophenol (30 mol %), K2CO3 (20 mol %) 3-chlorophenol (50 mol %), K2CO3 (50 mol %) 3-chlorophenol (10 mol %), K2CO3 (10 mol %) 3-fluorophenol (30 mol %), K2CO3 (30 mol %) 4-nitrophenol (50 mol %), K2CO3 (50 mol %) 3-nitrophenol (20 mol %), K2CO3 (20 mol %) 3-nitrophenol (20 mol %)
DMF MeTHF MeOH toluene CH3CN toluene toluene CH3CN CH3CN CH3CN CH3CN CH3CN CH3CN CH3CN CH3CN
70 °C 70 °C reflux 70 °C 70 °C 50 °C 20 °C 70 °C 70 °C 70 °C 70 °C 70 °C 70 °C 70 °C 70 °C
72 72 24 24 72 24 24 24 24 48 48 24 72 72 72
35 44 70b 72d 27 50 50 26 52 100 61 52 100 100 56
a
Determined by HPLC. bTransformation to the corresponding methyl ester of 18 occurred within 1 h. c1,5,7-Triazabicyclo[4.4.0]dec-5-ene. dA complex impure mixture was obtained.
API, and thus, a basic extractive workup was necessary after the BOC removal. Since we planned to perform a reverse quench into an aqueous Na2CO3 solution, an impractical operation was foreseen using HCl/dioxane to affect BOC removal.27 Thus, we wanted to find optimal conditions for this transformation in terms of ease of workup and impurity profile, and therefore, various solvents and a few acids were screened. Using formic acid instead furnished a homogeneous solution that after completion of the reaction could be transferred into an aqueous Na2CO3 solution. However, formylation byproducts along with tert-butyl adducts formed, forcing us to find alternative acids. Although TFA/CH2Cl2 is not the first choice for BOC removal on process scale, in our case we found that using this combination solved the problems of undesired precipitation of salts and byproduct formation, and we were able to isolate free amine 1 in almost quantitative yield with good quality after an extractive workup. Multihundred Gram Scale Synthesis of 1. After an extensive optimization phase of all steps on gram scale, we were ready to apply the methods on a larger scale to manufacture 1. Starting from 3.5 kg of the racemic dimethyl ester rac-20, the kinetic resolution mediated by Novozym 435 (15% w/w) yielded after an extractive workup 1.6 kg (95% w/w, 42% yield) of unreacted enantiomer 21 as an oil with >99% ee (Scheme 9). The purity of the crude product was considered good enough to be used as such in the next step. Ketone 21 was further reacted in a safe and dose-controlled manner with allylzinc reagent 23 prepared in situ by addition of 3 equiv of Zn(0) followed by dropwise addition of a slight excess (1.1 equiv) of 1-bromo-3methylbut-2-ene. After removal of excess zinc through filtration followed by an extractive workup, a mixture of lactone ester 24 and dimethyl ester 25 was obtained in a combined yield of 94%. The crude mixture was used as such in the following hydrolysis step, and diacid 28 was isolated as a solid in 97% yield.
conversion even after addition of more catalyst (entries 6 and 7).25 We continued to search for other additives for this transformation. We hoped that adding various phenols and other alcohols with appropriate acidity (pKa) would cause in situ transesterification of 18, forming a more reactive ester that could react with amine 17 (entries 8−15). Indeed, when differently substituted phenols were used as additives in the presence of K2CO3 as the base, a significant rate enhancement was observed, and good conversion of amine 17 to the desired amide 19 was obtained (entries 9−14). A significant decrease in rate was observed in the absence of K2CO3 (entry 15). Although 3chlorophenol as an additive in combination with K2CO3 gave clean and almost full conversion within a reasonable time frame, the pKa and hydrophilicity of this phenol were unfavorable in terms of ease of removal in an extractive workup. Instead, we found that 3-nitrophenol gave approximately the same rate enhancement, and unlike 3-chlorophenol, 3-nitrophenol can easily be removed in an extractive workup using Na2CO3 solutions.26 By the use of 3-nitrophenol as an additive (20 mol %) under the optimized conditions on a gram scale, amide 19 was obtained in good yield (∼90% by 1H NMR analysis) as a highly viscous oil. We found that it was possible to use the crude 19 as such in the subsequent step, followed by a final crystallization to obtain the pure API 1. However, starting with chemically pure 19 resulted in a significantly better yield in the final crystallization of 1, and to be prepared for both scenarios in the scale-up, a silica gel chromatography method for purification of the crude mixture of 19 was developed. Only the BOC removal from 19 remained to be optimized before we were ready for the scale-up of API 1. In the firstgeneration synthesis, the removal of the BOC protecting group was accomplished using HCl in dioxane solution, which resulted in precipitation of the corresponding HCl salt of 1 as a sticky semisolid. The free base 1 was preferred for formulation of the G
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
Scheme 9. Multihundred Gram Scale Synthesis of API 1
impurities accumulated in the six-step linear sequence. After purification by chromatography, we obtained diol 32 in 81% yield. Next, formation of the spiro-THF ring was a simple task, and after treatment of 32 with MsCl/Et3N followed by an extractive workup, we obtained compound 34 as an oil in 94% yield. The selective hydrolysis of the methyl ester in 34 was accomplished using 2.5 equiv of Et3N and 7 equiv of LiBr in wet CH3CN. No hydrolysis of the isopropyl ester was observed under these conditions. After extraction, carboxylic acid 35 was obtained in 97% yield as a highly viscous oil and was used as such in the subsequent Curtius rearrangement. This reaction was safely dose-controlled through slow addition of DPPA during 2 h to a hot (104 °C) mixture of 35 and NMM in toluene. Multiple 1 H NMR analyses were recorded during the course of the reaction to ensure that no accumulation of the potential hazardous acyl azide intermediate had occurred.24 After an aqueous workup, compound 36 was obtained in 96% yield as a viscous oil containing unreacted benzyl alcohol as the major impurity. In the optimization of the first-generation synthesis, we observed that this alcohol did not interfere in the subsequent steps, and therefore, the crude mixture of 36 was used as such in the next step. Standard hydrolysis of the isopropyl ester 36 using NaOH furnished 14 in 96% yield, and coupling of 14 with chiral
In the subsequent step, in the synthesis of isopropyl ester 30, we found that epimerization occurred to some extent (4%) at the C−H position next to the isopropyl ester moiety. However, product 30 was not unstable under these basic conditions, since no epimerization was observed over time. Therefore, we speculated that the epimerization occurred during the activation phase with CDI via ketene formation. This side reaction was not observed in the small-scale optimization work and can be explained by the shorter reaction time employed on this scale. Apart from the diastereomeric impurity, 30 was obtained with good quality in 96% yield, and we decided to use the crude mixture as such in the next step. Removal of the diastereomeric impurity in a later-stage chromatographic purification or crystallization was planned. Methanolysis of lactone 30 gave compound 31 as a viscous oil in 99% yield after an extractive workup. In the subsequent hydroboration−oxidation step, an excess of 9-BBN (2.5 equiv) was required to obtain full conversion of the hydroboration. After oxidation using H2O2 and treatment with NaOH, we obtained crude diol 32 as a viscous oil. Careful basification using NaOH was crucial to avoid hydrolysis of the ester moieties of 32. At this stage, we decided to purify the mixture to get rid of the undesired diastereomer obtained in the previous lactonization step as well as other H
DOI: 10.1021/acs.oprd.8b00179 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Article
°C (unless otherwise stated) at the frequency stated in each experiment. The chemical shifts (δ) are reported in parts per million (ppm), with the residual solvent signal used as a reference. Coupling constants (J) are reported in Hz. NMR abbreviations are used as follows: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. NMR peaks were assigned using MestReNova 8.0.1. Assays of intermediates and final products are given as weight/weight (w/w) and were determined by 1H NMR measurements using benzyl benzoate as an internal standard. Yields given for intermediates and the final product are corrected for actual assay. Analytic UHPLC/ MS experiments were performed using a Waters Acquity UPLC system combined with a Waters SQD mass spectrometer. The UHPLC was equipped with both a BEH C18 column (1.7 μm, 2.1 mm × 50 mm) in combination with a 46 mM ammonium carbonate/NH3 buffer at pH 10 and an HSS C18 column (1.8 μm, 2.1 mm × 50 mm) in combination with an 11 mM ammonium formate buffer at pH 3 with a flow rate of 1 mL/min. The mass spectrometer was operated with electrospray ionization (ESI) in both positive and negative mode. Enantiomeric excess was determined by analytical SFC (UPC2, Waters, Milford, MA, USA) using a Chiralpak ID 150 mm × 4.6 mm, 3 μm column (Chiral Technologies, Illkirch, France). The mobile phase was 5% ethanol in CO2 at 40 °C, 120 bar, and a flow rate of 3.5 mL/min. HRMS measurements were performed on a Bruker APEX mass spectrometer using ESI techniques. SFC of compounds 19 and 32 was performed on a Kromasil SIL 10 μm column (50 mm × 250 mm, flow rate 450 mL/min). Conditions for 32: 4 g of crude mixture in each injection as a solution in iPrOH (400 mg/mL), iPrOH in CO2 at 120 bar as the mobile phase (cycle time 260 s, 5% iPrOH for 110 s, 10% iPrOH 110−220 s, 5% iPrOH 220−260 s). Conditions for 19: 2.6 g of crude mixture in each injection as a solution in EtOH (320 mg/mL), MeOH in CO2 at 120 bar as the mobile phase (cycle time 400 s, 10% MeOH for 270 s, 25% MeOH 270−310 s, 10% MeOH 310−400 s). (1S,2S)-Dimethyl 4-Oxocyclopentane-1,2-dicarboxylate (21). K2HPO4·3H2O (6.38 kg, 28.0 mol) was charged to a 100 L jacketed vessel, followed by the addition of water (35 L). At a reaction temperature of 18 °C, DMSO (7 L) and transdimethyl 4-oxocyclopentane-1,2-dicarboxylate (rac-20) (3.5 kg, 17.5 mol) were added to the homogeneous solution, followed by the addition of Novozym 435 (525 g, 15% w/w). The mixture was stirred (92 rpm) at a temperature of 18 °C for 22 h, after which 50% conversion was obtained. The enzyme was removed by filtration using a 35 L Rosenmund pressure filter. The equipment was rinsed with 2-MeTHF (15 L) and the layers were allowed to settle overnight at 10 °C and then separated. The aqueous layer was extracted with 2-MeTHF (3 × 15 L), and the combined organic phase was concentrated under reduced pressure at 40 °C. Water was azeotropically removed using toluene (2 × 2.5 L) to give the title compound 21 (1.57 kg, 94.6% w/w, 7.42 mol, 42% yield, 99% ee). Analytical data were in accordance with those given in the literature.6 1H NMR (400 MHz, DMSO) δ 2.21−2.43 (m, 2H), 2.43−2.61 (m, 2H), 3.34 (p, J = 8.7 Hz, 2H), 3.62 (s, 6H). 13C NMR (101 MHz, DMSO) δ 39.5, 41.9, 50.9, 172.0, 211.5. (1R,4S,5S)-Methyl 1-(2-Methylbut-3-en-2-yl)-3-oxo-2oxabicyclo[2.2.1]heptane-5-carboxylate (24). Zinc dust (462 g, 7.07 mol) was suspended in dry THF (5 L) in a 10 L jacketed vessel, and the mixture was heated to 65 °C. 1,2Dibromoethane (16 mL, 189 mmol) was charged during 2 min, and the reaction mixture was stirred for 1 h at 65 °C and then
amine 15 using T3P furnished amide 16 in 92% yield. Reductive removal of the CBz protecting group from crude 16 using Pd/ C/H2 furnished primary amine 17 in 99% yield, and no purification was deemed necessary at this stage. We found that the hydrogenation was slow and required an extra charge of Pd/ C after 2 days of reaction to reach full conversion after an additional 2 days. The low rate was presumably due to poisoning of the catalyst by impurities and/or the substrate 16. Next, the coupling of crude amine 17 with ethyl ester 18 was performed using the optimized conditions described above (Table 1). Thus, by the use of a slight excess of ethyl ester 18, a catalytic amount of 3-nitrophenol, and K2CO3 as the base in CH3CN at 70 °C, full and clean conversion of the amine to compound 19 was obtained after 2 days of stirring. After an extractive workup using aqueous Na2CO3 solution, the 3-nitrophenol catalyst was removed, and compound 19 was obtained as a viscous oil. As mentioned above, silica gel chromatography was necessary at this stage to improve the yield in the final crystallization of API 1. Thus, after purification by supercritical fluid chromatography (SFC), compound 19 was obtained in 88% yield. After treatment of a solution of 19 in CH2Cl2 with TFA (7 equiv) followed by a reverse quench into an aqueous solution of Na2CO3 and crystallization of the crude product from acetonitrile, 833 g of the final compound 1 was obtained as a crystalline solid in 94% yield with 99% purity by HPLC.
■
CONCLUSION
Compared with the first-generation synthesis (16 steps, 0.7% overall yield), a more efficient and safer large-scale 14-step linear synthesis was developed that gave >800 g of the desired API 1 in 19% overall yield. The major improvement was the exclusion of chromatography in all but two steps. This was possible through the development of robust, high-yielding, and clean transformations in which crude intermediates in most of the steps could be used as such without further purifications. A practical early-stage enzymatic resolution instead of chiral chromatography was used to obtain the enantiomerically pure C2symmetric diester 21. The C2 symmetry was further utilized for a highly stereoselective introduction of the third chiral center of the spirocyclic THF ring of 34. In this way a single enzymatic resolution was used to control all three chiral centers in the cyclopentane core, in contrast to the first-generation synthesis, which required two applications of chiral chromatography. Other achievements were the development of a safe and highyielding allylzinc addition to ketone 21, the selective hydrolysis of diester 34, and a high-yielding Curtius rearrangement of 35 in which hazards associated with this transformation were minimized. Also worthy of mention is the development of the 3-nitrophenol-catalyzed aminolysis of ethyl ester 18.
■
EXPERIMENTAL SECTION All reactions were performed under an atmosphere of nitrogen. All of the reagents were commercially available and were used without further purification. trans-Dimethyl 4-oxocyclopentane1,2-dicarboxylate was purchased from Pharmablock (PBN20101010), zinc dust from Aldrich (>98%, dust
