Practical Synthesis of a S1P Receptor 1 Agonist ... - ACS Publications

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Practical Synthesis of a S1P Receptor 1 Agonist via a Guareschi− Thorpe Reaction Gunther Schmidt, Martin H. Bolli, Cyrille Lescop, and Stefan Abele* Chemistry Process R&D, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland S Supporting Information *

ABSTRACT: A practical synthesis of S1P receptor 1 agonist ACT-334441 (1) through late-stage convergent coupling of two key intermediates is described. The first intermediate is 2-cyclopentyl-6-methoxyisonicotinic acid whose skeleton was built from 1-cyclopentylethanone, ethyl oxalate, and cyanoacetate in a Guareschi−Thorpe reaction in 42% yield over five steps. The second, chiral intermediate, is a phenol ether derived from enantiomerically pure (R)-isopropylidene glycerol ((R)-solketal) and 3-ethyl4-hydroxy-5-methylbenzonitrile in 71% yield in a one-pot reaction. The overall sequence entails 18 chemical steps with 10 isolated intermediates. All raw materials are cheap and readily available in bulk quantities, the reaction conditions match with standard pilot plant equipment, and the route reproducibly afforded 3−20 kg of 1 in excellent purity and yield for clinical studies.

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INTRODUCTION ACT-334441 (1) is a synthetic sphingosine 1-phosphate 1 receptor (S1PR1) agonist which was developed to treat autoimmune diseases (Figure 1).1 The structure is similar to ACT-209905, another S1PR1 agonist found at Actelion Pharmaceuticals Ltd. The synthesis of ACT-209905 was described earlier in this journal.2 The new compound ACT334441 (1) differs in the isonicotinic acid moiety, which is decorated with a methoxy group and a cyclopentyl ring in the α positions of the nitrogen and in the chiral side chain, while the central part (oxadiazole and phenol moiety) is identical. Therefore, this account mainly deals with the route finding toward and the scale-up of the isonicotinic acid 5, the introduction of the polar, chiral side chain, and the convergent coupling to the active pharmaceutical ingredient (API) on kilogram scale. Two routes to the isonicotinic building block 5 are presented. The first route consists of four steps including a Negishi reaction as a key to introduce the cyclopentyl ring. This synthesis was smoothly scaled up to several kilograms. The second route comprises five steps and utilizes a Guareschi− Thorpe reaction to construct the isonicotinic acid core. Both routes are compared. The chiral side chain derived from L(−)-1,2-isopropylideneglycerol ((R)-solketal, 19) was efficiently attached to the phenol moiety and served in addition as an OH-protecting group.

ester was cleaved under aqueous acidic conditions to yield 2cyclopentyl-6-methoxyisonicotinic acid 5 as a white a solid. This acid was coupled with hydroxybenzamidine 62 employing O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU). Unfortunately, not only the hydroxybenzamidine but also the phenol function was acylated leading to considerable amounts (approximately 40%) of bis-acylated product. The crude mixture was cyclized in dioxane at elevated temperature to afford the oxadiazole 8 together with the phenol ester byproduct A (Figure 2). The crude product 8 was treated with aqueous NaOH to cleave the ester of byproduct A. The product 8 was isolated in 44% yield (from 7) after crystallization from acetonitrile with high purity (100% a/a, LC−MS). The free phenol group was alkylated with excess (S)-3-chloro-1,2-propandiol under basic conditions at 75 °C. The conversion reached only ca. 60%. The API 1 was isolated in pure form after chromatography and crystallization from EtOAc/heptane in a yield of 44%. The Risomer of API 1 was not detected by chiral HPLC analysis. For the rapid delivery of API 1 in kilogram amounts a safe and robust protocol for the Negishi reaction, an improved coupling of the isonicotinic acid 5 with a suitable hydroxybenzamide derivative, and a higher yielding method for the introduction of the chiral side chain were required. The low melting point of the API 1 (80 °C) and the good solubility in most organic solvents posed a further challenge to the purification and isolation.

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MEDICINAL CHEMISTRY ROUTE The first gram amounts of API 1 were synthesized in six steps from 2-chloro-6-methoxyisonicotinic acid 2 (Scheme 1). First, the chloro-isonicotinic acid 2 was protected as the methyl ester in high yield with methanol under acidic conditions. The cyclopentyl group was introduced via a Pd-catalyzed Negishi coupling of methyl ester 3 with commercially available cyclopentylzinc bromide at elevated temperature. The reaction was carried out in full batch mode by dissolving the reagents and the catalyst in dioxane and heating the resulting mixture to 85 °C. The pyridine derivative 4 was purified by flash chromatography and isolated as an oil in 61% yield. The © XXXX American Chemical Society

RESULTS AND DISCUSSION Isonicotinic Acid (5). We first addressed the scale-up of isonicotinic acid 5 (Scheme 2). Although the starting material 2 of the medicinal chemistry route was available, it was deemed too expensive for scale-up.3 Therefore, the less expensive dichloroisonicotinic acid 9 was chosen as starting point.4 The acid was converted to the methyl ester 10 in methanol and Received: June 13, 2016

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DOI: 10.1021/acs.oprd.6b00210 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Figure 1. ACT-334441 (1) and retrosynthetic bond breaks revealing the key building blocks, isonicotinic acid 5 and hydroxybenzamidine 23. Another similar S1PR1 agonist, ACT-209905,2 is shown for comparison.

Scheme 1. Route Used by Medicinal Chemistry for the Synthesis of the First Grams of the API 1

Scheme 2. First-Generation Route for the Synthesis of Building Block 5 for the Phase 1 Campaign

Figure 2. Phenol ester byproduct in the oxadiazole (8) formation step of the Medicinal Chemistry route.

sulfuric acid at an elevated temperature in the presence of the water scavenger trimethyl orthoformate.5 The ester crystallized from methanol/water and was isolated as a white solid in excellent yield (97%). One chloride of the dichloro isonicotinate 10 was selectively substituted with methoxide using NaOMe in methanol at reflux temperature.6 Again product 3 crystallized from methanol/water and was isolated as a white solid in excellent yield (90%) and good purity (100% a/ a, LC-MS). It was also possible to invert the reaction sequence by first installing the methoxy group and then the ester

function.7 We chose the first route because the workup was simpler and the yield was slightly higher. For the Negishi B



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Scheme 3. Second-Generation Route for the Synthesis of Isonicotinic Acid 5 for the Phase 2 Campaign

Phase 1 clinical studies. Still, the Negishi coupling presented a major pitfall for further scale-up, specifically, the high dilution, the high cost, and transportation restrictions of the organozinc reagent, and the disposal of concomitant zinc waste. Therefore, an alternative synthesis of the isonicotinic acid utilizing the Guareschi−Thorpe reaction was studied (Scheme 3).9 This strategy was already applied for the synthesis of ACT-209905.2 Thus, the reaction of cyclopentyl dioxobutanoate 14 with cyanoacetamide was expected to provide the isonicotinic acid framework, which could be further elaborated to the desired building block 5. Unfortunately, the simple cyclopentylmethyl ketone 13, the starting material of this route, was not available for a reasonable price.10 Several approaches to 13 are reported in the literature.11 All methods were not appropriate for our needs, due to low yields, the use of expensive starting materials, toxic reagents, or difficult-to-scale up protocols. A simpler and scalable synthesis of cyclopentylmethyl ketone 13 was in demand. An early publication by Goldsworthy11e described the reaction of ethyl acetoacetate and 1,4-dibromobutane in an ethanolic NaOEt solution to form ethyl 1-acetyl-cyclopentanecarboxylate, which was hydrolyzed and decarboxylized to yield the desired cyclopentyl methyl ketone 13. Although the process was low yielding and the decarboxylation reaction was sluggish, we speculated that the decarboxylation of the corresponding tert-butyl ester 12 under acidic conditions would provide the ketone 13 in a cleaner and higher yielding reaction.12 Following an alternative protocol for the cycloalkylation,13 the reaction of tert-butyl acetoacetate with 1,4dibromobutane under phase transfer catalysis (PTC) in a polar solvent like DMF or DMSO with K2CO3 at rt afforded the desired keto ester 12. As a phase transfer catalyst we utilized tetrabutyl ammonium iodide or bromide, or the ionic liquid [Bmim]BF4, and obtained similar results. The reaction was slow and did not reach full conversion even after several hours. Extractive work up and distillation afforded the product 13 in a

coupling we employed similar conditions as the precedent. Cyclopentylzinc bromide was purchased as a 0.5 M solution in THF from Rieke Metals.8 This reagent was also available in larger quantities for further scale-up. The catalyst of choice was [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with CH2Cl2 ((Pd(dppf)·DCM), and the loading was 0.008 equiv (1 g of catalyst per 33 g of substrate 3). Dioxane was replaced by the less toxic THF. Since the Negishi coupling was an exothermic reaction, we had to develop a dose controlled process. The addition of isonicotinic acid 3 to the organozinc bromide/Pd(dppf)·DCM mixture at elevated temperature resulted in incomplete conversion. Next, we added the cyclopentylzinc bromide solution (1.1 equiv) to the starting material and catalyst in THF at 60 °C. The addition was slightly exothermic and was easily controlled by the rate of addition. The conversion to 4 after addition of the reagent was usually 80%. After further stirring at elevated temperature, the conversion went to completion. Interestingly, the use of 1 M zinc reagent was less efficient, resulting in incomplete conversion. A further challenge was the workup and purification of the product. Most of the THF was distilled off. The residue was diluted with tert-butyl methyl ether (TBME) and extracted with aqueous HCl to remove the zinc salts. The TBME layer was clarified by filtration over charcoal and Celite. This afforded the crude ester 4 in good yield (95%) and purity (95% a/a, LCMS) as an orange oil. The hydrolysis of ester 4 with aqueous NaOH in ethanol was straightforward and provided a further purification possibility of the preceding Negishi coupling. The acid 5 could easily be purified by extraction into the basic aqueous layer and washing with TBME, back-extraction of 5 from the acidified aqueous solution, followed by crystallization of 5 from acetonitrile. This route delivered the nicotinic acid 5 in only four steps with an overall yield of 64% as an off-white solid with a purity of 100% a/a (LC-MS). This route was suitable for the production of 30 kg of building block 5 to support the GMP manufacturing of API for C



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dilution with water and removal of THF by distillation, the residue was added to 32% HCl and stirred at 100 °C for 22 h. Upon cooling the desired product 16 precipitated as a white solid in a yield of 69% and excellent purity (100% a/a, LC-MS). For the next steps, a direct transformation of isonicotinic acid 16 to the chloro ester 18 was tempting. Upon heating of 16 in excess POCl3 at reflux and quench into methanol, the desired product 18 was formed. However, the quench of the crude, concentrated POCl3 reaction mixture into methanol was very violent and exothermic. In addition, the subsequent aqueous workup gave product contaminated with trimethyl phosphate and some 2-chloro-6-cyclopentylisonicotinic acid E from hydrolysis of ester 18. This chloro acid did not react as fast as the ester 18 in the final methoxylation step and was not fully purged in the isolation of methoxy isonicotinic acid 5. Therefore, the acid 16 was first converted to the methyl ester 17 in methanol, trimethyl orthoformate, and catalytic H2SO4. After addition to water at 50 °C, 17 crystallized and was isolated by filtration in 97% yield. The direct methylation of 17 to 4 with methyl iodide in DMF with K2CO3 produced a 50:50 mixture of the N- and O-methylated regioisomers. No further investigations to improve this reaction were carried out. The triflate of pyridone 17 was reacted with KOMe in methanol yielding only the isonicotinic acid 16 upon workup. The twostep approach, i.e., chlorination−SNAr, seemed more promising. The chlorination of ester 17 with POCl3 was sluggish and required harsh conditions. In addition, the quench was violent and the workup not scalable.16 Therefore, POCl3 was exchanged with phenylphosphonic dichloride.17 This reagent is convenient because of its high boiling point (258 °C) allowing higher reaction temperatures than POCl3. The ester 17 was suspended in two equivalents of phenylphosphonic dichloride and heated to 130 °C for 4 h reaching a clean conversion to the product 18. The quench of the reaction mixture had to be carefully designed to prevent ester hydrolysis. To this end, the reaction mixture was diluted with acetonitrile and quenched onto 10% trisodium citrate solution (pH 8−9) and iPrOAc at 5−10 °C. The addition was only moderately exothermic.18 The ester was stable under these conditions (pH 1). A black slimy product was formed troubling the following extractive workup and necessitating several filtrations over Celite. Still, the dark-brown byproducts were partially soluble in iPrOAc and could not be removed completely during these treatments. Changing the solvent to TBME gave a less colored filtrate after Celite treatment. The slime was best removed by a filtration over Celite after the washing step with Na2CO3 solution affording two clear layers and a smooth ensuing aqueous extraction.19 The ester 18 was isolated as a brown oil in excellent yield (97%) and purity (100% a/a, LC-MS). The crude oil of 18 was treated with excess 25% KOMe in methanol and heated to reflux leading to rapid substitution of the chloride. Traces of water or KOH from the reagent converted the chloro ester 18 to the chloro acid E, which was much less reactive than the ester. Therefore, the mixture was concentrated by distillation to reach an internal temperature of 92 °C to accelerate the reaction. The reaction required approximately 7 h to reach a conversion of >99%. Chloro acid E was not removed during workup and even enriched during crystallization (up to 5%). Instead, it was easily removed in the following coupling step (Scheme 5). The lactam derivative 16 was formed as another minor impurity (99% a/ a, GC-MS). The 2,4-diketoester 14 was formed in a Claisen-Schmidt reaction by addition of a mixture of 13 and diethyl oxalate to a slight excess of KOtBu in THF at −20 °C. Usually, the reaction was complete after addition, and the product was formed in high purity. A workup or purification was not required. For the study of the envisioned Guareschi−Thorpe reaction, however, diketoester 14 was purified as colorless liquid by distillation (95 °C, 5 × 10−2 mbar). 14 was reacted with cyanoacetamide in the presence of triethylamine in ethanol at 65 °C for 1 h.14 Product 15 precipitated upon cooling and was isolated in 30% yield; the solubility in ethanol called for a rework of the mother liquor to get a second crop and a total yield of 60−70%. One of the detected byproducts was tentatively attributed to the acid B ([M + 1] = 233) (Figure 3).15 The water formed during the

Figure 3. Byproducts A−E in the Guareschi−Thorpe reaction route to 5 (according to LC-MS).

condensation could hydrolyze the ethyl ester. A second product ([M + 1] = 289) was detected and could correspond to the ethyl ether of 15 (C). When isopropanol was used as a solvent, this impurity was formed as well; the isopropyl ether could not be detected. To circumvent these problems THF and KOtBu (1.1 equiv) were chosen, the same conditions of the previous step. To our delight, the reaction performed well, and these two steps could be telescoped, the slight excess of base of the reaction to ketopyruvate 14 being sufficient to drive the Guareschi−Thorpe reaction to completion. The reaction temperature of the Guareschi−Thorpe reaction was set to 22 °C, since the reaction was too slow at −20 °C. The in-processcontrol (IPC) purity of the ethyl ester 15 was 63% a/a (LCMS) together with productive byproducts: while the impurity with the mass 288 was not detected anymore, the acid was still present together with a new impurity ([M + 1] = 251) which was the amide D derived from nitrile hydrolysis of B. After D



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removed easily under mild acidic conditions in the final step. The synthesis sequence started with the activation of the free hydroxy group of solketal as tosylate employing standard conditions.22 The tosylate 20 was isolated as crude oil in a yield of 95% and 99% a/a purity (GC-MS). Phenol 212 was cleanly alkylated with 20 in DMF employing K2CO3 as base at 120 °C. The liquid nitrile 22 was directly converted to the hydroxamidine 23 by treatment with hydroxylamine·HCl in methanol in the presence of NaHCO3. The right-hand side building block 23 was isolated as a white solid after crystallization from TBME and heptane. The reaction procedures allowed to telescope all three steps, avoiding the cumbersome isolation of oils. The overall yield for the sequence was 71%, and the LC-MS purity of the product 23 was 100% a/ a. Synthesis of the API (1). With the two stable and wellcharacterized building blocks 5 and 23 at hand, the stage was set for the convergent coupling to the API 1 (Scheme 5). A solution of the right-hand side building block 23 and Et3N in DCM was dosed to the acid chloride of isonicotinic acid 5, the latter being prepared using oxalyl chloride. Aqueous workup and solvent switch from DCM to ethanol resulted in precipitation of the coupled product 24 that was isolated as a white solid in yields of 77−82% and a purity of 97% a/a (HPLC). It was important to have an excellent quality at this stage since an upgrade was difficult to achieve at the later stages. For the formation of the oxadiazole ring, compound 24 was stirred in toluene at 110 °C for 4 h removing the generated water with a Dean−Stark trap. Without removal of water, the conversion was incomplete. The solvent was removed in the reactor under reduced pressure to isolate the product as a nonviscous yellow oil in quantitative yield. It was also possible to perform a solvent switch from toluene to ethanol and to telescope into the last step. Finally the protecting group had to be removed to liberate the API. The ketal 25 was easily cleaved with aqueous HCl at elevated temperature.23 Under strongly acidic conditions the methoxy group was slowly hydrolyzed; the ensuing impurity F (Figure 4) could not be purged, in contrast to unreacted starting material 25, during the workup and final crystallization. Stress tests were performed to determine the best conditions for the deprotection. The ketal 25 was dissolved in 2.5 vol ethanol and 1.5 vol 2 N HCl and heated to 60 °C. IPCs after 60 and 120 min showed 0.8 and 0.7% of 25 and 0.21 and 0.7% of the F, respectively. This showed that the reaction was proceeding rapidly, but reaching full conversion would lead to more byproduct. The reaction conditions were too harsh leading to an unacceptable degradation of the API. Lowering both the temperature to 50 °C and the acid concentration to 1 N HCl indicated 1.1% starting material and just 0.01% byproduct F after 1 h. After 120 min, the content of starting material was 0.7%, while 0.04% of F was formed. We chose slightly milder conditions for scale-up by reducing the volume of acid to 1.3 L/kg and lowering the temperature to 45 °C. In addition, the reaction was run under reduced pressure (400 mbar, 45 °C) to remove acetone, and the reaction time was extended to 4 h. Upon scale-up, the impurity F increased to 0.17%, while the starting material was at 0.5%. Extractive aqueous workup with EtOAc afforded the API 1 as a yellow oil in almost quantitative yield and high purity (98.8% a/a, HPLC). The API is very soluble in most common organic solvents and has a melting point of 80 °C (DSC analysis). The crude

mixture at 80 °C containing the methyl ester 4, thus triggering the hydrolysis to 5. After the removal of additional methanol, acidification with 32% HCl, and extraction with isopropyl acetate, the crude product was obtained quantitatively. Crystallization from acetonitrile afforded the isonicotinic acid 5 with a yield of 86% and excellent purity (100% a/a, LC-MS). A comparison of the two routes to 5 reveals a clear roadblock for further scale-up for route 1 (Table 1). Notably, the zinc Table 1. Comparison of the Two Approaches to Isonicotinic Acid 5 route 1, Scheme 2 Negishi steps/isolated intermediates yield metals cost driver robustness on scale

route 2, Scheme 3 Guareschi−Thorpe

4/3

8/4

63% Pd-ligand, zinc Rieke reagent (transport, waste) scale-up limitation (Negishi)

42% none phenylphosphonic dichloride all steps scalable, one distillation

reagent makes it a costly endeavor. Although the number of steps increased, the route 2 was chosen for Phase 2 clinical supplies as all steps are robust and use cheap reagents. Especially, no heavy metals are used, rendering it more environmentally benign. Synthesis of Hydroxybenzamidine 23. The strategy to couple the nonprotected hydroxybenzamidine phenol 6 with the isonicotinic acid 5 and to introduce the glycerol side chain at the last stage of the reaction sequence was sensible from a medicinal chemistry perspective to allow for the synthesis of diverse lead compounds with a similar synthetic scheme (see Scheme 1). It suffered from competing acylation at the phenolic hydroxy group. Moreover, the alkylation of the phenol 8 with a large excess of (S)-3-chloro-1,2-propandiol was low yielding and gave low purity crude API. The alkylation of phenol 8 with (S)-O-isopropylidene glycerol mesylate in DMF with NaH did not take place at rt, while at elevated temperature the starting material decomposed.21 The addition of KI or the use of K2CO3 as a base did not improve the reaction. We reckoned that an earlier introduction of the polar side chain was beneficial, with the additional asset of the phenol hydroxy group being protected. As a suitable chiral starting material, we chose (R)-solketal (19, Scheme 4). The protection group should be stable for the following steps and should be Scheme 4. New Three-Step Telescoped Sequence to RightHand Side Building Block 23 from (R)-Solketal 19

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Scheme 5. Convergent Coupling of Key Building Blocks 5 and 23 to ACT-334441 (1)

(er) was determined to be 99.7:0.3. The production of 3.3 kg of ACT-334441 was smooth, and the CRO was successfully reproducing it under GMP to deliver 20 kg of API 1 for Phase 1 clinical supply. Scheme 6 shows the entire synthetic sequence to the API 1 comprising 18 chemical transformations with 10 isolated intermediates. The longest linear chain (13 chemical steps, 7 isolated intermediates) starts from tert-butyl acetoacetate (11) and ends with the API 1 in 28% yield. Readily available 2-ethyl6-methyl aniline (26)24 and (R)-solketal (19) are the starting materials for the synthesis of the right-hand side building block 23. (R)-Solketal is the most expensive raw material.

Figure 4. Demethylated API impurity F, formed during deprotection of 25.

API could be crystallized from a mixture of EtOAc and nheptane in the presence of seed crystals. Without the addition of seeds the product usually separated as an oil. The water content must be controlled below 0.2% to prevent oiling out. Two protocols were developed. (1) The slow addition of nheptane to a solution of 1 in EtOAc at 20 °C followed by the addition of seeds, when the mixture got slightly turbid. (2) The slow addition of a solution of the API 1 in EtOAc to a suspension of a small quantity of 1 in n-heptane at 40 °C. After the suspension was formed, an aging time of at least 0.5 h at 40 °C was implemented. Both protocols produced fine needle-like particles. The particle size of the batches produced via the second method (Figure 5a) was slightly larger than that of the

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CONCLUSION

The success of the optimized large-scale synthesis of S1PR1 agonist ACT-334441 (1) resides on two pillars, i.e., (1) a scalable access to the strategic building block, isonicotinic acid 5, and (2) a new advanced building block (23) decorated with the chiral glycerol side chain. Both building blocks 5 and 23 are stable and well-characterized solids. Whereas the first route to 5 using a Negishi coupling with cyclopentylzinc bromide could be scaled up to 30 kg to support the API production for entryinto-man, a novel strategy had to be devised for the installation of the chiral side chain already for the first kilogram-makes. To this end, a phenol intermediate (21) from the manufacturing of another S1PR1 receptor agonist at Actelion Pharmaceuticals Ltd. (ACT-209905) could be used for the alkylation with the tosylate of (R)-solketal, leading, in a telescoped sequence to the second strategic building block, the hydroxamidine 23. Three high yielding steps lead to the API with high purity. As a testimony to the robustness of this novel convergent scheme, the scale-up to 100 g, 3.3 kg, and 20 kg of 1 proceeded well. FDA’s recent imperative for quality by design (QbD) calls for a robust process that delivers the API in a predictable and efficient way, in consistent purity at acceptable costs. To avoid later route changes and to profit from a longer learning curve, QbD should start as early as possible. Hence, the route to building block 5 was completely changed into more robust steps devoid of the deficiencies of the Negishi step like narrow operational windows and impediments on the environmental and cost side. A novel access to cyclopentyl methyl ketone (13)12 was decisive for the success of the Guareschi−Thorpe approach to 5 that is the preferred route at larger scales.

Figure 5. Microscope pictures of 1 showing the different particle sizes of batches obtained by two different crystallization protocols described in the text.

batches emerging from the first method (Figure 5b). The particle size distribution (PSD) showed that 90% of the particles were smaller than 59 and 18 μm for the second and first protocol, respectively. The filtration of the suspension was rather smooth causing no problems. The yield was 81%, and the HPLC purity increased from 98.8% a/a (crude) to 99.5% a/ a with single impurities

Practical Synthesis of a S1P Receptor 1 Agonist ... - ACS Publications

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