Development of Related HCV Protease Inhibitors: Macrocyclization of

Article pubs.acs.org/OPRD

Development of Related HCV Protease Inhibitors: Macrocyclization of Two Highly Functionalized Dienyl-ureas via Ring-Closing Metathesis Jeevanandam Arumugasamy, Kannan Arunachalam, David Bauer, Alan Becker, Catherine A. Caillet, Roberta Glynn, G. Mark Latham, Jinsoo Lim, Jia Liu, Benjamin A. Mayes,* Adel Moussa, Elodie Rosinovsky, Aurelien E. Salanson, Adrien F. Soret, Alistair Stewart, Jingyang Wang, and Xinghua Wu Idenix Pharmaceuticals Inc., 60 Hampshire Street, Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: A novel assembly of two structurally related 14-membered ring macrocyclic hepatitis C virus protease inhibitors is presented. Key to their successful construction was an ultimate ring-closing metathesis step on the respective highly functionalized dienyl-ureas. In the case of IDX316, this procedure significantly outperformed the original macrocyclizations in terms of reaction conditions, impurity profile, product isolation, and basic efficiency metrics. Simple nonchromatographic purification methods achieved sub-10-ppm ruthenium content in the isolated product. Overall yields to IDX316 from all five starting materials ranged from 11 to 40%, and the estimated process mass intensity was improved by a factor of 50 relative to the original unscalable discovery-based routes. Application of similar methodology in the case of IDX320 and first scale-up to halfkilogram batch sizes was demonstrated.

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INTRODUCTION In excess of 2% of the global population is estimated to be chronically infected with the hepatitis C virus (HCV).1 The existing standard of care therapy, consisting of pegylated interferon-α (IFN-α) and ribavirin, has been augmented by the recent introduction of the first two HCV direct acting antivirals: boceprevir and telaprevir.2 These agents are both linear inhibitors of HCV NS3 protease, which constitutes an integral component of the HCV replication machinery.3 Several highly potent and selective second-generation macrocyclic protease inhibitors (PIs) with improved resistance and clinical safety profiles are currently under development.4 The first of this class, ciluprevir (BILN2061),5 has been discontinued; however, it has since been followed by other more promising candidates,6 in particular danoprevir (ITMN191) 7 and semiprevir (TMC435),8 which are currently in phase 3 clinical trials. During the course of a broad HCV PI discovery program, IDX3169 (1) and subsequently IDX32010 (2) were identified as potential clinical candidates (Figure 1). Structurally, both compounds feature 14-membered ring macrocyclic lactams containing four stereogenic centers derived from a cyclopropylsulfonamide-substituted dehydrocoronamate and either a 4-(quinolinyloxy)proline (IDX316) or a 4-(quinolinyloxy)pipecolic acid (IDX320). The discovery synthesis to provide 1 utilized a synthetic strategy suitable for accessing structural analogs at the quinolinol and sulfonamide moieties: this included macrocyclization by ring-closing metathesis (RCM), Mitsunobu quinolinol coupling, and final sulfonamide formation.11 Assessment of this route identified multiple problematic areas for scale-up purposes. Consequently, in order to rapidly produce the first multigram quantities of 1 required for preclinical evaluation, two alternative routes were investigated. © XXXX American Chemical Society

Figure 1. Macrocyclic HCV PIs IDX316 (1) and IDX320 (2).

The preferred approach was subsequently applied to the analogous macrocyclic system 2. This report describes these development efforts and details the successful preparation of 2 on half-kilogram scale.

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RESULTS AND DISCUSSION The original discovery route to 1 consisted of nine linear synthetic steps, eleven in total, from five building blocks (Schemes 1 and 2). Four of these, dehydrocoronamate tosylate salt 3, Boc-proline 6, hexenylamine tosylate salt 7, and 1methylcyclopropylsulfonamide 17 were commercially available; however, the trifluoromethylthiazole-substituted quinolinol 14 was custom synthesized.12 Initial peptide coupling of proline 6 with 7 gave amide 8, and after Boc deprotection, the resulting amine 9 was coupled with the methyl iodide-activated Received: October 22, 2012

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dx.doi.org/10.1021/op300296t | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

Article

Scheme 1. Route 1 Discovery Synthesis of Macrocyclic Intermediate 13

Conditions: (i) CDI, TEA, THF, reflux (70%); (ii) MeI, MeCN, rt (quant); (iii) TBTU, DIPEA, DMF, 0 °C to rt (95%); (iv) TFA, DCM, rt (quant); (v) TEA, DCM, rt (70%); (vi) TBDMSCl, TEA, DCM, rt (70%); (vii) Hoveyda−Grubbs catalyst second generation (6 mol %), DCE, reflux (51%); (viii) TBAF, THF, rt (94%).

carbonylimidazole derivative of ethyl (1R,2S)-dehydrocoronamate 5 to give urea 10. After protection of the free hydroxyl as a TBDMS ether, the resulting diene 11 was subjected to ruthenium catalyzed RCM to yield the 14-membered ring macrocycle 12 with the requisite cis-alkene. Silyl deprotection of 12 was followed by Mitsunobu coupling with quinolinol 14 to provide ether 15, with inversion of stereochemistry at C-4 of the hydroxyproline. After ester hydrolysis, CDI activation of the resulting carboxylic acid provided an anhydro intermediate which was opened with cyclopropylsulfonamide 17 under basic conditions to give the target molecule 1 on a milligram scale. On assessment of the viability of this route for production of multigram and potentially kilogram quantities of 1, several problematic issues became apparent. The most obvious challenge was the scale-up of the RCM step due to the large volume of solvent involved (1242 g of DCE per g of diene, 1.9 mM) and the high loading of expensive Ru catalyst (1.17 g of Hoveyda−Grubbs catalyst second generation (H-G2) per g of 1). Despite this high dilution, a complex mixture of oligomeric and isomeric species was produced which required extensive chromatography to purify. The Mitsunobu etherification was also recognized to be an almost equally difficult obstacle due to the high dilution utilized (300 mL per g of quinolinol 14, 8.6 mM) and the formation of the epimeric ether side product. It was hypothesized that neighboring group participation from either the amide or urea carbonyl may lead to double inversion on introduction of the quinolinol, thus producing the undesired epimer with overall retention of configuration. Further, both steps in the final sulfonamide formation to give 1, initial CDI

activation and then displacement with 17, had been performed using microwave irradiation and resulted in a throughputhampering 40% yield. In total, nine chromatographic purifications were employed, necessitating the development of alternative isolation methods along the entire route. Initial calculations indicated this route had a PMI13 of over 59000 g per g of 1, with yields ranging from 1 to 10% from all five starting material building blocks.14 Route 2. A slightly shorter approach to 1 was investigated in which the use of silyl ether protection of the diene was avoided, eliminating the need for protection/deprotection steps and the requirement for the relatively expensive reagent TBDMSCl (Scheme 3). Thus, the RCM reaction was performed upon diene 18, bearing a free hydroxyl on the proline residue.15 Experience from related in house projects had indicated that Zhan Catalyst-1B16 was often a suitable alternative for H-G2 and also had the advantage of being available in bulk without intellectual property restriction. In order for the RCM reaction to become slightly more feasible for scale-up, the concentration was increased 2.5-fold relative to that of the original silyl protected route 1, from 1.9 mM (1242 g DCE per g of diene) to 4.9 mM. Even so, on 580 g scale, 364 kg of DCE was required and 30 g of Zhan Catalyst-1B was added portionwise over a reaction time of 40 h at 75 °C, which eventually resulted in >95% consumption of diene 18. Ru catalyst loading was reduced from 6 mol % or 72 mg per g of diene 11 in route 1 to 2.8 mol % or 52 mg per g of diene 18. Subsequent distillation of this volume of chlorinated solvent was far from ideal on both efficiency and environmental grounds, and after 17 h, just under B



Article

Scheme 2. Route 1 Discovery Synthesis of IDX316 (1)

Conditions: (i) Ph3P, DIAD, THF, 0−5 °C; (ii) LiOH, THF/H2O 1:1, rt (23% over two steps); (iii) (a) CDI, THF, MW 80 °C; (b) 17, DBU, MW 80 °C (40%).

1 kg of crude macrocycle 19 was obtained. Similarly to the RCM on the silyl ether diene 11, the profile of this crude material was complex, suffering from significant contamination with various macrocyclic oligomers. Extensive chromatographic purification was required using additional large volumes of solvent, and after the subsequent trituration to further upgrade purity, only 198 g of macrocycle 19 was isolated in 37% yield.17 This compared with 51% for the route 1 RCM, although the corresponding three-step protection/RCM/deprotection yield was 34%. Despite the reduction in number of steps, reaction solvent volume per g of 1, and amount of catalyst per g of 1 eventually output, the quantity of dienes 11 and 18 required

Scheme 3. Route 2 RCM on Unprotected Diene 18

Conditions: (i) Zhan Catalyst-1B (2.8 mol %), DCE, reflux (37%).

Scheme 4. Route 3 Formation of Sulfonamyl Dehydrocoronamate 24

Conditions: (i) Boc2O, NaHCO3, H2O, rt (quant); (ii) NaOH, THF/H2O 1:1, 60 °C (quant); (iii) (a) CDI, THF, 65 °C; (b) 17, DBU, rt (65− 77%); (iv) AcCI/MeOH, MeOH, 45 °C (quant). C



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Scheme 5. Route 3 Formation of IDX316 (1)

Conditions: (i) Ph3P, DIAD, THF, 0−5 °C (67%); (ii) LiOH, THF/H2O 1:1, 40 °C (92%); (iii) 7, TBTU, DIPEA, DMF, 5 °C to rt (90%); (iv) AcCl/MeOH, MeOH, 40 °C (quant); (v) CDI, DCM, rt (99%); (vi) 24, MeCN, 65−70 °C (58%); (vii) Zhan Catalyst-1B (1.4 mol %), DCE, 73− 77 °C (86%).

a seven linear step sequence rather than the original nine. The first example of a highly efficient macrocyclic RCM reaction on production scale was reported on the 15-membered ring lactam BILN2061, for which the key developmental challenges were clearly elucidated and successfully resolved.20 Rather than following the usual tactic of performing the RCM well before the ultimate synthetic step due to potential Ru contamination of the API, it was envisioned that there could be potentially significant benefits to the route 3 approach in this instance; namely, that the amount of diene, solvent volume, and catalyst would all be substantially reduced, whilst venturing that it would still be feasible to obtain acceptable Ru levels in the final product (sub-10-ppm Ru content). It remained to be investigated whether this was achievable and how the RCM reaction itself would perform relative to that for diene 11. The subsequent second-generation process to BILN2061 indicated that N-Boc substitution of the dienyl-amide substrate considerably improved the RCM concentration (from 10 mM to 0.2 M) and catalyst loading.21 Analogous methodology with this dienyl-urea was not pursued, however, due to the extra time associated with the development of additional protection/ deprotection steps and pressing material supply requirements.

per g of 1 was actually equal at 16 g. The difficult separation of oligomeric side products requiring a slow elution gradient during the chromatography actually greatly outweighed the 50% reduction in reaction solvent volume, such that the PMI for this single-step RCM reaction versus the silyl ether diene RCM reaction more than doubled to an untenable 7113 g per g of macrocycle. Only slight reductions (0−10%) in the calculated amounts of starting materials required to make 1 were observed, and combined with the impracticality of the RCM purification, neither route 1 nor 2 was pursued. Route 3. An alternative strategy was devised to tackle the considerable problems faced by the existing routes (Schemes 4 and 5). Clearly, performing the RCM reaction with the quinolinol unit already installed would negate the need for hydroxyproline protection/deprotection steps. Executing the quinolinol coupling early in the synthesis was predicted to be advantageous, as separation of the potential epimeric ether products would likely be more straightforward than had been observed at the macrocyclic ether 15. Although less convergent with respect to quinolinol 14 and sulfonamide 17, route 3 would be more convergent with respect to proline ester 25,18 hexenylamine salt 7, and dehydrocoronamate 20,19 resulting in D



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isocyanates;28 phosgene or its analogous reagents diphosgene/triphosgene, a method which was recently utilized for preparation of a proline-urea macrocycle;29 or phenyl and pnitrophenyl chloroformates.30 An initial approach to formation of urea 31 was attempted by treating the sulfonamyl dehydrocoronamate 24 with CDI followed by addition of proline 29. However, either with or without the presence of TEA base, activation of the carboxylate was promptly followed by intramolecular cyclization to give the undesired hydantoin 32 in good yield (86%, Scheme 6). Attempted opening of this hydantoin with a model quinolinyl-proline substrate was deemed to be futile, as even at 120 °C in DMF only a trace of urea was observed.

Finally, it was anticipated that performing the coupling of sulfonamide 17 with dehydrocoronomate 22 separately would potentially avoid the use of microwave radiation and remove the costly and highly inefficient loss of yield at the final step of the original route 1. Synthesis of Cyclopropylsulfonamyl Dehydrocoronamate 24. Commercially available methyl (1R,2S)-dehydrocoronamate tosylate 20 was Boc protected in aqueous NaHCO3 solution with di-tert-butyldicarbonate. After extraction into TBME and washing to remove the tosylate, TBME distillation provided the methyl Boc-dehydrocoronamate 21 in quantitative yield. Saponification of the methyl ester and neutralization afforded the Boc-dehydrocoronamic acid 22 as an oil, which was then subjected to activation with CDI. In contrast to the original route 1, which required the use of microwave irradiation, thermal heating at 65 °C was sufficient for complete conversion to the imidazole carbonyl species. Treatment of this activated ester with 1.5 equiv of cyclopropylsulfonamide 17 and DBU proceeded smoothly at room temperature: again, no microwave irradiation was necessary. After simple aqueous workup, the crude solid sulfonamide was dissolved in MeOH. Addition of six volumes of MeOH/water 1:10 at 60 °C and slow cooling to ambient temperature induced precipitation of the pure sulfonamide product 23 in 65% yield over the two steps on kilogram scale.22 Boc deprotection using dry HCl prepared in situ proceeded rapidly at 45 °C, and the desired hydrochloride 24 was obtained by distillation of the solvent in quantitative yield as a white solid.23 Synthesis of Urea Precursor 29. With the cyclopropylsulfonamyl dehydrocoronamate 24 in hand, attention was turned to the synthesis of the diene precursor 29. Mitsunobu conditions24 were employed to couple quinolinol 14 with 1.2 equiv of trans-4-hydroxyproline ester 25. Ether formation was found to proceed to completion within 1 h on 20 g scale, and contrary to the Mitsunobu reaction performed on macrocycle 13, the profile of this simple proline substrate was not found to have been complicated by epimeric ether formation, greatly simplifying purification. The commonly troublesome triphenylphosphine oxide (TPPO) byproduct was removed by successive triturations of the crude material in a three-solvent combination of MeOH/TBME/n-heptane in 1:3:5 ratio, eliminating the need for chromatography entirely. Proline ether 26 was isolated as a white powder in 67% yield from quinolinol 14 with residual TPPO and reduced-DIAD byproducts in sub-5-mol % levels by NMR analysis. These remaining byproducts were completely removed in the subsequent steps, and further purification was determined to be unnecessary to achieve production of clean IDX316, 1. Methyl ester 26 was saponified using LiOH, and after acidification, proline 27 was obtained in over 92% yield with >99% LCAP.25 Subsequent reaction with the readily available hexenylamine tosylate salt building block 7 was performed under standard solution phase amide coupling conditions using TBTU. Aqueous workup followed by digestion of the crude in EtOAc and precipitation using n-heptane gave analytically pure amide 28 in 90% yield. Boc deprotection using dry HCl was complete in 4 h at 45 °C, at which point the acidic methanolic solution was evaporated to provide 29.26 Dienyl-urea 31 Formation. Unsymmetrical urea formation between the hydrochlorides of proline 29 and the sulfonylamyl dehydrocoronamate 24 was not trivial. Multiple methods are known for the preparation of unsymmetrical ureas,27 for example, via the reaction of amines with

Scheme 6. Formation of Hydantoin 32

Conditions: (i) CDI, with TEA, THF, rt (86%).

A second approach utilized diphosgene and was modeled again with a less precious quinolinyl-proline substrate 33. It was determined that inverse addition of a solution of the proline to diphosgene was required to reduce the substantial amount of symmetrical proline urea side product which otherwise formed. Subsequent treatment with 24 required an excess (3.6 equiv) to achieve reasonable conversion but also resulted in a complex reaction profile. On 1 g scale, a mere 15% isolated yield of urea 34 was obtained after extensive chromatographic purification (Scheme 7). Scheme 7. Urea Formation with Model Quinolinylproline 33

Conditions: (i) (a) CI3CCOCI (1 equiv), Na2SO4 (s), DCM, rt; (b) 24 (3.6 equiv), DIPEA (3.6 equiv), DCM, rt (15%); (ii) (a) CDI (2.1 equiv), Na2SO4 (s), DCE, rt; (b) 24 (4 equiv), rt (47%).

Due to the poor performance and unfavorable safety aspects of the diphosgene approach, CDI was revisited as the carbonyl source, in this case activating the proline rather than dehydrocoronamate 24 (Scheme 7). Initially, a one-pot reaction was attempted, and it was found that 2.1 equiv of CDI was required for complete conversion of the model proline 33 to give the corresponding activated intermediate. Subsequent addition of 24, however, was not clean, and due to the presence of excess CDI, hydantoin formation was again observed, which demanded more of the advanced intermediate 24 (4 equiv). Symmetrical proline urea formation was also E



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observed, and after chromatographic isolation, 47% yield of urea 34 was obtained. A more practical two-step procedure was, therefore, implemented whereby excess CDI and imidazole were removed by mild aqueous workup prior to urea formation. It was envisioned that separate reaction of the activated proline would then be more efficient with respect to dehydrocoronamate 24. Proline 29 was therefore treated with 2 equiv of CDI at room temperature, and the imidazole carbonyl species was found to be quite stable toward aqueous workup, resulting in isolation of the activated ester 30 in 99% yield over 2 steps from Bocproline 28 with 98% LCAP. Subsequent treatment of carbonyl imidazole 30 with 2 equiv of dehydrocoronamate hydrochloride 24 in THF or MeCN at 60 °C successfully produced the desired unsymmetrical urea 31. Addition of TEA base was found to be highly detrimental, yielding no signs of product. It was presumed that protonation of the imidazole was required to improve its leaving group ability on reaction with the amine 24. Attempts to use less of 24 either with or without introduction of Et3N·HCl or imidazole·HCl were unsuccessful and merely led to incomplete coupling profiles. Success in purifying the crude after aqueous workup was not consistent, dependent upon slight variations in impurity profile which occasionally led to an unusable gummy solid. As an immediate solution, a silica gel plug filtration (at 8:1 silica/crude loading ratio) was introduced as a purity upgrade operation which then allowed for reproducible and effective isolation by precipitation from EtOAc/n-heptane. Urea formation was therefore achieved on 59 g scale in 58% yield with >98% LCAP. This dienyl-urea 31 was utilized directly for the ultimate macrocycle formation. Final RCM to Protease Inhibitor 1. With access to the requisite dienyl-urea 31 secured, investigations were performed into the final RCM reaction to produce the target proline urea macrocycle 1. It was immediately evident that this cyclopropylsulfonamide diene was a highly suitable substrate for RCM, considerably more so than either silyl ether diene 11 or unprotected diene 18. At a concentration of 250 mL per g of diene (4.9 mM)31 with 1.25 wt % Zhan Catalyst-1B, an 84:1 product/starting material ratio was observed by HPLC at 1 h and the total LCAP of all cyclic and linear oligomeric side products was found to be less than 1%. On the basis of this encouraging initial reaction profile, methods to ensure acceptable Ru levels in the isolated product were investigated (target sub-10-ppm).32 It is well recognized that removal of the Ru-containing byproducts of RCM reactions can be highly challenging. Several methods have been reported addressing this issue, involving, for example, the use of tris(hydroxymethyl)phosphine,33 lead tetraacetate,34 TPPO, or DMSO followed by silica gel filtration,35 absorption onto silica gel, activated carbon and silica gel chromatography,36 treatment with mercaptonicotinic acid (MNA) and washing with aqueous NaHCO3,37 and the use of supercritical fluid extraction.38 A variety of these operations was investigated, and the resulting Ru levels in 1 were analyzed by ICP-OES39 (Table 1). Initially, the RCM reaction mixture was treated with MNA and washed with aqueous NaHCO3 (entry 1). Subsequent absorption onto activated charcoal and filtration through silica gel gave an encouraging 14 ppm Ru level. Successive triturations with MeOH reduced this to 7.5 ppm. Elimination of the MNA and bicarbonate wash provided crude product with 10-fold higher Ru content (120 ppm), and subsequent MeOH triturations gave 1 with 20 ppm (entry 2). Although the Ru

Table 1. Effect of Reaction Conditions and Purification on Ru Content and Yield of 1 entry 1

2

3

4

5

reaction conditions 250 mL/g diene 31 4.9 mM 1.5 wt % catalyst 250 mL/g diene 31 4.9 mM 1.5 wt % catalyst 250 mL/g diene 31 4.9 mM 1.5 wt % catalyst 80 mL/g diene 31 15.2 mM 1.1 wt % catalyst 80 mL/g diene 31 15.2 mM 1.1 wt % catalyst

Ru reduction operation MNA/NaHCO3 wash; charcoal; silica gel filtration 1st MeOH trituration 2nd MeOH trituration charcoal; silica gel filtration 1st MeOH trituration 2nd MeOH trituration charcoal; silica gel filtration

Ru content (ppm)

yield (%)

14 12 7.5

63 58

120 34 20

81 75

120

MeOH trituration 5:4 v/v DCE/MeOH crystallization charcoal; silica gel filtration

48 4.6 880

MeOH trituration toluene crystallization

300 22

charcoal; silica gel filtration

880

MeOH trituration 2:1 v/v EtOAc/n-heptane crystallization

300 19

79 61

84 45

84 51

level was lower, it was clear that the use of MNA/bicarbonate led to a substantial decrease in product recovery by 15−20%, and this technique was not pursued. Employing a crystallization from DCE/MeOH in place of the second MeOH trituration gave an improved Ru content (4.6 ppm), again at the expense of product yield (61%, entry 3 vs entry 2). Interestingly, reaction concentration also appeared to impact Ru content: even utilizing a slightly lower catalyst loading, with a 3-fold higher diene concentration, similar charcoal treatment and silica gel filtration provided crude material with a 7-fold higher Ru content (880 ppm), and this ratio was maintained through the first MeOH trituration (300 ppm, entry 4 vs entry 3). Screening of alternate solvent systems for the second crystallization, using toluene (entry 4) or EtOAc/n-heptane (entry 5) provided substantial Ru level reductions (to ∼20 ppm); however, recoveries were inferior to the previous treatments with MeOH or DCE/MeOH. Evidently, obtaining an acceptable Ru content was indeed feasible using a combination of relatively simple purification techniques. Rather than employing charcoal treatment, it was investigated whether a combination of TPPO and a solid-supported metal scavenger would be a beneficial alternative purification methodology. Thus, reaction profiles and purification outcomes were assessed at three different RCM concentrations under otherwise identical conditions (Table 2). Using 1.25 wt % Zhan Catalyst-1B, on increasing the RCM diene concentration from 4.9 to 7.6 to 15.2 mM, the LCAP of total oligomeric side products at 1 h was observed to increase slightly from 1% to 3.3%. Relative to the amount of catalyst, 100 equiv of TPPO was then added at 75 °C, prior to DCE solvent distillation. The three crude products were then treated with refluxing MeOH to solubilize the TPPO-chelated Ru byproducts, and the contaminated waste filtrates were discarded. A significant yield decrease (from 95% to 78%) and higher Ru content (180−390 ppm) on increasing reaction concentration were F



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initial two routes.42 The amount of Ru catalyst required per g of 1 was reduced by 73-fold and 54-fold relative to routes 1 and 2, respectively, and the DCE solvent quantity was similarly decreased by 84-fold and 44-fold to 241 g per g of 1. Comparison of PMIs for the single-step RCM reactions alone showed nearly 12-fold improvement versus the original route 1 and over 26-fold improvement versus route 2. Despite the modest absolute values, initial calculations clearly indicated significant relative benefits in efficiency for route 3 compared to the first two methodologies: the total combined PMI over the 11 steps was 1273 g per g of 1 versus 59000 g per g of 1 for the 11-step route 1 and 65600 g per g of 1 for the 9step route 2, representing approximately 50-fold improvements. In addition, estimated quantities required of the five building blocks to make 1 via route 3 were substantially reduced: relative to routes 1 and 2, which were almost identical, quinolinol 14 was reduced by 3-fold; proline 6/25 was reduced by 10-fold; hexenylamine salt 7 was decreased by 20-fold; and dehydrocoronamate salt 3/20 was cut by 10-fold. Only sulfonamide 17 remained essentially unchanged across the three routes. Strategic Considerations for IDX320, 2. On the basis of the successful proof-of-concept achieved for IDX316 (1), a similar synthetic strategy was envisioned for IDX320 (2); namely, the construction of the requisite dienyl-urea 40 with quinolinyl, dehydrocoronamate, and sulfonamyl subunits already installed, followed by RCM as the ultimate step (Schemes 8 and 9). Due to the short time frame for material requirements, a suitably scalable synthetic route was employed which utilized nonoptimized reaction conditions and isolation methods. Conveniently, three of the four building blocks required, hexenylamine salt 7, thiazolylquinolinol 14, and sulfonamyl dehydrocoronamate 24, were used in the prior IDX316 campaign; therefore, access to these starting materials was already secured. The remaining starting material 4hydroxypipecolic ester 37 was commercially available as a solid in its hydrochloride form. Formation of Dienyl-urea 40. Initial unsymmetrical urea formation between the two secondary amines 7 and 37 was attempted using CDI. As such, hexenylamine tosylate 7 was treated with 1.2 equiv of CDI to give imidazolyl carboxamide 35 in quantitative yield after simple aqueous workup. Subsequent direct reaction of 35 with pipecolate 37, however, was found to produce only traces of desired urea 38. Activation of imidazolyl carboxamides by N-alkylation with MeI is a known method to prepare ureas with less reactive amines and, indeed, proved to be successful in this case.43 Hence, carbamoyl imidazolium salt 36 was formed by treating 35 with excess MeI, followed by distillation of the volatiles to give 36 in quantitative yield. Subsequent reaction of 1.5 equiv of 36 with pipecolate 37 in refluxing THF afforded the desired urea 38 without interference via potential urethane formation from the free hydroxyl at C-4. Aqueous workup provided relatively straightforward access to urea 38 in 71% yield from pipecolate 37. Etherification between quinolinol 14 and 4-hydroxypipecolate 38 with inversion of stereochemistry at C-4 was performed under Mitsunobu conditions in a similar fashion to 4hydroxyproline 25. Complete consumption of quinolinol 14 was not observed, and typical profiles consistently resulted in a 1:1 starting material-to-product ratio despite adjustments in stoichiometry; therefore, due to both being high-value components, 38 and 14 were used in equimolar quantities on

Table 2. Effect of RCM Reaction Concentration on HPLC Profile, Ru Content, Purity, and Yield of 1 entry

1

2

3

conc (mL DCE/g of diene 31) diene conca (mM) Zhan Catalyst-1B (wt %) diene remaining at 1 h (% LCAPb) product 1 at 1 h (% LCAP) combined oligomers at 1 h (% LCAP) Ru content after TPPO; MeOH trituration (ppm) yield after TPPO; MeOH trituration (%) purity after TPPO; MeOH trituration (% LCAP) Ru content after Siliabond-DMT (ppm) yield after Siliabond-DMT (%) purity after Siliabond-DMT (% LCAP)

80 15.2 1.25 1.3 94.2 3.3 380 78 98.0 66c 75 98.6

160 7.6 1.25 3.4 93.4 1.3 200 88 98.1 3.6 86 98.5

250 4.9 1.25 1.2 96.4 0.99 180 95 97.7 7.7 89 97.9

a

All reactions on 1 g scale. bAt 272 nm. cStirring was less efficient for this sample.

observed at this stage; however, HPLC purity was similar for all three experiments (∼98% LCAP). The resultant beige powders were then dissolved in 2-MeTHF and refluxed with 8 equiv (relative to the initial Ru catalyst) of Siliabond-DMT, a silicabound dimercaptotriazine solid-supported metal scavenger.40 After 16 h, filtration to remove the Siliabond-DMT and concentration of the respective product filtrates gave 1 as a pale yellow solid, with minimal yield losses (2−6%) and high purities (98% LCAP).41 Excellent Ru levels were observed (as low as 3.6 ppm), indicating the potential utility of this approach to produce macrocycle 1. During the course of this work, IDX316 was deprioritized and replaced by the more potent HCV PI candidate 2; therefore, further development and scale-up activities for target macrocycle 1 were not pursued. The intended subsequent crystallization process (potentially using n-PrOH or IPA) to provide a final purity upgrade was not employed. Although based on small scale experiments, the proof of concept for this new assembly of the proline-urea macrocyclic sulfonamide 1 via route 3 was achieved (Table 3). The RCM throughput was improved by 14-fold to 1.2 g diene per g of 1 relative to the Table 3. Comparison of RCM Reactions for Routes 1, 2, and 3 metric yield (%) catalyst loading (mg/g of diene) dilution (g of DCE/g of diene) conc (mM) throughput (g of diene/g of 1) catalyst required (g of catalyst/g of 1) solvent required (g of DCE/g of 1) RCM RMIa (g/g) RCM PMIb (g/g)

route 1 silyl ether diene 11 RCM

route 2 unprotected diene 18 RCM

route 3 sulfonamidediene 31 RCM

51 72

37 52

86 10

1242

628

201

1.9 16.3

4.9 16.7

7.6 1.2

1.172

0.868

0.016

20200

10500

241

2573 3152

1842 7113

242 269

a

Reaction Mass Intensity = total mass of reagents in process step (g)/ mass of product (g). bProcess Mass Intensity = total mass in process step (g)/mass of product (g). G



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Scheme 8. Formation of Pipecolic Acid 39

Conditions: (i) CDI, TEA, DCM, rt (quant); (ii) MeI, MeCN, rt (quant); (iii) TEA, THF, reflux (71%); (iv) Ph3P, DIAD, THF, 0−5 °C; (v) KOH, THF/H2O 1:1 (50% over two steps).

proceeding directly to the subsequent saponification. Accordingly, the crude ester was taken up in THF and hydrolyzed with aqueous KOH. Acid 39 was then precipitated as its potassium salt, which allowed for removal of both aqueous and organic soluble impurities. Subsequent ion exchange via acidification with aqueous HCl provided acid 39 in 50% yield from both quinolinol 14 and pipecolate 38 with >98% LCAP. Although somewhat laborious and far from ideal in terms of yield and efficiency, this protocol was sufficient to allow access to acid 39 on kilogram scale. With pipecolic acid 39 in hand, formation of the requisite dienyl-urea 40 was achieved via solution phase amide coupling using TBTU with 1.2 equiv of sulfonamyl dehydrocoronamate hydrochloride 24. As with acid 39, purification of the resulting dienyl-urea 40 was effected by precipitation of the corresponding potassium salt, via the acidic sulfonamide N−H. Ion exchange with aqueous HCl then provided the desired dienylurea 40 in its free acid form in 94% yield from acid 39 with excellent purity (99% LCAP).44 Macrocyclization to Protease Inhibitor 2. The highly functionalized dienyl-urea 40 proved to be an acceptable substrate for macrocyclization via RCM. As with dienyl-urea 31, initial investigations were performed with Zhan Catalyst-1B using DCE as reaction solvent; however, at a dilution of 240 mL per g of diene (5.0 mM), cyclodimerization was observed.45 After treatment of the reaction mixture with charcoal to quench the Ru catalyst and purification by silica gel chromatography, 10% of the cyclic dimer was isolated with a disappointingly low 34% yield of the desired macrocycle 2. Increasing the dilution further by a factor of 2 improved the isolated yield to the 60− 65% range (Table 4, entry 2); however, even at 790 mL per g dilution (1.5 mM), no improvement was observed, and 2 was isolated in 56% yield on 19 g scale (entry 4). In order to achieve superior results and avoid the use of large volumes of this ICH Class 1 chlorinated solvent, alternative reaction media were explored.46 In TBME at 500 mL per g of diene (2.4 mM), the RCM reaction was considerably slower, presumably in part

Scheme 9. Diene 40 Formation and Macrocyclization to IDX320 (2)

Conditions: (i) TBTU, DIPEA, DMF, 0−5 °C (94%); (ii) Zhan Catalyst-1B (3 mol %), EtOAc, 72−75 °C (45−66%).

scale-up. The triphenylphosphine oxide (TPPO) Mitsunobu byproduct was partially removed on trituration with TMBE, and silica gel chromatography was utilized as an additional purity upgrade operation to remove further TPPO and to recover the remaining unreacted quinolinol 14. The crude quinolinyl pipecolate ether was only 50% w/w purity at this stage due to the presence of the reduced-DIAD Mitsunobu byproduct; however, this impurity was successfully purged on H



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Table 4. Effect of Solvent and Reaction Conditions on RCM Yield for Dienyl-urea 40 entry

solvent

1 2 3 4 5 6 7 8 9 10 11 12

DCE DCE DCE DCE TBME EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc

dilution (mL/g; mM) 240; 480; 600; 790; 496; 120; 240; 400; 460; 512; 500; 500;

5.0 2.5 2.0 1.5 2.4 10.0 5.0 3.0 2.6 2.3 2.4 2.4

catalyst (mol %)

temp (°C)

scale (g)

yield* (%)

3.0 3.0 3.0 3.0 3.0 + 1.8 3.0 3.0 3.0 3.0 3.2 3.0 3.3

78−80 78−80 78−80 78−80 55−56 74−76 74−76 74−76 73−75 72−75 72−75 72−75

4.1 1 1 19 1.3 1 1 1 177 726 1344 1525

34 63 60 56 49 46 63 69 74 73 66 66

Table 5. Effect of Purification Operation on API Residual Impurity Content entry 1 2 3 4 *

purification operation silica gel chromatography 1st IPA crystallization 2nd IPA crystallization EtOH/water crystallization

residual diene 40 (% LCAP)

residual ru level (ppm)

1.6

130

0.22

30

residual solvent (% w/w)

yield* (%) 66

IPA, 1.27

53

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