Topics in Current Chemistry

, 374:77 | Cite as

Process Chemistry in Antiviral Research

  • Yong-Li Zhong
  • Nobuyoshi Yasuda
  • Hongming Li
  • Mark McLaughlin
  • David Tschaen
Part of the following topical collections:
  1. Antiviral Drug Research


This article reviews antiviral therapies that have been approved for human use during the last decade, with a focus on the process chemistry that enabled access to these important drugs. In particular, process chemistry highlights from the practical syntheses of the HCV drugs sofosbuvir (Gilead), grazoprevir (Merck), and elbasvir (Merck), the HIV therapy darunavir (Tibotec) and the influenza treatment peramivir (BioCryst) are presented.


Process chemistry Antivirals HCV HIV Influenza Practical synthesis Asymmetric synthesis Sofosbuvir Grazoprevir Elbasvir Darunavir Peramivir 

1 Introduction

Viruses are the most abundant type of biological entity on earth and they can be found in almost every ecosystem on the planet [1]. Although it is likely that there are millions of different types of viruses in existence, only around 5000 individual virus species have been fully characterized to date [2]. Viruses are small infectious agents that replicate themselves by hijacking the normal molecular processes that occur inside the cells of other organisms. Viruses can infect all types of life forms, including animals, plants, and microorganisms such as bacteria and archaea [3]. Many viruses can be seriously harmful to human health, including the human immunodeficiency virus (HIV), the hepatitis B and C viruses (HBV and HCV), the influenza virus, and the human papillomavirus (HPV).

Since the first antiviral medicine was discovered in the 1960s, the search for new and effective antiviral agents has continued with increasing intensity [4]. In the last decade alone, a total of 32 new human antiviral therapies including 26 small molecules and six biologics (Pegasys® (peginterferon alfa-2a) is a polyethylene glycol (PEG)-modified form of human recombinant interferon alfa-2a. also see: [5]; Pegintron® (peginterferon alfa-2b) is also a polyethylene glycol (PEG)-modified form of human recombinant interferon alfa-2b. The main difference between Peginterferon alfa-2a and -2b is in the dosing; Rebetol® (ribavirin) [6, 7, 8, 9, 10]; Infergen® (interferon alfacon-1), interferons are a family of small protein molecules with molecular weights of 15,000–21,000 Da, which are produced and secreted by cells in response to viral infections or to various synthetic and biological inducers, Gardasil® is a vaccine for the treatment of HPV type 6, 11, 16, 18; Gardasil® 9 is a vaccine for the treatment of HPV types 9; Cervarix® is a vaccine for the treatment of HPV types 16, 18) have been approved by regulatory authorities around the world, the majority of which fall in the small molecule category. Within this group of new small molecule therapies, 12 compounds are for the treatment of HCV infections (Fig. 1) (Viekira Pak™ (a combination of ombitasvir, paritaprevir, ritonavir, and dasabuvir): for ombitasvir [11, 12], for paritaprevir [13, 14], for ritonavir [15, 16, 17, 18, 19, 20, 21], for dasabuvir [22, 23]); Harvoni® (a combination of ledipasvir and sofosbuvir): for ledipasvir [24, 25, 26, 27, 28]; Olysio® (a combination of simeprevir and sofosbuvir): for simeprevir [29, 30, 31, 32, 33, 34, 35, 36]; Incivek™ (telaprevir) [37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49]; Victrelis® (boceprevir) [50, 51, 52, 53, 54, 55, 56, 57]; Sovaldi® (sofosbuvir), see [109, 110, 111, 112, 113, 114, 115], see also footnotes 1–3; Zepatier® (a combination drug of grazoprevir and elbasvir), see [116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130], see also footnotes 4, 5), ten compounds are for the treatment of HIV (Fig. 2) (Intelence® (etravirine) [58, 59]; Viramune® (nevirapine, NVP) [60, 61, 62]; Edurant® (rilpivirine, RPV) [63, 64, 65, 66]; Aptivus® (tipranavir) [67, 68, 69]; Selzentry® (maraviroc) [70, 71, 72]; Tivicay® (dolutegravir) [73, 74, 75, 76]; Vitekta™ (elvitegravir) [77, 78, 79, 80]; Isentress® (raltegravir) [81, 82, 83, 84]; Tybost™ (cobicistat) [85, 86, 87]; the four cocktail drugs are Triumeq® (a combination of abacavir, dolutegravir, and lamivudine); Atripla® (a combination of efavirenz, emtricitabine and tenofovir); Stribild® (a combination of elvitegravir, cobicistat, emtricitabine, and tenofovir), and Complera® (a combination of emtricitabine, rilpivirine, and tenofovir); Prezista® (darunavir), see [131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142]) and two additional compounds each are for the treatment of HBV infections (Baraclude® (entecavir) [88, 89, 90]; Tyzeka® (telbivudine) [91, 92, 93]) and influenza infections (Laninamivir [94, 95]; Rapivab™ (peramivir), see [143, 144, 145, 146, 147, 148, 149]) (Fig. 3). In this chapter, process development highlights from the published syntheses of selected examples (sofosbuvir, grazoprevir, elbasvir, darunavir, and peramivir) are presented.
Fig. 1

Compounds for the treatment of HCV infections approved in the past 10 years

Fig. 2

Compounds for the treatment of HIV infections approved in the past 10 years

Fig. 3

Compounds for the treatment of HBV and influenza infections approved in the past 10 years

Within the pharmaceutical industry, synthetic organic chemistry encompasses both medicinal chemistry and process chemistry activities. Medicinal chemists focus on the discovery of appropriate small molecules that interact with biological mechanisms in such a way as to provide a beneficial effect on a given disease state. Process chemists aim to design and develop efficient, practical, and economical chemical syntheses for the drug candidates identified by medicinal chemists. The differing goals of medicinal and process chemists have a significant bearing on the nature of the synthetic chemistry strategies employed by either group of chemists. Broadly, medicinal chemists typically design flexible synthetic routes that quickly access common building blocks from which many candidate compounds can be readily prepared, allowing structure–activity relationships to be determined rapidly and the appropriate molecular entity to be identified. Reaction yield, expense, robustness, and green chemistry considerations are of secondary importance relative to speed of compound synthesis and acquisition of data from biological assays. Conversely, the industrial manufacture of drug compounds at commercial scale puts a premium on those synthesis attributes relegated to lesser significance by medicinal chemists. Consequently, process chemists seek to devise an ideal route that has maximum synthetic efficiency and is high yielding. Often this necessitates the invention of new synthetic methods, providing an excellent venue for creative chemists to conceive novel applications of existing transformations or even to develop entirely new reactions. The commercial process must be operationally safe, cost-effective, and sufficiently robust to deliver API in consistent high quality. Intellectual property (IP) aspects must also be considered when designing and developing the synthesis to assure freedom to operate without infringing upon competitor patents [96]. Lastly, given the larger scale of operation relative to medicinal chemistry activities, minimizing the environmental impact of industrial drug production is a priority and drives the application of green-chemistry principles wherever possible.

In addition to the myriad of technical objectives faced by process chemists, attention must also be given to broader, cross-functional facets of development programs including program drivers and timelines. Typically, in the early stages of pre-clinical and clinical drug development, relatively small quantities of drug substance are required to support initial in vitro and in vivo toxicology studies and the speed of API delivery is normally prioritized over definition of an ideal synthesis. This being the case, it is not unusual to have an interim synthesis (“supply route”) used to support early API deliveries with a parallel (or staggered) effort to develop increasingly refined routes that are aligned with the overall program context, considering factors such as drug demand, synthesis complexity/cost and clinical timelines. In an ideal world, the best, most efficient synthesis is established as early as possible in the overall timeline but in practice it is common to observe an evolution in synthetic efficiency over the development cycle, culminating in the manufacturing route being ready as the program moves closer to regulatory filing (often in phase III) [97, 98]. The examples of drug development featured in the subsequent section were selected to relate various aspects of process chemistry alluded to in this introduction. In each case presented, the intent is to focus on key challenges and highlights encountered during process development. The examples presented in this chapter may not be the actual commercial routes, however they reflect the information that is currently available in the literature. The reader is directed to the original publications for further details on the complete syntheses if desired.

2 Medicinal Therapies for the Treatment of HCV

The management of HCV continues to present a global health problem. According to a report by the World Health Organization (WHO), it is estimated that 130–170 million people have chronic HCV infection worldwide. Approximately half a million deaths each year result from HCV-related diseases [99, 100]. For these reasons, the search for effective antiviral agents to combat HCV is an ongoing endeavor within the global medical/pharmaceutical community [101, 102].

2.1 Chemical Synthesis and Process Development of Sofosbuvir: An HCV NS5B Nucleoside Polymerase Inhibitor

2.1.1 Background

Sofosbuvir is a treatment for HCV marketed by Gilead Sciences under the trade name Sovaldi® (Fig. 1). Initially developed at Pharmasset, following acquisition by Gilead in 2011 for approximately $11 billion, further clinical development of sofosbuvir resulted in FDA approval of this drug in December 2013. The mechanism of action of sofosbuvir is via inhibition of the non-structural 5B (NS5B) nucleoside polymerase enzyme [103, 104]. Treatment duration for a course of Sovaldi® is 12 weeks and the cure rate is 90%. Gilead Sciences sold $10.3 billion of Sovaldi® in 2014, making it the second best-selling drug in the world in its first year on the market [105, 106]. The following sections present the medicinal chemistry route to sofosbuvir and highlights key synthetic advances made during process development.

2.1.2 Medicinal Chemistry Route

The medicinal chemistry route for the synthesis of sofosbuvir was carried out in ten linear chemical steps in approximately 0.5% overall yield (Scheme 1) [107, 108]. The synthesis of the API started from commercially available cytidine. Selective benzoylation of cytidine with benzoic anhydride followed by silylation with TIDPSCl2 in the presence of pyridine afforded alcohol 1 in 63% yield over two steps. Oxidation of the alcohol 1 under Swern oxidation conditions provided the corresponding ketone 2 in 78% yield. Addition of methyllithium to the ketone 2 at −78 °C in diethyl ether gave exclusively alcohol 3 in quantitative yield. De-silylation of 3 with TBAF-AcOH (61% yield) followed by benzoyl protection afforded intermediate 5 in 67% yield. Fluorination of the tertiary alcohol 5 with diethylaminosulfur trifluoride (DAST) delivered the desired product 6 in 19% isolated yield. Treatment of 6 in 80% acetic acid aqueous solution gave the corresponding uridine 7, which was converted to diol 8 by deprotection in the presence of methanolic ammonia with 78% yield over two steps.
Scheme 1

Medicinal chemistry route to sofosbuvir

The chlorophosphoramidate 9 was prepared by the reaction of isopropyl l-alanate hydrogen chloride with phenyl phosphoro-dichloridate in the presence of N-methylimidazole (NMI). With the two key fragments uridine nucleoside 8 and chlorophosphoramidate 9 in hand, the combination of these in the presence of NMI gave a 1:1 mixture of diastereomers (at the phosphorus center) epi-sofosbuvir and sofosbuvir. The desired sofosbuvir was isolated in high purity after crystallization and recrystallization. Thus, the medicinal route for the synthesis of sofosbuvir was carried out in ten linear chemical steps from cytidine in 0.5% overall yield.

2.1.3 Process Development Routes Retrosynthetic Analysis
The medicinal chemistry route shown previously was appropriate to provide the initial gram quantities of sofosbuvir to establish the compound as a drug candidate of significant interest, however an improved synthesis was required to support continued development activities. Sofosbuvir is comprised of three main structural fragments; the ribose derivative, the base and the phosphoramidate pro-drug attachment. Although alternate disconnections are conceivable, retrosynthetic cleavage at the anomeric and phosphorus centers presents three building blocks that can be linked together in covergent fashion. Consequently, in this case, the retrosynthetic analysis for the process route to sofosbuvir follows a similar synthetic strategy to that of the discovery route (Scheme 2). Nevertheless, within this overall analysis there remained significant scope for alternate approaches to these main building blocks and also how they could be linked together. Two key synthetic challenges presented by this molecule can be recognized in the medicinal chemistry route. First, the use of the DAST reagent for introduction of the 2′-fluorine substituent is costly. Moreover, the low yield of 19% for this step compounds the cost issue and significantly decreases the overall yield of the synthesis. A second major issue to be addressed for the production of sofosbuvir is the attachment of the phosphoramidate to the nucleoside fragment of the molecule. The phosporamidate functional group is used to create what is known as a “pro-drug”, a technique whereby the parent drug molecule is rendered more efficacious by forming a derivative that is more easily absorbed into the bloodstream (i.e., making the drug more bio-available)1 In this particular case, the phosphorus atom in the phosphoramidate pro-drug appendage is chiral and control of stereochemistry during installation of this group is a non-trivial synthetic transformation. Highlighted below are several approaches that address these key issues for process development of sofosbuvir.
Scheme 2

Retrosynthetic analysis of sofosbuvir Synthetic Approaches to Lactone 11
Lactone 11 represents a key synthon for the ribose fragment of sofosbuvir and two syntheses that address some of the previously highlighted issues with the discovery route have been described in the literature. The first approach described by Gilead Sciences (Scheme 3, route 1) follows a retrosynthesis where lactone 11 is disconnected to reveal (D)-glyceraldehyde acetonide 12 as a chiral pool starting material [109]. A second route published by the MacMillan group (Scheme 3, route 2) disconnects 11 back to a fluoropropionate derivative and an alternate glyceraldehyde synthon 15 2 [110, 111].
Scheme 3

Retrosynthetic analysis of lactone 11

The Gilead synthesis begins with Wittig olefination of d-glyceraldehyde 12 using 13 to provide enone 16 in 79% yield with E/Z ratio 97:3 (Scheme 4). Dihydroxylation of 16 with potassium permanganate in acetone gives the desired diol 17 in 67% isolated yield after crystallization. Cyclic sulfite 18 formation with thionyl chloride followed by oxidation in the presence of bleach and a catalytic amount of TEMPO affords the corresponding cyclic sulfate 19. Fluoride-induced opening of the cyclic sulfate 19 (notably at the fully substituted α-carbon) is achieved by tetraethylammonium fluoride hydrate and provides product 20. This obviates the use of DAST for the installation of the fluoride, which is a significant benefit to this approach. The utilization of DAST for large-scale operation could be potentially challenging from a safety perspective. Selective hydrolysis of the sulfate 20 to alcohol 21 is achieved by using concentrated hydrochloric acid in the presence of 2,2-dimethoxypropane, an additive that helps suppress undesired premature hydrolysis of the acetonide functionality. Alcohol 21 is finally converted to lactone 11 by further treatment with concentrated hydrochloric acid in 67% overall yield from diol 17. Thus, the lactone 11 can be prepared in a seven-step sequence in 35% overall yield [109].
Scheme 4

Synthesis of lactone 11 via Wittig reaction

The MacMillan approach involves a four-step linear synthesis to prepare lactone 11-OBn via route-2 as shown in Scheme 5. Enantioselective α-coupling of commercially available 3-(benzyloxy)-propanal with TEMPO in the presence of 10 mol% CuCl2 catalyst and 20 mol% of chiral organocatalyst 22 affords aldehyde 15 in 77% yield and 90% ee. Mukaiyama–aldol reaction of aldehyde 15 with silyl ketene acetal 14 in the presence of Lewis acid TiCl2(O i Pr)2 furnishes β-hydroxyester 23 in 79% yield as a single diastereomer after isolation. This approach involves the use of a fluoride-containing starting material (14) as an alternative to using DAST for the installation of fluoride. Zn-mediated reductive NO bond cleavage of the OTMP group provides alcohol 24, which is converted to lactone 11-OBn by treatment with TFA in 80% overall yield. It is possible to upgrade the stereochemical purity of lactone 11-OBn to 99% ee by recrystallization. Thus, the lactone 11-OBn can be prepared in four linear steps and 49% overall yield from 3-(benzyloxy)propanal [110, 111, also see footnotes 2].
Scheme 5

Synthesis of lactone 11-OBn via Mukaiyama–aldol Elaboration of Lactone 11 to Nucleoside 29
Benzoylation of the diol 11 in the presence of benzoyl chloride and pyridine gives bis-benzoate 25 in 70% yield (Scheme 6). Lithium tri-tert-butoxyaluminum hydride-mediated reduction of lactone 25 provides lactol 26 in a 2:1 β/α anomeric ratio, which is converted to acetate 27 in the presence of acetic anhydride and pyridine. The ratio of β/α anomers does not impact stereoselectivity in the next step, which occurs via a planar oxonium cation. Thus, tin chloride-mediated glycosylation of the acetate 27 with N-(2-((trimethylsilyl)oxy)pyrimidin-4-yl)benzamide affords a mixture of β/α (4:1) nucleoside 28, which was isolated by crystallization to give β-anomer nucleoside 28 in 29% yield from lactone 11. Deprotection of 28 in the presence of methanolic ammonia gives the key nucleoside 29 in 88% yield [109].
Scheme 6

Synthesis of key intermediate nucleoside 29 Attachment of the Phosphoramidate Pro-drug Functionality
As outlined above, the phosphoramidate pro-drug group of sofosbuvir contains a stereogenic phosphorus atom, which presents an interesting synthetic challenge. The stereoselective synthesis of P-chiral compounds has been documented. In the case of sofosbuvir, the phosphoramidate group contains another stereogenic center (in the isopropylalanine amino ester unit) and therefore one could envisage taking advantage of this pre-existing stereocenter to control the ultimate configuration at phosphorus. In practice, this is achieved via pre-formation of a diastereomerically pure phosphoramidate transfer reagent and reaction in a stereospecific manner with the 5′-hydroxy group of the sofosbuvir nucleoside. As shown in Scheme 7, reaction of phenyl phosphorodichloridate with alanine isopropylester generates a 1:1 diastereomeric mixture of intermediates 30a/30b. This mixture is reacted with pentafluorophenol to afford another 1:1 mixture of diastereomers of 31a/31b. At this stage, it is possible to crystallize the desired diastereomer 31a in 34% overall yield with excellent rejection of 31b (>98% de for isolated solid). Selection of pentafluorophenol as the derivatizing reagent here was critical to endow 31a with the ideal properties for subsequent use, namely adequate stability to allow crystallization (stereochemical upgrade) and sufficient reactivity to enable coupling with the sofosbuvir nucleoside via stereospecific displacement of pentafluorophenoxide3 [112, 113, 114].
Scheme 7

Preparation of chiral phosphoramidate 31

Hydrolysis of pyrimidine nucleoside 29 in refluxing 80% aqueous acetic acid converts it to uridine 33 in 88% yield (Scheme 8). Stereospecific displacement of pentafluorophenoxide from chiral phosphoramidate 31a by the magnesium alkoxide generated from 33 via deprotonation using tert-butylmagnesium chloride gives crude sofosbuvir in ~99% de. Recrystallization of the crude product from dichloromethane delivers the sofosbuvir drug substance in 68% isolated yield with 99.8% purity and 99.7% de [115].
Scheme 8

Final stages for the synthesis of the sofosbuvir

Overall, the process chemistry route to sofosbuvir successfully addressed two of the major issues identified in the medicinal chemistry route via the implementation of improved approaches for the introduction of fluorine and control of the phosphoramidate stereochemistry. By virtue of these improvements, it is possible to synthesize sofosbuvir (without recourse to chromatography) in approximately 5% overall yield, representing an approximate tenfold improvement from 0.5% yield in the discovery route. As described previously, this is a summary of the work published by the Process Chemistry groups of Pharmasset and Gilead to date. It is unclear which aspects of this work comprise the contemporary manufacturing route for sofosbuvir, but it is anticipated that further improvements have been achieved and may be communicated in due course.

2.2 Chemical Synthesis and Process Development of Zepatier: A Combination of HCV NS3/4A (Non-structural Protein 3/4A) (Grazoprevir) and HCV NS5A (Non-structural Protein 5A) (Elbasvir) Inhibitors

2.2.1 Background

Zepatier® is a two-drug combination therapy for the treatment of HCV that was developed at Merck and approved by the FDA in early 2016. Zepatier® is a combination of a HCV NS3/4A protease inhibitor (grazoprevir) and a HCV NS5A protein inhibitor (elbasvir) (Fig. 1). Similar to Sovaldi®, Zepatier® is intended to be prescribed orally and once-daily for 12 weeks. This provides a significant quality-of-life benefit for patients with respect to existing treatment regimens that typically require co-treatment with a small molecule (ribavarin) and an injectable biological agent (interferon); unpleasant flu-like symptoms are typically associated with this mode of therapy, often causing a failure in patient adherence to the treatment plan. Additionally, the ribavarin/interferon combination achieves only moderate cure rates (in the range 40–80%) depending, among other factors, on the genotype of HCV being treated. Notably, Zepatier has achieved a 92% cure rate in clinical trials without reports of the side effects typically reported for the ribavarin/interferon combination [116, 117]. The sections below outline the major synthetic challenges associated with both grazoprevir and elbasvir and how process chemistry innovation enabled development and commercialization of these critical new drugs.

2.2.2 Development of Grazoprevir Medicinal Chemistry Route to Grazoprevir
The medicinal chemistry route for the synthesis of grazoprevir is outlined in Schemes 9 and 10. This synthesis involves 14 linear chemical steps and delivers grazoprevir in approximately 3% overall yield [118]. Starting from commercially available acrolein, a Michael addition of 4-butenylmagnesium bromide in the presence of 5 mol% of CuBr2·SMe2 followed by in situ trapping with TMSCl gave the silyl enol ether 34 in 92:8 E/Z regioselectivity and 55% yield. This intermediate 34 required purification by distillation under reduced pressure before use in the downstream steps. Simmons–Smith cyclopropanation of 34 with ZnEt2 and CH2I2 afforded the desired trans-cyclopropane 35, which was converted to the racemic cyclopropanol (±)-36 in 91% overall yield following desilylation using TBAF. Enzymatic acetylation of the racemic alcohol (±)-36 with vinyl acetate gave the product in only 50% ee. The resulting acetate was purified by chromatography and then methanolysis with NaOMe provided chiral cyclopropanol 36 in 50% ee and 53% overall yield without optimization. Treatment of 36 with methyl (S)-2-isocyanato-3,3-dimethylbutanoate, which was prepared in situ from methyl 3-methyl-l-valinate hydrochloride and triphosgene, generated carbamate 37 in 60% yield as a single diastereomer after chromatographic purification. LiOH-mediated hydrolysis of 37 gave the corresponding carboxylic acid 38 in 98% yield.
Scheme 9

Synthesis of carbamate acid 38

Scheme 10

Synthesis of quinoxaline–pyrrolidinium chloride 43

The synthesis of key intermediate 43 started with the condensation of 4-methoxy-phenylenediamine hydrochloride with diethyl oxalate in the presence of triethylamine, giving 6-methoxyquinoxaline-2,3-diol 39 in 69% yield (Scheme 10). Regioselective chlorination of the diol 39 with thionyl chloride in DMF afforded 3-chloro-7-methoxyquinoxalin-2-ol 40. S N 2 displacement of bromobenzenesulfonate 41, which was prepared in one step from N-Boc-4-cis-hydroxyproline methyl ester with 40 in the presence of Cs2CO3, followed by a chromatographic purification led to product 42 in 70% overall yield from 39. N-Boc-deprotection with 4 M hydrogen chloride in dioxane provided the target intermediate 43 in 99% yield.

With the two key components 38 and 43 in hand, HATU-mediated amide coupling between these two intermediates in the presence of DIPEA afforded chloroquinoxaline 44 in 78% yield after chromatographic purification (Scheme 11). Cross-coupling of 44 with potassium trifluoro(vinyl)borate in the presence of 10 mol% of PdCl2(dppf)-CH2Cl2 adduct and triethylamine gave the bis-olefin 45, which was subjected to a ring-closing metathesis (RCM) reaction in 0.02 M dichloroethane solution with 10 mol% of the Zhan 1b catalyst to give the corresponding macrolactam 46 in 25% yield after chromatographic purification. Reduction of 46 in the presence of 10% Pd/C, followed by LiOH-mediated hydrolysis, gave the corresponding free acid 47, which was converted to grazoprevir in 89% overall yield from 46 by an amide coupling with commercially available 48 in the presence of TBTU and base.
Scheme 11

Final stages for the synthesis of grazoprevir Process Chemistry Route

Retrosynthetic Analysis As is often the case in pharmaceutical development, the medicinal chemistry route described above was determined inadequate to prepare the typical quantities of API needed for clinical evaluation of grazoprevir. Major issues for process development included the low yield for the key macrocycle-forming RCM reaction, together with uncertainty around freedom to operate using RCM catalysts for commercial production due to the extensive patent coverage in the methathesis space. Hydrolytic instability of TMS enol ether 34 and overall low efficiency/high cost of the synthesis were additional concerns in the context of increasing scale of operation. Although the size and molecular complexity of grazoprevir provides enormous opportunity for synthesis exploration, it was necessary to balance the use of interim chemistry against the search for the ultimate route in order to expedite clinical development. In this regard, the early process routes to grazoprevir had a focus not only on better chemistry but also on speed of access to clinically useful amounts of API. This approach resulted in several synthetic route variations that evolved over time as the compound advanced through clinical development. In this section, the chemistry used for a first-generation interim “supply route” will be discussed. Approaches to address the significant issues associated with the medicinal chemistry synthesis will be highlighted.

An obvious structural feature of grazoprevir is the 18-membered ring macrolactam ring and any potential process route would need to address the low yielding RCM reaction used to form this ring in the discovery route. Although numerous methods for formation of macrocycles have been reported in the literature, the most common and practical synthetic approaches to these large rings are RCM, macrolactamization, and transition metal-catalyzed coupling reactions [119]. In particular, macrolactamization is a well-documented strategy that is largely free of IP considerations, making it relatively attractive in comparison to RCM approaches. As such, the retrosynthetic approach taken for the first-generation process chemistry is outlined in Scheme 12. Grazoprevir can be derived from three key moieties: chloroquinoxaline 42, vinylcyclopropane 48 and alkyne acid 49. Further disassembly of the key alkyne acid 49 can reduce this fragment to commercially available pinacol vinyl boronate 50 and l-tert-leucine. Chloroquinoxaline 42 can disconnect via a S N Ar displacement of the dichloroquinoxaline (DCQ) and commercially available inexpensive N-Boc trans-l-hydroxyproline methyl ester (Boc HP)4 [120].
Scheme 12

Retrosynthetic analysis of grazoprevir

Synthesis of Alkyne Acid 49 Racemic cyclopropanation of the pinacol vinyl boronate 50 with diethylzinc and diiodomethane in the presence of TFA provides a 79% yield of the trans-isomer (Scheme 13). It is worth noting that the use of diethylzinc is not desirable in a manufacturing route due to safety concerns associated with this pyrophoric reagent, however with proper precautions it is feasible to use this cyclopropanation at fairly large scale5 Moreover, this chemistry was found rather quickly during development of grazoprevir and it is relatively robust, which was in alignment with the previously mentioned interim goal to rapidly develop a useable API synthesis that could provide meaningful quantities in short order. Hydrogen peroxide oxidation of the C–B bond of cyclopropane 51 affords the corresponding racemic trans-chlorocyclopropanol 52 in 69% yield. Next, S N 2 displacement of the lithium salt of 52 with 1.1 equiv of lithium acetylide-ethylene diamine complex gives racemic alkyne alcohol (±)-53, which is converted to racemic alkyne acetate (±)-54 by treatment with acetyl chloride in the presence of triethylamine. Enzyme-mediated selective hydrolysis of the acetate gave the corresponding chiral alkyne alcohol 53 in 96% ee and in 40% overall yield from (±)-53 [121]. Again, the use of an enzyme to conduct a kinetic resolution of the racemic mixture (losing half of the material in the process) reflects the desire for a quick answer to the synthetic problem posed by the alkyne acid. Last, CDI activation of enantio-enriched 53, followed by reaction with tert-l-leucine furnishes the alkyne acid 49 in 75% yield [120, also see footnote 4].
Scheme 13

Synthesis of alkyne acid 49

Final Stages for the Synthesis of Grazoprevir via the Synthesis of Chloroquinoxaline 42 Chloroquinoxaline 42 is prepared by a S N Ar displacement of dichloroquinoxaline (DCQ) with inexpensive Boc-HP. The reaction proceeds with 95:5 regioselectivity and a 71% isolated yield is observed after crystallization to reject the undesired regioisomer. The Sonogashira cross-coupling between chloroquinoxaline 42 and alkyne acid 49 is achieved with 3 mol% of Pd(OAc)2, 6 mol% of P(t-Bu)3BF4, and 2.5 equiv of K2CO3 (2.5 equiv) to afford product 55 in 98% HPLC assay (solution) yield. Intermediate 55 is not isolated but instead is processed forward into the downstream chemistry, which involves Pd-catalyzed hydrogenation of the triple bond of 55 was conducted with a catalytic amount of Pd/C to provide the saturated product 56 in 89% HPLC assay yield. The N-Boc group was removed by treatment of crude 56 with PhSO3H and the product benzene sulfonate salt was neutralized in situ by addition of Hünig’s base. The resulting mixture was slowly added to a solution of HATU (1.25 equiv) in MeCN to complete a one-pot process that gave a 65% isolated yield of marcolactam 57 in >98% purity after crystallization. Through this slow addition for the cyclization, the macrolactamization was allowed to run in ~0.2 M concentration, which is 10× more efficient than the RCM reaction (0.02 M). Lithium hydroxide promoted saponification of 57 leads to the corresponding acid 47 in 95% yield, which is converted to grazoprevir in 92% yield via amide coupling with commercially available 48 in the presence of EDC, Hünig’s base and catalytic amount of HOBt [120, also see footnote 4].

The first-generation process route to grazoprevir described here serves as an excellent example of how it is often impossible to simultaneously address every synthetic issue during the early stages of development while adequately supporting program demands that sometimes prioritize API supply over how that supply is obtained. In this case, the key problem of inefficient macrocycle formation was solved via a change in synthetic strategy from low yielding, solvent-intense RCM to a hugely preferable macrolactamization that occurs in high yield under much greener reaction conditions (lower solvent usage). However, other identified deficiencies such as the inefficient and unscalable approach to the alkyne acid 49 were only partially improved upon in this first-generation process route. For example, the unstable TMS-enol ether substrate for cyclopropanation was replaced by the more well behaved alkene-boronate but the need for pyrophoric diethylzinc remains a less attractive feature of this route. Furthermore, the racemic nature of the alkyne acid 49 synthesis is not optimal for long-term commercial manufacture due to the inherent waste associated with this approach. The description of an alternative approach for the synthesis of grazoprevir will be the subject of future reports from Merck Research Laboratories (Scheme 14).
Scheme 14

Final stages for the synthesis of grazoprevir

2.2.3 Development of Elbasvir Medicinal Chemistry Route
The synthesis of elbasvir by medicinal chemists at Merck Research Laboratories, in which the core benzoxazinoindole was synthesized via a racemic approach, was achieved in nine steps with 3.9% overall yield [122]. As shown in Scheme 15, acylation of 3-bromophenol, followed by Fries rearrangement, gave desired acetophenone 58 in 70% yield. The 2′-phenol-indole derivative 59 was obtained smoothly through traditional Fisher indole synthesis. Double alkylation of NH and OH with dibromomethylbenzene afforded the racemic benzoxazinoindole core 60 in moderate yield, which was directly used in next step. Next, a bis-borylation/bis- Suzuki–Miyaura cross-coupling sequence with bromoimidazole 61 (both catalyzed by Pd(dppf)Cl2) followed by N-Boc-deprotection with acid gave the corresponding bis-imidazole derivative 62 as the mixture of disastereomers. The chiral bromoimidazole 61 was synthesized in three steps from the chiral pool starting material N-Boc-l-proline aldehyde via a Radziszewski imidazole synthesis, bis-bromination with NBS and mono-des-bromination with Na2SO3 in 41% overall yield (Scheme 16). A final bis-amide formation between the free amine 62 with N-methoxycarbonyl-l-valine generated the product as the mixture of diastereomers, from which optically pure elbasvir was obtained by SFC separation.
Scheme 15

Medicinal chemistry route

Scheme 16

Synthesis of chiral bromo-proline imidazole 61 Process Chemistry Route
Retrosynthetic Analysis: Major Disconnections As alluded to in the introduction to this chapter, it is often the case that the synthetic route to a drug substance evolves over time in concert with gains in technical understanding of the chemistry and wider cross-functional demands/drivers. The development of elbasvir represents an excellent example of this synthetic evolution, where it took several years of in-depth study of the chemistry in parallel with supplying drug substance in support of time-critical clinical trials before the ultimate route to the API was identified. In the following sections the development of first- and second-generation process chemistry routes will be described, drawing comparisons between those and the original medicinal chemistry synthesis. In a retrosynthetic sense the approach taken for disconnection of elbasvir was broadly similar to that of the discovery chemistry analysis. As shown in Scheme 17, elbasvir can be cleaved into two building blocks; the central benzoxazinoindole core and side chain(s) 63 by cleavage of two sp2–sp2 C–C bonds. A major objective for process chemistry was the identification of an asymmetric approach to the core chiral benzoxazinoindole fragment, which would obviate the chiral chromatography in the medicinal chemistry synthesis. This objective was met in both iterations of the synthetic route although different strategies were employed for the synthesis of this fragment in each route.
Scheme 17

Retrosynthetic analysis of elbasvir—major disconnections

First-Generation Process Chemistry Route to Elbasvir In the first-generation approach, the general idea was to disconnect the benzoxazinoindole at the benzylic C–O and C–N bonds to reveal 2-(1H-indol-2-yl)phenol 59 and an appropriate electrophile such as a dihalomethylbenzene or benzaldehyde (Scheme 18). It was envisaged that a phase-transfer-catalyzed (PTC) double alkylation or an asymmetric condensation could be conducted using these synthons in conjunction with 59.
Scheme 18

Direct cyclization from Indole to the chiral benzoxazinoindole 64

Access to the 2-phenol-indole substrate 59 is relatively straightforward so the asymmetric synthesis of benzoxazinoindole 64 starting from this potential precursor was explored extensively. Initial work focused on the PTC approach under basic reaction conditions. As shown in Scheme 18, although this bond formation was viable in a racemic sense for the discovery route, attempts to render this process asymmetric using PTC were not successful. Switching tactics, the direct condensation of 2-phenol-indole 59 with benzaldehyde in presence of chiral Lewis acid or chiral Brønsted acid was also explored but this gave only the indole C-3 cyclization product due to the intrinsic higher reactivity of the indole C-3 position over the indole nitrogen (for chiral hemiaminal synthesis using chiral phosphoric acid catalysts, see [123, 124]. Other examples [125, 126, 127]).

Faced with the challenge presented by the unique benzoxazinoindole motif and failure of the initial attempts, the project team devised an entirely novel approach. Rather than attempting to form the desired chiral center starting from an achiral intermediate (59), it was proposed to utilize a related compound (66) at a different oxidation state (indoline vs. indole) that bears an additional stereocenter. In this strategy, it was anticipated that a stereochemical relay from chiral indoline 66 could be employed to control the benzoxazinoindole chiral center as depicted in Scheme 19. Meanwhile, this approach eliminated the issue related to the intrinsic C-3 reactivity of indole.
Scheme 19

Chiral relay approach via oxidation to the chiral benzoxazinoindole 64

In this scenario, chiral indoline 66 can be engaged in a diastereoselective cyclocondensation with benzaldehyde where the benzoxazinoindole stereocenter is controlled via 1,3-chiral induction from the indoline C-2 stereocenter (for a chiral relay strategy to elbasvir, see: [128, 129]). The inherent diastereoselectivity for this transformation is around 9:1 in favor of the desired compound 65 and the process is made even more efficient as a consequence of the widely differing solubilities of the diastereomeric products. This difference in solubility, coupled with the equilibrating (reversible) conditions employed for the condensation, enables a dynamic crystallization-driven process where the desired diastereomer 65 is crystallized directly from the reaction mixture with simultaneous re-equilibration of the mixture in solution to the thermodynamic 9:1 ratio. Overall, this remarkable transformation delivers 65 in excellent yield and with essentially complete control of stereochemistry. As shown in Scheme 20, this approach to controlling the benzoxazinoindole stereochemistry is the cornerstone of the synthesis, which starts from 2,5-dibromophenylacetic acid and bromophenol. Phenyl ketone 67 is obtained via an ester formation/Fries rearrangement sequence in good yield. Treatment of ketone 67 with NH3 in MeOH affords the desired imine 67a. It should be noted that reduction of imine 67a generates the stereocenter that ultimately controls the benzoxazinoindole stereocenter so it is critical to achieve good stereocontrol in this transformation. Application of asymmetric transfer hydrogenation conditions using the well-established (R, R)-Teth-TsDPEN-RuCl catalyst (0.3 mol% loading) furnishes the desired amine 68 in both excellent yield (96%) and stereoselectivity (98% ee). Further conversion of chiral amine 68 into indoline 66 via CuI-catalyzed intramolecular C–N bond formation delivers the requisite substrate for the stereochemical relay process described in detail above. Further oxidation of indoline 65 using conditions (KMnO4) carefully selected to avoid disturbance of the newly created benzoxazinoindole stereocenter generated the desired indole 64 in 83% yield and >99% ee. Definition of this effective asymmetric synthesis of the benzoxazinoindole core fragment met a key objective for process development and set the stage for the overall synthesis of elbasvir.
Scheme 20

Synthesis of elbasvir via chiral relay approach

Completion of the elbasvir first-generation process synthesis from this point onwards is similar to the discovery synthesis. The side-chains 63 are installed in stepwise fashion via a bis-borylation/bis-Suzuki–Miyaura cross-coupling sequence of 64 with Boc-protected bromoimidazole 61, followed by N-Boc deprotection final amide coupling with N-methoxycarbonyl-l-valine. During early development, the understanding of factors which influence reaction byproducts was incomplete and only weakly crystalline forms of the API were known. These deficiencies necessitated purity control at several crystalline intermediates prior to API isolation, requiring the stepwise approach to attaching the side chains 63. These issues were ultimately overcome as knowledge of the end-game chemistry improved and extensive work around the API crystal form identified more robust options for isolation of elbasvir. This understanding was put to good use in development of the second-generation synthesis described below. The first-generation process chemistry route delivers the API in 25% overall yield over an 11-step sequence. This is a significant improvement over the discovery synthesis and is largely due to the definition of a practical asymmetric approach to the benzoxazinoindole core.

Second-Generation Process Chemistry Route to Elbasvir In the first-generation process route described above, the key chiral center is established via a 1,3-stereochemical relay from the stereocenter already present in indoline intermediate 66. Although this approach represented a significant step forward from the discovery route, the process chemistry team sought to push the envelope and seek an even more efficient synthesis of elbasvir. It was recognized that two steps in the overall sequence were potentially redundant, namely the imine reduction and downstream indoline (re)-oxidation to the indole. To eliminate this redox chemistry it was desirable to identify an alternate means of controlling the benzoxazinoindole stereocenter that would maintain the appropriate oxidation state throughout the synthesis. To this end, the project team devised an innovative approach based on a novel dynamic kinetic resolution.

As shown Scheme 21, the bromo-benzoxazinoindole (70 or 71) can be formed via cyclization of imines 67ab with benzaldehyde under catalysis by Lewis or Brønsted acids. It was anticipated that 70 could be engaged in an intramolecular C–N bond formation to complete the indole construction. During a detailed study of intermediate 70, an observation that the compound was prone to racemization under certain conditions opened up the possibility of a unique novel transformation (Scheme 22).
Scheme 21

Comparison of the two approaches

Scheme 22

Racemization of hemiaminal derived from imine

Following the discovery of this stereochemical lability in 70, a dynamic kinetic resolution of the racemic imine-bromo-hemiaminal to the chiral indole-hemiaminal was proposed and ultimately realized (Scheme 23) [130]. After extensive development work it was established that potassium phosphate is the optimal base to promote both the racemization and cyclization, and the latter process is effectively catalyzed by Pd(OAc)2 in conjunction with QuinoxP*. This startling process delivers the desired chiral benzoxazinoindole 74 in 96% yield and 94% ee. The novelty of this process stimulates questions around the nature of the mechanism involved and plausible catalytic cycle is shown in Scheme 23. In the presence of K3PO4 and water, L1/Pd(OAc)2 undergoes intramolecular redox reaction to generate the active non-bis-phosphine-mono-oxide (BPMO) L2–Pd(0) catalyst. Starting material 73 can isomerize via its open form 72, and oxidative addition gives Pd(II) complex B which may also isomerize via its open form C. Deprotonation leads to the stereodefined imido–Pd complex A, and then reductive elimination affords 74 and regenerates the Pd(0) catalyst. The exact nature of the enantio-determining step in this process is currently under investigation and will be the subject of future publications from the Merck Research Laboratories.
Scheme 23

Dynamic kinetic resolution approach to chiral indole hemiaminal

Having identified a novel reaction to construct the key hemiaminal stereocenter, it was possible to design an improved synthesis of elbasvir that incorporated the new chemistry. Starting from commercially available 2-(2-bromo-5-chlorophenyl)acetic acid, direct acylation of the phenol occurs smoothly by heating reactants in neat triflic acid at 55 °C to give the corresponding ketone 75 with high regioselectivity (Scheme 24). Treatment of 75 with ammonia in MeOH affords the imine in 75% yield over two steps. Cyclization of imine with benzaldehyde in presence of catalytic amount of triflic acid and anisidine imine provides the racemic imine-hemiaminal 73, which sets the stage for the key DKR transformation described above.
Scheme 24

Synthesis of elbasvir via DKR approach

As alluded to in the discussion of the first-generation chemistry, completion of the elbasvir synthesis involves attachment of the side-chain(s) 63. The most convergent approach would couple the fully elaborated 63 directly to benzoxazinoindole 74 however complex reaction purity profiles together with limited availability of suitable crystalline phases of the API in early development hampered execution in this regard due to inadequate control of final drug substance purity. Consequently, in the first-generation synthesis it was necessary to use a stepwise approach that offered several crystalline intermediates for control of purity. With a better understanding of the cross-coupling chemistry and API crystallinity in the late development phase this challenge was overcome. Bis-borylation of 74 catalyzed by Pd(OAc)2/XPhos generated bis-boronate and double-Suzuki–Miyaura coupling with fully elaborated side chain 63 directly affords elbasvir in 82% isolated yield. Thus, the second-generation synthesis of elbasvir provides a 42% overall yield over six synthetic steps—a remarkably concise construction of this complex molecule [130].

To summarize the development of elbasvir, the variety of different routes described here represents an excellent example of how understanding of the chemistry around the synthetic target matures over time with continued study and leads to increasingly efficient access to the molecule of interest. In this case the chemistry evolved through three different versions of the synthetic route: a racemic discovery synthesis that required preparative chromatographic separation to provide the desired stereoisomer, a practical asymmetric stereochemical relay approach that supported clinical development and launch of elbasvir, and a final iteration based on a novel DKR process that represents the most ideal synthesis of this complex molecule identified to-date. The overall yield across these three syntheses increased from 4% to 25% to 42%, respectively, and the total number of steps was reduced from 11 to nine and finally to six steps in the DKR route.

3 Synthesis of a HIV Protease Inhibitor (Darunavir)

The retrovirus designated as human immunodeficiency virus (HIV) is etiologically linked to the immunosuppressive disease known as acquired immunodeficiency syndrome (AIDS) [131, 132, 133]. The HIV virus gradually attacks the immune system and destroys a type of white blood cell known as a T-helper cell, all the while replicating itself in the host cells. It may take around 10–15 years for AIDS to develop in the body if left untreated. The enzymes known as reverse transcriptase (RT), integrase (IN) and protease (PR) are each involved in key steps of the virus replication cycle. Small molecules that inhibit these enzymes can serve as antiviral agents and these are often taken together as “combination therapies”. The first effective medicine to become available for the treatment of HIV was zidovudine (a.k.a “AZT”), which was FDA approved in 1987. Since then we have witnessed tremendous gains in the control of HIV with the development of many different drugs, including the example discussed in this section (darunavir), which is in the protease inhibitor category. Despite these advances, in 2014 it was estimated by the World Health Organization (WHO) and the United Nations Programme on HIV and AIDS (UNAIDS) that 36.9 million people worldwide are living with HIV, of which 1.2 million unfortunately died of HIV-related causes. In view of these sobering facts, the case for continued research to discover new, even more effective therapies is clear, as is the need for elegant process chemistry solutions to the synthetic problems posed by these novel drug compounds.

3.1 Chemical Synthesis and Process Development of HIV Protease Inhibitor (Darunavir)

3.1.1 Background

Darunavir belongs to the class of HIV therapies known as protease inhibitors, which were first introduced in the 1990s. Improved understanding of the C-2 symmetrical active site of the protease enzyme made it possible to rationally design appropriate drug structures and the search for new compounds continues today. Consequently, darunavir is a second-generation protease inhibitor, intended to overcome problems with the older agents in this class. Early protease inhibitors often had severe side effects and drug toxicities, requiring a high therapeutic dose and exhibiting a disturbing susceptibility to drug resistant mutations. Such mutations can develop in as little as 1 year of use, and effectively render the drugs useless. Darunavir (trade name as Prezista®) was discovered and developed by Tibotec and is one of most recently FDA approved protease inhibitors (June 2006). Darunavir was designed to form robust interactions with the protease enzyme from many strains of HIV, including strains from treatment-experienced patients displaying multiple resistance mutations to previously available protease inhibitors. Darunavir is on the World Health Organization’s List of Essential Medicines, which defines the most important medications needed in a basic health system.

3.1.2 Medicinal Chemistry Route

The medicinal chemistry synthesis of darunavir was accomplished by carbamate formation between amine 79 and the activated bis-THF derivative 80 as shown in Scheme 25 [134]. Amine 79 was prepared in two steps from chiral amine 77, which in turn can be made according to an earlier report from G. D. Searle Company [135]. It is also noted that the synthesis of darunavir has been reported by Ghosh et al. [136], in which the corresponding azide intermediate was used instead of dibenzyl protected amine 77.
Scheme 25

Medicinal chemistry route

The Tibotec discovery synthesis for carbonate 80 is shown in Scheme 26. Starting from dihydrofuran, halohydrin formation gave racemic trans iodo-acetal (±)-81 with N-iodosuccinimide and 2 equiv. of propargyl alcohol in 91–95% yield. The next key reaction was a radical carbon–carbon bond formation using acetal 81, closing the second ring in bis-THF fragment 82 [137]. This radical cyclization was conducted in the presence of 10 mol% of cobaloxime and sodium borohydride in ethanol at 65 °C to give exo-methylene bicyclic compound (±)-82 in 72% yield. The exo-methylene functionality was ozonized and the resulting ketone was reduced to give racemic (±)-83 with high diastereoselectivity in 74% yield. The racemic mixture was kinetically resolved by immobilized lipase in the presence of acetic anhydride and, after chromatographic separation, the desired 83 was isolated in 42% yield. Thus, the synthesis of compound 83 was achieved in 20% overall yield from dihydrofuran.
Scheme 26

The synthesis of the 83 via radical cyclization followed by lipase resolution

3.1.3 Process Chemistry Route Retrosynthetic Analysis
Retrosynthetically, the discovery chemistry approach which involves cleavage of darunavir at the carbamate linker is a reasonable disconnection since it sets up a convergent late-stage coupling. The process chemistry team adopted this same convergent approach. The amine fragment 77 is relatively less complex and the synthetic strategy around this compound will not be discussed in detail here. On the other hand, bis-THF fragment 83 is a densely functionalized small molecule bearing three contiguous chiral centers and presents a far more significant synthetic challenge. A major inefficiency in the discovery route to 83 was the racemic nature of the synthesis and therefore a key objective for process development was identification of a stereoselective approach. As shown in Scheme 27, multiple disconnections can be considered here and the various options are discussed below.
Scheme 27

Retrosynthetic analysis Synthesis of Intermediate 83
In 2005, a joint team of DSM Pharma Chemicals and Tibotec reported a synthesis of 83 using the chiral pool starting material glyceraldehyde acetonide [138]. The chemistry is summarized in Scheme 28.
Scheme 28

Synthesis of intermediate 83 via lactone 92 (DSM/Tibotec route)

(R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde is condensed with dimethyl malonate in the presence of 0.5 equiv of pyridine and 3 equiv of acetic anhydride to yield the condensation product 88. Nitromethane is added to 88 in MeOH with 0.1 equiv of DBU at 0 °C, giving the conjugate addition adduct 89 with high diastereoselectivity (9:3 ratio). A Nef reaction converts the nitro-group into a carbonyl and the resulting aldehyde 90 is treated in situ with acidic methanol to simultaneously achieve acetonide removal/double cyclization to bicyclic lactone 91. Aldehyde intermediate 90 is stereochemically labile (via facile epimerization at the α-position) and any epimer generated results in formation of di-ester 93. This decreases the overall yield of the transformation but does not impact the final purity of 91 because trans-compound 93 cannot readily lactonize and is easily rejected to the aqueous layer during workup. Intermediate 91 is subject to basic solvolysis followed by acidification to obtain crystalline lactone acetal 92 as a single isomer. Lactone 92 is reduced to a lactol with LiBH4 and equilibration under acidic conditions provides the key intermediate 83 as the thermodynamic product in 77–80% isolated yield. The overall yield of this four-step synthesis is 14% (demonstrated at multi-kilogram scale), which compares very favorably with the discovery synthesis yield of 20%. This route was reported in a Tibotec patent application starting from >200 kg of the aldehyde [139].

In line with the theme highlighted with the earlier examples of process chemistry presented in this chapter, the development story of darunavir also highlights the concept of continuous improvement in synthetic routes. In this case, rather than design a completely new route, the Tibotec team elected to make refinements to what was already an efficient approach to bis-THF fragment 83 [140]. As shown in Scheme 29, a key modification in the second-generation route is replacement of α,β-unsaturated malonate derivative 88 with ethyl acrylate derivative 94. This change in synthetic tactics confers several advantages over the first-generation route: (1) the original Knoevenagel reaction (converting glyceraldehyde acetonide to 86) is sensitive to water and the maximum yield is 77–80%, (2) the decarboxylation-recyclization step (from 91 to 92) requires an extended reaction time with associated product degradation and (3) lactone ester 91 is somewhat sensitive to pH/temperature during aqueous work-up, again leading to potential degradation and reduced yield of the desired product.
Scheme 29

Modified synthesis of intermediate 83 via lactone 92 (Tibotec)

Using the same glyceraldehyde chiral pool raw material, a Horner–Wadsworth–Emmons olefination provides the (Z)-alkene 94 with excellent selectivity over the (E)-isomer. Similar to the first-generation route, a DBU-promoted conjugate addition of nitromethane into 94 followed by a Nef reaction affords 96, which does not require decarboxylation before conversion to common intermediate 97. Again, during extractive work-up for lactone 97, the trans isomer 98 is easily rejected to the aqueous layer because it cannot lactonize. Acetal 97 is obtained as a mixture of diastereomers and equilibration under acidic conditions funnels the material to the most thermodynamically stable β isomer 92. Taken as a whole, these modifications deliver significant gains in synthetic efficiency, increasing the overall yield to key intermediate 83 from 14 to 33%. Final Stages for the Synthesis of Darunavir
The medicinal route utilized hydroxysuccinate activated carbonate 80 for the final coupling reaction. The process route uses the same method, however 80 is prepared in situ and methylene chloride is replaced by a more environmentally friendly solvent combination of ethyl acetate and acetonitrile (Scheme 30). After aqueous work-up and recrystallization of crude material from ethanol, darunavir is isolated in 81% yield as ethanolate solvate.
Scheme 30

Final stages for the production of darunavir

To summarize the chemical development of darunavir, the overall route is highly convergent and utilizes two key building blocks 79 and 80. The approach to fragment 79 leverages the synthesis previously described in another drug (amprenavir). Therefore, the key challenge for the darunavir process chemistry team was the identification and development of an asymmetric approach to the bis-THF fragment 83. This was achieved via utilization of glyderaldehyde acetonide as a chiral pool raw material. As a result of improved access to intermediate 83 (since the bis-THP intermediate 83 is a privileged pharmacophore, Gilead and GSK independently reported Lewis acid-catalyzed synthesis of 83. see: [141, 142]), the overall yield of darunavir was vastly improved, ensuring a robust supply of this important antiviral drug to patients worldwide.

4 Synthesis of a Compound for the Treatment of Influenza (Peramivir)

Influenza is a RNA virus that has long history of causing health problems for the human race. Influenza is highly contagious and can present serious concerns for those with weakened immune systems (very young children, the elderly and people with certain medical conditions are particularly vulnerable). Of the three known types of influenza virus (A, B and C), type A is the most commonly encountered and was the cause of virulent human influenza pandemics such as Spanish flu outbreak in 1918, the Hong-Kong flu outbreak in 1968, through to more recent outbreaks of Bird flu and Swine flu in 2004 and 2009 respectively. Collectively, these pandemics resulted in more than a million deaths worldwide [143, 144].

Until relatively recently, options for treating the influenza virus were very limited, including vaccinations and a small molecule therapy called amantadine. In the United States, the use of amantadine as a flu therapy was discontinued after a study by the Centers for Disease Control and Prevention (CDC) indicated 100% of 2008/2009 flu virus samples were fully resistant to this treatment. The current state of the art treatment option for influenza is a class of small molecules which block the function of neuraminidase enzymes and prevent viral reproduction. Examples of these neuraminidase inhibitors include oseltamivir (Tamiflu®), zanamivir (Relenza®) and also peramivir (Rapivab™), which is the subject of the process development discussion below.

4.1 Synthesis of a Compound for the Treatment of Influenza (Peramivir)

4.1.1 Background

Peramivir was discovered and developed by BioCryst Pharmaceutical as a potent neuraminidase inhibitor for the treatment of influenza virus [145, 146]. BioCryst collaborated with Ortho-McNeil (a subsidiary of Johnson & Johnson) in 1998, but ultimately partnered with both Green Cross Pharmaceuticals in 2006 and Shionogi Pharmaceutical in 2007 to co-develop Peramivir, which is currently approved in Japan (2010), South Korea (2010), China (2013) and United States (2014).

4.1.2 Medicinal Chemistry Route

The medicinal chemistry route for the synthesis of Peramivir is summarized in Scheme 31. The key reaction in this synthesis was a [2 + 3] cycloaddition between an alkene and an in situ generated nitrile oxide [145, 146].
Scheme 31

Medicinal chemistry route for the synthesis of peramivir

Starting from commercially available bicyclic lactam 99, solvolysis in acidic MeOH resulted in conversion of the lactam to the ring-opened methyl ester. The amino group was subsequently protected as the Boc carbamate to form intermediate 100. Next, the key [2 + 3] cycloaddition reaction of 100 with nitrile oxide 101 in benzene delivered the cycloadduct as a mixture of four isomers (102ad). The desired isomer 102a was isolated as a crystalline solid in 61% yield after chromatographic separation. The isoxazoline ring in 102a was stereoselectively cleaved from the less hindered β face under hydrogenation conditions in the presence of PtO2/HCl. The resulting amine was acetylated to provide 103 in 67% isolated yield after chromatography. After N-Boc deprotection, introduction of the guanidine group was performed using 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea in the presence of mercury(II) chloride. Methyl ester in 106 was solvolyzed with NaOH and the corresponding acid 107 was isolated in 90% yield as a solid after acidification. Finally, the guanidine N-Boc group was removed by TFA in methylene chloride to provide peramivir as TFA salt.

4.1.3 Process Chemistry Route Retrosynthetic Analysis

From a synthetic strategy perspective, the approach used in the discovery synthesis is relatively efficient and therefore the process chemistry approach retained the [3 + 2] cycloaddition as a key bond formation. However, several characteristics of the discovery route were unsatisfactory with respect to commercial manufacture and these were targets for refinement/optimization in the process route [147, 148]. Specifically, the use of phenyl isocyanate with a nitroalkane to generate the key nitrile oxide intermediate combined with the use of benzene (a genotoxic carcinogen) as solvent was not acceptable. Additional problematic aspects were the need for chromatographic separation of isomeric products after the [3 + 2] cyclization, the cost associated with PtO2 for reduction of the isoxazoline ring and the use of highly toxic mercury(II) chloride for the installation of guanidine group. Process Improvement for the [3 + 2] Cycloaddition and Isolation
The preparation of 100 was improved to 92% from 86% through process optimization. A common method for the nitrile oxide formation is through oxidation of an oxime. As shown in Scheme 32, the nitrile oxide can be generated from 2-ethyl-N-hydroxybutanimidoyl chloride, which is easily prepared by chlorination of the corresponding oxime with NCS. Cycloaddition is now carried out in toluene rather than benzene in the presence of triethylamine. The crude cyclic product, a mixture of four isomers, is solvolyzed to the corresponding acid where isolation of the desired isomer can be achieved (66% isolated yield) in straightforward fashion via salt formation with tert-butylamine, thereby avoiding the tedious chromatography required in the discovery route. The salt is re-esterified with HCl in trimethyl orthoformate and MeOH to provide the methyl ester 102a in 97% yield (a more recent report by another company reported a similar method and the methyl ester 102a was crystallized via solvolysis/re-esterification sequence without formation of tert-butylamine salt. see: [149]).
Scheme 32

New [2 + 3] cycloaddition conditions and isolation of isoxazoline 102a Process Improvement for the Reduction of Isoxazoline
Of the possible conditions available for reduction of the isoxazoline 102a, the use of PtO2 is relatively expensive even though it is only used as a catalyst for the hydrogenation. The less expensive reducing reagents NaBH4/NiCl2 hexahydrate and LiAlH4 are equally effective for this transformation as shown in Scheme 33, although the ability to conduct a workup with sodium potassium tartrate after LiAlH4 reduction is convenient and the best yield (81%) is obtained through this procedure [149].
Scheme 33

Improvement on reduction of 102a Process Improvement for the Final Stages via Mercury-Free Guanidine Formation
The guanylation conditions were modified from the discovery chemistry as shown in Scheme 33. Treatment of amine 104 with pyrazole carboxamidine hydrochloride and Hünig’s base in DMF gives free guanidine 108, which is hydrolyzed to provide peramivir in 50% yield from 103 (Scheme 34).
Scheme 34

Improved synthesis of peramivir

The peramivir case study provides an example of process development where the retrosynthetic analysis and bond disconnections from the original medicinal chemistry synthesis were retained and modifications to the reagents/procedures were made to render the process more practical. The process route is chromatography-free, eliminates benzene/mercury reagents and delivers peramivir in 21% overall yield across a 10 linear-step sequences.

5 Conclusions

Process chemistry plays a pivotal role in the development and manufacture of drugs. The five antiviral process chemistry case studies presented in this chapter were chosen to reflect the varying approaches to synthetic development taken across the pharmaceutical industry. As alluded to in the introduction, cross-functional demands (e.g., expediting clinical supply) often intertwine with the broad goal of designing and developing ideal syntheses of APIs, sometimes influencing the direction of process development. It is clear however, that the search for the best synthetic routes to drug targets provides an excellent venue for scientific discovery and allows expansion of the current knowledge of organic chemistry as a whole. The development of elegant and practical routes to complex pharmaceutical targets sometimes necessitates the discovery of fundamentally new chemistry, which often has broader applications. Consequently, a mindset of continuous innovation is extremely valuable within process chemistry and paves the way for discovery and development of important new medicines for humankind.


  1. 1.

    The situation may be actually slightly more complex because the phosphoramidate hydrolyzes to leave the 5′-phosphate, which avoids the need for biological mono-phosphorylation of the 5′-alcohol, on the pathway towards triphosphate formation.

  2. 2.

    Employed a similar processes developed by Gilead Sciences, lactone 11-OBn was converted to 29 in 25% overall yield.

  3. 3.

    The chlorides in the chlorophosphates 30a and 30b are replaced by pentafluorophenol due to the instability of the chlorophosphate towards isolation/upgrade.

  4. 4.

    Intermediate 48 can be sourced.

  5. 5.

    Alternate, stereoselective approaches were ultimately developed by Merck Research Laboratories and will be the subject of future publications.


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Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Department of Process ChemistryMerck and Co., Inc.RahwayUSA

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