Discovery and Development of Doravirine - ACS Publications

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Chapter 7

Discovery and Development of Doravirine: An Investigational Next Generation Non-Nucleside Reverse Transcriptase Inhibitor (NNRTI) for the Treatment of HIV Jason D. Burch,1 Benjamin D. Sherry, Donald R. Gauthier Jr.,2 and Louis-Charles Campeau*,2 1Process

Research & Development, MRL, 126 E. Lincoln Avenue, P.O. Box 2000, Rahway, New Jersey 07065, United States 2Inception Sciences Canada, 7150 Frederick-Banting, Montreal, Quebec H4S 2A1, Canada *E-mail: [email protected].

The current standard of care for HIV is a combination of drugs, known as highly active antiretroviral therapy (HAART). A frequent component of HAART are non-nucleoside reverse transcriptase inhibitors (NNTRI) which prevent conversion of viral RNA into DNA. Herein we discuss the discovery, early and late development of doravirine, an investigational drug for the treatment of HIV. Key structure activity relationships, as well as critical optimized pharmacokinetic and physico-chemical parameters are presented. The evolution of the synthesis methodologies used throughout the lifetime of the program will also be elaborated, including the early development supply route and our efforts toward the establishement of the manufacturing process.

Introduction In 1981 the first clinical cases of what is now known as acquired immune deficiency syndrome (AIDS) were reported, when clusters of intravenous drug users and homosexual men displayed symptoms of previously rare Pneumocystis carinii (PCP) (1) infection and Kaposi’s sarcoma (KS) (2, 3). Two years later, © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


two independent research groups proposed that infection by a novel retrovirus may have been a precursor to development of AIDS (4, 5), and this virus was subsequently named human immunodeficiency virus (HIV). Since its discovery, AIDS has caused more than 35 million deaths worldwide, and more than 35 million people remain infected with the virus (6). In 2013 alone, the disease caused 1.34 million deaths (7), and AIDS is considered a global pandemic. AIDS is a health emergency in sub-Saharan Africa, where infection rates in countries such as Zimbabwe (15%), South Africa (19%) and Swaziland (27%) are astronomical (8). HIV is classified as a retrovirus since it replicates its genetic material through reverse transcription, the process of creating a DNA copy of the RNA genome – opposite of the usual pattern. The enzyme responsible for this reverse transcription is HIV reverse transcriptase (HIV RT), which quickly became a potential target for HIV treatment. Within two years of the identification of the HIV virus, 3’-azido3’-deoxythymidine (AZT; 1, Figure 1) was identified by scientists at the National Cancer Institute as a potent inhibitor of the virus in vitro and in vivo (9). AZT was approved by the FDA for the treatment of HIV infection on March 20, 1987 (10), and the four year timeline from viral identification to drug approval is arguably the most impressive success story in the history of targeted drug discovery.

Figure 1. Structures of HIV RT inhibitors AZT and efavirenz.

AZT belongs to a class of HIV RT inhibitors called nucleoside analog reverse-transcriptase inhibitors (NRTIs). These molecules mimic the naturally occurring nucleoside bases, and after phosphorylation inside the host cell they are incorporated into the growing DNA chain by HIV RT (11). Unlike natural nucleosides, however, NRTIs lack the 3’-hydroxyl group necessary for chain elongation. Since HIV RT lacks the “proof reading” function of human DNA polymerase, this chain termination results in a permanent inhibition of viral DNA synthesis, and thus viral replication is halted (9). Although AZT is remarkably effective against inhibiting wild type HIV replication, HIV RT is extremely error-prone, incorporating incorrect base pairs at an average rate of 1/1700 (12). These “errors” provide a mechanism for HIV to evolve resistance to replication inhibitors, and thus efficacy of treatment by single agents is generally short-lived. Today, long-lasting viral suppression is achieved through simultaneous treatment by a cocktail of three or more distinct drugs, and this approach has been dubbed highly active anti-retroviral therapy (HAART). 176

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


This combination can include multiple distinct inhibitors of HIV RT, but can also contain inhibitors of other important enzymes to the HIV life cycle, such as HIV integrase (which incorporates viral DNA into the host DNA) (13) or HIV protease (which produces mature virions in the final step of the life cycle) (14). This highly effective approach has resulted in a dramatic increase in life expectancy for an HIV patient: between 2000 and 2007 the average life expectancy of a North American infected with HIV increased by 15 years (15). The top selling HAART regimen, Atripla®, which has sales in excess of $3 billion annually, is a single once-daily pill HAART regimen combining two NRTIs (300 mg of tenofavir disoproxil fumerate and 200 mg of emtricitabine) with 600 mg of efavirenz (Sustiva®, 2, Figure 1). Like the NRTIs, efavirenz is also an inhibitor of HIV RT, but it is a member of a class of molecules which inhibit HIV RT through a different mechanism called non-nucleoside reverse transcriptase inhibitors (NNRTIs). NNRTIs bind to a lipophilic allosteric pocket 10 Å from the active site of HIV RT (Figure 2), causing a distortion of the catalytic aspartate triad (16).

Figure 2. X-ray crystal structure of efavirenz bound to wild type HIV RT (PDB code 1FK9) (17).

Efavirenz was discovered by chemists at Merck & Co., Inc. (Kenilworth, NJ, USA) in the early ‘90s (18, 19), and was approved by the FDA in September 1998. It has excellent human pharmacokinetic properties, with bioavailability above 40%, and terminal plasma half-life in excess of 40 h (20), albeit with high plasma protein binding (>99%) which accounts for the relatively high dose necessary to achieve maximal efficacy (600 mg). Unfortunately, since efavirenz does not interact directly with the catalytic machinery of HIV RT, and relies upon lipophilic interactions with amino acid side chains in the allosteric pocket (c.f. Figure 2), efavirenz presents a relatively low barrier to the evolution of resistant mutants. In particular, the K103N and Y181C mutants are observed 177 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


clinically in patients taking efavirenz (21), and in vitro inhibition of these RT mutants is diminished significantly (c.f. Table 1 below). Furthermore, a number of adverse events are associated with efavirenz treatment, such as effects on dyslipidemia (22), teratogenicity (23), and a host of CNS effects such as insomnia, nightmares, memory loss and depression (24), which are postulated to be driven through off-target modulation of 5-HT receptors (25, 26). Finally, efavirenz is an inducer of the 2B6 and 3A4 isoforms of cytochrome P450 (27), and thus cannot be co-dosed with HIV protease inhibitors, which are in general extensively metabolized by Cyp3A4. For these reasons, numerous companies have embarked on research efforts directed towards next-generation NNRTIs with improved potency versus clinically relevant mutants and improved safety profiles.

Medicinal Chemistry of Doravirine The Merck & Co., Inc. (Kenilworth, NJ, USA) effort towards development of a next-generation NNRTI began with the discovery of a class of tetrazole thioacetanilides (e.g. Compound 3, Figure 3) by high-throughput screening (28–30). While these inhibitors, discovered nearly simultaneously by others (28), were able to exhibit low nanomolar cellular inhibition of wild-type HIV reverse transcriptase, and minimal shift versus the K103N mutant, poor pharmacokinetic properties precluded advancement of this substrate class. Merger of this series with a related acetanilide para-sulfonamide series from the prior art (GW-8248, 4) (31) led to proposal of a novel class of diaryl ether NNRTIs 5 (32), in which the benzophenone moiety of 4 has been replaced by a 1,3-phenoxy relationship (33).

Figure 3. Genesis of the diaryl ether series of NNRTIs.

Selected structure activity relationships for this class of inhibitors are summarized in Table 1. Inhibitor 6 lacking additional substitution on rings A and B exhibited moderate enzymatic inhibition of wild type HIV reverse transcriptase, and significant shift toward the clinically relevant mutants K103N and Y181C. Dramatic improvement in potency, especially versus the Y181C mutant, was 178 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

achieved through introduction of a chlorine atom at the 3-position of ring B (7), with further potency improvements observed by mono- (8) and di-substitution (9) of ring A, that resulted in inhibitors with low nanomolar inhibition of the wild type, K103N and Y181C enzymes. Transposition of the chlorine atom from the 3- to the 2-position of ring A (10) resulted in sub-nanomolar inhibition of all three enzymes, and double-digit nanomolar cellular inhibition in the presence of 50% normal human serum (34).


Table 1. Structure-Activity Relationships for the Diaryl Ether Series of NNRTIs (32)

Unfortunately, all diaryl ether phenoxyacetanilides 6-10 showed rapid in vivo clearance, and metabolic profiling confirmed rapid metabolic hydrolysis of the anilide. Thus numerous heterocyclic amide isosteres were investigated, and ultimately indazole 11 (Table 2) proved to be an acceptable replacement for the acetanilide moiety, with excellent enzymatic potency versus wild type RT, minimal shift to the clinically relevant mutants, and acceptable potency in the high-serum Spread assay. Crystallographic studies of 11 in the presence of the wild type RT enzyme (Figure 4(a); PDB code 3C6U) revealed that the inhibitor binds in the same allosteric pocket occupied by efavirenz, with a few key differences which potentially explain the limited shift versus the K103N and Y181C mutants for 11 (Figure 4(b)): 179 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

•


•

while efavirenz is in close contact with the lipophilic portion of the lysine 103 side chain, inhibitor 11 forms a donor-acceptor hydrogen bonding interaction with the lysine 103 backbone, and thus would be expected to be more tolerant of mutations of the side chain residue. tyrosine 181 was rotated by 90° relative to its position in the efavirenz structure, thus moving it away from direct contact with inhibitor 11.

Figure 4. (a) X-ray crystal structure of inhibitor 11 bound to wild type HIV RT (PDB code 3C6U); (b) Superposition of the crystal structures of 11 (purple) and efavirenz 2 (blue; from PDB code 1FK9) bound to wild type HIV RT, with key residues shown.

While 11 showed improved metabolic stability in vivo relative to its acetanilide precursors (Cl < hepatic blood flow in rats, dogs and rhesus monkeys), minimal exposure was detected following oral dosing, which was attributed to its poor solubility. The crystal structure of 11 bound to HIV RT (Figure 4) revealed that the six-membered ring of the indazole projected toward a solvent-exposed, hydrophilic cavity, and thus the potential to install polar and/or solubilizing groups in this region was investigated (Table 2) (35). Ultimately, a 6-amino-7-azaindazole replacement was found to be optimal (16, Table 2), and this compound was eventually nominated as a pre-clinical candidate and labeled as MK-4965. Gratifyingly, MK-4965 demonstrated markedly improved bioavailability and in vivo clearance in rats and beagle dogs, although non-human primate oral absorption and clearance was a concern (Table 3). Furthermore, MK-4965 showed no significant issues in pre-clinical safety studies, and was shown to have a low potential for drug-drug interactions (as a victim or a perpetrator), and thus had the potential to be differentiated relative to efavirenz.

180 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Table 2. Introduction of Polarity into the Indazole Terminus of the Diaryl Ether Series of NNRTIs (35)

Table 3. Pharmacokinetic Parameters for MK-4965 (16) (35)

As MK-4965 advanced through pre-clinical development, the search continued for further improved chemical matter. In particular, to increase confidence in prediction of human pharmacokinetic properties, we hoped to improve upon the high clearance and poor bioavailability observed in non-human primates. While extensive optimization of the head and tail region of the inhibitors had been executed, the meta-resorcinol diether core remained largely unexplored (36). We hypothesized that decreasing the lipophilicity of the resorcinol core might improve pharmacokinetic properties, as the correlation between logP and clearance (37) and inverse correlation between logP and solubility (38) are well documented. At that time, a series related to our diaryl ethers was disclosed by Roche in which the “eastern” side chain had been truncated by removal of the oxygen atom (Figure 5, 17) (39). To expand on the pharmacophore of these direct 181 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


aryl-carbon linked analogs, we were intrigued to explore heteroaryl replacements of these truncated inhibitors. In particular, pyridones such as 19 were attractive as this replacement resulted in reduction of nearly two full units of calculated logP relative to the m-resorcinol starting point (Figure 5 inset).

Figure 5. Evolution from resorcinol to pyridone core NNRTIs. Prior to synthesis of pyridone inhibitors such as 19, torsional energy calculations (40) were carried out with simplified versions of the cores of 15, 17 and 19 (Figure 6) (41). These calculations demonstrated that different energy minima were predicted for O-linked (C) and C-linked (B) phenyl cores, and crystallographically observed bound conformations of these molecules (PDB codes 3DRS and 3DYA respectively) show dihedral angles at or near these minima in both cases. Furthermore, torsional minima for a pyridone core (A) was predicted to mimic that observed for the C-linked analogs such as 17.

Figure 6. Dihedral torsional strain for N-ethyl pyridone, ethyl benzene and methoxybenzene. 182 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Gratifyingly, 4-methyl pyridone 20 (Table 4) proved to be a potent inhibitor of both wild type and mutant forms of HIV RT. Extending the carbon chain by one atom (pyridone 21) led to a dramatic reduction in potency, suggesting that the pyridones are a more accurate isosteric replacement for truncated analogs 17 instead of the m-resorcinol analogs 15, thus validating the torsional predictions of Figure 6. Furthermore, pyridone analog 20 showed a markedly reduced shift between intrinsic enzyme potency and the high-serum Spread cellular assay relative to its m-resorcinol precursors, likely the consequence of reduced plasma protein binding for these less lipophilic inhibitors (42).

Table 4. Discovery of the Pyridone Class of NNRTIs

The first phase of optimization of the pyridone inhibitors involved optimization of the substituent at the 4-position of the core (Table 5) (43), where modifications were tested for maintenance of wild type and mutant RT inhibition, and in this case mutant potency was measured in the high-bar Spread assay as the K103N/Y181C double mutant. Further, in vivo stability was assessed in rats through determination of the terminal plasma half-life (44). A variety of small lipophilic substituents (halogens, alkyl groups, fluorinated alkyl groups and thioethers) were tolerated at the 4-position, with excellent enzyme and wild-type Spread inhibition, and limited shift toward the double mutant. The majority of the inhibitors demonstrated marginal plasma half-life, with the exception of the 4-trifluoromethyl analog 24, which exhibited markedly improved plasma stability. The 4-trifluoromethyl pyridone core was thus selected for further optimization. Re-examination of the phenol appendage was also undertaken, however the 3-chloro-5-cyano-phenol motif again proved optimal. Although the profile of pyridone 24 was initially promising, poor aqueous solubility (0.2 ng/mL) resulted in poor oral absorption (bioavailability of 15% in rats and 9% in dogs when dosed as a methocel suspension), and precluded further advancement of this inhibitor. We hypothesized that the pyrazolopyridine side chain was the source of this insolubility, as a small molecule crystal structure of a related inhibitor from the benzotriazole series (36) revealed a highly ordered intermolecular donor-acceptor relationship between the side chains of two monomers (Figure 7). 183



Table 5. Optimization of the 4-Position of the Pyridone Core

Figure 7. Intermolecular donor-acceptor hydrogen bonding for a pyrazolopyridine-containing NNRTI. (copyright Elsevier, 2014) (43). We thus sought to replace the pyrazolopyridine with an alternative side chain which could maintain the desirable in vitro potency profile of 24 without the possibility of forming this strong donor-acceptor pair. The challenge here was that the pyrazolopyridine forms a key donor-acceptor hydrogen bond with the backbone of K103 (c.f. Figure 4). Replacements were thus selected for exploration which contained at least one hydrogen bond donor or acceptor, and a 184 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

selection of the resulting inhibitors is presented in Table 6 (43). These molecules, which were available from direct alkylation of hydroxypyridine 28 (equation 1), were assessed for their enzymatic and cellular inhibition of wild type RT, as well as their kinetic solubility in aqueous media.


Table 6. Structure-Activity and Solubility Relationships for Side Chain Replacements

An early validation of this approach was inhibitor 29, which shows improved solubility relative to 24 despite being more lipophilic, presumably due to removal of the pyridine nitrogen of the donor-acceptor hydrogen bonding pair. The most dramatic solubility improvement was observed when switching to monocyclic heterocycles such as thiazole 31 (45). Although significant potency was lost relative to earlier bicycles, much of this potency could be recovered by addition of a methyl group at the 4-position (32), which presumably occupies a similar lipophilic space to the second ring of the bicyclic side chains. Ultimately, the isosteric methyltriazolinone 34 proved the optimal replacement, with dramatically improved kinetic solubility and no loss of cellular potency relative to pyrazolopyridine 24. 185 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Additional characterization revealed that inhibitor 34 demonstrated excellent potency versus clinically relevant mutants, and compared favorably when benchmarked versus the marketed NNRTIs efavirenz and rilpivirine (46) (Table 7). Furthermore, profiling of 34 versus an extensive panel of clinically relevant reverse transcriptase mutants by Monogram Bioscience identified the rare mutant Y188L (47) as the only single- or double-mutant against which 34 demonstrates a significant potency shift. Crystallographic analysis of 34 bound to wild type HIV RT (Figure 8) revealed a binding mode analogous to the diphenyl ether class (c.f. Figure 4): a donor-acceptor hydrogen bonding pair with the K103 backbone, and pi-stacking interactions with Y188 (face-to-face) and W229 (edge-to-face). As was observed for diphenyl ether 11, Y181 is rotated 90° relative to its orientation in the efavirenz structure.

Table 7. Wild Type and Mutant Cellular Potency of 34 Compared with Marketed NNRTIs

Figure 8. X-ray crystal structure of inhibitor 34 bound to wild type HIV RT (PDB Code: 4NCG). 186 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Evaluation of the pharmacokinetic profile of 34 revealed excellent absorption and stability across pre-clinical species (Table 8). Combined with the observation that 34 exhibited low metabolic turnover in human liver microsomes and hepatocytes resulted in prediction of once-daily, low dose regimen in human. Further, thermodynamic solubility of 34 was dramatically improved relative to the pyrazolopyridine precursor 24, whether in pH 4 buffer (6 μg/mL vs. 90% conversion was reached. Early deliveries of doravirine used a trituration of crude API from the methylation with hot acetone to provide material in 71% isolated yield which met specifications, rejecting all major impurities to below 1%. Later we found that direct precipitation of crude API from an NMP solution was possible, affording a more streamlined and productive option. The direct isolation protocol typically results in greater purity crude API (89-90%). This improved input crude API stream allowed us to develop a final recrystallization which afforded doravirine with even greater overall purity than the trituration process, in addition to simplifying unit operations. The new and improved protocol recrystallized the API from NMP:EtOH (~8:1) at ~75 ºC which yields doravirine in similar yield (69%) and improved purity (99.1%). This improved end-game process was executed on 130 kg batches to afford >92 kg of API per batch and purity typically >99% (Figure 13). A summary of the initial supply route to doravirine is presented in Figure 14. This route afforded quick entry into preclinical development to assess the safety and tolerability of the drug in preclinical safety studies and was suitable to support 191


the development program through Phase IIB. In all, over 400 kg of API batches as large as in ~90 kg were produced using this route (56).


Figure 13. Doravirine End-Game and Final Recrystallization. (Based on an existing figure. Copyright American Chemical Society publications, 2016) (56)

Figure 14. Supply Route to Doravirine (34).

Commercial Manufacturing Route to Doravirine However, while scalable and robust, the synthesis offered opportunities for improvement in order to provide sustainable chemistry for long-term manufacturing production: •

• •

the functional group manipulation for both the phenol appendage and the methyl-triazolinone could be streamlined into a more convergent approach. the halogenated pyridine 42, while commercially available, was expensive and not widely available. the end-game sequence as it existed occurred in modest selectivity and yield. 192


Consequently, significant gains in synthetic efficiency could be realized from a redesigned approach to doravirine where: • • •

the aryl nitrile is incorporated in a phenol raw material. pyridone 28 is either assembled de novo or prepared from an inexpensive pyridine. an N-methylated triazolinone is used as the alkylating agent in the final coupling.


Realization of these goals was expected to result in a more convergent, productive and sustainable synthesis of doravirine (Figure 15) (57).

Figure 15. Retrosynthetic Design for Commercial Route to Doravirine. (Based on an existing figure. Copyright American Chemical Society publications, 2016) (57)

First, a re-investigation of the use of cyanated phenol 43 as a key raw material was a focus of our route development work. A selective SNAr reaction of 43 with pyridine 42 occurred under similar conditions to those used for the iodinated compound and afforded biarylether 44 in 86% yield (Figure 16). As mentioned above, since direct hydrolysis led to the formation of benzoic acid 45, we attempted pyridinium formation but salt formation did not occur with suitable electrophiles. High-throughput screening of metal-catalyzed (Pd or Cu) C-O bond formation did not lead to suitable conditions for installation of the required 2-oxygenation. The direct addition of potassium tert-butoxide in toluene to hydrolyze the pyridinyl chloride provided a modest yield of pyridone 28 after in-situ deprotection of the tert-butyl group with trifluoroacetic acid. Unfortunately, the efficiency of this transformation could not be improved beyond 58% yield and therefore alternative avenues were pursued.

Figure 16. Attempted Hydrolysis of the 2-Chloropyridine. 193 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


We also investigated an approach where an SNAr reaction between an N-alkyl pyridone and phenol 43 akin to early SAR approaches (Figure 9, approach #2). Reversing the order of operations in this manner would obviate the need for selective hydrolysis of the 2-chloropyridine group in the presence of the nitrile. Securing a low cost route to pyridone 52 could thereby offer an efficient pathway to the target structure. The Fries re-arrangement approach used in lead-optimization to prepare 50 was deemed unsuitable for commercial scale and therefore alternative syntheses were considered. Jiang and co-workers reported an efficient synthesis of 4-trifluoromethyl-2(1H)-pyridinone by a Reformatsky reaction between vinylogous ester 47 and chloroacetonitrile (58). Efforts to adapt this approach to organozinc precursors bearing the aldehyde oxidation state at the alpha-carbon were examined. Ultimately, the reaction of 46 and ester 47 in the presence of zinc and trimethylsilyl chloride delivered Reformatsky adduct 48 in 78% assay yield (Figure 17). Treatment of 48 with methanolic ammonia provided primary amide 49, which was subjected to acid-mediated cyclization/dehydration to deliver the desired pyridone 50 in 70-75% isolated yield after crystallization from n-butanol / heptane. Alkylation with protected triazolinone 51 provided 52 in 90% assay yield. A final SNAr reaction between phenol 43 and 52 provided 34 in 72% isolated yield after in-situ deprotection of 53 under acidic conditions. Though this alternative route has considerable advantages over the initial route, raw materials cost estimates, final purity and process robustness concerns prompted additional route development work.

Figure 17. SNAr Route to Doravirine.

The Reformatsky disconnection used to assemble pyridone 50 was a distinctly simplifying transformation which enabled the use of low cost vinylogous ester 47 as a trifluoromethyl containing raw material. Exploiting this bond construction, but redesigning the nucleophile to incorporate the aryloxy group was identified as 194 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

a means to further improve access to doravirine. To investigate this approach the aldol reaction of 54 and 55 with 47 in the presence of alkali metal amide bases was examined (Table 10). The reactions were conducted by pre-forming the metal enolate at the indicated temperature followed by addition of 47.


Table 10. Batch Aldol Optimization

Executing the aldol addition on pilot plant scale was expected to be challenging due to the sensitivity of the reaction to temperature and time cycle. The power of this synthetic approach, however, merited consideration of alternative processing technologies capable of addressing many of these difficulties. Continuous reactions are being pursued with increasing frequency in both academic and industrial labs for the synthesis of small molecules, and one power of this technology is its ability to address in a robust way the very challenges posed by scale-up of the aldol addition (59–61). Prior to evaluating the chemistry in a flow reactor, reaction parameters were re-examined in 1-10 mg scale batch experiments where mixing, heat transfer and time-cycle concerns were minimal. Unlike in the optimization of the batch process, the reaction was run under Barbier conditions with the base added as the final component. The reaction of ethyl ester 54 with 47 mediated by potassium tert-amyloxide in toluene at -20 to -30 °C proved most efficient. The flow reactor illustrated in Figure 18 was then designed to test the continuous process. One feed was a toluene solution of ester 54 and vinylogous ester 47. The second feed was a commercially available 1.7 M solution of potassium tert-amyloxide in toluene. After the streams were combined at a T-mixer the reaction was quenched at a second T-mixer with aqueous phosphoric acid buffered with potassium phosphate monobasic. The continuous reaction provided the desired aldol adduct 56 in up to 85% assay yield as a mixture of diastereomers. 195



Figure 18. Flow Reactor Schematic for Continuous Aldol Addition.

The aqueous phase was removed from liquid/liquid biphasic exit stream of the flow reactor in a batch workup (Figure 19). The remaining toluene solution was azeotropically dried and the tertiary alcohol underwent elimination with trifluoroacetic anhydride and triethylamine to afford diene 58 as a mixture of alkene stereoisomers. This material proved unstable to storage when neat and was therefore used as the unpurified crude solution for downstream chemistry.

Figure 19. Aldol Addition and Elmination.

Diene 58 bears the appropriate terminal oxidation states for direct cyclization to a pyridone. Literature precedence for the heterocyclization of acyclic species such as 58 exists, though examples of substrates bearing alpha-oxygenation are scarce (62). Optimization of the heterocyclization reaction was carried out using a Multi-Max® reactor system and mass flow meter to precisely control the molar charge of ammonia gas (Table 11). At a reaction time of 20 h improved conversion was observed as the ammonia charge increased from 3 to 11 equivalents. Decreasing the reaction time to 6 h resulted in a slight decrease in conversion, which could be mitigated by increasing the ammonia charge up to 40 equivalents. Product formation increased with temperature up to 80 °C after which point decomposition pathways started to compete. The reaction was not sensitive to vessel fill indicating gas/liquid equilibrium has little impact on the robustness of the reaction. 196 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Table 11. Heterocyclization Reaction Optimization

At the completion of the cyclization reaction a homogeneous solution was obtained due to the large excess of dissolved ammonia in the reaction mixture. Compound 28 shows a steep pH dependent solubility in MeOH, as pH increases the solubility increases sharply. Crystallization of the product was induced by switching the solvent from a mixture of methanol, toluene and ammonia to pure methanol. Pyridone 28 was obtained in 60-65% isolated yield and high purity by filtration of the resulting slurry. Various opportunities to streamline the aldol process were considered and are illustrated in Figure 20. Attempting to engage the aldol addition product directly in a dehydration / cyclization reaction mediated by ammonium salts proved inefficient. An alternative means to realize significant gains in productivity was through direct elimination of the unquenched aldol adduct. In-line elimination was considered, but ultimately not pursued, as reagents which promote this reaction invariably produce an insoluble inorganic by-product and present a risk of clogging in the reactor. Rather the aldol condensation was carried out as a semi-continuous process where the unquenched aldol exit stream from the flow reactor was collected in a cooled receiver vessel to which trifluoroacetic anhydride and triethylamine were added continuously. In this manner the desired diene could be obtained, directly obviating the need for phase separation, azeotropic drying and a discrete elimination step. The product solution was heterogeneous owing to the precipitation of potassium trifluoroacetate but was formed in comparable purity to that observed in the two step process. Additionally, upon addition of methanol as a co-solvent, a homogeneous solution was obtained which was poised for the subsequent ammonia-mediated reaction. Extensive range finding and robustness studies were conducted in advance of a pilot plant execution of this sequence, a selection of which are shown for reference in Table 12. All reactions were conducted starting from 25-50 g of ester 54 and the isolated yield reflects three step sequence from 54 to 28. The continuous aldol reaction displayed a broad operating range for the reaction temperature (here defined as the bath temperature in which the flow reactor was submerged), showing only an 11% drop in isolated yield over the range 14 to -25 °C. Optimal conversion was achieved with a base charge of 1.5 equivalents or greater, where the decrease



in isolated yield at 1.3 equivalents is due to incomplete consumption of ester 54 in the aldol addition reaction.

Figure 20. Streamlined Continuous Aldol Process.

Table 12. Pyridone Process Summary and Select Range Finding Data

A more convergent approach to doravirine employs N-methylated electrophile 35 for the alkylation of pyridone 28. The reported synthesis of 35 starting from N-methylsemicarbazide (see Figure 10) could be feasible on commercial scale, the realization of a more direct and lower cost synthesis was deemed necessary. To this end various routes to the target heterocycle were evaluated (Figure 21). • •

Methylation of 36 did not provide any of the desired product. Condensation of 59-HX with orthoester 61 using a procedure analogous to that described for EMEND® provided only a low yield of 35 owing to the low regioselectivity of the cyclization. 198


•


•

Acylation of 59 with ethyl glycolate or (chloro)acetyl chloride did not produce encouraging results, however 59 reacted cleanly with glycolic acid in diglyme. The acylated product 60 crystallized from solution on cooling and was isolated in 86% yield. This material was advanced to the target electrophile 35 by a sequence similar to that previously described to achieve proof of concept for the new route.

Figure 21. Strategies for Methyl-Triazolinone Fragment.

As process development work was initiating we were unable to procure greater than gram-scale quantities of key semicarbazide raw material 59. To ensure continuous supply and a robust process to 35, a synthesis of semicarbazide 59 was included as part of the development work. Carbamate formation between phenyl chloroformate and aqueous methylamine provided 62 in 96% isolated yield (Figure 22). Semicarbazide 59 was generated by the addition of hydrazine in hot isopropanol and converted without isolation to acylated adduct 61 in 81% yield over the two steps. Base-mediated cyclization with sodium hydroxide in n-propanol afforded triazolinone 62 in 85% isolated yield. Chlorination of the primary alcohol with thionyl chloride in ethyl acetate provided the key fragment 35 in 87% isolated yield. The described route was suitable for scale-up and >1200 kg of hydroxy compound 62 have been prepared to date by this route. Having established a viable process to prepare triazolinone 35, we evaluated the final alkylation reaction to generate doravirine directly. Rigorous control of the impurity profile in the alkylation was of paramount concern as many impurities have a solubility similarly to doravirine 34 in the crystallization solvent. Conditions for the alkylation reaction were examined including solvent, temperature, base and reagents charges. Ultimately the most robust conditions 199



used Hunig’s base in a mixture of NMP and tert-amyl alcohol with a slight excess of alkylating agent 35. For ease of handling, triazolinone 62 was chlorinated in NMP and the crude stream can be used directly in the alkylation step (Figure 23). The product was generated in high solution yield with good impurity control under these conditions.

Figure 22. Process to Prepare N-Methyl Triazolinone 35. (Based on an existing figure. Copyright American Chemical Society publications, 2016) (57)

Doravirine crystallizes in two different anhydrate forms, of which anhydrate form II was desired. Anhydrate form I is kinetically preferred at moderate temperatures and thermodynamically more stable above 80 °C. Anhydrate form II is the thermodynamically more stable form below 80 °C, but growth of this form is complicated by uncontrolled nucleation of form I. The supply route to doravirine employed crystallization of the undesired anhydrate form I from the reaction mixture and a recrystallization to deliver the desired form II. To simplify processing and lower manufacturing costs, isolation of anhydrate form II with acceptable purity directly from the reaction mixture was desirable. The alkylation reaction stream after quenching with acetic acid was warmed to 70-75 °C. Water was charged and the batch seeded with anhydrate form II. Slow addition of the remaining water allowed controlled growth of anhydrate form II on the seed. Cooling to room temperature lowered to the liquor concentration of 34 to the range of 3-7 mg/mL and filtration of the resulting slurry provided anhydrate form II with chemical purity >99.5%. The final manufacturing route to doravirine is presented in Figure 23 (57). The longest linear sequence of five steps from phenol 43, with only three isolations was used to generate all phase III supplies and will support the launch of doravirine when commercialized. The new route reduces process mass intensity by greater than 85% and obviates the use of any precious metal. The overall yield is greater than 55% and it has been successfully demonstrated on batches as large as 180 kg. 200



Figure 23. Final Manufacturing Route to Doravirine.

Summary and Outlook As of this August 2016, doravirine is currently in phase III clinical trials for the treatment of HIV infection. Doravirine has demonstrated a favorable resistance profile in vitro as it remains active against viruses with K103N and Y181C mutations at clinically relevant concentrations (63, 64). In a phase 2B clinical study, similar efficacy to efavirenz was shown, but with fewer side effects, especially CNS-related, following treatment in combination with TDF/FTC (65). Development of better anti-retroviral combinations will be needed to help meet HIV as well as other health objectives worldwide. We hope that the discovery and development work described herein, in support of doravirine’s clinical program, will yield a novel medicine for patients (66).

Acknowledgments The authors would like to thank the countless smart, creative scientists with which we have had the privilege to work during the discovery and development of doravirine. This chapter is dedicated to the men and women of the Merck Frosst Center for Theraputic Research, Kirkland, Canada, where doravirine was first discovered. 201 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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