The Development and Manufacture of Azacitidine, Decitabine, and

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Chapter 9

The Development and Manufacture of Azacitidine, Decitabine, and Cladribine: Stereoselective Ribonucleoside Drug Synthesis Using the Vorbrüggen Glycosylation Erick W. Co and Julian P. Henschke* ScinoPharm Taiwan, Ltd., No. 1, Nan-Ke 8th Road, Tainan Science Industrial Park, Shan-Hua, Tainan, Taiwan 74144 *E-mail: [email protected].

The process development of 5-azacytosine-based nucleosides azacitidine and decitabine are described as a backdrop to a more detailed account of the discovery and development of cladribine, a chlorinated, deaminase-resistant derivative of the naturally occurring nucleoside, 2′-deoxyadenosine, that possesses both antineoplastic and immunosuppressive activity. To address the distinct challenges in the regio- and stereoselective syntheses of these molecules, and their stability, each process employs unique variations of the Vorbrüggen glycosylation for the key C-N bond-forming step.

The Discovery and Preclinical Development of Cladribine Background As a manufacturer of APIs, ScinoPharm receives a significant number of customer inquiries about the production of many agents, including nucleoside derivatives, both for use in generic and brand drugs. As a consequence of this interest in nucleoside drugs, the process, analytical development, and ultimately cGMP manufacture, of eight nucleoside derivatives and analogs were conducted in our company starting in the mid-2000s. In this chapter we will focus on the discovery and process development of cladribine (β-1). With the development of © 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.


azacitidine (2) and decitabine (β-3) occurring prior to cladribine (Figure 1), and with much having been learned about the Vorbrüggen glycosylation from these projects, their development will also be described here.

Figure 1. Ribonucleoside APIs produced using the Vorbrüggen glycosylation.

While the use of these drugs in the treatment of disease is much more recent, a relatively large amount of the academic and patent literature describing their syntheses already existed. These syntheses were often directed towards the preparation of collections of related nucleoside derivatives, rather than being tailored for single molecules. As a result, the synthetic methods were often inefficient and cumbersome, low yielding, and not directly scalable. Around the time that these molecules received FDA approval, [2004 for azacitidine (Vidaza®); 2006 for decitabine (Dacogen®); 1993 and 2010 for cladribine (Leustatin®)], reports of their use in the treatment of disease, as well as process and polymorph patent literature began to emerge. The existence of this literature meant that if we were to manufacture these drugs, we had to develop methods that provided us freedom-to-operate. This necessity spurred the innovation and the development of novel processes and intellectual property that will be discussed later in this chapter. The most significant lesson from the development of these compounds was that seemingly small differences between apparently very similar compounds often lead to different synthetic and process strategies for their manufacture. For example, the absence of the 2′-hydroxyl group of decitabine and cladribine resulted in lower stereoselectivity than azacitidine during the glycosylation step. The substitution of a carbon atom in the cytosine ring for nitrogen made the nucleobase rings of azacitidine and decitabine very susceptible to hydrolysis as compared to their natural counterparts, cytidine and 2′-deoxycytidine. These examples were only a few of the challenges of this set of related compounds. The Advent of Nucleoside Analogs as Viable Therapeutics The study and use of nucleoside analogs as agents for anticancer and antiviral therapeutics has spanned the last fifty years. The advent of the field can be traced back firmly to the emergence of cytarabine (for Acute Myeloid Leukemia (AML)) and edoxudine (for Herpes Simplex Virus), agents both approved by the FDA in 1969 (Figure 2). Though edoxudine since has been superseded by improved treatments and is no longer clinically relevant, more than 25 nucleoside or 272

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.


nucleotide analogs have been approved for antiviral applications such as hepatitis B and HIV (1). Cytarabine, on the other hand, has withstood the test of time and is listed on the World Health Organization’s Model List of Essential Medicines (2). Cytarabine has paved the way for six additional approved nucleosides in the arena of anticancer treatments (Table 1), including cladribine, the primary focus of this chapter.

Figure 2. Cytarabine and edoxudine signaled the start of the nucleoside era for cancer and viral therapeutics.

Nucleosides consist of a nucleobase, usually from the purine (adenine or guanine) or pyrimidine (cytosine, uracil, thymine) families, attached to a sugar derivative. Nucleotides, which comprise many of the antiviral agents, incorporate an extra diversity point by the installation of a variable number of phosphate or polar groups to the sugar moiety. Not surprisingly, this additional functionality is a primary reason for the greater number of antiviral therapies available on the market today. Figure 3 illustrates the nomenclature basis in this class of therapeutic agents.

Figure 3. Nomenclature of nucleosides and nucleotides. 273 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.

agent (trademark name)

current manufacturer or licensor

FDA approval

mechanism of action (target)

indication(s)

cytarabine (Aracytine®)

Pfizer

1969

DNA incorporation

Acute Myeloid Leukemia; Acute Lymphocytic Leukemia Lymphomas

gemcitabine (Gemzar®)

Eli Lilly

1996

DNA incorporation

Non-small cell lung cancer; Pancreatic, bladder, and breast cancers

azacitidine (Vidaza®)

Celgene

2004

DNA methyltransferase inhibitor

Myelodysplastic syndrome

decitabine (Dacogen®)

Eisai and JanssenCilag

2006

DNA methyltransferase inhibitor

Myelodysplastic syndrome; Acute Myeloid Leukemia

capecitabine (Xeloda®)

Hoffmann-La Roche (Genentech)

1998

Irreversible inhibitor of thymidylate synthase

Metastatic breast cancer; Metastatic colon cancer Gastric cancer (off-label) Esophageal cancer (off-label)

entecavir (Baraclude®)

Bristol-Myers Squibb

2004

Reverse Transcriptase Inhibitor

Hepatitis B

cladribine (Leustatin®, Movectro®)

Janssen-Cilag

1993 2010a

Adenosine Deaminase inhibitor

Hairy Cell Leukemia; Relapsing Remitting Multiple Sclerosisa

274


Table 1. Prominent Nucleoside Analogs, Approval Dates, and Indications


current manufacturer or licensor

FDA approval

mechanism of action (target)

indication(s)

clofarabine (Clolar®)

Genzyme

2004

DNA incorporation

Acute Lymphoblastic Leukemia

tegafurb

Merck-Serono

N/A

RNA & DNA incorporation

Advanced Colorectal Cancer

a Cladribine was approved for multiple sclerosis in 2010 in Russia and Australia; Merck consequently withdrew all marketing applications in 2011 voluntarily after failure to obtain additional approvals in Europe and North America. b Tegafur, a prodrug of 5-fluorouracil (5-FU), is a component of the combination drug tegafur/uracil (Uftoral®) approved in many countries, but excluding the United States.

275


agent (trademark name)



ScinoPharm’s Involvement with Cladribine ScinoPharm entered into the nucleoside class of therapeutics in 2005 with gemcitabine which had presented a promising opportunity for generic manufacturing. As a result, we developed a novel, patentable process for the synthesis of gemcitabine. Subsequent customer inquiries turned our focus onto the synthesis of the DNA demethylating agents: azacitidine and decitabine. Capitalizing on our academic, albeit pragmatic approach to development, we established efficient routes to the synthesis of both azacitidine and decitabine. (3, 4). In 2007, we became aware that cladribine, already approved in 1993 for Hairy Cell Leukemia (HCL), was being positioned as an oral multiple sclerosis treatment by Merck-Serono, having obtained fast-track status from the FDA that same year (5). The prospect of drug repositioning into an indication with a large patient population, coupled with the significant relevance of our platform synthesis technology, made the option to pursue cladribine very attractive. In addition to cladribine, ScinoPharm completed the preparation of a number of other nucleoside analogs. (Figure 4).

Figure 4. Various nucleoside analogs manufactured by ScinoPharm.

Rationale Toward the Discovery of Cladribine Cladribine (2-chloro-2′-deoxyadenosine a.k.a. 2-CdA, Leustatin®, ® Movectro ) is a synthetic nucleoside analog composed of a modified purine nucleobase and a deoxyribose component. Biological activity of ribose and various halogenated analogs, including the individual components of cladribine, had been studied as early as the 1950s (6). The first mention of 2-chloro-2′-deoxyadenosine was in the early 1960s as an uncharacterized intermediate (7) and as an isolated compound by Ikehara (8, 9). Ikehara and coworkers prepared the novel derivative 8,2′-cyclonucleoside 4 from chlorosugar 5 and studied its synthetic utility for the synthesis of 2′-deoxyadenosine (6) (Scheme 1). 276 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.


Scheme 1. Cladribine as a Synthetic Intermediate toward 2′-Deoxyadenosine

The earliest suggestion of 2-chloro-2′-deoxyadenosine as an in vitro anti-proliferative agent, however, was reported in 1972 by Broom et al. (10) In their studies, they described the structure-activity relationship (SAR) of various substituted 2′-deoxyadenosine derivatives at the 2-position of the purine. Table 2 aptly demonstrates that the β-anomer β-1 is equivalent to, or more potent than its corresponding α-anomer α-1. It is important to note that cladribine (first entry) is the most potent analog, with 7 x 10-8 M inhibitory activity against L-1210 cells. Cladribine is also several orders of magnitude more potent than its corresponding ribose derivative, 2-chloroadenosine 7, in the same cell line. The rationale for directed use of 2-chloro-2′-deoxyadenosine in its subsequently approved use in lymphoproliferative diseases, however, was pioneered through the drug design and research of Carson et al. (11) Rather than conducting a protracted high-throughput and medicinal chemistry approach to identifying hits and leads, they elected to approach drug discovery by exploiting the unique mechanism of action of deoxyadenosine analogs. They took note of a rare pediatric immunodeficiency in which lymphocyte levels were depleted by a malfunction of the enzyme adenosine deaminase (ADA) (12). They postulated that lymphospecific toxicity in this particular ailment may arise from the selective buildup of deoxyadenosine nucleotides by lymphocytes, which are known to have higher levels of deoxycytidine kinase, and conversely, low dephosphorylation activity (13). Armed with this hypothesis, Carson et al. sought to design an analog of deoxyadenosine that could induce similar lymphocytic effects as experienced in autoimmune diseases. Their strategy revolved around the chief principles that: • •

The agent should be a close analog of deoxyadenosine to retain activation properties. The agent should exhibit stability against key enzymes such as ADA and nucleotide phosphorylases. 277



They found that substitution of the hydrogen at the 2-position of adenine afforded resistance to ADA (Table 3). Furthermore, choice of a chlorine substituent effectively retained its substrate specificity for deoxycytidine kinase, allowing for proper phosphorylation inside the cell. Out of a focused set of 25 deoxyadenosine analogs, 2-chloro-2′-deoxyadenosine was determined to have the best potency and selectivity in in vitro experiments and in vivo with L-1210 leukemia in mice (14).

Table 2. Early SAR Studies on Nucleoside Components and Analogs

278 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 3. Toxicity of ADA-Resistant Analogs toward Various Lymphoblasts

Preclinical and Clinical Development of Cladribine Compared to other anticancer therapeutics, cladribine possesses a unique dual-mode of action in being able to act upon lymphocytes in both the dividing and dormant states (15). Figure 5 illustrates the cellular metabolism of cladribine. Upon internalization, cladribine, which is resistant to ADA, is sequentially phosphorylated by deoxycytidine kinase (dCK), which is present in higher levels than its counterpart, 5′-nucleotidase (5′-NT). Once cladribine triphosphate is generated, the mechanisms of both dividing and quiescent lymphocytes are disrupted, leading to apoptosis. 279 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.


Figure 5. Cellular uptake and activation of cladribine leads to apoptosis of both dividing and quiescent lymphocytes.

Figure 6. Hairy Cell Leukemia cells are characterized by a hair-like layer surrounding the cell surface. (Reproduced with permission from Dr. William Karkow, M.D., F.A.C.S. (16)) 280 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.


Regarding the selection of an optimal indication, hematological malignancies dependent on lymphocyte proliferation were quickly identified during preclinical evaluation. Though shown to be effective for diseases such as chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma, and acute myelogenous leukemia (AML), cladribine proved superior in a rare form of chronic leukemia, Hairy Cell Leukemia (HCL). HCL, whose cells have clear hair-like protrusions from its surface (Figure 6) (16), accounts for approximately 2% of the leukemic population. In one of the earliest clinical trials for cladribine, Piro et al. followed a 144 patient cohort for a median of 14 months. They reported (17) that 123 (85%) achieved complete responses, 17 (12%) partial responses, and 3 (2%) recorded no response. At 36 months post treatment, only 4 patients had relapsed. Cladribine for early clinical trials was produced via enzymatic methods. As shown in Scheme 2, a Lactobacillus helveticus-derived transdeoxyribosylase, with thymidine as the deoxyribose source, effectively glycosylated 2-chloroadenine (8). Subsequent purification by ion-exchange chromatography afforded pure cladribine in unspecified yields (18).

Scheme 2. Carson’s Enzymatic Synthesis of Cladribine for Clinical Trial Use Despite facing the uncommon patent challenges of a drug that has been described in the literature for decades prior to its approval, the determination and perseverance of Carson safely ushered cladribine through clinical trials (18). Under the Orphan Drug Act, cladribine was approved by the FDA in 1993, licensed as Leustatin®, and became a prominent standard of care for lymphoproliferative diseases. Current and Potential Use of Cladribine for Lymphoproliferative Indications Compared to its analogs, azacitidine and decitabine, cladribine’s current market demand and use is very small (Table 4) (19). Cladribine’s low demand, despite still being considered widely as the first option therapy for HCL (20), may be attributed to several factors: • • • •

HCL accounts for only 2% of total leukemic incidence. HCL symptoms may subside before medical intervention is required. The majority of patients require only a single course of therapy. The treatment cycle for an average 70 kg adult male is 1 equiv TfOH. As with TfOH in MeCN, the β/α ratio increased with decreasing temperature when TMSOTf in DCM was used instead (β/α was 1.3:1 at 20 °C, 2.0:1 at -20 °C and 2.6:1 at -40 °C). In contrast to TfOH, however, as long as the amount of TMSOTf was kept at 2:1). Further, increasing the reaction concentration or the molar equivalents of nucleophile 16 resulted in decreased selectivity for the β-nucleoside.


Stereoselectivity Explained When the completed glycosylation product mixture comprising 22a was allowed to stand at ambient temperatures for an extended period of time without work-up, the initial >2:1 β/α ratio degraded to a 90% HPLC-purity was conveniently isolated from the deprotection slurry by filtration of the mixture. The impurities were efficiently cleared into the filtrate. In fact, the β/α ratio was typically amplified from 2–3:1 in crude 22b to >60–105:1 of decitabine/α-3 due to the relatively high solubility of the α-anomer in MeOH.


Purification of Decitabine While MeOH proved to be an excellent solvent to obtain crystalline, high-purity (99.9%) decitabine (72, 73, 83, 84), its poor solubility (91) meant that ≥80 volumes were required for dissolution. Although small amounts of DMSO significantly reduced the amount of MeOH (≤20 volumes) required, the recovery yield was unsatisfactory. With the threat of residual DMSO contaminating the recovered API, anhydrous MeOH was therefore selected. Yields of API-grade decitabine were about 70% and another 15% could be recovered by partial concentration of the mother liquors, isolation and recrystallization.

cGMP Manufacture of Decitabine Prior to the first cGMP manufacture runs, further process development was conducted. The most significant process change was an increase in glycosylation temperature from -40 °C to ~ 0 °C. In addition to the warmer temperature being more suitable for manufacturing, it ensured complete and rapid conversion of 21 without impacting product quality. Despite the β/α ratio inevitably being reduced due to the rate of in situ anomerization being higher at 0 °C, the β-anomer still comprised around 50% of the crude 22b mixture, which was in fact similar if not slightly better than had been achieved in the exploratory phase of the development. Further changes to the process allowed enrichment of the β-anomer by partial precipitation of the less soluble α-anomer α-22b during work-up with β/α ratios of the isolated 22b being about 3:1. In fact, not only was the yield increased from around 50% up to 70%, based on β-22b, but the quality of crude decitabine was increased from around 90% up to ≥97%. The purification step was modified by incorporation of an activated charcoal treatment step, partial concentration, and holding at a cooler temperature, resulting in an improved 75% recovery yield. Together these changes resulted in process and operational simplification and reduced manufacturing times while increasing the overall yield to 15% (based on 21). The first cGMP manufacturing campaign proceeded smoothly. Silyl 5-azacytosine 16 was prepared as for the manufacture of azacitidine (see cGMP Manufacture of Azacitidine). Using 3.6–3.7 kg batches of 2-deoxyribosyl acetate 21, 4-chlorobenzoyl protected decitabine 22b was obtained as a mixture of anomers (β-purity was 48-55%) from the glycosylation of an equimolar amount 299



of 16 using 1.05 equiv TMSOTf in DCM at 0 °C (5 h). Stabilization of the glycosylation mixture was achieved using about 1 equiv MeNH2 in MeOH, prior to dilution with DCM and treatment with aqueous NaHCO3 at 25 °C. Of the 22b mixture, β-22b was produced in yields of about 40% based on 21. Following its isolation by evaporation, milling (FitzMill®) and drying, deprotection of 22b as a slurry on 5.4–6.2 kg batch scales using 30% NaOMe in MeOH furnished 96.4–97.1% HPLC area %-pure crude decitabine following filtration in yields of 59–66% (based on β-22b). Crystallization of the crude API on 1.1–1.2 kg scales from MeOH provided high purity decitabine meeting the established specification in ~ 66–72% yield. Overall yields of up to almost 20% were achieved reflecting improvements upon the laboratory process.

Conclusion As per azacitidine, decitabine was also produced using a two-step/two-pot silylation/Vorbrüggen glycosylation. Although the process strategy could be used on both nucleosides (4), modifications (TMSOTf in DCM) to the glycosylation step allowed better stereocontrol and complete conversion of the glycosyl donor 21. The lack of high stereoselectivity in the glycosylation step and the unexpected anomerization of protected decitabine 22b, inherently limited the efficiency of the process. Deactivation of the glycosylation catalyst with MeNH2 was helpful in inhibiting anomerization after the reaction. Unlike that for azacitidine, omission of an aqueous work-up after glycosylation was not feasible. The deprotection step proved the most challenging step during process development. Crude protected decitabine 22b had to be sufficiently exposed to aqueous NaHCO3 in the prior step to destroy the silicon residues, had to be devoid of residual DCM and moisture, and had to be ground into a fine powder to ensure reproducibility of the reaction. Heterogeneous conditions were preferred to homogeneous conditions but ultimately crude decitabine of 97% purity was achieved by filtration in up to about 70% yield (based on β-22b) due to excellent clearance of the α-anomer and other impurities into the filtrate. In all, only one dedicated crystallization step was required and chromatography was not necessary. The process was cost-effective and allowed the industrial scale manufacture of decitabine of very high quality. The novelty, non-obviousness and industrial applicability of the process resulted in the granting of our US patent application (90) in 2013 (4). With the next target, cladribine, also being a 2′-deoxyribonucleoside, it was anticipated that the use of low temperatures in the glycosylation step would improve stereoselectivity.

Process Development and Manufacture of Cladribine Reported Methods for the Synthesis of Cladribine Cladribine (Figure 8) has been prepared using a diverse range of approaches over the last fifty years. As compared to azacitidine and decitabine, for which 300 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.


much larger numbers of manufacturers exist, cladribine has received less attention. This may be due to the very small annual volume (1:1 (following dissolution with moist DMSO) were only witnessed when using combinations of strong catalysts (TMSOTf, TBSOTf or TfOH) with BSTFA (but not for weaker MsOH, p-TsOH, TFA or Ms2O with BSA or BSTFA). reactions conducted under homogeneous conditions were nonstereoselective.

We realized that selective precipitation of the β-anomer β-30a during glycosylation combined with anomerization of the α-anomer α-30a in the solution phase was responsible for the overall β-enrichment witnessed (Scheme 16). Glycosylation reversibility, presumably via oxocarbenium ion 11a, had already been implied in the isomerization of the N-7 (31) to the N-9 (30) regioisomers, and in the corresponding glycosylation in the preparation of decitabine (vide supra) and literature precedents (54, 66, 88, 89). Apparently the combination of TfOH, TMSOTf or TBSOTf with BSTFA was essential for this process to occur under the conditions tested. It is probable that the higher β-selectivity observed when using 20 mol% (Table 10, entry 6) as compared to when using 10 mol% (entry 7) was the result of more rapid anomerization and therefore conversion of α-30a to β-30a. We believe that the α-selectivity observed in tests using the weaker silylating agent BSA and/ or weaker glycosylation catalysts results from an absence of anomerization and therefore indicate that the α-anomer is kinetically favored over the β-anomer in the glycosylation of 29 with 21. 309



Scheme 16. Precipitation-Driven β-Enrichment via Anomerization. (Reproduced with permission from reference (119). Copyright 2013, ACS Publications)

Aging Step With anomerization being true, it followed that extending the reaction time would enhance selectivity and increase the isolatable yield of β-30a. Indeed, while immediate filtration upon consumption of deoxyribofuranose 21 (1 h at 60 °C) provided a 45% isolated yield of β-30a (β/α 28:1; Table 11, entry 1), leaving the product mixture to ‘age’ at 60 °C (entries 2-8) provided improved yields (53–68%) and greater β-enrichment (β/α 44–71:1), especially when the aging time was not less than 8 h. At 20 °C, glycosylation was complete within 19 h but proceeded in lower yields (39–42%; entries 9 and 10) along with relatively low β-enrichment that was, in fact, identical to that following filtration immediately upon consumption of 21 (entry 1). Notably, when the glycosylation product produced at 20 °C was subsequently aged at 60 °C for another 8 h (entry 11), improved β-selectivity and yield were obtained. This showed that while the glycosylation operated at ambient temperature, a discrete aging step at a raised temperature was necessary to produce higher yields and β-enrichment of the filtered solids. When the glycosylation/aging steps were conducted over an extended period at 80 °C, a low yield (38%) was obtained (entry 12). This displayed essentially no benefit over that of the exploratory phase of the project when the glycosylation was conducted for 30 min at 80 °C (Table 7, entry 1). We suspect that at 80 °C the solubility of β-30a is too high for effective precipitation to drive the equilibrium in the desired direction. Given these results, a target temperature of 60 °C was deemed suitable for both the glycosylation and aging steps. 310



Table 11. Study of the Aging Stepa

Using potency assay, the absolute amounts of the two anomers (Figure 12) present in the solution phase throughout the aging step at 60 °C were determined over 1 d. While the amount of the β-anomer in solution was constant at a 2–4% assay-determined yield (based on 21), the α-anomer fell from 29% to 9% yield. The isolated yields of β-30a from nine aging experiments for different periods (plotted in Figure 12) clearly show the correlation between the isolated yield of β-30a, aging time and the decreasing level of the α-anomer in the solution phase. At the end of the test period, 97.3% HPLC-pure β-30a (2.1% α-30a) in 68% yield was isolated by filtration. Including the β-anomer present in solution, the total reaction yield of β-30a was just over 70%. The remainder of the mass balance was accounted for by the ca. 10% total yield of α-anomer and 20% decomposition. 311 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 believe that more rapid anomerization, and therefore increased yields and process efficiency, is possible by further optimization of the conditions. The addition of more silylating agent and/or catalyst after completion of the glycosylation step would probably achieve this.

Figure 12. Solution phase assay of α-30b and β-30b and isolated yields of β-30a over the aging step. (Reproduced with permission from reference (119). Copyright 2013, ACS Publications)

Work-up As per the azacitidine process (vide supra), the relatively low catalyst loading meant that an aqueous work-up could be avoided altogether. In fact, the glycosylation slurry was simply filtered and washed (with MeCN) giving crude, albeit relatively high purity (>94%) β-30a. The silylated α-anomer α-30a, spent reagents and impurities were conveniently purged into the filtrate. Anhydrous conditions were essential, however, otherwise desilylation, co-precipitation and contamination of β-30a with the non-silylated α-anomer α-30b occurred.

Deprotection Deprotection of crude β-30a was effective using catalytic amounts of NaOMe in MeOH. Despite being heterogeneous as a result of the low solubility of β-30a and cladribine in MeOH, deprotection was rapid at 20 °C and furnished crude cladribine of 98.3–99.7% HPLC area % purity in up to 92% yield. Notably, on laboratory scales the crude product was contaminated with typically 80% yield that was contaminated with

The Development and Manufacture of Azacitidine, Decitabine, and

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