Discovery of Novel Protein Fucosylation Inhibitors and Development of

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Chapter 2

Discovery of Novel Protein Fucosylation Inhibitors and Development of a Manufacturing Process To Prepare Inhibitor 6,6,6-Trifluorofucose Seb Caille1 and John G. Allen*,2 1Process

Development – Drug Substance Technologies, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States 2Therapeutic Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States *E-mail: [email protected].

In the manufacture of therapeutic monoclonal antibody proteins the level of fucosylated glycan in the product can affect efficacy and is a critical product attribute. Particularly for biosimilar antibodies, the fucosylation level may need to substantially match that of the reference product. 6,6,6-Trifluorofucose (1, fucostatin I), is a metabolic inhibitor of fucosylation that when added to cell culture reduces the fucosylation of recombinantly expressed antibodies in a concentration-dependent manner. A robust process for the synthesis of 1 from D-arabinose in 11% overall yield and >99.5/0.5 diastereomeric ratio was developed based on a key diastereoselective ruthenium-catalyzed tandem ketal hydrolysis-transfer hydrogenation reaction.

Introduction Antibody-dependent cell-mediated cytotoxicity (ADCC) is an innate immune response in which natural killer (NK) cells and oesinophils are targeted to pathogen-infected or cancer cells by IgG antibodies. The antibody binds to the target cell via its variable region and to the FcγRIIIa receptor of the effector cells via its constant fraction crystallizable (Fc) region. Therapeutic IgG1 antibodies bearing Asn297 glycans with lowered levels of fucosylation have been shown © 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.

to exhibit enhanced ADCC due to increased binding of the Fc region to fraction crystallizable γ receptor IIIa (FcγRIIIa, Figure 1). Afucosylated Fc glycan forms stronger interactions with FcγRIIIa glycan and favors a high affinity conformation around the Fc residue Tyr296 (1). Increased ADCC has been associated with improved in vivo efficacy in animal oncology models and in the clinic (2, 3), and therefore fucosylation level is an important product attribute to control in the manufacturing of mAb products (4). Protein fucosylation may be reduced by: • •


• •

Manipulation of cell growth and production conditions. Screening for cell lines or clones producing protein with low fucosylation levels. Use of engineered cell lines in which a key enzyme involved in protein fucosylation is knocked out. Addition of selective or non-selective chemical inhibitors of fucosylation (5, 6).

Figure 1. Fucostatins and the ADCC response. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Chemical inhibition is an appealing approach to reduce fucosylation during antibody manufacture in as much as other protein or cell growth attributes remain undisturbed. In addition, this method permits fucose levels to be controlled based on inhibitor dosage using cell lines that had been previously optimized. Inhibitors of fucosylation (7, 8), and other glycosylations (9–11) have been previously described, however, reported potencies are in the micromolar range. In addition to weak potency, many glycosylation inhibitors carry the risk of incorporation into the antibody product (12–14). 38 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.


6,6,6-Trifluorofucose (1, fucostatin I, Figure 2) was discovered to be a fucosylation inhibitor and validated in our laboratories along with several other potent fucosylation inhibitors (15). The potential of fucostatin I was demonstrated in improved cell lysis activity of afucosylated anti-mesothelin (16) IgG1 monoclonal antibodies produced in the presence of this inhibitor. The wide-scale use of 1 in mAb manufacturing was anticipated and mandated the development of an efficient manufacturing process to prepare this molecule on several hundred gram scale. The target material (1) had previously been synthesized by Tokokuni and coworkers (17), however this route to the molecule did not meet our production requirements due to the use of stoichiometric toxic metals and the use of multiple chromatography steps, and due to the overall length of the synthetic route including several protection/deprotection steps. This chapter will focus on: • •

The identification and evaluation of 6,6,6-trifluorofucose (1) and other fucosylation inhibitors. A description of the manufacturing process developed to prepare 1 on several hundred gram scale.

Figure 2. Furanoside and pyranoside forms of 6,6,6-trifluorofucose (1).

Discovery of the Fucostatin Inhibitors of Fucosylation. Inhibition of GMD by GDP-Fucose and GDP-1 (1D) 6,6,6-Trifluorofucose (1, fucostatin I) was designed to function as a mechanism targeted inhibitor of fucosyltransferase wherein the oxocarbenium character of the transition state of the α-(1,6)-fucosyltransferase FUT8 would be destabilized by the electron withdrawing fluorine atoms (18). Fucose occurring in endogenous glycoproteins is generated starting from D-glucose by the de novo pathway shown in Figure 3. It is proposed that guanosine diphosphate-1 (GDP-1, 1D) is formed by the salvage pathway depicted in Figure 3 and in fact serves as an allosteric inhibitor of GDP-mannose 4,6-dehydratase (GMD), thus blocking the production of fucosylated glycoproteins. In the following sections we suggest that allosteric inhibition of GMD (19) by 1D rather than inhibition of fucosyl transferases is the primary mechanism of action. The acetylated carbohydrate 2 39

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.


was also prepared and evaluated as a fucosylation inhibitor. It is proposed that this material (2) diffuses into cells as a prodrug, undergoes deprotection to generate 1, and leverages the fucose salvage pathway to form the active metabolite 1D (Figure 3). Compound 1 on the other hand likely enters the cell by active transport (20).

Figure 3. Formation of 1D and Inhibition of GMD. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

40 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.


Inhibition and Rescue of Cell Surface Fucosylation Inhibitors were initially screened for their ability to inhibit cell surface fucosylation in Chinese hamster ovary (CHO) cells. In an automated assay, cell surface fucosylation was determined by Lens Culinaris A (LCA) lectin binding to fucose-containing glycans as detected with a fluorescein isothiocyanate (FITC)-antibody conjugate and measured by fluorescence assisted cell sorting (FACS, Figure 4a). Cell surface glycans could be completely defucosylated by treatment with 2. In treated cells, cell surface fucosylation could be restored (rescued) by addition of L-fucose or L-fucose per-O-acetate in a dose-dependent manner. Complete rescue resulted at 100 µM of L-fucose or L-fucose per-O-acetate, suggesting that FUT8 is not inhibited in cells treated with 2, although studies on purified FUT8 were not performed. Importantly, D-fucose, which is not a substrate for fucose transporters nor a substrate for fucose kinase, failed to rescue inhibition by 2 (Figure 4b).

Cocrystallization of GDP-Fucose and 1D with GMD Cocrystal structures of GDP-fucose (PDB 5IN5) and 1D (PDB 5IN4) with GMD were obtained, and for the first time showed the occupied allosteric fucose binding site of the enzyme (Figure 5). The crystal structure asymmetric unit contained four GMD monomers in a homotetramer with an overall appearance of a dimer of dimers. Each GMD monomer consists of an N-terminal cofactor binding domain containing an NADP/H cofactor and a C-terminal substrate binding domain containing GDP. The NADP/H and GDP binding sites formed at the interfaces between two GMD-GMD symmetrical dimers. Each GMD-GMD dimer also contains two molecules of 1D at allosteric sites formed at the dimer interface. 1D forms simultaneous direct and water-mediated hydrogen bond contacts with two different monomers, likely stabilizing this interface. 1D is bound in a horseshoe conformation, and the nucleobase and trifluoromethyl group of the inhibitor interact with one GMD monomer while the phosphate and ribose groups interact with the other monomer. A loop of residues 69-77 near the allosteric site is disordered in a known apo GMD structure (PDB 1T2A, unpublished) and in our other structures lacking inhibitors (data not shown). In the presence of 1D this loop becomes ordered and packs above the inhibitor. The guanine nucleobase forms a base stacking interaction between His75 in the allosteric loop and Phe60. The trifluoromethyl group of 1D is buried in a small pocket and compared to GDP-fucose forms more extensive Van der Waals (vdw) interactions with near-by hydrophobic residues. These increased vdw interactions are in all likelihood responsible for the gain in potency over the natural inhibitor GDP-fucose (KD 11 vs. 35 µM respectively) as both molecules bind in identical conformations with a slight 0.5 Å shift of the larger trifluoromethyl group.



Figure 4. (a) Dose-response inhibition of cell surface fucosylation by 2. (b) Rescue of cell surface fucosylation by L-fucose and L-fucose per-O-acetate, LCA lectin binding. 20 µM 2 gives 14.0 ± 3.3% cell surface fucosylation. Error bars represent the standard deviation. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

In order to explain the unexpectedly high potency of 2, the molecule was initially proposed to covalently bind to GMD (21–23), but biophysical data were not consistent with that hypothesis. The high potency of 2 and low dissociation constant of 1D were instead explained by contacts revealed in the 1D/GMD cocrystal structure as described above. Overlap with GDP-fucose was perfect, but for the exception of a noted deeper incursion of the trifluoromethyl group into a pocket coated with alanine and leucine. The additional vdw interactions result in a reduced dissociation constant for 1D compared to GDP-fucose. The activity of 2 is remarkable compared to the related difluoro (3) and monofluoro (4) analogs (Figure 6). These analogs were less potent in the cell surface assay (4.7±1.9 µM and 6.6±5.5 µM, respectively), underscoring the importance of the trifluoromethyl group in potently binding GMD. 42 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. (a) 1D (PDB 5IN4) cocrystallized with GMD. Four molecules of 1D bind to the GMD tetramer at the interface of a dimer pair as indicated by the arrows. (b) 1D in the GMD binding pocket, partial space-filling model. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society. 43 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 6. Difluoro and monofluoro analogs of 2.

Metabolite Analysis and Design of Non-Incorporating Fucosylation Inhibitors As shown in Figure 3, 2 diffused into cells upon dosing and was de-esterified to generate 1. This material (1) was subsequently converted to 1C by fucose kinase, and then to 1D by GDP-β-L-fucose pyrophosphorylase (GFPP). Both metabolites 1C and 1D were identified by selective reaction monitoring – mass spectrometry (SRM-MS) in cell lysate (Figure 7a for 2). 1D was synthesized by chemo-enzymatic methods and was shown to bind GMD by surface plasmon resonance (SPR, KD 11 µM, Figure 7d). GMD as the target of inhibition was consistent with the accumulation of GDP-mannose (Figure 7b for 2), as well as the disappearance of GDP 4-keto-6-deoxy-mannose (data not shown) and GDP-fucose in the cell. These data together with the rescue data vide supra suggested FUT8 inhibition is not a significant contributor to the overall inhibition of fucosylation. As will be discussed in more detail in an upcoming section, dosing CHO cells with 2 led to low levels of 1 being incorporated into the glycans of expressed proteins by replacement of fucose. Replacing the exoanomeric oxygen of 1 and 2 with a methylene group was expected to block inhibitor incorporation by preventing the corresponding GDP sugars from being substrates in glycosylation reactions. By preparing these methylene analogs as phosphonates we hoped they would enter the salvage pathway as substrates of GFPP. Thus fucose phosphonate analogs 5 and 6, and 6,6,6-trifluoromethylfucose phosphonate analogs 7 and 8 were prepared as suitably protected prodrugs (24) (Figure 8). We were pleased to find that the phosphonate prodrug 5 was deprotected in the cell and gave rise to a phosphonate GDP metabolite 5B as demonstrated by identification of these compounds in cell lysate by SRM-MS (Figure 7a for 5). The GDP-phosphonate 5B was synthesized and was found to bind to immobilized GMD by surface plasmon resonance (SPR, Figure 7d, KD 9 µM). Protein incorporation was not observed for phosphonate 5, and this inhibitor blocked protein and cell surface fucosylation (IC50 18.1±6.5 µM). The non-native α-anomer 6 is not considered to be a substrate for the fucose salvage pathway and it was inactive in the cell surface assay. Another analog of 5 that combined the phosphonate strategy and the 5-CF3 group of 1 and 2, β-trifluoromethylphosphonate 7, formed the corresponding GDP metabolite 7B but failed to inhibit fucosylation. This observation may have been due to inactivity at GMD as evidenced by a lack of GDP-mannose accumulation (Figure 7b for 7).





Figure 7. (a) Log(AUC) of metabolites observed by SRM-MS in lysate from cells treated with fucosylation inhibitor analogs 2, 5, 7 and 8. (b) Relative concentrations of GDP-mannose and GDP-fucose observed by SRM-MS in lysate from cells treated with fucosylation inhibitor analogs 2, 5, 7 and 8, and normalized to 100%. (c) Chromatographic data for metabolites observed by SRM-MS in cells treated with 2 and arbitrarily normalized to 100% intensity. (d) Derived steady-state SPR dissociation curves for 1D (circles, KD 11 µM), 5B (triangles, KD 9 µM), GDP-mannose (diamonds, KD 51 µM), GDP-fucose (open triangles, KD 35 µM),. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Figure 8. Exoanomeric CH2 fucosylation inhibitor analogs and metabolites. 46 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.


By contrast, the corresponding non-native α-anomer 8 was a potent inhibitor of cell surface (IC50 7.0±7.0 µM) and protein fucosylation. Surprisingly, the glycans of protein produced in the presence of 8 showed some incorporation of 1. SRM-MS analysis of lysates from these cells identified the corresponding deprotected phosphonate (8A), but relatively little of the GDP-phosphonate (8B). Instead, the phosphate metabolite was observed in this case (1D). Cellular (25) hydrolysis of the α-phosphonate 8 to reducing sugar 1 would explain the similarity in metabolic data provided by 8 and 2, where both 8 and 2 give rise to the same metabolite (1) (26). This interesting hypothesis has some precedent in the cleavage of C-glycosides in bacterial cell-free lysate (27) and in phosphonate hydrolysis by bacterial hydroxyethylphosphonate dioxygenase and the protein product of the bacterial PhpD gene (28, 29), and deserves further study.

Blocking Fucosylation of Expressed Proteins In order to study the effect of the fucostatins on fucosylation of expressed proteins, anti-TRAIL2 receptor (TR-2) immunoglobulin G1 monoclonal antibodies (IgG1 mAbs) (30) and anti-mesothelin (MSLN) IgG1 mAbs (16) were prepared in CHO cells using a single dose of 2 in DMSO on day 0. Protein fucosylation could be titrated with different doses of 2, with an EC50 of ~4 µM (Figure 9a). No changes were observed up to the top concentration of 20 µM of 2 in the cumulative growth, viability or productivity behavior of treated cells, or in other protein characteristics (Figure 9b). Selective inhibition of fucosylation was a design of the targeted fucostatins. As was seen for cell surface fucosylation (Figure 4a), fucosylation of produced protein could also be rescued by a single dose of fucose on day 0 via the GMD-independent salvage pathway (Figure 9d). Hydrophilic interaction liquid chromatography (HILIC) analysis found 1 incorporated in lieu of fucose in about 0.5% of total glycans (white triangles in Figure 9e). Both compounds 1 and 2 gave similar results in these studies. Although compound 2 slowly hydrolyzed to 1 in cell culture medium, this did not affect the results observed as both 1 and 2 are cell penetrant. ADCC activity was tested in vitro for anti-MSLN antibodies targeting natural killer (NK) cells from human donors to MSLN expressing cancer cells. Target cell lysis was improved for Abs produced in the presence of increasing amounts of 2 (Figure 9c), an observation consistent with reduced fucosylation levels. Additionally, NK cells expressing low affinity V158F fraction crystallizable γ receptor IIIa (FcγRIIIa, V158F polymorphism) showed good efficacy on activation by afucosylated anti-MSLN antibodies. The grey bars represent cell lysis EC50’s for lower antigen expression CAPAN2 (pancreatic adenocarcinoma) cells treated with natural killer (NK) cells isolated from a human donor harboring the low affinity FcγRIIIa allele. Efficacy is significantly reduced for this system compared to high antigen expressing N87 (gastric carcinoma) treated with high affinity Val158-FcγRIIIa allele NK cells (black bars). However, using antibodies expressed in the presence of 2, less than 0.1 µg/mL of anti-MSLN antibodies with 20% afucosylated glycans was needed to lyse 50% of CAPAN2 target cells 47


by low affinity NK cells. The 80% fucosylated antibodies resulted from a single 2 µM dose of 2 on day 0 of a 10 day production run. Patients with high and low affinity FcγRIIIa receptor alleles show different responses to ADCC therapeutics (31). One advantage of afucosylated therapeutic IgG1 antibodies is improved responses in all patient populations (32, 33), as modeled in this assay.


Glycan Incorporation Comparison for Fucosylation Inhibitors Besides reduced fucosylation, HILIC analysis of proteins expressed in the presence of 2 also displayed low levels of incorporation of 6,6,6-trifluorofucose in place of fucose (Figure 9e). No other aspects of cell growth including cumulative viable cell density, mAb titer, and specific productivity in the expression system were impacted by dosing with 2. Low incorporation of 2 suggests that 1D is a weak substrate of fucosyltransferase FUT8, possibly due to inductive electron withdrawing destabilization of the glycosylation transition state (34). Higher levels of inhibitor incorporation occurred with difluorofucose (3) and monofluorofucose (4, Figure 6 and Table 1), compounds that bear fewer electron-withdrawing fluorine atoms and may not be as effective in destabilizing the cationic FUT8 transition state. Functional enzymatic inhibition of fucosyl transferases by the fucostatins or their metabolites was not directly tested in this study, but seems unlikely, or at least less significant, since fucosylation was fully rescued in treated cells with the addition of fucose. Phosphonate sugars 5, 6, 7, and 8 (Figure 8) were designed to eliminate glycan incorporation by substituting a methylene group for the exoanomeric oxygen. This was expected to prevent the corresponding GDP sugars from being substrates in glycosylation reactions. The phosphonates were prepared as acetate/pivaloyloxymethyl (POM) esters that diffuse into the cells as prodrugs (24) and be cleaved by intracellular esterases. When prepared and tested, β-fucosephosphonate 5 (fucostatin II) u deprotection in cells, form the GDP derivative, 5B and, similar to 2, accumulate GDP-mannose and bind GMD (KD 9 µM, SPR, Figure 7). Unlike 1, 5 was only cell penetrant in its prodrug form. In protein production experiments, due to hydrolytic instability of the prodrug 5 in the production growth medium, 5 was added as a DMSO stock to fresh medium every day starting on day 3. Potent inhibition of mAb fucosylation occurred with an EC50 of ~30 µM. By this protocol (35) mAbs were produced with varying amounts of fucosylation and showed no indication of incorporated inhibitor (Figure 10a). Despite showing no influence on cell viability in a propidium iodide assay up to 100 µM, in the production assay, inhibition of cumulative cell density, titer and specific production was apparent at >40 µM due to loss of cell viability (Figure 10b). The non-native α-anomeric stereoisomer 6 was inactive in the cell surface assay and was not further tested.





Figure 9. (a) 2 was added on day 0 of production at the levels indicated on the x-axis to an anti-TR-2 mAb -expressing CHO cell-line grown under fed-batch production conditions. Glycan analysis was performed on the produced antibodies (circle = percent fucosylated glycans, square = percent afucosylated glycans, triangle = percent high mannose glycans; open circle = percent glycans with incorporated 6,6,6-trifluorofucose). (b) Cumulative viable cell density, titer and specific productivity obtained under different concentrations of 2 in three different cell lines (each data point represents an average of duplicates). (c) Anti-MSLN mAb antibodies produced in the presence of varying amounts of 2 and with varying levels of fucosylated glycans (filled circles) were purified and assayed in a calcein-based ADCC assay with cells containing high (N87; black bars) and lower (CAPAN2; gray bars) target expression. EC50 values represent the amounts of anti-MSLN mAb needed to lyse 50% of target cells at the indicated anti-MSLN mAb fucosylation levels. (d) CHO cells expressing anti-TR-2 mAb were grown under fed-batch production conditions. Compound 2 was added to the cells on day 0 at 20 µM concentration. Production cultures were also dosed with 1 mM fucose on either day 0 or day 6. The glycan profile of the produced anti-TR-2 mAb antibodies was measured (black bars = percent fucosylated glycans, gray bars = percent afucosylated glycans, white bars = percent high mannose). Each bar represents and average of duplicates. (e) HILIC Asn297 glycan analysis of Ab produced in the presence of 250 µM 2 showing 98% afucosylated glycans and 0.7% incorporation. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Although 5 effectively blocked fucosylation with the advantage of undetectable incorporation, it was about ten-fold less potent than 2 in the cell surface assay (FITC-LCA IC50 18.1±6.5 µM). To improve potency we attempted to take advantage of additional vdw contacts about a 5-CF3 group as for 1/1D. Compound 7, the trifluoromethyl analog of 5, was inactive, even though it did give rise to the required GDP metabolite 7B. This suggests that 7B is not a ligand of GMD, the combination of both exo-anomeric methylene and 5-CF3 perhaps being too dissimilar from native GDP-fucose to bind. Further mechanistic studies could not be completed due to the chemical instability of 7B.



Figure 10. (a) The impact of repeated additions of 5 on cell culture performance using medium exchanges initiated on day 3. Compound 5 was added to the cultures daily at the levels indicated on the x-axis. (b) Cumulative viable cell density, titer and specific productivity obtained in the presence of different concentrations of 5 (each data point represents an average of duplicates). Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Considering that compound 7 was inactive, it was surprising to observe potent inhibition of cell surface and protein fucosylation for the α-isomer of 7, compound 8 (FITC-LCA IC50 7.0±7.0 µM). An analysis of the metabolites from cells treated with 8 found deprotected 8A, but less of the 8B GDP derivative was detected. In addition to the phosphonate metabolite 8A, phosphate metabolite 1C was also observed. Consistent with the formation of the same metabolite, low levels of incorporation of 1 were observed in expressed proteins. These results suggested the phosphonate group of 8 may be hydrolyzed in the cell to give 1 in situ (27–29). Rigorous purification and characterization of 8 eliminated the consideration of contamination of the testing compound. Since similar metabolites were not found in cells treated with 7, it is proposed that hydrolysis of the phosphonate α-isomer may be accelerated compared with hydrolysis of the β-isomer 7 due to the anomeric effect. Similar in situ hydrolysis of the α-isomer 6 would not have been detected in these studies. 51



Table 1. Phosphate and Phosphonate Metabolite Data

Data for fucostatins and other analogs are summarized in Table 1. As described above, 6,6,6-trifluorofucose peracetate (2) was a potent inhibitor of protein fucosylation as measured by FITC-LCA staining of cell surface glycoproteins. Compound 2 also inhibited the fucosylation of the glycans of expressed proteins as determined by hydrophilic interaction chromatography (HILIC) analysis. Metabolite identification from the lysate of treated cells displayed that 2 was processed through the fucose salvage pathway as evidenced by formation of 1C and 1D as metabolites. The accumulation of intracellular GDP-mannose and loss of GDP-fucose, as well as the rescue of fucosylation by added fucose, suggests the inhibition of GMD by 1D. As corroborating evidence, 1D was found to bind to GMD (KD 11 µM, SPR) and was cocrystallized in 52 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.


the GMD allosteric site (19). It’s possible fucosyl transferases are also weakly inhibited by 1D as has been reported for other fucosylation inhibitors (7, 8), but inhibition of purified FUT8 was not determined for the fucostatins or their metabolites. The improved potency of the fucostatins compared to the previously reported fucosylation inhibitor, 2-deoxy-2-fluorofucose (2FF) is also shown in Table 1. GDP-2FF has been reported to have ~50 µM inhibition of GMD and ~200 µM inhibition of FUT8 (8). In the FITC-LCA assay 2FF had >100 µM activity in inhibiting cell surface fucosylation. A second reported fucosylation inhibitor, peracetylated 5-thiofucose (7) (5T-Fuc) inhibits sialyl-Lewisx on HepG2 cells with EC50 = 21 µM. Fucostatin potency of 0.87-18.1 µM compares favorably to previously reported inhibitors.

Development of a Process To Manufacture 6,6,6-Trifluorofucose. Published Route and a Novel Strategy. Among the several analogs characterized as potent and selective inhibitors of protein fucosylation, 6,6,6-trifluorofucose (1) was particularly attractive due to its chemical stability under cell culture conditions and high water solubility (>50 mg/mL). In contrast, DMSO was used to solubilize 2 but addition of DMSO can impact cell culture parameters (36). Besides improved solubility, compound 1 was the most potent of the inhibitors evaluated (FITC-LCA IC50 0.87 μM) and could be administrated via single dosing at the start of the cell culture cycle. In consideration of the factors above, 6,6,6-trifluorofucose (1) was pursued as a reagent for large-scale monoclonal antibody production. Wide ranging application of the fucostatin I inhibitor 6,6,6-trifluorofucose (1) for manufacture of protein therapeutics demanded a robust process to deliver several hundred grams of the inhibitor. The synthetic route to prepare 1 reported by Tokokuni and co-workers (17) was not suitable in this regard. The Tokokuni route involved: • • • • •

A nine step synthesis from expensive D-lyxose (37) (Scheme 1). The use of stoichiometric quantities of toxic mercury, lead, and chromium reagents. Non-crystalline synthetic intermediates. Multiple challenging silica gel chromatographies. Use of nine protecting groups.

Moreover, the Tokokuni approach is not stereoselective and yields a 50/50 mixture of epimers at C5. In planning the development of a novel approach to 1, multiple objectives were outlined. It was conceived that the new route would sustain the anomeric carbon of the starting material in 1 and would involve crystalline synthetic intermediates for purification. Furthermore, chromatographic separations and the extensive use of protecting groups would be avoided. Finally, the approach would be diastereoselective at C5 (38). 53 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. Tokokuni Route to 6,6,6-Trifluorofucose (1). Reproduced with permission from ref. (38). Copyright 2016, American Chemical Society.

Initial Factors D-(−)-Arabinose provided the advantages of low cost (39) and convenient availability on kilogram-scale and was selected as a starting material for an improved process to manufacture 1. The anomeric carbon of D-(−)-arabinose would be preserved in 1, thus providing an opportunity to carry out the sequence using rigid five-membered ring furanosides and significantly improving the odds of uncovering crystalline intermediates that might be used for purification. The enantioselective hydrogenation of trifluoromethyl ketones is precedented using hydrogen gas (40) or via transfer hydrogenation (41), and we planned to carry out such a diastereselective hydrogenation of ketone 9 (Scheme 2) using substrate control or by employing a chiral catalyst. Ketone 9 would be prepared by addition (42) of a trifluoromethyl group starting with known carboxylic acid 54 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.


10 (43). A procedure to prepare 10 from D-(−)-arabinose on small scale has been reported (43), however this process would need to be developed to enable manufacture of 10.

Scheme 2. Retrosynthetic Analysis of a Novel Approach to 1. Reproduced with permission from ref. (38). Copyright 2016, American Chemical Society.

Process To Prepare Furanoside Ester 12a The formation of methyl furanoside intermediates 11a/11b is the first step of our process (Scheme 3). This reaction must be quenched after ~4 h by the addition of ammonium bicarbonate at 20 °C due to isomerization of the product over longer reaction times. The methyl furanosides 11a/11b appear to be the kinetic product of the reaction while the thermodynamic pyranoside products 11c/11d are formed more slowly. Once formed, one of the pyranoside anomers (11d) crystallized out of the reaction medium. Thirty hours were required to convert more than 50% of the material to pyranosides 11c and 11d. This period of time was long enough to allow for robust control upon preparation of kilogram batches of 11a/11b which contained less than 10% of the undesired 11c/11d. Upon quenching the reaction mixture with ammonium bicarbonate and filtration of the ammonium chloride by-product, the bulk of the methanol was distilled and the remainder was dissolved in water in preparation for the next chemical transformation. The methyl furanosides 11a/11b underwent a selective Heyns oxidation (44) of the primary alcohol group in the presence of the secondary alcohols (Scheme 3). The oxidation was performed using platinum black and oxygen at elevated temperature in water. The rate of reaction was optimal between pH 8 and 9. 55



Addition of sodium bicarbonate (NaHCO3) base in portions throughout the process was effective to maintain the ideal pH. Despite a high metal catalyst loading (20%) and moderate yield of 10a/10b (60-63%), the downstream treatment consisted of filtration of the catalyst and use of the aqueous product solution directly in the next step, thus ensuring process practicality. Platinum black was recycled via reduction with hydrogen.

Scheme 3. Preparation of Benzyl Ester 12a Benzyl ester 12a was identified as an intermediate that had the capacity to be crystallized as a single diastereomer (>98/2 dr) and provided a valuable opportunity for purification. Crude acids 10a/10b were esterified using benzyl bromide in DMF with tetrabutylammonium bromide as a phase transfer catalyst. 56 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.

Crude compounds 12a/12b were crystallized from t-butylmethylether (MTBE), resulting in almost complete rejection of furanoside diastereomer 12b in the mother liquors. Unfortunately, anomer 12b is a productive intermediate to prepare 1 and thus the overall yield of the synthetic sequence is reduced by the selective isolation of anomer 12a. However, the study of the subsequent diastereoselective ketone hydrogenation was simplified by the use of a single furanoside species. Overall, starting from inexpensive D-(–)-arabinose material, 12a was isolated in 16% yield (45).


Process To Manufacture Trimethylsilyl Ketal 14 Trifluoromethyl ketones commonly form hydrates and thus are unreliable synthetic intermediates. Consequently, a strategy was devised involving the conversion of ester 12a into trimethylsilyl ketal 14 (46, 47). In situ hydrolysis of ketal 14 would subsequently provide the ketone substrate for the planned diastereoselective hydrogenation. The two hydroxyl groups of 12a were expected to interfere with the formation of the trimethylsilyl ketal from ester 12a, which prompted their transient protection. It was decided to protect these hydroxyl groups as trimethylsilyl ethers. Removal of the trimethylsilyl ether groups was expected during the acidic work-up and isolation of the transfer hydrogenation product.

Scheme 4. Preparation of Trifluoromethyl Furanoside 14 The 12a diol was silylated using TMSCl and imidazole in DMF (Scheme 4) to provide crude ester 13, that was used in the next step without further isolation. Trifluoromethyl ketal 14 was obtained by treatment of 13 with TMSCF3 in the presence of catalytic tetrabutylammonium fluoride (85-90% yield from 12a). Crude 14 was not submitted to an aqueous work-up due to its poor stability in the presence of water. Instead, excess fluorinating agent was quenched by 57 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.

addition of silica gel to the reaction mixture. The silica gel was filtered off, and the product solution containing stable intermediate 14 was used in the subsequent step without further purification.


Tandem Ketal Hydrolysis-Transfer Hydrogenation Process The in situ formation of the trifluoromethylketone hydrogenation substrate 15 from the trimethylsilyl ketal starting material 14 was carried out using KOH. This base also enabled the formation of the freebase (active form) of the transfer hydrogenation catalyst from the commercially available HCl salt. Both of these processes proceeded well in 2-propanol, a fitting solvent to conduct the transfer hydrogenation reaction. Upon treatment of 14 with KOH in 2-propanol, a four step desilylation sequence takes place resulting in the unstable hydrogenation substrate 17. In the absence of the catalyst, under the reaction conditions, 17 was found to decompose. Consequently, the timing of addition of the base and catalyst is important for designing a robust process to prepare the target secondary alcohol product 18. One possible sequence for the desilylation cascade is depicted in Scheme 5.

Scheme 5. A Proposed Base Catalyzed Desilylation Cascade. Reproduced with permission from ref. (38). Copyright 2016, American Chemical Society. In this proposal the desilylation starts with the hydrolysis of trimethylsilyl ketal 14 to form ketone 15. Ketone 15 is in equilibrium with the corresponding hydrate 16a. Spatial proximity of the ketone hydrate group and the C2 silyl ether enables a base catalyzed trimethylsilyl group transfer from the silyl ether function at C2 providing trimethylsilyl hemi-ketal 16b. This hemi-ketal then hydrolyzes 58 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.

under the reaction conditions to afford ketone 17. Consistent with this mechanistic proposal, alcohol 18 is the product of the hydrogenation process (48) prior to HCl quench of the reaction mixture. In the absence of transfer hydrogenation catalyst, the decomposition of 17 leads to the formation of multiple products.


Table 2. Optimization of the Diastereoselective Hydrogenation of Ketal 14

The transfer hydrogenation conditions reported by Noyori and co-workers in their pioneering report served as a starting point for the optimization of the synthesis of 19 from 14 (Table 2) (49). Using 2% RuCl[(R,R)-TsDPEN](mes) and 0.3 equivalents of KOH at 20 ºC in IPA (entry 1) gave 90% assay yield of 59 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.


desired alcohol 19 in 2 h after acidic quench. Interestingly, no attrition of the diastereomeric ratio (dr) of alcohols (19/20) occurred upon longer reaction times (14 h). Triethylamine-formic acid has been shown to give a kinetically controlled (50) mixture of products irrespective of the starting ketone’s structure. Although this reductant produced alcohols 19 and 20 in slightly higher dr (entry 2) compared to isopropyl alcohol, the use of this solvent conferred practicality to the process and these conditions were selected for further development. A slower reaction rate was observed using 24 h was necessary to reach the pre-filtration crystallization end-point. This was presumably caused by a correspondingly low equilibration rate of the four isomeric species of 1 in the absence of water. This caused the concentration of the single crystallizing species in solution to be low at any point 61



during the process. To allow a productive crystallization rate, one equivalent of water was added in order. Filtration of the crystallized product, cake wash with n-heptane, and drying of the solid provided 1 as a crystalline material in 74% corrected yield from ketal 14. The C5 epimeric diasteromer of 1 (see Scheme 1) was present in 7% in the crude hydrolysis reaction mixture but was largely reduced to 9/1 dr from a trimethylsilylketal intermediate is the key transformation of the sequence. A ruthenium catalyzed tandem ketal hydrolysis-transfer hydrogenation step effected this transformation in excellent yield and diastereoselectivity. The potent and selective fucosylation inhibitor 6,6,6-trifluorofucose (1) and the process to access quantities of this reagent enables the preparation of mAbs displaying improved ADCC and improved in vivo efficacy.

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