Process Development and GMP Production of a Potent NEDD8

Chapter 2

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Process Development and GMP Production of a Potent NEDD8-Activating Enzyme (NAE) Inhibitor: Pevonedistat Ian Armitage, Ashley McCarron, and Lei Zhu* Chemical Development Laboratories, Millennium Pharmaceuticals, Inc., a subsidiary of Takeda Pharmaceutical Company Limited, 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States *E-mail: [email protected].

Development efforts for a manufacturing process of a novel NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924) are described. Highlights include an enantioselective synthesis of an aminodiol cyclopentane intermediate containing three chiral centers and a novel, regioselective sulfamoylation using N-(tert-butoxycarbonyl)N-[(triethylenediammonium)sulfonyl]azanide. The linear process, involving six isolations, has been carried out in multiple cGMP productions on 15 to 30 kg scale to produce pevonedistat in 98% purity and 25% overall yield.

Section 1: Process Development for GLP and First GMP Production of Pevonedistat Introduction (((1S,2S,4R)-4-{4-[(S)-2,3-dihydro-1H-inden-1-ylamino]-7H-pyrrolo[2,3d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate hydrochloride) (pevonedistat, Figure 1), also known as MLN4924 and TAK-924, a novel NEDD8-activating enzyme (NAE) inhibitor, has demonstrated in vitro cytotoxic activity against a variety of human malignancies. It is currently being developed © 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.


by Takeda Pharmaceuticals Company Limited in phase I/II clinical trials for the treatment of hematological and solid tumor cancers (1–17). The five-membered ring, carrying three chiral centers and an acid/base sensitive terminal sulfamoyl group, presented considerable challenges for the development of a practical and scalable synthesis of pevonedistat. This report details our successful efforts in developing a laboratory-scale synthesis to a multikilogram, reproducible preparation.

Figure 1. Chemical Structure of Pevonedistat.

General Strategy The original medicinal chemistry synthetic route to produce pevonedistat involved 13 linear steps and multiple chromatographic purifications (Scheme 1). More than half of the chemical transformations in the synthesis were carried out to construct the chiral five-membered ring. The poor overall efficiency of this route resulted from: • • •

a low yielding resolution (step 4). removal of an extra chiral hydroxyl group (step 10). installation of an acetal protecting group (step 7).

Furthermore, the late stage terminal sulfamoylation on the primary hydroxyl group was poorly selective and low yielding. Although the original route was reproducible on gram scale and able to supply material needs for early drug metabolism and pharmacokinetics (DMPK), toxicological, and pharmacology studies, the process chemistry group assessed that it would not be viable for producing kilogram quantities to support further preclinical and clinical studies. It would have been a considerable challenge to complete the first GMP API campaign without incorporating significant process improvements. However, due to the time constrains from candidate selection to IND, we set our short-term goal to develop a synthetic route capable of delivering up to 200 g of API to supply IND enabling toxicology studies. Subsequently, we needed to prepare 1–2 kg of clinical material for clinical studies. This section describes our activities towards achieving these goals. 14

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. Original Medicinal Chemistry Synthesis of Pevonedistat

Research Towards a Scalable Route Several alternative syntheses were considered on a theoretical basis in the development of a scalable route. The selected bond disconnection strategy that enabled the cGMP production of multiple hundred gram and ultimately multi kilogram quantities of pevonedistat is displayed in Scheme 2.

15 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 2. Retrosynthetic Analysis of Pevonedistat

The basis of this strategy was supported by literature precedent of the synthesis of the acetal derivative of aldehyde 5 (18–20). Coupling of substrates similar to 4 and 5 was also demonstrated in the same reference, although it was not clear whether aldehyde and acetal would work equally in our case. Coupling of 6 with 3 and subsequent sulfamoylation were carried out previously by medicinal chemistry. The key development focuses envisioned were: • • •

establishment of a scalable route towards enantiomerically pure cyclopentane aminodiol intermediate 4. successful installation of the labile sulfamate group. optimization of the chemically inefficient process.

Research Strategies To Enable a Scalable Route To Produce up to 200 g for IND Enabling Toxicology Studies Focusing on the desired enantiomerically pure building block aminodiol 4, two routes were examined thoroughly based on retrosynthetic analysis (Scheme 3). The first one centered on a Sharpless epoxidation of an allyl alcohol intermediate and the other on a chiral lactone intermediate which would be then reduced to result in the diol.

Scheme 3. Retrosynthetic Analysis of Aminodiol 4 16 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.

Sharpless Epoxidation Route to Intermediate 4


The preparation of the allylic alcohol 11 necessary to test the Sharpless epoxidation route was inspired by the work from Bray et al. (Scheme 4) (21). A bulkier protecting group, trityl, was installed to aid steric control of the epoxidation.

Scheme 4. Sharpless Epoxidation Process to Intermediate 4 Thionyl chloride (2.05 equivalents) in methanol resulted in ring opening of commercially available lactam 7. Isolation of the desired product 8 was initially achieved by complete removal of solvent, but this method afforded a gum or a sticky solid and was not scalable. Subsequently, it was found that distillation of methanol to approximately half of the original volume followed by the addition of an antisolvent induced successful precipitation of aminoester 8. Toluene was initially used as the antisolvent, resulting in > 85% yields, but on a multi hundred gram scale, the use of toluene resulted in the isolation of unacceptably sticky solid. Thus, upon further investigation, we identified methyl t-butyl ether (MTBE) to be a more effective antisolvent. The product precipitated as a free-flowing white solid that was isolated easily by filtration on up to 500 g scale in quantitative yield. Trityl protection of 8 provided Tr-protected aminoester 9, which was isolated via solvent removal and afforded an oil that could be taken directly into the double bond migration step as crude material. On gram scale, once double bond migration 17



was complete by HPLC, the solvent was removed. The crude material was purified via a silica gel plug filtration followed by solvent removal to provide the α,βunsaturated ester 10. Subsequent process development led to an aqueous work-up whereby DBU was simply removed by washing the crude reaction mixture with water and telescoping the DCM extract into the next step of the synthesis. For the ester reduction step (Scheme 4, 10 to allylic alcohol 11), the amount of DIBAL-H (1.0 M solution in toluene) was optimized to 2.2 equivalents for a cleaner reaction profile. Isolation of this intermediate was accomplished by solvent removal resulting in a viscous oil. We later switched the reaction solvent from DCM to toluene to avoid this clumsy oil isolation and to allow telescoping into the key transformation of this strategy: the Sharpless epoxidation. The oxidation of allylic alcohol 11 to epoxide 12 was examined under the following conditions: 1) the standard Sharpless epoxidation conditions using (+)-diethyl L-tartrate. 2) the standard Sharpless epoxidation conditions using (–)-diethyl D-tartrate (22–25). 3) with m-chloroperbenzoic acid in the presence of triethylamine with DCM used as solvent. 4) with methyl trioxorhenium and t-butyl hydroperoxide. 5) with vanadyl acetylacetonate. The NMR studies indicated that the Sharpless conditions using (+)-diethyl Ltartrate (condition 1) produced the desired epoxide 12 whereas the corresponding diastereomer 13 (Figure 2) was the major product obtained from conditions 2–4. The epoxidation reaction in the presence of vanadyl acetylacetonate (condition 5) resulted in a mixture of 12 and 13.

Figure 2. Isomers from Epoxidation.

While the stereoselective Sharpless chemistry (condition 1) was effective, it involved extensive work-up, and the product required column purification. With the desired diastereomer epoxide 12 in hand, we were in a position to test the key transformation of the route: the regioselective ring opening of 12 to form 1,3-diol 14. We hypothesized that the use of Red-Al would provide the desired regioselectivity (26–28), but unfortunately little reaction occurred under these conditions. Thus, screening was initiated to find an alternative reducing agent (Table 1). 18 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 1. Ring-Opening Conditions for Epoxide 12 to Diol 14

a

Conditionsa

Solvent

Results

Red-Al, 20 °C, 2.5 equivalents

THF

No reaction

Red-Al, 1.0 equivalent

DME

Loss of the protecting group

BF3•Et2O, NaBH3•CN, 1.0 equivalent

THF

Decomposition

B2H6, 1.0 equivalent

HMPA

Multiple nonpolar products

NaBH4, 1.0 equivalent

THF

No reaction

Super-Hydride, 1.0 equivalent

THF

Formation of a less-polar product

LAH, 1.0 equivalent

THF

Formation of 15

BH3•THF, NaBH4, 1.0 equivalent

THF

1:2 ratio of 14/15

LiBH4, 2.0 equivalents

THF

Formation of 15

Na(OAc)3BH, 2.0 equivalents, 16 h

THF

Unknown compound

BH3•THF, 2.0 equivalents, 16 h

THF

1:1.3 ratio of 14/15


THF

1:1.4 ratio of 14/15


DCM

1:1.5 ratio of 14/15

BH3•DMS, 2.0 equivalents, 16 h

THF

1:1.5 ratio of 14/15

Reactions were carried out at room temperature overnight if not specified.

The use of BH3•THF appeared to be the most useful for maximizing the conversion to diol 14. After further investigation, it was determined that the simultaneous presence of two reductants (BH3•THF and NaBH4), was efficacious. The reaction was performed in the presence of sodium borohydride (1.0 equivalent) and BH3•THF (2.0 equivalents) in DCM at 35 °C, which provided the best balance of conversion to the required 1,3-diol regioisomer and isolated yield on small scale. After aqueous work-up, separation of regioisomers was achieved on small scale via preparative column chromatography; however, no crystallization conditions could be developed to separate the two isomers. Therefore, a different approach was needed to separate the isomers. Another method explored to affect this separation was to utilize protecting groups and exploit the potential difference in reactivity between isomeric diols 14 and 15. Two options were investigated: •

•

attempted acetonide formation with 10-camphorsulfonic acid/2,2dimethoxypropane did not reach completion and resulted in loss of the trityl protecting group (Scheme 5). selective reaction of the 1,3-diol 14 in the product mixture with 1,3-dichloro-1,1,3,3-tetra-isopropyldisiloxane, followed by a chromatographic separation of derivative 16 from the unreacted diol 15. The deprotection of compound 16 regenerated the desired 1,3-diol, 14. Removal of Tr group afforded the desired aminodiol 4 (Scheme 5). 19



Scheme 5. Purification and Separation of 14

On treating the diol mixture (1:2 ratio of 14/15) with 1,3-dichloro-1,1,3,3tetraisopropyldisiloxane in the presence of triethylamine (Et3N) and DCM, there was complete consumption of 14 to protected product while the isomeric 15 remained largely unreacted. The desired acyclic product 16 was purified by column chromatography but still retained 2–5% of the silylated undesired isomer 17. Reaction optimization focused on temperature, reaction time, and equivalents of silylating agent to further minimize the reaction of compound 15 and to reduce formation of cyclic compounds 18 and 19, which formed over extended reaction times. Reducing the charge of silylating agent in accordance with the mole ratio of desired diol in the mixture, halting the reaction once complete consumption of 14 was achieved, and performing the reaction at ambient temperature proved successful in reducing the amount of undesired compounds 17, 18, and 19. Ultimately, the product was purified by column chromatography. Simple deprotection conditions were developed using TBAF to afford the desired amine-protected diol, 14, in excellent yield (~90% based on moles available in starting mixture). Deprotection was also readily achieved in similar yields and purity using a solution of hydrofluoric acid in triethylamine but ultimately this method was not selected for scale-up due to concerns over the use of HF. Final trityl deprotection of 14 was effected by catalytic hydrogenation in methanol. Complete conversion was achieved, but column chromatography was required to obtain aminodiol 4 in desired quality. No further optimization was carried out for this step. 20


The Sharpless epoxidation route was successful in providing 550 g of material in a multibatch strategy to support development and scale-up work for later steps. However, due to several intermediates being isolated as oils and frequent column chromatography being required, the route was deemed unsuitable for the kilogram scale production of material. Instead, the significantly shorter bromolactonization route (discussed in the following section) was considered and proved to be ultimately the answer.


Bromolactonization Process to Aminodiol 4 The starting material, trans amino acid 20 for this route is a known compound, but not widely available commercially (Scheme 6) (29). We initiated our own development work to produce this desired starting material based on ring opening of Vince lactam and epimerization (30), while sourcing strategies for 20 occurred in parallel. While internal chemistry efforts were showing progress, a commercial supplier capable of producing 20 on multikilogram scale and delivering required amounts was identified to meet project timelines.

Scheme 6. Bromolactonization Process to Intermediate 4

Successful lactonization directed by the existing stereochemistry in Boc-amino acid 20 was considered the key transformation in this route (31–34). Similar to the Sharpless route, internal work began with the trityl protecting group. However, since the reaction outcomes between 20 and its trityl protected analog were identical, the Boc-protected variant was selected on the basis of better atom efficiency and lower cost. Only research on the Boc-protected synthesis is described below.

Scheme 7. Bromolactonization of Compound 20 21 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.


Bromolactonization to produce β-lactone 21 (Scheme 7) was first attempted on a small scale, utilizing bromine and tetrabutylammonium hydroxide in DCM under cryogenic conditions and varying the equivalents of bromine. This method proved successful on a laboratory scale with 2.0 equivalents of bromine showing optimal performance at 65–70% yield. The solid product was isolated by removal of solvent and this method was deemed suitable for the production of material necessary for toxicological studies. A further-developed, process-friendly isolation would be addressed at a later time. Screening efforts were undertaken to investigate alternatives to bromine for lactonization, e.g. iodine, NBS, silver triflate, and copper iodide. However, only bromine led to the desired product.

Scheme 8. Reduction of β-Lactone 21 We investigated the use of both NaBH4 and LiBH4 for the reduction of βlactone 21 (Scheme 8). LiBH4 in solution proved superior with respect to reaction rate and purity of product for the formation of Boc-bromo diol 22 as well as ease of use and the ability to add in a controlled manner. Reaction at 0–5 °C was necessary to control purity. The reaction was rapid and complete immediately after the addition of LiBH4 was finished. A simple NH4Cl aqueous quench followed by extractive work-up with MTBE and removal of solvent, yielded product in almost quantitative yield as a thick oil or glassy solid.

Scheme 9. Reductive Debromination of β-Lactone 21 Several attempts at this stage were also made for simultaneous reductive dehalogenation (Scheme 9) by modification of the reaction conditions, either through the addition of Lewis acids to the LiBH4, alternative reducing agents, or via elevating the temperature. If successful, this approach would shorten the 22 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.


synthesis to aminodiol 4 by one step. Unfortunately, most conditions yielded Boc-bromo diol 22, or instigated degradation of 22, with no desired des-bromo product seen. Nevertheless, the LiBH4 conditions were deemed suitable for the scale required of the chemistry to form the bromo-diol 23 and we considered telescoping this step into the following step for the first GMP production. A set of standard conditions for removal of the Boc group utilizing HCl in dioxane and IPA was employed to obtain bromo-diol 23, an HCl salt, and removal of Boc was typically complete within 3–5 h (Scheme 9). Isolation of intermediate 23 was not attempted at that time, and the resultant mixture was concentrated and hydrogenated under typical conditions with Pd/C as the catalyst (35). The debromination of 23 was clean, and the desired aminodiol 4 was formed exclusively. We initially pursued a combination of methanol and IPA as solvent in conjunction with sodium bicarbonate to quench any residual HCl from the deprotection reaction. Methanol provided high solubility for both the starting material as well as the product. Simple filtration to remove catalyst and inorganic salts followed by removal of solvent yielded the desired product, the HBr salt of 4 (36).

Coupling Steps Preparation of Dichloropyrimidine Acetal

Scheme 10. Preparation of Dichloropyrimidine Acetal

The literature synthesis of 27 is provided in Scheme 10. From a process chemistry perspective, the areas of concern were: • • •

the use of sodium methoxide solid or solution in step 1. the use of neat POCl3 in step 2. the availability of ozonolysis capabilities for step 3. 23



Synthetic procedures available from the literature (18–20) were utilized on small scale to produce both sufficient quantities of 25, ultimately from guanidine, and diester 24 for gram scale development work. For the production of compound 25, the use of either sodium methoxide solution or solids resulted in a similar reaction profile. Eventually, solid NaOMe was used in production. For step 2, the literature procedure called for reaction in neat POCl3 with diethylaniline. The transformation of 25 to dichloride 26 could be achieved using toluene/acetonitrile as a solvent system and DIPEA as the base. Yields after work-up using these cosolvents were generally ~15% lower than when neat POCl3 was utilized. The yield became much lower as the chemistry was scaled to > 100 g. Reaction in neat POCl3 was reassessed and successful conditions were developed using five volumes of neat POCl3. The work-up for this step was modified by introducing toluene at the end of reaction in order to facilitate azeotropic removal of residual POCl3. This azeotropic removal of POCl3 was followed by an extractive aqueous work-up. The product was isolated as an oil by concentration. Ozonolysis utilizing the literature conditions proved effective for the conversion of alkene 26 to aldehyde 5 (18). The work-up was modified from the ether extractions described in the literature to isolation by trituration with EtOAc/heptanes in order to obtain better product quality. Once product was formed, ethyl acetate was added to achieve phase separation, a series of aqueous washes was performed, and then the organic phase was concentrated to a low volume. Crystalline product was obtained by the addition of heptanes as antisolvent. The conversion of 26 to aldehyde 5 was also investigated using catalytic osmium tetroxide with NaIO4 or NMO. Although use of osmium tetroxide was successful in obtaining product in similar yield and quality to the ozonolysis process, the toxicity of osmium and potential for residual osmium in intermediates was a concern for large scale reactions. Ultimately, we identified vendors with the required large scale capabilities for ozonolysis to produce aldehyde 5. The osmium method henceforth was no longer considered. No development work was performed on the acetal formation step to produce 27. The chemistry described in Scheme 10 using solid sodium methoxide in step 1, five volumes of neat POCl3 in step 2, ozonolysis for step 3, and literature conditions for step 4 was successfully implemented at two different Contract Manufacturing Organizations (CMOs) to produce 1 kg of 5, 200 g of 27 for non-GMP work, and 6 kg of 27 for the first GMP campaign. Later, commercial suppliers were identified for 5 during the first GMP production campaign and no further development work to prepare 5 was performed by Takeda.

Preparation of Diol Intermediate 2 The conditions for the coupling of aminodiol 4 and acetal 27 are based on literature (18–20). The reference highlights the importance of using an acetal derivative to avoid unwanted imine formation. For this reason, acetal 27 was used in this reaction. A screen of alternative solvents and bases was performed. 24 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.


Triethylamine in IPA showed the cleanest reaction profile. After extractive workup with EtOAc, chloropyrimidine 3 was obtained by removal of solvent under reduced pressure. Crystallization conditions were not fully developed prior to production of material for toxicological studies, due to time constraints.

Scheme 11. Chemistry To Prepare Diol Intermediate 2

Coupling of the aminoindane 6 to chloropyrimidine 3 had been previously performed on other scaffolds by medicinal chemists utilizing microwave chemistry on small scale. However, during early process research on the coupling step, it proved challenging to drive the transformation fully to diol 2. The reaction was originally carried out in high boiling solvents such as DMAc and NMP in the presence of inorganic bases. The desired organic bases were prone to vaporization due to operating temperatures above the boiling point of the base. However, in order to drive this transformation to completion in a reasonable timeframe, a temperature of greater than 125 °C was necessary. After multiple failed attempts to perform the reaction at atmospheric pressure, a screen in a pressurized system was conducted. This revealed that 2-butanol with DIPEA provided a clean reaction profile with a completion time of ~70 h at 135 °C under 80 psig. While this appears extreme, all solvent and base combinations investigated required longer reaction time and higher temperatures in order to drive the reaction to completion. 25


With reaction completion achieved, extractive work-up was employed to remove excess aminoindane and the DIPEA•HCl salts. The organic solvent (EtOAc) was subsequently concentrated to a low volume and crystallization was initiated by the addition of DCM to yield crystalline diol 2 in high yield (> 70%) and purity (Scheme 11).


Final Steps

Scheme 12. Final Steps for the Synthesis of Pevonedistat The final steps include the sulfamoylation of diol 2 and salt formation of free base 1 to produce pevonedistat (Scheme 12). Sulfamoylation chemistry presented another significant challenge for the synthesis of pevonedistat. Both the primary and secondary hydroxyl groups of diol 2 had similar reactivities toward the two sulfamoylation reagents 29 and sulfamoyl chloride 30 (37–39) (Scheme 13) used in the original synthesis. A mixture of the desired monosulfamoylated product 1 with its regioisomer 31 and the bis-sulfamoylated byproduct 32 were produced, requiring purification of free base 1 by chromatography. Both approaches with the two different sulfamoylation reagents posed challenges to overcome with regards to efficiency (37, 40, 41) and safety (gas evolution and exothermicity) (40) in order to achieve successful scale-up.

Scheme 13. Sulfamoylation Chemistry with Reagents 29 and 30 26 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 adopted two main approaches to differentiate the two hydroxyl groups in the conversion of 2 to 1. The first is to use a protecting group strategy and the second is to modulate the reactivity of sulfamoyl chloride. The protecting group approach explored TBSCl and TBSOTf in conjunction with a variety of bases (Scheme 14). Poor mono- versus di-protection and modest regioselection necessitating chromatographic purification rendered this approach unattractive. Instead, we chose to pursue the alternative approach of modulating the reactivity of the sulfamoyl chloride.

Scheme 14. Sulfamoylation via Protecting Group

Sulfamoylation Using Sulfamoyl Chloride or Derivatives Thereof Early sulfamoylation work was based on the literature conditions for accomplishing this transformation (i.e. NH2SO2Cl/DMAc, with or without base) (40, 41). Use of sulfamoyl chloride alone with no base gave minimal to no selectivity between the desired free base 1, the regioisomer 31, and the undesired bis-sulfamated product 32. We subsequently screened the use of sulfamoyl chloride 30 in DMAc and THF (Scheme 13) as possible reaction solvents with DIPEA, Et3N, DBU, DABCO, or t-BuOLi as base. The reactions were carried out at both 0 °C and room temperature with sulfamoyl chloride and base combined together in either DCM or MeCN before addition to diol 2 in the main reaction. Many of these conditions yielded the desired free base 1, however in low purity (Table 2). Clearly a different approach was needed. To better control the formation of the desired free base 1 in the large scale sulfamoylation reaction, a protected variant of sulfamoyl chloride was investigated. This strategy was a focus for process development after the first GMP API campaign was complete. For this early investigation, Boc was selected as the protecting group of choice due to its simplicity (42) and the bases Et3N, 2,6-lutidine, DBU, DABCO, and DIPEA were examined (Scheme 15). We hypothesized that the bulkiness of a protecting group (e.g. Boc) would sterically block access of Boc-sulfamoyl chloride 33 to the secondary alcohol, improving the relative reactivity of the primary position. Although there was some evidence that the rate of addition of Boc-sulfamoyl chloride 33 to diol 2 would help to reduce the overall amount of bis-sulfamoyl impurity formation, poor stability of 33 in solution made this option less feasible. Along this line of thought, multiple 27


charges of the sulfamoylating agent were often required to consume all the starting material, thus necessitating further exploration.


Table 2. Sulfamoylation of 2 with Different Bases at 0 °C Solvent

Base

1:31

(1+31):32

DMA

none

6.5:1

5.9:1

DMA

DIPEA

HPLC not resolved

1:2.2

THF

tBuOLi

6.0:1

5.4:1

THF

DBU

8.7:1

3.9:1

THF

DABCO

7.1:1

9.7:1

Scheme 15. Sulfamoylation Chemistry with Boc-Sulfamoyl Chloride 33

Further studies of the bases utilized in this reaction revealed that DABCO worked best to modulate the reactivity of 33. This approach involved precomplexation of 2 equivalents of Boc-sulfamoyl chloride with 2 equivalents of DABCO, followed by the addition of 1 equivalent of diol 2. Under these conditions, the reaction with the diol was relatively slow, and thus allowed facile control of the reaction endpoint. Also, the need for adding multiple charges of sulfamoylating reagent via slow addition was eliminated. Instead, a relatively fast addition was possible, resulting in reduced overall reaction time. Once sulfamoylation was complete, the Boc group could be removed in situ by quenching the reaction mixture with an equal volume of 9 M HCl. Upon complete deprotection and extractive work-up, the solution was neutralized. Efforts were made to crystallize the desired product and remove the impurities. All crystallization attempts proved unsuccessful due to the similar physical properties of the compounds. Silica gel column purification was still required, but due to the minimal separation seen in a multitude of eluents investigated, the isolated product unfortunately still contained 1–2% of 32 and/or 31. 28


This procedure proved consistent up to 300 gram scale. While the ultimate goal in preparation for GMP production by using the same process to produce pure free base 1 was achieved, the challenges still remained to develop a manufacturing process with the ability to control the amounts of undesired regioisomer 31 or bis-sulfamate 32 in API.


Salt Formation To Provide the Final API The final step to produce pevonedistat was a salt formation (Scheme 12). From salt screening and polymorph studies, the HCl salt, an anhydrous form (Form 1), was selected. Form 1 was not readily crystallized directly, but rather was produced via drying of several intermediate solvated forms. Of the isostructural solvates identified, the ethanolate (Form 3) most readily converted to Form 1 upon drying. A crystallization method was developed by dissolving the free base of pevonedistat (1) in ethanol followed by the addition of one equivalent of ethereal HCl to provide pevonedistat in high yield. Unfortunately, the undesired regioisomer 31 or bissulfamate 32 impurities were not readily rejected in the salt formation, which highlighted the need to consider these impurities in further work on pevonedistat. During salt formation, one of the main impurities in the drug substance, a chloro derivative (Figure 3), was formed during the reaction. While this impurity was rejected by crystallization, it was unfortunately regenerated upon drying of the final API. Salt formation and drying conditions needed to be carefully studied later in order to minimize the formation of this impurity.

Figure 3. Chloro Impurity. The chemistry described thus far starting from the bromolactonization transformation to the protecting group mediated sulfamoylation outlined in Schemes 6–13 was successfully employed to produce ~200 g of material to support IND enabling toxicological studies and to provide material for the development of a phase 1 formulation. However, further scale-up required modified conditions to overcome residual impurities and other scalability concerns. Process Development for the First cGMP API Production The first cGMP production targeted a delivery of ~1.5 kg and was initiated almost immediately after the internal GLP campaign leaving minimal time for additional process development (Scheme 16). However, development work focusing on peripheral aspects (work-up, isolation, etc.) was performed to enable 29



further scale-up. There was also a continued effort to improve the selectivity and efficiency of the sulfamoylation chemistry on large scale. In order to meet the delivery timeline for the first cGMP API production, the team agreed to limit the scope of its development activities.

Scheme 16. GLP/First GMP Productions of Pevonedistat In the first GMP production, the bromolactonization (production of β-lactone 21) reaction proved robust; it was decided that only isolation of the product needed to be addressed for this stage of development. After reaction to form the bromolactone, excess bromine was quenched and aqueous washes were carried out to remove tetrabutylammonium bromide. The final ethyl acetate extracts were reduced to a low volume to initiate precipitation. After screening a number of potential antisolvents, 10% MTBE in heptanes afforded the product in desirable yield and purity in the form of a free-flowing solid. For the reductive opening of the β-lactone, we needed to address improving the work-up and isolation of the Boc-bromodiol 22 as a high priority to start the GMP campaign. Despite screening a multitude of solvents for crystallization, we could not identify any solvent system that precipitated the product as a filterable 30



free-flowing solid. In a few cases, we obtained solid products, however these solids had poor isolatable properties (e.g. a gum or sticky solid). Because of these difficulties, for this first GMP campaign, isolation of product was achieved by concentration of the final organic extracts to dryness. For the deprotection and subsequent dehalogenation steps there were concerns about telescoping the reaction. It was believed that isolation of the HCl salt of bromodiol 23 might be advantageous to aid in improving the purity of this intermediate. Precipitation of product occurred in the reaction media, but in suspension and upon isolation, the solid had a sticky morphology. Switching the main reaction solvent to MTBE and adding HCl/dioxane was successful in generating a free-flowing slurry. Unfortunately, upon isolation, the solid exhibited hygroscopic properties and melted together. This issue of hygroscopic solid was addressed through isolation of the solid under a positive nitrogen pressure and readily enabled air-free manipulation for short periods of time, sufficient to isolate the salt. The HCl salt of compound 23 was then subjected to hydrogenation to effect the debromination. A 1:1 ratio of MeOH and IPA was selected to afford a balance between the solubility of starting material and product, and carry-through of inorganic salts. Removal of inorganic impurities and the catalyst was initially performed by passing the reaction mixture through a pad of filter aid. However, during scale-up studies performed in preparation for GMP manufacture, several washes of the filter aid bed were required to fully elute the product which appeared to be stuck to the filter pad. Filtration through glass fiber paper readily solved this issue and afforded complete recovery of product. After filtration, the organic filtrate was concentrated to near dryness before adding a mixture of 1:1 IPA:ethyl acetate to resuspend the solid before the final isolation by filtration. The product, a mixture of 4•HBr and 4•HCl, from these development experiments contained higher amounts of residual inorganic impurities than desired. Fortunately, additional studies performed indicated that these impurities would not affect the subsequent coupling reaction. However, further investigation into the optimal base and isolation of product from the hydrogenation step was deemed to be necessary prior to subsequent API production campaigns. A single salt of aminodiol 4 was also preferred for compound characterization and pursued later. Coupling of aminodiol 4•HBr/4•HCl and acetal 27 to produce compound 3 was carried out under the same reaction conditions as described for the 200 g GLP run. However, one disadvantage was that the product was isolated by evaporating the solvent to dryness, a long procedure, which would require modification in the future. For the coupling of the aminoindane to chlorodiol 3, the only modification made from the GLP run (Scheme 16) was to concentrate the ethyl acetate extract, after the aqueous work-up, to a thick slurry/paste. Adding DCM to the mixture further reduced the solubility of 2. Compound 2 was readily isolable as a freeflowing solid with this improved work-up and isolation method. Sulfamoylation of diol 2, particularly with respect to selectivity of the sulfamoylation reagent and the ability to eliminate 31 and bis-sulfamate 32 impurities (Scheme 15) from the desired product, remained as a top concern 31


and significant challenge for the first GMP campaign. Poor selectivity resulted in mixtures of product and undesired byproducts, and required column chromatography for the isolation of free base 1. Yields for this step were inevitably low. Moreover, the inability to eliminate these impurities from the desired product in the final salt formation limited our options to purge the impurities. Thus, we focused on investigating alternative reagents to improve selectivity and minimize impurities. Factors examined were:


• • •

Bases: DABCO, imidazole, sparteine, 2,6-lutidine, DMAP, or DBU. Solvents: DMAc, THF, 2-Me THF or EtOAc. Ratio of base to Boc-sulfamoyl chloride 33.

THF proved to be the best solvent and DABCO remained the best base to use, with sparteine giving similar results. Excess base (beyond 3 equivalents) extended the reaction time and only provided a negligible improvement to the ratio of product and undesired byproducts. We decided to utilize these conditions for the internal 200 g preparation for the GMP production. Continued efforts to crystallize or recrystallize free base 1 after the initial column chromatography proved unsuccessful for removing the isomer 31 or bis-sulfamate 32 (Scheme 15). We did change the solvent elution mixture for column chromatography from MeOH/DCM to MeOH/EtOAc, and this eluent change reduced the overall amount of solvent required. We also noted that the MeOH/EtOAc eluent made it easier to separate bis-sulfamate impurity 32 from the desired product than the regioisomeric sulfamate impurity 31. To take advantage of this physiochemical property of the bis-sulfamate, the reaction time in the GMP production was lengthened to convert the regioisomeric impurity 31 to bis-sulfamate 32 prior to work-up, in order to increase the efficiency of the column chromatography. While this change did aid in purification, it reduced the yield by about 10–15% for the step compared to the prior internal results. This was considered a fair trade-off for the present maturation of the project. An atypical result was observed when isolated parent free base 1 was taken into the salt formation step. In the demonstration batch prior to the GMP production, the final HCl salt crystallized much slower and the yield was significantly lower than that obtained from previous development lots. After carefully examining the isolated free base 1, we discovered that there was some contamination with polybutylene glycol. This contamination was attributed to polymerization of THF during the Boc deprotection with HCl. Even after work-up of post-sulfamoylation and column chromatography, high amounts (visible by NMR) of polybutylene glycol were still present. This polymer contamination was the root cause for inhibiting crystallization and led to the low yields. A solution to the problem was urgently needed to prevent delays to manufacture and release of API. A study on recrystallization of free base 1 identified DCM as a suitable solvent to remove the polymeric impurity at this stage, and allowed production to continue on schedule. The following salting step provided API in acceptable quality with no extra development work. 32


Conclusions for First GMP Preparation


The chemistry and route utilized for the internal 200 g production with the modifications as described vide supra were successfully employed to produce 1.5 kg of cGMP material to support phase I clinical trials (Scheme 16). However, almost every step required concentration of the worked-up solutions for isolation of product, and the penultimate step required chromatographic purification. It was widely recognized within our group that further work was needed to improve the chemistry from an operational efficiency standpoint for future GMP productions on larger scales.

Section 2: Process Development for the Current GMP Preparation of Pevonedistat Overall Development Strategy It was rewarding to the process chemistry team that many aspects of the chemistry developed for the GLP API production were readily transferable to the first GMP campaign. This enabled us to produce enough API to initiate clinical trials. However, it was also clear that many parts of the process needed further development and optimization for robustness and efficiency if the process was to be successful on multikilogram scale. The yields of a number of steps needed to be improved. Concentration of reaction mixture to dryness needed to be replaced by larger scale/manufacturing process friendly isolation methods (e.g. crystallization). Column chromatography needed to be avoided (Scheme 16 GLP/first GMP). Post first GMP production, we focused our development efforts on the challenging areas discussed vide supra, as well as directed at improving process efficiency and lowering the production costs. Reaction conditions, choice of reagents, and isolation procedures were carefully examined for each step. This section details our progression from a process used to produce kilogram quantities of API to a potential commercial-scale process to support multikilogram preparation of pevonedistat. Process Development for the Current GMP Synthesis of β-Lactone 21 Main Issues with the First GMP Process The original laboratory scale procedure for the preparation of β-lactone 21 was unsuitable for a large scale production due to the cryogenic requirements (i.e. –25 °C) for the bromolactonization and the subsequent necessary multibatching due to size restrictions of the cryogenic plant equipment. In the first GMP production, the bromolactonization conditions were modified for larger scale manufacture and utilized NaHCO3 as base with bromine in an aqueous system at 0–5 °C (Scheme 17). These conditions caused significant foaming in the reaction mixture on the manufacturing scale during the addition of bromine and the aqueous sodium ascorbate quench. Another drawback of these conditions was 33



impurity formation. For this reason, the development efforts for this step focused on identifying alternative conditions that could eliminate these issues and provide the product in high yield and purity.

Scheme 17. Modified GMP Conditions To Prepare 21

Initial Attempts to Address Undesired Foaming Issues We investigated several approaches to suppress or eliminate the foaming without significantly changing the reaction conditions (e.g. replacing NaHCO3). Simple techniques were attempted such as the application of vacuum, nitrogen, and the introduction of antifoaming agents, all of which were ineffective. Additional studies were then carried out on the remaining parameters to see what effect each played on the foaming and if refining these parameters would offer a solution to the troublesome issue. Base amount, bromine addition rate, reaction temperature and time, quench solution amount, and rate of addition were each assessed. Several factors were found to offer improvement toward reducing foaming: • • •

an increase in base from 2 to 4 equivalents. a slow controlled addition of bromine at 5 °C over ≥ 2.5 h. a controlled addition of the sodium ascorbate quench over a minimum of 1.5 h.

A clean reaction profile was found to be heavily dependent on the reaction temperature and time. A reaction temperature of 0 ± 5 °C was determined to be critical and an increase in temperature above this range significantly impacted the purity profile. A prolonged reaction time, e.g. > 6 h, also led to notable impurity formation. Fortunately, it was discovered that water and heptanes reslurries were effective at reducing the levels of residual sodium bromide and impurities, respectively, in the product. Although these efforts helped to minimize the foaming and related processing issues, they did not completely eliminate the problems. For this reason, further development was deemed necessary. 34 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.

Further Process Development for the Conversion of Acid 20 to β-Lactone 21


Screening Organic Bases Attempts at redesigning the process sought to replace NaHCO3 with an organic base to eliminate the foaming. In purely aqueous conditions, pyridine and DIPEA proved to be acceptable, and produced the bromolactone in high purity but in low yield. In comparison, the use of Et3N led to an incomplete reaction. The introduction of an organic base helped to avoid the foaming issue, but unfortunately, the organic base also promoted the formation of solid agglomerates in the reaction mixture, which were identified as residual bromine and product. This agglomeration suppressed the reactivity of the bromine, which led to low yields and incomplete reaction. However, this effect was minimal with the use of pyridine which was chosen as base for further development efforts.

Organic Cosolvent The switch to an organic base prompted us to investigate the addition of an organic cosolvent as a way of improving the homogeneity of the reaction mixture and suppressing the agglomeration, which was still a problem with pyridine. 1,2Dimethoxyethane (DME) was studied at varying ratios with results showing only a small amount was necessary to enhance solubility, suppress agglomeration, and improve the isolated yield. In early work, 30% DME in water by volume gave the best results. This ratio of DME in water provided an appropriate solubility of starting acid 20, while precipitating the product, β-lactone 21. Under these conditions, the precipitation of 21 from the reaction mixture limited its interaction with other reagents in situ and increased the product purity. This change also offered the potential of isolation via filtration without a quench. Lower amounts of DME (10–20% v/v, DME/water) promoted the precipitation of pyridinium hydrobromide, while higher concentrations (> 30% DME) enhanced the solubility of product in the reaction mixture. Both of these events contributed to a decrease in product purity, so a more thorough investigation to define the optimal amount of DME was carried out. Ultimately, the yield and purity achieved from these solvent experiments did not offer an improvement over the 30% DME/water system. There was also a difference observed in the ease and manageability of the reaction as the amount of DME was increased. Greater DME content hindered bromine solubility causing it to oil out and settle at the bottom of the vessel. This immiscibility not only made it difficult for the reaction to reach completion, but also resulted in a higher level of residual bromine and an orange hue to the final solid. As was mentioned, limiting the solvent ratio to 30% DME/water allowed for β-lactone 21 to precipitate from the reaction mixture. Further study proved this solvent ratio did in fact allow for the successful isolation of the bromolactone solid without a sodium ascorbate quench. This process proceeded well and eliminated the second foaming event observed with the quench. Additionally, the earlier implementation of the water and heptanes reslurry/wash protocol was sufficient 35


for upgrading the product purity. This permitted scale-up of the lactonization step to multikilogram scale.


Bromine Charge The addition of DME to the reaction system suppressed the observed agglomeration of bromine and product, which occurred as a result of the use of pyridine. This improvement allowed for a decrease in the bromine charge from the standard 2.0 to 1.25 equivalents. A range of 1.1–1.3 equivalents was investigated to determine the potential point of failure around this parameter, yet comparable yields and purity were achieved across this span. An increase in the amount of residual bromine in β-lactone 21 was noted when this range was exceeded, so it was deemed beneficial to minimize the bromine content to avoid potential problems associated with carry-through in the downstream chemistry.

Outcome of Process Optimization Process redevelopment led to new conditions (Scheme 18) which utilized a 30% DME in water (v/v) solvent system, pyridine (2.5 equivalents) as base, and bromine (1.25 equivalents) at 0–5 °C, with isolation at –10 °C followed by water and heptanes washes. The new reaction rate was slightly slower than with the previous process, but the trade-off was a more process-friendly reaction and workup. This process has been successfully proven eight times to date, on scales up to 75 kg. Yields resulting from this process are typically in the 60–70% range with product purity ≥ 95%.

Scheme 18. Step 1 Current GMP Process To Prepare 21

Process Development for β-Lactone 21 to Boc-bromodiol 22 Main Issues with the First GMP Process By the time that this step reached GMP manufacture, the reduction of βlactone 21 to bromodiol 22 using lithium borohydride was a fairly well-established process (Scheme 19). Efforts for improvement of this step focused on increasing 36 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.


product yield and purity, as well as improving the cumbersome isolation of a sticky, glass-like solid which was undesirable from a process handling perspective. First to be examined was the choice of reducing agent.

Scheme 19. Modified GMP Conditions for 21 to 22

Screen of Reducing Agents A screen was carried out comparing our initial reducing agent, LiBH4/THF, with various other reducing agents including NaBH4, NaBH(OAc)3, BH3·THF, L-Selectride™, and Super Hydride™, in several different solvents: MeOH, IPA, MeCN, THF, and toluene. Of the alternative systems investigated, only NaBH4 in IPA at 25 °C showed formation of the desired Boc-bromodiol 22 while all others resulted in either no reaction or a complex mixture. However, upon scaling up the reduction with NaBH4 in IPA, it resulted in about 20–30% decrease in yield and up to a 20% decrease in purity. Based on this result, we returned our focus to improving the LiBH4 reduction conditions.

Water as Cosolvent Subsequently, we investigated whether the presence of a small amount of water would enhance the reaction rate and improve the purity profile (43). Our initially developed LiBH4 process in THF at 0 °C was attempted in the presence of 0 to 30% v/v water. Experimental results indicated that the presence of 5–15% water improved this reaction. The modified conditions provided a cleaner purity profile and a faster reaction rate than the standard anhydrous conditions. Increasing the water content above 15% resulted in the formation of a significant amount of an unknown impurity. Conditions utilizing 1.0 equivalent of LiBH4 in 10% water in THF proved optimal and were successfully scaled to produce multigram quantities of 22, giving excellent yields (> 99%) and high purity (> 96%). This change to the solvent system mandated a reassessment of the reaction parameters in order to understand their edges of failure. Using the 10% water in THF solvent system, LiBH4 was varied from 0.50 to 1.5 equivalents (compared to reactant) of 2.0 M LiBH4/THF to find the optimal operating range. This work 37



demonstrated that quantities at < 0.9 equivalent LiBH4 resulted in incomplete reaction, while 0.9 to 1.50 equivalents gave normal conversion. Longer reaction times were required at the lower end of the range of equivalents, while the upper end of the range, (1.25 –1.50 equivalents), displayed a decrease in purity. The yield in the experiment utilizing 1.50 equivalents was compromised, due to the formation of the same unknown impurity observed in the initial screen when using 20–30% water/THF. This impurity was isolated and identified to be the triol structure (Figure 4) using 1H NMR, 13C NMR, COSY, and HMQC analyses.

Figure 4. Impurity in Boc-Bromodiol 22. An investigation linked the triol formation to elevated temperatures (> 10 °C) experienced during an exotherm caused by the presence of excess LiBH4 and its reaction with water. Based on this information, the optimal amount of LiBH4 was further evaluated employing 5% water in THF as a solvent system in order to reduce the exotherm that occurred when 10% water was used (Table 3). These studies revealed that 1.05 equivalents of LiBH4 in 5% water/THF led to complete conversion, while suppressing the exotherm and minimizing triol formation, even allowing that a small portion of the reagent is quenched during addition on larger scale.

Table 3. Optimization of LiBH4 Content Using 5% Water in THF as Solvent

a

LiBH4 (equiv)

Conversion (%, a/a)

Triol (%, a/a)

Comments

0.50

95

0

Reaction stalled at 78% conversion after 2 h, reached 95% conversion after stirring at ambient temperature.

0.75

100

0

Completion in 2 h at 0–5 °C.

1.00

100

0

Completion in 2 h at 0–5 °C.

1.25

100

1.9

Completion in 2 h at 0–5 °C, 23% triola observed after 16 hours at ambient temp.

HPLC area %

This process was scaled multiple times to 100 g quantities, which provided yield and purity comparable to the 10% water/THF process. Good control of the exotherm was achieved through the appropriate amount of LiBH4, water, and a slow, controlled addition rate, resulting in low levels of the triol impurity. The Bocbromodiol product 22 resulting from these conditions was then carried through 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.

the subsequent two steps to ensure this material performed appropriately in the downstream processes. Quality was deemed sufficient by NMR and yields were essentially quantitative.


Work-up Modifications With the reduction process under control, work focused on improving the work-up. The initial work-up protocol utilized EtOAc extraction. Following a concentration to dryness, 22 was isolated as a sticky foam that settled into a glassy solid. This solid was difficult to handle at any scale, so efforts were made to improve the work-up through either modification of the product’s physical form, or elimination of the isolation along with an implementation of a telescoped process. Once attempts to attain crystallization conditions to avoid the sticky solids failed, several alternative extraction solvents were investigated. We sought to replace EtOAc in attempts to identify conditions that might improve the purity profile and allow a simplified isolation. MTBE, isopropyl acetate, and n-propyl acetate were screened with MTBE providing the highest yield based on NMR assay and a purity profile most similar to EtOAc. The decision was made to replace EtOAc with MTBE based on these results. Since the subsequent Boc deprotection is carried out in IPA, a solvent swap was implemented to substitute MTBE with IPA. We took into consideration the solubility of Boc-bromodiol 22 and investigated the distillation process to determine the amount of solvent required to keep 22 in solution while achieving the most effective removal of MTBE via the solvent swap. It was determined that 22 remained in solution in as little as 2 volumes of MTBE. Analysis proved concentrating to 2 volumes MTBE, implementing two IPA additions and subsequent concentrations to 2 volumes, sufficiently removed the residual THF and MTBE to a suitable extent that they would not have any impact on the downstream process.

Outcome of Process Scale-Up Improvements Process redevelopment efforts led to the following improvements: • • • • •

utilized 5% water in THF as the solvent system. charged 2.0 M LiBH4 in THF as the reducing agent (1.05 equivalents) slowly at –7.5 ± 2.5 °C. stirred at 0 ± 5 °C for 2 h. performed the work-up with MTBE. solvent exchanged to IPA in preparation for the next step.

This process (Scheme 20) was successfully carried out nine times to date, on scales up to 56 kg. Yields resulting from this process were typically ≥ 90% with purity ≥ 85%. 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.


Scheme 20. Current GMP Process To Prepare 22

Process Development for the Current GMP Process of des-Boc Bromo-Aminodiol 23 Main Issues with the First GMP Process The main concern for this transformation on large scale was the use of HCl as the acid source to affect carbamate deprotection, which could potentially produce mixed salt forms of bromo-aminodiol 23 (HCl and HBr salts) after the subsequent debromination (Scheme 21). While the formation of a mixed salt would create a problem in characterization and reproducibility, we believed that the formation of a homogeneous salt could be achieved by replacing HCl with HBr. A single HBr salt of aminodiol 4 would be formed.

Scheme 21. Initial GMP Conditions for 22 to 23•HCl

Replacement of HCl with HBr to Influence the Boc Deprotection of 22 Various hydrobromic acid reagents were studied for the carbamate deprotection including 48% aqueous HBr, 33% HBr in AcOH, and 20–30% HBr in EtOH. The reagents were furthermore investigated in IPA, EtOH, and MTBE from ambient to 50 °C. The 48% aqueous HBr and 20–30% ethanolic HBr in IPA and EtOH proved successful at elevated temperatures, while all others resulted in 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.

incomplete reaction and/or a poor purity profile. Further work revealed that the use of aqueous HBr resulted in slightly lower yields of aminodiol salt 4•HBr than the ethanolic HBr reagent. This was due to the presence of water, which resulted in higher solubility of 4•HBr. Although ethanolic HBr provided a higher yield, 48% aqueous HBr was still chosen for this reaction due to its wider commercial availability in bulk quantities.


Outcome of Process Optimization We determined that adding 48% aqueous HBr (1.3 equivalents) slowly to Boc-bromodiol 22 in IPA and heating the reaction mixture to 50 ± 5 °C were the optimal conditions for the deprotection. Once reaction completion was achieved, the reaction mixture containing 23•HBr was transferred directly into the debromination step as a solution in IPA. This process (Scheme 22) has been successfully carried out eight times to date, on large scales up to 127 kg. Because of the telescoped nature of this process, the yield is assumed to be quantitative and purity is in the range of 85–93%.

Scheme 22. Step 3 Current GMP Process To Prepare 23•HBr

Process Development for Bromo-Aminodiol 23•HBr to Aminodiol 4•HBr Main Issues with the First GMP Process During our initial GLP development work, we discovered that free base 4 darkened in color over six months, which raised concern for long term storage. Isolation of a salt was much more desirable from a stability standpoint (Scheme 23). Further process challenges with this debromination were: • •

NaHCO3 utilized as base caused CO2 evolution. crystallization was carried out by evaporating to dryness then adding a mixture of IPA and EtOAc (1:1). Significant amounts of inorganic salts precipitated and were isolated together with 4. 41


Scheme 23. Initial GMP Conditions for 23•HBr/HCl to 4•HBr/HCl


Use of Organic Base Initial Work to Replace Sodium Bicarbonate with N,N-Diisopropylethylamine The original method for the debromination utilized NaHCO3 as base and was performed as an isolated step (i.e. no telescoping). Due to the problematic issue of inorganic residue carry-through to downstream chemistry using NaHCO3, we decided that the use of an organic amine base was preferable. DIPEA was selected as the best from those screened. The optimal DIPEA stoichiometry (from 1.0 to 3.5 equivalents) for the debromination was investigated. Early work using isolated 23•HCl indicated that 1.0 equivalent of DIPEA was insufficient and led to poor conversion due to reaction with the acid equivalent. A slight excess of DIPEA (1.2 equivalents), however, drove the reaction to completion in good yield in only 8 h. Additional amounts of DIPEA (≥ 1.6 equivalents) resulted in no further benefits to the reaction rate and isolated yield of 4•HBr/HCl, and thus 1.2 equivalents of DIPEA was adopted as the new stoichiometry.

Telescoped One-Pot Process from 22 Through to 4 The telescoped process from 22 through to 4 was also being considered at this time. Thus, 23•HBr, obtained from the newly developed HBr deprotection conditions containing residual solvent and acid, was also investigated to determine how it would behave under the organic base debromination conditions. This work indicated that a minimum of 2.0 equivalents of DIPEA were required to drive the reaction to completion. If the reaction stalled and hydrogen uptake ceased, charging additional DIPEA was effective at driving the reaction to completion with no detriment to the yield or purity of 4•HBr. This data supported the feasibility of a telescoped process, but highlighted the need for further investigation. In order to determine whether the two-step deprotection/debromination of 22 to 4•HBr could be carried out in a one-pot process, several experiments were carried out. Keeping all other factors constant, the amounts of HBr (1.2–2.0 equivalents) for the deprotection and DIPEA (1.4–3.0 equivalents) in the dehalogenation were varied. This work indicated that the use of a 1:1 ratio of HBr and DIPEA led to poor conversion and a slight excess of DIPEA was necessary to drive the reaction to completion. Reducing the amount of 48% aqueous HBr in 42



the previous deprotection step significantly improved the isolated yield of 4•HBr. This is a result of minimizing the residual water content, which in turn, also minimized the solubility of 4•HBr in the reaction mixture. Based on these results, the optimal conditions for the two-step, one-pot process were 1.2 equivalents of HBr for the deprotection and 1.8 equivalents of DIPEA for the debromination, which led to an overall yield of ~60%. Unfortunately, on larger scale, this one-pot process of converting 22 to 4 resulted in a lower yield. Investigations demonstrated that this was a result of 4•HBr loss to the filtrate. Lost 4•HBr was not recoverable in acceptable purity due to the excess of DIPEA salts present. Previous work indicated that 1.8 equivalents of DIPEA were sufficient for the process, but later development work revealed that actually 3.0 equivalents were necessary for the optimal yield. Catalyst Issues Catalyst Removal from the Debromination Process A factor leading to the large amount of residual inorganic impurities and loss of product was the process for catalyst removal. At this time, palladium removal was achieved by filtration of the reaction mixture through Celite®, but this filtration required excessive rinsing of the filter cake with MeOH to fully remove the product from the filter medium. Catalyst removal by filtration through glass fiber filter paper with a 1:1 methanol/IPA rinse resulted in no loss of product when the mixed solvent wash was used. The combined mass balance of inorganic solids and isolated product was consistent with complete recovery. This filtration protocol provided a simple and high recovery method for catalyst removal.

Effect of Catalyst Loading, Pressure, and Base on the Debromination of 23 In efforts to improve both the yield and purity, other reaction parameters were investigated. An assessment of catalyst loading using 10% Pd/C indicated 2 wt% was optimal, with lower catalyst loadings resulting in minimal conversion to the desired aminodiol and 18% of a new impurity. ES-MS analysis supported an epoxide structure for this impurity (Figure 5). Further investigation of the process and impurity formation on larger scale highlighted inconsistencies in the application of hydrogen pressure. It was determined that the resulting low hydrogen pressure in combination with low catalyst loading and a side reaction between 23•HBr and DIPEA over extended periods of time contributed to the formation of this impurity and had a significant impact on the overall yield and purity.

Figure 5. Epoxide Impurity Observed During Debromination Optimization. 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.


In order to determine what catalyst loading and pressure were sufficient to avoid impurity formation, several experiments were carried out varying catalyst loading over 2 to 3 wt% and hydrogen pressure up to 55 psig. Although all reactions reached completion, longer reaction times were required and as much as 10% of the undesired epoxide was observed with 2 wt% catalyst loading at lower pressures, e.g. < 30 psig. This result was deemed unacceptable for large scale development. This study revealed that 3 wt% catalyst loading and 50 psig resulted in a faster reaction rate with no impurity formation. This process was found to be suitable for large scale and was therefore utilized in the future process.

Crystallization: Identification of an Antisolvent for the Isolation of Aminodiol 4•HBr Knowing that the inorganic content (from the previous steps) of the reaction mixture had such a significant impact on the isolation, yield, and purity of aminodiol 4•HBr, solubility studies were performed on both 4•HBr and the reaction byproduct DIPEA•HBr. We hoped to identify conditions that would provide the lowest 4•HBr solubility and best rejection of the base salt. DCM, MeCN, acetone, MTBE, and THF were all investigated but only DCM and MeCN were viable antisolvent options. Studies with DCM resulted in a slow, controlled crystallization of 4•HBr with better recovery and purity over any process using MeCN. It was decided to therefore utilize DCM as antisolvent in the isolation of 4•HBr.

Optimization of the DCM Crystallization Process The other crystallization parameters were investigated in efforts to optimize the new DCM process. This work looked into the concentrated volume of the crude reaction mixture prior to DCM addition; the rate, volume, and temperature of antisolvent addition; and finally the stir time after antisolvent addition. Once the debromination was complete, the reaction mixture was concentrated to remove the MeOH and most of the IPA to allow for a less hindered crystallization of 4•HBr. Based on this process, we first investigated the optimal volume to which the reaction mixture should be reduced before the DCM addition. When less than 3 volumes were utilized, an uncontrolled and spontaneous crystallization occurred and a thick slurry was formed. For this reason, 3 to 4 volumes were deemed optimal for the process. Additionally, the ratio of IPA/DCM added during the isolation of 4•HBr was investigated by slurrying mixtures of 4•HBr in solutions of IPA/DCM at 30 °C. The data indicated that when 2 volumes of IPA were used, there was a difference observed on recovery when varying the amount of DCM from 4 to 10 volumes. However, when 3 volumes of IPA were used, at least 8 volumes of DCM were necessary to achieve 90% recovery. Based on these results, it was decided to use 3:10 IPA/DCM as the optimal ratio for the isolation of 4•HBr. 44


The DCM addition rate to the IPA reaction mixture was also investigated. A comparison of a fast, one portion addition to a dropwise addition over 4 h showed no significant impact on yield. The purity, however, was greater when a longer addition time was applied. We decided to add the DCM over a minimum of 60 minutes. The temperature of aging was investigated from –5 to 20 °C and lower temperatures gave a higher yield due to the precipitation of DIPEA salts from solution. For this reason, the slurry was stirred for a period of time at 20 °C and cooled further to 5 °C just prior to filtration.


Outcome of Process Optimization Redevelopment of the debromination conditions resulted in a process utilizing DIPEA (3.0 equivalents) as base with Pd/C (3 wt%) under 50 psig hydrogen pressure in a solution of IPA and MeOH at 25 ± 5 °C. Once completion of the reaction was achieved, a solvent swap to IPA was performed to drive off MeOH (≤ 0.2%), followed by addition of DCM antisolvent, allowed for the isolation of 4•HBr as an off-white solid. This improvement provided yields in the range of 61–77% of aminodiol 4•HBr in ≥ 96% purity. The process (Scheme 24) has been successfully carried out ten times on scales up to 40 kg.

Scheme 24. Current GMP Process To Prepare 4•HBr

Process Development for Pyrimidine-Aldehyde 5 to Cyclopentane-diol 3 Main Issues with the First GMP Process Due to the time constraint for delivery of material, the first GMP process (Scheme 25) to prepare chloropyrimidine 3 was carried out based on a two-step procedure using literature conditions (18–20). The aldehyde 5 was converted to acetal 27, followed by reaction with aminodiol salt 4 and cyclization to give chloropyrimidine 3. Due to the switch from a mixture of HCl/HBr salts of 4 to 4•HBr as the other reactant, development efforts for this step (27 to 3) in post-first GMP production were focused on base, solvent, and reaction temperature. 45 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 25. Initial GLP/GMP Process To Convert Aldehyde 5 to Chloropyrimidine 3

Starting Material Switch from Acetal 27 to Aldehyde 5 We hypothesized if aldehyde 5 could react directly with aminodiol 4 and thus, the need for the acetal starting material 27 would be eliminated. However, unsuccessful reactions and undesired imine formation had been reported to occur with the aldehyde. (18, 20). The result was surprisingly good for the reaction of 5 with 4 using the Et3N, IPA conditions, and a similar reaction profile was obtained. This new one-step process eliminated the need for the additional step of converting aldehyde 5 to the acetal 27. Use of solid 5 versus the oily acetal 27 also made it easier to charge the starting material into the reactor.

Solvent and Base Screening Optimization work for this step included a comprehensive solvent and base screen in efforts to achieve a better reaction profile. For the solvent, toluene, THF, DMF, NMP, IPA, MeOH, EtOH, and 2-butanol were explored. Both organic and inorganic bases, such as Et3N, DIPEA, K2CO3, DABCO, DMAP, pyridine, and Cs2CO3 were examined. The best conversion and purity resulted from conditions using IPA or DMF as the solvent and Et3N or DIPEA as the base. Further study led to the findings that 3 could not be isolated by crystallization readily when DMF was used as the solvent, while IPA provided better results. It was determined that 8 to 12 volumes of IPA could be utilized with no significant impact on the extent of reaction, yield, or product purity. Several reactions were then carried out in IPA, investigating both the base options and the type and quality of 4, i.e. if 4 was 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.


more effective and stable as free base than its HBr salt and if additional purification was necessary before each use. The use of an alternative base (DIPEA, DABCO, DMAP, or pyridine) in IPA with 4•HBr did not offer any improvement to yield over use of Et3N and was therefore not investigated further. The reaction with 4•HBr and aldehyde 5 was also examined under acidic conditions (without addition of base). This resulted in the disappearance of starting material and formation of another compound, identified as the imine intermediate by MS (M+ 304) (Figure 6). However, further cyclization of the imine to the desired product 3 did not occur, indicating that base was required for this condensation.

Figure 6. Imine Intermediate When Base Was Not Charged.

Equivalents of Aldehyde 5 and Reaction Temperature Aldehyde 5 stoichiometry was initially reviewed between 0.8 and 1.1 equivalents versus the diol 4, with 0.9 equivalent being selected to ensure a complete reaction of aldehyde. Later, the ratio of starting materials was reinvestigated keeping other parameters constant as originally developed (2 equivalents of Et3N, 75–80 °C) to identify the optimal range. From this study, it was discovered that the isolated yield of product increased with the use of a slight excess of aldehyde. However, the benefit from increasing 5 was compromised by the decreased purity of the product, especially when more than 1.15 equivalents of aldehyde 5 was used (Table 4). The improved reaction profile using more aldehyde can be explained as related to stability concerns with the aldehyde. We hypothesized that thermal decomposition of the aldehyde competes with the desired reaction pathway, thus an excess of the aldehyde is beneficial to the overall reaction yield. However, more than 1.15 equivalents of aldehyde 5 used in the reaction led to lower product yields as more impurities were produced in the reaction (Table 4). Evaluation of the reaction temperature revealed 75 ± 5 °C as the optimal reaction temperature range for achieving a high isolated yield. Although high reaction temperatures are required for an acceptable reaction rate, thermal decomposition of 5 became significant when the reaction temperature exceeded 80 °C, thus defining the range. 47



Table 4. Screen for Optimal Amount of 5 4HBr (equiv)

5 (equiv)

Isolated Yield of 3 (%)

Purity (% a/a)

1.0

0.9

67

98

1.0

1.0

66

98

1.0

1.05

76

99

1.0

1.10

78

96

1.0

1.15

74

99

1.0

1.25

79

89

1.0

1.35

76

87

Isolation Water was used as the antisolvent to induce product crystallization from the reaction mixture in IPA. Slow addition of water after the reaction completion at 45–50 °C provided a cleaner product and more filterable crystals than addition at lower temperatures. The amount of water used for isolation was also investigated. Due to the minimal solubility of the product in water, the addition of 4–12 volumes had little impact on yield or product purity. Larger volumes of water, however, were found to be beneficial to remove the triethylamine salts and produce an overall better purity profile. The benefits of using larger volumes of water led to our final process.

Outcome of Process Optimization Process redevelopment led to the currently executed step 5 conditions (Scheme 26), which utilized IPA (20 volumes) as solvent and triethylamine (2.2 equivalents) as base to affect the reaction of 4•HBr (1.0 equivalent) and aldehyde 5 (1.1 equivalents) at 75 ± 5 °C for 18 ± 2 h. Isolation was carried out via crystallization at 45 ± 5 °C using water (15.5 volumes) as antisolvent, followed by filtration and a water wash. This process has been successfully proven six times to date, on scales up to 59 kg. Yields resulting from this process are typically in the 75–83% range with purity ≥ 98%.

Process Development for the Current GMP Process for Conversion of Chloropyrimidine 3 to Indane-Diol 2 The main issue with the first GMP process for conversion of chloropyrimidine 3 to indane-diol 2 was the long reaction time and low yield. As the high temperatures and high pressures required for the conversion were untenable in 48 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 plant, milder conditions were preferred. In the search for an alternative to this SNAr reaction, a cross-coupling strategy was explored. A number of literature reported catalysts with various ligands and bases were screened (Table 5) (44–46). However, none of the experiments provided results worthy of further effort. The initial GMP process was maintained (Scheme 27).

Scheme 26. Current GMP Process To Prepare Chloropyrimidine 3

Process Development for the Preparation of Free Base 1 Main Issues with the First GMP Process Burgess Reagent Development This sulfamoylation step represents the culmination of the addition of the final piece to produce pevonedistat. In the first GMP campaign, the Burgess-type reagent 33 was generated in situ in a separate vessel (Scheme 28). A large excess (4.0 equivalents) was prepared and used in order to ensure complete conversion of diol intermediate 2. Use of THF as solvent led to polymerization of the solvent during acidic deprotection, which produced an impurity that proved difficult to remove. Most problematic of all, the reaction was not clean and chromatographic purification was required to obtain pure free base 1. These problems were successfully addressed as detailed below.

Optimization of the First GMP Process An early strategy to improve the first GMP process was considered by retaining the use of in situ generated Boc-sulfamoyl chloride/DABCO reagent. These studies examined various solvents to replace THF and increase solubility of diol 2. DMAc was optimal over DMF, NMP, and DMSO. 49 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 5. Cross-Coupling Strategy To Prepare Diol 2

Most of our work on this transformation involved changes to the preparation and the manipulation of the Burgess-type reagent in order to improve the selectivity and efficiency of this reaction. Preliminary results indicated that having a small amount of acid present helped to speed up the reaction and improve selectivity. We hypothesized that the protonated form 38 is more electrophilic than 37 (Scheme 29). A series of reactions was also carried out in DMAc incorporating a group of selected acids (2 M HCl/THF, TFA, acetic acid and isobutyric acid) to determine their effect on the process. All acids increased 50 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 reaction rate; however, faster decomposition of the Burgess-type reagent also occurred simultaneously and a large excess of the Burgess-type reagent (5 equivalents) was required to drive the reaction to completion. Use of HCl was the most effective, as acetic acid and TFA resulted in the formation of a new impurity and isobutyric acid showed only a small increase in the reaction rate.

Scheme 27. Current GMP Process To Prepare Diol 2

Scheme 28. Initial GLP/GMP Process To Convert diol 2 to Free Base 1

Scheme 29. Acid Effect on Burgess-type Reagent Based on these results, development reactions were carried out in DMAc exploring: 51 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.


• • • • •

various reaction temperatures. order of addition. volume of solvent. amount of Burgess-type reagent. amount of HCl.

One possibility was that a low temperature might help with the selectivity, but it was found impractical due to the poor low temperature solubility of both diol 2 and the Boc-sulfamoyl chloride/DABCO reagent in DMAc. Better selectivity was seen with dilution of the reaction mixture and a higher ratio of Burgesstype reagent/HCl to 2. The Burgess-type reagent (or the activated form) was not stable under the reaction conditions and therefore an excess was required to reach completion. Portionwise addition of the Burgess-type reagent was initially considered to circumvent the degradation and need for excess reagent, but this strategy would add operational inconvenience and thus, was not pursued.

Development of a Solid Burgess-type Reagent Although the above improvements had addressed some of the problematic issues of the early process, they failed to provide a fundamental improvement in the process, which would allow the sulfamoylation to be carried out on a manufacturing scale. The ideal reaction conditions for this step would be to use a reagent that is solid and stable for the purposes of utility and storage. It would then be possible to charge the reaction mixture easily with small quantities of Burgess-type reagent when required. (47, 48). A second highly desirable property of the reagent would be for the reagent to selectively react with the primary hydroxyl group over the secondary hydroxyl group in diol intermediate 2. A reagent with these superior properties was in fact developed by Takeda’s process team as described below.

Preparation and Isolation of a t-Bu/DABCO Burgess-type Reagent Early in the process development phase, the unexpected discovery was made that the addition of DABCO to Boc sulfonyl chloride, resulted in a precipitation. This was later determined to be a mixture of 37 and DABCO•HCl salt, where the ratio depended on the type of reaction solvent (Scheme 30). Efforts were spent to isolate this solid in order to further develop a sulfamoylation reaction that would use a solid t-Bu/DABCO Burgess-type reagent. The first process to form solid t-Bu/DABCO Burgess-type reagent using acetone as solvent produced a mixture with 70% Burgess-type reagent and 30% DABCO•HCl salt byproduct by NMR. Apparently, DABCO•HCl salt has better solubility than t-Bu/DABCO Burgess-type reagent in acetone. Attempts to purify the Burgess-type reagent via an aqueous work-up to remove the DABCO•HCl salt via extraction failed since the reagent decomposed rather quickly in water (< 10 min). Due to the reactive nature of the reagent, isolation under nonaqueous 52



conditions with a minimum number of operational steps was targeted. The crude Burgess-type reagent reaction mixture obtained after solvent removal was slurried in various solvents (49) in the hope that a suitable recrystallization solvent could be identified. Unfortunately, none of the solvents worked; the reagent salt complex either decomposed slowly (> 18 h) in some solvents or did not result in clean separation of the t-Bu/DABCO Burgess-type reagent and DABCO•HCl. These many unsuccessful attempts forced us to consider a different approach. Isolation of the reagent as a fixed ratio with the byproduct, DABCO•HCl salt, was then pursued. Toluene was ultimately chosen as the reaction solvent since both the Burgess-type reagent and DABCO•HCl salt had very low solubility in toluene. The strategy resulted in collection of a 1:1 solid mixture of 37 and DABCO•HCl salt (39). The mixture was isolated from toluene in > 98% yield and > 98% purity by quantitative NMR analysis (Scheme 30) (48). The mixture was as effective in the sulfamoylation reaction as the original isolated reagent mixture that contained only 30% DABCO•HCl salt. It has been stored at ambient temperature for 12 months without a detectable decrease of activity. This synthesis proved to be robust and the desired solid Burgess-type reagent 39 has been prepared on 100–150 kg scale.

Scheme 30. Synthesis and Isolation of t-Bu/DABCO Burgess-type Reagent This optimized Burgess-type reagent, a solid, allowed a better control of the reaction with precise addition of reactants. We determined that the Burgess-type reagent/DABCO•HCl only needed to be used in slight excess. A solvent and temperature screen was carried out using the complex (2.0 equivalents) in MeCN. The key findings from this study were: •

•

Use of DMF or DMAc as a cosolvent in acetonitrile was examined for better solubility but neither was found beneficial to the rate of reaction or improvement of the product purity. The reaction rate at 30 °C was slow, while complete reaction was observed in 2 h at 60–70 °C. 53



•

The effect of solvent volume was studied from 5–10 volumes of solvent. When 5 volumes of acetonitrile were used, the reaction went to completion in 2 h. However, under these conditions, the reaction mixture was too thick to stir efficiently. An increase to 7.5 volumes of acetonitrile at 50 °C was optimal to achieve the desired rate of reaction and purity of product 34. Reaction at 10 volumes showed a similar reaction rate and product profile.

This work helped to define the extent and conditions for use of our Burgesstype reagent. While development of the t-Bu/DABCO Burgess-type reagent/DABCO•HCl mixture was ongoing, investigation into sulfamating reagents bearing other carbamate protecting groups (e.g. Boc, tertiary, or Bn-like) were considered. Unfortunately, none of the other Burgess-type reagent analogs demonstrated better results than the original reagent 39. The decision was made to remain with the current sulfamating agent.

Optimization of the Deprotection with the Newly Developed Burgess-Type Reagent Elimination of Impurities After effectively driving the reaction of diol 2 with 39 to a mixture of 34 and 36, the remaining challenge for a chromatography-free process would be the removal of the bis-sulfamate 36 side product (Scheme 31). Based on our previous experience from the first GMP production, separation of the free base 1 from the bis-sulfamate side product would not be an easy task due to the similarity of their structures. In early development work, use of an acid (HCl, TFA, or isobutyric acid) in the sulfamoylation was investigated as a way to improve the reaction rate and selectivity. In the course of these studies, we discovered that the Boc-protected bis-sulfamate impurity underwent elimination to a cyclopentene intermediate under aqueous acidic conditions at ambient temperature. This observation was not surprising as the Burgess-type reagent is known to be a mild reagent for alkene formation. (50, 51). Further investigation to find a better acid to perform this conversion identified 0.5 N aqueous HCl (6 volumes) to be optimal for this elimination reaction to produce the cyclopentene. We hoped that the structurally different cycloalkene impurities 40 or 41 compared to free base 1 would allow facile separation. Full conversion of the bis-sulfamate 36 to 40 was achieved in 1 h after adding 0.5 N aqueous HCl (6 volumes). No deprotection of 34 or 40 was observed during this transformation. Work-up procedures were investigated in attempts to identify conditions that would isolate Boc product 34 from the cyclopentene analog 40. Studies were carried out to determine if and how the cyclopentene impurity could be removed from the reaction mixture. 54



Scheme 31. Bis-Sulfamate Elimination to Cyclopentene Impurity After full conversion of 40 from 36, MTBE and water were introduced to the reaction system to allow separation of organics (mainly the product) from the inorganic salts (e.g. DABCO•HCl). We noticed that the cyclopentene impurity 40 was less soluble in MTBE; however, the amount of MTBE required to force precipitation of 40 from a MTBE/acetonitrile solution would be too high for large scale manufacture. We hypothesized that if a realistic amount of MTBE was added to the acetonitrile layer and the combined organic layer was then extracted portionwise with water, the cyclopentene impurity 40 could be separated from the organic layer when acetonitrile was washed away with water. This extractive process was tested and the results indicated that indeed the cyclopentene impurity 40 appeared as a solid at the interphase after water washes (2 × 10 volumes), and was filtered off from the organic layer. This process produced product 34 that retained a low level of impurities (5–10%). However, the aqueous washes also had a tendency to result in a gummy material coming out of the organic layer as acetonitrile was removed by water washes. The gummy material, likely being 34, made the work-up difficult, unless the washes were conducted at slightly elevated temperature (40 °C). Unfortunately, low levels of Boc deprotection of 34 now occurred at the higher temperatures. To circumvent this issue, we decided to telescope the MTBE solution without extensive water washes into the deprotection step, and utilize the crystallization operations in the downstream steps to reduce the levels of cyclopentene 40. This is described in the following section.

Deprotection Conditions To the solution of 34/40 in MTBE was added more acid for the deprotection. In the absence of THF in the system—and thus, no concern for polymerization—stronger acids were screened for this transformation. We also noted the cleavage of the indane moiety as a side reaction in the presence of aqueous solutions of strong acids. Thus, a balance of speedy Boc deprotection 55 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.


and minimum indane hydrolysis was required. To fine-tune the conditions for this step, the intermediate Boc-sulfamate 34 was isolated and a number of alternative deprotection conditions were investigated focusing on HCl of varying concentrations in EtOH, IPA, dioxane/MeCN, dioxane/EtOH, MeCN, and water. We determined that the use of 3.0 M HCl/MeCN system gave the cleanest reaction of > 99% deprotection, while retaining > 99% of the indane. It should be noted that higher concentrations of HCl also led to an increase in the formation of the chloride impurity (Figure 3), and therefore higher concentrations should be avoided. In order to further simplify the process and avoid MTBE extraction, a small amount of concentrated HCl was used to convert cyclopentane analog 36 to cyclopentene 40, followed by another addition of small amount of concentrated HCl to adjust the reaction pH to ~3 for deprotection of the Boc. Unfortunately, the bis-sulfamated byproduct underwent elimination easily, but the subsequent deprotection was extremely slow, taking multiple days and further acid additions to reach completion. Apparently, when DABCO salts and other inorganic impurities were not removed by aqueous work-up, deprotection was difficult. The reaction also resulted in only 35% yield, lower than the typical 45–55% achieved under the process with the MTBE/aqueous extractive work-up. The MTBE extraction of 34 was beneficial to the Boc deprotection and therefore was retained. After complete conversion of 34/40 to 1/41, the reaction mixture was diluted with water/EtOAc. The organic layer was separated and then washed with NaHCO3 solution. The level of cyclopentene impurity 41 was reduced to < 1% in the EtOAc layer, leaving free base 1 as the main component (> 90%).

Free Base 1 Crystallization The initial free base 1 purification/isolation process under the sulfamoyl chloride reaction conditions required the use of column chromatography for acceptable purification. In order to remove this column chromatography, efficient crystallization of free base 1 was required. An antisolvent screen identified EtOAc/DCM system to be optimal for the crystallization. As the recrystallization did not remove several polar impurities at borderline high levels, we sought to add an adsorbent treatment as an orthogonal purification step. The use of silica gel (100 wt%) and charcoal (100 and 50 wt%) showed comparable purity improvements, but the material loss at these loadings was slightly greater with charcoal. Thus, our recrystallization protocol included a silica gel filtration to remove impurities prior to the recrystallization.

Outcome of Process Optimization Using t-Bu/DABCO Burgess-type reagent/DABCO•HCl (1:1 mixture, 39), the current process for sulfamoylation of diol 2 was established (Scheme 32). Diol 2 was consumed with 2.0 equivalents of reagent 39 in 4–5 h at 50 °C. The end reaction mixture contained ~80% 34 and ~15% bis-byproduct 36. Byproduct 36 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.


was first hydrolyzed to mono-Boc-sulfamate olefin 40 under mild acidic conditions (44–46). Upon full conversion of 36, an increase in HCl concentration successfully removed the Boc groups to afford 1 and sulfamate olefin 41. Compound 41 was purged completely to the aqueous washes during work-up. Charcoal plug filtration followed by crystallization of the crude product from DCM afforded free base 1 in > 96% purity and > 50% yield. This procedure has been successfully carried out in multiple 30 kg API manufacturing campaigns.

Scheme 32. Current GMP Process To Prepare Free Base 1

Process Development for the Salt Formation of the API An early polymorph screening on pevonedistat identified eight forms, as follows: • • •

Among them, Form 1, being the most stable and with acceptable bulk drug physical properties, is the desired form for development. Form 2 is obtainable through interconversion of Form 1 under high moisture conditions. The other six forms were identified as solvates, many of which transform to Form 1 after desolvation.

In any event, we required a convenient process to isolate the desired Form 1. Initially, the salt formation was carried out in pure ethanol using ethereal HCl. However, this process produced a mixture of polymorphic forms. For one of them, comparison of the XRPD trace from the prequalification batch with known crystal forms, along with KF analysis, identified this mixture as consisting of Form 3, 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.


the ethanol solvate, and the hydrated Form 2. Unfortunately, Form 2 does not convert to Form 1 on drying, so a rework procedure and redevelopment of the crystallization process was necessary. It was known that a hot slurry in ethanol would force the conversion of the hydrated Form 2 to the EtOH solvate, Form 3. Form 3 converts to the desired crystal form (Form 1) under drying. Therefore, ethanolic HCl was chosen to replace ethereal HCl to allow a higher reaction temperature (Figure 7). Also, the use of pure Form 1 seed crystals was determined to be critical to prevent the formation of polymorphic mixtures.

Figure 7. Three Major Crystalline Forms of Pevonedistat. In summary, the desired polymorph was obtained from the following procedure making use of the crude pevonedistat obtained from our multikilogram procedure. Heating to reflux to achieve dissolution was implemented as higher purity free base 1 had lower solubility in EtOH at 50 °C. Addition of 1.25 M HCl in ethanol at 50 °C was carried out in two portions. Approximately half of the HCl was added rapidly and the remaining portion was added post seeding, over the course of an hour. After addition of HCl, the reaction mixture was then cooled to ambient over several hours. This procedure: • • •

aided in better control of the crystallization. provided the desired form. provided improved purity of the API.

A final issue after the first GMP campaign was the presence of ethyl chloride, a genotoxic impurity (GTI) in the final API (52). An investigation was carried out to determine the origin of ethyl chloride and effectiveness of drying to remove ethyl chloride in the API. Analytical methods could not detect ethyl chloride in ethanolic HCl. This suggested that ethyl chloride was generated during the HCl salt formation under heat. Drying at 35 °C for only one day resulted in an EtCl level comparable to drying at 25 °C for ten days. However, in both cases, the data showed that although there was EtOH and HCl trapped within the crystal structure, the close proximity and heating did not appear to generate any additional EtCl. While confusing, the levels of ethyl chloride were acceptable. All lots of API have met the acceptance criterion for ethyl chloride levels (NMT 4630 ppm) which was calculated based on the highest clinical daily dose. This procedure has been successfully carried out to afford multiple cGMP lots of pevonedistat. 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.


Summary A manufacturing process was developed for the synthesis of pevonedistat, a novel and potent NAE inhibitor. The chromatography free, six isolation process has been demonstrated on a 50 kg scale for multiple cGMP productions to afford drug substance with greater than 98% (a/a) chemical purity and 25% overall yield (Scheme 33). A high yielding chiral aminodiol 4·HBr synthesis was developed through bromolactonization of 20, followed by reductive lactone opening, deprotection, and removal of bromide. This work also showcased the development and use of a novel Burgess-type reagent 39 in the final selective sulfamoylation step. The increase in yield and removal of chromatography for this step also contributed to the overall improvement of the synthesis of pevonedistat. The total amount of API prepared by this process is > 100 kg.

Scheme 33. Current GMP Pevonedistat Manufacturing Process

Acknowledgments The authors thank the entire Takeda NAE team for helpful discussions. Special thanks go to Eric L. Elliott, Frederick Hicks, and Steve Langston for assistance in preparing the manuscript. 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.

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62 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.

Process Development and GMP Production of a Potent NEDD8

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