Synthesis of a Potent NAE Inhibitor: Pevonedistat - ACS Publications

Chapter 1

Synthesis of a Potent NAE Inhibitor: Pevonedistat Downloaded by 80.82.78.170 on January 13, 2017 | http://pubs.acs.org Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch001

Hirotaki Mizutani, Steven Langston,* and Stepan Vyskocil Medicinal Chemistry Pharmaceutical Research Division, Takeda Pharmaceuticals International Company, 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States *E-mail: [email protected].

Pevonedistat (MLN-4924, TAK-924) is a first-in-class inhibitor of Nedd-8 activating enzyme (NAE) currently undergoing clinical evaluation in oncology. This chapter will discuss the identification of pevonedistat and describe the synthetic routes undertaken by discovery chemists to provide material for pre-clinical studies.

Introduction Within the cellular environment, the levels of particular proteins at any given time are highly controlled by regulation of protein synthesis and also by regulation of protein degradation. The organelle responsible for the regulated decomposition of proteins within the cell is the proteasome, which acts as a multi-subunit protease with broad substrate specificity. Proteins tagged for proteolysis by the proteasome are first labeled with a poly-ubiquitin chain by way of a cascade of enzymes within the cell, a process known as ubiquitination. Ubiquitin (Ub) is a highly conserved, low molecular weight protein that is attached to the protein to be degraded through its carboxylic acid terminus and a side chain amine of a lysine residue of the protein. The enzyme responsible for initial activation of ubiquitin is known as ubiquitin activating enzyme (UAE), and is generically termed an E1. UAE utilizes ATP to activate the terminal carboxylic acid of ubiquitin to facilitate formation of a thioester bond through a © 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.

Downloaded by 80.82.78.170 on January 13, 2017 | http://pubs.acs.org Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch001

cysteine residue of UAE and release both adenosine 5′-O- monophosphate(AMP) and pyrophosphoric acid (PPi). Ubiquitin thus activated is transferred to an intermediary protein, known as a conjugating enzyme or E2, through thiol exchange with a cysteine residue on the E2 and then is finally attached, or ligated, to the protein substrate via an E3 ligase. This general pathway for regulated degradation of proteins is known as the ubiquitin-proteasome system or UPS (Figure 1) (1–3).

Figure 1. The Ubiquitin Proteasome System (UPS).

The E3 ligases are a broad family of enzymes which play a critical role in imparting substrate specificity to which proteins are ubiquitinated and ultimately degraded by the proteasome. Their activity is thus closely regulated. A subclass of E3 ligases are known as the Cullin-Ring-Ligases (CRLs) based on the make-up of subunits that compose the whole enzyme. For this class of ligases to be fully enzymatically active they are required to be modified (on the Cullin subunit) by a protein—Nedd-8. This is a protein closely related to ubiquitin and the process of attachment of Nedd-8 to its protein substrate is known as neddylation. The process of neddylation is analogous to ubiquitination with Nedd-8 activating enzyme (NAE) being the E1, and a protein known as UBC12 serving as the E2 before transferring to the CRL family of E3 ligases, (Figure 2) (1–3). Because neddylation is necessary for the activity of the CRLs it plays an important role in regulating which proteins are ubiquitinated and degraded by the proteasome. These proteins, such as phosphorylated IκB, Cdt-1 etc., often play critical roles in cellular processes such as DNA replication, cell division and cell signaling (1–3). 2



Figure 2. The ubiquitination and neddylation processes.

Identification of Pevonedistat (TAK-924, MLN4924) At the beginning of this program, the clinical success of the first proteasome inhibitor bortezomib (Velcade®) from our company gave us encouragement to investigate additional targets within the ubiquitin proteasome pathway. The inhibition of the proteasome essentially prevents the degradation of all protein substrates within the UPS. However, only a subset of those proteins would be expected to be stabilized by inhibition of NAE, i.e. those proteins targeted for proteasome degradation through ubiqutination via CRL E3 ligases, and thus dependent upon neddylation (and NAE activity) for activity, (Figure 2). It is known that AMP is a product of the NAE-catalyzed reaction and is a weak inhibitor of NAE (4, 5). The knowledge that ATP and AMP are recognized by E1 enzymes, including NAE, indicated that this class of enzymes may be druggable 3 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.


targets within the UPS. Indeed, a high throughput screen followed up by array synthesis identified N-6 substituted adenosine derivatives as μM inhibitors. Incorporating the 5’-O-phosphate with one of the preferred N-6 substituents, 1-(+)-(S)-aminoindane, gave a sub-μM inhibitor, compound 2. A key finding for us was that replacing the phosphate with the neutral sulfamate group (3, X=O, Figure 3 and Table 1) substantially boosted potency to low nM and exhibited good activity in cellular assays. However, the sulfamate group can act as a weak leaving group and analogs were prone to degradation upon storage through an internal cyclization reaction, (Scheme 1).

Figure 3. Compounds on route to the discovery of pevonedistat.

Scheme 1. Degradation of Adenosine Sulfamate Analogs through Internal Cyclization 4 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 Medicinal Chemistry effort was thus focused on stabilizing the compounds to intrinsic degradation and exploring the SAR (structure activity relationship) to identify more selective compounds for NAE over related E1 enzymes such as ubiquitin activating enzyme (UAE). Replacing the sulfamate group with the more stable sulfonamide (X = CH2, 5) or sulfamide (X = NH, 4) rendered reduced potency. A broad diversity of substituents at the 6-position of the adenine ring was also investigated with 1-(+)-(S)-aminoindane remaining one of the preferred groups. Replacing the ribose for a cyclopentane, and removal of the 2' OH group and N-7 of the adenine ring (adenosine numbering) were all well-tolerated. Combining these modifications gave compounds that were selective for NAE over UAE, for example 6 (Figure 4 and Table 1). However, the compounds remained somewhat prone to degradation through intramolecular cyclization. A breakthrough came with the surprising finding that inversion of the configuration at the methylene sulfamate position was tolerated (TAK-924, Figure 4, and Table 1). This places the sulfamate on the opposite face to the adenine type ring and thus immune to intramolecular attack by the adenine ring due to lack of proximity between the groups. These changes ultimately led to the identification of the candidate molecule pevonedistat (MLN4924, TAK-924) (Figure 4 and Table 1) (6).

Table 1. Inhibitory Activity against the Target Nedd-8 Activating Enzyme (NAE) and the Related Enzyme Ubiquitin Activating Enzyme (UAE) NAE *IC50 (nM)

UAE *IC50 (nM)

1

1600

>10000

2

120

>1000

3 X=O

0.5

2

4 X=NH

2

450

5 X=CH2

12

>1000

6

8

>1000

pevonedistat

3

>1000

Compound

*

HTRF assay. (8)

During the course of this Medicinal Chemistry effort, it was discovered that such adenosine sulfamate analogs, including pevonedistat, act as a unique class of mechanism based inhibitors, termed substrate assisted inhibition (7). The biological consequences of NAE inhibition through pevonedistat, including efficacy of cellular outcomes, pharmacodynamics markers and efficacy in in vivo xenograft models are described in detail elsewhere. (8, 9). 5 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 4. Structures of adenosine sulfamate analogs, including pevonedistat, in relation to adenosine mono-phosphate (AMP).

Scheme 2. Retrosynthesis of Pevonedistat

Synthesis of Pevonedistat Retrosynthesis of Pevonedistat The initial route for the synthesis for pevonedistat was designed to allow flexibility in the choice of a purine-like base and enantiomerically pure carbocycle, and utilized building blocks that could be prepared using chemistry precedented in the literature. Pyrrolopyrimidine 7 and cyclopentane building block 8 with an epoxide function were chosen as synthons (Scheme 2). While 7 could be easily accessed from commercially available materials, the construction of 8 with all cis-tetrasubstituted cyclopentane stereochemistry was significantly more challenging. We envisioned that it could be accessed 6 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.

from the alkene 9, which in turn could be derived from the precursor 10 via a metathesis reaction (10). Enantiomerically pure diene 10 could be then constructed using an Evans aldol reaction utilizing an oxazolidinone as the chiral auxiliary. Subsequently, a more direct route towards intermediate 9 was designed, utilizing a hetero-Diels-Alder reaction of cyclopentadiene with glyoxalic acid via hydroxylactone 11 (11), followed by enzymatic resolution of rac-9.


First Synthesis of Pevonedistat The first synthesis of pevonedistat used an Evans asymmetric aldol reaction as the key step for building the appropriate cyclopentane stereochemistry (Scheme 3) (12). (S)-Benzyloxazolidinone 12 was deprotonated using n-BuLi and treated with 4-pentenoyl chloride to form the oxazolidinone intermediate 13 in almost quantitative yield. Compound 13 underwent a highly stereoselective enolization using dibutylboron triflate in the presence of a base, and the corresponding boron enolate was then allowed to react with acrolein. Following oxidative workup, 14 was isolated in 61% yield in enantiomerically pure form (>99% ee) (12). Diene 14 was then cyclized to cyclopentene 15 using ring closing metathesis with Grubbs second generation catalyst (10) in 79% yield. The chiral auxiliary was then removed by reduction with lithium borohydride (13), to furnish the enantiomerically pure diol 9 in 61% yield. Diol 9 was protected and then subjected to diastereoselective epoxidation to cis-epoxydiol 16. High (>9:1) cis/trans selectivity could be rationalized by syn-stereodirecting effect of the allylic hydroxyl group on one of the oxygens of the peroxy group (14). The PMP-acetal protecting group with good basic pH stability was introduced using condensation with PMP-dimethylacetal and a catalytic amount of TsOH. This group was used in anticipation that the epoxide-opening step may require forcing basic conditions. The key coupling to produce the skeleton of pevonedistat was effected by treating the protected epoxide-diol 16 with pyrrolopyrimidine 7 under basic conditions with heating. The epoxide was opened regioselectively to give nucleoside precursor 17. The regiochemistry can be explained by steric factors associated with the acetal. The hydroxyl group at the 2-position was removed using a two-step sequence. This reaction proceeded through a thiocarbamoyl intermediate which underwent Barton-McCombie (15) radical deoxygenation to 18. Deprotection of the acetal-protecting group PMP was achieved using AcOH (16) to furnish diol 19. Initial attempts to introduce the sulfamoyl group with sulfamoyl chloride were unsuccessful as they led to mixtures of mono- and bis-sulfamoylated products. This final step required a sequence of protection/deprotection steps to selectively sulfamoylate the primary hydroxyl group, starting with TBS protection, followed by acetate protection of the secondary alcohol to yield 20. The primary TBS group was removed using HF•TEA, and the resulting alcohol was sulfamoylated using sulfamoyl chloride. Sulfamate 21 was then deacetylated using ammonia to furnish pevonedistat. 7 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

While this route enabled the synthesis of the initial quantities of pevonedistat, it had some drawbacks: • • •

long synthesis (15 synthetic steps). it required multiple chromatographic purifications. provided a low overall yield of product (5.5 %).


Thus, we needed an alternative route for the preparation of multigram quantities of pevonedistat required for in vivo testing.

Scheme 3. The First Synthesis of Pevonedistat

Gram Scale Synthesis of Pevonedistat The second synthetic iteration (Scheme 4) started with a hetero-Diels-Alder reaction of cyclopentadiene with glyoxalic acid (11). This [4+2] addition followed by rearrangement (17) produced hydroxylactone 11 which was converted to racemic cis-diol 9 via global reduction with LAH followed by periodate cleavage of the triol 22 (18). This sequence required only one chromatographic purification 8 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 was performed successfully on 250 g of hydroxylactone 11. Following selective TBS protection, racemic 23 was isolated by column chromatography in 75% overall yield for the 3 steps. This material was obtained as a single diastereosisomer with cis-orientation of cyclopentene substituents set by syn-addition in the first step.

Scheme 4. Gram Scale Synthesis of Pevonedistat Enantiomeric resolution of racemic 23 was achieved using enzyme-catalyzed acetylation with Candida antarctica lipase B on acrylic beads (19). The desired enantiomer 23 was separated from acetate 24, which possessed the opposite stereochemistry, in high yield (88% of theory) and high enantiomeric purity (>98% ee). Subsequent deprotection of 23 with TBAF yielded 9, which was then converted to 19 using the same chemistry as described in Scheme 3. It was desired to avoid the protection/deprotection protocol used in the first synthesis to differentiate the hydroxyls of 19. Since it would be difficult to functionalize the secondary in the presence of the primary alcohol, we sought to identify conditions for selective sulfamoylation at the desired primary position. Considering the steric hindrance of the secondary versus the primary alcohol, we studied bulky reagents to differentiate the two positions. We ultimately discovered that reagent 25 selectively sulfamoylated the primary hydroxyl group, and provided pevonedistat in 81% yield after hydrolysis. 9


This new process possessed a number of advantages: • • • •

fewer steps than the first synthesis (13 vs 15). higher overall yield (6.8 % vs 5.5%). this route provided robust access of to up to 50 g of key cyclopentene diol 9 in one synthetic sequence. it enabled the synthesis of multigram quantities of pevonedistat.


These two synthetic routes completed the Medicinal Chemistry synthesis of pevonedistat.

Conclusion The modular character of the synthetic routes described in Schemes 3-4 enabled modifications on both carbocyclic and purine-like portions of the molecule, helping to explore SAR of the series, and select pevonedistat for IND-enabling studies. The second generation route described in Scheme 4 provided a ~ 20 gram supply of pevonedistat for pharmacological and early toxicological evaluation of NAE inhibition. The main improvement over the initial approach (described in Scheme 3) was a robust and scalable supply of rac-23 cyclopentene. This second generation synthesis however had several segments with poor atom economy. While resolution of rac-23 provided the desired enantiomer in excellent enantiomeric purity, inability to utilize the undesired cyclopentene enantiomer 24 significantly reduced the overall yield. Also, the melding of pyrrolopyrimidine 7 with epoxide 16 required protecting group manipulations, and the removal of the undesired 2-hydroxyl group in PMP-acetal 17 which added two synthetic steps requiring chromatography purifications. While this was a suitable synthesis for Medicinal Chemistry purposes, the low yield, number of synthetic steps and need for multiple chromatography purifications prevented further scaling to provide larger quantities of pevonedistat. Therefore, a search for new, GLP/GMP-compatible routes was warranted to provide supply of pevonedistat for clinical development and is discussed in the next chapter.

Acknowledgments If it takes a village to raise a child it most certainly takes a team to discover and develop a drug candidate. We would like to express our thanks all those involved in the NAE program.

10 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.

References 1. 2. 3.


4. 5. 6.

7.

8.

9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Bedford, L.; Lowe, J.; Dick, L. R.; Mayer, R. J.; Brownell, J. E. Nat. Rev. Drug Discovery 2011, 10, 29–46. Nalepa, G.; Rolfe, M.; Harper, J. W. Nat. Rev. Drug Discovery 2006, 5, 596–613. Soucy, T. A.; Dick, L. R.; Smith, P. G.; Milhollen, M. A.; Brownell, J. E. Genes Cancer 2010, 1, 708–716. Haas, A. L.; Rose, I. A. J. Biol. Chem. 1982, 257, 10329–10337. Bohnsack, R. N.; Haas, A. L. J. Biol. Chem. 2003, 278, 26823–26830. Ciavarri, J.; Dick, Langston, S. P., The Discovery of First-in-Class Inhibitors of the Nedd8 Activating Enzyme (pevonedistat, TAK-924, MLN4924) and the Ubiquitin Activating Enzyme (TAK-243) Compr. Med. Chem. III Submitted for publication. Brownell, J. E.; Sintchak, M. D; Gavin, J. M.; Liao, H.; Bruzzese, F. J.; Bump, N. J.; Soucy, T. A.; Milhollen, M. A.; Yang, X.; Burkhardt, A. L.; Ma, J.; Loke, H. K.; Lingaraj, T.; Wu, D.; Hamman, K. B.; Spelman, J. J.; Cullis, C. A.; Langston, S. P.; Vyskocil, S.; Sells, T. B.; Mallender, W. D.; Visiers, I.; Li, P.; Claiborne, C. F.; Rolfe, M.; Bolen, J. B.; Dick, L. R. Mol. Cell 2010, 37, 102–111. Soucy, T. A.; Smith, P. G.; Milhollen, M. A.; Berger, A. J.; Gavin, J. M.; Adhikari, S.; Brownell, J. E.; Burke, K. E.; Cardin, D. P.; Critchley, S.; Cullis, C. A.; Doucette, A.; Garnsey, J. J.; Gaulin, J. L.; Gershman, R. E.; Lublinsky, A. R.; McDonald, A.; Mizutani, H.; Narayanan, U.; Olhava, E. J.; Peluso, S.; Rezaei, M.; Sintchak, M. D.; Talreja, T.; Thomas, M. P.; Traore, T.; Vyskocil, S.; Weatherhead, G. S.; Yu, J.; Zhang, J.; Dick, L. R.; Claiborne, C. F.; Rolfe, M.; Bolen, J. B.; Langston, S. P. Nature 2009, 458, 732–736. Milhollen, M. A.; Traore, T.; Adams-Duffy, J.; Thomas, M. P.; Berger, A. J.; Dang, L.; Dick, L. R.; Garnsey, J. J.; Koenig, E.; Langston, S. P.; Manfredi, M.; Narayanan, U.; Rolfe, M.; Staudt, L. M.; Soucy, T. A.; Yu, J.; Zhang, J.; Bolen, J. B.; Smith, P. G. Blood 2010, 116, 1515–1523. Monfette, S.; Fogg, D. E. Chem. Rev. 2009, 109, 3783–3816. Auge, J.; Gil, R.; Kalsey, S.; Lubin-Germain, N. Synlett 2000, 6, 877–879. Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem. Soc. 1981, 103, 2127–2109. Williams, D. R.; Nold, A. L.; Mullins, R. J. J. Org. Chem 2004, 69, 5374–5382. Ye, D.; Fringuelli, F.; Piermatti, O.; Pizzo, F. J. Org. Chem 1997, 62, 3748–3750. Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perkin Trans. 1. 1975, 16, 1574–1585. Toshima, K.; Jyojima, T.; Miyamoto, N.; Katohno, M.; Nakata, M.; Matsumura, S. J. Org. Chem 2001, 66, 1708–1715. Lubineau, A.; Auge, J.; Lubin, N. Tetrahedron Lett. 1991, 32, 7529–7530. An, G-I.; Rhee, H. Nucleosides, Nucleotides Nucleic Acids 2002, 21, 65–72. Johnson, C. R.; Bis, S. J. Tetrahedron Lett. 1992, 33, 7287–7290. 11


Synthesis of a Potent NAE Inhibitor: Pevonedistat - ACS Publications

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