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Chapter 4
Discovery and Chemical Development of JNJ-50138803, a Clinical Candidate BACE1 Inhibitor Harrie J. M. Gijsen,1 Jinguang Lin,2 and Yannis Houpis*,1 1Janssen
Research & Development, Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium 2STA Pharmaceuticals, A WuxiApptec Company, 589 N. Yulong Rd., Changzhou, 213127, China *E-mail:
[email protected] BACE1 inhibition is hypothesized to be a potential disease-modifying treatment for Alzheimer’s Disease (AD). Multiple BACE1 inhibitors have progressed into clinical trials, but several have been terminated due to off-target side effects or lack of therapeutic efficacy, presumably due to targeting an AD population progressed too far into the disease. The need for a next generation of BACE1 inhibitors has led to the discovery of JNJ-50138803. The initial medicinal chemistry synthesis is presented, as well as the evolution to more scalable synthesis routes, incorporating multiple improvements. This has culminated into a synthesis route, proven suitable to prepare a multikilogram GMP batch.
Introduction Alzheimer’s Disease (AD) is the most common form of dementia and leads to neurodegeneration of the brain, causing progressive problems with memory, thinking, and behavior. It is a devastating disease with enormous impact, not only on the patient but also on family and caregivers. With an aging population, its prevalence is increasing and, currently, no medication is available that can prevent, slow, or stop the disease progression. A defining pathological characteristic of AD is the presence of amyloid plaques and tau tangles in the brain. Data regarding both
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genetic causes and sporadic disease support targets and pathways associated with amyloid pathology as a key therapeutic focus. The β-amyloid cleaving enzyme-1 (BACE1) is the first and rate-limiting step in the formation of β-amyloid peptides and therefore has been a prime target for drug development since its discovery in 1999 (1). The evolution of BACE1 inhibitors toward brain-penetrant drugs able to significantly reduce β-amyloid production in the brains of animals and humans has been covered in multiple reviews (2, 3). Several compounds have moved into advanced Phase 2b or 3 clinical trials, including verubecestat (4), lanabecestat (5), elenbecestat (6), and atabecestat (7) (Figure 1).
Figure 1. Lead BACE inhibitors in the clinic. AD drug development has proven to be extremely challenging, with disappointing results in all late-stage clinical trials thus far (8). Among those, the studies of amyloid interventions have mostly targeted relatively late stages of disease, which may have been too late to successfully intervene in the disease process. For BACE, the recent termination of the Phase 3 trials of verubecestat (9, 10) as well as lanabecestat (11) based on futility analyses can be seen as examples of this. BACE1 inhibition has been shown to more effectively suppress initiation of β-amyloid pathology than progression, stressing the need for early intervention to be therapeutically efficacious (12). In line with this, several ongoing trials with BACE inhibitors are targeting an earlier, even asymptomatic population (13). Considering the need for long-term treatment in a relatively healthy but elderly population, compounds will require a very safe side-effect profile. With multiple BACE compounds already terminated due to off-target side effects, atabecestat being the most recent example (14), the need for next-generation BACE inhibitors remains strong.
Discovery of JNJ-50138803 By the time we initiated our BACE1 inhibitor program, researchers had identified several key pharmacophoric elements required for the design of an optimal BACE1 inhibitor (2, 3), as evidenced by the structural similarities of 92
the inhibitors in Figure 1. These key pharmacophoric elements play important roles in the optimal interactions between the inhibitor and the enzyme. For example, the presence of an amidine-containing heterocycle in the part of the BACE1 inhibitor known as the warhead achieves an optimal hydrogen-bond interaction with the two aspartate residues in the catalytic site of the aspartic protease. A tertiary carbon connected to the amidine endocyclic nitrogen atom, as depicted with an asterisk in Figure 2, allows for efficient filling of the enzyme pockets by positioning the aryl substituents directly into the S1 and S3 pockets and the methyl substituent into the S2′ pocket. The amide linker between the two (hetero)aromatic rings also helps direct the heteroaryl ring deeply into the S3 pocket, with the amide-NH additionally making a hydrogen bond to the Gly230 backbone carbonyl of the protein. The requirement of central penetration necessitates modulation of the amidine pKa to a value of 6–8, which will still allow protonation at the site of actual enzyme inhibition. In addition, the modulation of pKa has been proven to be key in mitigating liabilities such as cardiovascular issues via inhibition of the human ether-à-go-go-related gene (hERG) potassium ion channel inhibition and drug–drug interactions (especially cytochrome P450 2D6 inhibition).
Figure 2. Pharmacophore of BACE1 inhibitors.
Our initial work (15) targeted a combination of all of these characteristics and resulted in amino-piperazinone 1 (Figure 3). Suboptimal brain penetration prompted further modification of the warhead, with a 1,4-oxazine providing a template with an intrinsically reduced amidine basicity, as well as carbon atoms, which would allow for modification via further substitution (16). While 1,4-oxazine analog 2 was a potent BACE1 inhibitor in vitro (IC50 22 nM), the compound was not able to reduce central amyloid beta levels in mice due to poor brain penetration, which could be attributed to strong P-glycoprotein (PgP)-mediated efflux. 93
Figure 3. Medicinal chemistry evolution toward JNJ-50138803. Further reduction of the amidine pKa by substitution of the 1,4-oxazine with electron withdrawing groups (EWGs) initially led to 2-fluoro-1,4-oxazine 3 (16). This compound displayed a reduced PgP-mediated efflux and resulted in highly effective reduction of β-amyloid levels in the brain and cerebrospinal fluid (CSF) of mice and dogs, respectively. However, progression of 3 was halted due to a still significant hERG inhibition observed in a functional electrophysiology experiment, the hERG patch clamp assay (56% at 3 µM). Subsequent in vivo studies in an anesthetized guinea pig showed that this hERG inhibition translated 94
into an unacceptable QTc prolongation in the electrocardiogram, leading to an insufficient safety margin for 3 (17). In addition, there were concerns around the chemical stability of the fluoro substituent. A number of variations were synthesized bearing alternative EWGs, providing warheads covering a range of pKa’s, as exemplified by compounds 4–7. CF3-substituted 4 had a pKa similar to 3 and maintained enzymatic and cellular potency. The less electron-withdrawing CHF2 substituent resulted in a pKa of 8.4 for 5, translating into an increased PgP-mediated efflux. Adding multiple EWGs as in 6 reduced the amidine pKa to 6.8, which led to a reduced cellular potency, as measured by inhibition in cells of the formation of the highly amyloidogenic peptide of 42 amino acids long: Aß42. The olefinic CF2-substituted 7 resulted from a side reaction in the synthesis route toward 4 (vide supra) and was an intermediate in the synthesis of 5. This compound showed a significantly reduced enzymatic potency, probably related to an altered and suboptimal conformation of the 1,4-oxazine ring. An optimal balance of potency, minimal efflux, and reduced hERG inhibition was found in the CF3-substituted 1,4-oxazines, and multiple analogs of 4 with various S3 pocket–targeting heteroaryl groups were prepared, including compounds 8 and 9 (Figure 3). Compound 8 was co-crystallized with the BACE1 enzyme, and the crystal structure of 8, solved at 1.94 Å resolution, is shown in Figure 4 (PDB code 6E3Z). This structure confirmed a binding mode similar to that seen for other BACE1 inhibitors, with a strong network of interactions of the catalytic aspartate dyad and the amidine moiety in 8 and optimal filling of the S1 and S3 pockets.
Figure 4. Crystal structure of 8 in BACE1 (amino acids 1 - 454) Protein Data Bank (PDB) code 6E3Z. 95
Further in vitro and in vivo profiling of the most promising compounds led to the selection of CF3-substituted 1,4-oxazine 9, or JNJ-50138803, as the preferred candidate (17). The PK/PD relationship of 9 in dog is shown in Figure 5, and these data were modeled to provide an estimated EC50 to reduce ß-amyloid peptide Aβ42 levels in dog CSF of 105 ng/mL (18). The minimal hERG patch clamp inhibition for 9 of 22% at 3 µM resulted in a significantly improved cardiovascular safety margin for 9 compared with 3, and toxicity studies rendered 9 sufficiently safe to progress to GMP synthesis of JNJ-50138803 in order to prepare for GLP toxicity studies and clinical trials (17).
Figure 5. Beagle dog plasma levels of 9 and effect on CSF Aβ 1-42 levels on day 1 and 6, after repeated oral dosing (fasted state, once daily dosing). Avg + SEM, n = 6/dose group. MS = MesoScale.
Medicinal Chemistry Synthesis of JNJ-50138803 A key structural feature of all leading BACE1 inhibitors, including JNJ-50138803, is a tertiary amine chiral center (2, 3). In our case, this is introduced by the amino acid intermediate 12 (Scheme 1), which was readily obtained from acetophenone 10 via a Strecker reaction and subsequent hydrolysis of the resulting α-aminonitrile 11. The acid group of 12 provided a versatile entry toward multiple variations of substituted BACE1 inhibitor warheads in the early phase of lead optimization, including all variations present in compounds 1–7 (Figure 3). At this point in the program, the desired stereochemistry of the tertiary carbon center was already established, and racemic 12 was resolved easily on a 100 g scale via chiral supercritical fluid chromatography (SFC) to obtain the desired enantiomer 13. Cyclization of amino acid 13 to morpholinedione 14 was achieved in moderate yield by acylation of the amino group with chloroacetyl 96
chloride followed by lactonization. This intermediate proved to be a useful precursor to prepare a range of substituted morpholino warheads.
Scheme 1. Initial medicinal chemistry synthesis route of JNJ-50138803. The trifluoromethyl group was introduced by the addition of the Ruppert−Prakash reagent (TMS-CF3) (19) in the presence of a catalytic amount of tetra-n-butylammonium difluorotriphenylsilicate (TBAT) to 14, to afford 15 as an inseparable and unassigned 3:1 diastereomeric mixture. The hydroxyl group was replaced with hydrogen by chlorination and subsequent reductive dechlorination using metallic zinc in acetic acid to provide 18 in 4:1 diastereomeric ratio, favoring the desired stereochemistry. The reproducibility of this reaction was poor, forming various amounts of chlorofluoro-eliminated 17 as a side product depending on the zinc source. While this allowed us to explore -CHF2- and =CF2-substituted analogs such as 5 and 7, respectively (Figure 3), a more reproducible outcome was clearly desirable. After careful analysis of the reaction conditions and zinc sources, the presence of copper impurities was found to be responsible for the formation of 97
17. Thus, using fresh zinc dust in acetic acid at 100 °C resulted in the selective reductive dehalogenation to 18, whereas using zinc-copper couple in acetic acid at room temperature resulted in almost quantitative formation of the chloro-fluoro elimination product 17. Conversion of amide 18 into amidine 20 was achieved by sequential thionation with P2S5, followed by aminolysis of the intermediate thioamide 19 with ammonia in methanol in a closed, pressurized vessel at elevated temperature. Aniline 21 was obtained in a moderate yield (40–60%) via amination of the bromoarene 20 under copper-catalyzed, Buchwald-type conditions using benzophenone imine as the nitrogen source and 1,2-dimethylethylenediamine (DMEDA) as metal chelator. A significantly higher yield (90%) was achieved using a copper-catalyzed reaction employing sodium azide, in which the intermediate aryl azide was immediately reduced to the corresponding aniline through the use of stoichiometric Cu(I)I. In the last step, acylation of the aniline 21 with 5-cyanopicolinic acid was achieved selectively using the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) (20) to produce JNJ-50138803.
Alternative Routes to JNJ-50138803 While the initial medicinal chemistry route enabled the synthesis of gram quantities of JNJ-50138803 as well as the exploration of other EWG-substituted analogs, the number of steps (11) and the low overall yield (
