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Chemistry and Pharmacology of Drug Discovery
12. O'Brien, N. A.; Huang, H. K. T.; McDermott, M. S. J.; Madrid, A. M.;
Luo, T.; Ayala, R.; Issakhanian, S.; Gong, K. W.; Lu, M.; Zhang, J.; et
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significant combined activity with approved and novel breast cancer–
targeted therapies. Mol. Cancer Ther. 2022, 21, 751–761.
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1Orig1s000MultidisciplineR.pdf
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Abdulrasool, L. I.; Walker, L.; Endres, C. J. Evaluation of safety and
clinically relevant drug–drug interactions with tucatinib in healthy
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15. Sun, H.; Cardinal, K. A.; Wienkers, L.; Chin, A.; Kumar, V.; Neace,
C.; Henderson, C.; Endres, C. J.; Topletz-Erickson, A.; Regal, K.; et al.
Elimination of tucatinib, a small molecule kinase inhibitor of HER2, is
primarily governed by CYP2C8 enantioselective oxidation of gemdimethyl. Cancer Chemother. Pharmacol. 2022, 89, 737–750.
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metastatic breast cancer. Eur. J.
Cancer 2021, 154, 175–189.
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Hurvitz, S. A.; Lin, N. U.; Borges, V.; Abramson, V.; Anders, C.; et al.
Tucatinib, trastuzumab, and capecitabine for HER2-positive metastatic
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Tazemetostat (Tazverik): An EZH2 Inhibitor
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for Treatment of Epithelioid Sarcoma and
Follicular Lymphoma
Ruheng Zhao and
Timothy A. Cernak
10
1. Background
Enhancer of zeste homolog 2 (EZH2) is a core component of the polycomb repressive
complex 2 (PRC2) that imparts posttranslational modifications (PTMs) to histone
proteins, thereby regulating gene expression. Its critical roles in cellular processes such as
proliferation, apoptosis, and senescence have been well established.
cellular processes, overexpression, or mutations in EZH2 have been implicated in various
cancer-associated phenotypes, including tumorigenesis, metastasis, altered metabolism,
drug resistance, and immune evasion.
Due to the vast implications of EZH2 in cancer progression and maintenance,
pharmaceutical interventions targeting EZH2 emerged as a promising therapeutic
strategy. However, the development of specific and potent inhibitors has been
Chemistry and Pharmacology of Drug Discovery, First Edition. Edited by Jie Jack Li.
© 2025 John Wiley & Sons, Inc. Published 2025 by John Wiley & Sons, Inc.
1
Beyond these basic
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challenging, presenting obstacles such as achieving target specificity, managing off-target
effects, and optimizing pharmacokinetics.
Tazemetostat (1), developed by Epizyme (later acquired by Ipsen), represents a
paradigm shift in the field of EZH2 targeting. Unlike its predecessor, DZNep (2), which
inhibits EZH2 in a non-direct manner, tazemetostat (1) emerged as the first direct and
selective inhibitor of EZH2. Its development not only highlighted the importance of
methylation in PTMs but also is a beautiful art adopting “Magic Methyl” effect in drug
discovery. This phenomenon suggests that the strategic incorporation of a small methyl
group can substantially enhance drug properties such as potency, selectivity, and
pharmacokinetics by inducing conformational changes in the target protein.
In 2020, tazemetostat (1), marketed as Tazverik, received accelerated approval
for two clinical indications:
1. Treatment of metastatic or locally advanced epithelioid sarcoma, a rare and
aggressive soft tissue malignancy with limited treatment options.
2. Therapy for relapsed or refractory follicular lymphoma in patients harboring
EZH2 mutations who had undergone at least two prior systemic treatments.
Chemistry and Pharmacology of Drug Discovery
3, 4
5
5
While the approval of tazemetostat (1) represents a significant milestone in
EZH2-targeted therapies, a deeper understanding of the drug discovery process and its
underlying principles is essential for advancing the field of medicinal chemistry.
2. Pharmacology
2.1. Epigenetic Regulation in Gene Expression
Epigenetic regulation is foundational to the hallmarks of cancer. Disruptions in epigenetic
patterns or chromatin configurations can induce oncogenic properties or potentiate gene
repression, both propelling tumorigenesis. Central to this is the realm of PTMs, which
modulate gene expression without amending the DNA sequence. Identified PTMs include
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acetylation, methylation, ubiquitination, phosphorylation, and glycosylation. These
modifications are being targeted in drug development efforts to deliver more precise and
effective therapies.
Lysine methylation, one of the most common histone PTMs, plays a key role in
regulating gene expression.
specific lysine residue being modified and the state of methylation. Methylation states
involve the addition of one (me1), two (me2), or three (me3) methyl groups to the amine
tails of lysine residues in histones. The different methylation states instruct different
chromatin structure and the accessibility of transcription factors and other regulatory
proteins to DNA, ultimately influencing gene expression.
Chapter 10. Tazemetostat (Tazverik)
6
The impact of lysine methylation depends on both the
2.2. Posttranslational Modifications of EZH2 and
Cancer Progression
The nuanced world of methylation requires a series of enzymes for reading, writing, and
erasing the methyl code. A critical player here is the polycomb repressive complex 2
(PRC2), a multi-protein epigenetic regulatory complex, orchestrating transcription
regulation. EZH2, the catalytic engine of PRC2 and a representative of the Protein Lysine
Methyltransferase family, steers cellular processes like cell cycle progression, DNA
repair, and autophagy. Depending on context, EZH2 can suppress or co-activate
transcription either in a PRC2-dependent manner or through a PRC2-independent
pathway. A prime example is the methylation of Lys-27 on histone 3 (H3K27). A hotspot
mutation in the SET domain of EZH2 is Y641X (X = F, N, S, C, or H). The SET domain
responsible for transferring methyl groups to H3K27, and this mutation alters its catalytic
function.
EZH2’s specificity, escalating H3K27 di-methylation to tri-methylation.
H3K27me3, associated with gene repression, plays an instrumental role in cellular
decisions, often culminating in oncogenic outcomes. The mutation’s molecular genesis
possibly arises from the loss of tyrosine, reshaping the enzyme’s active site and
facilitating enhanced methylation.
7
This mutation, found in cancers such as DLBCL and melanoma, recalibrates
8
The resulting
2.3. Progress and Mechanisms of Action for EZH2
Inhibitors
EZH2’s SET domain functions by transferring methyl groups from S-adenosyl-
methionine (SAM) to target lysines. DZNep (2), the pioneer EZH2 inhibitor, indirectly
inhibits EZH2 by increasing levels of SAH, a byproduct that represses SAM-dependent
activity.
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Over recent years, potent SAM-competitive inhibitors, in addition to
tazemetostat (1), like GSK126 (3), PF-06821497 (4), and CPI-1205 (5), been
developed.
pocket, aiming to reduce H3K27 trimethylation and reverse the gene repression
associated with EZH2 mutation. New-age drugs also seek to perturb the PRC2 complex’s
integrity or triggering EZH2 degradation. However, to date, only tazemetostat (1) has
been approved approval for specific cancer types.
9–11
Chemistry and Pharmacology of Drug Discovery
These drugs, by binding to the SET domain, occupy the SAM binding
3. Structure–Activity Relationship (SAR)
3.1. The Discovery of Initial Hit Using HighThroughput Screening
The role of epigenetic regulator EZH2 in cancer progression has garnered significant
attention, highlighting the urgent need for targeted therapies. To address this, Epizyme
embarked on the discovery of potent EZH2 inhibitors. Utilizing high-throughput
screening (HTS) on a 175,000-compound library targeting the wild-type PRC2 complex,
they pinpointed an initial hit.
additional screenings and a pyridone-containing compound 6 emerged as a potential
1
Subsequent structural similarity assessments led to
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inhibitor. Despite its potency, further attempts to use this compound in cellular assays
was limited by poor solubility and low oral bioavailability.
Chapter 10. Tazemetostat (Tazverik)
3.2. Lead Optimization
The solubility issue was tackled first, by introducing polarity to the molecule. Polar
substitutions at the 6-position of the 7-azaindazole can be tolerated. Modifications were
made to the corresponding position, leading to a series of compounds with improved
solubility, oral bioavailability, and cellular activity. From the numerous synthesized
variants, it was found that the basic amine and small nonpolar group can be well tolerated
at this position without significant loss of potency, and the amine can link directly or via
an aryl spacer. Ultimately, a morpholine at the 4-position of the phenyl ring was chosen
as the candidate 7 because it significantly improved solubility, oral bioavailability, and
ensuring moderate clearance and modestly increase cellular activity. While considering
the ease of synthesis and the modest potency shift with this morpholine substitutions,
hydrogen or halogen was employed for subsequent SAR studies.
12
Considering the structure variations between the initial hits and compound 6
generated from the extended screening post-clustering, the central difference lies in the
core scaffold. Thus, the core pyrazolopyridine scaffold underwent an in-depth evaluation
(Table
1). Different heterocycle that bearing different electron densities and distinct
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target engagement activities was evaluated. A shift to indazole (9) enhanced potency,
while benzimidazole (10) and triazole (11) caused significant potency loss, highlighting
the loss of potency may attribute to the formation of intramodular hydrogen bond
between the amide and ortho N atom. Furthermore, the optimal substitution occurs at the
4-position when the amide is ortho substituted to the fused ring system. Taking both the
intramodular hydrogen bond and substitution position into account, conformation offers
the most plausible explanation for compounds performance. Specifically, a planer
conformation of amide relative to the ring adversely affects activity.
Chemistry and Pharmacology of Drug Discovery
Compound R EZH2 IC
8 3.1 ± 1.0
9 1.1 ± 0.3
10 >50
11 >50
12 22 ± 6
Table 1. Optimization of 5,6-bicylic core
50
(μM)
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Chapter 10. Tazemetostat (Tazverik)
The N1-substitution on the core scaffold also underwent evaluation, when
adopting pyrazolopyridine as the core motif, a bulkier, hydrophobic isopropyl group on
13 outperformed 10-fold than ethyl analog 14 and 100-fold than methyl counterpart 15,
showcasing the importance of substituent size. Furthermore, non-polar substituents were
generally more favorable than polar groups. However, when assessing more broader
range of synthesized analogs, further improvements were only marginal, particularly
when weighing the balance between physicochemical and drug-like attributes.
Since progress had stalled on the optimization of the nonpolar bulky substitution
on the indazole, the iterative optimization strategy was used, which returning to the
earlier phase to revisit and reevaluate the core scaffold.
The SAR data previously showed that planarity restricted by hydrogen bond or
lack of ortho substitution between the amide and the core rings diminished potency.
Recognizing this, the researchers opted for a less planar core scaffold, which could
potentially diversify the binding profile, and offering new avenues for functional group
additions. Accordingly, the disubstituted aniline structure was evaluated, achieved by
opening the 5-membered pyrazole ring (Table 2).
The concept of the “magic methyl” effect is fundamental to understanding the
optimizations in the lead compound. When a methyl group was introduced at the R
position, there was a significant enhancement in potency—a 1000-fold improvement
compared to the compounds 16 and 17.
Further understanding of the “magic methyl” effect’s mechanism, garnered from
reputable databases such as the Protein Data Bank and the Cambridge Structural
Database, elucidated its role beyond simple pocket occupation. The introduction of a
methyl group at the R
position instigated a conformational shift in the secondary amide,
1
twisting it away from a planar disposition and introducing a torsion angle in the range of
60º–140º or 220º–300º. Intriguingly, the influence of this methylation was not confined to
the amide. Computational studies highlighted that the ortho methylation induced
significant conformational changes in the disubstituted aniline. In this context, the methyl
group, precisely positioned ortho, serves a dual purpose to enhancing molecular potency:
it acts as a conformational lock to avoid planar structures for both amide and aniline. In
the presence of the methyl group, the molecule adopted a twisted conformation,
contrasting with the planar orientation that was preferred in its absence.
1
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Further refinements focused on the other substituents of the disubstituted aniline
(Table
presented a modest advantage over the methyl group (18), resulting in a 10-fold increase
in potency compared to monosubstituted aniline (20). The exploration of the R
substituent took cues from previous studies on N1-substituents of the indazole ring.
While the preference for bulkier groups was also observed, the removal of the rigid
indazole ring widened the possibilities, making polar groups an attractive alternative with
performances comparable to their nonpolar counterparts. Ultimately, the THP moiety
(17) was chosen for the R3 position, given its favorable influence on other
physicochemical properties, notably reducing logD and decreasing metabolic clearance.
2). The R
Chemistry and Pharmacology of Drug Discovery
substituent was eventually settled on ethyl derivative 17, which
2
Compound R1 R2 R3 R4 EZH2 IC50
3
16 H Et Cl 14 ± 2
17 Me Et Cl 0.01 ± 0.01
18 Me Me Br 0.03 ± 0.02
19 Me Me Br 0.03 ± 0.02
20 Me H Br 0.5 ± 0.2
21 Me Me H Cl 2.7 ± 1.2
Table 2. Optimization of substituted benzene core
Despite the enhanced potency achieved through extensive optimization, the
molecule still faced challenges regarding bioavailability. Insights from HTS brought the
4,6-dimethyl pyridone into the molecule, which was then assessed to identify further
optimization sites (Table
3). During this exploration, it became clear that preserving the
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integrity of the molecule’s “warhead” was crucial. Any modifications, such as the
addition of a benzylic carbon (23) or the methylation of the amide 22, drastically reduced
potency, with reductions ranging from 10- to 100-fold per alteration. Maintaining the 4,6dimethyl substitutions was essential; removing a single methyl group (24, 25) diminished
potency 10-fold, and removing both resulted in a significant 200-fold drop (26).
To balance the physicochemical properties, benzyl morpholine was finally
incorporated, leading to the creation of the compound now recognized as tazemetostat
(1). This optimized molecule not only showcases superior potency, minimal clearance,
and impressive bioavailability but also demonstrated its effectiveness in subsequent
clinical trials.
Chapter 10. Tazemetostat (Tazverik)
Compound R1 R2 R3 R4 EZH2 IC50
17 Me Me H H 0.01 ± 0.01
22 Me Me H Me 0.2 ± 0.1
23 Me Me Me H 3 ± 1
24 Me H H H 0.2 ± 0.01
25 H Me H H 0.1 ± 0.04
26 H H H H 3.3 ± 1.2
27 Me CF
3
H H 0.03 ± 0.01
Table 3. Optimization of pyridone warhead
4. Pharmacokinetics and Drug Metabolism
Epizyme marshaled a multidisciplinary team of scientists, dedicating many years of
focused research to develop tazemetostat (1). The challenge lay not just in achieving
potent inhibitory action but also in ensuring optimal physicochemical properties for oral
bioavailability.
Tazemetostat (1) is administered in its HBr salt form. The recommended oral
dose is 800 mg, taken twice daily. This dosing regimen was established based on rigorous
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