Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00515

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

Full text of DOI:10.1021/acs.jmedchem.6b00515

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Article
Design and synthesis of pyridone containing 3,4-dihydroisoquinoline-1(2H)-
ones as a novel class of enhancer of zeste homolog 2 (EZH2) inhibitors
Pei-Pei Kung, Eugene Rui, Simon Bergqvist, Patrick Bingham, John Braganza, Michael R. Collins,
Mei Cui, Wade Diehl, Dac M Dinh, Connie Fan, Valeria R. Fantin, Hovhannes J. Gukasyan, Wenyue
Hu, Buwen Huang, Susan Kephart, Cody T Krivacic, Robert A. Kumpf, Gary Li, Karen A. Maegley,
Indrawan McAlpine, Lisa Nguyen, Sacha Ninkovic, Martha A. Ornelas, Michael Ryskin, Stephanie Scales,
Scott C. Sutton, John H Tatlock, Dominique Verhelle, Fen Wang, Peter A Wells, Martin Wythes, Shinji
Yamazaki, Brian Yip, Xiu Yu, Luke Zehnder, Wei-Guo Zhang, Robert A. Rollins, and Martin P. Edwards
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Aug 2016
Downloaded from http://pubs.acs.org on August 11, 2016
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Yu, Xiu; Pfizer Global Research and Development La Jolla
Zehnder, Luke; Pfizer Global Research and Development La Jolla
Zhang, Wei-Guo; Pfizer Global Research and Development
Rollins, Robert; Pfizer Inc, Oncology
Edwards, Martin; Pfizer Global Research and Development La Jolla
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Design and synthesis of pyridone containing 3,4ꢀ
dihydroisoquinolineꢀ1(2H)ꢀones as a novel class of enhancer of zeste
homolog 2 (EZH2) inhibitors
a
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PeiꢀPei Kung *, Eugene Rui , Simon Bergqvist , Patrick Bingham , John Braganza , Michael
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Collins , Mei Cui , Wade Diehl , Dac Dinh , Connie Fan , Valeria R. Fantin , Hovhannes J.
Gukasyan , Wenyue Hu , Buwen Huang , Susan Kephart , Cody Krivacic , Robert A. Kumpf ,
Gary Li , Karen A. Maegley , Indrawan McAlpine , Lisa Nguyen , Sacha Ninkovic , Martha
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Ornelas , Michael Ryskin , Stephanie Scales , Scott Sutton , John Tatlock , Dominique
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Verhelle , Fen Wang , Peter Wells , Martin Wythes , Shinji Yamazaki , Brian Yip , Xiu Yu ,
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Luke Zehnder , WeiꢀGuo Zhang , Robert A. Rollins , and Martin Edwards
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Oncology Chemistry; Oncology Research Unit; Pharmacokinetics, Dynamics, and
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Metabolism; Pharmaceutical Science Research Enabling Group; Drug Safety Research and
Development;
Pfizer Global Research and Development, La Jolla Laboratories, 10770 Science Center Dr., San
Diego, CA 92121, USA
†
Pfizer Global Research and Development, 401 N. Middletown Rd., Pearl River, NY 10965,
USA
^
Mei Cui passed away on April 11, 2015.
Abstract. A new enhancer of zeste homolog 2 (EZH2) inhibitor series, comprised of a
substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage, has been
designed. A preferential amide torsion was identified for this series via computational analysis
that improved the binding properties of the compounds. Cyclization of the amide linker resulted
in a six member lactam analog, compound 18. This transformation significantly improved the
ligand efficiency/potency of the cyclized compound relative to its acyclic analog. Additional
optimization of the lactamꢀcontaining EZH2 inhibitors focused on lipophilic efficiency (LipE)
improvement which provided compound 31. Compound 31 displayed improved LipE and onꢀ
target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31
also displayed robust in vivo antiꢀtumor growth activity and doseꢀdependent deꢀrepression of
EZH2 target genes.
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INTRODUCTION
Epigenetic information is contained in living cells in many different forms that include
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DNA and histone modifications, nucleosome positioning, and microRNA expression.
Epigenetic dysregulation is a hallmark of many different cancer types, including both
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hematological malignancies and solid tumors.
Among possible histone modifications, lysine
methylation has gained increased attention due to the potential of the modified chromatin
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structures to regulate gene transcription, DNA replication, and DNA repair. This epigenetic
modification is controlled by families of methyltransferase and demethylase enzymes that
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coordinate the addition and removal of methyl groups to histone tails. The aberrant activity of
these epigenetic regulators in diseases such as cancer has made them attractive targets for the
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development of novel cancer therapies.
The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the catalytic
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subunit of the polycomb repressor complex 2 (PRC2). The PRC2 complex has been shown to
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initiate longꢀterm gene silencing by methylating lysine 27 on histone H3 (H3K27). Activating
point mutations, such as Y641N, Y641F, A677G, and A687V, in the catalytic SET (Su[var]3ꢀ9,
enhancer of zeste, trithorax) domain of EZH2 are responsible for the elevated H3K27Me3 levels
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in diffuse large Bꢀcell lymphoma (DLBCL) and follicular lymphoma (FL).
In addition,
several selective EZH2 inhibitors have been shown to effectively kill lymphoma cells bearing
these mutations, demonstrating that EZH2 enzymatic activity and elevated H3K27Me3 level are
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required for cell proliferation and survival in this setting.
In addition to EZH2ꢀmutant
lymphomas, small molecule inhibitors of EZH2 have also been reported to inhibit the
proliferation of tumors that harbor wild type EZH2 but which also contain different somatic
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mutations in subunits of the chromatin remodeling complex. These specific mutations in the
switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex are SNF5/INIꢀ
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1/SMARCB1 in malignant rhabdoid and synovial sarcoma
and ARID1A in ovarian cancer .
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These observations stimulated numerous EZH2 drug discovery programs, and the first examples
of such inhibitors have entered clinical trials for the treatment of diffuse large Bꢀcell lymphoma
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DLBCL), follicular lymphoma (FL), and SNF5/INIꢀ1/SMARCB1 genetically defined solid
tumors.
RESULT AND DISCUSSION
Our internal EZH2 inhibitor research program sought to identify small molecules that
blocked the enzyme’s SET domain catalytic function and therefore inhibited the formation of
trimethylated H3K27. EZH2 inhibitors reported in the literature typically contain a pyridone
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moiety attached via an amide linkage to a bicyclic heteroꢀaryl group [Figure 1, 1 (GSKꢀ126) , 2
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(
UNCꢀ1999) , 3 (EI1) , 4 (CPIꢀ169) ]. Unfortunately, the relatively large number of planar
aromatic rings contained in such structures tends to impair the solubility of the resulting
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compounds.
One notable exception is 5 (EPZꢀ6438, Tazemetostat) which incorporates a
phenyl ring in place of the bicylic group. This compound demonstrated impressive clinical
response and has currently progressed to phase II trials with a recommended dose of 800 mg,
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twice a day (BID). We were interested in identifying potent EZH2 inhibitors bearing a central
phenyl ring that were structurally distinct to those previously reported. We envisioned that the
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new molecules might combine good solubility with favorable pharmacokinetic properties.
We began our lead finding effort by incorporating the dimethylpyridone substituent
common to many potent EZH2 inhibitors into a library containing various substituted
heteroaromatic/phenyl/heterocyclic carboxyamides (Figure 2, total of 181 compounds, clogP
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ranged from ꢀ1 to 3). One compound exhibited encouraging properties and afforded a
measurable IC50 value in a lowerꢀthroughput biochemical assessment (compound 6, Table 1).
Accordingly, this compound was selected for additional modifications with the goal of further
improving EZH2 inhibition potency.
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As shown in Table 1, the diꢀsubstituted molecule 6 displayed improved EZH2 inhibition
properties relative to the monoꢀsubstituted analogs 7 and 8 (Table 1). Encouragingly, inclusion
of metaꢀethoxy and metaꢀisopropoxy substituents in the inhibitor designs further improved
inhibition potency (compounds 9 and 10, Table 1). Interestingly, replacement of the orthoꢀ
methyl contained in 6 with a variety of other substituents, such as F, OCH , and OH, afforded
3
compounds with significantly weaker EZH2 inhibition properties (compare 6 to 11, 12, and 13).
Of the additional ortho substituents investigated, only the Cl substituent retained acceptable
EZH2 inhibition levels (compound 14, Table 1). A similar trend was also observed with the
metaꢀisopropoxy substituent (compared 10 to 15, Table 1). These observations suggested that
the torsional angle between the phenyl group and the amide carbonyl moiety plays an important
role in EZH2 inhibition potency.
Small molecule crystal data from the Cambridge Crystallographic Database revealed a
dependence of the torsion angle between the carbonyl and the phenyl ring as a function of the
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size and polarity of the R substituent (Figure 3). A regression analysis was performed using
this distribution of conformational propensities and IC values for compounds 6, 8, and 11ꢀ14.
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This analysis indicated 50 ± 5 degrees as the optimal angle between the carbonyl and the phenyl
ring. This single descriptor was able to explain 96% of the variance in binding affinity
(p<0.0001) for this data set. The influence of this torsional angle on EZH2 inhibition can be
rationalized by three nonꢀexclusive hypotheses: (1) the linker orients the dimethyl pyridone and
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phenyl rings into an optimal binding geometry, (2) the carbonyl oxygen makes an important
hydrogen bond with a protein hydrogen bond donor that is located approximately 50 degrees
above or below the plane of the phenyl ring, (3) the NꢀH of the amide linker makes an important
hydrogen bond with a protein hydrogen bond acceptor that is located approximately 50 degrees
above or below the plane of the phenyl ring.
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To test these hypotheses, we explored rigidifying the inhibitor’s conformation in the
preferred binding mode as a means to improve EZH2 affinity. We initially employed a 7ꢀ
membered lactam in the new inhibitor design since such inclusion maintained the critical amide
torsion angle near the 50 degree optimum (compound 16, Table 2) (see Table 1S in
supplementary material for torsional angle analysis of the carbonyl moiety present in 7ꢀ
membered lactam). Encouragingly, 16 exhibited dramatically improved EZH2 biochemical
inhibition potency, roughly 50ꢀfold relative to the unꢀcyclized compound 14 (Table 2).
Importantly, this increased potency could not be simply ascribed to the improved binding
through higher lipophilicity as LipE increased by about a unit as compared to 14. This
observation provided some encouragement for our cyclization strategy. Unfortunately, 16 was
also highly unstable toward human liver microsomes and thus required additional optimization.
We next introduced an oxygen atom into the 7ꢀmembered lactam design with the
expectation that lowering compound lipophilicity should favorably impact human liver
microsome (HLM) stability. The resulting molecule (17, Table 2) exhibited improved HLM
clearance properties but also lost some EZH2 inhibition potency relative to 16 though LipE
minimally decreased (0.2). We next explored removing a methylene moiety from the lactam ring
present in 16 as an alternate strategy to lower lipophilicity and improve HLM stability. The
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resulting 6ꢀmembered lactam 18 displayed the desired improved HLM stability properties and
exhibited EZH2 inhibition activity that was only slightly weaker than that exhibited by 16.
Compounds 17 and 18 both displayed similar HLM clearance values (Table 2).
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However, 18 had a higher lipophilic metabolism efficiency (LipMetE) value (0.36) compared
with 17 (ꢀ0.27), suggesting that 17 contained an inherent metabolic soft spot. We therefore
decided to focus additional optimization efforts on the 6ꢀmembered lactam inhibitor series.
Accordingly, ethyoxy and isopropoxy substituents were used to replace the methoxy moiety
present in 18. The resulting compounds (19 and 20) displayed 3ꢀ10 fold potency improvement in
the biochemical assay relative to 18. Similar improvements were noted in the uncyclized
benzamide series described earlier in this work (Table 1). However, in contrast to the Table 1
SAR, replacement of the Cl group present in 20 with a methyl substituent resulted in a 7ꢀfold
potency loss (compound 21, Table 2). This difference may be due to subtle binding alterations
between the cyclized and unꢀcyclized inhibitor series. In order to confirm the hypothesis of the
importance of ortho substituents (such as Cl and Me) for the EZH2 inhibitory potency,
compound 22 was synthesized, and this molecule lost significant potency relative to either
compound 20 or 21.
One notable feature of the 6ꢀmembered lactam inhibitor series is that the carbonyl torsion
angle is calculated to be constrained to approximately 20° instead of the 50° optimum described
above (Table 1S, supplementary material). In a comparison of matched compound pairs, the
cyclized 6ꢀmembered lactam compounds exhibited consistentlyꢀimproved biochemical potencies
and LipE values relative to the corresponding unꢀcyclized analogs (Figure 4) suggesting a
possible contradiction to the previous hypotheses regarding the carbonyl torsion angle. We
suspect other binding improvements (for example: better contacts with the protein, and/or less
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overall strain in the protein and/or ligand) associated with recognition of EZH2 by this 6ꢀ
membered lactam series compensated for the less than ideal torsional angle in the 6ꢀmembered
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lactam inhibitor design.
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Enthalpy is a measure of changes in protonation state as well as the differential number
and quality of interactions (hydrogen bonds, van der Waals, electrostatic, and strain
contributions) between the protein, ligand, and solvent in the complex and unbound states. The
previous stated hypotheses essentially relate to the enthalpy of binding. To gain insight into the
EZH2 thermodynamic binding components, we performed isothermal titration calorimetry (ITC)
studies on compound 21 and its unꢀcyclized analog 10. As shown in Table 3, enthalpic
contributions dominated the binding of both compounds to EZH2. However, the improved
binding affinity of 21 was driven by an increase in favorable entropic contributions at the
expense of a diminished enthalpic component relative to compound 10.
There are numerous contributions to the energetic parameters determined from ITC
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experiments and it is generally difficult to assign specific binding energy components to a
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particular functional group or residue.
The generally ubiquitous enthalpyꢀentropy
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compensation further hinders efforts at interpretation.
Therefore, while the loss in binding
enthalpy for 21 relative to 10 is consistent with each of the three hypotheses regarding the
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carbonyl conformation, with currently available data only hypotheses (1) and (2) appear to be
candidates for explaining this difference.
Entropy differences arise from changes in solvent structure and residual mobility of the
protein and ligand between bound and unbound states. It is tempting to attribute the changes in
binding entropy to a greater rigidity for Compound 21 due to cyclization; however, as revealed
by data in Figure 3, the exocyclic bond to the carbonyl is already conformationally biased in the
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acyclic analog. This observation is consistent with calculations that estimate that the maximum
ꢀ1
differential conformational entropy contribution to be on the order of 0.14 kcal mol .
Compound 21, with two additional methylenes and one less NH than 10, is more lipophilic than
compound 10 as evidenced by the difference in logD at pH 7.4 of 0.8 log units. However, as has
been previously pointed out, a direct linkage between hydrophobicity and entropy cannot be
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established since many complexities exist.
Compound 20 displayed reasonable biochemical WT and mutant Y641N potency,
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moderate HLM clearance (53.8 ꢀL/min/mg), and permeability (19.6 x10 cm/sec). However,
the cellular IC values of compound 20 associated with inhibiting H3K27Me3 and reducing cell
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proliferation were about 1 ꢀM (Table 4). In order to utilize this novel class of EZH2 inhibitors
in in vivo studies, we sought to further improve the compound’s cell potency and biochemical
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lipophilic efficiency (LipE)
.
Toward this end, the isopropoxy moiety of compound 20 was selected as a site for further
exploration. A small array of 30 compounds which contained various alkoxy groups that
replaced the isopropoxy moiety present in 20 was designed and synthesized. Among these 30
molecules, compound 23, which contained a chiral THF moiety and thus possessed reduced
lipophilicity relative to 20, maintained similar biochemical potency and improved LipE by about
1.5 units (calculated based on Y641N IC ; Figure 5). Consequently, 23 displayed an improved
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HLM clearance value relative to 20 and also maintained good permeability (Table 5). In order to
further improve the lipophilic efficiency of 23 binding to EZH2, other positions on its phenyl
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moiety were also modified. These efforts led to the R position of the phenyl moiety being
identified as a promising vector. In order to maintain the desired high ligand binding efficiency,
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small groups, such as F, CH , and Cl, were introduced as new R substituents (compounds 24ꢀ26,
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Table 4). Compound 24, which contains a fluorine atom at the R position, lost about twoꢀfold
potency in both biochemical and cellular assays relative to inhibitor 23. However, compound 25,
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containing a CH group at the R position, improved potency by two fold and thus maintained
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similar LipE compared with 23 (Figure 5). Since compound 24 and 25 exhibited similar
measured logD values, the latter molecule displayed a higher LipE value (about 0.5 units, Figure
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). This result indicated that R CH was a better moiety to increase compound binding affinity
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relative to the R F substituent employed in 24. Encouragingly, substituting the H atom at R
with Cl resulted in compound 26 which displayed about 5ꢀfold and 3ꢀfold potency improvements
relative to 23 and 25, respectively. Compounds 23, 25, and 26 displayed similar LipE values due
to the increased lipophiliciy of 26 relative to 23 and 25 (Table 4 and Figure 5). Compound 26
was also about 3 to 5ꢀfold more potent in reducing cellular H3K27Me3 levels and cell
proliferation compared with 23.
Compounds 26 and 23 displayed similar HLM clearance and permeability values (Table
3
7
5
). However, 26 displayed lower kinetic solubility compared with 23 (Table 5). Mouse PK
data were collected on compounds 23 and 26. Unfortunately, mouse PK experiments indicated
that neither compound could afford free plasma exposures in excess of the corresponding cell
IC90 values for at least 6 hours following a 300 mg/kg orally administrated dose (data not
1
5
shown). As studies of other EZH2 inhibitors reported in the literature suggested that such
coverage was required to achieve in vivo efficacy, we sought to further improve both
H3K27Me3 reduction and antiꢀproliferation parameters (target cell IC50 = 20 nM for reducing
both cellular H3K27Me3 level and viability). Therefore, our optimization efforts continued via
2
exploring more diverse chemical moieties attached to the R position of the lactam scaffold.
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We decided to include heteroaryl moieties at the R position since they would probe a
very different chemical space compared with the alkoxy groups. We initially chose to employ
pyrazole moieties in this exploration since the pyrazole is a versatile heterocycle that could be
further derivatized via the nitrogen atom if desired. As shown in Table 4, attachment of a
substituted pyrazole via the 4ꢀposition to the inhibitor core afforded a compound (27, Table 4)
that exhibited WT and Y641N IC s of 51 nM and 271 nM, respectively. Addition of a methyl
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group to the pyrazole 5ꢀposition improved inhibition potency by about 2 fold (compared 28 with
27, Table 4) while incorporation of a second methyl group (compound 29, Table 4) further
enhanced the biochemical activity by 3ꢀ4 fold relative to 27. Compounds 27ꢀ29 increased the
associated lipophilicity relative to 26 (Table 4), but did not improve binding affinity. We next
attempted to elaborate compound 29 by alkylating the pyrazole NH present in its structure.
However, this modification was not tolerated (compound 30) suggesting either that the NH was
involved in an Hꢀbonding interaction with the EZH2 protein and/or that the additional steric bulk
could not be accommodated in the protein’s binding site. To help differentiate these
possibilities, we prepared the corresponding dimethylꢀsubstitued isoxazole compound 31 (Table
4). Encouragingly, this molecule displayed very potent EZH2 biochemical inhibition as well as
good activity in the cell H3K27Me3 reduction and antiꢀproliferation effect. Compound 31
displayed about 5ꢀ10 fold biochemical potency improvement relative to 26 and also increased
logD by 0.5 units (Table 4). These changes collectively resulted in 26 and 31 exhibiting similar
2
LipE values (Figure 5). Consistent with the SAR developed using R alkoxy substituents,
removal of the 4ꢀCl substituent from compound 31 resulted in a 4ꢀfold loss in inhibition activity
(
compound 32, Table 4). We were encouraged that compound 31 exhibited > 10ꢀfold
improvement in cell potency relative to 26 which maintaining similar biochemical LipE. We
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therefore profiled 31 in several additional in vitro experiments to better understand its biological
properties.
Compound 31 displayed biochemical and cellular potency in the Karpasꢀ422 cell line that
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was comparable to other reported EZH2 inhibitors.
The compound was also shown to be
competitive with the SAM substrate (Figure 1S, supplementary material) and thus displayed a
1
4ꢀ18
mechanism of inhibition that was similar to other reported EZH2 compounds
Given the
observation that EZH2 activity needs to be strongly inhibited for extended duration to improve
efficacy in animals, an inhibitor with a long residence time would be advantageous.
Unfortunately, traditional methods for measuring compound offꢀrates including jump dilution
and surface plasmon resonance (SPR) could not be applied to compound 31. Robust SPR
methods have not been developed due to the large size of the complex as well as the potential for
instability of the complex under continuous flow conditions. The jump dilution experiment is
38
difficult to perform properly with tight binding inhibitors and we were unable to use this
method. Therefore, we developed a method that measures the enzyme bound compound directly
by mass spectrometry (experimental methods and Figure 2S, supplemental material) following
separation from free compound. Using this method we were able to observe an offꢀrate for
3
9
compound 1 that is similar to the published value . Comparison of the offꢀrates for 1,
compounds 31 and 32 (Table 2S, supplementary material) suggests that both compounds 31 and
32 have residence times which are at least comparable to, and potentially superior to compound
1
. Compound 31 was also profiled in the CEREP histone methyltransferase selectivity panel and
Invitrogen kinase selectivity panel and was proven to be selective (Table 3S, supplementary
material). To provide further proof of cellular target modulation as well as additional
downstream biomarkers, compound 31 was tested for its ability to modulate the expression of
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TNFRSF21 and PRDM1, two PRC2 target genes that have previously been shown to be
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repressed by H3K27Me3.
Upon treatment with compound 31 for 7 days, both TNFRSF21
and PRDM1 were upꢀregulated in Karpasꢀ422 cells that contain both wild type and Y641N
mutant EZH2 proteins in a doseꢀdependent manner (Fig. 3S, supplementary material).
Conversely, minimal modulation of target gene expression was observed in the wild type EZH2ꢀ
containing OCIꢀLY19 cell line, indicating that gene repression was mediated by elevated
H3K27Me3 levels in the EZH2ꢀmutant cell line. These results correlated well with the cellular
phenotypic effects caused by compound 31 and provided robust cellular biomarkers for
subsequent in vivo studies.
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We also profiled compound 31 in various in vitro ADME assessments in preparation for
its use in vivo. As shown in Table 5, the molecule displayed reasonable solubility, good
permeability, and low efflux potential in these in vitro assays. On the other hand, compound 31
also displayed high clearance in a microsomal stability assay for both human and mouse species
(Table 5). As shown in Fig. 6, at a 300 mg/kg dose, compound 31 displayed free plasma
41
concentrations above the in vitro cell IC values for at least 6 h. However, at both 100 and 30
9
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mg/kg doses, the free plasma concentrations were either at or below the cell IC values at the 6
50
h time point. These results suggested that relatively high doses of 31 would be required to
maintain appropriate mouse plasma exposures. We therefore profiled the molecule in a mouse
tumor growth inhibition (TGI) experiment to confirm that the novel inhibitor series could
demonstrate in vivo efficacy.
Accordingly, an in vivo efficacy experiment with compound 31 was then designed with
dose groups of 200 and 300 mg/kg, twice a day (BID) for 20 days (Figure 7A). Treatment with
compound 31 was well tolerated at both doses with less than 10% body weight loss observed
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during the course of the experiment (Figure 7B). Tumor volumes were measured every 3ꢀ5 days
throughout the study. As shown in Figure 7A, on day 20, compound 31 demonstrated tumor
stasis or regression at the 200 and 300 mg/kg dose levels, respectively.
In addition, tumor
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growth inhibition was sustained for at least another three weeks after the last dose, demonstrating
the ability of compound 31 to mediate longꢀterm epigenetic reprogramming in cells.
On Day 20, the compound dosing was stopped and tumor samples were collected to
measure both H3K27Me3 and PRC2 target gene expression levels. Compound 31 induced
approximately 55.5% and 66.4% reduction of the H3K27Me3 level at doses of 200 mg/kg and
300 mg/kg, respectively (Figure 7C). In addition, the PRC2 target genes TNFRSF21 and
PRDM1 were both strongly upregulated in both the 200 mg/kg and 300 mg/kg dosing arms
(Figure 7D). The H3K27Me3 reduction coupled with the strong elevation of the two target
genes demonstrated the expected onꢀtarget effect for this novel class of EZH2 inhibitors.
Importantly, eventual tumor regrowth was strongly correlated with global increases in
H3K27Me3 levels and the reꢀsilencing of PRC2 target genes, further solidifying the relationship
between the cellular biomarkers and the antiꢀproliferative effect in this model (Figure 4S, A, B,
and C in supplementary material). The observed efficacy and safety profile associated with this
tool compound demonstrated proof of concept for this novel class of EZH2 inhibitor as a
therapeutic agent in DLBCL harboring EZH2 activating mutations.
CHEMISTRY
Benzoic acids 33aꢀi (Scheme 1) were either purchased or synthesized in two steps from
commercially available methylꢀ3ꢀhydroxy benzoates through phenol alkylation followed by ester
1
2
hydrolysis (see Table 1 for R and R ). Commercially available 3ꢀ(aminomethyl)ꢀ4,6ꢀ
dimethylpyridinꢀ2(1H)ꢀone (35a) was then coupled to the benzoic acids under standard amide
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coupling conditions (HATU, NMM, DMF) to give 6 and 8ꢀ15 as shown in Scheme 1.
Compound 7 was made in a similar fashion starting from commercially available 2ꢀ
methylbenzoic acid.
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Scheme 1. Synthesis of benzamides 6 and 8–15
Reagents and conditions: i. Cs CO , DMF, alkyl bromides or iodides; ii. LiOH, MeOH, 80 °C;
2
3
iii. HATU, NMM, DMF.
The benzyloxy protected chloromethyl pyridine intermediate 35b (Scheme 2) served as a
protected dimethylpyridone and allowed for late stage alkylation of various lactams. The
commercially available 3ꢀcyanoꢀ4,6ꢀdimethylpyridone was protected as the benzyl ether to
afford 2ꢀ(benzyloxy)ꢀ4,6ꢀdimethylnicotinonitrile (34a). Iterative reductions then transformed the
nitrile to the corresponding aldehyde (34b) and then to the primary alcohol (not shown). The
latter entity was subsequently converted to the chloromethyl pyridine 35b using standard
chemistries.
Scheme 2: Synthesis of 35b
OBn
N
O
OBn
N
OBn
N
O
NC
i
NC
ii
iii, iv
H
Cl
NH
34a
34b
35b
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Reagents and conditions: i. BnCl, Ag O, toluene, 100 °C; ii. DIBAL, DCM, 0 ꢀ 5 °C; iii. NaBH ,
2
4
MeOH, 25 °C; iv. SOCl , ꢀ40 °C, DCM.
2
1
Synthesis of R methylꢀsubstituted dihydroisoquinolinꢀ1(2H)ꢀone 21 (Scheme 3) was
42
accomplished starting from methyl 6ꢀbromoꢀ3ꢀisopropoxyꢀ2ꢀmethylbenzoate . Suzuki coupling
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with 2ꢀBocꢀaminoethyl trifluoroborate yielded compound 36. Treatment of 36 with HCl
deprotected the Boc group from the amine moiety and subsequent heating in the presence of
DIEA then generated lactam 37. Alkylation of the lactam nitrogen of 37 with 35b followed by
hydrogenolysis provided compound 21.
Scheme 3: Synthesis of 21
Reagents and conditions: i. Cs CO , cat. Pd(OAc) , cat. RuPhos, toluene, H O, 95 °C; ii. (a) HCl
2
3
2
2
1
,4ꢀdioxane, 25 °C; (b) DIEA, 80 °C; iii. NaH, THF, 35b; iv. Pd/C, H , MeOH.
2
Synthesis of various alkoxyꢀsubstituted dihydroisoquinolinꢀ1(2H)ꢀones 18–20 and 23
(Scheme 4) was accomplished using a common intermediate 38 that was prepared by regioꢀ
selective chlorination of commercially available 7ꢀmethoxyꢀ3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone.
The methyl ether present in 38 was cleaved using BBr in DCM to provide 39. Alkylation of 39
3
using various electrophiles (such as iodoethane, 2ꢀiodopropane, and (S)ꢀtetrahydrofuranꢀ3ꢀyl
3
methanesulfonate) under mildly basic condition provided 40aꢀ40c (see Table 2 for R ).
Subsequent alkylation of the lactam nitrogens present in 38 and 40aꢀ40c with 35b and deꢀ
protection of the benzyl ether using TFA afforded pyridone containing lactams 18ꢀ20 and 23.
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Scheme 4. Synthesis of 18–20 and 23
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Reagents and conditions: i. NCS, H SO , 0ꢀ25 °C; ii. NaH, DMF, 35b; iii. TFA, DCM, 25 °C; iv.
2
4
3
BBr , DCM, 5ꢀ25 °C; v. R X, DMF, K CO .
3
2
3
Synthesis of 24–26 (Scheme 5) began by using Sandmeyer chemistry to transform the
4
2
amino group present in 41 to various halogens (F, Cl, I) to provide 42aꢀc. The isopropoxy
groups of 42aꢀc were then removed by treatment with BBr in DCM to afford the corresponding
3
phenols (not shown).
These entities were alkylated with (S)ꢀtetrahydrofuranꢀ3ꢀyl
4
3
methanesulfonate to afford the 3ꢀ(R)ꢀTHF intermediates (43aꢀc) via SN2 inversion. In the case
of compound 25, the lactam present in 43c was condensed with 35b to give the corresponding
alkylated lactam (44). The iodo group of 44 was subsequently converted to a methyl group via a
Suzuki coupling reaction and the resulting product (not shown) was deprotected with TFA in
DCM to give 25. Lactam alkylation of the 3ꢀ(R)ꢀTHF intermediates (43a and 43b) with 35b
followed by benzyl ether deprotection afforded compounds 24 and 26 in modest overall yields.
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Scheme 5: Synthesis of 24ꢀ26
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Reagents and conditions: i. HBF , NaNO , H O, 0 °C; ii. isoamyl nitrite, CH CN, CH I , 80 °C;
4
2
2
3
2 2
iii. CuCl , LiCl, CH CN, isoamyl nitrite, 55 °C; iv. BBr , DCM, 5ꢀ25 °C; v., DMF, K CO ; vi.
2
3
3
2
3
NaH, DMF, 35b; vii. CH B(OH) , Cs CO , Pd(dppf)Cl , DME, H O, 90 °C; viii. TFA, DCM, 25
3
2
2
3
2
2
°
C.
2
Synthesis of R heteroarylꢀcontaining dihydroisoquinolinꢀ1(2H)ꢀones 27–31 (Scheme 6)
started with nitrile reduction (NiCl /NaBH ) of 2ꢀ(2,5ꢀdichlorophenyl)acetonitrile with
2
4
44
concomitant methyl carbamate protection to afford 45 in 54% yield. Intramolecular Friedelꢀ
4
5
Crafts acylation (TfOH, 80 °C) afforded 46 in 72% yield without chromatography.
Bromination of 46 with NBS in concentrated H SO cleanly afforded the 7ꢀbromo intermediate
2
4
(not shown) in 98% yield. Subsequent lactam nitrogen alkylation (35b, KOtꢀBu, DMF, 0 °C)
gave 47 in 85% yield. Introduction of heteroaromatic rings at the 7ꢀposition was accomplished
using Suzuki crossꢀcoupling reactions with various boronic esters followed by benzyl ether
deprotection to afford compounds 27, 29, and 31. In the case of compound 28, a THPꢀprotected
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pyrazole bromide was used in a Suzuki coupling reaction to prepare 48. Subsequent
simultaneous deprotection of both the THP and benzyl ether groups present in 48 then afforded
28. In the case of compound 30, the pyrazole present in 29 was selectively alkylated under
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mildly basic conditions to give the target molecule.
Scheme 6: Synthesis of 27ꢀ31
Reagents and conditions: i. NiCl •6H O, NaBH , MeOH, ClCO Me, 0 °C; ii. TfOH, 0ꢀ80 °C; iii.
2
2
4
2
NBS, H SO ; iv. 35b, KOtꢀBu, DMF, 0 °C; v. boronic esters, Pd catalysts, bases; vi. TFA, 25 °C;
2
4
vii. CH I, Cs CO , DMF, 25 °C; viii. (a) Bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, dioxane,
3
2
3
2
o
9
5 °C; (b) Pd(dppf)Cl , Na CO dioxane, 95 °C; ix. HCl, EtOH, 70 C.
2 2 3,
Synthesis of compound 32 (Scheme 7) started with commercially available 7ꢀaminoꢀ3,4ꢀ
dihydroisoquinolinꢀ1(2H)ꢀone which underwent electrophilic aromatic chlorination with NCS to
provide 49. The amino group present in 49 was transformed to the corresponding bromide using
Sandmeyer chemistry and the resulting intermediate (not shown) was alkylated on the lactam
nitrogen with compound 35b to give 50. Suzuki coupling of 50 with the 3,5ꢀdimethyloxazole
boronic ester and subsequent deprotection yielded compound 32.
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Scheme 7: Synthesis of 32
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Reagents and conditions: i. NCS, DMF, 55 °C; ii. CuBr, CH CN, isoamyl nitrite, 60 °C; iii. KOtꢀ
Bu, DMF, 35b; iv. Pd(PPh ) , CsF, 3,5ꢀdimethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
yl)isoxazole, dioxane, 110 °C; v. TFA, 25 °C.
3
3
4
Synthesis of the 7ꢀmembered ring compound 16 (Scheme 8) was accomplished via
chlorination of the commercially available 8ꢀmethoxyꢀ2,3,4,5ꢀtetrahydroꢀ1Hꢀ2ꢀbenzazepinꢀ1ꢀone
to give a mixture of regioꢀisomers 51a and 51b in a ratio of 5.3 to 1 (ratio was assessed based on
the individually isolated final products). This mixture was alkylated with compound 35b and the
resulting products (not shown) were subjected to acidic deꢀprotection conditions. Compound 16
was isolated from the resulting mixture via preparative HPLC.
The synthesis of compound 17 (Scheme 8) started with the previously described 34b
(Scheme 2). Reductive amination of 34b with ethanolamine yielded compound 52. Amide
coupling of 52 with 2ꢀchloroꢀ6ꢀfluoroꢀ3ꢀmethoxyꢀbenzoic acid followed by aromatic substitution
resulted in the 7ꢀmembered ring intermediate 53. Subsequent acidic deprotection of the benzyl
group provided compound 17.
Scheme 8: Synthesis of 7ꢀMembered Ring Analogs 16 and 17
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Reagents and conditions: i. NCS, HOAc, 100 °C; ii. NaH, DMF, 35b, 80 °C; iii. HCl, MeOH, 70
°
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Reagents and conditions: i. DIBAL, DCM, 0ꢀ5 °C; ii. HOCH CH NH , NaBH CN, MeOH; iii.
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2
3
HATU, Et N, DMF; iv. KOtꢀBu, THF/DMF, 100 °C; v. TFA, 60 °C.
3
CONCLUSION
In summary, a novel series of lactamꢀcontaining EZH2 inhibitors was created from an
initial benzamide lead (compound 6) using computational torsional angle analysis coupled with a
ligandꢀcyclization strategy. The ligand lipophilicity efficiency (LipE) and potency of the initial
lead were improved considerably during the course of these optimization activities. The 1ꢀ2 unit
LipE improvement was achieved via lead transformation of benzamides to lactams and by the
2
incorporation of the alkoxy and heteroaryl substituents at the R position (Figure 5). These
efforts culminated with the identification of an optimized molecule, compound 31, which
displayed good efficacy in a diffuse large Bꢀcell lymphoma Karpasꢀ422 tumor model and
exhibited onꢀtarget pharmacodynamic effects in vivo.
Further optimization of the
pharmaceutical properties of this class of compounds will be reported in due course.
EXPERIMENTAL METHODS
Chemistry
Unless otherwise noted, materials were obtained from commercial suppliers and were used
without further purification. Removal of solvent under reduced pressure or concentration refers
to distillation using Büchi rotary evaporator attached to a vacuum pump (3 mm Hg). Products
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obtained as solids or high boiling oils were dried under vacuum (1 mm Hg). Silica gel
chromatography was performed either by CombiFlash (Teledyne ISCO), SP4 or Isolera
(Biotage) purification systems. All reactions were performed under a positive pressure of
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nitrogen, argon, or with a drying tube, at ambient temperature (unless otherwise stated), in
anhydrous solvents, unless otherwise indicated. Analytical thinꢀlayer chromatography was
performed on glassꢀbacked Silica Gel 60_F 254 plates (Analtech, 0.25mm) and eluted with the
appropriate solvent ratios (v/v). The reactions were assayed by high performance liquid
chromatographyꢀmass spectrometry (LCꢀMS) or thinꢀlayer chromatography (TLC) and
terminated as judged by the consumption of starting material. LCMS utilized 254 and 220 nm
wavelengths and either electrospray ionization (ESI) positive mode or atmosphericꢀpressure
chemical ionization (APCI) in positive mode. The TLC plates were visualized by UV, pꢀ
anisaldehyde, phosphomolybdic acid, or iodine staining. Microwave assisted reactions were run
1
in a Biotage Initiator. H NMR spectra were recorded on a Bruker XWINꢀNMR (400 MHz)
spectrometer. Proton resonances are reported in parts per million (ppm) downfield from
1
tetramethylsilane (TMS). H NMR data are reported as multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; brs., broad singlet). For spectra
obtained in CDCl , DMSOꢀd , and CD OD, the residual protons (7.27, 2.50, and 3.31 ppm,
3
6
3
respectively) were used as the internal reference. The purity of final products was generally
1
>95% as determined by HPLC methods and/or H NMR.
General procedure for the synthesis of compounds 6ꢀ15:
Oꢀ(7ꢀazabenzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀtetramethyluronium hexafluorophosphate (HATU, 0.515
mmol) and Nꢀmethylmorpholine (NMM, 0.515 mmol) were added to a solution of 33aꢀi (0.515
mmol) in DMF. The solution was stirred at room temperature for 1 h, then 35a (0.515 mmol)
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and NMM (1.03 mmol) were added. The reaction was stirred at room temp for 2 h and diluted
with water. The resulting precipitate was collected by filtration, washed with water, transferred
to a vial with methanol/water, frozen and lyophilized to provide compounds 6ꢀ15.
10
11
12
13
14
15
16
17
18
19
20
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31
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Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ3ꢀmethoxyꢀ2ꢀmethylbenzamide
+
1
(6). 6 was isolated as a white solid. LCMS m/z 301 [M + H] ; H NMR (400 MHz, CDCl ) δ
3
ppm 10.81 (s, 1H), 7.06 (d, J = 8 Hz, 1H), 7.03ꢀ6.99 (m, 1H), 6.85 (d, J = 7.2 Hz, 1H), 6.77 (d, J
=
8.4 Hz, 1H), 5.85 (s, 1 H), 4.45 (d, J = 6.0 Hz, 2H), 3.74 (s, 3H), 2.32 (s, 3H), 2.16 (s, 3H),
2.13 (s, 3H).
Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀmethylbenzamide (7).
+
1
7
1
4
was isolated as a white solid. LCMS m/z 271 [M + 1] ; H NMR (400 MHz, DMSOꢀd ) δ ppm
6
1.38 (brs., 1H), 8.08 (t, J = 4.2 Hz, 1H), 7.22 ꢀ 7.31 (m, 2H), 7.13 ꢀ 7.22 (m, 2H), 5.85 (s, 1H),
.27 (d, J = 5.0 Hz, 2H), 2.29 (s, 3H), 2.19 (s, 3H), 2.11 (s, 3 H).
Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ3ꢀmethoxybenzamide (8). 8 was
+
1
isolated as a white solid. LCMS m/z 287 [M + H] ; H NMR (400 MHz, DMSOꢀd ) δ ppm
6
11.49 (s, 1H), 8.37 (s, 1H), 7.42ꢀ7.30 (m, 3H), 7.06ꢀ7.04 (m, 1H), 5.87 (s, 1H), 4.30ꢀ4.29 (m,
2H), 3.78 (s, 3H), 2.17 (s, 3H), 2.11 (s, 3H).
Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ3ꢀethoxyꢀ2ꢀmethylbenzamide (9).
+
1
9
1
4
was isolated as a white solid. LCMS m/z 315 [M + H] ; H NMR (400 MHz, CDCl ) δ ppm
3
1.68 (s, 1H), 7.04ꢀ7.00 (m, 2H), 6.83 (d, J = 7.6 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 5.86 (s, 1H),
.45 (d, J = 6 Hz, 2H), 3.96ꢀ3.91 (m, 2H), 2.32 (s, 3H), 2.17 (s, 3H), 2.12 (s, 3H), 1.34 (t, J = 6.8
Hz, 3H).
Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ3ꢀisopropoxyꢀ2ꢀmethylbenzamide
+
1
(
10). 10 was isolated as a white solid. LCMS m/z 329 [M + H] ; H NMR (400 MHz, CD OD)
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δ ppm 7.10 ꢀ 7.29 (m, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.13 (s, 1H), 4.61
(dt, J = 12 Hz, 6.1 Hz, 1H), 4.49 (s, 2H), 2.41 (s, 3H), 2.27 (s, 3H), 2.19 (s, 3H), 1.34 (d, J = 6.1
Hz, 6H).
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Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀfluoroꢀ3ꢀmethoxybenzamide
+
1
(11). 11 was isolated as a white solid. LCMS m/z 305 [M + H] ; H NMR (400 MHz, CDCl ) δ
3
ppm 11.5 (s, 1H), 8.26 (s, 1H), 7.24ꢀ7.12(m, 3H), 5.87 (s, 1H), 4.28 (d, J = 4.8 Hz, 2H), 3.84(s,
3H), 2.18 (s, 3H), 2.11 (s, 3H).
Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2,3ꢀdimethoxybenzamide (12). 12
+
1
was isolated as a white solid. LCMS m/z 339 [M + H + Na] ; H NMR (400 MHz, DMSOꢀd ) δ
6
ppm 11.6 (brs., 1H), 8.68 (t, J = 5.6 Hz, 1H), 7.34 (dd, J = 7.6, 2.0 Hz, 1H), 7.18 (d, J = 6.4 Hz,
1
H), 7.13 (t, 8.0 Hz, 1H), 5.88 (s, 1H), 4.31 (d, J = 6.0 Hz, 1H), 3.83 (s, 2H), 3.75 (s, 3H), 2.24
s, 3H), 2.11 (s, 3H).
Nꢀ((4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀhydroxyꢀ3ꢀmethoxybenzamide
(
+
1
(
13). 13 was isolated as a white solid. LCMS m/z 324 [M + H + Na] ; H NMR (400 MHz,
DMSOꢀd ) δ ppm 12.3 (brs., 1H), 11.6 (brs., 1H), 8.78 (t, J = 5.6 Hz, 1H), 7.46 (dd, J = 7.6, 2.0
6
Hz, 1H), 7.08 (d, J = 6.4 Hz, 1H), 6.77 (t, J = 8.0 Hz, 1H), 5.88 (s, 1H), 4.32 (d, J = 6.0 Hz, 1H),
3.78 (s, 2H), 3.35 (s, 3H), 2.19 (s, 3H), 2.12 (s, 3H).
2ꢀChloroꢀNꢀ((4,6ꢀdimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ3ꢀmethoxybenzamide
+
(
14). 14 was isolated as a white solid. LCMS m/z 321 [M + H] ; (400 MHz, DMSOꢀd ) δ ppm
6
1
1.45 (brs., 1H), 8.25 (t, J = 4.8 Hz, 1H), 7.23ꢀ7.34 (m, 1H), 7.14 (d, J = 8.1 Hz, 1H), 6.90 (d, J
= 7.6 Hz, 1H), 5.86 (s, 1H), 4.27 (d, J = 5.1 Hz, 2H), 3.85 (s, 3H), 2.18 (s, 3H), 2.11 (s, 3H).
ꢀChloroꢀNꢀ((4,6ꢀdimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ3ꢀisopropoxybenzamide
2
+
1
(
15). 15 was isolated as a white solid. LCMS m/z 350 [M + H] ; H NMR (400 MHz, DMSOꢀ
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d6) δ ppm 11.44 (brs., 1H), 8.23 (t, J = 4.8 Hz, 1H), 7.21ꢀ7.30 (m, 1H), 7.11ꢀ7.19 (m, 1H), 6.87
(dd, J = 1.4, 7.5 Hz, 1H), 5.85 (s, 1H), 4.65 (td, J = 6.0, 12.1 Hz, 1H), 4.26 (d, J = 5.1 Hz, 2H),
2.18 (s, 3H), 2.10 (s, 3H), 1.28 (d, J = 6.1 Hz, 6H).
10
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9
ꢀChloroꢀ2ꢀ((4,6ꢀdimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ8ꢀmethoxyꢀ2,3,4,5ꢀ
tetrahydroꢀ1Hꢀbenzo[c]azepinꢀ1ꢀone (16). A solution of commercial 8ꢀmethoxyꢀ2,3,4,5ꢀ
tetrahydroꢀ1Hꢀ2ꢀbenzazepinꢀ1ꢀone (25.0 mg, 0.154 mmol) and NCS (20.6 mg, 0.154 mmol) in
HOAc (3 mL) was stirred at 100 °C for 4.5 h. The reaction mixture was partitioned between
EtOAc (20 mL) and water (20 mL). The organic phase was dried over sodium sulfate, and
concentrated under vacuum to give a mixture of 9ꢀchloroꢀ8ꢀmethoxyꢀ2,3,4,5ꢀtetrahydroꢀ1Hꢀ2ꢀ
benzazepinꢀ1ꢀone (51a) and 7ꢀchloroꢀ8ꢀmethoxyꢀ2,3,4,5ꢀtetrahydroꢀ1Hꢀ2ꢀbenzazepinꢀ1ꢀone
(51b) (35 mg, 100%) as a colorless oil. To this mixture of 7ꢀring lactams (35.0 mg, 0.150 mmol)
in DMF (5 mL) was added 35b (47 mg, 0.180 mmol) and NaH (9 mg, 0.225 mmol, 60%
dispersion in mineral oil). The reaction mixture was heated at 80 °C for 24 h. The reaction
mixture was poured into a NaOAcꢀHOAc buffer (5 mL) and extracted with EtOAc (2 x 10 mL).
The combined organic layers were dried over sodium sulfate and concentrated under vacuum.
The resulting brown oil was dissolved in MeOH (1 mL) and HCl (0.05 mL of 3 M in nꢀbutanol)
was added. The reaction mixture was heated at 70 °C for 24 h. The solvent was removed in
vacuo and the residue was purified by preparative HPLC to give 16 as the first eluting product
+
1
(20 mg, 36%). LCMS m/z 361 [M + 1] ; H NMR (400 MHz, CD OD) δ ppm 7.09ꢀ7.14 (m,
3
1
H), 7.05ꢀ7.10 (m, 1H), 6.13 (s, 1H), 4.89 (s, 1H), 4.71ꢀ4.79 (m, 1H), 3.88 (s, 3H), 3.38 (dd, J =
5.9, 14.9 Hz, 1H), 2.92ꢀ3.01 (m, 1H), 2.57ꢀ2.73 (m, 2H), 2.35 (s, 3H), 2.25 (s, 3H), 2.06ꢀ2.14 (m,
H), 1.53ꢀ1.64 (m, 1H).
1
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ꢀChloroꢀ4ꢀ((4,6ꢀdimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ7ꢀmethoxyꢀ3,4ꢀ
dihydrobenzo[f][1,4]oxazepinꢀ5(2H)ꢀone (17). To a solution of 2ꢀchloroꢀ6ꢀfluoroꢀ3ꢀmethoxyꢀ
benzoic acid (1.00 g, 4.89 mmol) in anhydrous DMF (30 mL) were added HATU (2.30 g, 5.85
0
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mmol) and Et N (1.36 mL, 9.76 mmol). The reaction mixture was stirred for 5 min. and 2ꢀ[(2ꢀ
3
benzyloxyꢀ4,6ꢀdimethylꢀpyridinꢀ3ꢀylmethyl)ꢀamino]ꢀethanol (52, 1.47 g, 5.12 mmol) was added
as a solid in one portion. The resulting reaction mixture was stirred at room temperature for 2 h.
The reaction mixture was partitioned between ethyl acetate (200 mL) and water (200 mL). The
organic phase was separated, washed with brine (2 x 200 mL), dried over sodium sulfate, and
concentrated under vacuum to give Nꢀ(2ꢀbenzyloxyꢀ4,6ꢀdimethylpyridinꢀ3ꢀylmethyl)ꢀ2ꢀchloroꢀ6ꢀ
fluoroꢀNꢀ(2ꢀhydroxyethyl)ꢀ3ꢀmethoxybenzamide (2.31 g, 100%) as a gum. To a solution of Nꢀ
(2ꢀbenzyloxyꢀ4,6ꢀdimethylꢀpyridinꢀ3ꢀylmethyl)ꢀ2ꢀchloroꢀ6ꢀfluoroꢀNꢀ(2ꢀhydroxyꢀethyl)ꢀ3ꢀ
methoxyꢀbenzamide (2.31 g, 4.88 mmol) in anhydrous DMF (20 mL) was added KOtꢀBu (12.2
mL of 1 M solution in THF, 12.2 mmol). The reaction mixture was stirred at 100 °C for 3 h.
After cooling to room temperature, the reaction mixture was partitioned between EtOAc (200
mL) and water (200 mL). The organic phase was separated, washed with brine (2 x 200 mL),
dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column
chromatography (heptanes/EtOAc) to give 4ꢀ((2ꢀ(benzyloxy)ꢀ4,6ꢀdimethylpyridinꢀ3ꢀyl)methyl)ꢀ
6ꢀchloroꢀ7ꢀmethoxyꢀ3,4ꢀdihydrobenzo[f][1,4]oxazepinꢀ5(2H)ꢀone (53, 668 mg, 30% yield) as a
+
1
solid. LCMS m/z 453 [M + H] ; H NMR (400 MHz, CDCl ) δ ppm 7.41 ꢀ 7.46 (m, 2H), 7.29 ꢀ
3
7
.39 (m, 3H), 6.84 ꢀ 6.95 (m, 2H), 6.68 (s, 1H), 5.42 (s, 2H), 4.97 (s, 2H), 3.88 (s, 3H), 3.73 (t, J
= 5.4 Hz, 2H), 3.22 (t, J = 5.4 Hz, 2H), 2.44 (s, 3H), 2.39 (s, 3H). A mixture of 53 (636 mg, 1.40
mmol) in TFA (10 mL) was stirred at 60 °C for 3 h. The TFA was removed in vacuum and the
resulting residue was partitioned between ether (100 mL) and satd. aq. sodium bicarbonate (100
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mL). The organic phase was separated, and washed with brine (100 mL), dried over sodium
sulfate, and concentrated under vacuum to give 17 (503 mg, 99%) as a white solid. LCMS m/z
+
1
3
63 [M + 1] ; H NMR (400 MHz, CDCl ) δ ppm 12.59 (brs., 1H), 6.88 ꢀ 6.97 (m, 2H), 6.04 (s,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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48
49
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51
52
53
54
55
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60
1
H), 4.90 (s, 2H), 4.03 (brs., 2H), 3.89 (s, 3H), 3.54 (t, J = 5.4 Hz, 2H), 2.41 (s, 3H), 2.30 (s,
3H).
8ꢀChloroꢀ2ꢀ((4,6ꢀdimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ7ꢀmethoxyꢀ3,4ꢀ
dihydroisoquinolinꢀ1(2H)ꢀone (18). To a solution of compound 38 (80 mg, 0.38 mmol) in dry
DMF (2 mL) was added NaH (27 mg, 1.14 mmol, 60% in mineral oil) at 0 °C. The mixture was
stirred at 0 °C for 30 min. To the mixture was added compound 35b (198 mg, 0.76 mmol). The
mixture was stirred at room temperature for 14 h. To the mixture was added EtOAc (20 mL).
The mixture was washed with brine (4 x 20 mL), dried over Na SO and concentrated in vacuo.
2
4
The residue was purified by flash chromatography (petroleum ether/EtOAc= 2/1) to give 2ꢀ((2ꢀ
(benzyloxy)ꢀ4,6ꢀdimethylpyridinꢀ3ꢀyl)methyl)ꢀ8ꢀchloroꢀ7ꢀmethoxyꢀ3,4ꢀdihydroisoquinolinꢀ
1(2H)ꢀone (90 mg, 54%) as a yellow oil. Benzyl deprotection was conducted by dissolving the
intermediate (60 mg, 0.14 mmol) in DCM (3 mL) and adding TFA (3 mL). After stirring the
mixture at room temperature for 3 h, the reaction was concentrated in vacuo and the residue was
purified by flash chromatography (DCM/MeOH = 10:1, R = 0.4) to give 18 (26 mg, 54%) as a
f
+
1
white solid. LCMS m/z 347 [M + H] ; H NMR (400 MHz, CDCl ) δ ppm 7.14 (s, 2H), 6.11 (s,
3
1
H), 4.77 (s, 2H), 3.88 (s, 3H), 3.45 (t, J = 6.2 Hz, 2H), 3.21 (t, J = 6.2 Hz, 2H), 2.28 (s, 3H),
2
.24 (s, 3H).
8ꢀChloroꢀ2ꢀ((4,6ꢀdimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ7ꢀethoxyꢀ3,4ꢀ
dihydroisoquinolinꢀ1(2H)ꢀone (19). To a solution of 38 (0.50 g, 2.4 mmol) in dry DCM (20
mL) was added BBr (0.6 mL, 5.9 mmol) at 5 °C. The mixture was stirred at room temperature
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for 14 h. To the reaction mixture was added dropwise H O (20 mL). The mixture was extracted
2
with EtOAc (2 x 30 mL). The combined organic layers were washed with brine (40 mL), dried
over Na SO and concentrated in vacuo to give 8ꢀchloroꢀ7ꢀhydroxyꢀ3,4ꢀdihydroisoquinolinꢀ
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1(2H)ꢀone (39, 0.5 g, 100%) as a brown solid. This intermediate was taken to the next step
without further purification. To a mixture of crude 39 (0.47 g, 2.4 mmol) and K CO (0.99 g, 7.2
2
3
mmol) in dry DMF (10 mL) was added dropwise iodoethane (0.37 g, 2.9 mmol) at room
temperature. The mixture was stirred at room temperature for 14 h. The reaction mixture was
partitioned between brine (50 mL) and EtOAc (40 mL). The aqueous layer was extracted with
EtOAc (4 x 40 mL). The combined organic layers were dried (Na SO ) and concentrated in
2
4
vacuo. The residue was purified by flash chromatography (petroleum ether/EtOAc = 1/1, R ~
f
0.3) to give 8ꢀchloroꢀ7ꢀethoxyꢀ3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone (40a, 0.35 g, 64%) as a brown
solid. By an analogous procedure used to make 18 from 38, 19 was obtained as a white solid
+
1
from 40a. LCMS m/z 361 [M+H] ; H NMR (400 MHz, CDCl ) δ ppm 11.03 (s, 1H), 6.98ꢀ6.91
3
(m, 2H), 5.93 (s, 1H), 4.79 (s, 2H), 4.11ꢀ4.05 (m, 2H), 3.56 (t, J = 6.2 Hz, 2H), 2.76 (t, J = 6.2
Hz, 2H), 2.34 (s, 3H), 2.25 (s, 3H), 1.45 (t, J = 6.2 Hz, 3H).
8ꢀChloroꢀ2ꢀ((4,6ꢀdimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ7ꢀisopropoxyꢀ3,4ꢀ
dihydroisoquinolinꢀ1(2H)ꢀone (20). 20 was prepared from 40b using an analogous procedure
described for the synthesis of 19 but substituting iodoethane with 2ꢀiodopropane. 20 was
+
1
isolated as a white solid. LCMS m/z 375 [M+1] ; H NMR (400 MHz, DMSOꢀd ) δ ppm 11.53
6
(
brs., 1 H), 7.21 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 5.88 (s, 1H), 4.58ꢀ4.66 (m, 1H),
4.56 (s, 2H), 3.35ꢀ3.40 (m, 2H), 2.73 (t, J = 6.1 Hz, 2H), 2.15 (s, 3H), 2.12 (s, 3H), 1.27 (d, J =
.1 Hz, 6H).
6
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Page 29 of 72
Journal of Medicinal Chemistry
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ꢀ[(4,6ꢀDimethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl]ꢀ8ꢀmethylꢀ7ꢀ(propanꢀ2ꢀyloxy)ꢀ3,4ꢀ
dihydroisoquinolinꢀ1(2H)ꢀone (21). A mixture of methyl 6ꢀbromoꢀ2ꢀmethylꢀ3ꢀ(propanꢀ2ꢀ
4
1
yloxy)benzoate
(154
mg,
0.536
mmol),
potassium
{2ꢀ[(tertꢀ
10
11
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16
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60
butoxycarbonyl)amino]ethyl}(trifluoro)borate (269 mg, 1.07 mmol) and Cs CO (613 mg, 1.88
2
3
mmol) in a 3:1 mixture of toluene:water (2.2 mL) was degassed with N . Palladium acetate
2
(
Pd(OAc) , 7.2 mg, 0.032 mmol) and RuPhos (30 mg, 0.064 mmol) were added and the mixture
2
was degassed with N and heated at 95 °C for 19 h. The reaction mixture was cooled to room
2
temperature then acidified with 10% aqueous HCl to pH 6 and extracted with EtOAc (20 mL).
The organic layer was washed with brine (5 mL), dried over Na SO , filtered, and concentrated
2
4
under vacuum. After purification by flash chromatography (EtOAc/heptanes, 0ꢀ100%) methyl 6ꢀ
(2ꢀ((tertꢀbutoxycarbonyl)amino)ethyl)ꢀ3ꢀisopropoxyꢀ2ꢀmethylbenzoate (36) was obtained. 36
was then dissolved in DCM (2 mL) and 4N HCl in 1,4ꢀdioxane (0.3 mL) was added. After 16 h,
the reaction was concentrated to an oil under vacuum. The residue was dissolved in 1,4ꢀdioxane
(3 mL) and DIEA (0.025 mL) was added. The mixture was heated at 80 °C for 50 h,
concentrated under vacuum, and purified by column chromatography (EtOAc /heptanes, 0ꢀ100)
to give 8ꢀmethylꢀ7ꢀ(propanꢀ2ꢀyloxy)ꢀ3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone (37, 19 mg, 16%) as a
white solid. To a 0 °C solution of 37 (30 mg, 0.14 mmol) in THF (0.46 mL) was added 60%
sodium hydride (18 mg, 0.45 mmol). After 30 min., 2ꢀ(benzyloxy)ꢀ3ꢀ(chloromethyl)ꢀ4,6ꢀ
dimethylpyridine, 35b (43 mg, 0.16 mmol) was added and the resulting mixture was heated at 50
°
C for 16 h. The reaction mixture was quenched with water (1 mL) and extracted with EtOAc
20 mL), washed with brine (2 mL), dried over sodium sulfate, filtered, and concentrated under
vacuum. The residue was purified by column chromatography (EtOAc/heptanes, 0ꢀ100), to give
(
2ꢀ{[2ꢀ(benzyloxy)ꢀ4,6ꢀdimethylpyridinꢀ3ꢀyl]methyl}ꢀ8ꢀmethylꢀ7ꢀ(propanꢀ2ꢀyloxy)ꢀ3,4ꢀ
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