Design and Characterization of a Pyridone-Containing EZH2 Inhibitor Phosphate Prodrug

Article abstract of DOI:10.1021/acs.jmedchem.0c02112

A pyridone-derived phosphate prodrug of an enhancer of zeste homolog 2 (EZH2) inhibitor was designed and synthesized to improve the inhibitor's aqueous solubility. This prodrug (compound 5) was profiled in pharmacokinetic experiments to assess its ability to deliver the corresponding parent compound (compound 2) to animals in vivo following oral administration. Results from these studies showed that the prodrug was efficiently converted to its parent compound in vivo. In separate experiments, the prodrug demonstrated impressive in vivo tumor growth inhibition in a diffuse large B-cell lymphoma Karpas-422 cell line-derived xenograft model. The described prodrug strategy is expected to be generally applicable to poorly soluble pyridone-containing EZH2 inhibitors and provides a new option to enable such compounds to achieve sufficiently high exposures in vivo.

Full text of DOI:10.1021/acs.jmedchem.0c02112

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Article
Design and Characterization of a Pyridone-Containing EZH2
Inhibitor Phosphate Prodrug
Pei-Pei Kung,* Connie Fan, Hovhannes J. Gukasyan, Buwen Huang, Susan Kephart, Manfred Kraus,
Joseph H. Lee, Scott C. Sutton, Shinji Yamazaki, and Luke Zehnder
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ABSTRACT: A pyridone-derived phosphate prodrug of an enhancer of zeste homolog 2 (EZH2) inhibitor was designed and
synthesized to improve the inhibitor’s aqueous solubility. This prodrug (compound 5) was profiled in pharmacokinetic experiments
to assess its ability to deliver the corresponding parent compound (compound 2) to animals in vivo following oral administration.
Results from these studies showed that the prodrug was efficiently converted to its parent compound in vivo. In separate experiments,
the prodrug demonstrated impressive in vivo tumor growth inhibition in a diffuse large B-cell lymphoma Karpas-422 cell line-derived
xenograft model. The described prodrug strategy is expected to be generally applicable to poorly soluble pyridone-containing EZH2
inhibitors and provides a new option to enable such compounds to achieve sufficiently high exposures in vivo.
therapy may require high-dose regimens to sustain sufficient
target coverage. Therefore, the desired EZH2 inhibitors should
have either sufficient plasma exposure to ensure robust target
coverage or a long residence time as recently reported by
Constellation.10
We recently reported a novel class of lactam-containing
EZH2 inhibitors (exemplified by compound 1; Figure 1),11
which do not contain an amine functional group or any other
moiety that ionizes at physiological pH. This class of inhibitors
demonstrated impressive in vivo efficacy in the DLBCL Karpas-
422 xenograft model.11 However, compound 1 also displayed
high clearance in a human microsomal stability assay (Table
1). Subsequent optimization with the intention to reduce
compound lipophilicity resulted in the design of compound 2
(Figure 1), which displayed biochemical and cellular potency
comparable to that of 1 as well as an improved aqueous
solubility (Table 1). We subsequently profiled compound 2 in
INTRODUCTION
■
Epigenetic dysregulation is a hallmark of many different types
of cancers including hematological malignancies and solid
tumors.1a,b In particular, the methylation of lysine residues
present in histone proteins has gained increased attention due
to the potential of the modified chromatin structures to
regulate gene transcription, deoxyribonucleic acid (DNA)
replication, and DNA repair.2 Among the many lysine
methyltransferases that create these epigenetic changes, the
polycomb-repressive complex 2 (PRC2) has been associated
with oncogenesis and is an evolving therapeutic target.3a−d
Enhancer of zeste homolog 2 (EZH2) is the catalytic
component of the PRC2 complex, and several inhibitors of
its activity have entered clinical trials.4 Recently, both Epizyme
and Constellation Pharmaceuticals demonstrated impressive
therapeutic outcomes with the EZH2 inhibitors tazemetostat
(EPZ6438) and CPI-1205 in various clinical trials.5−7 The
former compound was also recently approved by the U.S. FDA
for the treatment of adults and pediatric patients with
metastatic or locally advanced epithelioid sarcoma and
follicular lymphoma.8a,b However, both compounds need to
be administered at relatively high doses (800 mg, twice daily
(BID)-three times daily (TID)) to obtain meaningful clinical
benefit.9 These findings suggest that successful EZH2 inhibitor
Received: December 7, 2020
Published: February 2, 2021
© 2021 The Authors. Published by
American Chemical Society
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mouse pharmacokinetic experiments and found that a
methylcellulose (MC) suspension formulation of its stable
crystalline form provided exposures that barely exceeded the
corresponding Karpas-422 cell in vitro antiproliferation IC90
value. In contrast, an oil-based self-emulsifying drug delivery
system (SEDDS)12 formulation helped solubilize 2 and
afforded increased (about 5×) and more sustained mouse in
vivo exposures relative to the MC formulation (Supporting
Information). An in vivo tumor growth inhibition (TGI) study
using the DLBCL Karpas-422 tumor model, which harbors an
EZH2 Y641N gain of function mutation, was subsequently
used to evaluate the therapeutic effect of compound 2 in the
SEDDS formulation. Under these conditions, compound 2
displayed encouraging dose-dependent TGI effects with near-
stasis observed at 120 mg/kg, twice daily (Figure 2). However,
the dose of 2 projected to achieve the targeted EZH2 IC90
inhibition in the clinic (about 200−400 mg, twice daily) was
not feasible for SEDDS formulations. We therefore explored
designing a phosphate-containing prodrug of compound 2 as
an alternative approach to achieve the in vivo exposures
required for strong efficacy outcomes.
RESULTS AND DISCUSSION
■
The clinical use of phosphate prodrugs to improve aqueous
solubility is well precedented.13 These examples include the
HIV protease inhibitor fosamprenivir14 and the antifungal
fosfluconazole.15 However, the chemical structure of com-
pound 2 does not offer a typical point of attachment for a
phosphate-containing moiety, such as hydroxyl group.
Accordingly, we decided to utilize the pyridone moiety present
in 2 as the location for prodrug attachment. This pyridone is
an important component of the EZH2 inhibitor pharmaco-
phore and is present in virtually all potent EZH2 SET domain
inhibitors reported to date. In addition, the existence of related
pyridone prodrugs in the patent literature16a−d,17a−c gave us
confidence that the designed prodrug molecule could be
successfully synthesized.
We began our exploration of pyridone-derived phosphate
prodrugs of compound 2 by applying a two-step trans-
formation that was first reported by Cannizzaro et al.17b As
shown in Scheme 1a, treatment of 2 with 1-chloromethyl
chloroformate/dimethylformamide (DMF) followed by ex-
posure of the crude product to di-tert-butyl potassium
phosphate in the presence of tetrabutylammonium iodide
afforded the derivatized compound 4a in modest yield (64%)
after purification on silica gel. The described transformation for
related pyridones was reported in the literature to proceed
through intermediates analogous to 3.17a−c However, little
evidence for such entities was provided in these prior literature
reports. We therefore used liquid chromatography-mass
spectrometry (LCMS) to analyze the reaction mixture
produced by treatment of 2 with 1-chloromethyl chlorofor-
mate/DMF and observed a new peak with the correct mass
corresponding to 3 (see the Experimental Section for
additional details). Unfortunately, attempts to isolate 3 in
purified form were unsuccessful, and our evidence for the
involvement of this intermediate in the transformation of 2 to
4a is supported solely by the described reaction mixture
analysis. Subsequent treatment of 4a with a warm acetone/
water mixture afforded the desired prodrug 5 following
literature precedent.18 Purification of 5 using preparative
high-pressure liquid chromatography (HPLC) provided an
83% isolated yield of 5 with a 98% purity. Compound 5 was
determined to be chemically stable in simulated gastric fluid
(SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8) for
at least 8 h. We subsequently obtained a crystal structure of
compound 5 (Figure 3), which confirmed that the prodrug was
attached to the pyridone N-atom (as opposed to the O-atom)
and thereby also confirmed the regio-chemistry associated with
compounds 3 and 4a. Compound 5 was determined to have a
solubility of approximately 21 640 μM in a 0.5% MC
formulation that contained 1 equivalent of NaOH. This
Figure 1. Chemical structures and in vitro data of compound 1 and
compound 2.
Figure 2. In vivo dose-dependent tumor growth inhibition observed
after treatment of 2 in SEDDS formulation (n = 10 mice/group).
Table 1. Metabolic Stability, Permeability, and Solubility Comparison between 1 and 2
a
log D
HLM (μL/(min mg) protein)
MDCK-LE (×10−6 cm/s)
app_sol pH 6.5 (μM)
thermodynamic solubility (μM)
1
2
3.1
2.4
107
27.8
15.7
9.0
140
338
4.5
41
a
A 0.5% MC/0.1% polysorbate 80 aqueous vehicle was used.
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Scheme 1. (a) Synthesis of N-Alkylated Pyridone Prodrug 5 from Compound 2. (b) Alternative Synthesis of N-Alkylated
Pyridone Prodrug 5
Figure 3. Small-molecule crystal structure of prodrug 5. Phosphate group was connected to the pyridone nitrogen atom via a CH2 linker, and a
water molecule was found to bind to the phosphate group.
Scheme 2. Synthesis of Compound 2
solubility was about 527× improved compared with the parent
compound 2 (Table 1).
Having successfully prepared prodrug 5 by the method
described above, we were curious to learn whether a more
direct (one-step) approach to intermediate 4a could also be
employed. As shown in Scheme 1b, treatment of 2 with
commercially available di-tert-butyl chloromethyl phosphate in
the presence of potassium tert-pentoxide in DMF afforded a
mixture of 4a and the corresponding O-alkylated isomer 4b
with the latter significantly dominating the outcome (4a:4b =
1:4). We sought to exploit the alternate pyridone alkylation
regio-chemistry observed in this transformation and therefore
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attempted to transform intermediate 4b into the corresponding
O-alkylated prodrug 6. However, deprotection of 4b using the
same neutral conditions that enabled conversion of 4a to 5
(Scheme 1a) provided only the original compound 2 in
relatively good yield. This outcome suggested that the
deprotected 4b (i.e., prodrug 6) was not stable to mild
aqueous conditions, and further synthetic pursuit of this
compound was therefore abandoned.
The synthesis of compound 2 (Scheme 2) commenced with
the condensation of 2-(2-bromo-5-chlorophenyl) ethanamine
with 2-nitrophenyl isocyanate in 1,2-dichloromethane to
provide the urea (compound 7). Exposure of 7 to
trifluoromethanesulfonic acid at elevated temperature then
formed lactam 8 via Friedel−Crafts acylation. A subsequent
acid-catalyzed iodination reaction introduced the iodide at the
7 position of the lactam ring (compound 9) while the
corresponding 6-iodo isomer was not observed. The alkylation
of the lactam nitrogen present in 9 with 2-(benzyloxy)-3-
(chloromethyl)-4,6-dimethylpyridine11 in the presence of
potassium t-butoxide afforded compound 10 in good yield.
Palladium-mediated coupling of 10 with dimethyltriazole19
then afforded compound 11. The O-benzyl group contained in
11 was subsequently removed by treatment with trifluoroacetic
acid (TFA) to give compound 2 in good yield.
prodrug 5 to mice at 147 mg/kg using an MC formulation
afforded plasma exposures of the parent compound 2 that
greatly exceeded those observed when 2 was similarly
administrated at an equivalent dose (120 mg/kg)22 (Figure
4b and Table S3). Prodrug 5 was not detected in the plasma of
the mice at any time point during the course of this experiment
(limit of quantitation (LOQ): 1 ng/mL) suggesting that rapid
and extensive conversion of 5 to 2 occurred. These phenomena
were similarly described in the literature for the conversion of
fosfluconazole to fluconazole.23 Importantly, the plasma
exposures of 2 achieved following administration of 5 exceeded
the levels obtained when 2 was administered using the SEDDS
formulation (Figure 4b and Table S3). This outcome indicated
that prodrug 5 was suitable for testing in in vivo efficacy
experiments.
We next compared the tumor growth inhibition effects of 5
(via in vivo conversion to 2) using a traditional MC
formulation with compound 2 using the previously described
MC and SEDDS formulations in the Karpas-422 cell-derived
xenograft model. Compound 5 was administered at three
different dose levels (60, 120, and 240 mg/kg daily doses
equivalent to compound 2), and compound 2 in both MC and
SEDDS formulations was dosed at a 240 mg/kg daily dose in
the same study. As shown in Figure 5, after a 30 day treatment,
The alkaline phosphatase-mediated conversion of phosphate
prodrugs to the corresponding parent compounds during
intestinal absorption is well precedented in the literature.20,21
We therefore tested compound 5 in mouse pharmacokinetics
(PK) experiments to investigate whether facile conversion to 2
was observed. As shown in Figure 4a, prodrug 5 was
Figure 5. TGI effect comparison using 5 and 2 in different
formulations (compare MC, SEDDS, and prodrug 5 in MC) (n =
10 mice/group).
compound 5 displayed dose-proportional TGI effects (37, 59,
and 121%, respectively). In contrast, the MC formulated 2
dosing group did not display any meaningful TGI effect. On
the other hand, when 2 was administered using the SEDDS
formulation, a strong TGI (98%) result was observed that was
consistent with the previous outcome. Importantly, the TGI
effects associated with the oral administration of 5 at 147 mg/
kg (equivalent to 120 mg/kg of 2) persisted long after dosing
was stopped (tumor regression was observed for up to 40 days
after dosing stopped).
To evaluate whether efficient conversion of the phosphate
prodrug 5 to the parent compound 2 occurred in rats (a
possible toxicology species) in addition to mouse, a rat PK
study was performed with the prodrug dosed at 2000 mg/kg
daily. As shown in Figure 6, high rat plasma exposures of 2
were observed following administration of 5, indicating good
conversion of the latter compound to the former molecule in
this species. These exposures were about 400× higher than
those noted when 2 was dosed directly to rats using the MC
formulation. In addition, only low levels of prodrug 5 could be
detected in rat plasma following oral administration.
Figure 4. (a) Dose-dependent increase of exposures in mouse plasma
of 2 after oral dosing of 5 at 30, 60, and 120 mg/kg BID dosing (daily
doses: 60, 120, and 240 mg/kg doses). (b) Comparison of mouse
exposures of 2 using MC, SEEDS formulations, and phosphate
prodrug 5 at 147 mg/kg (or 120 mg/kg equivalent), once daily (QD)
dosing.
administered to female mice orally at 30, 60, and 120 mg/kg
twice daily in MC formulation and afforded dose-dependent
increases in plasma exposures of 2. The Cmax, Tmax, and area
under the curve (AUC) values observed for 2 in these studies
were greater than dose-proportional over the tested dose range
(Table S2). In a separate experiment, oral administration of
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1
generally >95% as determined by HPLC methods and/or H NMR,
unless otherwise stated. Un-numbered reagents in the schemes are
commercially available.
(3-((5-Bromo-8-chloro-7-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-1-
oxo-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-4,6-dimethyl-2-oxo-
pyridin-1(2H)-yl)methyl Di-tert-butyl Phosphate (4a). To a
suspension of 2 (5.05 g, 10.3 mmol) in ethyl acetate (EtOAc) (90
mL) and DMF (35 mL) at room temperature (rt) was added
chloromethyl chloroformate (1.2 mL, 13.5 mmol). The reaction was
stirred at rt for 18 h. Additional chloromethyl chloroformate (0.5 mL,
5.6 mmol) was added, and the reaction was stirred for an additional
24 h. The EtOAc was removed under vacuum, and the resulting DMF
suspension was diluted with additional DMF (10.0 mL). The reaction
was cooled in an ice bath and then treated with triethylamine (6.5 mL,
47 mmol), potassium di-tert-butyl phosphate (5.1 g, 21 mmol), and
tetrabutylammonium iodide (190 mg, 0.514 mmol). The reaction was
removed from the ice bath and stirred at rt for 3 days. The reaction
was then diluted with 1:1 MTBE/EtOAc, washed with water and
brine, and concentrated. The resulting white foam was purified by
silica gel chromatography (eluting with a gradient of 50−100%
methylacetate in heptane) to afford the title compound (5.7 g, 64%)
as a clear oil. LCMS m/z 734/736 [M + Na]+; 1H NMR (400 MHz,
DMSO-d6) δ 7.90 (s, 1H), 6.07 (s, 1H), 5.80 (d, J = 6.6 Hz, 2H), 4.55
(s, 2H), 3.77 (s, 3H), 3.60 (t, J = 6.2 Hz, 2H), 2.99 (t, J = 6.1 Hz,
2H), 2.40 (s, 3H), 2.26 (s, 3H), 2.08 (s, 3H), 1.40 (s, 18H).
(3-((5-Bromo-8-chloro-7-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-1-
oxo-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-4,6-dimethyl-2-oxo-
pyridin-1(2H)-yl)methyl Dihydrogen Phosphate (5). A solution of 4a
(4.90 g, 6.87 mmol) in acetone (40.0 mL) and water (40.0 mL) was
stirred at 43 °C for 18 h. The acetone was removed under vacuum,
and the resulting aqueous suspension was frozen in a dry ice/acetone
bath and lyophilized to give a white powder. The crude powder was
purified by C18-bonded silica gel chromatography (eluting with a
gradient of 15−55% MeOH in water) to give the titled compound
(3.44 g, 83%) as a white powder. LCMS m/z 600/602 [M + H]+; 1H
NMR (400 MHz, DMSO-d6) δ 11.54 (br s, 2H), 7.90 (s, 1H), 6.06
(s, 1H), 5.75 (d, J = 5.8 Hz, 2H), 4.57 (s, 2H), 3.77 (s, 3H), 3.57 (t, J
= 6.2 Hz, 2H), 2.99 (t, J = 6.1 Hz, 2H), 2.40 (s, 3H), 2.24 (s, 3H),
2.08 (s, 3H); 31P NMR (162 MHz, DMSO-d6) δ -3.34 (s, 2P). Anal.
calcd for C22H24BrClN5O6P: C, 43.98; H, 4.03; N, 11.66; Br, 13.30;
Cl, 5.90. Found: C, 43.73; H, 3.92; N, 11.57; Br, 13.08; Cl, 5.89.
((3-((5-Bromo-8-chloro-7-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-1-
oxo-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-4,6-dimethylpyridin-
2-yl)oxy)methyl Di-tert-butyl Phosphate (4b). A solution of 2 (75.2
mg, 0.153 mmol) in DMF (2.0 mL) in an ice bath was treated with
potassium tert-butoxide (2.0 M in THF, 80 uL, 0.16 mmol) and then
di-tert-butyl chloromethyl phosphate (50.0 mg, 0.16 mmol). The
reaction was removed from the ice bath and stirred at rt for 3 h. The
reaction was poured into MTBE and washed with water. The organic
phase was concentrated and purified on silica gel (gradient of 60−
100% EtOAc in heptane) to give 4a (15 mg, 14%) and the title
compound 4b (58 mg, 53%) as a clear oil. Compound 4b: LCMS m/z
712/714 [M + H]+; 1H NMR (400 MHz, DMSO-d6) δ 7.92 (s, 1H),
6.87 (s, 1H), 5.92 (d, J = 11.1 Hz, 2H), 4.71 (s, 2H), 3.78 (s, 3H),
3.49 (t, J = 6.2 Hz, 2H), 3.01 (t, J = 6.2 Hz, 2H), 2.36 (s, 6H), 2.09 (s,
3H), 1.38 (s, 18H).
Figure 6. Rat plasma exposure comparison of 2 administrated with
0.5% MC and via 5 in the same formulation.
Collectively, these data suggest that (1) conversion of 5 to 2
was efficient in rats and (2) administration of prodrug 5 could
be used to achieve high rat exposures of 2 that could enable
toxicological evaluation.
CONCLUSIONS
■
In conclusion, we designed a phosphate prodrug derived from
the pyridone substituent commonly found in many EZH2
inhibitors. This phosphate prodrug (5) afforded high plasma
exposures of the parent molecule (2) following oral
administration to mice and rats and thereby demonstrated
impressive TGI effects in a mouse xenograft model.
Importantly, the use of the prodrug enabled oral dosing with
a standard formulation (MC) in the tumor growth inhibition
experiment. We believe that the described prodrug approach
will be generally applicable to most pyridone-containing EZH2
inhibitors and can thus be routinely used to increase the in vivo
exposures of such compounds in cases where poor solubility is
limiting.
EXPERIMENTAL SECTION
■
General Chemistry Methods. Compounds 2 and 5 were purified
by supercritical fluid chromatography (SFC) and silica gel
chromatography, respectively. Their purities were characterized by
proton nuclear magnetic resonance (NMR) and high-performance
liquid chromatography (HPLC) to be >95%. 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 a Buchi rotary
̈
evaporator attached to a vacuum pump (3 mm Hg). Products
obtained as solids or high boiling oils were dried under vacuum (1
mm Hg). Silica gel chromatography was performed by either
CombiFlash (Teledyne Isco), SP4, or Isolera (Biotage) purification
systems. All reactions were performed under a positive pressure of
nitrogen, argon, or with a drying tube; at an ambient temperature
(unless otherwise stated); and in anhydrous solvents, unless otherwise
indicated. Analytical thin-layer chromatography (TLC) was per-
formed on glass-backed silica gel 60 F254 plates (Analtech, 0.25 mm)
and eluted with the appropriate solvent ratios (v/v). The reactions
were assayed by high-performance liquid chromatography-mass
spectrometry (LCMS) or thin-layer 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 atmospheric-pressure chemical
ionization (APCI) in positive mode. The TLC plates were visualized
by UV, p-anisaldehyde, phosphomolybdic acid, or iodine staining. 1H
NMR spectra were recorded on a Bruker XWIN-NMR (400 or 700
MHz) spectrometer. Proton resonances are reported in parts per
1-[2-(2-Bromo-5-chlorophenyl)ethyl]-3-(2-nitrophenyl)urea (7).
A suspension of 2-(2-bromo-5-chloro-phenyl)-ethylamine hydro-
chloride (36.18 g, 133.5 mmol) in 1,2-dichloroethane (200 mL)
and 1.0 M aqueous sodium hydroxide solution (200 mL, 200 mmol)
was stirred vigorously at room temperature for 30 min, causing most
of the solid to dissolve. The layers were separated, and the aqueous
layer was extracted with dichloroethane (2 × 100 mL). The organic
layers were combined, dried over magnesium sulfate, filtered, and
concentrated to a colorless oil. The oil was dissolved in acetonitrile
(50 mL) and concentrated to dryness (2 cycles) to azeotrope off
residual water, leaving 2-(2-bromo-5-chlorophenyl)ethan-1-amine free
base (31.72 g) as a colorless oil. This oil was dissolved in 1,2-
dichloroethane (35 mL) and added dropwise over 10 min to a 25 °C
solution of 2-nitrophenyl isocyanate (21.88 g, 133.3 mmol) in
1
million (ppm) downfield from tetramethylsilane (TMS). H NMR
data are reported as multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; quint, quintuplet; sept, septuplet; dd, doublet of doublets; dt,
doublet of triplets; bs, broad singlet). For spectra obtained in CDCl3
or DMSO-d6, the residual protons (7.27 and 2.50 ppm, respectively)
were used as the internal reference. The purity of final products was
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dichloroethane (500 mL). About halfway through the addition, a
thick, bright yellow precipitate begins to form. The internal
temperature of the solution increased from 25 to 33 °C during the
addition. After addition was complete, mixing was continued for 5
min, and then the solid was collected by suction filtration. The flask
and the filter cake were rinsed with dichloroethane (2 × 50 mL). The
filtrate and rinses were concentrated to ∼150 mL, affording a second
crop of precipitate, which was collected along with the first. The
concentration of the filtrate to ∼50 mL affords a third crop of
precipitate, which was collected along with the other two crops. The
combined precipitate crops were dried under vacuum to give the title
compound (50.24 g, 94%) as a bright yellow solid. LCMS m/z 398/
To remove residual color, the solids were stirred in heptane (600
mL) for 1 h, the precipitate was collected by suction filtration, and the
filter cake was rinsed with heptane (3 × 100 mL). After air-drying
overnight, the precipitate was suspended in acetonitrile (200 mL) and
concentrated to dryness under vacuum, 3 times. The yellow solid
residue was suspended in acetonitrile (300 mL), stirred for 1 h, and
then the solids were collected by suction filtration. The filter cake was
air-dried until no longer damp and then dried in a 60 °C vacuum oven
overnight to give 9 (39.61 g, 90% pure) as a light brown solid. LCMS
1
m/z 386/388 [M + H]+; H NMR (DMSO-d6, 400 MHz) δ 8.33 (s,
1H), 8.31 (br s, 1H), 3.3−3.3 (m, 2H), 2.90 (t, 2H, J = 6.2 Hz).
2-{[2-(Benzyloxy)-4,6-dimethylpyridin-3-yl]methyl}-5-bromo-8-
chloro-7-iodo-3,4-dihydroisoquinolin-1(2H)-one (10). To a suspen-
sion of 5-bromo-8-chloro-7-iodo-3,4-dihydroisoquinolin-1(2H)-one
(9, 2.35 g, 6.08 mmol) in anhydrous DMF (30 mL, c = 0.20 M) in an
ice bath was added dropwise potassium tert-butoxide (7.30 mL, 7.30
mmol, 1.0 M). After stirring the mixture for 30 min, the reaction
mixture became clear, and 2-(benzyloxy)-3-(chloromethyl)-4,6-
dimethylpyridine (1.75 g, 6.69 mmol) in 10 mL anhydrous DMF
was added in one portion. The resulting reaction mixture was stirred
at 0 °C for 30 min. The reaction mixture was partitioned between
ethyl acetate (100 mL) and water (100 mL). The organic phase was
separated, washed with water (100 mL) and brine (100 mL), dried
over sodium sulfate, concentrated to dryness, and purified by silica gel
chromatography (0−40% ethyl acetate in heptane) to afford the title
compound as a gum (2.95 g, 79% yield). LCMS m/z 611/613 [M +
H]+. 1H NMR (400 MHz, chloroform-d) δ ppm 8.11 (s, 1H), 7.40−
7.47 (m, 2H), 7.27−7.37 (m, 3H), 6.62 (s, 1H), 5.42 (s, 2H), 4.85 (s,
2H), 3.25 (t, J = 6.2 Hz, 2H), 2.68 (t, J = 6.2 Hz, 2H), 2.41 (s, 3H),
2.32 (s, 3H).
2-{[2-(Benzyloxy)-4,6-dimethylpyridin-3-yl]methyl}-5-bromo-8-
chloro-7-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-3,4-dihydroisoquino-
lin-1(2H)-one (11). To a 40 mL septum vial was charged 2-{[2-
(benzyloxy)-4,6-dimethylpyridin-3-yl]methyl}-5-bromo-8-chloro-7-
iodo-3,4-dihydroisoquinolin-1(2H)-one (10, 2.90 g, 4.74 mmol), 1,4-
dimethyl-1H-[1,2,3]triazole20 (1.84 g, 18.9 mmol), palladium acetate
(162 mg, 0.722 mmol), xantphos (30.1 mg, 0.0520 mmol), cesium
pivalate (5.60 g, 23.9 mmol), and DMA (25 mL). The resulting
reaction mixture was degassed by bubbling nitrogen through for 10
min. The reaction was then heated at 100 °C for 24 h. After cooling
down to rt, the reaction mixture was partitioned between ethyl acetate
(200 mL) and water (200 mL). The organic phase was washed with
water (200 mL) and brine (200 mL), dried over sodium sulfate,
concentrated to dryness, and purified by silica gel chromatography
(0−80% ethyl acetate in heptane) to afford the title compound as a
foam-like solid (1.34 g, 49% yield). LCMS m/z 580/582 [M + H]+;
1H NMR (400 MHz, chloroform-d) δ ppm 7.43−7.48 (m, 3H),
1
400 [M + H]+, 420/422 [M + Na]+, 436/438 [M + K]+; H NMR
(DMSO-d6, 400 MHz) δ 9.31 (s, 1H), 8.27 (dd, 1H, J = 1.0, 8.6 Hz),
8.04 (dd, 1H, J = 1.5, 8.4 Hz), 7.5−7.7 (m, 3H), 7.45 (d, 1H, J = 2.6
Hz), 7.25 (dd, 1H, J = 2.6, 8.6 Hz), 7.12 (ddd, 1H, J = 1.2, 7.2, 8.3
Hz), 3.3−3.4 (m, 2H), 2.90 (t, 2H, J = 7.0 Hz); 13C NMR (DMSO-
d6, 101 MHz) δ 154.1, 140.7, 136.8, 135.7, 134.9, 134.0, 132.3, 130.6,
128.2, 125.2, 122.3, 122.0, 121.4, 35.5 (one peak obscured by DMSO-
d6).
5-Bromo-8-chloro-3,4-dihydroisoquinolin-1(2H)-one (8). A 500
mL flask containing 7 (50.24 g, 126.0 mmol) was cooled in an ice/
water bath for 30 min, and then trifluoromethanesulfonic acid (110 g,
733 mmol) was added from freshly opened ampules. High-quality
trifluoromethanesulfonic acid is necessary for the success of this
reaction. Stirring was continued at 0 °C for 10 min, and then the
mixture was heated in an 80 °C oil bath for 22 h. While the mixture
was still warm, it was carefully poured into a flask containing crushed
ice and water (∼800 mL), forming a gummy, red-brown precipitate.
Dichloromethane (600 mL) was added, and stirring was continued
until all of the ice had melted. Layers were separated, and the aqueous
layer was further extracted with dichloromethane (3 × 250 mL). The
combined organic layers were washed with 1 M sodium hydroxide
solution (500 mL). The basic aqueous layer was back-extracted with
dichloromethane (2 × 200 mL). The combined organics were dried
over magnesium sulfate, filtered, and concentrated to dryness, leaving
a red-brown sludge. Acetonitrile (300 mL) was added, the mixture
was stirred for 10 min, and the resulting precipitate was collected by
suction filtration. The filter cake was rinsed with acetonitrile (3 × 10
mL) and dried under vacuum to give the title compound (23.98 g,
73%) as a yellow solid. The concentration of the filtrate to ∼100 mL
afforded a second crop of precipitate, which was collected by suction
filtration and dried under vacuum to give a second batch (3.71 g,
11%) as a golden-brown solid. Though the batches differed in color,
both were >95% pure by LCMS, NMR, and combustion analysis.
1
LCMS m/z 260/262 [M + H]+; H NMR (DMSO-d6, 400 MHz) δ
8.23 (br s, 1H), 7.72 (d, 1H, J = 8.7 Hz), 7.35 (d, 1H, J = 8.6 Hz),
3.31 (dt, 2H, J = 3.8, 6.3 Hz), 2.95 (t, 2H, J = 6.4 Hz); 13C NMR
(DMSO-d6, 101 MHz) δ 161.4, 141.9, 135.3, 132.8, 131.3, 128.8,
120.8, 37.9, 30.1; anal. calcd for C9H7BrClNO: C, 41.49; H, 2.71; N,
5.38; Br, 30.67; Cl, 13.61; found (batch 1): C, 41.64; H, 2.59; N,
5.39; Br, 30.92; Cl, 13.75; found (batch 2): C, 41.66; H, 2.56; N,
5.41; Br, 30.94; Cl, 13.56.
7.28−7.39 (m, 3H), 6.64 (s, 1H), 5.44 (s, 2H), 4.86 (s, 2H), 3.84 (s,
3H), 3.37 (t, J = 6.3 Hz, 2H), 2.77−2.85 (m, 2H), 2.42 (s, 3H), 2.36
(s, 3H), 2.21 (s, 3H).
5-Bromo-8-chloro-2-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-
yl)methyl]-7-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-3,4-dihydroisoqui-
nolin-1(2H)-one (2). A mixture of 2-{[2-(benzyloxy)-4,6-dimethyl-
pyridin-3-yl]methyl}-5-bromo-8-chloro-7-(1,4-dimethyl-1H-1,2,3-tria-
zol-5-yl)-3,4-dihydroisoquinolin-1(2H)-one (11, 1.32 g, 2.27 mmol)
in TFA (15 mL) was stirred at 50 °C for 1 h. Excess TFA was
removed. The resulting residue was treated with a few mL of 7 N
NH3/MeOH and concentrated. The resulting residue was dissolved in
MeOH and subjected to SFC to afford the title compound as a solid
(823 mg, 74% yield). LCMS m/z 490/492 [M + H]+; 1H NMR (400
MHz, DMSO-d6) δ ppm 11.56 (br. s., 1H), 7.90 (s, 1H), 5.90 (s, 1H),
4.57 (s, 2H), 3.78 (s, 3H), 3.55 (t, J = 6.1 Hz, 2H), 2.98 (t, J = 6.1 Hz,
2H), 2.19 (s, 3H), 2.14 (s, 3H), 2.09 (s, 3H).
5-Bromo-8-chloro-7-iodo-3,4-dihydroisoquinolin-1(2H)-one (9).
A 500 mL flask containing 8 (28.72 g, 110.2 mmol) was cooled in an
ice/water bath. Concentrated sulfuric acid (220 mL) was added,
followed by N-iodosuccinimide (61.62 g, 273.9 mmol). The cooling
bath was removed, and the reaction mixture was stirred at 20 °C for 2
h. The reaction was quenched by pouring into a flask containing
crushed ice (∼500 mL) using cold water (2 × 50 mL) to quantitate
the transfer. The mixture was stirred until all of the ice had melted,
and then the resulting precipitate was collected by suction filtration.
The flask and filter cake were rinsed with cold water (2 × 50 mL), and
the precipitate was air-dried overnight in the filter funnel. The
partially dried precipitate was stirred vigorously in a biphasic mixture
of 0.5 M sodium hydroxide (400 mL) and heptane (200 mL) at room
temperature for 1 h, and then the solids were collected by suction
filtration. The filter cake was rinsed with deionized water (2 × 100
mL) and then with heptane (200 mL) and air-dried until no longer
damp.
In Vivo Studies. All procedures performed on these animals were
in accordance with regulations and established guidelines and were
reviewed and approved by Pfizer’s Institutional Animal Care and Use
Committee.
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
ASSOCIATED CONTENT
ACKNOWLEDGMENTS
■
■
sı
We thank Dr. Simon Bailey and Dr. Martin Wythes for their
helpful discussions during the course of this work. We
acknowledge Patrick Bingham and Lisa Nguyen for generating
biochemical and cell data. We also acknowledge the
Bioanalytical Group in Pharmacokinetics, Dynamics and
Metabolism (Pfizer Worldwide Research & Development,
Groton, CT) for bioanalytical analyses.
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02112.
Compound formulation methods; in vivo mouse
pharmacokinetic parameters associated with Figure
4a,b; general experimental details for Karpas-422 TGI
studies; protein binding and in vivo exposure methods;
single-crystal X-ray data for compound 5; H NMR
spectrum of compound 5; and HPLC spectra of
compounds 5 and 2 (PDF)
1
ABBREVIATIONS USED
■
APCI, atmospheric-pressure chemical ionization; AUC, area
under the curve; BID, twice daily; DMF, dimethylformamide;
DNA, deoxyribonucleic acid; EtOAc, ethyl acetate; EZH2,
enhancer of zeste homolog 2; HPLC, high-pressure liquid
chromatography; LCMS, liquid chromatography-mass spec-
trometry; LOQ, limit of quantitation; MC, methylcellulose;
NMR, nuclear magnetic resonance; PK, pharmacokinetics;
PRC2, polycomb-repressive complex 2; QD, once daily;
SEDDS, self-emulsifying drug delivery system; SET, Su(var)-
3-9, enhancer of zeste and trithorax; SFC, supercritical fluid
chromatography; TGI, tumor growth inhibition; TID, three
times daily; TLC, thin-layer chromatography
Molecular formula strings of compounds 5 and 2 (CSV)
Compound purity table_EZH2 prodrug (XLSX)
Accession Codes
The crystal structure of compound 5 has been deposited with
the Cambridge Crystallographic Data Centre (CCDC
Deposition Number 2027474).
AUTHOR INFORMATION
■
Corresponding Author
Pei-Pei Kung − Oncology Chemistry, Pfizer Global Research
and Development, La Jolla Laboratories, San Diego,
California 92121, United States; orcid.org/0000-0002-
9203-7374; Email: PKung1@its.jnj.com
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Joseph H. Lee − Oncology Research Unit, Pfizer Global
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Scott C. Sutton − Oncology Chemistry, Pfizer Global Research
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Shinji Yamazaki − Pharmacokinetics, Dynamics, and
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Luke Zehnder − Oncology Chemistry, Pfizer Global Research
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02112
Notes
(7) Taplin, M.-E.; Hussain, A.; Shah, S.; Shore, N. D.; Edeneld, W.;
Agrawal, M.; Clark, W.; Nordquist, L.; Sartor, A. O.; Wise, D. R.;
The authors declare no competing financial interest.
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