Discovery of A-893, A New Cell-Active Benzoxazinone Inhibitor of Lysine Methyltransferase SMYD2

Article abstract of DOI:10.1021/acsmedchemlett.5b00124

A lack of useful small molecule tools has precluded thorough interrogation of the biological function of SMYD2, a lysine methyltransferase with known tumor-suppressor substrates. Systematic exploration of the structure-activity relationships of a previous

Full text of DOI:10.1021/acsmedchemlett.5b00124

Letter
pubs.acs.org/acsmedchemlett
Discovery of A‑893, A New Cell-Active Benzoxazinone Inhibitor of
Lysine Methyltransferase SMYD2
Ramzi F. Sweis,*,† Zhi Wang,† Mikkel Algire,† Cheryl H. Arrowsmith,‡,§ Peter J. Brown,‡ Gary G. Chiang,†
Jun Guo,† Clarissa G. Jakob,† Steven Kennedy,‡ Fengling Li,‡ David Maag,† Bailin Shaw,†
Nirupama B. Soni,† Masoud Vedadi,‡,∥ and William N. Pappano†
†Discovery Research, AbbVie, Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States
‡Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada
§Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 2M9, Canada
∥Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada
S
* Supporting Information
ABSTRACT: A lack of useful small molecule tools has
precluded thorough interrogation of the biological function of
SMYD2, a lysine methyltransferase with known tumor-
suppressor substrates. Systematic exploration of the struc-
ture−activity relationships of a previously known benzox-
azinone compound led to the synthesis of A-893, a potent and
selective SMYD2 inhibitor (IC50: 2.8 nM). A cocrystal
structure reveals the origin of enhanced potency, and effective
suppression of p53K370 methylation is observed in a lung
carcinoma (A549) cell line.
KEYWORDS: Epigenetics, SMYD2, H3K36, p53, methyltransferase, lysine
SET and MYND domain-containing protein 2 (SMYD2) is a
lysine methyltransferase that is highly expressed in esophageal
squamous cell carcinoma and pediatric acute lymphoblastic
leukemia.1,2 Elevated expression in both of these cancers
correlates with a poor prognosis. Its function as a
methyltransferase has been reported to span a variety of
substrates including histone H3 (K363 and K44), retinoblasto-
ma tumor suppressor (Rb) (K860),5 and tumor suppressor p53
(K370).6,7 It is through these processes that SMYD2 is believed
to play a role in the network of post-translational modifications
that regulates tumor growth. In particular, SMYD2 may serve as
a putative oncogene by its methylation of lysine 370 of p53,
which is repressive to p53 function.
Despite the potential role of SMYD2 methylation in
epigenetic regulation of the aforementioned cancers, no small
molecule inhibitors of this enzyme were known until the
disclosure of AZ505 (1) a few years ago (Figure 1).8 This
Figure 1. Published inhibitors of SMYD2.
benzoxazinone was identified from a high-throughput screen
(HTS) and shown via a cocrystal structure to bind to the lysine
pocket of SMYD2. The potency of 1 toward SMYD2 was
disclosed to be moderate (IC50: 120 nM) despite being quite
selective (>700-fold) over six other methyltransferases. More
recently, an additional SMYD2 inhibitor representing a new
chemotype was published as a probe of the Structural
Genomics Consortium (Figure 1).9 LLY-507 (2) exhibited
much improved potency (IC50 < 15 nM) and inhibited
p53K370 methylation in cells.
As compound 1 represented the only known inhibitor of
SMYD2 until recently, we pursued an avenue of investigation
that systematically interrogated all binding elements of this
molecule in order to inspire development of a more potent
inhibitor.
Received: March 23, 2015
Accepted: April 29, 2015
© XXXX American Chemical Society
A
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At the outset of this endeavor, we sought to address
structural changes in three regions of the molecule (Figure 2).
Table 1. Structure−Activity Relationship of Benzoxazinone
Derivatives
Figure 2. Published binding features of SMYD2 inhibitor 1 (AZ505).
The first region comprises the benzoxazinone subunit. It
occupies the lysine binding channel and contributes to overall
binding via a water-bridged hydrogen bond of its hydroxyl and
ring amine functionalities to the backbone of residues V202 and
M205. In addition, its ether oxygen participates in another
water-bridged hydrogen bond interaction with the amine linker
of the molecule and N180, G183, and Y258 of the enzyme. The
second and third regions of the molecule contain the
cyclohexylamine and dichlorophenyl subunits, respectively.
These moieties fill two hydrophobic pockets outside of the
immediate lysine channel. Of note is a third water-bridged
interaction between the amine-linker, flanked by regions 1 and
2, and side-chain N180.
To establish structure−activity relationships (SAR) in these
three regions, a scintillation proximity assay (SPA) was utilized,
which measured methylation of the p53 peptide by SMYD2
(Table 1). At the outset, we postulated that the cyclohexyl and
dichlorobenzyl subunits of compound 1 (occupying regions 2
and 3) did not significantly contribute much to the overall
binding efficiency of the molecule. As such, the first targeted
analogues were made to evaluate the effect of subtle changes, or
omission, to these regions. Advanced intermediate 4 could be
generated from 2-nitrobenzene-1,3-diol, following a published
chemistry route, in order to enable SAR at these regions
(Scheme 1).10 This benzoxazinone was then subjected to
standard reductive amination conditions with a set of
aldehydes, represented by 5, utilizing sodium triacetoxybor-
ohydride. This installed variance at the R1 position on the
central amine. Following temporary Boc-protection of the
resultant secondary amine with di-tert-butyl dicarbonate, a 1,4-
addition with a second set of amines enabled divergence at the
R2 position as represented by compound 7.
Surprising sensitivity was observed to subtle changes at
positions R1 and R2. The potency of compound 1 (IC50: 230
nM) was in line with reported data (IC50: 120 nM). Shortening
the linker length by one carbon atom provided benzylamine
derivative 8. Over 10-fold potency was lost with this
modification (IC50: 3.1 μM). A more conservative change by
removing only the ortho-chlorine atoms resulted in compound
9, with slightly reduced but comparable potency (IC50: 440
nM). Using this as a template, the central region encompassing
R1 was evaluated. Substituting the cyclohexane from 9 with a
cyclopentane, as in 10, led to a significant loss in potency (IC50:
2.2 μM). This negative effect was further magnified by
cyclobutyl derivative 11, which was over 10-fold less potent
than 10. Finally, in the interest of reducing the molecular
weight, complete omission of the dichlorophenyl subunit of R2
Scheme 1. Synthesis of Benzoxazinone Derivatives of
Compound 1
a
a
Reagents and conditions: (a) Na(OAc)3BH, NMP, rt; (b) di-tert-
butyl dicarbonate, EtOAc, H2O, rt, 17−43% (over 2 steps); (c) R2OH
or R2NH2, EtOH, 50 °C, 42−88%; (d) trifluoroacetic acid, EtOH, rt,
48−92%.
was evaluated by installing a methoxy-group in its place (12).
This analogue was inactive against SMYD2.
This initial SAR made it apparent that reducing the size of
the subunits that occupy the hydrophobic pockets of regions 2
and 3 was generally deleterious to activity. As such, the
investigation shifted focus to changes at the benzoxazinone
subunit that occupied the lysine channel. A slightly altered
synthetic approach was employed to install diversity at the
lysine channel region at the end of the synthesis (Scheme 2).
This multistep sequence commenced with the addition of 2-
B
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observed (IC50: 5.0 μM). The ethyl-linked pyrrolidine was
likely too short to properly occupy the lysine channel.
Accordingly, propyl-pyrrolidine 18 was evaluated and found
to have more favorable IC50 of 1.4 μM. The slightly larger
piperidine replacement, 19, was less effective (IC50: 2.4 μM).
This survey revealed that replacements to the benzoxazinone
subunit of 1 were capable of maintaining SMYD2 inhibition,
albeit with less potency.
Scheme 2. Synthesis of Analogues with Alternative Subunits
to the Benzoxazinone of 1
a
Before embarking on a more extensive analysis of
benzoxazinone replacements, we first sought to address the
relative importance of each heteroatom in the benzoxazinone
itself in order to inspire a more promising structural change.
The first facet of this evaluation involved removing the
hydroxyl-group, which was reported to participate in a water-
bridged hydrogen bond with the backbone of residues V202
and M205 (Figure 1). Similar chemistry to that employed for
the previous benzoxazinone analogues was utilized, with the
exception that the hydroxyl-group in question was removed
(Scheme 3). This route led to intermediate azide 20, which we
attempted to reduce to the primary amine en route to the des-
hydroxy analogue 21.
a
Reagents and conditions: (a) 2-(3,4-dichlorophenyl)ethanamine,
ethanol, rt 85%; (b) Et3N, benzylchloroformate, CH2Cl2, rt, 71%;
(c) CF3CO2H, CH2Cl2, rt, 80%; (d) HOBt, EDC, (i-Pr)2NEt, N-(2,2-
dimethoxyethyl)-cyclohexanamine, DMF, rt, 61%; (e) p-toluenesul-
fonic acid, CH2Cl2, rt, 84%; (f) RNH2, Na(OAc)3BH, CH2Cl2, 0 °C−
rt, 68−87%; (g) BBr3, CH2Cl2, 0 °C−rt, 53−64%.
(3,4-dichlorophenyl)ethanamine to tert-butyl acrylate followed
by protection of the resultant secondary amine with
benzylchloroformate. Subsequent removal of the tert-butyl
ester with trifluoroacetic acid furnished carboxylic acid 14. This
was then coupled to N-(2,2-dimethoxyethyl)cyclohexylamine
using hydroxybenzotriazole (HOBt)/1-ethyl-3-(3-
(dimethylamino)propyl)carbo-diimide (EDC), followed by
acetal cleavage to yield aldehyde 15. This advanced
intermediate was then used for installing benzoxazinone
replacements (as in 16) via a two-step sequence: reductive
amination with sodium triacetoxyborohydride and deprotection
of the secondary amine with boron tribromide.
Scheme 3. Synthesis of A-893 Arising from Exclusive
Formation of Intermediate 22
The initial replacements of the benzoxazinone subunit of
compound 1 were envisioned to possess a simpler secondary
amine mimic of methyl lysine (Table 2).11 The first example of
this was pyrrolidine 17, as it represents a common motif of
methyltransferase inhibitors that effectively binds to the lysine-
peptide site.12−14 Unfortunately, a >20-fold loss in activity was
Table 2. Structure−Activity Relationship of Benzoaxazinone
Replacements
Not unexpectedly, hydroxyethyl amine 22 was the major
product observed from this step. The lack of a benzoxazinone
hydroxyl group removed the stabilization of a quinone-methide
intermediate during the reduction,15 rendering the newly
formed benzyl hydroxyl group, 22, with a lower propensity
toward further reduction. As a result, we chose to take forward
22, following chiral separation, via the same sequence as shown
in Scheme 1 to furnish separated enantiomers 23a and 23b.
The resultant potency of these compounds was remarkable.
The hydroxyl-substituted derivative 23a inhibited SMYD2 with
an IC50 of 2.8 nM. Its enantiomer was less efficacious, but
nonetheless far more potent than any analogue observed in this
series (IC50: 5.6 nM). The >80-fold enhancement in potency
was fortuitous, yet unexpected. We next investigated the
mechanism of action to assess whether this change in structure
affected its ability to be a peptide competitive inhibitor (Figure
3). The dependence of IC50 on peptide and SAM
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concentrations suggests that 23a retains the same mechanism
of inhibition (peptide-competitive) as inhibitor 1.
its relative importance to overall binding is eclipsed by the
enhanced coordination brought by the secondary alcohol.
In order to assess the ability of 23a to serve as a meaningful
probe of SMYD2 biology, we evaluated its selectivity against a
panel of 31 other methyltransferases (Figure 5). Three
Figure 3. Peptide-competitive and SAM-uncompetitive inhibition of
SMYD2 by 23a.
The dramatic increase in potency resulting from a modest
change in structure highlighted the possibility of a critical
hydrogen bond interaction accessed by the newly installed
secondary alcohol. To illuminate the key features of binding
with this new compound, we obtained a cocrystal structure of
SMYD2 with inhibitor 23a (Figure 4). Overall, the three
Figure 5. Selectivity of A-893 (23a). The inhibition of 30
methyltransferases at various concentrations of compound.
concentrations were chosen: 1, 10, and 50 μM. No significant
(>50%) inhibition was observed for any other methyltransfer-
ase at 1 μM. At 10 and 50 μM, inhibition values between 65%
and 80% were observed for PRMT3, PRMT7, SUV39H2, and
SMYD3. The exceptional selectivity observed with A-893 (23a)
rendered it as a suitable compound to evaluate inhibition of
SMYD2 in cells.
The functional consequence of SMYD2 inhibition was
interrogated using both A-893 (23a) and AZ505 (1) in a p53
cellular assay.16 Human A549 lung carcinoma cells were chosen
due to their high SMYD2 expression levels and wild-type p53
status.17 After 18 h of treatment with 10 μM of compound,
changes in p53K370me1 were measured along with changes to
overall p53 levels (Figure 6). The ratio of p53K370me1 to
overall p53 levels was reduced, as expected, by treatment with
either A-893 or AZ505 (Figure 6, part A). While this
unexpectedly depicts a more robust response with AZ505,
further dissection of the data provides clarity into the origin of
this. While overall p53 levels were unaffected by A-893, a
surprising >3-fold increase was observed with AZ505 (Figure
6B). Analysis of p53K370me1 levels reveals that inhibitor A-
893 exhibited 42% reduction in the methyl mark, while AZ-505
was slightly less effective at 28% reduction (Figure 6C).
Accordingly, while the p53K370me1/p53 ratio looks far
lower with AZ505 (Figure 6A), this is predominantly a result of
an overall increase in p53. This may reflect an off-target
phenotype that fortuitously is not present in A-893. The results
of this experiment suggest that A-893 cleanly targets p53K370
methylation, without convolution, and highlight its usefulness
as a cellular probe of SMYD2 function.
Figure 4. X-ray cocrystal structure of 23a and SMYD2, revealing the
orientation of key residues around the hydroxyethylamine linker and
the benzoxazinone moieties.
regions of SMYD2 that are occupied with 23a are generally
unchanged from compound 1. The central cyclohexylamine and
the terminal dichlorophenyl group occupy the two hydrophobic
pockets of aforementioned regions 2 and 3, respectively (from
Figure 2).
In the lysine channel, the ether oxygen of the benzoxazinone
accepts a hydrogen bond from the side-chain hydroxyl of Y258,
and the ring amine maintains a water-bridged interaction, albeit
without a nearby ring-hydroxyl group that was present in 1.
The secondary amine of the linker region also participates in an
electrostatic interaction with Y258. The newly installed benzylic
alcohol accesses a hydrogen bond with the backbone carbonyl
group of Y240. In evaluating this network of binding, the
enhanced potency of 23a over 1 reflects the more coordinated
nature of the flexible linker region, attained by additional
hydrogen bond of the alcohol. Conversely, the loss of the
benzoxazinone hydroxyl group that was present in 1 does not
have a deleterious impact on binding, perhaps due to its
redundancy in the water-bridged interaction engaged by the
amine segment of the benzoxazinone ring. Irrespective of this,
In summary, an analysis of the critical binding elements of
the first reported SMYD2 inhibitor, AZ505 (1), via structure−
activity relationships, has been conducted. Three regions of the
molecule were systematically varied (Figure 2), and the results
show large changes in activity from relatively modest changes in
structure for regions 2 and 3, which predominantly function by
D
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details are provided pertaining to X-ray
crystallography, biological assays, and the synthesis and
characterization of all compounds. The Supporting Information
is available free of charge on the ACS Publications website at
DOI: 10.1021/acsmedchemlett.5b00124.
AUTHOR INFORMATION
Corresponding Author
*E-mail: ramzi.sweis@abbvie.com. Tel: 847-937-0315.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
The design, study conduct, and financial support for this study
were provided by AbbVie and the SGC. AbbVie and the SGC
participated in the data generation, interpretation of data,
review, and approval of this publication. The SGC is a
registered charity (no. 1097737) that receives funds from
AbbVie, Bayer, Boehringer Ingelheim, Genome Canada
through Ontario Genomics Institute Grant OGI- 055,
GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research
Foundation, the Ontario Ministry of Economic Development
and Innovation, Pfizer, Takeda, and Wellcome Trust Grant
092809/Z/10/Z.
Notes
The authors declare the following competing financial interest:
R.F.S., Z.W., M.A., G.G.C., J.G., C.G.J., D.M., B.S., N.B.S., and
W.N.P. are employees of AbbVie, Inc. C.H.A., P.J.B., S.K., F.L.,
and M.V. receive funding from the Structural Genomics
Consortium, University of Toronto, Toronto, Canada.
REFERENCES
■
Figure 6. Changes to p53K370me1 and overall p53 levels in A549
cells after treatment with SMYD2 inhibitors.
(1) Komatsu, S.; Imoto, I.; Tsuda, H.; Kozaki, K.; Muramatsu, T.;
Shimada, Y.; Aiko, S.; Yoshizumi, Y.; Ichikawa, D.; Otsuji, E.; Inazawa,
J. Overexpression of SMYD2 relates to tumor cell proliferation and
malignant outcome of esophageal squamous cell carcinoma. Carcino-
genesis 2009, 30, 1139−1146.
(2) Sakamoto, L. H. T.; de Andrade, R. V.; Felipe, M. S. S.;
Motoyama, A. B.; Silva, F. P. SMYD2 is highly expressed in pediatric
acute lymphoblastic leukemia and constitutes a bad prognostic factor.
Leuk. Res. 2014, 38, 496−502.
(3) Brown, M. A.; Sims, R. J., III; Gottlieb, P. D.; Tucker, P. W.
Identification and characterization of Smyd2: a split SET/MYND
domain-containing histone H3 lysine 36-specific methyltransferase
that interacts with the Sin3 histone deacetylase complex. Mol. Cancer
2006, 5, 26.
(4) Abu-Farha, M.; Lambert, J.-P.; Al-Madhoun, A. S.; Elisma, F.;
Skerjanc, I. S.; Figeys, D. The tale of two domains: proteomics and
genomics analysis of SMYD2, a new histone methyltransferase. Mol.
Cell. Proteomics 2008, 7, 560−572.
(5) Saddic, L. A.; West, L. E.; Aslanian, A.; Yates, J. R., III; Rubin, S.
M.; Gozani, O.; Sage, J. Methylation of the retinoblastoma tumor
suppressor by SMYD2. J. Biol. Chem. 2010, 285, 37733−37740.
(6) Huang, J.; Perez-Burgos, L.; Placek, B. J.; Sengupta, R.; Richter,
M.; Dorsey, J. A.; Kubicek, S.; Opravil, S.; Jenuwein, T.; Berger, S. L.
Repression of p53 activity by Smyd2-mediated methylation. Nature
2006, 444, 629−632.
occupying hydrophobic pockets in the enzyme. Analysis of
region 1, the lysine channel binding site, reveals the importance
of several key hydrogen bond interactions provided by the
benzoxazinone subunit. Installation of a secondary alcohol in
the linker region of the molecule led to A-893 (23a), a
compound with an unexpectedly notable improvement in
potency (>80-fold). This molecule was found to function as a
peptide-competitive inhibitor and exhibited a high degree of
selectivity over a panel of 30 additional methyltransferases. An
analysis of the cocrystal structure of A-893 highlights the
contribution of the newly−installed hydroxyl group to an
intricate network of hydrogen bonds around the lysine pocket.
Most notably, A-893 represents a viable cell-active probe to
explore SMYD2 function. It reduced p53 methylation levels by
42% in A549 cells and is devoid of the confounding effect of
total p53 protein elevation as observed with AZ505. The
insights gained in the discovery of inhibitor A-893 (23a)
provide the foundation for continued refinement and develop-
ment of the next-generation of SMYD2 inhibitors
(7) Other histones (H2B and H4) have also been reported as
substrates of SMYD2 methylation: Wu, J.; Cheung, T.; Grande, C.;
Ferguson, A. D.; Zhu, X.; Theriault, K.; Code, E.; Birr, C.; Keen, N.;
Chen, H. Biochemical characterization of human SET and MYND
E
DOI: 10.1021/acsmedchemlett.5b00124
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
domain-containing protein 2 methyltransferase. Biochemistry 2011, 50,
6488−6497.
(8) Ferguson, A. D.; Larsen, N. A.; Howard, T.; Pollard, H.; Green,
I.; Grande, C.; Cheung, T.; Garcia-Arenas, R.; Cowen, S.; Wu, J.;
Godin, R.; Chen, H.; Keen, N. Structural basis of substrate methylation
and inhibition of SMYD2. Structure 2011, 19, 1262−1273.
(9) Nguyen, H.; Allali-Hassani, A.; Antonysamy, S.; Chang, S.; Chen,
L. H.; Curtis, C.; Emtage, S.; Fan, L.; Gheyi, T.; Li, F.; Liu, S.; Martin,
J. R.; Mendel, D.; Olsen, J. B.; Pelletier, L.; Shatseva, T.; Wu, S.;
Zhang, F. F.; Arrowsmith, C. H.; Brown, P. J.; Campbell, R. M.;
Garcia, B. A.; Barsyte-Lovejoy, D.; Mader, M.; Vedadi, M. LLY-507, a
cell-active, potent and selective inhibitor of protein lysine methyl-
transferase SMYD2. J. Biol. Chem. 2015, DOI: 10.1074/
jbc.M114.626861.
(10) Alcaraz, L.; Bonnert, R. V.; Connolly, S.; Cook, A. R.; Fisher, A.;
Humphries, A.; Raubo, P. Amine derivatives and their use in beta-2-
adrenoreceptor mediated diseases. WO 2008/075025 A1, June 26,
2008.
(11) Herold, J. M.; Wigle, T. J.; Norris, J. L.; Lam, R.; Korboukh, V.
K.; Gao, C.; Ingerman, L. A.; Kireev, D. B.; Senisterra, G.; Vedadi, M.;
Tripathy, A.; Brown, P. J.; Arrowsmith, C. H.; Jin, J.; Janzen, W. P.;
Frye, S. V. Small-molecule ligands of methyl-lysine binding proteins. J.
Med. Chem. 2011, 54, 2504−2511.
(12) Vedadi, M.; Barsyte-Lovejoy, D.; Liu, F.; Rival-Gervier, S.; Allali-
Hassani, A.; Labrie, V.; Wigle, T. J.; DiMaggio, P. A.; Wasney, G. A.;
Siarheyeva, A.; Dong, A.; Tempel, W.; Wang, S.-C.; Chen, X.; Chau, I.;
Mangano, T. J.; Huang, X.; Simpson, C. D.; Pattenden, S. G.; Norris, J.
L.; Kireev, D. B.; Tripathy, A.; Edwards, A.; Roth, B. L.; Janzen, W. P.;
Garcia, B. A.; Petronis, A.; Ellis, J.; Brown, P. J.; Frye, S. V.;
Arrowsmith, C. H.; Jin, J. A chemical probe selectively inhibits G9a
and GLP methyltransferase activity in cells. Nat. Chem. Biol. 2011, 7,
566−574.
(13) Sweis, R. F.; Pliushchev, M.; Brown, P. J.; Guo, J.; Li, F.; Maag,
D.; Petros, A. M.; Soni, N. B.; Tse, C.; Vedadi, M.; Michaelides, M. R.;
Chiang, G. G.; Pappano, W. N. Discovery and development of potent
and selective inhibitors of histone methyltransferase G9a. ACS Med.
Chem. Lett. 2014, 5, 205−209.
(14) Liu, F.; Barsyte-Lovejoy, D.; Li, F.; Xiong, Y.; Korboukh, V.;
Huang, X.; Allali-Hassani, A.; Janzen, W. P.; Roth, B. L.; Frye, S. V.;
Arrowsmith, C. H.; Brown, P. J.; Vedadi, M.; Jin, J. Discovery of an in
vivo chemical probe of the lysine methyltransferases G9a and GLP. J.
Med. Chem. 2013, 56, 8931−8942.
(15) Toteva, M. M.; Moran, M.; Amyes, T. L.; Richard, J. P.
Substituent effects on carbocation stability: The pKR for p-quinone
methide. J. Am. Chem. Soc. 2003, 125, 8814−8819.
(16) Total p53 Assay Whole Cell Lysate Kit (MSD). https://www.
mesoscale.com/en/products/k150dbd-3/.
(17) O’Connor, P. M.; Jackman, J.; Bae, I.; Myers, T. G.; Fan, S.;
Mutoh, M.; Scudiero, D. A.; Monks, A.; Sausville, E. A.; Weinstein, J.
N.; Friend, S.; Fornace, A. J., Jr.; Kohn, K. W. Characterization of the
p53 tumor suppressor pathway in cell lines of the National Cancer
Institute Anticancer Drug Screen and correlations with the growth-
inhibitory potency of 123 anticancer agents. Cancer Res. 1997, 56,
4285−4300.
F
DOI: 10.1021/acsmedchemlett.5b00124
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