Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit

Article abstract of DOI:10.1021/jm3010515

The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogat

Full text of DOI:10.1021/jm3010515

Article
pubs.acs.org/jmc
Identification of a Chemical Probe for Bromo and Extra C‑Terminal
Bromodomain Inhibition through Optimization of a Fragment-
Derived Hit
Paul V. Fish,†,⊥ Panagis Filippakopoulos,‡ Gerwyn Bish,† Paul E. Brennan,‡ Mark E. Bunnage,†,#
Andrew S. Cook,† Oleg Federov,‡ Brian S. Gerstenberger,§ Hannah Jones,∥ Stefan Knapp,‡
Brian Marsden,‡ Karl Nocka,∥ Dafydd R. Owen,*,†,# Martin Philpott,‡ Sarah Picaud,‡
Michael J. Primiano,∥ Michael J. Ralph,† Nunzio Sciammetta,† and John D. Trzupek∥
†Pfizer Worldwide Medicinal Chemistry, Pfizer Worldwide R&D, Ramsgate Road, Sandwich CT13 9NJ, United Kingdom
‡Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom
§Pfizer Worldwide Medicinal Chemistry, Pfizer Worldwide R&D, Eastern Pont Road, Groton, Connecticut 06340, United States
∥Pfizer Worldwide R&D, 35/200 Cambridgepark Drive, Cambridge, Massachusetts 02140, United States
S
* Supporting Information
ABSTRACT: The posttranslational modification of chroma-
tin through acetylation at selected histone lysine residues is
governed by histone acetyltransferases (HATs) and histone
deacetylases (HDACs). The significance of this subset of the
epigenetic code is interrogated and interpreted by an
acetyllysine-specific protein−protein interaction with bromo-
domain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a
small molecule is feasible, and this may represent an opportunity for disease intervention through the recently disclosed
antiproliferative and anti-inflammatory properties of such inhibitors. Herein, we describe the discovery and structure−activity
relationship (SAR) of a novel, small-molecule chemical probe for BET family inhibition that was identified through the
application of structure-based fragment assessment and optimization techniques. This has yielded a potent, selective compound
with cell-based activity (PFI-1) that may further add to the understanding of BET family function within the bromodomains.
range of functions.6 Bromodomains are the only modules that
can specifically recognize acetylated linear motifs. They are
found within large multidomain nuclear proteins tasked with
INTRODUCTION
■
Bromodomains are protein interaction modules that recognize
the ε-N-acetylation state of specific lysine residues found within
controlling processes such as methyl transfer, transcription
histone tails and other proteins.1 Histones are lysine-rich
coactivation, and motor protein (helicase) activity. There are 61
proteins that, left unmodified, are highly basic in character.
Modification of these hallmark lysine residues through
acetylation or iterative methylation causes changes in the
bromodomains in the human proteome and these are further
classified into eight families, one of which is the bromodomain
and extra C-terminal domain (BET) family.7 BET family
structural and physicochemical properties of the histone
protein,2,3 affecting the structure of nucleosomes that organize
function has been studied through protein expression
quantification or knockdown experiments of individual BET
the protein−DNA hybrid arrangement. Alteration of these ε-
family members. This has highlighted the control of processes
NH2 sites on lysine represents the protein-held part of the
that mediate cancer,8 inflammation,9 and viral infection,10
epigenetic code within the chromatin of each cell nucleus. The
among others. This renders the bromodomains within the BET
acetylation level of these initially basic residues is controlled by
protein family attractive targets for drug discovery, at least in
the action of histone acetyltransferases (HATs) and histone
the context of potential efficacy.1 Chemical probes against
deacetylases (HDACs); however, the significance of these
bromodomain families, or perhaps in time highly selective
modulations is relayed by the bromodomains and their histone
tail recognition function.4 The consequences of this acetylation
inhibitors of each of the 61 family members, will be useful tools
in fully establishing the role of these proteins. Chemical probes
reading process may trigger further remodeling at the
that can help validate the potential efficacy and, of equal
epigenetically modifiable sites within the protein or DNA
importance, safety of bromodomain inhibition will be of great
components of chromatin, ultimately manifesting themselves in
transcriptional activity control.5 The number of proteins
susceptible to lysine ε-NH2 acetylation state changes reaches
Received: July 18, 2012
into the thousands, and these have been shown to play a diverse
Published: October 25, 2012
© 2012 American Chemical Society
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utility in this emerging target class. The first chemotypes for
BET family inhibitors have recently been disclosed by members
of this group11 and others.12,13 The structurally related
triazolodiazepine compounds (+)-JQ1 1 and I-BET762 2
(Figure 1) are the most potent (nanomolar), BET family-
protein-coupled receptors (GPCRs), enzymes, and ion
channels.
RESULTS AND DISCUSSION
■
Despite the protein−protein interaction nature of the histone
and its associated bromodomain reader, the pockets for binding
acetylated lysine within the bromodomain proteins are
sufficiently deep and defined to potentially accommodate
inhibitory small molecules with high affinity and efficiency.7
Given this potential, and the knowledge that acetylated lysine is
the endogenous binding substrate for the pocket, we (and
others)15,17 embarked on screening fragment-sized potential
acetyllysine mimetics that may be weak, nonselective pan-
bromodomain binders. Much like starting points for Conway
and co-workers15 and Chung and co-workers,17 3,4-dihydro-
3methyl-2(1H)-quinazolinones seemed like attractive frag-
ments to test this hypothesis. Commercially available 3,4-
dihydro-3-methyl-2(1H)-quinazolinone 7 [also identified by
Chung as a bromodomain fragment, leading to a BRD2(1)
cocrystal deposition in the Protein Data Bank, accession code
4A9E]17 and its 6-brominated equivalent 8 both proved to be
active in a peptide displacement biochemical assay (Al-
phaScreen format) in the 30−90 μM range against BRD4
and cyclic AMP response binding element binding protein
(CREBBP).18 They were not universally active in all
bromodomains screened in this format, with no activity seen
at 200 μM in the BAZ2B and FALZ AlphaScreens.
Figure 1. First-generation BET family inhibitors.
selective, and cell-active BET family inhibitors disclosed thus
far. I-BET762 has recently entered clinical trials for NUT
midline carcinoma.14
(+)-JQ1 1 showed anti-cancer activity in patient-derived
xenografts,11 while I-BET762 exhibited anti-inflammatory
effects in mice.12 A small number of orthogonal chemotypes
have also been recently disclosed. Conway and co-workers15
(compounds 3 and 4, Figure 2) and Prinjha and co-workers16
With sub-30 μM pharmacology found in such small
molecules, the consequent ligand efficiencies19 of >0.45 made
for a very promising fragment-derived starting point. The 3,4-
dihydro-3-methyl-2(1H)-quinazolinone screening lead 3 of
Conway and co-workers15 (larger than conventional fragments)
had the ambiguity of two potential binding modes as it also
contained a 3,5-dimethylisoxazole at the 6-position of the
bicyclic core. This question was solved through their first crystal
structure in BRD4(1), which also showed the unexpected
oxidative incorporation of an ethylene glycol unit under the
conditions of crystallization. The compound to actually
cocrystallize was found to have the structure 4 (Figure 2).
This structure showed the 3,5-dimethylisoxazole making the
key interaction at the base of the binding pocket, accepting a
hydrogen bond from Asn140. The 3,5-dimethylisoxazole was
then optimized further by Conway and co-workers,15 moving
away from the dihydroquinazolinone substructure found in the
other half of the original screening lead 3.
Figure 2. Orthogonal bromodomain chemotype hits with BRD4
activity.
The smaller fragments 7 and 8 screened in our campaign
(featuring only H and Br at the core’s 6-position) contained the
cyclic urea as the only potential interaction functionality
available to Asn140. Chronologically in our study, a crystal
structure was first solved for 8 in CREBBP (not a BET family
member). This was followed up by crystallizing 8 in BRD4(1)
in a 1.63 Å structure, with no oxidative reactivity observed in
the crystallization protocol. Both the CREBBP and BRD4(1)
crystal structures for 8 showed the cyclic urea buried in the
pocket mimicking the acetyl NH of the endogenous histone
lysine. It also overlaid well in comparison to the BRD2(1)
structure of 7 (PDB code 4A9E).17 This reconfirms that both
binding modes screened for by Conway are indeed possible and
that 3,4-dihydro-3-methyl-2(1H)-quinazolinones (6-bromi-
nated or not) are viable N-acetyl mimetics. The binding of 8
in BRD4 (1) (Figure 3) is characterized by a lipophilic
sandwich of the bicyclic core between residues Val87, Leu92,
Leu94, Tyr97 and Phe83, Ile146 in the protein. The cyclic urea
(I-BET151, 5, Figure 2) have independently described 3,5-
dimethylisoxazole as a viable acetyllysine mimetic in identifying
novel BET binders. Bamborough and Chung and co-workers17
have also described fragment-based approaches to identifying
novel chemotypes (such as compound 6, Figure 2) through a
designed selection of fragments with the potential to be N-
acetyllysine mimetics. This yielded novel hits of moderate
efficiency in this proof-of-principle study.
Herein we describe the discovery and optimization of a novel
BET family inhibitor chemotype from similar fragmentlike
starting points as those of Chung and co-workers.17 The most
optimized compounds disclosed meet the criteria we defined at
the outset of the collaboration for a chemical probe, that being
∼100−300 nM activity in a biochemical assay; cell-based
activity; family selectivity within bromodomains; and broader
polypharmacological selectivity against a panel of kinases, G
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Figure 3. Two views (A and C) of fragment hit 8 (B) cocrystallized in BRD4(1).
Figure 4. First-generation sulfonamide cocrystal structures in BRD4(1). (A) Fragment hit 8; (B) ethylsulfonamide 12; (C) anilinesulfonamide 14
with change in NHSO2 conformation; (D) pyrrolidinesulfonamide 13.
meets Asn140 slightly offset with a hydrogen bond accepted by
the urea carbonyl from the amino acid carboxamide.
protein structures is found away from the N-acetyl binding
residues. Although the bromine atom represents a useful
synthetic handle for many bond formations in its own right, the
unoccupied regions of the BRD protein in the crystal structure
suggested that inducing a pronounced kink in the 6-substituent
directly off the template by design may be productive. This was
undertaken with access to a vacant lipohilic shelf (the so-called
WPF shelf) in mind at the opening of the pocket. Sulfonamides
were selected as a linker to explore this conformational plan.
The availability of the 6-brominated variant of the original 3,4-
dihydro-3-methyl-2(1H)-quinazolinone suggests that reactivity
of the unsubstituted parent is directed through the 6-position
The binding mode implication of the cyclic urea interaction
with Asn140 consequently presents the core’s brominated 6-
position in the general direction of solvent. The fragment hits 7
and 8, along with elucidation of the binding mode through X-
ray crystallography, efficiently directed design toward replacing
the bromine. The possibility for further productive interactions
before reaching solvent through extending a larger substituent
from the 6-position vector was the basis for the next round of
molecular design. This was also expected to improve selectivity
for the BET family targets, as greater variation in bromodomain
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a
Scheme 1. Synthesis of Sulfonamide Variants
a
(i) ClSO3H, DCM, 0 °C. (ii) R1R2NH, DCM, RT. (iii) H2SO4, KNO3, 0 °C. (iv) Cat. Raney Ni, H2 (1 atm), AcOH, RT. (v) R3SO2Cl, pyridine,
DCM, RT. (vi) Cs2CO3, cat. [(Pd2(dba)3)/X-Phos], R3SO2NH2, dioxane, 100 °C.
during electrophilic aromatic substitution reactions. To explore
this proposed reactivity, two templates were synthesized with
sulfonamide hypotheses in mind. Commercially available 7 was
reacted with chlorosulfonic acid to produce a 6-sulfonyl
chloride template 9, and the known 6-nitro-3,4-dihydro-3-
methyl-2(1H)-quinazolinone 10 was resynthesized and then
reduced to the corresponding aniline 11 by hydrogenation. The
6-sulfonyl chloride 9 and the 6-amino 11 synthesized as
templates are novel compounds and make for attractive
monomers in their own right. In this program they were used
as templates in relatively small library designs where diversity
was drawn from the corresponding partner amine or sulfonyl
chloride monomer sets, held within the Pfizer company
collection.
(15−24) in Table 1 show BRD4(1) activity (180 nM−2.3 μM)
across a selection of regioisomers of both electron-withdrawing
and -donating substituents on a phenyl ring.
Table 1. Structure−Activity Relationship for BRD4 Activity
a
within the Series
The sulfonamides derived from the 6-sulfonylchloride
template were profiled first. Pyrrolidine 12 and ethylamine 13
sulfonamides were 5−20-fold more active than the parent
bromide. The pyrrolidine sulfonamide 12 had a respectable
ligand efficiency of 0.38. The aniline-derived sulfonamide 14
was of similar activity, suggesting that this sulfonamide
regioisomer was not efficiently delivering the lipophilicity into
the open WPF shelf. Co-crystal structures of this series show
alkylamine sulfonamides (12 and 13) adopting highly
comparable conformations on binding, suggesting a tolerated
sulfonamide replacement of the 6-bromide from fragment hit 8
(Figure 4). The inefficiency of the anilinic sulfonamide 14 is
explained by the noticeable change in the SO2 group
conformation as a consequence of the phenyl group
accommodating itself on the shelf. The electron density on
solving the crystal structure also showed multiple conforma-
tions of similar occupancy for compound 14, suggesting that
there was no single ideal binding mode for the aryl group.
Reversing the sulfonamide by use of the 6-amino template 11
produced a series of compounds with significantly improved
BRD4 activity. The abundance of readily available arylsulfonyl
chlorides weighted design in favor of aryl substituents. These
compounds showed a structure−activity relationship (SAR)
implying that they were consistently accessing the lipophilic
shelf as targeted in the design hypothesis. A complementary
synthesis of this type of compound was also possible from the
bromide fragment hit 8 through palladium-catalyzed coupling
of a primary arylsulfonamide (Scheme 1). The 10 examples
a
IC50 values are expressed with a 95% confidence interval derived from
a minimum of two determinations.
Four out of five of the most active compounds are derived
from ortho-substituted arylsulfonyl chlorides, although the
entire structure−activity relationship in Table 1 falls within 10-
fold of each other for compounds 15−24, regardless of the aryl
group used. Given the position of this group, now occupying
the solvent-exposed WPF shelf, the key improvement in activity
has been attained primarily through the use of the reversed
sulfonamide linker. The binding of the now optimally
orientated aryl group is less sensitive to substitution patterns
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around the ring system. Compound 17 (PFI-1) was selected as
the nominated chemical probe from the series for further
profiling, as it struck the best compromise between lipophilicity
and pharmacology (Figure 5).
assessed on the more elaborated compound 17 from the best
sulfonamide series as a surrogate for broader bromdomain
selectivity. Surface plasmon resonance (SPR) determined a KD
of 49 μM for compound 17 in CREBBP. A Tm shift assay in
CREBBP also showed a significantly lower melting temperature
for 17 of 1.7 °C at 10 μM. The BET family protein members
showed Tm shifts of 2.1−6.5 °C for compound 17. Tm shifts for
BAZ2B, PB1(5), and PCAF were less than 1 °C as
representative members of bromodomains more distantly
related to the BET family.21 Compound 17 is a selective
chemical probe for the BET bromodomains.
Figure 5. Chemical probe selected for profiling.
Having studied the activity of 17 as a representative member
of this BET family inhibitor series in biochemical and
biophysical assays, we further studied the compound’s
credentials as a chemical probe for broader pharmacological
selectivity, a cell-based inflammatory end-point assay, and
rodent pharmacokinetics. The compound showed <50%
inhibition against a panel of 15 targets made up of GPCRs,
ion channels, and enzymes at 10 μM at Cerep and <20%
inhibition against 50 kinases at 1 μM at Invitrogen. Cell-based
activity was tested in an LPS challenge assay in PBMCs.
Compound 17 had an EC50 of 1.89 μM (n = 6) for the
inhibition of IL6 production from human blood mononuclear
cells stimulated by LPS. Rodent pharmacokinetics for 17 were
studied in both rat (1 mg/kg iv and 2 mg/kg po) and mouse (2
mg/kg sc). After iv administration in the rat, the volume of
distribution was 1 L/kg and the plasma clearance was 18/
mL·min−1·kg−1, giving a 1 h half-life. The volume of
distribution is consistent with the physicochemical properties
of the compound, and the clearance is in line with estimates
from in vitro rat liver microsome (RLM) data (RLM t1/2 >120
min).22 After oral dosing in the rat, the oral bioavailability was
low at 32%. This is inconsistent with the clearance and is
believed to be due to suboptimal compound solubility in the
gut. This was further supported by the oral exposure profile in
rat, where the compound was present at a steady yet low
A 1.52 Å cocrystal structure was obtained for 17 in BRD4(1).
Details of the crystal structure have been deposited in the PDB
by members of this collaboration.20 The view shown in Figure 6
reveals a right-angled turn in the molecule induced by the
sulfonamide. The key interaction with Asn140 is maintained
and the urea carbonyl also makes a water-bridged hydrogen-
bond interaction with the phenolic OH of Tyr97.
The most potent example 15 (2-Cl, 4-F) is >200-fold more
active than the fragment hit. The ligand efficiency of the initial
hit has been largely preserved in the series, and this indicates
that improvements in potency have been attained through the
incorporation of appropriately balanced lipophilicity. Beyond
the expectation for improved potency through functionalizing
the 6-position of the template, there was a BET family
selectivity requirement for the novel series. The fragment hits 7
and 8 were unselective starting points for bromodomain
selectivity with respect to CREBBP. This is attributed to the
fact that, due to their small size, the fragments only occupy the
region close to the conserved asparagine residue common to all
the bromodomains and are unable to exploit binding site
differences elsewhere within some of the more closely related
bromodomain proteins. As one of those most closely related
non-BET family bromodomains,7 CREBBP activity was
Figure 6. Two views (A and B) of 17 (PFI-1) cocrystallized to 1.52 Å in BRD4(1).
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the reaction [via thin-layer chromatography (TLC) with 10% MeOH
in dichloromethane (DCM)], the reaction mixture was filtered
through a Celite pad and evaporated to provided the crude product.
The crude material was purified by titration with ethyl acetate to
provide the desired material as a brown solid, which was an acetic acid
salt (4.1 g, 72%). 1H NMR (400 MHz, DMSO-d6) δ 2.83 (s, 3H), 4.23
(s, 2H), 6.29 (s, 1H), 6.35 (d, J = 8.36 Hz, 1H), 6.47 (d, J = 8.36 Hz,
1H), 8.73 (s, 1H); 13C NMR (DMSO-d6) δ 33.7, 50.1, 110.7, 113.6,
113.9, 118.2, 127.6, 142.8, 153.9; HRMS [M + H] for C9H11N3O,
calcd 178.0975, found 178.0977; LCMS [M + H] = 178, 99% (t = 1.33
min).
concentration (within 2-fold of the 1 h Cmax concentration of
120 ng/mL), out to the last time point of the study (62 ng/mL
at 7 h). This implies a depot of insoluble material in the gut
after oral dosing, gradually delivering low concentrations to the
systemic circulation over an extended time course. The
compound was 69% plasma protein-bound. The subcutaneous
mouse pharmacokinetics indicated exposure out to 4 h based
on the 2 mg/kg dose, giving a Cmax of 58 ng/mL with a Tmax of
1 h and a half-life of approximately 2 h.
In conclusion, 17 is a novel chemical probe for the BET
family of bromodomain epigenetic reader proteins. With a
molecular weight of 347, a cLogP of 1.3, and TPSA of 96, 17
has been optimized such that it has a low probability of
polypharmacology and conforms to most of the guidelines on
the design of druglike molecules.23 It is a small, nonlipophilic,
cell-penetrant compound with selectivity for the BET family
within the bromodomains. It has rodent oral bioavailability and
pharmacokinetics in line with its neutral template and
associated solubility profile. Compound 17 is structurally
orthogonal to the triazolodiazepines and isoxazoles first
discovered as selective BET family inhibitors and so offers a
complementary molecular approach for the use of chemical
probes in this field. It was discovered in two design cycles of
less than 250 compounds in total, based on an efficient
fragment-based starting point.
2-Methoxy-N-(3-methyl-2-oxo-1,2,3,4-tetrahydroquinazo-
lin-6-yl)benzenesulfonamide (17).24 To a stirred suspension of 6-
amino-3-methyl-3,4-dihydroquinazolin-2(1H)-one (11) (100 mg,
0.421 mmol, 1.0 equiv) in dichloromethane (10 mL) was added
pyridine (0.20 mL, 2.48 mmol, 5.9 equiv), followed by 2-
methoxybenzene-1-sulfonyl chloride (130 mg, 0.631 mmol, 1.5
equiv). After 2 h, the solvent was evaporated and the residue was
partitioned between ethyl acetate and aqueous 2 M HCl. The organic
layer was collected, washed with water and brine, dried over
magnesium sulfate, filtered, and concentrated to a residue. The
residue was dissolved in dimethyl sulfoxide (DMSO, ∼1 mL) and
purified by automated HPLC to provided the desired material (50 mg,
1
34%). H NMR (400 MHz, DMSO-d6) δ 2.78 (s, 3H), 3.91 (s, 3H),
4.25 (s, 2H), 6.59 (d, J = 9.16 Hz, 1H), 6.79−6.81 (m, 2H), 6.97 (t, J
= 7.5 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 7.52−7.56 (m, 1H), 7.68 (dd,
J = 1.4, 7.8 Hz, 1H), 9.09 (s, 1H), 9.64 (s, 1H); HRMS [M + H] for
C16H18N3O4S, calcd 348.1013, found 348.1019; LCMS [M + H] =
384.1, >99% (t = 1.23 min).
SELECTED EXPERIMENTAL PROCEDURES:
CHEMISTRY METHODS AND COMPOUND
CHARACTERIZATION
■
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional text describing biological methods and chemistry
experimental procedures; five figures showing rodent pharma-
cokinetic profiles and cellular activity; and two tables listing
bromodomain selectivity and X-ray statistics. This material is
available free of charge via the Internet at http://pubs.acs.org.
Proton (1H NMR) and carbon (13C NMR) magnetic resonance
spectra where obtained in DMSO-d6 at 400 and 100 MHz, respectively
unless otherwise noted. The following abbreviations were utilized to
describe peak patterns when appropriate: br = broad, s = singlet, d =
doublet, and m = multiplet. Accurate mass spectrometry (MS)
analyses were conducted on an Agilent 6220 TOF mass spectrometer
in positive or negative electrospray mode. The samples were separated
by UHPLC on an Agilent 1200 system prior to mass spectrometric
analysis. The mass accuracy was calculated for all observed isotopes
against the theoretical mass ions derived from the chemical formula by
use of MassHunter software. All air- and moisture-sensitive reactions
were carried out under an atmosphere of dry nitrogen with heat-dried
glassware and standard syringe techniques. Tetrahydrofuran (THF)
and acetonitrile were purchased from EMD anhydrous and were used
without further drying. Flash chromatography was performed on an
Analogix Intelliflash 280 with Sepra Si 50 silica gel with ethyl acetate/
heptane mixtures as solvent unless otherwise indicated. Certain
compounds from the examples described were purified on reversed-
phase via automated preparative high-performance liquid chromatog-
raphy (HPLC) on a Gilson GX281, Shimadzu CL-2010C, or Agilent
1200 system on an Agella Venusil ASB C18 column. Detection was
achieved by use of a Shimadzu SPD-20AV or Waters 2998 PDA
wavelength absorbance detector set at 220 or 200 nm, followed in
series by a Shimadzu MS2010EV or Waters 3100 mass spectrometer.
Quality control (QC) for purity analysis was performed by HPLC-MS
on a Shimadzu with XB-C18, X-Bridge, Gemini NX C18, or Gemini
NX C18 columns. Detection was achieved by use of a Shimadzu 10A
detector set at 220 or 260 nm, followed in series by a Shimadzu
MS2010EV or Applied Biosystems API 2000 mass spectrometer in
parallel. Compound purity was >95% other than intermediate 9 (91%)
which, as a reactive sulfonyl chloride, was used satisfactorily with no
further chromatographic purification.
Accession Codes
Coordinates for five compounds have been deposited with the
Protein Data Bank under the following accession codes: 8
(4HBV), 12 (4HBW), 13 (4HBX), 14 (4HBY), and 17
(4E96).
AUTHOR INFORMATION
Corresponding Author
*Telephone 1-617-665-5368; E-mail dafydd.owen@pfizer.com.
■
Present Addresses
⊥University College London School of Pharmacy, 29−39
Brunswick Square, London WC1N 1AX, U.K.
#Pfizer Worldwide Medicinal Chemistry, Pfizer Worldwide
R&D, 200 Cambridgepark Dr., Cambridge, MA 02140.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The Structural Genomics Consortium is a registered charity
(1097737) that receives funds from the Canadian Institutes for
Health Research, the Canada Foundation for Innovation,
Genome Canada, Pfizer, GlaxoSmithKline, Eli Lilly, the
Novartis Research Foundation, Abbott, the Ontario Ministry
of Research and Innovation, and the Wellcome Trust.
6-Amino-3-methyl-3,4-dihydroquinazolin-2(1H)-one (11).
To a solution of 3-methyl-6-nitro-3,4-dihydroquinazolin-2(1H)-one
(5.00g, 24.13 mmol, 1.0 equiv) in acetic acid (50 mL) was added
Raney nickel (500 mg), and the reaction mixture was stirred at room
temperature under H2 balloon (1 atm) for 16 h. After completion of
ABBREVIATIONS
BET, bromodomain and extra C-terminal domain; HAT,
histone acetyltransferase; HDAC, histone deacetylase; GPCR,
■
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(15) Hewings, D. S.; Wang, M.; Philpott, M.; Fedorov, O.; Uttarkar,
S.; Filippakopoulos, P.; Picaud, S.; Vuppusetty, C.; Marsden, B.;
Knapp, S.; Conway, S. J; Heightman, T. D. 3,5-Dimethylisoxazoles act
as acetyl-lysine-mimetic bromodomain ligands. J. Med. Chem. 2011, 54,
6761−6770.
(16) Dawson, M. A.; Prinjha, R. K.; Dittman, A.; Giotopoulos, G.;
Bantscheff, M.; Chan, W.-I.; Robson, S. C.; Chung, C.-w.; Hopf, C.;
Savitski, M. M.; Huthmacher, C.; Gudgin, E.; Lugo, D.; Beinke, S.;
Chapman, T. D.; Roberts, E. J.; Soden, P. E.; Auger, K. R.; Mirguet,
O.; Doehner, K.; Delwel, R.; Burnett, A. K.; Jeffrey, P.; Drewes, G.;
Lee, K.; Huntly, B. J. P.; Kouzarides, T. Inhibition of BET recruitment
to chromatin as an effective treatment for MLL-fusion leukaemia.
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(24) Prolonged solublized storage of 17 (for example, in DMSO)
should be avoided, as it is prone to oxidative insertion of nucleophilic
solvents such as alcohols or water at C-4. Cell-based experimentation
and in vivo work were reliably and reproducibly performed from fresh
solid sample. (17, PFI-1, Pfizer number PF-6405761, will be made
commercially available from Sigma Aldrich, Tocris Bioscience, and
Toronto Research Chemical.)
G protein-coupled receptor; cLogP, calculated LogP; CREB,
cyclic AMP response binding element; CREBBP, cyclic AMP
response binding element binding protein; BAZ2B, bromodo-
main adjacent to zinc finger domain 2B; FALZ, fetal Alzheimer-
50 clone 1 protein; SPR, surface plasmon resonance; DCM,
dichloromethane; RT, room temperature; cat., catalytic; dba,
dibenzylidene acetone; IL6, interleukin 6; LPS, lipopolysac-
charide; PBMC, peripheral blood mononuclear cell; sc,
subcutaneous; po, per os (orally); iv, intravenous; TPSA,
topological polar surface area
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