Identification and Development of 2,3-Dihydropyrrolo[1,2-a]quinazolin-5(1H)-one Inhibitors Targeting Bromodomains within the Switch/Sucrose Nonfermenting Complex

Article abstract of DOI:10.1021/acs.jmedchem.5b01997

Bromodomain containing proteins PB1, SMARCA4, and SMARCA2 are important components of SWI/SNF chromatin remodeling complexes. We identified bromodomain inhibitors that target these proteins and display unusual binding modes involving water displacement from the KAc binding site. The best compound binds the fifth bromodomain of PB1 with a K_D of 124 nM, SMARCA2B and SMARCA4 with K_D values of 262 and 417 nM, respectively, and displays excellent selectivity over bromodomains other than PB1, SMARCA2, and SMARCA4.

Full text of DOI:10.1021/acs.jmedchem.5b01997

Brief Article
pubs.acs.org/jmc
Identification and Development of 2,3-
Dihydropyrrolo[1,2‑a]quinazolin-5(1H)‑one Inhibitors Targeting
Bromodomains within the Switch/Sucrose Nonfermenting Complex
Charlotte L. Sutherell,†,‡ Cynthia Tallant,§,∥ Octovia P. Monteiro,§,∥ Clarence Yapp,§,∥ Julian E. Fuchs,†
Oleg Fedorov,§,∥ Paulina Siejka,§,∥ Suzanne Muller,§,∥ Stefan Knapp,§,∥,⊥ James D. Brenton,‡
̈
Paul E. Brennan,*,§,∥ and Steven V. Ley*,†
†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K.
‡Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, CB2 0RE, U.K.
§The Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington,
Oxford OX3 7DQ, U.K.
∥Target Discovery Institute, University of Oxford, NDM Research Building, Roosevelt Drive, Headington, Oxford OX3 7FZ, U.K.
⊥Department of Pharmaceutical Chemistry and Buchmann Institute for Life Sciences, Goethe University Frankfurt, 60438 Frankfurt
am Main, Germany
S
* Supporting Information
ABSTRACT: Bromodomain containing proteins PB1, SMARCA4, and
SMARCA2 are important components of SWI/SNF chromatin remodeling
complexes. We identified bromodomain inhibitors that target these proteins
and display unusual binding modes involving water displacement from the
KAc binding site. The best compound binds the fifth bromodomain of PB1
with a KD of 124 nM, SMARCA2B and SMARCA4 with KD values of 262
and 417 nM, respectively, and displays excellent selectivity over
bromodomains other than PB1, SMARCA2, and SMARCA4.
SMARCA2/4 and BRD7/9 contain one bromodomain each,
while PB1 has six distinct bromodomains. In yeast SWI/SNF
bromodomains are implicated in enhancing chromatin
remodeling efficiency, loci targeting, and protein−protein
interactions.9,10 In eukaryotes the SMARCA4 bromodomain
is reported to act in the DNA damage response.11,12 Mutations
in PB1’s bromodomains have been found in clear cell renal
carcinoma and linked to genome instability and aneuploidy.13,14
Bromodomains are protein modules of ∼110 amino acids
that recognize acetylated lysine (KAc) and share a common
structure of four α helices linked by flexible loop regions and
have been popular targets for chemical probe development.15,16
Sixty-one bromodomains are expressed in the human
proteome, across 42 BCPs, which can be grouped into
subfamilies based on amino acid sequence and structural
alignment.17 Chemical probes have now been developed for
multiple subfamilies and have helped identify new biology.18,19
In particular, (+)JQ1 and IBET-151 have demonstrated the
therapeutic potential of bromodomain and extraterminal (BET)
proteins in cancer, inflammation, and heart failure.20−22
INTRODUCTION
■
The structure of chromatin, the complex of proteins and DNA
through which DNA is packaged, is regulated by the post-
translational modification of histone tails and the action of
chromatin remodeling complexes such as the switch/sucrose
nonfermenting (SWI/SNF) complex. The multisubunit SWI/
SNF complex is based on the mutually exclusive helicase/
ATPase proteins SMARCA2 and SMARCA4, with additional
core subunits and regulatory or specificity proteins. Many
components contain domains that recognize histone post-
translational modifications, enabling cross-talk between the two
mechanisms of regulation.
The SWI/SNF complex is subject to considerable research
interest, since components are mutated in many cancers1−3 and
are implicated in developmental disorders.4 In some tumor
types, mutations within the SWI/SNF complex lead to context-
specific vulnerabilities, such as dependence on SMARCA2/4 in
SMARCB1 (Snf5) mutant rhabdoid tumors5 or the essential
presence of SMARCA2 in SMARCA4 mutant lung cancer.6,7
The biological roles of the complexes have led to interest in
generating chemical probes that target different domains within
the multisubunit complex, to assist the elucidation of their
function and therapeutic relevance.8 Bromodomain containing
proteins (BCPs) are prevalent in SWI/SNF complexes and are
present in SMARCA2/4 helicases, BRD7/9, and PB1.
The chemical probes I-BRD923 and LP99,24 targeting BRD9
and BRD7/BRD9, respectively, have been developed to study
Received: December 23, 2015
© XXXX American Chemical Society
A
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some bromodomains in the SWI/SNF complex. Recently a
chemical probe, PFI-3, targeting the bromodomains in
SMARCA2 and SMARCA4, as well as the fifth bromodomain
of PB1 (PB1(5)), has been developed based on a salicyclic acid
fragment hit and been used to demonstrate a role for
SMARCA4 in embryonic stem cell maintenance.25 These
results demonstrate that SWI/SNF BCPs are druggable, despite
being predicted to be more challenging to target due to a
featureless binding pocket lacking a clearly defined hydrophobic
groove and ZA binding channel as in BRD4(1) (Supporting
Information).17,26 A short ZA loop and a wider entrance than
BET bromodomains have been identified as factors that may
alter ligand binding mode in the structurally related
bromodomains PB1(5) and SMARCA2/4, while PB1(2−4)
are defined by an aromatic residue in the pocket that is
proposed to act as a “lid”.26 Given the varied structural nature
of the six bromodomains in PB1, one single probe is unlikely to
be able to target all simultaneously. Several compounds with
different chemotypes may be required.
and that the 2,3-dihydropyrrolo[1,2-a]quinazolin-5(1H)-one
core bound within the KAc binding site (Supporting
Information). We proposed that planarity of the compounds
with conjugated aromatic ring substituents contributed to the
poor solubility of the series and that analogues with greater sp3
character in their substituents, or the potential for ionization,
would have improved aqueous solubility.28
Compounds were synthesized by the routes shown in
Scheme 1. Amidation of 2-aminobenzamide 2 with 4-
chlorobutanoyl chloride29 gave amide 3, which could be
cyclized to the 2,3-dihydropyrrolo[1,2-a]quinazolin-5(1H)-
one core 4 using potassium tert-butoxide. The isomer was
confirmed on the basis of NOE correlations and subsequent
crystallography. The weak nucleophilicity of the core was then
exploited by reacting compound 4 with substituted N,N-
dialkylformamides using Vilsmeier−Haack conditions, giving
analogues incorporating nitrogen-based aliphatic side chains
solely as the E regioisomer. This chemistry was unsuitable for
piperazine-based formamides. For these side chains, analogues
12−15 were synthesized as single regioisomers by displacement
of the dimethylamino group in 5 using cyclic secondary amines
and DMAP catalysis. Due to the potential for hydrolysis of the
enamine-like moiety, the aqueous stability of 5 was assessed by
1H NMR in a D2O time course experiment and was stable for
>24 h. All analogues appeared to have increased aqueous
solubility compared to 1. Their binding to PB1(5) was assessed
by the operationally simple DSF assay (Table 1). Although the
simple scaffold 4 itself showed no interaction, aliphatic
analogues 5−15 generally showed improved binding to
PB1(5). The shape of the side chain was a key factor in
determining activity; inclusion of propyl side chains in 7 led to
moderate activity, but variation to isopropyl in 8 caused a loss
of binding. Six-membered aliphatic rings (10−14) were better
tolerated than analogues containing six-membered aromatic
side chains (see Supporting Information), likely due to their
greater flexibility. However, expansion to a seven-membered
ring reduced activity.
In the course of library screening, 1 (Figure 1) and other
related analogues were found to be weak PB1(5) inhibitors but
Figure 1. Initial hit from screening of a commercial library.
with poor aqueous solubility. In this work we describe the
optimization of 1, leading to a family of compounds with
increased potency, improved physicochemical properties, and
varied selectivity within the SWI/SNF bromodomains,
including the potential to target PB1 selectively over
SMARCA4 and SMARCA2 bromodomains.
RESULTS AND DISCUSSION
It was anticipated that compounds targeting PB1(5) might
interact with the SMARCA2 and SMARCA4 bromodomains
due to the similarity of their binding pockets. We confirmed the
binding of these analogues to the SMARCA4 bromodomain in
an AlphaScreen assay (Table 1), which established that the
compounds had affinities for this bromodomain ranging from
6.2 to 22.0 μM.
■
The library of commercial analogues of 1 showed varied
response by differential scanning fluorimetry (DSF) against
PB1(5). DSF has been demonstrated to be an operationally
simple assay, suitable for rapid assessment of potency and
selectivity for bromodomains.19,24,27 Limited SAR from the
library indicated sensitivity to the size of the aromatic side chain
a
Scheme 1. General Route to Core 2,3-Dihydropyrrolo[1,2-a]quinazolin-5(1H)-ones and Their Derivatives
a
t
Reagents and conditions: (a) 4-chlorobutanoyl chloride, Et3N, THF, 0 °C to rt, 12 h, 92%; (b) BuOK, THF, rt, 1 h, 77%; (c) N,N-
dialkylformamide, POCl3, CH2Cl2, 70 °C, 16−74%; (d) H-NR2, DMAP, EtOH, 70 °C, 24−96 h, 39−81%. For R see Table 1.
B
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Table 1. Effect of Aliphatic Side Chain Substitution on
Binding to PB1(5) and SMARCA4 by DSF and AlphaScreen
Figure 2. Binding modes of bromodomain inhibitors. (a) Cocrystal
structure of 10 with PB1(5) at 2.3 Å (PDB code 5FH6). Hydrogen
bonds are shown by black dashed lines. (b) Apo-crystal structure of
PB1(5) (PDB code 3G0J) showing conserved crystallographic waters
(red spheres) which are displaced in (a).
The crystal structure highlighted the narrowness of the PB1(5)
pocket, which the compound occupies well, and indicated that
the side chain is key in orienting the core within the pocket.
This helped explain the lack of activity of the unsubstituted
core. The piperidine side chain projects out from the pocket
forming a close interaction with the hydrophobic rim of the
binding site, explaining the loss in activity in 8 and 15, which
contain more sterically demanding side chains.
The carbonyl of Met731 was observed to be in an unsolvated
cavity in the structure, and it was hypothesized that substitution
on the aromatic ring ortho to the carbonyl group might enable
an additional interaction in this region. Compounds 16−20
could be accessed by incorporating substituted 2-amino-
benzamides into the synthetic methodology previously
developed. Compound 21 featuring hydroxyl substitution was
obtained by deprotection of the methoxy analogues using BBr3.
Detailed procedures are in the Supporting Information.
Screening these analogues against PB1(5) by DSF showed
that substitution of chlorine at R2 in 18 doubled the observed
ΔTm in comparison to unsubstituted 5, while bromine
substitution gave a smaller increase (Table 2). All other
substitutions reduced binding compared with analogue 5. This
SAR provides valuable compounds suitable as negative controls
a
Values shown are the average of three replicates and standard
b
deviation by DSF assay. Values shown are the average of two
replicates by AlphaScreen assay. IC50 not determined.
c
The improved solubility and activity of the compounds
allowed us to conduct isothermal calorimetry (ITC) experi-
ments with analogues 9 and 10. These had KD values of 2.3 and
1.2 μM, respectively, for the PB1(5) bromodomain. In both 9
and 10, the entropy change upon binding (TΔS) contributed
favorably to binding (TΔS values of 4.7 and 5.3 kcal/mol,
respectively). In addition, interaction of these two inhibitors
with PB1(5) was driven by enthalpic contributions (ΔH of
−2.9 and −2.7 kcal/mol, respectively). The underlying
molecular mechanism for the observed thermodynamics was
evident on analysis of the binding mode of 10, revealed by a
cocrystal with PB1(5) bromodomain (Figure 2).
Table 2. Effect of Aromatic Substitution on Binding to the
PB1(5) Bromodomain
This structure demonstrated that the rigid core of 10 sits
deep in the hydrophobic KAc binding site of PB1(5), displacing
the four waters observed in the apo-form and most BRD-ligand
crystal structures. The carbonyl of the 5,6-dihydropyrimidin-
4(1H)-one ring formed a direct hydrogen bond to Tyr696,
while the amide hydrogen-bonds to Asn739. This binding
mode is similar to that adopted by PFI-3 and its salicylic acid
precursor, which also displace conserved waters and form direct
hydrogen bonds to Tyr696 and Asn739.26 These interactions
are in marked contrast to those in most bromodomain
inhibitors, in which a KAc mimetic, directly hydrogen-bonding
to the asparagine residue, forms a water-mediated hydrogen
bond network to tyrosine using the structural conserved waters.
a
Values shown are the average of three replicates and standard
deviation.
C
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Journal of Medicinal Chemistry
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for the exploration of the biological activity of these
compounds.
(Table 3). Compounds 24, 26, and 27 had greatly improved
DMSO and aqueous solubility. The binding of these
compounds to PB1(5) was assessed by ITC (Table 4), and
all displayed similar KD values to 18.
A KD of 137 nM for PB1(5) was obtained for 18 by ITC,
with a significant increase in the enthalpic contribution to
binding (ΔH = −6.0 kcal/mol). Since Cl/Br inclusion also
increased binding of the core scaffold (ΔTm of 2.5 and 3.1 °C,
respectively; see Supporting Information), we proposed that
the improved interaction was due to formation of a halogen
bond to the carbonyl of Met731. While bromination would be
predicted to give a stronger halogen bond,30 the effect is
countered by steric constraints within the cavity.
This interaction was confirmed by a cocrystal structure of 18
with PB1(5), in which the chlorine occupies the cavity
previously observed with a separation of 3.2 Å from the
carbonyl oxygen of Met731. This is consistent with a halogen
bond (Figure 3).30 A slight twist in the scaffold, to maximize
Table 4. Binding of Key Compounds to PB1(5) Was
Assessed by Isothermal Calorimetry
K × 106
(M−1
ΔH
TΔS
compd
)
KD (nM)
(kcal/mol)
(kcal/mol)
18
24
26
27
28
7.29 0.91
7.86 0.89
8.09 0.66
7.05 0.68
5.88 0.60
137 (157−122)
127 (143−114)
124 (135−114)
142 (157−129)
170 (189−154)
−6.2
−5.2
−5.0
−4.4
−6.8
3.0
4.0
4.3
4.7
2.3
As shown in Table 3, the analogues were screened by DSF
against other bromodomains within the SWI/SNF complex,
including other structurally distinct bromodomains within
protein PB1, to determine their selectivity. When interpreting
this data, it should be noted that different proteins do not
behave identically in DSF assays, and ΔTm values should not be
viewed as an absolute scale. For example, probe PFI-3 has KD
values by ITC of 89 and 48 nM for SMARCA4 and PB1(5),
respectively, but ΔTm shifts of 5.1 and 7.5 °C.25
In our screening, compounds appeared to interact with the
bromodomains in both SMARCA2 isoforms, A and B, and in
SMARCA4 and PB1(5). ITC was used to further assess the
binding of 26 (Figure 4) to SMARCA2B and SMARCA4,
giving KD values of 262 and 417 nM, respectively. Binding to
the SMARCA bromodomains had not been observed prior to
inclusion of the chlorine substitution, suggesting that the
proposed halogen bond is again important for binding.
The bromodomain PB1(2) also demonstrated a moderate
response, despite containing a broader KAc binding pocket
than PB1(5). By contrast, minimal protein stabilization was
observed with bromodomains PB1(3) and PB1(4). This may
be due to the tyrosine residue in the ZA loop which acts as a
“lid” to the pocket in these bromodomains.26
Figure 3. Cocrystal structure of 18 with PB1(5) at 1.5 Å (PDB code
5FH7): (a) cut-through illustrating positioning of chlorine in cavity;
(b) surface view illustrating fit in PB1(5) pocket.
interactions with both Tyr696 and Asn739, can be observed.
Comparison with PFI-3 shows that the interaction with Met731
has not been previously exploited (Supporting Information).
To improve the aqueous solubility of the chlorinated
derivatives, we varied the enamine substituent using the
existing methodology to access analogues 22−27, the majority
of which retained their affinity for the target as assessed by DSF
Table 3. Structure−Activity Relationships of Chlorinated Derivatives against SWI/SNF Bromodomains
a
b
Values shown are the average of three replicates. Standard deviation is <0.6 °C. Values are a single measurement.
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Figure 4. Representative ITC trace, measured using 26 and the
bromodomain of PB1(5); 30 consecutive injections of PB1(5) into a
solution of 26 in 20 mM HEPES, 150 mM NaCl, 0.05 mM TCEP.
Raw heats (left) and normalized injection heats with a nonlinear least-
squares fit for a single binding site model (right) are shown.
Figure 5. (a) Cocrystal structure of 28 with PB1(5) at 1.6 Å (PDB
code 5FH8). (b) Overlay of PDB code 5FH7 (gray) of 18 and PDB
code 5FH8 (cyan) of 28 showing the shift in the ZA loop of the
PB1(5) domain on binding of 28.
We were interested to establish whether further side chain
variation could alter selectivity within SWI/SNF bromodo-
mains (Table 5). Analogues 28−31 incorporating alkene side
chains were synthesized by reaction of 6-chloro-2,3-dihydro-
pyrrolo[1,2-a]quinazolin-5(1H)-one (18c) with the appropri-
ate aliphatic aldehydes under basic conditions (see Supporting
Information for full methods). These were screened by DSF,
which suggested reduced binding to PB1(5) for the majority of
ligands except 28. When assessed by ITC, 28 had a KD of 170
nM with PB1(5). DSF results suggested an intriguing change in
selectivity among SWI/SNF bromodomains. In contrast to all
previous compounds tested in this report, and the chemical
probe PFI-3,26 28 showed a stronger interaction with PB1(2)
than with the SMARCA2/4 bromodomains. The weak binding
to SMARCA4 was confirmed by ITC, with a KD of 2.03 μM for
the interaction with 28.
Understanding the reason for the change in affinity would
help design of future chemical probes with selectivity for
bromodomains not yet targeted such as PB1(2−4) and offers
the intriguing possibility of targeting PB1 selectively over
SMARCA2/4. We obtained a cocrystal structure of 28 with
PB1(5) (Figure 5) to understand its unique features. This
revealed a shift in the ZA loop region of PB1(5), making the
KAc pocket wider and increasing its similarity to the PB1(2)
KAc pocket. The ethyl side chains interact with the
hydrophobic edges of the pocket and begin to occupy the ill-
defined peptide-binding channel in PB1(5). Although no
structure was obtained to confirm this, we propose that in
SMARCA4, Ile1543 blocks this channel, preventing the ethyl
side chains from being accommodated in the smaller pocket.
We assessed the selectivity of the most potent pan-PB1,
SMARCA2/4 analogue 26 against representative bromodo-
mains (PCAF, BRD4(1), CREBBP, TRIM33B) from other
subfamilies using DSF (Figure 6). No significant binding was
observed.
Finally, the ability of 26 to displace SMARCA2 from
chromatin was assessed using a fluorescence recovery after
photobleaching (FRAP) assay (Figure 7).31 U2OS cells were
transfected with a GFP-linked SMARCA2 construct. To
increase global histone acetylation and thereby the assay
window the broad-spectrum histone-deacetylase (HDAC)
inhibitor, suberoylanilide hydroxamic acid (SAHA) was used.
Cells transfected with a construct carrying the N1464F
mutation, unable to bind to chromatin via the bromodomain,
were used as a positive control. Treatment of cells with 26 at 1
or 5 μM reduced FRAP recovery times back to that of the
N1464F mutant and unstimulated levels, indicating that 26 was
able to displace full length SMARCA2 from chromatin.
CONCLUSIONS
■
We describe the optimization of an inhibitor series targeting
bromodomains found within the SWI/SNF complex from a
weakly potent hit with poor physicochemical properties.
Improvement of solubility has allowed cocrystal structures to
be obtained demonstrating the important role of water
a
Table 5. Structure−Activity Relationships of Compounds Bearing Alkene Based Side Chains against SWI/SNF Bromodomains
a
Values shown are the average of three replicates. Standard deviation is <0.6 °C. For PB1(3), values are a single measurement.
E
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from chromatin in cells, making it suitable as a chemical probe
with a distinct chemotype to PFI-3 and for further development
of SWI/SNF bromodomain inhibitors.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01997. In addition to the indicated Supporting
Information PDF and CSV files, additional data related to
this publication are available at https://www.repository.cam.ac.
uk/handle/1810/254994.
Additional structural images and screening data, ITC
traces, X-ray refinement statistics, additional text
describing biological methods and synthetic procedures,
characterization data, NMR (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*P.E.B.: e-mail, paul.brennan@sgc.ox.ac.uk; phone, +44 (0)
1865 612932.
■
*S.V.L.: e-mail, svl1000@cam.ac.uk; phone, +44 1223 336398.
Notes
Figure 6. Selectivity of 26. Inhibitor 26 was screened at 10 μM against
selected bromodomains by DSF assay. Temperature shifts for screened
proteins are shown on the phylogenetic tree.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.L.S. was funded by the Cambridge Ph.D. Training
Programme in Chemical Biology and Molecular Medicine.
We gratefully acknowledge the EPSRC (SVL, Grants EP/
K099494/1 and EP/K039520/1). The SGC is a registered
charity (No. 1097737) that received funds from AbbVie, Bayer
Pharma AG, Boehringer Ingelheim, the Canada Foundation for
Innovation, Genome Canada, GlaxoSmithKline, Janssen, Lilly
Canada, the Novartis Research Foundation, the Ontario
Ministry of Economic Development, and Innovation, Pfizer,
Takeda, and the Wellcome Trust (Grant 092809/Z/10/Z).
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; BCP, bromodomain containing
protein; BET, bromodomain and extraterminal domain; BRD7,
bromodomain containing protein 7; BRD9, bromodomain
containing protein 9; DMAP, N,N-dimethyl-4-aminopyridine;
DSF, differential scanning fluorimetry; FRAP, fluorescence
recovery after photobleaching; GFP, green fluorescent protein;
HDAC, histone deacetylase; ITC, isothermal titration calorim-
etry; KAc, acetyl-lysine; NOE, nuclear Overhauser effect; PB1,
polybromo-1; PB1(X), Xth bromodomain of PB1; PDB,
Protein Data Bank; SAHA, suberoylanilide hydroxamic acid;
SAR, structure−activity relationship; SMARCA2/4, SWI/SNF
related, matrix associated, actin dependent regulator of
chromatin, subfamily A, member 2/4; SWI/SNF, switch/
sucrose nonfermenting
Figure 7. Compound 26 inhibits SMARCA2 association with
chromatin in cells. (A) FRAP half recovery times of GFP-SMARCA2
are significantly decreased when treated with 26 at 1 or 5 μM as
indicated. Cells expressing the SMARCA2 bromodomain-inactivating
mutant (N1464) were analyzed as comparison. Significant differences
to cells treated with SAHA of p < 0.0001 are shown by ∗. (B) Time
dependence of fluorescence recovery in the bleached area of cells
expressing wt or mutant GFP-SMARCA2 with the corresponding
treatment.
displacement in the binding of these inhibitors. Chlorination of
the series has demonstrated the potential for exploitation of
previously unexplored interactions deep within the PB1(5) KAc
binding pocket through halogen bonding. Side chain variation
in 28 shows that the second and fifth bromodomains of PB1
can be targeted selectively over the SMARCA2/4 helicases, in
contrast to the selectivity shown by the chemical probe PFI-3.
Lead inhibitor 26 displays good affinity for PB1(5), SMARCA4,
and SMARCA2 as assessed by ITC, excellent selectivity within
the bromodomain family, and the ability to displace SMARCA2
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DOI: 10.1021/acs.jmedchem.5b01997
J. Med. Chem. XXXX, XXX, XXX−XXX