Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.0c00035

Bromodomain-containing protein 4 (BRD4) represents a promising drug target for anti-inflammatory therapeutics. Herein, we report the design, synthesis, and pharmacological evaluation of novel chromone derivatives via scaffold hopping to discover a new class of orally bioavailable BRD4-selective inhibitors. Two potent BRD4 bromodomain 1 (BD1)-selective inhibitors 44 (ZL0513) and 45 (ZL0516) have been discovered with high binding affinity (IC50 values of 67-84 nM) and good selectivity over other BRD family proteins and distant BD-containing proteins. Both compounds significantly inhibited the expression of Toll-like receptor-induced inflammatory genes in vitro and airway inflammation in murine models. The cocrystal structure of 45 in complex with human BRD4 BD1 at a high resolution of 2.0 ? has been solved, offering a solid structural basis for its binding validation and further structure-based optimization. These BRD4 BD1 inhibitors demonstrated impressive in vivo efficacy and overall promising pharmacokinetic properties, indicating their therapeutic potential for the treatment of inflammatory diseases.

Full text of DOI:10.1021/acs.jmedchem.0c00035

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Article
Discovery of Orally Bioavailable Chromone Derivatives
as Potent and Selective BRD4 Inhibitors: Scaffolding
Hopping, Optimization and Pharmacological Evaluation
Zhiqing Liu, Haiying Chen, Pingyuan Wang, Yi Li, Eric A. Wold, Paul
G. Leonard, Sarah Joseph, Allan R. Brasier, Bing Tian, and Jia Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00035 • Publication Date (Web): 07 Apr 2020
Downloaded from pubs.acs.org on April 8, 2020
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Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4
Inhibitors: Scaffolding Hopping, Optimization and Pharmacological Evaluation
†
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†
£
Zhiqing Liu, Haiying Chen, Pingyuan Wang, Yi Li, Eric A. Wold, Paul G. Leonard, Sarah
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Joseph, Allan R. Brasier, * Bing Tian, * and Jia Zhou†,§,ξ,*
£
∥,
‡,§,
†
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Chemical Biology Program, Department of Pharmacology and Toxicology, Department of
§
ξ
Internal Medicine, Sealy Center for Molecular Medicine, Institute for Translational Sciences,
University of Texas Medical Branch, Galveston, TX 77555, USA
£Core for Biomolecular Structure and Function, MD Anderson Cancer Center, 1881 East Road,
Houston, TX, 77054, USA
∥Institute for Clinical and Translational Research (ICTR), University of Wisconsin-Madison
School of Medicine and Public Health, 4248 Health Sciences Learning Center, Madison, WI
53705, USA
*
Corresponding authors: Dr. Jia Zhou, Chemical Biology Program, Department of
Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555,
United States. Tel: +1 (409) 772-9748; E-mail: jizhou@utmb.edu. Dr. Bing Tian, Tel: +1 (409)
7
72-1177; E-mail: bitian@utmb.edu. Dr. Allan R. Brasier, Tel: +1 (608) 263-7371; E-mail:
abrasier@wisc.edu.
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ABSTRACT
Bromodomain-containing protein 4 (BRD4) represents a promising drug target for anti-
inflammatory therapeutics. Herein, we report the design, synthesis, and pharmacological
evaluation of novel chromone derivatives via scaffold hopping to discover a new class of orally
bioavailable BRD4 selective inhibitors. Two potent BRD4 bromodomain 1 (BD1) selective
inhibitors 44 (ZL0513) and 45 (ZL0516) have been discovered with high binding affinity (IC50
values of 67~84 nM) and good selectivity over other BRD family proteins and distant BD-
containing proteins. Both compounds significantly inhibited the expression of Toll-like receptor
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(TLR3)-induced inflammatory genes in vitro and airway inflammation in murine models. The
cocrystal structure of 45 in complex with human BRD4 BD1 at a high resolution of 2.0 Å has
been solved, offering a solid structural basis for its binding validation and further structure-based
optimization. These BRD4 BD1 inhibitors demonstrated impressive in vivo efficacy and overall
promising pharmacokinetic properties, indicating their therapeutic potential for the treatment of
inflammatory diseases.
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INTRODUCTION
Epigenetic modifications on DNA, histones and other nuclear proteins regulate gene
expression, affect cellular differentiation and contribute to human diseases.1, 2 Lysine acetylation
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(KAc) is one of the most broadly studied post translational modifications occurring on histone
proteins. This highly dynamic process is regulated by opposing actions of histone acetyl
3
transferases (HATs) and histone deacetylases (HDACs). Histone acetylation also provides
binding sites for proteins, especially BD-containing proteins, to promote chromatin
reorganization and transcription. There are 61 structurally homologous BDs present in 46
different proteins in the human proteome. BRD4 belongs to BD and extra-terminal (BET) family
consisting of four members (BRD2, BRD3, BRD4 and BRDT). Similar to other subfamily of
BD-containing proteins, BET members function in epigenetic regulation of gene expression
through binding to KAc recognition pocket on histone tails and non-histone proteins.4-6 BET
family proteins, especially BRD4, have emerged as a promising epigenetic target for human
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HIV infection,15-17 heart failure,18
diseases and conditions, including cancers, inflammations,
and CNS disorders.19
A number of BET inhibitors with different chemotypes (e.g. azepines, 3,5-
dimethylisoxazoles, pyridones, diazobenzene) have been discovered and developed as depicted
in Figure 1.4, 20 Azepine (+)-JQ1 (1, Figure 1) has been the most widely used tool BRD4
inhibitor, and its analogue I-BET762 (2) has been advanced into phase II human clinical trials for
neoplasms.21, 22 RVX-208 (3), a BET inhibitor selective for the second BD, has been enrolled
into phase III clinical trials for high-risk cardiovascular disease patients with type 2 diabetes
mellitus and low levels of high-density lipoprotein. Despite the fact that compound 3
demonstrated tolerability and safety, it failed to meet the primary endpoint – reduction in major
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adverse cardiovascular events (MACE).24 Both ABBV075 (4) and BMS986158 (5) are
developed as pan-BET inhibitors in clinical trials for cancer therapy.25, 26 It has been reported
that BD2 inhibition (e.g., 3) only modestly affects BET-dependent gene transcription and BD1
selective inhibitors are urgently needed for elucidating bromodomain-specific functions. To date,
the availability of BD1 selective inhibitors is very limited, let alone BRD4 BD1 specific ones.23
To improve the selectivity and extend the application of BRD4 inhibitors, we recently designed
and synthesized a series of diphenyldiazene BRD4 inhibitors (e.g., ZL0454, 6 in Figure 1) that
suppress the TLR3-induced expression of proinflammatory genes (IL-6, ISG54 and Groβ) in
human small airway cells in vitro, as well as TLR3-induced airway inflammation and
neutrophilia in mouse models in vivo.8, 9, 12, 13 Although compound 6 displayed good BRD4
inhibition (IC50 values of 49 nM and 35 nM against BRD4 BD1 and BRD4 BD2, respectively)
and selectivity over other family members, its pharmacokinetic (PK) profile is not satisfactory
with poor orally bioavailability (F = 0.46%) and metabolic stability as well as poor aqueous
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solubility (12.8 µg/mL at pH = 7). To further improve the physicochemical properties of BRD4
inhibitors based on our first-generation lead compound 6, two approaches were utilized by either
replacing the N=N linker with moieties that have more favorable metabolic stability or tuning its
core structure to alternative privileged scaffolds with better oral bioavailability. Given our proof-
of-concept study of BRD4 in airway inflammation (3 as a positive control displaying moderate in
vivo efficacy) and the clinically validated safety profile of compound 3, we believe through
dedicated drug design and structural optimization, new analogues with unique scaffolds based on
compound 3 could be achieved to significantly improve their oral bioavailability and the in vivo
efficacy for the treatment of inflammatory diseases. Herein, we report our application of scaffold
hopping to discover a new class of potent, selective and orally available BRD4 inhibitors. The
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binding modes of them with BRD4 BD1 have been validated by the cocrystal structural analysis
of the inhibitor in complex with human BRD4 BD1 protein.
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Figure 1. Chemical structures of representative BET inhibitors 1~5 and recently identified
BRD4 selective inhibitor 6.
RESULTS AND DISCUSSION
Design. As depicted in Figure 2, the co-crystal structure of compound 3 in complex with
BRD4 BD1 (PDB code: 4MR4) exhibited the lactam of the quinazolin-4-one core, especially the
carbonyl oxygen atom, formed critical H-bonds with Asn140 and Tyr97. The 3,5-dimethylphenol
moiety of compound 3 does not reach the tryptophan-proline-phenylalanine (WPF) shelf but
extends to the upper area of four helix bundle. Interestingly, its analogue RVX-OH (7, Figure 2)
lacking a 2-hydroxyethyl group was found to interact with BRD4 BD1 in a completely reversed
2
3
conformation. The quinazolin-4-one core extends to the WPF shelf area while the oxygen atom
of phenyl group inserted to the critical site forms direct H-bonds with Asn140 and an indirect
interaction with Tyr97, exactly like compound 6. Such substantial difference may explain why
compound 3 is BD2 selective while compound 7 has no preference between BD1 and BD2.
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Given that its close analogue compound 3, is clinically proven both safe and metabolically stable,
we proposed to optimize substituents on the quinazolin-4-one core of compound 7 (Series I) and
the 3,5-dimethylphenol moiety of compound 3 (Series II) to explore whether we can improve the
BRD4 BD1 selectivity and achieve a paradigm shift in its clinical application from
cardiovascular disease to various inflammatory diseases such as chronic obstructive pulmonary
disease (COPD). To accomplish this objective, we used a scaffold hopping strategy to replace the
quinazolin-4-one core with chromen-4-one scaffold through fine tuning (Series III) to discover
structurally novel BRD4 inhibitors with enhanced potency and BD selectivity as well as an
improved drug metabolism pharmacokinetic (DMPK) profile. For series I compounds, they are
expected to form the critical interactions with Asn140 and Tyr97 through free -OH like
compound 7, and substitutions on the quinazolin-4-one core can reach the WPF shelf. The
relevant structure-activity relationship (SAR) will be helpful to explore the compatibility and
possibility to improve potency as well as selectivity at this site. For series II and III compounds,
we emphasize the investigations on the side chain which is oriented to a short sequence from
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Lys91 to Asp 96 in ZA loop which is believed critical for bromodomain selectivity. The short
sequence for BRD4 BD1 is KLNLPD (91~96), while it is ALGLHD for BRD4 BD2, KLGLPD
for BRD2 BD1, and ALGLHD for BRD2 BD2, respectively. They are not highly conserved,
especially Asn93 which is unique for BRD4 BD1. We envision that incorporating proper side
chains on the single phenyl ring of series II and III that can critically interact with KLNLPD of
BRD4 BD1 domain may achieve a better BD1 selectivity of BRD4.
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A sn140
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Tyr97
Figure 2. Design of three series of BRD4 inhibitors via a ligand structure-based drug design and
scaffold hopping strategy. First panel: overlay of cocrystal structures of compounds 3 (in light
blue, PDB code: 4MR4) and 7 (in pink, PDB code: 4MR3) with BRD4 BD1. Residues Asn140
and Tyr97 are labeled. Second panel: designed three series of novel compounds to explore the
structure-activity relationship (SAR).
Chemistry. The first series of compounds 10~16 were obtained from the reaction of 4-
hydroxy-3,5-dimethylbenzaldehyde 9 with various 2-aminobenzamides 8 via an efficient, metal-
2
8
free and I
around amide moiety of 7, a methyl group was introduced through two strategies. 4-Nitro-isotoic
anhydride 17 was substituted by CH NH to afford intermediate 18 which was readily
2
-mediated C-N bond formation method (Scheme 1). To explore the space tolerance
3
2
transformed into desired compound 19 with a methyl group on the amide. After construction of
the quinazolin-4-one core via allylic-protected benzaldehyde 20, compound 21 was methylated
by CH
3
I and deprotected in the presence of Pd(PPh
3
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and K
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Scheme 1. Synthetic Procedure of Series I.a
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aReagents and conditions: a) I
aminobenzamide, I , EtOH, 57% for two steps; d) CH
Pd(PPh , K CO , CH OH, reflux, 4 h, 72%.
, EtOH, reflux, 4 h, 89%~quant. b) CH
NH , THF, DIPEA; c) 2-
I, NaH, DMF, rt., overnight, 94%; e)
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The synthesis of the second series of compounds is depicted in Scheme 2. Compound 24
and 25 were obtained following a similar procedure to that of compounds 10~16 from
commercially available starting material 2-aminobenzamide 8a and various benzaldehydes 23.
Compounds 27~31 were obtained through the reaction of 2-amino-4,6-dimethoxybenzamide 8b
and substituted benzaldehydes 26 (commercially available or slight modification on 4-hydroxy-
3
,5-dimethylbenzaldehyde 9). Addition of 1-methylpiperazine with 31 produced 32 in a yield of
35%. Compound 3 was also resynthesized as the reference compound taking the same strategy in
a yield of 82% for two steps (Scheme 3), while the original synthetic method of compound 3 was
reported to take 8 steps with an overall yield of 5% and included harsh conditions like high
temperature (170 ˚C) and two gases in pure form (HCl and NH
).23
3
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Scheme 2. Synthetic Procedure of Series II.a
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aReagents and conditions: a) I
DMF, 90 ˚C, overnight, 35%.
, EtOH, reflux, 4 h, 86%~quant. b) 1-methylpiperazine, K
CO ,
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Scheme 3. Resynthesis of Compound 3.a
aReagents and conditions: a) 2-bromoethan-1-ol (33), K
EtOH, reflux, 2 h, 82% (two steps).
CO
, CH
CN, reflux, overnight. b) 8b, I ,
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The synthetic procedure of series III containing a new chromone core is depicted in Scheme
4. Aldol reaction of reagents 20 and 35 gave intermediate 36 which was cyclized under I in
DMSO leading to compound 37. Compound 37 was deprotected in presence of Pd(PPh and
CO affording critical intermediate 38. Compound 38 was substituted by 2-bromoethan-1-ol
33) to produce compound 39 which is exactly the same with compound 3 except the core. More
2
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3 4
K
2
3
(
derivatives 42~49 were obtained via a substitution reaction and cyclic addition. Compound 50
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was obtained through transformation of –OH (44) into –F under diethylaminosulfur trifluoride
(DAST) in a yield of 80%.
Scheme 4. Synthetic Procedure of Series III.a
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Reagents and conditions: a) 50% KOH, EtOH, rt., overnight, used directly for next step. b) I
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DMF, 80 ˚C, overnight, 55~72%. e) R -H, K
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3
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3
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Inhibitory Activities against TLR3-Induced Expression of Inflammatory Genes In
Vitro. All the newly synthesized compounds were first evaluated for inhibitory activity of TLR3
signaling in human small airway epithelial cells (hSAECs) at the concentration of 10 μM, an
assay to demonstrate cellular permeability and BRD4 inhibitory efficacy for further studies.
TLR3-inducible expression of CIG5 and IL-6 genes was determined by quantitative real-time
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PCR (qRT-PCR). Percentages of inhibition (%) were calculated based on the level of mRNA
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expression in poly(I:C)-stimulated cells without compounds. For the first series of compounds,
7 was tested as the positive control. Different halogens at diverse positions and bulky
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
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9
0
1
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7
8
9
0
substituents (e.g., 1-methyl-1H-pyrazol-3-yl and NO ) on the quinazolin-4-one core as well as a
2
methyl group on the amide were explored. Despite the finding that compound 7 displayed
impressive inhibitory activities of CIG5 and IL-6 mRNA induction with inhibition rates of 86%
and 75%, respectively, none of its newly synthesized analogues showed obvious improvement
(Table 1). This suggests that the electron-donating methoxy groups on the phenyl ring are
critical for the cellular effects.
Table 1. Inhibitory Rates of Series I Compounds on TLR3-induced Expression of
Inflammatory Genes in hSAECs at the concentration of 10 μMa
R1
R2
Compounds
CIG5 (%) IL-6 (%)
5
7
-OMe
-OMe
7
H
86 ± 11
75 ± 8.6
1
11
1
1
1
0
H
5-F
6-F
6-Cl
6-Br
7-Br
H
H
H
H
H
H
-26 ± 3.1
33 ± 4.8
-4.5 ± 0.57 62 ± 7.1
37 ± 4.9
43 ± 6.1
51 ± 6.3
81 ± 8.9
62 ± 7.2
2
3
4
53 ± 5.6
25 ± 3.4
15 ± 1.9
15
7
-(1-methyl-1H-
pyrazol-3-yl)
1
1
2
6
9
2
H
CH
CH
71 ± 8.5
-69 ± 7.4
18 ± 2.3
-24 ± 2.8
86 ± 9.1
55 ± 6.5
7-NO
2
3
3
H
aInhibitory rates (%) are reported as mean ± SD of expression with compound divided by that in
solvent (×100). Data are derived from three independent measurements (n = 3).
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5
6
The second series of compounds were tested using compounds 3 and 7 as the positive
controls, which had similar cellular activities (Table 2). We first validated the necessity of
electron-donating methoxy groups on the quinazolin-4-one core. Consistent with the results of
first series of small molecules, compounds 24 and 25 without methoxy groups exhibited
obviously decreased activity. Substituents on the single phenyl ring were then explored, and –
0
1
2
3
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5
6
7
8
9
0
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3
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9
0
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0
1
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3
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9
0
OH group at 4-position (29) was more favored than –CF (27 and 28). Replacement of 3,5-
3
dimethyl group with H (29) or 2-Cl (30) displayed slightly declined inhibitory activities against
both poly(I:C)-induced CIG5 and IL-6 expression. Further substituents on 4-OH were
investigated, and both compounds 31 and 32 showed equal or better inhibition than 3, indicating
that this position is tolerable for structural optimization.
Table 2. Inhibitory Rates of Series II Compounds on TLR3-induced Expression of Pro-
Inflammatory Genes at the concentration of 10 μMa
Compounds
R3
R4
CIG5 (%) IL-6 (%)
3
7
3,5-di Me
3,5-di Me
2,5-di F
3,5-di OMe
H
84 ± 8.6
86 ± 9.7
2.2 ± 0.37
61 ± 7.3
45 ± 4.2
-21 ± 2.9
81 ± 9.2
65 ± 7.3
75 ± 8.4
59 ± 6.8
59 ± 7.5
-33 ± 4.1
45 ± 5.7
63 ± 6.8
OH
H
24
2
5
H
27
CF
3
3
2
8
CH
3
CF
29
H
OH
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30
2-Cl
OH
42 ± 4.8
87 ± 9.9
66 ± 7.5
83 ± 8.4
3
1
3,5-di Me
3
2
3,5-di Me
83 ± 8.8
80 ± 9.2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aInhibitory rates (%) are reported as the geometric mean of expression with compound divided
by that in solvent (×100). Data are derived from three independent measurements.
The third series of compounds were obtained through a scaffold hopping approach (Table 3).
Compound 39 exhibited significantly improved inhibition of poly(I:C)-induced CIG5 and IL-6
expression (inhibitory rates of 97% and 98%, respectively) compared to compound 3, suggesting
that the new chromone core appears to be superior to the quinazolin-4-one scaffold. Based on
SAR of series II compounds, the side chain on 4-OH was found to be tolerant to modification.
Thus, we tried various hydrophilic side chains containing heterocyclic ring capable of increasing
the aqueous solubility of the small molecules and constraining its conformation at the same time.
Compared to 32, compounds 43 and 45 with new chromone core displayed a better inhibitory
activity (99% against both CIG5 and IL-6). Compounds incorporating piperidine (42 and 44) and
pyrrole (48) exhibited similarly potent effects to 1-methylpiperazine (43 and 45), while
compounds with moieties of morpholine (46), 4,4-difluoropiperidine (47) and dimethylamine (49)
displayed substantially decreased inhibitory activities. R- or S- configuration of -OH group on
the side chain was found to make no significant differences (42 vs 44 and 43 vs 45). Fluorine is
2
9
widely used to improve the metabolic stability in drug discovery process and the replacement
of –OH of 44 with –F (50) showed slightly declined inhibition against poly(I:C)-induced CIG5
and IL-6 expression compared to that of compound 44.
Table 3. Inhibitory Rates of Series III Compounds on TLR3-induced Expression of Pro-
a
Inflammatory Genes at the concentration of 10 μM
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2
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2
2
2
2
2
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3
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3
3
3
4
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4
4
4
4
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5
5
6
Compounds
R4
CIG5 (%)
IL-6 (%)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
3
84 ± 8.8
83 ± 7.7
97±10.5
100 ± 9.8
99 ± 11
99 ± 12
91 ± 10
42 ± 5.1
63 ± 7.5
92 ± 11
-19 ± 2.5
85 ± 9.3
65 ± 5.7
NTb
3
7
39
98±11.4
100 ± 11
99 ± 9.2
100 ± 11
97 ± 9.3
82 ± 9.6
88 ± 9.3
99 ± 11
82 ± 7.7
90 ± 8.6
4
2
43
4
4
45
4
4
4
4
5
6
7
8
9
0
a
Inhibitory rates (%) are reported as the geometric mean of expression with compound divided
by that in solvent (×100). Data are derived from three independent measurements. NT: Not
tested.
b
IC50 values were calculated for selected active compounds 39, 42-45 and 48 displaying
potent inhibition against both genes with inhibitory rates over 90% (Table 4). Compound 32
with quinazolin-4-one core was also included for comparison. Positive control 1 exhibited IC50
values of 0.92 μM and 1.02 μM against poly(I:C)-induced CIG5 and IL-6 expression,
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respectively. Compounds 32 and 39 displayed 2 to 4-fold decreased IC50 values which were
comparable to compound 3. Compounds 42~45 exhibited significantly improved effects with
submicromolar cellular IC50 values, while compound 48 displayed IC50 values around 1 μM
comparable to that of reference compounds 1 and 3. The most potent compound 45 showed
excellent IC50 values of 280 nM and 310 nM against poly(I:C)-induced CIG5 and IL-6
expression, respectively (Table 4).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. IC50 Values of Selected Compounds against TLR3-Induced Expression of
a
Inflammatory Genes
Compd
CIG5 (μM)
0.95 ± 0.11
1.66 ± 0.15
2.6 ± 0.22
IL-6 (μM)
1.02 ± 0.10
3.29 ± 0.41
3.5 ± 0.33
2.8 ± 0.32
0.54 ± 0.47
0.75 ± 0.08
1.1 ± 0.12
0.31 ± 0.02
0.97 ± 0.09
1
3
3
3
4
4
4
2
9
2
3
4
2.6 ± 0.21
0.58 ± 0.49
0.83 ± 0.07
0.52 ± 0.05
0.28 ± 0.03
1.48 ± 0.17
45
48
aIC50 values are reported as the mean concentration (M) derived from three independent
measurements. Each titration curve was generated from at least 8 different concentrations.
Binding Affinities of Selected Compounds towards BET BDs. Compounds 42, 43, 44 and
4
5 were selected for further studies to determine binding affinities with BET BDs and non-BET
proteins via time-resolved fluorescence energy transfer (TR-FRET) assay. Results of positive
controls 1 and 3 were consistent with reported data which reflected the reliability of our assay.
Compounds 42-45 showed about a 10-fold increase in potency over compound 3 for BRD4 BD1
binding. Compounds 42 and 43 are BRD4 selective without preference for BRD4 BD1 and
BRD4 BD2, while both compounds 44 and 45 exhibited a good selectivity for BRD4 BD1 over
other BET BDs, including BRD4 BD2. Besides, compound 45 displayed good selectivity over
non-BET bromodomain-containing proteins (Table S1). Meanwhile, no obvious off-target
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
effects of 45 were observed from the NIMH psychoactive drug screening program (PDSP) using
a wide panel of target screening (Table S2).
Table 5. Binding Affinities of Selected Compounds for the BET BDs and Non-BET Protein
a
CBP (nM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BDs
1
3
42
43
44
45
BRD4 BD1
BRD4 BD2
BRD2 BD1
BRD2 BD2
BRD3 BD1
BRD3 BD2
BRDT BD1
BRDT BD2
CBP
92 ± 7.8
62 ± 5.9
78 ± 7.2
52 ± 5.5
81 ± 7.4
69 ± 5.7
183 ± 16
217 ± 23
9,600 ± 785
1,142 ± 98
135 ± 14
86 ± 7.8
127 ± 14
67 ± 5.4
84 ± 7.3
93 ± 8.5
139 ± 12
684 ± 7.5
791±67
718 ± 69
886±75
5,780 ± 463
251 ± 186
3,962 ± 317
203 ± 19
1,205 ± 116
1,196 ± 97
1,906 ± 173
1,719 ± 156
2,502 ± 195
2,411 ± 207
>10,000
1,286±113
1,437 ± 156
2,631 ± 197
1,962 ± 159
2,765 ± 237
2,545 ± 236
>10,000
845 ± 73
914 ± 81
3,122 ± 285
2,495 ± 217
3,592 ± 293
3,176 ± 276
>10,000
2,395 ± 198
2,081 ± 186
2,869 ± 257
2,317 ± 206
>10,000
4,836 ± 457
708 ± 69
>10,000
aBinding affinity was measured using a TR-FRET assay with the isolated recombinant BD.
Reported as mean ± SD from three separate assay runs (n = 3).
Co-crystal Structure Analysis of Compound 45 in Complex with Human BRD4 BD1.
To gain more insight into the binding modes of compound 45 with BRD4 BD1 and validate the
efficiency of the scaffold hopping strategy, we have successfully solved a high resolution co-
crystal structure of 45 in complex with BRD4 BD1 at 2 Å (PDB code: 6UWU; Figure 3, S1 and
Table S3). Similar to the binding mode shown by compound 3, the new inhibitor 45 occupied
the classical KAc recognition site very well with the aid of several water molecules (Figure 3A).
The carbonyl O atom interacted with Asn140 directly and formed an indirect hydrogen bond
with Tyr97 mediated by a water molecule. One methoxy group on the chromone core was
accommodated in the pocket by a hydrogen bond mediated with water, while the other methoxy
group extended to the WPF shelf area which was consistent with SAR analysis indicating the
critical role of –OMe moieties. The 1-methylpiperazine was excluded from the pocket and
reached the solvent area consistent with the SAR and perfectly explaining why side chain
modifications were tolerated (Figure 3B). The chiral –OH on the side chain is in proximity of a
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water molecule where it forms a water-mediated hydrogen bond with Asn93 (Figure 3C). The
equivalent residue of Asn93 on BRD4 BD2 is a glycine, and thus this hydrogen bond interaction
is missing for BRD4 BD2 with 45. Similarly, the equivalent residues of Asn93 are glycine for
BRD2 BD1, glycine for BRD2 BD2, glutamic acid for BRD3 BD2, glutamine for BRDT BD1
and glycine for BRDT BD2, respectively. This may explain the selectivity of 45 over
bromodomains of BET family members. Although BRD3 BD1 has the same asparagine, the
overall ZA loop shift (as far as 5~8 Å) is obvious thereby allowing the KAc recognition pocket
much larger than BRD4 BD1 (Figure S2). Therefore, it is likely more difficult for the compound
adhering to the residues. This speculation is consistent with the previous reported results from
Morelli group.27 Overlay analysis of the crystal structures of 3 and 45 with BRD4 BD1
demonstrated a highly conserved pose except for the flexible side chain on 4-OH (Figure 3D).
These findings validated and supported the high reliability and feasibility of our scaffold hopping
strategy.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
2
3
4
5
6
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8
9
0
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0
1
2
3
4
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9
0
1
2
3
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6
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8
9
0
Figure 3. (A) Crystal structure of 45 (in yellow) in co-complex with human BRD4 BD1 (PDB
code: 6UWU) in representation of electrostatic potential surface. (B) Critical interactions of 45
with BRD4 BD1. Residues Asn140, Tyr97 and Pro82 are highlighted in light blue and hydrogen
bonds are colored in red. (C) Zoomed details around chiral –OH. Residues Asn93 and Leu92 are
highlighted in light blue. (D) Overlay analysis of 3 (in light blue, PDB code: 4MR4) and 45 (in
yellow) in co-complex with human BRD4 BD1 in ribbon representation.
Drug-like Properties of BRD4 BD1 Inhibitors 44 and 45. With promising in vitro
activities in hand, we then studied the in vivo PK profiles of compounds 44 and 45. As listed in
Table 6 and depicted in Figure S3, these inhibitors displayed similar but overall favorable PK
parameters (3 to 4 hours of half-life, maximum concentration around 600 ng/mL and AUC
values of about 5,000 ng∙h/mL). The ultimate oral bioavailability of compounds 44 and 45 are
3
8% and 35%, respectively, indicating both compounds are orally available. Moreover, both
compounds have an excellent aqueous solubility with concentrations > 50 mg/mL. Furthermore,
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compound 45 is metabolically stable in the liver microsomes stability assay and CYP450
inhibition study. The clearance rates were 25 and 5.6 μL/min/mg for mouse liver microsomes
(MLM) and human liver microsomes (HLM), respectively. The unbound fractions of compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5 are 12% and 17% in mouse and human plasma protein binding, respectively, which is
reasonable to drive the pharmacological effects. In addition, no obvious inhibitor effects were
found on either hERG or CYP450 enzymes including CYP 3A4, 1A2, 2C9, 2D6, 2C19, 2C8 or
2
B6 at the concentration of 10 μM (Table 7). Taken together, these BRD4 BD1 inhibitors
demonstrated impressive overall drug-like properties to support their further clinical
development.
Table 6. In Vivo PK Profiles of Compounds 44 and 45 in Rats.a
4
4
45
bt1/2 (h)
3.5 ± 0.71
2,861 ± 426
6,763 ± 590
1.48 ± 0.13
2.4 ± 0.55
575.1 ± 179
5,171 ± 3447
38.2
4.1 ± 0.43
2,090 ± 265
7,033 ± 2161
1.48 ± 0.38
2.8 ± 0.27
605.5 ± 182
4,966 ± 1772
35.3
cCmax (ng/mL)
AUC0-t (ng∙h/mL)
i.v. (10 mg/kg)
p.o. (20 mg/kg)
d
e
CL (L/hr/kg)
t
1/2 (h)
C
max (ng/mL)
AUC0f-t (ng∙h/mL)
F (%)
a
Compounds were formulated in 10%DMSO/60%PEG400/30%Saline and administrated
b
c
intravenously (i.v.) or per os (p.o.). t1/2, half-life. Cmax, maximum plasma concentration
achieved. AUC0-t, total exposure following single dose. CL, total clearance. F, oral
d
e
f
bioavailability.
Table 7. In Vitro Liver Microsomes Stability Assay, Cytochrome P450 Enzymes and hERG
Inhibition Assaya
Compound
MLM (CL, μL/min/mg)
45
25
HLM (CL, μL/min/mg)
5.6
12
17
Mouse plasma protein binding (free fraction, %)b
Human plasma protein binding (free fraction, %)
c
CYP 3A4(MDZ)
9.5 ± 5.6
8.2 ± 2.1
0.0 ± 0.0
CYP 3A4(Testo)d
CYP 1A2
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5
6
CYP 2C9
CYP 2D6
CYP 2C19
CYP 2C8
1.9 ± 0.25
0.0 ± 0.0
1.7 ± 0.29
17 ± 3.0
CYP 2B6
hERG (IC50, μM)
27 ± 4.3
> 30
0
1
2
3
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8
9
0
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a
c
b
Inhibitory rates (%) on CYP450 enzyme at the concentration of 10 μM. Concentration is 2 μM
Substrate is midazolam (MDZ). Substrate is testosterone (Testo).
d
In Vivo Efficacy of Selected BRD4 BD1 Inhibitors 44 and 45. Next, compounds 44 and
4
5 were investigated in our established murine model of TLR3-induced acute airway
inflammation.8, 12 Both oral administration (p.o.) and intraperitoneal administration (i.p.) were
employed to compare their in vivo efficacy. Vehicle alone and poly (I:C)-treated groups were
tested as healthy and pathological controls, respectively. Histologically, we observed that
poly(I:C) induced a profound neutrophilic inflammation around the small- and medium-sized
airways which was completely inhibited by two inhibitors at the dose of 10 mg/kg via p.o. or i.p.
administration respectively (Figure 4A). Meanwhile, elevated secretion of total cells and
neutrophils (Figure 4B) was suppressed, and inflammatory cytokines (IL-6, KC, MCP-1, and
RANTES, Figure 4C) in bronchoalveolar lavage fluid (BALF) were also significantly blocked.
We also measured inflammatory gene expression in lung tissue (Figure 4D). Administration of
poly(I:C)-induced 15- to 20-fold increase of inflammatory genes IL-6, KC, ISG54 and CIG5 in
lung tissue. Compounds 44 and 45 were found to inhibit these increases significantly. These
results validated the potent in vivo efficacy of our newly designed compounds achieved from the
scaffold hopping approach. To further validate their in vivo efficacy actually induced by
inhibiting BRD4 rather than any other off-target effects, we examined the level of H3K122Ac
which is verified as a BRD4 biomarker (Figure 5).10,12,13 Immunofluorescence staining of
H3K122Ac demonstrated that poly(I:C) induced significant increase of BRD4 activities in both
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hSAECs and lung tissue of mice, and this poly(I:C)-induced effect was effectively blocked by
BRD4 inhibitors 44 and 45 through the down-regulation of H3K122Ac.
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Figure 4. In vivo efficacy of BRD4 BD1 inhibitors 44 and 45 in poly(I:C)-induced airway
inflammation of mice. (A) C57BL6/J mice were pre-treated (i.p. or p.o.) with 44 and 45 at the
dose of 10 mg/kg before intranasal (i.n.) poly(I:C) exposure. Lung histology images of
Hematoxylin and Eosin (H&E) staining were performed on paraffin-embedded lung sections and
were shown at magnitude of × 10 and × 20 respectively. (B) BRD4 inhibitors 44 and 45 blocked
poly(I:C)-induced secretion of inflammatory cells in BALF. (C) BRD4 inhibitors blocked
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poly(I:C)-induced secretion of inflammatory cytokines in BALF. (D) BRD4 inhibitors blocked
poly(I:C)-induced inflammatory gene expression in lung tissue.
0
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Figure 5. BRD4 inhibitors 44 and 45 block poly(I:C)-induced H3K122Ac in hSAECs (A) and in
lung tissue of mice (B). A) Immunofluorescence staining of H3K122Ac (green) were performed
in hSAECs. B) Immunofluorescence staining of H3K122Ac (red) were performed on paraffin-
embedded lung sections of mice. Right panel, quantification of total fluorescence intensity
shown as fold changes on immunofluorescence staining of H3K122Ac. #p < 0.01, compared
with control; *p < 0.01 compared with poly(I:C) only, n = 5.
CONCLUSION
In summary, we have discovered a series of novel chromone derivatives as potent and selective
BRD4 BD1 inhibitors via a scaffolding hopping strategy. Among them, compounds 44 (ZL0513)
and 45 (ZL0516) displayed potent cellular effects with submicromolar IC50 values, nanomolar
binding affinity of 67~84 nM with BR4 BD1 and good selectivity as well as significant in vivo
efficacy in blocking poly(I:C)-induced airway inflammation and neutrophilia of mice. Co-crystal
structure of 45 in complex with human BRD4 BD1 validated its classical KAc recognition site
with critical hydrogen bonds to Asn140 and Tyr97. At the same time, both BRD4 BD1 inhibitors
4
4 and 45 demonstrated excellent DMPK properties including bioavailability >35%, aqueous
solubility, metabolic stability, and weak to low CYP450 enzymes and hERG inhibitory effects.
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Collectively, the findings support these BRD4 BD1 inhibitors with impressive overall profiles of
in vivo efficacy and drug-like properties as promising advanced lead compounds for the further
optimization and extensive clinical development towards epigenetic therapeutics for the
treatment of inflammatory diseases.
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EXPERIMENTAL SECTION
General Chemistry Information. All commercially available starting materials and
solvents were reagent grade, and used without further purification unless otherwise specified.
Reactions were performed under a nitrogen atmosphere in dry glassware with magnetic stirring.
Preparative column chromatography was performed using silica gel 60, particle size 0.063-0.200
mm (70-230 mesh, flash). Analytical TLC was carried out employing silica gel 60 F254 plates
(Merck, Darmstadt). Visualization of the developed chromatograms was performed with
1
detection by UV (254 nm). NMR spectra were recorded on a Bruker-600 or Bruker-300 ( H, 600
13
1
13
&
300 MHz; C, 150 & 75 MHz) spectrometer. H and C NMR spectra were recorded with
TMS as an internal reference. Chemical shifts were expressed in ppm, and J values were given in
Hz. High-resolution mass spectra (HRMS) were obtained from Thermo Fisher LTQ Orbitrap
Elite mass spectrometer. Parameters include the following: Nano ESI spray voltage was 1.8 kV;
o
Capillary temperature was 275 C and the resolution was 60,000; Ionization was achieved by
positive mode. Purities of final compounds were established by analytical HPLC, which was
carried out on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-20A UV/VIS).
HPLC analysis conditions: Waters Bondapak C18 (300 × 3.9 mm); flow rate 0.5 mL/min; UV
detection at 270 and 254 nm; linear gradient from 10% acetonitrile in water to 100% acetonitrile
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6
in water in 20 min followed by 30 min of the last-named solvent (0.1% TFA was added into both
acetonitrile and water). All biologically evaluated compounds are > 95% pure.
2-(4-Hydroxy-3,5-dimethylphenyl)quinazolin-4(3H)-one (10). To a solution of 2-
aminobenzamide (136 mg, 1.0 mmol) and 4-hydroxy-3,5-dimethylbenzaldehyde (180 mg, 1.2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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mmol) in 10 mL EtOH, I
2
(279 mg, 1.1 mmol) was added. After reflux for 4 h, 5% Na
2
S
2
4
O was
added to quench the reaction. Compound 10 (266 mg, quant.) was obtained through filtration as a
1
white solid. HPLC purity 95.7% (t
R
= 13.13 min). H NMR (300 MHz, DMSO-d
6
) δ 12.10 (s,
1
7
H), 8.11 (d, J = 7.9 Hz, 1H), 7.86 (s, 2H), 7.79 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H),
13
.45 (t, J = 7.7 Hz, 1H), 2.25 (s, 6H). C NMR (75 MHz, DMSO-d
6
) δ 162.7, 157.0, 152.8,
+
149.4, 134.9, 128.5, 127.4, 126.3, 124.7, 123.4, 121.0, 17.1. HR ESI-MS (M + H) m/z =
2
67.1134 (calcd for C16
H
15
N
2
O
2
: 267.1127).
5
-Fluoro-2-(4-hydroxy-3,5-dimethylphenyl)quinazolin-4(3H)-one (11). Compound 11
(71 mg, quant.) was obtained as a white solid following the procedure of 10. HPLC purity 95.4%
1
(t
R
= 13.97 min). H NMR (300 MHz, DMSO-d
6
) δ 12.15 (s, 1H), 9.00 (s, 1H), 7.85 (s, 2H), 7.79
1
3
–
7.67 (m, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.17 (dd, J = 10.9, 8.2 Hz, 1H), 2.24 (s, 6H). C NMR
(75 MHz, DMSO-d
6
) δ 162.8, 160.0, 159.3, 157.3, 153.7, 151.7, 135.4, 135.3, 128.6, 124.7,
+
1
23.7, 122.9, 112.6, 112.4, 110.4, 17.18. HR ESI-MS (M + H) m/z = 285.1029 (calcd for
C
16
H14FN
2
O
2
: 285.1039).
6
-Fluoro-2-(4-hydroxy-3,5-dimethylphenyl)quinazolin-4(3H)-one (12). Compound 12
(74 mg, quant.) was obtained as a white solid following the procedure of compound 10. HPLC
1
purity 96.1% (t
R
= 14.52 min). H NMR (300 MHz, DMSO-d6) δ 12.27 (s, 1H), 8.95 (d, J = 2.8
Hz, 1H), 7.83 (s, 2H), 7.76 (dq, J = 9.0, 5.7, 4.1 Hz, 2H), 7.67 (dd, J = 8.5, 2.5 Hz, 1H), 2.24 (s,
1
3
6H). C NMR (75 MHz, DMSO-d ) δ 162.1, 161.6, 158.4, 157.0, 152.2, 146.4, 130.4, 130.3,
6
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+
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2
28.5, 124.7, 123.5, 123.3, 123.2, 122.2, 122.1, 111.0, 110.7, 17.1. HR ESI-MS (M + H) m/z =
85.1031 (calcd for C16 : 285.1039).
6-Chloro-2-(4-hydroxy-3,5-dimethylphenyl)-3-methylquinazolin-4(3H)-one
H14FN
2
O
2
(13).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
4
4
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2
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Compound 13 (322 mg, quant.) was obtained as a yellow solid following the procedure of
1
compound 10. H NMR (300 MHz, DMSO-d
6
) δ 12.29 (s, 1H), 8.97 (s, 1H), 8.02 (d, J = 2.6 Hz,
1
3
1
H), 7.84 (s, 2H), 7.78 (dd, J = 8.7, 2.5 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 2.24 (s, 6H). C NMR
(
75 MHz, DMSO) δ 161.8, 157.2, 153.2, 148.2, 134.9, 130.4, 129.7, 128.6, 125.2, 124.7, 123.2,
+
1
22.2, 17.1. HR ESI-MS (M + H) m/z = 301.0749 (calcd for C17
H16ClN
2
O : 301.0744).
2
6
-Bromo-2-(4-hydroxy-3,5-dimethylphenyl)quinazolin-4(3H)-one (14). Compound 14
(384 mg, quant.) was obtained as a white solid following the procedure of compound 10. HPLC
1
purity 98.4% (t
R
= 17.23 min). H NMR (300 MHz, DMSO-d
6
) δ 12.33 (s, 1H), 8.99 (s, 1H),
8
.17 (d, J = 2.4 Hz, 1H), 7.91 (dd, J = 8.7, 2.4 Hz, 1H), 7.85 (s, 2H), 7.63 (d, J = 8.7 Hz, 1H),
1
3
2.24 (s, 6H). C NMR (75 MHz, DMSO-d
6
) δ 161.6, 157.2, 153.3, 148.6, 137.7, 130.0, 128.6,
+
1
3
28.6, 128.3, 124.7, 124.7, 123.2, 123.1, 122.6, 118.5, 17.09, 17.05. HR ESI-MS (M + H) m/z =
45.0225 (calcd for C16
H14BrN
2
O
2
: 345.0239).
6
-Bromo-2-(4-hydroxy-3,5-dimethylphenyl)-3-methylquinazolin-4(3H)-one
(15).
Compound 15 (340 mg, 94%) was obtained as a brown solid following the procedure of
1
compound 10. HPLC purity 96.1 % (t
R
= 17.54 min). H NMR (300 MHz, DMSO-d
6
) δ 8.00 (dd,
13
J = 8.4, 1.9 Hz, 1H), 7.86 (d, J = 4.3 Hz, 3H), 7.59 (d, J = 8.4 Hz, 1H), 2.29 – 2.16 (m, 6H). C
NMR (75 MHz, DMSO-d
6
) δ 162.4, 157.3, 154.2, 150.7, 129.7, 129.1, 128.7, 128.4, 128.3,
+
1
3
24.7, 123.1, 120.1, 17.1. HR ESI-MS (M + H) m/z = 345.0236 (calcd for C17
H
16BrN
2
2
O :
45.0239).
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2
-(4-Hydroxy-3,5-dimethylphenyl)-7-(1-methyl-1H-pyrazol-5-yl)quinazolin-4(3H)-
one (16). Compound 16 (70 mg, 89%) was obtained as a brown solid following the procedure of
1
compound 10. HPLC purity 95.8 % (t
R
= 14.74 min). H NMR (300 MHz, DMSO-d
6
) δ 12.25 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
H), 8.98 (s, 1H), 8.18 (d, J = 8.1 Hz, 1H), 7.89 (s, 2H), 7.81 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H),
1
3
.53 (d, J = 2.1 Hz, 1H), 6.61 (d, J = 2.3 Hz, 1H), 3.97 (s, 3H), 2.25 (s, 6H). C NMR (75 MHz,
DMSO-d
6
) δ 162.4, 157.2, 153.5, 149.8, 142.1, 138.6, 136.3, 128.6, 126.9, 126.2, 124.7, 123.3,
+
1
3
20.4, 107.2, 38.4, 17.1. HR ESI-MS (M + Na) m/z = 369.1333 (calcd for C20
H
19
N
4
O :
2
69.1327).
-(4-Hydroxy-3,5-dimethylphenyl)-3-methyl-7-nitroquinazolin-4(3H)-one (19). To a
solution of 7-nitro-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (416 mg, 2.0 mmol) in 20 mL THF,
DIPEA (516 mg, 4.0 mmol) and CH NH (544 mg, 8.0 mmol) were added at room temperature.
After 2 h, the solution was poured in to a 0.5 M HCl solution and extracted by CH Cl . The
organic phase was dried over anhydrous Na SO and concentrated to get crude 2-amino-N-
methyl-4-nitrobenzamide 18 which was used directly in the next step. Compound 19 (186 mg,
2
3
2
2
2
2
4
5
7% for two steps) was obtained from 2-amino-N-methyl-4-nitrobenzamide following the
1
procedure of 10 as a yellow solid. HPLC purity 98.8% (t
DMSO-d
R
= 16.66 min). H NMR (300 MHz,
6
) δ 8.84 (s, 1H), 8.31 (d, J = 8.6 Hz, 2H), 8.17 (d, J = 8.8 Hz, 1H), 7.27 (s, 2H), 3.43 (s,
13
3
1
H), 2.23 (s, 6H). C NMR (75 MHz, DMSO-d
6
) δ 161.5, 159.1, 155.7, 151.5, 147.9, 129.1,
+
28.9, 125.8, 124.6, 124.3, 122.5, 120.3, 35.1, 17.0. HR ESI-MS (M + H) m/z = 326.1147
(calcd for C17
-(4-(Allyloxy)-3,5-dimethylphenyl)quinazolin-4(3H)-one (22). Compound 21 was
obtained as a white solid following the procedure of 10. To a solution of compound 21 (153 mg,
H
16
N
3
O
4
:326.1141).
2
0.5 mmol) and CH
3
I (142 mg, 1.0 mmol) in 3 mL of DMF, NaH (24 mg, 1.0 mmol) was added.
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After 4 hours, the mixture was poured into iced water and extracted by CH
2
Cl . The combined
2
organic layer was concentrated and purified by PTLC to give 2-(4-(allyloxy)-3,5-
dimethylphenyl)-3-methylquinazolin-4(3H)-one as a white solid. To a solution of 2-(4-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
3
3
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3
4
4
4
4
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4
4
4
4
4
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5
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5
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5
6
0
1
2
3
4
5
6
7
8
9
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1
2
3
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5
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7
8
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0
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7
8
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(
allyloxy)-3,5-dimethylphenyl)-3-methylquinazolin-4(3H)-one (100 mg, 0.31 mmol) in 5 mL of
CH OH, Pd(PPh (11 mg, 0.01 mmol) and K CO (171 mg, 1.24 mmol) were added. After
reflux for 4 h, the mixture was poured in to a 0.5 M HCl solution and extracted by CH Cl . The
organic phase was dried over anhydrous Na SO and concentrated to get a crude product which
was purified by silica gel column chromatography (CH
3
3
)
4
2
3
2
2
2
4
2
Cl
2
/MeOH = 20/1 to 10/1) to provide the
1
desired product (62 mg, 72%) as a grey solid. HPLC purity 98.6 % (t
R
= 15.6 min). H NMR
(300 MHz, DMSO-d
6
) δ 8.15 (d, J = 7.9 Hz, 1H), 7.86 – 7.75 (m, 1H), 7.64 (d, J = 8.5 Hz, 1H),
13
7
.52 (d, J = 8.1 Hz, 1H), 7.25 (s, 2H), 3.42 (s, 3H), 2.23 (s, 6H). C NMR (75 MHz, DMSO-d
6
)
δ 162.4, 157.0, 155.7, 155.7, 147.7, 134.6, 129.0, 127.5, 126.8, 126.5, 126.1, 124.6, 120.2, 34.7,
+
17.1. HR ESI-MS (M + H) m/z = 281.1282 (calcd for C17
H
17
N
2
2
O : 281.1290).
2
-(2,5-Difluoro-4-hydroxyphenyl)quinazolin-4(3H)-one (24). Compound 24 (120 mg,
8
8%) was obtained as a light yellow solid following the procedure of 10. HPLC purity 95.7% (t
R
1
=
=
6.74 min). H NMR (300 MHz, DMSO-d
6
) δ 12.29 (s, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.83 (t, J
7.8 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.61 (dd, J = 11.4, 6.9 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H),
13
6
1
.89 (dd, J = 11.8, 7.2 Hz, 1H). C NMR (75 MHz, DMSO-d
6
) δ 162.0, 158.3, 155.0, 149.5,
49.2, 149.1, 146.3, 143.3, 135.0, 127.7, 127.2, 126.3, 121.4, 117.8, 117.5, 117.5, 112.5, 112.4,
+
106.0, 105.6. HR ESI-MS (M + H) m/z = 335.0794 (calcd for C14
H
9
F
2
N
2
O : 335.0843).
2
2
-(4-Hydroxy-3,5-dimethoxyphenyl)quinazolin-4(3H)-one (25). Compound 25 (153
mg, quant.) was obtained as a white solid following the procedure of 10. HPLC purity 95.3% (t
R
1
= 14.06 min). H NMR (300 MHz, DMSO-d
6
) δ 12.37 (s, 1H), 9.11 (s, 1H), 8.13 (dd, J = 8.0, 1.6
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Journal of Medicinal Chemistry
Page 28 of 50
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2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
Hz, 1H), 7.86 – 7.77 (m, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.58 (s, 2H), 7.52 – 7.43 (m, 1H), 3.89 (s,
1
3
6
H). C NMR (75 MHz, DMSO-d
6
) δ 162.9, 152.5, 149.3, 148.3, 139.6, 135.0, 127.6, 126.4,
+
126.3, 122.6, 121.1, 105.9, 56.7. HR ESI-MS (M + Na) m/z = 381.1105 (calcd for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
16
H
14
N
2
NaO
4
: 381.1163).
5
,7-Dimethoxy-2-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (27). Compound
2
9
2
5 (75 mg, 86%) was obtained as a white solid following the procedure of 10. HPLC purity
1
6.6% (t
R
= 17.17 min). H NMR (300 MHz, DMSO-d
6
) δ 12.23 (s, 1H), 8.35 (d, J = 8.2 Hz,
H), 7.90 (d, J = 8.1 Hz, 2H), 6.79 (s, 1H), 6.58 (s, 1H), 3.88 (d, J = 10.8 Hz, 6H). HR ESI-MS
+
(
M + H) m/z = 351.0947 (calcd for C17
H
14
F
3
N
2
O : 351.0957).
3
5,7-Dimethoxy-2-(3-methyl-4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one
(28).
Compound 28 (80 mg, 88%) was obtained as a white solid following the procedure of 10. HPLC
1
purity 97.5% (t
R
= 17.99 min). H NMR (300 MHz, DMSO-d
6
) δ 12.14 (s, 1H), 8.27 – 8.00 (m,
2H), 7.80 (d, J = 8.3 Hz, 1H), 6.78 (s, 1H), 6.57 (s, 1H), 3.88 (d, J = 11.0 Hz, 6H), 2.52 (s, 3H).
13C NMR (75 MHz, DMSO-d
) δ 164.8, 161.4, 160.1, 153.0, 152.2, 136.9, 136.4, 131.6, 126.3,
6
+
1
26.0, 101.9, 98.6, 56.5, 56.1, 19.3. HR ESI-MS (M + H) m/z = 365.1101 (calcd for
C
18
H
16
F
3
N
2
O
3
: 365.1113).
2
-(4-Hydroxyphenyl)-5,7-dimethoxyquinazolin-4(3H)-one (29). Compound 29 (84 mg,
quant.) was obtained as a yellow solid following the procedure of 10. HPLC purity 95.4% (t =
R
1
1
3.88 min). H NMR (300 MHz, DMSO-d
6
) δ 11.78 (s, 1H), 10.10 (d, J = 3.3 Hz, 1H), 8.21 –
7.91 (m, 2H), 7.06 – 6.82 (m, 2H), 6.69 (q, J = 2.2 Hz, 1H), 6.48 (q, J = 2.3 Hz, 1H), 4.05 – 3.65
13
(
m, 6H). C NMR (75 MHz, DMSO-d
6
) δ 164.6, 161.4, 161.0, 160.3, 153.6, 153.2, 129.9, 129.9,
+
1
23.3, 115.7, 115.7, 104.9, 101.4, 97.7, 56.4, 56.0. HR ESI-MS (M + H) m/z = 299.1021 (calcd
for C16
H
15
N
2
4
O : 299.1032).
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2
-(2-Chloro-4-hydroxyphenyl)-5,7-dimethoxyquinazolin-4(3H)-one (30). Compound
3
0 (94 mg, quant.) was obtained as a yellow solid following the procedure of 10. HPLC purity
1
97.9% (t
R
= 13.82 min). H NMR (300 MHz, DMSO-d
6
) δ 11.91 (s, 1H), 10.29 (s, 1H), 7.42 (d, J
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
=
7.9 Hz, 1H), 6.90 (s, 1H), 6.83 (s, 1H), 6.69 (s, 1H), 6.55 (s, 1H), 3.97 – 3.73 (m, 6H). C
NMR (75 MHz, DMSO-d
6
) δ 164.7, 161.5, 160.0, 159.8, 153.7, 153.3, 132.6, 132.3, 124.8,
+
1
16.6, 114.7, 105.3, 101.6, 98.3, 56.5, 56.1. HR ESI-MS (M + H) m/z = 333.0630 (calcd for
C
16
H
14ClN
2
O
4
: 333.0642).
(S)-2-(3,5-Dimethyl-4-(oxiran-2-ylmethoxy)phenyl)-5,7-dimethoxyquinazolin-4(3H)-
one (31). Compound 31 (215 mg, 94%) was obtained as a white solid following the procedure of
1
10. HPLC purity 95.6% (t
R
= 17.17 min). H NMR (300 MHz, DMSO-d
6
) δ 11.80 (s, 1H), 7.88
(
s, 2H), 6.72 (d, J = 2.2 Hz, 1H), 6.50 (d, J = 2.2 Hz, 1H), 5.59 (d, J = 3.7 Hz, 1H), 3.86 (d, J =
13
1
2.2 Hz, 6H), 3.79 (s, 2H), 3.49 (dd, J = 10.2, 3.9 Hz, 1H), 3.40 (s, 1H), 2.31 (s, 6H). C NMR
(75 MHz, DMSO-d
6
) δ 164.7, 161.4, 160.2, 158.3, 153.5, 152.9, 131.1, 128.8, 127.9, 105.1,
+
1
01.6, 98.0, 75.2, 69.4, 56.4, 56.1, 16.6, 12.1. HR ESI-MS (M + H) m/z = 383.1615 (calcd for
C
21
H
23
N
2
O
5
: 383.1607).
(
S)-2-(4-(2-Hydroxy-3-(4-methylpiperazin-1-yl)propoxy)-3,5-dimethylphenyl)-5,7-
dimethoxyquinazolin-4(3H)-one (32). Compound 32 (17 mg, 35%) was obtained as a white
1
solid following the procedure of 39. HPLC purity 96.1% (t
R
= 12.62 min). H NMR (300 MHz,
MeOD) δ 7.66 (s, 2H), 6.75 (d, J = 2.3 Hz, 1H), 6.50 (d, J = 2.3 Hz, 1H), 4.14 (dd, J = 7.7, 4.5
Hz, 1H), 3.91 (s, 6H), 3.82 (t, J = 4.2 Hz, 2H), 2.66 (dd, J = 13.3, 8.3 Hz, 10H), 2.35 (d, J = 2.8
13
Hz, 9H). C NMR (75 MHz, MeOD) δ 165.4, 161.6, 161.2, 158.6, 153.7, 153., 131.4, 128.1,
1
27.8, 104.1, 100.3, 97.4, 74.3, 67.6, 60.3, 55.0, 54.8, 54.4, 52.7, 44.4, 15.3. HR ESI-MS (M +
+
Na) m/z = 505.2426 (calcd for C26
H
35
N
4
5
O : 505.2427).
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