Optimization of potent and selective quinazolinediones: Inhibitors of respiratory syncytial virus that block RNA-dependent RNA-polymerase complex activity

Article abstract of DOI:10.1021/jm500902x

A quinazolinedione-derived screening hit 2 was discovered with cellular antiviral activity against respiratory syncytial virus (CPE EC₅₀ = 2.1 μM), moderate efficacy in reducing viral progeny (4.2 log at 10 μM), and marginal cytotoxic liability

Full text of DOI:10.1021/jm500902x

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Article
Optimization of Potent and Selective Quinazolinediones:
Inhibitors of Respiratory Syncytial Virus That Block
RNA-dependent RNA-polymerase Complex Activity
Daljit S. Matharu, Daniel Patrick Flaherty, Denise S. Simpson, Chad E. Schroeder,
Donghoon Chung, Dan Yan, James W. Noah, Colleen B. Jonsson, E. Lucile White,
Jeffrey Aubé, Richard K. Plemper, William E. Severson, and Jennifer E. Golden
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500902x • Publication Date (Web): 15 Nov 2014
Downloaded from http://pubs.acs.org on November 17, 2014
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Optimization of Potent and Selective
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Quinazolinediones: Inhibitors of Respiratory
Syncytial Virus That Block RNAꢀdependent RNAꢀ
polymerase Complex Activity
§
§
§
§
Daljit S. Matharu, Daniel P. Flaherty, Denise S. Simpson, Chad E. Schroeder, Donghoon
┴,‡
ɸ
‡
┴, ‡
‡
§
Chung, Dan Yan, James W. Noah, Colleen B. Jonsson, E. Lucile White, Jeffrey Aubé,
ɸ
┴, ‡
§,*
Richard K. Plemper, William E. Severson, and Jennifer E. Golden
§
University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, Kansas
66047, United States
┴
Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of
Louisville, Louisville, Kentucky 40202, United States
‡
Southern Research Specialized Biocontainment Screening Center, Southern Research Institute,
Birmingham, Alabama 35205, United States
ɸ
Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, United States
*
Corresponding author
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ABSTRACT: A quinazolinedioneꢀderived screening hit 2 was discovered with cellular antiviral
activity against respiratory syncytial virus (CPE EC = 2.1 ꢀM), moderate efficacy in reducing
50
viral progeny (4.2 log at 10 ꢀM), and marginal cytotoxic liability (selectivity index, SI ~ 24).
Scaffold optimization delivered analogs with improved potency and selectivity profiles. Most
notable were compounds 15 and 19 (EC = 300ꢀ500 nM, CC > 50 ꢀM, SI > 100) that
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significantly reduced viral titer (> 400,000ꢀfold), and several analogs were shown to block the
activity of the RNAꢀdependent RNAꢀpolymerase complex of RSV.
INTRODUCTION
Acute bronchiolitis, a common lower respiratory tract infection most seriously affecting infants
and the elderly, is predominately caused by the highly infectious human respiratory syncytial
1
ꢀ4
virus (hRSV). The virus belongs to the paramyxovirus family, which also includes mumps
5
and measles viruses; however, unlike these related pathogens for which vaccines have been
developed, a safe and effective vaccine remains elusive to prevent the contraction and
6,7
8
transmission of RSV. In fact, since the discovery of the virus over 50 years ago, the only
FDA approved small molecule inhibitor for treatment of the infection is ribavirin, a nucleoside
9
,10
antimetabolite, that is limited to use in critical cases due to its toxicological side effects. In the
United States, the prevalence of RSV infection in adults over the age of 65 results in
1
1
approximately 170,000 hospitalizations and 10,000 deaths annually while the global incidence
of RSV infection was estimated in 2005 to result in the hospitalization of 3.4 million children
12
under the age of 5. Furthermore, exposure does not impart full immunity from future infection,
and in fact, promotes an inflammatory response that can contribute to chronic lung complications
1
3,14
such as asthma.
These burdens, coupled with the absence of suitable therapeutic agents for
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susceptible populations, underscore the importance of identifying effective and safe
pharmacological countermeasures for RSV.
The scientific literature is replete with examples from translational development programs aimed
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15ꢀ17
18ꢀ22
at addressing this important need.
with several compounds that target RSV’s entryꢀenabling F protein,
the compounds were not pursued or clinical development was discontinued.
efforts, the search continues for RSV inhibitors that offer a superior pharmacological and safety
Replication inhibitors
have been investigated, along
2
3ꢀ27
though in most cases,
2
8,29
Despite these
3
0
profile compared to that of ribavirin.
As part of the National Institutes of Health Molecular Libraries Initiative, we pursued a subset of
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1,32,33
RSVꢀinhibiting hit scaffolds identified through a highꢀthroughput screen
of the national
3
4
compound repository. Optimization of a screening hit led to compound 1, probe ML232, a
sulfonamideꢀbased RSV inhibitor with singleꢀdigit micromolar in vitro activity and a proposed
1
5,35
entryꢀbased mechanism of inhibition based on timeꢀofꢀaddition studies (Fig. 1A).
In a
parallel effort, the team also launched an optimization campaign on a quinazolinedione
compound series for which we noted key differences with respect to the breadth of tunable
structureꢀactivity and structureꢀproperty relationships (SAR and SPR, respectively) and a
potentially different mechanism of action as compared to the ML232 compound series. The
quinazolinedione hit 2 was determined to inhibit a RSVꢀinduced cytopathic effect with an EC50
of 2.14 ꢀM and showed HEpꢀ2 cellular toxicity with a CC > 50 ꢀM, resulting in a selectivity
5
0
index (CC /EC ) of > 23.8 (Fig. 1B). In a titer reduction assay, hit 2 was also found to reduce
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0
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viral plaques by 4.2 log (~14,000ꢀfold as compared to control) at a concentration of 10 ꢀM. The
team undertook an optimization effort that focused on the five colored regions of the scaffold
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with the primary aims of broadening the selectivity index by enhancing potency and attenuating
cellular toxicity, amplifying the plaque reducing effect, and improving solubility (Fig. 1B).
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Figure 1. A. Structure and data for sulfonylpyrrolidineꢀderived probe 1, ML232. B. Structure and data for hit
quinazolinedione 2 with highlighted regions of structureꢀactivity relationship optimization.
CHEMISTRY
Analogs of hit 2 were generally prepared using standard peptide coupling conditions of 2ꢀamino
benzoic acid 3 with methyl 4ꢀ(aminomethyl)benzoate to afford an aminobenzamide intermediate
(Scheme 1). Subsequent cyclization with CDI generated quinazolinedione core intermediate 4.
Most analogs were made by ester hydrolysis of 4, followed by incorporation of the pendent
2
amido alkyl ether (R ) with routine amide coupling, followed by installation of the Nꢀbenzyl
1
appendage (R ) to afford products 5. In some cases, it was advantageous to affix the Nꢀbenzyl
portion prior to revealing the benzoic acid functionality for coupling to the preferred amine
1
2
component (i.e., shuffling the sequence of R vs R integration). In either case, the synthetic
route was flexible and offered selective, orthogonal, lateꢀstage diversification to prepare the
desired analog sets. Quinazolinones 7 were also prepared from anthranillic acid 3. Amide
coupling between 3 and methyl 4ꢀ(aminomethyl) benzoate afforded a 2ꢀaminobenzamide
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intermediate that was treated with an isopropylphenylacetic acid chloride. The resulting bisꢀ
amide 6 was treated with hydroxide base to reveal a benzoic acid that could be further
manipulated; however, these conditions fortuitously induced the intended hydrolysis and
necessary cyclization to generate the quinazolinone core in one step. Subsequent coupling of the
unmasked benzoic acid with 3ꢀmethoxypropylamine afforded the desired analogs 7.
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Scheme 1. Chemical Synthesis of Quinazolinedione and Quinazolinone Analogs
Reagents: (a) DIPEA, HATU, methyl 4ꢀ(aminomethyl) benzoate hydrochloride, DMF, rt, 2 h, 68%; (b) DIPEA, 1,1ꢀ
carbonyldiimidazole, CH Cl , reflux, 16 h, 95%; (c) LiOH·H O, THF, 40 °C, 1 h, 88%; (d) DIPEA, HATU, 3ꢀ
2
2
2
2
methoxypropylamine or other amine for R , DMF, rt, 2 h, 44ꢀ76%; (e) K CO , 4ꢀisopropylbenzyl bromide other aryl
2
3
1
halide for R , DMF, 40 °C, 16 h, 11ꢀ71%; (f) DIPEA, EDCI, HOBt, methyl 4ꢀ(aminomethyl) benzoate
hydrochloride, CH Cl , rt, 74%; (g) 3ꢀ or 4ꢀisopropylphenylacetic acid, (COCl) , cat. DMF, CH Cl , then pyridine,
2
2
2
2
2
CH Cl , 1.5 h, rt, 93ꢀ100%; (h) LiOH, THF, H O, 40 °C, 20 h; (i) 3ꢀmethoxypropylamine, EDCI, HOBt, DIPEA,
2
2
2
CH Cl , rt, 10ꢀ22% over 2 steps.
2
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RESULTS AND DISCUSSION
Medicinal Chemistry Optimization: For this stage of our program, 73 quinazolinedioneꢀ
derived analogs were prepared and analyzed. All compounds were evaluated for inhibition of an
RSVꢀinduced cytopathic effect and assessment of mammalian cell cytotoxicity. Both assays were
performed in a 10ꢀpoint dose response format using HEpꢀ2 cells (hRSV strain Long), and the
data from these screens was employed to drive iterative structural revision. A smaller subset of
the most promising analogs was subsequently tested for their ability to reduce viral plaques at a
compound concentration of 10 ꢀM. Solubility was determined for selected analogs as their
overall activity profiles were improved and as modifications were implemented with an
expectation of enhancing this particular parameter.
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Initially, changes in the quinazolinedione Nꢀbenzyl entity (R ) were examined while preserving
2
the hit structure’s 3ꢀmethoxypropylamine (R ) component (Table 1). Replacement of the 4ꢀ
methylbenzyl substituent with a hydrogen atom or use of truncated alkyl replacements such as Nꢀ
methyl, Nꢀnꢀpropyl or NꢀCH ꢀcyclohexyl were not tolerated, resulting in a loss of potency (EC50
2
1
>
50 ꢀM). Similarly, extension of the R benzyl linker by one additional methylene unit was not
advantageous (Nꢀ(CH ) ꢀ4ꢀtolyl, or Nꢀ(CH ) ꢀ4ꢀtrifluoromethylphenyl, or Nꢀ(CH ) ꢀ4ꢀ
2
2
2 2
2 2
bromophenyl, EC > 50 ꢀM). Generally, 4ꢀbenzyl substitution was preferred, as 3ꢀsubstituted
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benzyl derivatives lost potency compared to hit 2, and 2ꢀsubstituted benzyl analogs were inactive
(Table 1). Increasing steric bulk at the 4ꢀbenzyl position (methyl (2) ꢀ ethyl (18) ꢀ iꢀpropyl
(19)) resulted in improved potency. Importantly, cell toxicity was not observed at concentrations
exceeding 50 ꢀM for these analogs. Electron withdrawing groups incorporated at this same
position were acceptable, and those that mimicked the steric character of small branched
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aliphatic groups already known to be beneficial were the most promising. Consequently, the Nꢀ4ꢀ
nitrobenzyl derivative 15 and the Nꢀ4ꢀisopropylbenzyl analog 19 showed the best potency, viral
titer assay efficacy and cytotoxicity profile in the collection. Heterocyclic variants in this region
led to suboptimal potency, cytotoxicity and reduction in viral plaques (entries 14ꢀ17).
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Table 1. hRSV CPE Assay Potency, Cytotoxicity, Selectivity Index and Logarithmic Reduction
1
in Viral Plaques for Analogs with Structural Variations in the (R ) Region of Hit Compound 2
HEpꢀ2
RSV CPE
potency ±
standard
cellular
toxicity ±
standard
deviation
viral titer
reduction
at 10 ꢀM
(log)
selectivity
index
entry compound
deviation
(CC / EC )
50
50
a
EC (ꢀM)
b
50
1
CC (ꢀM)
50
R
c
c
1
2
3
4
5
6
7
8
9
2
CH ꢀ4ꢀmethylphenyl
2.1 ± 0.5
> 50.0
> 50.0
> 23.8
< 0.2
1.7
4.2
NT
NT
4.1
NT
NT
3.1
2.7
2
8
CH ꢀ2ꢀbromophenyl
2
8.2 ± 0.2
3.7 ± 0.4
> 50.0
9
CH ꢀ3ꢀbromophenyl
2
2.2 ± 0.1
0.9 ± 0.2
> 50.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CH ꢀ4ꢀbromophenyl
2
> 55.6
< 0.2
1.5
CH ꢀ2ꢀfluorophenyl
7.3 ± 1.0
7.6 ± 0.2
> 50.0
2
CH ꢀ4ꢀfluorophenyl
5.1 ± 0.4
6.7 ± 1.9
2.0 ± 0.9
0.5 ± 0.05
1.3 ± 0.1
1.3 ± 0.2
2
CH ꢀ4ꢀchlorophenyl
> 7.5
> 25.0
> 100.0
> 38.5
7.7
2
CH ꢀ4ꢀmethoxyphenyl
> 50.0
2
d
d
c
CH ꢀ4ꢀnitrophenyl
> 50.0
5.6
2
1
0
CH ꢀ4ꢀtrifluoromethylphenyl
> 50.0
12.5 ± 1.2
> 50.0
2.7
4.2
5.9
6.7
2.1
NT
0.1
0.9
2
11
CH ꢀ4ꢀnitrilephenyl
2
1
2
CH ꢀ4ꢀethylphenyl
1.0 ± 0.05
> 38.5
> 166.7
> 10.2
< 0.2
< 0.3
< 0.2
2
c
c,e
13
CH ꢀ4ꢀisopropylphenyl
0.3 ± 0.03
4.9 ± 0.7
> 50.0
> 50.0
2
1
4
CH ꢀ5ꢀbenzooxadiazole
> 50.0
7.7 ± 0.7
16.0 ± 0.3
8.0 ± 0.3
2
15
CH ꢀ3ꢀ(5ꢀmethylisoxazole)
2
1
1
6
7
CH ꢀ2ꢀpyridyl
> 50.0
2
CH ꢀ3ꢀpyridyl
> 50.0
2
a
b
c
Data were averaged from > 3 experiments. Data were averaged from > 2 experiments. Data were averaged from
d
e
two separate compound lots. Data were averaged from three separate compound lots. Data were obtained from a 3ꢀ
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day exposure experiment versus 5ꢀday duration due to precipitation of compound after 3 days. NT = not tested.
Data were analyzed using Microsoft Excel 2010.
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Alterations of the secondary amide (R ) were independently carried out in a parallel effort.
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Exchange of the methyl ether for the ethyl or isopropyl ether did not appreciably alter potency in
the CPE assay; however, the cytotoxic effects associated with those analogs increased relative to
the parent hit 2 (Table 2, entries 1ꢀ3). Replacement of the methyl ether with a tertiary amine for
the purpose of enhancing solubility was inferior in terms of both potency and toxicity (entry 4).
Elongation of the alkyl chain by one methylene unit only marginally enhanced potency and
reduced the therapeutic window (entry 5); however, truncating the linker afforded analog 28 with
comparable potency to hit 2 (entry 6). Introducing an oxetane as a cyclized version of the linear
ether chain resulted in a promising potency, cytotoxicity and viral plaque reduction profile (entry
7). Other modifications were explored to improve solubility or potency without inducing
cytotoxicity, but none were found to be more advantageous when considering multiparameter
optimization.
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Table 2. hRSV CPE Assay Potency, Cytotoxicity, Selectivity Index and Logarithmic Reduction
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in Viral Plaques for Analogs with Structural Variations in the (R ) Region of Hit Compound 2
HEpꢀ2
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11
12
13
14
15
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19
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RSV CPE
potency ±
standard
cellular
toxicity ±
standard
deviation
CC50
selectivity
index
viral titer
reduction
at 10 ꢀM
(log)
entry compound
(
CC /
50
deviation
EC )
a
50
EC (ꢀM)
5
0
b
2
(ꢀM)
R
c
c
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2
2
NH(CH ) OCH
3
2.1 ± 0.5
> 50.0
> 23.8
4.4
4.2
NT
NT
NT
2.1
NT
5.1
1.2
NT
NT
NT
NT
NT
5.5
NT
NT
NT
NT
NT
3.5
2
3
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
NH(CH ) OCH CH
3
2
3
2
1.9 ± 0.2
2.0 ± 0.2
9.5 ± 0.4
0.8 ± 0.05
2.2 ± 1.4
0.7 ± 0.1
1.0 ± 0.08
> 50.0
8.5 ± 0.3
19.3 ± 0.9
16.6 ± 1.5
6.5 ± 0.3
3
NH(CH ) OCH(CH )
3 2
9.8
2
3
4
NH(CH ) N(CH )
1.8
2 3
3 2
5
NH(CH ) OCH
3
7.8
2
4
d
6
NH(CH ) OCH3
> 50.0
22.7
66.1
7.9
2 2
7
NH(CH )ꢀ(3ꢀoxetane)
47.0 ± 1.9
7.6 ± 0.3
17.5 ± 1.1
5.4 ± 0.3
7.3 ± 1.3
45.3 ± 1.7
10.1 ± 0.2
8.4 ± 0.3
> 50.0
2
8
NH(CH )ꢀcyclobutane
2
9
Nꢀmorpholine
Nꢀpyrrolidine
Nꢀpiperidine
NHCH3
< 0.4
< 0.1
< 0.2
21.1
< 0.2
12.1
NA
10
> 50.0
1
1
> 50.0
12
2.2 ± 0.9
> 50.0
1
1
1
1
1
1
3
4
5
6
7
8
N(CH3)2
NHꢀtertꢀbutyl
NHꢀcyclohexyl
NHꢀphenyl
NHꢀbenzyl
0.7 ± 0.03
> 50.0
> 50.0
> 50.0
NA
> 50.0
41.8 ± 7.9
> 50.0
NA
NHꢀCH ꢀ2ꢀfuryl
> 50.0
NA
2
19
NHꢀ2ꢀthiazole
> 50.0
> 50.0
NA
2
0
NHꢀ4ꢀpyridyl
1.0 ± 0.04
1.8 ± 0.4
c
1.8
a
b
Data were an average of > 3 experiments. Data were an average of > 2 experiments. Data were an average of outcomes from
d
two separate lots of compound 2. Data were obtained from a 3ꢀday exposure experiment versus 5ꢀday duration due to
precipitation of compound after 3 days. NT = not tested; NA = not applicable. Data were analyzed using Microsoft Excel 2010.
1
2
Hybrid analogs derived from the most advantageous individual R or R modifications were then
prepared to assess synergistic effects, and additional SAR data was pursued using these
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7
8
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
5
5
5
5
5
5
5
5
5
6
compounds as templates. At this stage, several compounds were also evaluated for improvement
in solubility. The initial analog sets (Tables 1 and 2) revealed that analogs bearing the 4ꢀ
1
2
isopropylbenzyl or 4ꢀnitrobenzyl moiety for R and the R modification of a NHCH ꢀ3ꢀoxetane
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
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
were independently the most beneficial in terms of combined potency, cytotoxicity and plaque
reduction (compounds 15, 19, and 29, respectively). In pairing the 4ꢀnitrobenzyl functionality
1
2
(
R ) with the NHCH ꢀ3ꢀoxetane (R ) subunit, compound 43 (entry 2, Table 3) showed
2
comparable potency and toxicity to hit 2, but with a 14ꢀfold improvement in PBS solubility and
at least a 2 log increase in capacity to reduce viral titer. Combining the 4ꢀisopropylbenzyl group
1
2
(
R ) with the NHCH ꢀ3ꢀoxetane (R ) component resulted in potency comparable to the best
2
analogs in the series (44, entry 3, Table 3); however, the selectivity index was diminished due to
unwanted cytotoxicity. Alternatives to the 4ꢀnitrobenzyl element were examined, such as a 4ꢀ
benzoic acid derivative 45 which imparted a desirable solubility effect but also abrogated activity
which may be the result of reduced cellular permeability. The Nꢀ4ꢀdimethylaminobenzyl analog
46 was also found to have improved solubility, but did not robustly provide cytoprotection to the
extent observed with other architectural combinations (entry 5). Furyl and pyridyl variants in
2
place of the R appendage were also determined to be suboptimal overall (entries 7ꢀ9 and other
analogs not shown). Analogs bearing a substituent (halide, ꢀOCH , ꢀCH , or ꢀCF ) at the C6 or
3
3
3
C7 position of the quinazolinedione core resulted in loss of potency (EC > 50 ꢀM). The same
50
result was determined for analogs with an extra methylene spacer between the core and the
benzamide moiety or for compounds with the metaꢀpositioned amide functionality as opposed to
the paraꢀarrangement present in the hit (data not shown).
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Table 3. hRSV CPE Assay Potency, Cytotoxicity, Selectivity Index, Logarithmic Reduction in
Viral Plaques, and Solubility Assessments for Analogs with Tandem Structural Variations in the
1
2
(
R ) and (R ) Regions of Hit Compound 2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RSV
CPE
potency
±
standard
deviation
HEpꢀ2
cellular
toxicity ±
standard
deviation
solubility
ꢀM)
selectivity viral titer
(
index
reduction
entry compound
(CC / at 10 ꢀM
50
EC )
(log)
5
0
1
2
CC50
c
d
R
R
PBS media
EC50
b
a
(ꢀM)
(
ꢀM)
CH ꢀ4ꢀ
e
e
2
1
2
2
NH(CH ) OCH 2.1 ± 0.5
> 50.0
> 23.8
> 38.5
4.2
0.1
1.4
2.5
7.2
2
3
3
methylphenyl
CH ꢀ4ꢀ
NHCH ꢀ3ꢀ
oxetane
2
2
43
1.3 ± .05
> 50.0
> 6.2
nitrophenyl
CH ꢀ4ꢀiꢀ
propylphenyl
NHCH ꢀ3ꢀ
oxetane
2
2
3
4
44
45
0.4 ± .01 3.6 ± .08
> 50.0 > 50.0
9.0
5.8
NT
NT
NT
NT
CH ꢀ4ꢀCO Hꢀ
NHCH ꢀ3ꢀ
oxetane
2
2
2
NA
98.1
phenyl
CH ꢀ4ꢀ
2
NHCH ꢀ3ꢀ
oxetane
2
5
6
46
47
N(CH3)2
phenyl
1.1 ± 0.2 44.7 ± 7.2
1.6 ± .08 3.7 ± 0.3
41.8
2.3
> 6.2
3.4
11.2 18.5
ꢀ
CH ꢀ4ꢀtert
butylphenyl
NHCH ꢀ3ꢀ
oxetane
2
2
NT
NT
CH ꢀ4ꢀiꢀ
propylphenyl
NHCH ꢀ2ꢀ
2
2
7
8
9
48
49
50
1.2 ± 0.1
0.8 ± .01
0.5 ± .06
> 50.0
> 50.0
< 1.6
> 41.7
> 62.5
< 3.3
2.8
NT
NT
0.3
NT
NT
NT
10.2
NT
furyl
CH ꢀ4ꢀ
NHCH ꢀ2ꢀ
2
2
chlorophenyl
furyl
CH ꢀ4ꢀiꢀ
propylphenyl
NHꢀ2ꢀMeOꢀ
2
pyridyl
a
b
c
Data were an average of > 3 experiments. Data are an average of > 2 experiments. Kinetic solubility in 1x PBS, pH 7.4.
d
Kinetic solubility in CPE assay media: (DMEM/F12(r) (Sigma, Cat # D6434)/ 1x Pen/Strep/Glutamine (Gibco, Cat # 10378) /
2
2
% Heat Inactivated FBS (Gibco Cat # 10082)). NT = not tested; NA = not applicable. Data were analyzed using Microsoft Excel
e
010. Data were an average of outcomes from two separate lots of compound 2.
Given the idiosyncractic cytotoxicity profile for several of the compounds in the
3
6,37
quinazolinedione series and our experience with a related, antiꢀviral quinazolinone scaffold
that lacked any detectable mammalian cytotoxicity, two quinazolinone analogs were prepared to
explore if this modified core was beneficial against RSV (51-52, Fig. 2). Quinazolinone analogs
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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
5
5
5
5
5
5
5
5
5
6
5
1 and 52 were generated bearing peripheral components that had been shown to possess antiꢀ
RSV activity when integrated with the quinazolinedione core. While the compounds were not
protective against RSV (> 50 ꢀM), the effort underscored the importance of the carbonyl group
situated between the core nitrogens to retaining antiꢀRSV potency. Additionally, the impact of
introducing a nitrogen atom into the fused phenyl ring of the quinazolinedione core was studied
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
(53-54, Fig. 2). Only analog 54 was weakly active, but the compound also demonstrated some
appreciable toxicity. Though limited somewhat in scope, the core alterations represented by these
four analogs and those previously discussed in which functional groups were introduced at the
C6 or C7 position of the scaffold showed that no core modification made to date was well
tolerated.
OCH3
NH
O
N
O
N
O
X
Y
N
N
NH
O
O
H C
3
H C
3
H C
CH3
3
5
1, metaꢀisopropyl
52, paraꢀisopropyl
53, X = N, Y = CH
54, X = CH, Y = N
RSV CPE EC50 > 50
M
RSV CPE EC50 > 50
HEpꢀ2 CC50 > 50 M
M
RSV CPE EC50 = 13.2 ± 3.4
HEpꢀ2 CC50 = 35.3 ± 5.1 M
M
HEpꢀ2 CC50 > 50
M
Figure 2. Activity of quinazolinones and azaquinazolinediones
Profiling Assays: The purpose of this program was to identify and develop new compounds with
compelling in vitro antiꢀRSV activity that could be used as a platform for deriving suitable
probes for future in vivo efficacy studies. Towards this goal, several analogs emerged from the
SAR effort as interesting probe candidates worthy of further characterization based on
improvements in CPE potency, solubility, and viral titer. Nonetheless, limitations in aqueous
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solubility or the presence of functionality with suspected metabolic liability prompted the team
to assess passive permeability and hepatocyte toxicity for select analogs. Compounds 15, 19 and
46 were evaluated accordingly (Table 4).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Comparative SAR, Physiochemical and In Vitro ADME Data for Select Analogs
HEpꢀ2
cellular
toxicity ±
standard
deviation
CC50
aqueous
RSV CPE
potency
solubility
stability
g
selectivity viral titer
index reduction
(CC / at 10 ꢀM
Pe, PAMPA hepatocyte
(
ꢀM)
j,k
(
%)
±
standard
permeability toxicity
entry analog
ꢀ6
deviation
(x 10
cm/s)
LC50
5
0
1
:1
g,l
g,o
EC50
EC )
(log)
h
i
(ꢀM)
50
PBS media PBS PBS/
ACN
a
(
ꢀM)
d
(ꢀM)
1
2
2
2.1 ± 0.5
> 50.0
23.8
4.2
0.1
1.3
0.4
NT 88.1 94.0
NT
NT
b
c
b
f
m
n
15
0.5 ± 0.05
> 50.0
> 100
5.6
2.8 83.4 94.9 364/349/356
> 50
> 30
c,e
3
4
19
46
0.3 ± 0.03
> 50.0
> 166.7
41.8
6.7
10.2 27.0 100 718/631/703
m
1.1 ± 0.2
44.7 ± 7.2
> 6.2 11.2 18.5 90.2 100 780/686/514
> 50
c
a
b
Data were an average of > 3 experiments. Data were an average of outcomes from three separate lots of compound 15. Data
d
e
were an average of outcomes from two separate lots of compound 19. Data are an average of > 2 experiments. Data were
f
obtained from a 3ꢀday exposure experiment versus 5ꢀday duration due to precipitation of compound after 3 days. Data were an
g
average of outcomes from two separate lots of compound 15. Data collected by Ms. Arianna MangravitaꢀNovo at the Conrad
h
i
Prebys Sanford Burnham Medical Research Institute; Kinetic solubility in 1x PBS, pH 7.4. Kinetic solubility in CPE assay
media: (DMEM/F12(r) (Sigma, Cat # D6434)/1x Pen/Strep/Glutamine (Gibco, Cat # 10378) /2% Heat Inactivated FBS (Gibco
j
Cat # 10082)). Data collected by Mr. Patrick Porubsky at the University of Kansas Analytical Chemistry Core, Specialized
k
Chemistry Center; Results are represented as percent parent remaining after 48 h; Stability assessment was done independently
in PBS or with 1:1 PBS and acetonitrile, the latter was used to account for limitations in solubility affecting PBS results;
l
PAMPA donor pH: 5.0/6.2/7.4, acceptor pH: 7.4; controls: verapamil (222/1097/1936 – highly permeable), metoprolol
m
(
14/60/472 – moderately permeable), ranitidine (<10/<10/<10 – poorly permeable); PAMPA done in PBS, no additives;
n
PAMPA done with 20% acetontrile added to compensate for PBS solubility. Without acetonitrile, Pe was insignificant at each
o
pH. Fa2Nꢀ4 immortalized human hepatocytes. Data were analyzed using Microsoft Excel 2010; NT = not tested.
Aqueous solubility for isopropylbenzyl derivative 19 was the most limited of these three
compounds, resulting in a skewed result in aqueous stability (27% parent remaining, entry 2,
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7
8
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
5
5
5
5
5
5
5
5
5
6
Table 4). The addition of acetonitrile to the stability experiment to account for compound
precipitation in PBS alone reflected that compound 19 was stable to degradation. Solubility for
each compound in CPE assay media was improved compared to PBS buffer, likely due to protein
binding. Passive permeability was negligible for 19 in PBS, as expected from the solubility data.
Moderate to good permeability was observed for the nitrobenzyl and oxetaneꢀcontaining
derivatives 15 and 46, respectively, at each of 3 pH levels. Toxicity in hepatocytes, most
concerning for the nitrobenzyl analog 15, was not observed (> 50 ꢀM), nor was it significant
with the other two analogs tested. While each compound possessed desirable attributes,
compound 19 was selected as our lead, ML275, due to the overall profile which included the
most improved potency, selectivity window and reduction in viral titer. Probe candidates were
routinely assessed in a Eurofins PanLabs (formerly Ricerca) Hit LeadProfiling® screen against
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
6
7 discrete GPCRs, ion channels and transporters. All assays were performed in duplicate with
probe ML275 (19) at a concentration of 10 ꢀM, and > 50% inhibition was noted for five targets
Table 5). Inhibition of the human adenosine A receptor and the human platelet activating factor
(
3
was determined to be 44% and 43%, respectively; however, inhibition of all remaining targets
did not exceed 34%. A full list of the targets and percent inhibition by compound 19 is provided
in the supplemental material. The liability posed by significant inhibition of any given host target
depends on a multitude of factors that includes but is not limited to potency, metabolism, and
physiological compartmental exposure (e.g., CNS). These results, while not negligible, were
considered informational at this stage of development, as pursuit of individual IC values and
5
0
bioavailability data was costꢀprohibitive and revision of the scaffold architecture was expected
prior to finding a suitable tool for in vivo assessment. Nonetheless, the profiling outcome served
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as a useful alert to potential adverse effects associated with the series that will need to be
surveyed as development continues toward an advanced lead candidate.
Table 5. Off Target Profiling Results for Compound 19 at 10 ꢀM (> 50% Inhibition)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Eurofins
percent
Panlabs
entry
biological target
species
inhibition
assay
code
b
a
(%)
1
2
3
4
5
calcium channel, Lꢀtype, benzothiazepine
calcium channel, Lꢀtype, dihydropyridine
cannabinoid CB1
214510
214600
217030
271700
204410
rat
52
70
86
54
rat
human
human
human
serotonin (5ꢀhydroxytryptamine) 5ꢀHT2B
norepinephrine transporter
54
a
b
Detailed assay descriptions can be located at https://www.eurofinspanlabs.com/catalog; A full list of targets and
percent inhibition by compound 19 is provided in the supplemental material.
Mechanism of Action Studies: To gain an understanding of how this quinazolinedione class of
compounds was inhibiting RSV, a cellꢀbased, timeꢀofꢀaddition study was initially performed to
determine the ability of these compounds to inhibit different stages of a single round of viral
replication. The assay was performed by infecting cells with RSV and adding compound at a 10
ꢀM concentration at each of several timepoints up to 24 hours postꢀinfection and then tracking
cell viability over the course of the experiment. Compounds 15, 19, and 46 were evaluated as a
panel alongside ribavirin. While ribavirin treatment protected cells from RSVꢀinduced CPE
(
approximately 100%) for up to 7 hours postꢀinfection, indicating that it targets the period of
infection during which viral replication is in progress, none of the three quinazolinediones
showed dramatic changes in cell viability between 7ꢀ24 hours postꢀinfection, suggesting that
these compounds acted at a later stage of the viral life cycle (data not shown). This timeꢀofꢀ
addition profile suggested possible inhibition of the viral RNAꢀdependent RNAꢀpolymerase
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9
1
1
1
1
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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
(
RdRp) activity rather than interference with receptor binding and/or membrane fusion. To test
this hypothesis experimentally, compounds 15, 19 and 46 were subjected, along with a known
18
literatureꢀdescribed RdRp inhibitor 55, to a plasmidꢀbased RSV minigenome reporter assay that
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
38
specifically monitored bioactivity of the viral polymerase machinery (Fig 3). Luciferase
reporter interference was tested by comparing the activity of 19 in assays employing recRSVꢀ
luciferase or the equivalent measles virus recombinant (recMeVꢀluciferase), both of which rely
on luciferase activity as the assay readout. While recRSVꢀluciferase was efficiently inhibited,
compound 19 was inactive against recMeVꢀluciferase, excluding direct reporter interference. All
four analogs showed a doseꢀdependent inhibition of reporter expression with active
concentrations similar to those observed against live virus, indicating that the compounds
blocked RSV RdRp activity.
OCH3
H CO
O
N
3
H C
NO2
OCH3
3
O
N
N
O
NH
O
N
R
O
O
NH
O
NH
55
HN
N(CH
O
N
N
1
1
5, R = 4ꢀnitrobenzyl
9, R = 4ꢀisopropylbenzyl
46
3
)
2
H N
O
2
H C CH3
3
Figure 3. Transient RSV luciferase replicon reporter assay to determine RdRp activity in the presence of the
quinazolinedione analogs. Values were normalized for vehicle (DMSO) treated samples and represent averages of
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three independent experiments, each performed in duplicate. Error bars represent standard deviation, EC values
5
0
and 95% confidence intervals (in parentheses) are shown for each compound.
At this point the exact target with which these compounds interact leading to the observed block
of RdRp activity is unknown, but sequencing of RSVꢀresistant mutant viruses resulting from
compound treated cells is currently underway. These studies are expected to pinpoint the location
of mutation within the genome that may be responsible for resistance and identify a potential
target to investigate further. Attempts to unravel the nature of inhibition are being actively
pursued.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
In summary, we have identified a quinazolinedione class of compounds that show promising
cellular activity against RSV. While the SAR for this set of quinazolinediones was relatively
limited in texture, we have discovered distinct scaffold regions that tolerate structural change,
permit tuning of physiochemical properties and whose modification has led to a 7ꢀfold
improvement in selectivity over the hit compound 2. Several compounds in the series inhibited a
virusꢀinduced cytopathic effect in the submicromolar range without exhibiting significant
cellular toxicity, and importantly, these same compounds also significantly reduced viral plaque
formation at a concentration of 10 ꢀM. Furthermore, the mechanism of action for a subset of
quinazolinedione analogs was explored and demonstrated to block the activity of the viral RNAꢀ
dependent RNAꢀpolymerase complex which is responsible for RSV genome replication and
transcription. These results, combined with the insights from structural modifications of the
quinazolinedione scaffold and in vitro ADME data of select analogs, suggest that a favorable
pharmacological profile can be tuned to produce a lead antiviral compound suitable for in vivo
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1
1
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1
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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
efficacy assessment against RSV. As such, the current set of compounds will be useful tools in
establishing baseline pharmacokinetic parameters and further investigating how these agents
interact with the RdRp complex to inhibit viral replication.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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EXPERIMENTAL SECTION
Chemistry. Purity of all final compounds was > 95% and was confirmed by HPLC/MS analysis
employing an Agilent 1200 RRL chromatograph with photodiode array UV detection and an
Agilent 6224 TOF mass spectrometer. The chromatographic method utilized a Waters Acquity
BEH Cꢀ18 2.1 x 50 mm, 1.7 ꢀm column; UV detection wavelength = 214 nm; flow rate = 0.4
mL/min; gradient = 5 ꢀ 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100%
acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass
spectrometer utilized the following parameters: an Agilent multimode source which
simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and
hexakis(1H, 1H, 3Hꢀtetrafluoropropoxy) phosphazine; and a makeꢀup solvent of 90:10:0.1
MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist
1
13
ionization. H and C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating
at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126
MHz respectively) in CDCl with 0.03% TMS as an internal standard or DMSOꢀd . The
3
6
chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J)
are in Hertz (Hz). The spin multiplicities are reported as s = singlet, bs = broad singlet, d =
doublet, t = triplet, q = quartet, dd = doublet of doublet, hept = heptet, and m = multiplet.
Melting points were determined on a Stanford Research Systems OptiMelt apparatus.
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Compounds were generally prepared according to Scheme 1 and the protocols detailed for
compound 19, below, unless otherwise specified.
Synthesis of 4-((1-(4-isopropylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-
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(3-methoxypropyl)benzamide (19). Step 1: To a solution of 2ꢀaminobenzoic acid 3 (5.00 g,
36.50 mmol) in DMF (45 mL) was added methyl 4ꢀ(aminomethyl)benzoate hydrochloride (7.35
g, 36.5 mmol), HATU (15.25 g, 40.10 mmol) and N,Nꢀdiisopropylethylamine (18.08 mL, 109
mmol). The reaction mixture was stirred for 16 h at room temperature, then diluted with CH Cl2
2
(
200 mL) and washed sequentially with 1M HCl (150 mL), sat. aqueous NaHCO (150 mL) and
3
water (2 x 800 mL). The separated organic extract was dried (MgSO ), filtered, and concentrated
4
under reduced pressure to afford a crude product which was purified by silica gel flash column
chromatography (0 ꢀ 60% v/v EtOAc/Hexane), yielding the product, methyl 4ꢀ((2ꢀ
1
aminobenzamido)methyl)benzoate, as a white solid (5.00 g, 17.59 mmol, 48% yield). H NMR
(
400 MHz, CDCl ): δ 8.01 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.35 (dd, J = 7.9, 1.4
3
Hz, 1H), 7.25ꢀ7.19 (m, 1H), 6.70 (dd, J = 8.3, 0.9 Hz, 1H), 6.67ꢀ6.61 (m, 1H), 6.43 (broad s,
1H), 5.56 (broad s, 2H), 4.66 (d, J = 5.9 Hz, 2H), 3.91 (s, 3H).
Step 2: After stirring a solution of methyl 4ꢀ((2ꢀaminobenzamido)methyl)benzoate (5.00 g, 17.59
mmol) and N,Nꢀdiisopropylethylamine (14.53 mL, 88 mmol) in CH Cl (260 mL) for 10 minutes
2
2
at room temperature under nitrogen, 1,1'ꢀcarbonyldiimidazole (8.55 g, 52.80 mmol) was added,
and the reaction mixture was heated 16 h at reflux. The formed precipitate was filtered, dried
under vacuum and the desired product, methyl 4ꢀ((2,4ꢀdioxoꢀ1,2ꢀdihydroquinazolinꢀ3(4H)ꢀ
yl)methyl)benzoate 4, was furnished as a white solid without further purification (4.65 g, 14.99
1
mmol, 85% yield). H NMR (400 MHz, DMSOꢀd ): δ 11.58 (s, 1H), 7.94 (dd, J = 8.3, 1.4 Hz,
6
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H), 7.90 (d, J = 8.4 Hz, 2H), 7.72ꢀ7.64 (m, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.26ꢀ7.18 (m, 2H),
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.15 (s, 2H), 3.83 (s, 3H).
Step 3: To a solution of methyl 4ꢀ((2,4ꢀdioxoꢀ1,2ꢀdihydroquinazolinꢀ3(4H)ꢀyl)methyl)benzoate 4
(2.60 g, 8.38 mmol) in THF (50 mL) was added 1 M lithium hydroxide (50 mL, 50.3 mmol). The
reaction mixture was stirred at 40 °C for 1 hr, at which point TLC confirmed reaction
completion. Then 1 M HCl was cautiously added until the reaction mixture reached pH 2, at
which point the product precipitated out of solution. The precipitate was collected by filtration,
washed with water (2 x 70 mL), dried under high vacuum to afford the desired product, 4ꢀ((2,4ꢀ
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dioxoꢀ1,2ꢀdihydroquinazolinꢀ3(4H)ꢀyl)methyl)benzoic acid, as a white solid (2.19 g, 7.39 mmol,
1
8
8% yield). H NMR (400 MHz, DMSOꢀd ): δ 12.90 (broad s, 1H), 11.58 (s, 1H), 7.95 (d, J =
6
7.3 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.74ꢀ7.65 (m, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.28ꢀ7.18 (m,
2H), 5.15 (s, 2H).
Step 4: To a solution of 4ꢀ((2,4ꢀdioxoꢀ1,2ꢀdihydroquinazolinꢀ3(4H)ꢀyl)methyl)benzoic acid (1.00
g, 3.38 mmol) in DMF (15 mL) was added 3ꢀmethoxypropylamine (0.35 mL, 3.38 mmol),
HATU (1.41 g, 3.71 mmol) and N,Nꢀdiisopropylethylamine (1.67 mL, 10.13 mmol). The
reaction mixture was stirred for 16 h at room temperature, then diluted with CH Cl (90 mL) and
2
2
washed sequentially with 1 M HCl (60 mL), sat. aqueous NaHCO (60 mL) and water (2 x 180
3
mL). The organic extract was separated, dried (MgSO ), filtered, and concentrated under reduced
4
pressure to afford a crude product which was purified by silica gel flash column chromatography
(0 ꢀ 5% v/v MeOH/CH Cl ) yielding the desired product, 4ꢀ((2,4ꢀdioxoꢀ1,2ꢀdihydroquinazolinꢀ
2 2
3(4H)ꢀyl)methyl)ꢀNꢀ(3ꢀmethoxypropyl)benzamide, as a white solid (0.91 g, 2.48 mmol, 73%
1
yield), mp 244ꢀ246 °C. H NMR (400 MHz, DMSOꢀd ): δ 11.56 (s, 1H), 8.40 (t, J = 5.6 Hz, 1H),
6
7.95 (d, J = 7.3 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.71ꢀ7.63 (m, 1H), 7.36 (d, J = 8.4 Hz, 2H),
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.26ꢀ7.17 (m, 2H), 5.13 (s, 2H), 3.35 (t, J = 6.3 Hz, 2H), 3.31ꢀ3.24 (m, 2H), 3.22 (s, 3H), 1.78ꢀ
1.67 (m, 2H).
Step 5: To a solution of 4ꢀ((2,4ꢀdioxoꢀ1,2ꢀdihydroquinazolinꢀ3(4H)ꢀyl)methyl)ꢀNꢀ(3ꢀ
methoxypropyl)benzamide (0.050 g, 0.14 mmol) in DMF (2 mL) was added 4ꢀisopropylbenzyl
bromide (0.028 mL, 0.16 mmol) and potassium carbonate (0.056 g, 0.41 mmol). The resulting
reaction mixture was stirred at 40 °C for 16 h. The formed residue was dissolved in CH Cl (6
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2
2
mL) and sequentially washed with 1 M HCl (4 mL), water (3 x 20 mL) and brine (8 mL). The
organic layer was separated, dried (MgSO ), filtered, and concentrated under reduced pressure to
4
give a crude product which was purified by silica gel flash column chromatography (0 ꢀ 5% v/v
MeOH/CH Cl ) yielding 4ꢀ((1ꢀ(4ꢀisopropylbenzyl)ꢀ2,4ꢀdioxoꢀ1,2ꢀdihydroquinazolinꢀ3(4H)ꢀ
2
2
yl)methyl)ꢀNꢀ(3ꢀmethoxypropyl)benzamide (19) as a white solid (0.030 g, 0.060 mmol, 44%
1
yield), mp 178ꢀ180 °C. H NMR (500 MHz, CDCl ): δ 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.74 – 7.68
3
(m, 2H), 7.61 – 7.52 (m, 3H), 7.25 – 7.20 (m, 1H), 7.20 – 7.12 (m, 5H), 6.89 (t, J = 5.3 Hz, 1H),
5.36 (s, 2H), 5.33 (s, 2H), 3.59 – 3.52 (m, 4H), 3.37 (s, 3H), 2.87 (h, J = 6.9 Hz, 1H), 1.91 – 1.83
13
(m, 2H), 1.21 (d, J = 6.9 Hz, 6H). C NMR (126 MHz, CDCl ) δ 167.01, 161.92, 151.48,
3
148.54, 140.41, 140.16, 135.40, 134.15, 132.89, 129.25, 129.11, 127.19, 127.17, 126.57, 123.29,
115.74, 114.69, 72.59, 59.10, 47.36, 44.94, 39.31, 33.89, 28.95, 24.06. LCMS Retention time:
+
3.422 min. LCMS purity 98.8%. HRMS (ESI): m/z calcd for C H N O [M+H] 500.2544,
3
0
33
3
4
found 500.2540.
N-(3-methoxypropyl)-4-((1-(4-methylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-
1
yl)methyl)benzamide (2). Isolated as a white solid (36 mg, 56% yield), mp 197ꢀ199 °C. H
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NMR (500 MHz, CDCl ): δ 8.23 (dd, J = 7.9, 1.6 Hz, 1H), 7.74 – 7.68 (m, 2H), 7.60 – 7.51 (m,
3
3H), 7.22 (ddd, J = 8.0, 7.3, 0.9 Hz, 1H), 7.16 – 7.09 (m, 5H), 6.89 (t, J = 5.2 Hz, 1H), 5.36 (s,
1
3
2
H), 5.33 (s, 2H), 3.59 – 3.52 (m, 4H), 3.37 (s, 3H), 2.31 (s, 3H), 1.91 – 1.83 (m, 2H). C NMR
0
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(
126 MHz, CDCl ): δ 167.01, 161.92, 151.49, 140.41, 140.11, 137.59, 135.38, 134.16, 132.59,
3
129.80, 129.26, 129.11, 127.17, 126.54, 123.30, 115.76, 114.64, 72.58, 59.09, 47.37, 44.95,
39.31, 28.95, 21.23. LCMS Retention time: 1.98 min. LCMS purity 97.8%. HRMS (ESI): m/z
+
calcd for C H N O [M+H] 472.2158, found 472.2241.
2
8
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4
4-((1-(2-bromobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (8). Isolated as a white solid (31 mg, 71% yield), mp 187ꢀ189 °C.
1
H NMR (500 MHz, CDCl ): δ 8.27 (dd, J = 8.0, 1.6 Hz, 1H), 7.74 – 7.69 (m, 2H), 7.63 (dd, J =
3
7
.7, 1.5 Hz, 1H), 7.61 – 7.52 (m, 3H), 7.29 – 7.22 (m, 1H), 7.21 – 7.11 (m, 2H), 6.93 – 6.87 (m,
H), 6.85 – 6.79 (m, 1H), 5.41 (s, 2H), 5.37 (s, 2H), 3.60 – 3.52 (m, 4H), 3.37 (s, 3H), 1.91 –
2
1
3
1.83 (m, 2H). C NMR (126 MHz, CDCl ): δ 166.98, 161.83, 151.39, 140.28, 139.75, 135.65,
3
134.25, 134.09, 133.30, 129.37, 129.29, 129.21, 128.13, 127.20, 126.69, 123.63, 122.44, 115.76,
114.56, 72.60, 59.10, 48.02, 45.00, 39.33, 28.94. LCMS Retention time: 3.250 min. LCMS
+
purity 97.2%. HRMS (ESI): m/z calcd for C H BrN O [M+H] 538.1163, found 538.1159.
2
7
26
3
4
4-((1-(3-bromobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (9). Isolated as a white solid (28 mg, 64% yield), mp 172ꢀ174 °C.
1
H NMR (500 MHz, CDCl ): δ 8.26 (dd, J = 8.0, 1.6 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.61 – 7.54
3
(m, 3H), 7.44 – 7.36 (m, 2H), 7.29 – 7.23 (m, 1H), 7.20 (apparent t, J = 7.8 Hz, 1H), 7.17 – 7.12
(m, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.89 (t, J = 5.2 Hz, 1H), 5.36 (s, 2H), 5.33 (s, 2H), 3.61 – 3.51
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(
m, 4H), 3.37 (s, 3H), 1.93 – 1.81 (m, 2H). C NMR (126 MHz, CDCl ): δ 166.98, 161.76,
3
1
51.46, 140.25, 139.80, 138.05, 135.55, 134.24, 131.12, 130.74, 129.61, 129.49, 129.10, 127.22,
25.15, 123.60, 123.29, 115.82, 114.31, 72.59, 59.10, 47.02, 45.02, 39.32, 28.95. LCMS
1
10
11
12
13
14
15
16
17
18
19
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Retention time: 3.197 min. LCMS purity 99.4%. HRMS (ESI): m/z calcd for C H BrN O
4
2
7
26
3
+
[M+H] 538.1163, found 538.1159.
4-((1-(4-bromobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (10). Isolated as a white solid (23 mg, 49% yield), mp 179ꢀ181 °C.
1
H NMR (500 MHz, CDCl ): δ 8.25 (dd, J = 7.9, 1.6 Hz, 1H), 7.74 – 7.68 (m, 2H), 7.60 – 7.53
3
(m, 3H), 7.48 – 7.42 (m, 2H), 7.28 – 7.21 (m, 1H), 7.14 – 7.09 (m, 2H), 7.05 (d, J = 8.4 Hz, 1H),
6.89 (t, J = 5.4 Hz, 1H), 5.35 (s, 2H), 5.31 (s, 2H), 3.60 – 3.52 (m, 4H), 3.37 (s, 3H), 1.91 – 1.83
13
(
m, 2H). C NMR (126 MHz, CDCl ): δ 166.94, 161.76, 151.44, 140.24, 139.82, 135.48,
3
1
5
34.72, 134.26, 132.29, 129.46, 129.15, 128.32, 127.20, 123.55, 121.78, 115.80, 114.34, 72.62,
9.11, 47.06, 44.99, 39.34, 28.95. LCMS Retention time: 3.208 min. LCMS purity 99%. HRMS
+
(
ESI): m/z calcd for C H BrN O [M+H] 538.1163, found 538.1191.
2
7
26
3
4
4-((1-(4-fluorobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (12). Isolated as a white solid (27 mg, 70% yield), mp 173ꢀ175 °C.
1
H NMR (500 MHz, CDCl ): δ 8.25 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.61 – 7.54
3
(m, 3H), 7.26 – 7.19 (m, 3H), 7.10 (d, J = 8.4 Hz, 1H), 7.05 – 6.98 (m, 2H), 6.90 (t, J = 5.4 Hz,
1
3
1
H), 5.36 (s, 2H), 5.33 (s, 2H), 3.60 – 3.53 (m, 4H), 3.37 (s, 3H), 1.91 – 1.83 (m, 2H). C NMR
(
126 MHz, CDCl ): δ 166.97, 162.34 (d, J = 246.5 Hz), 161.80, 151.46, 140.28, 139.90, 135.44,
3
134.22, 131.38 (d, J = 3.2 Hz), 129.42, 129.14, 128.35 (d, J = 8.1 Hz), 127.19, 123.49, 116.11
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(
d, J = 21.7 Hz), 115.80, 114.38, 72.59, 59.09, 46.95, 44.98, 39.32, 28.94. LCMS Retention
+
time: 3.067 min. LCMS purity 99.4%. HRMS (ESI): m/z calcd for C H FN O [M+H]
27 26
3
4
4
76.1980, found 476.1972.
0
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4-((1-(4-chlorobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (13). Isolated as a white solid (37 mg, 55% yield), mp 185ꢀ187 °C.
1
H NMR (500 MHz, CDCl ): δ 8.25 (dd, J = 8.0, 1.6 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.61 – 7.53
3
(m, 3H), 7.34 – 7.28 (m, 2H), 7.28 – 7.21 (m, 1H), 7.20 – 7.14 (m, 2H), 7.06 (d, J = 8.5 Hz, 1H),
6
.91 (t, J = 5.3 Hz, 1H), 5.35 (s, 2H), 5.33 (s, 2H), 3.60 – 3.52 (m, 4H), 3.37 (s, 3H), 1.92 – 1.83
1
3
(m, 2H). C NMR (126 MHz, CDCl ): δ 166.94, 161.77, 151.44, 140.25, 139.83, 135.47,
3
134.25, 134.18, 133.73, 129.45, 129.34, 129.15, 127.99, 127.19, 123.54, 115.80, 114.35, 72.61,
5
9.10, 47.01, 44.99, 39.33, 28.95. LCMS Retention time: 3.171 min. LCMS purity 100%.
+
HRMS (ESI): m/z calcd for C H ClN O [M+H] 492.1685, found 492.1700.
27 26
3
4
4
-((1-(4-methoxybenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (14). Isolated as a white solid (33 mg, 50% yield), mp 171ꢀ173 °C.
1
H NMR (500 MHz, CDCl ): δ 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.60 – 7.53
3
(m, 3H), 7.22 (apparent t, J = 7.1 Hz, 1H), 7.20 – 7.14 (m, 3H), 6.90 (t, J = 5.2 Hz, 1H), 6.87 –
6.82 (m, 2H), 5.36 (s, 2H), 5.31 (s, 2H), 3.77 (s, 3H), 3.60 – 3.52 (m, 4H), 3.37 (s, 3H), 1.93 –
13
1
1
5
.82 (m, 2H). C NMR (126 MHz, CDCl ): δ 166.99, 161.89, 159.22, 151.48, 140.40, 140.07,
3
35.35, 134.16, 129.28, 129.10, 128.00, 127.62, 127.17, 123.29, 115.77, 114.60, 114.51, 72.58,
9.09, 55.42, 47.06, 44.93, 39.30, 28.95. LCMS Retention time: 3.002 min. LCMS purity 100%.
+
HRMS (ESI): m/z calcd for C H N O [M+H] 488.2180, found 488.2190.
2
8
29
3
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N-(3-methoxypropyl)-4-((1-(4-nitrobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-
yl)methyl)benzamide (15). Isolated as an offꢀwhite solid (21 mg, 48% yield), mp 170ꢀ173 °C.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
H NMR (500 MHz, CDCl ): δ 8.29 (dd, J = 7.9, 1.6 Hz, 1H), 8.23 – 8.17 (m, 2H), 7.75 – 7.69
3
(m, 2H), 7.61 – 7.54 (m, 3H), 7.43 – 7.37 (m, 2H), 7.31 – 7.24 (m, 1H), 6.98 (d, J = 8.4 Hz, 1H),
6
.91 (t, J = 5.3 Hz, 1H), 5.46 (s, 2H), 5.36 (s, 2H), 3.60 – 3.53 (m, 4H), 3.38 (s, 3H), 1.91 – 1.84
1
3
(m, 2H). C NMR (126 MHz, CDCl ): δ 166.89, 161.61, 151.41, 147.69, 143.14, 140.06,
3
139.57, 135.63, 134.37, 129.74, 129.21, 127.39, 127.23, 124.47, 123.86, 115.88, 113.97, 72.65,
5
9.11, 47.12, 45.07, 39.38, 28.94. LCMS Retention time: 2.973 min. LCMS purity 97.8%.
+
HRMS (ESI): m/z calcd for C H N O [M+H] 503.1925, found 503.1951.
27 26
4
6
4
-((2,4-dioxo-1-(4-(trifluoromethyl)benzyl)-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (16). Isolated as a white solid (27 mg, 59% yield), mp 174ꢀ177 °C.
1
H NMR (500 MHz, CDCl ): δ 8.27 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.62 – 7.53
3
(m, 5H), 7.34 (d, J = 7.9 Hz, 2H), 7.29 – 7.23 (m, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.90 (t, J = 5.3
1
3
Hz, 1H), 5.42 (s, 2H), 5.36 (s, 2H), 3.63 – 3.51 (m, 4H), 3.37 (s, 3H), 1.93 – 1.82 (m, 2H). C
NMR (126 MHz, CDCl ): δ 166.92, 161.72, 151.45, 140.18, 139.76, 135.57, 134.30, 130.26 (q, J
3
=
32.6 Hz), 129.56, 129.18, 127.21, 126.83, 126.18 (q, J = 3.8 Hz), 125.10, 123.67, 122.94,
115.83, 114.22, 72.63, 59.10, 47.21, 45.02, 39.36, 28.95. LCMS Retention time: 3.207 min.
+
LCMS purity 97.8%. HRMS (ESI): m/z calcd for C H F N O [M+H] 526.1948, found
2
8
26
3
3
4
5
26.1973.
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-((1-(4-cyanobenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (17). Isolated as a white solid (43.4 mg, 66% yield), mp 187ꢀ189
1
°
C. H NMR (500 MHz, CDCl ): δ 8.28 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.66 –
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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6
7
8
9
0
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9
0
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8
9
0
7.61 (m, 2H), 7.61 – 7.53 (m, 3H), 7.34 (d, J = 8.3 Hz, 2H), 7.30 – 7.24 (m, 1H), 6.98 (d, J = 8.4
Hz, 1H), 6.92 (t, J = 5.2 Hz, 1H), 5.41 (s, 2H), 5.35 (s, 2H), 3.62 – 3.50 (m, 4H), 3.38 (s, 3H),
13
1.93 – 1.82 (m, 2H). C NMR (126 MHz, CDCl ): δ 166.90, 161.63, 151.40, 141.16, 140.08,
3
139.60, 135.61, 134.32, 133.00, 129.66, 129.19, 127.23, 127.21, 123.80, 118.50, 115.83, 114.02,
111.96, 72.62, 59.09, 47.27, 45.04, 39.35, 28.92. LCMS Retention time: 2.910 min. LCMS
+
purity 99.6%. HRMS (ESI): m/z calcd for C H N O [M+H] 483.2027, found 483.2024.
2
8
26
4
4
4
-((1-(4-ethylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (18). Isolated as a white solid (39 mg, 59% yield) mp 170ꢀ172 °C.
1
H NMR (500 MHz, CDCl ): δ 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.61 – 7.52
3
(
m, 3H), 7.22 (ddd, J = 8.0, 7.3, 0.9 Hz, 1H), 7.18 – 7.12 (m, 5H), 6.91 (t, J = 5.4 Hz, 1H), 5.36
s, 2H), 5.34 (s, 2H), 3.60 – 3.52 (m, 4H), 3.37 (s, 3H), 2.61 (q, J = 7.6 Hz, 2H), 1.91 – 1.83 (m,
(
1
3
2H), 1.20 (t, J = 7.6 Hz, 3H). C NMR (126 MHz, CDCl ): δ 167.01, 161.91, 151.48, 143.91,
3
140.40, 140.12, 135.38, 134.14, 132.78, 129.24, 129.09, 128.60, 127.17, 126.58, 123.28, 115.74,
114.66, 72.56, 59.08, 47.36, 44.93, 39.29, 28.95, 28.59, 15.61. LCMS Retention time: 3.323 min.
+
LCMS purity 99.1%. HRMS (ESI): m/z calcd for C H N O [M+H] 486.2387, found
2
9
31
3
4
4
86.2383.
4
-((1-(benzo[c][1,2,5]oxadiazol-5-ylmethyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-
yl)methyl)-N-(3-methoxypropyl)benzamide (20). Isolated as an offꢀwhite solid (25 mg, 58%
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yield), mp 70ꢀ74 °C. H NMR (500 MHz, CDCl ): δ 8.31 (dd, J = 8.0, 1.6 Hz, 1H), 7.87 (dd, J =
3
9
7
3
.3, 1.0 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.63 – 7.54 (m, 4H), 7.36 (dd, J = 9.3, 1.5 Hz, 1H), 7.33 –
.27 (m, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.91 (t, J = 5.2 Hz, 1H), 5.44 (s, 2H), 5.37 (s, 2H), 3.61 –
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11
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14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
.52 (m, 4H), 3.37 (s, 3H), 1.92 – 1.84 (m, 2H). C NMR (126 MHz, CDCl ): δ 166.91, 161.57,
3
151.37, 149.12, 148.82, 140.02, 139.76, 139.49, 135.73, 134.39, 130.85, 129.82, 129.19, 127.25,
23.99, 117.97, 115.88, 113.88, 113.04, 72.61, 59.11, 47.41, 45.12, 39.35, 28.95. LCMS
Retention time: 2.955 min. LCMS purity 97.9%. HRMS (ESI): m/z calcd for C H N O
5
1
2
7
25
5
+
[M+H] 500.1928, found 500.1955.
N-(3-methoxypropyl)-4-((1-((5-methylisoxazol-3-yl)methyl)-2,4-dioxo-1,2
dihydroquinazolin-3(4H)-yl)methyl)benzamide (21). Isolated as a white solid (37 mg, 59%
1
yield), mp 164ꢀ166 °C. H NMR (500 MHz, CDCl ): δ 8.23 (dd, J = 7.9, 1.6 Hz, 1H), 7.74 –
3
7.69 (m, 2H), 7.65 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.59 – 7.53 (m, 2H), 7.46 (d, J = 8.4 Hz, 1H),
7.30 – 7.24 (m, 1H), 6.91 (t, J = 5.1 Hz, 1H), 5.97 (apparent d, J = 1.0 Hz, 1H), 5.34 (s, 2H),
5.33 (s, 2H), 3.60 – 3.52 (m, 4H), 3.37 (s, 3H), 2.37 (apparent d, J = 0.8 Hz, 3H), 1.92 – 1.82 (m,
1
3
2H). C NMR (126 MHz, CDCl ): δ 170.89, 166.95, 161.75, 159.51, 151.19, 140.17, 139.66,
3
135.68, 134.24, 129.32, 129.07, 127.19, 123.69, 115.69, 114.28, 101.28, 72.62, 59.09, 44.93,
39.78, 39.34, 28.94, 12.45. LCMS Retention time: 2.779 min. LCMS purity 100%. HRMS (ESI):
+
m/z calcd for C H N O [M+H] 463.1976, found 463.1994.
2
5
26
4
5
4-((2,4-dioxo-1-(pyridin-2-ylmethyl)-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
methoxypropyl)benzamide (22). Isolated as a white solid (23 mg, 53% yield), mp 191ꢀ193 °C.
1
H NMR (500 MHz, CDCl ): δ 8.57 (dt, J = 4.7, 1.5 Hz, 1H), 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.75
3
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2
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–
–
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1
7.69 (m, 2H), 7.63 (td, J = 7.7, 1.8 Hz, 1H), 7.61 – 7.53 (m, 3H), 7.30 (d, J = 8.5 Hz, 1H), 7.26
7.18 (m, 3H), 6.90 (t, J = 5.1 Hz, 1H), 5.48 (s, 2H), 5.37 (s, 2H), 3.62 – 3.51 (m, 4H), 3.37 (s,
1
3
H), 1.93 – 1.82 (m, 2H). C NMR (126 MHz, CDCl ): δ 167.00, 161.92, 155.77, 151.54,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
1
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9
0
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0
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9
0
49.69, 140.35, 140.15, 137.32, 135.46, 134.18, 129.19, 129.09, 127.18, 123.45, 122.94, 121.64,
115.73, 114.93, 72.60, 59.10, 49.51, 44.98, 39.32, 28.95. LCMS Retention time: 1.610 min.
+
LCMS purity 100%. HRMS (ESI): m/z calcd for C H N O [M+H] 459.2030, found
2
6
26
4
4
459.2100.
4-((2,4-dioxo-1-(pyridin-3-ylmethyl)-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-
1
methoxypropyl)benzamide (23). Isolated as a white solid (4.8 mg, 11% yield). H NMR (500
MHz, CDCl ): δ 8.62 (s, 1H), 8.55 (d, J = 4.9 Hz, 1H), 8.27 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 – 7.69
3
(m, 2H), 7.63 – 7.52 (m, 4H), 7.30 – 7.23 (m, 2H), 7.10 (d, J = 8.4 Hz, 1H), 6.90 (t, J = 5.2 Hz,
1
3
1H), 5.39 (s, 2H), 5.36 (s, 2H), 3.61 – 3.52 (m, 4H), 3.38 (s, 3H), 1.93 – 1.82 (m, 2H). C NMR
(126 MHz, CDCl ): δ 166.96, 161.68, 151.48, 149.48, 148.54, 140.16, 139.64, 135.59, 134.61,
3
134.29, 131.49, 129.63, 129.14, 127.23, 124.01, 123.69, 115.85, 114.05, 72.59, 59.10, 45.24,
45.03, 39.32, 28.95. LCMS Retention time: 1.510 min. LCMS purity 99%. HRMS (ESI): m/z
+
calcd for C H N O [M+H] 459.2030, found 459.2000.
2
6
26
4
4
N-(3-ethoxypropyl)-4-((1-(4-methylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-
yl)methyl)benzamide (24). Isolated as a white solid (24 mg, 44% yield), mp 174ꢀ176 °C. H
NMR (500 MHz, CDCl ): δ 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 – 7.70 (m, 2H), 7.61 – 7.51 (m,
1
3
3
H), 7.22 (ddd, J = 8.1, 7.3, 0.9 Hz, 1H), 7.16 – 7.11 (m, 5H), 7.08 (t, J = 5.1 Hz, 1H), 5.36 (s,
H), 5.33 (s, 2H), 3.64 – 3.54 (m, 4H), 3.51 (q, J = 7.0 Hz, 2H), 2.31 (s, 3H), 1.91 – 1.83 (m,
2
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H), 1.24 (t, J = 7.0 Hz, 3H). C NMR (126 MHz, CDCl ): δ 166.83, 161.90, 151.49, 140.38,
3
40.10, 137.57, 135.36, 134.14, 132.59, 129.79, 129.26, 129.09, 127.15, 126.54, 123.28, 115.76,
14.63, 70.71, 66.76, 47.36, 44.93, 39.63, 28.95, 21.23, 15.55. LCMS Retention time: 3.278 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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31
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33
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44
45
46
47
48
49
50
51
52
53
54
55
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57
58
59
60
+
LCMS purity 99.7%. HRMS (ESI): m/z calcd for C H N O [M+H] 486.2387, found
2
9
31
3
4
486.2389.
N-(3-isopropoxypropyl)-4-((1-(4-methylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-
1
yl)methyl)benzamide (25). Isolated as a white solid (35 mg, 62% yield), mp 163ꢀ165 °C. H
NMR (500 MHz, CDCl ): δ 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.76 – 7.70 (m, 2H), 7.61 – 7.51 (m,
3
3
H), 7.22 (ddd, J = 8.0, 7.3, 0.9 Hz, 1H), 7.16 (t, J = 4.5 Hz, 1H), 7.15 – 7.10 (m, 5H), 5.36 (s,
2H), 5.33 (s, 2H), 3.65 – 3.53 (m, 5H), 2.31 (s, 3H), 1.91 – 1.81 (m, 2H), 1.18 (d, J = 6.1 Hz,
13
6
1
4
H). C NMR (126 MHz, CDCl ): δ 166.77, 161.90, 151.49, 140.34, 140.10, 137.56, 135.36,
3
34.16, 132.59, 129.79, 129.26, 129.07, 127.17, 126.54, 123.28, 115.77, 114.62, 72.14, 68.29,
7.36, 44.93, 39.85, 29.15, 22.36, 21.23. LCMS Retention time: 3.374 min. LCMS purity 99.6%.
+
HRMS (ESI): m/z calcd for C H N O [M+H] 500.2544, found 500.2541.
3
0
33
3
4
N-(3-(dimethylamino)propyl)-4-((1-(4-methylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-
3
(4H)-yl)methyl)benzamide (26). Isolated as a white solid (12 mg, 22% yield), mp 185ꢀ187 °C.
1
H NMR (500 MHz, CDCl ): δ 8.44 (t, J = 4.8 Hz, 1H), 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.76 –
3
7.69 (m, 2H), 7.61 – 7.51 (m, 3H), 7.22 (ddd, J = 8.1, 7.3, 0.9 Hz, 1H), 7.17 – 7.09 (m, 5H), 5.36
(
s, 2H), 5.33 (s, 2H), 3.58 – 3.51 (m, 2H), 2.52 – 2.46 (m, 2H), 2.31 (s, 3H), 2.28 (s, 6H), 1.80 –
1
3
1
.70 (m, 2H). C NMR (126 MHz, CDCl ): δ 166.75, 161.93, 151.49, 140.19, 140.11, 137.58,
3
1
35.37, 134.16, 132.59, 129.79, 129.26, 129.06, 127.18, 126.55, 123.29, 115.77, 114.63, 59.64,
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