Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses

Article abstract of DOI:10.1016/j.bmc.2014.06.053

Human polyoma- and papillomaviruses are non-enveloped DNA viruses that cause severe pathologies and mortalities. Under circumstances of immunosuppression, JC polyomavirus causes a fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML) and the BK polyomavirus is the etiological agent of polyomavirus-induced nephropathy and hemorrhagic cystitis. Human papillomavirus type 16, another non-enveloped DNA virus, is associated with the development of cancers in tissues like the uterine cervix and oropharynx. Currently, there are no approved drugs or vaccines to treat or prevent polyomavirus infections. We recently discovered that the small molecule Retro-2^cycl, an inhibitor of host retrograde trafficking, blocked infection by several human and monkey polyomaviruses. Here, we report diversity-oriented syntheses of Retro-2^cycl and evaluation of the resulting analogs using an assay of human cell infections by JC polyomavirus. We defined structure-activity relationships and also discovered analogs with significantly improved potency as suppressors of human polyoma- and papillomavirus infection in vitro. Our findings represent an advance in the development of drug candidates that can broadly protect humans from non-enveloped DNA viruses and toxins that exploit retrograde trafficking as a means for cell entry.

Full text of DOI:10.1016/j.bmc.2014.06.053

Bioorganic & Medicinal Chemistry 22 (2014) 4836–4847
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural optimization of a retrograde trafficking inhibitor that
protects cells from infections by human polyoma- and
papillomaviruses
Daniel W. Carney a, , Christian D. S. Nelson b, , Bennett D. Ferris a, Julia P. Stevens a, Alex Lipovsky c,
Teymur Kazakov c, Daniel DiMaio c, Walter J. Atwood b, Jason K. Sello a,
⇑
a Department of Chemistry, Brown University, 324 Brook Street, Providence, RI 02912, United States
b Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, 185 Meeting Street, Providence, RI 02912, United States
c Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Human polyoma- and papillomaviruses are non-enveloped DNA viruses that cause severe pathologies
and mortalities. Under circumstances of immunosuppression, JC polyomavirus causes a fatal demyelinat-
ing disease called progressive multifocal leukoencephalopathy (PML) and the BK polyomavirus is the
etiological agent of polyomavirus-induced nephropathy and hemorrhagic cystitis. Human papillomavirus
type 16, another non-enveloped DNA virus, is associated with the development of cancers in tissues like
the uterine cervix and oropharynx. Currently, there are no approved drugs or vaccines to treat or prevent
polyomavirus infections. We recently discovered that the small molecule Retro-2cycl, an inhibitor of host
retrograde trafficking, blocked infection by several human and monkey polyomaviruses. Here, we report
diversity-oriented syntheses of Retro-2cycl and evaluation of the resulting analogs using an assay of
human cell infections by JC polyomavirus. We defined structure–activity relationships and also discov-
ered analogs with significantly improved potency as suppressors of human polyoma- and papillomavirus
infection in vitro. Our findings represent an advance in the development of drug candidates that can
broadly protect humans from non-enveloped DNA viruses and toxins that exploit retrograde trafficking
as a means for cell entry.
Received 17 May 2014
Revised 16 June 2014
Accepted 23 June 2014
Available online 10 July 2014
Keywords:
Polyomavirus
Papillomavirus
Retrograde trafficking
Dihydroquinazolinone
SAR
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
BKPyV-associated disease is most often observed during immuno-
modulatory therapy, and can lead to the development of
Polyomaviruses are small, non-enveloped, viruses with an icosa-
hedral capsid that contains a double-stranded DNA genome.1 These
viruses have established latent infections in the vast majority of the
human population.1,2 Primary infection often occurs early in life, and
it is estimated that as much as 90% of the adult population is sero-
positive for BK polyomavirus (BKPyV) and as much as 40% of adults
are seropositive for JC polyomavirus (JCPyV).2 Polyomaviruses estab-
lish an asymptomatic infection in humans, with polyomavirus-
associated disease seen only in the context of immunosuppression,
such as in AIDS patients or during immunomodulatory therapy.
During circumstances of reduced immune function, increased
replication and dissemination of JCPyV can lead to the development
of the neurodegenerative disease progressive multifocal leuko-
encephalopathy (PML), which affects 3–5% of AIDS patients.3,4
hemorrhagic cystitis and polyomavirus-associated nephropathy in
as many as 10% of transplant patients.1,5 Human papillomaviruses
(HPVs) are also non-enveloped, DNA viruses.6 HPV infection and
replication is limited to the squamous epithelial tissue7 and is
believed to be associated with as much as 5% of all cancers, most
notable of which are cancer of the cervix, other anogenital tissue,
and the oropharnyx.6,8 Prophylactic vaccination against certain
types of HPV has been successful. However, HPV related diseases
will remain a significant human health problem for at least several
decades for individuals who refuse vaccination or who become
infected before being vaccinated. Currently, there are no approved
small-molecule therapeutics for the treatment or prevention of
PyV and HPV infection. Therapeutic strategies that help to manage
the spread of these viruses would have significant value in medicine.
The development of antiviral agents is often guided by consid-
eration of viral life cycles. Many non-enveloped viruses, including
PyV and HPV, are unable to access the host cytoplasm directly from
⇑
Corresponding author. Tel.: +1 (401)863 1194.
E-mail address: jason_sello@brown.edu (J. K. Sello).
Authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2014.06.053
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4837
the cell surface or from endosomes after endocytosis.7,9 They
R2: Amide
Moiety
O
therefore tend to exploit host vesicular trafficking en route to the
Golgi apparatus or the endoplasmic reticulum, from which they
are released into the cytoplasm before reaching the nucleus for
replication.7,9–13 The movement of virus particles, macromolecules,
and metabolites from the cell surface to the endoplasmic reticulum
via the Golgi is known as retrograde trafficking. This phenomenon
is an important intracellular transport mechanism wherein pro-
tein, lipids, and small molecules are transported from endosomes
to the trans-Golgi network and Golgi membranes.11 Retrograde
trafficking is the primary mechanism for recycling chaperones,
receptors, and other cargo molecules that are targeted to the cell
membrane from the Golgi. In principle, small molecule modulation
of host intracellular trafficking could serve as a useful strategy for
the prevention of infections by non-enveloped viruses. Indeed, we
recently reported that Retro-2cycl, a dihydroquinazolinone (DHQZ)
inhibitor of retrograde trafficking14,15, blocked the infection of cells
by human and monkey polyomaviruses16 as well as by human
papillomaviruses.17
R3: Benzo
Moiety
N
S
R1: Heterocycle
Moiety
N
H
Retro-2cycl
O
O
R2
a, b
OH
N
H
R3
R3
NO2
NH2
2
1
c
O
O
O
R2
R2
R1
O
d
N
N
R3
R3
H
R3
N
H
O
N
N
H
DHQZ
Retro-2cycl apparently blocks toxin and viral retrograde trans-
port without significantly affecting endogenous trafficking.14–17
However, the specific host cellular factor that is targeted by
Retro-2cycl is not yet known. Here, we have used a diversity-
oriented synthetic strategy to prepare DHQZs that are structurally
related to Retro-2cycl. The capacities of the compounds to inhibit
the infection of human cells by JCPyV were assessed systemati-
cally. The experiments revealed critical structure activity-
relationships (SAR) and led to the discovery of Retro-2cycl analogs
with enhanced capacities to prevent both JCPyV and HPV
infections.
3
R1
Scheme 1. Reagents and conditions: (a) R2NH2, dicyclohexylcarbodiimide, 4-
dimethylaminopyridine, dichloromethane, room temperature, 16 h; (b) 10% Pd/C,
ammonium formate, methanol; (c) R1CHO, Sc(OTf)3, methanol, MW 100 °C, 1 h; (d)
R2NH2, tetrahydrofuran, reflux, then R1CHO, Sc(OTf)3.
primary amines (like anilines) necessitated preparation of DHQZs
from 2-nitrobenzoic acids (1).
The first moiety to be analyzed was the heterocycle substituent
of the dihydroquinazolinone aminal carbon (Table 1). In particular,
three structural aspects of the heterocycle moiety were investi-
gated: the identity of the heteroatom, the ring substitution pattern,
and the identity of the ring substituent. Compounds 4–6, which are
substituted with an unsubstituted thiophene, a pyrrole, or a furan,
all exhibited significantly attenuated anti-JCPyV activity relative to
Retro-2cycl. Interestingly, compound 7, bearing a 5-methylfuran
moiety, had similar activity to that of Retro-2cycl, which itself bears
a 5-methylthiophene moiety. Although the 5-methylthiophene
moiety confers greater potency, it is apparent that a 5-methylfuran
moiety can also be tolerated. The pattern and identity of the thio-
phene substituent also proved to be significant with respect to bio-
activity. Compound 8, bearing a methyl group at the thiophene 4
position, had nearly equal activity to that of Retro-2cycl. On the
other hand, the activity of compound 9, bearing a methyl group
at the 3 position of the thiophene, was attenuated to the same
extent as Retro-2cycl analogs with unsubstituted heterocycle moie-
ties. An analog that had a 5-ethyl thiophene (compound 10) in
place of the parent compound’s 5-methyl thiophene had markedly
improved potency. Interestingly, activity is severely compromised
when the heterocycle is a benzothiophene ring (compound 11).
Therefore, the 5-ethylthiophene moiety was held constant
throughout the remainder of the SAR analysis.
We next turned our attention to the amide moiety (Table 2) and
the effects of various aliphatic and aromatic amide groups on
JCPyV infectivity. Most of the compounds with substituents on
the amide nitrogen had activities that were similar those of com-
pound 10 and compound 12, which has only a hydrogen substitu-
ent. Dramatic improvements in activity were observed in
compounds wherein the phenyl group of Retro2cycl was replaced
by either benzyl (compound 17) or methylnapthyl (compound
19) groups. In the context of SAR analysis, our finding that the
incorporation of a benzyl group onto the amide moiety improved
potency was fortuitous because many substituted benzylamines
are commercially available. Using readily available building blocks,
we were able to prepare many DHQZs with structurally diverse
2. Results
2.1. Structure–activity relationship (SAR) analysis of the
dihydroquinazolinone Retro-2cycl
The suppression of virus infection by Retro-2cycl encouraged us
to pursue a SAR analysis to define critical structural elements for
bioactivity. We anticipated that such an analysis would also yield
compounds with superior potency as retrograde trafficking inhibi-
tors. For this analysis, we divided the Retro-2cycl structure into
three distinct elements (Scheme 1) a heterocycle moiety, an amide
moiety, and a benzo moiety. We therefore carried out compound
diversification in consecutive phases wherein one moiety was var-
ied independently of the other two. After each phase, the most
active compound served as a new lead structure for the subsequent
diversification phase. The activities of the compounds prepared in
each stage were systematically assessed in assays wherein human
SVGA cells were infected with JCPyV.
Two routes were used for the diversity-oriented synthesis of
DHQZs (Scheme 1). In one route, primary amines were coupled
to 2-nitrobenzoic acid (1) using dicyclohexylcarbodiimide (DCC)
and dimethylaminopyridine; subsequently, the nitro group was
reduced by transfer hydrogenation with ammonium formate and
10% palladium on carbon in methanol to afford anthranilamides
(2). The anthranilamide intermediates were condensed with aro-
matic aldehydes in the presence of scandium(III) triflate under
microwave irradiation to afford the desired DHQZs. Alternatively,
DHQZs were prepared in a one-pot, tandem reaction sequence
comprised of the decarboxylative condensation of an isatoic anhy-
dride (3) and primary amines in THF, followed by the scandium(III)
triflate-catalyzed condensation of the resulting amides with aro-
matic aldehydes. Preparation of DHQZs from isatoic anhydrides
(3) was the preferred route. However, in certain instances, the
availability of starting materials or the weak nucleophilicity of

4838
D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
Table 1
Anti-JCPyV activity of DHQZs at 25 l
M with varied heterocycle moietya
O
N
N
H
R1
Compound
R1
Infectivity (% DMSO control)
57.5 5.9
Compound
R3
Infectivity (% DMSO control)
55.6 14.4
S
S
S
S
Retro-2cycl
8
4
5
81.1 16.9
92.4 4.45
9
10
11
76.4 1.1
47.8 3.8
95.3 14.3
H
N
S
S
OO
6
7
69.9 27.8
64.4 12.7
O
a
HeLa M cells were pre-incubated with 25 lM of the DHQZs prior to inoculation with JC polyomavirus. Infections were scored and normalized to a DMSO-treated control.
The data represent the mean of three replicates with indicated standard error.
benzyl groups on the amide moiety. We investigated the effects of
ring substituents, including methoxy group(s) (compounds 20–23),
a fluorine atom (compounds 24–25), and nitro groups (compounds
26–27), on the bioactivity of the compounds. In most cases, the
addition of substituents on to the benzyl group was tolerated, aro-
matic nitro groups being a particular exception. A compound with
a para-fluorobenzylamide (compound 24) was more potent than
one with an unsubstituted benzyl group at the same position
(compound 17).
dihydroquinazolinone substructure into new chemical space. As a
proof of principle, n-butylamino analog 40 was prepared
(Scheme 2). A SNAr reaction of 5-fluoro-2-nitroamide 38 with
n-butylamine provided 5-(n-butylamino)-2-nitroamide 39. Reduc-
tion of the 2-nitro group followed by condensation/cyclization
with 5-ethyl-2-thiophenecarboxaldehyde provided DHQZ 40.
Unfortunately, incorporation of the n-butylamino group at C-6 of
the benzo moiety resulted in a severe attenuation of activity (i.e.,
infectivity at 25 lM: 71.0 8.28% DMSO Control).
In DHQZ syntheses that use achiral building blocks, the ring-
forming reaction yields racemic products. In two cases, chiral
Two final analogs were synthesized in order to probe SAR
around the DHQZ aromatic amine (Scheme 3). First, we examined
the significance of the heterocycle’s oxidation state by preparing
and evaluating the quinazolinone form of compound 24. The qui-
nazolinone species (41) has a more planar geometry than the cor-
responding DHQZ (24). Also, 41 lacks the N-1 amino hydrogen, a
potentially important hydrogen bond donor. Interestingly, quinaz-
olinone 41 was much less active than DHQZ 24. We also prepared
an N-methyl-DHQZ (43) and found that this compound was simi-
larly less active than DHQZ 24. From these observations, one could
predict that the N-1 amine is acting as hydrogen bond donor in the
molecule’s interaction with its cellular target.
a-methylbenzylamines (compounds 28–29) were used to generate
the amide moieties with the hopes of achieving stereochemical
induction at the DHQZ aminal stereocenter.18 The DHQZ-forming
reactions of substrates prepared from chiral
yielded configurationally stable products as single diastereomers.
The compounds with chiral -methylbenzylamide groups were
a-methylbenzylamines
a
more than four-fold less active than the compound with the unsub-
stituted benzylamide (compound 17). Interestingly, the DHQZ ana-
log with the (R)-a-methylbenzylamide was much less active than
its diastereomer derived from (S)-a-methylbenzylamine.
In the third phase of SAR analysis, the benzo moiety was varied
while the heterocycle and amide moieties were held constant as a
5-ethylthiophene group and a 4-fluorobenzyl group, respectively
(Table 3). We first examined the effect of methylation at each of
the four otherwise unsubstituted aromatic carbons of the benzo
moiety. Methyl groups were tolerated at C-5 (30) and C-6 (31).
However, compounds with methyl substituents at C-7 (32) and
C-8 (33) had severely attenuated activity. A chlorine atom could
likewise be tolerated at C-6 (34), but not a C-7 (35). Interestingly,
compounds with fluorine atoms at either C-6 (36) or C-7 exhibited
potent bioactivity (37). Although the former was more active, the
finding that a compounds with a fluorine, and not a methyl or
chloro-substituent, at C-7 is tolerated suggests that there substitu-
ent size at this atom of the benzo moiety is a critical factor for
binding to the cellular target.
The most potent Retro-2cycl analog discovered in this study was
DHQZ 36. In order to determine the fold improvement in potency
of our optimized compound over Retro-2cycl, dose response curves
were generated for both compounds against JCPyV and HPV16
pseudovirus, from which IC50s for inhibition of infectivity were
determined (Fig. 1). While JCPyV appeared to be more susceptible
to the DHQZs, there was an overall 6.7-fold improvement in
potency from Retro-2cycl to our optimized compound, DHQZ 36,
in the case of both viruses. This is consistent with the compounds’
targeting of a host cellular factor as opposed to viral proteins.
2.2. Optimized dihydroquinazolinone inhibitor interferes with
retrograde trafficking
In order to confirm that the optimized DHQZ (36) inhibited ret-
rograde trafficking, we conducted a proximity ligation assay, which
detects co-localization between the viral capsid proteins and local-
ized host cell marker proteins (Fig. 2). Marker proteins were cho-
sen from known intracellular sites where the virions are
delivered immediately downstream from their trafficking through
Since the SAR analysis of the benzo moiety suggested that C-5
and C-6 can be structurally elaborated without loss in potency,
we sought to explore this possibility. We envisioned that aromatic
substitution or cross-coupling reactions of the halogenated
species (compounds 34–37) could enable elaboration of the

D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4839
Table 2
Anti-JCPyV activity of DHQZs at 25 l
M with varied amide moietya
O
R2
N
S
N
H
Compound
R2
Infectivity (% DMSO control)
41.4 1.4
Compound
R2
Infectivity (% DMSO control)
OMe
12
H
21
27.5 1.8
13.7 0.5
OMe
OMe
13
14
76.4 1.1
22
23
69.9 27.8
16.4 3.9
OMe
15
16
17
18
42.4 2.6
45.7 10.3
13.3 1.1
50.4 2.3
24
25
26
27
7.62 1.0
14.0 2.6
30.6 3.2
44.5 8.8
F
F
NO2
NO2
19
14.5 0.7
21.2 5.1
28
29
32.4 13.7
52.7 1.5
20
OMe
HeLa M cells were pre-incubated with 25
a
l
M of the DHQZs prior to inoculation with JC polyomavirus. Infections were scored and normalized to a DMSO-treated control.
The data represent the mean of three replicates with indicated standard error.
Table 3
Anti-JCPyV activity of DHQZs at 25 l
M with varied benzo moietya
F
O
5
8
6
N
R3
S
N
H
7
Compound
R3
Infectivity (% DMSO Control)
Compound
R3
Infectivity (% DMSO Control)
30
31
32
33
5-Methyl
6-Methyl
7-Methyl
8-Methyl
10.2 5.68
8.34 2.76
67.1 9.12
44.5 2.51
34
35
36
37
6-Chloro
7-Chloro
6-Fluoro
7-Fluoro
13.7 16.2
21.3 2.82
4.45 0.613
8.82 7.52
a
HeLa M cells were pre-incubated with 25 lM of the DHQZs prior to inoculation with JC polyomavirus. Infections were scored and normalized to a DMSO-treated control.
The data represent the mean of three replicates with indicated standard error.
the retrograde pathway. For JCPyV, we immunostained for the viral
capsid protein, VP1, and the ER protein, PDI (protein disulfide
isomerase) in SVGA cells. For HPV16, we immunostained for viral
capsid protein, L1, and Golgi protein, TGN46, in HelaM cells. In
the proximity ligation assay, co-localization is indicated by a fluo-
rescent signal, which is detected only when the target proteins are
signal for both JCPyV and HPV16. Furthermore, in the case JCPyV,
DHQZ 36 appears to inhibit co-localization more potently than
Retro-2cycl. This trend is less clear in the case of HPV16. In any case,
we can conclude that the optimized DHQZ 36 inhibits retrograde
transport in a similar manner as Retro-2cycl
.
within 40 nm of each other. Cells were pretreated with Retro-2cycl
,
3. Discussion
DHQZ 36, or DMSO then inoculated with virus. Cells were fixed and
immunostained after an 8 h infection period for JCPyV or a 16 h
period for HPV16. In DMSO-treated cells, there was a strong co-
localization signal for both JCPyV and HPV16. In Retro-2cycl and
DHQZ 36 treated cells, there is a clear reduction in co-localization
Most anti-infective therapeutic strategies are based on target-
ing the pathogen with a small molecule that critically perturbs
its physiology. Since some pathogens use the host cell machinery
for infection or for delivery of toxins and other virulence factors,

4840
D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
F
F
F
O
O
O
a
b, c
n-BuHN
F
n-BuHN
N
H
N
N
H
S
NO2
NO2
N
H
38
39
40
JcPyV Infectivity at 25 µM:
71.0 8.28 % DMSO Control
Scheme 2. Reagents and conditions: (a) n-Butylamine, triethylamine, dimethoxyethane, room temperature, 16 h, 64%; (b) 10% Pd/C, ammonium formate, methanol, room
temperature, 2 h; (c) 5-ethylthiophene-2-carboxaldehyde, Sc(OTf)3, methanol, reflux, 2 h, 54% over 2 steps.
F
ricin, and Shiga-like toxins, due to their shared reliance on intracel-
lular trafficking.14,17 We suspect that the lethality of other
O
pathogens and toxins could be suppressed by DHQZ retrograde
O
a
trafficking inhibitors as well. Beyond their potentially broad appli-
cations in medicine, compounds that target the host cellular
machinery rather than the pathogen may have the added advan-
tage of being less prone to resistance-conferring mutations in the
pathogen.
O
N
N
H
O
S
N
3
41
Herein, we define structure–activity relationships associated
with DHQZ inhibitors of retrograde trafficking. Through our inves-
tigations, we have identified new analogs of Retro-2cycl with signif-
icantly improved potency. We employed a whole-cell based
polyomavirus infectivity assay to assess the efficacies of the com-
pounds as inhibitors of retrograde trafficking. The strength of this
assay is that it precludes the use of deadly toxins (e.g., ricin, Shiga
toxin) or cancer-causing viruses (e.g., HPV). Since the non-
enveloped viruses, ricin, and the Shiga toxins exploit the same ret-
rograde trafficking mechanism, we believe that the compounds
identified in this assay will have broad utility. This is evidenced
by the fact that a small molecule optimized to inhibit polyomavi-
rus infection exhibited similarly improved activity against human
papillomavirus as compared to Retro-2cycl. The drawbacks of our
PyV infectivity assay are its requirement of multiple replicates
for reliability and its incompatibility with high-throughput
screening technologies. Currently, we are working to address these
shortcomings.
JcPyV Infectivity at 25 µM:
63.3 1.64% DMSO Control
F
O
O
N
b
O
N
O
S
N
42
43
JcPyV Infectivity at 25 µM:
60.0 5.48% DMSO Control
Scheme 3. Reagents and conditions: (a) 4-Fluorobenzylamine, ethanol, reflux, then
5-ethylthiophenal, CuCl2, 94%; (b) 4-fluorobenzylamine, tetrahydrofuran, reflux,
then 5-ethylthiophenal, Sc(OTf)3, 88%.
The key DHQZ structural features that led to improvement in
activity were the incorporation of a 5-ethylthiophene as the het-
erocycle moiety, incorporation of a p-fluorobenzyl group as the
amide moiety, and addition of a fluorine atom onto C-6 of the
benzo moiety. During the late stages of our experiments, two
Retro-2cycl SAR papers focused on inhibition of Shiga-like toxin
trafficking were published by Cintrat and co-workers.19,20 While
our optimized DHQZ 36 differs in structure from the optimized
compounds reported by Cintrat, many of our general structure
activity relationships are in agreement. The combined SAR data
sets suggest additional analogs that may surpass the potency of
both our own and Cintrat’s optimized inhibitors. While our
findings concerning dihydroquinazolinone SAR analyses largely
agree with those that have been published, the only point of incon-
gruence is the consequences of methylation of the dihydroquinaz-
olinone N-1 amino nitrogen. We observed a severe attenuation of
activity resulting from methylation of the DHQZ N-1 amino
nitrogen, while the Cintrat and co-workers reported that
N-methyldihydroquinazolinones are highly potent inhibitors of ricin
intoxication. The only issue addressed by Cintrat and co-workers in
their studies20 that cannot be addressed in our work concerns
the configuration of the stereocenter in the bioactive
dihydroquinazolonine structure. After chiral separation, they were
able to evaluate the two DHQZ enantiomers and found that
the (S)-N-methyldihydroquinazolinones were significantly more
F
O
O
F
N
N
S
S
N
H
N
H
Retro-2cycl
JCPyV IC50 = 54 µM
HPV16 IC50 = 160 µM
DHQZ 36
JCPyV IC50 = 8.1 µM
HPV16 IC50 = 24 µM
Figure 1. Inhibition of JCPyV infectivity in SVG-A Cells and HPV16 infectivity in
HelaM Cells: cells were pre-incubated with increasing concentrations of Retro-2cycl
or DHQZ 36 prior to inoculation with virus.
one could also envision therapeutic agents that subtly perturb the
host’s physiology in ways that make it unsusceptible to pathogens
and/or their toxins. This phenomenon is compellingly illustrated
by the dihydroquinazolinone inhibitors of retrograde trafficking
described here and elsewhere.14–17 Remarkably, one of these mol-
ecules can protect cells from polyomaviruses, papillomaviruses,

D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4841
Figure 2. HPV16 and JCPyV Proximity Ligation Assay: cell nuclei are indicated by blue fluorescence. Green fluorescence indicates co-localization of target proteins: JCPyV—
capsid protein VP1 and ER protein PDI; HPV16—capsid protein L1 and Golgi protein TGN 46.
active than the corresponding (R)-N-methyldihydroquinazolinon-
es.20 We are unable to address the issue of configuration with
DHQZ 36 because the N-des-methyl-DHQZs are known to be
configurationally unstable.21
While there are obvious opportunities for additional structural
optimization, identification of the cellular target of the retrograde
trafficking inhibitors is the next crucial step. The SAR studies
described in this report, as well as by Cintrat and co-workers, will
guide the design of affinity reagents that can be used to identify the
sub-cellular target of the DHQZ retrograde trafficking inhibitors.
The preparation and use of these affinity reagents is currently
underway in these laboratories.
then pre-incubated for 0.5 h with 25 l
M of Retro-2cycl, the indi-
cated Retro-2cycl analog, or the vehicle control. The final DMSO
concentration was 0.04%. JCPyV was then added at an MOI of
0.25 and allowed to infect in the presence of the indicated com-
pound for 72 h. Samples were then processed for flow cytometry.
An infected culture that was not treated with (ꢀ20% total infected
cells) was then normalized to 100% and any reduction in infection
in the DHQZ treated samples were compared to this untreated con-
trol. Three independent experiments were performed and used to
calculate standard deviations.
For the IC50 analysis, 1:2 serial dilutions of a 2500Â stock of
Retro-2cycl or DHQZ 36 were performed in DMSO. These dilutions
were then added to complete media, such that the media con-
tained a 1Â concentration of the indicated concentration of com-
pound in a final DMSO concentration of 0.04%. Samples were
pretreated with the indicated compound and challenged with
JCPyV or HPV16 as above. Three independent experiments were
performed and used to calculate standard deviations.
4. Experimental procedures
4.1. Cells, viruses, plasmids, and antibodies
SVG-A cells were maintained in MEM complete media (minimal
essential media containing 10% fetal bovine serum, 1% penicillin,
1% streptomycin) and have been previously described.22 HelaM
cells were maintained in DMEM complete media (Dulbecco’s mod-
ification of minimal essential media containing 10% fetal bovine
serum, 1% penicillin, 1% streptomycin, 10 mM Hepes pH 7.5).
HelaM cells, a subclone of HelaS3 cells, were a kind gift from
4.3. Flow cytometric scoring of viral infection
JCPyV-infected SVG-A cells were detached from 12-well plates
by aspirating the growth media, washing once with phosphate buf-
fered saline (PBS), and detaching with Trypsin–EDTA. These cells
were then transferred to flow cytometry tubes and pelleted by cen-
trifugation at 600g for 5 min, washed with PBS and fixed in 0.5 mL
4% paraformaldehyde (PFA) for 10 min. Samples were pelleted and
washed with PBS, and permeabilized with 0.5 mL PBS containing
1% Triton X-100 for 10 min at 21 °C. Cells were then pelleted and
resuspended in 0.1 mL PBS containing 3% BSA and an Alexa
Fluor-labeled monoclonal antibody to VP1 (PAB 597-AF488). After
incubation for 1 h at 21 °C, cells were washed once with PBS and
infected cells were scored by flow cytometry (BD FACSCalibur).
HPV16-infected HelaM cells were detached from 12-well plates
by aspirating the growth media, washing once with phosphate
buffered saline (PBS), and detaching with Trypsin–EDTA. These
cells were then transferred to flow cytometry tubes and pelleted
by centrifugation at 600g for 5 min, washed with PBS and fixed
in 0.5 mL 4% paraformaldehyde (PFA) for 10 min. Infected cells
were then scored flow cytometry for HcRed expression.
Walther Mothes (Yale University). The Mad1-SVED strain of JCPyV
used in these experiments has previously been described.23 Stocks
of JCPyV were generated according to previously published stud-
ies.24 HPV16 pseudovirus expressing the reporter HcRed was gen-
erated from the p16sheLL plasmid, which expresses HPV L1 and L2,
and pCAG-HcRed were obtained from Addgene. HPV pseudovirus
stocks were generated according to previously published meth-
ods.17 The antibodies to PDI, SV40 VP1, and TGN46 were purchased
from Abcam. Polyclonal sera to HPV L1 was a kind gift from Pat Day
(National Institute of Health). The PAB597 anti-VP1 hybridoma
was a kind gift from Ed Harlow.
4.2. Inhibition of JCPyV infection by Retro-2cycl analogs
SVG-A cells were seeded in 12 welled plates at a density of
1 Â 105 cells per well and incubated overnight at 37 °C. Cells were

4842
D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4.4. Proximity ligation assays
4.6.3. N-(4-Fluorobenzyl)-5-methyl-2-nitrobenzamide
(2 mmol scale) from 5-methyl-2-nitrobenzoic acid, yield:
561 mg, 97%. HRMS (FAB) [C15H13FN2O3+Na]+ predicted:
311.0808, found: 311.0820. 1H NMR (400 MHz, DMSO-d6)
d = 9.16 (t, J = 5.8 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H), 7.49 (dd, J = 1.0,
8.3 Hz, 1H), 7.46–7.43 (m, 1H), 7.43–7.37 (m, 2H), 7.23–7.14 (m,
2H), 4.43 (d, J = 6.1 Hz, 2H), 2.43 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) d = 165.8, 162.5, 160.1, 144.8, 144.6, 135.1, 135.1,
132.7, 130.9, 129.5, 129.4, 129.3, 124.3, 115.2, 115.0, 41.9, 20.8.
Colocalization experiments between virus and ER or Golgi
markers was performed using proximity ligation assays, as previ-
ously described (Bethyl Labs) (24222489, 23569269). The primary
antibodies used for PLA were rabbit anti-VP1 and mouse anti-PDI
in the case of JCPyV, and rabbit anti-L1 and mouse anti-TGN46 in
the case of HPV16. Cells were pretreated with Retro-2cycl, DHQZ36,
or vehicle control for 0.5 h. SVGA cells (JCPyV) or HelaM cells
(HPV16) were then inoculated with virus at a MOI of 100 for 1 h
at 37 °C. Virus was removed by aspiration and any unbound virus
was removed by washing with media. Fresh media was added con-
taining either 0.1 mM Retro-2cycl or 0.05 mM DHQZ 36, or a vehicle
control. Cells were then incubated at 37 °C for 8 h (JCPyV) or 16 h
(HPV) prior to fixation with 4% PFA. Cells were permeabilized with
PBS containing 0.5% Triton X-100 for 0.5 h, washed three times in
PBS, then blocked in 5% Donkey Serum for 1 h at 37 °C. For JCPyV,
cells were then stained for VP1 (1:1000 dilution) and PDI (1:100
dilution) by overnight incubation at 4 °C. For HPV16, cells were
stained with L1 (1:1000 dilution) and TGN46 (1:200 dilution) by
incubation for 2 h at 37 °C. These cells were then immunostained
using the proximity ligation assay, following manufacturer’s
instructions. Cells were washed and the cell nuclei were counter-
stained using DAPI. Fluorescence micrographs were collected by
confocal microscopy and maximal z-projections were displayed.
4.6.4. N-(4-Fluorobenzyl)-4-methyl-2-nitrobenzamide
(2 mmol scale) from 4-methyl-2-nitrobenzoic acid, yield:
413 mg, 72%. HRMS (FAB) [C15H13FN2O3+Na]+ predicted:
311.0808, found: 311.0805. 1H NMR (400 MHz, DMSO-d6)
d = 9.18 (t, J = 5.8 Hz, 1H), 7.86 (s, 1H), 7.67–7.57 (m, 1H), 7.57–
7.51 (m, 1H), 7.39 (dd, J = 5.8, 8.3 Hz, 2H), 7.18 (t, J = 8.8 Hz, 2H),
4.42 (d, J = 5.8 Hz, 2H), 2.43 (s, 3H). 13C NMR (101 MHz, DMSO-
d6) d = 165.5, 162.5, 160.1, 147.4, 141.4, 135.2, 133.8, 129.5,
128.9, 124.2, 115.2, 115.0, 41.9, 20.5.
4.6.5. N-(4-Fluorobenzyl)-3-methyl-2-nitrobenzamide
(2 mmol scale) from 3-methyl-2-nitrobenzoic acid, yield:
458 mg, 80%. HRMS (FAB) [C15H13FN2O3+Na]+ predicted:
311.0808, found: 311.0801. 1H NMR (400 MHz, DMSO-d6)
d = 9.33 (t, J = 5.7 Hz, 1H), 7.59 (s, 3H), 7.46–7.29 (m, 2H), 7.26–
7.05 (m, 2H), 4.40 (d, J = 5.8 Hz, 2H), 2.30 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) d = 164.6, 162.5, 160.1, 149.1, 135.2, 135.2,
133.7, 130.7, 130.3, 129.6, 129.2, 129.2, 126.3, 115.2, 115.0, 41.9,
16.8.
4.5. Chemical synthesis-general
All commercially available reagents were used without further
purification. Reactions were carried out in oven dried glassware,
with dry solvent, and under ambient atmosphere. All spectra
were referenced to residual solvent signals in DMSO-d6
(d = 2.50 ppm for 1H and 39.51 ppm for 13C) and in chloroform-d
(d = 7.27 ppm for 1H-NMR and 77.00 ppm for 13C-NMR).
4.6.6. 5-Fluoro-N-(4-fluorobenzyl)-2-nitrobenzamide (38)
(2 mmol scale) from 5-fluoro-2-nitrobenzoic acid, yield:
477 mg, 82%. HRMS (FAB) [C14H10F2N2O3+Na]+ predicted:
315.0557, found: 311.0549. 1H NMR (400 MHz, DMSO-d6)
d = 9.25 (t, J = 5.4 Hz, 1H), 8.18 (dd, J = 4.8, 8.6 Hz, 1H), 7.67–7.49
(m, 2H), 7.41 (dd, J = 5.6, 8.3 Hz, 2H), 7.28–7.09 (m, 2H), 4.44 (d,
J = 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) d = 165.2, 164.3,
162.7, 162.6, 160.1, 143.2, 135.5, 135.4, 134.8, 134.8,
129.5, 129.4, 127.6, 127.5, 117.7, 117.4, 116.6, 116.4, 115.2,
115.0, 42.0.
4.6. Nitrobenzamide synthesis general procedure
Dicyclohexylcarbodiimide (5.00 mmol) and a nitrobenzoic acid
(5.55 mmol) were dissolved in DCM (20 mL) and allowed to stir
for 5 min before the addition of a primary amine (5.55 mmol)
and dimethylaminopyridine (0.055 mmol). The coupling reaction
was allowed to proceed for 16 h, after which the DCM was
removed in vacuo. The solid residue was resuspended in ethyl
acetate and filtered through a silica gel plug to remove the dicyclo-
hexylurea byproduct. The filtrate was then concentrated and the
desired nitrobenzamide isolated by silica gel flash chromatography
using a hexanes/ethyl acetate solvent gradient.
4.6.7. 4-Fluoro-N-(4-fluorobenzyl)-2-nitrobenzamide
(2 mmol scale) from 4-fluoro-2-nitrobenzoic acid, yield:
330 mg, 57%. HRMS (FAB) [C14H10F2N2O3+Na]+ predicted:
315.0557, found: 311.0565. 1H NMR (400 MHz, DMSO-d6)
d = 9.26 (t, J = 5.4 Hz, 1H), 8.03 (dd, J = 2.3, 8.6 Hz, 1H), 7.81–7.65
(m, 2H), 7.39 (dd, J = 5.7, 8.2 Hz, 2H), 7.27–7.10 (m, 2H), 4.43 (d,
J = 6.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) d = 164.6, 163.0,
162.5, 160.5, 160.1, 148.4, 148.3, 135.0, 135.0, 131.4, 131.3,
129.4, 129.3, 128.6, 128.6, 120.5, 120.3, 115.2, 115.0, 112.3,
112.0, 42.0.
4.6.1. N-(4-Fluorobenzyl)-2-methyl-6-nitrobenzamide
From 2-nitrobenzoic acid, yield 1.15 g, 95%. HRMS (FAB)
[C13H15N2O3+Na]+ predicted: 265.0589, found: 265.0589. 1H NMR
(400 MHz, DMSO-d6) d = 10.65 (s, 1H), 8.15 (d, J = 8.3 Hz, 1H),
7.91–7.83 (m, 1H), 7.80–7.73 (m, 2H), 7.67 (d, J = 8.0 Hz, 2H),
7.36 (t, J = 8.0 Hz, 2H), 7.16–7.09 (m, 1H). 13C NMR (151 MHz,
DMSO-d6) d = 164.1, 146.4, 138.8, 134.1, 132.7, 130.9, 129.3,
128.8, 124.2, 123.9, 119.6.
4.7. Dihydroquinazolinone synthesis
4.7.1. General procedure A
The nitrobenzamide intermediate (0.5 mmol) was dissolved in
methanol (2.5 mL). To the solution was added 10% Pd/C (75 mg)
and ammonium formate (32 mg, 0.5 mmol). After 1 h the reaction
was filtered, rinsing with methanol. The filtrate was adjusted to a
volume 1 mL in methanol and then treated with an aldehyde
(0.55 mmol) and scandium(III) triflate (0.05) mmol. The reaction
was microwave irradiated at 100 °C for 1 h, after which the solvent
was removed in vacuo. The product dihydroquinazolinones were
isolated by silica gel flash chromatography with a hexanes/ethyl
acetate solvent gradient. The chromatographed products were
subsequently purified by recrystallization.
4.6.2. 2-Methyl-6-nitro-N-phenylbenzamide
(2 mmol scale) from 2-methyl-6-nitrobenzoic acid, yield:
510 mg, 89%. HRMS (FAB) [C15H13FN2O3+Na]+ predicted:
311.0808, found: 311.0816. 1H NMR (400 MHz, DMSO-d6)
d = 9.15 (t, J = 5.9 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.51–7.46 (m,
1H), 7.46–7.43 (m, 1H), 7.40 (dd, J = 5.7, 8.5 Hz, 2H), 7.22–7.14
(m, 2H), 4.43 (d, J = 5.8 Hz, 2H), 2.43 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) d = 165.8, 162.5, 160.1, 144.8, 144.6, 135.1, 135.1,
132.7, 130.9, 129.5, 129.4, 129.3, 124.3, 115.2, 115.0, 41.9, 20.8.

D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4843
4.7.2. General procedure B
predicted: 343.0881, found: 343.0872. 1H NMR (400 MHz, DMSO-
d6) d = 7.73 (dd, J = 1.5, 7.8 Hz, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.42–
7.29 (m, 5 H), 7.27–7.22 (m, 1H), 6.94–6.90 (m, 1H), 6.82 (d,
J = 8.0 Hz, 1H), 6.80–6.74 (m, 2H), 6.44 (d, J = 2.8 Hz, 1H), 2.07 (d,
J = 0.8 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) d = 161.5, 146.3,
144.6, 140.5, 136.2, 133.8, 128.7, 128.3, 128.0, 126.3, 126.3,
120.9, 118.0, 115.6, 115.3, 69.4, 15.3.
An isatoic anhydride (1.20 mmol) was added to THF (6 mL) and
heated to 60 °C. To the hot solution of isatoic anhydride was added a
primary amine or aqueous ammonia (1.00 mmol), which was
allowed to react for 1–2 h. Once the amine had been completely
consumed, an aldehyde (1.20 mmol) and scandium triflate
(0.1 mmol) were added and allowed to react at 60 °C for an addi-
tional 3–5 h, after which the solvent was removed. The product
dihydroquinazolinones were isolated by silica gel flash chromatog-
raphy with a hexanes/ethyl acetate solvent gradient. The chromato-
graphed products were subsequently purified by recrystallization.
4.7.8. 2-(3-Methylthiophen-2-yl)-3-phenyl-2,3-
dihydroquinazolin-4(1H)-one (9)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(0.87 mmol scale), yield: 119 mg, 43%. HRMS (FAB) C19H16N2OSNa+
predicted: 343.0881, found: 343.0885. 1H NMR (400 MHz, DMSO-
d6) d = 7.74 (dd, J = 1.5, 7.8 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.37–
7.29 (m, 3H), 7.25–7.17 (m, 4H), 6.83–6.75 (m, 2H), 6.67 (d,
J = 5.0 Hz, 1H), 6.54 (d, J = 2.0 Hz, 1H), 1.91 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) d = 161.8, 146.6, 140.2, 137.0, 135.1, 133.8,
129.5, 128.6, 127.9, 127.5, 126.7, 124.2, 117.8, 115.0, 114.9, 67.9,
13.3.
4.7.3. 3-Phenyl-2-(thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-
one (4)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(1 mmol scale), yield: 181 mg, 59%. HRMS (FAB) [C18H14N2O2+Na]+
predicted: 313.0953, found: 313.0945. 1H NMR (400 MHz, DMSO-
d6) d = 7.72 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.58 (dd,
J = 0.8, 1.9 Hz, 1H), 7.43–7.35 (m, 2H), 7.35–7.28 (m, 3H), 7.28–
7.23 (m, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.79–6.70 (m, J = 1.0, 7.5,
7.5 Hz, 1H), 6.33 (dd, J = 1.9, 3.4 Hz, 1H), 6.25 (t, J = 3.3 Hz, 2H).
13C NMR (75 MHz, DMSO-d6) d = 161.8, 152.9, 146.5, 143.1, 140.6,
133.7, 128.7, 127.9, 126.3, 117.8, 115.5, 114.9, 110.4, 108.4, 67.3.
4.7.9. 2-(5-Ethylthiophen-2-yl)-3-phenyl-2,3-
dihydroquinazolin-4(1H)-one (10)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(1 mmol scale), yield: 125 mg, 37%. HRMS (FAB) C20H18N2OSNa+
predicted: 357.1038, found: 357.1028. 1H NMR (400 MHz, DMSO-
d6) d = 7.74 (dd, J = 1.5, 7.8 Hz, 1H), 7.63 (d, J = 2.8 Hz, 1H), 7.41–
7.34 (m, 2H), 7.34–7.29 (m, 3H), 7.27–7.21 (m, 1H), 6.83 (d,
J = 8.0 Hz, 1H), 6.80–6.76 (m, 1H), 6.75 (d, J = 3.5 Hz, 1H), 6.58
(td, J = 1.1, 3.3 Hz, 1H), 6.42 (d, J = 2.8 Hz, 1H), 2.67 (q, J = 7.4 Hz,
2H), 1.13 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d = 161.5, 146.7, 146.3, 141.7, 140.5, 133.8, 128.7, 128.0, 126.4,
126.3, 126.1, 122.7, 118.0, 115.5, 115.2, 69.7, 22.7, 15.6.
4.7.4. 3-Phenyl-2-(1H-pyrrol-2-yl)-2,3-dihydroquinazolin-
4(1H)-one (5)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(0.25 mmol scale), yield: 29 mg, 40%. HRMS (FAB) [C18H15N3O+Na]+
predicted: 312.1113, found: 312.1118. 1H NMR (400 MHz,
DMSO-d6) d = 10.72 (br s, 1H), 7.72 (dd, J = 1.5, 7.8 Hz, 1H), 7.34–
7.26 (m, 3H), 7.26–7.21 (m, 3H), 7.21–7.15 (m, 1H), 6.79 (dd,
J = 0.5, 8.3 Hz, 1H), 6.75 (ddd, J = 1.0, 7.1, 7.5 Hz, 1H), 6.63 (dt,
J = 1.5, 2.6 Hz, 1H), 6.18 (d, J = 2.3 Hz, 1H), 5.89 (t, J = 3.5 Hz, 1H),
5.81 (q, J = 2.7 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) d = 162.4,
146.8, 140.7, 133.4, 130.0, 128.5, 127.9, 126.6, 126.1, 118.2,
117.8, 115.9, 115.1, 107.6, 107.1, 68.1.
4.7.10. 2-(Benzo[b]thiophen-2-yl)-3-phenyl-2,3-
dihydroquinazolin-4(1H)-one (11)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(1 mmol scale), yield: 168 mg, 47%. HRMS (FAB) C22H16N2OSNa+
predicted: 379.0881, found: 379.0866. 1H NMR (400 MHz, DMSO-
d6) d = 7.88–7.82 (m, 1H), 7.79–7.71 (m, 3H), 7.42–7.37 (m, 4H),
7.37–7.33 (m, 1H), 7.33–7.28 (m, 3H), 7.28–7.22 (m, 1H), 6.86 (d,
J = 8.0 Hz, 1H), 6.80 (dt, J = 1.0, 7.5 Hz, 1H), 6.64 (d, J = 3.3 Hz,
1H). 13C NMR (101 MHz, DMSO-d6) d = 161.4, 146.2, 145.3, 144.8,
140.4, 138.3, 134.0, 128.8, 128.0, 126.5, 124.9, 124.5, 123.8,
123.0, 122.6, 118.2, 115.6, 115.3, 107.6, 69.9.
4.7.5. 2-(Furan-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-
one (6)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(0.25 mmol scale), yield: 61 mg, 84%. HRMS (FAB) C18H14N2O2Na+
predicted: 313.0953, found: 313.0945. 1H NMR (400 MHz, DMSO-
d6) d = 7.72 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.58 (dd,
J = 0.8, 1.9 Hz, 1H), 7.43–7.35 (m, 2H), 7.35–7.28 (m, 3H), 7.28–
7.23 (m, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.79–6.70 (m, J = 1.0, 7.5,
7.5 Hz, 1H), 6.33 (dd, J = 1.9, 3.4 Hz, 1H), 6.25 (t, J = 3.3 Hz, 2H).
13C NMR (75 MHz, DMSO-d6) d = 161.8, 152.9, 146.5, 143.1, 140.6,
133.7, 128.7, 127.9, 126.3, 117.8, 115.5, 114.9, 110.4, 108.4, 67.3.
4.7.11. 2-(5-Ethylthiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-
one (12)
Prepared via procedure B (1 mmol scale), yield: 148 mg, 57%.
HRMS (FAB) [C14H14N2OS+Na]+ predicted: 281.0725, found:
287.0730. 1H NMR (400 MHz, DMSO-d6) d = 8.39 (br. s, 1H), 7.60
(dd, J = 1.5, 7.8 Hz, 1H), 7.28–7.22 (m, 1H), 7.21 (br. s, 1H), 6.91
(d, J = 3.3 Hz, 1H), 6.74 (d, J = 8.1 Hz, 1H), 6.72–6.66 (m, 2H), 5.92
(t, J = 1.8 Hz, 1H), 2.74 (dq, J = 0.8, 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz,
3H). 13C NMR (151 MHz, DMSO-d6) d = 163.0, 147.2, 146.7, 143.4,
133.3, 127.2, 125.3, 122.7, 117.4, 115.0, 114.6, 62.8, 22.8, 15.8.
4.7.6. 2-(5-Methylfuran-2-yl)-3-phenyl-2,3-dihydroquinazolin-
4(1H)-one (7)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(0.25 mmol scale), yield: 42 mg, 55%. HRMS (FAB) C19H16N2O2Na+
predicted: 327.1109, found: 327.1125. 1H NMR (400 MHz, DMSO-
d6) d = 7.72 (dd, J = 1.0, 7.8 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.42–
7.33 (m, 4H), 7.30 (ddd, J = 1.8, 7.0, 8.3 Hz, 1H), 7.24 (tt, J = 1.8,
7.3 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.78–6.72 (m, 1H), 6.17 (d,
J = 3.3 Hz, 1H), 6.11 (d, J = 3.0 Hz, 1H), 5.93 (dd, J = 1.0, 3.0 Hz,
1H), 2.16 (s, 3H). 13C NMR (75 MHz, DMSO-d6) d = 161.8, 151.7,
150.8, 146.4, 140.6, 133.6, 128.7, 127.9, 126.3, 117.7, 115.5,
115.0, 109.4, 106.5, 67.3, 13.3.
4.7.12. 2-(5-Ethylthiophen-2-yl)-3-propyl-2,3-
dihydroquinazolin-4(1H)-one (13)
Prepared via procedure B (1 mmol scale), yield: 108 mg, 36%.
HRMS (FAB) [C17H20N2OS+Na]+ predicted: 323.1194, found:
323.1188. 1H NMR (400 MHz, DMSO-d6) d = 7.68–7.61 (m, 1H),
7.36 (br s, 1H), 7.24 (dt, J = 1.5, 7.7 Hz, 1H), 6.88 (d, J = 3.5 Hz,
1H), 6.74–6.67 (m, 2H), 6.63 (d, J = 3.5 Hz, 1H), 6.01 (d, J = 2.3 Hz,
1H), 3.88–3.74 (m, 1H), 2.81 (ddd, J = 5.7, 8.1, 13.5 Hz, 1H), 2.67
(q, J = 7.6 Hz, 2H), 1.67–1.55 (m, 1H), 1.55–1.42 (m, 1H), 1.13 (t,
J = 7.6 Hz, 3H), 0.85 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz,
4.7.7. 2-(4-Methylthiophen-2-yl)-3-phenyl-2,3-
dihydroquinazolin-4(1H)-one (8)
Prepared via procedure (A) from 2-nitro-N-phenylbenzamide
(0.87 mmol scale), yield: 145 mg, 52%. HRMS (FAB) C19H16N2OSNa+

4844
D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
DMSO-d6) d = 161.6, 146.4, 146.1, 142.1, 133.2, 127.4, 125.7, 122.6,
117.5, 115.2, 114.7, 66.8, 45.8, 22.7, 20.8, 15.7, 11.2.
(m, 1H), 2.81–2.71 (m, J = 5.1, 9.0, 9.0 Hz, 1H), 2.68 (q, J = 7.3 Hz,
2H), 1.13 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
d = 161.7, 146.7, 146.2, 141.8, 139.1, 133.3, 128.6, 128.4, 127.4,
126.2, 125.9, 122.6, 117.5, 114.9, 114.6, 67.0, 45.9, 33.6, 22.7, 15.7.
4.7.13. 2-(5-Ethylthiophen-2-yl)-3-isopropyl-2,3-
dihydroquinazolin-4(1H)-one (14)
Prepared via procedure B (1 mmol scale), yield: 142 mg, 47%.
HRMS (FAB) [C17H20N2OS+Na]+ predicted: 323.1194, found:
323.1180. 1H NMR (600 MHz, DMSO-d6) d = 7.66 (dd, J = 1.1,
7.7 Hz, 1H), 7.26 (d, J = 2.9 Hz, 1H), 7.22 (ddd, J = 1.5, 7.3, 8.2 Hz,
1H), 6.87 (d, J = 3.7 Hz, 1H), 6.71 (t, J = 7.5 Hz, 1H), 6.68 (d,
J = 8.1 Hz, 1H), 6.59 (d, J = 3.7 Hz, 1H), 6.06 (d, J = 2.9 Hz, 1H),
4.55 (spt, J = 6.8 Hz, 1H), 2.64 (q, J = 7.5 Hz, 2H), 1.23 (d,
J = 7.0 Hz, 3H), 1.11 (t, J = 7.5 Hz, 3H), 1.04 (d, J = 7.0 Hz, 3H). 13C
NMR (151 MHz, DMSO-d6) d = 161.2, 145.8, 145.7, 143.8, 132.9,
127.5, 125.2, 122.3, 117.6, 116.3, 114.8, 63.0, 45.6, 22.6, 20.3,
20.1, 15.5.
4.7.18. 2-(5-Ethylthiophen-2-yl)-3-(naphthalen-1-ylmethyl)-
2,3-dihydroquinazolin-4(1H)-one (19)
Prepared via procedure B (1 mmol scale), yield: 221 mg, 56%.
HRMS (FAB) [C25H22N2OS+Na]+ predicted: 421.1351, found:
421.1359. 1H NMR (400 MHz, DMSO-d6) d = 8.13–8.06 (m, 1H),
8.00–7.94 (m, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H),
7.58–7.46 (m, 4H), 7.34 (d, J = 2.3 Hz, 1H), 7.32–7.26 (m, 1H), 6.91
(d, J = 3.5 Hz, 1H), 6.80–6.74 (m, 1H), 6.68 (d, J = 8.3 Hz, 1H),
6.67–6.64 (m, 1H), 5.89 (d, J = 15.4 Hz, 1H), 5.80 (d, J = 2.5 Hz,
1H), 4.22 (d, J = 15.7 Hz, 1H), 2.69 (q, J = 7.5 Hz, 2H), 1.14 (dt,
J = 1.3, 7.4 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) d = 161.6,
146.6, 146.0, 140.8, 133.7, 133.5, 132.2, 131.1, 128.6, 128.2,
127.8, 126.5, 126.2, 126.0, 125.5, 123.6, 122.7, 117.8, 114.9,
114.7, 65.8, 44.4, 22.7, 15.7.
4.7.14. 3-Butyl-2-(5-ethylthiophen-2-yl)-2,3-
dihydroquinazolin-4(1H)-one (15)
Prepared via procedure B (1 mmol scale), yield: 167 mg, 53%.
HRMS (FAB) [C18H22N2OS+Na]+ predicted: 337.1351, found:
337.1366. 1H NMR (600 MHz, DMSO-d6) d = 7.64 (dd, J = 1.5,
8.1 Hz, 1H), 7.33 (d, J = 2.6 Hz, 1H), 7.27–7.21 (m, 1H), 6.88 (d,
J = 3.7 Hz, 1H), 6.73–6.67 (m, 2H), 6.64 (td, J = 1.0, 3.6 Hz, 1H),
6.00 (d, J = 2.6 Hz, 1H), 3.86 (ddd, J = 6.6, 8.4, 13.6 Hz, 1H), 2.83
(ddd, J = 5.5, 8.3, 13.7 Hz, 1H), 2.68 (dq, J = 1.1, 7.5 Hz, 2H), 1.59–
1.51 (m, 1H), 1.51–1.43 (m, 1H), 1.34–1.21 (m, 2H), 1.13 (t,
J = 7.5 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz, DMSO-
d6) d = 161.5, 146.4, 146.0, 142.0, 133.1, 127.4, 125.7, 122.5,
117.5, 115.2, 114.6, 66.7, 43.7, 29.5, 22.6, 19.5, 15.6, 13.6.
4.7.19. 2-(5-Ethylthiophen-2-yl)-3-(4-methoxybenzyl)-2,3-
dihydroquinazolin-4(1H)-one (20)
Prepared via procedure B (1 mmol scale), yield: 201 mg, 53%.
HRMS (FAB) [C22H22N2O2S+Na]+ predicted: 401.1300, found:
401.1285. 1H NMR (400 MHz, DMSO-d6) d = 7.69 (dd, J = 1.3,
7.6 Hz, 1H), 7.34 (d, J = 2.8 Hz, 1H), 7.32–7.19 (m, 3H), 6.90 (d,
J = 8.6 Hz, 2H), 6.86 (d, J = 3.5 Hz, 1H), 6.73 (t, J = 7.5 Hz, 1H), 6.69
(d, J = 7.8 Hz, 1H), 6.65 (d, J = 3.5 Hz, 1H), 5.84 (d, J = 2.5 Hz, 1H),
5.22 (d, J = 14.9 Hz, 1H), 3.80 (d, J = 14.9 Hz, 1H), 3.73 (s, 3H),
2.68 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) d = 161.6, 158.6, 146.6, 146.1, 141.3, 133.5, 129.3,
129.1, 127.6, 126.0, 122.7, 117.7, 114.8, 114.7, 113.9, 66.2, 55.1,
46.0, 22.7, 15.7.
4.7.15. 3-Cyclohexyl-2-(5-ethylthiophen-2-yl)-2,3-
dihydroquinazolin-4(1H)-one (16)
Prepared via procedure B (1 mmol scale), yield: 120 mg, 35%.
HRMS (FAB) [C20H24N2OS+Na]+ predicted: 363.1507, found:
363.1522. 1H NMR (400 MHz, DMSO-d6) d = 7.65 (dd, J = 1.2,
7.7 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 7.21 (ddd, J = 1.5, 6.8, 8.6 Hz,
1H), 6.85 (d, J = 3.7 Hz, 1H), 6.70 (t, J = 7.5 Hz, 1H), 6.66 (d,
J = 8.1 Hz, 163H), 6.58 (d, J = 3.4 Hz, 1H), 6.08 (d, J = 2.9 Hz, 1H),
4.23 (t, J = 11.9 Hz, 1H), 2.64 (q, J = 7.4 Hz, 2H), 1.76 (d,
J = 10.8 Hz, 2H), 1.68 (br s, 1H), 1.57 (br s, 3H), 1.39–1.15 (m,
4H), 1.11 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
d = 161.2, 145.7, 145.7, 144.0, 133.0, 127.6, 125.2, 122.4, 117.6,
116.4, 114.9, 63.0, 53.4, 30.3, 30.3, 25.7, 25.6, 24.9, 22.6, 15.5.
4.7.20. 2-(5-Ethylthiophen-2-yl)-3-(3-methoxybenzyl)-2,3-
dihydroquinazolin-4(1H)-one (21)
Prepared via procedure B (1 mmol scale), yield: 187 mg, 49%.
HRMS (FAB) [C22H22N2O2S+Na]+ predicted: 401.1300, found:
401.1282. 1H NMR (400 MHz, DMSO-d6) d = 7.69 (dd, J = 1.4,
7.7 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.32–7.22 (m, 2H), 6.92–6.82
(m, 4H), 6.78–6.69 (m, 2H), 6.64 (d, J = 3.3 Hz, 1H), 5.90 (d,
J = 2.8 Hz, 1H), 5.23 (d, J = 15.4 Hz, 1H), 3.90 (d, J = 15.4 Hz, 1H),
3.72 (s, 3H), 2.68 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) d = 165.2, 159.4, 146.6, 141.3, 139.1,
133.5, 129.6, 127.6, 126.0, 122.6, 119.6, 117.7, 114.8, 113.2,
112.4, 66.6, 55.0, 46.7, 22.7, 15.7.
4.7.16. 3-Benzyl-2-(5-ethylthiophen-2-yl)-2,3-
dihydroquinazolin-4(1H)-one (17)
Prepared via procedure B, HRMS (FAB) [C21H20N2OS+Na]+ pre-
dicted: 371.1194, found: 371.1186. 1H NMR (600 MHz, DMSO-d6)
d = 7.70 (dd, J = 1.8, 7.7 Hz, 1H), 7.37 (d, J = 2.6 Hz, 1H), 7.36–7.33
(m, 2H), 7.32–7.30 (m, 2H), 7.30–7.25 (m, 2H), 6.87 (d, J = 3.7 Hz,
1H), 6.74 (dt, J = 1.1, 7.5 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 6.65 (td,
J = 1.1, 3.3 Hz, 1H), 5.90 (d, J = 2.6 Hz, 1H), 5.26 (d, J = 15.4 Hz,
1H), 3.92 (d, J = 15.4 Hz, 1H), 2.69 (dq, J = 1.1, 7.5 Hz, 2H), 1.14 (t,
J = 7.5 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) d = 161.7, 146.6,
146.1, 141.2, 137.5, 133.4, 128.4, 127.6, 127.4, 127.1, 126.0,
122.6, 117.7, 114.8, 114.7, 66.5, 46.7, 22.7, 15.6.
4.7.21. 2-(5-Ethylthiophen-2-yl)-3-(2-methoxybenzyl)-2,3-
dihydroquinazolin-4(1H)-one (22)
Prepared via procedure B (1 mmol scale), yield: 234 mg, 62%.
HRMS (FAB) [C22H22N2O2S+Na]+ predicted: 401.1300, found:
401.1289. 1H NMR (400 MHz, DMSO-d6) d = 7.67 (dd, J = 1.4,
7.7 Hz, 1H), 7.41 (d, J = 2.8 Hz, 1H), 7.32–7.24 (m, 2H), 7.22 (d,
J = 7.3 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.93 (t, J = 7.5 Hz, 1H), 6.85
(d, J = 3.5 Hz, 1H), 6.77–6.69 (m, 2H), 6.67–6.63 (m, 1H), 5.92 (d,
J = 2.3 Hz, 1H), 5.11 (d, J = 15.9 Hz, 1H), 3.96 (d, J = 15.9 Hz, 1H),
3.81 (s, 3H), 2.69 (q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) d = 157.0, 146.2, 141.5, 133.5, 128.4,
127.7, 127.6, 125.9, 122.7, 120.4, 117.7, 114.9, 114.8, 110.7, 66.8,
55.4, 42.1, 22.7, 15.7.
4.7.17. 2-(5-Ethylthiophen-2-yl)-3-phenethyl-2,3-
dihydroquinazolin-4(1H)-one (18)
Prepared via procedure B (1 mmol scale), yield: 261 mg, 72%.
HRMS (FAB) [C22H22N2OS+Na]+ predicted: 385.1351, found:
385.1369. 1H NMR (400 MHz, DMSO-d6) d = 7.65 (d, J = 7.8 Hz,
1H), 7.37 (s, 1H), 7.33–7.25 (m, 3H), 7.25–7.17 (m, 3H), 6.92 (d,
J = 3.5 Hz, 1H), 6.77–6.68 (m, 2H), 6.68–6.62 (m, 1H), 6.04 (d,
J = 2.3 Hz, 1H), 4.09–3.93 (m, 1H), 3.14–3.01 (m, 1H), 2.98–2.84
4.7.22. 3-(2,4-Dimethoxybenzyl)-2-(5-ethylthiophen-2-yl)-2,3-
dihydroquinazolin-4(1H)-one (23)
Prepared via procedure B (1 mmol scale), yield: 202 mg, 50%.
HRMS (FAB)
C
23H24N2O3SNa+ predicted: 431.1405, found:
431.1405. 1H NMR (400 MHz, DMSO-d6) d = 7.69 (dd, J = 1.4,

D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4845
7.7 Hz, 1H), 7.36 (d, J = 2.8 Hz, 1H), 7.30–7.23 (m, 1H), 7.14 (d,
4.7.27. 2-(5-Ethylthiophen-2-yl)-3-((S)-1-phenylethyl)-2,3-
dihydroquinazolin-4(1H)-one (28)
J = 8.3 Hz, 1H), 6.84 (d, J = 3.5 Hz, 1H), 6.76–6.68 (m, 2H), 6.65 (d,
J = 3.5 Hz, 1H), 6.59 (d, J = 2.5 Hz, 1H), 6.52 (dd, J = 2.4, 8.5 Hz,
1H), 5.87 (d, J = 2.5 Hz, 1H), 5.08 (d, J = 15.4 Hz, 1H), 3.89 (d,
J = 15.4 Hz, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 2.69 (q, J = 7.5 Hz, 2H),
1.14 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) d = 161.6,
160.0, 158.1, 146.5, 146.0, 141.6, 133.4, 129.2, 127.6, 125.7,
122.7, 117.6, 117.0, 115.0, 114.7, 104.7, 98.4, 66.5, 55.5, 55.2,
41.5, 22.7, 15.7.
Prepared via procedure B (1 mmol scale), yield: 256 mg, 71%.
HRMS (FAB) [C22H22N2OS+Na]+ predicted: 385.135, found:
385.1342. 1H NMR (400 MHz, DMSO-d6) d = 7.71 (d, J = 7.8 Hz,
1H), 7.43–7.35 (m, 4H), 7.34–7.28 (m, 1H), 7.28–7.20 (m, 2H),
6.81 (d, J = 3.3 Hz, 1H), 6.74 (t, J = 7.5 Hz, 1H), 6.65 (d, J = 8.1 Hz,
1H), 6.61 (d, J = 3.3 Hz, 1H), 5.90 (q, J = 7.1 Hz, 1H), 5.78 (d,
J = 3.0 Hz, 1H), 2.66 (q, J = 7.7 Hz, 2H), 1.36 (d, J = 7.3 Hz, 3H),
1.12 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, CHLOROFORM-d)
d = 161.6, 146.0, 145.8, 143.6, 141.5, 133.3, 128.5, 127.8, 127.3,
126.8, 125.3, 122.5, 117.8, 115.9, 115.0, 63.0, 51.0, 22.6, 17.5, 15.6.
4.7.23. 2-(5-Ethylthiophen-2-yl)-3-(4-fluorobenzyl)-2,3-
dihydroquinazolin-4(1H)-one (24)
Prepared via procedure B (1 mmol scale) yield 130 mg, 36%.
HRMS (FAB) [C21H19FN2OS+Na]+ predicted: 389.1100, found:
389.1109. 1H NMR (400 MHz, DMSO-d6) d = 7.69 (dd, J = 1.6,
7.7 Hz, 1H), 7.40 (d, J = 2.8 Hz, 1H), 7.38–7.32 (m, 2H), 7.31–7.25
(m, 1H), 7.19–7.12 (m, 2H), 6.87 (d, J = 3.5 Hz, 1H), 6.79–6.69 (m,
2H), 6.64 (td, J = 1.0, 3.5 Hz, 1H), 5.94 (d, J = 2.5 Hz, 1H), 5.16 (d,
J = 15.4 Hz, 1H), 3.98 (d, J = 15.4 Hz, 1H), 2.68 (dq, J = 0.9, 7.5 Hz,
2H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
d = 161.8, 146.7, 146.2, 141.3, 133.8, 133.8, 133.5, 129.6, 129.5,
127.6, 127.4, 126.1, 122.6, 117.7, 115.3, 115.1, 114.8, 114.8, 66.7,
46.3, 22.7, 15.7.
4.7.28. 2-(5-Ethylthiophen-2-yl)-3-((R)-1-phenylethyl)-2,3-
dihydroquinazolin-4(1H)-one (29)
Prepared via procedure B (1 mmol scale), yield: 212 mg, 59%.
HRMS (FAB) [C22H22N2OS+Na]+ predicted: 385.1351, found:
385.1335. 1H NMR (400 MHz, DMSO-d6) d = 7.71 (dd, J = 1.5,
7.8 Hz, 1H), 7.42–7.35 (m, 4H), 7.34–7.28 (m, 1H), 7.28–7.22 (m,
2H), 6.81 (d, J = 3.5 Hz, 1H), 6.78–6.70 (m, 1H), 6.65 (d, J = 8.1 Hz,
1H), 6.61 (d, J = 3.5 Hz, 1H), 5.90 (q, J = 7.2 Hz, 1H), 5.78 (d,
J = 3.0 Hz, 1H), 2.66 (q, J = 7.5 Hz, 2H), 1.36 (d, J = 7.3 Hz, 3H),
1.12 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) d = 161.5,
146.0, 145.8, 143.6, 141.5, 133.3, 128.5, 127.8, 127.2, 126.8,
125.3, 122.5, 117.8, 115.9, 115.0, 63.0, 51.0, 22.6, 17.5, 15.6.
4.7.24. 2-(5-Ethylthiophen-2-yl)-3-(3-fluorobenzyl)-2,3-
dihydroquinazolin-4(1H)-one (25)
Prepared via procedure B (1 mmol scale), yield: 172 mg, 47%.
HRMS (FAB) [C21H19FN2OS+Na]+ predicted: 389.1100, found:
389.1082. 1H NMR (400 MHz, Acetone) d = 7.70 (dd, J = 1.1,
8.0 Hz, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.41–7.33 (m, 1H), 7.29 (dt,
J = 1.6, 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.14–7.05 (m, 2H),
6.88 (d, J = 3.3 Hz, 1H), 6.78–6.71 (m, 2H), 6.67–6.62 (m, 1H),
6.00 (d, J = 2.5 Hz, 1H), 5.16 (d, J = 15.7 Hz, 1H), 4.06 (d,
J = 15.7 Hz, 1H), 2.68 (q, J = 7.5 Hz, 2H), 1.13 (t, J = 7.5 Hz, 3H). 13C
NMR (101 MHz, Acetone) d = 163.5, 161.9, 161.0, 146.7, 146.3,
141.2, 140.8, 140.7, 133.6, 130.4, 130.3, 127.6, 126.2, 123.4,
123.4, 122.6, 117.8, 114.9, 114.7, 114.2, 114.0, 113.7, 67.0, 46.7,
22.7, 15.7.
4.7.29. 2-(5-Ethylthiophen-2-yl)-3-(4-fluorobenzyl)-5-methyl-
2,3-dihydroquinazolin-4(1H)-one (30)
Prepared via procedure A from N-(4-fluorobenzyl)-2-methyl-6-
nitrobenzamide (0.5 mmol scale), yield: 136 mg, 72%. HRMS (FAB)
[C22H21FN2OS+Na]+ predicted: 403.1256, found: 403.1262. 1H NMR
(400 MHz, DMSO-d6) d = 7.50 (s, 1H), 7.34 (dd, J = 5.6, 8.6 Hz, 2H),
7.21 (d, J = 2.5 Hz, 1H), 7.19–7.13 (m, 2H), 7.11 (dd, J = 2.0, 8.1 Hz,
1H), 6.85 (d, J = 3.5 Hz, 1H), 6.67–6.60 (m, 2H), 5.89 (d, J = 2.5 Hz,
1H), 5.16 (d, J = 15.2 Hz, 1H), 3.97 (d, J = 15.4 Hz, 1H), 2.67 (q,
J = 7.5 Hz, 2H), 2.21 (s, 3H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) d = 161.9, 146.6, 144.0, 141.4, 134.4, 129.6,
129.5, 127.5, 126.4, 126.1, 122.6, 115.3, 115.1, 115.0, 114.8, 66.8,
46.3, 22.7, 20.2, 15.8.
4.7.25. 2-(5-Ethylthiophen-2-yl)-3-(4-nitrobenzyl)-2,3-
dihydroquinazolin-4(1H)-one (26)
4.7.30. 2-(5-Ethylthiophen-2-yl)-3-(4-fluorobenzyl)-6-methyl-
2,3-dihydroquinazolin-4(1H)-one (31)
Prepared via procedure B (1 mmol scale), yield: 230 mg, 58%.
HRMS (FAB) [C21H19N3O3S+Na]+ predicted: 416.1045, found:
416.1061. 1H NMR (400 MHz, DMSO-d6) d = 8.27–8.12 (m, 2H),
7.69 (dd, J = 1.5, 8.1 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.49 (d,
J = 2.3 Hz, 1H), 7.36–7.25 (m, 1H), 6.89 (d, J = 3.5 Hz, 1H), 6.82–
6.71 (m, 2H), 6.63 (td, J = 0.9, 3.5 Hz, 1H), 6.07 (d, J = 2.5 Hz, 1H),
5.15 (d, J = 16.2 Hz, 1H), 4.29 (d, J = 16.2 Hz, 1H), 2.67 (q,
J = 7.4 Hz, 2H), 1.12 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-
d6) d = 162.1, 146.9, 146.6, 146.4, 146.0, 141.1, 133.7, 128.4,
127.6, 126.4, 123.5, 122.6, 117.8, 114.9, 114.6, 67.4, 47.0, 22.7,
15.8.
Prepared via procedure A from N-(4-fluorobenzyl)-5-methyl-
2-nitrobenzamide (0.5 mmol scale), yield: 88 mg 46%. HRMS
(FAB) C22H21FN2OSNa+ predicted: 403.1256, found: 403.1250. 1H
NMR (400 MHz, Acetone) d = 7.50 (s, 1H), 7.34 (dd, J = 5.6, 8.1 Hz,
2H), 7.23–7.07 (m, 4H), 6.85 (d, J = 3.5 Hz, 1H), 6.71–6.54
(m, 2H), 5.90 (d, J = 2.3 Hz, 1H), 5.16 (d, J = 15.4 Hz, 1H), 3.97
(d, J = 15.4 Hz, 1H), 2.67 (q, J = 7.4 Hz, 2H), 2.21 (s, 3H), 1.13 (t,
J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) d = 161.9, 146.6,
144.0, 141.4, 134.4, 129.6, 129.5, 127.5, 126.4, 126.1, 122.6,
115.3, 115.1, 115.0, 114.8, 66.8, 46.3, 22.7, 20.1, 15.8.
4.7.26. 2-(5-Ethylthiophen-2-yl)-3-(3-nitrobenzyl)-2,3-
dihydroquinazolin-4(1H)-one (27)
4.7.31. 2-(5-Ethylthiophen-2-yl)-3-(4-fluorobenzyl)-7-methyl-
2,3-dihydroquinazolin-4(1H)-one (32)
Prepared via procedure B (1 mmol scale), yield: 181 mg, 46%.
HRMS (FAB) [C21H19N3O3S+Na]+ predicted: 416.1045, found:
416.1038. 1H NMR (400 MHz, DMSO-d6) d = 8.17–8.04 (m, 2H),
7.76 (d, J = 7.8 Hz, 1H), 7.74–7.67 (m, 1H), 7.67–7.56 (m, 1H),
7.48 (d, J = 2.3 Hz, 1H), 7.30 (dt, J = 1.6, 7.6 Hz, 1H), 6.88 (d,
J = 3.5 Hz, 1H), 6.83–6.71 (m, 2H), 6.66–6.58 (m, 1H), 6.13 (d,
J = 2.5 Hz, 1H), 5.07 (d, J = 15.4 Hz, 1H), 4.39 (d, J = 15.7 Hz, 1H),
2.65 (q, J = 7.5 Hz, 2H), 1.10 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) d = 162.2, 147.7, 146.8, 146.4, 141.3, 140.4, 134.3,
133.7, 129.9, 127.6, 126.4, 122.6, 122.1, 117.8, 114.9, 114.6, 67.4,
46.9, 22.7, 15.7.
Prepared via procedure A from N-(4-fluorobenzyl)-4-methyl-2-
nitrobenzamide (0.5 mmol scale), yield: 157 mg, 82%. HRMS (FAB):
C
22H21FN2OSNa+ predicted: 403.1256, found: 403.1241. 1H NMR
(400 MHz, DMSO-d6) d = 7.57 (d, J = 7.8 Hz, 1H), 7.37–7.29 (m,
3H), 7.19–7.11 (m, 2H), 6.85 (d, J = 3.5 Hz, 1H), 6.63 (td, J = 0.9,
3.4 Hz, 1H), 6.56 (dd, J = 1.0, 8.1 Hz, 1H), 6.51 (s, 1H), 5.90 (d,
J = 2.5 Hz, 1H), 5.15 (d, J = 15.2 Hz, 1H), 3.95 (d, J = 15.4 Hz, 1H),
2.68 (q, J = 7.6 Hz, 2H), 1.16–1.11 (m, 3H). 13C NMR (101 MHz,
DMSO-d6) d = 162.1, 146.4, 143.9, 142.0, 134.1, 134.0, 133.9,
129.6, 129.6, 125.5, 125.5, 122.9, 122.7, 117.5, 115.4, 115.2,
115.2, 66.1, 46.5, 22.7, 16.8, 15.6.

4846
D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4.7.32. 2-(5-Ethylthiophen-2-yl)-3-(4-fluorobenzyl)-8-methyl-
2,3-dihydroquinazolin-4(1H)-one (33)
131.2, 131.1, 130.1, 130.0, 126.7, 123.2, 115.7, 115.6, 111.9, 105.7,
105.6, 101.1, 100.9, 67.2, 46.7, 23.2, 16.2.
Prepared via procedure A from N-(4-fluorobenzyl)-3-methyl-2-
nitrobenzamide (0.5 mmol scale), yield: 93 mg, 50%. HRMS (FAB)
[C22H21FN2OS+Na]+ predicted: 403.1256, found: 403.1265. 1H
NMR (400 MHz, DMSO-d6) d = 7.57 (d, J = 8.1 Hz, 1H), 7.45–7.32
(m, 2H), 7.18 (t, J = 7.8 Hz, 3H), 6.97 (d, J = 3.5 Hz, 1H), 6.81 (d,
J = 3.3 Hz, 1H), 6.71–6.65 (m, 1H), 6.62 (d, J = 3.3 Hz, 1H), 5.90 (d,
J = 3.8 Hz, 1H), 5.27 (d, J = 15.9 Hz, 1H), 4.04 (d, J = 14.9 Hz, 1H),
2.67 (q, J = 7.6 Hz, 2H), 2.07 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) d = 162.1, 146.4, 142.0, 134.1, 129.7,
129.6, 125.5, 125.5, 122.9, 122.8, 117.6, 115.4, 115.2, 66.1, 46.5,
22.7, 16.8, 15.7.
4.7.37. 5-(Butylamino)-N-(4-fluorobenzyl)-2-nitrobenzamide
(39)
5-Fluoro-N-(4-fluorobenzyl)-2-nitrobenzamide (605 mg, 2 mmol)
was dissolved in dimethoxy ethane (8 mL) and treated with
butylamine (217 lL, 2.2 mmol) and triethylamine (278 lL,
2 mmol). The reaction was allowed to proceed for 36 h, after which
the solvent was removed in vacuo and the product was isolated as
a bright yellow solid by flash chromatography. Yield: 440 mg, 64%.
HRMS (ESI) Predicted (C18H20FN3O3+H)+: 346.1567, found:
346.1565. 1H NMR (400 MHz, CHLOROFORM-d) d = 7.97 (d,
J = 9.1 Hz, 1H), 7.46–7.30 (m, 2H), 7.09–6.93 (m, 2H), 6.54–6.37
(m, 2H), 6.21 (t, J = 5.6 Hz, 1H), 4.94 (t, J = 5.2 Hz, 1H), 4.55 (d,
J = 5.8 Hz, 2H), 3.26–3.06 (m, 2H), 1.66–1.54 (m, 2H), 1.40 (qd,
J = 7.4, 14.9 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz,
CHLOROFORM-d) d = 167.9, 163.4, 161.0, 153.0, 135.8, 133.9,
133.4, 133.4, 129.8, 129.7, 127.8, 115.6, 115.4, 111.1, 110.9, 43.4,
43.0, 30.9, 20.1, 13.7.
4.7.33. 6-Chloro-2-(5-ethylthiophen-2-yl)-3-(4-fluorobenzyl)-
2,3-dihydroquinazolin-4(1H)-one (34)
Prepared via procedure B (1 mmol scale), yield: 335 mg, 84%).
HRMS (FAB) [C21H18FN2OS+Na]+ predicted: 423.0710, found:
423.0725. 1H NMR (400 MHz, DMSO-d6) d = 7.63 (dd, J = 2.5,
5.3 Hz, 2H), 7.42–7.29 (m, 3H), 7.20–7.12 (m, 2H), 6.88 (d,
J = 3.3 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.67–6.63 (m, 1H), 6.01 (d,
J = 2.5 Hz, 1H), 5.13 (d, J = 15.2 Hz, 1H), 4.01 (d, J = 15.2 Hz, 1H),
2.68 (q, J = 7.4 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) d = 160.7, 146.9, 145.0, 140.9, 133.4, 129.6, 129.6,
126.6, 126.3, 122.7, 121.4, 116.9, 115.8, 115.3, 115.1, 66.6, 46.4,
22.7, 15.7.
4.7.38. 6-(Butylamino)-2-(5-ethylthiophen-2-yl)-3-(4-
fluorobenzyl)-2,3-dihydroquinazolin-4(1H)-one (40)
5-(Butylamino)-N-(4-fluorobenzyl)-2-nitrobenzamide (368 mg,
1.1 mmol), was dissolved in methanol (5 mL). To the solution
was added 10% Pd/C (150 mg) and ammonium formate (64 mg,
1 mmol). After 1 h the reaction was filtered. The filtrate was
adjusted to a volume 2 mL in methanol and then treated with
4.7.34. 7-Chloro-2-(5-ethylthiophen-2-yl)-3-(4-fluorobenzyl)-
2,3-dihydroquinazolin-4(1H)-one (35)
5-ethyl-2-thiophenecarboxaldehyde (138 lL, 1.1 mmol) and scan-
Prepared via procedure B (1 mmol scale), yield: 172 mg, 43%).
HRMS (FAB) [C21H18FN2OS+Na]+ predicted: 423.0710, found:
423.0718. 1H NMR (400 MHz, DMSO-d6) d = 7.71–7.65 (m, 2H),
7.33 (dd, J = 5.7, 8.5 Hz, 2H), 7.15 (t, J = 8.8 Hz, 2H), 6.89 (d,
J = 3.5 Hz, 1H), 6.79–6.73 (m, 2H), 6.68–6.63 (m, 1H), 6.02 (d,
J = 2.5 Hz, 1H), 5.12 (d, J = 15.4 Hz, 1H), 3.99 (d, J = 15.4 Hz, 1H),
2.69 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) d = 161.1, 160.2, 147.2, 147.0, 140.9, 138.1, 133.6,
133.6, 129.7, 129.6, 129.5, 126.4, 122.8, 117.8, 115.3, 115.1,
113.9, 113.4, 66.7, 46.3, 22.7, 15.7.
dium(III) triflate (49 mg, 0.1) mmol. The reaction was microwave
irradiated at 100 °C for 1 h, after which the solvent was removed
in vacuo. The DHQZ (bright yellow solid) product was isolated by
silica gel chromatography with a solvent gradient of 30–50% ethyl
acetate in hexanes. The chromatographed product was subse-
quently purified by recrystallization from ethyl acetate. Yield
236 mg, 54%. HRMS (ESI) Predicted (C25H28FN3OS+H)+: 438.2015,
found: 438.2002. 1H NMR (400 MHz, CHLOROFORM-d) d = 7.34–
7.26 (m, 4H), 7.06–6.95 (m, 2H), 6.75–6.68 (m, 2H), 6.58–6.52
(m, 2H), 5.69 (s, 1H), 5.51 (d, J = 14.8 Hz, 1H), 4.14 (br s, 1H),
3.87 (d, J = 15.3 Hz, 1H), 3.17–3.09 (m, 2H), 2.73 (dq, J = 1.0,
7.5 Hz, 2H), 1.67–1.56 (m, 2H), 1.44 (qd, J = 7.3, 14.9 Hz, 2H), 1.23
(t, J = 7.5 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CHLO-
ROFORM-d) d = 163.3, 162.9, 160.9, 148.4, 139.8, 137.2, 132.9,
132.8, 129.6, 129.5, 126.0, 122.3, 120.9, 117.8, 117.1, 115.5,
115.3, 112.5, 67.5, 46.4, 45.3, 31.2, 23.4, 20.2, 15.6, 13.8.
4.7.35. 2-(5-Ethylthiophen-2-yl)-6-fluoro-3-(4-fluorobenzyl)-
2,3-dihydroquinazolin-4(1H)-one (36)
Prepared via procedure A from 5-fluoro-N-(4-fluorobenzyl)-2-
nitrobenzamide (0.5 mmol scale), yield: 187 mg, 97%. HRMS
(FAB) [C21H18F2N2OS+Na]+ predicted: 407.1006, found: 407.1018.
1H NMR (400 MHz, DMSO-d6) d = 7.43–7.37 (m, 2H), 7.34 (dd,
J = 5.6, 8.3 Hz, 2H), 7.23–7.12 (m, 3H), 6.87 (d, J = 3.5 Hz, 1H),
6.76 (dd, J = 4.5, 8.8 Hz, 1H), 6.66–6.62 (m, 1H), 5.97 (d,
J = 2.5 Hz, 1H), 5.12 (d, J = 15.4 Hz, 1H), 4.02 (d, J = 15.2 Hz, 1H),
2.68 (q, J = 7.6 Hz, 2H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (151 MHz,
DMSO-d6) d = 162.2, 161.0, 160.6, 155.8, 154.2, 146.7, 142.8, 140.9,
133.6, 133.6, 129.6, 129.5, 126.2, 122.6, 121.1, 120.9, 116.7, 116.6,
115.5, 115.5, 115.2, 115.1, 112.8, 112.6, 66.8, 46.4, 22.7, 15.6.
4.7.39. 2-(5-ethylthiophen-2-yl)-3-(4-fluorobenzyl)quinazolin-
4(3H)-one (41)
Prepared via procedure B (0.29 mmol scale) using ethanol as
solvent and CuCl2 (0.3 mmol) instead of ScOTf3, yield: 99 mg,
94%. HRMS (FAB) [C21H17FN2OS+Na]+ predicted: 387.0943, found:
387.0938. 1H NMR (400 MHz, DMSO-d6) d = 8.14 (dd, J = 1.3,
8.3 Hz, 1H), 7.85 (dt, J = 1.4, 7.6 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H),
7.59–7.50 (m, 1H), 7.19–7.07 (m, 5 H), 6.83 (dd, J = 0.8, 3.8 Hz,
1H), 5.45 (s, 2H), 2.81 (q, J = 7.5 Hz, 2H), 1.23 (t, J = 7.5 Hz, 3H).
13C NMR (101 MHz, DMSO-d6) d = 162.5, 161.8, 160.1, 152.0,
150.0, 147.0, 135.0, 134.0, 133.1, 133.1, 129.6, 128.1, 128.0,
127.2, 126.6, 124.7, 119.7, 115.8, 115.5, 48.2, 22.8, 15.8.
4.7.36. 2-(5-Ethylthiophen-2-yl)-7-fluoro-3-(4-fluorobenzyl)-
2,3-dihydroquinazolin-4(1H)-one (37)
Prepared via procedure A from 4-fluoro-N-(4-fluorobenzyl)-
2-nitrobenzamide (0.5 mmol scale), yield: 167 mg, 88%. HRMS
(FAB) [C21H18F2N2OS+Na]+ predicted: 407.1006, found: 407.1025.
1H NMR (600 MHz, DMSO-d6) d = 7.74 (dd, J = 6.6, 8.8 Hz, 1H),
7.66 (d, J = 2.6 Hz, 1H), 7.37–7.30 (m, 2H), 7.18–7.12 (m, 2H),
6.89 (d, J = 3.7 Hz, 1H), 6.65 (td, J = 1.0, 3.6 Hz, 1H), 6.54 (dt,
J = 2.4, 8.7 Hz, 1H), 6.49 (dd, J = 2.2, 10.6 Hz, 1H), 5.99 (d,
J = 2.6 Hz, 1H), 5.13 (d, J = 15.4 Hz, 1H), 3.98 (d, J = 15.4 Hz, 1H),
2.69 (q, J = 7.3 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H). 13C NMR (151 MHz,
DMSO-d6) d = 165.4, 162.7, 161.6, 148.7, 147.4, 141.5, 134.1, 134.1,
4.7.40. 2-(5-Ethylthiophen-2-yl)-3-(4-fluorobenzyl)-1-methyl-
2,3-dihydroquinazolin-4(1H)-one (43)
Prepared via procedure B (1 mmol scale), yield: 333 mg, 88%.
HRMS (FAB) [C22H21FN2OS+Na]+ predicted: 403.1256, found:
403.1266. 1H NMR (400 MHz, DMSO-d6) d = 7.80 (dd, J = 1.5,
7.6 Hz, 1H), 7.46–7.40 (m, 1H), 7.40–7.33 (m, 2H), 7.21–7.12
(m, 2H), 6.93 (d, J = 3.5 Hz, 1H), 6.88 (dt, J = 1.0, 7.5 Hz, 1H), 6.69

D. W. Carney et al. / Bioorg. Med. Chem. 22 (2014) 4836–4847
4. Boothpur, R.; Brennan, D. C. J. Clin. Virol. 2010, 47, 306.
4847
(d, J = 8.1 Hz, 1H), 6.65 (td, J = 1.0, 3.5 Hz, 1H), 5.92 (s, 1H), 5.13 (d,
J = 15.4 Hz, 1H), 3.95 (d, J = 15.4 Hz, 1H), 2.77 (s, 3H), 2.68–2.60 (m,
2H), 1.11 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
d = 161.3, 147.0, 146.4, 134.1, 133.5, 133.5, 129.6, 129.5, 127.8,
127.7, 122.6, 118.3, 115.3, 115.1, 112.9, 73.3, 46.3, 34.9, 22.6, 15.5.
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Acknowledgements
This project was funded in part by a J&J-Brown University Trans-
lational Award to J.K.S and W.J.A. D.C.W was funded by a dissertation
fellowship from the Chemistry Department at Brown University.
Research in the Atwood laboratory is funded by P01NS065719
(W.J.A.), R01NS043097 (W.J.A.), and Ruth L. Kirschstein National
14. Stechmann, B.; Bai, S.; Gobbo, E.; Lopez, R.; Merer, G.; Pinchard, S.; Panigai, L.;
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Research
Service
Awards
F32NS064870
(M.S.M.)
and
F32NS070687 (C.D.S.N.) from the National Institute of Neurological
Disorders and Stroke. Core facilities that support our work are
funded by P30GM103410 (W.J.A). Work in the DiMaio laboratory
is supported by P01CA16038 (D.D.) from the National Cancer
Institute.
18. Escalante, J.; Ortà z-Nava, C.; Flores, P.; Priego, J. M.; Garcà a-Martà nez, C.
Molecules 2007, 12, 173.
Supplementary data
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Supplementary data associated with this article can be found, in
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